A highly sensitive ELISA for the determination of polymyxin B sulfate (PMB) was developed which is capable of measuring as low as 32pg/ml. Anti-PMB antibody was obtained by immunizing rabbits wiht PMB conjugated with mercaptosuccinyl bovine serum albumin (MS. BSA) using N-(γ-maleimidobutyryloxy) succinimide (GMBS) as a heterobifunctional coupling agent. An enzyme marker was similarly prepared by coupling PMB with horseradish peroxidase (HRP) employing GMBS. This ELISA showed very low reactivity with the PMB analogue, polymyxin E (0.05%). The values for PMB concentration detected by this assay were comparable with those detected by the bioassay. Moreover, the ELISA was about 10000 times more sensitive in detecting PMB at lower concentrations. Serum PMB concentration after the oral administration of a PMB tablet to human subjects was determined by the ELISA. PMB was rapidly absorbed from the gastrointestinal tract after the administration. then slowly decreased. These results indicate that the ELISA may be a valuable tool for studies of the pharmacokinetics and pharmacodynamics of the anti-endotoxin drug, PMB.

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We have investigated the serum concentration of 16-dehydropregnenolone (3β-hydroxy-5, 16-pregnadien-20-one) 3-sulfate (16-DHPS) in the umbilical artery (U.A.), umbilical vein (U.V.) and maternal vein (M.V.) to discover the origin of 16-DHPS. Although there was no significant difference between the levels of 16-DHPS in U.A. (18±15ng/ml, mean±S.D., n=28) and U.V. (10±9ng/ml, n=28), these values were significantly higher (U.A., p<0.001; U.V., p<0.001) than that in M.V. (2±3ng/ml, n=28). These levels in the U.A. and U.V. did not fall in inafnts (30±18ng/ml, n=7) during the early neonatal period (2-7 d after birth). A significant correlation between the serum levels of 16-DHPS and 16-hydroxypregnenolone (3β, 16α-dihydroxy-5-pregnen-20-one) 3-sulfate (16-OH-PregS), which may be the precursor steroid for 16-DHPS, was observed in the U.A. (r=0.630, n=28, p<0.001), but not in the U.V. Moreover, this significant correlation persisted during the early neonatal period (p<0.05, r=0.842, n=7), although the neonate had been separated from the maternal milieu. These results suggest that 16-DHPS originates in the fetus.To confirm the metabolic pathway of 16-DHPS (i.e. pregnenolone (3β-hydroxy-5-pregnen-20-one) 3-sulfate (PregS)→16-OH-PregS→16-DHPS), we investigated the correlation between the serum concentrations of the precursor steroid and the product in both the U.A. and U.V. A significant correlation was obtained between the serum concentrations of PregS and 16-OH-PregS both in the U.A. (p<0.001, r=0.563, n=28) and U.V. (p<0.05, r=0.476, n=27). As described above, the serum levels of 16-DHPS and 16-OH-PregS only correlated significantly in the U.A. These findings support the existence of the pathway, PregS→16-OH-PregS→16-DHPS, in the fetus.

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TAK-778, ((2R, 4S)-(-)-N-[(4-diethoxyphosphorylmethyl)phenyl]-1, 2, 4, 5-tetrahydro-4-methyl-7, 8-methylene-dioxy-5-oxo-3-benzothiepin-2-carboxamide), is a novel osteoblast differentiation-promoting compound, and its sustained-release formulation is expected to be clinically used for the enhancement of fracture healing. To date, TAK-778 levels in serum have been measured using conventional reverse-phase HPLC with inferior sensitivity and time-consuming procedures. We have produced polyclonal antibodies against TAK-778 by using one of its derivatives coupled with a carrier protein, and developed a one-step ELISA for the determination of TAK-778 in serum. The antibodies had minimal cross-reactivities to the biologically inactive metabolites including the oxidized-form (0.36%) and the cleavaged-form (0.00083%). The competitive ELISA was accomplished within three hours with a detection limit of 0.5ng/ml serum, and the coefficients of variations for samples ranging from 1 ng/ml to 80 ng/ml were 1.1-4.4% in an intra-assay and 3.1-9.6% in an inter-assay. The present ELISA is so rapid, sensitive and selective for TAK-778 that it could be conveniently used in a clinical field for the determination of many serum specimens.

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Hypericin is a unique photosensitizing plant pigment and has been separately reported to induce differentiation and apotosis in neoplastic cells. In this study, we examined the relationship between activities to induce differentiation and apoptosis in human promyelocytic leukemia HL-60 cells, at a concentration range of 0.15 to 0.2μM. When treated with hypericin, the cell ratio reducible of nitroblue tetrazolium was significantly increased and the cell size was enlarged by flow cytometry analysis. Hypericin also significantly increased the ratio of the cells, which were of positive α-naphthyl acetate esterase activity and phagocytic activity, whereas it hardly influenced the naphthol AS-D chloroacetate esterase activity in the cells, as well as 1α, 25(OH)₂D₃ (10nM). In addition, hypericin increased hypodiploid nuclei and caused a nucleosomal ladder. These results indicate that hypericin induced both differentiation toward monocyte/macrophage lineage and apoptosis in HL-60 cells.

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The effects of ceramide on agonist-stimulated phospholipase A₂ (PLA₂) activity were studied in platelets. Cell-permeable C₆-ceramide (N-hexanoylsphingosine) exogenously added to platelet suspension enhanced U46619-stimulated arachidonic acid release and lysophosphatidylcholine production. Treatment of platelets with sphingomyelinase also led to an enhancement of the release. The enhanced arachidonic acid release by exogenous ceramide was completely inhibited by methyl arachidonyl fluorophosphonate, a cytosolic PLA₂ inhibitor. However, U46619-stimulated PLA₂ activity was not significantly potentiated by ceramide. These results suggest that enrichment of ceramide in membranes causes modification of intermolecular organization, leading to increased susceptibility of substrate phospholipids to PLA₂.

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The effects of toxic peakes (P-I, P-II and P-III eluted from Sephadex G-200 column) from the sea urchin Toxopneustes pileolus on time-dependent ⁴⁵Ca²⁺ uptake in chike P₂ fraction (crude synaptosome fraction) were studied under physiological ionic conditions. Time-dependent ⁴⁵Ca²⁺ uptake was inhibited by P-II and P-III, but not by P-I, P-II had the greatest inhibitory effect. The inhibitory effect of P-II was not due to the inhibition of ⁴⁵Ca²⁺ binding on P₂ fraction, because P-II did not affect ⁴⁵Ca²⁺ binding in osmotic-shocked P₂ fraction. P-II did not affect KCl-stimulated ⁴⁵Ca²⁺ uptake in P₂ fraction, (Ca²⁺-Mg-<2+>)-ATPase activity in the synaptic plasma membrane (SPM) fraction, or (Na⁺-K⁺)-ATPase and Mg²⁺-ATPase activities on osmotically-shocked P₂ fraction. In contrast, the Na⁺/Ca²⁺ exchanger blocker 2', 4'-dichlorobenzamil (DCB; 100μM), with a poor specificity, inhibited not only time-dependent ⁴⁵Ca²⁺ uptake but also KCl-stimulated ⁴⁵Ca²⁺ uptake, (Ca²⁺-Mg²⁺)-ATPase, Mg²⁺-ATPase and (Na⁺-K⁺)-ATPase. Involvement of Na⁺-Ca²⁺ exchanger in the time-dependent ⁴⁵Ca²⁺ uptake was ruled out, since in was not inhibited by replacement of Na⁺ with Li⁺ in reactio medium. These results suggested that the inhibition by P-II on time-dependent ⁴⁵Ca²⁺ uptake appeared to be more specific than the commercially available Na⁺/Ca²⁺ exchanger blocker DCB, although the mechanism is not clear yet.

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The antiplatelet and antithrombotic activities of a newly synthesized NQ301, 2-chloro-3-(4-acetophenyl)-amino-1, 4-naphthoquinone, were investigated on human platelet aggregation in vitro and rats ex vivo, and murine pulmonary thrombosis in vivo. NQ301 potently inhibited ADP-, collagen-, epinephrine- and calcium ionophore A23187-induced human platelet aggregation in a concentration-dependent manner in vitro. NQ301 significantly inhibited platelet aggregation in orally administered rats ex vivo. NQ301 prevented death due to pulmonary thrombosis in mice dose-dependently in vivo. NQ301 also showed significant prolongation of tail bleeding time in conscious mice. However, NQ301 did not alter such coagulation parameters as activated partial thromboplastin time, prothrombin time, and thrombin time in human plasma. These results suggest that NQ301 may be a promising antithrombotic agent, and the antithrombotic activity of NQ301 may be due to antiplatelet aggregation activity but not to in vitro anticoagulation.

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We investigated the role of histone acetylation in the promotion of antigen-specific antibody production in murine B cells induced by sodium butyrate (NaBu) plus interleukin 2 (IL-2). NaBu dose dependently increased the acetylation levels of histone H4 at concentrations which effectively enhanced anti-trinitrophenyl (TNP) antibody production in the presence of IL-2. Among other short-chain fatty acids and NaBu analogs, propionate, valerate and vinylacetate were effective in the presence of IL-2 in increasing both antibody production and the histone H4 acetylation level, but acetate, α-, β- and γ-hydroxybutyrates and α-, β- and γ-aminobutyrates were not effective, even in the presence of IL-2. The effect of the specific histone deacetylase inhibitor trichostatin A (TSA), which enhances anti-TNP antibody production without IL-2, was markedly inhibited by adding NaBu simultaneously. However, the effect of TSA was neither inhibited nor potentiated by NaBu in the presence of IL-2. Splenic B cells treated with NaBu, TSA and both together in the presence or absence of IL-2 showed almost the same increased acetylation level of histone H4. These results suggest that the NaBu-induced enhancement of anti-TNP antibody production in the presence of IL-2 is mediated through a moderate increase in the level of histone acetylation and that NaBu has both stimulating and inhibiting activities for anti-TNP antibody production, the latter of which is overcome by IL-2.

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Gastric mucosal pepsinogen and intrinsic factor (IF) contents, and their mRNA levels during starvation and refeeding were studied. During starvation for 4d, gastric mucosal pepsinogen and IF contents significantly decreased, whereas pepsinogen and IF mRNA levels increased by 30-50%. These results suggested that the mRNAs of pepsinogen and IF could be preserved for a long time so as to prepare for refeeding. After ceasing the starvation for 72h, gastric mucosal pepsinogen and IF contents were significantly decreased at 1h after refeeding, and their mRNA levels were increased by 20-30% at 30 min after refeeding. We examined whether the refeeding-induced changes in gastric mucosal pepsinogen and IF contents and their mRNA levels could be reproduced by the exogenous administration of secretagogues. They were not found to be affected by the administration of each secretagogue during starvation for 72h at 30 min. However, by the simultaneous administration of 2 or 3 secretagogues (carbachol, cholecystokinin octapeptide (CCK-8) or secretin), the contents of pepsinogen and IF decreased to 70-80% and 50-80% of the control, respectively. However, their mRNA levels increased to 140-160% and 120-135% of the control, respectively. Therefore, refeeding-induced changes in pepsinogen and IF contents and their mRNA levels were partially reproduced by exogenously administered secretagogues. This showed that food intake influences huge changes in neural, hormonal and physical conditions on the stomach. It was indicated that the secretagogues stimulated not only pepsinogen and IF secretion, but also had a tendency to increase their mRNA.

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To investigate whether direct oxidant damage induces hepatotoxicity via an apoptotic cell suicide pathway, we exposed primary cultured mouse hepatocytes to pro-oxidant hydrogen peroxide. We demonstrate that brief exposure to a concentration of hydrogen peroxide (3 mM) can induce hepatocyte cell death via apoptosis as shown by toxicity assays, specific DNA staining, and the appearance of DNA laddering on agarose gels. When hepatocytes were treated with N-acetylcysteine 15 min prior to hydrogen peroxide exposure, the cells were found to be protected from cytotoxicity and apoptosis. These results suggest that direct oxidative injury serves as a general trigger for apoptosis in the liver, and that other apoptotic stimuli, such as exposure to acetaminophen, also involve oxidative injury. Hydrogen peroxidase-induced apoptosis may serve as a valuable model for further studies of apoptosis in the liver.

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The protective effect of sodium-L-ascorbyl-2 phosphate (As-2P), a stable form of ascorbic acid (AsA), against photodamage induced by a single dose of UVB exposure (290-320 nm, Max 312 nm) was investigated using cultured mouse skin.When the cultured skin was treated with various As-2P concentrations, the cutaneous AsA level increased in proportion to the As-2P concentration. After 3 h of incubation, the AsA level in the cultured skin treated with 2, 20 and 100 mM As-2P increased 1.03-, 2.17- and 6.27-fold, respectively, compared with that of the control skin. These results suggest that As-2P was transported into the cultured mouse skin where it was converted to AsA. After 3h, the cutaneous AsA level in irradiated (20 kJ/m²) skin was depleted to a half of that in the control skin. However, the level in skin pretreated with 20 mM As-2P was maintained within normal limits, even after 24h. Pretreatment with 20 mM As-2P significantly prevented such photodamage as sunburn cell formation, DNA fragmentation and lipid peroxidation, which were caused by a single dose of UVB irradiation. These results suggest that the protective effect of 20 mM As-2P on UVB-induced cutaneous damage is due to the maintenance of a normal As level by conversion of As-2P to As in skin tissue.

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We have obtained evidence that co-incubation of thioglycollate-elicited peritoneal macrophages with an aqueous extract of Tinospora tuberculata inhibits lipopolysaccharide-stimulated excessive production of nitric oxide (NO) in vitro. This effect is concentration-dependent and appears to involve suppression of both inducible nitric oxide synthase (iNOS) activity and NADPH-diaphorase activity, thus altering NO production. As NO is one of the critical mediators in various disorders and iNOS inhibitors may have therapeutic potential, these results may explain some aspects of the multifunctional properties of Tinospora tuberculata, which has been used in various folk remedies in southeast Asia and China.

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To develop parasiticides, the nematocidal activity of 22 picrotoxane terpenoids, picrodendrins isolated from the Euphorbiaceae plant Picrodendron baccatum (L.) KLUG et URBAN, was measured using a species of Diplogastridae (Nematoda). Picrodendrin P displayed the most potent nematocidal activity with a minimum lethal concentration (MLC) value of 4.4×10^-3 M. The nematocidal activity of picrodendrin P was 9-fold more potent than that of kainic acid (4.0×10^-2 M) and santonin (4.0×10^-2 M) and 6-fold more potent than that of diethylcarbamazine citrate (3.0×10^-2 M). Thus, picrodendrin P may eventually be used as a leading parasiticide. In light of the relationship between the structure of picrodendrins and their nematocidal activities, potent nematocidal activity was found to require the following elements within the picrotoxane skeleton : the γ-lactone that bridges C-3 and C-5, the epoxide group that bridges between C-6 and C-8, a cis-OCH₃ group and a trans-CH₂OH group related to carbonyl at α-methylene-γ-lactone ring (C-18), and the absence of 4- and 8-hydroxyl groups. These results are the first demonstration of nematocidal activity for these terpenes and thus are important in furthering our understanding.

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6"-O-Xylosyltectoridin and tectoridin isolated from the flowers of Pueraria thunbergiana (Leguminosae), are metabolized to tectorigenin by human intestinal bacteria. Although tectoridin is metabolized to tectorigenin by most intestinal bacteria, 6"-O-xylosyltectoridin is metabolized to tectorigenin via tectoridin by only a few intestinal bacteria, such as Bifidobacterium breve K-110 and Eubacterium A-44. The metabolite, tectorigenin, had more potent hypoglycemic activity as well as in vitro cytotoxic activity against tumor cell lines than 6"-O-xylosyltectoridin and tectoridin. These results suggest that 6"-O-xylosyltectoridin and tectoridin. These results suggest that 6"-O-xylosyltectoridin and tectoridin are prodrugs which can be transformed to the active agents by human intestinal bacteria.

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Histamine release inhibitors in watercress (Nasturtium officinale) were isolated using a monitoring system with antigen-stimulated RBL-2H3 cells. Of the 15 compounds isolated, flavonols and megastigmanes significantly inhibited histamine release. Two flavonols, 3-O-sophorosides of rhamnetin and rhamnazin, were new compounds. To investigate the inhibitory mechanism, the effects of rhamnetin, rhamnetin 3-O-sophoroside and an isolated megatigmane glucoside on the increase in the intracellular free calcium concentration were examined at a concentration providing 60% inhibition of histamine release. The results suggest that these compounds did not affect the calcium influx at that concentration. The structure-activity relationships of the megastigmanes on histamine release were also investigated.

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The effect of Sanguisorbae Radix extract, a traditional crude drug, was investigated in renal dysfunction induced by lipopolysaccharide (LPS) endotoxin. Injection of LPS in rats resulted in a sharp rise in the serum levels of urea nitrogen and creatinine (Cr), indicating impairment of renal function. Nitrite and nitrate levels and the activity of inducible nitric oxide synthase (iNOS), an enzyme which participates in NO synthesis, were also significantly increased in the serum of LPS-treated rats compared with normal rats. In rats pretreated with Sanguisorbae Radix extract, renal dysfunction was attenuated and the increases in serum urea nitrogen and Cr induced by LPS were significantly reduced. The administration of Sanguisorbae Radix extract also effectively lowered serum nitrite/nitrate level. A similar effect was observed on the iNOS activity. These results indicate that Sanguisorbae Radix extract contributes to the regulation of renal function under conditions where there is excessive generation of NO.

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Emulsions containing a distearylamide (Gd-DTPA-SA) or a distearylester (Gd-DTPA-SE) of Gd (gadolinium)-diethylenetriaminepentaacetic acid (Gd-DTPA) were intraperitoneally injected in Greene's melanomabearing hamsters at a dose of 2.0 ml (3.0 or 6.0 mg Gd) per hamster. In the standard-Gd and high-Gd formulations used, the weight ratios of soybean oil, water, Gd-DTPA derivative (Gd-DTPA-SA or Gd-DTPA-SE), hydrogenated L-α-phosphatidylcholine from egg yolk (HEPC) and co-surfactant (HCO-60, Myrj 53, Myrj 59 or Brij 700) were 7.36 : 92 : 1 : 2 : 3 and 7.36 : 92 : 2 : 1 : 3, respectively. When the effects of the co-surfactants on the biodistribution of Gd from Gd-DTPA-SA-containing emulsions in the standard-Gd formulation were compared, the HCO-60 emulsion exhibited the highest Gd accumulation in tumors, possibly resulting from its fast and complete absorption, its small particle size (78nm) and the stable coat on the particle surfaces with polyoxyethylene. Brij 700 emulsion kept the highest blood Gd concentration for a prolonged period, possibly due to particle properties similar to those of HCO-60. However, it exhibited a slower Gd accumulation in tumors, only reaching an identical level, in comparison with the HCO-60 emulsion. This suggested the tumor to be saturated with lipid particles. When Gd-DTPA-SE was used instead of Gd-DTPA-SA, its HCO-60 emulsion exhibited only very poor Gd-accumulation due to its easy degradation. The HCO-60 emulsion particles containing Gd-DTPA-SA in the high-Gd formulation (6.0 mg Gd in 2 ml) exhibited in vivo behavior identical to those in the standard-Gd formulation; then the Gd level in tumors reached 107 μg Gd/g tumor (wet), and the tumor : blood (T/B) and tumor : skin (T/Sk) Gd concentration ratios were 13.2 and 5.6, respectively, at 48 h after intraperitoneal administration. These results suggest that when intraperitoneally administered, this HCO-60 emulsion, and possibly also the corresponding Brij 700 emulsion, may be an excellent delivery system for accumulating Gd in tumors in neutroncapture therapy (NCT).

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Recently, several reports of clinical cases of QT prolongation and torsades de pointes, associated with the use of tacrolimus (FK506), have come to light. We have previously demonstrated FK506-induced QT prolongation in guinea pigs [Minematsu T., et al., Life Sci., 65, PL197-PL202 (1999)]. We now examined the relationship between QTc prolongation and the pharmacokinetics of FK506 in guinea pigs, in order to evaluate the arrhythmogenicity of FK506 when compared with that of quinidine sulfate (QND). Thus, dose-response relationships for FK506 (0.01 or 0.1 mg/h/kg) or QND (30 mg/h/kg) were investigated during and after intravenous infusion and also following intravenous bolus administration of FK506 (0.2mg/kg). The dose-response relationship between plasma drug concentration and QTc prolongation for FK506 and QND were subsequently analyzed using an effect compartment model. The pharmacodynamic parameters thus obtained were as follows : k_E0 2.72×10^-4 (min^-1), E_max 27.1 (ms), EC₅₀ 0.376 (ng/ml) for FK506; and k_E0 0.148 (min^-1), K 8.41 (ms·ml/μg) for QND. The anti-clockwise hysteresis observed for FK506-induced QT prolongation was successfully analyzed by the present pharmacokinetic/pharmacodynamic model, which may provide a rational basis for developing a clinical dosing regimen to avoid possible QT prolongation induced by FK506.

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With the aim of overcoming the delivery problems (water-solubility and bioreductive activation problems) of phyllohydroquinone (PKH), an active form of phylloquinone (PK, vitamin K₁), the N, N-dimethylglycine esters of phyllohydroquinone (1-mono, 1; 4-mono, 2; and 1, 4-bis, 3) have been synthesized and assessed in vitro as a prodrug for the systemic bioreductive activation-independent delivery of PKH. The hydrochloride salts of the esters were found to be quite soluble in water. Hydrolysis of the esters in rat liver S9 fraction, rat plasma and phosphate buffer, pH 7.4, at 37°C, was kinetically studied in the presence and absence of an esterase inhibitor. The hydrolysis was catalyzed by esterases located in rat liver and rat plasma and quantitatively yielded PKH. The enzymatic cleavage and the vitamin K-dependent carboxylation activity of the esters in the rat liver microsome preparation at pH 7.2 and 25°C were studied. The regeneration of PKH from the esters was catalyzed by carboxylesterases located in the rat liver microsome, and the order was as follows : 1>3>2. The carboxylation was stimulated by selected ester 1 in the absence of dithiothreitol, an activator of the vitamin K cycle. The carboxylation activity of 1 was strongly inhibited in the presence of eserine, a carboxylesterase inhibitor. Compound 1 could also stimulate carboxylase under warfarin-poisoning conditions, where the vitamin K cycle was strongly inhibited. These results indicated that these highly water-soluble and liver-esterase hydrolyzable ester derivatives of PKH are potential candidates for parenteral prodrugs which can thus achieve the systemic bioreductive activation-independent delivery of PKH.

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The reversla effect of itraconazole on P-glycoprotein (P-gp)-mediated resistance of vinblastine, daunorubicin and doxorubicin was analyzed from a cellular pharmacokinetic point of view, namely by [³H]azidopine photoaffinity labeling, intracellular accumulation and transcellular transport experiments. The LLC-GA5-COL150 cells, which expressed human P-gp selectively on the apical membrane due to transfection of MDR1 cDNA into the porcine kidney epithelial cells (LLC-PK₁ cells), was used here, since this cell line constructs the monolayer with tight junction, being able to characterize the cellular pharmacokinetics. In LLC-GA5-COL150 cells, itraconazole caused a reversal from resistance as shown by a growth inhibition assay. [³H]Azidopine photoaffinity labeling demonstrated that itraconazole, vinblastine, daunorubicin and doxorubicin showed higher binding ability for P-gp compared with digoxin, suggesting the following results were via P-gp. The intracellular accumulation of [³H]vinblastine, [³H]daunorubicin and [¹⁴C]doxorubicin after their application on the basal and apical sides was increased by itraconazole. These changes were similar to the dose modifying factors determined by the growth inhibition assay. However, their basal-to-apical transport was hardly affected by itraconazole, and this was explained by the fact that itraconazole inhibited P-gp, and subsequently increased their intracellular concentration and then the non-P-gp mediated transport from the intracellular space to apical side. The apical-to-basal transport of [³H]vinblastine, [³H]daunorubicin and [¹⁴C]coxorubicin was increased by itraconazole, and this was reasonably explained by the inhibition of P-gp, and partly also by the increase of their intracellular concentration via the inhibition of P-gp.

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Fusion peptide P-9 was isolated from bovine serum albumin by controlled pepsin degradation in the presence of caprylic acid, followed by Sephadex G-75 gel filtration and ion-exchange chromatography of CM-cellulose. By this procedure, P-9 could be strictly separated from peptic fragment P-Phe, which has a molecular weight close to that of P-9. P-Phe has no fusogenic activity. The addition of P-9 to phosphatidylcholine (PC) liposomes containing cholesterol (Chol) gave rise to an increase of absorption intensity at aroung pH 4.0. The increase of turbidity by P-9 addition did not decrease with increasing pH, indicating P-9-mediated fusion of PC liposomes. The extent of the fusion of PC liposomes was strongly dependent on the PC chain length and temperature. The membrane fluidity close to the polar head groups of the fatty acyl chains of PC affected markedly the extent of P-9-mediated liposome fusion. However, there was no correlation between membrane fluidity near the hydrophobic end of the fatty acyl chains and the extent of liposome fusion. The rate of liposome fusion was dependent on both lipid composition and PC chain length. These results suggest that a contact or an interaction of P-9 with liposomal membrane occurs in the rigid regions. The character of the membrane-water interface region in the liposome controls a triggering effect for P-9-mediated fusion.

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We investigated the relationship between gastrointestinal (GI) transit and motility during postoperative ileus in dogs undergoing a single laparotomy. We combined X-ray radiography for a GI transit study with chronically implanted force transducers (FTs) for a GI motility study. Radio-opaque markers made of polyethylene and steel wires or barium sulfate were used to examine solid substance transit or liquid substance transit. For a while after the end of the operation, postoperative ileus was observed, with weak irregular contractions of the GI tract. Transmission of the contractions to the lower GI tract was then observed. The start point of interdigestive migrating contraction (IMC)-like motility was observed in the order of small intestine (I-IMC), duodenum (D-IMC), and stomach (G-IMC), and IMC proceeded gradually after the operation. The gastric emptying time of a solid marker was 73.6±2.3 h (n=5), and depended on the time of first occurrence of G-IMC (r=0.674, p=0.006). The gastric emptying of the liquid marker was finished before the time of the first occurrence of G-IMC, and its small intestinal transit time correlated with the time of the first occurrence of G-IMC (r=0.888, p=0.018). Using combined X-ray radiography and FTs we found that recovery from postoperative ileus was aided by GI motility in which contractions were transmitted from the stomach to the lower GI tract, like IMC.

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Twelve strains of Escherichia coli O157 which caused outbreaks in Japan were used as DNA sources. The sequences of the gene encoding the Shiga toxin 2 in all 12 strains were almost identical and the sequences downstream of this gene were similar to that of bacteriophage 933W.

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6-Phenyl-5-hydroxymethyl-4, 5-dihydro-3(2H)-pyridazinone (1) and 6-thienyl-5-hydroxymethyl-4, 5-dihydro-3(2H)-pyridazinone (2) inhibit platelet aggregation induced by thrombin (IC₅₀=0.25 and 0.26 mM, respectively) or by the calcium ionophore ionomycin (IC<50>=0.42 and 0.43 mM, respectively). Pyridazinones 1 and 2 also show concentration-dependent attenuation of the increases in platelet cytosolic free calcium concentration induced by thrombin and ionomycin, suggesting that their antiaggregatory activity may be due to their capacity to inhibit the passage of calcium through the cytoplasmic membrane. This effect may be implicated in other pharmacological activities of 6-aryl-5-substituted-pyridazinones.

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3, 6-Dimethylpyrazine-2-thiol administered at 10-70 mg/kg, p.o. was found to suppress androstenedione-in-duced increase of uterine weight in female rats. This action was weaker than that of aminoglutethimide (3-30 mg/kg, p.o.). After administration of androstenedione, increased plasma estradiol levels were reduced by 3, 6-dimethylpyrazine-2-thiol. Moreover, in vitro, production of estradiol in the pregnant mare serum gonadotropin (PMSG)-treated ovary was inhibited by 3, 6-dimethylpyrazine-2-thiol. These results suggest that 3, 6-dimethylpyrazine-2-thiol has an inhibitory action on aromatase activity.

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We have developed a new rat model of gangrenous peripheral vascular disease with vascular injury and occlusive thrombi. Rat hind limb gangrene was induced by injecting arachidonic acid (2 mg/leg) into the femoral artery. Using this model, we evaluated the effect of a thromboxane A₂ receptor antagonist, Z-335, on the progression of hind limb gangrene. Z-335 (10 mg/kg/d, p.o.) ameliorated arachidonic acid-induced hind limb gangrene. In contrast, daltroban (10 mg/kg/d, p.o.) and cilostazol (100 mg/kg/d, p.o.) tended to improve the hind limb gangrene but their effects failed to reach statistical significance. Z-335 (10 mg/kg. p.o.) inhibited U-46619-induced, but not collagen-induced, platelet aggregation in rat whole blood. Daltroban (10 mg/kg, p.o.) showed a tendency to inhibit U-46619-induced platelet aggregation. Cilostazol (100 mg/kg, p.o.) did not inhibit U-46619- or collageninduced platelet aggregation. Histopathological examination of the injured paws showed that Z-355 (10 mg/kg, p.o.) had partly inhibited the formation of occlusive thrombi. These results indicate that the thromboxane A₂ receptor antagonist Z-335 is effective arachidonic acid-induced hind limb gangrene in rats. Our experiments sugggest that Z-335 may be beneficial in the prevention of gangrenous peripheral vascular disease.

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There are several Kampo medicines (Chinese herbal medicines) containing both Bupleuri Radix and Scutellariae Radix, which are used for the treatment of inflammation. Saikosaponins are derived from Bupleuri Radix, and baicalein is from Scutellariae Radix. The present study was undertaken to investigate the pharmacological interaction of saikosaponin b1 and baicalein in prostaglandin E₂ (PGE₂) release from C6 rat glioma cells in vitro. Saikosaponin a, b1 and d potently stimulated PGE₂ release, while saikosaponin b2 and c moderately stimulated PGE₂ release. Saikosaponin b1 caused an irreversible elevation of intracellular Ca²⁺ concentration, which was eliminated by removing extracellular Ca²⁺. On the other hand, baicalein inhibited saikosaponin b1-induced PGE₂ release in a concentration-dependent manner. These results suggest that saikosaponins are activators of PGE₂ release, and baicalein is one of the functional inhibitors of PGE₂ release by saikosaponins.

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We found that epicatechin gallate, a constituent of an extract of tea leaves (green tea) markedly lowered the minimum inhibitory concentration (MIC) of oxacillin and other β-lactams, but not of other antibacterial agents tested, in strains of methicillin-resistant Staphylococcus aureus. The antibacterial action of epicatechin gallate plus oxacillin was a bactericidal one.

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BOF-4272, (±)-8-(3-methoxy-4-phenylsulfinylphenyl)pyrazolo[1, 5-a]-1, 3, 5-triazine-4(1H)-one, is a new drug intended for the treatment of hyperuricemia. This report describes the detailed metabolic pathways of BOF-4272 in the dog. The metabolic pathways were investigated using the metabolites found in plasma, urine, and feces after intravenous or oral administration of BOF-4272, as well as the metabolites found in the liver S9 incubation mixture after the addition of BOF-4272 or BOF-4269. BOF-4269 (the sulfide metabolite of BOF-4272) was the only metabolite detected in plasma and feces after the intravenous or oral administration of BOF-4272. BOF-4269 was detected in dog plasma after a lag time following the oral administration of BOF-4272, and the C_max and AUC_0-t of BOF-4269 were higher in fed dogs than in fasted dogs after the oral administration of BOF-4272. A small amount of BOF-4269 was detected in dog plasma immediately after the intravenous administration of BOF-4272. Only BOF-4276 (the sulfone metabolite of BOF-4272) was detected in the S9 incubation mixture after the addition of BOF-4272. Mainly BOF-4272 was detected and small amounts of BOF-4276 and M-1 (the hydroxy metabolite of BOF-4269) were detected in the S9 incubation mixture after the addition of BOF-4269. These findings suggest that BOF-4272 is mainly metabolized to BOF-4269 by the intestinal flora in dogs, whereas little of this drug is metabolized to BOF-4269 in the dog liver. In conclusion, this work has allowed us to formulate the proposed metabolic pathways of BOF-4272 in the dog.

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Menaquinone 7 (MW : 649, C₄₆H₆₄O₂), a natural electron acceptor for steroid ring A dehydrogenations, produced by Bacillus sp. SNU-299, was isolated as a rat prostate testosterone 5α-reductase inhibitor with an IC₅₀ value of 4.0×10^-5 M from the cultured broth. Phylloquinone was as active as the purified microbial metabolite with an IC₅₀ value of 6.6×10^-4 M. On the basis of this evidence, the inhibitory activities of electron carriers, menadione, phenazine methosulfate, and 2, 6-dichlorophenolindophenol, for rat prostate testosterone 5α-reductase were tested, and the IC₅₀ values were 3.1×10^-6 M, 4.9×10^-8 M, 8.9×10^-5 M, respectively. A product of the 5α-reductase enzyme reaction and an electron and proton carrier, NADP⁺, inhibited the 5α-reduction by rat prostate testerone 5α-reductase with an IC₅₀ value of 9.2×10^-5 M. However, the inhibition effect of a proton carrier, carbonylcyanide-m-chlorophenylhydrazone, for rat prostate testosterone 5α-reductase was substantially inactive.

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