John A. Hey

A novel, orally active CXCR1/2 receptor antagonist, sch527123, inhibits neutrophil recruitment, mucus production, and goblet cell hyperplasia in animal models of pulmonary inflammation

Sch527123 [2-hydroxy-N,N-dimethyl-3-[[2-[[1(R)-(5-methyl-2-furanyl)propyl]amino]-3,4-dioxo-1-cyclobuten-1-yl]amino]ben-zamide] is a potent, selective antagonist of the human CXCR1 and CXCR2 receptors (Gonsiorek et al., 2007). Here we describe its pharmacologic properties at rodent CXCR2 and at the CXCR1 and CXCR2 receptors in the cynomolgus monkey, as well as its in vivo activity in models demonstrating prominent pulmonary neutrophilia, goblet cell hyperplasia, and mucus production. Sch527123 bound with high affinity to the CXCR2 receptors of mouse (Kd = 0.20 nM), rat (Kd = 0.20 nM), and cynomolgus monkey (Kd = 0.08 nM) and was a potent antagonist of CXCR2-mediated chemotaxis (IC50 ∼3–6 nM). In contrast, Sch527123 bound to cynomolgus CXCR1 with lesser affinity (Kd = 41 nM) and weakly inhibited cynomolgus CXCR1-mediated chemotaxis (IC50 ∼1000 nM). Oral treatment with Sch527123 blocked pulmonary neutrophilia (ED50 = 1.2 mg/kg) and goblet cell hyperplasia (32–38% inhibition at 1–3 mg/kg) in mice following the intranasal lipopolysaccharide (LPS) administration. In rats, Sch527123 suppressed the pulmonary neutrophilia (ED50 = 1.8 mg/kg) and increase in bronchoalveolar lavage (BAL) mucin content (ED50 =<0.1 mg/kg) induced by intratracheal (i.t.) LPS. Sch527123 also suppressed the pulmonary neutrophilia (ED50 = 1.3 mg/kg), goblet cell hyperplasia (ED50 = 0.7 mg/kg), and increase in BAL mucin content (ED50 = <1 mg/kg) in rats after i.t. administration of vanadium pentoxide. In cynomolgus monkeys, Sch527123 reduced the pulmonary neutrophilia induced by repeat bronchoscopy and lavage (ED50 = 0.3 mg/kg). Therefore, Sch527123 may offer benefit for the treatment of inflammatory lung disorders in which pulmonary neutrophilia and mucus hypersecretion are important components of the underlying disease pathology.

Central antitussive activity of the NK1 and NK2 tachykinin receptor antagonists, CP-99,994 and SR 48968, in the guinea-pig and cat.

The purpose of this study was to investigate the antitussive activity and sites of action of the NK1 and NK2 tachykinin receptor antagonists, CP‐99,994, SR 48968, and the racemate of SR 48968, SR 48212A in the cat and guinea‐pig. Guinea‐pigs were dosed subcutaneously (s.c.) with CP‐99,994, SR 48212A or SR 48968 one hour before exposure to aerosols of capsaicin (0.3 mM) to elicit coughing. Coughs were detected with a microphone and counted. Intracerebroventricular (i.c.v.) cannulae were placed in the lateral cerebral ventricles of anaesthetized guinea‐pigs. Approximately one week later, the animals were dosed with CP‐99,994 or SR 48212A (i.c.v.) and exposed to aerosols of capsaicin (0.3 mM) to elicit coughing. Cough was produced in anaesthetized cats by mechanical stimulation of the intrathoracic trachea and was monitored from electromyograms of respiratory muscle activity. Cannulae were placed for intravenous (i.v.) or, in separate groups of animals, intravertebral arterial (i.a.) administration of CP‐99,994, SR 48212A or SR 48968. Dose‐response relationships for i.v. and i.a. administration of each drug were generated to determine a ratio of i.v. ED50 to i.a. ED50, known as the effective dose ratio (EDR). The EDR will be 20 or greater for a centrally active drug and less than 20 for a peripherally active drug. In the guinea‐pig, CP‐99,994 (0.1–30 mg kg−1, s.c.), SR 48212A (1.0–30 mg kg−1, s.c.), and SR 48968 (0.3–3.0 mg kg−1, s.c.) inhibited capsaicin‐induced cough in a dose‐dependent manner. Capsaicin‐induced cough was also inhibited by i.c.v. administration of CP‐99,994 (10 and 100 μg) or SR 48212A (100 μg). In the cat, both CP‐99,994 (0.0001–0.3 mg kg−1, i.a., n=5; 0.003–3.0 mg kg−1, i.v., n=5) and SR 48212A (0.003–1.0 mg kg−1, i.a., n=5; 0.01–3.0 mg kg−1, i.v., n=5) inhibited mechanically induced cough by either the i.v. or i.a. routes in a dose‐dependent manner. SR 48968 (0.001–0.3 mg kg−1, i.a., n=5; 0.03–1.0 mg kg−1, i.v., n=5) inhibited cough when administered by the i.a. route in a dose‐dependent manner, but had no effect by the i.v. route up to a dose of 1.0 mg kg−1. Intravenous antitussive potencies (ED50, 95% confidence interval (CI)) of these compounds were: CP‐99,994 (0.082 mg kg−1, 95% CI 0.047–0.126), SR 48212A (2.3 mg kg−1, 95% CI 0.5–20), and SR 48968 (>1.0 mg kg−1, 95% CI not determined). The intra‐arterial potencies of these compounds were: CP‐99,994 (1.0 μg kg−1, 95% CI 0.4–1.8), SR 48212A (25 μg kg−1, 95% CI 13–52), and SR 48968 (8.0 μg kg−1, 95% CI 1–32). The derived EDRs for each compound were: CP‐99,994, 82; SR 48212A, 92; and SR 48968, >125. We concluded that CP‐99,994 and SR 48968 inhibit cough in the guinea‐pig and cat by a central site of action. In the cat, the antitussive action of these compounds appears to be solely by a central site.

Nociceptin inhibits cough in the guinea‐pig by activation of ORL1 receptors

We studied the central and peripheral antitussive effect of ORL1 receptor activation with nociceptin/orphanin FQ in conscious guinea‐pigs. In guinea‐pig cough studies, nociceptin/orphanin FQ (10, 30, and 90 μg) given directly into the CNS by an intracerebroventricular (i.c.v.) route inhibited cough elicited by capsaicin exposure by approximately 23, 29 and 52%, respectively. The antitussive activity of nociceptin/orphanin FQ (90 μg, i.c.v.) was blocked by the selective ORL1 antagonist [Phe1γ(CH2‐NH)Gly2]nociceptin‐(1‐13)‐NH2 (180 μg, i.c.v.) and J113397 (10 mg kg−1, i.p.) but not by the opioid antagonist, naltrexone (3 mg kg−1, i.p.). Furthermore, intravenous (i.v.) nociceptin/orphanin FQ (1.0 and 3.0 mg kg−1) also inhibited cough approximately by 25 and 42%, respectively. These findings indicate that selective ORL1 agonists display the potential to inhibit cough by both a central and peripheral mechanism, and potentially represent a novel therapeutic approach for the treatment of cough.

Peripheral and central sites of action of GABA-B agonists to inhibit the cough reflex in the cat and guinea pig.

1 The GABA‐B receptor agonists baclofen and 3‐aminopropylphosphinic acid (3‐APPi) have antitussive activity in the cat and guinea pig. The purpose of this study was to investigate the sites of action of these GABA‐B receptor agonists to inhibit the cough reflex. 2 Single intracerebroventricular (i.e.v.) cannulas were placed in the lateral ventricles of anaesthetized guinea pigs. Approximately 1 week later, the animals were exposed to aerosols of capsaicin (0.3 μm) to elicit coughing. Coughs were detected with a microphone and counted. 3 Cough was produced in anaesthetized cats by mechanical stimulation of the intrathoracic trachea and was recorded from electromyograms of respiratory muscle activity. Cannulas were placed for intravenous (i.v.) or, in separate groups of animals, intravertebral arterial (i.a.) administration of baclofen, 3‐APPi, the centrally active antitussive drug codeine or the peripherally active antitussive drug BW443c. Dose‐response relationships for i.v. and i.a. administration of each drug were generated to determine a ratio of i.v. ED50 to i.a. ED50, known as the effective dose ratio (EDR). The EDR will be 20 or greater for a centrally acting drug. 4 In the guinea pig, baclofen (3 mg kg−1, s.c.) and 3‐APPi (10 mg kg−1, s.c.) inhibited capsaicin‐induced cough by 50% and 35% respectively. The antitussive activity of baclofen was completely blocked by i.e.v. administration of the GABA‐B receptor antagonist CGP 35348 (10 μg). Conversely, the antitussive effect of 3‐APPi was unaffected by i.e.v. CGP 35348. However, systemic administration of CGP 35348 (30 mg kg−1, s.c.) completely blocked the antitussive activity of 3‐APPi (10 mg kg−1, s.c). In separate experiments baclofen alone (1 μg, i.c.v.) inhibited capsaicin‐induced cough by 78%. 3‐APPi (10 and 100 μg, i.c.v.) had no effect on capsaicin‐induced cough in the guinea pig. 5 In the cat, potencies (ED50) of the standards and GABA‐B agonists by the i.v. route were: codeine (0.34 mg kg−1), BW443C (0.17 mg kg−1), baclofen (0.63 mg kg−1) and 3‐APPi (2.3 mg kg−1). Potencies of these drugs by the i.a. route were: codeine, 0.013 mg kg−1; BW443C, 0.06 mg kg−1; baclofen, 0.016 mg kg−1; and 3‐APPi, 0.87 mg kg−1. The EDRs for each drug were: codeine, 26; BW443C, 3; baclofen, 39; and 3‐APPi, 3. 6 We conclude that in both the cat and guinea pig baclofen inhibits cough by a central site of action, while 3‐APPi inhibits cough by a peripheral site of action.

Combined histamine H1 and H3 receptor blockade produces nasal decongestion in an experimental model of nasal congestion.

We studied the pharmacological actions of combined histamine H1/H3 receptor blockade on the increase in nasal airway resistance (NAR) and decrease in nasal cavity volume produced by nasal exposure to compound 48/80, a mast cell degranulator. In the anesthetized cat compound 48/80 (1%) produced a maximum increase in NAR of 9.1 ± 0.7 cmH20·L/minute. The increase in NAR in animals pretreated with a combination of the H1 antagonist, chlorpheniramine (CTM; 0.8 mg/kg i.v.) and increasing doses of the H3 antagonist, thioperamide (THIO; 1.0, 3.0, and 10.0 mg/kg i.v.) were 6.1 ± 2.1, 4.2 ± 1.0 and 2.2 ± 0.7 cmH20·L/minute, respectively. A second H3 antagonist, clobenpropit (CLOB; 0.03, 0.3, and 1.0 mg/kg i.v.) combined with CTM (0.8 mg/kg i.v.) also inhibited the nasal effects of compound 48/80. When the nonsedating H1 antihistamine, loratadine (3.0 mg/kg i.v.), was substituted for CTM, it also reduced nasal congestion when given in combination with THIO (10 mg/kg i.v.). In contrast, treatment with CTM (1.0 mg/kg i.v.) and the H2 antagonist, ranitidine (RAN; 1.0 mg/kg i.v.) were without activity. Loratadine, CTM, CLOB, RAN, or THIO administered alone were inactive. The α-adrenergic agonist, phenylpropanolamine (PPA; 1.0 mg/kg i.v.) demonstrated decongestant effects, but in contrast to H1/H3 blockade, PPA produced a significant hypertensive effect. Using acoustic rhinometry (AcR) we found that combined i.v. CTM (1.0 mg/kg) and THIO (10 mg/kg) and combined oral CTM (10 mg/kg) and THIO (30 mg/kg) blocked the decrease in nasal cavity volume produced by intranasal compound 48/80 (1%, 50 μL). We conclude that combined H1/H3 histamine receptor blockade enhances the efficacy of an H1 antagonist by conferring decongestant activity to the H1 antihistamine. We propose that the decongestant activity of combined H1/H3 blockade may provide a novel approach for the treatment of allergic nasal congestion without the hypertensive liability of current therapies.

Anandamide induces cough in conscious guinea‐pigs through VR1 receptors

Endogenous neuronal lipid mediator anandamide, which can be synthesized in the lung, is a ligand of both cannabinoid (CB) and vanilloid receptors (VR). The tussigenic effect of anandamide has not been studied. The current study was designed to test the direct tussigenic effect of anandamide in conscious guinea‐pigs, and its effect on VR1 receptor function in isolated primary guinea‐pig nodose ganglia neurons. Anandamide (0.3–3 mg·ml−1), when given by aerosol, induced cough in conscious guinea‐pigs in a concentration dependent manner. When guinea‐pigs were pretreated with capsazepine, a VR1 antagonist, the anandamide‐induced cough was significantly inhibited. Pretreatment with CB1 (SR 141716A) and CB2 (SR 144528) antagonists had no effect on anandamide‐induced cough. These results indicate that anandamide‐induced cough is mediated through the activation of VR1 receptors. Anandamide (10–100 μM) increased intracellular Ca2+ concentration estimated by Fluo‐4 fluorescence change in isolated guinea‐pig nodose ganglia cells. The anandamide‐induced Ca2+ response was inhibited by two different VR1 antagonists: capsazepine (1 μM) and iodo‐resiniferatoxin (I‐RTX, 0.1 μM), indicating that anandamide‐induced Ca2+ response was through VR1 channel activation. In contrast, the CB1 (SR 141716A, 1 μM) and CB2 (SR 144528, 0.1 μM) receptor antagonists had no effect on Ca2+ response to anandamide. In conclusion, these results provide evidence that anandamide activates native vanilloid receptors in isolated guinea‐pig nodose ganglia cells and induces cough through activation of VR1 receptors.

GABAB receptors in the lung

gamma-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in the mammalian CNS, is also found in peripheral tissues, including the lung. Recent pharmacological studies using selective ligands for GABAA and GABAB receptors demonstrate that of these two, the GABAB receptor is the important receptor subtype controlling lung functions. GABAB agonists inhibit a variety of responses in the airways, including neuronally induced cholinergic- and tachykinin-mediated smooth muscle contraction, microvascular leakage, anaphylactic bronchospasm and cough. Because these conditions are seen in certain respiratory diseases, such as asthma, a selective GABAB agonist may have therapeutic potential for the treatment of this respiratory disorder.

Coordination of Histamine H3 Receptor Antagonists with Human Adrenal Cytochrome P450 Enzymes

Optical difference spectroscopy was used to identify and quantify human adrenal microsomal and mitochondrial cytochrome P450 enzyme interactions with the histamine H3 receptor antagonists thioperamide, clobenpropit and ciproxifan. Addition of these structurally diverse imidazole H3 receptor antagonists to cytochrome-P450-containing human adrenal microsomal and mitochondrial preparations resulted in concentration-dependent type II optical difference spectra. Respective spectral dissociation constants (KS) for the drug interactions with human adrenal microsomal and mitochondrial cytochrome P450 were 1.5 and 1.6 µmol/l for thioperamide, 3.1 and 0.28 µmol/l for clobenpropit and 0.10 and 0.11 µmol/l for ciproxifan. The three compounds demonstrated a similar activity profile in cytochrome-P450-containing bovine adrenal microsomal and mitochondrial preparations. Findings indicate direct coordination of these imidazole-containing H3 receptor antagonists with the heme moiety of human adrenal cytochrome P450 isozymes.

Inhibition of sympathetic hypertensive responses in the guinea-pig by prejunctional histamine H3-receptors

1 The effect of (R)‐α‐methylhistamine, a selective H3‐histamine receptor agonist, was examined on the neurogenic hypertension and tachycardia that is induced by stimulation of areas in the medulla oblongata of guinea‐pigs. Electrical medullary stimulation (32 Hz, 3–5 s trains, 0.5–1.0 ms square pulse, 25–400 μA) produced intensity‐dependent increases in blood pressure and a more variable tachycardia. 2 (R)‐α‐methylhistamine inhibited the hypertension and tachycardia due to submaximal CNS stimulation. The inhibition of hypertension by (R)‐α‐methylhistamine was dose‐dependent (10–300 μg kg−1, i.v.) and was not seen at high intensities of stimulation. 3 (R)‐α‐methylhistamine (300 μg kg−1, i.v.) did not attenuate the pressor response to adrenaline (1 and 3 μg kg−1, i.v.), indicating that the effect of (R)‐α‐methylhistamine was not mediated by a postjunctional action on smooth muscle. 4 The inhibition of CNS‐induced hypertension by (R)‐α‐methylhistamine (300 μg kg−1, i.v.) was blocked by the H3 antagonists, thioperamide (ID50 = 0.39 mg kg−1, i.v.), impromidine (ID50 = 0.22 mg kg−1, i.v.) and burimamide (ID50 = 6 mg kg−1, i.v.). The rank order potency of these antagonists is consistent with activity at the H3B receptor subtype. Chlorpheniramine (30 μg kg−1, i.v.) and cimetidine (3 mg kg−1, i.v.) did not antagonize the inhibition of CNS‐hypertension by (R)‐α‐methylhistamine. 5 These results suggest that (R)‐α‐methylhistamine inhibits sympathetic hypertensive responses in guinea‐pigs by activation of prejunctional H3‐receptors, possibly located on postganglionic nerve terminals. Furthermore, on the basis of the rank order potency to different H3‐antagonists, it appears that the H3B‐receptor subtype is involved with H3‐receptor responses on vascular sympathetic nerves.

Nociceptin inhibits capsaicin-induced bronchoconstriction in isolated guinea pig lung.

The isolated perfused guinea pig lung was used to investigate the effect of nociceptin against bronchoconstriction elicited by endogenous and exogenous tachykinins. The opioid receptor-like 1 (ORL1) receptor agonist, nociceptin/orphanin FQ (0.001-1 microM) produced a dose-related inhibition of the capsaicin-induced bronchoconstriction (10(-5)-10(3) microg) in isolated guinea pig lung (P<0.05), a response mediated by the release of endogenous tachykinins from lung sensory nerves. The new ORL1 receptor antagonist 1-[(3R, 4R)-1-Cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1, 3-dihydro-2H-benzimidazol-2-one (J-113397) (0.3 microM) significantly blocked the inhibitory effect of nociceptin/orphanin FQ (0.01 microM) on capsaicin-induced bronchoconstriction, whereas the non-selective opioid receptor antagonist naloxone (1 microM) had no effect. Nociceptin/orphanin FQ (1 microM) did not affect the bronchoconstriction induced exogenously by the tachykinin NK2 receptor agonist neurokinin A. In conclusion, the present data provide evidence that nociceptin inhibits capsaicin-evoked tachykinin release from sensory nerve terminals in guinea pig lung by a prejunctional mechanism. This inhibitory action occurs independently from activation of opioid receptors. The present study also indicates that J-113397 is a potent ORL1 receptor antagonist.