Approach to predict the contribution of cytochrome P450 enzymes to drug metabolism in the early drug-discovery stage: the effect of the expression of cytochrome b(5) with recombinant P450 enzymes.
In order to evaluate the potential adverse effects due to genetic polymorphism and/or inter-individual variation, it is necessary to calculate the cytochrome P450 (CYP) contribution to the metabolism of new drugs. In the current study, the in vitro intrinsic clearance (CL(int)) values of marker substrates and drugs were determined by measuring metabolite formation and substrate depletion, respectively. Recombinant CYP microsomes expressing CYP2C9, CYP2C19 and CYP3A4 with co-expressed cytochrome b(5) were used, but those expressing CYP1A2 and CYP2D6 did not have co-expressed cytochrome b(5). The following prediction methods were compared to determine the CL(int) value using data from recombinant CYP enzymes: (1) relative CYP enzyme content in human liver microsomes; (2) relative activity factor (RAF) estimated from the V(max) value; and (3) RAF estimated from the CL(int) value. Estimating RAF from CL(int) proved the most accurate prediction method among the three tested, and differences in the CYP3A4 marker reactions did not affect its accuracy. The substrate depletion method will be useful in the early drug-discovery stage when the main metabolite and/or metabolic pathway has not been identified. In addition, recombinant CYP microsomes co-expressed with cytochrome b(5) might be suitable for the prediction of the CL(int) value.
Emoto
C
C
Department of Pharmacokinetics Dynamics Metabolism, Pfizer Global Research & Development, Nagoya Laboratories, Pfizer Japan Inc., Taketoyo, Aichi, Japan.
Iwasaki
K
K
D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism; D001711:Biotransformation; D002853:Chromatography, Liquid; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism; D065731:Cytochrome P-450 CYP2C19; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6 / Q000235:genetics / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D015195:Drug Design; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D015262:Xenobiotics / Q000378:metabolism / Q000493:pharmacokinetics
D011994:Recombinant Proteins; D015262:Xenobiotics; D015786:Cytochromes b5; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C045793:CYP2C19 protein, human; D019388:Cytochrome P-450 CYP1A2; D065731:Cytochrome P-450 CYP2C19; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human