IDENTIFICATION OF THE POLYMORPHICALLY EXPRESSED CYP2C19 AND THE WILD-TYPE CYP2C9-ILE359 ALLELE AS LOW-K-M CATALYSTS OF CYCLOPHOSPHAMIDE ANDIFOSFAMIDE ACTIVATION

Citation
Tkh. Chang et al., IDENTIFICATION OF THE POLYMORPHICALLY EXPRESSED CYP2C19 AND THE WILD-TYPE CYP2C9-ILE359 ALLELE AS LOW-K-M CATALYSTS OF CYCLOPHOSPHAMIDE ANDIFOSFAMIDE ACTIVATION, Pharmacogenetics, 7(3), 1997, pp. 211-221
Citations number
66
Categorie Soggetti
Pharmacology & Pharmacy","Genetics & Heredity
Journal title
ISSN journal
0960314X
Volume
7
Issue
3
Year of publication
1997
Pages
211 - 221
Database
ISI
SICI code
0960-314X(1997)7:3<211:IOTPEC>2.0.ZU;2-W
Abstract
Cyclophosphamide and ifosfamide are alkylating agent prodrugs that req uire activation by cytochrome P450 (CYP) to manifest their cancer chem otherapeutic activity, The present study investigates the activity of four individual human CYP2C enzymes and their allelic variants in cycl ophosphamide and ifosfamide activation as an initial attempt to gain i nsight into the underlying basis for the large interpatient difference s in the clinical pharmacokinetics and metabolism of these anticancer drugs, Recombinant CYP2C8, CYP2C19, two allelic variants of CYP2C18, a nd six variants of CYP2C9 expressed in a yeast cDNA expression system were each enzymatically active, as judged by the ability of the isolat ed microsomes to catalyse 7-ethoxycoumarin O-deethylation after recons titution with purified NADPH-cytochrome P450 reductase and cytochrome b(5). With cyclophosphamide as substrate, CYP2C19 had the lowest appar ent K-m, followed by CYP2C9, CYP2C18 and CYP2C8, whereas in the case o f ifosfamide, the rank order was: K-m CYP2C19 < CYP2C18 < CYP2C9 < CYP 2C8. CYP2C18 had the highest in vitro intrinsic clearance/catalytic ef ficiency (apparent V-max/K-m) in cyclophosphamide and ifosfamide activ ation, followed by 2C19 > 2C9 similar to 2C8. Examination of a panel o f CYP2C allelic variants revealed that CYP2C18-Thr(385) had both a hig her V-max and a higher apparent K-m toward cyclophosphamide than CYP2C 18-Met(385) with no difference in catalytic efficiency, whereas with i fosfamide the Thr(385) allele exhibited a strikingly lower apparent K- m resulting in a six-fold higher catalytic efficiency In the case of C YP2C9, a Ile(359) to Leu mutation associated with poor metabolism of t he hypoglycemic drug tolbutamide decreased catalytic efficiency toward cyclophosphamide by increasing the apparent K-m, whereas the same mut ation reduced the efficiency of this P450 toward ifosfamide by decreas ing the V-max. Substitution of CYP2C9-Gly(417) by Asp resulted in a. t wo-fold lower catalytic efficiency for cyclophosphamide metabolism but . a three-fold higher efficiency for ifosfamide metabolism. A His(276) to Gly substitution resulted in an increase in both V-max and apparen t K-m with no net change in catalytic efficiency for either oxazaphosp horine, Mutations at CYP2C9 residues 144 and 358 had little or no effe ct, Thus (a) wild type CYP2C19 and CYP2C9 are relatively low K-m catal ysts of cyclophosphamide and ifosfamide activation and (b) all four hu man CYP2C enzymes activate these two anticancer prodrugs with varying efficiencies and with striking differences among naturally occuring al lelic variants in the case of CYP2C9 and CYP2C18.