As a regulator of alveolo-capillary barrier integrity, Transient Receptor Potential Vanilloid 4 (TRPV4) antagonism represents a promising strategy for reducing pulmonary edema secondary to chemical inhalation. In an experimental model of acute lung injury induced by exposure of anesthetized swine to chlorine gas by mechanical ventilation, the dose-dependent effects of TRPV4 inhibitor GSK2798745 were evaluated. Pulmonary function and oxygenation were measured hourly; airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, and histopathology were assessed 24 h post-exposure. Exposure to 240 parts per million (ppm) chlorine gas for ≥50 min resulted in acute lung injury characterized by sustained changes in the ratio of partial pressure of oxygen in arterial blood to the fraction of inspiratory oxygen concentration (PaO2/FiO2), oxygenation index, peak inspiratory pressure, dynamic lung compliance, and respiratory system resistance over 24 h. Chlorine exposure also heightened airway response to methacholine and increased wet-to-dry lung weight ratios at 24 h. Following 55-min chlorine gas exposure, GSK2798745 marginally improved PaO2/FiO2, but did not impact lung function, airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, or histopathology. In summary, in this swine model of chlorine gas-induced acute lung injury, GSK2798745 did not demonstrate a clinically relevant improvement of key disease endpoints.

Administration of bolus intravenous fluids, common in pre-hospital and hospitalised patients, is associated with increased lung vascular permeability and mortality outside underlying disease states. In our laboratory, the induction of lung injury and oedema through rapid administration of intravenous fluid in rats was reduced by a non-specific antagonist of transient receptor potential vanilloid 4 (TRPV4) channels. The aims of this study were to determine the effect of selective TRPV4 inhibition on fluid-induced lung injury (FILI) and compare the potency of FILI inhibition to that of an established model of TRPV4 agonist-induced lung oedema. In a series of experiments, rats received specific TRPV4 inhibitor (GSK2789917) at high (15 μg/kg), medium (5 μg/kg) or low (2 μg/kg) dose or vehicle prior to induction of lung injury by intravenous infusion of TRPV4 agonist (GSK1016790) or saline. GSK1016790 significantly increased lung wet weight/body weight ratio by 96

Transient receptor potential vanilloid 4 (TRPV4) channels expressed on pulmonary endothelial cells are activated by elevated pulmonary vascular pressure, resulting in endothelial shape change, pulmonary barrier disruption, and edema. As such, TRPV4 blocker GSK2798745 was recently investigated in phase I/IIa trials to reduce pulmonary edema caused by heart failure (HF). In the absence of a suitable TRPV4 target engagement biomarker, we hypothesized that an ex vivo assay could be used to predict pharmacological activity at the intended site of action (endothelial cells) of subjects. In this assay, the ability of GSK2798745 to block TRPV4 agonist GSK1016790-induced impendence reduction in human umbilical vein endothelial cells (HUVECs) in the presence of human whole blood was assessed. Blood from healthy volunteers drawn 1–12 hours after single or repeated dose of GSK2798745 (5 mg) inhibited GSK1016790-induced impedance reduction by ≥85%. Similarly, blood samples from 16 subjects with HF dosed with GSK2798745 (2.4 mg) inhibited GSK1016790-induced HUVEC impedance reduction by ≥58% 1–24 hours after single dosing and ≥78% 1–24 hours after 7 days of repeated dosing. No inhibition was detected using blood from placebo subjects. Using matched GSK2798745 plasma levels, a pharmacokinetic/pharmacodynamic (PK/PD) relationship was calculated as 2.9 nM IC50, consistent with the 6.5 nM IC50 of GSK2798745 obtained from a rat in vivo PK/PD model of pulmonary edema after correcting for rat-to-human differences. These results indicate that circulating levels of GSK2798745 in the recently completed phase I/IIa trials were sufficient to block TRPV4 in lung vascular endothelial cells to a large extent, supporting this dosing regimen for assessing efficacy in HF. SIGNIFICANCE STATEMENT In the absence of a suitable target engagement biomarker, we developed an ex vivo assay to predict the pharmacological activity of the transient receptor potential vanilloid 4 (TRPV4) blocker GSK2798745 in healthy volunteers and subjects with heart failure (HF) from phase I/IIa trials. The potency values from the ex vivo assay were consistent with those predicted from a rat in vivo pharmacokinetic/pharmacodynamic model of pulmonary edema, strongly suggesting that circulating levels of GSK2798745 were sufficient to robustly block TRPV4, supporting use of GSK2798745 for assessing efficacy in HF.

GSK2798745, an antagonist of the transient receptor potential vanilloid 4 (TRPV4) ion channel, was recently investigated in clinical trials for the treatment of cardiac and respiratory diseases. Human plasma and urine samples collected from healthy volunteers following oral administration were analyzed to identify circulating and excreted metabolites of the parent drug. One major circulating metabolite (1) was found in pooled human plasma samples, accounting for approximately half of the observed drug-related material. Isolation of metabolite 1 from urine samples followed by MS and NMR studies led to a putative structural assignment of 1 where hydroxylation of GSK2798745 occurred on the central ring, producing a penta-substituted cyclohexane structure containing three stereocenters. Two unique chemical syntheses of the proposed structure were developed to confirm the identity of metabolite 1 and provide access to gram quantities for biological characterization.

Objective Airway sensory nerves involved in the cough reflex are activated by adenosine triphosphate (ATP) agonism of P2X purinoceptor 3 (P2X3) receptors. Transient receptor potential vanilloid 4 (TRPV4) channel activation causes ATP release from airway cells, and it is hypothesised that a TRPV4-ATP-P2X3 axis contributes to chronic cough. An adaptive study was run to determine if TRPV4 inhibition, using the selective TRPV4 channel blocker GSK2798745, was effective in reducing cough. Methods A two-period randomised, double blinded, placebo-controlled crossover study was designed with interim analyses for futility and sample size adjustment. Refractory chronic cough patients received either GSK2798745 or placebo once daily for 7 days with a washout between treatments. Pharmacokinetic samples were collected for analysis of GSK2798745 at end of study. The primary end-point was total cough counts assessed objectively during day-time hours (10 h) following 7 days of dosing. Results Interim analysis was performed after 12 participants completed both treatment periods. This showed a 32% increase in cough counts on Day 7 for GSK2798745 compared to placebo; the pre-defined negative criteria for the study were met and the study was stopped. At this point 17 participants had been enrolled (mean 61 years; 88% female), and 15 had completed the study. Final study results for posterior median cough counts showed a 34% (90% credible interval: −3%, +85%) numerical increase for GSK2798745 compared to placebo. Conclusion There was no evidence of an anti-tussive effect of GSK2798745. The study design allowed the decision on lack of efficacy to be made with minimal participant exposure to the investigational drug.

Introduction: Transient receptor potential vanilloid 4 (TRPV4) is an ion channel that is widely expressed and is activated by numerous chemical, osmotic and mechanical stimuli. By modulating Ca2+ entry, TRPV4 regulates cellular signaling associated with a variety of (patho)physiological processes and is a target of interest for treatment of human diseases including heart failure, respiratory diseases, gastrointestinal disorders, dermatological conditions, pain and cancer, among others.Areas covered: This article reviews small molecule TRPV4 antagonists and new therapeutic use claims disclosed in the patent literature from 2015 to 2020, including applications covering the first potent and selective TRPV4 clinical candidate and other advanced chemotypes.Expert opinion: TRPV4 has proven to be a tractable target and significant progress in discovery of TRPV4 antagonists has been realized in recent years. Several unique chemical templates with drug-like properties inhibit the channel and show efficacy in models that suggest their potential for treatment of a variety of diseases. While compelling clinical efficacy has not yet been seen in the limited early studies conducted with GSK2798745, evaluation of TRPV4 antagonists in larger trials across several indications is warranted given the availability of high-quality candidates and the promise of therapeutic benefit based on pre-clinical evidence.

TRPV4 is a ubiquitously expressed, non-selective cation channel activated by a range of stimuli including hypotonicity, temperature, pH, stretch and endogenous ligands. Agents that modulate TRPV4 are sought as potential therapeutics for the treatment of many diseases including osteoarthritis, respiratory illnesses, gastrointestinal disorders, pain and congestive heart failure. In recent years, significant advances in TRPV4 drug discovery have been realized as at least seven novel TRPV4 agonist or antagonist templates were reported and the first selective TRPV4 antagonist was evaluated in early clinical trials.

Lung congestion in patients with heart failure (HF) has traditionally been treated using interventions that reduce pulmonary capillary hydrostatic pressure. The transient receptor potential vanilloid 4 (TRPV4) channel regulates fluid transit across the pulmonary capillary‐interface, and represents a novel target to reduce lung water, independent of pulmonary capillary hypertension. This pilot study examined the safety and potential efficacy of TRPV4 blockade as a novel treatment for HF.

BACKGROUND Acute Respiratory Distress Syndrome (ARDS) is associated with increased pulmonary-vascular permeability. In the lung, transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable cation channel, is a regulator of endothelial permeability and pulmonary edema. We performed a Phase I, placebo-controlled, double-blind, randomized, parallel group, proof-of-mechanism study to investigate the effects of TRPV4 channel blocker, GSK2798745, on pulmonary-vascular barrier permeability using a model of lipopolysaccharide (LPS)-induced lung inflammation. METHODS Healthy participants were randomized 1:1 to receive 2 single doses of GSK2798745 or placebo, 12 hours apart. Two hours after the first dose, participants underwent bronchoscopy and segmental LPS instillation. Total protein concentration and neutrophil counts were measured in bronchoalveolar lavage (BAL) samples collected before and 24 hours after LPS challenge, as markers of barrier permeability and inflammation, respectively. The primary endpoint was baseline adjusted total protein concentration in BAL at 24 hours after LPS challenge. A Bayesian framework was used to estimate the posterior probability of any percentage reduction (GSK2798745 relative to placebo). Safety endpoints included the incidence of adverse events (AEs), vital signs, 12-lead electrocardiogram, clinical laboratory and haematological evaluations, and spirometry. RESULTS Forty-seven participants were dosed and 45 completed the study (22 on GSK2798745 and 23 on placebo). Overall, GSK2798745 was well tolerated. Small reductions in mean baseline adjusted BAL total protein (∼9%) and neutrophils (∼7%) in the LPS-challenged segment were observed in the GSK2798745 group compared with the placebo group; however, the reductions did not meet pre-specified success criteria of at least a 95% posterior probability that the percentage reduction in the mean 24-hours post LPS BAL total protein level (GSK2798745 relative to placebo) exceeded zero. Median plasma concentrations of GSK2798745 were predicted to inhibit TRPV4 on lung vascular endothelial cells by ∼70-85% during the 24 hours after LPS challenge; median urea-corrected BAL concentrations of GSK2798745 were 3.0- to 8.7-fold higher than those in plasma. CONCLUSIONS GSK2798745 did not affect segmental LPS-induced elevation of BAL total protein or neutrophils, despite blood and lung exposures that were predicted to be efficacious. CLINICALTRIALS. GOV IDENTIFIER NCT03511105.

Introduction and objectives Airway sensory nerves involved in the cough reflex may be mediated by adenosine triphosphate (ATP) agonism of P2X purinoceptor 3 (P2X3) receptors. Transient receptor potential vanilloid 4 (TRPV4) activation causes ATP release from airway macrophages and epithelial cells and it is hypothesised that a TRPV4-ATP-P2X3 axis contributes to chronic cough. The aim of this study was to evaluate, using an adaptive design, whether blockade of TRPV4 channels, using the selective TRPV4 channel blocker GSK2798745, is effective in reducing cough. Methods A placebo-controlled, double blind, randomised, two-period crossover study was designed with interim analyses for futility and to allow possible sample size adjustment during the study. Refractory chronic cough patients were recruited from four specialist clinics. Participants received either GSK2798745 or matching placebo once daily for 7 days with a 14–21 day wash out between treatments. Dose of GSK2798745 orally administered was predicted to give ∼65–72% TRPV4 inhibition over 24-hour period. Blood samples were collected for pharmacokinetic assessment. 24-hour cough count (VitaloJAK) was recorded before and after each treatment period. The primary endpoint was total cough counts during day-time hours following 7 days of dosing. Results Interim analysis was performed after 12 participants had completed both treatment periods and showed a 32% increase in cough counts on Day 7 for GSK2798745 compared to placebo. The negative criteria for the study were met and the study was subsequently stopped. At this point 17 participants had been enrolled (Mean 61yrs; 88% female), and 15 completed the study. Final study results for posterior median cough counts are shown in table 1. Conclusion There was no evidence of an anti-tussive effect of GSK2798745, despite cough frequency being highly reproducible within patients and expected drug exposure. Leicester Cough Questionnaire and severity and urge to cough VAS were consistent with this lack of change in cough counts. The design of the study allowed the decision on lack of efficacy to be made with minimal participant exposure to the molecule.

GSK2798745, a clinical candidate, was identified as an inhibitor of the transient receptor potential vanilloid 4 (TRPV4) ion channel for the treatment of pulmonary edema associated with congestive heart failure. We discuss the lead optimization of this novel spirocarbamate series and specifically focus on our strategies and solutions for achieving desirable potency, rat pharmacokinetics, and physicochemical properties. We highlight the use of conformational bias to deliver potency and optimization of volume of distribution and unbound clearance to enable desirable in vivo mean residence times.

GSK3527497, a preclinical candidate for the inhibition of TRPV4, was identified starting from the previously reported pyrrolidine sulfonamide TRPV4 inhibitors 1 and 2. Optimization of projected human dose was accomplished by specifically focusing on in vivo pharmacokinetic parameters CLu, Vdss(u), and MRT. We highlight the use of conformational changes as a novel approach to modulate Vdss(u) and present results that suggest that molecular-shape-dependent binding to tissue components governs Vdss(u) in addition to bulk physicochemical properties. Optimization of CLu within the series was guided by in vitro metabolite identification, and the poor FaSSIF solubility imparted by the crystalline properties of the pyrrolidine diol scaffold was improved by the introduction of a charged moiety to enable excellent exposure from high crystalline doses. GSK3527497 is a preclinical candidate suitable for oral and iv administration that is projected to inhibit TRPV4 effectively in patients from a low daily clinical dose.

Pulmonary edema is a common ailment of heart failure patients and has remained an unmet medical need due to dose-limiting side effects associated with current treatments. Preclinical studies in rodents have suggested that inhibition of transient receptor potential vanilloid-4 (TRPV4) cation channels may offer an alternative-and potentially superior-therapy. Efforts directed toward small-molecule antagonists of the TRPV4 receptor have led to the discovery of a novel sulfone pyrrolidine sulfonamide chemotype exemplified by lead compound 6. Design elements toward the optimization of TRPV4 activity, selectivity, and pharmacokinetic properties are described. Activity of leading exemplars 19 and 27 in an in vivo model suggestive of therapeutic potential is highlighted herein.

A novel series of pyrrolidine sulfonamide transient receptor potential vanilloid-4 (TRPV4) antagonists was developed by modification of a previously reported TRPV4 inhibitor (1). Several core-structure modifications were identified that improved TRPV4 activity by increasing structural rigidity and reducing the entropic energy penalty upon binding to the target protein. The new template was initially discovered as a minor regio-isomeric side product formed during routine structure-activity relationship (SAR) studies, and further optimization resulted in highly potent compounds with a novel pyrrolidine diol core. Further improvements in potency and pharmacokinetic properties were achieved through SAR studies on the sulfonamide substituent to give an optimized lead compound GSK3395879 (52) that demonstrated the ability to inhibit TRPV4-mediated pulmonary edema in an in vivo rat model. GSK3395879 is a tool for studying the biology of TRPV4 and an advanced lead for identifying new heart failure medicines.

Administration of bolus intravenous fluids, common in pre-hospital and hospitalised patients, has been associated with increased lung vascular permeability and mortality outside underlying disease states. In our laboratory, the induction of lung injury and oedema through rapid administration of intravenous fluid in rats was reduced by a non-specific antagonist of transient receptor potential vanilloid 4 (TRPV4) channels. The aims of this study were to determine the effect of selective TRPV4 inhibition on fluid-induced lung injury (FILI) and compare the potency of FILI inhibition to that of an established model of TRPV4 agonist-induced lung edema. In a series of experiments, rats received specific TRPV4 inhibitor (GSK2789917) at high (15 μg/kg), medium (5 μg/kg) or low (2 μg/kg) dose or vehicle prior to induction of lung injury by intravenous infusion of TRPV4 agonist (GSK1016790) or saline. GSK1016790 significantly increased lung wet weight/body weight ratio by 96% and lung wet-to-dry weight ratio by 43% in vehicle pre-treated rats, which was inhibited by GSK2789917 in a dose-dependent manner (IC 50 =3ng/mL). Similarly, in a single-dose study, bolus saline infusion significantly increased lung wet weight/body weight by 17% and lung wet-to-dry weight ratio by 15%, which was attenuated by high dose GSK2789917. However, in a final GSK2789917 dose-response study, inhibition did not reach significance and an inhibitory potency was not determined due to the lack of a clear dose-response. In the FILI model, TRPV4 may have a role in lung injury induced by rapid-fluid infusion, indicated by inconsistent amelioration with high dose TRPV4 antagonist.

In vitro studies of cardiac physiology and drug response have traditionally been performed on individual isolated cardiomyocytes or isotropic monolayers of cells that may not mimic desired physiological traits of the laminar adult myocardium. Recent studies have reported a number of advances to Heart-on-a-Chip platforms for the fabrication of more sophisticated engineered myocardium, but cardiomyocyte immaturity remains a challenge. In the anisotropic musculature of the heart, interactions between cardiac myocytes, the extracellular matrix (ECM), and neighboring cells give rise to changes in cell shape and tissue architecture that have been implicated in both development and disease. We hypothesized that engineered myocardium fabricated from cardiac myocytes cultured in vitro could mimic the physiological characteristics and gene expression profile of adult heart muscle. To test this hypothesis, we fabricated engineered myocardium comprised of neonatal rat ventricular myocytes with laminar architectures reminiscent of that observed in the mature heart and compared their sarcomere organization, contractile performance characteristics, and cardiac gene expression profile to that of isolated adult rat ventricular muscle strips. We found that anisotropic engineered myocardium demonstrated a similar degree of global sarcomere alignment, contractile stress output, and inotropic concentration–response to the β-adrenergic agonist isoproterenol. Moreover, the anisotropic engineered myocardium exhibited comparable myofibril related gene expression to muscle strips isolated from adult rat ventricular tissue. These results suggest that tissue architecture serves an important developmental cue for building in vitro model systems of the myocardium that could potentially recapitulate the physiological characteristics of the adult heart. Impact statement With the recent focus on developing in vitro Organ-on-Chip platforms that recapitulate tissue and organ-level physiology using immature cells derived from stem cell sources, there is a strong need to assess the ability of these engineered tissues to adopt a mature phenotype. In the present study, we compared and contrasted engineered tissues fabricated from neonatal rat ventricular myocytes in a Heart-on-a-Chip platform to ventricular muscle strips isolated from adult rats. The results of this study support the notion that engineered tissues fabricated from immature cells have the potential to mimic mature tissues in an Organ-on-Chip platform.

Background: Urotensin II (U-II) is highly expressed in the human lung and has been implicated in regulating respiratory physiology in preclinical studies. Our objective was to test antagonism of the urotensin (UT) receptor by GSK1440115, a novel, competitive, and selective inhibitor of the UT receptor, as a therapeutic strategy for the treatment of asthma. Methods: Safety, tolerability, and pharmacokinetics (PK) of single doses of GSK1440115 (1–750 mg) were assessed in a Phase I, placebo controlled study in 70 healthy subjects. In a Phase Ib study, 12 asthmatic patients were randomized into a two-period, single-blind crossover study and treated with single doses of 750 mg GSK1440115 or placebo and given a methacholine challenge. Results: Administration of GSK1440115 was safe and well-tolerated in healthy subjects and asthmatic patients. In both studies, there was a high degree of variability in the observed PK following oral dosing with GSK1440115 at all doses. There was a marked food effect in healthy subjects at the 50 mg dose. In the presence of food at the 750 mg dose, the time to maximal concentration was between 2 and 6 h and the terminal half-life was short at approximately 2 h. All asthmatic patients maintained greater than the predicted concentration levels necessary to achieve predicted 96% receptor occupancy for ≥3 h (between 4 and 7 h post-dose). There were no apparent trends or relationships between the systemic plasma exposure of GSK1440115 and pharmacodynamic endpoints, PC20 after methacholine challenge and FEV1, in asthmatics. Conclusion: While GSK1440115 was safe and well-tolerated, it did not induce bronchodilation in asthmatics, or protect against methacholine-induced bronchospasm, suggesting that acute UT antagonism is not likely to provide benefit as an acute bronchodilator in this patient population.

The small, highly conserved vasoactive peptide urotensin II (UII) is upregulated in atherosclerosis. However, its effects in diabetes-associated atherosclerosis have not been assessed.