Citation
Comparison of the pharmacokinetics and toxicity of sulfisoxazole in humans and two monogastric animal species

Material Information

Title:
Comparison of the pharmacokinetics and toxicity of sulfisoxazole in humans and two monogastric animal species
Creator:
Suber, Robert Lee, 1949-
Copyright Date:
1979
Language:
English
Physical Description:
xvi, 150 leaves : graphs ; 28 cm.

Subjects

Subjects / Keywords:
Administered dose ( jstor )
Albumins ( jstor )
Dogs ( jstor )
Dosage ( jstor )
Intravenous injections ( jstor )
Isoxazoles ( jstor )
Oral administration ( jstor )
Plasmas ( jstor )
Sulfonamides ( jstor )
Swine ( jstor )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Sulfisoxazole -- Physiological effect
Sulfonamides ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 144-148.
Additional Physical Form:
Also available on World Wide Web
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Robert L. Suber.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright Robert Lee Suber. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
020706386 ( ALEPH )
06082430 ( OCLC )
AAB7227 ( NOTIS )

Downloads

This item has the following downloads:


Full Text
COMPARISON OF THE PHARMACOKINETICS AND TOXICITY OF SULFISOXAZOLE
IN HUMANS AND TWO MONOGASTRIC ANIMAL SPECIES

By

ROBERT L. SUBER

A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY GF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1979



Copyright 1979
by

Robert L. Suber



ACKNOWLEDGEMENTS

The author wishes to express his sincerest appreciation and thanks
to Dr. George T. Edds, chairman of the supervisory committee, for his
assistance and guidance throughout this investigation.

The author also wishes to acknowledge the helpful criticism and
suggestions of Dr. Paul T. Cardeilhac for his assistance in pharmacology,
Dr. John C. Gudat for his collaboration in clinical chemistry, and
Dr. George Torosian and Dr. Charles Lee for their assistance in pharmaco-
kinetics.

Deepest appreciation and special thanks are expressed to Dr. Orlando
Osuna, Mr. Gary Neff, and Mrs. Rita Bellis Bortell for their assistance
and friendship throughout the investigation.

The author also wishes to thank Dr. John A. Cornell for his
assistance in the programming and interpretation of the statistical
analysis of the bilirubin data.

The author's deepest appreciation and love are extended to Mrs.
Christine Suber for her understanding, patience and assistance throughout

this investigation.

iii



TABLE OF CONTENTS

ACKNOWLEDGEMENTS. 2. 2 6 we ee we ee ee ee te ee
LIST OF TABLES. . 2. 2. 1. 6 ew we ee ee ee ee te ee
LIST OF FIGURES .. .. 1. 1 6 ee ew ew ee ee ee ee
ABSTRACT. . 2. 6 2 6 ee wee ee ewe ee we te te
INTRODUCTION. . 2. 2... 2 ee eee ee ee
REVIEW OF LITERATURE. . . 2. 1 1 1 1 ee we ee es
Sulfisoxazole. . . 1 2 6 ww ee ew we we ee ee eee
Blood Concentration of Sulfisoxazole. .....
Metabolism of Sulfisoxazole .........
Protein Binding of Sulfisoxazole. ...
Toxic Effects of Sulfisoxazole. ......
Urinary Excretion of Sulfisoxazole. .........
Pharmacokinetics . . 2... 1. 2 ee ee ee ee ee ee ee
Absorption Rate Constant and Bioavailability. ....

Distribution. . .... . ee ee we ew ee te ee

Elimination... 2... 6 6 ee we ew ee ee

Plasma Protein Binding. .......4..+e.2.-.

Two Compartment Model ........446-.
MATERIALS AND METHODS . . ..... ee ee we

High Performance Liquid Chromatographic Analysis of
Sulfonamides by Tonic Suppression. ..........

Materials .. . . 1 2 1 ww ee ee ee ee ee

iv

10

il

12

14

15



Page

Extraction and Separation .........+46+.+...... 18
Optimization of the Liquid Chromatographic Procedure. . . 30
Experimental Model . . 1. 1 ee ee ee ew we ww ew ew we ee) 680
Blood Samples . 2... ee ww ew ee eee eee we ee 8
Serum bilirubin. . .......-..24..66.42 +... 32

Serum albumin. . 2. 2-2 ee ee ew ee we we ww ew 8B

Serum sulfisoxazole. . . .. 1... 2 2 ee ew ee ee B34

Urinary Sulfisoxazole . . . 1. 1 6 6 ee ee ee ee ew B85

Analysis of Data. . . 1... 6 ee ee ew ee ee we ww we 36
In Vitro Plasma Protein Binding .........+.444.. 36

RESULTS AND DISCUSSION. . 2. 2. 1 1 2 eee ww eee we ew ew ee 8

Pharmacokinetics of Sulfisoxazole. ...... 4... 2... . 38
Administration of Sulfisoxazole to Dogs ......... 39
Intravenous administration . ....... 2... 6. 39

Oral administration. ............. 2... 43
Administration of Sulfisoxazole to Swine. ........ 45

Intravenous administration ........4. 6+... 45
Oral administration. ........ 6.6... 6«.e.. 5)
Administration of Sulfisoxazole to Humans ........ 54

Comparison of the Pharmacokinetics of Sulfisoxazole
in Dogs, Swine, and Humans. ........ 6.6.6.2... 58

Serum Bilirubin Concentrations ....... +... 6 « «© © « s 70

Dogs--Intravenous Administration. ............ 70
Dogs--Oral Administration .........4+. +2466... 72
Comparison of Bilirubin Levels in Dogs. ......... 74
Swine--Intravenous Administration ............ 81



Swine--Oral Administration.
Comparison of Bilirubin Levels in Swine
Humans--Oral Administration .

Comparison of Bilirubin Levels in Dogs, Swine,
and Humans. . ..... 56 2 se we ee

Conclusions Concerning Bilirubin Levels After
Administration of Sulfisoxazole .

Serum Albumin Concentrations
SUMMARY AND CONCLUSIONS
APPENDIX.
BIBLIOGRAPHY.

BIOGRAPHICAL SKETCH . ......+ 2...

vi

37

. 87

- 97

98

.103

.109

149



LIST OF TABLES

Table

1. Retention time (minutes) of sulfonamides in a water/
methanol (50/50) mobile phase with acetate buffer (PH
4.00) and without buffer (pH 7.45). ....... -. ss

2, Retention time (minutes) of sulfonamides in a water/
methanol (60/40) mobile phase with acetate buffer (pH
4.00) and without buffer (pH 7.45). ........4-..8.

3. Retention time (minutes) of sulfonamides in spiked human
plasma samples in a water/methanol (50/50) mobile phase
with acetate buffer (pH 4.00) and without buffer (pH 7.45).

4, Retention time (minutes) of sulfonamides in spiked human
plasma samples in a water/methanol (60/40) mobile phase
with acetate buffer (pH 4.00) and without buffer (pH 7.45).

5. Two compartment pharmacokinetic parameters in dogs
administered sulfisoxazole as a single intravenous dose. .

6. The mean amount of sulfisoxazole excreted in the urine of
6 dogs. 2. 6 6 6 we ee ee ee et et we ee we ee

7. Two compartment pharmacokinetic parameters in dogs adminis-~
tered sulfisoxazole as a single oral dose ........

8. Two compartment pharmacokinetic parameters in swine
administered sulfisoxazole as a single intravenous dose...

S. The mean amount of sulfisoxazole and acetylsulfisoxazole
(N+) excreted in the urine of 5 pigs as a percentage of

the dose. . . 2... ew ee eee we ew ee ke kk kk

10. The biological half-life of acetylsulfisoxazole in’) after
a single dose of sulfisoxazole. . ...... 2.206 2685068.

11. Two compartment pharmacokinetic parameters in swine
administered sulfisoxazole as a single oral dose. .....

12. Single compartment pharmacokinetic parameters in 6 humans
administered a 2.0 gm oral dose of sulfisoxazole. .....

13. The biological half-life (t,) of sulfisoxazole. ......
3

vil

LS
$=



Table

14.

15.

16.

22.

23.

24,

25.

26.

The biological half-life (t1,) of acetylsulfisoxazole,
the N4 metabolite of sulfisoxazole, .......

The mean distribution constants after intravenous
administration of sulfisoxazole ..........4.-

The ratio of distribution constants after administration
of sulfisoxazole. . . . 1 1 1 we ew ew ew ew ee ee et te

Comparison of the volumes of distribution in dogs, swine,
and humans after administration of sulfisoxazole. ...

Comparison of the bioavailability of sulfisoxazole in
dogs, swine, and humans ....... 2. 1 2 we ee eee

The fraction of sulfisoxazole bound (fg) to human serum
proteins in vitro . 6. 6. 1 ee ee wee ew ew ee ee

Mean control values of serum bilirubin and albumin in
humans, dogs, and swine .........-. 6+ -+. + © ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in dog 1 after intravenous and oral administration of
sulfisoxazole . . 1... 6 ee wee ee ew we et ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in dog 2 after intravenous and oral administration of
sulfisoxazole . 1. 6. 1 6 1 ew ee eee ee we we ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in dog 3 after intravenous and oral administration of
sulfisoxazole . . 1... 1 ee ew ew ee ee ew ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in dog 4 after intravenous and oral administration of
sulfisoxazole . . 1. 6. 1 6 eee we we eee ww ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of suifisoxazole
in dog 5 after intravenous and oral administration of
sulfisoxazole . 1. 1 16 6 6 ee ewe we ee ee et

Serum concentrations of albumin, bilirubin, and
sulfisoxazcle and urinary concentrations of sulfisoxazole
in dos 5 after intravenous and oral administration of
sulfisoxazole . . 6. 6 1 we ew ee ee ee ee ee ee

viii

63

64

66

67

89

109

113

115

117

119



Table

27.

28.

29,

30.

31.

32.

33%

34.

35.

36.

37.

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in pig 1 after intravenous and oral administration of
sulfisoxazole . 2... 1. 6 1 ew we ee we ee ee ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in pig 2 after intravenous and oral administration of
sulfisoxazole . . 1. 1. 1 6 ew ew ew we we ee ee ee es

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concantrations of sulfisoxazole
in pig 3 after intravenous and oral administration of
sulfisoxazole 2... 6 6 ew we ee we ew we ee ee es

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in pig 4 after intravenous and oral administration of
sulfisoxazole . 2. 2. 2. 2. 2 6 6 6 ww ew we ee we we

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in pig 5 after intravenous and oral administration of
sulfisoxazole . 2... 1 1 6 ew eee ee ee ee ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in pig 6 after intravenous and oral administration of
sulfisoxazole . 2... 1. 1 we ee we ew we ee ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in human 1 after oral administration of sulfisoxazole. .

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in human 2 after oral administration of sulfisoxazole . .

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in human 3 after oral administration of sulfisoxazole . .

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in human 4 after oral administration of sulfisoxazole . .

Serum concentrations of albumin, bilirubin, and

sulfisoxazole and urinary concentrations of sulfisoxazole
in human 5 after oral administration of sulfisoxazole. .

1X

Page

121

he
nN
ni

127

129

131

135

134

135

136

137



Table

38.

39.

40.

Al.

42.

-_
2

Serum concentrations of albumin, bilirubin, and

sulfisoxazole and urinary concentrations of sulfisoxazole

in human 6 after oral administration of sulfisoxazole .

Means and standard deviations of serum albumin, total
bilirubin, conjugated bilirubin, and indirect bilirubin

in 6 dogs after intravenous administration of sulfisoxazole

Means and standard deviations of serum albumin, total
bilirubin, conjugated bilirubin, and indirect bilirubin
in 6 dogs after oral administration of sulfisoxazole. .

Means and standard deviations of serum albumin, total
bilirubin, conjugated bilirubin, and indirect bilirubin
in 6 swine after intravenous administration of
sulfisoxazcle . .. 1. 6 ee ew ew ee ee ee ee

Means and standard deviations of serum albumin, total
bilirubin, conjugated bilirubin, and indirect bilirubin
in 6 swine after oral administration of sulfisoxazole .

Means and standard deviations of serum albumin, total
bilirubin, conjugated bilirubin, and indirect bilirubin
in 6 humans after oral administration of sulfisoxazole.

138

139

140

141

142

143



LIST OF FIGURES

Figure

1.

10.

li.

Standard concentration curves of sulfanilamide,
sulfaguanidine, sulfamerazine, sulfamethazine, sulfa-
pyridine, sulfisoxazole, n4 acetylsulfisoxazole, and
sulfathiazole in a water/methanol (50/50) mobile phase
with acetate buffer at pH 4.00. ..........4.4.2.4..

Standard concentration curves of sulfanilamide,
sulfaguanidine, sulfamerazine, sulfamethazine, sulfa-
pyridine, sulfisoxazole, n4 acetylsulfisoxazole, and
sulfathiazole in a water/methanol (50/50) mobile phase
with an isocratic pH 7.45 ........ 2.0.4.6 42.80.2084

Standard concentration surves of sulfanilamide,
sulfaguanidine, sulfamerazine, sulfamethazine, sulfa-
pyridine, sulfisoxazole, v4 acetylsulfisoxazole, and
sulfathiazole in a water/methanol (60/40) mobile phase
with acetate buffer at pH 4.00. ........4...

Standard concentration curves of sulfanilamide,
sulfaguanidine, sulfamerazine, sulfamethazine, sulfa-
pyridine, sulfisoxazole, n4 acetylsulfisoxazole, and
sulfathiazole in a water/methanol (60.40) mobile phase
with an isocratic pH 7.45 .........4464..

Serum concentrations of "free" sulfisoxazole in dogs. ...
Serum concentrations of "free" sulfisoxazole in swine. .

. . 4) :
Serum concentrations of acetylsulfisoxazole (N’) in swine .

4
Serum concentrations of "free" and acetylsulfisoxazole (N°)
in humans . . . 1 6 6 ee ew ee ee ee ee ee

Mean serum bilirubin concentrations in dogs after
intravenous administration of sulfisoxazole .....

Mean serum bilirubin concentrations in dogs after oral
administration of sulfisoxazole .........4...

Comparison of the mean total bilirubin concentrations in

dogs after intravenous and oral administration of
sulfisoxazole . 2... 6 6 we ew ee ee ew we we ee ee tw

xi

Page

28

55

71

73



Figure

12.

13.

14.

15.

16.

17.

18.

19.

20.

23.

24,

Comparison of the mean conjugated bilirubin concentrations
in dogs after intravenous and oral administration of
sulfisoxazole . . 1... 1. 1 6 ww ew we ew wt tt we el

Comparison of the mean indirect bilirubin concentrations
in dogs after intravenous and oral administration of
sulfisoxazole . 2... 1. 1 ee eee ew ee ee ee et ee

Mean serum bilirubin concentrations in swine after
intravenous administration of sulfisoxazole .........

Mean serum bilirubin concentrations in swine after oral
administration of sulfisoxazole .......4... 4.604668

Comparison of the mean total bilirubin concentrations in
swine after intravenous and oral administration of
sulfisoxazole .... 1. 2. 6 1 0 ee we we ww wt lw lt

Comparison of the mean conjugated bilirubin concentrations
in swine after intravenous and oral administration of
sulfisoxazole . . 1. 2. 6. 1 1 1 ee eee ee ew ee et es

Comparison of the mean indirect bilirubin concentrations
in swine after intravenous and oral administration of
sulfisoxazole . . . 1... 1 ee ew ew we we ww we ee te ee

Mean serum bilirubin concentrations in humans after oral
administration of sulfisoxazole ......4...464+.048c088.

Comparison of the mean total bilirubin concentrations in
dogs and swine after intravenous administration of
sulfisoxazole . 1. 1. 1. 1. we we we we we ee ee ee

Comparison of the mean total bilirubin concentrations in
dogs and swine after oral administration of sulfisoxazole .

Comparison of the mean conjugated bilirubin concentrations
in dogs and swine after intravenous administration of
sulfisoxazole . . 2. 1. 1 2 6 eee we ee ee ee

Comparison of the mean conjugated bilirubin concentrations
in dogs and swine after oral administration of sulfisoxazole.

Comparison of the mean indirect bilirubin concentrations
in dogs and swine after intravenous administration of

sulfisoxazole . . .. 1. 2 2 © © te © © ew ew tt tw le lt el

Comparison of the mean indirect bilirubin concentrations
in dogs and swine after oral administration of sulfisoxazole.

Mean serum albumin concentrations in dogs after intravenous
and oral administration of sulfisoxazole. ..........

xii

77

78

80

82

84

85

86

88

91

92

93

99



Figure Page

27.

Mean serum albumin concentrations in swine after intravenous
and oral administration of sulfisoxazole ......... .. L00

Mean serum albumin concentrations in humans after oral
administration of sulfisoxazole.

“

fis
fae
bh.



Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy

COMPARISON OF THE PHARMACOKINETICS AND TOXICITY OF SULFISOXAZOLE
IN HUMANS AND TWO MONOGASTRIC ANIMAL SPECIES

By
ROBERT L. SUBER
August 1979

Chairman: George T. Edds
Major Department: Animal Science

This experiment was designed to 1) develop a high performance liquid
chromatographic procedure for separation and quantitation of sulfonamides,
especiaily sulfisoxazole and its metabolites and 2) to compare the
toxicity and pharmacokinetics of sulfisoxazole, a water-soluble sulfona-
mide, in 3 monogastric species, humans, dogs, and swine.

An accurate, sensitive, and specific high performance liquid
chromatographic technique was developed for separation of sulfanilamide,
sulfaguanidine, sulfamerazine, sulfamethazine, sulfapyridine, sulfisoxa-
zole, acetylsulfisoxazole (n’)y, and sulfathiazole from a biological
matrix. Spiked serum samples were analyzed by injecting 20.0 ul onto au
Bondapak Cig column with absorbance measured at 254 nanometers. The
mobile phase was 50.0% double-distilled water/50.0% methanoi (pH 7.45)
for the separation and quantitation of sulfanilamide and sulfaguanidine.
The 6 other sulfonamides used a 60.0% double-distilled water/40.0%
methanol with acetate buffer (pH 4.00) mobile phase. Serum could be
injected directly in the system, equipred with an inline filter con-

taining Corasil Cig> deproteinated with methanol (1:1) before injection

Xiv



or filtered through 0.45 up filters without loss of resolution. Urinary
extraction of sulfisoxazole and its metabolite, acetylsulfisoxazole cnt) |
involved cooling 25.0 ml of urine to 4°C, acidifying to pH 3.00 with
concentrated hydrochloric acid and extraction into chloroform. Total
serum and urinary sulfisoxazole were determined by boiling the acid
hydrolysate for 1.0 hour at 100°C and performing the respective extrac-
tion and separation techniques previously described.

Six individuals from each species were administered a solution of
sulfisoxazole after a 12-hour fasting period. The trial was conducted
over a 72-hour period after intravenous administration and a 96-hour
period after oral administration in dogs and swine and over an 8-hour
period after oral administration in humans.

Analysis of the pharmacokinetic profiles of each species showed
that dogs were more similar to humans than swine. However, differences
did exist for biological half-lives, distribution constants, volumes of
distribution, bioavailability, metabolism to the acetyl (Nn) derivative,
and urinary excretion rates among all 3 species. Total and conjugated
bilirubin showed small but statistically significant increases (p < .01)
in dogs after oral and intravenous administration of sulfisoxazole
(100 mg/kg) and in swine after oral administration of sulfisoxazole
(100 mg/kg). The potentially toxic, indirect, or unconjugated bilirubin
showed small but statistically significant (p < .01) increases in dogs
after oral administration of sulfisoxazole. A similar increase was not
observed in swine or humans.

Total and conjugated bilirubin were significantly (p < .01) correlated
in dogs after oral and intravenous administration and in swine after oral

administration of sulfisoxazole. The increase in conjugated bilirubin,

xV



along with a concomitant increase in total bilirubin, could be due to
hepatic induction of glucuronidating capacity or regurgitation of con-
jugated bilirubin from the hepatocyte instead of excretion into the bile.
There was also a significant negative correlation (p < .01) in con-
jugated and indirect bilirubin, while total bilirubin increased, in

dogs after oral and intravenous administration of sulfisoxazole.

Dogs appear to be more similar to humans in the pharmacokinetics of
sulfisoxazole and the potential toxicity of bilirubin could be greatest
in dogs. The toxicity of indirect or unconjugated bilirubin is dependent
on a species difference with dogs being the most affected and ona
route of administration difference with the oral route being more likely

to induce toxicity than an intravenous route of administration.

°

KVL



INTRODUCTION

Sulfisoxazole, a pharmaceutical agent regularly used for urinary
tract infections, may be a potentially toxic compound due to the increase
in serum bilirubin levels after administration, has the potential for
inducing bacterial resistance due to long term, low level exposure
tarough the food chain and is a possible environmental contaminant,
especially in urban sewage. In order to evaluate the absorption, dis-
tribution, metabolism, and excretion of sulfisoxazole, it was necessary
to establish the pharmacckinetic parameters in human and animal model
systems.

This trial was to 1) find a rapid high performance liquid chromato-
graphic method for detecting sulfonamides, especially sulfisoxazole and
its metabolites, in a biological matrix, and 2) to compare the pharmaco-
kinetics and potential toxicity of sulfisoxazole in humans, dogs, and
swine in order to define a better animal model to correlate with human

research.



REVIEW OF LITERATURE

The medical and public health importance of the discovery of sulfona-
mides as the first effective chemotherapeutic agents was reflected by a
sudden decline in morbidity and mortality due to infectious diseases (24).

Paul Ehrlich, the founder of modern chemotherapy, initiated the
concept of the use of azo dyes as antibacterial agents (24). Sulfonila-
mide (aminobenzene sulfonamide) was first prepared by Gelmo in 1908 during
the investigation of azo dyes. A drug use patent was issued to Klarer
and Mietzsch in 1932 for Prontosil and other azo dyes containing a
sulfonamide radical. In the same year, Domagk, working with Klarer and
Mietzsch, observed that mice infected with streptococcal bacteria could
be protected by Prontosil (14, 15).

At the Pasteur Institute, researchers found that the azo linkage of
Prontosil was split in vivo to yield para-aminobenzenesulfonamide which
was thought to be the active chemotherapeutic agent (54). Colebrook and
Kenney (13) and Buttle et al. (8) reported favorable clinical results
with Prontosil which prompted the synthesis of more than 4500 sulfonamide
derivatives in the United States by 1943 (22).

The method of action of sulfonamides is based on the absorption into
the bacterial cell in its non-ionized form. After partial dissociation,
it competes with ionized para~aminobenzoic acid (PABA). This competition
inhibits bacterial growth by preventing PABA from being incorporated

into pteroylglutamic acid (folic acid) which is reduced to tetrahydrofolic



acid, a coenzyme essential for carbon metabolism (19, 59). Although
inhibition of pteroylglutamic acid synthesis is immediate, the effective-
ness of sulfonamides is restricted to bacteria which require or synthesize
pterovylglutamic acid during the growth phase. A log phase or latent
period, which is determined by the concentration of stored pteroyl-
glutamic acid, occurs between the administration of sulfonamide and its
bacteriostatic effect (52).

The structure-activity relationship of sulfonamides is such that the
para-amino group cn’) is essential and can only be replaced by such
radicals that can be converted in vivo into a free amino group. The
-SO,NH, (xt) group is not essential but the linkage of sulfur directly
to the benzene ring is of utmost importance (24). The more electro-
negative the -S0, group the greater is the bacteriostatic activity since
the tonic form is more active than the molecular form (3). This knowledge
led to the early synthesis of sulfisoxazole by Hoffman-LaRoche in 1947
(32) and its use patent for microbial infections. Since sulfisoxazole

is readily ionized and excreted by glomerular filtration, it is used

primarily for urinary tract infections (37

Sulfisoxazole

The therapeutic index of sulfonamides is relatively low so complete
bioavailability is important to maximize the percentage of patients who
will obtain a favorable therapeutic response from the drug (4). Sulfi-
soxazole or sulfafurazole (3,4 dimethyl-5-sulfanilamido-isoxazole) is a
white-yellowish, odorless, slightly bitter, crystalline powder with a PK,

of 4.9 (32), is relatively non-toxic, and is able to control experimental



~4=

bacterial infections (45). It is distributed in the extracellular fluid
and fails to enter cells (29, 58). Therefore, the administration of
sulfisoxazole results in a plasma concentration which is three times

higher than that preduced by an equal quantity of sulfanilamide (52).

Blood Concentration of Sulfisoxazole

Previous pharmacokinetic studies in dogs, swine, and cattle only
determined mean blood levels of "free" (unbound) sulfisoxazole (9, 21).
In these experiments, the mean blood level was highest 4 to 8 hours after
oral administration. In clinical trials with dogs given 0.78 to 2.33
grams per day orally, a maximum blood concentration, 2.8 to 29.0 mg/100 cc
blood, was attained. Intravenous administration to dogs (21), 199.6
mg/kg body weight, resulted in 22.5 mg of free drug/100 cc blood at
1 hour in 4 dogs; produced 26.4 mg of free drug/100 ce blood in 1 dog;
and 299.4 mg/kg produced 26.8 mg of free drug/100 cc blood in 3 dogs.
Subcutaneous administration to dogs (21) resulted in a maximum blood
concentration 4 hours after administration. Administration of 199.5
mg/kg body weight resulted in 14.5 mg of free drug/100 ml blood while
213.8 mg/kg resulted in 13.2 mg of free drug/100 ml blood. Oral adminis-
tration of 2.0 gm of sulfisoxazole to 4 dogs (40) resulted in a peak
plasma level of 13.6 mg/100 ml of whole blood.

The only administration reported in swine was by intraperitoneal
injection (21). The peak blood level was 25.4 mg of free drug/100 ml
blood at 1 hour after receiving a dose of 201.5 mg/kg body weight.

In desing children (39), administration of a single oral dose of
159 mg/kg body weight resulted in 13.5 mg of free sulfisoxazole/100 ml

ef serum after 3 hours and 3.8 mg/100 mi after 12 hours. An oral dose



of 100 mg/kg resulted in 9.2 mg of free drug/100 ml of serum at 3 hours
and 2.2 mg/100 ml at 12 hours. Svec et al. (53) observed that after oral
ingestion of a single 2.0 gm oral dose in adults, the mean blood concen-
tration of free sulfisoxazole was 9.0 mg/100 ml. Randall et al. (40)
administered an oral dose of 2.0 gm every 4 hours for 4 doses to 6 adults,
which produced a mean blood concentration of 21.0 mg of total sulfisoxa-
zole/100 ml at 10 hours after the initial dose; the free sulfisoxazole
concentration was 18.0 mg/100 ml at 10 hours. Loughlin and Mullin (28)
dosed 5 adults orally with 2.0 or 4.0 em of sulfisoxazole with whole
blood concentrations of free sulfisoxazole of 3.5 and 3.6 mg% at 2 and 4
hours, respectively, for the 2.0 gm dose; the 4.0 gm dose produced whole
blood concentrations of 7.4 and 7.3 mg% at 2 and 4 hours, respectively.
By 16 hours, the concentration was 40% of the 4 hour level for both the
2.0 and 4.0 gm dose and by 28 hours the blood level had decreased to 27%
of the 4 hour-2.0 gm dose and 22% of the 4 hour-4.0 gm dose.

The first complete pharmacokinetic profile in humans was conducted
by Kaplan et al. (26). Seven healthy adults administered a 2.0 gm single
oral dose had a mean absorption rate constant of 668.12 mg/hr, a half
life of 5.83 hr, peak plasma time of 2.5 hr with 168.7 ug/ml. Intravenous
administration of a single 2.0 gm dose to the same 7 volunteers resulted
in an initial plasma concentration of 259.03 ug/ml, a half life of
5.89 hr, and a volume of distribution of 16.37% of body weight. When
11 different sulfisoxazole tablets (500 mg) were administered to human
volunteers (49), the peak plasma level was 45.5 to 57.5 ug/ml in 3 hours

or less; the half life was 8.5 hours.



Metabolism of Sulfisoxazole

Saturable first pass conjugation of the aromatic amino group (n*)
of sulfisoxazole occurs during the initial passage of the drug from the
gastrointestinal lumen through the liver following oral administration.
The extent of conjugation increases as the oral dose decreases which is
consistent with saturable first pass conjugation. This does not occur
with intravenous administration which indicates that dose dependent con-
jugation occurs before the drug reaches the systemic circulation. By
inhibiting the motor activity of the stomach and small intestine to slow
drug absorption, one is able to increase conjugation of the drug (4).

Nelson and O'Reilly (33) reported that mean half-life for formation
of the acetyl conjugate from sulfisoxazole is 30 hours. The mean half-
life of the acetyl derivative was 9.0 hr which was longer than the half-
life of the parent compound (7.9 hr). Loughlin and Mullin (28) reported
14.2% of 2.0 and 4.0 gm oral doses were conjugated within 36 hours while
Weinstein (58) reported 28 to 35% is acetylated.

When acetyl (nt) sulfisoxazole is administered orally, the enzymes
responsible for Ne conjugation are saturated so the fraction metabolized
to the no acetyl derivative decreases as the oral acetyl (nt) sulfiscoxa-
zole dose increases (4). Flake et al. (20) supported this concept by
reporting 15% of sulfisoxazole is in the N’-acetylated form when acetyl
(nt) sulfisoxazole is administered. Bloedow (4) suggested that the high
degree of protein binding of sulfisoxazole may spare its first passage
metabolism to the Ne acetyl derivative, leaving more of the parent drug

available for its pharmacological action.



Protein Binding of Sulfisoxazole

The binding of a drug to plasma proteins affects its activity, dis-
tribution, rate of metabolism, and glomerular filtration (38). This
degree of binding is influenced by the molecular structure of the drug,
its lipid solubility, PK,» the concentration and affinity for plasma
proteins, the number of binding sites, the presence of competitively
binding drugs or endogenous compounds, and the physiological or patho-
logical state of the subject (7, 41, 44, 50). The degree of protein
binding increases near the pK, (1). With a low (acidic) pK.» a high
degree of drug binding occurs at physiological pH while unionized drugs
are slightly bound. The conjugation of methyl groups will increase the
binding tendency of a drug (1).

Albumin has a greater binding effect on sulfisoxazole than alpha-
or gamma-globulins (10). Experimental binding of sulfisoxazole in vitro
(5% albumin, pH 7.4, 25°C) resulted in 98.2% as bound, 1.8% as free base,
and 0.006% free acid (1) which is expected since sulfisoxazole has an
acidic pk, and 2 methyl groups. In vivo experiments in humans resulted
in approximately 25% of the total sulfisoxazole being bound to plasma
protein (40). The protein bound fraction acts as a reservoir between the
ineffective and toxic levels of the biologically active, unbound, un-
metabolized fraction (7) but there appears to be no correlation between

the half-life in man and the percentage which is protein bound (42).

Toxic Effects of Sulfisoxazole

Sulfonamides as a class of chemotherapeutic agents are considered to

be toxic due to precipitation in the kidney, producing crystalluria (24).



~38-

The infrequency of renal toxicosis (crystalluria) of sulfisoxazole is due
to the exceptionally high water solubility of the free and conjugated
(acetyl) fractions within the physiological pH range (28, 45).

Clinical toxicities have been induced by sulfisoxazole competing for
the same binding sites as warfarin (46) and furosemide (38, 47), inducing
hemolytic anemia due to glucose-6-phosphate dehydrogenase deficiency
(30), inhibition of anticoagulant factor VIII (56), hypersensitivity (9),
anorexia (60), agranulocytosis (60), aplastic anemia (60), and a case of
myocarditis, myositis, and vasculitis associated with severe eosinophilia
following sulfisoxazole therapy (18).

In young, 100 gm laboratory rats, hyperplasia of thyroid glands
occurred with diets of 0.5 and 1.0% sulfisoxazole for 13 weeks. These
rats did not exhibit any change in growth rate, agranulocytosis or
aplastic anemia. At 2.0% of the diet, a delayed growth rate, decreased
white blood cell count, and bone granulocytes occurred (40). The oral
lethal dose for 50% of mice tested (LD50) was 10.0 gm/kg of lithium and
sodium sait (45). The LD50 for an intravenous dose was 2.5 mg/kg and
2.3 mg/kg for the lithium and sodium salt, respectively; the subcutaneous
route of administration produced an LD50 at 5.0 and 2.8 mg/kg for the
lithium and sodium salt, respectively.

Kernicterus has been reported in infants with increased levels of
serum bilirubin in premature infants treated with sulfisoxazole (27, 34,
48, 51). Kernicterus occurred when unconjugated or indirect bilirubin
was less than 20 mg% in 24 infants, less than 17 mg4 in 15 infants, and
less than 15 mg%Z in 11 infants. These occurrences were enhanced by prior

acidosis, hypercapnia, and hypothermia (51).



~9-

Plasma samples from 6 adults showed that sulfisoxazole concentraticns
above 5 mg/100 ml had a significant displacing affect on bilirubin in
vitro (36). When compared to the displacing effects of salicylic acid,
salicyluric acid and aspirin to sulfisoxazoie at 10 mg/100 ml concentra-
tions, the most pronounced effect was observed when sulfisoxazole displaced
bilirubin from plasma samples of 6 adults and 13 infants in vitro (36).

The displacement bilirubin is due to competition for similar binding

sites on the albumin molecule (35).

Urinary Excretion of Sulfisoxazole

Sulfisoxazole is actively secreted in the proximal renal tubule in
dogs and humans. Tubular reabsorption is a passive process which depends
on urinary pH (12).

After single doses of sulfisoxazole, 83% was recovered in human
urine within 24 hours while 45% was excreted within 48 hours in dogs
(40). Kaplan et al. (26) reported a mean of 52.9% of sulfisoxazole was
recovered as the "free" drug within 48 hours after a 2.0 gm intravenous
dose in humans while 51.6% was recovered after a 2.0 gm oral dose. The
total sulfisoxazole recovered in 48 hours was 91.5% and 97.0% for the
2.0 gm intravenous and oral dose, respectively. Weinstein (58) reported

30% of the total sulfisoxazole excreted was in the acetylated form.

Pharmacokinetics

Pharmacokinetics has been defined as the quantitative study of the
absorption, distribution, metabolism, and excretion of drugs and their

pharmacologic, therapeutic, and toxic responses in animals and man (31).



-10-

The purposes of pharmacokinetics are to reduce the mathematical data
collected from an organism to meaningful parameters which can be used
to make predictions on the results of future experiments or of host
studies which would be too time consuming and costly if carried out

individually (57).

Absorption Rate Constant and Bioavailability

The first order absorption rate constant (k) is a mathematical
description of the rate at which the drug reaches the general circulatory
system after administration. A semi-logarithmic plot of serum concen-
tration (C,) versus time (t) is required with a second plot of the points
obtained by using the method of residuals. The slope (m) of this line
can then be used in determining the absorption rate constant by equation 1

(23, 57).

k= (-m) (2.3) Eq. 1

With sulfonamides, the calculated absorption rate constant (k.) can be
the elimination rate constant (ka). This can be checked by comparing the
Ka of an intravenous dose with the k, of an oral dose; if these are not
equal, the calculated ko the faster rate constant, is the kG (57).
Bioavailability (F) is a term used to indicate a measurement of both
the amount of administered drug which reaches the circulation and the
rate at which this occurs (25). The bioavailability depends on 1) the
rate and extent of release of the drug from the dosage form and 2) the
"first pass" effect where only a certain fraction of the drug presented

to the gastro-intestinal system reaches the general circulation intact.

The bioavailability (F) of a drug is calculated from a ratio of the oral



~ll-

blood concentration (Cc), volume of distribution (Va), the absorption

(k.) and elimination (k 4) constants, and the dose (D), equation 2.

Cc Vv, (k_ - k,) -k,t -k t =-1
_ d d d
re Oa } (e -e *) Eq. 2

A better measure of bioavailability (F) is determined by comparing
the area under the plasma curve after an oral dose (AUC, g.? with the
area under the plasma curve after the intravenous administration of the
same dose (AUC, |), equation 3. Alternatively, if the same dose is
administered by oral and intravenous routes, one can determine bio-
availability (F) by the ratio of the total drug excreted unchanged in
the urine after the oral dose Uno. to the total drug in the urine

after intravenous administration (ULs 7 ), equation 4.

AUC °
Fey Eq. 3
i.v
Veop Oo
F = _—— Eq. 4
ol.v.

Distribution

For a compound to exert its pharmacologic effect, distribution must
Occur into one or more volumes or compartments of the body. The highly
perfused or central compartment is characterized by the heart, liver,
and lungs and exchange between these tissues and the blood is very rapid.
The poorly perfused tissue characterized by muscle, fat, and skin and the
negligible perfused group characterized by the bone, teeth, hair, and

connective tissue compose the peripheral and deep compartments,



~12-

respectively, in which there is slow exchange between these tissues and
the blood (57). Since the blood is the common carrier for distribution
and urine is the most common method of excretion of a drug, monitoring of
the blood concentration and urinary excretion rate of the drug will allow
one to draw mathematical relationships as to the absorption, distribution,
and elimination of a compound (23).

The apparent volume of distribution (Vv) is a mathematical expression
for estimating or defining how extensively the drug is distributed
throughout the body. The lipid solubility, the degree of plasma protein
binding, and the cardiac output will affect the distribution of the
drug (57).

The apparent volume of distribution (Vy) of a drug is computed from
a semi-logarithmic plot of the plasma concentration (Cc) versus time (t)
after a known dose (D) of drug is administered intravenously, equation 5,

where the initial plasma concentration (c) is extrapolated.

D= (c) VD) Eq. 5

Elimination

The elimination rate constant (k) can be calculated from semi-
logarithmic plots of the plasma concentration versus time and from the
urinary excretion rate versus time by determining the slope of the
plotted line (m) and using equation 6 (57). The elimination rate

constant has the unit of reciprocal time (57).

ka = (2.3) (-m) Eq. 6



-13-

The biological or elimination half-life (ty) of a drug is derived
from a graphic plot of the logarithm of the plasma concentration (c)
versus time (t) in which the slope of the line (m) is used to calculate
a first order elimination or disposition rate constant (k,), equation 7

(57).

net Eq. 7

The biological half-life is defined as the time it takes for the drug
concentration to be reduced by one-half (23) and is usually referred to
in relation to the serum or blood concentration.
Urinary excretion is a major pathway for elimination for many drugs
and their metabolites. If the drug is totally excreted unchanged, the
renal clearance (CL) of the drug equals the plasma clearance (CL).
There are two ways of calculating clearance which involves the dose (D), the
total area under the plasma concentration curve (AUC), and the total

amount excreted in the urine (A), equations 8 and 9 (57).

DF

cL. = —— Eq. 8
P auc
P
A’

cL. =—+ Eq. 9
AUC
P

Renal clearance can also be calculated from the slope of the logarithm of
the urinary excretion rate (dA /dt) versus the plasma concentration at

midpoints of excretion intervals cc mid? (57), equation 10.

dA
u

dt



) Eq. 10

~ (CL) (C, mid



-14-

The elimination rate constant (ky) the excretion rate constant
(k); and the elimination half-life (ty) can also be calculated from a
"sigma~minus"” plot. This plot involves the logarithm of the difference
in total urinary excretion minus the amount in the urine at any one inter-
val [log (A, - ADI versus the excretion interval or by an excretion rate
plot of the concentration of drug excreted per unit time versus the mid-

time interval between sampling periods (57), equations 11 and 12,



respectively.
co co kA
log (A, - A, = log AL - 7.3 t Eq. 11
AA k
log —Y = log k D--%t Eq. 12
At e 2.3 “mid q-

Plasma Protein Binding

Plasma proteins, especially albumin, act as binding sites for acidic
and basic drugs. Acidic drugs, at normal body temperature and normal
therapeutic doses, appear to bind to albumin at a single binding site,
possibly at the N-terminal amino acid group (38). Protein binding in-
fluences the volume of distribution, the rate of metabolism, and the rate
of excretion (57).

The percent or fraction of protein bound drug (f,) is calculated
from the total serum concentration (c.) and the amount which exists as

free, unbound unmetabolized parent drug (C.) as in equation 13 (57).

Eq. 13



Two Compartment Model

A two compartment model is defined as a mathematical relationship of
the kinetics of distribution of substances in the body (57). When a two
compartment model does exist, one must calculate a volume of distribution

for both compartments (V, and V4), a first order rate constant for the

1
transfer of the drug from the central compartment to the peripheral
compartment (kj 5) and from the peripheral compartment to the central

compartment (ko4)> and a first order elimination constant by all pro-

cesses from the central compartment (k, or k

l 10)? assuming that etimina=

tion only occurs through the central compartment (57). This two compart-
ment model in which elimination occurs only from the central compartment
is represented by the following scheme:

Ki2

Central —~——~ Peripheral
Compartment ~———— Compartment

Koy

Kio

Elimination

In a two compartment model, a semi-logarithmic plot of serum or
plasma concentration (c) versus time (t) yields two straight lines by
least squares analysis, the distribution (a phase) and the elimination
(8 phase) phases. The rate constant of the first or distribution com-
partment (a) is calculated from the slope (m) of a second semi-logarithmic
plot of the serum concentration obtained by the method of residuals,
equation 14 (57). The rate constant for the second or elimination
compartment (8) is calculated from the slope (m) of the least squares
line of the semi-logarithmic serum concentration versus time graph,

equation 15 (57).



-16-

(2.3) (-m)

RQ
W

WD
Nl

(2.3) (-m)

time for the a phase and 8 phase, respectively.

concentration (c) is the sum of A and B.

C =A e +B et
P

co =A+B

P

(a) (8) = (ky1) (yg)

_ Dose
“1 7 A+B



(V1) (ky) = (Vy) (R54)

_ AB + Ba

Ko. = A+B

Eq. 14

Eq. 15

The remaining constants could then be calculated from equations 16

Eq.

Eq.

Eq.

Eq.

Eq.

Eq.

Eq.

through 22, where A and B are the extrapolated blood concentrations at 0

The total initial blood

16

17

18

19

I
nh



MATERIALS AND METHODS

High Performance Liquid Chromatographic Analysis of
Sulfonamides by Ionic Suppression

Materials

Ten milligram samples of sulfanilamide,* sulfaguanidine,* sulfamera-
zine,* sulfamethazine,* sulfapyridine,* sulfisoxazole, acetylsulfisoxa-
zole cn’), and sulfathiazole* were dissolved in methanol? and triple
distilled water (50/50) to give concentrations of 100, 50, and 25 ug/ml.
Twenty microliters of these standard solutions were injected onto the
column. Standard solutions (500 ug/ml) were added to 1.0 ml human plasma
samples to give in vitro plasma concentrations of 100, 50, and 25 ug/ml.
The in vitro plasma samples were incubated for 30 minutes in a 37°C water
bath, deproteinated with methanol? (1:1), centrifuged (2000 x g) for 10
minutes and 20 ul of the supernatent injected or 20 ul of the in vitro
plasma sample was injected directly onto the column without deproteina-
tion or extraction.

An ALC/GPC 204 liquid chromatograph * was equipped with a Model

440 3 ultraviolet absorbance detector (254 nm). A dual pen recorder**

*Fort Dodge Laboratories, Fort Dodge, Iowa 50501.
"Hoffman-LaRoche, Nutley, New Jersey 07110.

tpurdick Jackson Laboratories, Muskegon, Michigan 49442.
swaters Associates, Milford, Massachussetts 01757.

**Houston Instruments, Austin, Texas 78753.



-18-

was used to quantitate concentration as a function of peak height (mm).
A u Bondapak Cig reverse-phase column* was used with a water/methanol |
mobile phase and an in-line guard column* packed with Corasil/C,,.* The
mobile phase was either 60/40 or 50/50 water/methanol mixture, isocratic

7

pH of 7.45, or with acetate buffer' added to adjust the pH to 4.00 as
confirmed by a pH electrode and meter. * The water was triple distilled

and all analyses were conducted at ambient temperature (25°C) with a

0.8 ml/min flow rate.

Extraction and Separation

The retention times of 8 sulfonamides in standard solutions of
methanol/water are given in Tables 1 and 2. An examination of the data
shows that, in some cases, a change in retention time occurred when the
pH was reduced to 4.00 by the addition of acetate buffer to the mobile
phase. The retention time of sulfamethazine was increased while the
retention time of sulfamerazine, sulfapyridine, sulfisoxazole, and acetyl-
sulfisoxazole (N*) was decreased when the mobile phase was 50% water/50%
methanol (Table 1).

When the mobile phase was changed to 60% water/40% methanol (Table
2), the retention time was increased for sulfaguanidine, sulfamerazine,

sulfamethazine, and sulfapyridine. The retention times for the remaining

*Waters Associates, Milford, Massachussetts 01757.
‘Burdick-Jackson Laboratories, Muskegon, MI 49442.
*Glacial acetic acid, Fisher Scientific, Orlando, Florida 32809.

5 orion Model 407A, Oricn Research, Inc., Cambridge, Massachussetts 02139.



~19-

Table 1. Retention time (minutes) of sulfonamides in a water/methanol
(50/50) mobile phase with acetate buffer (pH 4.00) and without
buffer (pH 7.45).

pH 4.00 pH 7.45
Sulfanilamide 3.80 3.80
Sulfaguanidine 3.70 3.70
Sulfamerazine 4.90 4.50
Sulfamethazine 5.00 5.45
Sulfapyridine 4.75 4.45
Sulfisoxazole 5.20 3.20
Acetylsulfisoxazole in?) 7.00 2.90

Sulfathiazole 4.00 4.00



Table 2. Retention time (minutes) of sulfonamides in a water/methanol
(60/40) mobile phase with acetate buffer (pH 4.00) and without
buffer (pH 7.45).

pH 4.00 pH 7.45
Sulfanilamide 3.75 3.05
Sulfaguanidine 3.60 3.90
Sulfamerazine 4.10 5.40
Sulfamethazine 4.25 6.10
Sulfapyridine 4.00 5.00
Sulfisoxazole 4.20 4.20
Acetylsulfisoxazole (nt) 5.00 4.40

Sulfathiazole 3.75 3.30



sulfonamides were reduced except for sulfisoxazole which remained
constant.

When one compares the results presented in Tables 1 and 2 at pH
4.00, it is noted the retention times of six sulfonamides were increased
when the mobile phase was 50% water/50% methanol with acetate buffer.
There was no significant difference in the retention times of sulfanilamide
or sulfaguanidine in a mobile phase of 50% water/50% methanol with or
without the buffer or if the mobile phase was 60% water/40% methanol with
the buffer (pH 4.00). However, at pH 7.45, use of the 60% water/40%
methanol mobile phase resulted in an increase in the retention time of
sulfaguanidine while reducing that of sulfanilamide.

Human plasma samples were spiked with standard solutions of sulfona-
mides and the retention time recorded. Sulfanilamide and sulfaguanidine
were not separable from endogenous serum peaks in the 50% water/50Z
methanol (pH 4.00) mobile phase (Table 3). Sulfisoxazole and its no
metabolite, acetylsulfisoxazole, were not separable from endogenous
compounds at pH 7.45. Ionic suppression did decrease the retention time
of sulfapyridine and sulfathiazole in plasma while that of sulfamethazine
remained constant (Table 3).

When the mobile phase was changed to 60% water/40% methanol (Table
4), sulfanilamide, sulfaguanidine, and sulfisoxazole and acetylsulfisoxa-
zole were not detectable at pH 7.45. The remaining four sulfonamides
were detectable with an increased retention time at pH 7.45.

By comparing Tables 3 and 4, the retention time of the detectable
sulfonamides was increased by the 50/50 mobile phase at pH 4.00 and

decreased at pH 7.45.



-22-

Table 3. Retention time (minutes) of sulfonamides in spiked human plasma
samples in a water/methanol (50/50) mobile phase with acetate
buffer (pH 4.00) and without buffer (pH 7.45).

pH 4.00 pH 7.45

Sulfanilamide ND* 3.80
Sulfaguanidine ND* 3.70
Sulfamerazine 4.50 4.60
Sulfamethazine 4.80 4.80
Sulfapyridine 4.40 4.30
Sulfisoxazole 5.10 ND*

Acetylsulfisoxazole (n*) 6.90 ND*

Sulfathiazole 4.25 3.95

“ND = Not detectable due to endogenous serum components with similar
retention times.



-23-

Table 4. Retention time (minutes) of sulfonamides in spiked human plasma
samples in a water/methanol (60/40) mobile phase with acetate

buffer (pH 4.00) and without buffer (pH 7.45).

pH 4.00

Sulfanilamide ND*

Sulfaguanidine ND*

Sulfamerazine 4.10
Sulfamethazine 4.30
Sulfapyridine 4.00
Sulfisoxazole 4,20
Acetylsulfisoxazole (n°) 5.00
Sulfathiazole 3.75

pH 7.45

ND*

ND*

4.35

4ND = Net detectable due to endogenous serum components with similar

retention times.



-—oi-

The plasma matrix increased the retention time of sulfamerazine,
sulfamethazine, sulfapyridine, sulfisoxazole, and acetylsulfisoxazole
cx’) in the 50/50 mobile phase at pH 4.00 (Tables 1 and 3). The reten-
tion time of sulfanilamide, sulfaguanidine, and sulfathiazole were not
changed in the 50/50 mobile phase at pH 7.45. However, the retention time
of sulfamethazine and sulfapyridine was increased in the plasma matrix
at pH 7.45 while that for sulfamerazine decreased (Tables 1 and 3).

With the 60/40 mobile phase, there were no significant changes in
retention time by the plasma matrix at pH 4.00 (Tables 2 and 4). At pH
7.45, the retention time of sulfapyridine and sulfathiazole was increased
by the plasma matrix (Tables 2 and 4).

When serum is the biological matrix, four of the sulfonamides are
easily separated at either pH or mobile phase polarity. Sulfanilamide
and sulfaguanidine are only detectable from plasma when the mobile phase
is 50% water/50% methanol at pH 7.45, the pH value closest to their PK,
value. Sulfisoxazole and acetylsulfisoxazole cn’) are detectable only
after ion suppression at pH 4.00, regardless of either mobile phase. The
endogenous serum compounds which absorb at 254 nm can be separated from
the sulfonamides by changing pH or mobile phase polarity. Deproteination
of plasma samples with methanol did not change the retention time of any
sulfonamide as compared to the direct injection of the spiked plasma
sample. Separation of combinations of sulfonamides was easily accomplished
if the retention time differed by at least 0.1 minutes.

Construction of concentration curves by plotting peak height versus
10, 50, and 100 ng/ml and 10, 25, 50, and 100 ug/ml of standard solutions
of sulfonamides in methanol/water (Figures 1 through 4) demonstrated that

a difference in sensitivity occurred as a function of ion suppression



~25-

240 20
220 16
200 E 2
€
180 3
160 4
E b
— 140 10 50 100
E ng/ml
& 120
TT
~ 100 g
a e
WW
a 80 h
d
60 c
f

h
oO
0

tw
Oo

0 25 0 100
CONCENTRATION OF SULFONAMIDE ( pg/ml)

ca
ae
|

Figure 1. Standard concentration curves of sulfanilamide (a), sulfa-
guanidine (b), sulfamerazine (c), sulfamethazine (d),
sulfapyridine (e), sulfisoxazole (f), N* acetylsulfisoxazole
(g), and sulfathiazole (h) in a water/methanol (50/50) mobile
phase with acetate buffer to pH 4.00.



-26-

240 20

220 16

200 ¢ 12

€

{80 8

{60 4
= a
E 140
~ 10 50 {00
6 120 ng/ml b
ul
x
x {00
<

Oo @
°o o
fo gator.)

\

10 25 50 100
CONCENTRATION OF SULFONAMIDE ( jzg/m!)

Figure 2. Standard concentration curves of sulfanilamide (a), sulfa-
guanidine (b), sulfamerazine (c), sulfamethazine (d),
sulfapyridine (e), sulfisoxazole (f), N* acetylsulfisoxazole
(g), and sulfathiazole (h) in a water/methanol (50/50) mobile
phase with an isocratic pH 7.45.



-27-

and polarity of the mobile phase. The mean standard error of peak height
at these concentrations was t 0.017.

In Figure 1, the sensitivity of sulfaguanidine (b) at 254 nm is
greater than sulfanilamide (a) at pH 4.00 in a 50/50 mobile phase. If
the mobile phase remains at pH 7.45 (Figure 2), this sensitivity is re-
versed, with sulfanilamide peak height being greater than sulfaguanidine.
There is also a change in the slope of the other 6 sulfonamides as the
pH changes in the 50/50 mobile phase (Figures 1 and 2).

If the mobile phase is 60% water/40% methanol, sensitivity of the
assays for sulfanilamide (a) and sulfaguanidine (b) are the most sensitive
at either pH (Figures 3 and 4). Detection of the remaining 6 sulfona-
mides varies in sensitivity at 254 nm in the 60/40 mobile phase with the
pH change. The most sensitive mobile phase is 60/40 at pH 4.00 as shown
by the greater slopes of each sulfonamide (Figure 3). Additional dilu-
tion of standard solutions of sulfonamides and increased sensitivity
settings to 0.01 a.u.f.s. (absorbance units full scale) on the ulitra-
violet detector recorded peak heights equivalent to 10.0 ng/ml of
sulfonamide from a single 20 yl injection without concentration or
reconstitution of the extract (inset, Figures 1 to 4).

The slope of the sulfisoxazole (f) curve was the same for the 60/40
mobile phase regardless of pH (Figures 3 and 4). By comparing Figures
1 and 2, one observes that the slope of the sulfamethazine (d) curve was
the same for the 50/50 mobile phase at either pH 4.00 or pH 7.45.
Sulfaguanidine (b, Figures 1 and 3) and sulfapyridine (e, Figures 2 and
4) curves had the same slope at pH 4.00 and pH 7.45, respectively, re-

gardless of the polarity of the mobile phase.



~28-

260

240

200

180

160

i40

i20

100

PEAK HEIGHT (mm)
~anasr o

80

60

40

20



0 100
CONCENTRATION OF SULFONAMIDE ( 2g/mi)

Figure 3. Standard concentration curves of sulfanilamide (a), sulfa-
guanidine (b), sulfamerazine (c), sulfamethazine (d),
sulfapyridine (e), sulfisoxazole (f), N* acetylsulfisoxazole
(g), and sulfathiazole (h) in a water/methanol (60/40) mobile
phase with acetate buffer at pH 4.00.



~29~

220 16
200 € 12
é
(80 8
160 4
E
~ !40 10 50 100
E ng/ml
© 120 3
bon
100
x b
tad
a so f
e
60 g
h
d
40 EE c

20



10 25 50 100
CONCENTRATION OF SULFONAMIDE ( pg/ml)

Figure 4. Standard concentration curves of sulfanilamide (a), sulfa~
guanidine (b), sulfamerazine (c), sulfamethazine (d),
sulfapyridine (e), sulfisoxazole (f), N4 acetylsulfisoxazole
(g), and sulfathiazole (h) in a water/methanol (60/40) mobile
phase with an isocratic pH 7.45.



~30-

Optimization of the Liquid Chromatographic Procedure

Four sulfonamides (sulfamerazine, sulfamethazine, sulfapyridine, and
sulfathiazole) could be separated from a plasma matrix in a 50/50 or
60/40 methanol/water mobile phase at either pH. Sulfanilamide and sulfa-
guanidine were only separable from plasma in a 50% water/50% methanol
mobile phase without acetate buffer (pH 7.45) due to their increased pK,
of 10.5 and 11.3, respectively. Sulfisoxazole and acetylsulfisoxazole
(x) were separated with either mobile phase by ionic suppression with
acetate to reduce the mobile phase pH to 4.00.

Sensitivity of the assays to 10.0 ng/ml of sulfonamide from 20 ul
injections of spiked plasma samples without concentration or reconstitu-
tion of the extract. Assays for sulfanilamide and sulfaguanidine were
the most sensitive in the water/methanol mobile phase at 254 nm. A 60%
water/40% methanol with acetate buffer to pH 4.00 was the most sensitive

of the assays.

Experimental Model

The trial consisted of 6 male human volunteers from 25-30 years old,
weighing 70 to 80 kg; 3 female dogs approximately 2 years old, weighing
20.0 + 1.0 kg; and 6 female pigs approximately 3 months old, weighing
20.0 + 1.0 kg. The human volunteers were administered 2.0 gms of sul-
fisoxazole in a single dose and blood samples taken at 0, 1, 2, 4, 6, and
8 hours after administration. The dogs were administered i100 mg of
sulfisoxazole/kg body weight, by intravenous and oral routes in 2

replicates for each route with a 30 day rest period between each adminis-

tration. The pigs were also administered 100 mg of sulfisoxazole/kg of



-31-

body weight by intravenous and oral routes with a 21 day rest period
between administrations. Blood samples were taken at 0.5, 1, 2, 3, 4.5,
6, 9, 12, 22, 32, 44, 56, and 72 hours after intravenous administration
and 0, 1, 2, 4, 6, 8, 10, 12, 14, 23, 32, 44, 56, 76, and 96 hours after
oral administration in dogs and swine. All animals were maintained on a
commercial diet (Purina Dog Chow* or Swine Feed") with ad libitum access
to water. The animals were housed in metabolism cages and the urine was
collected as voided and frozen (0°C).

A 12.5% solution of sulfisoxazole* was prepared in our lab with
lithium hydroxide. The solutions were filtered and placed in sterile
50 ml ampules.” This solution was used for both the oral and intravenous
administration of the drug.

The partition coefficient of sulfisoxazole and acetylsulfisoxazole
from water (pH 5.0) into 2-octanol** after a one hour incubation period
was 0.441 for sulfisoxazole and 0.538 for acetylsulfisoxazole as quanti-
tated from the previous high performance liquid chromatographic procedure

using 60% water/40% methanol with acetate buffer (pH 4.00).

Blood Samples

ee

Blood samples were taken from the cephalic vein in dogs and humans

and via the anterior vena cava in swine. Human samples were drawn

*Ralston Purina, St. Louis, MO.

‘University of Florida Swine Unit, 18% protein feed, 72% yellow corn,
25% soybean oil meal, 3% salt, vitamin, calcium-phosphorus supplement.

THof fman-LaRoche, Nutley, N.J. 07110.
‘Wheaton, Scientific Products, Ocala, FL 32670.

*kEastman-Kodak, Rochester, N.Y. 14650.



-32+

directly into 7 ml sterile silicone~coated Vacutainer tubes. Ten milli-
liter samples were taken by a sterile syringe with a 20 gauge needle from
the dogs and pigs and transferred to sterile silicone-coated Vacutainer
tubes. The samples were centrifuged (2000 x g) for 10 minutes, the serum
extracted, protected from light, refrigerated, and analyzed within 30
minutes for total and conjugated (glucuronidated) bilirubin and albumin.
The remaining serum was frozen (0°C) for up to 14 days for analysis of
"free" sulfisoxazole, acetyl (x) -sulfisoxazole, and total (acid
hydrolyzed) sulfisoxazole.

Serum bilirubin. Serum was analyzed for total and conjugated (direct
or glucuronidated) bilirubin on E.I. DuPont's Automatic Clinical Analyzer*
(17) using a commercial standard."

The conjugated method is a modification of the Van den Bergh diazo

reaction (55):

Conjugated bilirubin + p-Nitrobenzendiazonium tetrafluoroborate (PNB)

~ |

Red chromophore absorbing at 540 nm

Under acidic conditions, the PNB is coupled to the glucuronidated bili-
rubin which is measured as an end point reaction at 540 and 600 nm. The
normal range in humans is considered to be 0.00 to 0.36 mg/dl.

The total bilirubin quantitation (the conjugated and unconjugated

fractions) is also a derivation cf the Van den Bergh reaction (55):

*E.L. DuPont, Instrument Products, Wilmington, Delaware.

“Dade Division, American Hospital Supply Corp., Miami, FL.



=33-

Total bilirubin + p-Nitrobenzenediazonium tetrafluoroborate (PNB)

H+ | Tween 20

Red chromophore at 540 nm

A surfactant (Tween 20)* is used to solubilize the unconjugated (free)
bilirubin; which along with the water soluble, glucuronidated bilirubin
reacts with PNB in an acid medium to measure an end point reaction at
540 and 600 nm. The normal total bilirubin concentration in humans is
less than 1.5 mg/dl (17); in dogs, the normal level is less than 0.5
mg/100 ce (5).

Interferences in these methods are due to hemolyzed samples and light
degradation of the bilirubin. All analyses were conducted within 30
minutes of sampling on serum which was kept in a cool, dark environment.

Serum albumin. Serum albumin was analyzed on E.I. DuPont's Automatic
Clinical Analyzer (17). This method is an adaptation of the bromocresol
green (BCG) dye binding method of Rodkey (43) which was later modified
by Doumas (16).

Albumin plus BCG dye at pH 4.2 yields an albumin-BCG complex which
elicits an absorbing spectrum at 600 nm. The end point reaction is
measured at 600 and 540 nm. The normal range in humans is 3.8 to 4.8
mg/dl.

Interferences are expected in icteric and hemolyzed samples. All

analyses were conducted within 30 minutes of sampling.

*Union Carbide Corporation, New York, New York.

+
‘E.I. DuPont, Instrument Products, Wilmington, Delaware.



—34-

Serum sulfisoxazole. Free (unbound, unmetabolized) serum sulfisoxa-
zole and serum acetyl (n’) sulfisoxazole were determined by deproteinating
1 part of serum with 2 parts methanol, centrifuging for 10 minutes
(2000 x g) and filtering the supernatent through Milex disposable
filters (SLHA 02505).*

Total serum sulfisoxazole (acid hydrolyzed) was determined by adding
2 parts water and 1 part 6N hydrocholric acid to 1 part serum and heated
in a boiling water bath (100°C) for 1 hour. The samples were allowed to
cool to room temperature and centrifuged for 10 minutes (2000 x g). The
supernatent was adjusted to pH with 2N NaOH, the final volume adjusted
to 1.5 ml with methanol and filtered with disposable Milex filters
(SLHA 02505) .*

Twenty microliters (1) of the Filtered supernatent were injected
into a Waters Model 6000A Liquid Chromatograph equipped with a Model 440
absorbance detector with a 254 nm filter, U6K injector, and a u-Bondapak
Cig column.* A Houston Instruments dual pen recorder was used to record
peak heights. A peak height ratio was used to determine serum concen-
tration with suifathiazeleâ„¢ being used as the internal standard. Serum
samples spiked with standard solutions of sulfisoxazole and acetyl-
sulfisoxazole** were injected and a standard curve of peak height of

drug to sulfathiazole was constructed.

*Millipore Corporation, Bedford, MA.

"purdick Jackson Laboratories, Muskegon, MI 49442.
Tatene Associates, Milford, MA 01757.

sport Dodge Laboratories, Fort Dodge, Iowa 50501.

**Hoffman-LaRoche, Nutley, N.J. 67110.



~35-

A mobile phase of 60% triple distilled water with 0.5% concentrated
acetic acid/40% methanol* (pH 4.00) was used with a flow rate of 0.8
ml/min. The water and methanol were filtered (NAWP047001 and FHUP647000),°
degassed by sonication and mixing maintained by using a Thermolyne

Â¥

stirrer.

Urinary Sulfisoxazole

Free and acetyl (x’)-sul fisoxazole were measured by cooling 25 ml
urine to 4°C in an ice bath; sufficient 6N hydrochloric acid was added
to reduce the pH to 3.0. After 5 minutes, 15 ml chloroform* was added,
the solution removed from the ice bath, and extraction ee completed in
5 minutes by swirling the solution once per minute. The chloroform* was
removed, evaporated under nitrogen and the residue reconstituted with
methanol.* Total sulfisoxazole was measured by heating the 25 ml of
urine in a boiling water bath for 1 hour and then extracted as previously
mentioned.

The reconstituted extract was then injected onto the liquid
chromatograph using the previously described technique. Peak height was
also used to determine the urinary concentration with sulfathiazole®
being used as the internal standard. Chloroform* extracts were utilized

to remove 94% of sulfisoxazole and 90% of acetyl (N’)-sulfisoxazole from

*Burdick Jackson Laboratories, Muskegon, MI 49442.
ok

‘Millipore Corporation, Bedford, MA.

oh

TScientific Products, Ocala, FL.

sport Dodge Laboratories, Fort Dodge, Iowa 50501.



-36-

spiked urine samples which had been incubated at 37°C for 1 hour. Benzene*

removed 60% of sulfisoxazole and 40% of acetyl (N*)-sulfisoxazole while

ether* removed 96% of sulfisoxazole and 50% of acetyl (n")-sulfisoxazole.
Both serum and urine samples were performed on thin-layer chroma-

tography plates (11) to determine if metabolites other than the acetyl

cn) metabolite existed. All spots were accounted for and analyzed

through the HPLC procedure previously mentioned without the appearance

of other metabolites, at 254 nm and 313 nm.

Analysis of Data

Total, conjugated, and indirect bilirubin and serum albumin were
analyzed for statistical differences by Barr and Goodnight (2) ANOVA
program at the Northeast Regional Computer Center, University of Florida.

"Free" sulfisoxazole after intravenous administration was analyzed
as a two-compartment model using a NONLIN program (57). Logarithmic-
least squares analysis and the method of residuals was used to calculate
a regression line for acetylsulfisoxazole after oral and intravenous

administration and for "free" sulfisoxazole after the oral dose.

In Vitro Plasma Protein Binding

In vitro human plasma samples were incubated in a water bath at
37°C for 1 hour with 25.0, 50.0, 100.0, 200.0, and 300.0 ug/ml of

sulfisoxazole standards in 1 part methanol with 9 parts double-distilied

*Burdick Jackson Laboratories, Muskegon, MI 49442.



-37-

water. Dialysis was conducted by membrane dialysis with a molecular
weight cutoff filter of 5000* after incubating 2.0 ml of spiked serum
with 2.0 ml of phosphate buffer, ? pH 7.41. The dialysate was collected
and analyzed by the previously described liquid chromatographic

method.

*Diachema Ag, CH 8803, Ruschklikon, Switzerland.

"visher Gram Pac Buffer, pH 7.41. Fisher Scientific, Orlando, Florida
32809,



RESULTS AND DISCUSSION

Pharmacokinetics of Sulfisoxazole

The pharmacokinetic profile of sulfisoxazole was determined following
intravenous and oral administration in dogs and swine and following oral
administration in humans. The intravenous blood level curves in dogs and
swine were observed to be biexponential and required a two-compartment

model system for data analyses (23, 57):



Kyo
Central Peripheral
Compartment < Compartment
k
21
X10
Elimination

Integration of the differential equations of a two-compartment model

yields the equation:

where 7 is the concentration of the drug in the plasma at time t, A and
B are ordinate axis intercepts , and the individual rate constants for
the compartments, kyo Koy and Kio are calculable from a and 8, the rate

constants for distribution and elimination, respectively (57).



~39-

Administration of Sulfisoxazole to Dogs

Intravenous administration. A least squares linear regression line

1

of the "free," unbound, non-metabolized sulfisoxazole in 6 dogs (Figure 5)
was calculated using a NONLIN program (57). The mean extrapolated serum
concentration at zero time (A) in the first or distribution compartment
was 189.42 + 38.45 ug/ml with a range of 126.89 to 228.39 ug/ml and a
mean extrapolated level at zero time (B) for the second or elimination
compartment was 2.56 ~ .39 ug/ml (Table 5).

The disposition rate from the first or distribution compartment (a)
ranged from 0.1381 to 0.1982 hours - with a mean of 0.1726 + .0604 hours +
which yields a mean half-life (try) of 4.08 £ .60 hours for sulfisoxa-
zole in the first compartment. Similar analysis of free sulfisoxazole
in the second or elimination compartment yielded a very slow mean dis-
position rate (8) of 0.0206 * .0014 hours or a mean half-life (ty 59)
of 33.74 * 2.17 hours (Table 5).

The mean rate of distribution between the central or distribution and
peripheral or elimination compartments (k, 9) was 0.0140 + .0049 hours ~
(Table 5) while the mean rate of distribution from the peripheral to the
central compartment (54) was 0.0228 + .0022 hours + (Table 5). This
indicated that free sulfisoxazole returned from the peripheral to the
central compartment at a faster rate than it had been distributed from
the central to the peripheral compartment. Calculation of the mean
K1/ky> ratio reflects this difference in distribution between the two
compartments. In these 6 dogs, the mean of the ko / Ky 9 ratio was 1.63,
indicating that the drug is returning rapidly from the distribution sites

for elimination from the body.



-40-

1000.0

SERUM "FREE" SULFISOXAZOLE IN DOGS ( j1g/mi )



10.0
B
1.0 70
10 20 30 40 50 60
TIME (hours)

Figure 5. Serum concentrations of "free" sulfisoxazole in dogs.



Table 5. ‘Two compartment pharmacokinetic parameters in dogs administered sulfisoxazole

as a single intravenous dose,

Sub ject A B 1 8 v v k k. k



( Tes
J 2 12 2) iD a ee ‘hg
- - | = am
Gig/ml) Gig/m)) (hours 1) (hours (liters) (ltters) (Qioure *) (hours ‘) (hours 4) (hours) (hours)

) 228.39 1.93 0.1503 0.0197 9.0) 2.9% 0.0068 0.0208 1.1424 2607.44 4.6) 35.18

2 160. 26 2,82 0,138t 0.0204 11.04 5.07 0.010% 0.0224 a.1258 2294.76 5.2 33.97

3 206.89 2.42 0.187) O.DLt 9.55 6.87 a.005) 0.0210 0.1701 2370.98 3.70 46. 28

4 213.96 2.61 0.1852 0.0207 10,07 6.34 0.01463 0,0227 0.1689 1995.91 3.74 33.48

5 126.24 3,10 0.1768 00,0232 14.07 10.77 0.0206 0,0269 0.3595 2298.98 3.92 29.87

6 200, 76 2.48 0.1982 0.0206 9.84 7.29 0.0109 0.0228 0.792 2161.60 3.50 33.04

Mean + 189.42 2.596 0.1726 0.1206 10.60 6.55 D.O0140 0.0228 9. 1565 2321.61 4.08 34.74

sb* 38.45 39 0604 0014 1.81 2.59 -0U49 0022 0200 196.49 60 2.17
*Mean 1 one standard devlatlon.

- Ty-



-42-

Table 6. The mean amount of sulfisoxazole excreted in the urine of

6 dogs.
Total Amount of
Sulfisoxazole 4 of
(mg) Dose
Intravenous
24 hours 0.792 39.59
48 hours 0.837 41.84
72 hours 0.843 42.17
Oral
24 hours 0.524% 26.20*
48 hours 0.582 29.10
96 hours 0.588 29.44

*Calculated from 5 dogs due to no collection of urine at 24 hours from
one individual.



-43-

The mean elimination rate (kK, 9) in the 6 dogs was 0.1565 = .0200
hours (Table 5). The ratio of B/k, 9 indicates the fraction of free
sulfisoxazole in the postdistributive phase which is available for
elimination. The mean B/G ratio was 0.13, indicating that 13.0% of
the drug in the body would be in the central compartment and available
for elimination.

The volume of the central compartment (Vv) ranged from 9.03 to 14.07
liters with a mean of 10.60 * 1.83 L (Table 5). The mean volume of the
second or peripheral compartment was 6.55 t 2.59 L (Table 5), indicating
that free sulfisoxazole is more widely distributed in the central than in
the peripheral compartment.

In 2 dogs, 3 and 6 (Table 23 and 26), the fraction bound was less
than 30% for the first 3 hours after intravenous administration but had
reached 40 to 50% at 4.5 hours after administration. In the other 4 dogs,
the fraction bound (£,) ranged from 30 to 50% throughout the trial period,
72 hours.

The mean amount of sulfisoxazole excreted in the urine (Table 6) was
39.59% of the dose within 24 hours after administration. By the end of
the trial, 72 hours, 42.17% of the dose was excreted in the urine.

Oral administration. From log-linear regression equations of the
serum concentration of free sulfisoxazole after oral administration, the
mean rate constants a and 8 were 0.1623 t .0361 hours! and 0.0296 £ .0172
hours! (Table 7), respectively. The mean half-life corresponding to the
faster disposition rate (ty was 4.37 t .76 hours and the mean elimina-
tion half-life corresponding to the slower disposition rate (ty 9? was

34.46 t 26.08 hours (Table 7).



Table 7. Two compartment pharmacokinetic parameters in dogs admlnistered sulfisoxazole as a single
oral dose.



Subject a B t

sa TB F oa
(hours!) Gioure *} (hours) (hours) (%)
1 0.1566 0.0299 4.33 23.14 100.92 2631.48
2 0.1543 0.0085 4.62 81.32 99.33 2279.43
3 0.1267 0.0145 5.33 47.76 99.14 2350.57
4 0.1497 0.0453 4.62 15.27 98.03 1956.60
5 0.1543 0.0530 4.33 12.86 99.02 2276.48
6 0.2326 0.0262 3.01 26.42 99.00 2338.14
SD* -0361 .0172 . 76 26.08 -94 215.69

*4Mean + one standard deviation.



-45-

The peak serum concentration had occurred by the first sampling
period, 1 hour (Tables 21 to 26). This rapid absorption was due to the
compound being given as a solution. The peak concentration of free
sulfisoxazole ranged from 122.25 to 165.00 ug/ml (Tables 21 to 26) and
the maximum total sulfisoxazole ranged from 152.78 to 229.22 ug/ml.

The degree of plasma protein binding was calculated to be 30 to 402
in 2 dogs in the first 6 hours of the trial (Tables 22 and 26). The
fraction bound was less than 30% in the remaining 4 dogs for the first 6
hours after oral administration. Between 6 and 44 hours, the fraction
pound was 40 to 50% but had increased to 70 to 90% in all dogs after 44
hours and continued at this level until the end of the trial.

The mean amount excreted in the urine of the 6 dogs was 26.20% of
the dose at 24 hours and 29.44% of the dose at the end of the trial, 96
hours (Table 6). Four of the 6 dogs excreted 43.78% of the dose in 24

hours while 2 dogs excreted 9.95 and 11.00% in 24 hours.

Administration of Sulfisoxazole to Swine

Intravenous administration. The "free," unbound, non-metabolized
sulfisoxazole in 6 pigs was analyzed as a two-compartment model (Figure
6) by a NONLIN program (57). At zero time, the mean extrapolated serum
concentration of A and B on the ordinate axis was 245.07 + 44.88 ug/ml
and 0.423 t .088 ug/ml (Table 8), respectively.

The disposition rate from the first compartment (a) ranged from
0.4961 to 9.5884 hours = with a mean of 0.5368 t .0362 hours which is
equivalent to a half-life (ty of 1.30 + .09 hours (Table 8). The
mean disposition rate from the second compartment was considerably longer,

0.0153 £ .0043 hours”” or 2 half-life (t, ,) of 53.33 $ 13.65 hours.
23



-~46-

1000.0

100.0

10.0

SERUM “FREE" SULFISOXAZOLE IN SWINE (pg/ml)

°



10 20 30 40 50 60 70

TIME (hours)

Figure 6. Serum concentrations of “free” sulfisoxazole in swine.



Table 8. Two compartment pharmacokinetic parameters in swine administered sulfisoxazole
as a Single intravenous dose.

Sub ject A B a K v Vv k, k k AUC t,



1 2 12 2} to r gytt
(ug/m)) (ug/ml) (igure) (hones!) (lhters) (Iters) (hours) (hours!) (hours?) (hours)

1 217.79 0.342 5732 0219 12.76 31.98 O.0214 0.0228 0.5509 696.10 1.21

2 186.07 0.495 0.531% O.OLA9 15.65 24 05 O.O317 0.0202 0.4782 1066.58 1.36

3 312.31 a. 300 0. 5884 G.012)3 8.20 15.70 0.0247 0.0129 0.563) 1339.20 1.18

4 250,45 0.459 0.5286 0.0107 9.58 33.32 0.0401 0.0116 0.4876 1287 86 1.32

5 275.07 0.439 0.5232 0.0113 7.5) 15.73 , 0.029) 0.0141 0.4931 1339, 38 1.32

6 233.92 0. 502 0.4961 O.0148 9.09 17.49 O.0304 D.O158 0.4647 1042.80 1.40
Mean ! 245.07 0.423 0.5368 0.0353 10.48 19.76 1.0296 0.0162 0.506} 1244.67 1.30
sh* 44.88 088 -0362 004 3 d.01 7.75 - 0064 0044 0406 280. 39 09

*Mean + one standard deviation.

{
55,8

(hours)

3 0%
36.07
56.34
64.7)
42.0)

46.82

53.33
13.65

-L9-



-48-

The mean distribution constant between the central and peripheral
compartments (k, >) was approximately twice as fast as the return of the

compound from the peripheral to the central compartment (k,,). The mean

21

k,> was 0.0296 + .0064 hours”* while k,, was 0.0162 + .0044 hours”

21
(Table 8). This is supported by the extended half-life of sulfisoxazole

). The ratio of k,./k,. was 0.55 which

i h d compartment (t
in the secon mp ( ky 21° 12

»8
reflects the distribution difference between the compartments with the
free sulfisoxazole readily entering the peripheral compartment but slowly
returning to the central compartment for elimination from the body.

The elimination rate (ks) ranged from 0.4647 to 0.5631 hours with
a mean of 0.5063 + .0406 hours ~ (Table 8). The ratio of B/k, 9 was 0.03
which indicated that only a very small fraction (3.0%) of free sulfisoxa-
zole was available to be excreted from the postdistributive phase.

The mean volume of distribution for the first compartment (v,)
(Table 8), 10.48 + 3.11 L, was one-half of the volume of the second
compartment (Vo); 19.76 + 7.75 L. This indicated that free sulfisoxa-
zole was much more widely distributed throughout the second compartment
than in the first compartment.

The degree of plasma protein binding in vivo (equation 10) ranged
from 40 to 60% throughout the trial period (Tables 27 to 32). Four of
the animals bound less than 20% of sulfisoxazole during the first hour
of the trial (Tables 29 to 32).

The mean amount of sulfisoxazole excreted in the urine as a percent
of the dose (Table 9) was 16.06% free sulfisoxazole and 19.17% acetyl-
sulfisoxazole at the end of 24 hours. By the end of the trial, 72 hours,
18.23% of the dose was excreted as free sulfisoxazole and 12.48% as

acetylsulfisoxazole.



-49-

Table 9. The mean amount of sulfisoxazole and acetylsulfisoxazole cn’)
excreted in the urine of 5 pigs* as a percentage of the dose.

% of Dose Excreted

Sulfisoxazole Acetylsulfisoxazole
Intravenous
24 hours 16.06 10.17
48 hours 18.08 12.37
72 hours 18.23 12.48
Oral
24 hours 15.24 10.62
48 hours 17.70 12.34
96 hours 18.27 12.78

*The urine from one animal was not recorded.



~50-

Table 10. The biological half-life of acetylsulfisoxazole cn) after a

single dose* of sulfisoxazole.

Chours)

Subject Humans
Oral a
(hours)
1 13.07 =
2 10.50 1.54
3 16.11 3.04
4 13.08 4.33
5 16.11 1.44
6 15.40 1.31
Mean + 14.05 2.33
spt 3.23 1.23

*Dogs did not metabolize sulfisoxazole to

a
‘Insufficient data for a log-linear regression plot.

£ . ue
"Mean t one standard deviation.

Pigs

37

27.

30.

35.

31.

.89

35

50

40

90

23.18

04
34

Intravenous
a B
(hours)

1.87 66.

0.71 27

1.93 5

1.61 15.

1.28 6

1.98 36.

1.56 26.
49 23.

the acetyl (4) metabolite.

86

86

.80

69

-47

60

55
15



The t, c and ¢t, of acetylsulfisoxazole (Table 10) were calculated
Bs “89

3,8

by log-linear regression. The t, ranged from 0.71 to 1.98 hours with
“2

oJ
9 &

a mean of 1.56 = .49 hours. The t, 8
Bs

of 5.80 to 66.86 hours and a mean of 26.55 + 23.15 hours. Three pigs had

was much more variable with a range

aty 2 less than 20 hours, 2 had a t, 3 between 20 and 40 hours, and 1
45 =
had a ty, 3 of 66.86 hours (Table 10). Acetylsulfisoxazole reached a

maximum level, 22.73 to 43.33 ug/ml (Tables 27 to 32), at 1 to 2 hours
after intravenous administration in all 6 pigs (Figure 7).

Oral administration. From log-linear regression equations of the
serum concentrations of free sulfisoxazole after oral administration, the
mean disposition rate constants, % and 8, were 0.5000 = .1706 heures ~ and
0.0148 + .0075 hours !, respectively (Table 11). The biological half-
life of free sulfisoxazole in the central or distribution compartment

(t

so ranged from 0.86 to 2.10 hours with a mean of 1.50 t .43 hours.
The mean half-life in the elimination compartment (ty, 9) was longer,
54.99 $ 21.84 hours with a range of 25.92 to 78.36 hours (Table 11).

Serum concentration of free sulfisoxazole reached a peak by the
first sampling period, 1 hour (Tables 27 to 32). This rapid absorption
was due to the compound being given as a solution. The maximum free
sulfisoxazole ranged from 32.77 to 100.64 ug/ml with 3 of the pigs having
less than 50.0 ug/ml of sulfisoxazole as a maximum serum concentration
(Tables 27, 28, and 31). Total sulfisoxazole attained a maximum of
68.62 to 202.70 ug/ml with the same 3 pigs having the lowest total
sulfisoxazole concentration (Tables 27, 28, and 31).

Acetylsulfisoxazcle reached a maximum of 12.11 to 20.02 ug/ml in 2
to 4 hours in all 6 pigs (Tables 27 to 32). The half-life of acetyl-

sulfisoxazole ranged from 1.31 to 4.33 hours for the central cr distribution



~52-

100.0

10.0

SERUM ACETYLSULFISOXAZOLE IN SWINE (j1g/ml)



10 20 30 40 50 60 70

TIME (hours)

. : : 4, , .
Figure 7. Serum concentrations of acetylsulfisoxazole (N_) in swine.



Table 11. Two compartment pharmacokinetic parameters in swine administered sulfisoxazole as a single
oral dose.

Subject ao 8 a CB F cre
(hours) (hours +) (hours) (hours) (4%)

L 0.8058 0.0217 0.86 31.48 33.33 232.00
2 0.4386 0.0267 1.58 25.92 30.42 324.46
3 0.3300 0.0088 2.10 78.36 38.97 521.93
4 0.4331 0.0091 1.60 75.60 52.08 670.76
5 0.4100 0.0117 1.69 59.00 18.16 243.29
6 0.5823 0.0110 1.19 59.20 58.50 610.07

Mean t 0.5000 0.0148 1.50 54.99 38.58 433.75

sp* .1706 .0075 -43 21.84 14.76 191.81

*Mean + one standard deviation.

-¢¢o-



-54-

compartment Coy with a mean of 2.33 + 1.32 hours (Table 10). For
the peripheral or elimination compartment (ty a)» the half-life ranged
from 23.18 to 37.89 hours with a mean of 31.04 + 5.34 hours (Table 10).

The bioavailability (F) of sulfisoxazole was calculated (equation 3)
from the area under the curve (AUC) after oral (Table 11) and intravenous
(Table 8) administration. The bioavailability ranged from 18.15 to
58.50% in swine with a mean of 38.58 t 14.76% (Table 11).

The degree of plasma protein binding (£,) in vivo ranged from 40 to
70% throughout the trial period (Tables 27 to 32). Three of the animals
bound less than 50% throughout the trial (Tables 27, 29, and 31) while
the fraction bound in the remaining 3 animals was more than 60% throughout
the trial (Tables 28, 30, and 32).

Swine excreted 15.24% of the dose as "free" sulfisoxazole into the
urine and 10.62% as acetylsulfisoxazole in the first 24 hours of the trial
after oral administration of the drug (Table 9). By the end of the trial,
96 hours, the amount of free sulfisoxazole excreted was 18.27% of the
dose as compared to the 48 hour level of 17.70%. The amount excreted as
acetylsulfisoxazole was 12.78% of the dose at the end of the trial, 96

hours (Table 9).

Administration of Sulfisoxazole to Humans

Following the oral administration of sulfisoxazole to humans, the
data were analyzed as a single compartment model (Figure 8). The half-
life (ty) following oral administration ranged from 5.97 to 8.77 hours
with a mean of 7.41 t .59 hours (Table 12). Kaplan et al. (26) reported

a mean half-life of 5.83 © .42 hours after oral administration of



-55-

1000.0

—— FREE SULFISOXAZOLE
— — ACETYLSULFISOXAZOLE

pg/ml)

i00.0

10.0

SERUM CONCENTRATIONS IN HUMANS (
(leg scale)



4 8 l2 16 20 24

TIME (hours)

. 4
Figure 8. Serum concentrations of "free" and acetylsulfisoxazole (N )
in humans.



~56-

sulfisoxazole (Gantrisin*) and 5.89 + .33 hours after intravenous
administration of the drug. The mean elimination constant (k,) was
0.095 + .013 hours + (Table 12).

The bioavailability (F) was calculated from the oral area under the
curve (Table 12) and the calculated area under the curve (705.92)
following intravenous administration (26). The bioavailability ranged
from 126.04 to 202.26% with a mean of 165.99 + 29.02% (Table 12). The
reason for F exceeding 100% could be due to 1) entero~hepatic recycling
or 2) the comparison of 2 different populations. Kaplan et al. (26) re-
ported a mean bioavailability of 123.0 t 11.0% with a range of 94.0 to
131.02.

The biological haif~life (ty) of acetylsulfisoxazole ranged from
10.50 to 16.11 hours with a mean of 14.05 + 2.23 hours (Table 10). The
maximum serum acetylsulfisoxazole concentration ranged from 29.73 to
34.94 ug/ml and reached maximum in all subjects 4 hours after oral
administration of 2.0 grams of sulfisoxazole (Tables 33 to 38).

The maximum "free" and total sulfisoxazole occurred before the
first sampling period dve to the drug being administered as a solution.
The maximum serum free sulfisoxazole levels ranged from 164.70 to
189.35 ug/ml (Tables 33 to 38) while the maximum total sulfisoxazole
ranged from 224.17 to 235.75 ug/ml. Kaplan et al. (26) reported a range
of 127.4 to 210.6 ug/ml of free sulfisoxazole after oral administration
of Gantrisin.*

The fraction of sulfisoxazole bound to plasma proteins (f,) was
less than 20.0% during the first hour for all 6 subjects (Tables 33 to

38). During the remainder of the trial period, the f. was 25 to 40%

*Gantrisin (active ingredient, sulfisoxazole), Hoffman~LaRoche, Nutley,
N.J. -



Table 12. Single compartment pharmacokinetic parameters in 6 humans
administered a 2.0 gm oral dose of sulfisoxazole.

Subject kK, ty AUC Fx
(hours!) (hours) (2)
1 0.079 8.77 1427.78 202.26
> 0.116 5.97 889.73 126.04
3 0.094. 7.37 1155.81 164.16
4 0.095 7.29 1127.38 159.70
5 0.099 7.00 1043.31 147.79
6 0.086 8.06 1386.45 196.40
Mean + 0.095 7.41 1171.74 165.99
spt 013 95 204.89 29.02

*Bioavailability is calculated using the mean AUC from Kaplan et al. (26)
of 6 humans after intravenous administration of 2.0 grams of Gantrisin
(sulfisoxazole); AUC = 705.92.

aa
Mean * one standard deviation.



-58-

which is less than the 85% previously reported (52) but similar to

another report of 25% bound in vivo (40).

Comparison of the Pharmacokinetics of Sulfisoxazole in Dogs,

Swine, and Humans

The mean extrapolated serum concentrations of sulfisoxazole in the
first compartment (A) was 189.42 t 38.45 ug/ml in dogs, 245.07 © 44.88
ug/ml in swine, and reported as 108.43 = 23.13 in humans (26). In the
second compartment, the extrapolated serum concentration (B) was 2.56 7
.39 ug/ml in dogs, 0.423 + .088 ug/ml in swine, and reported as 152.13 +
8.43 ug/ml in humans (26). The mean total initial serum concentration
(co = A+B) was 191.98 ug/ml in dogs, 245.49 ug/ml in swine, and re-
ported as 259.03 ug/ml in humans (26). The total and free initial
serum concentrations were not significantly different (2) in either
species. Kaplan et al. (26) reported a two-compartment model in humans;
the first compartment had a mean es of 33.6 minutes or 0.56 hours,
the second compartment had a mean ty 0 of 5.89 hours (Table 13) following

intravenous administration. The ty

4 in dogs was 4.08 hours and 1.30
29

hours in swine after intravenous administration. The shorter one in
humans was closer to the tho in swine after intravenous administration.
The half-life of sulfisoxazole in the second compartment (ty, 3? was
33.74 hours in dogs and 53.33 hours in swine (Table 13). Neither of
these half lives were comparable to the value previously reported in
humans (26), 5.89 hours. Due to the short term of this trial, no second
compartment was observed in the human subjects.

After oral administration, the t, was 7.41 hours in humans, 4.37
29

hours in dogs, and 1.50 hours in swine (Table 13). The ty 8 in dogs was
29



Table 13. The biological half-life (t,) of sulfisoxazole.
2



Dogs

Intravenous

Oral

Swine
Intravenous

Oral

Humans
Intravenous*

Oral

*Kaplan et al. (26).

t
X50.

(hours)

0.56*

t
4,8

(hours)

33.74

34.46

53.33

54.99

5.89%

5.83%



~60-

34.46 hours, 54.99 hours in swine, and reported as 5.83 hours in humans
(26) after oral administration of sulfisoxazole. There were no differences

in the half lives of sulfisoxazole, t due to the route of

B’

administration in dogs and swine. If the ty a of 7.41 hours in humans
2

>

1 . or ty
50 Bs

after oral administration is compared to the t, reported by Kaplan
“2

»8

et al. (26), there is no difference in the route of administration or in
the compartments. Kaplan et al. (26) postulated the two-compartment
model in humans due to taking 4 blood samples within the first hour
after intravenous administration of sulfisoxazole (Gantrisin*). There
were large differences between species. Humans had the longest mean

t » 7.41 hours, followed by dogs, 4.08 and 4.37 hours, and then swine,

1
BO

1.30 and 1.50 hours (Table 13). In the second compartment, swine had

the longest mean t, 53.55 and 54.99 hours, followed by the dogs,
3

» 8?
33.74 and 34.46 hours (Table 13).

Maximum serum acetylsulfisoxazole concentrations (Tables 27 to 38)
were higher in humans (30.0 to 35.0 ug/ml) than in swine (12.0 to 20.0
ug/ml). However, swine were able to acetylate (ny sulfisoxazole at a
faster rate as shown by the shorter time for maximum serum acetylsulfisoxa-
zole in swine (2 to 4 hours) as compared to humans (4 hours). Humans had
a mean th og of 14.05 hours (Table 14) after oral administration which is
longer than the 9.0 hours previously reported (33). Swine had a mean
theo of 2.33 hours after oral administration and 1.56 hours after

intravenous administration of sulfisoxazole. The t, of acetyl-

3,8

sulfisoxazole was 26.55 hours after oral administration and 31.04 hours

*Gantrisin (active ingredient, sulfisoxazole), Hoffman-LaRoche, Nutley,
N.J.



-61-

Table 14, The biological half-life (t,) of acetylsulfisoxazole, the N

metabolite of sulfisoxazole?

“te
(hours)
Dogs
Intravenous ND*
Oral ND*
Swine
Intravenous 1.56
Oral 2.33
Human
Oral 14.05

4

"1.48

(hours)

ND*

26.55

31.04

*ND = Non detectable. No (x) acetylsulfisoxazole was detected in dogs.



-62-

after intravenous administration of sulfisoxazole (Table 14) in swine.
Dogs did not metabolize sulfisoxazole to the (n*) acetyl metabolite.

The distribution constants of sulfisoxazole (Table 15) from the
central to the peripheral compartment (kK, 9) was greatest in humans (0.45
hours followed by swine (0.0296 hours"), and then dogs (0.0140
hours 3: The distribution from the peripheral compartment to the
central compartment (ko 1) was fastest in humans (0.87 hours‘), followed
by dogs (0.0228 hours “}, and then swine (0.0162 houve ~). The elimina-
tion constants (k, 9) were greatest in swine (0.5063 hoare “9. Eollowed
by humans (0.195 hours 7). and then dogs (0.1565 hours“). The order of
the elimination constant (ky) was the same as the oo of free
sulfisoxazole, shortest in swine, then dogs, and longest in humans.

The difference in distribution between the two compartments (k,)/K, 5)
was highest in humans (26), 2.3, followed by dogs, 1.63, and then swine,
0.55, indicating that the drug returned from the peripheral compartment
to the central compartment fastest in humans and slowest in swine

(Table 16). These values were concurrent with the mean half-life of

sulfisoxazole in the second compartment where t, was reported as shortest

e:
in humans (26), followed by dogs, and longest in swine (Table 13). The
ratio of B/K, 4 was reported as 0.66 in humans (26), 0.13 in dogs, and
0.03 in swine (Table 16). This is the same as the ko Ky (Table 16)
ratio so that more sulfisoxazole is available for elimination from the post-
distributive phase in humans, followed by dogs, and the least available
was in swine.

The mean volume of distribution for the central compartment (V,)

was approximately the same in dogs, 10.60 L, and swine, 10.48 L (Table 17).

The mean volume of the second compartment (V,) was much larger in swine,



~63-

Table 15. The mean distribution constants after intravenous administra-
tion of sulfisoxazole.*

Kio Koy Kio

(hours +) (hours +) (hours +)
Dogs 0.0140 0.0228 0.1565
Swine 0.0296 0.0162 0.5063
Humans" 0.45 0.87 0.195

*Mean of 6 individuals.

+
Kaplan et al. (26).



Table 16.

Dogs
Swine

Humans*

*Kaplan et al.

-64-

The ratio of distribution constants after administration
of sulfisoxazole.

ky4/K5 8/k,

1.63 0.13

0.55 0.03
2.30 0.66

(26).



-~65-

19.76 L, than in dogs, 6.55 L (Table 17). The calculated vy from pre-
vious data (26) was 7.67 L and 8.54 L for V5 in humans. Due to the
analytical procedure and the length of the trial (26), this Vo should not
be compared with the Vo reported in dogs and swine. From the calculated
Ya for dogs, swine, and humans, there is an approximate 40% difference

in the animal vy over the human volume of distribution. The volume of
distribution in the second compartment (V,) is much larger in swine than
in dogs.

The mean bioavailability (F) was greatest in humans, 165.99% when
the area under the intravenous curve from Kaplan et al. (26) was used
(Table 18). This value agreed with previous data (26) that F was 123.0%
in humans. The absolute bioavailability of sulfisoxazole was 99.24% in
dogs and 38.58% in swine (Table 18). The bioavailability in dogs is
closer to the calculated F in humans, and swine appear to be vastly
different from both humans and dogs. However, the higher bioavailability
in humans is due to enterohepatic circulation of sulfisoxazole which does
not occur in dogs and swine. The majority of sulfisoxazole is not
absorbed from the gastro-intestinal tract in swine and is excreted in
the feces.

The degree of protein binding in vivo was less than 30% in 2 dogs
for the first 3 hours and in 4 pigs for the first hour after intravenous
administration and in all 6 humans for the first hour after oral adminis-
tration of sulfisoxazole. More than 70% of sulfisoxazole was bound to
plasma proteins in dogs at 44 hours after oral administration of sulfisoxa-
zole. During the remainder of the trial, 30 to 50% of sulfisoxazole was

bound to plasma proteins in humans, dogs, and swine which compared

favorably with the 25% previously reported (40). This observation



~66-

Table 17. Comparison of the volumes of distribution in dogs, swine, and
humans after administration of sulfisoxazole.

Species Vi V5
(liters) (liters)
Dogs 10.60 6.55
Swine 10.48 19.76
Humans* 7.67 8.45

*Kaplan et al. (26).



-67-

Table 18. Comparison of the bioavailability* of sulfisoxazole in dogs,

swine, and

Species

Dogs
Swine

Humans

*Calculated

humans.

Bioavailability

99.24
38.58

165.99

from area under the plasma curve.



-68-

suggests that an individual difference occurs along with a species and
treatment difference. Fifty to sixty-three percent of sulfisoxazole was
bound to human serum proteins in vitro (Table 19) at 25 to 200 ug/ml.

At 300 yg/ml, only 41.0% of sulfisoxazole was bound. This suggests that
the degree of protein binding is capacity limited as the serum concen-
tration increases above 200 ug/ml. This hypothesis would explain the low
fraction bound which was observed during this experiment.

The amount of free sulfisoxazole excreted in the urine was 39.59%
of the dose in 24 hours after intravenous administration and 26.20% of
the dose in 24 hours after oral administration in dogs. The amount
excreted in the urine by swine was 25.86% of the dose and 26.23% of the
dose in 24 hours after oral and intravenous administration of sulfisoxa-
zole, respectively. By the end of the trial, 72 hours and 96 hours for
the intravenous and oral routes, respectively, more than 30.04 of the
dose was excreted in swine (Table 9) and in the dogs after oral adminis-
tration (Table 6). After intravenous administration, over 40.0% of the
dose was excreted in the urine by dogs. Kaplan et al. (26) reported
that 52.9% of the dose was excreted as free sulfisoxazole by humans.
Acetylsulfisoxazole accounted for less than half of the urinary level in
swine (Table 9) which confirmed the previously reported 30% (24)
excreted as the acetyl metabolite in humans. The differences between
the human and dog and pig excretion levels could be explained partly by
the analytical method used. Kaplan et al. (26) used a nonspecific method
for aromatic amines, the Bratton-Marshall method (6). This experiment

used a more specific method for both free and acetylsulfisoxazole.



-69-

Table 19. The fraction of sulfisoxazole bound (fp) to human serum
proteins in vitro.

Sulfisoxazole
Concentration
(ug/ml)

25.0
50.0
100.0
200.0

300.0

"Free" Serum
Concentration of Sulfisoxazole
(ug/ml)

7.57
16.67
27.27
57.57

112.12

57.00

50.00

63.00

60.00

41.00



-70-

Serum Bilirubin Concentrations

Dogs--Intravenous Administration

The mean total bilirubin concentration in 6 dogs administered
sulfisoxazole intravenously exhibited statistically significant (p < .01)
linear increases (2) at 4.5 (p < .05), 6.0 (p < .01), 9.0 (p < .01), and
12.0 (p < .01) hours with the maximum levels at 6 and 12 hours (Figure 9).
By the next sampling period, 22 hours, the mean total bilirubin concen-
tration had decreased to levels which were not significantly different
from control values until the 56 hour sampling period (p < .01).

The mean conjugated bilirubin levels also showed statistically
significant increases at 4 hours (p < .01) and continued throughout the
sampling period (p < .01), 72 hours. There was a significant (p < .01)
maximum mean conjugated bilirubin at 12 hours which coincided with the
maximum mean total bilirubin concentration (Figure 9). A second increase
was observed at 56 hours (p < .01) which coincided with the second in-
crease in total bilirubin (Figure 9). The mean indirect bilirubin con-
centration (Figure 9) was not significantly different from the control
or 0 level throughout the sampling period.

The mean total bilirubin was significantly correlated with mean
conjugated bilirubin (R = 0.75, p = .0001) and with mean indirect bili-
rubin (R = 0.31, p = .004) after intravenous administration. The
significant correlation of total and conjugated bilirubin is due to the
concomitant increase in glucuronidation activity as total bilirubin levels
increase. If the glucuronidation enzyme system is not capacity limited,
the possibility of kernicterus, due to an increase in indirect bilirubin,

is minimized in the dog after intravenous administration of sulfisoxazole.



0.60

—— TOTAL
— — - CONJUGATED

che —-— INDIRECT

0.40

MEAN SERUM BILIRUBIN CONCENTRATIONS(mg/dl)

0.30
—_————— or !
~
a
_7e~ {
- ~
0.20 ~ _ oe ~
~ =~ ~~
a ~~»
,@
0.10 Fee 2 o. te
~ eee
aes ~ e + OT



TIME (hours)

Figure 9. Mean serum bilirubin concentrations in dogs after intravenous administration of
sulfisoxazole.



-72-

This enzyme system acts to maintain a reduced or limited indirect bili-
rubin concentration and reduces the toxicity of displaced "protein-
bound" bilirubin or increased heme degradation. This was further empha-
sized by the significant negative correlation of conjugated and indirect
bilirubin (R = -0.39, p = .0002). As the level of conjugated bilirubin
increased, the indirect bilirubin level decreased.

A second explanation would be that sulfisoxazole alters the function
of the normal hepatocyte to increase conjugated bilirubin regurgitation
into the general circulation instead of being excreted into the bile.
The resulting increase in total bilirubin would be the result of the
increased level of regurgitated, conjugated bilirubin along with the

normal level of indirect bilirubin.

Dogs--Oral Administration

When sulfisoxazole was administered orally to dogs, there was a
significant linear increase in mean total (p < .01), conjugated (p < .001),
and indirect (p < .0001) bilirubin (Figure 10). Total bilirubin reached
its first significant peak at 12 hours (p < .01) and a higher concen-
tration at 76 hours (p < .01) (Figure 10). The mean total bilirubin
levels were significantly increased at 8 (p < .05), 10 (p < .01), and 12
(p < .01) hours. A second higher peak occurred at 76 hours (p < .01) and
remained greater than the control levels at the end of the sampling
period, 96 hours (p < .01) (Figure 10). The mean conjugated bilirubin
levels were also linearly increased (p < .001) during the sampling
period. Significant increases (p < .05) were observed at 8, 10, and 12

hours (Figure 10), the same periods in which total bilirubin was



Figure

0.60



0.50 —— TOTAL

~-- CONJUGATED
— ~— INDIRECT

0.40

0.30

0.20

MEAN SERUM BILIRUBIN CONCENTRATIONS (mg/d!)

TIME ( hours )

10. Mean serum bilirubin concentrations in dogs after oral administration of
sulfisoxazole.

-¢l-



-74-

significantly increased. After the 12 hour period, the mean conjugated
bilirubin had returned to levels which were not significantly different
from control levels. The mean conjugated bilirubin reached its maximum
concentration (0.22 mg/dl) at 10 hours while the maximum total bilirubin
(0.31 mg/dl) occurred 2 hours later, at the 12 hour sampling period
(Table 40). The mean indirect bilirubin was not significantly different
from control levels until the 76 and 96 hour periods (p < .01). Mean
indirect bilirubin reached a maximum of 0.28 mg/dl at 76 hours but had
begun to decline by the end of the trial, 96 hours.

Total bilirubin was significantly correlated with conjugated bili-
rubin (R = 0.60, p = .0001) and with indirect bilirubin (R = 0.56,
p = .00001) while conjugated bilirubin was also negatively correlated
with indirect bilirubin (R = -0.31, p = .003). The significant correla-
tion of total and conjugated bilirubin is explained by an increase in
glucuronidation as increased heme degradation or increased indirect
bilirubin occurs or due to regurgitation of conjugated bilirubin into the
general circulation. The significant correlation of indirect bilirubin
and total bilirubin illustrated that the glucuronidation activity could
not account for conjugation of all the bilirubin present. The negative
correlation of indirect and conjugated bilirubin is supportive evidence
that toxic, indirect bilirubin levels can be reduced, to prevent

kernicterus, if glucoronidation activity can be stimulated.

Comparison of Bilirubin Levels in Dogs

The mean total bilirubin reached maximum at 12 hours after intra-

venous (0.39 mg/dl) and oral (0.31 mg/dl) administration of sulfisoxazole



~75-

(Figure 11), with a second increase at 56 hours after intravenous adminis-
tration and 76 hours after oral administration. Conjugated bilirubin was
also maximum in the 10 and 12 hour period after oral (0.22 mg/dl) and
intravenous (0.32 mg/dl) administration of sulfisoxazole (Figure 12),
respectively. Conjugated bilirubin was significantly elevated at the

4.5 hour period and throughout the trial period after intravenous
‘administration; after oral administration, conjugated bilirubin was
significantly increased between 8 and 12 hours only. Indirect bilirubin
was not significantly increased after intravenous administration but was
significantly increased at 76 and 96 hours after oral administration
(Figure 13).

The total bilirubin increase, after oral or intravenous administra-~
tion of sulfisoxazole, was accompanied by an increase in glucuronidation
activity which increased the conjugated, water-soluble bilirubin levels.
This prevented toxicity by reducing the indirect bilirubin level and
increasing the water-soluble, conjugated bilirubin which is more easily
excreted. The glucuronidation activity was reduced during the later
periods after oral administration, allowing the potentially toxic, in-
direct bilirubin to increase.

There was a negative correlation of conjugated and indirect bilirubin
after intravenous (R = -0.39, p = .0001) and oral (R = -0.31, p = .003)
administration of sulfisoxazole. After intravenous administration,
conjugated bilirubin levels increased along with an increase in total
bilirubin (Figure 9). After oral sulfisoxazole administration, conjugated
bilirubin levels increased initially as total bilirubin levels increased
but later decreased, allowing indirect bilirubin levels to increase.

This result leads one to conclude that some pathological event is induced



0.50

—— INTRAVENOUS
— — ORAL

0.40

0.30

0.20

0.10

TOTAL BILIRUBIN CONCENTRATIONS (mg/dl)



5 10 20 30 40 50 60 70 80 90 100
TIME (hours)

Figure 11. Comparison of the mean total bilirubin concentrations in dogs after intravenous and
oral administration of sulfisoxazole.



0.50

0.40 —— INTRAVENOUS

—— ORAL

0.30

-{I-

0.20

MEAN CONJUGATED BILIRUBIN CONCENTRATIONS (ma/dl)



5 Te) 20 30 40 50 60 70 80 9390 {00
TIME (hours)

Figure 12. Comparison of the mean conjugated bilirubin concentrations in dogs after intravenous
and oral administration of sulfisoxazole.



0.50

0.40

0.30

—— INTRAVENOUS 7 ~

— — ORAL /

0.20

-~Sl-



MEAN INDIRECT BILIRUBIN CONCENTRATIONS (mg/d!)
\
?
/

5 10 20 30 40 50 60 70 80 390 100
TIME (hours)

Figure 13. Comparison of the mean indirect bilirubin concentrations in dogs after intravenous
and oral administration of sulfisoxazole.



during oral administration of sulfisoxazole but not after intravenous
administration which could allow indirect bilirubin to reach toxic levels.

This pathological event could be explained by the pharmacological
"first-pass" effect (57). In the "first-pass" effect, all of an absorbed
drug is presented to the liver before distribution whereas after intra~
venous administration, only 20% of the drug passes through the liver
before being distributed or excreted. In this case, the greater concen-
tration of sulfisoxazole presented to the liver, in addition to the lack
of acetylation of the drug, could be responsible for altering the
normal detoxification of bilirubin so that one observes an increase in
indirect bilirubin.

Total and conjugated bilirubin reached higher levels within 4 to 12
hours after intravenous administration than after oral administration
(Figures 11 and 12). After oral administration, conjugated bilirubin
reached its maximum within this time period (10 hours) but total and
indirect bilirubin did not reach maximum until 76 hours. The increased
indirect bilirubin levels should be expected after intravenous adminis-
tration due to the increased serum concentration of sulfisoxazole when
compared to levels following oral administration which would allow
sulfisoxazole to displace more bilirubin from albumin. The increased
total and indirect bilirubin levels at the end of the oral administration
trial supports the hypothesis that a pharmacological event is induced by
the oral route but not by the intravenous route which caused an increase
in total and indirect bilirubin, with possible toxic side effects at a
time much later after a single, oral dose of sulfisoxazole.

There were no statistical differences in serum total, conjugated or
indirect bilirubin between individual dogs after oral or intravenous

administration of sulfisoxazole.



0.60

——- TOTAL

--~-— CONJUGATED
0.50 —-— INDIRECT
0.40
0.30

MEAN SERUM BILIRUBIN CONCENTRATIONS (mg/dl)



4 8 16 24 32 40 48 56 64 72
TIME (hours)

Figure 14. Mean serum bilirubin concentrations in swine after intravenous administration of
sul Fisoxazole.

-08-



-81-

Swine-~Intravenous Administration

An analysis of variance, using mean bilirubin concentrations, re-~
vealed no statistical difference among the 6 pigs or during time intervals
for serum total, conjugated or indirect bilirubin after intravenous
administration of sulfisoxazole. The mean total conjugated and indirect
bilirubin were increasing at the end of the trial but were not signifi-
cantly different than control levels (Figure 14).

Total bilirubin was significantly correlated with conjugated bili-
rubin (R = 0.86, p = .0001) and with indirect bilirubin (R = 0.61,

p = .0001) after intravenous administration of sulfisoxazole.

Swine~--Oral Administration

After oral administration of sulfisoxazole, mean total bilirubin was
significantly increased (p < .01) to 0.52 mg/dl at 6 hours (Figure 15).
A parallel significant (p < .01) increase was also observed in mean con-
jugated bilirubin at 6 hours, 0.34 mg/dl (Figure 15). A significant
(p < .0001) linear decrease did occur over the trial period in conjugated
bilirubin. Mean indirect bilirubin was not significantly different from
control values at any time after oral administration of sulfisoxazole.
However, indirect bilirubin levels did exceed conjugated levels after
the 32 hour sampling period and remained at an increased level throughout
the remainder of the trial (Figure 15).

Total bilirubin was significantly correlated with conjugated bilirubin
(R = 0.81, p = .0001) and with indirect bilirubin (R = 0.42, p = .0001)

after oral administration of sulfisoxazole.



0.60

0.50 — TOTAL
——— CONJUGATED
—-— INDIRECT

MEAN SERUM BILIRUBIN CONCENTRATIONS (mg/d!)



TIME (hours)

Figure 15. Mean serum bilirubin concentrations in swine after oral administration of

sulfisoxazole.



-~33-

Comparison of Bilirubin Levels in Swine

It appears that an increase in total bilirubin in the pig is
accompanied by an increase in conjugated and indirect bilirubin regardless
of the route of administration, as shown by the positive correlation
coefficients. The higher serum "free" sulfisoxazole concentrations
(Tables 27 to 32) after intravenous administration did not significantly
affect bilirubin concentrations (Figure 14) but oral administration did
significantly increase total (p < .01) and conjugated (p < .0001) bili-
rubin at 6 hours but did not affect indirect bilirubin (Figure 15).
Indirect bilirubin was closely correlated with conjugated bilirubin
after intravenous administration (Figure 14) but exceeded conjugated
bilirubin after oral administration (Figure 15), which suggests that an
oral dose is handled differently than the intravenous one.

Intravenous administration of sulfisoxazole does not appear to
affect bilirubin levels in the pig. After oral administration, an in-
crease in total bilirubin could be accompanied by an increase in the
conjugated level which reduces the hazard of indirect bilirubin toxicity.
While the indirect bilirubin levels were not significantly different by
either treatment, one should note that indirect bilirubin levels did
exceed conjugated bilirubin levels only following oral administration
(Figure 15). While no toxic levels of bilirubin have been established
in swine, there was a significant increase in total (Figure 16) and
conjugated (Figure 17) bilirubin in 6 hours after oral administration
which did not occur after intravenous administration. This suggests
that an oral dose of sulfisoxazole may have a different effect in vivo

than an intravenous administration even though the intravenous route



0.60

0.50

0.30

—
—
—_—

-Q-

0.20



—— INTRAVENOUS
— — ORAL



MEAN TOTAL BILIRUBIN CONCENTRATION (mg/dl)

5S 10 20 30 40 50 60 70 80 90 100
TIME (hours)

Figure 16. Comparison of the mean total bilirubin concentrations in swine after intravenous and
8 P
oral administration of sulfisoxazole.



Full Text
COMPARISON OF THE PHARMACOKINETICS AND TOXICITY OF SULFISOXAZOLE
IN HUMANS AND TWO MONOGASTRIC ANIMAL SPECIES

By

ROBERT L. SUBER

A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY GF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1979
Copyright 1979
by

Robert L. Suber
ACKNOWLEDGEMENTS

The author wishes to express his sincerest appreciation and thanks
to Dr. George T. Edds, chairman of the supervisory committee, for his
assistance and guidance throughout this investigation.

The author also wishes to acknowledge the helpful criticism and
suggestions of Dr. Paul T. Cardeilhac for his assistance in pharmacology,
Dr. John C. Gudat for his collaboration in clinical chemistry, and
Dr. George Torosian and Dr. Charles Lee for their assistance in pharmaco-
kinetics.

Deepest appreciation and special thanks are expressed to Dr. Orlando
Osuna, Mr. Gary Neff, and Mrs. Rita Bellis Bortell for their assistance
and friendship throughout the investigation.

The author also wishes to thank Dr. John A. Cornell for his
assistance in the programming and interpretation of the statistical
analysis of the bilirubin data.

The author's deepest appreciation and love are extended to Mrs.
Christine Suber for her understanding, patience and assistance throughout

this investigation.

iii
TABLE OF CONTENTS

ACKNOWLEDGEMENTS. 2. 2 6 we ee we ee ee ee te ee
LIST OF TABLES. . 2. 2. 1. 6 ew we ee ee ee ee te ee
LIST OF FIGURES .. .. 1. 1 6 ee ew ew ee ee ee ee
ABSTRACT. . 2. 6 2 6 ee wee ee ewe ee we te te
INTRODUCTION. . 2. 2... 2 ee eee ee ee
REVIEW OF LITERATURE. . . 2. 1 1 1 1 ee we ee es
Sulfisoxazole. . . 1 2 6 ww ee ew we we ee ee eee
Blood Concentration of Sulfisoxazole. .....
Metabolism of Sulfisoxazole .........
Protein Binding of Sulfisoxazole. ...
Toxic Effects of Sulfisoxazole. ......
Urinary Excretion of Sulfisoxazole. .........
Pharmacokinetics . . 2... 1. 2 ee ee ee ee ee ee ee
Absorption Rate Constant and Bioavailability. ....

Distribution. . .... . ee ee we ew ee te ee

Elimination... 2... 6 6 ee we ew ee ee

Plasma Protein Binding. .......4..+e.2.-.

Two Compartment Model ........446-.
MATERIALS AND METHODS . . ..... ee ee we

High Performance Liquid Chromatographic Analysis of
Sulfonamides by Tonic Suppression. ..........

Materials .. . . 1 2 1 ww ee ee ee ee ee

iv

10

il

12

14

15
Page

Extraction and Separation .........+46+.+...... 18
Optimization of the Liquid Chromatographic Procedure. . . 30
Experimental Model . . 1. 1 ee ee ee ew we ww ew ew we ee) 680
Blood Samples . 2... ee ww ew ee eee eee we ee 8
Serum bilirubin. . .......-..24..66.42 +... 32

Serum albumin. . 2. 2-2 ee ee ew ee we we ww ew 8B

Serum sulfisoxazole. . . .. 1... 2 2 ee ew ee ee B34

Urinary Sulfisoxazole . . . 1. 1 6 6 ee ee ee ee ew B85

Analysis of Data. . . 1... 6 ee ee ew ee ee we ww we 36
In Vitro Plasma Protein Binding .........+.444.. 36

RESULTS AND DISCUSSION. . 2. 2. 1 1 2 eee ww eee we ew ew ee 8

Pharmacokinetics of Sulfisoxazole. ...... 4... 2... . 38
Administration of Sulfisoxazole to Dogs ......... 39
Intravenous administration . ....... 2... 6. 39

Oral administration. ............. 2... 43
Administration of Sulfisoxazole to Swine. ........ 45

Intravenous administration ........4. 6+... 45
Oral administration. ........ 6.6... 6«.e.. 5)
Administration of Sulfisoxazole to Humans ........ 54

Comparison of the Pharmacokinetics of Sulfisoxazole
in Dogs, Swine, and Humans. ........ 6.6.6.2... 58

Serum Bilirubin Concentrations ....... +... 6 « «© © « s 70

Dogs--Intravenous Administration. ............ 70
Dogs--Oral Administration .........4+. +2466... 72
Comparison of Bilirubin Levels in Dogs. ......... 74
Swine--Intravenous Administration ............ 81
Swine--Oral Administration.
Comparison of Bilirubin Levels in Swine
Humans--Oral Administration .

Comparison of Bilirubin Levels in Dogs, Swine,
and Humans. . ..... 56 2 se we ee

Conclusions Concerning Bilirubin Levels After
Administration of Sulfisoxazole .

Serum Albumin Concentrations
SUMMARY AND CONCLUSIONS
APPENDIX.
BIBLIOGRAPHY.

BIOGRAPHICAL SKETCH . ......+ 2...

vi

37

. 87

- 97

98

.103

.109

149
LIST OF TABLES

Table

1. Retention time (minutes) of sulfonamides in a water/
methanol (50/50) mobile phase with acetate buffer (PH
4.00) and without buffer (pH 7.45). ....... -. ss

2, Retention time (minutes) of sulfonamides in a water/
methanol (60/40) mobile phase with acetate buffer (pH
4.00) and without buffer (pH 7.45). ........4-..8.

3. Retention time (minutes) of sulfonamides in spiked human
plasma samples in a water/methanol (50/50) mobile phase
with acetate buffer (pH 4.00) and without buffer (pH 7.45).

4, Retention time (minutes) of sulfonamides in spiked human
plasma samples in a water/methanol (60/40) mobile phase
with acetate buffer (pH 4.00) and without buffer (pH 7.45).

5. Two compartment pharmacokinetic parameters in dogs
administered sulfisoxazole as a single intravenous dose. .

6. The mean amount of sulfisoxazole excreted in the urine of
6 dogs. 2. 6 6 6 we ee ee ee et et we ee we ee

7. Two compartment pharmacokinetic parameters in dogs adminis-~
tered sulfisoxazole as a single oral dose ........

8. Two compartment pharmacokinetic parameters in swine
administered sulfisoxazole as a single intravenous dose...

S. The mean amount of sulfisoxazole and acetylsulfisoxazole
(N+) excreted in the urine of 5 pigs as a percentage of

the dose. . . 2... ew ee eee we ew ee ke kk kk

10. The biological half-life of acetylsulfisoxazole in’) after
a single dose of sulfisoxazole. . ...... 2.206 2685068.

11. Two compartment pharmacokinetic parameters in swine
administered sulfisoxazole as a single oral dose. .....

12. Single compartment pharmacokinetic parameters in 6 humans
administered a 2.0 gm oral dose of sulfisoxazole. .....

13. The biological half-life (t,) of sulfisoxazole. ......
3

vil

LS
$=
Table

14.

15.

16.

22.

23.

24,

25.

26.

The biological half-life (t1,) of acetylsulfisoxazole,
the N4 metabolite of sulfisoxazole, .......

The mean distribution constants after intravenous
administration of sulfisoxazole ..........4.-

The ratio of distribution constants after administration
of sulfisoxazole. . . . 1 1 1 we ew ew ew ew ee ee et te

Comparison of the volumes of distribution in dogs, swine,
and humans after administration of sulfisoxazole. ...

Comparison of the bioavailability of sulfisoxazole in
dogs, swine, and humans ....... 2. 1 2 we ee eee

The fraction of sulfisoxazole bound (fg) to human serum
proteins in vitro . 6. 6. 1 ee ee wee ew ew ee ee

Mean control values of serum bilirubin and albumin in
humans, dogs, and swine .........-. 6+ -+. + © ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in dog 1 after intravenous and oral administration of
sulfisoxazole . . 1... 6 ee wee ee ew we et ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in dog 2 after intravenous and oral administration of
sulfisoxazole . 1. 6. 1 6 1 ew ee eee ee we we ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in dog 3 after intravenous and oral administration of
sulfisoxazole . . 1... 1 ee ew ew ee ee ew ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in dog 4 after intravenous and oral administration of
sulfisoxazole . . 1. 6. 1 6 eee we we eee ww ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of suifisoxazole
in dog 5 after intravenous and oral administration of
sulfisoxazole . 1. 1 16 6 6 ee ewe we ee ee et

Serum concentrations of albumin, bilirubin, and
sulfisoxazcle and urinary concentrations of sulfisoxazole
in dos 5 after intravenous and oral administration of
sulfisoxazole . . 6. 6 1 we ew ee ee ee ee ee ee

viii

63

64

66

67

89

109

113

115

117

119
Table

27.

28.

29,

30.

31.

32.

33%

34.

35.

36.

37.

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in pig 1 after intravenous and oral administration of
sulfisoxazole . 2... 1. 6 1 ew we ee we ee ee ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in pig 2 after intravenous and oral administration of
sulfisoxazole . . 1. 1. 1 6 ew ew ew we we ee ee ee es

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concantrations of sulfisoxazole
in pig 3 after intravenous and oral administration of
sulfisoxazole 2... 6 6 ew we ee we ew we ee ee es

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in pig 4 after intravenous and oral administration of
sulfisoxazole . 2. 2. 2. 2. 2 6 6 6 ww ew we ee we we

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in pig 5 after intravenous and oral administration of
sulfisoxazole . 2... 1 1 6 ew eee ee ee ee ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in pig 6 after intravenous and oral administration of
sulfisoxazole . 2... 1. 1 we ee we ew we ee ee

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in human 1 after oral administration of sulfisoxazole. .

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in human 2 after oral administration of sulfisoxazole . .

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in human 3 after oral administration of sulfisoxazole . .

Serum concentrations of albumin, bilirubin, and
sulfisoxazole and urinary concentrations of sulfisoxazole
in human 4 after oral administration of sulfisoxazole . .

Serum concentrations of albumin, bilirubin, and

sulfisoxazole and urinary concentrations of sulfisoxazole
in human 5 after oral administration of sulfisoxazole. .

1X

Page

121

he
nN
ni

127

129

131

135

134

135

136

137
Table

38.

39.

40.

Al.

42.

-_
2

Serum concentrations of albumin, bilirubin, and

sulfisoxazole and urinary concentrations of sulfisoxazole

in human 6 after oral administration of sulfisoxazole .

Means and standard deviations of serum albumin, total
bilirubin, conjugated bilirubin, and indirect bilirubin

in 6 dogs after intravenous administration of sulfisoxazole

Means and standard deviations of serum albumin, total
bilirubin, conjugated bilirubin, and indirect bilirubin
in 6 dogs after oral administration of sulfisoxazole. .

Means and standard deviations of serum albumin, total
bilirubin, conjugated bilirubin, and indirect bilirubin
in 6 swine after intravenous administration of
sulfisoxazcle . .. 1. 6 ee ew ew ee ee ee ee

Means and standard deviations of serum albumin, total
bilirubin, conjugated bilirubin, and indirect bilirubin
in 6 swine after oral administration of sulfisoxazole .

Means and standard deviations of serum albumin, total
bilirubin, conjugated bilirubin, and indirect bilirubin
in 6 humans after oral administration of sulfisoxazole.

138

139

140

141

142

143
LIST OF FIGURES

Figure

1.

10.

li.

Standard concentration curves of sulfanilamide,
sulfaguanidine, sulfamerazine, sulfamethazine, sulfa-
pyridine, sulfisoxazole, n4 acetylsulfisoxazole, and
sulfathiazole in a water/methanol (50/50) mobile phase
with acetate buffer at pH 4.00. ..........4.4.2.4..

Standard concentration curves of sulfanilamide,
sulfaguanidine, sulfamerazine, sulfamethazine, sulfa-
pyridine, sulfisoxazole, n4 acetylsulfisoxazole, and
sulfathiazole in a water/methanol (50/50) mobile phase
with an isocratic pH 7.45 ........ 2.0.4.6 42.80.2084

Standard concentration surves of sulfanilamide,
sulfaguanidine, sulfamerazine, sulfamethazine, sulfa-
pyridine, sulfisoxazole, v4 acetylsulfisoxazole, and
sulfathiazole in a water/methanol (60/40) mobile phase
with acetate buffer at pH 4.00. ........4...

Standard concentration curves of sulfanilamide,
sulfaguanidine, sulfamerazine, sulfamethazine, sulfa-
pyridine, sulfisoxazole, n4 acetylsulfisoxazole, and
sulfathiazole in a water/methanol (60.40) mobile phase
with an isocratic pH 7.45 .........4464..

Serum concentrations of "free" sulfisoxazole in dogs. ...
Serum concentrations of "free" sulfisoxazole in swine. .

. . 4) :
Serum concentrations of acetylsulfisoxazole (N’) in swine .

4
Serum concentrations of "free" and acetylsulfisoxazole (N°)
in humans . . . 1 6 6 ee ew ee ee ee ee ee

Mean serum bilirubin concentrations in dogs after
intravenous administration of sulfisoxazole .....

Mean serum bilirubin concentrations in dogs after oral
administration of sulfisoxazole .........4...

Comparison of the mean total bilirubin concentrations in

dogs after intravenous and oral administration of
sulfisoxazole . 2... 6 6 we ew ee ee ew we we ee ee tw

xi

Page

28

55

71

73
Figure

12.

13.

14.

15.

16.

17.

18.

19.

20.

23.

24,

Comparison of the mean conjugated bilirubin concentrations
in dogs after intravenous and oral administration of
sulfisoxazole . . 1... 1. 1 6 ww ew we ew wt tt we el

Comparison of the mean indirect bilirubin concentrations
in dogs after intravenous and oral administration of
sulfisoxazole . 2... 1. 1 ee eee ew ee ee ee et ee

Mean serum bilirubin concentrations in swine after
intravenous administration of sulfisoxazole .........

Mean serum bilirubin concentrations in swine after oral
administration of sulfisoxazole .......4... 4.604668

Comparison of the mean total bilirubin concentrations in
swine after intravenous and oral administration of
sulfisoxazole .... 1. 2. 6 1 0 ee we we ww wt lw lt

Comparison of the mean conjugated bilirubin concentrations
in swine after intravenous and oral administration of
sulfisoxazole . . 1. 2. 6. 1 1 1 ee eee ee ew ee et es

Comparison of the mean indirect bilirubin concentrations
in swine after intravenous and oral administration of
sulfisoxazole . . . 1... 1 ee ew ew we we ww we ee te ee

Mean serum bilirubin concentrations in humans after oral
administration of sulfisoxazole ......4...464+.048c088.

Comparison of the mean total bilirubin concentrations in
dogs and swine after intravenous administration of
sulfisoxazole . 1. 1. 1. 1. we we we we we ee ee ee

Comparison of the mean total bilirubin concentrations in
dogs and swine after oral administration of sulfisoxazole .

Comparison of the mean conjugated bilirubin concentrations
in dogs and swine after intravenous administration of
sulfisoxazole . . 2. 1. 1 2 6 eee we ee ee ee

Comparison of the mean conjugated bilirubin concentrations
in dogs and swine after oral administration of sulfisoxazole.

Comparison of the mean indirect bilirubin concentrations
in dogs and swine after intravenous administration of

sulfisoxazole . . .. 1. 2 2 © © te © © ew ew tt tw le lt el

Comparison of the mean indirect bilirubin concentrations
in dogs and swine after oral administration of sulfisoxazole.

Mean serum albumin concentrations in dogs after intravenous
and oral administration of sulfisoxazole. ..........

xii

77

78

80

82

84

85

86

88

91

92

93

99
Figure Page

27.

Mean serum albumin concentrations in swine after intravenous
and oral administration of sulfisoxazole ......... .. L00

Mean serum albumin concentrations in humans after oral
administration of sulfisoxazole.

“

fis
fae
bh.
Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy

COMPARISON OF THE PHARMACOKINETICS AND TOXICITY OF SULFISOXAZOLE
IN HUMANS AND TWO MONOGASTRIC ANIMAL SPECIES

By
ROBERT L. SUBER
August 1979

Chairman: George T. Edds
Major Department: Animal Science

This experiment was designed to 1) develop a high performance liquid
chromatographic procedure for separation and quantitation of sulfonamides,
especiaily sulfisoxazole and its metabolites and 2) to compare the
toxicity and pharmacokinetics of sulfisoxazole, a water-soluble sulfona-
mide, in 3 monogastric species, humans, dogs, and swine.

An accurate, sensitive, and specific high performance liquid
chromatographic technique was developed for separation of sulfanilamide,
sulfaguanidine, sulfamerazine, sulfamethazine, sulfapyridine, sulfisoxa-
zole, acetylsulfisoxazole (n’)y, and sulfathiazole from a biological
matrix. Spiked serum samples were analyzed by injecting 20.0 ul onto au
Bondapak Cig column with absorbance measured at 254 nanometers. The
mobile phase was 50.0% double-distilled water/50.0% methanoi (pH 7.45)
for the separation and quantitation of sulfanilamide and sulfaguanidine.
The 6 other sulfonamides used a 60.0% double-distilled water/40.0%
methanol with acetate buffer (pH 4.00) mobile phase. Serum could be
injected directly in the system, equipred with an inline filter con-

taining Corasil Cig> deproteinated with methanol (1:1) before injection

Xiv
or filtered through 0.45 up filters without loss of resolution. Urinary
extraction of sulfisoxazole and its metabolite, acetylsulfisoxazole cnt) |
involved cooling 25.0 ml of urine to 4°C, acidifying to pH 3.00 with
concentrated hydrochloric acid and extraction into chloroform. Total
serum and urinary sulfisoxazole were determined by boiling the acid
hydrolysate for 1.0 hour at 100°C and performing the respective extrac-
tion and separation techniques previously described.

Six individuals from each species were administered a solution of
sulfisoxazole after a 12-hour fasting period. The trial was conducted
over a 72-hour period after intravenous administration and a 96-hour
period after oral administration in dogs and swine and over an 8-hour
period after oral administration in humans.

Analysis of the pharmacokinetic profiles of each species showed
that dogs were more similar to humans than swine. However, differences
did exist for biological half-lives, distribution constants, volumes of
distribution, bioavailability, metabolism to the acetyl (Nn) derivative,
and urinary excretion rates among all 3 species. Total and conjugated
bilirubin showed small but statistically significant increases (p < .01)
in dogs after oral and intravenous administration of sulfisoxazole
(100 mg/kg) and in swine after oral administration of sulfisoxazole
(100 mg/kg). The potentially toxic, indirect, or unconjugated bilirubin
showed small but statistically significant (p < .01) increases in dogs
after oral administration of sulfisoxazole. A similar increase was not
observed in swine or humans.

Total and conjugated bilirubin were significantly (p < .01) correlated
in dogs after oral and intravenous administration and in swine after oral

administration of sulfisoxazole. The increase in conjugated bilirubin,

xV
along with a concomitant increase in total bilirubin, could be due to
hepatic induction of glucuronidating capacity or regurgitation of con-
jugated bilirubin from the hepatocyte instead of excretion into the bile.
There was also a significant negative correlation (p < .01) in con-
jugated and indirect bilirubin, while total bilirubin increased, in

dogs after oral and intravenous administration of sulfisoxazole.

Dogs appear to be more similar to humans in the pharmacokinetics of
sulfisoxazole and the potential toxicity of bilirubin could be greatest
in dogs. The toxicity of indirect or unconjugated bilirubin is dependent
on a species difference with dogs being the most affected and ona
route of administration difference with the oral route being more likely

to induce toxicity than an intravenous route of administration.

°

KVL
INTRODUCTION

Sulfisoxazole, a pharmaceutical agent regularly used for urinary
tract infections, may be a potentially toxic compound due to the increase
in serum bilirubin levels after administration, has the potential for
inducing bacterial resistance due to long term, low level exposure
tarough the food chain and is a possible environmental contaminant,
especially in urban sewage. In order to evaluate the absorption, dis-
tribution, metabolism, and excretion of sulfisoxazole, it was necessary
to establish the pharmacckinetic parameters in human and animal model
systems.

This trial was to 1) find a rapid high performance liquid chromato-
graphic method for detecting sulfonamides, especially sulfisoxazole and
its metabolites, in a biological matrix, and 2) to compare the pharmaco-
kinetics and potential toxicity of sulfisoxazole in humans, dogs, and
swine in order to define a better animal model to correlate with human

research.
REVIEW OF LITERATURE

The medical and public health importance of the discovery of sulfona-
mides as the first effective chemotherapeutic agents was reflected by a
sudden decline in morbidity and mortality due to infectious diseases (24).

Paul Ehrlich, the founder of modern chemotherapy, initiated the
concept of the use of azo dyes as antibacterial agents (24). Sulfonila-
mide (aminobenzene sulfonamide) was first prepared by Gelmo in 1908 during
the investigation of azo dyes. A drug use patent was issued to Klarer
and Mietzsch in 1932 for Prontosil and other azo dyes containing a
sulfonamide radical. In the same year, Domagk, working with Klarer and
Mietzsch, observed that mice infected with streptococcal bacteria could
be protected by Prontosil (14, 15).

At the Pasteur Institute, researchers found that the azo linkage of
Prontosil was split in vivo to yield para-aminobenzenesulfonamide which
was thought to be the active chemotherapeutic agent (54). Colebrook and
Kenney (13) and Buttle et al. (8) reported favorable clinical results
with Prontosil which prompted the synthesis of more than 4500 sulfonamide
derivatives in the United States by 1943 (22).

The method of action of sulfonamides is based on the absorption into
the bacterial cell in its non-ionized form. After partial dissociation,
it competes with ionized para~aminobenzoic acid (PABA). This competition
inhibits bacterial growth by preventing PABA from being incorporated

into pteroylglutamic acid (folic acid) which is reduced to tetrahydrofolic
acid, a coenzyme essential for carbon metabolism (19, 59). Although
inhibition of pteroylglutamic acid synthesis is immediate, the effective-
ness of sulfonamides is restricted to bacteria which require or synthesize
pterovylglutamic acid during the growth phase. A log phase or latent
period, which is determined by the concentration of stored pteroyl-
glutamic acid, occurs between the administration of sulfonamide and its
bacteriostatic effect (52).

The structure-activity relationship of sulfonamides is such that the
para-amino group cn’) is essential and can only be replaced by such
radicals that can be converted in vivo into a free amino group. The
-SO,NH, (xt) group is not essential but the linkage of sulfur directly
to the benzene ring is of utmost importance (24). The more electro-
negative the -S0, group the greater is the bacteriostatic activity since
the tonic form is more active than the molecular form (3). This knowledge
led to the early synthesis of sulfisoxazole by Hoffman-LaRoche in 1947
(32) and its use patent for microbial infections. Since sulfisoxazole

is readily ionized and excreted by glomerular filtration, it is used

primarily for urinary tract infections (37

Sulfisoxazole

The therapeutic index of sulfonamides is relatively low so complete
bioavailability is important to maximize the percentage of patients who
will obtain a favorable therapeutic response from the drug (4). Sulfi-
soxazole or sulfafurazole (3,4 dimethyl-5-sulfanilamido-isoxazole) is a
white-yellowish, odorless, slightly bitter, crystalline powder with a PK,

of 4.9 (32), is relatively non-toxic, and is able to control experimental
~4=

bacterial infections (45). It is distributed in the extracellular fluid
and fails to enter cells (29, 58). Therefore, the administration of
sulfisoxazole results in a plasma concentration which is three times

higher than that preduced by an equal quantity of sulfanilamide (52).

Blood Concentration of Sulfisoxazole

Previous pharmacokinetic studies in dogs, swine, and cattle only
determined mean blood levels of "free" (unbound) sulfisoxazole (9, 21).
In these experiments, the mean blood level was highest 4 to 8 hours after
oral administration. In clinical trials with dogs given 0.78 to 2.33
grams per day orally, a maximum blood concentration, 2.8 to 29.0 mg/100 cc
blood, was attained. Intravenous administration to dogs (21), 199.6
mg/kg body weight, resulted in 22.5 mg of free drug/100 cc blood at
1 hour in 4 dogs; produced 26.4 mg of free drug/100 ce blood in 1 dog;
and 299.4 mg/kg produced 26.8 mg of free drug/100 cc blood in 3 dogs.
Subcutaneous administration to dogs (21) resulted in a maximum blood
concentration 4 hours after administration. Administration of 199.5
mg/kg body weight resulted in 14.5 mg of free drug/100 ml blood while
213.8 mg/kg resulted in 13.2 mg of free drug/100 ml blood. Oral adminis-
tration of 2.0 gm of sulfisoxazole to 4 dogs (40) resulted in a peak
plasma level of 13.6 mg/100 ml of whole blood.

The only administration reported in swine was by intraperitoneal
injection (21). The peak blood level was 25.4 mg of free drug/100 ml
blood at 1 hour after receiving a dose of 201.5 mg/kg body weight.

In desing children (39), administration of a single oral dose of
159 mg/kg body weight resulted in 13.5 mg of free sulfisoxazole/100 ml

ef serum after 3 hours and 3.8 mg/100 mi after 12 hours. An oral dose
of 100 mg/kg resulted in 9.2 mg of free drug/100 ml of serum at 3 hours
and 2.2 mg/100 ml at 12 hours. Svec et al. (53) observed that after oral
ingestion of a single 2.0 gm oral dose in adults, the mean blood concen-
tration of free sulfisoxazole was 9.0 mg/100 ml. Randall et al. (40)
administered an oral dose of 2.0 gm every 4 hours for 4 doses to 6 adults,
which produced a mean blood concentration of 21.0 mg of total sulfisoxa-
zole/100 ml at 10 hours after the initial dose; the free sulfisoxazole
concentration was 18.0 mg/100 ml at 10 hours. Loughlin and Mullin (28)
dosed 5 adults orally with 2.0 or 4.0 em of sulfisoxazole with whole
blood concentrations of free sulfisoxazole of 3.5 and 3.6 mg% at 2 and 4
hours, respectively, for the 2.0 gm dose; the 4.0 gm dose produced whole
blood concentrations of 7.4 and 7.3 mg% at 2 and 4 hours, respectively.
By 16 hours, the concentration was 40% of the 4 hour level for both the
2.0 and 4.0 gm dose and by 28 hours the blood level had decreased to 27%
of the 4 hour-2.0 gm dose and 22% of the 4 hour-4.0 gm dose.

The first complete pharmacokinetic profile in humans was conducted
by Kaplan et al. (26). Seven healthy adults administered a 2.0 gm single
oral dose had a mean absorption rate constant of 668.12 mg/hr, a half
life of 5.83 hr, peak plasma time of 2.5 hr with 168.7 ug/ml. Intravenous
administration of a single 2.0 gm dose to the same 7 volunteers resulted
in an initial plasma concentration of 259.03 ug/ml, a half life of
5.89 hr, and a volume of distribution of 16.37% of body weight. When
11 different sulfisoxazole tablets (500 mg) were administered to human
volunteers (49), the peak plasma level was 45.5 to 57.5 ug/ml in 3 hours

or less; the half life was 8.5 hours.
Metabolism of Sulfisoxazole

Saturable first pass conjugation of the aromatic amino group (n*)
of sulfisoxazole occurs during the initial passage of the drug from the
gastrointestinal lumen through the liver following oral administration.
The extent of conjugation increases as the oral dose decreases which is
consistent with saturable first pass conjugation. This does not occur
with intravenous administration which indicates that dose dependent con-
jugation occurs before the drug reaches the systemic circulation. By
inhibiting the motor activity of the stomach and small intestine to slow
drug absorption, one is able to increase conjugation of the drug (4).

Nelson and O'Reilly (33) reported that mean half-life for formation
of the acetyl conjugate from sulfisoxazole is 30 hours. The mean half-
life of the acetyl derivative was 9.0 hr which was longer than the half-
life of the parent compound (7.9 hr). Loughlin and Mullin (28) reported
14.2% of 2.0 and 4.0 gm oral doses were conjugated within 36 hours while
Weinstein (58) reported 28 to 35% is acetylated.

When acetyl (nt) sulfisoxazole is administered orally, the enzymes
responsible for Ne conjugation are saturated so the fraction metabolized
to the no acetyl derivative decreases as the oral acetyl (nt) sulfiscoxa-
zole dose increases (4). Flake et al. (20) supported this concept by
reporting 15% of sulfisoxazole is in the N’-acetylated form when acetyl
(nt) sulfisoxazole is administered. Bloedow (4) suggested that the high
degree of protein binding of sulfisoxazole may spare its first passage
metabolism to the Ne acetyl derivative, leaving more of the parent drug

available for its pharmacological action.
Protein Binding of Sulfisoxazole

The binding of a drug to plasma proteins affects its activity, dis-
tribution, rate of metabolism, and glomerular filtration (38). This
degree of binding is influenced by the molecular structure of the drug,
its lipid solubility, PK,» the concentration and affinity for plasma
proteins, the number of binding sites, the presence of competitively
binding drugs or endogenous compounds, and the physiological or patho-
logical state of the subject (7, 41, 44, 50). The degree of protein
binding increases near the pK, (1). With a low (acidic) pK.» a high
degree of drug binding occurs at physiological pH while unionized drugs
are slightly bound. The conjugation of methyl groups will increase the
binding tendency of a drug (1).

Albumin has a greater binding effect on sulfisoxazole than alpha-
or gamma-globulins (10). Experimental binding of sulfisoxazole in vitro
(5% albumin, pH 7.4, 25°C) resulted in 98.2% as bound, 1.8% as free base,
and 0.006% free acid (1) which is expected since sulfisoxazole has an
acidic pk, and 2 methyl groups. In vivo experiments in humans resulted
in approximately 25% of the total sulfisoxazole being bound to plasma
protein (40). The protein bound fraction acts as a reservoir between the
ineffective and toxic levels of the biologically active, unbound, un-
metabolized fraction (7) but there appears to be no correlation between

the half-life in man and the percentage which is protein bound (42).

Toxic Effects of Sulfisoxazole

Sulfonamides as a class of chemotherapeutic agents are considered to

be toxic due to precipitation in the kidney, producing crystalluria (24).
~38-

The infrequency of renal toxicosis (crystalluria) of sulfisoxazole is due
to the exceptionally high water solubility of the free and conjugated
(acetyl) fractions within the physiological pH range (28, 45).

Clinical toxicities have been induced by sulfisoxazole competing for
the same binding sites as warfarin (46) and furosemide (38, 47), inducing
hemolytic anemia due to glucose-6-phosphate dehydrogenase deficiency
(30), inhibition of anticoagulant factor VIII (56), hypersensitivity (9),
anorexia (60), agranulocytosis (60), aplastic anemia (60), and a case of
myocarditis, myositis, and vasculitis associated with severe eosinophilia
following sulfisoxazole therapy (18).

In young, 100 gm laboratory rats, hyperplasia of thyroid glands
occurred with diets of 0.5 and 1.0% sulfisoxazole for 13 weeks. These
rats did not exhibit any change in growth rate, agranulocytosis or
aplastic anemia. At 2.0% of the diet, a delayed growth rate, decreased
white blood cell count, and bone granulocytes occurred (40). The oral
lethal dose for 50% of mice tested (LD50) was 10.0 gm/kg of lithium and
sodium sait (45). The LD50 for an intravenous dose was 2.5 mg/kg and
2.3 mg/kg for the lithium and sodium salt, respectively; the subcutaneous
route of administration produced an LD50 at 5.0 and 2.8 mg/kg for the
lithium and sodium salt, respectively.

Kernicterus has been reported in infants with increased levels of
serum bilirubin in premature infants treated with sulfisoxazole (27, 34,
48, 51). Kernicterus occurred when unconjugated or indirect bilirubin
was less than 20 mg% in 24 infants, less than 17 mg4 in 15 infants, and
less than 15 mg%Z in 11 infants. These occurrences were enhanced by prior

acidosis, hypercapnia, and hypothermia (51).
~9-

Plasma samples from 6 adults showed that sulfisoxazole concentraticns
above 5 mg/100 ml had a significant displacing affect on bilirubin in
vitro (36). When compared to the displacing effects of salicylic acid,
salicyluric acid and aspirin to sulfisoxazoie at 10 mg/100 ml concentra-
tions, the most pronounced effect was observed when sulfisoxazole displaced
bilirubin from plasma samples of 6 adults and 13 infants in vitro (36).

The displacement bilirubin is due to competition for similar binding

sites on the albumin molecule (35).

Urinary Excretion of Sulfisoxazole

Sulfisoxazole is actively secreted in the proximal renal tubule in
dogs and humans. Tubular reabsorption is a passive process which depends
on urinary pH (12).

After single doses of sulfisoxazole, 83% was recovered in human
urine within 24 hours while 45% was excreted within 48 hours in dogs
(40). Kaplan et al. (26) reported a mean of 52.9% of sulfisoxazole was
recovered as the "free" drug within 48 hours after a 2.0 gm intravenous
dose in humans while 51.6% was recovered after a 2.0 gm oral dose. The
total sulfisoxazole recovered in 48 hours was 91.5% and 97.0% for the
2.0 gm intravenous and oral dose, respectively. Weinstein (58) reported

30% of the total sulfisoxazole excreted was in the acetylated form.

Pharmacokinetics

Pharmacokinetics has been defined as the quantitative study of the
absorption, distribution, metabolism, and excretion of drugs and their

pharmacologic, therapeutic, and toxic responses in animals and man (31).
-10-

The purposes of pharmacokinetics are to reduce the mathematical data
collected from an organism to meaningful parameters which can be used
to make predictions on the results of future experiments or of host
studies which would be too time consuming and costly if carried out

individually (57).

Absorption Rate Constant and Bioavailability

The first order absorption rate constant (k) is a mathematical
description of the rate at which the drug reaches the general circulatory
system after administration. A semi-logarithmic plot of serum concen-
tration (C,) versus time (t) is required with a second plot of the points
obtained by using the method of residuals. The slope (m) of this line
can then be used in determining the absorption rate constant by equation 1

(23, 57).

k= (-m) (2.3) Eq. 1

With sulfonamides, the calculated absorption rate constant (k.) can be
the elimination rate constant (ka). This can be checked by comparing the
Ka of an intravenous dose with the k, of an oral dose; if these are not
equal, the calculated ko the faster rate constant, is the kG (57).
Bioavailability (F) is a term used to indicate a measurement of both
the amount of administered drug which reaches the circulation and the
rate at which this occurs (25). The bioavailability depends on 1) the
rate and extent of release of the drug from the dosage form and 2) the
"first pass" effect where only a certain fraction of the drug presented

to the gastro-intestinal system reaches the general circulation intact.

The bioavailability (F) of a drug is calculated from a ratio of the oral
~ll-

blood concentration (Cc), volume of distribution (Va), the absorption

(k.) and elimination (k 4) constants, and the dose (D), equation 2.

Cc Vv, (k_ - k,) -k,t -k t =-1
_ d d d
re Oa } (e -e *) Eq. 2

A better measure of bioavailability (F) is determined by comparing
the area under the plasma curve after an oral dose (AUC, g.? with the
area under the plasma curve after the intravenous administration of the
same dose (AUC, |), equation 3. Alternatively, if the same dose is
administered by oral and intravenous routes, one can determine bio-
availability (F) by the ratio of the total drug excreted unchanged in
the urine after the oral dose Uno. to the total drug in the urine

after intravenous administration (ULs 7 ), equation 4.

AUC °
Fey Eq. 3
i.v
Veop Oo
F = _—— Eq. 4
ol.v.

Distribution

For a compound to exert its pharmacologic effect, distribution must
Occur into one or more volumes or compartments of the body. The highly
perfused or central compartment is characterized by the heart, liver,
and lungs and exchange between these tissues and the blood is very rapid.
The poorly perfused tissue characterized by muscle, fat, and skin and the
negligible perfused group characterized by the bone, teeth, hair, and

connective tissue compose the peripheral and deep compartments,
~12-

respectively, in which there is slow exchange between these tissues and
the blood (57). Since the blood is the common carrier for distribution
and urine is the most common method of excretion of a drug, monitoring of
the blood concentration and urinary excretion rate of the drug will allow
one to draw mathematical relationships as to the absorption, distribution,
and elimination of a compound (23).

The apparent volume of distribution (Vv) is a mathematical expression
for estimating or defining how extensively the drug is distributed
throughout the body. The lipid solubility, the degree of plasma protein
binding, and the cardiac output will affect the distribution of the
drug (57).

The apparent volume of distribution (Vy) of a drug is computed from
a semi-logarithmic plot of the plasma concentration (Cc) versus time (t)
after a known dose (D) of drug is administered intravenously, equation 5,

where the initial plasma concentration (c) is extrapolated.

D= (c) VD) Eq. 5

Elimination

The elimination rate constant (k) can be calculated from semi-
logarithmic plots of the plasma concentration versus time and from the
urinary excretion rate versus time by determining the slope of the
plotted line (m) and using equation 6 (57). The elimination rate

constant has the unit of reciprocal time (57).

ka = (2.3) (-m) Eq. 6
-13-

The biological or elimination half-life (ty) of a drug is derived
from a graphic plot of the logarithm of the plasma concentration (c)
versus time (t) in which the slope of the line (m) is used to calculate
a first order elimination or disposition rate constant (k,), equation 7

(57).

net Eq. 7

The biological half-life is defined as the time it takes for the drug
concentration to be reduced by one-half (23) and is usually referred to
in relation to the serum or blood concentration.
Urinary excretion is a major pathway for elimination for many drugs
and their metabolites. If the drug is totally excreted unchanged, the
renal clearance (CL) of the drug equals the plasma clearance (CL).
There are two ways of calculating clearance which involves the dose (D), the
total area under the plasma concentration curve (AUC), and the total

amount excreted in the urine (A), equations 8 and 9 (57).

DF

cL. = —— Eq. 8
P auc
P
A’

cL. =—+ Eq. 9
AUC
P

Renal clearance can also be calculated from the slope of the logarithm of
the urinary excretion rate (dA /dt) versus the plasma concentration at

midpoints of excretion intervals cc mid? (57), equation 10.

dA
u

dt



) Eq. 10

~ (CL) (C, mid
-14-

The elimination rate constant (ky) the excretion rate constant
(k); and the elimination half-life (ty) can also be calculated from a
"sigma~minus"” plot. This plot involves the logarithm of the difference
in total urinary excretion minus the amount in the urine at any one inter-
val [log (A, - ADI versus the excretion interval or by an excretion rate
plot of the concentration of drug excreted per unit time versus the mid-

time interval between sampling periods (57), equations 11 and 12,



respectively.
co co kA
log (A, - A, = log AL - 7.3 t Eq. 11
AA k
log —Y = log k D--%t Eq. 12
At e 2.3 “mid q-

Plasma Protein Binding

Plasma proteins, especially albumin, act as binding sites for acidic
and basic drugs. Acidic drugs, at normal body temperature and normal
therapeutic doses, appear to bind to albumin at a single binding site,
possibly at the N-terminal amino acid group (38). Protein binding in-
fluences the volume of distribution, the rate of metabolism, and the rate
of excretion (57).

The percent or fraction of protein bound drug (f,) is calculated
from the total serum concentration (c.) and the amount which exists as

free, unbound unmetabolized parent drug (C.) as in equation 13 (57).

Eq. 13
Two Compartment Model

A two compartment model is defined as a mathematical relationship of
the kinetics of distribution of substances in the body (57). When a two
compartment model does exist, one must calculate a volume of distribution

for both compartments (V, and V4), a first order rate constant for the

1
transfer of the drug from the central compartment to the peripheral
compartment (kj 5) and from the peripheral compartment to the central

compartment (ko4)> and a first order elimination constant by all pro-

cesses from the central compartment (k, or k

l 10)? assuming that etimina=

tion only occurs through the central compartment (57). This two compart-
ment model in which elimination occurs only from the central compartment
is represented by the following scheme:

Ki2

Central —~——~ Peripheral
Compartment ~———— Compartment

Koy

Kio

Elimination

In a two compartment model, a semi-logarithmic plot of serum or
plasma concentration (c) versus time (t) yields two straight lines by
least squares analysis, the distribution (a phase) and the elimination
(8 phase) phases. The rate constant of the first or distribution com-
partment (a) is calculated from the slope (m) of a second semi-logarithmic
plot of the serum concentration obtained by the method of residuals,
equation 14 (57). The rate constant for the second or elimination
compartment (8) is calculated from the slope (m) of the least squares
line of the semi-logarithmic serum concentration versus time graph,

equation 15 (57).
-16-

(2.3) (-m)

RQ
W

WD
Nl

(2.3) (-m)

time for the a phase and 8 phase, respectively.

concentration (c) is the sum of A and B.

C =A e +B et
P

co =A+B

P

(a) (8) = (ky1) (yg)

_ Dose
“1 7 A+B



(V1) (ky) = (Vy) (R54)

_ AB + Ba

Ko. = A+B

Eq. 14

Eq. 15

The remaining constants could then be calculated from equations 16

Eq.

Eq.

Eq.

Eq.

Eq.

Eq.

Eq.

through 22, where A and B are the extrapolated blood concentrations at 0

The total initial blood

16

17

18

19

I
nh
MATERIALS AND METHODS

High Performance Liquid Chromatographic Analysis of
Sulfonamides by Ionic Suppression

Materials

Ten milligram samples of sulfanilamide,* sulfaguanidine,* sulfamera-
zine,* sulfamethazine,* sulfapyridine,* sulfisoxazole, acetylsulfisoxa-
zole cn’), and sulfathiazole* were dissolved in methanol? and triple
distilled water (50/50) to give concentrations of 100, 50, and 25 ug/ml.
Twenty microliters of these standard solutions were injected onto the
column. Standard solutions (500 ug/ml) were added to 1.0 ml human plasma
samples to give in vitro plasma concentrations of 100, 50, and 25 ug/ml.
The in vitro plasma samples were incubated for 30 minutes in a 37°C water
bath, deproteinated with methanol? (1:1), centrifuged (2000 x g) for 10
minutes and 20 ul of the supernatent injected or 20 ul of the in vitro
plasma sample was injected directly onto the column without deproteina-
tion or extraction.

An ALC/GPC 204 liquid chromatograph * was equipped with a Model

440 3 ultraviolet absorbance detector (254 nm). A dual pen recorder**

*Fort Dodge Laboratories, Fort Dodge, Iowa 50501.
"Hoffman-LaRoche, Nutley, New Jersey 07110.

tpurdick Jackson Laboratories, Muskegon, Michigan 49442.
swaters Associates, Milford, Massachussetts 01757.

**Houston Instruments, Austin, Texas 78753.
-18-

was used to quantitate concentration as a function of peak height (mm).
A u Bondapak Cig reverse-phase column* was used with a water/methanol |
mobile phase and an in-line guard column* packed with Corasil/C,,.* The
mobile phase was either 60/40 or 50/50 water/methanol mixture, isocratic

7

pH of 7.45, or with acetate buffer' added to adjust the pH to 4.00 as
confirmed by a pH electrode and meter. * The water was triple distilled

and all analyses were conducted at ambient temperature (25°C) with a

0.8 ml/min flow rate.

Extraction and Separation

The retention times of 8 sulfonamides in standard solutions of
methanol/water are given in Tables 1 and 2. An examination of the data
shows that, in some cases, a change in retention time occurred when the
pH was reduced to 4.00 by the addition of acetate buffer to the mobile
phase. The retention time of sulfamethazine was increased while the
retention time of sulfamerazine, sulfapyridine, sulfisoxazole, and acetyl-
sulfisoxazole (N*) was decreased when the mobile phase was 50% water/50%
methanol (Table 1).

When the mobile phase was changed to 60% water/40% methanol (Table
2), the retention time was increased for sulfaguanidine, sulfamerazine,

sulfamethazine, and sulfapyridine. The retention times for the remaining

*Waters Associates, Milford, Massachussetts 01757.
‘Burdick-Jackson Laboratories, Muskegon, MI 49442.
*Glacial acetic acid, Fisher Scientific, Orlando, Florida 32809.

5 orion Model 407A, Oricn Research, Inc., Cambridge, Massachussetts 02139.
~19-

Table 1. Retention time (minutes) of sulfonamides in a water/methanol
(50/50) mobile phase with acetate buffer (pH 4.00) and without
buffer (pH 7.45).

pH 4.00 pH 7.45
Sulfanilamide 3.80 3.80
Sulfaguanidine 3.70 3.70
Sulfamerazine 4.90 4.50
Sulfamethazine 5.00 5.45
Sulfapyridine 4.75 4.45
Sulfisoxazole 5.20 3.20
Acetylsulfisoxazole in?) 7.00 2.90

Sulfathiazole 4.00 4.00
Table 2. Retention time (minutes) of sulfonamides in a water/methanol
(60/40) mobile phase with acetate buffer (pH 4.00) and without
buffer (pH 7.45).

pH 4.00 pH 7.45
Sulfanilamide 3.75 3.05
Sulfaguanidine 3.60 3.90
Sulfamerazine 4.10 5.40
Sulfamethazine 4.25 6.10
Sulfapyridine 4.00 5.00
Sulfisoxazole 4.20 4.20
Acetylsulfisoxazole (nt) 5.00 4.40

Sulfathiazole 3.75 3.30
sulfonamides were reduced except for sulfisoxazole which remained
constant.

When one compares the results presented in Tables 1 and 2 at pH
4.00, it is noted the retention times of six sulfonamides were increased
when the mobile phase was 50% water/50% methanol with acetate buffer.
There was no significant difference in the retention times of sulfanilamide
or sulfaguanidine in a mobile phase of 50% water/50% methanol with or
without the buffer or if the mobile phase was 60% water/40% methanol with
the buffer (pH 4.00). However, at pH 7.45, use of the 60% water/40%
methanol mobile phase resulted in an increase in the retention time of
sulfaguanidine while reducing that of sulfanilamide.

Human plasma samples were spiked with standard solutions of sulfona-
mides and the retention time recorded. Sulfanilamide and sulfaguanidine
were not separable from endogenous serum peaks in the 50% water/50Z
methanol (pH 4.00) mobile phase (Table 3). Sulfisoxazole and its no
metabolite, acetylsulfisoxazole, were not separable from endogenous
compounds at pH 7.45. Ionic suppression did decrease the retention time
of sulfapyridine and sulfathiazole in plasma while that of sulfamethazine
remained constant (Table 3).

When the mobile phase was changed to 60% water/40% methanol (Table
4), sulfanilamide, sulfaguanidine, and sulfisoxazole and acetylsulfisoxa-
zole were not detectable at pH 7.45. The remaining four sulfonamides
were detectable with an increased retention time at pH 7.45.

By comparing Tables 3 and 4, the retention time of the detectable
sulfonamides was increased by the 50/50 mobile phase at pH 4.00 and

decreased at pH 7.45.
-22-

Table 3. Retention time (minutes) of sulfonamides in spiked human plasma
samples in a water/methanol (50/50) mobile phase with acetate
buffer (pH 4.00) and without buffer (pH 7.45).

pH 4.00 pH 7.45

Sulfanilamide ND* 3.80
Sulfaguanidine ND* 3.70
Sulfamerazine 4.50 4.60
Sulfamethazine 4.80 4.80
Sulfapyridine 4.40 4.30
Sulfisoxazole 5.10 ND*

Acetylsulfisoxazole (n*) 6.90 ND*

Sulfathiazole 4.25 3.95

“ND = Not detectable due to endogenous serum components with similar
retention times.
-23-

Table 4. Retention time (minutes) of sulfonamides in spiked human plasma
samples in a water/methanol (60/40) mobile phase with acetate

buffer (pH 4.00) and without buffer (pH 7.45).

pH 4.00

Sulfanilamide ND*

Sulfaguanidine ND*

Sulfamerazine 4.10
Sulfamethazine 4.30
Sulfapyridine 4.00
Sulfisoxazole 4,20
Acetylsulfisoxazole (n°) 5.00
Sulfathiazole 3.75

pH 7.45

ND*

ND*

4.35

4ND = Net detectable due to endogenous serum components with similar

retention times.
-—oi-

The plasma matrix increased the retention time of sulfamerazine,
sulfamethazine, sulfapyridine, sulfisoxazole, and acetylsulfisoxazole
cx’) in the 50/50 mobile phase at pH 4.00 (Tables 1 and 3). The reten-
tion time of sulfanilamide, sulfaguanidine, and sulfathiazole were not
changed in the 50/50 mobile phase at pH 7.45. However, the retention time
of sulfamethazine and sulfapyridine was increased in the plasma matrix
at pH 7.45 while that for sulfamerazine decreased (Tables 1 and 3).

With the 60/40 mobile phase, there were no significant changes in
retention time by the plasma matrix at pH 4.00 (Tables 2 and 4). At pH
7.45, the retention time of sulfapyridine and sulfathiazole was increased
by the plasma matrix (Tables 2 and 4).

When serum is the biological matrix, four of the sulfonamides are
easily separated at either pH or mobile phase polarity. Sulfanilamide
and sulfaguanidine are only detectable from plasma when the mobile phase
is 50% water/50% methanol at pH 7.45, the pH value closest to their PK,
value. Sulfisoxazole and acetylsulfisoxazole cn’) are detectable only
after ion suppression at pH 4.00, regardless of either mobile phase. The
endogenous serum compounds which absorb at 254 nm can be separated from
the sulfonamides by changing pH or mobile phase polarity. Deproteination
of plasma samples with methanol did not change the retention time of any
sulfonamide as compared to the direct injection of the spiked plasma
sample. Separation of combinations of sulfonamides was easily accomplished
if the retention time differed by at least 0.1 minutes.

Construction of concentration curves by plotting peak height versus
10, 50, and 100 ng/ml and 10, 25, 50, and 100 ug/ml of standard solutions
of sulfonamides in methanol/water (Figures 1 through 4) demonstrated that

a difference in sensitivity occurred as a function of ion suppression
~25-

240 20
220 16
200 E 2
€
180 3
160 4
E b
— 140 10 50 100
E ng/ml
& 120
TT
~ 100 g
a e
WW
a 80 h
d
60 c
f

h
oO
0

tw
Oo

0 25 0 100
CONCENTRATION OF SULFONAMIDE ( pg/ml)

ca
ae
|

Figure 1. Standard concentration curves of sulfanilamide (a), sulfa-
guanidine (b), sulfamerazine (c), sulfamethazine (d),
sulfapyridine (e), sulfisoxazole (f), N* acetylsulfisoxazole
(g), and sulfathiazole (h) in a water/methanol (50/50) mobile
phase with acetate buffer to pH 4.00.
-26-

240 20

220 16

200 ¢ 12

€

{80 8

{60 4
= a
E 140
~ 10 50 {00
6 120 ng/ml b
ul
x
x {00
<

Oo @
°o o
fo gator.)

\

10 25 50 100
CONCENTRATION OF SULFONAMIDE ( jzg/m!)

Figure 2. Standard concentration curves of sulfanilamide (a), sulfa-
guanidine (b), sulfamerazine (c), sulfamethazine (d),
sulfapyridine (e), sulfisoxazole (f), N* acetylsulfisoxazole
(g), and sulfathiazole (h) in a water/methanol (50/50) mobile
phase with an isocratic pH 7.45.
-27-

and polarity of the mobile phase. The mean standard error of peak height
at these concentrations was t 0.017.

In Figure 1, the sensitivity of sulfaguanidine (b) at 254 nm is
greater than sulfanilamide (a) at pH 4.00 in a 50/50 mobile phase. If
the mobile phase remains at pH 7.45 (Figure 2), this sensitivity is re-
versed, with sulfanilamide peak height being greater than sulfaguanidine.
There is also a change in the slope of the other 6 sulfonamides as the
pH changes in the 50/50 mobile phase (Figures 1 and 2).

If the mobile phase is 60% water/40% methanol, sensitivity of the
assays for sulfanilamide (a) and sulfaguanidine (b) are the most sensitive
at either pH (Figures 3 and 4). Detection of the remaining 6 sulfona-
mides varies in sensitivity at 254 nm in the 60/40 mobile phase with the
pH change. The most sensitive mobile phase is 60/40 at pH 4.00 as shown
by the greater slopes of each sulfonamide (Figure 3). Additional dilu-
tion of standard solutions of sulfonamides and increased sensitivity
settings to 0.01 a.u.f.s. (absorbance units full scale) on the ulitra-
violet detector recorded peak heights equivalent to 10.0 ng/ml of
sulfonamide from a single 20 yl injection without concentration or
reconstitution of the extract (inset, Figures 1 to 4).

The slope of the sulfisoxazole (f) curve was the same for the 60/40
mobile phase regardless of pH (Figures 3 and 4). By comparing Figures
1 and 2, one observes that the slope of the sulfamethazine (d) curve was
the same for the 50/50 mobile phase at either pH 4.00 or pH 7.45.
Sulfaguanidine (b, Figures 1 and 3) and sulfapyridine (e, Figures 2 and
4) curves had the same slope at pH 4.00 and pH 7.45, respectively, re-

gardless of the polarity of the mobile phase.
~28-

260

240

200

180

160

i40

i20

100

PEAK HEIGHT (mm)
~anasr o

80

60

40

20



0 100
CONCENTRATION OF SULFONAMIDE ( 2g/mi)

Figure 3. Standard concentration curves of sulfanilamide (a), sulfa-
guanidine (b), sulfamerazine (c), sulfamethazine (d),
sulfapyridine (e), sulfisoxazole (f), N* acetylsulfisoxazole
(g), and sulfathiazole (h) in a water/methanol (60/40) mobile
phase with acetate buffer at pH 4.00.
~29~

220 16
200 € 12
é
(80 8
160 4
E
~ !40 10 50 100
E ng/ml
© 120 3
bon
100
x b
tad
a so f
e
60 g
h
d
40 EE c

20



10 25 50 100
CONCENTRATION OF SULFONAMIDE ( pg/ml)

Figure 4. Standard concentration curves of sulfanilamide (a), sulfa~
guanidine (b), sulfamerazine (c), sulfamethazine (d),
sulfapyridine (e), sulfisoxazole (f), N4 acetylsulfisoxazole
(g), and sulfathiazole (h) in a water/methanol (60/40) mobile
phase with an isocratic pH 7.45.
~30-

Optimization of the Liquid Chromatographic Procedure

Four sulfonamides (sulfamerazine, sulfamethazine, sulfapyridine, and
sulfathiazole) could be separated from a plasma matrix in a 50/50 or
60/40 methanol/water mobile phase at either pH. Sulfanilamide and sulfa-
guanidine were only separable from plasma in a 50% water/50% methanol
mobile phase without acetate buffer (pH 7.45) due to their increased pK,
of 10.5 and 11.3, respectively. Sulfisoxazole and acetylsulfisoxazole
(x) were separated with either mobile phase by ionic suppression with
acetate to reduce the mobile phase pH to 4.00.

Sensitivity of the assays to 10.0 ng/ml of sulfonamide from 20 ul
injections of spiked plasma samples without concentration or reconstitu-
tion of the extract. Assays for sulfanilamide and sulfaguanidine were
the most sensitive in the water/methanol mobile phase at 254 nm. A 60%
water/40% methanol with acetate buffer to pH 4.00 was the most sensitive

of the assays.

Experimental Model

The trial consisted of 6 male human volunteers from 25-30 years old,
weighing 70 to 80 kg; 3 female dogs approximately 2 years old, weighing
20.0 + 1.0 kg; and 6 female pigs approximately 3 months old, weighing
20.0 + 1.0 kg. The human volunteers were administered 2.0 gms of sul-
fisoxazole in a single dose and blood samples taken at 0, 1, 2, 4, 6, and
8 hours after administration. The dogs were administered i100 mg of
sulfisoxazole/kg body weight, by intravenous and oral routes in 2

replicates for each route with a 30 day rest period between each adminis-

tration. The pigs were also administered 100 mg of sulfisoxazole/kg of
-31-

body weight by intravenous and oral routes with a 21 day rest period
between administrations. Blood samples were taken at 0.5, 1, 2, 3, 4.5,
6, 9, 12, 22, 32, 44, 56, and 72 hours after intravenous administration
and 0, 1, 2, 4, 6, 8, 10, 12, 14, 23, 32, 44, 56, 76, and 96 hours after
oral administration in dogs and swine. All animals were maintained on a
commercial diet (Purina Dog Chow* or Swine Feed") with ad libitum access
to water. The animals were housed in metabolism cages and the urine was
collected as voided and frozen (0°C).

A 12.5% solution of sulfisoxazole* was prepared in our lab with
lithium hydroxide. The solutions were filtered and placed in sterile
50 ml ampules.” This solution was used for both the oral and intravenous
administration of the drug.

The partition coefficient of sulfisoxazole and acetylsulfisoxazole
from water (pH 5.0) into 2-octanol** after a one hour incubation period
was 0.441 for sulfisoxazole and 0.538 for acetylsulfisoxazole as quanti-
tated from the previous high performance liquid chromatographic procedure

using 60% water/40% methanol with acetate buffer (pH 4.00).

Blood Samples

ee

Blood samples were taken from the cephalic vein in dogs and humans

and via the anterior vena cava in swine. Human samples were drawn

*Ralston Purina, St. Louis, MO.

‘University of Florida Swine Unit, 18% protein feed, 72% yellow corn,
25% soybean oil meal, 3% salt, vitamin, calcium-phosphorus supplement.

THof fman-LaRoche, Nutley, N.J. 07110.
‘Wheaton, Scientific Products, Ocala, FL 32670.

*kEastman-Kodak, Rochester, N.Y. 14650.
-32+

directly into 7 ml sterile silicone~coated Vacutainer tubes. Ten milli-
liter samples were taken by a sterile syringe with a 20 gauge needle from
the dogs and pigs and transferred to sterile silicone-coated Vacutainer
tubes. The samples were centrifuged (2000 x g) for 10 minutes, the serum
extracted, protected from light, refrigerated, and analyzed within 30
minutes for total and conjugated (glucuronidated) bilirubin and albumin.
The remaining serum was frozen (0°C) for up to 14 days for analysis of
"free" sulfisoxazole, acetyl (x) -sulfisoxazole, and total (acid
hydrolyzed) sulfisoxazole.

Serum bilirubin. Serum was analyzed for total and conjugated (direct
or glucuronidated) bilirubin on E.I. DuPont's Automatic Clinical Analyzer*
(17) using a commercial standard."

The conjugated method is a modification of the Van den Bergh diazo

reaction (55):

Conjugated bilirubin + p-Nitrobenzendiazonium tetrafluoroborate (PNB)

~ |

Red chromophore absorbing at 540 nm

Under acidic conditions, the PNB is coupled to the glucuronidated bili-
rubin which is measured as an end point reaction at 540 and 600 nm. The
normal range in humans is considered to be 0.00 to 0.36 mg/dl.

The total bilirubin quantitation (the conjugated and unconjugated

fractions) is also a derivation cf the Van den Bergh reaction (55):

*E.L. DuPont, Instrument Products, Wilmington, Delaware.

“Dade Division, American Hospital Supply Corp., Miami, FL.
=33-

Total bilirubin + p-Nitrobenzenediazonium tetrafluoroborate (PNB)

H+ | Tween 20

Red chromophore at 540 nm

A surfactant (Tween 20)* is used to solubilize the unconjugated (free)
bilirubin; which along with the water soluble, glucuronidated bilirubin
reacts with PNB in an acid medium to measure an end point reaction at
540 and 600 nm. The normal total bilirubin concentration in humans is
less than 1.5 mg/dl (17); in dogs, the normal level is less than 0.5
mg/100 ce (5).

Interferences in these methods are due to hemolyzed samples and light
degradation of the bilirubin. All analyses were conducted within 30
minutes of sampling on serum which was kept in a cool, dark environment.

Serum albumin. Serum albumin was analyzed on E.I. DuPont's Automatic
Clinical Analyzer (17). This method is an adaptation of the bromocresol
green (BCG) dye binding method of Rodkey (43) which was later modified
by Doumas (16).

Albumin plus BCG dye at pH 4.2 yields an albumin-BCG complex which
elicits an absorbing spectrum at 600 nm. The end point reaction is
measured at 600 and 540 nm. The normal range in humans is 3.8 to 4.8
mg/dl.

Interferences are expected in icteric and hemolyzed samples. All

analyses were conducted within 30 minutes of sampling.

*Union Carbide Corporation, New York, New York.

+
‘E.I. DuPont, Instrument Products, Wilmington, Delaware.
—34-

Serum sulfisoxazole. Free (unbound, unmetabolized) serum sulfisoxa-
zole and serum acetyl (n’) sulfisoxazole were determined by deproteinating
1 part of serum with 2 parts methanol, centrifuging for 10 minutes
(2000 x g) and filtering the supernatent through Milex disposable
filters (SLHA 02505).*

Total serum sulfisoxazole (acid hydrolyzed) was determined by adding
2 parts water and 1 part 6N hydrocholric acid to 1 part serum and heated
in a boiling water bath (100°C) for 1 hour. The samples were allowed to
cool to room temperature and centrifuged for 10 minutes (2000 x g). The
supernatent was adjusted to pH with 2N NaOH, the final volume adjusted
to 1.5 ml with methanol and filtered with disposable Milex filters
(SLHA 02505) .*

Twenty microliters (1) of the Filtered supernatent were injected
into a Waters Model 6000A Liquid Chromatograph equipped with a Model 440
absorbance detector with a 254 nm filter, U6K injector, and a u-Bondapak
Cig column.* A Houston Instruments dual pen recorder was used to record
peak heights. A peak height ratio was used to determine serum concen-
tration with suifathiazeleâ„¢ being used as the internal standard. Serum
samples spiked with standard solutions of sulfisoxazole and acetyl-
sulfisoxazole** were injected and a standard curve of peak height of

drug to sulfathiazole was constructed.

*Millipore Corporation, Bedford, MA.

"purdick Jackson Laboratories, Muskegon, MI 49442.
Tatene Associates, Milford, MA 01757.

sport Dodge Laboratories, Fort Dodge, Iowa 50501.

**Hoffman-LaRoche, Nutley, N.J. 67110.
~35-

A mobile phase of 60% triple distilled water with 0.5% concentrated
acetic acid/40% methanol* (pH 4.00) was used with a flow rate of 0.8
ml/min. The water and methanol were filtered (NAWP047001 and FHUP647000),°
degassed by sonication and mixing maintained by using a Thermolyne

Â¥

stirrer.

Urinary Sulfisoxazole

Free and acetyl (x’)-sul fisoxazole were measured by cooling 25 ml
urine to 4°C in an ice bath; sufficient 6N hydrochloric acid was added
to reduce the pH to 3.0. After 5 minutes, 15 ml chloroform* was added,
the solution removed from the ice bath, and extraction ee completed in
5 minutes by swirling the solution once per minute. The chloroform* was
removed, evaporated under nitrogen and the residue reconstituted with
methanol.* Total sulfisoxazole was measured by heating the 25 ml of
urine in a boiling water bath for 1 hour and then extracted as previously
mentioned.

The reconstituted extract was then injected onto the liquid
chromatograph using the previously described technique. Peak height was
also used to determine the urinary concentration with sulfathiazole®
being used as the internal standard. Chloroform* extracts were utilized

to remove 94% of sulfisoxazole and 90% of acetyl (N’)-sulfisoxazole from

*Burdick Jackson Laboratories, Muskegon, MI 49442.
ok

‘Millipore Corporation, Bedford, MA.

oh

TScientific Products, Ocala, FL.

sport Dodge Laboratories, Fort Dodge, Iowa 50501.
-36-

spiked urine samples which had been incubated at 37°C for 1 hour. Benzene*

removed 60% of sulfisoxazole and 40% of acetyl (N*)-sulfisoxazole while

ether* removed 96% of sulfisoxazole and 50% of acetyl (n")-sulfisoxazole.
Both serum and urine samples were performed on thin-layer chroma-

tography plates (11) to determine if metabolites other than the acetyl

cn) metabolite existed. All spots were accounted for and analyzed

through the HPLC procedure previously mentioned without the appearance

of other metabolites, at 254 nm and 313 nm.

Analysis of Data

Total, conjugated, and indirect bilirubin and serum albumin were
analyzed for statistical differences by Barr and Goodnight (2) ANOVA
program at the Northeast Regional Computer Center, University of Florida.

"Free" sulfisoxazole after intravenous administration was analyzed
as a two-compartment model using a NONLIN program (57). Logarithmic-
least squares analysis and the method of residuals was used to calculate
a regression line for acetylsulfisoxazole after oral and intravenous

administration and for "free" sulfisoxazole after the oral dose.

In Vitro Plasma Protein Binding

In vitro human plasma samples were incubated in a water bath at
37°C for 1 hour with 25.0, 50.0, 100.0, 200.0, and 300.0 ug/ml of

sulfisoxazole standards in 1 part methanol with 9 parts double-distilied

*Burdick Jackson Laboratories, Muskegon, MI 49442.
-37-

water. Dialysis was conducted by membrane dialysis with a molecular
weight cutoff filter of 5000* after incubating 2.0 ml of spiked serum
with 2.0 ml of phosphate buffer, ? pH 7.41. The dialysate was collected
and analyzed by the previously described liquid chromatographic

method.

*Diachema Ag, CH 8803, Ruschklikon, Switzerland.

"visher Gram Pac Buffer, pH 7.41. Fisher Scientific, Orlando, Florida
32809,
RESULTS AND DISCUSSION

Pharmacokinetics of Sulfisoxazole

The pharmacokinetic profile of sulfisoxazole was determined following
intravenous and oral administration in dogs and swine and following oral
administration in humans. The intravenous blood level curves in dogs and
swine were observed to be biexponential and required a two-compartment

model system for data analyses (23, 57):



Kyo
Central Peripheral
Compartment < Compartment
k
21
X10
Elimination

Integration of the differential equations of a two-compartment model

yields the equation:

where 7 is the concentration of the drug in the plasma at time t, A and
B are ordinate axis intercepts , and the individual rate constants for
the compartments, kyo Koy and Kio are calculable from a and 8, the rate

constants for distribution and elimination, respectively (57).
~39-

Administration of Sulfisoxazole to Dogs

Intravenous administration. A least squares linear regression line

1

of the "free," unbound, non-metabolized sulfisoxazole in 6 dogs (Figure 5)
was calculated using a NONLIN program (57). The mean extrapolated serum
concentration at zero time (A) in the first or distribution compartment
was 189.42 + 38.45 ug/ml with a range of 126.89 to 228.39 ug/ml and a
mean extrapolated level at zero time (B) for the second or elimination
compartment was 2.56 ~ .39 ug/ml (Table 5).

The disposition rate from the first or distribution compartment (a)
ranged from 0.1381 to 0.1982 hours - with a mean of 0.1726 + .0604 hours +
which yields a mean half-life (try) of 4.08 £ .60 hours for sulfisoxa-
zole in the first compartment. Similar analysis of free sulfisoxazole
in the second or elimination compartment yielded a very slow mean dis-
position rate (8) of 0.0206 * .0014 hours or a mean half-life (ty 59)
of 33.74 * 2.17 hours (Table 5).

The mean rate of distribution between the central or distribution and
peripheral or elimination compartments (k, 9) was 0.0140 + .0049 hours ~
(Table 5) while the mean rate of distribution from the peripheral to the
central compartment (54) was 0.0228 + .0022 hours + (Table 5). This
indicated that free sulfisoxazole returned from the peripheral to the
central compartment at a faster rate than it had been distributed from
the central to the peripheral compartment. Calculation of the mean
K1/ky> ratio reflects this difference in distribution between the two
compartments. In these 6 dogs, the mean of the ko / Ky 9 ratio was 1.63,
indicating that the drug is returning rapidly from the distribution sites

for elimination from the body.
-40-

1000.0

SERUM "FREE" SULFISOXAZOLE IN DOGS ( j1g/mi )



10.0
B
1.0 70
10 20 30 40 50 60
TIME (hours)

Figure 5. Serum concentrations of "free" sulfisoxazole in dogs.
Table 5. ‘Two compartment pharmacokinetic parameters in dogs administered sulfisoxazole

as a single intravenous dose,

Sub ject A B 1 8 v v k k. k



( Tes
J 2 12 2) iD a ee ‘hg
- - | = am
Gig/ml) Gig/m)) (hours 1) (hours (liters) (ltters) (Qioure *) (hours ‘) (hours 4) (hours) (hours)

) 228.39 1.93 0.1503 0.0197 9.0) 2.9% 0.0068 0.0208 1.1424 2607.44 4.6) 35.18

2 160. 26 2,82 0,138t 0.0204 11.04 5.07 0.010% 0.0224 a.1258 2294.76 5.2 33.97

3 206.89 2.42 0.187) O.DLt 9.55 6.87 a.005) 0.0210 0.1701 2370.98 3.70 46. 28

4 213.96 2.61 0.1852 0.0207 10,07 6.34 0.01463 0,0227 0.1689 1995.91 3.74 33.48

5 126.24 3,10 0.1768 00,0232 14.07 10.77 0.0206 0,0269 0.3595 2298.98 3.92 29.87

6 200, 76 2.48 0.1982 0.0206 9.84 7.29 0.0109 0.0228 0.792 2161.60 3.50 33.04

Mean + 189.42 2.596 0.1726 0.1206 10.60 6.55 D.O0140 0.0228 9. 1565 2321.61 4.08 34.74

sb* 38.45 39 0604 0014 1.81 2.59 -0U49 0022 0200 196.49 60 2.17
*Mean 1 one standard devlatlon.

- Ty-
-42-

Table 6. The mean amount of sulfisoxazole excreted in the urine of

6 dogs.
Total Amount of
Sulfisoxazole 4 of
(mg) Dose
Intravenous
24 hours 0.792 39.59
48 hours 0.837 41.84
72 hours 0.843 42.17
Oral
24 hours 0.524% 26.20*
48 hours 0.582 29.10
96 hours 0.588 29.44

*Calculated from 5 dogs due to no collection of urine at 24 hours from
one individual.
-43-

The mean elimination rate (kK, 9) in the 6 dogs was 0.1565 = .0200
hours (Table 5). The ratio of B/k, 9 indicates the fraction of free
sulfisoxazole in the postdistributive phase which is available for
elimination. The mean B/G ratio was 0.13, indicating that 13.0% of
the drug in the body would be in the central compartment and available
for elimination.

The volume of the central compartment (Vv) ranged from 9.03 to 14.07
liters with a mean of 10.60 * 1.83 L (Table 5). The mean volume of the
second or peripheral compartment was 6.55 t 2.59 L (Table 5), indicating
that free sulfisoxazole is more widely distributed in the central than in
the peripheral compartment.

In 2 dogs, 3 and 6 (Table 23 and 26), the fraction bound was less
than 30% for the first 3 hours after intravenous administration but had
reached 40 to 50% at 4.5 hours after administration. In the other 4 dogs,
the fraction bound (£,) ranged from 30 to 50% throughout the trial period,
72 hours.

The mean amount of sulfisoxazole excreted in the urine (Table 6) was
39.59% of the dose within 24 hours after administration. By the end of
the trial, 72 hours, 42.17% of the dose was excreted in the urine.

Oral administration. From log-linear regression equations of the
serum concentration of free sulfisoxazole after oral administration, the
mean rate constants a and 8 were 0.1623 t .0361 hours! and 0.0296 £ .0172
hours! (Table 7), respectively. The mean half-life corresponding to the
faster disposition rate (ty was 4.37 t .76 hours and the mean elimina-
tion half-life corresponding to the slower disposition rate (ty 9? was

34.46 t 26.08 hours (Table 7).
Table 7. Two compartment pharmacokinetic parameters in dogs admlnistered sulfisoxazole as a single
oral dose.



Subject a B t

sa TB F oa
(hours!) Gioure *} (hours) (hours) (%)
1 0.1566 0.0299 4.33 23.14 100.92 2631.48
2 0.1543 0.0085 4.62 81.32 99.33 2279.43
3 0.1267 0.0145 5.33 47.76 99.14 2350.57
4 0.1497 0.0453 4.62 15.27 98.03 1956.60
5 0.1543 0.0530 4.33 12.86 99.02 2276.48
6 0.2326 0.0262 3.01 26.42 99.00 2338.14
SD* -0361 .0172 . 76 26.08 -94 215.69

*4Mean + one standard deviation.
-45-

The peak serum concentration had occurred by the first sampling
period, 1 hour (Tables 21 to 26). This rapid absorption was due to the
compound being given as a solution. The peak concentration of free
sulfisoxazole ranged from 122.25 to 165.00 ug/ml (Tables 21 to 26) and
the maximum total sulfisoxazole ranged from 152.78 to 229.22 ug/ml.

The degree of plasma protein binding was calculated to be 30 to 402
in 2 dogs in the first 6 hours of the trial (Tables 22 and 26). The
fraction bound was less than 30% in the remaining 4 dogs for the first 6
hours after oral administration. Between 6 and 44 hours, the fraction
pound was 40 to 50% but had increased to 70 to 90% in all dogs after 44
hours and continued at this level until the end of the trial.

The mean amount excreted in the urine of the 6 dogs was 26.20% of
the dose at 24 hours and 29.44% of the dose at the end of the trial, 96
hours (Table 6). Four of the 6 dogs excreted 43.78% of the dose in 24

hours while 2 dogs excreted 9.95 and 11.00% in 24 hours.

Administration of Sulfisoxazole to Swine

Intravenous administration. The "free," unbound, non-metabolized
sulfisoxazole in 6 pigs was analyzed as a two-compartment model (Figure
6) by a NONLIN program (57). At zero time, the mean extrapolated serum
concentration of A and B on the ordinate axis was 245.07 + 44.88 ug/ml
and 0.423 t .088 ug/ml (Table 8), respectively.

The disposition rate from the first compartment (a) ranged from
0.4961 to 9.5884 hours = with a mean of 0.5368 t .0362 hours which is
equivalent to a half-life (ty of 1.30 + .09 hours (Table 8). The
mean disposition rate from the second compartment was considerably longer,

0.0153 £ .0043 hours”” or 2 half-life (t, ,) of 53.33 $ 13.65 hours.
23
-~46-

1000.0

100.0

10.0

SERUM “FREE" SULFISOXAZOLE IN SWINE (pg/ml)

°



10 20 30 40 50 60 70

TIME (hours)

Figure 6. Serum concentrations of “free” sulfisoxazole in swine.
Table 8. Two compartment pharmacokinetic parameters in swine administered sulfisoxazole
as a Single intravenous dose.

Sub ject A B a K v Vv k, k k AUC t,



1 2 12 2} to r gytt
(ug/m)) (ug/ml) (igure) (hones!) (lhters) (Iters) (hours) (hours!) (hours?) (hours)

1 217.79 0.342 5732 0219 12.76 31.98 O.0214 0.0228 0.5509 696.10 1.21

2 186.07 0.495 0.531% O.OLA9 15.65 24 05 O.O317 0.0202 0.4782 1066.58 1.36

3 312.31 a. 300 0. 5884 G.012)3 8.20 15.70 0.0247 0.0129 0.563) 1339.20 1.18

4 250,45 0.459 0.5286 0.0107 9.58 33.32 0.0401 0.0116 0.4876 1287 86 1.32

5 275.07 0.439 0.5232 0.0113 7.5) 15.73 , 0.029) 0.0141 0.4931 1339, 38 1.32

6 233.92 0. 502 0.4961 O.0148 9.09 17.49 O.0304 D.O158 0.4647 1042.80 1.40
Mean ! 245.07 0.423 0.5368 0.0353 10.48 19.76 1.0296 0.0162 0.506} 1244.67 1.30
sh* 44.88 088 -0362 004 3 d.01 7.75 - 0064 0044 0406 280. 39 09

*Mean + one standard deviation.

{
55,8

(hours)

3 0%
36.07
56.34
64.7)
42.0)

46.82

53.33
13.65

-L9-
-48-

The mean distribution constant between the central and peripheral
compartments (k, >) was approximately twice as fast as the return of the

compound from the peripheral to the central compartment (k,,). The mean

21

k,> was 0.0296 + .0064 hours”* while k,, was 0.0162 + .0044 hours”

21
(Table 8). This is supported by the extended half-life of sulfisoxazole

). The ratio of k,./k,. was 0.55 which

i h d compartment (t
in the secon mp ( ky 21° 12

»8
reflects the distribution difference between the compartments with the
free sulfisoxazole readily entering the peripheral compartment but slowly
returning to the central compartment for elimination from the body.

The elimination rate (ks) ranged from 0.4647 to 0.5631 hours with
a mean of 0.5063 + .0406 hours ~ (Table 8). The ratio of B/k, 9 was 0.03
which indicated that only a very small fraction (3.0%) of free sulfisoxa-
zole was available to be excreted from the postdistributive phase.

The mean volume of distribution for the first compartment (v,)
(Table 8), 10.48 + 3.11 L, was one-half of the volume of the second
compartment (Vo); 19.76 + 7.75 L. This indicated that free sulfisoxa-
zole was much more widely distributed throughout the second compartment
than in the first compartment.

The degree of plasma protein binding in vivo (equation 10) ranged
from 40 to 60% throughout the trial period (Tables 27 to 32). Four of
the animals bound less than 20% of sulfisoxazole during the first hour
of the trial (Tables 29 to 32).

The mean amount of sulfisoxazole excreted in the urine as a percent
of the dose (Table 9) was 16.06% free sulfisoxazole and 19.17% acetyl-
sulfisoxazole at the end of 24 hours. By the end of the trial, 72 hours,
18.23% of the dose was excreted as free sulfisoxazole and 12.48% as

acetylsulfisoxazole.
-49-

Table 9. The mean amount of sulfisoxazole and acetylsulfisoxazole cn’)
excreted in the urine of 5 pigs* as a percentage of the dose.

% of Dose Excreted

Sulfisoxazole Acetylsulfisoxazole
Intravenous
24 hours 16.06 10.17
48 hours 18.08 12.37
72 hours 18.23 12.48
Oral
24 hours 15.24 10.62
48 hours 17.70 12.34
96 hours 18.27 12.78

*The urine from one animal was not recorded.
~50-

Table 10. The biological half-life of acetylsulfisoxazole cn) after a

single dose* of sulfisoxazole.

Chours)

Subject Humans
Oral a
(hours)
1 13.07 =
2 10.50 1.54
3 16.11 3.04
4 13.08 4.33
5 16.11 1.44
6 15.40 1.31
Mean + 14.05 2.33
spt 3.23 1.23

*Dogs did not metabolize sulfisoxazole to

a
‘Insufficient data for a log-linear regression plot.

£ . ue
"Mean t one standard deviation.

Pigs

37

27.

30.

35.

31.

.89

35

50

40

90

23.18

04
34

Intravenous
a B
(hours)

1.87 66.

0.71 27

1.93 5

1.61 15.

1.28 6

1.98 36.

1.56 26.
49 23.

the acetyl (4) metabolite.

86

86

.80

69

-47

60

55
15
The t, c and ¢t, of acetylsulfisoxazole (Table 10) were calculated
Bs “89

3,8

by log-linear regression. The t, ranged from 0.71 to 1.98 hours with
“2

oJ
9 &

a mean of 1.56 = .49 hours. The t, 8
Bs

of 5.80 to 66.86 hours and a mean of 26.55 + 23.15 hours. Three pigs had

was much more variable with a range

aty 2 less than 20 hours, 2 had a t, 3 between 20 and 40 hours, and 1
45 =
had a ty, 3 of 66.86 hours (Table 10). Acetylsulfisoxazole reached a

maximum level, 22.73 to 43.33 ug/ml (Tables 27 to 32), at 1 to 2 hours
after intravenous administration in all 6 pigs (Figure 7).

Oral administration. From log-linear regression equations of the
serum concentrations of free sulfisoxazole after oral administration, the
mean disposition rate constants, % and 8, were 0.5000 = .1706 heures ~ and
0.0148 + .0075 hours !, respectively (Table 11). The biological half-
life of free sulfisoxazole in the central or distribution compartment

(t

so ranged from 0.86 to 2.10 hours with a mean of 1.50 t .43 hours.
The mean half-life in the elimination compartment (ty, 9) was longer,
54.99 $ 21.84 hours with a range of 25.92 to 78.36 hours (Table 11).

Serum concentration of free sulfisoxazole reached a peak by the
first sampling period, 1 hour (Tables 27 to 32). This rapid absorption
was due to the compound being given as a solution. The maximum free
sulfisoxazole ranged from 32.77 to 100.64 ug/ml with 3 of the pigs having
less than 50.0 ug/ml of sulfisoxazole as a maximum serum concentration
(Tables 27, 28, and 31). Total sulfisoxazole attained a maximum of
68.62 to 202.70 ug/ml with the same 3 pigs having the lowest total
sulfisoxazole concentration (Tables 27, 28, and 31).

Acetylsulfisoxazcle reached a maximum of 12.11 to 20.02 ug/ml in 2
to 4 hours in all 6 pigs (Tables 27 to 32). The half-life of acetyl-

sulfisoxazole ranged from 1.31 to 4.33 hours for the central cr distribution
~52-

100.0

10.0

SERUM ACETYLSULFISOXAZOLE IN SWINE (j1g/ml)



10 20 30 40 50 60 70

TIME (hours)

. : : 4, , .
Figure 7. Serum concentrations of acetylsulfisoxazole (N_) in swine.
Table 11. Two compartment pharmacokinetic parameters in swine administered sulfisoxazole as a single
oral dose.

Subject ao 8 a CB F cre
(hours) (hours +) (hours) (hours) (4%)

L 0.8058 0.0217 0.86 31.48 33.33 232.00
2 0.4386 0.0267 1.58 25.92 30.42 324.46
3 0.3300 0.0088 2.10 78.36 38.97 521.93
4 0.4331 0.0091 1.60 75.60 52.08 670.76
5 0.4100 0.0117 1.69 59.00 18.16 243.29
6 0.5823 0.0110 1.19 59.20 58.50 610.07

Mean t 0.5000 0.0148 1.50 54.99 38.58 433.75

sp* .1706 .0075 -43 21.84 14.76 191.81

*Mean + one standard deviation.

-¢¢o-
-54-

compartment Coy with a mean of 2.33 + 1.32 hours (Table 10). For
the peripheral or elimination compartment (ty a)» the half-life ranged
from 23.18 to 37.89 hours with a mean of 31.04 + 5.34 hours (Table 10).

The bioavailability (F) of sulfisoxazole was calculated (equation 3)
from the area under the curve (AUC) after oral (Table 11) and intravenous
(Table 8) administration. The bioavailability ranged from 18.15 to
58.50% in swine with a mean of 38.58 t 14.76% (Table 11).

The degree of plasma protein binding (£,) in vivo ranged from 40 to
70% throughout the trial period (Tables 27 to 32). Three of the animals
bound less than 50% throughout the trial (Tables 27, 29, and 31) while
the fraction bound in the remaining 3 animals was more than 60% throughout
the trial (Tables 28, 30, and 32).

Swine excreted 15.24% of the dose as "free" sulfisoxazole into the
urine and 10.62% as acetylsulfisoxazole in the first 24 hours of the trial
after oral administration of the drug (Table 9). By the end of the trial,
96 hours, the amount of free sulfisoxazole excreted was 18.27% of the
dose as compared to the 48 hour level of 17.70%. The amount excreted as
acetylsulfisoxazole was 12.78% of the dose at the end of the trial, 96

hours (Table 9).

Administration of Sulfisoxazole to Humans

Following the oral administration of sulfisoxazole to humans, the
data were analyzed as a single compartment model (Figure 8). The half-
life (ty) following oral administration ranged from 5.97 to 8.77 hours
with a mean of 7.41 t .59 hours (Table 12). Kaplan et al. (26) reported

a mean half-life of 5.83 © .42 hours after oral administration of
-55-

1000.0

—— FREE SULFISOXAZOLE
— — ACETYLSULFISOXAZOLE

pg/ml)

i00.0

10.0

SERUM CONCENTRATIONS IN HUMANS (
(leg scale)



4 8 l2 16 20 24

TIME (hours)

. 4
Figure 8. Serum concentrations of "free" and acetylsulfisoxazole (N )
in humans.
~56-

sulfisoxazole (Gantrisin*) and 5.89 + .33 hours after intravenous
administration of the drug. The mean elimination constant (k,) was
0.095 + .013 hours + (Table 12).

The bioavailability (F) was calculated from the oral area under the
curve (Table 12) and the calculated area under the curve (705.92)
following intravenous administration (26). The bioavailability ranged
from 126.04 to 202.26% with a mean of 165.99 + 29.02% (Table 12). The
reason for F exceeding 100% could be due to 1) entero~hepatic recycling
or 2) the comparison of 2 different populations. Kaplan et al. (26) re-
ported a mean bioavailability of 123.0 t 11.0% with a range of 94.0 to
131.02.

The biological haif~life (ty) of acetylsulfisoxazole ranged from
10.50 to 16.11 hours with a mean of 14.05 + 2.23 hours (Table 10). The
maximum serum acetylsulfisoxazole concentration ranged from 29.73 to
34.94 ug/ml and reached maximum in all subjects 4 hours after oral
administration of 2.0 grams of sulfisoxazole (Tables 33 to 38).

The maximum "free" and total sulfisoxazole occurred before the
first sampling period dve to the drug being administered as a solution.
The maximum serum free sulfisoxazole levels ranged from 164.70 to
189.35 ug/ml (Tables 33 to 38) while the maximum total sulfisoxazole
ranged from 224.17 to 235.75 ug/ml. Kaplan et al. (26) reported a range
of 127.4 to 210.6 ug/ml of free sulfisoxazole after oral administration
of Gantrisin.*

The fraction of sulfisoxazole bound to plasma proteins (f,) was
less than 20.0% during the first hour for all 6 subjects (Tables 33 to

38). During the remainder of the trial period, the f. was 25 to 40%

*Gantrisin (active ingredient, sulfisoxazole), Hoffman~LaRoche, Nutley,
N.J. -
Table 12. Single compartment pharmacokinetic parameters in 6 humans
administered a 2.0 gm oral dose of sulfisoxazole.

Subject kK, ty AUC Fx
(hours!) (hours) (2)
1 0.079 8.77 1427.78 202.26
> 0.116 5.97 889.73 126.04
3 0.094. 7.37 1155.81 164.16
4 0.095 7.29 1127.38 159.70
5 0.099 7.00 1043.31 147.79
6 0.086 8.06 1386.45 196.40
Mean + 0.095 7.41 1171.74 165.99
spt 013 95 204.89 29.02

*Bioavailability is calculated using the mean AUC from Kaplan et al. (26)
of 6 humans after intravenous administration of 2.0 grams of Gantrisin
(sulfisoxazole); AUC = 705.92.

aa
Mean * one standard deviation.
-58-

which is less than the 85% previously reported (52) but similar to

another report of 25% bound in vivo (40).

Comparison of the Pharmacokinetics of Sulfisoxazole in Dogs,

Swine, and Humans

The mean extrapolated serum concentrations of sulfisoxazole in the
first compartment (A) was 189.42 t 38.45 ug/ml in dogs, 245.07 © 44.88
ug/ml in swine, and reported as 108.43 = 23.13 in humans (26). In the
second compartment, the extrapolated serum concentration (B) was 2.56 7
.39 ug/ml in dogs, 0.423 + .088 ug/ml in swine, and reported as 152.13 +
8.43 ug/ml in humans (26). The mean total initial serum concentration
(co = A+B) was 191.98 ug/ml in dogs, 245.49 ug/ml in swine, and re-
ported as 259.03 ug/ml in humans (26). The total and free initial
serum concentrations were not significantly different (2) in either
species. Kaplan et al. (26) reported a two-compartment model in humans;
the first compartment had a mean es of 33.6 minutes or 0.56 hours,
the second compartment had a mean ty 0 of 5.89 hours (Table 13) following

intravenous administration. The ty

4 in dogs was 4.08 hours and 1.30
29

hours in swine after intravenous administration. The shorter one in
humans was closer to the tho in swine after intravenous administration.
The half-life of sulfisoxazole in the second compartment (ty, 3? was
33.74 hours in dogs and 53.33 hours in swine (Table 13). Neither of
these half lives were comparable to the value previously reported in
humans (26), 5.89 hours. Due to the short term of this trial, no second
compartment was observed in the human subjects.

After oral administration, the t, was 7.41 hours in humans, 4.37
29

hours in dogs, and 1.50 hours in swine (Table 13). The ty 8 in dogs was
29
Table 13. The biological half-life (t,) of sulfisoxazole.
2



Dogs

Intravenous

Oral

Swine
Intravenous

Oral

Humans
Intravenous*

Oral

*Kaplan et al. (26).

t
X50.

(hours)

0.56*

t
4,8

(hours)

33.74

34.46

53.33

54.99

5.89%

5.83%
~60-

34.46 hours, 54.99 hours in swine, and reported as 5.83 hours in humans
(26) after oral administration of sulfisoxazole. There were no differences

in the half lives of sulfisoxazole, t due to the route of

B’

administration in dogs and swine. If the ty a of 7.41 hours in humans
2

>

1 . or ty
50 Bs

after oral administration is compared to the t, reported by Kaplan
“2

»8

et al. (26), there is no difference in the route of administration or in
the compartments. Kaplan et al. (26) postulated the two-compartment
model in humans due to taking 4 blood samples within the first hour
after intravenous administration of sulfisoxazole (Gantrisin*). There
were large differences between species. Humans had the longest mean

t » 7.41 hours, followed by dogs, 4.08 and 4.37 hours, and then swine,

1
BO

1.30 and 1.50 hours (Table 13). In the second compartment, swine had

the longest mean t, 53.55 and 54.99 hours, followed by the dogs,
3

» 8?
33.74 and 34.46 hours (Table 13).

Maximum serum acetylsulfisoxazole concentrations (Tables 27 to 38)
were higher in humans (30.0 to 35.0 ug/ml) than in swine (12.0 to 20.0
ug/ml). However, swine were able to acetylate (ny sulfisoxazole at a
faster rate as shown by the shorter time for maximum serum acetylsulfisoxa-
zole in swine (2 to 4 hours) as compared to humans (4 hours). Humans had
a mean th og of 14.05 hours (Table 14) after oral administration which is
longer than the 9.0 hours previously reported (33). Swine had a mean
theo of 2.33 hours after oral administration and 1.56 hours after

intravenous administration of sulfisoxazole. The t, of acetyl-

3,8

sulfisoxazole was 26.55 hours after oral administration and 31.04 hours

*Gantrisin (active ingredient, sulfisoxazole), Hoffman-LaRoche, Nutley,
N.J.
-61-

Table 14, The biological half-life (t,) of acetylsulfisoxazole, the N

metabolite of sulfisoxazole?

“te
(hours)
Dogs
Intravenous ND*
Oral ND*
Swine
Intravenous 1.56
Oral 2.33
Human
Oral 14.05

4

"1.48

(hours)

ND*

26.55

31.04

*ND = Non detectable. No (x) acetylsulfisoxazole was detected in dogs.
-62-

after intravenous administration of sulfisoxazole (Table 14) in swine.
Dogs did not metabolize sulfisoxazole to the (n*) acetyl metabolite.

The distribution constants of sulfisoxazole (Table 15) from the
central to the peripheral compartment (kK, 9) was greatest in humans (0.45
hours followed by swine (0.0296 hours"), and then dogs (0.0140
hours 3: The distribution from the peripheral compartment to the
central compartment (ko 1) was fastest in humans (0.87 hours‘), followed
by dogs (0.0228 hours “}, and then swine (0.0162 houve ~). The elimina-
tion constants (k, 9) were greatest in swine (0.5063 hoare “9. Eollowed
by humans (0.195 hours 7). and then dogs (0.1565 hours“). The order of
the elimination constant (ky) was the same as the oo of free
sulfisoxazole, shortest in swine, then dogs, and longest in humans.

The difference in distribution between the two compartments (k,)/K, 5)
was highest in humans (26), 2.3, followed by dogs, 1.63, and then swine,
0.55, indicating that the drug returned from the peripheral compartment
to the central compartment fastest in humans and slowest in swine

(Table 16). These values were concurrent with the mean half-life of

sulfisoxazole in the second compartment where t, was reported as shortest

e:
in humans (26), followed by dogs, and longest in swine (Table 13). The
ratio of B/K, 4 was reported as 0.66 in humans (26), 0.13 in dogs, and
0.03 in swine (Table 16). This is the same as the ko Ky (Table 16)
ratio so that more sulfisoxazole is available for elimination from the post-
distributive phase in humans, followed by dogs, and the least available
was in swine.

The mean volume of distribution for the central compartment (V,)

was approximately the same in dogs, 10.60 L, and swine, 10.48 L (Table 17).

The mean volume of the second compartment (V,) was much larger in swine,
~63-

Table 15. The mean distribution constants after intravenous administra-
tion of sulfisoxazole.*

Kio Koy Kio

(hours +) (hours +) (hours +)
Dogs 0.0140 0.0228 0.1565
Swine 0.0296 0.0162 0.5063
Humans" 0.45 0.87 0.195

*Mean of 6 individuals.

+
Kaplan et al. (26).
Table 16.

Dogs
Swine

Humans*

*Kaplan et al.

-64-

The ratio of distribution constants after administration
of sulfisoxazole.

ky4/K5 8/k,

1.63 0.13

0.55 0.03
2.30 0.66

(26).
-~65-

19.76 L, than in dogs, 6.55 L (Table 17). The calculated vy from pre-
vious data (26) was 7.67 L and 8.54 L for V5 in humans. Due to the
analytical procedure and the length of the trial (26), this Vo should not
be compared with the Vo reported in dogs and swine. From the calculated
Ya for dogs, swine, and humans, there is an approximate 40% difference

in the animal vy over the human volume of distribution. The volume of
distribution in the second compartment (V,) is much larger in swine than
in dogs.

The mean bioavailability (F) was greatest in humans, 165.99% when
the area under the intravenous curve from Kaplan et al. (26) was used
(Table 18). This value agreed with previous data (26) that F was 123.0%
in humans. The absolute bioavailability of sulfisoxazole was 99.24% in
dogs and 38.58% in swine (Table 18). The bioavailability in dogs is
closer to the calculated F in humans, and swine appear to be vastly
different from both humans and dogs. However, the higher bioavailability
in humans is due to enterohepatic circulation of sulfisoxazole which does
not occur in dogs and swine. The majority of sulfisoxazole is not
absorbed from the gastro-intestinal tract in swine and is excreted in
the feces.

The degree of protein binding in vivo was less than 30% in 2 dogs
for the first 3 hours and in 4 pigs for the first hour after intravenous
administration and in all 6 humans for the first hour after oral adminis-
tration of sulfisoxazole. More than 70% of sulfisoxazole was bound to
plasma proteins in dogs at 44 hours after oral administration of sulfisoxa-
zole. During the remainder of the trial, 30 to 50% of sulfisoxazole was

bound to plasma proteins in humans, dogs, and swine which compared

favorably with the 25% previously reported (40). This observation
~66-

Table 17. Comparison of the volumes of distribution in dogs, swine, and
humans after administration of sulfisoxazole.

Species Vi V5
(liters) (liters)
Dogs 10.60 6.55
Swine 10.48 19.76
Humans* 7.67 8.45

*Kaplan et al. (26).
-67-

Table 18. Comparison of the bioavailability* of sulfisoxazole in dogs,

swine, and

Species

Dogs
Swine

Humans

*Calculated

humans.

Bioavailability

99.24
38.58

165.99

from area under the plasma curve.
-68-

suggests that an individual difference occurs along with a species and
treatment difference. Fifty to sixty-three percent of sulfisoxazole was
bound to human serum proteins in vitro (Table 19) at 25 to 200 ug/ml.

At 300 yg/ml, only 41.0% of sulfisoxazole was bound. This suggests that
the degree of protein binding is capacity limited as the serum concen-
tration increases above 200 ug/ml. This hypothesis would explain the low
fraction bound which was observed during this experiment.

The amount of free sulfisoxazole excreted in the urine was 39.59%
of the dose in 24 hours after intravenous administration and 26.20% of
the dose in 24 hours after oral administration in dogs. The amount
excreted in the urine by swine was 25.86% of the dose and 26.23% of the
dose in 24 hours after oral and intravenous administration of sulfisoxa-
zole, respectively. By the end of the trial, 72 hours and 96 hours for
the intravenous and oral routes, respectively, more than 30.04 of the
dose was excreted in swine (Table 9) and in the dogs after oral adminis-
tration (Table 6). After intravenous administration, over 40.0% of the
dose was excreted in the urine by dogs. Kaplan et al. (26) reported
that 52.9% of the dose was excreted as free sulfisoxazole by humans.
Acetylsulfisoxazole accounted for less than half of the urinary level in
swine (Table 9) which confirmed the previously reported 30% (24)
excreted as the acetyl metabolite in humans. The differences between
the human and dog and pig excretion levels could be explained partly by
the analytical method used. Kaplan et al. (26) used a nonspecific method
for aromatic amines, the Bratton-Marshall method (6). This experiment

used a more specific method for both free and acetylsulfisoxazole.
-69-

Table 19. The fraction of sulfisoxazole bound (fp) to human serum
proteins in vitro.

Sulfisoxazole
Concentration
(ug/ml)

25.0
50.0
100.0
200.0

300.0

"Free" Serum
Concentration of Sulfisoxazole
(ug/ml)

7.57
16.67
27.27
57.57

112.12

57.00

50.00

63.00

60.00

41.00
-70-

Serum Bilirubin Concentrations

Dogs--Intravenous Administration

The mean total bilirubin concentration in 6 dogs administered
sulfisoxazole intravenously exhibited statistically significant (p < .01)
linear increases (2) at 4.5 (p < .05), 6.0 (p < .01), 9.0 (p < .01), and
12.0 (p < .01) hours with the maximum levels at 6 and 12 hours (Figure 9).
By the next sampling period, 22 hours, the mean total bilirubin concen-
tration had decreased to levels which were not significantly different
from control values until the 56 hour sampling period (p < .01).

The mean conjugated bilirubin levels also showed statistically
significant increases at 4 hours (p < .01) and continued throughout the
sampling period (p < .01), 72 hours. There was a significant (p < .01)
maximum mean conjugated bilirubin at 12 hours which coincided with the
maximum mean total bilirubin concentration (Figure 9). A second increase
was observed at 56 hours (p < .01) which coincided with the second in-
crease in total bilirubin (Figure 9). The mean indirect bilirubin con-
centration (Figure 9) was not significantly different from the control
or 0 level throughout the sampling period.

The mean total bilirubin was significantly correlated with mean
conjugated bilirubin (R = 0.75, p = .0001) and with mean indirect bili-
rubin (R = 0.31, p = .004) after intravenous administration. The
significant correlation of total and conjugated bilirubin is due to the
concomitant increase in glucuronidation activity as total bilirubin levels
increase. If the glucuronidation enzyme system is not capacity limited,
the possibility of kernicterus, due to an increase in indirect bilirubin,

is minimized in the dog after intravenous administration of sulfisoxazole.
0.60

—— TOTAL
— — - CONJUGATED

che —-— INDIRECT

0.40

MEAN SERUM BILIRUBIN CONCENTRATIONS(mg/dl)

0.30
—_————— or !
~
a
_7e~ {
- ~
0.20 ~ _ oe ~
~ =~ ~~
a ~~»
,@
0.10 Fee 2 o. te
~ eee
aes ~ e + OT



TIME (hours)

Figure 9. Mean serum bilirubin concentrations in dogs after intravenous administration of
sulfisoxazole.
-72-

This enzyme system acts to maintain a reduced or limited indirect bili-
rubin concentration and reduces the toxicity of displaced "protein-
bound" bilirubin or increased heme degradation. This was further empha-
sized by the significant negative correlation of conjugated and indirect
bilirubin (R = -0.39, p = .0002). As the level of conjugated bilirubin
increased, the indirect bilirubin level decreased.

A second explanation would be that sulfisoxazole alters the function
of the normal hepatocyte to increase conjugated bilirubin regurgitation
into the general circulation instead of being excreted into the bile.
The resulting increase in total bilirubin would be the result of the
increased level of regurgitated, conjugated bilirubin along with the

normal level of indirect bilirubin.

Dogs--Oral Administration

When sulfisoxazole was administered orally to dogs, there was a
significant linear increase in mean total (p < .01), conjugated (p < .001),
and indirect (p < .0001) bilirubin (Figure 10). Total bilirubin reached
its first significant peak at 12 hours (p < .01) and a higher concen-
tration at 76 hours (p < .01) (Figure 10). The mean total bilirubin
levels were significantly increased at 8 (p < .05), 10 (p < .01), and 12
(p < .01) hours. A second higher peak occurred at 76 hours (p < .01) and
remained greater than the control levels at the end of the sampling
period, 96 hours (p < .01) (Figure 10). The mean conjugated bilirubin
levels were also linearly increased (p < .001) during the sampling
period. Significant increases (p < .05) were observed at 8, 10, and 12

hours (Figure 10), the same periods in which total bilirubin was
Figure

0.60



0.50 —— TOTAL

~-- CONJUGATED
— ~— INDIRECT

0.40

0.30

0.20

MEAN SERUM BILIRUBIN CONCENTRATIONS (mg/d!)

TIME ( hours )

10. Mean serum bilirubin concentrations in dogs after oral administration of
sulfisoxazole.

-¢l-
-74-

significantly increased. After the 12 hour period, the mean conjugated
bilirubin had returned to levels which were not significantly different
from control levels. The mean conjugated bilirubin reached its maximum
concentration (0.22 mg/dl) at 10 hours while the maximum total bilirubin
(0.31 mg/dl) occurred 2 hours later, at the 12 hour sampling period
(Table 40). The mean indirect bilirubin was not significantly different
from control levels until the 76 and 96 hour periods (p < .01). Mean
indirect bilirubin reached a maximum of 0.28 mg/dl at 76 hours but had
begun to decline by the end of the trial, 96 hours.

Total bilirubin was significantly correlated with conjugated bili-
rubin (R = 0.60, p = .0001) and with indirect bilirubin (R = 0.56,
p = .00001) while conjugated bilirubin was also negatively correlated
with indirect bilirubin (R = -0.31, p = .003). The significant correla-
tion of total and conjugated bilirubin is explained by an increase in
glucuronidation as increased heme degradation or increased indirect
bilirubin occurs or due to regurgitation of conjugated bilirubin into the
general circulation. The significant correlation of indirect bilirubin
and total bilirubin illustrated that the glucuronidation activity could
not account for conjugation of all the bilirubin present. The negative
correlation of indirect and conjugated bilirubin is supportive evidence
that toxic, indirect bilirubin levels can be reduced, to prevent

kernicterus, if glucoronidation activity can be stimulated.

Comparison of Bilirubin Levels in Dogs

The mean total bilirubin reached maximum at 12 hours after intra-

venous (0.39 mg/dl) and oral (0.31 mg/dl) administration of sulfisoxazole
~75-

(Figure 11), with a second increase at 56 hours after intravenous adminis-
tration and 76 hours after oral administration. Conjugated bilirubin was
also maximum in the 10 and 12 hour period after oral (0.22 mg/dl) and
intravenous (0.32 mg/dl) administration of sulfisoxazole (Figure 12),
respectively. Conjugated bilirubin was significantly elevated at the

4.5 hour period and throughout the trial period after intravenous
‘administration; after oral administration, conjugated bilirubin was
significantly increased between 8 and 12 hours only. Indirect bilirubin
was not significantly increased after intravenous administration but was
significantly increased at 76 and 96 hours after oral administration
(Figure 13).

The total bilirubin increase, after oral or intravenous administra-~
tion of sulfisoxazole, was accompanied by an increase in glucuronidation
activity which increased the conjugated, water-soluble bilirubin levels.
This prevented toxicity by reducing the indirect bilirubin level and
increasing the water-soluble, conjugated bilirubin which is more easily
excreted. The glucuronidation activity was reduced during the later
periods after oral administration, allowing the potentially toxic, in-
direct bilirubin to increase.

There was a negative correlation of conjugated and indirect bilirubin
after intravenous (R = -0.39, p = .0001) and oral (R = -0.31, p = .003)
administration of sulfisoxazole. After intravenous administration,
conjugated bilirubin levels increased along with an increase in total
bilirubin (Figure 9). After oral sulfisoxazole administration, conjugated
bilirubin levels increased initially as total bilirubin levels increased
but later decreased, allowing indirect bilirubin levels to increase.

This result leads one to conclude that some pathological event is induced
0.50

—— INTRAVENOUS
— — ORAL

0.40

0.30

0.20

0.10

TOTAL BILIRUBIN CONCENTRATIONS (mg/dl)



5 10 20 30 40 50 60 70 80 90 100
TIME (hours)

Figure 11. Comparison of the mean total bilirubin concentrations in dogs after intravenous and
oral administration of sulfisoxazole.
0.50

0.40 —— INTRAVENOUS

—— ORAL

0.30

-{I-

0.20

MEAN CONJUGATED BILIRUBIN CONCENTRATIONS (ma/dl)



5 Te) 20 30 40 50 60 70 80 9390 {00
TIME (hours)

Figure 12. Comparison of the mean conjugated bilirubin concentrations in dogs after intravenous
and oral administration of sulfisoxazole.
0.50

0.40

0.30

—— INTRAVENOUS 7 ~

— — ORAL /

0.20

-~Sl-



MEAN INDIRECT BILIRUBIN CONCENTRATIONS (mg/d!)
\
?
/

5 10 20 30 40 50 60 70 80 390 100
TIME (hours)

Figure 13. Comparison of the mean indirect bilirubin concentrations in dogs after intravenous
and oral administration of sulfisoxazole.
during oral administration of sulfisoxazole but not after intravenous
administration which could allow indirect bilirubin to reach toxic levels.

This pathological event could be explained by the pharmacological
"first-pass" effect (57). In the "first-pass" effect, all of an absorbed
drug is presented to the liver before distribution whereas after intra~
venous administration, only 20% of the drug passes through the liver
before being distributed or excreted. In this case, the greater concen-
tration of sulfisoxazole presented to the liver, in addition to the lack
of acetylation of the drug, could be responsible for altering the
normal detoxification of bilirubin so that one observes an increase in
indirect bilirubin.

Total and conjugated bilirubin reached higher levels within 4 to 12
hours after intravenous administration than after oral administration
(Figures 11 and 12). After oral administration, conjugated bilirubin
reached its maximum within this time period (10 hours) but total and
indirect bilirubin did not reach maximum until 76 hours. The increased
indirect bilirubin levels should be expected after intravenous adminis-
tration due to the increased serum concentration of sulfisoxazole when
compared to levels following oral administration which would allow
sulfisoxazole to displace more bilirubin from albumin. The increased
total and indirect bilirubin levels at the end of the oral administration
trial supports the hypothesis that a pharmacological event is induced by
the oral route but not by the intravenous route which caused an increase
in total and indirect bilirubin, with possible toxic side effects at a
time much later after a single, oral dose of sulfisoxazole.

There were no statistical differences in serum total, conjugated or
indirect bilirubin between individual dogs after oral or intravenous

administration of sulfisoxazole.
0.60

——- TOTAL

--~-— CONJUGATED
0.50 —-— INDIRECT
0.40
0.30

MEAN SERUM BILIRUBIN CONCENTRATIONS (mg/dl)



4 8 16 24 32 40 48 56 64 72
TIME (hours)

Figure 14. Mean serum bilirubin concentrations in swine after intravenous administration of
sul Fisoxazole.

-08-
-81-

Swine-~Intravenous Administration

An analysis of variance, using mean bilirubin concentrations, re-~
vealed no statistical difference among the 6 pigs or during time intervals
for serum total, conjugated or indirect bilirubin after intravenous
administration of sulfisoxazole. The mean total conjugated and indirect
bilirubin were increasing at the end of the trial but were not signifi-
cantly different than control levels (Figure 14).

Total bilirubin was significantly correlated with conjugated bili-
rubin (R = 0.86, p = .0001) and with indirect bilirubin (R = 0.61,

p = .0001) after intravenous administration of sulfisoxazole.

Swine~--Oral Administration

After oral administration of sulfisoxazole, mean total bilirubin was
significantly increased (p < .01) to 0.52 mg/dl at 6 hours (Figure 15).
A parallel significant (p < .01) increase was also observed in mean con-
jugated bilirubin at 6 hours, 0.34 mg/dl (Figure 15). A significant
(p < .0001) linear decrease did occur over the trial period in conjugated
bilirubin. Mean indirect bilirubin was not significantly different from
control values at any time after oral administration of sulfisoxazole.
However, indirect bilirubin levels did exceed conjugated levels after
the 32 hour sampling period and remained at an increased level throughout
the remainder of the trial (Figure 15).

Total bilirubin was significantly correlated with conjugated bilirubin
(R = 0.81, p = .0001) and with indirect bilirubin (R = 0.42, p = .0001)

after oral administration of sulfisoxazole.
0.60

0.50 — TOTAL
——— CONJUGATED
—-— INDIRECT

MEAN SERUM BILIRUBIN CONCENTRATIONS (mg/d!)



TIME (hours)

Figure 15. Mean serum bilirubin concentrations in swine after oral administration of

sulfisoxazole.
-~33-

Comparison of Bilirubin Levels in Swine

It appears that an increase in total bilirubin in the pig is
accompanied by an increase in conjugated and indirect bilirubin regardless
of the route of administration, as shown by the positive correlation
coefficients. The higher serum "free" sulfisoxazole concentrations
(Tables 27 to 32) after intravenous administration did not significantly
affect bilirubin concentrations (Figure 14) but oral administration did
significantly increase total (p < .01) and conjugated (p < .0001) bili-
rubin at 6 hours but did not affect indirect bilirubin (Figure 15).
Indirect bilirubin was closely correlated with conjugated bilirubin
after intravenous administration (Figure 14) but exceeded conjugated
bilirubin after oral administration (Figure 15), which suggests that an
oral dose is handled differently than the intravenous one.

Intravenous administration of sulfisoxazole does not appear to
affect bilirubin levels in the pig. After oral administration, an in-
crease in total bilirubin could be accompanied by an increase in the
conjugated level which reduces the hazard of indirect bilirubin toxicity.
While the indirect bilirubin levels were not significantly different by
either treatment, one should note that indirect bilirubin levels did
exceed conjugated bilirubin levels only following oral administration
(Figure 15). While no toxic levels of bilirubin have been established
in swine, there was a significant increase in total (Figure 16) and
conjugated (Figure 17) bilirubin in 6 hours after oral administration
which did not occur after intravenous administration. This suggests
that an oral dose of sulfisoxazole may have a different effect in vivo

than an intravenous administration even though the intravenous route
0.60

0.50

0.30

—
—
—_—

-Q-

0.20



—— INTRAVENOUS
— — ORAL



MEAN TOTAL BILIRUBIN CONCENTRATION (mg/dl)

5S 10 20 30 40 50 60 70 80 90 100
TIME (hours)

Figure 16. Comparison of the mean total bilirubin concentrations in swine after intravenous and
8 P
oral administration of sulfisoxazole.
0.50

0.40




——_ INTRAVENOUS

— — ORAL
0.30

-¢g-

0.20

0.10

MEAN CONJUGATED BILIRUBIN CONCENTRATIONS (mg/dl }

40 70 80 90 100
TIME (hours)

Figure 17. Comparison of the mean conjugated bilirubin concentrations in swine after intravenous
and oral administration of sulfisoxazole.
0.50

0.40




— INTRAVENOUS

0.30 — — ORAL

0.20

MEAN INDIRECT BILIRUBIN CONCENTRATIONS (mg/d!)
-98-

5 10 20 30 40 50 60 70 80 90 100
TIME (hours)

Figure 18. Comparison of the mean indirect bilirubin concentrations in swine after intravenous
and oral administration of sulfisoxazole.
~87-

created an increased serum concentration of sulfisoxazole when compared
to the serum level after oral administration, possibly due to a "first-
pass" effect (57). In swine, the route of administration does not
appear to affect the concentration of the potentially toxic, indirect
bilirubin (Figure 18). This species effect in swine could prevent

bilirubin toxicity induced by sulfonamides.

Humans--Oral Administration

An analysis of variance evaluation after oral administration of
sulfisoxazole resulted in no statistical difference in mean total,
conjugated, and indirect bilirubin levels (Figure 19). The lack of
significance could be due to 1) the low therapeutic dose of 2.0 gm or
approximately 30 mg/kg administered to the human subjects, 2) humans are
able to reduce the in vivo effects of sulfisoxazole on the hepato-biliary
system, or 3) the sampling period was too short and additional samples
should have been taken over a longer trial period.

The values recorded after oral administration of sulfisoxazole were

within the normal limits reported for the Automatic Clinical Analyzer (17).

Comparison of Bilirubin Levels in Dogs, Swine, and Humans

Control values for total and conjugated bilirubin were higher in
humans than in swine and lowest in dogs (Table 20). The indirect
bilirubin levels in all three species were similar (Table 20).

A significant increase in total bilirubin occurred in dogs at 12
hours (Figure 20) after intravenous administration of sulfisoxazole but

did not occur in swine (Figure 20) after administration of the same dose.
0.60

0.50
_—-~-
O.40F ~~ ~e~ ~~ ee ee
—— TOTAL
30
= ——~ CONJUGATED
—-+— INDIRECT

0.20

MEAN SERUM BILIRUBIN CONCENTRATION (mg/d!)



TIME (hours)

Figure 19. Mean serum bilirubin concentrations in humans after oral administration of
sulfisoxazole.

-8e-
~89-

Table 20. Mean* control values of serum bilirubin and albumin in humans,
dogs, and swine.

Total Conjugated Indirect
Bilirubin Bilirubin Bilirubin Albumin
(mg/d1) (mg/d1) (mg/dl) (gm/d1)
Humans 0.54 0.41 0.13 4.63
Dogs 0.21 0.08 0.13 3.02
Swine 0.35 0.19 0.15 3.75

*The mean value of 8 samples before oral and intravenous administration
of sulfisoxazole in 6 dogs and 6 swine. The mean values for humans are
calculated from the 0 sample time for 6 human subjects.
~90~

After oral administration of sulfisoxazole, a significant increase in
total bilirubin occurred in dogs (Figure 21) and swine (Figure 21) at 6
and 12 hours, respectively, with another increase in dogs at 76 hours.
In humans, this increase was not observed over the same time period,
probably due to the difference in the oral dosage (Figure 19).

Initially, total bilirubin levels were higher in swine than in dogs
(Table 20) but after intravenous administration of sulfisoxazole, total
bilirubin was significantly (p < .01) higher in dogs between 3 and 15
hours (Figure 20). Total bilirubin levels after oral administration were
higher in swine than in dogs until the 56 hour period when the levels in
dogs were significantly increased (p < .01) (Figure 21). Even after
administration of sulfisoxazole, the total bilirubin increases in dogs
and swine did not reach the control or treated levels in humans.

Conjugated bilirubin levels were not significantly increased in
swine after intravenous administration of sulfisoxazole even though the
control levels were greater in swine than in dogs (Figure 22). Conjugated
bilirubin levels were significantly increased in dogs after intravenous
administration of sulfisoxazole, exceeded the levels in swine after 3
hours and reached a maximum at 12 hours after administration (Figure 22).
After oral administration of sulfisoxazole, conjugated bilirubin levels
were elevated in swine at 6 hours and showed a statistically significant
increase in dogs at 10 hours after administration of the drug (Figure 23).
The conjugated bilirubin levels in dogs and swine after intravenous or
oral administration of sulfisoxazole did not reach the control or treated
conjugated bilirubin levels in humans at any of the sampling periods.

Potentially toxic, indirect bilirubin showed a statistically

significant, but clinically small, increase after oral administration
0.50

0.40

-T 6-

ee
-_<- oO
—

0.20

0.10 _—— Swine

MEAN TOTAL BILIRUBIN CONCENTRATIONS (mg/d!)
-
Ww
o



4 8 16 24 32 40 48 56 64 72

TIME (hours)

Comparison of the mean total bilirubin concentrations in dogs and swine after

Figure 20.
intravenous administration of sulfisoxazole.
0.60

0.30

-T6-

0.20

0.10

MEAN TOTAL BILIRUBIN CONCENTRATIONS (mg/dl)



10 20 30 40 50 60 70 80 90 100
TIME (hours)

Figure 21. Comparison of the mean total bilirubin concentrations in dogs and swine after oral
administration of sulfisoxazole.
0.50

0.40
—— DOGS

— — SWINE

0.30

~C6-

0.20

MEAN CONJUGATED BILIRUBIN CONCENTRATIONS (mg/d!)



4 8 16 24 32 40 48 56 64 72
TIME (hours)

Figure 22. Comparison of the mean conjugated bilirubin concentrations in dogs and swine after
intravenous administration of sulfisoxazole.
0.40

A — pocs
\ — — Swine

0.30 | \

0.20

~06-

0.10

100

MEAN CONJUGATED BILIRUBIN CONCENTRATIONS(mg/d! )



TIME (hours)

Comparison of the mean conjugated bilirubin concentrations in dogs and swine after

Figure 23.
oral administration of sulfisoxazole.
> 0.50

e

Zz

©

EF 0.40

Om

ft

=

O

=z

S 0.30 —— pocs

z — — SWINE

a

2

ax '
3 0.20 wn
a 1
ft

O

uJ

x

Q

=

Zz


uJ

=



4 8 16 24 32 40 48 56 64 72
TIME (hours)

Figure 24, Comparison of the mean indirect bilirubin concentrations in dogs and swine after
intravenous administration of sulfisoxazole.
0.40

0.30 —— DOG
— — SWINE

0.20



5 10 20 30 40 50 60 70 80 30 100
TIME (hours )

MEAN iNDIRECT BILIRUBIN CONCENTRATION (mg/d!)

Figure 25. Comparison of the mean indirect bilirubin concentrations in dogs and swine after oral
administration of sulfisoxazole.
=97=

of sulfisoxazole in dogs (Figure 25). There were no statistically
significant increases in indirect bilirubin after oral administration in
humans or swine or in either the dogs or swine after intravenous adminis-

tration of sulfisoxazole (Figure 24).

Conclusions Concerning Bilirubin Levels after Administration

of Sulfisoxazole

Small, but statistically significant, increases in total and con-
jugated bilirubin occurred when sulfisoxazole was administered orally to
dogs (Figure 10) and swine (Figure 15) and only when administered intra-
venously to dogs (Figure 9). The maximum increases occurred at different
points in time depending on species and route of administration. This
species variation is further reflected by the indirect bilirubin
exceeding conjugated bilirubin levels in dogs (Figure 10) and swine
(Figure 15) when sulfisoxazole was administered orally. While toxic
levels of bilirubin have not been established, these results show that
the oral route of administration does increase the potentially toxic,
indirect bilirubin concentration at a time far removed from the initial,
single dose in dogs and swine.

The small but statistically significant increase of total (Figure 8)
and conjugated (Figure 10) bilirubin, after oral administration of
sulfisoxazole, suggests that the absorption of the oral dosage form does
affect the hepatocyte to increase the normal rate of heme degradation
or to increase conjugated bilirubin regurgitation into the general
circulation. This is supported by the intravenous dose given to dogs
only increasing the total and conjugated bilirubin and not significantly

affecting the indirect bilirubin level.
-~98~

The lack of significant effects in humans and the lack of correla-
tion between humans and dogs or swine could be due to the shorter sampling
period in humans, the reduced dosage given to humans or the species
variation which exists even though all three are monogastric.

When comparing the correlation coefficients of total and conjugated
bilirubin in dogs (R = 0.75, p = .0001) and swine (R = 0.85, p = .0001)
after intravenous administration, these parameters are more closely
correlated in the pig. This is also true for the significant correlation
of indirect and total bilirubin in dogs (R = 0.31, p = .004) and swine
(R = 0.61, p = .0001) after intravenous administration of the drug.

When sulfisoxazole is administered orally, the total and conjugated

bilirubin levels are more closely correlated in swine (R = 0.81, p =

.0001) than in dogs (R = 0.60, p = .0001) but indirect bilirubin is

more closely correlated to total bilirubin in dogs (R = 0.56, p = .0001)
than in swine (R = 0.42, p = .0001). In addition, conjugated and in-
direct bilirubin are negatively correlated after intravenous (R = -0.39,

p = .0002) and oral (R = -0.31, p = .003) administration in the dog
only, not in the pig.

It is evident that the route of sulfisoxazole administration does
affect the bilirubin level differently in these 3 species of animals.
The potentially toxic, indirect bilirubin level is significantly in-

creased only after oral administration in one of the three species, dogs.

Serum Albumin Concentrations

There were no significant differences in the serum albumin levels

in either of the three species after intravenous or oral administration
3.10

3.00

-66-



—— INTRAVENOUS
— — ORAL




MEAN SERUM ALBUMIN CONCENTRATIONS (gm/d1)

10 20 30 40 50 60 70 80 30 100
TIME (hours)

Figure 26. Mean serum albumin concentrations in dogs after intravenous and oral administration
of sulfisoxazole.
3.90

3.80

— ea ei

3.70



ERUM ALBUMIN CONCENTRATIONS (gm/d!)

3.60 bu

—— INTRAVENOUS S

/ — — ORAL

w 3.50
z
a
tu
=
0 10 20 30 40 50 60 70 80 Yo) 100

TIME (hours)

Figure 27. Mean serum albumin concentrations in swine after intravenous and oral administration
of sulfisoxazole.
4.80

4.70

~TOT-

4.60

| 2° 3 4 5 6 7 8
TIME (hours)

MEAN SERUM ALBUMIN CONCENTRATIONS (gm/d!)

Figure 28. Mean serum albumin concentrations in humans after oral administration of sulfisoxazole.
-102-

of sulfisoxazole (Figures 26, 27, and 28). There was an initial decrease
in albumin levels in all three species after sulfisoxazole administration
with a return to control or normal levels within 4 to 6 hours. There
was a greater decrease after oral administration in dogs (Figure 26) and
swine (Figure 27).

Humans (Figure 28) had a higher initial concentration of albumin
(Table 20) than dogs or pigs and initial albumin levels were higher in

pigs than in dogs (Table 20). These differences were maintained throughout

the trial periods after intravenous and oral administration.
SUMMARY AND CONCLUSIONS

From the pharmacokinetic parameters which were calculated or pre-
viously reported (26), the mean initial total serum concentrations were
similar in all three species. However, the extrapolated serum concen-
tration in the first compartment was highest in swine, followed by dogs
and lowest in humans (26). In the second compartment, the extrapolated
serum concentration was higher in dogs than in swine.

The biological half-life of "free"' sulfisoxazole in the first
compartment was longest in humans, one-half this value in dogs, and one-
fifth the time of humans in swine. In the second compartment, the half-
life was twice as long in swine than in dogs. A second compartment was
not observed in humans during this trial. The half-life of the acetyl
(n*) metabolite in the first compartment was 8 times longer in humans
than in swine. A second compartment was not observed in humans but in
swine, the half-life was longer than 24 hours. Dogs did not acetylate
the drug or the metabolite was de-acetylated so rapidly that a serum
concentration was not observed.

Distribution of sulfisoxazole from the central to the peripheral
compartment was fastest in humans (26) followed by swine and slowest in
dogs. Distribution from the peripheral to the central compartment was
also fastest in humans (26), followed by dogs, and slowest in swine. The
elimination rate constant was fastest in swine, followed by humans, and

Slowest in dogs. More sulfisoxazole was available for excretion from

-103-
-104-

the central compartment in humans (26) than in dogs and the least in
swine. The volume of distribution in the first compartment was similar
in dogs and swine which was greater than the calculated value in humans
(26). The volume of distribution in the second compartment was 3 times
greater in swine than in dogs.

The bioavailability was higher in humans than in dogs. In swine,
the bioavailability was one-fourth of the human value or one-third the
value in dogs. Swine are physiologically similar to humans, especially
the gastrointestinal tract; however, from this trial, the amount of
sulfisoxazole available in swine was much less than in humans and sul-
fisoxazole undergoes enterohepatic recirculation in humans but not in
swine or dogs. The total sulfisoxazole excreted in the urine as a percent
of the dose was less in swine, followed by dogs, and reported as the
highest in humans (26).

The fraction of sulfisoxazole bound to plasma proteins was less than
30% in 2 dogs and 4 pigs after intravenous administration of the drug and
in all 6 humans after oral administration of sulfisoxazole during the
initial part of the trial. This result suggests that the fraction bound
is capacity limited due to the initially high serum concentrations and
is supported by the in vitro binding data. During the remainder of the
sampling period, the fraction bound was 30 to 50% in all subjects after
intravenous and oral administration except in dogs after the 44 hour
period when the fraction bound was 70 to 90% after oral administration
of sulfisoxazole.

There was a significant (p < .01) increase in total and conjugated
bilirubin in dogs after intravenous and oral administration of sulfisoxa-~

zole. The potentially toxic, indirect or free bilirubin was significantly
~-105-

(p < .01) increased in dogs only after oral administration of sulfisoxa-
zole.

In swine, there was an increase in total and conjugated bilirubin
only after oral administration of sulfisoxazole. The potentially toxic,
indirect bilirubin was not increased by either intravenous or oral
treatment.

There was no significant increase in total, conjugated, or indirect
bilirubin in humans after oral administration of sulfisoxazole due to
1) the low therapeutic dose used of approximately 30 mg/kg compared to
the animal dosage of 100 mg/kg, 2) the ability of humans to reduce the
in vivo effects on the hepato-biliary system, or 3) the sampling period
was too short and additional samples should have been taken over a longer
trial period.

There was a significant correlation (p < .91) of total and con-
jugated bilirubin in dogs after oral and intravenous administration and
in swine after oral administration of sulfisoxazole. This evidence
supports a hypothesis that sulfisoxazole is hemolytic and increases the
breakdown of heme to bilirubin. Glucuronidation could increase to com-
pensate for the increased bilirubin in order to prevent an increase in
indirect bilirubin. In the dog, a significant negative correlation
between indirect and conjugated bilirubin after intravenous and oral
administration supports the reduction of potentially toxic, free bilirubin
by glucuronidation.

From the observed changes in bilirubin and the significant correla-
tion (p < .01), it appears that dogs are more susceptible to possible
toxicity by indirect bilirubin only after oral administration of sulfisoxa-

zole. A treatment difference also occurs in swine since total and
~106-

conjugated bilirubin are increased only after oral administration of
sulfisoxazole.

From this trial, the three species studied are not pharmacokinetically
similar with respect to sulfisoxazole. The only pharmacokinetic parameters
which were similar were the initial total serum concentration of "free"
sulfisoxazole and the fraction bound to plasma proteins. The half-lives
of "free" sulfisoxazole and its acetyl (n*) metabolite, the distribution
constants, the volume of distribution, the bioavailability, and the
urinary excretion percentage were different. However, dogs appear to
have serum concentrations, distribution constants, volumes of distribu~
tion, bioavailability, and excretion rates more similar to humans than
swine. There was a difference in the physiological affects on the
hepato-biliary system due to the treatment and species differences.

Dogs could be more susceptible to the toxic effects of indirect bilirubin
after oral administration possibly due to the "first-pass" effect. The
lack of toxicity in swine and humans could be due to the metabolism of
sulfisoxazole to the acetyl cn’) derivative and the shorter half-life

of "free" sulfisoxazole in swine.

The increase in potentially toxic free and indirect bilirubin in
dogs could be due to the slower distribution of "free" sulfisoxazole from
the central to the peripheral compartment. This allows free sulfisoxazole
to remain in the central, highly-perfused compartment for a long time
period and exert its toxic effect on the hepato-biliary system. There
also appears to be a route of administration effect with the oral route
affecting the hepato-biliary system more than the intravenous route.

If dogs have pharmacokinetic profiles of sulfisoxazole similar

to humans and are more susceptible to the toxic effects of indirect
-107-

bilirubin, then dogs are a better model and should be used in additional

research to explain the toxicity of bilirubin.
APPENDIX
Table 21. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
concentrations of sulfisoxazole in dog 1 after intravenous and oral
administration of sulfisoxazole.



Serum
Total Conjugated indirect “Free! Acetyl- Total
Time Albumia Billrubin Bilirubin Bilirubin Sulflsoxazole Sulfisoxazole Sulfisoxazole
(hours) (gin/dt) (mg/dl) (ing /d1) (mg/dl) (ug/ml) (ug/ml) (ug/ml)
Intravenous: *
0.0 3.23 0.16 0.02 0.14 -- -- --
0.5 1.09 0.20 9,00 0.20 237.95 -- 333.13
1.0 2.96 0.14 0,02 0.10 221.90 -- 315.2)
2.0 2.90 0.14 0.02 0.12 163.27 - 228,58
3.0 3.03 0.16 0.00 0.16 148.97 -- 202.27
4.5 3.06 0.40 0,30 0.10 114.65 -- 167.78
6.0 3.10 0.34 0.00 0.34 106.36 -- 159.21
9.0 3.18 0.28 0.26 0.02 57.90 -- 81.06
12.0 3.11 0,28 0.20 0.08 36.55 “- 51.17
22.0 3.05 0.24 0.14 0.10 7,25 -- 11.47
33.0 3,08 0,32 0.24 0.08 3.90 ~~ 5.16
46.0 3.00 0.24 0,24 0.00 1.61 -- 2.09
56.0 3.04 0.34 0.32 0.02 0.78 -- 1.25
72.0 3.13 0,20 0.10 0.10 0.39 -- 0.55
Oral:
0.0 3.08 0.08 0.00 0.08 -- -- ~--
1.0 2.96 0.10 0.04 0.06 165.00 ~~ 205.35
2.0 2.95 0.02 0.00 0.02 161.16 -- 197.80
4.0 3.15 0,10 0,06 0.04 108.71 -- 119.11
6.0 3.18 0.16 0,06 0.10 42.98 -- 90.51
&.0 2.77 0.18 0.18 0.00 42.27 -- 88.45
10.0 3.22 0,18 0.14 0,04 30.13 -- 47.51
12.0 3.07 0.20 0.18 0.02 21.73 -~ 34.06
14.0 3.00 0.12 0.08 0.04 15.47 -- 27.38
23.0 3.03 0.04 0,00 0.04 8.09 -- 16.52
32.0 2.98 0.18 0.12 0.06 1.50 -- 3.78
44.0 3.01 0.18 0.10 0.08 1.04 -- 3.12
56.0 3.14 0.10 0.02 0,08 0.32 -- 2.05
76.0 3.04 0.52 0.18 0,34 0.11 -- 1.64
96.0 2.96 0.34 0,08 0.26 0.20 -- ‘ 1.01

*Dose based on 20.8 kg body welght.

‘Dose based on 20.6 kg body weight.
Table 21. continued



Urine
"Pree" Acetyl- Total Total
Time Sulfisoxazole Sulfisoxazole Sulfisoxazole Drug in Urine
(hours) (ug/ml) (ug/ml) (g/ml) (ug)
intravenous:
0.0 -- ~~ -- --
0.5 =~ -- -- --
1.0 -- -- -- --
2.0 -- -- -- --
3.0 -- -- -- -~
4.5 -- -- -- --
6.0 - -- -- --
9.0 -— -- -- --
12.0 -— -- -- --
22.0 -- -- alae =~
33.0 929,24 ~- 929,24 929.24
46.0 -- -~ -- --
56.0 17.62 -- 17.62 946.86
72.0 1.60 -- 1.60 948.46
Oral:
0.0 -~ -- -- --
1.0 -- -- -- --
2.0 -- -- -- --
4.0 -- -- -- ~~
6.0 391.40 -- 391.40 391.40
8.0 -- -- ~-- --
10.0 -- -- -- ‘ --
12.0 -- -- -- ~-
14.0 59.83 ~~ 59.83 451.23
23.0 - ~~ -- ~-
32.0 5.66 -- 5.66 456.89
44.0 -- -- -- --
56.0 3.57 -- 3.57 460.46
76.0 5.00 ~~ 5.00 465.46

-QTT-


Table 22. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
concentrations of sulfisoxazole in dog 2 after intravenous and oral
administration of sulfisoxazole.



serum

Yotal Conjogated Indirect “Free” Acetyl- Total
Time Albumin Billrublin Bilirubin Bilirubin Sulfisoxazole Sulfisoxazole Sulfisoxazole
(hours) (egm/dl) (mg/dl) (mg/d 1) (mg/d1) (ug/ml) (pg/ml) (ug/ml)
Intravenous :*
0.0 2.66 0.32 0,06 0.26 -- -- --
0.5 2.48 0.24 0,00 0.24 138,27 -- 245.65
1.0 2.50 0.20 0.02 0.18 140.91 “- 179.61
2.0 2.46 0.32 0.08 0.24 121.84 -- 201.94
3.0 2.43 0.16 0,02 0.14 114,98 -- 165.22
4.5 2.67 0.46 0.24 0.22 82.40 -- 143.02
6.0 2.60 0.46 0.22 0.24 68.69 -- 173.41
9.0 2.50 0.46 0.28 0.08 60.51 -- 127.03
12.0 2.60 0.54 0,32 0.22 49.13 -~ 72.41
22.0 2.47 0.36 0.22 0.14 10.94 -- 16,05
33.0 2.60 0.42 0.24 0.18 2.83 -- 3.92
46.0 2.62 0.30 0.16 0.14 0.97 -- 2.04
56,0 2.72 0. 38 0.12 0.26 1,37 -- 1.80
72.0 2.66 0.24 0.04 0.20 1.00 -- 1,60
oral:*
0.0 2.86 0.16 0.04 0.12 -- -- --
1.0 2.62 0.16 0.04 0.12 142,50 -- 229.22
2.0 2.65 0.16 0-04 0.1 117.31 -— 237.35
4.0 a --t = = 99.84 = 149.76
6.0 2.98 0.46 0,42 0.04 88.21 -- 135.77
8.0 2.78 0.38 0.28 0.10 60.88 -- 121.29
10.0 2.75 0.36 0.32 0.04 27.06 -- 58.00
12.0 2.72 0.30 0.30 0.00 23.42 -- 43.43
14.0 2.65 0,22 0.20 0.02 14.22 =~ 26.18
23.0 2.67 0.38 0.36 0.02 5.84 -- 13.45
32.0 2.77 0.10 0.06 0,04 1,68 -- 3.35
44.0 2.70 0.20 0.02 0.18 0.44 -- 3.51
56.0 2.79 0.10 0.02 0.08 0.43 -- 4.19
76.0 2.68 0.54 0.02 0,52 0.30 — ~- 3.96
96.0 2.78 0.40 0.08 0.32 0.30 -- 3.71

*Pose based on 18.0 kg body weight.
tose based on 19,0 kg body weight.

to valoe recorded due to excessive hemolysis of sample.

~TTt-
Table 22.



Time
(hours)

[ntravenous:

eoosoosooUscocOoUNS

Oral:

- st
oc

ae —-—-———

Re i
WErnoemearn

continued
Urine
"Free" Acetyl- Total Total
Sul fisoxazole Sulfisoxazole Sulflsoxazole Drug in Urine
(ug/ml) (ig/ml) (ug/ml) (ug)
717.40 ~~ 717.40 717.40
25.66 -- 25.66 743.06
2.90 -+ 2.90 745.96 \
2.07 -- 2.07 748.03 te
hk
to
t
219.98 -- 219,98 219.98
41.87 -- 41.87 261,85
2.48 -- 2.48 ° 264.33
1.40 -- 1.40 265.73

0.95 -- 0.95 266.68
Table 23. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
concentrations of sulfisoxazole in dog 3 after intravenous and oral
administration of sulfisoxazole.

Serum
Total Conjugated Indirece "Free" Acetyl- Total
Time Albumin Billrubin Bilirubin Billrubin Sulf}soxazole Sulffsoxazole Sul f lsoxazole
(hours) (gm/d1) (mg/d)) (mg/dl) (mg/d1) (ug/ml) (ug/ml) (8/01)
Int ravenous: *
0.0 1,26 0.22 0.10 0.12 -- -- =
0.5 3.20 0,26 0.14 0.12 198.35 -- 246.37
1.0 3.16 0.20 0.08 0.12 167.50 -- 214.40
2.0 3.05 0.18 0.10 0.08 160.93 -- 190,21
3.0 3.10 0.20 0.12 0.08 140.17 -- 175,32
4.5 3.36 0.38 0.30 0,08 300.73 -- 160.16
6.0 3.20 0.443 0.28 0.15 74.26 -- 118.11
9.0 2.98 0.38 0.32 0.06 48.04 -- 60.12
12.0 3.25 0.36 0,35 0.01 18.03 -- 31.68
22.0 3.05 0.24 0.16 0.08 5.01 ~- 10.23
33.0 3.12 0.14 0.14 0.00 2.97 -- 3.64
46.0 3.10 0.26 0.20 0.06 0.90 -- 1.34
56.0 3.14 0,28 0.28 0.00 0.78 -- 1.23
72.0 3.05 0.30 0.24 0.06 0.65 -- 1.18
Oral:
0.0 2.73 0.22 0.10 0.12 -~ -- --
1.0 2.66 0.22 0.12 0.10 147.25 -- 195.43
2.0 2.72 0.16 0.08 0.08 129.06 -- 179.55
4.0 2,61 0.10 0.06 0.04 92.94 -~ 150.69
6.0 2.58 0.24 0.16 0.08 85.56 -~ 133.37
8.0 3.10 0.14 0.10 0.04 68.04 -~ 90.48
10.0 2.72 0.26 0.18 0.08 41.73 -~ 72.30
12.0 2.70 0.22 0,12 0.10 38.13 -- 58.97
14.0 2.88 0.20 0.20 0.00 21.52 -- 48.39
23.0 2.81 0.16 0.08 0.08 14,19 -- 21.54
32.0 2.72 0.18 0.06 0.12 3.48 -~ 6.50
44.0 2.89 0,22 0.20 0.02 0.99 -- 2.59
56.0 3.00 0.04 0.02 0.02 0.63 -- 2.92
76.0 2.78 0.48 0.02 0.46 0.35 -- 2.02
96.0 2.80 0.34 0.08 0.26 0.49 -- 2.31

*Dose based on 20.0 kg body welght.

Tose based on 19.8 kg body weight.

-€TIt-
Table 23. continued

Urine
"free" Acetyl- Total Total
Time Sul fisoxazele Sulflsoxazole Sulfisoxazele Drug in Urine
(hours) (ug/ml) (ug/ml) Cug/m) Cig)
Intravenous:
0.0 cal a -- --
0.5 -- -~ = --
1.0 -- -- -- --
2.0 -- a -- --
3.0 -- -- -- --
4.5 -- ~- a --
6.0 -- cd -- --
9.0 -- -- =~ --
12.0 -- -- -- --
22.0 762.80 -- 762.80 762.80
33.0 25.30 -- 25.30 788.10
46,0 -- -- -- --
56.0 3.74 -- 3.74 791.84 I
72. 1.90 -- 1.90 793.74 i
&
Oral: !
0.0 -- -- -- --
1.0 -- -- -- --
2.0 -- -~ -- --
4.0 -- -- -- --
6.0 -- -- -- --
8.0 -- -- -- --
10.0 -- -- -- ~-
12.0 -- -- -- --
14.0 822.25 -- 822.25 822.25
23.0 — -_—— -- --
32.0 31.03 -- 31.03 853.28
44.0 -- -- -- ae
56.0 3.17 -- 3.17 856.45
76.0 2.19 -- 2.19 858.64
Table 24. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
concentrations of sulfisoxazole in dog 4 after intravenous and oral
administration of sulfisoxazole.









Serum
Total Conjugated indirect "Pree" Acetyl- Total
Time Albunin Bilirubin Bilirubin Bilirubin Sulfisoxazole Sulfisoxazole Sulfisoxazole
(hours) (gm/d1) (ug/d1) (mg/dl) (mg/dl) (ye /ml) (ye@/ml ) Cyeg/ml)
int cavenous: *
0.0 3.11 0.16 0.06 0.10 -- -- -~
0.5 2.81 0.12 0.04 0.08 228.57 -- 269.52
1.0 2.89 0.10 0.08 0.02 157.50 -- 245.25
2.0 2.85 0.06 0.02 0.04 151.57 -- 238.15
3.0 2.96 0.16 0.10 0.06 141.05 -~ 211.69
4.5 3.05 0.20 0.14 0.06 110.93 -- 146.86
6.0 3.10 0.24 0.24 0.00 78.11 -- 122.28
9.0 3.03 0.28 0.24 0.04 37.70 -- 60.08
12.0 3.05 0.28 0.26 0.02 22.19 -- 31.99
21.0 2.96 0.18 0.14 0.04 7.09 -- 9.55
32.0 2.83 0.24 0.10 0.14 1.75 -- 2.73
47.0 3.20 0.24 0.22 0.02 1.30 -- 1.94
56.0 2.94 0.28 Q.24 0.04 1.08 -- 1.60
72.0 2.99 0.28 0.22 0.06 0.49 -- 0.83
Oral: *
0.0 3.16 0,22 0.10 0.12 -- a -~
1.0 2.95 0.12 0.06 0.06 322.25 a 152.78
2.9 2.93 0.18 0.08 0.10 108.19 ~~ 130.45
4.0 3.13 0.18 0.10 0.08 73.97 -- 82.50
6.0 3.15 0.20 0.12 0.08 61.92 ~- 85.08
8.0 3.07 0.30 0.28 0.02 30.68 -- 35.26
10.0 3.15 0.24 0.14 0.10 23.22 -- 38.08
12.0 3.03 0,30 0.18 0.12 14.89 ~- 20.90
14.0 2.98 0.24 0.16 0.08 9.92 -- 10.16
23.0 3.12 0.34 0.22 0,12 4.18 -- 8.00
32.0 3.09 0,28 0.10 0.18 1.73 -- 4.61
47.0 3.00 0.22 0.14 0.08 1.03 -~ 3.84
56.0 3.03 0.36 0.28 0.08 1,39 -- 3.59
76.0 3.16 0.18 0.06 0.12 0.85 -- 2.17
96.0 3.11 0.22 0.10 0.12 0.2) -- 1.49

xDose based on 21.8 kg body weight.

those based on 22.3 kg body weight.

-STI-
Table

Time
(hours)

24. continued

Intravenous:

0.0

NORE WNHE OS
ooomocou

Or

~_

Sm WNre ee
SMW Kr OARDSNK DD
oocoooooceccnae:

wn wu
naAD
oof

Urine
“Free” Acetyl]~- Total Total
Sulfisoxazole Sulfisoxazole Sulf Lsoxazole Drug in Urine
(ug/ml) (ug/ml) Cug/in} ) (ug)
919.76 -- 919.76 919.76
1.24 -- 1.24 921.00
4.60 -- 4.60 925.60 I
= he
4.40 4.40 930.00 2
oN
t
929.10 -- 929.10 929.10
0.83 -~ 0.83 | 929.93
2.40 -- 2.40 932.33
Table 25. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
coucentrations of sulfisoxazole in dog 5 after intravenous and oral
administration of sulfisoxazole.





Serum
Total Conjogated Indirect "Free" Acetyl- Total
Time Atbumin Blilrubin Bilirebdin Billrubin Sulfisoxazole Sult Lrosazole Sulfisoxazole
(hours) (gin/d1) (mg/d) (my /d1) (mg/dl) (3 /mt) (e/a) Cpe/ml )
Intravenous: *
0.0 2.84 0.20 0,06 0.14 -- -- ~-
0.5 2.64 0.10 0.04 0.06 139.18 -- 194.79
1.0 2.66 0.10 0.08 0.02 104. 32 -~ 199.34
2.0 2.76 0.20 0.08 0.12 99.78 -- 179.55
3.0 2.78 0.24 0.10 0.14 94.13 -- 132.70
4.5 3.02 0.22 0.10 0.12 85.20 -- 146.55
6.0 2.77 0.34 0.24 0.10 53.47 -- 99.10
9.0 3.27 0.38 0.28 0.10 26.34 -- 52.61
12.0 2.82 0.46 0.42 0.04 12.33 -- 22.38
23.0 2.75 0.36 0.20 0.16 4.77 -- 8.10
32.0 3.20 0.30 0.18 0.12 2.92 -- 4.33
47.0 2.82 0,22 0.10 0.12 0.99 -- 1.38
56.0 2.74 0.20 0.20 0.00 0.87 -- 1.21
72.0 2.80 0.26 0.20 0.06 0.52 -- 1.07
Oral :1
0.0 2.97 0.26 0.12 0.14 -- -- --
1.0 2.72 0.26 0.14 0.12 142.2) -- 179.52
2.0 2.75 0.24 0.06 0.18 123.56 -+ 155.19
4.0 2.79 0.24 0.04 0.20 94.89 -- 128.77
6.0 2.78 0.24 0.08 0.16 71.07 -- 91.95
8.0 2.77 0.36 0.18 0.18 41.00 -- 56.96
10.0 2.83 0.38 0.24 0.14 32.49 -~ 64.34
12.0 2.73 0.38 0.22 0,16 17.37 -- 35.26
£4.0 2.74 0.32 0.24 0.08 11.58 -- 21.46
23.0 2.90 0,28 0.08 0.20 5.31 -- 10.17
32.0 2.84 0.26 0.08 0.18 1.70 -- 3.49
47.0 2.87 0.24 0.14 0.10 0.42 -- 3.07
56.0 2.81 0.44 0.22 0,22 0.25 -- 2.59
76.0 2.93 0.18 0.08 0.10 0.66 -- 2.30
96.0 2.97 0.26 0.04 0.22 0.22 -- 2.32

*Dose based on 20.0 kg body weight.

those based on 17.8 kg body weight.

-LTT-
Table 25. continued

"Free"
Time Sulfisoxazole
(hours) (ug/ml)
Intravenous:
0.0 --
0.5 --
1.0 --
2.0 --
3.0 ~~
4.5 --
6.0 --
9.0 --
12.0 833.12
23.0 --
32.0 --
47.0 11.17
56.0 --
72,0 2.37
Oral:
0,0 --
1.0 --
2.0 --
4.0 --
6.0 ~-
8.0 --
10.0 --
12.0 --
14.0 --
23.0 199,17
32.0 --
47.0 29.30
56.0 --
76,0 --
96.0 4.66

Urine

Acetyl-

Sul fisoxazole

(ug/ml)

Total
Sul fisoxazole
(ug/ml)

Total
Drug in Urine
(ug)

-8TI-
Table 26. Serum concentrations of albumin, bilirubin, and sulifisoxazole and urinary
concentrations of sulfisoxazole in dog 6 after intravenous and oral
administration of sulfisoxazole.

Serau
Total Conjugated Indirect “Free” Acetyl- Total
Tine Albunin Bllirubdn Bilirubin Bilirubin Sulfisoxazale Sulfisoxazole Sul fisoxazole
(hours) (gu/dl) (g/d) (mp/d}) (mp /d tb) (ug /imt) Cig/m) ) (ug/m))
Intravenous: *
0.0 3.27 0,22 0.14 0.08 -- -- --
0.5 4.12 0,22 0.16 0.06 188.21 -- 236.63
1.0 4.06 0.18 0.08 0.10 157.50 -- 208.40
2.0 4.04 0.16 0.10 0.06 150.00 ~~ 171.94
3.0 3.27 0.20 0.14 0.06 135.91 -- 155.62
4.5 4,38 0.18 0.14 0.04 95.91 -- 145.23
6.0 3.24 0. 38 0.24 0.14 61.37 -- 103.12
9.0 2.88 0.38 0.32 0,06 44,20 -~ 53.0)
[2.0 3.29 0.44 0.40 0.04 14.67 -- 29.47
23.0 3.00 0.16 0.14 0.02 4.58 -- 9.68
32.0 3.09 0.18 0.14 0.04 2.84 -- 3.12
47.0 3.34 0.34 0,32 0.02 0,86 -- 1.27
56.0 3.12 0,32 0.22 0.10 0,72 “= 1.33
72.0 3.00 0.28 0,22 0.06 0.60 -- 1.15
Oral:
0.0 3.18 0.24 0.14 0,10 - -~ ==
1.0 2.87 0,18 0,06 0.12 140.04 -- 225.59
2.0 3.08 O.18 0,02 0.16 124.04 —— 196.89
4.0 3.02 0.16 0.02 0.14 95.86 -- 185.30
6.0 3.14 0.32 0.02 0.30 82.86 -- 351.42
8.0 3.22 0.34 0.20 0.14 61.1L -- 91.13
10.0 3.16 0,38 0.30 0.038 44.20 -- 64.20
12.0 3.14 0.48 0.22 0.16 24.50 -- 49.14
14.0 3.00 Q.34 0.14 0.20 17.9t -- 31.74
23.0 3.93 0.28 0.18 0.50 4.6] -- 8.7)
32.0 2.97 0,30 0,26 0.04 1.2) -- 4.26
47.0 3.22 0.46 0.40 0.06 0.89 -- 3.06
56.0 3.30 0,36 0.24 0.12 0.58 -- 3.00
76.9 3.12 0,24 0.08 0.16 0.28 -- 2.74
96.0 3.06 0,26 0.02 0.24 0.25 -- 2.09

*Dose based on 20.0 kg body weight.

those based on 21.6 kg body welght.

-6TT-
Table 26. continued

Urine
"Free" Acetyl-
Time Sulfisoxazole Sulf isoxazole
(hours) (ne/ml) (ug/ml)

Intravenous:
0.0 — -

So
wa
'

t
[

t

weno nrwn—
oooownooco
|
I
1
4

cme)



Total Total
Sulfisoxazole Drug in Urine
(ng/ml) (vg)
253.34 253,34
527.12 780.46
10.83 791.29
2.27 793.56
0.87 794.43
0.71 795.14
763.08 763.08



-0¢T-
Table 27. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
concentrations of sulfisoxazole in pig 1 after intravenous and oral
administration of sulfisoxazole.



Se runt
Total Conjugated Tndd rect "Free" Acetyl- Yotal
Tine Albums Bilirubin Bilirubin Bhlirublo sullflsoxazole Sul fisoxazole Sul Fisoxazole
(hours) (gm/dt) (ng /d1) (mg/d) ) Crip Jd) (ye /id) (4s /ind ) (ug/ml)
Intravenous:*
0.0 3.93 0.44 0.32 0.12 -~ -- --
0.5 3.74 0.30 0.20 0.10 153.20 19.08 227.38
1.0 4.77 0.24 0.10 0.14 129,62 26.52 208.06
2.0 1.86 0.18 0.10 0.08 70.88 27.14 102.74
3.0 4.65 0.18 0.06 0.12 40.90 19.04 67.21
4.5 4.90 0.24 0.24 0.00 20.00 9.80 38.53
6.0 3.91 0.16 0.00 0.16 7.09 4.97 15.05
9.0 3.96 0.24 0.08 0.16 1.23 1.61 4.71
12.0 3.88 0.28 0,22 0,06 0.56 0.29 1.51
23.0 4.77 0.20 0.00 0.20 0.36 0.35 0.82
32.0 3.78 0,22 0.08 0.14 0.80 0.85 1.87
47.0 3.89 0.20 0.08 0.12 0.66 0.73 1.79
56.0 3.71 0.32 0.12 0.20 0.14 0.16 9.85
72.0 3.92 0.78 0.42 0.36 0.05 0.21 0.59
Oral:
0.9 3.62 0.40 0.22 0.18 -- ~~ --
1.0 4.52 0.40 0. 26 0.14 43.70 9.10 68.62
2.0 3,55 0.28 0.24 0.04 31.86 12.11 70.82
4.0 3.47 0.32 0.14 0.18 11.53 5.61 34.16
6.0 4.49 0.40 0.38 0.02 4.43 1.78 8.68
8.0 3.53 0.28 0.18 0.10 2.66 1.35 6.05
10.0 3.51 0.26 0.14 0.12 1.27 0.49 2.53
12.0 1.46 0.24 0.12 0.32 1.08 0.37 2.0)
14.0 3.48 0.22 0.12 0.10 0.84 0.36 1,67
23.0 3.54 0.30 0.28 0.02 0.78 0.25 1.40
32.0 3.70 0.18 0.00 0.18 0.53 0.1) 0.98
47.9 3.71 0.18 0.00 0.18 0.49 0.26 0.88
56.0 3.83 0.14 0.00 0.14 0.43 0.47 1.05
76.9 3.77 0.12 0.02 0.10 0.13 0.38 0.78
96.0 3.70 0.22 0,00 0.22 0.20 0.42 0.80

‘Dose based on 27.2 ky body weight.

aa
"Dose based on 24.5 kg body weight.

-T¢t-


Table 27. continued
"Pree"!
Time Sulfisoxazole
(hours) (ug/ml)
Intravenous:
0.0 --
0.5 --
1.0 --
2.0 -~
5.0 397.11
4.5 ~-
6.90 --
9.0 --
12.0 --
23.0 -~
32.0 38.07
47.0 --
56.0 1.65
72,0 0.91
Oral:
0.0 --
1.0 --
2.0 1.00
4.0 --
6.0 193.15
8.0 -~
10.0 --
12.0 --
14.0 138.73
23.0 --
32.0 15.21
47.0 ~~
56.0 --
76.0 -~
96.0 2.64

Urlne

Acetyl-
Sulflsoxazole
(ug/ml)



Total Total
Sulf lsoxazole Drug in Urine
Cug/m1) (ig)
664.01 664.01
69.77 733.78
3.85 737.63 An
1.41 739.04 ho
No
'
1.60 1.60
347.55 349.15
239.31 588 .48
63.21 651.69
3.04 654.73


Table 28. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
concentrations of sulfisoxazole in pig 2 after intravenous and oral
administration of sulfisoxazole.



Serum
Total Vou jupated (ndirect "Pree" Acetyi- Total
‘Time Albumin Bilirubin Bilirubin Bilirubin Sul fisoxazole Sulfisoxazole Sulfisoxazole
Chours) (pit/d 1) (ng /dl) (my/d1) (ng/dl) (p/m) ) (ng/ml) (ug/ml)
Lat ravenous: *
0.0 1.84 0.26 0.18 0.08 -- -- --
0.5 3.07 0.16 0.00 0.16 177.04 28.88 296.11
1.0 4.61 0.16 0.04 0.12 148.07 43.80 273.00
2.0 4.73 O.14 0.10 0,04 70.90 27.40 158,47
1.0 3.78 0.16 0.06 0.10 40.62 17.20 77.31
4.5 3.77 0.26 0.12 0.14 14.06 6.48 29.48
6.0 4.00 0.30 0.10 0.20 10,90 4.66 19.96
9.0 4.94 0,20 0.08 0.12 2.56 1.87 8.58
12.0 4.90 0.28 0.14 0.14 0.80 0.68 2.64
23.0 3.84 0.26 0.12 0.14 0.54 0.30 1.94
32.0 3.75 0.18 0,08 0.10 0.34 0.48 1.03
47.0 4.10 0.18 0.10 0.08 0.24 0.25 0.89
56.0 3.88 0.28 0.20 0.08 0.13 -- 0.40
72.0 4.22 0.44 0.22 0.22 0.13 -- 0.38
+
Oral:
0.0 4.05 0.40 0.22 0.18 -- -- --
1.0 3.46 0.32 0.16 0.16 48.04 14.05 117,09
2.0 3.59 0.34 0.14 0.20 48.74 14,14 193.76
4.0 3.54 0.30 0.28 0.02 23.73 13.67 91.15
6.0 3.45 0.38 0.30 0.08 17.90 7.71 52.56
8.0 43.64 0.26 0.12 0.14 8.53 2.50 35.62
10.0 3.58 0.28 0.08 0.20 2.97 0.89 11.50
12.0 4.41 -- -- --t 1.45 0.77 7.81
14.0 3.67 0.24 0.14 0.10 0.83 0.59 5.23
23.6 3.59 0. 32 0.22 0.10 0.54 0.40 3.88
32.0 3.83 0.18 0.14 0.04 0.87 0.59 2.38
47.0 4.71 0.18 0.00 0.18 0.25 0.19 1.21
546.0 4.00 0.26 0.12 0.14 0.09 Q.221 0.90
76.0 3.06 0.12 0.00 0.12 0.17 0.32 0.73
96.0 3.96 0.12 0.08 0.04 0.10 0.08 0.51

*Pose based on 22.9 ky body weight.
Dose based on 19.5 kg bady weight.

tho value recorded due to excessive hemolysis of sample.

-€?@Tt-
Table 28.

Tirw
(bours)

Intravenous:
0.0

NAN MWNHUODHEwWNHK—O
odvooocormvcoow

NOP WN —

Oral

we
Omar fSnrNrosd
oDdvooconco:.

-
nN

14.0
23.0
32.0
47.0
56.0
76.0
96.0



continued

Urine

"Free" Acetyl~ Total Total
Sul flsoxazole Sulfisoxazole Sulfisoxazole Drug in Urine
(ug/ml) (ug/ml) (ug/ml) (ug)

*No urine level was recorded after 10.0 hours due to a problem In collection.

-77T-
Table 29. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
concentrations of sulfisoxazole in pig 3 after intravenous and oral
administration of sulfisoxazole.





Seruo
Total Conjugated Indirect “Kree” Acetyl- Yotal
Time Albuniu Bhlirubiu Billrabin Biidrabian Sulflsoxazole Saltisoxazole Sulfisoxazole
(hours) (gm/al) (ing /d1) Gug/dl) (mg/d) Gug/ml ) (g/ml) (ug/ml)
Intravenous: *
0.0 3.89 0.30 0.10 0,20 -- -~ -~
0.5 3.72 0.26 0.18 0.08 222.64 33.95 288.93
1.0 3.62 0.30 0.10 0,20 180.16 40,63 256.06
2.0 3.70 0.18 0.14 0.04 102.27 43.33 188.92
4.0 3.66 0.22 O.14 0,08 61.24 40.88 125.29
4.5 3.74 0.28 0.14 0.14 20.19 13.58 63.53
6.0 3.74 0.32 0.26 0.06 7.60 5.30 20.90
9.0 3.68 0.28 0.10 0.18 1.93 1.12 6.69
12. 3.81 0.22 0.18 0.04 0.90 0.30 2.75
24.0 4,01 0.16 0.00 0.16 0.45 0.20 1.71
32.0 1.80 0.20 0.06 0.14 0.14 0.13 0.85
47.0 3.5) 0.16 0.08 0.08 0.14 -— 0.53
56.0 3.49 0.18 0.04 0.14 0.2) -~ 0.40
72.0 4.66 0.22 0.12 0,10 0.21 -~ 0.31
Oral: !
0.0 3.87 0.36 0.26 0.10 -- -- --
1.0) 3.65 0.28 0.20 0.08 73.48 16,38 127.50
2.0 3.49 0.40 0.26 0.04 56.11 17,36 102.63
4.0 3.57 0.28 0.08 0.20 70.03 18.70 135.87
6.0 3.8) 0.44 0,22 0.22 18.87 7.77 48.97
8.0 3.75 0.38 0,32 0.06 12.98 5.64 19.42
10.0 3.63 0.20 0.14 0.06 5.97 3.48 16.72
12.0 3.84 --f --f --4 1.82 0.78 3.98
14.0 ).81 0,30 0.16 0.14 1.17 0.47 2.54
23.0 3.76 0,30 0,14 0.16 0.63 0.09 1.74
32.0 3.78 0.32 0.12 0,20 0.30 0,26 1.0)
47.0 3.B3 0.34 0.10 0.24 0.211 0.48 0.94
56.0 4.04 0.24 0.02 0.22 0.19 0.39 0.88
76.0 3.93 0.12 0.00 0.12 0.15 0.116 0.79
96.0 3.88 0.24 0.14 0.10 0,25 0.44 0.78

*Dose based on 25.6 kg body welght.

1
Dose based on 19.5 kg body weight.
4

*No value recorded due to excessive hemolysis of sample.

~SéT-
Table 29. continued



"Tree!
Time Sulfjsoxazole
(hours) (ve /m1)
Intravenous:
0.0 --
0.5 --
1.0 a
2.0 --
3.0 242.43
4.5 --
6,0 --
9.0 --
12.0 75.07
23.0 -
32.0 4.15
47.0 --
54.0 0.79
72.0 0.96
Oral:
0.0 --
1.0 --
2.0 --
4.0 --
6.0 107,30
8.0 --
10.0 --
12.0 157.47
14.0 -~
23.0 --
32.0 --
47.0 --
56.0 9.97
76.0 --
96.0 4.22

Urine
Acetyl- Total
Sulfisoxazole Sulf Lsoxazole

(ug/ml) Gig/mL)

71.30 146.37
6,20 10.35
0.80 1.59
0,60 1,56
69.60 176.90
98.40 255.87
6.60 16.57

Total
Drug in Urine
(ug)

176.90

432.77

-9CT-
Table 30. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
concentrations of sulfisoxazole in pig 4 after intravenous and oral
administration of sulfisoxazole.





Serum
Total Conjugated Indirect "Free" Acetyl- Total
Time Albuorin Bilirubin Bilirubla Bilirubin Sulf lsoxazole Sulfisoxazole Sulf isoxazole
(hours (um/d1) (mg/d) ) (imy/d1) (mg /db) (ug/ml) (ug/ml) (ug/ml)
Intravenous :*
0.0 3.73 ). 36 0.18 0.18 -~ a ~~
0.5 3.60 0,34 0.20 0.14 212.81 27.46 261.64
1.0 3.52 0.40 0.28 0.12 170.69 32.61 219.02
2.0 3.48 0.32 0.08 0.24 82.93 30.41 142,08
3.0 3.40 0.26 0.12 0.14 56.89 24 62 100.58
4.5 3.64 0.26 0.10 0.16 20.87 10.17 49.78
6.0 3.67 0.38 0.14 0.24 9.52 4.03 15.7]
9.0 4.53 0.26 0.10 0.16 2.79 1.50 5.87
12.0 3.61 0.30 0.18 0.12 1.00 0.59 2.95
23.0 4.91 0.20 0.18 0.02 0.45 0.35 1.71
42.0 3.46 0.24 0.16 0.08 0.40 0,22 1.50
47.0 3.61 0,28 0.18 0.10 0.2L 0.05 0.80
56.0 4,66 0.30 0,28 0.02 0.22 0.04 0.50
72.0 3.55 0. 32 0.14 0.18 0.32 0.07 0.45
Oral
0.0 4.86 0.36 0.12 0.24 -- -- --
1.0 3.73 0.34 0,22 0.12 91.34 16.68 167.64
2.0 3.62 0.34 0.24 0.10 80,38 20.02 155.82
4.0 3.67 0.34 0.26 0.08 40.72 13.76 86.50
6.0 3.66 0.68 0.42 0,26 16.40 6.26 47.50
8.0 3.471 0.38 0.26 0.12 4.82 2.04 32.42
10.0 4.86 0,36 0.20 0.16 2.22 0.86 12.08
12.0 4.01 0.40 0,28 0.12 1,24 1.92 6.85
14.0 3.80 0.40 0.24 0.16 0.88 1,86 4.87
23.0 3.60 0.16 0.18 0.18 0.54 0.40 3.90
32.0 4.89 0.30 0.18 0.12 0.52 Q. 36 2.07
47.0 3.48 0.32 0.14 0.18 0.36 0.38 1.29
56,0 3.93 0.20 0.06 0.14 0,32 0.30 0.94
76.0 3.90 0.22 0.02 0,20 0.37 0.39 0.86
96.0 4.88 0.30 0,00 0.30 0.26 0.50 0.80

4Dose based on 24.0 ky body weight.

Those based on 20.6 ky body weight.

-LET-






Table 30. continued
Urine
"Â¥reeâ„¢ Acetyl- Total Total
Time Sul fisoxazole Sulfisoxazole Sul fisoxazole Drug In Urine
(hours) (ug/ml) (ug/m1) (ug/m1) (ug)
Intravenous:
0.0 -- -- -- --
0.5 -- -- -- --
1.0 ~~ - -- a
2.0 -- -- -~ ~~
3.0 -- -- -- a
4.5 -- -- -- --
6.0 ~~ -- -- --
9.0 -- ies -- --
12.0 300.00 169,00 469.00 469.00
23.0 -- a -- --
32.0 0.78 0.40 1.18 470.18
47,0 a -- -- --
56.0 2.48 1.10 3.58 473.76 a
72,0 1.36 1.50 2.86 476.62 nN
?
Oral:
9.0 -- -- -- --
1.0 -- -- ~-- --
2.0 -- -- -- --
4.0 -- -- -~ ~~
6.0 3.74 3.20 6.94 6.94
8.0 -- -~ a --
10.0 100.90 57.60 158.50 165.44
12.0 -- -- -- --
14.0 268.30 198.60 466.90 632.34
23.0 -- -- ~- --
32.0 14.65 8.10 22.75 655.09
47.0 -- -- -— --
56.0 2.55 1.90 4.45 659.54
76.0 -- ~~ -- --
96.0 1.52 1.20 2.72 662,26
fable 31. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
concentrations of sulfisoxazole in pig 5 after intravenous and oral
administration of sulfisoxazole.



Yotal Conjugated Indl rect "Bree" Acetyl- Total
Time Albumin Wilirabin Ritlerubla Bilirubin Sulfisoxazole SulFisoxazote Sulfisoxazole
(hours) Cam/dl) Gays /dd) Gog /dl) (ng /d1) (yg /int) Cug/inl ) (ny/ml )
Int mavenous:*
0.0 3.49 0,30 OG 0.16 -- -- --
0.5 3.42 0.50 0.38 0.12 222.36 31.25 277.47
1.0 3.52 0,56 O.44 0.22 160.26 39.30 228.51
2.0 3.494 0.60 0.44 0.16 94.85 34.16 156.21
400 3.4) 0. 16 0.20 0.16 59.07 2983 305.77
4.5 3.43 --t -~" --1 24.13 13.05 50.89
6.0 3.01 0.36 0,22 0.14 11.04 6.49 28.17
9.0 4.43 0.40 0.26 0.14 2.58 L.26 12.30
12.0 3.45 0.64 0.36 0.28 1.19 0.48 1.95
23.0 3.63 0.60 0.34 0.26 0.61 0.37 1.18
32.0 3.56 0.22 0.12 0,10 0,38 0.08 0.94
47.0 3.61 0.24 0.12 0.12 0.36 0.23 0.83
56.0 3.57 0.24 0.32 0.12 0.14 -- 0.50
72,0 3.56 0.28 0.16 0.12 0.22 -- 0.40
Oral: |
0.0 3.45 0.16 0.20 0.16 -- -- --
1.0 3.23 a. 30 0.20 0.10 32.77 9.36 72.76
2.0 1.30 0.29 0.18 0.10 34.14 13.54 53.53
4.0 3.25 0.48 0.24 0.24 15.85 8.54 38.37
6.0 3.32 0.86 0.50 0.36 6.17 4.05 16.48
8.0 3.28 0.08 0.46 0.22 2.27 1.51 6.35
10.0 3.56 0. 36 0.20 0.16 1.26 0.78 2.98
12.0 3.54 0.40 0.26 0.14 0.74 0.28 1.58
14.0 4.38 O.34 0.20 0.14 0.55 0.37 1.26
241.0 3.35 0.32 0.20 0.12 0.46 0.17 0.98
32.0 4.55 0.38 0.24 0.14 0.25 0.15 0.73
47.0 4.48 0.32 0.14 0.18 0.64 0.31 1.
56.0 3.51 0.22 0.18 0.04 0.37 0.19 0.84
76.0 3.52 0,22 0,02 0,20 0.65 0.31 1.28
96.0 3.60 0.28 0.06 0,22 0.11 0.20 0.45



*Dose based on 20.9 kg body weight.
+
‘Dose based ou 16.3 ky body weight.

tNo value recorded due to excessive hemolysis of sample.

~6¢2T-




Table 31. continued
Urine
"Free" Acetyl- Total Total
Time Sulfisoxazole Sul fisoxazole Sulfisoxazole Drug in Urine
(hours) (ug/m1) (ug/ml) (ug/ml) (ug)
intravenous:
0.0 -- -- -- --
0.5 -~ -- -- --
1.0 -- = -- --
2.0 -~ -- -- a
3.0 6.42 3.40 9.82 9.82
4.5 “= -- -~- =~
6.0 -- -- -- -~
9.0 -- — “— -
12.0 107.70 31.00 138.70 148.52
23.0 -- -- -- --
32.0 156,94 185.90 342.84 491,36
47.0 -~ -- -- --
56,0 2.46 1.90 4.36 495.72
72.0 3.05 1.90 4.95 500.67
Oral:
0.0 =~ -- -- --
1.0 -- -- -- --
2.0 -~ -- -- --
4.0 -~ -- -- --
6.0 194.79 136.60 331.39 331.39
8.0 -- -- -- --
10.0 -- -- -- --
12.0 -- -- -- --
14.0 -- -- -- --
23.0 -- ~- ~- ~~
32.0 175.94 70.40 246.34 577.73
47.0 -- a -- -—
56.0 2.65 1.60 4.25 581.98
76.0 -- -- -- --
96.0 1.66 1.60 3.06 585.04



-O€T-
Table 32. Serum concentrations of albumin, bilirubin, and sulfisoxazole and urinary
concentrations of sulfisoxazole in pig 6 after intravenous and oral
administration of sulfisoxazole.

fe ram



Total Con piagated Indirect “Free” Acetyl- Total
Time Al ful Bilirubin Bilirubin Billvabin Sulf isoxazole Sulflsoxazole Sulfisoxazele
(hours) (gm/dt) (my /dt) Ging /d1) (mp /d it) Gig f/m) Cag /int ) Cays/mt )
lnt ravenous: *
0.0 4.78 0.40 0.24 0.06 -- a --
0,5 4.53 0.28 0.16 0,12 171.98 19.88 207.87
1.0 3.45 0.16 0.12 0.04 125,50 22.73 199.40
2.0 3.55 0,24 0,12 0.12 83.55 19.93 129.52
4.0 3.53 --{ --| -4 56.45 15,50 84,29
4.5 3.49 0.20 0,08 0,12 30,26 , 8.26 50.41
6.0 3.50 0.20 0,20 0,00 14.30 4,50 20,59
9.0 3.61 0.30 0.08 0,22 $.60 1.76 7.05
i2.0 3.62 0.48 0.22 OL 16 0,89 0.60 2.03
23.0 3.81 0.56 0.42 0.14 0.25 0.33 0.89
42.0 4.59 0.26 0.12 O.14 0,18 0.31 0.81
47.0 3.51 0,42 0.22 0.20 0. $0 0.24 0.71
56.0 4.55 O.14 0.08 0.06 0.26 0.31 0.70
72.0 3.34 0.24 0,08 0,16 0,19 0.14 0.50
Oral: |
0.0 4.55 0.32 O14 0.18 -- -- -~
1.0 4.55 0.28 0.26 0,02 100.64 18,30 202,70
2.0 3.57 0.42 0,22 0.10 58.13 19.11 100.98
4.0 3.43 0.30 0.28 0,02 22.96 7,84 52.83
6.0 3.61 0.36 0.22 0.14 8.30 3.28 20.43
8.0 4.70 0.48 0.32 0.16 1.14 0.42 4.42
10,0 3.77 0, 48 0,38 0.00 2.26 0.85 7.58
12.0 4.61 0.26 0.24 0.02 0.46 0.20 1.37
14.0 3.67 0.38 0.20 0.18 0.49 0.15 1.35
23.0 3.64 0.44 0.18 0.16 0.17 0.14 0,72
32.0 4.68 0,20 0.10 0.10 0.17 0.28 0.80
47.0 3.66 0.18 0.06 0.32 oO.td 0.15 0.62
56.0 3.82 0.26 0.12 O14 0.19 0.23 0.79
76.0 3.74 0.16 0.14 0.02 0.20 -- 0.45
96.0 4.55 0.30 0.12 0.18 0.12 0.17 0.40

*Dase based on 212.3 kg body welght.
"hose based on 20.2 kg body weipht.

to value recorded due to excessive hemolysis.

~TEt-


Table 32. continued
Urine
"Free" Acetyl-
Vime Sullisoxazole Salfisoxazole
(hours) (ig/ml ) (ug/ml)
{utravenouss
0.0 -- =
9.5 -- --
1.0 -- --
2.0 -- --
3.0 1.31 0.60
4.5 -- --
6.0 -- --
9.0 -- --
12,0 312.67 338.40
23.0 -- --
32.0 13.07 31.60
47.0 -- --
56.0 ~~ --
72.0 1.01 1.20
Oral:
0.0 -- a
1.0 -- --
2.0 -- --
4.0 -- --
6.0 202.18 100.70
8.0 -- a
10,0 -- --
12.0 -- --
14.0 -- “+
23.0 155,65 71.50
32.0 =~ --
47.0 -- --
56.0 16.56 28.60
76.0 -- --
96.0 0.50 0.40



Total Total
Sulfisoxazole Drug in Urine
Cug/mL) (ig)
1.9) 1.91
651.07 652.98
34.67 687.65
~ ~ ‘
2.21 689.86 lo
Nh
I
302.88 302.88
227.15 530.03
45.16 575.19




Table 33. Serum concentrations of albumin, bilirubin, and sulfisoxazole in human 1 after oral administration
of sulfisoxazole.*

Serum
Total Conjugated Indirect "Free" Acetyl- Total

Time Albumin Bilirubin Bilirubin Bilirubin Sulfisoxazole Sulfisoxazole Sulfisoxazole

(hours) (gm/d1) (mg/d1) (mg/d1) (mg/dl) (ug/ml) (ug/ml) (ug/ml)

0.0 4.86 0.60 0.46 0.14 -- -- --

1.0 4.85 0.56 0.46 0.10 177.25 10.91 229.74

2.0 4.89 0.62 0.50 0.12 161.72 20.73 228.52 bo
Ww
uo

4.0 4.87 0.60 0.46 0.14 155.04 31.23 218.14

6.0 4.96 0.64 0.54 0.10 120.25 29.73 196.17

8.9 4.90 0.60 0.52 0.08 100.33 25.10 171.43

*Body weight of 79.5 kg.




Table 34. Serum concentrations of albumin, bilirubin, and sulfisoxazole in human 2 after oral administration
of sulfisoxazole.*

Serum
Total Conjugated Indirect "Free" Acetyl- Total
Time Albumin Bilirubin Bilirubin Bilirubin Sulfisoxazole Sulfisoxazole Sulfisoxazole
(hours) (gm/d1) (mg/d1) (mg /d1) (mg/d1) (ug/m1) (ug/ml) (ug/ml)
0.0 4.41 0.45 0.38 0.07 -- -~ --
1.0 4.35 0.45 0.40 0.05 183.06 16.90 231.45
2.0 4.61 0.38 0.30 0.08 164.75 28.16 230.27 be
~
4.0 4.44 0.40 0.38 0.02 136.27 34.94 210.34
6.0 4.67 0.38 0.34 0.04 101.20 29.52 190.17
8.0 4.60 0.42 0.38 0.04 81.92 26.10 165.37

*Body weight of 66.8 kg.
Table 35. Serum concentrations of albumin, bilirubin, and sulfisoxazole in human 3 after oral administration
of sulfisoxazole.*

Serum
Total Conjugated Indirect "Free" Acetyl- Total
Time Albumin Bilirubin Bilirubin Bilirubin Sulfisoxazole Sulfisoxazole Sul fisoxazole
(hours) (gm/d1) (mg/d1) (mg/d1) (mg/d1) (ug/ml) (ug/ml) (ve/ml1)
0.0 4.59 0.40 0.35 0.05 -- -- --
1.0 4.50 0.40 0.36 0.04 180.74 14.19 227.91
2.0 4.71 0.36 0.30 0.06 165.24 20.65 225.54 I
ni
4.0 4.65 0.38 0.36 0.02 138.72 31.39 210.15 '
6.0 4.75 0.38 0.34 0.04 110.26 28.95 187.79
8.0 4.79 0.42 0.38 0.04 93.67 25.59 160.72

*Body weight of 70.0 kg.
Table 36. Serum concentrations of albumin, bilirubin, and sulfisoxazole in human 4 after oral administration
of sulfisoxazole.*

Serum
Total Conjugated Indirect "Free" Acetyl- Total
Time Albunin Bilirubin Bilirubin Bilirubin Sulfisoxazole Sulfisoxazole Sulfisoxazole
(hours) (gm/d1) (mg/dl) (mg/d1) (mg/dl) (ug/ml) (ug/ml) (ug/ml)
0.0 4.76 0.62 0.45 0.17 -- -- --
1.0 4.80 0.60 0.40 0.20 176.91 10.08 225.16
2.0 4.85 0.64 0.48 0.16 160.24 19.76 215.18 na
Lo
On
4.0 4.83 0.58 0.45 0.13 130.26 30.37 206.15 ‘
6.0 4.90 0.58 0.45 0.13 109.38 28.79 179.95
8.0 4.88 0.62 0.46 0.16 91.36 25.53 159.27

*Body weight of 79.0 kg.
Table 37. Serum concentrations of albumin, bilirubin, and sulfisoxazole in human 5 after oral administration
of sulfisoxazole.%*

Serum
Total Conjugated Indirect "Pree" Acetyl- Total
Time Albumin Bilirubin Bilirubin Bilirubin Sul fisoxazole Sulfisoxazole Sulfisoxazole
(hours) (gm/d1) (mg/d1) (mg/dl) (mg/d1) (ug/ml) (ug/ml) (ug/ml)
0.0 4.65 0.64 0.46 0.18 ~~ -- --
1.0 4.60 0.66 0.45 0.21 164.70 12.86 224.17
2.0 4.75 0.64 0.40 0.24 147.05 19.51 209.15 bo
lo
wd
4.0 4.70 0.56 0.38 0.18 114.50 29.73 189.57 '
6.0 4.71 0.54 0.38 0.16 98.70 28.95 170.84
8.0 4.78 0.60 0.44 0.16 88.46 25.01 150.17

*Body weight of 82.0 kg.
Table 38. Serum concentrations of albumin, bilirubin, and sulfisoxazole in human 6 after oral administration
of sulfisoxazole.*

Serum
Total Conjugated Indirect "Free" Acetyl- Total
Time Albumin Bilirubin Bilirubin Bilirubin Sulfisoxazole Sulfisoxazole Sulfisoxazole
(hours) (gm/d1) (mg/d1) (mg/d1) (mg/d1) (ug/ml) (ug/m1) (ug/ml)
0.0 4.54 0.56 0.38 0.18 a -- --
1.0 4.50 0.58 0.36 0.22 189.35 11.91 235.75
i
2.0 4.64 0.56 0.36 0.20 170.42 23.74 228.47 5
Go
1
4,0 4.64 0.54 0.38 0.16 150.18 34.17 200.19
6.0 4.60 0.58 0.34 0.24 120.81 27.65 180.54
8.0 4.60 0.60 0.40 0.20 104 .62 24.98 160.18

*Body weight of 66.0 kg.
Table 39.

Means and standard deviations* of serum albumin, total bilirubin, conjugated
bilirubin, and indirect bilirubin in 6 dogs after intravenous administration
of sulfisoxazole.



Time Albumin

(hours) (gm/d})

0.0 3.06 + .25

0.5 2.89 + .29

1.0 2.87 + .24

2.0 2.84 + .22

3.0 2.96 + 133

4.5 3.09 + .26

6.0 3.00 + .25

9.0 2.96 © .25

12.0 3.02 & .27

22.0 (23.0)' 2.86 + .33 (2.90
32.0 (33.0)! 3.04 + .19 (2.93
46.0 (47.0)! 2.90 + .25 (3.05
56.0 2.95 + .18

72.0 2.93 4.17

*Means t 1 standard deviation.

I+

I+

-13)
.29)

.20)

Total
Bllirubin
(mp/d1)
0.21 £ .05
0.19 + .07
0.15 + .05
0.18 + .09
0.19 + .03
0.31 + .125
0.37 + .08t
0.34 4 .05t
0.39 + .107
0.28 +
0.24 £
0.27 4
0.30 4 .06°
0.26 + .04

t, :
The mean of 3 observations at each time Interval.

tp

§
P

<

<

OL.

05.

.07 (0.23
-06 (0.29

-03 (0.27



+

-11)
-14)

-06)

Con jugated
Bilirubin
(gm/d1)

0.06 + .07

0.06 + .03

0.07 4 .04

0.08 + .06

0.20 + .o9t
0.20 + .107
0.28 + .o3t
0.33 + .o8t
0.17 + .04 (0.16
0.14 + .04 (0.21
0.20 + .04 (0.21
0.23 + .o7*

0.17 + .o8t

|

.03)5

.06)*
yt

Indirect
Bilirubin
(mg/d1)
0.14 + .06
0.12 4 .08
0.09 t .06
0.11 4 .07
0.11 + .05
0.10 + .06
0.47 + .12
0.06 4 .03
0.07 + .08
0.11 4 .03
0.10 + .05
0.07 + .07
0.07 4 .10
0.09 + .06

(0.07
(0.09

(0.05

-08)
.09)

.06)

-6€T-
Table 40. Means and standard deviations* of serum albumin, total albumin, conjugated
bilirubin, and indirect bilirubin in 6 dogs after oral administration of



sulfisoxazole.

Total Conjugated Indirect











Time Atbumin Bilirubin Bilirubin Billrubin
(hours) (g/dl) (mg/d] ) (ing /d1) - (mg/dd)
0.0 3,00 § .18 0.20 + .07 0.08 + .05 O.11 + .02
1.0 2.80 + .15 0.17 1 .06 0.08 + .04 0.10 + .03
2.0 2.85 4 47 0.26 + .07 0.05 + .03 0.11 + .06
4.0 2.94 & £23 0.16 + .06 0.06 + .03 0.10 + .07
6.0 2.97 4 .24 0.27 4.11 0.14 + .14 0.12 + .09
8.0 2.95 4 .20 0.28 4 .10° 0.20 4 .075 0.08 + .07
10.0 2.97 4 .23 0.30 + .o8t 0.22 + .08° 0.08 + .04
12.0 2.90 + .20 o.31 4 .10t 0.20 4 .065 0.09 £ .07
14.0 2.89 £ 17 0.24 4 .08 0.17 + .06 0.07 + .07
24.0 2.94 + .18 0.25 4 .13 0.15 + ,13 0.09 + ,06
32.0 2.90 4 .14 0.22 + .08 0.11 + .08 0.10 + .07
44.0 (47.0)" 2.87 4 .16 (3.03 4 .18) 0.20 £ .02 (0.31 + .13) 0.11 4 .09 (0.23 £ .15) 0.09 4 .08 (0.08
56.0 2,98 4.15 0.23 4 .17 0.13 2.13 0.10 4 .07
76.0 2.95 § .19 0.36 + .17 0.07 + 0.06 0.28 + vist
96.0 , 2.95 4 .13 0.30 4 .07 0.07 + .03 0.24 + .o74

*Means * 1 standard deviatlon.
t
$

i, < .05.

The mean of 3 observations at each time interval.

p< .OL.

t

-02)

-O7T-
-141-

Table 41. Means and standard deviations* of serum albumin, total bilirubin,
conjugated bilirubin, and indirect bilirubin in 6 swine after
intravenous administration of sulfisoxazole.

Total Conjugated Indirect
Time Albumin Bilirubin Bilirubin Bilirubin

(hours) (gm/d1l) (mg/d1) (mg/d1) (mg/d1)
0.0 3.78 + .16 0.33 + .06 0.19 + .08 0.13 + .06
0.5 3.61 + .12 0.31 + .11 0.19 + .12 0.12 + .03
1.0 3.58 + .11 0.30 + .16 0.16 + .12 0.14 + .06
2.0 3.61 + .19 0.28 + .17 0.16 + .14 0.11 + .08
3.0 3.57 + .15 0.24 + .08 0.12 + .06 0.12 + .03
4.5 3.66 + .18 0.25 + .03 0.14 + .06 0.11 + .06
6.0 3.74 + .19 0.29 + .09 0.15 + .09 0.13 + .09
9.0 3.69 + .22 0.28 + .07 0.12 + .07 0.16 + .03
12.0 3.71 + .18 0.35 + .15 0.22 + .08 0.13 + .09
23.0 3.83 + .13 0.33 + .20 0.18 + .17 0.15 + .08
32.0 3.66 + .14 0.22 + .03 0.10 + .04 0.12 + .03
47.0 3.71 + .24 0.25 + .10 0.13 + .06 0.12 + .04
56.0 3.64 + .14 0.24 + .07 0.14 + .09 0.10 + .08
72.0 3.71 + .31 0.38 + .21 0.19 + .12 0.19 + .09

*Means + 1 standard deviation.
~142-

Table 42. Means and standard deviations* of serum albumin, total bilirubin,
conjugated bilirugin, and indirect bilirubin in 6 swine after
oral administration of sulfisoxazole.

Total Conjugated Indirect
Time Albumin Bilirubin Bilirubin Bilirubin
(hours) (gm/d1) (mg/dl) (mg/d1) (mg/dl)
0.0 3.73 + .23 0.37 + .03 0.19 + .05 0.17 + .05
1.0 3.52 + .17 0.32 + .05 0.22 + .04 0.10 + .05
2.0 3.52 + .12 0.31 + .03 0.21 + .05 0.10 + .06
4.0 3.49 + .14 0.34 + .07 0.21 + .08 0.12 + .10
6.0 3.56 + .17 0.52 + .20 0.344.117 0.18 + .12
8.0 3.61 + .18 0.41 + .15 0.28 + .12 0.13 + .05
10.0 3.65 + .14 0.31 + .07 0.19 + .10 0.12 + .07
12.0 3.65 + .23 0.33 + .09 0.23 + .07 0.10 + .05
14.0 3.64 + .17 0.31 + .07 0.18 + .04 0.14 + .03
23.0 3.61 + .16 0.32 + .02 0.20 + .05 0.12 + .06
32.0 3.74 + .12 0.26 + .08 0.13 + .08 0.13 + .06
47.0 3.65 + .14 0.25 + .08 0.07 + .06 0.18 + .04
56.0 3.69 + .38 0.22 + .05 0.08 + .07 0.14 + .06
76.0 3.75 + .15 0.16 + .05 0.03 + .05 0.13 + .07
96.0 3.76 + .17 0.24 + .07 0.07 + .06 0.18 + .09

*Means * 1 standard deviation.

- < O01.
-143-

Table 43. Means and standard deviations* of serum albumin, total bilirubin,
conjugated bilirubin, and indirect bilirubin in 6 humans after
oral administration of sulfisoxazole.

Time Albumin

(hours) (gm/d1)
0 4,64 +
1 4.60 +
2 4.74 +
4 4.68 +
6 4.77 +
8 4.76 +

*Means ¢ 1 standard

.16
-19
-1ll
15
14

-13

Total
Bilirubin
(mg/d1)
0.55 + .10
0.54 + .10
0.53 + .13
0.51 + .10
0.52 + .11
0.54 + .10

deviation.

Conjugated
Bilirubin
(mg/d1)

0.41 + .05

0.40

I+

04

0.39

I+

.09

0.40

I+

04
0.40 + .08

0.43

i+

.O5

Indirect
Bilirubin
(mg/d1)

0.13

I+

02

0.14

(+

.08
0.14 + .07

0.11

I+

.07

0.12

i+

08

0.11

(+

.07
10.

ll.

12.

13.

BIBLIOGRAPHY

Agren, A., Elofsson, R., and Nilsson, 5.0. Some physio-chemical
factors influencing the binding of sulfonamides to human
albumin in vitro. Acta Pharmacol. Toxicol. 29:48-56 (1971).

Barr, A.J. and Goodnight, J.H. Statistical Analysis System. North
Carolina State University, Raleigh, North Carolina (1972).

Bell, P.H. and Robbin, R.D. Studies in chemotherapy, VII. A theory
of the relation of structure to activity of sulfanilamide type
compounds. J. Am. Chem. Soc. 64:2905~2917 (1942).

Bloedow, D.C. and Hayton, W.L. Saturable first pass metabolism of
sulfisoxazole N+-acetyl in rats. J. Pharm. Sci. 65:334-338
(1976).

Bloom, F. The Blood Chemistry of the Dog and Cat. Gamma Publica-
tions, New York. pp. 102-110 (1960).

Bratton, C.A. and Marshall, E.K., Jr. A new coupling component for
sulfanilamide determination. J. Biol. Chem. 128:537-549
(1939).

Brodie, B.B. Displacement of one drug by another from carrier or
receptor sites. Proc. Roy. Soc. Med. 58:946-955 (1965).

Buttle, G.A.H., Gray, W.H., and Stephenson, D. Protection of mice
against streptococcal and other infections by p-aminobenzene
sulphonamide and related substances. Lancet 1:1286-1290 (1936).

Cairy, C.F. A new sulfonamide for veterinary use. J. Am. Vet. Med.
Assoc. 122:468-470 (1953).

Chrai, S.S. and Robinson, S.R. Binding of sulfisoxazole to protein
fractions of tears. J. Pharm. Sci. 65:437-439 (1976).

Clarke, E.G.C. Isolation and Identification of Drugs. Pharma~
ceutical Press, London. pp. 549-550 (1969).

Cohen, M. and Pocelinko, R. Renal transport mechanisms for the
excretion of sulfisoxazole. J. Pharmacol. Exp. Ther. 185:
703-712 (1973).

Colebrook, L. and Kenney, M. Treatment of human puerperal infections
and of experimental infections in mice, with Prontosil. Lancet
1:1279-1286 (1936).

-144-
14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24,

25.

26,

27.

28.

~145-

Domagk, G. Ein beitrag zur chemotherapie der bakteriellen infektionen.
Dt. med. Wschr. 61:250-253 (1935).

Domagk, G. Eine neue klasse von desinfektionsmitteln. Dt. med.
Wschr. 61:829-832 (1935).

Doumas, B.T., Watson, W.A., and Biggs, H.G. Albumin standards and
the measurement of serum albumin with bromcresol green. Clin.
Chim. Acta 31:87-96 (1971).

DuPont Instruments, Instrument Products, ACA Division, Wilmington,
Delaware 19898.

Ellman, L., Miller, L., and Rappeport, J. Leukopheresis therapy of
a hypereosinophilic disorder. JAMA 230:1004-1005 (1974).

Fildes, P. A rational approach to research in chemotherapy. Lancet
1:955-957 (1940).

Flake, R.E., Griffin, J., Townsend, E., and Yow, E.M. Studies on
the absorption, distribution and excretion of acetyl sulfisoxa-
zole an insoluble sulfonamide appearing in sulfisoxazole in the
blood. J. Lab. and Clin. Med. 44:582-585 (1954).

Florestano, H.J., Bahler, M.E., Blair, H.R., and Burch, G.R. Blood
concentrations of sulfonamides in dogs, swine and cattle,
N. Am. Vet. 34:17-20 (1953).

Frisk, A.R. 25 jahre chemotherapie mit antibiotics und sulfonamiden.
Antibiotica et Chemotherapia 9:1-18 (1961).

Gibladi, M. Biopharmaceutics and Clinical Pharmacokinetics. Lea and
Febiger, Philadelphia. pp. 45-96 (1977).

Goodman, L.S. and Gilman, A. The Pharmacological Basis of Thera-
peutics. Macmillan Co., New York. pp. 1177-1203 (1970).

Guidelines for Biopharmaceutical Studies in Man. American
Pharmaceutical Association, Academy of Pharmaceutical Sciences,
Washington, D.C. Appendix I:17 (1972).

Kaplan, S.A., Weinfield, R.E., Abruzzo, C.W., and Lewis, M.
Pharmacokinetic profile of sulfisoxazole following intravenous,
intramuscular and oral administration to man. J. Pharm. Sci.
61:773~778 (1972).

Khanna, N., Harper, E., and Stern, L. In vitro effect of sodium
phenobarbital and diethylnicotinamide (Coramine) on protein
binding of bilirubin. Clin. Biochem. 2:349-353 (1969).



Loughlin, E.H. and Mullin, W.G. The relative absorption and elimina-
tion of sulfisoxazole and acetyl sulfisoxazole. Antibiotic
and Chemotherapy V:609-615 (1955).
29.

30.

Sie

32.

33.

34.

aos

36.

37.

38.

39.

40.

42.

43.

-146-

Marshall, E.K. Determination of para-amino-salicylic acid in blood.
Proc. Soc. Exp. Biol. and Med. 68:472-473 (1948).

Meister, F., Endsley, L., and Schmidt, J.D. Drug induced anemia
associated with glucose-6-phosphate dehydrogenase deficiency.
J. Iowa Med. Soc. 65:465-468 (1975).

Mercer, H.D. The practical applications of pharmacokinetics in
veterinary medicine. First Annual Symposium in Veterinary
Pharmacology, Baton Rouge, Louisiana (1978).

Merck Index. Merck and Co., Inc., Rahway, N.J. Ninth Edition,

p. 8748 (1976).

Nelson, E. and O'Reilly, I. Kinetics of sulfisoxazole excretion in
humans. J. Pharmacol. Exp. Ther. 129:368-372 (1960).

Odell, G.B. The dissociation of bilirubin from albumin and its
clinical implication. J. Pediat. 55:268-279 (1959).

Odeil, G.B. Influence of binding on the toxicity of bilirubin.
Annals of N.Y. Acad. of Sci. 226:225-237 (1973).

Oie, S. and Levy, G. Interindividual differences in the effect of
drugs on bilirubin plasma binding in newborn infants and in
adults. Clin. Pharm. and Therap. 21(5):627-632 (1977).

Physicians Desk Reference. Medical Economics Company, Oradell, N.J.

29th Edition, pp. 1239-1240 (1975).

Prandota, J. and Pruitt, A.W. Furosemide binding to human albumin
and plasma of nephrotic children. Clin. Pharm. and Therap.
17:159-165 (1975).

Price, P.C. and Hansen, A.E. Blood levels in relation to dosage of
3,4 dimethyl-5-sulfanilamido-isoxazole (Gantrisin) in children
with clinical observation. Texas Rep. Biol. and Med. 9:764-
779 (1951).

Randall, L.O., Engelberg, R., Iliev, V., Row, M., Hoar, H., and
McGavack, T.H. Comparative studies on blood levels and urinary

excretion of sulfisoxazole and acetyl sulfisoxazole. Anti-
biotics and Chemotherapy 6:877-885 (1954).

Reidenberg, M.M. and Affrime, M. Influence of disease on binding
of drugs to plasma proteins. Ann. N.Y. Acad. Sci. 226:
115-126 (1973).

Reider, J. Physiklisch-chemische und biologische untersuchungen an
sulfonamiden. Arzeimettelforsch 13:81-103 (1963).

Rodkey, F.L. Direct spectrophotometric determination of albumin in
human serum. Clin. Chem. 11:478-487 (1965).
44,

45.

46.

47.

48.

49,

51.

52.

oie

56.

57.

-147-

Rudman, D., Bixler, T.J., and DelRio, A.E. Effect of free fatty
acids on binding of drugs by bovine serum albumin, by human
serum albumin and by rabbit serum. J. Pharmacol. Exp. Ther.
176:261-262 (1971).

Schnitzer, R.J., Foster, R.H.K., Ercoli, N., Soo-Hoo, G., Mangieri,
C.N., and Roe, M.D. Pharmacological and chemotherapeutic
properties of 3,4~dimethyi-5-sulfanilamido-isoxazole. J.
Pharmacol. Exp. Ther. 88:47~57 (1946).

Self, T.H., Evans, W., and Ferguson, T. Letter: Interaction of
sulfisoxazole and warfarin. Circulation 52:528 (1975).

Shankaran, S. and Poland, R.L. The displacement of bilirubin from
albumin by furosemide. J. of Pediatrics 90:642-646 (1977).

Silverman, W.A., Anderson, D.H., Blanc, W.A., and Crozier, D.N.
A difference in mortality rate and incidence of kernicterus
in infants allotted to two prophylactic bacterial regimes.
Pediatrics 18:614-624 (1956).

Slywka, G.W., Melikian, A.P., Straughn, A.B., Whyatt, P.L., and
Meyer, M.C. Bioavailability of 11 sulfisoxazole products in
humans. J. Pharm. Sci. 65:1494-1498 (1976).

Solomon, H.W. and Thomas, G.B. A rapid method for the estimation
of drug-albumin affinity constants in human plasma. Clin.
Pharmacol. Ther. 12:445~-448 (1971).

Stern, L., Doray, B., Chan, G., and Schiff, D. Bilirubin metabolism
and the induction of kernicterus. Birth Defects 12:255~-261
(1976).

Struller, Th. Long acting and short acting sulfonamides, recent
developments. Antibiotica et Chemotherapia 14:179-215 (1968).

Svec, F.A., Rhoads, P.S., and Rohr, J.H. New sulfonamide (Gantrisin)
studies on solubility, absorption and excretion. Arch. Int.
med. 85:83-90 (1950).

Tréfouél, J., Tréfouél, J. Mme., Nitti, F., and Bovet, D. Activité
du p~aminophenylsulfamide sur les infections streptococciques
de la souris et du lapin. Compt. Rend. 120:756-758 (1935).

Van den Bergh, A.A.H. and Miller, P. Uber eine direkte und eine
indirekte diazoreaction auf bilirubin. Biochem. Ztschr.
77:90-95 (1916).

Vera, J.C., Herzig, E.B., Sise, H.S., and Bauer, M.J. Acquired
circulating anticcagulant to factor VIII. JAMA 232:1038
(1975).

Wagner, J.G. Fundamentals of Clinical Pharmacokinetics. Drug
Intelligence Publications, Hamilton, Illinois. pp. 82-90 (1975).
-148-

58. Weinstein, L., Madoff, M.A., and Samet, C.M. The sulfonamides.
N.E. J. of Medicine 263:793-800 (1960).

59. Woods, D.D. Relationship of p-amino-benzoic acid to mechanism of
action of sulphanilamide. Br. J. Exp. Path. 21:74-90 (1940).

60. Yow, E.M. Observations on the use of sulfisoxazole (Gantrisin) in
1000 consecutive patients, with particular reference to the
frequency of undesirable side effects. Am. Practnr. Dig. Treat.
4:521-525 (1953).
BIOGRAPHICAL SKETCH

Robert Lee Suber was born May 16, 1949, in Quincy, Florida. In
June, 1967, he graduated from Quincy Junior-Senior High School, Quincy,
Florida, with high honors. He entered the University of Florida,
Gainesville, Florida, in September, 1967.

While attending the University of Florida, he was treasurer of Kappa
Alpha Order for three years, served as a Student Senator and was elected
to Gamma Sigma Delta Honorary. While an undergraduate, he was honored
on the Dean's List and the President's Honor Roll. He received the
Bachelor of Science degree from the University of Florida in June, 1971,
with a major in animal physiology.

After graduating from the University of Florida, he entered the U.S.
Army as a pathology specialist and served at Tripler Army Medical Center
in Hawaii.

In January, 1974, he entered the Graduate School at the University
of Florida and received a graduate research assistantship in the Depart-
ment of Animal Science. In March, 1975, he received the Master of Science
degree with a major in animal physiology.

After receiving the Master of Science degree, he worked as a
Laboratory Instructor in the Theodore Gildred Microsurgical Education
Center, Division of Neurological Surgery, University of Florida.

In September, 1976, he again entered the Graduate School at the

University of Florida to fulfill the requirements for the Dector of

-149~
~150-

Philosophy degree. He majored in toxicology and minored in pathology
(clinical chemistry).

Upon completion of the Doctor of Philosophy degree in August, 1979,
he has accepted a position as a Toxicologist and Clinical Chemist at the
National Center for Toxicological Research, Jefferson, Arkansas. He will
also hold appointments as an Assistant Professor in Pharmacology/
Toxicology and in Pathology, College of Medicine, University of Arkansas,
Little Rock, Arkansas.

Robert L. Suber is married to Christine Bonar. They were married
on August 12, 1972, and have one son, Robert Lee, Jr., born August 25,

1979.
Tcertity that 1 have read this study and that in my opinion it
conforms to acceptable standerds of scholarly presentation and ts fully
adequate,

in seope aad quility, as oa dissertation for the degree of
Dector of Philosophy.

ae th SG wth

George f. Udds, Chairman
Professor of Toxicology,
Professor of Animal Science

‘

cortify that To bave read this study and that ino ay opinicn it
cont ims te rceptuble standards of scholarly presentation and is tally
ule puate,

32 Scope and quality, as a dissertation for the deyree of
Dogtor of Phil saohy.

_) L a - 4
Keb A So be Ae Loe :

Pauol T. Cardeilhboc
Professor 0°

Veterinary Medicine

f certiiy thar T have read this study and that in my cpinion it
conforms to acceptable stamaards of scholarly presentation and is fully

afequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosaphy.

{

Let CO Plate ben

George Torosf%an

Assoctate Professer of Pharnacy
.
I certify that 1 have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully

adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.



ry .
AU thus {

John C. Gadat :
Assistant |Professor of Pathology

TI certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully

adequate, in scope and quality, as a dissertation for the devree of
Doctor of Philosopay.

f



a



Charles Lee
Assistant Professyr of Pharmacy

This dissertation was submitted to the Graduate Faculty of the College of
Agriculture and to the Graduate Council, and was accepted as partial
fulfillment cf the requirements for the degree of Doctor of Philosophy.

August, 1979

Dean, College of Agricultufe

iE those





Dean, Graduate School




PAGE 1

&203$5,621 2) 7+( 3+$50$&2.,1(7,&6 $1' 72;,&,7< 2) 68/),62;$=2/( ,1 +80$16 $1' 722 0212*$675,& $1,0$/ 63(&,(6 %\ 52%(57 / 68%(5 $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( &281&,/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 2

&RS\ULJKW E\ 5REHUW / 6¼EHU

PAGE 4

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

PAGE 5

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f§2UDO $GPLQLVWUDWLRQ &RPSDULVRQ RI %LOLUXELQ /HYHOV LQ 'RJV 6ZLQHf§,QWUDYHQRXV $GPLQLVWUDWLRQ Y

PAGE 6

3DJH 6ZLQHf§2UDO $GPLQLVWUDWLRQ &RPSDULVRQ RI %LOLUXELQ /HYHOV LQ 6ZLQH +XPDQVf§2UDO $GPLQLVWUDWLRQ &RPSDULVRQ RI %LOLUXELQ /HYHOV LQ 'RJV 6ZLQH DQG +XPDQV &RQFOXVLRQV &RQFHUQLQJ %LOLUXELQ /HYHOV $IWHU $GPLQLVWUDWLRQ RI 6XOILVR[D]ROH 6HUXP $OEXPLQ &RQFHQWUDWLRQV 6800$5< $1' &21&/86,216 $33(1',; %,%/,2*5$3+< %,2*5$3+,&$/ 6.(7&+

PAGE 7

/,67 2) 7$%/(6 7DEOH 3DJH 5HWHQWLRQ WLPH PLQXWHVf RI VXOIRQDPLGHV LQ D ZDWHU PHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU S+ f DQG ZLWKRXW EXIIHU S+ f 5HWHQWLRQ WLPH PLQXWHVf RI VXOIRQDPLGHV LQ D ZDWHU PHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU S+ f DQG ZLWKRXW EXIIHU S+ f 5HWHQWLRQ WLPH PLQXWHVf RI VXOIRQDPLGHV LQ VSLNHG KXPDQ SODVPD VDPSOHV LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU S+ f DQG ZLWKRXW EXIIHU S+ f 5HWHQWLRQ WLPH PLQXWHVf RI VXOIRQDPLGHV LQ VSLNHG KXPDQ SODVPD VDPSOHV LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU S+ f DQG [ALWKRXW EXIIHU S+ f 7ZR FRPSDUWPHQW SKDUPDFRNLQHWLF SDUDPHWHUV LQ GRJV DGPLQLVWHUHG VXOILVR[D]ROH DV D VLQJOH LQWUDYHQRXV GRVH 7KH PHDQ DPRXQW RI VXOILVR[D]ROH H[FUHWHG LQ WKH XULQH RI GRJV 7ZR FRPSDUWPHQW SKDUPDFRNLQHWLF SDUDPHWHUV LQ GRJV DGPLQLVn WHUHG VXOILVR[D]ROH DV D VLQJOH RUDO GRVH 7ZR FRPSDUWPHQW SKDUPDFRNLQHWLF SDUDPHWHUV LQ VZLQH DGPLQLVWHUHG VXOILVR[D]ROH DV D VLQJOH LQWUDYHQRXV GRVH 7KH PHDQ DPRXQW RI VXOILVR[D]ROH DQG DFHW\OVXOILVR[D]ROH 1f H[FUHWHG LQ WKH XULQH RI SLJV DV D SHUFHQWDJH RI WKH GRVH 7KH ELRORJLFDO KDOIOLIH RI DFHW\OVXOILVR[D]ROH 1Af DIWHU D VLQJOH GRVH RI VXOILVR[D]ROH 7ZR FRPSDUWPHQW SKDUPDFRNLQHWLF SDUDPHWHUV LQ VZLQH DGPLQLVWHUHG VXOILVR[D]ROH DV D VLQJOH RUDO GRVH 6LQJOH FRPSDUWPHQW SKDUPDFRNLQHWLF SDUDPHWHUV LQ KXPDQV DGPLQLVWHUHG D JP RUDO GRVH RI VXOILVR[D]ROH 7KH ELRORJLFDO KDOIOLIH W[f RI VXOILVR[D]ROH fµn 9ž

PAGE 8

7DEOH 3DJH 7KH ELRORJLFDO KDOIOLIH WLJf RI DFHW\OVXOILVR[D]ROH WKH PHWDEROLWH RI VXOILVR[D]ROH 7KH PHDQ GLVWULEXWLRQ FRQVWDQWV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7KH UDWLR RI GLVWULEXWLRQ FRQVWDQWV DIWHU DGPLQLVWUDWLRQ RI VXOI LVR[D]ROH &RPSDULVRQ RI WKH YROXPHV RI GLVWULEXWLRQ LQ GRJV VZLQH DQG KXPDQV DIWHU DGPLQLVWUDWLRQ RI VXOI LVR[D]ROH &RPSDULVRQ RI WKH ELRDYDLODELOLW\ RI VXOILVR[D]ROH LQ GRJV VZLQH DQG KXPDQV 7KH IUDFWLRQ RI VXOILVR[D]ROH ERXQG IJf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

PAGE 9

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

PAGE 11

/,67 2) ),*85(6 )LJXUH 3DJH 6WDQGDUG FRQFHQWUDWLRQ FXUYHV RI VXOIDQLODPLGH VXOIDJXDQLGLQH VXOIDPHUD]LQH VXOIDPHWKD]LQH VXOID S\ULGLQH VXOILVR[D]ROH DFHW\OVXOILVR[D]ROH DQG VXOIDWKLD]ROH LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU DW S+ 6WDQGDUG FRQFHQWUDWLRQ FXUYHV RI VXOIDQLODPLGH VXOIDJXDQLGLQH VXOIDPHUD]LQH VXOIDPHWKD]LQH VXOID S\ULGLQH VXOILVR[D]ROH DFHW\OVXOILVR[D]ROH DQG VXOIDWKLD]ROH LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DQ LVRFUDWLF S+ 6WDQGDUG FRQFHQWUDWLRQ VXUYHV RI VXOIDQLODPLGH VXOIDJXDQLGLQH VXOIDPHUD]LQH VXOIDPHWKD]LQH VXOID S\ULGLQH VXOILVR[D]ROH DFHW\OVXOILVR[D]ROH DQG VXOIDWKLD]ROH LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU DW S+ 6WDQGDUG FRQFHQWUDWLRQ FXUYHV RI VXOIDQLODPLGH VXOIDJXDQLGLQH VXOIDPHUD]LQH VXOIDPHWKD]LQH VXOID S\ULGLQH VXOILVR[D]ROH DFHW\OVXOILVR[D]ROH DQG VXOIDWKLD]ROH LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DQ LVRFUDWLF S+ 6HUXP FRQFHQWUDWLRQV RI IUHH VXOILVR[D]ROH LQ GRJV r 6HUXP FRQFHQWUDWLRQV RI IUHH VXOILVR[D]ROH LQ VZLQH 6HUXP FRQFHQWUDWLRQV RI DFHW\OVXOILVR[D]ROH 1 f LQ VZLQH 6HUXP FRQFHQWUDWLRQV RI IUHH DQG DFHW\OVXOILVR[D]ROH 1 f LQ KXPDQV 0HDQ VHUXP ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 0HDQ VHUXP ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH &RPSDULVRQ RI WKH PHDQ WRWDO ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH [L

PAGE 13

)LJXUH 3DJH 0HDQ VHUXP DOEXPLQ FRQFHQWUDWLRQV LQ VZLQH DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 0HDQ VHUXP DOEXPLQ FRQFHQWUDWLRQV LQ KXPDQV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOI LVR[D]ROH [LLL

PAGE 14

$EVWUDFW RI 'LVVHUWDWLRQ 3UHVHQWHG WR WKH *UDGXDWH &RXQFLO RI WKH 8QLYHUVLW\ RI )ORULGD LQ 3DUWLDO )XOILOOPHQW RI WKH 5HTXLUHPHQWV IRU WKH 'HJUHH RI 'RFWRU RI 3KLORVRSK\ &203$5,621 2) 7+( 3+$50$&2.,1(7,&6 $1' 72;,&,7< 2) 68/),62;$=2/( ,1 +80$16 $1' 7:2 0212*$675,& $1,0$/ 63(&,(6 %\ 52%(57 / 68%(5 $XJXVW &KDLUPDQ *HRUJH 7 (GGV 0DMRU 'HSDUWPHQW $QLPDO 6FLHQFH 7KLV H[SHULPHQW ZDV GHVLJQHG WR f GHYHORS D KLJK SHUIRUPDQFH OLTXLG FKURPDWRJUDSKLF SURFHGXUH IRU VHSDUDWLRQ DQG TXDQWLWDWLRQ RI VXOIRQDPLGHV HVSHFLDOO\ VXOILVR[D]ROH DQG LWV PHWDEROLWHV DQG f WR FRPSDUH WKH WR[LFLW\ DQG SKDUPDFRNLQHWLFV RI VXOILVR[D]ROH D ZDWHUVROXEOH VXOIRQDn PLGH LQ PRQRJDVWULF VSHFLHV KXPDQV GRJV DQG VZLQH $Q DFFXUDWH VHQVLWLYH DQG VSHFLILF KLJK SHUIRUPDQFH OLTXLG FKURPDWRJUDSKLF WHFKQLTXH ZDV GHYHORSHG IRU VHSDUDWLRQ RI VXOIDQLODPLGH VXOIDJXDQLGLQH VXOIDPHUD]LQH VXOIDPHWKD]LQH VXOIDS\ULGLQH VXOILVF[D ]ROH DFHW\OVXOILVR[D]ROH 1 f DQG VXOIDWKLD]ROH IURP D ELRORJLFDO PDWUL[ 6SLNHG VHUXP VDPSOHV ZHUH DQDO\]HG E\ LQMHFWLQJ SL RQWR D S %RQGDSDN & FROXPQ ZLWK DEVRUEDQFH PHDVXUHG DW QDQRPHWHUV 7KH PRELOH SKDVH ZDV b GRXEOHGLVWLOOHG ZDWHUb PHWKDQRO S+ f IRU WKH VHSDUDWLRQ DQG TXDQWLWDWLRQ RI VXOIDQLODPLGH DQG VXOIDJXDQLGLQH 7KH RWKHU VXOIRQDPLGHV XVHG D b GRXEOHGLVWLOOHG ZDWHUb PHWKDQRO ZLWK DFHWDWH EXIIHU S+ f PRELOH SKDVH 6HUXP FRXOG EH LQMHFWHG GLUHFWO\ LQ WKH V\VWHP HTXLSSHG ZLWK DQ LQOLQH ILOWHU FRQn WDLQLQJ &RUDVLO &/T GHSURWHLQDWHG ZLWK PHWKDQRO f EHIRUH LQMHFWLRQ [[Y

PAGE 15

RU ILOWHUHG WKURXJK S ILOWHUV ZLWKRXW ORVV RI UHVROXWLRQ 8ULQDU\ H[WUDFWLRQ RI VXOILVR[D]ROH DQG LWV PHWDEROLWH DFHW\OVXOILVR[D]ROH 1 fW LQYROYHG FRROLQJ PO RI XULQH WR r& DFLGLI\LQJ WR S+ ZLWK FRQFHQWUDWHG K\GURFKORULF DFLG DQG H[WUDFWLRQ LQWR FKORURIRUP 7RWDO VHUXP DQG XULQDU\ VXOILVR[D]ROH ZHUH GHWHUPLQHG E\ ERLOLQJ WKH DFLG K\GURO\VDWH IRU KRXU DW r& DQG SHUIRUPLQJ WKH UHVSHFWLYH H[WUDFn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f GHULYDWLYH DQG XULQDU\ H[FUHWLRQ UDWHV DPRQJ DOO VSHFLHV 7RWDO DQG FRQMXJDWHG ELOLUXELQ VKRZHG VPDOO EXW VWDWLVWLFDOO\ VLJQLILFDQW LQFUHDVHV S f LQ GRJV DIWHU RUDO DQG LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH PJNJf DQG LQ VZLQH DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH PJNJf 7KH SRWHQWLDOO\ WR[LF LQGLUHFW RU XQFRQMXJDWHG ELOLUXELQ VKHZHG VPDOO EXW VWDWLVWLFDOO\ VLJQLILFDQW S f LQFUHDVHV LQ GRJV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH $ VLPLODU LQFUHDVH ZDV QRW REVHUYHG LQ VZLQH RU KXPDQV 7RWDO DQG FRQMXJDWHG ELOLUXELQ ZHUH VLJQLILFDQWO\ S f FRUUHODWHG LQ GRJV DIWHU RUDO DQG LQWUDYHQRXV DGPLQLVWUDWLRQ DQG LQ VZLQH DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7KH LQFUHDVH LQ FRQMXJDWHG ELOLUXELQ [Y

PAGE 16

DORQJ ZLWK D FRQFRPLWDQW LQFUHDVH LQ WRWDO ELOLUXELQ FRXOG EH GXH WR KHSDWLF LQGXFWLRQ RI JOXFXURQLGDWLQJ FDSDFLW\ RU UHJXUJLWDWLRQ RI FRQn MXJDWHG ELOLUXELQ IURP WKH KHSDWRF\WH LQVWHDG RI H[FUHWLRQ LQWR WKH ELOH 7KHUH ZDV DOVR D VLJQLILFDQW QHJDWLYH FRUUHODWLRQ S f LQ FRQn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

PAGE 17

,1752'8&7,21 6XOILVR[D]ROH D SKDUPDFHXWLFDO DJHQW UHJXODUO\ XVHG IRU XULQDU\ WUDFW LQIHFWLRQV PD\ EH D SRWHQWLDOO\ WR[LF FRPSRXQG GXH WR WKH LQFUHDVH LQ VHUXP ELOLUXELQ OHYHOV DIWHU DGPLQLVWUDWLRQ KDV WKH SRWHQWLDO IRU LQGXFLQJ EDFWHULDO UHVLVWDQFH GXH WR ORQJ WHUP ORZ OHYHO H[SRVXUH WKURXJK WKH IRRG FKDLQ DQG LV D SRVVLEOH HQYLURQPHQWDO FRQWDPLQDQW HVSHFLDOO\ LQ XUEDQ VHZDJH ,Q RUGHU WR HYDOXDWH WKH DEVRUSWLRQ GLVn WULEXWLRQ PHWDEROLVP DQG H[FUHWLRQ RI VXOILVR[D]ROH LW ZDV QHFHVVDU\ WR HVWDEOLVK WKH SKDUPDFRNLQHWLF SDUDPHWHUV LQ KXPDQ DQG DQLPDO PRGHO V\VWHPV 7KLV WULDO ZDV WR f ILQG D UDSLG KLJK SHUIRUPDQFH OLTXLG FKURPDWRn JUDSKLF PHWKRG IRU GHWHFWLQJ VXOIRQDPLGHV HVSHFLDOO\ VXOILVR[D]ROH DQG LWV PHWDEROLWHV LQ D ELRORJLFDO PDWUL[ DQG f WR FRPSDUH WKH SKDUPDFRn NLQHWLFV DQG SRWHQWLDO WR[LFLW\ RI VXOILVR[D]ROH LQ KXPDQV GRJV DQG VZLQH LQ RUGHU WR GHILQH D EHWWHU DQLPDO PRGHO WR FRUUHODWH ZLWK KXPDQ UHVHDUFK

PAGE 18

5(9,(: 2) /,7(5$785( 7KH PHGLFDO DQG SXEOLF KHDOWK LPSRUWDQFH RI WKH GLVFRYHU\ RI VXOIRQDn PLGHV DV WKH ILUVW HIIHFWLYH FKHPRWKHUDSHXWLF DJHQWV ZDV UHIOHFWHG E\ D VXGGHQ GHFOLQH LQ PRUELGLW\ DQG PRUWDOLW\ GXH WR LQIHFWLRXV GLVHDVHV f 3DXO (KUOLFK WKH IRXQGHU RI PRGHUQ FKHPRWKHUDS\ LQLWLDWHG WKH FRQFHSW RI WKH XVH RI D]R G\HV DV DQWLEDFWHULDO DJHQWV f 6XOIRQLOD PLGH DPLQREHQ]HQH VXOIRQDPLGHf ZDV ILUVW SUHSDUHG E\ *HOPR LQ GXULQJ WKH LQYHVWLJDWLRQ RI D]R G\HV $ GUXJ XVH SDWHQW ZDV LVVXHG WR .ODUHU DQG 0LHW]VFK LQ IRU 3URQWRVLO DQG RWKHU D]R G\HV FRQWDLQLQJ D VXOIRQDPLGH UDGLFDO ,Q WKH VDPH \HDU 'RPDJN ZRUNLQJ ZLWK .ODUHU DQG 0LHW]VFK REVHUYHG WKDW PLFH LQIHFWHG ZLWK VWUHSWRFRFFDO EDFWHULD FRXOG EH SURWHFWHG E\ 3URQWRVLO f $W WKH 3DVWHXU ,QVWLWXWH UHVHDUFKHUV IRXQG WKDW WKH D]R OLQNDJH RI 3URQWRVLO ZDV VSOLW LQ YLYR WR \LHOG SDUDDPLQREHQ]HQHVXOIRQDPLGH ZKLFK ZDV WKRXJKW WR EH WKH DFWLYH FKHPRWKHUDSHXWLF DJHQW f &ROHEURRN DQG .HQQH\f DQG %XWWOH HW DO f UHSRUWHG IDYRUDEOH FOLQLFDO UHVXOWV ZLWK 3URQWRVLO ZKLFK SURPSWHG WKH V\QWKHVLV RI PRUH WKDQ VXOIRQDPLGH GHULYDWLYHV LQ WKH 8QLWHG 6WDWHV E\ f 7KH PHWKRG RI DFWLRQ RI VXOIRQDPLGHV LV EDVHG RQ WKH DEVRUSWLRQ LQWR WKH EDFWHULDO FHOO LQ LWV QRQLRQL]HG IRUP $IWHU SDUWLDO GLVVRFLDWLRQ LW FRPSHWHV ZLWK LRQL]HG SDUDDPLQREHQ]RLF DFLG 3$%$f 7KLV FRPSHWLWLRQ LQKLELWV EDFWHULDO JURZWK E\ SUHYHQWLQJ 3$%$ IURP EHLQJ LQFRUSRUDWHG LQWR SWHUR\OJOXWDPLF DFLG IROLF DFLGf ZKLFK LV UHGXFHG WR WHWUDK\GURIROLF

PAGE 19

DFLG D FRHQ]\PH HVVHQWLDO IRU FDUERQ PHWDEROLVP f $OWKRXJK LQKLELWLRQ RI SWHUR\OJOXWDPLF DFLG V\QWKHVLV LV LPPHGLDWH WKH HIIHFWLYHn QHVV RI VXOIRQDPLGHV LV UHVWULFWHG WR EDFWHULD ZKLFK UHTXLUH RU V\QWKHVL]H SWHUR\OJOXWDPLF DFLG GXULQJ WKH JURZWK SKDVH $ ORJ SKDVH RU ODWHQW SHULRG ZKLFK LV GHWHUPLQHG E\ WKH FRQFHQWUDWLRQ RI VWRUHG SWHUR\On JOXWDPLF DFLG RFFXUV EHWZHHQ WKH DGPLQLVWUDWLRQ RI VXOIRQDPLGH DQG LWV EDFWHULRVWDWLF HIIHFW f 7KH VWUXFWXUHDFWLYLW\ UHODWLRQVKLS RI VXOIRQDPLGHV LV VXFK WKDW WKH SDUDDPLQR JURXS 1 f LV HVVHQWLDO DQG FDQ RQO\ EH UHSODFHG E\ VXFK UDGLFDOV WKDW FDQ EH FRQYHUWHG LQ YLYR LQWR D IUHH DPLQR JURXS 7KH 6&A1A 1Af JURXS LV QRW HVVHQWLDO EXW WKH OLQNDJH RI VXOIXU GLUHFWO\ WR WKH EHQ]HQH ULQJ LV RI XWPRVW LPSRUWDQFH f 7KH PRUH HOHFWURn QHJDWLYH WKH 6 JURXS WKH JUHDWHU LV WKH EDFWHULRVWDWLF DFWLYLW\ VLQFH WKH LRQLF IRUP LV PRUH DFWLYH WKDQ WKH PROHFXODU IRUP f 7KLV NQRZOHGJH OHG WR WKH HDUO\ V\QWKHVLV RI VXOILVR[D]ROH E\ +RIIPDQ/D5RFKH LQ f DQG LWV XVH SDWHQW IRU PLFURELDO LQIHFWLRQV 6LQFH VXOILVR[D]ROH LV UHDGLO\ LRQL]HG DQG H[FUHWHG E\ JORPHUXODU ILOWUDWLRQ LW LV XVHG SULPDULO\ IRU XULQDU\ WUDFW LQIHFWLRQV f 6XOILVR[D]ROH 7KH WKHUDSHXWLF LQGH[ RI VXOIRQDPLGHV LV UHODWLYHO\ ORZ VR FRPSOHWH ELRDYDLODELOLW\ LV LPSRUWDQW WR PD[LPL]H WKH SHUFHQWDJH RI SDWLHQWV ZKR ZLOO REWDLQ D IDYRUDEOH WKHUDSHXWLF UHVSRQVH IURP WKH GUXJ f 6XOILn VR[D]ROH RU VXOIDIXUD]ROH GLPHWK\OVXOIDQLODPLGRLVR[D]ROHf LV D ZKLWH\HOORZLVK RGRUOHVV VOLJKWO\ ELWWHU FU\VWDOOLQH SRZGHU ZLWK D S. RI f LV UHODWLYHO\ QRQWR[LF DQG LV DEOH WR FRQWURO H[SHULPHQWDO

PAGE 20

EDFWHULDO LQIHFWLRQV f ,W LV GLVWULEXWHG LQ WKH H[WUDFHOOXODU IOXLG DQG IDLOV WR HQWHU FHOOV f 7KHUHIRUH WKH DGPLQLVWUDWLRQ RI VXOILVR[D]ROH UHVXOWV LQ D SODVPD FRQFHQWUDWLRQ ZKLFK LV WKUHH WLPHV KLJKHU WKDQ WKDW SURGXFHG E\ DQ HTXDO TXDQWLW\ RI VXOIDQLODPLGH f %ORRG &RQFHQWUDWLRQ RI 6XOILVR[D]ROH 3UHYLRXV SKDUPDFRNLQHWLF VWXGLHV LQ GRJV VZLQH DQG FDWWOH RQO\ GHWHUPLQHG PHDQ EORRG OHYHOV RI IUHH XQERXQGf VXOILVR[D]ROH f ,Q WKHVH H[SHULPHQWV WKH PHDQ EORRG OHYHO ZDV KLJKHVW WR KRXUV DIWHU RUDO DGPLQLVWUDWLRQ ,Q FOLQLFDO WULDOV ZLWK GRJV JLYHQ WR JUDPV SHU GD\ RUDOO\ D PD[LPXP EORRG FRQFHQWUDWLRQ WR PJ FF EORRG ZDV DWWDLQHG ,QWUDYHQRXV DGPLQLVWUDWLRQ WR GRJV f PJNJ ERG\ ZHLJKW UHVXOWHG LQ PJ RI IUHH GUXJ FF EORRG DW KRXU LQ GRJV SURGXFHG PJ RI IUHH GUXJ FF EORRG LQ GRJ DQG PJNJ SURGXFHG PJ RI IUHH GUXJ FF EORRG LQ GRJV 6XEFXWDQHRXV DGPLQLVWUDWLRQ WR GRJV f UHVXOWHG LQ D PD[LPXP EORRG FRQFHQWUDWLRQ KRXUV DIWHU DGPLQLVWUDWLRQ $GPLQLVWUDWLRQ RI PJNJ ERG\ ZHLJKW UHVXOWHG LQ PJ RI IUHH GUXJ PO EORRG ZKLOH PJNJ UHVXOWHG LQ PJ RI IUHH GUXJ PO EORRG 2UDO DGPLQLVn WUDWLRQ RI JP RI VXOILVR[D]ROH WR GRJV f UHVXOWHG LQ D SHDN SODVPD OHYHO RI PJ PO RI ZKROH EORRG 7KH RQO\ DGPLQLVWUDWLRQ UHSRUWHG LQ VZLQH ZDV E\ LQWUDSHULWRQHDO LQMHFWLRQ f 7KH SHDN EORRG OHYHO ZDV PJ RI IUHH GUXJ PO EORRG DW KRXU DIWHU UHFHLYLQJ D GRVH RI PJNJ ERG\ ZHLJKW ,Q GRVLQJ FKLOGUHQ f DGPLQLVWUDWLRQ RI D VLQJOH RUDO GRVH RI PJNJ ERG\ ZHLJKW UHVXOWHG LQ PJ RI IUHH VXOILVR[D]ROH PO RI VHUXP DIWHU KRXUV DQG PJ PO DIWHU KRXUV $Q RUDO GRVH

PAGE 21

RI PJNJ UHVXOWHG LQ PJ RI IUHH GUXJ PO RI VHUXP DW KRXUV DQG PJ PO DW KRXUV 6YHF BHW DO f REVHUYHG WKDW DIWHU RUDO LQJHVWLRQ RI D VLQJOH JP RUDO GRVH LQ DGXOWV WKH PHDQ EORRG FRQFHQn WUDWLRQ RI IUHH VXOILVR[D]ROH ZDV PJ PO 5DQGDOO HW DO f DGPLQLVWHUHG DQ RUDO GRVH RI JP HYHU\ KRXUV IRU GRVHV WR DGXOWV ZKLFK SURGXFHG D PHDQ EORRG FRQFHQWUDWLRQ RI PJ RI WRWDO VXOILVR[D ]ROH PO DW KRXUV DIWHU WKH LQLWLDO GRVH WKH IUHH VXOILVR[D]ROH FRQFHQWUDWLRQ ZDV PJ PO DW KRXUV /RXJKOLQ DQG 0XOOLQ f GRVHG DGXOWV RUDOO\ ZLWK RU JP RI VXOILVR[D]ROH ZLWK ZKROH EORRG FRQFHQWUDWLRQV RI IUHH VXOILVR[D]ROH RI DQG PJb DW DQG KRXUV UHVSHFWLYHO\ IRU WKH JP GRVH WKH JP GRVH SURGXFHG ZKROH EORRG FRQFHQWUDWLRQV RI DQG PJb DW DQG KRXUV UHVSHFWLYHO\ %\ KRXUV WKH FRQFHQWUDWLRQ ZDV b RI WKH KRXU OHYHO IRU ERWK WKH DQG JP GRVH DQG E\ KRXUV WKH EORRG OHYHO KDG GHFUHDVHG WR b RI WKH KRXU JP GRVH DQG b RI WKH KRXU JP GRVH 7KH ILUVW FRPSOHWH SKDUPDFRNLQHWLF SURILOH LQ KXPDQV ZDV FRQGXFWHG E\ .DSODQ BHWB DO f 6HYHQ KHDOWK\ DGXOWV DGPLQLVWHUHG D JP VLQJOH RUDO GRVH KDG D PHDQ DEVRUSWLRQ UDWH FRQVWDQW RI PJKU D KDOI OLIH RI KU SHDN SODVPD WLPH RI KU ZLWK XJPO ,QWUDYHQRXV DGPLQLVWUDWLRQ RI D VLQJOH JP GRVH WR WKH VDPH YROXQWHHUV UHVXOWHG LQ DQ LQLWLDO SODVPD FRQFHQWUDWLRQ RI \JPO D KDOI OLIH RI KU DQG D YROXPH RI GLVWULEXWLRQ RI b RI ERG\ ZHLJKW :KHQ GLIIHUHQW VXOILVR[D]ROH WDEOHWV PJf ZHUH DGPLQLVWHUHG WR KXPDQ YROXQWHHUV f WKH SHDN SODVPD OHYHO ZDV WR XJPO LQ KRXUV RU OHVV WKH KDOI OLIH ZDV KRXUV

PAGE 22

0HWDEROLVP RI 6XOILVR[D]ROH 6DWXUDEOH ILUVW SDVV FRQMXJDWLRQ RI WKH DURPDWLF DPLQR JURXS 1 f RI VXOILVR[D]ROH RFFXUV GXULQJ WKH LQLWLDO SDVVDJH RI WKH GUXJ IURP WKH JDVWURLQWHVWLQDO OXPHQ WKURXJK WKH OLYHU IROORZLQJ RUDO DGPLQLVWUDWLRQ 7KH H[WHQW RI FRQMXJDWLRQ LQFUHDVHV DV WKH RUDO GRVH GHFUHDVHV ZKLFK LV FRQVLVWHQW ZLWK VDWXUDEOH ILUVW SDVV FRQMXJDWLRQ 7KLV GRHV QRW RFFXU ZLWK LQWUDYHQRXV DGPLQLVWUDWLRQ ZKLFK LQGLFDWHV WKDW GRVH GHSHQGHQW FRQn MXJDWLRQ RFFXUV EHIRUH WKH GUXJ UHDFKHV WKH V\VWHPLF FLUFXODWLRQ %\ LQKLELWLQJ WKH PRWRU DFWLYLW\ RI WKH VWRPDFK DQG VPDOO LQWHVWLQH WR VORZ GUXJ DEVRUSWLRQ RQH LV DEOH WR LQFUHDVH FRQMXJDWLRQ RI WKH GUXJ f 1HOVRQ DQG 2n5HLOO\ f UHSRUWHG WKDW PHDQ KDOIOLIH IRU IRUPDWLRQ RI WKH DFHW\O FRQMXJDWH IURP VXOILVR[D]ROH LV KRXUV 7KH PHDQ KDOI OLIH RI WKH DFHW\O GHULYDWLYH ZDV KU ZKLFK ZDV ORQJHU WKDQ WKH KDOI OLIH RI WKH SDUHQW FRPSRXQG KUf /RXJKOLQ DQG 0XOOLQ f UHSRUWHG b RI DQG JP RUDO GRVHV ZHUH FRQMXJDWHG ZLWKLQ KRXUV ZKLOH :HLQVWHLQ f UHSRUWHG WR b LV DFHW\ODWHG :KHQ DFHW\O 1Af VXOI LVR[D]ROH LV DGPLQLVWHUHG RUDOO\ WKH HQ]\PHV UHVSRQVLEOH IRU 1 FRQMXJDWLRQ DUH VDWXUDWHG VR WKH IUDFWLRQ PHWDEROL]HG WR WKH 1 DFHW\O GHULYDWLYH GHFUHDVHV DV WKH RUDO DFHW\O 1 f VXOILVR[D ]ROH GRVH LQFUHDVHV f )ODNH HW DO f VXSSRUWHG WKLV FRQFHSW E\ UHSRUWLQJ b RI VXOILVR[D]ROH LV LQ WKH 1 DFHW\ODWHG IRUP ZKHQ DFHW\O 1Af VXOILVR[D]ROH LV DGPLQLVWHUHG %ORHGRZ f VXJJHVWHG WKDW WKH KLJK GHJUHH RI SURWHLQ ELQGLQJ RI VXOILVR[D]ROH PD\ VSDUH LWV ILUVW SDVVDJH PHWDEROLVP WR WKH 1 DFHW\O GHULYDWLYH OHDYLQJ PRUH RI WKH SDUHQW GUXJ DYDLODEOH IRU LWV SKDUPDFRORJLFDO DFWLRQ

PAGE 23

3URWHLQ %LQGLQJ RI 6XOILVR[D]ROH 7KH ELQGLQJ RI D GUXJ WR SODVPD SURWHLQV DIIHFWV LWV DFWLYLW\ GLVn WULEXWLRQ UDWH RI PHWDEROLVP DQG JORPHUXODU ILOWUDWLRQ f 7KLV GHJUHH RI ELQGLQJ LV LQIOXHQFHG E\ WKH PROHFXODU VWUXFWXUH RI WKH GUXJ LWV OLSLG VROXELOLW\ S.A WKH FRQFHQWUDWLRQ DQG DIILQLW\ IRU SODVPD SURWHLQV WKH QXPEHU RI ELQGLQJ VLWHV WKH SUHVHQFH RI FRPSHWLWLYHO\ ELQGLQJ GUXJV RU HQGRJHQRXV FRPSRXQGV DQG WKH SK\VLRORJLFDO RU SDWKRn ORJLFDO VWDWH RI WKH VXEMHFW f 7KH GHJUHH RI SURWHLQ ELQGLQJ LQFUHDVHV QHDU WKH S.A f :LWK D ORZ DFLGLFf S.A D KLJK GHJUHH RI GUXJ ELQGLQJ RFFXUV DW SK\VLRORJLFDO S+ ZKLOH XQLRQL]HG GUXJV DUH VOLJKWO\ ERXQG 7KH FRQMXJDWLRQ RI PHWK\O JURXSV ZLOO LQFUHDVH WKH ELQGLQJ WHQGHQF\ RI D GUXJ f $OEXPLQ KDV D JUHDWHU ELQGLQJ HIIHFW RQ VXOILVR[D]ROH WKDQ DOSKD RU JDPPDJOREXOLQV f ([SHULPHQWDO ELQGLQJ RI VXOILVR[D]ROH BLQ YLWUR b DOEXPLQ S+ r&f UHVXOWHG LQ b DV ERXQG b DV IUHH EDVH DQG b IUHH DFLG f ZKLFK LV H[SHFWHG VLQFH VXOILVR[D]ROH KDV DQ DFLGLF S.A DQG PHWK\O JURXSV ,Q YLYR H[SHULPHQWV LQ KXPDQV UHVXOWHG LQ DSSUR[LPDWHO\ b RI WKH WRWDO VXOILVR[D]ROH EHLQJ ERXQG WR SODVPD SURWHLQ f 7KH SURWHLQ ERXQG IUDFWLRQ DFWV DV D UHVHUYRLU EHWZHHQ WKH LQHIIHFWLYH DQG WR[LF OHYHOV RI WKH ELRORJLFDOO\ DFWLYH XQERXQG XQn PHWDEROL]HG IUDFWLRQ f EXW WKHUH DSSHDUV WR EH QR FRUUHODWLRQ EHWZHHQ WKH KDOIOLIH LQ PDQ DQG WKH SHUFHQWDJH ZKLFK LV SURWHLQ ERXQG f 7R[LF (IIHFWV RI 6XOILVR[D]ROH 6XOIRQDPLGHV DV D FODVV RI FKHPRWKHUDSHXWLF DJHQWV DUH FRQVLGHUHG WR EH WR[LF GXH WR SUHFLSLWDWLRQ LQ WKH NLGQH\ SURGXFLQJ FU\VWDOOXULD f

PAGE 24

7KH LQIUHTXHQF\ RI UHQDO WR[LFRVLV FU\VWDOOXULDf RI VXOILVR[D]ROH LV GXH WR WKH H[FHSWLRQDOO\ KLJK ZDWHU VROXELOLW\ RI WKH IUHH DQG FRQMXJDWHG DFHW\Of IUDFWLRQV ZLWKLQ WKH SK\VLRORJLFDO S+ UDQJH f &OLQLFDO WR[LFLWLHV KDYH EHHQ LQGXFHG E\ VXOILVR[D]ROH FRPSHWLQJ IRU WKH VDPH ELQGLQJ VLWHV DV ZDUIDULQ f DQG IXURVHPLGH f LQGXFLQJ KHPRO\WLF DQHPLD GXH WR JOXFRVHSKRVSKDWH GHK\GURJHQDVH GHILFLHQF\ f LQKLELWLRQ RI DQWLFRDJXODQW IDFWRU 9,,, f K\SHUVHQVLWLYLW\ f DQRUH[LD f DJUDQXORF\WRVLV f DSODVWLF DQHPLD f DQG D FDVH RI P\RFDUGLWLV P\RVLWLV DQG YDVFXOLWLV DVVRFLDWHG ZLWK VHYHUH HRVLQRSKLOLD IROORZLQJ VXOILVR[D]ROH WKHUDS\ f ,Q \RXQJ JP ODERUDWRU\ UDWV K\SHUSODVLD RI WK\URLG JODQGV RFFXUUHG ZLWK GLHWV RI DQG b VXOILVR[D]ROH IRU ZHHNV 7KHVH UDWV GLG QRW H[KLELW DQ\ FKDQJH LQ JURZWK UDWH DJUDQXORF\WRVLV RU DSODVWLF DQHPLD $W b RI WKH GLHW D GHOD\HG JURZWK UDWH GHFUHDVHG ZKLWH EORRG FHOO FRXQW DQG ERQH JUDQXORF\WHV RFFXUUHG f 7KH RUDO OHWKDO GRVH IRU b RI PLFH WHVWHG /'f ZDV JPNJ RI OLWKLXP DQG VRGLXP VDOW f 7KH /' IRU DQ LQWUDYHQRXV GRVH ZDV PJNJ DQG PJNJ IRU WKH OLWKLXP DQG VRGLXP VDOW UHVSHFWLYHO\ WKH VXEFXWDQHRXV URXWH RI DGPLQLVWUDWLRQ SURGXFHG DQ /' DW DQG PJNJ IRU WKH OLWKLXP DQG VRGLXP VDOW UHVSHFWLYHO\ .HUQLFWHUXV KDV EHHQ UHSRUWHG LQ LQIDQWV ZLWK LQFUHDVHG OHYHOV RI VHUXP ELOLUXELQ LQ SUHPDWXUH LQIDQWV WUHDWHG ZLWK VXOILVR[D]ROH f .HUQLFWHUXV RFFXUUHG ZKHQ XQFRQMXJDWHG RU LQGLUHFW ELOLUXELQ ZDV OHVV WKDQ PJb LQ LQIDQWV OHVV WKDQ PJb LQ LQIDQWV DQG OHVV WKDQ PJb LQ LQIDQWV 7KHVH RFFXUUHQFHV ZHUH HQKDQFHG E\ SULRU DFLGRVLV K\SHUFDSQLD DQG K\SRWKHUPLD f

PAGE 25

3ODVPD VDPSOHV IURP DGXOWV VKRZHG WKDW VXOILVR[D]ROH FRQFHQWUDWLRQV DERYH PJ PO KDG D VLJQLILFDQW GLVSODFLQJ DIIHFW RQ ELOLUXELQ LQ YLWUR f :KHQ FRPSDUHG WR WKH GLVSODFLQJ HIIHFWV RI VDOLF\OLF DFLG VDOLF\OXULF DFLG DQG DVSLULQ WR VXOILVR[D]ROH DW PJ PO FRQFHQWUDn WLRQV WKH PRVW SURQRXQFHG HIIHFW ZDV REVHUYHG ZKHQ VXOILVR[D]ROH GLVSODFHG ELOLUXELQ IURP SODVPD VDPSOHV RI DGXOWV DQG LQIDQWV LQ YLWUR f 7KH GLVSODFHPHQW ELOLUXELQ LV GXH WR FRPSHWLWLRQ IRU VLPLODU ELQGLQJ VLWHV RQ WKH DOEXPLQ PROHFXOH f 8ULQDU\ ([FUHWLRQ RI 6XOILVR[D]ROH 6XOILVR[D]ROH LV DFWLYHO\ VHFUHWHG LQ WKH SUR[LPDO UHQDO WXEXOH LQ GRJV DQG KXPDQV 7XEXODU UHDEVRUSWLRQ LV D SDVVLYH SURFHVV ZKLFK GHSHQGV RQ XULQDU\ S+ f $IWHU VLQJOH GRVHV RI VXOILVR[D]ROH b ZDV UHFRYHUHG LQ KXPDQ XULQH ZLWKLQ KRXUV ZKLOH b ZDV H[FUHWHG ZLWKLQ KRXUV LQ GRJV f .DSODQ HW DO f UHSRUWHG D PHDQ RI b RI VXOILVR[D]ROH ZDV UHFRYHUHG DV WKH IUHH GUXJ ZLWKLQ KRXUV DIWHU D JP LQWUDYHQRXV GRVH LQ KXPDQV ZKLOH b ZDV UHFRYHUHG DIWHU D JP RUDO GRVH 7KH WRWDO VXOILVR[D]ROH UHFRYHUHG LQ KRXUV ZDV b DQG b IRU WKH JP LQWUDYHQRXV DQG RUDO GRVH UHVSHFWLYHO\ :HLQVWHLQ f UHSRUWHG b RI WKH WRWDO VXOILVR[D]ROH H[FUHWHG ZDV LQ WKH DFHW\ODWHG IRUP 3KDUPDFRNLQHWLFV 3KDUPDFRNLQHWLFV KDV EHHQ GHILQHG DV WKH TXDQWLWDWLYH VWXG\ RI WKH DEVRUSWLRQ GLVWULEXWLRQ PHWDEROLVP DQG H[FUHWLRQ RI GUXJV DQG WKHLU SKDUPDFRORJLF WKHUDSHXWLF DQG WR[LF UHVSRQVHV LQ DQLPDOV DQG PDQ f

PAGE 26

7KH SXUSRVHV RI SKDUPDFRNLQHWLFV DUH WR UHGXFH WKH PDWKHPDWLFDO GDWD FROOHFWHG IURP DQ RUJDQLVP WR PHDQLQJIXO SDUDPHWHUV ZKLFK FDQ EH XVHG WR PDNH SUHGLFWLRQV RQ WKH UHVXOWV RI IXWXUH H[SHULPHQWV RU RI KRVW VWXGLHV ZKLFK ZRXOG EH WRR WLPH FRQVXPLQJ DQG FRVWO\ LI FDUULHG RXW LQGLYLGXDOO\ f $EVRUSWLRQ 5DWH &RQVWDQW DQG %LRDYDLODELOLW\ 7KH ILUVW RUGHU DEVRUSWLRQ UDWH FRQVWDQW N f LV D PDWKHPDWLFDO D GHVFULSWLRQ RI WKH UDWH DW ZKLFK WKH GUXJ UHDFKHV WKH JHQHUDO FLUFXODWRU\ V\VWHP DIWHU DGPLQLVWUDWLRQ $ VHPLORJDULWKPLF SORW RI VHUXP FRQFHQ REWDLQHG E\ XVLQJ WKH PHWKRG RI UHVLGXDOV 7KH VORSH Pf RI WKLV OLQH FDQ WKHQ EH XVHG LQ GHWHUPLQLQJ WKH DEVRUSWLRQ UDWH FRQVWDQW E\ HTXDWLRQ f ND Pff (T :LWK VXOIRQDPLGHV WKH FDOFXODWHG DEVRUSWLRQ UDWH FRQVWDQW N f FDQ EH WKH HOLPLQDWLRQ UDWH FRQVWDQW NAf 7KLV FDQ EH FKHFNHG E\ FRPSDULQJ WKH N RI DQ LQWUDYHQRXV GRVH ZLWK WKHN RI DQ RUDO GRVH LI WKHVH DUH QRW G G HTXDO WKH FDOFXODWHG NA WKH IDVWHU UDWH FRQVWDQW LV WKH NA f %LRDYDLODELOLW\ )f LV D WHUP XVHG WR LQGLFDWH D PHDVXUHPHQW RI ERWK WKH DPRXQW RI DGPLQLVWHUHG GUXJ ZKLFK UHDFKHV WKH FLUFXODWLRQ DQG WKH UDWH DW ZKLFK WKLV RFFXUV f 7KH ELRDYDLODELOLW\ GHSHQGV RQ f WKH UDWH DQG H[WHQW RI UHOHDVH RI WKH GUXJ IURP WKH GRVDJH IRUP DQG f WKH ILUVW SDVV HIIHFW ZKHUH RQO\ D FHUWDLQ IUDFWLRQ RI WKH GUXJ SUHVHQWHG WR WKH JDVWURLQWHVWLQDO V\VWHP UHDFKHV WKH JHQHUDO FLUFXODWLRQ LQWDFW 7KH ELRDYDLODELOLW\ )f RI D GUXJ LV FDOFXODWHG IURP D UDWLR RI WKH RUDO

PAGE 27

EORRG FRQFHQWUDWLRQ & f YROXPH RI GLVWULEXWLRQ 9f WKH DEVRUSWLRQ 3 G N f DQG HOLPLQDWLRQ Nf FRQVWDQWV DQG WKH GRVH 'f HTXDWLRQ D G ) & 9 N Nf NW N W O 3 G D f H G D @ N ' D (T $ EHWWHU PHDVXUH RI ELRDYDLODELOLW\ )f LV GHWHUPLQHG E\ FRPSDULQJ WKH DUHD XQGHU WKH SODVPD FXUYH DIWHU DQ RUDO GRVH $8& f ZLWK WKH S R DUHD XQGHU WKH SODVPD FXUYH DIWHU WKH LQWUDYHQRXV DGPLQLVWUDWLRQ RI WKH VDPH GRVH $8& f HTXDWLRQ $OWHUQDWLYHO\ LI WKH VDPH GRVH LV L Y DGPLQLVWHUHG E\ RUDO DQG LQWUDYHQRXV URXWHV RQH FDQ GHWHUPLQH ELRn DYDLODELOLW\ )f E\ WKH UDWLR RI WKH WRWDO GUXJ H[FUHWHG XQFKDQJHG LQ WKH XULQH DIWHU WKH RUDO GRVH 8 f WR WKH WRWDO GUXJ LQ WKH XULQH DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ 8 Y f HTXDWLRQ $8& ) A $8& O Y (T f± rrS R U f§r‘ X rrL Y (T 'LVWULEXWLRQ )RU D FRPSRXQG WR H[HUW LWV SKDUPDFRORJLF HIIHFW GLVWULEXWLRQ PXVW RFFXU LQWR RQH RU PRUH YROXPHV RU FRPSDUWPHQWV RI WKH ERG\ 7KH KLJKO\ SHUIXVHG RU FHQWUDO FRPSDUWPHQW LV FKDUDFWHUL]HG E\ WKH KHDUW OLYHU DQG OXQJV DQG H[FKDQJH EHWZHHQ WKHVH WLVVXHV DQG WKH EORRG LV YHU\ UDSLG 7KH SRRUO\ SHUIXVHG WLVVXH FKDUDFWHUL]HG E\ PXVFOH IDW DQG VNLQ DQG WKH QHJOLJLEOH SHUIXVHG JURXS FKDUDFWHUL]HG E\ WKH ERQH WHHWK KDLU DQG FRQQHFWLYH WLVVXH FRPSRVH WKH SHULSKHUDO DQG GHHS FRPSDUWPHQWV

PAGE 28

,Un UHVSHFWLYHO\ LQ ZKLFK WKHUH LV VORZ H[FKDQJH EHWZHHQ WKHVH WLVVXHV DQG WKH EORRG f 6LQFH WKH EORRG LV WKH FRPPRQ FDUULHU IRU GLVWULEXWLRQ DQG XULQH LV WKH PRVW FRPPRQ PHWKRG RI H[FUHWLRQ RI D GUXJ PRQLWRULQJ RI WKH EORRG FRQFHQWUDWLRQ DQG XULQDU\ H[FUHWLRQ UDWH RI WKH GUXJ ZLOO DOORZ RQH WR GUDZ PDWKHPDWLFDO UHODWLRQVKLSV DV WR WKH DEVRUSWLRQ GLVWULEXWLRQ DQG HOLPLQDWLRQ RI D FRPSRXQG f 7KH DSSDUHQW YROXPH RI GLVWULEXWLRQ 9Af LV D PDWKHPDWLFDO H[SUHVVLRQ IRU HVWLPDWLQJ RU GHILQLQJ KRZ H[WHQVLYHO\ WKH GUXJ LV GLVWULEXWHG WKURXJKRXW WKH ERG\ 7KH OLSLG VROXELOLW\ WKH GHJUHH RI SODVPD SURWHLQ ELQGLQJ DQG WKH FDUGLDF RXWSXW ZLOO DIIHFW WKH GLVWULEXWLRQ RI WKH GUXJ f 7KH DSSDUHQW YROXPH RI GLVWULEXWLRQ 9Af RI D GUXJ LV FRPSXWHG IURP D VHPLORJDULWKPLF SORW RI WKH SODVPD FRQFHQWUDWLRQ &Af YHUVXV WLPH Wf DIWHU D NQRZQ GRVH 'f RI GUXJ LV DGPLQLVWHUHG LQWUDYHQRXVO\ HTXDWLRQ ZKHUH WKH LQLWLDO SODVPD FRQFHQWUDWLRQ &rf LV H[WUDSRODWHG ' &rf 9 f (T 3 G (OLPLQDWLRQ 7KH HOLPLQDWLRQ UDWH FRQVWDQW Nf FDQ EH FDOFXODWHG IURP VHPL G ORJDULWKPLF SORWV RI WKH SODVPD FRQFHQWUDWLRQ YHUVXV WLPH DQG IURP WKH XULQDU\ H[FUHWLRQ UDWH YHUVXV WLPH E\ GHWHUPLQLQJ WKH VORSH RI WKH SORWWHG OLQH Pf DQG XVLQJ HTXDWLRQ f 7KH HOLPLQDWLRQ UDWH FRQVWDQW KDV WKH XQLW RI UHFLSURFDO WLPH f NG f Pf (T

PAGE 29

7KH ELRORJLFDO RU HOLPLQDWLRQ KDOIOLIH WAf RI D GUXJ LV GHULYHG IURP D JUDSKLF SORW RI WKH ORJDULWKP RI WKH SODVPD FRQFHQWUDWLRQ & f YHUVXV WLPH Wf LQ ZKLFK WKH VORSH RI WKH OLQH Pf LV XVHG WR FDOFXODWH D ILUVW RUGHU HOLPLQDWLRQ RU GLVSRVLWLRQ UDWH FRQVWDQW NAf! HTXDWLRQ f (T 7KH ELRORJLFDO KDOIOLIH LV GHILQHG DV WKH WLPH LW WDNHV IRU WKH GUXJ FRQFHQWUDWLRQ WR EH UHGXFHG E\ RQHKDOI f DQG LV XVXDOO\ UHIHUUHG WR LQ UHODWLRQ WR WKH VHUXP RU EORRG FRQFHQWUDWLRQ 8ULQDU\ H[FUHWLRQ LV D PDMRU SDWKZD\ IRU HOLPLQDWLRQ IRU PDQ\ GUXJV DQG WKHLU PHWDEROLWHV ,I WKH GUXJ LV WRWDOO\ H[FUHWHG XQFKDQJHG WKH UHQDO FOHDUDQFH &/Af RI WKH GUXJ HTXDOV WKH SODVPD FOHDUDQFH &/Af 7KHUH DUH WZR ZD\V RI FDOFXODWLQJ FOHDUDQFH ZKLFK LQYROYHV WKH GRVH 'f WKH WRWDO DUHD XQGHU WKH SODVPD FRQFHQWUDWLRQ FXUYH $8& f DQG WKH WRWDO 3 DPRXQW H[FUHWHG LQ WKH XULQH $ f HTXDWLRQV DQG f &/ 3 ') $8& 3 (T $ &/ f§A (T U $8&rr 3 5HQDO FOHDUDQFH FDQ DOVR EH FDOFXODWHG IURP WKH VORSH RI WKH ORJDULWKP RI WKH XULQDU\ H[FUHWLRQ UDWH G$AGWf YHUVXV WKH SODVPD FRQFHQWUDWLRQ DW PLGSRLQWV RI H[FUHWLRQ LQWHUYDOV & f f HTXDWLRQ S PLG G $ X G W 3 PLG f &/ f & (T

PAGE 30

7KH HOLPLQDWLRQ UDWH FRQVWDQW Nf WKH H[FUHWLRQ UDWH FRQVWDQW D N f DQG WKH HOLPLQDWLRQ KDOIOLIH W f FDQ DOVR EH FDOFXODWHG IURP D VLJPDPLQXV SORW 7KLV SORW LQYROYHV WKH ORJDULWKP RI WKH GLIIHUHQFH LQ WRWDO XULQDU\ H[FUHWLRQ PLQXV WKH DPRXQW LQ WKH XULQH DW DQ\ RQH LQWHUn YDO >ORJ $ $ f@ YHUVXV WKH H[FUHWLRQ LQWHUYDO RU E\ DQ H[FUHWLRQ UDWH SORW RI WKH FRQFHQWUDWLRQ RI GUXJ H[FUHWHG SHU XQLW WLPH YHUVXV WKH PLGn WLPH LQWHUYDO EHWZHHQ VDPSOLQJ SHULRGV f HTXDWLRQV DQG UHVSHFWLYHO\ RR N ORJ $c $Af ORJ $f° W (T 77 ‘ 9 f«PLG ( 3ODVPD 3URWHLQ %LQGLQJ 3ODVPD SURWHLQV HVSHFLDOO\ DOEXPLQ DFW DV ELQGLQJ VLWHV IRU DFLGLF DQG EDVLF GUXJV $FLGLF GUXJV DW QRUPDO ERG\ WHPSHUDWXUH DQG QRUPDO WKHUDSHXWLF GRVHV DSSHDU WR ELQG WR DOEXPLQ DW D VLQJOH ELQGLQJ VLWH SRVVLEO\ DW WKH 1WHUPLQDO DPLQR DFLG JURXS f 3URWHLQ ELQGLQJ LQn IOXHQFHV WKH YROXPH RI GLVWULEXWLRQ WKH UDWH RI PHWDEROLVP DQG WKH UDWH RI H[FUHWLRQ f 7KH SHUFHQW RU IUDFWLRQ RI SURWHLQ ERXQG GUXJ I f LV FDOFXODWHG IURP WKH WRWDO VHUXP FRQFHQWUDWLRQ &Af DQG WKH DPRXQW ZKLFK H[LVWV DV IUHH XQERXQG XQPHWDEROL]HG SDUHQW GUXJ &Af DV LQ HTXDWLRQ f I % & I F W (T

PAGE 31

7ZR &RPSDUWPHQW 0RGHO $ WZR FRPSDUWPHQW PRGHO ,V GHILQHG DV D PDWKHPDWLFDO UHODWLRQVKLS RI WKH NLQHWLFV RI GLVWULEXWLRQ RI VXEVWDQFHV LQ WKH ERG\ f :KHQ D WZR FRPSDUWPHQW PRGHO GRHV H[LVW RQH PXVW FDOFXODWH D YROXPH RI GLVWULEXWLRQ IRU ERWK FRPSDUWPHQWV 9A DQG 9Af D ILUVW RUGHU UDWH FRQVWDQW IRU WKH WUDQVIHU RI WKH GUXJ IURP WKH FHQWUDO FRPSDUWPHQW WR WKH SHULSKHUDO FRPSDUWPHQW NAf DQG IURP WKH SHULSKHUDO FRPSDUWPHQW WR WKH FHQWUDO FRPSDUWPHQW AOA DQG D ILUVW RUGHU HOLPLQDWLRQ FRQVWDQW E\ DOO SURn FHVVHV IURP WKH FHQWUDO FRPSDUWPHQW NJA RU NAf DVVXPLQJ WKDW HOLPLQDn WLRQ RQO\ RFFXUV WKURXJK WKH FHQWUDO FRPSDUWPHQW f 7KLV WZR FRPSDUWn PHQW PRGHO LQ ZKLFK HOLPLQDWLRQ RFFXUV RQO\ IURP WKH FHQWUDO FRPSDUWPHQW LV UHSUHVHQWHG E\ WKH IROORZLQJ VFKHPH N &HQWUDO ! 3HULSKHUDO &RPSDUWPHQW ‘r &RPSDUWPHQW (OLPLQDWLRQ ,Q D WZR FRPSDUWPHQW PRGHO D VHPLORJDULWKPLF SORW RI VHUXP RU SODVPD FRQFHQWUDWLRQ & f YHUVXV WLPH Wf \LHOGV WZR VWUDLJKW OLQHV E\ OHDVW VTXDUHV DQDO\VLV WKH GLVWULEXWLRQ D SKDVHf DQG WKH HOLPLQDWLRQ SKDVHf SKDVHV 7KH UDWH FRQVWDQW RI WKH ILUVW RU GLVWULEXWLRQ FRPn SDUWPHQW Df LV FDOFXODWHG IURP WKH VORSH Pf RI D VHFRQG VHPLORJDULWKPLF SORW RI WKH VHUXP FRQFHQWUDWLRQ REWDLQHG E\ WKH PHWKRG RI UHVLGXDOV HTXDWLRQ f 7KH UDWH FRQVWDQW IRU WKH VHFRQG RU HOLPLQDWLRQ FRPSDUWPHQW f LV FDOFXODWHG IURP WKH VORSH Pf RI WKH OHDVW VTXDUHV OLQH RI WKH VHPLORJDULWKPLF VHUXP FRQFHQWUDWLRQ YHUVXV WLPH JUDSK HTXDWLRQ f

PAGE 32

D f Pf (T f Pf (T 7KH UHPDLQLQJ FRQVWDQWV FRXOG WKHQ EH FDOFXODWHG IURP HTXDWLRQV WKURXJK ZKHUH $ DQG % DUH WKH H[WUDSRODWHG EORRG FRQFHQWUDWLRQV DW WLPH IRU WKH D SKDVH DQG SKDVH UHVSHFWLYHO\ 7KH WRWDO LQLWLDO EORRG FRQFHQWUDWLRQ &rf LV WKH VXP RI $ DQG % 3 & 3 $ HaDW % W &r $ % 3 D NL N Df f NLBf NLRA $ % 9[f Nf 9f Nf $ %D $ % (T (T (T (T (T (T (T

PAGE 33

0$7(5,$/6 $1' 0(7+2'6 +LJK 3HUIRUPDQFH /LTXLG &KURPDWRJUDSKLF $QDO\VLV RI 6XOIRQDPLGHV E\ ,RQLF 6XSSUHVVLRQ 0DWHULDOV 7HQ PLOOLJUDP VDPSOHV RI VXOIDQLODPLGHr VXOIDJXDQLGLQHr VXOIDPHUD ]LQHr VXOIDPHWKD]LQHr VXOIDS\ULGLQHr VXOILVR[D]ROH DFHW\OVXOILVR[D I L ]ROH 1 f DQG VXOIDWKLD]ROHr ZHUH GLVVROYHG LQ PHWKDQRO DQG WULSOH GLVWLOOHG ZDWHU f WR JLYH FRQFHQWUDWLRQV RI DQG \JPO 7ZHQW\ PLFUROLWHUV RI WKHVH VWDQGDUG VROXWLRQV ZHUH LQMHFWHG RQWR WKH FROXPQ 6WDQGDUG VROXWLRQV XJPOf ZHUH DGGHG WR PO KXPDQ SODVPD VDPSOHV WR JLYH LQ YLWUR SODVPD FRQFHQWUDWLRQV RI DQG \JPO 7KH BLQ YLWUR SODVPD VDPSOHV ZHUH LQFXEDWHG IRU PLQXWHV LQ D r& ZDWHU EDWK GHSURWHLQDWHG ZLWK PHWKDQROA f FHQWULIXJHG [ Jf IRU PLQXWHV DQG XO RI WKH VXSHUQDWHQW LQMHFWHG RU \O RI WKH MXL YLWUR SODVPD VDPSOH ZDV LQMHFWHG GLUHFWO\ RQWR WKH FROXPQ ZLWKRXW GHSURWHLQD WLRQ RU H[WUDFWLRQ i $Q $/&*3& OLTXLG FKURPDWRJUDSK ZDV HTXLSSHG ZLWK D 0RGHO A XOWUDYLROHW DEVRUEDQFH GHWHFWRU QPf $ GXDO SHQ UHFRUGHUrr r)RUW 'RGJH /DERUDWRULHV )RUW 'RGJH ,RZD +RIIPDQ/D5RFKH 1XWOH\ 1HZ -HUVH\ A%XUGLFN -DFNVRQ /DERUDWRULHV 0XVNHJRQ 0LFKLJDQ i :DWHUV $VVRFLDWHV 0LOIRUG 0DVVDFKXVVHWWV rr+RXVWRQ ,QVWUXPHQWV $XVWLQ 7H[DV

PAGE 34

ZDV XVHG WR TXDQWLWDWH FRQFHQWUDWLRQ DV D IXQFWLRQ RI SHDN KHLJKW PPf Mr $ \ %RQGDSDN & UHYHUVHSKDVH FROXPQr ZDV XVHG ZLWK D ZDWHUPHWKDQRO B/R PRELOH SKDVH DQG DQ LQOLQH JXDUG FROXPQr SDFNHG ZLWK &RUDVLO& r 7KH -BR PRELOH SKDVH ZDV HLWKHU RU ZDWHUPHWKDQRO PL[WXUH LVRFUDWLF S+ RI RU ZLWK DFHWDWH EXIIHUA DGGHG WR DGMXVW WKH S+ WR DV i FRQILUPHG E\ D S+ HOHFWURGH DQG PHWHU 7KH ZDWHU ZDV WULSOH GLVWLOOHG DQG DOO DQDO\VHV ZHUH FRQGXFWHG DW DPELHQW WHPSHUDWXUH r&f ZLWK D POPLQ IORZ UDWH ([WUDFWLRQ DQG 6HSDUDWLRQ 7KH UHWHQWLRQ WLPHV RI VXOIRQDPLGHV LQ VWDQGDUG VROXWLRQV RI PHWKDQROZDWHU DUH JLYHQ LQ 7DEOHV DQG $Q H[DPLQDWLRQ RI WKH GDWD VKRZV WKDW LQ VRPH FDVHV D FKDQJH LQ UHWHQWLRQ WLPH RFFXUUHG ZKHQ WKH S+ ZDV UHGXFHG WR E\ WKH DGGLWLRQ RI DFHWDWH EXIIHU WR WKH PRELOH SKDVH 7KH UHWHQWLRQ WLPH RI VXOIDPHWKD]LQH ZDV LQFUHDVHG ZKLOH WKH UHWHQWLRQ WLPH RI VXOIDPHUD]LQH VXOIDS\ULGLQH VXOILVR[D]ROH DQG DFHW\O VXOILVR[D]ROH 1 f ZDV GHFUHDVHG ZKHQ WKH PRELOH SKDVH ZDV b ZDWHUb PHWKDQRO 7DEOH f :KHQ WKH PRELOH SKDVH ZDV FKDQJHG WR b ZDWHUb PHWKDQRO 7DEOH f WKH UHWHQWLRQ WLPH ZDV LQFUHDVHG IRU VXOIDJXDQLGLQH VXOIDPHUD]LQH VXOIDPHWKD]LQH DQG VXOIDS\ULGLQH 7KH UHWHQWLRQ WLPHV IRU WKH UHPDLQLQJ r:DWHUV $VVRFLDWHV 0LOIRUG 0DVVDFKXVVHWWV n%XUGLFN-DFNVRQ /DERUDWRULHV 0XVNHJRQ 0, A*ODFLDO DFHWLF DFLG )LVKHU 6FLHQWLILF 2UODQGR )ORULGD i 2ULRQ 0RGHO $ 2ULRQ 5HVHDUFK ,QF &DPEULGJH 0DVVDFKXVVHWWV

PAGE 35

7DEOH 5HWHQWLRQ WLPH PLQXWHVf RI VXOIRQDPLGHV LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU S+ f DQG ZLWKRXW EXIIHU S+ f S+ S+ f« 6XOIDQLODPLGH 6XOIDJXDQLGLQH 6XOIDPHUD]LQH 6XOIDPHWKD]LQH 6XOIDS\ULGLQH 6XOILVR[D]ROH $FHW\OVXOILVR[D]ROH 1 f 6XOIDWKLD]ROH

PAGE 36

7DEOH 5HWHQWLRQ WLPH PLQXWHVf RI VXOIRQDPLGHV LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU S+ f DQG ZLWKRXW EXIIHU S+ f S+ S+ 6XOIDQLODPLGH 6XOIDJXDQLGLQH 6XOIDPHUD]LQH 6XOIDPHWKD]LQH 6XOIDS\ULGLQH 6XOILVR[D]ROH $FHW\OVXOILVR[D]ROH 1Af 6XOIDWKLD]ROH

PAGE 37

VXOIRQDPLGHV ZHUH UHGXFHG H[FHSW IRU VXOILVR[D]ROH ZKLFK UHPDLQHG FRQVWDQW :KHQ RQH FRPSDUHV WKH UHVXOWV SUHVHQWHG LQ 7DEOHV DQG DW S+ LW LV QRWHG WKH UHWHQWLRQ WLPHV RI VL[ VXOIRQDPLGHV ZHUH LQFUHDVHG ZKHQ WKH PRELOH SKDVH ZDV b ZDWHUb PHWKDQRO ZLWK DFHWDWH EXIIHU 7KHUH ZDV QR VLJQLILFDQW GLIIHUHQFH LQ WKH UHWHQWLRQ WLPHV RI VXOIDQLODPLGH RU VXOIDJXDQLGLQH LQ D PRELOH SKDVH RI b ZDWHUb PHWKDQRO ZLWK RU ZLWKRXW WKH EXIIHU RU LI WKH PRELOH SKDVH ZDV b ZDWHUb PHWKDQRO ZLWK WKH EXIIHU S+ f +RZHYHU DW S+ XVH RI WKH b ZDWHUb PHWKDQRO PRELOH SKDVH UHVXOWHG LQ DQ LQFUHDVH LQ WKH UHWHQWLRQ WLPH RI VXOIDJXDQLGLQH ZKLOH UHGXFLQJ WKDW RI VXOIDQLODPLGH +XPDQ SODVPD VDPSOHV ZHUH VSLNHG ZLWK VWDQGDUG VROXWLRQV RI VXOIRQDn PLGHV DQG WKH UHWHQWLRQ WLPH UHFRUGHG 6XOIDQLODPLGH DQG VXOIDJXDQLGLQH ZHUH QRW VHSDUDEOH IURP HQGRJHQRXV VHUXP SHDNV LQ WKH b ZDWHUb PHWKDQRO S+ f PRELOH SKDVH 7DEOH f 6XOILVR[D]ROH DQG LWV 1 PHWDEROLWH DFHW\OVXOILVR[D]ROH ZHUH QRW VHSDUDEOH IURP HQGRJHQRXV FRPSRXQGV DW S+ ,RQLF VXSSUHVVLRQ GLG GHFUHDVH WKH UHWHQWLRQ WLPH RI VXOIDS\ULGLQH DQG VXOIDWKLD]ROH LQ SODVPD ZKLOH WKDW RI VXOIDPHWKD]LQH UHPDLQHG FRQVWDQW 7DEOH f :KHQ WKH PRELOH SKDVH ZDV FKDQJHG WR b ZDWHUb PHWKDQRO 7DEOH f VXOIDQLODPLGH VXOIDJXDQLGLQH DQG VXOILVR[D]ROH DQG DFHW\OVXOILVR[Dn ]ROH ZHUH QRW GHWHFWDEOH DW S+ 7KH UHPDLQLQJ IRXU VXOIRQDPLGHV ZHUH GHWHFWDEOH ZLWK DQ LQFUHDVHG UHWHQWLRQ WLPH DW S+ %\ FRPSDULQJ 7DEOHV DQG WKH UHWHQWLRQ WLPH RI WKH GHWHFWDEOH VXOIRQDPLGHV ZDV LQFUHDVHG E\ WKH PRELOH SKDVH DW S+ DQG GHFUHDVHG DW S+

PAGE 38

7DEOH 5HWHQWLRQ WLPH PLQXWHVf RI VXOIRQDPLGHV LQ VSLNHG KXPDQ SODVPD VDPSOHV LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU S+ f DQG ZLWKRXW EXIIHU S+ f S+ S+ 6XOIDQLODPLGH 1'r 6XOIDJXDQLGLQH 1'r 6XOIDPHUD]LQH 6XOIDPHWKD]LQH 6XOIDS\ULGLQH 6XOILVR[D]ROH 1'r $FHW\OVXOILVR[D]ROH 1 f 1'r 6XOIDWKLD]ROH r1' 1RW GHWHFWDEOH GXH WR HQGRJHQRXV VHUXP FRPSRQHQWV ZLWK VLPLODU UHWHQWLRQ WLPHV

PAGE 39

7DEOH 5HWHQWLRQ WLPH PLQXWHVf RI VXOIRQDPLGHV LQ VSLNHG KXPDQ SODVPD VDPSOHV LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU S+ f DQG ZLWKRXW EXIIHU S+ f S+ S+ 6XOIDQLODPLGH 1'r 1'r 6XOIDJXDQLGLQH 1'r 1'r 6XOIDPHUD]LQH 6XOIDPHWKD]LQH 6XOIDS\ULGLQH 6XOILVR[D]ROH $ 1'r $FHW\OVXOILVR[D]ROH 1Af 1'r 6XOIDWKLD]ROH r1' 1HW GHWHFWDEOH GXH WR HQGRJHQRXV VHUXP FRPSRQHQWV ZLWK VLPLODU UHWHQWLRQ WLPHV

PAGE 40

7KH SODVPD PDWUL[ LQFUHDVHG WKH UHWHQWLRQ WLPH RI VXOIDPHUD]LQH VXOIDPHWKD]LQH VXOIDS\ULGLQH VXOILVR[D]ROH DQG DFHW\OVXOILVR[D]ROH 1Af LQ WKH PRELOH SKDVH DW S+ 7DEOHV DQG f 7KH UHWHQn WLRQ WLPH RI VXOIDQLODPLGH VXOIDJXDQLGLQH DQG VXOIDWKLD]ROH ZHUH QRW FKDQJHG LQ WKH PRELOH SKDVH DW S+ +RZHYHU WKH UHWHQWLRQ WLPH RI VXOIDPHWKD]LQH DQG VXOIDS\ULGLQH ZDV LQFUHDVHG LQ WKH SODVPD PDWUL[ DW S+ ZKLOH WKDW IRU VXOIDPHUD]LQH GHFUHDVHG 7DEOHV DQG f :LWK WKH PRELOH SKDVH WKHUH ZHUH QR VLJQLILFDQW FKDQJHV LQ UHWHQWLRQ WLPH E\ WKH SODVPD PDWUL[ DW S+ 7DEOHV DQG f $W S+ WKH UHWHQWLRQ WLPH RI VXOIDS\ULGLQH DQG VXOIDWKLD]ROH ZDV LQFUHDVHG E\ WKH SODVPD PDWUL[ 7DEOHV DQG f :KHQ VHUXP LV WKH ELRORJLFDO PDWUL[ IRXU RI WKH VXOIRQDPLGHV DUH HDVLO\ VHSDUDWHG DW HLWKHU S+ RU PRELOH SKDVH SRODULW\ 6XOIDQLODPLGH DQG VXOIDJXDQLGLQH DUH RQO\ GHWHFWDEOH IURP SODVPD ZKHQ WKH PRELOH SKDVH LV b ZDWHUb PHWKDQRO DW S+ WKH S+ YDOXH FORVHVW WR WKHLU S.A YDOXH 6XOILVR[D]ROH DQG DFHW\OVXOILVR[D]ROH 1 f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f GHPRQVWUDWHG WKDW D GLIIHUHQFH LQ VHQVLWLYLW\ RFFXUUHG DV D IXQFWLRQ RI LRQ VXSSUHVVLRQ

PAGE 41

3($. +(,*+7 PPf )LJXUH 6WDQGDUG FRQFHQWUDWLRQ FXUYHV RI VXOIDQLODPLGH Df VXOID JXDQLGLQH Ef VXOIDPHUD]LQH Ff VXOIDPHWKD]LQH Gf VXOIDS\ULGLQH Hf VXOILVR[D]ROH If DFHW\OVXOILVR[D]ROH Jf DQG VXOIDWKLD]ROH Kf LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU WR S+

PAGE 42

3($. +(,*+7 PPf )LJXUH 6WDQGDUG FRQFHQWUDWLRQ FXUYHV RI VXOIDQLODPLGH Df VXOID JXDQLGLQH Ef VXOIDPHUD]LQH Ff VXOIDPHWKD]LQH Gf VXOIDS\ULGLQH Hf VXOILVR[D]ROH If DFHW\OVXOILVR[D]ROH Jf DQG VXOIDWKLD]ROH Kf LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DQ LVRFUDWLF S+

PAGE 43

DQG SRODULW\ RI WKH PRELOH SKDVH 7KH PHDQ VWDQGDUG HUURU RI SHDN KHLJKW DW WKHVH FRQFHQWUDWLRQV ZDV ,Q )LJXUH WKH VHQVLWLYLW\ RI VXOIDJXDQLGLQH Ef DW QUD LV JUHDWHU WKDQ VXOIDQLODPLGH Df DW S+ LQ D PRELOH SKDVH ,I WKH PRELOH SKDVH UHPDLQV DW S+ )LJXUH f WKLV VHQVLWLYLW\ LV UHn YHUVHG ZLWK VXOIDQLODPLGH SHDN KHLJKW EHLQJ JUHDWHU WKDQ VXOIDJXDQLGLQH 7KHUH LV DOVR D FKDQJH LQ WKH VORSH RI WKH RWKHU VXOIRQDPLGHV DV WKH S+ FKDQJHV LQ WKH PRELOH SKDVH )LJXUHV DQG f ,I WKH PRELOH SKDVH LV b ZDWHUb PHWKDQRO VHQVLWLYLW\ RI WKH DVVD\V IRU VXOIDQLODPLGH Df DQG VXOIDJXDQLGLQH Ef DUH WKH PRVW VHQVLWLYH DW HLWKHU S+ )LJXUHV DQG f 'HWHFWLRQ RI WKH UHPDLQLQJ VXOIRQDn PLGHV YDULHV LQ VHQVLWLYLW\ DW QP LQ WKH PRELOH SKDVH ZLWK WKH S+ FKDQJH 7KH PRVW VHQVLWLYH PRELOH SKDVH LV DW S+ DV VKRZQ E\ WKH JUHDWHU VORSHV RI HDFK VXOIRQDPLGH )LJXUH f $GGLWLRQDO GLOXn WLRQ RI VWDQGDUG VROXWLRQV RI VXOIRQDPLGHV DQG LQFUHDVHG VHQVLWLYLW\ VHWWLQJV WR DXIV DEVRUEDQFH XQLWV IXOO VFDOHf RQ WKH XOWUDn YLROHW GHWHFWRU UHFRUGHG SHDN KHLJKWV HTXLYDOHQW WR QJPO RI VXOIRQDPLGH IURP D VLQJOH \O LQMHFWLRQ ZLWKRXW FRQFHQWUDWLRQ RU UHFRQVWLWXWLRQ RI WKH H[WUDFW LQVHW )LJXUHV WR f 7KH VORSH RI WKH VXOILVR[D]ROH If FXUYH ZDV WKH VDPH IRU WKH PRELOH SKDVH UHJDUGOHVV RI S+ )LJXUHV DQG f %\ FRPSDULQJ )LJXUHV DQG RQH REVHUYHV WKDW WKH VORSH RI WKH VXOIDPHWKD]LQH Gf FXUYH ZDV WKH VDPH IRU WKH PRELOH SKDVH DW HLWKHU S+ RU S+ 6XOIDJXDQLGLQH E )LJXUHV DQG f DQG VXOIDS\ULGLQH H )LJXUHV DQG f FXUYHV KDG WKH VDPH VORSH DW S+ DQG S+ UHVSHFWLYHO\ UHn JDUGOHVV RI WKH SRODULW\ RI WKH PRELOH SKDVH

PAGE 44

3($. +(,*+7 PPf )LJXUH 6WDQGDUG FRQFHQWUDWLRQ FXUYHV RI VXOIDQLODPLGH Df VXOID JXDQLGLQH Ef VXOIDPHUD]LQH Ff VXOIDPHWKD]LQH Gf VXOIDS\ULGLQH Hf VXOILVR[D]ROH If DFHW\OVXOILVR[D]ROH Jf DQG VXOIDWKLD]ROH Kf LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DFHWDWH EXIIHU DW S+

PAGE 45

3($. +(,*+7 PO &21&(175$7,21 2) 68/)21$0,'( WJPO f 6WDQGDUG FRQFHQWUDWLRQ FXUYHV RI VXOIDQLODPLGH Df VXOID JXDQLGLQH Ef VXOIDPHUD]LQH Ff VXOIDPHWKD]LQH Gf VXOIDS\ULGLQH Hf VXOILVR[D]ROH If DFHW\OVXOILVR[D]ROH Jf DQG VXOIDWKLD]ROH Kf LQ D ZDWHUPHWKDQRO f PRELOH SKDVH ZLWK DQ LVRFUDWLF S+ )LJXUH

PAGE 46

2SWLPL]DWLRQ RI WKH /LTXLG &KURPDWRJUDSKLF 3URFHGXUH )RXU VXOIRQDPLGHV VXOIDPHUD]LQH VXOIDPHWKD]LQH VXOIDS\ULGLQH DQG VXOIDWKLD]ROHf FRXOG EH VHSDUDWHG IURP D SODVPD PDWUL[ LQ D RU PHWKDQROZDWHU PRELOH SKDVH DW HLWKHU S+ 6XOIDQLODPLGH DQG VXOID JXDQLGLQH ZHUH RQO\ VHSDUDEOH IURP SODVPD LQ D b ZDWHUb PHWKDQRO PRELOH SKDVH ZLWKRXW DFHWDWH EXIIHU S+ f GXH WR WKHLU LQFUHDVHG S.A RI DQG UHVSHFWLYHO\ 6XOILVR[D]ROH DQG DFHW\OVXOILVR[D]ROH 1 f ZHUH VHSDUDWHG ZLWK HLWKHU PRELOH SKDVH E\ LRQLF VXSSUHVVLRQ ZLWK DFHWDWH WR UHGXFH WKH PRELOH SKDVH S+ WR 6HQVLWLYLW\ RI WKH DVVD\V WR QJPO RI VXOIRQDPLGH IURP \O LQMHFWLRQV RI VSLNHG SODVPD VDPSOHV ZLWKRXW FRQFHQWUDWLRQ RU UHFRQVWLWXn WLRQ RI WKH H[WUDFW $VVD\V IRU VXOIDQLODPLGH DQG VXOIDJXDQLGLQH ZHUH WKH PRVW VHQVLWLYH LQ WKH ZDWHUPHWKDQRO PRELOH SKDVH DW QP $ b ZDWHUb PHWKDQRO ZLWK DFHWDWH EXIIHU WR S+ ZDV WKH PRVW VHQVLWLYH RI WKH DVVD\V ([SHULPHQWDO 0RGHO 7KH WULDO FRQVLVWHG RI PDOH KXPDQ YROXQWHHUV IURP \HDUV ROG ZHLJKLQJ WR NJ IHPDOH GRJV DSSUR[LPDWHO\ \HDUV ROG ZHLJKLQJ  NJ DQG IHPDOH SLJV DSSUR[LPDWHO\ PRQWKV ROG ZHLJKLQJ NJ 7KH KXPDQ YROXQWHHUV ZHUH DGPLQLVWHUHG JPV RI VXOn ILVR[D]ROH LQ D VLQJOH GRVH DQG EORRG VDPSOHV WDNHQ DW DQG KRXUV DIWHU DGPLQLVWUDWLRQ 7KH GRJV ZHUH DGPLQLVWHUHG PJ RI VXOILVR[D]ROHNJ ERG\ ZHLJKW E\ LQWUDYHQRXV DQG RUDO URXWHV LQ UHSOLFDWHV IRU HDFK URXWH ZLWK D GD\ UHVW SHULRG EHWZHHQ HDFK DGPLQLVn WUDWLRQ 7KH SLJV ZHUH DOVR DGPLQLVWHUHG PJ RI VXOILVR[D]ROHNJ RI

PAGE 47

ERG\ ZHLJKW E\ LQWUDYHQRXV DQG RUDO URXWHV ZLWK D GD\ UHVW SHULRG EHWZHHQ DGPLQLVWUDWLRQV %ORRG VDPSOHV ZHUH WDNHQ DW DQG KRXUV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ DQG DQG KRXUV DIWHU RUDO DGPLQLVWUDWLRQ LQ GRJV DQG VZLQH $OO DQLPDOV ZHUH PDLQWDLQHG RQ D FRPPHUFLDO GLHW 3XULQD 'RJ &KRZr RU 6ZLQH )HHGAf ZLWK DG OLELWXP DFFHVV WR ZDWHU 7KH DQLPDOV ZHUH KRXVHG LQ PHWDEROLVP FDJHV DQG WKH XULQH ZDV FROOHFWHG DV YRLGHG DQG IUR]HQ r&f $ b VROXWLRQ RI VXOILVR[D]ROHn ZDV SUHSDUHG LQ RXU ODE ZLWK OLWKLXP K\GUR[LGH 7KH VROXWLRQV ZHUH ILOWHUHG DQG SODFHG LQ VWHULOH i PO DPSXOHV 7KLV VROXWLRQ ZDV XVHG IRU ERWK WKH RUDO DQG LQWUDYHQRXV DGPLQLVWUDWLRQ RI WKH GUXJ 7KH SDUWLWLRQ FRHIILFLHQW RI VXOILVR[D]ROH DQG DFHW\OVXOILVR[D]ROH IURP ZDWHU S+ f LQWR RFWDQROrr DIWHU D RQH KRXU LQFXEDWLRQ SHULRG ZDV IRU VXOILVR[D]ROH DQG IRU DFHW\OVXOILVR[D]ROH DV TXDQWLn WDWHG IURP WKH SUHYLRXV KLJK SHUIRUPDQFH OLTXLG FKURPDWRJUDSKLF SURFHGXUH XVLQJ b ZDWHUb PHWKDQRO ZLWK DFHWDWH EXIIHU S+ f %ORRG 6DPSOHV %ORRG VDPSOHV ZHUH WDNHQ IURP WKH FHSKDOLF YHLQ LQ GRJV DQG KXPDQV DQG YLD WKH DQWHULRU YHQD FDYD LQ VZLQH +XPDQ VDPSOHV ZHUH GUDZQ r5DOVWRQ 3XULQD 6W /RXLV 02 L 8QLYHUVLW\ RI )ORULGD 6ZLQH 8QLW b SURWHLQ IHHG b \HOORZ FRUQ b VR\EHDQ RLO PHDO b VDOW YLWDPLQ FDOFLXPSKRVSKRUXV VXSSOHPHQW A+RIIPDQ/D5RFKH 1XWOH\ 1i :KHDWRQ 6FLHQWLILF 3URGXFWV 2FDOD )/ rr(DVWPDQ.RGDN 5RFKHVWHU 1<

PAGE 48

GLUHFWO\ LQWR PO VWHULOH VLOLFRQHFRDWHG 9DFXWDLQHU WXEHV 7HQ PLOOLn OLWHU VDPSOHV ZHUH WDNHQ E\ D VWHULOH V\ULQJH ZLWK D JDXJH QHHGOH IURP WKH GRJV DQG SLJV DQG WUDQVIHUUHG WR VWHULOH VLOLFRQHFRDWHG 9DFXWDLQHU WXEHV 7KH VDPSOHV ZHUH FHQWULIXJHG [ Jf IRU PLQXWHV WKH VHUXP H[WUDFWHG SURWHFWHG IURP OLJKW UHIULJHUDWHG DQG DQDO\]HG ZLWKLQ PLQXWHV IRU WRWDO DQG FRQMXJDWHG JOXFXURQLGDWHGf ELOLUXELQ DQG DOEXPLQ 7KH UHPDLQLQJ VHUXP ZDV IUR]HQ r&f IRU XS WR GD\V IRU DQDO\VLV RI IUHH VXOILVR[D]ROH DFHW\O 1AfVXOILVR[D]ROH DQG WRWDO DFLG K\GURO\]HGf VXOILVR[D]ROH 6HUXP ELOLUXELQ 6HUXP ZDV DQDO\]HG IRU WRWDO DQG FRQMXJDWHG GLUHFW RU JOXFXURQLGDWHGf ELOLUXELQ RQ (, 'X3RQWnV $XWRPDWLF &OLQLFDO $QDO\]HUr M f XVLQJ D FRPPHUFLDO VWDQGDUG 7KH FRQMXJDWHG PHWKRG LV D PRGLILFDWLRQ RI WKH 9DQ GHQ %HUJK GLD]R UHDFWLRQ f &RQMXJDWHG ELOLUXELQ S1LWUREHQ]HQGLD]RQLXP WHWUDIOXRURERUDWH 31%f + 5HG FKURPRSKRUH DEVRUELQJ DW QP 8QGHU DFLGLF FRQGLWLRQV WKH 31% LV FRXSOHG WR WKH JOXFXURQLGDWHG ELOLn UXELQ ZKLFK LV PHDVXUHG DV DQ HQG SRLQW UHDFWLRQ DW DQG QP 7KH QRUPDO UDQJH LQ KXPDQV LV FRQVLGHUHG WR EH WR PJGO 7KH WRWDO ELOLUXELQ TXDQWLWDWLRQ WKH FRQMXJDWHG DQG XQFRQMXJDWHG IUDFWLRQVf LV DOVR D GHULYDWLRQ RI WKH 9DQ GHQ %HUJK UHDFWLRQ f r(, 'X3RQW ,QVWUXPHQW 3URGXFWV :LOPLQJWRQ 'HODZDUH I 'DGH 'LYLVLRQ $PHULFDQ +RVSLWDO 6XSSO\ &RUS 0LDPL )/

PAGE 49

7RWDO ELOLUXELQ S1LWUREHQ]HQHGLD]RQLXP WHWUDIOXRURERUDWH 31%f + 7ZHHQ 5HG FKURPRSKRUH DW QP $ VXUIDFWDQW 7ZHHQ fr LV XVHG WR VROXELOL]H WKH XQFRQMXJDWHG IUHHf ELOLUXELQ ZKLFK DORQJ ZLWK WKH ZDWHU VROXEOH JOXFXURQLGDWHG ELOLUXELQ UHDFWV ZLWK 31% LQ DQ DFLG PHGLXP WR PHDVXUH DQ HQG SRLQW UHDFWLRQ DW DQG QP 7KH QRUPDO WRWDO ELOLUXELQ FRQFHQWUDWLRQ LQ KXPDQV LV OHVV WKDQ PJGO f LQ GRJV WKH QRUPDO OHYHO LV OHVV WKDQ PJ FF f ,QWHUIHUHQFHV LQ WKHVH PHWKRGV DUH GXH WR KHPRO\]HG VDPSOHV DQG OLJKW GHJUDGDWLRQ RI WKH ELOLUXELQ $OO DQDO\VHV ZHUH FRQGXFWHG ZLWKLQ PLQXWHV RI VDPSOLQJ RQ VHUXP ZKLFK ZDV NHSW LQ D FRRO GDUN HQYLURQPHQW 6HUXP DOEXPLQ 6HUXP DOEXPLQ ZDV DQDO\]HG RQ (, 'X3RQWnV $XWRPDWLF &OLQLFDO $QDO\]HUA f 7KLV PHWKRG LV DQ DGDSWDWLRQ RI WKH EURPRFUHVRO JUHHQ %&*f G\H ELQGLQJ PHWKRG RI 5RGNH\ f ZKLFK ZDV ODWHU PRGLILHG E\ 'RXPDV f $OEXPLQ SOXV %&* G\H DW S+ \LHOGV DQ DOEXPLQ%&* FRPSOH[ ZKLFK HOLFLWV DQ DEVRUELQJ VSHFWUXP DW QP 7KH HQG SRLQW UHDFWLRQ LV PHDVXUHG DW DQG QP 7KH QRUPDO UDQJH LQ KXPDQV LV WR PJGO ,QWHUIHUHQFHV DUH H[SHFWHG LQ LFWHULF DQG KHPRO\]HG VDPSOHV $OO DQDO\VHV ZHUH FRQGXFWHG ZLWKLQ PLQXWHV RI VDPSOLQJ r8QLRQ &DUELGH &RUSRUDWLRQ 1HZ
PAGE 50

6HUXP VXOILVR[D]ROH )UHH XQERXQG XQPHWDEROL]HGf VHUXP VXOILVR[D ]ROH DQG VHUXP DFHW\O 1 f VXOILVR[D]ROH ZHUH GHWHUPLQHG E\ GHSURWHLQDWLQJ SDUW RI VHUXP ZLWK SDUWV PHWKDQRO FHQWULIXJLQJ IRU PLQXWHV [ Jf DQG ILOWHULQJ WKH VXSHUQDWHQW WKURXJK 0LOH[ GLVSRVDEOH ILOWHUV 6/+$ fr 7RWDO VHUXP VXOILVR[D]ROH DFLG K\GURO\]HGf ZDV GHWHUPLQHG E\ DGGLQJ SDUWV ZDWHU DQG SDUW 1 K\GURFKROULF DFLG WR SDUW VHUXP DQG KHDWHG LQ D ERLOLQJ ZDWHU EDWK r&f IRU KRXU 7KH VDPSOHV ZHUH DOORZHG WR FRRO WR URRP WHPSHUDWXUH DQG FHQWULIXJHG IRU PLQXWHV [ Jf 7KH VXSHUQDWHQW ZDV DGMXVWHG WR S+ ZLWK 1 1D2+ WKH ILQDO YROXPH DGMXVWHG WR PO ZLWK PHWKDQROn DQG ILOWHUHG ZLWK GLVSRVDEOH 0LOH[ ILOWHUV 6/+$ fr 7ZHQW\ PLFUROLWHUV \Of RI WKH ILOWHUHG VXSHUQDWHQW ZHUH LQMHFWHG LQWR D :DWHUV 0RGHO $ /LTXLG &KURPDWRJUDSK HTXLSSHG ZLWK D 0RGHO DEVRUEDQFH GHWHFWRU ZLWK D QUD ILOWHU 8. LQMHFWRU DQG D \%RQGDSDN &AJ FROXPQA $ +RXVWRQ ,QVWUXPHQWV GXDO SHQ UHFRUGHU ZDV XVHG WR UHFRUG SHDN KHLJKWV $ SHDN KHLJKW UDWLR ZDV XVHG WR GHWHUPLQH VHUXP FRQFHQ i WUDWLRQ ZLWK VXOIDWKLD]ROH EHLQJ XVHG DV WKH LQWHUQDO VWDQGDUG 6HUXP VDPSOHV VSLNHG ZLWK VWDQGDUG VROXWLRQV RI VXOILVR[D]ROH DQG DFHW\O VXOILVR[D]ROHrr ZHUH LQMHFWHG DQG D VWDQGDUG FXUYH RI SHDN KHLJKW RI GUXJ WR VXOIDWKLD]ROH ZDV FRQVWUXFWHG r0LO,LSRUH &RUSRUDWLRQ %HGIRUG 0$ A%XUGLFN -DFNVRQ /DERUDWRULHV 0XVNHJRQ 0, :DWHUV $VVRFLDWHV 0LOIRUG 0$ i )RUW 'RGJH /DERUDWRULHV )RUW 'RGJH ,RZD rr+RIIPDQ/D5RFKH 1XWOH\ 1

PAGE 51

$ PRELOH SKDVH RI b WULSOH GLVWLOOHG ZDWHU ZLWK b FRQFHQWUDWHG DFHWLF DFLGb PHWKDQROr S+ f ZDV XVHG ZLWK D IORZ UDWH RI POPLQ 7KH ZDWHU DQG PHWKDQRO ZHUH ILOWHUHG 1$:3 DQG )+83f GHJDVVHG E\ VRQLFDWLRQ DQG PL[LQJ PDLQWDLQHG E\ XVLQJ D 7KHUPRO\QH L VWLUUHU 8ULQDU\ 6XOILVR[D]ROH )UHH DQG DFHW\O 1 fVXOILVR[D]ROH ZHUH PHDVXUHG E\ FRROLQJ PO XULQH WR r& LQ DQ LFH EDWK VXIILFLHQW 1 K\GURFKORULF DFLG ZDV DGGHG WR UHGXFH WKH S+ WR $IWHU PLQXWHV PO FKORURIRUPr ZDV DGGHG WKH VROXWLRQ UHPRYHG IURP WKH LFH EDWK DQG H[WUDFWLRQ ZDV FRPSOHWHG LQ PLQXWHV E\ VZLUOLQJ WKH VROXWLRQ RQFH SHU PLQXWH 7KH FKORURIRUPr ZDV UHPRYHG HYDSRUDWHG XQGHU QLWURJHQ DQG WKH UHVLGXH UHFRQVWLWXWHG ZLWK PHWKDQROr 7RWDO VXOILVR[D]ROH ZDV PHDVXUHG E\ KHDWLQJ WKH PO RI XULQH LQ D ERLOLQJ ZDWHU EDWK IRU KRXU DQG WKHQ H[WUDFWHG DV SUHYLRXVO\ PHQWLRQHG 7KH UHFRQVWLWXWHG H[WUDFW ZDV WKHQ LQMHFWHG RQWR WKH OLTXLG FKURPDWRJUDSK XVLQJ WKH SUHYLRXVO\ GHVFULEHG WHFKQLTXH 3HDN KHLJKW ZDV i DOVR XVHG WR GHWHUPLQH WKH XULQDU\ FRQFHQWUDWLRQ ZLWK VXOIDWKLD]ROH EHLQJ XVHG DV WKH LQWHUQDO VWDQGDUG &KORURIRUPr H[WUDFWV ZHUH XWLOL]HG WR UHPRYH b RI VXOILVR[D]ROH DQG b RI DFHW\O 1 fVXOILVR[D]ROH IURP r%XUGLFN -DFNVRQ /DERUDWRULHV 0XVNHJRQ 0, n0LOOLSRUH &RUSRUDWLRQ %HGIRUG 0$ -/ A6FLHQWLILF 3URGXFWV 2FDOD )/ i )RUW 'RGJH /DERUDWRULHV )RUW 'RGJH ,RZD

PAGE 52

VSLNHG XULQH VDPSOHV ZKLFK KDG EHHQ LQFXEDWHG DW r& IRU KRXU %HQ]HQHr UHPRYHG b RI VXOILVR[D]ROH DQG b RI DFHW\O 1 fVXOILVR[D]ROH ZKLOH HWKHUr UHPRYHG b RI VXOILVR[D]ROH DQG b RI DFHW\O 1 fVXOILVR[D]ROH %RWK VHUXP DQG XULQH VDPSOHV ZHUH SHUIRUPHG RQ WKLQOD\HU FKURPDn WRJUDSK\ SODWHV f WR GHWHUPLQH LI PHWDEROLWHV RWKHU WKDQ WKH DFHW\O 1 f PHWDEROLWH H[LVWHG $OO VSRWV ZHUH DFFRXQWHG IRU DQG DQDO\]HG WKURXJK WKH +3/& SURFHGXUH SUHYLRXVO\ PHQWLRQHG ZLWKRXW WKH DSSHDUDQFH RI RWKHU PHWDEROLWHV DW QP DQG QP $QDO\VLV RI 'DWD 7RWDO FRQMXJDWHG DQG LQGLUHFW ELOLUXELQ DQG VHUXP DOEXPLQ ZHUH DQDO\]HG IRU VWDWLVWLFDO GLIIHUHQFHV E\ %DUU DQG *RRGQLJKW f $129$ SURJUDP DW WKH 1RUWKHDVW 5HJLRQDO &RPSXWHU &HQWHU 8QLYHUVLW\ RI )ORULGD )UHH VXOILVR[D]ROH DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ ZDV DQDO\]HG DV D WZRFRPSDUWPHQW PRGHO XVLQJ D 121/,1 SURJUDP f /RJDULWKPLF OHDVW VTXDUHV DQDO\VLV DQG WKH PHWKRG RI UHVLGXDOV ZDV XVHG WR FDOFXODWH D UHJUHVVLRQ OLQH IRU DFHW\OVXOILVR[D]ROH DIWHU RUDO DQG LQWUDYHQRXV DGPLQLVWUDWLRQ DQG IRU IUHH VXOILVR[D]ROH DIWHU WKH RUDO GRVH ,Q 9LWUR 3ODVPD 3URWHLQ %LQGLQJ ,Q YLWUR KXPDQ SODVPD VDPSOHV Z7HUH LQFXEDWHG LQ D ZDWHU EDWK DW r& IRU KRXU ZLWK DQG \JPO RI VXOILVR[D]ROH VWDQGDUGV LQ SDUW PHWKDQRO ZLWK SDUWV GRXEOHGLVWLOOHG r%XUGLFN -DFNVRQ /DERUDWRULHV 0XVNHJRQ 0,

PAGE 53

ZDWHU 'LDO\VLV ZDV FRQGXFWHG E\ PHPEUDQH GLDO\VLV ZLWK D PROHFXODU ZHLJKW FXWRII ILOWHU RI r DIWHU LQFXEDWLQJ PO RI VSLNHG VHUXP ZLWK PO RI SKRVSKDWH EXIIHU S+ 7KH GLDO\VDWH ZDV FROOHFWHG DQG DQDO\]HG E\ WKH SUHYLRXVO\ GHVFULEHG OLTXLG FKURPDWRJUDSKLF PHWKRG r'LDFKHPD $J &+ 5XVFKNOLNRQ 6ZLW]HUODQG A)LVKHU *UDP 3DF %XIIHU S+ )LVKHU 6FLHQWLILF 2UODQGR )ORULGD

PAGE 54

5(68/76 $1' ',6&866,21 3KDUPDFRNLQHWLFV RI 6XOILVR[D]ROH 7KH SKDUPDFRNLQHWLF SURILOH RI VXOILVR[D]ROH ZDV GHWHUPLQHG IROORZLQJ LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ LQ GRJV DQG VZLQH DQG IROORZLQJ RUDO DGPLQLVWUDWLRQ LQ KXPDQV 7KH LQWUDYHQRXV EORRG OHYHO FXUYHV LQ GRJV DQG VZLQH ZHUH REVHUYHG WR EH ELH[SRQHQWLDO DQG UHTXLUHG D WZRFRPSDUWPHQW PRGHO V\VWHP IRU GDWD DQDO\VHV f N &HQWUDO f§ &RPSDUWPHQW fµf§ 3HULSKHUDO &RPSDUWPHQW N (OLPLQDWLRQ ,QWHJUDWLRQ RI WKH GLIIHUHQWLDO HTXDWLRQV RI D WZRFRPSDUWPHQW PRGHO \LHOGV WKH HTXDWLRQ & $HaD& %H6W ZKHUH &A LV WKH FRQFHQWUDWLRQ RI WKH GUXJ LQ WKH SODVPD DW WLPH W $ DQG % DUH RUGLQDWH D[LV LQWHUFHSWV DQG WKH LQGLYLGXDO UDWH FRQVWDQWV IRU WKH FRPSDUWPHQWV Af¬ Af¬ DQFA A DUH FDAFXADAAH IURP D DQG WKH UDWH FRQVWDQWV IRU GLVWULEXWLRQ DQG HOLPLQDWLRQ UHVSHFWLYHO\ f

PAGE 55

$GPLQLVWUDWLRQ RI 6XOILVR[D]ROH WR 'RJV ,QWUDYHQRXV DGPLQLVWUDWLRQ $ OHDVW VTXDUHV OLQHDU UHJUHVVLRQ OLQH RI WKH IUHH XQERXQG QRQPHWDEROL]HG VXOILVR[D]ROH LQ GRJV )LJXUH f ZDV FDOFXODWHG XVLQJ D 121/,1 SURJUDP f 7KH PHDQ H[WUDSRODWHG VHUXP FRQFHQWUDWLRQ DW ]HUR WLPH $f LQ WKH ILUVW RU GLVWULEXWLRQ FRPSDUWPHQW ZDV XJPO ZLWK D UDQJH RI WR \JPO DQG D PHDQ H[WUDSRODWHG OHYHO DW ]HUR WLPH %f IRU WKH VHFRQG RU HOLPLQDWLRQ FRPSDUWPHQW ZDV s \JPO 7DEOH f 7KH GLVSRVLWLRQ UDWH IURP WKH ILUVW RU GLVWULEXWLRQ FRPSDUWPHQW Df UDQJHG IURP WR KRXUV A ZLWK D PHDQ RI A KRXUV A ZKLFK \LHOGV D PHDQ KDOIOLIH W f RI  KRXUV IRU VXOILVR[D nn &W ]ROH LQ WKH ILUVW FRPSDUWPHQW 6LPLODU DQDO\VLV RI IUHH VXOILVR[D]ROH LQ WKH VHFRQG RU HOLPLQDWLRQ FRPSDUWPHQW \LHOGHG D YHU\ VORZ PHDQ GLVn SRVLWLRQ UDWH f RI  KRXUV A RU D PHDQ KDOIOLIH WW 4f nfµnI 3 RI KRXUV 7DEOH f 7KH PHDQ UDWH RI GLVWULEXWLRQ EHWZHHQ WKH FHQWUDO RU GLVWULEXWLRQ DQG SHULSKHUDO RU HOLPLQDWLRQ FRPSDUWPHQWV NAf ZDV KRXUV A 7DEOH f ZKLOH WKH PHDQ UDWH RI GLVWULEXWLRQ IURP WKH SHULSKHUDO WR WKH FHQWUDO FRPSDUWPHQW N"Af ZDV KRXUV A 7DEOH f 7KLV LQGLFDWHG WKDW IUHH VXOILVR[D]ROH UHWXUQHG IURP WKH SHULSKHUDO WR WKH FHQWUDO FRPSDUWPHQW DW D IDVWHU UDWH WKDQ LW KDG EHHQ GLVWULEXWHG IURP WKH FHQWUDO WR WKH SHULSKHUDO FRPSDUWPHQW &DOFXODWLRQ RI WKH PHDQ NLINIL UDWLR UHIOHFWV WKLV GLIIHUHQFH LQ GLVWULEXWLRQ EHWZHHQ WKH WZR FRPSDUWPHQWV ,Q WKHVH GRJV WKH PHDQ RI WKH NANA UDWLR ZDV LQGLFDWLQJ WKDW WKH GUXJ LV UHWXUQLQJ UDSLGO\ IURP WKH GLVWULEXWLRQ VLWHV IRU HOLPLQDWLRQ IURP WKH ERG\

PAGE 56

6(580 )5(( 68/),62;$=2/( ,1 '2*6 [JPOf )LJXUH 6HUXP FRQFHQWUDWLRQV RI IUHH VXOILVR[D]ROH LQ GRJV

PAGE 57

7DEOH 7ZR FRPSDUWPHQW SKDUPDFRNLQHWLF SDUDPHWHUV LQ GRJV DGPLQLVWHUHG VXOILVR[D]ROH DV D VLQJOH LQWUDYHQRXV GRVH 6XE -L}F/ $ Q 9 9 N N NP $RU ,! , D 63 OLJPO f OLJPOf KRXUV rf OLRWX V rf ,WHUDf , KDV f OOn-OWO5 rf KRXUV Af KRXUV f KRXUVf KRXUVf L f " 0HDQ 62r r0HQQ RQH VL DPLD ,nG GHYLDWLRQ

PAGE 58

7DEOH 7KH PHDQ DPRXQW RI VXOILVR[D]ROH H[FUHWHG LQ WKH XULQH RI GRJV 7RWDO $PRXQW RI 6XOILVR[D]ROH PJf b RI 'RVH ,QWUDYHQRXV KRXUV KRXUV KRXUV 2UDO KRXUV r r KRXUV KRXUV A&DOFXODWHG IURP GRJV GXH WR QR FROOHFWLRQ RI XULQH DW KRXUV IURP RQH LQGLYLGXDO

PAGE 59

7KH PHDQ HOLPLQDWLRQ UDWH NAf LQ WKH GRJV ZDV KRXUV A 7DEOH f 7KH UDWLR RI NAJ LQGLFDWHV WKH IUDFWLRQ RI IUHH VXOILVR[D]ROH LQ WKH SRVWGLVWULEXWLYH SKDVH ZKLFK LV DYDLODEOH IRU HOLPLQDWLRQ 7KH PHDQ %NA4 UDWLR ZDV LQGLFDWLQJ WKDW b RI WKH GUXJ LQ WKH ERG\ ZRXOG EH LQ WKH FHQWUDO FRPSDUWPHQW DQG DYDLODEOH IRU HOLPLQDWLRQ 7KH YROXPH RI WKH FHQWUDO FRPSDUWPHQW 9Af UDQJHG IURP WR OLWHUV ZLWK D PHDQ RI W / 7DEOH f 7KH PHDQ YROXPH RI WKH VHFRQG RU SHULSKHUDO FRPSDUWPHQW ZDV / 7DEOH f LQGLFDWLQJ WKDW IUHH VXOILVR[D]ROH LV PRUH ZLGHO\ GLVWULEXWHG LQ WKH FHQWUDO WKDQ LQ WKH SHULSKHUDO FRPSDUWPHQW ,Q GRJV DQG 7DEOH DQG f WKH IUDFWLRQ ERXQG ZDV OHVV WKDQ b IRU WKH ILUVW KRXUV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ EXW KDG UHDFKHG WR b DW KRXUV DIWHU DGPLQLVWUDWLRQ ,Q WKH RWKHU GRJV WKH IUDFWLRQ ERXQG I f UDQJHG IURP WR b WKURXJKRXW WKH WULDO SHULRG ' KRXUV 7KH PHDQ DPRXQW RI VXOILVR[D]ROH H[FUHWHG LQ WKH XULQH 7DEOH f ZDV b RI WKH GRVH ZLWKLQ KRXUV DIWHU DGPLQLVWUDWLRQ %\ WKH HQG RI WKH WULDO KRXUV b RI WKH GRVH ZDV H[FUHWHG LQ WKH XULQH 2UDO DGPLQLVWUDWLRQ )URP ORJOLQHDU UHJUHVVLRQ HTXDWLRQV RI WKH VHUXP FRQFHQWUDWLRQ RI IUHH VXOILVR[D]ROH DIWHU RUDO DGPLQLVWUDWLRQ WKH PHDQ UDWH FRQVWDQWV D DQG 6 ZHUH KRXUV A DQG KRXUV A 7DEOH f UHVSHFWLYHO\ 7KH PHDQ KDOIOLIH FRUUHVSRQGLQJ WR WKH IDVWHU GLVSRVLWLRQ UDWH W f ZDV  KRXUV DQG WKH PHDQ HOLPLQDn ED WLRQ KDOIOLIH FRUUHVSRQGLQJ WR WKH VORZHU GLVSRVLWLRQ UDWH W Bf ZDV 3 L KRXUV 7DEOH f

PAGE 60

7DEOH 7ZR FRPSDUWPHQW RUDO GRVH SKDUPDFRNLQHWLF SDUDPHWHUV LQ GRJV DGPLQLVWHUHG VXOILVR[D]ROH DV D VLQJOH 6XEMHFW D % WM£D WM6% ) $8& 3 KRXUV ‘r‘f KRXUV nrf¬f KRXUVf KRXUVf bf 0HDQ  6'r r0HDQ B RQH VWDQGDUG GHYLDWLRQ

PAGE 61

7KH SHDN VHUXP FRQFHQWUDWLRQ KDG RFFXUUHG E\ WKH ILUVW VDPSOLQJ SHULRG KRXU 7DEOHV WR f 7KLV UDSLG DEVRUSWLRQ ZDV GXH WR WKH FRPSRXQG EHLQJ JLYHQ DV D VROXWLRQ 7KH SHDN FRQFHQWUDWLRQ RI IUHH VXOILVR[D]ROH UDQJHG IURP WR \JPO 7DEOHV WR f DQG WKH PD[LPXP WRWDO VXOILVR[D]ROH UDQJHG IURP WR \JPO 7KH GHJUHH RI SODVPD SURWHLQ ELQGLQJ ZDV FDOFXODWHG WR EH WR b LQ GRJV LQ WKH ILUVW KRXUV RI WKH WULDO 7DEOHV DQG f 7KH IUDFWLRQ ERXQG ZDV OHVV WKDQ b LQ WKH UHPDLQLQJ GRJV IRU WKH ILUVW KRXUV DIWHU RUDO DGPLQLVWUDWLRQ %HWZHHQ DQG KRXUV WKH IUDFWLRQ ERXQG ZDV WR b EXW KDG LQFUHDVHG WR WR b LQ DOO GRJV DIWHU KRXUV DQG FRQWLQXHG DW WKLV OHYHO XQWLO WKH HQG RI WKH WULDO 7KH PHDQ DPRXQW H[FUHWHG LQ WKH XULQH RI WKH GRJV ZDV b RI WKH GRVH DW KRXUV DQG b RI WKH GRVH DW WKH HQG RI WKH WULDO KRXUV 7DEOH f )RXU RI WKH GRJV H[FUHWHG b RI WKH GRVH LQ KRXUV ZKLOH GRJV H[FUHWHG DQG b LQ KRXUV $GPLQLVWUDWLRQ RI 6XOILVR[D]ROH WR 6ZLQH ,QWUDYHQRXV DGPLQLVWUDWLRQ 7KH IUHH XQERXQG QRQPHWDEROL]HG VXOILVR[D]ROH LQ SLJV ZDV DQDO\]HG DV D WZRFRPSDUWPHQW PRGHO )LJXUH f E\ D 121/,1 SURJUDP f $W ]HUR WLPH WKH PHDQ H[WUDSRODWHG VHUXP FRQFHQWUDWLRQ RI $ DQG % RQ WKH RUGLQDWH D[LV ZDV W \JPO DQG s \JPO 7DEOH f UHVSHFWLYHO\ 7KH GLVSRVLWLRQ UDWH IURP WKH ILUVW FRPSDUWPHQW Df UDQJHG IURP WR KRXUV A ZLWK D PHDQ RI  KRXUV ? ZKLFK LV HTXLYDOHQW WR D KDOIOLIH W[ f RI s KRXUV 7DEOH f 7KH & PHDQ GLVSRVLWLRQ UDWH IURP WKH VHFRQG FRPSDUWPHQW ZDV FRQVLGHUDEO\ ORQJHU L KRXUV A RU D KDOIOLIH WM f RI aW KRXUV

PAGE 62

6(580 )5(( 68/),62;$=2/( ,1 6:,1( LJPOf )LJXUH 6HUXP FRQFHQWUDWLRQV RI IUHH VXOILVR[D]ROH LQ VZLQH

PAGE 63

7DEOH 7ZR FRPSDUWPHQW SKDUPDFRNLQHWLF SDUDPHWHUV LQ VZLQH DGPLQLVWHUHG VXOILVR[D]ROH DV D VLQJOH LQWUDYHQRXV GRVH 6XE -HF/ $ 8.POf % On5POf D KRXUV 6 + KXQWV r 9M f H} Vf 9 WRUVf N OLQQ ,% rf N, OLRQWIO r f NR KRXUV $,,& O! f br KRX,6f KRXUVf L f f ! f f I! f 0HDQ VQr f f f r+FmLQ  RQH VWDQGDUG GHYLDWLRQ

PAGE 64

7KH PHDQ GLVWULEXWLRQ FRQVWDQW EHWZHHQ WKH FHQWUDO DQG SHULSKHUDO FRPSDUWPHQWV NAf ZDV DSSUR[LPDWHO\ WZLFH DV IDVW DV WKH UHWXUQ RI WKH FRPSRXQG IURP WKH SHULSKHUDO WR WKH FHQWUDO FRPSDUWPHQW fµ 7KH PHDQ NA ZDV s KRXUV A ZKLOH ZDV  KRXUV A 7DEOH f 7KLV LV VXSSRUWHG E\ WKH H[WHQGHG KDOIOLIH RI VXOILVR[D]ROH LQ WKH VHFRQG FRPSDUWPHQW W f 7KH UDWLR RI N N ZDV ZKLFK UHIOHFWV WKH GLVWULEXWLRQ GLIIHUHQFH EHWZHHQ WKH FRPSDUWPHQWV ZLWK WKH IUHH VXOILVR[D]ROH UHDGLO\ HQWHULQJ WKH SHULSKHUDO FRPSDUWPHQW EXW VORZO\ UHWXUQLQJ WR WKH FHQWUDO FRPSDUWPHQW IRU HOLPLQDWLRQ IURP WKH ERG\ 7KH HOLPLQDWLRQ UDWH NAJf UDQJHG IURP WR KRXUV A ZLWK D PHDQ RI W KRXUV A 7DEOH f 7KH UDWLR RI INAJ ZDV ZKLFK LQGLFDWHG WKDW RQO\ D YHU\ VPDOO IUDFWLRQ bf RI IUHH VXOILVR[Dn ]ROH ZDV DYDLODEOH WR EH H[FUHWHG IURP WKH SRVWGLVWULEXWLYH SKDVH 7KH PHDQ YROXPH RI GLVWULEXWLRQ IRU WKH ILUVW FRPSDUWPHQW 9Af 7DEOH f / ZDV RQHKDOI RI WKH YROXPH RI WKH VHFRQG FRPSDUWPHQW 9Af / 7KLV LQGLFDWHG WKDW IUHH VXOILVR[Dn ]ROH ZDV PXFK PRUH ZLGHO\ GLVWULEXWHG WKURXJKRXW WKH VHFRQG FRPSDUWPHQW WKDQ LQ WKH ILUVW FRPSDUWPHQW 7KH GHJUHH RI SODVPD SURWHLQ ELQGLQJ LQB YLYR HTXDWLRQ f UDQJHG IURP WR b WKURXJKRXW WKH WULDO SHULRG 7DEOHV WR f )RXU RI WKH DQLPDOV ERXQG OHVV WKDQ b RI VXOILVR[D]ROH GXULQJ WKH ILUVW KRXU RI WKH WULDO 7DEOHV WR f 7KH PHDQ DPRXQW RI VXOILVR[D]ROH H[FUHWHG LQ WKH XULQH DV D SHUFHQW RI WKH GRVH 7DEOH f ZDV b IUHH VXOILVR[D]ROH DQG b DFHW\O VXOILVR[D]ROH DW WKH HQG RI KRXUV %\ WKH HQG RI WKH WULDO KRXUV b RI WKH GRVH ZDV H[FUHWHG DV IUHH VXOILVR[D]ROH DQG b DV DFHW\OVXOILVR[D]ROH

PAGE 65

7DEOH 7KH PHDQ DPRXQW RI VXOILVR[D]ROH DQG DFHW\OVXOILVR[D]ROH 1 f H[FUHWHG LQ WKH XULQH RI SLJVr DV D SHUFHQWDJH RI WKH GRVH b RI 'RVH ([FUHWHG 6XOILVR[D]ROH $FHW\OVXOILVR[D]ROH ,QWUDYHQRXV KRXUV KRXUV KRXUV 2UDO KRXUV KRXUV KRXUV r7KH XULQH IURP RQH DQLPDO ZDV QRW UHFRUGHG

PAGE 66

7DEOH 7KH ELRORJLFDO KDOIOLIH RI DFHW\OVXOILVR[D]ROH 1 f DIWHU D VLQJOH GRVHr RI VXOILVR[D]ROH 6XEM HFW +XPDQV 2UDO 3LJV ,QWUDYHQRXV 2UDO KRXUVf D KRXUVf D KRXUVf % 0HDQ  6'" r'RJV GLG QRW PHWDEROL]H VXOILVR[D]ROH WR WKH DFHW\O 1 f PHWDEROLWH ; ,QVXIILFLHQW GDWD IRU D ORJOLQHDU UHJUHVVLRQ SORW r0HDQ  RQH VWDQGDUG GHYLDWLRQ

PAGE 67

7KH WM DQG W RI DFHW\OVXOILVR[D]ROH 7DEOH f ZHUH FDOFXODWHG n n6 3 E\ ORJOLQHDU UHJUHVVLRQ 7KH W UDQJHG IURP WR KRXUV ZLWK D PHDQ RI s KRXUV 7KH W 4 ZDV PXFK PRUH YDULDEOH ZLWK D UDQJH n 3 RI WR KRXUV DQG D PHDQ RI L KRXUV 7KUHH SLJV KDG D W R OHVV WKDQ KRXUV KDG D W[ 4 EHWZHHQ DQG KRXUV DQG KDG DW f± RI KRXUV 7DEOH f $FHW\OVXOILVR[D]ROH UHDFKHG D A \ 0 PD[LPXP OHYHO WR \JPO 7DEOHV WR f DW WR KRXUV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ LQ DOO SLJV )LJXUH f 2UDO DGPLQLVWUDWLRQ )URP ORJOLQHDU UHJUHVVLRQ HTXDWLRQV RI WKH VHUXP FRQFHQWUDWLRQV RI IUHH VXOILVR[D]ROH DIWHU RUDO DGPLQLVWUDWLRQ WKH PHDQ GLVSRVLWLRQ UDWH FRQVWDQWV D DQG ZHUH KRXUV A DQG L KRXUV UHVSHFWLYHO\ 7DEOH f 7KH ELRORJLFDO KDOI OLIH RI IUHH VXOILVR[D]ROH LQ WKH FHQWUDO RU GLVWULEXWLRQ FRPSDUWPHQW W f UDQJHG IURP WR KRXUV ZLWK D PHDQ RI L KRXUV D 7KH PHDQ KDOIOLIH LQ WKH HOLPLQDWLRQ FRPSDUWPHQW W f ZDV ORQJHU ‘L S L KRXUV ZLWK D UDQJH RI WR KRXUV 7DEOH f 6HUXP FRQFHQWUDWLRQ RI IUHH VXOILVR[D]ROH UHDFKHG D SHDN E\ WKH ILUVW VDPSOLQJ SHULRG KRXU 7DEOHV WR f 7KLV UDSLG DEVRUSWLRQ ZDV GXH WR WKH FRPSRXQG EHLQJ JLYHQ DV D VROXWLRQ 7KH PD[LPXP IUHH VXOILVR[D]ROH UDQJHG IURP WR \JPO ZLWK RI WKH SLJV KDYLQJ OHVV WKDQ \JPO RI VXOILVR[D]ROH DV D PD[LPXP VHUXP FRQFHQWUDWLRQ 7DEOHV DQG f 7RWDO VXOILVR[D]ROH DWWDLQHG D PD[LPXP RI WR \JPO ZLWK WKH VDPH SLJV KDYLQJ WKH ORZHVW WRWDO VXOILVR[D]ROH FRQFHQWUDWLRQ 7DEOHV DQG f $FHW\OVXOILVR[D]ROH UHDFKHG D PD[LPXP RI WR \JPO LQ WR KRXUV LQ DOO SLJV 7DEOHV WR f 7KH KDOIOLIH RI DFHW\On VXOILVR[D]ROH UDQJHG IURP WR KRXUV IRU WKH FHQWUDO RU GLVWULEXWLRQ

PAGE 68

6(580 $&(7
PAGE 69

7DEOH 7ZR FRPSDUWPHQW RUDO GRVH SKDUPDFRNLQHWLF SDUDPHWHUV LQ VZLQH DGPLQLVWHUHG VXOILVR[D]ROH DV D VLQJOH 6XEMHFW D W W R ) $8& nD 3 KRXUV Af KRXUV KRXUVf KRXUVf bf O 0HDQ 6,fr r0HDQ RQH VWDQGDUG GHYLDWLRQ

PAGE 70

FRPSDUWPHQW WM f ZLWK D PHDQ RI KRXUV 7DEOH f )RU AD WKH SHULSKHUDO RU HOLPLQDWLRQ FRPSDUWPHQW W[ f WKH KDOIOLIH UDQJHG f 3 IURP WR KRXUV ZLWK D PHDQ RI  KRXUV 7DEOH f 7KH ELRDYDLODELOLW\ )f RI VXOILVR[D]ROH ZDV FDOFXODWHG HTXDWLRQ f IURP WKH DUHD XQGHU WKH FXUYH $8&f DIWHU RUDO 7DEOH f DQG LQWUDYHQRXV 7DEOH f DGPLQLVWUDWLRQ 7KH ELRDYDLODELOLW\ UDQJHG IURP WR b LQ VZLQH ZLWK D PHDQ RI  b 7DEOH f 7KH GHJUHH RI SODVPD SURWHLQ ELQGLQJ If±f LQ YLYR UDQJHG IURP WR % b WKURXJKRXW WKH WULDO SHULRG 7DEOHV WR f 7KUHH RI WKH DQLPDOV ERXQG OHVV WKDQ b WKURXJKRXW WKH WULDO 7DEOHV DQG f ZKLOH WKH IUDFWLRQ ERXQG LQ WKH UHPDLQLQJ DQLPDOV ZDV PRUH WKDQ b WKURXJKRXW WKH WULDO 7DEOHV DQG f 6ZLQH H[FUHWHG b RI WKH GRVH DV IUHH VXOILVR[D]ROH LQWR WKH XULQH DQG b DV DFHW\OVXOILVR[D]ROH LQ WKH ILUVW KRXUV RI WKH WULDO DIWHU RUDO DGPLQLVWUDWLRQ RI WKH GUXJ 7DEOH f %\ WKH HQG RI WKH WULDO KRXUV WKH DPRXQW RI IUHH VXOILVR[D]ROH H[FUHWHG ZDV b RI WKH GRVH DV FRPSDUHG WR WKH KRXU OHYHO RI b 7KH DPRXQW H[FUHWHG DV DFHW\OVXOILVR[D]ROH ZDV b RI WKH GRVH DW WKH HQG RI WKH WULDO KRXUV 7DEOH f $GPLQLVWUDWLRQ RI 6XOILVR[D]ROH WR +XPDQV )ROORZLQJ WKH RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH WR KXPDQV WKH GDWD ZHUH DQDO\]HG DV D VLQJOH FRPSDUWPHQW PRGHO )LJXUH f 7KH KDOI OLIH W f IROORZLQJ RUDO DGPLQLVWUDWLRQ UDQJHG IURP WR KRXUV ZLWK D PHDQ RI W KRXUV 7DEOH f .DSODQ eW DO f UHSRUWHG D PHDQ KDOIOLIH RI KRXUV DIWHU RUDO DGPLQLVWUDWLRQ RI

PAGE 71

6(580 &21&(175$7,216 ,1 +80$16 ^ATPOf )LJXUH 6HUXP FRQFHQWUDWLRQV RI IUHH DQG DFHW\OVXOILVR[D]ROH 1 f LQ KXPDQV

PAGE 72

VXOILVR[D]ROH *DQWULVLQrf DQG s KRXUV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI WKH GUXJ 7KH PHDQ HOLPLQDWLRQ FRQVWDQW Nf ZDV G KRXUVn 7DEOH f 7KH ELRDYDLODELOLW\ )f ZDV FDOFXODWHG IURP WKH RUDO DUHD XQGHU WKH FXUYH 7DEOH f DQG WKH FDOFXODWHG DUHD XQGHU WKH FXUYH f IROORZLQJ LQWUDYHQRXV DGPLQLVWUDWLRQ f 7KH ELRDYDLODELOLW\ UDQJHG IURP WR b ZLWK D PHDQ RI L b 7DEOH f 7KH UHDVRQ IRU ) H[FHHGLQJ b FRXOG EH GXH WR f HQWHURKHSDWLF UHF\FOLQJ RU f WKH FRPSDULVRQ RI GLIIHUHQW SRSXODWLRQV .DSODQ HMW BDO f UHn SRUWHG D PHDQ ELRDYDLODELOLW\ RI b ZLWK D UDQJH RI WR b 7KH ELRORJLFDO KDOIOLIH W[f RI DFHW\OVXOILVR[D]ROH UDQJHG IURP nL WR KRXUV ZLWK D PHDQ RI s KRXUV 7DEOH f 7KH PD[LPXP VHUXP DFHW\OVXOILVR[D]ROH FRQFHQWUDWLRQ UDQJHG IURP WR \JPO DQG UHDFKHG PD[LPXP LQ DOO VXEMHFWV KRXUV DIWHU RUDO DGPLQLVWUDWLRQ RI JUDPV RI VXOILVR[D]ROH 7DEOHV WR f 7KH PD[LPXP IUHH DQG WRWDO VXOILVR[D]ROH RFFXUUHG EHIRUH WKH ILUVW VDPSOLQJ SHULRG GXH WR WKH GUXJ EHLQJ DGPLQLVWHUHG DV D VROXWLRQ 7KH PD[LPXP VHUXP IUHH VXOILVR[D]ROH OHYHOV UDQJHG IURP WR \JPO 7DEOHV WR f ZKLOH WKH PD[LPXP WRWDO VXOILVR[D]ROH UDQJHG IURP WR \JPO .DSODQ HW DO f UHSRUWHG D UDQJH RI WR \JPO RI IUHH VXOILVR[D]ROH DIWHU RUDO DGPLQLVWUDWLRQ RI *DQWULVLQr 7KH IUDFWLRQ RI VXOILVR[D]ROH ERXQG WR SODVPD SURWHLQV If ZDV ' OHVV WKDQ b GXULQJ WKH ILUVW KRXU IRU DOO VXEMHFWV 7DEOHV WR f 'XULQJ WKH UHPDLQGHU RI WKH WULDO SHULRG WKH I ZDV WR b D r*DQWULVLQ DFWLYH LQJUHGLHQW VXOILVR[D]ROHf +RIIPDQ/D5RFKH 1XWOH\ 1-

PAGE 73

7DEOH 6LQJOH FRPSDUWPHQW DGPLQLVWHUHG D SKDUPDFRNLQHWLF JP RUDO GRVH RI SDUDPHWHUV LQ VXOILVR[D]ROH KXPDQV 6XEMHFW NG KRXUV b KRXUVf $8& )r bf 0HDQ s 6' r%LRDYDLODELOLW\ LV FDOFXODWHG XVLQJ WKH PHDQ $8& IURP .DSODQ HW DO f RI KXPDQV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI JUDPV RI *DQWULVLQ VXOILVR[D]ROHf $8& ; 0HDQ RQH VWDQGDUG GHYLDWLRQ

PAGE 74

ZKLFK LV OHVV WKDQ WKH b SUHYLRXVO\ UHSRUWHG f EXW VLPLODU WR DQRWKHU UHSRUW RI b ERXQG LQL YLYR f &RPSDULVRQ RI WKH 3KDUPDFRNLQHWLFV RI 6XOILVR[D]ROH LQ 'RJV 6ZLQH DQG +XPDQV 7KH PHDQ H[WUDSRODWHG VHUXP FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ WKH ILUVW FRPSDUWPHQW $f ZDV \JPO LQ GRJV  \JPO LQ VZLQH DQG UHSRUWHG DV  LQ KXPDQV f ,Q WKH VHFRQG FRPSDUWPHQW WKH H[WUDSRODWHG VHUXP FRQFHQWUDWLRQ %f ZDV  \JPO LQ GRJV \JPO LQ VZLQH DQG UHSRUWHG DV s \JPO LQ KXPDQV f 7KH PHDQ WRWDO LQLWLDO VHUXP FRQFHQWUDWLRQ &r $ %f ZDV \JPO LQ GRJV \JPO LQ VZLQH DQG UHn SRUWHG DV \JPO LQ KXPDQV f 7KH WRWDO DQG IUHH LQLWLDO VHUXP FRQFHQWUDWLRQV ZHUH QRW VLJQLILFDQWO\ GLIIHUHQW f LQ HLWKHU VSHFLHV .DSODQ HW DO f UHSRUWHG D WZRFRPSDUWPHQW PRGHO LQ KXPDQV WKH ILUVW FRPSDUWPHQW KDG D PHDQ WA A RI PLQXWHV RU KRXUV WKH VHFRQG FRPSDUWPHQW KDG D PHDQ WM RI KRXUV 7DEOH f IROORZLQJ n 4 LQWUDYHQRXV DGPLQLVWUDWLRQ 7KH WA A LQ GRJV ZDV KRXUV DQG KRXUV LQ VZLQH DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ 7KH VKRUWHU W LQ `D KXPDQV ZDV FORVHU WR WKH W LQ VZLQH DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ Kr 7KH KDOIOLIH RI VXOILVR[D]ROH LQ WKH VHFRQG FRPSDUWPHQW WW 4f ZDV 3 KRXUV LQ GRJV DQG KRXUV LQ VZLQH 7DEOH f 1HLWKHU RI WKHVH KDOI OLYHV ZHUH FRPSDUDEOH WR WKH YDOXH SUHYLRXVO\ UHSRUWHG LQ KXPDQV f KRXUV 'XH WR WKH VKRUW WHUP RI WKLV WULDO QR VHFRQG FRPSDUWPHQW ZDV REVHUYHG LQ WKH KXPDQ VXEMHFWV $IWHU RUDO DGPLQLVWUDWLRQ WKH W[ ZDV KRXUV LQ KXPDQV n KRXUV LQ GRJV DQG KRXUV LQ VZLQH 7DEOH f 7KH W[ 4 n3 LQ GRJV ZDV

PAGE 75

7DEOH 7KH ELRORJLFDO KDOIOLIH WM f RI VXOILVR[D]ROH bD KRXUVf KRXUVf 'RJV ,QWUDYHQRXV 2UDO 6ZLQH ,QWUDYHQRXV 2UDO +XPDQV ,QWUDYHQRXVr r r 2UDO r r.DSODQ HBW DO f

PAGE 76

KRXUV KRXUV LQ VZLQH DQG UHSRUWHG DV KRXUV LQ KXPDQV f DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7KHUH ZHUH QR GLIIHUHQFHV LQ WKH KDOI OLYHV RI VXOILVR[D]ROH W RU W Q GXH WR WKH URXWH RI KD DGPLQLVWUDWLRQ LQ GRJV DQG VZLQH ,I WKH Wc RI KRXUV LQ KXPDQV FW DIWHU RUDO DGPLQLVWUDWLRQ LV FRPSDUHG WR WKH WW UHSRUWHG E\ .DSODQ n! 3 HW DO f WKHUH LV QR GLIIHUHQFH LQ WKH URXWH RI DGPLQLVWUDWLRQ RU LQ WKH FRPSDUWPHQWV .DSODQ HBW MA/ f SRVWXODWHG WKH WZRFRPSDUWPHQW PRGHO LQ KXPDQV GXH WR WDNLQJ EORRG VDPSOHV ZLWKLQ WKH ILUVW KRXU DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH *DQWULVLQrf 7KHUH ZHUH ODUJH GLIIHUHQFHV EHWZHHQ VSHFLHV +XPDQV KDG WKH ORQJHVW PHDQ W[ KRXUV IROORZHG E\ GRJV DQG KRXUV DQG WKHQ VZLQH D DQG KRXUV 7DEOH f ,Q WKH VHFRQG FRPSDUWPHQW VZLQH KDG WKH ORQJHVW PHDQ W[ DQG KRXUV IROORZHG E\ WKH GRJV ! 3 DQG KRXUV 7DEOH f 0D[LPXP VHUXP DFHW\OVXOILVR[D]ROH FRQFHQWUDWLRQV 7DEOHV WR f ZHUH KLJKHU LQ KXPDQV WR \JPOf WKDQ LQ VZLQH WR \JPOf +RZHYHU VZLQH ZHUH DEOH WR DFHW\ODWH 1Af VXOILVR[D]ROH DW D IDVWHU UDWH DV VKRZQ E\ WKH VKRUWHU WLPH IRU PD[LPXP VHUXP DFHW\OVXOILVR[Dn ]ROH LQ VZLQH WR KRXUVf DV FRPSDUHG WR KXPDQV KRXUVf +XPDQV KDG D PHDQ W RI KRXUV 7DEOH f DIWHU RUDO DGPLQLVWUDWLRQ ZKLFK LV KD ORQJHU WKDQ WKH KRXUV SUHYLRXVO\ UHSRUWHG f 6ZLQH KDG D PHDQ W RI KRXUV DIWHU RUDO DGPLQLVWUDWLRQ DQG KRXUV DIWHU +D LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7KH W RI DFHW\O S VXOILVR[D]ROH ZDV KRXUV DIWHU RUDO DGPLQLVWUDWLRQ DQG KRXUV r*DQWULVLQ DFWLYH LQJUHGLHQW VXOILVR[D]ROHf +RIIPDQ/D5RFKH 1XWOH\ 1-

PAGE 77

7DEOH 7KH ELRORJLFDO KDOIOLIH WAf RI DFHW\OVXOILVR[D]ROH WKH 1 PHWDEROLWH RI VXOILVR[D]ROH WW KRXUVf 6V KRXUVf 'RJV ,QWUDYHQRXV 1'r 1'r 2UDO 1'r 1'r 6ZLQH ,QWUDYHQRXV 2UDO +XPDQ 2UDO f§ r1' 1RQ GHWHFWDEOH 1R 1 f DFHW\OVXOILVR[D]ROH ZDV GHWHFWHG LQ GRJV

PAGE 78

DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7DEOH f LQ VZLQH 'RJV GLG QRW PHWDEROL]H VXOILVR[D]ROH WR WKH 1 f DFHW\O PHWDEROLWH 7KH GLVWULEXWLRQ FRQVWDQWV RI VXOILVR[D]ROH 7DEOH f IURP WKH FHQWUDO WR WKH SHULSKHUDO FRPSDUWPHQW NAf ZDV JUHDWHVW LQ KXPDQV KRXUV Af IROORZHG E\ VZLQH KRXUV Af DQG WKHQ GRJV KRXUV Af 7KH GLVWULEXWLRQ IURP WKH SHULSKHUDO FRPSDUWPHQW WR WKH FHQWUDO FRPSDUWPHQW ZDV IDVWHVW LQ KXPDQV KRXUV Af IROORZHG E\ GRJV KRXUV Af DQG WKHQ VZLQH KRXUV Af 7KH HOLPLQDn WLRQ FRQVWDQWV NAf ZHUH JUHDWHVW LQ VZLQH KRXUV Af IROORZHG E\ KXPDQV KRXUV Af DQG WKHQ GRJV KRXUV Af 7KH RUGHU RI WKH HOLPLQDWLRQ FRQVWDQW N f ZDV WKH VDPH DV WKH W RI IUHH 8 D VXOILVR[D]ROH VKRUWHVW LQ VZLQH WKHQ GRJV DQG ORQJHVW LQ KXPDQV 7KH GLIIHUHQFH LQ GLVWULEXWLRQ EHWZHHQ WKH WZR FRPSDUWPHQWV NANAf ZDV KLJKHVW LQ KXPDQV f IROORZHG E\ GRJV DQG WKHQ VZLQH LQGLFDWLQJ WKDW WKH GUXJ UHWXUQHG IURP WKH SHULSKHUDO FRPSDUWPHQW WR WKH FHQWUDO FRPSDUWPHQW IDVWHVW LQ KXPDQV DQG VORZHVW LQ VZLQH 7DEOH f 7KHVH YDOXHV ZHUH FRQFXUUHQW ZLWK WKH PHDQ KDOIOLIH RI VXOILVR[D]ROH LQ WKH VHFRQG FRPSDUWPHQW ZKHUH WA ZDV UHSRUWHG DV VKRUWHVW LQ KXPDQV f IROORZHG E\ GRJV DQG ORQJHVW LQ VZLQH 7DEOH f 7KH UDWLR RI 6NA4 ZDV UHSRUWHG DV LQ KXPDQV f LQ GRJV DQG LQ VZLQH 7DEOH f 7KLV LV WKH VDPH DV WKH AA Af UDWLR VR WKDW PRUH VXOILVR[D]ROH LV DYDLODEOH IRU HOLPLQDWLRQ IURP WKH SRVWn GLVWULEXWLYH SKDVH LQ KXPDQV IROORZHG E\ GRJV DQG WKH OHDVW DYDLODEOH ZDV LQ VZLQH 7KH PHDQ YROXPH RI GLVWULEXWLRQ IRU WKH FHQWUDO FRPSDUWPHQW 9Af ZDV DSSUR[LPDWHO\ WKH VDPH LQ GRJV / DQG VZLQH / 7DEOH f 7KH PHDQ YROXPH RI WKH VHFRQG FRPSDUWPHQW ZDV PXFK ODUJHU LQ VZLQH

PAGE 79

7DEOH 7KH PHDQ GLVWULEXWLRQ FRQVWDQWV DIWHU LQWUDYHQRXV DGPLQLVWUDn WLRQ RI VXOILVR[D]ROHr N KRXUV N KRXUV nrf¬f N KRXUV ‘rf 'RJV 6ZLQH +XPDQV A r0HDQ RI LQGLYLGXDOV .DSODQ DO f

PAGE 80

7DEOH 7KH UDWLR RI GLVWULEXWLRQ FRQVWDQWV RI VXOILVR[D]ROH DIWHU DGPLQLVWUDWLRQ NN N 'RJV 6ZLQH +XPDQVr r.DSODQ HBW DO f

PAGE 81

/ WKDQ LQ GRJV / 7DEOH f 7KH FDOFXODWHG IURP SUHn YLRXV GDWD f ZDV / DQG / IRU LQ KXPDQV 'XH WR WKH DQDO\WLFDO SURFHGXUH DQG WKH OHQJWK RI WKH WULDO f WKLV VKRXOG QRW EH FRPSDUHG ZLWK WKH UHSRUWHG LQ GRJV DQG VZLQH )URP WKH FDOFXODWHG IRU GRJV VZLQH DQG KXPDQV WKHUH LV DQ DSSUR[LPDWH b GLIIHUHQFH LQ WKH DQLPDO RYHU WKH KXPDQ YROXPH RI GLVWULEXWLRQ 7KH YROXPH RI GLVWULEXWLRQ LQ WKH VHFRQG FRPSDUWPHQW 9Af LV PXFK ODUJHU LQ VZLQH WKDQ LQ GRJV 7KH PHDQ ELRDYDLODELOLW\ )f ZDV JUHDWHVW LQ KXPDQV b ZKHQ WKH DUHD XQGHU WKH LQWUDYHQRXV FXUYH IURP .DSODQ HUW DO f ZDV XVHG 7DEOH f 7KLV YDOXH DJUHHG ZLWK SUHYLRXV GDWD f WKDW ) ZDV b LQ KXPDQV 7KH DEVROXWH ELRDYDLODELOLW\ RI VXOILVR[D]ROH ZDV b LQ GRJV DQG b LQ VZLQH 7DEOH f 7KH ELRDYDLODELOLW\ LQ GRJV LV FORVHU WR WKH FDOFXODWHG ) LQ KXPDQV DQG VZLQH DSSHDU WR EH YDVWO\ GLIIHUHQW IURP ERWK KXPDQV DQG GRJV +RZHYHU WKH KLJKHU ELRDYDLODELOLW\ LQ KXPDQV LV GXH WR HQWHURKHSDWLF FLUFXODWLRQ RI VXOILVR[D]ROH ZKLFK GRHV QRW RFFXU LQ GRJV DQG VZLQH 7KH PDMRULW\ RI VXOILVR[D]ROH LV QRW DEVRUEHG IURP WKH JDVWURLQWHVWLQDO WUDFW LQ VZLQH DQG LV H[FUHWHG LQ WKH IHFHV 7KH GHJUHH RI SURWHLQ ELQGLQJ LQL YLYR ZDV OHVV WKDQ b LQ GRJV IRU WKH ILUVW KRXUV DQG LQ SLJV IRU WKH ILUVW KRXU DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ DQG LQ DOO KXPDQV IRU WKH ILUVW KRXU DIWHU RUDO DGPLQLVn WUDWLRQ RI VXOILVR[D]ROH 0RUH WKDQ b RI VXOILVR[D]ROH ZDV ERXQG WR SODVPD SURWHLQV LQ GRJV DW KRXUV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[Dn ]ROH 'XULQJ WKH UHPDLQGHU RI WKH WULDO WR b RI VXOILVR[D]ROH ZDV ERXQG WR SODVPD SURWHLQV LQ KXPDQV GRJV DQG VZLQH ZKLFK FRPSDUHG IDYRUDEO\ ZLWK WKH b SUHYLRXVO\ UHSRUWHG f 7KLV REVHUYDWLRQ

PAGE 82

7DEOH &RPSDULVRQ RI WKH YROXPHV RI GLVWULEXWLRQ LQ GRJV VZLQH DQG KXPDQV DIWHU DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 6SHFLHV 9 9 OLWHUVf OLWHUVf 'RJV 6ZLQH +XPDQVr r.DSODQ BHWB DO f

PAGE 83

7DEOH &RPSDULVRQ RI WKH ELRDYDLODELOLW\r RI VXOILVR[D]ROH LQ GRJV VZLQH DQG KXPDQV 6SHFLHV %LRDYDLODELOLW\ 'RJV 6ZLQH +XPDQV &DOFXODWHG IURP DUHD XQGHU WKH SODVPD FXUYH

PAGE 84

VXJJHVWV WKDW DQ LQGLYLGXDO GLIIHUHQFH RFFXUV DORQJ ZLWK D VSHFLHV DQG WUHDWPHQW GLIIHUHQFH )LIW\ WR VL[W\WKUHH SHUFHQW RI VXOILVR[D]ROH ZDV ERXQG WR KXPDQ VHUXP SURWHLQV LQ YLWUR 7DEOH f DW WR \JPO $W \JPO RQO\ b RI VXOILVR[D]ROH ZDV ERXQG 7KLV VXJJHVWV WKDW WKH GHJUHH RI SURWHLQ ELQGLQJ LV FDSDFLW\ OLPLWHG DV WKH VHUXP FRQFHQn WUDWLRQ LQFUHDVHV DERYH \JPO 7KLV K\SRWKHVLV ZRXOG H[SODLQ WKH ORZ IUDFWLRQ ERXQG ZKLFK ZDV REVHUYHG GXULQJ WKLV H[SHULPHQW 7KH DPRXQW RI IUHH VXOILVR[D]ROH H[FUHWHG LQ WKH XULQH ZDV b RI WKH GRVH LQ KRXUV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ DQG b RI WKH GRVH LQ KRXUV DIWHU RUDO DGPLQLVWUDWLRQ LQ GRJV 7KH DPRXQW H[FUHWHG LQ WKH XULQH E\ VZLQH ZDV b RI WKH GRVH DQG b RI WKH GRVH LQ KRXUV DIWHU RUDO DQG LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D ]ROH UHVSHFWLYHO\ %\ WKH HQG RI WKH WULDO KRXUV DQG KRXUV IRU WKH LQWUDYHQRXV DQG RUDO URXWHV UHVSHFWLYHO\ PRUH WKDQ b RI WKH GRVH ZDV H[FUHWHG LQ VZLQH 7DEOH f DQG LQ WKH GRJV DIWHU RUDO DGPLQLVn WUDWLRQ 7DEOH f $IWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RYHU b RI WKH GRVH ZDV H[FUHWHG LQ WKH XULQH E\ GRJV .DSODQ HAW DA/ f UHSRUWHG WKDW b RI WKH GRVH ZDV H[FUHWHG DV IUHH VXOILVR[D]ROH E\ KXPDQV $FHW\OVXOILVR[D]ROH DFFRXQWHG IRU OHVV WKDQ KDOI RI WKH XULQDU\ OHYHO LQ VZLQH 7DEOH f ZKLFK FRQILUPHG WKH SUHYLRXVO\ UHSRUWHG b f H[FUHWHG DV WKH DFHW\O PHWDEROLWH LQ KXPDQV 7KH GLIIHUHQFHV EHWZHHQ WKH KXPDQ DQG GRJ DQG SLJ H[FUHWLRQ OHYHOV FRXOG EH H[SODLQHG SDUWO\ E\ WKH DQDO\WLFDO PHWKRG XVHG .DSODQ HW DO f XVHG D QRQVSHFLILF PHWKRG IRU DURPDWLF DPLQHV WKH %UDWWRQ0DUVKDOO PHWKRG f 7KLV H[SHULPHQW XVHG D PRUH VSHFLILF PHWKRG IRU ERWK IUHH DQG DFHW\OVXOILVR[D]ROH

PAGE 85

7DEOH 7KH IUDFWLRQ RI VXOILVR[D]ROH ERXQG IJf WR KXPDQ VHUXP SURWHLQV L[L YLWUR 6XOILVR[D]ROH &RQFHQWUDWLRQ SJPOf )UHH 6HUXP &RQFHQWUDWLRQ RI 6XOILVR[D]ROH SJPOf I% bf

PAGE 86

6HUXP %LOLUXELQ &RQFHQWUDWLRQV 'RJVf§,QWUDYHQRXV $GPLQLVWUDWLRQ 7KH PHDQ WRWDO ELOLUXELQ FRQFHQWUDWLRQ LQ GRJV DGPLQLVWHUHG VXOILVR[D]ROH LQWUDYHQRXVO\ H[KLELWHG VWDWLVWLFDOO\ VLJQLILFDQW S f OLQHDU LQFUHDVHV f DW S f S f S f DQG S f KRXUV ZLWK WKH PD[LPXP OHYHOV DW DQG KRXUV )LJXUH f %\ WKH QH[W VDPSOLQJ SHULRG KRXUV WKH PHDQ WRWDO ELOLUXELQ FRQFHQn WUDWLRQ KDG GHFUHDVHG WR OHYHOV ZKLFK ZHUH QRW VLJQLILFDQWO\ GLIIHUHQW IURP FRQWURO YDOXHV XQWLO WKH KRXU VDPSOLQJ SHULRG S f 7KH PHDQ FRQMXJDWHG ELOLUXELQ OHYHOV DOVR VKRZHG VWDWLVWLFDOO\ VLJQLILFDQW LQFUHDVHV DW KRXUV S f DQG FRQWLQXHG WKURXJKRXW WKH VDPSOLQJ SHULRG S f KRXUV 7KHUH ZDV D VLJQLILFDQW S f PD[LPXP PHDQ FRQMXJDWHG ELOLUXELQ DW KRXUV ZKLFK FRLQFLGHG ZLWK WKH PD[LPXP PHDQ WRWDO ELOLUXELQ FRQFHQWUDWLRQ )LJXUH f $ VHFRQG LQFUHDVH ZDV REVHUYHG DW KRXUV S f ZKLFK FRLQFLGHG ZLWK WKH VHFRQG LQn FUHDVH LQ WRWDO ELOLUXELQ )LJXUH f 7KH PHDQ LQGLUHFW ELOLUXELQ FRQn FHQWUDWLRQ )LJXUH f ZDV QRW VLJQLILFDQWO\ GLIIHUHQW IURP WKH FRQWURO RU OHYHO WKURXJKRXW WKH VDPSOLQJ SHULRG 7KH PHDQ WRWDO ELOLUXELQ ZDV VLJQLILFDQWO\ FRUUHODWHG ZLWK PHDQ FRQMXJDWHG ELOLUXELQ 5 S f DQG ZLWK PHDQ LQGLUHFW ELOLn UXELQ 5 S f DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ 7KH VLJQLILFDQW FRUUHODWLRQ RI WRWDO DQG FRQMXJDWHG ELOLUXELQ LV GXH WR WKH FRQFRPLWDQW LQFUHDVH LQ JOXFXURQLGDWLRQ DFWLYLW\ DV WRWDO ELOLUXELQ OHYHO LQFUHDVH ,I WKH JOXFXURQLGDWLRQ HQ]\PH V\VWHP LV QRW FDSDFLW\ OLPLWHG WKH SRVVLELOLW\ RI NHUQLFWHUXV GXH WR DQ LQFUHDVH LQ LQGLUHFW ELOLUXELQ LV PLQLPL]HG LQ WKH GRJ DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 87

0($1 6(580 %,/,58%,1 &21&(175$7,216PJGOf )LJXUH 0HDQ VHUXP ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 88

7KLV HQ]\PH V\VWHP DFWV WR PDLQWDLQ D UHGXFHG RU OLPLWHG LQGLUHFW ELOLn UXELQ FRQFHQWUDWLRQ DQG UHGXFHV WKH WR[LFLW\ RI GLVSODFHG SURWHLQ ERXQG ELOLUXELQ RU LQFUHDVHG KHPH GHJUDGDWLRQ 7KLV ZDV IXUWKHU HPSKDn VL]HG E\ WKH VLJQLILFDQW QHJDWLYH FRUUHODWLRQ RI FRQMXJDWHG DQG LQGLUHFW ELOLUXELQ 5 S f $V WKH OHYHO RI FRQMXJDWHG ELOLUXELQ LQFUHDVHG WKH LQGLUHFW ELOLUXELQ OHYHO GHFUHDVHG $ VHFRQG H[SODQDWLRQ ZRXOG EH WKDW VXOILVR[D]ROH DOWHUV WKH IXQFWLRQ RI WKH QRUPDO KHSDWRF\WH WR LQFUHDVH FRQMXJDWHG ELOLUXELQ UHJXUJLWDWLRQ LQWR WKH JHQHUDO FLUFXODWLRQ LQVWHDG RI EHLQJ H[FUHWHG LQWR WKH ELOH 7KH UHVXOWLQJ LQFUHDVH LQ WRWDO ELOLUXELQ ZRXOG EH WKH UHVXOW RI WKH LQFUHDVHG OHYHO RI UHJXUJLWDWHG FRQMXJDWHG ELOLUXELQ DORQJ ZLWK WKH QRUPDO OHYHO RI LQGLUHFW ELOLUXELQ 'RJVf§2UDO $GPLQLVWUDWLRQ :KHQ VXOILVR[D]ROH ZDV DGPLQLVWHUHG RUDOO\ WR GRJV WKHUH ZDV D VLJQLILFDQW OLQHDU LQFUHDVH LQ PHDQ WRWDO S f FRQMXJDWHG S f DQG LQGLUHFW S f ELOLUXELQ )LJXUH f 7RWDO ELOLUXELQ UHDFKHG LWV ILUVW VLJQLILFDQW SHDN DW KRXUV S f DQG D KLJKHU FRQFHQn WUDWLRQ DW KRXUV S f )LJXUH f 7KH PHDQ WRWDO ELOLUXELQ OHYHOV ZHUH VLJQLILFDQWO\ LQFUHDVHG DW S f S f DQG S f KRXUV $ VHFRQG KLJKHU SHDN RFFXUUHG DW KRXUV S f DQG UHPDLQHG JUHDWHU WKDQ WKH FRQWURO OHYHOV DW WKH HQG RI WKH VDPSOLQJ SHULRG KRXUV S f )LJXUH f 7KH PHDQ FRQMXJDWHG ELOLUXELQ OHYHOV ZHUH DOVR OLQHDUO\ LQFUHDVHG S f GXULQJ WKH VDPSOLQJ SHULRG 6LJQLILFDQW LQFUHDVHV S f ZHUH REVHUYHG DW DQG KRXUV )LJXUH f WKH VDPH SHULRGV LQ ZKLFK WRWDO ELOLUXELQ ZDV

PAGE 89

0($1 6(580 %,/,58%,1 &21&(175$7,216 PTGOf )LJXUH 0HDQ VHUXP ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 90

VLJQLILFDQWO\ LQFUHDVHG $IWHU WKH KRXU SHULRG WKH PHDQ FRQMXJDWHG ELOLUXELQ KDG UHWXUQHG WR OHYHOV ZKLFK ZHUH QRW VLJQLILFDQWO\ GLIIHUHQW IURP FRQWURO OHYHOV 7KH PHDQ FRQMXJDWHG ELOLUXELQ UHDFKHG LWV PD[LPXP FRQFHQWUDWLRQ PJGOf DW KRXUV ZKLOH WKH PD[LPXP WRWDO ELOLUXELQ PJGOf RFFXUUHG KRXUV ODWHU DW WKH KRXU VDPSOLQJ SHULRG 7DEOH f 7KH PHDQ LQGLUHFW ELOLUXELQ ZDV QRW VLJQLILFDQWO\ GLIIHUHQW IURP FRQWURO OHYHOV XQWLO WKH DQG KRXU SHULRGV S f 0HDQ LQGLUHFW ELOLUXELQ UHDFKHG D PD[LPXP RI PJGO DW KRXUV EXW KDG EHJXQ WR GHFOLQH E\ WKH HQG RI WKH WULDO KRXUV 7RWDO ELOLUXELQ ZDV VLJQLILFDQWO\ FRUUHODWHG ZLWK FRQMXJDWHG ELOLn UXELQ 5 S f DQG ZLWK LQGLUHFW ELOLUXELQ 5 S f ZKLOH FRQMXJDWHG ELOLUXELQ ZDV DOVR QHJDWLYHO\ FRUUHODWHG ZLWK LQGLUHFW ELOLUXELQ 5 S f 7KH VLJQLILFDQW FRUUHODn WLRQ RI WRWDO DQG FRQMXJDWHG ELOLUXELQ LV H[SODLQHG E\ DQ LQFUHDVH LQ JOXFXURQLGDWLRQ DV LQFUHDVHG KHPH GHJUDGDWLRQ RU LQFUHDVHG LQGLUHFW ELOLUXELQ RFFXUV RU GXH WR UHJXUJLWDWLRQ RI FRQMXJDWHG ELOLUXELQ LQWR WKH JHQHUDO FLUFXODWLRQ 7KH VLJQLILFDQW FRUUHODWLRQ RI LQGLUHFW ELOLUXELQ DQG WRWDO ELOLUXELQ LOOXVWUDWHG WKDW WKH JOXFXURQLGDWLRQ DFWLYLW\ FRXOG QRW DFFRXQW IRU FRQMXJDWLRQ RI DOO WKH ELOLUXELQ SUHVHQW 7KH QHJDWLYH FRUUHODWLRQ RI LQGLUHFW DQG FRQMXJDWHG ELOLUXELQ LV VXSSRUWLYH HYLGHQFH WKDW WR[LF LQGLUHFW ELOLUXELQ OHYHOV FDQ EH UHGXFHG WR SUHYHQW NHUQLFWHUXV LI JOXFRURQLGDWLRQ DFWLYLW\ FDQ EH VWLPXODWHG &RPSDULVRQ RI %LOLUXELQ /HYHOV LQ 'RJV 7KH PHDQ WRWDO ELOLUXELQ UHDFKHG PD[LPXP DW KRXUV DIWHU LQWUDn YHQRXV PJGOf DQG RUDO PJGOf DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 91

)LJXUH f ZLWK D VHFRQG LQFUHDVH DW KRXUV DIWHU LQWUDYHQRXV DGPLQLVn WUDWLRQ DQG KRXUV DIWHU RUDO DGPLQLVWUDWLRQ &RQMXJDWHG ELOLUXELQ ZDV DOVR PD[LPXP LQ WKH DQG KRXU SHULRG DIWHU RUDO PJGOf DQG LQWUDYHQRXV PJGOf DGPLQLVWUDWLRQ RI VXOILVR[D]ROH )LJXUH f UHVSHFWLYHO\ &RQMXJDWHG ELOLUXELQ ZDV VLJQLILFDQWO\ HOHYDWHG DW WKH KRXU SHULRG DQG WKURXJKRXW WKH WULDO SHULRG DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ DIWHU RUDO DGPLQLVWUDWLRQ FRQMXJDWHG ELOLUXELQ ZDV VLJQLILFDQWO\ LQFUHDVHG EHWZHHQ DQG KRXUV RQO\ ,QGLUHFW ELOLUXELQ ZDV QRW VLJQLILFDQWO\ LQFUHDVHG DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ EXW ZDV VLJQLILFDQWO\ LQFUHDVHG DW DQG KRXUV DIWHU RUDO DGPLQLVWUDWLRQ )LJXUH f 7KH WRWDO ELOLUXELQ LQFUHDVH DIWHU RUDO RU LQWUDYHQRXV DGPLQLVWUDn WLRQ RI VXOILVR[D]ROH ZDV DFFRPSDQLHG E\ DQ LQFUHDVH LQ JOXFXURQLGDWLRQ DFWLYLW\ ZKLFK LQFUHDVHG WKH FRQMXJDWHG ZDWHUVROXEOH ELOLUXELQ OHYHOV 7KLV SUHYHQWHG WR[LFLW\ E\ UHGXFLQJ WKH LQGLUHFW ELOLUXELQ OHYHO DQG LQFUHDVLQJ WKH ZDWHUVROXEOH FRQMXJDWHG ELOLUXELQ ZKLFK LV PRUH HDVLO\ H[FUHWHG 7KH JOXFXURQLGDWLRQ DFWLYLW\ ZDV UHGXFHG GXULQJ WKH ODWHU SHULRGV DIWHU RUDO DGPLQLVWUDWLRQ DOORZLQJ WKH SRWHQWLDOO\ WR[LF LQn GLUHFW ELOLUXELQ WR LQFUHDVH 7KHUH ZDV D QHJDWLYH FRUUHODWLRQ RI FRQMXJDWHG DQG LQGLUHFW ELOLUXELQ DIWHU LQWUDYHQRXV 5 S f DQG RUDO 5 S f DGPLQLVWUDWLRQ RI VXOILVR[D]ROH $IWHU LQWUDYHQRXV DGPLQLVWUDWLRQ FRQMXJDWHG ELOLUXELQ OHYHOV LQFUHDVHG DORQJ ZLWK DQ LQFUHDVH LQ WRWDO ELOLUXELQ )LJXUH f $IWHU RUDO VXOILVR[D]ROH DGPLQLVWUDWLRQ FRQMXJDWHG ELOLUXELQ OHYHOV LQFUHDVHG LQLWLDOO\ DV WRWDO ELOLUXELQ OHYHOV LQFUHDVHG EXW ODWHU GHFUHDVHG DOORZLQJ LQGLUHFW ELOLUXELQ OHYHOV WR LQFUHDVH 7KLV UHVXOW OHDGV RQH WR FRQFOXGH WKDW VRPH SDWKRORJLFDO HYHQW LV LQGXFHG

PAGE 92

727$/ %,/,58%,1 &21&(175$7,216 PJGO f )LJXUH &RPSDULVRQ RI WKH PHDQ WRWDO ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 93

0($1 fµ2 )LJXUH &RPSDULVRQ RI WKH PHDQ FRQMXJDWHG ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 94

0($1 ,1',5(&7 %,/,58%,1 &21&(175$7,216 PJG ,f )LJXUH &RPSDULVRQ RI WKH PHDQ LQGLUHFW ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 95

GXULQJ RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH EXW QRW DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ ZKLFK FRXOG DOORZ LQGLUHFW ELOLUXELQ WR UHDFK WR[LF OHYHOV 7KLV SDWKRORJLFDO HYHQW FRXOG EH H[SODLQHG E\ WKH SKDUPDFRORJLFDO ILUVWSDVV HIIHFW f ,Q WKH ILUVWSDVV HIIHFW DOO RI DQ DEVRUEHG GUXJ LV SUHVHQWHG WR WKH OLYHU EHIRUH GLVWULEXWLRQ ZKHUHDV DIWHU LQWUDn YHQRXV DGPLQLVWUDWLRQ RQO\ b RI WKH GUXJ SDVVHV WKURXJK WKH OLYHU EHIRUH EHLQJ GLVWULEXWHG RU H[FUHWHG ,Q WKLV FDVH WKH JUHDWHU FRQFHQn WUDWLRQ RI VXOILVR[D]ROH SUHVHQWHG WR WKH OLYHU LQ DGGLWLRQ WR WKH ODFN RI DFHW\ODWLRQ RI WKH GUXJ FRXOG EH UHVSRQVLEOH IRU DOWHULQJ WKH QRUPDO GHWR[LILFDWLRQ RI ELOLUXELQ VR WKDW RQH REVHUYHV DQ LQFUHDVH LQ LQGLUHFW ELOLUXELQ 7RWDO DQG FRQMXJDWHG ELOLUXELQ UHDFKHG KLJKHU OHYHOV ZLWKLQ WR KRXUV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ WKDQ DIWHU RUDO DGPLQLVWUDWLRQ )LJXUHV DQG f $IWHU RUDO DGPLQLVWUDWLRQ FRQMXJDWHG ELOLUXELQ UHDFKHG LWV PD[LPXP ZLWKLQ WKLV WLPH SHULRG KRXUVf EXW WRWDO DQG LQGLUHFW ELOLUXELQ GLG QRW UHDFK PD[LPXP XQWLO KRXUV 7KH LQFUHDVHG LQGLUHFW ELOLUXELQ OHYHOV VKRXOG EH H[SHFWHG DIWHU LQWUDYHQRXV DGPLQLVn WUDWLRQ GXH WR WKH LQFUHDVHG VHUXP FRQFHQWUDWLRQ RI VXOILVR[D]ROH ZKHQ FRPSDUHG WR OHYHOV IROORZLQJ RUDO DGPLQLVWUDWLRQ ZKLFK ZRXOG DOORZ VXOILVR[D]ROH WR GLVSODFH PRUH ELOLUXELQ IURP DOEXPLQ 7KH LQFUHDVHG WRWDO DQG LQGLUHFW ELOLUXELQ OHYHOV DW WKH HQG RI WKH RUDO DGPLQLVWUDWLRQ WULDO VXSSRUWV WKH K\SRWKHVLV WKDW D SKDUPDFRORJLFDO HYHQW LV LQGXFHG E\ WKH RUDO URXWH EXW QRW E\ WKH LQWUDYHQRXV URXWH ZKLFK FDXVHG DQ LQFUHDVH LQ WRWDO DQG LQGLUHFW ELOLUXELQ ZLWK SRVVLEOH WR[LF VLGH HIIHFWV DW D WLPH PXFK ODWHU DIWHU D VLQJOH RUDO GRVH RI VXOILVR[D]ROH 7KHUH ZHUH QR VWDWLVWLFDO GLIIHUHQFHV LQ VHUXP WRWDO FRQMXJDWHG RU LQGLUHFW ELOLUXELQ EHWZHHQ LQGLYLGXDO GRJV DIWHU RUDO RU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 96

0($1 6(580 %,/,58%,1 &21&(175$7,216 PJG ,f )LJXUH 0HDQ VHUXP ELOLUXELQ FRQFHQWUDWLRQV LQ VZLQH DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 97

6ZLQHf§,QWUDYHQRXV $GPLQLVWUDWLRQ $Q DQDO\VLV RI YDULDQFH XVLQJ PHDQ ELOLUXELQ FRQFHQWUDWLRQV UHn YHDOHG QR VWDWLVWLFDO GLIIHUHQFH DPRQJ WKH SLJV RU GXULQJ WLPH LQWHUYDOV IRU VHUXP WRWDO FRQMXJDWHG RU LQGLUHFW ELOLUXELQ DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7KH PHDQ WRWDO FRQMXJDWHG DQG LQGLUHFW ELOLUXELQ ZHUH LQFUHDVLQJ DW WKH HQG RI WKH WULDO EXW ZHUH QRW VLJQLILn FDQWO\ GLIIHUHQW WKDQ FRQWURO OHYHOV )LJXUH f 7RWDO ELOLUXELQ ZDV VLJQLILFDQWO\ FRUUHODWHG ZLWK FRQMXJDWHG ELOLn UXELQ 5 S f DQG ZLWK LQGLUHFW ELOLUXELQ 5 S f DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 6ZLQHf§2UDO $GPLQLVWUDWLRQ $IWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH PHDQ WRWDO ELOLUXELQ ZDV VLJQLILFDQWO\ LQFUHDVHG S f WR PJGO DW KRXUV )LJXUH f $ SDUDOOHO VLJQLILFDQW S f LQFUHDVH ZDV DOVR REVHUYHG LQ PHDQ FRQn MXJDWHG ELOLUXELQ DW KRXUV PJGO )LJXUH f $ VLJQLILFDQW S f OLQHDU GHFUHDVH GLG RFFXU RYHU WKH WULDO SHULRG LQ FRQMXJDWHG ELOLUXELQ 0HDQ LQGLUHFW ELOLUXELQ ZDV QRW VLJQLILFDQWO\ GLIIHUHQW IURP FRQWURO YDOXHV DW DQ\ WLPH DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH +RZHYHU LQGLUHFW ELOLUXELQ OHYHOV GLG H[FHHG FRQMXJDWHG OHYHOV DIWHU WKH KRXU VDPSOLQJ SHULRG DQG UHPDLQHG DW DQ LQFUHDVHG OHYHO WKURXJKRXW WKH UHPDLQGHU RI WKH WULDO )LJXUH f 7RWDO ELOLUXELQ ZDV VLJQLILFDQWO\ FRUUHODWHG ZLWK FRQMXJDWHG ELOLUXELQ 5 S f DQG ZLWK LQGLUHFW ELOLUXELQ 5 S f DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 98

0($1 6(580 %,/,58%,1 &21&(175$7,216 PJGOf )LJXUH 0HDQ VHUXP ELOLUXELQ FRQFHQWUDWLRQV LQ VZLQH DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 99

&RPSDULVRQ RI %LOLUXELQ /HYHOV LQ 6ZLQH ,W DSSHDUV WKDW DQ LQFUHDVH LQ WRWDO ELOLUXELQ LQ WKH SLJ LV DFFRPSDQLHG E\ DQ LQFUHDVH LQ FRQMXJDWHG DQG LQGLUHFW ELOLUXELQ UHJDUGOHVV RI WKH URXWH RI DGPLQLVWUDWLRQ DV VKRZQ E\ WKH SRVLWLYH FRUUHODWLRQ FRHIILFLHQWV 7KH KLJKHU VHUXP IUHH VXOILVR[D]ROH FRQFHQWUDWLRQV 7DEOHV WR f DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ GLG QRW VLJQLILFDQWO\ DIIHFW ELOLUXELQ FRQFHQWUDWLRQV )LJXUH f EXW RUDO DGPLQLVWUDWLRQ GLG VLJQLILFDQWO\ LQFUHDVH WRWDO S f DQG FRQMXJDWHG S f ELOLn UXELQ DW KRXUV EXW GLG QRW DIIHFW LQGLUHFW ELOLUXELQ )LJXUH f ,QGLUHFW ELOLUXELQ ZDV FORVHO\ FRUUHODWHG ZLWK FRQMXJDWHG ELOLUXELQ DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ )LJXUH f EXW H[FHHGHG FRQMXJDWHG ELOLUXELQ DIWHU RUDO DGPLQLVWUDWLRQ )LJXUH f ZKLFK VXJJHVWV WKDW DQ RUDO GRVH LV KDQGOHG GLIIHUHQWO\ WKDQ WKH LQWUDYHQRXV RQH ,QWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH GRHV QRW DSSHDU WR DIIHFW ELOLUXELQ OHYHOV LQ WKH SLJ $IWHU RUDO DGPLQLVWUDWLRQ DQ LQn FUHDVH LQ WRWDO ELOLUXELQ FRXOG EH DFFRPSDQLHG E\ DQ LQFUHDVH LQ WKH FRQMXJDWHG OHYHO ZKLFK UHGXFHV WKH KD]DUG RI LQGLUHFW ELOLUXELQ WR[LFLW\ :KLOH WKH LQGLUHFW ELOLUXELQ OHYHOV ZHUH QRW VLJQLILFDQWO\ GLIIHUHQW E\ HLWKHU WUHDWPHQW RQH VKRXOG QRWH WKDW LQGLUHFW ELOLUXELQ OHYHOV GLG H[FHHG FRQMXJDWHG ELOLUXELQ OHYHOV RQO\ IROORZLQJ RUDO DGPLQLVWUDWLRQ )LJXUH f :KLOH QR WR[LF OHYHOV RI ELOLUXELQ KDYH EHHQ HVWDEOLVKHG LQ VZLQH WKHUH ZDV D VLJQLILFDQW LQFUHDVH LQ WRWDO )LJXUH f DQG FRQMXJDWHG )LJXUH f ELOLUXELQ LQ KRXUV DIWHU RUDO DGPLQLVWUDWLRQ ZKLFK GLG QRW RFFXU DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ 7KLV VXJJHVWV WKDW DQ RUDO GRVH RI VXOILVR[D]ROH PD\ KDYH D GLIIHUHQW HIIHFW LQ YLYR WKDQ DQ LQWUDYHQRXV DGPLQLVWUDWLRQ HYHQ WKRXJK WKH LQWUDYHQRXV URXWH

PAGE 100

0($1 727$/ %,/,58%,1 &21&(175$7,21 PTGOf )LJXUH &RPSDULVRQ RI WKH PHDQ WRWDO ELOLUXELQ FRQFHQWUDWLRQV LQ VZLQH DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 101

0($1 &21-8*$7(' %,/,58%,1 &21&(175$7,216PJGOf )LJXUH &RPSDULVRQ RI WKH PHDQ FRQMXJDWHG ELOLUXELQ FRQFHQWUDWLRQV LQ VZLQH DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 102

0($1 ,1',5(&7 %,/L58%,1 &21&(175$7,216 PJGOf )LJXUH &RPSDULVRQ RI WKH PHDQ LQGLUHFW ELOLUXELQ FRQFHQWUDWLRQV LQ VZLQH DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 103

FUHDWHG DQ LQFUHDVHG VHUXP FRQFHQWUDWLRQ RI VXOILVR[D]ROH ZKHQ FRPSDUHG WR WKH VHUXP OHYHO DIWHU RUDO DGPLQLVWUDWLRQ SRVVLEO\ GXH WR D ILUVW SDVVf° HIIHFW f ,Q VZLQH WKH URXWH RI DGPLQLVWUDWLRQ GRHV QRW DSSHDU WR DIIHFW WKH FRQFHQWUDWLRQ RI WKH SRWHQWLDOO\ WR[LF LQGLUHFW ELOLUXELQ )LJXUH f 7KLV VSHFLHV HIIHFW LQ VZLQH FRXOG SUHYHQW ELOLUXELQ WR[LFLW\ LQGXFHG E\ VXOIRQDPLGHV +XPDQVf§2UDO $GPLQLVWUDWLRQ $Q DQDO\VLV RI YDULDQFH HYDOXDWLRQ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH UHVXOWHG LQ QR VWDWLVWLFDO GLIIHUHQFH LQ PHDQ WRWDO FRQMXJDWHG DQG LQGLUHFW ELOLUXELQ OHYHOV )LJXUH f 7KH ODFN RI VLJQLILFDQFH FRXOG EH GXH WR f WKH ORZ WKHUDSHXWLF GRVH RI JP RU DSSUR[LPDWHO\ PJNJ DGPLQLVWHUHG WR WKH KXPDQ VXEMHFWV f KXPDQV DUH DEOH WR UHGXFH WKH LQ YLYR HIIHFWV RI VXOILVR[D]ROH RQ WKH KHSDWRELOLDU\ V\VWHP RU f WKH VDPSOLQJ SHULRG ZDV WRR VKRUW DQG DGGLWLRQDO VDPSOHV VKRXOG KDYH EHHQ WDNHQ RYHU D ORQJHU WULDO SHULRG 7KH YDOXHV UHFRUGHG DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH ZHUH ZLWKLQ WKH QRUPDO OLPLWV UHSRUWHG IRU WKH $XWRPDWLF &OLQLFDO $QDO\]HU f &RPSDULVRQ RI %LOLUXELQ /HYHOV LQ 'RJV 6ZLQH DQG +XPDQV &RQWURO YDOXHV IRU WRWDO DQG FRQMXJDWHG ELOLUXELQ ZHUH KLJKHU LQ KXPDQV WKDQ LQ VZLQH DQG ORZHVW LQ GRJV 7DEOH f 7KH LQGLUHFW ELOLUXELQ OHYHOV LQ DOO WKUHH VSHFLHV ZHUH VLPLODU 7DEOH f $ VLJQLILFDQW LQFUHDVH LQ WRWDO ELOLUXELQ RFFXUUHG LQ GRJV DW KRXUV )LJXUH f DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH EXW GLG QRW RFFXU LQ VZLQH )LJXUH f DIWHU DGPLQLVWUDWLRQ RI WKH VDPH GRVH

PAGE 104

0($1 6(580 %,/,58%,1 &21&(175$7,21 PJG ,f )LJXUH 0HDQ VHUXP ELOLUXELQ FRQFHQWUDWLRQV LQ KXPDQV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 105

7DEOH 0HDQr FRQWURO YDOXHV RI VHUXP GRJV DQG VZLQH ELOLUXELQ DQG DOEXPLQ LQ KXPDQV 7RWDO %LOLUXELQ PJGOf &RQMXJDWHG %LOLUXELQ PJGOf ,QGLUHFW %LOLUXELQ PJGOf $OEXPLQ JPGOf +XPDQV 'RJV 6ZLQH r7KH PHDQ YDOXH RI VDPSOHV EHIRUH RUDO DQG LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH LQ GRJV DQG VZLQH 7KH PHDQ YDOXHV IRU KXPDQV DUH FDOFXODWHG IURP WKH VDPSOH WLPH IRU KXPDQ VXEMHFWV

PAGE 106

$IWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH D VLJQLILFDQW LQFUHDVH LQ WRWDO ELOLUXELQ RFFXUUHG LQ GRJV )LJXUH f DQG VZLQH )LJXUH f DW DQG KRXUV UHVSHFWLYHO\ ZLWK DQRWKHU LQFUHDVH LQ GRJV DW KRXUV ,Q KXPDQV WKLV LQFUHDVH ZDV QRW REVHUYHG RYHU WKH VDPH WLPH SHULRG SUREDEO\ GXH WR WKH GLIIHUHQFH LQ WKH RUDO GRVDJH )LJXUH f ,QLWLDOO\ WRWDO ELOLUXELQ OHYHOV ZHUH KLJKHU LQ VZLQH WKDQ LQ GRJV 7DEOH f EXW DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH WRWDO ELOLUXELQ ZDV VLJQLILFDQWO\ S f KLJKHU LQ GRJV EHWZHHQ DQG KRXUV )LJXUH f 7RWDO ELOLUXELQ OHYHOV DIWHU RUDO DGPLQLVWUDWLRQ ZHUH KLJKHU LQ VZLQH WKDQ LQ GRJV XQWLO WKH KRXU SHULRG ZKHQ WKH OHYHOV LQ GRJV ZHUH VLJQLILFDQWO\ LQFUHDVHG S f )LJXUH f (YHQ DIWHU DGPLQLVWUDWLRQ RI VXOILVR[D]ROH WKH WRWDO ELOLUXELQ LQFUHDVHV LQ GRJV DQG VZLQH GLG QRW UHDFK WKH FRQWURO RU WUHDWHG OHYHOV LQ KXPDQV &RQMXJDWHG ELOLUXELQ OHYHOV ZHUH QRW VLJQLILFDQWO\ LQFUHDVHG LQ VZLQH DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH HYHQ WKRXJK WKH FRQWURO OHYHOV ZHUH JUHDWHU LQ VZLQH WKDQ LQ GRJV )LJXUH f &RQMXJDWHG ELOLUXELQ OHYHOV ZHUH VLJQLILFDQWO\ LQFUHDVHG LQ GRJV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH H[FHHGHG WKH OHYHOV LQ VZLQH DIWHU KRXUV DQG UHDFKHG D PD[LPXP DW KRXUV DIWHU DGPLQLVWUDWLRQ )LJXUH f $IWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH FRQMXJDWHG ELOLUXELQ OHYHOV ZHUH HOHYDWHG LQ VZLQH DW KRXUV DQG VKRZHG D VWDWLVWLFDOO\ VLJQLILFDQW LQFUHDVH LQ GRJV DW KRXUV DIWHU DGPLQLVWUDWLRQ RI WKH GUXJ )LJXUH f 7KH FRQMXJDWHG ELOLUXELQ OHYHOV LQ GRJV DQG VZLQH DIWHU LQWUDYHQRXV RU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH GLG QRW UHDFK WKH FRQWURO RU WUHDWHG FRQMXJDWHG ELOLUXELQ OHYHOV LQ KXPDQV DW DQ\ RI WKH VDPSOLQJ SHULRGV 3RWHQWLDOO\ WR[LF LQGLUHFW ELOLUXELQ VKRZHG D VWDWLVWLFDOO\ VLJQLILFDQW EXW FOLQLFDOO\ VPDOO LQFUHDVH DIWHU RUDO DGPLQLVWUDWLRQ

PAGE 107

0($1 727$/ %,/,58%,1 &21&(175$7,216PJGOf )LJXUH &RPSDULVRQ RI WKH PHDQ WRWDO ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DQG VZLQH DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 108

0($1 727$/ %,/,58%,1 &21&(175$7,216 PJG ,f )LJXUH &RPSDULVRQ RI WKH PHDQ WRWDO ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DQG VZLQH DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 109

0($1 &21-8*$7(' %,/,58%,1 '2*6 7,0( KRXUVf )LJXUH &RPSDULVRQ RI WKH PHDQ FRQMXJDWHG ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DQG VZLQH DIW LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 110

0($1 &21-8*$7(' %,/,58%,1 )LJXUH &RPSDULVRQ RI WKH PHDQ FRQMXJDWHG ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DQG VZLQH DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 111

0($1 ,1',5(&7 %,/,58%,1 &21&(175$7,21 PJGOf )LJXUH &RPSDULVRQ RI WKH PHDQ LQGLUHFW ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DQG VZLQH DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 112

0($1 ,1',5(&7 %,/,58%,1 &21&(175$7,21 PJG ,f )LJXUH &RPSDULVRQ RI WKH PHDQ LQGLUHFW ELOLUXELQ FRQFHQWUDWLRQV LQ GRJV DQG VZLQH DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 113

RI VXOILVR[D]ROH LQ GRJV )LJXUH f 7KHUH ZHUH QR VWDWLVWLFDOO\ VLJQLILFDQW LQFUHDVHV LQ LQGLUHFW ELOLUXELQ DIWHU RUDO DGPLQLVWUDWLRQ LQ KXPDQV RU VZLQH RU LQ HLWKHU WKH GRJV RU VZLQH DIWHU LQWUDYHQRXV DGPLQLVn WUDWLRQ RI VXOILVR[D]ROH )LJXUH f &RQFOXVLRQV &RQFHUQLQJ %LOLUXELQ /HYHOV DIWHU $GPLQLVWUDWLRQ RI 6XOILVR[D]ROH 6PDOO EXW VWDWLVWLFDOO\ VLJQLILFDQW LQFUHDVHV LQ WRWDO DQG FRQn MXJDWHG ELOLUXELQ RFFXUUHG ZKHQ VXOILVR[D]ROH ZDV DGPLQLVWHUHG RUDOO\ WR GRJV )LJXUH f DQG VZLQH )LJXUH f DQG RQO\ ZKHQ DGPLQLVWHUHG LQWUDn YHQRXVO\ WR GRJV )LJXUH f 7KH PD[LPXP LQFUHDVHV RFFXUUHG DW GLIIHUHQW SRLQWV LQ WLPH GHSHQGLQJ RQ VSHFLHV DQG URXWH RI DGPLQLVWUDWLRQ 7KLV VSHFLHV YDULDWLRQ LV IXUWKHU UHIOHFWHG E\ WKH LQGLUHFW ELOLUXELQ H[FHHGLQJ FRQMXJDWHG ELOLUXELQ OHYHOV LQ GRJV )LJXUH f DQG VZLQH )LJXUH f ZKHQ VXOILVR[D]ROH ZDV DGPLQLVWHUHG RUDOO\ :KLOH WR[LF OHYHOV RI ELOLUXELQ KDYH QRW EHHQ HVWDEOLVKHG WKHVH UHVXOWV VKRZ WKDW WKH RUDO URXWH RI DGPLQLVWUDWLRQ GRHV LQFUHDVH WKH SRWHQWLDOO\ WR[LF LQGLUHFW ELOLUXELQ FRQFHQWUDWLRQ DW D WLPH IDU UHPRYHG IURP WKH LQLWLDO VLQJOH GRVH LQ GRJV DQG VZLQH 7KH VPDOO EXW VWDWLVWLFDOO\ VLJQLILFDQW LQFUHDVH RI WRWDO )LJXUH f DQG FRQMXJDWHG )LJXUH f ELOLUXELQ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH VXJJHVWV WKDW WKH DEVRUSWLRQ RI WKH RUDO GRVDJH IRUP GRHV DIIHFW WKH KHSDWRF\WH WR LQFUHDVH WKH QRUPDO UDWH RI KHPH GHJUDGDWLRQ RU WR LQFUHDVH FRQMXJDWHG ELOLUXELQ UHJXUJLWDWLRQ LQWR WKH JHQHUDO FLUFXODWLRQ 7KLV LV VXSSRUWHG E\ WKH LQWUDYHQRXV GRVH JLYHQ WR GRJV RQO\ LQFUHDVLQJ WKH WRWDO DQG FRQMXJDWHG ELOLUXELQ DQG QRW VLJQLILFDQWO\ DIIHFWLQJ WKH LQGLUHFW ELOLUXELQ OHYHO

PAGE 114

7KH ODFN RI VLJQLILFDQW HIIHFWV LQ KXPDQV DQG WKH ODFN RI FRUUHODn WLRQ EHWZHHQ KXPDQV DQG GRJV RU VZLQH FRXOG EH GXH WR WKH VKRUWHU VDPSOLQJ SHULRG LQ KXPDQV WKH UHGXFHG GRVDJH JLYHQ WR KXPDQV RU WKH VSHFLHV YDULDWLRQ ZKLFK H[LVWV HYHQ WKRXJK DOO WKUHH DUH PRQRJDVWULF :KHQ FRPSDULQJ WKH FRUUHODWLRQ FRHIILFLHQWV RI WRWDO DQG FRQMXJDWHG ELOLUXELQ LQ GRJV 5 S f DQG VZLQH 5 S f DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ WKHVH SDUDPHWHUV DUH PRUH FORVHO\ FRUUHODWHG LQ WKH SLJ 7KLV LV DOVR WUXH IRU WKH VLJQLILFDQW FRUUHODWLRQ RI LQGLUHFW DQG WRWDO ELOLUXELQ LQ GRJV 5 S f DQG VZLQH 5 S f DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI WKH GUXJ :KHQ VXOILVR[D]ROH LV DGPLQLVWHUHG RUDOO\ WKH WRWDO DQG FRQMXJDWHG ELOLUXELQ OHYHOV DUH PRUH FORVHO\ FRUUHODWHG LQ VZLQH 5 S f WKDQ LQ GRJV 5 S f EXW LQGLUHFW ELOLUXELQ LV PRUH FORVHO\ FRUUHODWHG WR WRWDO ELOLUXELQ LQ GRJV 5 S f WKDQ LQ VZLQH 5 S f ,Q DGGLWLRQ FRQMXJDWHG DQG LQn GLUHFW ELOLUXELQ DUH QHJDWLYHO\ FRUUHODWHG DIWHU LQWUDYHQRXV 5 S f DQG RUDO 5 S f DGPLQLVWUDWLRQ LQ WKH GRJ RQO\ QRW LQ WKH SLJ ,W LV HYLGHQW WKDW WKH URXWH RI VXOILVR[D]ROH DGPLQLVWUDWLRQ GRHV DIIHFW WKH ELOLUXELQ OHYHO GLIIHUHQWO\ LQ WKHVH VSHFLHV RI DQLPDOV 7KH SRWHQWLDOO\ WR[LF LQGLUHFW ELOLUXELQ OHYHO LV VLJQLILFDQWO\ LQn FUHDVHG RQO\ DIWHU RUDO DGPLQLVWUDWLRQ LQ RQH RI WKH WKUHH VSHFLHV GRJV 6HUXP $OEXPLQ &RQFHQWUDWLRQV 7KHUH ZHUH QR VLJQLILFDQW GLIIHUHQFHV LQ WKH VHUXP DOEXPLQ OHYHOV LQ HLWKHU RI WKH WKUHH VSHFLHV DIWHU LQWUDYHQRXV RU RUDO DGPLQLVWUDWLRQ

PAGE 115

0($1 6(580 $/%80,1 &21&(175$7,216 JPG ,f )LJXUH 0HDQ VHUXP DOEXPLQ FRQFHQWUDWLRQV LQ GRJV DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 116

0($1 6(580 $/%80,1 &21&(175$7,216 JPGf 7,0( KRXUVf )LJXUH 0HDQ VHUXP DOEXPLQ FRQFHQWUDWLRQV LQ VZLQH DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 117

0($1 6(580 $/%80,1 &21&(175$7,216 JPG ,f )LJXUH 0HDQ VHUXP DOEXPLQ FRQFHQWUDWLRQV LQ KXPDQV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 727

PAGE 118

RI VXOILVR[D]ROH )LJXUHV DQG f 7KHUH ZDV DQ LQLWLDO GHFUHDVH LQ DOEXPLQ OHYHOV LQ DOO WKUHH VSHFLHV DIWHU VXOILVR[D]ROH DGPLQLVWUDWLRQ ZLWK D UHWXUQ WR FRQWURO RU QRUPDO OHYHOV ZLWKLQ WR KRXUV 7KHUH ZDV D JUHDWHU GHFUHDVH DIWHU RUDO DGPLQLVWUDWLRQ LQ GRJV )LJXUH f DQG VZLQH )LJXUH f +XPDQV )LJXUH f KDG D KLJKHU LQLWLDO FRQFHQWUDWLRQ RI DOEXPLQ 7DEOH f WKDQ GRJV RU SLJV DQG LQLWLDO DOEXPLQ OHYHOV ZHUH KLJKHU LQ SLJV WKDQ LQ GRJV 7DEOH f 7KHVH GLIIHUHQFHV ZHUH PDLQWDLQHG WKURXJKRXW WKH WULDO SHULRGV DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ

PAGE 119

6800$5< $1' &21&/86,216 )URP WKH SKDUPDFRNLQHWLF SDUDPHWHUV ZKLFK ZHUH FDOFXODWHG RU SUHn YLRXVO\ UHSRUWHG f WKH PHDQ LQLWLDO WRWDO VHUXP FRQFHQWUDWLRQV ZHUH VLPLODU LQ DOO WKUHH VSHFLHV +RZHYHU WKH H[WUDSRODWHG VHUXP FRQFHQn WUDWLRQ LQ WKH ILUVW FRPSDUWPHQW ZDV KLJKHVW LQ VZLQH IROORZHG E\ GRJV DQG ORZHVW LQ KXPDQV f ,Q WKH VHFRQG FRPSDUWPHQW WKH H[WUDSRODWHG VHUXP FRQFHQWUDWLRQ ZDV KLJKHU LQ GRJV WKDQ LQ VZLQH 7KH ELRORJLFDO KDOIOLIH RI IUHH VXOILVR[D]ROH LQ WKH ILUVW FRPSDUWPHQW ZDV ORQJHVW LQ KXPDQV RQHKDOI WKLV YDOXH LQ GRJV DQG RQH ILIWK WKH WLPH RI KXPDQV LQ VZLQH ,Q WKH VHFRQG FRPSDUWPHQW WKH KDOI OLIH ZDV WZLFH DV ORQJ LQ VZLQH WKDQ LQ GRJV $ VHFRQG FRPSDUWPHQW ZDV QRW REVHUYHG LQ KXPDQV GXULQJ WKLV WULDO 7KH KDOIOLIH RI WKH DFHW\O 1 f PHWDEROLWH LQ WKH ILUVW FRPSDUWPHQW ZDV WLPHV ORQJHU LQ KXPDQV WKDQ LQ VZLQH $ VHFRQG FRPSDUWPHQW ZDV QRW REVHUYHG LQ KXPDQV EXW LQ VZLQH WKH KDOIOLIH ZDV ORQJHU WKDQ KRXUV 'RJV GLG QRW DFHWYODWH WKH GUXJ RU WKH PHWDEROLWH ZDV GHDFHW\ODWHG VR UDSLGO\ WKDW D VHUXP FRQFHQWUDWLRQ ZDV QRW REVHUYHG 'LVWULEXWLRQ RI VXOILVR[D]ROH IURP WKH FHQWUDO WR WKH SHULSKHUDO FRPSDUWPHQW ZDV IDVWHVW LQ KXPDQV f IROORZHG E\ VZLQH DQG VORZHVW LQ GRJV 'LVWULEXWLRQ IURP WKH SHULSKHUDO WR WKH FHQWUDO FRPSDUWPHQW ZDV DOVR IDVWHVW LQ KXPDQV f IROORZHG E\ GRJV DQG VORZHVW LQ VZLQH 7KH HOLPLQDWLRQ UDWH FRQVWDQW ZDV IDVWHVW LQ VZLQH IROORZHG E\ KXPDQV DQG VORZHVW LQ GRJV 0RUH VXOILVR[D]ROH ZDV DYDLODEOH IRU H[FUHWLRQ IURP

PAGE 120

WKH FHQWUDO FRPSDUWPHQW LQ KXPDQV f WKDQ LQ GRJV DQG WKH OHDVW LQ VZLQH 7KH YROXPH RI GLVWULEXWLRQ LQ WKH ILUVW FRPSDUWPHQW ZDV VLPLODU LQ GRJV DQG VZLQH ZKLFK ZDV JUHDWHU WKDQ WKH FDOFXODWHG YDOXH LQ KXPDQV f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f 7KH IUDFWLRQ RI VXOILVR[D]ROH ERXQG WR SODVPD SURWHLQV ZDV OHVV WKDQ b LQ GRJV DQG SLJV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI WKH GUXJ DQG LQ DOO KXPDQV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH GXULQJ WKH LQLWLDO SDUW RI WKH WULDO 7KLV UHVXOW VXJJHVWV WKDW WKH IUDFWLRQ ERXQG LV FDSDFLW\ OLPLWHG GXH WR WKH LQLWLDOO\ KLJK VHUXP FRQFHQWUDWLRQV DQG LV VXSSRUWHG E\ WKH LQ YLWUR ELQGLQJ GDWD 'XULQJ WKH UHPDLQGHU RI WKH VDPSOLQJ SHULRG WKH IUDFWLRQ ERXQG ZDV WR b LQ DOO VXEMHFWV DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ H[FHSW LQ GRJV DIWHU WKH KRXU SHULRG ZKHQ WKH IUDFWLRQ ERXQG ZDV WR b DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7KHUH ZDV D VLJQLILFDQW S f LQFUHDVH LQ WRWDO DQG FRQMXJDWHG ELOLUXELQ LQ GRJV DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[Dn ]ROH 7KH SRWHQWLDOO\ WR[LF LQGLUHFW RU IUHH ELOLUXELQ ZDV VLJQLILFDQWO\

PAGE 121

S f LQFUHDVHG LQ GRJV RQO\ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D ]ROH ,Q VZLQH WKHUH ZDV DQ LQFUHDVH LQ WRWDO DQG FRQMXJDWHG ELOLUXELQ RQO\ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7KH SRWHQWLDOO\ WR[LF LQGLUHFW ELOLUXELQ ZDV QRW LQFUHDVHG E\ HLWKHU LQWUDYHQRXV RU RUDO WUHDWPHQW 7KHUH ZDV QR VLJQLILFDQW LQFUHDVH LQ WRWDO FRQMXJDWHG RU LQGLUHFW ELOLUXELQ LQ KXPDQV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH GXH WR f WKH ORZ WKHUDSHXWLF GRVH XVHG RI DSSUR[LPDWHO\ PJNJ FRPSDUHG WR WKH DQLPDO GRVDJH RI PJNJ f WKH DELOLW\ RI KXPDQV WR UHGXFH WKH LQ YLYR HIIHFWV RQ WKH KHSDWRELOLDU\ V\VWHP RU f WKH VDPSOLQJ SHULRG ZDV WRR VKRUW DQG DGGLWLRQDO VDPSOHV VKRXOG KDYH EHHQ WDNHQ RYHU D ORQJHU WULDO SHULRG 7KHUH ZDV D VLJQLILFDQW FRUUHODWLRQ S f RI WRWDO DQG FRQn MXJDWHG ELOLUXELQ LQ GRJV DIWHU RUDO DQG LQWUDYHQRXV DGPLQLVWUDWLRQ DQG LQ VZLQH DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7KLV HYLGHQFH VXSSRUWV D K\SRWKHVLV WKDW VXOILVR[D]ROH LV KHPRO\WLF DQG LQFUHDVHV WKH EUHDNGRZQ RI KHPH WR ELOLUXELQ *OXFXURQLGDWLRQ FRXOG LQFUHDVH WR FRPn SHQVDWH IRU WKH LQFUHDVHG ELOLUXELQ LQ RUGHU WR SUHYHQW DQ LQFUHDVH LQ LQGLUHFW ELOLUXELQ ,Q WKH GRJ D VLJQLILFDQW QHJDWLYH FRUUHODWLRQ EHWZHHQ LQGLUHFW DQG FRQMXJDWHG ELOLUXELQ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ VXSSRUWV WKH UHGXFWLRQ RI SRWHQWLDOO\ WR[LF IUHH ELOLUXELQ E\ JOXFXURQLGDWLRQ )URP WKH REVHUYHG FKDQJHV LQ ELOLUXELQ DQG WKH VLJQLILFDQW FRUUHODn WLRQ S f LW DSSHDUV WKDW GRJV DUH PRUH VXVFHSWLEOH WR SRVVLEOH WR[LFLW\ E\ LQGLUHFW ELOLUXELQ RQO\ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[Dn ]ROH $ WUHDWPHQW GLIIHUHQFH DOVR RFFXUV LQ VZLQH VLQFH WRWDO DQG

PAGE 122

FRQMXJDWHG ELOLUXELQ DUH LQFUHDVHG RQO\ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH )URP WKLV WULDO WKH WKUHH VSHFLHV VWXGLHG DUH QRW SKDUPDFRNLQHWLFDOO\ VLPLODU ZLWK UHVSHFW WR VXOILVR[D]ROH 7KH RQO\ SKDUPDFRNLQHWLF SDUDPHWHUV ZKLFK ZHUH VLPLODU ZHUH WKH LQLWLDO WRWDO VHUXP FRQFHQWUDWLRQ RI IUHH VXOILVR[D]ROH DQG WKH IUDFWLRQ ERXQG WR SODVPD SURWHLQV 7KH KDOIOLYHV RI IUHH VXOILVR[D]ROH DQG LWV DFHW\O 1 f PHWDEROLWH WKH GLVWULEXWLRQ FRQVWDQWV WKH YROXPH RI GLVWULEXWLRQ WKH ELRDYDLODELOLW\ DQG WKH XULQDU\ H[FUHWLRQ SHUFHQWDJH ZHUH GLIIHUHQW +RZHYHU GRJV DSSHDU WR KDYH VHUXP FRQFHQWUDWLRQV GLVWULEXWLRQ FRQVWDQWV YROXPHV RI GLVWULEXn WLRQ ELRDYDLODELOLW\ DQG H[FUHWLRQ UDWHV PRUH VLPLODU WR KXPDQV WKDQ VZLQH 7KHUH ZDV D GLIIHUHQFH LQ WKH SK\VLRORJLFDO DIIHFWV RQ WKH KHSDWRELOLDU\ V\VWHP GXH WR WKH WUHDWPHQW DQG VSHFLHV GLIIHUHQFHV 'RJV FRXOG EH PRUH VXVFHSWLEOH WR WKH WR[LF HIIHFWV RI LQGLUHFW ELOLUXELQ DIWHU RUDO DGPLQLVWUDWLRQ SRVVLEO\ GXH WR WKH ILUVWSDVV HIIHFW 7KH ODFN RI WR[LFLW\ LQ VZLQH DQG KXPDQV FRXOG EH GXH WR WKH PHWDEROLVP RI VXOILVR[D]ROH WR WKH DFHW\O 1 f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

PAGE 123

ELOLUXELQ WKHQ GRJV DUH D EHWWHU PRGHO DQG VKRXOG EH XVHG LQ DGGLWLRQDO UHVHDUFK WR H[SODLQ WKH WR[LFLW\ RI ELOLUXELQ

PAGE 124

$33(1',;

PAGE 125

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ GRJ DIWHUn LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7LPH KRXUVf $OEXPLQ JPGOf 7R W D OLLOO UXE¯Q PJGOf &RQMXJDWHG %LOO UXE ,Q PJGOf 6HUXP QGLUHFW %LOLUXELQ PJGOf )UHH 6XOI,VR[D]ROH EJQLOf $FHW\O 6XOI,VR[D]ROH 0ILPOf 7R/DO 6XOILVR[D]ROH EJPOf ,QW UDYHQRXV r f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 2UDO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ r'RVH EDVHG RQ NJ ERG\ ZHLJKW A'RVH EDVHG RQ NJ ERG\ ZHLJKW

PAGE 126

7DEOH FRQWLQXHG 8ULQH )UHH $FHW\O 7RWDO 7RWDO 7LPH 6XOILVR[D]ROH 6XOILVR[D]ROH 6XOILVR[D]ROH 'UXJ ,Q 8L KRXUVf KJPOf QJPOf _LJLQOf KJf ,QWUDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 2UDO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§

PAGE 127

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ GRJ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 6HUXP 7LPH KRXUVf $OEXPLQ JLQGOf 7RWDO %LOLUXELQ PJLOOf &RQMXJDWHG %LOLUXELQ PJGf QGLUHFW %LOOUXELQ PJGf )UHH 6XOeVR[D]ROH XJQf $FHW\ 6XOILVR[D]ROH XJPOf 7RWDO 6XOILVR[D]ROH _MJPf ,QWUDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 2UDOI f§ f§ f§ f§ f§ I BBW f§ W 7 f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ r'RVH EDVHG RQ NJ ERG\ ZHLJKW , 'XVH EDVHG RQ NJ ERG\ ZHLJKW n1R YDOXH UHFRUGHG GXH WR H[FHVVLYH KHPRO\VLV RI VDPSOH

PAGE 128

7DEOH FRQWLQXHG 8ULQH )UHHf° $FHW\O 7RWDO 7RWDO 7 L PH 6XOILVR[D]ROH 6XOILVR[D]ROH 6XOIOVR[D]ROH 'UXJ ,Q 8ULQH KRXUVf XJPOf XJPOf XJPOf 0Jf ,QWUDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $ f§ f§ f§ f§ f§ f§ f§ 2UDO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ n f§ f§

PAGE 129

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ GRJ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 6HUXP 7R WDO &RQMXJDWHG ,QGLUHFW ,nUHH $FHW\O 7RWDO 7LPH $OEXPLQ +LOO UXE OX % L L UXO! W Q %LOO UXE¯Q 6XOI-VR[D]ROH 6XO IVR[D]ROH 6XOI,VR[D]ROH KRXUVf JPGOf PJGOf PJGf PJGOf 0 Q f JJOOOOf SWMf OLOW UDYHQRXV f§ f§ f§ f§ f§ f§ $ f§ f§ f§ f§ f§ f§ f§ f§ f§ 2UDO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ r'RVH EDVHG RQ NJ ERG\ ZHLJKW A'RVH EDVHG RQ NJ ERG\ ZHLJKW

PAGE 130

7DEOH FRQWLQXHG 7 L PH KRXUVf ) UHH 6XOILVR[D]XOH XJZOf 8ULQH $FHW\O 6XOIVR[D]ROH LLJPOf 7RWD 6XOILVR[D]ROH OLJPOf 7RWDO 'UXJ LQ OOUOQH LnJf ORW UDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ -2 f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ / f§ f§ f§ f§ f§ f§ $ f§ f§ f§ f§ $ f§ $ $ f§ $ 2UDO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ -22 BB f§ f§ f§ f§ f§ f§ f§ f $ f§ f§ f§ f§ f§ f§ $$ f§ f§ f§ f§ f§ $ f§ $ f§

PAGE 131

r'RVH EDVHG RQ NJ ERG\ ZHLJKW RRRRRRRRRRRRRRk fµfµ R 0 0 9' ref + URAVMIRL-1-?A 2222222k 8! 1, 2 2 A k k 2 FQ k IF FFRRRRFRRRkkkkk .L2RMnUZ&'UnA2A22&FFRZIR 22222222222222k 22A22Ln21fn22LA221f2&;!2Yn RRRRRRRRRRRRRR URWV-ZZUR0ZUR2nnn DFF!L!LRFRARDn&n202n kkkRRRRRRRRRRR URURURrf§ f§ URURUR f§ rf§ 222& URL!UR2¯!rA!L!RUR2'¯!Dn RRRRRRRRRRRRRRR U22Ark}f§ k k k fµf§ 2 Af§ URURFRF'221-2RUR2132RRRRFn1f RRRRRRRRRRRRRR RRR f§ RRRRRRRRR f§ ?LnIR!L!URLn2n&nenIRD2 + 1-: &G 2 : 22Lf§!rf§fµ¯f¯n¯!n;!&nnO-2rf§r8!&'1! , 1&':20+2&202nAY2+Efµf¯8&ZZFF0LURDURn-n28n !28¯A-2rf§ n, rf§ Ye!28L/+/Q Y2&&2/QYLnA2Lf§, : 8, 2 refµ ' Zen}}8nQ6 2'&12ARRUR28L ‘LnnWf§-2&f«n2&;&nn&'k 8 &' Z 2n UR 2++:\IL+21f &n?08e2Z 82A8f/Q?&&R&e LOO 7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ GRJ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH

PAGE 132

7DEOH FRQWLQXHG 8ULQH )UHH $FHW\O 7RWDO 7RWDO 7LPH 6WLO I LVR[DROH 6XOILVR[D]ROH 6XOILVR[D]ROH 'UXJ LQ 8ULQH KRXUVf XJPOf OLJPOf XJUDOf XJf LQWUDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ E f§ f§ f§ f§ 2UDO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 77

PAGE 133

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ GRJ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 6HUXP 7LPH $OEXPLQ KRXUVf JLQ-f 7RWDO UXE ,Q PJG f &RQMDEDWHG +LOO UXELQ PJG f QG UHQW %LOO UXE,Q PJGOf 7UHH 6XOILVR[D]ROH _LJPOf $FHW\O 6XOURVD]ROH JJPOf 7RWDO 6XOILVR[D]ROH _LJPOf ,QWUDYHQRXVr $ $ f§ f§ f§ $ $ f§ $ $ f§ $ f§ $ $ $ f§ $ f§ $ $ $ $ f§ $ f§ $ $ $ f§ $ f§ f§ $ $ f§ $ f§ f§ 2UDO $ f§ f§ f§ $ $f f§ $ f§ $ $ $ $ f§ $ f§ $ f§ $ $ $ f§ $$ f§ O$2 $ $ f§ $ f§ $ f§ $ $ $ $ $ f§ $$ f§ f§ $ f§ r'RVH EDVHG RQ NJ ERG\ ZHLJKW rLfRVH EDVHG RQ NJ ERG\ ZHLJKW

PAGE 134

7DEOH FRQWLQXHG )UHH 7LPH 6XOILVR[D]ROH KRXUVf XJPOf 8ULQH $FHW\ 6XOILVR[D]ROH XJPOf OQW UDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 2UDO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 7RWDO 7RWDO 6XOILVR[D]ROH 'UXJ LQ 8ULQH 8JPOf XJf RR

PAGE 135

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ GRJ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7LO &RQ MXJOWHG 6F UXP QGLUHHO )UHHf¬ $IHW\ 7RWD 7LPH $O ELQXL Q OLOL L UXOL ,Q %LOO QLOf ,Q ,6   UXE LQ 6XLLL62;8=2* 6X I L VR[D]ROH 6XOILVR[D]XH KRXUVf 5LLLG f QLJG QLJG f PJG f QVPOf QIMPO f 8ILPOf WLO UDYHQRXV r f§ f§ f§ f§ 2K f§ $ f§ $ f§ f§ \R f§ f f§ f§ f§ f§ f§ 2UDOr f§ f§ f§ f§ f§ f§ f§ VR f§ f§ f§ f§ f§ f§ f§ f§ ! f§ r_fRVH EDVHG RQ NJ /RLO\ ZHLJKW AIORVH EDVHG RQ NJ WRG\ ZHLJKW

PAGE 136

7DEOH FRQWLQXHG 8ULQH 7LPH KRXUVf )UHH 6XOILVR[D]ROH SJQLOf $FHW\O 6XLI,VR[D]ROH SJPOf ,QWUDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 2UD f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ E f§ f§ f§ f§ f§ 7RWDO 6XOILVR[D]ROH SJPOf 7RWDO 'UXJ LQ OOULQH SJf UR 2 ,

PAGE 137

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ SLJ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 6HW WLQW O2ODO &RQ MXJLW HG PO UL7 W WUHH $FHW \ 7XO D , 7 L PH $ KXP LL L QLOfOLW L L UXE Q WULOOf ,Q 6XOULVR[D]RH 6XOILVR[D]RM H 6XOILVR[D]ROH KRXUVf JPG ! f PJf OOOJGO f OOOJGO f WLJPOf OLJPLf OLJPOf OQW UDYHQRXV fµ r f§ f§ f§ ,OO 2UD r f§ f§ f§ r-!& EDVHG RQ NJ ERG\ ZHLJLOW 'RVH EDVHG RQ NJ ERG\ ZHLJKW

PAGE 138

7DEOH FRQWLQXHG 7LPH KRXUVf )UHH 6XOILVR[D]ROH XJPOf 8ULQH $FHW\O 6X /I,VR[D]ROH L-JPOf 7RWDO 6XOI,VR[D]ROH QJPOf 7RWDO 'UXJ ,Q 8ULQH OnJf ,QWUDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $ $ $ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $ f§ f§ f§ f§ $ $ 2UDO f§ f§ f§ f§ f§ f§ f§ $ f§ f§ f§ f§ $$ $ $ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $ $ f§ f§ f§ f§ $ $ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $ $ $ $

PAGE 139

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOI-VR[D]ROH LQ SLJ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 6HUXP 7 O PH $ EOOOOOL @f 7R/L +L LUXELQ &RQ M OLJDWHG +L LLXKLQ PLO UHHO UXE ,Q )UHHf° 6XOILVR[X[R9 $FH W\ L 6XOILVR[D]ROH 7RWDO 6XOILVR[D]ROH KRXUVf +LQLOO f LQILO f PX f LQJWOOf L}HLmLf OLJPOf OLMMPOf OXUDYHQRXV L! f§ f§ 2UDOn 28 f§ f§ f§ E BBW 7 rfRVH EXVHG RQ NJ ERG\ ZHLJKW 'RVH EXVHG RQ NJ ERG\ ZHLJKW A1R YDOXH UHFRUGHG GXH WR H[FHVVLYH KHPRO\VLV RI VDPSOH

PAGE 140

7DEOH FRQWLQXHG 8ULQH )UHH $FHW\O 7RWDO 7RWDO 7 L UWH 6XOIVR[D]ROH 6XOILVR[D]ROH 6XOILVR[D]ROH 'UXJ ,Q 8ULQH KRXUVf W-JPO f 0JQLOf XJLXOf OLJf ,QWUDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 2UDO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $ $ $ $ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $1R XULQH OHYHO ZDV UHFRUGHG DOWHU KRXUV GXH WR D SUREOHP ,Q FROOHFWLRQ

PAGE 141

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH LQ RI VXOILVR[D]ROH 6R QLP SLJ DIWHU LQWUDYHQRXV DQG RUDO 7RWD &RQ +JDWHG OPOL UHHO OLFHf° $FHW\O 7RWDO 7 O PH $f KWPLL LW ELO¯ UZEO X ,,, POf LQ +LOOUXKLQ 6X IVR[D]ROH 6Q LVR[D]R,H 6X ILVR[D]ROH KRXUVf JLLLDLf PJGLf QJGOf PJLOOf OLJPOf 8LJQLOf ,QWUDYHQRXVr f§ BB f§ $ $ $ $ $ $ $ $ $ $ $ ,$ $ \R $ $ $ $ $ $ $ f§ $ $ $ f§ $ f§ 2UD n f§ f§ $ $ $ $ $$ $ $ $ WRR $ $ +$ f§ I $ $ $ $ $ $ $ $ $ $ $ $$ $ $ $ $$ r'RVH EDVWnG RQ NJ ERG\ ZHLJKW r 'XVH EDVHG RQ NJ ERG\ ZHLJKW A1R YDOXH UHFRUGHG GXH WR H[FHVVLYH KHPRO\VLV RI VDPSOH

PAGE 142

7DEOH FRQWLQXHG OOUOQH 7 L LQH KRXUVf )UHH 6X IVR[D]ROH QJPOf $FHW\O 6XOILVR[D]ROH _MJPOf 7RW DO 6XOILVR[D]ROH XJQLOf 7RWDO 'UXJ LQ 8L 0Jf ,QW UDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 2UDO f§ f§ f§ f§ f§ f§ f§ a f§ f§ f§ f§ f§ f§ n f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§

PAGE 143

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ SLJ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 6F UXP 7QWLL &XQ@ LLI}L OXOO PO L UHFW ,7 HH $UHW\O 7RWDO 7 P H $OKXPLQ ,W L UXOLO Q f POf LL +LOO UXE¯Q 6XOIOVR[D]ROH 6LL ¯ L VX[D]QOLf 6XOI LVR[D]ROH KRXUV .+LG Of QLJGO f PJGOf PJGOf KJLQOf _OJOLOO f QJXLOf ,QWUDYHQRXVr RQ BB f§ R U! . $ 2UD _ r f§ f§ f§ r'XVH EDVHG RQ NJ ERG\ ZHLJKW AOORVH EDVHG RQ NJ ERG\ ZHLJKW

PAGE 144

7DEOH FRQWLQXHG 8ULQH OnUHH $FHW\O 7R/DO 7RWDO 7LPH 6XILVR[D]ROH 6XOILVR[D]ROH 6XOILVR[D]ROH 'UXJ LQ 8ULQH KRXUVf 0JPOf SJPOf XJPOf Of«Jf ,QWUDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 2UDO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ fµ f§ f§ f§ f§ f§

PAGE 145

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ SLJ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 6HUXP 7RW D &RQ LXJLLWXG ,QGUDFW ,r UHH0 $FHW\O 7RWDO 7LQX" $ EXQLL Q UXOLOLL +LOO UXEOQ GLOO UXE¯Q 6X LLVR[D]RH 6QILVQ[D]RLF 6LOO I L62;D=2 O Y ERPVf JPGOf PJGf PJG f PJGOf _LJLQ f KUPOf EJPOf , QL UDYHQRXV r f§ f§ f§ f§ W f§ I f§ f f§ f§ 2ULO f§ f§ f§ 8 rWf6& EDVHG RQ r NJ ERG\ ZHOJE/ 'RVH EDVHG RLO OE NJ ERG\ ZHLJO}W ,QR Y OLQH UHFRUGHG GXH WR H[FHVVLYH EHPRO\V-V RI VDPSOH

PAGE 146

7DEOH FRQWLQXHG OLU , QH )UHH $FHW\O 7RWDO 7RWDO 7 LPH 6XOILVR[D]ROH 6XOI,VR[D]ROH 6XOIOVR[D]ROH 'UXJ LQ 8ULQH KRXUVf XJPOf XJPOf XJPOf XJf LQWUDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ $ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 8UDO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ @

PAGE 147

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH DQG XULQDU\ FRQFHQWUDWLRQV RI VXOILVR[D]ROH LQ SLJ DIWHU LQWUDYHQRXV DQG RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 6Ln UXP 7LPH KRXUVf $O KXPQ InQL WO f 7ROD , ,W  OOO! ,, PY+f &XQ fXDWRO +LOO UXE ,Q LWLN+f PO L UHUW +LOOf OLE LL P\WO f 6XOLVR[D]RH L+WZO f $L LW \ 6XOILVR[D]ROH WLWWPO f 7RWDO 6XOILVR[D]ROH _LP, f ,QW UDYHQRXV r L LL , f§ $ 2UDO f§ f§ f§ 4 O ,6 f§ r'RmLL EDVHG RQ L NJ ERG\ ZHLJKW rOfRVm" EDVHG RQ NJ ERG\ ZHLJKW A1R YDOXH UHFRUGHG GXH WR H[FHVVLYH KHPRO\VLV

PAGE 148

7DEOH FRQWLQXHG f¯) UHH 8UL QH $FHW\O 7RWDO 7RWDO nULPH 6XO LVR[G]ROH 6XOILVR[D]ROH 6X ILVR[D]ROH 'UXJ LQ 8ULQH KRXUVf !JPf 0JPOf OLJPOf QJf LOO UDYHQRXV f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ 2UQO f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§ f§

PAGE 149

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH LQ KXPDQ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROHr 7LPH KRXUVf $OEXPLQ JPGOf 7RWDO %LOLUXELQ PJGOf &RQM XJDWHG %LOLUXELQ PJGOf 6HUXP ,QGLUHFW %LOLUXELQ PJGOf )UHH 6XOILVR[D]ROH MMJPOf $FHW\O 6XOILVR[D]ROH \JPLf 7RWDO 6XOILVR[D]ROH WLJPOf f§ f§ f§ r%RG\ ZHLJKW RI NJ

PAGE 150

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH LQ KXPDQ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROHr 7LPH KRXUVf $OEXPLQ JPGOf 7RWDO %LOLUXELQ PJGOf &RQMXJDWHG %LOLUXELQ PJGOf 6HUXP ,QGLUHFW %LOLUXELQ PJGOf )UHHf° 6XOILVR[D]ROH \JPOf $FHW\O 6XOILVR[D]ROH \JPOf 7RWDO 6XOILVR[D]ROH \JPOf f§ f§ f§ r%RG\ ZHLJKW RI NJ

PAGE 151

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH LQ KXPDQ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROHr 7LPH KRXUVf $OEXPLQ JPGOf 7RWDO %LOLUXELQ PJGOf &RQMXJDWHG %LOLUXELQ PJGf 6HUXP ,QGLUHFW %LOLUXELQ PJGOf )UHH 6XOILVR[D]ROH 3JPOf $FHW\O 6XOILVR[D]ROH 0JPOf 7RWDO 6XOILVR[D]ROH EJPOf f§ f§ f§ r%RG\ ZHLJKW RI NJ

PAGE 152

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH LQ KXPDQ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROHr 7LPH KRXUVf $OEXPLQ JPGOf 7RWDO %LOLUXELQ PJGOf &RQMXJDWHG %LOLUXELQ PJGOf 6HUXP ,QGLUHFW %LOLUXELQ PJGOf )UHH 6XOILVR[D]ROH \JPOf $FHW\O 6XOILVR[D]ROH \JPOf 7RWDO 6XOILVR[D]ROH 0JPOf f§ f§ f§ r%RG\ ZHLJKW RI NJ

PAGE 153

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH LQ KXPDQ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROHr 7LPH KRXUVf $OEXPLQ JPGOf 7RWDO %LOLUXELQ PJGOf &RQMXJDWHG %LOLUXELQ PJGOf 6HUXP ,QGLUHFW %LOLUXELQ PJGOf )UHH 6XOILVR[D]ROH _LJPOf $FHW\O 6XOILVR[D]ROH SJPOf 7RWDO 6XOILVR[D]ROH 0JPOf f§ f§ f§ r%RG\ ZHLJKW RI NJ

PAGE 154

7DEOH 6HUXP FRQFHQWUDWLRQV RI DOEXPLQ ELOLUXELQ DQG VXOILVR[D]ROH LQ KXPDQ DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROHr 7LPH KRXUVf $OEXPLQ JPGOf 7RWDO %LOLUXELQ PJGOf &RQM XJDWHG %LOLUXELQ PJGOf 6HUXP ,QGLUHFW %LOLUXELQ PJGOf )UHH 6XOILVR[D]ROH SJPOf $FHW\O 6XOILVR[D]ROH \JPOf 7RWDO 6XOILVR[D]ROH \JPOf f§ f§ f§ r%RG\ ZHLJKW RI NJ

PAGE 155

7DEOH 0HDQV DQG VWDQGDUG GHYLDWLRQVr RI VHUXP DOEXPLQ WRWDO ELOLUXELQ FRQMXJDWHG ELOLUXELQ DQG LQGLUHFW ELOLUXELQ LQ GRJV DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7RWDO &RQMXJDWHG ,QGLUHFW 7LPH $OEXPLQ %LOLUXELQ %LOLUXELQ %LOLUXELQ KRXUVf JPGOf PJGOf JPGOf PJGOf s s W L L s s i r s I s r I r L r r r r f s s f f s s f f f s L f L f L fr f fK s s f f fµ ,'r s f s s i r s s s s r r0HDQV ¯ VWDQGDUG GHYLDWLRQ I 7OLH PHDQ RI REVHUYDWLRQV DW HDFK WLPH ,QWHUYDO rS i S

PAGE 156

7DEOH 0HDQV DQG VWDQGDUG GHYLDWLRQVr RI VHUXP DOEXPLQ WRWDO DOEXPLQ FRQMXJDWHG ELOLUXELQ DQG LQGLUHFW ELOLUXELQ LQ GRJV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7RWDO &RQMXJDWHG ,QGLUHFW 7LPH $OEXPLQ %LOLUXELQ %LOLUXELQ %LOLUXELQ KRXUVf JLQGOf PJGOf PJGOf PJGLf s W L L O LR i L s r A V MB \ L s fW L s f s f s f L s s W s A s L s s r0HDQV VODQGDUO GHYLDWLRQ 7KH PHDQ RI REVHUYDWLRQV DW HDFK WLPH LQWHUYDO S S RQ

PAGE 157

7DEOH 0HDQV DQG VWDQGDUG GHYLDWLRQVr RI VHUXP DOEXPLQ WRWDO ELOLUXELQ FRQMXJDWHG ELOLUXELQ DQG LQGLUHFW ELOLUXELQ LQ VZLQH DIWHU LQWUDYHQRXV DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7RWDO &RQMXJDWHG ,QGLUHFW 7LPH $OEXPLQ %LOLUXELQ %LOLUXELQ %LOLUXELQ KRXUVf JPGOf PJGOf PJGOf PJGOf s s } s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s s r0HDQV s VWDQGDUG GHYLDWLRQ

PAGE 158

7DEOH 0HDQV DQG VWDQGDUG GHYLDWLRQVr RI VHUXP DOEXPLQ WRWDO ELOLUXELQ FRQMXJDWHG ELOLUXJLQ DQG LQGLUHFW ELOLUXELQ LQ VZLQH DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7RWDO &RQMXJDWHG ,QGLUHFW 7LPH $OEXPLQ %LOLUXELQ %LOLUXELQ %LOLUXELQ KRXUVf JPGOf PJGOf PJGOf PJGOf s s s s s s s s s s s s s n s s s s s s s s ; s s s s s s s s s s s s s s s s s s s s r0HDQV VWDQGDUG GHYLDWLRQ S

PAGE 159

7DEOH 0HDQV DQG VWDQGDUG GHYLDWLRQVr RI VHUXP DOEXPLQ WRWDO ELOLUXELQ FRQMXJDWHG ELOLUXELQ DQG LQGLUHFW ELOLUXELQ LQ KXPDQV DIWHU RUDO DGPLQLVWUDWLRQ RI VXOILVR[D]ROH 7RWDO &RQMXJDWHG ,QGLUHFW 7LPH $OEXPLQ %LOLUXELQ %LOLUXELQ %LOLUXELQ KRXUVf JPGOf PJGOf PJGOf PJGOf s s s s s s s s s s s s s s s r0HDQV VWDQGDUG GHYLDWLRQ

PAGE 160

%,%/,2*5$3+< $JUHQ $ (ORIVVRQ 5 DQG 1LOVVRQ 62 6RPH SK\VLRFKHPLFDO IDFWRUV LQIOXHQFLQJ WKH ELQGLQJ RI VXOIRQDPLGHV WR KXPDQ DOEXPLQ LQ YLWUR $FWD 3KDUPDFRO 7R[LFRO f %DUU $DQG *RRGQLJKW -+ 6WDWLVWLFDO $QDO\VLV 6\VWHP 1RUWK &DUROLQD 6WDWH 8QLYHUVLW\ 5DOHLJK 1RUWK &DUROLQD f %HOO 3+ DQG 5REELQ 5' 6WXGLHV LQ FKHPRWKHUDS\ 9,, $ WKHRU\ RI WKH UHODWLRQ RI VWUXFWXUH WR DFWLYLW\ RI VXOIDQLODPLGH W\SH FRPSRXQGV $P &KHP 6RF f %ORHGRZ '& DQG +D\WRQ :/ 6DWXUDEOH ILUVW SDVV PHWDEROLVP RI VXOILVR[D]ROH 1ADFHW\O LQ UDWV 3KDUP 6FL f %ORRP ) 7KH %ORRG &KHPLVWU\ RI WKH 'RJ DQG &DW *DPPD 3XEOLFDn WLRQV 1HZ
PAGE 161

'RPDJN * (LQ EHLWUDJ ]XU FKHPRWKHUDSLH GHU EDNWHULHOOHQ LQIHNWLRQHQ 'W PHG :VFKU f 'RPDJN * (LQH QHXH NODVVH YRQ GHVLQIHNWLRQVPLWWHOQ 'W PHG :VFKU f 'RXPDV %7 :DWVRQ :$ DQG %LJJV +* $OEXPLQ VWDQGDUGV DQG WKH PHDVXUHPHQW RI VHUXP DOEXPLQ ZLWK EURPFUHVRO JUHHQ &OLQ &KLP $FWD f 'X3RQW ,QVWUXPHQWV ,QVWUXPHQW 3URGXFWV $&$ 'LYLVLRQ :LOPLQJWRQ 'HODZDUH (OOPDQ / 0LOOHU / DQG 5DSSHSRUW /HXNRSKHUHVLV WKHUDS\ RI D K\SHUHRVLQRSKLOLF GLVRUGHU -$0$ f )LOGHV 3 $ UDWLRQDO DSSURDFK WR UHVHDUFK LQ FKHPRWKHUDS\ /DQFHW f )ODNH 5( *ULIILQ 7RZQVHQG ( DQG
PAGE 162

0DUVKDOO (. 'HWHUPLQDWLRQ RI SDUDDPLQRVDOLF\OLF DFLG LQ EORRG 3URF 6RF ([S %LRO DQG 0HG f 0HLVWHU ) (QGVOH\ / DQG 6FKPLGW -' 'UXJ LQGXFHG DQHPLD DVVRFLDWHG ZLWK JOXFRVHSKRVSKDWH GHK\GURJHQDVH GHILFLHQF\ ,RZD 0HG 6RF f 0HUFHU +' 7KH SUDFWLFDO DSSOLFDWLRQV RI SKDUPDFRNLQHWLFV LQ YHWHULQDU\ PHGLFLQH )LUVW $QQXDO 6\PSRVLXP LQ 9HWHULQDU\ 3KDUPDFRORJ\ %DWRQ 5RXJH /RXLVLDQD f 0HUFN ,QGH[ 0HUFN DQG &R ,QF 5DKZD\ 11LQWK (GLWLRQ S f 1HOVRQ ( DQG 2n5HLOO\ , .LQHWLFV RI VXOILVR[D]ROH H[FUHWLRQ LQ KXPDQV 3KDUPDFRO ([S 7KHU f 2GHOO *% 7KH GLVVRFLDWLRQ RI ELOLUXELQ IURP DOEXPLQ DQG LWV FOLQLFDO LPSOLFDWLRQ 3HGLDW f 2GHOO *% ,QIOXHQFH RI ELQGLQJ RQ WKH WR[LFLW\ RI ELOLUXELQ $QQDOV RI 1< $FDG RI 6FL f 2LH 6 DQG /HY\ * ,QWHULQGLYLGXDO GLIIHUHQFHV LQ WKH HIIHFW RI GUXJV RQ ELOLUXELQ SODVPD ELQGLQJ LQ QHZERUQ LQIDQWV DQG LQ DGXOWV &OLQ 3KDUP DQG 7KHUDS f f 3K\VLFLDQV 'HVN 5HIHUHQFH 0HGLFDO (FRQRPLFV &RPSDQ\ 2UDGHOO 1WK (GLWLRQ SS f 3UDQGRWD DQG 3UXLWW $: )XURVHPLGH ELQGLQJ WR KXPDQ DOEXPLQ DQG SODVPD RI QHSKURWLF FKLOGUHQ &OLQ 3KDUP DQG 7KHUDS f 3ULFH 3& DQG +DQVHQ $( %ORRG OHYHOV LQ UHODWLRQ WR GRVDJH RI GLPHWK\OVXOIDQLODPLGRLVR[D]ROH *DQWULVLQf LQ FKLOGUHQ ZLWK FOLQLFDO REVHUYDWLRQ 7H[DV 5HS %LRO DQG 0HG f 5DQGDOO /2 (QJHOEHUJ 5 ,OLHY 9 5RZ 0 +RDU + DQG 0F*DYDFN 7+ &RPSDUDWLYH VWXGLHV RQ EORRG OHYHOV DQG XULQDU\ H[FUHWLRQ RI VXOILVR[D]ROH DQG DFHW\O VXOILVR[D]ROH $QWLn ELRWLFV DQG &KHPRWKHUDS\ f 5HLGHQEHUJ 00 DQG $IIULPH 0 ,QIOXHQFH RI GLVHDVH RQ ELQGLQJ RI GUXJV WR SODVPD SURWHLQV $QQ 1< $FDG 6FL f 5HLGHU 3K\VLNOLVFKFKHPLVFKH XQG ELRORJLVFKH XQWHUVXFKXQJHQ DQ VXOIRQDPLGHQ $U]HLPHWWHOIRUVFK f 5RGNH\ )/ 'LUHFW VSHFWURSKRWRPHWULF GHWHUPLQDWLRQ RI DOEXPLQ LQ KXPDQ VHUXP &OLQ &KHP f

PAGE 163

5XGPDQ ' %L[OHU 7DQG 'HO5LR $( (IIHFW RI IUHH IDWW\ DFLGV RQ ELQGLQJ RI GUXJV E\ ERYLQH VHUXP DOEXPLQ E\ KXPDQ VHUXP DOEXPLQ DQG E\ UDEELW VHUXP 3KDUPDFRO ([S 7KHU f 6FKQLW]HU 5)RVWHU 5+. (UFROL 1 6RR+RR * 0DQJLHUL &1 DQG 5RH 0' 3KDUPDFRORJLFDO DQG FKHPRWKHUDSHXWLF SURSHUWLHV RI GLPHWK\OVXOIDQLODPLGRLVR[D]ROH 3KDUPDFRO ([S 7KHU f 6HOI 7+ (YDQV : DQG )HUJXVRQ 7 /HWWHU ,QWHUDFWLRQ RI VXOILVR[D]ROH DQG ZDUIDULQ &LUFXODWLRQ f 6KDQNDUDQ 6 DQG 3RODQG 5/ 7KH GLVSODFHPHQW RI ELOLUXELQ IURP DOEXPLQ E\ IXURVHPLGH RI 3HGLDWULFV f 6LOYHUPDQ :$ $QGHUVRQ '+ %ODQF :$ DQG &UR]LHU '1 $ GLIIHUHQFH LQ PRUWDOLW\ UDWH DQG LQFLGHQFH RI NHUQLFWHUXV LQ LQIDQWV DOORWWHG WR WZR SURSK\ODFWLF EDFWHULDO UHJLPHV 3HGLDWULFV f 6O\ZND *: 0HOLNLDQ $3 6WUDXJKQ $% :K\DWW 3/ DQG 0H\HU 0& %LRDYDLODELOLW\ RI VXOILVR[D]ROH SURGXFWV LQ KXPDQV 3KDUP 6FL f 6RORPRQ +: DQG 7KRPDV *% $ UDSLG PHWKRG IRU WKH HVWLPDWLRQ RI GUXJDOEXPLQ DIILQLW\ FRQVWDQWV LQ KXPDQ SODVPD &OLQ 3KDUPDFRO 7KHU f 6WHUQ / 'RUD\ % &KDQ * DQG 6FKLII ' %LOLUXELQ PHWDEROLVP DQG WKH LQGXFWLRQ RI NHUQLFWHUXV %LUWK 'HIHFWV f 6WUXOOHU 7K /RQJ DFWLQJ DQG VKRUW DFWLQJ VXOIRQDPLGHV UHFHQW GHYHORSPHQWV $QWLELRWLFD HW &KHPRWKHUDSLD f 6YHF )$ 5KRDGV 36 DQG 5RKU -+ 1HZ VXOIRQDPLGH *DQWULVLQf VWXGLHV RQ VROXELOLW\ DEVRUSWLRQ DQG H[FUHWLRQ $UFK ,QW PHG f 7U«IRXHO 7U«IRXHO 0PH 1LWWL ) DQG %RYHW ' $FWLYLW« GX SDPLQRSKHQ\OVXOIDPLGH VXU OHV LQIHFWLRQV VWUHSWRFRFFLTXHV GH OD VRXULV HW GX ODSLQ &RPSW 5HQG f 9DQ GHQ %HUJK $$+ DQG 0LOOHU 3 8EHU HLQH GLUHNWH XQG HLQH LQGLUHNWH GLD]RUHDFWLRQ DXI ELOLUXELQ %LRFKHP =WVFKU f 9HUD -& +HU]LJ (% 6LVH +6 DQG %DXHU 0$FTXLUHG FLUFXODWLQJ DQWLFRDJXODQW WR IDFWRU 9,,, -$0$ f :DJQHU -* )XQGDPHQWDOV RI &OLQLFDO 3KDUPDFRNLQHWLFV 'UXJ ,QWHOOLJHQFH 3XEOLFDWLRQV +DPLOWRQ ,OOLQRLV SS f

PAGE 164

:HLQVWHLQ / 0DGRII 0$ DQG 6DPHW &0 7KH VXOIRQDPLGHV 1( RI 0HGLFLQH f :RRGV '' 5HODWLRQVKLS RI SDPLQREHQ]RLF DFLG WR PHFKDQLVP RI DFWLRQ RI VXOSKDQLODPLGH %U ([S 3DWK f
PAGE 165

%,2*5$3+,&$/ 6.(7&+ 5REHUW /HH 6¼EHU ZDV ERUQ 0D\ LQ 4XLQF\ )ORULGD ,Q -XQH KH JUDGXDWHG IURP 4XLQF\ -XQLRU6HQLRU +LJK 6FKRRO 4XLQF\ )ORULGD ZLWK KLJK KRQRUV +H HQWHUHG WKH 8QLYHUVLW\ RI )ORULGD *DLQHVYLOOH )ORULGD LQ 6HSWHPEHU :KLOH DWWHQGLQJ WKH 8QLYHUVLW\ RI )ORULGD KH ZDV WUHDVXUHU RI .DSSD $OSKD 2UGHU IRU WKUHH \HDUV VHUYHG DV D 6WXGHQW 6HQDWRU DQG ZDV HOHFWHG WR *DPPD 6LJPD 'HOWD +RQRUDU\ :KLOH DQ XQGHUJUDGXDWH KH ZDV KRQRUHG RQ WKH 'HDQnV /LVW DQG WKH 3UHVLGHQWnV +RQRU 5ROO +H UHFHLYHG WKH %DFKHORU RI 6FLHQFH GHJUHH IURP WKH 8QLYHUVLW\ RI )ORULGD LQ -XQH ZLWK D PDMRU LQ DQLPDO SK\VLRORJ\ $IWHU JUDGXDWLQJ IURP WKH 8QLYHUVLW\ RI )ORULGD KH HQWHUHG WKH 86 $UP\ DV D SDWKRORJ\ VSHFLDOLVW DQG VHUYHG DW 7ULSOHU $UP\ 0HGLFDO &HQWHU LQ +DZDLL ,Q -DQXDU\ KH HQWHUHG WKH *UDGXDWH 6FKRRO DW WKH 8QLYHUVLW\ RI )ORULGD DQG UHFHLYHG D JUDGXDWH UHVHDUFK DVVLVWDQWVKLS LQ WKH 'HSDUWn PHQW RI $QLPDO 6FLHQFH ,Q 0DUFK KH UHFHLYHG WKH 0DVWHU RI 6FLHQFH GHJUHH ZLWK D PDMRU LQ DQLPDO SK\VLRORJ\ $IWHU UHFHLYLQJ WKH 0DVWHU RI 6FLHQFH GHJUHH KH ZRUNHG DV D /DERUDWRU\ ,QVWUXFWRU LQ WKH 7KHRGRUH *LOGUHG 0LFURVXUJLFDO (GXFDWLRQ &HQWHU 'LYLVLRQ RI 1HXURORJLFDO 6XUJHU\ 8QLYHUVLW\ RI )ORULGD ,Q 6HSWHPEHU KH DJDLQ HQWHUHG WKH *UDGXDWH 6FKRRO DW WKH 8QLYHUVLW\ RI )ORULGD WR IXOILOO WKH UHTXLUHPHQWV IRU WKH 'RFWRU RI

PAGE 166

3KLORVRSK\ GHJUHH +H PDMRUHG LQ WR[LFRORJ\ DQG PLQRUHG LQ SDWKRORJ\ FOLQLFDO FKHPLVWU\f 8SRQ FRPSOHWLRQ RI WKH 'RFWRU RI 3KLORVRSK\ GHJUHH LQ $XJXVW KH KDV DFFHSWHG D SRVLWLRQ DV D 7R[LFRORJLVW DQG &OLQLFDO &KHPLVW DW WKH 1DWLRQDO &HQWHU IRU 7R[LFRORJLFDO 5HVHDUFK -HIIHUVRQ $UNDQVDV +H ZLOO DOVR KROG DSSRLQWPHQWV DV DQ $VVLVWDQW 3URIHVVRU LQ 3KDUPDFRORJ\ 7R[LFRORJ\ DQG LQ 3DWKRORJ\ &ROOHJH RI 0HGLFLQH 8QLYHUVLW\ RI $UNDQVDV /LWWOH 5RFN $UNDQVDV 5REHUW / 6¼EHU LV PDUULHG WR &KULVWLQH %RQDU 7KH\ ZHUH PDUULHG RQ $XJXVW DQG KDYH RQH VRQ 5REHUW /HH -U ERUQ $XJXVW

PAGE 167

, FRU WLO\ WKDW FRQ I R UDV W R D R H L S W D f¬! DGHTXDWH LQ VFRSH DQG 'RFWRU R¯ 3KLORVRSK\ ‘ F f¬UW L I \ W OLLW URQ W L PV WR Ln2nL8LKOU LN S¯DWH L L 6L RSH DQG 'RFWRU Rf 3KLO VRLOLY FHUW L c \ W OLD U , FRQIRUPV WR QL F SWLKOH D cHTXDWH LQ VFRSH DQG 'RFWRU RI Of¬OQ ORVRSOLY LLYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ TXD@ L f¬ \ DV D GLVVHUWDWLRQ IRU OLWH GHJUHH RI *HRUJH 7 8GGV &KDLUPDQ 3URIHVVRU RI 7R[LFRORJ\ 3URIHVVRU RI $QLPDO 6FLHQFH VDYH UHQG WKLV VWXG\ DQG /K LW LQ U\ RS cQLFD LW VWDQGLUGV ‘! VFOLLLDUOY SUHVHQWDWLRQ DLG LV IXOO\ TXDOLW\ DV D GLVVHUWDWLRQ ORU WKH GHJUHH RI 2 3 LX , 7 &D U OH L OLQH 3URIHVVRU Rf¬ 9RWH U L QQ UY 0HGOLQH LDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW VW LQQDUGV RI VFKRODUO\ SUHVHQW DW LRQ DQG LV OXLO\ TXDOLW\ DV D GLVVHUWDWLRQ IRU KH GHJUHH RI & nn-nW/ *HRUJH 7RURVFDQ $VVRFLDWH 3URIHVVRU RI 3KDUPDF\

PAGE 168

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n3 'HDQ &ROOHJH RI $JULFXOWXUH 'HDQ *UDGXDWH 6FKRRO