FACTORS AFFECTING THE METABOLIC CONJUGATION

OF ARYLACETIC ACIDS

by

PATRICK FRANK AYODELE DIXON

a thesis submitted for the degree of Doctor of Philosophy

in the

University of London

May 1976 Department of Biochemistry

St. Mary's Hospital Medical School,

London, W2 1PG

2

ABSTRACT

The metabolic conjugation of I -naphthylacetic, diphenylacetic, and

hydratropic acid has been studied in man, some selected sub-human primates

and non-primate species, by examining the nature of the conjugates excreted

in the urine after administration of the 14

C-labelled acids. I-Naphthylacetic

acid forms glycine, taurine,glutamine and glucuronic acid conjugates but the

pattern of conjugation varied with species. Hydratropic acid is excreted

mainly as a glucuronic acid conjugate except in the cat which excretes also

the glycine and taurine conjugates. Diphenylacetic acid is excreted mainly

as its glucuronic acid conjugate irrespective of species. At low doses

1-naphthylacetic acid is excreted in the rat conjugated mainly with glycine but

at higher doses, with glucuronic acid. Dose level did not affect the metabolic

pattern of diphenylacetic and hydratropic acids in the rat which are conjugated

solely with glucuronic acid.

The pharmacokinetic behaviour of these acids and phenylacetic acid

was examined in the rabbit; and 1 -naphthylacetic, diphenylacetic and hydratropic

acids which are conjugated mainly with glucuronic acid, were shown to have a

low blood clearance, a low biological half-life and a high elimination rate

constant while the reverse is the case for phenylacetic acid.

The affinities of these four acids for the sites of conjugation (mitochondria

for amino acid and microsomes for glucuronic acid conjugations) and the

conjugating enzymes associated with these structures were investigated. It

was found that the pattern of conjugation of an arylacetic acid in the rat is

influenced by its affinity for uptake (as measured by binding)by mitochondria and

endoplasmic reticulum and affinity for the conjugating enzyme systems associated

with these structures (as measured by their abilities to conjugate with glycine and

glucuronic acid).

3

ACKNOWLEDGEMENTS -

The work described in this thesis was carried out in the Department

of Biochemistry, St. Mary's Hospital Medical School.

I wish to thank Professor R. T. Williams, F. R. S. , for the great interest

he has shown in this project.

To Dr. J. Caldwell and Professor R. L. Smith, I am deeply grateful

for their encouragement and helpful advice over the last three years. I will

always appreciate the willingness of my fellow research workers, especially

Jeff, members of staff and the technical staff to assist practically whenever

necessary and in providing helpful information.

My sincere thanks to Miss Sally Turner for so capably typing this

thesis.

Finally, words can never adequately express my appreciation to members

of my family especially my brother Rowland, who financed this project and for

the understanding, patience and encouragement shown to me at all times.

4

INDEX

Page

Abstract 2

Acknowledgements 3

Chapter One Introduction 5

Two Materials and Methods 52

Three Metabolism of 1-Naphthylacetic Acid 78

Four Metabolism of Diphenylacetic Acid 106

Five Metabolism of Hydratropic Acid 118

Six Pharmacokinetics and Subcellular Aspects 179

of Arylacetic Acid Conjugation

Seven General Discussion and Conclusion 164

Appendix 175

References 179

5

CHAPTER ONE

Introduction

Contents

Page

Introduction 7

Biological Factors Affecting Drug Metabolism 8

Effect of Chemical Structure on Drug Metabolism 14 Cyclohexanecarboxylic Acids 17 Phenols 17 Polychlorinated Phenols 19 Benzoic Acids 19 Hydratropic Acid and Related Compounds 22 Aryl Alkyl Sulphones 23

Conjugation Mechanisms 25

Glucuronide Formation 25

Amino Acid Conjugation 27

Conjugation Patterns of Some Aromatic- and Arylalkyl- 30 carboxylic A cids

Aromatic Carboxylic Acids 31 Benzoic Acid 31 2 - (4 ' -Aminobenzoyloxy)benz oic Acid 33 Quin ()line -2-carboxylic Acid 34

Primary Arylacetic Acids 34 Phenylacetic Acid and its Simple Derivatives 34 1-Naphthylacetic Acid 38 Indole-3-acetic Acid 38 Indomethacin 39 Myalex 39 p-(Cyclopropylcarbonyl)phenylacetic Acid 40 (SQ 20, 650)

Metiaz inic Acid 41

Imidazole-4-acetic Acid 41

Secondary Arylacetic Acids 42 Ibuprofen (2-[4-isobutylpheny1]-propionic Acid) 42

Fenoprofen (dl-2-[3-phenoxyphenyl]7propionic Acid) 42 a - [4-(1 -Oxo-2-isoindoliny1)-phenyl]-propionic 43

Acid Diphenylacetic Acid 43

6

Tertiary Arylacetic Acids 43 a, a -Dimethylphenylacetic Acid 43 Triphenylacetic Acid 44 Benzilic Acid (Diphenylglycollic Acid) 44

Compounds Metabolised to Arylacetic Acids 44

4-(2:4:5-Trichlorophenoxyl)-butyric Acid 44 Diphenhydramine 45 Brompheniramine 46 Haloperidol 46 o, p -DDD, [1- (o-Chlorophenyl) -1 -(p' -chlorophenyl) -2,2- 47 dichloroethane] DDT [1,1 -Bis (p -chlorophenyl) -2,2,2, -trichloroethane] 48

X-ray Contrast Media 49

Some Endogenous Arylacetic Acid Derivatives Normally Occuring in the 49 Urine

Scope of the Present Investigation 50

7

Introduction

Most organic compounds which enter the body undergo metabolic

transformation prior to their excretion, although a few compounds are not

metabolised and are therefore excreted unchanged. These are usually

strongly polar compounds e.g. strong acids or bases. In addition a few non-

polar compounds such as the chlorinated biphenyl 2,4,5 , 2' , 4', 5'- hexachloro

biphenyl are excreted unchanged but this is a relatively unusual situation.

Some examples of these three types of compounds are shown in Table 1.1

Table 1.1

Compounds Eliminated without undergoing biochemical transformation

(Smith, 1974).

Strong acids

Strong bases

Non-polar

Methotrexate 5,5'7Methylenedisalicylic acid 2,4,5 -Trichlorophenoxyacetic acid

Hexamethonium salts Methylglyoxal-bisguanylhydrazone

2,4,5,2' , 4', 5' -Hexachlorobiphenyl Ether Barbitone

The metabolism of a foreign compound is generally a biphasic phenomenon

(Williams, 1959). In the first stage (Phase I) the compound undergoes a bio-

chemical reaction which may be oxidation, reduction or hydrolysis and which

usually introduces into the molecule a functional group such as hydroxyl (OH),

carboxyl (-COOH), amino (-NH2) or thiol (SH). In the second stage (Phase II)

the metabolite so formed is combined, usually through the newly introduced

functional group, with a molecule provided by the body which is derived from

carbohydrate, amino acid or other sources.

aromatic hydroxylation

conjugations OH

HO

Benzene Phenol pKa 10

Phenyl-p-glucuronide, pKa 3.4

OH

8

The conjugated product is typically an acid, largely ionised at body pH and

readily excreted in the urine and / or the bile. This pattern is well illustrated

by the metabolic fate of benzene. This compound undergoes a number of

metabolic reactions, the major pathways being oxidation (aromatic hydroxy-

lation) to phenol, followed by conjugation of the latter with glucuronic acid to

form phenyl-p-glucuronide, a strong water-soluble acid (pKa 3. 4) which is

readily excreted.

COOH

If the compound already possesses a suitable functional group conjugation may

occur directly. A number of Phase I and Phase II (conjugation) reactions are

shown in Tables 1. 2 and 1.3 .

The metabolic route or routes which a particular compound may undergo

will be determined by a wide variety of factors, such as the following:

i) biological factors e.g. species, strain, age and sex.

ii) chemical factors e. g. chemical structure (including molecular

size and geometry), ionization and lipid solubility.

iii) other factors, such as dose, route of administration, presence

of another drug, diseases, nutritional status, temperature

and altitude.

Biological factors affecting drug metabolism

These factors are principally, species, strain, age and sex. Species

variations in metabolism can occur both in respect to the speed at which

9

Table 1.2

Phase 1 - Reactions of Foreign Compounds

Reaction Class Example

Oxidations

Reduction

Hydroxylation N- and 0-dealkylation Deamination Replacement of S by 0 Ether cleavage Aromatisation Oxidation of thioethers

to sulphoxides

Reduction of nitro and keto groups

Reductive cleavages of azo groups

Reduction of C=O, C=C

Hydrolysis Hydrolysis of esters Amide hydrolysis

10

Table 1.3

The Principal Phase II (Conjugation) Reactions

Conjugation Reaction Conjugating agent Source

Glucuronide synthesis glucuronic acid

Glycine conjugation glycine

Glutamine conjugation glutamine

Methylation methionine

Mercapturic acid synthesis cysteine

carbohydrate

amino acids

Ethereal sulphate synthesis sulphate

Acetylation acetyl miscellaneous

Thiocya.nate formation thio (S-SO3H)

11

metabolism occurs and in the metabolic pathways employed, and these arise

mainly because of interspecies differences in the enzymic control of Phase

I and Phase II reactions. The enzymes occur mainly in the liver but are also

found, usually to a lesser extent, in several other tissues, including the

intestine, kidney and lung. Species differences in the pattern of metabolism

commonly arise because of one or more of the following reasons (Williams, 1967) :

i) competing reactions for example, the hydroxylation and ring

cleavage in the metabolism of coumarin (Table 1.4)

Table 1.4

Competing Reactions : Metabolism of Coumarin

(Kaighen and Williams, 1961 ; Gangolli et al. , 1974)

% of dose excreted as :

Hydrox-y -coumarins Ring open products

69 - 92

low

3

50

41

23

Species

Man

Rat

Rabbit

ii) the occurrence of metabolic defects in some species appear

to lack the capacity to carry out certain metabolic reactions

and some examples are shown in Table 1.5.

Although the rat is defective in the N-hydroxylation of

chlorphentermine, this reaction takes place extensively in man

rhesus monkey, rabbit and guinea pig (Caldwell et al. , 1975 a).

(Table 1.6).

Table 1.5

Species Defects in Common Metabolic Reactions

Defective Reaction Species References

N-Hydroxylation of aliphatic amines Rat, Marmoset Caldwell et al (1975a)

Glucuronide formation Cat, Gunn rat Robinson and Williams (1958)

Sulphate formation Pig, Opossum Stekol (1936) ; Combs and Hele (1927)

Arylamine acetylation Dog, Fox Marshall (1954)

Mercapturic acid formation Guinea pig Bray , et al. (1959) Bray and James (1960)

Glycine conjugation Fruit Bat Bababunmi et al (1973)

13

The cat forms little or none of the glucuronic acid

conjugate of phenol (Capel et al. 1972) but this defective

characteristic seems to be substrate dependent (Millburn, 1974)

(Table 1. 7).

Table 1. 6

Species Difference in the N-Hydroxylation of Chlorphentermine

Cl

CH3

CH C-NH Cl 21 2 CH3

CH3 -CH C-NHOH 21

CH3

Species % of Urinary 14C as

N-Hydroxy-p-chlorphentermine

Rat 0

Guinea Pig 50

Rabbit 42

Marmoset 0

Rhesus monkey 76

Man 44

14

Table 1. 7

Glucuronic Acid Conjugation in Cats : Variation with Substrate

Compound % of 24 h excretion conjugated with:

Glucuronide Sulphate

Phenol 1 95

1-Naphthol 1 98

2-Naphthol 3 97

Pax acet am ol 3 86

Phenolphthalein 60 40

iii) The occurrence of unusual metabolic reactions may be

restricted to certain species or a group of species and also

to certain compounds (Table 1. 8).

Some metabolic reactions appear to be restricted in

their species occurrence to man and other primate species

(Table. 1. 9).

Effect of Chemical Structure on Drug Metabolism

The metabolism of a compound depends among other things on its

chemical structure, which influences its physico-chemical properties such as

ionization and lipid solubility. A change in the chemical structure may vary the

physico-chemical properties and also affect the affinity of the compound for the

metabolising enzymes, and hence induce a possible change in its metabolic pattern.

15

Table 1. 8

Some Thcommon Metabolic Conjugation Reactions

Conjugating Agents Species

Ornithine Certain birds and reptiles

Taurine Pigeon, Ferret

Amino-acids Serine Rat, Rabbit

Glycyltaurine Cat

Arginine Ticks and Spiders

Glucose Insects

Carbo-hydrates Ribose Rat, Mouse

N-Acetylglucos- Rabbit amine

Phosphate Cat, Dog, Man

Acids Formate Rat, Dog

Succinate Rat, Dog

Table 1. 9

Metabolic Reactions Apparently Restricted to Primate Species

Metabolic reaction Species occurrence

N1-Glucuronide formation

Glutamine conjugation

Aromatisation of quinic acid

Man, New and Old World Monkeys and prosimiaas

Man, New and Old World Monkeys

Man and Old World Monkeys

0-Methylation of 4-hydroxy-3, Man, Monkeys 5-diiodo-benzoic acid

16

OSO3H 0. C6I1

90

6

17

Cyclohexanecarboxylic acids

Certain cyclohexanecarboxylic acid derivatives undergo aromatization

in the body, but the extent to which the process occurs has been found to depend

upon the nature of substituents attached to the cyclohexane ring and the animal

species studied, (Williams, 1959). Table 1. 10shows the metabolic pattern of

some cyclohexanecarboxylic acids and related derivatives.

Phenols

Phenols undergo metabolic conjugation with both glucuronic acid and

sulphate.

Although monosubstituted phenols are metabolised in a similar manner

to phenol, there are quantitative differences related to the chemical nature and

position of the substituent groups (Williams, 1938).

The more complex phenols such as stilboestol, hexoestrol and dienoestrol j

OH OH

OH

CHC2H5

CHC2H5

C=CHCH3

C=CHCH3

OX

OX

OX

Hexoestrol (X=H)

Stilboestrol (X=H) Dienoestrol (X=H)

Table 1.10

The Metabolism of Cyclohexanecarboxylic Acid and Related Acids

(Adapted from Williams, 1959)

18

R = C6H11 or

Derivative Structure Aromatic Metabolite

Cyclohexanecarboxylic R. C 00H acid

Cyclohexylacetic acid R. CH2COOH

g-cyclohexylpropionic R. CH2CH2COOH

y-cyclohexylbutyric acid R. (CH2)3COOH

N-Methylhexahydrobenz- R. CONHCH3 amide

Hexahydrobenzoylalanine R. CONHCH(CH3)COOH

benzoic acid

no aromatization; complete oxidation

benzoic acid

no aromatization ; (oxalic acid ).

benzoic acid

no aromatisation; original compound ex-creted

acid

19

are conjugated mainly with glucuronic acid in the rabbit (Dodgson et al. , 1948,

Mazur and Shorr, 1942).

Another phenol which is highly conjugated with glucuronic acid in the

rabbit but not at all with sulphuric acid is p-hydroxybenzophenone (Robinson

and Williams, 1957).

CO OH

High glucuronic acid conjugation and almost negligible sulphate conjugation

seems to be characteristic of phenols containing two isolated benzene rings.

Polychlorinated phenols

The main reaction of these phenols is again conjugation, except in the

case of pentachlorophenol and other chlorinated phenols with pKa values of less

than 7. 2:4-Dichloro- 2:4:5-trichloro-and a tetrachloro-phenol have been

reported as being excreted conjugated with glucuronic acid in the rabbit

(Deichmann and Thomas, 1943). As far as chlorinated phenols are concerned,

those with pKa less than 7 do not form ethereal sulphates in the rabbit as shown

in Table 1.11.

Benzoic Acids

The pattern of conjugation of the monosubstituted benzoic acids is

qualitatively similar to that of benzoic acid, but there are considerable

quantitative variations in the relative extent of the glycine and glucuronic acid

conjugations with this series of acids. Thus in the rabbit 2-toluic acid is

conjugated solely with glucuronic acid and not at all with glycine, 2-nitrobenzoic

acid is largely excreted unchanged, whilst anisic acid gives rise to more of its

glucuronic acid conjugate than its glycine conjugate when given in doses at which

benzoic acid is conjugated mainly with glycine. The quantitative data on the

20

Table 1.11

Ethereal Sulphate Formation and pKa of Chlorinated Phenols

(taken from Dodgson et al. , 1950)

(dose about 0.2 g/kg )

Phenols % of dose excreted

as ethereal sulphate pKa

4-Chlorophenol 22 9.2

2:4-Dichlorophenol 16 7.7

2:6-Dichlorophenol 0 6.8

2:4:5-Trichlorophenol 10 7.7

2:4:6-Trichlorophenol 0 6.4

Pentachlorophenol 0 5.3

Table 1.12

The Conjugation of Monosubstituted Benzoic Acids in Rabbits

Sub stituent pKa Approximate dose g/ kg

% Conjugation

Reference Glycine glucuronic acid

(ester type) Free

None 4. 2 0.4 83 15 1 Bray et al. (1955) 4-Nitro * 3.4 0.1 - 0.2 0 3? 80 - 90 Bray et al. (1949 a) 2-Chloro 2. 9 0. 3 5 19 60 Bray et al. (1952) 4-Chloro 4. 0 0.3 63 21 7 Bray et al. (1952) 2-Methyl 3. 9 0. 3 0 73 Bray et al. (1949 b) 4-Methyl 4.4 0. 3 46 14 73 Bray et al. (1949 b) 3-Hydroxy + 4.1 0. 5 ? 6 70 Bray et al. (1955) 4-Methoxy 4. 5 0.4 38 57 1 Bray et al. (1955) 4-Acetamido 4. 3 0.4 2 0 77 Bray et al. (1955) 4-Amino 4. 9 0. 5 + + 13 - 30 ++ Bray et al. (1948) '

* 11-21% of these acids is reduced to aminobenzoic acids + 5-14% of 2-, 2-19% of 3- and 0-18% of 4-hydroxybenzoic acids form ether glucuronides +÷ This acid is partly acetylated.

22

metabolism of a number of substituted benzoic acids in the rabbit are given in

Table 1.12.

Similar studies on the dog have been made by Quick (1932 a,b) who

found that substituents at 3- and 4-positions in the benzoic acid molecule had

little influence on the extent of glycine conjugation when compared with benzoic

acid, except when they were hydroxyl group which caused a depression of glycine

conjugation. Substitution in 2-position always reduced the extent of glycine

conjugation. With glucuronic acid conjugation, substitution in 3- position had

little effect, while acidic groups in the 2- and 4- positions reduced it and basic

groups increased it. Here again, acid strength and alternative metabolic

reactions are important factors.

Hydratropic acid and related compounds

Early experiments showed that tropic acid (a -hydroxymethylphenylacetic

acid, see below) is not metabolised and is excreted unchanged in the urine of cat

(Kay and Raper, 1922). Later work in the rat and the mouse showed that 95-98%

of a dose of tropic acid was recovered unchanged in the urine within two hours

of dosing (Gosselin et al. , 1955). Its isomer, atrolactic acid, behaves

similarly in dogs and cats (Kay & Raper, 1922), but its dehydrated analogue,

atropic acid is believed to be completely destroyed in dogs (Kay and Raper, 1922).

In the rabbit

CHCOOH

CH2OH

C. COOH CH. COOH

CH2 CH3

Hydratropic acid (pKa 4. 6)

Tropic acid

(pKa 4.1) Atropic acid

(pKa 3. 85)

—COH. COOH

CH3 Atrolacetic acid

(pKa 3. 53)

CHCONH CH 3 2 2-\ CH 3

23

the racemic, dextro- and laevo- forms of the hydratropic acid are highly con-

jugated with glucuronic acid and are excreted as hydratropoylglucuronide

(Robinson et al. , 1955) but Kay and Raper (1922) have also isolated hydra-

tropoylglycine in the dog.

The metabolic route of tropic, atropic, atrolatic and hydratropic acids

seems to depend on the type of substituents in the a-carbon.

Aryl alkyl suiphones

Aminomethylphenyl methyl sulphone (V 335) possesses antibacterial

properties, and when administered to rabbits is excreted mainly as 4-methyl-

sulphonylbenzoic acid (Hartles and Williams, 1947).

NH2 02C113 oxidative

HOO 0 CHI 3 deamination

The acetylated derivative of V 335, 4-acetamidomethylphenyl methyl sulpbone. ,

has no antibacterial activity and on administration to rabbits is excreted

unchanged (Hartles and Williams, 1949). Table 1.13 shows the metabolic fate of

some other derivatives in the rabbit. The size of the acyl substituent seems to

determine whether or not it is excreted unchanged or metabolised, or excreted

in the faeces.

24

Table 1.13

Metabolism of 4-Acylaminomethylphenyl Methyl Sulphones

R. CONHCH2 C6 H4 . SO4. CH3 dose 1 g / rabbit

(Adapted from Williams, 1959)

Derivatives

Urinary Excretion Faecal excret-ion in 5 days of unchanged drug %

1

R unchanged

%

l metabolised*

%

V335 - 0 87

Formyl H 48.5 15.8

Acetyl CH3 61.1 0

Propionyl C2H5 42.9 13.1

Butyryl C3H7 3.0 62.1 0

Hexoyl C51111 0 56.8 2.3

Decoyl C9H19 0 22.2 40

Tetradecoyl C13H27 0 0 50

* Excreted in the urine as 4-methylsulphonylbenzoic acid.

25

Conjugation Mechanisms

The biosynthetic reactions between a foreign compound and a substance

provided by the body (see Table1.3)are catalysed by specific enzymes which

require certain activated nucleotides for their enzymic action (Williams, 1969).

The intermediate nucleotide may contain either the conjugating agent or the

foreign compound. These two types of conjugation may be represented as : -

1) Activated ener source

gy Foreign Compound> Conjugated Conjugating > conjugating

agent agent transferase product

Activated energy conjugating ■

2) Foreign > Foreign / Conjugated source agent

Compound Co mpound product + transferase

The first class of detoxication mechanism includes the formation of

glucuronides, acetylation and methylation.

Peptide conjugations with glycine, glutamine and ornithine come under

the second class of detoxication mechanism (Williams, 1969). In these cases, the

appropriate transferase enzyme is associated with the mitochondria. Table 1.14

shows some examples of these two classes of conjugation reactions.

Glucuronide Formation

Conjugation with glucuronic acid is widespread among species, occurring

in mammals (Teague, 1954), marsupials (Hinks and Bolliger, 1957), birds

(Baldwin, Robinson and Williams, 1960), reptiles and amphibia (Smith, 1964).

In insects, the mechanism appears to be replaced by p-glucoside formation. This

is a characteristic insect detoxication and probably occurs in other invertebrates

as well (Smith, 1968). Glucuronide formation can occur with compounds

possessing -OH, -COOH, -NH2 and - SH groups. The mechanism of glucuronide

Table 1.14

Mechanistic Classification of Conjugation Reactions

(Adapted from Williams, 1974)

Reaction Intermediate Transferring enzyme Location

1. Intermediate containing conjugating agent

Glucuronide synthesis

Glucoside

Ether sulphate sunthesis

Methylation

Acetylation

Cyanide detaNication

UDP-glucuronic acid

UDP -glucose

Adenosine 3'-phosphate 5'-sulphatophosphate

Adenosylmethionine

Acetyl-CoA

Thiosulphate (S-SO3H)

Glucuronyltransferase

UDP-glucose glucosyl-transferase

Sulphotransferase

Methyltransferase

Transferase

Sulphurtransferase

Liver, kidney, lung, spleen, urinary bladder, gastro-intestinal tract

In insects - hepatic caecum

Liver, kidney, intestine

Liver ,kidney, spleen, brain, small7dnte stine , lung, heart, muscle .

Liver, kidney, brain, lung, pancrease, spleen, blood.

Wide distribution in the body but high concentration in the liver.

2. Intermediate containing foreign compound

Glycine conjugation Aroyl-CoA Glycine acyltransferase Liver and /or kidney only

Glutamine conjugation Arylacetyl-CoA Not defined (glutamine acyltransferase ?).

In man - kidney and liver

Ornithine conjugation Aroyl-CoA Not defined (ornithine acyltransferase ? ).

In chickens - kidney only

Mercapturic acid Epoxide * of foreign Glutathione S-opoxide- Liver kidney, pancreas synthesis compound transferase *.

* Some compounds are sufficiently reactive to combine with glutathione without further changes ; others such as hydrocarbons require

activation by oxidation to epoxides ; several glutathione-S-transferases occur(see Boyland & Chasseaud, 1969);

27 s3rnthesis can be described as follows: -

1) a-Glucose-1- phosphate + UTP

Uridyl- UDP glucose + pyrophosphate transferase

2) UDP-glucose

UDP -glucose dehydrogenase

DPN

UDP glucuronic acid

3) UDP glucuronic UDP - glucuronyl Aglycone glucuronide +

acid +Aglycone UDP transferase

The enzyme UDP-glucuronyltransferase is in, or associated with-the

microsomes (Dutton, 1961). Table 1.15 shows some carboxylic acids that form

ester glucuronides.

Amino Acid Conjugation

The mechanisms of amino acid conjugation involve a three-stage process,

the first two stages result in the activation of the carboxylic acid ; and the third

in the conjugation of the ,activated carboxylic acid with an amino acid, the reaction

1) R. COOH +ATP R. CO. AMP + pyrophosphate

2) R. CO. AMP + CoA -SH .> R. COS CoA + adenylic acid

3) R. COS CoA + H2 NCHCOOH > R. CONH. CH. COOH I I R' IV

CoA-SH

being catalysed by the respective amino acid-N-acylase. , This process takes

place in the mitochondria (Schachter and Taggart, 1953). Table 1.16 shows the

amino acids used by some species in conjugating some carboxylic adds.

Birds classed as Galliformes (hens, turkeys) and Anseriformes (ducks

and geese) excrete aromatic acids and arylacetic acids as ornithine conjugates.

Monkey Lan et al. 1975

Dog, monkey Harman et al. 1964

Pig-tailed and Foulkes (1970) talapoin monkeys

Robinson et al. 1955

Rubin et al. 1972

Robinson .& Williams (1955)

McChesney and Hoppe 1954.

Rabbit

Man

Rabbit

Dog, cat

28

Table 1.15

Some carboxylic acids which form ester glucuronides

Compound Species References

2-Ethylbutyric acid Trimethylacetic acid 3:3-Dimethylbutyric acid 2:4:4-Trimethylpentoic

acid 2 -Ethylhexoic acid

Benzoic acid

[p-(Cyclopropylcarbonyl) -phenyl ] acetic acid

Indomethacin

Myalex

Hydratropic acid

Fenoprofen

a, a -Dimethylphenylacetic acid

Iopanoic acid

Rabbit

Dziewiatkowski et al. (1949)

Indian fruit bat Bridges et al. 1970

Table 1.16

The amino acids used in conjugation reactions

Amino acids Species Substrate References

Glycine rat, rabbit, man cat, monkey

benzoic acid Bridges et al., 1970

Glutamine man, monkey phenylacetic acid James et al., 1972 a

Taurine dog [p-(Cyclopropylcarbony1)- phenyl]acetic acid.

Lan et al., 1975

Ornithine chicken phenylacetic acid. James et al., 1972 a

Aspartic acid rat bis (p -chlorophenyl) acetic acid

Pinto et al.,1965

Alanine mouse, hamster bis(p-chlorophenyl)acetic acid

Wallcane et al 1973

Serine rat o,p'-dichlorodiphenylacetic acid bis(p-chlorophenyl) acetic acid

Reif and Sinsheimer, 1975 Pinto et al 1965

Glutamic acid fruit bat benzoic acid Idle et al. , (1975)

30

Ornithine conjugation is not characteristic of all birds since the pigeon and dove

(Columbiformes) excrete aromatic acid as glycine conjugates (Baldwin, Robinson

and Williams, 1960).

Reptiles, which have a close phylogenetic relationship to birds, are

also capable of conjugation with ornithine, though, in the species examined,

glycine conjugation is also found. Thus urine from tortoises dosed with aromatic

acids was found to contain both ornithine and glycine conjugates ; similar

results were found with a grass snake, two species of lizard and an alligator

(Smith, 1964).

Extensive conjugation of aromatic acids is found in Arachnids. Although

the excreta may contain conjugates with arginine, glutamine, glutamic acid,

citrulline, ornithine and agmatine, it is thought that, with the exception of the

glutamine conjugate, all these are derived from the original arginine conjugate

(Hitchcock and Smith, 1966).

Kaihara and Price (1965) have also reported glycyltaurine and glycyl-

glycine conjugates of quinoline-2-carboxylic acid in the cat.

Conjugation Patterns of Some Aromatic - and Arylalkyl - Carboxylic Acids

Aromatic - and short-chain arylalkyl - carboxylic acids are metabolised

in general by conjugation either with an amino acid or with glucuronic acid.

Some which have low pKa may be excreted unchanged (e. g. 2-nitrobenzoic acid

pKa 2.2). The nature of the amino acid used varies with species, the two main

ones being glycine, in sub-primate mammals, and glutamine in primates.

Conjugation with a variety of other amino acids, including taurine, serine,

aspartic acid, glutamic acid and alanine have also been reported (see Table

1.16).

COON

Benzoic acid

Hippuric acid

31

Aromatic Carboxylic Acids

Benzoic Acid

Benzoic acid generally forms hippuric acid and benzoylglucuronide in

the animal body, and the relative amounts of these conjugates depend on the

dose and the species.

CONHCH2COOH

COOC6H906

Benzoylglucuronide

Bridges et al. (1970) have investigate the metabolic pattern of benzoic acid in

a range of species and some of their results are shown in Table 1:17. They have also

reported that chicken, turtle (side-necked) and gecko excrete benzoic acid mainly

as ornithuric acid and with small amount of hippuric acid. Indian fruit bat produced

mainly benzoylglucuronide (Bridges et al. , 1970) and this possibily indicates a

defect in hippuric acid formation. This possible defect has been confirmed by

Bababunmi et al . (1973).

Idle et al. (1975) have recently reported that benzoylglutamic acid is one

of the metabolites of benzoic acid in the fruit bat.

32

Table 1.17

Metabolites of Benzoic Acid in Some Species

(Adapted from Bridges et al. ; 1970)

Species dose

mg/kg

% 24 h excretion found as

Hippuric acid

Benzoyl glucuronide

Man 1 100 0

Rhesus monkey 20 100 0

Rabbit, cat and capuchin

50 100 0

Dog, squirrel monkey, ferret, hedgehog and pigeon

50 80 20

Ferret 200 47 44

Ferret 400 30 49

— 0 - C 11 0

unchanged (50%)

COOH

OC6H903

COOC6H

90

6

OH

CONHCH2COOH

OH

33

2-(4'-Aminobenzoyloxy)benzoic acid

The metabolism of 2-(4'-am_inobenzoyloxy)benzoic acid has been studied

in man (Smyth et al. , 1974). It is metabolised to salicylic acid which is

excreted as salicyluric acid and salicyloyl glucuronides, and 4-aminobenzoic

acid which is excreted as N-acetyl-4-aminobenzoic acid.

COOH

2-(4'-Aminobenzoyloxy)benzoic acid

COOH

OH COOH

Salicylic acid

NH2

4-Aminobenzoic acid

N-acetylation

Salicyluric acid

Salicyloyl glucuronides ( 15 % ) (35%)

34

Quinoline-2-carboxylic acid

The aromatic acid quinoline-2-carboxylic acid (see below) and certain

hydroxylated derivatives of this acid, give rise to unusual conjugation reactions

in certain species. In the rat, the acid is conjugated with glycine. In the cat,

however, a major metabolite of this acid is quinaldylglycyltaurine, and small

amounts of quinaldylglycylglycine are also formed (Kaihara and Price, 1965).

Quinoline-2-carboxylic acid

The cat is also able to form the glycyltaurine conjugate of 4-hydroxy-

quinoline-2- carboxylic acid. The dihydroxylated derivative, 4, 8-dihydroxy-

quinoline-2-carboxylic acid, is excreted in the rat as double conjugate, giving

two ether glucuronides (8-monoglucuronide and 4, 8-diglucuronide) each with a

serine residue attached to the 2-carboxyl group, whereas in the rabbit, the

hydroxyl groups are conjugated with sulphate but again, a serine residue is

attached to the 2-carboxyl group (Rothstein and Greenberg, 1957).

Primary Arylacetic Acids

Phenylacetic acid and its simple derivatives

Some earlier workers showed that phenylacetic acid is excreted conjugated

with glycine in rabbits, dogs, sheep, horses and rhesus monkey (Salkowski and

Salkowsi, 1879 ; Salkowski, 1884 ; Vasiliu, 1909 ; Sherwin, 1917) and with

ornithine in the hen (Totani, 1910). Thierfelder and Sherwin (1914, 1915) showed

that phenylacetic acid is excreted as the glutamine conjugate by man , and the

same conjugate has also been found in the urine of chimpanzee (Power, 1936).

35

CH2COOH

Phenylacetic acid

-CH2CONHCH2

COOH

CH 2

H CONHCH \CH 2

COOH CONH2

Phenacetylglycine Phenacetylglutamine

COOH

CH2CONH(CH2

)3CHNHCOCH2

Diphenacetylornithine

Sherwin also studied the metabolism of some simple substituted

phenylacetic acids in man, dog and rabbit. He found that in man 2-, 3-, and

4-hydroxyphenylacetic acids, 2-,3- and 4-nitrophenylacetic acids were excreted

in the urine unchanged, and that 4-aminophenylacetic acid was acetylated but not

conjugated at the -COOH group. He also reported that 2- and 4-chloro-, bromo-

and iodo-phenylacetic acids were excreted as glycine conjugates by man and dog

(Sherwin 1918, Sherwin 1923 ; Cerecedo and Sherwin 1924 ; Muenzen, Cerecedo

and Sherwin, 1926).

In 1958 the glutamine conjugate of 3,4-dihydroxy-5-methoxyphenylacetic

acid was found as a metabolite of mescaline in man (Harley-Mason et al. 1958).

36

3,4,5-Trimethoxyphenylacetic acid was also reported to be excreted unconjugated

as a metabolite of mescaline (Charalampous et al. , 1964). Another substituted

phenylacetic acid, 4-methoxyphenylacetic acid is excreted as 4-methoxy-phenacetyl-

glutamine and its 0-deme,thylate metabolite 4-hydroxyphenacetylglutamine in man

(Oakley and Seakins, 1971).

The metabolic studies of phenylacetic acid by James et al. (1972 a) in

a number of species have shown the pattern of conjugation represented in Table

1.18.

The same workers also showed that two avian species differed in the

conjugates excreted. Pigeons excrete phenacetylglycine and phenacetyltaurine

while chickens excrete mainly diphenacetylornithine. They also showed that

phenacetyltaurine was found in significant amounts in the urine of ferret, bush-

baby, slow loris, squirrel monkey, capuchin and pigeon but small amounts

were also found in most other species.

James et al (1972 b ) also reported that 4-nitrophenylacetic acid is excreted

unchanged by man and rhesus monkey, but conjugated with glycine by rats, and

that 4-chlorophenylacetic acid is similar to phenylacetic acid in its species

distribution of glycine and glutamine conjugation.

Recently Ette et al (1974) have reported that phenacetylglycine is the

major metabolite, while phenacetylglucuronide is a minor metabolite of phenylacetic

acid in the Indian fruit bat. This is an indication that glycine conjugation in this

species is substrate dependent, since it does not occur with benzoic acid

(Bababunmi et al. , 1973).

37

Table 1.18

Metabolic Conjugation of Phenylacetic Acid

Class

% of 24 h excretion conjugated with

Glutamine Glycine

Man 93 0

Old World Monkeys (8) 30 - 90 0.1 - 1

New World Monkeys (3) 64 - 80 1 - 10

Lemurs (2) 0 85

Sub-primates (10) 0 80 - 100

(number of species tested shown in parentheses)

38

1 -Naphthylacetic Acid

Lethco and Brouwer (1966) have shown that when 1-naphthylacetic

acid is administered orally to the rat, it is conjugated with glycine and

glucuronic acid. With small doses, the conjugation with glycine is the major

metabolic route, whereas the glucuronide formation becomes the major route

with larger doses. Bernhard and Caflisch-Weill (1949) found the glycine

conjugate in the urine of a dog which had been fed 1-naphthylacetic acid but

they did not detect this metabolite in the urine of rats and rabbits receiving this

acid.

Indole-3-acetic Acid

CH 2COOH

H

Indole-3-acetic acid is excreted as its glutamine conjugate in man and

old world monkeys, as glutamine and glycine conjugates in new world monkeys

and as glycine conjugate in prosimian species and lower animals (Evans 1972).

Further work by Bridges et aL,(1974) indicates that in man, the glycine conjugate

was not formed, the metabolites being the glucuronic acid and glutamine

conjugates of indole -3-acetic acid. Indolylacetyltaurine - is the only metabolite in

the pigeon, while it is a substantial metabolite in the green monkey, squirrel

monkey, the capuchin monkey and the ferret.

Cl Cl

C=0

CH3 0

39

Indomethacin

The anti-inflammatory drug indomethacin is excreted in the urine in man

mainly as its ester glucuronide, but it is excreted mainly unchanged in the dog

(Harman et al. , 1964). It is partly metabolised in the rabbit to its N-deschloro-__

benzoyl derivative, and in the rat, guinea pig and monkey to both its N-deschloro-

benzoyl and 0-desmethyl derivatives. These metabolites are mostly excreted

as their ester glucuronides (Harman et al 1964).

Indomethacin 0-de smethylindomethacin

CH30 CH2COOH

CH3

N-deschlorobenzoylindomethacin

Mvalex

Another anti-inflammatory drug, Myalex (ICI 54450),has been reported to

undergo some metabolism in the aromatic ring by dogs and rats but no glycine

conjugate was detected (Foulkes, 1970). However, Myalex is excreted in the

urine of green and pigtailed monkeys as the ester glucuronide (Foulkes, 1970).

monkey (88%)

Glucuronic acid conjugate

Dog (27%)

Taurine conjugates

CH2COOH

Taurine CH2COOH

Dog og (30%) Glycine

Dog (31")> conjugate

CH2COOH

40

CH2COOC6H906

. Cl

Cl

Myalex Myalex ester glucuronide

p-(Cyclopropylcarbonyl)phenylacetic Acid (SQ 20, 650).

p-(Cyclopropylcarbonyl)phenylacetic acid a nonsteroidal anti-inflammatory

agent with analgesic activity, exhibits species variations in its metabolism

(Lan et al. 1975). In the rhesus monkey it is conjugated with glucuronic acid,

and in the dog with taurine. Its reduction product (a-cyclopropyl-a-hydroxy-p-

tolyl)acetic acid (SQ 21, 316) is conjugated with both taurine and glycine in the

dog.

( SQ 20, 650 )

Reduction Dog (53% ) Monkey (8-9%)

(SQ 21, 316)

41

Metiazinic Acid

Metiazinic acid is conjugated with both amino (-NH2) and methyl groups

in rabbit and man, and excreted as both amide and methylester (Populaire et al

1969). This is an unusual conjugation pattern since arylacetic acids are

usually conjugated with an amino acid or glucuronic acid.

Imidazole-4-acetic Acid

CH2 COOH

N CH

2COOH i 1 i

N j0

H V 0 CH-CHOH-CHOH-CHCH2OH

The metabolic conjugation of imidazole-4-acetic acid is unique when

compared with the other arylacetic acids already considered. The conjugation

does not involve the carboxylic acid group of the compound or the usual conjugating

agents utilised by arylacetic acids, but involves the use of ribose as the conjugating

agent. Schayer (1956) has reported that in rat and mice imidazole-4-acetic acid

is excreted as 1-ribosylimidazole-4-acetic acid. This riboside is also formed

as a metabolite of histamine which is degraded to imadazole-4-acetic acid. In

rats and mice the riboside is a major metabolite of small doses of histamine,

whereas in cats and dogs it is a very minor metabolite (Schayer 1956).

42

Secondary Arylacetic Acids

Ibuprofen (2-K-isobutylphenyll-propionic acid).

CH3

CH - CH

CH3

CH3

CH. C0011

Ibuprofen, an anti-inflammatory agent is metabolised by the oxidation of

the isobutyl side chain, but the carboxylic acid group is unaffected by its bio-

transformation in man (Adams et al. 1969) . This is unlike hydratropic acid

which is conjugated with glucuronic acid in the rabbit (Robinson et al. 1955) and

with glycine in the dog (Kay and Raper, 1922).

Fenoprofen (d1-2-[3-phenoxyphenyl]-propionic acid)

Fenoprofen, an anti-inflammatory analgesic agent is excreted mainly as

fenoprofen glucuroniceand 4'-hydroxyfenoprofen acyl-glucuronide in man (Rubin

et al. 1972). There are also very small amounts of unidentified acid -labile

conjugates of fenoprofen and 4'-hydroxyfenoprofen,

CH3 CH-COOH

fenoprofen

43

a - - (1 -Oxo-2-isoindoliny1)-phenyll-propionic Acid (K 4277)

CO\

N

CH2

CH3

CHCOOH

The metabolic fate of the anti-inflammatory agent K 4277 has been examined

in the rhesus monkey by Chasseaud et al. , (1974), who reported that it is excreted

mostly unchanged (78% of dose) with some 5% as the 5-hydroxy derivative and a

very small amount as glucuronide. K 4277 is excreted mainly as its ester

glucuronide conjugate by man and rabbit (Fuccella et al. , 1973 ; Goldaniga et al. ,

1973).

Diphenylacetic acid

Diphenylacetic acid (pKa 3. 94) is excreted as its ester glucuronide in man, dog

and rabbit (Miriam et al. , 1927a).

Tertiary Arylacetic Acids

a, a'-Dimethylphenylacetic Acid

a, a-Dimethylphenylacetic acid is metabolised exclusively to an ester

glucuronide in the rabbit (Robinson &Williams 1955).

44

Triphenylacetic Acid

Triphenylacetic acid , (pKa '3.96) has been shown by Miriam et al (1927b)

to be excreted totally unchanged by rabbit, dog and man.

Benzilic Acid (Diphenylglycollic acid)

Benzilic acid (pKa 3. 06) is excreted unchanged by rabbits (Sieberg and

Harloff, 1919).

Compounds Metabolised to Arylacetic Acids

4-(2:4:5-Trichlorophenoxy)-butyric Acid

Bohme and Grunow (1974) have examined the metabolism of 4-(2:4:5-

trichlorophenoxyl)-butyric acid, and 2:4:5-trichlorophenol was found as its

principal metabolite following oral administration to rats. 2:4:5-Trichlorophenoxy-

acetic acid, the final product of a-oxidation was also excreted in the urine. In a

45

different study, Grunow and Bohme (1974) have shown that 2:4:5-trichlorophenoxy-

acetic acid and 2:4-dichlorophenoxyacetic acid are excreted by rat and mice

as glycine and taurine conjugates.

Cl

Cl OCH2CH

2CH

2COOH

CI

OCH2COOH CI

4-(2:4:5 -Trichlorophenoxy) -butyric acid

Cl

Cl

V conjugated with glycine and

taurine

OH

Cl

2:4:5-Trichlorophenol

Diphenhydramine

Drach and Howell (1968) have shown that the antihistamine drug diphen-

hydramine (Benadryl: 2-diphenyl-methoxy-N,N-dimethylethylamine) is metabolised

to diphenylmethoxyacetic acid which is excreted as a glutamine conjugate by the

rhesus monkey.

CH 3 HC-OCH CH N

, oxidative 2 2 \CH3 deamination HC-OCH2COOH

Diphenhydramine Diphenylmethoxyacetic acid

conjugated with glutamine

CH 3 oxidative HC-CH CH N 2 2\ HC-CH

2C00:-I

deamination CH

3

46

Brompheniramine

Another antihistamine drug, brompheniramine which bears some structural

resemblance to diphenhydramine, is also metabolised to a substituted acetic acid

but this is reported to be excreted by man and dog partly as the glycine conjugate

(Bruce etal 1968).

Brompheniramine 47

conjugated with glycine

Haloperidol

Haloperidol, and other structurally related butyrophenone neuroleptic

drugs were reported to be metabolised in rats to p-fluorophenylacetic acid,

the glycine conjugate of which was the major urinary metabolite of the drug in

rats. (Braun, Saudijn and Poos, 1967).

CO-(CH2)„ -N/

>R CH2COOH

1

R' = -OH Cl

conjugated with

glycine

Haloperidol

Cl

(HO)2

Cl - - - - -> Cl

47

1 -( 0 -Chlorophenyl) -1 -(p'-chlorophenyl) -2,2 -dichloroethane (o, p' -DDD).

The metabolism of 0,pt-DDD has been investigated in the rat (Reif and

Sinsheimer, 1975). After 100 mg oral dose to rats, 7% is recovered in the

urine and 88% in faeces within 8 days. The urine was found to contain

o -p' -dichlorodiphenylacetic acid (0,p' -DDA) as well as 4-hydroxy-, 3-hydroxy-

and 3, 4-dihydroxy- substituted o -p'DDA. The serine and glycine conjugates

of 2,4' -DDA were also identified. In addition to the above metabolites the faeces

contained o-pi-DDD, 1-(2-chloropheny1)-1-(4-chloropheny1)- 2-chloroethylene,

and the aspartic acid conjugate of o,p'-DDA.

In the human studies, serine and glycine conjugates of o,p'-DDA have

been identified also (Reif and Sinsheimer, 1974).

Cl

3,4-Dihydroxy derivative 3-hydroxy- and 4-hydroxy

derivatives

48

DDT [1,1 -bis (p-chlorophenyl) -2,2,2 -trichloroethane I

DDT is partly metabolised to bis (p-chlorophenyl) acetic acid which is

excreted as serine and aspartic conjugates in rat (Pinto et al. 1965). The

mouse and hamster conjugate bis(p-chlorophenyl)- acetic acid with alanine and

glycine (Wallcane et al.1973). However, when bis(p-chlorophenyl) acetic acid

is administered intravenously to rat, it undergoes enterohepatic circulation

almost completely, and the only biliary metabolite is a glucuronide

(Gingell, 1975).

Cl -j Cl Cl

DDT

Cl H

COOH

bis(p-chtorophenyl)a cetic acid

49

X-ray Contrast Media

Compounds which are opaque to x-rays have been used as contrast media

after their injection to patients. Those which have high molecular weights

are excreted extensively in the bile, and produce gallbladder shadows which can

be used in diagnosis. Examples of such compounds are iopanoic acid, iophenoxic

acid and pheniodol.

Iopanoic acid is excreted in dog and cat as its ester glucuronide

(McChesney and Hoppe, 1958), and iophenoxic acid is excreted in dog as its

mono-ester-, mono-ether- and di-glucuronide (Wade et al., 1970) but pheniodol

seems to be excreted unchanged in rabbit and man (Junkman 1941).

C2H

5 1 CH

2CHCOOH

Iopanoic acid

C H 2 5

CH2CHCOOH

Iophenoxic acid

CH 2 —CHCOOH

Pheniodol

Some Endogenous Arylacetic Acid derivatives Normally Occuring in the Urine

A number of ring substituted arylacetic acids derived from the metabolism

of amino acids are found in normal urine with the carboxylic acid group unconjugated.

Included in this group are the phenolic acids which are normal constituents of

50

urine. Indeed the urinary levels of these acids can be important in diagnosis.

These include 3,4-dihydroxyphenylacetic acid (a metabolite of DOPA), and its

metabolites 3-methoxy-4-hydroxyphenylacetic acid and 3-hydroxyphenylacetic

acid, 2,5 -dihydroxyphenylacetic(homogenti dc acid) , 2 -hydroxyphenylacetic

acid, 4-hydroxyphenylacetic acid, 5-hydroxyindole-3-acetic acid (a metabolite

of tryptophol, and imidazoleacetic acid (a metabolite of histidine), (De Eds

et al. , 1955, Armstrong et al. , 1956, Neuberger et al. , 1947, Delvigs et al. ,

1965, Snyder et al. , 1964).

Scope of the present investigation

The literature survey indicates that there is enough evidence to suggest

that the structure of an aromatic - or arylalkyl - carboxylic acid greatly

influence its metabolic pattern of conjugation. The aromatic acids are con-

jugated with both amino acid and glucuronic acid, but the small primary

arylacetic acids are conjugated mainly with amino acids, however with increase

in complexity in the structure of the primary arylacetic acid there is a shift

from amino acid conjugation to glucuronic acid conjugation. Secondary arylacetic

acids and the small tertiary arylacetic acids are conjugated mainly with

glucuronic acid, but the large tertiary arylacetic acids are excreted unchanged.

The question arises as to what factors determine whether or not an amino

acid or glucuronic acid conjugation takes place. One or an interaction of two or

more of the following factors may be responsible : -

1) Ionization

2) Lipid solubility

3) Pharmacokinetic behaviour

4) Molecular size and geometry

5) Affinity for subcellular conjugation sites (mitochondria and

endoplasmic reticulum) and the associated enzymes.

51

Therefore this thesis describes investigation into the following: -

i) effect of chemical structure and dose on the metabolic

route of three arylacetic acids, namely 1-naphthylacetic,

diphenylacetic and hydratropic acids, in some selected

species.

ii) pharmacokinetic behaviour of these three acids and phenylacetic

acid in the rabbit.

iii) the affinities of these four acids to the conjugating sites

(mitochondria and microsomes) and the conjugating enzymes

associated with these structures.

52

CHAPTER TWO

Materials and Methods

Contents

Compounds

Radiochemical Synthesis

[Carboxyl-14C]diphenylacetic acid

[14 C -Methyl] - ( -Hydratropic acid

Synthesis of 1-Naphthylacetic Acid Conjugates

1 -Naphthylacetylglycine 1 -Naphthylacetyl-L -glutamine 1-Naphthylacetyltaurine 1-Naphthylacetylglucuronide

Synthesis of Diphenylacetic Acid Conjugates

Pages

54

54

54

55

56

56 56 57 58

58

Diphenylacetylglycine 58 Dipheny lacetyl -L -glutamine 59 Diphenylacetyltaurine 59 Diphenylacetylglucuronide 60

Synthesis of (±) Hydratropic Acid Conjugates 61

(±) -Hydratropoylglycine 61 (±) -Hydratropoyl-L-glutamine 62 (±) -Hydratropoyltaurine 62 (+) -Hydratropoylglucuronide 63

Benzylamine salt of (±)-Hydratropic acid 64

Metabolic Studies 65

Animals 65

Collection of carbon dioxide in the expired air 65 Paper and Thin-layer Chromatography 65 Location of Compounds on Chromatography 66

Ultra-violet (U. V.) light 66 Spray Reagents 66 Naphth ore sor cinol spray 66 4-Dimethylaminobenzaldehyde spray 66 Chlorine-starch/potassium iodide detection 67

reagent Ninhydrin spray 67

53

Chromatographic properties of taurine 67

Radiochemical Techniques 67 Spectra 71 Treatment of urine samples 71

Pharmacokinetic Studies 72

Animals 72

Cannulation of Marginal Vein 72

Administration of Compounds 72

Blood Analysis for 14C-content 73

In Vitro Studies 73

Preparation of Tissue Subfractions 73

Mitochondria 73 Microsomes 73

Binding Studies 74

Protein Determination 75

Enzyme Affinity 75

Glycine Conjugation 75 Glucuronic Acid Conjugation 75

Reverse Isotope Dilution Experiments 76

54

Materials and Methods

Compounds

[Carboxyl-14C]-1-naphthylacetic acid (specific activity, 44 mCi/mmol. ),

[Carboxyl u]-phenylacetic acid (specific activity, 59 mCi/mmol), sodium

[14

C]-cyanide (specific activity 55.5 mCi/mmol) and [14C]-methyl iodide (specific

activity 58 mCi/mmol) were purchased from the Radiochemical Centre,

Amersham, England. Phenylacetic, 1-naphthylacetic, diphenylacetic and

hydratropic acids were obtained from commercial sources and purified as

appropriate. Phenacetylglycine was a sample prepared by James et al (1972a).

Uridinediphosphoglucuronic acid (UDPGA) and adenosine triphosphate

(ATP) were purchased from Sigma Chemical Co, Surbiton, England, and co-

enzyme A (CoA) was obtained from Boehringer Corp. , Ealing, England.

Radiochemical Synthesis

[Carboxyl-14C]diphenylacetic acid

Chlorodiphenylmethane (1.06 g) was mixed with cuprous [14C]cyanide

(0.54 g ; 2 mei ; prepared from [14C] sodium cyanide, by the method of Reid et al. ,

(1951) ) and heated for 2 h in an oil-bath at 200-210°. The mixture was

allowed to cool and then extracted with acetone (30 ml). The acetone extract was

_14 filtered and evaporated to dryness. The residue of crude diphenylaceto- [ C]-

nitrile was hydrolysed by heating under reflux with stirring for 3 h with 48 % hydro-

bromic acid (50 ml) following which the hydrolysate was extracted with ether in a

continuous extractor for 3 h. The ether phase was extracted with N-NaOH

(25 ml), the latter separated and acidified with 2N-HCI. The mixture was

extracted once more into ether and then back again into N-NaOH (10 ml). The

latter was separated and acidified with 2N-HC1. The precipitate of [14C] —

diphenylacetic acid that separated was filtered and recrystallised from water to

55

give white crystals m. p. 147° specific activity 3jCi/mg (yield 0.28 g,

14

radio-

chemical yield from cuprous [ C]cyanide, 42%). It was shown by chromato-

graphy in solvent/D and F followed by radiochromatogram scanning to be

radiochemically pure as shown by the appearance of a single 14C peak at Rf

values 0.88 and 0.92 respectively corresponding to diphenylacetic acid.

Reverse isotope dilution analysis showed a radiochemical purity of 99. 5%.

14 r c-Methy1]-( 4 -hydratropic acid

To a suspension of NaH-mineral oil (51. 6%) in dry dimethyl sulphoxide

(25 ml) under nitrogen was added dropwise benzyl cyanide, (3 g) dissolved in

dimethyl sulphoxide (30 ml) and the reaction mixture was stirred at room

14 _ temperature for 4 h. [ C]Methyl iodide (3.6 g ; 1 mCi) was then added slowly,

the temperature being maintained at 10° with an ice/water bath. After stirring

for 2 h, a further portion of unlabelled methyl iodide (2.6 g) was added and the

solution stirred overnight. The reaction mixture was then treated with dilute

acetic acid and extracted twice with ether ( 2 x 50 ml). The ether extract was

washed with saturated sodium bicarbonate solution, evaporated to afford a residue

of crude [2-[14

C]-methyl]benzy1 cyanide.

The latter was dissolved in ethanol (200 ml) and refluxed for 7 h with a

solution of KOH (30 g) in water (70 ml). The reaction mixture was evaporated

to dryness and the residue acidified with 2N HC1 and extracted with ether (2 x 50 ml).

The latter was then extracted with saturated sodium bicarbonate solution (15 ml),

acidified with 2N-HC1 and reextracted with ether ( 2 x 50 m1). The ether was

evaporated leaving [14C]-hydratropic acid, specific activity 0.21 pCi/mg (yield

3.5 g, radiochemical yield, 73. 6%). It was shown by chromatography in solvents

D and F followed by radiochromatogram scanning to be radiochromatographically

pure as shown by the appearance of a single 14C peak at Rf values 0.75 and 0.87

56

respectively corresponding to hydratropic acid. Reverse isotope dilution

analysis showed a radiochemical purity of 96. 5%. However, as stated above,

radiochromatography of the product showed only one 14C peak, and since hydra-

tropic acid is an oil at room temperature, the apparently low value for the

radiochemical purity of this material determined by isotope dilution is probably

due to traces of solvent which remained after evaporation.

Synthesis of 1-naplithylacetic acid conjugates

1 -Naphthylacetylglycine was prepared from 1-naphthylacetylchloride

and glycine according to the method of Friedman and Masse(1910). It

was recrystallised from aqueous ethanol to give white crystals m. p.

148 - 149° (lit. 148 - 150°) and had an equivalent weight by titration

(0.1 N - NaOH) of 246 (requires 243) C14H1303 N requires C, 69.14 ; H, 5.35

and N, 5.76. Found C, 68.88 ; H, 5.44 and N, 5.77. The mass spectrum

of the methyl ester showed a molecular ion at m/e 257 (relative

intensity 22.6) with prominent peaks at 115 (24), 141 (76), 142 (95) and

168 (29).

1 -Naphthylacetyl-L-glutamine was prepared from 1-naphthylacetyl

chloride and L-glutamine according to the method of Thierfelder and

Sherwin (1914). It gave white needle crystals m. p. 185 - 186° when

recrystallised from methanol. Equivalent weight by titration (0.1 N - NaOH)

was 315 (requires 314). C17111804N2 requires C, 64. 97; H, 5.73 and N,

8.92. Found, C, 64. 81; H, 5. 91 and N, 9. 05.

The mass spectrum of 1 -naphthylacetyl-L-glutamine methyl ester pre-

pared by treatment of the free acid with ethereal diazomethane showed

a molecular ion at m/e of 328 (relative intensity 3.3) with prominent peaks

at 116 (29), 141 (78), 142 (56), 155 (17), 168 (78) and 187 (3.2). (cracking

pattern, see Appendix).

57

1-Naphthylacetyltaurine was synthesized as follows: -

1-naphthylacetyl chloride (17. 4 g) was added dropwise with stirring

over a period of 4 h to an ice-cold solution of taurine (9.2 g) dissolved

in N-NaOH (82 ml). The reaction was continued overnight after which

the mixture was adjusted to pH 2 with 2N-HC1 and extracted with ether

(3 x 30 ml) to remove 1-naphthylacetic acid. The aqueous layer was

separated and reduced.to dryness in a rotary evaporator and the residue

extracted with methanol (400 ml). The methanol extract was filtered and

reduced to 70 ml on a rotary evaporator. On addition of acetone to the

concentrate white crystals separated which were filtered and recrystallised

from the mixture of methanol and acetone (2:1 by vol.) to give 7 g of small

white crystals m. p. 228-229° of the sodium salt of 1-naphthylacetyltaurine.

The crystals gave a strong positive sodium flame test and did not react

with sodium bicarbonate. C14H14NO

4SNa requires C, 53. 33 ; H, 4.44 ;

N, 4.44 ; S, 10.16 ; Na, 7.30. Found C, 53.35; H, 4. 53 ; N, 4. 39 ;

S, 9.96 ; Na, 7.25.

The compound was shown to afford 1-naphthylacetic acid and taurine on

acid hydrolysis as follows : 10 mg of the compound was heated with 5N-HCI

(1 ml) in a sealed tube at 120° for 18 h. The hydrolysate was reduced to

dryness and the residue dissolved in water (0.25 ml). Portions (50 pd.)

of the latter were chromatographed on Whatman No. 4 paper using solvent

systems B and C. Spraying the chromatogram with ninhydrin revealed a

purple spot of Rf value 0.12 and 0.41 in solvents B and C respectively

which corresponded with taurine. Further portions of the hydrolysate

were chromatographed on thin-layer silica gel plates using solvent system

F. When viewed beneath u. v. light there appeared a dark purple spot Rf

0. 88 which corresponded to 1-naphthylacetic acid.

58

The infra-red spectrum (Nujol showed prominent absorption bands at

3260 cm-1 (N - H stretch), 1640 (Amide I band C = 0 stretch) 1555 (Amide

II band, C - N stretch), 1220 - 1165 (broad) and 1065 - 1020 (broad) (both

due to S = 0 stretch).

The mass spectrum of the methyl ester showed prominent peaks at m/e

185 (relative intensity 44%), 167 (12), 166 (9), 142 (74), 141 (100), 139

(23), 115 (46), 69 (20), 63 (11) and 44 (95) (cracking pattern shown in

Appendix).

1-Naphthylacetylglucuronide A total of 9 g of 1-naphthylacetic acid was

fed to three rabbits. The glucuronide gum was prepared from the

pooled 24 h urine, using the basic lead salt procedure of Kamil, Smith and

Williams (1952). The gum gave an intensely positive reaction with

naphthoresorcinol and was strongly reducing towards Fehlings and

Benedict's solution. When a small portion of the gum was incubated

over-night in pH 5 0. 5M-acetate buffer at 37° with 13 -glucuronidase or was

warmed with 2N-NaOH for 5 min it was shown chromatographically to

afford 1-naphthylacetic acid. The crude gum was used as standard

for chromatography. A small portion (about 1 g) was treated with

ethereal diazomethane and acetylated by method of Kamil et al. (1952)

but no crystals were obtained . No further characterisation was

undertaken.

Synthesis of diphenylacetic acid conjugates

Diphenylacetylglycine was prepared by reacting diphenylacetylchl.oride

(50 g) for 2 h with glycine (13 g) dissolved in N-NaOH (24 ml). The

reaction mixture was acidified with 2N-HC1 and the product that separated,

filtered washed with chloroform and recrystallised from aqueous ethanol to give

59

white needle crystals of diphenylacetylglycine (m. p. 141 - 142° ;

Miriam et al. ,1927a quote m. p. 157°). C16H1503N requires C, 71. 40;

H, 5. 62 ; N, 5. 20. Found C, 71. 16 ; H, 5. 71 ; N, 5. 12. Equivalent

weight by titration (0.1 N NaOH), 270 (required 269). The mass spectrum

of the methyl ester showed a molecular ion at m/e 283 (relative intensity 3)

with prominent peaks at 77 (22), 91 (49), 105 (68), 106 (100), 116 (8),

166 (11) and 167 (8) (for cracking pattern, see Appendix).

Diphenylacetyl-L-glutamine. Diphenylacetyl chloride (23 g), was added

gradually over a period of 3 h to a well stirred solution of L-glutamine

(13 g) dissolved in 100 ml of water containing sodium bicarbonate (23 g).

The reaction mixture after leaving overnight was filtered and acidifed with

2N-HCI. The precipitate was filtered, washed with 2N-HC1 and ether

and crystallized from water to give white crystals of diphenylacetyl-L-

glutamine (yield 12 g ; m. p. 149°). C19H2004N2 requires C, 67. 04 ;

H, 5. 92 ; N, 8.22. Found C, 67.1 ; H, 6. 07 ; N, 8. 31. Equivalent

weight by titration (0.1 N-NaOH) 342 (requires 342).

The mass spectrum of the methyl ester showed a molecular ion at rale of

355 (M +1; relative intensity, 0.2%) with prominent peaks at 155 (98),

165 (100), 166 (62), 167 (100), 168 (100), 169 (47) and 187 (62) (cracking

pattern, see Appendix).

Diphenylacetyltaurine . Diphenylacetyl chloride (25 g) was gradually added

with stirring to an ice-cold solution of taurine (11 g) dissolved in N-NaOH

(120 ml) over a period of 5 h and left to react overnight. The reaction

mixture was filtered and the filtrate acidified with 2N-HC1 and extracted

with ether ( 3 x 50 ml). The aqueous phase was separated and reduced to

60

dryness and extracted with hot methanol (250 ml). On cooling white needle

crystals of the sodium salt of diphenylacetyltaurine separated (24 g) m. p.

200 205 . The crystals gave a positive flame test for sodium and did not

give CO2

when treated with sodium bicarbonate. C16

H16

NO4

SNa requires,

C, 56.29 ; H, 4.73 ; N, 4.10 ; Na, 6.73 and S, 9.39. Found C, 56. 30 ; H,

4.74 ; N, 4. 04 ; Na, 6. 74 and S, 9. 38. The compound was shown to afford

diphenylacetic acid and taurine on acid hydrolysis as follows : the compound

(10 mg) was heated with 5N-HC1 (1 ml) in a sealed tube at 120 for 18 h. The

tube was broken, the hydrolysate removed and evaporated to dryness. The

residue was dissolved in water (0.25 ml) and portions (501/1) chromatographed

on Whatman No. 4 paper using solvent systems B and C. The dried chromato-

grams showed on treatment with ninhydrin a purple spot at Rf values 0. 11 and

0.42 in solvents B and C respectively which corresponded with taurine. Further

portions of the hydrolysate were subjected to t. 1. c. using solvent system F.

The developed chromatograms showed a dark purple spot Rf 0. 92 when

viewed beneath ultra violet light which corresponded to diphenylacetic acid.

Its infra red spectrum (Nujol) showed prominent absorption bands at 3380

N H stretch), 1650 (amide I band C = 0 stretch), 1510 (amide II band C - N

stretch), 1230 - 1170 (broad), and 1070 - 1050 (broad) (both due S = 0 stretch).

The mass spectrum of the methyl ester of diphenylacetyltaurine showed

prominent peaks at m/e 169 (relative intensity 81), 168 (53), 167 (94), 166 (17),

165 (48), 153 (8), 152 (29), 91 (6), 77 (8), 69 (17), 63 (9) and 44 (100)

(cracking pattern, see Appendix).

Dinhenylacetylglucuronide was isolated from the urine of rabbits dosed

with diphenylacetic acid. The glucuronide gum was prepared by the d

method of Kamil, Smith and Williams (1952) from the combine/24 11 urine

samples collected from three rabbits each fed 3 g of diphenylacetic acid.

61

The gum gave an intensely positive reaction with naphthoresorcinol and was

strongly reducing towards Fehling's and Benedict's solution. When

small portion of the gum was incubated overnight in pH 5 0. 5M-acetate

buffer at 37° with p-glucuronidase or was warmed with 2N-NaOH for 2 min

it was shown chromatographically to afford diphenylacetic acid. The

glucuronide gum was used as standard for chromatography.

A small portion of the gum was characterised as follows: about 1 g was

treated with ethereal diazomethane. Following removal of the solvent

the residue was recrystallised from water to give white crystalline needles

(200 mg) of diphenylacetylglucuronide methyl ester m. p. 175°. It gave a

positive reaction with -naphthoresorcinol and was strongly reducing

towards Fhlings and Benedicts reagents. C21H2208 requires C, 62. 68 ;

H, 5.51. Found C, 62.60 ; H, 5.44.

The mass spectrum of the diphenylacetylglucuronide methyl ester did not

show a molecular ion and gave peaks at m/e 207 (2), 173 (13), 168 (18),

167 (100), 166 (14), 165 (37), 91 (3), 90 (8) and 43 (100) (cracking pattern,

see Appendix).

Synthesis of (±) hydratropic acid conjugates

(±) - Hydratropoylglycine was prepared by treating a solution of glycine in

aqueous sodium bicarbonate with hydratropic acid chloride (Kay and Raper,

1922). It was recrystallised from aqueous ethanol to give white crystalline

needles m.p. 102 - 103° (lit. 103°). C11

l-11303N requires C, 63. 76 ; H, 6.32,

N, 6. 75. Found C, 63. 72 ; H, 6. 38 ; N, 6. 73. Equivalent weight by

titration (0. 1 N NaOH) 208 (requires 207).

The mass spectrum of the methyl ester showed a molecular ion at m/e

221 (relative intensity, 84%) with prominent peaks at m/e 116 (94) , 105 (96)

62

and 91 (100).(cracking pattern, see Appendix).

(±)-Hydratropoyl-L-glutamine Hydratropic acid chloride ( 34 g) was added

dropwise to a well-stirred solution of L-glutamine (24 g) in water (240

ml) containing sodium bicarbonate (50 g). After 3h the reaction mixture

was transferred to a separating funnel and the aqueous layer was

separated. The latter was acidified with 2N-HCI and reduced to dryness

using a rotary evaporator. The residue was extracted with hot methanol

(200 ml) and the extract reduced to dryness. The residue was

recrystallised from water to give white needle crystals (yield 30 g ; m. p.

139 -140 °).C141118 04N2 requires C, 60.42 ; H, 6. 52 ; N, 10. 06.

Found C, 60.50 ; H, 6.36 ; N, 10.43.

Equivalent weight by titration (0.1 N-NaOH) 280 (requires 278).

The mass spectrum of the methyl ester showed a molecular ion at m/e

of 292 (relative intensity 4%) with prominent peaks at 187 (54), 155 (98),

116 (96) and 91 (94) (cracking pattern, see Appendix).

(d)-Hydratropoyltaurine Hydratropic acid chloride (27 g) was added drop-

wise to an ice-cooled and well-stirred solution of taurine (15 g) in N-

NaOH (120 ml) and the stirred mixture allowed to react overnight. The

aqueous layer was separated from the oil that separated and acidified with

2N-HCI and evaporated to dryness on a rotary evaporator. The residue

was extracted with hot methanol (200 ml.) and the methanol extract

separated and reduced to dryness. The residue was recrystallised from

methyl ethyl ketone to give white crystals m. p. 88 - 89° (yield 20 g, 50%).

C11

H15

NO4S requires C, 51.35 ; H, 5.87 ; N, 5.44 ; S, 12.46. Found

C, 51.25; H, 5.89; N, 5.35 ; S, 12.42.

The compound was shown to afford hydrotropic acid and taurine on acid

63

hydrolysis as follows: 10 mg of the compound was heated with 5N-HC1

(1 ml) in a sealed tube at 120° for 18 h. The hydrolysate was reduced

to dryness and the residue dissolved in water (0.25 ml). Portions

(50 aul) of the latter were chromatographed on Whatman No. 4. paper

using solvent systems E and F. Spraying the chromatograms with

ninhydrin revealed a purple spot of Rf values 0.12 and 0.41 in solvents

B and C respectively which corresponded with taurine. Further

portions of the hydrolysate were chromatographed on thin-layer silica"

gel plates using solvent F. When viewed beneath ultra-violet light there

appeared a dark purple spot Rf 0. 87 which corresponded to hydratropic

acid.

Its infra-red spectrum (Nujol) showed prominent absorption bands at

3440 ( N - H stretch), 1650 (amide I band C = 0 stretch), 1570 (amide

II band C - N stretch), 1200 - 1140 (broad) and 1060 - 1010 (broad)

(both due to S = 0 stretch).

The mass spectrum of the methyl ester of hydratropoyltaurine gave a

molecular ion at m/e 271 (relative intensity 76%, with prominent peaks

at m/e 166 (90), 134 (80), 105 (96) and 91 (100) (cracking pattern, see

Appendix).

(±)-Hydratropoylglucuronide A total of 6 g. of hydratropic acid was fed

to three rabbits and the urine collected for 24 h. The glucuronide gum

was prepared from the pooled urine using the basic lead salt procedure

of Kamil, Smith and Williams (1952). The gum was dissolved in methanol

(5 ml) and ether (250 ml) followed by petroleum ether (b. p. 40-60°). After

standing for six months a white solid separated which was recrystallised

from water to give white needle crystals of (±)-hydratropoylglucuronide

and this was used as standard for chromatography m.p. 163° (lit.163-164°).

64

It reduced Benedict's and Fehling's solution readily and gave a

strongly positive naphthoresorcinol test. On warming with N-NaOH for 1 h

or incubating with f3 - glucur onidase in pH 5,0.5M - acetate buffer over-

night at 37° the conjugate was shown by chromatography to afford

hydratropic acid and glucuronic acid.

The conjugate was further characterised as its methyl ester following the

treatment of the remaining filtrate (see above) with diazomethane. The

solvents were removed by evaporation and the residue recrystallised

from water to give white needle crystals of (±)-hydratropoylglucuronide

methyl ester m.p. 164° (lit. 165 - 166°). It reduced both Fehlings and Benedicts

solution on warming and gave an intensely positive reaction with naphthore-

sorcinol. C16H2008 . H2O requires C, 53. 67 ; H, 6.18. Found C, 53. 89;

H, 5.76. Loss on drying at 110°, 5.08% (requires 5. 03%).

The mass spectrum showed a molecular ion at m/e of 356 (relative

intensity 2%) with major peaks at 191 (18), 173 (64), 105 (100) and 91 (16)

(cracking pattern, see Appendix).

Benzylamine salt of (±)-hydratropic acid was prepared for reverse

isotope dilution experiments with hydratropic acid. Hydratropic acid

(1 g) dissolved in ethyl acetate (3 ml) was added to benzylamine (1.5 g),

dissolved in hot ethanol. The mixture was heated for 5 min and the

solvent then removed using a rotary evaporator. The residue was

recrystallised from petroleum ether (b. p. 100-102°) to give white needle

crystals of the benzylamine salt of (±)-hydratropic acid m.p. 88° (yield 1. 8 g).

C16 H19 02 N requires C = 74. 68 ; H, 7.44 ; N, 5.44. Found C, 74.80 ;

H, 7. 37 ; N, 5. 24.

65

Metabolic Studies

Animals

All the species were obtained from dealers in the London area.

Equimolar amounts of _14 _14 C]-1-naphthylacetic acid (100 mg/kg) and C]-

diphenylacetic acid (114 mg-/kg) were given as aqueous solution dissolved in

the calculated amount of N-NaOH. [14Crilydratropic acid (81 mg/kg) was given

in a solution of /3-propylene glycol/water (2:1 v/v). For subhuman primates

the dose solution was sterilised by ultrafiltration prior to use. In the case

of human subjects the dose was 5 mg administered orally. The 0-24 and

24 - 48h urines were collected and adjusted to pH 5 with glacial acetic acid prior

to storage at 0°. Animals were kept in metabolism cages, which allowed the

separate collection of urine, and faeces, and maintained on an appropriate diet

with free access to water.

Bile-duct cannulated female rats were prepared as described by

Abou-El-Makarem et al. , (1967) and were injected intraperitoneally with the

dose solution (5-500 mg/kg). The bile was collected for 3 h and urine was

collected by bladder puncture.

Collection of carbon dioxide in the expired air

14 _ Rats dosed with either L C]-1-naphthylacetic acid or [14

Cj-diphenyl-

acetic acid were placed in Metabowl cages (Jencons) which allowed the collection

of expired air as well as urine and faeces. The expired air was drawn through a

drying trap of anhydrous CaC12 and then through two traps each containing 100 ml

of a 1:2 by vol. solution of ethanolamine (redistilled) in 2-methoxyethanol, (Jeffay

and Alvarez, 1961), at a rate which just prevented condensation inside the

Metabowls.

Paper and. Thin-Layer Chromatography

The solvent systems used were as follows : -

66

A. Butan-1-ol saturated with water

B. Butan-1-ol : acetic acid : water (4:1:1, by vol).

C. Propan-1-ol : ammonia (sp. gr. 0. 88) (7:3 v/v)

D. Benzene : acetone : acetic acid (2:2:1, by vol. )

E. Benzene : acetone : acetic acid (6:2:1, by vol).

F. Chloroform : methanol : acetic acid (24:8:1, by vol.)

Whatman No. 1 paper chromatograms were developed in solvent A

using descending technique , for identifying 1-naphthylacetic acid and its

conjugate. Whatman. No. 4 paper chromatograms were developed in solvent

systems B and C using the ascending technique for identifying taurine. Thin -

layer chromatograms, (aluminium backed silica gel 60 F254 plates, E. Merck

A. G. Darmstadt, Germany ; 0. 2 mm thick) were developed in solvent systems

D, E and F for identifying the arylacetic acids and their respective conjugates.

Location of Compounds on Chromatograms

Ultra-violet (U. V.) light

All the arylacetic acids and their conjugates considered were seen as

dark purple spots under U. V. lights (254 nm ; Hanovia Chromatolite, Slough,

Bucks. , U. K. ).

Spray Reagents

Naphthoresorcinol spray (Bridges, Kibby and Williams, 1965)

Chromatograms were sprayed with naphthoresorcinol (4% w/v) in acetone

to which phosphoric acid (10%) was added (4:1 , v/v) just before use. Glucuronides

showed up as blue spots on heating at 105° for 5 mins.

4-Dimethylaminobenzaldehyde spray (DMAB)

Chromatograms were sprayed with 4-dimethylaminobenzladehyde dissolved

in acetic anhydride (4% w/v) containing a little sodium acetate. After spraying

67

the chromatograms were gently heated with a hot-air blower, and glycine

conjugates showed up as orange spots.

Chlorine-starch/potassium iodide detection reagent

The chromatograms were exposed to chlorine generated from conc. HC1

and sodium hypochlorite for 30 min, aired and then sprayed with 1% (w/v)

aqueous solution of potassium iodide containing 1% (w/v) starch ; and amino acid

conjugates appeared as purplish brown spots.

Ninhydrin spray

Chromatograms were sprayed with 0. 3% ninhydrin in acetone. Amino

acids showed up as purple spots.

Chromatographic properties of taurine

Taurine has Rf values of 0.12 and 0.41 on Whatman No. 4 paper

chromatograms developed in solvent systems B and C respectively.

Radiochemical Techniques

The 14

C in the samples was determined using Packard Tri-Carb

Scintillation Spectrometers (models 3214 and 3320) and dioxan scintillator prepared

as described by Bridges et al. (1967). Urine (0.01 - 1 ml), bile (0. 01 - 0. 05 ml)

and cage washing (0. 05 - 1. 0 ml) were counted in triplicate. The radioactivity

in the expired carbon dioxide was estimated by counting 2 ml of the absorbent in

15 ml of a toluene : 2-methoxyethanol ( 2 : 1 v/v), scintillation medium containing

2, 5-diphenyloxazole (PPO, 5.5 g/l).

For the radiochromatogram scarmingthe urine (0. 01 - 0.2 ml) or bile

(0. 01 ml) containing about 1 x 104 d. p. m. was streaked on the chromatogram.

After development of the chromatograms they were scanned in a Packard

Radiochromatogram Scanner (Model, 7200). In some experiments the chromato-

grams were cut into sections (1 cm) and each counted in the scintillation counter

to enable a quantitation of the metabolites. Histograms were then plotted of the

Table 2. 1

Chromatographic Properties of 1 -Naphthylacetic Acid and its Conjugates

Technique: Compound

solvent:

Paper Thin-layer

A E F

1 -Naphthylacetic acid 0. 84 0. 86 0. 64

1 -Naphthylacetylglycine 0.46 0. 63 0. 37

1 -Naphthyl a cetyl glutamine 0. 24 0.40 0. 13

1 -Naphthylacetyltaurine 0.15 0.18 0. 0

1 -Naphthylacetylglucuronide 0.09 0. 0 0. 0

Rf value1

Colour reactions 1 i

Appearance Naphthoresorcinol Chlorine-

beneath U. V. spray DIVIAB starch/K1

reagent

dark purple

dark purple

dark purple

dark purple

dark purple

orange

blue

purplish brown

purplish brown

purplish brown

Table 2.2

Thin-Layer Chromatographic Properties of Diphenylacetic Acid and its Conjugates

Rf value Colour reactions

Compound Appearance

beneath U. V. solvent : D E F

Diphenylacetic acid 0.88 0.61 0.92

Diphenylacetylglycine 0.73 0.42 0.73

Diphenylacetylglutamine 0.54 0.21 0.35

Diphenylacetyltaurine 0.28 0.00 0.43

Diphenylacetylglucuronide 0.10 0.00 0.17

Naphthoresorcinol

spray DMAB

Chlorine - star ch/K1 reagent

dark purple

dark purple

dark purple

dark purple

dark purple blue

orange purplish brown

purplish brown

purplish brown

Appearance

beneath U. V.

Naphthore sorcinol

spray DMAB

Chlorine -starch/Kt reagent

dark purple

dark purple

dark purple

dark purple

dark purple

orange

blue

purplish brown

purplish brown

purplish brown

Table 2.3

Thin-Layer Chromatographic Properties of (±)Hydratropic Acid and its Conjugates

Rf value

Compound Solvent : D E F

(Li) -Hydratropie acid 0.75 0.75 0.87

(±)-Hydratropoylglycine 0.65 0.42 0.79

(±) -Hydratropoylglutamine 0. 53 0.18 0. 59

(1) -Hydratropoyltaurine 0. 30 0. 00 0. 33

(+)-Hydratropoylglucuronide 0.10 0. 00 0.13

Colour reactions

71

amount of C associated with each position on the original chromatogram. In

all the cases, quench correction was by channels ratio method.

Spectra

Infra-red (i. r.) spectra were recorded as liquid paraffin mulls on a

Perkin-Elmer Infracord 137 Spectrophotometer.

Mass spectra of compounds using direct insertion technique were

recorded on a Varian MAT CH5 Mass spectrometer : probe temperature

100 - 125°C ; chamber temperature, 160°C ; ionization energy, 70 eV ; ionization

current, 300 p.A.

Treatment of urine samples

Urine samples were chromatographed as obtained and following alkaline

hydrolysis or treatment with p-glucuronidase as follows:

(a) mild alkaline hydrolysis ; urine ( 1 ml ) was heated on a boiling water

bath with N-NaOH ( lml ) for 15 min. The solution was neutralised with 2N-

HCl and a portion chromatographed. This treatment was shown to

hydrolyse completely the glucuronic acid conjugates of the arylacetic

acids to their respective aglycones.

(b) j3-glucuronidase treatment ; urine ( 2 ml) was incubated with ketodase

( 1 ml ) and pH 5 0. 5 M-acetate buffer at 37° for 18 h. As a control

on the activity of the enzyme a tube was included containing phenolphthalein

glucuronide ( 1 mg ) dissolved in water ( 1 ml ) instead of urine.

At the end of the experiment the incubate was made alkaline with N-NaOH.

Appearances of a deep red-purple colour indicated that enzyme preparation

was active. Under these conditions arylacetylglucuronides were hydrolysed

to the free acids.

72

Pharmacokinetic Studies

Animals

Female Dutch rabbits weighing 2. 0 - 2. 5 kg ; (ay. wt. 2. 2 kg) were

used.

Cannulation of marginal vein

For intravenous administration of compounds and collection of blood the

marginal ear vein of the rabbit was cannulated as follows: The animal was

first restrained by being wrapped in a thick cotton cloth (100 x 100 cm) so that only

the head and the ears were exposed. The hairs around the vein were shaved

and xylene was used to dilate the blood vessel. A puncture was made in the vein

using a Gillette Scimitar disposable hypodermic needle ( 19 g x 5 cm ) and the

cannula inserted 2 - 3 cm into the vessel. The cannula was held in position

with an adhesive plaster.

The cannula was made from a 20 cm length of polythene tubing (Portex

Pp-60) which has been heated in an oven at 100°C and drawn out at one end. The

narrow end was cut at an angle to provide a sharp point. The wider end was

attached to a hypodermic needle (21 g x 3. 8 cm) and syringe containing heparinized

saline (500 I. U. /ml of heparin) (Paine s and Byrne Ltd. , Greenford, U.K.).

This enabled the cannula to be kept filled with saline medium without clotting.

After cannulation, the cannula was clamped with small (12 cm ) pair of artery

forceps to prevent the outflow of blood. The injection needle and the syringe

were disconnected from the cannula and replaced by a 2 cm length of a suitable

wire to act as a plug for the cannula. The artery forceps were then released.

Administration of compounds

After withdrawal of a blank blood sample, the [14q-arylacetic acids

69 ptmol/kg ; 6 teCi/kg ) were injected through the cannula.

73

Blood Analysis for 14C content

After administration of the compounds, blood samples (2 ml) were then

withdrawn at different time intervals up to 3 h. The cannula was kept full of

heparinized saline throughout the experiment. Blood samples (0.1 ml) were

counted in a dioxan based scintillation fluid for the 14C content. Blood samples

were also examined for the presence of metabolites as follows: blood (1 ml) was

mixed with acetone (1 ml) and centrifuged at 200 x g for 5 min. The supernatant

(100 pl) was then subjected to thin layer chromatography using solvents D and F.

In Vitro Studies

Preparation of tissue subfractions

Female Wistar albino rats , weighing 180-220 g, were killed with a blow

on the head, their livers excised and placed on ice. After weighing quickly, the

livers were homogenised in 3 volumes of ice-cold 0.25 M sucrose (for isolation

of mitochondria) or in ice-cold 1.15% w/v KCI (for isolation of microsomes)

using an Ultra-Turrax homogeniser. This homogenate was centrifuged for 10

min at 700 x g at 4°, using an MSE High -Speed 25 centrifuge, to remove nuclei

and cell debris.

Mitochondria

A 700 x g supernatant of liver in 0.25 M sucrose, was layered over an

equal volume of ice-cold 0.34M sucrose, and this centrifuged at 5000 x g for 10

min at 4°, using an MSE High Speed 25 centrifuge. The crude mitochondrial pellet

so obtained was redispersed in 1.15% KCI and centrifuged again using the above

conditions. This washed mitochondrial pellet was resuspended in 1.15% KC1 so that

1 ml contained mitochondria from 250 mg of liver.

Microsomes

A 700 x g supernatant, in 1.15% KCI was centrifuged at 5000 x g for 15

min at 4°. The supernatant from this was then centrifuged at 100,000 x g for

74 60 min at 4° in an MSE Superspeed 40 centrifuge, the pellet so obtained redispersed

in 1.15% KCl and centrifuged again under the same conditions as before. The

washed microsomal pellet so obtained was resuspended in 1.15% KCl so that 1 ml

contained microsomes from 250 mg of liver.

Binding studies

In order to distinguish between real binding of drugs to subcellular

fractions and non-specific extrapment, the approach of Goldstein, Lowney and

Pal (1971) was used. Two sets of tubes were set up, one of which (A) contained

14 only the . Cl-arylacetic acid and the other of which (B) was incubated with a

large excess of unlabelled acid prior to addition of the _14. Cl-arylacetic acid. In

the first set of tubes, A, the [14C]-arylacetic acid will participate in all possible

14 kinds of interaction with the organelles. In the second set, B, the _ Cl-arylacetic

acid will be blocked from entering saturable sites, but, their non-specific inter-

actions (trapped and dissolved) will be unaffected. The difference, A - B, there

fore measures the saturable binding of the arylacetic acid to the organelles.

In this experiment, the incubation, A, contained mitochondria or

microsomes (equivalent of 1 g liver), 0.2M Tris-HC1 buffer, pH 7.4 (2. ml)

together with the _14C]-arylacetic acid (10 nmol ; 50,000 d. p. m.) in final volume

of 4 ml and were incubated for 15 min at 37° with shaking.

The incubation B contained mitochondria or microsomes as above with

unlabelled arylacetic acid (1 p.mol) in a volume of 4 ml . After incubation at 37°

_ for 15 min with shaking, the 14C]-arylacetic acid (10 nmol) was added and then

incubated for a further 15 min at 37°.

After incubation the organelles were isolated by centrifugation as pre-

viously described and the supernatants removed and their [14C] content determined.

The incubation tubes were allowed to drain on tissue paper prior to resuspending

the pellet in 1.15% KCl (2 ml) for measurement of [14C] content.

75

Protein determination

The protein content of subcellular fractions were determined by the

Biuret method.

Enzyme Affinity

Glycine conjugation

The conjugation of the arylacetic acids with glycine was measured in

the rat liver homogenates and in an isolated mitochondria by an adaption of the

method of Caldwell et al (1976) for hippuric acid formation in human liver.

A typical incubation consisted of 14C-labelled acid ( 10nmol, 50,000 d. p. m. ),

glycine (60 mmol), MgC12 (3 pmol), glutathione ( 20 pmol) and mitochondria or

a whole liver homogenate (equivalent to 40m; of liver) in 0.2 M-Tris/HCl

buffer, pH 8.4 (1 ml ). After incubation for 30 min at 37° the reaction was

stopped by the addition of acetone ( 1 ml ) containing the appropriate carrier acid

( 5 mg) and its glycine conjugate (5 mg ). After centrifuging to remove the

protein (0. 05 ml) was chromatographed on thin-layer plates, and developed in

solvents E and F. The developed plate was viewed beneath U. V. light, and the

dark-quenching areas corresponding to the free acid and its glycine conjugate

were scraped from the plate, and the associated 14C was determined by liquid-

scintillation counting. For each compound optimal substrate and cofactor

conditions were determined.

Glucuronic acid conjugation

The liver microsomal conjugation with glucuronic acid of the arylacetic

acids was determined by the method of Dingell et al (1974). Typical incubation

mixture (3 ml) contained the [14q-arylacetic acid (10-500 nmol), UDPGA (1pmol)

and microsomes in 1. 15% KCI (corresponding to 100 mg liver ; 1. 5 mg protein

per incubation) in 0.2M phosphate buffer (pH as shown in results). These

76

mixtures were incubated in 50 ml ground-glass stoppered tubes at 37° for

various time intervals with shaking, and the reaction then stopped by adjusting

pH to 1.5 with 2M-HC1. The conjugation of the acids was then assayed by

solvent extraction of the free acid, leaving the glucuronide conjugates in the

aqueous phase.

With hydratropic and phenylacetic acids, extraction was with water-

saturated ether (2 x 15 ml) while with 1-naphthyl- and diphenyl-acetic acids,

water-saturated toluene ( 1x15 ml) was used. After centrifuging to separate

the phases, aliquots (2 ml) of each were counted for 14C as described earlier.

Reverse isotope dilution experiments

1 -Naphthylacetic acid, 1-naphthylacetyl-glycine and -glutamine,

diphenylacetic acid, diphenylacetyl-glycine and -glutamine, hydratropoyl-

glycine and -glutamine were all processed in the same way. 1 g of the appro-

priate acid, dissolved in aqueous NaHCO3 was added to urine samples containing

about 0. 5 - 1 /Xi of 14C. The urine was adjusted to pH 1 with 2N-HCl and the

precipitate that formed, filtered and recrystallised from water in the case of

1 --naphthylacetic acid m. p. 131; 1 -naphthylacetylglutamine m. p. 186°, hydra -

tropoyl-glycine m. p. 103° and -glutamine in. p. 139°, diphenylacetic acid m. p.

147° and diphenylacetylglutamine m. p. 149°, and from aqueous ethanol in the case

of 1 -naphthylacetylglycine m. p. 149°, and diphenylacetylglycine m. p. 142° until

constant specific activity was achieved.

In the case of 1-naphthylacetyltaurine, diphenylacetyltaurine and hydra-

tropoyltaurine, ig of the appropriate compound was added to urine samples and

the whole reduced to dryness on a rotary evaporator. The residue was treated

with hot methanol (5m1) and filtered. For diphenylacetyltaurine, the m3thanolic

extract was allowed to cool and the crystals that separated were recrystallised

77

from methanol to constant specific activity, m. p. 200-205°. In the case of

1-naphthylacetyltaurine the methanolic extract was treated with acetone (3 ml)

and the white crystals that separated were recrystallised from methanol/acetone

(2 : 1 v/v) to constant specific activity, m. p. 229°. The methanolic extract

for hydratropoyltaurine, was further reduced to dryness and the residue was

recrystallised from methyl ethyl ketone to constant specific activity m. p. 89°.

Hydratropic acid was treated differently. 1 g of the acid was added

to urine samples and the whole reduced to dryness. The residue was taken up

in ethylacetate (5 ml) and filtered. The filtrate was added to benzylamine (1.5g)

in boiling ethanol (25 ml) and allowed to boil for 5 min. Solvents were removed

using a rotary evaporator. The crystals of benzylamine salt of hydratropic acid

that formed were triturated with petroleum ether (b. p. 40-60°) filtered and

recrystallised from petroleum ether (b. p. 100-120°) to constant specific

activity, m. p. 88°.

78

CHAPTER THREE

Metabolism of 1 - Naphthylacetic Acid

Contents

Pages

Identification of Urinary Metabolites of 1-Naphthylacetic acid

79

Man 79 Rhesus and cynomolgus monkey 81 Capuchin, marmoset and squirrel monkey 84 Bushbaby 84 Cat 89 Rabbit and rat 91 Fruit bat 91

Influence of Dose on the Pattern of Metabolism and Excretion of 1-Naphthylacetic Acid in the Rat 94

Urine 94 Bile 94

Results

94

Man 95 Rhesus monkey 95 Cynomolgus monkey 95 Squirrel monkey 95 Capuchin 96 Marmoset 96 Bushbaby 96 Cat 96 Rabbit 96 Rat 97 Fruit bat 97

Influence of dose on the pattern of metabolism and excretion of 1-Naphthylacetic acid in the rat 97

Urinary excretion products 97 Biliary excretion products 100

Discus sion 100

79

Tne Metabolism of 1-Naphthylacetic Acid

The metabolic fate of 1-naphthylacetic acid has been studied in man,

6 sub-human primates and 4 non-primates . Additionally the influence of dose

on the pattern of metabolism and excretion of this acid has been studied in the

rat.

Identification of urinary metabolites of 1-naphthylacetic acid

The urines of animals dosed with 1-naphthylacetic acid were subjected to

paper and thin-layer chromatography as described in Chapter 2.

Man

Paper chromatography followed by radiochromatogram scanning of

human urine samples using solvent A revealed the presence of a large 14C peak

at Rf 0. 09 (Fig. 3:1). This gave a positive reaction with naphthoresorcinol

and disappeared when the urine was treated with p-glucuronidase or N-NaOH, to

be replaced by a new 14C peak at R

f 0. 84, corresponding to 1-naphthylacetic

acid. A portion of the glucuronide gum obtained from rabbits fed with 1-naphthyl-

acetic acid also showed on chromatography as above a naphthoresorcinol positive

spot at Rf 0. 09 which on treatment with p-glucuronidase or N-NaOH afforded

1-naphthylacetic acid (see chapter 2). The radioactive component found in the

urine with Rf 0. 09 was therefore identified as 1-naphthylacetylglucuronide.

There was also a small 14C peak at Rf 0.14 corresponding to 1-naphthylacetyl-

taurine. Similarly, thin-layer chromatography in solvent D showed the presence

on scanning of a single large 14C peak at the origin which gave a positive

reaction with naphthoresorcinol and was labile to p-glucuronidase or N-NaOH

treatment to afford 1-naphthylacetic acid (R1 0. 82). Reverse isotope dilution also

confirmed the presence of 1-naphthylacetyltaurine, and the presence of 1 -naphthyl - acid

acetic/was confirmed after the treatment of urine samples with p-glucuronidase or

N-Na0H.

0 S F.

2 3

after mild alkaline hydrolysis

S. F.

80

Fig. 3.1 Radiochromatogram scan of urine from man dosed orally

[14C]-1-naphthylacetic acid (5 mg ; 5 tiCi) and chromatographed

on Whatman No. 1 paper in solvent system A.

1. 1-Naphthylacetylglucuronide; 2. 1-naphthylacetyltaurine;

3. 1-naphthylacetic acid; 0 = origin; S. F. = solvent front

81

Rhesus and cynomolgus monkeys

Scans of chromatograms of rhesus monkey urine developed on Whatman

No. 1 paper in solvent A showed a single broad peak at Rf 0.09 and a minor peak

at Rf 0. 84 (Fig. 3.2). The major peak at Rf

0.09 gave a positive reaction with

naphthoresorcinol and disappeared when the urine was treated with g glucuronidase

or N-NaOH giving a 14C peak at Rf

0.85 which corresponded to 1-naphthylacetic

acid. The 14

C peak at Rf 0.09 was therefore identified as 1-naphthylacetyl-

glucuronide. There was a small 14C peak at R 0.14 corresponding to 1-

naphthylacetyltaurine, and the small peak in untreated urine at Rf

0. 84 was

identified as 1-naphthylacetic acid. Chromatography on thin-layer using solvent

D similarly revealed the main 14

C compound present to be 1-naphthylacetyl-

glucuronide together with small amounts of 1-naphthylacetyltaurine and 1-naphthyl-

acetic acid. The presence of these compounds was also confirmed by reverse

isotope dilution.

Chromatograms of cynomologus monkey urine developed on Whatman No.1

paper in solvent A showed on radiochromatogram scanning two major 14C peaks

(Fig. 3:3) ; at Rf values 0.09 and 0.84. The peak at Rf 0.09 gave positive

reaction with naphthoresorcinol and disappeared on treatment of urine samples

with j3-glucuronidase or N-NaOH and this was accompanied by an increase in

the size of the peak at Rf 0.84 due to 1-naphthylacetic acid. The peak at Rf

0.09 was therefore identified as 1-naphthylacetylglucuronide. There were also

two small peaks at Rf 0.14 and 0.26 corresponding to 1-naphthylacetyltaurine

and 1-naphthylacetylglutamine respectively.

14C scans on thin-layer chromatograms of cynomolgus monkey urine

developed in solvent D showed a large 14

C peak at the origin due to 1-naphthyl-

acetylglucuronide, and a second large 14C peak due to the unchanged acid at

after mild alkaline hydrolysis

3 S. F.

S. F.

82

Fig. 3.2 Radiochromatogram scan of urine from rhesus monkey

dosed intramuscularly with [14 C]-1-naphthylacetic acid

(100 mg /kg ; 17.2 pCi) and chromatographed on Whatman

NO. 1 paper in solvent system A.

1. 1 -Naphthylacetylglucuronide; 2, 1 -naphthylacetyltaurine;

3. 1-naphthylacetie acid; 0 = origin, S. F. = solvent front

0 S. F.

0 S. F.

after mild alkaline

hydrolysis 2 3 4

83

Fig. 3. 3 Radiochromatogram scan of urine from cynomolgus monkey

dosed intramuscularly with [14 C]-1-naphthylacetic acid

(100 mg/kg ; 21.2 pCi) and chromatographed on Whatman

No. 1paper in solvent system A.

1. 1 -Naphthy I acetylglucuronide ; 2, 1 -naphthylacetyltaurine ;

3. 1-naphthylacetylglutamine; 4. 1-naphthylacetic acid; 0 = origin ;

S. F. = solvent front

84

Rf 0. 86. Two minor

14C peaks appeared at R

f values 0.18 and 0. 38

corresponding to 1-naphthylacetyltaurine and-glutamine respectively. Reverse

isotope dilution also confirmed the presence of 1 -naphthylacetic acid, 1-

naphthylacetyl-taurine and - glutamine.

Capuchin, marmoset and squirrel monkey

Chromatography of urine samples on Whatman No. 1 paper using solvent

A revealed on radiochromatogram scanning the presence of five 14C peaks of Rf

values O. 09, 0.16, 0.25, 0.48 and 0. 86 (Figs. 3:4, 3:5 and 3:6). The 14

C

peak at 0. 09 gave a positive reaction with naphthoresorcinol and disappeared

when urine was treated with P-glucuronidase or N-NaOH to be replaced by a

larger 14

C peak at Rf 0.86, which corresponded to 1-naphthylacetic acid. This

peak at Rf 0.09 was therefore identified as 1-naphthylacetylglucuronide. The

14C peak at Rf 0.48 gave a positive reaction with the 4-dimethylaminobenzaldehyde

reagent and was identified as 1-naphthylacetylglycine. The 14C peaks of Rf

values, 0.16, and 0.25 corresponded to 1 -naphthylacetyl-taurine and-glutamine

respectively.

Similarly, chromatography on thin-layers using solvent D showed on

radiochromatogram scanning the presence of five 14C peaks, one at the origin

and others at Rf 0.18' 0. 40, 0.63 and 0.86 which corresponded to 1-naphthyl-

acetyl- glucuronide, taurine, glutamine, glycine and 1-naphthylacetic acid

respectively. The 14C peak at the origin disappeared from urine samples

treated with [3-glucuronidase or N-NaOH as above and it gave a positive

naphthoresorcinol reaction. The presence of 1 -naphthylacetyl- taurine, glutamine,

glycine and 1-naphthylacetic acid was also confirmed by reverse isotope dilution.

Bushbaby

14C scans of paper chromatograms of bushbaby urine developed with

solvent A showed three major radioactive peaks of R 0.08, 0.14 and 0. 44

0 S F.

0 S. F.

after mild alkaline hydrolysis

2 3 4 5

85

Fig. 3.4 Radiochromatogram scan of urine from capuchin monkey

dosed intramuscularly with [14C]-naphthylacetic acid

(100 mg/kg ; 8. 5 pCi) and chromatographed on Whatman

No.1 in solvent system A.

1. 1 -Naphthylacetylglucuronide; 2. 1-naphthylacetyltaurine

3. 1 -naphthylacetylglutamine; 4. 1-naphthylacetylglycine

5. 1-naphthylacetia acid . 0 = origin; S. F. = solvent front

0 S. F.

after mild alkaline hydrolysis

86

Fig. 3. 5

Radiochromatogram scan of urine from squirrel monkey

dosed intramuscularly with [14C]-1 -naphthylacetic acid

(100 mg/kg ; 5. 9 12 Ci) and chromatographed on Whatman

No.1 paper in solvent system A.

1. 1 -Naphthylacetylglucuronide ; 2. 1 -naphthylacetyltaurine ;

3. 1 -naphthylacetylglutamine; 4. 1 -naphthylacetylglycine

5. 1 -naphthylacetic acid. 0 = origin ; S. F. = solvent front

0 S. F.

after mild alkaline

hydrolysis

87

Fig. 3.6 Radiochromatogram scan of urine from marmoset

dosed intramuscularly with [14 C]-1-naphthylacetic

acid (100 mg/kg ; 5.9 iCi) and chromatographed on Whatman

No. 1 paper in solvent system A.

1. 1 -Naphthylacetylglucuronice; 2. 1-n.aphthylacetyltaurine

3. 1 -naphthylacetylglutaraine ;. 4. 1-naohthylacetylglycine

5. 1-naphthylacetic acid. 0 = origin ; S. F. solvent front

88

0

S. F.

0 Fig. 3. 7

10 cm 20 S F. Radiochromatogram scan and histogram of urine from bushbaby dosed intramuscularly with [14C]-1-naphthylacetic acid (100 mg/kg; 23. 6 p.Ci) and chromatographed on Whatman No. 1 paper in solvent system A.

1. 1- Naphthylacetylglucuronide ; 2. 1 -naphthylacetyltaurine

3. 1-naphthylacetylglycine ; 4. 1-naphthylacetic acid. 0 = origin ;

S. F. = solvent front

89

(Fig. 3:7) corresponding to 1-naphthylacetyl-glucuronide, -taurine and

-glycine respectively. The peak at Rf 0. 08 gave a positive naphthoresorcinol

reaction and disappeared from urine treated with 13-glucuronidase or N-NaOH

and was replaced by an enlarged peak at Rf 0. 84 corresponding to 1-naphthyl-

acetic acid. The peak at Rf 0. 08 was therefore identified as 1-naphthylacetyl-

glucuronide. The peak at Rf 0. 44 gave a positive reaction for a glycine

conjugate with 4-dimethylaminobenzaldehyde reagent and the peak at Rf 0.14

corresponded to 1-naphthylacetyltaurine. In addition to these three conjugates

urine also contained a small amount of 1-naphthylacetic acid as revealed by

the appearance of a minor 14

C peak at Rf 0. 84. Scans of thin-layer chromato-

grams developed in solvent D also revealed three major 14C peaks, Rf

0.0,

0.16, 0. 62 and a minor peak at 0. 84 corresponding to 1-naphthylacetyl-

glucuronide, -taurine, and-glycine and 1-naphthylacetic acid respectively.

Reverse isotope dilution also confirmed the presence of 1-naphthylacetyl-taurine

and - glycine and 1-naphthylacetic acid.

Cat

Radiochromatogram scanning of paper chromatograms of cat urine

developed in solvent A showed two major radioactive peaks of Rf

0.14 and 0.44

(Fig. 3:8) which corresponded to 1 -naphthylacetyl-taurine and -glycine respectively.

There also appeared a very minor peak at Rf 0. 84 corresponding to 1 -naphthylacetic

acid. Similarly, scans of thin-layer chromatograms developed with solvent D

revealed two major peaks of Rf

0. 15 and 0.60 corresponding 1-naphthylacetyl-

taurine and -glycine respectively and a minor peak at Rf 0. 85 due to unchanged

compound. Reverse isotope dilution also confirmed the presence of 1 -naphthyl-

acetyl-taurine and -glycine and 1-naphthylacetic acid.

90

0 S. F.

10 cm 20

S. F.

Fig. 3.8 Radiochromatogram scan and histogram of urine from cat dosed intraperitoneally with [14C]-1-naphthylacetic acid (100 mg/kg ; 14.8 pCi) and chromatographed on Whatman No. 1 paper in solvent system A.

1. 1 -Naphthylacetyltaurine; 2. 1 -naphthylacetylglycine

3. 1-naphthylacetic acid ; 0 = origin; S. F. = solvent front

91

Rabbit and rat

Radiochromatogram scans of rabbit and rat urine samples chromato-

graphed on paper and developed in solvent A revealed the presence of a large

14C peak at Rf 0. 09 and two smaller 14C peaks at Rf 0.46 and 0. 84 respectively

(Figs. 3:9, 3:10). The peak at 0. 09 gave a positive reaction with naphthoresorcinol

and disappeared when urine samples were treated with 13-glucuronidase or N-NaOH

Its disappearance was accompanied by an increase in the size of the peak at Rf

0. 84 which corresponded to 1-naphthylacetic acid. The peak at Rf 0.46 gave an

orange colour with the 4-dimethylaminobenzaldehyde reagent and was identified

as 1-naphthylacetylglycine. Similarly, scans of chromatograms developed in

solvent D showed a single large 14C peak at the origin and two smaller peaks at

Rf 0. 60 and 0. 86. As above the large peak at the origin disappeared when the

urine was treated with g-glucuronidase or N-NaOH to be replaced by an enlarged

peak at Rf 0. 86 corresponding to 1-naphthylacetic acid. The peaks at Rf 0.60

and 0. 86 were identified as 1-naphthylacetylglycine and 1-naphthylacetic acid and

there presence was also confirmed by reverse isotope dilution.

Fruit Bat

Paper chromatography of fruit bat urine followed by radiochromatogram

scanning showed the presence of a large 14C peak at R

f 0. 09 corresponding to

1-naphthylacetylglucuronide and a small peak at 111 0. 84 due to the unchanged

acid (Fig. 3:10). The peak at Rf 0. 09 gave a positive reaction with naphthore-

sorcinol and disappeared when urine samples were treated with (3-glucuronidase or

N-NaOH and this was accompanied by a large increase in the size of the peak at

Rf 0. 84 due to 1 -naphthylacetic acid. Chromatography on thin-layer in solvent D

showed the presence of 1-naphthylacetylglucuronide together with a small amount

of the unchanged acid.

92

Fig. 3. 9 Radiochromatogram scan and histogram of urine from

rat dosed intraperitoneally with [14C]-1-naphthylacetic

acid (100 mg/kg ; 5. 9 MCi) and chromatographed on Whatman

No. 1 paper in solvent system A.

1. 1-Naphtylacetylglucuronide; 2. 1-naphthylacetylglycine

3. 1-naphthylacetic acid 0 = origin; S.F. = solvent front

Fruit bat (dosed intraperitoneally, 10 ACi)

1

S. F. 0

93

Rabbit (dosed orally, 22. 5 ;lei)

2

S. F.

Fig. 3. 10 Radiochromatogram of urine from rabbit and fruit bat 14 _

dosed with [ C]-1-naplithylacetic acid (100 mg/kg )

and chromatographed on Whatman No. 1 paper in

solvent system A.

1. 1-Naphthylacetylglucuronide; 2. 1-naphthylacetylglycine;

3. 1-naphthylacetic acid; 0 = origin; S. F. = solvent front

94

Influence of dose on the pattern of metabolism and excretion of 1-naphthylacetic

acid in the rat

Bile and urine sample were collected over the first 3 h after dosing from

bile-duct cannulated rats dosed intraperitoneally with [14C1-1-naphthylacetic

acid (5 - 500 mg/kg) and were analysed for 14C and metabolites as described

in Chapter 2.

Urine - The thin-layer chromatograms of the urine samples developed in

solvent D revealed the presence of two major 14C peaks at Rf 0. 0 and 0. 62 and

a minor peak at Rf 0. 86. The peak at the origin gave positive reaction with

naphthoresorcinol reagent and disappeared when the urine samples were treated

with /3-glucuronidase or N-NaOH and this was accompanied by an increase in

the size of the peak at Rf 0. 86 corresponding to 1-naphthylacetic acid. The peak

at the origin was therefore identified as 1-naphthylacetylglucuronide. The 14C

peak at 0.62 gave an orange colour with 4-dimethylaminobenzaldehyde and was

identified as 1-naphthylacetylglycine. Thin-layer chromatograms developed

in solvent E confirmed the presence of 1-naphthylacetyl-glucuronide and -glycine

with very small amounts of unchanged acid.

Bile - The thin-layer chromatograms of bile samples developed in solvent D

revealed the presence of one major peak at the origin and a minor peak at Rf 0. 86.

The peak at the origin gave a positive reaction with naphthoresorcinol reagent

and disappeared on treatment of the bile samples with 13-glucuronidase or N-NaOH

and this was accompanied by an increase in the size of the peak at Rf 0. 86 due

to 1-naphthylacetic acid. The peak at the origin was therefore identified as

1 -naphthylacetylglucuronide.

Results

The quantitative and qualitative aspects of the excretion of 1-naphthylacetic

95

acid and its metabolites by the 11 species studied are shown in Table 3:1 and

those in rats at various dose levels are shown in Table 3:2.

Man - The two human subjects excreted in the urine about 97% of the radio-

activity in 24 h after an oral dose of 5 mg of [14

CJ-1-naphthylacetic acid. The

major excretion product was 1-naphthylacetylglucuronide (88 and 94% of the

dose respectively for the two subjects) with small amounts (7.8 and 3.4%) of

1-naphthylacetyltaurine. The glycine and glutamine conjugates of the acid

were not detected.

Rhesus monkey - The two rhesus monkeys excreted almost all the radioactivity

(99 and 100%) in 24 h after an intramuscular dose of 100 mg/kg of [140]-1-naphthyl-

acetic acid. The major excretion product was 1-naphthylacetylglucuronide

(83 and 94% of the dose respectively) with small amounts of 1-naphthylacetic acid

(12 and 6. 0%) and 1-naphthylacetyltaurine (3. 5 and 1%) but there were no

glycine or glutamine conjugate detected.

Cynomolgus monkey - The single cynomolgus monkey examined excreted 70% of

an injected dose (100 mg/kg, intramuscularly) of [14C]-1-naphthylacetic acid in

the urine within 24 h. The major excretion products were unchanged 1-naphthyl-

acetic acid (35% of the dose) and 1-naphthylacetylglucuronide (29%) with small

amounts of glutamine (2.6%) and taurine (3.5%) conjugates of the acid but the

glycine conjugate was not detected.

Squirrel monkey - The two squirrel monkeys examined excreted 39 and 22% of the

radioactivity in the urine within 24 h, after an intramuscular dose of 100 mg /kg

.14 f C1-1-naphthylacetic acid. The main radioactive compounds in the urine

were unchanged drug (13 and 7. 3% of the dose respectively), and glycine (7. 5 and

5.0), tauririe (7.5 and 5. 0), glucuronic acid (9.4 and 3.7%) and glutamine (2. 0 and

0.9%) conjugates of 1-naphthylacetic acid.

96

Capuchin The two Capuchin xamined excreted in the urine, 35 and 44% of

the injected dose (100 mg/kg, intramuscularly) of [14C]-1-naphthylacetic acid.

The major excretion products were the unchanged compound (16 and 4. 5% of

the dose respectively) taurine (14 and 15%) and glucuronic acid (2.2 and 13%)

conjugates with small amounts of glycine (2.2 and 8.8%) and glutamine (0.9 and

3.0%) conjugates of 1-naphthylacetic acid.

Marmoset - The single marmoset examined excreted 70% of an injected dose

(100 mg/kg, intramuscularly) of [14C]-1-naphthylacetic acid in the urine within

24 h. The major excretion products were 1-naphthylacetylglucuronide (49% of

the dose) 1-naphthylacetyltaurine (9.0%) and the unchanged compound (5.4%) the

with small amounts of/glutamine (1.6%) and glycine (1. 8%) conjugates of

1-naphthylacetic acid.

Bushbaby - The two bushbabies examined excreted 41 and 26% of the dose of

[14 C]-1-naphthylacetic acid (100 mg/kg, intramuscularly) respectively in the

urine in 48 h. The major excretion products were 1-naphthylacetylglycine

(21 and 4. 0% of the dose respectively), 1 -naphthylacetylglucuronide (11 and 3. 9%),

1 -naphthylacetyltaurine (4. 6 and 7. 5%) and some unchanged 1-naphthylacetic acid

(3.9 and 10.5%) but no glutamine conjugate of 1-naphthylacetic acid was detected.

Cat - Cats injected intraperitoneally with [14 C]-1-naphthylacetic acid (100 mg/kg)

excreted about 62% of the dose of radioactivity in the urine in 24 h. The major

excretion products were glycine (37% of dose) and taurine (25%) conjugates with

a small amount of the unchanged compound (1. 8%). 1- Naphthylacetylglucuronide

was not detected.

Rabbit - Rabbits dosed orally with [14

C]-1-naphthylacetic acid (100 mg/kg)

excreted about 81% of the dose of radioactivity in the urine in 24 h. The major

excretion product was 1-naphthylacetylglucuronide (71% of the dose) with small

97

amounts of 1 -naphthylacetylglycine (4. 5%) and 1-naphthylacetic acid (6.2%),

but no taurine conjugate of 1-naphthylacetic acid was detected.

14 . Rat - Rats injected intraperitoneally with [ C1-1- naphthylacetic acid (100 mg/kg)

excreted about 64% of the dose of radioactivity in the urine in 24 h. The major

excretion products were 1-naphthylacetylglucuronide (33% of the dose), 1-naphthyl-

acetylglycine (15%) and 1--naphthylacetic acid (17%). There was no taurine

conjugate detected.

In the experiment for the collection of expired carbon dioxide, there was

no radioactivity detected in the entrapped carbon dioxide indicating therefore,

that there was no metabolism by decarboxylation.

Fruit bat - The two fruit bats examined excreted 69 and 58% of the dose of

[14C]-1-naphthyIacetic acid (100 mg/kg, intraperitoneally) respectively in the

urine in 24 h. The major excretion product was 1-naphthylacetylglucuronide (62

and 56% of the dose respectively) with small amounts of the unchanged compound

(6.6 and 2. 0%). Neither the glycine nor the taurine conjugate was detected.

Influence of dose on the pattern of metabolism and excretion of 1-naplathylacetic

acid in the rat

Bile duct °annulated rats were injected intraperitoneally with [14C]-1-

naphthylacetic acid (5 to 500 mg/kg). From the dose level of 5 to 500 mg/kg,

the total amount of the radioactivity excreted in the urine in 3 h fell from 32 to 0.6%,

while the amount excreted in the bile fell from 37 to 10% of the dose respectively.

Urinary excretion products 1-Naphthylacetylglycine formed 88% of the excretion

products at 5 mg/kg dose level, but its proportion of the products fell to 11% at

the 500 mg/kg dose level. 1-Naphthylacetylglucuronide on the other hand formed

the minor metabolite at the lowest dose level (11%) but was the major metabolite

(88%) at the highest dose level. The unchanged acid formed a very small fraction

Species (NO. & Sex)

Primates

Man* (2M)

Rhesus monkey (1M,1F)

Cynomolgus monkey (1F)

Squirrel monkey (2F)

Capuchin (2F)

Marmoset (1M)

Busbaby (1M,1F)

Non-Primates

Cat (2F,1M)

Rabbit (3F)

Rat (3F)

Fruit bat (11', 1M)

Genus and Species Route of administration

Homo sapiens p. o.

Macaca mulatta i. m.

Macaca fascicularis (irus) i.m.

Saimiri sciureus i. m.

Cebus albifrons i. m.

Sanguinus oedipus i. m.

Galago crassicaudatus . i. m.

Fells cattus i. p.

Lepus caniculus p. o.

Rattus norvegicus i. p.

Pteropus giganteus i. p.

Dose of 14C /Xi/animal

14C excreted % of dose

5. 0 17.2

21.2

96, 97 99, 100

70

5. 9 39, 22

8. 5 35, 44

5.9 70

23.6 41, 26

14.8 62(57-69)

22. 5 81(76-87)

5. 9 64(59-70)

10.0 69, 58

* Dose 5mg + 48h excretion

Table 3 : 1

Conjugates of [14C1-1-naphthylacetic acid in various species

14 [ C]-1-naphthylacetic acid (100 mg/kg) dissolved in the appropriate amount of N-NaOH was administered as described in chapter two. The urine was collected for 2 days and the 0-24 h urine analysed for metabolites. Results are means for three animals with ranges in parentheses. Where only one or two animals were used individual values are given.

Amount of [14C] -1 -naphthylacetic acid excreted conjugated with various compounds in 24 h as % dose

Unconjugated Glutamine Glycine Taurine Glucuronic Acid

- - 7.8, 3.4 88, 94 12, 6 - - 3.5, 1 83, 94

35 2.6 - 3.5 29 13, 7.3 2.0, 0.9 7.5, 5.0 7.5, 5.0 9.4, 3.7 16, 4.5 0.9, 3.0 2.2, 8.8 14, 15 2. 2, 13

5.4 1.6 1.8 9.0 49 3.9, 10.5 - 21, 4 4.6, 7.5 11, 3.9

1. 8(1. 0-2. 2) - 37(29-48) 25(17-39) 6. 2(3. 6-10. 3) - - 4. 5(4. 0-5. 2) - 71(68-73) 17(15-18) - 15(10-21) - 33(31-34) 6.6, 2.0 - = - 62, 56

co

Table 3 :2

Influence of dose on the pattern of metabolism and excretion of 1-naphthylacetic acid it the rat

Bile-duct cannulated female rats were injected intraperit one ally with [14C I -1 -naphthylacetic acid (dissolved in the calculated amount of N-NaOH and diluted with water) and the bile collected for 3 h and urine removed from the bladder. Bile and urine were analysed for 14C and metabolites-as described in the text.

Urine Bile 14 I

% C excreted conjugated with: Dose (mg/kg) % dose excreted Unchanged Glycine Glucuronic

Acid

5 32 (31-33) 1.0 (0.8-1.2) 88 (84-91) 11 (8.2-15)

25 30 (27-37) 1.0 (0.9-1.1) 55 (49-60) 44 (38-49)

50 14 (11-16) 2.0 (1.8-2.1) 48 (43-52) 52 (47-56)

250 12 (8.2-13 5.1 (2.3-6.2) 12 (7.4-20) 85 (78-90)

500 0.6 (0.5-1.0) 2.4 (1.5-3.1) 11 (8.1-13) 88 (84-91)

r I

% dose

% 14C excreted conjugate

Unchanged Glycine

t

Glucuronic excreted Acid

37 (32-42) 5.3 (3.1-7.0) 4.1 (3.2-5.6) 91 (87-94)

34 (29-39) 5.9 (5.0-8.2) 1.0 (1. 0-1. 0) 92 (90-94)

44 (40-46) 5.1 (5.0-5.3) 1.0 (0.7-1.2) 94 (93-95)

15 (7.5-21) 8.4 (7.0-9.2) 1.0 (0.9-1.1) 91 (90-92)

10 (8.2-13) 16 (15-17) 1.1 (0.8-1.4) 84 (83-85)

100

of the urinary excretion products.

Binary excretion products. Irrespective of the dose level 1-naphthylacetyl-

glucuride was the major excretion product with only traces of 1-naphthylacetyl-

glycine. The unchanged acid rose from 5. 3% at the lowest dose level to 16%

at the highest level.

Discus sion

The metabolic fate of 1-naphthylacetic acid has been studied in eleven

species and the results show that 1-naphthylacetic acid can be conjugated at the

carboxyl group with amino acids and/or glucuronic acid, the pattern of which

varies with species. Table 3:3 summarises the conjugation pattern of 1-naphthyl-

acetic acid in the species studied and it suggests that man and the Old World

monkeys conjugate it mainly with glucuronic acid, the New World monkeys

(except the marmoset) and the bushbaby mainly with amino acids and to a small

extent with glucuronic acid, the cat with amino acids extensively, the rat and

rabbit principally with glucuronic acid and the fruit bat entirely with glucuronic

acid. The amino acids used were mainly glycine and taurine while the very

small amount of glutamine conjugation is restricted to the New World monkeys

and thecynomolgus monkey, a conjugation reaction which James et al (1972 a)

have reported to be restricted to man, New and Old World monkeys.

The taurine conjugation is predominant in the cat and the New World

monkeys and glycine conjugation also in the cat and bushbaby. James et al ,

(1972 a) have also reported that the taurine conjugate of phenylacetic acid is

a major metabolite in the New World monkeys, (except the marmoset), the

prosimians and in the ferrets. The quantitative distribution of the taurine

conjugate of 1-naphthylacetic acid in the species studied although haphazard,

was not detected in the fruit bat, rat and rabbit. Taurine conjugation has

previously been known to occur only with bile acids (see Sobotka 1937; Haselwood 1967),

Table 3:3

Species Variations in the pattern of conjugation of 1-Naphthylacetic acid.

1-naphthylacetic acid conjugates as % of the 14C excreted in the urine

Species Unchanged Glutamine Glycine Taurine Total conjugated with Glucuronic acid

Primates

amino acid

Man - - - 5.8 5.8 95

Rhesus monkey 8. 8 - - 2. 3 2. 3 89

Cynomolgus monkey 50 3. 7 - 5.0 8. 7 41

Squirrel monkey 33 4. 6 21 21 47 21

Capuchin 2. 9 4. 5 4. 2 36 45 17

Marmoset 7.7 2. 3 2.6 13 18 70

Bushbaby 25 - 53, 16 11, 29 64, 45 27, 15

Non-Primates

Cat 1.8 - 59 40 99 0

Rabbit 7. 6 - 5. 5 - 5. 5 87

Rat 26 - 23 <1 24 51

Fruit Bat 6.5 - - - 0 94

102

although a conjugate containing taurine, quinaldylglycyltaurine has been reported

as a metabolite of quinoline-2-carboxylic acid (quinaldic acid) and of 4-hydroxy-

quinoline-2-carboxylic acid (kynurenic acid) in the cat (Kaihara & Price 1961).

The cat formed glycine and taurine conjugates of 1-naphthylacetic acid but did

not form a glucuronic acid conjugate which may indicate a possible defect in

glucuronide formation with this compound, a characteristic which is highly

substrate dependent in the cat (Capel et al. , 1972 ; Millburn, 1974). Some

Carnivores such as the lion, civet and genet form high amounts of glycine and/or

taurine conjugates of 1-naphthylacetic acid but are defective in the glucuronide

formation (French etall, 1974) whereas others such as dog, ferret and hyaena

form glycine, taurine and glucuronic acid conjugates of the acid (Caldwell et al. ,

1975b; Idle et al, 1975). Table 3:4 shows conjugation of three compounds in

some carnivores and cat, and it shows the ability to form Nt-glucuronide of

sulphadimethoxine in the dog but not in the others. It also shows that the dog

and ferret form glucuronic acid conjugate with phenol and not in the rest but all

do form the sulphate conjugate. This finding suggests that the defect in the glucur-

onide formation do occur in some other carnivores other than the cat. The

fruit bat does not form a glycine conjugate with 1-naphthylacetic acid, and the

defect in glycine conjugation with benzoic acid has been reported by Bridges et al,

(1970) and confirmed by Bababunmi et al, (1973) but Ette et al, (1974) have

shown that glycine conjugation with phenylacetic acid does take place in this

species. They therefore suggest that glycine conjugation in the fruit bat is

highly substrate dependent.

The metabolic conjugation pattern discussed so far has been on one dose

level (100 mg/kg, except in the human subjects which received 5 mg). The

effect of dose on the pattern of conjugates in the urine and bile of bile duct-cannulated

rats receiving lrnaphthylacetic acid was shown in Table 3:2. It is clear from the

Table 3:4

Conjugation reactions in some Carnivores (Adapted from Caldwell et al, 1975 b)

Compound Conjugate found % of 24 h excretion in :

in urine DOG

50 18

56 7

25

0 19

FERRET

58 40

6 26 63

27 0

HYAENA

90 tr

46 40 11

0 4

CAT

90 tr

59 0

37

18 0

CIVET

97 0

74 tr 6

66 0

GENET

99 0

70 tr 18

50 0

Phenol

1 -Naphthylacetic acid

Sulphadimethoxine

Sulphate Glucuronide

Glycine Glucuronic acid Taurine

N4-Acetyl

1 N -Glucuronide

1

104

figures for urine that glucuronic acid conjugation is taking over from glycine

conjugation as the dose rises, but for the bile the pattern is practically

unchanged for all the doses. The bile pattern is explained by the fact that

1-naphthylacetylglucuronide (M. W. 362) is extensively excreted in the bile in

the rat whereas 1-naphthylacetylglycine (M. W. 231) is not. At saturation doses

the glucuronidation capacity in 3 h as derived from the data on Table 3:2 is

70nmol/kgThe molecular weight of the glucuronide is within the range 325+ 50

given by Hirom et al (1972) for extensive biliary excretion in the rat whereas

that of the glycine conjugate is below this range and this conjugate is unlikely

to be excreted in the bile in large quantities. This result is consistent

with examples in the literature which show that the size of the dose of a compound

could alter the pattern of its metabolism in so far as a large dose could exhaust the

mechanism by which a small dose is metabolised (Williams, 1959). The

compound could then be partly excreted unchanged or undergo another metabolic

reaction.

- For example it has been known for a considerable time that in man and

the pig, small doses of benzoic acid are metabolised entirely by conjugation

with glycine, but with large doses glucuronic acid conjugation become predominant

(see Williams, 1959). The cat, as already mentioned has a defective glucuronic

acid conjugation for certain compounds including benzoic acid. Consequently

benzoic acid is more toxic to the cat than most common species of animals

(Bedford & Clarke, 1971, 1972), for the cat conjugates benzoic acid entirely with

glycine which is limited in supply and glucuronic acid conjugation is not available

to take over when large doses of benzoic acid are administered. Another example

is 4-acetamidophenol which produces liver necrosis in high doses. This drug

forms a sulphate, a glucuronide and a mercapturic acid. The toxic effect of high

105

doses appears to be related to the depletion of the liver of glutathione which is

used to form the mercapturic acid. Studies in the hamster, a species most

sensitive to the necrotic effects of 4-acetamidophenol, have shown that at low

doses more of the drug is excreted as sulphur conjugates - sulphate and

mercapturic acid - than glucuronide, but at high doses the reverse is true

(Jollow et al. , 1973).

It is clear from the study of the effect of dose on glycine, sulphate

and glutathione conjugations that these mechanisms have a limited capacity

unless supplemented from outside sources and that when they are exhausted

the glucuronic acid mechanism takes over. It follows from this that with any

compound which is conjugated with glucuronic acid only the pattern of metabolites

will not change very much with dose unless this is excessive.

In general, it seems probable that the extent of metabolic pathways will

change with increase in dose, since it is to be expected that different mechanisms

have different capacities, and furthermore, some compounds can induce their

own metabolism and possibly one pathway more than another. These changes,

however, may also vary with species as in the case of the cat with its defective

glucuronic acid mechanism.

106

CHAPTER FOUR

Metabolism of Diphenylacetic Acid

Contents Page s

Chromatographic identification of urinary metabolites 107

Chromatographic identification of urinary and binary metabolites in the rat. 108

Results

108

Influence of dose on the pattern of metabolism and excretion of diphenylacetic acid in the rat

113

Discussion 113

107

The Metabolism of Diphenylacetic Acid

The metabolic fate of diphenylacetic acid has been examined in man,

6 sub-human primates and 4 non-primate species. Additionally the influence

of dose level in its pattern of metabolism and excretion in the rat has also

been studied.

Chromatographic identification of urinary metabolites

14 _ Urine samples from the 11 species dosed with [ Cidiphenylacetic

acid were subjected to thin-layer chromatography (see Chapter 2, Table 2:2).

Thin-layer chromatography in solvent F of urine samples from man,

rhesus,cynomolgus, squirrel and capuchin monkeys, marmoset, bushbaby, cat,

14 _ rabbit, rat and fruit bat dosed with [ C]diphenylacetic acid revealed on radio-

chromatogram scanning the, presence of two 14

C peaks at R f 0.17 and 0.90

respectively (see Figs. 4:1, 4:2 and 4:3). The peak at R1 0.17 gave a positive

naphthoresorcinol reaction and disappeared from urine samples treated with p

glucuronidase or N-NaOH and this was accompanied by an enlarged peak at Rf

0.92 which corresponded with diphenylacetic acid. A portion of the glucuronide

gum obtained from rabbits fed with diphenylacetic (shown to contain diphenyl -

acetylglucuronide following its conversion to its methyl ester) also showed on

chromatography as above a naphthoresorcinol positive spot at Rf 0.17 which on

treatment with fl-glucuronidase or N NaOH afforded diphenylacetic acid ( see

chapter 2). The radioactive component found in the urine of the species dosed

with [14C]diphenylacetic acid and showing an Rf 0.17 was therefore identified as

the ester glucuronide conjugate of the acid. Similarly, thin-layer chromatography

of urine samples in solvent D followed by radiochromatogram scanning showed

two 14C peaks at Rf 0.10 and 0.88 which corresponded to diphenylacetylglucuronide

and diphenylacetic acid respectively.

108

There were marked species differences in the relative sizes of the

two 14C peaks found on radiochromatogram scanning. Thus, thin-layer

chromatogram of urine samples obtained from the fruit bat, rabbit and rat

and developed in solvent F showed a large peak at Rf

0.17 corresponding to

diphenylacetylglucuronide and only a minor peak at Rf 0. 90 due to the free

acid. In the case of the cynomolgus and squirrel monkeys the size of the two

peaks were about the same whereas for the capuchin and rhesus monkeys the

cat and man the peak at Rf 0.17 was about twice the size of that at Rf 0.90.

In the case of the bushbaby, however, there occurred a major peak at Rf 0. 90

with minor one at Rf 0.17.

Chromatographic identification of urinary and biliary metabolites in the rat

3 h urine and bile samples from bile-duct cannulated rats dose intra-

peritoneally with [14C]diphenylacetic acid (5-500 mg/kg) were subjected to

thin-layer chromatography and developed in solvent F followed by radio-

chromatogram scanning. This revealed a major 14C peak at R

f 0.16, and

a very small peak at Rf 0. 92. The peak at 0.16 gave a positive naphthoresorcinol

reaction and disappeared from urine and bile samples treated with fl-glueuronidase

or g-Na0}1 and this was accompanied by an enlarged peak at Rf 0. 92 which

corresponded with diphenylacetic acid. The peak at Rf 0.16 was therefore

identified as diphenylacetylglucuronide.

Similarly chromatograms developed in solvent D revealed the presence

of diphenylacetylglucuronide and diphenylacetic acid.

Results

The quantitative and qualitative aspects of the excretion of diphenylacetic

acid and its metabolites by the 11 species studied are shown in Table 4:1 and those

in rats at various dose levels are shown in Table 4:2.

Table 4:1 shows a low urinary excretion of the radioactivity of the administered

0 S. F.

Man (5 orally)

S. F.

t 1

• t Rhesus Monkey 414 rng/ikgi ! (intramilsc-utarly

• • 2 ; ; 2

•

109

S. F.

Fig. 4:1 Radiochromatogram scans of urine after a dose of [

14C]

diphenylacetic acid. 1. Diphenylacetylglucuronide.

2. Diphenylacetic acid

Solvent system F. 0 = origin. S. F. = solvent front

0 S. F.

• Squirrel monkey

110

Fig 4:2 Radiochrornatogram scans of urine after a dose of [1 C] diphenylacetic acid. 1. Diphenylacetylglucuronide

2. Diphenylacetic acid

Solvent system F. 0 = origin. S. F. = solvent front

111

0

S. F.

Solvent system F. 0 = origin S. F. solvent front

Fig 4:3

Radiochromatogram scans of urine after a dose of [14

C] diphenylacetic acid. 3 . Diphenylacetylglucuronide

2. Diphenylacetic acid

0 S. F.

Fruit bat

(intraperitoneally)

S. F.

Rat . (intraperitoneally)

112

0 S. F.

Rabbit (Orally)

2. Diphenylacetic acid Solvent system F. 0 = origin S. F. = solvent front

Fig 4:4 Radiochromatogram scans of urine after a dose of [

14c]

diphenylacetic acid 1. Diphenylacetylglucuronicle

113

dose of [14C]diphenylacetic acid in 48 h in squirrel monkey (14% of the dose),

capuchin (15 and 21%), bushbaby (23 and 34%) fruit bat (29 and 35%), cat (40%),

rhesus monkey (45 and 49%) and rat (48%) but relatively higher values for

cynomolgus (57%) and marmoset (66%) monkeys. The rabbit excretes about 84%

whereas all the radioactivity is excreted by man. The major excretion products

are diphenylacetylglucuronide and diphenylacetic acid but the quantities varied

with species. Diphenylacetylglucuronide is the major excretion product in

most of the species except in the cynomolgus monkey and bushbaby which have

the unchanged acid as the major excretion product. There are traces of the glycine

conjugate in the marmoset, bushbaby, cat and rat, and also of the taurine conjugate

in the cat as shown by reverse isotope dilution.

Influence of dose on the pattern of metabolism and excretion of diphenyl-

acetic acid in the rat.

At 5 mg/kg dose of [14C]diphenylacetic acid (intraperitoneally) about 86%

of the radioactivity was excreted in the bile in 3 h while about 11% was in the

urine. The percentage excreted decreased with increasing dose and at 500 mg/kg

dose level, 20% is excreted in the bile , with some 8 % in the urine. The major

excretion product was diphenylacetylglucuronide with only small amounts of the un-

changed compound irrespective of the dose level.

Discussion

The metabolic fate of diphenylacetic acid has been studied in eleven species

and the results show that diphenylacetic acid is only conjugated with glucuronic

acid irrespective of species but the quantities varied with species. Table 4:3

summarises the conjugation pattern of diphenylacetic acid in the species studied

and it suggests among other things that bushbaby has a low capacity in forming a

glucuronic acid conjugate with diphenylacetic acid and bearing in mind the low

glucuronic acid conjugation with 1-naphthylacetic acid (see Chapter 3, Table 3:1)

Amount of [14C]-diphenylacetic acid excreted conjugated with various compounds in 48 h as % dose

Unconjugated Glutamine Glycine Taurine Glucuronic Acid

30, 34

12, 17

29

n.d.

n. d.

n. d.

n. d.

n. d.

n. d.

5. 7 n. d. n. d.

2.6, 6.7 n.d. n. d.

17 n. d. trace

29, 17 n. d. trace

9.6 (7.4-13) n.d. trace

6. 9 (5. 6-9.2) n. d. n. d.

2.6 (2: 2-2.9) n. d. trace

2.8, 1.5 n. d. n. d.

n. d. 70, 65

n. d. 30, 36 n. d. 27 n. d. 8.4

n. d. 18, 10

n. d. 49

n. d. 3.2, 8.7

trace 30 (26-32)

n. d. 77 (75-79)

n. d. 45 (45-45)

n. d. 26, 34

Table 4:1

Conjugates of [14C]-diphenylacetic acid in various species

14 [ C]-diphenylacetic acid (114 mg/kg) dissolved in the appropriate amount of N-NaOH was administered as described in the text. The urine was collected for 48 h and analysed for metabolites. Results are means for three animals with ranges in parentheses. animals were used individual values are given.

Species (No. & Sex) Genus and Species Dose of 14C Route of administration

Where only one or two

14C excreted %

Primates pCi/animal of dose

Man * (2M) Homo sapiens P. 0. 5. 0 100, 100 Rhesus monkey (1M,1F) Macaca mulatta i. m. 13. 0 45, 49 Cynomolgus monkey (1F) Macaca fascicularis (Iris) i. m. 11.4 57 Squirrel monkey (1F) Saimiri sciureus i.m. 14. 0 14 Capuchin (2F) Cebus albifrons i.m. 11.4 15, 21 Marmoset (1M) Sanguinus oedipus i. m. 14,0 66 Bushbaby(1M, 1F) Galago crassicaudatus i. m. 6. 0 23, 34

Non-Primates

Cat (2F, 1M) Felis cattus i. m. 11.4 40 (36-42) Rabbit (3F) Lepus canaliculus P.O. 21. 0 84 (88-91) Rat (3F) Rattus norvegicus i.p. 1.4 48 (42-53) Fruit bat (1F,1M) Pteropus giganteus 1. p. 11. 3 29, 35

* Dose 5 mg as shown by reverse isotope dilution n. d. not detected

Table 4:2

Influence of dose on the pattern of metabolism and excretion of diphenylacetic acid in the rat

Bile-duct cannulated female rats were injected intraperitoneally with diphenylacetic acid (dissolved in the calculated amount of N-NaOH and diluted with water) and the bile collected for 3h and urine removed from the bladder. Bile and urine were analysed for 14C and metabolites as described in the text.

Bile Urine I I

14C excreted conjugated with: % 14 % C excreted conjugated with:

Dose (mg/kg) % dose excreted Unchanged Glucuronic % dose excreted Unchanged Glucuronic Acid Acid

5 86 (74-95) 7.1 (6.2-8.4) 93 (92-94) 11 (4.4-19) 13 (2.1-34) 87 (66-98)

25 73 (72-74) 16.0 (12-19) 84 (88-89) 12 (7.3-17) 2.3 (2.1-3.3) 98 (97-98)

50 30 (27-36) 9.0 (8. 9-9. 2) 91 (91-92) 9.3 (4. 5-15) 2.9 (1.1-4.2) 98 (96-99)

250 39 (16-53) 7.2 (6. 8-10) 93 (91-93) 19 (13-22) 1.9 (1.7-3.0) 98 (97-98)

500 20 (15-44) 9.1 (8.3-10) 91 (90-92) 8.2 (7.3-9.4) 4.9 (4.0-6.2) 95 (94-96)

Table 4:3

Species Variation in the pattern of conjugation of Diphenylacetic acid

Diphenylacetic abid conjugates as % of the 14C excreted in the

urine

Species

Primates

Unchanged Glycine Glucuronic acid

Man 32 - 68

Rhesus monkey 31 - 69

Cynomolgus monkey 52 - 49

Squirrel monkey 40 - 60

Capuchin 17, 31 - 83, 69

Marmoset 25 < 1 75

Bushb aby 87, 74 <1 14, 26

Non Primates

Cat * 24 <1 76

Rabbit 8.2 - 92

Rat 5.4 <1 95

Fruit Bat 9.7 - 93

* 1% conjugated with taurine

117

one wonders if this is not characteristic with this species. The ability of

the cat to form glucuronic acid conjugateAurther substantiates the fact that

the glucuronide formation in this species is highly substrate dependent. (see

Chapter 3, Capel et al, 1972 ; Millburn 1974). This report is in agreemnt with

the finding of Miriam et al (1927a) which has shown that dog, man and rabbit

conjugate this acid with glucuronic acid.

The conjugation of diphenylacetic acid at different dose levels in the bile

duct-cannulated rats was also investigated (see Table 4:2). The figures show

that higher amounts were excreted in the bile than in the urine irrespective of

the dose and that diphenylacetylglucuronide was the only conjugated excretion

product in both urine and bile. This is understandable since this is the only

metabolic conjugation route employed by the rat, unlike in the case of

1-naphthylacetic acid (see Chapter 3) where there was a shift from glycine

conjugation at low doses to glucuronide conjugation at higher doses. At saturation

doses the glucuronidation capacity in 3h as derived from the data on Table 4:2 is

140nmol/kg.Finally the molecular weight of the diphenylacetyl glucuronide is 388

and this value is a prequisite for extensive biliary excretion in the rat. A

structurally related compotmd,bis (p-chlorophenyl)acetic acid has been reported

to undergo extensive enterohepatic circulation when administered intravenously

to rat (Gingell, 1975). Comparing the amount of diphenylacetic acid excreted in the

intact rat in 48 h and the high biliary excretion in the bile duct-cannulated rat in 3h.

It is suggested that the metabolite excreted in the bile is reabsorbed and excreted

in the urine slowly. The only metabolite is diphenylacetylglucuronide which

could be reabsorbed as such or after hydrolysis to the free acid by the gut flora and/

or secretions (see Smith and Williams, 1966). p-Glucuronidase activity is widely

distributed among the intestinal bacteria (Hawksworth et al. , 1971).

118

CHAPTER FIVE

Metabolism of Hydratropic Acid

Contents Pages

Chromatographic Identification of Urinary Metabolites 119

Man 119 Rabbit, Rat and Rhesus Monkey 119 Cat 122

Chromatographic Identification of Urinary and Biliary Excretion 122 in the Rat

Results 123

Man 123 Rhesus Monkey, Rabbit and Rat 123 Cat 123

Influence of Dose on the Pattern of Metabolism and Excretion of Hydratropic Acid in the Rat 126

Discussion 126

119

The Metabolism of Ilydratropic Acid

The metabolic fate of hydratropic acid has been studied in man, rhesus

monkey, cat, rabbit and rat. Additionally the influence of dose level on its

pattern of metabolism and excretion in the rat has been examined.

Chromatographic identification of urinary metabolites

Urine samples from the 5 species dosed with [14C]hydratropic acid were

subjected to thin-layer chromatography using the systems described in Chapter 2.

(see Table 2 : 3).

Man Chromatography of urine samples on thin-layers using solvent F showed on radio-

chromatogram scanning the presence of one major 14C peak of R

f 0.13 (Fig. 5:1).

This peak gave a positive reaction with naphthoresorcinol and disappeared when

urine samples were incubated with fl-glucuronidase or warmed with N-Na0H. Its

disappearance was accompanied by an appearance of a new peak at Rf 0. 88.

corresponding to hydratropic acid. The peak at Rf 0.13 therefore was identified

as hydratropoylglucuronide. Chromatograms developed in solvent D showed a

single large peak at the origin which corresponded with hydrotropoylglucuronide.

As described above this large peak at the origin disappeared when the urine was

treated with j3-glucuronidase or N-NaOH to be replaced by a new peak at R1 0. 76

corresponding to hydratropic acid.

Rabbit, rat and rhesus monkey . Thin-layer chromatography of urine samples

using solvent F, followed by radiochromatogram scanning showed the presence

of a large 14C peak at R

f 0.14 corresponding to hydratropoylglucuronide and a

small 14C peak at Rf 0.86 due to the unchanged compound (Fig. 5:2). The peak

at Rf 0.14 gave a positive reaction with naphthoresorcinol and disappeared when

urine samples were treated with f3 - g lu c u rani dase or N-NaOH and this was

accompanied by a large increase in the size of the peak at Rf 0. 86 due to hydra--

120

0

S. F.

S. F.

Fig 5:1 Hydratropic acid conjugates in urine chromatograms were developed in solvent F. 1. Hydratropoylgrucuronide. 9, . Hydratropoyltaurine 3. Hydratropoylglycine, 4. Hydratropic acid.

121

Rhesus monkey

ri

0

0

0

S. F.

Fig 5:2 Hydratropic acid conjugates in urine Chromatograms were developed in solvent F. 1. Hydratropoylglucuronide 2. Hydratropic acid

122

tropic acid. Chromatography on thin-layers in solvent D showed the presence

of hydratropoylglucuronide together with a small amount of the unchanged acid.

The presence of hydratropic acid before and after P-glucuronidase or N-NaOH

treatment was confirmed by reverse isotope dilution.

Cat 14C-scans of thin-layer chromatogram of cat urine developed in solvent F

showed four radioactive peaks of Rf values 0.12, 0. 33, 0.72 and 0. 87

respectively (Fig. 5:1). The peak at Rf 0.12 gave a positive reaction with

naphthoresorcinol and disappeared when urine samples were treated with

p-glucuronidase or N-NaOH and was accompanied by a large increase in the

size of the peak at Rf 0. 87 corresponding to hydratropic acid. The peaks at R1

0.33 and 0.72 gave a purple brown colour reaction with chlorine-potassium

iodide/starch reagent, and the peak at Rf 0. 72 gave also positive reaction with

the 4-dimethylaminobenzaldehyde reagent. Therefore, these peaks at Rf

0.12, 0.33, 0.72 and 0.87 were identified as hydratropoylglucuronide, -taurine

and -glycine and hydratropic acid respectively. Similarly chromatograms

developed in solvent D revealed four 14C peaks of Rf values 0.1, 0. 30, 0.63

and 0. 75 corresponding to hydratropoyl- glucuronide, -taurine and -glycine

and hydratropic acid respectively. The presence of hydratropoyl- taurine and

-glycine and hydratropic acid was also confirmed by reverse isotope dilution.

Chromatographic identification of urinary and biliary excretion in the rat

Urine and bile samples were collected over the first 3 h from bile-duct

cannulated rats dosed intraperitoneally with [14 C]+hydratropi c acid (5-500 mg/kg)

were streaked on thin-layers and developed in solvent F. Radiochromatogram

scanning revealed a major 14C peak at Rf 0.13 and a minor peak at Rf 0. 87. The

123

peak at Rf 0.13 gave a positive reaction with naphthoresorcinol and disappeared

when urine and bile samples were treated with p-glucuronidase or N-NaOH and

this was accompanied by a large increase in the size of the peak at R 0.87

due to hydratropic acid. The peak at R 0.13 therefore was identified as hydra-

tropoylglucuronide. Similarly chromatograms developed in solvent D reveals

the presence of hydratropoylglucuronide and hydratropic acid.

Results

The quantitative and qualitative data on the excretion of hydratropic acid

and its metabolites in the five species studied are shown in Table 5:1 and those

in rats at various dose levels are shown in Table 5:2.

Man. The two human subjects excreted 97 and 100% of the radioactivity in the

urine in 24 h after an oral dose of 5 mg [ CJ±hydratropic acid. The major

excretion product was hydratropoylglucuronide (95 and 100% of the dose respect-

ively) with 2.1% of the unchanged hydratropic acid in one of the subjects.

Rhesus monkey, rabbit and rat

The urinary excretion of the radioactivity of the administered dose of [14

C}-1-

hydratropic acid (81 mg/kg) in 24 h is about 97% in the rhesus monkey, 82% in the

rabbit and 81% in the rat. The major excretion product was hydratropoylglucuronide,

the quantities varied in the species, the rhesus monkey excreted 82 and 71.1% ( of

the dose in the two examined), rabbit 73.1% and rat 64.1%. There were also

small amounts of the unchanged compound.

Cat. Cats injected intraperitoneally with [14C1-thydratropic acid (81 mg/kg)

ex_creted about 58% in the urine within 48 h. The major excretion products were

hydratropoylglucuronide (24.1% of the dose), hydratropoyltaurine (13.1%),

hydratropoylglycine (8. 7%) and some unchanged hydratropic acid (12. 1%).

Table 5:1

Conjugates of [14C]-hydratropic acid in various species

[14C]-hydratropic acid (81 mg/kg) in solution of propylene glycol/water (2:1 by vol.) was administered as described in the text. The urine was collected for 24 h and analysed for metabolites. Results are means for three animals with ranges in parentheses. Where only one or two animals were used individual values are given.

Species (No. & Sex) Dose of 14C 14C excreted %

Genus and Species Route of administration tCi/animal of dose

Amount of [14C]-hydratropic acid excreted conjugated with various compounds in 24 h as % dose

Unconjugated Glycine Taurine Glucuronic Acid

Primates *

Man (2M) Homo sapiens p. o. 2. 5 97, 100 2.1, 0 95.0, 100

9.6

8. 5

9. 6

5. 2

Rhesus monkey (1M ,1F) Macaca mulatta i. m.

Non-Primates

Cat (2F, 1M) Fells cattus i. p.

Rabbit (3F) Lepus caniculus p. o.

Rat (3F) Rattus norvegicus i.p.

97, 97.5

58 (53-64)

82 (72-90)

81 (63-90)

15.0, 26.9 82.0, 70.6,

11.9 (8.3-13.9) 8.7(7.9-9.9) 13.3 (8. 1-17. 6) 24.1 (22. 3-26. 1)

9.3 (0. 0-16.6) 72.7 (65.4-82. 0)

16.6 (15.6-17.9) <1 63.9 (63.1-65.4)

* dose was 5 mg/man - = not detected + 48 h excretion

Table 5:2

Influence of dose on the pattern of metabolism and excretion of hydratropic acid in the rat

Bile-duct cannulated rats were injected intraperitoneally with [14C1-hydratropic acid (in solution of propyleneglycol/water, 2:1 v/v) and the bile collected for 3 h and urine removed from the bladder. Bile and urine were analysed for 14C and metabolites as described in the text

Urine Bile i

% 14C excreted conjugated with:

Dose (mg/kg) % dose excreted Unchanged Glucuronic % dose excreted Unchanged Glucuronic Acid Acid

5 26 (16-39) 7.2 (6.3-8.2) 93 (92-94) 32 (21-46) 28 (18-38) 73 (62-81)

25 23 (20-25) 8.6 (5. 8-19) 90.1 (81-94) 28 (20-37) 25 (22-28) 74.2 (72-78)

50 21 (19-23) 14 (3.2-26) 85.7 (74-97) 23 (14-32) 24 (17-25) 76.1 (75-78)

250 22 (7.3-31) 4.2 (2.1-7.0) 95.6 (94-98) 29 (5.5-36) 23 (15-32) 77.8 (68-83)

500 2.6 (2.2-3.0) 15.3 (8.6-25) 84.4 (73-91) 23 (7.9-41) 19 (8.9-25) 80.9 (75-91)

14 % excreted conjugated with:

*.hydratropoylglycine not detected in urine or bile

126

Influence of dose on the pattern of metabolism and excretion of hydratropic

acid in the rat

Bile-duct cannulated rats injected intraperitoneally with [ C]iydra-

tropic acid (5-500 mg/kg) excreted at 5 mg/kg dose level about 32% of the

radioactivity in the bile and 28% in the urine. These values fell with increasing

dose, and at the dose level of 500 mg/kg while 23% of the dose was excreted

in the bile only 2.6% was excreted in the urine. The major excretion product

was hydratropoylglucuronide in both urine and bile irrespective of the dose

level, and small amounts of the free acids were also excreted.

Discussion

The metabolic fate of hydratropic acid has been studied in the five species.

Table 5:2 shows the conjugation pattern of this acid in the species studied and it

suggests that hydratropic acid can be conjugated with amino acids and / or

glucuronic acids the pattern depending on the species. Man, rhesus monkey,

rabbit and rat form glucuronic acid conjugate of hydratropic acid, and earlier

Robinson et al (1955) have found this conjugate in the rabbit. The cat on the

other hand forms glucuronic acid, glycine and taurine conjugates of this acid and

Kay and Raper, (1922) have reported that another carnivore, the dog, conjugates

this acid with glycine. The ferret also conjugates the compound mainly with glycine

and taurine (unpublished data of J. R. Idle). The ability to form glucuronides in the

cat is substrate dependent (Millburn, 1974) and hydratropic acids seems a very

important substrate in the shift from amino acid to glucuronic acid conjugation

since this species forms only amino acid conjugates with phenylacetic (James et al

1972 a) and 1-naphthylacetic (see Chapter 3) acids but glucuronic acid conjugate

with diphenylacetic acid (see Chapter 4. )

The effect of dose on the metabolism and excretion of hydratropic acid in

bile duct cannulated rats has also been examined and the results shown in Table

5:2, suggests a fairly even excretion in the bile and urine except at the dose level

Table 5:3

Species Variation in the pattern of Conjugation of Hydratropic acid

Hydratropic acid conjugates as % of the 14C excreted in the urine

Species Unchanged Glycine Taurine Total conjugated with Glucuronic acid

Primates

amino acids

Man 1 - - 0 99

Rhesus monkey 16, 28 - - 0 85, 72

Non-Primates

Cat 21 15 23 38 42

Rabbit 11 - - 0 89

Rat 21 L. 1 - 1 79

128

of 500 mg/kg where the biliary excretion was higher. Hydratropoylglucuronide

was the only conjugated excretion product in bile and urine. This is to be

expected since this is the only metabolic conjugation route for hydratropic acid

in the rat. At saturation doses the glucuronidation capacity in 3 h as derived from

the data on Table 5:2 is 145nmol/kg. Hydratropoylglucuronide (M. W. 326) has a

molecular weight with the range 325 ± 50 given by Hirom et al. (1972) for

extensive biliary excretion in the rat. The excretion in intact rats and the bile

duct-cannulated rats suggest a reabsorption of the glucuronic acid conjugate as

stated in Chapter 4.

129

CHAPTER SIX

Pharmacokinetic and Subcellular Aspects of Arylacetic

Acid Conjugation

Contents Pages

Introduction 130

Drug Distribution 130

Membranes 131

Results

Discussion

Mitochondria 131 Microsomes 132

Drug Binding 134

Lipid Solubility and Metabolism 135

Affinities of Conjugation Sites for Drugs and 136 their Polar Metabolites

Pharmac °kinetics 137

Enzyme Affinity 140

Scope of the Present Investigation 141

141

PharmacokMetics 141

1-Naphthylacetic Acid 141 Diphenylacetic Acid 141 Hydratropic Acid 149 Phenylacetic Acid 149

Binding Studies 149

Enzyme Affinity 152

Glycine C onjugation 152

Glucuronic Acid Conjugation 152

152

Summary and Conclusion 162

130

Pharmacokinetics and subcellular Aspects of Arylacetic Acid Conjugation

Introduction The results of the in vivo studies (see Chapter 3, 4 and 5) and the work

of others reported elsewhere in the literature strongly suggest that the structure

of an arylacetic acid is the most important factor influencing its pattern of metabolic

conjugation. The pharmacokinetic behaviour of a compound may give information

as to its manner of distribution in the body , binding to body components and

more especially its access to the metabolic conjugation sites, in this case the

mitochondria (site of amino acid conjugation) Schachter and Taggart 1954) and the

endoplasmic reticulum (microsomes, site of glucuronic acid conjugation, Dutton,

1966). The overall metabolism would be a result of the affinity of the arylacetic

acid for the conjugating enzymes associated with these structures. The differential

sites for conjugation may be related to the ability of the mitochondria to absorb

ionic forms, whereas metabolism by microsomes requires in the main lipid

soluble nonpolar molecules.

Drug distribution

After a drug is absorbed or injected into the blood stream, it can enter or

pass through the various body fluid compartments — plasma, interstitial fluid,

transcellular fluids anc cellular fluids. Some drugs cannot pass cell membranes

and therefore, are restricted in their distribution and in their sites of action,

whereas others penetrate into cells and thereby distribute throughout all fluid

compartments. The important factors that influence distribution include the extent

of binding of drug to plasma and tissue proteins and other components of cells

such as mitochondria and endoplasmic reticulum (Burns et al. , 1953 ; Dingell

et al. 1964) ; regional differences in pH ; the permeability characteristics of — —

special membranes to drug ; and the lipid content of the cells (Melmon and Morrelli,

1972). Tissue uptake of a drug is dependent upon the mass of the tissue and the

131

rate of blood flow to it, as well as the partition coefficient of the drug between

blood and tissue (Gillette, 1973).

Membranes

For a drug introduced into the body to getto its metabolic site it has to

penetrate some membranes. The degree of penetration depends on the

constitution of the membrane, type of transport mechanism employed and the

partition coefficient of the drug between the membrane and its environment.

It is a widely accepted view that biological membranes possess both

physical and functional plasticity and this had led to the hypothesis of a membrane

structure in which mobile arrays of proteins and lipids are bound together by

non-covalent hydrophobic bonds (Singer, 1971 ; Gilter, 1972). In this context,

it has been shown by paramagnetic resonance and x-ray diffraction that fatty

acid chains in a lipid bilayer and in some biological membranes exhibit a high

degree of molecular motion (Luzzati, 1968 ; Hubbell and McConnell, 1971).

Furthermore, Branton et al. (1972) have shown with freeze-etched preparations

that the organisational changes associated with the fluidity of the membrane are

caused both by intrinsic modifications of the membrane and by changes in its

surrounding environment. More recent work on freeze-etched tetrahymena

membranes (Hubbell and Wunderlich, 1973) has further suggested that membrane

intercalated particles may be capable of transme,mbranal mobility.

Mitochondria

The penetration of drugs into subcellular particles such as mitochondria

appears to be an important aspect of drug distribution and action ; and the

penetration of drugs follows the same principles as for cell membranes. In

contrast to indications of membrane plasticity, the results of biochemical

studies on mitochondria have given a rather static picture of enzyme localization

132

in mitochondrial membranes (Waksman and Rendon, 1974). It has been shown

that some macromolecular components (enzymes and proteins) of mitochondrial

inner membrane are capable of large amplitude movements allowing them specific

and reversible escape from the field of membrane interactions (Waksman and

Rendon, 1971 ; Rendon and Waksman, 1971).

Waksman and Rendon (1974) have also reported that an integrated mito -

chondrial membrane system is endowed with the potential capacity to "read and

recognize" defined variations in the extramitochondrial environment and to

transduce this information by specific and reversible, large amplitude inter-

membranal protein movements. Since the behaviour of a protein is defined by

a set of interactions with its environment, the mobile protein in its membrane-

bound state should be related to its membrane partners by a characteristic set

of interactions. Once severed from these connections, it will become available

for new interactions in a new environment. At least two extreme possibilities ,

both of which depend on specific ligand-protein interactions are offered to the

released proteins. A first possibility is the environmentally induced metabolic

expression of the released enzymatic activities (Estrada-0 et al 1970) Thus some

mitochondrial enzymes may be modulated, by their shuttling to and away from

their substrates and ligands. A second possibility is that the protein shuttling

may be associated with the transport of small molecules, with some advantage

to cell economy. If these proteins are able to bind ligands, without necessarily

metabolizing them, the bound ligand might then be transported from one compart-

ment to another (Waksman and Rendon, 1974).

Microsomes

Many drugs are bound to microsomes, for example imipramine (Gillette,

1973) and halothane (Uehleke et al, 1973 ; Van Dyke and Wood, 1975). The

semipermeability of microsomal membrane/has certain important functional

133

implications, mainly since the accessibility of a number of substrates to the

membrane-bound enzymes is shown to be a limiting factor (Hers and deDuve, 1950 ;

Ernster and Jones, 1962 ; Mulder, 1970) . Changes in membrane permeability

by external or internal agents may be decisive for regulating the activity of

various enzymic systems (Blether, 1966 ; Rodbell, 1966).

Since the enzyme activity depends on the integrity of the membrane

structure, it follows that :

1) a membrane bound enzyme may as a result of interaction with

other membrane components have a different conformation ( and hence

different catalytic activity) from that which, it would possess in free

solution (Laidler and Bunting, 1973).

ii) depending on the location of the enzyme in the membrane its inter-

action with substrates may occur in an environment which is different

(e. g. in pH) from that outside the microsomal vesicles (Katchalski et al .

1972)

iii) if the enzyme is located in the microsomal membrane so that

a permeability barrier is interposed between substrates and active site,

partition of substrates between the aqueous and membrane phases,

permeability of the microsomal membrane and the nature of substrate

transport mechanisms may affect the overall rate of the enzyme reaction.

The size and the surface charge of the microsomal vesicles may be important.

Lysophosphatides, unsaturated fatty acids, phospholipase A and detergents

increase the permeability of microsomal membranes (Fiehn and Hasselbach,

1970 ; Kreibich et al. , 1973) and as a result might effect the apparent

enzyme activity e. g. rat liver UDP-glucuronyltransferase.

Nilsson et al (1971) studied the permeability of microsome,s

134

to different compounds and found that unchanged molecules freely

penetrated the microsomal membrane while smaller charged species did

not. They also demonstrated a high partition by a lipophilic substrate

like naphthalene into the microsomal phase by comparing its concentration

in the pellet and supernatant fraction after separation of the two phases.

Furthermore, Stier and Sackmann (1973) examined the interaction of

microsomal lipid and substrates with spin-label techniques. They have

concluded that the microsomal system consists of a heterogeneous lipid

matrix in which the enzymes are imbedded. This lipid matrix influences

the binding of substrate as well as the interaction of the enzymes since

the substrate is incorporated irto the membrane.

Drug binding

A drug can bind with tissue and subcellular components in a variety of

ways. Goldstein et al. (1971) have distinguished between non-saturable and

saturable interactions.

Non-saturable interactions are of two kinds. First, particles surrounded

by an osmotic membrane (Qg. synaptosomes) or having a spongy matrix can

contain entrapped drug in aqueous solution. Second, membranes will contain

dissolved drug, in amounts determined by the lipid/water partition coefficient

and the ambient aqueous concentration.

Non-specific saturable interaction arises through ionic bonds, hydrogen

bonds, and hydrophobic forces, but specific saturable interaction arises at the

receptor sites, where drug binding triggers the chain of events that leads to

the characteristic pharmacological effect. A measure of specific saturable

binding of an arylacetic acid to mitochondria and microsomes could be used as

an index of correlation as to the degree of amino acid (in the mitochondria) or

glucuronic acid (in the microsomes) conjugation. It has also been shown that

135

the parent drug and its nonpolar metabolite have more affinity for its metabolic

site than its polar metabolite (Bickel and Borner, 1974 ; Glauman et al. 1970 ;

Nilsson et al. 1971).

Lipid solubility and metabolism

The lipid solubility of a compound seems not only to determine its

penetration into cells and metabolic sites but also influences its metabolism.

The oxidative dealkylation of foreign N-alkylamines by rabbit liver microsomes

appears to be limited to compounds which are lipid soluble ; and since the micro-

somal hydroxylation of aromatic compounds also appears to be limited to lipid

soluble substances as well, Gaudette and Brodie (1959) suggested that an intra-

cellular fat-like boundary separates normally occuring polar substances from

the highly non-specific microsomal enzymes.

The microsomal system dealkylates a variety of highly lipid soluble N-

alkylamines,such as methylamphetamine, pethidine, ephedrine and codeine

(Gaudette and Brodie, 1959 ; Axelrod, 1956), but normally occurring N -alkyl-

amines such as sarcosine, dimethylglycine and epinephrine, which have very

low lipid solubility , are not demethylated by liver microsomes. The dimethyl-

ation of sarcosine and dimethylglycine, however, is accomplished by highly specific

systems in the mitochondria (Mackenzie and Frisell, 1958 ; Gaudette and Brodie,

1959) . A similar situation exists for the hydroxylation of aromatic compounds.

Despite the non-specificity of the microsomal hydroxylation system with respect

to foreign compounds, it does not promote the hydroxylation of naturally occuring

compounds such as L-phenylalanine, L--tryptophane, kynurenine and anthranilic acid,

which are hydroxylated by quite specific systems localised in other parts of the

liver cell (Mitoma et al. , 1956).

Other examples of the importance of lipid solubility are found in substrates

136

of the deamination enzyme present in rabbit microsomes, but absent in a number

of other mammalian species (Axelrod, 1954) e This enzyme deaminates the lipid

soluble substances amphetamine, methamphetamine and ephedrine, but does not act

on substances like tyramine or serotonin (Axelrod, 1954) , which are substrates

of monoamine oxidase.

The ability of certain drugs to increase the fluidity of lipid region in a

membrane may also affect the metabolism of that drug. Stier and Sackmann,

(1973) have suggested that the cytochrome P450 system, which is the drug

metabolising system situated in the microsomal fraction of the liver, may be

enclosed in a' gather rigid phospholipid halo'! A critical factor in the metabolism

of a substrate by the cytochrome P450 system would seem to be its lipid solubility.

However, the rate of metabolism of a drug could be determined not by its lipo-

philic nature alone but also by its ability to fluidise the phospholipid halo

surrounding the cytochrome P450 complex (see Cater et al. , 1974).

This lipophilic characteristic could be one of the vital factors among other things

operating in the metabolic conjugation of the arylacetic acids already examined, the

ones with high lipid solubility, or able to fluidise the lipid region of the micro-

somes would be, expected to be conjugated there.

Affinities of conjugation sites for drugs and their polar metabolites

Little is known on the uptake of drugs into cells, their binding to cellular

structures, as well as the nature of intracellular and intercompartmental trans-

location of drugs and their polar metabolites from binding and metabolic sites

to sites of incretion or excretion.

Bickel and Borner (1974) studied pharmacokinetic processes using

recirculating rat liver perfusion system, and determined the drug imipramine

and its major metabolites at various times in perfus ate, liver, bile and subcellular

137

liver fractions. They found that imipramine undergoes a rapid hepatic uptake,

the initial extraction being close to 100% ; and that most of the unchanged drug

is then localized in the microsomal fraction. Metabolism of imipramine is

not limited by uptake but by enzymatic factors. Like imipramine, its lipophilic

metabolite, desmethylimipramine is bound to microsomes, but the polar

glucuronides are easily released from endoplasmic reticulum, their site of

formation, into the cytosol and presumably from there excreted into the bile.

This would agree with the direct demonstration of this pathway for the polar

metabolites and conjugates of naphthalene (Glaumann et al. 1970) and morphine

(Nilsson et al. , 1971). The same general pattern of intra-hepatocytic binding,

and distribution has been observed by Bahr and Borga (1971) (desmethylimipramine --->

2-hydrov-desmethyl -imipramine ---)2-hydroxy-desmethylimipramine-

glucuronide) and by Levine and Singer (1972) with other compounds, where

glucuronides in contrast to the parent drugs, were mainly localized in the cytosol

fraction. Bickel and Steele (1974) also have demonstrated high affinities of liver

microsomes for imipramine and desmethylimipramine, but not their glucuronidated

derivatives. This finding could also apply to parent drugs and their polar

conjugates in the mitochondria.

Pharmacokinetics

A drug which when administered intravenously rapidly equilibrates between

the plasma and other body fluids and tissues, and when administered orally is not

subject to metabolism prior to reaching the systemic circulation, can usually be

adequately described by a one compartment model with first-order elimination

as depicted in scheme 1.

K input V

Scheme 1

output

138

In this scheme, K is the first-order rate constant for hepatic elimination and

V is the apparent volume of distribution of the drug. The apparent volume of

distribution is dependent on the size of drug molecule, lipid solubility and its

binding to plasma and tissue proteins ; and can be defined as that volume of

fluid into which the drug appears to distribute with concentration equal to that

in the blood.

Amount of drug in the body

Apparent Vol, of distribution - (1) Concentration of drug in the blood

The biologic half-life (T1/2) and clearance (Q) of a drug which confers

single compartment characteristics to the body are directly related to K as

follows:

T1/2 = 0.693 / K (2)

and

KV (3)

Since the liver is considered an integral part of this "homogeneous" one-

compartment system, the direct relationship between half-life and clearance, and

hepatic elimination as expressed in equations 2 and 3, would be anticipated.

Consequently, half-life and clearance should reflect the elimination characteristics

of this class of drugs (Perrier and Gibaldi, 1974).

A large number of drugs, however confer on the body the characteristics

of a multicompartment system rather than a simple one-compartment system.

For this class of drugs the body does not behave as a single kinetically "homogeneous"

compartment, but rather as two or more such compartments. The simplest

multicompartment system, a two-compartment system is illustrated in the scheme

2 where K12 and K21 are inte])..compartmental transfer rate constants and Ke is the

Central Compartment

V1

output \toe Ke

input

139

elimination rate constant.

K12

K21

Peripheral Compartment

V2

Scheme 2

Unless there is evidence to the contrary, the liver is usually assumed to be an

integral part of thehomogeneous central compartment. Hence, the rate

constant, Ke, is analogous to K in a one-compartment system in that it is also

the rate constant for hepatic elimination. The biologic half-life of a drug which confers

multicompartment characteristics on the body is not only a function of hepatic

elimination (Ke) but also of drug distribution (K12

and K21), and for a two

compartment model,

T1 = 0.693/ 2 [ (K12+ K21 Ke)- (K12 +K21 +Ke) 2 - 4 K2iKei (4)

-2-

The result is that the biologic half-life of this class of drugs is not

linearly related to the reciprocal of Ke, the hepatic elimination rate constant

(Gibaldi and Perrier, 1972). Clearance can be determined by employing the

relationship

Q = Dose AUC

where 'Dose' is the administered dose and AUC is the total area (from time zero to

infinity) under the plasma concentration-time curve. Clearance as calculated

previously for one-compartment model (equation 3) can also be determined employing

this relationship. For a drug which can be characterized by scheme 2

(5)

140

AUC = Dose = Dose Ke

pvd

. Q = KeV1 = ord (6)

where V1 is the apparent volume of the central compartment, Vd is the apparent

volume of distribution of the drug and 13 equals 0. 693/T1 where T1, the biologic

half -life is defined by equation 4. Equation 6 illustrates the direct relationship

between clearance and hepatic elimination. Therefore, clearance measurements

in multi-compartment systems should yield insight into the intrinsic elimination

characteristics of a drug, and hence serve as an index of hepatic elimination.

There exists a third group of drugs whose pharmacokinetics cannot be

explained adequately by the conventional single or multicompartment systems

discussed above. These are drugs which are subject to metabolism on their

initial pass through the liver after oral administration. This group includes,

among other drugs, propranolol (Shand et al. 1970) and propoxyphene (Perrier

and Gibaldi, 1972).

Enzyme affinity

The kinetic parameters of an enzymic reaction are defined by two

quantities namely Vmax and K . While K (Michaelis rate constant) is the m m

equilibrium constant of reaction, and represent the reciprocal of the affinity

of the enzyme for the substrate, Vmax is the measure of the velocity constant

of breakdown of the enzymes-substrate complex. These two quantities are bound

to influence the pharmacokinetic behaviour of a compound introduced into the

body.

141

Scope of the present investigation

Phenylacetic acid is conjugated mainly with amino acid (James et al. 1972 a)

1-na.phthyla.cetic acid with both amino acid and glucuronic acid (see Chapter 3),

hydratropic acid (except in the cat, see Chapter 5) and diphenylacetic acid (see

Chapter 4) are mainly conjugated with glucuronic acid. In this study an attempt

is made to correlate the metabolic patterns of these four arylacetic acids with

their pharmacokinetic behaviour, after intravenous administration, in the rabbit

and also their affinity for the conjugation sites (mitochondria and microsomes)

and the conjugating enzymes associated with these structures.

Results

Pharmacokinetics. The mean blood concentrations of 14C-labelled 1-naphthylacetic,

diphenylacetic and hydratropic acids at different time intervals in three rabbits

after an intravenous administration of these acids (69 i/mol/kg) are shown in

Tables 6:1, 6:2, 6:3 and 6:4 respectively, and after plotting the results on semi-

log paper, calculation of some of the pharmacokinetic parameters of these acids

were undertaken, the values of which are shown in Table 6:5. The blood samples

revealed on analysis the absence of any conjugates of these acids.

1 -Naphthylacetic acid. The semi-log plot of the mean blood concentration of

1 -naphthylacetic acid against time is shown in Fig. 6:1. This shows a mono-

phasic profiles and thus representative of one compartment system. Table 6:5

shows low values for blood clearance (Q, 0.72 ml. min 1), apparent volume of

distribution (VD, 0.137 1/kg) and elimination rate constant (Ke 9 0. 316 h-1

).

Diphenylacetic acid . The semi-log plot of the mean blood concentration of

diphenylacetic acid against time is shown in Fig. 6:2. This shows a biphasic pro -

file and thus representation of a multicompartment model (in this case taken

essentially as a two compartment system). Diphenylacetic acid has low clearance

142

Table 6:1

Mean blood concentrations of 1-naphthylacetic acid (pg/m1) in three rabbits

following intravenous administration of 28. 3 mg.

Time (min. )

5

10

15

Mean blood concentrations of 1 -naphthylacetic acid (µg/ml)

80.6 (59.9 - 102. 5)

90. 8 (75. 5 - 105. 9)

73.1 (68.7 - 81.7)

20 67. 4 (56. 7 - 80. 6)

30 55. 5 (45. 3 - 68. 0)

45 48. 3 (38. 9 - 57. 7)

60 34. 0 (28. 3 - 39. 7)

90 22.0 (17.3 -25.5)

120 13. 5 (9. 3 - 16. 0)

180 4. 0 ( 2. 6 - 5. 4 )

Table 6:2

Mean blood concentrations of diphenylacetic acid (µg/ml) in three rabbits

following intravenous administration of 32. 2 mg

Time Mean blood concentrations of diphenylacetic (min. ) acid (µg/m1)

5

10

44. 7

30.2

(42. 1 - 47. 2)

(30.1 - 30.4)

15 23. 5 (21. 8 - 26. 8)

20 15. 5 (12.1 - 18. 8)

30 9. 5 ( 8.1 - 10.1)

45 6. 1 ( 5. 8 - 6. 3 )

60 2.6

90 1.0

120 0. 3

143

Table 6:3

Mean blood concentrations of hydratropic acid (tteml) in three rabbits

following intravenous administration of 22. 7 mg

Time (min. )

5

Mean blood concnetrations of hydratropic acid (µg/m1)

59.4 (50.3 68. 5)

10 53. 0 (47. 0 - 59. 0)

15 38. 8 (30. 3 - 49. 5)

30 34. 5 (25. 0- 50. 5)

45 22. 2 (21. 0 - 23. 9)

60 19.9 (17. 8 - 22. 8)

90 14. 0 (12. 6 -16. 0)

120 9. 7 ( 9. 2 - 10.1)

180 4.5 ( 4.2 - 4.8)

Table 6:4

Mean blood concentration of phenylacetic acid (µg/m1) in three rabbits

following intravenous administration of 20. 5 mg.

Time (min. )

Mean blood concentrations of phenylacetic acid (pg/m1).

5 26. 2 (22. 4 - 29. 4)

10 19. 8 (14. 8 - 25. 2)

15 13.1 ( 7.4 - 20. 5)

20 11. 0 ( 8. 8 - 15. 0)

30 8.4 ( 7.0 - 9.7)

45 4. 2 ( 3. 5 - 5.3)

60 2. 2 ( 1. 8 - 2. 6)

90 O. 6 ( O. 5 - O. 7)

120 0. 4 ( O. 3 - 0.5)

180 0. 2

144

100 M

ean

bloo

d co

ncen

trat

ion

of 1

-nap

hthy

lace

tic

acid

(pg/

m1)

10

1.0

1 3

Time (h)

Fig. 6:1 Pharmacokinetic analysis of mean blood concentrations in three rabbits (ay. wt. 2. 2 kg) following intravenous administration of 28. 3 mg of [14C1-1-naphthylacetic acid.

145

Time ( h )

1

2

Fig 6:2 Pharmacokinetic analysis of mean blood concentrations in three rabbits (ay. wt. 2.2 kg) following intravenous administration of 32.2 mg of [14C]diphenylacetic acid.

Mea

n bl

ood

conc

entr

atio

n of

hydr

atro

pic

acid

(µg/

m1)

20

1.0

80 60

40

beta phase

alpha phase

0.1 -

1

146

Total (h) 1 2 3

Fig 6:3 Pharmacokinetic analysis of mean blood concentrations in three rabbits (ay. wt. 2.2 kg ) following intravenous administration of 22.7 mg of [14C]hydratropic acid.

Fig 6:4 Pharmacokinetic analysis of mean blood concentrations in three rabbits (ay. wt. 2.2 kg) following intravenous administration of 20.5 mg of 44 Cjphenylacetic acid.

blo

o d c

once

ntra

tion

of

phen

ylac

e tic

aci

d (d

ig/m

1)

alpha phase \

beta phase

Time (h )

147

Table 6:5

Pharmacokinetic parameters of arylacetic acids in three rabbits following intravenous administration of 69 pmolikg

Compound Compart - meat Model

Vc 1/kg

VD 1/kg

Ke h-1

K12

h-1 K21 h-1

Alpha (a)

h-1 aTi

2- h

Beta (g) h-1

gi- 1 2 h

Q

ml/min

1 -Naphthylacetic acid 1 - 0.137 0.316 - - - - O. 316 2.19 0.72

Diphenylacetic acid 2 0. 393 0.485 0.47 0. 053 1.57 1.492 0.469 0.495 1.40 4. 0

Hydratropic acid 2 0.320 0.256 0.21 0.604 2.44 1.75 0.396 0.296 3.3 1.33

Phenylacetic acid 2 0.301 5.481 0.086 2.0 3.07 0.835 0.83 0.314 8.66 28.7

VC = volume of central compartment ; VD = apparent volume of distribution ; Q = blood clearance

K12 = (a + 13) - (K21 + Ke) ; Ke = c /3/K21 ; K21 = (Aa(0)/3 + Ba(o)a) / (Aa(0) + Ba(o)) where Aa(o) = The intercept obtained

after extrapolating the alpha phase back to time zero. Ba(o) = The intercept obtained by extrapolating the beta phase back to time

zero.

149

(4.0 ml min-1) low apparent volume of distribution (0.485 1/kg) and low

elimination rate constant (0.47 h-1) although these values are higher than

in the case of 1-naphthylacetic acid (Table 6:5). The rate constant of distribution

represented by alpha is 1.492 h-1, and alpha half life ( aTi) is 0.469 h. The 2

ratio K12

: K21 is 1:30.

Hydratropic acid The semi-log plot of the mean blood concentration of hydra-

tropic acid against time is shown in Fig. 6:3. This shows a biphasic profile

and thus representative of a two-compartment model as stated above. Table 6:5

shows that hydratropic has a low clearance (1.33 ml. min-1), a low apparent

volume of distribution (0.256 1/kg) and low elimination rate constant (0.21 11-1).

The rate constant of distribution represented by alpha is 1.75 h-1 and the alpha

half life ( aTi) is 0.396 h-1. The ratio K12

: K21

is 1:4.

Phenylacetic acid The semi-log plot of the mean blood concentration of phenyl-

acetic acid against time is shown in Fig. 6.4. This shows a biphasic profile and

thus representative of a two-compartment model. Table 6:5 shows that phenyl-

acetic acid has a high clearance (28.5 ml. min-1), a high apparent volume of

distribution (5.481 1/kg) and a low elimination rate constant (0. 086 11-1). The

rate constant of distribution represented by alpha is 0.835 h-1 and the alpha

half life (aT 1) is 0.83 h. The ratio K12

: K21

is 2:3.. 2

Binding studies The binding of 1-naphthylacetic, diphenylacetic, hydratropic

and phenylacetic acids to initochondrial and microsomal fractions in 0.21VI Tris-HCI

buffer was investigated as described in Chapter 2, and the results are shown in

Table 6:5 and 6:7. The binding of these acids to mitochondria was in the proportion

phenylacetic acid, 40 ; 1-naphthylacetic acid, 10 ; diphenylacetic acid, 2 ; hydra-

tropic acid 1 (Table 6:6) and their binding to microsomes was in the proportion

hydratropic acid, 30 ; diphenylacetic acid, 27 ; 1-naphthylacetic acid, 20; phenyl-

acetic acid, 10 (Table 6:7).

Table 6:6

The binding of some arylacetic acids to mitochondria

Substrate concentration = 10 nmol

Binding in pmol / 10 mg protein

A B A -B

Compound Total Non-saturable Saturable (trapped and dissolved)

1 -Naphthylacetic acid 525 (521 - 527) 515 (509 - 519) 10 ( 8 - 12)

Diphenylacetic acid 330 (328 - 331) 328 (325 - 331) 2( 0- 3)

Hydratropic acid 599 (598 - 600) 598 (596 - 600) 1( 0- 2)

Phenylacetic acid 256 (251 - 264) 216 (203 - 229) 40 (35 - 48)

Table 6:7

The binding of some arylacetic acids to microsomes

Substrate concentration = 10nmol

Binding in pmol / 10 mg protein

A B A-B

Compound Total Novi saturable Saturable (trapped and dissolved)

1-Naphthylacetic acid 652 (648 - 654) 552 (546 - 558) 100 (96 - 102)

Diphenylacetic acid 400 (395 - 403) 265 (259 - 273) 135 (130 - 138)

Hydratropic acid 690 (681 - 696) 540 (525 - 555) 150 (141 - 156)

Phenylacetic acid 110 (101 - 115) 60 ( 46 - 74) 50 ( 41 - 55)

152

Enzyme affinity The conjugation of 1-naphthylacetic, diphenylacetic, hydra-

tropic and phenylacetic acids with glycine and glucuronic acid in vitro has been

examined as described in Chapter 2.

Glycine conjugation The experiment was performed in 0. 2M Tris-HC1 buffer pH 8.4

using both liver and kidney homogenates respectively as the enzyme sources. The

results are shown in Table 6:8. With kidney homogenate/there was 19% glycine

conjugate formed with phenylacetic acid as substrate (10 nmol), and none for the

other acids, but with liver homogenate it was 89% for phenylacetic acid, 1% for

1-naphthylacetic acid and none for both diphenylacetic and hydratropic acids.

Table 6:9 shows the kinetics of phenacetylglycine formation, while Fig. 6. 5 shows

the Lineweaver Burk plot of the results. The KM for the phenacetylglycine

formation was found to be 1.3 x 10-6M and the Vmax 5 nmol/ 40 mg liver / min.

Glucuronic acid conjugation The glucuronic acid conjugation of 1-naphthylacetic,

diphenylacetic, hydratropic and phenylacetic acids was studied using liver s the

microsomal fraction/as/enzyme source as described in Chapter 2. There was no

glucuronide formation detected with phenylacetic acid as substrate. The

glucuronidation kinetics for 1-naphthylacetic acid is shown in Table 6:10, diphenyl-

acetic acid, Table 6:11 and hydratropic acid Table 6:12 and the Lineweaver Burk

plots of these results are shown in Figs. 6:6, 6:7 and 6:8 respectively. The Km

for these acids were 1-naphthylacetic acid, 6.1 x 10-8

M ; diphenylacetic acid

2.3 x 10-8

M and hydratropic acid 2. 6 x 10-8

M. The V were 1-naphthylacetic max

acid, 11.1 nmol/mg protein/ min, diphenylacetic acid 1. 7 nmol/mg protein/ min and

hydratropic acid 1.4 nmol/mg protein/min.

Discussion

and The clearance / half-life of a drug can be used as indices of the intrinsic

capacity of the liver to metabolise drugs (Perrier and Gibaldi, 1974). The use

Table 6:8

Glycine conjugation

Substrate concentration = 10 nmol ; 0. 2M Tris-HC1 buffer pH 8.4

Glycine conjugation as % substrate/ 30min

Compound Kidney Liver

1 -Naphthylacetic acid 0 1.0

Diphenylacetic acid 0 0

Hydratropic acid 0 0

Phenylacetic acid 19 (16-23) 89 (80-93)

Table 6:9

Kinetics of phenacetylglycine formation

Buffer = 0.2 Tris-HC1 buffer pH 8.4.

S (nM) V 1/V 1 S x 0-5M nmo1/40mg liver / min

10 0.344 2.9 10

12.5 0.42 2.4 8

50.0 0.63 1.6 5

100.0 2.0 0.5 1

500.0 4.0 0.3 0.2

1000.0 5.0 0.2 0.1

153

154

Table 6:10

Kinetics of 1 -naphthylacetylglucuronide formation

Buffer = 0.2M

Phosphate buffer pH 5. 9

Substrate Initial

1/v 1/s I0-8M velocity (V)

Concentration (nmol/mg protein/ s (nM) minim

3. 3 0.59 1.7 3.0

4.2 0.75 1.3 2.4

8. 3 1.54 0. 7 1.2

16. 7 2.56 0.4 0. 6

33. 3 3.33 0.3 0. 3

Table 6:11

Kinetics of diphenylacetyl glucuronide formation

Buffer = 0.2M phosphate buffer pH 6. 2

S (nM)

V (nmol/mg protein/min)

1/V I/S x 10-8M

3.3 0.22 4.5 3.0

4.2 0.27 3.8 2.4

8.3 0.50 2.0 1.2

16.7 0.72 1.4 0.6

33.3 1.11 0.9 0.3

Table 6:12

Kinetics of hydratropoylglucuronide formation

Buffer = 0.2M phosphate buffer pH 5. 7

S (nM)

V (nmol/mg protein/min)

I/V I/S x 10-8M

3. 3 0.172 5. 8 3. 0

6. 7 0.308 3. 3 1.5

16. 7 0.60 1.7 0. 6

33. 3 0. 82 1.2 0. 3

50. 0 0.92 1.1 0. 2

66.7 1.06 0.9 0.15

155

156

Q

O

0

x 10 -5M

Fig. 6.5 Lineweaver-Burk plot of phenacetylglycine formation concentrations of phenylacetic acid were from 0. 01 - (50, 000 d. p. m. ) in the presence of 60 pmol of glycine. Reaction mixtures contained 3pmol MgCli , 20pmol giutathione, 0. 2M Tris/HCI buffer, pH 8.4 and 40 mg of rat liver homo - genate.

Km =1.3 x 10-6M V = 5nmo1/40 mg liver/mm

• 2-7

71) 0

0

-0.17 0

x 10-8M

Fig 6:6 Lineweaver-Burk plot of 1-naphthylacetylglucuronide formation. Concentrations of 1-naphthylacetic acid 3. 3 - 33.3 nM Incubation mixtures contained ltanol TJDPGA, 1. 5 mg microsomal protein and 0.2 M phosphate buffer pH 5. 9. Km = 6.1 x 10-8M, Vm = 11.1 nmol / mg protein/min.

157

158

6-

Fig. 6:7 Lineweaver -Burk-plot of diphenylacetyl.glucuronide formation. Concentrations of diphenylacetic acid were from 3. 3 to 33. 3 nM. The incubation mixtures contained Tµmol UDPGA, 1. 5

microsomal protein and 0.2 M phosphate buffer , pH 6.2 Km = 2. 3 x 10-8 M V = 1. 7 nmol/mg protein/min.

6- !•

0 -039

i/v

[nm

ol/m

g pr

otei

n/m

in].

6

2-

2 3

1 x 10 M

159

Fig 6:8 Lineweaver-Burk-plot of hydratropoylglucuronide formation. Concentrations of hydratropic acid were from 3. 3 - 66.7 nM. The incubation, mixtures contained linno1 UDPGA, 1.5 mg , mic.rosomal protein and Q. 2 M phosphate buffer, pH 5.7. K 2.6 x 10-8M V =1.4 nmol/mg protein/min. m

160

of these parameters as indices of hepatic elimination is highly dependent on the

pharmacokinetic characteristics of a particular drug. For drugs which confer

the pharmacokinetic characteristics of a single compartment model on the body,

both half-life and clearance serve a meaningful measures of hepatic elimination.

However, for a drug which confers multicompartment characteristics on the

body, biologic half-life does not reflect properly the intrinsic metabolic activity

of the liver. Clearance on the other hand, is a direct measure of this activity

regardless of the number of compartments conferred upon the body by a drug

provided the liver is an integral part of the central compartment (Perrier and

Gibaldi, 1974).

1-Naphthylacetic acid confers a one compartment model, while diphenyl-

acetic, hydratropic and phenylacetic acids confer a two compartment model on

the rabbit, but the rate of distribution (alpha) of diphenylacetic and hydratropic

acids is almost twice that of phenylacetic acid a factor which is reflected on the

alpha half life (aT1). The total body water of 2.2 kg rabbit is about 1. 7 litres 2

(extrapolated from the data of West et al. (1948) on 3. 5 kg infant with 2. 7 litres

body water). The apparent volume of distribution for phenylacetic acid is more

than the total body water of the rabbit and indicates that this acid is highly bound

to extravascular tissues. This acid has also a relatively higher blood clearance

compared with the other acids. 1-Naphthylacetic and hydratropic acids are

distributed into 17 and 32% respectively of body water and have very low blood

clearance and thus indicating a high vascular tissue binding for these acids.

Diphenylacetic acid on the other hand is 62% distributed into the body water and

has a fairly moderate blood clearance. The elimination rate constant is very

low for phenylacetic and this is also reflected on its very high biologic half-life

(flT1) when compared with the other acids. These pharmacokinetic parameters

161

in general may be influenced by the degree of binding of these acids to the

metabolic sites (in this case the conjugation sites, mitochondria and endoplasmic

reticulum), their affinity for the conjugating enzymes associated with these

structures and also their rate of conjugation with amino acid and / or glucuronic

acid.

The binding of 1-naphthylacetic, diphenylacetic, hydratropic and phenylacetic

acids to mitochondria and microsomes suggests a different binding capacities.

This differential binding capacity may arise as a result of a possible difference

in the mitochondrial and microsomal partition coefficient of these acidsdegree

of ionization, size and molecular geometry. These may also be the factors

influencing the glycine conjugation and the glucuronidation affinity of these acids.

Diphenylacetic and hydratropic acids have low binding capacity for mitochondria

and these did not form glycine conjugates while phenylacetic and 1-naphthylacetic

acids have a higher binding capacity and formed glycine conjugates although much

more in the case of phenylacetic acid. On the other hand phenylacetic acid has

the least binding capacity for the microsomes and it does not form a glucuronie

acid conjugate, whereas diphenylacetic, hydratropie and 1-naphthylacetic acids

with high binding capacity formed glucuronic acid conjugates although at different

rates and have different affinities for the conjugating enzymes as well. The

lipid solubility of a compound is known to determine not only its penetration into

cells and metabolic sites but also influences its metabolism (Gaudette and Brodie,

1959). Highly lipid soluble N-alkylamines are dealkylated in the microsomes

while the low lipid soluble ones in the mitochondria (Gaudette and Brodie, 1959 ;

Axelrod, 1956 ; Mackenzie and Frisell, 1958). The arylacetic acids seem to fall

into this discrimination pattern as regards conjugation sites and type of conjugation.

1-Naphthylacetic and diphenylacetic acids which have high binding capacity for

162

the microsomal fractions and form glucuronides have high lipid solubilities

while phenylacetic which has low lipid solubility has low binding capacity for

microsomes but high binding capacity for mitochondria does notform a glucuronic

acid but an amino acid conjugate. Although the difference in the lipid solubilities

of phenylacetic and hydratropic acids is small, it may be suggested that hydra-

tropic acid is able to fluidise the lipid region of the microsomes, a character -

istic Cater et al. (1974) have indicated may qualify a compound for micro-

somal metabolism.

The binding of the arylacetic acids to the subcellular fractions as reported

here, is rather a static reaction but correlating the binding to the conjugation

reaction (the kinetics) which is a dynamic reaction it may be possible to have

an insight to what happens in vivo when these compounds are administered to

an animal. Since non-polar compounds and their lipophilic metabolites have

more affinities for the metabolic sites than their polar conjugates (see Bickel

and Bonier, 1974 ; Glaumann et al. 1970 ; Nilsson et al, 1971) it follows

therefore that amino acid and glucuronic acid conjugates of these arylacetic acids

do have lower affinities for the conjugation sites than the parent compounds.

The conjugates therefore are excreted as soon as they are formed in the body

setting up a dynamic process which may be representative of the pharmaco-

kinetic study in the rabbit. It may be argued therefore that the pharmacokinetic

parameters calculated represent a combination of the binding to subcellular

fractions, metabolic conjugation and the influence the physico-chemical properties

of these arylacetic acids have on their metabolism in the rabbit.

Summary and Conclusion

1-Naphthylacetic acid confers a one-compartment model, while phenylacetic,

diphenylacetic and hydratropic acids confer a two-compartment model on the

rabbit. While phenylacetic acid has a high apparent volume of distribution, a high blood

163

clearance (Q) a high biologic half-life and a very low elimination rate constant,

1-naphthylacetic, diphenylacetic and hydratropic acids have low apparent volume

of distribution, relatively lower blood clearance, a low biologic half- life and a

high elimination rate constant. It may be suggested that the pharmacokinetic

parameters of phenylacetic acid may represent the index for arylacetic acids

which are conjugated mainly with an amino acid whereas those of the other three

acids for arylacetic acids which are conjugated mainly with glucuronic acid.

On the basis of the in vitro work it is also suggested that the pattern

of conjugation of an arylacetic acid in the rat is influenced by its affinity for

uptake (as measured by binding) by mitochondria and the endoplasmic reticulum

and affinity for the conjugating enzyme systems associated with these -structures.

164

CHAPTER SEVEN

Page

General Discussion and Conclusion 165

165

General Discussion and Conclusion

The previous chapters constitute an examination of the metabolic

conjugation pattern of 1-naphthylacetic, diphenylacetic and hydratropic acids

in some selected species and the effect of dose level on the pattern of

conjugation in the rat of these acids. Additionally the pharmacokinetic behaviour

of these acids and phenylacetic acid in the rabbit, and their relative affinity

for rat mitochondrial and microsomal fractions and the conjugating enzymes

associated with these structures have been studied.

1 -Naphthylacetic acid showed a distinct species variation in its metabolic

conjugation pattern in all the species studied, man and the Old World monkeys

(except the marmoset) and bushbaby mainly with amino acids and to a small

extent with glucuronic acid,the eat with amino acids and to a small extent with

glucuronic acid the cat with amino acids extensively, the rat and rabbit

principally with glucuronic acid and the fruit bat entirely with glucuronic acid.

Diphenylacetic acid on the other hand is conjugated with glucuronic acid irrespective

of the species, but hydratropic acid is conjugated mainly with glucuronic acid in

man, rhesus monkey, rat and rabbit and with both amino acids and glucuronic

acid in the cat. This study and similar work reported elsewhere in the

literature suggest that there is a shift from amino acid to glucuronic acid

conjugation with increase in complexity of the chemical structure of arylacetic

acids in most species. This concept applies to a limited degree to cat and some

other carnivores that are defective in glucuronic acid conjugation (French et al, 1974 ;

Caldwell et al, 1975b). Thus, arylacetic acids of relatively simple chemical

structure such as phenylacetic acid and its simple derivatives form mainly amino

acid conjugates (James et al, 1972 a,b) where as other more complex arylacetic

acids such as diphenylacetic acid (see Chapter 4), indomethacin (Harman et al, 1964)

166

and iopanoic acid (McChesney and Hoppe, 1954) form glucuronic acid

conjugates.

Arylcarboxylic acids such as benzoic and salicylic acids are conjugated

in man, and sub-human primates and non primate species with amino acid and

glucuronic acid the quantities differing with species. The simple primary

arylacetic acid such phenylacetic acid is conjugated mainly with amino acid

irrespective of species, but there is discrimination in the type of amino acid utilised.

The glutamine conjugation is restricted to man, Old and New World monkeys

while glycine conjugation is utilised by the rest. Taurine conjugation seems to be

predominate in the New World monkeys (except the marmoset) and the bushbaby

although their is a haphazard quantitative distribution in the other species (James

et al , 1972 a). In the more complex primary arylacetic acids such as

1-naphthylacetic acid there is a shift from amino acid to glucuronic acid

conjugation in most species except the cat, some other carnivores (see Chapter 3 ;

Caldwell et al, 1975b)the New World monkeys (except the marmoset) and the

bushbaby which largely conjugate this acid with glycine and taurine. For indolyl-

acetic acid the shift towards glucuronic acid conjugation was observed in man and not

in the other species, but there was glutamine conjugation in man, New and Old

World monkeys, a conjugation reaction which seems to be restricted to these species

(Bridges et al, 1974). With further increase in the complexity of the chemical

structure of primary arylacetic acids, there appears a distinctive discrimination

in the metabolism and conjugation pattern between the carnivores and the rest

of the species. Man conjugates indomethacin with glucuronic acid but the dog ex-

cretes it unchanged while, rat, guinea pig and the monkey conjugate its metabolised

derivative (N-deschlorobenzoyl and 0-desmethyl) with glucuronic acid(Harman et al

1964). Another example is p-(cyclopropylcarbonyl) phenylacetic acid (SQ 20, 650)

167

which is conjugated with glucuronic acid by the monkey but with taurine

by the dog (Lan et al. , 1975).

There has been little information in the metabolic conjugation pattern

of secondary arylacetic acids inmany species, but the available information

for hydratropic acid suggest a total shift from amino acid to glucuronic acid

conjugation in the species examined except in the carnivores. Although

hydratropic acid is conjugated with glucuronic acid by man, rhesus monkey,

rabbit, rat, the cat still conjugates this acid with glycine and taurine (38% of

the dose) and glucuronic acid acid (40%) (see Chapter 5). The dog is also

reported to conjugate this acid with glycine (Kay and Raper, 1922). It is

interesting to note that with the more complex secondary arylacetic acid,

diphenylacetic acid there is a total shift from amino acid to glucuronic acid

conjugation irrespective of species (see Chapter 4) a result which is in agree-

ment with the finding of Miriam et al , (1927a)in man, dog and rabbit.

The only information available on the metabolic conjugation of simple

tertiary arylacetic acid is the work of Robinson & Williams (1955) which showed

that a, a -dimethylphenylacetic acid is conjugated with glucuronic acid by the

rabbit. Miriam et al (1927 b) have also reported that the more complex chemical

structure, triphenylacetic is totally excreted uncharged by rabbit, dog and man.

This shift in the conjugation pattern with increase in the complexity of

the chemical structure of arylacetic acids has confirmed the highly substrate-

dependency of some conjugation reactions which are known to be deficient in

some species. For example, the fruit bat does not form glycine conjugate

with benzoic, 1-naphthylacetic and diphenylacetic acids but does with phenylacetic

acid (see Bababunmi et al, 1973 ; Ette et al , 1974 ; Chapters 3 and 4). The

cat is known to be deficient in glucuronide formation, and does not form glucuronic

168

acid conjugates with benzoic, phenylacetic and 1-naphthylacetic acids but

does with hydratropic and diphenylacetic acids (Bridges et al, 1970 ; James et al. ,

1972 a; Chapters 3, 4 and 5).

The effect of dose on the metabolic conjugation pattern of 1-naphthylacetic,

diphenylacetic and hydratropic acids has also been studied in bile duct- cannulated

rats (Chapters 3,4 and 5). At low doses the glycine conjugate is the major

urinary metabolite of 1-naphthylacetic acid but with increase in dose glucuronic acid

conjugation takes over, but at both low and high doses diphenylacetic and hydra-

tropic acids are conjugated extensively with glucuronic acid. This finding suggests the

that/glycine conjugation mechanism has limited capacity and that when it is exhausted

the glucuronic acid mechanism takes over, but with any compound which is con -

jugated with glucuronic acid only the pattern of metabolites will not change very

much with dose unless this is excessive. The cat as already mentioned has a

defective glucuronic acid conjugation for certain compounds including benzoic

acid. Consequently benzoic acid is more toxic to the cat than most common species

of animals (Bedford & Clarke, 1971, 1972), for the cat conjugates benzoic acid

entirely with glycine which is limited in supply, and glucuronic acid conjugation is

not available to take over when large doses of benzoic acid are administered. The

effect of dose on the shift from sulphate and glutathione conjugations to

glucuronic acid conjugation was discussed in Chapter 3.

These differences in the metabolic conjugation pattern of the aryl -

carboxylic and --acetic acids may be due to differences in their physico- chemical

properties which may influence their pharmaco-kinetic behaviour, when

administered to an animal, degree of penetration to the conjugation sites

(mitochondria and microsomes) and the affinity for the conjugating enzymes

associated with these structures. Some physico-chemical properties of some

169

selected acids are shown in Table 7:1. The acids are 99.9% ionized at pH 7.4

and thus the ionization does not play a significant role in there different metabolic

conjugation pattern, but Table 1:11 shows the influence of pKavalues on the pattern

of metabolism of chlorophenols. The data in Table 7:1 suggests that log P (P is

the molar partition coefficients between octanol and water) and the substitution

at the a-carbon and molecular geometry are the most influencing factors in

determining the metabolic conjugation pattern of arylacetic acids. Phenylacetic

acid with the least log P value (1.43) has planar molecular geometry and no a-carbon

substitution and is conjugated mainly with amino acids in all the species, but

the smaller molecule, benzoic acid with slightly higher log P (1.87), though

with planar molecular geometry has its a-carbon totally substituted and this

acid is conjugated with amino acid and glucuronic acid except in the carnivores

which conjugate mainly with amino acid. 1-Mphthylacetic acid on the other hand

has a high log P value (3.14), and is conjugated with both amino acid and

glucuronic acid (except in some carnivores) but like phenylacetic acid it has a

planar molecular geometry. Hydratropic and diphenylacetic acids have log P

values of 1.93 and 3.09 respectively, and have non-planar geometry with an

a-carbon substitution (one proton at a-C) are conjugated mainly with glucuronic

acid except in the case of hydratropic acid in the cat, ferret and dog. a, a-

Dimethylphenylacetic and triphenylacetic acids, although with high log P values

(2.46 and 5.08 respectively), with non-planar geometry and have their a-carbons

totally substituted, only a, a-dimethylphenylacetic acid has been reported to

form a glucuronic acid conjugate (Robinson & Williams, 1955) whereas triphenylacetic

acid is excreted unconjugated (Miriam et al. , 1972 b). This suggests that the

very high log P value is not a favourable factor for glucuronic acid conjugation,

and one wonders if the relationship between glucuronic acid conjugation and log P

Table 7:1

Characteristics of some Arylacetic Acids

Compound Log P ++

p-Ka

Molecular geometry

a -Carbon Molecular

Conjugation with :

Planar Non-planar Amino Glucuronic substitution(No. weight acid acid

Benzoic acid

Phenylacetic acid

1 -Naphthylacetic acid

Hydratropic acid

Diphenylacetic acid

a, a-Dime.thylphenylacetic acid

Triphenylacetic acid

1.87

1.43

3.14

1.93

3.09

2.46

5.08

4.21

4.32

4.21

4.6

3.94

3.96

+

+

+

-

-

of protons on a C)

122

136

186

150

212

164

288

+

+ **

+ *

-

-

-

+

-

+

+

+

+

+ (0)

- (2)

- (2)

+ (1)

+ (1)

+ (0)

+ (0)

except in the carnivores 4+ data obtained or calculated from Fujita et al . (1964) and Nys & Rekker (1973)

** except in the fruit bat

171

values is parabolic since at low and very high log P values there is no

glucuronic acid conjugation but at moderately high log P values glucuronic acid

conjugation seems to be favoured, or in the case of triphenylacetic acid whether

a steric effect is the inhibitory factor in this acid not being conjugated. The

glucuronyl acceptor substrates are lipid soluble and the affinity of enzymes

increase with their lipophilicity (Hanninen and Alanen, 1966), but from the

relationship between glucuronic acid conjugation and log P values shown above,

it is suggested that the compounds which are too lipophilic become trapped in

the external lipids of the cell walls and do not enter the cell and tim'efore have

no access to the metabolic conjugation sites (endoplasmic reticulum). A

similar concept was used to explain the parabolic relationship between lipo-

philic character and isoeffective doses of ethers and alkanes (Jeppsson, 1975)

and partition coefficient and narcosis (Hansch et al. , 1968). The conjugation

pattern shown by arylacetic acids is also seen in other compounds, for example

phenols of small molecular size are mainly conjugated with sulphate (Williams 1938 ;

Capel et al. , 1972) while those with large molecular size are conjugated mainly

with glucuronic acid (see Dodgson et al. , 1948 ; Mazur and Shorr, 1942). The

physico-cheinical properties shown in Table 7:1 may also be the factors directing

the pharmacokinetic behaviour of these acids when administered to an animal.

The pharmacokinetic behaviour of 1-naphthylacetic, diphenylacetic,

hydratropic and phenylacetic acids has been studied in the rabbit. The first

three acids are conjugated mainly with glucuronic acid, while phenylacetic acid

is conjugated mainly with glycine in this species. The result (see Table 6:5)

shows that phenylacetic acid has a high apparent volume of distribution, a

relatively high blood clearance, a low elimination rate constant and a high

biological half life (beta half life) while the reverse is true for 1-naphthylacetic,

172

hydratropic and diphenylacetic acids. This indicates that phenylacetic acid

is more quickly cleared from the blood and is heavily bound to extravasctlar

tissues and also has a relatively poor metabolic elimination mechanism as

compared with the other acids which seem also to be restricted in their binding

to either the vascular tissues or a preferential target site (es. endoplasmic

reticulum). From the binding studies (see Chapter 6) 1-naphthylacetic,

diphenylacetic and hydratropic acids have a higher affinity for the rat m5.crosomal

fraction than phenylacetic acid and the reverse is the case for the rat mIto-

chondrial fraction. The lipid solubility of a compound is known to determine

not only its penetration into cells and metabolic sites but also influences in. its

metabolism ( Gaudette and Brodie, 1959). Highly lipid soluble N-alkylamines are

dealkylated in the microsomes while the low lipid soluble ones in the mitochondria

(see, Gaudette and Brodie, 1959 ; Axelrod, 1956 ; Mackenzieand Frisell, 1958).

The in vitro conjugation study of 1-naphthylacetic, diphenylaceticlhydratropic

and phenylacetic acids with glucuronic acid and glycine (see Chapter 6) is in

agreement with the in vivo glucuronidation and amino acid conjugation capabilities

of these acids and has a direct relationship to their affinities for the metabolic

conjugation sites (as measured by binding to mitochondria and microsomal

fractions). 1-NaphthylaceticIdiphenylacetic and hydratropic acids are more lipid

soluble than phenylacetic acid thus the result of this study is in agreement with the

concept of Gaudette and Brodie, (1959). But non-polar compounds and their

lipophilic metabolites have more affinities for their metabolic sites than their

polar conjugates (see Bickel and Borner, 1974 ; Glaumann et al. , 1970) ; it follows

therefore that amino acid and glucuronic acid conjugates of these arylacetic acids

do have lower affinities for the conjugation sites than the parent compounds. The

conjugates therefore are excreted as soon as they are formed in the body setting

173

up a dynamic process which may be representative of the pharmacokinetic

study in the rabbit.

Amino acids and glucuronic acid have different conjugation mechanisms

as described in Chapter 1. For amino acid conjugation the substrate has to be

activated first (R. COSCoA) before reacting with the conjugating agent, whereas

for glucuronic acid conjugation the substrate reacts directly with the activated

(UDPGA) conjugating agent. The question arises as to whether or not the

arylacetic acids which do not form amino acid conjugates do form the activated

intermediate (R. COSCoA). These intermediates have been reported for benzoic,

phenylacetic , salicylic and.bile acids (Schacter and Taggart, 1953 ; Moldave and

Meister, 1957 ; Elliott, 1956 ; Tishler and Goldman, 1970) which are subsequently

converted to the respective amino acid conjugates by the specific amino acid N-

acylase . It may be interesting therefore to investigate at what stage in the

amino acid conjugation mechanism compounds like diphenylacetic acid are

defective. For example if the activated intermediates diphenylacetyl-AMP and

-CoA are formed then the deficiency is at the amino acid N-acylase enzyme stage

which may be explained in the terms that the activated intermediate -enzyme binding

forces may be too small to force the enzyme into the catalytically active conformation

and hence no conjugation takes place. On the other hand if these activated inter -

mediates are not formed it may be suggested that this reaction is not thermodyna-

mically feasible which may be a result of the chemical structure and the intrinsic

energy of the compound) and therefore prefer a different metabolic conjugation

pathway which is thermodynamically feasible.

In conclusion the metabolic conjugation pattern of arylacetic acids is

greatly influenced by the physico-chemical properties of the acid (such as lipid

solubility, chemical structure and molecular size and geometry) and the affinity

174

for the conjugation sites (mitochondria and endoplasmic reticulum) and the

conjugating enzymes associated with these structures.

APPENDIX.

175

Contents

Pages

176

Mass Spectral Fragmentation Pattern

1-Naphthylacetylglycine Methyl Ester 176

1 -Naphthy lac ety I -L -glutamine Methyl Ester 176

1-Naphthylacetyltaurine Methyl Ester 176

Diphenylacetyiglycine Methyl Ester 176

Diphenylacetyl -L -glutamine Methyl Ester 177

Diphenylacetyltaurine Methyl Ester 177

Diphenylacetylglucuronide Methyl Ester 177

Hydratropoylglycine Methyl Ester 178

Hydratropoyl-L-glutamine Methyl Ester 178

Hydratropoyltaurine Methyl Ester 178

Hydratropoylglucuronide Methyl Ester 178

105 cHpONFICH

2COOCH

3.

91\1

176 Mass Spectral Fragmentation Pattern

1 -Naphthylacetylglycine methyl ester

142 11681 1

CH IC*11CH2 COOCH3

1-Naphthylacetyl-L-glutamine methyl ester

142I164 I 1

CH 14THCHCOOCH 2I,_ ,_ L _ _ _ 3 — —116

0 - - --1 T H2

I CH 1 2 I CONH2 I

187

1 -Naphthylacetyltaurine methyl ester

1421 166

I 115 I I

CH21CONH1CH2CH2S03ICH3

185

Diphenylacetylglycine methyl ester

167

167i

I 1107

I

I ; CHpOp

COCH3

43

OH

177

Diphenylacetyl-L-glutamine methyl ester _

167

10 I CHICONHCHCOOCH3

1\ 1 CH 2

I CH, 1187 1 - CONH2

Diphenylacetyltaurine methyl ester

167 I 166

152;

CHFONHCH2CH2S031CH3

Diphenylacetylglucuronide methyl ester

178

Hydratropoylglycine methyl ester

I 116

1

- I-1- I CH4ONHCH2COOCH3

I 91

1051

Hydratropoyl-L-gliitamine methyl ester

1 187 CH

i 3 CH;

I

C ONHCHCOOCH3 1

H2 —

C — 1 I

I CH2

bONH2

— 116

Hydrotropoyltaurine methyl ester

1166 CH I

r „3-1, CHCONHCH CH SO CH

I I 2 2 3 3 I

911 1051 1131

Hydratropoyleucuronide methyl ester

j 191

I COOCH3

CH / I e -p xpo° I I I OH

91 OBI

101 173

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