New Prodrug Activation Gene Therapy for Cancer Using Cytochrome P450 4B1 and 2-Aminoanthracene/4-Ipomeanol

Abstract

Vector-mediated transfer of prodrug-activating genes provides a promising means of cancer gene therapy. In a search for more selective and more potent bioactivating enzymes for gene therapy of malignant brain tumors, the toxicity-generating capacity of the rabbit cytochrome P450 isozyme CYP4B1 was investigated. Rabbit CYP4B1, but not rat or human isozymes, efficiently converts the inert prodrugs, 2-aminoanthracene (2-AA) and 4-ipomeanol (4-IM), into highly toxic alkylating metabolites. Toxicity of these two prodrugs was evaluated in culture in parental and genetically modified rodent (9L) and human (U87) glioma cell lines stably expressing CYP4B1, and in vivo in a subcutaneous 9L tumor model in nude mice. The most sensitive CYP4B1-expressing glioma clone, 9L4B1-60, displayed an LD₅₀ of 2.5 μM for 2-AA and 4-IM after 48 h of prodrug incubation, whereas 20 times higher prodrug concentrations did not cause any significant toxicity to control cells. Substantial killing of control tumor cells by 2-AA was achieved by co-culturing these cells with CYP4B1-expressing cells at a ratio of 100:1, and toxic metabolites could be transferred through medium. In both CYP4B1-expressing cells and co-cultured control cells, prodrug bioactivation was associated with DNA fragmentation, as assayed by fluorescent TUNEL assays and by annexin V staining. Alkaline elution of cellular DNA after exposure to 4-IM revealed extensive protein-DNA crosslinking with single-strand breakage. Growth of 9L-4B1 tumors in nude mice was inhibited by intraperitoneal injection of 4-IM with minimal side effects. Potential advantages of the CYP4B1 gene therapy paradigm include: the low concentrations of prodrug needed to kill sensitized tumor cells; low prodrug conversion by human isozymes, thus reducing toxicity to normal cells; a tumor-killing bystander effect that can occur even without cell-to-cell contact; and the utilization of lipophilic prodrugs that can penetrate the blood–brain barrier.

Overview summary

This is the first report of a new prodrug activation scheme for cancer gene therapy. Rabbit cytochrome P450 4B1 efficiently converts the prodrugs, 2-aminoanthracene (2-AA) and 4-ipomeanol (4-IM), to alkylating metabolites, which are especially toxic to tumor cells, whereas the equivalent human isozymes have low activity against these compounds. Human and rat glioma cells transfected with this gene showed high sensitivity to these prodrugs both in culture and as subcutaneous tumors in nude mice. The toxicity had a strong bystander effect and was associated with extensive DNA–protein crosslinking. This prodrug activation scheme allows the generation of toxic drugs within the tumor, with low toxicity to other tissues. The lipophilicity of these prodrugs should also facilitate transport across the blood–brain barrier to allow access to brain tumors.