Human multidrug and toxin extrusion 1 (hMATE1) is a major candidate for being the molecular identity of organic cation/proton (OC/H+) exchange activity in the luminal membrane of renal proximal tubules (RPT). Although physiological function of hMATE1 supports luminal OC efflux, the kinetics of hMATE1-mediated OC transport have typically been characterized through measurement of uptake i.e., the interaction between outward-facing hMATE1 and OCs. To examine kinetics of hMATE1-mediated transport in a more physiologically relevant direction i.e., an interaction between inward-facing hMATE1 and cytoplasmic substrates, I measured the time course of hMATE1-mediated efflux of the prototypic MATE1-substrate, [3H]1-methyl-4-phenylpyridinium ([3H]MPP), under a variety of conditions, including different values for intra- and extracellular pH, from CHO cells that stably expressed hMATE1. I showed that an IC50/Ki for interaction between extracellular H+ and outward-facing hMATE1 determined from conventional uptake experiments [12.9 ± 1.23 nM (pH 7.89); n = 9] and from the efflux protocol [14.7 ± 3.45 nM (pH 7.83); n = 3] were not significantly different (P = 0.6). To test a hypothesis that H+ interacts symmetrically with each face of hMATE1, kinetics of interaction between intracellular H+ and inward-facing hMATE1 were determined using the efflux protocol. The IC50 for interaction with H+ was 11.5 nM (pH 7.91), consistent with symmetrical interactions of H+ with the inward-facing and outward-facing aspects of hMATE1. The efflux protocols demonstrated in this study are a potential means to examine kinetics at cytoplasmic face of hMATE1 and also a practical tool to screen uptake of substrates at extracellular face of hMATE1.

Insects accumulate various xenobiotics and toxic molecules through feeding and environmental exposure. This study examines the toxicology and regulation of a class of toxic molecules, organic cations, in Drosophila melanogaster. The results of this thesis demonstrate that transepithelial tetraethylammonium (TEA) secretion across the main segment of the Malpighian tubules is increased in response to diuretic factors. Both cAMP and cGMP, which increase transepithelial potential (TEP), as well as tyramine and LK-1, which decrease TEP, all enhanced TEA secretion. Both inc~eases and decreases ofTEP may enhance proton transport into the lumen of the tubule thus increasing the rate of organic cation/proton exchange across the apical membrane. These findings suggest that factors previously referred to as diuretic factors may in fact :let primarily or secondarily as stimulants of organic cation excretion. Haemolymrh concentrations of TEA increased linearly with the concentration of TEA in the diet and declined rapidly upon transfer of the larvae to TEA-free diet. The rate of decline was reduced by slowing the metabolic rate or by the addition of cimetidine to a diet containing TEA. Although larvae tolerated high levels of TEA in the diet, mortality increased when TEA was combined with either quinidine or cimetidine. It is suggested that inhibition of TEA transport by cimetidine or quinidine results in prolonged exposure to higher levels of TEA in the haemolymph and a consequent increase in toxicity. Surprisingly, TEA flux and fluid secretion rate were both reduced in Malpighian tubules isolated from adult flies raised on TEA-enriched diet. This suggests that the high concentration of TEA in the diet produced a non-lethal yet deleterious effect on the Malpighian tubules of Drosophila. / Thesis / Master of Science (MS)

Organic cations (OCs) make up ~40% of all prescribed drugs and renal secretion plays a major role in clearing these (and other OCs), from the plasma. The active and rate-limiting step of renal OC secretion is mediated by luminal OC/H+ exchange, the molecular basis of which is suspected to involve two homologous transport proteins, Multidrug And Toxin Extruders 1&2-K (MATE1 and MATE2-K). This study has two aims to resolve outstanding issues dealing with the mechanism of MATE-mediated OC transport: (Aim 1) develop predictive models of ligand interaction with hMATE1; (Aim 2) establish the kinetic mechanism(s) of ligand interaction with MATE transporters and the extent to which inhibitory ligands serve as transported substrates of MATE transporters. Transport was measured using human MATE1 and MATE2-K stably expressed in Chinese Hamster Ovary cells. Both MATEs had similar affinities for the prototypic OC substrate, 1-methyl-4-phenylpyridinium (MPP), and had overlapping selectivity for most of the test inhibitors. The IC50 values for 59 structurally diverse inhibitory ligands were used to generate a common features (HIPHOP) pharmacophore and three quantitative pharmacophores for hMATE1 (each displaying a significant correlation between predicted and measured IC50 values). The models identified (i) structural features that influence ligand interaction with hMATE1, including hydrophobic regions, H-bond donor and acceptor sites and an ionizable feature; and (ii) novel high affinity inhibitors of MATE-mediated transport from 13 new drug classes. Whereas metformin and creatinine were shown to be competitive inhibitors of MPP, the inhibition of MATE1-mediated MPP transport produced by pyrimethamine (PYR) and related analogs was not competitive but, instead, had a "linear, mixed-type" inhibitory profile suggestive of a MATE binding surface rather than a singular binding site. "Competitive exchange diffusion" showed that selected inhibitory ligands (including quinidine, caffeine, and the organic anion, PAH) also serve as transported substrates for MATE1. In conclusion, these data are consistent with the presence of a MATE binding surface with multiple, non-overlapping binding sites that can display different kinetic interactions with structurally distinct substrates. The creation of hMATE1 pharmacophores offers insight into development and interpretation of predictive models of drug-drug interaction in the kidney.

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.

The objective of this study was to investigate the role of organic cation transporters (OCTs) in the uptake of hydrophilic drugs into the olfactory bulb and subsequently to the brain. Two OCT2 substrates, amantadine and cimetidine were used as model drugs for this purpose. Bovine nasal explants (olfactory and respiratory tissue) were used as an in vitro model for preliminary screening to identify the role of transporters involved in the uptake of drug across these tissues. It was observed from both PCR and immunohistochemistry that OCTs, OCT2, OCTN1 and OCTN2 were present in the bovine respiratory and olfactory mucosa. Transport studies of amantadine in the presence and absence of OCT2 and OCTN2 inhibitors indicated that both these transporters play a role in the transport of amantadine across the bovine respiratory mucosa, whereas transport across the olfactory mucosa was predominantly via OCT2. This was followed by in vivo studies in rats where the blood, striatum and olfactory bulb concentrations of amantadine were determined following intranasal and intra-arterial administration. Shortly after nasal administration, the olfactory bulb concentrations exceeded the concentrations in the striatum suggesting the olfactory pathway to be the major route of uptake. Co-administration of the drug with an OCT2 inhibitor intranasally showed statistically significant reductions in the brain uptake of amantadine. A synergistic inhibitory effect on amantadine uptake was observed with the combined inhibition OCT2 and OCTN2. Additionally, the CNS exposure of these drugs following intranasal administration in the presence and absence of the OCT inhibitors was evaluated using the ratio of the free drug concentrations in the brain compared to plasma. While the plasma concentration profiles were similar both in the presence and absence of inhibition, the free drug ratios were highest when no inhibitor was included. Additionally similiar in vivo studies were also carried out for a second model drug, cimetidine, where cimetidine uptake into the rat brain was found to be significantly reduced in the presence of the OCT2 inhibitor, pentamidine. This demonstrates that there was a greater CNS exposure to each drug when OCT transporters were active, confirming their role in their direct CNS distribution from the nasal cavity to the brain. The results of this study suggest that OCT substrates might be good candidates for the delivery to the brain via the olfactory route.

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京都大学 / 新制・論文博士 / 博士(工学) / 乙第13542号 / 論工博第4205号 / 新制||工||1984(附属図書館) / (主査)教授 杉野目 道紀, 教授 大江 浩一, 教授 松原 誠二郎 / 学位規則第4条第2項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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