Inhibitory interaction of flavonoids with organic cation transporter 2 and their structure-activity relationships for predicting nephroprotective effects.

Organic cation transporter 2 (OCT2) is mainly responsible for the renal secretion of various cationic drugs, closely associated with drug-induced acute kidney injury (AKI). Screening and identifying potent OCT2 inhibitors with little toxicity in natural products in reducing OCT2-mediated AKI is of great value. Flavonoids are enriched in various vegetables, fruits, and herbal products, and some were reported to produce transporter-mediated drug-drug interactions. This study aimed to screen potential inhibitors of OCT2 from 96 flavonoids, assess the nephroprotective effects on cisplatin-induced kidney injury, and clarify the structure-activity relationships of flavonoids with OCT2. Ten flavonoids exhibited significant inhibition (>50%) on OCT2 in OCT2-HEK293 cells. Among them, the six most potent flavonoid inhibitors, including pectolinarigenin, biochanin A, luteolin, chrysin, 6-hydroxyflavone, and 6-methylflavone markedly decreased cisplatin-induced cytotoxicity. Moreover, in cisplatin-induced renal injury models, they also reduced serum blood urea nitrogen (BUN) and creatinine levels to different degrees, the best of which was 6-methylflavone. The pharmacophore model clarified that the aromatic ring, hydrogen bond acceptors, and hydrogen bond donors might play a vital role in the inhibitory effect of flavonoids on OCT2. Thus, our findings would pave the way to predicting the potential risks of flavonoid-containing food/herb-drug interactions in humans and optimizing flavonoid structure to alleviate OCT2-related AKI.

Lenvatinib causes reduced expression of carnitine/organic cation transporter 2 and carnitine deficiency in the skeletal muscle of rats.

Lenvatinib, an oral tyrosine kinase inhibitor, is widely used to treat several types of advanced cancers but often causes muscular adverse reactions. Although carnitine supplementation may prevent these effects, the mechanism underlying lenvatinib-induced skeletal muscle impairment remains poorly understood. To this end, we aimed to investigate the impact of lenvatinib on carnitine disposition in rats. Once-daily administration of lenvatinib repeated for two weeks did not affect urinary excretion or serum concentration of carnitines throughout the treatment period but ultimately decreased the L-carnitine content in the skeletal muscle. The treatment decreased the expression of carnitine/organic cation transporter (OCTN) 2, a key transporter of carnitine, in skeletal muscle at the protein level but not at the mRNA level. In cultured C2C12 myocytes, lenvatinib inhibited OCTN2 expression in a dose-dependent manner at the protein level. Furthermore, lenvatinib dose-dependently decreased the protein levels of carnitine-related genes, adenosine triphosphate content, mitochondrial membrane potential, and markers of mitochondrial function in vitro. These results reveal the deleterious effects of lenvatinib on OCTN2 expression, carnitine content, and mitochondrial function in skeletal muscle that may be associated with muscle toxicity.

Novel natural scaffold as hURAT1 inhibitor identified by 3D-shape-based, docking-based virtual screening approach and biological evaluation.

As a promising therapeutic target for gout, hURAT1 has attracted increasing attention. In this work, we identified a novel scaffold of hURAT1 inhibitors from a personal natural product database of verified herb-treated gout. First, we constructed more than 800 natural compounds from Chinese medicine that were verified to treat gout. Following the application of both shape-based and docking-based virtual screening (VS) methods, taking into account the shape similarity and flexibility of the target, we identified isopentenyl dihydroflavones that might inhibit hURAT1. Specifically, 9 compounds with commercial availability were tested with biochemical assays for the inhibition of 14C-uric acid uptake in high-expression hURAT1 cells (HEK293-hURAT1), and their structure-activity relationship was evaluated. As a result, 8-isopentenyl dihydroflavone was identified as a novel scaffold of hURAT1 inhibitors since isobavachin (DHF3) inhibited hURAT1 with an IC50 value of 0.39 ± 0.17 µM, which was comparable to verinurad with an IC50 value of 0.32 ± 0.23 µM. Remarkably, isobavachin also displayed an eminent effect in the decline of serum uric acid in vivo experiments. Taken together, isobavachin is a promising candidate for the treatment of hyperuricemia and gout.

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.

Polymorphisms in SLC44A1 are associated with cognitive improvement in children diagnosed with fetal alcohol spectrum disorder: an exploratory study of oral choline supplementation.

BACKGROUND: The essential nutrient choline provides one-carbon units for metabolite synthesis and epigenetic regulation in tissues including brain. Dietary choline intake is often inadequate, and higher intakes are associated with improved cognitive function. OBJECTIVE: Choline supplements confer cognitive improvement for those diagnosed with fetal alcohol spectrum disorder (FASD), a common set of neurodevelopmental impairments; however, the effect sizes have been modest. In this retrospective analysis, we report that genetic polymorphisms affecting choline utilization are associated with cognitive improvement following choline intervention. METHODS: Fifty-two children from the upper midwestern United States and diagnosed with FASD, ages 2-5 y, were randomly assigned to receive choline (500 mg/d; n = 26) or placebo (n = 26) for 9 mo, and were genotyped for 384 choline-related single nucleotide polymorphisms (SNPs). Memory and cognition were assessed at enrollment, study terminus, and at 4-y follow-up for a subset. RESULTS: When stratified by intervention (choline vs. placebo), 14-16 SNPs within the cellular choline transporter gene solute carrier family 44 member 1 (SLC44A1) were significantly associated with performance in an elicited imitation sequential memory task, wherein the effect alleles were associated with the greatest pre-/postintervention improvement. Of these, rs3199966 is a structural variant (S644A) and rs2771040 is a single-nucleotide variant within the 3' untranslated region of the plasma membrane isoform. An additive genetic model best explained the genotype associations. Lesser associations were observed for cognitive outcome and polymorphisms in flavin monooxygenase-3 (FMO3), methylenetetrahydrofolate dehydrogenase-1 (MTHFD1), fatty acid desaturase-2 (FADS2), and adiponectin receptor 1 (ADIPOR1). CONCLUSIONS: These SLC44A1 variants were previously associated with greater vulnerability to choline deficiency. Our data potentially support the use of choline supplements to improve cognitive function in individuals diagnosed with FASD who carry these effect alleles. Although these findings require replication in both retrospective and prospective confirmatory trials, they emphasize the need to incorporate similar genetic analyses of choline-related polymorphisms in other FASD-choline trials, and to test for similar associations within the general FASD population. This trial was registered at www.clinicaltrials.gov as NCT01149538.

Baicalein alleviates hyperuricemia by promoting uric acid excretion and inhibiting xanthine oxidase.

BACKGROUND: Insufficient renal urate excretion and/or overproduction of uric acid (UA) are the dominant causes of hyperuricemia. Baicalein (BAL) is widely distributed in dietary plants and has extensive biological activities, including antioxidative, anti-inflammatory and antihypertensive activities. PURPOSE: To investigate the anti-hyperuricemic effects of BAL and the underlying mechanisms in vitro and in vivo. METHODS: We investigated the inhibitory effects of BAL on GLUT9 and URAT1 in vitro through electrophysiological experiments and 14C-urate uptake assays. To evaluate the impact of BAL on serum and urine UA, the expression of GLUT9 and URAT1, and the activity of xanthine oxidase (XOD), we developed a mouse hyperuricemia model by potassium oxonate (PO) injection. Molecular docking analysis based on homology modeling was performed to explain the predominant efficacy of BAL compared with the other test compounds. RESULTS: BAL dose-dependently inhibited GLUT9 and URAT1 in a noncompetitive manner with IC50 values of 30.17 ± 8.68 µM and 31.56 ± 1.37 µM, respectively. BAL (200 mg/kg) significantly decreased serum UA and enhanced renal urate excretion in PO-induced hyperuricemic mice. Moreover, the expression of GLUT9 and URAT1 in the kidney was downregulated, and XOD activity in the serum and liver was suppressed. The docking analysis revealed that BAL potently interacted with Trp336, Asp462, Tyr71 and Gln328 of GLUT9 and Ser35 and Phe241 of URAT1. CONCLUSION: These results indicated that BAL exerts potent antihyperuricemic efects through renal UA excretal promotion and serum UA production. Thus, we propose that BAL may be a promising treatment for the prevention of hyperuricemia owing to its multitargeted inhibitory activity.

Regulation of organic anion transporters: Role in physiology, pathophysiology, and drug elimination.

The members of the organic anion transporter (OAT) family are mainly expressed in kidney, liver, placenta, intestine, and brain. These transporters play important roles in the disposition of clinical drugs, pesticides, signaling molecules, heavy metal conjugates, components of phytomedicines, and toxins, and therefore critical for maintaining systemic homeostasis. Alterations in the expression and function of OATs contribute to the intra- and inter-individual variability of the therapeutic efficacy and the toxicity of many drugs, and to many pathophysiological conditions. Consequently, the activity of these transporters must be highly regulated to carry out their normal functions. This review will present an update on the recent advance in understanding the cellular and molecular mechanisms underlying the regulation of renal OATs, emphasizing on the post-translational modification (PTM), the crosstalk among these PTMs, and the remote sensing and signaling network of OATs. Such knowledge will provide significant insights into the roles of these transporters in health and disease.

Assessing Transporter-Mediated Natural Product-Drug Interactions Via In vitro-In Vivo Extrapolation: Clinical Evaluation With a Probe Cocktail.

The botanical natural product goldenseal can precipitate clinical drug interactions by inhibiting cytochrome P450 (CYP) 3A and CYP2D6. Besides P-glycoprotein, effects of goldenseal on other clinically relevant transporters remain unknown. Established transporter-expressing cell systems were used to determine the inhibitory effects of a goldenseal extract, standardized to the major alkaloid berberine, on transporter activity. Using recommended basic models, the extract was predicted to inhibit the efflux transporter BCRP and uptake transporters OATP1B1/3. Using a cocktail approach, effects of the goldenseal product on BCRP, OATP1B1/3, OATs, OCTs, MATEs, and CYP3A were next evaluated in 16 healthy volunteers. As expected, goldenseal increased the area under the plasma concentration-time curve (AUC0-inf ) of midazolam (CYP3A; positive control), with a geometric mean ratio (GMR) (90% confidence interval (CI)) of 1.43 (1.35-1.53). However, goldenseal had no effects on the pharmacokinetics of rosuvastatin (BCRP and OATP1B1/3) and furosemide (OAT1/3); decreased metformin (OCT1/2, MATE1/2-K) AUC0-inf (GMR, 0.77 (0.71-0.83)); and had no effect on metformin half-life and renal clearance. Results indicated that goldenseal altered intestinal permeability, transport, and/or other processes involved in metformin absorption, which may have unfavorable effects on glucose control. Inconsistencies between model predictions and pharmacokinetic outcomes prompt further refinement of current basic models to include differential transporter expression in relevant organs and intestinal degradation/metabolism of the precipitant(s). Such refinement should improve in vitro-in vivo prediction accuracy, contributing to a standard approach for studying transporter-mediated natural product-drug interactions.

In Vitro Investigation, Pharmacokinetics, and Disposition of Imeglimin, a Novel Oral Antidiabetic Drug, in Preclinical Species and Humans.

Imeglimin is a novel oral antidiabetic drug for treatment of type 2 diabetes that targets mitochondrial bioenergetics. Its pharmacokinetics absorption characteristics, metabolism, distribution, and elimination were assessed through several in vitro and in vivo experiments in both animals and humans. Its potential to induce drug-drug interactions was also extensively assessed. Imeglimin is a small cationic compound with an intermediate intestinal permeability. Its absorption mechanism involves an active transport process in addition to passive paracellular absorption. Absorption was good (50%-80%) in vivo across several species but decreased with increasing dose, probably because of saturation of active transport. After absorption, imeglimin was rapidly and largely distributed to internal organs. Plasma protein binding was low, which can explain the rapid distribution to organs observed in all species. In animals and humans, imeglimin was largely excreted unchanged in urine, indicating a low extent of metabolism. Unchanged drug was the main circulating entity in plasma, and none of the identified metabolites were unique to human. Imeglimin renal clearance was higher than creatinine clearance, indicating that it was actively secreted into urine. There was no evidence that it had the potential to cause cytochrome P450 inhibition or induction. It was shown to be a substrate of organic cation transporter (OCT) 1, OCT2, multidrug and toxin extrusion (MATE) 1, and MATE2-K and an inhibitor of OCT1, OCT2, and MATE1; as a consequence, corresponding clinical drug-drug interaction studies were performed and confirmed the absence of relevant interactions with substrates or inhibitors of these transporters. SIGNIFICANCE STATEMENT: Imeglimin is absorbed through a passive and active mechanism, which can be saturated. It is rapidly and largely distributed to internal organs and mainly excreted unchanged in urine. It is poorly metabolized and has no cytochrome P450 inhibition or induction potential. Imeglimin is a substrate of MATE2-K and also a substrate and an inhibitor of OCT1, OCT2, and MATE1 transporters; however, there are no clinically significant interactions when imeglimin is coadministered with either a substrate or an inhibitor of these transporters.

A Multicompartment Human Kidney Proximal Tubule-on-a-Chip Replicates Cell Polarization-Dependent Cisplatin Toxicity.

Drug-induced kidney injury is a major clinical problem and causes drug attrition in the pharmaceutical industry. To better predict drug-induced kidney injury, kidney in vitro cultures with enhanced physiologic relevance are developed. To mimic the proximal tubule, the main site of adverse drug reactions in the kidney, human-derived renal proximal tubule epithelial cells (HRPTECs) were injected in one of the channels of dual-channel Nortis chips and perfused for 7 days. Tubes of HRPTECs demonstrated expression of tight junction protein 1 (zona occludens-1), lotus lectin, and primary cilia with localization at the apical membrane, indicating an intact proximal tubule brush border. Gene expression of cisplatin efflux transporters multidrug and toxin extrusion transporter (MATE) 1 (SLC47A1) and MATE2-k (SLC47A2) and megalin endocytosis receptor increased 19.9 ± 5.0-, 23.2 ± 8.4-, and 106 ± 33-fold, respectively, in chip cultures compared with 2-dimensional cultures. Moreover, organic cation transporter 2 (OCT2) (SLC22A2) was localized exclusively on the basolateral membrane. When infused from the basolateral compartment, cisplatin (25 µM, 72 hours) induced toxicity, which was evident as reduced cell number and reduced barrier integrity compared with vehicle-treated chip cultures. Coexposure with the OCT2 inhibitor cimetidine (1 mM) abolished cisplatin toxicity. In contrast, infusion of cisplatin from the apical compartment did not induce toxicity, which was in line with polarized localization of cisplatin uptake transport proteins, including OCT2. In conclusion, we developed a dual channel human kidney proximal tubule-on-a-chip with a polarized epithelium, restricting cisplatin sensitivity to the basolateral membrane and suggesting improved physiologic relevance over single-compartment models. Its implementation in drug discovery holds promise to improve future in vitro drug-induced kidney injury studies. SIGNIFICANCE STATEMENT: Human-derived kidney proximal tubule cells retained characteristics of epithelial polarization in vitro when cultured in the kidney-on-a-chip, and the dual-channel construction allowed for drug exposure using the physiologically relevant compartment. Therefore, cell polarization-dependent cisplatin toxicity could be replicated for the first time in a kidney proximal tubule-on-a-chip. The use of this physiologically relevant model in drug discovery has potential to aid identification of safe novel drugs and contribute to reducing attrition rates due to drug-induced kidney injury.

Tofacitinib and Baricitinib Are Taken up by Different Uptake Mechanisms Determining the Efficacy of Both Drugs in RA.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic autoimmune disease in which synovial fibroblasts (SF) play a key role. Baricitinib and Tofacitinib both act intracellularly, blocking the ATP-binding side of JAK proteins and thereby the downstream signalling pathway via STAT-3. Therefore, we investigated the role of organic cation transporters (OCTs) in Baricitinib and Tofacitinib cellular transport. METHODS: OCT expression was analysed in SF isolated from RA and osteoarthritis (OA) patients, as well as peripheral blood mononuclear cells. The interaction of Baricitinib and Tofacitinib with OCTs was investigated using quenching experiments. The intracellular accumulation of both drugs was quantified using LC/MS. Target inhibition for both drugs was tested using Western blot for phosphorylated JAK1 and STAT3 upon stimulation with IL-6. RESULTS: MATE-1 expression increased in OASF compared to RASF. The other OCTs were not differentially expressed. The transport of Baricitinib was not OCT dependent. Tofacitinib; however, was exported from RASF in a MATE-1 dependent way. Tofacitinib and Baricitinib showed comparable inhibition of downstream signalling pathways. CONCLUSION: We observed different cellular uptake strategies for Baricitinib and Tofacitinib. Tofacitinib was exported out of healthy cells due to the increased expression of MATE1. This might make Tofacitinib the favourable drug.

An LC-MS/MS- and hURAT1 cell-based approach for screening of uricosuric agents.

Urate anion exchanger 1 (URAT1) expressed in the proximal renal tubules is responsible for about 90% of the reabsorption of uric acid. URAT1 is identified as an important target of uricosuric drugs. Here we present an LC-MS/MS-based approach, combined with URAT1-transgenic MDCK cells, for the assessment of uric acid. Cell lysis was executed with 50 mM NaOH to release uric acid. 1,3-15N2 uric acid was employed as the internal standard. The harvested uric acid, along with the stable isotope-labeled uric acid, was analyzed by LC-MS/MS in multiple reactions monitoring and negative modes. Validation, i.e. determination of selectivity, precision, accuracy, extraction recovery, and matrix effect, and feasibility was evaluated by use of the approach developed. The linearity was observed in the range of 1.0-250 µM (r = 0.9960) with limit of detection of 50 nM and limit of quantitation of 200 nM. The precision and accuracy were found to be RSD ≤ 20% and 80-120% of the nominal value, respectively. Uric acid uptake showed concentration and time dependency in URAT1-transgenic cells. The observed inhibitory effects of three URAT1-targeted uricosuric drugs were consistent with those reported in literature. The stable isotope dilution-based approach was proven to be selective, sensitive, and convenient, which is a good in vitro model for URAT1-targeted drug candidate screening.

Organic Cation Transporter 1 and 3 Contribute to the High Accumulation of Dehydrocorydaline in the Heart.

Dehydrocorydaline (DHC), one of the main active components of Corydalis yanhusuo, is an important remedy for the treatment of coronary heart disease. Our previous study revealed a higher unbound concentration of DHC in the heart than plasma of mice after oral administration of C. yanhusuo extract or DHC, but the underlying uptake mechanism remains unelucidated. In our investigations, we studied the transport mechanism of DHC in transgenic cells, primary neonatal rat cardiomyocytes, and animal experiments. Using quantitative real-time polymerase chain reaction and Western blotting, we found that uptake transporters expressed in the mouse heart include organic cation transporter 1/3 (OCT1/3) and carnitine/organic cation transporter 1/2 (OCTN1/2). The accumulation experiments in transfected cells showed that DHC was a substrate of OCT1 and OCT3, with K m of 11.29 ± 3.3 and 8.96 ± 3.7 µM, respectively, but not a substrate of OCTN1/2. Additionally, a higher efflux level (1.71-fold of MDCK-mock) of DHC was observed in MDCK-MDR1 cells than in MDCK-mock cells. Therefore, DHC is a weak substrate for MDR1. Studies using primary neonatal rat cardiomyocytes showed that OCT1/3 inhibitors (quinidine, decynium-22, and levo-tetrahydropalmatine) prevented the accumulation of DHC, whereas OCTN2 inhibitors (mildronate and l-carnitine) did not affect its accumulation. Moreover, the coadministration of OCT1/3 inhibitors (levo-tetrahydropalmatine, THP) decreased the concentration of DHC in the mouse heart. Based on these findings, DHC may be accumulated partly by OCT1/3 transporters and excreted by MDR1 in the heart. THP could alter the distribution of DHC in the mouse heart. SIGNIFICANCE STATEMENT: We reported the cardiac transport mechanism of dehydrocorydaline, highly distributed to the heart after oral administration of Corydalis yanhusuo extract or dehydrocorydaline only. Dehydrocorydaline (an OCT1/3 and MDR1 substrate) accumulation in primary cardiomyocytes may be related to the transport activity of OCT1/3. This ability, hampered by selective inhibitors (levo-tetrahydropalmatine, an inhibitor of OCT1/3), causes a nearly 40% reduction in exposure of the heart to dehydrocorydaline. These results suggest that OCT1/3 may contribute to the uptake of dehydrocorydaline in the heart.

Hypouricemic Effects of Extracts from Urtica hyperborea Jacq. ex Wedd. in Hyperuricemia Mice through XOD, URAT1, and OAT1.

Urtica L. has been long used for gout in traditional Tibetan medicine and is closely related to the effect of reducing uric acid. This study aimed to investigate the effect of Urtica hyperborea Jacq. ex Wedd. (UW) on lowering uric acid and its mechanism by using HK2 cells and hyperuricemia mouse model. Petroleum ether extract (UWP), ethyl acetate extract (UWE), n-butanol extract (UWB), and alcohol-soluble extract (UWA) from UW were prepared, and HK2 cells were treated with various parts extracts to observe the expression of uric acid transporter at 25, 50, and 100 µg/mL for 24 h. Moreover, hyperuricemia mice were administered orally various parts extracts at 0.78 and 2.34 g/kg (crude drug dose converted by extraction rate) to observe the change of hepatic XOD, serum ADA, renal function, and uric acid transporter. In vitro experiments showed that UWA can remarkably elevate OAT1 expression and decrease URAT1 expression in HK2 cells. In vivo experiments showed that UWP, UWE, UWB, and UWA showed remarkable activity in reducing uric acid, rendering a substantial decline in the SUA level in hyperuricemia mice. Compared with the hyperuricemia and allopurinol groups, UWB and UWA had significant protective effects on renal injury. At the same time, UWA can significantly reduce the activity of XOD and ADA, reduce the expression of URAT1, and increase the expression of OAT1. These results indicated that UWA had an outstanding uric acid lowering effect and did not affect renal function. This may be related to increased uric acid excretion and decreased uric acid production, mediated by renal OAT1, URAT1, liver XOD, and serum ADA. UWA may be a potential drug against hyperuricemia.

The biology of ergothioneine, an antioxidant nutraceutical.

Ergothioneine (ERG) is an unusual thio-histidine betaine amino acid that has potent antioxidant activities. It is synthesised by a variety of microbes, especially fungi (including in mushroom fruiting bodies) and actinobacteria, but is not synthesised by plants and animals who acquire it via the soil and their diet, respectively. Animals have evolved a highly selective transporter for it, known as solute carrier family 22, member 4 (SLC22A4) in humans, signifying its importance, and ERG may even have the status of a vitamin. ERG accumulates differentially in various tissues, according to their expression of SLC22A4, favouring those such as erythrocytes that may be subject to oxidative stress. Mushroom or ERG consumption seems to provide significant prevention against oxidative stress in a large variety of systems. ERG seems to have strong cytoprotective status, and its concentration is lowered in a number of chronic inflammatory diseases. It has been passed as safe by regulatory agencies, and may have value as a nutraceutical and antioxidant more generally.

A proton-coupled organic cation antiporter is involved in the blood-brain barrier transport of Aconitum alkaloids.

ETHNOPHARMACOLOGICAL RELEVANCE: The herbs of Aconitum are the essential Traditional Chinese medicine and have played an indispensable role in many Asian countries for thousands of years to treat critical illnesses, and chronic, stubborn diseases. However, Aconitum may induce severe neurotoxicity and even death. So far the mechanism of Aconitum penetrating the blood-brain barrier (BBB) is still unclear. AIM OF THE STUDY: To determine whether influx transporters contribute to the brain uptake of the highly toxic alkaloids in Aconitum including aconitine (AC), mesaconitine (MA) and hypaconitine (HA). MATERIALS AND METHODS: The uptake of AC, MA and HA was characterized using in vitro hCMEC/D3 model and in situ mouse brain perfusion. In hCMEC/D3 cells, the effect of incubation temperature, time, initial drug concentration, energy (NaN3), extracellular and intracellular pH (FCCP and NH4Cl), the prototypical substrates/inhibitors of known organic cation transporting carriers and trans-stimulation (pre-incubating with pyrilamine and diphenhydramine) on the cellular uptake were studied. In addition, the effect of silencing OCTN1, OCTN2 and PMAT by specific siRNA was investigated. In mice, the contribution of the proton-coupled antiporter on the brain uptake of Aconitum was investigated by chemical inhibition. RESULTS: In hCMEC/D3 cells, AC, MA and HA were each taken up in a temperature-, time- and concentration-dependent manner, which were reduced by NaN3 and FCCP. Regulation of extracellular and intracellular pH as well as trans-stimulation studies showed that AC, MA and HA were transported by a proton-coupled antiporter expressed at the plasma membrane that could also transport pyrilamine and diphenhydramine. Each uptake was markedly inhibited by various cationic drugs, but insensitive to the prototypical substrates/inhibitors of identified organic cation transporting carriers, such as OCTs, PMAT, MATEs and OCTNs. In addition, silence of OCTN1, OCTN2 and PMAT had no significant inhibitory effect on the uptake of AC, MA and HA. In mice, the brain uptake of each alkaloid measured by in situ brain perfusion was suppressed by diphenhydramine when the transport capacity of P-gp/Bcrp at the BBB was chemically inhibited. CONCLUSIONS: A novel proton-coupled organic cation antiporter plays a predominant role in the blood to brain influx of AC, MA and HA at the BBB, and thus affect the safety of Aconitum species.

Upregulated OCT3 has the potential to improve the survival of colorectal cancer patients treated with (m)FOLFOX6 adjuvant chemotherapy.

PURPOSE: To investigate the influence of organic cation transporter 3 (OCT3) expression on the effect of the combination regimen of 5-fluorouracil, folinic acid and oxaliplatin ((m)FOLFOX6) in colorectal cancer (CRC) patients. METHODS: This is a retrospective study conducted at a single centre (Sichuan Academy of Medical Sciences & Sichuan Provincial People's Hospital, China). Patients with stage IIb-IV resectable CRC who were being postoperatively treated with (m)FOLFOX6 as a first-line adjuvant chemotherapy regimen for at least 5 cycles and had resected primary tumour samples available were eligible for the study. Patients who preoperatively received chemotherapy and/or radiotherapy or were treated with targeted drugs or other anticancer drugs were excluded from the study. Immunohistochemical staining and digital image analysis were used to assess OCT3 expression in tumour samples. According to OCT3 expression level, the receiver operating characteristic curve (ROC curve) was used to divide the patients into two groups. Cox proportional risk regression was performed with the forward LR (forward stepwise regression based on maximum likelihood estimation) method using SPSS17.0 software. The primary endpoint was the 2-year progression-free survival. RESULTS: In total, 57 patients were included between 2014 and 2016 according to the inclusion and exclusion criteria (22 had low OCT3 expression, and 35 had high OCT3 expression). The mean age was 55.7 (30-74) years, and 37 of the total patients were male. According to TNM stage, 5 patients had stage IV disease, 44 patients had stage III disease, and 8 patients had stage II disease. Through Cox regression analysis, we found that among patients receiving the (m)FOLFOX6 regimen, those with higher OCT3 expression had a higher two-year progression-free survival rate than those with lower OCT3 expression (P = 0.038). The hazard ratio of patients with high OCT3 expression compared with patients with low OCT3 expression was 0.247. Besides, it was found that the age of patients was negatively correlated with expression level of OCT3, which can explain why patients over 70 years do not benefit from oxaliplatin-containing chemotherapy. CONCLUSIONS: High OCT3 expression in CRC tissues may be a protective factor for CRC patients treated with (m)FOLFOX6.

An in vitro study on interaction of anisodine and monocrotaline with organic cation transporters of the SLC22 and SLC47 families.

Current study systematically investigated the interaction of two alkaloids, anisodine and monocrotaline, with organic cation transporter OCT1, 2, 3, MATE1 and MATE2-K by using in vitro stably transfected HEK293 cells. Both anisodine and monocrotaline inhibited the OCTs and MATE transporters. The lowest IC50 was 12.9 µmol·L-1 of anisodine on OCT1 and the highest was 1.8 mmol·L-1 of monocrotaline on OCT2. Anisodine was a substrate of OCT2 (Km = 13.3 ± 2.6 µmol·L-1 and Vmax = 286.8 ± 53.6 pmol/mg protein/min). Monocrotaline was determined to be a substrate of both OCT1 (Km = 109.1 ± 17.8 µmol·L-1, Vmax = 576.5 ± 87.5 pmol/mg protein/min) and OCT2 (Km = 64.7 ± 14.8 µmol·L-1, Vmax = 180.7 ± 22.0 pmol/mg protein/min), other than OCT3 and MATE transporters. The results indicated that OCT2 may be important for renal elimination of anisodine and OCT1 was responsible for monocrotaline uptake into liver. However neither MATE1 nor MATE2-K could facilitate transcellular transport of anisodine and monocrotaline. Accumulation of these drugs in the organs with high OCT1 expression (liver) and OCT2 expression (kidney) may be expected.

Pharmacological Evaluation of Dotinurad, a Selective Urate Reabsorption Inhibitor.

The effect of dotinurad [(3,5-dichloro-4-hydroxyphenyl)(1,1-dioxo-1,2-dihydro-3H-1λ 6-1,3-benzothiazol-3-yl)methanone] was compared with that of commercially available uricosuric agents-namely, benzbromarone, lesinurad, and probenecid. Its effect on urate secretion transporters was evaluated using probe substrates for respective transporters. Dotinurad, benzbromarone, lesinurad, and probenecid inhibited urate transporter 1 (URAT1) with IC50 values of 0.0372, 0.190, 30.0, and 165 µM, respectively. Dotinurad weakly inhibited ATP-binding cassette subfamily G member 2 (ABCG2), organic anion transporter 1 (OAT1), and OAT3, with IC50 values of 4.16, 4.08, and 1.32 µM, respectively, indicating higher selectivity for URAT1. The hypouricemic effects of dotinurad and benzbromarone were evaluated in Cebus monkeys. Dotinurad, at doses of 1-30 mg/kg, concomitantly decreased plasma urate levels and increased fractional excretion of urate (FEUA) in a dose-dependent manner. On the contrary, benzbromarone, at a dose of 30 mg/kg, showed a modest effect on plasma urate levels. The inhibitory effect of dotinurad on urate secretion transporters was evaluated in Sprague-Dawley rats, with sulfasalazine and adefovir as probe substrates of ABCG2 and OAT1, respectively. Drugs, including febuxostat as a reference ABCG2 inhibitor, were administered orally before sulfasalazine or adefovir administration. Dotinurad had no effect on urate secretion transporters in vivo, whereas benzbromarone, lesinurad, probenecid, and febuxostat increased the plasma concentrations of probe substrates. These results suggested dotinurad is characterized as a selective urate reabsorption inhibitor (SURI), which is defined as a potent URAT1 inhibitor with minimal effect on urate secretion transporters, including ABCG2 and OAT1/3, because of its high efficacy in decreasing plasma urate levels compared with that of other uricosuric agents. SIGNIFICANCE STATEMENT: Our study on the inhibitory effects on urate transport showed that dotinurad had higher selectivity for urate transporter 1 (URAT1) versus ATP-binding cassette subfamily G member 2 (ABCG2) and organic anion transporter (OAT) 1/3 compared to other uricosuric agents. In Cebus monkeys, dotinurad decreased plasma urate levels and increased fractional excretion of urate in a dose-dependent manner. To determine the inhibitory effect of dotinurad on urate secretion transporters, we studied the movement of substrates of ABCG2 and OAT1 in rats. Dotinurad had no effect on these transporters, whereas the other uricosuric agents increased the plasma concentrations of the substrates. These results suggested dotinurad as a potent and selective urate reabsorption inhibitor is characterized by increased efficacy with decreasing plasma urate levels.

Modified Chuanhu anti-gout mixture, a traditional Chinese medicine, protects against potassium oxonate-induced hyperuricemia and renal dysfunction in mice.

OBJECTIVE: Acute gout is a painful, inflammatory arthritis that features a rapidly escalating inflammatory response resulting from the formation of monosodium urate crystals in the affected joint space. Previously, we found that Chuanhu anti-gout mixture (CAGM) had similar effects as colchicine against gout in the clinic. Subsequently, to improve its effectiveness and efficacy, we modified the original formulation of CAGM. The current study evaluated the effectiveness of the modified formulation in mice. METHODS: Potassium oxonate (PO) was used to establish a mouse model of hyperuricemia. Plasma levels of uric acid and creatine were determined using the respective test kits. Hepatic xanthine oxidase (XOD) expression was examined by enzyme-linked immunosorbent assay. To explore the underlying mechanism, renal urate transporter 1 (URAT1) mRNA levels were evaluated by quantitative real-time PCR. Allopurinol and benzbromarone were used as reference drugs. RESULTS: The original CAGM and its modified high-dose formulation significantly reduced serum uric acid and creatine levels in hyperuricemic mice. In addition, the CAGM-treated groups displayed lower mRNA levels of hepatic XOD and renal URAT1. CONCLUSIONS: CAGM and its modified formulation significantly ameliorated PO-induced hyperuricemia in mice, which might be partially attributable to reductions of hepatic XOD and renal URAT1 levels.