Inhibitors Of Organic Cation Transporters Research Articles

Susceptibility of mouse cochlear hair cells to cisplatin ototoxicity largely depends on sensory mechanoelectrical transduction channels both Ex Vivo and In Vivo

Cisplatin, a highly effective chemotherapeutic drug for various human cancers, induces irreversible sensorineural hearing loss as a side effect. Currently there are no highly effective clinical strategies for the prevention of cisplatin-induced ototoxicity. Previous studies have indicated that short-term cisplatin ototoxicity primarily affects the outer hair cells of the cochlea. Therefore, preventing the entry of cisplatin into hair cells may be a promising strategy to prevent cisplatin ototoxicity. This study aimed to investigate the entry route of cisplatin into mouse cochlear hair cells. The competitive inhibitor of organic cation transporter 2 (OCT2), cimetidine, and the sensory mechanoelectrical transduction (MET) channel blocker benzamil, demonstrated a protective effect against cisplatin toxicity in hair cells in cochlear explants. Sensory MET-deficient hair cells explanted from Tmc1Δ;Tmc2Δ mice were resistant to cisplatin toxicity. Cimetidine showed an additive protective effect against cisplatin toxicity in sensory MET-deficient hair cells. However, in the apical turn, cimetidine, benzamil, or genetic ablation of sensory MET channels showed limited protective effects, implying the presence of other entry routes for cisplatin to enter the hair cells in the apical turn. Systemic administration of cimetidine failed to protect cochlear hair cells from ototoxicity caused by systemically administered cisplatin. Notably, outer hair cells in MET-deficient mice exhibited no apparent deterioration after systemic administration of cisplatin, whereas the outer hair cells in wild-type mice showed remarkable deterioration. The susceptibility of mouse cochlear hair cells to cisplatin ototoxicity largely depends on the sensory MET channel both ex vivo and in vivo. This result justifies the development of new pharmaceuticals, such as a specific antagonists for sensory MET channels or custom-designed cisplatin analogs which are impermeable to sensory MET channels.

Facilitated Transport of EGFR Inhibitors Plays an Important Role in Their Cellular Uptake.

Epidermal growth factor receptor (EGFR) is a transmembrane protein commonly targeted by tyrosine kinase inhibitors (TKIs) as a front-line therapy for patients with many cancers including nonsmall cell lung cancer (NSCLC). Effective treatment requires efficient intracellular drug uptake and target binding. However, despite the recent success in the development of new TKI drugs, the mechanisms of uptake for many TKIs are still poorly understood due to the difficulty in imaging and measuring nonfluorescent drug molecules at a subcellular resolution. It has previously been shown that weakly basic TKI drugs are sequestered in lysosomes. Leveraging this property, we apply hyperspectral stimulated Raman scattering imaging to directly visualize and quantify two Food and Drug Administration-approved EGFR inhibitor drugs (lapatinib and afatinib) inside living cells and the changes in their cellular uptake upon the addition of organic cation transporter inhibitors. These single-cell quantitative measurements provide new insight into the role of membrane transporters in the uptake of TKI drugs in living cells.

Effect of Cimetidine on Metformin Pharmacokinetics and Endogenous Metabolite Levels in Rats.

Tubular secretion is a primary mechanism along with glomerular filtration for renal elimination of drugs and toxicants into urine. Organic cation transporters (OCTs) and multidrug and toxic extrusion (MATE) transporters facilitate the active secretion of cationic substrates, including drugs such as metformin and endogenous cations. We hypothesized that administration of cimetidine, an Oct/Mate inhibitor, will result in increased plasma levels and decreased renal clearance of metformin and endogenous Oct/Mate substrates in rats. A paired rat pharmacokinetic study was carried out in which metformin (5 mg/kg, intravenous) was administered as an exogenous substrate of Oct/Mate transporters to six Sprague-Dawley rats with and without cimetidine (100 mg/kg, intraperitoneal). When co-administered with cimetidine, metformin area under the curve increased significantly by 3.2-fold, and its renal clearance reduced significantly by 73%. Untargeted metabolomics was performed to investigate the effect of cimetidine on endogenous metabolome in the blood and urine samples. Over 8,000 features (metabolites) were detected in the blood, which were shortlisted using optimized criteria, i.e., a significant increase (P value < 0.05) in metabolite peak intensity in the cimetidine-treated group, reproducible retention time, and quality of chromatogram peak. The metabolite hits were classified into three groups that can potentially distinguish inhibition of i) extra-renal uptake transport or catabolism, ii) renal Octs, and iii) renal efflux transporters or metabolite formation. The metabolomics approach identified novel putative endogenous substrates of cationic transporters that could be tested as potential biomarkers to predict Oct/Mate transporter mediated drug-drug interactions in the preclinical stages. SIGNIFICANCE STATEMENT: Endogenous substrates of renal transporters in animal models could be used as potential biomarkers to predict renal drug-drug interactions in early drug development. Here we demonstrated that cimetidine, an inhibitor of organic cation transporters (Oct/Mate), could alter the pharmacokinetics of metformin and endogenous cationic substrates in rats. Several putative endogenous metabolites of Oct/Mate transporters were identified using metabolomics approach, which could be tested as potential transporter biomarkers to predict renal drug-drug interaction of Oct/Mate substrates.

Stereoselective Inhibition of High- and Low-Affinity Organic Cation Transporters.

Many drugs have chiral centers and are therapeutically applied as racemates. Thus, the stereoselectivity in their interactions with membrane transporters needs to be addressed. Here, we studied stereoselectivity in inhibiting organic cation transporters (OCTs) 1, 2, and 3 and the high-affinity monoamine transporters (MATs) NET and SERT. Selectivity by the inhibition of 35 pairs of enantiomers significantly varied among the three closely related OCTs. OCT1 inhibition was nonselective in almost all cases, whereas OCT2 was stereoselectively inhibited by 45% of the analyzed drugs. However, the stereoselectivity of the OCT2 was only moderate with the highest selectivity observed for pramipexole. The (R)-enantiomer inhibited OCT2 4-fold more than the (S)-enantiomer. OCT3 showed the greatest stereoselectivity in its inhibition. (R)-Tolterodine and (S)-zolmitriptan inhibited OCT3 11-fold and 25-fold more than their respective counterparts. Interestingly, in most cases, the pharmacodynamically active enantiomer was also the stronger OCT inhibitor. In addition, stereoselectivity in the OCT inhibition appeared not to depend on the transported substrate. For high-affinity MATs, our data confirmed the stereoselective inhibition of NET and SERT by several antidepressants. However, the stereoselectivity measured here was generally lower than that reported in the literature. Unexpectedly, the high-affinity MATs were not significantly more stereoselectively inhibited than the polyspecific OCTs. Combining our in vitro OCT inhibition data with available stereoselective pharmacokinetic analyses revealed different risks of drug-drug interactions, especially at OCT2. For the tricyclic antidepressant doxepine, only the (E)-isomer showed an increased risk of drug-drug interactions according to guidelines from regulatory authorities for renal transporters. However, most chiral drugs show only minor stereoselectivity in the inhibition of OCTs in vitro, which is unlikely to translate into clinical consequences.

Goldenseal-Mediated Inhibition of Intestinal Uptake Transporters Decreases Metformin Systemic Exposure in Mice.

Goldenseal is a perennial plant native to eastern North America. A recent clinical study reported goldenseal decreased metformin Cmax and area under the blood concentration versus time curve (AUC) by 27% and 23%, respectively, but half-life and renal clearance were unchanged. These observations suggested goldenseal altered processes involved in metformin absorption. The underlying mechanism(s) remain(s) unknown. One mechanism for the decreased metformin systemic exposure is inhibition by goldenseal of intestinal uptake transporters involved in metformin absorption. Goldenseal extract and three goldenseal alkaloids (berberine, (-)-β-hydrastine, hydrastinine) were tested as inhibitors of organic cation transporter (OCT) 3, plasma membrane monoamine transporter (PMAT), and thiamine transporter (THTR) 2 using human embryonic kidney 293 cells overexpressing each transporter. The goldenseal extract, normalized to berberine content, was the strongest inhibitor of each transporter (IC50: 4.9, 13.1, and 5.8 μM for OCT3, PMAT, and THTR2, respectively). A pharmacokinetic study in mice compared the effects of berberine, (-)-β-hydrastine, goldenseal extract, and imatinib (OCT inhibitor) on orally administered metformin. Goldenseal extract and imatinib significantly decreased metformin Cmax by 31% and 25%, respectively, and had no effect on half-life. Berberine and (-)-β-hydrastine had no effect on metformin pharmacokinetics, indicating neither alkaloid alone precipitated the interaction in vivo. A follow-up murine study involving intravenous metformin and oral inhibitors examined the contributions of basolateral enteric/hepatic uptake transporters to the goldenseal-metformin interaction. Goldenseal extract and imatinib had no effect on metformin AUC and half-life, suggesting lack of inhibition of basolateral enteric/hepatic uptake transporters. Results may have implications for patients taking goldenseal with drugs that are substrates for OCT3 and THTR2. SIGNIFICANCE STATEMENT: Goldenseal is used to self-treat respiratory infections and digestive disorders. We investigated potential mechanisms for the clinical pharmacokinetic interaction observed between goldenseal and metformin, specifically inhibition by goldenseal of intestinal uptake transporters (OCT3, PMAT, THTR2) involved in metformin absorption. Goldenseal extract inhibited all three transporters in vitro and decreased metformin systemic exposure in mice. These data may have broader implications for patients co-consuming goldenseal with other drugs that are substrates for these transporters.

Strong inhibition of organic cation transporter 2 by flavonoids and attenuation effects on cisplatin-induced cytotoxicity

Organic cation transporter 2 (OCT2) is predominantly expressed in the basolateral membrane of renal proximal tubule cells and contributes to the renal excretion of various drugs such as metformin, cisplatin, oxaliplatin, cimetidine, and lamivudine. Cisplatin, an anticancer agent for various cancers, is a substrate of OCT2, and cisplatin-induced nephrotoxicity is in part attributed to OCT2 activity in the kidney, which increases the renal accumulation of cisplatin. In this study, we aimed to identify flavone derivatives with strong inhibitory effects on OCT2 transport. Among the 80 flavonoids tested, 24 showed moderate to strong inhibitory effects against OCT2 transport activity. The IC50 values were less than 5 μM for 10 flavonoids. All 10 compounds alleviated cisplatin-induced cytotoxicity in cells expressing OCT2, even though the magnitude of the effects varied depending on the functional moieties in each position. Multiple factor analysis revealed that the methyl group at the R1 position and methoxy group at the R6 position of the flavonol backbone are important for OCT2 inhibition. Information on the functional moieties in the flavonol backbone would help develop effective OCT2 inhibitors by providing a structural association with OCT2 inhibitory effects. In addition, the compounds with strong inhibitory effects on OCT2 identified in this study may be potential candidates for clinical use to mitigate cisplatin-induced nephrotoxicity.

Selective Inhibition of Organic Cation Transporter 1 by Benzoylpaeoniflorin Attenuates Hepatic Lipid Accumulation through AMPK Activation.

Organic cation transporter 1 (OCT1) is a liver-specific transporter and plays an essential role in drug disposition and hepatic lipid metabolism. Therefore, inhibition of OCT1 may not only lead to drug-drug interactions but also represent a potential therapy for fatty liver diseases. In this study, we systematically investigated the inhibitory effect of 200 natural products on OCT1-mediated uptake of 4,4-dimethylaminostyryl-N-methylpyridinium (ASP+) and identified 10 potent OCT1 inhibitors. The selectivity of these inhibitors over OCT2 was evaluated using both in vitro uptake assays and in silico molecular docking analyses. Importantly, benzoylpaeoniflorin was identified as the most potent OCT1 inhibitor with the highest selectivity over OCT2. Additionally, benzoylpaeoniflorin prevented lipid accumulation in hepatocytes, with concomitant activation of AMPK and down-regulation of lipogenic genes, such as acetyl-CoA carboxylase (ACC) and fatty acid synthase (FASN). To conclude, our findings are of significant value in understanding OCT1-based natural product-drug interactions and provide a natural source of OCT1 inhibitors which may hold promise for treating fatty liver diseases.

Pharmacokinetics and transplacental transfer of codeine and codeine metabolites from Papaver somniferum L.

Ethnopharmacological relevancePapaveris Pericarpium, which is the dried husk of Papaver somniferum L., has been used as a phytomedicine to relieve cough, diarrhea and pain. The alkaloid codeine contained therein via biotransformation converts to morphine and potentially produces addictive and toxic effects. Due to the healthy concern for a pregnant woman, our hypothesis is that codeine and its metabolites can penetrate the placental barrier to reach the foetus and amniotic fluid, and these processes may be modulated by the transporter. Aim of the studyBecause codeine is also considered a prodrug of morphine, it has a good analgesic effect. It is often used by pregnant women but may expose the foetus to the risk of morphine harm. The aim of this study is to investigate the metabolic rate, distribution and transplacental transfer mechanism of codeine and its metabolites morphine and morphine-3-glucuronide (M3G) in pregnant rats and to assess the risk of medication for pregnant women. Materials and methodsUltrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) combined with a microdialysis system was developed to monitor codeine, morphine and M3G in multiple sites of maternal blood, placenta, foetus and amniotic fluid after codeine administration. A compartmental model was used to calculate the pharmacokinetic parameters of codeine in blood after codeine administration (10 mg/kg, i.v.). The area under the concentration (AUC) ratio of AUCmetabolite/AUCcodeine and AUCtissue/AUCblood was used to represent the metabolic biotransformation ratio and the drug from blood-to-tissue transfer ratio, respectively. ResultsThe pharmacokinetic results demonstrated that codeine fit well with a two-compartment model and went through rapid metabolism to morphine and M3G in pregnant rats after codeine administration (10 mg/kg, i.v.). The biotransformation ratios of AUCmorphine/AUCcodeine, AUCM3G/AUCmorphine and AUCM3G/AUCcodeine were 0.12 ± 0.03, 54.45 ± 20.61 and 6.53 ± 2.47, respectively, after codeine administration (10 mg/kg, i.v.), which suggested that codeine was easily metabolized into M3G through morphine. The tissue distribution results demonstrated that all of the analytes penetrated into the foetus through the placenta; however, the blood-to-tissue transfer ratio (AUCtissue/AUCblood) of morphine and M3G was relatively lower than that of codeine after codeine administration (10 mg/kg, i.v.), which suggested that the blood-placenta barrier blocks the penetration of morphine and M3G into the foetus. Thus, the tissue transfer of morphine in the placenta and foetus was significantly enhanced by treatment with corticosterone, an inhibitor of organic cation transporter (OCT). ConclusionBased on microdialysis coupled to a validated UHPLC–MS/MS system, the pharmacokinetics and metabolic biotransformation of codeine and its metabolites were analyzed and clarified. The potential mechanism of morphine placental transfer was modulated by OCT transporters.

High Throughput Screening of a Prescription Drug Library for Inhibitors of Organic Cation Transporter 3, OCT3

IntroductionThe organic cation transporter 3 (OCT3, SLC22A3) is ubiquitously expressed and interacts with a wide array of compounds including endogenous molecules, environmental toxins and prescription drugs. Understudied as a determinant of pharmacokinetics and pharmacodynamics, OCT3 has the potential to be a major determinant of drug absorption and disposition and to be a target for drug-drug interactions (DDIs).GoalThe goal of the current study was to identify prescription drug inhibitors of OCT3.MethodsWe screened a compound library consisting of 2556 prescription drugs, bioactive molecules, and natural products using a high throughput assay in HEK-293 cells stably expressing OCT3.ResultsWe identified 210 compounds that at 20 μM inhibit 50% or more of OCT3-mediated uptake of 4-Di-1-ASP (2 μM). Of these, nine were predicted to inhibit the transporter at clinically relevant unbound plasma concentrations. A Structure-Activity Relationship (SAR) model included molecular descriptors that could discriminate between inhibitors and non-inhibitors of OCT3 and was used to identify additional OCT3 inhibitors. Proteomics of human brain microvessels (BMVs) indicated that OCT3 is the highest expressed OCT in the human blood-brain barrier (BBB).ConclusionsThis study represents the largest screen to identify prescription drug inhibitors of OCT3. Several are sufficiently potent to inhibit the transporter at therapeutic unbound plasma levels, potentially leading to DDIs or off-target pharmacologic effects.

The Study of the Transport Mechanism of Isorhynchophylline in Liver.

To investigate the transport mechanism of isorhynchophylline (IRN) by using the specific inhibitors of organic cation transporters (OCTs) and organic anion transporting polypeptides (OATPs) and attempt illustrate the metabolic mechanism of IRN in the liver. All animals were randomly divided into three groups: control group (only inject IRN), RIF group (inject IRN and rifampicin), and ADR group (inject IRN and adrenalone). The control group was injected with IRN via the caudal vein. The RIF group was injected with rifampicin (RIF) by gavage, and after 1 h, IRN was injected into the caudal vein. Similarly, the ADR group received adrenalone by the caudal vein, and after 0.5 h, IRN was injected into the caudal vein. Thereafter, blood samples were obtained by the heart punctures at 90 min, 180 min, and 300 min following drug administration. Rats were sacrificed at 300 min after drug administration; then, the liver tissue was harvested. The level of IRN was measured by using high-performance liquid chromatography (HPLC), and the Kp values were calculated. After RIF administration (OATPs inhibitors), the Kp value of IRN was slightly decreased when compared with that of the control group. Meanwhile, the Kp value of IRN was dramatically reduced compared to that of the control group following ADR administration (OCTs inhibitors). The results suggested that OCTs have mainly participated in the hepatic uptake process of IRN.

Trans-placental transfer of nicotine: Modulation by organic cation transporters.

Nicotine is a highly addictive substance and harmful to the developing foetus. However, few studies have investigated the transporter mechanism responsible for regulating the transfer of nicotine across the blood-placental interface. A multiple in-vivo microdialysis system coupled to ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed to monitor simultaneously nicotine and cotinine in the blood, placenta, foetus, and amniotic fluid of pregnant rats. The pharmacological mechanism of nicotine transfer across the placenta was investigated by co-administering corticosterone, an inhibitor of organic cation transporters (OCTs) that partly mediate the exchange of nicotine across the placenta. The results revealed that intravenously administered nicotine (1mg/kg) was rapidly metabolised to cotinine with a transformation ratio (AUCcotinine/AUCnicotine) of 0.67±0.08, 0.21±0.05, 0.25±0.12, 0.31±0.05 in maternal blood, placenta, amniotic fluid, and foetus, respectively. The tissue transformation ratios (AUCtissue/AUCblood) were 0.83±0.16, 0.65±0.17, 0.57±0.13 for nicotine, and 0.25±0.06, 0.24±0.12, 0.26±0.04 for cotinine at placenta, amniotic fluid and foetus, respectively. Following the co-administration of corticosterone (2mg/kg), the tissue transformation ratio of nicotine was significantly reduced in the placenta but was significantly increased in the foetus. Levels of cotinine were not significantly altered by the administration of corticosterone. These findings implicate OCT in mediating the transfer of nicotine across the blood-placenta barrier. Understanding the mechanism of nicotine transfer through the placenta may inform therapeutic strategies to lessen the exposure of the developing foetus to nicotine in the maternal bloodstream.

Biological Distribution of Orally Administered [123I]MIBG for Estimating Gastrointestinal Tract Absorption.

Gastrointestinal tract absorption of cationic anticancer drugs and medicines was estimated using whole-body imaging following oral [123I]MIBG administration. [123I]MIBG was added to cultures of human embryonic kidney (HEK)293 cells expressing human organic anion transporting polypeptide (OATP)2B1, carnitine/organic cation transporter (OCTN)1 and OCTN2, and organic cation transporter (OCT)1, OCT2, and OCT3 with and without cimetidine (an OCTN and OCT inhibitor) and L-carnitine (an OCTN inhibitor). Biodistribution analyses and single-photon emission computed tomography (SPECT) imaging in normal and dextran sodium sulfate (DSS)-induced experimental colitis mice were conducted using [123I]MIBG with and without cimetidine. [123I]MIBG uptake was significantly higher in HEK293/OCTN1, 2, and OCT1-3 cells than in mock cells. Uptake via OCTN was inhibited by L-carnitine, whereas OCT-mediated uptake was inhibited by cimetidine. Biodistribution analyses and SPECT imaging studies showed significantly lower accumulation of [123I]MIBG in the blood, heart, liver, and bladder in DSS-induced experimental colitis mice and mice with cimetidine loading compared with normal mice, whereas significantly higher accumulation in the stomach and kidney was observed after [123I]MIBG injection. [123I]MIBG imaging after oral administration can be used to estimate gastrointestinal absorption in the small intestine via OCTN and/or OCT by measuring radioactivity in the heart, liver, and bladder.

Wedelolactone protects against cisplatin-induced nephrotoxicity in mice via inhibition of organic cation transporter 2.

The balance of cisplatin uptake and efflux, mediated mainly by organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1), respectively, determines the renal accumulation and nephrotoxicity of cisplatin. Using transporter-mediated cellular uptake assay, we identified wedelolactone (WEL), a medicinal plant-derived natural compound, is a competitive inhibitor of OCT2 and a noncompetitive inhibitor of MATE1. Wedelolactone showed a selectivity to inhibit OCT2 rather than MATE1. Cytotoxicity studies revealed that wedelolactone alleviated cisplatin-induced cytotoxicity in OCT2-overexpressing HEK293 cells, whereas it did not alter the cytotoxicity of cisplatin in various cancer cell lines. Additionally, wedelolactone altered cisplatin pharmacokinetics, reduced kidney accumulation of cisplatin, and ameliorated cisplatin-induced acute kidney injury in the Institute of Cancer Research mice. In conclusion, these findings suggest a translational potential of WEL as a natural therapy for preventing cisplatin-induced nephrotoxicity and highlight the need for drug-drug interaction investigations of WEL with other treatments which are substrates of OCT2 and/or MATE1.

The Organic Cation Transporter 2 Inhibitor Quinidine Modulates the Neuroprotective Effect of Nerve Growth Factor and Memantine on Cholinergic Neurons of the Basal Nucleus of Meynert in Organotypic Brain Slices

Introduction: Alzheimer’s disease (AD) is a severe neurodegenerative disorder of the brain characterized by degeneration of cholinergic neurons which is directly linked to cognitive decline. Nerve growth factor (NGF) is the most potent protective factor for cholinergic neurons, additionally the NMDA antagonist memantine blocks glutamate-mediated excitotoxic activity. Quinidine is an inhibitor of organic cation transporter 2 (OCT2). OCT2 is located on cholinergic neurons and plays a role in presynaptic reuptake and recycling of acetylcholine in the brain. We hypothesize that quinidine can modulate the protective effects of NGF and memantine on cholinergic neurons in organotypic brain slices of the nucleus basalis of Meynert (nBM). Methods: Organotypic brain slices of nBM were incubated with 100 ng/mL NGF, 10 µM memantine, 10 µM quinidine, and combinations of these treatments for 2 weeks. Cholinergic neurons were immunohistochemically stained for choline acetyltransferase (ChAT). Results: Our data show that NGF as well as memantine counteracted the cell death of cholinergic nBM neurons. Quinidine alone had no toxic effect on cholinergic neurons but inhibited the protective effect of NGF and memantine when applied simultaneously. Discussion/Conclusion: Our data provide evidence that quinidine modulates the survival of cholinergic nBM neurons via OCT2.

Organic cation transporters mediate pharmacokinetic interaction between dopamine receptor agonists and dopamine

Purpose Parkinson's disease (PD) is typically characterized by progressive damage to the nigrostriatal dopaminergic neurons with the enormous depletion of striatal dopamine (DA). DA receptor agonists such as pramipexole (PPX) and ropinirole (RPR) have been widely used in clinic. However, long-term treatment with DA receptor agonists often results in declined therapeutic effect. Recent research has shown that uptake-2 transporters including organic cation transporters (OCT) 2/3 play an important role in the regulation of DA homeostasis in brain. The aim of the present study was to investigate the underlying mechanism by which DA receptor agonists modulate DA homeostasis. Methods Transfected cells were incubated with HBSS containing serial concentration of PPX/RPR/DA with or without OCT2/3 inhibitors. LC-MS/MS was used to quantify the level of substrates in cell lysates. Then, male C57BL/6J mice received MPTP subcutaneously to construct PD model. Synaptosomes were extracted by sucrose density gradient centrifugation, incubated with uptake buffer containing predefined substrate and inhibitor and precipitated after 5 min. Lysed followed by LC-MS/MS quantification. Striatal Oct2/3 gene and protein levels were measured by RT-qPCR and western blotting, respectively. Finally, the tertiary structure models of OCT2/3 were predicted and optimized using bioinformatics technology. The molecular docking between OCT2/3 and substrates of interest were predicted by ClusPro servers. Results Both MDCK-OCT2 and MDCK-OCT3 cells demonstrated significantly higher cellular uptake of DA/PPX compared with MDCK mock cells. Also, RPR uptake was increased when OCT2 was overexpressed. A variety of classical OCT2/3 inhibitors decreased cellular uptake of DA/PPX/RPR. Furthermore, PPX/RPR could inhibit OCT2/3 mediated DA uptake following distinct kinetic processes. On the other hand, OCT2-mediated PPX/RPR uptake could also be restrained by DA. The interaction between DA and PPX/RPR was further confirmed at the synatosomes level. Besides, OCT2/3 expression and function were obviously elevated under PD status and thus the transport efficiency was increased. The molecular docking results revealed the OCT2/3 binding pockets as well as affinity difference between DA receptor agonists and DA. The affinity results were in good agreement with the findings of uptake assay. Conclusion This study not only offers a novel perspective on anti-PD mechanism of PPX and RPR as organic cation transporter inhibitor, but also demonstrated that up-regulated OCT2 and OCT3 might be associated with the declined therapeutic effect of PPX and RPR.

Inhibition of organic cation transporter 3 activity by tyrosine kinase inhibitors.

Organic cation transporter (OCT) 3 (SLC22A3) is a widely expressed drug transporter, handling notably metformin and platinum derivatives, as well as endogenous compounds like monoamine neurotransmitters. OCT3 has been shown to be inhibited by a few marketed tyrosine kinase inhibitors (TKIs). The present study was designed to determine whether additional TKIs may interact with OCT3. For this purpose, the effects of 25 TKIs toward OCT3 activity were analyzed using OCT3-overexpressing HEK293 cells. 13/25 TKIs, each used at 10µM, were found to behave as moderate or strong inhibitors of OCT3 activity, that is, they decreased OCT3-mediated uptake of the fluorescent dye 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide by at least 50% or 80%, respectively. This OCT3 inhibition was correlated to some molecular descriptors of TKIs, such as the percentage of H atoms and that of cationic forms at pH=7.4. It was concentration-dependent, notably for brigatinib, ceritinib, and crizotinib, which exhibited low half maximal inhibitory concentration (IC50 ) values in the 28-106nM range. Clinical concentrations of these three marketed TKIs, as well as those of pacritinib, were next predicted to inhibit in vivo OCT3 activity according to regulatory criteria. Cellular TKI accumulation experiments as well as trans-stimulation assays, however, demonstrated that OCT3 does not transport brigatinib, ceritinib, crizotinib, and pacritinib, thus discarding any implication of OCT3 in the pharmacokinetics of these TKIs. Taken together, these data suggest that some TKIs may act as potent inhibitors of OCT3 activity, which may have consequences in terms of drug-drug interactions and toxicity.

Substrates and Inhibitors of Organic Cation Transporters (OCTs) and Plasma Membrane Monoamine Transporter (PMAT) and Therapeutic Implications.

The gene products of the SLC22A gene family (hOCT1, hOCT2, and hOCT3) and of the SLC29A4 gene (hPMAT or hENT4) are all polyspecific organic cation transporters. Human OCTs (including hPMAT) are expressed in peripheral tissues such as small intestine, liver, and kidney involved in the pharmacokinetics of drugs. In the human brain, all four transporters are expressed at the blood-brain barrier (BBB), hOCT2 is additionally expressed in neurons, and hOCT3 and hPMAT in glia. More than 40% of the presently used drugs are organic cations. This chapter lists and discusses all known drugs acting as substrates or inhibitors of these four organic cation transporters, independently of whether the transporter is expressed in the central nervous system (CNS) or in peripheral tissues. Of interest is their involvement in drug absorption, distribution, and excretion as well as potential OCT-associated drug-drug interactions (DDIs), with a focus on drugs that act in the CNS.

1315. No Dose Adjustment of Metformin with Fostemsavir Coadministration Based on Mechanistic Static and Physiologically Based Pharmacokinetic Models

BackgroundFostemsavir (FTR) is an oral prodrug of the first-in-class attachment inhibitor temsavir (TMR) which is being evaluated in patients with multidrug resistant HIV-1 infection. In vitro studies indicated that TMR and its 2 major metabolites are inhibitors of organic cation transporters (OCT)1, OCT2, and multidrug and toxin extrusion transporters (MATEs). To assess the clinical relevance, of OCT and MATE inhibition, mechanistic static DDI prediction with calculated Imax,u/IC50 ratios was below the cut-off limits for a DDI flag based on FDA guidelines and above the cut-off limits for MATEs based on EMA guidelines.MethodsMetformin is a commonly used probe substrate for OCT1, OCT2 and MATEs. To predict the potential for a drug interaction between TMR and metformin, a physiologically based pharmacokinetic (PBPK) model for TMR was developed based on its physicochemical properties, in vitro and in vivo data. The model was verified and validated through comparison with clinical data. The TMR PBPK model accurately described AUC and Cmax within 30% of the observed data for single and repeat dose studies with or without food. The SimCYP models for metformin and ritonavir were qualified using literature data before applications of DDI prediction for TMRResultsTMR was simulated at steady state concentrations after repeated oral doses of FTR 600 mg twice daily which allowed assessment of the potential OCT1, OCT2, and MATEs inhibition by TMR and metabolites. No significant increase in metformin systemic exposure (AUC or Cmax) was predicted with FTR co-administration. In addition, a sensitivity analysis was conducted for either hepatic OCT1 Ki, or renal OCT2 and MATEs Ki values. The model output indicated that, a 10-fold more potent Ki value for TMR would be required to have a ~15% increase in metformin exposureConclusionBased on mechanistic static models and PBPK modeling and simulation, the OCT1/2 and MATEs inhibition potential of TMR and its metabolites on metformin pharmacokinetics is not clinically significant. No dose adjustment of metformin is necessary when co-administered with FTRDisclosuresXiusheng Miao, PhD, GlaxoSmithKline (Employee) Mindy Magee, Doctor of Pharmacy, GlaxoSmithKline (Employee, Shareholder) Peter D. Gorycki, BEChe, MSc, PhD, GSK (Employee, Shareholder) Katy P. Moore, PharmD, RPh, ViiV Healthcare (Employee)

L-tetrahydropalmatine attenuates cisplatin-induced nephrotoxicity via selective inhibition of organic cation transporter 2 without impairing its antitumor efficacy

Cisplatin is a first-line chemotherapeutic agent that is widely used for treatment of various solid tumors. However, cisplatin-induced adverse effects, particularly severe nephrotoxicity, preclude its application. In this study, we showed that L-tetrahydropalmatine (L-THP) could selectively inhibit organic cation transporter 2 (OCT2), which plays a crucial role in renal cisplatin uptake from the circulation. Additionally, we demonstrated that L-THP attenuated cisplatin-induced toxicity in mouse primary renal tubular cells. Subsequently, we verified that L-THP reduced the renal accumulation of cisplatin and alleviated cisplatin-induced renal injury in healthy and tumor-bearing nude mice. In healthy mice, co-treatment of L-THP at 5–40 mg/kg reduced cisplatin renal accumulation to 75.0%-49.9% of that in cisplatin alone group (10 mg/kg), and alleviated cisplatin-induced nephrotoxicity. Additionally, it did not alter Pt concentration in the tumor tissue and did not impair its antitumor efficacy in tumor bearing nude mice. The tumor inhibitory rates of cisplatin (10 mg/kg) co-treated with L-THP at 10, 20 and 40 mg/kg were 71.4%, 70.4% and 69.4%, respectively, in H460 tumor bearing nude mice, higher than that of in cisplatin alone group (60.6%), while in HCT116 tumor bearing nude mice, the tumor inhibitory rates in co-treated with 20 mg/kg L-THP was 34.7% (vs 26.3% in cisplatin alone group). Moreover, L-THP reduced cisplatin accumulation and alleviated cisplatin-induced cytotoxicity in human primary renal tubular cells. Therefore, our findings suggested that concomitant administration of L-THP could attenuate cisplatin-induced renal injury via selective inhibition of OCT2 without impairing its antitumor efficacy.

Investigating Plasma Membrane Monoamine Transporter (PMAT) as a Target for Development of Novel Antidepressant Treatments for Juveniles

Depression is a psychiatric illness that affects individuals of all ages, yet only two antidepressants are approved to treat depression in children – both belong to the selective serotonin reuptake inhibitor (SSRI) class. Moreover, children are less effectively treated by SSRIs than adults. SSRIs block reuptake of serotonin via the high‐affinity, low‐capacity serotonin transporter (SERT). The resulting increase in extracellular serotonin is thought to initiate a cascade of downstream effects, which underlie the therapeutic utility of SSRIs. However, other transporters also clear serotonin from extracellular fluid, including the low‐affinity, high‐capacity organic cation transporters (OCTs) and plasma membrane monoamine transporter (PMAT). Our lab has shown that, in adults, decynium‐22 (D22), an inhibitor of OCTs and PMAT, produces antidepressant‐like effects when SERT function is either genetically or pharmacologically impaired. However, it is unknown whether OCTs and/or PMAT may be useful targets for therapeutic interventions in juveniles. In contrast to adults, our preliminary studies show that D22 has antidepressant‐like effects in juvenile (postnatal day 21 (P21)) wild‐type mice, suggesting that OCTs and/or PMAT may be functionally upregulated in juvenile mice. Since PMAT has a higher affinity for serotonin compared to OCTs, we investigated the antidepressant‐like effects of D22 in male and female P21 PMAT wild‐type (+/+) and constitutive PMAT knockout (−/−) mice and found the antidepressant‐like effects of D22 were lost in P21 PMAT−/− mice. Using in vivo chronoamperometry, we examined serotonin clearance kinetics in CA3 region of hippocampus in P21 PMAT+/+ and PMAT−/− mice. Across serotonin concentrations ranging from 0.25 to 1.5 micromolar, we found no difference in serotonin clearance rate between genotypes. These data suggest that either a) these concentrations of serotonin are insufficient to engage PMAT, and/or b) SERT, or other OCT isoform, is upregulated in constitutive PMAT−/− mice. In either case, detection of PMAT‐dependent serotonin clearance would be obscured. Ongoing studies are investigating the effect of D22 on serotonin clearance in juveniles and adults over a range of serotonin concentrations, as well as evaluating PMAT expression in brain as a function of age and PMAT genotype. Together, these studies will expand our understanding of differences in serotonin transport mechanisms between juvenile and adult brain, and will aid in discovery of novel targets for the development of antidepressants with improved therapeutic efficacy for children suffering from depression.Support or Funding InformationThis work was supported by the National Institutes of Health R01 MH106978 to LCD