Substrates Of Organic Cation Transporters Research Articles

No dose adjustment of metformin or substrates of organic cation transporters (OCT)1 and OCT2 and multidrug and toxin extrusion protein (MATE)1/2K with fostemsavir coadministration based on modeling approaches.

Fostemsavir is an approved gp120-directed attachment inhibitor and prodrug for the treatment of human immunodeficiency virus type 1 infection in combination with other antiretrovirals (ARVs) in heavily treatment-experienced adults with multi-drug resistance, intolerance, or safety concerns with their current ARV regimen. Initial invitro studies indicated that temsavir, the active moiety of fostemsavir, and its metabolites, inhibited organic cation transporter (OCT)1, OCT2, and multidrug and toxin extrusion transporters (MATEs) at tested concentration of 100 uM, although risk assessment based on the current Food and Drug Administration invitro drug-drug interaction (DDI) guidance using the mechanistic static model did not reveal any clinically relevant inhibition on OCTs and MATEs. However, a DDI risk was flagged with EMA static model predictions. Hence, a physiologically based pharmacokinetic (PBPK) model of fostemsavir/temsavir was developed to further assess the DDI risk potential of OCT and MATEs inhibition by temsavir and predict changes in metformin (a sensitive OCT and MATEs substrate) exposure. No clinically relevant impact on metformin concentrations across a wide range of temsavir concentrations was predicted; therefore, no dose adjustment is recommended for metformin when co-administered with fostemsavir.

Cardiac Uptake of the Adrenergic Imaging Agent meta-Iodobenzylguanidine (mIBG) Is Mediated by Organic Cation Transporter 3 (Oct3).

Heart failure (HF) is a chronic disease affecting 1%-2% of the global population.123I-labeled meta-iodobenzylguanidine (mIBG) is US Food and Drug Administration-approved for cardiac imaging and prognosis risk assessment in patients with HF. As a norepinephrine analog, mIBG is believed to be transported into adrenergic nerve terminals by the neuronal norepinephrine transporter (NET) and hence image sympathetic innervation of the myocardium. We previously showed that mIBG is an excellent substrate of organic cation transporter 3 (OCT3), an extraneuronal transporter expressed in cardiomyocytes. Here, we evaluated the in vivo impact of Oct3 on mIBG disposition and tissue distribution using Oct3 knockout mice. Oct3 +/+ and Oct3 -/- mice were administered with mIBG intravenously, and mIBG plasma pharmacokinetics and tissue exposures were determined. In Oct3 +/+ mice, mIBG exhibited extensive accumulation in multiple tissues (heart, salivary gland, liver, and adrenal gland). No difference was observed in overall plasma exposure between Oct3 +/+ and Oct3 -/- mice. Strikingly, cardiac mIBG was depleted in Oct3 -/- mice, resulting in 83% reduction in overall cardiac exposure (AUC0-24 h: 12.7 vs. 2.1 μg × h/g). mIBG tissue exposure (AUC0-24 h) was also reduced by 66%, 36%, and 31% in skeletal muscle, salivary gland, and lung, respectively, in Oct3 -/- mice. Our data demonstrated that Oct3 is the primary transporter responsible for cardiac mIBG uptake in vivo and suggested that cardiac mIBG imaging mainly measures OCT3 activity in cardiomyocytes but not NET-mediated uptake in adrenergic nerve endings. Our findings challenge the current paradigm in interpreting cardiac mIBG imaging results and suggest OCT3 as a potential genetic risk marker for HF prognosis. SIGNIFICANCE STATEMENT: 123I-labeled meta-iodobenzylguanidine is used for cardiac imaging and risk assessment in heart failure patients. Contrary to the current belief that meta-iodobenzylguanidine (mIBG) tracks cardiac sympathetic innervation due to its uptake by the neuronal norepinephrine transporter, the authors demonstrated that cardiac mIBG uptake is mediated by the extraneuronal transporter Oct3. Their findings warrant a re-evaluation of the scientific rationale behind cardiac mIBG scan and further suggest organic cation transporter 3 as a risk factor for disease progression in heart failure patients.

Regulation of Transporters for Organic Cations by High Glucose.

Endogenous positively charged organic substances, including neurotransmitters and cationic uremic toxins, as well as exogenous organic cations such as the anti-diabetic medication metformin, serve as substrates for organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs). These proteins facilitate their transport across cell membranes. Vectorial transport through the OCT/MATE axis mediates the hepatic and renal excretion of organic cations, regulating their systemic and local concentrations. Organic cation transporters are part of the remote sensing and signaling system, whose activity can be regulated to cope with changes in the composition of extra- and intracellular fluids. Glucose, as a source of energy, can also function as a crucial signaling molecule, regulating gene expression in various organs and tissues. Its concentration in the blood may fluctuate in specific physiological and pathophysiological conditions. In this work, the regulation of the activity of organic cation transporters was measured by incubating human embryonic kidney cells stably expressing human OCT1 (hOCT1), hOCT2, or hMATE1 with high glucose concentrations (16.7 mM). Incubation with this high glucose concentration for 48 h significantly stimulated the activity of hOCT1, hOCT2, and hMATE1 by increasing their maximal velocity (Vmax), but without significantly changing their affinity for the substrates. These effects were independent of changes in osmolarity, as the addition of equimolar concentrations of mannitol did not alter transporter activity. The stimulation of transporter activity was associated with a significant increase in transporter mRNA expression. Inhibition of the mechanistic target of rapamycin (mTOR) kinase with Torin-1 suppressed the transporter stimulation induced by incubation with 16.7 mM glucose. Focusing on hOCT2, it was shown that incubation with 16.7 mM glucose increased hOCT2 protein expression in the plasma membrane. Interestingly, an apparent trend towards higher hOCT2 mRNA expression was observed in kidneys from diabetic patients, a pathology characterized by high serum glucose levels. Due to the small number of samples from diabetic patients (three), this observation must be interpreted with caution. In conclusion, incubation for 48 h with a high glucose concentration of 16.7 mM stimulated the activity and expression of organic cation transporters compared to those measured in the presence of 5.6 mM glucose. This stimulation by a diabetic environment could increase cellular uptake of the anti-diabetic drug metformin and increase renal tubular secretion of organic cations in an early stage of diabetes.

Role of Mouse Organic Cation Transporter 2 for Nephro- and Peripheral Neurotoxicity Induced by Chemotherapeutic Treatment with Cisplatin.

Cisplatin (CDDP) is an efficient chemotherapeutic agent broadly used to treat solid cancers. Chemotherapy with CDDP can cause significant unwanted side effects such as renal toxicity and peripheral neurotoxicity. CDDP is a substrate of organic cation transporters (OCT), transporters that are highly expressed in renal tissue. Therefore, CDDP uptake by OCT may play a role in causing unwanted toxicities of CDDP anticancer treatment. In this study, the contribution of the mouse OCT2 (mOCT2) to CDDP nephro- and peripheral neurotoxicity was investigated by comparing the effects of cyclic treatment with low doses of CDDP on renal and neurological functions in wild-type (WT) mice and mice with genetic deletion of OCT2 (OCT2-/- mice). This CDDP treatment protocol caused significant impairment of kidneys and peripherical neurological functions in WT mice. These effects were significantly reduced in OCT2-/- mice, however, less profoundly than what was previously measured in mice with genetic deletion of both OCT1 and 2 (OCT1-2-/- mice). Comparing the apparent affinities (IC50) of mOCT1 and mOCT2 for CDDP, the mOCT1 displayed a higher affinity for CDDP than the mOCT2 (IC50: 9 and 558 µM, respectively). Also, cellular toxicity induced by incubation with 100 µM CDDP was more pronounced in cells stably expressing mOCT1 than in cells expressing mOCT2. Therefore, in mice, CDDP uptake by both OCT1 and 2 contributes to the development of CDDP undesired side effects. OCT seem to be suitable targets for establishing treatment protocols aimed at decreasing unwanted CDDP toxicity and improving anticancer treatment with CDDP.

The Role of Organic Cation Transporters in the Pharmacokinetics, Pharmacodynamics and Drug-Drug Interactions of Tyrosine Kinase Inhibitors.

Tyrosine kinase inhibitors (TKIs) decisively contributed in revolutionizing the therapeutic approach to cancer, offering non-invasive, tolerable therapies for a better quality of life. Nonetheless, degree and duration of the response to TKI therapy vary depending on cancer molecular features, the ability of developing resistance to the drug, on pharmacokinetic alterations caused by germline variants and unwanted drug-drug interactions at the level of membrane transporters and metabolizing enzymes. A great deal of approved TKIs are inhibitors of the organic cation transporters (OCTs). A handful are also substrates of them. These transporters are polyspecific and highly expressed in normal epithelia, particularly the intestine, liver and kidney, and are, hence, arguably relevant sites of TKI interactions with other OCT substrates. Moreover, OCTs are often repressed in cancer cells and might contribute to the resistance of cancer cells to TKIs. This article reviews the OCT interactions with approved and in-development TKIs reported in vitro and in vivo and critically discusses the potential clinical ramifications thereof.

Using a competitive counterflow assay to identify novel cationic substrates of OATP1B1 and OATP1B3.

OATP1B1 and OATP1B3 are two drug transporters that mediate the uptake of multiple endo- and xenobiotics, including many drugs, into human hepatocytes. Numerous inhibitors have been identified, and for some of them, it is not clear whether they are also substrates. Historically radiolabeled substrates or LC-MS/MS methods were needed to test for transported substrates, both of which can be limiting in time and money. However, the competitive counterflow (CCF) assay originally described for OCT2 and, more recently, for OCT1, OATP2B1, and OATP1A2 does not require radiolabeled substrates or LC-MS/MS methods and, as a result, is a more cost-effective approach to identifying substrates of multidrug transporters. We used a CCF assay based on the stimulated efflux of the common model substrate estradiol-17β-glucuronide (E17βG) and tested 30 compounds for OATP1B1- and OATP1B3-mediated transport. Chinese Hamster Ovary (CHO) cells stably expressing OATP1B1 or OATP1B3 were preloaded with 10 nM [3H]-estradiol-17β-glucuronide. After the addition of known substrates like unlabeled estradiol-17β-glucuronide, estrone-3-sulfate, bromosulfophthalein, protoporphyrin X, rifampicin, and taurocholate to the outside of the preloaded CHO cells, we observed efflux of [3H]-estradiol-17β-glucuronide due to exchange with the added compounds. Of the tested 30 compounds, some organic cation transporter substrates like diphenhydramine, metformin, and salbutamol did not induce [3H]-estradiol-17β-glucuronide efflux, indicating that the two OATPs do not transport them. However, 22 (for OATP1B1) and 16 (for OATP1B3) of the tested compounds resulted in [3H]-estradiol-17β-glucuronide efflux, suggesting that they are OATP substrates. Among these compounds, we further tested clarithromycin, indomethacin, reserpine, and verapamil and confirmed that they are substrates of the two OATPs. These results demonstrate that the substrate spectrum of the well-characterized organic anion transporting polypeptides includes several organic cations. Furthermore, as for other drug uptake transporters, the CCF assay is an easy-to-use screening tool to identify novel OATP substrates.

High Affinity Decynium-22 Binding to Brain Membrane Homogenates and Reduced Dorsal Camouflaging after Acute Exposure to it in Zebrafish.

Organic cation transporters (OCTs) are expressed in the mammalian brain, kidney, liver, placenta, and intestines, where they facilitate the transport of cations and other substrates between extracellular fluids and cells. Despite increasing reliance on ectothermic vertebrates as alternative toxicology models, properties of their OCT homologs transporting many drugs and toxins remain poorly characterized. Recently, in zebrafish (Danio rerio), two proteins with functional similarities to human OCTs were shown to be highly expressed in the liver, kidney, eye, and brain. This study is the first to characterize in vivo uptake to the brain and the high-affinity brain membrane binding of the mammalian OCT blocker 1-1′-diethyl-2,2′cyanine iodide (decynium-22 or D-22) in zebrafish. Membrane saturation binding of [3H] D-22 in pooled zebrafish whole brain versus mouse hippocampal homogenates revealed a high-affinity binding site with a KD of 5 ± 2.5 nM and Bmax of 1974 ± 410 fmol/mg protein in the zebrafish brain, and a KD of 3.3 ± 2.3 and Bmax of 704 ± 182 fmol/mg protein in mouse hippocampus. The binding of [3H] D-22 to brain membrane homogenates was partially blocked by the neurotoxic cation 1-methyl-4-phenylpyridinium (MPP+), a known OCT substrate. To determine if D-22 bath exposures reach the brain, zebrafish were exposed to 25 nM [3H] D-22 for 10 min, and 736 ± 68 ng/g wet weight [3H] D-22 was bound. Acute behavioral effects of D-22 in zebrafish were characterized in two anxiety-relevant tests. In the first cohort of zebrafish, 12.5, 25, or 50 mg/L D-22 had no effect on their height in the dive tank or entries and time spent in white arms of a light/dark plus maze. By contrast, 25 mg/L buspirone increased zebrafish dive tank top-dwelling (p < 0.05), an anticipated anxiolytic effect. However, a second cohort of zebrafish treated with 50 mg/L D-22 made more white arm entries, and females spent more time in white than controls. Based on these findings, it appears that D-22 bath treatments reach the zebrafish brain and have partial anxiolytic properties, reducing anti-predator dorsal camouflaging, without increasing vertical exploration. High-affinity binding of [3H] D-22 in zebrafish brain and mouse brain was similar, with nanomolar affinity, possibly at conserved OCT site(s).

Lack of impact of OCTN1 gene polymorphisms on clinical outcomes of gabapentinoids in Pakistani patients with neuropathic pain.

Gabapentinoids are the first-line drugs for neuropathic pain. These drugs are the substrate of organic cation transporter (OCTN1) for renal excretion and absorption across the intestinal epithelium. Gabapentinoids exhibit wide interindividual variability in daily dosage and therapeutic efficacy which makes titration regimens prolonged for optimal efficacy. The present study aimed to investigate the possible influence of the single nucleotide polymorphism (SNP) of OCTN1 on therapeutic efficacy and safety of gabapentinoids in neuropathic pain patients of the Pakistani population. Four hundred and twenty-six patients were enrolled in the study. All participants were genotyped for OCTN1 rs1050152 and rs3792876 by PCR-RFLP method and followed up for eight weeks. The therapeutic outcomes of gabapentinoids, reduction in pain score, inadequate or complete lack of response, adverse events (AEs) in responders and discontinuation of treatment on account of AEs were recorded for all patients. There was no significant association of genotypes and alleles of both SNPs on the clinical response of gabapentinoids (P ˃ 0.05). Similarly, significant differences were not found in the reduction of pain scores and AEs among different genotypes in the responders. The present study has reported the association of OCTN1 rs1050152 and rs3792876 polymorphisms with clinical outcomes of gabapentinoids for the first time in the real-world clinical setting. Our results suggest a lack of influence of OCTN1 genetic variants in the determination of clinical response to gabapentinoids in patients with neuropathic pain in the Pakistani population. These findings signify the role of renal functions in predicting the interindividual variability to therapeutic responsiveness of gabapentinoids.

Effects of 1α,25-Dihydroxyvitamin D3 on the Pharmacokinetics of Procainamide and Its Metabolite N-Acetylprocainamide, Organic Cation Transporter Substrates, in Rats with PBPK Modeling Approach.

In this study, possible changes in the expression of rat organic cationic transporters (rOCTs) and rat multidrug and toxin extrusion proteins (rMATEs) following treatment with 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) were investigated. Rats received intraperitoneal administrations of 1,25(OH)2D3 for four consecutive days, and the tissues of interest were collected. The mRNA expression of rOCT1 in the kidneys was significantly increased in 1,25(OH)2D3-treated rats compared with the control rats, while the mRNA expressions of rOCT2 and rMATE1 in the kidneys, rOCT1 and N-acetyltransferase-II (NAT-II) in the liver, and rOCT3 in the heart were significantly decreased. Changes in the protein expression of hepatic rOCT1 and renal rOCT2 and rMATE1 were confirmed by western blot analysis. We further evaluated the pharmacokinetics of procainamide (PA) hydrochloride and its major metabolite N-acetyl procainamide (NAPA) in the presence of 1,25(OH)2D3. When PA hydrochloride was administered intravenously at a dose 10 mg/kg to 1,25(OH)2D3-treated rats, a significant decrease in renal and/or non-renal clearance of PA and NAPA was observed. A physiological model for the pharmacokinetics of PA and NAPA in rats was useful for linking changes in the transcriptional and translational expressions of rOCTs and rMATE1 transporters to the altered pharmacokinetics of the drugs.

MPEG2k-PCLx Polymeric Micelles Influence Pharmacokinetics and Hypoglycemic Efficacy of Metformin through Inhibition of Organic Cation Transporters in Rats.

Increasing evidence has shown that nanocarriers have effects on several efflux drug transporters. To date, little is known about whether influx transporters are also modulated. Herein, we investigated the impact of amphiphilic polymer micelles on the uptake function of organic cation transporters (OCTs) and the influence on the pharmacokinetics and pharmacodynamics of metformin, a well-characterized substrate of OCTs. Five types of polymeric micelles (mPEG2k-PCL2k, mPEG2k-PCL3.5k, mPEG2k-PCL5k, mPEG2k-PCL7.5k, and mPEG2k-PCL10k) were prepared to evaluate the inhibition of hOCT1-3-overexpressing Madin-Darby canine kidney cells. The mPEG2k-PCLx micelles played an inhibitory role above the critical micelle concentration. The inhibitory potency could be ranked as mPEG2k-PCL2k > mPEG2k-PCL3.5k > mPEG2k-PCL5k > mPEG2k-PCL7.5k > mPEG2k-PCL10k, which negatively declined with the increase of molecular weight of the hydrophobic segment. The inhibitory effects of polymeric micelles on the hOCT1 isoform were the most pronounced, with the lowest IC50 values ranging from 0.106 to 0.280 mg/mL. The mPEG2k-PCL2k micelles distinctly increased the plasma concentration of metformin and significantly decreased Vss by 35.6% (p < 0.05) after seven consecutive treatments in rats, which was interrelated with the restrained metformin distribution in the liver and kidney. The uptake inhibition of micelles on hepatic and renal rOcts also diminished the glucose-lowering effect of metformin and fasting insulin levels in the oral glucose tolerance test. Consistent with the inhibitory effects, the mRNA and protein levels of rOct1 and rOct2 were decreased in the liver, kidney, and small intestine. The present study demonstrated that mPEG2k-PCLx micelles could inhibit the transport function of OCTs, indicating a potential risk of drug-drug interactions during concomitant medication of nanomedicine with organic cationic drugs.

Characterization of cytochrome P450 (CYP) 2D6 drugs as substrates of human organic cation transporters and multidrug and toxin extrusion proteins.

The metabolic activity of cytochrome P450 (CYP) 2D6 is highly variable and CYP2D6 genotypes insufficiently explain the extensive and intermediate metabolic phenotypes, limiting the prediction of drug response plus adverse drug reactions. Since CYP2D6 prototypic substrates are positively charged, the aim of this study was to evaluate the organic cation transporters (OCTs) and multidrug and toxin extrusion proteins (MATEs) as potential contributors to the variability of CYP2D6 hydroxylation of debrisoquine, dextromethorphan, diphenhydramine, perhexiline and sparteine. OCT1/SLC22A1-, OCT2/SLC22A2-, OCT3/SLC22A3-, MATE1/SLC47A1-, and MATE2K/SLC47A2-overexpressing cell lines were used to investigate the transport of the selected drugs. Individuals from a study cohort, well defined with respect to CYP2D6 genotype and sparteine pharmacokinetics, were genotyped for the common OCT1 variants rs12208357 (OCT1-R61C), rs34130495 (OCT1-G401S), rs202220802 (OCT1-Met420del), rs34059508 (OCT1-G465R), OCT2 variant rs316019 (OCT2-A270S) and MATE1 variant rs2289669. Sparteine pharmacokinetics was stratified according to CYP2D6 and OCT1, OCT2 or MATE1 genotype. OCTs and MATE1 transport sparteine and debrisoquine with high affinity in vitro, but OCT- and MATE1-dependent transport of dextromethorphan, diphenhydramine and perhexiline was not detected. Sparteine and debrisoquine transport depends on OCT1 genotype; however, sparteine pharmacokinetics is independent from OCT1 genotype. Some drugs that are substrates of CYP2D6 are also substrates of OCTs and MATE1, suggesting overlapping specificities. Variability in sparteine hydroxylation in extensive and intermediate metabolizers cannot be explained by OCT1 genetic variants indicating presence of other factors. Dose-dependent toxicities of dextromethorphan, diphenhydramine and perhexiline appear to be independent from OCTs and MATEs.

Cellular Uptake of Psychostimulants – Are High- and Low-Affinity Organic Cation Transporters Drug Traffickers?

Psychostimulants are used therapeutically and for illegal recreational purposes. Many of these are inhibitors of the presynaptic noradrenaline, dopamine, and serotonin transporters (NET, DAT, and SERT). According to their physicochemical properties, some might also be substrates of polyspecific organic cation transporters (OCTs) that mediate uptake in liver and kidneys for metabolism and excretion. OCT1 is genetically highly polymorphic, with strong effects on transporter activity and expression. To study potential interindividual differences in their pharmacokinetics, 18 psychostimulants and hallucinogens were assessed in vitro for transport by different OCTs as well as by the high-affinity monoamine transporters NET, DAT, and SERT. The hallucinogenic natural compound mescaline was found to be strongly transported by wild-type OCT1 with a K m of 24.3 µM and a v max of 642 pmol × mg protein−1 × min−1. Transport was modestly reduced in variants *2 and *7, more strongly reduced in *3 and *4, and lowest in *5 and *6, while *8 showed a moderately increased transport capacity. The other phenylethylamine derivatives methamphetamine, para-methoxymethamphetamine, (-)-ephedrine, and cathine ((+)-norpseudoephedrine), as well as dimethyltryptamine, were substrates of OCT2 with K m values in the range of 7.9–46.0 µM and v max values between 70.7 and 570 pmol × mg protein−1 × min−1. Affinities were similar or modestly reduced and the transport capacities were reduced down to half in the naturally occurring variant A270S. Cathine was found to be a substrate for NET and DAT, with the Km being 21-fold and the v max 10-fold higher for DAT but still significantly lower compared to OCT2. This study has shown that several psychostimulants and hallucinogens are substrates for OCTs. Given the extensive cellular uptake of mescaline by the genetically highly polymorphic OCT1, strong interindividual variation in the pharmacokinetics of mescaline might be possible, which could be a reason for highly variable adverse reactions. The involvement of the polymorphic OCT2 in the renal excretion of several psychostimulants could be one reason for individual differences in toxicity.

Pharmacokinetics of Imeglimin in Subjects with Moderate Hepatic Impairment.

Imeglimin is a novel oral antidiabetic drug used to treat type 2 diabetes, targeting the mitochondrial bioenergetics. Imeglimin is mainly excreted unchanged by the kidneys and is a substrate of organic cation transporters, which are expressed in the kidney and the liver. The aim of this study was to assess the effect of hepatic impairment on the pharmacokinetics of imeglimin. An open-label, single-dose, parallel-group study was carried out in seven subjects with normal hepatic function and seven subjects with moderate hepatic impairment who received a single dose of imeglimin 1000mg. Blood and urine samples were collected up to 48h after imeglimin administration. Pharmacokinetics were determined using non-compartmental methods. Imeglimin maximum observed plasma concentration (Cmax) and area under the plasma concentration-time curve (AUC) in subjects with moderate hepatic impairment was 1.3-fold (90% confidence interval [CI] 1.05-1.60) and 1.5-fold (90% CI 1.19-1.82) higher than in subjects with normal hepatic function, but was not considered as clinically meaningful. Higher plasma exposure and amount of imeglimin renally excreted in moderate hepatic impaired subjects, associated with an unchanged elimination rate, suggests that this increase could be linked to a higher oral absorption and/or lower hepatic uptake in this population. Imeglimin was safe and well tolerated in all subjects. EudraCT 2018-001950-83.

A Comprehensive Whole-Body Physiologically Based Pharmacokinetic Drug–Drug–Gene Interaction Model of Metformin and Cimetidine in Healthy Adults and Renally Impaired Individuals

BackgroundMetformin is a widely prescribed antidiabetic BCS Class III drug (low permeability) that depends on active transport for its absorption and disposition. It is recommended by the US Food and Drug Administration as a clinical substrate of organic cation transporter 2/multidrug and toxin extrusion protein for drug–drug interaction studies. Cimetidine is a potent organic cation transporter 2/multidrug and toxin extrusion protein inhibitor.ObjectiveThe objective of this study was to provide mechanistic whole-body physiologically based pharmacokinetic models of metformin and cimetidine, built and evaluated to describe the metformin-SLC22A2 808G>T drug–gene interaction, the cimetidine-metformin drug–drug interaction, and the impact of renal impairment on metformin exposure.MethodsPhysiologically based pharmacokinetic models were developed in PK-Sim® (version 8.0). Thirty-nine clinical studies (dosing range 0.001–2550 mg), providing metformin plasma and urine data, positron emission tomography measurements of tissue concentrations, studies in organic cation transporter 2 polymorphic volunteers, drug–drug interaction studies with cimetidine, and data from patients in different stages of chronic kidney disease, were used to develop the metformin model. Twenty-seven clinical studies (dosing range 100–800 mg), reporting cimetidine plasma and urine concentrations, were used for the cimetidine model development.ResultsThe established physiologically based pharmacokinetic models adequately describe the available clinical data, including the investigated drug–gene interaction, drug–drug interaction, and drug–drug–gene interaction studies, as well as the metformin exposure during renal impairment. All modeled drug–drug interaction area under the curve and maximum concentration ratios are within 1.5-fold of the observed ratios. The clinical data of renally impaired patients shows the expected increase in metformin exposure with declining kidney function, but also indicates counter-regulatory mechanisms in severe renal disease; these mechanisms were implemented into the model based on findings in preclinical species.ConclusionsWhole-body physiologically based pharmacokinetic models of metformin and cimetidine were built and qualified for the prediction of metformin pharmacokinetics during drug–gene interaction, drug–drug interaction, and different stages of renal disease. The model files will be freely available in the Open Systems Pharmacology model repository. Current guidelines for metformin treatment of renally impaired patients should be reviewed to avoid overdosing in CKD3 and to allow metformin therapy of CKD4 patients.Electronic supplementary materialThe online version of this article (10.1007/s40262-020-00896-w) contains supplementary material, which is available to authorized users.

Organic Cation Transporter Blocker Decynium 22 and Substrates Metformin and Berberine Enhance Social Interaction Preference in Adult Male Mice, Perhaps by Modulating Corticosterone Release

Social withdrawal in psychiatric disorders such as schizophrenia, depression and dementia resemble impaired preference for social interaction, which is a treatment‐resistant core symptom of autism. Deficient serotonin (5‐HT) signaling is strongly implicated as a contributing factor common to these psychiatric and neurodegenerative disorders. The relative lack of clinical efficacy of selective 5‐HT reuptake inhibitors (SSRIs) to improve social behavior deficits in these conditions has brought into question their therapeutic utility for this debilitating symptom. Given the limitations associated with treating sociability deficits by targeting the 5‐HT transporter (SERT), our initial goal was to characterize the effects of blocking auxiliary ‘uptake 2’ transporters of 5‐HT on social behaviors in mice. Organic cation transporters (OCTs) are among the ancillary 5‐HT transporters found in brain, and they are blocked endogenously by steroid hormones such as corticosterone. Male BTBR T+Itpr3tf/J (BTBR) mice have elevated serum corticosterone, particularly in adolescence, and they exhibit inherent deficits in preference for social interaction. In three‐chamber sociability preference tests the OCT blocker decynium‐22 (D‐22) enhanced social interactions in otherwise socially‐deficient BTBR mice, and was without adverse effect on the social behaviors of C57BL/6J (B6) mice (p &lt; 0.05, N= 8–9). Furthermore, D‐22, and OCT substrates, berberine and metformin, all similarly attenuated the social dominance of BTBR mice in tube tests. However in the course of our studies we also found that the alkaloid antibiotic berberine and the anti‐diabetic drug metformin also had sociability enhancing properties similar to D‐22 in BTBR mice. Yet as compared to D‐22 these substrates are poor blockers of synaptosomal 5‐HT uptake. For example, a lower concentration of D‐22 is required to inhibit cortical [3H] 5‐HT uptake, with a Michaelis constant (Km) ≈ 0.5 μM, while 500x more berberine is required to equivalently inhibit it (Km ≈ 240 μM), and metformin is an even less potent 5‐HT uptake inhibitor (Km &gt; 1000 μM, N = 3). However we and others have observed that D‐22, berberine and metformin can attenuate circulating corticosterone levels. Our findings confirm that systemic OCT blockade by D‐22 may be a promising strategy for improving social behavior, but the mechanism of action of berberine and metformin to similarly do so remain unclear. This warrants further investigation into how these compounds modulate corticosterone release, and how serum corticosterone relates to sociability impairments.Support or Funding InformationAutism Idea Award (AR110109), Department of Defense ‐ Congressionally Directed Medical Research Programs; South Texas Advanced Research Training: Undergraduate Program (R25GM097632), National Institutes of HealthD‐22 is a more potent 5‐HT uptake blocker than berberine.Figure 1

Drug-drug interaction between crizotinib and entecavir via renal secretory transporter OCT2

Both entecavir and crizotinib are substrates of organic cation transporter 2 (OCT2). The aim of present study was to investigate the mechanisms of drug interactions between these two drugs. Kinetic analysis of entecavir on crizotinib uptake was conduct. Plasma concentration of crizotinib in rats and lung cancer patients, uptake of crizotinib in kidney slices and OCT2 transfected cells, were determined by LC-MS/MS. The clinical pharmacokinetic interactions and impact on adverse reaction of crizotinib in lung cancer patients were investigated. Steady-state through concentration of crizotinib was measured. The crizotinib-related adverse reactions were recorded in lung cancer patients with and without entecavir. Entecavir and 1-methyl-4-phenylpyridinium iodide significantly inhibited the uptake of crizotinib in kidney slices. Kinetic constants for crizotinib uptake by OCT2 were Km 1.16 ± 0.26 µM, Vmax 12.05 ± 0.53 µmol/min mg-1 protein and Ki 9.711 nM. Entecavir can inhibit crizotinib transport by OCT2 in kidney. Co-administration of entecavir significantly reduced the elimination of crizotinib in rats. In lung cancer patients, the steady-state AUCss of crizotinib increased approximately 1.2 fold (p < 0.05) but clearance was decreased by approximately 15% in the presence of entecavir. Steady-state through concentration of crizotinib significantly increased 1.3-fold when co-administrated with entecavir (p>0.001). Co-medication of entecavir significantly (p < 0.05) increased the risks of vision disorders, diarrhea and vomiting 1.6-, 2.3- and 1.8-fold. Entecavir could increase the exposure and reduce the elimination of crizotinib in lung cancer patients. Moreover, the presence of entecavir could significantly increase the incidences of adverse reaction of crizotinib.

Sulfenamide derivatives can improve transporter-mediated cellular uptake of metformin and induce cytotoxicity in human breast adenocarcinoma cell lines

Metformin, the most frequently administered oral anti-diabetic drug, is a substrate for organic cation transporters (OCTs). This determines not only its pharmacokinetic properties but also its biochemical effects in humans, including its recently-discovered antiproliferative properties. The aim of the study was to verify the hypothesis whether chemical modification of its biguanide backbone may increase the cellular uptake and antiproliferative efficacy of metformin.The study examines five sulfenamide derivatives of metformin with differing lengths of alkyl chains. It determines their cellular uptake and the role of OCTs in their transport in human breast adenocarcinoma cells (epithelial-like MCF-7, and MDA-MB-231). It also evaluates whether increased cellular uptake of metformin derivatives is associated with their cytotoxic properties.Sulfenamide derivatives were characterized by a greater ability to bind to OCTs than metformin. Compound 2 with n-octyl alkyl chain was found to possess the greatest affinity towards OCTs, as measured by determination of [14C]choline uptake inhibition (IC50 = 236.1 ± 1.28 μmol/L, and 217.4 ± 1.33 μmol/L, for MCF-7 and MDA-MB-231 respectively). Sulfenamides were also found to exhibit better cellular uptake in comparison with the parent drug, metformin. For instance, the uptake of cyclohexyl derivative 1 was 1.28 ± 0.19 nmol/min/mg of proteins and thus was 12-fold higher than the metformin in MCF-7 cells. Furthermore, higher uptake was associated with the greatest antiproliferative properties expressed as the lowest IC50 value i.e. inhibiting the growth of 50% of the cells (IC50 = 0.72 ± 1.31 μmol/L).Collectively, chemical modification of metformin into sulfenamides with different alkyl substituents obtains better substrates for OCTs, and subsequently higher cellular uptake in MCF-7 and MDA-MB-231 cells. Additionally, the length of alkyl chain introduced to the sulfenamides was found to influence selectivity and transport efficiency via OCT1 compared to other possible transporters, as well as potential intracellular activity and cytotoxicity.

Physiologically Based Pharmacokinetic Modeling of Transporter-Mediated Hepatic Clearance and Liver Partitioning of OATP and OCT Substrates in Cynomolgus Monkeys.

In the present investigations, we evaluate in vitro hepatocyte uptake and partitioning for the prediction of in vivo clearance and liver partitioning. Monkeys were intravenously co-dosed with rosuvastatin and bosentan, substrates of the organic anion transporting polypeptides (OATPs), and metformin, a substrate of organic cation transporter 1 (OCT1). Serial plasma and liver samples were collected over time. Liver and plasma unbound fraction was determined using equilibrium dialysis. In vivo unbound partitioning (Kpu,u) for rosuvastatin, bosentan, and metformin, calculated from total concentrations in the liver and plasma, were 243, 553, and 15, respectively. A physiologically based pharmacokinetic monkey model that incorporates active and passive hepatic uptake was developed to fit plasma and liver concentrations. In addition, a two-compartment model was used to fit in vitro hepatic uptake curves in suspended monkey hepatocyte to determine active uptake, passive diffusion, and intracellular unbound fraction parameters. At steady-state in the model, in vitro Kpu,u was determined. The results demonstrated that in vitro values under-predicted in vivo active uptake for rosuvastatin, bosentan, and metformin by 6.7-, 28-, and 1.5-fold, respectively, while passive diffusion was over-predicted. In vivo Kpu,u values were under-predicted from in vitro data by 30-, 79-, and 3-fold. In conclusion, active uptake and liver partitioning in monkeys for OATP substrates were greatly under-predicted from in vitro hepatocyte uptake, while OCT-mediated uptake and partitioning scaled reasonably well from in vitro, demonstrating substrate- and transporter-dependent scaling factors. The combination of in vitro experimental and modeling approaches proved useful for assessing prediction of in vivo intracellular partitioning.

Glycosylated Platinum(IV) Complexes as Substrates for Glucose Transporters (GLUTs) and Organic Cation Transporters (OCTs) Exhibited Cancer Targeting and Human Serum Albumin Binding Properties for Drug Delivery.

Glycosylated platinum(IV) complexes were synthesized as substrates for GLUTs and OCTs for the first time, and the cytotoxicity and detailed mechanism were determined in vitro and in vivo. Galactoside Pt(IV), glucoside Pt(IV), and mannoside Pt(IV) were highly cytotoxic and showed specific cancer-targeting properties in vitro and in vivo. Glycosylated platinum(IV) complexes 5, 6, 7, and 8 (IC50 0.24-3.97 μM) had better antitumor activity of nearly 166-fold higher than the positive controls cisplatin (1a), oxaliplatin (3a), and satraplatin (5a). The presence of a hexadecanoic chain allowed binding with human serum albumin (HSA) for drug delivery, which not only enhanced the stability of the inert platinum(IV) prodrugs but also decreased their reduction by reductants present in human whole blood. Their preferential accumulation in cancer cells compared to noncancerous cells (293T and 3T3 cells) suggested that they were potentially safe for clinical therapeutic use.

Effect of Organic Cation Transporter Genetic Polymorphism on Ethambutol Pharmacokinetics Using Physiologically Based Pharmacokinetic (PBPK) Model

Ethambutol is a potent antimycobacterial agent used in tuberculosis (TB) treatment. Renal elimination is the major route and recently reported as a substrate of organic cation transporters. We previously reported that genetic polymorphism in OCT transporters has decreased metformin clearance. Therefore, we aimed to evaluate effect of OCT2 variants on ethambutol transport in vitro and prediction of pharmacokinetics using physiologically based pharmacokinetic modeling. The transport experiments were conducted using human embryonic kidney cells stably transfected with human OCT2 transporter wild type and variants (T199I, T201M and A270S) in vitro. A full PBPK model for ethambutol was developed following permeability limited mechanistic kidney model (MechKim) in the SIMCYP simulator (Ver 15.1) using in vitro data. A 25mg/kg single dose for the healthy subjects in the simulator was used to build the model. It was then optimized and verified using clinical pharmacokinetic data. The verified PBPK model was then used to predict the effect of OCT2 genetic variants on ethambutol pharmacokinetics in healthy subjects in the SIMCYP population database. The ethambutol uptake was saturable and active for OCT2. The genetic variants of OCT2-T199I (1% in KOR), -T201M (2% in KOR) and -A270S (14% in OR) were reduced 3.2-fold, 7-fold, and 1.7-fold ethambutol transport compared with wild-type, respectively. The predicted pharmacokinetics showed the unchanged amount of ethambutol excretion in urine and the total amount in kidney cells were 2-5 fold difference between the wild and mutant groups. The AUC ratio were 1.42, 1.40 and 1.36 fold higher and CL were 30.2, 29.3 and 28.7 % lower for having T199I, T201M and A270S subjects compared to wild type. This in vitro to in vivo extrapolation study showed genetic polymorphism in OCT2 transporter affected on ethambutol pharmacokinetics which may explain interindividual response.