[Advance in eosinophil chemotaxis assay method--automatic count of migrated cells by the image analyzing system in the Boyden method].

Eosinophils, which are prominent inflammatory cells in the asthmatic airway, are attracted to the airway by several chemoattractants. At present, eosinophil chemotaxis is studied employing the Boyden Millipore chamber system in vitro. In the Boyden method, the number of migrated cells are evaluated by direct microscopic observation of individual cells. This microscopic procedure usually requires much time and it is difficult to obtain objective results. Therefore, we have developed a new fractional measurement technique, using the image analyzing system connected with a color video camera and a computer for counting the eosinophils which have migrated into a filter. Using this system with the Boyden method, migrated cells were observed more objectively and quickly. Therefore, this image analyzing system with the Boyden method, may be a useful technique for the fractional measurement of migrated eosinophils in a sample mixture of eosinophils and neutrophils.

Suppression of the Arthus reaction by Y-24180, a potent and specific antagonist of platelet-activating factor.

A novel and potent antagonist of platelet-activating factor (PAF), Y-24180 (4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dimethyl-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4] diazepine) was investigated for the effects on the skin reactions induced by chemical mediators and the Arthus reactions. In the rat dorsal skin, Y-24180 (0.1-10 mg/kg, p.o.) inhibited increase in vascular permeability by the intradermal PAF injection in a dose dependent manner and the inhibitory activity was 60 times more potent than that of WEB 2086. While even at doses as large as 10 mg/kg, p.o., it had no effect on vascular permeability in the rat skin induced by histamine, serotonin, bradykinin and leukotriene D4. On a reversed passive Arthus reaction in rat dorsal skin, Y-24180 (0.1-1 mg/kg, p.o.) markedly inhibited vascular permeability in a dose dependent manner and the inhibitory activity was 15 times more potent than that of WEB 2086. Y-24180 also inhibited the Arthus dermal reaction in rabbits (0.03-0.3 mg/kg, p.o.) and guinea pigs (0.1-1 mg/kg, p.o.). In addition, Y-24180 (0.1-10 mg/kg, p.o.) significantly reduced the exudate volume and the number of infiltrated inflammatory cells in the reversed passive Arthus pleural reaction in rats. Furthermore, in rat passive Arthus pancreatitis, Y-24180 (0.3-10 mg/kg, p.o.) significantly inhibited the dye extravasation from the pancreas. These results provide strong evidence that endogenous PAF plays an important role as a mediator in the type III allergic inflammation.

[Anti-inflammatory effect of topically applied pranoprofen-gel].

The anti-inflammatory activity and mode of action of topically applied pranoprofen-gel were investigated in comparison with indomethacin-gel in experimental animals. Applied topically, 0.3 approximately 3% pranoprofen-gel inhibited carrageenin-induced paw edema, fracture-induced paw edema, carrageenin-induced increase in vascular permeability and adjuvant arthritis in rats and ultraviolet ray-induced erythema in guinea pigs dose-dependently, with a potency slightly greater than that of indomethacin-gel. In addition, pranoprofen-gel inhibited dose-dependently the formation of granuloma caused by a cotton pellet implantation, without affecting the weight of the thymus or adrenals and without inducing gastrointestinal lesions. Moreover, applied topically to carrageenin-treated rats, pranoprofen-gel inhibited dose-dependently, and more potently than indomethacin-gel, the production of a prostaglandin E2 (PGE2)-like substance in both the exudate of the carrageenin air pouch and the inflamed synovial membrane. Both drugs inhibited the potentiation of bradykinin-induced vascular permeability in rabbit skin by arachidonic acid, but not by PGE2. Pranoprofen was only one third as potent as indomethacin in inhibiting the production of PGE2 from the phagocytosing of killed bacteria by rat peritoneal leucocytes in vitro. These results show that pranoprofen-gel, applied topically, permeates well from the skin to the deep inflammatory site, relieving potently and long lastingly the inflammation by inhibiting PG production. As a topical anti-inflammatory agent, pranoprofen-gel is at least as effective as indomethacin-gel, so it may have good clinical use.

Effect of Y-24180, a platelet-activating factor-receptor antagonist, on the antigen-induced airway microvascular leakage in guinea pigs.

The inhibitory effect of Y-24180 ((+/-)-4-(2-chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6, 9-dimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine) on platelet-activating factor (PAF)- or antigen-induced airway microvascular leakage was studied in guinea pigs by oral administration. The tissue content of extravasated Evans blue dye was used as an index of plasma exudation in the trachea, main bronchi, central intrapulmonary airways and peripheral intrapulmonary airways. In all of these tissues, Y-24180 potently inhibited the leakage induced by PAF. The ED50 value of Y-24180 determined in each of the tissues was approximately 0.02 mg/kg, demonstrating that the inhibitory potency of Y-24180 is 4-6 times that of WEB 2086, another PAF antagonist. Even at a dose of 10 mg/kg, however, Y-24180 showed no inhibitory effect on the leakage induced by leukotriene (LT) D4, histamine or bradykinin. In the antigen-induced model of guinea pigs sensitized with aerosolized ovalbumin, Y-24180 (0.1-10 mg/kg) and WEB 2086 (1-100 mg/kg) potently inhibited the microvascular leakage in all of the examined airway tissues except for the trachea. At 1-100 mg/kg, however, both ONO-1078, an LT-receptor antagonist, and OKY-046, a thromboxane A2 synthetase inhibitor, prevented partially but not significantly the antigen-induced leakage. These results provide evidence that endogenous PAF partially mediates the antigen-induced airway microvascular leakage in guinea pigs.

Suppressive effects of Y-24180, a receptor antagonist to platelet activating factor (PAF), on antigen-induced asthmatic responses in guinea pigs.

OBJECTIVE AND DESIGN: Effects of Y-24180 on antigen-induced asthmatic responses were evaluated in actively sensitized guinea pigs and the effects were compared with those of several anti-asthmatic drugs. MATERIALS: Male Hartley guinea pigs were used. TREATMENT: Guinea pigs were actively sensitized with ovalbumin and were pretreated with pyrilamine Y-24180 was orally administered to the animals 3 h and others were 1 h before the antigen challenge. METHODS: The airway hyperresponsiveness was measured according to the method of Konzett and Rössler with some modifications. The immediate asthmatic response (IAR) and late asthmatic response (LAR) were measured by the oscillation method. Inflammatory cells infiltrated into the lungs were counted after the bronchoalveolar lavage. RESULTS: Under oral administration before or after the challenge with antigen, Y-24180, OKY-046, and ONO-1078 suppressed the antigen-induced airway hyperresponsiveness. Moreover, Y-24180, ONO-1078, AA-2414, and theophylline suppressed both the IAR and LAR, but OKY-046 only suppressed the LAR. Among the test drugs, only Y-24180 and theophylline suppressed the antigen-induced accumulation of eosinophils in the bronchoalveolar lavage fluid. CONCLUSIONS: The data indicate practical participation of PAF in the development of antigen-induced asthmatic responses in animals, and usefulness of Y-24180 in the clinical treatment of asthma as well as other anti-asthmatic drugs.

Effects of Y-24180, a long-acting and potent antagonist to platelet-activating factor, on immediate asthmatic response in guinea pigs.

The effect of Y-24180, a potent antagonist to-platelet-activating factor (PAF), was evaluated on the antigen-induced immediate asthmatic response (IAR) in actively sensitized guinea pigs that were pretreated with an antihistaminic agent, pyrilamine. Then, the effect was compared with that of other antiasthmatic agents. In a dose-dependent manner, Y-24180 (0.01-1 mg/kg, p.o.) suppressed the IAR, and WEB 2086 (0.1-10 mg/kg, p.o.), another PAF antagonist, also suppressed IAR in the same fashion as Y-24180. In contrast, AA-2414 (1-100 mg/kg,p.o.), a thromboxane A2 (TXA2) antagonist, was effective only at the beginning of the IAR and ONO-1078 (1-100 mg/kg, p.o.), a peptide leukotriene (pLT) antagonist, was effective only in the latter period, OKY-046, a TXA2 synthetase inhibitor, showed no significant suppression of the IAR at doses up to 100 mg/kg. Thus, PAF antagonists were more effective than the other agents tested in the present model for IAR. In a subsequent test, Y-24180 (1 mg/kg, p.o.) was confirmed to enhance the suppressive effects of theophylline (10 and 30 mg/kg, p.o.), procaterol (0.1 and 1 microgram/kg, i.v.), OKY-046 (100 mg/kg, p.o.) and ONO-1078 (100 mg/kg, p.o.) on the IAR. A combination of three agents, namely Y-24180 with OKY-046 and ONO-1078, completely suppressed the IAR. The results demonstrate that Y-24180 not only suppresses the IAR, but also enhances the suppressive effect of other antiasthmatic agents. Therefore, Y-24180 would be a clinically promising drug for the treatment of bronchial asthma.

[Analgesic and anti-inflammatory effect of (+/-)-N,N- dimethylcarbamoylmethyl 2-[7-(2-methyl-5H-[1] benzopyrano [2,3-b] pyridyl)] propionate (Y-23023), a new non-steroidal anti-inflammatory drug].

The analgesic and anti-inflammatory activities of Y-23023, a new nonsteroidal analgesic and anti-inflammatory compound, were investigated in acute, subacute and chronic pain models in rats. In the carrageenin paw edema test, Y-23023 (0.3-3 mg/kg, p.o.) dose-dependently inhibited hyperalgesia assessed by the Randall-Selitto method and paw edema. Y-23023 (1 mg/kg, p.o.), when administered at 2 hr after carrageenin injection, significantly elevated the reduced pain threshold without affecting paw edema. Therefore, the antinociception activity induced by Y-23023 was not the result of its anti-inflammatory activity. In addition, Y-23023-induced antinociception was resistant to post-treatment with naloxone (2 mg/kg, s.c.), but was antagonized by intraplantar injection of prostaglandin E2 (1 microgram/paw). These results suggest that Y-23023 produces a peripheral analgesic effect mediated by inhibition of prostaglandin production. Y-23023 (0.3-10 mg/kg, p.o.) also had a potent inhibitory effect on the silver nitrate-induced arthritic pain. In suppressing acute and subacute pain, Y-23023 was more potent than diclofenac sodium, indomethacin and loxoprofen sodium. The analgesic and anti-inflammatory activities of Y-23023 (0.1-1 mg/kg/day, p.o.) on the adjuvant-induced hyperalgesia and the paw swelling were nearly equipotent to diclofenac sodium and indomethacin, but were more potent than loxoprofen sodium. Therefore, Y-23023 would be regarded to show predominantly strong analgesic activity in acute and subacute pain when compared with reference drugs. The above results suggest that Y-23023 is a novel analgesic compound with an anti-inflammatory activity in the clinical field.

[Analgesic effect of topically applied pranoprofen-gel].

The analgesic effect of topically applied pranoprofen-gel (1% and 3%) was investigated in comparison with indomethacin-gel in experimental animals. Applied topically, 1% and 3% pranoprofen-gel inhibited the inflammatory pain induced by Randall and Selitto's method and the pain response (abnormal gait) of concanavalin A-induced arthritis in rats dose-dependently. Furthermore, in antigen (methylated bovine serum albumin)-induced arthritis in rats, pranoprofen-gel had a concentration and application-dependent therapeutic effect on knee joint swelling and the pain response. Pranoprofen-gel had a stronger analgesic effect than indomethacin-gel in these experimental models. Both drugs inhibited the flexor reflexes of the hind limb induced by injecting bradykinin (BK) in combination with arachidonic acid into the common iliac artery of the spinal rat, but failed to do so with BK combined with prostaglandin E2 (PGE2). Moreover, pranoprofen-gel inhibited the BK-induced increase in the firing rate of the saphenous nerve of the spinal cat. These results show that pranoprofen-gel, applied topically, permeates well from the skin to the nociceptor site, relieving the hyperalgesia caused by PGs-induced sensitization of pain receptors by inhibiting their production. As a topical anti-inflammatory and analgesic agent, pranoprofen-gel is at least as effective as indomethacin-gel, so that it should have good clinical potential.

[Mechanism of the inhibitory effect of pranoprofen on sodium urate crystal-induced inflammation].

The mechanism of the inhibitory effect of pranoprofen on sodium urate crystal-induced inflammation was investigated with several inflammatory parameters using leucocytes stimulated with sodium urate crystals in vitro. At 10(-5) M, pranoprofen tended to inhibit the production of chemotactic factor in guinea pig polymorphonuclear leucocytes (PMNL) stimulated with sodium urate crystals and at 10(-4) M, significantly inhibited it. At 10(-3) M and 10(-4) M, it potentiated the chemotaxis of guinea pig PMNL. Furthermore, it inhibited the production of superoxide anion (O2-) in guinea pig PMNL stimulated with sodium urate crystals, with an IC50 value of 5.0 X 10(-4) M, comparable to that of indomethacin. At 10(-3) M, it inhibited the release of beta-glucuronidase stimulated by sodium urate crystals. From doses as low as 10(-6) M, it inhibited dose-dependently the production of PGE2-like substance by the phagocytosis of sodium urate crystals by rat peritoneal leucocytes, with an IC50 value of 7.5 X 10(-6) M. These results suggest that in inhibiting the production of PGE2 stimulated by sodium urate crystals, pranoprofen shows an inhibitory effect on sodium urate crystal-induced inflammation.

Effect of a potent PAF antagonist, Y-24180, on an allergen-induced late asthmatic response in guinea-pigs.

The effect of Y-24180, a potent and specific PAF antagonist, on the antigen-induced late asthmatic response in guinea-pigs was investigated. We used an asthmatic guinea-pig model which had a high incidence of dual-phase reaction and high reproducibility. Y-24180 (1 mg/kg, p.o.) had a significant inhibitory effect on the late asthmatic response and eosinophil infiltration in bronchoalveolar lavage fluid. These results suggest that PAF may play, at least partially, an important role in the development of the late asthmatic response. Thus, Y-24180 may be useful for the treatment of asthma.

[Effect of pranoprofen on sodium urate crystal-induced inflammation].

Effect of a non-steroidal anti-inflammatory drug, pranoprofen (PPF), on sodium urate crystal-induced inflammation was investigated in comparison with standard drugs for treating acute gout in experimental animals. PPF inhibited sodium urate crystal-induced paw edema in both rats (1-10 mg/kg, p.o.) and mice (5-25 mg/kg, p.o.) in a dose-dependent manner. On rat sodium urate crystal-induced paw edema, PPF was found to be almost equally active as indomethacin (IM) and colchicine. In addition, PPF (2.5-10 mg/kg, p.o.) inhibited the accumulation of exudate and decreased the leucocyte numbers and the amount of prostaglandin E2 (PGE2)-like substance in sodium urate crystal-induced pleurisy in rats dose-dependently, with a potency slightly greater than that of IM. The specific anti-gout agent colchicine (5 mg/kg, p.o.) also suppressed the accumulation of exudate and decreased the leucocyte numbers, without affecting the amount of PGE2-like substance. Moreover, in mouse peritonitis, PPF (1-10 mg/kg, p.o.) suppressed the sodium urate crystal-induced increase in vascular permeability in a dose-dependent manner. Furthermore, in experimental models of articular gout, PPF inhibited the pain response (abnormal gait) of sodium urate crystal-induced arthritis in both rats (0.25 and 1 mg/kg, p.o.) and dogs (3 mg/kg, p.o.), with a potency greater than that of IM and phenylbutazone, respectively. These results indicate that as an anti-gout agent, PPF is at least as effective as other standard drugs, so that it should have good clinical potential.

Effect of Y-24180, a long-acting antagonist to platelet-activating factor (PAF), on PAF-induced reactions: a relationship between the partial structure of the compound and its duration of the action.

(+/-)-4-(2-Chlorophenyl)-2-[2-(4-isobutylphenyl)ethyl]-6,9-dime thy l-6H- thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepine (Y-24180) is a platelet-activating factor (PAF) antagonist, being similar to WEB 2086. One of the structural differences between the two PAF antagonists is the presence of a methyl substituent in Y-24180 at the 6-position of its ring system. Orally administered Y-24180 and WEB 2086 both dose-dependently prevented PAF-induced airway hyperresponsiveness (ED50: 0.0010 and 0.019 mg/ kg, respectively) and bronchoconstriction (ED50: 0.0014 and 0.024 mg/kg, respectively) in guinea pigs. Against the bronchoconstriction, here, the inhibitory effect of Y-24180 was significantly more potent and longer acting than that of WEB 2086. On the other hand, Y-24180 (10 mg/kg, p.o.) showed insignificant effects on the bronchoconstriction induced by leukotriene D4, histamine, serotonin, acetylcholine, arachidonic acid, or bradykinin. In an in vitro test, Y-24180 and WEB 2086 inhibited the PAF-induced aggregation of the rabbit washed platelets in a concentration-dependent manner (IC50: 1.2 and 64 nmol/l, respectively). Compared with desmethyl-Y-24180 and WEB 2086, Y-24180 and methyl-WEB 2086, both of which have a methyl substituent on the 6-position of their thienodiazepine ring, exhibited a longer acting suppressive effect on PAF-induced bronchoconstriction and significantly more stable binding to the PAF receptor after the washing-out procedure of the test compounds from platelets. Therefore, the 6-methyl substituent should be responsible for the PAF receptor binding stability of Y-24180, namely, for its long-acting anti-PAF effects in vivo. These results indicate that Y-24180 possesses the specific and long-acting PAF antagonistic effects in the airway.