Current Drug Metabolism - Volume 3, Issue 6, 2002

Cytochrome P450s comprise a superfamily of heme-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. Having been found in every class of organism, including Archaea, the P450 superfamily is believed to have originated from an ancestral gene that existed over 3 billion years ago. Repeated gene duplications have subsequently given rise to one of the largest of multigene families. These enzymes are notable both for the diversity of reactions that they catalyze and the range of chemically dissimilar substrates upon which they act. Cytochrome P450s support the oxidative, peroxidative and reductive metabolism of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and fatty acids. In humans, cytochrome P450s are best know for their central role in phase I drug metabolism where they are of critical importance to two of the most significant problems in clinical pharmacology: drug interactions and interindividual variability in drug metabolism. Recent advances in our understanding of cytochrome P450-mediated drug metabolism have been accelerated as a result of an increasing emphasis on functional genomic approaches to P450 research. While human cytochrome P450 databases have swelled with a flood of new human sequence variants, the functional characterization of the corresponding gene products has not kept pace. In response researchers have begun to apply the tools of proteomics as well as homology-based and ab initio modeling to salient questions of cytochrome P450 structure / function. This review examines the latest advances in our understanding of human cytochrome P450s.

Drugs and other chemicals that contain one or more heterocyclic nitrogen atoms are most widely known for their ability to both inhibit and induce cytochrome P450s. Their ability to affect a wide range of Phase II drug metabolizing enzyme activities has received much less attention and exposure. For some N-heterocycles, induction of Phase II metabolism occurs in the absence of induction effects on cytochrome P450, a property with potential utility in reducing drug and chemical toxicity (chemoprotection). The mechanism of this induction, and an accounting of the Phase II selectivity, has not been rigorously investigated. This review gathers and documents existing information on the simultaneous inductive effects of N-heterocycles on glucuronidation, sulfation (sulfonation) and glutathione conjugation reactions, as well epoxide hydrolase and quinone oxidoreductase activities. Basic information on cytochrome P450 induction is provided for comparative purposes. The review will serve as a base for any future interest in the phenomenon. The existing information on enzyme changes derives largely from laboratory animal studies. Chemicals investigated range from both simple and complex pyridines and imidazoles to benzoquinolines and phenanthrolines. Isomeric differences can have a major impact on induction characteristics and Phase II selectivity. Promising indications for the effectiveness of N-heterocycle elicited enzyme changes in chemoprotection in vivo awaits investigation.

In the last 16 years, more than a dozen gene-directed enzyme prodrug therapies for cancer treatment have been evaluated in preclinical studies. However, only few of them have evolved to the stage of clinical trial. This review assesses current knowledge in the area of cancer gene therapy, emphasizing cytochrome P450 (CYP)-based prodrug activation systems. This approach is intuitively highly suitable for the treatment of cancers, since several major anticancer drugs are activated by liver CYP enzymes. Important features of this strategy include: 1) use of human CYP genes to avoid immune complications that may hamper expression of therapeutic genes of non-human origin and thereby inhibit prodrug activation, 2) use of well established and clinically effective anticancer prodrugs, 3) strong bystander cytotoxic effect seen with all liver-activated CYP prodrugs, 4) the potential to inhibit liver CYP activity and expression to increase the bioavailability of prodrugs for CYP-transduced tumors, 5) possible extension to many CYP enzymes and their potential anticancer prodrug substrates, and 6) it can be used to arm therapeutic conditionally replicating viruses. Historically, this strategy utilized CYP 2B1 to activate oxazaphosphorines. It is now becoming clear that the repertoire of prodrugs is expandable and that CYP gene candidates are not limited to naturally occurring CYP genes, but may also encompass engineered CYP enzymes, improved by site directed mutagenesis or other approaches. Encouraging results from a recent phase I / II clinical trial that have implemented this strategy, as well as emerging problems related to gene delivery are discussed in this review.

Glucuronidation is responsible for the clearance of a diverse range of drug and chemicals whose topology confers properties that complicate in vitro-in vivo clearance correlations as compared to those possible for oxidative metabolism. The active site of the UGTs faces the inside of the luminal space of the endoplasmic reticulum, thus presenting diffusional barriers for substrates, the cosubstrate, UDPGA, and resultant glucuronide products. Transport processes for the cosubstrate UDPGA and glucuronidated products likely contribute to the well-known latency phenomena in which exogenous detergents or alamethicin are required for maximal UGT activity in microsomes. This complicates the extrapolation of results of in vitro clearance studies to the in vivo situation. Even with activation, the microsomal-based clearance values still underestimate the actual in vivo UGT-mediated clearance, therefore latency is not the only explanation for the poor correlation. Recent data indicate that hepatocytes are a promising in vitro system that can be used for the early evaluation of human clearance behavior of drug candidates. Both induction and inhibition of UGT-mediated clearance are a source of clinical drug-drug interactions. Emerging evidence indicates that the same mechanisms identified in the regulation of CYP enzymes also are involved in regulation of the UGTs, i.e., CAR, AH and probably PXR mediate regulation of UGT1A1, 1A6 and UGT2B7, respectively. In contrast to CYP-mediated interactions, with a few exceptions, the magnitude of UGT-mediated interactions are less than 2-fold because of the relatively high UGT Km values and substrate overlap among the multiple isozymes.

The amount of drug achieved and maintained in the brain after systemic administration is determined by the agent's permeability at blood-brain barrier (BBB), potential involvement of transport systems, and the distribution, metabolism and elimination properties. Passive diffusion permeability may be predicted by an in silico method based on a molecule's structure property. In vitro cell culture is another useful tool for the assessment of passive permeability and BBB transports (e.g. PGP, MRP). In situ or in vivo techniques like carotid artery single injection or perfusion, brain microdialysis, autoradiography, and others are used at various stages of drug discovery and development to estimate CNS penetration and PK / PD correlation. Each technique has its own application with specific advantages and limitations.