Cl Cl 4-hydroxy CPA

Phosphoramide Mustard

FIGURE 23.3 Cytochrome P450 (CYP); cyclophosphamide (CPA).

cell exposure to cytotoxic drug metabolites by targeting expression of enzymes to tumor cells. To date, this area has been dominated by two prodrugs, cyclophosphamide (CPA) and ifosfamide (IFA). CPA is a prodrug that is activated by liver-specific enzymes of the CYP family (see Fig. 23.3). The rat cytochrome P450 2B1 (CYP2B1), activates CPA with high efficiency

[32]. The active form of CPA, phosphoramide mustard, is an alkylating agent that generates DNA cross-links and consecutively DNA strand breaks and results in cell death.

The efficacy of CPA in treating brain tumors has been limited by the fact that although CPA crosses the blood-brain barrier, its active metabolites can be generated only by liver P450, and these metabolites are poorly transported across the blood-brain barrier

[33]. Gene therapy using CYP2B1 to activate CPA was designed primarily for use in brain tumors since other malignancies already have access to CPA's active metabolites. The implantation of CYP2B1 expressing retroviral vectors was shown to induce regression of intracerebral rat glioma cells after intratumoral or intrathecal CPA administration [34]. Like the CD/5-FC approach, CPA metabolites do not require cell-cell contact for a bystander effect, distributing by passive diffusion [35]. The transient immunosuppression provided by activated CPA metabolites has also been shown to favor viral replication and anticancer effects in vivo [36].

It has been reported that nucleoside analogs are synergistic in their anticancer action with alkylating agents [37]. While CPA metabolites are alkylating agents, GCV metabolites are nucleoside analogs. One approach uses the replacement of the large subunit of the HSV-1 genome with the CYP2B1 gene to generate a HSV-1 vector (rRp450) that is able to kill tumor cells through three modes: (1) using viral oncolysis and rendering infected cell sensitive to (2) CPA and (3) GCV [38]. Subcutaneous tumors established from glioma cell lines in immunodeficient mice regress only when they are treated with rRp450, CPA, and GCV [26]. This has lead to the hypothesis that after DNA chain alkylation by CPA metabolites, DNA repair mediated by DNA polymerases, is affected by GCV metabolites.

The cytochrome P450 system actually comprises two polypeptide components, the P450 and the P450 reductase (RED). RED expression is required to provide full catalytic activity of the rat CYP2B1 for gene therapy. Studies performed with rat gliosarcoma cells stably transfected with CYP2B1, RED, or both cDNAs, showed that further supplementation of RED by gene transfer enhanced the CYP/CPA effects, although tumor cells express enough RED to fulfill the transferred CYP gene's capability of CPA conversion [39]. The addition of RED not only improves CYP2B1-mediated conversion of CPA but also provides the ability to convert other prodrugs such as tirapazamine into active anticancer agents [40]. Another method to improve the therapeutic index of CYP/CPA is the inhibition of hepatic metabolism of the prodrug [41]. This might decrease systemic toxicity of CPA metabolites as well as increasing prodrug availability to tumor cells expressing CYP. One of the essential steps in tumorigenesis is the active recruitment of a neovascular supply by the neoplasm. A modified CPA regime showed an anti-angiogenic effect which also increased the therapeutic index of the CYP/CPA approach [42, 43]. Three CYP/ CPA clinical trials are underway, none of them in brain tumors.

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