Xiomara Fernandez

TRPV1 antagonists attenuate antigen-provoked cough in ovalbumin sensitized guinea pigs

We examined the molecular pharmacology and in vivo effects of a TRPV1 receptor antagonist, N-(4-Tertiarybutylphenyl)-4(3-cholorphyridin-2-yl)-tetrahydro-pyrazine1(2H) – carboxamide (BCTC) on the guinea pig TRPV1 cation channel. BCTC antagonized capsaicin-induced activation and PMA-mediated activation of guinea pig TRPV1 with IC50 values of 12.2 ± 5.2 nM, and 0.85 ± 0.10 nM, respectively. In addition, BCTC (100 nM) completely blocked the ability of heterologously expressed gpTRPV1 to respond to decreases in pH. Thus, BCTC is able to block polymodal activation of gpTRPV1. Furthermore, in nodose ganglia cells, capsaicin induced Ca2+ influx through TRPV1 channel was inhibited via BCTC in a concentration dependent manner. In in vivo studies capsaicin (10 – 300 μM) delivered by aerosol to the pulmonary system of non-sensitized guinea pigs produced an increase in cough frequency. In these studies, the tussigenic effects of capsaicin (300 μM) were blocked in a dose dependent fashion when BCTC (0.01–3.0 mg/kg, i.p.) was administered 30 minutes before challenge. The high dose of BCTC (3.0 mg/kg, i.p) produced a maximum inhibition of capsaicin-induced cough of 65%. We also studied the effects of BCTC (0.03 and 3.0) when administered 60 minutes before capsaicin. Under these conditions, BCTC (3.0 mg/kg, i.p) produced a maximum decrease in capsaicin-induced cough of 31%. In ovalbumin passively sensitized guinea pigs, we found that BCTC (1 and 3 mg/kg, i.p.) attenuated antigen ovalbumin (0.3%) cough responses by 27% and 60%, respectively. We conclude that TRPV1 channel activation may play role in cough mediated by antigen in sensitized guinea pigs. Our results supports increasing evidence that TRPV1 may play a role in the generation of the cough response.

Antitussive Profile of the NOP Agonist Ro-64-6198 in the Guinea Pig

We have previously shown that N/OFQ, the endogenous peptide ligand for the ‘opioid-like’ NOP receptor, inhibits cough in guinea pigs and cats. In the present study we sought to continue our characterization of the cough-suppressant effects of NOP stimulation by profiling the pulmonary and antitussive effects of a novel non-peptide NOP agonist, Ro-64-6198, in guinea pigs. In receptor-binding assays, we confirmed that Ro-64-6198 selectively binds to NOP receptors over other opioid receptors. The Ki values for Ro-64-6198 at NOP, MOP, KOP and DOP receptors was 0.3, 36, 214 and 3,787 nmol/l, respectively. In GTPγS-binding assays, Ro-64-6198 displayed >900-fold functional selectivity at NOP relative to MOP receptors. We evaluated the effects of Ro-64-6198 (3 and 10 µmol/l) in isolated guinea pig nodose ganglia cells on the increases in intracellular Ca2+ concentration evoked by capsaicin stimulation (1 × 10–8–1 × 10–6 mol/l). Similar to previously reported data with N/OFQ, Ro-64-6198 (3 and 10 µmol/l) significantly attenuated Ca2+ responses in nodose ganglia cells produced by exposure to capsaicin. The effect of Ro-64-6198 (3 µmol/l) on capsaicin-induced intracellular Ca2+ responses was blocked by the NOP antagonist, J113397 (3 µmol/l). In guinea pig in vivo studies, aerosolized capsaicin (10–300 µmol/l) produced a dose-dependent increase in cough number. Ro-64-6198 given i.p. significantly inhibited cough due to capsaicin (300 µmol/l) exposure. In a duration study we found that the maximum antitussive effect (42 ± 8% inhibition) of Ro-64-6198 (3 mg/kg) was observed at 1 h after i.p. administration. Also at 1 h after administration, Ro-64-6198 (0.003–3.0 mg/kg, i.p.) produced a dose-dependent inhibition of cough. The antitussive effect of Ro-64-6198 (3 mg/kg, i.p.) was blocked by J113397 (12 mg/kg, i.p.) but not by the classical opioid antagonist naltrexone (10 mg/kg, i.p.). Although the antitussive action of Ro-64-6198 may be mediated by a central and/or a peripheral site of action, we hypothesize that selective oral NOP agonists that do not penetrate the blood-brain barrier may provide a novel approach for the treatment of cough. Moreover, because these drugs do not interact at MOP receptors, they may be devoid of codeine-like side effects such as respiratory depression, sedation, constipation or proclivities for addictive liabilities.

Inhibition of Spleen Tyrosine Kinase Attenuates Allergen-Mediated Airway Constriction

Spleen tyrosine kinase (SYK) is a key activator of signaling pathways downstream of multiple surface receptors implicated in asthma. SYK function has been extensively studied in mast cells downstream of the high-affinity IgE receptor, FcεR1. Preclinical studies have demonstrated a role for SYK in models of allergic inflammation, but a role in airway constriction has not been demonstrated. Here, we have used a potent and selective pharmacological inhibitor of SYK to determine the role of SYK in allergen-mediated inflammation and airway constriction in preclinical models. Attenuation of allergic airway responses was evaluated in a rat passive anaphylaxis model and rat and sheep inhaled allergen challenge models, as well as an ex vivo model of allergen-mediated airway constriction in rats and cynomolgus monkeys. Pharmacological inhibition of SYK dose-dependently blocked IgE-mediated tracheal plasma extravasation in rats. In a rat ovalbumin-sensitized airway challenge model, oral dosing with an SYK inhibitor led to a dose-dependent reduction in lung inflammatory cells. Ex vivo analysis of allergen-induced airway constriction in ovalbumin-sensitized brown Norway rats showed a complete attenuation with treatment of a SYK inhibitor, as well as a complete block of allergen-induced serotonin release. Similarly, allergen-mediated airway constriction was attenuated in ex vivo studies from nonhuman primate lungs. Intravenous administration of an SYK inhibitor attenuated both early- and late-phase allergen-induced increases in airway resistance in an Ascaris-sensitive sheep allergen challenge model. These data support a key role for SYK signaling in mediating allergic airway responses.

Pharmacological profile of the NOP agonist and cough suppressing agent SCH 486757 (8-[Bis(2-Chlorophenyl)Methyl]-3-(2-Pyrimidinyl)-8-Azabicyclo[3.2.1]Octan-3-Ol) in preclinical models

We describe the pharmacological and pharmacokinetic profiles of SCH 486757, a nociceptin/orphanin FQ peptide (NOP) receptor agonist that has recently entered human clinical trials for cough. SCH 486757 selectively binds human NOP receptor (K(i)=4.6+/-0.61nM) over classical opioid receptors. In a guinea pig capsaicin cough model, SCH 486757 (0.01-1mg/kg) suppressed cough at 2, 4, and 6h post oral administration with a maximum efficacy occurring at 4h equivalent to codeine, hydrocodone, dextromethorphan and baclofen. The antitussive effects of SCH 486757 (3.0mg/kg, p.o.) was blocked by the NOP receptor antagonist J113397 (12mg/kg, i.p.) but not by naltrexone (10mg/kg, p.o.). SCH 486757 does not produce tolerance to its antitussive activity after a 5-day BID dosing regimen. After acute and chronic dosing paradigms, SCH 486757 (1mg/kg) inhibited capsaicin-evoked coughing by 46+/-9% and 40+/-11%, respectively. In a feline mechanically-evoked cough model, SCH 486757 produces a maximum inhibition of cough and expiratory abdominal electromyogram amplitude of 59 and 61%, respectively. SCH 486757 did not significantly affect inspiratory electromyogram amplitude. We examined the abuse potential of SCH 486757 (10mg/kg, p.o.) in a rat conditioned place preference procedure which is sensitive to classical drugs of abuse, such as amphetamine and morphine. SCH 486757 was without effect in this model. Finally, SCH 486757 displays a good oral pharmacokinetic profile in the guinea pig, rat and dog. We conclude that SCH 486757 has a favorable antitussive profile in preclinical animal models.

Inhibitory activity of nociceptin/orphanin FQ on capsaicin-induced bronchoconstriction in the guinea-pig.

In vivo studies were conducted in the guinea-pig to investigate the activity of the selective ORL1 receptor agonist nociceptin/orphanin FQ against capsaicin-induced bronchoconstriction, a response mediated by the release of tachykinins from pulmonary sensory nerves. Anesthetized guinea-pigs were ventilated with a rodent ventilator and placed in a whole-body plethysmograph, and pulmonary resistance (R(L)) and dynamic lung compliance (C(Dyn)) were monitored. Intravenous administration of nociceptin/orphanin FQ (0.3 mg/kg) inhibited the capsaicin-induced bronchoconstriction. The new nonpeptide ORL1 receptor antagonist 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) administered intravenously (1 mg/kg) produced a significant blockade of the inhibitory effect of nociceptin/orphanin FQ (0.3 mg/kg) on capsaicin-induced bronchoconstriction, whereas the nonselective opioid receptor antagonist naloxone (1 mg/kg) had no effect. Nociceptin/orphanin FQ (0.3 mg/kg) did not affect the bronchoconstriction induced exogenously by the tachykinin NK2 receptor agonist [beta-ala8]-neurokinin A (4-10). We conclude that nociceptin inhibits in vivo capsaicin-evoked tachykinin release from sensory nerve terminals in the guinea-pig by a prejunctional mechanism. This inhibitory action does not involve activation of opioid receptors.

The discovery of tropane derivatives as nociceptin receptor ligands for the management of cough and anxiety

The discovery of 1 as a high-affinity ligand for the nociceptin receptor has led to the synthesis of a series of tropane (8-methyl-8-azabicyclo[3.2.1]octane) derivatives as optimized ligands. These compounds exhibit high affinity for the nociceptin receptor, moderate to excellent selectivity over the opioid mu receptor, and behave as full agonists. In this Letter, we present the synthesis and highlight the structure-activity relationship of tropane derivatives culminating in the identification of 24 and 32 as potent and orally active antitussive and anxiolytic agents. The in vitro and in vivo activities, pharmacokinetic profile, and the hPXR activity, which predicts the potential 3A4 induction in human, are disclosed.

Discovery of orally active 3-pyridinyl-tropane as a potent nociceptin receptor agonist for the management of cough.

A series of 3-pyridinyl-tropane analogues based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. From the SAR study and our lead optimization efforts, compound 10 was found to possess potent oral antitussive activity in the capsaicin-induced guinea pig model. The rationale for compound selection and the biological profile of the optimized lead (10) are disclosed.

Identification of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the management of cough and anxiety.

A series of nortropane analogs based on previously reported compound 1 have been synthesized and shown to bind to the nociceptin receptor with high affinity. The synthesis and structure-activity relationships around the C-3 nortropane substitution are described. From the SAR study and hPXR screening effort, compound 15 was identified to possess potent oral antitussive and anxiolytic-like activities in the guinea pig models.

Structure-activity relationships of 3-substituted N-benzhydryl-nortropane analogs as nociceptin receptor ligands for the treatment of cough

A series of 3-axial-aminomethyl-N-benzhydryl-nortropane analogs have been synthesized and identified to bind to the nociceptin receptor with high affinity. Many of these analogs showed high binding selectivity over classic opioid receptors such as mu receptor. The synthesis and structure-activity relationships around the C-3 nortropane substitution are described. Selected compounds with potent oral antitussive activity in the guinea pig model are disclosed.

Effect of a novel NOP receptor agonist (SCH 225288) on guinea pig irritant-evoked, feline mechanically induced and canine infectious tracheobronchitis cough.

Background: Previous studies have demonstrated that nociceptin/orphanin FQ (N/OFQ), the endogenous peptide ligand for the G-protein-coupled NOP receptor, inhibits cough in experimental models. SCH 225288 is a nonpeptide, orally active NOP agonist that may provide the foundation for the development of novel treatments for cough. Methods: First we characterized the selectivity of SCH 225288 in human receptor binding assays. Afterwards, the antitussive activity of SCH 225288 was studied in three mechanistically distinct cough models. Specifically, we observed the cough-suppressant effect of SCH 225288 in a guinea pig capsaicin irritant-evoked cough model, a feline mechanically induced cough model and finally in a canine Bordetella bronchiseptica disease model. Results: SCH 225288 selectively binds human NOP receptor (Ki = 0.38 ± 0.02 nmol/l) over classical opioid receptors (COR). In a guinea pig capsaicin cough model, SCH 225288 (0.1–1 mg/kg) suppressed cough at 2, 4, and 6 h after oral administration. The antitussive effect of SCH 225288 (3.0 mg/kg, p.o.) was blocked by the NOP antagonist J113397 (12 mg/kg, i.p.) but not by the classical opioid receptor (COR) antagonist, naltrexone (3.0 mg/kg, i.p.). In the anesthetized cat, we evaluated the effects of SCH 225288 given either intravenously or via the intravertebral artery against the increases in cough number and respiratory expiratory and inspiratory muscle (rectus abdominis and parasternal) electromyographic (EMG) activities due to perturbations of the intrathoracic trachea. SCH 225288 (0.03–3.0 mg/kg, i.v.) inhibited both cough number and abdominal EMG amplitudes. Similarly, SCH 225288 (0.001–0.3 mg/kg) administered intra-arterially also diminished cough number and abdominal EMG amplitudes. No significant effect of the drug was noted on parasternal EMG activity. Finally, we studied the antitussive actions of SCH 225288 (1.0 mg/kg) in a canine B. bronchiseptica disease model. In this model, dogs were challenged intranasally with B. bronchiseptica. Comparisons were made between a vehicle group, an SCH 225288 (1.0 mg/kg, p.o., q.d.) and a butorphanol (0.6 mg/kg, p.o., b.i.d.) group on the mean change in cough scores from baseline values and days 6–9 after B. bronchiseptica challenge. SCH 225288 (1.0 mg/kg, p.o., q.d.) displayed a positive antitussive tendency (p = 0.06) to inhibit B. bronchiseptica cough whereas butorphanol (0.6 mg/kg, p.o., b.i.d.) was devoid of antitussive activity. Conclusions: Taken together, the present data show that SCH 225288 is a potent and effective antitussive agent in animal models of cough. Furthermore, these findings indicate that NOP agonists represent a promising new therapeutic approach for the treatment of cough without the side effect liabilities associated with opioid antitussives.