n-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2h)-carboxamide and Disease-Models--Animal

When Zika virus emerged as a public health emergency there were no drugs or vaccines approved for its prevention or treatment. We used a high-throughput screen for Zika virus protease inhibitors to identify several inhibitors of Zika virus infection. We expressed the NS2B-NS3 Zika virus protease and conducted a biochemical screen for small-molecule inhibitors. A quantitative structure-activity relationship model was employed to virtually screen ∼138,000 compounds, which increased the identification of active compounds, while decreasing screening time and resources. Candidate inhibitors were validated in several viral infection assays. Small molecules with favorable clinical profiles, especially the five-lipoxygenase-activating protein inhibitor, MK-591, inhibited the Zika virus protease and infection in neural stem cells. Members of the tetracycline family of antibiotics were more potent inhibitors of Zika virus infection than the protease, suggesting they may have multiple mechanisms of action. The most potent tetracycline, methacycline, reduced the amount of Zika virus present in the brain and the severity of Zika virus-induced motor deficits in an immunocompetent mouse model. As Food and Drug Administration-approved drugs, the tetracyclines could be quickly translated to the clinic. The compounds identified through our screening paradigm have the potential to be used as prophylactics for patients traveling to endemic regions or for the treatment of the neurological complications of Zika virus infection.

Topics: Animals; Antiviral Agents; Artificial Intelligence; Chlorocebus aethiops; Disease Models, Animal; Drug Evaluation, Preclinical; High-Throughput Screening Assays; Immunocompetence; Inhibitory Concentration 50; Methacycline; Mice, Inbred C57BL; Protease Inhibitors; Quantitative Structure-Activity Relationship; Small Molecule Libraries; Vero Cells; Zika Virus; Zika Virus Infection

Reported herein is the design, synthesis, and pharmacologic characterization of a class of TRPV1 antagonists constructed on a phenylquinoline platform that evolved from Cinchophen lead. This design composes three sections: a phenylquinoline headgroup attached to an aliphatic carboxamides, which is tethered at a phenyl tail group. Optimization of this design led to the identification of 37, comprising a pyrrolidine linker and a trifluoromethyl-phenyl tail. In the TRPV1 functional assay, using cells expressed hTRPV1, 37 antagonized capsaicin-induced Ca

Topics: Analgesics; Animals; Capsaicin; Disease Models, Animal; Drug Evaluation, Preclinical; Humans; Male; Mice; Pain; Pyrrolidines; Solubility; Structure-Activity Relationship; TRPV Cation Channels

Treatment with the TRPV1 agonist, capsaicin, was previously shown to protect against experimental colitis in the severe combined immunodeficiency (SCID) T-cell transfer model. Here, we investigate trpv1 gene expression in lymphoid organs and cells from SCID and BALB/c mice to identify a potential target for the anti-inflammatory effect of capsaicin.. The trpv1 expression was studied by real-time PCR in lymphoid tissues and gut of untreated and capsaicin-treated colitic SCID mice. Effects of capsaicin and a TRPV1 antagonist on T cells were studied in vitro.. In contrast to BALB/c mice, spleen, lymph nodes, and rectum of colitic and noncolitic SCID mice express trpv1 mRNA. Capsaicin treatment in vivo attenuated T-cell transfer colitis and capsaicin in vitro also attenuated T-cell proliferation induced by enteroantigen, mitogen, and anti-CD3/CD28 beads in BALB/c, C57BL/6 mice, and B6.129X1-trpv1tm1Jul/J trpv1 knockout mice. Proliferation and cytokine secretion were fully comparable in mice with and without trpv1 expression. Likewise, enteroantigen- and mitogen-stimulated T cells from wild-type and trpv1 knockout mice were equally inhibited by capsaicin. Surprisingly, the TRPV1 antagonist BCTC also inhibited enteroantigen- and mitogen-induced T-cell proliferation.. The trpv1 mRNA expression in lymphoid organs and the rectum of SCID mice suggests that the TRPV1 signaling in these organs could play a role in capsaicin-mediated attenuation of colitis. In addition, capsaicin-induced inhibition of T-cell proliferation of wild-type T cells lacking trpv1 expression suggests that capsaicin inhibits colitogenic T cells in a TRPV1 receptor-independent way, which might be linked to its anti-inflammatory effect.

Topics: Animals; Antigens; Antipruritics; beta 2-Microglobulin; Capsaicin; CD4-Positive T-Lymphocytes; Cell Proliferation; Colitis; Cytokines; Disease Models, Animal; Ligands; Lymphoid Tissue; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Mice, SCID; Pyrazines; Pyridines; Signal Transduction; TRPV Cation Channels

A novel series of N-aryl-3,4-dihydro-1'H-spiro[chromene-2,4'-piperidine]-1'-carboxamides was identified as transient receptor potential melastatin 8 (TRPM8) channel blockers through analogue-based rational design, synthesis and screening. Details of the synthesis, effect of aryl groups and their substituents on in-vitro potency were studied. The effects of selected functional groups on the 4-position of the chromene ring were also studied, which showed interesting results. The 4-hydroxy derivatives showed excellent potency and selectivity. Optical resolution and screening of alcohols revealed that (R)-(-)-isomers were in general more potent than the corresponding (S)-(+)-isomers. The isomer (R)-(-)-10e (IC50: 8.9nM) showed a good pharmacokinetic profile upon oral dosing at 10mg/kg in Sprague-Dawley (SD) rats. The compound (R)-(-)-10e also showed excellent efficacy in relevant rodent models of neuropathic pain.

Topics: Administration, Oral; Amides; Analgesics; Animals; Disease Models, Animal; Half-Life; Male; Mice; Mice, Inbred C57BL; Neuralgia; Piperidines; Protein Binding; Rats; Rats, Sprague-Dawley; Spiro Compounds; Stereoisomerism; Structure-Activity Relationship; TRPM Cation Channels

We determined if transient receptor potential melastatin 8 (TRPM8) channels are involved in the detrusor overactivity induced by menthol, or exposure to low temperature (LT).. Two days prior to cystometric investigation, the bladders of 10-week-old Sprague-Dawley rats were cannulated to measure bladder pressure. After a 20 min baseline cystometry period, the TRPM8 channel antagonist, N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide (BCTC), or vehicle, was administered through a jugular vein catheter (n = 6). A 90% menthol solution was sprayed onto bare leg skin once every 5 min for 20 min, and then cystometric measurements were repeated. After a 30-min recovery period, the rats were intravenously administered 0.1 µmol/kg BCTC. Five minutes later, they were again sprayed and cystometry recorded. In separate experiments, cannulated rats were intravenously administered 0.001, 0.01, or 0.1 µmol/kg BCTC (n = 6 each dose). Five minutes later, they were exposed to LT (4 ± 2°C) for 20 min of cystometry.. Menthol spray decreased voiding interval, micturition volume, and bladder capacity in the BCTC-free rats. However after BCTC administration, these effects were prevented. Exposure to LT elicited detrusor overactivity that caused decreased voiding interval, micturition volume, and bladder capacity. However, at 0.01 and 0.1 µmol/kg, BCTC inhibited this cold stress-induced detrusor overactivity.. Since the TRPM8 channel agonist, BCTC, inhibited detrusor overactivity in rats sprayed with the TRPM8 channel agonist, menthol, and the drug also inhibited cold stress-induced detrusor overactivity, we conclude that TRPM8 channels mediate, at least partially, detrusor overactivity elicited by exposure to LT.

Topics: Administration, Intravenous; Animals; Cold Temperature; Consciousness; Disease Models, Animal; Female; Methanol; Pressure; Pyrazines; Pyridines; Rats; Rats, Sprague-Dawley; Time Factors; TRPM Cation Channels; Urinary Bladder; Urinary Bladder, Overactive; Urination; Urodynamics

Heart failure is becoming a global epidemic. It exerts a staggering toll on quality of life, and substantial medical and economic impact. In a pre-clinical model of cardiac hypertrophy and heart failure, we were able to overcome loss of heart function by administering the TRPV1 antagonist BCTC (4-(3-Chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide). The results presented here identify TRPV1 antagonists as new treatment options for cardiac hypertrophy and heart failure.

Topics: Animals; Apoptosis; Cardiomegaly; Disease Models, Animal; Heart Failure; Humans; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Natriuretic Peptide, Brain; Pyrazines; Pyridines; TRPV Cation Channels

This study reports the findings of two classes of corneal afferents excited by drying of the cornea (dry responses) in isoflurane-anesthetized rats: cold-sensitive (CS; 87%) and cold-insensitive (CI; 13%) neurons. Compared with CI neurons, CS neurons showed significantly higher firing rates over warmer corneal temperatures (~31-15°C) and greater responses to menthol, drying, and wetting of the cornea but lower responses when hyperosmolar solutions were applied to the ocular surface. We proposed that the dry responses of these corneal afferents derive from cooling and an increased osmolarity of the ocular surface, leading to the production of basal tears. An ocular application of the transient receptor potential channel TRPM8 antagonist BCTC (20 μM) decreased the dry responses by ~45-80% but failed to completely block them, whereas the TRPA1 antagonist HC030031 did not influence the responses to drying of the cornea or hyperosmolar tears. Furthermore, the responses produced by cold stimulation of the cornea accounted for only 28% of the dry responses. These results support the view that the stimulus for basal tearing (corneal dryness) derives partly from cooling of the cornea that activates TRPM8 channels but that non-TRPM8 channels also contribute significantly to the dry responses and to basal tearing. Finally, we hypothesized that activation of TRPM8 by cooling in CS corneal afferents not only gives rise to the sensation of ocular coolness but also to the "wetness" perception (Thunberg's illusion), whereas a precise role of the CI afferents in basal tearing and other ocular dryness-related functions such as eye blink and the "dryness" sensation remain to be elucidated.

Topics: Acetanilides; Action Potentials; Analysis of Variance; Animals; Cornea; Disease Models, Animal; Dose-Response Relationship, Drug; Dry Eye Syndromes; Eye; Male; Mannitol; Neurons, Afferent; Osmolar Concentration; Purines; Pyrazines; Pyridines; Rats; Rats, Sprague-Dawley; Reaction Time; Stimulation, Chemical; Tears; Temperature; Transient Receptor Potential Channels; Vision, Ocular

Abnormalities of primary afferent nerve fibers are strongly associated with the visceral hypersensitivity state in inflammatory bowel disease. Hypersensitivity of afferent fibers occurs during inflammation. Therefore, to gain an insight into the alterations to receptors and channels expressed in primary afferent neurons, the current study aimed to investigate the time-dependent dynamic changes in levels of 5-hydroxytryptamine (5-HT)(3) receptors, 5-HT(4) receptors, transient receptor potential vanilloid type 1 (TRPV1) channels, and 5-HT regulatory factors in dextran sulfate sodium (DSS)-induced colitis model mice. 5-HT signaling molecules were detected by indirect staining with specific antibodies. TRPV1-immunoreactivity was detected by staining with fluorescein-conjugated tyramide amplification. To assess nociception, visceromotor responses (VMRs) to colorectal distension were measured by electromyography of abdominal muscles. Immunohistochemical analysis and VMRs to colorectal distention were measured during induction of DSS colitis (days 4 and 7). Inflammation led to downregulation of serotonin transporter immunoreactivities with concomitant increases in 5-HT and tryptophan hydroxylase-1-positive cell numbers. TRPV1-expressing nerve fibers gradually increased during DSS treatment. Abundant nonneuronal TRPV1-immunopositive cell-like structures were observed on day 7 of DSS treatment but not on day 4. The number of 5-HT(3) receptor-expressing nerve fibers in the mucosa was increased on day 7. On the other hand, the number of 5-HT(4) receptor-expressing nerve fibers in the mucosa decreased on day 7. We made the novel observation of increased expression of neuronal/nonneuronal TRPV1 channels and 5-HT(3) receptors, and decreased expression of 5-HT(4) receptors in the mucosa in a DSS-induced colitis model. Visceral hyperalgesia was observed on day 7 but not on day 4. A TRPV1 antagonist and a 5-HT(3) receptor antagonist attenuated the visceral hyperalgesia to the control level. The alterations of 5-HT signaling via 5-HT(3) receptors and of TRPV1 channels in mucosa may contribute to the visceral hypersensitivity in colitis model mice.

Topics: Animals; Carbolines; Dextran Sulfate; Disease Models, Animal; Electromyography; Hyperalgesia; Inflammatory Bowel Diseases; Intestinal Mucosa; Male; Mice; Mice, Inbred C57BL; Microscopy, Confocal; Nociception; Pyrazines; Pyridines; Receptors, Serotonin, 5-HT3; Receptors, Serotonin, 5-HT4; Serotonin; Serotonin Antagonists; Time Factors; TRPV Cation Channels; Tryptophan Hydroxylase; Visceral Afferents

The transient receptor potential vanilloid 1 (TRPV1) receptor is activated by noxious heat, various endogenous mediators and exogenous irritants. The aim of the present study was to compare three TRPV1 receptor antagonists (SB705498, BCTC and AMG9810) in rat models of heat hyperalgesia. The behavioural noxious heat threshold, defined as the lowest temperature evoking nocifensive reaction, was measured with an increasing-temperature water bath. The effects of TRPV1 receptor antagonists were assessed in thermal hyperalgesia induced by the TRPV1 agonist resiniferatoxin (RTX), mild heat injury (51 degrees C, 20s) or plantar incision in rats. The control heat threshold was 43.2+/-0.4 degrees C. RTX induced an 8-10 degrees C decrease in heat threshold which was dose-dependently inhibited by oral pre-treatment with any of the TRPV1 receptor antagonists with a minimum effective dose of 1mg/kg. The mild heat injury-evoked 7-8 degrees C heat threshold drop was significantly reversed by all three antagonists injected i.p. as post-treatment. The minimum effective doses were as follows: SB705498 10, BCTC 3 and AMG9810 1mg/kg. Plantar incision-induced heat threshold drop (7-8 degrees C) was dose-dependently diminished by an oral post-treatment with any of the antagonists with minimum effective doses of 10, 3 and 3mg/kg, respectively. Assessment of RTX hyperalgesia by measurement of the paw withdrawal latency with a plantar test apparatus yielded 30 mg/kg minimum effective dose for each antagonist. In conclusion, measurement of the noxious heat threshold with the increasing-temperature water bath is suitable to sensitively detect the effects of TRPV1 receptor antagonists in thermal hyperalgesia models.

Topics: Acrylamides; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cold Temperature; Disease Models, Animal; Diterpenes; Dose-Response Relationship, Drug; Female; Hot Temperature; Hyperalgesia; Pain; Pyrazines; Pyridines; Pyrrolidines; Rats; Rats, Sprague-Dawley; TRPV Cation Channels; Urea

A series of 1,2,3,6-tetrahydropyridyl-4-carboxamides, exemplified by 6, have been synthesized and evaluated for in vitro TRPV1 antagonist activity, and in vivo analgesic activity in animal pain models. The tetrahydropyridine 6 is a novel TRPV1 receptor antagonist that potently inhibits receptor-mediated Ca2+ influx in vitro induced by several agonists, including capsaicin, N-arachidonoyldopamine (NADA), and low pH. This compound penetrates the CNS and shows potent anti-nociceptive effects in a broad range of animal pain models upon oral dosing due in part to its ability to antagonize both central and peripheral TRPV1 receptors. The SAR leading to the discovery of 6 is presented in this report.

Topics: Administration, Oral; Analgesics; Animals; Arachidonic Acids; Calcium; Capsaicin; Disease Models, Animal; Dopamine; Dose-Response Relationship, Drug; Hydrogen-Ion Concentration; Hyperalgesia; Pain Measurement; Pyridines; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; TRPV Cation Channels

Rats with experimental colitis suffer from impaired gastric emptying (GE). We previously showed that this phenomenon involves afferent neurons within the pelvic nerve. In this study, we aimed to identify the mediators involved in this afferent hyperactivation. Colitis was induced by trinitrobenzene sulfate (TNBS) instillation. We determined GE, distal front, and geometric center (GC) of intestinal transit 30 min after intragastric administration of a semiliquid Evans blue solution. We evaluated the effects of the transient receptor potential vanilloid type 1 (TRPV1) antagonists capsazepine (5-10 mg/kg) and N-(4-tertiarybutylphenyl)-4-(3-cholorphyridin-2-yl)tetrahydropyrazine-1(2H)carboxamide (BCTC; 1-10 mg/kg) and the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8-37) (150 microg/kg). To determine TRPV1 receptor antagonist sensitivity, we examined their effect on capsaicin-induced relaxations of isolated gastric fundus muscle strips. Immunocytochemical staining of TRPV1 and RT-PCR analysis of TRPV1 mRNA were performed in dorsal root ganglion (DRG) L6-S1. TNBS-induced colitis reduced GE but had no effect on intestinal motility. Capsazepine reduced GE in controls but had no effect in rats with colitis. At doses that had no effects in controls, BCTC and CGRP-(8-37) significantly improved colitis-induced gastroparesis. Capsazepine inhibited capsaicin-induced relaxations by 35% whereas BCTC completely abolished them. TNBS-induced colitis increased TRPV1-like immunoreactivity and TRPV1 mRNA content in pelvic afferent neuronal cell bodies in DRG L6-S1. In conclusion, distal colitis in rats impairs GE via sensitized pelvic afferent neurons. We provided pharmacological, immunocytochemical, and molecular biological evidence that this sensitization is mediated by TRPV1 receptors and involves CGRP release.

Topics: Animals; Calcitonin Gene-Related Peptide; Calcitonin Gene-Related Peptide Receptor Antagonists; Capsaicin; Colitis; Disease Models, Animal; Dose-Response Relationship, Drug; Ganglia, Spinal; Gastric Emptying; Gastrointestinal Motility; Gastroparesis; Intestinal Mucosa; Intestines; Male; Muscle Relaxation; Neurons, Afferent; Peptide Fragments; Pyrazines; Pyridines; Rats; Rats, Wistar; Receptors, Calcitonin Gene-Related Peptide; RNA, Messenger; Signal Transduction; Time Factors; Trinitrobenzenesulfonic Acid; TRPV Cation Channels

Cold allodynia is a frequent clinical symptom of patients with neuropathic pain. Despite numerous studies of cold allodynia, using animal models of neuropathic pain, little is known about its underlying mechanisms. This study was performed to establish a method for the pharmacologic evaluation of cold allodynia using several analgesics in a chronic constriction injury (CCI) rat model of neuropathic pain. Compared with the results obtained before the CCI operation, the CCI rats placed on a cork plate at 20 degrees C exhibited a slight change in the paw withdrawal latency because of the mechanical stimulus mediated by the injured paw touching the plate. By contrast, there was a significant reduction in the paw withdrawal latency on a cold metal plate compared with that on the cork plate after the CCI surgery, with the maximum decrease occurring on postoperative day 7. This reduction is thought to specifically reflect cold-induced pain behavior. In addition, both naïve and CCI rats showed behavioral changes at 5 and 0 degrees C, but not at 10 degrees C or higher. Interestingly, a subcutaneous morphine dose of 6 mg/kg completely inhibited cold allodynia induced at 10 degrees C on postoperative day 7. Under this condition, both the sodium channel blocker mexiletine (10 and 30 mg/kg, subcutaneously) and the calcium channel alpha2delta subunit blocker pregabalin (30 and 100 mg/kg, orally) significantly suppressed cold allodynia. Additionally, both resiniferatoxin (0.3 mg/kg, subcutaneously), an ultrapotent analog of capsaicin that desensitizes C fibers, and the VR1 channel antagonist N-(4-tertiarybutylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide (10 and 30 mg/kg, orally) significantly prolonged the paw withdrawal latency. In conclusion, our data suggest that the activation of C fibers mediates cold allodynia.

Topics: Analgesics, Opioid; Animals; Behavior, Animal; Calcium Channel Blockers; Chronic Disease; Cold Temperature; Constriction, Pathologic; Disease Models, Animal; Diterpenes; gamma-Aminobutyric Acid; Male; Mexiletine; Morphine; Nerve Fibers, Unmyelinated; Pain; Pain Measurement; Peripheral Nervous System Diseases; Pregabalin; Pyrazines; Pyridines; Rats; Rats, Sprague-Dawley; Reaction Time; Sciatic Nerve; Sodium Channel Blockers; TRPV Cation Channels

RNA interference (RNAi) has proven to be a powerful technique to study the function of genes by producing knock-down phenotypes. Here, we report that intrathecal injection of an siRNA against the transient receptor potential vanilloid receptor 1 (TRPV1) reduced cold allodynia of mononeuropathic rats by more than 50% over a time period of approximately 5 days. A second siRNA targeted to a different region of the TRPV1 gene was employed and confirmed the analgesic action of a TRPV1 knock-down. Furthermore, siRNA treatment diminished spontaneous visceral pain behavior induced by capsaicin application to the rectum of mice. The analgesic effect of siRNA-mediated knockdown of TRPV1 in the visceral pain model was comparable to that of the low-molecular weight receptor antagonist BCTC. Our data demonstrate that TRPV1 antagonists, including TRPV1 siRNAs, have potential in the treatment of both, neuropathic and visceral pain.

Topics: Animals; Base Sequence; Capsaicin; Chlorocebus aethiops; Colitis; COS Cells; Disease Models, Animal; Humans; Male; Mice; Molecular Sequence Data; Nervous System Diseases; Pain; Pyrazines; Pyridines; Rats; RNA Interference; Sequence Alignment; Sequence Homology, Amino Acid; TRPV Cation Channels