Drug Metabolism Reviews
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Drug Metabolism: Prospects for the Future Kenneth C. Leibman To cite this article: Kenneth C. Leibman (1979) Drug Metabolism: Prospects for the Future, Drug Metabolism Reviews, 10:2, 299-309, DOI: 10.3109/03602537908997475 To link to this article: http://dx.doi.org/10.3109/03602537908997475
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 4
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idmr20 Download by: [University of Otago]
Date: 15 November 2015, At: 01:45
DRUG METABOLISM REVIEWS, 10(2), 299-309 (1979)
Downloaded by [University of Otago] at 01:45 15 November 2015
Drug Metabolism: Prospects for the Future* KENNETH C . LEIBMAN Department of Pharmacology University of Florida Gainesville, Florida 32610
When I first started working in the a r e a of drug metabolism, just about 20 y e a r s ago, someone asked why I wanted to get into a field that had been so thoroughly worked over, and in which there was probably nothing of importance still to discover, After all, he said, the major pathways of metabolism of drugs had been elucidated, most of them in the 19th century (Table 1). Even the mechanism of drug oxidation was known, said my questioner. It occurs in the liver microsomes, requires NADPH and oxygen, and is therefore to be classified as a mixed-function oxidase; this had been discovered a decade before by a postdoctoral fellow in the Millers' laboratory, and had been extended to the oxidation of a large number of drugs by Brodie's group (Table 2). The increase in amount of drug-metabolizing enzyme after treatment with various drugs had also been discovered in the Millers' laboratory, this time by a graduate student, and had just been shown to be general for a number of substrates and inducers. Surely there was little more worthwhile to be done in the field; the r e s t would just be mopping up. It appeared, however, that there were a few more things to be discovered about drug metabolism within the following 20 years.
*Presented a t Third Annual Symposium on "Drug Metabolism Today and Tomorrow,'' held in Fort Washington, Pennsylvania, May 2 and 3, 1979, under the auspices of the Drug Metabolism Discussion Group. 299 Copyright 0 1980 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical. including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
300
LEIBMAN TABLE 1
Downloaded by [University of Otago] at 01:45 15 November 2015
Discovery of Drug-Metabolic Pathwaysa Hippuric acid formation from benzoic acid
Keller 1842
Aromatic hydroxylation of benzene and sidechain hydroxylation of toluene
Schultzen and Naunyn 1867
Sulfate conjugation of phenol
Baumann 1876
Glucuronide conjugation of o-nitrotoluene and camphor metabolites
Jaffe' 1874 Schmiedeberg and Meyer 1879
Mercapturic acid formation from halobenzenes
Baumann and Preusse, Jaffk 1879
Reduction of chloral hydrate
von Mering 1884
Methylation of pyridine
H i s 1887
Nitroreduction and arylamine ace tylation
Cohn 1893
N-Dealkylation of methylamine
Pohl 1893
0-Demethylation of isoform
Rohmann 1905
N-Hydroxylation of acetanilide
Ellinger 1920
N-Oxidation of trimethylamine
Linzel, Hoppe-Seyler 1934
Azoreduction of prontosil
Tr6foue1, Trkfouel, Nitti, and Bovet 1935
acornpiled from Refs. 1 and 2. TABLE 2 Microsomal Mixed-Function Oxidation of Drugs Discovery
Mueller and Miller 1949 [31 Brodie e t al. 1954 [4]
Induction
Conney, Miller, and Miller 1956 [51 Conney and Burns 1959 [6]' Remmer 1959 [71
PROSPECTS FOR THE FUTURE
301
TABLE 3 Epoxidation Proposed
Boyland 1950 [8]
Heptachlor (in vivo)
Radomski and Davidow 1953 [9]
Downloaded by [University of Otago] at 01:45 15 November 2015
Microsomal epoxidation: Aldrin
Wong and T e r r i e r e 1965 [ l o ]
Trichloroethylene (proposed)
Byington and Leibman 1965 [ l l ]
Styrene, cycloalkenes
Leibman and Ortiz 1968 [12)
Octene
Watabe and Maynert 1968 [131
Naphthalene, toluene
Jerina e t al. 1968 [141
Indeed, a major metabolic pathway has been elucidated within that time. Epoxidation had been proposed many years ago by Boyland a s an intermediate step in the metabolism of naphthalene to dihydrodiols (Table 3). The great reactivity of most epoxides, however, made the demonstration of such a pathway impossible with the techniques available at the time. However, one type of epoxide that is quite stable is that of the polychlorinated cyclodiene insecticides; within a few years it was shown that the epoxide of one of these insecticides, heptachlor, could be extracted from the fat of animals given the substance. Over a decade went by before it w a s shown that the epoxidation of the cyclodiene insecticides was a typical mixed-function oxidase reaction occurring in liver microsomes. At that time we were trying to explain how a chlorine atom could be moved from one carbon atom to another during the microsomal oxidation of trichloroethylene to chloral hydrate. It appeared to u s that trichloroethylene oxide would be a very likely candidate for the first product of oxidation; this could rearrange to chloral either directly or after hydration to the respective glycol. Because of the great instability of such an epoxide, containing both oxygen and halogen functions on the same carbon atoms, we elected to investigate the oxidation of simpler unsaturated compounds, and were finally able to demonstrate the microsomal epoxidation of styrene and of several cycloalkenes; in the same year, epoxidation was shown to occur with long-chain aliphatic olefins and aromatic compounds, Epoxidation has since been shown to be a most important
Downloaded by [University of Otago] at 01:45 15 November 2015
302
LEIBMAN
reaction of compounds containing unsaturated bonds ; the reactive epoxide group is able to react with nucleophilic centers in various tissue constituents, with the consequent risk of altering an essential function and initiating a chain of events leading to cell death o r mutation [Is]. Only a few epoxides have been found to be excreted metabolites; in these cases the structure of the metabolites is such that the epoxide bridge is quite stable. Such metabolites a r e not very toxic, Certainly elucidation of the unstable, toxic epoxide metabolites would never have been possible by the classical method of searching for drug metabolites in the excreta, During these 20 y e a r s the nature of the enzymatic apparatus that catalyzes drug-oxidative reactions has been elucidated (Table 4). Within a 10-year period the carbon monoxide-binding pigment of liver microsomes was discovered and characterized, its function as the terminal oxidase-oxygenase of the microsomal drug-oxidizing enzyme system was confirmed, its heterogeneity began to be mapped, and i t s solubilization and purification was accomplished. Also during these 20 y e a r s much information has been gained on the events that occur during the induction of the liver microsomal drug-metabolizing enzyme system [ 171. The transcription and translation processes were shown to operate independently of each other, and both to be affected by the induction process, with the production of more messenger RNA in the nucleus, which then operates more efficiently at the ribosomes. Finally, a beginning has been made during these 2 0 years in the study of the relationship of drug metabolism to toxicity. In former times, drug metabolism was considered to be merely part of the elimination process for drugs, Neumeister [18] coined the t e r m "Entgiftung" for the process in 1895, and drug metabolism was commonly referred to a s 'ldetoxicationll during the first half of this century, Indeed, Williams' seminal volume [19] on drug metabolism (1947, 1959) was entitled Detoxication Mechanisms despite the fact that there had been many indications by that time that pharmacologic o r toxicologic activity may be due to metabolites rather than to the parent drugs. Thus the reduction of inactive pentavalent arsenicals to active trivalent arsenicals had been studied by Ehrlich in the first decade of this century [ZO], and the classical discovery of the reductive cleavage of prontosil to give sulfanilamide [21] had broken the German patent and ushered in the age of the sulfa drugs. At about the time of the appearance of Williams' first edition, Brodie and Axelrod [22] were demonstrating that the metabolic interrelationships among acetanilide, acetophenetidine, acetaminophen, and aniline
PROSPECTS FOR THE FUTURE
303
TABLE 4
Downloaded by [University of Otago] at 01:45 15 November 2015
Cytochrome P-450a Discovery
Klingenberg, Garfinkel 1958
Characterization
Mason 1962 Omura and Sato 1964
Function
Estabrook, Cooper e t al. 1963-5
Multiplicity
Sladek and Mannering 1966
Solubilization, purification
Lu and Coon 1968
a
Compiled from Ref. 16.
provided examples of the metabolic formation of substances more active than the parent compounds both pharmacologically and toxicologically, as well as their eventual detoxication. Between the two editions of the Williams monograph, Peters [23] studied the toxicologic implications of the conversion of fluoroacetic acid to fluorocitrate and coined the term "lethal synthesis," and Kiese [24] was finally able to demonstrate the formation from aromatic amines of phenylhydroxylamines which had long been known to cause methemoglobin production. During this time a s w e l l the first demonstration of covalent binding of an active metabolite of a carcinogenic compound to cell constituents was offered, again by the Millers [25]; in the last decade there has been a great deal of investigation and discussion of the role of covalent binding of reactive intermediates in the toxicity of foreign compounds [26]. Quite recently, enzymologists have been considering a special case of metabolic activation in which the metabolite inactivates the enzyme that formed it-the so-called "suicide inactivator s 'I [2 71. Yesterday and today lead inexorably to tomorrow, as Macbeth observed. We a r e now asked to cast the knucklebones to predict what might be some of the advances that a r e likely to be made in some of the a r e a s that we have been discussing. First, a s to pathways of drug metabolism: Has the book of important metabolic pathways been closed o r may we expect to see more such reactions to be revealed in future? If we examine recent reviews
Downloaded by [University of Otago] at 01:45 15 November 2015
304
LEIBMAN
on novel pathways of drug metabolism [28, 291, we find that most of the reactions described are extensions of those known previously. Thus new kinds of glucuronide linkages have been discovered within the past few years, involving linkages with tertiary amines and quaternary ammonium compounds, and direct linkages with carbon atoms. Many metabolites have been isolated that arise from quite unusual metabolic reactions, However, each of these appears to be unique, at least as is s o far known, to the particular compoundwith which it was discovered, or to a very small group of very closely related compounds. A s medicinal chemists synthesize compounds of more and more unusual structure in their search for new pharmacophores, it is inevitable that we shall be discovering some rather bizarre metabolic reactions. It thus appears that the major new pathway discovered in the past 20 y e a r s is the epoxidation of carbon-carbon double bonds, together with the epoxide hydrase reaction elucidated a t the same time. Why was this pathway, anticipated for s o long, s o late in becoming evident? The answer lies in the high degree of reactivity and short lifetime of most of the epoxides. It required quite sensitive methodology to detect the small amounts of epoxide metabolites that were present for short periods of time. Twenty y e a r s ago the well-equipped worker in drug metabolism had at his o r h e r disposal equipment for paper chromatography, ion-exchange chromatography, countercurrent distribution, radioisotope counting (largely from solid samples), ultraviolet and infrared spectrophotometry, and fluorometry. These two decades have seen the adoption into common usage of thin-layer, gas, and high-pressure liquid chromatography and mass and nuclear magnetic resonance spectrometry. Studies on enzyme systems may involve the use of high-gain split-beam spectrophotometry of turbid samples, electron spin-resonance spectrometry, optical rotatory dispersion/circular dichroism studies, and even more sophisticated methodology. Some of the major discoveries of the past 20 years could have been made, albeit much more slowly and with much greater difficulty, without the use of these expensive Black Boxes, However, many of them would have remained hidden from view without the delicate, specific, and sensitive probes afforded by advanced technology. Some of the most interesting and significant questions that can now be asked a r e going to have to await the development of even more sophisticated apparatus for their answers. A s the major pathways of detoxicative metabolism of more and more compounds become known, we shall be showing increasing interest in the minor, reactive, ephemeral
Downloaded by [University of Otago] at 01:45 15 November 2015
PROSPEC'I'S FOR THE FUTURE
305
metabolites that are responsible for toxicity. Many of these appear t o be very reactive products formed nonenzymatically from rather unstable metabolites that a r e products of well-known metabolic pathways, The methodology required to follow these rapid changes of minute amounts of material will probably require instrumentation totally different in principle from that available today. In addition to identification of the toxic intermediates, we need more knowledge of how they interact with tissue constituents to produce their damaging effects. Essentially our sole probe a t present is the measurement of covalent binding. How accurate an index of toxic interactions is covalent binding? Recently i t has been shown that m-hydroxyacetanilide, an isomer of acetaminophen, is metabolically converted to a form that is covalently bound to microsomes, similarly to acetaminophen [30]. The m-hydroxy isomer, however, does not cause hepatotoxicity a s does acetaminophen. This experiment will certainly stimulate further work on the significance of covalent binding in the hepatotoxicity of reactive intermediates, Besides the products of metabolism, we need to know more about the enzyme systems involved in their formation. Figure 1 shows current thinking on the mechanism of oxidation of foreign compounds by the microsomal cytochrome P-450 system. This represents a synthesis of the work of scores of scientists in a half-dozen countries over the last 20 years. Only the heme iron of the cytochrome is depicted here. Two electrons must be applied to the system: one to reduce the ferriheme to a ferroheme capable of reacting with oxygen while already bound to the compound that is to serve a s oxygen acceptor, A, and the other to reduce the oxygen and prepare one of the oxygen atoms for reaction with hydrogen atoms. The resulting monoxygen iron is the active oxygenating complex, believed to be a ferry1 group because of its spectral properties and because, similarly to the Compound I formed in peroxidase systems, it appears when substrate oxidation is driven by certain organic hydroperoxides, as shown by the broken arrow, in the absence of oxygen o r reducing equivalents. Breakdown of the substrate-ferry1 complex then results in the formation of the oxygenated substrate and the free ferriheme. The first electron is provided by the NADPH-cytochrome P-450 reductase flavoprotein enzyme, The second electron may come under certain circumstances via cytochrome b, [32]. However, we need to know more about the sources and mechanism of transfer of this second electron. We also need to know more about how different substrates bind to the different kinds of cytochrome P-450 that have been demonstrated. What determines the specificity of the various cytochromes
LEIBMAN
306
I
,
A
A
Downloaded by [University of Otago] at 01:45 15 November 2015
I I I I I
w
o q ferraur
1-
ROOH
+e
I I
[ir.
++++0-
i - ;'+++I 2H+
e
c
A fel.rY2
A
ferric monoxygen
HZO
femic hgdroperow
ferrom superoxide
FIG, 1. Current concepts of reactions a t the heme iron of cytochrome P-450 during mixed-function oxidation (adapted from Ref. 3 1 ) .
P-4501 How a r e substrates oriented for reaction with the heme iron? W e may look forward to answers to some of these questions in the next decade as more information on the chemical nature of cytochrome P-450 becomes available. Although much is now known about the events that occur during the induction of drug-metabolizing enzyme systems, the most basic questions remain unanswered, We know that the production of messenger RNA at the DNA templates in the nuclei is enhanced [IT]. What, however, is the impulse for this process, so that a certain lipidsoluble compound that can be metabolized by the microsomal enzymes, o r a metabolite thereof, can initiate a process that results i n the increased synthesis of a specific s e t of messenger RNA molecules ? Is this a direct action on the nucleus o r is it a feedback effect transmitted by some substance released from the microsomes? How much is enzyme catabolism affected by inducing agents? We may expect that answers to some of these questions will be forthcoming. In addition to the pharmacologic alteration of drug-metabolizing enzymes, we need to know more about the biological regulation of these systems. Why, for instance, do s e x differences in the r a t e of many
Downloaded by [University of Otago] at 01:45 15 November 2015
PROSPECTS FOR THE FUTURE
307
drug-metabolizing reactions occur in some species and not in others? We know something of the development of the drug-metabolism apparatus in very early life, but we a r e only beginning to measure the changes that occur at the other end of the life span, and are still far from understanding them. We must know more about the alterations in drug metabolism that occur in different pathological o r physiological states, and about the status of drug metabolism in various models of disease. Thus it has recently been demonstrated that in a chemically induced arthritis of r a t s that is commonly used for the testing of anti-inflammatory drugs, the metabolism of many of these drugs is markedly altered [33]. The implications of this finding for the use of such a model for screening purposes a r e obvious. This will probably not be the last such metabolic alteration to be demonstrated in disease models. A s t a r t has been made in understanding the relationship between diet and drug metabolism, but much is not understood. A spicy diet may induce the hepatic drug-metabolizing enzymes [34], whereas eating the lowly cabbage induces those of the intestine [%I. How many other agents a r e present in common foods that perturb the drug-metabolizing systems ? What can the specificity of these agents tell u s about the similarities and differences among the drug-metabolizing enzymes of the different organs ? Thus, just a s there has been enough to do to occupy a large comunity of scientists in the study of the many facets of drug metabolism during the last score of years, we may be sure that there will be enough to occupy the hands and minds of researchers in this field in the next score. We need new ideas, new approaches, and new insights, but a s long a s devoted workers continue to ask the questions, answers will eventually be forthcoming.
REFERENCES
[ll c21
C31
.
A. Conti and M.. H.- Bickel. Drug Metab. Rev.. , 6.. 1 (1977). Y
L. Young, in Drug Metabolism-From Microbe t i Man (D. V. Parke and R. L. Smith, eds.), Taylor and Francis, London, 1977, pp. 1-11. G. C. Mueller and J. A. Miller, J. Biol. Chem., 180, 1125 (1949).
c41
c 51
B. B. Brodie, J. Axelrod, J. R. Cooper, L. Gaudette, B. N. LaDu, C. Mitoma, and S. Udenfriend, Science, 121, 603 (1955). A . H. Conney, E. C. Miller, and J. A. Miller, Cancer Res., 16, 450 (1956).
LEIBMAN
308 C6 3
"7 1 [8 1 [9 1
Downloaded by [University of Otago] at 01:45 15 November 2015
ClO1 El1 1
Cl8 1 C19 1 L20 1 [211 [22
1
184, 363 (1959). H. Remmer, Naturwissenschaften, 46, 580 (1959). E. Boyland, Biochem. SOC. Symp., 2, 40 (1950). J. L. Radomski and B. Davidow, J. Pharmacol. Exp. Ther., 107, 266 (1953). K T . Wong and L. C. Terriere, Biochem. Pharmacol., 1_4, 375 (1965). K. H. Byington and K. C. Leibman, Mol. Pharmacol., 1, 247 (1965). K. C. Leibman and E. Ortiz, Pharmacologist, lo, 203 (1968). T. Watabe and E. W. Maynert, X d . , l_o, 203 (1968). D. M. Jerina, J. W. Daly, B. Witkop, P. Zaltzman-Nirenberg, and S. Udenfriend, J. Am, Chem. Soc., 90, 6525 (1968). R. C. Garner, Prog. Drug Metab., 1, 77 (1976). T. Omura, in Cytochrome P-450 (R. Sat0 and T. Omura, eds.), Kodansha, Tokyo, 1978, pp. 1-21. H. V. Gelboin, in Concepts in Biochemical Pharmacology, P a r t 2 (B. B. Brodie and J. R. Gillette, eds.), Springer, Berlin, 1971, pp. 431-451. R. Neumeister, Lehrb. Physiol. Chem., 2, 346 (1895); cited in Ref. 2. R. T. Williams, Detoxication Mechanisms, 1st ed., Chapman and Hall, London, 1947; 2nd ed., 1959. P. Ehrlich, Ber. Dtsch. Chem. Ges., 42, 17 (1909). J. Tre'fouel, Mme. J. Tre'fouel, F. Nitti, and D. Bovet, C. R. SOC. Biol., K O , 756 (1935). B. B. Brodie and J. Axelrod, J. Pharmacol. Exp. Ther., 94, 29 (1948). R. A. Peters, Bull, Johns Hopkins Hosp., 97, 21 (1955). M. Kiese, Naunyn-Schmiedebergs Arch, Exp. Pathol. Pharmakol., 235, 360 (1959). E. C. Miller and J. A. Miller, Cancer Res., '7, 468 (1947). D. J. Jollow, J. J. Kocsis, R. Snyder, and H. Vainio (eds.), Biological Reactive Intermediates, Plenum, New York, 1977. R. H. Abeles, in Enzyme-Activated Irreversible Inhibitors (N. Seiler, M. J. Jung, and J. Koch-Weser, eds.), Elsevier/ North-Holland, Amsterdam, 1978, pp. 1-12; R, R. Rando, Wd., pp. 13-26. Z. H. Israili, P. G. Dayton, and J. R. Kiechel, Drug Metab. Dispos., 2, 411 (1977).
A. H. Conney and J. J. Burns, Nature (London),
PROSPECTS FOR THE FUTURE
[29] [30] [31 ] [32]
Downloaded by [University of Otago] at 01:45 15 November 2015
[33
3
[34] [35]
309
B. Testa and P. Jenner, Drug Metab. Rev., I, 325 (1978). S. A. Roberts and D. J. Jollow, Fed. Proc., Fed, Am. SOC. Exp. Biol., 38, 4260 (1979). E. G. H r y c a E J. -A. Gustafsson, M. Ingelmann-Sundberg, and L. Ernster, Eur. J. Biochem., g ,43 (1976). G . J. Mannering, S. Kuwahara, and T. Omura, Biochem. Biophys. Res. Commun., 57, 476 (1974). K. F. Swingle, S. F. Chang, and E. H. Erickson, Biochem. Pharmacol., 27, 2395 (1978). A. E. M . McLean and H. E. Driver, E d . , 3,1299 (1977). E. J. Pantuck, K. C. Hsiao, W. D. Loub, L. W. Wattenberg, R. Kuntzman, and A. H. Conney, J. Pharmacol. Exp. Ther., 198, 278 (1976).
ISSN: 0360-2532 (Print) 1097-9883 (Online) Journal homepage: http://www.tandfonline.com/loi/idmr20
Drug Metabolism: Prospects for the Future Kenneth C. Leibman To cite this article: Kenneth C. Leibman (1979) Drug Metabolism: Prospects for the Future, Drug Metabolism Reviews, 10:2, 299-309, DOI: 10.3109/03602537908997475 To link to this article: http://dx.doi.org/10.3109/03602537908997475
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 4
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=idmr20 Download by: [University of Otago]
Date: 15 November 2015, At: 01:45
DRUG METABOLISM REVIEWS, 10(2), 299-309 (1979)
Downloaded by [University of Otago] at 01:45 15 November 2015
Drug Metabolism: Prospects for the Future* KENNETH C . LEIBMAN Department of Pharmacology University of Florida Gainesville, Florida 32610
When I first started working in the a r e a of drug metabolism, just about 20 y e a r s ago, someone asked why I wanted to get into a field that had been so thoroughly worked over, and in which there was probably nothing of importance still to discover, After all, he said, the major pathways of metabolism of drugs had been elucidated, most of them in the 19th century (Table 1). Even the mechanism of drug oxidation was known, said my questioner. It occurs in the liver microsomes, requires NADPH and oxygen, and is therefore to be classified as a mixed-function oxidase; this had been discovered a decade before by a postdoctoral fellow in the Millers' laboratory, and had been extended to the oxidation of a large number of drugs by Brodie's group (Table 2). The increase in amount of drug-metabolizing enzyme after treatment with various drugs had also been discovered in the Millers' laboratory, this time by a graduate student, and had just been shown to be general for a number of substrates and inducers. Surely there was little more worthwhile to be done in the field; the r e s t would just be mopping up. It appeared, however, that there were a few more things to be discovered about drug metabolism within the following 20 years.
*Presented a t Third Annual Symposium on "Drug Metabolism Today and Tomorrow,'' held in Fort Washington, Pennsylvania, May 2 and 3, 1979, under the auspices of the Drug Metabolism Discussion Group. 299 Copyright 0 1980 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical. including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
300
LEIBMAN TABLE 1
Downloaded by [University of Otago] at 01:45 15 November 2015
Discovery of Drug-Metabolic Pathwaysa Hippuric acid formation from benzoic acid
Keller 1842
Aromatic hydroxylation of benzene and sidechain hydroxylation of toluene
Schultzen and Naunyn 1867
Sulfate conjugation of phenol
Baumann 1876
Glucuronide conjugation of o-nitrotoluene and camphor metabolites
Jaffe' 1874 Schmiedeberg and Meyer 1879
Mercapturic acid formation from halobenzenes
Baumann and Preusse, Jaffk 1879
Reduction of chloral hydrate
von Mering 1884
Methylation of pyridine
H i s 1887
Nitroreduction and arylamine ace tylation
Cohn 1893
N-Dealkylation of methylamine
Pohl 1893
0-Demethylation of isoform
Rohmann 1905
N-Hydroxylation of acetanilide
Ellinger 1920
N-Oxidation of trimethylamine
Linzel, Hoppe-Seyler 1934
Azoreduction of prontosil
Tr6foue1, Trkfouel, Nitti, and Bovet 1935
acornpiled from Refs. 1 and 2. TABLE 2 Microsomal Mixed-Function Oxidation of Drugs Discovery
Mueller and Miller 1949 [31 Brodie e t al. 1954 [4]
Induction
Conney, Miller, and Miller 1956 [51 Conney and Burns 1959 [6]' Remmer 1959 [71
PROSPECTS FOR THE FUTURE
301
TABLE 3 Epoxidation Proposed
Boyland 1950 [8]
Heptachlor (in vivo)
Radomski and Davidow 1953 [9]
Downloaded by [University of Otago] at 01:45 15 November 2015
Microsomal epoxidation: Aldrin
Wong and T e r r i e r e 1965 [ l o ]
Trichloroethylene (proposed)
Byington and Leibman 1965 [ l l ]
Styrene, cycloalkenes
Leibman and Ortiz 1968 [12)
Octene
Watabe and Maynert 1968 [131
Naphthalene, toluene
Jerina e t al. 1968 [141
Indeed, a major metabolic pathway has been elucidated within that time. Epoxidation had been proposed many years ago by Boyland a s an intermediate step in the metabolism of naphthalene to dihydrodiols (Table 3). The great reactivity of most epoxides, however, made the demonstration of such a pathway impossible with the techniques available at the time. However, one type of epoxide that is quite stable is that of the polychlorinated cyclodiene insecticides; within a few years it was shown that the epoxide of one of these insecticides, heptachlor, could be extracted from the fat of animals given the substance. Over a decade went by before it w a s shown that the epoxidation of the cyclodiene insecticides was a typical mixed-function oxidase reaction occurring in liver microsomes. At that time we were trying to explain how a chlorine atom could be moved from one carbon atom to another during the microsomal oxidation of trichloroethylene to chloral hydrate. It appeared to u s that trichloroethylene oxide would be a very likely candidate for the first product of oxidation; this could rearrange to chloral either directly or after hydration to the respective glycol. Because of the great instability of such an epoxide, containing both oxygen and halogen functions on the same carbon atoms, we elected to investigate the oxidation of simpler unsaturated compounds, and were finally able to demonstrate the microsomal epoxidation of styrene and of several cycloalkenes; in the same year, epoxidation was shown to occur with long-chain aliphatic olefins and aromatic compounds, Epoxidation has since been shown to be a most important
Downloaded by [University of Otago] at 01:45 15 November 2015
302
LEIBMAN
reaction of compounds containing unsaturated bonds ; the reactive epoxide group is able to react with nucleophilic centers in various tissue constituents, with the consequent risk of altering an essential function and initiating a chain of events leading to cell death o r mutation [Is]. Only a few epoxides have been found to be excreted metabolites; in these cases the structure of the metabolites is such that the epoxide bridge is quite stable. Such metabolites a r e not very toxic, Certainly elucidation of the unstable, toxic epoxide metabolites would never have been possible by the classical method of searching for drug metabolites in the excreta, During these 20 y e a r s the nature of the enzymatic apparatus that catalyzes drug-oxidative reactions has been elucidated (Table 4). Within a 10-year period the carbon monoxide-binding pigment of liver microsomes was discovered and characterized, its function as the terminal oxidase-oxygenase of the microsomal drug-oxidizing enzyme system was confirmed, its heterogeneity began to be mapped, and i t s solubilization and purification was accomplished. Also during these 20 y e a r s much information has been gained on the events that occur during the induction of the liver microsomal drug-metabolizing enzyme system [ 171. The transcription and translation processes were shown to operate independently of each other, and both to be affected by the induction process, with the production of more messenger RNA in the nucleus, which then operates more efficiently at the ribosomes. Finally, a beginning has been made during these 2 0 years in the study of the relationship of drug metabolism to toxicity. In former times, drug metabolism was considered to be merely part of the elimination process for drugs, Neumeister [18] coined the t e r m "Entgiftung" for the process in 1895, and drug metabolism was commonly referred to a s 'ldetoxicationll during the first half of this century, Indeed, Williams' seminal volume [19] on drug metabolism (1947, 1959) was entitled Detoxication Mechanisms despite the fact that there had been many indications by that time that pharmacologic o r toxicologic activity may be due to metabolites rather than to the parent drugs. Thus the reduction of inactive pentavalent arsenicals to active trivalent arsenicals had been studied by Ehrlich in the first decade of this century [ZO], and the classical discovery of the reductive cleavage of prontosil to give sulfanilamide [21] had broken the German patent and ushered in the age of the sulfa drugs. At about the time of the appearance of Williams' first edition, Brodie and Axelrod [22] were demonstrating that the metabolic interrelationships among acetanilide, acetophenetidine, acetaminophen, and aniline
PROSPECTS FOR THE FUTURE
303
TABLE 4
Downloaded by [University of Otago] at 01:45 15 November 2015
Cytochrome P-450a Discovery
Klingenberg, Garfinkel 1958
Characterization
Mason 1962 Omura and Sato 1964
Function
Estabrook, Cooper e t al. 1963-5
Multiplicity
Sladek and Mannering 1966
Solubilization, purification
Lu and Coon 1968
a
Compiled from Ref. 16.
provided examples of the metabolic formation of substances more active than the parent compounds both pharmacologically and toxicologically, as well as their eventual detoxication. Between the two editions of the Williams monograph, Peters [23] studied the toxicologic implications of the conversion of fluoroacetic acid to fluorocitrate and coined the term "lethal synthesis," and Kiese [24] was finally able to demonstrate the formation from aromatic amines of phenylhydroxylamines which had long been known to cause methemoglobin production. During this time a s w e l l the first demonstration of covalent binding of an active metabolite of a carcinogenic compound to cell constituents was offered, again by the Millers [25]; in the last decade there has been a great deal of investigation and discussion of the role of covalent binding of reactive intermediates in the toxicity of foreign compounds [26]. Quite recently, enzymologists have been considering a special case of metabolic activation in which the metabolite inactivates the enzyme that formed it-the so-called "suicide inactivator s 'I [2 71. Yesterday and today lead inexorably to tomorrow, as Macbeth observed. We a r e now asked to cast the knucklebones to predict what might be some of the advances that a r e likely to be made in some of the a r e a s that we have been discussing. First, a s to pathways of drug metabolism: Has the book of important metabolic pathways been closed o r may we expect to see more such reactions to be revealed in future? If we examine recent reviews
Downloaded by [University of Otago] at 01:45 15 November 2015
304
LEIBMAN
on novel pathways of drug metabolism [28, 291, we find that most of the reactions described are extensions of those known previously. Thus new kinds of glucuronide linkages have been discovered within the past few years, involving linkages with tertiary amines and quaternary ammonium compounds, and direct linkages with carbon atoms. Many metabolites have been isolated that arise from quite unusual metabolic reactions, However, each of these appears to be unique, at least as is s o far known, to the particular compoundwith which it was discovered, or to a very small group of very closely related compounds. A s medicinal chemists synthesize compounds of more and more unusual structure in their search for new pharmacophores, it is inevitable that we shall be discovering some rather bizarre metabolic reactions. It thus appears that the major new pathway discovered in the past 20 y e a r s is the epoxidation of carbon-carbon double bonds, together with the epoxide hydrase reaction elucidated a t the same time. Why was this pathway, anticipated for s o long, s o late in becoming evident? The answer lies in the high degree of reactivity and short lifetime of most of the epoxides. It required quite sensitive methodology to detect the small amounts of epoxide metabolites that were present for short periods of time. Twenty y e a r s ago the well-equipped worker in drug metabolism had at his o r h e r disposal equipment for paper chromatography, ion-exchange chromatography, countercurrent distribution, radioisotope counting (largely from solid samples), ultraviolet and infrared spectrophotometry, and fluorometry. These two decades have seen the adoption into common usage of thin-layer, gas, and high-pressure liquid chromatography and mass and nuclear magnetic resonance spectrometry. Studies on enzyme systems may involve the use of high-gain split-beam spectrophotometry of turbid samples, electron spin-resonance spectrometry, optical rotatory dispersion/circular dichroism studies, and even more sophisticated methodology. Some of the major discoveries of the past 20 years could have been made, albeit much more slowly and with much greater difficulty, without the use of these expensive Black Boxes, However, many of them would have remained hidden from view without the delicate, specific, and sensitive probes afforded by advanced technology. Some of the most interesting and significant questions that can now be asked a r e going to have to await the development of even more sophisticated apparatus for their answers. A s the major pathways of detoxicative metabolism of more and more compounds become known, we shall be showing increasing interest in the minor, reactive, ephemeral
Downloaded by [University of Otago] at 01:45 15 November 2015
PROSPEC'I'S FOR THE FUTURE
305
metabolites that are responsible for toxicity. Many of these appear t o be very reactive products formed nonenzymatically from rather unstable metabolites that a r e products of well-known metabolic pathways, The methodology required to follow these rapid changes of minute amounts of material will probably require instrumentation totally different in principle from that available today. In addition to identification of the toxic intermediates, we need more knowledge of how they interact with tissue constituents to produce their damaging effects. Essentially our sole probe a t present is the measurement of covalent binding. How accurate an index of toxic interactions is covalent binding? Recently i t has been shown that m-hydroxyacetanilide, an isomer of acetaminophen, is metabolically converted to a form that is covalently bound to microsomes, similarly to acetaminophen [30]. The m-hydroxy isomer, however, does not cause hepatotoxicity a s does acetaminophen. This experiment will certainly stimulate further work on the significance of covalent binding in the hepatotoxicity of reactive intermediates, Besides the products of metabolism, we need to know more about the enzyme systems involved in their formation. Figure 1 shows current thinking on the mechanism of oxidation of foreign compounds by the microsomal cytochrome P-450 system. This represents a synthesis of the work of scores of scientists in a half-dozen countries over the last 20 years. Only the heme iron of the cytochrome is depicted here. Two electrons must be applied to the system: one to reduce the ferriheme to a ferroheme capable of reacting with oxygen while already bound to the compound that is to serve a s oxygen acceptor, A, and the other to reduce the oxygen and prepare one of the oxygen atoms for reaction with hydrogen atoms. The resulting monoxygen iron is the active oxygenating complex, believed to be a ferry1 group because of its spectral properties and because, similarly to the Compound I formed in peroxidase systems, it appears when substrate oxidation is driven by certain organic hydroperoxides, as shown by the broken arrow, in the absence of oxygen o r reducing equivalents. Breakdown of the substrate-ferry1 complex then results in the formation of the oxygenated substrate and the free ferriheme. The first electron is provided by the NADPH-cytochrome P-450 reductase flavoprotein enzyme, The second electron may come under certain circumstances via cytochrome b, [32]. However, we need to know more about the sources and mechanism of transfer of this second electron. We also need to know more about how different substrates bind to the different kinds of cytochrome P-450 that have been demonstrated. What determines the specificity of the various cytochromes
LEIBMAN
306
I
,
A
A
Downloaded by [University of Otago] at 01:45 15 November 2015
I I I I I
w
o q ferraur
1-
ROOH
+e
I I
[ir.
++++0-
i - ;'+++I 2H+
e
c
A fel.rY2
A
ferric monoxygen
HZO
femic hgdroperow
ferrom superoxide
FIG, 1. Current concepts of reactions a t the heme iron of cytochrome P-450 during mixed-function oxidation (adapted from Ref. 3 1 ) .
P-4501 How a r e substrates oriented for reaction with the heme iron? W e may look forward to answers to some of these questions in the next decade as more information on the chemical nature of cytochrome P-450 becomes available. Although much is now known about the events that occur during the induction of drug-metabolizing enzyme systems, the most basic questions remain unanswered, We know that the production of messenger RNA at the DNA templates in the nuclei is enhanced [IT]. What, however, is the impulse for this process, so that a certain lipidsoluble compound that can be metabolized by the microsomal enzymes, o r a metabolite thereof, can initiate a process that results i n the increased synthesis of a specific s e t of messenger RNA molecules ? Is this a direct action on the nucleus o r is it a feedback effect transmitted by some substance released from the microsomes? How much is enzyme catabolism affected by inducing agents? We may expect that answers to some of these questions will be forthcoming. In addition to the pharmacologic alteration of drug-metabolizing enzymes, we need to know more about the biological regulation of these systems. Why, for instance, do s e x differences in the r a t e of many
Downloaded by [University of Otago] at 01:45 15 November 2015
PROSPECTS FOR THE FUTURE
307
drug-metabolizing reactions occur in some species and not in others? We know something of the development of the drug-metabolism apparatus in very early life, but we a r e only beginning to measure the changes that occur at the other end of the life span, and are still far from understanding them. We must know more about the alterations in drug metabolism that occur in different pathological o r physiological states, and about the status of drug metabolism in various models of disease. Thus it has recently been demonstrated that in a chemically induced arthritis of r a t s that is commonly used for the testing of anti-inflammatory drugs, the metabolism of many of these drugs is markedly altered [33]. The implications of this finding for the use of such a model for screening purposes a r e obvious. This will probably not be the last such metabolic alteration to be demonstrated in disease models. A s t a r t has been made in understanding the relationship between diet and drug metabolism, but much is not understood. A spicy diet may induce the hepatic drug-metabolizing enzymes [34], whereas eating the lowly cabbage induces those of the intestine [%I. How many other agents a r e present in common foods that perturb the drug-metabolizing systems ? What can the specificity of these agents tell u s about the similarities and differences among the drug-metabolizing enzymes of the different organs ? Thus, just a s there has been enough to do to occupy a large comunity of scientists in the study of the many facets of drug metabolism during the last score of years, we may be sure that there will be enough to occupy the hands and minds of researchers in this field in the next score. We need new ideas, new approaches, and new insights, but a s long a s devoted workers continue to ask the questions, answers will eventually be forthcoming.
REFERENCES
[ll c21
C31
.
A. Conti and M.. H.- Bickel. Drug Metab. Rev.. , 6.. 1 (1977). Y
L. Young, in Drug Metabolism-From Microbe t i Man (D. V. Parke and R. L. Smith, eds.), Taylor and Francis, London, 1977, pp. 1-11. G. C. Mueller and J. A. Miller, J. Biol. Chem., 180, 1125 (1949).
c41
c 51
B. B. Brodie, J. Axelrod, J. R. Cooper, L. Gaudette, B. N. LaDu, C. Mitoma, and S. Udenfriend, Science, 121, 603 (1955). A . H. Conney, E. C. Miller, and J. A. Miller, Cancer Res., 16, 450 (1956).
LEIBMAN
308 C6 3
"7 1 [8 1 [9 1
Downloaded by [University of Otago] at 01:45 15 November 2015
ClO1 El1 1
Cl8 1 C19 1 L20 1 [211 [22
1
184, 363 (1959). H. Remmer, Naturwissenschaften, 46, 580 (1959). E. Boyland, Biochem. SOC. Symp., 2, 40 (1950). J. L. Radomski and B. Davidow, J. Pharmacol. Exp. Ther., 107, 266 (1953). K T . Wong and L. C. Terriere, Biochem. Pharmacol., 1_4, 375 (1965). K. H. Byington and K. C. Leibman, Mol. Pharmacol., 1, 247 (1965). K. C. Leibman and E. Ortiz, Pharmacologist, lo, 203 (1968). T. Watabe and E. W. Maynert, X d . , l_o, 203 (1968). D. M. Jerina, J. W. Daly, B. Witkop, P. Zaltzman-Nirenberg, and S. Udenfriend, J. Am, Chem. Soc., 90, 6525 (1968). R. C. Garner, Prog. Drug Metab., 1, 77 (1976). T. Omura, in Cytochrome P-450 (R. Sat0 and T. Omura, eds.), Kodansha, Tokyo, 1978, pp. 1-21. H. V. Gelboin, in Concepts in Biochemical Pharmacology, P a r t 2 (B. B. Brodie and J. R. Gillette, eds.), Springer, Berlin, 1971, pp. 431-451. R. Neumeister, Lehrb. Physiol. Chem., 2, 346 (1895); cited in Ref. 2. R. T. Williams, Detoxication Mechanisms, 1st ed., Chapman and Hall, London, 1947; 2nd ed., 1959. P. Ehrlich, Ber. Dtsch. Chem. Ges., 42, 17 (1909). J. Tre'fouel, Mme. J. Tre'fouel, F. Nitti, and D. Bovet, C. R. SOC. Biol., K O , 756 (1935). B. B. Brodie and J. Axelrod, J. Pharmacol. Exp. Ther., 94, 29 (1948). R. A. Peters, Bull, Johns Hopkins Hosp., 97, 21 (1955). M. Kiese, Naunyn-Schmiedebergs Arch, Exp. Pathol. Pharmakol., 235, 360 (1959). E. C. Miller and J. A. Miller, Cancer Res., '7, 468 (1947). D. J. Jollow, J. J. Kocsis, R. Snyder, and H. Vainio (eds.), Biological Reactive Intermediates, Plenum, New York, 1977. R. H. Abeles, in Enzyme-Activated Irreversible Inhibitors (N. Seiler, M. J. Jung, and J. Koch-Weser, eds.), Elsevier/ North-Holland, Amsterdam, 1978, pp. 1-12; R, R. Rando, Wd., pp. 13-26. Z. H. Israili, P. G. Dayton, and J. R. Kiechel, Drug Metab. Dispos., 2, 411 (1977).
A. H. Conney and J. J. Burns, Nature (London),
PROSPECTS FOR THE FUTURE
[29] [30] [31 ] [32]
Downloaded by [University of Otago] at 01:45 15 November 2015
[33
3
[34] [35]
309
B. Testa and P. Jenner, Drug Metab. Rev., I, 325 (1978). S. A. Roberts and D. J. Jollow, Fed. Proc., Fed, Am. SOC. Exp. Biol., 38, 4260 (1979). E. G. H r y c a E J. -A. Gustafsson, M. Ingelmann-Sundberg, and L. Ernster, Eur. J. Biochem., g ,43 (1976). G . J. Mannering, S. Kuwahara, and T. Omura, Biochem. Biophys. Res. Commun., 57, 476 (1974). K. F. Swingle, S. F. Chang, and E. H. Erickson, Biochem. Pharmacol., 27, 2395 (1978). A. E. M . McLean and H. E. Driver, E d . , 3,1299 (1977). E. J. Pantuck, K. C. Hsiao, W. D. Loub, L. W. Wattenberg, R. Kuntzman, and A. H. Conney, J. Pharmacol. Exp. Ther., 198, 278 (1976).
