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Gemcitabine (dFdC, 2′,2′-difluorodeoxycytidine) is metabolized by cytidine deaminase (CDA) and deoxycytidine kinase (DCK), but the contribution of genetic variation in these enzymes to the variability in systemic exposure and response observed in cancer patients is unclear. Wild-type enzymes and variants of CDA (Lys27Gln and Ala70Thr) and DCK (Ile24Val, Ala119Gly, and Pro122Ser) were expressed in and purified from Escherichiacoli, and enzyme kinetic parameters were estimated for cytarabine (Ara-C), dFdC, and its metabolite 2′,2′-difluorodeoxyuridine (dFdU) as substrates. All three CDA proteins showed similar Kmand Vmaxfor Ara-C and dFdC deamination, except for CDA70Thr, which had a 2.5-fold lower Kmand 6-fold lower Vmaxfor Ara-C deamination. All four DCK proteins yielded comparable metabolic activity for Ara-C and dFdC monophosphorylation, except for DCK24Val, which demonstrated an approximately 2-fold increase (P< 0.05) in the intrinsic clearance of dFdC monophosphorylation due to a 40% decrease in Km(P< 0.05). DCK did not significantly contribute to dFdU monophosphorylation. In conclusion, the Lys27Gln substitution does not significantly modulate CDA activity toward dFdC, and therefore would not contribute to interindividual variability in response to gemcitabine. The higher in vitro catalytic efficiency of DCK24Val toward dFdC monophosphorylation may be relevant to dFdC clinical response. The substrate-dependent alterations in activities of CDA70Thr and DCK24Val in vitro were observed for the first time, and demonstrate that the in vivo consequences of these genetic variations should not be extrapolated from one substrate of these enzymes to another.

The mechanism by which CYP2B6*6 allele alters drug metabolism in vitro and in vivo is not fully understood. To test the hypothesis that altered substrate binding and/or catalytic properties contribute to its functional consequences, efavirenz 8-hydroxylation and bupropion 4-hydroxylation were determined in CYP2B6.1 and CYP2B6.6 proteins expressed without and with cytochrome b5 (Cyt b5) and in human liver microsomes (HLMs) obtained from liver tissues genotyped for the CYP2B6*6 allele. The susceptibility of the variant protein to inhibition was also tested in HLMs. Significantly higher Vmaxand Kmvalues for 8-hydroxyefavirenz formation and ∼2-fold lower intrinsic clearance (Clint) were noted in expressed CYP2B6.6 protein (–b5) compared with that of CYP2B6.1 protein (–b5); this effect was abolished by Cyt b5. The Vmaxand Clintvalues for 4-hydroxybupropion formation were significantly higher in CYP2B6.6 than in CYP2B6.1 protein, with no difference in Km, whereas coexpression with Cyt b5 reversed the genetic effect on these kinetic parameters. In HLMs, CYP2B6*6/*6 genotype was associated with markedly lower Vmax(and moderate increase in Km) and thus lower Clintvalues for efavirenz and bupropion metabolism, but no difference in catalytic properties was noted between CYP2B6*1/*1 and CYP2B6*1/*6 genotypes. Inhibition of efavirenz 8-hydroxylation by voriconazole was significantly greater in HLMs with the CYP2B6*6 allele (Ki= 1.6 ± 0.8 μM) than HLMs with CYP2B6*1/*1 genotype (Ki= 3.0 ± 1.1 μM). In conclusion, our data suggest the CYP2B6*6 allele influences metabolic activity by altering substrate binding and catalytic activity in a substrate- and Cyt b5-dependent manner. It may also confer susceptibility to inhibition.

Tamoxifen, an antiestrogen used in the prevention and treatment of breast cancer, is extensively metabolized by cytochrome P450 enzymes. Its biotransformation to α-hydroxytamoxifen (α-OHT), which may be genotoxic, and to N-desmethyltamoxifen (N-DMT), which is partially hydroxylated to 4-hydroxy-N-DMT (endoxifen), a potent antiestrogen, is mediated by CYP3A enzymes. However, the potential contribution of CYP3A5 and the impact of its low-expression variants on the formation of these metabolites are not clear. Therefore, we assessed the contributions of CYP3A4 and CYP3A5 and examined the impact of CYP3A5 genotypes on the formation of α-OHT and N-DMT, by using recombinant CYP3A4 and CYP3A5 and human liver microsomes (HLM) genotyped for CYP3A5 variants. We observed that the catalytic efficiency [intrinsic clearance (CLint)] for α-OHT formation with recombinant CYP3A4 was 5-fold higher than that with recombinant CYP3A5 (0.81 versus 0.16 nl · min–1· pmol cytochrome P450–1). There was no significant difference in CLintvalues between the three CYP3A5-genotyped HLM (*1/*1, *1/*3, and *3/*3). For N-DMT formation, the CLintwith recombinant CYP3A4 was only 1.7-fold higher, relative to that with recombinant CYP3A5. In addition, the CLintfor N-DMT formation by HLM with CYP3A5*3/*3 alleles was approximately 3-fold lower than that for HLM expressing CYP3A5*1/*1. Regression analyses of tamoxifen metabolism with respect to testosterone 6β-hydroxylation facilitated assessment of CYP3A5 contributions to the formation of the two metabolites. The CYP3A5 contributions to α-OHT formation were negligible, whereas the contributions to N-DMT formation ranged from 51 to 61%. Our findings suggest that polymorphic CYP3A5 expression may affect the formation of N-DMT but not that of α-OHT.