William Kreutner

Characterization of a murine model of allergic pulmonary inflammation.

Pulmonary inflammation with eosinophil (EOs) infiltration is a prominent feature of allergic respiratory diseases such as asthma. In order to study the cellular response during the disease development, an animal model of IgE-mediated pulmonary inflammation with characteristic eosinophilia is needed. We developed a method for inducing severe pulmonary eosinophilia in the mouse and also studied the numbers of EOs in blood and bone marrow and the response to corticosteroid treatment. Animals were sensitized with alum-precipitated ovalbumin (OVA) and challenged with aerosolized OVA 12 days later when serum IgE levels were significantly elevated. Four to eight hours after challenge there were moderate increases in the number of EOs in the bone marrow and peripheral blood, but only a few EOs were observed in the lung tissue and in bronchoalveolar lavage (BAL) fluid. Twenty-four hours after challenge, there was a marked reduction of EOs in bone marrow, while the number of EOs peaked in the perivascular and peribronchial regions of the lung. Forty-eight hours after challenge, the highest number of EOs was found in the BAL fluid, making up > 80% of all cells in that compartment. The high levels of EOs in the lung tissue and BAL fluid lasted for 2-3 days and was followed by a more moderate but persistent eosinophilia for another 10 days. Nonsensitized animals showed no significant changes in the number of EOs in BAL fluid, lungs, blood or bone marrow. Histopathological evaluation also revealed epithelial damage, excessive mucus in the lumen and edema in the submucosa of the airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical characterization of desloratadine, a potent antagonist of the human histamine H1 receptor

We have characterized desloratadine (5H-benzo[5,6]cyclohepta[1,2-b]pyridine, 8-chloro-6,11-dihydro-11-(4-piperidinylidene), CAS 100643-71-8) as a potent antagonist of the human histamine H(1) receptor. [3H]Desloratadine bound to membranes expressing the recombinant human histamine H(1) receptor in Chinese hamster ovary cells (CHO-H(1)) in a specific and saturable manner with a K(d) of 1.1+/-0.2 nM, a B(max) of 7.9+/-2.0 pmol/mg protein, and an association rate constant of 0.011 nM(-1) x min(-1). The K(d) calculated from the kinetic measurements was 1.5 nM. Dissociation of [3H]desloratadine from the human histamine H(1) receptor was slow, with only 37% of the binding reversed at 6 h in the presence of 5 microM unlabeled desloratadine. Seventeen histamine H(1)-receptor antagonists were evaluated in competition-binding studies. Desloratadine had a K(i) of 0.9+/-0.1 nM in these competition studies. In CHO-H(1) cells, histamine stimulation resulted in a concentration-dependent increase in [Ca(2+)](i) with an EC(50) of 170+/-30 nM. After a 90-min preincubation with desloratadine, the histamine-stimulated increase in [Ca(2+)](i) was shifted to the right, with a depression of the maximal response at higher concentrations of antagonist. The apparent K(b) value was 0.2+/-0.14 nM with a slope of 1.6+/-0.1. The slow dissociation from the receptor and noncompetitive antagonism suggests that desloratadine may be a pseudoirreversible antagonist of the human histamine H(1) receptor. The mechanism of desloratadine antagonism of the human histamine H(1) receptor may help to explain the high potency and 24-h duration of action observed in clinical studies.

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.

Evaluation of the CNS properties of SCH 29851, a potential non-sedating antihistamine.

SCH 29851 [8-chloro[6,11-dihydro-11-(1-carboethoxy-4-piperidylidene)-5-H-benzo[5,6]cyclohepta[1,2-b]-pyridine] was discovered as part of a search for a new antihistamine without effects on the central nervous system (CHS). Antihistaminic potency and duration of action of SCH 29851 and other antihistamines were assessed by inhibition of histamine-induced lethality in guinea pigs and histamine-induced paw edema in mice. Evaluation of possible CNS effects included gross observation of mice, rats, dogs and monkeys, prevention of electroshock-induced convulsions, acetic acid-induced writhing and physostigmine-induced lethality in mice and biochemical measures related to sedative liability such as displacement ofin vivo3H-mepyramine binding in mouse brain andin vitro3H-WB 4101 binding in guinea pig cortex. Comparisons were made to several antihistamines considered to be sedative to varying degrees, including diphenhydramine, promethazine, chlorpheniramine and azatadine and to the newer antihistamines terfenadine and astemizole which are reported to be non-sedating in man at doses that antagonize the effects of histamine peripherally.SCH 29851 had antihistamine activity in the tests used with a potency at least comparable to most standards and was devoid of activity in all the functional and biochemical models used as indices of CNS activity. It is expected that SCH 29851 should be an effective, long acting, antihistamine in man without sedative effects at therapeutic doses.

Antiallergic activity of loratadine, a non-sedating antihistamine.

Loratadine is a new non‐sedating antihistamine. The present studies compared loratadine and terfenadine, another non‐sedating antihistamine, for their ability to inhibit the bronchial response to histamine and other autacoids which have been implicated as contributing to the symptoms of an allergic reaction. In addition, the two antihistamines were evaluated in models of immunologically mediated allergic reactions. Loratadine is a more potent inhibitor of histamine‐induced bronchospasm in guinea pigs than is terfenadine. Both antihistamines exhibit marked antiserotonin activity at doses 10 times their antihistamine ED50 values. In contrast, loratadine and terfenadine produce little or no inhibition of the bronchial responses to methacholine, leukotriene C4 or platelet‐activating factor. An allergic bronchospasm in guinea pigs is inhibited by loratadine (ED50= 0.40 mg/kg, p.o.) and terfenadine (ED50= 1.7 mg/kg, p.o.). The bronchospasm associated with allergic anaphylaxis in rats is significantly inhibited by 10 mg/kg, p.o. loratadine and 30 mg/kg, p.o. terfenadine. Loratadine exhibits antiallergy activity in vitro. At micromolar concentrations, loratadine inhibits the release of histamine from Con A and A23187‐stimulated rat peritoneal mast cells and the release of histamine and leukotrine C4 from a Con A‐stimulated cloned murine mast cell line

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.

Antitussive effects of GABAB agonists in the cat and guinea-pig.

1 GABAB agonists inhibit neuronal processes which are important in the pathogenesis of airway disease, such as bronchospasm. Cough is a prominent symptom of pulmonary disease, but the effects of GABAB agonists on this airway reflex are unknown. Experiments were conducted to determine the antitussive effect of GABAB receptor agonists in comparison to the known antitussive agents, codeine and dextromethorphan. 2 Unanaesthetized guinea‐pigs were exposed to aerosols of 0.3 mm capsaicin to elicit coughing, which was detected with a microphone and counted. Cough also was produced in anaesthetized cats by mechanical stimulation of the intrathoracic trachea and was recorded from electromyograms of respiratory muscle activity. 3 In guinea‐pigs, the GABAB agonists baclofen and 3‐aminopropyl‐phosphinic acid (3‐APPi) produced dose‐dependent inhibition of capsaicin‐induced cough when administered by subcutaneous or inhaled routes. The potencies of baclofen and 3‐APPi compared favourably with codeine and dextromethorphan. 4 The GABAB antagonist, CGP 35348 (0.3– 30 mg kg−1, s.c.) inhibited the antitussive effect of baclofen (3.0 mg kg−1, s.c). However, CGP 35348 (10 mg kg−1, s.c.) had no effect on the antitussive activity of codeine (30 mg kg−1, s.c). The antitussive effect of baclofen was not influenced by the GABAA antagonist, bicuculline (3 mg kg−1, s.c.) or naloxone (0.3 mg kg−1, s.c). 5 In the cat, baclofen (0.3–3.0 mg kg−1, i.v.) decreased mechanically‐induced cough in a dose‐dependent manner. In this model, baclofen (ED50 = 0.63 mg kg−1) was less potent than either codeine or dextromethorphan. The antitussive effect of baclofen in the cat was antagonized by the GABAB antagonists, CGP 35348 (10 mg kg−1, i.v.) and 3‐aminopropylphosphonic acid (3 mg kg−1, i.v.). 6 We show that baclofen and 3‐APPi have antitussive effects in the guinea‐pig and cat and these effects are mediated by GABAB receptors.

Inhibition of experimental acute pulmonary inflammation by pirfenidone.

Pirfenidone, a putative tumor necrosis factor-alpha (TNF-alpha) inhibitor, has recently gained recognition for its therapeutic use in the treatment of idiopathic pulmonary fibrosis. As pulmonary fibrosis may be the result of lung inflammatory processes, we examined the anti-inflammatory potential of pirfenidone in several models of acute pulmonary inflammation. In antigen-induced allergic paradigms, 24 h after antigen challenge, sensitized mice or guinea pigs develop a prominent pulmonary inflammation, reflected by a significant increase in the number of recoverable bronchoalveolar lavage (BAL) total cells and eosinophils. In both species, the pretreatment of animals with pirfenidone (10 and 30 mg/kg) resulted in a dose-dependent inhibition of the antigen-induced pulmonary inflammation, which was reflected by a significant decrease in the BAL eosinophils and total cells by the 30 mg/kg dose. In a non-allergic model of pulmonary inflammation, rats challenged with intratracheal LPS develop a significant increase in BAL neutrophils and total cells, along with significant increases in TNF-alpha and IL-6. Pretreatment with pirfenidone (3 and 30 mg/kg) showed a dose-dependent inhibition of the LPS-induced pulmonary inflammation, reflected by a significant decrease in the number of BAL total and neutrophilic cells at both the 3 and 30 mg/kg dose. However, pirfenidone had no effect on the peak BAL levels of TNF-alpha. In contrast, pirfenidone significantly inhibited BAL levels of IL-6. In summary, we have shown that pirfenidone can inhibit allergic and non-allergic inflammatory cell recruitment and that its pulmonary anti-inflammatory activity is independent of TNF-alpha inhibition.

Prejunctional GABA-B inhibition of cholinergic, neurally-mediated airway contractions in guinea-pigs

GABA is a known inhibitory neurotransmitter in the CNS. Recent studies have also demonstrated the presence of GABA in peripheral tissue, including lung. To delineate a role for GABA in lung, the effect of GABA and selective GABA agonists and antagonists on neuronally-induced airway contractions in guinea pigs were studied. In vitro, tracheal contractions induced by electrical field stimulation (EFS) were inhibited by tetrodotoxin and atropine indicating that the contractions were mediated by neuronal release of acetylcholine. The contractions caused by EFS, but not those by exogenous acetylcholine, were inhibited by GABA (EC50 = 4.5 microM) and the selective GABA-B agonist baclofen (EC50 = 9 microM), but not by the GABA-A agonist, muscimol. The inhibitory effect of baclofen was not affected by the GABA-A antagonist, bicuculline, but was significantly reversed with the GABA-B antagonists, 3-aminopropylphosphonic acid (3-APPA) (pA2 = 4.5) and 2-hydroxysaclofen (pA2 = 4.1). In vivo, vagal nerve stimulation (5 V, 20 Hz, 0.5 ms, 5 s) in anesthetized, mechanically ventilated guinea-pigs caused cholinergic-dependent bronchospasms that were inhibited by intravenous GABA (3 and 10 mg/kg) and baclofen (1-10 mg/kg), but not by muscimol. The inhibitory effects of GABA and baclofen against vagal bronchospasm were blocked by 3-APPA (5 mg/kg, i.v.), but not by bicuculline. Responses to the GABA-B agonists were unaltered after the treatment of animals with phentolamine or propranolol to block alpha-adrenergic and beta-adrenergic receptors, respectively. Bronchospasm due to intravenous methacholine was also unchanged by GABA and baclofen.(ABSTRACT TRUNCATED AT 250 WORDS)