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Pharmacological Properties of a New Anti-Inflammatory Compound, α-(3,5-Di-Tert-Butyl-4-Hydroxybenzylidene)-γ-Butyrolactone (KME-4), and Its Inhibitory Effects on Prostaglandin Synthetase and 5-Lipoxygenase

The pharmacological effects of a new anti-inflammatory compound, α-(3, 5-di-tert-butyl-4-hydroxybenzylidene)-γ-butyrolactone (KME-4), and its inhibitory effects on arachidonate prostaglandin synthetase and 5-lipoxygenase activities were examined. KME-4 showed anti-inflammatory activity. It was less active than indomethacin, but more active than naproxen and ibuprofen in carrageenin-induced paw edema in rats; and it was less active than indomethacin, equipotent as naproxen, but more active than ibuprofen in granuloma formation in rats. The ulcerogenic activity of KME-4 was weaker than indomethacin and naproxen, but stronger than ibuprofen in starved rats. The ratio of UD50 stomach to ED30 carrageenin edema or to ED25 granuloma for KME-4 showed higher values than those of the reference drugs. KME-4 showed antipyretic activity in yeast-induced fever in rats. It also inhibited platelet aggregation induced by arachidonic acid and protected rabbits from arachidonic acid-induced death. Furthermore, KME-4 was found to be equipotent in inhibiting both prostaglandin synthetase and 5-lipoxygenase activities of rat basophilic leukemia cells, unlike indomethacin, naproxen and ibuprofen. It also inhibited the prostaglandin synthetase activity of bovine seminal vesicle. The present findings indicate that KME-4 may be a new type of anti-inflammatory drug with dual prostaglandin synthetase and 5-lipoxygenase inhibition.

Abstract-The pharmacological effects of a new anti-inflammatory compound, a (3,5-di-tert-butyl-4-hydroxybenzylidene)-r-butyrolactone (KIVI E-4), and its inhibitory effects on arachidonate prostaglandin synthetase and 5-lipoxygenase activities were examined. KME-4 showed anti-inflammatory activity. It was less active than indomethacin, but more active than naproxen and ibuprofen in carrageenin-induced paw edema in rats; and it was less active than indomethacin, equipotent as naproxen, but more active than ibuprofen in granuloma formation in rats. The ulcerogenic activity of KME-4 was weaker than indomethacin and naproxen, but stronger than ibuprofen in starved rats. The ratio of UD50 stomach to ED30 carrageenin edema or to ED25 granuloma for KME-4 showed higher values than those of the reference drugs. KME-4 showed antipyretic activity in yeast induced fever in rats. It also inhibited platelet aggregation induced by arachidonic acid and protected rabbits from arachidonic acid-induced death. Furthermore, KME-4 was found to be equipotent in inhibiting both prostaglandin synthetase and 5-lipoxygenase activities of rat basophilic leukemia cells, unlike indomethacin, naproxen and ibuprofen.
It also inhibited the prostaglandin synthetase activity of bovine seminal vesicle.
The present findings indicate that KME-4 may be a new type of anti-inflammatory drug with dual prostaglandin synthetase and 5 lipoxygenase inhibition.
During a search for anti-inflammatory compounds, a series of 3,5-di-tert-butyl styrene derivatives were found to have anti inflammatory activity and inhibition of platelet aggregation in our laboratories (manuscript in preparation). Of them, a (3,5-di-tert-butyl-4-hydroxybenzylidene) -r butyrolactone (KME-4), with a chemically new structure (Fig. 1), was the most active. We further found that it inhibited not only prostaglandin synthetase but also 5 lipoxygenase, unlike known nonsteroidal anti inflammatory drugs such as indomethacin, naproxen and ibuprofen. Chemical structure of KME-4 In this paper, we describe pharmacological properties of KME-4 and its inhibitory effects on arachidonate prostaglandin synthetase and 5-lipoxygenase. All the test compounds were suspended in 2.5% arabic gum solution for oral adminis tration, and for in vitro assays, they were used after dissolving them in ethanol.

Materials and Methods
Carrageenin-induced paw edema: The assay was done by the method of Winter et al. (1). Ten rats weighing 150-200 g were used in each group. Test compounds were administered p.o. 1 hr before s.c. injection of 0.1 ml of 1 % carrageenin (picnin A®, Zushi Chemical Laboratory) in 0.9% NaCI solution into right hind paw. The foot volume was measured with a plethysometer 5 hr after carrageenin.
Paper disk-induced granuloma formation: The assay was done by the method of Hara and Tomizawa (2). Six rats weighing 150-200 g were used in each group. A sterilized paper disk (Toyo Roshi, 29±1 mg) was implanted s.c. into bilateral area of axilla under anesthesia with amobarbital sodium. Test compounds were administered p.o. once daily for 7 days, starting on the day of surgery. The animals were killed 7 days after the implantation, and the paper disks with granulomatous tissues were removed, dried at 60°C overnight and weighed.
Gastric damage: The assay was carried out essentially based on the methods of Okabe et al. (3) and Hitchens et al. (4). Six rats weighing 150-200 g were used in each group. The animals were starved for 18 hr, and then test compounds were administered p.o. Four hr later, the animals were killed by decapitation 10 min after the injection of 5% pontamine sky blue solution (0.5 ml/rat) via the tail vein, and their stomachs were removed, fixed with 2% formalin for 10 min and opened along the greater curvature. The mucosal membranes were examined for the lesions under a dissecting microscope. The animals showing at least two lesions (erosions and/or ulcers) that were 0.5 mm and over in length were regarded as positive.
Yeast-induced fever: The assay was done by the method of Yanagi et al. (5). Rats weighing 180-200 g were used. A 20% baker's yeast (Wako Pure Chemical Co.) suspension in 0.9% NaCI was injected s.c. into the dorsal region of animals in a volume of 1.0 ml/100 g body weight. Eighteen hr later, the animals that had a rectal temper ature of 39.2'C and over were selected, divided into groups of six each, and then test compouds were administered p.o. The rectal temperature was measured with a ther mometer every hour for 5 hr after the admin istration of test compounds.
Platelet aggregation: The assay was carried out based on the method of Silver et al. (6). Platelet-rich plasma (PRP) was prepared from citrated rabbit blood (blood: 3.8% sodium citrate=9:1, by volume). PRP was preincubated with or without test compounds (ethanol concentration: 1%) at 37 °C for 1 min, and then sodium arachidonate (final concentration: 100 /cg/ml) or ADP (final concentration: 2 /eM) was added to the mixture to induce platelet aggregation. Platelet aggregation was monitored as the change in light transmission using an ag gregometer.
Arachidonic acid-induced sudden death: Five rabbits weighing 2.5-3.5 kg were used in each group. Test compounds were adminis tered p.o. 2 hr before i.v. injection of 1 .4 mg/kg of arachidonic acid dissolved in 0.9% saline containing 7% HCO-60. The animals were observed for death within 5 min after arachidonic acid.
Assay of prostaglandin synthetase activity of BSVM: The assay was carried out based on the methods of Flower et al. (7) and Taylor and Salata (8). The reaction mixture (0.5 ml) consisted of 100 mM tris-HCI (pH 8.2), 0.2 mM [1-14C] arachidonic acid (0.2 ,iCi), 3 mM epinephrine bitartrate, 3 mM reduced glutathione, 2 mg lyophilized BSVM and test compound. The reaction was started by the addition of enzyme, and the mixture was incubated with or without test compounds (ethanol concentration: 4%) at 37T for 4 min with shaking. The reaction was terminated by the addition of 3N HCI (20 iii) and extracted twice with ethyl acetate (2 ml). After the extracts were evaporated under a N2 gas stream, the residues were redissolved in ethanol , spotted quantitatively on silica gel coated plastic sheets (Eastman Kodak) and developed in solvent system A (ethyl acetate/water/2,2,4 trimethyl-pentane/acetic acid=11 /10/5/2 by volume, upper phase). Authentic arachidonic acid and PGE2 were run on the same sheet and were located by iodine vapor. The radioactive bands corresponding to PGE2 were cut out and counted in a liquid scintil lation counter. The radioactive PGE2 formed from [1 -14C] arachidonic acid was designated as prostaglandin synthetase activity.
Assays of prostaglandin synthetase and 5 lipoxygenase activities of RBL-1 cells: The assays were carried out based on the methods of Jakschik and Lee (9) and Steinhoff et al.
(10). RBL-1 cells were grown in Eagle's minimum essential medium (Nissui) sup plemented with 15% fetal bovine serum (Flow Laboratories) in a C02 incubator at 37'C for 3 days. The harvested cells were washed twice with buffer A (50 mM phos phate buffer, pH 7.4, 1.0 mM EDTA and 0.1% gelatin). The washed cells were resuspended in buffer A at 5 x 107 cells/ml, sonicated and centrifuged at 10,000xg for 20 min. The supernatant fraction was used as the enzyme source for both assays.
The assay conditions (0.5 ml) for pro staglandin synthetase activity were as follows: The supernatant (0.4 ml) was preincubated with or without test compounds (ethanol concentration: 2%) at 370C for 1 min. After the addition of [1-14C] arachidonic acid (0.1 riCi, 10 tlM), the mixture was incubated at 37°C for 20 min with shaking. For the assay of 5-lipoxygenase, to the above incubation mixture, 2 mM CaCl2 and 20 ,uM indomethacin were added, and then it was incubated as described in the assay of prostaglandin synthetase activity. Both reactions were terminated by the addition of acetone (1.0 ml) and 0.9% NaCI (0.5 ml). The mixture was acidified with 5N formic acid and extracted twice with chloroform (2 ml). After each extract was evaporated under a N2 gas stream, the residue was redissolved in chloroform: methanol (2:1, by volume), spotted quantitatively on a silica gel glass plate (Kieselgel F254, Merck) and developed in solvent A. Radioactive bands were located by radioautography, scraped off and counted in a liquid scintillation counter. The sum of radioactivity of PGD2 and PGF2a formed from [1-14C] arachidonic acid was designated as prostaglandin synthetase activity, and the sum of radioactivity of 5 HETE and 5,12-diHETE formed was desig nated as 5-lipoxygenase activity. The pro ducts were identified by cochromatography with authentic standards on silica gel plates.
Statistical analysis: Statistical significance was evaluated by Student's t-test.

Results
Effect on carrageenin-induced paw edema of rats: As shown in Fig. 2, KME-4 was found to inhibit carrageenin-induced paw edema at 5 hr in a dose-dependent manner, as well as the other reference compounds. The ED30 values calculated for indome thacin, KME-4, naproxen and ibuprofen were 4, 8, 1 3 and 42 mg/kg, respectively.
Effect on paper disk-induced granuloma formation: KME-4 showed significantly a dose-dependent inhibition of granuloma formation at doses of 2-10 mg/kg/day ( Table 1). The ED25 for KME-4 was 2.2 mg/ kg/day, and it was 0.3 times as active as indomethacin, equipotent as naproxen and 6.4 times as active as ibuprofen. KME-4 at the indicated doses did not reduce body weight gain or increase adrenal weights (data not shown).
Ulcerogenic activity in rats: KME-4 caused gastric lesions in starved rats. The UD50 value for KME-4 was 43 mg/kg, and it was about 0.2 times as irritating as indomethacin and naproxen and 1.7 times as irritating as ibuprofen (Table 2).
Antipyretic activity in rats: Figure 3 shows the comparison of antipyretic activity of KME-4 and indomethacin. KME-4 showed a significant reduction of the increase in body temperature induced by yeast in rats at doses of 0.5-10 mg/kg. Indomethacin did not significantly reduce it at 0.5 mg/kg, but it was effective at 2-10 mg/kg. In addition, KME-4 did not affect the body temperature of normal rats at doses up to 50 mg/kg (data not shown).

Discussion
In this study, we have demonstrated that KME-4 possesses anti-inflammatory, anti pyretic and anti-platelet aggregatory activi ties, and it inhibits both prostaglandin synthetase and 5-lipoxygenase.
The anti-inflammatory activity of KME-4 was weaker than indomethacin, but stronger than naproxen and ibuprofen in the rat carrageenin edema test. Its activity was weaker than indomethacin, similar to naproxen, but stronger than ibuprofen in the rat granuloma test. As most non-steroidal anti -inflammatory drugs (NSAIDs) cause gastrointestinal damage, it is important to clarify the distinction between an anti inflammatory dose and an ulcerogenic dose. KME-4 also produced gastric lesions in starved rats, and it was less irritanting than indomethacin and naproxen, but more ir ritanting than ibuprofen.
On the basis of these results, when the ratio of UD50 stomach to ED30 carrageenin edema or to ED25 granuloma was compared for KME-4 and the other anti-inflammatory drugs, KME-4 showed higher values than those of the reference drugs (Table 4). This suggests that KME-4 shows anti-inflammatory activity at a lower dose which is far from the dose producing ulcerogenic activity.
In the antipyretic activity test, KME-4 was effective in reducing fever induced by yeast at relatively low doses (0.5 to 10 mg/ kg), but did not affect the body temperature of normal rats at a higher dose (50 mg/kg). This characteristic of KME-4 is considered to be desirable as a drug. KME-4 was found to inhibit platelet aggregation induced by arachidonic acid and prostaglandin synthetase activity of bovine seminal vesicle. The NSAIDs which are the inhibitors of prostaglandin synthetase and platelet aggregation have been shown to prevent respiratory distress and death induced by i.v. injection of arachidonic acid (11, 12). As for this, we also confirmed that KME-4 had preventive effects on arachidonic acid-induced acute death in rabbits as well as indomethacin.
This effect may be mainly due to the inhibition of thromboxane A2 formation from arachidonic acid via cyclo oxygenase.
(19, 20), have been shown to have an unusual profile of anti-inflammatory effects which may account for the inhibition of both cyclooxygenase and lipoxygenase. In these respects, the present results indicate that KME-4 is also a dual prostaglandin synthetase and 5-lipoxygenase inhibitor with anti inflammatory activity, and it is suggested that KME-4 offers more different pharma cological effects than selective prostaglandin synthetase inhibitors. Especially, whether KME-4 shows the inhibitory effect on leukocyte migration should be demonstrated. This, however, is now under investigation.
In addition, slow reacting substance of anaphylaxis (SRS-A) that has recently been identified as leukotrienes C4, D4 and E4, which are also 5-lipoxygenase products, has been recognized as the important mediator in bronchial asthma and anaphylaxis (for review, 21). Therefore, 5-lipoxygenase in hibitors including KME-4 may be effective in the treatment of allergic asthma. The inhibitory effects of KME-4 on SRS-A biosynthesis and allergic reactions also remain to be examined.