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We used the Ascaris-sensitive primate model to assess the effect of oral doses of the 2-aminochroman (U-83836E) lazaroid on the antigen-induced late phase (24 h) bronchoconstriction (LPBC), eosinophilia and methacholine hyperreactivity. Following establishment of consistent lung resistance measurements and bronchoalveolar lavage eosinophilia in 4 Ascaris reactor primates, we determined the baseline aerosol mecholyl provocative challenge for 50% increase (PC50 in mg/ml) in these animals. Twenty-four hours following antigen challenge, we again determined the PC50 (mg/ml). In all 4 animals, there was a statistically significant decrease in PC50 (5.4- to 32-fold, n = 5-7; p0.029). Bronchoconstriction at 24 h increased in all 4 (49-86% over saline aerosol, p0.05). Eosinophilia increased from 21 to an average of 33% of total cells (p0.05 compared to saline). Repeating the antigen challenge in the presence of oral doses of 10 mg/kg U-83836E 18 and 3 h before and 6 h after challenge resulted in 53-70% inhibition of LPBC, 53-81% inhibition of eosinophilia (p0.05 compared to Ascaris) and return of the mecholyl PC50 (mg/ml) to before antigen levels (p = NS) thus blocking increased hyperreactivity. These results indicate U-83836E, like steroids, would be an effective drug for asthma and lung inflammation.

Inflammation of the airways contributes to the multicomponent disease known as asthma. The primary cells that infiltrate the airway in response to antigen exposure are neutrophils and eosinophils. Eosinophils have been implicated in much of the histopathology of the airway following infiltration and degranulation. We used topical antigen exposure in the trachea of sensitized beagle dogs to study the kinetics of eosinophil infiltration with a modified double balloon endotracheal tube technique. After establishment of the eosinophilia, we used inhibitors with known actions to implicate certain mediators in the cellular response. Beagle dogs were sensitized to ascaria ova by feeding them orally by gavage. After 6 wk, challenge with 2.0 micrograms/ml ascaris antigen protein via the tracheal chamber resulted in a rapid (maximal in 4 h) and repeatable influx (p less than 0.05 versus vehicle) of eosinophils that was faster and larger than that of exogenously added LTB4 or PAF exposure. After 4 to 6 separate (every 2 wk) antigen challenges in which the response varied by (SEM +/- 13 to 50%), the individual dogs were rechallenged in the presence of various inhibitors administered either topically in the perfusate or systemically. The inhibitors that were effective in blocking the eosinophil influx by a statistically significant amount were: (1) the lazaroid U-78517F orally 0.5 to 30 mg/kg-18 h (2) Medrol acetate 5 mg/kg intramuscularly-18 h, (3) H1-antihistamines topically (10(-6) M) Astemizole, Cetirizine, and Mepyramine, also orally 30 mg/kg, and Azelastine. Inhibitors falling to inhibit influx topically were a LTB4 receptor antagonist (U-75302) and U-80271 (Merck L-652731), a PAF antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway obstruction and hyperreactivity are characteristics of human asthma and of "heaves," a naturally occurring respiratory disorder of horses and ponies. We measured pulmonary function and plasma immunoreactive 15-hydroxyeicosatetraenoic acid (i15-HETE) concentrations in simultaneously collected carotid artery and right ventricle blood samples in five affected ponies and their age- and gender-matched control ponies. Measurements and sampling were performed before (Period A), during (Period B), and following recovery from (Period C) acute airway obstruction precipitated by housing ponies in a barn and exposing them to hay dust. Pulmonary resistance increased significantly, and dynamic compliance and PaO2 decreased significantly in affected ponies at Period B. Plasma i15-HETE concentrations were greater in carotid artery samples compared with right ventricle samples in affected ponies at each measurement period, suggesting that the lung was a source of i15-HETE. Carotid artery i15-HETE concentrations were significantly greater in affected ponies than in control ponies and increased at Period B. There was a significant negative correlation between changes in plasma i15-HETE and changes in dynamic compliance between measurement Periods A and B. We conclude that the lung is a source of i15-HETE in ponies with heaves, that these ponies produce greater quantities of i15-HETE than control ponies, and that exposing affected ponies to a barn environment produces acute airway obstruction and increased plasma concentrations of i15-HETE.

Activated eosinophils are believed to be major contributors to the chronic inflammatory sequelae of asthma, but the details of the mechanism of eosinophil activation in vivo are unknown. In our search for physiologically important modes of eosinophil activation, we studied the effects of recombinant human platelet-derived growth factor (PDGF) on human peripheral blood eosinophils. We compared two activation end-points: secretion of granule contents, exemplified by the release of eosinophil peroxidase (EPO), and eosinophil-derived neurotoxin (EDN), and the generation of active oxygen metabolites (O2- production). PDGFc-sis dose dependently stimulated the secretion of large amounts of EPO and EDN from eosinophils. Higher concentrations of PDGF induced a dose-dependent O2- production, especially if the cells were first primed with low concentrations of phorbol ester. These activities were not seen with the AA homodimer of PDGF, suggesting that the activation was receptor dependent. However, several attempts to directly demonstrate the existence of such receptors were unsuccessful. The magnitude of the secretory response to PDGF, and the realization that eosinophils could be easily exposed to this substance as they travel towards the lung, suggests the possibility that this growth factor may be a physiologically important activator of eosinophils in the pulmonary inflammation which is associated with asthma.

This investigation focuses on the changes in the concentrations of cyclooxygenase (CO) products present within the crevicular fluid in naturally-progressing periodontitis in the beagle and the effects of various non-steroidal anti-inflammatory drugs (NSAIDs) on these metabolite levels and disease progression. Six groups of 5-6 beagles with periodontitis were followed for 6 months to determine the pretreatment rate of radiographic bone loss. At baseline, groups of animals were placed on soft chow to promote disease progression. Groups were treated with either placebo, three different formulations of systemic ibuprofen, systemic naproxen or topical flurbiprofen. During the 6-month treatment phase, crevicular fluid (CF) samples and radiographs were taken at regular intervals. Radioimmunoassay of CF samples from untreated animals demonstrated a steady increase in prostaglandin E2 (PGE2) over baseline values. At 1 month, CF-PGE2 levels increased 2-fold over baseline and, by 6 months, had reached a 5- to 6-fold elevation. Crevicular fluid thromboxane B2 (CF-TxB2) levels rapidly reached a 4- to 5-fold peak over baseline at 1 month and subsequently dropped to a 2-fold elevation for the remainder of the study. The rate of bone loss (BLOSS) in untreated animals increased 38% during the 6-month period, as compared to baseline pretreatment BLOSS rates. Overall, there was a significant depression in the CF levels of both PGE2 and TxB2 in all NSAID-treated groups. All NSAID treatments significantly retarded BLOSS, ranging from 21.0-36.9% of the control BLOSS rate. Furthermore, CO activation represents a major regulatory step in bone destruction and may thereby serve as an important site for pharmacological modulation.

Inflammation of the airways contributes to the multicomponent disease known as asthma. The primary cells that infiltrate the airways in response to antigen exposure are PMNs and eosinophils, cells that can release cellular components, and damage the airways. We adapted a double-balloon endotracheal tube to study the cellular response to three de novo synthesized lipid mediators (LTB4, PAF-acether and 15 HETE) found in respiratory fluids following antigen exposure. In random repeat challenges in groups of 7 dogs using mongrel dogs at 240 min following exposure to 10(-6) M agonists, the PMN content of the perfused fluid was 870 +/- 240, 1632 +/- 883, 515 +/- 395, and 1575 +/- 214 cells/ml/5 high power fields for vehicle, LTB4, PAF, and 15 HETE respectively. Eosinophils that infiltrated the lumen at 240 min were 162 +/- 23, 608 +/- 287, 502 +/- 23, 115 +/- 14 cells/ml/5 HPF for vehicle, LTB4, PAF, and 15 HETE respectively. Thus LTB4 and PAF-acether significantly (p less than 0.05) increased eosinophils, and LTB4 and 15 HETE increased PMNs (p less than 0.05). After determining the agonist response for the 3 agonists we included 2 specific antagonists in the perfusate. The LTB4 antagonist U-75,302 10(-5) M, and the PAF antagonist L 652,731 10(-5) M in chambers containing LTB4 and PAF-acether respectively blocked significantly the influx of PMNs and eosinophils compared to vehicle (p less than 0.01). Methylprednisolone 5 mg/kg i.m.--18 hrs blocked eosinophilia to PAF and LTB4. Oral U-78,517F a Trolox amine lazaroid, active as an inhibitor of lipid peroxidation, 30 mg/kg--18 hrs significantly blocked eosinophilia to PAF-acether and LTB4 directed chemotaxis compared to vehicle (p less than 0.05) but not 15 HETE. Specificity was shown for each antagonist since the PAF and LTB4 antagonists did not block the opposite agonist. Use of this novel in vivo chemotaxis model allows the additional advantage of studying chemotaxis in living tissue.

We are interested in the physiologic mechanisms of eosinophil activation because of the presumed participation of activated eosinophils in the inflammatory sequelae of asthma. Suspecting that other formed elements of the blood may contribute to such an activation, we examined the capacity of platelet-derived growth factor (PDGF), a product of activated platelets, to activate eosinophils. We found that highly purified monkey and human eosinophils, but not guinea pig eosinophils, were activated by PDGF (superoxide anion production) in a dose-dependent fashion. Moreover, this activation was further dependent on a prior ‘priming’ of the cells by a brief exposure to subthreshold concentrations of phorbol ester. The response was specific for the BB homodimer of PDGF suggesting it is receptor-dependent.

Leukotrienes (C4, D4) have been shown to enhance mucus secretion in both isolated human airway tissue and intact canine trachea in vivo. They also have been implicated as putative mediators in several airways diseases. In previous canine studies the mucus enhancing effect of leukotriene-C4 was blocked by atropine, FLP 55,712, and hexamethonium but not by cutting the superior laryngeal and vagus nerves. We anesthetized mongrel dogs with chloralose (100 mg/kg) and urethane (500 mg/kg) and ventilated them on a pump. To visualize the secretions from submucosal glands, we exposed the mucosa of the upper trachea and coated its surface with powdered tantalum. Secretions from the glands formed elevation in the tantalum layer (hillocks) with time: the number of tracheal hillocks (an index of mucus secretion) was measured at one or more of the four time points on six dogs after each treatment of the treatment sequence: no LTC4, LTC4, no LTC4 + blocker, and LTC4 + blocker. The potential blocker was diphenhydramine, an H1 antagonist for histamine. LTC4 was injected into the cranial thyroid artery which directly feeds the tracheal segment. We observed hillocks through a dissecting microscope, and the number of hillocks per 1.2 cm2 were counted for a 1-4 min interval. In 6 dogs with 12 responses, LTC4 (10 micrograms) gave a positive response that was significantly different from control (p less than 0.01-0.05) at 2-4 min. Diphenhydramine (n = 6), 0.5 mg/kg, a dose which blocked a histamine challenge without blocking an acetylcholine challenge of secretion, gave a statistically significant (p less than 0.01-0.05) reduction in mucus secretion at 1-4 min. These results support the conclusion that leukotriene C4 induces mucus secretion in dogs that is blocked by prior diphenhydramine administration. This would indicate histamine has a role, but as yet an unknown mechanism in the action of leukotriene-C4 in enhancing mucus.

A new inhibitor of IgE-mediated allergic reactions has been studied in rats. This new inhibitor is chemically different from cromoglycate and shows qualitative and quantitative advantages over it. It is 2,500 times more active in rats than disodium cromoglycate and shows oral activity. This new inhibitor possesses no bronchodilator or end-organ antagonism activity and does not raise intracellular cAMP levels but as cromoglycate acts by inhibition of mediator release. Multiple high (25 mg/kg) doses in rats lead to tachyphylaxis. This effect is reversed both by decreasing the first dose and increasing the time between doses. Over a 5- to 10-fold concentration range, this inhibitor in mast cells can be stimulatory or inhibitory to 45Ca++ flux into cells. This ability to inhibit ionophore-induced 45Ca++ flux correlates well with inhibition of histamine release.

Highly purified rat peritoneal mast cells were prelabeled with tritiated 5-hydroxytryptamine and mediator release was induced with compound 48/80, polymyxin B, concanavalin A, immunoglobulin E followed by antibody to IgE, and by the calcium ionophore, A 23187. In none of these cases was it possible to achieve a dependence of mediator release on exogenously added calcium. A brief preincubation of the cells with L-palmitylcarnitine chloride (PC) before the addition of the secretion-inducing agent (secretagogue) resulted in inhibition of mediator release which differed in extent depending on the nature of the secretagogue which was used. This inhibition, which occurred in the absence of effects of the inhibitor on spontaneous mediator release or on cell viability, persisted even if the inhibitor was removed by centrifugation and washing of the cells before addition of the secretagogue. Other acyl carnitines also inhibited mediator release provided the acyl group was at least 14 carbon atoms long. Atractyloside, an inhibitor of acyl carnitine-dependent fatty acid β-oxidation, and several calcium ATPase inhibitors (at non-inhibitory concentrations) failed to affect the inhibition of mediator release caused by PC. On the other hand, the extent of inhibition of compound 48/80-induced mediator release (but not the release which was induced by the other secretagogues) was markedly affected by the calcium concentration in the medium. The 50% inhibitory dose for PC decreased from 12 μM in the absence of calcium of 5 μM in the presence of 3 mM calcium. Spontaneous uptake of radiolabeled calcium by purified mast cells was inhibited by preincubation with 10 μM PC and enhanced by preincubation with 1 μM PC. On the other hand, calcium uptake which was induced by compound 48/80 was inhibited by 1 μM PC. These results suggest that acylcarnitine esters may interfere with the intracellular calcium economy of mast cells, thereby inhibiting mediator release. In this respect, these results are reminiscent of recently reported observations on the effect of PC on calcium transport in sarcoplasmic reticulum.

Leukotrienes have been implicated as putative mediators in several air way diseases. In previous canine studies it was shown that leukotriene C4 (LTC4) enhanced fluid secretion over baseline values and this enhancement could be blocked by hexamethonium. This indicates that leukotrienes have as one of their actions, stimulation of ganglionic motor neurons. In the present study, we determined that LTC4 acts at a similar site as the specific nicotinic receptor agonist dimethylphenylpiperazinium (DMPP). Both LTC4 and DMPP when given alone enhanced mucus secretion and induced tracheal muscle contraction over control baseline (p 4 enhanced the DMPP effect of muscle contraction at 5 and 8 μg by a synergistic amount, while the secretion was only additive. The slopes of the dose-response curves for DMPP+ LTC4 did not differ by a statistically significant amount. LTC4 and DMPP act on a similar, if not the same, ganglionic receptor.

Ab initio Hartree-Fock SCF calculations with the molecular fragment technique have been performed on several drugs which exhibit activity in the rat passive cutaneous anaphylaxis (PCA) assay. Representative molecules of the following types were included in the series: oxanilic acids, 1,4-dihydro-4-oxoquinaldic acids, and 4-oxo-4H-1-benzopyran-2-carboxylic acids. A quantitative relationship has been established between the observed biological activity and an electronic index obtained from the calculations. The correlation is rationalized in terms of charge-transfer stabilization of the drug-receptor complex.

By the use of close arterial injection of leukotrienes into the circulation supplying the upper cervical canine trachea, it has been possible to assess the secretogogue effects of leukotriene C4, and D4 on mucus secretion. Both LTC4 and LTD4 increased mucus secretion over baseline levels by a statistically significant level (p = less than 0.05). LTD4 was more potent than C4 with relative potencies of 2500, 320, 630, and 500 based on hillock formation (a measure of secretion) at 1, 2, 3, and 4 minutes after injection. The overall difference in potency in this animal model of mucus production was LTD4 greater than C4 by 1000-fold.

The pyrrole analog of prostacyclin, 6,9-deepoxy-6,9-N-phenylimino-delta 6,8-prostaglandin I1 was synthesized from PGF2 alpha methyl ester. This pyrroloprostacyclin (U-60,257) and its methyl ester (U-56,467) have been shown to inhibit leukotriene C/D biosynthesis and antagonize leukotriene C/D contractions in vitro. Antigen induced bronchopulmonary changes in monkeys and guinea pigs are inhibited by U-60,257 in vivo.

A series of dialkyl N,N'-(m-phenylene)dioxamates was synthesized by treatment of the requisite m-phenylenediamines with an alkyloxalyl chloride in the presence of triethylamine. Hydrolysis with sodium hydroxide solution gave the corresponding N,N'-(m-phenylene)dioxamic acids. Several N,N'-(p-phenylene)dioxamic acids were synthesized also in the same manner starting with the requisite p-phenylenediamines. These compounds were tested in the rat passive cutaneous anaphylaxis (PCA) assay. When tested iv, activity was found in the N,N'-(m-phenylene) dioxamic acids up to 2500 times that shown by disodium cromoglycate [50% inhibition at 0.001 mg/kg for N,N'-(2-chloro-5-cyano-m-phenylene)dioxamic acid (compound 61)]. Oral activity was seen in this series of compounds with duration of activity up to 120 min. Oral activity was detected in diethyl N,N'-(2-chloro-5-cyano-m-phenylene)dioxamate (compound 38) at levels of drug as low as 0.1 mg/kg.

Adenosine alone at 0.1 and 1.0 mg per tracheal segment stimulated mucus secretion by 52% and 88%, respectively, compared to baseline (P less than 0.0001). The site of the potent secretagogue effect of adenosine in canine trachea was consistent with A2 activation. A2 site activation and enhanced secretion were also induced by N-ethylcarboxamide adenosine and dipyridamole. N6-R-phenylisopropyl adenosine (PIA) and 2',5'-dideoxyadenosine (ddAdo) inhibited the adenosine-induced secretion (35% and 42%, respectively). However, when PIA or ddAdo were administered in conjunction with the potent phosphodiesterase inhibitor, methylisobutylxanthine (MIX), the effects of PIA were potentiated and the effects of ddAdo were reversed, yielding stimulation (A2) and antagonism (A1) of secretion, respectively. 8-Phenyltheophylline by aerosol was a very potent antagonist of the secretagogue effect of adenosine (70% inhibition; P less than 0.00001).

The IgE-mediated hypersensitivity to Ascaris antigen in reactor rhesus primates was used to assess the pharmacologic profile of U-60,257, a pyrrole analog of prostacyclin. Whether the compound was given by aerosol or intravenously, it effectively inhibited the bronchopulmonary effects of antigen challenge. Dose responses by the aerosol route showed a dose-dependent inhibition of resistance (RL) and compliance (Cdyn) changes (100% ± 0 to 10.2% ± 14.5 for RL and 79.9% ± 20 to 0% for Cdyn after 15 breaths of 1.0–0.01 % solutions delivered into the lungs; n = 16). When administered at 1.0% by aerosol the duration of the inhibitory effect was 6 h (n = 3). Dose-dependent inhibition in both RL (93.3% ± 5.6 to 69.4% ± 34.8) and Cdyn (51.4 ± 40.4 to 47.0% ± 28.5) was also seen when the compound was given intravenously (5.0–0.01 mg/kg; n = 16). U-60,257 is a selective inhibitor of leukotriene synthesis in in vitro systems. Therefore, the finding of synergism in the inhibition of response of primates to Ascaris between this compound and the H1 histamine blocker diphenhydramine suggests that the leukotrienes may play an etiologic role in this response.

The IgE-mediated hypersensitivity to Ascaris antigen in reactor rhesus primates was used to assess the pharmacologic profile of U-66,858 (1-acetoxy-2-n-butyl-4-methoxy-naphthalene). When the compound was given by the oral route, it showed dose-related inhibition of resistance (RL) and compliance (Cdyn) changes. When the compound was given by the aerosol route, it showed dose independent inhibition. In 15 animals, aerosols (52 ± 32 to 53 ± 10% for RL, p = 0.05 and 45 ± 19 to 28 ± 19% Cdyn inhibitions, p = 0.05) for 5.0–0.1% aerosol. By the oral route, inhibition was seen at 1–4 h following administration. In 5 animals, oral doses of 10 and 5 mg/kg inhibited (RL by 98 ± 2 to 78 ± 1.5 %, p = 0.01 and Cdyn by 75 ± 17 to 60.9 ± 9.1 %, p = 0.05) by 10 and 5 mg/kg U-66,858, respectively. The in vivo demonstration of inhibition of pulmonary bronchoconstriction by this compound, in a model known to be leukotriene sensitive, coupled with its potent in vitro inhibition of 5-lipoxygenase enzymes, suggests this compound may be of use in 5-lypoxygenase-mediated models of asthma.

Immediate type hypersensitivity to Ascaris antigen in Rhesus reactor monkeys was used to assess the pharmacologic profile of cromolyn Na (DSCG) and a new inhibitor of IgE-mediated lung function changes (U-42,585E). Two parameters used to measure lung function, respiratory rate increase and tidal volume decrease, were significantly altered in a dose-related fashion by U-42,585E when the latter was administered either by the intravenous, intrabronchial, or the oral route. Oral activity of U-42,585E was demonstrated with doses as low as 5 mg/kg in Ascaris reactor primates.

A new inhibitor of IgE-mediated allergic reactions has been studied in rats. This new inhibitor is chemically quite different from cromoglycate and shows qualitative and quantitative advantages over it. This new inhibitor possesses no bronchodilator or end-organ antagonism activity, but as cromoglycate acts by inhibition of mediator release. Multiple doses in rats lead to tachyphylaxis. This effect is reversed both by decreasing the first dose and increasing the time between doses.

Orally administered iodoxamide ethyl (U-42,718) inhibited anaphylactic reactions in a dose-related manner in the following test animals: (1) in the rat PCA reaction, excellent activity (75% inhibition at 0.1 mg/kg) was seen with a duration of activity of 30 min, (2) In the ascaris-sensitive primate (45% inhibition at 1.0 mg/kg) in lung parameters related to increased resistance and decreased compliance which persisted for up to 3 h, and (3) 50 mg/kg protected guinea pigs, sensitized to egg albumin, from lung function changes. Activity in these animal systems indicates that this orally active drug may hold promise in clinical asthma.

Incubation of antigen with normal mouse peritoneal exudate cells in vitro and subsequent reinjection of the washed cells into syngeneic mice resulted in increased antibody titers as compared to mice injected with antigen alone. Several of the variables influencing this system were studied with and without the stimulus of complex homoribopolynucleotides (poly A:U or poly I:C) as adjuvants to determine the cellular site of action of the latter. It was found that addition of poly A:U or poly I:C caused a further rise in circulating antibody levels which correlated with increased RNA synthesis, suggesting that the macrophage was one cell affected by this adjuvant. Actinomycin D was found to inhibit the rise in titer induced by PEC and this inhibition could be overcome by poly A:U. Injection of the polynucleotides 18 hr before antigen resulted in depression of circulating antibody levels, and poly A:U or poly I:C injected 18 hr before harvesting PEC and incubation with antigen also inhibited the capacity of the PEC to increase antibody levels. A 4S RNA-rich fraction was purified after treatment with phenol of PEC exposed to antigen in vitro, and under the stimulus of poly A:U this RNA was capable of inducing specific antibody titers and rosette-forming cells on injection into mice. Antigen contamination of Pronase-treated RNA, active biologically, was below 10–11 g as determined isotopically.

Discussion and Summary Our results confirm the observations of Foreman et al. (10) that calcium-specific ionophores can initiate histamine release. In an accompanying communication (17) the production of a substance resembling SRS-A under the influence of A23, 187 is also reported. In this study we addressed ourselves to the question: “Can these ionophores be used as a tool to study DSCG's mechanism of inhibition of secretory histamine release?” DSCG does not chelate divalent cations very well (2, 12), but this conclusion is based on measurements of Ca in the free medium. Under similar conditions in which the EDTA shows significant evidence of complex formation with divalent cations the pH titration curve of DSCG is unaffected. The calcium involved in histamine release may be cell bound in a specific spatial arrangement that lends itself to “tying” up by DSCG at the cell surface. Thus, it is possible that one of the ways that DSCG inhibits is by preventing the increased uptake of calcium by cells under the influence of histamine release initiators such as compound 48/80 or antigen-antibody reactions, and thereby inhibiting mediator release. Physicochemical studies on binding to cell-bound ions by DSCG will be required to answer this point. DSCG can exist in a water lattice form that may have a specific binding capacity when divalent cations like calcium are present (18). It may thus exert its effect by existing in juxtaposition to the cell surface and binding ions, rather than actually binding to the mast cells. This juxtaposition could result in decreased calcium movement and diminished release of histamine. These studies do not answer the questions of peculiar self-inhibition effects seen with DSCG, nor do they explain the biphasic nature of the drug's dose response in most in vitro cells systems. They are consistent with DSCG being involved in Ca economy of the cells, but it may also inhibit some unidentified enzyme needed for release. Self-inhibition effects of DSCG in ionophore-induced release are being studied as well as desensitization as a consequence of calcium deprivation to the cell and will be reported at a later date.

Single low doses of lodoxamide tromethamine or lodoxamine ethyl inhibit histamine release in animals by inhibiting calcium flux into the cell. Single high doses enhance histamine release because they stimulate guanylate cyclase and inhibit guanylate phosphodiesterase which breaks down cyclic GMP. As cyclic GMP increases in the cell membrane, calcium is forced into the cell. The calcium influx stimulates the mast cell to release histamine which could be responsible for some of the undesirable effects that have occurred in patients who have received 100 to 1000 times the doses required to produce the desired inhibition of histamine release. Second and subsequent LT or LE doses administered 30–60 min after the preceding dose do not inhibit histamine release in animals. Prevention of the expected effect is tachyphylaxis of the second dose by the first. Because atropine completely blocks the tachyphylaxis, cholinergic receptors may mediate that phenomenon.

SummaryThe predosing of rats with different doses of DSCG followed by a sufficient time interval so that the drug's effect was gone, then injection of another DSCG dose with antigen led to a high dose inhibition and low dose sparing of the second DSCG dose. The degree of sparing was concentration related and so was the degree of self-inhibition. These effects persisted for a 20-hr dosing regimen but were gone if one gave the last dose 48 hr before challenge.

Summary After a single 5-min exposure to an aerosol containing ovalbumin, rats produced a heat labile, skin sensitizing antibody by 14 days. By radioiodination of the protein the amount of antigen deposited in the respiratory tract during this immunization was calculated to be 12.8 µg. The amount of protein swallowed by the animals (80 µg) was incapable of elaborating an IgE response. The synthesis of IgE under these conditions was absolutely dependent on heat-killed Bordetella pertussis vaccine given i.p. before exposure. Second exposures to antigen resulted in renewed and accelerated IgE production. Repeated aerosolization with a low dose of antigen (0.1%) caused no detectable antibody synthesis.

Inhibition of canine mucus secretion in vivo induced by arachidonic acid administration was correlated with a reduction of 15 Hete levels in canine mucus. Antioxidants and inhibitors of lipid peroxidation were effective inhibitors of both mucus secretion and 15 Hete production. This same series of inhibitors also dose dependently inhibited Fe2+ dependent oxidation of arachidonic acid in vitro as assessed by an inhibition of thiobarbituric acid reactive material and conjugated diene formation. These data argue for an involvement of reactive oxygen species and lipid peroxidation in the generation and elaboration of mucus secretion.

A group of (2-carboxy-1,4-dihydro-4-oxoquinolyl)oxamic acids (5) containing the oxamic acid group in the 5,6, or 7 positions were synthesized and investigated for antiasthma activity as indicated by the passive cutaneous anaphylaxis (PCA) reaction in rats. Also synthesized and investigated were two (2-carbosy-1,4-dihydro-4-oxobenzo[h]-quinolyl)oxamic acids (9 and 10). Several of the compounds synthesized (viz. 5e, 5f, and 10) showed activity in the PCA test approximately 25 times that shown by disodium cromoglycate (1), as measured by the ID50 doses.

Adenosine injected via the cranial thyroid artery route into canine tracheas was a very potent and quick acting secretagogue in a unique model of mucus production. It is not known if adenylate cyclase activation was involved, but adenosine caused a statistically significant, dose related enhancement over baseline secretion rates in seventeen mongrel dogs (0.01 mg 18.4 +/- 5.04% increase N.S.; 0.1 mg 62.5 +/- 9.00% P less than 0.001; and 1.0 mg 91.2 +/- 14.1% P less than 0.001). The response was complete in less than two minutes and muscle relaxation usually accompanied the response. Adenosine activation of submucosal glands, mast cells or airway tissue may mean that adenosine is an agonist in pathophysiological abnormalities of the airways.

A series of compounds containing two or more 4-oxo-1,4-dihydropyridine-2-carboxylic acid units fused to a central aromatic nucleus was synthesized and tested in the rat passive cutaneous anaphylaxis (PCA) assay for potential antiallergy activity. Most of the compounds of this series showed significant activity in the PCA assay. Three of these compounds. 11d, 13f, and 21, were more than 250 times as active as the standard drug, cromolyn sodium. The synthesis and biological activity are discussed.

The synthesis and biological activity in the rat passive cutaneous anaphylaxis (PCA) test of a new class of compounds, 1-[(ethoxyamino)methyl]-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins, are reported. These compounds are synthesized from the adduct of 1-(bromomethyl)-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins, are reported. These compounds are synthesized from the adduct of 1-(bromomethyl)-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepin and ethylene glycol or chloroethanol. The influence of the amine function on activity in the rat PCA is discussed. Aryl- or heteroarylpiperazines favor activity with this class of compounds.

A series of compounds containing two or more 4-oxo-1,4-dihydropyridine-2-carboxylic acid units fused to a central aromatic nucleus was synthesized and tested in the rat passive cutaneous anaphylaxis (PCA) assay for potential antiallergy activity. Most of the compounds of this series showed significant activity in the PCA assay. Three of these compounds. 11d, 13f, and 21, were more than 250 times as active as the standard drug, cromolyn sodium. The synthesis and biological activity are discussed.

The hypothesis that active immunization of primates to give airway allergic responses would also confer on them a hyperreactivity to a nonspecific stimulus such as histamine was tested in 29 normal rhesus primates. At 6 wk after immunization, specific primate IgE (Rast) to Ascaris antigen had increased from 0.35 +/- 0.17 to 0.98 +/- 0.35 units/ml x 10(2) (p less than 0.05). Histamine released from bronchial alveolar lavage cells in response to antigen increased from 5.4 +/- 0.67 to 24 +/- 1.8ng/10(6) cells (p less than 0.05). In a subgroup of seven animals, airway resistance RL and compliance before and after feeding embryonated Ascaris ova increased from RL 3.5 +/- 3.1 to 275 +/- 212 cm H2O/L/s (p = 0.02) and Cdyn fell from 81 +/- 10 to 11.3 +/- 12 ml/cm H2O (p less than 0.05). The bronchial lavage fluid contained a very high percentage of eosinophils after infection, 8 +/- 3.1 to 24 +/- 13% per 500 cells counted, and did not increase appreciably upon later antigen challenge, 24 +/- 13 to 31 +/- 11% of the cells at 9.5 h after antigen challenge (p = NS). When a group of seven of these 29 animals were compared for their histamine responsiveness before and after acquired Ascaris airway reactivity, there was no difference in 19 animals. Pulmonary response to histamine delivered from freon canisters at doses of 0.005, 0.01, 0.025, and 0.05% did not change (RL, 141 +/- 102 to 93 +/- 62; Cdyn, 49 +/- 7 to 46 +/- 11% change before and after, respectively) (p = NS).(ABSTRACT TRUNCATED AT 250 WORDS)

A series of dialkyl N,N'-(m-phenylene)dioxamates was synthesized by treatment of the requisite m-phenylenediamines with an alkyloxalyl chloride in the presence of triethylamine. Hydrolysis with sodium hydroxide solution gave the corresponding N,N'-(m-phenylene)dioxamic acids. Several N,N'-(p-phenylene)dioxamic acids were synthesized also in the same manner starting with the requisite p-phenylenediamines. These compounds were tested in the rat passive cutaneous anaphylaxis (PCA) assay. When tested iv, activity was found in the N,N'-(m-phenylene) dioxamic acids up to 2500 times that shown by disodium cromoglycate [50% inhibition at 0.001 mg/kg for N,N'-(2-chloro-5-cyano-m-phenylene)dioxamic acid (compound 61)]. Oral activity was seen in this series of compounds with duration of activity up to 120 min. Oral activity was detected in diethyl N,N'-(2-chloro-5-cyano-m-phenylene)dioxamate (compound 38) at levels of drug as low as 0.1 mg/kg.

Because leukotrienes have been implicated as putative mediators in upper airways disease, we studied whether leukotriene C4 (LTC4) might also have a mucus enhancing effect on submucosal glands. We anesthetized mongrel dogs with chloralose (100 mg/kg) and urethane (500 mg/kg) and ventilated them on a pump. To help visualize the secretions from submucosal glands, we exposed the mucosa of the upper trachea and coated its surface with powdered tantalum. Secretions from the glands (hillocks) were measured with time: The number of hillocks was measured at four time points on 19 dogs after each treatment in the sequence: no LTC4, LTC4, no LTC4 and LTC4 + blocker. The potential blockers were nerve cutting, atropine, FPL-55,712, and hexamethonium. Each potential blocker was used on 3-5 dogs. LTC4 was injected into the cranial thyroid artery. In 19 dogs with 27 responses, LTC4(8.6-11.0 micrograms) gave a positive response that was significantly different from control (P less than 0.01) at 1-4 min. These effects were not abolished in 5 dogs by cutting the superior laryngeal (SLN) and the vagus nerves (P less than 0.01). Pretreatment of the dogs (n = 5) with atropine, hexamethonium and the specific SRS-A (LTC4) antagonist FPL 55,712 (n = 3) gave a statistically significant (P less than 0.01-0.05) reduction in mucus secretion at all times for atropine, hexamethonium, and at all times except 4 min for (FPL 55,712). These results indicate that leukotriene C4 induces mucus secretion in dogs. This secretion does not depend on an intact reflex pathway but is altered at the individual gland by agents which block ganglionic motor pathways.

Normal and Ascaris reactor primates were compared for their bronchial pulmonary response to aerosolized leukotriene D4 (LTD4). When 10 micrograms/ml LTD4 was aerosolized (total amount delivered to endotracheal tube was 1.0 micrograms) into the lungs of 6 normal primates, a small increase in total lung resistance (RL) was noted (4.4 +/- 4.5% increase, in 19 separate challenges). However, a larger effect was seen in compliance (27.6 +/- 15.8% decrease, n = 19). Ascaris reactors (n=4) demonstrated a larger RL effect than normals with almost an identical Cdyn change (RL 36.1 +/- 27.7% increase, Cdyn 32.8 +/- 18.8% decrease n = 12). When the pharmacological blockers diphenhydramine, 0.5 mg/kg and atropine, 0.5 mg/kg were administered iv separately before LTD4 challenge, significant antagonist activity was seen. Diphenhydramine inhibited the LTD4 response in normal primates (RL 64.2 +/- 44.3% and Cdyn 50.5 +/- 40.9% n = 6) and in reactors (RL 47.8 +/- 43.1% and Cdyn 19.2 +/- 20.8% n = 4). Atropine inhibited normals (RL 100% and Cdyn 73.1 +/- 32.7% n = 2) and reactors (RL 96.3 +/- 7.7 and Cdyn 47.4 +/- 35.1% n = 3). These results indicate that the LTD agonist action is partially mediated through histamine, primarily acting on lung resistance (large airways) and, in addition, may have a reflex atropine-sensitive component. The difference between the response of normal and reactor primates to LTD4 is primarily a histamine-mediated large airway response.

A group of (2-carboxy-1,4-dihydro-4-oxoquinolyl)oxamic acids (5) containing the oxamic acid group in the 5,6, or 7 positions were synthesized and investigated for antiasthma activity as indicated by the passive cutaneous anaphylaxis (PCA) reaction in rats. Also synthesized and investigated were two (2-carbosy-1,4-dihydro-4-oxobenzo[h]-quinolyl)oxamic acids (9 and 10). Several of the compounds synthesized (viz. 5e, 5f, and 10) showed activity in the PCA test approximately 25 times that shown by disodium cromoglycate (1), as measured by the ID50 doses.

A most active biologue of disodium cromoglycate (DSCG) available, lodoxamide tromethamine (LT), has been studied and characterized pharmacologically, in animal and human models of asthma. It has self-tachyphylaxis, but has oral activity (lodoxamide ethyl) in rats, primates, and man. In rats (LT) was 2,500 X more active than DSCG (ID50 = 0.001 mg/kg), in primates the drug was also active by several routes (inhalation 1 microgram/kg, IV 0.001 mg/kg, and oral 10 mg/kg). In isolated rat peritoneal mast cells, the compound displayed a biphasic dose response inhibition to histamine release initiated by (48/80, anti-IgE, and the calcium ionophore A23,187) with IC50 values of 0.1-50 microM. The consistent finding relating to its mode of action was its ability to inhibit 45calcium flux into the mast cell in response to antigen or A23,187. Clinical evaluations of lodoxamide tromethamine showed that at aerosol doses of 1.0 mg or less, it demonstrated significant inhibitory activity against antigen or exercise induced bronchospasm. However, in pilot evaluation studies in clinical asthma settings, the compound could not be shown to spare bronchodilator usage, relative to placebo, or be shown to be more effective than placebo treated patients based on other clinical endpoints. The reason for rat and primate models not being predictive for human long-term clinical asthma in the characterization of anti-release compounds is not known.

The synthesis and biological activity in the rat passive cutaneous anaphylaxis (PCA) test of a new class of compounds, 1-[(ethoxyamino)methyl]-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins, are reported. These compounds are synthesized from the adduct of 1-(bromomethyl)-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins, are reported. These compounds are synthesized from the adduct of 1-(bromomethyl)-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepin and ethylene glycol or chloroethanol. The influence of the amine function on activity in the rat PCA is discussed. Aryl- or heteroarylpiperazines favor activity with this class of compounds.

A novel leukotriene synthesis inhibitor, piriprost (U-60,257) was characterized in three quantitated assays of secretion in the canine in vivo trachea. The normal baseline secretion rate was inhibited in a dose-related fashion when the inhibitor was given via the cranial thyroid artery (decrease compared to control dogs: 27 ± 3.11% at 0.1 mg, n = 3, p < 0.05; 56 ± 4.85% at 0.5 mg, n = 3, p < 0.0l; 61.5 ± 6.8% at 1.0 mg, n = 14, p < 0.001). Hypoxia gas mixtures (8% O2 and 92% N2 4 min) delivered into the lung caused a mean increase in secretion of 160 ± 11.2% (p < 0.05). When the leukotriene inhibitor (1.0 mg) was given into the close arterial segment before hypoxia, this increased secretion response was blocked (160 ± 11.2 to 40 ± 16.7%; p < 0.05). Finally, arachidonic acid caused a potent and long-lasting enhancement in secretion (179 ± 9.0% of control; p < 0.01) that was partially blocked by 5 mg/kg indomethacin 30 min before (90 ± 12.7% of arachidonic acid; p < 0.01), and when 1.0 mg U-60,257 preceded the arachidonic acid, an additional inhibition of 50% of the indomethacin inhibition was seen (p < 0.01; n = 14). These findings are consistent with the hypothesis that canine secretion is modulated by arachidonic acid metabolites that act as agonists for secretion both by the cyclooxygenase and lipoxygenase pathways.

Cells of all organisms contain one or more basic substances. In the bacteria and the viruses these are polyamines while in animal cells the polyamines are usually supplemented by basic proteins such as the histones. These basic molecules have been implicated in a large number of biochemical processes. They are known to affect RNA synthesis and protein synthesis (Muench, 1969, 1967; Nicholson and Peacocke, 1966; Scholtissek and Brecht, 1966; O’Brien, Olenick and Hahn, 1966, and many others). In the viruses they are presumed to play a key role in the folding of the viral nucleic acids to permit their encapsulation in the limited space afforded inside the viral capsule. Similarly, the histones appear to be synthesized in mammalian cells in perfect balance with the synthesis of DNA (Bazill and Philpot, 1963; Butler and Cohn, 1963; Littlefield and Jacobs, 1965) and they occur as complexes with DNA. They are likely to play an important role in the regulation of the availability of the DNA genome for translation into RNA and protein (Bonner and Huang, 1966).