Involvement of IL-5 in a murine model of allergic pulmonary inflammation: prophylactic and therapeutic effect of an anti-IL-5 antibody.

Interleukin-5 (IL-5) is important in the control of differentiation, migration, and activation of eosinophils. In order to study the role of IL-5 in the development of eosinophilic inflammation of the airways, we have used a monoclonal antibody to murine IL-5 (TRFK-5) in a murine model of allergic pulmonary inflammation. B6D2F1 mice were sensitized with alum-precipitated ovalbumin and were challenged with aerosolized ovalbumin on day 12 after sensitization. Samples of bronchoalveolar lavage (BAL) fluid, lung tissue, blood, and bone marrow aspirate were collected at different times after ovalbumin challenge. Twenty-four hours after challenge there were significant increases in the number of eosinophils in the BAL fluid, lung tissue, and blood while bone marrow eosinophils were decreased. Treatment of sensitized mice with TRFK-5 (0.01-1 mg/kg, i.p.) 2 h before ovalbumin challenge reduced the numbers of eosinophils in the BAL fluid and lung tissue and prevented the decrease in bone marrow eosinophils in a dose-dependent fashion. The number of eosinophils in the BAL fluid, peribronchial and alveolar regions of the lung was also reduced when TRFK-5 (2 mg/kg, i.p.) was given up to 5 d after ovalbumin challenge. Furthermore, there was no evidence of increased epithelial damage, edema, or the presence of mucus that could have resulted from eosinophil apoptosis and release of toxic proteins after neutralization of IL-5. These results demonstrate an important role for IL-5 in the development of eosinophilic inflammation of the airways and for the migration of eosinophils from the bone marrow into blood in response to antigen challenge.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of a murine model of allergic pulmonary inflammation.

Pulmonary inflammation with eosinophil (EOs) infiltration is a prominent feature of allergic respiratory diseases such as asthma. In order to study the cellular response during the disease development, an animal model of IgE-mediated pulmonary inflammation with characteristic eosinophilia is needed. We developed a method for inducing severe pulmonary eosinophilia in the mouse and also studied the numbers of EOs in blood and bone marrow and the response to corticosteroid treatment. Animals were sensitized with alum-precipitated ovalbumin (OVA) and challenged with aerosolized OVA 12 days later when serum IgE levels were significantly elevated. Four to eight hours after challenge there were moderate increases in the number of EOs in the bone marrow and peripheral blood, but only a few EOs were observed in the lung tissue and in bronchoalveolar lavage (BAL) fluid. Twenty-four hours after challenge, there was a marked reduction of EOs in bone marrow, while the number of EOs peaked in the perivascular and peribronchial regions of the lung. Forty-eight hours after challenge, the highest number of EOs was found in the BAL fluid, making up > 80% of all cells in that compartment. The high levels of EOs in the lung tissue and BAL fluid lasted for 2-3 days and was followed by a more moderate but persistent eosinophilia for another 10 days. Nonsensitized animals showed no significant changes in the number of EOs in BAL fluid, lungs, blood or bone marrow. Histopathological evaluation also revealed epithelial damage, excessive mucus in the lumen and edema in the submucosa of the airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effect of the TRFK-5 anti-IL-5 antibody in a guinea pig model of asthma.

To investigate the role of IL-5 in airway hyperreactivity and pulmonary eosinophilia, we used a model of allergic asthma in guinea pigs and a neutralizing monoclonal antibody (TRFK-5) directed against murine IL-5. Sensitized guinea pigs were challenged with 1% ovalbumin (OVA) aerosol and assessed for airway eosinophilia (by bronchoalveolar lavage [BAL] and histologic evaluation of airway tissue) and bronchoconstrictor responsiveness to substance P (SP) (as RL100 and Cdyn40) 24 h later. OVA challenge of sensitized animals caused a significant increase in airway responsiveness to SP, with a 4.9-fold decrease in RL100 and a 4.7-fold decrease in Cdyn40. Accompanying this increased sensitivity to SP was a 9-fold increase in eosinophils recovered in BAL and a 4- to 5-fold increase in eosinophils in intrapulmonary bronchial tissue. Intraperitoneal treatment with 10 mg/kg of the IL-5 antibody 2 h before OVA challenge blocked BAL and lung tissue increases in eosinophils but had no effect on the development of airway sensitivity to SP. In contrast, similar treatment with 30 mg/kg of this antibody blocked OVA-induced increased sensitivity to SP as well as BAL and lung tissue eosinophilia. These data suggest a critical and possibly independent role for IL-5 in allergic airway hyperresponsiveness and the accumulation of eosinophils within the lung of the guinea pig.

In vitro and in vivo immunopharmacological profile of Sch 40120.

Sch 40120 (10-(3-chlorophenyl) - 6,8,9,10- tetrahydrobenzo [b] [1,8] naphthyridin-5 (7H)-one) is a leukotriene inhibitor that is also a potent inhibitor of acute inflammatory responses in rodent systems. In the present study, we have evaluated the effects of this drug on immune function as well as its activity in models of immune mediated chronic inflammatory disease. Sch 40120 was particularly effective in suppressing T cell proliferative responses in vitro. Antigen-specific and poly-clonally-induced in vitro antibody responses were also inhibited by the drug. However, the in vivo potency of Sch 40120 in suppressing immune responses and in inhibiting the pathological changes seen in rodent models of autoimmune disease (EAE and adjuvant arthritis) was somewhat less than that previously observed in models of acute inflammation. Nevertheless, the spectrum of activities exhibited by Sch 40120 suggests that it will be particularly useful in the treatment of psoriasis where T lymphocytes have been implicated in the development of disease and leukotrienes appear to have a role in the persistence of psoriatic plaques.

Interleukin-5 modulates eosinophil accumulation in allergic guinea pig lung.

Based on its involvement in eosinophil biology, interleukin 5 (IL-5) may play a role in the pulmonary eosinophilia associated with allergic reactions. We have examined that hypothesis using a neutralizing antibody to IL-5 in ovalbumin-sensitized guinea pigs challenged with aerosolized antigen. The extent of eosinophilia has been quantitated in bronchoalveolar lavage (BAL) and by histologic evaluation of lung tissue sections. Acute intraperitoneal administration of a rat IgG, monoclonal antibody to murine IL-5 derived from TRFK-5 cells prevented lung and BAL eosinophilia in a dose-dependent fashion at and above 10 micrograms per guinea pig. Treatment with either an experimentally irrelevant, isotype-matched antibody from GL113 cells or with heat-denatured IL-5 antibody was without effect. These studies demonstrate the importance of IL-5 to pulmonary eosinophilia in challenged, allergic guinea pigs.

Sch 37370: a new drug combining antagonism of platelet-activating factor (PAF) with antagonism of histamine.

Multiple mediators are involved in the pathophysiology of allergic and inflammatory disorders. Drugs that affect the action of more than one mediator may, therefore, be particularly effective in these disorders. Two such mediators are platelet-activating factor (PAF) and histamine. From a structural series with documented antihistamine activity, Sch 37370 has been identified as a dual antagonist of PAF and histamine. In vitro, Sch 37370 selectively inhibits PAF-induced aggregation of human platelets (IC50 = 0.6 microM) and also competes with PAF binding to specific sites in membrane preparations from human lungs (IC50 = 1.2 microM). Sch 37370 also blocks the binding of [3H]pyrilamine to histamine H1 receptors in rat brain membranes. In guinea pigs, orally administered Sch 37370 is effective against bronchospasm to histamine (ED50 = 2.4 mg/kg), PAF (ED50 = 6.0 mg/kg) or serotonin (ED50 = 9.6 mg/kg). In contrast, it only weakly antagonizes methacholine-induced bronchospasm (ED50 = 51 mg/kg) and is totally inactive at 50 mg/kg against bronchospasm due to leukotriene C4 or substance P. Sch 37370 blocks hypotension in rats and a cutaneous reaction in monkeys induced by either PAF or histamine, as well as PAF-induced edema in the rat pleural cavity. In addition, Sch 37370 blocks bronchospasm induced by either antigen in sensitized guinea pigs or hyperventilation in nonsensitized guinea pigs. Sch 37370 also inhibits antigen-induced lung eosinophilia in sensitized guinea pigs and a reverse passive Arthus reaction in rats. Although Sch 37370 is not the most potent PAF antagonist or antihistamine, it is the first compound that combines these pharmacologically relevant activities and may offer important advantages over currently available antihistamine therapies.

Substituted 1,3-dihydro-2H-pyrrolo[2,3-b]pyridin-2-ones as potential antiinflammatory agents.

A series of analogues based on the 1,3-dihydro-2H-pyrrolo[2,3-b]pyridin-2-one ring system have been synthesized and shown to possess oral antiinflammatory activity in both the reverse passive Arthus reaction (RPAR) pleural cavity assay in rats and in the adjuvant-induced arthritic rat model (AAR). Several members of this series additionally exhibit an inhibitory effect on the in vivo production of prostaglandin- and leukotriene-derived products or arachidonic acid metabolism although these compounds exhibit no significant inhibitory activity against the cyclooxygenase and 5-lipoxygenase enzymes in vitro. Structure-activity relationships in this series are discussed.

Anaphylactic challenge causes eosinophil accumulation in bronchoalveolar lavage fluid of guinea pigs. Modulation by betamethasone, phenidone, indomethacin, WEB 2086, and a novel antiallergy agent, SCH 37224.

Eosinophil infiltration into bronchoalveolar areas of the lung has been assessed in guinea pigs sensitized to ovalbumin (OA) and then challenged with the aerosolized antigen. Cell content, histamine, and guinea pig albumin (GPA) have been measured in bronchoalveolar lavage (BAL) fluid from these animals. Extensive eosinophil accumulation resulted from sensitization followed by OA challenge; monocytes that initially accounted for greater than 80% of the BAL cells remained essentially constant, and neutrophils comprised less than 3% of the population throughout. Eosinophils were elevated at 3 h, peaked with a fivefold increase at 24 h, and remained elevated for at least 7 days. Histopathologic changes observed in lungs taken from sensitized guinea pigs 24 h after OA challenge confirm this eosinophilia. Increased histamine and GPA were detected only at 5 min. Oral treatment with betamethasone (ED50 = 0.4 mg/kg), phenidone (ED50 = 15 mg/kg), Sch 37224 (ED50 = 0.5 mg/kg), and WEB 2086 (ED50 = 4 mg/kg) decreased eosinophil accumulation in the BAL fluid, indicating roles for 5-lipoxygenase products and PAF in this multimediator-dependent model of allergic inflammation. On the other hand, 4 mg/kg of indomethacin increased total cells with no effect on eosinophils, precluding a major role for cyclooxygenase products. Sch 37224, an antileukotriene agent and an orally active novel antiallergy agent in sheep, guinea pigs, and humans, is as potent as betamethasone at blocking eosinophil infiltration, suggesting that it may also suppress human pulmonary inflammation.

Antiinflammatory activity of a series of substituted 2,3-dihydro-6-hydroxypyrimido[2,1-f]purine-4,8(1H,9H)-diones.

A series of substituted analogues based on the novel 2,3-dihydro-6-hydroxypyrimido[2,1-f]purine-4,8(1H,9H)-dione ring system have been synthesized and shown to exhibit antiinflammatory activity in the adjuvant-induced arthritis rat model (AAR). The activity exhibited by the pyrimidopurinediones in this model of chronic inflammation is comparable to that of their previously studied 2-oxo congeners, the 6-hydroxypyrimido[2,1-f]purine-2,4,8-(1H,3H,9H)-triones, the best of which show potency levels approximately equal to that of naproxen. On the basis of its potency in the AAR assay, 9-benzyl-2,3-dihydro-1,3-dimethyl-6-hydroxy-7-(3-methyl-2-butenyl) pyrimido-[2,1-f]purine-4,8(1H,9H)-dione was selected for further evaluation and found to exhibit cyclooxygenase inhibitory activity in the in vitro rat neutrophil model. With respect to side-effect liability, this prenylated derivative has been shown to be devoid of gastric ulcer inducing potential, as well as the ocular toxicity observed previously with the 2-oxo series.

Effect of adrenalectomy, flutamide, and leuprolide on the growth of the Dunning rat R-3327 prostatic carcinoma.

The R-3327 prostatic tumor implanted in the male Copenhagen x Fischer F1 rat continues to grow because androgen is being supplied from an endogenous source. It follows that regimens which decrease the availability of androgen will retard the growth rate of the tumor. These experiments showed that castration, the antiandrogen flutamide, and the luteinizing hormone-releasing hormone (LHRH) agonist leuprolide inhibited tumor growth. Adrenalectomy alone had no significant effect on tumor size and did not further retard the growth of the tumor in castrated rats. Further, under the conditions of this study, there was no significant difference in tumor growth rates between the groups of rats treated with either flutamide alone or flutamide combined with leuprolide. Total ablation of androgen may not be needed for maximal inhibition of tumor growth.

17-Heteroaroyl esters of corticosteroids. 2. 11 beta-Hydroxy series.

The preparation and topical antiinflammatory potencies of a series of 17-furoyl and -thenoyl esters of 9 alpha-fluoro-11 beta-hydroxy-16 methyl and 9 alpha-chloro-11 beta-hydroxy-16-methyl corticosteroids are described. The 17 alpha-esters were introduced to the 9 alpha-fluoro 11-ketones or to the appropriate delta 9(11) compounds by direct acylation with the appropriate heteroaryl carbonyl chloride in the presence of 4-(dimethylamino)pyridine. Functionalization of the C ring was completed by standard methods. The most extensively studied heterocyclic acyl group was 2-furoyl, but 3-furoyl and 2- and 3-thenoyl derivatives were also investigated. Antiinflammatory potencies were measured in mice by a 5-day modification of the Tonelli croton oil ear assay. The most potent topical antiinflammatory agents were 1e, dexamethasone 17-(2'-furoate) 21-propionate, and 2c, the 21-chloro 17-(2'-furoate) in the 9 alpha-chloro series, both being 6 times as potent as betamethasone 17-valerate. Several other 9 alpha-chloro-11 beta-hydroxy-17-heteroaryl carboxylates (2a, 2b, 2d, and 2g) were at least 4 times as potent as betamethasone 17-valerate. Evaluation of 2c in the clinic confirmed that the compound is a potent topical antiinflammatory agent in humans.

Synthesis and structure-activity studies of corticosteroid 17-heterocyclic aromatic esters. 1. 9 alpha, 11 beta-dichloro series.

The preparation and topical antiinflammatory potencies of a series of 9 alpha, 11 beta-dichloro-16-methyl corticosteroid 17-heteroaryl carboxylates are described. The 17-acyl group was introduced to the 9 alpha, 11 beta-dichloro 21-acetate by direct acylation with the appropriate heteroaryl carbonyl chloride in the presence of 4-(dimethylamino)pyridine. Alternatively, the 21-functionalized 17-hydroxy delta 9(11) compound was acylated at 17, followed by C-ring chlorination. The most extensively studied heterocyclic acyl functionality was the 2-furoyl, but the 3-furoyl, and 2- and 3-thenoyl derivatives were also investigated. Antiinflammatory potencies were measured in mice by a 5-day modification of the Tonelli croton oil ear assay. The most potent topical antiinflammatory compounds were 17-heteroaryl esters in the 16 alpha-methyl series where the 21-substituent was chloro or fluoro. Thus 2p [21-chloro 17-(2'-furoate)] was 8 times as potent as betamethasone valerate, while 2s [21-fluoro 17-(2'-furoate)], 2r [21-chloro 17-(2'-theonate)], and 2v [6 alpha-fluoro 21-chloro 17-(2'-furoate)] were 3 times as potent as betamethasone valerate.

Structure-activity relationships of a series of novel topical corticosteroids.

The effect of various heteroaroyl groups in the 17-position of topical corticosteroids has been studied. The corticosteroids esterified at C17 were of 9 alpha,11 beta-dichloro, 9 alpha-chloro 11 beta-hydroxy and 9 alpha-fluoro 11 beta-hydroxy series. Among the 17-acyl groups 2'-furoates were most extensively investigated, although 2'-thenoates, 3'-thenoates and 3'-furoates were also examined. Many of these esters exhibited enhanced topical anti-inflammatory potencies. The most potent compounds investigated were the 21-chloro 17(2'-furoates) either in the 9 alpha,11 beta-dichloro, or in the 9 alpha-chloro 11 beta-hydroxy series. These compounds were at least 6 times as potent as betamethasone 17-valerate. Among 16-substituents studied 16 alpha-methyl compounds had the highest potency. Topical anti-inflammatory potencies were determined by using a 5-day modification of the croton oil ear assay in mice. The more potent compounds were also evaluated in the P. ovale induced chronic psoriaform lesion in the guinea-pig.

Inhibition of collagen II arthritis by simultaneous administration of concanavalin A and other substances with antigen emulsion.

Several pharmacological agents, some of which are known to have effects on the immune system, decrease the incidence of collagen II-induced arthritis when added to the antigen emulsion. Concanavalin A, which has been reported to exert suppressive effects on the immune system in vivo, consistently reduced the immune response to the collagen antigen. These effects were dose and time dependent. The suppressive effects of pokeweed mitogen, tilorone and carrageenan on anti-collagen II responses were somewhat variable. Suppressive activity could be observed with concanavalin A and levamisole when the drugs were injected at a site distant from the collagen emulsion. These studies indicate that local administration of drugs is an effective approach for demonstrating the activity of some agents that may alter the course of collagen II disease through an effect on the immune system.

A study of the mechanism of Con A-induced immunosuppression in vivo.

The plaque-forming cell (PFC) response to sheep erythrocytes (SRBC) is suppressed in a dose-related manner when concanavalin A (Con A) is administered intravenously to mice prior to or after immunization with antigen. The magnitude of suppression as well as the duration of the Con A effect greatly depends on the concentration of antigen used for immunization. Although profound suppression of the anti-SRBC PFC response is observed in intact mice pretreated with Con A for 4-24 hr, spleen cells from these mice do not exhibit suppressive activity when transferred into normal recipients or when cotransferred with normal spleen cells into irradiated recipients. Moreover, the cells from Con A-treated mice respond as normal spleen cells to SRBC when transferred alone into irradiated hosts. Suppression of the anti-SRBC PFC is only observed when adoptive hosts of cells from Con A-treated mice are also injected with Con A within 48 hr (but not 72 hr) of cell transfer and immunization. This time course of responsiveness to the suppressive effects of Con A is similar to that observed in normal mice and in irradiated recipients of normal spleen cells. The immune response to SRBC is also suppressed in adoptive hosts of normal spleen cells that are pretreated with Con A 4-24 hr prior to irradiation and cell transfer. Although functionally inactive when transferred into adoptive hosts, spleen cells from mice pretreated with Con A for 4-24 hr can suppress a primary antibody response to SRBC in vitro. The suppressive activity, which cannot be detected in the spleens of mice when the interval between pretreatment and assay is longer than 24 hr, is present in a subpopulation that bears the Thy 1.2 and Lyt 2 phenotype. Taken together the results obtained in in vivo and in vitro functional assays suggest that a suppressor cell population is activated following in vivo treatment with Con A, but that the cells rapidly lose their state of activation when removed from a Con A environment. This phenomenon is in all probability responsible for the failure to demonstrate suppressive activity in the spleens of Con A-treated mice using in vivo functional assays.

Selective effects of 7 alpha-halogeno substitution on corticosteroid activity: Sch 22219 and Sch 23409.

Two 7 alpha-halogeno substituted corticosteroids, 7 alpha-chloro-16 alpha-methylprednisolone-17,21 dipropionate (Sch 22219) and 7 alpha-bromo-16 alpha-methylprednisolone-17-benzoate-21-acetate (Sch 23409) were compared to other clinically utilized topical corticosteroids for local and parenteral antiinflammatory and glucocorticosteroid activity. Both compounds were at least equivalent to the most potent comparison corticosteroids in topical antiinflammatory activity, and exhibited favorable ratios of local to systemic effects. In mice, Sch 22219 showed a greater dissociation of antiinflammatory activity from side effects than Sch 23409, although the reverse was true in rats. On the basis of the data available, both compounds possess enhanced topical antiinflammatory potency, with the potential for reduced side effects in man.

Edema formation and neutrophil mobilization in the neutropenic rat.

The relationship between leucocyte mobilization and edema formation was evaluated in the carrageenan pleurisy model. In normal rats carrageenan was able to mobilize between 80 and 100 million cells per ml of fluid. In neutropenic rats the concentration fell to between 20 and 50 million cells per ml, suggesting the edema formed after carrageenan injection is not directly correlated with cellular mobilization.