Anti-asthmatic activity of newly synthesized calcium antagonists: 2-n-butyl-1 (N-methyl-n-[2-(N',N'-dimethylamino)ethyl]amino) -5,6-methylenedioxy-indene and -indane

The anti-asthmatic activity of the newly synthesized methylenedioxyindene (MDI) derivatives, 2-n-butyl-1 (N-methyl-N-[2-(N', N' -dimethylamino)ethyl]amino)-5, 6-methylenedioxy-indene (MDI-C) and -indane (MDI-D), were investigated in vitro and in vivo in guinea pigs. The in vitro pharmacological activity of both derivatives was compared to that of 8-(diethylamino)octyl-3,4, 5-trimethyoxybezoate hydrochloride. All three agents caused a concentration-dependent inhibition of the antigen-induced contraction of sensitized guinea pig tracheal smooth muscle. In histamine and leukotriene D4-induced contractions of guinea pig tracheal smooth muscle, each agent showed clear antagonistic actions. Additionally, all three agents demonstrated potent calcium antagonistic actions via inhibition of guinea pig tracheal smooth muscle contractions caused by CaCl2 in a high potassium and Ca-free medium and by compound 48/80 in a solution of ethylene glycol bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid in a contained Ca-free medium. MDI-C and MDI-D inhibited the antigen-induced release of histamine and slow reacting substance of anaphylaxis from sensitized guinea pig lung tissue. Lastly, both MDI is clearly inhibited asthmatic respiratory disorders without affecting blood pressure in guinea pigs.     

Inhibition of antigen- and calcium ionophore A23187-induced contractions of guinea pig isolated airways with 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8)

Both antigen- and calcium ionophore A23187-induced airways contractions are dependent on increased concentrations of intracellular calcium. Challenge of isolated tracheal spirals and parenchymal strips from sensitized guinea pigs with antigen or calcium ionophore A23187 in the presence of the intracellular calcium antagonist, 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), resulted in significantly reduced contractions. The results demonstrated that TMB-8 inhibits events that are dependent on either mobilization of intracellular calcium (i.e. antigen) or entry of calcium from extracellular sources (i.e. calcium ionophore A23187). The efficacy of TMB-8 in this model suggests that intracellular calcium antagonists may be potentially useful in the therapy of asthma.     

Effect of cinnarizine on IgE antibody-mediated experimental allergic reactions in guinea pigs

The anti-allergic activity and mechanism of cinnarizine was investigated in guinea pigs. Nifedipine, a calcium antagonist, and tranilast, a potent, orally active anti-allergic agent, were used as comparative drugs. Cinnarizine protected against fatal systemic anaphylactic shock in guinea pigs passively sensitized with IgE antibody. Cinnarizine reduced many of the features of severe respiratory disorders. Nifedipine and tranilast showed similar effects. Cinnarizine and nifedipine inhibited the contractile response to antigen of sensitized tracheal smooth muscle when the challenge was carried out at low antigen concentrations. Tranilast showed a tendency to inhibit the antigen-induced contraction of tracheal smooth muscle. Cinnarizine and nifedipine inhibited Ca-induced contraction in potassium-depolarized tracheal smooth muscle, tranilast had no effect. Cinnarizine showed antagonistic action to the contraction by histamine or leukotriene D4 (LTD4) of tracheal muscle. Nifedipine showed similar antagonistic action, although its potency is lower than cinnarizine. Tranilast showed slight antagonistic action to LTD4. Antigen-induced release of histamine and slow reacting substance of anaphylaxis (SRS-A) from sensitized lung tissues was inhibited by nifedipine and tranilast but not by cinnarizine. The release of histamine and SRS-A from lung tissues by calcium ionophore A23187 was inhibited by nifedipine and tranilast but not by cinnarizine. These results suggest that the anti-allergic action of cinnarizine is mainly due to the antagonistic action to allergic mediators and not by interfering with the release of mediators. Cinnarizine's mechanism seems to be related to its antagonistic action to Ca in smooth muscle, but not to the transport of Ca in releasing the anaphylactic chemical mediators in mast cells and other target cells.     

Reduction of antigen-induced contraction of sensitized guinea-pig tracheal smooth muscle in vitro by calmodulin inhibitors

The effect of two calmodulin inhibitors, 1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-beta-(2,4-dichlorobenzylox y) phenethyl]imidazolinium chloride (R 24571) and chlorpromazine (CPZ) on antigen-induced contraction of guinea-pig tracheal smooth muscle was studied. Ketotifen, an anti-allergic compound, was used as a comparative drug. Contraction of sensitized guinea-pig trachea induced by antigen challenge was reduced by all three agents. The two calmodulin inhibitors effected relaxation of contraction of guinea-pig trachea induced by histamine and leukotriene D4 (LTD4). Ketotifen relaxed histamine-induced contraction, but hardly affected LTD4-induced tone. Contrary to chemical mediator induced tone, all examined agents had no effect on resting tone. These three agents shifted histamine- and LTD4-induced concentration-contraction curves to the right. They produced a downward displacement of the maximum, without a parallel shift in histamine- and LTD4-induced concentration-contraction curves. The two calmodulin inhibitors did not affect the antigen-induced release of histamine and SRS-A from sensitized guinea-pig lung tissue. Ketotifen slightly inhibited the release of histamine. These results suggest that R 24571 and CPZ, calmodulin inhibitors, reduced an antigen-induced contraction of sensitized guinea-pig trachea in vitro mainly by affecting the contractility of tracheal smooth muscle by chemical mediators but not by interfering with the release of mediators.     

Effect of TMB-8 on calcium transport in mast cells in relation to histamine secretion

We have previously reported an inhibition of histamine release by TMB-8 both in the presence and absence of calcium and with glucose in the medium. In the present investigation we have studied the effect of TMB-8 on calcium transport. The observations show that TMB-8 inhibits calcium uptake and enhances calcium efflux in mast cells. As antigen-induced histamine release from sensitized mast cells is primarily dependent on extracellular calcium, the inhibition of anaphylactic histamine release by TMB-8 is probably mainly due to an inhibition of calcium influx into the mast cells. We have shown an increased calcium efflux during histamine release from mast cells induced by compound 48/80 in the absence of calcium in the medium, suggesting the release of intracellular calcium stores. The increased calcium efflux was not inhibited by TMB-8. On the contrary, the enhanced calcium efflux caused by compound 48/80, was added to that by TMB-8. TMB-8 thus had no effect on the calcium release from intracellular stores by compound 48/80 but the enhanced calcium efflux by TMB-8 would tend to inhibit histamine release.     

Inhibition by auranofin of pharmacologic and antigen-induced contractions of the isolated guinea pig trachea

The effects of an orally active gold complex, auranofin, were investigated on the isolated guinea pig trachea contracted pharmacologically and antigenically. Pretreatment of the tracheal tissues for 15 minutes with auranofin (10(-5) and 10(-4) mol/L) produced a significant rightward shift of a histamine dose-response curve (p less than 0.001), whereas a 30-minute pretreatment with auranofin (10(-4) mol/L) inhibited LTD4-induced (but not LTC4-induced) contraction of the tracheal spirals. In tracheas from animals actively sensitized to ovalbumin, auranofin (10(-4) mol/L) significantly inhibited contractions elicited by 0.01 micrograms/ml of this antigen. Auranofin does not appear to behave like a nonspecific suppressant of tracheal muscle contraction since it failed to antagonize a potassium chloride-induced (6 X 10(-2) mol/L) contraction. Therefore, auranofin appears to alter the in vitro response of the guinea pig trachea not only to histamine but also, more importantly, to LTD4 and specific antigen. These results characterize and extend further the pharmacology of auranofin in airway tissue.     

Quercetin inhibits anaphylactic contraction of guinea pig ileum smooth muscle

Certain flavonoids inhibit antigen-induced release of histamine from mast cells and basophils and also inhibit contraction of guinea pig ileum induced by histamine, acetylcholine, and PGE2. We examined the effect of one flavonoid, quercetin, on anaphylactic smooth muscle contraction of ileum from guinea pigs sensitized to egg albumin. Quercetin inhibited both the phasic and tonic components of anaphylactic contraction in a concentration-dependent fashion (IC50 approximately 10 microM). Whether this is primarily an effect on mast cell mediator release or inhibition of mediator effects on smooth muscle has not been established.     

Responses of isolated Japanese monkey tracheal muscle to allergic mediators.

The responsiveness of isolated Japanese monkey (Macaca fuscata) tracheal muscle to antigen, carbachol, histamine, leukotriene C4 (LTC4), U-46619 and substance P (SP) was compared to that of isolated human trachea. Weak but persistent contraction was observed after the addition of antigen to isolated Japanese monkey tracheal muscle passively sensitized with monkey serum containing IgE antibody against Japanese cedar (Cryptomeria japonica) antigen. Unlike monkey tracheal muscle, a fair amount of contraction was caused by the antigen in human tracheal muscle passively sensitized with human atopic serum. When chopped, passively sensitized monkey or human lung tissue was challenged with antigen, a significant level of histamine was released from these tissues. In Japanese monkey tracheal muscle, histamine and SP produced no contraction of tracheal muscle, whereas carbachol, LTC4 and U-46619 caused contraction in a dose-dependent fashion. Contrary to the Japanese monkey, histamine and carbachol caused distinct contraction in tracheal muscle obtained from the cotton-headed tamarin (Saguinus oedipus). In human tracheal muscle, all test substances (carbachol, histamine, LTC4, U-46619 and SP) induced clear contraction. In lung parenchyma obtained from Japanese monkey, histamine induced a weak contraction, and this histamine-induced contraction was also inhibited by pyrilamine (H1 receptor antagonist). These results indicate that antigen-induced contraction of isolated Japanese monkey tracheal muscle, passively sensitized with monkey atopic serum, is not a useful model for human allergic bronchoconstriction in vitro because of the unresponsiveness of tracheal muscle to histamine and SP.     

The influence of cyclic nucleotides on histamine release from lungs and on the histamine-induced contraction of guinea pig ileum

Conclusion In conclusion, exogenous cAMP and cGMP exert certain opposite actions on antibody-induced histamine release from guinea pig lungs as well as on histamine or antigen-induced contraction of guinea pig ileum smooth muscle.     

Antiasthmatic activity of a novel thromboxane A2 antagonist, S-1452, in guinea pigs.

We examined the effect of a potent thromboxane (Tx) A2 receptor antagonist, calcium (1R, 2S, 3S, 4S)-(5Z)-7-(((phenylsulfonyl)amino)bicyclo[2.2.1] hept-2-yl)-5-heptenoate dihydrate (S-1452), on antigen- and various allergic-spasmogen-induced contractions of guinea pig lung parenchymal strips and on the increase in insufflation pressure, an index of bronchoconstriction, in anesthetized guinea pigs. In isolated guinea pig lung parenchymal strips, S-1452 showed competitive antagonism of the contractile activity of U-46619, a TxA2 mimetic, with a pA2 value of 8.9. The compound also inhibited the contraction induced by prostaglandin (PG) D2 and PGF2 alpha, but a TxA2 synthetase inhibitor, OKY-046, did not. In contrast, both drugs inhibited not only leukotriene (LT) D4-induced contraction but also antigen-induced contraction in the presence of a histamine antagonist. In anesthetized guinea pigs, oral administration of S-1452 markedly inhibited the bronchoconstrictions induced by intravenous injection of U-46619, PGD2, PGF2 alpha, LTD4 and platelet-activating factor (PAF) with ED50 values of 0.006, 0.031, 0.112, 0.033 and 0.115 mg/kg, respectively, but OKY-046 inhibited only that by LTD4 and PAF. Additionally, bronchoconstriction following intravenous injection of antigen was almost completely suppressed by S-1452 (0.1 mg/kg) and partially by OKY-046 (300 mg/kg) in passively sensitized guinea pigs which were treated with diphenydramine and propranolol. The inhibitory effect of S-1452 against U-46619-induced broncho-constriction persisted up to 7 h after oral administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anterior hypothalamic lesions decrease anaphylactic contractions in guinea pig trachea in vitro by reducing histamine and LTC4 reactivity

Discrete lesions in the anterior hypothalamus (AHA) of the guinea pig brain reduce the anaphylactic contraction of the trachea in vitro after active in vivo sensitization by 40%. This difference in anaphylactic contraction does not correlate with a difference in homocytotropic antibodies but coincides with a decreased smooth muscle response to the anaphylactic mediators histamine and leukotriene C4. No difference in the beta-adrenoceptor function of the tracheal preparations can be found. The results suggest that AHA lesions afford protection against anaphylaxis in actively sensitized guinea pigs at least in part through a reduced smooth muscle response to anaphylactic mediators.     

Effect of a novel leukotriene D4 antagonist with 5-lipoxygenase and cyclooxygenase inhibitory activity, Wy-45,911, on leukotriene-D4-and antigen-induced bronchoconstriction in the guinea pig

Wy-45,911 (4-[hydroxy-[3-(2-quinolinylmethoxy)phenyl] amino]-4-oxabutanoic acid, methyl ester) was found to inhibit competitively leukotriene D4 (LTD4)-induced contractions of the isolated guinea pig trachea but not those of leukotriene C4 (LTC4), even in the presence of a gamma-glutamyltranspeptidase inhibitor, reduced glutathione (GSH). Tracheal contractions induced by histamine or pilocarpine were also not significantly altered by Wy-45,911. The drug inhibited the tracheal contractions induced by antigen, even in the presence of GSH. This latter effect resulted from inhibition of 5-lipoxygenase (5-LO), as the synthesis of 5-LO products by rat polymorphonuclear leukocytes and by mouse macrophages was markedly reduced by Wy-45,911. The drug inhibited both LTD4-induced and antigen-induced bronchoconstriction when injected intraduodenally or intragastrically into intact guinea pigs though it was more potent against LTD4-induced bronchoconstriction. We conclude that Wy-45,911 is a novel, orally active LTD4 antagonist in the guinea pig, with some 5-LO inhibitory activity.     

Effect of NZ-107, a newly synthesized pyridazinone derivative, on antigen-induced contraction of human bronchial strips and histamine release from human lung fragments or leukocytes.

The effects of a newly synthesized pyridazinone derivative, NZ-107, 4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone, and two well-known antiasthmatic drugs, amlexanox (orally active disodium cromoglycate-like drug) and disodium cromoglycate (DSCG) on antigen-, histamine- and leukotriene C4 (LTC4)-induced constriction of isolated human tracheal muscle, and histamine release from human lung tissues and leukocytes were investigated in vitro. In some experiments, salbutamol was used as a reference drug. NZ-107 inhibited antigen-, histamine- and LTC4-induced contraction of tracheal muscle. Amlexanox and DSCG did not affect the contractile response of tracheal muscle caused by each stimulant. Salbutamol inhibited antigen-induced contraction of tracheal muscle. NZ-107, amlexanox, DSCG and salbutamol clearly inhibited the antigen-induced release of histamine and LTC4 from human lung tissue. The antigen-induced histamine release from atopic human leukocytes was inhibited by NZ-107 and amlexanox, but not by DSCG. Pretreatment with IL-3 did not alter antigen-induced contraction of tracheal muscle and histamine release from lung tissue, but antigen- or calcium ionophore A 23187-induced histamine release from leukocytes was clearly enhanced. Amlexanox inhibited the IL-3-induced enhancement of histamine release from leukocytes in the case of both stimuli, but NZ-107 and DSCG had no effect. These data suggest that NZ-107 has potent anti-allergic actions based on the inhibition of antigen-induced contraction of human tracheal muscle and mediator release from human lung tissue and leukocytes.     

Possible role of slow sodium-calcium channels in the mechanism of changes in electrical and mechanical activity of guinea pig heart cells in local anaphylaxis (the isoptine effects)

The local anaphylactic reaction and the effects of histamine during blocking of the slow sodium-calcium channels by isoptine were investigated in the spontaneously contracting oracle of the atrium of a guinea pig previously sensitized to egg albumin. Simultaneously with the intracellular recording of the potentials, isometric contractions of the preparation were recorded by means of a mechanotron. The investigation showed that egg albumin (0.2 mg/ml) and histamine (0.1 mg/ml) are antagonists of isoptine (2–16 mg/liter) as regards its effect on automatic contraction and duration of the action potential. It is postulated that this anaphylatic reaction is based on activation of the slow sodium-calcium channels in the surface membrane of the myocardial fibers.     

Physiological and immunological effects of chronic antigen exposure in immunized guinea pigs

Antigenic tolerance was induced in previously sensitized guinea pigs by challenging with ovalbumin (OA) aerosol 1 h/day, 5 days/week for 6 weeks. Reactivity was assessed visually and by lung mechanics. Sera from tolerant and sensitized animals showed comparable titers of antigen-specific antibody by passive cutaneous anaphylaxis with 6-hour, 4-day (heated serum) and 7-day sensitizations. In vitro contractile responses of airway smooth muscle revealed comparable histamine responses in sensitized and tolerant guinea pigs but decreased OA sensitivity in smooth muscle from tolerant animals. Although lung histamine content was equivalent in the two groups, antigen-induced histamine release from chopped lung preparations was significantly less in tolerant animals at a low antigen concentration. We conclude that antigen-induced histamine release is impaired in tolerant animals.     

Relative activities of three calcium channel antagonists on histamine,acetylcholine and antigen-induced contractions of guinea pig ileum

The molar concentrations of verapamil, diltiazem, nifedipine, tripelennamine and atropine necessary to block histamine, acetylcholine (ACH) and ovalbumin0induced contractions of the isolated guinea pig ileum were compared. The calcium blockers inhibited ovalbumin contractions in previously sensitized ilea at lower concentrations than those required to block the phasic component of histamine and ACH contractions. These compounds were, however, more active in blocking the tonic phase of histamine and ACH concentrations than ovalbumin contractions. Tripelennamine was almost as active against ovalbumin contractions as it was against histamine concentrations, while atropine was inactive against ovalbumin. The results are interpreted as being supportive of continued efforts to develop calcium channel antagonists which selectively block the smooth muscle effects of inflammatory mediators.     

Inhibition of interleukin 1-induced biosynthesis of stromelysin by the calcium antagonist TMB-8 (8-(N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl)

This study was done to identify agents that can inhibit interleukin 1 (IL1)-induced stromelysin biosynthesis and to gain insight into the mechanism of IL1 action. For this purpose, various agents known to modulate calcium-dependent signal transduction pathway were evaluated in rabbit synovial fibroblast (RSF) cultures. Only the conditioned medium from RSF treated with the intracellular calcium antagonist TMB-8 (8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride) had significantly lower proteoglycan-degrading metalloproteinase activity than controls. Biosynthetic labeling, immunoprecipitation and immunohistochemical studies, using a polyclonal antibody against rabbit stromelysin, demonstrated that TMB-8 inhibited synthesis stromelysin, the proteoglycan-degrading matrix metalloproteinase. Further evaluation of the TMB-8 effect revealed that the compound had no effect on secretion and that it was not acting by preventing activation of the proenzyme or by inhibiting the enzyme activity. These results suggest that TMB-8 may be inhibiting stromelysin synthesis by limiting intracellular calcium levels.     

C5a and antigen-induced tracheal contraction: Effect of a combination of an antihistamine and cyclo-oxygenase inhibitors

Our previous studies with C5a, a cleavage product of the fifth component of complement, have shown that the antihistamine diphenhydramine and the cyclo-oxygenae inhibitor aspirin do not inhibit the C5a-induced contraction of isolated guinea pig trachea (Regal, Eastman & Pickering, 1980; Regal & Pickering, 1981). We investigated the effect of cyclo-oxygenase inhibitors in the presence of diphenhydramine to determine if cyclo-oxygenase products were contributing to the contraction beyond any effect they might have on histamine release. A combination of a cyclo-oxygenase inhibitor and diphenhydramine caused a delay in onset and decrease in magnitude and duration of the C5a-induced contraction. Indomethacin itself also caused a slight inhibition. In contrast, a combination of aspirin and diphenhydramine did not inhibit the initial portion of antigen-induced tracheal contraction any more than diphenhydramine alone and enhanced the later portion just as aspirin alone. Cross tachyphylaxis experiments demonstrated that antigen pretreatment significantly inhibited a subsequent C5a-induced tracheal contraction, though C5a pretreatment did not affect a subsequent antigen-induced contraction. Thus, cyclo-oxygenase products do contribute to C5a-induced tracheal contraction, and histamine participation in the presence of cyclo-oxygenase inhibitors is suggested. Our studies demonstrate the dissimilarities of C5a and antigen-induced contraction as regards inhibition by aspirin plus diphenhydramine, yet suggest common pathways leading to the contractile response as evidenced by cross tachyphylaxis experiments.     

Decreased sensitivity and response of isolated tracheal muscle to methacholine and histamine without changing the activity of pulmonary muscarinic receptors in the egg albumin-sensitized guinea pig

Guinea pigs were sensitized by daily intraperitoneal injections of 100 mg of egg albumin for 3 consecutive days and sacrificed at 30-35 days after the last injection. The in vitro sensitivity and response of the sensitized tracheal muscle to methacholine and histamine were significantly less than those of littermate controls. However, there was no difference between the dissociation constants and concentrations of pulmonary muscarinic receptor binding sites of the control and sensitized guinea pigs. We conclude that subsensitivity and hyporesponsiveness to methacholine and histamine occur in the airway muscle of this guinea pig model of experimental asthma and suggest that the decreased reactivity to methacholine might be due to defects beyond the receptor.     

Mechanism of the action of amoxanox (AA-673), an orally active antiallergic agent

Amoxanox inhibited immunologically stimulated and LTD4-induced bronchoconstriction in laboratory animals. Amoxanox, like DSCG, inhibited rat IgE-mediated PCA and histamine release from rat peritoneal mast cells, and suppressed immunologically stimulated or calcium ionophore A23187-induced SRS-A generation in rat peritoneal cavity and guinea pig lung fragments. This compound also reduced the contractile response of guinea pig lung parenchymal and ileal strips to LTD4, but did not significantly affect the response of the ileum to either histamine or acetylcholine. Therefore, the antiallergic action of amoxanox seems to be associated with inhibition of chemical mediator release and antagonistic activity on SRS-A.