The protective effect of a new antiallergic agent,KP-136 on mast cell activation: A comparison with disodium cromoglycate

The protective effects on mast cell activation were compared between a new antiallergic agent, KP-136 and disodium cromoglycate (DSCG), both of which inhibited the immunological degranulation of rat peritoneal mast cells. The IC₅₀ was 0.03 g/ml for KP-136 and 4.7 g/ml for DSCG. KP-136 predominantly acted on the early stage of mast cell activation processes and inhibited the immunological increase in⁴⁵Ca uptake. KP-136 also inhibited A23187-and heat-induced degranulation and heat-induced hemolysis. In addition, KP-136 was effective on phospholipase A₂-induced degranulation, although the compound did not directly affect the enzyme activity. In all tests for comparison, KP-136 and DSCG had similar profiles of action and the parallel experiments indicated that KP-136 was a more potent inhibitor of mast cell activation than DSCG, having a DSCG-like membrane stabilizing activity.     

Inhaled lodoxamide tromethamine in the treatment of perennial asthma: a double-blind placebo-controlled study

The efficacy of lodoxamide tromethamine in the treatment of asthma was studied in a 16-week double-blind, placebo-controlled study of 68 perennial allergic subjects with asthma. Patients received either lodoxamide tromethamine, 0.25 mg four times daily, or placebo, administered by metered-dose inhaler. Response to treatment was assessed by analyzing changes in asthma symptoms, inhaled bronchodilator requirements, and pulmonary function when compared to a 2-week baseline period. Patients treated with lodoxamide tromethamine demonstrated an improvement in daytime breathing difficulty, cough, sputum production, and sleep (p less than 0.01 to 0.05), but improvement was not significantly different from that demonstrated by placebo-treated patients. Patients from both treatment groups were able to reduce their inhaled bronchodilators (p less than 0.01), but again no significant difference was apparent between lodoxamide tromethamine and placebo treatment, nor were there any differences in peak expiratory flow rate or FEV1 between the two groups. Seven patients who received lodoxamide tromethamine withdrew because of a sensation of heat and gastrointestinal symptoms. Thus, although lodoxamide tromethamine possesses potent mast cell-stabilizing activity in vitro, we have failed to demonstrate any useful long-term effect in the treatment of mild allergic asthma.     

Modulation of bradykinin responses in airway smooth muscle by epithelial enzymes

We have studied the effect of epithelium removal on responses of guinea pig trachea to bradykinin (BK). BK (1 nM–10 M) gave a concentration-dependent relaxation when epithelium was present (E+: EC₅₀=10±3 nM). Epithelium removal resulted in a biphasic response to BK with relaxation at low concentrations (E–: EC₅₀=3.0±1.0 nM) and a recontraction to baseline at higher concentrations (EC₅₀=2.0±1 M). Phosphoramidon (10 M), an inhibitor of neutral endopeptidase (NEP), which cleaves BK into inactive peptides, potentiated relaxation (EC₅₀=1.0±0.9 nM and 0.1±0.1 nM in E+ and E respectively) and contraction in trachea with intact epithelium (EC₅₀=0.08±0.03 M). Inhibition of cyclooxygenase by indomethacin (5 M), inhibited relaxation to BK in E+ tracheal segments, resulting in a slight contraction (EC₅₀=1.0 M), whereas a potent contractile response was observed in E–segments (EC₅₀ 1.6 M, maximal contraction >1 g). In the presence of both indomethacin and phosphoramidon BK caused contraction, even in the presence of epithelium (EC₅₀=0.2±0.11 M), and the response in the absence of epithelium was similar to the response observed in trachea with intact epithelium (EC₅₀=0.25±0.1 M). The contractile effect of BK on airway smooth muscle may be inhibited by a protective role of epithelium, due to release of relaxant prostanoids and by degradation by epithelial NEP. In asthma, bronchoconstrictor responses to BK may be partly explained by loss of airway epithelium.     

Assessment of thein vivo biochemical efficacy of orally active leukotriene biosynthesis inhibitors

In man, the therapeutic effectiveness of specific inhibitors of leukotriene (LT) biosynthesis against allergen-induced bronchoconstriction appears to be related to thein vivo biochemical efficacy of these compounds, as measured by inhibition of whole blood LTB₄ generation (upon A23187 stimulus) and, particularly, urinary LTE₄ excretion. Accordingly, we have assessed the ability of two clinically documented LT biosynthesis inhibitors, zileuton and MK-886, and the structurally novel 5-lipoxygenase activatig protein antagonist, MK-0591, to inhibit the production of these inflammatory arachidonic acid metabolites in laboratory dogs. Zileuton (2 mg/kg) was extremely bioavailable in dogs (>10 M plasma concentrations), and inhibited the A23187-inducedex vivo production of LTB₄ by venous blood by >90%, in concordance with its potency in canine bloodin vitro (IC₅₀=1.1 M). Despite this degree of inhibition in whole blood, urinary LTE₄ excretion was reduced by only 52%, a profile of activity similar to that seen in clinical studies. MK-886 was less well absorbed, with plasma concentrations of 3 M being achieved only at 25 mg/kg. These levels resulted in <45% inhibition of LTB₄ production, but a significant (p<0.05) 47% inhibition of urinary LTE₄ excretion. MK-0591 was similarly bioavailable (compared with MK-886), but 10-fold more activein vivo as a 2 mg/kg dose resulted in 41–62% inhibition of urinary LTE₄ excretion (p<0.05 vs controls;n=4, 28). Significant inhibition ofex vivo LTB₄ synthesis was also observed at this dose (49%), in accord with peak plasma concentrations of 0.5 M and anin vitro potency of 0.2–0.4 M (IC₅₀) in whole blood from these animals. At higher dose (10 mg/kg), MK-0591 inhibited LTE₄ excretion by 69%, with 88% inhibition of the LT biosynthetic capacity of whole blood. These data demonstrate that the biochemical efficacy of structurally diverse leukotriene biosynthesis inhibitors can be assessedin vivo in normal laboratory dogs. Such measurements, combined with bioavailability data from other species, may be useful for predicting biochemical activity in man.     

Adrenaline-stimulated,aspirin-sensitive synthesis of histamine in the rat

The intravenous injection of 40 g/kg of adrenaline raised total rat lung histamine from 5.3±0.7 g, to 8.4±0.7 g and rat skin histamine from 624±51 g to 835±85 within 5 min. These changes were no longer apparent after 10 min. Stomach histamine was unaffected. Blood drawn 2 min after the injection of adrenaline failed to show an increased content of histamine. Rats given 1 mg/kg of compound 48/80, had greatly elevated levels of histamine in blood, but exhibited no increase in lung histamine. This result, as well as the extent of the increase of histamine observed in skin, which cannot be accounted for in any other way, point towards stepped-up local synthesis as the origin of the effect of adrenaline. Aspirin (20 mg/kg, intravenously 10 min prior to adrenaline), prevented increases of skin histamine. Evidence suggesting mast cells as the site of action of adrenaline, is discussed.     

Oxatomide inhibits the release of bronchoconstrictor arachidonic acid metabolites (iLTC4 and PGD2) from rat mast cells and guinea-pig lung

The effect of oxatomide, an orally active antiallergic drug, on immunoreactive LTC₄ (iLTC₄) production has been studied in rat peritoneal exudate cells (PEC) and guinea-pig lung fragments using the calcium ionophore A23187 and specific antigen in vitro. Oxatomide (10^–5 M) inhibited iLTC₄ release by 70% with A23187 from rat PEC, and by 48% with antigen from guinea-pig lung. Oxatomide is supposed to affect the biosynthesis pathway of leukotrienes, because oxatomide inhibits 5-lipoxygenase from guinea-pig peritoneal leukocytes with an IC₅₀ 17 M. Oxatomide also depressed the release of PGD₂ from rat peritoneal mast cells stimulated by A23187 (IC₅₀ 4.2 M). The effects of oxatomide on iLTC₄ and PGD₂ release were more potent than other antiallergic drugs (DSCG, ketotifen, tranilast).     

Inhibitory effect of DS-4574, a peptidoleukotriene antagonist with mast cell stabilizing action,on compound 48/80-induced gastric mucosal lesions in rats

We evaluated the inhibitory effect of DS-4574, a peptidoleukotriene antagonist with mast cell stabilizing action, on rat gastric mucosal lesions induced by compound 48/80 (C48/80: a mast cell degranulator), in comparison with those of disodium cromoglycate (DSCG: a mast cell stabilizer), LY171883 (a peptidoleukotriene antagonist) and cimetidine (a histamine H₂ receptor antagonist). Subcutaneous administration of C48/80 (1 mg/kg) once daily for four consecutive days produced extensive gastric lesions in the fundic mucosa. DS-4574 (20, 50 and 100 mg/kg/day, oral) and DSCG (200 mg/kg/day, intraperitoneal) treatment markedly inhibited formation of these mucosal lesions, but LY171883 (100 and 200 mg/kg/day, oral) and cimetidine (400 mg/kg/day, oral) treatment did not. Moreover, DS-4574 and DSCG significantly suppressed both hyperhistaminemia and histamine release from rat peritoneal mast cells induced by C48/80. These results indicate that the inhibitory effect of DS-4574 on gastric lesions induced by C48/80 may be related to its mast cell stabilizing action, but to neither its antisecretory nor its peptidoleukotriene antagonistic activity.     

Activation of hamster mast cells for IgE — Mediated histamine release

The conditions for active sensitization of hamster peritoneal and pleural mast cells and IgE-induced histamine release as well as cell desensitization were defined. Immunization of hamsters with ovalbumin (5 g) in Al/OH/₃ gel (5 mg) with several boosters resulted in sensitization of peritoneal and pleural mast cells; in the presence of extracellular Ca⁺⁺, pH of medium 7.2 and at 37°C these cells released up to 70% of histamine on the challenge with specific antigen. Partial release was observed when the cells were challenged with antigen in the absence of extracellular calcium. The rate of release is high during the first seconds of activation and is complete at 1 min. 30 min preincubation of peritoneal and pleural mast cells in calcium-free conditions (in the presence of 4 mM EDTA) resulted in complete desensitization of cells to subsequent action of antigen in potimal conditions. The present experiments demonstrate, that hamster peritoneal and pleural mast cells can be a useful model system forin vitro studies of the mechanisms of IgE-induced cell activation.     

Evidence for Na+/Ca2+ exchange in isolated smooth muscle cells: a fura-2 study

Isolated smooth muscle cells (SMC) from guinea pig taenia coli were employed. Suspension of cells were externally loaded in saline with the fluorescent calcium indicators quin-2/AM or fura-2/AM at 20–40 M or 4 M respectively, resulting in an estimated intracellular concentration of 100–200 M for quin-2 or 10–20 M fura-2 (free acid). On addition of 100 M carbachol or high K _o ⁺ (80 mM) depolarization, fura-2 loaded cells contracted (104±47 m,n=121 rest: 39±13 m,n=59 contracted) identically to control (103±35 m,n=232 rest: 39±16 m,n=89 contracted) cells, whereas quin-2 loaded cells were unresponsive to these protocols and there was no significant length change. The Ca _i ²⁺ of fura-2 loaded cells was 100±18 nM (mean±SD,n=15) and was not significantly different from quin-2 loaded cells 107±26 nM (n=13). Treatment of fura-2 loaded cells with 100 M ouabain saline for 10–60 min progressively elevated the Ca _i ²⁺ to a mean of 266±83 nM (n=15). Reduction of Na _p ⁺ (96% Li⁺ replaced) significantly increased Ca _i ²⁺ to 317±77 nM (n=8). After pretreatment with ouabain (100 M), Na _o ⁺ replacement (Li⁺) increased Ca _i ²⁺ at a significantly faster rate [3.6 nM min^–1 (control) cf. 19.8 nM min^–1 (ouabain)].     

LG 30435, a new bronchodilator/antiallergic agent,inhibits PAF-acether induced platelet aggregation and bronchoconstriction

LG 30435 is a quaternary phenothiazinic antihistamine, endowed with bronchodilator and antiallergic activity. Since PAF-acether (PAF) is a potential mediator of asthma, LG 30435 was assayed for its ability to counteract PAF-induced platelet aggregation (PA) in rabbit platelet rich plasma and bronchoconstriction (BC) in anaesthetized guinea-pigs, in comparison with other antihistamines. LG 30435 was the most potent and selective inhibitor of PAF-induced PA (IC₅₀66 M), concentrations three and more than fifteen fold higher being needed to inhibit PA induced by collagen and arachidonic acid respectively. The other antihistamines, namely mepyramine, promethazine, mequitazine, thiazinamium methyl sulfate and ketotifen were less potent inhibitors of PAF-induced PA, while interfered at lower concentrations with collagen-induced PA. LG 30435 and thiazinamium, administered intravenously, inhibited dose-dependently PAF-induced BC, starting from the dose of 0.1 mol/kg. The travenously, inhibited dose-dependently PAF-induced BC, starting from the dose of 0.1 mol/kg. The ED₅₀ of LG 30435 was 0.28 mol/kg, while the inhibition obtained with thiazinamium did not reach 50% even at 3 mol/kg. Ketotifen and promethazine were partially active only at 3 mol/kg, while mequitazine and mepyramine were inactive up to this dose. These results show that LG 30435 is endowed with a peculiar anti-PAF action, which may be advantageous in the treatment of asthma.A preliminary report on some of these results was presented at the 2nd World Conference on Inflammation, Montecarlo, March 1986.     

Inhibition of C5a-induced basophil degranulation by disodium cromoglycate

Injection of purified porcine C5a into 24-hr basophil-rich cutaneous basophil hypersensitivity sites in the dose range 10^–12–10^–10 moles/site produced cutaneous basophil anaphylaxis (CBA). The H₁ anti-histamine antagonist mepyramine, given orally (3.0–30 mg/kg), inhibited the vasopermeability, but not the basophil degranulation, characteristic of CBA. The antiallergy agent disodium cromoglycate (DSCG), administered intravenously (3.0–30 mg/kg), inhibited vasopermeability and basophil degranulation. DSCG inhibition of mast cell degranulation was not important in the inhibition of CBA, since intact mast cells were found to be depleted at basophil-rich sites and absent at C5a-induced CBA sites from animals treated with DSCG.C5a at 10^–11 moles/site also induced vasopermeability and mast cell degranulation in normal guinea pig skin. Vasopermeability, but not mast cell degranulation, was inhibited by mepyramine at 30 mg/kg p.o. However, DSCG at 10 mg/kg i.v. failed to inhibit either the vasopermeability or the mast cell degranulation of this reaction. These results indicate that C5a induces the degranulation of both basophils and mast cells in the guinea pig, and that C5a-induced degranulation of basophils, but not mast cells, is inhibited by DSCG.     

Effect of auranofin,a new antiarthritic agent,on immune complex-induced release of lysosomal enzymes from human leukocytes

Auranofin, an oral chrysotherapeutic agent effective in the treatment of rheumatoid arthritis (RA), was found to be a potent, noncytotoxic inhibitor of IgG-RF immune complex-induced lysosomal enzyme release (LER) from human leukocytes. At a concentration of 1 g Au/ml (5M), auranofin produced a marked reduction in-glucuronidase (100%), acid phosphatase (88%), and lysozyme (72%) release. In contrast, gold sodium thiosulfate (GST, an injectable gold compound) had no inhibitory activity on LER at equivalent gold concentrations (i.e., 1g Au/ml) and only modest activity (< 36% inhibition) at concentrations as high as 40g Au/ml. The 50% inhibitory dose (ID₅₀) of auranofin on LER was calculated to be 3–4M (0.6–0.8g Au/ml). Blood gold levels in auranofin-treated RA patients were found to be within the range required for in vitro inhibition of LER, and correlated with decreases in IgG, RF titers, and IgG-RF immune-complex formation in vitro. These results suggest that the therapeutic action of auranofin may be caused, at least in part, by inhibition of LER and/or decreases in immune-complex formation.SK&F D-39162 (2,3,4,6-tetra-O-acetyl-1-thio--D-glucopyranosato-S) (triethylphosphine) Gold.     

Effect of the tachykinin receptor antagonists,SR 140333, FK 888, and SR 142801, on capsaicin-induced mouse ear oedema

We examined the effect of SR 140333, a nonpeptide NK₁ receptor antagonist, FK 888, a peptide NK₁ antagonist, and SR 142801, a non-peptide NK₃ antagonist, on ear oedema induced by topical application of capsaicin (250 g/ear) in mice. SR 140333 (ED₅₀: 39 g/kg, i.v.) dose-dependently inhibited the oedema response to capsaicin, whereas FK 888 (1.0 mg/kg, i.v.) and SR 142801 (3.0 mg/kg, i.v.) had no effect. Furthermore, SR 140333 significantly (p<0.001) suppressed ear oedema in response to intradermal injection of substance P (SP) (100 pmol/site) by i.v. administration (0.1 mg/kg), and co-injection (50 pmol/site). In contrast, FK 888 (1.0 mg/kg, i.v. and 500 pmol/site) was ineffective in the response to SP. The present results suggest that the difference in effects of the two NK₁ receptor antagonists on the oedema response to capsaicin is due to species differences in affinities for the NK₁ receptor in the mouse skin. Moreover, it seems unlikely that the NK₃ receptor is involved primarily in capsaicin-induced mouse ear oedema.accepted by G. W. Carter     

Comparison of histamine release from peritoneal mast cells derived from diabetic and control rats

Clinical and experimental diabetes are associated with an increased number of mast cells and elevated tissue histamine concentrations. This study compared histamine release from peritoneal mast cells derived from diabetic and control rats. Experimental diabetes was induced by a single i.v. injection of streptozotocin (50 mg/kg body weight). Measurement of plasma glucose levels confimed the diabetic state. Peritoneal mast cells were stimulated for 10 min with the lectin concanavalin A (0.5–100 g/ml) in the presence or absence of phosphatidylserine, clinical dextran (0.6–1200 g/ml) in the presence of phosphatidylserine, the calcium ionophore A23187 (0.1–1 M) or the basic releasing agent compound 48/80 (0.1–10 g/ml). Histamine release induced by these agents was similar in both populations. Further studies will compare the differences in histamine release from mast cells isolated from different tissues, e.g. heart and lung. In addition, physiological stimuli which are altered in the diabetic state (e.g. hyperosmolalar solutions and free radical generating systems) are under investigation.     

An open, controlled, crossover study on the effects of cimetidine on the steady-state pharmacokinetics of trovafloxacin

Twelve healthy male volunteers participated in this open, randomized, placebo-controlled, two-way crossover study to investigate the effects of cimetidine on the steady-state pharmacokinetics of oral trovafloxacin. Volunteers were randomized to receive either 400 mg cimetidine twice daily or placebo for 5 days. From day 3–5, volunteers received 200 mg trovafloxacin once daily in addition to either cimetidine or placebo. After a minimum 7-day washout period, the study was repeated; those volunteers who received placebo during the first study period were administered cimetidine, and vice versa. The maximum observed serum trovafloxacin concentration, the area under the concentration-time curve of trovafloxacin within the dosing interval of 24 h and the earliest time to the maximum serum concentration for trovafloxacin in volunteers receiving concomitant cimetidine were 2.4 (g/ml, 27.8 g·h/ml and 1.4 h, respectively, compared with 2.5 g/ml, 27.1 g·h/ml and 1.5 h, respectively, in volunteers receiving concomitant placebo. Thus, multiple dosing with cimetidine had no significant effect on the absorption or disposition of trovafloxacin at steady state. Co-administration of cimetidine and trovafloxacin was also well tolerated and without serious adverse effects.     

Forskolin inhibition of hexose transport in cardiomyocytes

The effects of insulin, forskolin, isoproterenol, and epinephrine on 3-O-methylglucose (hexose) transport and cell cyclic AMP levels were determined in adult rat cardiomyocytes. Insulin stimulated hexose transport in these cells an average of 2.5-fold. Initial hexose transport rates at 1 mM hexose were 3.75×10^–2 nmol/mg cell protein/second in the absence of insulin, and 8.25×10^–2 nmol/mg cell protein/second in the presence of 12.3 M insulin. Forskolin at 5 M nearly abolished hexose transport within 3 s of exposure, but did not increase cell cyclic AMP concentrations within 9 s. The apparentK _i for hexose transport inhibition was about 0.3 M forskolin. Epinephrine and isoproterenol at 50 M increased cell cyclic AMP 4-fold during 9 s exposure, but did not affect hexose transport. Treatment of cells with these catecholamines of forskolin for up to 99 s increased cell cyclic AMP, but only forskolin inhibited hexose transport. We coclude from these results that forskolin acts on hexose transport independent of its action on adenyl cyclase, and that cyclic AMP does not inhibit or stimulate hexose transport.Supported by NIH-HL 07094     

The role of intracellular Ca2+ in the degranulation of skinned mast cells

The intracellular pH of rat peritoneal mast cells was slightly acidic and compound 48/80 induced a decrease in the cytoplasmic pH of these cells. By means of chemical skinning, it was revealed that perfusion with Ca²⁺ or inositol 1,4,5-trisphosphate (IP₃) induced degranulation dose-dependently in mast cells at concentrations higher than 10 M and 0.1 M, respectively. Na⁺ was essential for the release of histamine from mast cells. An assay based on the binding of⁴⁵Ca to mast cell fragments revealed that the intracellular Ca store of the mast cell is located in the endoplasmic reticulum. IP₃ liberated Ca from the endoplasmic reticulum.     

Antiinflammatory benzimidazole derivative with inhibitory effects on neutrophil function

5-Methyl-2,2,2-trifluoroethylsulfonyl-1H-benzimidazole (BI-L-45 XX) inhibits both neutrophil enzyme release and chemotaxisin vitro and also inhibits chemotaxisin vivo. BI-L-45 XX has an IC₅₀ between 16M and 25 M in inhibiting lysosomal enzyme release from human peripheral blood neutrophils. In a Boyden chamber experiment, BI-L-45 XX inhibited migration in response to fMLP with an IC₅₀ of 5 M. When given orally to passively sensitized rats at doses of 0.1 to 1.0 mg/kg, it inhibited migration of neutrophils to the pleural cavity in response to an antigen (ovalbumin) challenge. BI-L-45 XX also shows activity in the developing adjuvant arthritis model, with an ED₅₀ of 45 mg/kg, while exhibiting no significant inhibition of cyclooxygenase in a human platelet assay. This suggests the possibility that its antiinflammatory activity may be in part mediated by its effect on neutrophil function.     

Protective effect of lodoxamide tromethamine on allergen inhalation challenge

Lodoxamide tromethamine (U-42,585E) is a new drug intended for prophylaxis of mast cell-mediated allergic disease. It is a water-soluble, cromolyn-like agent with demonstrated activity in rat peritoneal mast cell assay, rat percutaneous anaphylaxis (rat PCA) and sensitized rhesus monkey airway system. Ten allergen-sensitive asthmatics were pretreated with lodoxamide (0.01, 0.1, or 1.0 mg) or placebo, then challenged with serial dilutions of allergen extract. Analysis of allergen dose-response curve parameters shows that pretreatment with lodoxamide offers significant protection against experimental allergen-induced bronchoconstriction. At 0.01 mg, lodoxamide was effective in over half the subjects tested. Administration of lodoxamide by inhalation at doses of 0.1 and 1.0 mg uniformly allowed subjects to tolerate significantly larger doses of inhaled allergen. Side effects observed at these doses were minimal.     

Hyperosmolar induced histamine release from mast cells: A mechanism for the pathogenesis of exercise-induced asthma?

Hyperosmolar buffer solutions produced by the addition of mannitol (0.1–1.0M) induced histamine release from mast cells from dispersed human lung (DL), bronchoalveolar lavage (BAL) and the rat peritoneal cavity (RPMC). The maximal releases wereca 60% (RPMC), 40% (BAL) and 15% (DL), respectively.Under defined conditions, the release from the RPMC was shown to be non-cytotoxic with biphasic kinetics. The process was inhibited by disodium cromoglycate, nedocromil sodium and theophylline, with IC₅₀ values ofca 100 M, 100 M and greater than 10 mM, respectively. On the basis of these results, the role of hyperosmolar induced mast cell activation in exercise-induced asthma is discussed.