In pharmacokinetic studies, ordinary methods of identifying or quantitatively measuring metabolites have been complicated and become difficult to handle. Here, a novel TLC-autoradioluminography (TLC-ARLG) is reported. This technique is especially suited for quantitatively measuring a minute amount, below-1 ng, of many radioactive metabolites in plasma, tissues or organs, thus eliminating cumbersome extraction procedures. The method has an extremely wide dynamic range and could offer a powerful analytical technique for studying a radioactive compound and its metabolites in a biological specimen. Autoradioluminography is a new quantitative technology developed by combining autoradiography with radioluminography. Radioluminography is a kind of radiography in which a specially designed radio-sensor plate, called an "Imaging plate (IP), "developed by Fuji Photo Film Co., Ltd., is used. The radio-sensor is made of fine crystals of BaF : Eu²⁺, and the technology is based on photo-luminescence phenomena.

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Hybridoma clones producing monoclonal antibodies specific for staphylokinase were isolated. A competitive assay revealed that the monoclonal antibodies studied could be divided into at least two groups. Representatives of these groups, AS22 and B3E6, recognized quite different epitopes on staphylokinase. This finding led us to develop an assay system for the quantitative analysis of staphylokinase by enzyme-linked immunosorbent assay using AS22 as the capturing antibody and biotinylated B3E6 as the "detector". The lower limit of sensitivity of the assay was 20 pg of staphylokinase per ml. The assay exhibited good reproducibility, with values of 5.8 and 3.8% for the intra-and inter-assay coefficients of variation, respectively. Staphylokinase could be assayed in the presence of human plasma when the plasma was diluted more than 320-fold, and the measurement was unaffected by the presence of physiological concentrations of human plasminogen. Hence, this assay was considered useful for the detection and quantification of staphylokinase in clinical samples.

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We have studied lysosomal proteases capable of degrading a major form of cytochrome P450 (CYP2B1) which was purifled from the liver microsomes of phenobarbital-treated rats. After incubation of CYP2B1 with extracts of triton-filled lysosomes (tritosomes), its proteolysis was measured by a quantitative immunoblot procedure. A CYP2B1 protein band with an apparent molecular mass of 53 kilodaltons (kDa) was degraded to 42-, 38-, and 29-kDa polypeptides after a short period of incubation. These proteolytic fragments disappeared on prolonged incubation. One milligram of tritosomal protein contained enough activity to hydrolyze approximately 67.3μg CYP2B1 protein/min at pH 4.5. Approximately 85% of the hydrolyzing activity was localized in a soluble fraction of the tritosomes. The degradation of CYP2B1 was effectively inhibited by pepstatin A, an aspartic protease inhibitor, but not by phenylmethanesulfonyl fluoride (PMSF), o-phenanthroline and leupeptin. CYP2B1-hydrolyzing activity was coeluted with cathepsin D when the soluble fraction was chromatographed by means of Ultrogel Ac44 gel filtration. Cathepsin D, purified from rat livers, was able to degrade CYP2B1 at pH 4.5 which corresponds to the intralysosomal pH. These results indicate that cathepsin D is responsible for the major CYP2B1-hydrolyzing enzyme activity in lysosomes.

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An acidic polysaccharide, called alisman PII, was isolated from the tuber of Alisma orientale JUZEPCZ. It was homogeneous on electrophoresis and gel chromatography, and its molecular mass was estimated to be 5.25×10⁴. It is composed of L-arabinose : D-galactose : D-glucuronic acid in the molar ratio of 4 : 9 : 2, in addition to some of O-acetyl groups. Reduction of carboxyl groups, methylation analysis, nuclear magnetic resonance and controlled Smith degradation indicated that the core structural features include a backbone chain composed of β-1, 3-linked D-galactose units. Some of the galactose units in the backbone carry β-D-galactosyl side chains at position 6. Both α-1, 5-linked L-arabinosyl side chains and terminal β-D-glucuronic acid residues are linked to the core galactan units. Alisman PII showed significant potentiation of the reticuloendothelial system using a carbon clearance test and also potent anti-complementary activity.

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The release of hepatic triacylglyceride lipase [EC 3.1.1.3] has been examined in isolated hepatocytes in primary culture. The stimulatory release of activity from the hepatocytes into the medium by sodium orthovanadate (vanadate) was observed in a time-and dose-dependent manner. However, insulin failed to have this stimulatory action. Moreover, vanadate rapidly increased the cyclic adenosine monophosphate (cyclic AMP) content in hepatocytes in a time-and dose-dependent manner. The treatment of hepatocytes with H-89, which is a potent cyclic AMP-dependent protein kinase inhibitor, decreased the stimulatory release of hepatic lipase activity by vanadate. The vanadate-stimulated release of the enzyme activity was suppressed by uncouplers. In addition, the incorporation of [³H] leucine into protein was increased in the presence of vanadate. Under the marked inhibition of protein synthesis by cycloheximide, vanadate still showed a full effect on the release of the enzyme activity. These results suggest that the vanadatestimulated release of hepatic lipase activity from the cultured hepatocytes is associated with a rapid increase in intracellular cyclic AMP content, probably due to an activation of cyclic AMP-dependent protein kinase which requires a metabolic energy process rather than an elevation in enzyme molecule synthesis.

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Among the calcium-binding proteins from chicken gizzard smooth muscle, smooth muscle calcium-binding protein 11 (SMCaBP 11), which is an EF-hand type calcium-binding protein, can prevent Ca²⁺ dependent histamine release from permeabilized rat peritoneal mast cells. This activity appears to be specific since other calcium-binding proteins (CaBPs), including h-calcimedin, l-calcimedin, calmodulin and S100, had no inhibitory action. Approximately 40% of the histamine release was inhibited by SMCaBP 11 at 48 nM and the half-maximal inhibition occurred at 20 nM. Immunological techniques revealed that permeabilized mast cells elicited by Ca²⁺ also released actin into the medium, but not tubulin. The addition of SMCaBP 11 prevented the secretion of actin from the mast cells, though the distribution of F-actin was only slightly affected. These findings suggest that SMCaBP 11 may modulate Ca²⁺ and actin-mediated exocytosis from permeabilized mast cells.

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To determine the mechanism of the growth inhibition associated with the induction of erythroid differentiation in K562 cells by hemin, we used two K562 subclones with different hemoglobin (Hb)-producing activity. Hemin strongly inhibited the growth of K562-L, which had a low Hb-producing activity, but not that of K562-H, which had a high Hb-producing activity. When the cell growth of K562-L was inhibited by hemin, the S phase of the cell cycle decreased and the G₂/M phase increased. In contrast, hemin had no effect on the cell cycle of K562-H. Without hemin treatment, the α-globin mRNA level was related to the degree of Hb production in K562-L and -H but the γ-globin mRNA level was not. With hemin treatment, there was no increase in the α-globin mRNA level in K562-L but there was an increase in K562-H. The difference in α-globin mRNA levels correlated with the Hb production in K562-L and -H induced by hemin. The levels of c-myc and c-myb mRNAs in K562-L decreased when cell growth was strongly inhibited by hemin. These findings indicate that the growth inhibition of K562 cells by hemin is due to a suppression of the progression from the G₀/G₁ phase to the S phase and a delay in the G₂/M phase, caused by the inhibition of c-myc and c-myb transcription. It is also affected by the Hb production, reflected in α-globin transcription.

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We determined the copper (Cu) and metallothionein (MT) concentrations in the liver and kidney supernatants of Long-Evans rats with a cinnamon-like coat color (LEC rats), and also measured the Cu and MT levels in the serum of these rats. Seven-week-old rats had abnormally high levels of both substances in the liver. The levels in the liver supernatant were over 80-and 16-fold higher, respectively, in LEC rats than in normal 7-week-old Wistar rats. LEC rats suffering from acute hepatitis or hepatoma had a much higher level of hepatic MT, but the Cu level was higher only in the liver of those with hepatoma. The serum levels of Cu and MT in LEC rats with acute hepatitis were more than 10-fold higher than those in normal LEC rats. These levels were decreased in the rats with chronic hepatitis or hepatoma. In the liver of LEC rats with hepatoma, the area of hepatocellular carcinoma and of noncancerous liver showed over twice higher Cu and MT levels than the area of chloangiofibrosis. The Sephadex G-75 elution profile from the liver supernatant of a normal LEC rat showed that the peak of Cu closely corresponded to that of MT recognized with anti-MT antiserum. The levels of Cu and MT in the kidney supernatant of LEC rats with acute hepatitis were more than 25-fold higher than in that of normal LEC rats. However, there were no marked increases in the levels in the kidney supernatant of LEC rats with chronic hepatitis or hepatoma.

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β-Fructofuranosidase activities of eight strains of Bifidobacteria, intestinal bacteria, were assayed and Bifidobacterium infantis was selected for purification of the enzyme. β-Fructofuranosidase activity was recovered in the supernatant fraction after disruption of B. infantis cells with sonication and was purified to homogeneity by ammonium sulfate fractionation, and DEAE-cellulose, butyl-Toyopearl and Sephacryl S-300 column chromatographies. The enzyme (molecular weight (M.W.) 232000) was composed of three identical subunits (M.W.75000) whose NH₂-terminal amino acids were threonine. The enzyme was stable at pH 6-8, having the optimum activity at pH 6.0-6.2. The enzyme activity was stable under 40°C and the optimal temperature was 55°C. This enzyme catalyzed the hydrolysis of sucrose, 1-kestose, nystose, inulin and raffinose at the relative velocities of 100, 297, 365, 140 and 3.8, respectively, but did not catalyze the hydrolysis of maltose or cellobiose. These results indicated that this fructooligosaccharide hydrolyzing enzyme is a novel type of β-fructofuranosidase.

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The activity of flavin-containing monooxygenase (FMO) in rat brain microsomes was measured by fluorometrical determination of benzydamine (BZY) N-oxygenation with HPLC. The apparent K_m value for the formation of BZY N-oxide from BZY by brain microsomes was similar to that by hepatic microsomal fraction or purified FMO, but the V_max value for brain microsomes was about one-hundredth of that for hepatic microsomes. BZY N-oxygenation activity by brain microsomes was at a maximum near pH 8.5, slightly more acidic than the optimum pH for liver FMO. BZY N-oxygenation activity was inhibited completely by heat inactivation and markedly in the presence of 1mM thiourea, but slightly in the presence of 1 mM SKF-525A, and it was only barely activated in the presence of 5 mM n-octylamine, a positive effector of liver FMO. The addition of rabbit antisera raised against rat liver FMO resulted in 30% inhibition of BZY N-oxygenation by solubilized brain microsomes. Compared with microsomes from five different brain regions, the activity was highest in microsomes of olfactory bulbs. These results show that the activity of FMO in rat brain is distinctly determined by BZY N-oxygenation.

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The activities of glutathione-related enzymes in isolated rat small intestine were investigated under oxidative stress mediated by Fe²⁺-EDTA. The isoelectric points and approximate molecular weights of the enzymes investigated were first determined. The reduced and oxidized glutathione contents, and low molecular weight thiols in isolated rat small intestine were also studied under oxidative stress. Significant reducing activities of glutathione S-transferase and lactate dehydrogenase were observed accompanied by increases in oxidized glutathione content, whereas thioltransferase, glutathione reductase, glutathione peroxidase and thioredoxin reductase retained the same levels of activity as controls. First-order inactivation of purified rat small intestime glutathione S-transferase including class-α, μ and π was observed and the isozyme class-π was inactivated at several pH's under Fe²⁺-EDTA-mediated oxidative stress. Leakage of protein and both reduced and oxidized forms of glutathione was significantly increased during incubation with Fe²⁺-EDTA. In conclusion, enzymes which were not inactivated under Fe²⁺-EDTA-mediated oxidative stress may play an important role in cellular antioxidant defenses in the small intestine. Furthermore, enzymes such as glutathione S-transferase, mainly a class-π isozyme, and lactate dehydrogenase which were inactivated may form part of the barrier against oxidative stress similar to reduced glutathione.

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The structural stability due to a disulfide bridge between Cys77 and Cys95 of the wild-type human lysozyme is partly recovered by a putative hydrogen bond introduced in to the mutant human lysozyme C77/95S, where Cys77 and Cys95 have been replaced by serines (Yamada et al. (1994) Biol. Pharm. Bull., 17, 192 (1994). In order to understand quantitatively the role of the hydrogen bond in the thermal stability of the mutant human lysozyme, we constructed further mutant proteins, C77SC95A in which Cys77 and Cys95 were replaced by serine and alanine, respectively, and C77AC95S, in which Cys77 and Cys95 were replaced by alanine and serine, respectively. From the thermal unfolding studies of these mutant proteins, both C77SC95A and C77AC95S were shown to be destabilized up to -0.81 and -1.32 kcal/mol, respectively, as far as the free energy changes of unfolding were concerned by compared with C77/95A, where both Cys77 and Cys95 were replaced by two alanines. Considering that these decreases in conformational stability are attributable to hydrophobic effects, the hydrogen bond between Ser77 and Ser95, buried in the hydrophobic cavity in C77/95S, was estimated as 3.0kcal/mol.

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In this study, we showed that systemic administration of SSG, a highly branched soluble (1→3)-β-D-glucan obtained from Sclerotinia sclerotiorum, induced immunological changes in the alveolar space of mice in vivo, assessed by analysing some immune mediators in bronchoalveolar lavage (BAL) fluid. A single i.v. administration of SSG (250μg/mouse) induced a rapid but transient leakage of the serum components, IgG and flbronectin, into the alveolar space. This was apparent 12 h post-administration and reached a peak on day 2. Similar kinetic changes were found for lysosomal enzyme activities and interferon γ (IFNγ) concentrations in BAL which are markers of activated alveolar macrophages (AMs) or pulmonary T cells. BAL prepared from SSG-treated mice stimulated lysosomal enzyme release from AMs in vitro. However, SSG did not provoke the chronic accumulation of serum proteins in alveoli and did not induce the release of detectable amounts of nitric oxide and the inflammatory cytokines, IL-1, IL-6 and TNFα, into BAL. However, their mRNAs were detected in lung tissue using the reverse-transcriptase polymerase chain reaction (RT-PCR) technique. Similar results were observed for multiple i.v. administration (250 μg, once a day for 10 consecutive days), and there were a little differences between single and multiple administration. In summary, systemic administration of SSG induces immune responses, including activation of AMs and lymphocytes, but does not provoke chronic inflammation in the alveolar space when administered either as single or multiple doses. This finding is very important for the clinical application of SSG in immunocompromised hosts as a biological response modifier (BRM) without toxic-side effects on lung tissue.

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The effect of carboxamidemethylated Fc fragment (CM-Fc) from human immunoglobulin G (IgG) on type II collagen (C II)-induced arthritis (CIA) in DBA/1J mice was studied. CM-Fc suppressed the development of CIA while Fc fragment did not. CM-Fc inhibited the increase in serum anti-C II IgG. CM-Fc suppressed C II-induced delayed type hypersensitivity (DTH) in DBA/1J mice. Moreover, the C II-induced proliferation of splenocytes from mice immunized with C II was suppressed by the administration of CM-Fc. Following an analysis of the lymphocyte population, a decreased percentage of B220⁺ cells and ratio of L3T4⁺/Ly-2⁺ cells was observed in lymph nodes of CM-Fc administered mice as compared with those of control group. These results suggest that CM-Fc inhibits CIA by regulating lymphocyte function.

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Using the [³H] thymidine incorporation technique, the anti-proliferative effects of benidipine, a long-acting calcium antagonist, on porcine cultured vascular smooth muscle cells (VSMCs) were determined and compared with those of other calcium antagonists. Benidipine inhibited serum-stimulated [³H] thymidine incorporation into VSMCs (IC₅₀, 0.2 μM), and this inhibitory effect was significantly more potent than that of nitrendipine, felodipine, nisoldipine, manidipine, amlodipine, nifedipine, verapamil and diltiazem. When growth-arrested cells were stimulated with platelet-derived growth factor followed by insulin, benidipine, administered with either stimulation, inhibited [³H] thymidine incorporation into VSMCs. This suggests that it acts in both the G₀/G₁ and G₁/S phases. In another series of experiments, the anti-proliferative effect in vivo was investigated using rats subjected to balloon catheterinduced endothelial denudation of the aorta. Benidipine (5mg/kg, p.o., b.i.d.) siguiflcantly reduced the incorporation of [³H] thymidine into aortic DNA 48 h after balloon injury, whereas it did not affect incorporation into bone marrow, suggesting that it inhibits arterial DNA synthesis. From our results, benidipine was shown to exert antiproliferative effects on VSMCs in vivo as well as in vitro. The drug may be useful for the treatment of vascular proliferative diseases such as restenosis following percutaneous transluminal angioplasty and atherosclerosis.

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The ability of a lipopolysaccharide from Pantoea agglomerans (LPSp) to relieve hyperalgesia was examined by observing its inhibition of the decrease in the threshold for nociceptive perception, as determined by the tail-pinch test, in streptozotocin-induced diabetic mice. Subcutaneous injection of LPSp suppressed hyperalgesia in streptozotocin-induced diabetic mice and also exerted a therapeutic effect on hyperalgesia in these animals. The present data suggest that LPSp may be effective in relieving the pain associated with diabetic neuropathy.

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We studied the effect of ginsenoside-Rb2 extracted from Panax ginseng on angiogenesis and metastasis produced by B16-BL6 melanoma cells in syngeneic mice. Intravenous administration of ginsenoside-Rb2 on day 1, 3 or 7 after tumor inoculation achieved a remarkable reduction in the number of vessels oriented toward the tumor mass, but did not cause a significant inhibition of tumor growth. The anti-angiogenic effect was dose-dependent ranging from 10 to 500 μg/mouse. In contrast, intra-tumoral or oral administration of ginsenoside-Rb2 caused a marked inhibition of both neovascularization and tumor growth. ginsenoside-Rb2 did not affect the growth of rat lung endothelial (RLE) cells, B16-BL6 melanoma cells or various types of murine normal cells in vitro. The invasion of RLE cells into the reconstituted basement membrane (Matrigel), which is considered to be an essential event in tumor neovascularization, was inhibited by ginsenoside-Rb2 in a concentration-dependent fashion, while ginsenoside-Rb2 did not inhibit the haptotactic migration of endothelial cells to fibronectin-substrate. Multiple administrations of ginsenoside-Rb2 after the intravenous inoculation of B16-BL6 melanoma cells resulted in a significant inhibition of lung metastasis as compared with the untreated control. These results suggest that the inhibition of tumor-associated angiogenesis by ginsenoside-Rb2 may partly contribute to the inhibition of lung tumor metastasis.

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The effect of the membrane fluidity of lactosylceramide (LacCer)-bearing liposomes on their liver uptake was investigated in rats. Liposomes consisting of phosphatidylcholine (PC) : cholesterol : dicetylphosphate : LacCer (7 : 2 : 1 : 1, molar ratio) were prepared with various fluidities using dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC) and egg PC. These liposomes were all equally stable in serum and were small enough to pass freely through the fenestrae and be taken up easily by liver cells. The LacCer modification of DPPC-liposomes markedly facilitated blood clearance, whereas no enhancing effect of LacCer was observed with egg PC-and DMPC-liposomes. Tissue distribution studies showed the preferential liver uptake of LacCer-bearing DPPC-liposomes, which was largely compatible with the rapid clearance induced by the LacCer modification. In addition, electron spin resonance (ESR) spectroscopic analysis revealed that the LacCer modification of DPPC-liposomes significantly enhanced the order parameter S, indicating that LacCer-bearing DPPC-liposomes were the most rigid of those used in this study. These observations suggest that the membrane fluidity of liposomes in vivo is a crucial factor for their preferential liver uptake.

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We investigated the profiles of sudden death and hemoconcentration induced by endothelin-1 (ET-1) and big endothelin-1 (big ET-1) in mice using various compounds as pharmacological tools. In ET-1-induced sudden death (5 nmol/kg, i.v.), pretreatment with the Ca²⁺-channel blockers, diltiazem, nifedipine or verapamil at a dose of 2 mg/kg, i.v. significantly inhibited the mortality and prolonged the latency to death. These Ca²⁺-channel blockers, however, failed to inhibit the rise in hematocrit (Ht), namely hemoconcentration, induced by ET-1 (2.5 nmol/kg). A beta-adrenoceptor agonist, isoproterenol (1 mg/kg) tended to prolong the latency, whereas, a beta-adrenoceptor blocker, propranolol (2 mg/kg), and an alpha-and beta-adrenoceptor blocker, labetalol (5 mg/kg), aggravated the sudden death. Esculetin (10 mg/kg) and fenbufen (10 mg/kg), which are enzyme inhibitors in the arachidonate cascade, prevented only the hemoconcentration. Anti-arrythmic drugs, lidocaine (1 mg/kg) and disopyramide (20 mg/kg) did not improve any parameters. Big ET-1 also caused sudden death (20 and 25 nmol/kg, i.v.) and hemoconcentration (10 nmol/kg, i.v.). Of several proteinase inhibitors, only a metalloproteinase inhibitor, phosphoramidon (2 mg/kg i.v.), prevented the sudden death and the hemoconcentration induced by big ET-1 but not by ET-1. Ca²⁺-channel blockers exerted their protective effects only when a lower dose of big ET-1 was employed. These results indicate that the sudden death caused by both peptides is mainly due to myocardial ischemia and respiratory disorder, and that hemoconcentration caused by them is due not to their vasoconstrictor action but to their effects on the vascular permeability via secondary endogenous factors.

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Aloe-emodin-9-anthrone (AE-anthrone), produced from barbaloin in the rat large intestine, caused not only an increase in the intestinal water content but also stimulated mucus secretion. This might play an important role in the occurrence of diarrhea. It was demonstrated that the amount of AE-anthrone produced in the rat large intestine (maximal amount : 568 μg/rat at 4 h after injection) was enough to cause both of these effects, which were observed following intracecal administration of barbaloin (31.1 mg/kg). These results together with our previous data, which showed a relationship between increase in the intestinal water content and the stimulation of peristalsis, confirm that AE-anthrone is the principal agent responsible for the cathartic effect of barbaloin. We also propose that the increase in water content is a more important factor than stimulation of peristalsis in the induction of diarrhea by barbaloin.

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The DNA strand-breaking activities of 9 quinolones, widely used as antimicrobial agents and suspected to induce photo-dermatological disorders in humans, were examined under irradiation using standard domestic fluorescent lamps. Two quinolones, tosufloxacin and enoxacin, converted the supercoiled covalently closed circular plasmid DNA to the open circular form under visible light irradiation. The maximum of photodynamic single strand-breaking activity for tosufloxacin was found to be 12% at 50 μM and that of enoxacin was 33% at a concentration of 2mM. The DNA strand-breaking activities of photoirradiated tosufloxacin and enoxacin were markedly inhibited by NaN₃ but only partially by D-mannitol, superoxide dismutase (SOD) and catalase did not inhibit these activities. These results suggest that some quinolone antimicrobial agents can be activated by visible light and induce phototoxic DNA damage in various organisms. We should carefully investigate such phototoxic activities during the development of new quinolones to avoid producing phototoxic disorders in man.

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Primary cultures of dog hepatocytes sensitively responded to various inducers of cytochrome P-450 ; phenobarbital (PB) significantly elevated the activity of 7-ethoxycoumarin O-deethylase (ECOD) and progesterone 6β-hydroxylase (6β-OHP). β-Naphthoflavone (β-NF) and rifampicin (Rif) elevated the levels of 7-ethoxyresorufin O-deethylase (EROD) and 6β-OHP, respectively. When primary cultures of dog hepatocytes were incubated with bromobenzene, cellular reduced glutathione (GSH) levels decreased time-and dose-dependently. Pretreatment of the cultures with PB enhanced GSH depletion by bromobenzene, while β-NF and Rif had little effect, suggesting that the 2B type cytochrome P-450 is responsible for the primary oxidation of bromobenzene to GSH-reactive metabolite (s). Bromobenzene-dependent GSH depletion was completely prevented by 10 μM SKF-525A both in the control and PB-treated primary cultures. Treatment of dog primary cultures with PB analogues also elevated the level of drug-metabolizing activity leading the cells to be more susceptible to GSH depletion by bromobenzene.

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We measured the plasma concentrations of 7-ethyl-10-[4-(1-piperidino)-1-piperidine] carbonyloxycamptothecin (CPT-11) and the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), after treatment with CPT-11 to rats pretreated with bis-p-nitrophenylphosphate (BNPP) which is a specific inhibitor of carboxylesterase, and non-pretreated rats. The plasma level of SN-38 was decreased in the BNPP-pretreated group compared with these of non-pretreated group, indicating that the esterase involved in CPT-11 metabolism is a carboxylesterase. We also characterized the molecular species of carboxylesterase involved in CPT-11 metabolism using enzyme preparations purified from liver microsomes. Thirteen carboxylesterase isozyme activities towards CPT-11 were compared and guinea pig GLP1 was found to have the highest activity, while human HU1 isozyme had relatively lower activity than those of animal species. In studies on the kinetic parameters of the hydrolysis of CPT-11 by the purified carboxylesterase isozymes the highest V_max value of the isozymes was found in human HU1 and the smallest was seen in rat RL1. The V_max/K_m for RL1 showed the largest value of 21.7 nmol/mg protein/mM.

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The Et₂O-soluble fraction of the MeOH extract of the leaves of Aucuba japonica THUNB. showed an anti-inflammatory effect when topically applied to rats. E-Phytol, phytone, bis (2-ethylhexyl) phthalate and friedelin were isolated as active anti-inflammatory components inhibiting carrageenin-induced paw edema. The most effective, E-phytol, inhibited histamine-induced paw edema more potently than the compound 48/80-induced edema, suggesting that the mechanism of action is due to inhibition of the H₁ receptor.

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To search for possible anti-tumor-promoters, we carried out a primary screening of 21 cucurbitane triterpenoids using an in vitro assay system. Of these triterpenoids, scandenoside R6 (6), 23, 24-dihydrocucurbitacin F (14), 25-acetyl-23, 24-dihydrocucurbitacin F (15), 2-O-β-D-glucopyranosyl-23, 24-dihydrocucurbitacin F (17) and cucurbitacin F (18) exhibited significant inhibitory effects on Epstein-Barr virus (EBV) activation induced by the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Further, compounds 14 and 17 exhibited remarkable anti-tumor-promotion effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.

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The pharmacokinetics of RK-28 [1-(4-hydroxy-2-butenoxy) methyl-2-nitroimidazole], a new hypoxic cell radiosensitizer, was studied following intravenous, oral, and rectal administration to rabbits. After an oral administration of RK-28 solution, the plasma concentration of RK-28 was considerably lower than that after intravenous administration through all time periods, and the absolute bioavailability was a mere 4.2%. It was presumed that a specific acid-catalized decomposition of RK-28 progressed in the stomach, and also, the absorbed RK-28 suffered first-pass effects in the liver. In contrast, the absolute bioavailability following the rectal administration of a solidified RK-28 suppository preparation was significantly increased in comparison with that obtained by other administration routes. Also, RK-28 emulsion suppositories were prepared by emulsifying various amounts of the drug with 1-hexadecanol and hydrogenated castor oil (HCO 60) at 80°C, and these were administered into the rabbit rectum. The resulting absolute bioavailability was 91% for the RK-28 emulsion suppository and 86.9% for an RK-28 emulsion suppository containing small amounts of Eudispert hv. These values were better than those in rats. The rectal administration of the RK-28 emulsion suppository containing small amounts of Eudispert hv showed a preferable plasma concentration-time pattern that reflects on radiation therapy.

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The blood-brain barrier (BBB) permeability of mepyramine was measured by the carotid injection technique to elucidate the transport mechanism of an H₁-antagonist in the central nervous system. Mepyramine was found to enter the brain by saturable and carrier-mediated transport. The in vivo kinetic parameters were estimated as follows : the maximum uptake rate (J_max) was 7.12±1.37μmol/min/g of brain, the Michaelis constant (K_d) was 4.40±2.00mM, and the nonsaturable first order rate (K_d) was 0.28±0.02ml/min/g of brain. The mepyramine transport was not inhibited either by nutrients or by choline, hemicholinium-3, though it was inhibited by the classical H₁-antagonists such as diphenhydramine, diphenylpyraline, and also by propranolol. The above inhibitory effects suggest that a transport system different from the amine transport system exists for the BBB transport of mepyramine, and that this transporter is common not only for H₁-antagonists but also for basic drugs.

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The effects of liposomes, bearing galactosyl ligands on their surface, on their clearance from circulation and on tissue distribution were studied in rats. The ligands were obtained through coupling 2, 3, 4, 6-tetraacetyl-{2-[2-(2-aminoethoxy) ethoxy] ethyl}-β-D-galactoside p-toluenesulfonate with hydrophobic anchors, followed by deprotection. We introduced mid-chain or long, branched or non-branched aliphatic chains, and cholesterol as anchors. Among various anchors tested, that with a dihexadecyl branch caused the greatest accumulation of liposomes in the liver. On the other hand, liposome bearing these ligands were aggregated by Ricinus communis agglutin. This showed that these ligands were similarly incorporated to liposomes. These results show the importance of the balance between lipophilicity and the structure of ligands.

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The correlation of chemical structures with the volume of distribution of 373 miscellaneous medicinals was studied to construct an expert system for predicting the pharmacokinetic properties of organic chemicals. The compounds studied were classified into three groups ; non-aromatics, aromatics, and heteroaromatics. Their total body volumes of distribution observed in human adults were allotted into three ratings, and the relationships with chemical structure were analyzed using the fuzzy adaptive least-squares method recently developed in our laboratory. Quantitative relationship models formulated for the three structure groups gave significant information about factors influencing the volume of distribution, and were statistically reliable both in recognition and leave-one-out prediction despite the diversity and complexity of the structures of the compounds investigated.

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The pharmacokinetics of tolbutamide (TB) following intravenous and oral administration was compared between normal rats and rats with experimentally-induced obstructive jaundice (OJ). The plasma concentration-time curve of i.v. administered TB was lower in rats with OJ, in comparison with normal rats. The result of the pharmacokinetic analysis showed no change in the elimination rate constant, but a significant increase in the volume of distribution in the OJ state. The increase in the volume of distribution of TB could be explained by the decreased protein binding in plasma. In the case of oral administration, the elevation of the plasma concentration was slow and the plasma concentration profile was remarkably low in rats with OJ. The rate of TB disappearance from the small-intestinal lumen was delayed in the OJ state, and its marked accumulation in the tissues of the small intestine and the liver was observed. This retarded uptake by the small-intestinal mucosa and subsequent pre-systemic accumulation might, at least in part, be the reason for the slow appearance in the systemic plasma in the OJ state.

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The characteristics of pirarubicin transport have been investigated in polymorphonuclear leukocytes isolated from rats. The uptake of pirarubicin by leukocytes was time-, temperature- and concentration-dependent with the maximum velocity (V_max) of 4.84 nmol/5×10⁶ cells/min and the Michaelis constant (K_m) of 13.4μM. The uptake depended on the extracellular pH, indicating that the molecular form of pirarubicin is more permeable than its ionic form and/or that the transporter of pirarubicin has optimal pH range. When the intracellular space was changed by increasing the medium osmolarity with sucrose, the uptake was altered by osmolarity changes and it appeared that about 27% of the drug was bound to the membrane surface. The initial uptake was inhibited by the metabolic inhibitors rotenone, 2, 4-dinitrophenol and sodium cyanide. Pirarubicin accumulation in the intracellular glucose-depleted cells diminished significantly compared with that in normal cells. The efflux of pirarubicin from leukocytes was also temperature-dependent and inhibited by a metabolic inhibitor. These results indicate that a specific mechanism is concerned in the transport of pirarubicin in polymorphonuclear leukocytes.

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We have reported a stable superoxide dismutase (SOD)-like copper complex, Cu-ATSM, which shows high membrane permeability and distribution to the brain or heart. In this study, we evaluated the protective effects of Cu-ATSM on superoxide-mediated tissue damage caused by ischemia-reperfusion using an isolated perfused rat heart model. Lipid peroxidation levels in the Cu-ATSM treated group were lower than those in the non-treated group. Furthermore, released creatine phosphokinase into the perfusate, a marker of tissue damage, was reduced by Cu-ATSM treatment. These results indicated the possibility of Cu-ATSM being an effective SOD-like drug for the treatment of superoxide-mediated damage, such as ischemia-reperfusion injury.

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The effect of long chain fatty acid (FA) and its analogs on the accumulation of thiamine disulfide (TDS) in rat skin using propylene glycol as a vehicle was studied in vitro. Lauric acid (12 : 0) increased the accumulation of TDS in skin, while myristic acid and stearic acid caused a slight decrease in accumulation. Lauryl alcohol and lauric acid methyl ester did not change the accumulation of TDS in the skin. The ratio of the amount of TDS accumulated in skin to the solubility of TDS in the vehicle increased dependent on the concentration of 12 : 0 added in the vehicle. It was suggested that the increase in the permeability coefficient of TDS by 12 : 0 results from the enhanced transport of TDS from the vehicle to skin.

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To adapt the monitoring of cyclosporin A (CyA) to clinical practice, we have developed a model to predict the CyA erythrocyte-to-plasma distribution ratio (CyA-EP), and evaluated its utility in clinical practice. We monitored CyA trough concentrations in whole blood and performed a series of biochemical tests during disease states in patients undergoing immunosuppressive therapy with CyA after transplantation. An estimate of CyA-EP (EP_pr) was thus given by the following equation : EP_pr=6.0831-0.2944×(TG+CHO)-0.0037×(CyA_blood)-0.0553×(HCT)+0.0463×(BW)+0.4447×(CRE)-0.0366×(AGE). In this predictive model, EP_pr is given as a function of the plasma lipid levels (TG+CHO, mM), the CyA concentration in whole blood (CyA_blood, ng/ml), and the hematocrit (HCT, %), as well as the patient's body weight (BW, kg), serum creatinine (CRE, mg/dl) and age (AGE, years). The parameters TG+CHO, CyA_blood, HCT, and AGE were negatively correlated with the CyA-EP, whereas BW and CRE exhibited a positive correlation. The predictive performance of this model was satisfactory for clinical use and changes in CyA-EP in transplant patients were obtained from monitoring CyA in whole blood and routine biochemical tests, without directly measuring CyA-EP. Since CyA-EP is an useful indicator for predicting a shift of CyA into tissues and its systemic clearance in plasma, our model to predict the CyA-EP will help the physician select a CyA regimen during immunosuppressive therapy in a variety of disease states after trans plantation.

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The inhibitory effect of clindamycin on β-lactamase biosynthesis was investigated in β-lactam resistant bacteria with inducible and/or constitutive production of the enzyme (s). Clinical isolates of Enterobacter cloacae (E. cloacae) and Pseudomonas aeruginosa (P. aeruginosa) were examined. Three strains of each, against which sulbactam at 8μg/ml reduced the minimum inhibitory concentration (MIC) of cefoperazone (CPZ) at least 4 fold, were studied in greater detail. Although clindamycin had no appreciable effect on bacteria growth at concentrations up to 50μg/ml, it suppressed inducible β-lactamase biosynthesis almost completely at a concentration of 20μg/ml. There was also a significant reduction of constitutive enzyme biosynthesis by clindamycin at 50 μg/ml. Thus, it was estimated that a high concentration of clindamycin was required to suppress β-lactamase biosynthesis in β-lactamase producing bacteria of the constitutive type.

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We developed a rapid and simple quantitative method for measuring bile alcohol glucuronides in serum involving high-performance liquid chromatography without group separation and hydrolysis. The assay of 5β-cholestane-3α, 7α, 12α, 25-tetrol 3-glucuronide, the major bile alcohol component in serum from cerebrotendinous xanthomatosis patients, with the present method was useful for the diagnosis of cerebrotendinous xanthomatosis.

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Lipoprotein lipase (LPL), which is responsible for the hydrolysis of lipoprotein triacylglyceride, has been examined in Ehrlich ascites tumor cells. Low molecular weight dextran sulfate (DXS, M.W. 3.2kDa) stimulates the release of the enzyme activity from the tumor cells into the incubation medium in a time-dependent manner. The stimulatory effect of DXS was markedly decreased by incubation with protein tyrosine kinase (TK) inhibitors, such as ST 638, biochanin A and amiloride. The activity of the partially purified TK preparation from the tumor cells was found to be increased following incubation with DXS in a manner which was both time-and dose-dependent. These results suggest that the stimulatory release of LPL activity by DXS is associated with the activation of TK in the tumor cells.

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Among various fluorogenic substrates for trypsin-like proteinases, tert-butyloxycarbonyl-L-valyl-L-prolyl-L-arginine 4-methylcoumarin-7-amide was strongly hydrolyzed by a crude extract of rabbit platelets. The proteinase was partially purified (92-fold) from rabbit platelets by successive chromatographic separations on phenyl-Sepharose CL-4B, L-arginine-Sepharose 4B and Sephadex G-200 columns. Its molecular mass was found to be greater than 200 kDa by analytical gel filtration and its optimal pH was approximately 9. The proteinase activity was strongly inhibited by diisopropylfluorophosphate, phenylmethanesulfonyl fluoride, tosyl-L-lysine chrolomethyl ketone, leupeptin, p-nitrophenyl-p-guanidinobenzoate, and also by the 2, 4-dimethylphenyl ester of amidinopiperidine-4-propionic acid and the 4-tert-butylphenyl ester of trans-4-guanidinomethylcyclohexanecarboxylic acid which strongly inhibit platelet aggregation induced by various stimuli.

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The effects of 2, 5-dimethylpyrazine (DMP) on plasma testosterone and levels of polyamines and acid phosphatase in the prostate of rat were studied. A high dose of DMP administered once daily for two weeks to juvenile male rats significantly decreased plasma testosterone and levels of polyamines and acid phosphatase in the prostate. These effects were not obtained by administration to mature male rats, and the inhibition was thus recognized to occur during the growth period. These findings suggest that a high dose of DMP inhibits the biosynthesis of polyamines and acid phosphatase in the prostate by decreasing the circulating testosterone level.

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We investigated the properties of superoxide anion (O^-₂)-induced histamine release from rat peritoneal mast cells using superoxide anion radicals obtained from potassium superoxide (KO₂). KO₂ elicited a rapid histamine release in a dose-dependent fashion, without lactate dehydrogenase (LDH) release. The KO₂-induced release was temperature-and energy-dependent. KO₂ rapidly increased the intracellular free Ca²⁺ concentration, accompanied by a marked increase of Ca²⁺-uptake. These findings indicate that the increase in intracellular Ca²⁺ concentration is involved in the initiation of KO₂-induced histamine release, and KO₂ could be used as an agent of O^-₂-induced biological reactions.

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Neuron-glia interaction is considered to be important for the establishment and maintenance of neuronal shape and polarity during development. Glial cells release a number of trophic factors, many of which have not been fully characterized. In the present study, the trophic influences of conditioned media (CMs) of four glioma cell lines (T98G, Hs683, AC, C6) were tested on cultured hippocampal neurons dissociated from fetal rat brain. The survival of hippocampal neurons was enhanced by C6-CM, but inhibited by T98G-CM. The elongation of neurites was remarkably potentiated by C6-CM and T98G-CM. The other two CMs had little effect on neuronal survival and neurite elongation. These results suggest that different glioma cell lines produce and release different kinds and/or amounts of neurotrophic factors. Our experimental method may provide a suitable assay system for the identification of unknown trophic factors.

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Sulfated schizophyllans and sulfated oligosaccharides were prepared and their inhibitory effects on coagulation and fibrinolysis were examined. Some sulfated schizophyllans, which had a sulfur content of more than 10%, prolonged prothrombin time (PT) and thrombin time (TT). Interestingly, some sulfated schizophyllans and related oligosaccharides were able to inhibit plasmin toward fibrinolysis and amidolysis.

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The effect of various structural/functional tubulin disruptors (including colchicine-type disruptors, vinblastine, rhizoxin, maytansine, peptide-type disruptors, and taxol) on the chemically induced differentiation of human leukemia cell lines (HL-60 and K562) was examined. As differentiation-inducing agents, 12-O-tetradecanoylphorbol-13-acetate (TPA) was used for the differentiation of both HL-60 and K562 to monocyte/macrophages, retinoids were used for the differentiation of HL-60 to mature granulocytes, and hemin was used for the erythroid differentiation of K562. All the tubulin disruptors investigated increased the chemically-induced differentiation of HL-60 and K562 cell lines to the cognate mature cell types, regardless of the nature of the differentiation.

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Bioadhesive tablets for intraoral drug delivery were prepared by directly compressing the drug with a mixture of chitosan and sodium alginate in weight ratios of 4 : 1, 1 : 1 and 1 : 4, and the adhesion and release characteristics of the prepared systems were evaluated in vitro and in vivo. Ketoprofen was used as a model drug. The magnitudes of the adhesion force of chitosan/alginate tablets were observed to be comparable to that of Aftach^TM, which is a typical commercial preparation of an oral mucosal adhesive tablet. Increasing the chitosan content in the tablets resulted in a decrease in the release rate of ketoprofen. When the tablets were administered to the sublingual site of rabbits, ketoprofen from the tablets with chitosan/alginate was rapidly absorbed without an initial sharp peak. Furthermore, the plasma concentration curves for the tablet with a 1 : 4 chitosan/alginate ratio showed a sustained release 3 h after administration. The data presented suggest that tablets prepared from chitosan and alginate are potential candidates for intraoral drug delivery.

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A conjugate of bleomycin (BLM) and the mannan of bakers' yeast (Saccharomyces cerevisiae wild type strain) (WNM) was synthesized. The assay of its antimetastatic effect on Lewis lung carcinoma (3LL) implanted in C57BL/6 mice showed that this conjugate exhibited a higher antimetastatic effect and longer life-span elongation than those of free bleomycin and mannan with corresponding doses. This conjugate was also found to kill the 3LL cells in vitro. ¹⁴C-Labeled mannan-bleomycin conjugate was much more bound that ¹⁴C-labeled dextran-bleomycin conjugate to the 3LL. It was concluded that the anti-cancer mechanism of this conjugate, WNM-BLM possessed a specific binding effect to the tumor cells and exhibited a cytocidal effect on the 3LL target cells.

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The effects of sodium caprate (C10) and disodium ethylenediaminetetraacetate (EDTA) on the epithelial permeability of polyethylene glycol 4000 (PEG4000) were examined in Caco-2 cell monolayers on the polycarbonate membrane (filter) of a Transwell cluster dish. Under serosal Ca²⁺-free conditions irrespective of the presence or absence of mucosal Ca²⁺, the permeability of PEG4000 was remarkably enhanced, even in the absence of enhancer. For control experimental conditions with a permeation enhancer in Caco-2 cells, Ca²⁺ was added to both the mucosal and serosal sides. Under the control conditions, EDTA increased the permeability of PEG4000 to 14 times the control and C10 by 3.5 times. Mucosal Ca²⁺-free conditions further increased the EDTA-enhancing effect, to 29 times the control value. In contrast to the EDTA effect, however, the C10 effect was unchanged in the absence of mucosal Ca²⁺. These results suggest that the EDTA effect is induced by chelation with Ca²⁺ but that C10 has some other action in addition to chelation.

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The performance of milk fat-globule membrane (MFGM) emulsion as an oral dosage form was evaluated in rats using [¹⁴C] α-linolenic acid as a lipophilic model solute. For emulsions prepared by homogenization alone, the area under the plasma concentration versus time curve (AUC) after oral administration tended to be larger for MFGM emulsion, 35.0±2.5μg eq·h/ml (mean±S.E., n=3), than for Tween 80 emulsion, 28.5±0.6μg eq·h/ml at p<0.1, though the peak plasma concentration (C_max) and the time required to reach C_max (T_max) were not significantly different. The absorption in the intestinal loop was not significantly different between the two emulsions, either. Thus, MFGM emulsion, compared with Tween 80 emulsion, did not show an obvious advantage or enhancement in the oral and intestinal absorption of α-linolenic acid, except for a slight increase in AUC. However, MFGM could be a good alternative to a synthetic emulsifier for oral use, considering that it is of natural origin and may be safer. In addition, it was shown that the AUC, as well as the absorption in the intestinal loop, was decreased for a MFGM emulsion in which the droplet size was reduced by sonication, presumably because of the observed decrease in α-linolenic acid concentration in the water phase, which was assumed to be the result of an increased distribution of α-linolenic acid, due to its amphipathic nature, to the increased oil-water interface.

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The enhancement of nitric oxide (NO) production in glycyrrhizin (GL)-induced macrophages (Mφ) in response to lipopolysaccharide (LPS) was investigated. NO production in GL-induced macrophage culture supernatants was stimulated in response to LPS (10 μg/ml) for 24-or 48-h cultures, and these levels were compared three times with the levels in saline-induced peritoneal exudate cell cultures. Furthermore, Mφ induced with proteose peptone (PP) containing GL could generate greater NO production than Mφ induced with PP alone. However, no stimulation of NO production was observed by addition of GL in the cultures of Mφ induced with thioglycollate or Bacillus Calmette Guerin. Moreover, GL-induced Mφ showed cytostasis against such tumor target cells as L 1210 and P 388 lymphoma cell lines. These observations indicate that GL can activate the Mφ in vivo system and stimulate NO production in response to LPS.

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