The aggregability of rabbit platelets was studied under various cytoplasmic pHs (pH_i). Nigericin, a K⁺/H⁺ ionophore, which can induce a decrease in pH_i, at 2-10 μM in 2 min incubation reduced both platelet aggregation and an increase in cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) stimulated with thrombin or U46619. The reduced aggregability recovered 10 min after incubation with nigericin in parallel with an increase in pH_i. In contrast, when pH_i was increased by simultaneous addition of NH₄Cl, methylamine or monensin, aggregation in response to a low concentration of thrombin, U46619, arachidonic acid or A23187 was enhanced significantly. The enhancing effect of NH₄Cl was lowered by prolonged incubation with NH₄Cl, by which the increased pH_i was improved concomitantly. Indomethacin, an inhibitor of cyclooxygenase, failed to inhibit the enhancement of aggregation by NH₄Cl under stimulation with U46619. In addition, treatment with NH₄Cl enhanced an increase in [Ca²⁺]_i in response to U46619 in a concentration-dependent manner, although the treatment by NH₄Cl alone did not affect [Ca²⁺]_i. When pH_i was artificially altered during the ranges of 6.6-7.4 by treatment with nigericin in K⁺-rich medium, aggregation by low concentration of thrombin was dependent on the pH_i. These data indicate that pH _i is an important factor for platelet activation including intracellular Ca²⁺ mobilization and aggregation.

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Three effective phosphate acceptors (35, 15 and 13 kDa polypeptides) for casein kinase II (CK-II) in the Superdex CK-II fraction prepared from a 0.5 M NaCl extract of bamboo shoots were selectively purified by glycyrrhizin (GL)-affinity column chromatography (HPLC). These three proteins (p35, p15 and p13) were identified as 60S acidic ribosomal P proteins by determination of their partial N-terminal sequences CK-II was associated with p35 since the GL-affinity fraction was coprecipitated with an anti-serum against Drosophila CK-IIβ. Moreover, a derivative (oGA) of glycyrrhetinic acid (GA) and several polyphenol-containing anti-oxidative compounds [quercetin, epigallocatechin gallate (FGCG) and two isoflavones, i.e., 3', 4', 7-trihydroxyisoflavone (3', 4', 7-THI) and 8-chloro-3', 4', 5, 7-tetrahydroxyisoflavone (8C-3', 4', 5, 7-THI)] inhibited the CK-II-mediated phosphorylation of 60S acidic ribosomal P proteins in vitro. Quercetin was found to be the most effective compounds on CK-II activity since its ID₅₀ was approx. 50 nM. These results suggest that (i) GL-affinity column chromatography is useful for the selective purification of 60S acidic ribosomal P proteins as a heterocomplex associated with CK-II from various cell sources; (ii) natural anti-oxidative compounds with polyphenols, but not GL and GA, act as potent CK-II suppressors; and (iii) CK-II mediates the regulation of the physiological functions of 60S acidic ribosomal P proteins in growing plant cells.

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Calprotectin is a calcium- and zinc-binding protein complex that is abundant in cytosol of neutrophils. The concentration of calprotectin in extracellular fluids is greatly increased under various inflammatory conditions in vivo. We recently demonstrated that calprotectin inhibited cell growth and induced apoptosis of vairous cell types including tumor cells and normal fibroblasts; therefore, extracellular calprotectin might cause tissue destruction in severe inflammatory diseases. We previously found that an alkaloid, lycorine inhibits induction of apoptosis by calprotectin. In this paper, we examined the inhibitory activities of other Amaryllidaceae alkaloids, namely, lycoricidinol, hippeastrine and ungerine against the cytotoxicity of calprotectin. Lycoricidinol (narciclasine) inhibited calportectin-induced cytotoxicity at more than 10-fold lower concentration (IC₅₀=0.001-0.01μg/ml) than lycorine, while the effects of the latter two alkaloids were very weak. Therefore, we next checked the prophylactic effect of lycorine and lycoricidinol on the adjuvant arthritis model in rats. Lycoricidinol, but not lycorine, significantly suppressed the degree of swelling of adjuvant-treated as well as untreated feet, suggesting that lycoricidinol might be a candidate as a the drug having marked suppressive activity for inflammation which might be influenced by calprotectin.

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Extension of dendrites and axons in neurons may compensate and rescue damaged neuronal networks in the dementia brain. Our aim is to isolate and identify constituents of coffee beans exhibiting neurite outgrowth activity. Among the extracts of raw and roasted coffee beans, a methanol fraction of the ethanol extract (1 μg/ml) of raw beans increased significantly the percentage of cells with neurites in human neuroblastoma SK-N-SH cells. Among subfractions of this methanol fraction was a basic fraction (5 μg/ml) wich exhibited significant neurite outgrowth activity. Finally, trigonelline in the basic fraction was identified to be active as far neurite extension was concerned. Treatment with trigonelline (30 μM) increased the percentage of cells with neurites at 3 and 6 d after treatment. In addition, the number of neurites reacting positively to phosphorylated neurofilament-H was increased by treatment with 30 μM trigonelline for 6d, suggesting enhancement of axonal extension. These results show that trigonelline promotes functional neurite outgrowth.

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The effects of newly synthesized pyridothiazepines MM 4 (1-[N-[2-(3, 4-dimethoxy-phenyl)ethyl]-N-methyl-aminoacetyl]-1, 2, 3, 4-tetrahydro-pyrido[2, 3-b][1, 4]thiazepine fumarate), MM 6 (1-[N-[2-(3, 4-dimethoxyphenyl)-ethyl]-N-methylaminopropionyl]-1, 2, 3, 4-tetrahydro-pyrido[2, 3-b][1, 4] thiazepine fumarate) and the novel pyridothiazines MM 10 (2, 3-dihydro-1-[N-[2-(3, 4-dimethoxypheny)ethyl]-N-methylaminoacetyl]-1H-pyrido[2, 3-b]-[1, 4]thiazine fumarate) and MM 11 (2, 3-dihydro-1-[N-[2-(3, 4-dimethoxy-phenyl)ethyl-N-methylaminopropionyl]-1H-pyrido[2, 3-b][1, 4]thiazine fumarate) on the contracility of isolated papillary muscles and aortic preparations of guinea pigs were studied using isometric contraction force measurements. The EC₅₀values for the negative inotropic effect were 27 μmol/l MM 4), 19 μmol/l (MM 6), 32 μmol/l (MM 10) and 24 μmol/l (MM 11). In K⁺-precontracted aortic rings ([K⁺]₀ 60 mmol/l), the compounds induced relaxation with EC₅₀ values of 27μmol/l (MM 4), 24 μmol/l (MM 6), 84 μmol/l MM 10) and 68 μmol/l (MM 11). Pyridothiazepines as well as pyridothiazines (100 μmoll/l) were able to depress norepinephrine bitartrate (NE 10 μmol/l)-induced contraction of aortic rings in a calcium-free solution. It was concluded that the investigated compounds exert calcium antagonistic properties in both cardiac and smooth muscle. This antagonistic effect be due to the inhibition of transmembrane calcium influx and/or intracellular calcium release.

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In vitro, ex vivo and in vito α₁-adrenoceptor binding of JTH-601 (3{N-[2-(4-hydroxy-2-isopropyl-5-methylphenoxy)ethyl]-N-methylaminomethyl}-4-methoxy-2, 5, 6-trimethyl-phenol hemifumarate), a novel α_1L-adrenoceptor antagonist, in rat tissues was investigated. JTH-601 competed in a concentration-dependent manner with [³H]prazosin for binding sites in the prostate, submaxillary gland and spleen of rats in vitro, and the inhibitory effect was not largely different among these tissues, as shown by the K_i values of 2-3nM. At 0.25, 0.5 and 3 h after oral administration of JTH-601 (6.5 μmol/kg) in rats, there was a significant (57, 64 and 28%, respectively) increase in the apparent dissociation constant (K_d) for prostatic [³H]prazosin binding, compared to the control value. The administration of a higher dose (21.8 μmol/kg) of this agent produced greater (67-99%) increase in K_d values for prostatic [³H]prazosin binding at 0.5-12 h later. Similar significant increases in K_d values, as with the prostate, were seen in the submaxillary gland and heart 0.25-12 h after the oral administration of JTH-601 (6.5 and 21.8 μmol/kg), but significant increases in the spleen and cerebral cortex were seen only at 0.25-3 h and 0.5 h, respectively. At 10 min of i.v. injection of [³H]JTH-601 in rats, in vivo specific binding was observed in the prostate, cerebral cortex, submaxillary gland, spleen and heart but not in the aorta. The binding in the prostate, submaxillary gland and heart, but not in the cerebral cortex and spleen, lasted until 120 min. It is concluded that JTH-601 may exert a considerably sustained blockade of α₁-adrenoceptors in the prostate of rats. This finding may be important in characterizing the therapeutic effect of JTH-601 for bladder outlet obstruction in patients with benign prostatic hyperplasia.

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To elucidate the mechanisms of analgesic action of elcatonin, a synthetic analog of eel calcitonin, the effect of centrally injected elcatonin on acetic acid-induced writhing behavior was examined in mice. Intracisternal or intracerebroventricular injection of elcatonin significantly inhibited acetic acid-induced writhing behavior, while the intrathecal injection of elcatonin did not inhibit it. The inhibitory effect of intracisternal elcatonin was significantly attenuated by subcutaneous pretreatment with methysergide and (±)-propranolol or by intrathecal pretreatment with methysergide, (±)-propranolol, 1-(2-methoxyphenyl)-4-[4-(2-phthalimido) butyl]-piperazine hydrobromide (NAN-190) and granisetron, but not with (±)-atenolol or butoxamine. Further, the depletion of spinal 5-hydroxytryptamine (5-HT, serotonin) by pretreatment with 5, 7-dihydroxytryptamine (5, 7-DHT) significantly attenuated the inhibitory effect of intracisternally injected elcatonin on acetic acid-induced writhing behavior. These results suggest that the inhibitory descending serotonergic systems may be involved, through 5-HT_1A and 5-HT₃ receptors, in the production of an antinociceptive effect by centrally injected elcatonin.

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Effects of the administration of a water-soluble prodrung of vitamin E on doxorubicine (DXR)-induced lethal and oxidative toxicity in mice were studied. The prodrug used was d-α-tocopheryl N, N-dimethylaminoacetate hydrochloride (TDMA). It was intravenously administered to animals 2 h prior to an intraperitoneal administration of DXR (15 mg/kg). The single preadministration of the prodrug (10-50mg/kg equivalent for d-α-tocopherol) delayed the DXR-induced death and the ameliorative effect was TDMA-dose dependent. The extent of total lipid peroxidation of the heart and liver was assessed by 2-thiobarbituric acid reactant substance levels. DXR significantly accelerated lipid peroxidation in the liver but not in the heart. The elevation of liver lipid peroxide was significantly suppressed to a normal range by a single preadministration of TDMA (50 mg/kg equivalent for d-α-tocopherol). TDMA did not significantly affect the antitumor activity of DXR in mice inoculated with L1210 leukemia cells.

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Radiation injury to the skin is one of the major limiting factors in radiotherapy. We designed this study using Sprague-Dawley rats to evaluate the reduction in skin injury achieved using natural products from plant extracts as protection. The acute skin reaction in tetrandrine- and Madecassol-treated animals appeared earlier, but was significantly less severe, than in the control group. The peak skin reactions in the tetrandrine group were less serious than those of the control group at three different radiation doses. At a high dose irradiation, the healing effect of tetrandrine is better than Madecassol and vaseline. The histologic findings indicate that tetrandrine and Madecassol are able to reduce acute radiation reactions by their anti-inflammatory activity.

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We examined the stereoselective membrane permeation of new fluorinated quinolone derivatives (NQs) across LLC-PK₁ cell monolayers, using levofloxacin (LVFX) and its R-(+) isomer. LVFX permeation was 1.6-fold greater in the basal-to-apical direction than that in the apical-to-basal direction, suggesting that LVFX permeated LLC-PK₁ cell monolayers in a secretory-oriented manner. In contrast to LVFX, the permeation of the R-(+) isomer was almost identical in both directions. LVFX permeation in the basal-to-apical direction was significantly reduced in the presence of guanidine, enoxacin, and L-arginine, whereas tetraethylammonium, D-arginine, D-and L-lysine had no effect on the basal-to-apical permeation of LVFX. Basal-to-apical permeation of the R-(+) isomer was not affected by these compounds. Cellular accumulation of LVFX was inversely increased when guanidine suppressed the appearance of LVFX in the apical medium in a concentration-dependent manner. These results imply that the inhibitory effect of guanidine on the basal-to-apical permeation of LVFX involves the permeation process across the apical membrane. Guanidine trans-stimulated the efflux of LVFX from LLC-PK₁ cells but did not affect cimetidine efflux. These results suggest that some NQs, like LVFX and its R-(+) isomer, are stereoselectively secreted across LLC-PK₁ cell monolayers and that an organic cation transport system, which favors guanidine as a typical substrate, may be involved in the secretory-oriented permeation of some NOs.

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We report a very promising approach for liver site-selective drug delivery through drug instillation on liver surface. Phenol red, which was selected as a model drug, was accumulated in the instillation site after instillation on the rat liver surface. The site-selective localization was enhanced by gradually and continuously instilling a small amount of drug solution on the liver surface.

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We previously encountered a patient with epilepsy who exhibited rapid elimination of sustained-release valproic acid (VPA) administered at the dose of 2.8 g/d as a sodium salt. The purpose of this study was to clarify the relationship between the VPA elimination rate and the proportion of the dose excreted in urine as its glucuronide conjugate (VPA-G) in epileptic patients. Twenty-four-hour urine was collected from four epileptic patients who had taken VPA orally (age : 16-39y, weight : 50-63 kg, dose : 1.0-2.8 g/d). VPA and its metabolites were detected by gas chromatography-mass spectrometry. The amounts of VPA, VPA-G, 3-keto VPA, and 3-OH VPA excreted in the 24-h urine were 1.8-13.2, 178-2158, 125-320, and 8.6-18.7 mg (converted into VPA), respectively, and 0.2-0.5, 20.5-88.7, 5.8-18.7, and 0.6-1.0% of the dose administered, respectively. The dose of VPA correlated well with the proportion of the dose excreted in urine as VPA-G in each patient, and the patients administered a high dose excreted a large amount of VPA-G in the urine. Thus, differences in the VPA-G production rate may be one of the major variable factors affecting the elimination of administered VPA. We also present a dynamic model of VPA in the kidney which may explain the VPA elimination phenomena in humans on the basis of the data obtained here regarding the concentrations of VPA and its metabolites in plasma and their urinary excretion levels.

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The inhibitory effects of five angiotensin-converting enzyme (ACE) inhibitors on the uptake of an aminocephalosporin antibiotic, cefroxadine, by rabbit small intestinal brush-border membrane vesicles were examined in the presence of an inward H⁺ gradient. Dixon plot analysis showed that all these ACE inhibitors inhibited the uptake of cefroxadine, which is transported by a H⁺/oligopeptide transporter in the membrane, in the order of enalapril<quinapril, benazepril<temocapril, trandolapril at extravesicular pH 6.0. These drugs except for enalapril, which are relatively hydrophobic, exhibited a mix of competitive and noncompetitive inhibition. We also examined the inhibitory effects of quinapril and temocapril at extravesicular pH 5.5, at which the ratio of the zwitterionic species of the drugs increased. The inhibition occurred in a nearly competitive manner at this pH and the inhibitory effects were stronger than at pH 6.0. Regression analysis of the inhibitory effects suggested that the affinity of these ACE inhibitors for the transporter was regulated by the hydrophobicity of these ACE inhibitors and the ratio of the zwitterionic species of the drugs.

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Notopterol, 5[(2E)-5-hydroxy-3, 7-dimethyl-2, 6-octadienyloxy]psoralen showed an inhibitory effect on aminopyrine N-demetylase activity in liver microsomes. In addition, notopterol has been found to be metabolized by cytochrome P450, and two kinds of metabolites were formed from notopterol. Furthermore, specific cytochrome P450 3A4 inhibitors which were isolated from grapefruit juice had the same furocoumarin structure as notopterol. Two metabolites of notopterol were separated by HPLC, and the chemical structures of the hydroxylated metabolites were estimated by ¹H-NMR spectra and liquid chromatography-mass spectrometry.

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Glucocorticoids ameliorate erosion in animal osteoarthritis (OA) models and suppress synthesis of matrix matalloproteinases (MMP). However, in in vitro studies, their inhibitory effects on matrix degradation of cartilage have not been well documented by monitoring aggrecan. Collagen was monitored in this study to examine the effects of dexamethasone in cartilage explant culture. Dexamethasone cleary blocked collagen degradation induced by the combination of interleukin-1 (IL-1) and plasminogen at the concentration of 10^-9M, whcih is much lower than the concentrations reportedly required to inhibit matrix synthesis. In addition, MMP-1 and MMP-3 were suppressed by dexamethasone treatment in a similar range of concentrations. The conversion of plasminogen to plasmin, however, was not blocked by treatment with dexamethasone. These results suggest that the inhibitory effect of dexamethasone on collagen degradation may be due to suppression of MMP production rather than suppression of fibrinolytic cascade. Thus, the ability of glucocorticoids to inhibit matrix degradation in vitro, which could be clearly shown by monitoring collagen degradation, may endorse their efficacy in animal OA models and suggest potential therapeutic effectiveness.

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The inhibition of rabbit heart carbonyl reductase (PHCR) by fatty acids was examined using 4-benzoylpyridine (4BP) as a substrate. The inhibitory potency of saturated fatty acids increased with elongation in the carbon chain from caprylic acid to myristic acid, but decreased with further elongation. Myristic acid with 14 carbon atoms most strongly inhibited RHCR. All of the unsaturated fatty acids tested strongly inhibited RHCR; the cisisomers were more potent inhibitors than the corresponding trans-isomers. The methyl esters and alcohols, which lack a carboxyl group, derived from fatty acids did not exert a significant inhibitory effect on RHCR. These results indicate that the existence of a proper length of carbon chain, double bond(s), and a carboxyl group in a fatty acid molecule is important for RHCR inhibition. We also propose the possibility that myristic acid at low concentrations inhibits the reduction of 4BP by interacting with a binding site other than the coenzyme- and substrate-binding sites of RHCR.

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To obtain an antagonist antibody against human growth hormore receptors (hGHRs), we prepared monoclonal antibodies against the recombinant hGHR extracellular domain. One of the clones, GHBP116, exhibited binding activity to intact human IM-9 cells and effectively immunoprecipitated the receptors in cell lysate. GHBP116 competitively inhibited ¹²⁵I-human growth hormore (hGH) binding to the cells. The antagonist activity of GHBP116 was assessed in terms of ligand-induced receptor internalization, degradation, and phosphorylation of signal transducer and activator of transcription (STAT) 5. The antibody alone did not cause internalization or degradation of hGHRs, but a 1 : 25000 dilution of ascitic fluid almost completely inhibited ligand (1 nM hGH)-induced internalization and degradation of surface hGHRs. Moreover, GHBP116 alone did not stimulate the phosphorylation of STAT5, used as an indicator of Janus kinase (JAK)-STAT signaling, but almost completely inhibited hGH-induced phosphorylation of STAT5. These results suggest that GHBP116 acts as a specific antagonist of hGH.

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We investigated the mechanism of adhesion of highly malignant ascites hepatoma AH66F cells to mesothelial cells. The adhesion rate of AH66F cells to mesentery-derived mesothelial cells (M-cells) was about 46% at 37°C, but it decreased to about 27% at 4°C. The adhesion rate of AH66F cells was about 25% in the presence of leukocyte function-associated antigen 1 (LFA-1) mAb at both 4°C and 37°C. When M-cells were treated with hyaluronidase, the AH66F/M-cell adhesion was decreased to half at 37°C and had nearly disappeared at 4°C. The residual adhesion of AH66F cells to M-cells treated with hyaluronidase almost disappeared in the presence of LFA-1 mAb. AH66F cells strongly adhered to a hyaluronate (HA)-coated plate, but not to a bovine serum albumin-coated plate. AF66F cells expressed a CD44 molecule (a HA receptor) in the plasma membrane, with a molecular size of about 85 to 90 kDa, corresponding to the CD44H isoform.These results indicated that the adhesion of AH66F cells to mesothelial cells is composed of pathways of CD44/HA and LFA-1/ICAM-1.

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Drugs for testing the function of liver and kidney (sulfobromophthalein, phenolsulfonphthalein, indigo carmine and indocyanine green) and other organic anions (rose bengal and haematin) were found to potently inhibit human liver aldehyde reductase that is involved in the detoxification of 3-deoxyglucosone and methylglyoxal, reactive intermediates, during the formation of advanced glycation end products. The inhibition patterns by the compounds were non-competitive with respect to both coenzyme (NADPH) and substrate (D-glucuronate). The kinetics of the inhibition by a mixture of the 2 inhibitors suggests that all the inhibitory compounds bind to overlapping sites on the enzyme. The binding of rose bengal, sulfobromophthalein and phenylsulfonphthalein to the free enzyme was detected by ultrafiltration assay. However, in the reverse reaction, the enzyme was inhibited competitively with respect to the alcohol substrate by rose bengal, haematin, phenolsulfonphthalein, sulfobromophthalein, indigo carmine and indocyanine green, which showed K_i values of 0.1, 1, 3, 4, 4 and 10 μM, respectively. The results suggest that these potent inhibitors bind weakly to the free enzyme and tightly to the enzyme-NADP binary complex.

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To investigate the effects of vasoactive intestinal peptide (VIP) on Cl^- transport across normal human bronchial epithelial (NHBE) cells grown in a monolayer, changes in short-circuit current (Isc) were measured in Ussing chamber systems. In the presence of 10^-4M amiloride, the addition of VIP to the serosal solution led to an increase in the Isc in a concentration-dependent manner, the 50% effective concentration (EC₅₀) being 2.6×10^-11M. However, the addition of 10^-5M forskolin had little effect on the increase in Isc. On the other hand, in the intracellular cAMP measurement, 10^-5M forskolin remarkably increased the cAMP levels, but 10^-7M VIP did not.This result suggests that Cl^- secretion by VIP is not related to the raised intracellular cAMP levels in NHBE cells.

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When quercitrin was anaerobically incubated with human intestinal becteria, quercetin, 3, 4-dihydroxyphenylacetic acid and 4-hydroxybenzoic acid were found as metabolites. The main metabolite was quercetin. The bacterium transforming quercitrin to quercetin was Fusobacterium K-60. However, Bacteroides, JY-6, which produced α-L-rhamnosidase, did not transform quercitrin to quercetin. Among quercitrin and its metabolites, 3, 4-dihydroxyphenylacetic acid and 4-hydroxylphenylacetic acid had more potent activity than quercitrin on in vitro anti-platelet aggregation activity, and quercetin and 3, 4-dihydroxyphenylacetic acid showed more potent cytotoxicity against tumor cell lines than quercitrin and 4-hydroxylphenylacetic acid.

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It is very important to search for natural compounds possessing nerve growth factor (NGF)-potentiating activity. Extracts of 7 Chinese and 10 Paraguayan medicinal plants were examined for their effects on the NGF-mediated neurite outgrowth from PC12D cells to evaluate their NGF-potentiating activities. In the methanol extracts, Gymmopteris rufa (LINN.) BERNH, Ruta graveolens LINN. and Picrorhiza scrophulariiflora PENNELL markedly increased the proportion of neurite-bearing cells. In the case of ethyl acetate fractions, Equisetum giganteum LINN. produced the most powerful enhancement of the proportion of the neurite-bearing cells, and the activities were in the following decreasing order : Equisetum giganteum LINN., Gymmopteris rufa (LINN.) BERNH, Ruta graveolens LINN., and Picrorhiza scrophulariiflora PENNELL. In the water fractions, Imperata cylindrica, Ginseng Radix, Gymmopteris rufa (LINN.) BERNH, Gochnatia polymorpha (LESS) CAB and Picrorhiza scrophulariiflora PENNELL caused a weak enhancement of the proportion of PC12D cells with neurites. Of all extracts and fractions, the methanol extract of Picrorhiza scrophulariiflora PENNELL induced the longest neurites in PC12D cells. In the ethyl acetate and water fractions of Nardostachys chinensis, long neurites were observed although only a small proportion of PC12D cells had neurites. On the other hand, in the ethyl acetate fraction of Equisetum gigantheum LINN., while the length of the neurites was short, the proportion of neurite-bearing cells was largest among all the extracts and fractions.

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In vitro release behavior and cytotoxic activity, and in vivo plasma disposition of newly synthesized macromolecular derivatives of cisplatin (CDDP) were investigated and compared with CDDP. The derivatives included oxidized dextran conjugate of CDDP (OX-Dex/CDDP) and dicarboxymethylated dextran conjugate of CDDP (DCM-Dex/CDDP).In vitro release of platinum complex from dextran conjugated CDDP was determined by an equilidrium dialysis method. These dextran conjugates showed sustained release of the platinum complex. In vitro release half-life for DCM-Dex/CDDP was significantly longer (4.5 times) than that for OX-Dex/CDDP. In vitro cytotoxic activity of CDDP and dextran conjugated CDDP against colon 26, mouse colon cancer cell line, was measured using the MTT assay method. OX-Dex/CDDP showed a similar cytotoxic activity to CDDP. However, both cytotoxic activities were markedly decreased when preincubated with the medium containing serum. On the other hand, DCM-Dex/CDDP retained residual cytotoxic activity at a significantly higher level than OX-Dex/CDDP after preincubation with the medium containing serum, although it showed the lowest cytotoxic activity. This indicated longer maintenance of the in vitro antitumor activity of DCM-Dex/CDDP in serum compared with OX-Dex/CDDP. Plasma disposition of CDDP and dextran conjugated CDDP was determined by intraveous administration to rats. Although the total platinum plasma concentration-time profile for OX-Dex/CDDP was similar to that for CDDP, its markedly higher profile was achieved when DCM-Dex/CDDP was administered. The values of the total platinum AUC and MRT, where AUC is the area under the platinum concentration-time curve and MRT is the mean residence time, for DCM-Dex/CDDP were 11.2 times and 4.8 times significantly higher than with OX-Dex/CDDP in plasma, respectively. DCM-Dex/CDDP also showed a significantly lower total clearance compared with OX-Dex/CDDP. These results from the in vivo experiments revealed that retention of DCM-Dex/CDDP in blood circulation was much greater than thet for OX-Dex/CDDP.DCM-Dex/CDDP thus has potential as a macromolecular derivative of CDDP for passive tumor targeting.

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Uptake of the angiotensin-converting enzyme (ACE) inhibitor enalapril by rabbit small intestinal brush border membrane vesicles was examined. In the presence of an inward H⁺ gradient the uptake of this peptide mimetic drug was accelerated and an overshoot phenomenon was observed. The uptake was more stimulated by higher H⁺ gradient. Initial uptake rate was saturable in the presence of an inward H⁺ gradient, with apparent K_m value of 4.2 mM. These findings suggest the involvement of the H⁺-coupled carrier-mediated transport in the uptake of enalapril. The uptake was inhibited by ACE inhibitor trandolapril but hardly or only slightly inhibited by aminocephalosporins.

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A protocol for rapid purification of DNA from a variety of herbal crude drugs, starting from samples as small as 1 mg, has been developed. Neither phenol extraction nor alcohol precipitation is involved in the procedure. Total nucleic acids are first extracted with the lysis buffer containing SDS. After CHCl₃ extraction, DNA is bound to glass fiber fleece in the presence of guanidine thiocyanate, while unbound impurities are washed away. The entire procedure can be carried out in 1.5-ml microtubes. The purified DNA obtained serves as a template for PCR.

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A new metabolic pathway of (24R)-24, 25-dihydroxyvitamin D₃ [24, 25(OH)₂D₃] was clarified in the vivo experiments. After the administration of 24, 25(OH)₂D₃ to rats, a new monoglucuronide of a vitamin D metabolite was obtained from the bile together with 24, 25(OH)₂D₃ 3- and 24-glucuronides. The genin of the metabolite was identified as 3-epi-24, 25(OH)₂D₃ in comparison with the synthetic sample based on the data from ¹H-NMR, GC/MS, and LC/atmospheric pressure chemical ionization-MS. The conjugation position was determined to be the 24-hydroxy group by the LC/electrospray ionization-MS and -MS/MS/MS combined with derivatization. To our knowledge, this is the first reported instance of the epimerization of the 3-hydroxy group of vitamin D compound with no hydroxy group at the 1α-position.

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The enzymatic breakdown of β-(isoxazolin-5-on-2-yl)-L-alanine (BIA) with formation of 2, 3-diaminopropionic acid (DAPA), the direct precursor of the neurotoxin β-ODAP in Lathyrus sativus, was confirmed in vitro. Some properties of the enzyme responsible for the biosynthesis of DAPA are described.

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