26510318-Bio-Transformation-of-Xenobiotics

CHAPTER 6 BIOTRANSFORMATION OF XENOBIOTICS

183

P450 enzymes, namely CYP2C19 and CYP3A4. However, these reactions are catalyzed by CYP3A4 with such low affinity that the N-demethylation of diazepam and the 5-hydroxylation of omeprazole in vivo appear to be dominated by CYP2C19 (Kato and Yamazoe, 1994a). When several P450 enzymes catalyze the same reaction, their relative contribution to xenobiotic biotransformation is determined by the kinetic parameter, Vmax Km, which is a measure of in vitro intrinsic clearance at low substrate concentrations ( 10 percent of Km) (Houston, 1994).

Inasmuch as the biotransformation of a xenobiotic in humans is frequently dominated by a single P450 enzyme, considerable attention has been paid to defining the substrate specificity of the P450 enzymes expressed in human liver microsomes (a process commonly referred to as reaction phenotyping or enzyme mapping). Three in vitro approaches have been developed for reaction phenotyping. Each has its advantages and disadvantages, and a combination of approaches is usually required to identify which human P450 enzyme is responsible for metabolizing a xenobiotic (Wrighton et al., 1993; Rodrigues 1999; Madan et al., 2000). The three approaches to reaction phenotyping are as follows:

1.Correlation analysis, which involves measuring the rate of xenobiotic metabolism by several samples of human liver microsomes and correlating reaction rates with the variation in the level or activity of the individual P450 enzymes in the same microsomal samples. This approach is successful because the levels of the P450 enzymes in human liver microsomes vary enormously from sample to sample (up to 100-fold) but vary independently from each other.

2.Chemical and antibody inhibition, which involves an evaluation of the effects of known P450 enzyme inhibitors or inhibitory antibodies on the metabolism of a xenobiotic by human liver microsomes. Chemical inhibitors of cytochrome P450, which are discussed later, must be used cautiously because most of them can inhibit more than one P450 enzyme. Some chemical inhibitors are metabolism-dependent (mecha- nism-based) inhibitors that require biotransformation to a metabolite that inactivates or noncompetitively inhibits cytochrome P450.

3.Biotransformation by purified or recombinant human P450 enzymes, which can establish whether a particular P450 enzyme can or cannot biotransform a xenobiotic, but it does not address whether that P450 enzyme contributes substantially to reactions catalyzed by human liver microsomes. The information obtained with purified or recombinant human P450 enzymes can be improved by taking into account large differences in the extent to which the individual P450 enzymes are expressed in human liver microsomes, which is summarized in Table 6-4. Some P450 enzymes, such as CYP1A1 and CYP1B1, are expressed at such low levels in human liver microsomes that they contribute negligibly to the hepatic biotransformation of xenobiotics. Other P450 enzymes are ex-

pressed in some but not all livers. For example, CYP3A5 is expressed in 25 percent of human livers.

These in vitro approaches have been used to characterize the substrate specificity of several of the P450 enzymes expressed in human liver microsomes. Examples of reactions catalyzed by human P450 enzymes are shown in Figs. 6-36 through 6-43, and examples of substrates, inhibitors, and inducers for each P450

Table 6-4

Concentration of Individual P450 Enzymes

in Human Liver Microsomes

Specific Content (pmol/mg protein)

P450 ENZYME	SOURCE (1)			SOURCE (2)			SOURCE (3)
CYP1A2	45			42			15
CYP2A6	68			42			12
CYP2B6	39			1.0			3.0
CYP2C8	64
CYP2C9	96
CYP2C18	2.5
CYP2C19	19
CYP2D6	10			5.0			15
CYP2E1	49			22
CYP3A4	108			98			40
CYP3A5		1.0
TOTAL	534			344

SOURCE: Rodrigues, 1999.

enzyme are given in Table 6-5. Additional examples appear in a comprehensive review by Rendic and Di Carlo (1997), and clinically important examples can be found on the Internet at http://www.dml.georgetown.edu/depts/pharmacology/davetab.html (Abernathy and Flockhart, 2000). It should be emphasized that reaction phenotyping in vitro is not always carried out with pharmacologically or toxicologically relevant substrate concentrations. As a result, the P450 enzyme that appears responsible for biotransforming the drug in vitro may not be the P450 enzyme responsible for biotransforming the drug in vivo. This may be particularly true of CYP3A4, which metabolizes several drugs with high capacity but low affinity. The salient features of the major P450 enzymes in human liver microsomes are summarized below (subsequent sections, this chapter).

CYP1A1/2 All mammalian species apparently possess two inducible CYP1A enzymes, namely CYP1A1 and CYP1A2. Human liver microsomes contain relatively high levels of CYP1A2 but not of CYP1A1, even though this enzyme is readily detectable in the human lung, intestine, skin, lymphocytes, and placenta, particularly from cigarette smokers. In addition to cigarette smoke, inducers of the CYP1A enzymes include charcoal-broiled meat (a source of polycyclic aromatic hydrocarbons), cruciferous vegetables (a source of various indoles), and omeprazole, a proton-pump inhibitor used to suppress gastric acid secretion. In contrast to CYP1A1, CYP1A2 is not expressed in extrahepatic tissues. CYP1A1 and CYP1A2 both catalyze the O-dealkylation of 7-methoxyresorufin and 7-ethoxyresorufin (see Fig. 6-40). Reactions preferentially catalyzed by CYP1A1 include the hydroxylation and epoxidation of benzo[a]pyrene (see Fig. 6-6) and the epoxidation of the leukotriene D4 receptor antagonist, verlukast (Fig. 6-38). CYP1A2 catalyzes the N-hydroxylation of aromatic amines, such as 4-aminobiphenyl and 2-aminonaphthalene, which in many cases represents the initial step in the conversion of aromatic amines to tumorigenic metabolites (see Fig. 6-9). CYP1A2 also catalyzes the O-dealkylation of phenacetin and the 4-hydroxylation of acetanilide, both of which produce acetaminophen, which can be converted by CYP1A2 and other P450 enzymes

Table 6-5

Examples of Substrates, Inhibitors, and Inducers of the Major Human Liver Microsomal P450 Enzymes Involved in Xenobiotic Biotransformation

	CYP2A6	CYP2B6	CYP2C8	CYP2C9	CYP2C19	CYP2E1
Substrates	Coumarin	Benzphetamine	Arachidonic acid	Celecoxib	Citalopram	Acetaminophen
	Butadiene	7-Benzyloxyresorufin	Carbamazepine	Diclofenac	Diazepam	Alcohols
	Nicotine	Bupropion	Paclitaxel (Taxol)	Phenacetin	Diphenylhydantoin	Aniline
		Cyclophosphamide		Phenobarbital	Hexobarbital	Benzene
		7-Ethoxy-4-trifluoro-		Phenytoin	Imipramine	Caffeine
		methylcoumarin		Piroxicam	Lansoprazole	Chlorzoxazone
		Ifosphamide		Tenoxicam	S-Mephenytoin	Dapsone
		S-Mephenytoin		Tetrahydrocannabinol	Mephobarbital	Enflurane
				Tienilic acid	Omeprazole	Halogenated alkanes
				Tolbutamide	Pentamidine	Isoflurane
				Torsemide	Phenobarbital	Methylformamide
				S-Warfarin	Proguanil	4-Nitrophenol
					Propranolol	Nitrosamines
						Styrene
						Theophylline
Inhibitors	Diethyldithiocarbamate	9-Ethynylphenathrene	Etoposide	Sulfaphenazole	Fluconazole	3-Amino-1,2,4-triazole*
	Letrozole	Methoxychlor	Nicardipine	Sulfinpyrazone	Teniposide	Diethyldithiocarbamate
	8-Methoxypsoralen*	Orphenadrine*	Quercetin		Tranylcypromine	Dihydrocapsaicin
	Pilocarpine		Tamoxifen			Dimethyl sulfoxide
	Tranylcypromine		R-Verapamil			Disulfiram
						4-Methylpyrazole
						Phenethylisothiocyanate*
Inducers	Barbiturates?	Phenobarbital	Not known	Rifampin	Artemisinin?	Ethanol
		Phenytoin			Rifampin	Isoniazid
		Rifampin
		Troglitazone

184

TOXICANTS OF DISPOSITION 2 UNIT

*Metabolism-dependent (mechanism-based) inhibitor.

Table 6-5	(continued)
		CYP1A2		CYP2D6			CYP3A4
Substrates		Acetaminophen	Amiflamine	Dolasetron	Miniaprine	Acetaminophen	Erythromycin	Rapamycin
		Acetanilide	Amitriptyline	Encainide	Nortriptyline	Aldrin	Ethinylestradiol	Retinoic acid
		Aminopyrine	Aprindine	Flecainide	Ondansetron	Alfentanil	Etoposide	Saquinavir
		Antipyrine	Brofaromine	Fluoxetine	Paroxetine	Amiodarone	Flutamide	Steroids (e.g., cortisol)
		Aromatic amines	Bufurolol	Flunarizine	Perhexiline	Aminopyrine	Hydroxyarginine	Tacrolimus (FK 506)
		Caffeine	Captopril	Fluphenazine	Perphenazine	Amprenavir	Ifosphamide	Tamoxifen
		Estradiol	Chlorpramazine	Guanoxan	Propafenone	Antipyrine	Imipramine	Taxol
		Ethoxyresorufin	Cinnarizine	Haloperidol	Propranolol	Astemizole	Indinavir	Teniposide
		Imipramine	Citalopram	(reduced)	N-Propylajmaline	Benzphetamine	Lansoprazole	Terfenadine
		Methoxyresorufin	Clonipramine	Hydrocodone	Remoxipride	Budesonide	Lidocaine	Tetrahydrocannabinol
		Phenacetin	Clozapine	Imipramine	Sparteine	Carbamazepine	Loratadine	Theophylline
		Tacrine	Codeine	Indoramin	Tamoxifen	Celecoxib	Losartan	Toremifene
		Theophylline	Debrisoquine	Methoxyamphetamine	Thioridazine	Cisapride	Lovastatin	Triazolam
		Trimethadone	Deprenyl	Methoxyphenamine	Timolol	Cyclophosphamide	Midazolam	Trimethadone
		Warfarin	Desmethylcitalopram	Metoprolol	Tomoxetine	Cyclosporin	Nelfinavir	Troleandomycin
			Despiramine	Mexiletene	Trifluperidol	Dapsone	Nicardipine	Verapamil
			Dextromethorphan	Mianserin	Tropisetron	Delavirdine	Nifedipine	Warfarin
						Digitoxin	Omeprazole	Zatosetron
						Diltiazem	Quinidine	Zonisamide
						Diazepam
Inhibitors		Ciprofloxacin	Ajmalicine	Fluoxetine	Quinidine	Amprenavir	Indinavir	Ritonavir
		Fluvoxamine	Celecoxib	Lobelin	Trifluperidol	Clotrimazole	Itraconazole	Saquinavir
		Furafylline*	Chinidin	Propidin	Yohimbine	Delavirdine	Ketoconazole	Troleandomycin*
		-Naphthoflavone	Corynanthine			Ethinylestradiol*	Miconazole	Verapamil
						Fluoxetine	Nelfinavir	Activator:
						Gestodene*	Nicardipine	-Naphthoflavone
Inducers		Charcoal-broiled	None known			Carbamazepine	Phenytoin	Sulfadimidine
		beef				Dexamethasone	Rifabutin	Sulfinpyrazone
		Cigarette smoke				Glutethimide	Rifampin	Troglitazone
		Cruciferous				Nevirapine	Ritonavir?	Troleandomycin
		vegetables				Phenobarbital	St. John’s Wort
		Omeprazole

XENOBIOTICS OF BIOTRANSFORMATION 6 CHAPTER

*Metabolism-dependent (mechanism-based) inhibitor.

186	UNIT 2 DISPOSITION OF TOXICANTS

to a toxic benzoquinoneimine (Fig. 6-28). The anticholinesterase agent tacrine, which is used in the treatment of Alzheimer’s disease, is similarly converted by CYP1A2 to a reactive quinoneimine/ quinone methide (Pirmohamed and Park, 1999). As shown in Fig. 6-40, CYP1A2 catalyzes the N3-demethylation of caffeine to paraxanthine. By measuring rates of formation of paraxanthine in blood, urine, or saliva or by measuring the exhalation of isotopically labeled CO2 from 13C- or 14C-labeled caffeine, the N3- demethylation of caffeine can be used as an in vivo probe of CYP1A2 activity, which varies enormously from one individual to the next. CYP1A1 and CYP1A2 are both inhibited by -naph- thoflavone. Ellipticine preferentially inhibits CYP1A1, whereas the metabolism-dependent inhibitor furafylline is a specific inhibitor of CYP1A2.

Although CYP1A1 and CYP1A2 are expressed in all mammals, there are species differences in their function and regulation. For example, although CYP1A1 is not expressed in human liver (or in the liver of most other mammalian species), it appears to be constitutively expressed in rhesus monkey and guinea pig liver. Conversely, although CYP1A2 is expressed in human liver (and in most other mammalian species), it does not appear to be constitutively expressed in cynomolgus monkey liver. Polycyclic and polyhalogenated aromatic hydrocarbons appear to induce CYP1A enzymes in all mammalian species. In contrast, omeprazole is an inducer of CYP1A enzymes in humans, but not in mice or rabbits (Diaz et al., 1990). The function of the CYP1A enzymes is fairly well conserved across species, although there are subtle differences. For example, in some species, such as the rat, CYP1A1 is considerably more effective than CYP1A2 as a catalyst of 7-ethoxyresorufin O-dealkylation, whereas the opposite is true in other species, such as rabbit. In mice, CYP1A1 and CYP1A2 catalyze the O-dealkylation of 7-ethoxyresorufin at comparable rates. In the rat, CYP1A1 preferentially catalyzes the O-dealkylation of 7-ethoxyresorufin whereas CYP1A2 preferentially catalyzes the O-dealkylation of 7-methoxyresorufin. However, in other species, such as the mouse and the human, CYP1A2 catalyzes the O-dealky- lation of 7-ethoxyresorufin and 7-methoxyresorufin at about the same rate. There are also species differences in the affinity with which CYP1A2 interacts with xenobiotics. For example, furafylline is a potent, metabolism-dependent inhibitor of human CYP1A2 but a weak inhibitor of rat CYP1A2. Although the levels of CYP1A2 vary enormously from one individual to the next, genetic defects in CYP1A2 are rare ( 1 percent).

CYP2A6 Enzymes belonging to the CYP2A gene family show marked species differences in catalytic function. For example, the two CYP2A enzymes expressed in rat liver, namely CYP2A1 and CYP2A2, primarily catalyze the 7 - and 15 -hydroxylation of testosterone, respectively. In contrast, the CYP2A enzyme expressed in human liver, namely CYP2A6, catalyzes the 7-hydrox- ylation of coumarin, as shown in Fig. 6-37. Just as rat CYP2A1 and CYP2A2 have little or no capacity to 7-hydroxylate coumarin, human CYP2A6 has little or no capacity to hydroxylate testosterone. Mouse liver microsomes contain three CYP2A enzymes: a testosterone 7 -hydroxylase (CYP2A1), a testosterone 15 - hydroxylase (CYP2A4), and a coumarin 7-hydroxylase (CYP2A5). Functionally, CYP2A5 can be converted to CYP2A4 by a single amino acid substitution (Phe209 Leu209). In other words, this single amino substitution converts CYP2A5 from a coumarin 7-hydroxylating to a testosterone 15 -hydroxylating enzyme (Lindberg and Negishi, 1989). The fact that a small change in primary structure can have a dramatic effect on substrate specificity

makes it difficult to predict whether orthologous proteins in different species (which are structurally similar but never identical) will catalyze the same reactions.

Differences in CYP2A function have important implications for the adverse effects of coumarin, which is hepatotoxic to rats but not humans. Whereas coumarin is detoxified in humans by conversion to 7-hydroxycoumarin, which is subsequently conjugated with glucuronic acid and excreted, a major pathway of coumarin biotransformation in rats involves formation of the hepatotoxic metabolite, coumarin 3,4-epoxide, as shown in Fig. 6-38. In addition to catalyzing the 7-hydroxylation of coumarin, CYP2A6 converts 1,3-butadiene to butadiene monoxide and nicotine to nicotine1 ,5 -iminium ion, which is further oxidized by aldehyde oxidase to cotinine, as shown in Fig. 6-43. 8-Methoxypsoralen, a structural analog of coumarin, is a potent, metabolism-dependent inhibitor of CYP2A6. Although the levels of CYP2A6 vary enormously from one individual to the next, genetic defects in this enzyme are rare ( 1 percent). Nevertheless, those individuals who lack CYP2A6 appear to have an aversion to cigarette smoking, presumably because they are poor metabolizers of nicotine (Pianezza et al., 1998). CYP2B6 Enzymes belonging to the CYP2B subfamily have been studied extensively in many species, although only recently was it appreciated that, despite its low levels in human liver, CYP2B6 contributes significantly to the biotransformation of certain xenobiotics (Ekins and Wrighton, 1999). CYP2B6 appears to be the functional gene expressed in human, with CYP2B7 (a splice variant of CYP2B6) unable to encode for a functional enzyme. CYP2B6 catalyzes the O-dealkylation of 7-ethoxy-4-trifluromethyl- coumarin, the N-demethylation of benzphetamine, and the O-dealkylation of benzyloxyresorufin; however, these reactions are not selective for CYP2B6. More specific reactions are S-mephenytoin N-demethylation and the cyclopentyl ring hydroxylation of the phosphodiester inhibitor 3-cyclopentyloxy- N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide (RP 73401). Orphenadrine inhibits CYP2B6, but not selectively. 9-Ethynylphenan- threne has been shown to be a potent metabolism-dependent (mechanism-based) inhibitor of CYP2B6, and appears to be selective. CYP2B6 levels in adult human liver are low, but the enzyme is inducible by rifampin, troglitazone and various anticonvulsant drugs (phenobarbital, phenytoin), which also induce CYP3A4. Phenobarbital (and a host of other xenobiotics) primarily induce the corresponding CYP2B enzymes in other species. For example, CYP2B10, CYP2B1/2, CYP2B11 and CYP2B17 are the major phenobarbital-inducible P450 enzymes in mouse, rat, dog, and cynomolgus monkey, respectively.

CYP2C8 The 6 -hydroxylation of the taxane ring of paclitaxel (which generates a metabolite known as M5, VIII and HM3) is a specific marker for CYP2C8 (Rahman et al., 1994). Another pathway of paclitaxel metabolism, aromatic hydroxylation (3 -p-hy- droxylaton) to M4 (also known as VII ), is catalyzed by CYP3A enzymes. CYP2C8 catalyzes the 10,11-epoxidation of carbamazepine, although this reaction in human liver microsomes is dominated by CYP3A4/5. The 4-hydroxylation of all-trans retinoic acid appears to be catalyzed primarily by CYP2C8, however, members of the CYP3A subfamily may be minor participants as well. CYP2C8 also metabolizes endogenous arachidonic acid to form epoxyeicosatrienoic acids, which are converted by cytosolic epoxide hydrolase dihydroeicosatrienoic acids. It has been speculated that the biologically active eicosanoids may be important in maintaining homeostasis in the liver. The multidrug resistance (MDR) reversing agents R-verapamil, tamoxifen, and etoposide (VP-16)

CHAPTER 6 BIOTRANSFORMATION OF XENOBIOTICS

187

inhibit the CYP2C8-dependent metabolism of paclitaxel but also inhibit its metabolism by CYP3A4. Quercetin has been shown to inhibit the CYP2C8-dependent hydroxylation of paclitaxel; however, it may not inhibit CYP2C8 selectively.

CYP2C9 A genetic polymorphism for tolbutamide metabolism was first described in 1978–1979, although its incidence is still unknown (Back and Orme, 1992). Poor metabolizers are defective in CYP2C9, which catalyzes the methyl-hydroxylation of this hypoglycemic agent. Poor metabolizers of tolbutamide are also poor metabolizers of phenytoin, which is consistent with in vitro data suggesting that CYP2C9 catalyzes both the methyl-hydroxylation of tolbutamide and the 4-hydroxylation of phenytoin. The antimalarial naphthoquinone 58C80 is converted to a t-butyl hydroxylated metabolite by CYP2C9. CYP2C9 also catalyzes the 7-hydroxylation and, to a lesser extent, the 6-hydroxylation of S-warfarin; it also appears to catalyze the 7-hydroxylation of1-tetrahydrocannabinol. Although CYP2C9 (Arg144, Tyr358, Ile359, Gly417) and its allelic variant CYP2C9 (Arg144 Cys144) both catalyze the methyl-hydroxylation of tolbutamide (the former being twice as active as the latter), the latter enzyme is virtually devoid of S-warfarin 6- and 7-hydroxylase activity (Rettie et al., 1994). This suggests that individuals who express the allelic variant CYP2C9 (Arg144 Cys144) could be poor metabolizers of warfarin but near normal metabolizers of tolbutamide. CYP2C9 also appears to be responsible for metabolizing several NSAIDs, including the 4 -hydroxylation of diclofenac and the 5 -hydroxyla- tion of piroxicam and tenoxicam. Tienilic acid is also metabolized by CYP2C9, but with potentially deleterious effects. CYP2C9 converts tienilic acid to an electrophilic thiophene sulfoxide, which can react either with water to give 5-hydroxytienilic acid or with a nucleophilic amino acid (Ser365) in CYP2C9 to form a covalent adduct, which inactivates the enzyme (Koenigs et al., 1999). Antibodies directed against the adduct between CYP2C9 and tienilic acid are thought to be responsible for the immunoallergic hepatitis that develops in about 1 out of every 10,000 patients treated with this uricosuric diuretic drug. Sulfaphenazole is a potent inhibitor of CYP2C9, both in vitro and in vivo.

CYP2C18 The functions of CYP2C18 are largely unknown. The mRNA encoding this protein is expressed in all human livers, although the mean value is one-seventh to one-eighth that of the mRNAs encoding CYP2C8 and CYP2C9. The levels of CYP2C18 mRNA vary widely from one liver to the next and vary independently of the mRNAs encoding CYP2C8 and CYP2C9, which tend to be coregulated.

CYP2C19 A genetic polymorphism for the metabolism of S-mephenytoin was first described in 1984 (reviewed in Wilkinson et al., 1989). The deficiency affects the 4 -hydroxylation (aromatic ring hydroxylation) of this anticonvulsant drug. The other major pathway of S-mephenytoin metabolism, namely N-demethylation to S-nirvanol, is catalyzed by CYP2B6, so it is not affected. Consequently, poor metabolizers excrete little or no 4 -hydrox- ymephenytoin in their urine but excrete increased amounts of the N-demethylated metabolite, S-nirvanol (S-phenylethylhydantoin). Interestingly, the P450 enzyme responsible for this genetic polymorphism, namely cytochrome CYP2C19, is highly stereoselective for the S-enantiomer of mephenytoin. In contrast to the S-enantiomer, the R-enantiomer is not converted to 4 -hydroxymephenytoin but is N-demethylated to R-nirvanol (R-phenylethylhydantoin). The formulation of mephenytoin used clinically is mesantoin, which is a racemic mixture of S- and R-enantiomers. An exaggerated central response has been observed

in poor metabolizers administered mephenytoin at doses that were without effect in extensive metabolizers of mephenytoin.

There is considerable interethnic variation in the incidence of the poor-metabolizer phenotype for S-mephenytoin. In Caucasians, CYP2C19 is defective in 2 to 5 percent of the population, but it is defective in 12 to 23 percent of Japanese, Chinese, and Korean subjects. On Vanuatu and some other Pacific islands, CYP2C19 is deficient in 70 percent of the population (Kaneko et al., 1999). Based on clinical observations and/or in vitro analysis, CYP2C19 also appears to metabolize diphenylhydantoin (dilantin), mephobarbital, hexobarbital, propranolol, imipramine, diazepam, omeprazole, and lansoprazole. CYP2C19 also converts proguanil to cycloguanil, its active antimalarial metabolite. Artemisinin, another antimalarial drug, is an effective inducer of CYP2C19 and dramatically induces its own metabolism (Mihara et al., 1999). The monoamine oxidase inhibitor tranylcypromine is a potent but not specific inhibitor of CYP2C19.

CYP2C Enzymes in Other Species The multiplicity, function, and regulation of the CYP2C enzymes vary enormously from one species to the next. For example, whereas the 4 -hydroxylation of S-mephenytoin in humans is catalyzed by CYP2C19, this same reaction in rats is catalyzed by a CYP3A enzyme, not a CYP2C enzyme. Conversely, one of the reactions catalyzed by rat CYP2C11, namely the 2 -hydroxylation of testosterone, is not catalyzed by the human CYP2C enzymes (or any of the other P450 enzymes in human liver microsomes).

CYP2D6 In the late 1950s, clinical trials in the United States established that sparteine was as potent as oxytocin for inducing labor at term. However, the duration and intensity of action of sparteine were dramatically increased in 7 percent of all patients tested. The exaggerated response to sparteine included prolonged (tetanic) uterine contraction and abnormally rapid labor. In some cases, sparteine caused the death of the fetus. The drug was not recommended for clinical use because these side effects were unpredictable and occurred at doses of 100 to 200 mg/kg, which were well tolerated by other patients. The antihypertensive drug debrisoquine was subsequently found to cause a marked and prolonged hypotension in 5 to 10 percent of patients, and a genetic polymorphism for the metabolism of debrisoquine and sparteine was discovered in 1977–1979 (Gonzalez, 1989; Meyer, 1994). Poor metabolizers lack CYP2D6, which catalyzes the 4-hydroxylation of debrisoquine and the 2- and 5-oxidation of sparteine (see Fig. 6-43).

In addition to debrisoquine and sparteine, CYP2D6 biotransforms a large number of drugs, as shown in Table 6-5. Individuals lacking CYP2D6 have an exaggerated response to most but not all of these drugs. For example, even though debrisoquine and propranolol are both biotransformed by CYP2D6, the effects of propranolol are not exaggerated in poor metabolizers of debrisoquine for two reasons. First, 4-hydroxypropranolol is a -adrenoceptor antagonist, so the 4-hydroxylation of propranolol by CYP2D6 does not terminate the pharmacologic effects of the drug. Second, CYP2D6 is not the only P450 enzyme to biotransform propranolol. As mentioned above, CYP2C19 catalyzes the side-chain oxidation of propranolol to naphthoxylactic acid (see Fig. 6-23). Because CYP2D6 and CYP2C19 both contribute significantly to the biotransformation of propranolol, a deficiency in either one of these enzymes does not markedly alter the pharmacokinetics of this beta blocker. However, in one individual who lacked both enzymes, the total oral clearance of propranolol was markedly reduced (Wilkinson et al., 1989). CYP2D6 catalyzes the O-demethylation of

188	UNIT 2 DISPOSITION OF TOXICANTS

codeine to the potent analgesic morphine. Pain control with codeine is reduced in individuals lacking CYP2D6. The biotransformation of substrates for CYP2D6 occurs 5 to 7.5Å from a basic nitrogen, which interacts with an anionic residue (Glu301) in the enzyme’s substrate-binding site (Strobl et al., 1993). Quinidine is a potent inhibitor of CYP2D6 because it interacts favorably with the anionic site on CYP2D6, but it cannot be oxidized at sites 5 to 7.5Å from its basic nitrogen atoms. Fluoxetine (and several other selective serotonin-reuptake inhibitors), ajmalicine, and yohimbine are also potent competitive inhibitors of CYP2D6. A poor-metabolizer phenotype can be induced pharmacologically with these potent inhibitors of CYP2D6. Quinine, the levorotatory diasteriomer of quinidine, is not a potent inhibitor of CYP2D6, and neither drug is a potent inhibitor of the CYP2D enzymes expressed in rats. CYP2D6 is one of the few P450 enzymes that efficiently uses the peroxide shunt, so that reactions catalyzed by CYP2D6 can be supported by cumene hydroperoxide.

As shown in Fig. 6-40, CYP2D6 catalyzes the O-demethyla- tion of dextromethorphan to dextrorphan, which is glucuronidated and excreted in urine. Dextromethorphan can also be N-demethy- lated, a reaction catalyzed predominantly by CYP3A4 and CYP2B6, but this metabolite is not glucuronidated and excreted in urine. Because the urinary excretion of dextromethorphan is dependent on O-demethylation, this over-the-counter antitussive drug can be used to identify individuals lacking CYP2D6, although most poor metabolizers can be identified by DNA analysis (genotyping). There is considerable interethnic variation in the incidence of the poor metabolizer phenotype for debrisoquine/sparteine. In Caucasians, CYP2D6 is defective in 5 to 10 percent of the population, but it is defective in less than 2 percent of African Americans, Africans, Thai, Chinese, and Japanese subjects. Individuals lacking CYP2D6 have an unusually low incidence of some chemically induced neoplastic diseases, such as lung cancer, bladder cancer, hepatocellular carcinoma, and endemic Balkan nephropathy (Idle, 1991; Taningher et al., 1999). It has been hypothesized that CYP2D6 may play a role in the metabolic activation of chemical carcinogens, such as those present in the environment, in the diet, and/or in cigarette smoke. According to this hypothesis, individuals lacking CYP2D6 have a low incidence of cancer because they fail to activate chemical carcinogens. However, CYP2D6 appears to play little or no role in the activation of known chemical carcinogens to DNA-reactive or mutagenic metabolites with the possible exception of the tobacco-smoke specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), which is also activated by other P450 enzymes. Therefore, it remains to be determined whether a deficiency of CYP2D6 is causally or coincidentally related to a low incidence of certain cancers.

CYP2E1 As shown in Fig. 6-20, CYP2E1 was first identified as MEOS, the microsomal ethanol oxidizing system (Lieber, 1999). In addition to ethanol, CYP2E1 catalyzes the biotransformation of a large number halogenated alkanes (Guengerich et al., 1991). CYP2E1 is expressed constitutively in human liver and possibly in extrahepatic tissues, such as the kidney, lung, lymphocytes, and bone marrow, and the enzyme is inducible by ethanol and isoniazid. CYP2E1 catalyzes the N1- and N7-demethylation of caffeine to theobromine and theophylline, as shown in Fig. 6-40, and it can activate acetaminophen to the hepatotoxic metabolite N-acetyl- benzoquinoneimine, as shown in Figs. 6-28 and 6-43. The mechanism by which alcohol potentiates the hepatotoxic effects of acetaminophen (Tylenol) is thought to involve increased activation of

acetaminophen due to the induction of CYP2E1 and decreased inactivation due to a lowering of glutathione levels. However, the degree to which ethanol consumption increases the risk of aceta- minophen-induced hepatotoxicity remains a controversial issue (Prescott, 1999). Induction of CYP2E1 by isoniazid stimulates the dehalogenation of the volatile anesthetics enflurane and isoflurane. In human liver microsomes, CYP2E1 activity can be conveniently measured by the 6-hydroxylation of chlorzoxazone and the hydroxylation of 4-nitrophenol. The 6-hydroxylation of chlorzoxazone can also be catalyzed by CYP1A1, but this enzyme is rarely expressed in human liver. Chlorzoxazone is an FDA-approved muscle relaxant (Paraflex), and the urinary excretion of 6-hydroxy- chlorzoxazone and the plasma ratio of 6-hydroxychlorzoxazone to chlorzoxazone have been used as noninvasive in vivo probes of CYP2E1. The levels of CYP2E1 are by no means constant among individuals, but they do not exhibit the marked interindividual variation characteristic of other P450 enzymes. CYP2E1 is one of the P450 enzymes that requires cytochrome b5, which lowers the apparent Km for several substrates biotransformed by CYP2E1. The function and regulation of CYP2E1 are well conserved among mammalian species.

CYP3A The most abundant P450 enzymes in human liver microsomes belong to the CYP3A gene subfamily, which includes CYP3A4, CYP3A5, and CYP3A7. CYP3A7 is considered a fetal enzyme, whereas the others are considered to be adult forms, although livers from some adults contain CYP3A7 and some fetal livers contain CYP3A5. All human livers appear to contain CYP3A4, although the levels vary enormously ( 10-fold) among individuals (Wrighton and Stevens, 1992; Shimada et al., 1994). CYP3A5 is expressed in relatively few livers (10 to 30 percent). One or more of these enzymes is expressed in extrahepatic tissues. For example, CYP3A4 is expressed in the small intestine, whereas CYP3A5 is expressed in 80 percent of all human kidneys.

The CYP3A enzymes biotransform an extraordinary array of xenobiotics and steroids, as shown in Table 6-5 (Thummel and Wilkinson, 1998; Dresser et al., 2000). CYP3A enzymes also catalyze the oxidation of hydroxyarginine to citrulline and nitric oxide. Factors that influence the levels and/or activity of the CYP3A enzymes influence the biotransformation of many of the drugs listed in Table 6-5, and many of these drugs have been shown to inhibit each other’s metabolism (Pichard et al., 1990).

In humans, CYP3A enzymes are inducible by numerous drugs, such as rifampin, phenobarbital, phenytoin, and troglitazone (Pichard et al., 1990). Inhibitors of human CYP3A include azoletype antimycotics (e.g., ketoconazole and clotrimazole), macrolide antibiotics (e.g., erythromycin and troleandomycin), HIV protease inhibitors (especially ritonavir), the ethynylprogesterone analog gestodene, and certain flavones or other component(s) present in grapefruit juice (although other flavones, such as-naphthoflavone, are activators of CYP3A4). Several noninvasive clinical tests of CYP3A activity have been proposed, including the [14C-N-methyl]-erythromycin breath test, the plasma clearance of midazolam and nifedipine, and the urinary excretion of 6 - hydroxycortisol (Watkins, 1994). However, for reasons that are not yet understood, there can be marked differences in the results obtained with different tests in the same individuals. Nevertheless, the various noninvasive tests suggest that CYP3A activity varies widely among individuals ( 10-fold), but there appear to be no individuals who are completely devoid of CYP3A activity, possibly due to the multiplicity of enzymes in the human CYP3A subfamily.

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The function and regulation of the CYP3A enzymes is fairly well conserved among mammalian species, with some notable exceptions. For example, rifampin is an inducer of the CYP3A enzymes in humans and rabbits but not rats or mice, whereas the opposite appears to be true of pregnenolone-16 -carbonitrile (Pichard et al., 1990). In adult rats, the levels of CYP3A2 in males are much greater ( 10-fold) than in females, whereas no marked sex difference in CYP3A levels is observed in humans (if anything, the levels of CYP3A enzymes are higher in females than in males).

CYP4A9/11 The CYP4A enzymes in human and other species catalyze the - and -1 hydroxylation of fatty acids and their derivatives, including prostaglandins, thromboxane, prostacyclin, and leukotrienes. With lauric acid as substrate, the CYP4A enzymes preferentially catalyze the -hydroxylation to 12-hydroxylauric acid (see Fig. 6-36), which can be further oxidized to form a dicarboxylic acid. Although CYP4A enzymes also catalyze the -1 hydroxylation of lauric acid, other P450 enzymes, including CYP2E1, can contribute significantly to the formation of 11hydroxylauric acid. The CYP4A enzymes are unusual for ability to preferentially catalyze the -hydroxylation of fatty acids over the thermodynamically more favorable reaction leading to -1 hydroxylation. Despite their physiologic importance, the CYP4A enzymes appear to play a very limited role in the metabolism of drugs and other xenobiotics.

Activation of Xenobiotics by

Cytochrome P450

Biotransformation by cytochrome P450 does not always lead to detoxication, and several examples have been given previously where the toxicity or tumorigenicity of a chemical depends on its activation by cytochrome P450. The role of individual human P450 enzymes in the activation of procarcinogens and protoxicants is summarized in Table 6–6 (adapted from Guengerich and Shimada, 1991). A variety of cytochrome P450–dependent reactions are involved in the activation of the chemicals listed in Table 6-6. The conversion of polycyclic aromatic hydrocarbons to tumor-forming metabolites involves the formation of bay-region diolepoxides, as shown in Fig. 6-6 for the conversion of benzo[a]pyrene to benzo[a]pyrene 7,8-dihydrodiol-9,10, epoxide. Epoxidation generates hepatotoxic metabolites of chlorobenzene and coumarin (Fig. 6-38), and generates an hepatotumorigenic metabolite of aflatoxin B1 (Fig. 6-27).

The initial step in the conversion of aromatic amines to tumorforming metabolites involves N-hydroxylation, as shown for 2-amino-6-nitrobenzylalcohol (Fig. 6-9) and 2-acetylaminofluo- rene (Fig. 6-33A). In the case of acetaminophen, activation to an hepatotoxic metabolite involves dehydrogenation to N-acetyl- benzoquinoneimine, as shown in Fig. 6-28. A similar reaction converts butylated hydroxytoluene to a toxic quinone methide, as shown in Fig. 6-30. The myelotoxicity of benzene depends on its conversion to phenol and hydroquinone (Fig. 6-29). The toxicity of several organophosphorus insecticides involves oxidative group transfer to the corresponding organophosphate, as shown for the conversion of parathion to paraoxon in Figs. 6-41 and 6-42. The hepatotoxicity of carbon tetrachloride involves reductive dechlorination to a trichloromethyl free radical, which binds to protein and initiates lipid peroxidation, as shown in Fig. 6-15. The hepatotoxicity and nephrotoxicity of chloroform involves oxidative dechlorination to phosgene (Fig. 6-15). Oxidative and reductive dehalo-

Table 6-6

Examples of Xenobiotics Activated by Human P450 Enzymes

CYP1A1

Benzo[a]pyrene and other polycyclic aromatic hydrocarbons

CYP1A2

Acetaminophen 2-Acetylaminofluorene 4-Aminobiphenyl 2-Aminofluorene 2-Naphthylamine NNK*

Amino acid pyrrolysis products (DiMeQx, MelQ, MelQx, Glu P-1, Glu P-2, IQ, PhlP, Trp P-1, Trp P-2) Tacrine

CYP2A6

N-Nitrosodiethylamine NNK*

CYP2B6

6-Aminochrysene Cyclophosphamide Ifosphamide

CYP2C8, 9, 18, 19

Tienilic acid Valproic acid

CYP2D6

NNK*

CYP2E1

Acetaminophen Acrylonitrile Benzene

Carbon tetrachloride Chloroform Dichloromethane 1,2-Dichloropropane Ethylene dibromide Ethylene dichloride Ethyl carbamate Halothane N-Nitrosodimethylamine Styrene Trichlorothylene

Vinyl chloride

CYP3A4

Acetaminophen Aflatoxin B1 and G1 6-Aminochrysene

Benzo[a]pyrene 7,8-dihydrodiol Cyclophosphamide Ifosphamide

1-Nitropyrene Sterigmatocystin Senecionine

Tris(2,3-dibromopropyl) phosphate

CYP4A9/11

None known

*NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific nitrosamine.

sOURCE: Adapted from Guengerich and Shimada, 1991.

190	UNIT 2 DISPOSITION OF TOXICANTS

genation both play a role in the activation of halothane, although hepatotoxicity in rats is more dependent on reductive dehalogenation, whereas the immune hepatitis in humans is largely a consequence of oxidative dehalogenation, which leads to the formation of neoantigens (Pohl et al., 1989). Formation of neoantigens (by covalent binding to CYP2C9) is also the mechanism by which the uricosuric diuretic drug tienilic acid causes immune hepatitis (Koenigs et al., 1999).

Some of the chemicals listed in Table 6-6 are activated to toxic or tumorigenic metabolites by mechanisms not mentioned previously. For example, N-nitrosodimethylamine, which is representative of a large class of tumorigenic nitrosamines, is activated to an alkylating electrophile by N-demethylation, as shown in Fig. 6-44. The activation of ethyl carbamate (urethan) involves two sequential reactions catalyzed by cytochrome P450 (CYP2E1): dehydrogenation to vinyl carbamate followed by epoxidation, as shown in Fig. 6-44. CYP2E1 is one of several P450 enzymes that can catalyze the epoxidation of tetrachloroethylene. The rearrangement of this epoxide to a carbonyl is accompanied by migration of chlorine, which produces the highly reactive metabolite,

trichloroacetylchloride, as shown in Fig. 6-44. The toxic pyrrolizidine alkaloids, such as senecionine, are cyclic arylamines that are dehydrogenated by cytochrome P450 (CYP3A4) to the corresponding pyrroles. Pyrroles themselves are nucleophiles, but electrophiles are generated through the loss of substituents on the pyrrolizidine nucleus, as shown in Fig. 6-44 (Mabic et al, 1999).

Cyclophosphamide and ifosphamide are examples of chemicals designed to be activated to toxic electrophiles for the treatment of malignant tumors and other proliferative diseases. These drugs are nitrogen mustards, which have a tendency to undergo intramolecular nucleophilic displacement to form an electrophilic aziridinium species. In the case of cyclophosphamide and ifosphamide, the nitrogen mustard is stabilized by the presence of a phosphoryl oxygen, which delocalizes the lone pair of nitrogen electrons required for intramolecular nucleophilic displacement. For this reason, formation of an electrophilic aziridinium species requires hydroxylation by cytochrome P450, as shown in Fig. 6-44 for cyclophosphamide. Hydroxylation of the carbon atom next to the ring nitrogen leads spontaneously to ring opening and elimination of acrolein. In the resultant phosphoramide mustard, delo-

Figure 6-44. Additional mechanisms of cytochrome P450–dependent activation of xenobiotics to reactive

(electrophilic) metabolites.

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191

calization of the lone pair of nitrogen electrons to the phosphoryl oxygen is now disfavored by the presence of the lone pair of electrons on the oxygen anion, hence the phosphoramide undergoes an intramolecular nucleophilic elimination to generate an electrophilic aziridinium species. This reaction is catalyzed by CYP3A4 and CYP2B6. Cyclophosphamide is also activated by CYP2B enzymes in rats, one of which (CYP2B12) is expressed in the skin (Friedberg et al., 1992). Activation of cyclophosphamide by P450 enzymes in the skin would generate a cytotoxic metabolite at the base of hair follicles, which may be the reason why hair loss is one of the side effects of cyclophosphamide treatment.

Many of the chemicals listed in Table 6-6 are also detoxified by cytochrome P450 by biotransformation to less toxic metabolites. In some cases, the same P450 enzyme catalyzes both activation and detoxication reactions. For example, CYP3A4 activates aflatoxin B1 to the hepatotoxic and tumorigenic 8,9-epoxide, but it also detoxifies aflatoxin B1 by 3-hydroxylation to aflatoxin Q1. Similarly, CYP3A4 activates senecionine by converting this pyrrolizidine alkaloid to the corresponding pyrrole, but it also detoxifies senecionine through formation of an N-oxide (a reaction mainly catalyzed by FMO3). Epoxidation of trichloroethylene by CYP2E1 appears to be both an activation and detoxication pathway, as shown in Fig. 6-44. Rearrangement of trichloroethylene epoxide can be accompanied by migration of chlorine, which produces chloral (trichloroacetaldehyde), or hydrogen, which produces dichloroacetylchloride. Chloral is much less toxic than dichloroacetylchloride, hence, migration of the chlorine during epoxide rearrangement is a detoxication reaction, whereas migration of the hydrogen is an activation reaction. These few examples serve to underscore the complexity of factors that determine the balance between xenobiotic activation and detoxication. Further details of xenobiotic activation are available in recent review articles (Bolton et al., 2000; Mabic et al., 1999; Pirmohamed and Park, 1999; Purohit and Basu, 2000).

P450 Knockout Mice

Several strategies have been developed to explore the role of cytochrome P450 enzymes in the activation of xenobiotics. Cytochrome P450 levels in rodents can be increased by a variety of inducers, which would be expected to enhance xenobiotic toxicity. Alternatively, cytochrome P450 activity can be decreased by various inhibitors, which would be expected to have a protective effect. (Inducers and inhibitors of cytochrome P450 are discussed later in this section.) Various in vitro techniques can be employed, similar to those described earlier in this section for determining the role of human P450 enzymes in drug metabolism. Transgenic mice that lack one or more P450 enzymes, which are commonly referred to as knockout mice or null mice, provide a relatively new strategy to evaluate the role of specific P450 enzymes in xenobiotic activation (Gonzalez and Kimura, 1999; Buters et al., 1999). It should be noted, however, that such transgenic mice often have abnormal phenotypes due to disruption of genes other than the target gene. Moreover, deletion of the same gene can produce markedly different phenotypes, as has been observed in different transgenic mice lacking the Ah receptor.

Knockout mice have confirmed the role of several P450 enzymes in the activation of specific xenobiotics. For example, CYP2E1 knockout mice are relatively resistant to the myelotoxic effects of benzene (as previously discussed under “PeroxidaseDependent Cooxidation”). These same mice are relatively resist-

ant to the toxic effects of chloroform and the lethal effects of acetaminophen. CYP1A2 knockout mice are also relatively more resistant to acetaminophen toxicity, whereas mice lacking both CYP1A2 and CYP2E1 are highly resistant. These results were largely expected based on other evidence implicating CYP2E1 in the activation of benzene, chloroform, and acetaminophen and CYP1A2 in the activation of acetaminophen. However, some results with knockout mice were unexpected. For example, contrary to expectation, CYP1A2 knockout mice were not resistant to hepatotumorigenic effect of 4-aminobiphenyl, even though CYP1A2 was thought to be primarily responsible for catalyzing the initial step in the activation of this carcinogenic amine (i.e., N-hydroxylation). On the other hand, the demonstration that CYP1B1 knockout mice are resistant to the toxic and tumorigenic effects of 7,12-dimethylbenzanthracene provides compelling evidence that CYP1B1, which is expressed in numerous extrahepatic tissues, plays a major role in the activation of polycyclic aromatic hydrocarbons to carcinogenic metabolites (Buters et al., 1999).

These studies in knockout mice are relevant to humans because their counterpart can be found in those individuals who lack certain P450 enzymes or other xenobiotic-biotransforming enzymes. Experiments in knockout mice underscore how genetic polymorphisms in the human population are risk modifiers for the development of chemically induced disease.

Inhibition of Cytochrome P450

In addition to predicting the likelihood of some individuals being poor metabolizers due to a genetic deficiency in P450 expression, information on which human P450 enzyme metabolizes a drug can help predict or explain drug interactions (Peck et al., 1993). For example, when administered with azole antifungals (e.g., ketoconazole and itraconazole) or macrolide antibiotics (e.g., erythromycin and troleandomycin), the antihistamine terfenadine (Seldane) can cause torsades de pointes, which in some individuals has apparently led to lethal ventricular arrhythmias (Kivsito et al., 1994). This drug interaction can be rationalized on the basis that terfenadine is normally converted by intestinal and liver CYP3A4 to a tertiary-butyl alcohol, which is further oxidized to a carboxylic acid metabolite. This latter metabolite blocks H1 receptors and does not cross the blood–brain barrier, which is why terfenadine is a nonsedating antihistamine. When formation of the carboxylic acid metabolite is blocked by CYP3A4 inhibitors — such as ketoconazole, itraconazole, erythromycin, or troleandomycin — the plasma levels of the parent drug, terfenadine, become sufficiently elevated to block cardiac potassium channels, which can lead to arrhythmias. In some cases, inhibition of cytochrome P450 is advantageous. For example, ritonavir blocks the CYP3A-dependent metabolism of sequinavir and thereby improves its pharmacokinetic profile. Both these drugs are HIV protease inhibitors, and combined therapy is better than monotherapy in helping to curtail the development of drug-resistant strains of HIV. Ketoconazole and erythromycin inhibit the biotransformation of cyclosporine by intestinal and liver CYP3A4 and consequently increase the bioavailability of cyclosporine and decrease the rate of elimination of this expensive immunosuppressant. However, much higher doses of cyclosporine must be given to patients taking the CYP3A4 inducer rifampin in order to achieve therapeutic levels and immune suppression.

Inhibitory drug interactions generally fall into three categories. The first involves competition between two drugs that are

192	UNIT 2 DISPOSITION OF TOXICANTS

metabolized by the same P450 enzyme. For example, omeprazole and diazepam are both metabolized by CYP2C19. When the two drugs are administered simultaneously, omeprazole decreases the plasma clearance of diazepam and prolongs its plasma half-life. The inhibition of diazepam metabolism by omeprazole is presumed to involve competition for metabolism by CYP2C19 because no such inhibition occurs in individuals who, for genetic reasons, lack this polymorphically expressed P450 enzyme. The second inhibitory drug interaction is also competitive in nature, but the inhibitor is not a substrate for the affected P450 enzyme. The inhibition of dextromethorphan biotransformation by quinidine is a good example of this type of drug interaction. Dextromethorphan is O-demethylated by CYP2D6, and the clearance of dextromethorphan is impaired in individuals lacking this polymorphically expressed enzyme. The clearance of dextromethorphan is similarly impaired when this antitussive agent is taken with quinidine, a potent inhibitor of CYP2D6. However, quinidine is not biotransformed by CYP2D6, even though it binds to this enzyme with high affinity (Ki 100 nM). Quinidine is actually biotransformed by CYP3A4, and is a weak competitive inhibitor of this enzyme (Ki 100 M). The COX-2 inhibitor celecoxib (Celebrex) is also a CYP2D6 inhibitor, even though it is metabolized by other P450 enzymes.

The third type of drug interaction results from noncompetitive inhibition of cytochrome P450, and it often involves metabo- lism-dependent inhibition of cytochrome P450 (also known as mechanism-based or suicide inactivation) (Halpert et al., 1994). The inhibition of terfenadine metabolism by macrolide antibiotics appears to be an example of this type of drug interaction. CYP3A4 converts macrolide antibiotics to a metabolite that binds so tightly (but noncovalently) to the heme moiety of CYP3A4 that it is not released from the enzyme’s active site. The noncompetitive inhibition of a P450 enzyme by a metabolism-dependent inhibitor can completely block the metabolism of a drug. As the fatal interactions between macrolide antibiotics and terfenadine indicate, noncompetitive inhibition of cytochrome P450 can have profound consequences. Numerous compounds are activated by cytochrome P450 to metabolites that bind covalently to the heme moiety or surrounding protein. These compounds, known as suicide inactivators, include various halogenated alkanes (CCl4), halogenated alkenes (vinyl chloride, trichloroethylene), allylic compounds (allylisopropylacetamide and secobarbital), and acetylenic compounds (ethinylestradiol and the ethynylprogesterone, gestodene). Ethinyl derivatives of various P450 substrates have been synthesized as potential selective metabolism-dependent inhibitors of individual P450 enzymes. For example, polycyclic aromatic hydrocarbons are preferred substrates for CYP1A1, and this enzyme can be inactivated by various ethynyl derivatives of naphthalene and pyrene. 9-Ethynylphenanthrene is a metabolism-dependent inhibitor of CYP2B6. Furafylline is a metabolism-dependent inhibitor of CYP1A2, for which the structurally related xanthine, caffeine, is a substrate. Similarly, tienilic acid is metabolism-dependent inhibitor of CYP2C9, whereas 8-methoxypsoralen, which is a derivative of coumarin, is a metabolism-dependent inhibitor of CYP2A6 (Table 6-5).

Induction of Cytochrome P450

In contrast to inhibitors, inducers of cytochrome P450 increase the rate of xenobiotic biotransformation (Conney, 1967, 1982; Batt et al., 1992). Some of the P450 enzymes in human liver microsomes

are inducible, as summarized in Table 6-5 (Pichard et al., 1990). Clinically important consequences of P450 enzyme induction include the enhanced biotransformation of cyclosporine, warfarin, and contraceptive steroids by inducers of the CYP3A and CYP2C enzymes and enhanced activation of acetaminophen to its hepatotoxic metabolite N-acetylbenzoquinoneimine, by the CYP2E1 inducers ethanol and isoniazid, and possibly by CYP3A enzyme inducers. As an underlying cause of serious adverse effects, P450 induction is generally less important than P450 inhibition, because the latter can cause a rapid and profound increase in blood levels of a drug, which can cause toxic effects and symptoms of drug overdose. In contrast, cytochrome P450 induction lowers blood levels, which compromises the therapeutic goal of drug therapy but does not cause an exaggerated response to the drug. An exception to this rule is the potentiating effect of alcohol and isoniazid on acetaminophen hepatotoxicity, which is in part because of cytochrome P450 induction.

However, even this drug interaction is complicated by the fact that ethanol and isoniazid are inhibitors as well as inducers of CYP2E1 (Zand et al., 1993). Consequently, increased activation of acetaminophen by ethanol and isoniazid is a delayed response, due to the time required for increased synthesis of CYP2E1 and for the inducers to be cleared to the point where they no longer cause an overall inhibition of CYP2E1 activity. CYP3A4 and CYP1A2 are also inducible enzymes capable of activating acetaminophen to a reactive quinoneimine. By inducing CYP3A4, rifampin and barbiturates would be expected to enhance the hepatotoxicity of acetaminophen, and there is some clinical evidence that this does occur. In contrast, induction of CYP1A2 by cigarette smoking or dietary exposure to polycyclic aromatic hydrocarbons (in charcoalbroiled beef) or indole-3-carbinol derivatives (in cruciferous vegetables) has been reported to have no effect on the hepatotoxicity of acetaminophen, even though CYP1A2 induction in rodents potentiates the hepatocellular necrosis caused by acetaminophen.

Induction of cytochrome P450 would be expected to increase the activation of procarcinogens to DNA-reactive metabolites, leading to increased tumor formation. Contrary to expectation, there is little evidence from either human epidemiologic studies or animal experimentation that P450 induction enhances the incidence or multiplicity of tumors caused by known chemical carcinogens. In fact, most evidence points to a protective role of enzyme induction against chemical-induced neoplasia (Parkinson and Hurwitz, 1991). On the other hand, transgenic (knockout) mice lacking the Ah receptor were recently shown to be resistant to the carcinogenic effects of benzo[a]pyrene (Shimizu et al., 2000), which suggests that induction of CYP1A1 plays an important role in the carcinogenicity of polycyclic aromatic hydrocarbons (PAH). However, the Ah receptor may be critical to PAH carcinogenicity for reasons other than its role in CYP1A1 induction. For example, the Ah receptor may transport DNA-reactive metabolites, such as ortho- quinones, to the nucleus (see Fig. 6-6; Burczynski and Penning, 2000), or it might regulate the expression of genes that influence tumor promotion, as has been proposed for other receptors that mediate cytochrome P450 induction (discussed later in this section). In this regard it is significant that benzo[a]pyrene is a complete carcinogen (both an initiator and promoter), and that TCDD and other Ah receptor ligands are potent tumor promoters.

Cytochrome P450 induction can cause pharmacokinetic tolerance, as in the case of artemisinin [which induces CYP2C19 (Mihara et al., 1999)], in which case larger doses of drug must be administered to achieve therapeutic blood levels due to increased drug