Pharmacogenetics, pharmacogenomics, and individualized medicine.

Individual variability in drug efficacy and drug safety is a major challenge in current clinical practice, drug development, and drug regulation. For more than 5 decades, studies of pharmacogenetics have provided ample examples of causal relations between genotypes and drug response to account for phenotypic variations of clinical importance in drug therapy. The convergence of pharmacogenetics and human genomics in recent years has dramatically accelerated the discovery of new genetic variations that potentially underlie variability in drug response, giving birth to pharmacogenomics. In addition to the rapid accumulation of knowledge on genome-disease and genome-drug interactions, there arises the hope of individualized medicine. Here we review recent progress in the understanding of genetic contributions to major individual variability in drug therapy with focus on genetic variations of drug target, drug metabolism, drug transport, disease susceptibility, and drug safety. Challenges to future pharmacogenomics and its translation into individualized medicine, drug development, and regulation are discussed. For example, knowledge on genetic determinants of disease pathogenesis and drug action, especially those of complex disease and drug response, is not always available. Relating the many gene variations from genomic sequencing to clinical phenotypes may not be straightforward. It is often very challenging to conduct large scale, prospective studies to establish causal associations between genetic variations and drug response or to evaluate the utility and cost-effectiveness of genomic medicine. Overcoming the obstacles holds promise for achieving the ultimate goal of effective and safe medication to targeted patients with appropriate genotypes.

Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity.

The majority of marketed small-molecule drugs undergo metabolism by hepatic Cytochrome P450 (CYP) enzymes (Rendic 2002). Since these enzymes metabolize a structurally diverse number of drugs, metabolism-based drug-drug interactions (DDIs) can potentially occur when multiple drugs are coadministered to patients. Thus, a careful in vitro assessment of the contribution of various CYP isoforms to the total metabolism is important for predicting whether such DDIs might take place. One method of CYP phenotyping involves the use of potent and selective chemical inhibitors in human liver microsomal incubations in the presence of a test compound. The selectivity of such inhibitors plays a critical role in deciphering the involvement of specific CYP isoforms. Here, we review published data on the potency and selectivity of chemical inhibitors of the major human hepatic CYP isoforms. The most selective inhibitors available are furafylline (in co-incubation and pre-incubation conditions) for CYP1A2, 2-phenyl-2-(1-piperidinyl)propane (PPP) for CYP2B6, montelukast for CYP2C8, sulfaphenazole for CYP2C9, (-)-N-3-benzyl-phenobarbital for CYP2C19 and quinidine for CYP2D6. As for CYP2A6, tranylcypromine is the most widely used inhibitor, but on the basis of initial studies, either 3-(pyridin-3-yl)-1H-pyrazol-5-yl)methanamine (PPM) and 3-(2-methyl-1H-imidazol-1-yl)pyridine (MIP) can replace tranylcypromine as the most selective CYP2A6 inhibitor. For CYP3A4, ketoconazole is widely used in phenotyping studies, although azamulin is a far more selective CYP3A inhibitor. Most of the phenotyping studies do not include CYP2E1, mostly because of the limited number of new drug candidates that are metabolized by this enzyme. Among the inhibitors for this enzyme, 4-methylpyrazole appears to be selective.

Metabolic bioactivation and drug-related adverse effects: current status and future directions from a pharmaceutical research perspective.

Retrospective studies indicate that many drugs that cause clinical adverse reactions, such as hepatotoxicity, undergo metabolic bioactivation, resulting in the formation of electrophilic intermediates capable of covalently modifying biological macromolecules. A logical extension of these findings is a working hypothesis that compounds with reduced levels of bioactivation should be inherently safer drug molecules and thus have a greater likelihood of success in drug development. Whereas some research-based pharmaceutical companies have adopted a strategy of addressing metabolic bioactivation early in drug discovery, much skepticism remains on whether such an approach would enable the industry to reach the desired objectives. The debate is centered on the question of whether there is a quantitative correlation between bioactivation and the severity of drug-treatment-related toxicity, and whether covalent protein modification represents only one of several possible mechanisms underlying observed tissue injury. This communication is intended to briefly review the current understanding of drug-induced hepatotoxicity and to discuss the controversy and future directions with respect to the effort of minimizing the probability of clinical adverse reactions.

Drug metabolism and pharmacokinetics in support of drug design.

Pharmacokinetics has been recognized as one of the elements determining the probability of success in pharmaceutical research. As a result, compounds are routinely evaluated in drug discovery for their absorption, distribution, metabolism and elimination properties. The primary objective of these studies is to eliminate "flawed" molecules or a structural class based on preset selection criteria, while building a knowledge base for compilation of structure-activity relationships to guide chemistry synthesis efforts. This article is intended to provide a brief overview combined with critical evaluation on several strategies employed during lead optimization processes, and the analyses are supported by case studies. Future directions are discussed in the context of overcoming deficiencies in the current practice by developing tools enabling better prediction of clinical outcomes.

Antibodies as a probe in cytochrome P450 research.

Cytochrome P450 (P450) is the superfamily of enzymes responsible for biotransformation of endobiotics and xenobiotics. However, their large isoform multiplicity, inducibility, diverse structure, widespread distribution, polymorphic expression, and broad overlapping substrate specificity make it difficult to measure the precise role of each individual P450 to the metabolism of drugs (or carcinogens) and hamper the understanding of the relationship between the genetic/environmental factors that regulate P450 phenotype and the responses of the individual P450s to drugs. The antibodies against P450s have been useful tools for the quantitative determination of expression level and contribution of the epitope-specific P450 to the metabolism of a drug or carcinogen substrate in tissues containing multiple P450 isoforms and for implications in pharmacogenetics and human risk assessment. In particular, the inhibitory antibodies are uniquely suited for reaction phenotyping that helps to predict human pharmacokinetics for clinical drug-drug interaction potential in drug discovery and development.

The challenges of dealing with promiscuous drug-metabolizing enzymes, receptors and transporters.

Unlike classical enzymes, drug-metabolizing enzymes (DMEs), such as the liver microsomal cytochrome P450, UDP-glucuronyltransferase, epoxide hydrolase, and flavin-containing monooxygenase, all exhibit broad substrate specificities, low turnover rates, atypical kinetics, and other unusual properties. Receptors (the pregnane X receptor, NR1I2; the constitutive androstane receptor, NR1I3; and the aromatic hydrocarbon receptor) responsible for the induction of DMEs and transporters (P-glycoprotein) responsible for drug transport also have broad substrate specificities. These promiscuous proteins are all intimately involved in drug disposition. Promiscuous proteins, by definition, are known for diversity, but not specificity, in their interaction with drugs. In this review, we analyzed recent advances on the three dimensional structures and kinetic properties of DMD proteins from crystallography, mutational, and kinetic studies to gain insights into the structural and biochemical basis for the promiscuous ligand-protein interactions of the proteins. Large substrate-binding cavities (SBCs), binding of more than one substrate/effector and binding of substrates in alternative orientations and locations within the SBCs, rotation of a substrate at the active site, and substantial substrate-induced conformational changes of the SBCs are common features of the promiscuous DMEs, receptors, and transporters, and therefore, are important parameters to be considered in dealing with drug metabolism issues and safety evaluation of drugs and environmental chemicals.

Metabolism of dietary polyphenols and possible interactions with drugs.

Polyphenolic compounds are abundant in the human diet and gram quantities are ingested daily. The consumption of polyphenols is expected to rise due to the use of dietary supplements and public health initiatives promoting the consumption of more fruits and vegetables. It is known that these dietary polyphenols are extensively metabolized. Many of these compounds are therefore are expected to compete with other substrates of Phases I, II, III enzymes and transporters. In addition, some dietary polyphenols may induce certain drug metabolizing enzymes and affect the metabolism of important therapeutic agents. This review will discuss 1) the metabolism of dietary polyphenols using green tea polyphenols (catechins) as an example, 2) inhibition of drug metabolism by polyphenols, and 3) induction of drug metabolizing enzymes by dietary polyphenols. The potential consequences of these effects on drug metabolism will also be discussed.

CYP1A induction and human risk assessment: an evolving tale of in vitro and in vivo studies.

CYP1A1 and 1A2 play critical roles in the metabolic activation of carcinogenic polycyclic aromatic hydrocarbons (PAHs) and heterocyclic aromatic amines/amides (HAAs), respectively, to electrophilic reactive intermediates, leading to toxicity and cancer. CYP1As are highly inducible by PAHs and halogenated aromatic hydrocarbons via aryl hydrocarbon receptor-mediated gene transcription. The impact of CYP1A induction on the carcinogenic and toxic potentials of environmental, occupational, dietary, and therapeutic chemicals has been a central focus of human risk evaluation and has broadly influenced the fields of cancer research, toxicology, pharmacology, and risk assessment over the past half-century. From the early discovery of CYP1A induction and its role in protection against chemical carcinogenesis in intact animals, to the establishment of CYP1A enzymes as the principal cytochromes P450 for bioactivation of PAHs and HAAs in in vitro assays, to the recent realization of an essential protective role of CYP1A in benzo[a]pyrene-induced lethality and carcinogenesis with CYP1A knockout mice, the understanding of the interrelation between CYP1A induction and chemical safety has followed a full circle. This unique path of CYP1A research underscores the importance of whole animal and human studies in chemical safety evaluation.

Utility of recombinant cytochrome p450 enzymes: a drug metabolism perspective.

An important role of human cytochrome P450s (P450s) has been well recognized in the area of drug metabolism and pharmacokinetics. It has become possible in recent years to express catalytically active forms of these enzymes in various host systems. The resulting recombinant human P450s are either purified for studies of protein structure and the mechanism of catalysis or isolated in microsomal forms to serve the purposes of P450 phenotyping, metabolic stability screening and inhibitory potential evaluation. Intact mammalian cells expressing human enzymes may also be used to test the mutagenic and toxicity potential of drug candidates. The issue remains, however, that the data derived from recombinant P450s are not always consistent with those generated from human tissue preparations. The aim of this communication is to discuss applications of recombinant P450s in the drug discovery and development setting, with an emphasis on comparison of recombinant and human liver microsomal systems.

Metabolism-based drug-drug interactions: what determines individual variability in cytochrome P450 induction?

Individual variability in cytochrome P450 (P450) induction comprises an important component contributing to the difficulties in assessing and predicting metabolism-based drug-drug interactions in humans. In this article, we outline the major factors responsible for the individual variability in P450 induction, including variable transporter activity and metabolism of inducers in vivo, genetic variations of P450 genes and their regulatory regions, genetic variations of receptors and regulatory proteins required for induction, and different physiological and environmental elements. With a better understanding of the major determinants in P450 induction and a profile of the phenotypes of these determinants in each individual, it is believed that the individual variability in induction-mediated drug-drug interactions can be adequately evaluated.

Identification of critical amino acid residues of human CYP2A13 for the metabolic activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, a tobacco-specific carcinogen.

Among all the known human cytochrome P450 enzymes, CYP2A13 has the highest efficiency in catalyzing the metabolic activation (keto aldehyde and keto alcohol formation) of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent lung carcinogen in animals and a suspected human lung carcinogen. As part of the structure-activity relationship (SAR) study, the present work was done to identify the key amino acid residues in CYP2A13 that are responsible for this high catalytic efficiency by using a series of mutants (Ala117Val, His164Gly, Ser208Ile, His372Arg, and Pro465Ser). In these CYP2A13 mutants, the amino acid residues were substituted by the residues at the corresponding positions of CYP2A6, which shares 93.5% amino acid sequence identity with CYP2A13 but is significantly less active (<5%) than CYP2A13 in NNK alpha-hydroxylation. We demonstrated that, except for the His164Gly mutant, all the CYP2A13 mutant proteins showed a significant decrease in the catalytic efficiency (Vmax/Km) for NNK alpha-hydroxylation. The His372 to Arg substitution resulted in a 20-fold increase in the Km value and a 7-fold decrease in the Vmax value for keto aldehyde formation as well as a total loss of detectable keto alcohol formation. The Ala117 to Val substitution, however, only caused a selective decrease in the Vmax value for keto aldehyde formation. The role of these amino acid residues in CYP2A13-catalyzed reactions is clearly substrate-dependent, since the same Ala117Val and His372Arg mutants showed a 9-fold increase in the catalytic efficiency for coumarin 7-hydroxylation. Together with the computational substrate docking, our study provides new SAR in formation of human CYP2A13.

Identification of Val117 and Arg372 as critical amino acid residues for the activity difference between human CYP2A6 and CYP2A13 in coumarin 7-hydroxylation.

Human cytochrome P450 (CYP) 2A6 and 2A13 play an important role in catalyzing the metabolism of many environmental chemicals including coumarin, nicotine, and several tobacco-specific carcinogens. Both CYP2A6 and CYP2A13 proteins are composed of 494 amino acid residues. Although CYP2A13 shares a 93.5% identity with CYP2A6 in the amino acid sequence, it is only about one-tenth as active as CYP2A6 in catalyzing coumarin 7-hydroxylation. To identify the key amino acid residues that account for such a remarkable difference, we generated a series of CYP2A6 and CYP2A13 mutants by site-directed mutagenesis/heterologous expression and compared their coumarin 7-hydroxylation activities. In CYP2A6, the amino acid residues at position 117 and 372 are valine (Val) and arginine (Arg), respectively; whereas in CYP2A13, they are alanine (Ala) and histidine (His). Kinetic analysis revealed that the catalytic efficiency (Vmax/Km) of the CYP2A6 Val(117)--> Ala and Arg(372)--> His mutants was drastically reduced (0.41 and 0.64 versus 3.23 for the wild-type CYP2A6 protein). In contrast, the catalytic efficiency of the CYP2A13 Ala(117) --> Val and His(372) --> Arg mutants was greatly increased (2.65 and 2.60 versus 0.31 for wild-type CYP2A13 protein). These results clearly demonstrate that the Val at position 117 and Arg at position 372 are critical amino acid residues for coumarin 7-hydroxylation. Based on the crystal structure of CYP2C5, we have generated the homology models of CYP2A6 and CYP2A13 and docked the substrate coumarin to the active site. Together with the kinetic characterization, our structural modeling provides explanations for the amino acid substitution results and the insights of detailed enzyme-substrate interactions.

Cytochrome P450 in vitro reaction phenotyping: a re-evaluation of approaches used for P450 isoform identification.

Marker substrates, chemical inhibitors, and inhibitory antibodies are important tools for the identification of cytochrome P450 (P450) isoform responsible for the metabolism of therapeutic agents in vitro. In view of the versatile and nonspecific nature of P450 enzymes, many of the marker substrates and chemical inhibitors used for P450 in vitro reaction phenotyping are isoform selective but not specific. Recently, the use of marker substrate and chemical inhibitors in CYP2D6 in vitro reaction phenotyping was questioned by Granvil et al. (2002). In comparison of a panel of 15 recombinant P450 enzymes, they found that in addition to CYP2D6, CYP1A1 is also capable of catalyzing the formation of 4-hydroxylated metabolite of debrisoquine and that the intrinsic clearance of debrisoquine by CYP2D6-mediated 4-hydroxylation is only about twice that by CYP1A1. In their study, they have also demonstrated that quinidine inhibits both CYP2D6- and CYP1A1-mediated debrisoquine 4-hydroxylation. In view of these important findings, we have reevaluated various approaches used to identify P450 isoform(s) responsible for the metabolism of therapeutic agents. While acknowledging the value of inhibitory antibodies in P450-phenotyping studies, it is our opinion that in well conducted in vitro experiments, isoform-selective chemical inhibitors can also provide valuable and reliable information. Hopefully, future efforts may produce even better P450 isoform-selective marker substrates and inhibitors.