Mark Vandenbranden

Examination of purported probes of human CYP2B6.

7-Ethoxy-4-trifluoromethylcoumarin (7-EFC) was examined as a substrate for cytochrome P450 (P450) in microsomes from human livers and expressed in B-lymphoblastoid cells. The O-deethylation of 7-EFC to 7-hydroxy-4-trifluoromethylcoumarin (7-HFC) varied over a liver bank (n = 19) by a factor of 13 (40-507 pmol min-1 mg-1 protein). When compared with the ability of the bank of human liver samples to metabolize form-selective substrates of the P450, 7-HFC formation correlated strongly with the formation of the S-mephenytoin metabolite, nirvanol (r2 = 0.86, p < 0.0001). alpha-Napthoflavone (ANF), diethyldithiocarbamate (DDC) and chloramphenicol (CAP) inhibited the O-deethylation of 7-EFC by microsomes from human livers by greater than 60%. Orphenadrine (ORP), a reported specific CYP2B6 inhibitor, was a less potent inhibitor of 7-HFC formation by microsomes from human liver than DDC or ANF. Using microsomes from B-lymphoblastoid cells expressing specific P450s, CYP2B6 and CYP1A2 were found to produce substantial levels of 7-HFC whereas CYP2E1 and CYP2C19 produced detectable amounts of this metabolite. ORP inhibited expressed CYP2E1 and CYP2B6 mediated 7-HFC formation to a greater extent than the inhibition observed for CYP1A2. Methoxychlor and S-mephenytoin inhibited expressed CYP2B6 but not CYP1A2 mediated 7-EFC O-deethylation. Livers (n = 5) with high relative rates of 7-HFC formation displayed biphasic enzyme kinetics with the low K(m) site (average K(m) = 3.3 microM) demonstrating allosteric activation. Five livers with low relative rates of 7-HFC formation also exhibited biphasic kinetics but lacked evidence of an allosteric mechanism being involved in the low K(m) component (average K(m) = 2.4 microM). Furthermore, expressed CYP2B6 and CYP2E1 converted 7-EFC to 7-HFC with allosteric activation indicated, while CYP1A2 mediated metabolism of 7-EFC to 7-HFC best fit the classic Michaelis-Menten model. A commercially available antibody to rat CYP2B, suggested to be specific for CYP2B6, was found to cross react with all members to the CYP2 family examined including CYP2C19, which possessed a nearly identical electrophoretic mobility to that of CYP2B6 in the system examined. In total, the evidence presented indicates that multiple P450s are involved in the formation of 7-HFC from 7-EFC, therefore this does not appear to be a useful or a selective probe of CYP2B6 catalytic activity. Furthermore, the specificity of both antibody and chemical inhibitor (ORP) probes previously suggested to be specific for CYP2B6 is also questioned.

Isolation and characterization of human fetal liver cytochrome P450HLp2: a third member of the P450III gene family.

We purified from human fetal livers a form of cytochrome P450, termed HLp2, related to adult human liver cytochrome P450HLp by monitoring the purification procedures by immunoblots developed with a monoclonal antibody to HLp. The preparation of HLp obtained was judged to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and found to have an apparent monomeric molecular weight slightly greater than that of HLp, 51,500 versus 51,000. Immunoblot and Ouchterlony double-diffusion analyses indicated that HLp and HLp2 are immunochemically related. However, ferrous-CO versus ferrous difference spectra yielded different Soret maxima for HLp and HLp2 (448.5 and 449.5 nm, respectively). In addition, structural comparisons between HLp and HLp2 indicated that they were distinct isozymes. Specifically, the 28 amino acid amino-terminal sequence determined for HLp2 was found to be 79% homologous to that of HLp. In addition, peptide mapping of HLp and HLp2 by limited proteolysis with three proteases yielded similar but different patterns indicating that HLp and HLp2 are structurally distinct. These results demonstrate that HLp2 and HLp are highly related but distinct proteins and that HLp2 is a member of the steroid-inducible P450III gene family.

The Use of In Vitro Metabolism Techniques in the Planning and Interpretation of Drug Safety Studies

An important issue in toxicology is the suitability of the data obtained with experimental animals for human risk assessment. Because it is not possible to use humans in long-term toxicological studies, the use of animals will continue. However, the data obtained in animal studies can be better extrapolated to the patient by utilizing bridging studies with in vitro models of human drug metabolism. There are 2 basic categories of in vitro methods for the examination of human liver drug metabolism. The first group of in vitro methods consists of the cellular models, which include primary hepatocytes, liver slices, and cell lines. The second group is the use of preparations of the drug-metabolizing enzymes such as tissue homogenates, subcellular fractions, and isolated enzymes. Studies modeling both the human and experimental animal metabolism of a drug are useful in the design of toxicological studies. In vitro studies can identify metabolites, species-specific metabolic routes, and the experimental animal model that best reflects the potential human exposure to the drug and its metabolites. This information can also be useful in the design of the clinical studies by identifying human metabolites, the enzymes responsible for the metabolic clearance of the drug, the effects of genetics and other host factors on the metabolism of the drug, and potential drug-drug interactions. An example of how such information is generated and interpreted is presented.