Wistar strain rats as the model for IgE antibody experiments.

The amount of plasma IgE antibody formed and its change over time were investigated by enzyme-linked immunosorbent assay (ELISA) in male and female Sprague-Dawley (SD), Donryu, and Wistar strain rats. IgE antibody formation was initiated by injecting a mixture of 2,4-dinitrophenylated ascaris extract (DNP-As) as antigen and killed Bordetella pertussis as adjuvant into the paws of the animals. The amount of IgE antibody formed was low on day 10 in both male and female SD (40-80 ng/ml) and Donryu (20-40 ng/ml) strain rats, and an increase in the amount was observed on day 20. The peak value of IgE antibody was observed day 10 in Wistar strain rats and was 130 and 200 ng/ml in the male and female rats, respectively. These results suggest that Wistar strain rats produce the most IgE antibody when DNP-As is used as antigen and they can serve as a model for allergic diseases.

Synthesis and physiological activity of novel tocopheryl glycosides.

Vitamin E glycosides were synthesized and enzymatic hydrolysis was examined for use as potential pro-drugs, however, the glycoside bond was found to be stable. On the other hand, among the glycosides synthesized, dl-alpha-tocopherylglucoside (6b) and dl-alpha-tocopherylmannoside (6c) showed strong inhibitory action on histamine release from mast cells. In addition, 6c also showed a suppressive action on IgE antibody formation. Thus, tocopheryl glycoside showed new properties compared to tocopherol (vitamin E). In particular, 6c was shown to be a novel lead compound with excellent manifold anti-allergic activity and anti-inflammatory activity.

Antioxidative and 5-lipoxygenase inhibiting activities of novel bis(4-hydroxy-2,3,5-trimethylphenoxy)alkyl derivatives.

Novel bis(4-hydroxy-2,3,5-trimethylphenoxy)alkyl derivatives were synthesized and evaluations were made of their inhibiting action on Fe3+-ADP induced lipid peroxidation in rat liver microsome and reducing action on alpha,alpha-diphenyl-beta-picrilhydrazyl (DPPH), a stable radical, in addition to their inhibiting action on 5-lipoxygenase (5-LO), an enzyme that synthesizes leukotrienes. We performed a structure-activity correlation study on these derivatives. A strong Fe3+-ADP induced lipid peroxidation preventing activity was observed for the derivatives with an odd number of methylene groups including 1,3-bis(4-hydroxy-2,3,5-trimethylphenoxy)propane (3b) and 3a. No change in the DPPH reducing activity was found with change in the number of methylene groups. 5-LO inhibiting activity among the derivatives was the highest for 1,6-bis(4-hydroxy-2,3,5-trimethylphenoxy)hexane (3e). MM2 calculations were performed to find a stable steric structure for the derivatives, and 1,5-bis(4-hydroxy-2,3,5-trimethylphenoxy)pentane (3d) showed a strong activity in both antioxidative action and 5-LO inhibiting action.

Synthesis and biological activities of novel antiallergic agents with 5-lipoxygenase inhibiting action.

Novel benzimidazole derivatives were synthesized and their pharmacological activities were examined. These compounds showed a good suppressive action on histamine release from rat peritoneal mast cells produced by antigen-antibody reaction, an antagonistic action on guinea pig ileum contraction caused by histamine, an inhibitory action on 5-lipoxygenase in rat basophilic leukemia-1 (RBL-1) cells, and a preventive action on NADPH dependent lipid peroxidation induced by Fe3+-ADP in rat liver microsomes. In addition, 1-[2-[2-(4-Hydroxy-2,3,5-trimethylphenoxy)ethoxy]-ethyl]-2-(4-meth yl-1-homopiperazino)-1H-benzimidazole difumarate (BOM1006) exhibited a dose dependent suppressive action on 48 h homologous passive cutaneous anaphylaxis (PCA) reaction in rats orally administered the drug.

Synthesis of benzimidazole derivatives as antiallergic agents with 5-lipoxygenase inhibiting action.

Syntheses were conducted of novel benzimidazole derivatives that suppress histamine release from mast cells, inhibit 5-lipoxygenase, and possess antioxidative action. Among the compounds synthesized, 1-[2-[2-(4-hydroxy- 2,3,5-trimethylphenoxy)ethoxy]ethyl]-2-(4-methyl-1- homopiperazino)benzimidazole (22) potently suppressed histamine release from rat peritoneal mast cells triggered by the antigen-antibody reaction, inhibited 5-lipoxygenase in rat basophilic leukemia-1 (RBL-1) cells, and prevented the NADPH-dependent lipid peroxidation induced by Fe(3+)-ADP in rat liver microsomes, in addition to an antagonizing the contraction of guinea pig ileum caused by histamine.

Inflammatory action of 8-methoxypsoralen-spermine photoproduct (8-MOP-Spm-P(GFC)) and effects of various drugs on rat paw edema induced by 8-MOP-Spm-P(GFC).

The photoproducts produced by irradiating 8-methoxypsoralen (8-MOP) in the presence of spermine (Spm) were fractionated using gel filtration chromatography (GFC) on a Sephadex G-25 column. As a result, two bands which were characterized by the effects on hyaluronidase activity were obtained. The first band strongly activated the hyaluronidase, but a second band did not exhibit any effect on the enzyme activity. The first and second bands contained photoproducts with molecular weights (MW)>2700 and MW<728, respectively, determined by the GFC method. The photoproducts, 8-MOP-Spm-P(GFC) obtained from the first band, but not the photoproducts with lower MW from the second band, showed enzyme activating action. 8-MOP-Spm-P(GFC) induced paw edema, which was stronger in the first phase than the second one in rats, differing from that induced by carrageenin. This photoproduct was a substance with lower cell toxicity because it did not cause hemolysis on red blood cells or the release of lactic dehydrogenase from mast cells in rats. The effects of various drugs on 8-MOP-Spm-P(GFC)-induced edema were investigated. As a result, edema formation was inhibited by drugs with an anti-histaminic action, such as alimemazine, dl-chlorpheniramine, promethazine, ketotifen and azelastine, and with anti-serotonin action such as cyproheptadine. On the other hand, tranilast did not show significant inhibition and indomethacin showed a tendency to increase its formation. These results suggested that 8-MOP-Spm-P(GFC) is a new inflammatory substance and is very useful as an agent to develop new anti-inflammatory drugs without cyclooxygenase inhibitory action.

Synthesis of trimethylhydroquinone derivatives as anti-allergic agents with anti-oxidative actions.

A novel series of trimethylhydroquinone derivatives was synthesized and evaluated for their anti-lipid peroxidation activity in rat liver microsomes, inhibition of rat basophilic leukemia-1 (RBL-1) cell 5-lipoxygenase and 48 h homologous passive cutaneous anaphylaxis (PCA) activity in rats. 4-[4-[4-(Diphenylmethyl)-1-piperazinyl]-butoxy]-2,3,6-trimethyl phenol (9c) exhibited the ability to inhibit Fe(3+)-ADP induced NADPH dependent lipid peroxidation (IC50 = 5.3 x 10(-7) M), 5-lipoxygenase ((IC50 = 3.5 x 10(-7) M) and PCA reaction (57% inhibition at 100 mg/kg p.o.).

A study of novel antiallergic agents with eosinophilic infiltration inhibiting action.

The antiallergic action of a series of novel mono-O-substituted trimethylhydroquinones was investigated. Among this series of the compounds, 4-[4-[4-(diphenylmethyl)-1-piperazinyl]butoxy]-2,3,6- trimethylphenol (compound 3) showed a potent antihistaminic action (pA2 = 7.11) and an antiasthmatic action (100 mg/kg. p.o) on sensitized guinea pigs. Moreover, this compound exhibited a strong eosinophilic infiltration inhibiting action on sensitized mice (100 mg/kg p.o.).

Post-initiation effects of a super critical extract of propolis in a rat two-stage carcinogenesis model in female F344 rats.

Post-initiation modifying effects of dietary administration of a super critical extract of propolis on major organs were examined using a two-stage carcinogenesis model. Groups of 21 or 22 F344 female rats were treated sequentially with 2,2'-dihydroxy-di-n-propylnitrosamine (DHPN, i.g.), 7,12-dimethylbenz[a]anthracene (DMBA, i.g.), 1,2-dimethylhydrazine (DMH, s.c.) and N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN, in drinking water) during the first 3 weeks for initiation, and then administered diet containing 0.1 or 0.01% propolis for 33 weeks. Further groups were treated with the carcinogens alone, 0.1% propolis alone or basal diet alone. All surviving animals were killed at week 36, and major organs were examined histopathologically for development of preneoplastic and neoplastic lesions. The incidence and multiplicity of mammary carcinomas were significantly decreased by the 0.1 and 0.01% propolis treatments. In the urinary bladder, the incidence of PN hyperplasia but not tumors was, in contrast, significantly increased by 0.1% propolis. Similarly, the number and area of glutathione S-transferase placental form (GST-P)-positive liver foci were significantly elevated with this high dose. The results indicate that a low dose of a super critical extract of propolis may find application as a potent chemopreventor of mammary carcinogenesis.

Evaluation of propolis (II): effects of Brazilian and Chinese propolis on histamine release from rat peritoneal mast cells induced by compound 48/80 and concanavalin A.

To establish a biological method for evaluating propolis and to reveal their anti-allergic action, the effects of the ethanol and water extracts (EA-ET and WA-WT, respectively) from Brazilian, Chinese and Japanese propolis on the histamine release induced by compound 48/80 and concanavalin A (Con A) were investigated. The relation between the inhibitory activities of these extracts on the histamine release and their E(1 cm)1% values were also examined. As a result, the following was found: 1) 0.003-0.01% ethanol and 0.03-0.1% water extracts inhibited the histamine release induced by compound 48/80 and Con A, and the inhibitory potencies of the former extracts were more than 10 times stronger than those of the latter extracts, making it clear that both the ethanol and water extracts possess an anti-allergic action; 2) most of the ethanol and water extracts responded to the histamine release induced by both the histamine releasers in a concentration-dependent manner; 3) the inhibitory activities of 0.003% EM from Hebei Province, EP from Sichuan Province, EQ from Zhejiang Province and ER from Anhui Province in China were weaker than those of 0.01% corresponding extracts, whereas 0.001% ED-EH from Brazilian propolis, EM, EN from Henan Province in China and EP-ER promoted the Con A-induced histamine release of more than 10%, suggesting that such extracts must be carefully given to humans; 4) the inhibitory potencies of only 0.03-0.1% water extracts from Chinese propolis on the Con A-induced histamine release related excellently with their E(1 cm)1% values; 5) from the results of the relation between the inhibitory potencies of the propolis extracts and their E(1 cm)1% values, it was suggested that an unknown compound, being a poorly water-soluble compound which is a non-flavonoid, with an anti-allergic action is contained in propolis; 6) to precisely evaluate the anti-allergic action of the propolis, the biological method, which measures the inhibitory activities of the propolis extracts on histamine release, was markedly superior to the physicochemical method.

Evaluation of propolis. I. Evaluation of Brazilian and Chinese propolis by enzymatic and physico-chemical methods.

To establish a method of evaluating propolis, the effects of the ethanol and water extracts from various collecting of propolis from different countries and plant sources on hyaluronidase activity were investigated along with their absorption spectra and specific absorbance (E(1%)1 cm value). The relations between the hyaluronidase inhibitory activities of these extracts and their E(1%)1 cm values were also examined, and the following was found: 1) the enzyme inhibitory activities of the ethanol extracts were more potent than those of the water extracts; 2) the enzyme inhibitory activities of the ethanol extracts from Araucaria angustifolia (BERT.) O. KTZE were low compared with those of other ethanol extracts; 3) the enzyme inhibitory activities of all the ethanol extracts correlated excellently with their E(1%)1 cm values, but in the water extracts, they decreased with increase in E(1%)1 cm values; 4) the water extracts of Chinese propolis from Hebei, Jiangsu, Sichuan and Zhejiang Provinces inhibited weakly compared with that from Brazilian and other Chinese propolis; 5) the shapes of absorption bands of the propolis extracts and the E(1%)1 cm values were approximately dependent on the place or the plant source from which propolis was collected. These experimental results indicated that, for the exact evaluation of propolis, the enzymatic method, measuring the hyaluronidase inhibitory activity, was superior to the physicochemical method.

Pharmaceutical properties of freeze-dried formulations of egg albumin, several drugs and olive oil.

The freeze-dried ternary formulations of meclizine (MZ, an anti-motion sickness drug), prednisolone (PRED, an anti-inflammatory drug) and norfloxacin (NFLX, an anti-microbial drug) which are poorly water-soluble and are low bioavailability drugs, were prepared using egg albumin and olive oil. The powder X-ray diffractions, the dissolution rate and the bioavailabilities in vivo of these formulations were studied in comparison with each drug alone. By forming ternary formulations of these drugs, the dissolution rates of the drugs from the formulations were significantly improved compared with each drug alone. The results of their powder X-ray diffraction measurements showed that these drugs in the ternary formulations presented in an amorphous form, indicating increased dissolution rates. On the other hand, the plasma concentrations of these drugs increased significantly after oral administration in formulations to rats, except for the NFLX formulation, and the areas under the concentration-time curves (AUC) of the ternary formulations of MZ, PRED and NFLX were 2.1, 1.6 and 1.3 times those of the drugs alone, respectively. From these results, it was proven that formulations consisting of egg albumin, olive oil and poorly water-soluble drugs were useful preparations for improving the drug's disadvantageous pharmaceutical properties.

Synthesis and anti lipid-peroxidation activity of hydroquinone monoalkyl ethers.

A series of hydroquinone monoalkyl ethers was synthesized and evaluated for anti lipid-peroxidation activity in rat liver microsomes. 4-Hexyloxy-2,3,6-trimethylphenol (9), having a low redox potential, as well as ascorbic acid exhibited the strongest anti lipid-peroxidation activity (IC50 = 4.2 x 10(-7) M). Structure-activity relationship studies demonstrated that the inhibitory effect of hydroquinone monoalkyl ethers on lipid peroxidation was increased by the acquisition of an optimum hydrophobicity and decreased by an insufficient or excessive hydrophobicity.

Syntheses and inhibitory effects on gastric lesions of 4-guanidinomethylbenzoic acid arylamides.

A novel series of 4-guanidinomethylbenzoic acid (GMBA) arylamides was synthesized. Several showed more potent inhibitory effects on stress-induced gastric lesion in rats than cetraxate. We selected 4-guanidinomethylbenzoic acid (2'-ethoxycarbonyl)phenylamide 3 for further pharmacological assessments because it had low toxicity. Compound 3 showed significant inhibitory effects on stress-, HCl-ethanol- and indomethacin-induced gastric lesions and gastric secretion, the ED50 values being 34.4, 45.0 and 23.0 mg/kg (p.o.) and 240 mg/kg (i.d.), respectively. Furthermore, this compound restored the reduction of gastric mucus caused by the stress-loading and inhibited compound 48/80-induced ulcer.

Pharmacological and pharmaceutical properties of freeze-dried formulations of egg albumin, indomethacin, olive oil, or fatty acids. II.

To confirm the increased bioavailability of indomethacin (IND) when incorporated in a preparation with egg albumin and olive oil, we studied the detailed pharmaceutical characteristics of a ternary formulation consisting of egg albumin, IND and olive oil. From the results of X-ray powder diffraction measurements, the drug in the formulation was found to be in an amorphous form. When orally administered to rats, the ternary formulation significantly increased the plasma concentration and cumulative biliary and urinary excretion of IND alone as well as the urinary excretion of its major metabolite, desmethylindomethacin, compared with the drug alone. In addition, the dissolution rate of IND from the formulation was higher than that of the drug alone. These results clearly suggest that the bioavailability of IND was markedly improved by incorporating it in a protein-drug formulation containing olive oil as an absorbefacient element, and this effect may be due to an increased absorption of IND.

Pharmacological and pharmaceutical properties of freeze-dried formulations of egg albumin, indomethacin, olive oil, or fatty acids.

Formulations consisting of egg albumin, indomethacin (IND), and olive oil or fatty acids, were prepared by vigorous stirring using a high-speed homogenizer and subsequent freeze-drying. To confirm the anti-inflammatory properties and ulcerogenic effects of the formulations, we examined the action of the formulations on carrageenan-induced edema in rats as well as their ulcerogenic actions in the same species. Compared with IND alone, albumin-IND-olive oil (9:1:4.3), albumin-IND-linolenic acid (9:1:4.3), albumin-IND-linolic acid (9:1:4.3), albumin-IND-oleic acid (9:1:4.3), albumin-IND-stearic acid (9:1:4.3), and albumin-IND-tristearin (9:1:4.3) formulations all exhibited a more potent inhibitory effect on carrageenan-induced edema. In addition, the inhibitory effects on edema formation of an albumin-IND (9:1) complex was as strong as that of IND alone. These results suggested that the bioavailability of IND was increased by olive oil, fatty acid, and tristearin as absorbefacient agents. The increase in the bioavailability was evident from the fact that the mean plasma levels, maximum plasma levels (Cmax), and area under plasma concentration-time curve (AUC) values after oral administration of the albumin-IND-olive oil (9:1:4.3) formulation was significantly greater than that after administration of the drug alone. With respect to their ulcerogenic properties, the formulations were significantly less active than IND alone, suggesting that a reduction in the ulcerogenic activity of IND was by produced complexation with egg albumin.

Inflammatory activity of polymeric photoproducts of chlorpromazine.

To confirm the inflammatory activity of polymeric photoproducts (CPZ-polymers) of chlorpromazine (CPZ), which were obtained by gel filtration of a UV-pre-irradiated CPZ aqueous solution, the histamine release from rat peritoneal exudate cells was studied and the paw-inflammation in mice induced by these CPZ-polymers was examined. CPZ-polymers induced a dose-dependent histamine release at concentrations of 1, 3 and 10 mg/ml. This effect was approximately one-tenth of that of compound 48/80. Furthermore, CPZ-polymers markedly induced lasting paw-edema in a dose-dependent manner, the swellings remaining for at least 96 h. When intraperitoneally injected into mice, CPZ-polymers induced a significant elevation of histamine release in the peritoneal cavity 0.5 h after the injection, compared with a control group. The histamine levels in the cavities returned to normal within the next 0.5 h, and remained normal for at least 23 h, indicating that histamine release may be caused only in the early stages of CPZ-polymer-induced inflammation. The inflammatory activity of the CPZ-polymers suggests that they are inflammatory substances formed from CPZ by UV-irradiation.

Syntheses and inhibitory effects on gastric lesions of trans-guanidinomethylcyclohexane carboxylic acid arylamides.

A novel series of trans-guanidinomethylcyclohexanecarboxylic acid (trans-GMCHA) arylamides was synthesized. The several trans-GMCHA arylamide derivatives showed more potent inhibitory effects on the stress- and HCl-ethanol-induced gastric ulcers than cetraxate in rats. In acute toxicity studies in mice, most amides showed such severe toxicity that all mice injected with these compounds (50 mg/kg, i.p.) died. However, mice injected with the trans-GMCHA (2'-,3'- and 4'-ethoxycarboxy)phenylamide (7, 8 and 9) which bear an alkyloxycarbonyl group at benzene ring survived. From these results, trans-GMCHA (2'-ethoxycarbonyl)phenylamide (7) was selected as a promising anti-ulcer agent.

Activation of hyaluronidase by 8-methoxypsoralen-polyamine photoproducts.

Ultraviolet radiation of 8-methoxypsoralen (8-MOP) in the presence of various polyamines resulted in stable photoproducts that were very soluble in water and showed hyaluronidase-activating properties. Among them, the photoproducts obtained from the reaction systems of 8-MOP-spermine and 8-MOP-spermidine markedly activated hyaluronidase. The enzyme activity was not affected by 8-MOP alone and the photoproduct of 8-MOP (8-MOP-P). From these facts, it was suggested that the photoproducts with hyaluronidase-activating properties might play an important role in the onset of 8-MOP-induced photosensitivity.

3-O-alkylascorbic acids as free-radical quenchers: synthesis and inhibitory effect on lipid peroxidation.

A novel series of 3-O-alkylascorbic acids (3-RASA, 3a-n) was synthesized to act as radical scavengers for active oxygen species and free radicals, and their redox potentials and inhibitory effects on lipid peroxidation in rat liver microsomes were evaluated. The redox potentials of the 3-RASA compounds were increased by the substituent group to 90-190 mV above the potential for ascorbic acid (i.e., 3-RASA compounds were harder to oxidize). Although 3-O-dodecylascorbic acid (3c) and 3-O-(decylcarbomethyl)ascorbic acid (3i) differed in their redox potentials, they both markedly inhibited lipid peroxidation in rat liver microsomes to a similar extent (IC50 = 3.1 and 3.3 X 10(-6) M, respectively). Structure-activity relationship studies demonstrated that the anti lipid peroxidation activity of the 3-RASA compounds was markedly dependent upon their hydrophobicity.