Discovery of GS-7682, a Novel 4'-Cyano-Modified C-Nucleoside Prodrug with Broad Activity against Pneumo- and Picornaviruses and Efficacy in RSV-Infected African Green Monkeys.

Acute respiratory viral infections, such as pneumovirus and respiratory picornavirus infections, exacerbate disease in COPD and asthma patients. A research program targeting respiratory syncytial virus (RSV) led to the discovery of GS-7682 (1), a novel phosphoramidate prodrug of a 4'-CN-4-aza-7,9-dideazaadenosine C-nucleoside GS-646089 (2) with broad antiviral activity against RSV (EC50 = 3-46 nM), human metapneumovirus (EC50 = 210 nM), human rhinovirus (EC50 = 54-61 nM), and enterovirus (EC50 = 83-90 nM). Prodrug optimization for cellular potency and lung cell metabolism identified 5'-methyl [(S)-hydroxy(phenoxy)phosphoryl]-l-alaninate in combination with 2',3'-diisobutyrate promoieties as being optimal for high levels of intracellular triphosphate formation in vitro and in vivo. 1 demonstrated significant reductions of viral loads in the lower respiratory tract of RSV-infected African green monkeys when administered once daily via intratracheal nebulized aerosol. Together, these findings support additional evaluation of 1 and its analogues as potential therapeutics for pneumo- and picornaviruses.

Utilization of OATP1B Biomarker Coproporphyrin-I to Guide Drug-Drug Interaction Risk Assessment: Evaluation by the Pharmaceutical Industry.

Drug-drug interactions (DDIs) involving hepatic organic anion transporting polypeptides 1B1/1B3 (OATP1B) can be substantial, however, challenges remain for predicting interaction risk. Emerging evidence suggests that endogenous biomarkers, particularly coproporphyrin-I (CP-I), can be used to assess in vivo OATP1B activity. The present work under the International Consortium for Innovation and Quality in Pharmaceutical Development was aimed primarily at assessing CP-I as a biomarker for informing OATP1B DDI risk. Literature and unpublished CP-I data along with pertinent in vitro and clinical DDI information were collected to identify DDIs primarily involving OATP1B inhibition and assess the relationship between OATP1B substrate drug and CP-I exposure changes. Static models to predict changes in exposure of CP-I, as a selective OATP1B substrate, were also evaluated. Significant correlations were observed between CP-I area under the curve ratio (AUCR) or maximum concentration ratio (Cmax R) and AUCR of substrate drugs. In general, the CP-I Cmax R was equal to or greater than the CP-I AUCR. CP-I Cmax R < 1.25 was associated with absence of OATP1B-mediated DDIs (AUCR < 1.25) with no false negative predictions. CP-I Cmax R < 2 was associated with weak OATP1B-mediated DDIs (AUCR < 2). A correlation was identified between CP-I exposure changes and OATP1B1 static DDI predictions. Recommendations for collecting and interpreting CP-I data are discussed, including a decision tree for guiding DDI risk assessment. In conclusion, measurement of CP-I is recommended to inform OATP1B inhibition potential. The current analysis identified changes in CP-I exposure that may be used to prioritize, delay, or replace clinical DDI studies.

Interpretation of Protein-Mediated Uptake of Statins by Hepatocytes Is Confounded by the Residual Statin-Protein Complex.

Inclusion of plasma (or plasma proteins) in human hepatocyte uptake studies narrows, but does not close, the gap in in vitro to in vivo extrapolation (IVIVE) of organic anion transporting polypeptide (OATP)-mediated hepatic clearance (CLh) of statins. We have previously shown that this "apparent" protein-mediated uptake effect (PMUE) of statins by OATP1B1-expressing cells, in the presence of 5% human serum albumin (HSA), is mostly an artifact caused by residual statin-HSA complex remaining in the uptake assay. We determined if the same was true with plated human hepatocytes (PHH) and if this artifact can be reduced using suspended human hepatocytes (SHH) and the oil-spin method. We quantified the uptake of a cocktail of five statins by PHH and SHH in the absence and presence of 5% HSA. After terminating the uptake assay, the amount of residual HSA was quantified by quantitative targeted proteomics. For both PHH and SHH, except for atorvastatin and cerivastatin, the increase in total, active, and passive uptake of the statins, in the presence of 5% HSA, was explained by the estimated residual stain-HSA complex. In addition, the increase in active statin uptake by SHH, where present, was marginal (<50%), much smaller than that observed with PHH. Such a marginal increase cannot bridge the gap in IVIVE of CLh of statins. These data disprove the prevailing hypotheses for the in vitro PMUE. A true PMUE should be evaluated using the uptake data corrected for the residual drug-protein complex. SIGNIFICANCE STATEMENT: We show that the apparent protein-mediated uptake (PMUE) of statins by human hepatocytes is largely confounded by residual statin when plated or suspended human hepatocytes are used. Therefore, mechanisms other than PMUE need to be explored to explain the underprediction of the in vivo human hepatic clearance of statins by human hepatocyte uptake assays.

Comparison of the usefulness of the cardio-ankle vascular index and augmentation index as an index of arteriosclerosis in patients with essential hypertension.

Arteriosclerosis leads to various serious diseases that substantially reduce the quality of life. When treating hypertension, it is important to evaluate the degree of arteriosclerosis. In recent years, the cardio-ankle vascular index and augmentation index have been frequently used as indicators of arterial wall sclerosis. However, the superiority of either the cardio-ankle vascular index or the augmentation index as an index of arteriosclerosis remains unclear. Therefore, the present study compared the usefulness of these two indices as an index of arteriosclerosis. Associations between the cardio-ankle vascular index or augmentation index and risk factors for arteriosclerosis and other indices of arteriosclerosis in 535 consecutive patients with essential hypertension were evaluated. The cardio-ankle vascular index was significantly correlated with age, hemoglobin A1c, brain natriuretic peptide, and estimated glomerular filtration rate. In contrast, the augmentation index showed significant correlations only with age, brain natriuretic peptide, and estimated glomerular filtration rate. In addition, these correlations with the augmentation index were generally weaker than those with the cardio-ankle vascular index. The cardio-ankle vascular index, but not the augmentation index, was significantly correlated with flow-mediated dilation, an index of vascular endothelial function, and carotid intima-media thickness, an index of carotid atherosclerosis. Similar results were observed in subgroups stratified by sex and age. These data indicate that the cardio-ankle vascular index is more closely associated with risk factors for arteriosclerosis and other indices of arteriosclerosis than the augmentation index, suggesting that the cardio-ankle vascular index may be superior to the augmentation index as an index of arteriosclerosis.

Clearance prediction for Amgen molecules against Extended Clearance Classification System (ECCS) and future directions.

Early prediction of elimination pathways for new chemical entities can have a profound impact on drug discovery programs. The recently proposed Extended Clearance Classification System (ECCS) is a step in the right direction, providing a framework to help identify the major elimination pathway of a drug. A list of 42 Amgen small molecules was evaluated against the ECCS framework to assess its performance in retrospectively predicting their major elimination pathway. Here, we present a critical analysis of the chemical space defined by the ECCS framework with the aim of identifying its applicability and constraints. This evaluation highlights the critical need for periodic review and revision of ECCS, given that target constraints are moving molecules away from the traditional 'drug-like' physicochemical space.

Prediction of Transporter-Mediated Rosuvastatin Hepatic Uptake Clearance and Drug Interaction in Humans Using Proteomics-Informed REF Approach.

Suspended, plated, or sandwich-cultured human hepatocytes are routinely used for in vitro to in vivo extrapolation (IVIVE) of transporter-mediated hepatic clearance (CL) of drugs. However, these hepatocyte models have been reported to underpredict transporter-mediated in vivo hepatic uptake CL (CL uptake,in vivo ) of some drugs. Therefore, we determined whether transporter-expressing cells (TECs) can accurately predict the CL uptake,in vivo of drugs. To do so, we determined the uptake CL (CL int,uptake,cells ) of rosuvastatin (RSV) by TECs (organic anion transporting polypeptides/Na+-taurocholate cotransporting polypeptide) and then scaled it to that in vivo by relative expression factor (REF) (the ratio of transporter abundance in human livers and TEC) determined by liquid chromatography tandem mass spectrometry-based quantitative proteomics. Both the TEC and hepatocyte models did not meet our predefined success criteria of predicting within 2-fold the RSV CL uptake,in vivo value obtained from our positron emission tomography (PET) imaging. However, the TEC performed better than the hepatocyte models. Interestingly, using REF, TECs successfully predicted RSV CL int,uptake,hep obtained by the hepatocyte models, suggesting that the underprediction of RSV CL uptake,in vivo by TECs and hepatocytes is due to endogenous factor(s) not present in these in vitro models. Therefore, we determined whether inclusion of plasma (or albumin) in TEC uptake studies improved IVIVE of RSV CL uptake,in vivo It did, and our predictions were close to or just fell above our lower 2-fold acceptance boundary. Despite this success, additional studies are needed to improve transporter-mediated IVIVE of hepatic uptake CL of drugs. However, using REF and TEC, we successfully predicted the magnitude of PET-imaged inhibition of RSV CL uptake,in vivo by cyclosporine A. SIGNIFICANCE STATEMENT: We showed that the in vivo transporter-mediated hepatic uptake CL of rosuvastatin, determined by PET imaging, can be predicted (within 2-fold) from in vitro studies in transporter-expressing cells (TECs) (scaled using REF), but only when plasma proteins were included in the in vitro studies. This conclusion did not hold when plasma proteins were absent in the TEC or human hepatocyte studies. Thus, additional studies are needed to improve in vitro to in vivo extrapolation of transporter-mediated drug CL.

Albumin-Mediated Uptake Improves Human Clearance Prediction for Hepatic Uptake Transporter Substrates Aiding a Mechanistic In Vitro-In Vivo Extrapolation (IVIVE) Strategy in Discovery Research.

This study focused on exploring various in vitro to in vivo extrapolation (IVIVE) approaches with the primary goal of improving human hepatic clearance (CL) prediction for OATP substrates. To that effect, the impact of albumin-mediated uptake in human hepatocytes was investigated. In vitro hepatic uptake assay using suspended human hepatocytes was performed with 16 selected OATP substrates to determine the uptake CL in the absence and presence of 4% BSA and unbound hepatocyte to media partition coefficient (Kpuu). Substantial enhancement of the unbound uptake CL (PSu,inf) was observed in the presence of 4% BSA, demonstrating "albumin-mediated" uptake. Prediction of human hepatic CL was performed using two non-traditional IVIVE approaches: initial uptake CL (PSu,inf) and intrinsic metabolic CL (CLint,met) corrected by Kpuu based on extended clearance concept. Compared to traditional IVIVE using CLint,met only, the two tested IVIVE approaches significantly improved the prediction of human hepatic CL. Particularly, direct extrapolation from PSu,inf (+BSA) showed the most robust correlation with in vivo human hepatic CL for all 16 compounds with bias of 1.9-2.0 for two lots of human hepatocytes, respectively. In addition, PSu,inf (+BSA) and Kpuu were also determined in suspended cynomolgus monkey hepatocytes. Prediction of monkey hepatic CL was improved by both approaches, although with more bias compared to human. These results suggested supplementing 4% BSA in human hepatocyte uptake assay provides a useful tool to characterize hepatic uptake CL for OATP substrates, enabling more accurate human CL prediction without any empirical scaling factor (ESF).

Pitfalls in Predicting Hepatobiliary Drug Transport Using Human Sandwich-Cultured Hepatocytes.

During drug development, in vivo human biliary drug clearances (CL) are usually predicted using human sandwich-cultured hepatocytes (SCH). To do so, SCH are pre-incubated with Ca2+-containing or Ca2+-free buffer to maintain or disrupt canalicular tight junctions (CTJ), respectively. Drug uptake into SCH is then conducted in the presence of Ca2+ (up to 20 min). Under this standard protocol, two key assumptions are made: first, that the CTJ are not reformed during the uptake phase when Ca2+ is repleted, and second, disruption of CTJ by the Ca2+-free buffer does not affect the activity of any of the transporters present in the sinusoidal or canalicular membrane. Here we investigated the validity of these assumptions using rosuvastatin (RSV) and taurocholic acid (TCA) as our model drugs. In human SCH, the disrupted CTJ were "reformed" with just 10-min Ca2+ repletion as reflected in a significant increase in TCA cell accumulation. To avoid CTJ reformation and cell toxicity, the standard SCH protocol was modified by conducting the uptake in the absence of Ca2+ for 10 min. Surprisingly, using this protocol, RSV uptake into SCH, plated hepatocytes, and transporter-expressing cells confirmed that Ca2+ depletion substantially decreased NTCP and not OATP1B1 activity. Collectively, this study provides the first evidence of reformation of CTJ in human SCH with 20-min Ca2+ repletion, whereas Ca2+ depletion, during the uptake phase, leads to a significant reduction in NTCP uptake. Thus, the entire SCH protocol needs to be re-examined and optimized to correctly estimate hepatobiliary CL of drugs including those that are NTCP substrates.

[Transcriptomics-driven Evaluation on Liver Toxicity Using Adverse Outcome Pathways (AOP)].

Because the liver is the primary target organ for chemicals and pharmaceuticals, evaluation of these substances' liver toxicity is of critical importance. New evaluation methods without animal testing (i.e., in vitro and/or in silico) are eagerly anticipated, both for animal welfare and for decreasing cost. Also, the importance of mechanistic interpretation of the output derived from non-animal testing has been increasing. Accordingly, we investigated the potential for evaluating liver toxicity by applying the adverse outcome pathway (AOP) concept using gene set enrichment analysis (GSEA) from gene expression (GEx) data. A case study targeting hepatocellular fatty degeneration (HFD) is reported and discussed. We first identified the events detectable in an in vitro system by comparing the GEx data from the rat primary hepatocyte (in vitro) and rat liver (in vivo) treated with a chemical with the ability to induce HFD as one of the phenotypes in a 28-day repeated-dose toxicity test. Then, the scores based on GSEA were calculated after establishing the gene sets for each event leading to HFD. As a result, the mechanistic information leading to HFD was obtained from the score calculated based on the GSEA and the usefulness of the transcriptome-driven evaluation using AOP was demonstrated.

Positron Emission Tomography Imaging of [11 C]Rosuvastatin Hepatic Concentrations and Hepatobiliary Transport in Humans in the Absence and Presence of Cyclosporin A.

Using positron emission tomography imaging, we determined the hepatic concentrations and hepatobiliary transport of [11 C]rosuvastatin (RSV; i.v. injection) in the absence (n = 6) and presence (n = 4 of 6) of cyclosporin A (CsA; i.v. infusion) following a therapeutic dose of unlabeled RSV (5 mg, p.o.) in healthy human volunteers. The sinusoidal uptake, sinusoidal efflux, and biliary efflux clearance (CL; mL/minute) of [11 C]RSV, estimated through compartment modeling were 1,205.6 ± 384.8, 16.2 ± 11.2, and 5.1 ± 1.8, respectively (n = 6). CsA (blood concentration: 2.77 ± 0.24 µM), an organic-anion-transporting polypeptide, Na+ -taurocholate cotransporting polypeptide, and breast cancer resistance protein inhibitor increased [11 C]RSV systemic blood exposure (45%; P < 0.05), reduced its biliary efflux CL (52%; P < 0.05) and hepatic uptake (25%; P > 0.05) but did not affect its distribution into the kidneys. CsA increased plasma concentrations of coproporphyrin I and III and total bilirubin by 297 ± 69%, 384 ± 102%, and 81 ± 39%, respectively (P < 0.05). These data can be used in the future to verify predictions of hepatic concentrations and hepatobiliary transport of RSV.

Comparison of Direct and Mediated Electron Transfer in Electrodes with Novel Fungal Flavin Adenine Dinucleotide Glucose Dehydrogenase.

Direct and mediated electron transfer (DET and MET) in enzyme electrodes with a novel flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from fungi are compared for the first time. DET is achieved by placing a single-walled carbon nanotube (CNT) between GDH and a flat gold electrode where the CNT is close to FAD within the distance for DET. MET is induced by using a free electron transfer mediator, potassium hexacyanoferrate, and shuttles electrons from FAD to the gold electrode. Cyclic voltammetry shows that the onset potential for glucose response current in DET is smaller than in MET, and that the distinct redox current peak pairs in MET are observed whereas no peaks are found in DET. The chronoamperometry with respect to a glucose biosensor shows that (i) the response in DET is more rapid than in MET; (ii) the current at more than +0.45V in DET is larger than the current at the current-peak potential in MET; (iii) a DET electrode covers the glucose concentration range for clinical requirements and is not susceptible to interfering agents at +0.45 V; and (iv) a DET electrode with the novel fungal FAD-GDH does not affect sensing accuracy in the presence of up to 5 mM xylose, while it often shows a similar response level to glucose with other conventionally used fungus-derived FAD-GDHs. It is concluded that our DET system overcomes the disadvantage of MET.

Transport Kinetics, Selective Inhibition, and Successful Prediction of In Vivo Inhibition of Rat Hepatic Organic Anion Transporting Polypeptides.

For successful in vitro-to-in vivo extrapolation of hepatic drug uptake and drug-drug interactions (DDI), it is important to characterize the kinetic properties of the individual transporters involved, their fraction (ft) contribution to hepatic uptake, and their selective inhibitors. Here, we characterized the in vitro transport kinetics of two model drugs, rosuvastatin (RSV) and olmesartan acid (OLM), by rat hepatic organic anion transporting polypeptides (Oatp1a1, 1a4, and 1b2) and identified selective inhibitors of these transporters. [3H]-RSV was transported by Oatp1a1, 1a4, and 1b2, and their Michaelis-Menten constant (Km) values were estimated to be 9.61, 67.2, and 28.1 µM, respectively. In contrast, [3H]-OLM was transported by only Oatp1b2 (Km: 72.8 µM). Digoxin (IC50: 0.107 µM) and rifamycin SV (IC50: 0.140 and 0.088 µM for RSV and OLM, respectively) were potent and selective inhibitors of Oatp1a4 and 1b2, respectively, and glyburide (100 µM) completely inhibited all three rat hepatic Oatps. These inhibitors can therefore be used alone and in combination to determine the contribution of each Oatp to hepatic influx. In addition, the magnitude of in vivo inhibition of sinusoidal uptake clearance of RSV by rifampin was well predicted using rifampin IC50 profiles for each Oatps and RSV ft by each Oatp. This is the first report to 1) detail the transport kinetics of RSV and OLM by rat hepatic Oatps, 2) identify selective inhibitor concentrations of rat Oatps, and 3) demonstrate successful prediction of the magnitude of transporter-mediated in vivo DDI from IC50 profiles of an inhibitor and ft of a drug by each transporter.

Successful Prediction of In Vivo Hepatobiliary Clearances and Hepatic Concentrations of Rosuvastatin Using Sandwich-Cultured Rat Hepatocytes, Transporter-Expressing Cell Lines, and Quantitative Proteomics.

We determined whether in vivo transporter-mediated hepatobiliary clearance (CL) and hepatic concentrations of rosuvastatin (RSV) in the rat could be predicted by transport activity in sandwich-cultured rat hepatocytes (SCRHs) and/or transporter-expressing cell lines scaled by differences in transporter protein expression between SCRHs, cell lines, and rat liver. The predicted hepatobiliary CLs and hepatic concentrations of RSV were compared with our previously published positron emission tomography imaging data. Sinusoidal uptake CL ([Formula: see text]) and efflux (canalicular and sinusoidal) CLs of [3H]-RSV in SCRHs were evaluated in the presence and absence of Ca2+ and in the absence and presence of 1 mM unlabeled RSV (to estimate passive diffusion CL). [Formula: see text] of RSV into cells expressing organic anion transporting polypeptide (Oatp) 1a1, 1a4, and 1b2 was also determined. Protein expression of Oatps in SCRHs and Oatp-expressing cells was quantified by liquid chromatography tandem mass spectrometry. SCRHs well predicted the in vivo RSV sinusoidal and canalicular efflux CLs but significantly underestimated in vivo [Formula: see text]. Oatp expression in SCRHs was significantly lower than that in the rat liver. [Formula: see text], based on RSV [Formula: see text] into Oatp-expressing cells (active transport) plus passive diffusion CL in SCRHs, scaled by the difference in protein expression in Oatp cells versus SCRH versus rat liver, was within 2-fold of that observed in SCRHs or in vivo. In vivo hepatic RSV concentrations were well predicted by Oatp-expressing cells after correcting [Formula: see text] for Oatp protein expression. This is the first demonstration of the successful prediction of in vivo hepatobiliary CLs and hepatic concentrations of RSV using transporter-expressing cells and SCRHs.

Effects of acupuncture at the ST-36 point on muscle sympathetic nerve activity and blood pressure in normal adults.

The aim of the present study was to determine the effects of acupuncture on post-ganglionic muscle sympathetic nerve activity (MSNA) in humans. MSNA was measured in 8 healthy adult males by microneurography evaluation of the left peroneal nerve. Blood pressure (BP) and heart rate (HR) were simultaneously recorded. MSNA was evaluated as the burst rate, with total MSNA, BP and HR normalized to their respective baseline values. After 10min of rest in the supine position, acupuncture was applied to the right ST-36 point in the tibialis anterior muscle for 15min, with recovery then monitored over a 20-min period. While the burst rate and total MSNA remained constant throughout the study, there was a significant decrease in BP during the real but not sham acupuncture procedure (p<0.05). HR did not significantly change throughout the study. The results rule out the role of MSNA in the BP fall during acupuncture at the ST-36 point, and suggest possible involvement of other factors in the fall of BP.

Molecular Diagnostic Techniques for Onychomycosis: Validity and Potential Application.

BACKGROUND: Diagnosis of onychomycosis requires positive findings by direct microscopy and fungal culture. Fungal culture is slow and difficult, with low yields. We compared two dermatophyte identification methods, one using a real-time polymerase chain reaction (PCR) method, and the other using fungal culture to validate the molecular method. METHODS: Nail specimens were collected from 149 patients with distal and lateral subungual onychomycosis who were positive for fungal elements by direct microscopy using potassium hydroxide. Each specimen was subjected to the modified real-time PCR assay of Miyajima et al. and fungal culture. RESULTS: Of 149 specimens, 142 (95.3%) were positive for Trichophyton rubrum or Trichophyton mentagrophytes including Trichophyton interdigitale by PCR, while only 104 (69.8%) were positive by fungal culture performed simultaneously. No specimen was negative by PCR, but positive by culture. All specimens positive for T. rubrum or T. mentagrophytes by culture were also positive by PCR, showing complete concordance for Trichophyton species. The culture of 17 specimens yielded fungi other than T. rubrum or T. mentagrophytes, whereas PCR identified T. rubrum in 11 of these specimens. Among 28 culture-negative specimens, 23 showed T. rubrum and four showed T. mentagrophytes by PCR. PCR allowed more rapid identification of causative fungi (≤2 days vs. ≤28 days). CONCLUSION: Real-time PCR achieved a higher dermatophyte identification rate and showed complete concordance with conventional culture for two Trichophyton species. Specimens never yielded both T. mentagrophytes and T. rubrum simultaneously, suggesting that mixed infection is uncommon.

Simultaneous profiling of 17 steroid hormones for the evaluation of endocrine-disrupting chemicals in H295R cells.

AIM: There is urgent need to develop a new protocol for the evaluation of chemical substances to potentially interact with the endocrine system and induce numerous pathological issues. The recently validated in vitro screening assay is limited on monitoring two steroid hormones. Methodology & results: The H295R model cell was exposed to seven endocrine disrupting chemicals (EDCs). The levels of 17 steroid hormones in cell extracts were subsequently determined by a quantitative targeted GC/MS/MS method. Through wide coverage, this system managed to capture the effects of exposure to increasing EDCs concentrations in the entire steroidogenic pathways. CONCLUSION: The developed approach could be beneficial for the mechanistic investigation of EDCs.

The role of the equilibrative nucleoside transporter 1 on tissue and fetal distribution of ribavirin in the mouse.

Ribavirin is used for the treatment of hepatitis C virus (HCV) infection. The equilibrative nucleoside transporter 1 (ENT1) expressed in hepatocytes transports ribavirin into the liver, the site of efficacy of the drug. However, it is still unclear whether ENT1 plays a dominant role in the hepatic distribution of the drug in vivo. In addition, due to fetal toxicity, administration of ribavirin to pregnant women with HCV infection is contraindicated. ENT1 might play a role in the fetal distribution and therefore the fetal toxicity of ribavirin. The aim of the present study was to investigate the in vivo contribution of ENT1 to the tissue distribution of ribavirin. When compared with that in Ent1(+/+) mice, the ribavirin tissue to plasma concentration ratio (including phosphorylated metabolites) in Ent1(-/-) mice at 15 min and 6 h after intravenous [(3) H]-ribavirin (3 mg/kg) administration was consistently and significantly decreased in the liver and the pancreas. Likewise, when compared with the Ent1(+/+) mice, the fetal distribution of ribavirin at 15 min after administration was significantly reduced in Ent1(-/-) fetuses and placenta. In contrast, there was no significant difference between Ent1(+/+), Ent1(+/-) and Ent1(-/-) mice in the fetal or placental to maternal plasma ribavirin concentration ratio at 2 h after ribavirin administration. The findings in the present study suggest that ENT1 plays a pivotal role in the distribution of ribavirin into tissues including the liver and pancreas, but affects only the rate, but not the extent, of ribavirin distribution into the fetus. Copyright © 2016 John Wiley & Sons, Ltd.

Suppression of activation of muscle sympathetic nerve during non-noxious local cooling after the end of local cooling in normal adults.

PURPOSE: While non-noxious local cooling is widely used in physical medicine, its effect on muscle sympathetic nerve activity (MSNA) and cardiovascular regulation are not clear. The purpose of the present study was to assess the responses of MSNA, blood pressure (BP), heart rate (HR) and local blood flow during non-noxious local cooling. METHODS: The study included two protocols. Both protocols consisted of 10-min rest in supine position, followed by 15-min local cooling (15 °C) of the shin and anterior foot, and 20-min recovery. MSNA of the right common peroneal nerve, BP, HR, and shin skin temperature (TSK) were recorded in eight men in the first protocol, while leg blood flow (LBF) was measured in the same subjects by strain-gauge plethysmography in the second protocol. RESULTS: TSK gradually decreased from 31.5 ± 0.02 to 16.0 ± 1.01 °C (mean ± SEM) during local cooling, and gradually increased after the end of local cooling. No subject complained of pain, and BP and HR remained constant. The MSNA burst rate increased significantly (p < 0.05) to 141.1 ± 12.5 % during local cooling, but decreased significantly (p < 0.05) to 73.6 ± 5.9 % during the recovery period. Total MSNA also increased to 148.0 ± 14.2 % (p < 0.05) during local cooling, and decreased to 74.0 ± 13.9 % (p < 0.05) at recovery. LBF remained constant through the experiment. CONCLUSIONS: The results suggest that MSNA is activated by non-noxious local cooling, and attenuated after the end of local cooling without any changes in HR and BP.

Membrane transport mechanisms of choline in human intestinal epithelial LS180 cells.

The aim of the present study was to investigate the membrane transport mechanisms of choline using human intestinal epithelial LS180 cells. The mRNA of choline transporter-like proteins (CTLs) was expressed significantly in LS180 cells, and the rank order was CTL1 > CTL4 > CTL3 > CTL2 > CTL5. In contrast, the mRNA expression of other choline transporters, organic cation transporter (OCT) 1, OCT2 and high-affinity choline transporter 1 (CHT1), was considerably lower in LS180 cells. Five mm unlabelled choline, hemicolinium-3 and guanidine, but not tetraethylammonium, inhibited the cellular uptake of 100 µm choline in LS180 cells. The uptake of choline into LS180 cells was virtually Na(+)-independent. The uptake of choline was significantly decreased by acidification of the extracellular pH; however, it was not increased by alkalization of the extracellular pH. In addition, both acidification and alkalization of intracellular pH decreased the uptake of choline, indicating that the choline uptake in LS180 cells is not stimulated by the outward H(+) gradient. On the other hand, the uptake of choline was decreased by membrane depolarization along with increasing extracellular K(+) concentration. In addition, the Na(+)-independent uptake of choline was saturable, and the Km value was estimated to be 108 µm. These findings suggest that the uptake of choline into LS180 cells is membrane potential-dependent, but not outward H(+) gradient-dependent.