Triple-tyrosine kinase inhibition by BIBF1000 attenuates airway and pulmonary arterial remodeling following chronic allergen challenges in mice.

BACKGROUND: Airway remodeling is an important pathological feature of chronic airway diseases, which leads to a progressive decline in lung function. The present study examined the anti-remodeling and anti- inflammatory effect of BIBF1000, a triple-tyrosine kinase inhibitor that targets VEGF, PDGF, and FGF receptor signaling in a mouse model of repeated ovalbumin (OVA) challenges. METHODS: Female Balb-c mice were immunized intraperitoneally on days 0 and 12 with 50 µg ovalbumin plus 1 mg of Al(OH)3 in 200 µl saline. Intranasal OVA challenges (20 µg/50 µl in PBS) were administered on days 26, 29, and 31, and were repeated twice a week for 3 months. Animals received vehicle or BIBF1000 (25 mg/kg, b.i.d.) through gavage from day 26 to the end of fourth month. On day 120, bronchoalveolar lavage (BAL) and lung tissue were collected for biochemical and immunohistological analysis. RESULTS: Compared to vehicle controls, treatment with BIBF1000 reduced the numbers of BAL eosinophils, macrophages, neutrophils, and lymphocytes by 70.0%, 57.9%, 47.5%, and 63.0%, respectively, and reduced IL-5 and IL-13 in BAL. Treatment with BIBF1000 reduced airway mucus secretion, peribronchial fibrosis, small airway, and pulmonary arterial wall thickness, compared to vehicle controls. Furthermore, treatment with BIBF1000 also reduced the expression of inflammatory mediators (TNF-α, IL-1ß, IL-5, IL-13, MMP-2, MMP-9, COX-2, and iNOS) and inhibited ERK and AKT phosphorylation. CONCLUSIONS: The protective effect afforded by triple-tyrosine kinase inhibition with BIBF1000 in reducing allergen-induced airway and arterial remodeling was associated with down-regulation of inflammatory mediators, as well as inhibition of ERK and AKT signaling pathways.

Kinin B1 receptor blockade attenuates hepatic fibrosis and portal hypertension in chronic liver diseases in mice.

BACKGROUND AND AIMS: Kinin B1 receptors (B1Rs) are implicated in the pathogenesis of fibrosis. This study examined the anti-fibrotic effects of B1R blockade with BI 113823 in two established mouse models of hepatic fibrosis induced by intraperitoneal carbon tetrachloride (CCl4) injection or bile duct ligation (BDL). The mechanisms underlying the protection afforded by B1R inhibition were examined using human peripheral blood cells and LX2 human hepatic stellate cells (HSCs). METHODS: Fibrotic liver diseases were induced in mice by intraperitoneal carbon tetrachloride (CCl4) injection for 6 weeks, and by bile duct ligation (BDL) for 3 weeks, respectively. Mice received daily treatment of vehicle or BI 113823 (B1R antagonist) from onset of the experiment until the end of the study. RESULTS: B1Rs were strongly induced in fibrotic mouse liver. BI 113823 significantly attenuated liver fibrosis and portal hypertension (PH), and improved survival in both CCl4 and BDL mice. BI 113823 significantly reduced the expression of fibrotic proteins α-SMA, collagens 1, 3, 4, and profibrotic growth factors PDGF, TGFß, CTGF, VEGF, proliferating cell nuclear antigen; and reduced hepatic Akt phosphorylation in CCl4- and BDL-induced liver fibrosis. BI 113823 also reduced expression of Cytokines IL-1, IL-6; chemokines MCP-1, MCP-3 and infiltration of inflammatory cells; and inhibited human monocyte and neutrophil activation, transmigration, TNF-α & MPO production in vitro. BI 113823 inhibited TGF-ß and B1R agonist-stimulated human-HSC activation, contraction, proliferation, migration and fibrosis protein expression, and inhibited activation of PI3K/Akt signalling pathway. CONCLUSIONS: B1Rs merits consideration as a novel therapeutic target for chronic liver fibrosis and PH.

Reversal of pulmonary arterial hypertension and neointimal formation by kinin B1 receptor blockade.

BACKGROUND: This study examined whether BI113823, a novel selective kinin B1 receptor antagonist can reverse established pulmonary arterial hypertension (PAH), prevent right heart failure and death, which is critical for clinical translation. METHODS: Left pneumonectomized male Wistar rats were injected with monocrotaline to induce PAH. Three weeks later, when PAH was well established, the rats received daily treatment of BI113823 or vehicle for 3 weeks. RESULTS: Treatment with BI113823 from day 21 to day 42 after monocrotaline injection reversed established PAH as shown by normalized values of mean pulmonary arterial pressure (mPAP). BI113823 therapy reversed pulmonary vascular remodeling, pulmonary arterial neointimal formation, and heart and lung fibrosis, reduced right ventricular pressure, right heart hypertrophy, improved cardiac output, and prevented right heart failure and death. Treatment with BI113823 reduced TNF-α and IL-1ß, and macrophages recruitment in bronchoalveolar lavage, reduced CD-68 positive macrophages and expression of proliferating cell nuclear antigen (PCNA) in the perivascular areas, and reduced expression of iNOS, B1 receptors, matrix metalloproteinase (MMP)-2 and MMP-9 proteins, and the phosphorylation of ERK1/2 and AKT in lung. Treatment with BI113823 reduced mRNA expression of ANP, BNP, ßMHC, CGTF, collange-I and IV in right heart, compared to vehicle treated controls. In human monocytes cultures, BI113823 reduced LPS-induced TNF-α production, MMP-2 and MMP-9 expression, and reduced TNF-α-induced monocyte migration. CONCLUSIONS: We conclude that BI113823 reverses preexisting severe experimental pulmonary hypertension via inhibition of macrophage infiltration, cytokine production, as well as down regulation of matrix metalloproteinase proteins.

Inhibition of microsomal prostaglandin E synthase-1 ameliorates acute lung injury in mice.

BACKGROUND: To examine the effects of BI 1029539 (GS-248), a novel selective human microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor, in experimental models of acute lung injury (ALI) and sepsis in transgenic mice constitutively expressing the mPGES1 (Ptges) humanized allele. METHODS: Series 1: Lipopolysaccharide (LPS)-induced ALI. Mice were randomized to receive vehicle, BI 1029539, or celecoxib. Series 2: Cecal ligation and puncture-induced sepsis. Mice were randomized to receive vehicle or BI 1029539. RESULTS: Series 1: BI 1029539 or celecoxib reduced LPS-induced lung injury, with reduction in neutrophil influx, protein content, TNF-ɑ, IL-1ß and PGE2 levels in bronchoalveolar lavage (BAL), myeloperoxidase activity, expression of mPGES-1, cyclooxygenase (COX)-2 and intracellular adhesion molecule in lung tissue compared with vehicle-treated mice. Notably, prostacyclin (PGI2) BAL concentration was only lowered in celecoxib-treated mice. Series 2: BI 1029539 significantly reduced sepsis-induced BAL inflammatory cell recruitment, lung injury score and lung expression of mPGES-1 and inducible nitric oxide synthase. Treatment with BI 1029539 also significantly prolonged survival of mice with severe sepsis. Anti-inflammatory and anti-migratory effect of BI 1029539 was confirmed in peripheral blood leukocytes from healthy volunteers. CONCLUSIONS: BI 1029539 ameliorates leukocyte infiltration and lung injury resulting from both endotoxin-induced and sepsis-induced lung injury.

Inhibition of human microsomal PGE2 synthase-1 reduces seizure-induced increases of P-glycoprotein expression and activity at the blood-brain barrier.

The cause of antiseizure drug (ASD) resistance in epilepsy is poorly understood. Here, we focus on the transporter P-glycoprotein (P-gp) that is partly responsible for limited ASD brain uptake, which is thought to contribute to ASD resistance. We previously demonstrated that cyclooxygenase-2 (COX-2) and the prostaglandin E receptor, prostanoid E receptor subtype 1, are involved in seizure-mediated P-gp up-regulation. Thus, we hypothesized that inhibiting microsomal prostaglandin E2 (PGE2) synthase-1 (mPGES-1), the enzyme generating PGE2, prevents blood-brain barrier P-gp up-regulation after status epilepticus (SE). To test our hypothesis, we exposed isolated brain capillaries to glutamate ex vivo and used a combined in vivo-ex vivo approach by isolating brain capillaries from humanized mPGES-1 mice to study P-gp levels. We demonstrate that glutamate signaling through the NMDA receptor, cytosolic phospholipase A2, COX-2, and mPGES-1 increases P-gp protein expression and transport activity levels. We show that mPGES-1 is expressed in human, rat, and mouse brain capillaries. We show that BI1029539, an mPGES-1 inhibitor, prevented up-regulation of P-gp expression and transport activity in capillaries exposed to glutamate and in capillaries from humanized mPGES-1 mice after SE. Our data provide key signaling steps underlying seizure-induced P-gp up-regulation and suggest that mPGES-1 inhibitors could potentially prevent P-gp up-regulation in epilepsy.-Soldner, E. L. B., Hartz, A. M. S., Akanuma, S.-I., Pekcec, A., Doods, H., Kryscio, R. J., Hosoya, K.-I., Bauer, B. Inhibition of human microsomal PGE2 synthase-1 reduces seizure-induced increases of P-glycoprotein expression and activity at the blood-brain barrier.

Triple-tyrosine kinase inhibition attenuates pulmonary arterial hypertension and neointimal formation.

The present study examined the effects of simultaneous inhibition of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) receptor signaling with BIBF1000, a novel triple tyrosine kinase inhibitor on preventing and reversing the progression of severe pulmonary arterial hypertension (PAH) in an experimental model in rats. Left pneumonectomized male Wistar rats were injected with monocrotaline to induce PAH. Treatment with BIBF1000 from day 1 to day 21 after monocrotaline injection attenuated PAH development, as evidenced by lower values for pulmonary artery pressure (mPAP), right ventricular pressure (RVSP), pulmonary arterial neointimal formation, and the ratio of right ventricular weight to left ventricular and septum weight [RV/(LV+S)] on day 21 compared to control rats. Treatment with BIBF1000 from day 21 to day 42 after monocrotaline injection reversed established PAH as shown by normalized values for mPAP and RVSP, RV/(LV+S) ratio, pulmonary arterial occlusion scores, levels of heart and lung fibrosis, as well as improved survival. Treatment with BIBF1000 reduced inflammatory cell recruitment in bronchoalveolar lavage and lung tissues, reduced CD-68 positive macrophages and expression of proliferating cell nuclear antigen in the perivascular areas, and reduced TNF-α and growth factor productions, and inhibited the phosphorylation of AKT and GSK3ß in lungs. In addition, BIBF1000 inhibited pulmonary artery smooth muscle cells migration and proliferation from rat pulmonary artery explant cultures. Simultaneous inhibition of VEGF, PDGF, and FGF receptor signaling by BIBF1000 prevents and reverses the progression of severe pulmonary arterial hypertension and vascular remodeling in this experimental model.

Standard analgesics reverse burrowing deficits in a rat CCI model of neuropathic pain, but not in models of type 1 and type 2 diabetes-induced neuropathic pain.

BACKGROUND: Burrowing is a rodent behavior validated as a robust and reproducible outcome measure to infer the global effect of pain in several inflammatory pain models. However, less is known about the effect of analgesics on burrowing in neuropathic pain models and no studies have determined burrowing performance in models of diabetes-associated neuropathic pain. OBJECTIVE: To compare the sensitivity of the burrowing assay in different neuropathic pain models: mononeuropathic pain and diabetic polyneuropathy. METHODS: Burrowing performance was determined by the amount of substrate left in a hollow tube by rats with chronic constriction injury (CCI). In addition, burrowing performance, locomotion and pain development was assessed in the Zucker diabetic fatty (ZDF) rat model, resembling type-2 diabetes. Efficacy of clinically-active reference drugs (opioids, gabapentin and/or pregabalin) were investigated in these models. Burrowing behavior was additionally assessed in a second model, induced by streptozotocin (STZ) treatment, resembling type-1 diabetes. RESULTS: In the CCI model, moderate but consistent burrowing deficits were observed that persisted over a period of ≥20 days. Systemic administration of morphine, pregabalin and gabapentin reversed this deficit. In contrast, none of the reference drugs improved marked burrowing deficits detected in ZDF rats, and pregabalin did not reverse severe burrowing deficits observed in STZ rats. CONCLUSIONS: Burrowing performance cannot necessarily be used as pain-related readout across pain models and largely depends on the model used, at least in models of neuropathy. Specifically, analgesic drug effects might be masked by general diabetes-associated alteration of the animals' well-being, resulting in false negative outcomes.

Kinin B1 receptor blockade and ACE inhibition attenuate cardiac postinfarction remodeling and heart failure in rats.

INTRODUCTION: The aim of the present study was to evaluate the effects of the novel kinin B1 receptor antagonist BI113823 on postinfarction cardiac remodeling and heart failure, and to determine whether B1 receptor blockade alters the cardiovascular effects of an angiotensin 1 converting enzyme (ACE) inhibitor in rats. METHODS AND RESULTS: Sprague Dawley rats were subjected to permanent occlusion of the left coronary artery. Cardiovascular function was determined at 6weeks postinfarction. Treatment with either B1 receptor antagonist (BI113823) or an ACE inhibitor (lisinopril) alone or in combination significantly reduced the heart weight-to-body weight and lung weight-to-body weight ratios, and improved postinfarction cardiac function as evidenced by greater cardiac output, the maximum rate of left ventricular pressure rise (±dP/dtmax), left ventricle ejection fraction, fractional shorting, better wall motion, and attenuation of elevated left ventricular end diastolic pressure (LVEDP). Furthermore, all three treatment groups exhibited significant reduction in cardiac interstitial fibrosis, collagen deposition, CD68 positive macrophages, neutrophils, and proinflammatory cytokine production (TNF-α and IL-1ß), compared to vehicle controls. CONCLUSION: The present study shows that treatment with the novel kinin B1 receptor antagonist, BI113823, reduces postinfarction cardiac remodeling and heart failure, and does not influence the cardiovascular effects of the ACE inhibitor.

Duloxetine and 8-OH-DPAT, but not fluoxetine, reduce depression-like behaviour in an animal model of chronic neuropathic pain.

The current study assessed whether antidepressant and/or antinociceptive drugs, duloxetine, fluoxetine as well as (±)-8-hydroxy-2-[di-n-propylamino] tetralin (8-OH-DPAT), are able to reverse depression-like behaviour in animals with chronic neuropathic pain. Chronic constriction injury (CCI) of the sciatic nerve in rats was selected as neuropathic pain model. Mechanical hypersensitivity and depression-like behaviour were evaluated 4 weeks after surgery by "electronic algometer" and forced swimming test (FST), which measured the time of immobility, and active behaviours climbing and swimming. The selective noradrenergic and serotonergic uptake blocker duloxetine (20mg/kg) and the selective 5-HT1A agonist 8-OH-DPAT (0.5mg/kg) significantly reversed both mechanical hypersensitivity and depression-like behaviour in CCI animals. Duloxetine significantly reversed depression-like behaviour in CCI rats by increasing the time of climbing and swimming, while 8-OH-DPAT attenuated depression-like behaviour mainly by increasing the time of swimming. However, the selective serotonergic uptake blocker fluoxetine (20mg/kg) failed to attenuate mechanical hypersensitivity and depression-like behaviour, possibly due to confounding pro-nociceptive actions at 5-HT3 receptors. These data suggest to target noradrenergic and 5-HT1A receptors for treatment of chronic pain and its comorbidity depression.

Kinin B1 receptor antagonist BI113823 reduces allergen-induced airway inflammation and mucus secretion in mice.

Kinin B1 receptors are implicated in asthmatic airway inflammation. Here we tested this hypothesis by examining the anti-inflammatory effects of BI113823, a novel non-peptide orally active kinin B1 receptor antagonist in mice sensitized to ovalbumin (OVA). Male Balb-c mice were randomly assigned to four study groups: (1) control, (2) OVA+vehicle, (3) OVA+BI113823, (4) OVA+dexamethasone. Mice were sensitized intraperitoneally with 75µg ovalbumin on days 1 and 8. On days 15-17, mice were challenged intranasally with 50µg of ovalbumin. Mice received vehicle, BI113823, or dexamethasone (positive control) on days 16-18. On day 19, bronchoalveolar lavage (BAL) and lung tissue were collected for biochemical and immuno-histological analysis. Compared to controls treatment with BI113823 significantly reduced the numbers of BAL eosinophils, macrophages, neutrophils and lymphocytes by 58.3%, 61.1%, 66.4% and 56.0%, respectively. Mice treated with dexamethasone showed similar reductions in BAL cells. Treatment with BI113823 and dexamethasone also significantly reduced total protein content, IgE, TNF-α and IL-1ß in lavage fluid, reduced myeloperoxidase activity, mucus secretion in lung tissues, and reduced the expression of B1 receptors, matrix metalloproteinase (MMP)-2 and cyclooxygenase (COX)-2 compared to vehicle-treated mice. Only BI113823 reduced MMP-9 and inducible nitric oxide synthase (iNOS). BI113823 effectively reduced OVA-induced inflammatory cell, mediator and signaling pathways equal to or greater than that seen with steroids in a mouse asthma model. BI113823 might be useful in modulating inflammation in asthma.

Kinin B1 Receptor Inhibition With BI113823 Reduces Inflammatory Response, Mitigates Organ Injury, and Improves Survival Among Rats With Severe Sepsis.

BACKGROUND: This study examined the therapeutic effects of an orally active nonpeptide kinin B1 receptor antagonist, BI113823, in a clinically relevant experimental model of polymicrobial sepsis in rats. METHODS: Sepsis was induced by cecal ligation and puncture (CLP). Animals received treatment with either vehicle or BI113823. The experiment was terminated in the first set of animals 15 hours after CLP. Seven-day survival following CLP was determined in the second set of animals. RESULTS: Compared with vehicle treatment, administration of BI113823 reduced neutrophil and macrophage infiltration, reduced cytokine production, attenuated intestinal mucosal hyperpermeability, prevented hemodynamic derangement, and improved cardiac output. Furthermore, administration of BI113823 reduced inducible nitric oxide synthase expression and the injury score in the lung and attenuated nuclear factor ĸB activation and apoptosis in the liver. Treatment with BI113823 also reduced plasma levels of cardiac troponin, aspartate aminotransferase, alanine aminotransferase, urea, and lactate, as well as proteinuria. Finally, administration of BI113823 improved the 7-day survival rate following CLP in rats. CONCLUSIONS: Administration of BI113823 reduced systemic and tissue inflammatory responses, prevented hemodynamic derangement, attenuated multiorgan injury, and improved overall survival.

Pharmacological characterization of intraplantar Complete Freund's Adjuvant-induced burrowing deficits.

BACKGROUND: It has recently been suggested that non-reflex behavioral readouts, such as burrowing, may be used to evaluate the efficacy of analgesics in rodent models of pain. OBJECTIVE: To confirm whether intraplantar Complete Freund's Adjuvant (CFA)-induced pain reliably results in burrowing deficits which can be ameliorated by clinically efficacious analgesics as previously suggested. METHODS: Uni- or bilateral intraplantar CFA injections were performed in male Wistar Han rats. The time- and concentration-response of burrowing deficits and the ability of various analgesics to reinstate burrowing performance were studied. An anxiolytic was also tested to evaluate the motivational cue that drives this behavior. RESULTS: Burrowing deficits were dependent on the concentration of CFA injected, most pronounced 24h after CFA injections and even more pronounced after bilateral compared with unilateral injections. Celecoxib and ibuprofen reversed CFA-induced burrowing deficits whereas indomethacin failed to significantly reinstate burrowing performance. Morphine and tramadol failed to reinstate burrowing performance, but sedation was observed in control rats at doses thought to be efficacious. An antibody directed against the nerve growth factor significantly improved CFA-induced burrowing deficits. Neither gabapentin nor the anxiolytic diazepam reinstated burrowing performance and the opportunity to find shelter did not modify burrowing performance. CONCLUSION: Burrowing is an innate behavior reliably exhibited by rats. It is suppressed in a model of inflammatory pain and differently reinstated by clinically efficacious analgesics that lack motor impairing side effects, but not an anxiolytic, suggesting that this assay is suitable for the assessment of analgesic efficacy of novel drugs.

Kinin B1 Receptor Antagonist BI113823 Reduces Acute Lung Injury.

OBJECTIVES: This study was undertaken to examine the effects of BI113823, a potent small molecule orally active nonpeptide B1 receptor antagonist, in an experimental model of endotoxin-induced direct lung injury in mice and indirect lung injury and survival in cecal ligation and puncture-induced polymicrobial sepsis in rats. DESIGN: Experimental, prospective study. SETTING: University research laboratory. SUBJECTS: Male BALB/c mice and male Wistar rats. INTERVENTIONS: Series 1: acute lung injury was induced in mice by intratracheal injection of lipopolysaccharide. Mice were then randomly assigned to receive treatment of vehicle, BI113823, or dexamethasone. Bronchoalveolar lavage fluid and lung tissues were analyzed for inflammatory cell influx and various histologic variables. Series 2: sepsis was induced by cecal ligation and puncture in anesthetized rats. Animals were then randomly assigned to receive treatment of vehicle or BI113823. Experiments were terminated at 20 hours and 7 days following cecal ligation and puncture, respectively. MEASUREMENTS AND MAIN RESULTS: Series 1: treatment with BI113823 significantly reduced lipopolysaccharide-induced neutrophil influx in bronchoalveolar lavage fluid. The BI113823 group had significantly lower lung vascular permeability, lung water content, myeloperoxidase activity, lung apoptosis and lung injury scores, total protein content, and tumor necrosis factor-α and interleukin-1ß levels compared with vehicle controls. In addition, nuclear factor-κB phosphorylation, nuclear translocation, and cyclooxygenase-2 and inducible nitric oxide synthase expression in the lung were attenuated in BI113823-treated animals compared with vehicle controls. Series 2: BI113823 significantly reduced sepsis-induced macrophage recruitment, protein content, and tumor necrosis factor-α and interleukin-1ß levels in lavage fluid and also reduced lung water content and plasma levels of tumor necrosis factor-α and interleukin-6 compared with vehicle controls. Most importantly, treatment with BI113823 significantly improved survival following severe sepsis in rats. CONCLUSIONS: Administration of B1 receptor antagonist BI113823 significantly reduced endotoxin-induced direct lung injury and also reduced sepsis-induced lung inflammatory response. Most importantly, BI113823 improved survival following severe polymicrobial sepsis.

Inhibition of kinin B1 receptors attenuates pulmonary hypertension and vascular remodeling.

This study examined whether the kinin B1 receptor is involved in the pathogenesis of pulmonary hypertension, and whether its inhibition could reduce inflammation, pulmonary hypertension, vascular remodeling, and right heart dysfunction. Male Wistar rats underwent left pneumonectomy. Seven days later, the rats were injected subcutaneously with monocrotaline (60 mg/kg). The rats were then randomly assigned to receive treatment with vehicle or with BI113823 (a selective B1 receptor antagonist, 30 mg/kg, twice per day) via oral gavage from the day of monocrotaline injection to day 28. By day 28, BI113823-treated rats had significantly lower mean pulmonary artery pressure, less right ventricular hypertrophy, and pulmonary arterial neointimal formation than that of the vehicle-treated rats. Real-time polymerase chain reaction revealed that there was a significant increase in mRNA expression of B1 receptors in the lungs of monocrotaline-challenged pneumonectomized rats. Treatment with BI113823 significantly reduced macrophage recruitment, as measured via bronchoalveolar lavage. It also markedly reduced CD-68 positive macrophages and proliferating cell nuclear antigen positive cells in the perivascular areas, reduced expression of inducible nitric oxide synthase, matrix metalloproteinase 2 and 9, and B1 receptors compared with measurements in vehicle-treated rats. These findings demonstrate that kinin B1 receptors represent a novel therapeutic target for pulmonary arterial hypertension.

The somatostatin receptor 4 agonist J-2156 reduces mechanosensitivity of peripheral nerve afferents and spinal neurons in an inflammatory pain model.

Somatostatin (SST) is a peptide hormone that regulates the endocrine system and affects neurotransmission via interaction with G protein-coupled SST receptors and inhibition of the release of different hormones. The aim of this study was to investigate whether the analgesic properties of the selective SSTR4 agonist J-2156 are mediated via peripheral and/or spinal receptors. Effect on mechanical hyperalgesia in the Complete Freund׳s Adjuvant (CFA) model was measured after intraperitoneal application of J-2156. Electrophysiological neuronal recordings were conducted 24 h after injection of CFA or vehicle into the paw of Wistar rats. Mechanosensitivity of peripheral afferents of the saphenous nerve as well as of spinal wide dynamic range (WDR) and nociceptive-specific (NS) neurons were measured after systemic or spinal application of J-2156. In CFA animals J-2156 dose dependently reduced hyperalgesia in behavioral studies. The minimal effective dose was 0.1 mg/kg. Mechanosensitivity of peripheral afferents and spinal neurons was significantly reduced by J-2156. NS neurons were dose dependently inhibited by J-2156 while in WDR neurons only the highest concentration of 100 µM had an effect. In sham controls, J-2156 had no effect on neuronal activity. We demonstrated that J-2156 dose-dependently reduces peripheral and spinal neuronal excitability in the CFA rat model without affecting physiological pain transmission. Given the high concentration of the compound required to inhibit spinal neurons, it is unlikely that the behavioral effect seen in CFA model is mediated centrally. Overall these data demonstrated that the analgesic effect of J-2156 is mediated mainly via peripheral SST4 receptors.

Des-Arg9-bradykinin causes kinin B1 receptor mediated endothelium-independent contractions in endotoxin-treated porcine coronary arteries.

This study examined responses of isolated pig coronary arteries after kinin B1 receptor induction by endotoxin. Des-Arg9-bradykinin (DBK) induced concentration-dependent, endothelium-independent contractions in lipopolysaccharide (LPS)-treated but not untreated arterial rings. The B1-receptor antagonist SSR240612, but not the B2-receptor antagonist HOE140, prevented the endothelium-independent contractions to DBK. The DBK-induced contractions were blocked by indomethacin (nonselective cyclooxygenase [COX] inhibitor), celecoxib (selective COX-2 inhibitor), and terbogrel (thromboxane-prostanoid [TP] receptor antagonist) but not valeryl salicylate (selective COX-1 inhibitor), AH6809 (an E prostanoid [EP] and PGD2 receptor [DP1] receptor antagonist), AL 8810 (a selective PGF2α [FP] receptor antagonist), or RO1138452 (a selective I prostanoid [IP] receptor antagonist). They were attenuated by N-(p-amylcinnamoyl) anthranilic acid (ACA), and by DETCA plus tiron but not by l-NAME. Quantitative RT-PCR revealed excessive up-regulations of mRNA expressions of B1 receptors, COX-2, and thromboxane A synthase 1 (TBXAS1) following LPS incubation, but not of B2 receptors or COX-1. The present data demonstrate that B1 receptors are coupled to COX-2 in causing endothelium-independent contractions in endotoxin-treated pig coronary arteries. Accordingly, kinin B1 receptor induction during inflammation may have a pathological significance in the vasculature, particular in coronary arteries with dysfunctional endothelial cells.

COX-2 mediated induction of endothelium-independent contraction to bradykinin in endotoxin-treated porcine coronary artery.

This study examined the vascular effects of bradykinin in health and vascular inflammation comparing responses of isolated pig coronary arteries in the absence and presence of endotoxins. Bradykinin induced contractions in lipopolysaccharide-treated, but not untreated, arterial rings without endothelium. The B2-receptor antagonist HOE140, but not the B1-receptor inhibitor SSR240612, blocked these endothelium-independent contractions in response to bradykinin. The bradykinin-induced contractions were blocked by indomethacin, celecoxib, and terbogrel but not valeryl salicylate, AH6809, AL 8810, or RO1138452. They were attenuated by N-(p-amylcinnamoyl) anthranilic acid, and by diethyldithiocarbamate plus tiron but not by L-NAME. Quantitative reverse-transcription polymerase chain reaction revealed significant upregulations of messenger RNA expressions of B1 receptors, COX-2, and thromboxane A synthase 1 (TBXAS1) following lipopolysaccharide incubation but not of B2 receptors or COX-1. The present data demonstrate that bradykinin induces contractions mediated by the COX-2 pathway in endotoxin-treated pig coronary arteries. These results support differential roles of bradykinin in health and disease.

Somatostatin 4 receptor activation modulates TRPV1[correction of TPRV1] currents in dorsal root ganglion neurons.

Somatostatin (sst) is a cyclic neuropeptide known to have inhibitory roles in the central nervous system. It exerts its biological effects via the activation of the 5 sst receptor subtypes, which belong to the family of G-protein coupled receptors (GPCR). This peptide has analgesic properties, specifically via the activation of the sst4 receptor subtype. Although this is established, the precise molecular mechanisms causing this have not yet been fully elucidated. This research aimed to identify a possible anti-nociceptive mechanism, showing functional links to the transient receptor potential vanilloid type 1 (TRPV1) within the pain processing pathway. Calcium imaging and whole cell voltage clamp experiments were conducted on DRG neurons prepared from adult rats, utilizing capsaicin stimulations and the sst4 receptor specific agonist J-2156. The complete Freund's adjuvant (CFA) inflammatory pain model was used to examine if effects are augmented in pain conditions. The sst4 receptor agonist J-2156 was able significantly to inhibit capsaicin induced calcium and sodium influx, where the effect was more potent after CFA treatment. This inhibition identifies a contributory molecular mechanism to the analgesic properties of sst4 receptor activation.

Somatostatin 4 receptor activation modulates G-protein coupled inward rectifying potassium channels and voltage stimulated calcium signals in dorsal root ganglion neurons.

Somatostatin has a wide biological profile resulting from its actions on the five receptor subtypes (sst1-5). Recently somatostatin was shown to exert analgesic effects via activation of the sst4 receptor. Although the analgesia in pain models is established, the precise molecular mechanism has yet to be fully elucidated. This research aimed to identify possible anti-nociceptive mechanisms, showing functional links of the sst4 receptor to G-protein coupled inward rectifying potassium (GIRK) channels and reduction of voltage stimulated calcium influx within the pain processing pathway. Whole cell voltage clamp experiments and calcium imaging experiments were conducted on DRG neurons prepared from adult rats. Application of an sst4 receptor selective agonist, J-2156, on DRG neurons induced a GIRK modulated potassium current, and inhibited voltage sensitive calcium current. Both mechanisms are thought to contribute to the analgesic properties of sst4 receptor agonists.

Sabiporide reduces ischemia-induced arrhythmias and myocardial infarction and attenuates ERK phosphorylation and iNOS induction in rats.

The aim of the present study was to investigate the effects of sabiporide, a potent and selective NHE1 inhibitor, on myocardial ischemia-induced arrhythmias and myocardial infarction and the possible pathways related to the cardioprotection afforded by sabiporide treatment. Anesthetized rats were subjected to myocardial ischemia via left main coronary artery occlusion for 30 minutes, followed by 2 hours of reperfusion. Administration of sabiporide (0.01-3.0 mg/kg) prior to coronary artery occlusion dose-dependently reduced ischemia-induced arrhythmias and infarct size with an ED50 value of 0.14 mg/kg. Administration of sabiporide (1.0 mg/kg) prior to reperfusion also reduced infarct size by 38.6%. The reduction in infarct size was accompanied by a decrease in circulating levels of creatine phosphokinase and troponin I. In addition, sabiporide (1.0 mg/kg) given prior to coronary artery occlusion or immediately before reperfusion significantly reduced phosphorylation of the extracellular signal-regulated kinase (ERK1/2) and the expression of the inducible nitric oxide synthase (iNOS) following myocardial ischemia-reperfusion. This study demonstrates that sabiporide is a potent and effective cardioprotective agent during myocardial ischemia and reperfusion, by reducing serious ventricular arrhythmias and myocardial infarct size. The cardioprotection afforded by sabiporide is attributed in part to inhibition of ERK1/2 phosphorylation and suppression of iNOS expression.