TRPC6, a Therapeutic Target for Pulmonary Hypertension

Idiopathic pulmonary arterial hypertension (PAH) is a fatal and progressive disease. Pulmonary vasoconstriction due to pulmonary arterial smooth muscle cell (PASMC) contraction and pulmonary arterial remodeling due to PASMC proliferation are causes for increased pulmonary vascular resistance in patients with PAH. We and others observed upregulation of TRPC6 channels in PASMC from patients with PAH. An increase in cytosolic Ca2+ concentration ([Ca2+]cyt) in PASMC triggers PASMC contraction and vasoconstriction, while Ca2+-dependent activation of PI3K/AKT/mTOR pathway is pivotal for cell proliferation and gene expression. Despite evidence supporting a pathological role of TRPC6, no selective and orally bioavailable TRPC6 blocker has yet been developed and tested for treatment of PAH. We sought to investigate whether block of receptor-operated Ca2+ channels or TRPC6 can reverse established PH in mice via inhibiting Ca2+-dependent activation of AKT/mTOR signaling. Here we report that intrapulmonary application of 2-aminoethyl diphenyl borniate (2-APB), a non-selective blocker of cation channels or BI-749237, a selective blocker of TRPC6, significantly and reversibly inhibited acute hypoxic pulmonary vasoconstriction. Intraperitoneal injection of 2-APB significantly attenuated the development of PH and partially reversed established PH. Oral gavage of the selective TRPC6 blocker BI-749237 reversed established PH by 50% via regression of pulmonary vascular remodeling. Furthermore, 2-APB and BI-749237 both inhibited PDGF- and serum-mediated phosphorylation of AKT and mTOR in PASMC. These results indicates that the receptor-operated and mechanosensitive TRPC6 channel is a good target for developing novel treatment for PAH. BI-749237, a selective TRPC6 blocker, is potentially a novel and effective drug for treating PAH.

The role of parasympathetic mechanisms in the infarct-limiting effect of SGLT2 inhibitor ertugliflozin

Introduction: Based on data that outcome in patients with acute myocardial infarction is predicted by final infarct size (IS), reducing IS is of paramount importance. Recent experimental studies have demonstrated a strong infarct-sparing effect of SGLT2 inhibitors – a class of drugs which have proved to be safe and beneficial in patients with heart failure. Repurposing SGLT2 inhibitors for the benefit of patients presenting with acute myocardial infarction should be preceded by investigation of the underlying mechanisms of this infarct limitation. Experimental and clinical data indicate a potential role for autonomic modulation in these mechanisms, specifically sympatho-inhibition. The aim of this study was to investigate the role of the parasympathetic mechanism in the infarct-limiting effect of SGLT2 inhibition. Methods: Fortyeight Sprague Dawley male rats were fed with a standard diet containing either the SGLT2 inhibitor ertugliflozin or vehicle, for 5-7 days. Myocardial ischaemia/reperfusion injury was initiated by a 40-min occlusion of the left anterior descending coronary artery followed by a 2hr period of reperfusion under isoflurane anaesthesia. Bilateral cervical vagotomy was performed 10min prior to myocardial ischaemia. Alternatively, muscarinic receptors were blocked systemically with the non-selective blocker atropine sulphate (2 mg/kg bolus, then 1 mg/kg/h) or the M3-selective blocker 4-DAMP (2 mg/kg bolus). Results: Pre-treatment with ertugliflozin reduced IS in comparison with the vehicle-treated controls (p<0.001). Bilateral vagotomy, atropine sulphate and 4-DAMP all abolished this infarct-limiting effect (IS 35±10%, 44±8%, and 35±4% respectively; P<0.01 vs. Ertu for vagotomy, P<0.001 vs. Ertu for both atropine sulphate and 4-DAMP). Conclusion: These results suggest that the Infarct-limiting effect of the SGLT2 inhibitor ertugliflozin, may be mediated via activation of the vagus nerve and M3-cholinoreceptors.

Molecular Targets for Biological Therapies of Severe Asthma: Focus on Benralizumab and Tezepelumab

Asthma is a heterogeneous respiratory disease characterized by usually reversible bronchial obstruction, which is clinically expressed by different phenotypes driven by complex pathobiological mechanisms (endotypes). In recent years several molecular effectors and signaling pathways have emerged as suitable targets for biological therapies of severe asthma, refractory to standard treatments. Indeed, various therapeutic mono-clonal antibodies currently allow one to intercept at different levels the chain of pathogenic events leading to type 2 (T2) airway inflammation. Pro-allergic immunoglobulin E (IgE) is the first molecule against which an anti-asthma monoclonal antibody (omalizumab) was developed; today other targets are successfully being exploited by biological treatments for severe asthma. In particular, pro-eosinophilic interleukin 5 (IL-5) can be targeted by mepolizumab or reslizumab, whereas benralizumab is a selective blocker of IL-5 receptor, and IL-4 and IL-13 can be targeted by dupilumab. Besides these drugs, which are already available in medical practice, other biologics are under clinical development such as those targeting innate cytokines, including the alarmin thymic stromal lymphopoietin (TSLP), which plays a key role in the pathogenesis of type 2 asthma. Therefore, ongoing and future biological therapies are significantly changing severe asthma management on a global level. These new therapeutic options make it possible to implement phenotype/endotype-specific treatments, which are delineating personalized approaches precisely addressing the individual traits of asthma pathobiology. The aim of the study is to review the immunopathology and treatment efficacy for severe asthma and focused on new biological agents with benralizumab (anti-IL-5) and tezepelumab (anti-TSLP).

Arrhythmogenic and antiarrhythmic actions of late sustained sodium current in the adult human heart

Late sodium current (late INa) inhibition has been proposed to suppress the incidence of arrhythmias generated by pathological states or induced by drugs. However, the role of late INa in the human heart is still poorly understood. We therefore investigated the role of this conductance in arrhythmias using adult primary cardiomyocytes and tissues from donor hearts. Potentiation of late INa with ATX-II (anemonia sulcata toxin II) and E-4031 (selective blocker of the hERG channel) slowed the kinetics of action potential repolarization, impaired Ca2+ homeostasis, increased contractility, and increased the manifestation of arrhythmia markers. These effects could be reversed by late INa inhibitors, ranolazine and GS-967. We also report that atrial tissues from donor hearts affected by atrial fibrillation exhibit arrhythmia markers in the absence of drug treatment and inhibition of late INa with GS-967 leads to a significant reduction in arrhythmic behaviour. These findings reveal a critical role for the late INa in cardiac arrhythmias and suggest that inhibition of this conductance could provide an effective therapeutic strategy. Finally, this study highlights the utility of human ex-vivo heart models for advancing cardiac translational sciences.

NFκB-Activated COX2/PGE2/EP4 Axis Controls the Magnitude and Selectivity of BCG-Induced Inflammation in Human Bladder Cancer Tissues

Bacillus Calmette-Guérin (BCG) is commonly used in the immunotherapy of bladder cancer (BlCa) but its effectiveness is limited to only a fraction of patients. To identify the factors that regulate the response of human BlCa tumor microenvironment (TME) to BCG, we used the ex vivo whole-tissue explant model. The levels of COX2 in the BCG-activated explants closely correlated with the local production of Treg- and MDSCS attractants and suppressive factors, while the baseline COX2 levels did not have predictive value. Accordingly, we observed that BCG induced high levels of MDSC- and Treg-attracting chemokines (CCL22, CXCL8, CXCL12) and suppressive factors (IDO1, IL-10, NOS2). These undesirable effects were associated with the nuclear translocation of phosphorylated NFκB, induction of COX2, the key enzyme controlling PGE2 synthesis, and elevation of a PGE2 receptor, EP4. While NFκB blockade suppressed both the desirable and undesirable components of BCG-driven inflammation, the inhibitors of PGE2 synthesis (Celecoxib or Indomethacin) or signaling (EP4-selective blocker, ARY-007), selectively eliminated the induction of MDSC/Treg attractants and immunosuppressive factors but enhanced the production of CTL attractants, CCL5, CXCL9 and CXCL10. PGE2 blockade allowed for the selectively enhanced migration of CTLs to the BCG-treated BlCa samples and eliminated the enhanced migration of Tregs. Since the balance between the CTLs and suppressive cells in the TME predicts the outcomes in patients with BlCa and other diseases, our data help to elucidate the mechanisms which limit the effectiveness of BCG therapies and identify new targets to enhance their therapeutic effects.

Electroanalysis Applied to Compatibility and Stability Assays of Drugs: Carvedilol Study Case

Carvedilol (CRV) is a non-selective blocker of α and β adrenergic receptors, which has been extensively used for the treatment of hypertension and congestive heart failure. Owing to its poor biopharmaceutical properties, CRV has been incorporated into different types of drug delivery systems and this necessitates the importance of investigating their compatibility and stability. In this sense, we have investigated the applicability of several electroanalytical tools to assess CRV compatibility with lipid excipients. Voltammetric and electrochemical impedance spectroscopy techniques were used to evaluate the redox behavior of CRV and lipid excipients. Results showed that Plurol® isostearic, liquid excipient, and stearic acid presented the greatest anode peak potential variation, and these were considered suitable excipients for CRV formulation. CRV showed the highest stability at room temperature and at 50 °C when mixed with stearic acid (7% w/w). The results also provided evidence that electrochemical methods might be feasible to complement standard stability/compatibility studies related to redox reactions.

Effect of Cardioselective Beta Blocker on Lisinopril Treated Isolated Rabbit’s Heart

Background: Certain drugs produce unpredictable responses when used in emergency conditions. These variable outcomes may be harmful or beneficial for the patient. Objective: This study has been conducted to evaluate the pharmacodynamic interaction between angiotensin converting enzyme inhibitor and metoprolol, a selective blocker of β1 receptors. Cardioselective beta blockers are commonly used to treat hypertension, arrhythmias and ischemic heart disease. Method: In this study, 20 healthy male rabbits were selected and divided into two groups. Effective dose of Lisinopril (10 mg/kg) was administered orally via oral feeding, for 9 days. By using Langendroff’s technique, the effects of metoprolol were observed in isolated hearts. Result: The data showed that the effective dose of Lisinopril (10 mg/kg daily orally) increases the inotropic and chronotropic effects of metoprolol significantly (p<0.05). Conclusion: Therefore, lisinopril, an inhibitor of angiotensin converting enzyme may increase the response of cardioselective beta blocker metoprolol in isolated rabbit’s heart.

Periodic syndrome associated with the mutation of the receptor gene of the tumor necrosis factor (clinical case)

The paper deals with a case of TRAPS in a Ukrainian family. The manifestations of this syndrome appeared at the age of 2,5 years and gradually the attacks of fever became more frequent and the recurrence was typical of this diagnosis. Classically, besides fever, there was an intense abdominal pain, such as an “acute abdomen”, arthralgia in the right hip and headache. Micropoliadenia was also detected. This patient did not have any other symptoms. A genetic study found a mutation in the TNFRSF1A gene (substitution in exon 4 with 3449T> G: p.C117G). This mutation has not been recorded in the international electronic database INFEVERS. The child was administered pathogenetic therapy with a selective blocker of interleukin (IL-1) receptors (anakinra) at a dose of 1-5 mg / kg of body weight subcutaneously daily. After the first injection of anakinra the patient got rid of fever, joint syndrome and of abdominal pain. After 1 week of therapy, laboratory parameters of the disease activity (ESR, CRP) became normal. The child has taken anakinra for two years, there were no exacerbations of the disease or side effects due to the treatment. The variety of clinical manifestations of congenital periodic fever and the presence of previously unknown genetic mutations that lead to the development of auto-inflammatory syndromes, indicate the need for a detailed study of these diseases.

Sildenafil Citrate Influences Production of TNF-α in Healthy Men Lymphocytes

The aim of our study was to determine whether sildenafil citrate influences the production of Th1- (TNF-α, INF-γ) or Th2-type (TGF-β, IL-10) cytokines by lymphocytes of healthy men. Sildenafil citrate (SC) is a selective blocker of phosphodiesterase 5, by competing for the binding site with cGMP. It was reported that a higher risk of sexually transmitted diseases (STD) could be correlated with a recreational use of sildenafil, especially when combined with another drug. While behavioral causes of these findings are understood, it is worth considering other causes of that phenomenon that might rely on the influence of sildenafil on the immune system. Material and Methods. Peripheral blood mononuclear cells (PBMCs) were isolated from 27 healthy men donors and cultured in the presence of SC at a concentration of 400 ng/ml. The first set of research was performed on cells stimulated, for at least 4 hours, by incubation with phorbol myristate acetate (PMA), ionomycin, and Golgi-Stop. Subsequently, we determined cytokine production in cells stimulated with phytohemagglutinin (PHA) for 12 hours in the presence of Golgi-Stop. Flow cytometry immunophenotyping of PBMC was performed towards the surface marker of T cells: CD3 and intracellular cytokine expression: TNF-α, IFN-γ, TGF-β, and IL-10. Our findings show that SC significantly decreased the percentage of T cells producing TNF-α and displayed tendency to decrease IFN-γ, when stimulated with PMA. Frequent usage of SC might strengthen this effect. That could partially explain the impaired immune response to the pathogens of men using the drug.