Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles.

Article Details

Citation

Roy P, Yu LJ, Crespi CL, Waxman DJ

Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles.

Drug Metab Dispos. 1999 Jun;27(6):655-66.

PubMed ID
10348794 [ View in PubMed
]
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 approximately 16-fold higher in vitro intrinsic clearance (apparent Vmax/Km) than IFA, whereas 3A4 demonstrated approximately 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.

DrugBank Data that Cites this Article

Drug Enzymes
DrugEnzymeKindOrganismPharmacological ActionActions
CyclophosphamideCytochrome P450 2A6ProteinHumans
Unknown
Substrate
Details
CyclophosphamideCytochrome P450 2C18ProteinHumans
Unknown
Substrate
Details
IfosfamideCytochrome P450 2A6ProteinHumans
Unknown
Substrate
Details
IfosfamideCytochrome P450 2B6ProteinHumans
Unknown
Substrate
Details
IfosfamideCytochrome P450 2C18ProteinHumans
Unknown
Substrate
Details
IfosfamideCytochrome P450 2C19ProteinHumans
Unknown
Substrate
Details
IfosfamideCytochrome P450 3A4ProteinHumans
Unknown
Substrate
Inhibitor
Inducer
Details
IfosfamideCytochrome P450 3A5ProteinHumans
Unknown
Substrate
Details