Received for publication August 7, 2008.
Revised November 17, 2008.
Accepted for publication November 17, 2008.

New ifosfamide analogues designed for lower associated neurotoxicity and nephrotoxicity with modified alkylating kinetics leading to enhanced in vitro anticancer activity

Abstract

Ifosfamide is an useful prodrug with CYP450 metabolism associated to both antitumor activity and toxicities. Isophosphoramide mustard is the bisalkylating active metabolite and acrolein, the urotoxic one. Since acrolein toxicity is limited by co-administration of mesna, the incidence of urotoxicity has been lowered. Current evidence suggests that chloroacetaldehyde, a side-chain oxidation metabolite is responsible for neurotoxicity and nephrotoxicity. The aim of our research is to prevent chloroacetaldehyde formation using new enantioselective synthesized ifosfamide analogues i.e. C7,C9-dimethyl-ifosfamide. We hypothesise that reduced toxicogenic catabolism may induce less toxicity without change in the anticancer activity. Metabolites determination of the dimethyl-ifosfamide analogues was performed using liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) after in vitro biotransformation by drug-induced rat liver microsomes and human microsomes expressing the main CYP 3A4 and minor CYP2B6 enzymes. Both human and rat microsomes incubations produced the same N-dechloroalkylated and 4-hydroxylated metabolites. Then, a co-culture assay of 9L rat glioblastoma cells and rat microsomes was performed to evaluate their cytotoxicity. And finally, a mechanistic study using Phosphorus Nuclear Magnetic Resonance (³¹P-NMR) kinetics allowed estimating the alkylating activity of the modified mustards. The results showed that, analogues were still metabolized through the same N-deschloroalkylation pathway but they showed an increased activity. They were 4 to 6-fold more cytotoxic than ifosfamide on 9L and the generated dimethylated mustards were 28-fold faster alkylating agents than ifosfamide mustards. Among these new ifosfamide analogues, the 7S,9R enantiomer will be assessed for further in vivo investigations for its anticancer activity and its toxicological profile.

Key words: 31P-NMR, Ifosfamide, anticancer drug, kinetic, metabolism, microsome