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.

Impact of Sex and Genetic Variation in Relevant Pharmacogenes on the Pharmacokinetics and Safety of Valsartan, Olmesartan and Hydrochlorothiazide.

Drug combination therapy is the most common pharmacological strategy for hypertension management. No pharmacogenetic biomarkers for guiding hypertension pharmacotherapy are available to date. The study population were 64 volunteers from seven bioequivalence trials investigating formulations with valsartan, olmesartan and/or hydrochlorothiazide. Every volunteer was genotyped for 10 genetic variants in different transporters' genes. Additionally, valsartan-treated volunteers were genotyped for 29 genetic variants in genes encoding for different metabolizing enzymes. Variability in pharmacokinetic parameters such as maximum concentration (Cmax) and time to reach it (tmax), the incidence of adverse drug reactions (ADRs) and blood pressure measurements were analyzed as a function of pharmacogenetic and demographic parameters. Individuals with the ABCB1 rs1045642 T/T genotype were associated with a higher valsartan tmax compared to those with T/G and G/G genotypes (p < 0.001, ß = 0.821, R2 = 0.459) and with a tendency toward a higher postural dizziness incidence (11.8% vs. 0%, p = 0.070). A higher hydrochlorothiazide dose/weight (DW)-corrected area under the curve (AUC∞/DW) was observed in SLC22A1 rs34059508 G/A volunteers compared to G/G volunteers (p = 0.050, ß = 1047.35, R2 = 0.051), and a tendency toward a higher postural dizziness incidence (50% vs. 1.6%, p = 0.063). Sex impacted valsartan and hydrochlorothiazide pharmacokinetics, showing a lower exposure in women, whereas no significant differences were found for olmesartan pharmacokinetics.

Role of human organic cation transporter-1 (OCT-1/SLC22A1) in modulating the response to metformin in patients with type 2 diabetes.

BACKGROUND: Organic cation transporter 1 primarily governs the action of metformin in the liver. There are considerable inter-individual variations in metformin response. In light of this, it is crucial to obtain a greater understanding of the influence of OCT1 expression or polymorphism in the context of variable responses elicited by metformin treatment. RESULTS: We observed that the variable response to metformin in the responders and non-responders is independent of isoform variation and mRNA expression of OCT-1. We also observed an insignificant difference in the serum metformin levels of the patient groups. Further, molecular docking provided us with an insight into the hotspot regions of OCT-1 for metformin binding. Genotyping of these regions revealed SNPs 156T>C and 1222A>G in both the groups, while as 181C>T and 1201G>A were found only in non-responders. The 181T>C and 1222A>G changes were further found to alter OCT-1 structure in silico and affect metformin transport in vitro which was illustrated by their effect on the activation of AMPK, the marker for metformin activity. CONCLUSION: Taken together, our results corroborate the role of OCT-1 in the transport of metformin and also point at OCT1 genetic variations possibly affecting the transport of metformin into the cells and hence its subsequent action in responders and non-responders.

Relationships between Inhibition, Transport and Enhanced Transport via the Organic Cation Transporter 1.

The organic cation transporter 1 (OCT1, SLC22A1) transports a large number of structurally diverse endogenous and exogenous substrates. There are numerous known competitive and non-competitive inhibitors of OCT1, but there are no studies systematically analyzing the relationship between transport, stimulation, and inhibition. Here, we tested in vitro OCT1 inhibition by OCT1 substrates and transport of OCT1 inhibitors under uniform analytical conditions. Beyond inhibition testing with two model substrates, we tested nine additional OCT1 substrates for their mutual inhibition. Inhibition of ASP+ uptake by most OCT1 substrates was weak. The model substrate sumatriptan, with its moderately stronger inhibitability, was used to confirm this. Interestingly, OCT1 substrates exhibiting stronger OCT1 inhibition were mainly biaromatic ß-agonistic drugs, such as dobutamine, fenoterol, ractopamine and ritodrine. Biaromatic organic cations were both, strong inhibitors and good substrates, but many OCT1 substrates showed little pairwise inhibition. Surprisingly, sumatriptan did significantly enhance dobutamine uptake. This effect was concentration dependent and additional experiments indicated that efflux inhibition may be one of the underlying mechanisms. Our data suggests, that OCT1 substrates are mainly weak OCT1 inhibitors and among those inhibiting well, noncompetitive inhibition could be responsible. Weak competitive inhibition confirms that OCT1 inhibition screenings poorly predict OCT1 substrates. Additionally, we showed that the OCT1 substrate sumatriptan can enhance uptake of some other OCT1 substrates. OCT1 transport stimulation was already observed earlier but is still poorly understood. Low OCT1 uptake inhibition and strong OCT1 efflux inhibition could be mechanisms exploitable for enhancing transport.

Cloning and Functional Characterization of Dog OCT1 and OCT2: Another Step in Exploring Species Differences in Organic Cation Transporters.

OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.

MPP+-Induced Changes in Cellular Impedance as a Measure for Organic Cation Transporter (SLC22A1-3) Activity and Inhibition.

The organic cation transporters OCT1-3 (SLC22A1-3) facilitate the transport of cationic endo- and xenobiotics and are important mediators of drug distribution and elimination. Their polyspecific nature makes OCTs highly susceptible to drug-drug interactions (DDIs). Currently, screening of OCT inhibitors depends on uptake assays that require labeled substrates to detect transport activity. However, these uptake assays have several limitations. Hence, there is a need to develop novel assays to study OCT activity in a physiological relevant environment without the need to label the substrate. Here, a label-free impedance-based transport assay is established that detects OCT-mediated transport activity and inhibition utilizing the neurotoxin MPP+. Uptake of MPP+ by OCTs induced concentration-dependent changes in cellular impedance that were inhibited by decynium-22, corticosterone, and Tyrosine Kinase inhibitors. OCT-mediated MPP+ transport activity and inhibition were quantified on both OCT1-3 overexpressing cells and HeLa cells endogenously expressing OCT3. Moreover, the method presented here is a valuable tool to identify novel inhibitors and potential DDI partners for MPP+ transporting solute carrier proteins (SLCs) in general.

Fine Mapping and Functional Analysis Reveal a Role of SLC22A1 in Acylcarnitine Transport.

Genome-wide association studies have identified a signal at the SLC22A1 locus for serum acylcarnitines, intermediate metabolites of mitochondrial oxidation whose plasma levels associate with metabolic diseases. Here, we refined the association signal, performed conditional analyses, and examined the linkage structure to find coding variants of SLC22A1 that mediate independent association signals at the locus. We also employed allele-specific expression analysis to find potential regulatory variants of SLC22A1 and demonstrated the effect of one variant on the splicing of SLC22A1. SLC22A1 encodes a hepatic plasma membrane transporter whose role in acylcarnitine physiology has not been described. By targeted metabolomics and isotope tracing experiments in loss- and gain-of-function cell and mouse models of Slc22a1, we uncovered a role of SLC22A1 in the efflux of acylcarnitines from the liver to the circulation. We further validated the impacts of human variants on SLC22A1-mediated acylcarnitine efflux in vitro, explaining their association with serum acylcarnitine levels. Our findings provide the detailed molecular mechanisms of the GWAS association for serum acylcarnitines at the SLC22A1 locus by functionally validating the impact of SLC22A1 and its variants on acylcarnitine transport.

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.

Organic Cation Transporters in the Lung-Current and Emerging (Patho)Physiological and Pharmacological Concepts.

Organic cation transporters (OCT) 1, 2 and 3 and novel organic cation transporters (OCTN) 1 and 2 of the solute carrier 22 (SLC22) family are involved in the cellular transport of endogenous compounds such as neurotransmitters, l-carnitine and ergothioneine. OCT/Ns have also been implicated in the transport of xenobiotics across various biological barriers, for example biguanides and histamine receptor antagonists. In addition, several drugs used in the treatment of respiratory disorders are cations at physiological pH and potential substrates of OCT/Ns. OCT/Ns may also be associated with the development of chronic lung diseases such as allergic asthma and chronic obstructive pulmonary disease (COPD) and, thus, are possible new drug targets. As part of the Special Issue "Physiology, Biochemistry and Pharmacology of Transporters for Organic Cations", this review provides an overview of recent findings on the (patho)physiological and pharmacological functions of organic cation transporters in the lung.

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.

Multiple binding sites in organic cation transporters require sophisticated procedures to identify interactions of novel drugs.

In vitro evaluation of drugs for interaction with transporters is essential during drug development. As polyspecific organic cation transporters (OCTs) are critical for pharmacokinetics of many cationic drugs, in vitro testing of human OCT1 and human OCT2 is recommended. In the currently applied tests it is determined whether uptake of one model cation in stably transfected epithelial cells is inhibited using a substrate concentration in the micromolar range. In this review experimental evidence for the existence of low- and high-affinity cation binding sites in OCTs that may interact with drugs is compiled. Most data were obtained from studies performed with rat Oct1. Whereas overlapping low-affinity cation binding sites are directly involved in transport, the high-affinity cation binding sites may induce allosteric inhibition of transport. Remarkably, high-affinity inhibition is only observed when uptake is measured using nanomolar substrate concentrations far below the respective Km values. Affinities of inhibitors are dependent on molecular structure and concentration of the employed substrate. Because the currently applied in vitro tests for identification of interaction of novel drugs with OCTs do not consider the influence of substrate structure and are not capable of identifying high-affinity inhibition, more sophisticated testing protocols are proposed.

Functional transport of organic anions and cations in the murine mesonephros.

The mesonephros of mammals is a transient renal structure that contributes to various aspects of mammalian fetal development, including the male reproductive system, hematopoietic stem cells, and vascular endothelial cells. The mesonephros develops from the intermediate mesoderm and forms tubules that are segmented in a similar way to the nephrons of the permanent kidney (but lacking loops of Henle). Early studies have suggested that the mesonephros in marsupials and some placental mammals may perform an excretory function, but these studies have not directly shown active transport of organic anions and cations. Excretory function in the rodent mesonephros has not been investigated. Functional characterization of the earliest stages of mammalian renal development is important for our understanding of congenital disease and may help to inform the growing field of renal tissue engineering. Here, we use live uptake and efflux assays in vitro to show that the murine mesonephros is able to transport organic anions and cations through specific transporters from early in its development. Transcript analysis suggests that there are subtle differences between the transporters involved in uptake and efflux by the murine permanent metanephric tubules and by the mesonephric tubules. These data suggest that the mammalian mesonephros can provide an excretory function for the early developing embryo, in addition to the excretory function provided by the placenta.

Impact of SLC22A1 and CYP3A5 genotypes on imatinib response in chronic myeloid leukemia: A systematic review and meta-analysis.

Contrasting results have been reported on the role of rs628031 and rs683369 polymorphisms of SLC22A1 and rs776746 of CYP3A5 on imatinib treatment response in patients with chronic myeloid leukemia (CML). In the present study, we conducted a systematic review and meta-analysis of published studies to estimate the impact of the above-mentioned gene variants on major molecular response (MMR) or complete cytogenetic response (CCyR) in imatinib-treated CML patients. We performed a comprehensive search through PubMed, Web of Knowledge, and Cochrane databases up to September 2017. The pooled analyses showed association between carriers of SLC22A1 rs628031A allele (GA + AA vs GG, OR: 0.58, 95% CI: 0.38-0.88, P = 0.011) or rs683369G allele (CG + GG vs CC, OR: 0.64, 95% CI: 0.42-0.96, P = 0.032) and a lower MMR rate. The combined analyses also revealed a correlation between the dominant (GG + AG vs AA, OR: 2.43, 95%CI: 1.12-5.27, P = 0.024) or the allelic model (G vs A, OR: 1.72, 95% CI: 1.09-2.72, P = 0.020) of CYP3A5 rs776746 with higher CCyR rates. The subsequent sensitivity analysis confirmed the statistical significance of CYP3A5 rs776746 among Asian CML patients (dominant model OR: 3.90; 95%CI: 2.47-6.14, P < 0.001; allelic model OR: 2.08; 95% CI: 1.47-2.95, P < 0.001). In conclusion, the present meta-analysis supports the association of SLC22A1 and CYP3A5 genotypes with clinical imatinib response rates of CML patients, nevertheless further large studies, particularly in Caucasians, are still warranted to provide conclusive evidences.

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.

Association of the Reduced Function Met420del Polymorphism of SLC22A1 with Metformin-Induced Gastrointestinal Intolerance in Ethiopian Patients with Type 2 Diabetes Mellitus.

Background: Despite its widespread use and favored profile, there are extensive variations in the treatment outcome of metformin therapy. Furthermore, studies reported that the inter-individual variability in the occurrence of metformin treatment associated side effects were related to the differences in individual genetic profiles. Thus, this study aimed to evaluate whether the reduced function methionine deletion at codon 420 (Met420del) variant of SLC22A1 (rs72552763) is associated with metformin induced gastrointestinal intolerance in Ethiopian patients with type 2 diabetes mellitus (T2DM). Patients and Methods: A retrospective observational study was conducted on 47 T2DM patients on metformin treatment for <3 years to assess the association of SLC22A1 (rs72552763) polymorphism with metformin induced gastrointestinal intolerance. Accordingly, 24 metformin tolerant and 23 metformin intolerant individuals with T2DM were recruited. Genotyping of rs72552763 was performed using TaqMan® Drug Metabolism Enzyme Genotyping Assay and its association to metformin induced gastrointestinal intolerance was assessed based on switching to a new class of glucose lowering agents or failure to up titrate dose due to metformin induced gastrointestinal intolerance. Chi-square, logistic regression and Mann-Whitney statistical tests were used as appropriate. Statistical significance was set at p < 0.05. Results: In our study, no significant association was observed between rs72552763 and metformin induced gastrointestinal intolerance. We found that the female gender and physical inactivity were risk factors for metformin gastrointestinal intolerance. Conclusion: Our study found that the Met420del variant of SLC22A1 (rs72552763) was not associated with metformin induced gastrointestinal intolerance in Ethiopian patients with T2DM. This is the study first to investigate the association of rs72552763 with metformin intolerance in the Ethiopian population with T2DM. However, the findings need to be cautiously interpreted given the relatively small sample size. In addition, a more complete investigation of SLC22A1 variants would be required to fully assess the effect of the gene on metformin induced gastrointestinal intolerance as several variants with a more severe loss of function have been described.

Impact of SLC22A1 variants rs622342 and rs72552763 on HbA1c and metformin plasmatic concentration levels in patients with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is a major global health problem. Response to first-line therapy is variable. This is partially due to interindividual variability across those genes codifying transport, metabolising, and drug activation proteins involved in first-line pharmacological treatment. Single nucleotide polymorphisms (SNPs) of genes SLC22A1, SLC22A2 and SLC22A3 affect metformin therapeutic response in patients with T2DM patients. The present study investigated allelic and genotypic frequencies of organic cation (OCT)1, OCT2, and OCT3 polymorphisms among metformin-treated patients with type 2 diabetes mellitus (T2DM). It also reports the association between clinical and genetic variables with glycated haemoglobin (HbA1c) control in 59 patients with T2DM. Patients were genotyped through real-time PCR (TaqMan assays). Metformin plasmatic levels were determined by mass spectrometry. Neither the analysis of HbA1c control by SNPs in SLC22A1, SLC22A2 and SLC22A3, nor the dominant genotypic model analysis yielded statistical significance between genotypes in polymorphisms rs72552763 (P=0.467), rs622342 (P=0.221), rs316019 (P=0.220) and rs2076828 (P=0.215). HbA1c levels were different in rs72552763 [GAT/GAT, 6.0 (5.7-6.6), GAT/del=6.5 (6.2-9.0), del/del=6.5 (6.4-6.8); P=0.022] and rs622342 [A/A=6.0 (5.8-6.5), A/C=6.4 (6.1-7.7), C/C=6.8 (6.4-9.3); P=0.009] genotypes. The dominant genotypic model found the lowest HbA1c levels in GAT/GAT (P=0.005) and A/A (P=0.010), in rs72552763 (GAT/GAT vs. GAT/del + del/del) and rs622342 (A/A vs. A/C + CC), respectively. There was a significant correlation between HbA1c levels and metformin dosage amongst del allele carriers in rs72552763 (ß1=0.14, P<0.001, r2=0.387), as opposed to GAT/GAT in rs72552763. There were no differences between HbA1c values in the test set and those predicted by machine learning models employing a simple linear regression based on metformin dosage. Therefore, rs72552763 and rs622342 polymorphisms in SLC22A1 may affect metformin response determined by HbA1c levels in patients with T2DM. The del allele of SNP rs72552763 may serve as a metformin response biomarker.

Genetic Variation in CYP2D6 and SLC22A1 Affects Amlodipine Pharmacokinetics and Safety.

Amlodipine is an antihypertensive drug with unknown pharmacogenetic biomarkers. This research is a candidate gene study that looked for associations between amlodipine pharmacokinetics and safety and pharmacogenes. Pharmacokinetic and safety data were taken from 160 volunteers from eight bioequivalence trials. In the exploratory step, 70 volunteers were genotyped for 44 polymorphisms in different pharmacogenes. CYP2D6 poor metabolizers (PMs) showed higher half-life (t1/2) (univariate p-value (puv) = 0.039, multivariate p-value (pmv) = 0.013, ß = -5.31, R2 = 0.176) compared to ultrarapid (UMs), normal (NMs) and intermediate metabolizers (IMs). SLC22A1 rs34059508 G/A genotype was associated with higher dose/weight-corrected area under the curve (AUC72/DW) (puv = 0.025; pmv = 0.026, ß = 578.90, R2 = 0.060) compared to the G/G genotype. In the confirmatory step, the cohort was increased to 160 volunteers, who were genotyped for CYP2D6, SLC22A1 and CYP3A4. In addition to the previous associations, CYP2D6 UMs showed a lower AUC72/DW (puv = 0.046, pmv = 0.049, ß = -68.80, R2 = 0.073) compared to NMs, IMs and PMs and the SLC22A1 rs34059508 G/A genotype was associated with thoracic pain (puv = 0.038) and dizziness (puv = 0.038, pmv = 0.014, log OR = 10.975). To our knowledge, this is the first work to report a strong relationship between amlodipine and CYP2D6 and SLC22A1. Further research is needed to gather more evidence before its application in clinical practice.

Association of Met420del Variant of Metformin Transporter Gene SLC22A1 with Metformin Treatment Response in Ethiopian Patients with Type 2 Diabetes.

Objective: This study aimed to evaluate whether the M420del variants of SLC22A1 (rs72552763) is associated with metformin treatment response in Ethiopian patients with type 2 diabetes mellitus (T2DM). Patients and Methods: A prospective observational cohort study was conducted on 86 patients with T2DM who had been receiving metformin monotherapy for <1 year. Patients showing ≥0.5% reduction in HbA1c levels from baseline within 3 months and remained low for at least another 3 months were defined as responders while those patients with <0.5% reduction in HbA1c levels and/or those whom started a new class of glucose-lowering drug(s) because of unsatisfactory reduction were defined as non-responders. In addition, good glycemic control was observed when HbA1c ≤7.0%, and the above values were regarded as poor. Genotyping of rs72552763 SNP was performed using TaqMan® Drug Metabolism Enzyme Genotyping Assay and its association with metformin response and glycemic control were assessed by measuring the change in HbA1c and fasting blood glucose levels using Chi-square, logistic regression and Mann-Whitney U-test. Statistical significance was set at p <0.05. Results: The minor allele frequency of the rs72552763 SNP of SLC22A1 was 9.3%. Metformin response was significantly higher in deletion_GAT (del_G) genotypes as compared to the wild-type GAT_GAT (G_G) genotypes. Furthermore, a significantly lower median treatment HbA1 level was found in del_G genotypes as compared to G_G genotypes. However, the association of rs72552763 with metformin response was not replicated at the allele level. In contrast, the minor del_allele was significantly associated with good glycemic control compared to the G_allele, though not replicated at del_G genotypes level. Conclusion: This study demonstrated that metformin response was significantly higher in study participants with a heterozygous carrier of M420del variants of SLC22A1 as compared to the wild-type G_G genotypes after 3 months of treatment.

Differences in the pharmacokinetics and steady-state blood concentrations of orally administered lenvatinib in adult and juvenile rats.

Objective: The aim of this study was to compare the pharmacokinetics and steady-state serum concentrations of lenvatinib in adult and juvenile rats. Experimental study: An ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method was developed to quantify lenvatinib in the serum and liver of rats. Six juvenile and six adult rats in each group were orally administered with a single dose of 7.0 mg/kg lenvatinib suspension for pharmacokinetics. Another 12 juvenile and adult rats were subjected to oral gavage with 7.0 mg/kg lenvatinib once daily for 5 days. Biofluild samples were pre-treated by protein precipitation and sorafenib was used as the internal standard for UPLC-MS analysis. The pharmacokinetic parameters were estimated by compartment and statistical model. The mRNA expression of CYP3A2 and SLC22A1 in liver of adult and juvenile rats was measured by real-time fluorescence quantitative PCR (RT-qPCR). Results: The UPLC-MS method met the requirements for quantitative analysis of lenvatinib in serum and liver. The pharmacokinetic results showed that the mean retention time (MRT(0-∞)) was 19.64 ± 7.64 h and 126.38 ± 130.18 h, with AUC(0-∞) values of 3.97 ± 0.73 µg‧mL-1 h and 5.95 ± 2.27 µg mL-1 h in adult and juvenile rats, respectively. When comparing adult rats (0.35 ± 0.15 µg/mL) to juvenile rats, no significant differences were observed in steady-state serum lenvatinib (0.32 ± 0.11 µg/mL), but a noteworthy decrease to one-third of steady-state liver lenvatinib was observed after multiple oral doses of lenvatinib in juvenile rats. Additional findings revealed that the mRNA expression of CYP3A2 and SLC22A1 was notably increased by 6.86 and 14.67 times, respectively, in juvenile rats compared to adult rats. Conclusion: Juvenile rats exhibit lower levels of lenvatinib in the liver's steady-state, potentially due to the disparity in CYP3A2 mRNA expression. These results imply that the dosage of lenvatinib for pediatric patients may need to be augmented in order to attain the desired clinical outcome.

Genetic variants in SLC22A1 are related to serum lipid levels in Mexican women.

Dyslipidemia is the main risk factor for coronary artery disease and is characterized by alterations in concentrations of lipids, including low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol (HDL-c), and triacylglycerols. The participation of several genes in the development of dyslipidemia has been evidenced. Genetic variants in SLC22A1 have been associated with elevated cholesterol and LDL-c levels. The aim of this study was to evaluate the association between single-nucleotide polymorphisms (SNPs) in the SLC22A1 gene with atherogenic risk lipid levels in Mexican women. Anthropometric and biochemical measurements were performed, and four SNPs in SLC22A1 were genotyped by real-time polymerase chain reaction. The Hardy-Weinberg equilibrium was verified, and haplotype frequencies were calculated. We found significant differences between the allele frequencies of the SNPs analyzed with those reported in Mexico and in the world, which could be due to differences in the historical admixture of the women studied. Generalized linear models were evaluated to determine the association between genotypes and haplotypes with lipids levels. We identified a significant increase in total cholesterol and LDL-c levels in women who were carriers of the GA and AG genotypes of the polymorphisms rs628031 and rs594709, respectively, significant effect that is also shown in a dominant inheritance model. Interestingly, we identified an important relationship of the AGC-GAT haplotype with the elevation in LDL-c levels and AGA-GAT haplotype with the elevation in HDL-c levels. On the other hand, we found a strong linkage disequilibrium between the polymorphisms studied. Our results show that variants in the SLC22A1 gene influence serum levels of atherogenic risk lipids, suggesting that these variants probably affect the function of organic cation transporter-1 and therefore, on the regulation of lipid metabolism.