Polymorphisms in organic cation transporters SLC22A4, SLC22A23 and IBD5 locus have been associated with pathogenesis of inflammatory bowel disease (IBD). We sought to investigate the association of polymorphisms in these genes to IBD risk in a Canadian population, subclone and express human SLC22A23 gene to determine the localization in the cell. DNA samples from 160 patients with Crohn´s disease (CD), 149 patients with ulcerative colitis (UC) and 142 healthy controls were genotyped by PCR-RFLP analysis or TaqMan system. Gateway® recombination technology was used to transform and express SLC22A23 gene in HEK 293 cell line. Polymorphisms in the IBD5 locus rs17622208-AA genotype and rs11739135-CC genotype increase the risk of CD. Moreover, carriers of SLC22A23 polymorphisms rs4959235-TT genotype and rs9503518-GG genotype increase dramatically the risk of UC. We confirm that SLC22A23 polymorphisms are important in the pathogenesis of IBD and they can ultimately be used as biomarkers of the disease risk.

Polymorphisms in organic cation transporters SLC22A4, SLC22A23 and IBD5 locus have been associated with pathogenesis of inflammatory bowel disease (IBD). We sought to investigate the association of polymorphisms in these genes to IBD risk in a Canadian population, subclone and express human SLC22A23 gene to determine the localization in the cell. DNA samples from 160 patients with Crohn´s disease (CD), 149 patients with ulcerative colitis (UC) and 142 healthy controls were genotyped by PCR-RFLP analysis or TaqMan system. Gateway® recombination technology was used to transform and express SLC22A23 gene in HEK 293 cell line. Polymorphisms in the IBD5 locus rs17622208-AA genotype and rs11739135-CC genotype increase the risk of CD. Moreover, carriers of SLC22A23 polymorphisms rs4959235-TT genotype and rs9503518-GG genotype increase dramatically the risk of UC. We confirm that SLC22A23 polymorphisms are important in the pathogenesis of IBD and they can ultimately be used as biomarkers of the disease risk.

Studies in this dissertation were conducted to explore the roles of organic cation transporters (OCTs) in the disposition of N-butylpyridinium Chloride (NBuPy-Cl) and structurally related ILs. Following a single i.v. dose to rats, the blood concentration of NBuPy-Cl and 1-butyl-1-methylpyrrolidinium chloride (BmPy-Cl) decreased in a biphasic manner with a clearance of 3.3 and 7 ml/min, respectively. More than 84% of dosed compounds were excreted in the urine. Depending on the vehicle, the dermal absorption of BmPy-Cl and NBuPy-Cl (5 mg/kg, 125 μg/cm²) was 10-35% at 96 h. Following a single oral (50 mg/kg) administration to rats, the maximum blood concentrations of both ILs were reached in less than 90 min in rats. Most of the orally dosed NBuPy-Cl (62-68 %) was excreted in the urine in 72 h. However, more of the dosed BmPy-Cl was eliminated in the feces Its oral bioavailability was only 47%. The elimination differences between BmPy-Cl and NBuPy-Cl were not altered by the size (0.5, 5, or 50 mg/kg) or frequency (1 or 5 administrations) of oral doses. In all urine and blood samples, only parent compounds were detected. Co-administration of NBuPy-Cl and inulin intravenously to rats revealed that the clearance of NBuPy-Cl exceeded the rat glomerular filtration rate, suggesting a renal secretion processing. The in vitro transport studies demonstrated that NBuPy-Cl, BmPy-Cl and 1-butyl-3-methylimidazolium chloride are substrates (Kt, 9~277 μM), as well as inhibitors (IC₅₀: 0.2~7.5 μM), of rOCT1/2 and hOCT2. Their inhibitory effects increased dramatically with increasing the alkyl chain length. The IC₅₀ values were 0.1, 3.8, 14 and 671 μM (hexyl-, butyl-, ethyl-pyridinium and pyridinium chloride) for rOCT2 mediated metformin transport. Similar structurally related inhibitory kinetics were observed for rOCT1 and hOCT2. In vivo co-administration of NBuPy-Cl prolonged the plasma half-life and reduced renal clearance of the diabetic drug, metformin. In summary, BmPy-Cl and NBuPy-Cl are partially absorbed from gastrointestinal tract. The present in blood is eliminated rapidly in the urine as parent, by renal filtration and OCT-mediated secretion. ILs also compete with other substrates of OCTs and have the potential to alter their pharmacokinetic profiles.

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The organic cation transporters (OCTs) play a critical role in the absorption, distribution and elimination of many drugs, hormones, herbal medicines, and environmental toxins. Given the broad substrate specificity of OCTs, they fall victim to the high susceptibility for contributing to harmful drug-drug interactions. Further defining how human (h)OCTs mechanistically bind to its broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCTs. The goal of the current study was to elucidate the critical amino acid residues for transporter-substrate binding interactions on human (h)OCT1 and 2 utilizing in silico molecular modeling techniques (homology modeling and automated docking), as well as in vitro mutagenesis and kinetic transport experiments. Three-dimensional homology models were generated for hOCT1 and 2 using Piriformospora indica phosphate transporter (PiPT) serving as template. A putative binding pocket was identified and used to dock the prototypical substrate MPP+. Docking studies revealed five residues for each transporter (hOCT1 and hOCT2) that may be critical for substrate-transporter interactions. The in silico data was used to guide subsequent in vitro site-directed mutagenesis and kinetic analysis. Four hOCT1 mutants (Gln241Lys, Thr245Lys, Tyr361Ala, and Glu447Lys) and three hOCT2 mutants (Gln242Lys, Tyr362Phe, and Tyr362Ala) showed complete loss of MPP+ transporter activity. Decreased affinity for MPP+ was observed for Phe244Ser and Thr245Ser in hOCT1, and Tyr245Ala in hOCT2. All amino acid residues highlighted in the in vitro experiments may be potentially critical for substrate-transporter interactions particularly Tyr361, Phe244 and Thr245 in hOCT1; and Tyr362 and Tyr245 in hOCT2. Docking of known structurally divergent hOCT1 and hOCT2 substrates revealed similar binding interactions as that identified for MPP+, albeit with some unique residues, suggesting the presence of a large central cavity within both transporters. Through the combination of in silico and in vitro experiments, a putative binding pocket was defined and several residues important for substrate-transporter interaction were identified and verified for hOCT1 and hOCT2. Further defining how OCTs biochemically interact with their broad array of substrates will provide significant insight to the understanding and prediction of drug-drug interactions in polypharmacy patients and the advancement of future rational drug design for therapeutics targeting OCT1 and OCT2.

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