The methylimidazolium ionic liquid M8OI is a substrate for OCT1 and p-glycoprotein-1 in rat.

The methylimidazolium ionic liquid M8OI was recently found to be present in both the environment and man. In this study, M8OI disposition and toxicity were examined in an established rat progenitor-hepatocyte model. The progenitor B-13 cell was approx. 13 fold more sensitive to the toxic effects of M8OI than the hepatocyte B-13/H cell. However, this difference in sensitivity was not associated with a difference in metabolic capacities. M8OI toxicity was significantly decreased in a dose-dependent manner by co-addition of the OCT1 (SLC22A1) inhibitor clonidine, but not by OCT2 or OCT3 inhibitors in B-13 cells. M8OI toxicity was also dose-dependently increased by the co-addition of p-glycoprotein-1 (ABCB1B, multi drug resistant protein 1 (MDR1)) substrates/inhibitors. Excretion of B-13-loaded fluorophore Hoechst 33342 was also inhibited by the p-glycoproteins substrate cyclosporin A and by M8OI in a dose-dependent manner. Comparing levels of OCT and p-glycoprotein transcripts and proteins in B-13 and B-13/H cells suggest that the lower sensitivity to M8OI in B-13/H cells is predominantly associated with their higher expression of p-glycoprotein-1. These data together therefore suggest that a determinant in M8OI toxicity in rats is the expression and activity of the p-glycoprotein-1 transporter.

The influence of metformin transporter gene SLC22A1 and SLC47A1 variants on steady-state pharmacokinetics and glycemic response.

Interindividual variation is important in the response to metformin as the first-line therapy for type-2 diabetes mellitus (T2DM). Considering that OCT1 and MATE1 transporters determine the metformin pharmacokinetics, this study aimed to investigate the influence of SLC22A1 and SLC47A1 variants on the steady-state pharmacokinetics of metformin and the glycemic response. This research used the prospective-cohort study design for 81 patients with T2DM who received 500 mg metformin twice a day from six primary healthcare centers. SLC22A1 rs628031 A>G (Met408Val) and Met420del genetic variants in OCT1 as well as SLC47A1 rs2289669 G>A genetic variant in MATE1 were examined through the PCR-RFLP method. The bioanalysis of plasma metformin was performed in the validated reversed-phase HPLC-UV detector. The metformin steady-state concentration was measured for the trough concentration (Cssmin) and peak concentration (Cssmax). The pharmacodynamic parameters of metformin use were the fasting blood glucose (FBG) and glycated albumin (GA). Only SLC22A1 Met420del alongside estimated-glomerular filtration rate (eGFR) affected both Cssmax and Cssmin with an extremely weak correlation. Meanwhile, SLC47A1 rs2289669 and FBG were correlated. This study also found that there was no correlation between the three SNPs studied and GA, so only eGFR and Cssmax influenced GA. The average Cssmax in patients with the G allele of SLC22A1 Met408Val, reaching 1.35-fold higher than those with the A allele, requires further studies with regard to metformin safe dose in order to avoid exceeding the recommended therapeutic range.

Amino acids in transmembrane helix 1 confer major functional differences between human and mouse orthologs of the polyspecific membrane transporter OCT1.

Organic cation transporter 1 (OCT1) is a membrane transporter that affects hepatic uptake of cationic and weakly basic drugs. OCT1 transports structurally highly diverse substrates. The mechanisms conferring this polyspecificity are unknown. Here, we analyzed differences in transport kinetics between human and mouse OCT1 orthologs to identify amino acids that contribute to the polyspecificity of OCT1. Following stable transfection of HEK293 cells, we observed more than twofold differences in the transport kinetics of 22 out of 28 tested substrates. We found that the ß2-adrenergic drug fenoterol was transported with eightfold higher affinity but at ninefold lower capacity by human OCT1. In contrast, the anticholinergic drug trospium was transported with 11-fold higher affinity but at ninefold lower capacity by mouse Oct1. Using human-mouse chimeric constructs and site-directed mutagenesis, we identified nonconserved amino acids Cys36 and Phe32 as responsible for the species-specific differences in fenoterol and trospium uptake. Substitution of Cys36 (human) to Tyr36 (mouse) caused a reversal of the affinity and capacity of fenoterol but not trospium uptake. Substitution of Phe32 to Leu32 caused reversal of trospium but not fenoterol uptake kinetics. Comparison of the uptake of structurally similar ß2-adrenergics and molecular docking analyses indicated the second phenol ring, 3.3 to 4.8 Å from the protonated amino group, as essential for the affinity for fenoterol conferred by Cys36. This is the first study to report single amino acids as determinants of OCT1 polyspecificity. Our findings suggest that structure-function data of OCT1 is not directly transferrable between substrates or species.

Oral epigallocatechin gallate reduces intestinal nadolol absorption via modulation of Oatp1a5 and Oct1 transcriptional levels in spontaneously hypertensive rats.

BACKGROUND: Concurrent use of epigallocatechin-3-gallate (EGCG) and medication may lead to botanical-drug interactions, subsequently therapeutic failure or drug toxicity. It has been reported that EGCG reduces plasma nadolol bioavailability in normotensive models. Nevertheless, evidence on the effects of EGCG on hypertensive model, and the possible underlying mechanism have not been elucidated. OBJECTIVES: This study aims (i) to investigate the effects of EGCG on nadolol pharmacokinetics (maximum plasma concentration, time to achieve maximum concentration, area under the time-plasma concentration curve, plasma half-life and total clearance) and subsequently its impact on blood pressure control; and (ii) to identify transcriptional regulatory roles of EGCG on the nadolol intestinal and hepatic drug-transporters in SHR. METHODS: Male SHR were pre-treated with a daily dose of EGCG (10 mg/kg body weight, i.g.) for 13 days. On day-14, a single dose of nadolol (10 mg/kg body weight) was given to the rats 30 min after the last dose of EGCG administration. Systolic blood pressure (SBP) was measured at 6-h and 22-h post-nadolol administration. Plasma and urinary nadolol concentrations were quantified using high-performance liquid chromatography, and pharmacokinetic parameters were analyzed by using non-compartmental analysis. Hepatic and ileal Oatp1a5, P-gp, and Oct1 mRNA expressions were determined by real-time PCR. RESULTS: SBP of SHR pre-treated with EGCG and received nadolol was significantly higher than those which were not pre-treated with EGCG but received nadolol. Pre-treatment of EGCG resulted in a marked reduction of plasma nadolol maximum concentration (Cmax) and area under the time-plasma concentration curve (AUC) by 53% and 51% compared to its control. The 14-day treatment with oral EGCG led to a significant downregulation of mRNA levels of ileal Oatp1a5, P-gp, and Oct1 genes by 4.03-, 8.01- and 4.03-fold; and hepatic P-gp, and Oct1 genes by 2.61- and 2.66-fold. CONCLUSION: These data concluded that exposure to EGCG could lead to reduced nadolol bioavailability and therefore, uncontrolled raised blood pressure and higher risks of cardiovascular events. Our data suggest that the reduced nadolol bioavailability is associated with the downregulation of ileal Oatp1a5 and Oct1 mRNA levels that subsequently lead to poor absorption of nadolol to the systemic circulation.

Influence of Cation Transporters (OCTs and MATEs) on the Renal and Hepatobiliary Disposition of [11C]Metoclopramide in Mice.

PURPOSE: To investigate the role of cation transporters (OCTs, MATEs) in the renal and hepatic disposition of the radiolabeled antiemetic drug [11C]metoclopramide in mice with PET. METHODS: PET was performed in wild-type mice after administration of an intravenous microdose (<1 µg) of [11C]metoclopramide without and with co-administration of either unlabeled metoclopramide (5 or 10 mg/kg) or the prototypical cation transporter inhibitors cimetidine (150 mg/kg) or sulpiride (25 mg/kg). [11C]Metoclopramide PET was also performed in wild-type and Slc22a1/2(-/-) mice. Radiolabeled metabolites were measured at 15 min after radiotracer injection and PET data were corrected for radiolabeled metabolites. RESULTS: [11C]Metoclopramide was highly metabolized and [11C]metoclopramide-derived radioactivity was excreted into the urine. The different investigated treatments decreased (~2.5-fold) the uptake of [11C]metoclopramide from plasma into the kidney and liver, inhibited metabolism and decreased (up to 3.8-fold) urinary excretion, which resulted in increased plasma concentrations of [11C]metoclopramide. Kidney and liver uptake were moderately (~1.3-fold) reduced in Slc22a1/2(-/-) mice. CONCLUSIONS: Our results suggest a contribution of OCT1/2 to the kidney and liver uptake and of MATEs to the urinary excretion of [11C]metoclopramide in mice. Cation transporters may contribute, next to variability in the activity of metabolizing enzymes, to variability in metoclopramide pharmacokinetics and side effects.

Amphetamine-like Neurochemical and Cardiovascular Effects of α-Ethylphenethylamine Analogs Found in Dietary Supplements.

Dietary supplements often contain additives not listed on the label, including α-ethyl homologs of amphetamine such as N,α-diethylphenethylamine (DEPEA). Here, we examined the neurochemical and cardiovascular effects of α-ethylphenethylamine (AEPEA), N-methyl-α-ethylphenethylamine (MEPEA), and DEPEA as compared with the effects of amphetamine. All drugs were tested in vitro using uptake inhibition and release assays for monoamine transporters. As expected, amphetamine acted as a potent and efficacious releasing agent at dopamine transporters (DAT) and norepinephrine transporters (NET) in vitro. AEPEA and MEPEA were also releasers at catecholamine transporters, with greater potency at NET than DAT. DEPEA displayed fully efficacious release at NET but weak partial release at DAT (i.e., 40% of maximal effect). In freely moving, conscious male rats fitted with biotelemetry transmitters for physiologic monitoring, amphetamine (0.1-3.0 mg/kg, s.c.) produced robust dose-related increases in blood pressure (BP), heart rate (HR), and motor activity. AEPEA (1-10 mg/kg, s.c.) produced significant increases in BP but not HR or activity, whereas DEPEA and MEPEA (1-10 mg/kg, s.c.) increased BP, HR, and activity. In general, the phenethylamine analogs were approximately 10-fold less potent than amphetamine. Our results show that α-ethylphenethylamine analogs are biologically active. Although less potent than amphetamine, they produce cardiovascular effects that could pose risks to humans. Given that MEPEA and DEPEA increased locomotor activity, these substances may also have significant abuse potential. SIGNIFICANCE STATEMENT: The α-ethyl homologs of amphetamine have significant cardiovascular, behavioral, and neurochemical effects in rats. Given that these compounds are often not listed on the ingredient labels of dietary supplements, these compounds could pose a risk to humans using these products.

Polysaccharides of vinegar-baked radix bupleuri promote the hepatic targeting effect of oxymatrine by regulating the protein expression of HNF4α, Mrp2, and OCT1.

ETHNOPHARMACOLOGICAL RELEVANCE: Vinegar-baked Radix Bupleuri (VBRB) is a processed form of Bupleurum chinense DC. As a well-known meridian-guiding drug, it is traditionally used as a component of traditional Chinese medicine formulations indicated for the treatment of liver diseases. However, the liver targeting component in VBRB remains unclear. Therefore, this study aims to explore the efficacy and mechanism of PSS (polysaccharides in Vinegar-baked Radix Bupleuri) in enhancing liver targeting. MATERIALS AND METHODS: Drug distribution of OM alone or combined with PSS was investigated in vivo. Relative uptake efficiency (RUE) and relative targeting efficiency (RTE) were calculated to evaluate liver targeting efficiency. The mRNA and protein expression of organic cation transporter 1 (OCT1), multi-drug resistance protein 2 (Mrp2), and hepatocyte nuclear factor 4α (HNF4α) in the liver were determined by q-PCR and Western blot. Then, AZT, the inhibitor of OCT1 and BI6015, the inhibitor of HNF4α were used to investigate regulatory mechanisms involved in the uptake of OM in the cell. At last, the role of PSS in the anti-hepatitis B virus (HBV) was explored on HepG2.2.15. RESULTS: PSS increased the AUC of OM in the liver and increase the RUE and RTE in the liver which indicated a liver targeting enhancing effect. The mRNA and protein expression of OCT1 was increased while Mrp2 and HNF4α decreased. PSS could increase the uptake of OM in HepG2 by increasing the protein expression of HNF4α and OCT1, while inhibited Mrp2. Moreover, PSS combined with OM could enhance the anti-HBV effect of OM. CONCLUSION: PSS enhanced the liver targeting efficiency and the underlying mechanism related to up-regulating the expression of OCT1 and HNF4α, while down-regulating of Mrp2. These results suggest that PSS may become a potential excipient and provide a new direction for new targeted research.

Trospium Chloride Transport by Mouse Drug Carriers of the Slc22 and Slc47 Families.

BACKGROUND: The muscarinic receptor antagonist trospium chloride (TCl) is used for pharmacotherapy of the overactive bladder syndrome. TCl is a hydrophilic positively charged drug. Therefore, it has low permeability through biomembranes and requires drug transporters for distribution and excretion. In humans, the organic cation transporters OCT1 and OCT2 and the multidrug and toxin extrusion MATE1 and MATE2-K carriers showed TCl transport. However, their individual role for distribution and excretion of TCl is unclear. Knockout mouse models lacking mOct1/mOct2 or mMate1 might help to clarify their role for the overall pharmacokinetics of TCl. METHOD: In preparation of such experiments, TCl transport was analyzed in HEK293 cells stably transfected with the mouse carriers mOct1, mOct2, mMate1, and mMate2, respectively. RESULTS: Mouse mOct1, mOct2, and mMate1 showed significant TCl transport with Km values of 58.7, 78.5, and 29.3 µM, respectively. In contrast, mMate2 did not transport TCl but showed MPP+ transport with Km of 60.0 µM that was inhibited by the drugs topotecan, acyclovir, and levofloxacin. CONCLUSION: TCl transport behavior as well as expression pattern were quite similar for the mouse carriers mOct1, mOct2, and mMate1 compared to their human counterparts.

CP-25 improves nephropathy in collagen-induced arthritis rats by inhibiting the renal inflammatory response.

Paeoniflorin-6'-O-benzene sulfonate (CP-25) is a derivative of paeoniflorin. We previously confirmed that CP-25 inhibits inflammatory responses in several arthritis animal models. The aim of the present study was to investigate the beneficial effects of CP-25 on renal damage in rats with collagen-induced arthritis (CIA). CIA was induced in rats, which were orally administered CP-25 (25, 50 and 100 mg/kg/day) for 24 days. The levels of plasma blood urea nitrogen (BUN) and urine protein in CIA rats were measured. Pathological changes in renal tissues and joints were observed, and inflammatory cell infiltration was evaluated by immunohistochemistry. Moreover, renal inflammatory mediators and transporters were measured by western blotting. We found that CP-25 not only inhibited arthritis manifestations but also improved renal pathological manifestations and kidney injury by decreasing serum BUN and urine protein levels. Further study revealed that CP-25 treatment reduced the number of renal CD68+ cells and downregulated the levels of MCP-1, TNF-α and IL-6 in CIA rats. On the other hand, we noted that CP-25 decreased the ratios of phosphorylated NF-κB p65 (p-p65) to total p65 and p-IκBα to total IκBα in CIA rats, suggesting that CP-25 blocked NF-κB activation. Finally, we observed that CP-25 restored the abnormal expression of OAT1 and OCT1 in the renal tissues of CIA rat. Our data indicate that CP-25 ameliorates kidney damage in CIA rats, and this beneficial effect is closely related to inhibiting renal inflammation and the abnormal expression of transporters.

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.

The fatty acid amide hydrolase inhibitor URB597 modulates splenic catecholamines in chronically stressed female and male rats.

The changes in sympathetic innervations in lymphoid organs could be a key factor in immune dysregulation. The endocannabinoid system has been shown to exhibit potent immunomodulatory effects that may differ between males and females, representing a potential therapeutic target for peripheral and central inflammatory disorders. Thus, in the present study, an examination was made of the effect of fatty acid amide hydrolase inhibitor URB597 treatment on splenic catecholamine content, synthesis, uptake and degradation in chronically unpredictably stressed (CUS) female and male rats. The results show that CUS increases anxiety-like behaviors and that URB597 had an anxiolytic effect on chronically stressed animals of both sexes. CUS induced the expression of plasma interleukin - 6 (IL-6), interleukin - 10 (IL-10) and IL-6 in the spleen, whereas the expression of IL-10 was reduced in the spleen of both sexes. URB597 treatment did not cause changes in IL-6 in plasma or the spleen, whereas it increased IL-10 in the spleen in CUS animals of both sexes. CUS caused a significant depletion of noradrenaline content in the spleen of female rats and a reduction in noradrenaline uptake in the spleen of female rats, while stressed males had a small but insignificant decrease of splenic noradrenaline levels and an enhanced uptake. The FAAH inhibitor URB597 enhances reduced noradrenaline content, affecting its uptake directly at the level of the spleen. It gives rise to the possibility that endocannabinoids exert a neurorestorative effect on the sympathetic nerve system and cell-mediated immune responses in the spleen of chronically stressed rats.

Metabolic Acidosis Alters Expression of Slc22 Transporters in Mouse Kidney.

INTRODUCTION: The kidneys play a central role in eliminating metabolic waste products and drugs through transporter-mediated excretion along the proximal tubule. This task is mostly achieved through a variety of transporters from the solute carrier family 22 (SLC22) family of organic cation and anion transporters. Metabolic acidosis modulates metabolic and renal functions and also affects the clearance of metabolites and drugs from the body. We had previously shown that induction of metabolic acidosis in mice alters a large set of transcripts, among them also many transporters including transporters from the Slc22 family. OBJECTIVE: Here we further investigated the impact of acidosis on Slc22 family members. METHODS: Metabolic acidosis was induced for 2 or 7 days with NH4Cl, some animals also received the uricase inhibitor oxonic acid for comparison. Expression of transporters was studied by qPCR and immunoblotting. RESULTS: NH4Cl induced no significant changes in plasma or urine uric acid levels but caused downregulation of Slc22a1 (Oct1), Slc22a6 (Oat1), Slc22a19 (Oat5), and -Slc22a12 (Urat1) at mRNA level. In contrast, Slc22a4 mRNA (Octn1) was upregulated. On protein level, NH4Cl increased Octn1 (after 7 days) and Urat1 (after 2 days) abundance and decreased Oat1 (after 2 days) and Urat1 (after 7 days). Oxonic acid had no impact on protein abundance of any of the transporters tested. CONCLUSION: In summary, metabolic acidosis alters expression of several transporters involved in renal excretion of metabolic waste products and drugs. This may have implications for drug kinetics and clearance of waste metabolites.

Pharmacological profiles of compounds in preworkout supplements ("boosters").

Preworkout supplements ("boosters") are used to enhance physical and mental performance during workouts. These products may contain various chemical substances with undefined pharmacological activity. We investigated whether substances that are contained in commercially available athletic multiple-ingredient preworkout supplements exert amphetamine-type activity at norepinephrine, dopamine, and serotonin transporters (NET, DAT, and SERT, respectively). We assessed the in vitro monoamine transporter inhibition potencies of the substances using human embryonic kidney 293 cells that expressed the human NET, DAT, and SERT. The phenethylamines ß-phenethylamine, N-methylphenethylamine, ß-methylphenethylamine, N-benzylphenethylamine, N-methyl-ß-methylphenethylamine, and methylsynephrine inhibited the NET and less potently the DAT similarly to D-amphetamine. ß-phenethylamine was the most potent, with IC50 values of 0.05 and 1.8 µM at the NET and DAT, respectively. These IC50 values were comparable to D-amphetamine (IC50 = 0.09 and 1.3 µM, respectively). The alkylamines 1,3-dimethylbutylamine and 1,3-dimethylamylamine blocked the NET but not the DAT. Most of the phenethylamines interacted with trace amine-associated receptor 1, serotonin 5-hydroxytryptamine-1A receptor, and adrenergic α1A and α2A receptors at submicromolar concentrations. None of the compounds blocked the SERT. In conclusion, products that are used by athletes may contain substances with mainly noradrenergic amphetamine-type properties.

Deletion of organic cation transporter Oct3 promotes hepatic fibrosis via upregulation of TGFß.

Organic cation transporters (OCT) are responsible for the intracellular uptake and detoxification of a broad spectrum of endogenous and exogenous substrates. OCTs are downregulated in cholestasis, fibrosis, and hepatocellular carcinoma, but the underlying molecular mechanisms and downstream effects of OCT deletion are unknown. Oct3-knockout (Oct3-/-; FVB.Slc22a3tm10pb) and wild-type (WT; FVB) mice were subject to escalating doses of carbon tetrachloride (CCl4) or thioacetamide (TAA) for 6 wk to induce advanced parenchymal liver fibrosis. Secondary biliary fibrosis was generated by bile duct ligation. Liver fibrosis was assessed by hydroxyproline determination, quantitative Sirius red morphometry, and quantitative real-time PCR for fibrosis and inflammation-related genes. Ductular reaction was assessed by bile duct count per field of view in hematoxylin and eosin staining. General gene expression analyses were performed in liver tissue from untreated Oct3-/- and WT mice. Finally, primary murine hepatocytes were treated with the nonselective OCT inhibitor quinine, and transforming growth factor-ß1 (Tgfß1) protein expression was quantified by quantitative real-time PCR and Western blot. Oct3-/- mice developed significantly more fibrosis after bile duct ligation and CCl4 treatment compared with WT mice. Ductular reaction was enhanced in the long-term model. Concomitantly, Oct1 mRNA expression was downregulated during cholestatic and chemically (TAA and CCl4) induced fibrogenesis. The downregulation of Oct1 mRNA in fibrotic liver tissue reversed within 4 wk after TAA cessation. Gene expression analysis by next-generation sequencing revealed an enrichment of Tgfß1 target genes in Oct3-/- mice. Tgfß1 mRNA expression was significantly upregulated after chemically induced fibrosis (P < 0.001) in Oct3-/- compared with WT mice. Accordingly, in primary murine hepatocytes functional inhibition of OCT led to an upregulation of Tgfß1 mRNA expression. Loss of Oct3 promotes fibrogenesis by affecting Tgfß-mediated homeostasis in mice with chronic biliary and parenchymal liver damage and fibrosis.NEW & NOTEWORTHY We show for the first time that organic cation transporter 3 (Oct3) is not only downregulated in fibrosis but loss of Oct3 also leads to an upregulation of transforming growth factor-ß contributing to fibrosis progression.

In vivo pharmacodynamic and pharmacokinetic effects of metformin mediated by the gut microbiota in rats.

AIMS: The gut microbiota plays a crucial role in the efficacy of metformin in T2DM treatment. We evaluated whether the pharmacodynamics and pharmacokinetics of metformin are mediated by gut microbiota. MAIN METHODS: We used conventional diabetic and pseudo-germ-free rats. After 6 weeks of metformin treatment, pharmacodynamic indexes were determined. Metformin concentrations were measured with a validated HPLC-MS/MS method after the first oral administration. KEY FINDINGS: Most endpoints were similar between vehicle-treated diabetic and vehicle-treated pseudo-germ-free diabetic rats. However, after 6 weeks of metformin treatment, compared with conventional diabetic rats, pseudo-germ-free diabetic rats exhibited significantly increased FBG, decreased oral glucose, reduced GSP, worsened insulin resistance, increased hyperlipidemia, and increased hepatic steatosis severity. Moreover, the Cmax of pseudo-germ-free rats increased significantly, while the t1/2α decreased significantly. These pharmacodynamic and pharmacokinetic changes were probably due to a decrease in Oct1 expression in the liver, resulting in altered hepatic uptake of metformin in vivo. SIGNIFICANCE: These results implied that the gut microbiota may play an important role in the pharmacodynamics and pharmacokinetics of metformin and that the changes in these properties are probably due to Oct1 downregulation in the livers of pseudo-germ-free rats.

Comparison of uptake transporter functions in hepatocytes in different species to determine the optimal model for evaluating drug transporter activities in humans.

A thorough understanding of species-dependent differences in hepatic uptake transporters is critical for predicting human pharmacokinetics (PKs) from preclinical data. In this study, the activities of organic anion transporting polypeptide (OATP/Oatp), organic cation transporter 1 (OCT1/Oct1), and sodium-taurocholate cotransporting polypeptide (NTCP/Ntcp) in cultured rat, dog, monkey and human hepatocytes were compared. The activities of hepatic uptake transporters were evaluated with respect to culture duration, substrate and species-dependent differences in hepatocytes. Longer culture duration reduced hepatic uptake transporter activities across species except for Oatp and Ntcp in rats. Comparable apparent Michaelis-Menten constant (Km,app) values in hepatocytes were observed across species for atorvastatin, estradiol-17ß-glucuronide and metformin. The Km,app values for rosuvastatin and taurocholate were significantly different across species. Rat hepatocytes exhibited the highest Oatp percentage of uptake transporter-mediated permeation clearance (PSinf,act) while no difference in %PSinf,act of probe substrates were observed across species. The in vitro hepatocyte inhibition data in rats, monkeys and humans provided reasonable predictions of in vivo drug-drug interaction (DDIs) between atorvastatin/rosuvastatin and rifampin. These findings suggested that using human hepatocytes with a short culture time is the most robust preclinical model for predicting DDIs for compounds exhibiting active hepatic uptake in humans.

Rat Organic Cation Transporter 1 Contains Three Binding Sites for Substrate 1-Methyl-4-phenylpyridinium per Monomer.

Organic cation transporters OCT1 (SLC22A1) and OCT2 (SLC22A2) are critically involved in absorption and excretion of diverse cationic drugs. Because drug-drug interactions at these transporters may induce adverse drug effects in patients, in vitro testing during drug development for interaction with the human transporters is mandatory. Recent data performed with rat OCT1 (rOCT1) suggest that currently performed in vitro tests assuming one polyspecific binding site are insufficient. Here we measured the binding and transport of model substrate 1-methyl-4-phenylpyridinium+ (MPP+) by cell-free-expressed fusion proteins of rOCT1 and rOCT1 mutants with green fluorescent protein that had been reconstituted into nanodiscs or proteoliposomes. The nanodiscs were formed with major scaffold protein (MSP) and different phospholipids, whereas the proteoliposomes were formed with a mixture of cholesterol, phosphatidylserine, and phosphatidylcholine. In nanodiscs formed with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine or cholesterol, phosphatidylserine, and phosphatidylcholine, two low-affinity MPP+ binding sites and one high-affinity MPP+ binding site per transporter monomer were determined. Mutagenesis revealed that tryptophan 218 and aspartate 475 in neighboring positions in the modeled outward-open cleft contribute to one low-affinity binding site, whereas arginine 440 located distantly in the cleft is critical for MPP+ binding to another low-affinity site. Comparing MPP+ binding with MPP+ transport suggests that the low-affinity sites are involved in MPP+ transport, whereas high-affinity MPP+ binding influences transport allosterically. The data will be helpful in the interpretation of future crystal structures and provides a rationale for future in vitro testing that is more sophisticated and reliable, leading to the generation of pharmacophore models with high predictive power.

Organic cation transporter and multidrug and toxin extrusion 1 co-mediated interaction between metformin and berberine.

Metformin and berberine are often combined for treating diabetes. In the present study, we evaluated the drug-drug pharmacokinetic interaction between metformin and berberine after oral co-administration in vivo and the underlying mechanism. As revealed by comparison with the metformin-only group, berberine significantly decreased the maximum plasma concentration (Cmax), area under the curve from 0 to 4 h (AUC0-4h), and urinary and bile excretion, and increased the kidney tissue concentration of metformin in rats. The non-everted intestinal sac study showed that berberine inhibited the absorption of metformin, and in transfected Madin-Darby canine kidney (MDCK)-rat organic cation transporter 1 (MDCK-rOCT1), MDCK-rat organic cation transporter 2 (MDCK-rOCT2), and MDCK-rat multidrug and toxin extrusion 1 (MDCK-rMATE1) cells, berberine significantly inhibited metformin transport mediated by OCT1, OCT2, and MATE1 in a concentration-dependent manner with half-maximal inhibitory concentration (IC50) values of 18.8, 1.02, and 10.7 µM, respectively. In contrast, co-administration of metformin increased the Cmax and AUC0-4h of berberine with no significant difference in pharmacokinetics parameters between co-administration and berberine-only groups. Furthermore, metformin increased kidney and liver concentrations and reduced the urinary and biliary excretion of berberine. Metformin (≥1 or ≥0.3 mM) decreased berberine transport in MDCK-rOCT1, MDCK-rOCT2, and MDCK-rMATE1 cells. However, metformin did not affect berberine concentration in MDCK-multidrug resistance protein 1 cells. These results suggest that the combination of metformin and berberine induced a pharmacokinetic interaction by cooperatively inhibiting OCT and MATE1-mediated transport.

Atorvastatin modulates drug transporters and ameliorates nicotine-induced testicular toxicity.

We studied the changes in mRNA expressions of influx and efflux transporters, blood-testis-barrier proteins (BTB) and key apoptotic genes in the testis of rats coadministered with nicotine and atorvastatin. Rats were divided into four groups: (i) control, (ii) atorvastatin (10 mg/kg b.wt), (iii) nicotine (0.6 mg/kg b.wt) and (iv) atorvastatin (10 mg/kg b.wt) + nicotine (0.6 mg/kg b.wt). Atorvastatin was given by oral intubation and nicotine by intraperitoneal injection. After 60 days of treatment, expressions of key apoptotic genes involved in both intrinsic and extrinsic pathways; solute carrier influx transporters SLCOB1, SLC22A1 and efflux transporter ABCB1 associated with transport of atorvastatin and nicotine, and proteins of BTB were assayed. Nicotine administration activated apoptosis and downregulated SLCOB1, which transport atorvastatin. Atorvastatin administration suppressed apoptotic pathway and downregulated SLC22A1, transporter of nicotine. Coadministration of atorvastatin with nicotine downregulated expressions of apoptotic genes. The combined administration of atorvastatin and nicotine reduced the influx of both atorvastatin and nicotine and enhanced the efflux of these drugs thereby altering the microenvironment of testis and improving testicular function. We conclude that atorvastatin-mediated alterations of BTB and drug transporters might have played a significant role in ameliorating nicotine-induced testicular toxicity.

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.