A neutrophil elastase inhibitor reduces cigarette smoke-induced remodelling of lung vessels.

Cigarette smoking produces pulmonary hypertension (PHT) through unknown mechanisms. In animal models acute smoke exposure induces cell proliferation in the small arteries adjacent to the alveolar ducts, and chronic exposure results in muscularisation of these vessels, with changes related to the development of PHT. Studies indicate that serine-elastase inhibitors can prevent experimental monocrotaline-induced PHT. This study examined whether they could also prevent cigarette smoke-induced pulmonary vascular disease. Guinea-pigs were exposed to cigarette smoke or air for 6 months. Some animals also received ZD0892, an orally active, synthetic, selective, serine-elastase inhibitor. The percentage of muscularised, small, pulmonary arteries was determined by morphometric analysis of histological sections and vascular cell proliferation by proliferating cell nuclear antigen staining. Vascular cell proliferation was markedly increased in the smoke-exposed animals and the percentage of completely muscularised small vessels was increased four-fold. Cell proliferation indices correlated with muscularisation indices. In the animals treated with ZD0892, the number of completely muscularised vessels was reduced by 50% and cell proliferation was reduced by 61%. These data suggest that smoke-induced cell proliferation leads to pulmonary arterial muscularisation. Serine-elastase inhibitors appear to be able to reduce cell proliferation and vascular remodelling.

Racemic salbutamol administration to guinea-pigs selectively augments airway smooth muscle responsiveness to cholinoceptor agonists.

1. An aim of this study was to investigate whether continuous in vivo administration of a low dose of salbutamol to guinea-pigs alters the responsiveness of airway smooth muscle in vitro. 2. Osmotic minipumps containing a solution of racemic salbutamol were implanted subcutaneously in guinea-pigs. The drug was infused at a dose of 0.2 mg kg(-1) day(-1) for 10 days and, at the end of that time, the trachea was isolated and concentration-response relationships to several contractile agonists were examined. 3. This treatment resulted in significant increases in the maximum tension developed by tracheal preparations in response to cholinoceptor agonists, carbachol and methacholine. 4. Cumulative concentration-response curves for histamine, leukotriene D4, and KCl were similar in tracheal segments from saline-control and salbutamol-infused animals. 5. Time course experiments showed that augmented airway contractile responsiveness to cholinoceptor agonists was reversible within 3 days after cessation of the 10 day salbutamol infusion. 6. Our findings support the hypothesis that beta2-adrenoceptor agonist drugs, administered over time in vivo, induce a transient hyperresponsiveness of airway smooth muscle to cholinergic bronchoconstrictor stimuli.

Cloning, sequencing and functional expression of a guinea pig lung bradykinin B2 receptor.

Kinin receptors are classified as B1 and B2 based upon agonist and antagonist potencies and cloning and expression studies. Using sequences from human and rat bradykinin B2 receptors, polymerase chain reaction (PCR) was utilized to isolate cDNA from guinea pig lung. The receptor obtained is predicted to have 372 amino acids and shares > 80% sequence homology with human, rat, rabbit and mouse B2 receptors. In competition binding experiments in Chinese hamster ovary (CHO-K1) cells in which the guinea pig cDNA was expressed, [3H]bradykinin was displaced by kinin receptor ligands with an order of potency consistent with a B2 subtype. In CHO cells expressing the guinea pig receptor, bradykinin caused a concentration 45Ca2+ efflux. A B1 receptor agonist, desArg9-bradykinin, also caused 45Ca2+ efflux but with a potency several orders of magnitude lower than bradykinin. Curiously, several B1 and B2 receptor antagonists induced 45Ca2+ efflux, indicating that this receptor may be coupled differently in CHO cells than in native tissues.

Pharmacology and structure--activity relationships of the nonpeptide bradykinin receptor antagonist WIN 64338.

A series of competitive, nonpeptide bradykinin receptor antagonists based on an alpha-amino acid scaffold have been developed and biologically characterized. The lead compound in the series, WIN 64338, demonstrates competitive inhibition of bradykinin-mediated functional responses through B2 receptors in a variety of tissues and species. WIN64338 is a specific for the bradykinin B2 receptor; it is inactive at both the B1 and B2 kinin receptors. In conscious guinea pigs, WIN 64338 inhibits kinin-mediated bronchoconstriction but does not attenuate a similar response to acetylcholine. A series of WIN 64338 analogues display a well-defined structure-activity relationship, strongly suggesting binding in a specific manner to the B2 receptor. Structure-activity data suggest that a hydrophobic binding pocket that prefers large aromatic groups in a specific conformational orientation exists in the receptor ligand binding domain. This class of nonpeptide bradykinin receptor antagonists may lead to the design of other compounds with enhanced receptor affinity and optimal in vivo biological activity.

Effects of bradykinin receptor agonists, and captopril and thiorphan in ferret isolated trachea: evidence for bradykinin generation in vitro.

Bradykinin B2 receptor agonists, but not a B1 receptor agonist, were potent spasmogens of ferret isolated trachea. Bradykinin-induced contractions were unaffected by several pharmacological agents, indicating a direct effect on airway smooth muscle B2 receptors. Captopril plus thiorphan caused contractions in approximately 70% of preparations that were abolished by B2 receptor antagonists, but not by B1 receptor antagonist. Thus, ferret tracheal tissues appear capable of releasing and degrading kinins in vitro. Inhibition of peptide degradation with captopril and thiorphan may allow the endogenous kinins to accumulate in concentrations sufficient to elicit tracheal contraction via activation of B2 receptors.

Effects of a novel nonpeptide bradykinin B2 receptor antagonist on intestinal and airway smooth muscle: further evidence for the tracheal B3 receptor.

1. We examined the effects of phosphonium, [[4-[[2- [[bis(cyclohexylamino)methylene]amino]-3-(2-naphthalenyl) 1-oxopropyl]amino]-phenyl]-tributyl, chloride, monohydrochloride (WIN 64338), a novel, nonpeptide bradykinin B2 receptor antagonist, on bradykinin-induced contractions of guinea-pig isolated ileum, and guinea-pig and ferret trachea. 2. WIN 64338 potently and competitively antagonized ileal contractions, in response to bradykinin, exhibiting a pA2 value of 7.97 +/- 0.10. The compound was without effect on contractions elicited by methacholine, a muscarinic receptor antagonist. Thus, WIN 64338 is a competitive and selective antagonist of ileal B2 receptors. 3. In contrast, WIN 64338 was completely without effect on bradykinin-induced contractions of guinea-pig or ferret trachea. Thus, even at a concentration of 1 microM, which was sufficient to cause a 100 fold decrease in ileal sensitivity to bradykinin, WIN 64338 failed to shift the bradykinin log concentration-response curves in trachea isolated from either species. 4. These data confirm that WIN 64338 represents the first reported nonpeptide antagonist of guinea-pig ileal B2 receptors. They also provide additional evidence for heterogeneity of bradykinin receptors within the same species (guinea-pig) and, furthermore, indicate that the tracheal bradykinin receptor (B3?) is different from that in ileal tissue (B2).

Effects of bradykinin receptor antagonists on antigen-induced respiratory distress, airway hyperresponsiveness and eosinophilia in guinea-pigs.

1. We examined effects of bradykinin (BK) receptor antagonists on airway hyperresponsiveness and eosinophilia in sensitized guinea-pigs that had been administered single, as well as repeated (chronic) challenges with inhaled ovalbumin. In addition, the effects of BK antagonists on antigen-induced respiratory distress during the chronic study were noted. 2. At 24 h following single antigen challenge, guinea-pigs exhibited airway hyperresponsiveness to the bronchoconstrictor effect of i.v. histamine, characterized by a left shift in the dose-response curve. In addition, responses to the maximum dose of histamine that could be used were significantly increased in hyperresponsive guinea-pigs. The percentages of bronchoalveolar fluid, eosinophil and neutrophils also increased. 3. A BK B1 receptor antagonist, desArg9-[Leu8]-BK, significantly inhibited airway hyperresponsiveness induced by single antigen challenge. A B2 receptor antagonist, D-Arg-[Hyp3, Thi5,8,D-Phe7]-BK (NPC 349) had a small, but statistically significant inhibitory effect on responsiveness to the highest histamine dose in challenged animals. DesArg9-[Leu8]-BK significantly inhibited the neutrophilia, whereas NPC 349 inhibited infiltration by both cell types. 4. Chronic antigen challenge also caused airway hyperresponsiveness to i.v. acetylcholine (ACh), distinguished by an increase in the slope of the dose-response curve. Thus, the magnitude of the bronchoconstrictor responses to the maximum dose of ACh that could be used was significantly increased. No change in sensitivity to ACh was evident. Marked eosinophilia was also noted in the trachea, bronchi and lung parenchyma. 5. Airway hyperresponsiveness and eosinophilia, induced by chronic antigen challenge, were markedly inhibited by the B2 antagonists, D-Arg-[Hyp3,D-Phe7]-BK (NPC 567) or D-Arg-[Hyp3,Thi5d-Tic7,Tic8]-BK (NPC 16731).NPC 16731 also abolished antigen-induced cyanosis, and delayed the onset of dyspnoea,doubling the time taken for animals to exhibit respiratory distress.6. The ability of BK receptor antagonists to inhibit antigen-induced airway hyperresponsiveness, in addition to eosinophilia, indicates an important role for endogenous kinins. Moreover, the abrogation of eosinophil infiltration suggests that BK has a significant function in maintaining allergic inflammation of the airways.

Enzymatically active Peptostreptococcus magnus: association with site of infection.

Fifty-four strains of Peptostreptococcus magnus (11 were recovered from abdominal infections, 18 were from nonpuerperal breast abscesses, and 21 were from diabetic foot infections; the type strain and three other strains were from the American Type Culture Collection, Rockville, Md.) and the type strain of Peptostreptococcus micros were tested for their ability to produce various enzymes, including catalase, hippurate hydrolase, serine dehydratase, threonine dehydratase, collagenase, gelatinase, alkaline phosphatase, and esterase C4. The data were analyzed by cluster analysis. The results showed that all but one strain could be assigned to either of two distinct, valid clusters. The first cluster of 11 strains was composed of strains that were relatively inactive, having produced one or two of the eight strain-dependent enzymes. The second was a large cluster of strains (n = 43) that were considerably more active, all having produced at least three enzymes; the vast majority of strains (89%) produced four or more enzymes. The unclustered strain produced one enzyme that was different from that produced by the strains in the first cluster. The chi 2 test of homogeneity applied to the clustering solution indicated that greater enzyme activity was significantly associated with the site of infection (P less than 0.001). The more enzymatically active P. magnus strains were recovered significantly more often from nonpuerperal breast abscesses and diabetic foot infections than they were from abdominal infections. These results may provide insight into the nature of certain polymicrobial soft tissue infections and suggest that (i) P. magnus may participate more in nonpuerperal breast and diabetic foot infections than in abdominal infections and that (ii) peptostreptococcal production of proteolytic enzymes may have an important adjunctive effect on the pathogenesis of certain soft tissue infections.

Induction of vascular smooth muscle bradykinin B1 receptors in vivo during antigen arthritis.

Antigen arthritis in rabbits was associated with induction of bradykinin B1 receptors in isolated aorta smooth muscle 24 h following intra-articular injection of antigen in sensitized animals. Control tissues developed responsiveness to desArg9-bradykinin or bradykinin during 3 h incubation, but failed to respond to either kinin at the beginning of experiments. Aorta from rabbits 24 h after induction of arthritis not only developed responsiveness to kinins more rapidly than controls, but also responded at the outset of experiments. Antigen arthritis was characterized by acute phase protein synthesis and joint swelling. This is the first demonstration of induction of smooth muscle responsiveness to desArg9-bradykinin during an immune complex disease.

Aminoalkynyldithianes. A new class of calcium channel blockers.

Several dithiane derivatives, prepared as intermediates for compounds structurally related to the therapeutically useful antimuscarinic agent oxybutynin, were effective inhibitors of calcium ion induced contraction of guinea pig ileal strips and of KCl-induced calcium entry into neuronal cells. Although the first member of this series, 2-[5-(diethylamino)-3-pentynyl]-1,3-dithiane (2a), was only marginally effective, its condensation product with diphenyl ketone, i.e. 2-[5-(diethylamino)-3-pentynyl]-2-(a,a-diphenyl-a- hydroxymethyl)-1,3-dithiane (3a), demonstrated weak, but significant, calcium channel antagonist activity. As part of a structure-activity relationship (SAR) study, various structural analogues of 2a and 3a were prepared and examined for calcium antagonist properties. In addition to these structural types, ring bridged (tricyclic) congeners of 3, i.e. 4, related bicyclic compounds 5, dehydroxylated derivatives 6, some homologous 2-[[[(N,N-disubstituted-amino)methyl]2- phenyl-1,3-dithianes (7), and a series of 2-[6-[N,N-disubstituted-amino)methyl]-1-hydroxy-1-phenyl- 4-hexynyl]-1,3-dithianes (8) were prepared and studied for calcium channel blocking activity. In general, greatest potency was noted in the tricyclic series 4; however, a definitive SAR could not be established. A structural similarity between several potent calcium antagonists having the structures 7c, 8b, and 8d and the well-known calcium channel blockers verapamil and tiapamil suggests these compounds may act at the same site. Compounds in the other classes (2-6) failed to show clearly defined SAR and their potency differed markedly in two tests for calcium channel antagonist activity. These results may indicate that the dithiane derivatives 2-6 produce their effects in a manner differing from that of the calcium channel antagonists diltiazem, verapamil, and nitrendepine.

Anaerobic pathogenesis: collagenase production by Peptostreptococcus magnus and its relationship to site of infection.

Fifty isolates of Peptostreptococcus magnus from intraabdominal sepsis, nonpuerperal breast abscess, and diabetic foot infections were examined for collagenase activity using bovine type I collagen. Collagenase production was detected in a higher percentage of strains from nonpuerperal breast and diabetic foot specimens (P less than .001). This enzyme may be responsible for P. magnus playing a more central role in the pathogenesis of nonpuerperal breast abscess and diabetic foot disease than in intraabdominal sepsis.

A bradykinin antagonist modifies allergen-induced mediator release and late bronchial responses in sheep.

We assessed the role of bradykinin (BK) in allergen-induced early and late bronchial responses, airway inflammation, mediator release, and antigen-induced airway hyperresponsiveness in allergic sheep by studying the effects of the BK B2 receptor antagonist, NPC-567 (D-Arg-[Hyp3, D-Phe7]-BK), on these parameters. Antigen challenge was performed on two occasions greater than 3 wk apart, once with placebo (control) and once after high-dose (10 mg/ml) and low-dose (5 mg/ml) treatments with aerosol NPC-567. In the control trials (n = 14) antigen challenge resulted in an early and late increase in specific lung resistance (SRL). The early response was associated with increases (p less than 0.05) in prostaglandin (PG) D2, immunoreactive kinin, tosyl-L-arginine methyl ester (TAME)-esterase, and PGE2 in bronchoalveolar lavage (BAL) fluid. The late response was associated with increases (p less than 0.05) in leukotrienes (LT) B4 and C4, thromboxane (TX) B2, 6-keto-PGF10, and PGE2. There was a significant influx of neutrophils in the BAL fluid during the late response, and airway hyperresponsiveness to carbachol aerosol was apparent 4 h after challenge. In six sheep the high-dose NPC-567 treatment (given before, during, and 4 h after antigen challenge) did not attenuate the early bronchoconstrictor response or the early release of mediators but caused a significant reduction in the late response (p less than 0.05). This protective effect was accompanied by reductions (p less than 0.05) in both the concentrations of all the mediators associated with the late response and the severity of the BAL neutrophilia. High-dose NPC-567 did not attenuate the airway hyperresponsiveness or the cellular inflammatory response seen 24 h after challenge. In eight sheep treated with the low dose of NPC-567 (given before, during, and 4, 8, and 24 h after challenge) the early response was not blocked but the late response was again inhibited, as were the mediators associated with the late response. At the low dose the drug did not prevent the airway inflammation at 8 or 24 h. The additional treatments did, however, prevent the 24 h hyperresponsiveness. These data suggest that kinin generation during antigen-induced airway anaphylaxis may be important for controlling the release of arachidonic acid metabolites from airway inflammatory cells that contribute to the development of the late response in the allergic sheep model.

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin, a potent antagonist of smooth muscle BK2 receptors and BK3 receptors.

D-Arg[Hyp3-Thi5-D-Tic7-Tic8]-bradykinin (NPC 16731) inhibited bradykinin (BK) binding and BK-induced contraction in guinea-pig ileum, being markedly more potent than D-Phe7-BK analogues as a BK2 receptor antagonist. In isolated trachea NPC 16731, unlike other BK2 antagonists, inhibited BK binding and BK-induced contraction, and 45Ca2+ efflux in tracheal smooth muscle cells. That NPC 16731 potently inhibits BK effects in trachea provides further evidence for the existence of the airway BK3 receptor.