CONFIDENTIAL

"THE MECHANISM OF HEPATOTOXICITY OF AN ANTI-ALLERGY COMPOUND"

by

Charles Thomas Eason, M .I.B io l.

A thesis submitted in accordance

with the requirements of the University of Surrey for the degree of Doctor of Philosophy

Department of Biochemistry

University of Surrey Guildford, Surrey, September, 1981

ProQuest Number: 10798409

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SUMMARY

The an ti-a lle rgy drug FPL 52757 (Tisons L td .,) 6 ,8 -d ie thy l-5 -

hydroxy-^oxo-AH^-benzopyran^-carboxylic acid produced mild and reversible hepatotoxicity in some patients during c lin ica l t r ia ls . The purpose of this work was to elucidate the mechanism by which FPL 52757 caused hepato- to x ic ity . However, since further humans could not be tested the work is lim ited to animal models. Toxicity studie s ; showed that the beagle dog was

the only animal species of ten tested which was readily susceptible to the

hepatotoxic'effect o f the compound. In this project hepatotoxicity could not be demonstrated in ferrets or rats in special studies designed to

reduce resistance to drug-induced hepatotoxicity. S im ilarly , hepatic microsomal enzyme induction in dogs by pretreatment with phenobarbitone and protection against cytotoxicity by pretreatment with methionine provided no evidence for involvement of a reactive metabolite in the hepatoxicity.

Work by Fisons and additional studies in this project showed that the compound was extensively metabolised and rapidly eliminated by most species. Plasma clearance by the dog and man was in comparison slow,and due mainly to elimination of unchanged compound. As the dog and man are

particu larly susceptible to FPL 52757 induced hepatotoxicity the parent compound was implicated as the hepatotoxic agent. Species which cleared

the compound rapidly compared with the dog did not readily show a hepato­toxic response.

The tox ic ity o f FPL 52757 tn retrograde b ilia ry infusion studies in ra t and in the lysis of erythrocytes was studied and related to the

detergent properties o f the drug. Other chromones were also examined and a detergency/toxicity/structure/activity relationship was established.

Pharmacokinetic studies in dog were carried out and showed that FPL 52757 resulted in increases in b ilia ry excretion of alkaline phosphatase, gamma glutamyl-transpeptidase and 5 '-nucleotidase which parallelled the b ilia ry

concentration of FPL 52757 and was accompanied by reduction in b ile flow. The excretory mechanism of FPL 52757 in the b ilia ry trac t was shown to be saturable

and could result in high hepatocellular drug concentration and subsequent l iv e r damage in the dog. By implication from this work- the mild hepatotoxic e ffec t in man was lik e ly to have been caused by the slow clearance and

consequent build up in the liv e r and b ilia ry trac t of unchanged FPL 52757, the to x ic ity observed being mediated by the membrane damaging potential o f high concentrations of the compound. As a consequence of this work i t could

confidently be predicted that lower concentrations would be perfectly safe in man.

- i i i -

acknowledgements

I should lik e to thank a ll my colleagues and friends at the University o f Surrey and Fison's Pharmaceutials for a ll th e ir help and advice. In particu lar I am indebted to my supervisors,Professor D.V. Parke, Dr. Bruce Clark and his associate a t Fison's,Dr. D. Smith.

My thanks are also due to Dr. R. Hinton and Dr. B. Mullock for th e ir advice and assistance with b ile enzymes and the electro­phoresis experiments, and to Dr. G. Wilson of Fison's for his s k ilfu l! surgical assistance in the pharmacokinetic dog studies.

Last, but not least, my thanks go to Mrs. Margaret Whatley for typing this thesis and guidance throughout my stay at Guildford.

To Ruth, Tom, Mickie, Rod and Candy.

- iv -

CONTENTSPage

Summary . . . . . . . . . . . . . . . iAcknowledgements . . . . . . . . . . . . i i iCo n to nts »«. ~ . . . . . . . . . . . . i v •

CHAPTER ONE Introduction . . . . . . . . . ]

CHAPTER TWO Experiments designed to elucidate the role of a reactive metabolite in FPL 52757 hepatotoxicity . . . . . . • •• 24

CHAPTER THREE The pharmacokinetics o f FPL 52757 . . . ^

CHAPTER FOUR The physico-chemical properties o f chromones 63

CHAPTER FIVE The relationship between chromone to x ic ityand detergency . . . . . . . . . 71

CHAPTER SIX The hepatobiliary toxicokinetics of FPL 52757in rat and dog . . . . . . "- ••• ' 87

CHAPTER SEVEN Summary and conclusions . . . . . . 100

BIBLIOGRAPHY . . . . . . . . . . . . . . . 109

Appendix I . . . . . . . . . . . . . . . . 115

Appendix 2 . . . . . . . . . . . . . . . 124

Appendix 3 (to Chapter 6 ) . . . . . . • • • 1 5 0

Appendix 4 . . . . . . . . . . . . . . . 161

CHAPTER ONE

Introduction

DRUGS, POISONS AND TOXICITY

A drug, according to the W.H.O. is "any substance which, when taken into the liv in g organism may modify one of its functions".Drugs, in i t ia l ly o f herbal origin-have been taken by man from ancient time for medicinal (e .g . Cinchona), pleasurable (e .g . opium and cocaine) and even religious (e .g . mescaline) reasons. Alongside the beneficialeffects of medicines come the problems of intoxication, addiction, and

side-effects .

The naive division o f xenobiotics into drugs and poisons is man made and does not take into account Ehlich's concept o f selective to x ic ity . This misunderstanding has been propagated despite an apparent grasp of the fundamentals o f toxicology from as early as the fourteenth

century. Paracelsus (1493-1541) notes that, "All things are poison for there is nothing without poisonous qu a lities . I t is only the dose which makes a thing a poison".

Most modern drugs are selectively toxic compounds, exercising the ir

therapeutic effects by the selective poisoning o f some infectious or invasive agent or by the selective inhib ition of some enzyme system, subcellular membrane or endogenous substrate. Their c lin ica l response is frequently dramatic; the sp ecific ity of pharmacological action is high, and because of the frequently close proximity of the therapeutically effective dose to toxic leve l, dosage is often c r i t ic a l . When considering treatment the risks must be weighed against the possible benefits to the

patients. For drugs'with a high potential for generalised to x ic ity the prognosis fo r the untreated patient should be poor.

The twentieth century heralds the beginning of the drug explosion, starting slowly [e.g. asparine (Dreser, 1899 ) , novocain (Einhorn, 1905) and Salvarsan (Ehlich and Bartheim, 1912] and quickening considerably a fte r 1935 with the discovery of prontosil for the treatment of systemic

bacterial infections. An ever increasing l is t of toxic or adverse effects has accompanied this pro liferation of drugs. Commonly the adverse effects

are minor, and lim ited to diarrhoea, nausea or vomiting and the benefits to the patient outweigh the discomfort experienced. However, numerous

cases of serious toxic si de-effects have occurred over the decades.

More recently an increased awareness of diseases has accompanied drug

development, but this has not eliminated the chance that unpredicted serious side-effects may occur.

Causes of Drug Toxicity

The following causes o f to x ic ity lis ted below are b rie fly elaborated in this introductory section:-

- Inappropriate drug formulations- Drug formulation affecting absorption rates- Pre-existing pathological conditions affecting drug excretion

ratesPre-existing pathological conditions affecting drug metabolism

- Drug interactions (general)- Drug interactions caused by induction or inh ib ition of drug

metabolizing enzymesHypersensitivity and iodiosyncratic reactions to drugs

The examples chosen (see Table 1 .l)to illu s tra te the causes of to x ic ity show how one or more factors may combine or how a chain of events might escalate to result in a toxic e ffe c t. They are not intended

as a comprehensive review, but are presented to form a general introduction to the possible types of mechanism involved in drug to x ic ity .

Inappropriate drug formulations

Early drug to x ic ity resulted not only from the active components but also from impurities and inappropriate formulations. This was highlighted in 1937 in the U.S.A. when an e l ix i r of sulphanilamide dissolved in d i­ethylene glycol was marketed. The recommended dose contained su ffic ie n t diethylene glycol to cause death.

Changes in drug formulation affecting absorption rates

Digoxin is a classic example of a drug with therapeutic and toxic

doses only marginally separated. Changes in the formulation o f a digoxin preparation have been shown to lead to major changes in plasma levels o f the drug with consequent toxic si de-effects.

Pre-existing pathological conditions affecting drug excretion rates

In patients on streptomycin therapy who are suffering from impaired

kidney function, the corresponding slow drug clearance can result in severe damage to the eighth cranial nerve.

Pre-existing pathological conditions affecting drug metabolism

The effects o f any drug undergoing metabolism in the liv e r w ill be affected by hepatic disease. Drug metabolism w ill be impaired and the

risk of drug to x ic ity is increased, e.g . severe haemorrhage can follow dicumerol therapy. An extreme example of this phenomenon occurs in patients with congenital deficiency o f glucose-6 -phosphate dehydrogenase. Haemolysis can follow treatment with phenacetin, primaquine and n itro ­furantoin in these individuals.

Drug interactions

Interactions between drugs may cause unpredictable toxic side effects

and may take place simultaneously a t a number of s ites . For example, the anti-inflammatory agent phenylbutazone competes with the anticoagulant warfarin for plasma binding, metabolic deactivation and renal tubular excretion. The overall effects are a reduction of the plasma level o f warfarin and its more rapid clearance from the body, yet because more of the drug is available at the receptor sites in the liv e r the anticoagulant a c tiv ity is increased (O 'Reilly and Levy, 1970).

Drug interactions caused by induction and inhibition of drug metabolising enzymes

The toxic consequences of inducing hepatic drug-metabolising enzymes can be illu s tra te d by the interactions of phenobarbitone and bis-hydroxy- coumarin. The anticoagulant is more rapidly metabolised when administered

with phenobarbitone and withdrawal of phenobarbitone w ill eventually lead to restoration of drug metabolising enzymes to th e ir normal leve ls . The

corresponding reduction in the metabolic deactivation of the anticoagulant may resu lt in spontaneous haemorrhage i f the dose of bis-hydroxycoumarin is not simultaneously reduced (Cuci.nel],,et al_, 1965). Metabolic inh ib ition by monoamine oxidase in h ib ito r w ill result in decreased

metabolism of a number of drugs, e.g . pethidine which can lead to intoxication (Clark and Thompson, 1972).

TABL

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x> CU <U f0 X I tO E ' fO fO E U tO<c E O O (0 Q O n r n r < c to n r

i -P CUo E to S Z-P . • r - -PC L tO x j o x j -P E

to CU «— O E o CU •r- Er— E CU o cu o cj E *r—CU •P > i— E ■ 1— to .to> to CU X) n X I E 4 - tocu i— o CU E <u

r— 4- <4- -r- 4 - S Z fO +->cu O XJ ° * ° o a S *r-o XJ E O

E t -to

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LU £r~ CJ 1— cu SZ CJ - r -

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x : x cu X cu C l cu CU E= CU to CJ CO cu CJ -Q O XJ x :o E O E CU E E Z3 E E n 5 E 3 to -P to ■p

CU -p U CO O E O u O U CU *a o ■a -p to -P•I- E E -1— CU ■P X CU -r- cu x: cu *P E cu cu CU CJ oQ -r- i—i -O Q to cu Q Q CL o r o •i— cc E o r £ -p

E -P .to E

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i— >> i— x : -o x : - a •r— l > x :O r— 3 ■P cu •P <u o a E XT •PE CD E to E to E •i— »r— O -P E

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Hypersensitivity and idiosyncratic to x ic ity

These reactions occur in a small number o f patients. This type of response occurs with p e n ic illin . The mammalian to x ic ity is so low that i t can be regarded as the most innocuous o f a ll an ti-bacteria l drugs. However,.a small proportion of patients become sensitized to i t . These two unpredictable classes include severe and dangerous toxic effects such as liv e r necrosis, cholestatic jaundice, blood dyscrasias and anaphylactic shock.

"The extent and rate o f absorption, d istribu tion , metabolism and

excretion of a drug may a ffec t not only the pharmacological response to the drug but also its potential toxic ity" (Parke, 1972). From the l is t

of causes of drug to x ic ity a ll but the f i r s t and las t had in common a pharmacokinetic basis of to x ic ity , illu s tra tin g the importance of this

phenomenon in drug to x ic ity . The subject o f toxico-kinetics and mechanisms of to x ic ity are further explored la te r in this introductory chapter in the section on metabolism and species variation in pharmacokinetics.

Safety Evaluation of Drugs in Animals

Dreser in 1899 evaluated the to x ic ity of acetyl s a lic y lic acid on "one small fish and two freshly captured frogs'. Animal to x ic ity testing has

become increasingly sophisticated; however, the aims are s t i l l the same, namely, to reduce the risk o f adverse drug-effects in man. The testing

of potential drugs in animal species is now well established. Prior to

marketing, a new drug must undergo successful c lin ica l t r ia ls and before th is the metabolic and pharmacokinetic p ro file o f a drug must be intensively explored in animals in both acute and chronic to x ic ity tests. Administration

of the drug to a small number of human volunteers a t this stage to elucidate the pharmacokinetic p ro file in man can greatly improve the choice o f animal species for long term testing, thus increasing the chance o f finding toxic side-effects in animal species that may eventually occur in man.

No species exactly mimics man in the way i t handles a ll chemical substances. Nevertheless, for every chemical there may be an animal species with sim ilar metabolism and excretion to man. In some instances past experience with a compound w ill enable reasoned choice of suitable species for testing a sim ilar drug. The use of a number of species including a t least one non-rodent type is ju s tif ie d , since a drug action that occurs

in a number of species probably involves a common physiological

mechanism which is lik e ly to be present in man. Conversely, a

s ide-e ffect seen in only one o f a number of species might be thought to be an e ffec t peculiar to that species and less lik e ly to occur in

man, unless i t is this species that man resembles more closely than any other.

Even when animal species handle a drug sim ilarly to man, other major differences, such as re la tive organ weight, l i f e span and

gestation period w ill provide d iff ic u lt ie s in extrapolation of toxicological data from animals to man. Hence, in protecting the human population from drug to x ic ity , animal studies should be regarded

as risk-reducing and not risk-elim inating. There s t i l l remains the

chance that unpredicted side-effects may occur as with thalidomide

(Curran, 1971), practolol (Wright, 1974), and clo fibrate (O liver,1978). Nevertheless, with current comprehensive practices, predict­a b ility is high and in addition to protecting man from drastic toxic effects

animal studies can give information on lesser undesirable effects which

might occur during c lin ica l tr ia ls and la te r when a drug is used more widely.

Pharmacokinetics and the Consequences of Species Variation

Drug metabolism

The important role o f metabolism in the determination o f the pharmaco­kinetic p ro file of a drug has been alluded to in the preceding pages.Apart from elimination from the body o f unchanged compounds, fo r example

by the kidney, the pharmacological and therapeutic actions o f a drug are lim ited by the metabolism brought about mainly by enzymes o f the liv e r .This metabolic deactivation is purely fortuitous, resulting from the action of enzymes whose function is presumably to protect against the accumulation

and undesirable effects o f anutrients, naturally present in the food and

in the environment (Parke, 1968). The behaviour is the same towards drugs as towards the natural xenobiotics and although leading ultim ately to the

deactivation and excretion of the drugs, may a t f i r s t resu lt in enhanced therapeutic a c tiv ity (activation ), changed a c tiv ity or increased to x ic ity

(in tox ication ). In ter and intra-species variatipn in the pharmacokinetic response to drugs may be great. Within a given population there w ill be

a normal distribution in the pharmacokinetic p ro file of a drug and in

some individuals variation can be extreme. (This has already been

illu s tra te d by the example of defective phenacetin metabolism in

individuals with defic ient glucose-6-phosphate dehydrogenase). The consequences o f inter-species variations in toxicology and th e ir under­standing are a pre-requisite for sound drug safety evaluation.

Species variation in anutrient metabolism

Pharmacokinetic differences, that is , differences in absorption, distribu tion , metabolism and excretion w ill account for species variation

in response to anutrients. However, the type of mechanism o f to x ic ity and how an animal species ‘ handles' a compound w ill d ictate the response

of the individual species e ither promoting or lim iting the toxic

potential o f a drug. Since the extent o f metabolism w ill be shown to

play a key role in the to x ic ity o.f the drug under investigation in this thesis, thfs section includes a lim ited outline on the metabolism and excretion of foreign compounds (fo r comprehensive reviews see, Parke 1968;La Du e t aT, 1971; Goldstein e t £l^, 1974; Gerok e t a\_9 1975 and Parke,1980).

Drug metabolism undertaken mainly in the liv e r is an enzymic process, usually involving two phases -

Phase I BiotransformationsPhase I I Conjugations

Phase I reactions can be classified as oxidations, reductions and hydrolyses in which hydroxyl, carboxylic, amino and occasionally sulphydry! groups are

introduced, which increase the po larity of the molecule and act as a centre

for conjugation. Phase I I conjugations occur when a foreign compound or

a product of Phase I combines with naturally occurring molecules in the l iv e r , such as glucuronic acid, generally making the molecule more polar

and more readily excreted. In most animal species anutrients may be metabolised by e ither one or both phases. Either way, metabolism usually

leads to detoxification. Species variation in the amounts o f specific enzymes w ill a ffect both Phase I and Phase I I metabolism. However, Phase I I species variation is mainly dependent on the a v a ila b ility o f conjugating agents, for example, taurine conjugation is a minor process in most species

but is important fo r the fe rre t (James et; al_, 1972). Numerous examples

of Phase I variation ex is t, e .g . amphetamine metabolism occurs in ra t but

not in guinea-pig (_wi^ 1 i ams> I 96? ), guinea-pig metabolism of 6-chloro-5- cyclotoxylindane-I-carboxylic acid is minimal in guinea-pig compared with

mouse (Parke, 1980), and the dog is unable to hydroxylate the a lic y c lic and aliphatic carbon atoms of compounds readily hydroxylated by a number of other species (Dayton e t a l, 1973; Gros e£ al_, 1974 and Zacchei e t a l ,1978). Reviews in this f ie ld document a great many examples o f this sort (Hucker, 1970; Kato, 1979; Parke and Williams, 1969; Walker, 1978; Williams, 1967 and Williams, 1974).

Within the cell the enzymes which catalyse these reactions may be found in the mitochondria, the cytosol, and particu larly in the endoplasmic

reticulum where the enzyme system responsible for oxidizing most anutrients the microsomal mixed-function oxidase (M.F.O.) system is membrane bound. Enzyme a c tiv ity , particu larly of the M.F.O. system o f d iffe ren t species, may largely influence species differences in drug metabolism. Many investigators have studied the components of the M.F.O. system in a number of species, to correlate differences in component levels with metabolic differences. For example, Kato (1966) showed that an iline hydroxylase a c tiv ity in d iffe ren t species corresponded with levels of cytochrome P-450

and cytochrome c reductase a c tiv ity . Although his recent review (Kato,1979) illu s tra tes the d iff ic u lt ie s other workers have found in establishing a clear relationship between xenobiotic metabolism in d iffe ren t species and

th e ir hepatic content of cytochrome P-450 or other M.F.O. system components

i t is generally agreed that a relationship must exist between species differences in component levels of the M.F.O. system and the metabolic

capability of each species and this relationship probably consists o f multiple variables and w ill be influenced by the a c tiv ity of other

detoxifying microsomal enzymes e.g . epoxide hydrase and glucuronyl transferase.

Metabolic capability ini vij/o will be affected by the b io ava ila b ility

of a drug. Anatomical and physiological species differences w ill cause

differences in absorption, distribution and excretion. Recent studies indicate the importance o f protein binding, hepatic blood flow and the

volume o f distribution (Wilkinson and Shand, 1975) in hepatic bio­a v a ila b ility and clearance. Some other in flu en tia l features affecting b io ava ilab ility in d ifferen t species are lis te d and/or b r ie f ly discussed

below:-

( i ) Concentrating capacity o f the liv e r and differencesin glutathione levels

( i i ) Presence or absence o f a gall bladder

( i i i ) Bile flow rate

(iv ) Threshold molecular weight for b ilia ry excretion

(v) ' Relative organ s iz e . ' Large vertebrates tend to havere la tiv e ly small livers (Munro, 1969), which leads

into the observation that large mammals show low re la tive mono-oxygenase ac tiv ity when measured in

terms of body weight (Walker, 1978).

Species variation in k inetic aspects of metabolism

Walker correlates metabolic rate and body size for a number of compounds metabolised by the M.F.O. system. I t was Brodie who f i r s t presented the concept that species differences in the pharmacological response to a drug correlate with its rate of metabolism and its plasma level (Quinn e t al_, 1958; Brodie, 1964; Brodie and Reid, 1967). Marked

species differences in the h a lf - l i fe of hexobarbital metabolism among mouse, rabbit, ra t , dog and man were observed. A relationship between duration of drug action and biological h a lf - l i fe was established, as

well as inverse relationship between enzyme a c tiv ity and duration of response (see Table 1 .2 .overleaf).

In this work Walker's hypothesis on body size and metabolic rate

holds well with the exception o f rabbits. However, his 's ize -ra te

relationship' does not hold fo r xenobiotics where additional factors or metabolic routes other than the mono-oxygenase system are equally important. I t does not generally apply to conjugation reactions or to

non-cytochrome P-450 oxidative reactions. Hence, there w ill be numerous occasions where this relationship does not hold, as with isoxepac ( I l l in g and Fromson, 1978) and the oxidatively metabolised compound phenylbutazone (Burns e t 1965). I t is evident that i f any compound is given to more

than one species, then a species difference in the amounts and rates of metabolism are lik e ly , often with differences in the route of metabolism. Although, these differences are largely unpredictable, once a difference is found with a compound i t is lik e ly that related substances w ill behave

sim ilarly and exhib it sim ilar patterns of species varia tion .

Table 1.2 Species differences in the duration of action andin the metabolism of hexobarbital

Species Duration o f action (min)

Biologicalh a lf - l i fe

(min)

Relative enzymea c tiv ity(mg/g/hr)

Mouse 12 19 598Rabbi t 49 60 196Rat 90 140 134Dog 315 260 36Man

"

360'

Data from Quinn e t al_ (1958)

Toxicokinetic aspects of species variation in metabolism (and the mechanisms

involved in reactive metabolite to x ic ity ).

Although metabolism generally results in detoxification, i t is now realised that the same metabolic systems can produce reactive intermediates in the form o f epoxides, hydroxylamines, free radicals or carbenes, with

the possib ility of a toxic manifestation. The variations in response to hepatotoxins w ill be in tr in s ic a lly linked to the metabolic capabilities

of d ifferen t species. For instance, i f a compound (A) exerts a hepato­toxic e ffec t via a reaction metabolite (B), metabolism to th is intermediate w ill be a pre-requisite for to x ic ity .

(A) Ki (B) K2 (C) >

A = parent compound B = reactive metabolite C = non-toxic end metabolite

Figure 1. A sim plified metabolite pathway

Species which convert A to B rapidly ( i . e . Ki, reaction rate is high) resulting in sustained high plasma and tissue levels o f B w ill be

susceptible particu larly those in which the enzymic conversion from B

to C is slow ( i .e . K2 reaction rate is low). The mouse illu s tra tes this toxicokinetic principle since i t possesses high mono-oxygenase

a c tiv ity (Ki ) but low epoxide hydrase (K2 ) and as a result is

comparatively susceptible to toxic effects mediated by epoxides (Oesch e t aj_, 1977). In less susceptible species Ki may be comparatively low

and K2 o f an equivalent rate . Numerous examples of compounds requiring metabolic activation ex ist; probably amongst the most well documented is paracetamol (Davies e t a l , 1974; Jo llow et cf[, 1974; Potter e ta l_ , 1974

and G ille tte , 1977) currently thbught to produce to x ic ity via an imidoquinone (G ille tte ■ e t'a l>,’l 980) I f the parent compound (A) is toxic species suscept­ib i l i t y w ill be reversed. In this situation those species capable of metabolising the parent compound rapidly w ill be protected. Species

unable to detoxify the compound w ill be at greater r is k . Thus metabolic capability and metabolic rate play a major toxicokinetic ro le , often

determining whether toxic levels of anutrients or th e ir metabolites are achieved in an animal species. The consequences of microsomal metabolism have been summarised by Parke, 1980, as follows:-

1. Detoxification and accelerated excretion o f metabolites.

2. Activation and reaction with ce llu la r components, e lic it in g toxic reactions.

3. Ligand complex binding to cytochrome P-450 with inh ib ition or

destruction of th is enzyme, resulting in autoxidation and non­specific oxidation of environmental chemical substrates.

Reactive metabolites in to x ic ity are well documented (Parke, 1980). Selected mechanisms w ill be described here because of th e ir relevance to this thesis. These are best illu s tra ted by specific examples (see Figure 2 ).

Hepatotoxicity mediated by reactive metabolites is produced through

attack on v ita l hepatic macromolecules by e ither one electron (rad ical) or two electron (Cations,invidoquinones epoxides) e lectrophilic reactions. These reactive intermediates are potent oxidising agents (Calder et al_, 1975

depleting tissue glutathione, oxidising and binding with other v ita l tissue components resulting in hepatocellular necrosis. Some reactive metabolites may exhibit spec ific ity in th e ir a f f in ity for sub-cellu lar

C12C=CC12/ 0 \

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components, for example, diol epoxides form stabilized internal ion-pair compounds that react as carbonium ions with DNA even in the presence of

epoxide need exist for DNA arylation and consequent damage o f DNA and

carcinogenesis (Hulbert, 1975). The more usual sequence of events in the development of to x ic ity is envisaged as glutathione-reactive metabolite

interaction resulting in depletion o f glutathione levels . Once this has occurred the reactive intermediate is free to bind with other subcellular components to produce tox ic ity and necrosis.

Thus glutathione and additional microsomal enzyme systems (e .g . epoxide hydrase) can be viewed as secondary defence lines should the

primary defence lines in the form of the M.F.O. system activate (rather than detoxify) a foreign compound.

Cytochrome P-450 is the target fo r another type o f specific binding of reactive metabolites or metabolic by-products. Safrole, for example, following hydroxylation, loses water molecules to form carbenes which ligand complex with cytochrome P-450. Many halogenated compounds such

as carbon-tetrachloride, chloroform and halothane are thought to act in

a sim ilar manner (Parke, 1979). Sulphur containing compounds can lose sulphur in exchange for oxygen following M.F.O. a c tiv ity . The sulphur so released can bind with cytochrome P-450, resulting in a lowering of M.F.O. a c tiv ity and eventually in hepatocellular damage (Parke, 1980).

glutathione and proteins, so that no threshold concentration of diol

I t is recognised that vu lnerab ility of the liv e r to toxins is a result o f its s ite in the body. Situated between the in testina l trac t and the general circulation makes i t the1target organ1 fo r foreign compounds. The liv e r receives foreign compounds via the hepatic

portal vein, on th e ir f i r s t pass before they enter the systemic circu lation . Exposure of the liv e r w ill be maintained a fte r a substance is in the general circulation by the hepatic artery or by the hepatic

portal vein i f enterohepatic circulation occurs. Hence toxic compounds may be concentrated in the liv e r prior to metabolism and elimination or

i f hepatic bio transformations produce toxic metabolites primary exposure of this organ to the toxic insu lt w ill s t i l l occur.

Species Dependent Toxicity o f FPL 52757

A chromone, 6 ,8-diethyl-5-hydroxy-4-oxo-4 Hr T-benzopyran-2 -carboxylic acid

(FPL 52757) was developed as an effective an ti-a lle rgy agent (Augstein, e t a l , 1977). Animal to x ic ity data was obtained in several species to support human volunteer tolerance and c lin ica l studies. No adverse effects were found in human volunteers treated for 14 days a t 300 mg day"^ (equivalent to 4.5 mg kg~^day~^) . Subsequently one month tr ia ls were carried out inasthmatic patients. No abnormalities were seen in patients treated at

-1 -1100 mg day but some who received 300 mg day (approximately 20% ofthose treated) showed significant increases in serum alanine amino­transferase (ALT) and serum aspartate aminotransferase (AST) indicative

of an e ffe c t on the l iv e r . No other liv e r function tests were abnormal and no other adverse effects were seen. These findings, although mild and readily reversible were su ffic ien t to in h ib it the further development of the compound a t this dose for long term use in asthma. The animal data

accumulated prior to c lin ica l tr ia ls had fa iled to predict this apparent sensitiv ity of the human liv e r to the compound. Consequently, additional studies in a variety o f species were undertaken and the species-dependent nature and mechanism of FPL 52727 to x ic ity is elucidated in this thesis.A summary of a ll the animal toxicology data follows the next section on chromones.

THE CHROMONES) -

History o f chromones

FPL 52757 is a member o f the chromone group of drugs, of which disodium; cromoglycate# marketed as 'In ta l ' since the late 1960's, is

the most well known. Altounyan (1967) and Pepys et al_ (1968) described

the action of the bischromone derivative disodium- cromoglycate■ in

preventing experimental asthma in man. Administration by inhalation effec tive ly reduced the asthmatic response to inhaled antigen in

sensitized individuals and this protective e ffec t lasted fo r several hours. No a c tiv ity was found when the compound was given o ra lly . The

chemical approach to this discovery was based on the long-known smooth- muscle relaxing effects of plant extracts containing K hellin , to which i t is chemically related. However, the anti-asthmatic properties o f disodium (cromoglycate are not attributed to these effects which the

drug does not possess, but to another action involving suppression of autacoid release. Since its introduction in 1968 disodium; cromoglycate j

has been widely used in Europe in the prophylaxis of severe bronchial asthma.

Pharmacological effects

D i s o d i u m cromoglycatev inh ib its the release of histamine and slow-

reacting substance (SRS-A) in anaphylaxis from human lung during a lle rg ic response. Both these substances, especially SRS-A, are potent bronchial spasmogens. An inhibitory action on antigen-induced production o f histamine

and SRS-A has been demonstrated in peritoneal mast cells in vivo and in

v itro (Garland and Mongar, 1974). Disodium chromoglycate does not in h ib it

fixation of antibodies, nor does i t seem to in terfere with the anti gen- anti body reactions, rather i t suppresses the response to th is reaction.

The need for the oral-acting chromone derivative

Despite the undoubted success o f disodium cromoglycate in asthma

therapy and its lack of toxic side-effects there is an even greater potential for an o ra lly -e ffec tiv e anti-asthma drug. Disodium cromoglycate

is poorly absorbed ora lly and is only e ffective c lin ic a lly when administered

d irectly to the target organ. D iffic u ltie s arise from this route of administration, despite the development o f increasingly sophisticated

and effective inhalers.

The problems encountered with this route include the following:-

(1) Patient compliance is variable; some patients find s e lf­administration with the aerosols uncomfortable and d islike

using them.

(2) Patients may misunderstand the instructions of th e ir physician.

(3) Other patients fu lly understand these instructions and are w illing

to comply with the therapeutic regime. Nevertheless, amongst this group misuse w ill s t i l l occur, e.g . accidentally swallowing the

drug instead of inhaling i t .

An o ra lly -e ffec tive an ti-a llergy compound should reduce the problem of patient compliance and enable the drug dose level to be better regulated.

FPL 52757 has been shown to be active by the oral route in both experimental and c lin ica l asthma. Like disodium cromoglycate , FPL 52757 given by inhalation before antigen challenge affords protection. However, unlike disodium chromoglycate,FPL 52757 given in single or multiple oral doses of 50-100mg from 2-12 hours before challenge,5 affords protection from anaphylactic

broncho constriction. Both the immediate and the late reactions arising

from antigen challenge appeared to be susceptible to treatment. In addition to these experimental results, further studies have shown that FPL 52757, given ora lly (50 mg b .d .) over a period of one month, has afforded c lin ica l improvement.

ANIMAL TOXICOLOGY AND'SAFETY EVALUATION STUDIES ON FPL 52757

Methods

Conventional toxicological techniques and study design (as outlined in ‘Guidelines for Preclinical and C linical Testing o f New Medical Products', Part I Preclinical by the Association of the British Pharma­ceutical Industry, 1977) were followed including observation of behaviour, monitoring of body weight, food and water intake, comprehensive blood biochemistry and haematology screens, autopsy and microscopic examination

of a range of tissues. Most of these studies generated negative data and are not described in d e ta il. The experiments carried out following c lin ica l tr ia ls were designed specifica lly for an evaluation of hepatotoxicity.

Dose levels for the longer repeated-dose studies were based on acute to x ic ity data and on short duration (4 to 5 days) repeated-dose to x ic ity studies. Some studies were conducted by contract research laboratories

as indicated in Table 3.

Results

Animal to x ic ity stucjies prior to c lin ica l tria .ls .

Median lethal dose.The intravenous single LD^ o f FPL 52757 following 14 days

observation was approximately 100 mg kg~ in rats and mice. The single

oral dose LD^q (mg.. kg~^) following 14 days observation in various species was as follows - mouse 1800-1900; hamster 500-1000; ra t 700-900; rabbit 250-500 and cat 100-300.

Multi dose studies

A summary o f the repeated-dose studies with major and relevant findings are presented in Table T.3,and further details are given in Table 1, Appendix 1. Reproductive studies are included and although these were not designed as conventional tox ic ity studies they provide

additional evidence for the lack of hepatotoxicity and in many cases the livers were subsequently examined microscopically with negative findings.

None of the five species - ra t, hamster, rabbit, squirrel monkey and cynoraolgus monkey, which were studied for periods of five days to

six months, showed any defin ite evidence o f hepatotoxicity even when

lethal or near lethal dose levels were administered. In some studies an increase in l iv e r weight was found. (Details of liv e r weight changes are shown in Table 2, Appendix I ) . There was no evidence o f treatment- related pathological change in these cases.

Repeated dose to x ic ity studies carried out subsequent to c lin ica l t r ia ls

A fter the results o f the c lin ica l tr ia ls became known, c lin ica l experiments were done to investigate further the hepatotoxic p o te n tia l, of FPL 52757. These additional experiments included studies in rodents

and other species including the beagle dog.

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High doses of compound produced minimal liv e r changes. In mice

there was a possible minor increase in aniline hydroxylase and s ligh t changes in the glycogen pattern. In ra t, electron microscopy o f the l iv e r revealed a s lig h t degree of pro liferation of smooth endoplasmic

reticulum. In rabbit there were no dicernable changes in the l iv e r .

Studies in the beagle dog (see Table 1.5 §nd Appendix 1 (Tables 4 and 5) fo r fu lle r d e ta ils ). •

Contrasting with the other species studied, FPL 52757 was re la tive ly toxic in dogs and caused death a fte r as few as six oral doses of 20 mg k g ~ \A number of sequential studies were done which involved a to tal o f 33 beagle dogs. Eight were control dogs and 25 were dosed ora lly with FPL 52757 at various dose levels. Most:FPL 52757 treated animals showed

evidence o f drug-related hepatotoxicity. The to x ic ity was accompanied by gross abnormalities in liv e r function, confirmed by histochemistry and histopathology. Eight o f the animals died. Others lost considerable body

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and summarizes the dosing regimen and findings.

Severe gastrointestinal symptoms were observed in the dog, inaddition to hepatotoxicity. In several there was biochemical evidenceof liv e r damage occurring very early and i t may be inferred that FPL52757 has a primary hepatotoxic action in dogs, with a theshold dose

- 1 - 1(in the range 20-40 mg kg day ) and a steep dose response with 100 mg kg” day" being invaribaly le th a l. In some dogs hepatotoxicity appeared a fte r about one week of treatment with subsequent rapid deterioration, and

in these cases there may have been gradual accumulation of the drug in the l iv e r which eventually exceeded the threshold for hepatotoxicity. The release of the liv e r-sp ec ific mitochondrial glutamate dehydrogenase (GLDH) and the rapid increases in plasma ALT, and to a lesser extent AST, were consistent with hepatocellular necrosis. Histopathology showed hepato­b ilia ry in jury with centrilobular necrosis and distended b ile ducts; histochemical analysis revealed substantial increases in alkaline phosphatase (ALP) despite only comparatively s ligh t increases in serum alkaline phosphatase. When dosing of seven dogs was discontinued (see Appendix 1 ,Table

l iv e r function serum levels returned to normal in six animals; but in the seventh animal ALP continued to rise a fte r cessation o f treatment, u n til

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death, despite substantial fa lls in ALT, AST and GLDH. This

observation and the high levels of ALP in other severely affected dogs emphasizes the importance of hepatobiliary injury in the escalation of hepatotoxicity. The pathological changes were

generally consistent with a chronic toxic e ffe c t. Of the seven

species the dog is uniquely susceptible to a hepatotoxic e ffect of FPL 52757. A relationship between these findings and the mild hepatic changes seen in man with the compound is suggestive but unproven. ‘

THE ORIGINAL HYPOTHESIS FOR THE MECHANISM OF FPL 52757 INDUCED

HEPATOTOXICITY

The original hypothesis for FPL 52757-induced hepatotoxicity was based around metabolism to reactive intermediates. I t was assumed at the outset o f this investigation in to the mechanism o f FPL 52757

induced hepatotoxicity, that the drug was well metabolised in most species. Hence i t was postulated that metabolism of the chromone was

necessary. A hypothetical metabolic pathway is shown in Figure 3, which includes iden tified metabolites and possible intermediates in

parentheses. With the discovery of the 6 - ethinyl metabolite [FPL 58280} in man i t was thought this might be the hepatotoxic agent. However, in animal species the occurrence of FPL 58280 did not correspond to the incidence o f to x ic ity .

A quinone methide could arise from the secondary alcohol [FPL 57704)

possibly non-enzymically but perhaps catalysed by liv e r enzymes. This tautomer of the vinyl metabolite would equilibrate with FPL 58280 or react with ce llu la r macromolecules. A lternatively , an epoxide formed

from FPL 58280 could act in a sim ilar fashion. Examples of this type of interaction for both quinone methides [Hanson et aj[, 1970 and Turner, 1966) and epoxides [Je'rina e t a l , 1974) are fam ilia r.

In the following chapters studies designed to elucidate the mechanism of FPL 52757-induced hepatotoxicity are described. The

order of the chapters is based on the natural progression of the

research programme.

FIGUR

E 3.

FPL

5275

7 AN

D ME

TABO

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S

As data accumulated areas of in terest developed and certain facets

of the mechanism are elaborated.

THE REASONS FOR UNDERTAKING THIS STUDY OF HEPATOTOXICITY

Prior to c lin ica l tr ia ls with FPL 52757, five species, including two simians, received repeated oral doses for a period from 5-180 days. The chromone did not produce hepatotoxicity in any of these species even a t the maximum tolerated doses. The s u ita b ility of these species as

experimental models fo r man had been previously established on metabolic grounds; a ll of the species readily hydroxylated the drug, as did man. The clinico-pathological findings in man, although re la tive ly mild, were nevertheless surprising. Further studies in rodents and rabbits

fa iled to demonstrate hepatotoxicity. Thus, in a l l , seven species were

looked at carefully for evidence of hepatotoxicity. The toxic potential of this compound was realised only in dog (showing l iv e r disturbances at doses approximately four times higher than those used in man). The mechanism in man and dog is not necessarily the same.

Hence the study o f the mechanism of hepatotoxicity of the chromone was in itia te d for the following reasons -

(a) An understanding of the mechanism of biochemical to x ic itymight be helpful in the design of new and non-toxic drugs

(b) I t seems probable that a new type of biochemical mechanismo f to x ic ity might be uncovered

Cc) Chemical hepatotoxicity occurred in man which was notpreviously observed in animal to x ic ity studiesvan understanding

of this species variation is important'for future drug evaluation.

CHAPTER TWO

EXPERIMENTS DESIGNED TO ELUCIDATE THE

ROLE OF A REACTIVE METABOLITE IN FPL 52757

HEPATOTOXICITY.

SUMMARY

Further species treated with FPL 52757 in multidose to x ic ity

studies include the guinea-pig, fe rre t and baboon. FPL 52757 fa iled to produce more than mild (and in the most part inconsistent) liv e r disturbances at doses 10-20 times greater than those causing hepato­toxic side effects in man. The beagle dog was the only animal species of tenexamined which was readily susceptible to the hepatotoxic effect of the compound. Liver disturbances could be induced in in i t ia l ly normal dogs a t doses approximately 4 times higher than those used in man. Hepatotoxicity could not be demonstrated in ferrets or rats in special studies including pretreatment with 3-methylcholanthrene or phenobarbitone,

defic ient d ie t and neo-natal animals, designed to reduce resistance to drug- induced hepatotoxicity and elucidate the mechanism involved.S im ilarly , hepatic microsomal enzyme induction in dogs by pretreatment with phenobarbitone and protection against cytotoxicity by pretreatment with methionine provided no evidence for involvement of a reactive metabolite

in the hepatotoxicity induced by FPL 52757.

INTRODUCTION

Chapter one includes a summary of safety evaluation toxicology carried out by Fisons Pharmaceuticals on a potential anti-asthma

compound, FPL 52757. Subsequent experiments (conducted by the author unless otherwise stated) are described in th is and the following

chapters which consist of supplementary studies beyond the normal scope of an industria lly based pharmaceutical/company, nevertheless deemed both of academic and practical importance, for the reasons

outlined in the previous Discussion. The aims of the multi-dose to x ic ity studies described in this chapter were threefold: f i r s t ly , to investigate the uniqueness o f the animal hepatotoxicity by carrying out further multidose to x ic ity tests in untried ' species; secondly, to find a new small animal species susceptible to FPL 52757 to use as

a suitable model fo r investigational studies by manipulation of conventional conditions for to x ic ity testing i f necessary; and th ird ly , to elucidate the original hypothesis for the mechanism of chromone- induced hepatotoxicity. These experimental designs were based on previously proven methods for demonstrating the role of a reactive metabolite in drug induced hepatotoxicity, by affecting the severity

of damage (M itchell, et al_, 1973; Angeli-Creaves and McLean, 1979, and

Parke, 1979). Detoxification by metabolism is commonplace; however, where drugs or toxic chemicals are activated instead of being detoxicated

by metabolism, enzyme induction w ill lead to increased to x ic ity (Parke, 1979). The a b ility of hepatic microsomal enzymes to produce toxic metabolites is altered by age (Kato et a l , 1964) and nutritional status (McLean and Day, 1975). Hepatotoxicity mediated by reactive metabolites

can be decreased by selective pretreatment (e.g. methionine) aimed at protecting the hepatocytes (McLean and Day, 1975).

MATERIALS AND METHODS

FPL compounds

FPL 52757 (6 )8-diethy1~5-hydroxy-4-oxo-4H-1-benzopyran-2-carboxy.'Hc

acid) is a stable crysta lline solid with the following physical properties

M. wt. = 262, melting point 219-221°C, pK 1.4 , soluble in organic solventsav ir tu a lly insoluble in water. I t was synthesized by Fisons Limited, Pharmaceutical Division, Loughborough, U.K. (Cairns, et al_, 1974) as were the metabolic standards(FPL 57704) 8 -e th y l-5-hydroxy-6-(i~hydroxy-

ethyl)-4-oxo-4H-l-benzopyran-2-carboxylic acid, the secondary alcohol, .

8 ethyl-5-hydroxy-6-(2-hydroxyethyl-4-oxo-4H-l-benzopyran-2-carboxylic acid

the primary alcohol? and(FPL 58280) 6-e th in y l-8-ethyl-5-hydroxy-4-oxo-4H- 1-benzopyran-2-carboxy1ic acid the ethinyl metabolite. The major metabolite FPL 57704 was prepared by addition of S^ethyl'resacetaphenone to

dimethyl-acetylenedicarboxyl ate, cyclysation w ith ’ chloroSulphonic acid ’ was followed by reduction to the secondary alcohol with borohydride. The primary alcohol was formed by reacting 3-ethyl- 2 ,6-dihydroxyacetophenone

with a lly l bromide and the resulting 6-allyloxy-3-ethyl-2-hydroxyaceto- phenone condensed with diethyl oxalate to give the chromone ester.Claisen re-arrangement to the a lly ! chromone, oxidative cleavage of the a lly l double bond to the 6-formylmethyl derivative and reduction with sodium borohydride gave the chromone ester with the desired substituents

Alkaline hydrolyses of the ester afforded the required primary alcohol.The ethinyl metabolite was prepared by dehydration of the secondary alcohol with dimethyl suIphoxide.

Their structures and that of FPL 52757 were confirmed by standard

physical chemical techniques ( i . r . , u .v ., n .m .r.) and elemental analysis (see Table 2 .T ).

Other chemicals: 3-methylcholanthrene, phenobarbitone, methionine, sodiumchloride, methyl cellulose (Sigma Chemical Co., St. Louis, Missouri, USA). Corn o il (J. Sainsbury Ltd, London, England).

Animals: These were obtained as follows:- guinea-pig 340-390 gm, males

(Duncan-Hartley, Animal Virus Research In s titu te , P irbright, Surrey, England); fe rre t 1.0-2.0 kg, males (A.S.. Roe, Norfolk, England); baboon,8 kg, 1 male, 1 female (Shamrock Farms L td ., Henfield, Sussex, England); neonatal rats (Sprague-Dawley, Charles River, Manston, Kent, U .K ., and Surrey University Colony); rats 125 gm, males (Sprague-Dawley, Charles

River, Manston, Kent, U .K., Fison's Colony); beagle dog 14-16 kg, male

(Fisons Colony).

Animal Husbandry and Treatment

Prior to the study, a ll animals were c lin ic a lly examined and any

animal below the necessary quality was rejected. Animal room temperature

was controlled a t 21°C + 2°C, with a 50% humidity and lighting controlled to give 12 hours lig h t and 12 hours darkness per 24 hours. Water was free ly

available to a ll animal species via automatic drinking systems. Appropriate housing, d ie t, dose levels, dosing and sampling techniques varied with

species and are described in detail for each study.(* The primary alcohol was not designated an FPL number)

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Assessment of to x ic ity

Conventional toxicology techniques and study design were followed

with special emphasis on assessment of liv e r in jury.

Observation: Animals were observed periodically during the day forchanges in conditions and behaviour. Any animals showing signs of acute intoxication were k ille d .

Body weight:- Body weights were recorded on a regular basis. The

regularity varied with species and intended duration of the study.

Food and water consumption:. Food and water intake was estimated on a

daily basis where possible.

Necropsy: A ll major organs from a ll animals which died or were k illedwere examined fo r gross pathology. Routine weighing of livers was under­taken. Histopathological examination included several sections from each

liv e r and other major organs. Sections were stained with haemotoxylin and eosin (H and E).

C linical chemistry: Appropriate tests designed to pick up adverse effectson the liv e r were carried out where possible at regular in tervals. The following enzymes are known to be markers for liv e r damage: Isocitra te

dehydrogenase (ICDH), alanine aminotransferase (ALT); alkaline phosphatase (ALP) aspartate aminotransferase (AST) and glutamate dehydrogenase (GLBH). A

combination of at least two enzymes were investigated in each test (see Table 2 .2 ).

Table 2.2 Enzyme Analyses to Assess Hepatotoxicity

Experiment No. Species Enzymes

1 Guinea-pig ICDH, ALT, and ALP2 Ferret ICDH, ALT, and ALP3 Baboon GLDH, ALT, AST and ALP

4 Neonatal ra t5 Rat6 Ferret7 Dog

ICDH, ALT and ALP

GLDH and ALT ICDH, ALT and ALP

GLDH, ALT, AST, and ALP

Plasma enzyme analysis is established as a sensitive means of predicting the progression of hepatotoxicity. To this end regular blood

samples were taken throughout the fe rre t , baboon and dog experiments.Only terminal bleeds were possible in certain species (e.g. guinea-pig

and neonatal ra t) whose physiology and anatomy prevented regular sampling without considerable trauma to the animals.

The experiments were conducted by the author at the following locations: Experiment 1, 2, 4 and 6 a t the Animal Unit, University of Surrey, Guildford, Surrey, and Experiments 5 and 7 at the Hi 11 crest Research Station, Fisons Ltd ., Loughborough, Leicestershire. Experiment 3, the baboon study, was

contracted to Hazelton Laboratories, Harrogate, West Yorkshire, under the

direction of the author.

C lin ical Chemistry Methods

Measurements were carried out at the University of Surrey using the

manual procedures outlined by Bernt and Bergmeyer (1974); a t Hazelton Laboratories, Boehringer Test Kits were employed, and at Fisons, analyses were achieved using established automated methods using a Vitatron AKES Enzyme Analyser. Fundamentally,the techniques and optimised standard

methods are based on the following princip les:- ( i ) AST converts L-aspartate and a-oxoglutarate into glutamate and oxaloacetate. In an indicator reaction

with MDH, oxaloacetate and NADH are converted into malate and NAD. AST

ac tiv ity is measured by the rate of decrease in absorbance o f NADH a t 340 nm (Karman et al_, 1955) - ( i i ) ALT converts L-alanine and a-oxoglutarate

into glutamate and pyruvate. In an indicator reaction with LDH, pyruvate and NADH are converted into lactate and NAD. ALT a c tiv ity is measured by the rate of decrease in absorbance of NADH at 340 nm (Wroblewski and La Du, 1956). ( i i i ) Alkaline phosphatase hydrolyses p-nitrophenol phosphate to

phosphate and the p-nitrophenol liberated is proportional to ALP a c tiv ity , which is measured by the rate of increase in absorbance of p- nitrophenol at 405 nm (Hausaman£t al,1967(iv) GLDH, with the addition of ammonia and NADH, catalyses the ami nation of 2 -oxoglutarate, producing L-glutamate and NAD. * .The decrease in"-NADH concentration per minute (measured at 340 nm) is d irec tly proportional to GLDH a c tiv ity (Wallnofer

e t .a l , 1974). (y) Isocitra te is oxidised by ICDH using NADP ascoenzyme. The amount of isocitra te oxidised is determined by the increase in extinction due to the formation of NADPH, which in turn is a measure of

'ICDH ac tiv ity (Bernt and Bergmeyer, 1974).

Quantitative Analysis of FPL Compounds

Levels of FPL 52757 and known metabolites were assessed from

s e ria lly obtained samples where possible and at autopsy from b ile ,

plasma, urine and faecal homogenates, using the following technique:

Known volumes. (0 .2: 2 ml ) were taken into

stoppered tubes arid acid ified with an equal volume of 2N hydrochloric

acid. Ten times the volume of hexane/ether (1:1 v/v ) was added and the tubes were shaken fo r ten minutes. The hexane/ether layer was removed, fresh hexane/ether wasadded and the extraction was repeated.The combined hexane/ether layers were blown to dryness in a stream of a ir . The extracts were then redissolved in a known volume of ethyl acetate

(50-lOOyl} and known volumes (5 -20yl) were spotted on to Merck glass- backed s ilic a gel T.L.C. plates. The TLC plates were then developed in chloroform, diethyl ether, glacial acetic acid (6:2:2 v/v ) . The TLC

plates were then examined under UV lig h t before scanning on a Vitatron

TLD using the transmittance mode and a 348 nm f i l t e r . . - For comparison, standards of FPL 52757 and the common metabolites FPL 57704 and FPL58280 were run through the procedure and used to estimate the levelsof compound present.

Part 1, Further studies in previously untested species.

The oral to x ic ity of FPL 52757 in guinea-pig, fe rre t and baboon was

exami ned.

Guinea-pig experiment: Groups of three male guinea-pigs were treatedorally for up to 19 days with vehicle or FPL 52757 at 100, 200 and 300ng

-1 -1kg day . FPL 52757 was suspended in vehicle (1% methyl cellu lose).A ll animals were dosed at 10 ml kg via a ball-headed dosing needleinserted through the mouth into the stomach. The guinea-pigs were allowed

free access to p e lle t d iet (B.P. Nutrition (U.K.) L td ., Witham, Essex.The guinea-pigs were housed 3 to a cage. Blood samples were taken by cardi puncture prior to euthanation, to assess drug-induced hepatotoxicity.

Ferret experiment: Three male ferrets were treated for up to fiv e dayseither with vehicle or FPL 52757 at 200 or 400 mg kg day FPL 52757was administered orally as an aqueous suspension in 1% methyl cellulosevia a ball-headed dosing needle through the mouth into the stomach.

-1In i t ia l ly animals were dosed at 5 ml kg ; this was reduced to 2.5 ml

-1kg on day 2 , and f in a lly the ferrets were dosed twice daily with half the daily dose of 2.5 ml~H9“ to reduce the adverse effects of dosing.The ferrets were allowed free access to pelleted d iet (B.P. Nutrition (U.K.) L td ., Witham, Essex) mixed with cat food and housed one to a cage. Blood samples were taken by cardiac puncture prior to euthanation. A novel method for ta il-b leeding ferrets (Bleakley, 1980) enabled serial samples to be collected for assessment of to x ic ity .

Baboon experiment: Two baboons, one male and one female were treatedfor 28 days with FPL 52757 at 40 mg kg" day" (Days 1-7) 80 mg kg~ day"^

- 1 - 1(Days 8-14, 16-28) and 160 mg kg day inadvertently given on day 15.No control animals were included since pretreatment data was used as

control values. Doses were prepared at the required concentration each day as suspensions in 1% methyl cellulose and were administered at a

volume of 2 ml kg via a rubber catheter inserted through the mouth into the stomach. The monkeys were offered a Bonio biscuit (Spratts, Barking) weighing approximately 25g in the morning (a fte r dosing) and approximately lOOg of Old World Monkey expanded primate d ie t (B.P. Nutrition

(U.K.) L td ., Witham, Essex) and a fresh f r u i t supplement in the afternoon.A vitamin B-^ compound (Cytacon, Glaxo Laboratories L td ., Greenford, Middlesex, England) was added to the drinking water once a week. The

monkeys were housed individually. Blood was withdrawn from the femoral vein, serial samples were regularly analysed to assess drug-induced

to x ic ity .

Part 2. Investigational studies in species other than dog.

The potential of the chromone to cause hepatotoxicity in ra t and

fe rre t was further investigated.

Neo-natal rat experiment: Groups of ten male, five-day old rats weretreated ora lly with vehicle or with FPL 52757 at 100 mg kg ^day“ suspended

-1in vehicle (1% methyl cellulose) dosed at 10 ml kg (via a ball-headed dosing needle inserted through the mouth to the stomach) fo r four days.The rats were housed ten to a cage and allowed free access to a lactating mother. Blood samples were collected following decapitation, for

assessment of to x ic ity .

Experiment to demonstrate the e ffect of deficient d iet and phenobarbitonepretreatmentonTPL 52757 in the r a t : A d ie t deficient in selenium, vitaminE and methionine (see Appendix 2) was provided by B.P. Nutrition (U .K .)L td ., Witham, Essex. Groups of rats were divided as follows: Sub-group1; four male rats received defic ient d ie t and vehicle o ra lly . Sub-group2; four male rats received normal rodent d ie t (B.P. Nutrition L td ., Witham,

- 1 - 1Essex) and FPL 52757 at 200 mg kg day ora lly . Sub-group 3; sixteen-1 -1male rats received the defic ient d ie t and FPL 52757 at 200 mg kg day

o ra lly . Sub-group 4; eight male rats received the defic ient d ie t, FPL- 1 - 1 - 1 52757 at 200 mg kg day and phenobarbitone (1 mg ml ) in th e ir drinking

water. Sub-group 5, eight male rats received deficient d ie t, phenobarbitone(1 mg ml"^) in th e ir drinking water and oral treatment with FPL 52757 at

-1 -1400 mg kg day . The defic ient d ie t and phenobarbitone were given forten days prior to FPL 52757 or vehicle (1% methyl cellulose) dosed a t 10 mlkg~ via a ball-headed needle through the mouth into the stomach. The combined treatment was then maintained for 14 days. Serial blood samples

were taken via the caudal vein fo r assessment of to x ic ity .

Experiment to demonstrate the e ffect of phenobarbitone and 3-methylcholanthrenepretreatment on FPL 52757 in the fe rre t: Two groups of three male ferretswere treated for up to 28 days ora lly with FPL 52757 at 100 mg kg day ^(suspended in 1% methyl cellulose) via a ' ba'll-headed needle through themouth into the stomach. One group was pretreated with phenobarbitone (40 mg

-1kg i .p . fo r two days prior to and during the study) and the other with3-methylcholanthrene (20 mg kg i .p . fo r two days prior to and during thestudy). The inducers were dissolved in sodium chloride and corn o ilrespectively. Three control groups of 2 ferrets were treated for 28 days

-1ora lly with vehicle alone or with phenobarbitone (40 mg kg i .p . fo r two days prior to and during the study) and vehicle o ra lly , or with 3-methyl­cholanthrene (20 mg kg i .p . for two days prior to and during the study) and vehicle ora lly . Phenobarbitone and 3-methylcholanthrene were administered

i .p . at 1ml kg~ and 2 ml kg~ respectively. FPL 52757 was administered

o ra lly a t 1 ml kg \ The ferrets were allowed free access to a pelleted

d iet (B.P. Nutrition (U.K.) L td ., Witham, Essex) mixed with cat food supplemented with a lOOg dead ra t (a fte r dosing). The animals were housed

one per cage. Blood samples were taken either by cardiac puncture (terminal bleed only) or via the caudal vein, again making use of the recently developed technique for se ria lly sampling in th is species (Bleakley, 1980) which enabled the severity of any drug-induced to x ic ity to be assessed

throughout the posing period.

Part 3. Investigational studies in the dog.

Effects of phenobarbitone and methionine on the to x ic ity of FPL 52757 to dogs.

Four male beagle dogs were treated ora lly with the chromone in 1%aqueous methyl cellulose (v ia a rubber catheter inserted through the mouthinto the stomach, in a volume o f 0.5 ml kg” flushed through with 6 ml water)in i t ia l ly a t 30 mg kg” day~ rising to 40 a fte r ten days in the absenceof hepatotoxicity. Two dogs received additional pretreatment. One receivedphenobarbitone [20 mg kg" day” o ra lly in water, 18 days prior to , and30 days during treatment with FPL 52757. Another dog received methionine

- 1 - 1at 300 mg kg day ora lly in water 7 days prior to , and 42 days during treatment with FPL 52757. Induction was established in the phenobarbitone

pretreated dog by measurement o f the antipyrine h a lf - l i fe (t^?)* The two control dogs received FPL 52757 only. The dogs were offered 400g o f standard pelleted dog food (B.P. Nutrition (U.K.) L td ., Witham, Essex) a fte r dosing. The dogs were housed ind ividually . Blood was withdrawn from the femoral vein and seria l samples were regularly analysed to assess to x ic ity .

RESULTS

The results section summarises the experimental detail relating to hepato­to x ic ity . Tabulated Results are provided in Appendix 2.

Part 1. Studies on previously untested species (see Table 2 .3 ) .

Findings related to the liv e r .Guinea-pig experiment: Following treatment of guinea-pigs for 19 days at100 mg kg” day" ICDH levels were s ign ifican tly raised (83%.p < 0 .1 ) , butALT and ALP were only s lig h tly elevated. Small isolated areas o f s ligh tnecrosis with vacuolation in the necrotic areas were accompanied bylocalized inflammatory responses in two 100 mg kg~ day" animals (seeFigure i ) . The liv e r o f the th ird guinea-pig was normal. In the higher

-1 -1dose groups (200 and 300 mg kg day ) FPL 52757 was highly toxic and the animals died or were k illed before any significant hepatotoxic effects were manifested, although vacuolation of the liv e r cytoplasm was seen.Liver weight did not d iffe r s ign ifican tly between the control and the

100 mg kg" 1 group.

*(The t | for antipyrine was reduced from 3.6 to 2.3 hr)

Ferret experiment: Following treatment of ferrets for up to fiv e dayseither with vehicle or FPL 52757 at 200 or 400 mg kg day no adverse effects on the livers were detected even though the highest dose was

le th a l. Liver weight, plasma enzyme and microscopic analysis a ll revealed no abnormalities.

Baboon experiment: Treatment of baboons with FPL 52757 (up to 160 mg

for twenty-eight days raised levels of GLDH, AST, ALT and ALP

up to 2 x normal levels. S light centrilobular necrosis, particu larly in the liv e r from the male was .accompanied'by some b ile duct erosion

in both. j '._ • .

Part 2 Investigational studies in species other than dog (see Table 2.4)

Findings related to the liv e r .

Neo-natal ra t experiment: Treatment of neo-natal rats fo r four days withTj ..1

FPL 52757 100 mg kg day or vehicle produced no differences in ALP, ALT and ICDH levels between the survivors in the test and

control groups. Macroscopic and microscopic examination of liv e r sections (including those from neo-nates which died during the study) revealed no

abnormalities.

Experiment to demonstrate the effect of deficient diet and phenobarbitone

pretreatment of FPL 52757 in the r a t : Dosing of rats (on deficient d ietand those on a combination of phenobarbitone in th e ir drinking water and a defic ient d ie t) with FPL 52757 at 200 and 400 mg kg"1 was maintained for

14 days. Some m ortalities occurred amongst rats which received the experimental d iet and FPL 52757 particu larly in the high dose group where six out of eight died. Analysis of plasma transaminase, GLDH and micro­scopic examination of livers fa iled to indicate any adverse effects on the liv e r even in moribund animals. (A possible slight increase in liv e r weight occurred in rats receiving experimental d iet and FPL 52757 compared

with those receiving experimental d iet alone. A larger increase (x 2) in l iv e r weight resulted from phenobarbitone and FPL 52757 treatment of rats

on the deficient d ie t) .

Experiment to demonstrate the effect of phenobarbitone and 3-methylcholanthren

pretreatment of FPL 52757 in the fe r r e t : Following treatment of fe rretsreceiving either phenobarbitone or 3-methylcholanthrene pretreatment with

FPL 52757 100 mg kg~! day*^ plasma enzyme levels (ALP, ALT, ICDH) were

elevated particularly ICDH (up to 6 x normal levels ). Macroscopic and microscopic examination of the livers showed no general abnormalities

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except in one fe rre t which showed marked reversible toxic changes in centrilobular hepatocytes. This particular fe rre t,in the 3-methylcholanthrene pretreatment group became anorectic during

th is study. This may have altered the sens itiv ity of the animal to

FPL 52757. Liver weights were apparently normal in the group receiving phenobarbitone pretreatment but there was a possible.slight increase amongst those receiving 3-methylcholanthrene.

Part 3 Investigational studies in the dog

Experiment to demonstrate the e ffect of phenobarbitone and methionine

on FPL 52757 to x ic ity in dog (see Table 2 .5 ) : The two dogs receiving FPL 52757 only were autopsied on day 13 and 14, a fte r substantial increases in th e ir plasma enzymesfe.g. GLDH and ALT were increased 50 to 200 fo ld ). Hepatotoxic damage was confirmed by microhistopathology; centrilobular necrosis was extensive with enlargement, pro liferation and congestion of b ile ducts accompanied by erosion of b ile duct w a l l s ( s e e F ig .2.1 , 2 .2 ,2 f3 and 2 .4 .The dog receiving FPL 52757 plus, phenobarbitone pretreatment survived fo r 30 days of drug treatment (see Table 2 .5 ). Despite two bouts of mild hepatic dysfunction, plasma enzymes were near normal when the dog

was k ille d . Hepatotoxicity developed slowly in the dog receiving daily methionine (200 mg kg"^) pretreatment. However, severe FPL/52757-induced damage occurred twenty-seven days a fte r concomitant pretreatment with

methionine was withheld. Trace amounts of metabolites were found in some

plasma and b ile samples from three of the dogs with no obvious correlation to

pretreatment. High concentrations of the parent drug (5-8 mg ml”^) were

found in gall bladder b ile samples at post-mortem.

DISCUSSION

PART 1 .FURTHER STUDIES IN PREVIOUSLY UNTESTED SPECIES

This chapter describes toxicological assessment of the chromone in novel species, bringing the to tal number of species subjected to th is oral chromone

treatment up to ten. v

Guinea-pig and fe rre t experiments: Studies in guinea-pig and fe rre t wereundertaken with expectations of identifying a new small and convenient animal species for investigative studies on the mechanism of hepatotoxicity of FPL 52757. Results in fe rre t and guinea-pig were'equivocal, although

-39-

Figure 2.1 Shows a section-iof l i v e r from 30 mg kg day i l lu s t r a te s (Magnification x 160, stained

a male beagle erosion of b i l w ith H & E).

dog receiving e duct wa lls .

f ig u re 2.2 Shows a section of normal dog l i v e r .(Magnification x 160, stained w ith H & E).

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Figure 2.3 Shows a section o f l i v e r from a male beagle dog receiving 30 mg k g "1 day : C en tr i lobu la r necrosis is evident withenlargement p ro l i fe ra t io n and congestion of b i le ducts. (Magnification x 40, stained with H & E).

Figure 2.4 Shows a section of l: jver from a male beagle dogreceiving 30 mg kg"'day- ' . C en tr i lobu la r necrosis is evident. Necrosis occurs in a star-shaped pattern around the central vein.

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indications of disturbed liv e r function occurred only a t nearly lethal doses. Hence, these species were deemed as probably being unsuitable

animal models,for further investigations. Nevertheless, one further attempt to induce consistent reproducible hepatotoxicity in fe rre t was considered worthwhile, since this species could have great potential as a non-rodent species in special toxicological investigations and in routine

safety evaluation toxicology.(This study is discussed la te r in this section).

Baboon experiment: The baboon study was directed in case this speciesshowed a susceptib ility sim ilar to man and greater than that o f the dog.The baboon showed some disturbances of l iv e r function at 80 mg kg~ day”\

This dose level may be compared with sim ilar changes occurring in man at4-5 mg kg~ day"^.

Only the dog, out of a ll the animal species investigated, is re la tive ly sensitive to a hepatotoxic e ffec t of FPL 52757 showing l iv e r disturbances at doses about 4 times higher than those used in man.

PART 2. INVESTIGATIONAL STUDIES IN SPECIES OTHER THAN THE DOG

A number of special studies are described in this chapter, based on

previously proven methods for demonstrating the role of a reactive metabolite

in hepatotoxicity (M itch e ll, e t a l , 1973; Angeli-Greaves and McLean, 1979 and Parke, 1979) and reports that age affects to x ic ity (Parke and Williams

1969).

Neonatal ra t experiment: New born rats have low levels of microsomalenzymes including cytochrome P-450. These enzymes begin to appear in thef i r s t few days a fte r b irth but do not reach maximum levels un til 30 days a fte rbirth (Kato e t 'a l ; 1964) The rationale for the f i r s t study on neonatalrats was based on the hypothesis that impaired metabolism in these immatureanimals could lead to toxic manifestations, particu larly as the LD^q inneonatal rats is approximately 0.25 of that found in adult ra ts . Therewere no signs of hepatotoxicity in neonatal rats a fte r 4 days of dosing

-1 -1with FPL 52757 at 100 mg kg day despite some m orta lities . I t is

evident that neither adult nor neonatal rats are susceptible to FPL 52757

irrespective of th e ir varying a b ility to metabolise foreign compounds.The resistance of neonatal rats could be viewed as suggestive evidence

for the compound i ts e l f being innocuous. However, subsequent experiments

described in this chapter and la te r do not substantiate this view.

Experiment to demonstrate the effect of deficient diet and phenobarbitone

pretreatment on FPL 52757 in the r a t : Low protein diets reduce liv e r gluta­

thione levels(_Leef & Nauberger,1947). A d iet deficient in selenium, vitamin E and methionine has been shown to increase the susceptib ility of rats to hepatotoxins. Phenobarbitone pretreatment makes the protein-depleted

animals even more sensitive (McLean and Day, 1975). As FPL 52757 hepato- to x ic ity was not produced in rats on a deficient d iet with phenobarbitone

pretreatment, there was no evidence to support the existence of a toxic

metabolite. The study emphasized the high degree of resistance of the rat to FPL 52757 induced hepatotoxicity. Even when lethal or near lethal doses were given to weakened animals with lowered resistance to hepatotoxins

no pathological changes occurred in th e ir liv e rs .

Experiment to demonstrate the effect of phenobarbitone and 3 -methylcholanthrene

pretreatment on FPL 52757 in the fe r re t : An additional attempt to produce consistent reproducible hepatotoxicity in fe rre t involved pretreatment with the two types of inducers, increasing the probability of FPL 52757 causing

hepatotoxicity in this species, should a particular metabolic pathway be favoured. Phenobarbitone is regarded as a re la tiv e ly non-specific inducer of microsomal enzymes, including cytochrome P-450 and increases the binding

of Type I and Type I I substrates whereas polycyclic hydrocarbons lik e

3-methylcholanthrene replace cytochrome P-450 with cytochrome P-448 and

promote Type I I binding (Parke, 1975). Despite the toxic symptoms exhibited by some ferrets in the chromone treated groups, th is 28-day multi-dose study fa iled to demonstrate the male fe rre t to be a useful model for FPL 52757 induced hepatotoxicity. Serum enzyme elevation indicated slight FPL 52757-induced hepatotoxicity in both the chromone-treated groups. Histological examination of liv e r sections revealed no pathological changes in livers from the phenobarbitone treated group. However, in the 3-methylcholanthrene group changes in blood biochemistry correlated with

histological findings. Nevertheless, damage was not severe and was restricted to reversible toxic change. Anorexia in the most severely affected animal may have contributed to the manifestations of hepatotoxicity

by producing hepatocellular changes which effective ly sensitized the liv e r or influenced the pharmacokinetics of the chromone in this sick animal, allowing accumulation of the chromone in the liv e r . As no consistent hepatotoxicity occurred in the fe rre t , despite high doses of FPL 52757 for 28 days in combination with metabolic inducers, no clear evidence for a reactive metabolite was provided.

Since species which metabolise and excrete the chromone rapidly

(see Chapter 3) are not susceptible to the hepatotoxic e ffec t of the chromone and exhaustive attempts to implicate a reactive metabolite fa ile d , i t may be implied that the parent compound is responsible for

the hepatotoxicity. This conclusion must be viewed with caution when considering the rat and fe rre t investigational studies since neither of these species were susceptible to compound-induced hepatotoxicity.However, a sim ilar study in the beagle dog, the animal species most susceptible to FPL 52757 induced hepatotoxicity provides further

evidence for parent compound involvement,,

PART 3. INVESTIGATIONAL STUDIES IN THE DOG

Experiment to demonstrate the e ffec t o f phenobarbitone and methionine in FPL 52757 to x ic ity in dog: Both phenobarbitone and methioninedecreased the hepatotoxicity of FPL 52757 in the dog. Methionine protects the liv e r from hepatotoxicity by maintaining glutathione levels

(McLean and Day, 1975), and the phenobarbitone-induced increase o f cytochrome P-450 usually results in an increased formation of a reactive

metabolite (s'-) with an accompanying increase in hepatotoxicity. As the metabolism in dogs was nearly non-existent, despite induction of cytochrome P-450 with phenobarbitone, i t is apparent that protection by both phenobarbitone and methionine must involve other e ffects . B ile flow rate is increased by both phenobarbitone (Maxwell, 1973) and methionine

(Narodefskaya and Nasterin, 1974). The choleretic e ffec t of these compounds may have increased the chromone clearance rate and thereby protected the liv e r .

I t has been observed on a number of d iffe ren t occasions that b ilia ry excretion of a compound can be increased by stimulating b ile flow

(Angelucci ejt _aT_, 1970; Klassen and Plaa, 1968; Klassen, 1970; Roberts

and Plaa, 1967; Hart_et aT, 1969, and Goldstein and Taurog, 1968). The assumption for this possible method o f protection seems ju s tif ia b le in

the lig h t of the particu larly high concentrations of FPL 52757 found at post-mortem in b ile from the gall bladder of dogs receiving no pre­treatment or where pretreatment had been withheld. Analysis o f b ile

from the gall bladder of the dog receiving phenobarbitone pretreatment s t i l l revealed a high b ilia ry concentration of FPL 52757 (5 mg ml*" ) but this level was approximately ha lf that o f the other dogs. Furthermore,

microscopic examination of the liv e r revealed congested enlarged b ile ducts with erosion of b ile duct walls in severely affected dogs. The threshold

molecular weight for b ilia ry excretion varies between d ifferen t species (Hircm et 1972} and is dependent on the excretory process i ts e lf and

not differences in b ile flow (Smith, 1973). However, where a b ilia ry excretory mechanism exists for a compound its toxicokinetics may be altered by alterations in b ile flow rate .

Species variation in the pharmacokinetics of FPL 52757 w ill be shown

to be particu larly important in . ( i ) explaining species variation in the incidence of to x ic ity and ( i i ) adding further weight to*the development of an

alternative hypothesis, fo r the mechanism of hepatotoxicity involving the parent compound. Species variations in the metabolism and kinetics of FPL 52757 are described in the following chapter.

CHAPTER THREE

THE PHARMACOKINETICS OF FPL 52757

SUMMARY

The disposition of FPL 52757 in d ifferen t species has been investigated. The compound is extensively metabolised

by the following species:- mouse, ra t , hamster, rabb it, fe rre t, stump­ta iled macaque, squirrel monkey, cynomolgus monkey and baboon (e.g. 50-100% in ra t , cynomolgus monkey and squirrel monkey). The plasmaclearance of FPL 52757 in ra t , rabbit and squirrel monkey was 138, 44

- 1 - 1and 56 ml 'kg hr respectively. Plasma clearance by the dog and-1 -1man was in comparison slow (13 and 15 ml kg hr respectively, and

due mainly to elimination of unchanged compound in dog. In man

minimal or partia l metabolism occurred (4-50%). As the dog and man are particu larly susceptible to FPL 52757 induced hepatotoxicity the parent compound may be responsible. Species which clear the compound

rapidly compared with dog and man did not readily show a hepatotoxic response. A sim ilar mechanism for dog and man is probable though not proven. In v itro isolated rat liv e r perfusion experiments are described. This technique enabled high concentrations of the chromone to reach the

liv e r without the complications of generalised FPL 52757-induced to x ic ity .

INTRODUCTION

In the preceding chapters the toxicology of FPL 52757 and the unlikely role o f a reactive metabolite in hepatotoxicity have been described.This chapter outlines the metabolism and clearance of FPL 52757 and

links the • two e a rlie r chapters on a pharmacokinetic basis. Pharmaco­kinetic analysis for a number of species, including man, was achieved

by extrapolation from plasma concentration data accumulated during Fison's Safety Evaluation Programme. No such data existed for the dog., the one animal species readily susceptible to FPL 52757-induced hepato­to x ic ity . Experiments are described in th is chapter which enable toxicokinetic comparisons to be made between the susceptible and

resistant species on the basis of the length of exposure to the chromone. These investigations study the clearance of FPL 52757 in dog. The

toxicological implications of metabolism and clearance of the chromone are considered.

I t has been demonstrated that the ra t in vivo is highly resistant to FPL 52757 (see Chapter 2). The in v itro isolated perfused ra t liv e r technique is employed to allow exposure of the liv e r to high levels of the drug without the problems of generalised to x ic ity . In i t ia l ly intended as a 'new animal model system' in which to study the role of reactive metabolites, the technique contributes toxicokinetic data.As data accumulated implicating the parent compound in hepatotoxicity

the isolated perfused ra t liv e r programme was curtailed in favour of studies on the physico-chemical nature of chromones (see Chapter 4 ). Nevertheless, the limited data obtained from these few perfusion studies areincluded in this chapter. As only a small number of experiments were conducted the results must be viewed merely as further suggestive evidence of the importance of pharmacokinetics in FPL 52757“induced hepatotoxicity. More conclusive evidence comes with the in vivo

results in this chapter and the more sophisticated toxicokinetic

experiments in dog conducted at a la te r stage of this project (see

Chapter 6 ).

MATERIALS AND METHODS

FPL compounds

FPL 52757 (micronised free ac id ), FPL 52758 (water soluble Na+:sa lt o f FPL 52757), (14C) FPL 52757 (specific a c tiv ity 2.23yCi mg'1) ,( C) FPL 52758 (specific a c tiv ity 3.10 yCi mg 1) were synthesized by Fison L td .,Pharmaceuticals Division, Loughborough, U .K., as were \

the. metabolic/standards, . th e secondary alcohol (FPL 57704) and the ethinyl compound (FPL 5.8280) (see Chapter 2 for further d e ta ils ).

Pharmacokinetic Studies

Animal species

Animals were obtained as follows - mouse, males and females, 40g (CFW, Fisons Colony), ra t , males and females, T25g (Sprague-Dawley,Fisons Colony), hamster, males, lOOg (BioResearch Consultants, Boston,Bio 15-16 inbred s tra in ), guinea-pig^ males, 350g (Duncan-Hartley,Animal Virus Research In s titu te , P irbright, Surrey), fe rre t , males,1 to 2kg; A.S. Roe, Norfolk), squirrel monkey, males and females,0.5 to 0.9 kg, stumped-tailed macaque, males and females, 9kg, and

cynomologus monkey, males and females, 2 to 4 kg (Fisons stock originated from the w ild ), baboon, male and female, 8 kg (Shamrock Farms Ltd, Herefield, Sussex), beagle dog, females 15 kg (Fisons stock).

Animal d iet

Water was free ly available to a ll animal species via automatic

drinking systems. Each species received a commercially available pe lle t diet (provided by B.P. N utrition, Stepfield, Witham, Essex) with

appropriate supplements as follow s:-

Hamster, rats and mice - Oxoid Breeding Diet (Oxoid L td ., Basingstoke, Hants). Rabbits, Coney rabbit pellets No. 350 (B.O.C.M. Si lock Ltd, made up by T y rre ll, Byford and Pallet L td ., Station Road, Attleborough, Norfolk); ferrets , FDL pelleted d iet with a meat supplement (BP N utrition

U.K. L td ., Witham, Essex); dogs Spratts No. 2 d ie t (Spratts L td ., Barking, Essex, England); monkeys, mixed d iet with fre s h -fru it and Newmarket cubes

(Spillers-Spratts L td ., Barking, Essex); baboon, f r u i t supplements, Bonio biscuits (Spratts, Barking, Essex, England); old world monkey expanded

primate d iet (B.P. Nutrition (U.K.) L td ., Witham, Essex), Vitamin B^

(Cytacon, Glaxo Laboratories L td ., Greenford, Middlesex, England) was

added to the drinking water.

Dosing of animals and man

A ll animals received oral doses of FPL 52757 by stomach tube orvia a ball-headed dosing needle in a suspension of 1% aqueous methylcellulose (M420 British Celanese). Human volunteers received oraldoses in rice paper cachets or gelatin capsules. Urine and faeceswere collected from animals in metabolism cages. Intravenous dosing

14was carried out using water soluble sodium sa lt ( C) FPL 52758

(specific a c tiv ity 3.10 yCi mg"1) or { 14C) FPL 52757 (specific a c tiv ity 2.23 yCi mg"1) dissolved in 2% sodium bicarbonate (Sigma Chemical Co.,St. Louis, M.O., USA,).

14For clearance studies ( C) FPL 52758 was administered intravenouslyat 10 mg kg 1 to the ra t, rabbit and squirrel monkey. The dog receiveda range of doses (1-50 mg kg"1) 1ZC FPL 52757. Human volunteers receivedan intravenous dose of approximately 1 mg ml 1 of FPL 52758. Bloodsamples were taken at regular intervals up to 72 hours a fte r administrationof the compound. Clearance, which is the volume of plasma cleared ofrad ioactiv ity in one minute was calculated for animals from the area

14under the plasma ( C) concentration versus time curve,

i .e . Plasma Clearance Dose

(where AUC is the area under the plasma concentration versus time curve extrapolated-to t= oo). in the case of man the plasma clearance

of parent compound was determined. The values given for animals under­estimate the actual clearances of parent compound depending on the degree of metabolism

Analysis of FPL 52757 and metabolites

Total urinary and faecal excretion and plasma concentration of radio­

ac tiv ity in species receiving (^ C ) FPL.52757 and FPL 52758 were determined from the concentrations of rad ioactiv ity obtained by liquid s c in tilla tio n

counting of aliquots of urine, faeces homogenates and plasma. Samples

were dissolved in s c in tilla tio n flu id (Shellsol A 2 vo ls ., Triton X100,- i

1 v o l., plus 6g l i t r e of 2,5 diphenyloxazole). Radioactivity determinations

were carried out using a Packard Liquid S c in tilla tio n Spectrometer.Metabolic patterns and concentrations of the chromone in human- samples and other animal species receiving 'cold' FPL 52757 were determined

with extracts of urine, plasma and faecal homogenates using standard

t . l . c . techniques (see Chapter 2 ). Autoradiographs were prepared by placing t . l . c . plates in contact with an x-ray film (Blue, Brand Kodak L td .,) in a lig h t-t ig h t cassette for fiv e days, developing (Ilfo rd PQ Universal Developer) and fix ing (Kodafix, Kodak L td .).

Isolated Perfused Rat Liver Studies

Perfusion media.

A medium comprising 100 ml tissue culture medium 199 without phenol red (Difco Laboratories, D etro it, Michigan, USA), 2.5 bovine serum

albumin, fraction v (Sigma Chemical Company, St. Louis, Missouri, USA),500 units of heparin (BDH Chemicals L td ., Poole, Dorset) and 75 mg genta- mycin (Flow Laboratories L td ., Irv in e , Ayrshire) was prepared and 65 ml of th is medium made up to 100 ml with erythrocytes (washed with isotonic saline) to give the perfusion medium. The remaining ce ll free medium

(35 ml) was used as the pre-perfusion medium.

Animals

Rats, 250g, male (Charles River, Manston, U.K.) were anaesthetized-1with sodfum pentobarbitone (Nembutal, 60 mg kg ) and the b ile duct and

hepatic portal vein of each cannulated with pp 25 and pp 60 polypropylenetubing (Portex L td ., Hythe, Kent) respectively. An in situ infusion ofthe liv e r with the pre-perfusion medium 10 ml min*^ was performed duringhepatectomy a fte r which the liv e r was connected into a perfusionsystem sim ilar to that described by Curtis et al (1970). In th is systemthe medium was oxygenated by a continuous stream of a moistened mixtureof 95% 09 and 5% C09 passing over a multibulb oxygenator. The liv e r was

- 1 - 1perfused at a rate o f 1 ml min 9' liv e r under a constant head of 30 cm perfusate ( i .e . 30 cm hydrostatic pressure) at 37°C. The perfusion medium containing substrate (FPL 52758) was allowed to equilibrate in thesystem for 5 minutes before the introduction of the liv e r .

Table 3.1. Isolated Rat Liver Perfusion Experiments

Perfusion Number Treatment

PI Control, no addition of chromone.

P2 Control, no addition of chromone.

P3 100 mg of FPL 52758 was added to give a perfusate-1

concentration of lOOOyg ml .

P4' 75 mg of FPL 52758 was added to give a perfusate-1

concentration of 750 yg ml .

P5 40 mg of FPL 52758 was added to give a perfusate

concentration of 400 yg ml ^.

P6 40 mg of FPL 52758 was added to give a perfusate

concentration of 400yg m l~ \ The ra t liv e r had

prior exposure to SKF 525A 50 mg kg p.o. (Smith, Kline & French, Welwyn Garden C ity) one

hour before hepatectomy.

Analyses and assessment of to x ic ity from the perfusion experiments.

Perfusate samples were collected regularly for analyses of the serum enzymes isocitra te dehydrogenase (ICDH), alanine aminotransferase (ALT) and alkaline phosphatase C.ALP] using the manual techniques as described by Bernt and Bergmeyer (1974) and analysis of FPL 52758 and

metabolites by t . l . c . and Vitatron densitometry (see Chapter 2 fo r further details). Bile flow measurement was undertaken and samples were

collected at hourly in tervals.

RESULTSClearance and metabolic studies

The to ta l excreted rad ioactiv ity was measured fo r some of the species

in both urine and faeces (see Table 3 .2)and showed considerable interspecies

variation. The metabolic p ro file of the compound was compared in twelve species. Of the major metabolites in urine and plasma, quantitative

estimates are given where data is available (see Table 3 .3 ). The major metabolites are shown in Figure 3 .1 .Most species metabolised the compound

Tabl

e 3.2

Ex

cret

ion

of ra

dioa

ctiv

ity

in ur

ine

and

faec

es

afte

r or

al

and

intr

aven

ous

adm

inis

trat

ion

of FP

L 52

757

in va

rious

an

imal

sp

ecie

s

r .1

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•r— .4-5 •r— O CMO CO LO CMCOO

•r-*oCO ^ -— ^s- CM O CD CM

l o LO CD CO r .r— r— i 1 i 1 iCO CO r"» CO CD i— LO4-5 £- CM CM *3* CM LOO o -----' -—' -■—-* ^ '4->

CO LO *3- CO CD4- CO *3" d - co LOO

CD CDCD CCO •i— '•4-> S—C 3 ---s, *■— s, *—CD CD CM CDO r^. CO LOS- i 1 1CD . CM 1 i CD LOD_ > «3* - r— LO

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ham

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mal

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4 fe

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rabb

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8 fe

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m

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m Fi

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s.

Tabl

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P

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FPL

5275

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etab

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s in

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fd

CD CD CDI I1 1 U 1 1 1 O + O

fd tO fdE E E40 40 40

CD CD CD CDO U o O

+ 1i fd fd i + fd + + fdE E E E4-> +-> +o 4 0

o<u LOo ---fd + I + I +E

40

■ + CD + IuI fd I I I . s • ■ + I I ------

^ E ^ ^LO 4-> O Oco cmI I ILO CD o cd oCM O LO o r—

to to+ + E + + 1 ' + + E + +

4-> 4 0

^CO o+

CD3

> c r>> fdCD 0

>> fdCD E S I

OE E X JO CD

co E to 1—•1— Z3 •1—CL 1— CO fd

E 1 CD 40CD CD fd 40 4-> E 0 1 Eto 40 CD •r— CD E E Cl O3 ■ 40 CO E JO E • r “ 0 E OO fd E •1— JO E =3 E . zs CD JO

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m Fi

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s.

% =

Perc

enta

ge

of ad

min

iste

red

dose

. Fi

gure

3,1

sh

ows

chem

ical

st

ruct

ures

of

met

abol

ites

I to

IV,

OH 0

0/^C O O H

/

OH OH o

CHH3C

O^COOH

Cl) FPL 52757 ( I I ) The secondary alcohol (FPL 57704)

OH 0

HO.H,C

0X^COOH

( I I I ) Primary alcohol (IV ) Ethinyl compound (FPL 58280)

Figure 3.1 FPL 52757 and Major Metabolites

readily . Although the data is largely qualitative in nature the species

variation in the degree and pattern of metabolism is evident. The compound was metabolised extensively 50-100% in the ra t, cynomolgus monkey and squirrel monkey. To a lesser extent in man (4-51%) and to a negligible

degree in dogs. Most species produced the secondary alcohol ( I I ) as the major metabolite in urine. However, the squirrel monkey produced large amounts of primary alcohol ( I I I ) and no significant amounts of metabolite

( I I ) , The occurrence of the ethinyl metabolite (IV) was inconsistent.In general/ only parent compound ( I ) was found in significant amounts

in the plasma of a ll species.Table 3.4 gives values for the plasma clearance of FPL 52757 in ra t , rabb it, squirrel monkey, dog and man.Dog and man cleared the compound slowly compared with other species. The slow plasma clearance of FPL 52757 in the dog results in a sustained high plasma

concentration (see Figure 3 .2 ).

Isolated perfused ra t liv e r studies

C lin ical chemistryFigure 3.3 shows the results obtained for perfusions 1 - 4. Back­

ground hepatic deterioration is estimated by analysis of the control perfusates (PI and P2). A concentration of 750yg ml FPL 52758 (P4)in the perfusion media had l i t t l e effect on ALT or ICDH. However, ALP

-1was elevated. FPL 52758 at lOOOyg ml (P3) produced marked elevationof a ll three enzymes. Pretreatment with SKF 525A caused an apparentincrease in ALP when combined with a perfusion media concentration of400vtg ml~ FPL 52758 (P6). ICDH, ALT and ALP levels from the perfusion

_1receiving FPL 52758 (400 yg ml ) alone (P5) were within control lim its (see Figure 3 .4 ); , ' V }r / ' y

Bile flow

FPL 52758 reduced b ile flow rate . SKF 525A pretreatment did not a ffec t this reduction (see Table 3 .5 ).

FPL 52758 and metabolite levels in the perfusate

A slight reduction in FPL 52758 metabolism following SKF 525A pretreatment (see Table 3 .6 ).

Table 3.4 Plasma Clearance of FPL 52757

Species Number of Animals Investigated

Dose, mg kg"

Clearance, ml kg" hr

*Rat 4 10 138+

*Rabbit 4 10 44+

*Squirrel Monkey 2 10 59+

Dog 1 20'

5 12f

10 13+

20 10+

35 13f

50 13+

*Man 3 approx. 1 15

+ 14Denotes clearance of the ( C )-label, Clearance values fo r the parentcompound in these species w ill be greater depending upon the concentrationof (14C) metabolites present in the plasma. Thus the values given for dogw ill approximate to the clearance of the parent compound. The clearancegiven for man is for parent compound.

* Calculated from blood level data in Fisons Safety Evaluation F iles .

-57-

Plasma concentration (u g .m l-1 )

200150

100 H

5 0 -

25 -

1 0 -

5 -

2 -

dog

rabbit

ratsquirrel - monkey

0 ------------- 1------------ r2 A 6

T i m e ( h )

” i10

Intravenous infusions were given to the following species:-ih ® dog, 0 - - - - - - - - - - - 8 rabb it, A- - - - - - - - - - - - - - A squirrel monkey and o----- -o ra t ,and the subsequent levels are shown above.

1A -1Figure 3.2 Plasma concentration of C-FPL 52757 (pg ml )

following an intravenous infusion of 10 mg kg ^.

Figu

re

3.3

ICDH

, AL

T an

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Table 3.5 Bile Flow yl hr ^

Time(in hours)

Perfusion(No)

Pi P 2 P 3 P4 P 5 P 6

0 - 1 500 300 250(,K

trace 200 1501 - 2 400 250 trace 50 1502 - 3 400 250 trace 150 1503 - 3| 150 ' 125 trace 100 50

Bile duct cannulated with pp 25 polypropylene tubing; b ile drained and

collected in a graduated LP3 tube for hourly measurement.

Table 3.6 FPL 52757 and FPL 57704 levels yg -1ml in plasmasamples from P5 and P6

Perfusion(No)

Time(in hours)

P 5

FPL57704

FPL52757

P6FPL57704

FPL52757

1 37 390 0 4402 55 410 35 5753 70 400 55 460

°2 80 370 50 540

Two ml perfusate samples were collected at hourly intervals from a

sampling duct below the liv e r .

DISCUSSION

FPL 52757 was extensively metabolised and eliminated as parent compound

and metabolites in the urine and faeces of most species. Metabolism was principally by a-hydroxylation of the 6-ethyl substituent. There was no evidence for hydroxylation of the 8-ethyl group. Hydroxylation to the primary alcohol (designated I I I in Figure 3.1) was a minor metabolite in

most species with the exception of the squirrel monkey. The dog appeared to be atypical being unable to hydroxylate FPL 52757 to any appreciable

degree. The poor a b ility o f the dog to hydroxylate a lic y c lic or a liphatic carbon atoms which are readily metabolised by a number of species, including

rodents and primates, has been noted with a number of compounds • These include probenecid (Dayton et e\_9 1973), bucloxic acid (G ros^t al_, 1974), debrisoquine (Allen e t al_, 1970), dichloro-2-methyl-l-oxo-sindanyloxy acetic acid (Zacchei et al_, 1978) and proxicromil (Smith, 1980). Whilst this

seems to indicate a trend there are examples where the dog more readily hydroxylates a lic y c lic and aliphatic carbon atoms. For instance 3a, 4, 5 ,

7, 7a-Hexahydro-3-(l-methyl-5-nitro-lH-im idazol-2yl)l,2-benzisoxazole is

extensively hydroxylated at the a lic yc lic carbons of its hexahydrobenziso- oxazole ring by the dog (Vandenleuvel et'al_, 1976). S im ilarly , one o f the tertiarybutyl groups of terbucromil (Augstein £ t al_., 1976) which is related

in structure to proxicromil and FPL 52757, is also readily hydroxylated by the dog (unpublished data). Recently substantial differences have been demonstrated between the microsomal components of the liv e r in ra t and dog (Golan £ ta j_ , 1979) . The total cytochrome P-450 level o f dog liv e r re la tive

to microsomal protein is about h a lf that of ra t , yet the rate o f in v itro 0-dealkylation of 7-ethoxycoumarin in dog is more than twice that o f ra t re la tiv e to microsomal protein. The types of individual cytochrome P-450 present in dog liv e r are therefore lik e ly to d iffe r substantially from those present in the ra t and these differences may be reflected in the substantial variations in carbon hydroxylation observed for FPL 52757 between species.

The ethinyl compound (designated IV) in Figure 3.1 was found in small irregular amounts in some species, but more consistently in human samples.I t probably arises via dehydration from the secondary alcohol. Dehydration

has been shown to occur with other compounds, for example, quinic acid is converted to benzoic acid in the gut of old world monkeys (Adamson e t a l , 1970).

The slow plasma clearance of FPL 52757 in the dog and man (see Table 3.4) results in a sustained high plasma concentration following

dosing (see Figure 3 .2 ). This facto r, together with a high degree of hepatobiliary clearance of the parent compound in the dog may explain

the particular sensitiv ity of this species to the hepatotoxic action of the chromone. Hepatotoxicity due to FPL 52757 in the dog and

possibly man may therefore be related to the amount of parent compound

accumulating in the hepatobiliary system.

The isolated perfused ra t liv e r experiments, because of th e ir

incompleteness, cannot be regarded as conclusive. However, i f to x ic ity is considered to be related to drug treatment, i t is apparent that FPL 52757 reduced b ile flow and produced hepatobiliary damage. Evidence for chromone-induced to x ic ity would have been more convincing i f a clear dose-response had occurred. Nevertheless, even the lim ited data obtained provides further evidence for the high degree of resistance of the rat liv e r to FPL 52757 induced-hepatotoxicity.Additional experiments were planned to elucidate the role of metabolism in to x ic ity . These studies were terminated (as data accumulated from the

in vivo studies described in Chapter two implicating the parent compound in hepatotoxicity) in favour of a simple novel method for the assessment of the hepatotoxic effect of FPL 52757 and other chromones on the ra t liv e r by retrograde b ilia ry infusion (see Chapter 5 ) and experiments to elucidate the physico-chemical value of chromones (see Chapter 4);

CHAPTER FOUR

The physico-chemical properties of chromones.

SUMMARY

The degree of detergency of a series of chromones is linked to structural properties and a detergency/structure a c tiv ity relationship established. .. • ,

INTRODUCTION

The results in the previous chapters implicate the parent compound

as the hepatotoxic agent and species variation in susceptib ility is

explained on a toxicokinetic basis. Although these findings contribute

towards an understanding of chromone induced to x ic ity , the actual mechanism of hepatotoxicity remained obscure. In th is chapter the physico­chemical properties of FPL 52757, and a number of related chromones are

considered. The structure of FPL 52757 suggests that i t should have detergent a c tiv ity (Schwartz and Perry, 1949). To investigate th is

possib ility the surfactant properties of FPL 52757 have been compared with a number of other chromones. Surface action could account fo r the hepatotoxicity of FPL 52757 i f accumulation in the liv e r occurred.

To complement surface a c tiv ity data a study on the a b ility of chromones to form micelles in solution is reported.

MATERIALS AND METHODS

FPL compounds and other chemicals.FPL 55689 (6 ,8-diethyl-4-oxo-4H-l-benzopyran-2-carboxylate), FPL 52758

(Na* salt)ofFPL 52757 (6 ,8-diethyl-5-hydroxy 4-oxo-4H-l-benzopyran-2-carboxyl

ac id ), FPL 57704 (8-ethyl-5-hydroxy-6-(1-hydroxyethyl)-4-oxo-4H-l-benzopyran- 2-carboxylic ac id ), FPL 58280 (6-ethinyl-8-ethyl-5-hydroxy-4-oxo-4H-l-benzo- pyran-2-carboxylic a c id ), FPL 57731 (4-oxo-6,8-dipropyl-4H-l-benzopyran-2- carboxylic acid) , FPL 57498 (5-hydroxy-4-oxo-6,8-dipropyl-4H-1-benzopyran-2- carboxylic a c id ), FPL 57579 (6,7,8,9-tetrahydro-4-oxo-10-propyl-4H-l-naptho- pyran-2-carboxylic ac id ), FPL 57787 (Proxicromil) (6 ,7 ,8 ,9-tetrahydro-5- hydroxy-4-oxo-10-propyl-4H-naptho 2,3-6 pyran-2-carboxylic acid) and FPL

59665 (6,7,8,9-tetrahydro-5,7-hydroxy-10-propyl-4H-naptho 2,3-6 pyran-2- carboxylic acid) were provided by Fisons Pharmaceuticals, Loughborough, Leicestershire, U.K. A ll FPL compounds were more than 98% pure, th e ir structures were established by unequivocal synthesis confirmed by n .m .r., i .v . and u.v. spectrometry (personal communication). Lauryl sulphate and Rhodamine 6G (benzoic acid 0-(6-ethylam ino-3-(ethylim ino-2,7 dimethyl 3H- xanthen-9-yl) ethyl ester monohydrochloride were obtained from the Sigma Chemical Company, St. Louis, M.O., U.S.A.

N.B. The structure of each of the above FPL compounds is given in the Results Section (Table 4 .1 ).

Surface a c tiv ity evaluation

The surface tension of solutions of FPL 55689, FPL 52757 (or its sodium s a lt, FPL 52758), FPL 57704, FPL 58280, FPL 57731, FPL 57498,FPL 57579 (Proxicromil) , FPL 57787 and FPL 59665 were measured at 22°C

+ 2 C with a Du Novy tensiometer. A measure of surfactant a c tiv ity is the surface pressure | ( i .e , surface tension of control solution of 1% aqueous NaHCO minus the surface tension of the drug-containing solution (Dujovne, 1975)|

C ritica l micelle concentration'(CMC) measurements

The CMC was measured by adapting a technique by Carey and Small(1969), Rhodamine 6G was prepared as a stock solution with 1%> aqueous

- f iNaHCOg to give a fin a l concentration of 2,5 x 10 M. A range of concentrations (between 0 - 76 mM) of FPL 52757, FPL 57787 and FPL 58280 wei*e prepared in Rhodamine 6G and scanned on a Varian spectrophotometer from 580 to 500 nm, to determine the wavelength of maximum absorption at room temperature.

The CMC is extrapolated from the lower break point of the plot of the wavelength of maximum absorption of Rhodamine 6G versus log concentration,

RESULTS

Surfactant properties of FPL compounds

The surface a c tiv ity expressed as the surface pressure is given in Table 4.1 . FPL 57498, 57787, 57579 and 52757 had surface a c tiv ity effects sim ilar to that of lauryl sulphate. The major hydroxylated metabolite (FPL 57704), the ethinyl compound (FPL 58280), FPL 55689 and FPL 57731 were shown to have l i t t l e or no effec t. From the structures

of FPL 52757 and other chromones (Table 4 .1 ), a s tructure-activ ity relationship is apparent. FPL 52757 exhibited a concentration related

increase in surface a c tiv ity , for FPL 58280 and FPL 57787 the increase was reversed above a c r it ic a l concentration (see Table 4 .2 ).

C ritica l micelle concentration (CMC)

The spectral maximum of the dye is plotted against the log concentrati of the chromone (see Figure 4 .1 ) , and the spectral sh ift which occurred

over a range of chromone concentration is illu s tra ted . The lower break

Table 4.1 The structure and surface pressure of FPL compounds

FPL 57579

FPL 57787 (Proxicromil)

FPL 59665

Lauryl sulphate

COOH

Compound Ri R2 R3 R4 Surface Pressure

FPL 52689 ii C2H5 H C2H5 4* (+ 3 .0 )

FPL 52757 OH C2H5 H C2H5 12 (+ 1 .0 )

FPL 57704 OH c2h. oh H C2H5 4* (+ 2.0)

FPL 58280 OH C2H4 H C2H5 6 (+ 2 .0 )

FPL 52731 H C3H7 H C3H7 6 (+ 2 .0 )

FPL 57498 OHC3H7

H C3H7 33 (+ 1 .0 )

OH

OH

C3H7

C3H7

C3H7

14 (+ 5 .0)

20 (+ 3 .0)

4* (+ 4 .0)

30 (+ 2 .0)

The surface pressures of the FPL compounds and lauryl sulphate were-1measured a t 1 mg ml in ]% aqueous NaHCO . The surface tension of 1%

aqueous NaHCOg was 65 (+ 4 ).

* Change in surface tension within control values.Standard deviations from 6 readings are given in brackets.

Table 4 .2 . Additional surface pressure (dynes cnT^) measurements.

Compound

Concentrationsmg/ml

Surface pressure dynes cm

FPL 52757 FPL 58280 FPL 57787 Proxicromi

0.5 10 (+ 1 ) 3* (+ 1) 10 (+ 1)

1.0 12 (+ 1 ) 4* (+ 2) 20 (+ 1)

2.0 15 (± 1 ) 10 (± 1) 23 (+ 1)

4.0 16 (+ 1 ) 13 (± 1 ) 24 (+ 1 )

8.0 19 (± 1 ) 15 (+ 1 ) 22 (+ 1)

i—i o o 21 (+ 1 ) 22 (+ 0) 21 (± 1)

20.0 20 (+ 3) 11 (+ 1 ) 20 (+ 2)

The surface pressure of FPL 52757, FPL 58280 and FPL 57787 was measured"1over a range of concentrations (0.5 - 20.0 mg ml ).

The surface tension of 1 % aqueous NaHCOg was 6.5 (+ 4 ).

*Change in surface tension within control values.

Standard deviations fo r 6 readings are given in brackets.

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point a t A is taken as the arb itrary CMC value, and gave the following

values - FPL 52757 0.30 mM; FPL 58280 0.22 mM and FPL 57787 0.19 mM.

DISCUSSION

The structu re -activ ity /surface-activ ity relationship of the FPL compounds.

The surface-activity of FPL compounds (having a strongly polar

carboxyligroup at one end of the chromone nucleus and a lipoph ilic chain or ring at the other end) is in agreement with that suggested by direct observation of the number of linear carbon atoms comprising the lipoph ilic

portion of each molecule. The 5-hydroxyl group substituent (a t R^) .e ffec tive ly increases the lip o p h ilic ity (by strong hydrogen binding to the adjacent keto function, i t negates its own polarity and that of the keto group) and is essential fo r surfactant properties in a chromone.(see Table 4 .1 ). Hence the potency of the parent chromones is as follows - FPL 57498 > FPL 57787 > FPL 52757. The metabolites of FPL 52757, FPL

57704 and FPL 58280 were comparatively inactive as was FPL 59665 the

metabolite of FPL 57787. The analogues of FPL 52757 and FPL 57787; FPL 55689

and FPL 55731 respectively, also lacked surface a c tiv ity . However, FPL 57579, the analogue of FPL 57787, retained some of the parent compounds

surfactant properties despite the absence of the 5-hydroxyl group. As

a general rule either the insertion of an additional hydroxyl group into the lipoph ilic region or the deletion of the original 5-hydroxyl group

appears d rastica lly to reduce the detergency of the parent chromones.

The surface a c tiv ity of FPL 527^7 in solution continued to increase

with increased drug concentration. However, th is phenomenon •‘did not hold

for the other chromones investigated (see Table 4 .2 ).

The c r it ic a l m icelle concentration of FPL 52757, FPL 57787 and FPL 58280.

At the outset of this experiment i t was believed that the a b il ity of the chromones to form micelles would correlate with reduced surfactant and

toxic properties. The comparatively low CMC of FPL 57787 and FPL 58280 could explain the surface tension results obtained with increasing

chromone concentration i f m icelle formation reduced surface a c tiv ity .Howeverj Chapter 5 w ill show there is no relationship between to x ic ity and the CMC values reported in th is chapter.

Carey and Small (1969) suggest that wavelength changes are produced by

dye binding to micelles and as more micelles form more dye is bound, and

the top break point is where the dye partition is strongly in favour of micelles, thus the spectrum ceases to s h ift. This implies that the top break point represents the concentration where micelle formation is at its height. Values were as follows: FPL 52757 9.4 mM; FPL 57787 12.5 mM and FPL 58280 5.0 mM. The smaller the above value the lower the

concentration at which the micelle form of the chromone w ill predominate,A correlation with to x ic ity FPL 58280 < FPL 52737 < FPL 57787 occurs in a number of in v itro tests (see Chapter 5 ), which'may'imply that the existence of the chromones in the largely micelle form reduces the ir

surfactant properties and to x ic ity .

Coleman & Hoidsworth(l976)relate micelle formation to erosion and damage of the b ile canalicular membrane. They suggest that as b ile sa lt concentration exceeds CMC in the canalicular lumen the b ile salts form micelles which withdraw lip id from the outer face of the canalicular

membrane resulting in membrane perturbation and loss of externally orientated enzymes (e.g. 5' nucleotidase and alkaline phosphatase).

In relation to chromone to x ic ity , the role of micelle formation

remains unclear. Suffice i t to say that thethree chromones had sim ilar

CMC and sim ilar physicochemical properties to b ile salts (Carey and

Small, 1969), and lik e these endogenous detergents the chromones can form micelles which may in terfere with the plasma membrane of the

b ilia ry trac t in the manner postulated for b ile salts.

In the following chapter the physico-chemical characteristics of the FPL compounds are related to to x ic ity , and the a b ility of FPL 52757

to damage the hepatobiliary trac t is investigated in both Chapters 5 and 6.

CHAPTER FIVE

The relationship between chromone to x ic ity and detergency

-71-

SUMMARY

The to x ic ity of FPL 52757 in retrograde b ilia ry infusion studies in ra t and in the lysis of erythrocytes in v itro is related to the

detergent properties of the drug. Other chromones have also been

examined. The greater the detergent properties of the chromones the

greater th e ir to x ic ity in both biological tes t systems.

INTRODUCTION

In the previous chapter a detergency/structure-activity relationship was presented. This surface-action could account for

the hepatotoxicity of the drug i f accumulation in the liv e r occurred.To investigate th is possib ility the to x ic ity of FPL 52757 has beencompared with a number of other chromone compounds in erythrocytelysis and retrogade b ilia ry infusion studies in ra t. The erythrocytelysis experiments give a measure of the actual membrane damagingpotential of each chromone. The retrograde b ilia ry infusion studiescan be regarded as a novel method of assessing the hepatotoxicpotential of drugs, i f the hepatobiliary system is involved inprogression of drug-induced to x ic ity . This method enables high concentrationsof a drug to be administered to the ra t liv e r with considerable ease via thehepatobiliary trac t thus largely avoiding metabolism.

The toxicology of FPL 52757 in the hepatobiliary trac t is ofparticular interest for a number of reasons:

- FPL 52757 was found in high concentrations (approx. 8 mg ml

in gall bladder b ile from dogs at autopsy (see Chapter 2).

- Gall-bladder b ile samples from these dogs were abnormally th ick, viscous and dark.

- Excretion of FPL 52757 via the b ile duct appears to be saturable in dog (see the following chapter).

- Following oral FPL 52757 treatment in dogs histopathological examination of liv e r sections revealed enlarged, congested and damaged b ile ducts (Chapter 2).

- Coleman e t aj[ (1976) havesuggested that the endogenous detergent bile salts cause membrane perturbation*, i t seemsprobable that exogenous detergents in the b ile may have a sim ilar and possibly more drastic e ffe c t.

Following retrograde b ilia ry infusion of chromones toxicological assessment by measurement of b ile flow, b ile enzyme levels and b ile ' electrophoresis is complemented by conventional micro-histopathology.These unconventional but sensitive means of assessing to x ic ity are used again in the following chapter in detailed toxicokinetic studies

in dog and ra t (see Chapter 6 ). The results from Chapter 6 are pertinent to those presented in this chapter.

MATERIALS AND METHODS

FPL Compounds

FPL 55689, FPL 52758 (Na+ sa lt of FPL 52757), FPL 52757,FPL 57704, FPL 55731, FPL 57498, FPL 57579, FPL 57787 and FPL 59665. (The fu ll chemical names and structures are given in the

previous chapter).

METHODS

Erythrocyte Lysis

Erythrocytes were prepared by centrifugation (2,500 x g for 10 minutes) of freshly drawn heparinised ra t blood. The separated erythrocytes were washed twice by resuspension in isotonic saline

and recentrifuged and then suspended in a volume equal to twice the

original blood volume. Solutions (0.95 ml) in isotonic saline of the chromone compounds under te s t a t various concentrations were incubated at 37°C for 5 minutes. Aliquots (0.05 ml) of the erythrocyte suspension were

added to the chromones, rapidly mixed and incubated at 37°C for 15 minutes. Incubations were terminated by centrifugation (2,500 x g for 10 min.) and an aliquot (0.1 ml) of the supernatant was removed, diluted with

d is tille d water to 1 ml and the absorption measured at 416 nm in a spectrophotometer (Pye Unicam SP 1800, Cambridge, England). Reference samples to determine the degree of lysis were produced by a suspension

of erythrocytes in isotonic saline with no drug (0% lys is ) and in d is tille d water (100% ly s is ).

Erythrocytes rather than isolated liv e r cells were chosen as the model ce ll system for the reasons given by Coleman dt ^ l (1979) in his 'studies

on mammalian b ile composition and its membrane damaging properties.

Retrograde b ilia ry infusion

The method of retrograde b ilia ry infusion was adapted from Takadaet al ( 1976). Male Wistar ra ts , 250 g, (Charles-River, Manston, U.K.)were anaesthetized by an fntraperitoneal in jection of phenobarbitone

-1(Nembutal, 60 mg kg ). The common b ile duct was cannulated using

pp 25 Portex polypropylene tubing (Fisons S cientific Apparatus). Bile

was collected and the flow rate measured. The chromone compounds were

dissolved in fresh ra t b ile ( i f they were insoluble in b ile 1% aqueous NaHCOg was used) and were injected (250 y l) into the b ile ducts followed by a further 250 yl of b ile , the syringe being held in place for one

minute post-infusion. The e ffect of th is retrograde infusion on the b ile flow rate was compared with an infusion of control b ile or 1%

aqueous NaHCOg containing no chromone. The flow rate was measured for 1 hr prior to infusion and two hours post infusion. Bile samples were analysed fo r 5 '-nucleotidase and gamma-glutamyl transpeptidase levels.Liver damage was assessed by lig h t microscopy. Bile protein content was assessed by immunoelectrophoresis.

C lin ical chemistry (on b ile samples)

Gamma-glutamyl transpeptidase (G-GT): G-GT catalyses the transfer ofthe glutamyl residue from y-glutamyl-p-i n itroanilide to glycyl-glycine. The

liberated p-n itroaniline is proportional to the G-GT a c tiv ity . The absorbance increase^caused by its yellow colour can be measured at 410 nm.A manual method was adapted from the two established techniques described by Rosalki (1975). Two ml of 6.25 mM L-glutam yl-p-nitroanilide in 0.1 M

Tris with 0.05 M glycyl-glycine was mixed with 0.5 ml of b ile (diluted 1 in 5 ). This was incubated a t 37°C for 1 hr. The reaction was stopped with .1 ml of acetic acid, and solutions were read at 410 nm.

5'-Nucleotidase: 5'-nucleotidase liberates phosphate from the substrate5’ -adenosine monophosphate (5'-AMP), The liberated phosphate is

proportional to 5'-nucleotidase a c tiv ity .

A manual nucleotidase assay was based on the method of Jjoldsworthand Coleman (1975). 0.4 ml of buffer cons i s ti ng of 0.25 M Tris^ 12.5 mMMgClg and 25 mM Na K ta rtra te (fpH 7. 8^at~~37 °Cj was mixed with 0.5 ml b ile(diluted 1:5) and 0.1 ml of 0.05 M adenosine monophosphate, and incubatedfo r 1 hr at 37°C. 1.5 ml of 6% Trichloracetic acid was added and proteinremoved by centrifugation. To measure phosphate in 2.5 ml of supernatant,1.5 ml of 6% ammonium molybdate in 5% perchloric acid was added and le f t

-1fo r 5 min. F inally 0.5 ml of ascorbic acid (2 mg ml ) was added and

the solution read at 700 nm a fte r 20 min.

Electrophoresis of b ile samples: Inmunoelectrophoresis (based on thetechnique of Axel senret aj_, 1973),was used to characterise b ile proteins from ra t b ile samples following retrograde b ilia ry infusion. Agarose 1%

was mixed with a tris/barbitone buffer pH 8 .6 , boiled gently un til

completely dissolved then maintained at 55°C. 2.5 ml agarose was runon to a plate in a measured s tr ip , a well was cut and 5 yl b ile sample was applied. A current of 80 volts was maintained across this s trip for 1 hr. 7.5 ml agarose containing antisera was applied before the

next run of 12 volts overnight, which was followed by a staining with Coomassic B r il l ia n t Blue R. Plasma proteins were removed from selected

b ile ra t samples prior to electrophoresis by conjugation on to a Sepharose slurry on to which anti-serum to plasma protein had been bound overnight (Mullock et aj_, 1977). Bile samples were gently shaken in a tumble mix overnight with the slurry. Preparation of antisera was by the method described by Mullock eit aj[ (1977).

RESULTS

Erythrocyte lysis

Incubation of the various chromones with erythrocytes gives an indication of th e ir actual membrane damaging potential in terms of the

concentrations required to lyse 5.0% of the erythrocytes (EL^q) (see Table

5 .1 ). The potencies of the chromones to cause erythrocyte lysis correlated

d irec tly with th e ir surface a c tiv ity in solution. The major hydroxyl metabolite of FPL 52757 (contrasting with the parent compound), shows no membrane damaging effects under the conditions of the tes t. The pattern

is repeated for other chromones, and metabolites.

Retrograde b ilia ry infusion

To reproduce the effect of high concentrations of FPL 52757 and other chromones occurring in the b ilia ry system, compounds were

introduced into in tact anaesthetised rats by retrograde infusion into the

b ile ducts. The to x ic ity of each of the compounds was then assessed according to impairment of b ile flow and the degree of liv e r damage. Infusion of surfactant chromones produces dose-related cholestasis

(see Table 5.2) (e .g . FPL 52757 infusion at 5 mg, 7.5 mg and 10 mg gave

23%, 54% and 100% decreasesin b ile flow respectively) which is accompanied by dose-related hepatocellular and bile-duct damage (see Table 5 .2 ).D irect damage has been assessed by conventional lig h t microscopy of l iv e r sections. Both b ilia ry flow impairment and the degree of necrosis correlated with the surfactant a c tiv ity of the chromone compounds.

Table 5.1. Aqueous surface a c tiv ity (detergency) and erythrocyte

lysis for a series of chromones

Compound El -1 50 mg ml 1 Degree of surface a c tiv ity (Detergency)

FPL 55689 > 2 slight

FPL 52757 1.05 moderate

FPL 57704 > 2 slight

FPL 58280 not measured slight

FPL 55731 1.5 sligh t

FPL 57498 0.17 high

FPL 57579 1.02 moderate

FPL 57787 0.18 high •

FPL 59665 > 2 V s ligh t

Lauryl sulphate 0.04 high

EL q (concentration to lyse 50% of the erythrocytes in test solution) value given alongisde the degree of surface a c tiv ity (data derived from

previous chapter). The erythrocyte was incubated with solutions of compound under test for 15 min. at 37°C. Lysis was assayed by absorption a t 415 nm.

(N.B, Lauryl sulphate is a compound with known detergent properties)

Tabl

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FPL 52757, in company with the other surface active chromone (FPL 57498) and lauryl sulphate, markedly impaired b ile flow and

damaged the b ilia ry system and surrounding hepatocytes. The remaining

chromones, including the major hydroxyl metabolite of FPL 52757 (FPL 57704) and the ethinyl compound (FPL 58280) showed no effect (see Table 5 .2 ). Qualitative and quantitative data on hepatobiliary damage was obtained by measurement of 5 '-nucleotidase and G-GT levels and immunoelectrophoretic studies.

B ilia ry enzyme analysis

Retrograde infusion of FPL 52757 into the b ile duct of the ra t produced no change in 5 '-nucleotidase levels in subsequent b ile samples. G-GT showed a significant dose-related elevation (see Table 5 .3 ).

Immunoelectrophoresis of b ile samples

Bile against b ile-protein anti sera: Plates 1 and 2 show the results obtained following 2-dimensional electrophoresis of b ile samples in the presence of b ile protein antisera (2%). The b ile sample on plate

2 was obtained following in tra b ilia ry retrograde infusion of FPL 52757 at 5 mg. Plate 1 is a control. The test sample was shown to contain greater amounts of protein.

Bile against b ile-protein and serum-protein anti sera: Plates 3 and 4

show the results obtained following 2-dimensional electrophoresis of b ile samples in the presence 0f b ile protein antisera (2%). Plates 5 and 6

show the results obtained following 2-dimensional electrophoresis of bile samples in the presence of 2% rabbit immunoglobulins to ra t serum protein. The b ile samples on plates 4 and 6 were obtained following

retrograde infusion of FPL 52757 at 7.5 mg. Plates 3 and 5 were obtained

from control b ile samples. Plates 3 and 4 confirm the results obtained above. Comparing the two experiments a dose-response increase of protein

in the b ile is evident. Bile run against antisera to plasma proteins indicates a massive leakage of serum proteins into the b ile .

Conjugated and unconjugated b ile experiment: The following two dimensionalelectrophoresis plates were run, using b ile samples obtained a fte r b ilia ry retrograde infusion of 5 mg FPL 52757:

Table 5.3. S'-Nucleotidase and gamma-glutamyl transpeptidasea c tiv ity in b ile following retrograde b ilia ry infusionof FPL 52757

Total infusion dose in mg.

5 '-Nucleotidase _-] ymol product formed min

G-glutanyl transpeptidase yg product formed hour

0 5 (+ 1.2) 3.9 (+ 1.3)

1.25 7.2 (+ 2 .0 ) 5.6 (+ 2.5)

2.5 7.3 (+ 1 .0 ) 7.0 (+ 2.7)

5.0 5.5 (+ 2.2) 9 .3* (+ 4 .3)

10.0 not measured 12.5* (+ 6 .0 )

Each result represents the mean of at least fiv e samples. Standard deviation for each result is given in brackets.

^Results d iffe r from control p > 0.02.

Plate 1 Control B i le .

-Plate 2 B ile fo llow ing RBI o f FPL 52757 (5 mg)

B ile against b i le -p ro te in anti sera

%

Plate 3 Control B ile

Plate 4 B ile from RBI o f FPL 52757 (7.5 mg)

■ 'ilje against b i le serum-protein a n t iw a

Plate 5, Control B fle ,

P la te 6. B ile from RBI of FPL 52757 (7.5 mg)

Plate 7. Bile run against ra t anti-sera to plasma protein (2%).Plate 8. B ile against antisera to b ile proteins (2%).Plate 9. Conjugated b ile against anti-sera to b ile protein (2%)Plate 10. Conjugated b ile against anti-sera to b ile canalicular

membrane (5% ).^Plate 11. Conjugated b ile against ra t anti-sera to plasma protein (2%).

Analysis of plates showed massive leakage of plasma protein. Plate 8 showed

a low IgA component. The apparent removal of the secretory component from the b ile run against a n ti-b ile anti-sera further indicates severe damage to

hepatocyte function. There was a complete absence of protein (from canal­icu lar membrane) on plate jo t The presence of plasma proteins in plate d

indicated that to ta l removal during conjugation was not achieved.

DISCUSSION

In the previous chapter the physico-chemical properties of the chromones were compared and a 'detergency/structure-activity ' relationship is established. The to x ic ity of the FPL compounds, assessed

by the methods described in this chapter, is in agreement with that suggested by the degree of detergency of each compound. The novel method fo r assessing hepatobiliary to x ic ity allowed the lik e ly e ffec t of FPL 52757 in the dog to be reproduced in the ra t, since the hepatobiliary

trac t of the dog is lik e ly to be exposed to high concentrations of FPL 52757 following oral dosing. (This concept is verified in the next chapter). The retrograde b ilia ry infusion allows the hepatobiliary

damaging potential of FPL compounds to be compared in a specific manner in a convenient small animal species.

Analysis of b ile samples obtained following retrograde b ilia ry infusions of FPL 52757 showed a dose-related increase in G-GT w hilst 5'-nucleotidase levels remained constant (see Table 5 .3 ). Since G-GT

and 5*-nucleotidase are marker enzymes for ce lls lin ing the b ilia ry ductules and ducts (A!bert et cH, 1964; Roslaki, 1975) and the b ile canal­icu lar membranes (Toda et a l , 1964; Rosalki, 1975) respectively, i t is

apparent that FPL 52757-induced damage in th is study was restric ted to the b ile ducts and ductules. This contrasts with a previous study in ra t

(Nekae et 1977) where retrograde infusion of another surfactant (Triton X-100) caused 5'-nucleotidase elevation.

*B'1$nk plate pot included,

Conjugated and unconjugateo DiJe experiments

Plate No.7. B ile fo llow ing RBI FPL 52757 (5 mg) against an ti-sera to plasma p ro te in .

Plate Mo.8. B ile fo llow ing RBI FPL 52757 (5 mg) against b i le prote in an ti-se ra .

•Conjugated and unconjugated b i le experiments

Plate No.9. Conjugated b i le fo llow ing RBI FPL 52757 (5 mg) against an ti-se ra to b i le prote ins.

# v - . . ' \ 6 ?

. -V

X

No 11 ‘Conjugated b i le fo llow ing RBI FPL 52757 (5 mg) Plate no. . a n t i_sera tQ plasma p ro te in .

The infusate was maintained in place for 15 min. compared with

only 1 min. in the current study. This difference in experimental conditions probably allowed the surfactant to penetrate further up the b ilia ry tract and a ffec t the canalicular membrane in the Nekae

study. The lack o f an e ffec t on the b ile canaliculi in this study was verified by 2-dimensional immunoelectrophoresis of b ile against anti-sera to b ile canalicular membranes. The absence o f protein bands on this plate indicated a complete lack of protein derived from b ile canalicular membranes. These enzymes and electrophoretic results may be viewed as evidence for the s ite specific nature of 5'-nucleotidase.

In general the electrophoretic results demonstrated a massive leakage of serum proteins into the b ile following retrograde b ilia ry infusion o f FPL 52757. Leakage o f such large amounts sabotage attempts to completely remove the serum proteins, and masked changes in other

specific b ile proteins.

The liv e r damage produced by retrograde b ilia ry infusion clearly

showed that the more surface active chromones lik e FPL 52757 have the potential to damage both the b ilia ry system i ts e l f and the surrounding hepatocytes. However, these studies were conducted with high concentrations o f compound introduced as a bolus.. The 'in s u lt' to the b ilia ry system a fte r oral or i . v . administration is lik e ly to involve exposure to the compound at lower levels for longer periods. Nevertheless, the retrograde infusion toxicology is validated in view o f the sim ilar pattern of FPL compound to x ic ity established in the erythrocyte lysis

experiments. Further validation is provided by the 'hepatobiliary toxicokinetic' studies in dog and ra t in the following chapter and the observation that levels of up to approximately 8 mg ml” o f FPL 52757

have been found in the gall-bladders of dogs following multi dose oral treatment.

CHAPTER SIX

The hepatobiliary toxicokinetics of FPL 52757 in ra t and dog

-87-

SUMMARY

Toxicokinetic studies in dog showed that intravenous infusion of_1 _1

FPL 52757 at 4, 8 and 12 mg kg hr for 2| hours resulted in increased b ilia ry excretion of alkaline phosphatase (up to 17-fo ld ), gamma-glutamyl transpeptidase (up to 9 -fo ld ), and 5 '-nucleotidase (up to 13 fo ld ) which

paralleled the b ilia ry concentration of FPL 52757 (up to 1.3 mg ml~^) and was accompanied by alterations in b ile flow, jhe excretory mechanism of FPL 52757 into the b ilia ry trac t was shown to be saturable and could

result in high hepatocellular drug concentration and subsequent liv e r damage. In a paralle l study in ra t , sim ilar early indications of a potential hepatotoxic e ffect did not occur following an intravenous bolus

. . iof 50 mg kg FPL 52757 and the study demonstrates yet again the high degree of resistance of this species to FPL 52757-induced hepatotoxicity.

INTRODUCTION

Previous to x ic ity studies have demonstrated that FPL 52757 {6,8- diethyl-5-hydroxy-4-oxo-4H-T-benzopyran-2-carboxylic acid) is hepato- toxic to the dog but not the ra t following oral dosing (see Chapters .1 and 2). Centrilobular necrosis occurs in dogs accompanied by some

damage to b ile ducts. The ra t is protected by fast metabolism and

elimination of FPL 52757 whereas the clearance from the dog is slow

compared with other species (see Chapter 3) and high concentrations of FPL 52757 have been found in the b ile contained in the gall-bladder

at autopsy. FPL 52757 has been shown to be a detergent (see Chapter 4) and its to x ic ity is related to its detergent properties (see Chapter 5). Elevation of b ile enzymes have been reported following oral treatment with b ile salts in man (Bode et cH, 1973)

and following retrograde b ilia ry infusion of FPL 52757 in the ra t (see Chapter 5). The objectives of th is study were to gain further information on the pharmacokinetics of FPL 52757 in the dog and ra t following intravenous dosing and to re la te drug levels in blood and

b ile to early physiological and pathological drug-induced changes in

the hepatobiliary trac t. Attempts were made to slow the rate of metabolism and clearance of the chromone in rats by pretreatment of animals with the metabolic inh ib itor SKF 525A,

MATERIALS AND METHODS

Materials

FPL 52758 (Na+ s a lt of FPL 52757) was provided by Fisons Pharmaceuticals (Loughborough, Leicestershire, U .K .). SKF 525A was provided by Smith,Kline and French L td ., (Welwyn Garden C ity , Hertfordshire, U .K .).

Hepatobiliary toxicokinetics of FPL 52758 in beagle dogs.

Three male dogs (12-16.6 kg) were anaesthetized intravenously with Sagital and by inhalation of halothane. The b ile duct of each was cannulated (3 mm Portex polyethylene tubing, Fisons S c ien tific Apparatus, Loughborough) for sample collection a fte r the gall baldder had been ligated. The ureters were cannulated (2 mm Portex tubing) for urine

collection , and an additional 2 mm cannula was inserted in the femoral vein (2 mm Portex tubing) for blood sampling. FPL 52758 in 0.9% aqueous

NaCl was given by i .v . infusion to individual dogs over a period of 2\ hours. Bile samples (every 15 min.) and urine and blood samples (every 30 min.) were taken prior to , during and 2 hours post-infusion

of FPL 52758. Three dogs were treated with doses of 4, 12 or 36 mg kg* hr~ respectively.

Hepatobiliary toxicokinetics of FPL 52758 in the r a t .

Six male rats (350 g Fisons House stra in ) were anaesthetized i .p . with Sagital. The femoral vein, carotid artery and b ile duct of each were cannulated (pp 25 Portex tubing) fo r i .v . dosing, blood sampling

and b ile sample collection respectively. FPL 52758 in 0 .91 aqueous

NaCl was given as an i .v . bolus injection to fiv e animals. Four received SKF 525A in 0.9% aqueous NaCl i .p . 30 min before FPL 52757. Each individual treatment is oultined.below:-.

Table 6.1. The dosing schedule for the six rats in the

FPL 52758 hepatobiliary toxicokinetic study

Rat Number Treatment FPL 52758 SKF 525A mg kg" i .v . mg kg" i .p .

1 20 -

2 20 503 20 504 30 505 ■; 50 506

- no treatment.

In both studies the following analyses were undertaken using conventional techniques or methods described previously unless otherwise

stated: a) concentrations of drug in plasma, b ile and urine; b) b ile flow rate; c) alkaline phosphatase (ALP), 5 '-nucleotidase* and gamma- glutamyl transpeptidase*(G-GT) a c tiv itie s in b ile ; d) immunoelectro- phoresis of b ile samples*; e) serum glutamate dehydrogenase (GLDH)*; f ) serum alanine aminotransferase (ALT)* and serum alkaline phosphatase (ALP). (* Assay undertaken on dog b ile samples only).

A Boehringer test k it was used in conjunction with a Vitatron automatic analyser AKED with a 410 nm interference f i l t e r (Szasz,1969) for G-GT measurement.

RESULTS

Hepatobiliary toxicokinetics of FPL 52758 (the soluble Na+ sa lt of FPL 52757) in the dog.

No metabolites of FPL 52757 were detected in any sample of plasma, urine or b ile analysed. The parent compound was found in the plasma and b ile , but not in the urine. Chromone concentrations o f about 1 mg

ml” were attained in the b ile o f a ll 3 dogs (see Figure 6 .1 ).

The b ile flow was accelerated by i .v . infusion of FPL 52758; within 30 mini of dosing the flow rate was increased by 50-200% inthe 3 dogs. This increase was maintained throughout the experiment

-1 -1 -1 -1at the lowest dose (4 mg kg hr ); a t 12 mg kg hr the flow rateslowly decreased to control levels; at the highest dose (36 mg kg hr ^) the decline was rapid and the rate eventually became slower than

controls (see Figure 6 .1 ).

The enzyme ALP, G-GT and 5'-nucleotidase excreted in the b ile were a ll increased, a fte r an in i t ia l f a l l , in the 3 dogs. The increase in these enzymes paralleled the chromone concentration in the b i le , and the onset o f release o f these enzymes depended on the dosage administered

(see Figure 6 .2 ). Some serum enzymes were s lig h tly raised e.g . ALP was increased 2-fold in a ll 3 dogs. Other serum enzymes were near normal with

the exception of AST (2-fo ld increase) and GLDH (9-fo ld increase) which

was s ign ifican tly raised in the dog receiving the highest dose (see

Appendix 3, Tables 1 -3 ).

The rate of b ilia ry excretion o f FPL 52757 did not vary much with dose (see Table 6.2) and as only 19% was excreted in 4| hours.even,at .

the lowest dose i t is lik e ly that the excretion rates recorded represent the maximum excretion rate (V ) .

U lC l/\

The percentage o f the dose given in the tissues at d iffe re n t times

during intravenous infusion o f FPL 52757 was estimated by subtracting the amount excreted plus the amount in the plasma from the to ta l quantity

of drug given.

°/o change in b ile volume

ooCOL_

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Table 6.2 The effect of d iffe ren t doses of FPL 52758 administeredby intravenous infusion on the rate of b ilia ry secretion

of the drug from the dog

FPL 52758 , (mg kg hr )

Dose of Excretion rate*(mg hr"1 kg"1)

Amount of drug found in b ile

(% dose)**

4 0.43 19.2

36

12 0.51

0 . ^

7 .?

1.4

* Excretion rate - Total amount of drug excreted in the b ile ‘Dog wt. x duration of collection period i

* * Amount of drug = Total excreted in t he b ile x 100found in the b ile {%) L)ose given —

The to ta l amount in the tissues was calculated from the product of the plasmaconcentration at time ( t ) and the volume of in it ia l d ilu tion (VID) (seeTable 6 .3 ). The apparent volume of distribution at a given time wascalculated from:

Extrapolation from the line of best f i t gave the volume of in it ia l d ilution (VID) (see Table 6 .3 ).

Immunoelectrophoresis of dog b ile samples.Two dimensionsional electrophoresis of dog b ile samples in the

presence of anti-sera to dog: serum protein{6%) showed that d ilu tion of b flia ry protein occurred at chloresis. Plates 1 - 4 illu s tra te the effect with b ile samples from the top dose dog. Similar results were

obtained with the other dogs. The time in relation to the s tart of the infusion and the volume of the sample is given adjacent to each plate.

Hepatobiliary toxicokinetics of FPL 52758 (the soluble Na+ sa lt of FPL 52757 in the r a t .

Early indications of a potential hepatotoxic e ffec t, such as an alteration in the b ile flow and enzyme levels did not occur in the ra t

following an i .v . bolus of FPL 52758 (of up to 50 mg kg"1) with concomitant SKF 525A treatment. The b ile flow was approximately

20 (+ 5) |a.1 min in a ll animals. B ilia ry ALP levels did not vary greatly and there was no correlation with either the dose given or

Total amount injected (mg) - total amount excreted (mg)Plasma concentration mg ml ” x Dog

body wt in kg

C ontinued p . " 97

Tabl

e 6.

3.

The

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Immunoelectrophoresis o f dog b i le samples.

Two dimensional electrophoresis of dog b ile samples in the presence of anti-sera to dog serum protein (6%) showed that dilution of b ilia ry

protein occurred at chloresis. Plates 1-4 illu s tra te the e ffec t with

b ile samples from the top dose dog. Sim ilar results were obtained from the other dogs. The time in relation to the s ta rt of the infusion and the volume of the sample is given adjacent to each plate.

v s . 4 / .

PLATE 1(Time -0 hr) Volume 2.4 ml.

a|/io7%o PO * j; ,vs. 6 / A-fcO* £

PLATE 2(Time 0- I hr) Volume 3.6 ml.

/ \

2#/* ©/to COCrTobhe I 10 i ' / . f i - P 0 4 - S

PLATE 3I H ( . U . - l J h r )

Volume 2.4 mlJs&

H

I \

The resu lts on Plates 1 and 3 are derived from b i le samples o f equal

volume. A higher concentration o f b i l i a r y pro te in is present in Plate 3 b i le .

A*

PLATE 4(Time 2 i , - 2 j hr)

Volume 1.3 m l.

I

The highest concentration o f b i l i a r y prote in coincides w ith a reduction in b i le volume (see Plate 4).

chromone levels in the b ile . B ilia ry ALP levels fluctuated between 40-90 IU and the chromone reached a maximum b ilia ry concentration

of 500 yg ml” but this concentration was short-lived (approximately 20 min.) and occurred only in the ra t receiving the highest dose with SKF 525A pretreatment (see Appendix 3 9 Tables 3.4 to 3 .6 ).

DISCUSSION

Hepatobiliary toxicokinetics in the dog

The concentrations of FPL 52757 in gall bladder b ile (5-8 mg ml"^) was higher than that found in primary excreted b ile (approx. 1.0 mg ml~ as was expected, and the gall bladder concentrates hepatic b ile some 10-fold (Diamond, 1965) The concentration o f FPL 52757 in the b ile

showed a close correlation with the release of ALP, G-GT and 5 '- nucleotidase into the b ile , and immunoelectrophoresis demonstrates an

increased amount of b ile protein (ml“^) is present following FPL 52757

infusion (see Plates 1 and 3 ). Since a ll three enzymes in the b ile

were increased i t is lik e ly that the chromone affected the cell lining the whole o f the b ilia ry tra c t, as 5 '-nucleotidase and G-GT are marker enzymes for b ile canaliculi membranes (Hoidsworth and Coleman, 1975; Mullock, 1977) and ductal membranes respectively (Rosalki, 1975).Sim ilar enzyme increases in ra t b ile have been produced by retrograde b ilia ry in jection o f FPL 52757 (see Chapter 5 ), of other surfactants

(Nakae ejt 1977) and following treatment with b ile salts (Bode e t a l ,1973) / . Dujovne (1977) suggests-that for drugswhich are excreted in b ile , surfactant a c tiv ity could affect the integri of hepatocellular canalicular membranes and exert toxic e ffec ts , and

surface ac tiv ity has been shown to be correlated with hepatotoxicity

in numerous other compounds (Dujovne, 1978; Yasuhara e t al_, 1979; and Dujoyjne et a l, 1977). The surface a c tiv ity of FPL 52757 has been demonstrated (see Chapter 4 ), and its a b ility to act in the manner suggested by DujoVne has now been ve rifie d .

The results in Figures 6.1 and 6.2 illu s tra tes that there was an apparent threshold concentration o f approx. 500-600^ ml” below which

release of enzymes into b ile remained at near normal levels , above this level enzyme release escalated. The chromone w ill be active a t these sites as i t w in be available as 'fre e ' drug. Normally i t is more than

99$ bound to proteins at concentrations up to 300 yg ml"'* in plasma

from dog. The major route of elimination in the dog is unchanged material in the b ile . B ilia ry excretion of anionic material involves an active transport mechanism situated at the hepatocyte membrane, which

w ill enable attainment of the high drug concentrations' in the b ile .Hence, in i t ia l ly the canalicular membrane and at a la te r stage the ductal membrane of the b ilia ry tract in dog w ill be exposed to high

concentrations of the free chromone.

Under normal conditions, the liv e r has the a b ility to secrete high concentrations of naturally-occurring detergent b ile salts into the b ilia ry trac t without damaging i ts e lf . This has been explained (Coleman

et al_, 1977) as follows: b ile salts are present in extremely low concentrations inside hepatocytes and are pumped out by active transport; as the b ile sa lt concentration in the b ile canaliculus approaches or exceeds

m icellar lev e l, materials are removed from the outer face of the plasma membrane, but do not result in su ffic ien t damage to cause leakage of in tra ce llu la r m aterials; the health of the membrane of the outer surface

of the canalicular and other duct cells is maintained by continuous biosynthetic repair mechanisms. I t seems lik e ly that the presence of the chromone in the b ilia ry trac t leads to a situation where the damaging

effects overtake the repair mechanisms. The chromone in i t ia l ly increases then decreases b ile flow. Immunoelectrophoresis shows that during chloresis proteins in the b ile are diluted. As cholestasis develops the b ile proteins

become concentrated by an amount disproportionate to the reduction in b ile volume.

Since fo r a ll 3 doses of FPL 52758 the maximum excretory rate of the

chromone into the dog b ile was sim ilar (see Table 6.2) and independent of the dosage given, i t is concluded that the excretory mechanism for the chromone into the b ile is saturable. This important finding revealed thatthe Vmav fo r excretion into the b ile fo r a ll 3 dose levels was approx.max n _ rj _ -1 n0.5 mg kg hr" (equivalent to 12 mg kg day" ). Saturation of the

excretory mechanism for FPL 52757 leads to tissue accumulation (see Table 6 .3 ). Coincident with this phenomenon the apparent volume of d istribution (Table 6.3) increases markedly with time, at the highest dose. The larger

increase in at the top dose may be due to saturation of protein binding

leading to a greater tissue distribution,including the l iv e r . Estimates of the amount of the chromone which is lik e ly to accumulate in the tissue

(see Table 6,3) show that there was a non-linear increase in the amount

accumulated at the top dose. This agrees with the data on percentagerecovery (see Table 6.2) of the drug and its apparent volume of d istribution.Much of this accumulation is lik e ly to have been in the liv e r , the primarytarget organ fo r the compound in the dog. Such an uptake, followed byexcretion via the b ile without reabsorption from the gut probably explainsthe lack of compound in the urine. Although hepatocellular damage inthe dog was s light in the present study, repeated-dose to x ic ity studiesin the dog showed extensive hepatocellular damage. This is understandable

- 1 - 1in view of the maximum b ilia ry excretion of approximately 12 mg kg day in dog which would lead to accumulation of drug in the liv e r when the daily

dose exceeded this rate.

Hepatobiliary toxicokinetics in the r a t .

The metabolic inh ib itor SKF 525A had only a partia l e ffec t on FPL 52758 metabolism in the ra t. I t had been anticipated that a considerable

inh ib ition of metabolism would occur resulting in the elevation of chromone concentrations in b ile and plasma to the extent that to x ic ity could be observed in the ra t. This, however, was not the case and the study demonstrates yet again the high degree of resistance of this

species to the hepatotoxic effects of th is chromone,

The fin a l hypothesis for the mechanism of the chromone hepatotoxicity and the species anomalies in pharmacokinetics which results in the species- specific to x ic ity w ill be further developed in the fin a l chapter.

Acknowledgements

Thanks are due to Dr. G. Wilson fo r his s k illfu l assistance with

surgical procedures in the dog, and Dr. R, Hinton for running the electrophoretic plates presented in this chapter.

CHAPTER SEVEN

SUMMARY AND CONCLUSIONS

THE MECHANISM OF FPL 52757 XKDUCED HEPATOTOXICITY

Original hypothesis versus fin a l hypothesis

At the outset of th is project the original favoured hypothesis for the mechanism of FPL 52757-induced hepatotoxicity was that: "The hepato- to x ic ity of FPL 52757 results from metabolic activation of parent compound to a hepatotoxic reactive metabolite which can bind to v ita l hepatocellular constituents causing cell death". The current and fin a l hypothesis is as follows: "The hepatotoxicity of FPL 52757 is due to an

accumulation of parent compound in the liv e r and hepatobiliary system, and i t is a combination of the in trin s ic detergent properties of the drug, its pharmacokinetics, and species anomalies in metabolism which result in the species-specific to x ic ity" .

The early investigations to elucidate the mechanism of hepatotoxicity set out to verify the original hypothesis (Chapter 2), These special - studies fa iled to implicate reactive metabolite involvement. This, coupled with pharmacokinetic data revealing that species resistant to FPL 52757-induced to x ic ity rapidly metabolise and excrete the chromone

(Chapter 3) implicated the parent compound as the hepatotoxic agent. Chapters 4 and 5 demonstrate that detergency is related to to x ic ity and

that FPL 52757 is more detergent and toxic than its metabolites. In Chapter 6 the presence of the chromone in the hepatobiliary trac t is

closely correlated with the incidence of hepatotoxicity in the dog, and

hepatobiliary excretion is shown to be saturable.

The pattern of FPL 52757 hepatotoxicity does not f i t into the classical schemes for mechanisms of to x ic ity . Most toxicants act either by acute lethal in jury or through autoxidation via free radicals. In acute lethal injury energy starvation leads to fa ilu re of the "sodium pump" and to cell death. Autoxidation via free radicals leads f i r s t to

glutathione depletion then oxidative damage of ce llu la r membranes.This classic type of to x ic ity is described fu lly

in Chapter 1 as i t ties in with the original hypothesis for FPL 52757- induced hepatotoxicity.

Drug detergency and toxic ity

Following verifica tion o f a structure-activ ity relationship for a series of chromones i t became apparent that the hepatotoxicity of FPL 52757 was probably related to its detergent properties. This is a comparatively l i t t l e known mechanism of drug-induced to x ic ity despite

work in the fie ld for a number o f years, explaining the membrane damaging e ffec t of a number o f drugs and endogenous compounds (see Table 7 .1 ). In this thesis and in these previous reports, the degree of detergency of a group of compounds is related to to x ic ity in v it ro , providing evidence for a probable mechanism of in vivo to x ic ity .

Table 7.1 Previous reports of to x ic ity related to detergency

Drug or group of compounds Reference

Dialose Plus (a laxative) ; i .Erythromycin derivatives 2, 3, 4 , 5 and 15.Oral contraceptives 6.Bile salts 3, 7, 8, 9, 10, 11, 12,

13, 14.Chlorpromazine 3, 5, 15 and 16.Tricyclic antidepressants 7 and 16.

1. Dujovne and Shoeman (1972);2. Dujovne (1975); 3. Dujovne (1978);4. Dujovne (1978); 5. Dujovne jet al_ (1977a); 6. Dujovne and Pentikainen

[1973); 7. Dujovne e jt^ [ [1977b); 8. Dujovne (1977); 9. Coleman and Holdsworth (1976); 10. Holdsworth and Coleman (1976); Coleman e t a l , (1976); 12. Vyvoda ejt al (1977); 13. Coleman e t ail,, (1979); 14. B illington et _al_ (1980); 15. Dujovne and Salhab (1980); 16. Salhab £ t aV (1979a);17. Salhab e t a l (1979b), 18. Yasuhura et .art (1979).

A dose related increase in detergency and in vivo and in v itro

to x ic ity has been observed with FPL 52757 (see Chapter 4 ) . The effects

on b ile flow and the erosion o f membrane cells lin ing the b ile canaliculi and ductules may be related and can be envisaged in three

stages.

Membrane a lteration as a hypothetical basis for chromone-inducedliv e r injury

Stage 1: Modification of Membrane: The chromone passes into b ile ducts by active transport, modifying the membrane in tran s it to allow increased permeability, thus increasing b ile flow. The duration of this stage decreases with dose, e.g.

Dog 1 - 36 mg kg~ hr~^; 0 - |hr of the experiment- 1 - 1Dog 2 - 12 mg kg hr ; 0 - 2hr of the experiment-1 -1Dog 3 - 4 mg kg hr ; 0 - 4hr of the experiment.

There is l i t t l e indication of severe b ilia ry tract damage during thisstage. B ilia ry enzyme levels are near normal and electrophoreticevidence shows d ilu tion of b ile protein at th is stage (see Chapter 6,Plates 1 and 2).

Stage 2: Mediation of Membrane Damage: The)cholere t ic |effect of the drug is transient. The increased drug presence in the membrane and/or progressive damage, reduces thejcholeresis1, which further enhances erosion

of membrane cells lin ing the b ilia ry trac t as the drug becomes more concentrated in the b ile . At this stage the compound is s t i l l beingtransported into the b ile at the same approximate rate , hence the chromoneconcentration increases in the b ile to higher levels which coincides with an escalation of b ile canalicular membrane damage. B ile flow continues to decline a fte r th is stage and the decline does not appear to correlate with the concentration of drug in the b ile ; i t may relate

to the concentration in the b ile canalicular membrane and surrounding hepatocytes. The duration o f stage 2 is as follows:-

Dog 1 - 36 mg kg hr~^; H - 2?hoursDog 2 - 12 mg kg” hr"^; 2 - 4-|hoursDog\3 - 4 mg kg"l hr"^; stage 2 is not reached at th is dose.

During this stage enzyme release escalated and electrophoretic evidence shov/s an increase of b ilia ry protein (per m i l l i l i t r e ) in the b ile .Despite a normal or greater than normal b ile flow rate (compare Plates 1 and 3 in Chapter 6).

Stage 3: Maceration of the Bile Canalicular Membrane: Bile flow continuesto decline u n til i t is below normal levels, correspondingly the drug concentration in the b ile rises. This exacerbates the damage and eventually

the b ile flow completely stops as the architecture of the b ilia ry tract has become irre trievab ly injured, leading to escalating damage to the b ile ducts canaliculi and surrounding hepatocytes as the compound continues to accumulate in the liv e r . The duration of stage 3, which is only reached in dog 1 (36 mg kg“ hr“1) is from f to 3 hours.

Electrophoretic evidence shows that a massive increase in b ilia ry protein coincides with a reduction in the volume which appears to be disproportionately greater than can be accounted for simply by the reduction in b ile volume (see Chapter 6, Plates 1 and 4).

The pattern of enzyme release into the b ilia ry trac t following oral dosing in dog was d ifferen t from that obtained in rat following retrograde b ilia ry infusion. This probably reflects damage to the whole of the b ilia ry trac t following oral dosing, whilst following retrograde b ilia ry infusion the penetration of the toxicant is lim ited.

Bfle sa lt dependent choleresis j

An a lternative hypothesis might also account for the chromone-induced Icholeresis;, Since the chromones are structually sim ilar to b ile salts i t is possible that a build up of drug in the b ile stimulates b ile s a lt dependent secretion leading to1 choleresis^

Conceivably both membrane permeability a lteration and b ile sa lt dependent secretion act synergistically , un til more drastic b ile canalicular and duct membrane alterations counteract those effects to produce cholestasis , i t is recognised that the interactions betweenin itia tin g processes and consequences may reinforce each other making

interpretation of the progression of events d i f f ic u lt . Nevertheless, i t seems lik e ly that cholestasis accompanies and accentuates the hepatotoxic effect on the liv e r .

The response of the hepatobiliary trac t to endogenous and exogenous

detergents

Although both the species-variation to chromones and th e ir varying hepatotoxic potential are explained in th is thesis, certain anomalies

raise the following fundamental questions. Why is the b ilia ry trac t affected by these exogenous detergents but not normally by the endogenous detergents, and is the chromone-containing b ile more surfactant and cytotoxic than normal b ile ? Similar effects on b ile flow and b ilia ry

transport maxima have recently been reported following infusion of endogenously detergent b ile salts (Kitani and Kanai, 1981) in rats . Coleman's concept of a continuing pattern of erosion and repair

of :a chromone-induced imbalance in this natural pattern, has been alluded

to in the previous Chapter. Attempts to elucidate this phenomenon wereundertaken, based on the simple premise that normal dog b ile might beless detergent than dog b ile containing FPL 52757. Surface tensionmeasurements and cytotoxicity were assessed using previously describedtechniques (see Chapters 4 and 5). These unpublished results fa iled todistinguish between normal b ile and b ile containing the drug and arenot described in d e ta il. Suffice i t to say that i f this premise is notan over-simplication, then either the methods were insensitive and thenatural detergency of the b ile masked increased surface tension anddetergency or that i t is not the concentration of detergent moleculesin the b ile but th e ir concentration in the cells lin ing the hepatobiliarytrac t prior to passage into the b ile that is crucial. Saturation ofb ilia ry excretion and subsequent accumulation of the drug in thehepatocytes is suggested as the probable cause of to x ic ity (see Chapter 6).I t could be further postulated that the cells o f the hepatobiliary trac twould be in the 'f ro n t- l in e 1 of this accumulation, and highest ce llu la rdrug concentrations would be found in the membrane ; ;. Furtherevidence' fo r the importance of the V„ ■ for excretion into the b ile isr maxprovided by comparison of FPL 52757 and FPL 57787 (Proxicromil).

Inter-drug variations in hepatobiliary excretion and th e ir toxicological im plication.

The b ilia ry excretion in dog for FPL 52757 is approximately1 -I nlaX0.5 mg kg" hr" (see Chapter 6 ), This is about half that of FPL 57787 which ranged from 0.09-1.25 mg kg" hr"^ (Smith, 1980). Of the two,FPL 5,2757, following oral dosing, is the more potent hepatotoxin in the dog (unpublished data from Fisons Safety Evaluation F iles ). However,FPL 57787 has greater surfactant properties and is more toxic in v itro

(see Chapter 5 ). The anomaly between in v itro and in vivo results might be explained by the greater hepatic accumulation of FPL 52757 resulting

from slower b ilia ry excretion (see Table 7 .2 ).

Table 7.2 A comparison between two hepatotoxic chromones

^max ^or b ilia ry excretion FPL 52757 < FPL 57787

In vivo to x ic ity in dog FPL 52757 > FPL 57787

Detergency per se. FPL 52757 < FPL 57787

In v itro to x ic ity FPL 52757 < FPL 57787

Alternative or contributory reasons for species variation

Interspecies variation in hepatobiliary excretion: The molecular weight threshold for b ilia ry excretion has been shown to vary in ra t, rabbit and guinea-pig (Hirom e t a]_, 1972) apparently dependent on the excretory process i ts e l f and not to

differences in b ile flow. Associated with this the Vmax for b ilia ry excretion

is lik e ly to vary. This could be offered as an explanation for species variation in to x ic ity amongst slow metabolizers. However, i t certainly cannot be extrapolated further as metabolism and subsequent renal excretion o f polar

metabolites o f FPL 52757 by fast metabolizers w ill reduce the significance of b ilia ry excretion in these species.. The concentration of unchanged FPL 52757 in the b ilia ry trac t appears to be an important factor in the development of to x ic ity . Thus in the ra t even a fte r i .v . bolus ( a mode of administration lik e ly to exaggerate the b ilia ry concentration)injections o f FPL 52757 b ile concentrations of unchanged compound were only a fraction o f those achieved indogs receiving infusions (e .g . Rat: i .v . bolus 20 mg kg~^, max. concn <200pg ml ^

-1 -1 -1 of b ile ; Dog: 4 mg kg h" i .v . infusion; max concn > 800yg ml of b i le ) .Interspecies differences is susceptib ility of the hepatobiliary trac t: Inpublications by Coleman: .et aj_ (1980a .&Jb) the susceptib ility of erythrocytes fromd ifferen t species is related to species variation in phospholipid content, inparticu lar resistance to membrane damage is related to high sphingomyelin content.Coleman suggests that since b ile canalicular membranes have re la tiv e ly high totalsphingomyelin content ; . th is may contribute to their resistance to b ile s a ltattack. Correspondingly species variation in sphingomyelin levels in the b ilecanalicular membrane may be related to d iffering species susceptib ility todetergent drugs which are readily excreted in the b ile .Idosyncratic to x ic ity : In a number of accounts where idosyncratic hepatotoxicityhas been reported (Dujovne, 1977) i t is inferred that detergency o f the drug may be the cause of to x ic ity and that a better understanding of the pharmacokinetics of the drug might enable prediction of to x ic ity . This concept is exemplified by

the toxicokinetic comparison of the two detergent chromones FPL 57787 (Proxicromil) and FPL 52757 (see above).

V a ria b ility in human response to FPL 52757In the introductory chapter hypersensitivity and idiosyncratic responses

to drugs are mentioned in the section on causes of to x ic ity . I t should be considered that as the mechanisms involved in drug-induced to x ic ity are

9enerally better understood the incidence o f adverse drug effects defined as idiosyncratic w ill decline. Certainly the hepatotoxicity that occurred in

20I o f patients with FPL 52757 may have appeared at f i r s t sight to be idiosyncratic. However, as a result o f the investigationsin both man and animals in this thesis i t .is possible to

speculate that the incidence of hepatotoxicity could have been predicted. The animal data shows that those species which metabolise the compound slowly are susceptible and fast metabolisers are not. I f the implications of these species variations in metabolism are extrapolated to man one might expect that the 20% of patients susceptible to to x ic ity represent a group of the population that are slow hydroxylators and the remaining 80% metabolise the chromone su ffic ien tly rapidly so as to avoid the

adverse reactions. This is mere conjecture and i t would equally be

suggested that these individuals have vulnerable hepatobiliary tracts

or a particu larly low V for b ilia ry excretion, or that a completelyIlia a

d iffe ren t mechanism operates in man. However, as numerous other examples

exist where sub-groups ' (10-30%) in given human, populations respondabnormally to drugs because of differences in metabolic capacity, defective hydroxylation of FPL 52757 in 20% of the patient population

would seem a lik e ly explanation for the incidence of FPL-induced hepato­to x ic ity seen in man.

Of the previous examples of individual variations in the human

population with toxic consequences isoniazid acetylation is probably the best documented. Slow acetylators are more lik e ly to develop peripheral neuritis (Devadetta et a1_, 1960), and fast metabolisers are lik e ly to develop hepatotoxicity (M itch e ll, et aj_ (1975).

*More recently, and of greater relevance to the present problem, i t

has been observed that 91% of a British white population effect rapid 4-hydroxylation of debrisoquine (see Figure 7.1)-

Tls .NH2CINH

4-Hydroxydeb r i soqui ne

NH.

Debrisoquine

Figure 7.1

*(since debrisoquine is poorly metabolised by the dog (Allen et jr t , 1976)

whereas 9% were characterised by an impaired a b ility to hydroxylate

(Mahigoub e t al_, 1977). In this small group there was subsequent greater b io ava ila b ility and a greater tendency to develop postural hypotension. Furthermore, these individuals, typecast for th e ir

re la tiv e ly poor a b ility to metabolise debrisoquine, were re la tive ly

poor oxidisers o f other compounds, for example, phenacetin, guanoxan (Sloan £ t jf [ , 1978) and s-carboxymethyl-L-cysteine (Waring, et al_, J981)

Those findings may have practical implication in the development of new drugs and the use o f drugs in the future. Discoveries of this nature may^open up many new possib ilities of increasing our understanding of the inter-individual differences in the metabolic handling and resultant responses to drugs and toxic substances. I f a population could be profiled prior to therapy for metabolic capacity many useful drugs

may be retained safely. For example, i f fast metabolisers of FPL 52757 could be distinguished from slow metabolisers prior to medication, 80%

of a given human population might benefit from the anti-asthmatic properties

of this chromone without serious side-effects, and as a consequence o f this work i t could be confidently predicted that lower concentrations of FPL 52757 would be perfectly safe in man.

In a parallel yet converse situation the hepatotoxicity o f two detergent erythromycin derivatives might have been avoided. Dujovne

(1978) has related the hepatotoxicity o f erythromycin estolate (ee) and erythromycin cetyl sulphate (ec)to th e ir surfactant properties. Both ee and e (Robinson, 1962; Dujovne, 1978) have been reported to produce

hepatotoxicity in man. The complete drugs, ee and ecconsist o f a combination of the erythromycin base and a detergent molecule, e ither lauryl sulphate or cetyl sulphate respectively. The detergents are

added to enhance gastrointestinal absorption o f the a n tib io tics . Since ee is now thought to be hydrolysed by an esterase (unpublished communication), i t seems probable that the resultant release o f the detergent molecules lauryl or cetyl sulphate and subsequent accumulation in the hepatocytes

and hepatobiliary system could account for hepatotoxicity. I f this is the case i t would be the fastest metaboliser in the human patient population that would be a t r isk . Again, i t is possible that these individuals could be identified prior to therapy by pre-screening and the drug used safely.

HAVE THE ORIGINAL OBJECTIVES OF THIS RESEARCH PROGRAMME BEEN MET ?

In Chapter 1 the reasons for the study of the mechanism of hepatotoxicity of FPL 52757 were lis ted as follows:-

a) An understanding of the mechanism of biochemical to x ic ity might be helpful in the design of new and non-toxic drugs.

b) I t seemed probable that a new type of biochemical mechanism

of to x ic ity might be uncovered.

c) Chemical hepatotoxicity occurred in man which was not previously observed in animal to x ic ity studies, an under­standing of this species variation is important for future drug safety evaluation.

To summarise, i t is clear that these questions have been answered

to varying degrees. Taking them in reverse order:-

Species variation in hepatotoxicity response to FPL 52757

is d irec tly related to metabolic capacity and the bio­a v a ila b ility of the parent compound. Species which metabolise

the compound slowly are more lik e ly to be susceptible to

to x ic ity .

Although a novel biochemical mechanism for hepatotoxicity

has not been uncovered, a re la tive ly l i t t l e known mechanism

has been highlighted.

- An understanding of the mechanism of to x ic ity may help in the

design of new and non-toxic drugs for administration by oral routes. For such compounds the detergency aids absorption.Whilst retaining this property and efficacy i t could be important to look fo r chromone structures, which by virtue of th e ir physical structural properties are accessible to substantial and rapid

metabolism and excretion. As further information accumulates on the pharmacokinetic response of man and other species to anutrients

such predictions may become a re a lity .

-109-

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APPENDIX 2

Table A2.1

GUINEA-PIG EXPERIMENT

SUMMARY OF LIVER MICRO HISTOPATHOLOGY

Guinea-PigTreatment, Group & No.

Liver Pathology

1 N

Control ■ 2 s N

3 N (21.4)

1 ■ Isolated areas of s light necrosis. Vaculation in necrotic areas. Chronic inflammation.

Test 100 , mg kg, day 1

2

3

As above, but only a very s light e ffect.

N (20.0)

1 Cytoplasmic vaculation

Test 200 , mgkgT day

2

3 Cytoplasmic vaculation

, 1 V . .. ■:

Test 300 , mgkgT day

2 ;

: 3

Marked generalised cytoplasmic vaculation

N = normal- = no slide prepared

() = mean liv e r weight gm.

TABLE A2.2 PLASMA ENZYME ANALYSIS

Individual values. Mean values for each in parenthesis.

EnzymesGroup and Treatment

Aik. Phos.

(u/ l )

ICDH

(u/ l )

GPT

(u/ l )

Control (Day 19)

423 433

. 460

(438.8 + 6.37)

29.7. 29.7

29.0*

(29.3 + 0.21)

23.8 23.726.9

(24.8 + 0 .6 )

Test 1 613.2 59.8 39.7(Day 19) , , (200 mgkg day )

388.8 65.0 36.0436.5 39.7

*25.5

(479 + 55.7) (54.8 + 6 .3 ) (53.4 + 3.34)

Test 2 343 36.0 10.7(Day 3) , , (200 mgkg day )

561.9 31.2 7.11

(452,45) (33.6) (8.92)

Test 3(Day 3) , (300 mgkg day )

396.8) - 10.7)

-kTreatment group sign ifican tly d iffers from the control at a probability of less than 0.1.Standard Error +

Plasma samples were collected 4-5 hours a fte r the fin a l treatment fo r the control and test groups. Samples collected from groups 2 and 3 were taken following the onset of acute toxic symptoms.

TABLE A2.3 MEAN LEVELS OF FPL 52757 PER GROUP IN FAECES URINE, BILE AND PLASMA

CompoundUrine Faeces Bile Plasma

Group and Treatment

Control

Test 1 _•] 245yg/ml 866yg/gm 161.5yg/ml 125yg/ml(lOO-.mgkg"oay 1)

day"1)

Test 3 NS NS NS NS(200 .mg kg”1 day ‘ )

NS - No sample

*A trace of another metabolite was found in the b ile samples of tes t group 2.

Urine and faeces samples were collected for 24 hrs a fte r a dose of FPL 52757. Plasma and b ile samples were collected from test group 1^4-5 hours a fte r the fina l dose. Samples from test group 2 were taken following death or onset of symptoms of acute to x ic ity .

Test 2 (200 mgkg'

NS NS 765yg/ml* 36Cyg/mT

FERRET EXPERIMENT

Table A2.4 LIVER WEIGHT AND PATHOLOGY

Ferret and Treatment

Liver Weight gm

Liver Pathology

1. Control 47.5 Normal

2. 200 mgkg"1 day"’ 1 42 Normal

3. 300 mgkg"1 day 1 45 Normal

Table A2.5 PLASMA ENZYME LEVELS - results illu s tra ted (U/L)

Enzymes ICDHru/L)

GPT _ (U/L)

Aik. Phos. (U/L)

Day No. 0 3 4 5 0 3 4 5 0 3 4 5

Ferret and treatment

1 (Control) 17 12 > 14* 50 26 40 51.0 30 - 22*

2(200mgkg"1 day ) 10 20 10 16.5 56

(8)9.5 - 27*

(12)52 33 22 11*

3(400mgkg”1 day ) 18 20 - 40 14.5 - - 52 30

* Values from terminal aorta bleed () Values from ta i l bleed

- no values obtained.

TABLE A2.6 LEVELS OF FPL 52757 IN PLASMA, URINE AND FAECES

Sample

Ferret, treatment and day of sample

Urine Plasma Faeces Bile

1. Control ; - ■ ~ .

2. Day 4 (200mgkgr1ciayr I 1)' 95 yg/ml* . • ' - '■■■■•■■ ■

Day 5 (200mgkg-lday~ 1) 270 yg/ml* 95 yg/ml 1700 yg/gm 1360yg/ml

3. Day 4 (400mgkgr^day’^ ) 400 yg/ml* NS NS NS

NS = No sample

* 2 other metabolites present in these urine samples which were unidentified , one was probably FPL 57704. Trace amounts of this metabolite were also found in b ile and faeces.

(Urine and faeces samples were collected 24 hours a fte r the previous dose. Plasma and b ile samples were collected approximately 3 hours a fte r the las t half daily dose of FPL 52757).

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NEO-NATAL RAT EXPERIMENT

TABLE A2.9 LIVER HISTOLOGY. MICROSCOPIC EXAMINATION OF

LIVER SECTIONS FOLLOWING STAINING WITH H AND E.

Animals and treatment group Description of Livers

Control No. Prominent occasional chronic inflammatory1 - 10 cell in f ilt ra t io n in parenchyma and portal

tracts .

lOOmgkg^day”1,

Test No.4 - 1 0 As above, however inflammatory foci in the

pyrenchyma were less numerous.

3 Prominent occasional chronic inflammatory ce llin f ilt ra t io n in parenchyma and portal trac ts . Frequency of foci sim ilar to control. Mid- zonal vacuolation throughout most lobes. A small focus of acute inflammation beneath the capsule of one lobe.

1-2 Inflammatory cell in f ilt ra t io n as in controlswith congested blood within the lobes.

-131-

TABLE A2.10 MEAN PLASMA ENZYME LEVELS (UL)

Treatment Enzyme Group

AlkalinePhosphatase

(U/l)

ICDH(U/L)

OPT(U/L)

1. Control 441.5 (2) [437-448]

16.2 (5) [8-20]

10.9 (3)

P -18]

2. 100 mgkg^day-^ 439.1 (3)

[431-450]

14.4 (5)

[12-17]

20.3 (3)

[18-24]

() = number of animals

|J = range of values

RAT DATA

Experiment to demonstrate the effect of defic ient d iet and phenobarbitone treatment.

TABLE A 2.ll HISTOLOGY REPORT ON RAT LIVERS

Group Rat Comments

1Experimental d iet

control A ll livers normal

Normal d ie t plus -1 -1 200 mgkg day .

ii ii

Experimental d iet + 200 mgkg"1 day"1.

91011121314151617181920 21 222324

Most livers appeared normal. Livers from rats 20 and 23 had pale stained hepatocytes in centrilobular areas with some vacuo!ation. No signs of necrosis

25& 26 Most livers appeared normal.

27 Livers from rats 26 and 31 hadExperimental d ie t 28 paled stained hepatocytes inwith phenobarbitone 29 centrilobular areas with somein the drinking water 30 vacuolation. No signs of necrosis.+ 200 mgkg day . 31

32

5 34 One liv e r was normal. 5 livers hadpale stained hepatocytes in cen tri-

Experimental d iet with 36 lobular areas with some vacuolation.phenobarbitone in the 37 No signs of necrosis,drinking water + 38400 mgkg" day" . 39

TABLE A2.12. PLASMA.ENZYMES

a) Average G.L.D.H. Levels (IU). .in Rats per Group

Group No. Prior to Treatment Day 0 Day 6 Day 14/15

1 5 (4-6) 6.25 (4-11) 4.3 (0.13) 3.5 (3-4)Experimental d iet

ControlM [4j M [4]

2 ; 5 (4-6) 6.0 (5-7) 3.8 (3-5) 5.5 (5-6)

Normal d iet , 200 mgkg day

[4] M M

3 5.4(3 .7) 9.4 (1-25) 4.6 (3-10) 7.0 (1-20)Experimental d ie t, + 200 mgkg" day

[14] 0 *1 P4J [ " ]

4 5 (4-6) 9.3 (4-21) 5.5 (3-8) 7.0 (2-15)Experimental d iet with phenobarbitone in the drinking water + 200 mgkg" day"

[s] [8] [8] [8]

5Experimental d iet with phenobarbitone in the drinking water + 400 mgkg" day"

5.5 (4-6)

[8]

5.3 (0-12)

[8]

5 (4-6)

[8]

11

[8]

() range of values[J number of animals in each group

TABLE A2.13 PLASMA ENZYMES

b) Average G.P.T. Levels (IU) in Rats

Group No. Prior to Treatment

Day 0 Day 6 Day 14/15

1Experimental d iet

control

8 0 .3 (66 -91 )

M

54.5 (47-61) [4]

68.8(51-75)

r a

64 (57-69)

W

2Normal d ie t ' , ' 200 mgkg” day”

70.5 (66-78)

p j

62.3 (57-70)

P I

52 (38-67)

P ]

54.3 (48-60)

PJ

3Experimental d ie t, + 200 mgkg"1 day"

63.6 (40-75)

P 5]

51.6 (37-73)

[15]

68.6(48-105) 68.5 (48-98)

P * ] . P 2,

4Experimental d iet with phenobarbitone in the drinking water + 200 mgkg day"

57.9 (49-69)T8]

72,6 (60-83) [8]

97.1 (58-125) 63.1 (40-91 [8] [8

5Experimental d ie t with phenobarbitone in the drinking water +400 mgkg” day”

60.1 (48-74)

[8]

57.6 (29-92)

r a

96.6(49-153)

P ]

101

P I

() range of values[] No. of animals in each group

TABLE A2.14 LEVEL OF FPL 52757 AND METABOLITES

IN yg/ml PLASMA and pg/mg LIVER

Group Number RatNumber Plasma Liver

FPL 52757 FPL 57704 FPL 52757 FPL 577041

Experimental diet 1 0 0 0 0Control 2 0 0 0 0

3 0 0 0 0 -4 0 0 0 0

2 5 15 0 - —Normal d iet 200 mgkg" day

67

12725

200

360

100

8 6 0 (6 1 *) 0 10 0

9 9* . ’10 145 25 42 011 no trace 18 012 150 trace 72 36

3 13 165 trace 28 0Experimental d iet + 200 mgkg" day"

1415

*—32 10

16 145 trace 60 617 195 25 58 1618 - . -r t19 215 25 84 1620 — - — ■21 235 25 76 1222 — — \23 165 * 20 55 824 180 (171.0) 60 70 8

Experimental d iet with phenobarbitone in the drinking water ‘plus 200 mgkg" day" .

25 60 trace 10 026 35 0 16 027 90 trace 28 428 —f ■ i 70 829 40 0 8 030 15 15 20 031 240 25 0 032 55(76.4) 0 0 0

33 120 500 0 05 34 4. . - f

Experimental d iet with 35 - - ■ — -rr-

phenobarbitone in the 36 . 40drinking water plus 37 - \ — 80 80400 mgkg" day" . 38 \

- 7

39 ; - r - r

40 . *T* —

The presence of FPL 58280 was not c learly demonstrated, appeared to be present in plasma samples.mean per group. — no sample obtained.

However, some

C .A p C f ' | l l | C | | u l*u u o i ; u | p u) q u c c | | cv»i» v»| jji i<_i ( v ' - ' i f t MMM o i i j c i d i j r i ^ n u i u n v i u u | i u

pretreatment and deficient d ie t.

TABLE A2.15. HISTOLOGY REPORT AND LIVER WEIGHT gm ,

Group FerretNumber

Appearance of Liver Weight in gm.

11 Occasional foci of chronic

inflammatory cells in the liv e r , parenchyma and portal tracts.

41.3

Control 2 Minimal amount of chronic inflammatory cell in f iltra t io n mainly in the portal tra c t.

35.0

2Phenobarbitone

Control

3

10

Usual amount of chronic inflammatory cell in f iltra t io n mainly in portal areas.

44.7

34.2

3 - i FPL 52757 lOOmgkg 1.-I

day plus

456

Usual amount of chronic inflammatory cell in f iltra t io n mainly in portal areas.

31.643.938.0

phenobarbitone

7 Occasional prominent foci of chronic in f ilt ra t io n in the liv e r , parenchyma and portal areas.

75.6

4FPL 52757, ■ , 100 mgkg" day’ + 3-methyl-

cholanthrene.

In one lobe a large area of b ile duct pro liferation or possibly a primary tumour was found. Enlarged foamy hepatocytes surrounded this odd area.Mineralisation had occurred in a few isolated cells near to the odd area. Appeared to be metastatic ca lc ifica tio n and some necrotic ce lls .

8 Minimal amount of chronic inflammatory 45.6 cell in f ilt ra t io n in portal areas.Vacuolation around portal areas.

9 Minimal chronic inflammatory ce ll 48.8 in f ilt ra t io n in l iv e r , parenchyma and portal areas, with marked centrilobular pre-necrosis a c tiv ity

53-methyl-

11 Minimal chronic inflammatory ce ll in f ilt ra t io n in multiple small foci in the parenchyma and in portal tracts

42.4

•cholanthrene

Control 12 Occasional multi-nucleated giant ce lls 44.4 were noted.

TABLE A2.16 PLASMA ICDH LEVELS (U/1)

Day -14 1 8 12 14 22 28

Group FerretNo.

(24) (5) (3) (3-4) (2)

11 - 19.0 13.0 11.0 - 13.9 17.6

2 27.9 19.0 12.0 13.7 17.4 16.3

23 - 27.0 - - 14.9 20.9 15.0

10 ■* — 25.0 ** 25.9 20.0

4 28.9 - 16.0 8.0 8.0 25.9 37.6

3 5 35.5 - - 10.0 - 18.9 34.5

6 31.9 - 14.0 14.9 10.0 25.9 21.9

7 24.9 - 34 30.0 39.8 50.0

4 8 - - 9 - 15.0 23.9 15.2

9 33.9 20.0 12.0 22.0 25.0 41.8 132.0

511 28.0 16.0 13.0 - . 10.0 27.4 23.4

12 19.9 14.0 10.4 21.9 21.4

() hours a fte r las t dose o f chromone or vehicle. Treatment per group as for Table A2.15.

-138-

TABLE A2.17 PLASMA GPT LEVELS (U /l)

Day -14 1 8 12 14 22 28

Group FerretNo.

(24) (5) (3) (5-4) (2)

11 - - - 38.7 - 58.0

2 - 62.5 38.4 38.7 28.6 32 28.6

23 - 65.5 ■ - - - 86.3 34.5

10 - - 40 - - 142 89.2

4 59.5 _ 16.6 41.7 169 333 -

5 - 29.8 - 32.7 59

6 65.5 - •18.6 71.4 16.6 32.7 23.2

7 - 123 107.1 74.0 109 -

4 8 - - . 43 - 47.6 32.5 21.7

9 71.4 16.6 56 30.9 60.0 217.5

11 74.3 60.0 30.9 59.7 80.35 ■■

12 40.0 36.0 54.5 97

() hours a fte r las t dose.Treatment per group as for Table A2.15

-139-

TABLE A2.18 PLASMA ALKALINE PHOSPHATASE LEVELS (U /l)

Day -14 1 8 12 14 22 28

Group . •• (24) (5) (3) (3-4) (2)

1 ■ _ 38.2 26 22.0 59.01

2 33.0 33.0 27 16,4 65.5 18.0 39.6

3 36.0 . . . . ' 49.1 19.1 60.82

10 33.0 38.2 21.8 43.7 16.4 54.6

4 76,0 76,4 11,0 21,8 13.1 32.8 57.0

3 5 87.0 - 27.3 49.1 30.0 55.8

6 52,0 - 18.0 . 32.8 27.3 55.6

7 50 - 103,7 70,4 109.2 240.2 -

4 8 - 44 - - 43.7 87.4

9 98.0 - 50.1 - 76.4 54.6 90.0

11 52.4 54.6 11.0 21.8 88.65

12 54.6 65.5 33.0 49.1 27.3 87.9

Q hours a fte r las t doseTreatment per group as fo r Table A2.15.

-140-

TABLE A2.19. ANALYSIS OF SERUM SAMPLES

Group Ferret No. FPL 52757 iig/ml

(8 hours^after las t dose) 30

5 2503 .

6 280

8 300

49 190

7 170(week 2 sample)

Trace amounts of FPL 57704 were also found in a ll samples. Samples taken 1.5 hrs a fte r fin a l dose

TABLE A2.20 ANALYSIS OF URINE SAMPLES*

(A) FPL 52757 Levels in Urine -ug/ml

Group Ferret No. Wkl Wk2 Wk3 Wk4

4 340 330 -

3 5 272 195 260 2156 354 160 220 250

7 300 230 280 2174 8 330 300 340 70

9 375 - 195 290

(B) FPL 57704 Levels in Urine -ug/ml

Group Ferret No. Wkl Wk2 Wk3 Wk4

4 150 180

3 5 155 155 170 1006 120 45 70 220

7 55 45 70 -

4 8 75 50 420 109 90 - 200 200

*NB One additional unidentified metabolite was found in a ll the urine samples close to the origin.

TABLE A2.21 ANALYSIS OF FAECES SAMPLES*

FPL 52757 Levels in Faeces - yg/gm

Group Ferret No. Wkl Wk2 Wk3 Wk4

4 400 600 500 1100

3 5 640 1000 900 2400

6 2600 700 500 2100

7 •1640 1100 700 NF

4 8 1100 1000 800 NF

9 2600 2200 2600 NF

NF = No Faeces

Urine and faeces collected for 24-hour period following dosing.

TABLE A2.22 (A) ANALYSIS OF LIVER - yg/gm

Group Ferret Compound 52757 Compound 57704Number (yg/gm) (yg/gm)

; 4 .60 ■ ■■■ 94 57 95 42 9

3 5 51 66 60 126 60 9

7 54 -

7 54

4 8 66 68 66 6

9 36 69 50 12

TABLE A2.22 (B) ANALYSIS OF BILE* - yg/ml

Group FerretNumber

Compound 52757 (yg/ml)

Compound 57704 (yg/ml)

3 5 750 3606 • 720 315

7 570 904 8 630 130

9 630 620

*NB Two unidentified peaks. F irs t peak corresponded to one found in urine. Second not previously analysed (found a t higher levels in 5 and 5, almost negligible in 7, 8 and 9).Samples taken 2 hours a fte r fin a l dose.

Experiment to demonstrate the effect of phenobarbitone and methionine on

FPL 52757 to x ic ity in dogs.

HISTOLOGY REPORT*

Positive Control: Centrilobular necrosis in "seastar" pattern around central vein.Normal hepatocytes around portal areas. Bile duct enlargement and pro life ra tio n , with signs of erosion of b ile duct walls.

Phenobarbitone pretreated dog: Isolated necrotic hepatocytes, mainlyin centrilobular region of many lobes.

Positive Control dog: Liver congested with blood. Marked areas of "seastar"

bridging necrosis. Nuclear fragmentation-Fatty change occurred with increased deposits in a "seastar" centrilobular

pattern. Normal hepatocytes were found around portal areas.

Bile ducts. Numerous b ile ducts showed extreme d ila tio n ,swollen with m aterial. P ro liferation of b ile ducts around the swollen ducts.

Methionine pretreated dog: Slight centrilobular necrosis. Some b ile ductenlargement with erosion of b ile duct walls.

*(see Rappaport, 1976)

TABLE A2.22 PLASMA ENZYME LEVELS IN DOGS

Day of Study Pretreatment

AnimalNo.

ALPIU

GOT.IU

GPTIU

GLDHIU

C1ii192 22 30 1

1 222 33 37 22 ii 241 33 34 13 » '329 32 31 34 it 275 29 33 15 a 278 25 34 68 ii 329 29 31 29 ii 367 27 33 0

n ii 304 23 28 112 ii 284 25 40 615 ii 317 30 49 616 ii 297 46 83 2417 ii 295 29 63 1318 H 289 23 56 119 it 319 29 49 322 ii 323 25 36 323 •• 311 22 35 126 ii 289 49 162 4130 ii 262 26 113 7

Cl = Control Dog

TABLE A2.23

Day of Study Pretreatment

AnimalNo.

ALPIU

GOTIU

GPTIU

GLDHIU

P 154 41 35 11 P 155 50 44 22 P 165 51 45 13 P 168 43 38 14 P 162 48 40 15 P 182 44 43 38 P 150 55 38 39 P 175 55 59 811 P 224 85 257, 5312 P 231 119 309 5313 P 251 144 425 52

P = Phenobarbitone pretreatment

TABLE A2.24 PLASMA ENZYME.LEVELS IN DOGS(CONTINUED)

Day of Study Animal ALPIU

GOTIU

GPTIU

GLDHIU

M 129 35 44 11. «i 190 70 74 12 ii 177 31 63 23 ii 179 25 51 25 ii 155 31 41 36 ii 180 37 30 18 ii 164 31 36 4

9 ii 143 27 32 210 it 152 26 30 7n ii 135 31 32 512 H 134 31 29 115 ii 138 36 37 518 ii 143 42 92 1520 ii 145 43 110 1223 ii 140 52 114 1224 ii 129 29 95 5

25 ii 126 29 80 326 ii 126 29 78 529 ii 122 34 52 430 u 116 32 47 432 n 113 36 42 436 ii 114 24 34 338 ii no 28 38 540 ii 126 32 39 843 ii 115 26 34 244 a 128 32 35 346 ii 121 26 30 247 ii 119 36 31 150 ii 130 34 30 151 ii 122 29 30 152 ii QNS QNS 34 853 it QNS QNS 60 2654 ii 141 42 61 1457 ii 138 64 58 1958 ii 148 39 85 2759 ii 162 41 113 3960 n 153 46 123 1561 ii 148 37 107 1364 ii 145 33 71 365 H 152 32 58 367 u 151 31 51 268 ii 181 91 211 15169 ii 261 149 672 187

M = methionine pretreated

TABLE A2.25 PLASMA ENZYME LEVELS IN DOGS (CONTINUED)

Day of Study Animal ALPIU

GOTIU

GPTIU

GLDHIU

V 192 33 33 1

1 II 224 35 38 12 II 215 32 34 53 II 225 26 35 25 II 204 54 99 54

6 II 223 80 162 958 II 208 25 89 16

9it 201 21 65 3

10 ii 208 24 58 3

11 »• 208 73 157 9812 ii 263 141 410 14315 ii 2550 4700 9140 175

= Control dog 2.

TABLE A2.26 LEVELS OF FPL 52757 AMD FPL 57704 IN PLASMA,LIVER AND BILE

DOG CompoundMaterial

FPL 52757 FPL 57704

Cl Plasma 190 yg/ml Trace

Liver - Lobe 1 58 yg/gm -- Lobe 2 60 yg/gm —

Bile 8,8000 yg/ml -

P Plasma - 2hr 192.5yg/ml -- 9hr 125.5yg/ml -

Liver - Lobe 1 32 yg/ml- Lobe 2 44 yg/ml —- Lobe 3 68 yg/ml -

Bile 5,000 yg/ml -

C2 Bile 8,000 yg/ml Trace

Faeces 900 yg/gm -

Urine 205 yg/ml -

M Plasma 180(100-260 yg/ml)

Trace

Liver - Lobe 1 60 yg/gm- Lobe 2 50 yg/gm -

Bile 7,000 yg/ml - ”

Faeces 900 yg/gm -

Urine 175 yg/ml 45 yg/ml

CT =■ Control; P = Phenobarbitone pretreated .02= Control; M = Methionine pretreated.

Urine and faeces samples collected over a twenty-four hour period.

Plasma samples taken 2 hours a fte r dosing.

The presence or absence of an additional metabolite FPL 58280 in the faeces was not c learly demonstrated.

SPECIAL RODENT DIET defic ient in selenium, Vitamin E and methionine

contained these components at the following levels:-

Selenium > 0.005 mg kg**l

-1Vitamin E 2.5 mg kg

Methionine 0.21% of to ta l d iet

1.5% of to ta l protein.

In addition cysteine was deficient ( i .e . 0.15% of to ta l d ie t or

1.1% of to ta l protein). The minimum protein requirement fo r rodents

is 4.5% of to tal d ie t. The special d iet had a total protein content

of 2.6%.

The d iet was supplied by B.P, Nutrition (U .K .), S tepfield, Witham, Essex, CM8 3AB.

APPENDIX 3 (Appendix to Chapter 6)

DOG RESULTS

TABLE A3.1. CLINICAL CHEMISTRY AND DRUG CONCENTRATIONS FROM PLASMA SAMPLES FOLLOWING INTRAVENOUS INFUSION OF FPL 52758 at 36 mg kg"1 hr"1

Time ALP GOT GPT GLDH FLP 52758

vg ml”1

0 218 44 82 1 —

I hr 224 (1.02) 49 (1 .1) 87 (1 .1) 5 (5 ) 3301 hr 231 (1.05) 53 (1.2) 89 (1 .1) 5

(5)425

11 hrs 242 (1.11) 57 (1.3) 108 (1 .3) 7 (7 ) 480

2 hrs 253 (1.16) .64 (1.5) 100 (1 .2) 7 (7) 53o

2\ hrs 265 (1.21) 73 (1 .7) 98 (1 .2) 8 (8) 345

3 hrs 300 (1.4) 90 (2 .1) 109 (1 .3) 9 (9 ) 220

3| hrs 342 (1.6) 100 (2 .3) 116 (1 .4) 9 (9 ) 240

() Increase

* FPL 52758

expressed as x

is the soluble

normal level,

sodium s a lt o f FPL 52757.

TABLE A3.2. CLINICAL CHEMISTRY AND DRUG CONCENTRATIONS FROM PLASMA SAMPLESFOLLOWING INTRAVENOUS INFUSION OF FPL 52758 at 12 mg kg-1 hr-1

Time GLDH ALP GOT GPT FPL 52758

vg mg”1

0 3 150 52 57 - ■

§ hr 2 (0 .7) 146 (1 .0) 53 1.0) 55 1.0) 135

1 hr 2 (0.7) 160 (1.1) 57 1.1) 58 1.0) 2501| hrs 3 (1 .0) 181 (1 .2) 54 1.0) 60 1.6) 2352 hrs 4 (1.3) 162 (1.1) 52 1.0) 53 1.0) 3602| hrs 3 (1 .0) 187 1.3) 51 1.0) 56 1.0) 2853 hrs 5 (1.7) 208 (1.4) 52 1.0) 56 1.0) 3303| hrs 5 (1.7) 236 (1 .6) 57 1.1) 61 1.1) 2254 hrs 5 (1.7) 266 (1.8) 59 1.2) 56 1.0) 2504§ hrs 5 (1 .7) 291 (1.9) 64 1.2) 51 0.9) 250

() Increase expressed as x normal' le v e l.

TABLE A3.3. CLINICAL PATHOLOGY AND DRUG CONCENTRATIONS FROM PLASMA SAMPLESFOLLOWING INTRAVENOUS INFUSION OF FPL 52758 at 4 mg k g '1 hr~1

Time ALP GOT GPT GLDH FPL 52758 yg ml’

o 184 123 186 5 _

i hr 197 (1.1) 116 < 1 198 (1 .1) 6 (1 .2) 70

1 hr 1 9 0 (1 .0 ) 110 < 1 193 (1 .0) 4 (0 .8) 1401| hrs 198 (1 .1) 104 < 1 197 (1 .1) 6 (1 .0 ) 1402 hrs 2 0 4 (1 .1 ) 96 < 1 193 (1 .0) 8 ( 1 .6 ) 16021 hrs 224 (1 .2) 94 < 1 196 (1.1) 11 (2 .2) 2053 hrs 248 (1 .3) 93 < 1 198 (1 .1) 13 (2 .6) 19031 hrs 275 (1 .5) 94 < 1 198 (1 .1) 14 (2 .8) 180

4 hrs 308 (1 .7) 100 < 1 205 (1 .1) 15 (3) 16041 hrs 337 (1 .8) 97 < 1 202 (1 .1) 15 (3) 160

() Increase expressed as x normal level

RAT RESULTS

Bile Flow

TABLE A3.4 (a ) : RAT 1 TABLE A3.4 (b ): RAT 1

+ SKF 525A 50mg kg"1

Prior to 27.2 Prior to 25.0Dosing Dosing 21.4

19.4

1.45 28.6 2.15 22.23.40 26.1 4.30 22.25.45 24.0 6.45 22.27.45 24.0 9.00 22.29.30 28.6 11.15 22.2

11.45 22.2 13.28 22.513.50 24.0 15.45 21.916.00 23.0 18.15 — 20.018.00 25.0 20.45 20.020.10 23.1 24.00 15.422.30 21.4 27.00 16.630 21.345 23.3

60 21.1

90 19.1

TABLE A3.4 (c) RAT 3 . TABLE A3.4 (d) RAT 4FPL 52758 20 mg kg-1 FPL 52758 30mg kg'+ SKF 525A. + SKF 525A_.

Timein Min Flow ra t yl rain Time in Min. Flow Rate jiT rain 1

Prior to 21.4 Prior to 27.3Dosing Dosing 23.1

24.02.50 17-6. 1.48 27.85.45 12.8 3.46 24.29.15 18.18 5.50 . 23.0

11.40 11.8 8.00 24.015.55 14.8 10.05 24.019.30 22.2 12.10 23.121.45 15.3 15.20 23.123.20 21.4 17.30 24.025.40 21.4 19.40 24.0

28.00 20.7 21.45 24.030.25 21.4 23.50 24.032.25 21.4 25.55 24.035.05 21.4 27.00 24.037.40 19.4 29.00 24.040.20 20.7 31.30 20.0 "42.45 17.6 34.35 16.248.50 17.6 37.45 15.8

51.40 16.6 40.30 17.634.40 17.6 45.00 11.137.30 ' ; 17.660.30 16.675.5 v 15.0 ■'90.5 ; 16.6105 16.0

TABLE A3.4 (e) RAT 5FPL 52758 50 mg kg"1 + SKF S25A 50 mg kg

TABLE A3.4 ( f ) RAT 6No treatment

Time in Min Flow Rat pi min Time in Min. Flow Rate pi min"1

Prior to 18.2Treatment 18.2

2.25 20.7 1.50 27.3

4.45 20.7 3.45 26.1

7.15 21.4 5.40 26.1

9.40 20.7 7.35 26.1

12.00 21.4 9.30 26.1

14.20 21.4 11.25 26.1

16.40 21.4 13.20 26.1

19.00 21.4 15.20 25.0

21.20 21.4 17.20 25.0

23.30 23.1 19.20 25.0

25.40 23.1 21.20 25.0

28.00 21.4 23.20 25.0

30.00 25.0 23.20 25.0

32.05 24.0 25.20 25.0

34.05 25.0 27.20 25.0

36.10 24.0 29.25 25.0

38.15 24.0 31.35 25.0

40.10 25.0 34.40 25.0

42.10 25.0 50.20 24.0

44.10 27.3 65.10 23.1

45.50 27.3 80.20 24.0

47.40 28.5

51.20 26.5

TABLE A3.5 (a) FPL 52757 AND FPL .57704 CONCENTRATIONS IN THEBILE FOLLOWING I.V . BOLUS OF 20 mg kg"1 FPL 52758 (Rat 1)

Time in Mins

FPL 52757 Concentration in p9 ml"

FPL 57704

ng ml

5.45 100 126

16.0 126 144

30 88 158

45 106 207

60 88 185

90 74 118

100 82 40

TABLE A3.5 (b) FPL 52757 AND FPL 57704 CONCENTRATIONS IN THEBILE FOLLOWING I.V . BOLUS OF 20 mg kg" 1 FPL 52758 ANDPP*ETREATMENT WITH SKF 525A 50 mg kg-1 .

Time in Mins

FPL 52757. Concentration in ng ,-1 FPL 57704 m 1 >

vg ml"

10 164 1023 164 10235 no 5650 100 7260 100 8075 120 7490 66 -

105 69 45125 78 28

TABLE A3.5 (c) FPL 52757 AND FPL 57704 CONCENTRATIONS IN THE MBILE FOLLOWING I .V. BOLUS INJECTION'OF 50 mg k g '1 FPL 52758AND PRETREATMENT WITH SKF 525A.

Time in Mins

FPL 5275' Concentration in yg ml”

FPL 57,7^4 yg ml"

7 500 68

20 400 108

25 196 76

35 268 220

47 200 200

57 316 220

70 190 148

TABLE A3.5 (d) FPL 52757 AND FPL 57704 CONCENTRATIONS IN THE i, PLASMA FOLLOWING AN I.V . BOLUS INJECTION OF 20 mg kg FPL 52758

Time in Mins

FPL 52757 Concentration yg ml"

in FPL 57704 yg ml"1

2 197 1

6 119 v - ' 1

11.0 79 -

15,0 115 45

22.0 70 70

40.0 42 42

45.0 47 47

60.0 37 37

90,0 15 17

TABLE A3.5 (el FPL 52758 CONCENTRATIONS IN THE PLASMA* FOLLOWING I.V .BOLUS INJECTION OF 20 ma kg"1 FPL 52758 + PRETREATMENT WITH SKF 525A 50 mg kg T i.p . ____________

Time in Mins FPL 52757 Concentration in yg ml

2. ; 100

: ..4 . . 88

. 6 90

8 80

11 38

15 55

20 88

30 26

60 14

90 20

120 15

*No -OH metabolite present

TABLE A3.5 ( f ) FPL 52757 AND FPL 57704 CONCENTRATIONS IN THE .PLASMA FOLLOWING AN I.V . BOLUS INJECTION OF 50 mg kg’ 1 FPL 52758 + PRETREATMENT WITH SKF 525A 50 mg kg"1 i.p .

Time in Mins

FPL 52757 Concentration in yg ml"

FPL 57704 yg ml

2 300 trace4 312 trace8 240 21

11 250 trace22 250 2830 146 18.645 220 060 146 o90 85.3 0

TABLE A3.6 (a) ALKALINE PHOSPHATASE (ALP) CONCENTRATIONS IN THE , BILE FOLLOWING AN I.V . BOLUS INJECTION OF 20 mg k g '1 OF FPL 52758

Time (in mins) ALP ( I .U .)

10 45

30 55

45 65

60 60

90 72

TABLE A3,6 (b) ALP CONCENTRATIONS IN THE INJECTION OF 20 mg.kg"1 OF SKF 525A 50 mg kg"1 i .p .

BILE FOLLOWING AN I.V . BOLUS FPL 52758 + PRETREATMENT WITH

Time (in mins) ALP ( I .U .)

15 80

46 80

60 85

75 75

90 80

125 75

TABLE A3.6 (c) ALP CONCENTRATIONS IN THE BILE FOLLOWING AN I.V . BOLUSINJECTION OF 50 mg.kg’*1 OF FPL 52758 + PRETREATMENT WITH SKF 525A 50 mg kg i.p ._____________________________ __

Time (in mins) ALP ( I .U .)

10 55

22 65

30 50

42 40

50 70

65 90

77 75

TABLE A3.6 (d) ALP CONCENTRATION IN THE PLASMA FOLLOWING AN I.V . BOLUS INJECTION OF 50 mg kg’ 1 FPL 52758 AND SKF 525A 50 mg kg

I.P .

Time(in mins)

0

11

45

ALP ( I .U .)

394

334

420

Meeting on ’’Extrapolation from Animals to Man”

TITLE Toxicity studies on EPL 52757 an oral anti-allergy compound.

ABSTRACT EPL 52757 (6, 8 -diethyl-5-hydroxy- 4 -oxo-4H-l- benzopyran - 2 -carboxylic acid) was devloped as an orally effective anti-allergic drug Treatment of-human volunteers for 2 weeks at 5mg kg"~* day~* did not reveal any evidence of toxicity. However, the drug was found to cause an incidence of abnoimal liver function in man after treatment for one month at daily doses of approximately 5 mg kg""*. The effects were generally minor and were readily reversible when treatment stopped. Nevertheless they were sufficient to halt the development of the compound for chronic use at this dose level. Previous extensive animal studies in five species, rat, hamster, rabbit, squirrel monkey and cynomolgus monkey, of up to six months duration, had failed to indicate any hepatotoxicity due to the drug. Subsequent further animal studies in rat, mouse, rabbit, ferret, guinea-pig, baboon and dog confirmed that most species are not readily susceptable to a hepatutoxic effect of EPL 52757, with the exception cf the dog. Deatn primarily due to liver failure could be induced in dogs by repeated doses of 20 mg kg~* day""*. The cause of the species variation in response to this compound has been investigated and it is apparent that metabolic and pharmacokinetic differences affect toxicity. The dog is a species which is incapable of clearing the compound rapidly by metabolism and as a consequence is susceptible to its hepatotoxic effects. The parent compound appears to be responsible for the hepatotoxicity and the susceptibility of a species is probably related to its ability to clear the compound.

Authors: C.T. Eason, B. Clark, D.A. Snith and D.V. Parke