Identification of the polymorphically expressed CYP2C19 and the wild-type CYP2C9-ILE359 allele as low-Km catalysts of cyclophosp

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Pharmacogenetics. 1997 Jun;7(3):211-21.
of the polymorphically expressed CYP2C19 and the wild-type
CYP2C9-ILE359 allele as low-Km catalysts of cyclophosphamide and
ifosfamide activation.
Chang TK, Yu L, Goldstein JA, Waxman DJ.
Department of Biology, Boston University, MA 02215, USA.
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
O-deethylation 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: Km
CYP2C19 < CYP2C18 < CYP2C9 < CYP2C8. CYP2C18 had the highest
in vitro intrinsic clearance/catalytic efficiency (apparent Vmax/Km) in
cyclophosphamide and ifosfamide activation, followed by 2C19 > 2C9
approximately 2C8. Examination of a panel of CYP2C allelic variants
revealed that CYP2C18-Thr385 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 Ile359 to Leu
mutation associated with poor metabolism of the hypoglycemic drug
tolbutamide decreased catalytic efficiency toward cyclophosphamide by
increasing the apparent Km, 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 Km 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 occurring
allelic variants in the case of CYP2C9 and CYP2C18.