Probing the World of Cytochrome P450 Enzymes

Introduction

Patients’ responses to medications are often variable and sometimes unpredictable. Response variability can arise from genetic and nongenetic factors that affect either drug-metabolizing enzymes, drug targets (receptors), or both (1). Variability in drug metabolism contributes to interindividual variability in response to medications by altering steady-state plasma drug concentrations. Thus, a tremendous amount of research has focused on trying to elucidate factors that contribute to variability in human drug metabolism. In particular, there has been much interest in metabolism mediated by the cytochrome P450 (CYP) enzyme system.

The cytochrome P450 (CYP) enzyme superfamily, one of the most important drug-metabolizing enzyme systems in humans, is responsible for the oxidative metabolism of a large number of endogenous compounds (e.g., steroids) and xenobiotics (e.g., drugs) (2). The activity of CYP enzymes has been reported to vary up to 50-fold between individuals for some index metabolic reactions (3). Several factors affect CYP enzyme activity, including genetic polymorphisms, age, gender, disease states, and environmental influences such as smoking or exposure to environmental chemicals.

CYP enzymes are primarilylocated in the liver, although some are distributed in other tissues, such as intestine, lung, kidney, and brain. A standard nomenclature system has been developed in which CYP enzymes are named by the root “CYP,” followed by an Arabic number designating the enzyme family to which enzymes are assigned if they share ≥ 40% amino acid sequence identity a letter for the enzyme subfamily (those sharing ≥ 55% sequence identity) and another number denoting the individual CYP enzyme (e.g., CYP2C19) (http://drnelson.utmem.edu/CytochromeP450.html) (4). For each enzyme, the most common or “wild-type” allele is denoted as *1. Allelic variants (i.e., alleles having one or more single nucleotide polymorphisms or SNPs) are sequentially numbered as they are identified (i.e., *2, *3, etc.). A CYP allele nomenclature committee has been established, and current-information on genetic variants can be found at http://www.imm.ki.se/CYPalleles.

A total of 270 CYP gene families found in various organisms-have been described to date (2). The eighteen gene families that exist in humans encode fifty-seven individual CYP genes (2). Despite the large number of CYP genes and enzymes, it appears that only the CYP1, CYP2, and CYP3 families of enzymes have a major role in drug metabolism. The remaining CYP families all have essential roles in intermediary metabolism. For example, the CYP4 family is involved in the oxidation of fatty acids, prostaglandins, and steroids (2). A partial listing of drugs metabolized by the most important CYP enzymes is presented in Table 1⇓ (5). Comprehensive and current information regarding substrates, inhibitors, and inducers of CYP enzymes can be found at http://medicine.iupui.edu/flockhart/.

Variability in CYP Enzyme Activity

One of the major causes of interindividual variability in drug response is the inherent genetic variation among individuals in the major CYP enzymes that contribute to human drug and xenobiotic metabolism (6). For some enzymes (e.g., CYP2D6 and CYP2C19), allelic variants that have been identified are the result of SNPs that create an altered splice site, frameshift mutation, premature stop codon, gene deletion, or missense mutation, each of which produces nonfunctional alleles. SNPs may also cause an amino-acid substitution that results in altered catalytic activity as compared to the fully functional *1 allele (e.g., CYP2C9*2) (7).

Although the genetics of CYP enzymes are important and can have a profound impact on the ability of an individual to metabolize some drugs or environmental chemicals (i.e., xenobiotics), genetics alone does can explain all of the variability. In fact, a large degree of variability is still observed within any particular genotype group. Indeed, other factors including age, diet, disease states, and concomitant drug treatment can substantially alter CYP enzyme activity. Thus, the ability to measure the activity (i.e., the phenotype) of an individual enzyme is an important experimental tool used to evaluate how various factors affect CYP-mediated metabolism. Quantifying in vivo enzyme activity has also proved useful to study the relationship between activity of the drug-metabolizing enzymes and disease (e.g., cancer); to predict possible therapeutic failures or toxic reactions to conventional drug doses; or to characterize drug interactions. The approach to measure enzyme activity that has been applied successfully both in vitro and in vivo is the use of “probe” substrates.

CYP Probe Drugs

Probe substrates (or probe drugs) are compounds that are predominately or exclusively metabolized in vitro by an individual CYP enzyme (8). The metabolism of the candidate probe is generally characterized through the use of preparations containing individually expressed human CYP enzymes or preparations of human liver microsomes (9). Drugs that are selectively metabolized and that can be safely administered to humans may be used as in vivo probe drugs for the purposes of estimating enzyme activity (i.e., phenotyping). For example, the nonsteroidal anti-inflammatory drug flurbiprofen is selectively metabolized in vitro by CYP2C9 to 4-hydroxyflurbiprofen (10), providing the necessary justification for exploring the use of flurbiprofen as an in vivo probe of CYP2C9 activity (11, 12).

The phenotyping procedure typically involves the administration of the probe drug and the collection of blood and/or urine in order to determine some measure of the enzyme’s functional activity. Typically, as small a dose of the probe drug as possible is administered so as to avoid or minimize undesirable clinical effects. An index of enzyme activity, also referred to as a phenotypic trait measure, is chosen to reflect the catalytic activity of a single pathway of metabolism. The intrinsic clearance of a probe or of the metabolite(s) produced, termed formation clearance, is the most appropriate measure of enzyme activity (13). For example, the short-acting benzodiazepine midazolam is frequently used as an in vivo probe of CYP3A activity. Midazolam is predominantly metabolized to 1′-hydroxymidazolam by both CYP3A4 and CYP3A5 (collectively referred to as CYP3A) (14). After intravenous or oral administration of subtherapeutic, lowdose midazolam, multiple blood samples are obtained in order to calculate midazolam clearance, which serves as an index of CYP3A4/5 activity (15). Midazolam has been used extensively to characterize factors (e.g., genetics and drug interactions) that affect CYP3A-mediated metabolism (3, 8).

Probe drugs have been used to gain important insight into the clinical relevance of genetic variation (i.e., pharmacogenetics) and to characterize nongenetic factors that influence metabolism, including demographic characteristics (e.g., age, sex, weight, etc.), as well as, drug–drug or drug–herb interactions. For example, studies providing the first indication that genetics are a major determinant of interindividual variability used probe drugs to evaluate the impact of inheritance on drug metabolism through the use of family studies or twin studies, an experimental paradigm in which metabolism in monozygotic and dizygotic twins is compared (16–18). Thus, in early studies probe drugs provided an important tool to identify subpopulations of subjects, who could be categorized according to their inherent capacity to metabolize the probe drug. DNA obtained from individual members of each phenotype group could then be evaluated and compared. More recently, probe drugs continue to provide an important means by which to explore the clinical relevance of SNPs in drug-metabolizing enzymes and also to evaluate the interaction between genes and the environment. Several probe drugs for specific CYP enzymes have been identified and the advantages and disadvantages associated with each have been reviewed elsewhere (8). A partial listing of probe drugs is provided in Table 2⇓.

Combinations of Probe Drugs: The “Cocktail” Approach

Probe drugs have been used alone and also in various combinations, offering several advantages, provided that metabolic or analytical interactions do not occur with simultaneous administration. Administering multiple probe compounds concomitantly, termed the “cocktail” strategy, is a useful method in the assessment of drug-metabolizing enzyme activities because it allows for the in vivo assessment of multiple pathways of drug metabolism in a single experiment (19). The utility of the cocktail strategy, first demonstrated by Schellens and Breimer in multiple investigations (19), offers several potential advantages in that it reduces participation time for the study subjects and increases efficiency for the investigators by decreasing time and expense. More importantly, this approach minimizes intraindividual variability since the evaluation will occur on one day rather than separate days. Early applications of the cocktail approach used probe drugs that are no longer considered selective for individual enzymes, whereas more recent applications have utilized enzyme-selective probe drugs (20, 21). Thus, the cocktail strategy appears to be an invaluable method to investigate differential modulation of CYP activity (22).

The most common use of the cocktail approach to date has been in the evaluation of drug interactions. Because information on multiple pathways is obtained in a single study, the cocktail strategy provides an efficient means by which to screen for potential drug interactions (9). For example, the cocktail approach has recently been used to evaluate the potential for herbal preparations to alter CYP enzyme activity (23, 24). However, the cocktail strategy is also likely to be useful in the study of genetic variation for many of the same reasons. Phenotypic data (i.e., enzyme activity) can be obtained simultaneously on multiple pathways while genetic material obtained can be examined for known or novel SNPs in the drug-metabolizing enzyme genes.

Probe Drugs to Study the Clinical Consequences of Genetic Variation

The inability of certain individuals to metabolize the antihypertensive drug debrisoquine led to the discovery of the first genetic polymorphism in the metabolism of a drug associated with the expression of a CYP enzyme in man (17). Debrisoquine subsequently became a standard probe drug used to investigate CYP2D6-mediated metabolism. Thus, the use of probe drugs has provided important information on the functional significance of SNPs in drug-metabolizing enzymes. A SNP may result in either a change (nonsynonymous SNP) or no change (silent polymorphism or synonymous SNP) in the coded amino acid. Probe drugs have been used to provide valuable information on the functional relevance of SNPs that result in a change in an encoded amino acid. The impact such a change may have on enzyme function depends on the location of the amino-acid substitution. For example, an amino-acid change in the substrate recognition site is likely to be more important than a change elsewhere. Nonsynonymous SNPs in CYP2C9 and CYP2D6 yield allelic variants with altered catalytic activity. A representative listing of nonsynonymous polymorphisms in CYP genes is provided in Table 3⇓.

CYP2C9 Allelic Variants

CYP2C9 is the major CYP2C isoform found in human liver and it is involved in the metabolism of several clinically important drugs including phenytoin and warfarin (25, 26). SNPs that have been identified within the coding region produce variant alleles CYP2C9*2, which encodes an Arg144Cys substitution, CYP2C9*3, which encodes an Ile359Leu substitution, and CYP2C9*5, which encodes an Asp360Glu substitution (25–27).

The L³⁵⁹ and E³⁶⁰ substitutions would be expected to produce-more significant changes in metabolism because these amino acids appear to reside within the CYP2C9 active site, whereas the C¹⁴⁴ substitution (*2) is not located in the active site (26). The magnitude of decrease in activity caused by expression of the CYP2C9 variant proteins may also be substrate dependent. For example, the Ile359Leu substitution produced a decrease in intrinsic clearance that ranged from 3-fold for diclofenac 4-hydroxylation to 27-fold for piroxicam 5′-hydroxylation (28). The in vitro metabolism of S-warfarin, flurbiprofen, and tolbutamide by the CYP2C9*3 variant (L359 substitution) is markedly decreased as compared to the metabolism by the wild-type enzyme. These findings are consistent with observations in vivo with tolbutamide, warfarin, and other CYP2C9 substrates (12, 26).

The clinical relevance of CYP2C9 genetic variation has been shown with warfarin. Warfarin 7-hydroxylation is reduced in individuals with the CYP2C9*1/*3 genotype, which results in a much smaller dose of the drug being needed to achieve the same effect in individuals having the CYP2C9*1/*3 genotype as compared to the CYP2C9*1/*1 genotype (3.78 mg vs. 5.28 mg per day) (26). For comparison, the dose needed for individuals having the rare CYP2C9*3/*3 genotype is approximately 0.5 mg per day. Observations in vitro with CYP2C9*5 suggest the E³⁶⁰ substitution may have an important effect on metabolism, but the effects in vivo have not been reported to date (27).

CYP2D6 Allelic Variants

The pharmacogenetics of CYP2D6 have been extensively studied since polymorphic hydroxylation of the well-known substrate debrisoquine was first reported over twenty-five years ago. CYP2D6 metabolizes 25–30% of all clinically used drugs, including β-blockers (e.g., metoprolol, carvedilol), antiarrhythmics, antidepressant (e.g., nortriptyline, fluoxetine), antipsychotics (e.g., haloperidol), and codeine (5). CYP2D6 has a high degree of genetic variability, with more than 75 allelic variants identified to date (29). Multiple nonfunctional alleles have been identified and approximately 5–10% of Caucasians and 1% of Asians do not express the enzyme and are thus not able to metabolize CYP2D6 substrates (6, 29). There are two allelic variants that have been associated with decreased catalytic activity: CYP2D6*10, which is common in Asians, and CYP2D6*17, which is found in African-Americans (6). Metabolism of the probe drugs debrisoquine and dextromethorphan is decreased in individuals with the CYP2D6*17/*17 genotype as compared to the CYP2D6*1/*1 genotype, but metabolism of the CYP2D6 substrates codeine or metoprolol is not affected (30). The CYP2D6*10 allele has also been associated with reduced activity, but the clinical importance, especially for heterozygotes, has not been shown consistently (31, 32).

Summary

Multiple factors including genetics, age, sex, disease, and environmental exposures contribute to variability in drug response. Probe drugs have provided valuable information on the relevance of various genetic and nongenetic factors on the activities of CYP enzymes. Although probe drugs represent an important tool with which to characterize enzyme activity in vivo, there currently is not a consensus as to what probe drug is best for a given enzyme. For example, there is controversy with respect to which probe is ideal for characterizing CYP3A activity in vivo and there is in vitro evidence to suggest that a single probe may not be feasible (33). Despite the limitations of some currently available probes, their use will continue to provide an invaluable tool to explore the clinical relevance of genetic and nongenetic factors that affect CYP enzyme activity.