The antioxidant potential of albumin-bound sulfur (SBA) was investigated in rat liver microsomes using lipid peroxidation systems in vitro. Sulfur bound to protein is a reduced metabolite which is produced from cystine by γ-cystathionase. Lipid peroxidation was induced either chemically by ferrous ions and ascorbate or enzymatically by carbon tetrachloride or tert-butyl hydroperoxide as indicated by the increase in thiobarbituric acid reactive substances (TBA-RS) and oxygen consumption. Although the antioxidant effect of SBA was weak on the non-enzymatic lipid peroxidation system, the addition of SBA significantly inhibited TBS-RS formation and oxygen consumption compared with non-treated bovine serum alubumin (BSA) in a microsomal lipid peroxidation system induced enzymatically.The sulfur bound to albumin disappeared during incubation with liver microsomes. However, slight differences in the disapperance were observed depending on whether or not lipid peroxidation was induced in the enzymatic systems. In the CCl₄-induced lipid peroxidation system, the cytochrome P-450 level was significantly decreased by the addition of SBA. Therefore, in cytochrome P-450 dependent lipid peroxidation system, the potential effects of sulfur bound to albumin are due to an inhibition of cytochrome P-450 rather than by the oxidation itself caused by radical trapping.

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Sulfotransferases (ST) are contained as multiple forms in human tissues with overlapping substrate specificities. To identify a form which contributes to the metabolism of 3, 3', 5-triiodothyronine (T₃), the functional properties of human STs were compared using recombinant STs, ST1A3, ST1A5, ST1B2, ST1E4 and ST2A3.ST1B2 showed a high affinity (K_m 46.2 μM) for T₃ sulfation, whereas ST1A3, ST1A5, ST1E4 and ST2A3 showed high affinities to p-nitrophenol (K_m 0.4 μM), dopamine (K_m 7.1 μM), β-estradiol (K_m 0.3 μM) and dehydroepiandrosterone (K_m 3.3 μM), respectively. In Western blotting using antibodies raised against an individual ST, hepatic absolute amounts of these STs were determined. The content of ST1B2 in human liver correlated well with T₃ sulfation activities in human liver (r=0.96). These results indicate that ST1B2 is biochemically distinct from other forms of ST, and is involved in the metabolism of T₃ in human. In addition, studies of thermal stability and 2, 6-dichloro-4-nitrophenol (DCNP) inhibition showed that ST1B2 was thermostable and more DCNP resistant than other forms of ST. Affinities for a co-factor, phosphoadenosine 5'-phosphosulfate, also differed 9-fold among 5 different forms of ST.

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New thienothiazine derivatives which differ in their side chain on the nitrogen atom of the thienothiazine molecule were studied in guinea pig papillary muscles and terminal ilea using isometric contraction force measurements. Compounds with a heterocyclic ring in their side chain like MS 57 (pyrrolidinylethylcarboxamide side chain), MS 58 (piperidinoethylcarboxamide side chain) and MS 55 (morpholinoethylcarboxamide side chain) had the most potent negative inotropic effect on isolated paplillary muscles. The relaxing effect on smooth muscle was more pronounced with compounds carrying an aromatic ring in their side chain like MS 25(dimethoxyphenylethyl-N-aminopropionyl side chain), MS 24 (dimethoxyphenylethyl-N-methylaminoacetyl side chain) and MS 27 (dimethoxyphenyl-N-methylethylaminoethylcarboxamide side chain). Our results show a tissue selectivity of the thienothiazine compounds.

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The present study was conducted to develop an experimental model for evaluation of chlorpromazine-induced orthostatic hypotension in rabbits. In addition, the α-adrenoceptor blocking effect of chlorpromazine was investigated in isolated rabbit aorta and saphenous vein in comparison with prazosin. Chlorpromazine (0.1 and 1 mg/kg, i.v.) potentiated significantly a decrease in mean blood pressure at 1 min after the onset of head-up tilt in rebbits anesthetized with urethane alone, urethane+α-chloralose or nitrous oxide alone, but not in conscious and morphine+urethane+α-chloralose-anesthetized rabbits. There was a negative correlation (r=-0.986, p<0.01) between the extent of chlorpromazine-induced orthostatic hypotension and the amplitude of tilt-induced reflex tachycardia before chlorpomazine treatment. Both prazosin and pentolinium elicited orthostatic hypotension under all four anesthetic conditions. The pA₂ value for chlorpromazine to antagonize norepinephrine-induced contraction in aorta was significantly larger than that in saphenous vein, whereas prazosin blocked aortic and venous contractions to a similar extent. These results suggest that a rabbit under an anesthesia which impairs tilt-induced reflex tachycardia may be useful for evaluation of orthostatic hypotension by chlorpromazine.The relatively low potential of chlorpromazine to produce orthostatic hypotension may be partly due to its weak venodilating action.

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In our continuous work on the enhancement of the antibacterial activity of β-lactam antibiotics against the cells of methicillin-resistant Staphylococcus aureus (MRSA) stains by Keggin-structural polyxotungstates and their lacunary species, Wells-Dawson, double-Keggin, and Keggin-sandwich polyoxotungstates are also found to be synergistic but highly cytotoxic. The coexistence of polylysine or protamine sulphate decreased the synergistic potency of the polyoxotungstates, due to their electrostatic interaction with negatively charged polyoxotungstates.Inductively coupled plasma atomic emission spectrometry (ICP) analysis of the polyoxotungstate-treated cells indicated that the polyoxotungstates uptaken in the cell are preferentially located at the membrane fraction with intact compositio. The polyoxotungstates depressed not only the production of PBP2', but also the production of β-lactamase which hydrolyzed β-lactam antibiotics on the membrane. This leads to the synergistic effect of polyoxotungstates against the MRSA cells in the coexistence of β-lactam antibiotics which have high affinities to PBPs 1-4. MRSA cells which were modified to be susceptible to β-lactam antibiotics during incubation in the presence of polyoxotungstates recovered their resistance to β-lactam antibiotics when they were subcultured in the absence of the polyoxotungstate.

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In this study, the cytotoxic activity of gallic acid derivatives (GDs) was studied using some cancer cell lines.Among them, 3, 4-methylenedioxyphenyl 3, 4, 5-trihydroxybenzoate (GD-1) and S-(3, 4-methylenedioxyphenyl)-3, 4, 5-trihydroxy-thiobenzoate (GD-3) were found to induce cell death in cancer cell lines with IC₅₀s ranging from 2.9 to 114.4 μM, a concentration comparable with or lower than that of gallic acid. On the other hand, although gallic acid did not show any cytotoxicity against primary cultured rat hepatocytes and human keratinocytes, GD-1 and -3 showed slightly higher sensitivity against such normal cells, when compared with gallic acid. The cell death induced by gallic acid and GD-1 was accompanied by internucleosomal DNA fragmentation characteristic of apoptosis, whereas only smear DNA degradation was detected following GD-3 treatment. When the mechanism by which GD-1 and -3 caused cell death in HL-60RG cells was examined, GD-1 and -3-induced cell death was inhibited by the intracellular Ca²⁺ chelator, bis-(o-aminophenoxy)-N, N, N, N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM), calmodulin inhibitor, W-7, and the Ca²⁺/MG²⁺-dependent endonuclease inhibitor zinc sulfate. In contrast, catalase, N-acetylcysteine (NAC), and ascorbic acid inhibited gallic acid-induced apoptosis in HL-60RG cells, whereas they had no effect on GD-1- and -3-induced cell death. This result suggests that GD-1 and -3 induced cell death in a different manner to gallic acid. In conclusion, esterification of gallic acid with a 3, 4-methylenedioxyphenyl group yielded potent agents to treat cancer with a different signaling pathway from gallic acid, although selectivity was lost.

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The effects of a water extract of perilla (Perilla frutescens BRITTON) leaves on nitric oxide (NO) production by cultured murine mesangial cells were investigated. Perilla extract significantly induced NO production from mesangial cells, which was enormously augmented without cytotoxity by combination with interferon (IFN)-γor tumor necrosis factor-α. On the other hand, perilla extract suppressed a large amount of NO production induced by IFN-γ combined with lipopolysaccharide. Northern blot analysis revealed that such effects of perilla extract were dependent on inducible NO synthase mRNA expression. Perilla extract exhibited an inhibitory effect on cytokine-induced mesangial cell proliferation, and this effect was significantly decreased upon combination with N^G-monomethyl-L-arginine (L-NMMA), a non-specific NO synthase inhibitor, suggesting that perilla extract inhibits mesangial cell proliferation partially through the induction of NO production. Such results indicate that perilla may be a promising agent for the prevention of the progression of glomerulonephritis.

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The phylogenetic relationship of Acorus gramineus and three types of Acorus calamus was analyzed by comparing the 700 bp sequence of a 5S-rRNA gene spacer region. Although there was no intra-specific variation in the essential oil profile of A. gramineus which contained a phenylpropanoid (Z-asarone) as a predominant constituent, A. calamus was classified into two chemotypes : chemotype A in which Z-asarone is a major essential oil constituent and chemotype B which contained sesquiterpenoids predominantly. An intermediate type (M) of these two chemotypes in various ratios was also observable. The NJ tree constructed based on the sequences revealed that A. gramineus was clearly distinguished from any of the chemotypes of A. calamus and that the phylogenetic relationship predicted by the spacer region data correlated well with the essential oil chemotype pattern of A. calamus.

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The effect of Ogi-Keishi-Gomotsu-To-Ka-Kojin (OKGK), a Japanese traditional herbal medicine (Kampo medicine), has been studied in a cyclophosphamide (CPM)-induced hyperlipidemia model in fasted rabbits. In this model, the accumulation of chylomicrons and very low density lipoprotein (VLDL) was known to occur as a result of a reduction in lipoprotein lipase (LPL) activity in the heart and heparin-releasable heart LPL. Oral administration of OKGK for 4 weeks was found to reverse the increase in serum triglycerides and cholesterol produced by CPM treatment especially in chyromicrons and VLDL. In addition, OKGK treatment led to a recovery in postheparin plasma LPL activity and heparin-releasable heart LPL activity which were reduced markedly by CPM treatment. We previously reported that OKGK increased LPL activity in postheparin plasma in rats. In this study, we have also found that OKGK improved hyperlipidemia in the CPM-induced hyperlipidemia model in rabbits, mainly due to an increase in heparin-releasable heart LPL activity and postheparin plasma LPL activity.

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Seventeen thiopaeonimetabolin-I adducts were obtained as mixtures of diastereoisomers after incubation of paeoniflorin with Lactobacillius brevis in the presence of various thiols. The anticonvulsant activity of the adducts was investigated in mice using the maximal subcutaneous pentylenetetrazol seizure test and sodium valproate (1.5 mmol/kg) as a positive control. Thirteen adducts showed dose-dependent prolongation of latencies of clonic and tonic convulsions. Maximal protection against convulsions was effectively demonstrated by 8-(n-hexylthio)paeonimetabolin I (8) and 8-benzoylthiopaeonimetabolin I (18) at doses of 0.125 and 0.25 mmol/kg, respectively, while 100% protection was only achieved at 0.5 mmol/kg of 8-cyclopentylthiopaeonimetabolin I and 8-(p-tolylthio)paeonimetabolin I. The principal anticonvulsant activity of the diastereoisomers of 8 and 18 was attributed to their 7S-isomers [ED₅₀ values of 0.09 and 0.12 mmol/kg, and protective indices of 5.0 and 4.0 for 8(7S) and 18 (7S), respectively], while the 7R counterparts [8 (7R) and 18 (7R)] showed a muscle relaxation effect.

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RS-8359, (±)-4-(4-cyanoanilino)-5, 6-dihydro-7-hydroxy-7H-cyclopenta[d]-pyrimidine, inhibits, selectively and reversely, A-type monoamine oxidase (MAO-A). In order to clarify the stereoselective metabolism of this drug, plasma concentrations of the [R] and [S]-enantiomers of RS-8359 were determined by chiral column HPLC after oral administration of each enantiomer to rats, mice, dogs, and monkeys. After administration of the [R]-enantiomer, high levels were retained in all animal species studied. On the other hand, when the [S]-enantiomer was administered, plasma concentrations decreased rapidly in rats and mice, and extremely rapidly in dogs, while in monkeys, only a trace amount was detected immediately after dosing. Thus, it was found, as a common phenomenon in rats, mice, dogs, and monkeys, that plasma concentrations of the [S]-enentiomer were markedly lower than those of the [R]-enantiomer. Secondly, the [R]-enantiomer was observed in plasma after administration of the [S]-enantiomer, and the [S] to [R] chiral inversion rate was estimated from AUC([R] after[S])/AUC([R] after [R]). The percentage was 45.8% in rats, 3.8% in mice, 0.8% in dogs, and 4.2% in monkeys.Further, the [S]-enantiomer was detected in plasma of SD rats dosed with the [R]-enantiomer, suggesting [R] to[S] shiral inversion in rats. These results show marked species differences in the chiral inversion of the cyclopentanol group of RS-8359. A mechanism of chiral inversion is discussed based on experiments using isolated rat hepatocytes.

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The purpose of this study was to evaluate a possible interaction between lansoprazole and clarithromycin as well as other macrolides in dogs. Lansoprazole (30 mg) was orally administered to male beagle dogs, with or without oral pretreatment with 200-mg clarithromycin twice a day for 5 d. The experiments had a randomized cross-over design with a two-week washout period between dosing regimens. Clarithromycin pretreatment for 5 d resulted in a significant increase in the area under the serum lansoprazole concentration-time curve (AUC), whereas the area for a lansoprazole metabolite, lansoprazole sulfone, was significantly reduced, as was the maximum serum concentration (C_max) of lansoprazole sulfone. When the effects of clarithromycin on the metabolism of lansoprazole were studied using dog liver microsomes, it was found that clarithromycin significantly inhibited the formation of lansoprazole sulfone but not another metabolite, 5-hydroxylansoprazole. These results suggest that co-medication of lansoprazole with clarithromycin may produce a synergistic effect caused by the increased serum levels of lansoprazole of benefit in Helicobacter pylori eradication.

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The permeability of model hydrophilic compounds with defferent molecular weights and model dipeptides were examined to characterize the tracheal epithelial barrier in in vitro experiments using excised rabbit trachea.6-Carboxyfluorescein (6-CF; 376 Da) and fluorescein isothiocyanate (FITC)-dextrans (FDs) with varying molecular weights (4 to 70 kDa) were used as model hydrophilic and macromolecular compounds, and glycyl-D-phenylalanine (Gly-D-Phe) and glycyl-L-phenylalanine (Gly-L-Phe) were used as model dipeptides in this experiment.The apparent permeability coefficients (P_app) of 6-CF and FDs with Mw 376 Da to 70 kDa ranged form 2.35×10^-7 to 4.05×10^-10 cm/s and exhibited a good inverse correlation with their molecular weights. The tracheal permeability of 6-CF, FD-4 (4 kDa) and FD-10 (10 kDa) were increased over three fold by 10 mM glycocholate, which is an absorption enhancer. The P_app of Gly-D-Phe was 1.03×10^-6 cm/s and there was no metabolism during tracheal permeation. Gly-L-Phe was immediately degraded in the mucosal fluid and its intact form was not detected in serosal fluid during the 150 min experimental period. These results suggest that absorption of some peptide drugs via the respiratory tract may contribute to their systemic delivery following pulmonary administration by intratracheal insufflation and instillation.

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The ability of complement (C) system to remove liposomes from blood circulation was examined in vivo using rat and guinea pig as models. Although the liposomes were not degraded in guinea pig serum in vitro, they were degraded remarkably in guinea pig circulation, as assessed by the urinary excretion of [³H]inulin released from liposomes. The suppression of rat C system to 64% normal C hemolytic activity by treating animals with K76COOH agent resulted in a significant decrease in both the uptake of liposomes by liver and the release of[³H]inulin, providing in vivo evidence for C-mediated clearance of liposomes in rats via uptake by macrophages and degradation in blood circulation, respectively. On the other hand, the K76COOH-induced suppression of C(70% normal hemolytic activity) in guinea pigs slightly increased both the hepatic uptake and the release of [³H]inulin. In addition, the hepatic uptake and in vivo degradation in guinea pigs varied in an opposite manner when the animals were preloaded by empty liposomes or when the liposome size and cholesterol content varied.These results suggest there is a difference between the factors involved in liposome degradation and the factors involved in hepatic uptake and also support the likelihood that there is no C-mediated degradation in guinea pigs.

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The rectal absorption of a platinum anti-tumor agent, [bis (acetato) ammine dichloro (cyclohexylamine)platinum(IV)](BNS-182751), was investigated in rats. BMS-182751 was co-ground with various carriers to improve its poor aqueous solubility. The highest drug dissolution was observed for the co-ground mixture of BMS-182751 and low molecular (LM) gelatin (1 : 9, w/w), followed by β-cyclodextrin and polyvinylpyrrolidone. The influence of a suppository base or additive on the rectal absorption of BMS-182751 in the drug state of crystalline powder or co-ground mixture was examined in vitro using excised rat rectum. A macrogol base gave much higher BMS-182751 permeation across the rat rectum than that from a Pharmasol base. The addition of sodium caprylate or caprylic acid to the macrogol base markedly enhanced the drug permeation, and a 3% addition of sodium caprylate to the base afforded maximum drug permeation. Two rectal formulations, the co-ground mixture with LM-gelatin plus 3% sodium caprylate in macrogol and the crystalline drug alone plus 3% sodium caprylate in macrogol, as well as an oral aqueous drug suspension, were administered to rats. The C_max and AUC_0-24h values for platinum from the former suppository were 5.1-and 4.1-fold greater than those from the oral suspension, respectively. The values from the latter suppository were almost comparable to those from the suspension. These results suggest that the suppository may provide a promising therapeutic means for cancer treatment using this platinum agent.

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The effects of oral co- and pre-administration of Sho-seiryu-to extract powder (TJ-19, 1 g/kg), a widely used Kampo (traditional Chinese herbal) medicine, on the pharmacokinetics of an anti-epileptic drug, carbamazepine(CBZ), and its active metabolite (carbamazepine-10, 11-epoxide, CBZ-E) after oral administration of CBZ(50 mg/kg) were examined in male rats. The simultaneous administration of TJ-19 significantly lengthened the time to reach the peak plasma concentration (T_max), but did not influence the peak plasma concentration, area under the plasma concentration-time curve or terminal elimination half-life (t_1/2). Each parameter for CBZ or CBZ-E with a single pretreatment with TJ-19 was not significantly different from that with the vehicle. T_max and the elimination rate constant for CBZ were significantly increased by 1-week repeated pretreatment with TJ-19, by 83% (p<0.01) and 88% (p<0.001), respectively.t_1/2 and the mean residence time from zero to infinity (MRT_0-∞)in the TJ-19 pretreatment group were significantly shortened, by 52 and 34% (p<0.005), respectively. No significant difference in the bound fraction of each drug at two concentrations (1 and 10 μg/ml) was observed between the control and TJ-19 pretreatment groups. These results indicate that simultaneous oral administration of TJ-19 delays the oral absorption of CBZ, while 1-week repeated pretreatment with TJ-19 accelerates the metabolism of CBZ in rats, without affecting the protein binding of CBZ.

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Cycloprodigiosin hydrochloride (cPrG·HCl) is a stable fluorescent red pigment obtained from the marine bacterium Pseudoalteromonas denitrificans. It was found that the compound was incorporated into Plasmodium falciparum cells upon incubation and exhibited a potent antimalarial activity with the concentration required for 50% of the activity being 11 nM, which is stronger than that of chloroquine, a well-known antimalarial agent.The compound did not affect growth rate of mammalian cells. Antimalarial activity of cPrG·HCl was also observed in vivo. These results indicate that cPrG·HCl is a potent antimalarial drug.

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The intestinal absorption of ten steroid hormones was evaluated in the rat small intestine, especially focusing on the interaction with intestinal P-glycoprotein (P-gp). Hydrocortisone, prednisolone, 6α-methylprednisolone, and dexamethasone (adrenocortical steroid hormones) all disappeared in a regional-dependent manner(duodenum>jejunum>ileum). The decreased rate of disappearance in the lower small intestine seemed to be due to the involvement of absorption barriers like P-gp. In contrast, all sex hormones including progesterone exhibited very high absorbability in the entire small intestine (duodenum=jejunum=ileum), possibly demonstrating the absence of restricted absorption by intestinal P-gp. Progesterone enhanced the rate of disappearance of vinblastine but did not affect 6α-methylprednisolone. In the presence of vinblastine and verapamil, on the other hand, the rate of disappearance of 6α-methylprednisolone increased significantly. It was demonstrated that there was a plural P-gp family, which had different substrate specificities, in the rat intestive and that steroid hormones interacted with them as substrates or inhibitors in a very complex manner.

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We have developed a simple, sensitive and reliable assay procedure for cyclosporin A (CyA), a modified fluorescence polarization immunoasay method incorporating fat emulsion (FE-FPIA), to determine the CyA content in rat skin. The conventional fluorescence polarization immunoassay (FPIA) method for CyA using a commercially available FPIA kit, TDX^[○!R] cyclosporine monoclonal whole blood, was modified. A fat emulsion for intravenous infusion, Intralips^[○!R], was incorporated for dissolving the CyA extracted from the skin tissue, and a mixture of MeOH/purified water was used as the sample pretreatment medium instead of the precipitation reagent in the conventional FPIA kit intended for whole blood samples. These modifications enabled us to produce a reliable and the sensitive assay of CyA in skin tissue. The reproducibility (coefficient of variation), detection limit, and assay time for FE-FPIA were below 2%, 25 ng/ml, and about 24 min/24 samples, respectively, and were comparable with those for the whole blood samples determined by the conventional FPIA. Pre-purification of samples required by the HPLC assay is not needed in the FE-FPIA method. The usefulness of the FE-FPIA method in evaluating the topical pharmacokinetics of CyA in skin is discussed.

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The effect of the Dynorphin A analog, E2078, on the physicochemical properties of lipid membranes was investigated. E2078 induces the leakage of calcein, especially out of negatively-charged vesicles. The peptide binds to dipalmitoylphosphatidylglycerol sonicated vesicles according to the Lamgmuir isotherm, with a binding constant of 4.0×10⁵ M^-1 and a binding -site number of 0.14 per lipid molecule. The leakage seems to occur at a critical binding-site number of approx. 0.025 per lipid molecule. In addition, E2078 increased the membrane fluidity of the acidic lipid bilayer. In conclusion, E2078 interacts with acidic lipids through electrostatic interactions, inducing leakage and increasing membrane fluidity.

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A new cytotoxic substance was isolated following acid treatment of fermented broth of a Streptomyces strain numbered MJ915-WF12. The structure was elucidated by spectroscopic analyses to be 5-dihydro-4-formyl-6-hydroxy-2-hydroxymethyl-6-methyl-2H-pyran. It was thought to be generated by dehydration and ring reformation of the precursor material in cultured broth by acid.

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Asp-hemolysin is a human low density lipoprotein (LDL)binding protein. We found evidence that Asp-hemolysin binds to oxidized LDL (oxLDL) as well as to LDL in a concentration-dependent manner. This result suggests that Asp-hemolysin is a novel protein, which shares binding abilities to LDL and oxLDL.

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