Modulation of cyclophosphamide-based cytochrome P 450 gene therapy using liver P450 inhibitors

Abstract

The sensitivity of tumors to cyclophosphamide ( CPA ) and other anticancer prodrugs can be substantially enhanced by transduction of tumors with a prodrug -activating mammalian cytochrome P450 ( CYP ) enzyme in combination with the flavoenzyme P450 reductase. This gene therapy strategy provides for intratumoral prodrug activation, but is also associated with a high level of hepatic prodrug activation, which reduces the extent of intratumoral prodrug activation and contributes to systemic drug toxicity. To address this issue, five P450 inhibitors were tested for their ability to block liver CYP2C -catalyzed CPA activation selectively, i.e., without inhibiting the corresponding intratumoral activation of CPA catalyzed by a transduced CYP2B enzyme. In vitro studies revealed that the P450 inhibitors 1 -aminobenzotriazole and DDEP were preferentially inhibitory toward CYP2C -dependent liver microsomal CPA activation, whereas the P450 inhibitor SKF -525A inhibited CYP2C -and CYP2B -dependent CPA activation without P450 form selectivity. By contrast, the P450 inhibitors chloramphenicol and metyrapone preferentially inhibited CYP2B -dependent CPA activation. Rat pharmacokinetic studies confirmed the inhibitory action of these compounds in vivo, with up to a 4 -fold decrease in C max and a 7 -fold increase in apparent half -life of the activated CPA metabolite, 4 -hydroxy -CPA, seen in the case of 1 -aminobenzotriazole. Although the rate of hepatic CPA activation could thus be decreased substantially by P450 inhibitor treatment, the net extent of hepatic CPA activation was only modestly decreased, as judged by plasma area -under -the -curve values for 4 -hydroxy -CPA. Moreover, P450 inhibitor treatment did not decrease CPA's host toxicity and did not enhance the tumor growth delay response to CPA in rats bearing CYP2B1 -transduced gliosarcomas. These findings are discussed in the context of P450 -based gene therapy strategies and ongoing efforts to enhance anticancer drug activity by increasing the exposure of P450 -expressing tumors to the P450 -activated prodrug CPA. Cancer Gene Therapy ( 2001 ) 8, 450 ± 458

Suicide genes for cancer therapy

“…Various inhibitors of liver P450 activity have been shown to substantially reduce CPA activation in liver microsomes in vitro and to a minor extent in vivo in rat liver (Huang and Waxman, 2001). However, treatment of subcutaneous gliosarcoma cell xenografts expressing recombinant Cyp2B1 with CPA and selected P450 inhibitors did not reveal an improvement in tumour growth delay as compared to CPA treatment alone, suggesting that these inhibitors lack a sufficient specificity for the liver P450 enzymes (Huang and Waxman, 2001).…”

“…Various inhibitors of liver P450 activity have been shown to substantially reduce CPA activation in liver microsomes in vitro and to a minor extent in vivo in rat liver (Huang and Waxman, 2001). However, treatment of subcutaneous gliosarcoma cell xenografts expressing recombinant Cyp2B1 with CPA and selected P450 inhibitors did not reveal an improvement in tumour growth delay as compared to CPA treatment alone, suggesting that these inhibitors lack a sufficient specificity for the liver P450 enzymes (Huang and Waxman, 2001). In a different approach, inhibition of transcription of the endogenous NADPH cytochrome P450 reductase (CPR) gene in the rat liver by anti-thyroid drugs such as methimazole resulted in a decreased P450 mediated activation of CPA, since the CPR is essential for P450 activity by providing redox equivalents for hydroxylation of P450 substrates (Huang et al, 2000).…”

In Vitro Inhibitory Effect of 1-Aminobenzotriazole on Drug Oxidations in Human Liver Microsomes: a Comparison with SKF-525A

“…Some laboratories used ABT without a preincubation, but others used it with the preincubation. [13][14][15] Therefore, we investigated the inhibitory eŠect of ABT with and without preincubation in this study. With respect to CYP3A, it is recommended to use multiple CYP3A probes for the in vitro evaluation of CYP3A-dependent drug interactions, and we used midazolam, nifedipine, and testosterone.…”

Oxidation and rearrangements of flavanones by mammalian cytochrome P450

“…In the microsomal reaction, the formation of these metabolites was markedly inhibited by two inhibitors of CYP2B, SKF-525A and metyrapone (Huang and Waxman 2001), whereas the inhibitor of CYP1A, a-naphthoflavone (Liu et al 2003) had no significant inhibitory effect (figure 4). These results suggest that CYP2B is mainly involved in the conversion of (AE)-4 0 -methoxyflavanone to 4 0 -methoxyflavone and (AE)-2,3-trans-4 0 -methoxyflavanonol.…”