Pharmacokinetics of sodium dichloroacetate after repetitive intravenous infusion

MISSING IMAGE

Material Information

Title:
Pharmacokinetics of sodium dichloroacetate after repetitive intravenous infusion
Physical Description:
vi, 80 leaves : ill. ; 29 cm.
Language:
English
Creator:
Chu, Pei-i, 1959-
Publication Date:

Subjects

Subjects / Keywords:
Infusions, Parenteral   ( mesh )
Acetic Acids -- metabolism   ( mesh )
Pharmacy thesis M.S.P   ( mesh )
Dissertations, Academic -- Pharmacy -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (M.S.)--University of Florida, 1984.
Bibliography:
Bibliography: leaves 77-80.
Statement of Responsibility:
by Pei-i Chu.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000550875
oclc - 13342463
notis - ACX5338
sobekcm - AA00004889_00001
System ID:
AA00004889:00001

Full Text









/PHARMACOKINETICS OF SODIUM DICHLOROACETATE
AFTER REPETITIVE INTRAVENOUS INFUSION/










BY

PEI-I CHU


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF MASTER OF SCIENCE
IN PHARMACY


UNIVERSITY OF FLORIDA


1984


















ACKNOWLEDGEMENTS

The author wishes to thank her major professor, Dr.

Stephen H. Curry, for his strong support and guidance. His

wonderful personality has also been appreciated. The author

would also like to thank the committee members, Dr. Kamlesh

M. Thakker, Dr. Stephen H. Schulman, and Dr. Margaret 0.

James, for all of their advice and suggestions during the

preparation for this thesis. The author must not forget to

acknowledge the typist Lynn for typing her thesis and having

the patience in producing the final draft. Finally, with

much joy and gratitude, the author would like to give

special thanks to her parents. Without them, she would not

have been able to become what she is today.













TABLE OF CONTENTS


P
ACKNOWLEDGEMENTS ......................... ...............

ABSTRACT ...........................................

CHAPTER


age

ii
v


I. INTRODUCTION ..................................

Dichloroacetic Acid ........................

Lactic Acidosis ............................

Known Kinetics of DCA ......................

Technique for Assaying DCA .................

II. PURPOSE OF THIS RESEARCH ......................

III. MATERIALS AND METHODS .........................

Chemicals ..................................

Apparatus .................................

Preparation of Reagents ..................

Human Subjects .............................

Assay of DCA ...............................

Study of the Effect of DCA Metabolites
(Oxalate, Glyoxalate, Glycolic Acid) to
DCA Assay ...................... ......

Study of DCA Binding Using a Dianorm
Equilibrium Dialyser ...................

Red Blood Cell Localization Study and
Partition Coefficient ..................

Oxalate Assay ............................

Creatinine Assay ...........................

iii.









IV. DATA ANALYSIS ................................. 23

Compartment Model .......................... 23

Elimination Rate Constant and Half Life .... 23

Volume of Distribution ..................... 24

Protein Binding ............................ 25

Red Blood Cell Localization and Partition
Coefficient ............................. 26

Area Under the Blood Level Curve ........... 27

Statistical Analysis ....................... 28

V. RESULTS ...................................... 29

Identity of DCA and DCA Assay .............. 29

Concentrations in Plasma ................... 30

Protein Binding ............ ............... 31

Red Cell Localization ...................... 31

VI. DISCUSSION ................................... 66

Binding and Red Cell Localization .......... 66

Pharmacokinetic Analysis ................... 68

APPENDIX ........................................... 74

REFERENCES ...................................... ..... 77

BIOGRAPHICAL SKETCH ................................. 81












Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree Master of Science
in Pharmacy


PHARMACOKINETICS OF SODIUM DICHLOROACETATE
AFTER REPETITIVE INTRAVENOUS INFUSION


By
Pei-I Chu

August 1984



Chairman: Dr. Stephen H. Curry W
Major Department: Pharmacy

Twelve healthy volunteers each received one or two

series of five intravenous infusion doses of sodium

dichloroacetate (DCA) at two hour intervals. Dose levels

were 10 mg/kg (three subjects), 15 mg/kg (one subject), 25

mg/kg (five subjects), and 50 mg/kg (4 subjects)(one male

subject was studied twice). Plasma DCA showed the expected

rise and fall during and after each dose, with higher

levels induced by higher doses. Twenty-four hour urinary

oxalate excretion was also measured and there was a linear

relationship between urinary oxalate excretion and DCA dose.

Protein binding was found by equilibrium dialysis to be
constant up to a total DCA concentration of 300u g/ml.

Individual protein binding fractions ranged from 0.2 to 0.42







in nine volunteers at 100 pg/ml DCA concentration. There

was no significant difference in the fraction bound when

dialysis was at 25 degrees or 37 degrees for one hour. The

partition coefficient of DCA between red blood cells and

plasma water was constant within any one batch of blood up

to 1000 ug/ml DCA. Individual partition coefficients in nine

volunteers ranged from 0.17 to 0.78 at a whole blood DCA

concentration of 100v g/ml.

The plasma levels were analyzed by comparison with zero

order infusion/first order elimination pharmacokinetic

models. The mean half life values following the first,

second, third, fourth and fifth infusions were 90.0 min,

110.0 min, 188.4 min, 210.0 min, and 451.8 min.

respectively. The area under the curve of concentration

versus time to infinity was linearly related to dose. There

was no significant change in apparent volume of distribution

after each dosing. The mean apparent volume of distribution

of DCA was 0.3 1/kg.

The average rates of metabolism of DCA following each

infusion were 1187.4, 1089.1, 881.7, 950.2, and 803.6

mg/hour. Because of the large standard deviations on these

means, there were no significant differences among these

data except for between the second and the last infusions.

This might indicate enzyme poisoning during DCA metabolism.

More volunteers need to be studied to draw more definite

conclusions.











CHAPTER I
INTRODUCTION

Dichloroacetic Acid


Dichloroacetic acid has been known for many years as an

escharotic, topical keratolytic and topical astringent

compound [1]. Dichloroacetic acid is a relatively strong

organic acid. Its dissociation constant is 5140 x 10(-5)

which lies between that of monochloroacetic acid 155 x

10(-5) and trichloroacetic acid 12100 x 10(-5). Chemically,

it can be obtained through the reaction of chloral hydrate

and aqueous sodium cyanide:

CI3C CHOH + NaCN
OH

boil C1 C COOH
CT

The decomposition of dichloroacetic acid is thought to

occur through the following pathway:
Dichloroacetic Acid H20 (HO > CHCOOH + OHCCOOH + H20
(glyoxalic acid)

Glyoxalic acid boil/alkali COOH + COOH
CH20H COOH
Glycollic Acid Oxalic Acid







This decomposition involves dechlorination. This

reaction also occurs enzymatically. The enzymatic mechanism

for dechlorination of dichloroacetate is unknown, but [4 C]

oxalate is formed from [14C] dichloracetate by isolated rat

liver mitochondria [2], suggesting that the necessary

enzymes are in these organelles. Bacterial dehalogenases

that substitute hydroxyl groups for the chlorine of

dichloroacetate have been described [3]. Dehalogenation of

dichloroacetate has not been demonstrated in mammalian

tissues other than liver.

Dichloracetate possesses systemic pharmacological

activity. The LDso orally in rats is 2.82g/kg. The effects

of DCA on intermediary metabolism have also been extensively

studied. Dichloroacetate reduces blood glucose

concentrations in both diabetic and fasted animals but not in

healthy, fed animals [4-7]. The possible mechanism is

thought to occur through the pathway shown in Fig. 1 [8].

Dichloroacetate accelerates pyruvate, lactate and

alanine oxidation by activation of pyruvate dehydrogenase

via direct inhibition of pyruvate dehydrogenase kinase.

Consequently, release of lactate and alanine from peripheral

tissues into circulation is reduced [9], and fewer

three-carbon precursors are available for hepatic glucose

synthesis.

In a previous report [10] it was shown that orally
administered sodium dichloroacetate, at a daily dose of 50

mg/kg, rapidly and significantly decreased plasma lactate















Lactate Metabolism
Glucose


Alanine Pyruvate -, Lactate
S .. ..... .. Mitochondrial
Membrane
Pyruvate
Pyruvate
Dehydrogenase DCA

Acetyl CoA

Lipid Krebs Cycle Ketone Bodies


Fig. 1. Probable Mechanism of DCA on Glucose Metabolism.







and alanine concentrations in maturity-onset diabetic

patients who had normal or slightly elevated basal plasma

lactate concentration. Other workers have shown that

intravenously administered DCA can prevent or even reverse

hyperlactatemia and lactic acidosis induced in animals by

phenformin administration [11-13], acute hepatitis [14],

functional hepatectomy [15-16], epinephrine infusion [17],

or exercise [18,19].

Dichloroacetate reportedly diminishes the

hyperlactatemia of endotoxin shock in rabbits [20,21] and

chickens [22]. Pretreatment of rabbits with dichloroacetate

reduces both the degree of hyperlactatemia and mortality due

to E. Coli endotoxin. Loubatieres et al. first reported that

dichloroacetate has no effect on the hyperlactatemia of

hypoxic dogs [23], but later found that lactate values

returned to normal more quickly in dichloracetate treated

animals, but only after normal oxgenation was restored.

Dichloroacetate diminishes lactate levels after intense

muscular work in dogs [23]. Dichloroacetate also increases

the time rats can swim before exhaustion, an effect which

may be explained by dichloroacetate accumulation in muscle

[24].

Irsigler et al. first used dichloroacete to treat human

phenformin-related lactic acidosis [25]. Their first two

patients were severely acidotic (pH<6.95) and did not

survive. Their third patient was only slightly acidotic

(pH=7.3) and administration of dichloroacetate, fluids,







glucose, insulin, furosemide, tris buffer and oxygen brought

about marked clinical improvement.

Stacpoole et al. [26] administered dichloroacetate to

thirteen patients with lactic acidosis of various causes.

The metabolic effects of dichloroacetate were evaluated in

eleven patients. Seven patients had a mean decrease in

lactate of 80 percent. Despite the improvement in the lactic

acidemia, all patients but one died of their underlying

disease.

Dichloroacetate has potential toxicity when given for

prolonged periods. In rats, it is metabolized by the liver

to the potentially toxic metabolites, oxalate and

glyoxylate [2]. Chronic oral doses of DCA 50 mg/kg/day

for several weeks produced a reversible neuropathy in

animals and in one human subject [27]. In contrast, acute

and sub-acute intravenous toxicity studies of DCA in animals

at doses even greater than 50mg/kg/day showed no evidence

of toxicity. In the study of Wells et al. [28], at an

intravenous dose of 50mg/kg, one healthy subject complained

of drowsiness up to 24 hours after drug administration. The

greater potential toxicity of DCA administered orally than

intravenously may be due to the fact that more of the drug

is initially converted in the liver to toxic metabolites when

it is given orally, since a greater proportion of a single

drug dose reaches the liver when given by mouth than when

given by vein. Toxicological evaluation was necessary for

higher and longer DCA dosing.








Lactic Acidosis


Lactic acidosis is a disorder of intermediary

metabolism. It can be defined as a metabolic acidosis due

totally or in part to an accumulation of blood lactate and

accompanying hydrogen ion.

The accumulation of lactate in the blood has always

been recognized as a normal response to vigorous exercise

and as a manifestation of acute hypoxia secondary to shock

or asphyxia. In 1961, Huckabee called attention to the

occurrence of hyperlactatemia in a variety of clinical

circumstances lacking evidence of either circulatory failure

or hypoxemia [29,30]. Since then, a large and still growing

literature on this subject has appeared and some experts now

suspect that lactic acidosis may be the most frequent form

of acute metabolic acidosis.

Lactate is produced and consumed in all cells, but

there is net production or consumption only in certain

tissues. Lactate is normally added to the plasma by the

skin, gut, skeletal muscles, blood cells and brain at a rate

of 10 to 20 mmol per kilogram of body weight per day. The

net rate of production of lactate in all cells is determined

by the rate of formation of pyruvate (with which the lactate

is in equilibrium) and by the cytoplasmic redox potential,

which influences the equilibrium relation between lactate and

pyruvate. The formation of pyruvate, in turn, is determined







mainly by the factors regulating glycolysis. Lactic

acidosis results whenever the rate of production of lactate

exceeds the capacity of the lactate utilizing reactions. The

primary disturbance may be an increase in production or a

decrease in utilization, or most frequently, a combination

of the two. In any case, the result is a rise in the blood

level of lactate and pyruvate, a fall in plasma bicarbonate

and usually some reduction in blood pH. Most clinicians

establish the diagnosis of lactic acidosis with a blood

level greater than 5 meq/1.

By far the most common clinical form of lactic acidosis

is that accompanying severe acute circulatory or respiratory

failure, in which the sudden rise in blood lactate is a

biochemical sign of tissue hypoxia. Sometimes, the lactate

level begins to rise even before the clinical signs of shock

appear, but the ominous implications of a rapidly increasing

blood lactate in injured or bleeding patients and in those

with sepsis or acute cardiac failure are by now all too

familiar. Unless their circulatory failure can be rapidly

reversed most such patients will die with severe lactic

acidosis.

It is well established that the first and most

important step in treating lactic acidosis is to attempt to

correct the underlying cause (e.g., infection with

antibiotics, heart failure with pressor drugs). Beyond

this, all other methods remain controversial. Use of sodium

bicarbonate is standard practice. Raising the arterial pH







above 7.0 will, in theory, convert the splanchnic bed from

a lactate-producing to lactate-consuming site. But rapid

intravenous boluses of NaHC03 may produce acute

intra/extra-cellular potassium and calcium shifts which, in

addition to alkalosis, may be arrythmogenic, and

intracellular pH is a complex function of arterial pH, pC02

and intracellular bicarbonate concentration. Thus one cannot

rationalize correcting intracellular pH merely by decreasing

extracellular acidemia. Animal experiments involving NaHC03

administration to dogs with experimentally-induced lactic

acidosis does not increase survival or improve arterial pH

and may, in fact, worsen cardiac function [31]. A limited

number of controlled clinical studies exist, which show that

treatment with NaHC03 significantly reduces morbidity or

mortality in patients with lactic acidosis, compared to

patients receiving only general supportive care. Thus

treatment of lactic acidosis needs to be reconsidered with

the use of NaHC03.


Known Kinetics of DCA


Dichloroacetate plasma levels have been studied only in

human volunteers given single intravenous doses. Plasma drug

concentrations were assayed by gas chromatography. Evidence

has shown that peak plasma concentrations were linearly

related to the dose up to 30 mg/kg [28]. At higher doses

some subjects showed disproportionately higher







concentrations than predicted by the linear relation seen at

lower doses. Nonlinear disposition was also indicated by

the time course of plasma DCA concentrations following drug

administration. Evidence had been obtained of applicability

of a Michaelis-Menten elimination model to DCA plasma level

data above 100 ug/ml, while a first order elimination model

was applicable at lower levels, with a mean DCA half-life of

approximately 30 minutes. For the patient study [26], an

apparent increase in the apparent volume of distribution of

DCA with second and third doses was observed and also there

was an apparent slowing of elimination, also with second and

third doses. Lukas et al. [32] also observed a decreased

intrinsic clearance and plasma clearance for the higher dose

infusion case and concluded "dose dependent pharmacokinetics

of DCA."


Technique for Assaying DCA


Dichloroacetate is conventionally assayed by gas

chromatography and detected by electron capture detector.

Gas chromatography is one type of partition chromatography.

The components of a vaporized sample are fractionated as a

consequence of partition between a mobile gaseous phase and

a stationary phase held in a column. The stationary phase

of the column can be either an adsorbent or a liquid

distributed in the form of a thin film on the surface of a

solid support. The solid support consists of porous inert







particles. In this study, Chromasorb 101 was used as the

packing material. Chromosorb 101 consists of porous

particles. At the end of the column, the individual

components emerged separated in time. They were then

detected and the detector signal was displayed on a

recorder. Here the electron capture detector was used.

The effluent from the column was passed over an

emmiter-nickel-63. An electron from the emitter caused

ionization of the carrier gas and the production of a burst

of electrons. The electron capturing species eluting from

the column reacted with the electrons to form ions, which

were swept from the cell. The net result was a reduction in

the number of electrons found at steady state or a drop in

the standing current.

Quantitative analysis was based upon a comparison of

either the height or the area of chromatographic peak of the

analyte with the standards. A carefully measured quantity

of TCA as an internal standard was introduced into each DCA

standard and sample, and the ratio of DCA to TCA peak height

served as the analytical parameter. The calibration curve

was linear up to 200 vg/ml and the coefficient of variation

varied from 2.5 to 4.03%.












CHAPTER II
PURPOSE OF THIS RESEARCH


The purpose of this research is as follows.

(1) To confirm the identity of DCA.

(2) To evaluate the relationship between plasma levels of

DCA and dose.

(3) To characterize the apparent volume of distribution and

elimination rate constant on multiple dosing.

(4) To study the red blood cell localization and distribution

coefficient of DCA.

(5) To determine the protein binding of DCA at different dose

levels.

(6) To find out if the metabolites of DCA will interfere with

DCA analysis.












CHAPTER III
MATERIALS AND METHODS

Chemicals

Dichloroacetic acid was as the sodium salt obtained as

a gift from Tokyo Kasei Kogyo Company.

Trichloroacetic acid was purchased from Sigma Chemical

Company Lot 102F-0440, No. T-488 5.

Boron Trifluororide-Methanol was purchased from Sigma

Chemical Company Lot No. 23F 7180, No. B-1252.

Benzene was purchased from Burdick & Jackson

Laboratories Inc, LC Grade Lot No. AD815.

Potassium monobasic phosphate was Analytical Grade

purchased from Mallinckrodt Inc., Lot WCPS. No. 7100.

Sodium chloride was also from Mallinckrodt Chemical

Works, Lot YGVB, No. 7581.

Sodium oxalate reagent grade was purchased from J.T.

Baker Chemical Company Lot No. 37256.

Sodium Glyoxylate was purchased from Sigma Chemical

Company Lot. 67C-5027, No. G-4502.

Glycolic acid was purchased from Sigma Chemical Company

Lot 99C-0243, No. G-1884.

Sodium phosphate dibasic reagent grade scientific

products distributed by Mallinckrodt, No. 7917-5.

Human plasma was obtained from pooled sources (blood

bank supplies).







Dialysis membrane was purchased from Diachema AG, No.

10.15, diameter 64 nm, M.W. cut off 10,000.

Nitrogen gas used was from Liquid Air Corporation

(UN1066).



Apparatus


The gas chromatography system used was a Varian 3700

Model, with Ni Electron capture detector kit part no.

02-001972-00. The recorder was Model 9176 by Varian.

The centrifuge used was a Dynac II Centrifuge from Clay

Adams, Division of Becton, Dickinson and Company.

The pH meter used was Beckman Model 4500 digital pH

meter.

The rotator used was a Tube Rotator form Scientific

Equipment Products.

The Dianorm Equilibrium Dialyser used was from Diachema

A.G. (Ruschikon/Zurich). A Gilson pipetman was used for

injection. A Micro 2 cell type was used.



Preparation of Reagents

Isotonic Phosphate Buffer Solution

Isotonic buffer solution was made by adding 1.9g

potassium monobasic phosphate, 8.1g sodium dibasic phosphate

and 4.11g of sodium chloride to 1000ml volumetric flask and

adding deionized H 0 up to the mark. The pH was measured

with a Beckman pH meter as mentioned before.







Trichloroacetic Acid (TCA) Stock Solution and Working
Solution

The TCA stock solution was made by dissolving 250 mg of

TCA in 250 ml of deionized water. A one in ten dilution of

the stock solution (100p g/ml) was used as the internal

standard.

DCA Stock Solution and Standard Sample of Calibration Curve

The DCA stock solution was made by dissolving 100 mg

sodium dichloroacetate in 100 ml plasma. A series of

dilutions was made with a micropipette to give 20, 40, 60,

80, 100, 150, and 200 g/ml solutions as standards for the

calibration curve.

Preparation of DCA Injection Solution

Two and one half grams of sodium dichloroacetate were

dissolved in 19 ml isotonic saline and 6 ml of sodium

phosphate injection solution (276 mg of monobasic sodium

phosphate monohydrate and 142 mg of dibasic sodium phosphate

per mililiter of H20) to give a final concentration of 100

mg/ml. The solution was sterilized by autoclaving at 250

degrees for fifteen to twenty minutes after passage through

an 0.22 microfilter (Milipore). Samples of the injection

solution were assayed by gas chromatography. Limulus Lysate

Test was also performed to test for pyrogenicity.







Human Subjects


Seven male and five female healthy volunteers

participated. Ages ranged from 22 to 57 yrs (Table 1). One

male subject was studied twice, each time at a different

drug dose. Subjects were initially screened as outpatients

by means of medical history, physical examination, complete

blood count and routine chemical analysis of renal, hepatic

and coagulation function. They were then admitted as

inpatients on the morning of study to the Clinical Research

Center of Shands Hospital, University of Florida. The

subjects were fasted overnight. A small diameter catheter

was inserted into a large superficial vein in each forearm.

A heparin lock was attached to one catheter for blood

withdrawal, while the other catheter was kept open with a

slow saline infusion between DCA doses.

After the catheter placement, the first DCA infusion

was begun. Dichloroacetate in phosphate-buffered saline (pH

5.2) was diluted to a total volume of 50 ml and administered

over 30 minutes using a Harvard infusion pump. On each

occasion, the subject received five identical drug doses,

each spaced two hours apart. Repeated doses of 10 mg/kg DCA

were administered in the first three investigations. One

subject received 15 mg/kg doses. Five subjects, including

one of the first three, received 25 mg/kg doses. The last

four investigations were at the 50 mg/kg dose level. Blood

samples were obtained at 30 minute intervals for the first





16

8.5-9 hours, and subsequently at 3-4 hour intervals up to 24

hours after starting the first infusion. Two ml of blood

were collected in citrate-containing Vacutainer tubes for

DCA assay. The blood was stored in an ice bucket until

centrifuged. After centrifugation, the supernatant was

frozen at -20 degrees C until assayed. Immediately before

the first drug infusion, subjects voided and then commenced

a 24 hr urine collection for oxalate determination. This

project was approved by the applicable Institutional Review

Board (see Appendix). The subjects gave informed consent to

participate. An Initial New Drug certificate for the study

of DCA has been issued by the FDA to Dr. P.W. Stacpoole, the

physician in charge of the volunteers.


Assay of DCA


A sample (0.1 ml) of plasma (unknown or standard) was

placed in a Hypo-vial sealablee thick glass vial). If the

plasma concentration was too high, 0.1 ml of diluted plasma

was ued. Then 50 ul of a 100 yg/ml TCA solution and 2 ml of

14% boron trifluride in methanol were added. The vial was

closed with a rubber septum and then the septum was covered

with an aluminum cap and agitated briefly and heated in a

boiling water bath for ten minutes. The vial was then

cooled to room temperature. One mililiter of water and two

mililiters of benzene were then added to the vial through

the rubber septum. The vial was then rotated on the rotator







for two hours and then the vial was opened. The contents of

the vial were then poured into a test tube and centrifuged

at 2000 rpm for two minutes. Samples (2 pl) of the

supernatant (benzene layer) were then injected into the gas

chromatograph. The Chromosorb 101 column was 6 ft x 2mm

i.d. at a temperature of 180 degrees. The carrier gas was

nitrogen at a flow rate of 60 ml/min. The Ni-63 electron

capture detector was set at 300 degrees.


Study of the Effect of DCA Metabolites
(Oxalate, Glyoxalate, Glycolic Acid) to DCA Assay



Solutions were prepared containing 100 p g/ml DCA, with

1000, 100, and 10 pg/ml of oxalate, glyoxalate and glycolic

acid individually. Each sample was assayed in duplicate by

gas chromatography following the procedure described in the

DCA assay.


Study of DCA Binding Using
a Dianorm Equilibrium Dialyser


The Dianorm equilibrium dialyser consists of a set of

four racks of cell holders, each containing five dialysis

cells located between spring-loaded metal spacers to avoid

leakage. One rack of 5 cells was shown in Figure 2.

The cells are made of Teflon to minimize absorption of

the drug into the material of the Dialyser. The cells are

divided into two halves (half cell A and B, respectively)



























2 23 4 t

1 5





Fig. 2. Diagram of Cell Unit of Dianorm Equilibrium
Dialyser
1. Driving Flange with Guide Rods
2. Teflon Cell BASE or Half-Cell A) 5 Pair in
3. Teflon Cell LID or Half-Cell B) Complete Stack'
4. Spring Loaded Cell Spacers, 6 in Complete Stack
5. Bearing Flange secured with 3 Knurled Nuts







which are separated by a semi-permeable membrane. The

solutions under study are injected into half cell. The

dialysis membranes used were previously soaked in deionized

water for 30 minutes. Each half cell was injected with

1.5ml of plasma or pH 7.4 isotonic buffer solution. For

consistency and convenience, the plasma was always put into

the thick half cell and buffer into the thin half cell. The

racks of the cells were rotated at 4 revolutions per minute.

To study the time to equilibrium, 100 yg/ml DCA was put

on buffer side and the cell was rotated. Samples were

withdrawn after 1, 2, 4, 8 and 16 hrs of dialysis. Both

buffer and plasma were assayed for DCA content. To study

whether DCA equilibrium was affected by adding it to either

the buffer side or the plasma side, a solution, (100 yg/ml)

of DCA was also put into the plasma side initially and

dialysed for 1, 2, 4, 8 and 16 hrs. After dialysis, the DCA

concentration on both sides were measured.

To study the effect of temperature on binding, dialysis

was performed at 37 degrees C and 25 degrees C in a water

bath. A solution (100 ug/ml) of DCA was added to the buffer

side and dialysed for one hour. The DCA concentrations on

both sides were assayed.

To study the effect of DCA concentration on protein

binding, DCA concentrations of 100, 150, 200, 250 and 300

pg/ml were dialyzed at 37 degrees C for one hour. 'The

sodium dichloroacetate was assayed on both sides

after dialysis.







Individual plasma samples were also dialyzed.

Dichloroacetic acid was added to blank plasma samples from

some of the subjects at a concentration of 100 ug/ml to

study the individual variability of protein binding. The

DCA concentrations on both sides after dialysis were assayed

and fraction of protein binding of DCA was calculated and

listed.


Red Blood Cell Localization Study
and Partition Coefficient



Eighteen capped test tubes, each with 10 ml of whole

blood inside containing DCA at 100 yg/ml were put on a tube

rotator. Samples were withdrawn at 5, 10, 30, 60, 90, and

120 minutes to study the time to reach equilibrium. Three

tubes were used at each time point.

Centrifugation was at 2000 rpm for five minutes after

rotation. Plasma was separated from the red blood cells and

the plasma content of DCA was measured.

To study the effect of DCA concentration on red blood

cell localization, whole blood concentrations of 50, 100,

300, 600 and 1000 ug/ml DCA were prepared. These samples

were rotated for two hours to make sure equilibrium had been

reached. After centrifugation at 2000 rpm for 5 minutes,

separating plasma from the red blood cells, the plasma

content of DCA was assayed.







The partition coefficients were determined in the

following way. Fifteen capped test tubes, each with 5 ml

whole blood inside, were taken and washed with pH 7.4

isotonic buffer solution three times in order to remove all

the plasma protein. The washed blood cells were

reconstituted by adding pH 7.4 isotonic buffer solution to

give the original blood volume. This formed "pseudoblood."

Sodium dichloroacetate was added to the pseudoblood,

resulting, in triplicate, in whole "blood" concentrations of

50, 100, 300, 600 and 1000 pg/ml. After centrifuging at

2000 rpm and separating the buffer and red blood cells, the

DCA content in the buffer was assayed.


Oxalate Assay


Twenty four hour urine was collected and adjusted with

HC1 to a pH value less than 3, and mixed thoroughly. A 60

ml aliquot was submitted to the Clinical Chemistry

Laboratory in Shands Hospital of the J. Hillis Miller

Health Center, University of Florida. Analysis was done by

atomic absorption spectrophotometry [33].


Creatinine Assay


Twenty four hour urine was refrigerated until'analysis.

The creatinine assay was carried out by the Clinical

Chemistry Laboratory in Shands Hospital. Creatinine





22

concentration was determined by the red color development on

reacting with picric acid [34].











CHAPTER IV
DATA ANALYSIS

Compartment Model


The concentration-time data for most drugs can be

analyzed with the use of compartmental models. The one

compartment model assumes the drug to be homogeneously

distributed throughout the body. The two compartment model

assumes a central compartment containing the blood volume

and the highly perfused tissues such as liver, kidneys and

lungs. The peripheral compartment then consists of the

poorly perfused tissues such as muscle and fat. Previous

studies [28,32] have shown that DCA follows a one compartment

distribution model in its distribution. So in this study, a

one compartment model was adopted.


Elimination Rate Constant and Half Life


The elimination rate constant assesses the speed of

drug elimination from the systemic circulation. It can be

calculated from the slope of the concentration-time profile.

If the drug follows first order kinetics, then the

elimination rate constant can be obtained by calculation of

the slope of the graph of natural logarithm of the

concentration versus time. If the drug follows a zero order







kinetics, then the elimination rate constant can be obtained

by calculating the slope of a concentration versus time

profile. In this study, the elimination rate constant of

each volunteer following termination of each infusion was

calculated in this way for both zero and first order

models and the goodness of fit (R2) was also calculated.

The elimination half-life (first order model) was also

calculated following termination of each infusion using the

equation:


t = 1n2 Eq. (1)


Volume of Distribution


Volume of distribution is not a real volume but a

hypothetical volume of body fluid that would be required for

the body content of the drug to distribute at the same

concentration as that found in the blood. Using the first

order rate constants, the apparent volume of distribution of

DCA was calculated using the formula:



-kel*t -kel*t
Cpt = ko [1-e ] + Cp e Eq. (2)
Vd*kel t-1


where:

Cpt is the peak plasma concentration following each

I.V. infusion,
















If the

the Cp
t


is the infusion rate,

is the time of infusion, in this work it is always

equal to 30 minutes or 0.5 hours),

is the plasma concentration prior to starting the

infusion.

plasma concentration was obtained after the first dos

is equal to zero.
-1


Protein Binding


Protein binding is the phenomenon occurring when a drug

combines with plasma protein or tissue protein to form a

reversible complex. Drug can be displaced from binding by

other compounds having higher affinity for the binding sites.

The pharmacologic effect is thought to be from the free drug

only. So the extent of binding is an important feature of

the drug, and the fraction of protein bound is defined:


f = [D-P]
[D]
tot


[D-P]
[D] + [D-P]
free


Eq. (3)


In this study, drug concentration on the plasma side is

given by [D] + [D-P], and drug concentration in the buffer

side is the free drug concentration, since only free drug

passes the membrane. So:


e,









f = [Drug concentration in plasma side]
[Drug concentration in plasma side]

Eq. (4)
[Drug concentration in buffer side]
[Drug concentration in plasma side]

Red Blood Cell Localization and Partition Coefficient


The purpose of studying red cell localization is to

find out the amount of DCA that is bound to red blood cells.

The partition coefficient can be defined as:


D = Drug Concentration in Erythrocyte Eq. (5)
Drug Concentration in Plasma Water


This is significant in at least two ways. First blood

is often hemolyzed on collection. Hemolysis will then cause

inflation of plasma levels. Second, red blood cell

localization can be a significant factor in calculation of

volume of distribution. Variations in red blood cell

localization can possibly cause variation in drug response.

The partition coefficient can also be used to calculate

the fraction of protein binding if the drug concentrations

in red cells and in the plasma are obtained [35], since:


D = { Cb f 1} 1-H) Eq. (6)
Cp*(l-f)*(R-H) T-H HTr







Multiplication of both sides by H, and rearrangement gives:


H*D = Cb (1-H Eq. (7)
Cp*((1-f)- l-fT


where H is the hematocrit which can be determined

experimentally, Cb stands for whole blood concentration

which can be determined before the experiment, Cp is the

plasma concentration after equilibrium with red cells, and f
is the calculated fraction of protein binding.


Area Under the Blood Level Curve


Area under the curve (AUC) is an estimate of the total

amount of drug absorbed for first order elimination. In

this study, it is calculated from:




AUC = AUC T + Eq. (8)
o o 0el Eq. (8)




where AUCT is the area under the curve from time zero to the

last sampling time T, which can be obtained by a trapezoidal
method, and Ct is the plasma concentration for the last

sample. Kel is the elimination rate constant of the drug

for each individual.









Statistical Analysis


The elimination rate constant was obtained by best fit

of the slope of linear regression using a TI55-II hand-held

calculator. The comparison of observed half-life, volume of

distribution, etc., of each individual after each dosing was

done by analyses of variance (ANOVA), the SAS statistical

program package, and Duncan's multiple range test [36]. A P

value of 0.05 was chosen as the standard of whether

treatment is of statistical difference or not. The

comparison of goodness of fit by using I- where R is

correlation coefficient for the linear model or the log.

linear model was done using student's t test.












CHAPTER V
RESULTS

Identity of DCA and DCA Assay


The DCA sample was shown to be of required identity and

purity first by NMR spectroscopy, which showed the CH bond

of the CHC12 grouping (Fig. 3). The IR spectrum showed

bands consistent with C=O, COOH and C-C1 bonds (Fig. 4).

Elemental analysis gave a content which agreed with the

putative formula (Table 15). The gas chromatography of DCA

is shown in Fig. 5. The first peak is the methylester of

DCA on reaction of DCA with BF /methanol. That was proved

by injection of an authentic sample of the ester. The

second and also the last peak was the methylester of TCA.

Injection solutions typically assayed at 99.5% of the stated

concentration using the specific gas-chromatographic method

and both the DCA sample and the authentic ester sample as

standards. A typical calibration curve of peak height ratio

versus DCA concentration is presented in Fig. 6. The square

of the correlation coefficient for the relationship was

0.992. The relation was linear up to 200 ug/ml.

It has been known for some years that DCA is

metabolized to glyoxalic acid which is then converted to

oxalic acid. However, the present research was the first to

examine the relation of oxalic acid excretion to DCA dosing





30
(Fig. 7). There was no evidence in the literature to prove

that the putative DCA metabolites did not interfere with the

DCA assay. By adding various amounts (10, 100, 1000 g/ml)

of oxalic, glyoxalic and glycolic acids separately to 100

,g/ml DCA sample, it was shown that there was no
relationship between contaminant and found DCA concentration

(Table 2). Besides, there were no interfering peaks on gas

chromatography.

Concentrations in Plasma

The concentration-time profiles in the volunteers

plasma are shown in Figs. 8-20. Concentrations showed the

expected rises during each infusion and the expected falls

following the end of each infusion, and a steady decline to

or towards zero after the first dose. There was, however, a

large degree of variation within the data. The area under

the curve for each set of data from zero to the 24 hour

point using the trapezoidal rule was calculated, and the

extrapolated plasma concentration to time infinity was used

to calculate the area under curve to infinity (Equation 8).

The correlation coefficient for a linear relationship

between area under curve to infinity and dose was 0.72,

(p<0.05).

Protein Binding

The time to reach equilibrium of protein binding was

one hour (Table 3). The effect of temperature on protein

binding was also studied. There was no significant

difference between 37 degrees C and 25 degrees C after







dialysis for one hour (Table 4). The DCA was added to both

plasma and buffer sides of the membrane separately, and

there was no significant difference in the results of the

two experiments (Tables 3 and 5). This means protein

binding of DCA was reversible. The study of the

relationship between protein binding and concentration

showed that binding was linear up to 300v g/ml (Table 6).

Red Cell Localization

It was shown that five minutes was enough for the

distribution of DCA into red blood cells to reach

equilibrium (Table 7). So a time of five minutes was used

for later experiments. It was shown that the partition

coefficient between red cells and plasma water was a

constant up to 1000 ~g/ml DCA whole blood concentration

(Table 8).




























CD


C>
0O




CV)
C C







CD )



ci 4-
4-
0
CL







3
c,
C)


C)




t







a. 0

o 4-
0
u



CL
UU)



.3-
C,,
0*
0)1





U)
0






U
@3

C,-


z

0




.9-






























C)







C)


0
0

I4-3


C> 0



LUJ
< d





=C E
M






0
V)Q






44-
0
E


Co u
occz



CD3


--
C)C











cc '.o 19t

NOISSIWSNVU.i 1NJ3d













Blank Plasma










Fig. 5. Gas Chromatogram of
Dichloroacetate and
the Internal Standard
Trichloroacetic Acid,
both as Methyl
Esters.


TCA


Plasma









DCA


TCA







4 min


F-H






















2.0



1.6




o1.2



0.8


C-
0.4



0.0 I I I
0 40 30 120 160 200
Concentration ( q/ml)

Fig. 6. Calibration Graph for Dichloroacetate in
Plasma. Y = 0.07 + 0.0085x, r2 = 0.992,
where Y is the Peak Height Ratio and x is
the concentration.




















"' 400





S300



o 3
0
0 *
. 200


4-J


U
100


0 0



0 10 20 30 40 50

Dose (mg/kg)



Fig. 7. Graph of 24 hours Urinary Oxalate Excretion
vs. Dose in Eleven Subjects Given Various
Doses of DCA. Y = 29 + 3.47x, R = 0.67,
where Y is mg oxalate/mg Creatinine.
x is the Dose of DCA. Subject #4 was not
Included because the Dose was in Doubt.




















12.0



10.0-



S8.0




6.0



4.0




2.0-



0.Oi
0 10 20 30 40 50
Dose (mg/kg)


Fig. 8. Graph of Area Under the Curve of Plasma
Dichloroacetate Concentration vs. Time
against Dose in Eleven Subjects given
Various Doses. Y = 0.]6x 0.24, R =
0.73, where Y is the Area Under the Curve
in mg-h/ml and x is the Dose in mg/kg.
Subject #4 was in Doubt.



























Q)


E

0
5-l


ci
4e-
ca




re
I-i

O*







4- *0
U-
c)0)
0
c/) 0











4-
O 0
I-


0 -
E4-
0-4
C-



Q*1

-:
O th





0)(
EC



0-



a-'~


(I u/,n) I uo oJs ue, uo3





























2

0

L
SC-
4-
C4



Go








S wJ



0
"" UC
0

S*1Q











4C
e- I
o a -E






-20I











/* 2 QE c
CI- 6-4-




b--- j 0





CO


EC






S- E
I-I>





1 --o L



(Lw/bn)uonej;uaouo3








40




















CD
0,






4-
0





co



64-


UL
0




CLO
0








uS-o
.I-

4- o









0 *r


I -



Co
0 'r


Ec





U-
-4




( (NLd -








41



















CN =

e-4
1-




0

o*-





c-
Q

/ 0




S-
0E
4-4-


L

4- 0








(* r-u o
O- to






C 0
CL
-H 05


-)4



c I













y --O
































































































o 0 0 0
-0 C


0 0 0 0
o o o


01





to
CC
4-)

10


o



UL
0





c0

S .r-
U 0-



0 :
.000





4--




S-O




o **-
;-







c 0
o-i



EC
10






CD







*-
cn.
La.


(LW/6i5)uooLejJUauo3











43











c'n






0,
E
Ll)
o Ct





%0
C;

0.




(n 0
-o




4-'4-


(tJ C#.. 0
0 4-0
\o GJ








S-',




-- 0


--4-0
Q)



uC
c o
03






a
r1









0)-










a,-
o L.

















to CD 0 0 000
tO C.' 0 tO C.',
-
~O



























Cr>

en
E

OJ



'4-





0





OS.
*













S-
-
v,-0








S-4-.-
)- 0





4-,
C=






*r-
Oc













0*-



S a
EC




u- 4-

(A "

C-L
10,
St-


(LW//6r) uOL 4BjuaO3uo3
































E
Cr,


LO


(1)
4-3
'4-
co
co


0
CL
0






-o


4-4-

-c

EE 4- 0
04-




4L-
V)Q











go 0



C
a 0v)











'0
CO
0*'-
L).
'u 4-



c.,l
-4L

U...


(Lw/65()uopvjjuouuo3
















46
















SLd
*C,









O*
4M-^











co
S-











4- 0
(i'













0-
tU
)-I




4.,
4-



















*r-o
C0

Q*










C
CO
0 *r-










4-0
E- 0









r- O
4*

















o-
Li.


(Lw/6Br)uanunue0uo


\o









47





















O
.)
M ,-^








0
S.
o i


.4-
(a




0

0 .
P L













O
z-o















4-4-
(1)




4--


0
*r-






03





cc**







-O..
M 4-










Q,. i-






o




l-i
O i

S^-

00"
-1
*,

Q S


(iiW/6r)uoLjePjua3uo3








48


















E-


LO

ci
4e
4-





O
go



0





V) 0
s- cn

a- t--4
US-
0










co





0 *













4-4-
C
0) '4








r cc
z. '-c0












CLC
0
S0

-- O w=



<-6

C O


(TI/2r) uo!TjBaua9uoD









49



















C)



m
0
U,


4-









V)0
S- C





a4-


4-0




04-
l-
co S










.:-*
c












a0





3-,
0).

O14










(TM/Srf)~C. Inq-4u~o
EC

I-,.




0)o
-o
o 0 0 0
o 05 0
'.0 ~

(pn/~~rf) UOCUUO








50















01

cn
CN3
0
3 C



4--
0


u -
)(



*r



4--0
0*O
0










-o
00

O -3

I-





to




0 i






CO
o *-
V I--V







S4-
C.








*0,





*,
CD~L


o' 0 0
0' 0 0

(Tw-i/Srw) uot:BJU3uoo










TABLE 1. Demographic Data.


Wt Hematocrit Dose
Patient No. Sex Age (kg) (%) (mg/kg)


1

2

3a

4

5

6

7

8

3b

9

10

11

12


74.8

76.0

72.4

61.4

80.6

67.8

73.3

56.8

72.4

101.4

57.6

65.0

58.0


44.4

49.0

39.2

41.3

45.5

42.6

42.8

39.9

39.2

47.5

41.5

44.1

40.9









TABLE 2.


Determination of the Interference of DCA
Metabolite in DCA Assay. 100 ig/ml DCA was
Initially Added to Three Different Concentrations
of DCA Metabolite. Results were Expressed as
. g/ml DCA.


DCA Plus: 1000 p g/ml 100 yg/ml 101 g/ml
metabolite metabolite metabolite


Metabolite:

Oxalate 103.6 99.9 102.3

104.8 99.9 102.3

Glycolate 97.5 105.9 101.1

101.1 97.5 98.9

Glycoxylate 110.8 103.6 104.8

97.5 107.2 105.9









TABLE 3. Determination of Time to Reach Dialysis Equilibrium
Using Dianorm Equilibrium Dialyser. Dichloro-
acetate 100 pg/ml was Initially Added to the Plasma
Side. Concentrations of DCA on Both Sides were
Assayed.


Time (hr)

1 2 4 8 16


Plasma 55.1 49.9 54.2 54.6 58.1
Concentration
(pg/ml) 53.4 50.8 62.9 57.9 53.6

Buffer 35.8 33.4 38.4 36.0 41.2
Concentration
(pg/ml) 36.2 36.2 33.7 36.2 31.6









Studies of the Effect of Temperature on Protein
Binding. Dichloroacetate (100 ug/ml) Was
Initially Added to Buffer Side. Both Buffer and
Plasma Side were Assayed After Dialysis. "f"
Stands for Fraction of Protein Bindina.


37 C


25 C


Plasma Buffer Plasma Buffer
Side Side Side Side
Conc. Conc. Conc. Conc.
(u g/ml) (, g/ml) f (ug/ml) (pg/ml) f


0.20 59.5


0.27


58.9


0.26 53.3


AVE 56.9 43.1 0.24 57.2 44.8 0.21


SE 0.57 1.21 0.02 1.96 0.46 0.03


TABLE 4.


Dialysis
After
One
Hour


57.1

57.8

55.8


45.4

42.4

41.4


43.9

45.1

45.5


0.26

0.23

0.15









TABLE 5. Determination of Time to Reach Dialysis
Equilibrium. Dichloroacetate (100 lg/ml) was
Initially Added to the Buffer Side. Concentrations
of DCA on Both Sides were Assayed.



Time (hr)

1 2 4 8 16


Plasma 58.2 59.4 52.6 52.9 53.4
(pg/ml)
47.4 52.2 46.3 55.0 47.1

Buffer 35.5 39.2 35.6 33.8 37.4
(ug/ml)
34.0 33.5 33.8 39.8 30.9












56

0




V)
30 00
t3 C






+j +
uc IIt-
a0 *-




w) r0 4 04 V-4 r'-
0 >
04 .







L* 0 0 C 0 0
04 r- 4 O r-4 *-








-3 4-) 4 0000 0 0 CM
V U) 0 cn ( S
C 0 0 0 CO Lm rC 0 LC r
O L .0
0 4-+ 0 0 c 0C 0
O- Lo c- C 0) LOC






4-) U) r-4
0-" 4 LO m. R C- r
M 4- COM 4N C 0) 0
0 -N c 4 -



4U) 4-M CM C) (D D C o
Oc 00 0 0 0 o 0
/)n 0)








to- t 0o 0 0 0 0 CV 0
0 O N N COO LAO 0 0



4- 4r- r 0V C' 0 D LA L
4- II c
*-C. o4- C( e4 CM en Co)
o x004- .. 0 .
S*- 0 0 0 0

V-1 V r-4 (: )
+C* C 0 0 rD OO O LO O 0




4- I M .0O 1 M0 0 (0



9 4-3 (-f to co rq N% r-4

X (4- 0A 0 0
4-* c0 Q. c o





C- *- C0 0 C D C



00 U)M4
4- 0 O 0C 0 cr 01 C'


CN k- *.4 Cd 0 cO
On T i- k10 to Ur %0 % r





0c 4 %. 00 r-
cVal5 C') CV) C)0
*r- 4- 04) 4- *
0) t4- 0 0 0 0 C
4D VCIC c C e-1 a C
05- 0C 0 i LA






-C5- 6 C LA




c) 4-) LA 40 CM CM LA 9
c) 4-r rL LOo LA LA Lo






*i- 0 rU
*i** e( n 4 .




Ui0> 0 0 0 0 0
pt- o -o en c5 --i Oo L
'.0 Ol r0 CO O O CO*







Sv 4 -
-*r- or 4-



CD0 0 < a
I- *0- 0C *r)
I- O aeCo Q-- S:








TABLE 7. Determination of Time to Reach Equilibrium for Red
Cell Localization. Dichloroacetate (100, g/ml)
was Initially Added to the Whole Blood. Plasma
Concentration was Assayed. Blood was Obtained
from Volunteer 3a.



Time (min)

5 10 30 60 90 120


140.7 130.6 131.6 131.6 130.1 133.9

135.2 139.9 125.9 120.9 127.0 120.5
Concentraton
(vg/ml) 143.0 132.2 136.1 134.0 127.9 136.2

130.8 129.1 145.3 127.9 124.2 115.3

140.7 126.4 140.7 129.4 130.3 146.6

138.8 135.3 135.9 123.4 135.5 152.7

Mean 138.2 132.2 135.9 127.9 129.2 134.2

SE 1.8 2.0 2.8 2.0 1.6 5.9









TABLE 8.


58
Partition Coefficient (P) of DCA Between Red Blood
Cells and Isotonic Buffer up to 1000 ug/ml Whole
Pseudoblood Concentration. Blood was Obtained From
Subject 3a.


DCA Concentration ,g/ml


50 100 300 600 1000


0.19 0.43 0.46 0.22 0.39

0.32 0.39 0.35 0.58 0.44

P 0.47 0.46 0.39 0.73 0.45

0.41 0.33 0.48 0.79 0.41

0.23 0.32 0.52 0.45 0.42

0.47 0.52 0.36 0.71 0.42


Mean 0.35 0.41 0.42 0.58 0.42


SE 0.05 0.03 0.03 0.09 0.01










TABLE 9. Protein Binding (f), Red Blood Cell/Plasma Ratio
(RBC/P), and Partition Coefficient for Red Blood
Cell/Plasma Water Distribution in Nine Volunteers.
Data were Obtained at 100 pg/ml Initial DCA
Concentration.


Subject No.

3a 4 5 7 8 9 10 11 12


f 0.30 0.39 0.33 0.32 0.20 0.35 0.42 0.40 0.20


RBC/P 0.08 0.47 0.20 0.06 0.32 0.42 0.17 0.47 0.57

Partition
Coefficient 0.17 0.78 0.42 0.36 0.42 0.57 0.29 0.66 0.62









TABLE 10. Sodium Dichloroacetate in Plasma: Calculated R2 and kel Values
for Repeated I.V. Infusions in Different Volunteers, Using a
First Order Kinetics Model.


Infusion Number

I II III IV V


kel R2 kel R2 kel R2 kel R2 kel R2
Volunteer

1 1.54 0.72 0.92 0.94 0.38 0.94 0.25 0.89 0.25 0.89
2 0.01 0.06 0.56 1.00 0.07 0.55 0.06 0.11 0.03 0.85
3a 2.09 0.98 2.45 0.96 1.90 0.93 1.13 0.89 1.10 1.00
4 0.13 0.74 0.02 0.02 0.18 0.63 0.01 0.02 0.26 0.84
5 0.74 0.92 0.30 0.70 0.17 0.83 0.20 0.97 0.26 0.88
6 0.90 0.97 0.36 0.48 1.56 0.66 0.18 0.92 0.23 0.97
7 0.47 0.80 0.35 0.90 0.05 0.01 0.16 0.70 0.12 0.95
8 2.76 0.90 0.62 0.99 0.20 0.74 0.20 0.70 0.03 0.84
3b 0.41 0.81 0.31 0.94 0.21 0.91 0.22 0.98 0.20 0.98
9 0.37 0.97 0.31 0.99 0.18 0.29 0.30 0.76 0.14 0.98
10 0.25 0.55 0.19 0.55 0.12 0.87 0.29 0.87 0.10 0.87
11 0.14 0.08 0.45 0.90 0.11 0.29 0.03 0.19 0.07 0.86
12 0.41 0.08 0.45 0.90 0.11 0.29 0.03 0.19 0.07 0.86


Mean 0.98 0.71 0.64 0.80 0.50 0.63 0.30 0.68 0.22 0.91

SE 0.27 0.09 0.05 0.08 0.19 0.08 0.08 0.09 0.08 0.02










TABLE 11.


Calculated DCA Volume of Distribution (1/kg) Using
a First Order Kinetic Model Applied to Repeated IV
Infusion Data in 11 Subjects. Subject 4 was not
Included Because the Dose was not Certained. R <
0.81 Was Not Listed (Expressed in "--").


No. of Infusion


Volunteer


1
3a
5
6
7
8
3b
9
10
11
12


0.13
0.20
0.25
0.36
0.37
0.47
0.24
0.24


0.23


0.31
0.10
0.29

-1.18
0.28
0.45
0.23

0.23
0.30


0.32
0.11
0.43
0.25

0.28
0.43

0.49


0.28
0.19
0.34
0.20
-14.2
0.37
0.39
0.27
0.16

0.42


0.35
0.25
0.49
0.6

0.27
0.39
0.25
0.09
0.22
0.22


AVE 0.28 0.27 0.30 0.29 0.31


SE 0.03 0.04 0.04 0.03 0.05


Subject 2 was not included because data was not available.


Negative data was not included in average.










TABLE 12.


Calculated Half Life for Volunteer After Each
Dose. R Value Smaller Than 0.81 in Line Fit was
not Included (Expressed in "--").


No. of Infusion


Volunteer


1
2
3a
4
5
6
7
8
3b
9
10
11
12


0.45

0.33
5.5
0.94
0.77
1.47
0.25
1.69
1.87


1.69


0.75
1.24
0.28

2.31

1.98
1.15
2.24
2.24

1.54
4.62


1.82

0.36

4.10


3.46
3.3

5.78
--
--


2.77

0.61

3.46
3.85
4.33
3.46
3.15
2.31
2.39

8.66


1.87
23.10
0.63
2.67
2.66
3.01
5.77
23.1
3.46
4.95
6.93
9.9
9.9


AVE 1.50 1.84 3.14 3.50 7.53


SE 0.46 0.36 0.66 0.63 2.07










TABLE 13.


Dichloroacetate in Plasma: Rates of Metabolism
of Each Subject After Repeated IV Infusions
at 2-Hour Intervals (Numbered Consecutively
I-V). Subject 2 was not Included Because
Data Was Not Available. R < 0.81 was not
Listed (Expressed in "--").


Interval

I II III IV V


Subject

1 829.2 901.7 334.5 273.9 383.0
3a 953.3 1037.7 874.6 634.2 706.8
4 110.2 -- -- -- 1522.2
5 1259.9 738.4 677.6 805.1 1460.2
6 1230.2 -- 2266.0 241.9 1029.2
7 766.1 *
8 3271.4 757.3 293.3 523.7 82.9
9 1730.6 2020.7 -- 2987.2 1400.1
10 -- -- 1004.8 1478.4 474.5
11 -- 1621.4 -- -- 466.6
12 1046.7 678.8 -- 781.5 506.1


AVE 1187.4 1089.1 881.7 950.2 803.6


SE 267.6 169.5 250.9 282.2 146.8


* Data was not available.










TABLE 14.


Sodium Dichloroacetate in Plasma: Calculated R2 and Rate
Constants for Repeated I.V. Infusion in 12 Volunteers,
Using a Zero Order Kinetics Model.


Infusion Number

I II III IV V

kel R2 kel R2 kel R2 kel R2 kel R2
Volunteer

1 +3.07 0.71 19.8 0.86 10.7 0.97 10.6 0.86 5.56 0.95
2 1.30 0.25 55.5 0.99 10.0 0.51 9.1 0.11 4.80 0.78
3a +18.9 0.75 35.4 0.70 33.6 0.78 21.5 0.81 4.63 0.73
4 +16.6 0.75 3.42 0.01 42.8 0.70 1.72 0.05 10.5 0.84
5 +39.4 0.95 24.2 0.82 17.7 0.83 25.4 0.97 9.81 0.97
6 +15.8 0.66 15.4 0.47 15.6 0.99 15.5 0.93 8.10 0.95
7 -38.8 0.83 -24.4 0.86 6.34 0.02 -21.4 0.70 5.68 0.85
8 +29.1 0.96 48.6 0.95 15.0 0.73 21.3 0.71 4.58 0.78
3b +28.4 0.77 24.0 0.95 20.3 0.93 24.6 0.99 9.11 0.93
9 +55.2 0.98 70.3 0.997 49.2 0.33 84.4 0.77 22.0 0.97
10 +48.6 0.57 38.2 0.52 30.6 0.87 114.8 0.84 37.7 0.72
11 +12.5 0.04 86.1 0.93 27.4 0.29 11.1 0.22 21.2 0.80
12 +59.6 0.77 35.2 0.99 81.0 0.63 29.6 0.79 23.8 0.84
Average 29.7 0.69 38.01 0.77 27.7 0.66 30.8 0.67 12.5 0.85


SE 4.9 0.08 6.86 0.080 5.7 0.08 9.72 0.09 2.56 0.03


Negative kels were not included in average.











TABLE 15. Identity of Sodium Dichloroacetate by Elemental
Analysis.



ELEMENT FOUND THEORY


C 15.71 15.8

H 1.39 1.3

0 21.1

Cl 46.35 46.7

Na 15.1










CHAPTER VI
DISCUSSION

Binding and Red Cell Localization


In this research, attention was particularly focused

on plasma concentrations of dichloroacetate. A major factor

in interpreting plasma concentrations of drugs is bindg of

the drugs to plasma protein. This was studied for

dichloroacetate using the Dianorm apparatus. Preliminary

experiments concerned factors such as time to reach

equilibrium.

Times to reach dialysis equilibrium are different for

individual ligands. Also, the dialysis time increases with

decreasing temperature, increasing dialysis volume,

increasing thickness of membrane, and increasing molecular

weight of ligand.

The Dianorm apparatus is designed for optimal geometry

and a high concentration gradient is maintained across the

membrane. In this research, slow rotation was used (4 rpm)

to make sure mixing was thorough. The thin wall of the

cell assured rapid heat exchange between water bath and cell

content. By using this apparatus DCA equilibrium was

reached within one hour (Table 3). There was no significant

difference for equilibrium time between 25 degrees C and 37

degrees C.

Binding of drugs to plasma protein may have a marked

effect on the distribution and pharmacologic effects of a

66









drug, and on the rate at which it is eliminated from the

body. This is because distribution of drugs to body tissues

takes place from the blood compartment. Also, it is the

free drug concentration which equilibrates with the body

tissues. The fraction of drug in the plasma bound to plasma

protein is not immediately available for distribution into

the extravascular space or for certain modes of elimination.

Because of this, the binding of dichloroacetate to plasma

proteins from individual subjects was studied. The fraction

bound at a whole plasma dichloroacetate concentration of 100

Pg/ml varied from 0.2 to 0.42 (Table 9). At this level,

protein binding is generally considered to be of no great

clinical significance.

Several drugs enter the erythrocyte quite rapidly and

others do so at a slower rate. The extent of penetration

can often be correlated with the solvent/water partition

coefficients of these drugs highly lipid soluble drugs

show greater accumulation in the erythrocytes.

Dichloroacetate is a highly water soluble compound, so the

predicted partition coefficient between erythrocytes and

water is very low. This was proved to be so by the present

experiments. It has been suggested that determination of the

RBC/plasma concentration ratio may serve as a simple and

rapid technique for the indirect measurement of plasma

protein binding. This would be applicable in the

large-scale screening of abnormal plasma binding in routine

clinical blood samples, thereby facilitating








68
individualization of drug therapy. However, in our case,

the RBC/plasma concentration ratio did not show very large

variation (Table 9).

The calculated fractions of DCA protein binding in two

subjects using equation 7 were 0.60 (subject 3a) and 0.59

(subject 5). Each of these figures is a mean of six

determinations. The standard deviation in subject 3a was

0.23. These means are higher than the means for fraction

bound obtained by equilibrium dialysis. However, they were

obtained by using the mean partition coefficient and the

standard deviation of the partition coefficient was not

taken into consideration. It has been shown in the

literature that buffer systems, ionic strength and pH value

will affect binding properties [37,38,39]. It is necessary

to conduct further investigations concerning the various

factors affecting DCA protein binding. But from the

clinical point of view, 0.3 versus 0.60 was not of

physiological significance.


Pharmacokinetic Analysis


The decay of DCA concentrations in plasma was examined

following termination of each infusion. In each patient,

the four available concentration-time points from dose I to

IV and five or six available points following the last

infusion were compared with a linear model (concentration

versus time) and then with a logarithmic concentration versus









time model. The mean values of t (where R is the

correlation coefficient) for the relationship using both

models are listed in Table 10 and Table 14. Although the

goodness of fit was comparable for the two models, the zero

order model was rejected for two reasons. First, other

investigators have shown first order kinetics to apply at

the level of this data seen in this work after the initial

doses. There was a trend that more data points gave better

Re (where R is the correlation coefficient) for the first

order than zero order model. This was found by comparing

the ie for the data after the fifth doses using first order

or zero order models. The first order model is

significantly better than the zero order model at p=0.1

level. Second, the putative zero order slopes were

calculated to range from 4.58 rg/ml/h to 114.8 pg/ml/h. That

means the output rate would be very close or even greater

than input rate, which is inconceivable.

Using only the data for which R was greater than 0.81,

which is the lower limit for significance of a four-point

correlation, the mean half-life calculation was found to be

90.0 min for first dosing, 110.4 min, 188.4 min, 210.0 min,

and 451.8 min for 2nd, 3rd, 4th, and 5th infusions (Table

12). The mean half-life rose steadily from the first dose

to the fifth dose, and the differences are statistically

different (p<0.005).

Data of this kind could have several mechanistic

implications. For example, first order decay kinetics are a









special case of Michaelis-Menten (MM) kinetics, applicable

when the substrate concentration (S) is low. In this work S

was clearly low enough for the first order simplification of

the MM model to apply, even when S increased from the first

to the fifth dose. However, it can be argued that the data

should have shown gradual deviation from the first order

case at higher S values if In and Vmax had remained

constant. Since this devation did not occur, it may be that

changes in either ~n or Vmax occurred as dosage progressed,

but with retention of applicability of the first order

model. Mechanisms for such a change could include slowing of

dichloroacetate metabolism following enzyme poisoning by the

substrate, and/or product inhibition. Definition of the

mechanism must await suitable further studies, including in

vitro metabolism studies, and development of zero order

input/first order output pharmacokinetic models appropriate

to intermittent intravenous infusion dosing. Additionally,

models for zero order input Michaelis-Menten output will be

needed.

The calculated rates of metabolism after each dose are

listed in Table 13. These rates were obtained by multiplying

plasma DCA concentrations by volumes of distribution and

rate constants of elimination. There was a small trend

towards a reduced rate of metabolism with time. Statistics

showed that the rate of metabolism after the fifth dose was

significantly less than that after the second dosing

(p<0.05). These calculations were conducted because three









possible observations could have been made:

(1) an increase in the rate of metabolism with time

and/or level might have indicated adherence to first order

or Michaelis-Menten kinetics, with a tendency towards

saturation of the enzyme concerned (the normal for enzyme

kinetics);

(2) a confirmed decrease in the rate of metabolism

would have indicated a reduction in enzyme activity; and

(3) no change would have been equivocal. While failing
to confirm a decrease, the calculations do rule out an

increase. There is support for the theory that DCA

metabolism involves enzyme poisoning or product inhibition

but more experiments are needed to confirm this idea.

The applications of the term "volume of distribution"

are many, including those to dosage regimen calculation of

drugs based on the characteristics of an individual patient.

It is possible to observe different plasma concentration

values in different individuals after the administration of

the same dose because of the differences in the volume of

distribution. That is the rationale for the calculation of

dosage adjustment according to body weight.

No evidence was obtained for a volume of distribution

change with repeated dosing (Table 11). This observation

disagreed with earlier observations in patients [26].

However, all of the patients in the other study were in shock

at the time of commencing DCA infusion, and a rise in blood

pressure, possibly caused by a positive inotropic effect of







72

DCA occurred. This is being investigated further, but it is

not to be expected to be seen in healthy volunteers, who

showed no cardiovascular changes when treated with DCA.

Obviously, a change in the DCA volume of distribution might

well be linked to cardiac output and related cardiovascular

status.




































APPENDIX













UNIVERSITY OF FLORIDA J. HILLS MILLER HEALTH CENTER BOX J-14
Vice President for Health Affairs Gainsville, Florida zip 32610
HEALTH CENTER INSTTITIONAL REVIEW BOARD MAY 11, 1983
TO: Peter W. Stacpoole, M. D., Ph.D.
Assistant Professor, College of Medicine
PFRa: B. Joe Wilder, M. D. J-14
Chairman, Institutional Review Board l
SUBJECT: Approval of the Institutional Review Board
Project I 133-83 entitled Clinical Pharmacology of.
Sodium Dichloroacetate (DCA)."

The Institutional Review Board has recommended the approval of your protocol,
identified above, for a period of 12 months, and has determined that
human subjects will be at risk.
Approval of your research project is, therefore, granted until 5/11/84
By the end of this period you will be asked to inform the Board on the status
of your project. If this has not been completed, you may request renewed
approval at that time.
You are reminded that a change in protocol in this project requires its resub-
mission to the Board. Also, the principle investigator must report o the Chair
of the Institutional Review Board promptly, and in writing, any unanticipated
problems involving risks to the subjects or others, such as adverse reactions
to biological drugs, radio-isotopes or to medical devices.
If it is anticipated that VA patients will be included in this project, or if
the project is to be conducted in part on VA premises or performed by an) VA
employee during VA-cmpensated time, final approval should be obtained by appli-
cation to the Veterans Administration Hospital Research Office.
Ba copy of this memorandum the chairman of your department is reminded that
his responsible for being informed concerning research projects involving
human subjects in his department. He should review the protocols of such
investigations as often as he thinks is necessary to insure that the experiment
is being conducted in compliance with our institution and with DIMS regulations.
cc: James E. McGuigan, M. D. co-PIs
B. Joe Wilder, M. D. JStephen H. Curry, Ph.D.
Thomas G. Baumgartner, M. Ed., Pharm.D. T. G. Baumgartner, Pharm.D.
William C. Thomas, Jr., M. D.
Clinical Research Center
Becky Stevens, Ph.D.
Cellee of Medicine College of Nrsing College of Phiemucy College of Helh Relted Perolssions Cellee of Dentistry
College of Vterinry Medicinel Shnds Teehing Hospital end Clinics Veterons Admnistrati n Hospitel
QUAL UICLOMYLNWT OLPORTUNITYV/APWIMATIVI ACTIOeN t) MWI.etC











HEALTH CENTER INSTITUTIONAL REVIEW BOARD

jq


UNIVERSITY OF FLORIDA J. HILLIS MILLER HEALTH CENTER BOX J-14
Vice President for Health Affairs Gainesville, Florida zip 32610


May 11, 1983

Peter W. Stacpoole, M. D., Ph.D.
Assistant Professor
College of Medicine
Department of Medicine
Box J-226

SUBJECT: Protocol #133-83, Clinical Pharmacology of
Sodium Dichloroacetate (DCA)

Dear Dr. Stacpoole:

Your protocol, referenced above, was reviewed at our
meeting this date.

It was the decision of the Committee that this study can be
approved if the following condition is met:

In the three (3) Informed Consent Forms, clearly state
that there is remote possibility of added risks because
of the use of this drug. (state what these are).- If
there are none, state so.

You will find enclosed the Approval Letter for this study.

Please send four copies (4) each of the three (3) revised
Informed Consent Forms to this office (J-14) at your earliest
convenience, since approval was contingent upon this condition
being met.

We are glad to be of service to you in this capacity, and we
appreciate your cooperation in this matter.

Sincerely, Q


B. Joe Wilder, M. D.
Chairman
BJW/ddo
continued.....
College of Medicine College of Nursing College of Pharmacy College of Health Related Professions College of Dentistry
College of Veterinory Medicine Shonds Teaching Hospital and Clinics Veterans Administration Hospital
I KQUAL EMPLOYMENT OPPORTUNITY/IAPIRMATIVI ACTION EMPLOYEE










Page 2 May 11, 1983
Protocol #133-83 76
P. W. Stacpoole, M. D.,
Ph.D.


cc: vtephen H. Curry, Ph.D.,
co-PI
Thomas G. Baumgartner, M.Ed.,
Pharm.D., co-PI
File
Enclosure: Approval Letter (#133-83)














REFERENCES


(1) Windhol, M., Budavari, S., Stroumtsos, L.Y., and Fertig,
M.N., Merck Index, Merck & Co., Inc., Rahway, N.J., Ninth Edition, 1976.

(2) Demaugre, F., Cepanec, C., and Leroux, J.P., "Characterization
of Oxalate as a Catabolite of Dichloroacetate Responsible for the
Inhibition of Glyconeogenesis and Pyruvate Carboxylation in Rat Liver
Cells." Biochem. Biophys. Res. Commun. 85:1180-1185, 1978.

(3) Little, M., and Williams, P.A., "A Bacterial Halidohydrolase:
Its Purification, Some Properties and Its Modification by Specific
Amino Acid Reagents." Euro. J. Biochem. 21:99-109, 1971.

(4) Eichner, H.G., Stacpoole, P.W., and Forsham, P.H., "The
Metabolic Effects of Sodium Dichloroacetate in the Starved Rat."
Biochem. J. 142:279-286, 1974.

(5) Lorini, M., and Ciman, M., "Hypoglycaemic Action of
Diisopropylammonium Salts in Experimental Diabetes." Biochem.
Pharmacol. 11:823-827, 1962.

(6) Stacpooke, P.W., and Felts, J.M., "Diisopropylammonium
Dichloroacetate (DIPA) and Sodium Dichloroacetate (DCA) Effect
on Glucose and Fat Metabolism in Normal and Diabetic Tissue."
Metabolism 9:71-78, 1970.

(7) Eichner, H.L., Stacpoole, P.W., and Forsham, P.H.,
"Treatment of Streptozotocin Diabetes with Diisopropylammonium
Dichloroacetate (DIPA)." Diabetes 23:179-182, 1974.

(8) Whitehouse, S., and Randle, P.J., "Activation of Pyruvate
Dehydrogenase in Perfused Rat Heart by Dichloroacetate." Biochem.
J. 141:761-774, 1974.

(9) Goodman, M.N., Ruderman, N.B., and Aoki T.T., "Glucose and
Amino Acid Metabolism in Perfused Skeletal Muscle: Effect of
Dichloroacetate." Diabetes 27:1065-1074, 1975.

(10) Stacpoole, P.W., Moore, G.W., and Kornhauser, D.M.,
"Metabolic Effects of Dichloroacetate in Patients with Diabetes
Millitus and Hyperlipoproteinemia." N. Engl. J. Med. 298:
526-630, 1978.










(11) Holloway, P.A.H., and Alberti, K.G.M.M., "Reversal
of Phenformin-Induced Hyperlactaemia by Dichloroacetate in
Normal and Diabetic Rats." Diabetologia 11:250-251, 1975.

(12) Holloway, P.A.H., and Alberti, K.G.M.M.,
"Phenformin-Induced Lactic Acidosis: Prevention by
Dichloroacetate." Clin. Sci. Mol. Med. 50:33, 1976.

(13) Arieff, A.I., Leach, W., and Lazarowitz, V.,
"Treatment of Experimental Lactic Acidosis with
Dichloroacetate (DCA)." Clin. Res. 26:410A, 1978.

(14) Johnson, G.A.H., and Alberti, K.G.M.M., "The
Metabolic Effects of Sodium Dichloroacetate in Experimental
Hepatitis in the Rat." Biochem. Soc. Trans. 5:1387-1388,
1977.

(15) Park, R., Leach, W., and Arieff, A.,
"Dichloroacetate (DCA) Prevents Hyperlactatemia Following
Functional Hepatectomy (HPX)." Clinical Research 27:48A,
1979.

(16) Park, R., Leach, W., and Arieff, A.,
"Dichloroacetate (DCA) Decreases Extrahepatic Lactate
Production in Diabetic Dogs." Clin. Res. 27:374A, 1979.

(17) Loubatieres, A.L., Ribes, G., and Valette, G.,
"Pharmacological Agents and Acute Experimental
Hyperlactataemia in the Dog." Br. J. Pharmacol. 58:429p,
1976.

(18) Zambraski, E.J., and Merrill, G.F.,
"Dichloroacetate Sodium (DCA) Decreases Plasma Lactic Acid
(LA) Observed During Exercise in Dogs." Clin. Res. 27:380A,
1979.

(19) Schneider, S.H., Komanicky, P.M., and Goodman,
M.N., "Enhancement of Exercise Performance by Dichloroacetate
(DCA) in Rats." Diabetes 28(Suppl.2):61, 1979.

(20) Merrill, G.F., Rosolowsky, M., and Young, M.A.,
"The Influence of Dichloroacetate Sodium on the
Hyperlactacidemia of Endotoxin Shock." Fed. Proc., Fed. Am.
Soc. Exp. Biol. 38:1192, 1979.

(21) Merrill, G.F., and Rosolowsky, M., "Effect of
Dichloroacetate Sodium on the Lactacidemia of Experimental
Endotoxin Shock." Circ. Shock 7:13-21, 1980.

(22) Grover, G.J., Scanes, C.G., and Merrill, G.F.,
"Effects of Dichloroacetate Sodium on Circulating Levels of
Corticosterone, Glucose and Lactate during Endotoxemia in the











Domestic Fowl Gallus
Exp. Biol. 38:1123,


domesticus." Fed. Proc., Fed. Am. Soc.
979.


(23) Loubatieres, A.U., Ribes, G., and Valette, G.,
"Pharmacological Agents and Acute Experimental Hyperlactatemia
in the Dog." Br. J. Pharmacol. 58: 429, 1976.

(24) Schneider, S.H., Komanicky, P.M., and Goodman, M.N.,
"Enhancement of Exercise Performance by Dichloroacetate (DCA) in
Rat." Diabetes 28(Suppl 2):360, 1979.

(25) Irsigler, K., Brandle, J., Kaspar, L., Kritz, H., Lageder, H.,
and Regal, H., "Treatment of Biguanide-Induced Lactic Acidosis with
Dichloroacetate: 3 Case Histories." Arzneim-Forsch 29(I):555-558,
1979.

(26) Stacpoole, P.W., Harman, E.M., Curry, S.H., Baumgartner, T.G.,
and Misbin, R.I., "Treatment of Lactic Acidosis with Dichloroacetate."
N. Engl. J. Med. 309:390-396, 1983.


Stacpoole,
of Chronic


P.W., Moore, G.W., and Kornhauser, D.M.,
Dichloroacetate." N. Engl. J. Med. 300:372,


(28) Wells, P.G., Moore, G.W., Rabin, D., Wilkinson, G.R.,
Oates, J.A., and Stacpoole, P.W., "Metabolic Effects and Pharmacokinetics
of Intravenously Administered Dichloroacetate in Humans." Diabetologia
19:109-113, 1980.


(29) Idem: "Abnormal Resting Blood Lactate II.
Am. J. Med. 30:840-848, 1961.


(
Signi
Med.


Lactic Acidosis."


30) Huckabee, W.E., "Abnormal Resting Blood Lactate I. The
ficance of Hyperlactatemia in Hospitalized Patients." Am. J.
30:833-839, 1961.


(31) Park, R., and Arieff,
with Dichloroacetate in Dogs."


A., "Treatment of Lactic Acidosis
J. Clin. Inves. 90:853-862, 1982.


(32) Lukas, G., Vyas, K.H., Brindle, S.D., LeSher, A'.R., and
Wagner, W.E. Jr., "Biological Disposition of Sodium Dichloroacetate
in Animals and Humans after Intravenous Administration." J. Pharm.
Sci. 69(4):419-421, 1980.

(33) Henry, R.J., Cannon, D.C., and Winkelman, J., Clinical
Chemistry, Principles and Technics, 2nd edition. New York, Harper
and Row, 1349, 1974.


(34)
Methods,


Israel, D., and Henry, J.B., Clinical Diagnosis by Laboratory
Philadelphia, W.B. Saunders Company, .88, 1974.


(27)
"Toxicity
1979.










(35) Garrett, E.R., and Lambert, H.J., "Pharmacokinetics of
Trichloroethanol and Metabolites and Trichloroethanol and Metabolites
and Interconversions among Variously Referenced Pharmacokinetic
Parameters." J. Pharm. Sci. 62(4):550-572, 1973.

(36) Barr, A.J., Goodnight, J.H., Sail, J.P., and Helwig, J.T.,
1976. A User's Guide to SAS 76. Raleigh, N.C.; SAS Institute, Inc.

(37) Fleitman, J., and Perrin, J.H., "The Effects of pH, Calcium
and Chloride Ions on the Binding of Benoxaprofen to Human Serum
Albumin, Circular Dichroic and Dialysis Measurements." Int. J. Pharm.
11:227-236, 1982.

(38) Klotz, I.M., and Urquhart, J.M., "The Binding of Organic Ions
by Protein and Buffer Effects." J. Phy. Coll. Chem. 53:100-109, 1949.

(39) Henry, J.K., Dunlop, A.Q., Mitchell, S.N., Turner, P., and
Adams, P., "A Model for the pH Dependence of Drug Protein Binding."
J. Pharm. Pharmacol. 33:179-182, 1981.




















BIOGRAPHICAL SKETCH

Pei-I Chu was born in Cang Shang, Taiwan, Republic of

China, on November 29, 1959. She graduated from the

Department of Pharmacy of National Taiwan University in

June, 1981, and passed the National Pharmacy board in July

that year. She worked as a teaching assistant in the

Department of Biochemistry in the College of Medicine of

National Taiwan University from 1981 to 1982. She joined

the graduate program in the College of Pharmacy, University

of Florida, in September, 1982, to work towards the degree

of Master of Science in Pharmacy with a major in

pharmaceutics, under the guidance of Dr. Stephen H. Curry.
















I 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 degree of M r of Science.
in Pharmacy. ,


Stephen H. Curry/ Chairman
Professor of Pharmacy

I 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 degree of M ~eriof Sciee..
in Pharmacy.


Stephen H. Schulman
Professor of Pharmacy
I 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 degree of Mase of Science.
in Pharmacy. 4aJL [


Kamlesh M. Thakker
Assistant Professor of
Pharmacy
I 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 degree of Master of Science
in Pharmacy.


MargarVt 0. James
Assistant Professor of
Medicinal Chemistry










This Thesis was submitted to the Graduate Faculty of the College
of Pharmacy and to the Graduate School, and was accepted as partial
fulfillment of the requirements for e degree of Master of Science
in Pharmacy.

August, 1984 i

Dean, Colle of Pharmacy


Dean for Graduate Studies and
Research

























UNIVERSITY OF FLORIDA
II 1ll ll tlllllllllllll111111ll11111111111111111111
3 1262 08554 4004