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DRUG METABOLISM REVIEWS, 34(1&2), 17–35 (2002) CHARACTERISTICS AND COMMON PROPERTIES OF INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES Paul F. Hollenberg The University of Michigan, Ann Arbor, Michigan HISTORICAL BACKGROUND The modification of the enzymatic activity of the cytochrome P450 (CYP) enzymes by inhibition, induction, or activation is of great interest to enzymologists, pharmacologists, and chemists who are interested in mechanisms relating to these three effectors of enzyme activity. When the activity of a CYP is modified in vivo in humans, these effects are a major concern for clinicians and patients due to the potential for these alterations to greatly change the metabolism of the drug substrate(s) for that enzyme and thereby alter the biological activity of the drug leading to the potential for harmful drug– drug interactions. The inhibition and induction of the CYP enzymes are probably the most common causes for most drug interactions that have been documented in the literature (1). The inhibition of the metabolism of one drug, as a result of competition between two different drugs for metabolism by the same CYP, may result in unexpected elevations in the plasma concentrations of one or both drugs that can ultimately result in a variety of minor as well as serious adverse effects. These types of interactions have led to very serious adverse events including fatalities and ultimately have resulted in several prominent drugs being withdrawn from the market (2,3). The onset of inhibition is usually rapid following a single administration of the inhibitory compound. However, as will be discussed later, some types of inhibition are not manifested rapidly. On the other hand, enzyme induction, which results in an increase in the total amount of an enzyme with a concomitant increase in its drug metabolizing ability, may attenuate the pharmacological effect of a drug as a result of increasing the metabolism and subsequent elimination leading to marked decreases in plasma concentrations. 17 Copyright q 2002 by Marcel Dekker, Inc. www.dekker.com 18 HOLLENBERG Unlike inhibition, the onset of induction is generally relatively slow and it may take days or even weeks for the full effects to be manifested. Although inhibitory effects generally involve the direct interaction of the inhibitory drug with the CYP it is effecting, the effects of inducers are generally indirect in that in most cases they do not interact physically with the enzyme in order to cause induction. Significant drug interactions leading to limited utility of a given drug entity or even its withdrawal from the market may result in significant economic losses for the pharmaceutical companies. Therefore, pharmaceutical companies are developing and utilizing many new approaches that can be used to predict the possibility that new drug candidates will cause significant drug interactions through inhibition, induction, or activation of the CYPs. During the last decade, our knowledge of the structures and the regulatory mechanisms of the CYP enzymes have grown remarkably as a result of the many major advances in biochemical technology and molecular biology. Although the mechanistic aspects of both CYP inhibition and induction are much better understood than previously, the accurate prediction of the occurrence and consequences of drug– drug interactions continues to be an important unsolved challenge. INHIBITORS OF THE CYTOCHROME P450 ENZYMES As can be seen from the tabular presentation included as part of this issue of Drug Metabolism Reviews, many inhibitors of the CYP enzymes have been identified and although some are inhibitory for a number of different CYPs, a fair number are very selective for only one enzyme. This property is particularly important for the identification of the role that a particular enzyme plays in the metabolism of a given drug substrate and for the development of drugs that may target a specific enzyme. The nature of the catalytic cycle of the CYP enzymes presents a number of potential points at which inhibition of substrate metabolism may occur. The primary steps in the catalytic cycle for the reactions catalyzed by the CYPs are: 1) substrate binding; 2) one-electron reduction of the ferric (Fe+3) enzyme to the ferrous (Fe+2) enzyme; 3) binding of molecular oxygen to the ferrous (Fe+2) iron; 4) transfer of the second electron to the ferrous-oxy-substrate complex leading to the release of water and the formation of an activated oxygen intermediate; 5) the catalytic insertion of the activated oxygen into the substrate to form the oxygenated product; and 6) the release of the oxygenated product resulting in the release of the native ferric (Fe+3) form of the enzyme that can then undergo another catalytic cycle (4). Three of these steps: 1) substrate binding; 2) the binding of molecular oxygen to the ferrous (Fe+2) enzyme; and 3) the catalytic step in which the activated oxygen is transferred from the heme iron to the substrate, appear to be particularly susceptible to inhibition (5). An additional target for inhibitors is the transfer of electrons from the CYP reductases to the CYP, which occurs following substrate addition and after the binding of molecular INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES 19 oxygen to the ferrous CYP. These inhibitors generally interfere with the transfer of electrons to the CYP by accepting electrons directly from the reductase and consequently do not cause inhibition by interacting directly with the CYP. Therefore, they are generally nonspecific with respect to the CYP forms they inhibit. The basic types of enzyme inhibitors include: 1) competitive; 2) noncompetitive; 3) uncompetitive; 4) product inhibition; 5) transition-state analogs; 6) slow, tight binding inhibitors; and 7) irreversible inhibitors (6). Inhibitors of the CYPs can be divided into three general categories that differ in their mechanisms (5). They are: 1) compounds that bind reversibly; 2) compounds that form quasi-irreversible complexes with the iron of the heme prosthetic group; and 3) compounds that bind irreversibly to the prosthetic heme or to the protein, or that cause covalent binding of the heme prosthetic group or its degradation product to the apoprotein. In general, those inhibitors that interfere with the catalytic cycle prior to the formation of the activatedoxygen intermediate are reversible inhibitors that act as competitive, noncompetitive, uncompetitive, product, or transition-state inhibitors. Those compounds that act during or subsequent to the formation of the activated oxygen intermediate are generally either quasi-irreversible or irreversible inhibitors and, in many cases, have been shown to be mechanism-based inactivators (5). Since the inhibitors of the human CYPs are indicated in the Table in this issue, they will not be discussed individually here but we will focus on the types and mechanisms of inhibition. Generally, reversible inhibition is thought to be the most common cause of drug– drug interactions. Reversible inhibition of CYPs is transient and the normal metabolic functions of the enzymes will continue following elimination of the inhibitor from the body. As opposed to the irreversible and quasi-irreversible inhibitors that exhibit both dose-dependent and time-dependent inhibition of substrate metabolism, reversible inhibitors exhibit only dose-dependent inhibition (7). As already indicated, reversible inhibition be can further classified as competitive, noncompetitive, and uncompetitive and may involve product inhibition, or inhibition by transition-state analogs or slow, tight binding inhibitors. In competitive inhibition, binding of the inhibitor to the enzyme prevents the binding of the substrate to the active site of the enzyme. This generally is believed to be the result of the inhibitor sharing some degree of structural similarity with the substrate(s) of that CYP. Depending on the substrate specificity of the CYP, some of which exhibit relatively little apparent specificity, the structural similarities between the competitive inhibitor and the substrate whose metabolism is inhibited may or may not be apparent. Although the competitive inhibitor may be a substrate also for the CYP that it inhibits, that is not necessarily the case. The competition by the inhibitor for occupancy of the CYP active site may involve simple competition for binding to a lipophilic domain in the active site or it may involve hydrogen bonding or ionic bonds with specific amino acid residues in the 20 HOLLENBERG active site (5). This type of inhibition is most commonly observed when two different substrates of the same CYP enzyme are present. In the case of classic competitive inhibition where the inhibitor is not a substrate of the enzyme, the Km apparent for the substrate increases in the presence of the inhibitor; however, there is no change in the Vmax. The standard approach to characterizing competitive inhibition involves performing metabolism studies with varying concentrations of both the substrate, S, and the inhibitor, I, and analyzing the data using a Lineweaver – Burk plot (1/v vs. 1/S ) or other transformations of the Michaelis – Menten equation. In the Lineweaver– Burk plot, competitive inhibition is identified by a common intersection point for all the lines on the ordinate axis. The Dixon plot in which 1/v is plotted vs. S is an alternative method which is often used (8). Since all of the linear transforms of kinetic data have some problems with the weighting of the data points (9), a variety of nonlinear regression programs have been developed and are readily available for the data from studies on the effects of inhibitors on metabolism. One approach that has been developed for the analysis of enzyme inhibitor mechanisms involves the use of virtual kinetics (10). When the inhibitor is not metabolized by the CYP being studied, Ki is the actual binding constant for the inhibitor, unlike the Km, which is usually not equal to the binding constant of the substrate (8). However, when the inhibitor is also metabolized by the CYP, then Ki may not be a valid measure of the binding constant. In this case, the two substrates are exhibiting mutually competitive inhibition of each other. Competitive inhibition is encountered relatively frequently in drug metabolism studies both when using microsomal preparations or purified reconstituted enzyme systems in vitro as well as in metabolism studies performed in vivo. Approaches have been developed for characterizing inhibitory interactions in vivo through analysis of various pharmacokinetic parameters (11,12). Since many of the CYPs have numerous drugs as substrates, competition of various drugs for metabolism by a specific CYP is a common occurrence leading to drug– drug interactions in patients who are simultaneously administered several different drugs. In noncompetitive inhibition, the inhibitor binds to the enzyme at a site other than the active site of the enzyme and has no effect on substrate binding. However, the enzyme – substrate– inhibitor complex is unable to function catalytically. In this case, a decrease in the Vmax is observed without a concomitant change in the Km. Although this type of inhibition is discussed routinely in any presentation of enzyme kinetics, examples of such inhibition are relatively uncommon and they are rarely observed, if at all, in studies of metabolism by CYPs. In studies with microsomes or the expressed enzyme, this type of kinetic behavior would be expected to be observed also when some of the enzyme involved in metabolism is inactivated, e.g., by a mechanism-based inactivator or by the formation of an MI complex (to be discussed later). INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES 21 Uncompetitive inhibition, like noncompetitive inhibition, is readily defined but it also is seldom seen in drug metabolism. In this case, the inhibitor, instead of binding to the free enzyme, binds to the enzyme– substrate complex resulting in the formation of a nonproductive enzyme –substrate– inhibitor complex. In this case, both Vmax and Km are decreased proportionately so that the ratio Vmax/Km remains constant. In a Lineweaver –Burk plot of the data, parallel lines would be observed with different concentrations of the inhibitor. Some reversible inhibitors act by coordinating with the prosthetic heme iron atom. The coordination of a strong ligand to the heme iron shifts the iron from the high- to the low-spin form giving rise to the “Type II” difference spectrum (13). This change in the spin state occurs concomitantly with a change in the redox potential of the CYP that makes reduction by the CYP reductase more difficult (14). Thus, the inhibition of CYPs by strong iron ligands is a result not only of the occupation of the sixth coordination site of the heme iron but also the change in the reduction potential of the iron. Inhibitors that bind to both a hydrophobic binding site in the CYP active site and to the prosthetic heme-iron are generally much more effective inhibitors than those that utilize only one of those interactions (5). In general, these relatively potent reversible inhibitors are nitrogen-containing aliphatic and aromatic compounds. Some of the most widely used compounds in this class are derivatives of imidazole and pyridine. A second category of inhibitors includes those compounds that form quasiirreversible complexes with the heme iron-atom. These compounds generally require catalytic activation by the enzyme to transient intermediates that then coordinate very tightly to the prosthetic heme in the CYP active site leading to inhibition. This coordination is generally so tight that the inhibitory complexes can only be broken down to release the native catalytically active enzyme under special experimental conditions. The formation of these metabolic intermediates that can coordinate so effectively with the CYP to convert it to a catalytically nonfunctional state is associated with several different classes of compounds including those containing a dioxymethylene function and nitrogen-containing compounds including 1,1-disubstituted hydrazines, acyl hydrazines, and a variety of alkylamines that may be converted to nitroso metabolites. Alkyl and aryl methylenedioxy compounds are oxidized by CYPs to yield species that coordinate tightly to the prosthetic heme iron leading to metabolite intermediate complexes (MI complexes) that are extremely stable. A requirement for an initial catalytic event has been unequivocally demonstrated (15). The stability of the ferrous MI complex is demonstrated by the fact that it can be isolated intact from rats treated with isosafrole. The ferric MI complex is much less stable than the ferrous complex and incubation with lipophilic compounds that can displace the inhibitor from the active site lead to regeneration of the native catalytically active enzyme (16). The nature of the side-chain substituent on the methylenedioxy compound is an important determinant of the formation of the MI complex and its stability. Both the size and lipophilicity of the alkyl group are important since analogues 22 HOLLENBERG with alkyl chains of one to three carbons give relatively unstable complexes while those with larger alkyl groups are stable (17). A second class of agents that forms MI complexes with the CYP heme iron is the relatively large class composed of alkyl and aromatic amines. This class includes a number of clinically important antibiotics such as erythromycin and troleandomycin (18). The oxidation of these amines yields intermediates that coordinate tightly to the ferrous heme resulting in an optical spectrum with an absorbance maximum at 445– 455 nm. Primary amines, but not secondary or tertiary amines, are capable of forming the MI complexes following oxidation. Secondary and tertiary amines may also form MI complexes following N-dealkylation to the primary amines. The primary amines appear to be hydroxylated initially followed by a two-electron oxidation of the resulting hydroxylamine to give the nitroso group, which currently is thought to be involved in chelation with the iron to form the MI complex (18). A final category of inhibitors are those compounds that bind irreversibly to the prosthetic heme or to the protein, or that cause covalent binding of the heme prosthetic group, or its degradation product to the apoprotein. These compounds require metabolic activation by the CYPs and are part of a class of inhibitors commonly referred to as “catalysis-dependent”, “suicide”, or “mechanism-based” inactivators (19,20,21). Mechanism-based inactivation is generally regarded to be a relatively unusual occurrence with most enzymes. However, it is observed in reactions catalyzed by CYPs in somewhat higher frequency than would normally be expected, possibly as a result of the reactivity of the oxygenated intermediates formed during the course of the oxygenation reactions. The utility of mechanismbased inactivators in the design of new drugs that are highly selective for a given CYP enzyme has attracted great interest recently since, in principle, these compounds could be designed so that they would only inhibit the target enzyme (21). These inactivators also have attracted considerable interest as a result of their utility in elucidating enzyme mechanisms. Finally, a great deal of effort has been expended on the development of mechanism-based inactivators as inhibitors of specific CYP enzymes that can be used as diagnostic tools to identify which form(s) of the CYPs are involved in catalyzing a particular reaction in microsomal preparations. These compounds result in the formation of covalent bonds that cannot be broken to regenerate catalytically active enzyme. Although the onset of inhibition by these compounds may occur in vivo more slowly than that observed with reversible or quasi-irreversible inhibitors, the final effect of the mechanismbased inactivators is generally much more profound and inhibition of drug metabolism can be reversed only by the synthesis of new, catalytically active CYPs. A variety of compounds have been shown to be good mechanism-based inactivators for CYPs. These include: 1) acetylenes and terminal alkyl and aryl olefins such as 2-ethynylnaphthalene, 9-ethynylphenanthrene, 5-phenyl-1-pentyne, 10-undecynoic acid, and 17a-ethynyl-estradiol; 2) a variety of organosulfur INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES 23 compounds such as disulfiram, cimetidine, dialkylsulfides, parathion, diethyldithiocarbamate, isothiocyanates, thioureas, and xanthates; 3) halogenated compounds such as chloramphenicol and N-monosubstituted dichloroacetamides; 4) 1-aminobenzotriazole and its N-aralkylated derivatives; and 5) furanocoumarins such as methoxypsoralins, L -754, 394 (a Merck compound synthesized as a potential HIV protease inhibitor) and bergamottin (5,21,22). Reactions that result in the mechanism-based inactivation of CYPs involve the formation of a complex between the CYP and a reactive intermediate that can then either react with the CYP to form a covalent adduct resulting in inactivation or it can dissociate resulting in product formation and the regeneration of the native catalytically active enzyme. The derivation of the various kinetic constants for this type of reaction has been described (20). Key to this type of inactivation is the concept of the partition ratio (23). The partition ratio can be thought of as the number of latent activator molecules metabolized and released as product for each molecule metabolized to give the inactivated form of the enzyme. This can be thought of also as the number of cycles of metabolism the enzyme can traverse, on the average, before it is inactivated. The partition ratio is generally considered a measure of the efficiency of the inactivator. The most efficient mechanism-based inactivator would have a partition ratio of zero and in a standard assay where metabolite formation is measured it would not be considered a substrate since product formation could not be measured. Mechanism-based inactivators having partition ratios ranging from almost zero to several thousand have been reported. As pointed out by Ortiz de Montellano and Correia (5), mechanism-based inactivators may exhibit better enzyme specificity than reversible inhibitors since: 1) the initial binding of the inhibitor to the specific CYP enzyme must satisfy all of the constraints imposed on reversible inhibitors; 2) the inhibitor must be acceptable as a substrate for that CYP due to the requirement for catalytic activation to a reactive species; and 3) the formation of the reactive species during metabolism leads to an irreversible modification of the enzyme that permanently removes it from the pool of active enzymes. The apparent flexibility of the active sites of many of the CYPs, as evidenced by the sizes and numbers of different substrates they can metabolize, offers the possibility of designing many different mechanism-based inactivators having a variety of potentially reactive moieties. Due to their broad substrate specificities, a major problem in designing mechanism-based inactivators for CYPs lies in designing ones that will be specific for a single CYP. The development of reversible, quasi-irreversible, and irreversible inhibitors of CYPs and our understanding of the mechanisms of action of these various types of inhibitors have increased remarkably over the past few years and have provided important insights for the development of highly selective isozyme-specific inhibitors of the CYPs. These inhibitors are of substantial interest not only for studies probing the structures, mechanisms of action, and biological roles of 24 HOLLENBERG specific P450s, but also because of their potential as modulators of CYP activity that can be used as therapeutic agents in a manner analogous to the way inhibitors of the steroidogenic P450s have been used to treat endocrine disorders and as anticancer agents. Since different CYP enzymes play major roles in the metabolic activation and detoxication of various chemical carcinogens and other toxins, the development of agents that can be used to selectively inactivate these enzymes in order to shift the balance between the various metabolic pathways such that metabolic activation is minimized whereas detoxication is enhanced would be of great value. INDUCERS OF THE CYTOCHROME P450 ENZYMES The induction of the drug metabolizing enzymes was recognized first because of the profound effects that it had on the pharmacological responses to drugs and other xenobiotics (24). For example, animals exposed chronically to barbiturates were shown to exhibit “tolerance” to the hypnotic effect of the barbiturates as a result of the induction of the CYPs responsible for their metabolism. Induction of CYPs with specific inducers also was shown to decrease tumor formation in animals exposed to chemical carcinogens. These two examples illustrate two important aspects of induction that were understood early on. First, inducers are substrates for the enzymes they induce and secondly, enzyme induction oftentimes may enhance detoxication; particularly for exposure to relatively low concentrations of substrate. Depending on the CYP enzyme(s) being induced and the compounds that the animal is exposed to, induction may be extremely deleterious also. For example, if the induced CYP catalyzes the metabolic activation of a toxin or carcinogen, exposure of the organism to that compound following induction may greatly enhance its toxicity or carcinogencity (24). The effect of an inducer is to increase the activity of one or more enzymes by causing an increase in the intracellular concentration of the induced enzyme(s). Although the increase in the level of the induced protein is oftentimes the result of an increase in the transcription of the gene associated with the induced protein, this is not always the case. Enzyme induction generally exhibits typical doseresponse relationships. Although a single CYP enzyme can often account for most of the induction by a given inducer, there may be significant inductive effects on one or more additional CYPs. Induction of forms that are either absent or at extremely low levels constitutively may have profound effects on the metabolism of some substrates. The term induction in general has been restricted to cases in which protein synthesis is stimulated. However, it is important to recognize that the steady-state concentration of a protein is determined by both its rate of synthesis and its rate of degradation. Therefore, any change in the steady-state levels of a CYP due to exposure to an inducer could reflect changes in either process or even both INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES 25 synthesis and degradation. Although there are a few exceptions, inducers of the CYP enzymes have generally been demonstrated to stimulate de novo synthesis of the protein. The rate of CYP synthesis is dependent on the concentration of the mRNA for that enzyme. As was already indicated to be the case for the steadystate concentrations of the proteins, the mRNA concentrations for a given CYP also reflect the rate of transcription of the gene as well as the rate of degradation of the mRNA. In most cases, CYP induction involves an increase in gene transcription. The sequence of events for induction due to increases in gene transcription usually involves a transient increase in the rate of transcription that begins soon after exposure to the inducer with maximum transcriptional activity observed at about 10 –12 hr and the activity returns to basal within approximately 18 hr, depending on the dose of the inducer. The concentration of the specific message increases in parallel with the increase in transcriptional activity. However, the mRNA is generally degraded relatively slowly and therefore, it will accumulate and remain elevated following the return of transcription activity to basal levels. The time required for a protein to reach a new steady-state level as a consequence of an increase in its rate of synthesis is determined by its rate of degradation. The degradation of proteins generally has been shown to be a firstorder process and, depending on the specific CYP, the half-life is anywhere from 8 to 30 hr. As a consequence, the increase in the concentration of the CYP will lag behind the rise in synthesis due to increases in the message. When induction is due to a decrease in the rate of protein degradation, the concentration of the CYP may increase more rapidly than when it is due solely to transcriptional activation. Erythromycin and troleandomycin appear to induce primarily by decreasing the degradation rate for the 3A protein (25). They form MI complexes with the CYP that are relatively resistant to degradation and have been shown to extend the half-life of 3A1 to more than 60 hr. Induction of the CYPs increases the capability for metabolic detoxication and elimination, and thus is considered an important part of the defense system against exposure to xenobiotics. It was noted in the early 1950s that feeding 3-methylcholanthrene to rats reduced the carcinogenic activity of some aminoazo dyes by increasing their N-demethylation (26) and that phenobarbital (PB) elicited sleep times were shortened following chronic administration of barbiturates due to increases in the metabolism of the barbituates. Although induction of the CYPs may be advantageous in most cases, it can have a variety of pharmacological consequences including alterations in drug efficacy, drug – drug interactions, and increases in the metabolic activation of procarcinogens. Consequently induction of the CYPs can be viewed as a “double-edged sword” for the organism involved. Most of the CYP enzymes appear to be inducible, although to various extents. Human CYP 1A1/2, 2A6, 2C9, 2C19, 2E1, and 3A4 are all known to be inducible (27). The molecular mechanisms for CYP induction have been 26 HOLLENBERG extensively studied in rodents beginning with the pioneering studies of Poland and co-workers (28). The enzymes in the CYP1 family are involved in the metabolism of many planar aromatic compounds and aromatic amines. The induction of the genes in this family (CYP1A1, CYP1A2, and CYP1B1) is under the control of the arylhydrocarbon (Ah) receptor. In the absence of ligand, most of this protein exists in the cytosol in a complex with Hsp90, a heat-shock chaperone protein. Binding of an inducing agent to the receptor dissociates this complex leading to translocation of the Ah receptor into the nucleus, where it forms a second heterodimer with the Ah-receptor nuclear transporter (ARNT), a nuclear basic helix – loop– helix protein. This heterodimer then binds to a response element, the xenobiotic response element (XRE), present in multiple copies in the 50 flanking region of the CYP1 genes and functions as a transcriptional enhancer to stimulate gene transcription (29,30). Although this scheme is widely accepted as a general scheme for the induction of CYP1 family members, it is clear that the induction mechanisms may be much more complex under a variety of conditions (27). In addition to the XRE, human CYP1A promoters have a number of other regulatory elements including a glucocorticoid response element (GRE) that mediates glucocorticoid potentiation of CYP1A1 induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs) (31). There is also a negative regulatory element on the 50 -flanking region of the human and rat CYP1A1 genes (32,33) that binds a member of the NF-Y transcription family (33) and the human CYP1A2 gene has an AP-1 site (34). The induction of CYP1 enzymes in humans has been well established (27). The ability of PB to cause marked induction of liver microsomal drug- and steroid-metabolizing enzymes was one of the fundamental observations leading to interest in the P450s. Subsequently, numerous compounds which are structurally unrelated to PB (e.g., allylisopropylacetamide, chlordane, DDT, trans-stilbene oxide, diortho-substituted polychlorinated biphenyls) were shown to induce similar patterns of enzyme activities and are considered to induce in a “PB-like” fashion (35). In rat liver, PB induces a number of different P450s including 2A1, 2B1/2, 2C6/7/11, and 3A1/2. Induction responses range from 50 to 100-fold (2B1/ 2) to 2- to 4-fold (2A1 and 2C6). The CYP2B family of enzymes in rodents has been shown to play important roles in catalyzing the metabolism of a large number of drugs and other xenobiotics in laboratory animals. However, the information on the metabolic capabilities of CYP 2B6, the human orthologue, and its role in metabolism is relatively limited. Hepatic expression of 2B6 is low, averaging about 1% of the total P450, and there is no evidence for inducibility in humans. In contrast to the detailed mechanistic understanding developed for the CYP1 family over the past 20– 30 years the induction mechanisms responsible for increases in CYP2B expression in mammalian systems following exposure to “PB-like” inducers remained largely unknown until recently when several key INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES 27 discoveries were made regarding the mechanisms by which hepatic CYPs are induced by xenochemicals (36,37). Three members of the “orphan” nuclear receptor family of ligand-activated transcription factors (CAR, PXR, and PPAR) have been shown to play crucial roles in the induction of the hepatic CYP2, CYP3, and CPA4 families, respectively, following exposure to the prototypical inducers phenobarbital (CAR), rifampicin and pregnenolone 16a-carbonitrile (PXR), and clofibric acid (PPAR). The acronym CAR stands for constitutive androstane receptor, PXR for pregnane X receptor, and PPAR for peroxisome proliferatoractivated receptors. Liver X receptor (LXR) and Farnesol X receptor (FXR), two other nuclear receptors, are activated by oxysterols and bile acids, respectively, and are involved in regulating the expression of the cholesterol 7a-hydroxylase, a key enzyme in bile acid biosynthesis. All five of these receptors involved in the regulation of CYPs belong to the same family of nuclear receptors (NR1). They share the same heterodimerization partner , the retinoid X-receptor (RXR) and they interact with other nuclear receptors as well as with a variety of other intracellular signaling pathways. Endogenous ligands have been identified recently for each of the nuclear receptors and information has been obtained on their physiological receptor functions (36). The CAR receptor is constitutively active and its endogenous ligands include androstanol and androstenol, which both are inhibitory. Stimulatory ligands for PXR include corticosterone and pregnenolone and for PPAR they include linoleic acid and arachidonic acid (36). To exert their effects the receptors bind to the DNA response elements indicated: CAR (DR4), PXR (DR3, ER6), PPAR (DR1), LXR (DR4), and FXR (IR1) (36). Because of its important role in the metabolic activation of a variety of drugs, chemical carcinogens, and other toxicants, the regulation of CYP2E1 has been the subject of intensive investigation and these studies have demonstrated the involvement of multiple induction mechanisms (27). Induction in rodents has been shown to involve effects on essentially all regulatory levels including increases in transcription, mRNA stabilization, translational efficiency increases, and post-translational protein stabilization (27,38). Starvation, higher levels of ethyl alcohol, and hypophysectomy appear to be associated with increased transcription of 2E1 in rats. CYP2E1 induction observed in diabetic rats may be due to mRNA stabilization. Acetone and pyridine have been reported to exert effects at the level of translational efficiency. This is accompanied by a shift of the polysomal distribution to a higher density along with an increase in incorporation into the newly synthesized protein of radiolabeled amino-acids (39,40). Ethyl alcohol as well as a variety of other small organic molecules appear to induce by post-transcriptional stabilization of the CYP 2E1 apoprotein as evidenced by significant changes in the protein half-life (41). Since these separate induction mechanisms can all occur simultaneously, it has been suggested that the theoretical maximum induction of 50- to 100-fold could be observed (38). 28 HOLLENBERG Enzyme induction studies in cells in culture or in experimental animals can be performed readily since the amounts of the CYPs induced and their catalytic activities can be measured directly. Direct evidence of CYP induction in humans is much more difficult to obtain due to practical and ethical considerations. However, there are some reports that demonstrate large interindividual variability in CYP protein levels (or mRNA) as well as catalytic activity in response to inducers. Ged and co-workers (42) reported results from liver biopsies collected before and after treatment with rifampicin (600 mg/day) for four days. They observed increases due to induction ranging from 160 to 2900%. Kolars et al. (43) also observed considerable interindividual variability in rifampicin induction of CYP3A4 in the small intestine in five healthy volunteers where the increase in message ranged from 0 to 1200%. As mentioned previously, reports of direct evidence of the induction of CYPs in vivo based on measurements of protein levels and catalytic activity are limited. However, there are numerous studies in which the reduction in plasma areas under the curve (AUCs) have been determined as indirect measures of enzyme induction. This approach is based on the belief that induction causes a change in the level of the enzymes involved in metabolizing a probe substrate but not in the identities or the properties of the CYPs. Although these beliefs may be true generally, they are probably not universally true. However, assuming the general applicability of this hypothesis, then enzyme induction leads to an increase in the intrinsic clearance (Vmax/Km) due to an increase in the Vmax that is directly proportional to the increase in the CYP activity due to induction. Thus, the concept of intrinsic clearance is used to relate enzyme induction to changes in the AUC. Large individual variations in enzyme induction have been reported based on this indirect approach. In eight healthy volunteers, induction by rifampicin caused decreases in the oral AUC for S-verapamil ranging from 5- to 60-fold with a mean value of 30-fold (44). Rifampicin treatment also was shown to decrease the AUCs for midazolam by 11.6- to 55-fold in another study of 10 subjects (45). Increases in the oral clearance of cyclosporin ranging from 2.5- to 6.6-fold were seen following treatment with rifampicin (46). Major reasons for these large interindividual variabilities in enzyme induction probably include environmental, dietary, age, and genetic factors. Although there is significant variability among individuals with respect to the magnitude of the inductive effect, there does appear to be a limiting value above which there is no greater increase in activity. That is to say that among individuals, the enzyme levels following maximal induction are quantitatively similar. For example, the maximally induced values of CYP3A in six human hepatocyte cultures were essentially identical, probably representing the normal level to which CYP3A could be induced in those cells. Interestingly, the induction of the activity for the 4-hydroxylation of oxazaphosphorine was inversely related to the basal activity in human hepatocytes (47), suggesting that those individuals with lower basal levels of a given CYP might exhibit a greater degree of enzyme induction. INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES 29 ACTIVATORS OF THE CYTOCHROME P450 ENZYMES A variety of compounds have been identified that enhance the catalytic activity of the CYPs by some type of activation or stimulation mechanism as opposed to induction. Although examples of in vivo stimulation of drug metabolism have been reported, they are rare and the relevance of these types of interactions resulting in enzyme activation in vivo leading to drug –drug interactions is not clear. If CYP stimulation occurs in vivo, the outcome would be likely to be the same as induction. However, one difference that might be expected would be that the effect of an activator would be extremely rapid as opposed to induction, which may require days and multiple doses before the effects of the inducer on drug metabolism are manifested. Examples of activation or stimulation of drug metabolizing enzyme activity in microsomes or in the reconstituted system using purified enzymes of the CYPs are relatively abundant. A variety of compounds are known to stimulate CYP activity in either of these two systems and they are listed in the accompanying tabular presentation included as part of this issue of Drug Metabolism Reviews. In order to stimulate the rate of an enzyme-catalyzed reaction, the stimulatory compound must cause an increase in the rate-limiting step for the overall catalytic reaction. Given the multitude of different CYP enzymes and substrates, it is likely that a variety of rate-limiting steps may be targets for the stimulation of various reactions catalyzed by the CYPs. These would include the first electron transfer, substrate binding, oxygen binding, transfer of the second electron, oxygen activation, insertion of the activated oxygen into the substrate, and product release (4). Depending on the site of action of the activator and its mechanism of action, one might observe increases in the Vmax, decreases in the Km, or both. Possible mechanisms would include allosteric effects on substrate binding, effects on the redox potential of the heme ion, alterations in the interactions between the reductase and the CYP, shunting of electrons from one CYP enzyme to another in microsomes, alterations in the fluidity or other physical or chemical characteristics of the microsomal membrane, or destabilization of the enzyme – product complex. Stimulation of microsomal drug-metabolism involving the CYPs was an area of great interest and experimental activity in the 1970s and some of the key results have been reviewed in excellent reviews by Anders (48), Cinti (49), and Holtzman (50). It is interesting to note that some compounds that inhibit the microsomal metabolism of certain substrates cause marked stimulation of the metabolism of other substrates. For example, metyrapone, which inhibits the oxidative metabolism of tyramine, morphine, hexobarbital, and aminopyrine, significantly increased the microsomal metabolism of acetanilide and trichloroethylene (51). The enhancement of liver microsomal drug oxidations by acetone was first reported by Anders (52). Acetone was shown to stimulate the para-hydroxylation of aniline, acetanilide, and N-butylaniline in microsomal preparations. The acetone stimulation exhibited increased activity with increasing pH and it caused increases in both the Km and Vmax with increasing concentrations. 30 HOLLENBERG Although a number of investigators have attempted to elucidate the mechanism of the acetone enhancement of aniline hydroxylation, the detailed mechanism is still not known (49). Some of the most interesting and enlightening studies involving activation of microsomal drug metabolism have been associated with 7,8-benzoflavone (anaphthoflavone), a synthetic flavonoid that was used widely in studies of chemical carcinogenesis in the 1970s and 1980s (49). Studies by Conney and co-workers showed that the addition of 7,8-benzoflavone to homogenates or microsomes from human liver increased the rates of metabolism of a variety of substrates including benzo[a]pyrene, aflatoxin B1, antipyrine, and zoxazolamine, but had little or no effect on the metabolism of coumarin, 7-ethoxycoumarin, or hexobarbital. This specificity for certain substrates suggested that the flavonoids may exert their stimulatory effects on specific CYP enzymes. This was subsequently demonstrated using purified CYP enzymes in the reconstituted system (53). The CYP3A4 was shown to be activated by several different flavonoids during the bioactivation of benzo[a]pyrene and aflatoxin B1, in studies using the purified recombinant enzyme and specific antibodies (54). A particularly intriguing aspect of the stimulation of CYP3A4 is the regioselectivity exhibited with substrates that can undergo multiple routes of metabolism. One example involves the metabolism of aflatoxin B1 by 3A4. Although 7,8-benzoflavone enhances the 8,9-epoxidation of this substrate, it inhibits its 3a-hydroxylation (55). Detailed studies on purified CYPs in the reconstituted system have lead to the suggestion that the stimulatory influences of xenobiotics on the various CYP enzymes may be due to either homotropic or heterotropic cooperativity (reviewed in Refs. 56– 58). Homotropic cooperativity, also referred to as substrate activation, is an activating effect due to the substrate itself. In this case, a plot of enzyme activity vs. substrate concentration would exhibit a sigmoidal pattern. Heterotropic cooperativity refers to the situation in which the activity for the metabolism of one substrate is increased by the presence of another compound, often referred to as the effector. By definition, homotropic cooperativity would not lead to drug– drug interactions whereas heterotropic cooperativity has the potential for significant drug– drug interactions, the outcome of which would be very similar to those seen with enzyme induction. The mechanisms for homotropic and heterotropic cooperativity of the CYPs are not well understood at this time. However, several models have been proposed describing possible interactions between the CYP and the substrate(s) and/or effector (58 – 60). The stimulation of benzo[a]pyrene metabolism by 7,8-benzoflavone was initially suggested to be due to increased efficiency of the NADPHCYP reductase. However, this explanation for 7,8-benzoflavone stimulation lost credibility following the observation that although 7,8-benzoflavone stimulated CYP3A4 catalyzed 8,9-epoxidation of aflatoxin B1, it inhibited its 3ahydroxylation. In addition, it has been suggested that the CYP3A4 active site may have multiple ligand-binding sites such that the substrate molecule and the INHIBITORS, INDUCERS, AND ACTIVATORS OF CYP ENZYMES 31 effector (which may be a second substrate molecule) may be present simultaneously in the CYP 3A4 active site. Based on studies of several sitespecific mutants, a model has been developed to explain the cooperativity of CYP3A4 that consists of two substrate-binding domains and an effector-binding site (60– 62). A recent model to explain the cooperativity of CYP3A4 is the “nested allosterism” model in which it is proposed that there is a complex of two conformers of the enzyme with each conformer being able to accommodate two binding domains (63). When considered in conjunction with the concept of multiple binding domains, the conformation hypothesis appears to be a reasonable mechanism that can explain both homotropic and heterotropic cooperativity. However, it must be recognized that this is a very abstract concept with no physical representation. In addition, it is based on studies using recombinant enzymes in a reconstituted system that may not reflect concentrations of drugs, effectors or cofactors, interactions, etc., experienced by the drug-metabolizing enzyme systems in vivo in human liver or in extra-hepatic tissues. Thus, the clinical significance of the stimulatory effects (homotropic or heterotropic cooperativity) of any xenochemicals on CYP3A4 or any other CYP remains to be determined. Because of the complexities involved in dealing with human subjects and performing drug metabolism in vivo as well as the large interindividual variability which may constantly change in response to changes in diet, etc., it may not be possible to demonstrate stimulation of a specific CYP activity in vivo. However, this does not negate the importance of understanding the mechanism for the cooperative effects of various compounds in the CYPs and identifying those compounds that may act as potent effectors of one or more CYP enzymes. 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