Improving the spectrophotometric determination of the alkylating activity of anticancer agents: a new insight into the mechanism of the NBP method.

In this paper, the mechanism of the nitrobenzylpyridine (NBP) method to measure the alkylating activity of drugs originally described by Epstein et al. [J. Epstein, R.W. Rosenthal, R.J. Ess, Anal. Chem. 27 (1955) 1435-1439] and modified later by others was revisited using melphalan, m-sarcolysin, chlorambucil, cyclophosphamide and ifosfamide. Its direct application to determine the activity of these drugs in human serum and aqueous media is described and discussed. This method, based on the formation of a chromophore due to the reaction between the alkylating agent and NBP, was significantly improved by extracting as quickly as possible the reaction product(s) into chloroform before adding alkali to develop the color. This significantly limited the degradation by hydrolysis of the products and enhanced the yield of the end chromophore in the organic phase. The reaction time was optimized by monitoring each compound color development. The best reaction time for each compound was selected and a higher stability of the extracted color over at least 1h was obtained (compared to a couple of minutes in previous studies). Most interestingly, water evaporation due to heating had little or no effect on the linearity of standard curves evaluated in the micromolar concentration range. Both the sensitivity and reproducibility of the method were therefore significantly improved. There appears to be a direct correlation between compound hydrolysis and alkylation activity; the relative reactivity is different among the compounds owing to the rate of (i) production, (ii) the relative proportions and (iii) the hydrolysis of the intermediates. A general mechanism for the nucleophilic competitive substitution is proposed.

A novel transport and delivery mechanism underpins the effectiveness of prolyl-m-sarcolysyl-p-fluorophenylalanine (PSF) in a human melanoma xenograft nude-mouse model.

The alkylating peptide PSF shows very promising results in vitro on different cancer cells but its efficacy in animals has not been assessed. Here we evaluate the efficacy of PSF in human melanoma-bearing nude mice and examine the underlying mechanism. In melanoma-bearing nude mice, escalating doses of PSF showed dose-dependent responses and reached tumor regression with an optimal dose of 20 mg/kg for 1 month. A comparison of PSF with its free moiety m-sarcolysin and melphalan showed a highly significant advantage of PSF. Furthermore, dose fractionation yielded an even better control of tumor regrowth. In vitro studies unraveled an original delivery mechanism based on the rapid binding of PSF mainly due to red blood cells to form a pro-drug complex and the subsequent release of active metabolites by tumor-associated proteolytic enzymes. Blood kinetics showed one major metabolite partially released over time, while in the presence of melanoma cells three additional metabolites are generated. Interestingly, tumor-shed proteases also induce the production of these metabolites and varying combinations of enzyme inhibitors indicate the involvement of metallo- and other families of proteases in the delivery process. This particular transport and delivery of such an alkylating agent may have several benefits, mainly lowering the drug-free moiety in plasma and at the same time increasing its concentration in protease rich areas such as tumors.