Novel dual inhibitors of 5-lipoxygenase and thromboxane A2 synthetase: synthesis and structure-activity relationships of 3-pyridylmethyl-substituted 2-amino-6-hydroxybenzothiazole derivatives.

As part of our search for novel antiinflammatory drug candidates, we have designed and synthesized a series of 3-pyridylmethyl-substituted 2-amino-6- hydroxybenzothiazoles. Introduction of a 3-pyridylmethyl group into the 2-amino group (type-A) or the benzene ring (type-B) of 2-amino-6-hydroxybenzothiazoles imparted dual inhibitory activity against the production by glycogen-induced peritoneal cells of rat (in vitro) of leukotriene B4 (LTB4) and thromboxane A2 (TXA2), while not significantly inhibiting that of prostaglandin E2 (PGE2). The observed inhibition of the former two arachidonic acid metabolites was indicated to be the result of a direct action on 5-lipoxygenase and TXA2 synthetase by a cell-free in vitro assay. On the other hand, the inhibitory activities against PGE2 production were for most compounds very weak, indicating that they did not inhibit cyclooxygenase. Structure-activity relationship studies concerning the position of the 3-pyridylmethyl group revealed that type-B compounds generally showed about 10-fold stronger inhibitory activity against TXA2 synthetase than type-A compounds. The position of the 3-pyridylmethyl group played an important role in TXA2 synthetase inhibition. When some of these compounds (8, 13a, 26a (E3040), 26b, 27b, and 28b) were orally administered in the rat TNB/ethanol-induced chronic colitis model (100 mg/kg), the production of both LTB4 and TXB2 in the rat colon was reduced (ex vivo). In addition, one type-B compound, 6-hydroxy-5,7-dimethyl-2-(methylamino)-4-(3-pyridylmethyl)benzothiazole (26a), demonstrated a therapeutic effect at treatments of 100 mg/kg po once daily for 11 days and showed almost comparable activity to sulfasalazine at a dose of 500 mg/kg, the reference drug for inflammatory bowel diseases, in this in vivo model.

Effects of taurocholic acid/HCl alone or after pretreatment with geranylgeranylacetone on phospholipid metabolism in rat gastric mucosa.

Changes in phospholipid metabolism in gastric mucosa caused by instillation of taurocholic acid (TCA)/HCl (80 mM/300 mM) into the stomach of rats and the effects of pretreatment with an antiulcer agent, geranylgeranylacetone (GGA), were studied after intravenous injection of radioisotope-labeled precursors. The instillation of TCA/HCl rapidly reduced the incorporation of labeled fatty acids and glycerol into phosphatidylcholine and phosphatidylethanolamine, indicating the inhibition of de novo synthesis of phospholipids. These changes were restored by 120-150 min after the TCA/HCl treatment. Pretreatment with GGA enhanced the incorporation of precursors into phosphatidylcholine immediately after the instillation of TCA/HCl. Experiments in which the mucosal lipids were labeled with fatty acids prior to the instillation of TCA/HCl showed that the degradation of cellular lipids and release of the products into the gastric lumen were induced by TCA/HCl and that these changes were not prevented by GGA. Since GGA almost completely inhibited the gastric lesions induced by TCA/HCl, the enhancement of synthesis of mucosal phosphatidylcholine induced by GGA may be involved in the prevention of gastric damage. The incorporation of labeled fatty acids into free fatty acid fraction and diacylglycerol was increased quickly by the TCA/HCl treatment, suggesting early damage to the blood vessels of the gastric mucosa; these changes were inhibited significantly by GGA.

Inhibitions of acid secretion by E3810 and omeprazole, and their reversal by glutathione.

A substituted benzimidazole ([4-(3-methoxypropoxy)-3-methylpyridine-2-yl]methylsulfinyl)- 1H-benzimidazole sodium salt (E3810), is a gastric proton pump (H+, K(+)-ATPase) inhibitor. E3810 and omeprazole inhibited acid accumulation dose dependently as measured with aminopyrine uptake in isolated rabbit gastric glands, their IC50 values being 0.16 and 0.36 microM, respectively. The addition of exogenous reduced glutathione (GSH) to the gland suspension reactivated dose dependently the acid secretion which had been inhibited by 2 microM E3810 or omeprazole as a function of the incubation time. Furthermore, GSH at 1 and 3 mM reversed the antisecretory effect of E3810 more quickly than it did that of omeprazole. The antisecretory effect of E3810 was slightly greater than that of omeprazole in histamine-stimulated fistula dogs in vivo. The duration of the antisecretory activity of E3810 at concentrations of 2 and 4 mg/kg was shorter than that of omeprazole at the same concentrations in pentagastrin-stimulated fistula dogs. The reversal of the antisecretory activity of the inhibitors in dogs is suggested to be due to the action of endogenous extracellular GSH, in addition to de novo synthesis of the proton pump, because bullfrog gastric mucosae were found in the present study to secrete GSH into the mucosal solution at the rate of about 0.25 nmol/min/g tissue.

Effect of E3040, an inhibitor of 5-lipoxygenase and thromboxane synthase, on rat bowel damage induced by lipopolysaccharide.

Intravenous administration of lipopolysaccharide to rats that had been immunized with lipopolysaccharide induced hemorrhagic damage in the large intestine. We investigated the role of 5-lipoxygenase and thromboxane synthase products in the damage of the large intestine induced by lipopolysaccharide. In the large intestine of lipopolysaccharide-immunized rats, intravenous injection of lipopolysaccharide increased the vascular permeability, production of leukotriene B(4), leukotriene C(4)/D(4), thromboxane B(2) and prostaglandin E(2), and also increased the activity of myeloperoxidase, a marker enzyme of neutrophils. Oral administration of E3040 (6-hydroxy-5,7-dimethyl-2-(methylamino)-4-(3-pyridylmethyl)benzothiazole), a novel dual inhibitor of 5-lipoxygenase and thromboxane synthase, at 30 and 100 mg/kg inhibited the increase in vascular permeability induced by lipopolysaccharide in the large intestine. E3040 inhibited the production of leukotriene B(4) and thromboxane B(2) and tended to increase the production of prostaglandin E(2) in the large intestine. Sulfasalazine (500 mg/kg) and prednisolone (10 mg/kg), drugs used for the treatment of inflammatory bowel disease, had no significant effect on eicosanoid production and vascular permeability. These results indicate that E3040 inhibits the production of both leukotriene B(4) and thromboxane B(2) and prevents lipopolysaccharide-induced damage in the large intestine of lipopolysaccharide-immunized rats.

In vitro effects of E3040, a dual inhibitor of 5-lipoxygenase and thromboxane A(2) synthetase, on eicosanoid production.

In vitro pharmacological profiles of E3040, 6-hydroxy-5, 7-dimethyl-2-(methylamino)-4-(3-pyridylmethyl) benzothiazole were investigated. Against the 5-lipoxygenase activity of rat basophilic leukemia cells, E3040 and zileuton (a 5-lipoxygenase inhibitor) had an IC(50) of 0.23 and 0.93 microM, respectively. Against the thromboxane A(2) synthetase activity of human platelets, E3040 had an IC(50) of 0.01 microM, which was comparable to that of OKY-1581 (sodium (E)-3-[4-(3-pyridylmethyl) phenyl]-2-methylacrylate, a thromboxane A(2) synthetase inhibitor). Against cyclooxygenase activity of sheep seminal vesicles, E3040 showed no inhibition (IC(50), >300 microM). Sulfasalazine and 5-aminosalicylic acid, therapeutic drugs for inflammatory bowel disease, inhibited 5-lipoxygenase activity with an IC(50) of 293 and 970 microM, respectively. Sulfasalazine inhibited thromboxane A(2) synthetase activity with an IC(50) of 20 microM. In rat peritoneal leukocytes, E3040 inhibited leukotriene B(4) and thromboxane B(2) production with an IC(50) of 0.17 and 0.24 microM, respectively. E3040 inhibited leukotriene B(4) production in human neutrophils and thromboxane B(2) production in human platelets (IC(50) of 0.21 and 0.09 microM, respectively). These results indicated that E3040 potently inhibited 5-lipoxygenase and thromboxane A(2) synthetase and blocked leukotriene B(4) and thromboxane B(2) production in rat peritoneal and human blood cells.

Low level wild-type and pre-core mutant hepatitis B viruses and HBeAg negative reactivation of chronic hepatitis B.

A qualitative and a quantitative mutation-site specific polymerase chain reaction assay (MSSA) was used to detect low level wild-type and pre-core mutant hepatitis B virus (HBV)-DNA. Serum samples from 11 anti-hepatitis B e (anti-HBe)-positive asymptomatic HBV carriers (Group A) and 10 anti-HBe-positive chronic hepatitis B patients who achieved alanine transaminase (ALT) normalization after antiviral therapy (Group B) were tested. Eleven patients had both wild-type and pre-core mutant HBV-DNA (52%, 4 from Group A and 7 from Group B), whereas 3 patients had only pre-core mutant HBV-DNA (14%, 2 from Group A and 1 from Group B) by qualitative MSSA assay. During a 3-year follow-up period, relapses were observed in 3 patients from Group B and intermittent ALT elevation was observed in 4 patients from Group A and 3 patients from Group B. The wild-type HBV-DNA concentration in the patients with reactivation was 10(2.06+/-2.62) copies/ml, whereas that in all patients without reactivation was below 10(2) copies/ml (P < .05). The pre-core mutant HBV-DNA concentration in the patients with reactivation was also significantly higher than that in the patients without reactivation (10(3.94+/-2.25) vs. 10(0.65+/-1.45) copies/ml, P < .001). All patients with both HBV-DNA concentrations below 10(2) copies/ml did not exhibit reactivation. Our result suggest that a high prevalence of coexistence of low level wild-type and pre-core mutant HBV-DNA has the potential for reactivation in anti-HBe-positive patients. Furthermore, quantification of wild-type and pre-core mutant HBV-DNA was useful to predict the prognosis of anti-HBe-positive infection and evaluate the efficacy of antiviral therapy.

Effect of secretin on stress-induced gastric bleeding in rats.

The effect of secretin on gastric bleeding induced by water (20 degrees C) immersion stress was studied by means of either perfusion of hydrochloric acid (0.13 N) or instillation of hydrochloric acid solution (0.4 N) into the rat stomach. When the stomach was perfused with acid solution, gastric bleeding occurred 20 min after the stress of water immersion and the amount of bleeding increased with time. It was found that integrated amounts of gastric bleeding after stress were more significantly decreased in the secretin-treated group (7.5 clinical units/kg/hr) than the saline control. On the other hand, intraduodenal administration of cimetidine (100 mg/kg) or propantheline (30 mg/kg) did not affect gastric bleeding. It was also revealed that the total amount of glycoprotein in the 4-hr perfusate was decreased 30% by the water immersion, but that secretin treatment prevented the 30% decrease in glycoprotein in the perfusate. In experiments on intragastric instillation of hydrochloric acid solution, it was found that gastric bleeding was seen only when the concentration of hydrochloric acid solution was higher than 0.4 N. However, the bleeding was also significantly prevented by treatment with secretin (2.5 and 7.5 clinical units/kg/hr). In conclusion, secretin prevents stress-induced gastric bleeding at least in part by increasing glycoprotein secretion in the gastric mucosa in the rat.

Effects of rabeprazole, a gastric proton pump inhibitor, on biliary and hepatic lysosomal enzymes in rats.

The effects of rabeprazole (E3810), omeprazole and chloroquine on hepatic lysosomal function were studied. After chloroquine (50 mg/kg), rabeprazole (5 mg/kg) or omeprazole (5 mg/kg) was given intraperitoneally to rats for 6 days, the bile was collected via a bile duct cannula for 5 hr, and hepatic and biliary lysosomal enzyme (N-acetyl-beta-glucosaminidase and beta-galactosidase) activities were measured. The latency (an index for the hepatic lysosomal membrane integrity) was calculated from the N-acetyl-beta-glucosaminidase activity. The biliary constituents and plasma concentrations of lipids were also measured. The administration of chloroquine significantly increased hepatic and biliary lysosomal enzyme activities, but did not affect the lysosomal enzyme latency, hepatic and biliary protein content or bile flow. It significantly decreased the bile acid level. On the other hand, the administration of rabeprazole and omeprazole did not alter the lysosomal enzyme activities, lysosomal enzyme latency, protein content in liver or liver weight. Furthermore, no significant differences were observed in biliary lysosomal enzyme activity, protein content, bile flow, biliary constituents or in the plasma concentrations of lipids between the drug groups (rabeprazole or omeprazole) and the control group. The results of the present study indicate that rabeprazole, like omeprazole, does not influence hepatic lysosomal function.

Antiulcer effect of geranylgeranylacetone, a new acyclic polyisoprenoid on experimentally induced gastric and duodenal ulcers in rats.

Antinuclear effects of geranylgeranylacetone (GGA), new acyclic polyisoprenoid, on several types of experimental gastric and duodenal ulcers were studied in rats. The prophylactic administration of GGA (50--200 mg/kg p.o. or 12.5--50 mg/kg i.p.) reduced the gastric ulcers induced by the exposure to cold-restraint stress and by the administration of indomethacin, acetylsalicylic acid (ASA), prednisolone or reserpine and the duodenal ulcer after the administration of cysteamine, although it was not effective against Shay's ulcer. The curative treatment with GGA accelerated the healing process of the gastric ulcers induced by the topical application of acetic acid or thermocautery and by the administration of ASA with the exposure to cold-restraint stress. The antinuclear effect of GGA was more distinct than that of gefarnate in all types of experimental models studied. Carbenoxolone effectively reduced the gastric ulcer formation by cold-restraint stress when it was administered i.p. but not p.o., whereas GGA was effective either i.p. or p.o. GGA and gefarnate did not affect the gastric secretion in pylorus-ligated rats, whereas carbenoxolone definitely reduced the secretion of gastric juice and acid. Hexosamine content in the stomach was reduced by the exposure to cold-restraint stress. The pretreatment with GGA prevented the reduction in hexosamine contents in the superepithelial mucous layer and mucosal layer. These results may suggest a high possibility that GGA is useful for clinical treatment of peptic ulcers, probably through a mechanism of increasing defence force of the gastric mucosa.

Effects of the antiulcer drug geranylgeranylacetone on aspirin-induced gastric ulcers in rats.

Antiulcer effects of geranylgeranylacetone (GGA) on aspirin-induced gastric ulcers in rats were studied, comparing them with those of gefarnate. The oral administration of GGA prevented the development of gastric ulcer induced by a single or repeated oral administration (5 consecutive days) of aspirin. The effects of GGA were more potent and more definite than those of gefarnate. The intraduodenal administration of GGA, but not the intragastric administration, also inhibited the ulceration induced by aspirin in pylorus-ligated rats, while the intraduodenal administration of gefarnate did not. GGA prevented the reduction of the H+ concentration and the increment of Na+ concentration in the gastric juice induced by aspirin. In addition, the decrease of hexosamine content in the gastric mucosa induced by aspirin was restored to a normal level by GGA, but not by gefarnate. From these results, it was concluded that the protective actions of GGA on aspirin-induced gastric ulcers might be due to its protection from the weakening of gastric mucosal resistances.

Effect of geranylgeranylacetone on aspirin-induced changes in gastric glycoproteins.

The effects of aspirin and treatment with geranylgeranylacetone (GGA), an antiulcer drug, on the content of gastric glycoproteins were investigated in pylorus-ligated rats. In normal rats, the amount of gastric glycoproteins in the mucous layer was about 1.5 times higher than that in the gastric mucosa, indicating that the glycoproteins were distributed in the mucous layer as a highly concentrated state. Aspirin (100 mg/kg, p.o.) decreased the content of gastric glycoproteins both in the mucous layer and in the gastric mucosa. The amount of the macromolecular fraction in the gastric juice, which corresponded to the gastric glycoproteins on the basis of molecular size, was not affected by aspirin. GGA (300 mg/kg, i.d.) could prevent the decreases of the total amount of gastric glycoproteins in the mucous layer plus gastric mucosa. These results indicated that the glycoproteins coating the surface of the gastric mucosa may play a role as a defensive mechanism and that GGA exerted an antiulcer effect on aspirin-induced mucosal damage through preventing the decreases in gastric glycoproteins.

[Inhibitory action of E3810 on H+,K(+)-ATPase and gastric acid secretion in vitro].

The inhibitory action of (+-)-sodium 2-[(4-(3-methoxypropoxy)-3-methylpyridine-2-yl) methylsulfinyl]-1H- benzimidazole (E3810) on H+,K(+)-ATPase and gastric acid secretion in vitro was investigated, and it was compared with those of omeprazole (OPZ). E3810 concentration-dependently inhibited the H+,K(+)-ATPase activity of hog gastric vesicles. Its IC50 was 0.26 microM at pH 6.1. The inhibition was irreversible in nature and reversed by dithiothreitol. The potency of E3810 was 10-times that of omeprazole. Acidification of the intravesicular (luminal) space increased 1000-fold the potency of E3810, indicating that E3810 is a specific inhibitor which binds to the luminal cysteine residue of H+,K(+)-ATPase. Prolonged incubation of up to 180 min in the absence of thiol reagents of rabbit gastric glands which had been inhibited by a low concentration of E3810 (0.3 and 0.5 microM) time-dependently and completely reversed the inhibition, as determined by aminopyrine uptake, whereas it did not recover the acid secretion in omeprazole-treated glands. These results suggest that the acid-activated E3810 is a potent specific inhibitor of H+,K(+)-ATPase, and that the duration of the inhibitory action of E3810 is much shorter than that of omeprazole in isolated gastric glands.

Effect of synthetic acyclic polyisoprenoids on the cold-restraint stress induced gastric ulcer in rats.

The antiulcer effect of a series of synthetic acyclic polyisoprenoids on cold-restraint stress induced ulcer in female rats was determined, and the relationship between their antiulcer effect and chemical structure was also investigated. As a result, the following findings were obtained: The antiulcer effect of acyclic polyisoprenoids closely correlated with the number of intramolecular isoprene units, and geranylgeranyl derivatives showed a particularly marked antiulcer effect. The terminal polar groups such as 2-oxopropyl and 2-hydroxypropyl groups in geranylgeranylacetone seemed to play an important role in the antiulcer activity of acyclic polyisoprenoids. Terminal bulky groups decreased their antiulcer activity, however. The antiulcer activity of geranylgeranylacetone correlated with the number of intramolecular double bonds. There was no significant difference in antiulcer activity between all-trans-geranylgeranylacetone, 5-cis-geranylgeranylacetone and the mixture of these isomers (1:1 and 3:2). The results of this experiment suggested that the antiulcer activity of acyclic polyisoprenoids might be governed by such factors as the number of isoprene units, terminal polar groups, and number of intramolecular double bonds.