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Pharmacogenetics: Past and future
Life Sciences, vol. 47, pp. 1385-1397 Printed in the U.S.A.
Pergamon Press
The Pennsylvania State University College of Medicine 1990 Bernard B. Brodie Lecture PHARMACOGENETICS: PAST AND FUTURE Werner Kalow Department of Pharmacology University of Toronto Toronto, Ontario (Received in final form August 10. 1990) HOMAGE TO BERNARD B. BRODIE It is the greatest honor for a pharmacologist to be invited to pay homage ._. to Bernard B. Brodie by giving a lecture in his honor. Drs. Conney (1) and Costa (2) who presented the Bernard Brodie Lectures in preceding years, had both been working under his direct guidance, and both have experienced his foresight in daily contact with him. I was not that lucky, but my personal development as a pharmacologist was nevertheless decisively influenced by him. Brodie had the gift to see the large designs of nature revealed by humble evidence. He learned from every experience, he taught effectively, and he gathered around him young scientists who could benefit from his teaching. From his beginning in horse racing chemistry, he generalized his knowledge to eventually establish the study of drug metabolism as a cornerstone of pharmacology and toxicolgy; then, he adapted the chemical methodology for measuring and studying neurotransmitters, thereby laying a new basis for modern neuropharmacology. We have to think of Bernard B. Brodie with reverence but all who knew him will think of him also with affection. LOOKING BACK THE VARIANTS OF PLASMA CHOLINESTERASE A oersonal beginning. One of Brodie's early papers was instrumental in getting me into the cholinesterase field and thereby into pharmacogenetics. Let me explain the connection. In 1948, Brodie with Lief and Poet (3) investigated the fate of procaine in man; they found that its hydrolysis by "procaine esterase" in human plasma was the determinant of its elimination. (Procaine had been in vogue as an illegal stimulant of horses; I therefore suppose that Brodie's study of procaine in human beings came from his experience with that drug in race horses.) In January 1947, some time before Brodie's paper came out, I joined the Department of Pharmacology in Berlin as a Research Assistant. It then happened that Professor Herken in that department learned of two cases of death following a routine use of procaine as a local anesthetic (4). Procaine had been introduced in 1905; it was considered a most reliable drug, stemming from the time of search for substitutes of the local anaesthetic cocaine. The clinicians responsible felt that these fatalities could have occurred only on a basis of abnormal susceptibility of the patients. Herken wondered whether the fatal procaine poisoning could have been due to a nutritionally caused reduction of procaine esterase activity in plasma. 0024-3205/90 $3.00 + .oo Copyright (c) 1990 Pergamon Press plc
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Nutritional protein deficiencies were common in post-war Berlin, and Herken had established that one of the early consequences was a decrease in plasma He suggested to me to volume and an alteration of plasma proteins (5,6). investigate procaine hydrolysis in human serum, and he proposed to utilize the ultraviolet spectrum of procaine for these investigations (7). Ultraviolet spectrophotometry was an expensive technique at that time, made possible for us by the donation of a photomultiplier by the US Army in Berlin. I had barely initiated these studies in Berlin when I receiv;trea; invitation by Carl F. Schmidt to study pharmacology in Philadelphia. had the opportunity not only to continue my studies of procaine hydrolysis but to improve the methodology with the help of a marvelous new instrument, a I furthermore had the good luck of receiving Beckman Spectrophotometer. I ended up by demonstrating instruction in enzymology from Britton Chance. the identity of procaine esterase and plasma cholinesterase, and by having developed a spectrophotometric method to assay plasma cholinesterase activity in vitro. My report was declined publication in the Journal of Biological Chemistry on the erroneous grounds that human serum was known to contain only a single esterase and that my work was therefore superfluous. The paper was published without change in the Journal of Pharmacology and experimental Therapeutics in 1952 (8). The discoverer of plasma cholinesterase was Professor Bruno Mendel in Toronto (9). He called it "pseudo-cholinesterase" in distinction from "true cholinesterase", the acetylcholinesterase. One of my reasons for moving from Philadelphia to Toronto was my expectation to meet him, but when I arrived in Toronto in September 1951, he had just left for his native Holland. However, a research project initiated by him was still being pursued: The project was an investigation of the elevation of cholinesterase activity in patients with hyperthyroidism. (I believe the investigation was never completed nor ever pursued by anyone else.) I was proud about my method, and I took pity on the investigator of Mendel's project who used the Warburg apparatus to measure cholinesterase activity - the then accepted method. It was a cumbersome, gasometric method, based on the liberation of carbon dioxide from a bicarbonate buffer when acid was added to the system through ester hydrolysis. My spectrophotometric method was much simpler and faster and could yield a precise measurement of cholinesterase activity within a few minutes. When I proposed a change-over to my method, I was asked to first produce a rigorous comparison of the two methods. For this comparison of methods, I used blood from student volunteers (10) but I did not get pathological samples with very low esterase activity as I had hoped to include into my series. In order for me to obtain such specimens, Professor J.K.W. Ferguson' brought me in contact with the Psychiatrist Dr. D.R. Gunn who had patients with very low cholinesterase activity, as he knew from his studies with succinylcholine. Succinylcholine was an old drug but its muscle-relaxing properties were a recent discovery at that time, and the Nobel Prize winner Daniel Bovet had explained its short duration of action in man by its rapid destruction by plasma cholinesterase. Dr. Gunn was a forward looking physician of exacting habits who tested and used succinylcholine to ameliorate muscle contraction during electroconvulsive When injecting succinylcholine, he invariably controlled his speed therapy. of injection by observing a stopwatch; the quality of his observations is
1J.K.W. Ferguson is the discoverer of the obstetrically important Ferguson-Reflex; later as Director of the Connaught Research Laboratories he managed the smooth introduction of the Salk vaccine.
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unsurpassed (11). Dr. Gunn had some patients who always responded to succinylcholine with a grossly prolonged effect; he therefore had established with the help of the provincial laboratories that their cholinesterase activity was low. I received a specimen of serum from one of these patients for my comparison of methods for measuring cholinesterase activity. When testing this serum with my spectrophotometric method, it hydrolysed benzoylcholine but I saw that the time-concentration record showed an unusual curvature. I was sufficiently familiar with the system that I immediately recognized a gross elevation of the Michaelis constant (Km);
t FIG 1 Benzovlcholine Hvdrolvsis bv Plasma Cholinesterase Spectrophotometric observation at 240 nm. Re-drawing of characteristic substrate decay cuL_ves.u - "usual", a * "atypical" cholinesterase. Methods according to refs. 10, 12,0r 13. I could quickly exclude the presence of a competitive inhibitor that might have caused the Km elevation. I then knew that I was facing an enzyme variant which could only have been genetic. The cholinesterase in plasma from the parents of Dr. Gunn's patient was neither normal nor like that of the patient; I concluded that they were heterozygous carriers of the variant, but I found our demonstration of their deviant enzymology unsatisfactory. I therefore wanted a method suitable for detecting the heterozygotes, and I worked for about a year with my colleagues, the anesthetist Dr. Natalie 'Staron (12) and Mr. Klaus Genest (13), a descendent of German Huguenots, until we could propose "Dibucaine Numbers" as a means to recognize homozygotes and heterozygotes for the enzyme variant. I named the variant "atypical" in order to avoid any stigma of abnormality. While we were completing the dibucaine method, a paper by Lehmann and Ryan (14) appeared which indicated heritability of "low cholinesterase activity'. I quickly sent a letter to Lancet to indicate that there was more to the genetics of cholinesterase than merely a lowering of activity (15).
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follow-UD. It was gratifying to notice the life-saving effect of the discovery of genetic variation of plasma cholinesterase activity. As far as I could judge, most fatalities due to succinylcholine had been produced by wrong emergency treatments administered by frightened anesthetists: Most had never before seen or heard of the prolonged apnea which succinylcholine produced in the functional absence of cholinesterase activity. To give artificial ventilation until the patient was breathing spontaneously was usually all that was needed; the key to the adoption of this therapeutic restraint was an understanding of the cause and nature of prolonged succinylcholine action.
The
Following our definition of atypical plasma cholinesterase came discoveries of other variants. TABLE I lists the variants in a compilation adapted from Lockridge (16). The listing contains 11 entries, 7 of these are variants with reduced activity; of these, the atypical and fluoride-resistant variants have an activity reduction which is only a reflection of an increase of Km, that There are probably several silent is, a decrease of substrate affinity. variants, reflecting different mutations that render the molecule inactive or prevent its formation. The resulting complexity of the biochemistry and genetics, and the clinical significance of the diagnosis, led to the formation of specialized units for cholinesterase investigations in England (17) and in Denmark (18). TABLE I Genetic Variants of Cholinesterase Normal activity Usual
Genotvne UU
Reduced activity Atypical (Asp70-->Gly) Silent-l (Glyll7-->frame shift) Fluoride Quantitative Variant J Quantitative Variant K (Ala539-->Thr) Quantitative Variant H
M ss FF JJ KK HH
Newfoundland Increased activity c5+ Cynthiana Variant South African Variant from Lockridge (16) During recent years, Professor Bert LaDu and Dr. Oksana Lockridge in Ann Arbor have, together with some students and colleagues (19), clarified much of the molecular genetics of the cholinesterase variants, as indicated in TABLE They furthermore established by amino acid comparisons that there is I. human homology to plasma cholinesterase of for 54% Torpedo acetylcholinesterase, for rabbit liver microsomal 30% esterase, andinterestingly 28% to bovine thyroglobulin (20). Is one to speculate that the unexplained relationship between thyroid function and cholinesterase activity has molecular ties?
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To round off the cholinesterase picture, let me mention that LaDu has now developed a method of identifying persons with the atypical and the K variants, by multiplying the mutant portions of the gene derived from There is a promise to lymphocytes, using the polymerase chain reaction. expand the methodology to allow the recognition of all genotypes. This will have a number of medical consequences. It will be useful in toxicology if one can readily distinguish between genetically low cholinesterase activity and that from organophosphate It will also be useful in medicine since the cholinesterase level exposure. is one of the most sensitive indicators of liver function. TABLE II REASONS FOR LOW PLASMA CHOLINESTERASE ACTIVITY 1) Presence of a low activity mutant 2) Liver damage (Reduced protein synthesis) 3) Esterase inhibition (e.g. organophosphate insecticide) As I have seen repeatedly, it is a fatal prognosis in hepatitis if the level of activity drops to about 25% of a given patient's normal value; it probably indicates a breakdown of protein synthesis. However, cholinesterase tests for liver function were generally useless in the past because it was impossible to distinguish with one measurement between pathologically and genetically low esterase levels. THE RISE OF PHARMACOGENETICS The concent.
The reports on the heritable variation of plasma cholinesterase activity were a major stimulus for Motulsky to write in 1957 a seminal article on "Drug Reactions, Enzymes, and Biochemical Genetics" (21). Other principal examples for this article were primaquine hemolysis in black American soldiers dehydrogenase (G-6-PD) glucose-6-phosphate deficiency), variable (now atropinesterase in rabbits, and - last but not least - Brodie's work (22) on species differences in the duration of hexobarbital narcosis, as caused by variable detoxication capacities. Vogel printed the term Pharmacogenetics in 1959 (23). I was writing a monograph which appeared in 1962 (24). I described above the development of the cholinesterase story from the beginning to the present. G-6-PD deficiency (25) is now known to affect approximately 400 million people worldwide; it is genetically heterogeneous, and over 300 variants have been identified (26). The N-acetyltransferase polymorphism which was also discovered in the 1950s (27) had a whole monograph devoted to it (28). A defect predisposing to malignant hyperthermia (29) has been located in the rhyanodin receptor (30,31). In short, all the human examples that gave rise to the concept of pharmacogenetics in the late 1950's and early 1960's have been exploited and are part of the lore which medical students have to learn. All of these dramatic examples were monogenic traits, showing Mendelian inheritance. During the 196Os, the most stimulating work in pharmacogenetics was that of Elliot Vesell. It brought a change of concept which firmly linked genetics and drug metabolism. He started investigations in twins in order to check the heritability of the elimination rates of different drugs. In 1968 together with Page, he tested antipyrine (32), dicoumarol (33), and phenylbutazone (34). Stimulated by the success, he and other investigators used twin studies to test the elimination rates also of ethanol, halothane, phenytoin,
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At least a theophylline, desipramine, nortriptyline and amobarbital (35). significant hereditary component in drug elimination capacity was discovered In in all cases, in some cases it was a virtually complete heritability. short, the twin studies by Vesell and by his followers have shown that drug elimination capacity is under genetic control, with built-in opportunities for adaptive changes and other modifications. L. Following the spate of pharmacogenetic discoveries between 1950 and 1960, there was hope for a continued high discovery rate - which never came. There were some interesting observations regarding specific drugs (36,37), but between 1960 and there were only two discoveries of enzyme variants in 1977, pharmacogenetics (see Table 3). One might call this a pause in discovery which deserves some analysis because there was no lack of opportunity, as shown subsequently; there was a lack of proper search. TABLE III PHARMACOGENETIC VARIANTS OF HUMAN ENZYMES. DATES OF KEY REPORTS 1937
1952 1953 1954 1956 1960 1965 1968 1977 1977 1979 1980 1981 1984
Acute intermittent porphyria (uroporphyrin I synthetase deficiency) Acatalasemia Acetylation polymorphism G-C-PD deficiency Atypical plasma-cholinesterase Methemoglobin reductase deficiency Atypical alcohol dehydrogenase Paraoxonase polymorphism Debrisoquine hydroxylase deficiency Catechol-0-methyltransferase variation Aldehyde dehydrogenase deficiency Thiopurine methyltransferase deficiency Glutathione-S-transferase deficiency Mephenytoin hydroxylase deficiency
Waldenstrom (38)
Takahara (39) Boenicke & Reif (27) Dern et al. (25) Kalow (40) Scott (41) von Wartburg et al. (42) Krisch (43) Dengler & Eichelbaum (44) Mahgoub et al. (45) Weinshilboum et al. (46) Goedde et al. (47) Weinshilboum & Sladek (48) Board (49) Kuepfer et al. (50)
Let us consider the fact that most drugs are not eliminated by one single route but are metabolized in various parallel and consecutive reactions, so that failure of one reaction does not necessarily alter the drug elimination. Besides metabolism, there may be excretion via urinary, biliary, intestinal, If each of the elimination steps is genetically or pulmonary routes. controlled, and if one measures the overall elimination rate of the drug, one may find it to be a multigenic but not a monogenic trait; a monogenic inheritance would be observed only if one of the genetically controlled pathways is decisive for the fate of the drug. As a corollary, it follows that there is an increased chance of discovering a single-gene defect if the appearance of a metabolite is measured instead of the disappearance of the parent drug. The debrisoquine/sparteine metabolic defect was discovered when Dengler and Eichelbaum (44) searched for metabolites of sparteine and Mahgoub et al. (45) for metabolites of debrisoquine. The variable enzyme is now classified as cytochrome P450IID6 (51) but it is often called cytochrome dbl, or
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Both sparteine and debrisoquine are exceptional debrisoquine hydroxylase. drugs in that their metabolism depends overwhelmingly on only one single enzyme, namely the debrisoquine hydroxylase. There are very numerous papers on debrisoquine hydroxylase, covering such different aspects as the molecular basis of the deficiency (52), the many drugs affected by it (53), the enzyme inhibition produced by quinidine or by neuroleptics (54), and the frequencies of the deficiency in different ethnic groups (55). I will not delve into any of these findings. My points are the importance of the search for metabolites, and the tenuous relationship between a metabolic defect and any clinical impact that it may have. Drue metabolism and drue tareets. Why do we know so many more variants of drug metabolism than of receptor function? One reason may well be that direct investigations of drug metabolizing enzymes are easier to perform than those of receptors; there may have been fewer opportunities to find variant However, there could also be a genetic reason. A receptor is a receptors. transducer made to respond to some internal messenger; the system may become inoperative by any alteration of either receptor or messenger; in other words, there are inherent constraints on the variability of the system. On the other hand, a chemical defence system against intruding toxicants does not have such constraints; rather, constant adaptation and optimization of the system against different intruders is to be expected. In short, variability of drugmetabolizing enzymes could perhaps remain of greater concern than variability of receptors. GAZING INTO THE FUTURE Two of the massive developments of our age, information technology and molecular genetics, will continue to bring innumerable changes in medicine. For pharmacogenetics, Meyer (56) considered the three most important techniques of molecular genetics to be 1) restriction analysis of genomic DNA, 2) enzymatic amplificaton of DNA by the polymerase chain reaction, and 3) the expression of cDNAs in cell culture. More important than the technological opportunities themselves will be conceptual changes, and the changing goals which one may expect as a sequence to the improved understanding of human biology. I will scrutinize four topics. PHARMACOANTHROPOLOGY Pharmacoanthropology is the medical science which deals with inter-ethnic differences of pharmacological or toxicological significance (57,58). The coming of massive work in this area is foreseeable for various reasons. One reason is simple and human: There are enough examples of interethnic genetic differences in drug metabolism that striving for additional information is scientifically sound. As research laboratories are erected in more and more countries, the question will repeatedly come up: "Are we different from anyone else?" Members of the pharmaceutical industry in Japan - thinking of export - have begun a more systematic scrutiny of population differences than I have seen in Western firms. In the past, almost all of the precisely defined interethnic differences were those of gene frequencies: it is widely known that there are fewer slow acetylators (59) and fewer persons with debrisoquine hydroxylase deficiency (55) in the Orient than in Europe. However, counting the number of subjects in a population who have a particular enzyme deficiency is only a part of the problem; there are also shifts in mean values. Past studies with debrisoquine hydroxylase offer a simple illustration of the problem: In all studies conducted in Europe - be it in Sweden (60),
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Hungary (62), or elsewhere - the metabolic ratio (the ratio of Spain( debrisoquine and of 4-OH debrisoquine excreted in urine) of most samples is smaller than unity; the ratio measured in most Chinese subjects (63) is Larger than unity (signifying a comparatively low activity of that enzyme); among the Venda in Africa, 75 percent of the population have ratios larger than one (64). (FIG 2) It could be that these differences have environmental causes. It also could mean that different alleles serve as the predominant entities in different populations, analogous to the situation with alcohol dehydrogenase. Of this enzyme, the beta-l and a beta-2 variants predominate, respectively, in Europe and in Asia, while a beta-3 variant seems peculiar to Blacks, as seen Through DNA comparisons, the clarification of this in North America (65). kind of problem will be much easier than it was in the past. Nevertheless, functional comparisons of drug-metabolizing enzymes in human cells or tissue fluids derived from different populations offer insufficiently used scientific opportunities.
15
1
10
5 0:
9J 0.1
1
80 60 1
40
MR = 12.6
20 0
A
1
L
10
100
1000
MetabolicRatio FIG 2 Distribution of Debrisoauine Metabolic Ratios in an African and an Euronean Ponulation, The upper curve represents an adaptation of an illustration by Sommers et al. (64) of measurements in a population of 98 Venda. The lower curve represents an adaptation of an illustration by Steiner et al. (60) of measurements in a Swedish population of 752 subjects; the black bars indicate "poor metabolizers". The vertical Line at unity metabolic ratios is drawn to show up the different medians of metabolic activity in the two populations; furthermore, the distinction between poor and extensive metabolizers in the Swedish population is meaningless in the Venda.
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DRUG METABOLISM IN BRAIN It has long been known that heroin is converted in the brain into the and that the neurotransmitters monoacetyl morphine, active opioid acetylcholine and noradrenaline are metabolically inactivated in the brain. Vice versa, it has been established that the brain contains a number of drugenzymes, as for instance a number of cytochromes P450 metabolizing (66,67,68); while the concentrations are seemingly low when compared with those in liver, they may have high concentrations in certain groups of cells. A new interest in drug-metabolizing enzymes in the brain was stimulated by the unsubstantiated suspicion of Barbeau et al (69) that debrisoquine hydroxylase deficiency was a predisposition to Parkinson's disease. Otton et al. (70) had noted the high affinity of that enzyme for neuroleptics. FonnePfister et al. (71) observed that the Parkinson-producing "recreational" drug MPTP was a strong inhibitor of that enzyme, and they obtained evidence for the occurrence of that enzyme in brain. We have recently seen that debrisoquine hydroxylase in dog brain is identical with the so-called "piperazine acceptor" site, a protein distinct from the dopamine transporter but with some similar binding characteristics (72). This raises many questions, among them whether the functional variation of debrisoquine hydroxylase in liver is also relevant in brain. Bertilsson et al (73) in Sweden administered psychological tests to 45 poor metabolizers (PM) and 83 extensive metabolizers of debrisoquine. The authors arrived at the verdict that there were some personality differences, such as a relative ease of decision making by PMs who had a higher frequency of extreme responses (p
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important proteins. The task then becomes to search for any biological significance of such a variant. It means that pharmacogenetics will become increasingly dependent on biochemical epidemiology, and that there will have to develop increased connection between pharmacologists and epidemiologists (75,76). LIFE WITH PRE-TESTING OF PHARMACOGENETIC VARIANTS Imagine the situation after discovery of all or most major enzyme or protein polymorphisms that may affect a drug's action. Imagine further that diagnostic technology for the variant genes in a person's lymphocytes or hair roots is sufficiently economical to pre-screen all candidates for serious drug therapy. The consequences for a patient would be plainly beneficial if through these tests the intake of unsuitable drugs would be avoided. The consequences would also benefit any health care system since a substantial proportion of hospitalizations are needed because of adverse drug reactions. There are already examples of pharmacogenetic pre-testing, as e.g. the avoidance of halothane in patients predisposed to malignant hyperthermia, or the avoidance of the antiarrhythmic drug perhexiline in patients with debrisoquine hydroxylase deficiency. TABLE IV CONSEQUENCES OF DNA-BASED TESTING OF PATIENTS FOR THE PRESENCE OF PHARMACOGENETIC ENZYME VARIANTS Individuals:
Intake of unsuitable drugs avoided
Public Health: Reduction of adverse drug effects diminishes hospitalisation. Industry:
a) Avoid the production of drugs whose fate depends on polymorphic enzymes. b) Chemicals otherwise discarded can become drugs when reserved for pre-tested patients - increased therapeutic scope.
The genetic screening should be sufficiently broad to detect persons with double defects as these are not necessarily very rare. For instance, propanolol might be hazardous for a person with a deficiency of both debrisoquine hydroxylase and mephenytoin hydroxylase (77). Each enzyme catalyzes a different biotransformation pathway of that drug; while a single deficiency is immaterial for the fate of the drug, this is not so in the presence of the double deficiency. At least one per 400 Caucasians are expected to have the double defect. The consequences for the pharmaceutical industry could be substantial. It is usually side effects in only a few individuals that kill a prospective new If one can avoid giving the drug to these few individuals through drug. knowledge of their biochemical peculiarities, the benefits of that drug could be made available for everybody else. Many chemicals that are now discarded could become drugs so that our therapeutic armamentarium would be expanded. However, for this to happen extensively, drug medication would have to be better targeted than is usually the case, that is, decisions for medication should be weighed similarly as is any surgical intervention. This change of therapeutic philosophy is not likely near at hand but is worth much effort.
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In the meantime, it seems logical for the pharmaceutical industry to utilize Human drug-metabolizing knowledge of pharmacogenetics in a different way. enzymes will become increasingly available in cell culture through targeted These will allow in vitro testing of enzyme formation after gene transfer. the metabolism of a prospective drug in order to assess the contribution of a If so, the chemical may be polymorphic enzyme to its biotransformation. discarded prior to the use in human beings. Alternatively, during phase I studies, the drug could be deliberately tested in a person with the defective enzyme, in order to see whether other elimination pathways can compensate for Finally, the structure of the drug might be deliberately the deficiency. altered so as to change its biotransformation pathways.
CONCLUSION Pharmacogenetics has become too important to be relegated to a position of neglect. Indeed it is foreseeable that pharmacogenetics will play an increasingly important role in clinical pharmacology and in medicine. Brodie had a hand in this development when he stressed species differences. I quoted two of his studies, that of hexobarbital metabolism in different species, and that of the metabolism of procaine which in humans occurs in plasma while other species metabolize it in liver. For a student of variation, it is only an additional step from the recognition of differences between species to We have to learn that differences between individuals within one species. differences between people can be discovered only by studying humans, and not by investigating rats, mice, or monkeys.
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R.J. DERN. E. BEUTLER and A.S. ALVING, J. Lab. & Clin. Med. 44 171-176 (1954). ' L. LUZZATTO, Ethnic Differences in Reactions to Drues and Xenobiotics, W. Kalow, H.W. Goedde and D. Agarwal (eds), 385-399, Liss, New York (1986). R. BONICKE and W. REIF, Arch. exper. Path. u. Pharmakol. 220 321-333 (1953). WENDELL W. WEBER, The Acetvlator Genes and Drug Resoonse, Oxford University Press (1987). M.A. DENBOROUGH and R.R.H. LOVELL, Lancet 11 45 (1960). D.H. MACLENNAN, C. DUFF, R. ZORZATO, J. FUJII, M. PHILLIPS, R.G. KORNELUK, W. FRODIS, B.A. BRITT and R.G. WORTON, Nature 343 559-561 (1990). T.V. MCCARTHY, J.M.S. HEALLY, J.J.A. HEFFRON, M. LEHANE, T. DEUFEL, F. LEHMANN-HORN, M. FARRALL and K. JOHNSON, Nature 343 562-564 (1990). E.S. VESELL and J.G. PAGE, Science 161 72-73 (1968). E.S. VESELL and J.G. PAGE, J. Clin. Invest. 42 2657-2663 (1968). E.S. VESELL and J.G. PAGE, Science 159 1479-1480 (1968). E.S. VESELL, Clin. Pharmacol. Ther. 18 603-612 (1985). H. KUTT, M. WOLK, R. SCHERMAN and F. MCDOWELL, Neurology 14 542-548 (1964). N.T. SHAHIDI, Annals of the New York Aca. of Sci., 151 822-832 (1968). J. WALDENSTROM, Acta. Med. Stand. 82. (suppl) l-254 (1937). S. TAKAHARA, Lancet u 1101-1104 (1952). W. KALOW, Can. Anaes. Sot. J. 3 22-30 (1956). E.M. SCOTT, J. Clin. Invest. 39 1176 (1960). J.-P. VON WARTBURG, J. PAPENBERG and H. AEBI, Can. J. Biochem. 42 889898 (1964). K. KRISCH, Z. Klin. Chem. Klin. Biochem. 5 41-45 (1968). H.J. DENGLER and M. EICHELBAUM, Arzneimitt. Forsch. 27 1836-1844 (1977). A. MAHGOUB, J.R. IDLE, L.G. DRING, R. LANCASTER and R.L. SMITH, Lancet 11 584-586 (1977). R.M. WEINSHILBOUM, F.A. RAYMOND, L.R. ELVEBACK and W.H. WEIDMAN, Nature 252 490-491 (1974). H.W. GOEDDE, S. HARADA and D.P. AGARWAL, Human Genet. 51 331-334 (1979). R.M. WEINSHILBOUM and S.L. SLADEK, Am. J. Human Genet. 22. 651-662 (1980). P.G. BOARD, Am. J. Human Genet. 33 36-43 (1981). A. KUPFER, P. DESMOND, R. PATWARDHAN, S. SCHENKER and R.A. BRANCH, Clin. Pharmac. Ther. 21. 33-39 (1984). S. KIMURA, M. UMENO, R.C. SKODA, U.A. MEYER and F.J. GONZALEZ, Am. J. Hum. Genet. 45 889-904 (1989). U.A. MEYER, R.C. SKODA and U.M. ZANGER, Pharmac. Ther. 46 297-308 (1990). W. KALOW, Clinical Pharmacologv in Psvchiatrv, S.G. Dahl and L.F. Gram (eds.) 148-162 Springer-Verlag, Berlin Heidelberg (1989). K. BROSEN and L.F. GRAM, Eur. J. Clin. Pharmacol. 36 537-547 (1989). M. EICHELBAUM and A.S. GROSS, Pharmac. Ther. 46 377-394 (1990). U.A. MEYER, Pharmac. Ther. 46 349-355 (1990). W. KALOW, Federation Proc. 43 2314-2318 (1984). W. KALOW, H.W. GOEDDE and D.P. AGARWAL (Eds.) Ethnic Defferences in reactions to Drues and Xenobiotics, Alan R. Liss, Inc., New York (1986). D.A.P. EVANS, Pharmac. Ther. 42 157-234 (1989). E. STEINER, L. BERTILSSON, J. SAWE, I. BERTLING and F. SJOQVIST, Clin. Phannacol. Ther. 44 431-435 (1988). J. BENITEZ, A. LLERENA and J. COBALEDA, Clin. Pharmacol. Ther. 44 74-77 (1988).
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Pergamon Press
The Pennsylvania State University College of Medicine 1990 Bernard B. Brodie Lecture PHARMACOGENETICS: PAST AND FUTURE Werner Kalow Department of Pharmacology University of Toronto Toronto, Ontario (Received in final form August 10. 1990) HOMAGE TO BERNARD B. BRODIE It is the greatest honor for a pharmacologist to be invited to pay homage ._. to Bernard B. Brodie by giving a lecture in his honor. Drs. Conney (1) and Costa (2) who presented the Bernard Brodie Lectures in preceding years, had both been working under his direct guidance, and both have experienced his foresight in daily contact with him. I was not that lucky, but my personal development as a pharmacologist was nevertheless decisively influenced by him. Brodie had the gift to see the large designs of nature revealed by humble evidence. He learned from every experience, he taught effectively, and he gathered around him young scientists who could benefit from his teaching. From his beginning in horse racing chemistry, he generalized his knowledge to eventually establish the study of drug metabolism as a cornerstone of pharmacology and toxicolgy; then, he adapted the chemical methodology for measuring and studying neurotransmitters, thereby laying a new basis for modern neuropharmacology. We have to think of Bernard B. Brodie with reverence but all who knew him will think of him also with affection. LOOKING BACK THE VARIANTS OF PLASMA CHOLINESTERASE A oersonal beginning. One of Brodie's early papers was instrumental in getting me into the cholinesterase field and thereby into pharmacogenetics. Let me explain the connection. In 1948, Brodie with Lief and Poet (3) investigated the fate of procaine in man; they found that its hydrolysis by "procaine esterase" in human plasma was the determinant of its elimination. (Procaine had been in vogue as an illegal stimulant of horses; I therefore suppose that Brodie's study of procaine in human beings came from his experience with that drug in race horses.) In January 1947, some time before Brodie's paper came out, I joined the Department of Pharmacology in Berlin as a Research Assistant. It then happened that Professor Herken in that department learned of two cases of death following a routine use of procaine as a local anesthetic (4). Procaine had been introduced in 1905; it was considered a most reliable drug, stemming from the time of search for substitutes of the local anaesthetic cocaine. The clinicians responsible felt that these fatalities could have occurred only on a basis of abnormal susceptibility of the patients. Herken wondered whether the fatal procaine poisoning could have been due to a nutritionally caused reduction of procaine esterase activity in plasma. 0024-3205/90 $3.00 + .oo Copyright (c) 1990 Pergamon Press plc
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Nutritional protein deficiencies were common in post-war Berlin, and Herken had established that one of the early consequences was a decrease in plasma He suggested to me to volume and an alteration of plasma proteins (5,6). investigate procaine hydrolysis in human serum, and he proposed to utilize the ultraviolet spectrum of procaine for these investigations (7). Ultraviolet spectrophotometry was an expensive technique at that time, made possible for us by the donation of a photomultiplier by the US Army in Berlin. I had barely initiated these studies in Berlin when I receiv;trea; invitation by Carl F. Schmidt to study pharmacology in Philadelphia. had the opportunity not only to continue my studies of procaine hydrolysis but to improve the methodology with the help of a marvelous new instrument, a I furthermore had the good luck of receiving Beckman Spectrophotometer. I ended up by demonstrating instruction in enzymology from Britton Chance. the identity of procaine esterase and plasma cholinesterase, and by having developed a spectrophotometric method to assay plasma cholinesterase activity in vitro. My report was declined publication in the Journal of Biological Chemistry on the erroneous grounds that human serum was known to contain only a single esterase and that my work was therefore superfluous. The paper was published without change in the Journal of Pharmacology and experimental Therapeutics in 1952 (8). The discoverer of plasma cholinesterase was Professor Bruno Mendel in Toronto (9). He called it "pseudo-cholinesterase" in distinction from "true cholinesterase", the acetylcholinesterase. One of my reasons for moving from Philadelphia to Toronto was my expectation to meet him, but when I arrived in Toronto in September 1951, he had just left for his native Holland. However, a research project initiated by him was still being pursued: The project was an investigation of the elevation of cholinesterase activity in patients with hyperthyroidism. (I believe the investigation was never completed nor ever pursued by anyone else.) I was proud about my method, and I took pity on the investigator of Mendel's project who used the Warburg apparatus to measure cholinesterase activity - the then accepted method. It was a cumbersome, gasometric method, based on the liberation of carbon dioxide from a bicarbonate buffer when acid was added to the system through ester hydrolysis. My spectrophotometric method was much simpler and faster and could yield a precise measurement of cholinesterase activity within a few minutes. When I proposed a change-over to my method, I was asked to first produce a rigorous comparison of the two methods. For this comparison of methods, I used blood from student volunteers (10) but I did not get pathological samples with very low esterase activity as I had hoped to include into my series. In order for me to obtain such specimens, Professor J.K.W. Ferguson' brought me in contact with the Psychiatrist Dr. D.R. Gunn who had patients with very low cholinesterase activity, as he knew from his studies with succinylcholine. Succinylcholine was an old drug but its muscle-relaxing properties were a recent discovery at that time, and the Nobel Prize winner Daniel Bovet had explained its short duration of action in man by its rapid destruction by plasma cholinesterase. Dr. Gunn was a forward looking physician of exacting habits who tested and used succinylcholine to ameliorate muscle contraction during electroconvulsive When injecting succinylcholine, he invariably controlled his speed therapy. of injection by observing a stopwatch; the quality of his observations is
1J.K.W. Ferguson is the discoverer of the obstetrically important Ferguson-Reflex; later as Director of the Connaught Research Laboratories he managed the smooth introduction of the Salk vaccine.
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unsurpassed (11). Dr. Gunn had some patients who always responded to succinylcholine with a grossly prolonged effect; he therefore had established with the help of the provincial laboratories that their cholinesterase activity was low. I received a specimen of serum from one of these patients for my comparison of methods for measuring cholinesterase activity. When testing this serum with my spectrophotometric method, it hydrolysed benzoylcholine but I saw that the time-concentration record showed an unusual curvature. I was sufficiently familiar with the system that I immediately recognized a gross elevation of the Michaelis constant (Km);
t FIG 1 Benzovlcholine Hvdrolvsis bv Plasma Cholinesterase Spectrophotometric observation at 240 nm. Re-drawing of characteristic substrate decay cuL_ves.u - "usual", a * "atypical" cholinesterase. Methods according to refs. 10, 12,0r 13. I could quickly exclude the presence of a competitive inhibitor that might have caused the Km elevation. I then knew that I was facing an enzyme variant which could only have been genetic. The cholinesterase in plasma from the parents of Dr. Gunn's patient was neither normal nor like that of the patient; I concluded that they were heterozygous carriers of the variant, but I found our demonstration of their deviant enzymology unsatisfactory. I therefore wanted a method suitable for detecting the heterozygotes, and I worked for about a year with my colleagues, the anesthetist Dr. Natalie 'Staron (12) and Mr. Klaus Genest (13), a descendent of German Huguenots, until we could propose "Dibucaine Numbers" as a means to recognize homozygotes and heterozygotes for the enzyme variant. I named the variant "atypical" in order to avoid any stigma of abnormality. While we were completing the dibucaine method, a paper by Lehmann and Ryan (14) appeared which indicated heritability of "low cholinesterase activity'. I quickly sent a letter to Lancet to indicate that there was more to the genetics of cholinesterase than merely a lowering of activity (15).
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follow-UD. It was gratifying to notice the life-saving effect of the discovery of genetic variation of plasma cholinesterase activity. As far as I could judge, most fatalities due to succinylcholine had been produced by wrong emergency treatments administered by frightened anesthetists: Most had never before seen or heard of the prolonged apnea which succinylcholine produced in the functional absence of cholinesterase activity. To give artificial ventilation until the patient was breathing spontaneously was usually all that was needed; the key to the adoption of this therapeutic restraint was an understanding of the cause and nature of prolonged succinylcholine action.
The
Following our definition of atypical plasma cholinesterase came discoveries of other variants. TABLE I lists the variants in a compilation adapted from Lockridge (16). The listing contains 11 entries, 7 of these are variants with reduced activity; of these, the atypical and fluoride-resistant variants have an activity reduction which is only a reflection of an increase of Km, that There are probably several silent is, a decrease of substrate affinity. variants, reflecting different mutations that render the molecule inactive or prevent its formation. The resulting complexity of the biochemistry and genetics, and the clinical significance of the diagnosis, led to the formation of specialized units for cholinesterase investigations in England (17) and in Denmark (18). TABLE I Genetic Variants of Cholinesterase Normal activity Usual
Genotvne UU
Reduced activity Atypical (Asp70-->Gly) Silent-l (Glyll7-->frame shift) Fluoride Quantitative Variant J Quantitative Variant K (Ala539-->Thr) Quantitative Variant H
M ss FF JJ KK HH
Newfoundland Increased activity c5+ Cynthiana Variant South African Variant from Lockridge (16) During recent years, Professor Bert LaDu and Dr. Oksana Lockridge in Ann Arbor have, together with some students and colleagues (19), clarified much of the molecular genetics of the cholinesterase variants, as indicated in TABLE They furthermore established by amino acid comparisons that there is I. human homology to plasma cholinesterase of for 54% Torpedo acetylcholinesterase, for rabbit liver microsomal 30% esterase, andinterestingly 28% to bovine thyroglobulin (20). Is one to speculate that the unexplained relationship between thyroid function and cholinesterase activity has molecular ties?
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To round off the cholinesterase picture, let me mention that LaDu has now developed a method of identifying persons with the atypical and the K variants, by multiplying the mutant portions of the gene derived from There is a promise to lymphocytes, using the polymerase chain reaction. expand the methodology to allow the recognition of all genotypes. This will have a number of medical consequences. It will be useful in toxicology if one can readily distinguish between genetically low cholinesterase activity and that from organophosphate It will also be useful in medicine since the cholinesterase level exposure. is one of the most sensitive indicators of liver function. TABLE II REASONS FOR LOW PLASMA CHOLINESTERASE ACTIVITY 1) Presence of a low activity mutant 2) Liver damage (Reduced protein synthesis) 3) Esterase inhibition (e.g. organophosphate insecticide) As I have seen repeatedly, it is a fatal prognosis in hepatitis if the level of activity drops to about 25% of a given patient's normal value; it probably indicates a breakdown of protein synthesis. However, cholinesterase tests for liver function were generally useless in the past because it was impossible to distinguish with one measurement between pathologically and genetically low esterase levels. THE RISE OF PHARMACOGENETICS The concent.
The reports on the heritable variation of plasma cholinesterase activity were a major stimulus for Motulsky to write in 1957 a seminal article on "Drug Reactions, Enzymes, and Biochemical Genetics" (21). Other principal examples for this article were primaquine hemolysis in black American soldiers dehydrogenase (G-6-PD) glucose-6-phosphate deficiency), variable (now atropinesterase in rabbits, and - last but not least - Brodie's work (22) on species differences in the duration of hexobarbital narcosis, as caused by variable detoxication capacities. Vogel printed the term Pharmacogenetics in 1959 (23). I was writing a monograph which appeared in 1962 (24). I described above the development of the cholinesterase story from the beginning to the present. G-6-PD deficiency (25) is now known to affect approximately 400 million people worldwide; it is genetically heterogeneous, and over 300 variants have been identified (26). The N-acetyltransferase polymorphism which was also discovered in the 1950s (27) had a whole monograph devoted to it (28). A defect predisposing to malignant hyperthermia (29) has been located in the rhyanodin receptor (30,31). In short, all the human examples that gave rise to the concept of pharmacogenetics in the late 1950's and early 1960's have been exploited and are part of the lore which medical students have to learn. All of these dramatic examples were monogenic traits, showing Mendelian inheritance. During the 196Os, the most stimulating work in pharmacogenetics was that of Elliot Vesell. It brought a change of concept which firmly linked genetics and drug metabolism. He started investigations in twins in order to check the heritability of the elimination rates of different drugs. In 1968 together with Page, he tested antipyrine (32), dicoumarol (33), and phenylbutazone (34). Stimulated by the success, he and other investigators used twin studies to test the elimination rates also of ethanol, halothane, phenytoin,
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At least a theophylline, desipramine, nortriptyline and amobarbital (35). significant hereditary component in drug elimination capacity was discovered In in all cases, in some cases it was a virtually complete heritability. short, the twin studies by Vesell and by his followers have shown that drug elimination capacity is under genetic control, with built-in opportunities for adaptive changes and other modifications. L. Following the spate of pharmacogenetic discoveries between 1950 and 1960, there was hope for a continued high discovery rate - which never came. There were some interesting observations regarding specific drugs (36,37), but between 1960 and there were only two discoveries of enzyme variants in 1977, pharmacogenetics (see Table 3). One might call this a pause in discovery which deserves some analysis because there was no lack of opportunity, as shown subsequently; there was a lack of proper search. TABLE III PHARMACOGENETIC VARIANTS OF HUMAN ENZYMES. DATES OF KEY REPORTS 1937
1952 1953 1954 1956 1960 1965 1968 1977 1977 1979 1980 1981 1984
Acute intermittent porphyria (uroporphyrin I synthetase deficiency) Acatalasemia Acetylation polymorphism G-C-PD deficiency Atypical plasma-cholinesterase Methemoglobin reductase deficiency Atypical alcohol dehydrogenase Paraoxonase polymorphism Debrisoquine hydroxylase deficiency Catechol-0-methyltransferase variation Aldehyde dehydrogenase deficiency Thiopurine methyltransferase deficiency Glutathione-S-transferase deficiency Mephenytoin hydroxylase deficiency
Waldenstrom (38)
Takahara (39) Boenicke & Reif (27) Dern et al. (25) Kalow (40) Scott (41) von Wartburg et al. (42) Krisch (43) Dengler & Eichelbaum (44) Mahgoub et al. (45) Weinshilboum et al. (46) Goedde et al. (47) Weinshilboum & Sladek (48) Board (49) Kuepfer et al. (50)
Let us consider the fact that most drugs are not eliminated by one single route but are metabolized in various parallel and consecutive reactions, so that failure of one reaction does not necessarily alter the drug elimination. Besides metabolism, there may be excretion via urinary, biliary, intestinal, If each of the elimination steps is genetically or pulmonary routes. controlled, and if one measures the overall elimination rate of the drug, one may find it to be a multigenic but not a monogenic trait; a monogenic inheritance would be observed only if one of the genetically controlled pathways is decisive for the fate of the drug. As a corollary, it follows that there is an increased chance of discovering a single-gene defect if the appearance of a metabolite is measured instead of the disappearance of the parent drug. The debrisoquine/sparteine metabolic defect was discovered when Dengler and Eichelbaum (44) searched for metabolites of sparteine and Mahgoub et al. (45) for metabolites of debrisoquine. The variable enzyme is now classified as cytochrome P450IID6 (51) but it is often called cytochrome dbl, or
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Both sparteine and debrisoquine are exceptional debrisoquine hydroxylase. drugs in that their metabolism depends overwhelmingly on only one single enzyme, namely the debrisoquine hydroxylase. There are very numerous papers on debrisoquine hydroxylase, covering such different aspects as the molecular basis of the deficiency (52), the many drugs affected by it (53), the enzyme inhibition produced by quinidine or by neuroleptics (54), and the frequencies of the deficiency in different ethnic groups (55). I will not delve into any of these findings. My points are the importance of the search for metabolites, and the tenuous relationship between a metabolic defect and any clinical impact that it may have. Drue metabolism and drue tareets. Why do we know so many more variants of drug metabolism than of receptor function? One reason may well be that direct investigations of drug metabolizing enzymes are easier to perform than those of receptors; there may have been fewer opportunities to find variant However, there could also be a genetic reason. A receptor is a receptors. transducer made to respond to some internal messenger; the system may become inoperative by any alteration of either receptor or messenger; in other words, there are inherent constraints on the variability of the system. On the other hand, a chemical defence system against intruding toxicants does not have such constraints; rather, constant adaptation and optimization of the system against different intruders is to be expected. In short, variability of drugmetabolizing enzymes could perhaps remain of greater concern than variability of receptors. GAZING INTO THE FUTURE Two of the massive developments of our age, information technology and molecular genetics, will continue to bring innumerable changes in medicine. For pharmacogenetics, Meyer (56) considered the three most important techniques of molecular genetics to be 1) restriction analysis of genomic DNA, 2) enzymatic amplificaton of DNA by the polymerase chain reaction, and 3) the expression of cDNAs in cell culture. More important than the technological opportunities themselves will be conceptual changes, and the changing goals which one may expect as a sequence to the improved understanding of human biology. I will scrutinize four topics. PHARMACOANTHROPOLOGY Pharmacoanthropology is the medical science which deals with inter-ethnic differences of pharmacological or toxicological significance (57,58). The coming of massive work in this area is foreseeable for various reasons. One reason is simple and human: There are enough examples of interethnic genetic differences in drug metabolism that striving for additional information is scientifically sound. As research laboratories are erected in more and more countries, the question will repeatedly come up: "Are we different from anyone else?" Members of the pharmaceutical industry in Japan - thinking of export - have begun a more systematic scrutiny of population differences than I have seen in Western firms. In the past, almost all of the precisely defined interethnic differences were those of gene frequencies: it is widely known that there are fewer slow acetylators (59) and fewer persons with debrisoquine hydroxylase deficiency (55) in the Orient than in Europe. However, counting the number of subjects in a population who have a particular enzyme deficiency is only a part of the problem; there are also shifts in mean values. Past studies with debrisoquine hydroxylase offer a simple illustration of the problem: In all studies conducted in Europe - be it in Sweden (60),
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Hungary (62), or elsewhere - the metabolic ratio (the ratio of Spain( debrisoquine and of 4-OH debrisoquine excreted in urine) of most samples is smaller than unity; the ratio measured in most Chinese subjects (63) is Larger than unity (signifying a comparatively low activity of that enzyme); among the Venda in Africa, 75 percent of the population have ratios larger than one (64). (FIG 2) It could be that these differences have environmental causes. It also could mean that different alleles serve as the predominant entities in different populations, analogous to the situation with alcohol dehydrogenase. Of this enzyme, the beta-l and a beta-2 variants predominate, respectively, in Europe and in Asia, while a beta-3 variant seems peculiar to Blacks, as seen Through DNA comparisons, the clarification of this in North America (65). kind of problem will be much easier than it was in the past. Nevertheless, functional comparisons of drug-metabolizing enzymes in human cells or tissue fluids derived from different populations offer insufficiently used scientific opportunities.
15
1
10
5 0:
9J 0.1
1
80 60 1
40
MR = 12.6
20 0
A
1
L
10
100
1000
MetabolicRatio FIG 2 Distribution of Debrisoauine Metabolic Ratios in an African and an Euronean Ponulation, The upper curve represents an adaptation of an illustration by Sommers et al. (64) of measurements in a population of 98 Venda. The lower curve represents an adaptation of an illustration by Steiner et al. (60) of measurements in a Swedish population of 752 subjects; the black bars indicate "poor metabolizers". The vertical Line at unity metabolic ratios is drawn to show up the different medians of metabolic activity in the two populations; furthermore, the distinction between poor and extensive metabolizers in the Swedish population is meaningless in the Venda.
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DRUG METABOLISM IN BRAIN It has long been known that heroin is converted in the brain into the and that the neurotransmitters monoacetyl morphine, active opioid acetylcholine and noradrenaline are metabolically inactivated in the brain. Vice versa, it has been established that the brain contains a number of drugenzymes, as for instance a number of cytochromes P450 metabolizing (66,67,68); while the concentrations are seemingly low when compared with those in liver, they may have high concentrations in certain groups of cells. A new interest in drug-metabolizing enzymes in the brain was stimulated by the unsubstantiated suspicion of Barbeau et al (69) that debrisoquine hydroxylase deficiency was a predisposition to Parkinson's disease. Otton et al. (70) had noted the high affinity of that enzyme for neuroleptics. FonnePfister et al. (71) observed that the Parkinson-producing "recreational" drug MPTP was a strong inhibitor of that enzyme, and they obtained evidence for the occurrence of that enzyme in brain. We have recently seen that debrisoquine hydroxylase in dog brain is identical with the so-called "piperazine acceptor" site, a protein distinct from the dopamine transporter but with some similar binding characteristics (72). This raises many questions, among them whether the functional variation of debrisoquine hydroxylase in liver is also relevant in brain. Bertilsson et al (73) in Sweden administered psychological tests to 45 poor metabolizers (PM) and 83 extensive metabolizers of debrisoquine. The authors arrived at the verdict that there were some personality differences, such as a relative ease of decision making by PMs who had a higher frequency of extreme responses (p
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important proteins. The task then becomes to search for any biological significance of such a variant. It means that pharmacogenetics will become increasingly dependent on biochemical epidemiology, and that there will have to develop increased connection between pharmacologists and epidemiologists (75,76). LIFE WITH PRE-TESTING OF PHARMACOGENETIC VARIANTS Imagine the situation after discovery of all or most major enzyme or protein polymorphisms that may affect a drug's action. Imagine further that diagnostic technology for the variant genes in a person's lymphocytes or hair roots is sufficiently economical to pre-screen all candidates for serious drug therapy. The consequences for a patient would be plainly beneficial if through these tests the intake of unsuitable drugs would be avoided. The consequences would also benefit any health care system since a substantial proportion of hospitalizations are needed because of adverse drug reactions. There are already examples of pharmacogenetic pre-testing, as e.g. the avoidance of halothane in patients predisposed to malignant hyperthermia, or the avoidance of the antiarrhythmic drug perhexiline in patients with debrisoquine hydroxylase deficiency. TABLE IV CONSEQUENCES OF DNA-BASED TESTING OF PATIENTS FOR THE PRESENCE OF PHARMACOGENETIC ENZYME VARIANTS Individuals:
Intake of unsuitable drugs avoided
Public Health: Reduction of adverse drug effects diminishes hospitalisation. Industry:
a) Avoid the production of drugs whose fate depends on polymorphic enzymes. b) Chemicals otherwise discarded can become drugs when reserved for pre-tested patients - increased therapeutic scope.
The genetic screening should be sufficiently broad to detect persons with double defects as these are not necessarily very rare. For instance, propanolol might be hazardous for a person with a deficiency of both debrisoquine hydroxylase and mephenytoin hydroxylase (77). Each enzyme catalyzes a different biotransformation pathway of that drug; while a single deficiency is immaterial for the fate of the drug, this is not so in the presence of the double deficiency. At least one per 400 Caucasians are expected to have the double defect. The consequences for the pharmaceutical industry could be substantial. It is usually side effects in only a few individuals that kill a prospective new If one can avoid giving the drug to these few individuals through drug. knowledge of their biochemical peculiarities, the benefits of that drug could be made available for everybody else. Many chemicals that are now discarded could become drugs so that our therapeutic armamentarium would be expanded. However, for this to happen extensively, drug medication would have to be better targeted than is usually the case, that is, decisions for medication should be weighed similarly as is any surgical intervention. This change of therapeutic philosophy is not likely near at hand but is worth much effort.
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In the meantime, it seems logical for the pharmaceutical industry to utilize Human drug-metabolizing knowledge of pharmacogenetics in a different way. enzymes will become increasingly available in cell culture through targeted These will allow in vitro testing of enzyme formation after gene transfer. the metabolism of a prospective drug in order to assess the contribution of a If so, the chemical may be polymorphic enzyme to its biotransformation. discarded prior to the use in human beings. Alternatively, during phase I studies, the drug could be deliberately tested in a person with the defective enzyme, in order to see whether other elimination pathways can compensate for Finally, the structure of the drug might be deliberately the deficiency. altered so as to change its biotransformation pathways.
CONCLUSION Pharmacogenetics has become too important to be relegated to a position of neglect. Indeed it is foreseeable that pharmacogenetics will play an increasingly important role in clinical pharmacology and in medicine. Brodie had a hand in this development when he stressed species differences. I quoted two of his studies, that of hexobarbital metabolism in different species, and that of the metabolism of procaine which in humans occurs in plasma while other species metabolize it in liver. For a student of variation, it is only an additional step from the recognition of differences between species to We have to learn that differences between individuals within one species. differences between people can be discovered only by studying humans, and not by investigating rats, mice, or monkeys.
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