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Genetics of drug transformation
Genetics of Drug Transformation W. KALOW
Department of Pharmacology, University of Toronto, Toronto, Canada Biotransformations of drugs are controlled or strongly affected by genetic factors. During the past few years several genetic deficiencies of drug-metabolizing reactions catalyzed by members of the family of cytochrome P-450 were observed. Choice of the appropriate drug to study and attention to urinary metabolites have been the essential ingredients for the recent discovery of genetic deficiencies of drug metabolism in man which include recessive deficiency of debrisoquine/sparteine metabolism and of mephenytoin metabolism. The clinical significance of these defects is discussed. Ethanol after metabolism to acetaldehyde is further metabolized to acetic acid by aldehyde dehydrogenase. Numerous isozymes of aldehyde dehydrogenase exist, one of which possesses a high affinity for acetaldehyde. Approximately 40% of the Oriental population lack this high affinity isozyme so that in these individuals who may have symptoms of flushing and other unpleasant effects the acetaldehyde formed is destroyed only at high plasma concentrations.
KEY WORDS: genetics; drug metabolism; debrisoquine; sparteine; mephenytoin; alcohol, ethyl; cytochrome P-450 t seems likely that all biotransformations of all drugs .are either controlled or at least strongly affected by Igenetic factors. There may be numerous gene products contributing simultaneously, directly or indirectly, to a given transformation process, giving rise to multifactorial control; or there could be a single, genetically variable enzyme leading to a Mendelian segregation in families and populations of a particular drug-metabolizing reaction (1-3). The following brief review will deal entirely with this latter, Mendelian or monogenic control of drug metabolism. The 1950's saw the first discoveries of monogenic variations in man which affected drug metabolizing capacity. B5nicke and Reif (4) in Germany, and Hughes et al. (5) in the United States, discovered independently that there were good and poor metabolizers of isoniazid. These discoveries were the consequence of efforts to associate plasma levels with antibacterial effects of the drug. Another discovery was that of a structural variant of plasma cholinesterase (6), the presence of which had a profound effect on the action of succinylcholine This discovery was a chance observation made in pursuance of a different aim. However, for a long time, no further monogenic variations of drug transformation were discovered (7). During the past few years, several genetic deficiencies of drug-metabolizing reactions were observed, reactions catalysed by the polysubstrate monooxygenases of human liver, that is, by members of the family of cytochrome P-450-containing microsomal enzymes. It is Correspondence: Dr. W. Kalow, Department of Pharmacology, University of Toronto, Toronto, ON M5S 1A8. This paper is based on a presentation at the Symposium, ~'Frontiers in Clinical Pharmacology and Therapeutic Drug Monitoring", The Hospital for Sick Children, Toronto, ON, June 6-7, 1985. 76
interesting to note why these discoveries were made recently, although intensive investigations of human drug metabolizing capacity have been going on for many years. An impediment to discovery had been the fact that the cytochrome P-450 system is a multi-enzyme system containing cytochromes with overlapping substrate specificities (8, 9), and most drugs are metabolized simultaneously in parallel along several routes. Direct elimination via bile or urine may be going on in addition. As long as the emphasis of investigations was on the determination of pharmacokinetic parameters of the parent drug such as half-life, clearance, or area under concentration-time curves, variation appeared to be multifactorial and not particularly striking. Research emphasis on the testing for urinary metabolites is more likely to lead to the discovery of monogenic defects (10), and indeed the recent discoveries all arose from measurements of metabolites. A second factor helping the discovery of clinically important deficiencies depends on finding the right drug for investigation. These right drugs would be those exceptional ones which happen to be metabolized by a single member of the cytochrome family of enzymes, at least predominantly so. Debrisoquine, which was a tool for discovery, and which will be discussed shortly, is a predominantly mono-metabolized substrate (11-13), and thus, variability in its metabolism is due to a single gene defect (14). On the other hand, the barbiturate amobarbital can be easily shown to be hydroxylated by at least two cytochromes (15); an absence of both of these should be a rare event and in any case has never been observed. In short, choice of the right drug and attention to urinary metabolites have been the ingredients for the recent discovery of genetic deficiencies of drug metabolism in man. We will now take a closer look at the two most important of these recently discovered metabolic defects.
Recessive deficiency of debrisoquine-sparteine metabolism Let me give you first a brief summary of some salient facts which will be my point of departure. The defect was discovered independently by Eichelbaum in Germany when investigating sparteine (16, 17) and by Smith, Idle and their colleagues in Britain when investigating debrisoquine (18). The British group was particularly active in following up their discovery and in freely publishing their observations, so that the debrisoquine story is better known than that of sparteine. Debrisoquine is an antihypertensive drug of HoffmannLaRoche sold in Canada under the trade name of Declinax ® (the drug has not been available in the United States). Sparteine was at some time important CLINICAL BIOCHEMISTRY,VOLUME 19, APRIL 1986
GENETICS OF DRUG TRANSFORMATION
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240
2.50
540
720
1440 I ~ l t ]
Figure 2 - - Plasma sparteine concentrations. Levels found in a metabolizer (lower curve) and a non-metabolizer (upper curve) after intravenous administration of 200 mg sparteine sulfate. (As demonstrated separately for metabolizers of sparteine, plasma concentrations after oral and intravenous administration are very similar). Eichelbaum et al. (25), with permission of authors and Eur. J. Clin. Pharmacol.).
8
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Figure 1 - - Plasma debrisoquine concentrations. Mean and standard deviations in 4 extensive metabolizers (open circles) and 3 poor metabolizers (closed circles) following a 10 mg oral dose. (Sloan et al. (24), with permission of authors and Br. J. Clin. Pharmacol.). in North America as an oxytocic but about 7% of w o m e n experienced serious overdose effects including fetal mortality (19) so t h a t the drug is no longer used for t h a t purpose. I will l a t e r t a l k about the m a n y other drugs affected by this deficiency. All pertinent information is compatible with the notion t h a t the deficiency represents a structural alteration r a t h e r t h a n the absence of one particular cytochrome (20-22). Almost 10% of Europeans have this defect but it is distinctly more r a r e in Egypt and Saudi Arabia (23). In order to u n d e r s t a n d the clinical significance of the defect, a closer look at the model drugs debrisoquine and sparteine is worthwhile. Although both drugs are strongly affected by this metabolic defect, the w a y they are affected is completely different. Figure 1 shows p l a s m a levels of poor and extensive metabolizers of debrisoquine after an oral intake of a small dose of the drug (24). There is a striking difference in p l a s m a level but the shapes of the time concentration curves are the same. After the same dose, poor metabolizers of debrisoquine have a much higher plasma concentration t h a n extensive metabolizers but the disappearance rates from p l a s m a are not altered. This p a t t e r n m e a n s t h a t the difference is one of presystemic metabolism, often referred to as first-pass effect. In other words, in extensive metabolizers m u c h of the drug is destroyed when entering the liver from the portal vein, and before it reaches general circulation. One could say t h a t there is a grossly reduced bioavailability of the drug in most people who .are extensive metabolizers while poor metabolizers h a v e full bioavailability. The antihypertensive effect of the drug CLINICAL BIOCHEMISTRY, VOLUME 19, APRIL 1986
is related to its p l a s m a concentration. Hence, poor metabolizers h a v e the full effects from this drug, but these full effects a p p e a r to be excessive because they occur only in relatively few people. The p l a s m a levels explain why, even after a single dose of the drug, the clinical responses of poor and extensive metabolizers are very different. Figure 2 shows p l a s m a concentration time curves of sparteine (25). The upper curve is t h a t of a poor metabolizer, the lower curve of an extensive metabolizer, who both received the same small oral dose of the drug. The p e a k p l a s m a levels are nearly identical in these two subjects but the subsequent half-lives or elimination r a t e s are very different. In this case, the same single dose of the drug would have about the same intensity of effect but the duration of effect would differ. The m a i n danger of this drug for a poor metabolizer would be its tendency to cumulate on repeated intake of doses suitable for extensive metabolizers. The frequencies of poor metabolizers of debrisoquine h a v e been tested in a variety of populations (23). Figure 3 shows a frequency distribution in a British population (14). The metabolizing capacity of each person is expressed as a metabolic ratio, t h a t is, as a ratio of the amounts of p a r e n t drug divided by those of metabolites excreted in urine during eight hours. Therefore, the poor metabolizers are indicated by the black bars at the right h a n d end of the distribution curves; they represent about 8% of the populations. T h e r e a p p e a r to be very few poor metabolizers a m o n g Arabs (26, 27). It turned out recently t h a t the poor metabolizers of debrisoquine in G h a n a are not poor metabolizers of sparteine (28, 29). In Caucasian populations these two defects went together regularly. Also, in vitro studies of h u m a n liver indicate t h a t it is one enzyme t h a t metabolizes both drugs (20, 22, 30). Hence, the d e b r i s o q u i n e - s p a r t e i n e dissociation in G h a n a suggests t h a t there are in existence probably not two kinds 77
KALOW TABLE 1 Drugs Whose Biotransformation is not Impaired in Poor Metabolizers of Sparteine and/or Debrisoquine
50-
40.
Acetanilide Antipyrine Amobarbital Caffeine Carbocysteine
¢' 30"
Clozapin Guanethidine Mephenytoin Metiamide Methaqualone
Nicotine Phenytoin Sulphamethazine Tolbutamide
"6 20. o z
TABLE 2 Drugs Whose Biotransformation is Impaired in Poor Metabolizers of Debrisoquine -1"0 Loglo melabolic ratio
10
2'0
Figure 3 - - Frequency distribution of log metabolic ratio of debrisoquine in a British population. The metabolic ratio refers to the 8-hour urinary excretion of debrisoquine per that of 40H-debrisoquine. The population consisted of 258 unrelated white subjects. The poor metabolizers are represented by the black bars. (Price Evans et al. (14), with permission of authors and J. Med. Genet.). of this particular cytochrome, giving rise to poor and extensive metabolizers among Europeans, but that there is a different deficiency variant in Ghana. It seems to me that Chinese subjects tend to have again a somewhat different allele, requiring not only a reinvestigation of poor metabolizers but also of extensive metabolizers (23). This is a matter of current investigations, and further detail is not yet warranted. The point is that we cannot take for granted that metabolizing capacities observed in one population will be exactly the same in another.
Clinical significance of the d e b r i s o q u i n e sparteine metabolic defect Clinical investigations have shown that the fate of many drugs has nothing to do with poor or extensive metabolizing capacity for debrisoquine or sparteine. Table 1 shows a list of such drugs. Of these mephenytoin (also called Mesantoin ®) is subject to a separate defect to which I will refer later on. The presence of phenytoin (that is, Diphenylhydantoin or Dilantin ®) on this list of negatives (31-33) deserves emphasis as it was postulated at some time that its metabolism was covariant with that of debrisoquine, but this initial claim proved to be incorrect. Table 2 shows the list of drugs which are affected by this particular defect (34). Let me comment on some items of this list. Debrisoquine and sparteine are the prototype drugs but neither is available in the U.S.A. There are tests underway to see whether the old antitussive drug dextromethorphan could be used in the future for phenotyping (35). Dextromethorphan is ordinarily used as a component of cough mixtures but it might be made available for use on its own. Another candidate as a phenotyping probe is methoxyphenamine (36). Most strongly affected by the defect was the antiarrhythmic drug perhexiline (37). Elimination of that 78
Amiflamine Amitriptyline Desipramine Nortriptyline Dextromethorphan Encainide
Guanoxan Captopril Alprenolol Bufuralol Metoprolol Propranolol
Timolol Methoxyphenamine Penicillamine Perhexiline Phenformin Sparteine
drug depends almost completely on the debrisoquine metabolizing cytochrome. In a non-metabolizer, virtually every molecule of that drug tends to stay in the body, thus accumulating during therapy and eventually causing disastrous consequences, either in the form of liver disease or nerve damage. The drug has been withdrawn from the market so that the defect has lost its clinical importance for perhexiline, except for past cases with persisting damage. Phenformin (38) and sparteine (19) have been withdrawn. Debrisoquine is used rarely. Thus, the clinical significance of the defect for these drugs has become almost nil. However, this elimination of clinical significance was achieved at a high therapeutic cost, namely the elimination of some uniquely valuable drugs whose serious side effects were restricted to a group of independently definable subjects. For amiflamine (39), captopril (40), the amphetamines and methoxyphenamine, as well as penicillamine, additional investigations on the clinical significance have to be awaited (34). Of interest is the new antiarrhythmic encainide (41) which requires biological activation in order to become highly effective. This activation is due to the debrisoquine metabolizing enzyme and does not take place in non-metabolizers in whom the normal dose of the drug is therefore insufficient. For the remaining drugs in this list, an undoubted importance of the defect has been shown for metoprolol (42). The importance has been demonstrated by plasma level determinations, by metabolite analysis, and by measuring its therapeutic effects in terms of reduction in exercise heart rate. There is good reason for the suspicion that plasma levels in non-metabolizers may be high enough to reduce the benefit of its specificity for blocking beta1 receptors; one should therefore postulate a disproportionally increased danger of metoprolol in asthmatics. On the other end of the scale is propranolol: some of its metabolism is dependent on the debrisoquine metabolizing enzyme but other pharmacokinetic factors determine its intensity and duration of action (43). The clinical significance of this defect for propranolol thus is minimal. For the other three beta CLINICAL BIOCHEMISTRY,VOLUME 19, APRIL 1986
GENETICS OF DRUG TRANSFORMATION 10 8
Caucasians (n = 118)
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Chinese (n = 39)
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Figure 4 -- Frequency distribution of urinary excretion ofphydroxymephenytoin in three populations. The abscissa indicates the 12-hour excretion of this metabolite of mephenytoin in log percent of ingested mephenytoin. All three populations are Canadian, of ethnic origin as indicated. (Ref 56 with permission of the Br. J. Clin. Pharmacol.). blockers named here, the consequences of poor metabolizing capacity are probably all in between those for metoprolol and propranolol. Exact comparisons are not yet available for alprenolol. For the fate of timolol (42), the influence of the debrisoquine/sparteine metabolic defect was roughly 2/3 as strong as for that of metoprolol. The defect is a strong determinant for the metabolism of bufuralol (44), and there is stereoselectivity of metabolism as for metoprolol. However, some bufuralol metabolites are biologically active so that the clinical consequences of deficient metabolism seem to be less incisive than for metoprolol (42). There are two interesting biochemical sidelines: first, metoprolol undergoes three separate biotransformations; two of these, the alpha hydroxylation and the O-dealkylation, are deficient in poor metabolizers (42). The same pattern is found in the triple metabolism of methoxyphenamine (36). It seems to be true that - - in the cytochrome P-450 system - - there can be one enzyme acting on one substrate to form two different products. Second, metoprolol is a racemate of which the (S)-enantiomer provides all or most of the beta blockade (45). Extensive metabolizers eliminate most rapidly the inactive (R)-enantiomer so that the plasma level represents mostly the highly active moiety. The R - S enantiomeric ratio is reversed in poor metabolizers. Hence, a given plasma level of the drug, measured without attention to the optical isomerism, represents less pharmacological activity in poor than in extensive metabolizers. For amitriptyline the defect is not very important (46). However, for desipramine and nortriptyline there is a substantial difference in drug elimination between CLINICALBIOCHEMISTRY,VOLUME 19, APRIL 1986
extensive and poor metabolizers of debrisoquine (47). The anti-depressive action of these drugs depends critically on the plasma level in the sense that too much or too little prevents therapeutic benefit. There is every reason to suspect that, prior to long-term treatment with these antidepressants, phenotyping of the patients with debrisoquine may well be worthwhile as a means to simplify dose adjustments - - at least in Caucasian patients. The debrisoquine-sparteine metabolizing defect may have clinical significance from an entirely different point of view. First, in vitro screening tests with potential inhibitors of debrisoquine or sparteine by human liver preparations indicated quinidine is an extraordinarily strong inhibitor of this enzyme (48). What, if anything, this means for the metabolism of quinidine itself remains to be seen. In the meantime, it has become clear that patients receiving quinidine are susceptible to enzyme inhibition, so much so that they behave metabolically like poor metabolizers of debrisoquine (49). In short, all drugs known to be affected by the genetic defects of debrisoquine hydroxylation may also have inhibited metabolism in recipients of quinidine. Finally, there is a possibility of an association between susceptibility to bronchial carcinoma and extensive metabolizing phenotype for debrisoquine (50). There is no biochemical rationale at the present time in so far as none of the known precarcinogens were metabolically activated by preparations of human or rat liver (51); perhaps the right precarcinogens have not yet been tested. On the other hand, Cartright in Britain could not find any association between debrisoquine metabolism and chemically induced bladder cancer (52). Further research on this metabolic defect deserves watching.
The defect of mephenytoin metabolism The anticonvulsant mephenytoin is a racemic drug of which the two enantiomers are normally undergoing different biotransformations (53-55). The S-isomer is selectively hydroxylated and rapidly eliminated. Thereby, the R-isomer is left over and undergoes a slow demethylation to an active metabolite which has a plasma half-life of many days. This slow elimination of the R-isomer causes its accumulation in plasma and it thereby becomes the therapeutically active moiety of the drug. If the hydroxylation reaction of the S-isomer is missing, both isomers are demethylated and accumulate, causing overdose effects. Thus, the defect is important for the relatively few epileptics who receive mephenytoin. The deficiency of hydroxylation capacity for mephenytoin is an autosomal recessive trait, like that for debrisoquine, but the two defects clearly involve different genes. Whether they are on the same chromosome and thereby linked is not yet known. Extensive data on mephenytoin hydroxylation in other than Caucasian populations are not yet available. However, a recent observation which is indicated in Figure 4 suggests a high frequency of non-metabolizers of mephenytoin in Japan, that is, 22% in a small sample 79
KALOW of Japanese vs 5% of Caucasians in the Toronto population (56). This is a significant difference. In the m e a n t i m e we received confirmation of our observation in form of an abstract, estimating 24% of poor metabolizers of mephenytoin in J a p a n (57). There is as yet no evidence that any drug other t h a n mephenytoin is metabolized by the same enzyme t h a t hydroxylates S-mephenytoin. However, Inaba et al. (58) have screened a large number of drugs for competitive inhibition of the mephenytoin hydroxylase of h u m a n liver. The following items represent selected data from this study. The high Ki of phenytoin indicates very weak binding to the mephenytoin hydroxylase as we should expect from the fact that the metabolism of phenytoin and of mephenytoin are clinically unrelated (55). Tranylcypromine is able to bind quite strongly to this genetically variable enzyme, and so it might be a substrate. Of interest is the binding capacity of the two benzodiazepines: flurazepam has been shown capable of binding to both the mephenytoin and the sparteine metabolizing enzyme. This raises the interesting possibility t h a t some of the different metabolic pathways of t h a t drug may depend on these two cytochromes. It is certainly worth investigating whether persons with the double defect, that for mephenytoin hydroxylation and t h a t for debrisoquine-sparteine hydroxylation, are particularly sensitive to flurazepam. If all estimates are correct, about 1% of the Japanese population might be expected to carry both defects.
due to the genetic lack of aldehyde dehydrogenase isozyme-I, the high-affinity isozyme (62, 63). Flushing subjects have much higher acetaldehyde levels in blood t h a n non-flushing controls, while ethanol levels tend to be identical. Second, Harada et al. investigated 175 Japanese alcoholics and almost all possessed the aldehyde dehydrogenase-I, while this isozyme was absent in 4 1 - 4 8 % of the various control groups (64). In short, a person lacking the high affinity form of aldehyde dehydrogenase is not likely to become an alcoholic. Since this deficiency is relatively rare in Western populations, it cannot be the main explanation for genetic variation in the occurrence of alcoholism in North America. However, it could well be a major factor in other countries.
Outlook and conclusions This account of recent discoveries was exclusively concerned with drug oxidations, that is, the most important category of ~¢phase I reactions". The past history of these discoveries promises further, similar observations. One conclusion to be drawn is that striking biochemical deficiency of drug metabolizing enzymes may or may not have important clinical consequences. This brief review did not cover any of the new developments in research on conjugation CPhase II") reactions which also find new pharmacogenetic attention.
Alcohol sensitivity There are two main areas of inter-individual and inter-ethnic differences in respect to alcohol: First, the susceptibility to alcoholism and second, the appearance of flushing and other unpleasant effects of even small doses of ethanol in some people. I believe it is fair to say that in both respects, past investigations of alcohol dehydrogenase and its genetic variants have proved disappointing. However, new attention has to be paid to aldehyde dehydrogenase (59-60). We recall that ethanol is converted by alcohol dehydrogenase (61) into acetaldehyde which in t u r n is metabolized to acetic acid by aldehyde dehydrogenase. Acetaldehyde is a substance of well-known toxicity (62). There are several isozymes of aldehyde dehydrogenase with varying affinities for both substrate and cofactor. One isozyme has a particularly high affinity for acetaldehyde (59). If this isozyme is present, the aldehyde never reaches high blood levels. If this enzyme is missing, or if it is inhibited, the acetaldehyde will also be destroyed but only at a high plasma level. It happens t h a t approximately 40% of most Oriental populations lack this high-affinity-isozyme of'aldehyde dehydrogenase. This was first established with autopsy material. However, as Goedde and his co-workers have shown, the presence of this crucial isozyme of aldehyde dehydrogenase can also be assessed through the study of hair roots (59) so that the relationship between aldehyde dehydrogenase activity and biological effects of ethanol can be investigated in individual subjects. To date, these studies have yielded two striking results. First, the circulatory effects of ethanol like flushing are 80
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GENETICS OF DRUG TRANSFORMATION 11. Kahn GC, Boobis AR, Murray S, Brodie MJ, Davies DS. Assay and characterisation of debrisoquine 4-hydroxylase activity of microsomal fractions of human liver. Br J Clin Pharmacol 1982; 13: 637-45. 12. Inaba T, Nakano M, Otton SV, Mahon WA, Kalow W. A human cytochrome P-450 characterized by inhibition studies as the sparteine-debrisoquine monooxygenase. Can J Physiol Pharmacol 1984; 62: 860-2. 13. Distlerath LM, Guengerich FP~ Characterization of a human liver cytochrome P-450 involved in the oxidation of debrisoquine and other drug antibodies raised to the analogous rat enzyme. Proc Natl Acad Sci USA 1984; 81: 7348-52. 14. Price Evans DA, Mahgoub A, Sloan TP, Idle JR, Smith RL. A family and population study of the genetic polymorphism of debrisoquine oxidation in a white British population. J Med Gen 1980; 17: 102-5. 15. Reilly PA, Tang BK, Stewart DJ, Kalow W. The occurrence of two hepatic microsomal sites for amobarbital hydroxylation. Can J Physiol Pharmacol 1983; 61: 67- 71. 16. Eichelbaum M. Ein neuentdeckter Defekt im Arzneimittelstoffwechsel des Menschen: Die fehlende n-Oxydation des Spartein. Bonn: Habilitations schrift, 1975. 17. Eichelbaum M, Spannbrucker N, Steincke B, Dengler HJ. Defective N-oxidation of sparteine in man: A new pharmacogenetic defect. Eur J Clin Pharmacol 1979; 16: 183-7. 18. Mahgoub A, Dring LG, Idle JR, Lancaster R, Smith RL. Polymorphic hydroxylation of debrisoquine in man. Lancet 1977; 2: 584-6. 19. Newton BW, Benson RC, McCarriston CC. Sparteine sulfate: a potent capricious oxytocic. A m J Obstet Gynecol 1966; 94: 234-41. 20. Guengerich FP, Distlerath LM, Reilly PEB, et al. Human liver cytochromes P-450 involved in polymorphisms of drug oxidation. Xenobiotica (in press). 21. Meyer UA. Genetic variants of hepatic microsomal polysubstrate monooxygenases (cytochrome P-450). In: Brunner H, Thaler H, Eds. Hepatology: A Festschrift for Hans Popper. Pp. 75-8. New York: Raven Press, 1985. 22. Distlerath LM, Reilly PEB, Martin MV, Davis GG, Wilkinson GR, Guengerich FP. Purification and characterization of the human liver cytochromes P-450 involved in debrisoquine 4-hydroxylation and phenacetin O-deethylation, two prototypes for genetic polymorphism in oxidative drug metabolism. J Biol Chem 1985; 260: 9057-67. 23. Kalow W. Pharmacogenetics and Anthropology. In Lemberger L, Reidenberg MM, Eds. Proceedings of the Second World Conference on Clinical Pharmacology and Therapeutics. Pp. 264-86. Am Soc Pharmacol Exp Ther, 1983.
24. Sloan TP, Lancaster R, Shah RR, Idle JR, Smith RL. Genetically determined oxidation capacity and the disposition of debrisoquine. Br J Clin Pharmacol 1983; 15: 443-50. 25. Eichelbaum M, Spannbrucker N, Dengler HJ. Influence of the defective metabolism of sparteine on its pharmacokinetics. Eur J Clin Pharmacol 1979; 16: 189-94. 26. Islam SI, Idle JR, Smith RL. The polymorphic 4-hydroxylation of debrisoquine in a Saudi Arab population. Xenobiotica 1980; 10: 819-25. 27. Mahgoub A, Idle JR, Smith RL. A population and familial study of the defective alicyclic hydroxylation of debrisoquine among Egyptians. Xenobiotica 1979; 9: 51-6. 28. Eichelbaum M, Woolhouse EM. Inter-ethnic difference in sparteine oxidation among Ghanaians and Germans. Eur J Clin Pharmacol 1985; 28: 79-83. 29. Woolhouse NM, Eichelbaum M, Oates NS, Idle JR, Smith RL. Dissociation of co-regulatory control of debrisoquineCLINICALBIOCHEMISTRY, VOLUME 19, APRIL 1986
phenformin and sparteine oxidation in Ghanaians. Clin Pharmacol Ther 1985; 37: 512-21.
30. Inaba T, Nakano M, Otton SV, Mahon WA, Kalow W. A human cytochrome P-450 characterized by inhibition studies as the sparteine-debrisoquine monooxygenase. Can J Physiol Pharmacol 1984; 62: 860-2. 31. Sloan TP, Idle JR, Smith RL. Influence of DH/DL alleles regulating debrisoquine oxidation on phenytoin hydroxylation. Clin Pharmacol Ther 1981; 29: 493-7. 32. Kadar D, Fecycz D, Kalow W. The fate of orally administered [4-14C]phenytoin in two healthy male volunteers. Can J Physiol Pharmacol 1983; 61: 403-7. 33. Roots I, Otte F, Berchtold C, Heinemeyer G, Schmidt D, Cornaggia C. Debrisoquine phenotyping in epileptic patients treated with phenytoin and carbamazepine. Biochem Pharmacol 1985; 34: 447-8. 34. Lennard MS, Ramsay LE, Silas JH, Tucker GT, Wood HF. Protecting the poor metabolizer: clinical consequences of genetic polymorphism of drug oxidation. Pharm Intern 1983; 4: 61-5. 35. Kfipfer A, Schmid B, Preisig R. Dextromethorphan as a safe probe for debrisoquine hydroxylation polymorphism. Lancet 1984; 2: 517-8. 36. Roy SD, Hawes EM, McKay G, Korchinski ED, Midha KK. Metabolism of methoxyphenamine in extensive and poor metabolizers of debrisoquin. Clin Pharmacol Ther 1985; 38: 128-33. 37. Cooper RG, Evans DAP, Whibley EJ. Polymorphic hydroxylation of perhexiline maleate in man. J Med Gen 1984; 21: 27-33. 38. Oates NS, Shah RR, Idle JR, Smith RL. Influence of oxidation polymorphism kinetics and dynamics. Clin Pharmacol Ther 1983; 34: 827-34. 39. Alvan G, Grind M, Graffner C, Sjoqvist F. Relationship of N-demethylation of amiflamine and its metabolite to debrisoquine hydroxylation polymorphism. Clin Pharmacol Ther 1984; 36: 515-9. 40. Oates NS, Shah RR, Drury PL, Idle JE, Smith RL. Captopril-induced agranulocytosis associated with an impairment of debrisoquine hydroxylation. Br J Clin Pharmacol 1982; 14: 3129. 41. Wang T, Roden DM, Wolfenden HT, Woosley RL, Wood AJJ, Wilkinson GR. Influence of genetic polymorphism on the metabolism and disposition of encainide in man. J Pharmacol Exp Ther 1984; 228: 605-11. 42. Lennard MS. Oxidation phenotype and the metabolism and action of beta-blockers. Kiln Wochenschr 1985; 63: 285-92. 43. Raghuram TC, Koshakji RP, Wilkinson GR, Wood AJJ. Polymorphic ability to metabolize propranolol alters 4-hydroxypropranolol levels but not beta blockade. Clin Pharmacol Ther 1984; 36: 51-6. 44. Minder EI, Meier PJ, Mfiller HK, Minder C, Meyer UA. Bufuralol metabolism in human liver: a sensitive probe for the debrisoquine-type polymorphism of drug oxidation. Eur J Clin Invest 1984; 14: 184-9. 45. Lennard MS, Tucker GT, Silas JH, et al. Differential stereoselective metabolism of metoprolol in extensive and poor debrisoquin metabolizers. Clin Pharmacol Ther 1983; 34: 732-7. 46. Balant-Gorgia AE, Schulz P, Dayer P, et al. Role of oxidation polymorphism on blood and urine concentrations of amitriptyline and its metabolites in man. Arch Psychiatr Nervenkr 1982; 232: 215-22. 47. Sj6qvist F, Bertilsson L. Clinical pharmacology of antidepressant drugs: pharmacogenetics. In: Usdin E, et al., Eds. Frontiers in Biochemical and Pharmacological Research in Depression. Pp. 359-72. New York: Raven Press, 1984.
48. Otton SV, Kalow W, Seeman P. High affinity of quinidine 81
KALOW
49.
50. 51. 52. 53.
54. 55. 56.
57.
82
for a stereoselective microsomal binding site as determined by a radioreceptor assay. Experientia 1984; 40: 973. Leeman T, Dayer P, Fabre J, Meyer UA. Quinidine treatment mimicks the debrisoquine "poor metaboliser" phenotype of drug oxidation. Eur J Clin Pharmacol; in press. Ayesh R, Idle JR, Ritchie JC, Crothers MJ, Hetzel MR. Metabolic oxidation phenotypes as markers for susceptibility to lung cancer. Nature 1984; 312: 160-70. Hunt PG. I n vitro characterization of sparteine monooxygenases present in rat liver. University of Toronto, Thesis, 1984. Cartwright RA, Philip PA, Rogers HJ, Glashan RW. Genetically determined debrisoquine oxidation capacity in bladder cancer. Carcinogenesis 1984; 5: 1191-2. Ktipfer A, Desmond PV, Schenker S, Branch RA. Stereoselective metabolism and disposition of the enantiomers of mephenytoin during chronic oral administration of the racemic drug in man. J Pharmacol Exp Ther 1982; 221: 590-7. Kiipfer A, Preisig R. Pharmacogenetics ofmephenytoin: a new drug hydroxylation polymorphism in man. Eur J Clin Pharmacol 1984; 26: 753-9. Kalow W. The genetic defect of mephenytoin hydroxylation. Xenobiotica (in press). Jurima M, Inaba T, Kadar D, Kalow W. Genetic polymorphism of mephenytoin p(4')-hydroxylation: difference between Orientals and Caucasians. Br J Clin Pharmacol 1985; 19: 483-7. Jacqz E, Goto F; Nakamura K, Wilkinson GR, Branch RA.
58.
59.
60. 61.
62. 63. 64.
Interethnic difference in polymorphism of s-mephenytoin hydroxylation between Japanese and Caucasians. Clin Pharmacol Ther 1985; 37: 202. Inaba T, Jurima M, Mahon WA, Kalow W. In vitro inhibition studies of two isozymes of human liver cytochrome P-450; Mephenytoin p-hydroxylase and sparteine monooxygenase. A m Soc Pharmacol Exp Ther 1985; 13: 443-8. Goedde HW, Agarwal DP, Harada S. The role of alcohol dehydrogenase and aldehyde dehydrogenase isozymes in alcohol metabolism, alcohol sensitivity, and alcoholism. Isozymes: Current Topics in Biological and Medical Research 1983; 8: 175-93. Von Wartburg J-P, Biihler R. Biology of Disease: Alcoholism and aldehydism: new biomedical concepts. Lab Invest 1984; 50: 5-15. Von Wartburg JP, Biihler R, Maring J-A, Pestalozzi D. The polymorphisms of alcohol and aldehyde dehydrogenase and their significance for acetaldehyde toxicity. Pharmacol Biochern Behav 1983; 18: 123-5. Von Wartburg JP. Acetaldehyde. In: Sandler M, Ed. Psychopharmacology of Alcohol. New York: Raven Press, 1980. Mizoi Y, Ijiri I, Tatsuno Y, et al. Relationship between facial flushing and blood acetylaldehyde levels after alcohol intake. Pharmacol Biochem Behav 1978; 10: 303-11. Harada S, Agarwal DP, Goedde HW, Tagaki S, Ishikawa B. Possible protective role against alcoholism for aldehyde dehydrogenase isozyme deficiency in Japan. Lancet 1982; 2: 628.
CLINICAL BIOCHEMISTRY, VOLUME 19, APRIL 1986
Department of Pharmacology, University of Toronto, Toronto, Canada Biotransformations of drugs are controlled or strongly affected by genetic factors. During the past few years several genetic deficiencies of drug-metabolizing reactions catalyzed by members of the family of cytochrome P-450 were observed. Choice of the appropriate drug to study and attention to urinary metabolites have been the essential ingredients for the recent discovery of genetic deficiencies of drug metabolism in man which include recessive deficiency of debrisoquine/sparteine metabolism and of mephenytoin metabolism. The clinical significance of these defects is discussed. Ethanol after metabolism to acetaldehyde is further metabolized to acetic acid by aldehyde dehydrogenase. Numerous isozymes of aldehyde dehydrogenase exist, one of which possesses a high affinity for acetaldehyde. Approximately 40% of the Oriental population lack this high affinity isozyme so that in these individuals who may have symptoms of flushing and other unpleasant effects the acetaldehyde formed is destroyed only at high plasma concentrations.
KEY WORDS: genetics; drug metabolism; debrisoquine; sparteine; mephenytoin; alcohol, ethyl; cytochrome P-450 t seems likely that all biotransformations of all drugs .are either controlled or at least strongly affected by Igenetic factors. There may be numerous gene products contributing simultaneously, directly or indirectly, to a given transformation process, giving rise to multifactorial control; or there could be a single, genetically variable enzyme leading to a Mendelian segregation in families and populations of a particular drug-metabolizing reaction (1-3). The following brief review will deal entirely with this latter, Mendelian or monogenic control of drug metabolism. The 1950's saw the first discoveries of monogenic variations in man which affected drug metabolizing capacity. B5nicke and Reif (4) in Germany, and Hughes et al. (5) in the United States, discovered independently that there were good and poor metabolizers of isoniazid. These discoveries were the consequence of efforts to associate plasma levels with antibacterial effects of the drug. Another discovery was that of a structural variant of plasma cholinesterase (6), the presence of which had a profound effect on the action of succinylcholine This discovery was a chance observation made in pursuance of a different aim. However, for a long time, no further monogenic variations of drug transformation were discovered (7). During the past few years, several genetic deficiencies of drug-metabolizing reactions were observed, reactions catalysed by the polysubstrate monooxygenases of human liver, that is, by members of the family of cytochrome P-450-containing microsomal enzymes. It is Correspondence: Dr. W. Kalow, Department of Pharmacology, University of Toronto, Toronto, ON M5S 1A8. This paper is based on a presentation at the Symposium, ~'Frontiers in Clinical Pharmacology and Therapeutic Drug Monitoring", The Hospital for Sick Children, Toronto, ON, June 6-7, 1985. 76
interesting to note why these discoveries were made recently, although intensive investigations of human drug metabolizing capacity have been going on for many years. An impediment to discovery had been the fact that the cytochrome P-450 system is a multi-enzyme system containing cytochromes with overlapping substrate specificities (8, 9), and most drugs are metabolized simultaneously in parallel along several routes. Direct elimination via bile or urine may be going on in addition. As long as the emphasis of investigations was on the determination of pharmacokinetic parameters of the parent drug such as half-life, clearance, or area under concentration-time curves, variation appeared to be multifactorial and not particularly striking. Research emphasis on the testing for urinary metabolites is more likely to lead to the discovery of monogenic defects (10), and indeed the recent discoveries all arose from measurements of metabolites. A second factor helping the discovery of clinically important deficiencies depends on finding the right drug for investigation. These right drugs would be those exceptional ones which happen to be metabolized by a single member of the cytochrome family of enzymes, at least predominantly so. Debrisoquine, which was a tool for discovery, and which will be discussed shortly, is a predominantly mono-metabolized substrate (11-13), and thus, variability in its metabolism is due to a single gene defect (14). On the other hand, the barbiturate amobarbital can be easily shown to be hydroxylated by at least two cytochromes (15); an absence of both of these should be a rare event and in any case has never been observed. In short, choice of the right drug and attention to urinary metabolites have been the ingredients for the recent discovery of genetic deficiencies of drug metabolism in man. We will now take a closer look at the two most important of these recently discovered metabolic defects.
Recessive deficiency of debrisoquine-sparteine metabolism Let me give you first a brief summary of some salient facts which will be my point of departure. The defect was discovered independently by Eichelbaum in Germany when investigating sparteine (16, 17) and by Smith, Idle and their colleagues in Britain when investigating debrisoquine (18). The British group was particularly active in following up their discovery and in freely publishing their observations, so that the debrisoquine story is better known than that of sparteine. Debrisoquine is an antihypertensive drug of HoffmannLaRoche sold in Canada under the trade name of Declinax ® (the drug has not been available in the United States). Sparteine was at some time important CLINICAL BIOCHEMISTRY,VOLUME 19, APRIL 1986
GENETICS OF DRUG TRANSFORMATION
sot
100 -
/ 30
E
0-01"
~10 E
60
1
i
I
2
i
1
i
4 Time
I
6
i
I
240
2.50
540
720
1440 I ~ l t ]
Figure 2 - - Plasma sparteine concentrations. Levels found in a metabolizer (lower curve) and a non-metabolizer (upper curve) after intravenous administration of 200 mg sparteine sulfate. (As demonstrated separately for metabolizers of sparteine, plasma concentrations after oral and intravenous administration are very similar). Eichelbaum et al. (25), with permission of authors and Eur. J. Clin. Pharmacol.).
8
(h)
Figure 1 - - Plasma debrisoquine concentrations. Mean and standard deviations in 4 extensive metabolizers (open circles) and 3 poor metabolizers (closed circles) following a 10 mg oral dose. (Sloan et al. (24), with permission of authors and Br. J. Clin. Pharmacol.). in North America as an oxytocic but about 7% of w o m e n experienced serious overdose effects including fetal mortality (19) so t h a t the drug is no longer used for t h a t purpose. I will l a t e r t a l k about the m a n y other drugs affected by this deficiency. All pertinent information is compatible with the notion t h a t the deficiency represents a structural alteration r a t h e r t h a n the absence of one particular cytochrome (20-22). Almost 10% of Europeans have this defect but it is distinctly more r a r e in Egypt and Saudi Arabia (23). In order to u n d e r s t a n d the clinical significance of the defect, a closer look at the model drugs debrisoquine and sparteine is worthwhile. Although both drugs are strongly affected by this metabolic defect, the w a y they are affected is completely different. Figure 1 shows p l a s m a levels of poor and extensive metabolizers of debrisoquine after an oral intake of a small dose of the drug (24). There is a striking difference in p l a s m a level but the shapes of the time concentration curves are the same. After the same dose, poor metabolizers of debrisoquine have a much higher plasma concentration t h a n extensive metabolizers but the disappearance rates from p l a s m a are not altered. This p a t t e r n m e a n s t h a t the difference is one of presystemic metabolism, often referred to as first-pass effect. In other words, in extensive metabolizers m u c h of the drug is destroyed when entering the liver from the portal vein, and before it reaches general circulation. One could say t h a t there is a grossly reduced bioavailability of the drug in most people who .are extensive metabolizers while poor metabolizers h a v e full bioavailability. The antihypertensive effect of the drug CLINICAL BIOCHEMISTRY, VOLUME 19, APRIL 1986
is related to its p l a s m a concentration. Hence, poor metabolizers h a v e the full effects from this drug, but these full effects a p p e a r to be excessive because they occur only in relatively few people. The p l a s m a levels explain why, even after a single dose of the drug, the clinical responses of poor and extensive metabolizers are very different. Figure 2 shows p l a s m a concentration time curves of sparteine (25). The upper curve is t h a t of a poor metabolizer, the lower curve of an extensive metabolizer, who both received the same small oral dose of the drug. The p e a k p l a s m a levels are nearly identical in these two subjects but the subsequent half-lives or elimination r a t e s are very different. In this case, the same single dose of the drug would have about the same intensity of effect but the duration of effect would differ. The m a i n danger of this drug for a poor metabolizer would be its tendency to cumulate on repeated intake of doses suitable for extensive metabolizers. The frequencies of poor metabolizers of debrisoquine h a v e been tested in a variety of populations (23). Figure 3 shows a frequency distribution in a British population (14). The metabolizing capacity of each person is expressed as a metabolic ratio, t h a t is, as a ratio of the amounts of p a r e n t drug divided by those of metabolites excreted in urine during eight hours. Therefore, the poor metabolizers are indicated by the black bars at the right h a n d end of the distribution curves; they represent about 8% of the populations. T h e r e a p p e a r to be very few poor metabolizers a m o n g Arabs (26, 27). It turned out recently t h a t the poor metabolizers of debrisoquine in G h a n a are not poor metabolizers of sparteine (28, 29). In Caucasian populations these two defects went together regularly. Also, in vitro studies of h u m a n liver indicate t h a t it is one enzyme t h a t metabolizes both drugs (20, 22, 30). Hence, the d e b r i s o q u i n e - s p a r t e i n e dissociation in G h a n a suggests t h a t there are in existence probably not two kinds 77
KALOW TABLE 1 Drugs Whose Biotransformation is not Impaired in Poor Metabolizers of Sparteine and/or Debrisoquine
50-
40.
Acetanilide Antipyrine Amobarbital Caffeine Carbocysteine
¢' 30"
Clozapin Guanethidine Mephenytoin Metiamide Methaqualone
Nicotine Phenytoin Sulphamethazine Tolbutamide
"6 20. o z
TABLE 2 Drugs Whose Biotransformation is Impaired in Poor Metabolizers of Debrisoquine -1"0 Loglo melabolic ratio
10
2'0
Figure 3 - - Frequency distribution of log metabolic ratio of debrisoquine in a British population. The metabolic ratio refers to the 8-hour urinary excretion of debrisoquine per that of 40H-debrisoquine. The population consisted of 258 unrelated white subjects. The poor metabolizers are represented by the black bars. (Price Evans et al. (14), with permission of authors and J. Med. Genet.). of this particular cytochrome, giving rise to poor and extensive metabolizers among Europeans, but that there is a different deficiency variant in Ghana. It seems to me that Chinese subjects tend to have again a somewhat different allele, requiring not only a reinvestigation of poor metabolizers but also of extensive metabolizers (23). This is a matter of current investigations, and further detail is not yet warranted. The point is that we cannot take for granted that metabolizing capacities observed in one population will be exactly the same in another.
Clinical significance of the d e b r i s o q u i n e sparteine metabolic defect Clinical investigations have shown that the fate of many drugs has nothing to do with poor or extensive metabolizing capacity for debrisoquine or sparteine. Table 1 shows a list of such drugs. Of these mephenytoin (also called Mesantoin ®) is subject to a separate defect to which I will refer later on. The presence of phenytoin (that is, Diphenylhydantoin or Dilantin ®) on this list of negatives (31-33) deserves emphasis as it was postulated at some time that its metabolism was covariant with that of debrisoquine, but this initial claim proved to be incorrect. Table 2 shows the list of drugs which are affected by this particular defect (34). Let me comment on some items of this list. Debrisoquine and sparteine are the prototype drugs but neither is available in the U.S.A. There are tests underway to see whether the old antitussive drug dextromethorphan could be used in the future for phenotyping (35). Dextromethorphan is ordinarily used as a component of cough mixtures but it might be made available for use on its own. Another candidate as a phenotyping probe is methoxyphenamine (36). Most strongly affected by the defect was the antiarrhythmic drug perhexiline (37). Elimination of that 78
Amiflamine Amitriptyline Desipramine Nortriptyline Dextromethorphan Encainide
Guanoxan Captopril Alprenolol Bufuralol Metoprolol Propranolol
Timolol Methoxyphenamine Penicillamine Perhexiline Phenformin Sparteine
drug depends almost completely on the debrisoquine metabolizing cytochrome. In a non-metabolizer, virtually every molecule of that drug tends to stay in the body, thus accumulating during therapy and eventually causing disastrous consequences, either in the form of liver disease or nerve damage. The drug has been withdrawn from the market so that the defect has lost its clinical importance for perhexiline, except for past cases with persisting damage. Phenformin (38) and sparteine (19) have been withdrawn. Debrisoquine is used rarely. Thus, the clinical significance of the defect for these drugs has become almost nil. However, this elimination of clinical significance was achieved at a high therapeutic cost, namely the elimination of some uniquely valuable drugs whose serious side effects were restricted to a group of independently definable subjects. For amiflamine (39), captopril (40), the amphetamines and methoxyphenamine, as well as penicillamine, additional investigations on the clinical significance have to be awaited (34). Of interest is the new antiarrhythmic encainide (41) which requires biological activation in order to become highly effective. This activation is due to the debrisoquine metabolizing enzyme and does not take place in non-metabolizers in whom the normal dose of the drug is therefore insufficient. For the remaining drugs in this list, an undoubted importance of the defect has been shown for metoprolol (42). The importance has been demonstrated by plasma level determinations, by metabolite analysis, and by measuring its therapeutic effects in terms of reduction in exercise heart rate. There is good reason for the suspicion that plasma levels in non-metabolizers may be high enough to reduce the benefit of its specificity for blocking beta1 receptors; one should therefore postulate a disproportionally increased danger of metoprolol in asthmatics. On the other end of the scale is propranolol: some of its metabolism is dependent on the debrisoquine metabolizing enzyme but other pharmacokinetic factors determine its intensity and duration of action (43). The clinical significance of this defect for propranolol thus is minimal. For the other three beta CLINICAL BIOCHEMISTRY,VOLUME 19, APRIL 1986
GENETICS OF DRUG TRANSFORMATION 10 8
Caucasians (n = 118)
6
41
21 I
IlL ~
I L
Chinese (n = 39)
Z 0
I
I I
Japanese (n = 31)
0 -0.5
i
|
L
i
i
0
O_5
1.0
1.5
2.0
Log ( % H M )
Figure 4 -- Frequency distribution of urinary excretion ofphydroxymephenytoin in three populations. The abscissa indicates the 12-hour excretion of this metabolite of mephenytoin in log percent of ingested mephenytoin. All three populations are Canadian, of ethnic origin as indicated. (Ref 56 with permission of the Br. J. Clin. Pharmacol.). blockers named here, the consequences of poor metabolizing capacity are probably all in between those for metoprolol and propranolol. Exact comparisons are not yet available for alprenolol. For the fate of timolol (42), the influence of the debrisoquine/sparteine metabolic defect was roughly 2/3 as strong as for that of metoprolol. The defect is a strong determinant for the metabolism of bufuralol (44), and there is stereoselectivity of metabolism as for metoprolol. However, some bufuralol metabolites are biologically active so that the clinical consequences of deficient metabolism seem to be less incisive than for metoprolol (42). There are two interesting biochemical sidelines: first, metoprolol undergoes three separate biotransformations; two of these, the alpha hydroxylation and the O-dealkylation, are deficient in poor metabolizers (42). The same pattern is found in the triple metabolism of methoxyphenamine (36). It seems to be true that - - in the cytochrome P-450 system - - there can be one enzyme acting on one substrate to form two different products. Second, metoprolol is a racemate of which the (S)-enantiomer provides all or most of the beta blockade (45). Extensive metabolizers eliminate most rapidly the inactive (R)-enantiomer so that the plasma level represents mostly the highly active moiety. The R - S enantiomeric ratio is reversed in poor metabolizers. Hence, a given plasma level of the drug, measured without attention to the optical isomerism, represents less pharmacological activity in poor than in extensive metabolizers. For amitriptyline the defect is not very important (46). However, for desipramine and nortriptyline there is a substantial difference in drug elimination between CLINICALBIOCHEMISTRY,VOLUME 19, APRIL 1986
extensive and poor metabolizers of debrisoquine (47). The anti-depressive action of these drugs depends critically on the plasma level in the sense that too much or too little prevents therapeutic benefit. There is every reason to suspect that, prior to long-term treatment with these antidepressants, phenotyping of the patients with debrisoquine may well be worthwhile as a means to simplify dose adjustments - - at least in Caucasian patients. The debrisoquine-sparteine metabolizing defect may have clinical significance from an entirely different point of view. First, in vitro screening tests with potential inhibitors of debrisoquine or sparteine by human liver preparations indicated quinidine is an extraordinarily strong inhibitor of this enzyme (48). What, if anything, this means for the metabolism of quinidine itself remains to be seen. In the meantime, it has become clear that patients receiving quinidine are susceptible to enzyme inhibition, so much so that they behave metabolically like poor metabolizers of debrisoquine (49). In short, all drugs known to be affected by the genetic defects of debrisoquine hydroxylation may also have inhibited metabolism in recipients of quinidine. Finally, there is a possibility of an association between susceptibility to bronchial carcinoma and extensive metabolizing phenotype for debrisoquine (50). There is no biochemical rationale at the present time in so far as none of the known precarcinogens were metabolically activated by preparations of human or rat liver (51); perhaps the right precarcinogens have not yet been tested. On the other hand, Cartright in Britain could not find any association between debrisoquine metabolism and chemically induced bladder cancer (52). Further research on this metabolic defect deserves watching.
The defect of mephenytoin metabolism The anticonvulsant mephenytoin is a racemic drug of which the two enantiomers are normally undergoing different biotransformations (53-55). The S-isomer is selectively hydroxylated and rapidly eliminated. Thereby, the R-isomer is left over and undergoes a slow demethylation to an active metabolite which has a plasma half-life of many days. This slow elimination of the R-isomer causes its accumulation in plasma and it thereby becomes the therapeutically active moiety of the drug. If the hydroxylation reaction of the S-isomer is missing, both isomers are demethylated and accumulate, causing overdose effects. Thus, the defect is important for the relatively few epileptics who receive mephenytoin. The deficiency of hydroxylation capacity for mephenytoin is an autosomal recessive trait, like that for debrisoquine, but the two defects clearly involve different genes. Whether they are on the same chromosome and thereby linked is not yet known. Extensive data on mephenytoin hydroxylation in other than Caucasian populations are not yet available. However, a recent observation which is indicated in Figure 4 suggests a high frequency of non-metabolizers of mephenytoin in Japan, that is, 22% in a small sample 79
KALOW of Japanese vs 5% of Caucasians in the Toronto population (56). This is a significant difference. In the m e a n t i m e we received confirmation of our observation in form of an abstract, estimating 24% of poor metabolizers of mephenytoin in J a p a n (57). There is as yet no evidence that any drug other t h a n mephenytoin is metabolized by the same enzyme t h a t hydroxylates S-mephenytoin. However, Inaba et al. (58) have screened a large number of drugs for competitive inhibition of the mephenytoin hydroxylase of h u m a n liver. The following items represent selected data from this study. The high Ki of phenytoin indicates very weak binding to the mephenytoin hydroxylase as we should expect from the fact that the metabolism of phenytoin and of mephenytoin are clinically unrelated (55). Tranylcypromine is able to bind quite strongly to this genetically variable enzyme, and so it might be a substrate. Of interest is the binding capacity of the two benzodiazepines: flurazepam has been shown capable of binding to both the mephenytoin and the sparteine metabolizing enzyme. This raises the interesting possibility t h a t some of the different metabolic pathways of t h a t drug may depend on these two cytochromes. It is certainly worth investigating whether persons with the double defect, that for mephenytoin hydroxylation and t h a t for debrisoquine-sparteine hydroxylation, are particularly sensitive to flurazepam. If all estimates are correct, about 1% of the Japanese population might be expected to carry both defects.
due to the genetic lack of aldehyde dehydrogenase isozyme-I, the high-affinity isozyme (62, 63). Flushing subjects have much higher acetaldehyde levels in blood t h a n non-flushing controls, while ethanol levels tend to be identical. Second, Harada et al. investigated 175 Japanese alcoholics and almost all possessed the aldehyde dehydrogenase-I, while this isozyme was absent in 4 1 - 4 8 % of the various control groups (64). In short, a person lacking the high affinity form of aldehyde dehydrogenase is not likely to become an alcoholic. Since this deficiency is relatively rare in Western populations, it cannot be the main explanation for genetic variation in the occurrence of alcoholism in North America. However, it could well be a major factor in other countries.
Outlook and conclusions This account of recent discoveries was exclusively concerned with drug oxidations, that is, the most important category of ~¢phase I reactions". The past history of these discoveries promises further, similar observations. One conclusion to be drawn is that striking biochemical deficiency of drug metabolizing enzymes may or may not have important clinical consequences. This brief review did not cover any of the new developments in research on conjugation CPhase II") reactions which also find new pharmacogenetic attention.
Alcohol sensitivity There are two main areas of inter-individual and inter-ethnic differences in respect to alcohol: First, the susceptibility to alcoholism and second, the appearance of flushing and other unpleasant effects of even small doses of ethanol in some people. I believe it is fair to say that in both respects, past investigations of alcohol dehydrogenase and its genetic variants have proved disappointing. However, new attention has to be paid to aldehyde dehydrogenase (59-60). We recall that ethanol is converted by alcohol dehydrogenase (61) into acetaldehyde which in t u r n is metabolized to acetic acid by aldehyde dehydrogenase. Acetaldehyde is a substance of well-known toxicity (62). There are several isozymes of aldehyde dehydrogenase with varying affinities for both substrate and cofactor. One isozyme has a particularly high affinity for acetaldehyde (59). If this isozyme is present, the aldehyde never reaches high blood levels. If this enzyme is missing, or if it is inhibited, the acetaldehyde will also be destroyed but only at a high plasma level. It happens t h a t approximately 40% of most Oriental populations lack this high-affinity-isozyme of'aldehyde dehydrogenase. This was first established with autopsy material. However, as Goedde and his co-workers have shown, the presence of this crucial isozyme of aldehyde dehydrogenase can also be assessed through the study of hair roots (59) so that the relationship between aldehyde dehydrogenase activity and biological effects of ethanol can be investigated in individual subjects. To date, these studies have yielded two striking results. First, the circulatory effects of ethanol like flushing are 80
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