Enhanced antitumor activity of P450 prodrug-based gene therapy using the low Km cyclophosphamide 4-hydroxylase P450 2B11

Abstract

Gene therapy using the prodrug-activating enzyme P450 2B6 has shown substantial promise in preclinical and initial clinical studies with the P450 prodrugs cyclophosphamide and ifosfamide. We sought to optimize this therapy using the canine P450 enzyme 2B11, which activates cyclophosphamide and ifosfamide with K m of 80 to 160 Mmol/L, f10-to 20-fold lower than the K m of P450 2B6. Retrovirus encoding a P450 2B11-internal ribosome entry signal-P450 reductase expression cassette induced marked cyclophosphamide and ifosfamide cytotoxicity toward 9L gliosarcoma cells and exhibited an impressive bystander killing effect at micromolar prodrug concentrations, where P450 2B6 displayed low activity. Adeno-2B11, a replication-defective, E1/E3 region-deleted adenovirus engineered to coexpress P450 2B11 and P450 reductase, dramatically increased tumor cell-catalyzed cyclophosphamide 4-hydroxylation and cytotoxicity compared with Adeno-2B6 and effected strong bystander killing at low (20 Mmol/L) cyclophosphamide concentrations. Further increases in cyclophosphamide cytotoxicity were obtained in several human cancer cell lines, including a 4-hydroperoxycyclophosphamide-resistant MCF-7 breast cancer cell line, when Adeno-2B11 was combined with Onyx-017, an E1b-55-kDa gene-deleted, tumor cellreplicating adenovirus that coamplifies and facilitates tumor cell spread of Adeno-2B11. To evaluate the therapeutic effect of P450 2B11 expression in vivo, 9L gliosarcoma cells transduced with P450-expressing retrovirus were grown as solid s.c. tumors in immunodeficient mice. Cyclophosphamide treatment on a metronomic, 6-day repeating schedule led to full regression of 9L/2B11 tumors but not P450-deficient control tumors, resulting in a tumor-free period lasting up to f100 days. 9L/2B6 tumors regressed more slowly and exhibited a tumor-free period of only 21 to 39 days. Thus, P450 genedirected enzyme prodrug therapy can be greatly improved by using the low K m P450 enzyme 2B11, which catalyzes intratumoral activation of cyclophosphamide and ifosfamide at pharmacologically relevant drug concentrations.

Enhancement of intratumoral cyclophosphamide pharmacokinetics and antitumor activity in a P450 2B11-based cancer gene therapy model

“…A similar result was obtained in 9L/2B1 tumors treated with IFA, an isomer of CPA that is also activated by 4-hydroxylation (Figure 2b). We conclude that the levels of P450 2B6 and P450 2B1 expressed in these tumors, while very effective at enhancing CPA's antitumor activity, 24 are too low to measurably increase the 4-hydroxy metabolite above the high background level that results from hepatic P450 metabolism.…”

“…[37][38][39] In the case of cytochrome P450, the low catalytic efficiency of the high K m CPA 4-hydroxylase enzymes P450 2B1 and P450 2B6 toward the anticancer prodrug substrate CPA (K m B500-1500 mM) can be greatly improved by the use of P450 2B11, a low K m CPA 4-hydroxylase (K m B70 mM). 24 Presently, we used scid mice bearing 9L gliosarcomas transduced with retrovirus encoding either high K m or low K m CPA-activating P450 enzymes as a model system to characterize the metabolic and pharmacokinetic factors that underlie this improved activity and to investigate the impact of i.t. CPA delivery using a slow release polymer.…”

“…24 9L cells infected with an empty pBabe-based retrovirus were used as P450-deficient controls. Immunochemical analysis using anti-P450 2B antibodies revealed P450 2B expression in B90-95% of the cultured 9L/2B cells.…”

“…24 Given this low K m , the therapeutic advantage of P450 2B11 is expected to be manifest at CPA concentrations that are comparatively low (p100 mM), as they are in CPA-treated cancer patients. 30,31 Indeed, although tumor-associated 4-OH-CPA levels were lower in 9L/2B1 and 9L/2B6 tumors than in liver (Figure 2), i.t.…”

“…CPA activation. Intratumoral CPA activation also occurs in the case of the high K m enzymes P450 2B6 and P450 2B1, as evidenced by the enhanced apoptotic response (Figure 1) and antitumor effect compared to P450-deficient tumors, 24 but could not be discerned in the context of the high background level of hepatic P450 metabolism. The P450 2B11-dependent increase in i.t.…”