Higenamine inhibits acute and chronic inflammatory pain through modulation of TRPV4 channels.

Pain is the cardinal symptom of many debilitating diseases and results in heavy health and economic burdens worldwide. Asarum (Asarum sieboldii Miq.) is a commonly used analgesic in Chinese medicine. However, the analgesic components and mechanisms of asarum in acute and chronic pain mice model remain unknown. In this study, we first generated asarum water extract and confirmed strong analgesic properties in mice in both the acute thermal and mechanical pain models, as well as in the complete Freund's adjuvant (CFA) induced chronic inflammatory pain model. Second, we identified higenamine as a major component of asarum and found that higenamine significantly inhibited thermal and mechanical induced acute pain and CFA induced chronic inflammatory pain. Then, using Trpv4-/- mice, we found that TRPV4 is necessary for CFA induced thermal and mechanical allodynia, and demonstrated that higenamine analgesia in the CFA model is partly through TRPV4 channel inhibition. Finally, we found that GSK1016790A, a TRPV4 agonist, induced calcium response was significantly inhibited by higenamine in both cultured DRG neurons and TRPV4 transfected HEK293 cells. Consistent with calcium imaging results, higenamine pretreatment also dose-dependently inhibited GSK1016790A induced acute pain. Taken together, our behavior and calcium imaging results demonstrate that the asarum component higenamine inhibits acute and chronic inflammatory pain by modulation of TRPV4 channels.

Mechanosensitive TRPV4 is required for crystal-induced inflammation.

Crystal structures activate innate immune cells, especially macrophages and initiate inflammatory responses. We aimed to understand the role of the mechanosensitive TRPV4 channel in crystal-induced inflammation. Real-time RT-PCR, RNAscope in situ hybridisation, and Trpv4eGFP mice were used to examine TRPV4 expression and whole-cell patch-clamp recording and live-cell Ca2+ imaging were used to study TRPV4 function in mouse synovial macrophages and human peripheral blood mononuclear cells (PBMCs). Both genetic deletion and pharmacological inhibition approaches were used to investigate the role of TRPV4 in NLRP3 inflammasome activation induced by diverse crystals in vitro and in mouse models of crystal-induced pain and inflammation in vivo. TRPV4 was functionally expressed by synovial macrophages and human PBMCs and TRPV4 expression was upregulated by stimulation with monosodium urate (MSU) crystals and in human PBMCs from patients with acute gout flares. MSU crystal-induced gouty arthritis were significantly reduced by either genetic ablation or pharmacological inhibition of TRPV4 function. Mechanistically, TRPV4 mediated the activation of NLRP3 inflammasome by diverse crystalline materials but not non-crystalline NLRP3 inflammasome activators, driving the production of inflammatory cytokine interleukin-1ß which elicited TRPV4-dependent inflammatory responses in vivo. Moreover, chemical ablation of the TRPV1-expressing nociceptors significantly attenuated the MSU crystal-induced gouty arthritis. In conclusion, TRPV4 is a common mediator of inflammatory responses induced by diverse crystals through NLRP3 inflammasome activation in macrophages. TRPV4-expressing resident macrophages are critically involved in MSU crystal-induced gouty arthritis. A neuroimmune interaction between the TRPV1-expressing nociceptors and the TRPV4-expressing synovial macrophages contributes to the generation of acute gout flares.

High-Throughput Screening of Transient Receptor Potential Channel 1 Ligands in the Light of the Bioluminescence Resonance Energy Transfer Technique.

Ion channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening. SIGNIFICANCE STATEMENT: This study screened a chemical library against TRPV1 ion channel using bioluminescence resonance energy transfer (BRET) molecular probes and compared the results with the ones obtained using reference techniques such as automated calcium assay and automated patch-clamp. Multiparametric analysis of our results shed light on the capability of calmodulin antagonists to inhibit chemical activation of TRPV1 and indicates that BRET probes may advantageously be included in ion channel drug screening campaigns.

Pharmacological studies on aqueous extract of Launaea arborescens from southwest Algeria.

ETHNOPHARMACOLOGICAL RELEVANCE: Launaea arborescens, its vernacular name is Mol-albina belonging to asteracaea family origin of the southwest of Algeria. This plant is used in folk medicines to treat gastroenteritis, diabetes, child aliment and other diseases; it is taken macerated or boiled. AIM: This study aims to evaluate the anti-inflammation an analgesic activity of the aqueous extract of Launaea arborescens (AqELA) and its pathway of action. METHODS: the investigation of anti-inflammatory and analgesic effects were done using formalin test, acetic acid test. For mechanism investigation, it was used hot plate test to induce opioid receptors, a histamine and serotonin test to induce edema paw, finally, for the TRPV1 receptor, it was used the capsaicin test. RESULTS: The aqueous extract of Launaea arborescens showed a significant inhibition of abdominal writhing test 95% and 100% inhibition of licking paw using acid acetic test and formalin test respectively (EC: 47 mg/kg and 104 mg/kg). The analgesic effect of the aqueous extract of Launaea arborescens showed inhibition of sensation of pain after 120 min compared to morphine effect. The aqueous extract of Launaea arborescens reduced paw volume after 180 min and 120 min for histamine and serotonin respectively with dose-dependent. Concerning of TRPV1 receptors, the inhibition was showed at doses 100 mg and 300 mg. CONCLUSION: Our results contribute towards validation of the traditional use of Launaea arborescens for inflammation ailment.

TRPV1 Ion Channel: Structural Features, Activity Modulators, and Therapeutic Potential.

Although TRPV1 ion channel has been attracting researchers' attention for many years, its functions in animal organisms, the principles of regulation, and the involvement in pathological processes have not yet been fully clarified. Mutagenesis experiments and structural studies have identified the structural features of the channel and binding sites for its numerous ligands; however, these studies are far from conclusion. This review summarizes recent achievements in the TRPV1 research with special focus on structural and functional studies of the channel and on its ligands, which are extremely diverse in their nature and interaction specificity to TRPV1. Particular attention was given to the effects of numerous endogenous agonists and antagonists that can fine-tune the channel sensitivity to its usual activators, such as capsaicin, heat, acids, or their combination. In addition to the pain sensing not covered in this review, the TRPV1 channel was found to be involved in the regulation of many important physiological and pathological processes and, therefore, can be considered as a promising therapeutic target in the treatment of various diseases, such as pneumonia, ischemia, diabetes, epilepsy, schizophrenia, psoriasis, etc.

Injectable Capsaicin for the Management of Pain Due to Osteoarthritis.

Capsaicin is a potent agonist of the TRPV1 channel, a transduction channel that is highly expressed in nociceptive fibers (pain fibers) throughout the peripheral nervous system. Given the importance of TRPV1 as one of several transduction channels in nociceptive fibers, much research has been focused on the potential therapeutic benefits of using TRPV1 antagonists for the management of pain. However, an antagonist has two limitations. First, an antagonist in principle generally only affects one receptor. Secondly, most antagonists must have an ongoing presence on the receptor to have an effect. Capsaicin overcomes both liabilities by disrupting peripheral terminals of nociceptive fibers that express TRPV1, and thereby affects all of the potential means of activating that pain fiber (not just TRPV1 function). This disruptive effect is dependent on the dose and can occur within minutes. Thus, unlike a typical receptor antagonist, continued bioavailability at the level of the receptor is not necessary. By disrupting the entire terminal of the TRPV1-expressing nociceptive fiber, capsaicin blocks all the activation mechanisms within that fiber, and not just TRPV1 function. Topical capsaicin, an FDA approved treatment for neuropathic pain, addresses pain from abnormal nociceptor activity in the superficial layers of the skin. Effects after a single administration are evident over a period of weeks to months, but in time are fully reversible. This review focuses on the rationale for using capsaicin by injection for painful conditions such as osteoarthritis (OA) and provides an update on studies completed to date.

A plant-derived TRPV3 inhibitor suppresses pain and itch.

BACKGROUND AND PURPOSE: Itching is the most frequent pathology in dermatology that has significant impacts on people's mental health and social life. Transient receptor potential vanilloid 3 (TRPV3) channel is a promising target for treating pruritus. However, few selecetive and potent antagonists have been reported. This study was designed to identify selective TRPV3 antagonist and elucidate its anti-pruritus pharmacology. EXPERIMENTAL APPROACH: FlexStation and calcium fluorescence imaging were conducted to track the functional compounds. Whole-cell patch clamp was used to record itch-related ion channel currents. Homologous recombination and site-directed mutagenesis were employed to construct TRPV3 channel chimeras and point mutations for exploring pharmacological mechanism. Mouse models were used for in vivo anti-pruritus assay. KEY RESULTS: An acridone alkaloid (citrusinine-II) was purified and characterized from Atalantia monophylla. It directly interacts with Y564 within S4 helix of TRPV3 to selectively inhibit the channel with a half maximal inhibitory concentration (IC50 ) of 12.43 µM. Citrusinine-II showed potential efficacy to attenuate both chronic and acute itch. Intradermal administration of citrusinine-II (143 ng/skin site) nearly completely inhibited itch behaviours. It also shows significant analgesic effects. Little side effects of the compound are observed. CONCLUSION AND IMPLICATIONS: By acting as a selective and potent inhibitor of TRPV3 channel, citrusinine-II shows valuable therapeutic effects in pruritus animal models and is a promising candidate drug and/or lead molecule for the development of anti-pruritus drugs.

Effect of San'ao decoction with scorpio and bombyx batryticatus on CVA mice model via airway inflammation and regulation of TRPA1/TRPV1/TRPV5 channels.

ETHNOPHARMACOLOGICAL RELEVANCE: Cough variant asthma (CVA) is characterized with its long-lasting cough symptom on clinic. The mechanism of CVA is related to chronic persistent airway inflammation, airway hyperresponsiveness, etc. The traditional Chinese prescription has achieved good curative effect on CVA treatment through reducing cough counts, decreasing airway hyperresponsiveness and alleviating airway inflammation. The mechanism is associated with reducing IL4, IL-13, NGF and CGRP levels, as well as down-regulating TRPA1/TRPV1/TRPV5 channels in both lung and brain tissues. AIM OF THE STUDY: The Chinese prescription, San'ao decoction with scorpio and bombyx batryticatus (SSB), is well known in treating cough in asthmatic patients. In this study, the anti-tussive and anti-asthmatic role of SSB, as well as its mechanism on CVA mice model were explored and evaluated via alleviating airway inflammation and regulation of TRP channels. MATERIALS AND METHODS: The major chemical components in SSB were detected and analyzed by UPLC-QTOF-MS under an optimized chromatographic and MS condition. 60 BALB/c mice were randomly divided into six groups: normal group, model group, dexamethasone group (0.1178 mg/kg/d), SSB high dose group (9.74 g/kg/d), SSB middle dose group (4.87 g/kg/d) and SSB low dose group (2.435 g/kg/d). The cough variant asthma mice model was established by ovalbumin sensitization and challenge. The protective role of SSB on CVA mice model was studied through inducing cough counts by capsaicin, assessing inflammatory cells in peripheral blood and bronchoalveolar lavage fluid (BALF), measuring airway responsiveness, detecting histopathological changes in lung tissues, analyzing cytokines and neuropeptides levels in BALF, as well as examining the mRNA and protein expressions of TRPA1, TRPV1 and TRPV5 in both lung and brain tissues. RESULTS: 17 signal peaks of the chemical components in SSB were identified by using UPLC-QTOF-MS. SSB (especially the high dose and middle dose), showed significantly effects on mice model by reducing mice cough counts (P ＜ 0.01), decreasing eosinophil (EOS) counts in blood (P ＜ 0.01) and inflammatory cell numbers in BALF (P ＜ 0.01), decreasing airway hyperresponsiveness (P ＜ 0.05), reducing the levels of IL-4 (P ＜ 0.05), IL-13 (P ＜ 0.01), NGF (P ＜ 0.01) and CGRP (P ＜ 0.01) in BALF, as well as down regulating the mRNA and protein expressions of TRPA1, TRPV1 and TRPV5 in both lung and brain tissues (P ＜ 0.01). CONCLUSIONS: SSB showed anti-tussive and anti-asthmatic effects on cough variant asthma mice model by reducing cough counts, improving lung function, alleviating lung injury and airway inflammation. The mechanism of SSB might be associated with the regulation of cytokines and neuropeptides in BALF, as well as the regulation of TRPA1, TRPV1, TRPV5 channels in both lung and brain tissues.

Resolvin D3 controls mouse and human TRPV1-positive neurons and preclinical progression of psoriasis.

Rationale: Psoriasis is a chronic inflammatory disease caused by a complex interplay between the immune and nervous systems with recurrent scaly skin plaques, thickened stratum corneum, infiltration and activation of inflammatory cells, and itch. Despite an increasing availability of immune therapies, they often have adverse effects, high costs, and dissociated effects on inflammation and itch. Activation of sensory neurons innervating the skin and TRPV1 (transient receptor potential vanilloid 1) are emerging as critical components in the pathogenesis of psoriasis, but little is known about their endogenous inhibitors. Recent studies have demonstrated that resolvins, endogenous lipid mediators derived from omega-3 fatty acids, are potent inhibitors of TRP channels and may offer new therapies for psoriasis without known adverse effects. Methods: We used behavioral, electrophysiological and biochemical approaches to investigate the therapeutic effects of resolvin D3 (RvD3), a novel family member of resolvins, in a preclinical model of psoriasis consisting of repeated topical applications of imiquimod (IMQ) to murine skin, which provokes inflammatory lesions that resemble human psoriasis. Results: We report that RvD3 specifically reduced TRPV1-dependent acute pain and itch in mice. Mechanistically, RvD3 inhibited capsaicin-induced TRPV1 currents in dissociated dorsal root ganglion (DRG) neurons via the N-formyl peptide receptor 2 (i.e. ALX/FPR2), a G-protein coupled receptor. Single systemic administration of RvD3 (2.8 mg/kg) reversed itch after IMQ, and repeated administration largely prevented the development of both psoriasiform itch and skin inflammation with concomitant decreased in calcitonin gene-related peptide (CGRP) expression in DRG neurons. Accordingly, specific knockdown of CGRP in DRG was sufficient to prevent both psoriasiform itch and skin inflammation similar to the effects following RvD3 administration. Finally, we elevated the translational potential of this study by showing that RvD3 significantly inhibited capsaicin-induced TRPV1 activity and CGRP release in human DRG neurons. Conclusions: Our findings demonstrate a novel role for RvD3 in regulating TRPV1/CGRP in mouse and human DRG neurons and identify RvD3 and its neuronal pathways as novel therapeutic targets to treat psoriasis.

Qingfei oral liquid alleviates airway hyperresponsiveness and mucus hypersecretion via TRPV1 signaling in RSV-infected asthmatic mice.

Pediatric asthma is exacerbated by Respiratory Syncytial Virus (RSV) infection, and Transient Receptor Potential Vanilloid 1 (TRPV1) promotes production of inflammatory cytokines and mucus hypersecretion in the pathology of this disease. Our previous research revealed that Qingfei oral liquid (QF) inhibited airway inflammation and mucus hypersecretion in RSV-infected asthmatic mice models and that this may be associated with the TRPV1-regulation of NF-κB and Mucin 5AC (MUC5AC) expression, but the exact mechanism is unknown. In the present study, LC-MS was used for analyzing the chemicals in QF, ovalbumin (OVA)-induced asthmatic mice inhaled RSV three consecutive times to create an RSV-infected asthmatic model. We found treatment from QF alleviated airway hyperresponsiveness (AHR) and reduced congestion, edema, and infiltration of inflammatory cells into pulmonary tissues. Additionally, QF was found to decrease expression of NF-κB and its downstream inflammatory cytokines IL-1ß, IL-4, IL-5, and IL-13, as well as a decrease in MUC5AC and pro-inflammatory cytokines in PKC via a reduction in Protein Kinase C-dependent signaling. These findings suggest that QF can alleviate AHR and mucus hypersecretion caused by RSV infection in asthmatic mice, and its mechanism may be associated with the regulation of the TRPV1 signaling pathway.

Identification of Transient Receptor Potential Vanilloid 3 Antagonists from Achillea alpina L. and Separation by Liquid-Liquid-Refining Extraction and High-Speed Counter-Current Chromatography.

Bioassay-guided fractionation of the ethanol extract of whole herbs of Achillea alpina led to the isolation of isochlorogenic acids A and B as transient receptor potential vanilloid 3 (TRPV3) channel antagonists by using a calcium fluorescent assay. The structures were identified by spectroscopic analysis and the inhibitory activities of isochlorogenic acids A and B were confirmed by whole-cell patch clamp recordings of human embryonic kidney 293 (HEK293) cells expressing human TRPV3. Molecular docking results revealed that these two compounds reside in the same active pocket of human TRPV3 channel protein with lower binding energy than the agonist 2-aminoethoxydiphenyl borate (2-APB). High-speed counter-current chromatography (HSCCC) coupled with a liquid-liquid extraction approach was successfully established for the separation of isochlorogenic acids A and B from the whole herbs of A. alpina. Ethyl acetate and n-hexane-ethyl acetate-water (3:3:4 and 1:5:4, v/v/v) were selected as liquid-liquid extraction solvent systems to remove high- and low-polarity impurities in the mixture. Sixty g of ethanol extract was refined by solvent partition to yield 1.7 g of the enriched fraction, of which 480 mg in turn obtained 52.5 mg of isochlorogenic acid B (purity 98.3%) and 37.6 mg isochlorogenic acid A (purity 96.2%) after HSCCC with n-hexane-ethyl acetate-water containing 1% acetic acid (1:4:8, v/v/v).

Desensitization of transient receptor potential vanilloid type-1 (TRPV1) channel as promising therapy of irritable bowel syndrome: characterization of the action of palvanil in the mouse gastrointestinal tract.

TRPV1 are involved in the control of the gastrointestinal (GI) functions and pain sensation. Their activation induces pain but it is followed by desensitization, which in turn causes analgesia. The studies from the last two decades indicate that TRPV1 are involved in visceral hypersensitivity in the GI tract and pathogenesis of irritable bowel syndrome (IBS). Therefore, the aim of this study is to assess the action of fast desensitizing agonist of TRPV1, palvanil (N-palmitoyl-vanillamine), in the murine GI tract and on nociception to evaluate its potential application in the therapy of IBS. The effect of palvanil on smooth muscle contractility was evaluated using organ baths. The impact of palvanil on intestinal secretion was assessed in Ussing chambers. In vivo, the action of palvanil (0.1-1 mg/kg) was assessed in whole GI transit, fecal pellet output, and colonic bead expulsion tests. The antinociceptive potency of palvanil was tested in the mustard oil-induced pain test. Palvanil inhibited colonic contractions (evoked by electrical field stimulation, EFS) and decreased the ion transport in the colon stimulated with forskolin. It did not affect secretion in experiments with veratridine. In vivo, palvanil prolonged whole GI transit at all doses tested. At the lower dose tested, it accelerated colonic motility during first 60 min following injection. By contrast, at the dose of 1 mg/kg, colonic motility was inhibited. Palvanil induced antinociceptive action at all tested doses in mustard oil-induced pain test. TRPV1 fast-desensitizing compounds, i.e., palvanil, may be promising agents in the therapy of IBS since it modulates intestinal motility and reduces visceral pain.

Discovery of indane propanamides as potent and selective TRPV1 antagonists.

A series of indane-type acetamide and propanamide analogues were investigated as TRPV1 antagonists. The analysis of structure-activity relationship indicated that indane A-region analogues exhibited better antagonism than did the corresponding 2,3-dihydrobenzofuran and 1,3-benzodioxole surrogates. Among them, antagonist 36 exhibited potent and selective antagonism toward capsaicin for hTRPV1 and mTRPV1. Further, in vivo studies indicated that antagonist 36 showed excellent analgesic activity in both phases of the formalin mouse pain model and inhibited the pain behavior completely at a dose of 1 mg/kg in the 2nd phase.

Electroacupuncture Alleviates Paclitaxel-Induced Peripheral Neuropathic Pain in Rats via Suppressing TLR4 Signaling and TRPV1 Upregulation in Sensory Neurons.

Paclitaxel-induced peripheral neuropathy is a common adverse effect during paclitaxel treatment resulting in sensory abnormalities and neuropathic pain during chemotherapy and in cancer survivors. Conventional therapies are usually ineffective and possess adverse effects. Here, we examined the effects of electroacupuncture (EA) on a rat model of paclitaxel-induced neuropathic pain and related mechanisms. EA robustly and persistently alleviated paclitaxel-induced pain hypersensitivities. Mechanistically, TLR4 (Toll-Like Receptor 4) and downstream signaling MyD88 (Myeloid Differentiation Primary Response 88) and TRPV1 (Transient Receptor Potential Vallinoid 1) were upregulated in dorsal root ganglion (DRGs) of paclitaxel-treated rats, whereas EA reduced their overexpression. Ca2+ imaging further indicated that TRPV1 channel activity was enhanced in DRG neurons of paclitaxel-treated rats whereas EA suppressed the enhanced TRPV1 channel activity. Pharmacological blocking of TRPV1 mimics the analgesic effects of EA on the pain hypersensitivities, whereas capsaicin reversed EA's effect. Spinal astrocytes and microglia were activated in paclitaxel-treated rats, whereas EA reduced the activation. These results demonstrated that EA alleviates paclitaxel-induced peripheral neuropathic pain via mechanisms possibly involving suppressing TLR4 signaling and TRPV1 upregulation in DRG neurons, which further result in reduced spinal glia activation. Our work supports EA as a potential alternative therapy for paclitaxel-induced neuropathic pain.

The Role of the Activation of the TRPV1 Receptor and of Nitric Oxide in Changes in Endothelial and Cardiac Function and Biomarker Levels in Hypertensive Rats.

The purpose of the present study was to analyze the actions of transient receptor potential vanilloid type 1 (TRPV1) agonist capsaicin (CS) and of its antagonist capsazepine (CZ), on cardiac function as well as endothelial biomarkers and some parameters related with nitric oxide (NO) release in L-NG-nitroarginine methyl ester (L-NAME)-induced hypertensive rats. NO has been implicated in the pathophysiology of systemic arterial hypertension (SAHT). We analyzed the levels of nitric oxide (NO), tetrahydrobiopterin (BH4), malondialdehyde (MDA), total antioxidant capacity (TAC), cyclic guanosin monophosphate (cGMP), phosphodiesterase-3 (PDE-3), and the expression of endothelial nitric oxide synthase (eNOS), guanosine triphosphate cyclohydrolase 1 (GTPCH-1), protein kinase B (AKT), and TRPV1 in serum and cardiac tissue of normotensive (118±3 mmHg) and hypertensive (H) rats (165 ± 4 mmHg). Cardiac mechanical performance (CMP) was calculated and NO was quantified in the coronary effluent in the Langendorff isolated heart model. In hypertensive rats capsaicin increased the levels of NO, BH4, cGMP, and TAC, and reduced PDE-3 and MDA. Expressions of eNOS, GTPCH-1, and TRPV1 were increased, while AKT was decreased. Capsazepine diminished these effects. In the hypertensive heart, CMP improved with the CS treatment. In conclusion, the activation of TRPV1 in H rats may be an alternative mechanism for the improvement of cardiac function and systemic levels of biomarkers related to the bioavailability of NO.

Discovery of GSK3527497: A Candidate for the Inhibition of Transient Receptor Potential Vanilloid-4 (TRPV4).

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, Vdssu, and MRT. We highlight the use of conformational changes as a novel approach to modulate Vdssu and present results that suggest that molecular-shape-dependent binding to tissue components governs Vdssu 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.

Intrathecal lentivirus-mediated RNA interference targeting nerve growth factor attenuates myocardial ischaemia-reperfusion injury in rat.

BACKGROUND: Nerve growth factor (NGF) has been implicated in hyperalgesia by sensitising nociceptors. A role for NGF in modulating myocardial injury through ischaemic nociceptive signalling is plausible. We examined whether inhibition of spinal NGF attenuates myocardial ischaemia-reperfusion injury and explored the underlying mechanisms. METHODS: In adult rats, lentivirus-mediated short-hairpin RNA targeted at reducing NGF gene expression (NGF-shRNA) or a transient receptor potential vanilloid 1 (TRPV1) antagonist (capsazepine) was injected intrathecally before myocardial ischaemia-reperfusion. Infarct size (expressed as the ratio of area at risk) and risk of arrhythmias were quantified. Whole-cell clamp patch electrophysiology was used to record capsaicin currents in primary dorsal root ganglion neurones. The co-expression of substance P (SP) and calcitonin gene-related peptide (CGRP), plus activation of TRPV1, protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were also quantified. RESULTS: NGF levels increased by 2.95 (0.34)-fold in dorsal root ganglion and 2.12 (0.27)-fold in spinal cord after myocardial ischaemia-reperfusion injury. Intrathecal injection of NGF-shRNA reduced infarct area at risk from 0.58 (0.02) to 0.37 (0.02) (P<0.01) and reduced arrhythmia score from 3.67 (0.33) to 1.67 (0.33) (P<0.01). Intrathecal capsazepine was similarly cardioprotective. NGF-shRNA suppressed expression of SP/CGRP and activation of Akt/ERK and TRPV1 in spinal cord. NGF increased capsaicin current amplitude from 144 (42) to 840 (132) pA (P<0.05), which was blocked by the TRPV1 antagonist 5'-iodoresiniferatoxin. Exogenous NGF enhanced capsaicin-induced Akt/ERK and TRPV1 activation in PC12 neuroendocrine tumour cells in culture. CONCLUSIONS: Spinal NGF contributes to myocardial ischaemia-reperfusion injury by mediating nociceptive signal transmission.

Evaluation of cationic channel TRPV2 as a novel biomarker and therapeutic target in Leukemia-Implications concerning the resolution of pulmonary inflammation.

Patients treated during leukemia face the risk of complications including pulmonary dysfunction that may result from infiltration of leukemic blast cells (LBCs) into lung parenchyma and interstitium. In LBCs, we demonstrated that transient receptor potential vanilloid type 2 channel (TRPV2), reputed for its role in inflammatory processes, exhibited oncogenic activity associated with alteration of its molecular expression profile. TRPV2 was overexpressed in LBCs compared to normal human peripheral blood mononuclear cells (PBMCs). Additionally, functional full length isoform and nonfunctional short form pore-less variant of TRPV2 protein were up-regulated and down-regulated respectively in LBCs. However, the opposite was found in PBMCs. TRPV2 silencing or pharmacological targeting by Tranilast (TL) or SKF96365 (SKF) triggered caspace-mediated apoptosis and cell cycle arrest. TL and SKF inhibited chemotactic peptide fMLP-induced response linked to TRPV2 Ca2+ activity, and down-regulated expression of surface marker CD38 involved in leukemia and lung airway inflammation. Challenging lung airway epithelial cells (AECs) with LBCs decreased (by more than 50%) transepithelial resistance (TER) denoting barrier function alteration. Importantly, TL prevented such loss in TER. Therefore, TRPV2 merits further exploration as a pharmacodynamic biomarker for leukemia patients (with pulmonary inflammation) who might be suitable for a novel [adjuvant] therapeutic strategy based on TL.

Cannabidiol Increases Proliferation, Migration, Tubulogenesis, and Integrity of Human Brain Endothelial Cells through TRPV2 Activation.

The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca2+ cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca2+ levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC50 0.3 ± 0.1 µM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.

Lactobacillus reuteri DSM 17938 Protects against Gastric Damage Induced by Ethanol Administration in Mice: Role of TRPV1/Substance P Axis.

This study aimed to evaluate the effect of Lactobacillus reuteri DSM 17938 (DSM) on ethanol-induced gastric injury, and if its possible mechanism of action is related to inhibiting the transient receptor potential vanilloid type 1 (TRPV1). We evaluated the effect of supplementing 108 CFU•g body wt-1•day-1 of DSM on ethanol-induced gastric injury. DSM significantly reduced the ulcer area (1.940 ± 1.121 mm²) with 3 days of pretreatment. The effects of DSM supplementation were reversed by Resiniferatoxin (RTX), TRPV1 agonist (3 nmol/kg p.o.). Substance P (SP) (1 µmol/L per 20 g) plus 50% ethanol resulted in hemorrhagic lesions, and DSM supplementation did not reverse the lesion area induced by administering SP. TRPV1 staining intensity was lower, SP, malondialdehyde (MDA) and nitrite levels were reduced, and restored normal levels of antioxidant parameters (glutathione and superoxide dismutase) in the gastric mucosa in mice treated with DSM. In conclusion, DSM exhibited gastroprotective activity through decreased expression of TRPV1 receptor and decreasing SP levels, with a consequent reduction of oxidative stress.