The activity of latent benzoperimidine esters to inhibit P-glycoprotein and MRP1 dependent efflux of pirarubicin from several lines of multidrug resistant tumour cells.

D. Glowacka-Rogacka, M. Arciemiuk, A. Kupiec, M.M. Bontemps-Gracz, E. Borowski, J. Tarasiuk 1

Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, Poland. 1Department of Biochemistry, University of Szczecin, Poland

AIM: Multidrug resistance to antitumour agents structurally dissimilar and having different intracellular targets is the major problem in cancer therapy. Multidrug resistance phenotype is associated with drug accumulation failure resulting from enhanced drug export via energy-dependent efflux pumps belonging to the ABC transporter family (e.g. P-glycoprotein, MRP1). One approach to overcome multidrug resistance is to use efficient modulators (chemosensitizers) being good substrates for exporting pumps. In consequence, the inhibition of antitumour drugs efflux leading to the restoration of their accumulation in resistant cells could be achieved. It is suggested that substrate spectrum of P-glycoprotein and MRP1 is in part the same but some significant differences exist, especially that MRP1 is the efficient transporter of organic anions. Recently we obtained a novel group of antitumour compounds benzoperimidines. We have found that these derivatives had very fast cellular uptake kinetics and the reduction of their accumulation in resistant cells was not observed1. For this reason, benzoperimidine derivatives were rationally chosen to obtain modulators of P-glycoprotein and MRP1 mediated efflux. They have been synthesised in latent ester forms for the reason to facilitate the passive diffusion across the plasma membrane. METHOD:A general procedure was followed in their synthesis. The 3-((tert-butoxycarbonyl)-2-[(7-oxo-7H-benzo[e]perimidin-6-yl)amino]ethylamino)propanoic acid was treated in boiling benzene with DBU and an excess of halomethyl ester to form the corresponding esters. The halomethyl esters were prepared in the reaction of the appropriate acid halide with paraformaldehyde. Removing of protective Boc group gave a desirable ester of benzoperimidines. Using spectrofluorometric method carried out in HEPES buffer, the ability of obtained benzoperimidine esters to effectively inhibit P-glycoprotein and MRP1 dependent efflux of anthracycline - pirarubicin and to restore its accumulation in resistant cells was studied. The reversal activity of benzoperimidine esters was examined in in vitro assays. RESULTS: In this study biological data for a series of bezoperimidine esters modified at the side chain are presented for properly selected seven cell lines: sensitive (K562, GLC4, HL60), P-gp resistant (K562/DX, HL60/VINC), MRP1/LRP resistant (GLC4) and MRP1 resistant (HL60/DOX) lines. These compounds had no cytotoxic activity towards sensitive and MDR resistant tumour cells used in this study. The ability of obtained benzoperimidine esters to effectively inhibit MRP1 dependent efflux of anthracycline - pirarubicin and to restore its accumulation in resistant cells overexpressing MRP1 (HL-60/DX) and MRP1/LRP (GLC4/DX) was demonstrated. It was evidenced using resistant cells overexpressing P-glycoprotein (K562/DX, HL-60/VINC) that examined esters were effective also in inhibiting P-glycoprotein dependent efflux of pirarubicin.In contrast to the high effectiveness in pirarubicin active efflux inhibition and restoration of its cellular accumulation obtained in HEPES buffer, our study revealed the lack of latent compounds reversal activity in vitro because in the cell culture an enzymatic cleavage of esters occurs due to the presence of blood serum esterases. CONCLUSION: We suggest that the effective inhibition of pirarubicin efflux could be obtained only under conditions of stability of latent compounds. It could be realized maybe by an encapsulation of latent esters into liposome vesicles1 K. Tkaczyk-Gobis et al., (2001) Eur. J. Pharmacol. 413, 131-141.

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Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Multidrug Resistance - MDR.