The development and application of a new enzyme immunoassay for general assay of the Glycyrrhizae radix (GR) component in Chinese traditional medicines is described. Three commercial GR-based medicines, Tohoku kanzo (GRTK), Seihoku Kanzo (GRSEK) and Shinkyo kanzo (GRSK) were used as GR specimens. Anti-GRSK serum was elicited from rabbits immunized with GRSK fragments. The presence of common proteins as specific antigens of GR was first established by Western blot analysis of extracts of GRSK, GRSEK or GRTK using anti-GRSK. The specific antigens were applied to develop an ELISA for the assay of GR extract. Anti-GRSK was put in sompetition with a sample or standard GR extract and immobilized GRSK components in microtiter plate wells. The proportion of antibody binding to the solid-phase GRSK component wa detected using na enzyme-labeled second antibody. The ELISA method was specific to GRSK extract and showed low sensitivity for the assay of GRTK extract. The technique of selected antibody enzyme immunoassay (SAEIA) was applied to develop a sensitive general assay method. Solid-phase GRTK extract, rather than immobilized GRSK extract, was used in the SAEIA. The SAEIA possessed the same quantitative working range of between 1 and 100 μg/ml for the asssay of each extract of GRTK, GRSK and GRSEK. The SAEIA was successful in the detection and quantitative measurement of GR component cntents in Chinese traditional medicines.

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Previously, we reported that a high α-linolenate [18 : 3(n-3)] diet compared with a high linoleate [18 : 2(n-6)] diet suppressed the anti-egg albumin (EA) immunoglobulin E (IgE) antibody response in mice. Because of relatively high background values obtained with the method used previously, we used an improved ELISA and once again determined serum IgE levels. In contrast to our previous results, the serum levele of anti-dinitrophenyl specific (anti-DNP) as well as total IgE in mice immunized with DNP-antigen was slightly but significantly heigher in the high α-linolenate diet group than in the high linoleate diet group. Anti-DNP IgG₁ and IgG_2a antibody responses were not significantly different in mice fed these diets. Indomethacin administration during immunization tended to enhance the IgE antibody responses. The mortality of mice from antigen-induced anaphylatic shock was significantly lower in the high α-linolenate diet group than in the high linoleate diet group; however, there was no difference between the groups in terms of vascular permeability and histamine levels. Thus, the high α-linolenate diet enhances the IgE antibody response slightly without affecting either the IgG antibody response, vascular permeability or histamine release. The high α-linolenate diet possibly suppress anaphylactic shock by reducing the synthesis of lipid mediators such as eicosanoids and platelet-activating factor.

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p33/41 (annexin IV) is a member of the family of Ca²⁺-dependent phospholipid binding proteins known as annexins. We previously described that bovine kidney p33/41 (annexin IV) has Ca²⁺-dependent carbohydrate binding acrivity. In this study, we purified human p33/41 (annexin IV) from the HT29, human colon adenocarcianoma cell line, as well as the bovine kidney annexin by affinity chromatography. Then, we prepared recombinant human p33/41 (annexin IV) expressed in Escherichia coli. The apparent size and the Ca²⁺-dependent carbohydrate binding properties of purified recombinant p33/41 (annexin IV) were indistinguishable from those of the bovine kidney protein. We also performed inhibition assays of carbohydrate binding and of phosphatidylserine/phosphatidylcholine liposome binding of recombinant p33/41 (annexin IV) with anti-p33/41 monoclonal antibodies (AS11 and AS17). We determined the epitopes recognized by the monoclonal antibodies by Western blot analysis using deleted-recombinant p33/41 (annexin IV). The monoclonal antibodies recognized domain 1 and/or 2 of p33/41 (annexin IV). The results of the inhibition assays and the determination of the epitope showed that a carbohydrate binding site is located at domains 3 and 4 of p33/41 (annexin IV) and on the cell surface.

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We evaluated the cardiovascular effects of YM430, a novel 1, 4-dihydropyridine derivative with β-adrenoceptor blocking activity, in dogs and rats. In anesthetized dogs, YM430 (0.01-0.3 mg/kg, i.v.) dose-dependently decreased mean blood pressure, total peripheral resistance and double product without increasing the heart rate. YM430 (0.01-0.3 mg/kg, i.v.) increased coronary artery as well as vertebral artery blood flow, whereas its effects on carotid, mesenteric, femoral and renal blood flow were small. At the same dose range as that which induced vasodilation effects, YM430 had little effect on the max. dp/dt or PQ-interval. In conscious dogs, YM430 (0.1-1 mg/kg, i.v.) produced dose-dependent hypotension with tachycardia. In conscious rats, oral dosing of YM430 (100 mg/kg p.o.) produced a long-lasting hypotensive effect with slight tachycardia. YM430 also inhibited isoproterenol (0.1 μg/kg i.v.)-induced tachycardia. These two effects of YM430 may be attributable to its calcium entry blocking and β₁-adrenoceptor blocking activity, respectively. The time course of the hypotensive (calcium entry blocking) effect of YM430 after oral dosing was very similar to that of its inhibition of isoproterenol-induced tachycardia (β₁-adrenoceptor blocking effect). These results indicate that the ratio of the two activities (calcium entry blocking and β₁-adrenoceptor blocking) of YM430 is constant after oral administration. In conclusion, YM430 could be both an antianginal and antihypretensive agent, because of its dual activities.

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Stimulation of collagen synthesis prevents the aging process. We found such a synergistic effect by using the leaves of Eucommia ulmoides Oliver, Eucomiaceae (Du-Zhong leaf) and the roots of Panax ginseng C. A. MEYER (Ginseng). The formula consists of amounts which exert no effect when used individually. We tested several formula ratios of Ginseng and Du-Zhong leaf, 1 : 1, 1 : 2, 1 : 3 and 1 : 4, and concluded that the last two formulas were effective. However, we did not observe a significant difference between 1 : 3 and 1 : 4. Thus, it was demonstrated that the formula ratio of Ginseng to Du-Zhong leaf of 1 : 3 was the most effective for the stimulation of collagen synthesis and the prevention of decreased protein metabolism in aging.

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The effects of 70% methanol extract (EA-ext) from Evodiae Fructus (EA) consisting of dried fruits of Evodia rutaecarpa var. bodinieri (Rutaceae) on nocicetive responses were investigated. Oral administration of 50 or 200 mg.kg EA-ext had the same antinociceptive effect on writhing responses as induced by acetic acid. Its major alkaloidal constituents, evodiamine and rutaecarpine also had the antinociceptive effect. EA-ext significantly decreased the frequency of licking behavior within a unit of time at the late phase without affecting that of the early phase in the formalin test. EA-ext also increased nociceptive threshold of the inflamed paw without increasing that in the non-inflamed paw in the Randall-Selitto test. Although EA-ext inhibited the rise of vascular permeability induced by acetic acid and the increase of paw edema induced by carrageenin, it was ineffective on nociceptive response in the hot plate test and on locomotor activity. These results suggest that EA possesses antinociceptive effects and its mode of action may be mediated by anti-inflammatory action, and that the antinociceptive constituents are only partially attributable to alkaloidal components mentioned above.

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Skin permeation rates of five non-steroidal anti-inflammatory drugs, three of them lipophilic (ibuprofen, indomethacin and ketoprofen) and two hydrophilic (sodium diclofenac and antipyrine), through Yucatan hairless micropig (YMP) full-thickness skin were determined in vitro using Franz-type diffusion cells, and the usefulness of YMP skin as an animal model skin was investigated. Five-month-old YMP skin showed smal variation with respect to drug permeation rates, if the region of skin site was the same. But skin from 6-month-old YMPs showed faster and varied permeation rates due to skin defects. In the case of hydrophilic drugs, no effects on permeation due to the region from which the skin was obtained were observed, although higher permeation rates were observed through the flank than through dorsal skin in the case of ibuprofen and ketoprofen. For YMP skin, the permeation coefficients of drugs were lower than those for hairless rat skin, especially for hydrophilic drugs. Compared with reported human data on permeation coefficients, the permeation coefficients of drugs through YMP skin were 1/2-1/8 of those through human skin, regardless of whether the drugs were hydrophilic or lipophilic. YMP skin could be used as an in vitro animal skin model because of the reproducibility of the permeation rate of drugs through this skin and its similarity to human skin.

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We previously demonstrated that IgG1 plasmacytosis in human interleukin-6 transgenic mice (hIL-6 Tgm) was suppressed by the implantation of SK2 hybridoma cells (SK2 cells, which secrete anti-hIL-6 monoclonal antibodies) microencapsulated in a semipermeable and biocompatible device. In this study, we demonstrated that the mesangioproliferative glomerulonephritis in hIL-6 Tgm was also improved by the same treatment. These results strongly support the concept of cytomedicine, which is a novel drug delivery system (DDS) using living cells. However, an electron microscopy study showed that cytomedicine has a limited duration of effectiveness because of the disappearance of space for cell proliferation in the microcapsule. Thus, the control of cell proliferation in a device must be developed to prolong the function and effectiveness of cytomedicine.

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We synthesized branched type galactosyllipid derivatives for liposome modification for the targeting of asialoglycoprotein receptors on the surface of liver cells. Galactose was coupled to the α- and γ-carboxyl groups of glutamic acid via a triethyleneglycol spacer, then this glutamic moiety was bound to the lipid anchor. Ricinus communis agglutinin (RCA₁₂₀) induced the agglutination of liposomes modified with mono-, bi- and tri-antennary neogalactosyllipid. With the bi- or tri-antennary derivatives, agglutination was observed at fewer galactosyl residues on the liposomes.We examined the effect of the branching structure in vivo. The difference in accumulation of liposomes between non-branched type neogalactosyllipid and branched type neogalactosyllipid was not large. Liver accumulation of liposomes depended on the galactosyl residues. The number of galactosyl residues was more effective for accumulation in the liver than for branching.We studied the effect of asialofetuin preinjection on the hepatic accumulation of neogalactosyllipid modified liposomes. Hepatic accumulation of liposomes was inhibited by preinjection of asialofetuin. The effect of preinjection was almost equal among the ligands. These results show that the saccharide density on the liposome surface seemed to be a more important factor than the branching structure of the ligand for liver targeting.

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Magainin 2 is an antimicrobial peptide found in the skin of Xenopus laevis. To find a reversed peptide comparable to the antibacterial activity of magainin 2 analogs, we have synthesized three reversed analogs, the peptide 53D, 87-ISM and A87-ISM, corresponding to the normal peptide D35, MSI-78 and MSI-78A, respectively. We examined their ability to inhibit the growth of Escherichia coli and Staphylococcus aureus. Among the analogs, the A87-ISM, that is, the reverse of MSI-78A enhanced the amphiphilicity and the α-helical tendency of magainin 2, showed not only almost the same antibacterial activity against the bacteria as MSI-78A, but also stronger activity than other magainin 2 analogs. In addition, at the MIC (minimum inhibitoy concentration) value, A87-ISM shows no hemolysis to human red blood cells, while both MSI-78 and MSI-78A cause strong hemolysis at the MIC value. This result indicates that a novel reversed peptide comparable or superior to normal magainin 2 analogs is available.

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The cytotoxicity and lipid peroxidation of pesticides containing a halogen group were examined in isolated rat hepatocytes. We examined 9 pesticides of 3 different representative chemical families, chlorinated aromatic fungicides (pentachlorophenol (PCP), pentachloronitrobenzene (PCNB), chlorothalonil, fthalide), polyhaloalkylated thio fungicides (dichlofluanid, captan) and diphenyl ether herbicide (2, 4-dichlorophenyl 4-nitrophenyl ether (NIP), 4-nitrophenyl 2, 4, 6-trichlorophenyl ether (CNP), chlomethoxynil). The contents of the hydroperoxides in phospholipid, phosphatidylcholine hydroperoxide (PCOOH) and phosphatidylethanolamine hydroperoxide (PEOOH) were determined by the HPLC-chemiluminescence (CL-HPLC) method, which is sensitive and specific for lipid hydroperoxide. Chlorothalonil, dichlofluanid and captan were the most potent cytotoxicants evaluated by lactate dehydrogenase (LDH) leakage. PCP, NIP and CNP exhibited intermediate cytotoxicity. PCNB, fthalide and chlomethoxynil showed low cytotoxicity. The cellular phospholipid hydroperoxide (PCOOH and PEOOH) levels were remarkably increased by chlorothalonil (PCOOH, 23 times and PEOOH, 7 times), dichlofluanid (PCOOH, 523 times and PEOOH, 22 times) and captan (PCOOH, 518 times and PEOOH, 16 times) as compared with the control group. The PCOOH content was slightly increased by PCP (4.8 times) and NIP (6.3 times), whereas the other 4 pesticides did not change the phospholipid hydroperoxide level.Severe cytotoxicity was observed with a remarkable increase of phospholipid hydroperoxide by chlorothalonil, dichlofluanid and captan.

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The in vivo decalcification of calcium polycarbophil was examined. The decalcification ratio of [⁴³Ca]calcium polycarbophil in the stomach after oral dosing to rats was more than 70% at each designated time and quite closely followed the in vitro decalcification curve, indicating that hte greater part of the calcium ion is released from calcium polycarbophil under normal gastric acidic conditions. The residual radioactivity in rat gastrointestine was nearly equal to that after oral administration of either [⁴⁵Ca]calcium chloride or [⁴⁵Ca]calcium chloride+polycarbophil. The serum level of radioactivity was nearly equal to that after oral dosing of [⁴⁵Ca]calcium lactate. These results indicate that the greater part of orally administered calcium polycarbophil released calcium ions to produce polycarbophil in vivo.

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This study was designed to assess a local drug delivery system of an anticancer agent, doxorubicin (DOX), using fibrin glue (Beriplast P^[○!R]) as a drug carrier. In vivo release of DOX from the fibrin glue was examined by a dialysis method in the presence and absence of sodium alginate. The in vitro mean dissolution times of DOX with solution, fibrin glue, and fibrin glue containing sodium alginate were 3.7 h, 8.7 h, and 81 h, respectively, indicating a sustained release of DOX from fibrin glue, especially in the presence of sodium alginate. Fibrin glue containing 6 mg of DOX and 2.5 mg of sodium alginate was applied on the surface of an AH60C tumor at the back of rats, DOX concentrations in the tumor extracellular fluid were monitored by a microdialysis method. Local application of DOX using fibrin glue containing sodium alginate to the tumor resulted in extremely higher concentrations in the tumor extracellular fluid than those in plasma (AUC ratio>800), indicating an advantage of the site-specific delivery of DOX using fibrin glue with sodium alginate. The tumor volumes were inversely correlated with tumor extracellular fluid-to-plasma AUC ratios (r=0.882), suggesting the relevance of tumor size in the drug efflux from tumor to blood. In conclusion, the site-specific delivery of DOX using fibrin glue with sodium alginate to the tumor was demonstrated to be advantageous with regard to the extent and duration of drug concentrations in the tumor extracellular fluid, as assessed by a microdialysis technique.

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A suppository containing an ozagrel tablet was prepared using Witepsol H-15 as a base, and its rectal absorption was studied in male human volunteers. In comparison, a commercially available ozagrel tablet was administered orally to all the individuals in a cross-over design. After rectal dosing, ozagrel was absorbed rapidly at a T_max of 0.75 h, and its elimination half-life was longer than after oral dosing. The extent of absorption of ozagrel after both administration routes was similar. However, the bioavailability of the rectal suppository is 93±37% (mean±S.D.; n=6) relative to the oral tablet. The tablet-containing suppository is easy to prepare, with its content being accurate and reproducible. Thus, the present study suggests that the rectal administration of an ozagrel suppository is a practical and promising alternative to oral administration, especially for patients who cannot take tablets orally. This study demonstrated for the first time the possibility of an ozagrel suppository in human subjects.

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An injectable solution of Danshen was prepared and its in vivo disposition was examined in rabbits. The presence of Danshensu, one of the active components of Danshen, in the obtained solution was confirmed by a simple high-performance liquid chromatographic (HPLC) method. The pharmacokinetics of Danshensu in rabbits was evaluated by the HPLC method for plasma Danshensu. The calibration curve for Danshensu was linear (r=0.998) over the concentration range of 0.25-40.0 μg/ml. The intra-assay coefficients of variation (CV_s) were 3.8, 3.1, and 3.1% at 1, 10, and 50 μg/ml, respectively, and the inter-assay CV_s were 5.3, 5.3, and 2.9% at 1, 10, and 50 μg/ml, respectively. The analytical recovery of Danshensu in plasma averaged 95.2%. From the plasma concentraion profile of Danshensu after its intravenous administration, the t_1/2, mean residence time (MRT), V_dss, and Cl<tot> were determined as 32 min, 48 min, 149 ml/kg, and 3.13 ml/min/kg, respectively.

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The effect of pulsed output ultrasound (1 MHz) with on/off ratios of 1 : 2, 1 : 4 and 1 : 9 on transdermal absorption of indomethacin from an ointment was studied in rats. Ultrasound energy was supplied for between 10 and 19 min at a range of intensities (1.0-2.5 W·cm^-2), energy levels commonly used for therapeutic purposes. The on/off pulsed ratio, intensity and the time of application were found to play an important role in the transdermal phonophoretic delivery system of indomethacin; 1 : 2 pulsed output ultrasound appeared to be the most effective in improving the transdermal absorption. The highest penetration was observed at an intensity of 1.0 W·cm^-2 and application time of 15 min. With pulsed output it was possible to use higher intensities of ultrasound without increasing skin temperature or damaging skin.

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(RS)-[3-³H]-2, 3-Oxidosqualene (1) was converted into (20S)-dammarenediol (2) and not to (20R)-dammarenediol by a microsomal fraction preparaed from the hairy root of Panax ginseng. The enzyme activity was highest at pH 6.0 and was not increased by the addition of any detergents. These properties differed significantly from those of other 2, 3-oxidosqualene cyclases reported from higher plants and animals.

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The absolute configuration of 3α, 7α, 12α, 24-tetrahydroxy-5β-cholestan-26-oic acid CoA ester (V-CoA) produced by the incubation of (24E)-3α, 7α, 12α-trihydroxy-5β-cholest-24-en-26-oic acid CoA ester (24E-THC-CoA) with D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase (D-bifunctional protein) was investigated. When 24E-THC-CoA was incubated with D-bifunctional protein the formation of only one isomer (24R, 25R-isomer) of four possible stereoisomers of V-CoA was observed, which suggested the cis-addition of water to a side chain double bond of 24E-THC-CoA. The dehydration reaction of V-CoA catalyzed by D-bifunctional protein was also observed when (24R, 25R)-V-CoA was used as a substrate. The other three isomers (24R, 25S-, 24S, 25R- and 24S, 25S-isomers) were not dehydrated with D-bifunctional protein. These results showed that D-bifunctional protein catalyzes stereospecifically the hydration and dehydration step in bile acid biosynthesis.

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