Five kinds of rabbit antisera produced against five kinds of crude drugs, Pinella Tuber, Glycyrrhizae Radix (GR), Trichosanthes Root, Hoelen, and Panax Ginseng, and five kinds of extracts of the same crude drugs were prepared. Specificity of each antiserum was demonstrated by two immunological methods for analyses, the selected antibody enzyme immunoassay (SAEIA) and western blotting, and five crude drug extracts as specimens. Each crude drug extract contained characteristic antigen specific to the corresponding antiserum. Characteristic antigens were suggested as protein components. Characteristic antigen of the crude drug GR was separated from GR extract by three chromatographic procedures and a protein component was separated. The separated protein was successfully applied to develop a SAEIA method applicable for specific assays of both the separated protein and GR extract.

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Sucrase-isomaltase complex was purified from human intestinal mucosa. Immunostaining shows that sucrase-isomaltase is confined to the area of the striated cell borders of human small intestinal absorptive cells of the villus. Inhibition of sucrase and isomaltase activity by ethanolamine derivatives was investigated. Tris inhibits both types of enzyme activity and is the strongest inhibitor of the ethanolamine derivatives investigated. Bis-Tris inhibited sucrase more than isomaltase. On the other hand, mono-, di- and tri-ethanolamine were weak inhibitors of sucrase but not isomaltase.

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The culture of hepatocytes isolated from C3H mouse liver results in the spontaneous development of colonies of liver epithelial cells that possess some features of the hepatocytes. These liver epithelial cells frequently have a loss of chromosome 4, and become neoplasms which are hepatocellular carcinomas by transfection with the activated c-Ha-ras gene. The suppression of malignant phenotypes by mouse chromosome 4 has already been shown by fusion between normal and malignant mouse cells. We established a total of six liver epithelial cell lines from C3H mice in order to investigate the presence of a tumor suppressor gene(s) on chromosome 4 in mouse hepatocarcinogenesis, and performed an allelotype analysis in seven microsatellites on chromosome 4 by the comparative multiplex PCR method. The result of analysis revealed that three of the six liver epithelial cells had allelic imbalances in four microsatellite loci, especially, two liver epithelial cell lines showed homozygous deletion in the D4MIT77 locus. Then, we investigated the status of the mouse homolog of p16/CDKN2 gene (mouse p16) on chromosome 4 by the comparative multiplex PCR method, and detected the homozygous deletion in two liver epithelial cell lines. Our result thus supports the theory that alterations of tumor suppressor gene(s) located on chromosome 4 may play a role in mouse hepatocarcinogenesis. Mouse p16, which is an inhibitor of cyclin dependent kinase 4, may suppress the cancer development in mouse hepatocarcinogenesis, or suppress liver cell immortalization.

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Agelasphin-11 (AGL-11), a novel α-galactosylceramide isolated from an extract of a marine sponge, Agelas mauritianus, markedly prolonged the life span of mice intraperitoneally inoculated with B16 cells. Since AGL-11 did not show any direct cytotoxic activity against B16 cells, this compound is considered to be a biological response modifier (BRM). We focused on the enhancing effect of this compound on in vivo natural killer (NK) cell activity because several BRMs have already been determined to enhance the in vivo natural killer (NK) cell activity. When we evaluated the enhancing activity of AGL-11 using normal mice, AGL-11 enhanced in vivo NK cell activity more potently than Poly I : C, which is a positive control. In addition, we examined the effect of this compound on the NK cell activity of tumor-bearing mice, and found that AGL-11 recovers the reduced NK cell activity in a tumor-bearing condition to a higher level than that of normal mice. These results suggest that AGL-11 shows antitumor activity by the activation of antitumor effector cells such as NK cells.

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The effect of γ-ray irradiation and camptothecin (CAM) on human colorectal adenocarcinoma (LS 174T) growth was examined. On inhibition of DNA synthesis of LS 174T cells, almost all combined treatments presented a synergistic effect, excepting that the additive effects were shown at the lowest or the highest doses of CAM prior to the γ-ray (¹³⁷Cs) irradiation. The in vivo antitumor activity of ¹³¹I-NCC-CO-411 monoclonal antibody against carcinoembryonic antigen in combination with CAM was assessed by a tumor growth rate in LS 174T tumor-bearing nude mice. The tumor growth of the combined group was supra-additively suppressed in comparison with that of the individual group. The results suggest that the efficacy of radioimmunotherapy may be enhanced by combination treatment with CAM. A clinical protocol involving a combination treatment with CAM is underway.

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The weights of prostate and seminal vesicle were decreased by treatment with 3, 6-dimethylpyrazine-2-thiol (3, 6-DMP-SH) but the weights of testes and the plasma testosterone levels were not changed. The recovery of the weight of prostate and seminal vesicles of testosterone-treated orchidectomized rats were inhibited by treatment with 3, 6-DMP-SH. Direct injection of 3, 6-DMP-SH into accessory reproductive organs also induced the decrease in the weight of relevant organs. Administration of oxendrone or chlormazinone along with 3, 6-DMP-SH decreased the weights of accessory reproductive organs more than administration of only one substance. In vitro uptake of testosterone into prostate and seminal vesicle were blocked by the presence of 3, 6-DMP-SH. These results suggest that 3, 6-DMP-SH directly inhibited testosterone uptake into the accessory reproductive organs.

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Hypoglycemic effect of water extract of the root of Pandanus odorus RIDL. (Thai name : Toei-hom, Pandanaceae) was examined in normal and streptozotocin-diabetic rats. In the hypoglycemic test without glucose load, an administration of the extract at doses of 0.125-0.5g/kg p.o. did not affect significantly the plasma glucose level in normal rats, whereas the extract significantly lowered the plasma glucose level at a dose of 0.5g/kg p.o. in diabetic rats. In oral glucose tolerance test, an administration of the extract at a dose of 0.5g/kg p.o. significantly lowered the plasma glucose level in normal rats. The extract at doses of 0.5 and 1.0g/kg p.o. also significantly lowered the plasma glucose level in diabetic rats. A reference drug, glibenclamide at a dose of 5mg/kg p.o. showed a significant hypoglycemic effect in both normal and diabetic rats. Repeated administration of the extract at doses of 0.25 and 0.5g/kg p.o. for 7d produced a significant hypoglycemic effect in diabetic rats. Glibenclamide (5mg/kg p.o.) also caused a significant hypoglycemia in the diabetic rats. LD₅₀ (95% confidence limit) after intraperitoneal injection was 1.87 (1.26-2.76) g/kg in male and female rats and 1.62 (1.18-2.24) g/kg in male and female mice, respectively. The LD₅₀ after oral administration was over 8g/kg in both sexes of rat and mice.

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The effects of oral administration of cryomilled powder of the soft-shelled turtle (SST powder) on solid and ascites tumor-bearing C3H/He mice were studied. The SST powder was administered orally at a dose of 100 or 500mg/kg/d for two or four weeks, and then MH134 hepatoma cells were inoculated; oral administration was continued after inoculation. Treatment with the SST powder attenuated the increase in diameter and weight of the tumor and prolonged the survival of mice with solid tumors. However, the same treatment did not show any significant effect in mice with ascites tumor, indicating that the SST powder has no direct killing action on tumor cells. These results suggest that oral administration of the SST powder decreases the growth of solid tumors possibly by activating the host immune system.

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The effects of intracerebroventricular (i.c.v.) injection of the μ-selective opioid receptor agonist [D-Ala², N-MePhe⁴, Gly-ol] enkephalin (DAMGO) and the κ-selective opioid receptor agonist dynorphin A-(1-13) on the development of methamphetamine-induced behavioral sensitization in the mouse were determined using multidimensional behavioral analyses based upon a capacitance system. Methamphetamine (2mg/kg, s.c.) was administered to mice on 6 occasions at 3-or 4-d intervals. The methamphetamine-induced increase in linear locomotion and circling was markedly augmented by repeated administrations (3 or more times) of the drug, showing behavioral sensitization. Although repeated administrations of DAMGO (0.003 and 0.01μg, i.c.v.) or dynorphin A-(1-13) (3 and 12.5μg, i.c.v.) alone did not produce any significant effects on behavior, repeated administrations of DAMGO (0.003 and 0.01μg, i.c.v.) and dynorphin A-(1-13) (3 and 12.5μg, i.c.v.) attenuated the behavioral sensitization induced by methamphetamine (2mg/kg, s.c.). The attenuating effects of DAMGO (0.003 and 0.01μg, i.c.v.) and dynorphin A-(1-13) (3 and 12.5μg, i.c.v.) were fully reversed by withdrawal of these drugs for 3 weeks. Additionally, a single administration of DAMGO (0.003 and 0.01μg, i.c.v.) or dynorphin A-(1-13) (3 and 12.5μg, i.c.v.) alone did not produce any significant effects on behavior ; DAMGO (0.003 and 0.01μg, i.c.v.) and dynorphin A-(1-13) (3 and 12.5μg, i.c.v.) only attenuated the behavioral sensitization which had previously been developed by methamphetamine (2 mg/kg, s.c.). These results suggest that opioid receptor agonists selective for μ and κ receptors play an inhibitory role in the development of methamphetamine-induced behavioral sensitization.

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The effects of some amino acids such as L-glutamine (Gln), L-arginine (Arg) and L-methionine (Met) on rectal irritation caused by sodium caprate were studied in rats. Rectal irritation was assessed by the balloon method in fasting conscious rats. This method is based on measuring rectal contractions due to possible irritation caused by the presence of drugs and/or adjuvants in the rectum. Strong contractions were observed after rectal administration of an aqueous solution of 100 mM sodium caprate. However, the presence of Gln, Arg or Met (100 mM) in sodium caprate (100 mM) solution resulted in a significant decrease in the intensity of the rectal contraction caused by sodium caprate. The rectal absorption-promoting effect of sodium caprate on 6-carboxyfluorescein (6-CF) was examined following administration with amino acids in rats. The absorption of 6-CF was not influenced by the concurrent administration of amino acids. In addition, the rectal tissue interaction of sodium caprate, with or without Gln, was examined. The concentration of sodium caprate in rectal tissue was reduced by the presence of Gln.

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The effects of Pluchea indica LESS root extract (PI-E) on locomotor activity and pentobarbital-induced sleep, social isolation-induced aggressive behavior, motor coordination in the rotarod test, pentylenetetrazole-induced convulsion and nociceptive responses in the tail-pinch test were examined in mice. Socially isolated mice showed higher locomotor activity and shorter duration of pentobarbital sleep than group-housed mice. PI-E (50-100mg/kg, p.o.) significantly decreased locomotor activity and prolonged pentobarbital sleep in a dose-dependent manner in isolated mice but not in group-housed mice. At a large dose (400mg/kg, p.o.), PI-E not only decreased locomotor activity but also prolonged pentobarbital sleep in group-housed mice. The reference drug diazepam, at 0.5mg/kg, also suppressed the locomotor activity in isolated mice but not in group-housed mice. Moreover, diazepam, at 0.1 and 0.5mg/kg, significantly prolonged pentobarbital sleep in both isolated mice and group-housed mice. The effects of PI-E and diazepam on pentobarbital sleep in isolated mice were significantly attenuated by flumazenil (1mg/kg, i.v.). PI-E (50-100 mg/kg), as well as diazepam (0.5-5mg/kg, p.o.), dose-dependently suppressed social isolation-induced aggressive behavior, but it had no effect on pentylenetetrazole-induced convulsion, motor coordination in the rotarod test, or nociceptive response in the tail pinch test in group-housed mice. These results suggest that PI-E attenuates pathophysiological changes caused by social isolation stress in mice, and that the GABA ergic system is partly involved in the action of PI-E on a social isolation-induced decrease in pentobarbital sleep.

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We investigated the influence of the streptozocin (STZ)-diabetic state on the dose-response curves for salivary flow and protein content in saliva stimulated by pilocarpine and noradrenaline in mice. The diabetic state increased the relative weights of parotid and sublingual salivary glands but not the weight of submandibular glands, despite body weight loss. In the dose-response curves, (1) the maximal responses to stimulation with pilocarpine and noradrenaline on salivary flow, and with noradrenaline on protein content in saliva, were depressed by the diabetic state, and (2) the value of the 50% effective dose for salivary flow with pilocarpine, but not with noradrenaline, was decreasingly altered by diabetic mice. These results suggest that xerostomia, one of the complications of non-insulin-dependent diabetes mellitus, is caused in part by muscarinic and adrenergic receptor dysfunction in the salivary glands.

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The effect of an herbal prescription, S-113m, consisting of biota, ginseng and schizandra, on learning and memory performance was studied in the senescence accelerated mouse (SAM). A solid diet containing 1% (w/w) S-113m was given to SAM from 1 month of age. A behavioral experiment, started 4 or 9 months later, revealed prominent learning impairment in SAMP8, a senescence accelerated-prone mouse. Chronic ingestion of S-113m improved the memory retention disorder of SAMP8 in a passive avoidance test and increased the conditioned avoidance rate in a lever-press test at the age of 10 months. The preparation also facilitated the memory retention deficit in the passive avoidance test in 10-month-old SAMR1, a senescent substrain. These results raise the possibility that S-113m might be useful for treating physiological aging and age-related memory deficits in human.

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The effects of a crude methanol extract, butanol- and chloroform-fractions, and a pure compound, corymine, extracted from the leaves of H. zeylanica on locomotor activity and rearing, pentobarbital-induced sleep, and drug-induced convulsions were studied in mice. The methanol extract dose-dependently decreased rearing without a significant effect on locomotor activity at doses of 15, 60 and 120mg/kg. It did not significantly prolong the sleeping time but potentiated the convulsions induced by strychnine, but not that by either picrotoxin or pentylenetetrazole, at a dose of 120mg/kg. The butanol-fraction significantly prolonged sleeping time at a dose of 125mg/kg but did not affect either of the convulsive drugs. The chloroform fraction prolonged sleeping time at doses of 62.5 and 125 mg/kg and potentiated the convulsions induced by either strychnine or picrotoxin, but not that by pentylenetetrazole, at doses of 15, 30, 60 and 120mg/kg. Corymine did not significantly prolong sleeping time, but potentiated the convulsions induced by either strychnine or picrotoxin, not by pentylenetetrazole, at doses of 2, 8 and 15mg/kg. These results suggest that crude alkaloidal extracts of H. zeylanical leaves produce biphasic effects on the central nervous system (CNS), depression and stimulation, while the pure compound, corymine, has a unique central stimulatory effect in mice.

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Previously, we noted significant differences in the behavioral patterns of mice fed safflower oil with a very low α-linolenate/linoleate ratio and perilla oil with a high α-linolenate/linoleate ratio from mothers to offsprings. In this report, we compared the behavior and drug responses in mice fed diets containing six different vegetable oils-corn, rapeseed, soybean, safflower, perilla and a mixture of perilla and safflower oils-for a relatively short period : 8 months after weaning. Soybean oil is a component of most conventional diets and was used as a control. The α-linolenate/linoleate ratios of the oils appeared to affect the locomotor activities in a wheel cage : the activity decreased in the order of safflower, the mixture (1 : 1) and the perilla oil groups. However, the rapeseed oil group exhibited much higher locomotor activity than that expected from the α-linolenate/linoleate ratio. Additionally, the rapeseed oil group exhibited unusual behavior patterns, including higher ambulation and rearing activities, faster acquisition of the water maze task and slower habituation behavior as compared with the control group. Susceptibility to pentobarbital anesthesia tended to be higher in the rapeseed oil group. The differences in the α-linolenate/linoleate ratios of these oils alone do not account for the observed differences in the behavioral patterns among the six dietary groups. Although we cannot exclude the possibility that the observed behavioral anomaly is due to the unique fatty acid composition of rapeseed oil, we speculate that a factor(s) other than fatty acids in rapeseed oil affected nervous system functions.

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A novel tricyclic 21-amino-acid peptide, FR901724, was isolated from the cultured broth of Streptomyces sp. No. 73264. This peptide appears to possess potent anti-human immunodeficiency virus (HIV) activity in vitro and might represent a lead to a new class of anti-HIV agents ; it qualifies as an HIV-cell fusion inhibitor because of its weak inhibition of virus-cell binding and strong inhibition of syncytium formation. From the time-of-addition experiments, the mode of action of FR901724 was found to definitely differ from that of KNI-272, a peptide mimetic allophenylnorstatine-derivative HIV protease inhibitor. FR901724 appears to interact with a stage of the virus replicative cycle that may well correspond to virus-cell fusion. We also found that FR901724 was synergistic or had a strong tendency toward synergism when combined with other antiviral drugs, such as KNI-272, AZT, ddI and dextran sulfate.

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Examination was made of the urinary and biliary excretion of the metabolites of genistein and genistin, the major components of Glycine and Sophora genus in rats. The urine of rats administered genistein orally contained eight metabolites. Three of these metabolites, genistein 4'-O-sulfate (M-1), genistein 7-O-β-D-glucuronide (M-3), genistein 4'-O-sulfate 7-O-β-D-glucuronide (M-6), were identified from spectroscopic and chemical data. The bile of rats administered genistein orally contained M-2, M-3 and M-6. M-6, a major biliary metabolite, was isolated and identified from spectroscopic and chemical data. The urine or bile of rats treated with genistin, the glycoside of genistein, contained M-1-M-8 or M-2, M-3, M-6 in the above metabolites. These findings suggest that genistin is absorbed as genistein after hydrolysis in the gastrointestinal tract. The total cumulative amounts of the two metabolites and genistein excreted in the urine during 48h, or of M-6 excreted in the bile during 36h following the oral administration of genistein, were approximately 5.7% or 16.0% of the doses administered, respectively. The results show that M-1, M-3 and M-6, having a free hydroxyl, glucuronide-or sulfate-conjugated hydroxyls at the C-7 or C-4' position, are excreted in the urine and bile as parts of the metabolites of genistein.

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We studied the in vivo behavior of galactosyllipid-modified liposomes for targeting the asialoglycoprotein receptor present on the surface of liver parenchymal cells. We examined the effects of the lipid composition of liposomes on the in vivo behavior of galactosyllipid-modified liposomes, and found good accumulation in the liver of liposomes of a high cholesterol content and liposomes made of lipids of a high gel-liquid crystalline phase transition temperature. The amount of modification with the galactosyllipid derivative required for effective targeting to the liver was found to be more than 5% of the total lipids. The concentration of galactosyllipid-modified liposomes was lower than that of control liposomes in all the organs except for the liver, showing high selectivity of galactosyllipid-modified liposomes for the liver. Hepatic accumulation of liposomes was inhibited by preinjection of asialofetuin. This result suggests that hepatic accumulation of {8-(2-hexadecyloctadecanoylamido)-3, 6-dioxaoctyl}-β-D-galactoside (Gal-t-psa) liposome was involved with the asialoglycoprotein receptor in the liver. Therefore, it was concluded that our neogalactolipid-modified liposomes are useful for selective delivery to the liver.

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To characterize the pharmacokinetic properties of a new antirheumatic drug, KE-298, the metabolic fate of [¹⁴C] labeled KE-298 in rats was investigated, focussing especially on the identification of metabolites and its physiological properties. [¹⁴C] KE-298 was rapidly and almost completely absorbed after oral administration, and was well distributed throughout the body. In plasma, only a small amount of unchanged KE-298 was detected and the major component was an active metabolite, deacetyl-KE-298, which accounted for approximately 50% of the radioactivity in the plasma. Further evidence was obtained by ¹H-NMR analysis that deacetyl-KE-298 existed as ketone-thiol and thiohemiacetal forms in a tautomeric equilibrium. As the second main metabolite in plasma, S-methyl-KE-298, a methyl conjugate of deacetyl-KE-298, was detected. Neither deacetyl-KE-298-amino acid mixed disulfide nor any disulfide of the drugs was found. Though a thiol-containing drug generally remains in the body due to the formation of mixed disulfide with protein, no evidence of retained radioactivity was found in any tissues after the administration of [¹⁴C] KE-298. Further, in the ex vivo studies of plasma protein binding, the formation of drug-protein conjugate was scarcely detected. These results suggest that the metabolic pattern of deacetyl-KE-298 is different from that of common thiol-containing drugs, and that the reactivity of the thiol moiety of deacetyl-KE-298 to protein is extremely low. This property of deacetyl-KE-298 may be principally responsible for the nonaccumulation of radioactivity in the tissues after the administration of [¹⁴C] KE-298.

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The relationships between plasma drug concentration and antihypertensive effect of eight calcium channel antagonists (nicardipine, nifedipine, nilvadipine, benidipine, manidipine, barnidipine, nitrendipine and efonidipine) in Japanese essential hypertensive patients were analyzed. Based on the effect compartment model, we could explain the long duration of the pharmacological effect, and there was significant correlation (r=0.876, p<0.05) between estimated EC₅₀ values and the dissociation constants (K_d) obtained from in vitro binding studies. We also developed the ion-channel binding model to understand the pharmacodynamics of long acting calcium antagonists. The model was also well fitted to antihypertensive effect data. A significant correlation between the apparent in vivo dissociation constants and in vitro K_d values was observed with a slope of 1.45 (r=0.913), suggesting that the mechanism of long-lasting antihypertensive effect of newer developed calcium antagonists is due to their high binding affinity at ion-channel sites.

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The disposition of adenosine 5'-phosphosulfate (APS), an endogenous nucleotide, was investigated in rats. The degradation of APS in rat plasma was very rapid. APS was degraded in rat plasma to AMP, and ATP, and these nucleotides were further degraded through adenosine. The degradation kinetics was examined. For the in vivo study, the method to protect APS from degradation in blood was examined, and it was found that the addition of EDTA to APS-containing blood and storage at 4°C can protect against APS degradation. After intravenous bolus injection, APS in plasma declined rapidly and the rate of elimination was dose-dependent : the biological half-life was about 2s at the dose of 0.3mg/kg and was longer at 3mg/kg. When APS was administered by intravenous infusion, the plasma level rapidly reached a steady-state, which then rapidly declined after the infusion was stopped. The total body clearance of APS could not be fully explained by metabolism in plasma or glomerular filtration, therefore the contribution of other elimination processes to the total body clearance was suggested.

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The population pharmacokinetic parameters of phenytoin were estimated using routine therapeutic drug monitoring data from 116 epileptic patients. The 531 serum concentration values at steady-state after repetitive oral administration were analyzed using a nonlinear mixed effects model (NONMEM) program designed for estimation of population pharmacokinetic parameters. A one-compartment model with dose-dependent clearance was used for the pharmacokinetic analysis of phenytoin. The volume of distribution (V) was estimated to be 1.23l/kg in a typical 42-kg patient, assuming that the bioavailability of orally administered phenytoin is 100%. The maximal elimination rate (V_max) and the Michaelis-Menten constant (K_m) were 9.80mg/d/kg and 9.19μg/ml, respectively. The parameter of power function of weight to adjust V and V_max was estimated to be 0.463. In addition, K_m for phenytoin appeared to be 16% increased in patients receiving zonisamide concurrently. The population pharmacokinetic parameters of phenytoin will be useful for designing dosage regimens in epileptic patients.

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We previously demonstrated that ligands of macrophage scavenger receptors such as acetylated low density lipoprotein (LDL), oxidized LDL and advanced glycation-end products (AGE) of the Maillard reaction induce the growth of peritoneal exudate macrophages, and that the activity of AGE is inhibited by the presence of an antibody for granulocyte/macrophage colony-stimulating factor (GM-CSF). To evaluate the suggested role of the scavenger receptor in the induction of macrophage growth, we compared the effect of various polyanionic compounds which were reported to either have or not to have competent activity for the binding of acetylated LDL to scavenger receptors on macrophage DNA synthesis. Among the polyanions exhibiting such activity, polyguanilic acid (poly G) and dextran sulfate strongly augmented macrophage DNA synthesis, although they did not increase macrophage cell number. On the other hand, polyanions which are not ligands for the scavenger receptors did not show a significant augmenting effect, suggesting that the binding of polyanions to the scavenger receptor is important but not, by itself, sufficient. The augmentation of DNA synthesis in macrophages cultured with dextran sulfate or poly G was inhibited by the co-presence of anti-GM-CSF antibody, suggesting that the reaction is mediated by GM-CSF. However, dextran sulfate did not augment the production of GM-CSF in macrophages. Therefore, GM-CSF spontaneously present in macrophages might be a prerequisite for the induction of DNA synthesis.

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1, 1'-(m-Xylenediyl)-bis (1, 4, 7, 10-tetraazacyclododecane)-Zn^II₂ complex (m-xylenediyl-bicyclen-Zn^II₂), a potent inhibitor of human immunodeficiency virus (HIV), was obtained from cyclen by a combination of dimerization and metal complexation. The ratio of median cytotoxic concentration against host cells (CC₅₀) and median effective concentration against HIV cytopathogenicity (EC₅₀), referred to as the selectivity index (SI), was regarded to be a measure of anti-HIV activity. These two chemical modifications contributed to a potent, in vitro anti-HIV activity of m-xylenediyl-bicyclen-Zn^II₂ by respectively increasing the CC₅₀ and decreasing the EC₅₀ in comparison with those of cyclen.

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The in vitro antibacterial effect of the combination of various polyoxometalates with β-lactam antibiotics on methicillin-resistant Staphylococcus aureus (MRSA) strains is investigated by the use of both the National Committee for Clinical Laboratory Standards (NCCLS) disk method and the agar dilution method. Keggin-structural polyoxotungstates such as K₇ [PTi₂W₁₀O₄₀]·6H₂O (5) and K₇ [BVW₁₁O₄₀]·7H₂O (8) and their lacunary species formulated by [XW₁₁O₃₉]^n- and [XW₉O₃₄]^n- potentiated the antibacterial activity of β-lactam antibiotics such as oxacillin, piperacillin and cefazolin on MRSA with high selectivity. The depression of bacterial growth with the coexistence of polyoxotungstates and oxacillin was confirmed by the measurement of the bacterial turbidity at 660nm. Polyoxomolybdates and polyoxovanadates, on the other hand, exhibited hardly any synergisitic effect in combination with oxacillin. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the membrane proteins separated from MRSA revealed that polyoxotungstates depressed the formation of penicillin-binding protein 2' (PBP2'), an enzyme which is essential for cell wall construction in the MRSA growth. It is concluded that polyoxotungstates make the MRSA strains susceptible to β-lactam antibiotics.

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Two types of fragmented keratin were prepared from buffalo horn and hoof using savinase and Na₂S, and their physicochemical and biopharmaceutical properties were examined in mice. The number-average molecular weight of enzymatically fragmented keratin (E-FK), chemically fragmented keratin (C-FK), and fragmented gelatin (FG) were 8000, 33000, and 6600, respectively. The systematic acute toxicity of FKs was significantly low. Moreover, the immunogenicity of FKs was significantly lower than that of superoxide dismutase. FKs and FG were partially hydrolyzed by trypsin. FKs were digested easily by α-chymotrypsin, but FG underwent less hydrolysis under the same conditions. FKs were bound to plasma proteins, including albumin, and also to some proteins in liver and kidney homogenates. In plasma, E-FK was hydrolyzed slowly, but in liver and kidney homogenates it showed slightly faster hydrolysis. In contrast, FG was not hydrolyzed in any of the media used here. After intravenous administration of FKs and FG to mice, these molecules were rapidly eliminated from the plasma. E-FK and C-FK were taken up into the kidneys (CL_{uptake, kidney} ; 10400, 11600μl/h/g), and then gradually excreted in urine. FG was excreted rapidly into urine (CL_urine ; 6360μl/h). Interestingly, C-FK was also taken up into the liver (CL_liver ; 4820μl/h). These results indicated that fragmented keratins are biodegradable materials and might be used as new types of liver-and kidney-specific targeting carriers.

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We have previously shown that exogenous type II phospholipase A₂ (PLA₂) alone elicits degranulation of mast cells, including rat serosal mast cells (SMC) and mouse bone marrow-derived mast cells (BMMC). Here we report that lysophosphatidylserine (lysoPS), a co-factor for activation of rodent SMC in response to some tyrosine kinase-coupled agonists, enhanced type II PLA₂-elicited histamine release from rat SMC. In contrast, mouse BMMC was insensitive to lysoPS. Our findings demonstrate a novel route for activation of SMC in that type II PLA₂ can act as a direct activator of SMC with enhancement by lysoPS, which is generated from membrane phosphatidylserine possibly by the action of the same enzyme.

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Using various routes of administration, the antinociceptive effects of dihydroetorphine (DHE) and morphine were measured in mice. Regardless of the route of systemic and local administration, DHE (1-20μg/kg, i.p. ; 1-10μg/kg, s.c. ; 1-10μg/kg, i.v. ; 10-1000μg/kg, p.o. ; 10-100ng/mouse, intracerebroventricularly (i.c.v.) ; and 10-100ng/mouse, intrathecally (i.t.)) and morphine (1-20mg/kg, i.p. ; 1-10mg/kg, s.c. ; 1-10mg/kg, i.v. ; 10-100mg/kg, p.o. ; 1-10μg/mouse, i.c.v. ; and 0.5-3μg/mouse, i.t.) produced an antinociceptive effect in a dose-dependent manner, as evaluated by the tail pinch method. However, the duration of the antinociceptive effect of DHE was shorter than that of morphine. The efficacy ratio of the antinociceptive effect between DHE and morphine was approximately 1000 to 1500 : 1 by parenteral administration (i.p., s.c., or i.v.) and about 100 : 1 by the oral route. Meanwhile, using direct application into the central nervous system (CNS) (i.c.v. or i.t.), the effect of DHE was only 10 to 20 times that of morphine. These data suggest that DHE has an ideal quality as an analgesic by systemic administration which is a more convenient application than local injection, since only a minimum dose of DHE is needed to induce suitable potency of antinociception, and the duration of the effect is short. Further, these unique characteristics of DHE might lead to the prevention of the development of dependence by avoiding accumulation of the drug in the CNS.

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The effect of lysosomal enzyme inhibition on the cytotoxic activity of an immunoconjugate composed of anti-α-fetoprotein monoclonal antibody and vindesine analog (VDS) was studied in vitro using human tumor clonogenic assay (HTCA). Addition of the lysosome enzyme inhibitors, leupeptin and ammonium chloride, to the HTCA system had little influence on the cytotoxicity of this immunoconjugate. In separate experiments, no released VDS was detected by HPLC after incubation with the supernatant of rat liver homogenate without inhibitor. These results show that the immunoconjugate may by pass the lysosomal process and exert its activity as an intact or similar form.

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Transient permeabilization was readily achieved by vortex-stirring cultured mammalian cells for a few seconds in the presence of 1 to 10 μg/ml of high molecular weight polyacrylic acid (PAA). This was demonstrated by an appreciable uptake by murine leukemia L1210 cells of Lucifer Yellow (LY), which is known to be a non-permeant except under conditions in which micropores are induced in the plasma membrane. In addition, the cytotoxicity of bleomycin (BLM), a very poor permeant, was markedly enhanced by vortex-stirring with PAA. Scope and limitation of this permeabilization procedure is preliminarily described.

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