Isophosphoramide Mustard-Lysine (ZIO-201): Active Moiety of Ifosfamide (IFOS) without Kidney Toxicity.

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Ifosfamide (IFOS) and cyclophosphamide (CPA) are widely used anti-cancer drugs. Both are pro-drugs which must be metabolized before their metabolites cross-link DNA. Use of these drugs, especially high-dose IFOS, is complicated by substantial adverse effects of metabolites not directly involved in DNA-cross-linking including kidney, bladder and CNS toxicities. Recently, a lysine-stabilized form of isophosphoramide mustard (IPM-lysine; ZIO-201), an IFOS-metabolite, was developed. ZIO-201 directly cross-links DNA and is active against human cancer cell lines and in mice with human cancer xenografts. To evaluate nephrotoxicity of ZIO-201 we used an in vitro primary rabbit kidney proximal tubule (RPT) cell culture system, which retains many of the characteristics of renal proximal tubule cells including a polarized morphology, a Na+/glucose cotransport system and a p-aminohippurate transport system, glutathione status, and hormone responses (including a parathyroid hormone sensitive adenylate cyclase). We studied effects of ZIO-201 and other IFOS metabolites on primary RPT cells. IFOS is activated by the cytochrome P450 system producing a mixture of 2- and 3-dechloroethylifosfamide and resulting in formation of chloroacetaldehyde (CAA). Also, metabolism of IFOS by the cytochrome P450 system produces 4-hydroxyifosfamide, ultimately resulting in the production of acrolein (ACR). CAA and ACR are IFOS metabolites implicated in kidney toxicity in humans. Effects of ZIO-201 and CAA on the viability of confluent monolayers of primary rabbit kidney proximal tubule (RPT) cells were tested at 15–100 μM. Viability was determined by neutral red dye uptake by the primary cultures after 8 h incubation. ZIO-201 did not significantly reduce viability of primary RPT cells; the LD50 for CAA was about 40 μM with complete inhibition at 75 μM. Effects of 4-hydroperoxyifosfamide (HIFA), a synthetic prodrug of 4-hydroxyifosfamide and ACR were also studied. Although ACR had an LD50 of 80 μM HIFA did not significantly affect viability. The IFOS metabolite 4-hydroxyifosfamide, was prepared from 4-hydroperoxyifosfamide (1). At 200 μM, 4-hydroxyifosfamide resulted in extensive cell death after 4 h incubation. These data show that ZIO-201 avoids the kidney toxicity caused by metabolites of pro-drugs like IFOS. Whereas 2 IFOS metabolites, CAA and ACR were cytotoxic to kidney cells, ZIO-201 was not. An explanation for the toxic effects of 200 μM 4-hydroxyifosfamide t might be metabolism to ACR in vitro.