6,11-Dihydro-11-oxodibenz [b,e] oxepinacetic acids with potent antiinflammatory activity.

A series of 6,11-dihydro-11-oxodibenz[b,e]oxepinacetic acids was synthesized and the antiinflammatory activity determined. Studies on 29 compounds revealed certain structure-activity relationships. In the carrageenan edema test, eight compounds exhibited higher antiinflammatory activities than did indomethacin. Several compounds (2, 9, 14, 22, 25) also proved to have activities superior or comparable to indomethacin in suppressing chronic as well as acute inflammation and carrageenan-induced hyperesthesia. Gastric irritation and lethality rates were less frequently observed with these compounds.

Nonsteroidal antiinflammatory agents. 2. Derivatives/analogues of dibenz[b,e]oxepin-3-acetic acid.

6,11-Dhydro-11-oxodibenz[b,e]oxepins and some related compounds have been synthesized and evaluated for antiinflammatory effect according to the carrageenan paw edema method in rats. The structure-activity relationships have been discussed among acetic acid, carboxylic acid, alcohol, and tetrazole derivatives of dibenzoxepins and acetic acid derivatives of thienobenzoxepins and of the corresponding thiepins. The 3-isopropyl alcohol 9 and 11-deoxo-3-propionic acid (49) were more active than indomethacin but not as active as the title compound (i.e., 43). Carboxylic acids, tetrazoles, esters, amides, and ketones were less active than the corresponding acetic acids. Three compounds (31, 33, and 34) were evaluated for ulcerogenicity and lethality but none surpassed 6,11-dihydro-11-oxodibenz[b,e]oxepin-3-acetic acid (41) in therapeutic ratio.

Effects of DP-1904, a thromboxane synthetase inhibitor, on the antigen-induced airway hyperresponsiveness and infiltration of inflammatory cells in guinea-pigs.

The effect of DP-1904, a novel thromboxane (TX) synthetase inhibitor, on airway hyperresponsiveness was studied in actively sensitized guinea-pigs. Airway hyperresponsiveness to intravenous ACh was observed at 3 and 7 h after aerosolized antigen challenge. In the model, a significant correlation between increases of respiratory resistance and microvascular leakage was observed, corresponding to the elevation of TXB2 in bronchoalveolar lavage fluid (BALF) in the early phase. DP-1904, at doses of 3 mg/kg or higher given orally one hour prior to the antigen challenge, inhibited the TXB2 production and the development of airway hyperresponsiveness in the early phase. Further, DP-1904 significantly suppressed the accumulation of lymphocytes in BALF and airway hyperresponsiveness in the late phase, although it only slightly decreased the mobilization of eosinophils and neutrophils. The results suggest that TXA2 is possibly involved in the development of airway hyperresponsiveness, and DP-1904 prevented the airway hyperresponsiveness via inhibition of TXA2 production and regulation of inflammatory cells.

A possible involvement of thromboxane A2 and peptide leukotrienes in hyperresponsiveness of Sephadex-treated rat lung parenchyma.

An augmented contraction and elevated thromboxane (TX) B2 release were observed, when the isolated parenchyma from Sephadex-treated rats was stimulated by 5-hydroxytryptamine (5-HT). Release of peptide leukotrienes (pLTs) was also increased by the stimuli. In the Sephadex-induced hyperresponsiveness model, DP-1904, a novel TX synthetase inhibitor, at the concentrations of 3 x 10(-7) to approximately 3 x 10(-6) M, reduced the augmented contraction. Also, indomethacin (3 x 10(-6) M), a histamine H1 antagonist and AA-2414 (10(-6) M, a TXA2 antagonist, significantly attenuated the hyperresponsiveness to 5-HT. ICI-198,615 (10(-7) M), a leukotriene receptor antagonist, partially but significantly reduced the augmented contraction. In an ex vivo study, oral DP-1904 significantly inhibited both the augmented contraction and elevated TXB2 release from Sephadex-treated rat parenchyma, but did not affect the blood eosinophilia induced by Sephadex-treatment. These results suggested that the ability to synthesize newly generated lipid mediators such as TXA2 and pLTs to exogenous 5-HT was altered upward by Sephadex injection, and so could lead to augmented contraction of established hyperresponsiveness in rats.

Effect of DP-1904, a thromboxane synthetase inhibitor, on antigen- and spasmogen-induced bronchoconstriction in rodents.

The effect of DP-1904 [6-(1-imidazolylmethyl)-5,6,7,8-tetra-hydronaphthalene-2-car boxylic acid hydrochloride], a selective thromboxane synthetase inhibitor, was examined on antigen- and spasmogen-induced bronchoconstriction in rodents. Oral administration of DP-1904 (1, 3, 10 mg/kg) as well as OKY-046 (sodium (E)-3[4-(1-imidazolylmethyl)-phenyl]-2-propanoate, 100 mg/kg), significantly inhibited immunoglobulin G-mediated bronchoconstriction in actively sensitized guinea pigs. Immunoglobulin E-mediated bronchoconstriction in actively sensitized rats was also inhibited by both DP-1904 (1, 10 mg/kg) and OKY-046 (100 mg/kg). DP-1904 (3-30 mg/kg) and OKY-046 (30 mg/kg) suppressed leukotriene D4-induced bronchoconstriction in guinea pigs. In these models, the endogenous levels of thromboxanes significantly increased following the stimulus (antigen and leukotriene D4). DP-1904 (10 mg/kg) inhibited the increase in thromboxane level in both plasma and bronchial alveolar lavage fluid. These actions of DP-1904 persisted for more than 12 h, indicating a long-lasting effect of DP-1904 on bronchoconstriction. The results showed that the biological activity of DP-1904 in our rodents models is more potent than that of OKY-046 (Ozagrel), which is available as an anti-asthma agent in Japan.

Correlative alteration of thromboxane A2 with antigen-induced bronchoconstriction and the role of platelets as a source of TXA2 synthesis in guinea pigs: effect of DP-1904, an inhibitor of thromboxane synthetase.

A marked and sustained bronchoconstriction after antigen challenge was produced in actively sensitised guinea pigs, and correlated with increments of thromboxane (TX) A2 level in both the plasma and bronchoalveolar lavage fluid. DP-1904 given orally relieved the bronchoconstriction and increase in TXA2 in a dose-dependent manner. In platelet-depleted animals, antigen-induced bronchoconstriction and TXA2 release in the plasma were significantly reduced compared to those of non-platelet-depleted animals, indicating that platelets are a major cell source of TXA2 production, the remainder originating from the other cells excluding platelets. In the platelet-deprived animal, DP-1904 showed further significant inhibition of the constriction and plasma TXA2 level, and therefore likely inhibits TXA2 synthesis of various cells, including platelets, in the bloodstream. The results suggested that TXA2 is an important mediator responsible for producing antigen-induced bronchoconstriction, and endogenously originated from various cells including platelets in guinea pigs.

In vitro effect of DP-1904, a novel anti-asthma agent, against antigen-induced constriction and TXB2 release from the isolated guinea-pig lung parenchymal tissue.

The contractile activity and mobilisation of arachidonic acid metabolites in response to the antigen challenge were studied in isolated lung parenchymal tissue from the actively sensitised guinea pig. The sustained constriction of the lung tissue was evoked by the antigen, associated with significant liberation of TXB2, histamine and p-LTs. Other prostanoids (PGF2 alpha, PGD2, PGE2 and 6-keto-PGF1 alpha) were also released by the antigen challenge. DP-1904, an inhibitor of TX synthetase, significantly suppressed the late phase of the antigen-induced constriction. DP-1904 was potent to inhibit the production of TXB2, while DP-1904 accelerated the formation of PGF2 alpha, PGE2 and 6-keto-PGF1 alpha, presumably indicating the alternative changes of dilatory metabolites to the spasmogenic component. Mepyramine and FPL-77512 augmented the effect of DP-1904. AA-861 inhibited the antigen-induced constriction of the lung parenchymal tissue by inhibiting the release of p-LTs and TXB2. Pretreatment of the lung parenchymes with anti-guinea pig platelet serum, in order to deplete the platelets, did not affect the generation of TXB2 both in resting and also in the antigen-stimulated status, indicating that TXA2 is produced in the topical pulmonary tissue. It is concluded that DP-1904 inhibits the parenchymal contraction through potent inhibition of TXA2 generation, associated with significant elevation in PGE2 and PGI2.

Induction of colony-stimulating factor and stimulation of stem cell proliferation by injection of muroctasin.

Modulation of myelopoiesis by anomeric mixture of N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutamyl]-N6-stearoyl-L-lysin e (MDP-Lys(L18), muroctasin), a muramyl dipeptide analog, was investigated in mice. When BDF1 mice were subcutaneously treated with a single dose of 100 micrograms of MDP-Lys(L18), an increase in the number of peripheral blood leukocytes, a rise in the serum levels of colony-stimulating factor (CSF), proliferation of multipotential stem cells in the bone marrow and the spleen, and an expansion of granulocyte-macrophage progenitors in the bone marrow were observed. These findings suggest that the increase in the number of peripheral blood leukocytes in BDF1 mice by MDP-Lys(L18) is due to CSF production and stimulation of stem cell proliferation.

Inhibitory effect of DS-4574 on leukotriene- or antigen-induced bronchoconstriction in guinea pigs.

We studied the leukotriene (LT) antagonistic activity of DS-4574 in vivo and the inhibitory effect of this compound on antigen-induced bronchoconstriction in actively sensitized guinea pigs. Bronchoconstriction induced by LTD4 was inhibited by intravenous and oral treatment with DS-4574 in a dose-dependent manner. Orally administered DS-4574 was also able to inhibit the bronchoconstriction mediated by intravenous administration of LTC4 and E4 and that by endogenous LTs. The inhibitory effect of DS-4574 showed similar potency to those of FPL-55712 and LY171883. In contrast, histamine-, acetylcholine- or 5-hydroxytryptamine-induced bronchoconstriction was not significantly affected by DS-4574. Moreover, DS-4574 given orally or intravenously inhibited antigen-induced bronchoconstriction in actively sensitized guinea pigs and this compound prevented antigen-induced mediator release from actively sensitized guinea-pig lung fragments. The anti-asthmatic effect of this compound appears to be associated with LT antagonism and inhibition of the release of chemical mediators. This study therefore shows DS-4574 to have orally effective LT antagonistic and anti-asthmatic activities. This compound may prove useful in the treatment of bronchial asthma.

The effect of treatment with interferon-gamma on type II collagen-induced arthritis.

We have investigated the effects of recombinant murine interferon-gamma (rIFN-gamma) on type II collagen-induced arthritis (CA) in DBA/l mice. Therapeutic as well as prophylactic treatment with subcutaneous rIFN-gamma, at 10(5) U/mouse six times a week, inhibited the development of CA without any obvious side effects. The accompanying suppression of anti-CII antibody responses may partly explain the inhibition of CA by rIFN-gamma. The possible role of the anti-inflammatory effect of systemic IFN-gamma in the inhibition of CA is discussed.

The effect of treatment with recombinant gamma-interferon on adjuvant-induced arthritis in rats.

We have investigated the effects of recombinant rat gamma-interferon (rIFN gamma) on adjuvant-induced arthritis (AA). Lewis rats, inoculated in the left hind-paw with adjuvant (day 0), were given 10(5) U/rat of rIFN gamma daily (days 0 to 20), subcutaneously and intramuscularly on alternate days. rIFN gamma suppressed the secondary phase of swelling of both hind-paw on and after day 18 without influencing the earlier phases, both primary and secondary, of swelling. rIFN gamma also reduced the hind-paw bone lesions, the degree of splenomegaly, and the increase in erythrocyte sedimentation rate and plasma fibrinogen. These results indicate a new aspect of the regulatory role of IFN gamma in chronic inflammation.

Production of colony-stimulating factor from macrophages by muroctasin.

The effect of a muramyl dipeptide analog, N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutamyl]-N6-stearoyl-L-lysin e (MDP-Lys(L18), muroctasin), on the colony-stimulating factor (CSF) production was investigated in vitro. MDP-Lys(L18) stimulated murine peritoneal macrophages to induce CSF. However, the compound produced more CSF in the presence of both macrophages and T cell enriched fraction than in the absence of T cells. This phenomenon was found to be attributed to the fact that MDP-Lys(L18) also stimulated macrophages to induce interleukin 1 (IL-1) and the IL-1 induced CSF production by T cells. These findings suggest that macrophages may be the first target cells of MDP-Lys(L18) to produce CSF.

Antigenicity study of muroctasin.

Antigenicity study of N2-[(N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysi++ + ne (MDP-Lys(L18), muroctasin) was carried out in mice, guinea pigs and rabbits with passive cutaneous anaphylaxis (PCA), systemic anaphylaxis (SA), Arthus and delayed skin reaction and enzyme-linked immunosorbent assay (ELISA). Mice were sensitized intraperitoneally, twice at 3-week intervals, with MDP-Lys(L18) (40, 400 or 4000 micrograms/kg) or MDP-Lys(L18)-ovalbumin (OA) conjugate (500 micrograms/kg) in alumina gel. No IgE antibodies to MDP-Lys(L18) were detected in the sera obtained from the sensitized mice by 24-h PCA in rat. Guinea pigs were sensitized subcutaneously with MDP-Lys(L18) (4, 40 or 400 micrograms/kg) or MDP-Lys(L18)-OA (2 mg/kg) emulsified in Freund's complete and incomplete adjuvant, 3 times at 2-week intervals. No SA was observed in the sensitized animals after the intravenous injection of MDP-Lys(L18) (1 mg/kg). Rabbits were sensitized subcutaneously with MDP-Lys(L18) (2 or 20 micrograms/kg) or MDP-Lys(L18)-OA (2 mg/kg) emulsified in Freund's incomplete adjuvant, 3 times at 2-week intervals. Neither Arthus nor delayed skin reaction was observed in the sensitized animals after the intradermal injection of MDP-Lys(L18) (2 micrograms/site). Although antibodies to MDP-Lys(L18) were not detected in MDP-Lys(L18) sensitized animals, the antibodies were detected in two out of 9 guinea pigs and all of the rabbits in the MDP-Lys(L18)-OA sensitized groups by 4-h PCA after the intravenous injection of MDP-Lys(L18) (1 mg/kg). These results suggest that MDP-Lys(L18) may possess antigenic potential by PCA reaction in guinea pigs.

Specific binding sites of muroctasin on murine macrophages.

The specific binding sites for N2-[N-acetylmuramoyl)-L-alanyl-D-isoglutaminyl]-N6-stearoyl-L-lysine (MDP-Lys(L18), muroctasin), a synthetic muramyl dipeptide analog, on murine macrophages were investigated using [3H]-MDP-Lys(L18). The specific binding sites were found to be located on macrophage membrane and not to cross-react with MDP. It was also found that MDP-Lys(L18) was not metabolized by macrophages.

Prostaglandin synthetase systems in rat and rabbit renal medulla and inhibition by non-steroidal anti-inflammatory drugs.

The properties of prostaglandin synthetase systems (PSSs) in the renal medulla of the rat and rabbit, and inhibition by ketoprofen, indomethacin, ibuprofen, phenylbutazone and aspirin were investigated in relation to their anti-inflammatory, analgesic, antipyretic and ulcerogenic activities. Rat and rabbit PSSs produced prostaglandin (PG)E and PGF from arachidonic and dihomo-gamma-linolenic acids and had an optimal pH of 8.5 and 7.5 for PGE formation, respectively. Only a slight loss of activity occurred with lyophilization. In the rat PSS, all drugs tested were inhibitory in the order of ketoprofen, ibuprofen, indomethacin and aspirin, respectively. In the rabbit PSS, the same potency relationship was also found. Drug sensitivity of the rat PSS was remarkably lower than that of the rabbit PSS. Significant correlations were noted between the inhibitory potencies of the drugs against both PSSs and other in vivo pharmacological activities within the same species.

Cell-mediated transfer of collagen-induced arthritis in mice and its application to the analysis of the inhibitory effects of interferon-gamma and cyclophosphamide.

We developed a convenient and reliable procedure for the cell-mediated passive transfer of type II collagen (CII)-induced arthritis (CA). Spleen cells from DBA/1 mice with CA were intravenously transferred into syngeneic recipient mice. Arthritis developed only in those recipients which had received whole-body x-irradiation (8 Gy) just before cell transfer and intraperitoneally given soluble CII without adjuvant immediately after transfer. Non-immunized splenocytes could not induce arthritis even in irradiated recipients given soluble CII. Development of arthritis depended on the number of cells transferred; 5 x 10(7) cells induced severe and long-lasting arthritis in every recipient approximately 10 days after transfer. Severity of this arthritis was clinically and histologically similar to classical CA in donors. Arthritogenic splenocytes were generated in donors no later than 20 days after priming with CII in Freund's complete adjuvant, when arthritis had yet to occur, and were detected for more than 5 weeks. One splenocyte population responsible for transferring arthritis was CD4+ T cells. We then applied this system to show that prophylactic treatment of CII-immunized mice with cyclophosphamide (CY, 7 mg/kg), but not interferon-gamma (IFN-gamma, 10(5) U/mouse), suppressed the arthritogenic ability of their spleen cells, although both treatments inhibited the development of CA. Treatment of recipients with IFN-gamma, however, inhibited the development of arthritis upon transfer with CII-immunized splenocytes. These results indicate that CY and IFN-gamma act at the induction and effector phases of arthritogenic lymphocytes, respectively. Thus, this system facilitates investigation of pathological mechanisms of CA, and of mechanisms of anti-arthritics.