Common bacterial metabolite indole directly activates nociceptive neuron through transient receptor potential ankyrin 1 channel.

ABSTRACT: Nociceptors are known to directly recognize bacterial cell wall components or secreted toxins, thereby leading to pain induced by bacterial infection. However, direct activation of nociceptors by bacterial metabolites remains unclear although bacteria produce numerous metabolites related to health and disease. In this study, we investigated whether and how a common bacterial metabolite, indole, which is produced by normal microflora of the gastrointestinal tract and oral cavity, can directly activate nociceptive sensory neurons. We found that indole elicits calcium response and evokes inward currents in subsets of dorsal root ganglia (DRG) neurons. Intraplantar (i.pl.) injection of indole produced nocifensive behaviors in adult mice, which were enhanced in complete Freund's adjuvant-induced chronic inflammatory condition. Indole increased calcitonin gene-related peptide release in DRG neurons, and i.pl. injection of indole increased hind paw thickness, suggesting its role in generation of neurogenic inflammation. These in vitro and in vivo indole-induced responses were pharmacologically blocked by transient receptor potential ankyrin 1 (TRPA1) antagonist, HC-030031, and significantly abolished in TRPA1 knockout (KO) mice, indicating that indole targets TRPA1 for its action in DRG neurons. Nocifensive licking behavior induced by the injection of live Escherichia coli was significantly decreased in tryptophanase mutant (TnaA KO) E. coli- injected mice that lack indole production, further supporting the idea that bacteria-derived indole can induce pain during infection. Identifying the mechanism of action of indole through TRPA1 provides insights into bacteria-neuron interactions and the role of bacterial metabolites in pain signaling, especially in inflammation-accompanied bacterial infection.

Inhibitory effect of polysulfide, an endogenous sulfur compound, on oxidative stress-induced TRPA1 activation.

Polysulfide (PS), an endogenous sulfur compound, generated by oxidation of hydrogen sulfide, has a stimulatory action on the nociceptive TRPA1 channel. TRPA1 is also activated by reactive oxygen species such as hydrogen peroxide (H2O2) produced during inflammation. Here, we examined the effect of PS on H2O2-induced responses in native and heterologously expressed TRPA1 using a cell-based calcium assay. We also carried out behavioral experiments in vivo. In mouse sensory neurons, H2O2 elicited early TRPA1-dependent and late TRPA1-independent increases of [Ca2+]i. The former was suppressed by the pretreatment with PS. In cells heterologously expressed TRPA1, PS suppressed [Ca2+]i responses to H2O2. Simultaneous measurement of [Ca2+]i and the intracellular PS level revealed that scavenging effect of PS was not related to the inhibitory effect. Removal of extracellular Ca2+, a calmodulin inhibitor and dithiothreitol attenuated the inhibitory effect of PS. Pretreatment with PS diminished nociceptive behaviors induced by H2O2. The present data suggest that PS suppresses oxidative stress-induced TRPA1 activation due to cysteine modification and Ca2+/calmodulin signaling. Thus, endogenous sulfurs may have regulatory roles in nociception via functional changes in TRPA1 under inflammatory conditions.

Translational and pharmacokinetic-pharmacodynamic application for the clinical development of GDC-0334, a novel TRPA1 inhibitor.

GDC-0334 is a novel small molecule inhibitor of transient receptor potential cation channel member A1 (TRPA1), a promising therapeutic target for many nervous system and respiratory diseases. The pharmacokinetic (PK) profile and pharmacodynamic (PD) effects of GDC-0334 were evaluated in this first-in-human (FIH) study. A starting single dose of 25 mg was selected based on integrated preclinical PK, PD, and toxicology data following oral administration of GDC-0334 in guinea pigs, rats, dogs, and monkeys. Human PK and PK-PD of GDC-0334 were characterized after single and multiple oral dosing using a population modeling approach. The ability of GDC-0334 to inhibit dermal blood flow (DBF) induced by topical administration of allyl isothiocyanate (AITC) was evaluated as a target-engagement biomarker. Quantitative models were developed iteratively to refine the parameter estimates of the dose-concentration-effect relationships through stepwise estimation and extrapolation. Human PK analyses revealed that bioavailability, absorption rate constant, and lag time increase when GDC-0334 was administered with food. The inhibitory effect of GDC-0334 on the AITC-induced DBF biomarker exhibited a clear sigmoid-Emax relationship with GDC-0334 plasma concentrations in humans. This study leveraged emerging preclinical and clinical data to enable iterative refinement of GDC-0334 mathematical models throughout the FIH study for dose selection in subsequent cohorts throughout the study. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? GDC-0334 is a novel, small molecule TRPA1 inhibitor and a pharmacokinetic-pharmacodynamic (PK-PD) modeling strategy could be implemented in a systematic and step-wise manner to build and learn from emerging data for early clinical development. WHAT QUESTION DID THIS STUDY ADDRESS? Can noncompartmental and population-based analyses be used to describe the PK and PD characteristics of GDC-0334 in preclinical and clinical studies? WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? GDC-0334 exposure generally increased with dose in rats, dogs, and monkeys. The starting dose (25 mg) in the clinical study was determined based on the preclinical data. GDC-0334 exhibited linear PK in humans and the bioavailability was increased with food. The inhibitory effect of GDC-0334 on dermal blood flow induced by the TRPA1 agonist allyl isothiocyanate in humans indicates a clear PK-PD relationship. HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? The models developed based on TRPA1 agonist-induced dermal blood flow inhibition data can be used to predict PK-PD relationships in future preclinical and clinical studies evaluating new drug entities that target TRPA1.

Identification of herbal components as TRPA1 agonists and TRPM8 antagonists.

Transient receptor potential (TRP) channels are non-selective cation channels that are implicated in analgesia, bowel motility, wound healing, thermoregulation, vasodilation and voiding dysfunction. Many natural products have been reported to affect the activity of TRP channels. We hypothesize that numerous traditional herbal medicines (THMs) might exert their pharmacological activity through modulating the activity of TRP channels. The present study aimed to evaluate the effects of flavonoid aglycones and their glycosides, which are the main components of many THMs, on the TRP channel subtypes. A Ca2+ influx assay was performed using recombinant human TRPA1, TRPV1, TRPV4 and TRPM8 cell lines. Our findings showed that flavonoid aglycones and glycycoumarin activated TRPA1. In particular, isoflavone and chalcone compounds displayed potent TRPA1 agonistic activity. Furthermore, flavone aglycones showed concomitant potent TRPM8 inhibiting activity. Indeed, flavone, isoflavone aglycones, non-prenylated chalcones and glycycoumarin were found to be TRPM8 inhibitors. Hence, flavonoid aglycones metabolized by lactase-phlorizin hydrolase and ß-glucosidase in the small intestine or gut microbiota of the large intestine could generate TRPA1 agonists and TRPM8 antagonists.

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.

Analgesic effects of the CTK 01512-2 toxin in different models of orofacial pain in rats.

BACKGROUND: Orofacial pain is clinically challenging, having therapeutic failures and side effects. This study evaluated the antinociceptive activities of the CTK 01512-2 toxin, the TRPA1 channel antagonist, and the selective inhibitor of the N-type voltage-gated calcium channels (N-type VGCC), in different pain models. MATERIALS AND METHODS: The trigeminal ganglia were stimulated in vitro with capsaicin. The in vivo models received subcutaneous (sc) injections of formalin into the upper lip of the rats, Freund's Complete Adjuvant (FCA) into the temporomandibular joint (TMJ), and infraorbital nerve constrictions (IONC). CTK 01512-2 at concentrations of 30, 100, and 300 pmol/site, intrathecally (ith), and MVIIA at 10, 30, and 100 pmol/site in the formalin test, guided the doses for the models. The glutamate levels in the CSF of the rats that were submitted to IONC were analyzed. RESULTS: CTK 01512-2 decreased the nociceptive behavior in the inflammatory phase of the formalin test (65.94 ± 7.35%) and MVIIA in the neurogenic phase (81.23 ± 3.36%). CTK 01512-2 reduced facial grooming with FCA in the TMJ (96.7 ± 1.6%), and in the IONC neuropathy model, it decreased heat hyperalgesia (100%) and cold hyperalgesia (81.61 ± 9.02%). The levels of glutamate in the trigeminal ganglia in vitro (81.40 ± 8.59%) and in the CSF in vivo (70.0 ± 9.2%) were reduced. CONCLUSIONS: The roles of TRPA1 in pain transduction and the performance of CTK 01512-2 in the inhibition of the N-type VGCCs were reinforced. This dual activity may represent an advantage in clinical treatments.

Moringin, A Stable Isothiocyanate from Moringa oleifera, Activates the Somatosensory and Pain Receptor TRPA1 Channel In Vitro.

Moringa oleifera Lam. is a tropical plant widely used in traditional medicines and as a food supplement. It is characterized by the presence of glucosinolates and isothiocyanates; the stable isothiocyanate 4-[(α-l-rhamnosyloxy)benzyl]isothiocyanate (moringin) has been widely studied for its bioactivity as hypoglycemic, antimicrobial, anticancer and in particular for its involvement in nociception and neurogenic pain. Moringa extracts and pure moringin were submitted to in vitro assays with the somatosensory TRPA1 ion channel, proving that moringin is a potent and effective agonist of this receptor involved in nociceptive function and pain states. Moringin do not activate or activates very weakly the vanilloids somatosensory channels TRPV1,2,3 and 4, and the melastatin cooling receptor TRPM8. The comparison of moringin's activity with other known agonists of natural origin is also discussed.

Topical transient receptor potential ankyrin 1 antagonist treatment attenuates nociception and inflammation in an ultraviolet B radiation-induced burn model in mice.

BACKGROUND: Ultraviolet B (UVB) radiation exposure promotes sunburn and thereby acute and chronic inflammatory processes, contributing to pain development and maintenance. New therapeutic alternatives are necessary because typical treatments can cause adverse effects. An attractive alternative would be to target the transient receptor potential ankyrin 1 (TRPA1), a calcium-permeable, non-selective cation channel, which is involved in a variety of inflammatory pain models. OBJECTIVE: Evaluate the peripheral participation of TRPA1 using a topical treatment (HC030031 gel formulation; a selective TRPA1 antagonist) in nociception and inflammation caused by a UVB radiation-induced burn model in male mice (25-30 g). METHODS: The mice were anaesthetised, and just the right hind paw was exposed to UVB radiation (0.75 J/cm2). Topical treatments were applied immediately after irradiation and once a day for 8 days. RESULTS: HC030031 gel presented suitable pH and spreadability factor, ensuring its quality and the therapeutic effect. HC030031 0.05 % reversed UVB-induced mechanical and cold allodynia, with maximum inhibition (Imax) of 69 ± 13 % and 100 % (on day 4), respectively. HC030031 0.05 % also reduced the paw edema and MPO activity, with Imax of 77 ± 6 % (on day 5) and 69 ± 28 %, respectively. Likewise, UVB radiation increased the H2O2 levels (a TRPA1 agonist) and the Ca2+ influx in mice spinal cord synaptosomes. UVB radiation-induced Ca2+ influx was reduced by HC030031. CONCLUSION: These findings confirm the activation of the TRPA1 channel by UVB radiation, suggesting that topical TRPA1 antagonists can be a new strategy for the adjuvant treatment of sunburn-associated pain and inflammation.

Identification and characterization of saikosaponins as antagonists of transient receptor potential A1 channel.

Neuropathic pain is associated with an increased sensitivity to painful stimuli or abnormal sensitivity to otherwise innocuous stimuli. However, in addition to adverse effects, currently available drugs have shown limited response in patients with neuropathic pain, which provides a rationale to explore new drug classes acting on novel targets and with better efficacy and safety profiles. Here, we found that saikosaponins potently inhibit agonist-induced activation of the transient receptor potential A1 (TRPA1) channel, which has been reported to mediate neuropathic pain by sensing a variety of chemical irritants. Molecular docking and site-directed mutagenesis analyses suggested that saikosaponins bind to the hydrophobic pocket in TRPA1 near the Asn855 residue, which, when mutated to Ser, was previously associated with enhanced pain perception in humans. In support of these findings, saikosaponin D significantly attenuated agonist-induced nociceptive responses and vincristine-induced mechanical hypersensitivity in mice. These results indicate that saikosaponins are TRPA1 antagonists and provide a basis for further elaboration of saikosaponin derivatives for the development of new therapeutics for neuropathic pain.

TRPA1 Channel as a Regulator of Neurogenic Inflammation and Pain: Structure, Function, Role in Pathophysiology, and Therapeutic Potential of Ligands.

TRPA1 is a cation channel located on the plasma membrane of many types of human and animal cells, including skin sensory neurons and epithelial cells of the intestine, lungs, urinary bladder, etc. TRPA1 is the major chemosensor that also responds to thermal and mechanical stimuli. Substances that activate TRPA1, e.g., allyl isothiocyanates (pungent components of mustard, horseradish, and wasabi), cinnamaldehyde from cinnamon, organosulfur compounds from garlic and onion, tear gas, acrolein and crotonaldehyde from cigarette smoke, etc., cause burning, mechanical and thermal hypersensitivity, cough, eye irritation, sneezing, mucus secretion, and neurogenic inflammation. An increased activity of TRPA1 leads to the emergence of chronic pruritus and allergic dermatitis and is associated with episodic pain syndrome, a hereditary disease characterized by episodes of debilitating pain triggered by stress. TRPA1 is now considered as one of the targets for developing new anti-inflammatory and analgesic drugs. This review summarizes information on the structure, function, and physiological role of this channel, as well as describes known TRPA1 ligands and their significance as therapeutic agents in the treatment of inflammation-associated pain.

Ongoing pain in streptozotocin model of diabetes in the rat: correlation with cutaneous cheminociception.

Streptozotocin (STZ) is commonly used to induce diabetes mellitus in experimental animal studies on peripheral diabetic neuropathy (PDN). Animals with STZ model of diabetes commonly develop changes in test stimulus-evoked pain behavior. However, it is still unclear whether rats with STZ model of diabetes have ongoing pain. Here we assessed whether STZ-induced diabetes induces ongoing pain-like behavior in male rats using conditioned place-preference (CPP) paradigm. CPP was tested in the fourth week of diabetes by pairing one chamber of the CPP device with vehicle and another chamber with either pregabalin (an established analgesic; 30 mg/kg i.p.; n = 9) or Chembridge-5861528 (a TRPA1 channel antagonist; 30 mg/kg i.p.; n = 9). After drug-pairings, the animals were allowed to choose which chamber they preferred. Mechanical sensitivity was assessed with monofilaments and chemonociception in the skin by determining mustard oil-induced pain behavior. Diabetic animals developed in two weeks mechanical hypersensitivity that changed into hyposensitivity by the fourth week. Mustard oil-induced sustained pain was reduced by the 4th week. After 4 weeks of diabetes, neither pregabalin nor the TRPA1 antagonist induced a significant overall change in the median CPP, although both drugs significantly reduced median withdrawal responses evoked by noxious mechanical stimulation. Pregabalin-induced CPP, however, had a significant positive correlation with the sustained pain behaviour induced by topical mustard oil. In conclusion, the present results suggest that the response to topical mustard oil may predict ongoing pain-like behavior in the STZ model of diabetes.

Inflammatory pain control by blocking oxidized phospholipid-mediated TRP channel activation.

Phospholipids occurring in cell membranes and lipoproteins are converted into oxidized phospholipids (OxPL) by oxidative stress promoting atherosclerotic plaque formation. Here, OxPL were characterized as novel targets in acute and chronic inflammatory pain. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) and its derivatives were identified in inflamed tissue by mass spectrometry and binding assays. They elicited calcium influx, hyperalgesia and induced pro-nociceptive peptide release. Genetic, pharmacological and mass spectrometric evidence in vivo as well as in vitro confirmed the role of transient receptor potential channels (TRPA1 and TRPV1) as OxPAPC targets. Treatment with the monoclonal antibody E06 or with apolipoprotein A-I mimetic peptide D-4F, capturing OxPAPC in atherosclerosis, prevented inflammatory hyperalgesia, and in vitro TRPA1 activation. Administration of D-4F or E06 to rats profoundly ameliorated mechanical hyperalgesia and inflammation in collagen-induced arthritis. These data reveal a clinically relevant role for OxPAPC in inflammation offering therapy for acute and chronic inflammatory pain treatment by scavenging OxPAPC.

Sensitization of TRPV1 and TRPA1 via peripheral mGluR5 signaling contributes to thermal and mechanical hypersensitivity.

Peripheral tissue inflammation or injury causes glutamate release from nociceptive axons, keratinocytes, and Schwann cells, resulting in thermal hypersensitivity. However, the detailed molecular mechanisms underlying glutamate-induced thermal hypersensitivity are unknown. The aim of this study was to clarify the involvement of peripheral transient receptor potential (TRP) TRP vanilloid 1 (TRPV1), TRP ankyrin 1 (TRPA1), and protein kinase C epsilon (PKCε) in glutamate-induced pain hypersensitivity. The amount of glutamate in the facial tissue was significantly increased 3 days after facial Complete Freund's adjuvant injection. The head-withdrawal reflex threshold to heat, cold, or mechanical stimulation was significantly decreased on day 7 after continuous glutamate or metabotropic glutamate receptor 5 (mGluR5) agonist (CHPG) injection into the facial skin compared with vehicle-injected rats, and glutamate-induced hypersensitivity was significantly recovered by mGluR5 antagonist MTEP, TRPA1 antagonist HC-030031, TRPV1 antagonist SB366791, or PKCε translocation inhibitor administration into the facial skin. TRPV1 and TRPA1 were expressed in mGluR5-immunoreactive (IR) trigeminal ganglion (TG) neurons innervating the facial skin, and mGluR5-IR TG neurons expressed PKCε. There was no significant difference in the number of GluR5-IR TG neurons among glutamate-injected, saline-injected, and naive rats, whereas that of TRPV1- or TRPA1-IR TG neurons was significantly increased 7 days after continuous glutamate injection into the facial skin compared with vehicle injection. PKCε phosphorylation in TG was significantly enhanced following glutamate injection into the facial skin. Moreover, neuronal activity of TG neurons was significantly increased following facial glutamate treatment. The present findings suggest that sensitization of TRPA1 and/or TRPV1 through mGluR5 signaling via PKCε is involved in facial thermal and mechanical hypersensitivity.

TRPA1 and TRPV1 Antagonists Do Not Inhibit Human Acidosis-Induced Pain.

Acidosis occurs in a variety of pathophysiological and painful conditions where it is thought to excite or contribute to excitation of nociceptive neurons. Despite potential clinical relevance the principal receptor for sensing acidosis is unclear, but several receptors have been proposed. We investigated the contribution of the acid-sensing ion channels, transient receptor potential vanilloid type 1 (TRPV1) and transient receptor potential ankyrin type 1 (TRPA1) to peripheral pain signaling. We first established a human pain model using intraepidermal injection of the TRPA1 agonist carvacrol. This resulted in concentration-dependent pain sensations, which were reduced by experimental TRPA1 antagonist A-967079. Capsaicin-induced pain was reduced by the TRPV1 inhibitor BCTC. Amiloride was used to block acid-sensing ion channels. Testing these antagonists in a double-blind and randomized experiment, we probed the contribution of the respective channels to experimental acidosis-induced pain in 15 healthy human subjects. A continuous intraepidermal injection of pH 4.3 was used to counter the buffering capacity of tissue and generate a prolonged painful stimulation. In this model, addition of A-967079, BCTC or amiloride did not reduce the reported pain. In conclusion, target-validated antagonists, applied locally in human skin, have excluded the main hypothesized targets and the mechanism of the human acidosis-induced pain remains unclear. PERSPECTIVE: An acidic milieu is a trigger of pain in many clinical conditions. The aim of this study was to identify the contribution of the currently hypothesized sensors of acid-induced pain in humans. Surprisingly, inhibition of these receptors did not alter acidosis-induced pain.

Systemic desensitization through TRPA1 channels by capsazepine and mustard oil - a novel strategy against inflammation and pain.

We demonstrate a novel dual strategy against inflammation and pain through body-wide desensitization of nociceptors via TRPA1. Attenuation of experimental colitis by capsazepine (CPZ) has long been attributed to its antagonistic action on TRPV1 and associated inhibition of neurogenic inflammation. In contrast, we found that CPZ exerts its anti-inflammatory effects via profound desensitization of TRPA1. Micromolar CPZ induced calcium influx in isolated dorsal root ganglion (DRG) neurons from wild-type (WT) but not TRPA1-deficient mice. CPZ-induced calcium transients in human TRPA1-expressing HEK293t cells were blocked by the selective TRPA1 antagonists HC 030031 and A967079 and involved three cysteine residues in the N-terminal domain. Intriguingly, both colonic enemas and drinking water with CPZ led to profound systemic hypoalgesia in WT and TRPV1(-/-) but not TRPA1(-/-) mice. These findings may guide the development of a novel class of disease-modifying drugs with anti-inflammatory and anti-nociceptive effects.