Fig. 5. Metabolism of cyclophosphamide. The mammalian enzyme cytochrome P450 hydroxylates cyclophosphamide (CPA). 4-Hydroxycyclophosphamide then spontaneously decomposes into the toxic metabolite and alkylating agent phosphoramide mustard (PM).
poorly transported across the blood-brain barrier (61). Gene therapy using CYP2B1 to activate CPA was designed primarily for use in brain tumors because other tumor types have ready access to CPA's active metabolites produced by the liver when CPA is administered systemically.
Intratumoral implantation of VPCs releasing retroviral vectors expressing CYP2B1 caused regression of intracerebral rat glioma cells in nude mice after intratumoral or intrathecal CPA administration (62). Development of lep-tomeningeal tumor infiltration was prevented in most animals, whereas control tumors lacking CYP2B1 exhibited extensive leptomeningeal infiltration. In fact, the regression of parenchymal tumor mass caused by CPA treatment of CYP2B1-transduced cells was not nearly as dramatic as the inhibition of lep-tomeningeal infiltration observed, suggesting better access of CPA to lep-tomeningeal infiltrates than to the main tumor mass. Different modes of drug delivery, such as intraarterial administration or intratumoral implantation of polymer-based wafers containing CPA (63), should allow better access of CPA to parenchymal tumors.
When 10% of cultured glioma cells express CYP2B1, CPA causes a 75% decrease in cell proliferation (64). Separating CYP2B1-expressing cells from parental cells by a membrane still allows a bystander effect, and PM appears to be the diffusible metabolite responsible (65).
The P450 system consists of two protein components, the heme-containing P450 and the flavoprotein NADPH-P450 reductase (RED), both embedded in the phospholipid bilayer of the endoplasmic reticulum. RED catalyzes the transfer of electrons required for all microsomal P450-dependent enzyme reactions. Transfecting tumor cells with the RED cDNA enhances CPA sensitivity after infection with recombinant adenovirus expressing CYP2B1 (66). The same group has also used a retroviral vector to deliver various human cytochrome P450 genes to a rat gliosarcoma cell line, with CYP2B6 causing the most dramatic enhancement in CPA sensitivity. These human P450s should prove less immunogenic in human tumor gene therapy.
Three P450/CPA clinical trials are under way, none in brain tumors. Seventeen patients are enrolled as of September 2001, with results pending (3).
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