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Vol. 27, Issue 6, 655-666, June 1999
Division of Cell and Molecular Biology, Department of Biology,
Boston University, Boston, Massachusetts (P.R., L.J.Y., D.J.W.); and
Gentest Corp., Woburn, Massachusetts (C.L.C.)
The contributions of specific human liver cytochrome P-450 (CYP)
enzymes to the activation, via 4-hydroxylation, of the oxazaphosphorine anticancer prodrugs cyclophosphamide (CPA) and ifosfamide (IFA) were
investigated. Analysis of a panel of 15 human P-450 cDNAs expressed in
human lymphoblasts and/or baculovirus-infected insect cells
(Supersomes) demonstrated that CYPs 2A6, 2B6, 3A4, 3A5, and three CYP2C
enzymes (2C9, 2C18, 2C19) exhibited significant oxazaphosphorine
4-hydroxylase activity, with 2B6 and 3A4 displaying the highest
activity toward CPA and IFA, respectively. CYP2B6 metabolized CPA at a
~16-fold higher in vitro intrinsic clearance (apparent
Vmax/Km) than
IFA, whereas 3A4 demonstrated ~2-fold higher
Vmax/Km toward
IFA. A relative substrate-activity factor (RSF)-based method was
developed to calculate the contributions of individual P-450s to total
human liver microsomal metabolism based on cDNA-expressed P-450
activity data and measurements of the liver microsomal activity of each
P-450 form. Using this method, excellent correlations were obtained
when comparing measured versus predicted (calculated) microsomal
4-hydroxylase activities for both CPA (r = 0.96, p < .001) and IFA (r = 0.90, p < .001) in a panel of 17 livers. The RSF method
identified CYP2B6 as a major CPA 4-hydroxylase and CYP3A4 as the
dominant IFA 4-hydroxylase in the majority of livers, with CYPs 2C9 and
2A6 making more minor contributions. These predicted P-450 enzyme
contributions were verified using an inhibitory monoclonal antibody for
2B6 and the P-450 form-specific chemical inhibitors troleandomycin for
3A4 and sulfaphenazole for 2C9, thus validating the RSF approach. Finally, Western blot analysis using anti-2B6 monoclonal antibody demonstrated the presence of 2B6 protein at a readily detectable level
in all but one of 17 livers. These data further establish the
significance of human liver CYP2B6 for the activation of the clinically
important cancer chemotherapeutic prodrug CPA.
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