Received for publication March 26, 2004.
Revised April 28, 2004.
Accepted for publication April 29, 2004.

Metabolism of N,N',N'-Triethylenethiophosphoramide by CYP2B1 and CYP2B6 Results in the Inactivation of Both Isoforms by Two Distinct Mechanisms

Abstract

The anti-cancer drug N,N",N"-triethylenethiophosphoramide (tTEPA) inactivated CYP2B6 and CYP2B1 in the reconstituted system in a time-, concentration-, and NADPH- dependent manner indicative of mechanism-based inactivation. The K_I for the pseudo-first order inactivation of CYP2B1 was 38 µM, the k_inact, was 0.3 min^-1, and the t_1/2 was 2.5 minutes. Spectral CO binding- and HPLC heme studies of the tTEPA-inactivated CYP2B1 suggest that the loss in the enzymatic activity was primarily due to the binding of a reactive tTEPA intermediate to the 2B1 apoprotein. Inactivation by tTEPA in the presence of 7-ethoxycoumarin (7-EC), an alternate substrate, reduced the rate of inactivation of CYP2B1. Incubations with tTEPA and NADPH resulted in greater than 90% loss in the 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC) O-deethylation activity and testosterone hydroxylation activity of CYP2B1. In contrast, benzphetamine metabolism was significantly less inhibited (47%). CYP2B6 was inactivated by tTEPA with a K_I of 50 µM, a k_inact of 0.1 min^-1, and a t_1/2 of 14 min. However, unlike CYP2B1, the tTEPA-inactivated human isoform showed losses in the P450 CO spectrum, the pyridine hemochrome spectrum, and in the amount of native heme that were comparable to the loss in the 7-EFC and benzphetamine activity, suggesting that activity loss was brought about by a tTEPA reactive intermediate damaging the CYP2B6 heme. CYP2B6 could only be protected from the tTEPA-dependent inactivation by the 2B6 specific substrate bupropion but not by other substrates of CYP2B such as benzphetamine, testosterone, or 7-ethoxycoumarin. The data indicate that tTEPA metabolism by these two 2B isoforms results in inactivation of the CYPs by two distinct mechanisms.

Key words: CYP2B1, CYP2B6, Cytochrome P450, drug interaction, mechanism-based inactivation, thioTEPA

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