Pharmacogenetic studies of membrane transporters relevant to anti-cancer drug therapy.

摘要

Membrane transporters are important determinants of drug disposition and response. The goals of this dissertation are to examine the interaction of membrane transporters with anti-cancer drugs, in particular cladribine, fludarabine, and methotrexate, and to analyze their genetic and functional diversity. We showed that the human concentrative nucleoside transporter CNT3 (SLC28A3), which transports nucleoside analogs, is under negative selective pressure. The rare variant, protein reference sequence (p.) Gly367Arg, exhibited reduced transport of nucleosides, whereas common non-synonymous variants of CNT3 exhibited no change in their transport of nucleosides, suggesting that common coding region variants of CNT3 do not contribute to common variation in the efficacy of cladribine and fludarabine. Methotrexate exhibits variable pharmacokinetics and renal toxicity. We showed that the human organic anion transporting polypeptide, OATP1A2 (SLCO1A2 ) transports methotrexate in a pH-dependent manner. Since the human organic anion transporter OAT3 (SLC22A8) and OATP1A2 are expressed in the renal tubule, they may play a role in the elimination and nephrotoxicity of methotrexate. We identified four protein-altering variants in OATP1A2 and six in OAT3 that had altered transport of methotrexate, suggesting that these variants may contribute to variation in the renal disposition and toxicity of methotrexate. We characterized the mechanism of transport and identified critical amino acid residues in OATP1A2 that are responsible for methotrexate uptake. The enhanced transport at acidic pH resulted from increased substrate affinity and did not depend on the proton gradient. Conserved cationic residues in OATP1A2 were important in methotrexate recognition and uptake. Lastly, we initiated collaborative studies to examine the characteristics of methotrexate elimination in patients with acute lymphoblastic leukemia (ALL). Methotrexate pharmacokinetics were non-linear and highly variable. Increased exposure to methotrexate was associated with delayed elimination. In future studies, we will genotype ALL patients for 11 variants and 16 haplotypes in 6 kidney transporters to analyze the contribution of kidney transporters to variation in MTX pharmacokinetics and renal toxicity. These studies have enhanced our understanding of how membrane transporters interact with anti-cancer drugs and, thereby, control their systemic and tissue levels. Genetic variation in these transporters may contribute to variation in the efficacy and toxicity of anti-cancer drugs.