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With a great quantity of solid dosage tested by dissolution technology, developing a rapid and sensitive method to access the content of drug within dissolution media is highly desired by analysts and scientists. Traditionally, dissolution media is not compatible with mass spectrometry since the inorganic salts in the media might damage the mass spectrometer. Here, paper spray ionization mass spectrometry (PSI-MS), one of the ambient mass spectrometry technologies, is developed to characterize the content of drugs in dissolution media. The porous structure of paper can effectively retain salts from entering mass spectrometer. This makes the measurement of drug content within dissolution media by mass spectrometer possible. After the experimental parameters were optimized, calibration curves of model drugs - enalapril, quinapril and benazepril were established by using corresponding deuterated internal standards. PSI-MS was then deployed to characterize the content of enalapril from the dissolution testing of enalapril tablets. The results from PSI-MS are comparable to those from HPLC characterization. More importantly, the analysis time of 6 samples is shortened from 90min to 6min. Detection limit of enalapril maleate tablets by PSI-MS is 1/300 of LC. PSI-MS is rapid, sensitive and accurate in analyzing drug content from dissolution tests.

Osteosarcoma is one of the most common primary malignant tumors of bone in adolescents. Human umbilical vein endothelial cells (HUVECs) derived exosomes are associated with osteosarcoma cell stemness. Little is known about the function of HUVECs-exosomes in osteosarcoma cell stemness. This work aimed to investigate the mechanism of action of HUVECs-exosomes in regulating stem cell-like phenotypes of osteosarcoma cells. HUVECs were treated with GW4869 (exosome inhibitor). Human osteosarcoma cells (U2OS and 143B) were treated with HUVECs supernatant, HUVECs-exosomes with or without RO4929097 (γ secretase inhibitor, used to block Notch signaling pathway). We found that HUVECs supernatant and HUVECs-exosomes enhanced the proportions of STRO-1+CD117+ cells and the expression of stem cell-related proteins Oct4 and Sox2. Both HUVECs supernatant and HUVECs-exosomes promoted the sarcosphere formation efficiency of U2OS and 143B cells. These stem-like phenotypes of U2OS and 143B cells conferred by HUVECs-exosomes were repressed by GW4869. Moreover, HUVECs-exosomes promoted the expression of Notch1, Hes1 and Hey1 in the U2OS and 143B cells. RO4929097 treatment reversed the impact of HUVECs-exosomes on Notch1, Hes1, and Hey1 expression by inhibiting Notch1 signaling pathway. In conclusion, this work demonstrated that HUVECs-exosomes promoted cell stemness in osteosarcoma through activating Notch signaling pathway. Thus, our data reveal the mechanism of HUVECs-exosomes in regulating cell stemness of osteosarcoma, and provide a theoretical basis for osteosarcoma treatment by exosomes.

To investigate the suppressive function of RO4929097, a potent -secretase inhibitor, on RANKL-induced osteoclastogenesis. The cytotoxicity of RO4929097 was evaluated. The suppressive effect and possible molecular mechanism of RO4929097 on RANKL-induced osteoclastogenesis was evaluated both in vitro and in vivo. The IC50 of RO4929097 was 2.93 μM. Treatment with different doses of RO4929097 (100 nM, 200 nM, and 400 nM) effectively reduced osteoclast formation (number and resorption area) in a dose-dependent manner. The qPCR results revealed that RO4929097 attenuates RANKL-induced osteoclast formation and NFATc1 protein expression. The in vivo experiments demonstrated that RO4929097 had an inhibitory effect on LPS-induced bone resorption. Our in vitro experiments showed that RO4929097 can potently inhibit osteoclastogenesis and bone resorption by down-regulating the Notch/MAPK/JNK/Akt-mediated reduction of NFATc1. In accordance with these in vitro observations, RO4929097 attenuated LPS-induced osteolysis in mice. In conclusion, our findings indicate that Notch may represent a potential therapeutic target for the treatment of osteolytic diseases.

Rationale: Osteosarcoma (OS), the most common type of bone tumor, which seriously affects the patients' limb function and life quality. OS has a strong tendency of lung metastasis, and the five-year survival rate of patients with metastatic osteosarcoma is less than 20%. Thus, new treatment targets and strategies are urgently needed. Methods: The expression of the histone demethylase KDM6B and H3K27me3 levels in OS specimens were analyzed using quantitative PCR and immunohistochemical assays. The biological functions of KDM6B were determined using in vitro transwell, wound healing assays, and an in vivo orthotopic injection-induced lung metastasis model. Subsequently, chromatin immunoprecipitation sequencing (ChIP-seq) combined with transcriptomic RNA sequencing (RNA-seq), and subsequent ChIP-qPCR, western blot, and aerobic glycolysis assays were used to explore the mechanism of KDM6B function and validate the candidate target gene of KDM6B. Results: KDM6B expression was significantly upregulated in OS patients, and high KDM6B expression was associated with poorer prognosis in OS patients. Targeting KDM6B significantly inhibited OS cell migration in vitro and lung metastasis in vivo. RNA-seq and ChIP-seq analysis revealed that KDM6B increases lactate dehydrogenase LDHA expression in OS cells by directly mediating H3K27me3 demethylation. The phenotypes of inhibited cell metastasis in KDM6B-knockdown OS cells was reversed upon overexpression of LDHA. Finally, a small molecule inhibitor targeting KDM6B significantly inhibited OS cell migration in vitro and lung metastasis in vivo. Conclusions: Collectively, we elucidated that upregulated KDM6B facilitates tumor metastasis in OS via modulating LDHA expression. Our findings deepen the recognition of OS metastasis mechanism and suggest that KDM6B might be a new potential therapeutic target for the treatment of OS (especially highly metastatic OS).

Rationale: As the central hallmark of liver fibrosis, transdifferentiation of hepatic stellate cells (HSCs), the predominant contributor to fibrogenic hepatic myofibroblast responsible for extracellular matrix (ECM) deposition, is characterized with transcriptional and epigenetic remodeling. We aimed to characterize the roles of H3K27 methyltransferase EZH2 and demethylase JMJD3 and identify their effective pathways and novel target genes in HSCs activation and liver fibrosis. Methods: In primary HSCs, we analyzed effects of pharmacological inhibitions and genetic manipulations of EZH2 and JMJD3 on HSCs activation. In HSCs cell lines, we evaluated effects of EZH2 inhibition by DZNep on proliferation, cell cycling, senescence and apoptosis. In CCl4 and BDL murine models of liver fibrosis, we assessed in vivo effects of DZNep administration and Ezh2 silencing. We profiled rat primary HSCs transcriptomes with RNA-seq, screened the pathways and genes associated with DZNep treatment, analyzed EZH2 and JMJD3 regulation towards target genes by ChIP-qPCR. Results: EZH2 inhibition by DZNep resulted in retarded growth, lowered cell viability, cell cycle arrest in S and G2 phases, strengthened senescence, and enhanced apoptosis of HSCs, decreased hepatic collagen deposition and rescued the elevated serum ALT and AST activities of diseased mice, and downregulated cellular and hepatic expressions of H3K27me3, EZH2, α-SMA and COL1A. Ezh2 silencing by RNA interference in vitro and in vivo showed similar effects. JMJD3 inhibition by GSK-J4 and overexpression of wild-type but not mutant Jmjd3 enhanced or repressed HSCs activation respectively. EZH2 inhibition by DZNep transcriptionally inactivated TGF-β1 pathway, cell cycle pathways and vast ECM components in primary HSCs. EZH2 inhibition decreased H3K27me3 recruitment at target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, and increased their expressions, while Jmjd3 overexpression manifested alike effects. Conclusions: EZH2 and JMJD3 antagonistically modulate HSCs activation. The therapeutic effects of DZNep as epigenetic drug in liver fibrosis are associated with the regulation of EZH2 towards direct target genes encoding TGF-β1 pseudoreceptor BAMBI, anti-inflammatory cytokine IL10 and cell cycle regulators CDKN1A, GADD45A and GADD45B, which are also regulated by JMJD3. Our present study provides new mechanistic insight into the epigenetic modulation of EZH2 and JMJD3 in HSCs biology and hepatic fibrogenesis.

Rationale: The Notch and transforming growth factor-β (TGFβ) signaling pathways are two intracellular mechanisms that control fibrosis in general but whether they play a major role in retinal fibrosis is less clear. Here we study how these two signaling pathways regulate Muller cell-dominated retinal fibrosis in vitro and in vivo. Methods: Human MIO-M1 Muller cells were treated with Notch ligands and TGFβ1, either alone or in combination. Western blots were performed to study changes in γ-secretase proteases, Notch downstream effectors, endogenous TGFβ1, phosphorylated Smad3 (p-Smad3) and extracellular matrix (ECM) proteins. We also studied the effects of RO4929097, a selective γ-secretase inhibitor, on expression of ECM proteins after ligand stimulation. Muller cell viability was studied by AlamarBlue and cytotoxicity by lactate cytotoxicity assays. Finally, we studied changes in Notch and TGFβ signaling and tested the effect of intravitreal injections of the Notch pathway inhibitor RO4929097 on retinal fibrosis resulted from Sodium iodate (NaIO3)-induced retinal injury in mice. We also studied the safety of intravitreal injections of RO4929097 in normal mice. Results: Treatment of Muller cells with Notch ligands upregulated γ-secretase proteases and Notch downstream effectors, with increased expression of endogenous TGFβ1, TGFβ receptors and p-Smad3. TGFβ1 upregulated the expression of proteins associated with both signaling pathways in a similar manner. Notch ligands and TGFβ1 had additive effects on overexpression of ECM proteins in Muller cells which were inhibited by RO4929097. Notch and TGFβ ligands stimulated Muller cell proliferation which was inhibited by RO4929097 without damaging the cells. NaIO3-induced retinal injury activated both Notch and TGFβ signaling pathways in vivo. Intravitreal injection of RO4929097 prevented Muller cell gliosis and inhibited overexpression of ECM proteins in this murine model. We found no safety concerns for up to 17 days after an intravitreal injection of RO4929097. Conclusions: Inhibiting Notch signaling might be an effective way to prevent retinal fibrosis. This study is of clinical significance in developing a treatment for preventing fibrosis in proliferative vitreoretinopathy, proliferative diabetic retinopathy and wet age-related macular degeneration.

Trypanothione synthetase (TryS) produces N1,N8-bis(glutathionyl)spermidine (or trypanothione) at the expense of ATP. Trypanothione is a metabolite unique and essential for survival and drug-resistance of trypanosomatid parasites. In this study, we report the mechanistic and biological characterisation of optimised N5-substituted paullone analogues with anti-TryS activity. Several of the new derivatives retained submicromolar IC50 against leishmanial TryS. The binding mode to TryS of the most potent paullones has been revealed by means of kinetic, biophysical and molecular modelling approaches. A subset of analogues showed an improved potency (EC50 0.5–10 µM) and selectivity (20–35) against the clinically relevant stage of Leishmania braziliensis (mucocutaneous leishmaniasis) and L. infantum (visceral leishmaniasis). For a selected derivative, the mode of action involved intracellular depletion of trypanothione. Our findings shed light on the molecular interaction of TryS with rationally designed inhibitors and disclose a new set of compounds with on-target activity against different Leishmania species.

Tumor-initiating cells (TICs) maintain heterogeneity within tumors and seed metastases at distant sites, contributing to therapeutic resistance and disease recurrence. In colorectal cancer (CRC), strategy that effectively eradicates TICs and is of potential value for clinical use still remains in need. Methods: The anti-tumorigenic activity of a small-molecule inhibitor of KDM6 histone demethylases named GSK-J4 in CRC was evaluated by in vitro assays and in vivo imaging of xenografted tumors. Sphere formation, flow cytometry analysis of cell surface markers and intestinal organoid formation were performed to examine the impact of GSK-J4 on TIC properties. Transcriptome analysis and global profiling of H3K27ac, H3K27me3, and KDM6A levels by ChIP-seq were conducted to elucidate how KDM6 inhibition reshapes epigenetic landscape and thereby eliminating TICs. Results: GSK-J4 alleviated the malignant phenotypes of CRC cells in vitro and in vivo, sensitized them to chemotherapeutic treatment, and strongly repressed TIC properties and stemness-associated gene signatures in these cells. Mechanistically, KDM6 inhibition induced global enhancer reprogramming with a preferential impact on super-enhancer-associated genes, including some key genes that control stemness in CRC such as ID1. Besides, expression of both Kdm6a and Kdm6b was more abundant in mouse intestinal crypt when compared with upper villus and inhibition of their activities blocked intestinal organoid formation. Finally, we unveiled the power of KDM6B in predicting both the overall survival outcome and recurrence of CRC patients. Conclusions: Our study provides a novel rational strategy to eradicate TICs through reshaping epigenetic landscape in CRC, which might also be beneficial for optimizing current therapeutics.

Notch pathways have important roles in carcinogenesis including pathways involving the Notch1 and Notch2 oncogenes. Pan-Notch inhibitors, such as gamma secretase inhibitors (GSIs), have been used in the clinical trials, but the outcomes of these trials have been insufficient and have yielded unclear. In the present study, we demonstrated that GSIs, such as MK-0752 and RO4929097, inhibit breast tumor growth, but increase the breast cancer stem cell (BCSC) population in Notch3-expressing breast cancer cells, in a process that is coupled with IL6 induction and is blocked by the IL6R antagonist Tocilizumab (TCZ). IL6 induction results from inhibition of Notch3-Hey2 signaling through MK-0752. Furthermore, HIF1α upregulates Notch3 expression via direct binding to the Notch3 promoter and subsequently downregulates BCSCs by decreasing the IL6 levels in Notch3-expressing breast cancer cells. Utilizing both breast cancer cell line xenografts and patient-derived xenografts (PDX), we showed that the combination of MK-0752 and Tocilizumab significantly decreases BCSCs and inhibits tumor growth and thus might serve as a novel therapeutic strategy for treating women with Notch3-expressing breast cancers.

Background Poor response to diuretics is associated with worse prognosis in patients with acute heart failure (AHF). We hypothesized that treatment with tolvaptan improves diuretic response in patients with AHF. Methods We performed a secondary analysis of the AQUAMARINE open-label randomized study in which a total of 217 AHF patients with renal impairment (eGFR

Although the role of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in neuronal excitability and synaptic transmission is still unclear, it is postulated that the HCN channels may be involved in seizure activity. The aim of this study was to assess the effects of ivabradine (an HCN channel inhibitor) on the protective action of four classical antiepileptic drugs (carbamazepine, phenobarbital, phenytoin and valproate) against maximal electroshock-induced seizures in mice. Tonic seizures (maximal electroconvulsions) were evoked in adult male albino Swiss mice by an electric current (sine-wave, 25 mA, 0.2 s stimulus duration) delivered via auricular electrodes. Acute adverse-effect profiles of the combinations of ivabradine with classical antiepileptic drugs were measured in mice along with total brain antiepileptic drug concentrations. Results indicate that ivabradine (10 mg/kg, i.p.) significantly enhanced the anticonvulsant activity of valproate and considerably reduced that of phenytoin in the mouse maximal electroshock-induced seizure model. Ivabradine (10 mg/kg) had no impact on the anticonvulsant potency of carbamazepine and phenobarbital in the maximal electroshock-induced seizure test in mice. Ivabradine (10 mg/kg) significantly diminished total brain concentration of phenytoin and had no effect on total brain valproate concentration in mice. In conclusion, the enhanced anticonvulsant action of valproate by ivabradine in the mouse maximal electroshock-induced seizure model was pharmacodynamic in nature. A special attention is required when combining ivabradine with phenytoin due to a pharmacokinetic interaction and reduction of the anticonvulsant action of phenytoin in mice. The combinations of ivabradine with carbamazepine and phenobarbital were neutral from a preclinical viewpoint.

Background and Purpose Effective anti‐respiratory syncytial virus (RSV) agents are still not available for clinical use. Current major targets are virus surface proteins, such as a fusion protein involved in viral entry, but agents effective after RSV infection is established are required. Here we have investigated the effects of late therapeutic intervention with a novel inhaled RSV polymerase inhibitor, PC786, on RSV infection in human airway epithelium. Experimental Approach Air liquid interface‐cultured bronchial or small airway epithelium was infected with RSVA2. PC786 was applied apically or basolaterally once daily following peak virus load on Day 3 post inoculation. Apical wash was collected daily for determination of viral burden by PCR and plaque assay (primary endpoints) and biomarker analyses. The effects were compared with those of ALS‐8112, an anti‐RSV nucleoside analogue, and GS‐5806, a fusion‐protein inhibitor, which were treated basolaterally. Key Results Late intervention with GS‐5806 did not show significant anti‐viral effects, but PC786 produced potent, concentration‐dependent inhibition of viral replication with viral load falling below detectable limits 3 days after treatment commenced in airway epithelium. These effects were superior to those of ALS‐8112. PC786 showed inhibitory activities against RSV‐induced increases of CCL5, IL‐6, double‐strand DNA and mucin. The effects of PC786 were also confirmed in small airway epithelium. Conclusion and Implications Late therapeutic intervention with the RSV polymerase inhibitor, PC786, reduced the viral burden quickly in human airway epithelium. Thus, PC786 demonstrates the potential to be an effective therapeutic agent to treat active RSV infection.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults. The current standard of care includes surgery followed by radiotherapy (RT) and chemotherapy with temozolomide (TMZ). Treatment often fails due to the radiation resistance and intrinsic or acquired TMZ resistance of a small percentage of cells with stem cell-like behavior (CSC). The NOTCH signaling pathway is expressed and active in human glioblastoma and NOTCH inhibitors attenuate tumor growth in vivo in xenograft models. Here we show using an image guided micro-CT and precision radiotherapy platform that a combination of the clinically approved NOTCH/γ-secretase inhibitor (GSI) RO4929097 with standard of care (TMZ + RT) reduces tumor growth and prolongs survival compared to dual combinations. We show that GSI in combination with RT and TMZ attenuates proliferation, decreases 3D spheroid growth and results into a marked reduction in clonogenic survival in primary and established glioma cell lines. We found that the glioma stem cell marker CD133, SOX2 and Nestin were reduced following combination treatments and NOTCH inhibitors albeit in a different manner. These findings indicate that NOTCH inhibition combined with standard of care treatment has an anti-glioma stem cell effect which provides an improved survival benefit for GBM and encourages further translational and clinical studies.

Pimobendan (PIMO) can cause adverse effects, such as mitral valve degeneration, in dogs; however, it is unclear whether these effects occur in cats. Therefore, we aimed to determine whether PIMO or benazepril produces adverse cardiac effects in healthy cats. This was a blinded, randomized, prospective parallel study. Twelve cats were randomly divided into two groups of six cats, namely, an angiotensin-converting-enzyme inhibitor group that received benazepril and a PIMO group. Cats were administered their respective treatments for 506 days, and we evaluated cardiac parameters, blood biochemistry and glomerular filtration rates during that time. At the end of the trial, the cats were euthanized, and histopathological examinations were performed by a pathologist who was blinded to the treatment groups. No significant changes were observed in any of the parameters measured in either of the groups. In particular, no significant cardiac lesions were observed in either of the groups. In healthy cats, neither PIMO nor benazepril appears to cause cardiac lesions, but future studies are needed to examine the effects of PIMO in cats with heart disease.

BACKGROUND Tolvaptan, an antagonist of the vasopressin V2 receptor is a novel oral diuretic that promotes water excretion selectively. We have used furosemide as a primary diuretic and added human atrial natriuretic peptide (hANP) if necessary for fluid management postoperatively in living-donor liver transplantation (LDLT) recipients. Recently we introduced tolvaptan and used both tolvaptan and furosemide as primary diuretics. MATERIAL AND METHODS Clinical outcomes were compared between LDLT recipients whose postoperative fluid management was performed before (control group, n=10) and after (tolvaptan group, n=16) introduction of tolvaptan. RESULTS Preoperative and intraoperative demographic data did not differ significantly between the groups except for the period of post-surgical follow-up and total ischemic time. Urine volume was 1,242±692, 2,240±1307, and 2,268±1262 mL on postoperative day 1, 3, and 7, respectively, in the tolvaptan group. These volumes did not significantly differ from those in control group (1,027±462, 1,788±909, and 2,057±1216 mL on day 1, 3, and 7 postoperatively, respectively). Body weight gain and fluid volume from abdominal drainage tubes postoperatively did not differ significantly between groups. The time from hANP initiation to discontinuation and the time to removal of central vein catheters were significantly reduced in tolvaptan-treated patients. No severe side effects directly related to tolvaptan were observed. The survival rate at month 6 was 90.0% in control patients versus 93.8% in tolvaptan-treated patients. CONCLUSIONS The outcomes of this investigation indicate that tolvaptan in combination with furosemide provides an adequate diuretic for fluid management subsequent to LDLT without causing adverse effects.

The Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure (JNC 7) recommended a thiazide-like diuretic, alone or in combination with other antihypertensive drug classes, as initial therapy for hypertension. JNC 7, however, did not specify preferred combinations. The Avoiding Cardiovascular Events through Combination Therapy in Patients Living with Systolic Hypertension (ACCOMPLISH) trial was completed five years after the JNC 7 and demonstrated a 20 % advantage in cardiovascular risk reduction when blood pressure was lowered using the single-pill combination of benazepril-amlodipine compared to benazepril-hydrochlorothiazide (Jamerson et al. 359(23):2417-28 [1]). This new and significant finding provided compelling evidence that the long-standing preference for diuretics as initial therapy could be refuted, but it may also be relevant to the lower-than-expected reduction in coronary disease related events (compared to stroke) observed for decades prior to the ACCOMPLISH approach to therapy. The JNC 8 panel members recently published their recommendations, and while the group did not recommend benazepril-hydrochlorothiazide over other combinations, they did highlight the findings of ACCOMPLISH, rating the primary ACCOMPLISH paper as "good." The American Society of Hypertension position paper and the European Hypertension Society guidelines endorse such combinations as a first-line agent for patients with stage 2 hypertension. We review the current position of ACCOMPLISH in the guidelines regarding treatment of stage 2 hypertension.

Homeobox (HOX) genes are frequently dysregulated in leukemia. Previous studies have shown that aberrant HOX gene expression accompanies leukemogenesis and affects disease progression and leukemia patient survival. Patients with acute myeloid leukemia (AML) bearing PML-RARα fusion gene have distinct HOX gene signature in comparison to other subtypes of AML patients, although the mechanism of transcription regulation is not completely understood. We previously found an association between the mRNA levels of HOX genes and those of the histone demethylases JMJD3 and UTX in PML-RARα- positive leukemia patients. Here, we demonstrate that the release of the PML-RARα-mediated block in PML-RARα-positive myeloid leukemia cells increased both JMJD3 and HOX gene expression, while inhibition of JMJD3 using the specific inhibitor GSK-J4 reversed the effect. This effect was driven specifically through PML-RARα fusion protein since expression changes did not occur in cells with mutated RARα and was independent of differentiation. We confirmed that gene expression levels were inversely correlated with alterations in H3K27me3 histone marks localized at HOX gene promoters. Furthermore, data from chromatin immunoprecipitation followed by sequencing broaden a list of clustered HOX genes regulated by JMJD3 in PML-RARα-positive leukemic cells. Interestingly, the combination of GSK-J4 and all-trans retinoic acid (ATRA) significantly increased PML-RARα-positive cell apoptosis compared with ATRA treatment alone. This effect was also observed in ATRA-resistant NB4 clones, which may provide a new therapeutic opportunity for patients with acute promyelocytic leukemia (APL) resistant to current treatment. The results of our study reveal the mechanism of HOX gene expression regulation and contribute to our understanding of APL pathogenesis.

Exposure to opiates induces locomotor sensitization in rodents, which has been proposed to correspond to the compulsive drug-seeking behaviour. Numerous studies have demonstrated that locomotor sensitization can occur in a dopamine transmission-independent manner; however, the underlying mechanisms are unclear.Co-immunoprecipitation, BRET and cross-antagonism assays were used to demonstrate the existence of receptor heterodimers. Function of heterodimers was evaluated by behavioural studies of locomotor sensitization.The dopamine DOur results suggest that μ receptor-D

Multiple myeloma (MM), a blood cancer characterized by the uncontrolled proliferation of plasma cells, remains incurable by current therapy. Notch signaling has been implicated in the growth and chemoresistance of various cancer types including MM, and therefore we hypothesized that targeting the Notch pathway could be beneficial for the treatment of this disease. Here, we report an anti-tumor effect of Notch/γ-secretase inhibitor RO4929097 in a pre-clinical model of MM. We demonstrate that this effect was associated with decreased angiogenesis and significant down-regulation of TGF-β1. In addition, we also show that treatment with RO4929097 results in decreased number and functional activity of osteoclasts. Taken together, our data indicate that targeting Notch may be considered as a new strategy to be tested for MM therapy.

Background and Purpose Varenicline, a neuronal nicotinic acetylcholine receptor (nAChR) modulator, decreases ethanol consumption in rodents and humans. The proposed mechanism of action for varenicline to reduce ethanol consumption has been through modulation of dopamine (DA) release in the nucleus accumbens (NAc) via α4*-containing nAChRs in the ventral tegmental area (VTA). However, presynaptic nAChRs on dopaminergic terminals in the NAc have been shown to directly modulate dopaminergic signalling independently of neuronal activity from the VTA. In this study, we determined whether nAChRs in the NAc play a role in varenicline's effects on ethanol consumption. Experimental Approach Rats were trained to consume ethanol using the intermittent-access two-bottle choice protocol for 10 weeks. Ethanol intake was measured after varenicline or vehicle was microinfused into the NAc (core, shell or core-shell border) or the VTA (anterior or posterior). The effect of varenicline treatment on DA release in the NAc was measured using both in vivo microdialysis and in vitro fast-scan cyclic voltammetry (FSCV). Key Results Microinfusion of varenicline into the NAc core and core-shell border, but not into the NAc shell or VTA, reduced ethanol intake following long-term ethanol consumption. During microdialysis, a significant enhancement in accumbal DA release occurred following systemic administration of varenicline and FSCV showed that varenicline also altered the evoked release of DA in the NAc. Conclusion and Implications Following long-term ethanol consumption, varenicline in the NAc reduces ethanol intake, suggesting that presynaptic nAChRs in the NAc are important for mediating varenicline's effects on ethanol consumption.

Item does not contain fulltext Glioblastoma is the most common malignant primary brain tumor. Temozolomide (TMZ) is the standard chemotherapeutic agent for this disease. However, intrinsic and acquired TMZ-resistance represents a major obstacle for this therapy. In order to identify factors involved in TMZ-resistance, we engineered different TMZ-resistant glioblastoma cell lines. Gene expression analysis demonstrated that EFEMP1, an extracellular matrix protein, is associated with TMZ-resistant phenotype. Silencing of EFEMP1 in glioblastoma cells resulted in decreased cell survival following TMZ treatment, whereas overexpression caused TMZ-resistance. EFEMP1 acts via multiple signaling pathways, including gamma-secretase-mediated activation of the Notch pathway. We show that inhibition of gamma-secretase by RO4929097 causes at least partial sensitization of glioblastoma cells to temozolomide in vitro and in vivo. In addition, we show that EFEMP1 expression levels correlate with survival in TMZ-treated glioblastoma patients. Altogether our results suggest EFEMP1 as a potential therapeutic target to overcome TMZ-resistance in glioblastoma.

Background and Purpose Airway microvascular leak (MVL) involves the extravasation of proteins from post‐capillary venules into surrounding tissue. MVL is a cardinal sign of inflammation and an important feature of airway inflammatory diseases such as asthma. PGE2, a product of COX‐mediated metabolism of arachidonic acid, binds to four receptors, termed EP1–4. PGE2 has a wide variety of effects within the airway, including modulation of inflammation, sensory nerve activation and airway tone. However, the effect of PGE2 on airway MVL and the receptor/s that mediate this have not been described. Experimental Approach Evans Blue dye was used as a marker of airway MVL, and selective EP receptor agonists and antagonists were used alongside EP receptor‐deficient mice to define the receptor subtype involved. Key Results PGE2 induced significant airway MVL in mice and guinea pigs. A significant reduction in PGE2‐induced MVL was demonstrated in Ptger2 −/− and Ptger4 −/− mice and in wild‐type mice pretreated simultaneously with EP2 (PF‐04418948) and EP4 (ER‐819762) receptor antagonists. In a model of allergic asthma, an increase in airway levels of PGE2 was associated with a rise in MVL; this change was absent in Ptger2 −/− and Ptger4 −/− mice. Conclusions and Implications PGE2 is a key mediator produced by the lung and has widespread effects according to the EP receptor activated. Airway MVL represents a response to injury and under ‘disease’ conditions is a prominent feature of airway inflammation. The data presented highlight a key role for EP2 and EP4 receptors in MVL induced by PGE2.

Homeostatic eating cannot explain overconsumption of food and pathological weight gain. A more likely factor promoting excessive eating is food reward and its representation in the central nervous system (CNS). The anorectic hormones leptin and insulin reduce food reward and inhibit related CNS reward pathways. Conversely, the orexigenic gastrointestinal hormone ghrelin activates both homeostatic and reward-related neurocircuits. The current studies were conducted to identify in rats the effects of intracerebroventricular ghrelin infusions on two distinct aspects of food reward: hedonic valuation (i.e., “liking”) and the motivation to self-administer (i.e., “wanting”) food. To assess hedonic valuation of liquid food, lick motor patterns were recorded using lickometry. Although ghrelin administration increased energy intake, it did not alter the avidity of licking (initial lick rates or lick-cluster size). Several positive-control conditions ruled out lick-rate ceiling effects. Similarly, when the liquid diet was hedonically devalued with quinine supplementation, ghrelin failed to reverse the quinine-associated reduction of energy intake and avidity of licking. The effects of ghrelin on rats' motivation to eat were assessed using lever pressing to self-administer food in a progressive-ratio paradigm. Ghrelin markedly increased motivation to eat, to levels comparable to or greater than those seen following 24 h of food deprivation. Pretreatment with the dopamine D1 receptor antagonist SCH-23390 eliminated ghrelin-induced increases in lever pressing, without compromising generalized licking motor control, indicating a role for D1 signaling in ghrelin's motivational feeding effects. These results indicate that ghrelin increases the motivation to eat via D1 receptor-dependent mechanisms, without affecting perceived food palatability.

In order to understand the structure–activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structure of the binary complex of human dihydro­folate reductase (hDHFR) with the potent and selective inhibitor 2,4-diamino-6-­{2′-O-(3-carboxypropyl)oxydibenz[b,f]-azepin-5-yl}methylpteridine (PT684) was determined to 1.8 A resolution. These data revealed that the carboxylate side chain of PT684 occupies two alternate positions, neither of which interacts with the conserved Arg70 in the active-site pocket, which in turn hydrogen bonds to water. These observations are in contrast to those reported for the ternary complex of mouse DHFR (mDHFR) with NADPH [Cody et al. (2008 ▶), Acta Cryst. D64, 977–984], in which the 3-carboxypropyl side chain of PT684 was hydrolyzed to its hydroxyl derivative, PT684a. The crystallization conditions differed for the human and mouse DHFR crystals (100 mM K2HPO4 pH 6.9, 30% ammonium sulfate for hDHFR; 15 mM Tris pH 8.3, 75 mM sodium cacodylate, PEG 4K for mDHFR). Additionally, the side chains of Phe31 and Gln35 in the hDHFR complex have a single conformation, whereas in the mDHFR complex they occupied two alternative conformations. These data show that the hDHFR complex has a decreased active-site volume compared with the mDHFR complex, as reflected in a relative shift of helix C (residues 59–64) of 1.2 A, and a shift of 1.5 A compared with the ternary complex of Pneumocystis carinii DHFR (pcDHFR) with the parent dibenz[b,f]azepine PT653. These data suggest that the greater inhibitory potency of PT684 against pcDHFR is consistent with the larger active-site volume of pcDHFR and the predicted interactions of the carboxylate side chain with Arg75.

Background: Evidence-based smoking cessation guidelines recommend nicotine replacement therapy (NRT), bupropion SR and varenicline as first-line therapy in combination with behavioural interventions. However, there are limited data to guide clinicians in recommending one form over another, using combinations, or matching individual smokers to particular forms. Objective: To develop decision rules for clinicians to guide differential prescribing practices and tailoring of pharmacotherapy for smoking cessation. Methods: A Delphi approach was used to build consensus among a panel of 37 international experts from various health disciplines. Through an iterative process, panellists responded to three rounds of questionnaires. Participants identified and ranked “best practices” used by them to tailor pharmacotherapy to aid smoking cessation. An independent panel of 10 experts provided cross-validation of findings. Results: There was a 100% response rate to all three rounds. A high level of consensus was achieved in determining the most important priorities: (1) factors to consider in prescribing pharmacotherapy: evidence, patient preference, patient experience; (2) combinations based on: failed attempt with monotherapy, patients with breakthrough cravings, level of tobacco dependence; (3) specific combinations, main categories: (a) two or more forms of NRT, (b) bupropion + form of NRT; (4) specific combinations, subcategories: (1a) patch + gum, (1b) patch + inhaler, (1c) patch + lozenge; (2a) bupropion + patch, (2b) bupropion + gum; (5) impact of comorbidities on selection of pharmacotherapy: contraindications, specific pharmacotherapy useful for certain comorbidities, dual purpose medications; (6) frequency of monitoring determined by patient needs and type of pharmacotherapy. Conclusion: An algorithm and guide were developed to assist clinicians in prescribing pharmacotherapy for smoking cessation. There appears to be good justification for “off-label” use such as higher doses of NRT or combination therapy in certain circumstances. This practical tool reflects best evidence to date of experts in tobacco cessation.

The pathogenesis of cardiac failure involves activation of the neurohumoral axis including stimulation of the sympathetic nervous system, the renin-angiotensin-aldosterone, and nonosmotic vasopressin systems. While these responses are critical in maintaining arterial pressure, they are associated with renal vasoconstriction, as well as sodium and water retention. In advanced circumstances, renal dysfunction and hyponatremia occur with cardiac failure. Even a modest rise in serum creatinine related to diminished renal function in heart failure patients is associated with increased risk for cardiovascular morbidity and mortality. Similarly, increased thirst and the nonosmotic stimulation of vasopressin in advanced cardiac failure leads to hyponatremia, which is also a major risk factor for mortality. Currently, V2 vasopressin receptor antagonists have been shown to correct hyponatremia in cardiac failure. One such agent, conivaptan, also is a V1 receptor antagonist which could theoretically benefit heart failure patients by decreasing cardiac afterload and remodeling. The effect of V2 receptor antagonists to correct hyponatremia in heart failure patients appears to be quite safe. However, to date no effect on mortality has been demonstrated.

The structures of mouse DHFR holo enzyme and a ternary complex with NADPH and a potent inhibitor are described., It has been shown that 2,4-diamino-6-arylmethylpteridines and 2,4-diamino-5-arylmethylpyrimidines containing an O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from opportunistic pathogens such as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV/AIDS patients. In order to understand the structure–activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structures of mouse DHFR (mDHFR; a mammalian homologue) holo and ternary complexes with NADPH and the inhibitor 2,4-diamino-6-(2′-hydroxy­dibenz[b,f]azepin-5-yl)methylpteridine were determined to 1.9 and 1.4 Å resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed no electron density for the O-carboxylalkyloxy side chain. The side chain was either cleaved or completely disordered. The electron density fitted the less potent hydroxyl compound PT684a. Additionally, cocrystallization of mDHFR with NADPH and the less potent 2′-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holoenzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and P. carinii DHFR (pcDHFR) indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit into the active site or that the inhibitor side chain would have to adopt an alternative binding mode to that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active-site volume as reflected in the relative shift of helix C (residues 59–64) by 0.6 Å compared with pcDHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.

Application of orthosteric sigma-1 receptor agonists as anti-seizure drugs has been hindered by questionable efficacy and potential adverse effects. Here, we have investigated the anti-seizure effects of the novel and potent allosteric modulator of sigma-1 receptors, SKF83959 and its derivative SOMCL-668 (3-methyl-phenyl-2,3,4,5-tetrahydro-1H-benzo[d]azepin-7-ol).The anti-seizure effects of SKF83959 were investigated in three mouse models, maximal electroshock seizures, pentylenetetrazole-induced convulsions and kainic acid-induced 'status epilepticus'. Also, in rats, the cortical epileptiform activity induced by topical application of picrotoxin was recorded in electrocorticograms. In rat hippocampal brain slices, effects of the drugs on the high potassium-evoked epileptiform local field potentials were studied. Anti-seizure activities of SOMCL-668, a newly developed sigma-1 receptor selective allosteric modulator, were also investigated.SKF83959 (20, 40 mg·kg(-1) ) exhibited anti -seizure actitity in the three mouse models and reduced the cortical epileptiform activity without alteration of spontaneous motor activity and motor coordination. These effects were blocked by the sigma-1 receptor antagonist BD1047, but not the dopamine D1 receptor antagonist SCH23390. SKF83959 alone did not directly inhibit the epileptiform firing of CA3 neurons induced by high potassium in hippocampal slices, but did potentiate inhibition by the orthosteric sigma-1 receptor agonist SKF10047. Lastly, a selective sigma-1 receptor allosteric modulator SOMCL-668, which does not bind to dopamine receptors, exerted similar anti-seizure activities.SKF83959 and SOMCL-668 displayed anti-seizure activities, indicating that allosteric modulation of sigma-1 receptors may provide a novel approach for discovering new anti-seizure drugs.

In pigs, ivabradine reduces infarct size even when given only at reperfusion and in the absence of heart rate reduction. The mechanism of this non-heart rate-related cardioprotection is unknown. Hence, in the present study we assessed the pleiotropic action of ivabradine in more detail.Anaesthetized mice were pretreated with ivabradine (1.7 mg · kg(-1) i.v.) or placebo (control) before a cycle of coronary occlusion/reperfusion (30/120 min ± left atrial pacing). Infarct size was determined. Isolated ventricular cardiomyocytes were exposed to simulated ischaemia/reperfusion (60/5 min) in the absence and presence of ivabradine, viability was then quantified and intra- and extracellular reactive oxygen species (ROS) formation was detected. Mitochondria were isolated from mouse hearts and exposed to simulated ischaemia/reperfusion (6/3 min) in glutamate/malate- and ADP-containing buffer in the absence and presence of ivabradine respectively. Mitochondrial respiration, extramitochondrial ROS, mitochondrial ATP production and calcium retention capacity (CRC) were assessed.Ivabradine decreased infarct size even with atrial pacing. Cardiomyocyte viability after simulated ischaemia/reperfusion was better preserved with ivabradine, the accumulation of intra- and extracellular ROS decreased in parallel. Mitochondrial complex I respiration was not different without/with ivabradine, but ivabradine significantly inhibited the accumulation of extramitochondrial ROS, increased mitochondrial ATP production and increased CRC.Ivabradine reduces infarct size independently of a reduction in heart rate and improves ventricular cardiomyocyte viability, possibly by reducing mitochondrial ROS formation, increasing ATP production and CRC.

Sensorimotor gating is a perceptual process aimed at filtering out irrelevant information. In humans and animal models, this function can be operationally measured through the prepulse inhibition (PPI) of the acoustic startle reflex. Notably, PPI deficits are associated with numerous neuropsychiatric conditions characterized by gating disturbances, including schizophrenia and Tourette syndrome. Ample evidence has shown that dopamine plays a key role in PPI regulation and, in particular, rodent studies indicate that this neurotransmitter modulates PPI through D1 and D2 dopamine receptors. In mice, the relative contributions of these two families of receptors are strain-dependent. Conversely, the role of D1 receptors in the regulation of PPI across different rat strains remains unclear.We tested the effects of selective D1 and D2 receptor agonists and antagonists on the startle reflex and PPI of Sprague-Dawley, Wistar and Long-Evans rats.In contrast with Sprague-Dawley and Wistar rats, the full D1 receptor agonist SKF82958 elicited significant PPI deficits in Long-Evans rats, an effect sensitive to the selective D1 antagonist SCH23390.Our results suggest that, in Long-Evans rats, D1 receptor activation may be sufficient to significantly impair PPI. These data emphasize the role of D1 receptors in the pathophysiology of neuropsychiatric disorders featuring alterations in sensorimotor gating, and uphold the importance of the genetic background in shaping the role of dopamine receptors in the regulation of this key information-processing function.This article is part of a themed section on Updating Neuropathology and Neuropharmacology of Monoaminergic Systems. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.13/issuetoc.

© Impact Journals, LLC. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited., Advances in the molecular biology of medulloblastoma revealed four genetically and clinically distinct subgroups. Group 3 medulloblastomas are characterized by frequent amplifications of the oncogene MYC, a high incidence of metastasis, and poor prognosis despite aggressive therapy. We investigated several potential small molecule inhibitors to target Group 3 medulloblastomas based on gene expression data using an in silico drug screen. The Connectivity Map (C-MAP) analysis identified piperlongumine as the top candidate drug for non-WNT medulloblastomas and the cyclin-dependent kinase (CDK) inhibitor alsterpaullone as the compound predicted to have specific antitumor activity against Group 3 medulloblastomas. To validate our findings we used these inhibitors against established Group 3 medulloblastoma cell lines. The C-MAP predicted drugs reduced cell proliferation in vitro and increased survival in Group 3 medulloblastoma xenografts. Alsterpaullone had the highest efficacy in Group 3 medulloblastoma cells. Genomic profiling of Group 3 medulloblastoma cells treated with alsterpaullone confirmed inhibition of cell cycle-related genes, and down-regulation of MYC. Our results demonstrate the preclinical efficacy of using a targeted therapy approach for Group 3 medulloblastomas. Specifically, we provide rationale for advancing alsterpaullone as a targeted therapy in Group 3 medulloblastoma., This study was supported by the Canadian Cancer Society (Grant #2011-70051), the Pediatric Brain Tumor Foundation of the United States, the Brain Tumour Foundation of Canada, Meagan’s Walk, b.r.a.i.n.child and the Wiley Fund at the Hospital for Sick Children.

Evidence suggests that renin‐angiotensin‐aldosterone system inhibition ameliorates endothelial dysfunction. The authors examined the effect of amlodipine besylate/benazepril HCl combination treatment compared with amlodipine besylate monotherapy in modulating endothelial dysfunction. This multicenter, double‐blind, 12‐week study randomized 70 hypertensive subjects with at least one other endothelial dysfunction risk factor to amlodipine besylate/benazepril HCl (5/20 mg/d force‐titrated to 5/40 mg/d) or amlodipine besylate monotherapy (5 mg/d force‐titrated to 10 mg/d). Both the combination and monotherapy produced significant median increases from baseline in percentage flow‐mediated vasodilation (2.0% and 1.2%, respectively) and percentage change in percent flow‐mediated vasodilation (25% and 16%, respectively). These improvements were numerically larger with combination treatment, but between‐group differences did not achieve statistical significance. Reductions in systolic and diastolic blood pressure were significantly greater (P=.0452/P=.0297) with combination treatment (−18.6/−12.3 mm Hg) than with monotherapy (−14.8/−9.1 mm Hg). A highly positive correlation between change in systolic blood pressure and change in percent of flow‐mediated vasodilation was demonstrated only for combination treatment.

A formal enantioselective synthesis of benazepril·HCl (4), an anti- hypertensive drug, is reported. Our synthesis employed an asymmetric aza-Michael addition of L-homophenylalanine ethyl ester (LHPE, 1) to 4-(2-nitrophenyl)-4-oxo- but-2-enoic acid methyl ester (6) as the key step to prepare (2S,3’S)-2-(2-oxo-2,3,4,5- tetrahydro-1H-benzo[b]azepin-3-ylamino)-4-phenylbutyric acid ethyl ester (8), which is the key intermediate leading to benazepril·HCl (4).

Pulse pressure (PP) is an independent risk factor for cardiovascular (CV) disease and death but few studies have investigated the effect of antihypertensive treatments in relation to PP levels before treatment. The Avoiding Cardiovascular Events Through Combination Therapy in Patients Living With Systolic Hypertension (ACCOMPLISH) trial showed that the combination of benazepril+amlodipine (B+A) is superior to benazepril+hydrochlorothiazide (B+H) in reducing CV events. We aimed to investigate whether the treatment effects in the ACCOMPLISH trial were dependent on baseline PP. High-risk hypertensive patients (n=11,499) were randomized to double-blinded treatment with single-pill combinations of either B+A or B+H and followed for 36 months. Patients were divided into tertiles according to their baseline PP and events (CV mortality/myocardial infarction or stroke) were compared. Hazard ratios (HRs) for the treatment effect (B+A over B+H) were calculated in a Cox regression model with age, coronary artery disease, and diabetes mellitus as covariates and were compared across the tertiles. The event rate was increased in the high tertile of PP compared with the low tertile (7.2% vs 4.4% P

Neuronal nitric oxide (NO) plays an important role in gastric motor activity and modulates the mechanosensitivity of gastro-oesophageal vagal afferents. Effects of NO on food intake are dependent on feeding status. We sought to determine the effect of NO on gastro-oesophageal vagal afferent activity in the normally fed and food-restricted states and the second messenger pathways mediating these effects. Eight week old female C56BL/6 mice were fed ad libitum or food restricted for 14 h. An in vitro preparation was used to determine the functional effects of NO and the second messenger pathways involved. Expression of NO signal transduction molecules in vagal afferents was determined by reverse-transcription polymerase chain reaction (RT-PCR). Endogenous NO and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibited vagal mucosal afferent responses to tactile stimuli in mice fed ad libitum. After a 14 h fast endogenous NO and SNAP potentiated tension and mucosal afferent responses to mechanical stimulation. The excitatory effect of NO was blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin. After a 14 h fast expression of NADPH oxidase 2 (NOX2) mRNA in whole nodose ganglia was significantly reduced and the excitatory effect of NO on gastro-oesophageal vagal afferents was lost. Under fasting conditions the inhibitory effect of NO was blocked with the hyperpolarisation-activated cyclic nucleotide-gated (HCN) channel blocker ivabradine and mRNA expression of HCN3 in the nodose ganglia was elevated. In conclusion, the role of NO in the peripheral modulation of gastro-oesophageal vagal afferents is dynamic and dependent on feeding status.

A regulatory effect of arginine vasopressin (AVP) on sweat water conservation has been hypothesized but not definitively evaluated. AVP-mediated insertion of sweat and salivary gland aquaporin-5 (AQP5) water channels through activation of the vasopressin type 2 receptor (V2R) remains an attractive, yet unexplored, mechanism that could result in a more concentrated sweat with resultant decreased water loss. Ten runners participated in a double-blind randomized control treadmill trial under three separate pharmacological conditions: a placebo, V2R agonist (0.2 mg desmopressin), or V2R antagonist (30 mg tolvaptan). After a familiarization trial, runners ran for 60 min at 60% of peak speed followed by a performance trial to volitional exhaustion. Outcome variables were collected at three exercise time points: baseline, after the steady-state run, and after the performance run. Body weight losses were +]) [ F = 38.9; P < 0.0001], which verified both pharmacological activation and inhibition of the V2R at the kidney collecting duct. Plasma osmolality and [Na+] demonstrated significant exercise ( F = 26.0 and F = 11.1; P < 0.0001) and condition ( F = 5.1 and F = 3.8; P < 0.05) effects (osmolality and [Na+], respectively). No significant exercise or condition effects were noted for either sweat or salivary [Na+]. Significant exercise effects were noted for plasma [AVP] ( F = 22.3; P < 0.0001), peak core temperature ( F = 103.3; P < 0.0001), percent body weight change ( F = 6.3; P = 0.02), plasma volume change ( F = 21.8; P < 0.0001), and thirst rating ( F = 78.2; P < 0.0001). Performance time was not altered between conditions ( P = 0.80). In summary, AVP acting at V2R does not appear to regulate water losses from body fluids other than renal excretion during exercise.

Subtypes of the hyperpolarization-activated cyclic nucleotide-gated (HCN) family of cation channels are widely expressed on nerves and smooth muscle cells in many organ systems, where they serve to regulate membrane excitability. Here we have assessed whether HCN channel inhibitors alter the function of airway smooth muscle or the neurons that regulate airway smooth muscle tone.The effects of the HCN channel inhibitors ZD7288, zatebradine and Cs(+) were assessed on agonist and nerve stimulation-evoked changes in guinea pig airway smooth muscle tone using tracheal strips in vitro, an innervated tracheal tube preparation ex vivo or in anaesthetized mechanically ventilated guinea pigs in vivo. HCN channel expression in airway nerves was assessed using immunohistochemistry, PCR and in situ hybridization.HCN channel inhibition did not alter airway smooth muscle reactivity in vitro to exogenously administered smooth muscle spasmogens, but significantly potentiated smooth muscle contraction evoked by the sensory nerve stimulant capsaicin and electrical field stimulation of parasympathetic cholinergic postganglionic neurons. Sensory nerve hyperresponsiveness was also evident in in vivo following HCN channel blockade. Cs(+) , but not ZD7288, potentiated preganglionic nerve-dependent airway contractions and over time induced autorhythmic preganglionic nerve activity, which was not mimicked by inhibitors of potassium channels. HCN channel expression was most evident in vagal sensory ganglia and airway nerve fibres.HCN channel inhibitors had a previously unrecognized effect on the neural regulation of airway smooth muscle tone, which may have implications for some patients receiving HCN channel inhibitors for therapeutic purposes.

This study aimed to investigate the relationship between the plasma concentration (PK) of the novel histamine H3 receptor antagonist, GSK239512, and the brain occupancy of H(3) receptors (RO) in healthy human volunteers.PET scans were obtained after i.v. administration of the H(3) -specific radioligand [(11) C]GSK189254. Each subject was scanned before and after single oral doses of GSK239512, at 4 and 24 h after dose. PET data were analysed by compartmental analysis, and regional RO estimates were obtained by graphical analysis of changes in the total volumes of distribution of the radioligand, followed by a correction for occupancy by the high affinity radioligand. The PK/RO relationship was analysed by a population-modelling approach, using the average PK of GSK239512 during each scan.Following administration of GSK239512, there was a reduction in the brain uptake of [(11) C]GSK189254 in all regions, including cerebellum. RO at 4 h was higher than at 24 h, and the PK/RO model estimated a PK associated with 50% of RO of 0.0068 ng·mL(-1) . This corresponds to a free concentration of 4.50 × 10(-12 ) M (pK = 11.3).The affinity of GSK239512 for brain H3 receptors in humans in vivo is much higher than that expected from studies in vitro, and higher than that observed in PET studies in pigs. The study illustrates the utility of carrying out PET studies in humans early in drug development, providing accurate quantification of GSK239512 RO in vivo as a function of time and dose.

To investigate the role of oral ivabradine as a heart rate reducing agent in patients undergoing CT coronary angiography (CTCA). Despite the routine use of β-blockers prior to CTCA studies, it is not uncommon to have patients with heart rates persistently above the target range of 65 bpm. Ivabradine is a selective inhibitor of the I(f) current, which primarily contributes to sinus node pacemaker activity, and has no significant direct cardiovascular effects such as reduction of blood pressure, cardiac contractility or impairment of cardiac conduction.We investigated 100 consecutive patients who had been referred for CTCA for the evaluation of suspected coronary artery disease (CAD). Patients were randomised to receive either of the following two pre-medication protocols: oral metorprolol or oral ivabradine.Ivabradine was significantly more effective than metorprolol in lowering the heart rate; the mean percentage reduction in heart rate with ivabradine vs metorpolol was 23.89+6.95% vs 15.20+4.50%, respectively (p=0.0001). Metoprolol significantly lowered both systolic and diastolic blood pressure while ivabradine did not. The requirement of additional doses to achieve a target heart rate of65 beats per min was also significantly more frequent with metoprolol.Ivabradine is a potentially attractive alternative to currently used drugs for reduction of heart rate in patients undergoing CTCA.

Dopamine released from the endings of descending dopaminergic nerve fibres in the spinal cord may be involved in modulating functions such as locomotion and nociception. Here, we examined the effects of dopamine on spinal synaptic transmissions in rats.Spinal reflex potentials, monosynaptic reflex potential (MSR) and slow ventral root potential (sVRP), were measured in the isolated spinal cord of the neonatal rat. Dopamine release was measured by HPLC.Dopamine at lower concentrations (1 µM) depressed sVRP, which is a C fibre-evoked polysynaptic response and believed to reflect nociceptive transmission. At higher concentrations (1 µM), in addition to a potent sVRP depression, dopamine depolarized baseline potential and slightly depressed MSR. Depression of sVRP by dopamine was partially reversed by dopamine D(1) -like but not by D(2) -like receptor antagonists. SKF83959 and SKF81297, D(1) -like receptor agonists, and methamphetamine, an endogenous dopamine releaser, also caused the inhibition of sVRP. Methamphetamine also depressed MSR, which was inhibited by ketanserin, a 5-HT(2A/2C) receptor antagonist. Methamphetamine induced the release of dopamine and 5-HT from spinal cords, indicating that the release of endogenous dopamine and 5-HT depresses sVRP and MSR respectively.These results suggested that dopamine at lower concentrations preferentially inhibited sVRP, which is mediated via dopamine D(1) -like and other unidentified receptors. The dopamine-evoked depression is involved in modulating the spinal functions by the descending dopaminergic pathways.

BACKGROUND AND PURPOSE Selective hyperpolarization activated, cyclic nucleotide-gated channel (HCN) blockers represent an important therapeutic goal due to the wide distribution and multiple functions of these proteins, representing the molecular correlate of f- and h-current (I(f) or I(h) ). Recently, new compounds able to block differentially the homomeric HCN isoforms expressed in HEK293 have been synthesized. In the present work, the electrophysiological and pharmacological properties of these new HCN blockers were characterized and their activities evaluated on native channels. EXPERIMENTAL APPROACH HEK293 cells expressing mHCN1, mHCN2 and hHCN4 isoforms were used to verify channel blockade. Selected compounds were tested on native guinea pig sinoatrial node cells and neurons from mouse dorsal root ganglion (DRG) by patch-clamp recordings and on dog Purkinje fibres by intracellular recordings. KEY RESULTS In HEK293 cells, EC18 was found to be significantly selective for HCN4 and MEL57A for HCN1 at physiological membrane potential. When tested on guinea pig sinoatrial node cells, EC18 (10 µM) maintained its activity, reducing I(f) by 67% at -120 mV, while MEL57A (3 µM) reduced I(f) by 18%. In contrast, in mouse DRG neurons, only MEL57A (30 and 100 µM) significantly reduced I(h) by 60% at -80 mV. In dog cardiac Purkinje fibres, EC18, but not MEL57A, reduced the amplitude and slowed the slope of the spontaneous diastolic depolarization. CONCLUSIONS Our results have identified novel and highly selective HCN isoform blockers, EC18 and MEL57A; the selectivity found in recombinant system was maintained in various tissues expressing different HCN isoforms.

Preclinical pharmacological characterization of GSK1004723, a novel, dual histamine H(1) and H(3) receptor antagonist.GSK1004723 was characterized in vitro and in vivo using methods that included radioligand binding, intracellular calcium mobilization, cAMP production, GTPγS binding, superfused human bronchus and guinea pig whole body plethysmography.In cell membranes over-expressing human recombinant H(1) and H(3) receptors, GSK1004723 displayed high affinity, competitive binding (H(1) pKi = 10.2; H(3) pKi = 10.6). In addition, GSK1004723 demonstrated slow dissociation from both receptors with a t(1/2) of 1.2 and 1.5 h for H(1) and H(3) respectively. GSK1004723 specifically antagonized H(1) receptor mediated increases in intracellular calcium and H(3) receptor mediated increases in GTPγS binding. The antagonism exerted was retained after cell washing, consistent with slow dissociation from H(1) and H(3) receptors. Duration of action was further evaluated using superfused human bronchus preparations. GSK1004723 (100 nmol·L(-1) ) reversed an established contractile response to histamine. When GSK1004723 was removed from the perfusate, only 20% recovery of the histamine response was observed over 10 h. Moreover, 21 h post-exposure to GSK1004723 there remained almost complete antagonism of responses to histamine. In vivo pharmacology was studied in conscious guinea pigs in which nasal congestion induced by intranasal histamine was measured indirectly (plethysmography). GSK1004723 (0.1 and 1 mg·mL(-1) intranasal) antagonized the histamine-induced response with a duration of up to 72 h.GSK1004723 is a potent and selective histamine H(1) and H(3) receptor antagonist with a long duration of action and represents a potential novel therapy for allergic rhinitis.

Recent clinical data suggest beneficial effects of ivabradine, a specific heart rate (HR)-lowering drug, in heart failure patients. However, the mechanisms responsible for these effects have not been completely clarified. Thus, we investigated functional/molecular changes in I(f), the specific target of ivabradine, in the failing atrial and ventricular myocytes where this current is up-regulated as a consequence of maladaptive remodelling.We investigated the effects of ivabradine (IVA; 10 mg·kg(-1) ·day(-1) for 90 days) on electrophysiological remodelling in left atrial (LA), left ventricular (LV) and right ventricular (RV) myocytes from post-mycardial infarcted (MI) rats, with sham-operated (sham or sham + IVA) rats as controls. I(f) current was measured by patch-clamp; hyperpolarization-activated cyclic nucleotide-gated (HCN) channel isoforms and microRNA (miRNA-1 and miR-133) expression were evaluated by reverse transcription quantitative PCR.Maximal specific conductance of I(f) was increased in MI, versus sham, in LV (P0.01) and LA myocytes (P0.05). Ivabradine reduced HR in both MI and sham rats (P0.05). In MI + IVA, I(f) overexpression was attenuated and HCN4 transcription reduced by 66% and 54% in LV and RV tissue, respectively, versus MI rats (all P0.05). miR-1 and miR-133, which modulate post-transcriptional expression of HCN2 and HCN4 genes, were significantly increased in myocytes from MI + IVA.The beneficial effects of ivabradine may be due to the reversal of electrophysiological cardiac remodelling in post-MI rats by reduction of functional overexpression of HCN channels. This is attributable to transcriptional and post-transcriptional mechanisms.