Functional and Pharmacological Comparison of Human, Mouse, and Rat Organic Cation Transporter 1 toward Drug and Pesticide Interaction.

Extrapolation from animal to human data is not always possible, because several essential factors, such as expression level, localization, as well as the substrate selectivity and affinity of relevant transport proteins, can differ between species. In this study, we examined the interactions of drugs and pesticides with the clinically relevant organic cation transporter hOCT1 (SLC22A1) in comparison to the orthologous transporters from mouse and rat. We determined Km-values (73 ± 7, 36 ± 13, and 57 ± 5 µM) of human, mouse and rat OCT1 for the commonly used substrate 1-methyl-4-phenylpyridinium (MPP) and IC50-values of decynium22 (12.1 ± 0.8, 5.3 ± 0.4, and 10.5 ± 0.4 µM). For the first time, we demonstrated the interaction of the cationic fungicides imazalil, azoxystrobin, prochloraz, and propamocarb with human and rodent OCT1. Drugs such as ketoconazole, clonidine, and verapamil showed substantial inhibitory potential to human, mouse, and rat OCT1 activity. A correlation analysis of hOCT1 versus mouse and rat orthologs revealed a strong functional correlation between the three species. In conclusion, this approach shows that transporter interaction data are in many cases transferable between rodents and humans, but potential species differences for other drugs and pesticides could not be excluded, though it is recommendable to perform functional comparisons of human and rodent transporters for new molecular entities.

Assay Conditions Influence Affinities of Rat Organic Cation Transporter 1: Analysis of Mutagenesis in the Modeled Outward-Facing Cleft by Measuring Effects of Substrates and Inhibitors on Initial Uptake.

The effects of mutations in the modeled outward-open cleft of rat organic cation transporter 1 (rOCT1) on affinities of substrates and inhibitors were investigated. Human embryonic kidney 293 cells were stably transfected with rOCT1 or rOCT1 mutants, and uptake of the substrates 1-methyl-4-phenylpyridinium+ (MPP+) and tetraethylammonium+ (TEA+) or inhibition of MPP+ uptake by the nontransported inhibitors tetrabutylammonium+ (TBuA+), tetrapentylammonium+ (TPeA+), and corticosterone was measured. Uptake measurements were performed on confluent cell layers using a 2-minute incubation or in dissociated cells using incubation times of 1, 5, or 10 seconds. With both methods, different apparent Michaelis-Menten constant (Km) values, different IC50 values, and varying effects of mutations were determined. In addition, varying IC50 values for the inhibition of MPP+ uptake and varying effects of mutations were obtained when different MPP+ concentrations far below the apparent Km value were used for uptake measurements. Eleven mutations were investigated by measuring initial uptake in dissociated cells and employing 0.1 µM MPP+ for uptake during inhibition experiments. Altered affinities for substrates and/or inhibitors were observed when Phe160, Trp218, Arg440, Leu447, and Asp475 were mutated. The mutations resulted in changes of apparent Km values for TEA+ and/or MPP+ Mutation of Trp218 and Asp475 led to altered IC50 values for TBuA+, TPeA+, and corticosterone, whereas the mutation of Phe160 and Leu447 changed the IC50 values for two inhibitors. Thereby amino acids in the outward-facing conformation of rOCT1 could be identified that interact with structurally different inhibitors and probably also with different substrates.

Tropane alkaloids as substrates and inhibitors of human organic cation transporters of the SLC22 (OCT) and the SLC47 (MATE) families.

Tropane alkaloids and their derivatives are anticholinergic drugs with narrow therapeutic range. Here we characterize the organic cation transporters from the SLC22 (OCT1, OCT2, and OCT3) and the SLC47 families (MATE1 and MATE2-K) as potential mediators of the renal and extra-renal excretion, the two major roads of elimination of these substances. All analyzed compounds inhibited and the quaternary amine derivatives ipratropium and trospium were strongly transported by OCTs and MATEs. Overexpression of OCTs or MATEs in HEK293 cells resulted in an up to 63-fold increase in the uptake of ipratropium (Km of 0.32 µm to OCT2 and Vmax of 3.34 nmol×mg protein-1×min-1 to MATE1). The transcellular transport of ipratropium was 16-fold higher in OCT2-MATE1 and 10-fold higher in OCT1-MATE1 overexpressing compared to control MDCKII cells. Genetic polymorphisms in OCT1 and OCT2 affected ipratropium uptake and clinically relevant concentration of ondansetron and pyrithiamine inhibited ipratropium uptake via MATEs by more than 90%. This study suggests that OCT1, OCT2 and MATEs may be strongly involved in the renal and extra-renal elimination of ipratropium and other quaternary amine alkaloids. These substances have a notoriously narrow therapeutic range and the drug-drug interactions suggested here should be further critically evaluated in humans.

Pharmacology of novel synthetic stimulants structurally related to the "bath salts" constituent 3,4-methylenedioxypyrovalerone (MDPV).

There has been a dramatic rise in the abuse of synthetic cathinones known as "bath salts," including 3,4-methylenedioxypyrovalerone (MDPV), an analog linked to many adverse events. MDPV differs from other synthetic cathinones because it contains a pyrrolidine ring which gives the drug potent actions as an uptake blocker at dopamine and norepinephrine transporters. While MDPV is now illegal, a wave of "second generation" pyrrolidinophenones has appeared on the market, with α-pyrrolidinovalerophenone (α-PVP) being most popular. Here, we sought to compare the in vitro and in vivo pharmacological effects of MDPV and its congeners: α-PVP, α-pyrrolidinobutiophenone (α-PBP), and α-pyrrolidinopropiophenone (α-PPP). We examined effects of test drugs in transporter uptake and release assays using rat brain synaptosomes, then assessed behavioral stimulant effects in mice. We found that α-PVP is a potent uptake blocker at dopamine and norepinephrine transporters, similar to MDPV. α-PBP and α-PPP are also catecholamine transporter blockers but display reduced potency. All of the test drugs are locomotor stimulants, and the rank order of in vivo potency parallels dopamine transporter activity, with MDPV > α-PVP > α-PBP > α-PPP. Motor activation produced by all drugs is reversed by the dopamine receptor antagonist SCH23390. Furthermore, results of a functional observational battery show that all test drugs produce typical stimulant effects at lower doses and some drugs produce bizarre behaviors at higher doses. Taken together, our findings represent the first evidence that second generation analogs of MDPV are catecholamine-selective uptake blockers which may pose risk for addiction and adverse effects in human users. This article is part of the Special Issue entitled 'CNS Stimulants'.

Effect of cimetidine on pentamidine induced hyperglycemia in rats.

The antiprotozoal agent pentamidine, used for the treatment of Pneumocystis jirovecii pneumonia (PCP), is known to cause abnormalities in blood glucose homeostasis, such as hypoglycemia and hyperglycemia. Pentamidine has been reported to be a substrate of organic cation transporter 1 (OCT1). We investigated the combination effects of cimetidine, an OCT1 inhibitor, on the pharmacokinetics of pentamidine and on pentamidine-induced hyperglycemia. Pentamidine was infused intravenously to rats for 20 min at a dose of 7.5 or 15 mg/kg and serum samples were obtained periodically. The serum concentration of glucose did not change significantly after pentamidine infusion at 7.5mg/kg, while it increased with pentamidine at 15 mg/kg, and the maximal concentration of glucose was 167 ± 36 mg/dl, 30 min after the start of pentamidine infusion. Cimetidine (50mg/kg) enhanced the pentamidine-induced elevation of glucose concentration and the maximal concentration of glucose was 208 ± 33 mg/dl in the pentamidine 15 mg/kg treated group. Cimetidine combination significantly reduced total body clearance of pentamidine and increased pentamidine concentrations in the liver, kidneys, and lungs. A significant correlation was found between changes in serum glucose concentrations and serum concentrations of pentamidine 30 min after the start of pentamidine infusion. These results suggest that the hyperglycemic effect of pentamidine is dependent on the concentration of pentamidine and can be enhanced by cimetidine combination.

Characterization of the inhibitory effects of N-butylpyridinium chloride and structurally related ionic liquids on organic cation transporters 1/2 and human toxic extrusion transporters 1/2-k in vitro and in vivo.

Ionic liquids (ILs) are a class of salts that are expected to be used as a new source of solvents and for many other applications. Our previous studies revealed that selected ILs, structurally related organic cations, are eliminated exclusively in urine as the parent compound, partially mediated by renal transporters. This study investigated the inhibitory effects of N-butylpyridinium chloride (NBuPy-Cl) and structurally related ILs on organic cation transporters (OCTs) and multidrug and toxic extrusion transporters (MATEs) in vitro and in vivo. After Chinese hamster ovary cells expressing rat (r) OCT1, rOCT2, human (h) OCT2, hMATE1, or hMATE2-K were constructed, the ability of NBuPy-Cl, 1-methyl-3-butylimidazolium chloride (Bmim-Cl), N-butyl-N-methylpyrrolidinium chloride (BmPy-Cl), and alkyl substituted pyridinium ILs to inhibit these transporters was determined in vitro. NBuPy-Cl (0, 0.5, or 2 mg/kg per hour) was also infused into rats to assess its effect on the pharmacokinetics of metformin, a substrate of OCTs and MATEs. NBuPy-Cl, Bmim-Cl, and BmPy-Cl displayed strong inhibitory effects on these transporters (IC(50) = 0.2-8.5 µM). In addition, the inhibitory effects of alkyl-substituted pyridinium ILs on OCTs increased dramatically as the length of the alkyl chain increased. The IC(50) values were 0.1, 3.8, 14, and 671 µM (hexyl-, butyl-, and ethyl-pyridinium and pyridinium chloride) for rOCT2-mediated metformin transport. Similar structurally related inhibitory kinetics were also observed for rOCT1 and hOCT2. The in vivo coadministration study revealed that NBuPy-Cl reduced the renal clearance of metformin in rats. These results demonstrate that ILs compete with other substrates of OCTs and MATEs and could alter the in vivo pharmacokinetics of such substrates.

The transmembrane transport of metformin by osteoblasts from rat mandible.

Previous studies have demonstrated that metformin, one of systemic antihyperglycemic drugs, can slow bone loss caused by diabetes mellitus and has an osteogenic action on osteoblasts in vitro. It is tempting to speculate that metformin would be transported into bone tissues around dental implant by topical administration to improve the bone-implant contact in diabetic patients. In this study, the osteoblasts from rat mandible were cultured with 5.5 mM (control) or 16.5 mM d-glucose, then the uptake of metformin by osteoblasts was detected with high performance liquid chromatography (HPLC). Rat organic cation transporter (rOct) expression was characterized by immunocytochemistry, RT-PCR and Western blotting. It was found that, the uptake of metformin was saturable, Na(+)-dependent, affected by extracellular pH and inhibited by both phenformin and cimetidine (an inhibitor of Octs). rOct1 but no rOct2 was expressed extensively in osteoblasts and the protein level of rOct1 could be up-regulated by metformin. The uptake of metformin and phosphorylated-rOct1 at hyperglycaemic cell culture (16.5 mM d-glucose) significantly increased versus 5.5 mM control (p < 0.05). In conclusion, rat osteoblasts have the ability to transport the metformin intra-cellularly, the uptake of metformin by osteoblasts is a secondary active transportation mediated by rOct1 and high-glucose can improve the uptake of metformin by osteoblasts through phosphorylation of rOct1. The current results suggest that metformin could be used for dental implant topically in type 2 diabetic patients to increase the bone formation, therefore, to enhance the success rate of dental implants clinically.

Is the inhibition of nicotinic acetylcholine receptors by bupropion involved in its clinical actions?

In this mini review we will focus on those molecular and cellular mechanisms exerted by bupropion (BP), ultimately leading to the antidepressant and anti-nicotinic properties described for this molecule. The main pharmacological mechanism is based on the fact that BP induces the release as well as inhibits the reuptake of neurotransmitters such as a dopamine (DA) and norepinephrine (NE). Additional mechanisms of action have been also determined. For example, BP is a noncompetitive antagonist (NCA) of several nicotinic acetylcholine receptors (AChRs). Based on this evidence, the dual antidepressant and anti-nicotinic activity of BP is currently considered to be mediated by its stimulatory action on the DA and NE systems as well as its inhibitory action on AChRs. Considering the results obtained in the archetypical mouse muscle AChR, a sequential mechanism can be hypothesized to explain the inhibitory action of BP on neuronal AChRs: (1) BP first binds to AChRs in the resting state, decreasing the probability of ion channel opening, (2) the remnant fraction of open ion channels is subsequently decreased by accelerating the desensitization process, and (3), BP interacts with a binding domain located between the serine (position 6') and valine (position 13') rings that is shared with the NCA phencyclidine and other tricyclic antidepressants. This new evidence paves the way for further investigations using AChRs as targets for the action of safer antidepressants and novel anti-addictive compounds.

Purification and functional reconstitution of the rat organic cation transporter OCT1.

The rat organic cation transporter rOCT1 with six histidine residues added to the C-terminus was expressed in Sf9 insect cells, and expression of organic cation transport was demonstrated. To purify rOCT1 protein, Sf9 cells were lysed with 1% (w/v) CHAPS [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate], centrifuged, and subjected to sequential affinity chromatography using lentil-lectin Sepharose and nickel(II)-charged nitrilotriacetic acid-agarose. This procedure yielded approximately 70 microg of purified rOCT1 protein from 10 standard culture plates. Using a freeze-thaw procedure, purified rOCT1 was reconstituted into proteoliposomes formed from phosphatidylcholine, phosphatidylserine, and cholesterol. Proteoliposomes exhibited uptake of [3H]-1-Methyl-4-phenylpyridinium ([3H]MPP) that was inhibited by quinine and stimulated by an inside-negative membrane potential. MPP uptake was saturable with an apparent K(m) of 30 +/- 17 microM. MPP uptake (0.1 microM) was inhibited by tetraethylammonium, tetrabutylammonium, and tetrapentylammonium with IC50 values of 197 +/- 11, 19 +/- 1, and 1.8 +/- 0.03 microM, respectively. With membrane potential clamped to 0 mV using valinomycin in the presence of 100 mM potassium on both sides of the membrane, uptake of 0.1 microM MPP was trans stimulated 3-fold by 2.5 mM intracellular choline, and efflux of 0.1 microM MPP was trans stimulated 4-fold by 9.5 mM extracellular choline. The data show that rOCT1 is capable and sufficient to mediate transport of organic cations. The observed trans stimulation under voltage-clamp conditions shows that rOCT1 operates as a transporter rather than a channel. Purification and reconstitution of functional active rOCT1 protein is an important step toward the biophysical characterization and crystallization.