Separation of five glycerophospholipids having different polar groups, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylglycerol (PG) and cardiolipin (CL), was investigated by means of solid-phase extraction (SPE) cartridges. First, the phospholipids were retained in an aminopropylbonded phase (NH₂) cartridge and subsequently eluted as neutral (PC and PE) and acidic (PS, PG and CL) glycerophospholipid fractions. Secondly, a combination of silica gel (SI) cartridge and NH₂ cartridge was employed to separate five glycerophospholipids. The polarity of the eluent was responsible for neutral glycerophospholipid separation. Concerning acidic glycerophospholipids, the separation of PG and CL from PS depended mainly on the pH of the eluents, and the separation of PG and CL was affected by the solvent, depending on eluent polarities. Favorable recovery (not less than 95%, for five authentic phospholipids, 10-100 μg each) and repeatability (σ=2.3 for 10 μg ranges) were attained by the present method. This method of separation was applicable to the analysis of phospholipids in biological samples.

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The purpose of this study was to evaluate the hydroxyl radical scavenging activities of hydroxybenzoic acids and their esters from both chemical and biological aspects. These activities of hydroxybenzoic acids and their related compounds were estimated by ESR-spin trapping method, in which 3, 4, 5-trihydroxybenzoic acid and its ethyl and propyl esters showed the highest activities as estimated by IC₅₀ value (50% inhibition concentration of hydroxyl radicals generated in the system) : 78.04±11.23, 95.95±2.64, and 86.46±2.31 μM, respectively. In addition, 3, 4, 5-trihydroxybenzoic acid (gallic acid) at a concentration of 25 μM, protected against dermal fibroblast cell damage induced by H₂O₂, and enhanced the survival to 83.8±3.1%, in which the survival of control was 44.2±1.0%. Based on these results, the pretreatment effects of 3, 4, 5-trihydroxybenzoic acid n-alkyl esters on cell damage induced by H₂O₂ were examined. The survival of fibroblasts pretreated with the esters increased depending on the alkyl chain-length. Both C₁₂ and C₁₆ alkyl esters gave almost complete cell survival of 89.5±2.0% and 91.3±1.0%, respectively. The order of the protective effects of the compounds was in good agreement with that of their partition coefficients, suggesting that 3, 4, 5-trihydroxybenzoic acid alkyl esters are incorporated into fibroblasts, and thus prevent the cells from the toxicity caused by H₂O₂. In addition, an increase of intracellular peroxide formation in fibroblasts induced by UVA-irradiation, was suppressed to 2.27±0.41 nmol/10⁴ cells by pretreatment with C₁₆ alkyl ester at a concentration of 25 μM. Since 3, 4, 5-trihydroxybenzoyl group has been demonstrated to possess a potent scavenging activity of hydroxyl radicals, this moiety was indicated to be important in preventing cell damage induced by UVA or H₂O₂ : in turn, these produce hydroxyl radicals in the presence of trace metal ions such as iron and copper in cells.

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A homogeneous fluorescence polarization immunoassay (FPIA) was developed to measure levels of progesterone in urine using a TDx analyzer in photocheck mode (Abbott Labs). Two tracers of ethylenediamine fluorescein thiocarbamyl (EDF) were employed; one was synthesized from 11α-hydroxyhemisuccinate progesterone (Prog-11OH-HS) and the other was synthesized from 3-(o-carboxymethyl)oxime progesterone (Prog-3CMO). Each derivative of progesterone was conjugated with bovine serum albumin and used as an immunogen which produced monoclonal antibody clone 15A (MAb 15A, anti-Prog-11OH-HS) and clone 2B7 (MAb 2B7, anti-Prog-3CMO), respectively. Different combinations of tracers and antibodies were investigated in the FPIA system. Similar sensitivity was observed when using the pair, MAb 2B7 and its homologous tracer, Prog-3CMO-EDF, or MAb 15A and its homologous tracer, Prog-11OH-HS-EDF. In this immunoassay, no separation step was required and the total time for an assay of 10 samples was approximaterly 7 min. The progesterone detection limit in a 10 μl sample was 3 ng/ml. The cross-reactivity results indicate that the A-, B- and D-ring of a steroid are buried in the binding pocket of MAb 15A, while the C-ring faced outward, resulting in cross-reactivity with 11-αhydroxy progesterone. The A-, B- and C-ring of a steroid of MAb 2B7, in contrast, are buried deep in the pocket leaving the D-ring facing outward, resulting in some different degrees of cross-reactivity with C17 position substituted steroids.

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The effect of gemfibrozil, an analogue of clofibric acid, on the centrifugal behavior of peroxisomes and activity of peroxisomal enzymes involved in lipid metabolism was studied. Rats were fed chow containing 0.2% gemfibrozil for 2 weeks. Nycodenz gradient centrifugation of the light mitochondrial fraction revealed that peroxisomes of gemfibrozil-treated rats were concentrated in fractions of higher density compared with control rats. The activity of fatty acyl-CoA oxidase, crotonase, β-hydroxybutyryl-CoA dehydrogenase, and thiolase (individual enzymes of the peroxisomal fatty acid β-oxidation system) were enhanced 9.6, 2.3, 3.4 and 9.1 times respectively compared with controls, by treatment. The hydroxymethylglutaryl-CoA (HMG-CoA) reductase (rate-limiting enzyme of cholesterol synthesis) activity of peroxisomes and microsomes was greatly increased by in vivo treatment with gemfibrozil, but was decreased by addition of the agent to the assay mixture of the enzyme. Gemfibrozil directly inhibited the reductase activity and did so at a lower concentration than clofibric acid. Peroxisomal reductase was more resistant to damage by the agent than the microsomal enzyme. The HMG-CoA reductase activity of peroxisomes and microsomes of hyperlipidemic rats was also increased by in vivo treatment with gemfibrozil, whereas the serum cholesterol level was hardly changed.These results indicate that the effect of gemfibrozil differs from that of clofibric acid, the main difference being the effect on HMG-CoA reductase. Gemfibrozil increased reductase activity in vivo, unlike clofibric acid, but inhibited the enzyme in vitro to a greater extent than clofibric acid.

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In a mouse macrophage-like cell line, RAW264.7, arachidonic acid was cleaved within 30 min of lipopolysaccharide (LPS)-treatment. However, prostaglandin (PG) synthesis did not accompany this cleavage, being delayed by 4h, although significant PGH synthase (PGHS) activity was detected in the lysate of LPS-nontreated cells. The K_m value of this basal PGHS activity toward arachidonic acid was more than one hundred-fold higher than that for the lysate of cells treated with LPS for 4h. Changes in the sensitivity of the PGHS activity to nonsteroidal antiinflammatory drugs after LPS-treatment also suggested induction of PGHS with different properties from that in the case of basal PGHS. The concomitant increase in PGH synthase (PGHS) activity with the induction of PGHS-2 protein after LPS-treatment suggested a contribution by PGHS-2 to the delayed synthesis of PGs in LPS-treated macrophage cells. As for PGHS in the control cells without LPS-treatment, a different K_m value from that of PGHS-1 and the lack of cross-reactivity to anti-PGHS-1 antibodies suggested that this basal PGHS was different from the typical PGHS-1. The lower affinity of this enzyme for arachidonic acid might be the reason for the failure to release PGs by the cells without LPS-treatment.

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We examined the expression of the fliC, fliA and flhD genes in the dnaA46 mutant. In Northern blot analysis, the amounts of fliC, fliA, and flhD mRNA decreased in the dnaA46 mutant growing at 37°C but not at 28°C. The promoter activity of each gene also decreased in this mutant at 37°C, but not at 28°C. The decrease in expression of the flhD gene was not related to the cAMP-catabolite activator protein (CAP) pathway. We tentatively conclude that DnaA protein is involved in the expression of the flhD gene by a mechanism independent of the cAMP-CAP pathway.

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In guinea pigs, a marked increase in airway responsiveness to acetylcholine (Ach) was observed at 2h after lipopolysaccharide (LPS) inhalation. To examine the mediators responsible for the airway hyperresponsiveness, the changes of peptide-leukotrienes (LTs), tumor necrosis factor (TNF), interleukin-1 (IL-1), histamine and 5-hydroxytryptamine (5-HT) levels in bronchoalveolar lavage fluid (BALF) were measured. Airway responsiveness to Ach reached a peak 2h after LPS inhalation. The influx of neutrophil into BALF increased gradually and reached a peak 24h after LPS inhalation. After the inhalation of LPS, LTD₄ and TNF contents in BALF increased within the first 2h after LPS inhalation. However, other mediators were not detected or increased 6h after LPS inhalation. Aeroinhalation of LTD₄ and murine recombinant TNF-α caused airway hyperresponsiveness in guinea pigs. In addition, a LTD₄ antagonist, BAYx7195, and an inhibitor of TNF, pentoxifylline, inhibited the LPS-induced airway hyperresponsiveness. These results suggest that LTs and/or TNF play an important role in the onset of airway hyperresponsiveness in guinea pigs.

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Mitragynine, a major constituent of the young leaves of Mitragyna speciosa KORTH., has been reported to exert antinociceptive activity in mice. To determine the mechanism the influence of mitragynine on cAMP content was measured in NG108-15 cells which possess δ opioid receptors and α2B-adrenoceptors. Mitragynine inhibited the forskolin-stimulated cAMP content in a concentration dependent manner as well as morphine and noradrenaline. Mitragynine- and morphine-induced inhibition of cAMP content were blocked by naloxone. Although idazoxane inhibited noradrenaline-induced inhibition of the cAMP content, idazoxane had no effect on mitragynine-induced inhibition. These results suggest that mitragynine acts directly on opioid receptors, but not on α2-adrenoceptors, to show antinociceptive activity.

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Two types of calcium antagonists, diltiazem and nicardipine, were separately infused in 23-28 week-old spontaneously hypertensive (SHR) and age-matched normotensive Wistar-Kyoto (WKY) rats (under sodium thiobutabarbital anesthesia and ventilation, n=4) through the left femoral vein, resulting in the reduction of blood pressure. In each rat, mean arterial blood pressure, heart rate and the concentration of plasma catecholamines (CAs), norepinephrine (NE) and epinephrine (E), were concomitantly determined and the correlations between these three values were studied for each calcium antagonist. Plasma concentration of CAs were measured in blood samples collected during the infusion from the right femoral artery of each rat by the automatic sensitive and selective detection system. The reduction of blood pressure induced by the calcium antagonists brought about an increase in plasma CAs levels. The blood pressure correlated well with the logarithm of plasma NE and E concentration and the relations were expressed as Y=-αlog(X)+m (Y, blood pressure; X, concentration of plasma NE or E; α, slope; and m, intercept). The αs of SHR rats were greater than those of WKY rats for the calcium antagonists employed, meaning that the increment of plasma CAs responding to a decrease in blood pressure was smaller in SHR than in WKY rats. It was concluded that the contribution of the sympathetic nervous system to maintaining blood pressure reduced by diltiazem and nicardipine is less in SHR than in WKY rats.

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In order to screen potent inhibitors of tumor invasion and metastasis, we here devised a simple and reproducible in vitro assay for tumor invasion and migration. A conventional cell-counting assay using a Transwell chamber with a microporous membrane filter is troublesome and time-consuming, involving visually counting the cells under a microscope, and the invaded or migrated cells are sometimes distributed unevenly in predetermined fields on the lower surface of the filter. Therefore, it is difficult to evaluate the invasive and migratory abilities of tumor cells easily and quantitatively by the cell counting method. In the present study, crystal violet dye was used for staining the invaded cells and colorimetrically assessing the invasive ability per filter as an absorbance. In this crystal violet assay, tumor cell invasion into a reconstituted basement membrane Matrigel was proportional to both the cell number added into the chamber and the incubation period, and inversely proportional to the amount of Matrigel barrier on the upper surface of filter. The results obtained by this dye-uptake method were highly consistent with those of a conventional cell-counting assay. Using this crystal violet assay, the anti-invasive effect of doxorubicin (DOX) was detected more easily and found to be highly proportional to that by the conventional cell-counting method. We therefore applied this convenient assay method to screen anti-invasive and anti-metastatic compounds. As a result, caffeic acid was found to be more active in the inhibition of both tumor cell invasion and migration without showing direct cytotoxicity in vitro than other related compounds.

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Emotional disturbances, such as lack of motivation or depression, are common after stroke. The drugs mainly used to treat these syndromes in Japan are the cerebral metabolic enhancers whose biochemical and pharmcological profiles are similar to those of antidepressant drugs. In order to examine the possible therapeutic effect of T-794 [(5R)-3-(6-(cyclopropylmethoxy) 2-naphthalenyl)-5-(methoxymethyl) 2-oxazolidone], a new reversible inhibitor of monoamine oxidase (MAO) type A, on those emotional disturbances, its antidepressant activity was compared with those of major cerebral metabolic enhancers in rodents with or without treatment of cerebral ischemia. Oral administration of T-794 potently prevented reserpine-induced ptosis (ED₅₀=4.41 mg/kg), akinesia (ED₅₀=3.29 mg/kg), and hypothermia (minimum effective dose=3 mg/kg) in mice. It was at least 3.7, 13.0, and 3.3 times more potent than cerebral metabolic enhancers tested (indeloxazine, bifemelane, amantadine and idebenone) in antagonism of the ptosis, the skinesia, and the hypothermia, respectively. Effect of T-794 was also examined in the behavioral despair test in rats subjected to forebrain ischemia. The ischemia was induced by a combination of bilateral common carotid artery occulusion (15 min) and systemic hypotension (sodium nitroprusside 5 mg/kg, s.c.). From 13d after the surgery, drugs were orally administered twice daily 7 times, and following the last administration rats were assessed for their behavior. T-794 reduced the duration of immobility in the behavioral despair test at 30 mg/kg without affecting spontaneous motor activity, whereas indeloxazine showed no significant effect. Antidepressant-like activity of T-794 was suggested in rodents with as well as those withotu cerebral ischemia. The results suggest that T-794 may make an important contribution to the treatment of emotional disturbances following stroke.

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(-)-N-(trans-4-Isopropylcyclohexanecarbonyl)-D-phenylalanine (A-4166) is a new nonsulfonylurea hypoglycemic agent that lowers blood glucose by stimulating insulin release. In the present study, we examined the effects of A-4166, voglibose (an α-glucosidase inhibitor), and glibenclamide (a sulfonylurea) on the postprandial glycemic increase in rats with or without diabetes millitus. Oral administration of A-4166 (25-100 mg/kg) dose-dependently decreased blood glucose with a rapid onset and short duration in normal rats. On the other hand, glibenclamide (1-4 mg/kg) showed a slower onset of its hypoglycemic action, and voglibose (0.2 mg/kg) had no effect. In the case of postprandial glucose excursion, the carbohydrate-induced increase in blood glucose was reduced by oral administration of either A-4166 or voglibose without causing sustained hypoglycemia in both normal and neonatal streptozotocin-induced diabetic rats. However, the efficacy of voglibose varied with the type of carbohydrate load. Glibenclamide produced a prolonged decrease in blood glucose without any appreciable effect on the initial glucose excursion. After sucrose loading, plasma insulin levels during the initial 1 h were significantly higher in A-4166-treated rats than in control rats, while voglibose completely inhibited the insulin response to sucrose. In glibenclamide-treated rats, an augmented insulin response was not seen. In conclusion, unlike other hypoglycemic agents, A-4166 suppresses postprandial glucose excursions by stimulating the early phase of insulin secretion.

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Effects of nebracetam, a novel nootorpic agent, on the synaptosomal uptake of neurotransmitter monoamines of the brain regions were examined. Striatal and hippocampal synaptosomes were isolated by the Percoll gradient method, and the striatal dopamine uptake and hippocampla serotonin uptake were measured in the presence of different concentrations (1 to 1000 μM) of nebracetain in vitro. A significant reduction in dopamine uptake in the striatum and serotonin uptake in the hippocampus was seen at concentrations of 100 μM or above. In in vivo microdialysis study, there were no appreciable changes in the extracellular concentrations of striatal dopamine and hippocampal serotonin when this agent at a dose of 30 mg/kg, which was effective in improving ischemic brain energy metabolism, was applied i.p. to the rat. The ineffectiveness of nebracetam in the in vivo microdialysis may be due to low levels of the concentration of nebracetam when the agent was administered i.p. at a dose of 30 mg/kg, since the brain blood concentration of this agent is pharmacokinetically estimated to be no more than 15 μM when this dose of nebracetam is employed. Thus, it is unlikely that this agent at a pharmacologically effective dose alters dopamine or serotonin uptake in the brain nerve terminal under normal conditions.

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The induction and subsequent intracellular distribution of the 72-kDa heat shock (stress) protein (Hsp72) by exposure of cultured human alveolar (L-132) cells to dimethylarsinic acid (DMAA), a main metabolite of inorganic arsenics in mammals, were examined. A significant induction of Hsp72 in the cells was observed by exposure to 10 mM DMAA for 6h. The induction was similar to the case by arsenite for 6h or by heat treatment at 42°C for 3h. However, the nuclear distribution of Hsp72 differed greatly between DMAA and 42°C treatment or arsenite exposure, i.e., Hsp72 induced by exposure to DMAA accumulated not only in the nucleoli but also in the nucleoplasm, whereas that by 42°C or arsenite exposure did not accumulate in the nucleoplasm. Furthermore, the Hsp72 accumulated in the nucleus by DMAA exposure hardly diffused with the addition of ATP, suggesting that the DMAA-induced Hsp72 strongly binds to some macromolecules in the nucleus. The fact that Hsp72 induced by DMAA exposure accumulated in the nucleus of the cells may reflect a protective response toward the nucleus-specific damaging action of dimethylarsenics.

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A newly synthesized conjugate of 7-hydroxy-3-O-phosphonocholic acid (ursodeoxycholic acid monophosphate) with 5-aminosalicylic acid (5-ASA-UDCA monophosphate) was investigated to determine its suitability for the evaluation of enteric bacteria. This compound, 5-ASA-UDCA monophosphate, was efficiently deconjugated by cholylglycine hydrolase to release 5-ASA, whereas it was completely resistant to deconjugation by pancreatic and intestinal mucosal enzymes. In everted gut sac experiments, 5-ASA-UDCA monophosphate was not actively absorbed from any part of the small intestine. In animal experiments, urinary excretions of N-acetyl-5-ASA (Ac-5ASA) were measured for 24h following the oral administration of 20 mg of 5-ASA-UDCA monophosphate. Control rats excreted 276.3±89.0 μg (mean±S.E.) of Ac-5ASA, whereas the rats with intestinal bacterial overgrowth excreted more (1224.1±231.5 μg; p<0.01). These basic studies indicate that this compound is likely to offer a simple method for the evaluation of microbial overgrowth without the use of radioisotopes or expensive, special apparatus.

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To evaluate the usefulness of a traditional Chinese medicine (Kampo prescription), Keishi-bushi-to (KBT), which is composed of five medicinal plants derived from Kampo prescriptions used to treat rheumatoid arthritis, we investigated the effect of KBT on the development of arthritis induced by type II collagen (CII). Oral administration of KBT at a dose of 500 mg/kg from 7d before intradermal injection of CII significantly reduced the severity from 7d after the onset of arthritis. The reduction in body weight resulting from the development of arthritis was not seen in rats treated with KBT.Plasma IgG and IgM anti-CII antibody levels were lower in KBT-treated rats than control rats. In addition, the clearane of IgG anti-CII-antibody from circulating blood after intravenous injection was faster in KBT-treated rats than control rats. These results indicate that KBT is effective in suppressing collagen-induced arthritis and its effect is at least partly due to the suppression of humoral and cellular immunity.

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The hepatoprotective effects of the fruits of Hovenia dulcis THUNB. on chemically or immunologically induced experimental liver injury models were examined. The methanol extract showed significant hepatoprotective activity against CCl₄-toxicity in rats and D-galactosamine (D-GaIN)/lipopolysaccharide-induced liver injury in mice. The methanol extract also significantly protected against CCl₄-toxicity in primary cultured rat hepatocytes. Hepatoprotective activity-guided fractionation and chemical analysis led to the isolation of an active constituent, (+)-ampelopsin (1) from the methanol extract.

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The protective effects of betamipron (BP, N-benzoyl-β-alanine) on the nephrotoxicity of cisplatin were examined as indicated by body weight gain, ratio of kidney weight to body weight, blood urea nitrogen and serum creatinine levels in tumor-bearing rats. The results showed clearly that administration of BP 1h after cisplatin treatment affords protection against the nephrotoxicity of cisplatin. Furthermore, the addition of BP to cisplatin had no apparent effect on the efficacy of cisplatin against Walker 256 carcinosarcoma cells in rats. In addition, no observed significant difference in plasma cisplatin concentration between cisplatin with alkaline solution and cisplatin with BP may be partly attributed to the decrease in the cisplatin exsorption to the intestine and its excretion to bile, and to an increase in cisplatin excretion to the urine.

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The first-pass metabolism in the intestine and liver of ONO-5046 (N-[2-[4-(2, 2-dimethylpropionyloxy)phenylsulfonylamino]benzoyl]aminoacetic acid), a newly synthesized elastate inhibitor, was separately estimated in rats. When ONO-5046 solution was administered into the whole intestine via the bile duct at a dose of 5 μmol/rat, the extent of bioavailability was only 1.5%. A small but significant increase in the bioavailability with an increase in the dose suggested marked first-pass metabolism with a saturable process. Hepatic first-pass metabolism was estimated by determining the hepatic extraction ratio of ONO-5046 after administration into the portal vein at two different infusion rates (5 μmol/kg/9 min or 5 μmol/kg/20 s). The extraction ratio was relatively small and constant (about 20%) under 2 different infusion rates of the drug. Intestinal first-pass metabolism was estimated by determining the drug recovery in the measenteric plasma after administering the drug into the intestinal loop in situ (mesenteric blood collecting method in situ). The recovery percentage of ONO-5046 in the mesenteric plasma was small (2.58±0.04% at a dose of 1 μmol/rat), and the remaining ONO-5046 recovered in the mesenteric plasma and in the intestinal loop was a metabolite of ONO-5046 (EI-601, N-[2-[(4-hydroxyphenyl)sulfonylamino]benzoyl]aminoacetic acid). Recovery percentage of ONO-5046 in the mesenteric plasma increased significantly with an increase in the dose, although the recovery percentage was still low, even at a higher dose (9.55±1.17% of dose at a dose of 5 μmol/rat). These results indicate that the low oral bioavailability of ONO-5046 in vivo is mainly due to the marked intestinal first-pass metabolism, including the metabolism in the intestinal fluid, and the dose-dependent oral bioavailability was derived from the saturable intestinal first-pass metabolism.

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Molecularly imprinted polymer (MIP) was propared against phenobarbital using methacrylic acid as the functional monomer and ethylene glycol dimethacrylate as the cross linking monomer. We analyzed the recognition properties of the phenobarbital MIP. In some organic solvents, imprinted polymer showed selective binding to phenobarbital. Two dissociation constants of binding were calculated by Scatchard plot analyses; K_d values were 1.8, 121.7 μM, and the number of binding sites was 8.3, 92.3 μmol/g MIP in toluene-heptane-acetic acid (25 : 75 : 1, v/v), respectively. The relationship between the binding affinity to phenobarbital MIP and the polarity of the solvent system, as well as the structure of the template molecule is also discussed.

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Examination of the EtOH extract of the fruiting bodies of Terminalia chebula RETZ. led to the isolation of two potent antimicrobial substances against even methicillin-resistant strains of Staphylococcus aureus. On the basis of spectroscopic evidence, the two isolates have been identified as gallic acid and its ethyl ester.

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This study was designed to evaluate the enterohepatic circulation of racemic etodolac in rats. Additionally, the effect of hepatic and renal failure on the pharmacokinetics was estimated. The biliary excretion and the reabsorption of the drug excreted in bile were examined in order to clarify the effect of enterohepatic circulation on the disposition, and a pharmacokinetics model was applied to describe the enterohepatic circulation. The relatively rapid elimination of etodolac was seen in the bile-exteriorized rats (BE rat) compared with that in control rats. Total biliary excretion of etodolac, mainly as glucuronides, after intravenous administration was about 45% of the dose, indicating extensive enterohepatic circulation of the drug. The plasma concentrations of the drug in bile duct-linked rats approximately agreed with the simulation curve by the model, with the peak concentration 6-7h after dosing. The elimination of the drug was markedly retarded in rats with hepatic (CCl₄-induced) and renal (uranyl acetate-induced) failure, and high plasma levels were maintained over the longer times, due to greatly decreased distribution volume. The biliary excretion of etodolac enantiomers was not significantly different between the control and CCl₄-groups, suggesting that hepatic glucuronyl transferase activity was preserved in rats impaired by CCl₄.

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The effects of erythromycin, clarithromycin and rokitamycin on the metabolism of nifedipine were studied in vitro and in situ. Erythromycin, clarithromycin or rokitamycin added to nifedipine did not inhibit the formation of metabolite, M-1, of nifedipine, whereas pretreatment with erythromycin or clarithromycin significantly (p<0.05) inhibited its formation. Only erythromycin significantly (p<0.05) inhibited the formation of M-2 from M-1. These observations agreed with the results obtained using an in situ rat intestinal loop technique. As assessed by the concentrations of nifedipine and M-2 in jugular and portal vein blood, the inhibition of nifedipine metabolism by erythromycin was greater after multiple doses than after a single dose.Moreover, our results suggest that rokitamycin is a less potent inhibitor of nifedipine metabolism.

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For the past few years, we have been investigating polysaccharides from Ganoderma lucidum as antifibrotic agents. In a previous study, we discovered that polysaccharides extracted from G. lucidum lowered the collagen content in liver but had no effect on serum biochemical parameters in rats subjected to bile duct ligation and scission-induced fibrosis. In this study, we changed the extraction method and obtained polysaccharides extracted from G. lucidum. The polysaccharide from G. lucidum reduced the serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and total bilirubin and also reduced the collagen content in liver and improved the morphology. Pentoxifylline, which is reported to exhibit an antifibrotic effect in pigs with fibrosis induced by yellow phosphorus, did not have any antifibrotic effects in fibrosis induced by biliary obstruction. Glycyrrhizin, which is used in the treatment of hepatitis, reduced serum ALT ans AST values but there was no significance. It has no effect on liver hydroxyproline content which implies that glycyrrhizin has no antifibrotic effect in the rats with fibrosis induced by bile duct ligation and scission. These data suggest that the polysaccharide from Ganoderma lucidum could be a promising antifibrotic agent. However, further study is needed to understand the inhibition mechanism of collagen deposition of polysaccharides from Ganoderma lucidum and its clinical applicability remains to be established.

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We evaluated the relationship between the cumulative amounts of 5 kinds of β-blockers (alprenolol, oxprenolol, timolol, acebutolol and atenolol) permeating through the stratum corneum and a^* values obtained by measuring the formation of erythema, a skin irritation reaction, with a chromameter after transdermal application of adhesive patches containing a β-blocker to the skin of guinea pigs.The cumulative amount of β-blocker released from each adhesive patch to the skin increased with the increase in application time. The contents of alprenolol, oxprenolol and timolol in the stratum corneum and in the stripped skin increased markedly up to 4h after application and thereafter were maintained at high levels up to 24h. The contents of acebutolol and atenolol, on the other hand, increased up to 24h, but these values were low. a^* values of all adhesive patches 24h after application were higher than those before application.The correlation coefficients between the cumulative amounts of alprenolol, oxprenolol, timolol, acebutolol or atenolol permeating through the stratum corneum and (Δa^*-Δa^*_Pleacbo) values were 0.739, 0.717, 0.722, 0.551 and 0.633, respectively. The correlation coefficient calculated by averaging the cumulative amounts of 6 kinds of β-blockers permeating through the stratum corneum [including propranolol which was reported previously (Kobayashi I., et al., Biol. Pharm. Bull., 19, 839-844 (1996))] was 0.731, higher than the correlation coefficient between contents of these β-blockers in the stripped skin and (Δa^*-Δa^*_Placebo) values (r=0.552). This suggests that there was a high correlation between the cumulative amounts of β-blockers permeating through the stratum corneum and (Δa^*-Δa^*_Placebo) values.

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The tension of isolated ring preparation of the aorta from rats was measured isometrically to study the influence of OSW-1 [3β, 16β, 17α-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-β-D-xylopyranosyl)-(1→3)-(2-O-acetyl-α-L-arabinopyranoside)], a steroidal saponin, on the endothelium dependent and independent relaxation caused by acetylcholin (ACh) and sodium nitroprusside (SNP), respectively. OSW-1 (10^-7 M), which has more than 100 times higher concentration for anti-tumor activity, had no influence on either the endothelium dependent or independent relaxation. OSW-1 (10^-6M, 0.9 μg/ml) slightly reduced the endothelium dependent relaxation caused by ACh but did not affect the SNP-induced relaxation. In contrast to OSW-1, 1 mg/ml of saponin significantly suppressed the ACh-induced relaxation and shifted the dose-relaxation curve for SNP to the right. OSW-1 (10^-7 and 10^-6 M) did not affect the norepinephrine-induced contraction but 1 mg/ml of saponin significantly attenuated it. The results suggest that though the higher concentration of OSW-1 shows weaker influence on the endothelium function compared with saponin, OSW-1 at an anti-tumor dose has no influence on either endothelium or smooth muscle function.

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An alkaloidal component, dehydrocorydaline (DHC) isolated from Corydalis Tuber (tuber of Corydalis turtschaninovii forma yanhusuo), has been screened for ctivity against types I-IV allergic reactions. In a type I allergic models, DHC at a dose of 0.5 mmol/kg, p.o. inhibited 48h homologous passive cutaneous anaphylaxis (PCA) in rats, which is related to IgE. DHC also exhibited an inhibitory effect on antigen-induced histamine release from peritoneal mast cells. In a type II allergic model, DHC did not inhibit reversed cutaneous anaphylaxis (RCA) in rats. In a type III allergic model, DHC showed weak inhibition on direct passive arthus reaction (DPAR) in rats. Furthermore, in a type IV allergic model, DHC had inhibitory effects on the induction phase and effector phase in picryl chloride-induced contact dermatitis (PC-CD). These results indicated that DHC not only inhibits antibody-mediated allergic reactions but also influences cell-mediated allergic reactions, and the inhibitory effect of Corydalis Tuber on allergic reactions may be partially attributed to DHC.

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We studied the effect of karasurin-A, a type-I ribosome-inactivating protein (RIP) isolated from fresh root tubers of Trichosanches kirilowii MAX. var. japonica KITAMURA, on the mitogenic response of murine splenocytes and nitric oxide (NO) production by murine peritoneal macrophages in vitro. Karasurin-A inhibited the lymphocyte proliferation by LPS, ConA and PHA and NO production by LPS at non-cytotoxic concentrations (10-1000 ng/ml for splenocytes and 10-100 ng/ml for macrophages, respectively). These data suggest that karasurin-A has immunosuppressive activity in vitro.

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The relationship between the pharmacokinetic properties and the analgesic effect of indomethacin (IDM) was evaluated on a carrageenin-induced inflammation model in the rat. Rats were administered the drug in one of two ways : intravenous (i.v.) IDM bolus or i.v. IDM infusion. The analgesic activity was measured by Randall-Selitto test. No correlation was observed between the analgesic effect and the plasma IDM concentration after i.v. bolus administration of IDM. However, in the case of infusion, IDM produced a dose-dependent analgesic effect. In this paper, we demonstrated that the plasma concentration of IDM maintained by i.v. infusion had a prolonged analgesic effect on the carrageenin-induced inflammation model.

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Capric acid (C10) enhanced the absorption of cefoxitin sodium in a concentration-dependent manner following the rectal administration as a suppository in rats. The optimal concentration of C10 was 13%. C10 administered as a suppository also reduced rectal membrane resistance (R_m), showing that the above enhancing effect was induced by widening the paracellular pathway. Both the enhancing effect on the absorption and the reducing effect on R_m were inhibited by W7, an inhibitor of myosin light chain kinase. These results supported that, as shown in the in vitro Caco-2 cell system, the C10 effect on the paracellular pathway is due to activating the contraction of Ca²⁺-calmodulin-dependent actin filament.

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Effects of angiotensin-converting enzyme (ACE) inhibitors, captopril, enalapril maleate and quinapril, on the uptake of aminocephalosporin antibiotic, cefroxadine, by rabbit small intestinal brush border membrane vesicles were examined. These ACE inhibitors significantly inhibited the uptake of cefroxadine, which is transported by H⁺/dipeptide transporter in the membrane, in the order of captopril<enalapril<quinapril in the presence of an inward H⁺ gradient. Inhibitory effect of quinapril was more marked than that of aminocephalosporin cepharadine, while in the absence of an inward H⁺ gradient inhibition by these ACE inhibitors was much less. Dixon plot analysis showed that the inhibition by enalapril and quinapril in the presence of an inward H⁺ gradient occurred in a competitive manner and estimated inhibition constants of these two drugs to be 5.3 mM and 0.46 mM, respectively. These results suggested the strong affinity of these ACE inhibitors, especially quinapril, on the H⁺/dipeptide transporter.

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The effect of pH on the microenvironment surrounding ³⁴Cys in human serum albumin (HSA) has been studied using acrylodan, a Cys-specific fluorescence probe. The reactivity of ³⁴Cys with 5, 5'-dithiobis(2-nitro benzoic acid) (DTNB) followed a pseudo-first-order reaction, and the increase in reactivity was dependent on pH and oleate content. Compared with the N-form of HSA-acrylodan conjugate (pH 6.2), the B-form (pH 8.4) has a blue-shifted Em_max and enhanced fluorescence intensity derived from acrylodan covalently attached to ³⁴Cys suggesting that the exposure around ³⁴Cys in the B-form was less than that in the N-form. The conformational change induced by fatty acid increased the exposure around ³⁴Cys, while that induced by an increase in pH decreased it. Further, since the effect of oleate on the fluorescence of acrylodan was nealy the same for both conformers, the effects of pH and oleate on the microenvironment surrounding ³⁴Cys should be independent and additive. We concluded that the increase of reactivity of ³⁴Cys as a function in increasing pH may well be related to an increase in mercaptide ion content.

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The release behavior of phosphorothioate oligodeoxynucleotide, 5'-GCCGAGGTCCATGTCGTACGC-3' (ODN), from poly (lactic acid-co-glycolic acid) (PLGA) implant was studied. The pillar shape implant was fabricated by the heating mold method. About 20% of ODN were released initially, and the subsequent pseudo-zero-order release lasted for more than 20d from a PLGA10000 implant loaded with 8.4% ODN in phosphate buffered saline, pH 7.4. The duration of ODN release did not depend on the molecular weights of PLGA, and pseudo-zero-order release may be achieved by changing the loaded amount of ODN in fabrication of the PLGA implant. Almost the same release profiles were obtained in the pH range of 7.2 to 7.6, which is known as the physiological pH of a vitreous body. Furthermore, the duration of intact ODN release in bovine vitreous was found. The implant in the present study may possibly be applicable to intravitreal implantation for the treatment of oculr disease.

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We investigated the usefulness and efficiency of the co-grinding method with D-mannitol to improve the bioavailability of a sparingly water-soluble drug, (±)-5-[[2-(2-naphthalenylmethyl)-5-benzoxazolyl]methyl]-2, 4-thiazolidinedione (174), and compared it with those of the single-grinding method.The co-ground mixtures in drug/carrier weight ratios up to 1 : 5 gave fine particle sizes of less than about 3 μm, which showed a marked increase in the dissolution rate with reduction of particle size, compared with the single-ground powder, even with a similar particle size. The oral bioavailability study of co-ground powders in beagle dose exhibited a dramatic increase, as did the dissolution rate, according to finer particle size. Finally, complete bioavailability was obtained at the finest particle size of 1.2 μm (drug/carrier ratio of 1 : 5, w/w) as was a solution of the drug. Bioavailability had a good linear correlation with the dissolution rate. These findings suggested that the co-grinding method with D-mannitol dramatically increased the available surface area, caused by a reduction of particle size, which not only accelerated the dissolution rate but also resulted in greater enhancement of the bioavailability of 174.

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A novel bioassay system based on RT-PCR has been established to search for biologically active compounds which can modulate the expression of genes encoding important regulatory proteins. Changes in target mRNA level in the treated cell cultures were quantitatively determined by competitive PCR using internal standard DNA. The compounds discovered using this bioassay will be useful tools to elucidate the mechanisms of cellular signaling guiding the expression of the target genes. As the first application of the new bioassay strategy, the expression of the interleukin-2 gene in Jurkat cells, a human T cell line, was investigated. Screening of several crude drugs used in traditional Chinese medicines demonstrated that the aqueous acetone extract of Coptidis Rhizoma enhanced the expression of the interleukin-2 gene by about 5-fold as compared to the control.

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