Identification of the polymorphically expressed CYP2C19 and the wild-type CYP2C9-ILE359allele as low-Kmcatalysts of cyclophosphamide and ifosfamide activation

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Abstract
Cyclophosphamide and ifosfamide are alkylating agent prodrugs that require activation by cytochrome P450 (CYP) to manifest their cancer chemotherapeutic activity. The present study investigates the activity of four individual human CYP2C enzymes and their allelic variants in cyclophosphamide and ifosfamide activation as an initial attempt to gain insight into the underlying basis for the large interpatient differences in the clinical pharmacokinetics and metabolism of these anticancer drugs. Recombinant CYP2C8, CYP2C19, two allelic variants of CYP2C18, and six variants of CYP2C9 expressed in a yeast cDNA expression system were each enzymatically active, as judged by the ability of the isolated microsomes to catalyse 7-ethoxycoumarin Odeethylation after reconstitution with purified NADPH-cytochrome P450 reductase and cytochrome b5. With cyclophosphamide as substrate, CYP2C19 had the lowest apparent Km, followed by CYP2C9, CYP2C18 and CYP2C8, whereas in the case of ifosfamide, the rank order was:K m CYP2C192C9 ~2C8. Examination of a panel of CYP2C allelic variants revealed that CYP2C18-Thr3S5 had both a higher Vmax and a higher apparent Km toward cyclophosphamide than CYP2C18-Met385 with no difference in catalytic efficiency, whereas with ifosfamide the Thr385 allele exhibited a strikingly lower apparent Km resulting in a six-fold higher catalytic efficiency. In the case of CYP2C9, a Ile35- J to Leu mutation associated with poor metabolism of the hypoglycemic drug tolbutamide decreased catalytic efficiency toward cyclophosphamide by increasing the apparent Kin, whereas the same mutation reduced the efficiency of this P450 toward ifosfamide by decreasing the Vmax. Substitution of CYP2C9-Gly417 by Asp resulted in a two-fold lower catalytic efficiency for cyclophosphamide metabolism but a three-fold higher efficiency for ifosfamide metabolism. A His276 to Gly substitution resulted in an increase in both Vmax and apparent Kin with no net change in catalytic efficiency for either oxazaphosphorine. Mutations at CYP2C9 residues 144 and 358 had little or no effect. Thus (a) wild type CYP2C19 and CYP2C9 are relatively low Km catalysts of cyclophosphamide and ifosfamide activation, and (b) all four human CYP2C enzymes activate these two anticancer prodrugs with varying efficiencies and with striking differences among naturally occuring allelic variants in the case of CYP2C9 and CYP2C18.

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