Sustained P450 expression and prodrug activation in bolus cyclophosphamide-treated cultured tumor cells. Impact of prodrug schedule on P450 gene-directed enzyme prodrug therapy

Abstract

Cytochrome P450-based gene therapy can substantially increase the sensitivity of tumor cells to P450-activated cancer chemotherapeutic prodrugs such as cyclophosphamide (CPA) without increasing host toxicity. While the role of 4-OH-CPA, the primary active metabolite of CPA, in eliciting tumor cell death is well established, the effect of 4-OH-CPA exposure on the capacity of P450-expressing tumor cells for continued metabolism and activation of CPA has not been investigated. The present study addresses this question and characterizes the impact of CPA dose and treatment schedule on the ability of P450-expressing tumor cells to sustain prodrug activation over time. 9L gliosarcoma cells expressing human P450 2B6 and treated with CPA in a continuous manner exhibited a time-and CPA dose-dependent decrease in P450-catalyzed CPA 4-hydroxylase activity. This decrease reflects a selective, 4-OH-CPA-induced loss of cellular P450 protein content. By contrast, when the P450-expressing tumor cells were treated with CPA as a single 8 hours exposure, cellular CPA 4-hydroxylase activity and P450 protein expression were substantially prolonged when compared to continuous prodrug treatment. This schedule-dependent effect of CPA was influenced by the level of P450 protein expressed in the tumor cells. At high P450 protein and activity levels, which could be achieved by culturing the tumor cells at high cell density, net production and release of 4-OH-CPA into the culture media was increased substantially. This increase fully offset the decline in CPA 4-hydroxylase activity as the tumor cells underwent CPAinduced apoptotic death. These findings demonstrate the impact of CPA dose and treatment schedule on the efficacy of P450 genedirected enzyme prodrug therapy, with bolus CPA treatment being compatible with sustained expression of P450 protein and maintenance of P450-dependent prodrug activation by the target tumor tissue.

The constitutive androstane receptor is a novel therapeutic target facilitating cyclophosphamide-based treatment of hematopoietic malignancies

“…In this effort, several studies have shown that using retroviral, replicating herpes viral, and adenoviral vectors, regional delivery of CYP2B expression cassette into tumor tissues significantly increased cancer cell cytotoxicity and intracellular 4-OH-CPA formation. 18,19,45,46 Whereas this strategy may benefit patients with localized solid tumors, in the case of hematologic malignancies, systemic treatment is required and bioactivation of CPA primarily relies on hepatic drugmetabolizing enzymes. Our HPH-leukemia cell coculture model represents an excellent in vitro system mimicking human in vivo condition that allows simultaneous investigation of hepatic metabolism and extrahepatic anti-cancer activity under a shared cellular environment.…”

“…18,19 Although this strategy appears to be attractive in CPA-based treatment of localized solid tumors, it may not be applicable to hematologic malignancies, such as leukemia and lymphoma, in which systemic chemotherapy is necessary. Therefore, drugs affecting hepatic expression of CYP2B6 may profoundly influence the systemic exposure of CPA metabolites, as well as the efficacy and safety of CPA in the treatment of leukemia and lymphoma.…”

Differential Regulation of Hepatic CYP2B6 and CYP3A4 Genes by Constitutive Androstane Receptor but Not Pregnane X Receptor

“…Cyclophosphamide (CPA) is an example of such a drug. This antineoplastic undergoes N-dechloroethylation to an inactive toxic metabolite exclusively by CYP3A4 and 4-hydroxylation to a therapeutically active metabolite primarily by CYP2B6 (Roy et al, 1999;Schwartz et al, 2003). Assuming that hCAR selectivity for CYP2B6 also occurs in vivo, concurrent administration of CPA with a selective hCAR activator should facilitate enhanced production of its beneficial metabolite without simultaneously increasing formation of its toxic metabolite.…”

Nuclear Receptor CAR Requires Early Growth Response 1 to Activate the Human Cytochrome P450 2B6 Gene

“…Human CYP2B6 is a clinically important enzyme that metabolizes many commonly used therapeutics and activates anti-cancer pro-drugs, such as cyclophosphamid and ifosfamide (6,7). Two distinct DNA sequences within the CYP2B6 promoter are required for CAR to synergistically activate the promoter activity: the distal phenobarbital-responsive enhancer module (PBREM) and the proximal OA response element now called OARE KI (5,8).…”