Pharmacokinetic studies of acepromazine in the cat and the horse, studies in lipophilicity, red blood cell partitioning ...

MISSING IMAGE

Material Information

Title:
Pharmacokinetic studies of acepromazine in the cat and the horse, studies in lipophilicity, red blood cell partitioning and protein binding
Alternate title:
Pharmacokinetic studies of acepromazine in the horse and the cat studies in lipophilicity, red blood cell partitioning and protein binding
Studies in lipophilicity, red blood cell partitioning and protein binding
Physical Description:
xviii, 140 leaves : ill. ; 29 cm.
Language:
English
Creator:
Marroum, Patrick John, 1960-
Publication Date:

Subjects

Subjects / Keywords:
Research   ( mesh )
Acepromazine -- pharmacokinetics   ( mesh )
Protein Binding   ( mesh )
Phenothiazines -- pharmacology   ( mesh )
Erythrocytes -- drug effects   ( mesh )
Horses -- physiology   ( mesh )
Cats -- physiology   ( mesh )
Department of Pharmaceutics thesis Ph.D   ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Pharmaceutics -- UF   ( mesh )
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1990.
Bibliography:
Bibliography: leaves 137-139.
Statement of Responsibility:
by Patrick J. Marroum.
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 - 001581149
oclc - 24641740
notis - AHK5056
sobekcm - AA00006100_00001
System ID:
AA00006100:00001

Full Text











PHARMACOKINETIC STUDIES OF ACEPROMAZXNE
IN THE CAT AND THE HORSE,
STUDIES IN LIPOPHILICITY, RED BLOOD CELL PARTITIONING
AND PROTEIN BINDING

4.


BY


PATRICK J. MARROUM


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


UNIVERSITY OF FLORIDA
1990


































To my parents

Mrs Linda Kattan Marroum
Mr Edward Marroum
for 29 years of inspiration and guidance















ACKNOWLEDGMENTS
I express my deepest gratitude to Professor Dr. Stephen

H. Curry, chairman of my supervisory committee, for his

invaluable guidance during my graduate career and the course

of this research project. I am also grateful to him for

accepting me and providing me with training support and

facilities after Dr. Garrett's retirement.

My very special thanks go to Professor Dr. Edward R.

Garrett for his guidance and patience for more than three

years. I am very grateful for the moral support and for the

sound scientific education he provided me and also for the

personal kindness he showed me during my troubled time in

1986.

I also wish to thank the members of my supervisory

committee, especially Professor Dr. Alistair Webb whose

assistance, skill and knowledge made these studies possible.

I gratefully acknowledge the financial assistance

provided by the College of Pharmacy and the Palestine Student

Fund for providing me funds for more than ten years.

Special thanks go to Mrs Patricia Khan for all her help
and kindness during the past six years and specially in the

preparation of this dissertation. Dr. Gina Aeschbacher and

Mr. John Bliss should not be forgotten because his skill in

iii














handling the animals and assistance in performing the studies

were invaluable in the success of this project.

My deepest thanks go to Dr Jurgen Venitz for all his
advice, friendship and moral support for the past four

years.

Last but not least, my sincerest thanks to Miss Jaimini
Patel for her friendship and kind company and support during

the last four years of my studies.
















TABLE OF CONTENTS

page
ACKNOWLDGMENTS ....................................... iii

LIST OF TABLES ...................................... v

LIST OF FIGURES ..................................... viii

ABSTRACT................................................... xii

CHAPTERS
1 INTRODUCTION...................................

Historical Background.......................2

Pharmacology.................................5

Pharmacology and Clinical Applications of
Acepromazine..................................6

Toxicity of Acepromazine....................7

Metabolism .......... ....................... 10

Pharmacokinetics ........................... 10

Analytical Methods for the Assay of
Phenothiazines .............................15

2 MATERIALS AND METHODS. ........................

Chemicals .............................. ......17

Food .......................... .... ....... 18

General Apparatus................ .......... 18

Stability Studies of Acepromazine in Water
and 1 N Hcl................................21

Determination of the Partition Coefficient
between Hexane and Phosphate Buffer pH 7.4
for some selected Phenothiazines ........... 21

v
















Analysis of Acepromazine and Other
Phenothiazines from Biological Fluids
Mainly Plasma, Urine and Whole Blood.......22

Red Blood Cell/Plasma Distributiom
Coefficient ................................ 23

Extent of Protein Binding Calculated from
the Red Blood Cell Partitioning Studies....25

Bioavailabilty of Acepromazine in the Cat..26

Experimental Animals................. 26

Vascular Access Port Implantation.....27

Norfolk Vascular Access Port.........27

Maintenance of the Vascular Port......31

Administration, Collection and
Treatment of Samples..................31

Pharmacodynamics-Pharmacokinetics of
Acepromazine in the Horse..................33

Blood Pressure Measurements...........33

Heart Rate........................... 34

Blood Gas Analysis.....................34

Electrocardiogram.......................35

Urinary Catheter ......................35

Venous Sampling Line....................36

CNS and Sedative Effects.............. 36

Evaluation and Fitting of Pharmacokinetic
Data...... ... .. ...... ..... ..... ........... 37

Pharmacokinetic Symbols and Equations......38

vi



















3 RESULTS

*t


Calculation of the Various Pharmacokinetic
Parameters ................................. 39


AND DISCUSSION ......................... .... 40

Analysis of Acepromazine in Plasma, Whole
Blood and Red Blood Cells.................40

Analysis of Other Phenothiazines in Plasma,
Whole Blood and Red Blood Cells............47

Stabilty of Acepromazine in 1 N NaOH and
1 N HC1 at 90 C............................47

Partition Coefficient Between Hexane and
Phosphate Buffer pH 7.4 for some Clinically
Relevant Phenothiazines.................... 47

Lipophilicity and Red Blood Cell
Partitioning ............................... 48

Extent of Protein Binding..................54

Pharmacokinetics-Pharmacodynamics of
Acepromazine in the Horse..................60

Pharmacodynamic Effects of Acepromazine
in the Horse...............................76

Effect on the Equine Hematocrit....... 76

Cardiovascular and Hemodynamic Effect
of Acepromazine .......................78

CNS and Sedative Effects of
Acepromazine in the Horse.............85

Pharmacodynamic-Pharmacokinetic
Correlation ..........................91

Pharmacokinetics of Acepromazine
in the cat ............... ................. 92


vii















Bioavailability of Acepromazine after
Oral, Subcutaneous and Intramuscular
Administration .................... 100

Bioavailability of Acepromazine after
SC and IM Administration in the Cat. 103

Effect of Acepromazine in the Cat........ 110

General Observations .................. 114

Conclusions and Clinical Significance... 115

.PPENDIX ...................... ....... ............... 119

BIBLIOGRAPHY.............................................. 137

BIOGRAPHICAL SKETCH.................................... 140


viii



















Table 1.


Table 2:


LIST OF TABLES
Page
Summary of phenothiazine structure
activity relationships .....................8

Interday variability in the HPLC assay
of acepromazine in biological fluids......43


Table 3: Intraday variability in the HPLC assay
of acepromazine Maleate in biological
fluids ............. ...................... 44
Table 4; The extraction recovery of acepromazine
from biological fluids....................45

Table 5: Summary of the equations describing the
calibration curve for each phenothiazine
assayed by the HPLC system .................46

Table 6: Partition coefficients between hexane and
phosphate buffer for the various
phenothiazines of interest................56
Table 7: Red blood cell partition coefficient as
a function of lipophilicity and
concentration for the phenothiazines of
interest.................................. 57

Table 8: Analysis of variance table for the red
blood cell partition coefficients
for the phenothiazines studied as a
function of concentration................58
Table 9: Fraction of drug bound to plasma proteins
as calculated from the partition
coefficient between red blood cells and
phosphate buffer and red blood cells and
plasma....................................59
Table 10: Summary of the pharmacokinetic parameters -
for all the horses after IV administration
of acepromazine ...........................71
















Table 11: Confidence intervals for the distribution
and elimination rate constants
after IV administration in the horse..... 72

Table 1i: Summary of the pharmacokinetic parameters
after IV administration of 0.3 mg/kg doses
of acepromazine in the four cats..........97

Table 13: Confidence intervals for the distribution
and elimination rate constants
after IV administration in the cat........98

Table 14: Summary of the pharmacokinetic parameters
after oral administration of 10 mg tablets
of acepromazine to cats.................104

Table 15: Summary of the pharmacokinetic parameters
after subcutaneous administration of
0.3 mg/kg doses
of acepromazine to cats................... 115

Table A-l: Physical history of the cats............120

Table A-2: Physical history of the horses..........121


Table A-3:


Table A-4:



Table A-5:


Summary of the plasma concentration for
all the Horses after IV administration
of acepromazine ........................ 123

Summary of the Blood Pressure (mm Hg)
vs Time (min) After IV Administration
of a 0.15 mg/kg dose of Acepromazine
Maleate in the Horse ................... 124

Heart rate as a function of time after
an IV administration of 0.15 mg/kg
dose of acepromazine...................125


Table A-6A,B: Sedative and CNS effects as a
function of time after an IV
administration of a 0.15 mg/kg
of acepromazine .... ............ ......126
Table A-7: Blood gases vs time after an IV















administration of a 0.15 mg/kg
of acepromazine......................... 128


Table A- 8:


Hematocrit vs Time (min) After an IV
Administration of 0.15 mg/kg
of Acepromazine Maleate
to the Horse ..........................129


Table A-9: Plasma concentration in ng/ml vs time
in minutes after IV administration
of a 0.3 mg/kg dose of acepromazine
for the 4 cats studied .................. 130


Table A-10:


Plasma concentration in ng/ml as
a function of time (min) after
an oral administration of a 10 mg
tablet of acepromazine maleate.........131


Table A-ll: Plasma concentration in ng/ml as a
function of time after an
intramuscular administration of
a 0.3 mg/kg of acepromazine............132

Table A-12: Plasma concentration in ng/ml as a
function of time after an intramuscular
administration of a 0.3 mg/kg
of acepromazine ........... ............ 133

Table A-13: Summary of the hematocrit as a function
of time for all the hematocrit for
all the routes of administration of
acepromazine in the 4 cats............134
Table A-14: Rating scale for the CNS and sedative
effects ........ .......................135













List of Figures


Figure 1: Tricyclic basic structure
for phenothiazines ..............................3

Figure 2: Liquid chromatographic traces of blank cat
C plasma and cat plasma containing 100 ng/mL
of acepromazine with trimepazine
(150ng/ml) ..................................... 41

Figure 3: Sample calibration curve for the HPLC
assay of acepromazine..........................42

Figure 4: Plot of the red blood cell partition
coefficient vs lipophilicity
for the phenothiazines of interest.
( 0 ) 300 ng/mL; ( 0 )500 ng/mL; (A ) 1000
ng/mL; ( A ) whole blood 1000 ng/mL...........55

Figure 5: Fitted arterial plasma concentrations
vs time (min) for horse Letren
after IV administration of
an 0.15 mg/kg dose of acepromazine.............61

Figure 6: Fitted venous plasma concentrations
vs time (min) for horse Letren after IV
administration of an 0.15 mg/kg dose of
acepromazine................................... 62

Figure 7: Fitted arterial plasma concentrations
vs time (min) for horse Dappler Arab
after IV administration of an 0.15 mg/kg
dose of acepromazine ......................... 63

Figure 8: Fitted venous plasma concentrations
vs time (min) for horse Dappler Arab
after IV administration of an 0.15 mg/kg
dose of acepromazine...........................64

Figure 9: Fitted plasma concentrations vs time (min)
for horse Chestnut after IV administration
of an 0.15 mg/kg dose of acepromazine.........65

Figure 10: Fitted plasma concentrations vs time (min)
for horse Sara after IV administration of
an 0.15 mg/kg dose of acepromazine.............66


xii














Figure 11: Fitted plasma concentrations vs time (min)
for horse Juanita after IV administration
of an 0.15 mg/kg dose of acepromazine..........67

Figure 12: Fitted plasma concentrations vs time (min)
for horse Raisin after IV administration
of an 0.15 mg/kg dose of acepromazine..........68
Figure'13: Fitted plasma concentrations vs time (min)
for horse Roan after IV administration
of an 0.15 mg/kg dose of acepromazine..........69
Figure 14: Fitted plasma concentrations vs time (min)
for horse Roan after IV administration
of an0.3 mg/kg dose of acepromazine...........70
Figure 15: Plot of hematocrit vs time (min) for all
5 horses after IV administration of
0.15 mg/kg doses of acepromazine.
( ) Dappler; ( ) Juanita;
( A) Sara; ( A ) Chestnut; ( [) Letren ......77
Figure 16: Plot of the systolic blood pressure
(in mm Hg) vs time (min) for all 5 horses
after IV administration of 0.15 mg/kg doses
of acepromazine. ( O ) Dappler;
( ) Juanita; ( A ) Sara;
( A ) Chestnut; (C ) Letren .................. 80


Figure 17:





Figure 18:




Figure 19:


Plot of the diastolic blood pressure
(in mm Hg) vs time (min) for all 5 horses
after IV administration of 0.15 mg/kg doses
of acepromazine. ( ) Dappler;
( 0) Juanita; ( A ) Sara; ( A ) Chestnut;
( 3 ) Letren ...................................81
Plot of the mean blood pressure for
all 5 horses after IV administration
of 0.15 mg/kg doses of acepromazine.
(0) Dappler; ( ) Juanita;
( ) Sara; ( ) Chestnut; ( ) Letren.......82
Plot of the heart rate vs time (min)
for all 5 horses after IV administration
of 0.15 mg/kg doses of. acepromazine.
( 0) Dappler; ( *) Juanita;
( A) Sara; (A ) Chestnut; ( [) Letren.......83


xiii














Figure 20: Plot of the degree of eyelid droop
vs time (min) for all 5 horses after IV
administration of 0.15 mg/kg doses of
acepromazine. ( ) Dappler;
( *) Juanita; ( & ) Sara; ( A ) chestnut;
,( 0) Letren ...................................86
Figure ,21: Plot of the reaction to the pin prick test
vs time (min) for all 5 horses after IV
administration of 0.15 mg/kg doses of
acepromazine. ( ) Dappler;
( ) Juanita; ( A ) Sara; ( A ) Chestnut;
( Letren ............... .. ......... ... ...... 87
Figure 22: Plot of the head carriage vs time (min)
for all 5 horses after IV administration
of 0.15 mg/kg doses of acepromaz;ne.
( ) Dappler; ( ) Juanita; ( A) Sara;
( A) Chestnut; ( ) Letren ................ 88
Figure 23: Plot of the extent of movement vs time (min)
for all 5 horses after IV administration of
0.15 mg/kg doses of acepromazine.
( ) Dappler; ( ) Juanita; ( A) Sara;
( A) Chestnut; ( 0 ) Letren. .................89
Figure 24: Plot of the fitted plasma concentration
vs time (min) for cat Green 1, after IV
administration of an 0.3 mg/kg of
acepromazine...................................93

Figure 25: Plot of the fitted plasma concentration
vs time (min) for cat Red 1, after IV
administration of an 0.3 mg/kg dose of
acepromazine... ........................ ...94


Figure 26:



Figure 27:
,


Plot of the fitted plasma concentration
vs time (min) for cat Green 2, after IV
administration ofan 0.3 mg/kg dose of
acepromazine ...................................95

Plot of the fitted plasma concentration
vs time (min) for cat Red 2,after IV
administration of an 0.3 mg/kg dose of
acepromazine ........ ........................96


xiv












Figure 28:


Plot of plasma concentration after an oral
administration of 10 ag of acepromazine,
CatGreen 1 (Panel A); Cat Red 1 (Panel B);
Cat Green2 (Panel C)...........................105


Figure 29: Plot of plasma concentration vs time (min)
After subcutaneous administration of
S0.3 mg/kg doses ofacepromazine to:
Cat Green 1 (Panel A); Cat Redl (Panel B);
Cat Green 2 (Panel C); and Cat Red2
(Panel D) ..... .......... ......... ............ 108


Figure 30:




Figure 31:


Figure 32:


Plot of plasma concentration vs time (min)
after intramuscular administration of
0.3 mg/kg dosesof acepromazine to :
Cat Green 1 (Panel A); CatRed 1 (Panel B);
Cat Red 2 (Panel C) ......................... 109

Plot of the fraction remaining to be absorbed
vs time (min) for: Cat Green 1 (Panel A);
Cat Green2 (PanelB)..........................111

Plot of the fraction remaining to be absorbed
vstime (min) for Cat Red 1......................112














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

PHARMACOKINETIC STUDIES OF ACEPROMAZINE IN THE HORSE AND THE
CATSTUDIES IN LIPOPHILICITY, RED BLOOD CELL PARTITIONING AND
PROTEIN BINDING


By
Patrick J Marroum

August 1990
Chairman: Dr. Stephen Curry
Major Department: Pharmaceutics

The pharmacokinetics of acepromazine were investigated

in the horse and the cat. The overall objective of this work

were 1) to study the physicochemical characteristics of six

clinically relevant phenothiazines; 2) to study the

pharmacokinetics and pharmacodynamics of acepromazine in the

horse; and 3) to study the bioavailability of acepromazine in

the cat after oral, subcutaneous and intramuscular

administration.

The various compounds were assayed by means of an HPLC

system with electrochemical detection developed specifically

for this research. Partition coefficients were measured

between hexane and phosphate buffer (pH 7.4), between red

blood cells and plasma and between red blood cells and

phosphate buffer (pH 7.4). Studies in horses involved


xvi














intravenous doses, collection of frequent blood samples and

assay of the acepromazine content of these samples. The

pharmacodynamic measurements consisted of measuring the

hematocrit, blood pressure, heart rate, blood gases and the

sedative effects of the drug. Studies in cats involved

intravenous, oral, subcutaneous and intramuscular doses,

collection of frequent blood samples and assay of them for

their drug content as well as measurement of the hematocrit.

The bioavailabilty of acepromazine in the cat was determined

for all the administered routes.

After intravenous administration, acepromazine followed

biphasic pharmacokinetics in both the cat and the horse. In

general,"the pharmacodynamic effects of acepromazine persisted

much longer than detectable plasma concentrations of the drug.

Acepromazine markedly decreased the hematocrit in both animal

species tested. Additionally, there was a decrease in the

blood pressure and a marked sedation in the horse after an

intravenous dose of 0.15 mg/kg. Intravenous and subcutaneous

doses gave concentrations much higher than oral doses. In two

cats, intamuscular doses gave plasma concentrations higher

than subcutaneous doses. It seems likely that oral doses of

acepromazine undergo a very high first pass effect as observed

with other related compounds in humans.


xvii














There was no concentration dependency for the red blood

cell partitioning between phosphate buffer (pH 7.4) and the

red cells. Additionally, there was no relationship between the

lipophilicity of the phenothiazines studied as measured by

their hexane- phosphate buffer (pH 7.4) partition coefficient

and the extent of the red blood cell partitioning.





























I


xviii


















CHAPTER 1

INTRODUCTION
The use of drugs for the treatment of psychiatric
disorders has become widespread since the mid-1950s. Today,

20% of the prescriptions written in the US are for medications

intended to change the mood or behavior. Phenothiazines,

which are major tranquilizers or neuroleptics, are mainly used

to treat psychosis. Classification of these drugs can be on

the basis of chemistry (e.g. benzodiazepines, phenothiazines,

tricyclic antidepressants), neuropharmacology (e.g.,

neuroleptics, amine re-uptake inhibitors, GABA antagonists)

or use (major tranquilizers, minor tranquilizers,

antidepressants). One particular phenothiazine (acepromazine)

is used only in animals as a sedative preanesthetic drug.

The specific objective of this research was to describe

the pharmacokinetic properties of acepromazine in certain

domestic animal species. A method of analysis using high

pressure liquid chromatography (HPLC) with electrochemical

detection was developed. Other relevant pharmacokinetic

properties such as red blood cell partitioning and plasma

protein binding were investigated.















In addition, because of the significance of the

physicochemical properties of drugs in drug disposition, an

investigation of the relationship between lipophilicity and

RBC partitioning was undertaken.

Historical Backcround

The first phenothiazine was synthesized by Bersthen in
1883 while synthesizing dyes related to methylene blue (1).

However, it was until the late 1940s that a derivative of

phenothiazine promethazine was found to have antihistaminic

and strong sedative properties. Also in that period, Gilman,

Shirly, and Charpentier independently synthesized a series of

10-dialkylaminoalkyl derivatives which had weak anthelmintic,

trypanocidal and antimalarial properties. In 1951, Charpentier

and coworkers, while investigating the central nervous system

action of phenothiazines, were successful in synthesizing

chlorpromazine (2). Courvoisier in 1953 described the many

pharmacological actions of chlorpromazine (3) and Laborit

reported that chlorpromazine potentiated anaesthesia and

induced a state of artificial hibernation consisting of

hypothermia, decreased metabolism and reduced oxygen

requirements in patients (4).

The basic phenothiazine nucleus is tricyclic, two benzene

rings are attached to each other by a nitrogen and a sulfur
























RI


I
R2


Figure 1: Tricyclic basic structure for phenothiazines



,'s:














4
as can be seen in Figure 1. The important therapeutically

useful phenothiazines are substituted in positions 2 and 10.

The 10'aminoalkyl phenothiazines are classified on the basis

of their side chain: (5)

-Aliphatic substitution:

(1) prototype activity: chlorpromazine.

(2) pronounced sedative effects: most useful in agitated

schizophrenics.
B-Piperidine group:

(1) similar antipsychotic activity to chlorpromazine.

(2) reduced extrapyramidal effects.

C-Piperazine group:

(1) most potent antipsychotic activity.

(2) insignificant sedative effects; therefore useful in

depressed or withdrawn schizophrenics.

(3) increased extrapyramidal effects.
Substitution at Rl includes:

A-Chlorine or methoxy group:

(1) increased potency against psychotic behavior.

(2) depression of motor activity.


B-Thiomethyl group:

(1) increased potency against psychotic behavior.















C-Trifluoromethylene group:

(1) greatly increased potency against psychotic behavior.
(2) increased antiemetic potency.

(3) increased tendency to produce extrapyramidal

symptoms.

(4) less sedation (6).


A summary of the structure-activity relationship is given in

Table 1.

Pharmacology
The pharmacological properties of phenothiazines can be
summarized as follows:

1-sedation.

2-decreased anxiety.

3-decreased spontaneous motor activity.

4-complex behavior is disrupted; difficulty with

intellectual tasks.

5-antihistaminic and hypotensive activity (1).
The above pharmacologic effects are thought to be mediated by

the blockade of postsynaptic dopamine receptors resulting in

an increase in the rate of production of dopamine metabolites

and therefore interfering with the actions of dopamine as a

synaptic neurotransmitter in the brain. (7)
















Pharmacoloav and Clinical AoDlications of Acenromazine

Acepromazine (Promace, Atravet, Notensil) has most of

the pharmacological effects typical of phenothiazines (8).

It is generally considered more potent than chlorpromazine and

promazine and is effective at very low doses. It has been

shown that a dose of acepromazine as low as 0.1 mg/kg

decreases the mean arterial blood pressure in dogs as well as

in horses and in cats (9-13). The extent of hypotension seems

to be dose independent since the same degree was observed with

the high dose or low dose.

However, the duration of hypotension was much longer with

the high dose (1.1 mg/kg) compared to a dose of 0.1 mg/kg

(14).

In the dog, and to a lesser extent in the horse,

acepromazine induces bradycardia due, most probably, to its

adrenergic blocking actions (10). However, this bradycardia

might be insignificant due to the reflex tachycardia induced

by the hypotensive effects of the drug.

In the horse as well as the dog, acepromazine markedly

decreases the respiratory rate (11, 15). This decrease in

respiration rate, however, has no effect on either blood gases

or blood pH. The most sensitive response to the action of

acepromazine in the horse is the decrease in hematocrit or














7
packed cell volume (PCV) (12). This effect is dose dependent

and can be induced by a dose as small as 0.01 mg/kg The

decrease in hematocrit can be as high as 50 % and can last
*-
up to 12 hours with higher doses. The decrease in hematocrit

is primarily due to a splenic sequestration of red blood

cells.

Acepromazine is primarily used as a preanesthetic agent

in the dog, cat and horse (15, 16, 18) It markedly

potentiates barbiturates facilitating handling and restraint

of animals. Additionally, acepromazine is used in the

treatment of equine colic (17). Partial blockade of

adrenergic receptors may possibly explain this antispasmodic

effect. Unfortunately, adequate blood volume and arterial

pressure must exist before the drug can be administered

because acepromazine can cause cardiovascular collapse or

shock.

Acepromazine is not approved for use in cattle and should

not be used in animals that are consumed by humans.

Toxicity of Aceoromazine

The toxic reactions of phenothiazines can be divided into

3 types:'











t
I'




II
U
61
*i
m.r



0
f-4
I
-1
do


cn



I
'a-
ow



i
I


w '-4
~'-4


--1L

04


ii


if


El
5a>
1^



ii
0











I


sJ ..
-4 -it
'U^s


v 4


if


s _, P
















A-Extrapyramidal effects:

1-Parkinsonian syndrome: rigidity, tremors, etc...

2-Akithisia: need for a constant motor activity.

3-Dystonia: facial grimaces.

4-Dyskinesia.

B-Sensitivity reactions:

1-Jaundice.

2-Dermatological reactions: rash, hives,

photosensitivity.

C-Blood dyscriasias: leukocytosis (1).

In certain cases, sudden collapse has been observed

manifested by apnea, slow pulse and unconsciousness. Some

adverse behavioral alterations have been observed in the dog

(19, 20). Aggression and vicious behavior have been

manifested soon after administration (21). The CNS seizure

threshold may be lowered leading to seizures in susceptible

animals. Additionally, acepromazine can cause syncope

associated with high vagal tone and subsequent bradycardia.

In horses, acepromazine was noted to cause priapism or penile

prolapse. This effect seems to be dose related and can last

up to 10'hours with a dose of 0.4 mg/kg. The penile prolapse

may be due in part to relaxation of the retractor penis

muscles, which are innervated by adrenergic nerve fibers (22).
















Metabolism
Both the phenothiazine nucleus and the side chain undergo

substantial metabolic transformations (23-25). The main route
9.
of metabolism of the phenothiazines is by oxidation largely

mediated by hepatic microsomal enzymes (26). Conjugation

with glucuronic acid is very important. Most of these

metabolites being very hydrophilic are excreted in the urine

but to some extent also in the bile. Some of the

phenothiazines have biologically active metabolites which

complicate the correlation of concentrations in blood levels

with biological effects (29). Scheme 1 summarizes the major

metabolic pathways for the phenothiazine nucleus.

Concomitant with the metabolic changes in the nucleus,

various biotransformations occur in the side chain,

illustrated in Scheme 2. Sequentially, they are N-oxidation

and hydroxylation, monodesmethylation, didesmethylation,

desamination and beta-oxidation (28).

As for acepromazine specifically, the only reported study

about its metabolism is by Dewey et al where they administered

acepromazine to normal mares in the range of 5 to 50 mg. The

major metabolite isolated from the urine of these horses was

unconj ugated 2 (1-hydroxyethyl) promazine sulfoxide. Conjugated

7 hydroxy-acetylpromazine and conjugated 2-(l-hydroxyethyl)















7 hydroxy promazine were also isolated and identified.

Additionally, 2(1-hydroxyethyl) promazine was isolated in some

urine but in very minor quantities and thus the authors

suggested that this route is very minor and can be considered
negligible. Scheme 3 summarizes the metabolic scheme in the

horse for acepromazine (30).

Pharmacokinetics
In general, pharmacokinetic studies of phenothiazines are
few and usually inconclusive (31-37). In spite of the fact

that the prototype, chlorpromazine, has been extensively

studied; its renal excretion is almost totally unknown. To
date, there is not a single report on the renal clearance of

the parent drug or its metabolites in humans and animals.

The plasma concentration-time profiles usually follow a

multiphasic pattern. Chlorpromazine has a large variation in

the terminal half life (6.64 to 118.9 hours) in humans (31-

37).
Large variations were also observed in the RBC/plasma
concentration ratios (38, 39). Over 90 % of the

phenothiazines in blood are bound to plasma proteins (40, 41).

There is only one pharmacokinetic study on acepromazine
published to date (42). The drug was detectable up to 8 hours

postinfusion after intravenous injection of a 0.3 mg/kg dose














I
(CHM),
DCPZ I
NHCH3


t
Oeethylation


Sulfoxidatto


CH), \
CPZ N
N(CH3) e 0eulnation

Hydroxylation

1


OC1
"Taci


(CHe3)
N(CH3)2


7-OH-CPZ


I
(CHI)3
I


CPZNO


0-N(CH,3)




fACl
R
R


CHS0~,o CI


Conjugation


N(CH3)8


7-06-CPZ


Principal metabolic reactions of chlorpromazine (CPZ).
DCPZ demonomethylchlorpromazine; DDCPZ dedimethyl-
chlorpromazine; 7-OH-CPZ 7-hydroxychlorpromazine;
CPZSO chlorpromazine sulphoxide; CPZNO chlorpromazine N-
oxide; 7-OG-CPZ 7-hydroxychlorpromazine glucuronide.
(Reprinted with permission, from Curry, 1976 b.)


Scheme I


I
(CHO)3
NHI


DCPZ




PZSO


/


(CHM)3 C
I


in


N(C He


a .ci


0D



















0


OH OH
C-CN, -CH,
I I I
H H
(CH),3 (CH1),3 (CH3),
I I I
N N N
CH, CH3 CH3 CH3 CH3 CH3

II -

Acepromazine Major Metabolite





0 OH
10 C-CCHI
(CHZg) (CHZg) H
N N
CH3 Ck1 CH3 CH3


III IV'


III and IV uere Isolated as Conjugates with Glucuronic Acid



Scheme II: Metabolic Pathuays of Rcepromazihe in the Horse















14
of acepromazine. The plasma decay was biexponential with an

alpha phase half life of 4.2 min and a beta phase half life

of 184.8 min. The volume of distribution was 6.6 1/kg
*-
indicating that acepromazine was widely distributed in the

horse.

It was also extensively bound to plasma proteins

(> 99 %). In blood acepromazine partitioned in the plasma

(46%) and in the erythrocyte phase (54%).

It is notable that, although that the study of Ballard and

coworkers provided us with valuable information about the

pharmacokinetics of this drug, the study's major drawback was

the dose given to the horses. An acepromazine dose of 0.3 mg

/kg is considered too high and will cause a lot of toxicities

in the horse and therefore will be of little clinical value.

Thus the need for other studies where a more clinically

applicable dose is given and the results obtained would be of

value to the practitioner in the clinic. The oral,

subcutaneous and intramuscular bioavailability of acepromazine

was not determined. For all these reasons, more thorough

studies are needed to elucidate the pharmacokinetic properties

of acepromazine.















Analytical Methods for the Assay of Phenothiazines

Successful pharmacokinetic studies depend on sensitive
and specific analytical methods for both the parent compound

and its'metabolites.

Phenothiazines are difficult to assay because they are
present in low quantities in body fluids. Their extreme

lipophilicity leads to variable glass binding.

To date, various analytical methods have been described
e.g. spectroscopy, fluorometry, radioimmunoassay, etc... (24)

Sufficient specificity and appropriate sensitivity was

achieved by Curry and Brodie in 1968 who assayed

chlorpromazine at na

nogram levels (43-44) using a gas chromatograph equipped with

an electron capture detector after extraction with heptane.

Most of the subsequent published assays involved gas

chromatography with either electron capture or nitrogen

detectors with a lower limit of detection of 10 ng/ml.

Although this limit has been satisfactory with the higher

doses of phenothiazines, it was not enough for the more potent

congeners.

There are 2 published assays for acepromazine to date,
both using gas chromatography. Ballard and coworkers used GLC

with a nitrogen detector. The column was 6 foot 3 % OV 101














16
glass column (42). The drug of interest was extracted from

plasma with saturated tetrahydroborate buffer and

dichloromethane (45).

Courtot used a flame ionization detection. The column was

also 6 foot packed with either OV 1 or OV 17. Acepromazine

was extracted from biological fluids mainly equine saliva with

diethyl ether after alkalinization with 2 N NaOH.

Unfortunately, in both these papers, no statistics were

included and thus no conclusions about the sensitivities or

the limit of detection could be drawn (46).

This dissertation presents a specific and sensitive assay

for acepiomazine using HPLC with an electrochemical detection.













CHAPTER 2

MATERIALS AND METHODS

Chemicals

Acetonitrile, hexane, ammonium acetate, sodium acetate,

disodium phosphate and sodium hydrogen phosphate, sodium

hydroxide, hydrochloric acid, toluene were LC or analytical

grade from Fisher Scientific (Pittsburgh PA, USA).

Hexamethyldisilazane- SCM Speciality Chemicals, Gainesville,

Fla.

Acepromazine maleate as a powder was obtained from Fort

Dodge Laboratories, Fort Dodge Iowa. Reference samples of

marketed drugs were obtained from the manufacturers:

ChlorpT-omazine Hydrochloride.Trimeprazine

Tartarate.Trifluoroperazine dihvdrochloride- Smith Kline and

French Laboratories, Philadelphia Pa.

Promethazine Hydrochloride- Aldrich Chemical Company, Milwauke

Wisconsin.

Mesoridazine. Thioridazine Hcl- Sandoz Pharmaceuticals, E

Hanover NJ.














18
Fluphenazine 2 Hcl- The Squibb Institute for Medical Research,

Chicago II.

Isoflurane-Forane, Anaquest, Madison, Wisconsin 53713

Na heparin-LyphoMed inc. Rosemont, Illinois 60018.

Polvflex (amDicillin suspension)- Avco Co., Inc., Fort Dodge,

Iowa 50501.

0.9 % sodium chloride infection USP: Kendall McGaw

Laboratories Inc. Irvine, Ca 92714.

Food
Purina Cat Chow: Ralston Purina Co., Saint Louis, Missouri

63164

General ADparatus:
Mettler Balance-Metler Instrument Corporation Hightstown N.J.

Metler Balance P1210-Metler Instrument Corporation Hightston

N.J.

Tube ShakEer- bach Corporation, Ann Arbor Michigan.

Beckman Model TJ6 Centrifuqe-Beckman Palo Alto Ca.

Microhematocrit Centrifuae-Damon/IEC Division, Needham Hts,

Mass.

MicrocaDillarv Reader-Damon/IEC Division, Needham Ets, Mass.

Micro-Hematocrit Capillary Tubes- Fisher Scientific,

Pittsburgh Pa.














19
Thermolvne Maxi Mix 2 Mixer-Thermolyne Corporation, Dubuque

Iowa.

Pierce Reacti-Therm Heatina Module- Pierce Chemical Company,

Rockford Illinois.

Cornina DH Meter model 140- Corning Corporation Medfield,

Mass.

CArv model 219 UV Spectrophotometer. Varian Corporation,

Sugarland Texas.

Vacutainer- Vacutainer Systems, Rutherford, N.J.

Precision Microliter Diettte Pinetman- Rainin Instrument

Company, Woburn Mass.

Aauamatic K module K-20: American Medical Systems, Cincinnati

Ohio 45238.

Vascular-Access-Port Model SLA with five French silicone

rubber outlet catheter (0.8 mm ID and 1.7 mmODI:Access

Technologies, Skokie, Illinois 60078.

Catheter Introducer:Becton Dickinson, Rutherford, New Jersey

07070.

Abbocath-T: Abbott Hospital inc, North Chicago, Illinois.

Anaiocath 20 GA 2" #2818: Desert Medical Inc. Sandy Utah

84070.

Datascone Model 870: Datascope Corporation, Paramus, NJ 07653-


0005.














20
P23-d model Transducer: Gold-Statham medical products

division, Oxnard Ca 93030.

IL813 Blood aas and Acid-Base Analyzer: Instrumentation

Laboratory Inc., Lexington Massachsetts.


The HPLC System consisted of:

-Waters Solvent Delivery System Model 6000- Waters Associates,

Millford Mass.

-WISP automatic Injector Model 710 A- Waters Associates,

Millford Mass.

-Data Module Model M730 Waters Associates, Millford, Mass.

-Fisher Recordal 5000 series recorder- Fisher Scientific,

Pittsburgh Pa.

-ESA Model 5100A Coulochem Electrochemical Detector-ESA Inc,

Bedford, Mass.

The Oxidation Potential was set at .7 V for the Analytical

Cell and .75 V for the Guard Cell.

The Column was a Zorbax Dupont CN bonded 13 cm column with a

5 um particle size diameter Obtained from Mac-Mod Analytical

Inc, Chadds Ford, Pa.

The mobile phase consisted of either 90:10 acetonitrile: .2

M ammonium acetate pH 6.9 or 75:25 acetonitrile: 0.1 M acetate

buffer pH 4.75. The only exception for these conditions was














21
with mesoridazine where the mobile phase consisted of 75:25

acetonitrile 0.1 M phosphate buffer pH 6.

The flow rate was 1.2 ml/min.
0.
Stability Studies of Aceoromazine in Water and 1 N HC1

Five mls of a 0.1 mg/ml aquous stock solution of

acepromazine were added to two tubes, one containing 45 mis

of pure deionized water, the other, 1 N HC1 so that the final

concentration was 10 ug/ml. These solutions were covered by

aluminum foil to protect them from the ultraviolet light and

were incubated at 90 o C. The UV absorbance of various

aliquots of these cooled (tO room temperature) incubated

solutions taken at different times were measured.

The spectrophotometric settings were:

-scan rate: 2 nm/sec.

-chart speed: 10 nm/sec.

-range: 500-200 nm.

-the full scale of the recorder was 1 absorbance unit.

Determination of the Partition coefficient between Hexane and
Phosphate Buffer oH 7.4 for Some Selected Phenothiazines

Seven solutions of the different phenothiazines

(thioridazine, fluphenazine, chlorpromazine,

trifluoroperazine, mesoridazine, acepromazine, promazine) were

prepared by adding 0.1 ml of a 100 ug/ml aquous stock solution

to 10 mls of phosphate buffer pH 7.4. The concentration of














22
each solution was measured by HPLC before and after extraction

with various volumes of hexane ranging from 0.1 ml to 10 mls

(0.1 ml for thioridazine, chlorpromazine, trifluoroperazine,

promazine, 1 ml for acepromazine and fluphenazine and 10 mis

for mesoridazine).

Additionally, 0.5 ml of the hexane were taken and dried

under a constant stream of nitrogen and reconstituted in

mobile phase.

The concentration of the phenothiazine in the buffer
andthe mobile phase was determined by HPLC from a standard

calibration curve in buffer and mobile phase respectively.

The partition coefficient was determined in 2 ways:

D =[Cc]/[Cbu] (1)

where Ch is the concentration in hexane and Cu is the

concentration in buffer solution, and

D (([C]b [Cbl])/(CC(].)) (V/Vo) (2)
where [C,]b is the buffer concentration before extraction,

[C,], is the buffer concentration after extraction, V, is the
volume of the aqueous phase, and Vo is the volume of the

organic phase.

Analysis' of AceDromazine and Other Phenothiazines from
Biological Fluids Mainly Plasma. Urine and Whole Blood

To a sample of 0.5 to 2ml of biological fluid such as
plasma, urine or whole blood 0.1 mls of 1 N NaOH are added














23
to make the pH alkaline. An appropriate amount of a suitable

sample is added. This alkaline sample is then extracted with

5 mls of hexane for 1 hour. After centrifugation, the hexane

phase was removed and evaporated to dryness at 25C under a

constant stream of nitrogen. If any emulsion still persisted

after centrifugation, gentle stirring with a glass rod

followed by further centrifugation solved the problem. The

residue was redissolved in an appropriate volume of mobile

phase usually 250 ul and an aliquot of this reconstituted

residue was injected into the chromatographic system.

Red Blood Cell/Plasma Distribution Coefficient

Since phenothiazines as a class have a relatively high

lipophilicity, it seems that a distribution between red blood

cells (rbc) and plasma would occur when the drugs were

introduced into a volume of whole blood. To analyze for such

distribution, the red blood cell concentration/plasma ratio was

determined.

From fresh blood obtained from the blood bank, packed rbcs

was obtained by centrifugation, these rbcs was washed three

times with isotonic saline and finally resuspended in isotonic

phosphate buffer pH 7.4.

Either these pseudoblood samples or whole blood was

spiked with different amounts of the phenothiazine of interest














24
so that the final concentration is 300, 500 and 1000 ng/ml

respectively. The hematocrit was measured after equilibration

of the samples for 60 minutes at 37C.

After centrifugation for 20 minutes at 3000 rpm, the

hematocrit of the red blood cell phase was measured to

determine how much supernatant was left after centrifugation.

Additionally, an aliquot of the rbc phase was taken and

diluted with equal amounts of water in order to lyse the cells

so that it was possible to measure their drug content.

Both the supernatant phase and the rbc phase were

analyzed for their drug content.

Appropiate calibration curves in both supernatant and rbc
were constructed by spiking blank supernatant and blank rbc

solutions with different amounts of the corresponding

phenothiazine and the internal standard so that the final

concentrations would be between 1000 ng/ml and 25 ng/ml.

RBC partitioning was evaluated in 3 different ways:

D [CI c/[C,] (3)

C =/Cp [ (A,- (Cp. VP))/(Vb*H]/Cp (47) (4)
D (Cre (Cpw(l-H))/(H/Cpw) 5)
where D 'is the RBC-supernatant partitioning coefficient, CrW

is the concentration of the drug in the red blood cells, CP

is the concentration of the drug in the supernatant, Ato. is















25
the total amount of drug added to the blood or pseudoblood,

Vb is the volume of blood or pseudoblood, Vpw is the volume

of supernatant calculated as (l-Hb)*Vb with Hb being the

hematocrit of the blood

or pseudoblood before spiking with the drug solution and H is

the hematocrit of the red blood cell phase after separation

of the two phases.

Concentration dependency of the red blood cell

partitioning for the various phenothiazines was challenged by

determining this coefficient at various concentrations mainly

at 1000, 500 and 300 ng/ml respectively.

The three results calculated from the three different

methods were compared to determine significant glass binding

and the extent it affected the results.

Extent of Protein Bindina Calculated from the Red Blood Cell
Partitioning Studies

The extent of protein binding was calculated from the

differences between the red blood cell partition coefficients

between plasma and the red blood cells and between phosphate

buffer and red blood cells. The fraction of drug bound to

proteins was calculated from:

f- 1- Kd/D (47)
where f is the fraction of drug bound to proteins, Kd is the

red blood cells partition coefficient between plasma and the














26
red blood cell phase, D is the red blood cell partition

coefficient between the phosphate buffer and the red blood

cells. This extent of protein binding was determined at a

concentration of 1 ug/ml.

Bioavailabiltv of AceDromazine in the Cat

Experimental Animals
The studies performed were approved in advance by the

University of Florida's Institutional Animal Use and Care

Committee. The laboratory facilities were also approved by

that committee for the performance of minor survival surgical

procedures including placement of the vascular access ports.

The animals used in these studies were adult male short
haired domestic cats with an average age of 12.6 +/- 0.44

months (range: 6-33) and which weighed an average 4.2 +/- 0.55

Kg (range:2.6-5.8). The cats were purchased from a USDA

Licensed Animal Dealer who had bred the cats in a minimal

disease colony.

Following the investigators' stipulations, none of the cats

had received any drugs or medical treatment other than routine

vaccinations as kittens and, in the housing at the University

of Florida.

The cats were housed and maintained in the University of

Florida's Health Center Animal Resources Division's facilities















27
which are approved by the American Association for the

Accreditation of Laboratory Animal Care. The cats were fed

commercial dry cat food ad libitum and water.
9-.
Vascular Access Port Implantation

Frequent blood sampling required direct access to the

systemic venous system. This was acheived by implanting

vascular ports into the femoral and jugular vein of the cat

which allowed percutaneous access.

Norfolk Vascular Access Port:

The unit implanted consisted of a silicone rubber

cathether connected to a blind reservoir. The reservoir was

encased at its base and sides in a more rigid plastic which

had flanges and holes to facilitating in anchoring it in the

body. The top of the reservoir was a rubber septum through

which access can be got to the bore of the catheter. The

catheter itself can be of varied sizes but a 5 French gauge

(1.7 mm OD and 0.8 mm ID) catheter was chosen for insertion

into the cat's femoral vein. The catheter was shipped by the

manufacturer at a 12 cm length. The size port used for

insertion in the cat was the SLA model with a reservoir

approximately 15 mm in diameter and 10 mm in height.

Upon receipt of the catheter from the manufacturer it was

wiped with alcohol and washed with soapy water to remove any














28
grease or foreign material. It was then tested by injecting

distilled water through the reservoir and occluding the distal

end of the catheter whilst pressurizing the system. Leaks

would then be visible. No leaks were seen in new ports but

were found in occasional ports that had been removed from

animals and were being recycled. Approximately 15 to 17 cm

from the reservoir, a bead of silicone rubber sealant was

placed around the catheter to allow suturing of the catheter

to the vessel it was being implanted into without risking

damage to the catheter itself. The bead was allowed to cure

for 24 hours. The ports were then thoroughly washed with

distilled water and packaged for autoclaving.

Implantation of the Port

The cats were anesthesized (usually with isoflurane).

Once anesthetized, and at a surgical plane of anesthesia, the

medial and lateral left thigh was prepared for aseptic

surgery. The port to be implanted was flushed with

heparinized saline (1 IU heparin /ml 0.9 % saline for

injection USP).

With the cat in right lateral recumbency, the left leg

was raised and a stab incision made through the skin just

medial to the patella. A pair of long hemostats was then

passed through the incision, tunneling subcutaneously upwards















29
to the lateral flank where a skin incision was made to

exteriorize the tips.

The distal end of the vascular access port's catheter was
*>
grasped by the hemostats and the catheter drawn subcutaneously

to emerge at the knee incision. Sufficient catheter was drawn

through the subcutaneous tunnel so that the reservoir was

pulled against the skin. The cat was rolled into lateral

recumbency of the opposite side exposing the sterile prepared

groin area.

The femoral vein was palpated high in the cat's groin and

a 2-4 cm incision made over it. The fascia covering the

femoral vein nerve and artery lifted away and dissected.

The femoral vein was separated from the accompanying femoral

nerve and artery. Two pieces of suture were looped under the

vein to facilitate lifting it and ocluding it during puncture

and initial catheter insertion. At this time a subcutaneous

tunnel was made down to the knee incision to bring the the

catheter up to the exposed vein. The silicone rubber catheter

was then cut so it was 5 cm long from bead to tip. The cut

was done with a scalpel blade so the catheter had an

untraumatized bevel of about 45. It was important that

the tip not be roughened or damaged as it was thought that

such a damage could act as a nidus for a thrombus formation.














30
When all preparations were complete, the vein was raised

and the proximal suture used to occlude the vein. A 19 gauge

needle was then placed at an acute angle cranially into the

vein to the extent that about half the bevel had entered the

vein and then it was withdrawn. To confirm penetration into

the venous lumen, the proximal suture loop was momentarily

lowered so blood could flow from the puncture site. A venous

dilator was then placed into the puncture hole and moved up

in the venous lumen. The catheter was grasped lightly with

blunt forceps and passed under the dilator into the vein

towards the heart. As the catheter approached the proximal

suture, the loop was relaxed whereupon usually some visible

confirmation of placement could be seen as a faint blood pulse

wave in the catheter lumen. The catheter was then rapidly

passed up the remainder of its length until the bead reached

the venous puncture site. The bleb was sutured to the artery

wall and surrounding fascial and muscle tissue. The groin

wound was closed.

The cat was returned to lateral recumbency and the

reservoir sutured to muscle fascia under a skin pocket and

the skin wound closed. The cat was given ampicillin

antibiotic coverage prophylactically for the next three days.
















Maintenance of the Vascular Port

The vascular access port was maintained by a heparin lock

that was removed and replaced, two to three times a week,

after first flushing vigorously with physiological saline.

All injections into or out of the port were performed

aseptically.

Administration. Collection and Treatment of Samples:

The bioavailabilty of Acepromazine in the cat after

oral, intramuscular and subcutaneous as compared to

intravenous administration was studied as following:

The study was a cross over design whereby each animal served

as his own control.

Initially, two cats were given 0.3 mg/kg of acepromazine

IV (the concentration of the solution was 0.5 mg/ml). The two

other cats were each given orally a 10 mg tablet. After a

washout period of two weeks the treatments were switched so

that the first two cats received the oral treatment and the

second two cats received the IV treatment. After a period of

approximately three weeks, the same four cats were

administered a dose of 0.3 mg/kg subcutaneously and 0.3 mg/kg

IM with 'a washout period of three to four weeks in between

treatments.















32
Two mis of blood were withdrawn from the implanted port

per sampling time. The hematocrit for each blood sample was

measured. After the hematocrit measurement, the blood sample
6.
was centrifuged immediately at 300 rpm to separate plasma and

red blood cells.

After each blood sample withdrawal, the cats were

reinjected with 2 mls of isotonic saline solution to prevent

hypovolemia and shock in the cats.

The initial protocol for blood sampling after IV

administration was: 0, 1.5, 3, 4.5, 6, 7.5, 9, 12, 15, 18,

24, 30, 45, 60, 75, 90, 120, 150, 180, 210, 240, 300, and 360

minutes respectively.

The protocol for PO, SQ and IM was modified so that the

sampling times were as following: 0, 4, 8, 12, 16, 20, 23,

26, 30, 35, 40, 50, 60, 75, 90, 120, 150, 180, 210, 240, 300,

360 minutes respectively.

No urine was collected from these cats since

catheterization of unsedated cats was not possible.

Prior to experimentation, the animals were given enough

time to acclimate to their surroundings. Their full medical

history was well characterized.















33
Pharmacodvnamics-Pharmacokinetics of AceDromazine in the Horse

All these studies were conducted using seven donated

horses whose medical histories are summarized in

Appendix 1.

These horses were given each 0.15 mg/kg of acepromazine

intravenously (the concentration of the solution was 0.5

mg/ml).

The acepromazine was injected into the right jugular vein

through an 18 gauge two inch teflon over-the-needle catheter.

Blood samples for acepromazine assay were withdrawn from the

left jugular vein through a 14 G five inch teflon over the-

needle catheter. Both catheters were kept flushed with

heparinized saline. The sampling protocol was exactly the

same one used in the iv cat studies. The hematocrit and the

blood samples were treated the same way as described in the

cat studies.

The following pharmacodynamics parameters were measured

as described in the following:

Blood Pressure Measurements
Systemic systolic, diastolic and mean blood pressures

were obtained by transducing the pressures obtained by the

percutaneous catheterization of the transfacial artery. The

artery was cannulated with a 20 G two inch over-the-needle














34
catheter and this was connected to a pressure transducer by

means of a physiological saline filled pressure manometer

tube. The transducer was powered by a multichannel

oscilloscope with digital pressure and heart rate displays.

After an adequate warm-up period was allowed to elapse, the

transducer system was calibrated against a mercury column

manometer at zero (atmospheric pressure only), 50, 100, 150

and 200 mm Hg.

Heart Rate
Heart rate was determined by the rate counter on the

multichannel oscilloscope that counted the number of arterial

pulse wave per ten second period and gave a beats per minute

count output.

Blood Gas ANalysis
Systemic arterial blood gas tensions and pH were measured

from samples collected anaerobically from the arterial

catheter into a heparinized plastic syringe. The sample was

placed into either an IL813 or IL1304 Blood Gas Analyzer

within two hours minutes of collection. Immediately after

collection the sample was stoppered and stored in ice slush

until analysis could be performed.

The blood gas analyzer was calibrated each according to

the manufacturer's instructions with two sets of standards.














35
Additionally the analyzer's performance was checked on the

day of each study with the manufacturer's quality control

samples.

Electrocardiogram
Electrocardiographic (ECG) tracings were obtained using

Lead II where the left and right arm electrodes were placed

laterally at the level of the shoulder and about three inches

caudal to it on the left and right sides respectively, while

the leg electrode was placed on the left thorax at the level

of the 10th rib about 5 inches lateral to the spinal column.

The "electrodes" were commercial pre-jelled self-sticking

electrodes placed on skin that had been thoroughly cleaned

with isopropyl alcohol. The ECG was displayed on the

multichannel analyzer described above.

Urinary Catheter

The mare's urinary bladders were cathetherized using

sterile 30 G French Foley catheters placed by manual

palpation. The catheters balloons were inflated using water

and a weight tied to the catheter to keep it lodged against

the neck of the bladder. The catheters were attached to

plastic 'collection bags which were emptied into measuring

cylinders when required so urine output was to be measured.

The urine tended not to be free flowing and was usually














36
obtained by stimulating the mare to urinate by manipulating

the catheter.

Venous Sampling Line

The hair is clipped over the jugular groove mid neck and

the skin sterilized with alcohol.

A 14 gauge 5.25 inch catheter was placed in the jugular vein.

On the other side of the neck the same procedure was repeated

but with 18 gauge 2 inch catheter. An adhesive ECG electrode

was placed and patched over each shoulder and on the mid left

thorax. These electrodes were connected using an ECG lead II

configuration. A strain-gauge pressure transducer was

connected to the oscilloscope and allowed to warm up 20

minutes then calibrated against a mercury column manometer.

A 20 gauge 2 inch catheter was placed in either the

transfacial or ocular artery and connected through a saline

filled manometer tubing to a strain gauge transducer.

CNS and Sedative Effects

The sedative and central nervous system effects were

evaluated by rating the degree of sedation, the general

behavior, the posture and the general alertness of the

horse according to a scale developed jointly by the principal

investigators. This scale or rating is presented in appendix

2.

















All. the horses were rated by the same person to avoid any

subjective differences from one person to the other.

Evaluation and Fitting of Pharmacokinetic Data

The observed plasma concentrations of acepromazine were

separately fitted using a commercial softwear package (RSTRIP)

to a sum of exponentials and also using the Lotus 123

spreadsheet.

In the case of first order input and excretion, the

linear sum of exponentials were fitted to:

C = Ae-kit + Aek2t

where C is the expected value of plasma concentration, Ai and

Ki are constants and t is time in minutes. Methods for

estimating Ai and Ki were discussed by Riggs (47) and Gibaldi

and Perrier (48). The methods of analysis of weighted

residual and weighted residual sum of squares were used to

minimize the number of exponentials and obtain the best

estimates of each Ai and Ki. The residuals could be defined

as the difference between estimated values of C and the

observed values at a given time.

The' absorption rate constants were determined using the

Loo-Riegelman method (49) where the fraction absorbed or the

fraction remaining to be absorbed is plotted vs time.














38
The bioavailabity of acepromazine was determined by

comparing the areas under the plasma concentration time curve

for the different routes of administration with the area after

intravenous administration after adjusting for the different

doses.

Pharmacokinetic Symbols and Definitions
Alpha: distribution rate constant (min') describing the

distribution of the drug from the central compartment to

peipheral tissues in the body.

Beta: disposition rate constant (min"') summarizing the

complexity of elimination and re-equilibration that describes

the ultimate disposition of the drug in the central

compartment.

CO: plasma concentration at time 0 (ng/ml).

MRT: mean residence time (minutes). It represents the time

for 63.2% of the administered dose to be eliminated

Vdss: volume of distribution at steady state (liters). This

volume relates drug concentration in plasma or blood to the

total amount of drug in the body during steady state.

Vdpss: volume of distribution at pseudo steady state

(liters). It is an estimate of the volume of distribution on

the assumption of a one compartment body model when in fact

you have two compartments and is usually an overestimate of














39
the volume of distribution. This volume relates drug

concentration in plasma or blood to the total amount of drug

in the body during the terminal exponential phase for any

multicompartment model where elimination occurs from the

central compartment.

Vdcc: volume of distribution of central compartment

(liters). This volume term may be useful for estimating peak

concentrations in plasma or blood for drugs that distribute

relatively slowly in the body and are absorbed relatively

rapidly after oral or intramuscular administration.

Cltot: clearance total (ml/min). It is the hypothetical

volume of blood that is completely cleared from the drug per

unit of time.


Calculation of the Various Pharmacokinetic Parameters

MRT- AUMCg/AUC,

Vdcc- Dose/CO

Vdpss- Cltot/beta

Vdss= Cltot*MRT

Cltot- Dose/AUCOO

AUCtra(tn)- sum i-l to n ((c,+c,.,)*(t,-t.1)/2)

AUMCtrp(tn) sumi-1 ton( (c,*t,+ci.1*t_.1)* (t,-t,.)/2)

AUCtr, c./beta.
















AUNMCr.e c.*tn/beta +cn/beta2

AUCAo = AUCp(tn) + AUCtr

A MCoo AUMCtr (t) + AMCtr








0,


















CHAPTER 3

RESULTS AND DISCUSSION
Analysis of Acepromazine in Plasma. Whole Blood and Red Blood
Cells

The HPLC chromatogram of blank plasma and plasma

containing 100 ng/ml acepromazine with trimeprazine (150

ng/ml) from a cat receiving 0.3 mg /kg of acepromazine IV is

shown in Figure 2 At worst the coefficient of variation on

repeated assay of plasma containing known quantities of

acepromazine was 16.5 % At best this value was 3.89 %.

The type of calibration curve presented in Figure 3 is

y- -0.01044+/-0.0279 + (0.595+/-0.0258)x

where y is the peak height ratio and x is the concentration

of acepromazine in the plasma. The correlation coefficient

was 0.9943. The reproducibilty of analysis for interday and

intraday variability is shown in tables 2 and 3 respectively.

The acpromazine was reproducibly recovered (see Table 4 ).

The coefficient of variation is relatively high. This

is due to the fact that phenothiazines being very lipophilic

exhibit a large degree of glass binding and this renders their

assay more difficult due to the introduction of a large degree

of variabilty from assay to assay.


A f










Trimepazine


Figure 2: Liquid chromatographic traces of blank cat plasma
and cat plasma containing 100 ng/mL of Acepromazine with
trimepazine (150 ng/mL)

















2.5-



2.0
o
0

.-C
.C

-Y
I 1.0




a
0.5



0.0 III
0 25 50 75 100 125

Concentration

Figure 3 Sample calibration curve for the HPLC assay of
Acepromazine














*bhle 2: Interday Variability Pbr t HPLC Assay of Ap Eazine Maleate 43

frua Biological Fluids



M an OMYS VARuABLM


PFl PEMEAK EQGH1 PATIO

(nr/nl) Rl R2 R3 MEAN SD CV(%)


100 1.57 1.69 1.59 1.61 0.06 3.99

90 1.33 1.41 1.41 1.38 0.05 3.33

80 1.17 1.26 -

70 1.06 1.05 1.11 1.07 0.03 3.00

60 0.83 0.93 0.96 0.91 0.07 7.50

50 0.71 0.87 0.80 0.79 0.08 10.3

40 0.65 0.72 0.65 0.67 0.04 5.94

30 0.33 0.55 0.50 0.46 0.11 25.07

20 0.29 0.35 0.35 0.33 0.03 10.44

10 0.21 -0.27 0.20 0.22 0.04 17.20


























4.8"i '0 4' 0
o\ ~ ~ ~ ~ u r "4' 0`d c ;c~~ ,


0
0




o

*


0








*0
d
r
o


0



In
d


tn





0
o




d









4.


S8 2 00 0
4' qw N 4


>1
oS~


0
I


"4


a s
01 9


S* (

S U
r4


_4 '
a "4


"-4


a5 ?
ci r(


4"


M
**
<1 ^M


8











45












doi co i t* o r -.4
0






.4 r N w

0 m i A *
O N 0 0' 0 0 04

b co co % N m n in) % 0
r in %0 w- n %0

C0 0 4 4 N % 4
IV 0 W O 04 C4 r


IA n'
o 0% u4 N r% %0 N M
m 0 # N 0 0






*
'1 o %0 A 0 0 o A
f-4 0 co 0% f m I (' N N I-V







0 % 0 r
S co r- cn tn C" C14 03



S S o
1 N




0 '4 N a 0' 'i O
0M w o o Qi IV c c V4



) In
i # N m
N %0 r4 N %0 tl 6 o o o
co at co r% IV qv C4 r at
ato c co m C4
Mt C ) C













TABLE 5: Summary of the Equaticmn Describing the Calibration
curve for Each h othiazine Asayed by the HPC
System. 46






e-
CRDGCa EswDPe Sa fSSCEPP SEia B



ACEPRMAZINE 0.72 0.03 0.133 0.046 0.993

ROCHIOPERAZINE 1.85 0.16 -0.07 0.05 0.988

FUIHENAZINE 0.007 0.0005 0.686 0.68 0.992

HIORIDAZINE 0.0036 0.0002 0.408 0.135 0.989

TRIFIIUOOPERAZINE 0.0002 0.0001 0.017 0.005 0.993

MESORIDAZINE 0.18 0.009 0.033 0.047 0.994

PRMAZINE 0.002 0.00013 0.07 0.12 0.984

CHKIERPRCAZINE 8.41 0.194 -0.155 0.178 0.997

















Analysis of Other Phenothiazines in Plasma. Whole Blood and
Red Blood Cells


In general all the 6 other phenothiazines studied for

their red blood cell partitioning were analyzed using the same

chromatographic conditions as described for acepromazine.

Their retention times ranged between 6 and 12 minutes

respectively. The equations of the calibration curve for each

drug are summarized in Table 5. The extraction procedures

with hexane were exactly as described for acepromazine except

for mesoridazine which is more polar and had to be extracted

with toluene.

Stability of Acepromazine in 1 N NaOH
and 1 N HC1 at 90 C

Acepromazine was found to be stable from acid and base

degradation for up to 24 hours at 90 0 centigrade since there

was no significant change in its concentration as measured by

UV. Thus, for our purposes

acepromazine can be considered stable enough for analysis.

Partition Coefficient Between Hexane and Phosphate Buffer oH
7.4 for some Clinically Relevant Phenothiazines

The apparent partition coefficients between hexane and

phosphate buffer pH 7.4 were determined by measuring the

concentration in the aqueous and organic phase and by














48
differences in the concentrations in the aqueous phase before

and after extraction. The results are summarized in Table 6.

As expected mesoridazine, being a polar metabolite of

thioridazine had the lowest partitioning in hexane (K=.0141).

The most lipophilic was trifluoroperazine (K- 193) which had

a partitioning slightly higher than thioridazine or

chlorpromazine (K=129-152). Fluphenazine and acepromazine

could be considered of intermediate lipophilicity with a

partition coefficient of around 10.

It is to be noted that greater variability in the results

was observed with the more lipophilic phenothiazines, ie

thioridazine and chlorpromazine. This is most probably due

to the fact that the more lipophilic the phenothiazine, the

greater is the glass binding thus introducing more difficulty

and' more variabilty into assessment techniques.

Lipophilicity and Red Blood Cell Partitioning

It is an established fact that lipid solubility is a very

important factor in the actions of drugs affecting the brain

and central nervous system. Thus for a drug to exert its

pharmacological activity in the brain it should be able to

cross the blood brain barrier. Fundamentally, the more lipid

soluble the drug is, the more likely and the easier it is to

cross this barrier (50).














49
This is the basis of the Ferguson principle concerning

central nervous depressants which states that the depressant

effect of unrelated substances increases with increasing oil-

water partition coefficient. In other words, the higher the

partition coefficient, the greater the depressant action.

However, this relationship is not linear but parabolic because

substances that are very lipophilic will accumulate first in

oily sites of loss and will be trapped there and will not be

able to exert their action in the targeted tissues.

This is typically exemplified by the barbiturates.

Thiopental, the most lipophilic of the group enters the brain

most rapidly after IV injection. An intermediate rate of

penetration is shown by amobarbital. Relatively polar

barbiturates such as barbital penetrate the brain quite slowly

such that it has no utility in clinical situations.

The influence of lipid solubility is not limited to brain

penetration. It is a factor in absorption from the

gastrointestinal tract, reabsorption in the renal tubule and

in metabolism. Metabolism itself confers polarity but there

is a stong evidence that a certain level of lipophilicity is

needed if a drug is to bind to microsomal P-450 enzymes. This

fact raises the question of whether other binding reactions

are similarly related. Indeed, binding to plasma proteins














50
often correlates with lipophilicity as if the chemistry

controlling lipophilicity also controls binding affinity. For

instance, there is a nearly perfect correlation between the

binding of some phenothiazines to bovine serum albumin and the

octanol/pH 7.4 buffer logarithm of the partition coefficient

(51). Binding to dopamine receptors of the D-2 type also

correlates with lipid solubilty and therefore with protein

binding. However, 7 hydroxychlorpromazine has pharmacological

activity in excess of that predicted from its lipid

solubility. In contrast, the plasma protein binding of this

active metabolite is considerably below that of the parent

compound* and below the inactive chlorpromazine N oxide.

Presumably, the hydroxyl group inhibits the entry of the

molecule into the hydrophobic interior of the albumin

molecule. Therefore lipid solubility and protein binding seem

to be poor predictors of potency. It was thought that the

ease or ability of a drug to enter or partition into the RBCs

would correlate well with the ability of the drug to cross the

blood brain barrier and enter the brain. Thus the RBC

partition coefficient might be a better indicator or predictor

of the potency of phenothiazines. For that purpose the

relationship between red blood cell partitioning and

lipophilicity was investigated. The red blood cell partition














51
coefficients for the various phenothiazines studied as a

function of concentration are summarized in Table 7. Figure

4 shows the plot of the red blood cell partition as a function

of lipophilicity for the phenothiazines of interest at the

three concentartions investigated.

As can be seen this partition coefficient for all the

phenothiazines studied except for mesoridazine lie within the

same values. Typically, the red blood partition coefficient

was around five which meant that these phenothiazines were

mostly found in the red blood cells. It was also observed

that these values were independent of the concentration (see

analysis of variance in Table 8). These results are contrary

to what was expected because it was thought that that the more

lipophilic the phenothiazine is the easier it is going to bind

or partition into the red blood cell. The nondependence of

this partitioning on lipophilicity might be explained by the

fact that at pH 7.4 all these phenothiazines having a pKa of

9 (being basic amines) will be in the ionized form. The

existence of the positive charge on the amine will impart a

certain degree of polarity which will be the same for this

class of drugs and thus it will overule the lipophilicity

parameter in partitioning. Thus because of the positive

charge they will most probably have the same physical














52
characteristics and thus will all partition in the same way.

Another reason for the nondependence of the red blood cell

partitioning on lipophilicity might be due to the fact that

the drug is not partitioning into the cell itself but binding

to either the membrane or to some component inside the cell.

In this case, lipophilicity will not be a major factor in
contributing to the magnitude of this partition coefficient

and other factors will play a much more important role since

hydrophylic compounds can also be bound to the membrane of

the cell and thus will have a relatively large red blood cell

partition coefficient (greater than 1). It is noteworthy to
mention that Ballard and coworkers found out that the red

blood cell coefficient was around 11. This value was

calculated from the protein binding which was found to be 90

% at a concentration of 1000 ng/ml and the partition

coefficient in whole blood at the same concentration which was

found to be 1.12. The value that Ballard obtained seem to be
excessively large since it was almost double than what we had
obtained in our studies.

It is notable that for drugs that are very lipophilic and
that exhibit a great degree of glass binding, the red blood
cell partition coefficient cannot be calculated by just

measuring the concentration in the buffer phase and














53
calculating the concentration or amount in the red blood cells

by difference. This will result in an overestimation of the

red blood cell coefficient as it is seen and confirmed from

the results obtained in our studies. This overestimation is

due to the fact that the drug that is not found in the

supernatant phase is assumed to be bound to the red cells.

However, for drugs that undergo glass binding, there is a

three way partition between the glass, the red blood cells and

the glass walls. From the results presented in Table 7, it

can be seen that the red blood cell partition coefficient was

overestimated for the phenothiazines studied as compared to

the value obtained by the actual measurement of both phases.

This overestimation was almost 100 % in certain cases such

as thioridazine where the partition coefficient was 6.3 by

actual measurement and 11.8 by difference.

On the other hand, the red blood cell partition

coefficient can be underestimated if it is assumed that the

red blood cell phase, is completely made of red blood cells

and there was no plasma water present between the cells. It

was found that the red blood cells phase is not made of 100

% red cells but that around 10 to 20 % of the volume was

plasma water. This resulted in an underestimation of the

concentration in the red blood cell phase because the














54
concentration measured was a combination of the concentration

in the blood cells and the concentration in the plasma water.

This underestimation is found only in the case where the drugs
4,
partition highly in the red blood cells such as seen with the

phenothiazines because the actual concentration measured is

smaller than what the concentration would be if the red blood

cell phase was completely or 100 % red cells.

On the other hand, an overestimation would result for

drugs that would poorly partition in the red blood cells

because the concentration measured would be higher than the

actual value if it is assumed that the red blood cell phase

is only red cells because more drug would be present per unit

volume in the plasma phase than the red cell phase.

Extent of Protein Bindina

The extent of protein binding for the phenothiazines

studied are summarized in Table 9. The fraction bound was

determined at a concentration of 1000 ng/ml. The results

obtained agree with what was reported in the literature for

at least one compound promazine using totally different

methods.

As *'for the other phenothiazines, no information was

available for their protein binding was available at the

concentration range studied. From our studies acepromazine












10.0

.6-6
C
. 7.5


*O O
5.0 1
S*0
c .0 5 0 A
oso



o 2.5
m


o.oT-,- ---. ...-----
0 50 100 150 200

Lipophilicity



Figure 4: Plot of the red blood cell partition coefficient
vs lipophilicity for the phenothiazine of interest. (0) 300
ng/ml; (0) 500 ng/ml; (A) 1000 ng/ml; (A) whole blood
1000 ng/ml.


















a S H
1.1 S :; 2 *
u. 0 = a 3

a *
s a



w 4 .
0 a > .s .0



4w o. s


: o s I 9
0 *0


E- 0


CC 9S 0 -
gg a





BO C




55 5i 2 S !



g e

ie 0j
a 5 j e e
-si

















i U WO 0-04 'e *- 0 lp "a a



i I 01c 0 0p 09. 0I 0


61






Id
EU








'I



iie
ii


f
.4
a

an
$SS

ex=
i33

SXR

.a
0
9109.

d(O
.444


"3 lot

a ass.
. a~s

* :

0 09

as*
06094


9,
fit

.a
of


* t
9i.



* 0


. gs



24
s is
s Is

* *.4
2 S1


8 iS

* 0*
i io
* 00~


6% wt .4 A we 0 4je 4 4OW
A4> a nOf wo So A* w 1% I

I QI I -!! *C

Sa I I I I


l|it Ijslisli


* a* *



* ** 0

0a




I


3 52 2 |
* ** *





: g; S:

a as 3 S
.* "q* i

i Ii i i|||j

3i~


*os ..
P. 0
4

s 4 *
4224 .
j, SS ^
ass3 s~

adid a





















TABLE 8: Analysis of Variance for the Concentration Dependency of
the Red Blood Cell Partitioning for the Phenothiazines
Studied


Drug


SOURCE df


Acepromazine



Fluphenazine



Trifluoroperazine



Thioridazine





Mesoridazine
Promazine


between
within
Total

between
within
total

between
within
total

between
within
total


tcalc- 0.789 <1
tcalc- 0.96 <1


59.25
13.04


0.71
54


46.71
93.49


108.41
316.3
"'f


29.62
0.72


0.355
3.19


23.35
9.35


54.2
24.33


41.13



0.11



2.5



2.22













Table 9: Extent of Protein Binding for the
Phenothiazines Studied as Determined 59
from their Red Blood Cell Partitioning.

Drug % bound to proteins Rangea

Acepromazine 74 70 79

Fluphenazineb 67.5 60 -64

Thioridazine 95.5 94.9 -96.16

Trifluoroperazine 82.61 80.94-84

Promazine 78.32 76-80.62


a The range was calculated as follows:
The lower range was: 1- (Kd+SE)/(D-SE)
The upper range was: 1- (Kd-SE)/(D+SE)
b- The concentration for fluphenazine was 333 ng/ml.














60
was found to be 75 % bound to plasma proteins at a

concentration of 1000 ng/ml. Ballard and coworkers found that

for the same concentration range the fraction bound was almost

90 %. This result most probably is not accurate because it

means that acepromazine would have a partition coefficient

between phosphate buffer pH 7.4 and the red blood cells of

over 15 which is very unlikely and very seldomly encountered

with any drug. Hu determined the plasma protein binding of

promazine by ultracentrifugation and found that only 25 % of

the drug was unbound. From our calculations, the free fraction

of drug was 22 % (53).

It is notable that these results are of little

significance clinically because 1000 ng/ml is too high a

concentration and would seldomly be encountered in the

clinical practice.

Pharmacokinetics-Pharmacodvnamics of Aceromazine in the Horse

The respective acepromazine plasma concentrations for

all the horses after administration of intravenous doses of

0.15 mg/kg are presented in Table A-3. Figures 5 to 14 show

the fitted plasma time profiles for each horse. The maximum

Co concentration ranged from 1701 ng/ml to 67 ng/ml with a

mean value of 476 ng/ml.










61


103- .







Tim
C


1 \
+ 102-:4
Ga






10u- i

0 20 40 60 80

Time

Figure 5: Fitted arterial plasma concentration vs time (min)
for horse Letren after an IV administration of 0.15 mg/kg
dose of Acepromazine.








62


10'





C 1
"-I
(0






*"^ *"*^*----
102
UJ







101- I I I
0 20 40 60 80 100

Time

Figure 6: Fitted venous plasma concentration vs time (min)
for horse Letren after IV administration of 0.15 mg/kg dose
of Acepromazine.








63








C

CO

o 101





II I:



0 10 20 30 40 50 60

Time



Figure 7: Fitted arterial plasma concentration vs time (min)
for horse Dappler Arab after IV administration of 0.15 mg/kg
dose of Acepromazine.
dose of Acepromazine.













103




C
0 102
-I
C .

a-i
0 101

-- -o' ---.--




100
0 S 10 20 30 40 50

Time



Figure 8: Fitted venous plasma concentration vs time (min)
for horse Dappler Arab after IV administration of 0.15 mg/kg
dose of Acepromazine.











103 ...





0



IU
C
O 0

-- ----- .. .
0 0


101- I I I I
0 40 80 120 160 200

Time


Figure 9: Fitted plasma concentration vs time (min) for
horse Chestnut after IV administration pf 0.15 mg/kg dose
of Acepromazine.









66








oC


L
0 0 .


0 101 ..
U
S10' --0
.) 0 -----..





100 1111111
0 20 40 60

Time

Figure 10: Fitted plasma concentration vs time (min) for
horse Sara after IV administration of 0.15 mg/kg dose of
Acepromazine.









67

103 ,





I 102- .

Timi
-.t



I -- -
0 10""-
*ro





0 10 20 30 40 50 60 70

Time
Figure 11: Fitted plasma concentration vs time (min) for
horse Juanita after IV administration of 0.15 mg/kg dose of
Acepromazine.




















HL
4 -0-








101 I -I I I
0 20 40 60 80 100 120

Time

Figure 12: Fitted plasma concentration vs time (min) for
horse Raisin after IV administration of 0.15 mg/kg dose of
Acepromazine.









69









102
+-j
CO



0 i

U



10 10

0 10 20 30 40 50 60 70

Time


Figure 13: Fitted plasma concentration vs time (min) for
horse Roan after IV administration of 0.15 mg/kg dose of
Acepromazine.



















C
0 102+\





4-+



100 I I I I
0 20 40 60 80 100 120

Time






Figure 14: Fitted plasma concentration vs time (min) for
horse Roan after IV administration of 0.3 mg/kg dose of
Acepromazine.

















%* # i r q In V
%0 moo^ r4 M N 4 M on o O N 71V
co 0%e 0 q 4; 71i M 1 v V
%D M *0 Me 6 Qr4 V

A W M O A 9 *
0 0M w r N 0 w B '0 M N
S' O
v N OiOn m* 0 %* i at*
m w N N 4 w C


m w tfO 0 0 w cc CO *% 0 o
% O (* 00 0 00 C co


;0 % N M *
r- 0 %a co R^ co wo -I ai 4 .l 6 0%




ar tn fn 0 r. C4 In o





O0 N N fN (9 N N M at N- r4 (m9Q in
S ; A 'i w!
0 & N N
% c 6 w % 0%
MN M V V m in w









O m e *( 9I L% ol
U .co A 0 A in N N
S| o N Nr m N % Oen












Of m c! 0 0 m 0 CI m
(n M W o W
r0 Ml 1fNO l C O m 0



r n tr in in m %0
m co M % Ch co % < in 4


en m p I n oo w M



S* r r W w m N O *






w 04 (9 0 v IC ( mN
I *a



m !h w 0 r m 4 In M M c4
l C; n r o o
(n o t .4 c o In c4 66 ral
cl oi oo n ri i o .0 V 41







~ ~ r ? -


& si a^$ 5^5 *^'p8 2 ?^ 00 ^











r- 0
o 0 r N
- 0
4- 0 0
N o 0
0 0 '


d 0
CN N
N M
8 88
u) C; .


CO
o a
0 0 N


a







0 N
o




.u

in





0~
C o



10 d


* tn 0%

o o o


o 0
0 0 0








o a n







So 0 S 0


-5









-r4












49
^1


la


N S
I. P
N f
o O
*^ *
0 0
0
M o a

a 8
o P


i in w M [I C4
9 *
< **m 0 0 0 0


















The plasma concentration time profile for all the horses

after administration of 0.15 mg /kg doses of acepromazine was
*-
fitted best to a two compartment open body model.

Acepromazine pharmacokinetic parameters obtained by fitting

a two compartment model are summarized in Table 10. The

distribution phase with values ranging from 0.13 min -1 to

3.03 min-1 i.e. half-life from 0.23 to 4.43 min indicated

the existence of a relatively fast distribution into a shallow

compartment after the drug entered the systemic blood stream.

The mean terminal half-life, at 67 +/- 38 min, ranging from

18 to 148 min showed larger variation and relatively slower

elimination from the body. The confidence intervals for both

rate constants are presented in Table 11. Due to the large

variation in the values one can only estimate the magnitude

of the rate constants and the half lives. The volume of

distribution of the central compartment ranged from 137 1 to

503 1 with a mean value of 290 liters. These values are much

larger than the volume of blood in the horse (70 ml/Kg of body

weight) indicating that acepromazine is widely distributed in

the horse. The total clearance of acepromazine in the horse

after IV administration was found to be 23.5 +/-11 1/min

(52.49+/-24.62 ml/min/kg). This value was determined by















74
taking the ratio of the dose divided by the area under the

curve up to time infinity. This total clearance value is not

significantly different than the cardiac output in the horse
0.
which is around 18 1/min (54) indicating that this drug does

not undergo any non flow dependent metabolism. Therefore the

plasma clearance or metabolism contribution to the total

clearance is negligible. However, the contribution of each

organ to the total clearance is unknown since the rena

clearance of acepromazine could not be determined.

The only other study about the pharmacokinetics of

acepromazine in the horse was done by Ballard and Coworkers

where they administered 0.3 mg/kg. They obtained an alpha

phase half life of 4.2 min and an elimination half life of

185 min. Both these values are almost triple that obtained

in the present work. This might be due to the fact that they

gave a dose double the present dose. The difference might

also be due to the fact that the analytical method might be

different enabling us to measure lower concentration. Another

difference might be that their reported value was not be the

terminal phase but a value of a second compartment that they

were able to see because of their higher dose. Interestingly

enough, the values for the total clearance agree well with

each other, their value was 25 1/min while the one obtained














75
here was 23.5 1/min even though the dose in this study was

half of theirs. This strongly suggests that the

pharmacokinetics of acepromazine in the horse behave in a

linear fashion.

It is notable that even though horse Letren suffered

from a mitral valve insufficiency in its heart affecting

somewhat its cardiac output. Although showing no sign of frank

cardiac failure, this deficiency did not affect the

disposition of acepromazine in this horse since the

pharmacokinetic parameters obtained from this horse were very

similar to what was obtained with other horses. Horse Letren

but not horse Dappler Arab showed the same pharmacokinetic

profile when venous and arterial plasma concentrations were

plotted versus time. All the pharmacokinetic parameters were

the same for the horse Letren but not for horse Dappler Arab

(refer to Table 8). The terminal half life for horse Letren

was 82 min for the venous plasma levels and 90 min for the

arterial samples. As for horse Dappler Arab, the terminal

half life was 32 and 59 min for the venous and arterial

samples respectively. This is not to say that there were no

differences between the venous and arterial plasma samples.

For the first three minutes, the venous plasma samples (the

drug was injected into the venous side) showed higher














76
concentrations of drug than the arterial samples taken at the

same time points. After this initial time of three minutes

the plasma concentrations were the same on either side

indicating that the drug had been redistributed and been

circulated all over the body and thus reached equilibrium

between the two sides.

PHARMACODYNAMIC EFFECTS OF ACEPROMAZINE IN THE HORSE

A-Effect on the Eauine Hematocrit

Table A-8 summarizes the hematocrit values as a

function of time for all the five horses that were given

0.15 mg/iCg. A plot of the hematocrit values vs time is given

in Figure 15. It can be seen that at this dose level,

acepromazine has a profound effect on the hematocrit. In

all the horses studied the hematocrit dropped by more than

20 %. This drop in hematocrit was not immediate but was

gradual and reached the lowest value after six hours. This

is not to say that the biggest effect on the hematocrit

occurred at six hours, the study was stopped at this time

and it is possible that a further reduction might have

occurred' after this time period. Also it was not possible

to determine how long this reduction might have lasted.

These results agree very well with other investigators who









45


40


35o o
000
U El
0
| 0-^A f a

AA ft20 2 0 0 0
25 AA A A
A 25

20 A1 I I,
0 100 200 300
Time (minutes)
Figure 15: Plot of hematocrit vs time for all 5 horses after
IV administration of 0.15 mg/kg dose of Acepromazine. ( O)
Dappler; ( @ ) Juanita; ( A ) Sara; ( A ) Chestnut;
( 0) Letren.














78
found that the duration and not the degree of the decrease

in the hematocrit was dose related. Parry and Anderson

found that the hematocrit returned to its control value

twenty one hours after acepromazine administration.

The mechanism of action of this decrease is thought to

be due to the alpha-adrenolytic activity of acepromazine

together with a depression of the vasomotor centre causing

splenic relaxation with consequent erythrocyte sequestration

causing a drop in the hematocrit values. The fact that this

decrease is not dose dependent might be due to the fact that

it is dependent on the splenic storage capacity.

B-Cardiovacular and Hemodvnamic Effect of AceDromazine

A summary of the systolic, diastolic and mean blood

pressure as a function of time are given in Table A-4 while

the heart rates for all the horses after acepromazine

administration are given in Table A-5. Figure 16, 17 and 18

show the plot of the systolic, diastolic and mean blood

pressure as a function of time while Figure 19 show the plot

of the heart rate as a function of time for all the horses

given 0,15 mg/kg of acepromazine. Table A-7 summarizes the

blood gases as a function of time for the five horses

studied.














79
In all the horses studied (except for horse Letren)

there was a marked decrease in systolic blood pressure. As

seen with the hematocrit, this drop in systolic blood

pressure was not sudden but was gradual and reached its

maximum decrease between 60 and 90 minutes post

administration of the acepromazine dose. In four out the

five horses, the systolic blood pressure dropped from a

value of around 140 am of Hg to values around 100 in a time

period of approximately 100 minutes. These values remained

depressed even after the elapse of up to six hours. Horse

Letren did not show any marked changes in its systolic blood

pressure. This might be due to the fact that Letren

suffered already from some hemodynamic problems and thus

would not be expected to react normally to the

pharmacological actions of acepromazine.

The same trend was also observed for both the

diastolic and mean blood pressure. They exhibited the same

pattern as was observed with the systolic blood pressure.

The drop was gradual over a certain period of time and its

maximal decrease at about the same time. Again in horse

Letren the changes in these blood pressures were not as

obvious as with the other horses. This decrease in blood

pressure might be due to a direct effect on the heart and









80
175
0'
S 165-
E 155 D
E O
0 145, 0

135

I 125 0 A 0



85-
m' n


0 100 200 300
Time (minutes)



Figure 16: Plot of the systolic blood pressure (um BG) vs
time (minutes) for all 5 horses after IV administration of
0.15 mg/kg dose of acepromazine. (0) Dappler; (0 ) Juanita;
(A) Sara; (A) Chestnut; (Q) Letren.









100 81





70 0
n Ai0 A
E 90

0 50o
0 100 200 300
Mo b I -

o 40( D M ,
-S 306 0

1 20 0 ,n A--'A

0 100 200 300
Time (minutes)




Figure 17: Plot of the diastolic blood pressure (mm Hg) vs
time for all 5 horses after IV administration of 0.15mg/kg
dose of Acepromazine. ( O ) Dappler; ( 0 ) Juanita;
( A ) Sara; ( A ) Chestnut; ( [t ) Letren.