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Studies of carbamazepine metabolism Webster, Donald Shaw 1989

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STUDIES OF CARBAMAZEPINE METABOLISM By DONALD SHAW WEBSTER B.Sc. (Honours), The Un ivers i ty of B r i t i s h Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Pharmacology 3 Therapeutics Facul ty of Medicine We accept t h i s thes i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1989 ©DONALD SHAW WEBSTER, 1989 lo; In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Pharmacology & Therapeutics The University of British Columbia Vancouver, Canada Date August 21. 1989 _ DE-6 (2/88) i i ABSTRACT The ob jec t ive of t h i s study was to examine aspects of carbamazepine metabolism, in order to contr ibute to a long term goal of a thorough examination of how the metabolism of carbamazepine i s inf luenced by other drugs. The f i r s t set of experiments were designed with the i n t e n t of determining values fo r the pharmacokinetic parameters of carbamazepine metabolism in male New Zealand white r a b b i t s . Values were obtained f o r t m a x (60-90 min), t l / 2 (90-122 min), c learance (46.2-142.4 ml/min/kg), and the e l i m i n a t i o n constant (0.0057-0.0077 min" 1 ) in f i v e t e s t cases. In the remainder of cases , unexpected r e s u l t s were observed which did not al low c a l c u l a t i o n of these parameters. The plasma carbamazepine concentrat ion was e i t h e r delayed in reaching i t s peak concentrat ion or i t reached an apparent peak, but maintained that leve l for an extended period of t ime. I t i s thought that these d i f fe rences between rabb i ts may have been due to d i f fe rences in the rates of g a s t r i c emptying, a f a c t o r that may have been inf luenced by the food eaten by the animals in a per iod in excess of the 12 hours that some of the rabb i ts were fasted p r i o r to the experiments. A l t e r n a t i v e l y , the time per iod of required sampling may have been underestimated. In a d d i t i o n , i t i s a lso poss ib le that some degree of enterohepatic c i r c u l a t i o n i s taking p l a c e . The r e l a t i v e pos i t ions of the curves for carbamazepine and for carbamazepine-10, l l -epoxide suggest that there may be d i f fe rences in the a c t i v i t i e s of hepatic monooxygenases and glucuronysyl t ransferases responsib le fo r the metabolic fates of carbamazepine. i i i The second set of experiments examined the in f luence of i s o n i a z i d and some of i t s p r i n c i p a l metabol ites on the conversion of carbamazepine to carbamazepine-10, l l -epoxide in the S9 f r a c t i o n of rat l i v e r homogenate. This study i s a prelude to planned j_n v ivo studies of the i n t e r a c t i o n in r a b b i t s . Three concentrat ions of each of i s o n i a z i d , acetyl hydrazine, a c e t y l i s o n i a z i d , hydrazine, and l ' sonicot in ic ac id were tested in a system conta in ing constant concentrat ions of carbamazepine and of e s s e n t i a l c o - f a c t o r s . The r e s u l t s ind icated that there was a concentrat ion dependent i n h i b i t i o n of carbamazepine metabolism by i s o n i a z i d , hydrazine, and i son i ' co t in i c a c i d . These types of experiments should expanded to inc lude a range of carbamazepine concentrat ions so that an eva luat ion of the type of i n h i b i t i o n can be determined, as can be done Michaelis-Menton k i n e t i c s . iv TABLE OF CONTENTS CHAPTER Page ABSTRACT rr LIST OF TABLES v LIST OF FIGURES vi ACKNOWLEDGEMENT v i i i 1 PHARMACOKINETICS OF CARBAMAZEPINE IN RABBITS 1 1.1 INTRODUCTION: 1 1.1.1 PHARMACOKINETICS: 1 1.1.2 CARBAMAZEPINE: 3 1.1.3 PHARMACOKINETICS OF CARBAMAZEPINE: 17 1.1.4 INDUCTION OF CARBAMAZEPINE METABOLISM: 18 1.1.5 STATEMENT OF PROBLEM: 19 1.2 EXPERIMENTAL: 20 1.2.1 MATERIALS: 20 1.2.2 ADMINISTRATION AND SAMPLING: 20 1.2.3 EXTRACTION: 21 1.2.4 ANALYSIS OF S A B L E S : 23 1.3 RESULTS: 23 1.4 DISCUSSION: 34 2 THE INFLUENCE OF ISONIAZID ON THE IN VITRO METABO 43 2.1 INTRODUCTION: 43 2.1.1 CARBAMAZEPINE: 45 2.1.2 ISONIAZID: 45 2.1.3 STATEMENT OF PROBLEM: 54 2.2 EXPERIMENTAL: 54 2.2.1 MATERIALS: 54 2.2.2 MICROSOME PREPARATION: 55 2.2 .3 INCUBATION: 56 2.2.4 ANALYSIS OF SAMPLES: 57 2.3 RESULTS: 57 2.4 DISCUSSION: 67 3 REFERENCES: 73 V LIST OF TABLES TABLE Page I Pharmacokinetic parameters for those t r i a l s for which the 34 parameters could be determined. II Estimates of a and b for i s o n i a z i d t e s t s . 65 III Estimates of a and b for hydrazine t e s t s . 66 IV Estimates of a and b fo r i s o n i c o t i n i c ac id t e s t s . 66 V P-values from run ana lys i s of in v i t r o i n t e r a c t i o n s tud ies . 67 vi LIST OF FIGURES FIGURE Page 1 Imipramine 4 2 Imi nodi benzyl 4 3 Iminosti lbene 4 4 Carbamazepine 4 5 Three-dimensional s t ructure of carbamazepine as 6 revealed by X-ray d i f f r a c t i o n . 6 Structures of carbamazepine metabol i tes i s o l a t e d from 11 human ur ine and major pathways of b iotransformat ion . 7 Serum carbamazepine and carbamazepine-10,11-epoxide 24 in rabb i ts administered carbamazepine (25 mg/kg) and fed ad l i b . 8 Serum carbamazepine and carbamazepine-10, l l -epoxide 27 in rabb i ts administered carbamazepine (25 mg/kg) and f a s t e d . 9 Serum carbamazepine and carbamazepine-10,11-epoxide 30 in rabb i ts administered carbamazepine (12.5 mg/kg) and f a s t e d . 10 Sample chromatogram from carbamazepine pharmacokinetics 32 study in r a b b i t s . 11 Ison iaz id 48 12 Relevant aspects of i s o n i a z i d metabolism. 48 13 Sample chromatogram from in v i t r o i n t e r a c t i o n study. 58 14 Influence of i s o n i a z i d on the conversion of carbamazepine 59 to carbamazepine-10,11-epoxide in the S9 f r a c t i o n of ra t l i v e r homogenate. 15 Influence of a c e t y l i s o n i a z i d on the conversion of 60 carbamazepine to carbamazepine-10,11-epoxide in the S9 f r a c t i o n of rat l i v e r homogenate. V I 1 LIST OF FIGURES (cont 'd) FIGURE Page 16 Influence of acetyl hydrazine on the conversion of 61 carbamazepine to carbamazepine-10,11-epoxide in the S9 f r a c t i o n of ra t l i v e r homogenate. 17 Influence of hydrazine on the conversion of carbamazepine 62 to carbamazepine-10,11-epoxide in the S9 f r a c t i o n of r a t l i v e r homogenate. 18 Influence of i s o n i c o t i n i c ac id on the conversion of 63 carbamazepine to carbamazepine-10,11-epoxide in the S9 f r a c t i o n of r a t l i v e r homogenate. v i i i ACKNOWLEDGEMENTS I would l i k e express my s inceres t apprec iat ion to Dr. Richard A. Wall fo r g iv ing me the opportunity to work in h is laboratory and for h is cont inuing advice and guidance. His extensive knowledge and dedicat ion made completion of t h i s degree a pleasant experience. Thanks must a lso be extended to Ms. Maureen Murphy and to Mr. Stephen Adams for t h e i r technica l ass is tance and al lowing me the chance to tap t h e i r many years of experience. Thank-you to the facu l ty members of the department for t h e i r commitment to teaching, which enabled to learn what I d id about pharmacology, and to the s e c r e t a r i a l s t a f f , Margaret Wong, E la ine Jan, and J a n e l l e H a r r i s , without whose ass i s tance I would never have been able to complete t h i s t h e s i s . I must a lso express my grat i tude to the members of my examining committee for working under the time const ra in ts that were requ i red . F i n a l l y , thank-you to my fami ly . Mere words cannot descr ibe how impor-tant they are to me. Without t h e i r support, I could never have made i t as f a r as I have. ix LIST OF ABBREVIATIONS AcHz Acetyl hydrazine AcINH A c e t y l i s o n i a z i d CBZ Carbamazepine CE Carbamazepine-10,11-epoxide Cl Clearance Hz Hydrazine INA I s o n i c o t i n i c ac id INH Isoniaz id H/2 Apparent h a l f - l i f e V d Volume of d i s t r i b u t i o n WEBSTER, D. S. 1 1 PHARMACOKINETICS OF CARBAMAZEPINE IN RABBITS; 1.1 INTRODUCTION: The f i r s t por t ion of t h i s t h e s i s examines the pharmacokinetics of carba-mazepine in r a b b i t s . In our laboratory , there i s an ongoing study of the hepatotox ic i ty of i s o n i a z i d in r a b b i t s . One of the future goals of that study i s to examine the in f luence of carbamazepine. To do so, a knowledge of the pharmacokinetics of carbamazepine, in the same animal model, i s requ i red . S ince the appropriate pharmacokinetic parameters were not a v a i l -able in the l i t e r a t u r e and s ince an appropr iate l i q u i d chromatographic assay procedure had been developed, the study was done as part of t h i s p r o j e c t . 1.1.1 PHARMACOKINETICS: The study of pharmacokinetics involves the i n v e s t i g a t i o n of the k i n e t i c s of drug absorpt ion , d i s t r i b u t i o n , and e l i m i n a t i o n ( i . e . , metabolism and e x c r e t i o n ) . There are s e n s i t i v e , accurate and prec ise a n a l y t i c a l methods for the d i r e c t measurement of drugs in b i o l o g i c a l samples, such as plasma and ur ine . These measurements of drug concentrat ions can be used to deter-mine pharmacokinetic parameters such as b i o a v a i l a b i l i t y , the e l i m i n a t i o n rate constant , the apparent volume of d i s t r i b u t i o n , and the e l im inat ion h a l f - l i f e . B i o a v a i l a b i l i t y i s def ined as the f r a c t i o n of unchanged drug reaching the systemic c i r c u l a t i o n fo l lowing admin ist rat ion by any route (Benet, 1987). Thus, f o r an intravenous dose, the b i o a v a i l a b i l i t y w i l l be equal to one. For an oral dose, the b i o a v a i l a b i l i t y may be less than one for any of several reasons. The most obvious reason i s the incomplete absorption of WEBSTER, D. S. 2 the drug. As w e l l , the drug may be metabolized during absorpt ion. The i n t e s t i n a l mucosa contains sulphate-conjugating enzymes which may i n a c t i v a t e c e r t a i n drugs during t h e i r absorpt ion . The drugs may a lso be suscept ib le to the e f f e c t s of g a s t r o i n t e s t i n a l secret ions and they may be subject to meta-bol ism by bac ter ia r e s i d i n g in the i n t e s t i n e s . Drugs may a lso undergo meta-bol ism in the porta l blood or in the l i v e r without ever being able to reach the systemic c i r c u l a t i o n . As a r e s u l t , the b i o a v a i l a b i l i t y can be s i g n i f i -cant ly decreased from un i ty . The apparent volume of d i s t r i b u t i o n (V^) of a drug i s the volume of f l u i d i t would occupy i f the to ta l amount in the body were in a so lu t ion a t the same concentrat ion as in the plasma. The volume of d i s t r i b u t i o n may vary widely depending on the pK of the drug, the degree of plasma prote in a b i n d i n g , the p a r t i t i o n c o e f f i c i e n t of the drug in the f a t t y t i s s u e s , and the degree of b inding to other t i s s u e s with in the body. For example, a drug that i s t i g h t l y bound to plasma prote in may have a volume of d i s t r i b u t i o n of 0.06 L/kg, which corresponds to the plasma volume per ki logram of body weight. In cont ras t , some drugs that are s e l e c t i v e l y bound to const i tuents of t i s s u e s or are taken up s e l e c t i v e l y by c e l l s can have an apparent volume of d i s t r i b u t i o n that i s several hundred times body volume. The e l i m i n a t i o n h a l f - l i f e ( t ^ ) > a n expression of the r e l a t i o n s h i p between volume of d i s t r i b u t i o n and c learance (t jy 2 =0.693 x V d / C l ) , i s a useful k i n e t i c parameter in that i t ind icates the time required to a t t a i n steady state or to decay from steady state condi t ions a f t e r a change ( i . e . , s t a r t i n g or stopping) in a p a r t i c u l a r rate of drug admin is t rat ion (dosing regimen). However, i t has l i t t l e value as an i n d i c a t o r of drug e l iminat ion or d i s t r i b u t i o n . Clearance i s the measure of the body's a b i l i t y to e l iminate a drug. The WEBSTER, D. S. 3 organs of e l i m i n a t i o n can only c l e a r drug from the blood or plasma that i s in d i r e c t contact with the organ. Thus, the time course of drug in the body w i l l depend on both the volume of d i s t r i b u t i o n and the c learance. 1.1.2 CARBAMAZEPINE: Carbamazepine [5-carbamyld ibenz-(b , f ) -azep in] , an iminost i lbene der iva-t i v e r e l a t e d to imipramine (Figure 1 ) , was f i r s t synthesized by Sch ind ler in the l a t e 1950's and i t was patented in 1961 (Sch ind ler , 1961a). I t was f i r s t introduced for the treatment of t r igemina l neura lg ia (Blom, 1962, 1963), and i t was approved for use as an ant iconvulsant agent in the United States in 1974. Carbamazepine has been studied e x t e n s i v e l y , p a r t i c u l a r l y with respect to i t s metabolism and pharmacokinetics, i t s t o x i c i t i e s , and i t s potent ia l mechanisms of a c t i o n . 1 .1 .2 .1 DEVELOPMENT AND CHEMISTRY: Iminodibenzyl ( 1 0 , l l - d i h y d r o - 5 H - d i b e n z o [ b , f ] a z e p i n e ) , shown in F igure 2, f i r s t descr ibed by Thei l and Holzinger in 1899, may be considered h i s t o r i -c a l l y as the precursor of carbamazepine. Sch ind ler and H a f l i g e r (1954) synthesized a number of iminodibenzyl d e r i v a t i v e s that possessed loca l anaesthet ic and a n t i h i s t a m i n e propert ies and some modest ant iconvulsant a c t i v i t y . When a carbamyl (carboxamide) group was added at the 5-pos i t ion of iminodibenzyl , considerable ant iconvulsant a c t i v i t y was observed. The strongest ant iconvulsant propert ies were observed when a carbamyl s ide chain was combined with iminost i lbene (Figure 3 ) , a s t ructure analagous to imino-WEBSTER, D. S. 4 Figure 3: Iminosti lbene Figure 4 : Ca rbamazep ine WEBSTER, D. S. 5 d i b e n z y l , but having a double bond between the 10 and 11 p o s i t i o n s . This s t ructure has become known as carbamazepine, and i t s synthesis was described in 1961 (Sch ind ler , 1961b). Carbamazepine (5-carbamyl-5H-dibenzo[b,f]azepine; 5H-dibenzo[b,f]azepine-5-carboxamide) (Figure 4 ) , an iminost i lbene d e r i v a t i v e with an empir ica l formula C ^ H ^ ^ O and a molecular weight of 236.26 g/mol, appears as a white c r y s t a l l i n e compound with a melt ing point between 190°C and 193°C (Kutt and P a r i s - K u t t , 1982). I t i s a neutral l i p o p h i l i c substance. It i s v i r t u a l l y inso lub le in water, but i t d i s so lves in ethanol , chloroform, dichloromethane, and other so lvents . X-ray d i f f r a c t i o n studies of carbamazepine have produced measurements that are c h a r a c t e r i s t i c of t r i c y c l i c psychoactive drugs. In the three dimensional s t ructure the angle of f l e x u r e , a , i s 53°, the angle of annela-t i o n , 6, i s 30°, and the angle of t o r s i o n , y, i s 3" (Figure 5 ) . The d istance between the centres of the benzene r ings measured 4.85 Angstroms (Gagneux, 1976). The s i m i l a r i t y to imipramine i s obvious in the s t e r i c parameters, except fo r the t o r s i o n angle which i s 20° fo r imipramine. 1.1.2.2 THERAPEUTIC USES: Carbamazepine was f i r s t introduced in the ear ly 1960's, when i t was administered to pat ients su f fe r ing from tr igeminal neuralgia (Blom, 1962, 1963), a d isorder that i s a lso re fer red to as t i c douloureux. By f a r the most dramatic , i f not the most common, of a l l the pa infu l d isorders that a f f l i c t the human face , t r igeminal neura lg ia was descr ibed in d e t a i l by F o t h e r g i l l (1773), who compiled, with thoroughness, the c l i n i c a l features of fourteen pat ients with tr igeminal neura lg ia . In a short time fo l lowing Blom's r e p o r t s , a number of c l i n i c a l inves t igators publ ished s i m i l a r obser-WEBSTER, D. S. 6 < \n n = 53* '5 = 4.85 A X i 0 = 30* y = 3-Figure 5: Three-dimensional structure of carbamazepine as revealed by X—ray diffraction. Above: frontal view Middle: top view Below: side view Note that the carbamazepine molecule is bent and slightly twisted, (from Gagneux. 1976) WEBSTER, D. S. 7 vations (Tay lor , 1963; Bonduel le , et a l . , 1963; Da less id and Abbott, 1966; Amols, 1966). Carbamazepine i s now the drug of choice in the treatment of t r igeminal neura lg ia . Later inves t igat ions ind icated that carbamazepine i s a lso e f f e c t i v e in the treatment of glossopharyngeal neura lg ia (Ekborn and Westberg, 1966). Carbamazepine has a lso been used to t r e a t l i g h t n i n g pains associated with tabes dorsal i s (Ekborn, 1966, 1972). The bas is of carbamazepine use in t h i s d isorder was the s i m i l a r i t y between tabet i c l i g h t n i n g pains and the pains in t r igemina l neura lg ia . The pains are paroxysmal, b r i e f , and very intense. Tabes dorsal i s presents symptoms and signs of demyelination of the p o s t e r i o r columns, dorsal root , and dorsal root columns of the spinal c o r d . In 1974, carbamazepine was approved for use in the United States as an ant i convu lsant . I t i s useful in pat ients with genera l ized t o n i c - c l o n i c and both simple and complex p a r t i a l se izures . I ts e f f i c a c y i s comparable to that of phenytoin and phenobarbital f o r the treatment of grand mal ep i lepsy and e s p e c i a l l y fo r psychomotor attacks (Meinardi , 1972; Cereghino, et a l . , 1974; L i v i n g s t o n , et a l . , 1974). Carbamazepine i s now a f i r s t - l i n e drug in the treatment of most forms of e p i l e p s y . In recent y e a r s , the therapeut ic i n d i c a t i o n s for carbamazepine have broadened. Carbamazepine has been suggested for use in cases of b i p o l a r depression (Post, et a l . , 1984), exc i ted psychosis ( K l e i n , et a l . , 1984), and alcohol withdrawal syndrome ( R i t o l a and Malinen, 1981). 1.1.2.3 MECHANISMS OF ACTION: The e l u c i d a t i o n of the mechanism of act ion of carbamazepine i s not an easy task. Carbamazepine has several c l i n i c a l e f f e c t s , but the time course of onset of a c t i o n and the dose required for these e f f e c t s var ies g r e a t l y . WEBSTER, D. S. 8 This suggests that there may be d i f f e r e n t mechanisms of act ion for the d i f f e r e n t c l i n i c a l e f f e c t s . In f a c t , carbamazepine exerts a p lethora of biochemical e f f e c t s on a v a r i e t y of neurotransmitter , neuromodulator, second messenger, and neuropeptide systems (Post, 1988). The d i f f e r e n t i a l time course of carbamazepine's ant iconvulsant , a n t i n o c i c e p t i v e , ant imanic, and ant idepressant e f f e c t s may provide ins ight into the mechanisms of act ion re levant to each syndrome. The ant iconvulsant and ant inoc i cept ive e f f e c t s of carbamazepine are r e a d i l y apparent with in hours to days, whereas improvement in sleep occurs in the f i r s t week, and maximal antimanic and ant idepressant e f f e c t s tend to occur with in two and three weeks, respect ive ly (Post, 1988). The a v a i l a b i l -i t y of su i tab le animal models f o r se izure and pain d isorders has allowed p a r t i a l e l u c i d a t i o n of the ant iconvulsant e f f e c t s of carbamazepine. The ant iconvulsant e f f e c t s appear to be c l o s e l y t i e d to mechanisms invo lv ing "per iphera l - type" benzodiazepine receptors (Weiss, et a l . , 1986), i n h i b i t i o n of sodium currents (Willow, et a l . , 1984; MacDonald, et a l . , 1985), noradrenergic potent ia t ion (Post, et a l . , 1985), and, p o s s i b l y , decreased glutamate respons iv i ty (Olpe, et a l . , 1985; Volger and Zeiglgansberger, 1985). As w e l l , chronic treatment with carbamazepine has been assoc iated with a s i g n i f i c a n t reduct ion in cerebrospinal f l u i d somatostatin in a f f e c t i v e l y i l l pat ients (Rubinow, et a l . , 1985) and in e p i l e p t i c pat ients (Steardo, et a l . , 1986). I t has been suggested that the ant inoc i cept ive e f f e c t s of carbamezepine are exerted v i a a mechanism that involves GABAg receptors (Terrence, e t a l . , 1983). Foong and Satoh (1985) a lso impl icated noradrenergic and dopa-minergic mechanisms. WEBSTER, D. S. 9 The r e l a t i v e lack of su i tab le animal models for mania and depression makes the task of l i n k i n g the psychotropic e f f e c t s of carbamazepine to s p e c i f i c biochemical mechanisms d i f f i c u l t . Carbamazepine a f f e c t s almost every neurotransmitter-modulator system hypothesized to be involved in mania and depression (Post, 1988), though a s i g n i f i c a n t dose range i s requ i red , ranging from therapeutic to high to t o x i c . The time course ana lys i s performed by Post (1988) suggests that adenosine and substance P should be added to the large range of candidates for i t s putat ive antimanic and a n t i -depressant e f f e c t s . 1 .1.2.4 BIOTRANSFORMATION: The term "biotransformation" i s appl ied to the chemical changes which substances undergo in b i o l o g i c a l systems. These changes are almost i n v a r i -ably cata lysed by enzymes, so the form they take and the rate at which they occur are dependent on the physico-chemical p roper t ies of the substance concerned and the enzymatic complement of the b i o l o g i c a l system ( F a i g l e , et a l . , 1976). As stated prev ious ly , carbamazepine i s c l a s s i f i e d as a neutral l i p o p h i l i c substance based on the c h a r a c t e r i s t i c s of i t s s o l u b i l i t y and p a r t i t i o n in organic and aqueous media. Since the l i p o p h i l i c i t y of carbamazepine i s a property conducive to d i f f u s i o n of the act ive substance through the body's var ious l i p i d membranes and b a r r i e r s , i t a lso f a c i l i t a t e s the transport of the drug to i t s s i t e s of a c t i o n . The body possesses no mechanism by which exogenous l i p o p h i l i c substances, p a r t i c u l a r l y those of a neutral character , can be r e a d i l y excreted in unchanged form (Weiner, 1967). In f a c t , at most 2 percent of a dose of carbamazepine can be recovered unchanged in human WEBSTER, D. S. 10 ur ine or b i l e (Levy, et a l . , 1975; P i t l i c k , 1975). As a r e s u l t , such substances must f i r s t be transformed with in the organism to more highly hydrophi l i c metabol i tes which can be more r e a d i l y excreted through the kidney. Thus, b iotransformat ion i s important fo r both the i n t e n s i t y and the durat ion of pharmacologic e f f e c t s , s ince the e l i m i n a t i o n of carbamazepine from the organism i s c o n t r o l l e d by the primary metabolic react ions rather than by renal or b i l i a r y excret ion of the unchanged drug (Fa ig le and Feldmann, 1982). Carbamazepine was introduced in the ear ly 1960's but the f i r s t i d e n t i f i -ca t ion of a metabol i te was in 1972, when the 10,11-epoxide was i d e n t i f i e d ( F r i g e r i o , et a l . , 1972). Since then, more than t h i r t y metabol i tes have been i d e n t i f i e d (Lertratanangkoon and Horning, 1982). While the o v e r a l l rate of b iotransformat ion in man i s d r a s t i c a l l y d i f f e r e n t from that in animal spec ies , as r e f l e c t e d by the e l i m i n a t i o n h a l f - l i v e s of carbamazepine determined in plasma, i t would appear that they metabolize carbamazepine by the same basic mechanisms, making i t apparently permiss ib le to extrapolate c e r t a i n biochemical f ind ings from animal models to man (Fa ig le and Feldmann, 1982). Radiotracer studies (Fa ig le and Feldmann, 1975; F a i g l e , et a l . , 1976; R i c h t e r , et a l . , 1978) of carbamazepine admin ist rat ion reveal metabol i te s t ructures that suggest that the b iotransformation of carbamazepine in man proceeds by four major pathways (Figure 6 ) . Taking the to ta l ur inary r a d i o a c t i v i t y as 100 percent, the fo l lowing approximate percentages are a t t r i b u t a b l e to the d i f f e r e n t pathways or the corresponding metabol i tes : epoxidat ion of the 10,11 double bond of the azepine r i n g , 40 percent; hydroxylat ion of the six-membered aromatic r i n g s , 25 percent; d i r e c t N-glucuronidat ion at the carbamoyl s ide cha in , 15 percent; and s u b s t i t u t i o n WEBSTER, D. S. 11 Figure 6: Structures of carbamazepine metabolites isolated from human urine and major pathways of biotransformation, (from Faigle and Feldmann. 1982) WEBSTER, D. S. 12 of the six-membered r ings with s u l f u r - c o n t a i n i n g groups, 5 percent. The remaining r a d i o a c t i v i t y excreted by the kidneys can be a t t r i b u t e d to i n t a c t drug and to products of combined metabolic a t tack . For example, there may be metabol i tes that have been both hydroxylated and N-glucuronidated. The react ion producing carbamazepine-10, l l -epoxide (the f i r s t i n t e r -mediate of pathway 1) i s cata lyzed by hepatic monooxygenase ( F r i g e r i o , et a l . , 1976). Most of the epoxide i s enzymatica l ly converted to t rans-10,11-dihydro-10,11-dihydroxycarbamazepine in the l i v e r by epoxide hydrase (Oesch, 1973). The epoxide accounts fo r only one percent of excreted r a d i o a c t i v i t y , while the d i o l accounts for approximately 35 percent. In ur ine , the d i o l i s p a r t l y present as such and p a r t l y as i t s mono-O-glucuronide. A smaller por t ion of the epoxide intermediate i s converted to a r ing-contracted compound, 9-hydroxymethyl-10-carbamoylacridan by a mechanism that has y e t to be e l u c i d a t e d . In a d d i t i o n , i t i s not y e t known whether t h i s react ion proceeds d i r e c t l y or v ia the d i o l . The acr idan i s almost completely conjugated with glucuronic ac id at the hydroxymethyl group before excret ion ( F a i g l e , et a l . , 1976). The second pathway, a lso thought to be cata lyzed by monooxygenases, s t a r t s with the hydroxylat ion of carbamazepine at various pos i t ions of the six-membered r ings . S ingle s u b s t i t u t i o n r e s u l t s in a l l four poss ib le phenols, i . e . , 1-, 2-, 3 - , and 4-hydroxycarbamazepine. Two other intermediates of t h i s pathway carry a hydroxy group in post ion 2 and, a d d i t i o n a l l y , a methoxy group in p o s i t i o n 1 or in p o s i t i o n 3 (R ichter , et a l . , 1978). The bulk of these metabol i tes are excreted by the kidney as O-glucuronate and 0-su l fa te conjugates in a r a t i o of about 2:1, while only t race amounts of the phenols are excreted unconjugated. Add i t iona l WEBSTER, D. S. 13 metabol i tes have been found by other i n v e s t i g a t o r s , inc lud ing three hydroxymethoxy compounds and three dihydroxy compounds (Lynn, et a l . , 1977, 1978), a l l of which were g lucuronidated. The t h i r d important route of b iotransformat ion i s d i r e c t conjugation of carbamazepine with g lucuronic a c i d . In the conjugate, the l igand i s bound to the amino group of the carbamoyl s ide cha in . I t has been assumed that the conjugation i s metabolized by a hepatic glucuronyl t ransferase (Fa ig le and Feldmann, 1982). The enzyme e-glucuronidase i s able to c leave the glucuronide from most conjugated spec ies , but i t i s unable to do so in t h i s case. The fourth major pathway involves the in t roduct ion of a s u l f u r - c o n t a i n i n g subst i tuent into one of the six-membered r ings of the carbamazepine molecule. Four products r e s u l t i n g form t h i s pathway were found in human u r i n e : 2- and 3-methylsulf inylcarbamazepine and 2- and 3-methylsulfonylcarbamazepine (Fa ig le and Feldmann, 1982). The mechanism by which these conjugates are generated i s unknown. Add i t iona l products formed by pathways 1, 2, and 4 have been described by Lertratanangkoon and Horning (1982). Such products w i l l reduce the u n i d e n t i f i e d f r a c t i o n in ur ine , but they are r e l a t i v e l y minor components. A study has been done to examine the s t e r i c course of the enzymatic hydro lys i s of carbamazepine-10,11-epoxide, a primary metabol i te of carbama-zepine ( B e l l u c c i , et a l . , 1987). During the study, the in tent ion was to subject the epoxide to the a c t i o n of microsomal epoxide hydrolase from animal l i v e r in v i t r o . However, attempts with microsomes from r a b b i t , rat and guinea pig and with c y t o s o l i c f r a c t i o n s from rat and guinea pig showed that t h e i r hydro ly t i c a c t i v i t y on the epoxide was very low, with only t races of the d i o l being formed a f t e r protracted incubat ions. As a r e s u l t , the WEBSTER, D. S. 14 inves t igators were forced to i s o l a t e the d io l from the urine of pat ients under carbamazepine treatment. Both the f ree d i o l , and that obtained a f ter treatment with e -g lucuron idase/ary l su l fa tase , were found to be formed in an enantiomeric excess of 80 percent, the prevalant enantiomer having the ( - ) - 1 0 S , l l S absolute c o n f i g u r a t i o n . This f i n d i n g i s an i n d i c a t i o n of pronounced e n a n t i o s e l e c t i v i t y of the microsomal epoxide hydrolase toward meso and racemic substrates , but i s in contrast with the prevalent formation of ( R , R ) - d i o l s in most other known cases of enzymatic hydro lys i s of epoxides ( B e l l u c c i , et a l . , 1987). 1 .1 .2 .5 ADVERSE EFFECTS: I t has been estimated that 33 to 50 percent of adul ts and c h i l d r e n being t rea ted with carbamazepine experience side e f f e c t s and/or t o x i c i t i e s . S ide e f f e c t s seem to be more common with polytherapy than with monotherapy (Masland, 1982). In genera l , the adverse e f f e c t s of carbamazepine can be d iv ided in to two c lasses - c l i n i c a l s ide e f f e c t s and laboratory abnormali-t i e s . Most of the c l i n i c a l s ide e f f e c t s are mi ld , t r a n s i e n t , and r e v e r s i b l e i f the dosage i s reduced or i f i n i t i a t i o n of treatment i s gradual . The most common s ide e f f e c t s inc lude nausea, drowsiness, v e r t i g o , a t a x i a , b lurred v i s i o n , d i p l o p i a , and s lu r red speech. A l l but the f i r s t are neurotoxic in o r i g i n . These s ide e f f e c t s are ser ious enough to warrant d i scont inuat ion of therapy in only f i v e percent of cases (Pe l lock , 1987). The carbamazepine-assoc iated s ide e f f e c t that i s most f requent ly reported to Geigy Pharmaceu-t i c a l s and the FDA in the United States are skin and a l l e r g i c react ions such as Stevens-Johnson syndrome, L y e l l ' s syndrome, e x f o l i a t i v e dermat i t i s , and WEBSTER, D. S. 15 erythema mult i forme. Certain movement d isorders and seizure increases have been a t t r i b u t e d to carbamazepine therapy. The movement d i sorders , which include chorea, dyston ia , a s t e r i x i s , and myoclonus, are very rare and have usua l ly been observed in conjunct ion with toxic plasma l e v e l s of carbamaze-p ine , most commonly in pat ients rece iv ing polytherapy for hard-to-contro l se izures and having s i g n i f i c a n t neurologic dysfunct ion (Masland, 1982). There have been a number of reports of carbamazepine assoc iated seizure increase (Shie lds and Saslow, 1983; Johnson, et a l . , 1984; Sachedo and Chokroverty, 1985; Snead and Hosey, 1985; Hurst, 1985; Horn, et a l . , 1986). Most of these involved pat ients with general ized nonconvulsive se izures with slow spike and wave genera l ized e lect roencepha lograph^ abnormal i t ies . The se izures that a r i s e are genera l ly a ton i c , myoclonic, and absence-type se i zures , but these can progress to genera l ized t o n i c - c l o n i c se i zures . A var ie ty of laboratory s ide e f f e c t s have been observed with carbamaze-pine therapy, the most important of which are hematologic and hepatic abnor-m a l i t i e s . Although hematologic react ions to carbamazepine are ra re , they are very important, s ince carbamazepine can produce ser ious and p o t e n t i a l l y f a t a l cases of protracted bone marrow depression. Agranulocytos is and a p l a s t i c anemia are perhaps the most ser ious hematologic side e f f c t s , but they are not the most common. Those most f requent ly reported to Geigy Pharmaceuticals, in order of frequency, include thrombocytopenia, a p l a s t i c anemia, agranulocytos is , pancytopenia, and bone marrow depression. As w e l l , leukopenia i s a cond i t ion estimated to occur in approximately 10 percent of c h i l d r e n and adults t reated with carbamazepine (Hart and Easton, 1982). The most common hepatic and pancreat ic abnormal i t ies reported to Geigy Pharma-c e u t i c a l s inc lude h e p a t i t i s , abnormal l i v e r funct ion t e s t s , jaundice/chole-s t a t i c i c t e r u s , l i v e r dys funct ion , hepatomegaly/hepatosplenomegaly, and WEBSTER, D. S. 16 p a n c r e a t i t i s . The most common observations are t r a n s i e n t e l e v a t i o n of l i v e r enzymes, reported to occur in 5 to 10 percent of pat ients rece iv ing carbama-zepine (Pe l lock , 1987). In view of the known c y t o t o x i c , te ratogen ic , mutagenic, and carc inogenic propert ies of some aromatic p o l y c y c l i c hydrocarbons (Oesch, 1976), carbama-zepine and some of i t s metabol i tes have been examined with respect to these f a c t o r s . Carbamazepine-10,11-epoxide was found to be neither cy to tox i c to human c e l l s in v i t r o and to mice bearing leukemia L1210 ( i . e . , there was no e f f e c t on the surv iva l rate of the mice) ( F r i g e r i o and M o r s e l l i , 1975) nor mutagenic to b a c t e r i a l t e s t e r s t r a i n s ( G l a t t , et a l . , 1975). In teratogeni -c i t y studies in mice, some studies reported p o s i t i v e r e s u l t s (Eluma, et a l . , 1981) while other studies y i e l d e d negative r e s u l t s ( F r i t z , et a l . , 1976; Wray, et a l . , 1982). In humans, no increase in congenital malformations was observed in o f f s p r i n g of mothers treated with carbamazepine during pregnancy (L iv ings ton , et a l . , 1974; Nakane, et a l . , 1980). Recent ly , the mutagenic e f f e c t s of carbamazepine have been studied by examining i t s a b i l i t y to induce s i s ter -chromat id exchanges (SCE) and s t ruc tura l aberat ions in the chromosomes (Schaumann, et a l . , 1985). In such s tud ies , there was no c o r r e l a t i o n observed between chromosome breaks and SCE in e i t h e r J_n v ivo or in v i t r o s tud ies . The studies produced negative _i_n v ivo r e s u l t s , i n d i c a t i n g an absence of detectable chromosome damaging e f f e c t s of carbamazepine in monotherapy in e p i l e p t i c human subjects who had been administered carbamaze-pine at therapeut ic l e v e l s fo r a minimum of 18 months. The serum carbamaze-pine concentrat ions of the subjects in the study ranged from 4.3 to 9.0 u g/ml. WEBSTER, D. S. 17 1.1.3 PHARMACOKINETICS OF CARBAMAZEPINE: The pharmacokinetics of carbamazepine in humans has been studied exten-s i v e l y , and very thorough reviews of the information are a v a i l a b l e (Morse l l i and F r i g e r i o , 1975; B e r t i l s s o n and Tomson, 1986). In a d d i t i o n , the pharma-c o k i n e t i c s of carbamazepine have been examined in the rhesus monkey (Wedlund and Levy, 1983; Levy, et a l . , 1984), in the rat (Chang and Levy, 1986) and in the rabb i t (S iegers , et a l . , 1982; Sumi, et a l . , 1987). In healthy human subjects , the apparent plasma h a l f - l i f e , fo l lowing s ing le doses, has been reported to be 35-37 hours (Palmer, et a l . , 1973), 40-41 hours (Fa ig le and Feldmann, 1975), 31-55 hours ( M o r s e l l i , et a l . , 1975), a mean of 35 hours with a range of 20-65 hours (Strandjord and Johannessen, 1975), and 26.2 ± 6.1 hours (Eichelbaum, et a l . , 1985). The immense v a r i a t i o n of t h i s parameter between i n d i v i d u a l s i s obvious. The volume of d i s t r i b u t i o n has been reported as 1.3 L/kg (Palmer, et a l . , 1973) and as 0.82-1.04 L/kg ( M o r s e l l i , et a l . , 1975). This value can be explained in part by the l e v e l of plasma prote in binding of carbamazepine being 70 to 80 percent ( B e r t i l s s o n , 1978) and the hydrophobicity of the drug which could lead to i t s p a r t i t i o n i n g in to f a t t y t i s s u e s . There have been two studies invo lv ing the pharmacokinetics of carbamaze-pine in r a b b i t s . In one study carbamazepine was administered intravenously (Sumi, et a l . , 1987) and in the other i t was administered o r a l l y (S iegers , e t a l . , 1982). The intravenous admin ist rat ion produced a h a l f - l i f e of 0.648 ± 0.143 hours, a c learance rate of 1.219 ± 0.470 L/hr/kg, and a volume of d i s t r i b u t i o n of 1.086 * 0.287 L/kg. When carbamazepine was administered o r a l l y , the h a l f - l i f e was 2.35 * 1 hour. S ince these studies e i t h e r used WEBSTER, D. S. 18 the wrong route of admin ist rat ion or d id not determine a l l of the des i red parameters for the experiments planned f o r our laboratory , the current study was i n i t i a t e d . 1.1.4 INDUCTION OF CARBAMAZEPINE METABOLISM: I t i s well estab l i shed that carbamazepine i s subject to autoinduct ion and to hetero induct ion . During long term therapy, carbamazepine induces i t s own metabolism ( B e r t i l s s o n , et a l . , 1980; Eichelbaum, et a l . , 1975). Con-comitant treatment with phenobarbitone or phenytoin fu r ther induces the metabolism (Chr is t iansen and Dam, 1973; Eichelbaum, et a l . , 1979, 1985). The epoxide-dio l pathway i s the metabolic route that i s induced during both auto- and heteroinduct ion (Eichelbaum, et a l . , 1985). There are i n d i c a t i o n s t h a t i t i s not only the epoxidat ion but a l so the formation of the t r a n s - d i o l metabol i te that i s induced (Bourgeois and Wad, 1984; Eichelbaum, et a l . , 1985; Tybr ing , et a l . , 1981; Wedlund, et a l . , 1982). The time course of autoinduct ion of carbamazepine k i n e t i c s has been studied in three c h i l d r e n with a recent ly developed psychomotor ep i lepsy ( B e r t i l s s o n , et a l . , 1980). Tetradeuter ium-label led carbamazepine (CBZ-D^) was given as a s ing le dose before maintenance therapy, and on three occas ions, part of the regular carbamazepine dose was replaced by CBZ-D^. On day 6 (second dose of carbamazepine given during maintenance therapy) , the c learance of CBZ-D 4 was greater than i t was for the i n i t i a l CBZ-D 4 dose. The c learance of CBZ-D 4 was doubled from 21 to 36 days and was not fu r ther increased a f t e r f i v e months, when the l a s t CBZ-D 4 dose was g iven . Thus, in c h i l d r e n at l e a s t , the autoinduct ion of carbamazepine metabolism seems to be complete during the f i r s t 3-5 weeks of treatment. WEBSTER, D. S. 19 Autoinduct ion of carbamazepine metabolism has a lso been studied in human adults (Eichelbaum, et a l . , 1985). The f i r s t group in the study consisted of healthy volunteers that received a s ing le ora l dose of 200 mg of carbamazepine. The second group cons isted of e p i l e p t i c pat ients who had received carbamazepine monotherapy f o r at l e a s t s ix months. Compared with healthy subjects , the plasma c learance was 3 - fo ld higher in pat ients on carbamazepine monotherapy. The increased plasma c learance was mainly a t t r i b u t e d to the induct ion of the epoxide-dio l pathway. Thus, in human a d u l t s , a s i g n i f i c a n t degree of induct ion occurs with in s ix months. Unfortunately , the in f luence p r i o r to the s ix month time point i s not as c l e a r l y de f ined . The induct ion of microsomal enzymes in rat l i v e r by carbamazepine has a l so been examined (Wagner and Schmid, 1987). The animals were t reated for four days with d a i l y ora l equimolar doses of 315 nmol/kg. The rats were k i l l e d by decap i ta t ion 24 hours a f t e r the l a s t dose of carbamazepine. The l i v e r s were exc ised , and i t was found that carbamazepine s i g n i f i c a n t l y increased the l i v e r weight and the concentrat ion of cytochrome P4gQ» but not the concentrat ion of microsomal p r o t e i n . This ind icates t h a t , in r a t s , an induct ive e f f e c t i s occurr ing wi th in four days. 1.1.5 STATEMENT OF PROBLEM: As stated at the beginning of t h i s in t roduct ion , the goal of t h i s study was to determine the pharmacokinetic parameters assoc iated with carbamaze-pine metabolism in r a b b i t s . The information presented in the in t roduct ion ind icates that knowledge of the parameters fo r t h i s species i s sparse, desp i te the f a c t that the metabolism has been studied extens ive ly in man and in other spec ies . Thus, the basis of t h i s study i s j u s t i f i e d . WEBSTER, D. S. 20 1.2 EXPERIMENTAL: 1.2.1 MATERIALS: Carbamazepine, heparin, and ethanol were obtained from Sigma Chemical Co. A c e t o n i t r i l e and methanol were obtained from BDH Chemicals. Dextrose (5 percent) was obtained from Abbott Laborator ies , and Hoffman-LaRoche was the suppl ier of nitrazepam. Nar^PO^ was obtained from F i sher S c i e n t i f i c Co. Male New Zealand white rabb i ts (2-3 kg) were obtained from the Animal Care Unit of the Un ivers i ty of B r i t i s h Columbia. 1.2.2 ADMINISTRATION AND SAMPLING: Carbamazepine, suspended i n lmL of Tween 20 (0.5 mg/mL) and 5 percent dextrose as requ i red , was administered o r a l l y to male New Zealand white rabb i ts (2-3 kg) . This s i z e of rabb i t was chosen because animals from the loca l supp l ier weighing under 2 kg are suscept ib le to a f requent ly l e tha l s u b c l i n i c a l resp i ra tory i n f e c t i o n when transported and handled. Administra-t i o n of the suspension was by syringe to the back of the throat of the r a b b i t . The rabb i ts were administered doses of 12.5 mg/kg or 25 mg/kg, and they were e i t h e r fasted or fed ad l i b . p r i o r to admin is t rat ion of the drug. The doses were chosen based on the f a c t that ra ts are often administered a dose in the range of 80 mg/kg and that ra ts have a much more rapid rate of metabolism. In the one a v a i l a b l e reference in which a comparable study was done (S iegers , et a l . , 1982), the carbamazepine dose was 40 mg/kg. I n c i -dent ly , the existence of the study by t h i s group was not discovered u n t i l the current study was e s s e n t i a l l y complete. In studies in which WEBSTER, D. S. 21 carbamazepine was administered intravenous ly , the dose was general ly about 10 mg/kg (Rimerman, et a l . , 1979; Sumi, et a l . , 1987). As fur ther j u s t i f i -cat ion for the doses used, in one experiment of the current study a dose of 250 mg/kg was acc ident ly administered to a r a b b i t , but the maximum plasma carbamazepine obtained was very s i m i l a r to the concentrat ion obtained as a r e s u l t of a 25 mg/kg dose, as was the res t of the time p r o f i l e of carbamaze-pine concentrat ions . Each rabb i t underwent two admin is t rat ions , with at l e a s t seven days separat ion between the admin is t rat ions . Th is per iod allowed complete e l i m i n a t i o n of the drug so that there was no carryover e f f e c t , a l lowing each animal to be used as i t s own c o n t r o l . Ten blood samples were taken over a per iod of up to s ix hours, to monitor the l e v e l s of carbamazepine and of i t s primary metabol i te , carbama-z e p i n e - 1 0,ll - e p o x i d e , over t ime. Blood samples (approximately 0 .7-0.8 mL) were taken from the ear vein through a catheter (Je lco 22g) that remained in place for the durat ion of the experiment. Blood volume was kept r e l a t i v e l y constant by f lush ing the catheter with a volume of hepar inized sa l ine that was approximately equivalent to the volume of blood removed. The blood samples were centr i fuged in microcentr i fuge tubes fo r one minute using an Eppendorf Centr i fuge 3200. The plasma was c o l l e c t e d and was stored at -20"C u n t i l ex t rac ted . 1.2.3 EXTRACTION: Carbamazepine and carbamazepine-10,ll-epoxide were extracted from the plasma samples using a s o l i d phase ext rac t ion procedure descr ibed by Hart ley , et a l . (1986). This type of e x t r a c t i o n was chosen over a l i q u i d - l i q u i d ext rac t ion because the l a t t e r have a tendency to form emulsions that decrease WEBSTER, D. S. 22 the e f f i c i e n c y of the e x t r a c t i o n s . On the other hand, the column method was a r e l a t i v e l y simple procedure that was quick and reasonably e f f i c i e n t . Hart ley and co-workers reported recover ies in the range of 90 to 93 percent , and i t was these values and the ease of the technique that led to the dec i -s ion to use t h i s method. The recover ies obtained during the current study were s i m i l a r to those reported by H a r t l e y ' s group. Reversed-phase octade-c y l s i l a n e bonded s i l i c a columns, with a 2.8 n i c a p a c i t y , were used. Both Bond-Elut (manufactured by Analytichem I n t e r n a t i o n a l , Harbor C i t y , CA) and Clean-Up (manufactured by Worldwide Monitor ing, Horsham, PA) C l g columns were u t i l i z e d , with no s i g n i f i c a n t d i f f e r e n c e in e f f i c i e n c y between column types . The vacuum apparatus used was a Baker-10 SPE system ( J . T . Baker Chemical Co . , P h i l l i p s b u r g , NJ). The columns were condit ioned immediately p r i o r to use by drawing through, under vacuum, two column volumes each of a c e t o n i t r i l e and water. With the vacuum re leased , 250 nL of the plasma sample and 25nL of in te rna l standard (nitrazepam, 100 ng/mL, in ethanol) were appl ied and allowed to e q u i l i b r a t e f o r one minute. The vacuum was appl ied to t ransport a l l of the plasma into the column. Upon re lease , there was a two minute e q u i l i b r a t i o n period before washing with one volume of water and one volume of water/aceton i t r i l e (80:20). The compounds of i n t e r e s t (Carbamazepine and i t s epoxide metabol i te) were e luted with 750 nL of ethanol , and t h i s ext rac t was evaporated to dryness under a n itrogen stream at 55"C. The residue was reconst i tu ted in 250 uL of mobile phase and stored at -20"C u n t i l analysed. Nitrazepam was chosen as the standard in t h i s procedure s ince i t i s s i m i l a r to carbamazepine and the epoxide with respect to hydrophobicity and w i l l , as a r e s u l t , e lu te from the ext rac t ion column under the same condi t ions and provide a s i m i l a r recovery. In WEBSTER, D. S. 23 a d d i t i o n , the compounds of i n t e r e s t have s i m i l a r e x t i n c t i o n c o e f f i c i e n t s at the wave length used for detect ion fo l lowing t h e i r separat ion by l i q u i d chromatography. 1.2.4 ANALYSIS OF S A B L E S : The samples were analysed by reversed-phase high performance l i q u i d chromatography (HPLC) using a Spectra-Physics SP8000B l i q u i d chromatograph. A 125 x 4 .6 mm bore column was packed with Spherisorb 5 um 0DS2. The oven temperature was set at 48"C. The mobile phase was 42 percent methanol with a 0.01M NaH 2 P0 4 bu f fer . The f low rate was lmL/minute. Detect ion was by a Spectra-Physics SP8400 uv/vis detector set at 210 nm, al lowing the detec-t i o n of both carbamazepine and carbamazepine-10, l l -epoxide, i t s primary metabol i te (Rambeck, et a l . , 1981). The r e s u l t s were obtained as peak areas as determined by the data system of the chromatograph. 1.3 RESULTS: The r e s u l t s of the study of carbamazepine pharmacokinetics in rabb i ts are shown in F igures 7, 8, and 9, with a sample chromatogram being d isp layed in Figure 10. F igure 7 shows the r e s u l t s in animals administered a dose of 25 mg/kg and allowed f ree access to food. For those t r i a l s shown in F igure 8, the animals were a lso given a dose of 25 mg/kg, but they were fas ted . The animals used to get the data in F igure 9 were a lso f a s t e d , but they were administered a dose of only 12.5 mg/kg. I n i t i a l l y , the per iod over which samples were taken was f i v e hours. When t h i s did not seem adequate for rabbi t Dl (25 mg/kg, fed ad l i b . ) , the time per iod was extended to s ix hours fo r rabb i t D2 (25 mg/kg, fed ad l i b . ) , Plasma Concentration <uo/ml) Plasma Concentration (ug/ml) Plasma Concentration (ug/ml) WEBSTER, D. S. 26 Figure 7: Seurm carbamazepine and carbamazepine-10.11—epoxide in rabbits administered carbamazepine (25 mg/kg) and fed ad lib. The data for each animal is presented in an individual frame. ro Plasma Concentration (ug/ml) Plasma Concentration (ug/ml) Plasma Concentration (ug/ml) co —I m o GO ro co e | a i csz RABBIT D 8 25 mg/kg: fasted - • - CE WEBSTER, D. S . 29 O 3 0 6 0 9 0 120 150 180 2 1 0 2 4 0 2 7 0 3 0 0 3 3 0 3 6 0 Time (minj RABBIT D9 25 mg/kg: fasted —*— cez - • - CE O 3 0 6 0 9 0 120 150 180 2 1 0 2-40 2 7 0 3O0 3 3 0 3 6 0 Time (minj RABBIT D1 1 25 mg/kg: fasted —*— C8Z -•- ce a O 3 0 6 0 9 0 120 150 180 2 1 0 2 4 0 2 7 0 3 0 0 3 3 0 3 6 0 Time <minj Figure 8: Serum carbamazepine and carbamazepine— 10,11 —epoxide in rabbits administered carbamazepine (25 mg/kg) and fasted. The data for each animal is presented in an individual frame. Plasma Concentration (ug/ml) Plasma Concentration (ug/ml) Plasma Concentration (ug/ml) Plasma concentration (ug/ml) Plasma Concentration (ug/ml) Plasma Concentration (ug/ml) WEBSTER, D. S. 32 Figure 9: Serum carbamazepine and carbamazepine-10,1 1—epoxide in rabbits administered carbamazepine (12.5 mg/kg) and f a s t e d The data for each animal is presented in an individual frame. Figure 10: Sample chromatogram from carbamazepine pharmacokinetics study in rabbits. The sample shown was taken at 120 min from a rabbit fed ad lib. and administered a dose of 25 mg/kg. The chromatogram shows carbamazepine-10.11 -epoxide (293), nitrazepam (579). and carbamazepine (632). WEBSTER, D. S. 33 at which time an appropriate p r o f i l e was obtained. This was a lso observed for rabbit D3. But, as more animals were tes ted , i t became obvious that a usable concentrat ion-t ime p r o f i l e could not be obtained in a l l cases. Subsequently, the t e s t s were repeated with the rabb i t s fasted overnight p r i o r being administered carbamazepine, and for at l e a s t one hour a f t e r drug admin is t rat ion (most animals did not eat during the per iod of blood sampling). The animals were allowed f ree access to water fo r the durat ion of the experiment. The idea was that the presence of food in the stomach may have delayed g a s t r i c emptying, and as a r e s u l t delayed presentat ion of the drug to the s i t e of absorpt ion, namely the small i n t e s t i n e . But, an extended plateau was again seen in the pharmacokinetic p r o f i l e fo r several animals (F igure 8 ) . Tests at a lower dose (12.5 mg/kg) d isp layed a s i m i l a r phenomenon (Figure 9 ) . As a r e s u l t of the phenomenon that was observed, i t was not poss ib le to accurate ly determine the pharmacokinetic parameters, o u t l i n e d in the objec-t i v e s of t h i s study, fo r a l l of the animals in the study groups. In the group that was fed ad l i b . and administered a dose of 25 mg/kg (Figure 7) , rabb i ts D2 and D3 produced a usable type of p r o f i l e . For these t e s t s , the values f o r t m a w were 60 and 90 minutes, f o r the apparent h a l f - l i f e ^1/2^ w e r e 9 0 a n < * ^ minutes, f o r c learance were 91.6 and 46.2 mL/min/kg, and for the e l i m i n a t i o n constant ( k e l ) were 0.0077 and 0.0071 m i n " 1 , r e s p e c t i v e l y . Of the rabb i ts administered a dose of 25 mg/kg and fas ted (Figure 8 ) , rabb i ts D3 and D8 gave usable p r o f i l e s . The values fo r t m a v were 60 and 90 minutes, f o r the apparent h a l f - l i f e were 90 and 122 minutes, f o r c learance were 142.4 and 101.0 mL/min/kg, and for the e l i m i n a t i o n constant were 0.0077 and 0.0057 m i n - 1 fo r rabb i ts D3 and D8, r e s p e c t i v e l y . In the group administered a dose of 12.5 mg/kg (Figure 9 ) , only rabb i t D8 WEBSTER, D. S. 34 gave a standard p r o f i l e . The t v was 60 minutes, as in the prev ious ly max mentioned t e s t . The h a l f - l i f e was 110 minutes. The c learance rate was 121.8 ni/min/kg, and the e l i m i n a t i o n constant was 0.0063 m i n " 1 . These r e s u l t s are summarized i n Table I . Table I: Pharmacokinetic parameters for those t r i a l s fo r which the para-meters could be determined. Treatment Rabbit t max min K min c learance mL/min/kg e l i m i n a t i o n constaijit min" 25 mg/kg D2 60 90 91.6 0.0077 Ad l i b . D3 90 98 46.2 0.0071 25 mg/kg D3 60 90 142.4 0.0077 Fasted D8 90 122 101.0 0.0057 12.5 mg/kg D8 60 110 121.8 0.0063 Fasted 1.4 DISCUSSION: The pharmacokinetics of carbamazepine in rabb i ts had been prev ious ly been examined by two teams of i n v e s t i g a t o r s . The f i r s t (S iegers , et a l . , 1982) administered the drug o r a l l y (40 mg/kg), as was done in the current study. But, the only parameters that were determined were the time at which the maximum plasma concentrat ion was achieved (t^ ) , which was 2 hours, and the h a l f - l i f e ( t ^ ) ' which was 2.35 hours. The values fo r t m £ ( X were s i m i l a r to those obtained from animals e x h i b i t i n g conventional one compartment pharmacokinetic p r o f i l e s in the current study. This held true WEBSTER, D. S. 35 for each of the dosing/feeding regimens. The h a l f - l i v e s measured in the current t e s t s were a l l l e s s than the average reported by Siegers and co-workers, but they s t i l l f e l l with in the reported range. The p r o f i l e s used to determine the values of the pharmacokinetic parameters in t h i s study c l o s e l y resembled those obtained by the Se igers group, which they descr ibed as conforming to the s ing le compartment model. In s ing le dose studies in man, those studies in which so lut ions or suspensions have been administered have d isp layed r e s u l t s that could be described by the one compartment model ( B e r t i l s s o n , 1978; Pynnonen, 1979), whereas when commercially a v a i l a b l e t a b l e t s have been administered the d i s p o s i t i o n of the drug fo l lows the two compartment model (Ronfeld and Benet, 1977). The second team of i n v e s t i -gators (Sumi, et a l . , 1987) administered the carbamazepine intravenously (10 mg/kg) and they determined values fo r c learance (1.219 L/hr/kg), volume of d i s t r i b u t i o n (1.086 L/kg), and h a l f - l i f e (0.684 h r ) . These values are a l l s i g n i f i c a n t l y less than those in the current s t u d i e s . The d i f f e r e n c e s in the values obtained in these var ious studies may be a t t r i b u t e d , in par t , to d i f fe rences in s t r a i n s of animals used. When an intravenous dose i s administered the e n t i r e dose enters the blood stream at once, g iv ing an immediate peak concentrat ion . From t h i s p o i n t , the only a c t i v i t y occurr ing i s e l i m i n a t i o n . On the other hand, when the drug i s administered o r a l l y , there i s a delay before the peak plasma concentrat ion i s a t t a i n e d . As w e l l , e l i m i n a t i o n i s occurr ing at the same time as uptake for a per iod of t ime. Normally, when there i s rapid uptake of an o r a l l y administered drug, the e l i m i n a t i o n w i l l be the same for both the oral and intravenous routes of admin is t ra t ion . I t i s poss ib le t h a t , in the current s i t u a t i o n , the uptake of the drug was slowed, r e s u l t i n g in concurrent uptake and e l i m i n a t i o n . I f t h i s i s occur r ing , the values WEBSTER, D. S. 36 obtained for c learance , volume of d i s t r i b u t i o n , and h a l f - l i f e f o r the intravenous dose would a l l appear to be l e s s than for an o r a l l y administered dose. This i s the r e s u l t that was a c t u a l l y observed, with respect to the values of the parameters. A recurrent pattern emerged i n several animals which ind icated that the s ing le compartment model was not s a t i s f a c t o r y in a l l cases. In several cases, the plasma concentrat ion of carbamazepine reached a peak at 60-90 minutes, began to decrease, and e i t h e r plateaued or increased again . This pattern was observed for animals in each of the dosing/feeding groups that were t e s t e d . The most l i k e l y explanation for t h i s observat ion may be obvious i f the experiments were to be repeated employing a longer time course of sampling. The reasoning behind t h i s statement may be obvious i f one considers the sequence of steps involved in the uptake of an o r a l l y administered drug. Fol lowing ingest ion of the drug, i t must be d isso lved in the stomach, in the case of t a b l e t s . This i s not a problem in the current case since the carba-mazepine was administered as a suspension of f i n e p a r t i c l e s . The stomach i s l ined by a t h i c k , mucus-covered membrane with small surface and high e l e c t r i c a l r e s i s t a n c e . I ts primary funct ion i s d i g e s t i o n . On the other hand, the ep i the l ium of the small i n t e s t i n e has an extremely large surface area. I t i s t h i n , has low e l e c t r i c a l res i s tance , and i t s primary funct ion i s f a c i l i t a t i n g the absorpt ion of n u t r i e n t s . Carbamazepine, being a hydrophobic drug, should be absorbed quite read i l y in the small i n t e s t i n e s ince i t i s h ighly l i p i d s o l u b l e . Thus, i t w i l l be quick ly taken by the e p i t h e l i a l c e l l s of the small i n t e s t i n e . The drug must then d i f f u s e through the c e l l , again pass through the c e l l membrane, and then make i t s way across the membrane of the porta l vasculature and into the porta l c i r c u l a t i o n . WEBSTER, D. S. 37 From t h i s point the drug i s t ransported to the l i v e r , where i t undergoes f i r s t pass metabolism, and then on to the systemic c i r c u l a t i o n . Of the processes descr ibed , the only one that d i sp lays any s i g n i f i c a n t degree of v a r i a b i l i t y i s the rate of g a s t r i c emptying. Since the stomach i s the organ responsib le for the major i ty of d iges-t i o n , while the rest of the g a s t r o i n t e s t i n a l t r a c t i s responsib le pr imar i l y fo r absorpt ion , the presence of food in the stomach w i l l necessar i ly slow the rate of g a s t r i c emptying. Should t h i s process be delayed, the rate at which the drug enters the small i n t e s t i n e and i s able to be absorbed w i l l a l so be slowed. I t was thought that f a s t i n g the animals overnight p r i o r to the experiment would be adequate to a l l e v i a t e any worries about d i f f e r e n t i a l g a s t r i c emptying between animals, but i t i s poss ib le that rabb i t s require a long period for the emptying of food contents from the stomach. As a r e s u l t , the observed r e s u l t s may have been inf luenced by the feeding of the animals p r i o r to the per iod of f a s t i n g . I t i s a lso poss ib le that carbamazepine i t s e l f may have been responsib le fo r slowing g a s t r i c emptying in some of the animals. I t i s well known that f a t t y foods genera l ly require a greater period to be passed through the stomach than do carbohydrates and p r o t e i n s . This has been a t t r i b u t e d to the c a l o r i c content of the food, but the exact mechanism by which the s ignal to slow g a s t r i c emptying i s i n i t i a t e d i s unknown. I t i s poss ib le that there are "detectors" that are s p e c i f i c fo r some chemical feature of the d i f f e r e n t food types. I f t h i s i s the case, perhaps a p a r a l l e l can be drawn to carbamazepine. Since i t i s very hydrophobic, carbamazepine shares a key chemical property with f a t . As a r e s u l t , carbamazepine i t s e l f may be responsib le fo r delayed g a s t r i c emptying. A l t e r n a t i v e l y , the presence of the surfactant Tween in the suspension may have been r e s p o s i b l e , due i t s WEBSTER, D. S. 38 s t ructura l s i m i l a r i t y to f a t t y ac ids . I f c a l o r i c content i s r e a l l y a major determinant of the rate of g a s t r i c emptying, i t i s poss ib le that the dextrose used to make the suspension was s u f f i c i e n t to slow emptying. One of the metabolic fates of carbamazepine i s g lucuron idat ion . Glucur-onidated compounds can be excreted in the b i l e . Based on t h i s f a c t , the p o s s i b i l i t y that enterohepatic c i r c u l a t i o n i s occurr ing must be considered. Due to the l i p o p h i l i c i t y of carbamazepine, most of i t s uptake occurs in the small i n t e s t i n e . I f the glucuronide of carbamazepine i s excreted in the b i l e , i t i s poss ib le that i n t e s t i n a l m i c r o f l o r a are able to hydrolyze the glucuronides using glucuronidase enzymes. Th is being the case, the parent drug can be regenerated in the small i n t e s t i n e and i t can again be f ree f o r uptake. The r e s u l t w i l l be an apparently much slower e l i m i n a t i o n (the observed plateau) or even an increase in the c i r c u l a t i n g concentrat ion of the drug. This postu la t ion can and should be tested in the future . The amount of carbamazepine in the b i l e , presumably as the glucuronide conjugate, can be determined by cannulat ing the b i l e duct and c o l l e c t i n g samples of b i l e . A technique for t h i s type of procedure has been descr ibed for ra ts (Johnson and R i s i n g , 1978), wherein b i l i a r y excret ion and enterohepatic c i r c u l a t i o n can be assessed. This technique could e a s i l y be adapted for use in r a b b i t s . The excret ion of carbamazepine in the b i l e of man has been evaluated and only about 1 percent of the administered dose of carbamazepine was excreted in the b i l e (Terhaag, et a l . , 1978). Based on t h i s in format ion, enterohepatic c i r c u l a t i o n might ru led n e g l i g i b l e in man. In f a c t , i t has not been descr ibed as occurr ing to any measurable extent in man. Enterohepatic c i r c u l a t i o n cannot be ru led out in other spec ies , such as the rabb i ts being used here, p a r t i c u l a r l y s ince the b i l e measurements note the level of carbamazepine only and neglect to re fe r to conjugated WEBSTER, D. S. 39 metabol i tes . In a d d i t i o n , the i n t e s t i n a l f l o r a of the rabb i t i s very l i k e l y d i f f e r e n t from that in man, and i t may be better able to hydrolyze the conjugating connect ion. This f a c t o r would increase the importance of there being a high glucuronysyl t ransferase a c t i v i t y , s ince i f there were more conjugate excreted in the b i l e the degree to which enterohepatic c i r c u l a t i o n i s occurr ing would be more pronounced. I f one were to compare the p r o f i l e s d i s p l a y i n g a p lateau with those that show the t r a d i t i o n a l type of p r o f i l e , i t would be reasonable to estimate that 15 to 20 percent of the dose i s experiencing enterohepatic c i r c u l a t i o n in order to produce the observed p lateau. The d i f fe rences between rabb i ts in t h i s study are quite c l e a r , in that some d isp lay usable s ing le compartment concentrat ion-t ime p r o f i l e s , while others obviously do not. While the reasons for t h i s observat ion are not e n t i r e l y c l e a r , i t i s quite poss ib le that a genetic f a c t o r may be respons ib le . For example, those animals which are suspected of experiencing enterohepatic c i r c u l a t i o n may have a genetic complement that produces a greater rate of glucuronide conjugate formation ( i . e . , the a c t i v i t y of glucuronysyl t rans ferase i s g r e a t e r ) . A l t e r n a t i v e l y , those rabb i ts that have a more conventional pharmacokinetic p r o f i l e may have a slower rate of conjugation (or , poss ib ly a f a s t e r rate of o x i d a t i o n ) . Since the reac t ion forming the glucuronide conjugates i s under enzymatic c o n t r o l , i t i s qu i te poss ib le that a genetic in f luence on a c t i v i t y can e x i s t . Thus, a rabb i t producing a greater proport ion of glucuronidated metabol i tes w i l l excrete more metabol i te in the b i l e and w i l l experience a greater degree of entero-hepatic c i r c u l a t i o n . On the other hand, a rabb i t with a greater proport ion of the metabol i tes excreted in the ur ine w i l l not experience r e c i r c u l a t i o n of carbamazepine and w i l l d i sp lay the conventional s ing le compartment WEBSTER, D. S. 40 p r o f i l e . The presence of a genetic polymorphism, of the nature descr ibed above, i s not often found wi th in a s t r a i n of a given species . By d e f i n i -t i o n , a s t r a i n should have a f a i r l y high degree of genetic uni formity which would, in e f f e c t , negate the proposal presented in t h i s paragraph. Despite the supposed uni formity of a s t r a i n , i n v e s t i g a t o r s in our laboratory have been able to obta in rabb i t s , from the same s t r a i n that was used in t h i s study, that have had d i f f e r e n t i a l phenotypes with respect to rates of a c e t y l a t i o n a c t i v i t y . Th is ind icates that genetic polymorphisms do e x i s t amongst the populat ion of rabb i ts that were used in t h i s study and that the explanat ion put f o r t h i s p o s s i b l e . The apparent genetic polymorphism may a lso a r i s e from the a c t i v i t y of the hepatic monooxygenases that convert carbamazepine to carbamazepine-10, 11-epoxide. Using Figure 7 as an example, d i f fe rences in the r e l a t i o n s of the epoxide curves to the carbamazepine curves can be seen. For rabb i ts D2, D3, and D4 the concentrat ion of epoxide increases to a l e v e l that i s greater than the concentrat ion of carbamazepine at the same time and does not decrease in a manner that p a r a l l e l s the decrease of plasma carbamazepine concentrat ion . I t i s poss ib le that the animals noted above have a f a s t e r rate of o x i d a t i v e metabolism which may be due to genetic d i f fe rences in the cytochromeCs) P 4 5 0 that i s (are) responsib le f o r the metabolism of carbamazepine in i n d i v i d u a l animals. I t would be qu i te l i k e l y , i f these polymorphisms do e x i s t f o r glucuronysyl t rans ferase and f o r hepatic monooxygenases, that the resu l tant observations are due to the combined inf luences of two genetic f a c t o r s . A l t e r n a t i v e l y , i t i s poss ib le that auto- induct ion i s occurr ing over the time course of the experiment, though the time course seems to be too short fo r such an occurrence. That i s , i t i s poss ib le that carbamazepine i s able to induce i t s own metabolism in the WEBSTER, D. S. 41 rabbi ts noted. This e f f e c t may a lso have a genetic p r e d i s p o s i t i o n , s ince the apparent induct ion occurs in some animals and not in others , despite the f a c t that they were a l l administered the same dose. However an examination of the i n i t i a l rates of react ion ind icates that the former explanat ion i s more l i k e l y to be t r u e , s ince those animals d i s p l a y i n g an apparently greater rate of ox idat ive metabolism over time a lso had the f a s t e s t i n i t i a l rates of r e a c t i o n . In a d d i t i o n , i f , as stated in the i n t r o d u c t i o n , the autoinduct ion of carbamazepine metabolism can take over 3 weeks in human c h i l d r e n , the l i k e l i h o o d of i t occurr ing over a s ing le dose in rabb i t s i s s l im at best . In f a c t , S iegers and co-workers (1982) attempted to examine the carbamaze-pine autoinduct ion phenomena in rabb i ts and found no d i f f e r e n c e s in the maximum concentrat ions at the expected t . The problem with t h e i r study was that only a s ing le d a i l y oral dose (10 mg/kg) was administered and blood samples were only taken p r i o r to and 2 hours a f t e r drug admin is t ra t ion . As a r e s u l t , e s s e n t i a l l y complete c learance of carbamazepine i s allowed between doses, and any induct ive e f f e c t s may have time to reverse . These phenomena must be examined f u r t h e r , s ince any i n s i g h t can only be gained by increas ing the data base s i g n i f i c a n t l y . As t h i s d i scuss ion i n d i c a t e s , the work in t h i s f i e l d i s by no means complete. A great deal of research must be done to answer the questions that remain. One might be tempted to exp la in the observations for which F igure 7 was used as an example by c la iming t h a t , s ince the animals were fed ad l i b . , there may have been d i f f e r e n t i a l uptake of the drug, based on the food content of the g a s t r o i n t e s t i n a l t r a c t . But, i f F igures 8 and 9 are examined, i t i s obvious that s i m i l a r patterns are seen for these t r i a l s where the animals were fasted overnight p r i o r to and for at l e a s t one hour fo l lowing drug admin is t rat ion (most animals did not eat during the period of WEBSTER, D. S. 42 blood sampling a f t e r the carbamazepine was administered) . Thus, as stated prev ious ly , the data base must be expanded using the method employed in the current study, a longer time course, and a longer per iod of f a s t i n g p r i o r to the admin is t rat ion of carbamazepine. I f a s e r i e s of experiments were done under these c o n d i t i o n s , the issue of the e f f e c t of g a s t r i c contents on the r e s u l t s can be c o n c l u s i v e l y s e t t l e d . The s i t u a t i o n regarding enterohepatic c i r c u l a t i o n in the rabb i t should be examined in d e t a i l . In a d d i t i o n , the time course of carbamazepine auto- induct ion must be e l u c i d a t e d . WEBSTER, D. S. 43 2 THE INFLUENCE OF ISONIAZID ON THE IN VITRO METABOLISM OF CARBAMAZEPINE: 2.1 INTRODUCTION: The second port ion of t h i s thes i s examines the in f luence of i s o n i a z i d on carbamazepine metabolism in rat l i v e r microsomes, rather than the o r i g i n a l l y planned extension of the pharmacokinetic study to the examination of in v ivo metabolic i n t e r a c t i o n s between i s o n i a z i d and carbamazepine in the r a b b i t . The change in plans was a r e s u l t of an i l l n e s s that prevented me from working the in tens ive 12-15 hour days that would have been requ i red . Instead, an rn v i t r o study using rat l i v e r microsomes was done. The ser ies of experiments are c l o s e l y r e l a t e d to the o r i g i n a l route of progress ion, but they did not require such extremely long working days. The in v i t r o model had been developed in our laboratory by undergraduate summer students C h r i s t i a n Band and Lawrence Selby. This study was able to provide a more complete ana lys i s of the scenario than d id t h e i r pre l iminary observat ions. I t has been suggested that i s o n i a z i d i s an i n h i b i t o r of carbamazepine metabolism, as ind icated by c l i n i c a l reports wherein coadminist rat ion of normally therapeutic doses of carbamazepine and i s o n i a z i d produced signs of carbamazepine i n t o x i c a t i o n and/or i s o n i a z i d hepatotox ic i ty (Block, 1982; Wright, et a l . , 1982; Va lsa lan and Cooper, 1982; Barbare, et a l . , 1986). The observed s igns of carbamazepine i n t o x i c a t i o n inc luded headaches, nausea, vomit ing, b lurred v i s i o n , drowsiness, and confus ion. The onset of these symptoms of t o x i c i t y occurred in conjunct ion with an increase in serum carbamazepine concentrat ion to l e v e l s well above that which i s general ly considered s u f f i c i e n t fo r therapeut ic e f f e c t i v e n e s s . Thus, i t would appear v a l i d to postu late that i s o n i a z i d e i t h e r i n h i b i t s carbamazepine metabolism WEBSTER, D. S. 44 or a l t e r s the d i s t r i b u t i o n of carbamazepine. I n h i b i t i o n of metabolism i s a much more l i k e l y postu la t ion s ince d i s t r i b u t i o n tends to be a d i f fus ionary process that i s d i f f i c u l t to a l t e r . Carbamazepine i s metabolized by a hepatic microsomal system that can be r e a d i l y i n h i b i t e d (Pippenger, 1987). Drug c learance i s decreased, drug h a l f - l i v e s are prolonged, and steady state serum drug concentrat ions are e levated as a r e s u l t of i n h i b i t i o n . Symptoms of drug i n t o x i c a t i o n appear as soon as the serum concentrat ion r i s e s above the minimum toxic concentrat ion . The changes in metabolic pattern begin as soon as the i n h i b i t i n g drug i s added to the p a t i e n t ' s therapeutic regimen. Th is s i t u a t i o n occurred in the reports ind ica ted above where the pat ients were undergoing chronic carbama-zepine therapy for the contro l of se izure-caus ing d i sorders , and the admini-s t r a t i o n of i s o n i a z i d , in add i t ion to the carbamazepine, led to the observed symptoms of i n t o x i c a t i o n . Wright and co-workers (1982) reported a 45 percent decrease in carbamazepine c learance 3-5 days a f t e r i s o n i a z i d admini-s t r a t i o n . In v i t r o studies of the inf luence of i s o n i a z i d in rat l i v e r microsomes (S9 f r a c t i o n ) ind icated that the rate of metabolism of carbamazepine, in t h i s system, i s dependent on the concentrat ion of i s o n i a z i d , with the rate being slowed by greater concentrat ions of i s o n i a z i d (Webster, et a l . , 1989). Th is thes i s w i l l examine the r e s u l t s of the studies with i s o n i a z i d and those of experiments with the primary metabol i tes of i s o n i a z i d , inc lud ing acety l i s o n i a z i d , acetyl hydrazine, hydrazine, and i s o n i c o t i n i c a c i d . The goal of t e s t i n g the metabol i tes was to determine which were responsible fo r the i n h i b i t i o n of carbamazepine metabolism and, p o s s i b l y , to r e l a t e t h i s information to the production of hepatotox ic i ty by i s o n i a z i d . WEBSTER, D. S. 45 2.1.1 CARBAMAZEPINE: The development and chemistry, therapeutic uses, mechanisms of a c t i o n , b iotransformat ion, and t o x i c i t i e s of carbamazepine were discussed in the in t roduct ion to the previous sec t ion . As such, t h i s information w i l l not be repeated here. 2.1.2 ISONIAZID: Ison iaz id i s the primary drug f o r the chemotherapy of t u b e r c u l o s i s , and a l l pat ients with tubercu los i s caused by i s o n i a z i d - s e n s i t i v e s t r a i n s of tuberc le b a c i l l u s should rece ive the drug i f they can t o l e r a t e i t (Mandell and Sande, 1985). I s o n i a z i d , the hydrazide of i s o n i c o t i n i c ac id was i n t r o -duced in 1952 (Bernste in , et a l . , 1952). Despite a great deal of work s ince then, i t s mechanism of act ion has not been c o n c l u s i v e l y e l u c i d a t e d . Isonia-z i d has been tested for i t s therapeutic value in a number of d i sorders . The extensive use and t e s t i n g of i s o n i a z i d has provided the opportunity f o r the observat ion and d e s c r i p t i o n of many t o x i c i t i e s and adverse e f f e c t s , one of the most important of which i s i son iaz id - induced hepatotox ic i ty . 2 .1 .2 .1 DEVELOPMENT AND CHEMISTRY: The synthet ic tubercu los ta ts , i . e . , those which were devised in the chemical l aboratory , lend themselves r e a d i l y to c r e a t i v e manipulation and l i m i t l e s s p o t e n t i a l . For t h i s reason, the f i r s t ha l f of the twentieth century saw a great deal of synthet ic chemistry performed in an attempt to produce a t h e r a p e u t i c a l l y e f f e c t i v e agent against t u b e r c u l o s i s (Fox, 1953). WEBSTER, D. S. 46 The f i r s t compound to e x h i b i t marked vn v ivo a c t i v i t y against the tuberc le b a c i l l u s was 4,4-diaminophenylsulfone, but i t proved to be less e f f e c t i v e in man than in laboratory animals. In a d d i t i o n , i t exh ib i ted considerable t o x i c i t y . Subsequently, many attempts were made to synthesize sulfones with increased a c t i v i t y and s o l u b i l i t y and decreased t o x i c i t y . Most of the compounds that were synthesized were more soluble and less t o x i c , but they a lso lacked the necessary a c t i v i t y . In the ear ly 1940's, c e r t a i n benzoates and s a l i c y l a t e s were found to decrease the oxygen uptake and i n h i b i t the growth of tuberc le b a c i l l u s , the organism responsib le fo r t u b e r c u l o s i s . The most common of these was p-aminosa l i cy l i c a c i d , commonly known as PAS (Lehmann, 1944). Many v a r i a t i o n s in the PAS s t ructure were inves t iga ted , but none were found to be super ior to the parent compound. In the l a t e 1940's, the t u b e r c u l o s t a t i c a c t i v i t y of the thiosemicarba-zones was d iscovered as a r e s u l t of a systematic i n v e s t i g a t i o n of the s u l f a drugs in t u b e r c u l o s i s . Behnisch and h i s co-workers (1950) tested a s e r i e s of thiosemicarbazones and found them to be strongly t u b e r c u l o s t a t i c . The best known compound of the se r ies i s p-acetamido benzaldehyde thiosemicarba-zone (TbI) , which was the most ac t ive synthet ic tubercu los tat in use u n t i l the advent of the hydrazides (Fox, 1953). A ser ies of pyr id ine carboxy l i c ac id d e r i v a t i v e s were synthesized and tested in the l a t e 1940's. The t u b e r c u l o s t a t i c a c t i v i t y of n icot inamide, the parent compound of t h i s group, was discovered by Kushner and h is co-workers in 1948. This was followed by the discovery of pyrazinamide, a compound with approximately three times the t u b e r c u l o s t a t i c a c t i v i t y of nicot inamide or PAS. But, r e s i s t a n t s t r a i n s rap id ly emerged. The desire to study pyr id ine carboxy l i c ac id d e r i v a t i v e s c l o s e l y re la ted to nicotinamide WEBSTER, D. S. 47 led to attempts to prepare isonicot ina ldehyde thiosemicarbazone (Fox, 1952). Since isom'cot inoyl hydrazide ( i son iaz id) (Figure 11) i s a pyr id ine carboxy l i c ac id d e r i v a t i v e , and thus re la ted to the structures being s tud ied , i t was invest igated for tubercu los ta t i c a c t i v i t y and was found to be more ac t ive than any known substance - whether synthet ic or a n t i b i o t i c . Extensive chemical and chemotherapeutic studies showed that , unl ike 3-amino-i s o n i c o t i n i c a c i d or pyrazinamide, i som'cot inoyl hydrazide could be modif ied in many ways without abo l i sh ing i t s a c t i v i t y . 2 .1 .2 .2 THERAPEUTIC USES: Ison iaz id i s the primary drug for the chemotherapy of t u b e r c u l o s i s , and i t has been s ince i t s in t roduct ion by B e r n s t e i n ' s group in 1952. In a d d i t i o n , i s o n i a z i d has been tested f o r i t s therapeut ic value in several other d i s o r d e r s . The j u s t i f i c a t i o n f o r t e s t i n g i s o n i a z i d in d i f f e r e n t s i t u -at ions a r i s e s from the phys io log i ca l in f luences of the drug in the subject to which i t i s administered. To an extent, t h i s takes advantage of the s t ruc tura l r e l a t i o n s h i p between i s o n i a z i d and endogenous compounds. For example, i s o n i a z i d has been tested for i t s value in the treatment of Huntington disease (Perry, et a l . , 1979, 1982). In t h i s d isease, there i s a marked loss of small neurons in the caudate nucleus and putamen, most of which probably belong to a populat ion of c e l l s that u t i l i z e y-aminobutyric ac id (GABA) as an i n h i b i t o r y neurotransmitter . A marked decrease in GABA content in the a f fected areas has been observed in pat ients with the disease (Perry , et a l . , 1973). When large doses of i s o n i a z i d are given to animals, b ra in GABA content increases (Perry and Hansen, 1973; Perry, et a l . , 1974), a r e s u l t that was thought to be due to the i n h i b i t i o n of GABA-aminotrans-f e r a s e , the f i r s t of two sequential enzymes that degrade GABA in the bra in . ONHNH. WEBSTER, D. S. 48 Figure 11: Isoniazid N Figure 12: Relevant aspects of isoniazid metabolism. WEBSTER, D. S. 49 However, in a double-b l ind c l i n i c a l t r i a l (Perry, et a l . , 1982), while the cerebrospinal f l u i d GABA concentrat ions were markedly increased during i s o n -i a z i d therapy, there was a lack of c l i n i c a l improvement in most Huntington disease p a t i e n t s . In recent years , i s o n i a z i d has been examined for i t s usefulness against tremors assoc iated with m u l t i p l e s c l e r o s i s (Sabra, et a l . , 1982; Duquette, et a l . , 1985; H a l l e t t , et a l . , 1985; F ranc i s , et a l . , 1986; Bozek, et a l . , 1987). The e a r l y studies of t h i s type gave cont rad ic tory r e s u l t s that were due, in par t , to the mechanism of eva luat ion , which was subject to pat ient and observer b i a s . The l a t e r studies used techniques such as p o l a r i s e d l i g h t goniometry (Franc is , et a l . , 1986) and tremograms (Bozek, et a l . , 1987) to evaluate the e f fec t iveness of i s o n i a z i d . Po lar i sed l i g h t gonio-metry demonstrated a two to t h r e e - f o l d reduct ion of tremor when standard methods of c l i n i c a l assessment showed only marginal improvement. Bozek's group found that , although i s o n i a z i d appears to have a l im i ted therapeutic r o l e , a c l i n i c a l t r i a l i s warranted in mu l t ip le s c l e r o s i s pat ients with postural tremor. In add i t ion to those d isorders already d iscussed, i s o n i a z i d has been tested for i t s value in the therapy of Parkinson's disease (Gershanik, et a l . , 1988) and rheumatoid a r t h r i t i s (when administered with r i fampic in) (McConkey and Situnayake, 1988) 2 .1 .2 .3 MECHANISMS OF ACTION: The mechanism by which i s o n i a z i d i s e f f e c t i v e in the treatment of tuber-c u l o s i s i s unknown, but there are several hypotheses. For example, the c e l l u l a r mycolate synthetase a c t i v i t y of Mycobacterium tubercu los i s H37Ra WEBSTER, D. S. 50 has been shown to be very s e n s i t i v e to i s o n i a z i d (Wang and Takayama, 1972). a-Mycolic a c i d , a major mycolate component of Mycobacterium t u b e r c u l o s i s , i s one of a homologous se r ies of C^-Cg^ f a t t y acids conta in ing a long a l i p h a t i c cha in at the a - p o s i t i o n , a hydroxyl group at the e - p o s i t i o n , and two cyclopropane r ings (Minnikin and Polgar , 1967). These f a t t y ac ids are present in the c e l l w a l l . (Lederer, 1971). I t has been shown that i s o n i a z i d i n h i b i t s the synthesis of saturated fa t ty ac ids greater than C 2 6 and of unsaturated f a t t y acids greater than C 2 4 (Takayama, et a l . , 1975). These f a t t y acids are thought to be precursors of the mycolic a c i d s , and thus i n h i b i t i o n of t h e i r formation w i l l d i s rupt the c e l l wa l l s of growing c e l l s . Because of the s t ruc tura l s i m i l a r i t y between i s o n i a z i d and nicot inamide, many of the proposed mechanisms of ac t ion involve pathways where n i c o t i n -amide adenine d inuc leot ide (NAD+) or reduced NAD (NADH) are important intermediates. For example, r e l a t i v e l y high concentrat ions of i s o n i a z i d i n h i b i t NAD synthesis by c e l l - f r e e ext racts of both i s o n i a z i d s e n s i t i v e and r e s i s t a n t Mycobacterium tubercu los i s var . homonis H37Rv, the human v i r u l e n t s t r a i n (Sr iprakash and Ramakrishnan, 1969). In a d d i t i o n , i s o n i a z i d i s able to i n a c t i v a t e the i n h i b i t o r of NAD glycohydrolase of H37Rv, the enzyme responsible fo r the degradation of NAD. The r e s u l t i s the re lease of NADase (Gopinthan, et a l . , 1966). The i n h i b i t o r present in an i s o n i a z i d r e s i s t a n t s t r a i n of H37Rv, however, i s not inac t i va ted by i s o n i a z i d (Bekierkunst and B r i c k e r , 1967). Thus, a d i r e c t c o r r e l a t i o n e x i s t s between the l e t h a l i t y of i s o n i a z i d and the lowering of i n t r a c e l l u l a r concentrat ions of NAD. DNA l i g a s e of bac ter ia depends on NAD as a co fac tor ( O l i v e r a , 1971) and since DNA l i g a s e i s e s s e n t i a l fo r e longat ion of polydeoxynucleotide chains formed in DNA synthes is , the u n a v a i l a b i l i t y of NAD for t h i s v i t a l react ion may lead to i n h i b i t i o n of DNA synthes is . WEBSTER, D. S. 51 I t has been postulated that the b a c t e r i o s t a t i c e f f e c t of i s o n i a z i d i s re la ted to i t s a b i l i t y to complex c e r t a i n e s s e n t i a l heavy metals such as Cu and Fe. The i n h i b i t o r y e f f e c t s of i s o n i a z i d on hepatic cata lase (Middlebrook, 1954; Arora and Krishna M u r t i , 1960), on the succinoxidase system of pigeon breast muscle (Arora and Krishna M u r t i , 1954), or on the organic n i t roreductase of gram-negative b a c t e r i a (Arora, et a l . , 1959) are presumably r e l a t e d to sequestering of e s s e n t i a l metal ion moiet ies from the enzymes. 2 .1 .2 .4 BIOTRANSFORMATION: Since the in t roduct ion of i s o n i a z i d fo r the treatment of tubercu los i s in the ear ly 1950's, there has been a great deal of work done to e luc idate i t s metabolic pathway, p a r t i c u l a r l y as i t r e l a t e s to i son iaz id- induced l i v e r i n j u r y . The re levant aspects of the cur rent ly accepted scheme of the metabolic fa te of i s o n i a z i d are shown in F igure 12. The f i r s t publ ished i d e n t i f i c a t i o n of i s o n i a z i d metabol i tes occurred in the same year as the in t roduct ion of the drug, when studies in dogs i d e n t i f i e d i s o n i c o t i n i c ac id as a major metabol i te ( K e l l y , et a l . , 1952). This was fo l lowed, in 1953, by the f i r s t studies in man (Cuthbertson, et a l . , 1953), i n which n i c o t i n i c a c i d , i s o n i c o t i n i c a c i d , n icot inamide, i son icot inamide , n i c o t i n i c ac id hydrazide, i s o n i a z i d , n i c o t i n o y l g l y c i n e , and isom'cot inoyl g lyc ine were i d e n t i f i e d in u r ine . By the mid 1960's i t had been estab l i shed that a c e t y l a t i o n i s the major route of i s o n i a z i d i n a c t i v a t i o n in man, with work having been done to p u r i f y and charac ter i ze the N-acety l t ransferase enzymes responsible fo r cata lyz ing the conversion (Jenne, 1965). Production of a c e t y l i s o n i a z i d was found to be WEBSTER, D. S. 52 the step that i s s o l e l y responsib le fo r the d i f f e r e n c e s in i s o n i a z i d metabo-l ism between rapid and slow i n a c t i v a t o r s (Peters , et a l . , 1965). I t was estab l i shed that these d i f fe rences in i n a c t i v a t i o n c a p a c i t i e s are under genetic c o n t r o l . In the mid 1970's, when much work was being done with respect to i s o n i a -z id- induced hepatotox ic i ty , a -ketog lutarate i s o n i a z i d hydrazone and pyruvate i s o n i a z i d hydrazone had been i d e n t i f i e d as metabol i tes ( M i t c h e l l , et a l . , 1975). At the same t ime, hydrazide metabol i tes , p a r t i c u l a r l y acetyl hydra-z i n e , were i d e n t i f i e d as the metabol ites responsible fo r production of the " react ive intermediate" which led to hepatic i n j u r y . Hydrazines and t h e i r d e r i v a t i v e s were known to be hepatotoxins, mutagens, and carcinogens (Black and Thomas, 1970; Druckrey, 1973). Subsequently, i t was observed that acety l hydrazine i s e l iminated from the body by at l e a s t three routes ( T i m b r e l l , et a l . , 1977). F i r s t , i t i s excreted in the urine as f ree acety l hydrazine and as the a -oxog lutar i c ac id and pyruvic ac id hydrazones. Second, i t i s acety lated to d iacety l hydrazine, which i s excreted in the ur ine . T h i r d , the acetyl hydrazine i s e l iminated by metabolism by the hepatic microsomal enzyme system, a pathway that i s thought to produce the reac t ive intermediate which i s responsib le fo r the hepatotox ic i ty ( M i t c h e l l , et a l . , 1976; Nelson, et a l . , 1975). 2 . 1 . 2 . 5 ADVERSE EFFECTS: The adverse e f f e c t s of i s o n i a z i d are dose r e l a t e d , with a rate of occur-rence of 1 to 2 percent at conventional low dose therapy (3 to 5 mg/kg/day) and 15 percent at 10 mg/kg/day (Goldman and Braman, 1972). The most common react ions are rash, fever , jaund ice , and per ipheral n e u r i t i s (Mandell and WEBSTER, D. S. 53 Sande, 1985). The range of react ions to i s o n i a z i d i s quite broad, i n c l u -d i n g : h y p e r s e n s i t i v i t y react ions , such as fever , sk in erupt ions , h e p a t i t i s , and m o b i l l i f o r m , maculopapular, purpur i c , and u r t i c a r i a l rashes; hematologi-ca l react ions such as agranulocytos is , eosonophi l ia , thrombocytopenia, anemia, and v a s c u l i t i s ; and a r t h r i t i c symptoms such as back pa in , b i l a t e r a l proximal interphalangeal j o i n t involvement, and a r t h r a l g i a of the knees, elbows, and w r i s t s . There have a lso been reports of opt i c n e u r i t i s , muscle twi tch ing , d i z z i n e s s , a t a x i a , parasthes ias , stupor, tox ic encephalopathy (may terminate f a t a l l y ) , and mental abnormal i t ies such as euphor ia , t r a n s i e n t impairment of memory, separat ion of ideas and r e a l i t y , loss of s e l f - c o n t r o l , and f l o r i d psychoses (Mandell and Sande, 1985). One of the most common, and c e r t a i n l y the most extens ive ly s tud ied , of the side e f f e c t s of i s o n i a z i d i s t o x i c h e p a t i t i s , which occurs in less than 5 percent of those treated ( G i r l i n g , 1982). I t i s manifested as hepatic necros i s , with increased l e v e l s of l i v e r enzymes in the serum being ev ident . I t was f i r s t suggested, in 1974, that the species responsib le for the l i v e r necros is i s a c e t y l i s o n i a z i d (Snodgrass, et a l . , 1974). I t was l a t e r sug-gested that the hepatoce l lu la r injury i s due to the metabolic a c t i v a t i o n of acetyl hydrazine to a react ive intermediate (Nelson, et a l . , 1976; M i t c h e l l , et a l . , 1976; T i m b r e l l , et a l . , 1980). D e t o x i f i c a t i o n i s by a fur ther a c e t y l a t i o n to the non-toxic d iacety l hydrazine (Wright and T i m b r e l l , 1978). Thus, the key determinant of the t o x i c i t y of i s o n i a z i d may be the balance between metabolic a c t i v a t i o n and a c e t y l a t i o n , i . e . , between acetyl hydrazine product ion and metabolism to d iacety l hydrazine (Lauterburg, et a l . , 1985). Based on t h i s , one would expect acety la tor phenotype to play a s i g n i f i c a n t r o l e in the incidence of t o x i c i t y , with rapid a c e t y l a t o r s being at a greater r i s k ( M i t c h e l l , et a l . , 1975). But, there i s abundant c l i n i c a l evidence to WEBSTER, D. S. 54 show that the r i s k of hepatic react ions during treatment with i s o n i a z i d i s no greater in rapid than in slow acety la tors (Dickinson, et a l . , 1981; R i ska , 1976). In f a c t , some studies have shown that slower acety la tors may be at higher r i s k ( L a i , et a l . , 1972; Smith, et a l . , 1972), inc lud ing one publ ished by Lauterburg and co-workers (1985) who were involved in the studies proposing the opposite hypothesis. In support of the l a t t e r obser-vat ions , there has been a recent report in which hepatic necros is was h i s t o -l o g i c a l l y demonstrated in rabb i ts as a r e s u l t of induct ion by hydrazine i t s e l f , rather than acetyl hydrazine (Noda, et a l . , 1983). This i s of i n t e r e s t s ince hydrazine i s produced by a d i f f e r e n t metabolic pathway whose r e l a t i v e proport ion of i s o n i a z i d metabolism i s increased in slow a c e t y l a t o r s . 2.1.3 STATEMENT OF PROBLEM: As stated at the beginning of t h i s in t roduct ion , the goal of t h i s study was to examine the in f luence of i s o n i a z i d and i t s major metabol i tes on the metabolism of carbamazepine in rat l i v e r microsomes. Th is w i l l , hopefu l ly , provide a better understanding of the nature of the c l i n i c a l i n t e r a c t i o n and r e s u l t a n t t o x i c i t i e s that were noted. 2.2 EXPERIMENTAL: 2.2.1 MATERIALS: Carbamazepine, NADP+, glucose-6-phosphate, and glucose-6-phosphate dehydrogenase were obtained from Sigma Chemical Co. I s o n i a z i d , a c e t o n i t r i l e , methanol, and KC1 were obtained from BDH Chemicals. F i sher S c i e n t i f i c was the suppl ier of MgCl^, hydrazine, t r i s (hydroxymethyl) aminomethane (TRIS) b u f f e r , and NaH 2 P0 4 . Acetyl hydrazine was obtained from A l d r i c h Chemical WEBSTER, D. S. 55 Co. and i s o n i c o t i n i c ac id was obtained from Eastman Kodak Co. A c e t y l i s o n i -az id was synthesized from mater ia ls suppl ied by BDH, according to the method of von Sassen, et a l . (1985), wherein i s o n i a z i d was reacted with a f o u r - f o l d excess of a c e t i c ac id anhydride at room temperature fo r 1.5 hours with continous s t i r r i n g , and the synthesized product was r e c r y s t a l l i z e d in methanol - d ie thy l ether ( 1 : 4 ) . Male Sprague-Dawley rats (265-340g) were obtained from the Animal Care Unit of the Un ivers i ty of B r i t i s h Columbia. 2.2.2 MICROSOME PREPARATION: The microsome preparat ion and the subsequent incubat ion were based on the methods of Grasela and Rocci (1984). Rats were s a c r i f i c e d by a blow to the head, an i n c i s i o n through the neck to the spinal cord , and thorough exsanguinat ion. The mid-sect ion was exposed and the l i v e r was slowly perfused (0.1M NaHoPO^ buf fe r , pH 7.4) v ia the porta l v e i n . When the l i v e r ceased to expand, the super ior vena cava was occluded. Further perfus ion allowed the l i v e r to expand completely, a t t a i n i n g a tan c o l o u r . The l i v e r was excised and was homogenized using a Potter-Elvejhem apparatus, with a 4:1 r a t i o of homogenizing buffer (20mM TRIS; 1.15 percent KC1) to l i v e r (v:w). The homogenate was centr i fuged at 10,000 g fo r 15 minutes at 4"C using a Beckman Model JA21 r o t o r . The S9 microsome f r a c t i o n was obtained as the supernatant and kept on i ce u n t i l used. The cytochrome P ^ ^ Q content of the prepara-t i o n s was determined using the method descr ibed by Mazel (1971). In t h i s method, 2 mL of the microsome suspension was placed in each of two matched cuvettes with t e f l o n stoppers. An LKB Ultrospec 4050 spectrophotometer was WEBSTER, D. S. 5 6 used to determine the basel ine absorptions at 4 5 0 and at 4 8 0 nm. Carbon monoxide was gently bubbled in to the sample cuvette f o r 2 0 seconds, a few mi l l igrams of s o l i d sodium d i t h i o n i t e ( N a 2 S 2 0 4 ) were added, the cap was placed on secure ly , the cuvette was i n v e r t e d , and then the sample was gassed again with carbon monoxide fo r an add i t iona l 2 0 seconds. The reference was t reated only with a few mi l l igrams of sodium d i t h i o n i t e and mixed w e l l . The absorptions of each cuvette were again measured at 4 5 0 and at 4 8 0 nm. The quantity of cytochrome P ^ Q was c a l c u l a t e d from the o p t i c a l densi ty d i f fe rence ( 4 5 0 - 4 8 0 nm) and the molar e x t i n c t i o n c o e f f i c i e n t - 1 - 1 of 9 1 mM cm . Total p rote in concentrat ion was determined using the method of Bradford ( 1 9 7 6 ) . 2 . 2 . 3 INCUBATION: Each incubat ion mixture cons is ted of 1 5 0 uL of lOmM NADP+ ( 0 . 7 2 1 mM f i n a l concent rat ion) , 1 5 0 uL of 6 0 mM glucose -6-phosphate ( 4 . 3 2 7 mM f i n a l concent ra t ion) , 3 6 uL of 2 5 0 mM MgCl 2 ( 4 . 3 2 7 mM f i n a l concent ra t ion) , 1 5 0 uL of 1 0 I . U . / n i glucose -6-phosphate dehydrogenase ( 0 . 7 2 1 I.U./mL f i n a l concent ra t ion) , 3 4 uL of 6 0 mM carbamazepine ( 0 . 9 8 1 mM f i n a l concent rat ion) , and one of 6 0 nl of homogenizing b u f f e r , or 6 0 uL of 4 mM ( 0 . 1 1 5 mM f i n a l concentrat ion) or 1 7 mM ( 0 . 4 9 0 mM f i n a l concentrat ion) of i s o n i a z i d , or of one of i t s metabol i tes ( a c e t y l i s o n i a z i d , hydrazine, acetyl hydrazine, or i s o n i c o t i n i c a c i d ) . For i s o n i a z i d and each metabol i te there were three assays ( c o n t r o l , 0 . 1 1 5 mM, and 0 . 4 9 0 mM) on microsome samples from each of four male Sprague-Dawley rats (that i s , the sample s i ze was f o u r ) . The incubat ion mixtures were incubated in a shaking water bath ( 3 7 "C) fo r 4 0 minutes, with 1 0 0 \il samples being removed at 0 , 5 , 1 0 , 1 5 , 2 0 , 3 0 , and 4 0 WEBSTER, D. S. 57 minutes and being placed into microcentr i fuge tubes conta in ing 200 uL of a c e t o n i t r i l e . The samples were then centr i fuged in an Eppendorf Centr i fuge 3200 for one minute. The supernatants were removed, placed in storage v i a l s , and stored at -20°C u n t i l analysed. 2.2.4 ANALYSIS OF SAMPLES: The samples were analysed using the same apparatus, so lvents , and temperature as descr ibed prev ious ly fo r the r a b b i t pharmacokinetic study. The sole d i f f e r e n c e was that standards were run f requent ly to a l low the product ion of standard curves and the determination of concentrat ions of the samples. An example of the chromatograms obtained i s shown in F igure 13. 2.3 RESULTS: The epoxide metabolic ra t io - t ime p r o f i l e s in the S9 l i v e r microsome preparat ions at three d i f f e r e n t concentrat ions of i s o n i a z i d , a c e t y l i s o n i -a z i d , acety l hydrazine, hydrazine, and i s o n i c o t i n i c ac id are presented in F igures 14 to 18. In the t r i a l with i s o n i a z i d (Figure 14), the metabolism of carbamazepine was apparently i n h i b i t e d by i s o n i a z i d in a dose dependent manner. The l i n e s representing the three concentrat ions on the f i g u r e diverge with the progression of t ime, a very good i n d i c a t o r of the t rend. S i g n i f i c a n t d i f fe rences were observed between the blank and 0.490 mM i s o n i -az id samples at the 30 and the 40 minute marks (using s tudent ' s t - t e s t ) . There were no points d i s p l a y i n g s i g n i f i c a n t d i f f e r e n c e s in the t r i a l s with a c e t y l i s o n i a z i d (Figure 15) or acety l hydrazine (Figure 16). In f a c t , the use of three concentrat ions of a c e t y l i s o n i a z i d d id not even produce a concentrat ion re la ted pat tern . Instead, there i s some degree of v a r i a b i l i t y WEBSTER, D. S. 58 D O r •"I f M i S . . . (Nji ; CO CS" in 13: Sample chromatogram from the in vitro interact ion study. C the left is a standard sample of carbamazepine (769) and c a r b a m a z e p i n e - 1 0 . 1 1 - e p o x i d e (339) and on the right is a sample taken at 4 0 minutes. WEBSTER, D. S. 59 C B Z / I N H I N T E R A C T I O N S — ' — Control -•€>-• 0.115 mM — 0 . 4 9 0 mM INH INH _ 1.00 r N T 0 .80 -Time (min) Figure 14: Influence of isoniazid (INH) on the conversion of carbamazepine (CBZ) to carbamazepine-10,1 1 -epoxide (CE) in the S9 fraction of rat liver homogenate. Data represent mean +/— S £ M (n=4) WEBSTER, D. S. 60 C B Z / A c l N H I N T E R A C T I O N S — • — Control --©-• 0.115 mM — ± — 0.490 mM AclNH AclNH _ 0.14 r N CD U 0.12 " + 0 5 10 15 2 0 2 5 3 0 3 5 4 0 Time (min) Figure 15: Influence of acetylisoniazid (AclNH) on the conversion of carbamazepine (CBZ) to carbamazepine-10.11-epoxide (CE) in the S 9 fraction of rat liver homogenate. Data represent mean +/- S.EM (n=4) WEBSTER, D. S. 61 CBZ/AcHz INTERACTIONS — i — Control --©-• 0.115 mM — 0 . 4 9 0 mM AcHz AcHz _ 0.12 r N CD 0 5 10 15 2 0 2 5 3 0 3 5 4 0 Time (min) Figure 16: Influence of acetylhydrazine (AcHz) on the conversion of carbamazepine (CBZ) to carbamazepine-10,1 1 -epoxide (CE) in the S9 fraction of rat liver homogenate. Data represent mean +/- S.EM (n=4) WEBSTER, D. S. 62 C B Z / H z I N T E R A C T I O N S • Control 0.115 mM — A — 0.490 mM Hz Hz _ 0.14 p N CO 5 10 15 20 25 30 35 40 Time (min) F , g u r e 1 7 : Influence of hydrazine (Hz) on the conversion of carbamazepine (CBZ) to carbamazepine-10.1 1-epoxide (CE) in the S9 fraction of rat liver homogenate. Data represent mean +/- SE.M. (n=4) WEBSTER, D. S. 63 C B Z / I N A I N T E R A C T I O N S Time (min) Figure 18: Influence of isonicotinic acid (INA) on the conversion of carbamazepine (CBZ) to carbamazepine-10.11-epoxide (CE) in the S9 fraction of rat liver rK>mogenate. Data represent mean +/- S E M (n=4) WEBSTER, D. S. 64 in the r e l a t i v e p o s i t i o n s of the points at each time of measuremment. This i s not true in the case of acety l hydrazine, where the contro l sample c l e a r l y has the greatest values fo r the metabolic r a t i o over the complete time course, although there are no s i g n i f i c a n t d i f fe rences between s p e c i f i c po ints . In the cases of hydrazine (Figure 17) and i s o n i c o t i n i c ac id (Figure 18), the l i n e represent ing the contro l i s c l e a r l y d i s t i n c t from those represen-t ing incubat ion mixtures contain ing metabol i te . In f a c t , i n the hydrazine t r i a l s , there are s i g n i f i c a n t d i f fe rences at 30 and at 40 minutes, while in the i s o n i c o t i n i c ac id t r i a l s , there i s a s i g n i f i c a n t d i f fe rence observed at the 20 minute po in t . Thus, i t would appear that both hydrazine and i s o n i c o -t i n i c ac id are capable of i n h i b i t i n g the metabolism of carbamazepine. In an e f f o r t to achieve a comparison of the whole spectrum of points between doses, another type of ana lys i s was a lso attempted. A nonl inear est imat ion a n a l y s i s was performed using the Systat software package with the modell ing equation being M R = a ( l - e b t ) , where MR represents the metabolic r a t i o and t i s the t ime. The var iab les a and b are the values f o r which estimates were obtained, with a representing the t h e o r e t i c a l maximum metabolic r a t i o atta ined ( i . e . , the plateau value) and b represent ing a combined rate constant of several f i r s t order rate processes that i n f e r s the rate of exhaustion of essent ia l components in the react ion mixture. The f i r s t order process was chosen because the r e s u l t s d isp layed what appears to be a satur- at ing funct ion ing and f i r s t order funct ions are the usual observed for t h i s type of experiment. One would not expect to see a great deal of v a r i a t i o n in the value obtained f o r a , s ince the leve l of i s o n i a z i d or of one of i t s metabol i tes would not be the l i m i t i n g f a c t o r i n the r e a c t i o n . In c o n t r a s t , one would expect to see d i f fe rences in the value of WEBSTER, D. S. 65 b, s ince i f any i n h i b i t i o n i s occurr ing the rate of react ion w i l l necessar i l y be a l t e r e d . Values were obtained for a and b fo r those tes ts in which the t - t e s t s showed s i g n i f i c a n c e at one or more p o i n t s . The estimates obtained using the software package are shown in Tables II - IV. I f the numbers are examined c l o s e l y , i t i s obvious that the software package was unable to make appropr iate approximations. The values fo r a appeared to be l e s s than the values of the metabolic r a t i o at the 40 minute time p o i n t . That i s , the program seems to be ignor ing the increase in the value of the metabolic r a t i o that occurs between 30 and 40 minutes. The values of a and b fo r each ind iv idua l run were tested by ana lys i s of variance on each i n t e r a c t i o n group. By t h i s method of a n a l y s i s , s i g n i f i c a n t d i f f e r e n c e s were observed only fo r the group in which carbamazepine - i s o n i -az id i n t e r a c t i o n s were t e s t e d . For t h i s group, the a n a l y s i s of a y i e l d e d p.0.25, but fo r the a n a l y s i s of b, p,0.0005. For the t e s t s with hydrazine, the a n a l y s i s y i e l d e d 0 .10 .p .0 .05 f o r both parameters. For the t e s t s with Table I I : Estimates of a and b fo r t e s t s with i s o n i a z i d . T r i a l Parameter Control Concentration of 0.115 mM i s o n i a z i d 0.490 mM 1 a 0.041 0.041 0.037 b -6.897 -6.903 -6.892 2 a 0.052 0.053 0.046 b -6.927 -6.919 -6.896 3 a 0.043 0.034 0.032 b -6.912 -6.888 -6.875 4 a 0.042 0.044 0.044 b -6.911 -6.911 -6.907 WEBSTER, D. S. 66 Table I I I : Estimates of a and b fo r t e s t s with hydrazine. Concentration of hydrazine T r i a l Parameter Control 0.115 mM 0.490 mM 1 a 0.084 0.056 0.059 b -7.027 -6.940 -6.955 2 a 0.059 0.049 0.042 b -6.948 -6.929 -6.903 3 a 0.073 0.047 0.036 b -6.987 -6.916 -6.899 4 a 0.048 0.038 0.045 b -6.924 -6.895 -6.927 Table IV: Estimates of a and b fo r t e s t s with i s o n i c o t i n i c a c i d . Concentration of i s o n i c o t i n i c ac id T r i a l Parameter Control 0.115 mM 0.490 mM 1 a 0.069 0.049 0.043 b -6.970 -6.924 -6.923 2 a 0.040 0.036 0.046 b -6.915 -6.908 -6.926 3 a 0.060 0.043 0.045 b -6.953 -6.903 -6.914 § a 0.039 0.035 0.038 b -6.980 -6.885 -6.896 i s o n i c o t i n i c a c i d , the r e s u l t s of the a n a l y s i s gave p=0.25 and p.0.25 for a and b, r e s p e c t i v e l y . This i s somewhat unexpected. I f the f igures for the t e s t s done with each of these compounds are examined v i s u a l l y , i t i s qui te apparent that over the course of the experiment there are dose re la ted d i f f e r e n c e s developing. The problem i s that these d i f fe rences are not r e f l e c t e d in the s t a t i s t i c a l procedures presented thus f a r . WEBSTER, D. S. 67 If the p r o b a b i l i t y procedure of run ana lys i s i s employed, the r e s u l t s are somewhat d i f f e r e n t . In t h i s type of a n a l y s i s , the p r o b a b i l i t y of two points of adjacent l i n e s being d i f f e r e n t i s 0 .5 . These p r o b a b i l i t i e s can be m u l t i p l i e d over the course of the l i n e . For example, i f t h i s procedure were done on the curves from the carbamazepine/isoniazid i n t e r a c t i o n study, the p-value for the comparison of the contro l to the 0.490 mM curve would be 5 (0.5) or 0.03, s ince the points of the contro l l i n e are above the points of the 0.490 mM l i n e at 5, 15, 20, 30, and 40 minute p o i n t s . Table V: P-values from run a n a l y s i s of i n v i t r o i n t e r a c t i o n s tud ies . Treatment Control vs 0.490 mM P-value f o r : Control vs 0.115 mM 0.115 mM vs 0.490 mM Ison iaz id 0.03 0.03 0.02 A c e t y l i z o n i a z i d 0.03 0.03 0.06 Acety l hydrazone 0.02 0.02 0.06 Hydrazine 0.02 0.02 0.13 I s o n i c o t i n i c ac id 0.02 0.02 0.13 2.4 DISCUSSION: The r e s u l t s presented in t h i s study would appear to provide an j_n v i t r o conf i rmat ion of the c l i n i c a l observations that i s o n i a z i d i s able to i n h i b i t carbamazepine metabolism, as reported by Wright and co-workers (1982), by Block (1982), and by Valsalan and Cooper (1982). In a d d i t i o n , the r e s u l t s suggest that hydrazine, i s o n i c o t i n i c a c i d , and poss ib ly even acetyl hydrazine are metabol i tes of i s o n i a z i d that i n h i b i t carbamazepine metabolism by S9 l i v e r microsome f r a c t i o n s . WEBSTER, D. S. 68 I t i s not poss ib le to determine the exact nature of the i n h i b i t i o n of carbamazepine metabolism at t h i s point i n time, but the concentrat ion dependent nature of the i n h i b i t i o n would suggest a competit ive i n t e r a c t i o n , p a r t i c u l a r l y s ince i s o n i a z i d metabolism has been reported to be impaired when carbamazepine and i s o n i a z i d are coadministered in a c l i n i c a l s i t u a t i o n (Barbare, et a l . , 1986). I t i s a lso poss ib le that an i r r e v e r s i b l e i n t e r -act ion i s taking p l a c e , but a d e f i n i t e conc lus ion cannot be made without doing binding s tud ies . An i r r e v e r s i b l e i n t e r a c t i o n i s less l i k e l y , though, s ince plasma concentrat ions of carbamazepine seem to return to normal with in a few days a f t e r i s o n i a z i d admin is t rat ion i s ceased (Block, 1982; Wright, et a l . , 1982). Despite t h i s observat ion , subsequent observat ions of the e f f e c t s of i s o n i a z i d on acetaminophen metabolism suggest that i s o n i a z i d i s a s u i c i d e i n h i b i t o r of c e r t a i n cytochrome P45Q' s capable of causing a 70 percent decrease in acetaminophen metabolism (Epste in , et a l . , in p r e s s ) . The potent i n h i b i t i n g c a p a b i l i t y of i s o n i a z i d described above was not observed in the current study. The carbamazepine concentrat ion used in t h i s s e r i e s of experiments was chosen because i t provided the best response in pre l iminary studies done in the absence of any i n h i b i t i n g spec ies . The lack of a more potent d i f f e r e n c e i n the presence of i s o n i a z i d suggests that the chosen carbamazepine concentrat ion may have been too high and not a r e f l e c -t i o n of what i s occurr ing in the c l i n i c a l s i t u a t i o n . In f a c t , when compared with other studies where a s i m i l a r procedure was used, the concentrat ion used i n t h i s study were towards the high end, but not out of range. The concentrat ions of carbamazepine used in some other studies ranged from 0.05 mM (Tybr ing, et a l . , 1981) to 1.0 mM (van Boxte l , et a l . , 1977). The higher carbamazepine may have resu l ted in the systems adopting a zero-order , or c lose to zero-order , k i n e t i c p r o f i l e , with the i n h i b i t o r having very l i t t l e e f f e c t . WEBSTER, D. S. 69 When d i scuss ing these experiments, one must remain aware that the conversion of carbamazepine to carbamazepine-10, l l -epoxide i s not the only process that i s able to occur in the system. In the S9 preparat ion epoxide hydrolase, the enzyme responsible fo r the conversion of the epoxide to the d i o l , i s s t i l l present . As w e l l , the enzymes responsib le for g lucuronid-at ion and for the conversion of the epoxide to the acr idan are a lso present, but these are minor. Each of these processes i s f i r s t order with respect to the epoxide. Thus, a plateau of the metabolic r a t i o curve has to a r i s e from a balance of processes. In f a c t , i t i s the a c t i v i t y of the epoxide hydrolase that i s responsib le fo r the curve not qu i te being exponent ia l . I f the react ion had been allowed to proceed f o r long enough, i t i s quite poss ib le that the metabolic r a t i o may have decreased, s ince the r a t i o i s the consequence of a balance of several enzymic processes. Based on an examination of the s t ructures of the two compounds, one might expect the aromatic r ing s t ructure to be an important recogni t ion f a c t o r at the ac t i ve s i t e of the enzyme responsib le fo r metabolism of the drugs, provided that the mechanism of i n h i b i t i o n i s a competit ive process . I t i s poss ib le that the s ide chains of the two drugs, namely the carbamoyl side chain of carbamazepine and the hydrazide group of i s o n i a z i d , are , in p a r t , responsib le for pos i t ion ing the compounds on the enzyme. I f t h i s combination of in f luences was o c c u r r i n g , one would expect to see i n h i b i t o r y a c t i v i t y from the metabol i tes that r e s u l t from the simple breakdown of the i s o n i a z i d compound, namely hydrazine and i s o n i c o t i n i c a c i d . Such an act ion was observed. I f the mechanism of i n h i b i t i o n i s not a competit ive process, the same postu lat ions about the r e l a t i v e importance of the s t ructura l features of i s o n i a z i d and i t s metabol i tes would l i k e l y hold true for a regu-l a t o r y s i t e , rather than for the ac t ive s i t e . WEBSTER, D. S. 70 The s l i g h t e f f e c t d isp layed by acetyl hydrazine may be due to one of two reasons. F i r s t , the s t r u c t u r a l s i m i l a r i t y between acety l hydrazine and hydrazine may play a r o l e , with the presence of the acetyl group decreasing the e f fec t iveness of acety l hydrazine at i n h i b i t i n g carbamazepine metabo-l i s m . The second poss ib le reason i s that a small proport ion of the a c e t y l -hydrazine i s being converted to hydrazine in the preparat ion . Such a conversion has not y e t been descr ibed , but i t i s poss ib le that such a reac t ion may occur. A c e t y l i s o n i a z i d , on the other hand, d isplayed no apparent i n h i b i t i o n of carbamazepine metabolism. This i s l i k e l y due to the prevention of access to the required recogni t ion s i t e s by the acety l group, a f a c t o r not i n f l u e n c i n g the act ions of i s o n i a z i d or i s o n i c o t i n i c a c i d . An eva luat ion of the r e l a t i v e e f fec t i veness of each of the compounds tested for t h e i r a b i l i t y to i n h i b i t carbamazepine metabolism reveals that the hydrazine moiety i s l i k e l y to be most responsib le fo r the a c t i v i t y . I f the t r i a l s with hydrazine and i s o n i c o t i n i c ac id (Figures 15 and 16) are compared, i t i s obvious that hydrazine exerted a greater e f f e c t . Th is might lead one to surmise that the hydrazide moiety of i s o n i a z i d i s responsib le fo r a greater proport ion of the observed i n h i b i t o r y a c t i v i t y . The r e s u l t s obtained, with regard to the i d e n t i t y of the species p r i m a r i l y responsib le fo r the i n h i b i t i o n of carbamazepine metabolism, do not c o r r e l a t e well with the r e s u l t s of studies examining the tox i c e f f e c t s of i s o n i a z i d and i t s metabol i tes . In studies of i son iaz id- induced hepatoxic-i t y , acetyl hydrazine has been shown to be the metabol i te that i s p r imar i l y responsib le ( M i t c h e l l , et a l . , 1975; Nelson, et a l . , 1976). There i s evidence that acetyl hydrazine i s ox id ized by a microsomal cytochrome P ^ Q enzyme to produce the react ive acy la t ing agents in in v i t r o studies with r a t and human l i v e r microsomes (Nelson, et a l . , 1976). Subsequent r e s u l t s WEBSTER, D. S. 71 showed that the e n t i r e acetyl group of acetyl hydrazine i s trapped by c y s t e i n e , thereby e l iminat ing ketene as the r e a c t i v e a c e t y l a t i n g agent formed during the ox idat ion of acetyl hydrazine by l i v e r microsomes (Nelson, Hinson, and M i t c h e l l , 1976). Based on t h i s informat ion, acety l hydrazine would be expected to be the most e f f e c t i v e of the metabol i tes tested at i n h i b i t i n g carbamazepine metabolism, due to i t s a b i l i t y to cova lent ly bind p r o t e i n s , such as cytochrome ^ Q ' S that metabolize carbamazepine. But, i n the experiments being presented c u r r e n t l y , acety l hydrazine did not have a s i g n i f i c a n t e f f e c t . Thus, i t would appear that there i s some degree of s p e c i f i c i t y with respect to the prote in species that can be cova lent ly bound as a r e s u l t of acetyl hydrazine r e a c t i v i t y . The enzyme responsib le f o r carbamazepine metabolism would seem to be one such p r o t e i n . The most l i k e l y reason for the exc lus ion of a prote in from being cova lent ly bound by the mechanism described would be the lack of exposed cyste ine moei t ies , or at l e a s t of any that are pos i t ioned so as to a l t e r carbamazepine metabolism i f a c y l a t e d . I t cannot be confirmed i f t h i s in f a c t i s the case, s ince the s t ructure of the enzyme responsib le f o r carbamazepine epoxidat ion has y e t to be e l u c i d a t e d . I t i s reasonable to postu la te , though that there are no cyste ines present in the ac t i ve or regulatory s i t e s of the enzyme, s ince a c y l a t i o n at such a s i t e would l i k e l y lead to the i n h i b i t i o n of carbamaze-pine metabolism. The r e s u l t s obtained in t h i s study are cons is tent with a number of s tudies wherein a metabolic i n t e r a c t i o n or competit iveness has been observed between carbamazepine and other drugs with c y c l i c moeit ies that undergo hepatic ox idat ive metabolism, such as erythromycin (Barzaghi , et a l . , 1987), imipramine (Daniel and Netter , 1988), and c imet id ine (Grasela and Rocc i , 1984). The obvious route to pursue in the future would be to continue WEBSTER, D. S. 72 studying the i n t e r a c t i o n s between compounds that f i t the c r i t e r i a stated above. I t i s hopeful that information derived from these types of s tudies w i l l a s s i s t c l i n i c i a n s in making wise dec i s ions about the co-admin is t rat ion of drugs. This may r e s u l t in fewer pat ients that experience the adverse e f f e c t s that sometimes r e s u l t from drug i n t e r a c t i o n s . With respect to the system examined in the current study, i t i s necessary to examine the in te rac t ions over a wide range of carbamazepine concentra-t i o n s . I f t h i s i s done, i t w i l l be poss ib le to examine the k i n e t i c s of the react ion more thoroughly by p l o t t i n g Michaelis-Menten and Lineweaver-Burke p l o t s . This should provide more information about the nature of the i n h i b i -t i o n that i s observed, i . e . , the question of whether the i n h i b i t i o n by i s o n i a z i d i s competit ive or of a su ic ide- type should be answered. In a d d i t i o n , i f s tudies are done with chemica l ly synthesized compounds that are s p e c i f i c a l l y designed f o r s p e c i f i c types of i n t e r a c t i o n s at the a c t i v e s i t e of the enzyme, i t should be poss ib le to get a better idea about the s t ruc-ture of the ac t i ve s i t e . WEBSTER, D. S. 73 3 REFERENCES: Amols W. 1966. 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