A comparison of microfluidic methods for high-throughput cell deformability measurements.

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.

Limiting tumor cells comprehensively at micro and macro levels to improve the therapeutic effect of chemotherapy.

Clinical data shows that antitumor treatments are often ineffective if tumor cells have metastasized. To gain an effective antitumor therapeutic effect, in this report, the tumor cell was limited to the primary site and simultaneously ablated by chemotherapy. Considering the extremely complicated process of cancer metastasis, we seek to comprehensively suppress tumor metastases at both micro and macro levels, which closely link to migration and interact with each other. At the micro level, the motility of the tumor cell was decreased via accelerating mitochondria fusion. At the macro level, the unfavorable hypoxia environment was improved. A liposome-based multifunctional nanomedicine was designed by coloading latrunculin B (LAT-B), an inhibitor of actin polymerization, and doxorubicin (DOX) into the hydrophobic bilayers and aqueous cavity, respectively. Meanwhile, an oxygen reservoir named perfluoropentane (PFP) was encapsulated into the liposome core to fulfill synergistic treatment of metastatic tumors. In this paper, we demonstrated that the metastasis of the tumor cell could be effectively inhibited by LAT-B through promoting mitochondria fusion without affecting its function, making it as an encouraging candidate for effective anti-metastasis therapy. Meanwhile, we found that the combination of LAT-B and DOX shows a synergistic effect against tumors because the combined effect of these two drugs cover the entire cell proliferation process. In a word, this report presents a potential improvement in the treatment of metastatic cancer.

Teneligliptin Promotes Bile Acid Synthesis and Attenuates Lipid Accumulation in Obese Mice by Targeting the KLF15-Fgf15 Pathway.

Bile acids (BAs) play essential physiological roles not only by facilitating the absorption and transport of nutrients but also by acting as a complex molecular signaling system. Reduced levels of BAs have been observed in obesity and other metabolic disorders. In the present study, we explored the effect of the dipeptidyl peptidase-4 (DPP-4) inhibitor teneligliptin on BA synthesis, both in vitro and in vivo. In our in vivo experiments, we found that teneligliptin increased the liver, ileal, and serum BA concentrations in mice undergoing teneligliptin treatment for 10 weeks. We further found that in mice fed a high-fat diet, teneligliptin prevented an increase in markers of obesity (body weight, total cholesterol, total triglyceride, adipocyte size) while increasing the total serum and ileal levels of BA. Mechanistically, teneligliptin increased BA synthesis through the alternative synthesis pathway, as the levels of both 7α-hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) along with downstream oxysterol 7α-hydroxylase (CYP7B1) but not sterol 12α-hydroxylase (CYP8B1) were increased. Importantly, teneligliptin suppressed the expression of the BA synthesis inhibitory factor Fgf15, which was mediated through phosphatidylinositol 3-kinase (PI3K)/AKT/Krüppel-like factor 15 (KLF15) signaling. Inhibition of KLF15 abolished this effect. Together, our results provide evidence of the potential benefit of teneligliptin in the treatment of metabolic disorders via increased BA production.

Small molecule-induced simultaneous destabilization of ß-catenin and RAS is an effective molecular strategy to suppress stemness of colorectal cancer cells.

BACKGROUND: Cancer stem cells (CSCs), the major driver of tumorigenesis, is a sub-population of tumor cells responsible for poor clinical outcomes. However, molecular mechanism to identify targets for controlling CSCs is poorly understood. METHODS: Gene Set Enrichment Analyses (GSEA) of Wnt/ß-catenin and RAS signaling pathways in stem-like subtype of colorectal cancer (CRC) patients were performed using two gene expression data set. The therapeutic effects of destabilization of ß-catenin and RAS were tested by treatment of small molecule KYA1797K using CRC patient derived cells. RESULTS: Treatment with KYA1797K, a small molecule that destabilizes both ß-catenin and RAS via Axin binding, effectively suppresses the stemness of CSCs as shown in CRC spheroids and small intestinal tumors of ApcMin/+/K-RasG12DLA2 mice. Moreover, KYA1797K also suppresses the stemness of cells in CRC patient avatar model systems, such as patient-derived tumor organoids (PDTOs) and patient-derived tumor xenograft (PDTX). CONCLUSION: Our results suggest that destabilization of both ß-catenin and RAS is a potential therapeutic strategy for controlling stemness of CRC cells. Video abstract.

Multicenter prospective observational study of teneligliptin, a selective dipeptidyl peptidase-4 inhibitor, in patients with poorly controlled type 2 diabetes: Focus on glycemic control, hypotensive effect, and safety Chikushi Anti-Diabetes Mellitus Trial-Teneligliptin (CHAT-T).

Objective: We purpose to confirm the effect of teneligliptin (Tenelia), a selective dipeptidyl peptidase-4 (DPP-4) inhibitor, on glycemic control and non-glucose risk factors for macroangiopathy, including blood pressure, lipid metabolism, and body weight.Methods: In a prospective, multicenter, open-label, observational study, teneligliptin (20 mg/day) was administered to type 2 diabetic patients with poor glycemic control (HbA1c ≥ 6.5% to <10%) at our hospitals. The safety of teneligliptin and its impact on blood glucose, blood pressure, and the lipid profile were assessed after administration for 3 and 6 months.Results: One hundred and sixty-two patients were enrolled between February 2014 and August 2015. HbA1c was 7.6% at baseline and showed significant reduction to 7.1% after 3 months of treatment and to 6.9% after 6 months (both p < 0.01). Patients with poorly controlled hypertension (systolic blood pressure [SBP] ≥130 mmHg and/or diastolic blood pressure [DBP] ≥80 mmHg) at study initiation were extracted to investigate the effect of teneligliptin on blood pressure. SBP showed a significant decrease from 141.2 ± 9.8 mmHg at baseline to 131.1 ± 14.3 mmHg after 3 months and 133.9 ± 11.5 mmHg after 6 months (both p < 0.001). DBP also decreased significantly from 85.8 ± 5.7 mmHg at baseline to 78.4 ± 10.0 mmHg after 3 months and 79.7 ± 10.1 mmHg after 6 months (both p < 0.001). Adverse events were pruritus in four patients, and cerebral infarction was reported as a cerebrovascular event in one patient.Conclusions: Teneligliptin therapy was safe and improved glycemic control irrespective of baseline HbA1c. Blood pressure was also improved in patients with concomitant hypertension.

Hyperglycemia-Triggered Sphingosine-1-Phosphate and Sphingosine-1-Phosphate Receptor 3 Signaling Worsens Liver Ischemia/Reperfusion Injury by Regulating M1/M2 Polarization.

Hyperglycemia aggravates hepatic ischemia/reperfusion injury (IRI), but the underlying mechanism for the aggravation remains elusive. Sphingosine-1-phosphate (S1P) and sphingosine-1-phosphate receptors (S1PRs) have been implicated in metabolic and inflammatory diseases. Here, we discuss whether and how S1P/S1PRs are involved in hyperglycemia-related liver IRI. For our in vivo experiment, we enrolled diabetic patients with benign hepatic disease who had liver resection, and we used streptozotocin (STZ)-induced hyperglycemic mice or normal mice to establish a liver IRI model. In vitro bone marrow-derived macrophages (BMDMs) were differentiated in high-glucose (HG; 30 mM) or low-glucose (LG; 5 mM) conditions for 7 days. The expression of S1P/S1PRs was analyzed in the liver and BMDMs. We investigated the functional and molecular mechanisms by which S1P/S1PRs may influence hyperglycemia-related liver IRI. S1P levels were higher in liver tissues from patients with diabetes mellitus and mice with STZ-induced diabetes. S1PR3, but not S1PR1 or S1PR2, was activated in liver tissues and Kupffer cells under hyperglycemic conditions. The S1PR3 antagonist CAY10444 attenuated hyperglycemia-related liver IRI based on hepatic biochemistry, histology, and inflammatory responses. Diabetic livers expressed higher levels of M1 markers but lower levels of M2 markers at baseline and after ischemia/reperfusion. Dual-immunofluorescence staining showed that hyperglycemia promoted M1 (CD68/CD86) differentiation and inhibited M2 (CD68/CD206) differentiation. Importantly, CAY10444 reversed hyperglycemia-modulated M1/M2 polarization. HG concentrations in vitro also triggered S1P/S1PR3 signaling, promoted M1 polarization, inhibited M2 polarization, and enhanced inflammatory responses compared with LG concentrations in BMDMs. In contrast, S1PR3 knockdown significantly retrieved hyperglycemia-modulated M1/M2 polarization and attenuated inflammation. In conclusion, our study reveals that hyperglycemia specifically triggers S1P/S1PR3 signaling and exacerbates liver IRI by facilitating M1 polarization and inhibiting M2 polarization, which may represent an effective therapeutic strategy for liver IRI in diabetes.

Mechanistic insights from sequential combination therapy with a sodium glucose co-transporter-2 inhibitor and a dipeptidyl peptidase-4 inhibitor: Results from the CANARIS Trial using canagliflozin and teneligliptin.

AIM: To elucidate the mechanisms involved in the sequential use of SGLT2 and DPP4 inhibitors (SGLT2i and DPP-4i). METHODS: Twenty-six type-2 diabetes mellitus patients were recruited into a stepped regimen of 100 mg of canagliflozin daily from day 1, supplemented with 20 mg of teneligliptin daily from day 4. Glucose (Glu), insulin and glucagon were measured at fasting and after ingesting a mixed meal on days 1, 4 and 6. RESULTS: Canagliflozin decreased fasting plasma glucose to an extent inversely proportional to the change in the glucagon-to-insulin (G/I) ratio. This correlation at fasting was maintained when adding teneligliptin, while the change in the area under the curve of Glu (GluAUC) correlated closely with that in the G/I ratio at fasting and 60 min with canagliflozin. Moreover, these correlations persisted at 60 and 120 min postprandially, but not at fasting on day 6 when teneligliptin was added. CONCLUSION: The result suggested that the dominant mechanism responsible for the glucose metabolism reflected in the G/I ratio was attributable to SGLT2i and that its active mechanism persisted, despite adding a DPP-4i.

Coadministration of the prostaglandin F2α receptor antagonist preterm labour drug candidate OBE022 with magnesium sulfate, atosiban, nifedipine and betamethasone.

AIMS: To investigate presence or absence of clinically relevant drug interactions (pharmacokinetic and safety/tolerability) of OBE022 with standard-of-care medicines for preterm labour, enabling coadministration and further clinical development. METHODS: Part A: open-label, randomized, 3-period crossover assessing coadministration of single doses of OBE022 (1100 mg) and MgSO4 . Part B: open-label, single-sequence crossover assessing the interactions following administration of OBE022 (1000 mg/day) at steady state coadministered with single doses of atosiban, nifedipine and betamethasone. Twenty-five healthy nonpregnant women of reproductive age were enrolled (Part A: n = 12; Part B: n = 13). RESULTS: OBE022, alone or in combination with standard-of-care medications, was well tolerated. Headache and dizziness were the most frequently reported adverse events; dizziness occurred more often with the nifedipine/OBE022 combination. There were no clinically significant pharmacokinetic interactions when coadministered with MgSO4 . Co-administration had no notable effect on atosiban exposure. Atosiban reduced exposure to OBE002 (peak concentration [Cmax ] 22%, area under the concentration-time curve [AUC] 19%). Coadministration with betamethasone slightly increased betamethasone exposure (Cmax  + 18%, AUC +27%) and OBE002 exposure (Cmax  + 35%, AUC +15%). These changes were not considered clinically significant. Coadministration with nifedipine slightly increased OBE002 exposure (Cmax  + 29%, AUC +24%) and markedly increased nifedipine exposure (Cmax by 2-fold and AUC by 2-fold), which may be clinically significant. CONCLUSIONS: The use of OBE022, a PGF2α antagonist prodrug, in combination with standard-of-care medicines may provide new treatment alternatives for preterm labour. All tested combinations were well tolerated. Nifedipine doses could potentially be reduced or staggered when coadministered with OBE022.

Novel hybrids of benzothiazole-1,3,4-oxadiazole-4-thiazolidinone: Synthesis, in silico ADME study, molecular docking and in vivo anti-diabetic assessment.

A series of new benzothiazole-1,3,4-oxadiazole-4-thiazolidinone hybrid analogs (Tz1-Tz28) were synthesized in search of potential anti-diabetic agents. Molecular docking study was conducted with binding pocket of peroxisome proliferator activated receptor-gamma to elucidate the binding interactions of newly synthesized targets. Seven selected compounds with best docking scores were further screened for in vivo anti-hyperglycemic efficacy by oral glucose tolerance test in non-diabetic rats and on streptozotocin induced diabetic rat models. All the tested compounds demonstrated excellent to moderate reduction in blood glucose levels. Three of the compounds (Tz21, Tz7 and Tz10) showed excellent anti-diabetic effect by reducing concentration of glucose to 157.15 ±â€¯1.79 mg/dL, 154.39 ±â€¯1.71 mg/dL, 167.36 ±â€¯2.45 mg/dL, respectively better than the standard drug, pioglitazone, 178.32 ±â€¯1.88 mg/dL. Moreover, three derivatives Tz21, Tz4 and Tz24 with IC50 values of 0.21 ±â€¯0.01 µM, 9.03 ±â€¯0.12 µM and 11.96 ±â€¯0.40 µM respectively also showed better inhibitory activities on alpha-glucosidase even more than the standard acarbose (IC50 = 18.5 ±â€¯0.20 µM), indicating Tz21 has the highest inhibitory effect among the seven tested derivatives. Prediction of Drug like properties using molinspiration online software suggests that all the synthesized compounds have potential of becoming the orally active molecules. Thus, these novel hybrids could serve as potential candidates to become leads for the development of new drugs eliciting anti-hyperglycemic effect orally.

Phase I studies of AZD1208, a proviral integration Moloney virus kinase inhibitor in solid and haematological cancers.

BACKGROUND: Proviral integration Moloney virus (PIM) kinases (PIM1, 2 and 3) are overexpressed in several tumour types and contribute to oncogenesis. AZD1208 is a potent ATP-competitive PIM kinase inhibitor investigated in patients with recurrent or refractory acute myeloid leukaemia (AML) or advanced solid tumours. METHODS: Two dose-escalation studies were performed to evaluate the safety and tolerability, and to define the maximum tolerated dose (MTD), of AZD1208 in AML and solid tumours. Secondary objectives were to evaluate the pharmacokinetics, pharmacodynamics (PD) and preliminary efficacy of AZD1208. RESULTS: Sixty-seven patients received treatment: 32 in the AML study over a 120-900 mg dose range, and 25 in the solid tumour study over a 120-800 mg dose range. Nearly all patients (98.5%) in both studies experienced adverse events, mostly gastrointestinal (92.5%). Dose-limiting toxicities included rash, fatigue and vomiting. AZD1208 was not tolerated at 900 mg, and the protocol-defined MTD was not confirmed. AZD1208 increased CYP3A4 activity after multiple dosing, resulting in increased drug clearance. There were no clinical responses; PD analysis showed biological activity of AZD1208. CONCLUSIONS: Despite the lack of single-agent clinical efficacy with AZD1208, PIM kinase inhibition may hold potential as an anticancer treatment, perhaps in combination with other agents.

OBE022, an Oral and Selective Prostaglandin F2α Receptor Antagonist as an Effective and Safe Modality for the Treatment of Preterm Labor.

Preterm birth is the major challenge in obstetrics, affecting ∼10% of pregnancies. Pan-prostaglandin synthesis inhibitors [nonsteroidal anti-inflammatory drugs (NSAIDs)] prevent preterm labor and prolong pregnancy but raise concerns about fetal renal and cardiovascular safety. We conducted preclinical studies examining the tocolytic effect and fetal safety of the oral prodrug candidate OBE022 [(S)-2-amino-3-methyl-butyric acid (S)-3-{[(S)-3-(biphenyl-4-sulfonyl)-thiazolidine-2-carbonyl]-amino}-3-(4-fluoro-phenyl)-propyl ester] and its parent OBE002 [(S)-3-(biphenyl-4-sulfonyl)-thiazolidine-2-carboxylic acid [(S)-1-(4-fluoro-phenyl)-3-hydroxy-propyl]-amide], both potent and highly selective antagonist of the contractile prostaglandin F2α (PGF2α ) receptor (FP). Efficacy of OBE022 and OBE002, alone and in combination with other tocolytics, was assessed in human tissues and pregnant animal models for inhibition of uterine contraction and delay of parturition. Selective safety of OBE022 and/or OBE002, compared with NSAID indomethacin, was assessed on renal function, closure of the ductus arteriosus, and inhibition of platelet aggregation. In in vitro studies, OBE002 inhibited spontaneous, oxytocin- and PGF2α -induced human myometrial contractions alone and was more effective in combination with atosiban or nifedipine. In in vivo studies, OBE022 and OBE002 reduced spontaneous contractions in near-term pregnant rats. In pregnant mice, OBE022 delayed RU486 [(8S,11R,13S,14S,17S)-11-[4-(dimethylamino)phenyl]-17-hydroxy-13-methyl-17-prop-1-ynyl-1,2,6,7,8,11,12,14,15,16-decahydrocyclopenta[a]phenanthren-3-one] -induced parturition and exerted synergistic effects in combination with nifedipine. OBE022 and/or OBE002 did not show the fetal side effects of ductus arteriosus constriction, impairment of kidney function, or inhibition of platelet aggregation observed with indomethacin. Orally active OBE022 and OBE002 exhibits potent tocolytic effects on human tissues ex vivo and animal models in vivo without causing the adverse fetal side effects seen with indomethacin. Selectively targeting the FP receptor in combination with existing tocolytics may be an effective strategy for preventing or delaying preterm delivery.

Pharmacokinetics, safety and tolerability of OBE022, a selective prostaglandin F2α receptor antagonist tocolytic: A first-in-human trial in healthy postmenopausal women.

AIMS: Preterm birth remains a significant risk for later disability. The selective inhibition of the prostaglandin F2α receptor has significant advantages for a tocolytic. The prodrug OBE022 and its metabolite OBE002 are novel prostaglandin F2α receptor antagonists under development for treating preterm labour. METHODS: We performed a prospective, first in human, Phase I, dose escalation, placebo-controlled, randomized trial at a clinical trial site in the UK. Placebo, single ascending doses of 10, 30, 100, 300, 1000 or 1300 mg, and multiple ascending doses over 7 days of 100, 300 or 1000 mg day-1 ; were administered to postmenopausal female volunteers. Food interaction was additionally evaluated. RESULTS: Subjects tolerated OBE022 well at all single and multiple doses. No clinically relevant changes in safety parameters were shown and there were no serious adverse events. Observations showed that prodrug OBE022 was readily absorbed and rapidly converted into its equally active stable metabolite OBE002. The plasma level of OBE002 rose with increasing doses, reaching exposure levels that were anticipated to be clinically relevant within 1 h following administration. There was no clinically significant food interaction, with peak exposures reduced to 80% and area under the curve staying bioequivalent. The mean half-life of OBE002 ranged between 8 and 11 h following administration of a single dose and 22-29 h after multiple doses. CONCLUSIONS: Administration of OBE022 was safe and had favourable pharmacokinetic characteristics and no clinically relevant interaction with food. Our results allow further investigation of OBE022 in preterm labour patients.

Pharmacokinetics of pidotimod in broiler chickens by UHPLC-MS/MS after oral and intravenous administration.

Pidotimod is widely used in children as an immune promoter but it has not been fully evaluated in animals. The pharmacokinetics of pidotimod and its oral bioavailability have not been described in broiler chickens. We developed a simple and sensitive UHPLC-MS/MS assay for rapid determination of pidotimod levels in chicken blood. Recoveries were nearly 100% and the coefficients of accuracy and precision were minimal. Healthy broiler chickens were given 10 mg/kg pidotimod either orally or intravenously. The oral pidotimod was rapidly absorbed (time to reach maximum concentration, 1.25 h) and rapidly eliminated (the mean residence time was 3.2 h). A noncompartmental analysis of the intravenous route indicated a mean plasma clearance of 2.2 L (h kg)-1 with an estimated mean volume of distribution at steady state of 12.69 L/kg. The bioavailability of pidotimod after oral dosing was 27%.

AF710B, an M1/sigma-1 receptor agonist with long-lasting disease-modifying properties in a transgenic rat model of Alzheimer's disease.

INTRODUCTION: AF710B (aka ANAVEX 3-71) is a novel selective allosteric M1 muscarinic and sigma-1 receptor agonist. In 3×Tg-AD mice, AF710B attenuates cognitive deficits and decreases Alzheimer-like hallmarks. We now report on the long-lasting disease-modifying properties of AF710B in McGill-R-Thy1-APP transgenic (Tg) rats. METHODS: Chronic treatment with AF710B (10 µg/kg) was initiated in postplaque 13-month-old Tg rats. Drug or vehicle was administered orally daily for 4.5 months and interrupted 5 weeks before behavioral testing. RESULTS: AF710B long-term treatment reverted the cognitive deficits associated with advanced Alzheimer-like amyloid neuropathology in Tg rats. These effects were accompanied by reductions in amyloid pathology and markers of neuroinflammation and increases in amyloid cerebrospinal fluid clearance and levels of a synaptic marker. Importantly, these effects were maintained following a 5-week interruption of the treatment. DISCUSSION: With M1/sigma-1 activity and long-lasting disease-modifying properties at low dose, AF710B is a promising novel therapeutic agent for treating Alzheimer's disease.

[Clinical effect of pidotimod oral liquid as adjuvant therapy for infectious mononucleosis].

OBJECTIVE: To study the clinical effect of pidotimod oral liquid as adjuvant therapy for infectious mononucleosis and its effect on T lymphocyte subsets. METHODS: A total of 76 children with infectious mononucleosis, who were admitted to the hospital between July 2016 and June 2017, were enrolled and randomly divided into two groups: conventional treatment and pidotimod treatment (n=38 each). The children in the conventional treatment group were given antiviral therapy with ganciclovir for injection and symptomatic treatment. Those in the pidotimod treatment group were given pidotimod oral liquid in addition to the treatment in the conventional treatment group. The course of treatment was two weeks for both groups. The two groups were compared in terms of the recovery of clinical indices and the changes in peripheral blood T lymphocyte subsets. RESULTS: Compared with the conventional treatment group, the pidotimod treatment group had significantly shorter fever clearance time, time to the disappearance of isthmopyra, time to the relief of lymph node enlargement, time to the relief of hepatosplenomegaly, and length of hospital stay (P<0.05). After treatment, the pidotimod treatment group had significant reductions in the percentages of CD3+ and CD8+ T cells and had significantly lower percentages of CD3+ and CD8+ T cells than the conventional treatment group (P<0.001). The pidotimod treatment group had significant increases in the percentage of CD4+ T cells and CD4+/CD8+ ratio after treatment, which was significantly higher than those in the conventional treatment group (P<0.001). The conventional treatment group had no significant changes in T lymphocyte subsets after treatment (P>0.05). CONCLUSIONS: Pidotimod oral liquid has a good clinical effect as the adjuvant therapy for infectious mononucleosis and can improve cellular immune function, so it holds promise for clinical application.

Efficacy and safety of canagliflozin as add-on therapy to teneligliptin in Japanese patients with type 2 diabetes mellitus: Results of a 24-week, randomized, double-blind, placebo-controlled trial.

AIMS: To investigate efficacy and safety of the sodium-glucose co-transporter 2 (SGLT2) inhibitor canagliflozin administered as add-on therapy to the dipeptidyl peptidase-4 (DPP-4) inhibitor teneligliptin in patients with type 2 diabetes mellitus (T2DM). MATERIALS AND METHODS: We conducted a multicentre, randomized, double-blind, placebo-controlled, phase 3 clinical trial in Japanese patients with T2DM who had inadequate glycaemic control with teneligliptin. Patients were randomized to receive teneligliptin 20 mg plus either canagliflozin 100 mg (T + C, n = 70) or placebo (T + P, n = 68) once daily. The primary endpoint was the change in glycated haemoglobin (HbA1c) from baseline to week 24. Other endpoints included changes in fasting plasma glucose, body weight, proinsulin/C-peptide ratio, homeostatic model assessment 2-%B and adverse events. Patients also underwent mixed-meal tolerance tests. RESULTS: The difference between the T + C and T + P groups for HbA1c change from baseline to week 24 was -0.88% (least-squares mean, P < .001). Fasting plasma glucose, body weight and the proinsulin/C-peptide ratio were significantly lower in the T + C group than in the T + P group. Homeostatic model assessment 2-%B improved with T + C compared with T + P. The T + C group exhibited a decrease in the 2-hour postprandial plasma glucose and plasma glucose area under the curve (AUC)0-2h in a mixed-meal tolerance test. No significant between-group differences were observed for C-peptide AUC0-2h or glucagon AUC0-2h after meals. Incidences of adverse events were 60.0% and 47.1% in the T + C and T + P groups, respectively. No hypoglycaemia was observed. CONCLUSIONS: Canagliflozin administered as add-on therapy to teneligliptin was effective and well tolerated in Japanese T2DM patients.

Immunomodulatory effects of pidotimod in adults with community-acquired pneumonia undergoing standard antibiotic therapy.

The morbidity and mortality of community-acquired pneumonia (CAP) are still elevated and two aspects seem to contribute to a worse outcome: an uncontrolled inflammatory reaction and an inadequate immune response. Adjuvants, including corticosteroids and intravenous immunoglobulins, have been proposed to counterbalance these effects but their efficacy is only partial. We examined the immunomodulatory activity of Pidotimod (PDT), a synthetic dipeptide molecule in adult patients hospitalized for CAP. Sixteen patients with a diagnosis of CAP and a PSI score III or IV and/or a CURB-65 0-2 were randomized to receive either levofloxacin 500 mg b.i.d. alone or levofloxacin plus PDT (800mg, 2 daily doses). Blood samples were drawn at baseline (T0), before initiation of therapy, as well as 3 (T3), and 5 (T5) days after initiation of therapy. Immunologic and clinical parameters were analyzed at each time point. Supplementation of antibiotic therapy with PDT resulted in an upregulation of antimicrobial and of immunomodulatory proteins as well as in an increased percentage of Toll like receptor (TLR)2- and TLR4, and of CD80- and CD86-expressing immune cells. Notably, Pidotimod supplementation was also associated with a robust reduction of TNFα-producing immune cells. No significant differences were observed in clinical parameters. These results confirm that supplementation of antibiotic therapy with Pidotimod in patients with CAP results in a potentially beneficial modulation of innate immunity.

Increasing taurine intake and taurine synthesis improves skeletal muscle function in the mdx mouse model for Duchenne muscular dystrophy.

KEY POINTS: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease associated with increased inflammation, oxidative stress and myofibre necrosis. Cysteine precursor antioxidants such as N-acetyl cysteine (NAC) and l-2-oxothiazolidine-4-carboxylate (OTC) reduce dystropathology in the mdx mouse model for DMD, and we propose this is via increased synthesis of the amino acid taurine. We compared the capacity of OTC and taurine treatment to increase taurine content of mdx muscle, as well as effects on in vivo and ex vivo muscle function, inflammation and oxidative stress. Both treatments increased taurine in muscles, and improved many aspects of muscle function and reduced inflammation. Taurine treatment also reduced protein thiol oxidation and was overall more effective, as OTC treatment reduced body and muscle weight, suggesting some adverse effects of this drug. These data suggest that increasing dietary taurine is a better candidate for a therapeutic intervention for DMD. ABSTRACT: Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease for which there is no widely available cure. Whilst the mechanism of loss of muscle function in DMD and the mdx mouse model are not fully understood, disruptions in intracellular calcium homeostasis, inflammation and oxidative stress are implicated. We have shown that protein thiol oxidation is increased in mdx muscle, and that the indirect thiol antioxidant l-2-oxothiazolidine-4-carboxylate (OTC), which increases cysteine availability, decreases pathology and increases in vivo strength. We propose that the protective effects of OTC are a consequence of conversion of cysteine to taurine, which has itself been shown to be beneficial to mdx pathology. This study compares the efficacy of taurine with OTC in decreasing dystropathology in mdx mice by measuring in vivo and ex vivo contractile function and measurements of inflammation and protein thiol oxidation. Increasing the taurine content of mdx muscle improved both in vivo and ex vivo muscle strength and function, potentially via anti-inflammatory and antioxidant effects of taurine. OTC treatment increased taurine synthesis in the liver and taurine content of mdx muscle, improved muscle function and decreased inflammation. However, OTC was less effective than taurine treatment, with OTC also decreasing body and EDL muscle weights, suggesting that OTC had some detrimental effects. These data support continued research into the use of taurine as a therapeutic intervention for DMD, and suggest that increasing dietary taurine is the better strategy for increasing taurine content and decreasing severity of dystropathology.

Selectivity of commonly used inhibitors of clathrin-mediated and caveolae-dependent endocytosis of G protein-coupled receptors.

Among the multiple G protein-coupled receptor (GPCR) endocytic pathways, clathrin-mediated endocytosis (CME) and caveolar endocytosis are more extensively characterized than other endocytic pathways. A number of endocytic inhibitors have been used to block CME; however, systemic studies to determine the selectivity of these inhibitors are needed. Clathrin heavy chain or caveolin1-knockdown cells have been employed to determine the specificity of various chemical and molecular biological tools for CME and caveolar endocytosis. Sucrose, concanavalin A, and dominant negative mutants of dynamin blocked other endocytic pathways, in addition to CME. In particular, concanavalin A nonspecifically interfered with the signaling of several GPCRs tested in the study. Decreased pH, monodansylcadaverine, and dominant negative mutants of epsin were more specific for CME than other treatments were. A recently introduced CME inhibitor, Pitstop2™, showed only marginal selectivity for CME and interfered with receptor expression on the cell surface. Blockade of receptor endocytosis by epsin mutants and knockdown of the clathrin heavy chain enhanced the ß2AR-mediated ERK activation. Overall, our studies show that previous experimental results should be interpreted with discretion if they included the use of endocytic inhibitors that were previously thought to be CME-selective. In addition, our study shows that endocytosis of ß2 adrenoceptor through clathrin-mediated pathway has negative effects on ERK activation.

Morning vs evening dosing of the cathepsin K inhibitor ONO-5334: effects on bone resorption in postmenopausal women in a randomized, phase 1 trial.

UNLABELLED: The cathepsin K inhibitor, ONO-5334, improves bone mineral density in postmenopausal women with osteoporosis. The effects of morning versus evening administration of ONO-5334 were investigated by measuring bone turnover marker levels in healthy postmenopausal women. Morning administration of ONO-5334 showed a more consistent suppressive effect on bone resorption than evening administration. INTRODUCTION: Bone turnover is thought to be subject to circadian variation, and the efficacy of osteoporosis treatments may be optimized by regulating the time of dosing. This study assessed whether evening administration of the cathepsin K inhibitor, ONO-5334, had a differential effect on the bone turnover marker, C-terminal telopeptide of type I collagen (CTX-I), compared with morning administration. METHODS: This was a single-center, single blind crossover study. Fourteen healthy postmenopausal women were assigned to receive ONO-5334 150 mg once daily for 5 days in each period; they were randomized to receive either evening doses in the first period and morning doses in the second or vice versa. Serum and urinary levels of CTX-I were measured throughout the study. RESULTS: Both regimens showed similar patterns of reduction in serum and urinary CTX-I; however, CTX-I suppression was more consistently >60% over 24 h following morning administration. Morning administration led to 6% greater suppression of 24-h serum CTX-I area under the effect curve (AUE; 69 vs 63%; P < .05) and 7% greater suppression of urinary CTX-I/creatinine AUE (93 vs 86%; P < .01) than evening administration. Higher plasma ONO-5334 concentrations were observed between 12 and 24 h postdose following morning administration, with mean trough concentrations for the morning and evening regimens at 9.4 and 4.0 ng/mL, respectively. There were no safety findings of concern. CONCLUSION: Morning dosing of ONO-5334 is more efficacious at reducing markers of bone turnover in healthy postmenopausal women than evening dosing. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01384188 , registered on June 27, 2011 EudraCT: 2008-006284-37.