Measurement of radiographic changes in adjuvant-induced arthritis in rats by quantitative image analysis.

OBJECTIVE: To apply quantitative analytical methods to the evaluation of radiographic images in experimental arthritis. METHODS: Adjuvant was used to induce arthritis in rats. Arthritis progression was followed by conventional methods. In addition, digitized images of radiographs of the calcaneus were examined for changes in the mean and in the distribution pattern of gray values. Periosteal new bone formation was measured as an increase in image area of the calcaneus. RESULTS: Significant changes in the gray value profile and increases in periosteal bone formation occurred in arthritic rats. More extensive changes occurred in Lewis rats than in Sprague-Dawley rats. Analysis of serial radiographs revealed an initial decrease in the density of juxtaarticular bone, followed by progressive increases in gray value variation due to concurrent bone loss and bone formation. Eventually, bone formation in arthritic rats resulted in increased gray values above those in nonarthritic rats. CONCLUSION: Image analysis represents a sensitive, quantitative method for detecting radiographic changes in experimental arthritis.

Inhibition of human neutrophil elastase with peptidyl electrophilic ketones. 2. Orally active PG-Val-Pro-Val pentafluoroethyl ketones.

Valylprolyvalyl pentafluoroethyl ketones with different N-protecting groups were evaluated in vitro and in vivo as inhibitors of human neutrophil elastase (HNE). Several of these compounds were found to be orally active in HNE-induced rat and hamster lung hemorrhage models. The compound with 4-(4-morpholinylcarbonyl)benzoyl as the protecting group, 71 (MDL 101,146), was studied in greater detail. Hydration and epimerization studies were performed on 71 and related compounds in various media, including human blood serum. High-performance liquid chromatography studies on a reversed-phase system as a measure of the lipophilicity of 71 and related compounds revealed a small range of relative retention times wherein the orally active compounds fell. The Ki value determined for 71 vs HNE was 25 nM.

Parainfluenza virus type 3 induced alterations in tachykinin NK1 receptors, substance P levels and respiratory functions in guinea pig airways.

We have investigated the effects of parainfluenza virus type 3 (PI-3) on sensory neuropeptide levels, tachykinin receptors and their functions in guinea pig airways during the course of respiratory viral infection. PI-3 infected guinea pigs were hyperresponsive to methacholine and substance P aerosols as determined by earlier onset of dyspnea in these animals as compared with control on post-inoculation day (PID) 7 but not 19. In addition, plasma protein extravasation produced in response to the tachykinin was increased in infected airways during the first week post inoculation. Infected guinea pig trachea did not respond any differently to methacholine when smooth muscle contraction and [3H]inositol phosphate accumulation were measured although the magnitude of substance P effects using in vitro tests was significantly greater than control on post-inoculation day 7 but not 19. Trachea from PI-3 infected animals were characterized by reductions in substance P-like immunoreactivity, tachykinin NK1 receptor number and agonist affinity during the first post-inoculation week. Substance P levels or tachykinin NK1 receptor numbers or affinity were not altered in trachea of guinea pigs 4 days after treatment with lipopolysaccharide. These data suggest substance P release occurs during critical periods of respiratory viral infection which are temporally correlated with airway hyperresponsiveness. Despite apparent down-regulation of tachykinin NK1 receptors, substance P-mediated functions remained enhanced suggesting some alterations in post-receptor mechanisms.

A time-course study of airway hyperresponsiveness in conscious parainfluenza virus type 3-infected guinea pigs.

The study of virus-induced airway hyperresponsiveness may provide insight into mechanisms that contribute to respiratory diseases such as asthma. We examined changes induced by parainfluenza virus type 3 (PI-3) in lung lesions, tissue weights, and airway responsiveness to aerosols of histamine, methacholine, or citric acid in conscious guinea pigs, using modified whole body plethysmography. During the first week after inoculation, infected lung tissue had peribronchiolitis and airway hyperresponsiveness to various agents when dyspnea and significant respiratory events were measured; these effects persisted throughout postinoculation weeks 2 and 3. Airway hyperresponsiveness was defined by reductions in the onset of dyspnea or significant respiratory events. Throughout the course of the study, PI-3 infected animals had resting respiratory patterns that reflected labored breathing and may have been related to the edema indicated by increased lung weights. Furthermore, increased numbers of inflammatory cells were observed in lung tissue as well as bronchoalveolar lavage fluid of infected animals at these times. Unlike PI-3 infection, exposure to gram-negative endotoxin resulted primarily in airway hyporesponsiveness to histamine aerosol. Hence, we have shown PI-3 infection in guinea pigs causes time-dependent alterations in airway responsiveness to diverse bronchoactive agents as well as in normal breathing patterns, which may persist up to several weeks after inoculation in animals that may otherwise appear normal.

Tachykinin-mediated respiratory effects in conscious guinea pigs: modulation by NK1 and NK2 receptor antagonists.

Tachykinins, in particular neurokinin A and substance P, produce a number of airway effects which may contribute to respiratory diseases such as asthma. We examined the ability of aerosolized substance P, neurokinin A or capsaicin to produce respiratory alterations in conscious guinea pigs using modified whole body plethysmography. Substance P-mediated dyspnea and significant respiratory events were inhibited by the NK1 receptor antagonist, CP-96,345. Neurokinin A-mediated respiratory effects were ablated by the NK2 receptor antagonists: MEN 10207, MDL 29,913 and SR 48,968, the latter being the most potent. The peptide-based antagonist, MEN 10207, produced respiratory effects itself suggesting partial agonist activity. The cyclic hexapeptide, MDL 29,913, relaxed airway smooth muscle via mechanisms other than tachykinin antagonism. NK2 but not NK1 receptor antagonists were able to delay the onset of capsaicin-induced dyspnea, although alone they did not usually (in approximately 10% of the animals) eliminate the response. However, when NK2 receptor antagonists were combined with CP-96,345, the incidence of dyspnea induced by capsaicin decreased significantly (40%) suggesting that both tachykinins contribute to dyspnea in this system.

Acute hypersensitivity to aerosolized histamine induced by aerosolized ovalbumin in guinea pigs.

Acute airway hyperresponsiveness can be induced after exposure to aerosolized ovalbumin in sensitized guinea pigs. The purpose of the present studies was to determine if "pro-inflammatory agents" would potentiate and prolong antigen-induced pulmonary hyperresponsiveness to histamine in guinea pigs. Guinea pigs were sensitized to aerosolized ovalbumin by exposing them to a 3 min aerosol, generated ultrasonically from a 10% ovalbumin solution on day 0 and day 7. On day 13 the guinea pigs were exposed to a 3 min aerosol of deionized water or a pro-inflammatory agent (1 microgram/ml PAF, 1 mg/ml LPS, or 4% B. pertussis vaccine). Twenty-four hours later, on day 14, the conscious guinea pigs were challenged with a 3 min aerosolized ovalbumin exposure (under isoproterenol cover) and the individual guinea pig responsiveness to aerosolized histamine was determined 2 and 24 h later in an anesthetized modified Konzett-Rossler preparation. Under these experimental conditions, ovalbumin challenge to sensitized guinea pigs produced only an acute hyperresponsiveness (about a 3-10-fold shift) to aerosolized histamine, which lasted less than 24 h. The pro-inflammatory agents neither potentiated nor prolonged the duration of the hyperresponsiveness.

Airway effects of inhaled bradykinin, substance P, and neurokinin A in sheep.

The effects of inhaled bradykinin (BK), substance P (SP), and neurokinin A (NKA) on pulmonary resistance and airway responsiveness to carbachol were studied in conscious allergic sheep. Inhaled BK (20 breaths, 0.1 to 5.0 mg.ml-1) caused dose-dependent increases in pulmonary resistance. Neither inhaled SP nor NKA (20 breaths, 0.1 to 1.0 mg.ml-1) produced significant bronchoconstriction in allergic sheep. However, the response to SP could be enhanced (p less than 0.05) by pretreatment with the neutral endopeptidase inhibitor, thiorphan (40 breaths, 1 mg.ml-1). Sheep that were allergic to Ascaris suum antigen were 5.9 times (p less than 0.05) more sensitive to the constrictor effects of BK than nonallergic sheep. BK-induced bronchoconstriction was blocked in a dose-dependent fashion by the BK beta 2-receptor antagonist, NPC 567 (D-arginine[hydroxyproline3,D-phenylalanine7]BK). Atropine (0.2 mg.kg-1, intravenously) and nedocromil sodium (1 mg.kg-1 in 3 ml of saline, aerosolized) significantly inhibited the BK-induced bronchoconstriction by 97% and 43%, respectively. Chlorpheniramine (2 mg.kg-1, intravenously) had no effect. NKA caused a transient increase in airway responsiveness in allergic sheep, producing a mean 1.9-fold leftward shift in dose-response curves to aerosolized carbachol (p less than 0.05). This hyperresponsiveness was not evident 24 hours after NKA challenge. Neither SP nor BK changed airway responsiveness. Thus, in allergic sheep, inhaled BK caused a more pronounced bronchoconstriction than that observed in nonallergic sheep. The bronchoconstriction was blocked by a BK-receptor antagonist and appeared to be partially mediated via cholinergic reflexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of 5-lipoxygenase by substituted 3,4-dihydro-2H-1,4-thiazines.

A series of substituted 3,4-dihydro-2H-1,4-thiazines inhibit 5-lipoxygenase from rat leukocytes and exhibit submicromolar IC50 values. A novel synthesis of these compounds was developed based on the formation of hydroxymethyleneamine 13 and its cyclization to the title compounds. The dihydrothiazines have low oxidation potentials, typically E1/2 is near 0.3 V, and a representative compound reduces Fe(III)(phen)3, with k = 10(5) M-1s-1. We propose that these lipophilic compounds bind to 5-lipoxygenase and reduce the iron in the active site, thus inactivating the enzyme.

3-(1H-tetrazol-5-yl)-4(3H)-quinazolinone sodium salt (MDL 427): a new antiallergic agent.

Syntheses for 3-(1H-tetrazol-5-yl)-4(3H)-quinazolinone sodium salt monohydrate (9; MDL 427) and the related formamido compound, 2-(formylamino)-N-1H-tetrazol-5-ylbenzamide (10), are described. Both compounds are active in the rat passive cutaneous anaphylaxis and passive peritoneal anaphylaxis tests. A 94:6 equilibrium mixture of 9 and ionized 10, respectively, forms in aqueous buffer systems at a pH-dependent rate. In addition, analogues of 3-(1H-tetrazol-5-yl)-4(3H)-quinazolinone (8) bearing substituents on the benzene ring, substituents at the 2-position, and heteroaryl groups at the 3-position other than tetrazole were prepared. These analogue sets demonstrated that an accessible electrophilic center and an acidic functionality were requirements for good antiallergic activity.

Synthesis and antiallergic activity of some quinolinones and imidazoquinolinones.

A group of 1,4-dihydro-4-oxoquinoline-2- and -3-carboxylic acid esters with nitrogen functionality at the 8-position was synthesized, and 6-oxo-6H-imidazo[4,5,1-ij]quinoline-4- and -5-carboxylic acid esters were elaborated from these. Several of the compounds displayed activity in the rat passive cutaneous anaphylaxis (PCA) test for antiallergic activity. However, PCA activity in this series was accompanied by rat toxicity, as measured by a decrease in percent of normal weight gain over a 2-week period, following a single oral dose.

Contrasting mechanisms of adrenal catecholamine release by acetylcholine and high potassium.

Continuous twenty minute exposure of isolated perfused bovine adrenal glands to high potassium concentrations produced shorter durations of catecholamine release than twenty minute continuous stimulation with acetylcholine. Catecholamine release during continuous exposure to potassium decreased linearly on a semilog plot, whereas acetylcholine caused a biphasic release of catecholamine. Glands previously treated with high potassium responded poorly to acetylcholine; however, glands previously treated with acetylcholine showed a maximum response to high potassium concentrations. Full recovery of the response to acetylcholine after high potassium treatment occurred in twenty minutes. High potassium concentrations appear to evoke secretion of "readily releasable" material only, whereas acetylcholine has access to a larger catecholamine store in the adrenal.