On the basis of the results obtained in previous research, three series of compounds (A-C), derived from verapamil, were designed and synthesized to obtain drugs able to revert multidrug resistance (MDR), an acquired resistance that frequently impairs cancer chemotherapy. The ability of the obtained compounds to revert MDR was evaluated on anthracycline-resistant erythroleukemia K 562 cells, measuring the uptake of THP-adriamycin (pirarubicin) by continuous spectrofluorometric monitoring of the decrease of the fluorescence signal of the anthracycline at 590 nm (lambdaex = 480 nm), after incubation with cells. Cardiovascular activity, which is responsible for unwanted side effects, was also evaluated. The results obtained show that many of the compounds studied are potent reverters of MDR and are endowed with reduced cardiovascular activity. One of the compounds (7, MM36) presents a pharmacological profile (unprecedented nanomolar potency, high reversal of MDR, low cardiovascular activity) that makes it a promising drug candidate to treat MDR and a useful tool for studying P-glycoprotein.
The multidrug resistant (MDR) tumor phenotype, characterized by a decreased cellular drug accumulation is achieved by ATP-dependent extrusions of drugs from cells by P-glycoprotein (P-gp) and/or by multidrug resistance protein (MRP1). Despite the huge amount of research that has been performed on the mechanisms of P-gpmediated efflux of drug, it is not yet known what the molecular parameters are required for a molecule to be recognized and pumped out by P-gp. Anthracyclines are weak bases and, depending on the pH, can exist either in the neutral or in the positively charged form. The aim of the work reported here was to determine which molecular form is actively pumped out by P-gp (the neutral form, the protonated form, or both), and if both, the relative efficiencies of pumping. We used spectrofluorometric methods to determine the efflux of anthracyclines in K562/Adr cells, at different intracellular and extracellular pH levels. Using 3 H -deamino, 3 H -hydroxyl doxorubicin (OH-DOX), which is permanently neutral, we first verified that our methodologies were accurate and that the P-gp-mediated efflux of OH-DOX would not depend on the pH being in the range 6.6±8.4. The P-gp-mediated efflux of daunorubicin (DNR) and 3 H -hydroxy-4-amino (WP608) was determined at different pH values. These two drugs were chosen because: (a) the lipophilicity of the neutral forms of these two molecules is so similar that any difference in the P-gp-mediated efflux cannot be assigned to lipohilicity variation, and (b) their pK a values are different (8.4 and 7.7 for DNR and WP608, respectively), which makes it easy to obtain a large variation in the proportions of the neutral and positively charged forms. Our data show that both forms are recognized by Pgp but the neutral form is pumped about three times more efficiently than the charged form. This is corroborated by results showing the active efflux (checked at pH i 7.3 only) of five other anthracycline containing a basic center. We interpret these data to mean that: (a) the positive charge of anthracycline is not a necessary requirement for P-gp recognition, but that (b) the presence of a protonable basic nitrogen facilitates the processing of these compounds by MDR efflux system.
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