The metabolism of the immunosuppressive drug azathio-prine (Imuran) to 1-methyl-4-nitro-5-(S-glutathionyl)imi-dazole and 6-mercaptopurine is an example of hetero-aromatic nucleophilic substitution involving GSH.433-435 Interestingly, 6-mercaptopurine formed in this reaction appears to be responsible for azathioprine's immunosuppressive activity.436
Arene oxides and aliphatic epoxides (or oxiranes) represent a very important class of substrates that are conjugated and detoxified by GSH.437 The three-membered oxygen-containing ring in these compounds is highly strained and, therefore, reactive toward ring cleavage by nucleophiles (e.g., GSH, H2O, or nucleophilic groups present on cellular macromolecules). As discussed previously, arene oxides and epoxides are intermediary products formed from CYP oxidation of aromatic compounds (arenes) and olefins, respectively. If reactive arene oxides (e.g., benzo[a]pyrene-4,5-oxide, 4-bromobenzene oxide) and aliphatic epoxides (e.g., styrene oxide) are not "neutralized" or detoxified by GSH S-transferase, epoxide hydrase, or other pathways, they ultimately covalently bind to cellular macromolecules and cause serious cytotoxicity and carcinogenicity. The isolation of GSH or mercapturic acid adducts from benzo[a]pyrene, bromobenzene, and styrene clearly demonstrates the importance of GSH in reacting with the reactive epoxide metabolites generated from these compounds.
GSH conjugation involving substitution at heteroatoms, such as oxygen, is seen often with organic nitrates. For example, nitroglycerin (Nitrostat) and isosorbide dinitrate (Isordil) are metabolized by a pathway involving an initial GSH conjugation reaction. The GSH conjugate products, however, are not metabolized to mercapturic acids but instead are converted enzymatically to the corresponding alcohol derivatives and glutathione disulfide (GSSG).438
The nucleophilic addition of GSH to electron-deficient carbon-carbon double bonds occurs mainly in compounds with a,S-unsaturated double bonds. In most instances, the double bond is rendered electron deficient by resonance or conjugation with a carbonyl group (ketone or aldehyde), ester, nitrile, or other. Such a,S-unsaturated systems undergo so-called Michael addition reactions with GSH to yield the corresponding GSH adduct.421-428 For example, in rats and dogs, the diuretic agent ethacrynic acid (Edecrin) reacts with GSH to form the corresponding GSH or mercap-tunc acid derivatives.439 Not all a,jS-unsaturated compounds are conjugated with GSH. Many steroidal agents with a ,S -unsaturated carbonyl moieties, such as prednisone and digitoxigenin, have evinced no significant conjugation with GSH. Steric factors, decreased reactivity of the double bond, and other factors (e.g., susceptibility to metabolic reduction of the ketone or the C=C double bond) may account for these observations.
Occasionally, metabolic oxidative biotransformation reactions may generate chemically reactive a,j-unsaturated systems that react with GSH. For example, metabolic oxidation of acetaminophen presumably generates the chemically reactive intermediate N-acetylimidoquinone. Michael addition of GSH to the imidoquinone leads to the corresponding mercapturic acid derivative in both animals and hu-mans.245,248 2-Hydroxyestrogens, such as 2-hydroxy-17j-estradiol, undergo conjugation with GSH to yield the two isomeric mercapturic acid or GSH derivatives. Although the exact mechanism is unclear, it appears that 2-hydroxyestro-gen is oxidized to a chemically reactive orthoquinone or semiquinone intermediate that reacts with GSH at either the electrophilic C-1 or C-4 position.440,441
In most instances, GSH conjugation is regarded as a detoxifying pathway that protects cellular macromolecules such as protein and DNA against harmful electrophiles. In a few cases, GSH conjugation has been implicated in causing toxic-ity. Often, this is because the GSH conjugates are themselves electrophilic (e.g., vicinal dihaloethanes) or give rise to metabolic intermediates (e.g., cysteine metabolites of haloalkenes) that are electrophilic.424-428 1,2-Dichloroethane, for example, reacts with GSH to produce S-(2-chloroethyl)glutathione; the nucleophilic sulfur group in this conjugate can internally dis place the chlorine group to give rise to an electrophilic three-membered ring episulfonium ion. The covalent interaction of the episulfonium intermediate with the guanosine moiety of DNA may contribute to the mutagenic and carcinogenic effects observed for 1,2-dichloroethane.425-427 The metabolic conversion of GSH conjugates to reactive cysteine metabolites is responsible for the nephrotoxicity associated with some halogenated alkanes and alkenes.428 The activation pathway appears to involve y-glutamyl transpeptidase and cysteine conjugate j-lyase, two enzymes that apparently target the conjugates to the kidney.
Acetylation constitutes an important metabolic route for drugs containing primary amino groups.408,442,443 This encompasses primary aromatic amines (ArNH2), sulfonamides (H2NC6H4SO2NHR), hydrazines (—NHNH2), hydrazides (—CONHNH2), and primary aliphatic amines. The amide derivatives formed from acetylation of these amino functionalities are generally inactive and nontoxic. Because water solubility is not enhanced greatly by N-acetylation, it appears that the primary function of acetylation is to terminate pharmacological activity and detoxification. A few reports indicate, however, that acetylated metabolites may be as active as (e.g., N-acetylprocainamide),444,445 or more toxic than (e.g., N-acetylisoniazid),446 447 their corresponding parent compounds.
The acetyl group used in N-acetylation of xenobiotics is supplied by acetyl-CoA.408 Transfer of the acetyl group from this cofactor to the accepting amino substrate is carried out by soluble N-acetyltransferases present in hepatic reticuloen-dothelial cells. Other extrahepatic tissues, such as the lung, spleen, gastric mucosa, red blood cells, and lymphocytes, also show acetylation capability. N-Acetyltransferase enzymes display broad substrate specificity and catalyze the acetyla-tion of several drugs and xenobiotics (Fig. 3.16).442,443 Aromatic compounds with a primary amino group, such as aniline,408 p-aminobenzoic acid,448449 ^-aminosalicylic acid,418 procainamide (Pronestyl),444,445,448,449 and dapsone (Avlosulfon),450 are especially susceptible to N-acetylation. Aromatic amine metabolites resulting from the reduction of aryl nitro compounds also are N-acetylated. For example, the anticonvulsant clonazepam (Klonopin) undergoes nitro reduction to its 7-amino metabolite, which in turn is N-acetylated.315 Another related benzodiazepam analog, nitrazepam, follows a similar pathway.316
The metabolism of several sulfonamides, such as sulfanil-amide,451 sulfamethoxazole (Gantanol),452 sulfisoxazole (Gantrisin),452 sulfapyridine453 (major metabolite from azo reduction of sulfasalazine, Azulfidine), and sulfameth-azine,408 occurs mainly by acetylation at the N-4 position. With sulfanilamide, acetylation also takes place at the sulfamido N-1 position.451 N-Acetylated metabolites of sulfonamides tend to be less water soluble than their parent compounds and have the potential of crystallizing out in renal tubules (crystalluria), thereby causing kidney damage. The frequency of crystalluria and renal toxicity is especially high with older sulfonamide derivatives, such as sulfathiazole.1,420 Newer sulfonamides, such as sulfisoxazole and sulfamethox-azole, however, are metabolized to relatively water-soluble acetylated derivatives, which are less likely to precipitate out.
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The biotransformation of hydrazine and hydrazide derivatives also proceeds by acetylation. The antihypertensive hydralazine (Apresoline)454,455 and the MAO inhibitor phenelzine (Nardil)456 are two representative hydrazine compounds that are metabolized by this pathway. The initially formed N-acetyl derivative of hydralazine is unstable and cyclizes intramolecularly to form 3-methyl-s-tria-zolo[3,4-a]phthalazine as the major isolable hydralazine metabolite in humans.454,455 The antituberculosis drug iso-niazid or isonicotinic acid hydrazide (INH) is metabolized extensively to N-acetylisoniazid.446447
The acetylation of some primary aliphatic amines such as histamine,457 mescaline,208,209 and the bis-N-demethylated metabolite of a( —)-methadol458-460 also has been reported. In comparison with oxidative deamination processes, N-acetylation is only a minor pathway in the metabolism of this class of compounds.
The acetylation pattern of several drugs (e.g., isoniazid, hydralazine, procainamide) in the human population displays a bimodal character in which the drug is conjugated either rapidly or slowly with acetyl-CoA.461,462 This phenomenon is termed acetylation polymorphism. Individuals are classified as having either slow or rapid acetylator phe-notypes. This variation in acetylating ability is genetic and is caused mainly by differences in N-acetyltransferase activity. The proportion of rapid and slow acetylators varies widely among different ethnic groups throughout the world. Oddly, a high proportion of Eskimos and Asians are rapid acetylators, whereas Egyptians and some Western European groups are mainly slow acetylators.462 Other populations are intermediate between these two extremes. Because of the bimodal distribution of the human population into rapid and slow acetylators, there appears to be significant individual variation in therapeutic and toxicological responses to drugs displaying acetylation polymorphism.408,461,462 Slow acetylators seem more likely to develop adverse reactions, whereas rapid acetylators are more likely to show an inadequate therapeutic response to standard drug doses.
The antituberculosis drug isoniazid illustrates many of these points. The plasma half-life of isoniazid in rapid acetylators ranges from 45 to 80 minutes; in slow acetylators, the half-life is about 140 to 200 minutes.463 Thus, for a given fixed-dosing regimen, slow acetylators tend to accumulate higher plasma concentrations of isoniazid than do rapid acetylators. Higher concentrations of isoniazid may explain the greater therapeutic response (i.e., higher cure rate) among slow acetylators, but they probably also account for the greater incidence of adverse effects (e.g., peripheral neuritis and drug-induced systemic lupus erythematosus syndrome) observed among slow acetylators.462 Slow acetyla-tors of isoniazid apparently are also more susceptible to certain drug interactions involving drug metabolism. For example, phenytoin toxicity associated with concomitant use with isoniazid appears to be more prevalent in slow acetylators than in rapid acetylators.464 Isoniazid inhibits the metabolism of phenytoin, thereby leading to an accumulation of high and toxic plasma levels of phenytoin.
Interestingly, patients who are rapid acetylators appear to be more likely to develop isoniazid-associated hepatitis.446,447 This liver toxicity presumably arises from initial hydrolysis of the N-acetylated metabolite N-acetylisoniazid to acetylhy-
drazine. The latter metabolite is further converted (by CYP enzyme systems) to chemically reactive acylating intermediates that covalently bind to hepatic tissue, causing necrosis. Pathological and biochemical studies in experimental animals appear to support this hypothesis. Therefore, rapid acetylators run a greater risk of incurring liver injury by virtue of producing more acetylhydrazine.
The tendency of drugs such as hydralazine and pro-cainamide to cause lupus erythematosus syndrome and to elicit formation of antinuclear antibodies (ANAs) appears related to acetylator phenotype, with greater prevalence in slow acetylators.465,466 Rapid acetylation may prevent the immunological triggering of ANA formation and the lupus syndrome. Interestingly, the N-acetylated metabolite of procainamide is as active an antiarrhythmic agent as the parent drug444,445 and has a half-life twice as long in humans.467 These findings indicate that N-acetylprocainamide may be a promising alternative to procainamide as an antiarrhythmic agent with less lupus-inducing potential.
Methylation reactions play an important role in the biosynthesis of many endogenous compounds (e.g., epinephrine and melatonin) and in the inactivation of numerous physiologically active biogenic amines (e.g., norepinephrine, dopamine, serotonin, and histamine).468 Methylation, however, constitutes only a minor pathway for conjugating drugs and xenobiotics. Methylation generally does not lead to polar or water-soluble metabolites, except when it creates a quaternary ammonium derivative. Most methylated products tend to be pharmacologically inactive, although there are a few exceptions.
The coenzyme involved in methylation reactions is S-adenosylmethionine (SAM). The transfer of the activated methyl group from this coenzyme to the acceptor substrate is catalyzed by various cytoplasmic and microsomal methyltransferases (Fig. 3.17).468,469 Methyltransferases of particular importance in the metabolism of foreign compounds include catechol-O-methyltransferase (COMT), phenol-O-methyltransferase, and nonspecific ^-methyltransferases and S-methyltransferases.358 One of these enzymes, COMT, should be familiar because it carries out O-methylation of such important neurotransmitters as norepinephrine and dopamine and thus terminates their activity. Besides being present in the central and peripheral nerves, COMT is distributed widely in other mammalian tissues, particularly the liver and kidney. The other methyltransferases mentioned are located primarily in the liver, kidney, or lungs. Transferases that specifically methylate histamine, serotonin, and epinephrine are not usually involved in the metabolism of xenobiotics.468
Foreign compounds that undergo methylation include catechols, phenols, amines, and ^-heterocyclic and thiol compounds. Catechol and catecholamine-like drugs are metabolized by COMT to inactive monomethylated catechol products. Examples of drugs that undergo significant O-methylation by COMT in humans include the antihypertensive (S)( —)a-methyldopa (Aldomet),470471 the antiparkinsonism agent (S)(—)-dopa (Levodopa),472 isoproterenol (Isuprel),473 and dobutamine (Dobutrex).474 The student should note the marked structural similarities between these drugs and the endogenous catecholamines such as norepinephrine and dopamine. In the foregoing four drugs, COMT selectively O-methylates only the phenolic OH at C-3. Bismethylation does not occur. Catechol metabolites arising from aromatic hydroxylation of phenols (e.g., 2-hydroxylation of 17a-ethinylestradiol)54,55 and from the arene oxide dihydrodiol-catechol pathway (see section on oxidation of aromatic moieties, e.g., the catechol metabolite of phenytoin)475 also undergo O-methylation. Substrates undergoing O-methylation by COMT must contain an aromatic 1,2-dihydroxy group (i.e., catechol group). Resorcinol (1,3-dihydroxybenzene) or p-hydroquinone (1,4-dihydroxybenzene) derivatives are not substrates for COMT. This explains why isoproterenol undergoes extensive O-methylation473 but terbutaline (which contains a resorcinol moiety) does not.396
Occasionally, phenols have been reported to undergo O-methylation but only to a minor extent.468 One interesting
example involves the conversion of morphine to its O-methylated derivative, codeine, in humans. This metabolite is formed in significant amounts in tolerant subjects and may account for up to 10% of the morphine dose.476
Although N-methylation of endogenous amines (e.g., his-tamine, norepinephrine) occurs commonly, biotransformation of nitrogen-containing xenobiotics to N-methylated metabolites occurs to only a limited extent. Some examples reported include the N-methylation of the antiviral and an-tiparkinsonism agent amantadine (Symmetrel) in dogs477 and the in vitro N-methylation of norephedrine in rabbit lung preparations.468 N-methylation of nitrogen atoms present in heterocyclic compounds (e.g., pyridine derivatives) also takes place. For example, the pyridinyl nitrogens of nicotine187,188 and nicotinic acid478 are N-methylated to yield quaternary ammonium products.
Thiol-containing drugs, such as propylthiouracil,479 2,3-dimercapto-1-propanol (BAL),480 and 6-mercaptopu-rine,481,482 also have been reported to undergo 5-methylation.
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