Interactions of antiviral agent PMEG and its prodrug with selected SLC transporters

J Mandikova1, M Volkova1, Z Novy1, P Pavek1, Z Janeba2, F Trejtnar1. 1Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Department of Pharmacology and Toxicology, Czech Republic, 2Academy of Sciences of the Czech Republic, Institute of Organic Chemistry and Biochemistry, Czech Republic

Acyclic nucleoside phosphonates (ANPs) are nucleotide analogs with significant antiviral, cytostatic and antiproliferative activities. ANPs are eliminated predominantly by the kidney and may be involved in renal failure and nephrotoxicity. Solute carrier family (SLC) drug transporters located in the renal proximal tubular cells have been shown to interact with numerous frequently prescribed drugs. Although it is known that the antiviral agent 9-(2-phosphonylmethoxyethyl)guanine (PMEG) is a substrate for the human organic anion transporter 1 (hOAT1), there is no information regarding other SLC transporters abundantly expressed in the kidney such as organic cation transporters (OCTs) or nucleoside transporters (NTs). The purpose of our study was to investigate the affinity of antiviral agent PMEG and its N6 substituted prodrug 9-(2-phosphonylmethoxyethyl)-N6-cyclopropyl-2,6-diaminopurine (cPrPMEDAP) to hOAT1, hOCT2 and hCNT3 and compare the results with other antiviral tenofovir and its disoproxil fumarate prodrug (TDF) in an appropriate cellular in vitro model. Human cervical cancer cell line (HeLa) transiently transfected with hOAT1 and Madin-Darby Canine Kidney II cells (MDCKII) transiently transfected with hOCT2 or hCNT3 were used for the experiments. The transporter overexpression was confirmed by western blot analysis. The cytotoxic effect of the studied substances was proved using a standard MTS assay. In functional accumulation studies, all three used cellular models (HeLahOAT1, MDCKIIhOCT2 or MDCKIIhCNT3) showed a strong uptake of specific substrates [3H]para-aminohippuric acid, [3H]1-methyl-4-phenylpyridinium or [3H]uridine respectively, compared with control cells transiently transfected with empty vector. These findings confirmed proper functioning of our cellular models. In the affinity transport studies, the rate of inhibition of intracellular accumulation of tritium labeled prototypical substrate was induced by gradually increasing concentration of the tested compound. The found affinity of PMEG to hOAT1 transporter was much lower than the affinity of tenofovir. For the comparison, prodrugs of tested substances showed either no (cPrPMEDAP) or very low affinity (TDF) to hOAT1. The affinity value of tenofovir to hOAT1 was approximately ten times higher than the affinity of its ester prodrug TDF. In affinity transport studies we did not observe any significant interaction of tested compounds with transporters in the transiently transfected MDCKIIhOCT2 and MDCKIIhCNT3 models. The founded cytotoxicity of tested antivirals was very low. Although the cytotoxicity curve didn´t allow the calculation of the standard cytotoxicological parameter IC50, PMEG substance appeared to be more cytotoxic than its prodrug cPrPMEDAP. In conclusion, we demonstrated significant differences in the interaction on hOAT1 between the tested antiviral drugs and their prodrugs. The interaction of the studied drugs and prodrugs with hOCT2 and hCNT3 was not proved. The study was supported by Charles University in Prague (Project SVV 265003), grant GAUK No. 360811/FaF/C-LEK and grant IGA MZ No. NT12398-4/2011.