Abstract
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 (apparentVmax/Km) than IFA, whereas 3A4 demonstrated ∼2-fold higherVmax/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.
Footnotes
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Send reprint requests to: Dr. David J. Waxman, Department of Biology, Boston University, 5 Cummington St., Boston, MA 02215. E-mail: djw{at}bio.bu.edu
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This work was supported in part by National Institutes of Health Grant CA49248 (D.J.W.).
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↵2 RSF calculations based on the Supersomes expression system were based on activities determined in the presence of cytochrome b5, with the exception of 2B6, where Supersomes minus cytochrome b5 data was used. 2B6 + cytochrome b5 Supersomes showed ∼5-fold higher 2B6 diagnostic substrate activity (7-EFC O-deethylation) compared with 2B6 without cytochrome b5, whereas CPA 4-hydroxylase activity was only ∼35% higher with cytochrome b5, indicating that there is a significant substrate dependence to the cytochrome b5 stimulation of 2B6. This differential stimulation of test versus diagnostic substrate activity significantly alters the results of RSF-based calculations and is recognized as a limitation of this method (SeeDiscussion).
- Abbreviations used are::
- CYP
- cytochrome P-450
- CPA
- cyclophosphamide
- IFA
- ifosfamide
- HLS
- human liver microsomal sample
- 7-EFC
- 7-ethoxy-4-trifluoromethylcoumarin
- RSF
- relative substrate-activity factor
- RAF
- relative activity factor, TAO, troleandomycin
- 2C9*1
- 2C9-Arg144 allele
- 2C9*2
- 2C9-Cys144 allele,2C9*3, 2C9-Leu359 allele
- MAB-2B6
- monoclonal antibody specific to 2B6
- Received December 23, 1998.
- Accepted March 18, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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