A HPLC determination method for methamphetamine (MA) and its metabolites in the urine samples of abusers has been developed. MA, amphetamine (AP), norephedrine (NE), p-hydroxymethamphetamine (pOHMA), p-hydoroxyamphetamine (pOHAP) and an internal standard, namely β-phenylethylamine (PEA) were derivatized with dansyl chloride. They were separated on a reversed phase column with gradient elution using an acetonitrile/tetrahydrofuran/imidazole buffer mobile phase and chemilumigenically determined using bis(2, 4, 6-trichlorophenyl)-oxalate/hydrogen peroxide as post column reagents. The lower determination limits were as low as 1×10^-14-3×10^-14mol. AP, NE, pOHAP and PEA were derivatized with naphthalene-2, 3-dicarboxaldehyde, and were separated on a reversed phase column using an acetonitrile/imidazole buffer mobile phase and chemilumigenically determined. The lower determination limits were 3×10^-16-1.5×10^-15mol. Enzymatic hydrolysis of glucuronides of pOHMA (pOHMAG) and pOHAP (pOHAPG) allowed them to be determined as pOHMA and pOHAP, respectively. After adjusting the pH of the urine samples to 10.5 and adding PEA, all metabolites except glucuronides were extracted quantitatively into chloroform-isopropanol (3 : 1). Utilizing the two methods, MA and all metabolites were determined in urine samples of MA abusers. The tendency, in order of decreasing concentration was : [MA]>[A]>[pOHMAG]>[pOHMA]>[NE]>[pOHAPG]>[pOHAP]. Although ephedrine (EP) was detected in several samples, it was not considered to be a metabolite of MA but rather a component derived from cough medicine.

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The reactivity of factor G mediated coagulation pathway in limulus amebocyte lysate which is triggered by (1→3)-β-D-glucans is thought to depend on the structure of the glucans, especially on the ultrastructure : triple helix, single helix and random coil. We used Sonifilan (SPG) and grifolan (GRN) as parent compounds to compare the reactivities of these three conformers. Under a meutral condition, alkaline treated SPG (SPG-OH, single helix) and polycarboxylated SPG (PC-SPG, random coil) showed significantly stronger reactivity than untreated SPG (triple helix). After the alkaline treatment, all three conformers showed comparable reactivities. It is suggested that the pretreatment of the glucan preparations by sodium hydroxide is quite important to compare quantitatively the reactivity of the glucans by limulus test, and comparing the data of untreated and alkaline treated glucans would provide information about their conformations. Using this approach, it was found that after heat treatment at around 150°C, the conformation of GRN was changed to rich in the triple helix, and that following sodium hydroxide treatment and dialysis of GRN, the conformation of GRN was changed to single helix rich conformer. About half of the single helix conformer was gradually changed to triple helix conformer over one week at 4°C.

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Release of HeLa cells arrested at the G₁/S boundary by double-thymidine block immediately coused uptake of [³H]thymidine into DNA. The duration of the cell cycle time was 23 h and definite changes in cell density were observed between 12 h and 13 h and also between 35 h and 36 h after removal of thymidine. Addition of trans-4-amidinocylohexanevarboxylic acid 4-tert-butylphenyl ester (ACHCA-OPhBu^t), immediately after removal of the arrest had no effect on DNA synthesis, although it dose-dependently suppressed the first mitosis and the next round of DNA synthesis. While the addition of ACHCA-OPhBu^t at any time from 0 to 10 h after removal of thymidine suppressed mitosis, its addition after 11 h did not. A trypsin-like proteinase sharply appeared around 10 h 30 min and vanished within a few minutes. The proteinase activity seemed to be density dependent and was strongly inhibited by ACHCA-OPhBu^t. The effects of trans-4-amidinocyclohexanepropionic acid 4-tert-butylphenyl ester (ACHCA-OPhBu^t), another trypsin inhibitor, on the proteinase activity and mitosis were more potent than those of ACHCA-OPhBu^t. These results suggest the involvement of the proteinase in the entry of HeLa cells from the G₂ late phase into mitosis. The proteinase was named late G₂ proteinase.

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We investigated the alteration of glycosaminoglycans (GAGs) induced by recombinant human tumor necrosis factor alpha (rhTNFα) using confluent cultures of bovine aortic smooth-muscle cells, bovine aortic endothelial cells, Chang liver cells and porcine kidney LLC-PK1 cells. It was found that the incorporation of both [³⁵S]sulfate and [³H]glucosamine into GAGs in the trypsinate fraction of the cell layer was significantly decreased by rhTNFα in vascular smooth-muscle cells and vascular endothelial cells; the incorporation of [³⁵S]sulfate was increased but that of [³H]glucosamine was unchanged in Chang liver cells; the incorporation of both [³⁵S]sulfate and [³H]glucosamine was increased by rhTNFα in LLC-PK1 cells. In the conditioned medium, the incorporation of both [³⁵S]sulfate and [³H]glucosamine was not greatly changed by rhTNFα in all tested cell types. Characterization of GAGs revealed that each cell type uniquely altered its GAGs after rhTNFα treatment; the cytokine-induced alteration of each GAG component was not necessarily the same among different cell types. It was therefore concluded that rhTNFα-induced alteration of GAGs is dependent upon cell type.

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Thioltransferase was partially purified from bovine leukocyte using sonication, heat treatment at pH 5.1, Sephadex G-50 gel filtration and isoelectric focusing techniques. Isoelectric point (pI) of 8.3 for leukocyte thioltransferase was quite different from pI of 6.5 for erythrocyte enzyme. Bovine leukocyte thioltransferase was employed in a study on the influence of 12 anti-inflammatory drugs and 7 anti-histaminic drugs. Piroxicam which is a well-known anti-inflammatory drug demonstrated the most powerful inhibitory effect on enzyme activity. Inhibition of piroxicam was noncompetitive, and the K_i value measured 55.0 μM in the experiment involving bovine leukocyte enzyme. Tranilast which is a typical anti-histaminic drug most strongly inhibited the enzyme activity and the K_i value of the medicine was 20.3 μM (noncompetitive). Bovine liver and erythrocyte thioltransferases also were effectively inhibited by both medicines similar to leukocyte enzyme.

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The effects of two calcium channel blockers, nifedipine and nicardipine, on glucagon-stimulated glycogenolysis in primary cultures of rat hepatocytes were examined in vitro. When nifedipine and nicardipine (10^-7-10^-6M) were added to the incubation mixture with various concentrations of glucagon (10^-10-10^-6M), these dihydropyridine calcium channel blockers significantly potentiated the glycogenolytic action of glucagon by increasing intracellular cAMP levels. 1-Methyl-3-isobutylxanthine (IBMX), caffeine and papaverine, which is known to inhibit cAMP phosphodiesterase, also potentiated the stimulatory effect of glucagon on the glycogenolysis in a dose-dependent manner. Parallel to the potentiation of glycogenolysis, IBMX also increased the glucagon-stimulated intracellular cAMP levels in a dose-dependent manner. These results suggest that the mechanism of potentiation of the glucagon-stimulated glycogenolysis by nifedipine and nicardipine is related to the known inhibition of cAMP phosphodiesterase by these agents.

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A new method of assaying deaminase activity was established in which methylamine and/or dimethylamine formed from drugs containing N, N-dimethyl or N-methyl group were derivatized with phenylisothiocyanate to phenylthiourea derivatives. After purification with Sep-PAK C₁₈ cartridge, the derivatives were separated by a reversed phase high-performance liquid chromatography monitored by ultraviolet absorption. The recoveries and determination limits of methylamine and dimethylamine were over 55% and about 0.4 nmol/ml of incubation mixture, respectively. The method was used to measure the deaminase activities of liver microsomes of rats, rabbits and guinea pigs for 11 drugs. Of the compounds tested, diphenhydramine and diltiazem are deaminated with microsomes from all the above animal species; rat and rabbit liver microsomes also well deaminated promethazine. Most other drugs such as chlorpromazine, promazine, imipramine, amitriptyline and tetracaine were found to be poor substrates. In general, dimethylamine but not methylamine was the predominant metabolite formed from drugs containing N, N-dimethylamino group. The results also suggested that the deamination of these compounds takes place mainly via a one step mechanism, thus implying that the sequential reaction consisting of N-demethylation and elimination of ammonia is of minor importance. The relation between in vitro deaminase activity and the extent of the in vivo deamination for drugs is discussed.

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We studied in vitro metabolites of 2, 4, 6, 2', 4', 6'-hexachlorobiphenyl (HCB, IUPAC PCB No. 155) produced by liver microsomes of a phenobarbital (PB)-treated beagle dog. The major metabolites were 3-hydroxy-2, 4, 6, 2', 4', 6'-HCB (M-1), 4-hydroxy-2, 6, 2', 4', 6'-pentachlorobiphenyl (PenCB, M-2) and 3, 4-dihydroxy-2, 6, 2', 4', 6'-PenCB (M-3). Furthermore, 4-hydroxy-2, 3, 6, 2', 4', 6'-HCB (M-4), which could be formed via the 3, 4-epoxidation and the subsequent NIH-shift of the chlorine from the 4 to the 3 position, was also detected. We found that M-3 is a common secondary metabolite of the two major monohydroxy metabolites, M-1 and M-2. These results indicate that the dog seems to metabolize and eliminate this congener not only by a mechanism involving direct insertion of a hydroxyl group but also via an arene oxide intermediate.

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The anti-allergic activity of the carboxamidemethylated Fc fragment (CM-Fc) from human serum immunoglobulin G (IgG) was studied using sheep red blood cell-induced delayed type hypersensitivity in mice (SRBC-DTH). CM-Fc suppressed the DTH response when administered 30 min before, or 4 h after the SRBC challenge, but not when administered 8 h or more after the challenge. The Fc fragment showed no activity. CM-Fc administration 30 min before the challenge was unable to suppress the DTH response in the cyclophosphamide (CY)-treated mice. However, adoptive transfer of splenocytes from mice treated with CM-Fc to CY-pretreated mice caused suppression of the SRBC-DTH response.These results suggest that CM-Fc suppressed the DTH response by mediating the function of CY-susceptible cells.

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Rats given an intrathecal injection of substance P (0.3-10 nmol) or any of the excitatory amino acid agonists, N-methyl-D-aspartate (NMDA, 1-10 nmol), kainate (1 and 3 nmol) or α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA, 0.3-3 nmol), showed biting or licking the hind paws, scratching with the hind paws (only after substance P) and vocalization (only after excitatory amino acid agonists). The intrathecal co-administration of the NMDA antagonist, 2-amino-5-phosphonovaleric acid (APV, 10 nmol), inhibited behavioral responses to NMDA (10 nmol) and substance P (10 nmol) but not to kainate (3 nmol). Co-administration of the non-NMDA antagonist, 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX, 10 nmol), suppressed responses to kainate (3 nmol), AMPA (3 nmol) and substance P (10 nmol) but not to NMDA (10 nmol). Co-administration of the substance P antagonist, CP-96, 345 (3 nmol), inhibited the behavioral responses to substance P (10 nmol), but not to NMDA (10 nmol), kainate (3 nmol) and AMPA (3 nmol). The results suggest that the aversive behavior induced by intrathecal NMDA and non-NMDA agonists is mediated by activation of the corresponding glutamate receptors, but not by NK-1 receptors, and that the behavioral action of intrathecal substance P is mediated not only by direct activation of NK-1 receptors but also indirectly by NMDA and non-NMDA receptors for glutamate.

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Microsomal aldehyde oxygenase (MALDO) activity for 9-anthraldehyde (9-AA) was significantly higher in the male than in the female adult rat liver. 9-AA MALDO activity was also significantly enhanced by pretreatment with dexamethasone and phenobarbital, whereas it was not significantly changed by 3-methylcholanthrene or acetone. Several cytochrome P450 isozymes purified from rat hepatic microsomes were able to catalyze the oxdation of 9-AA to 9-anthracene carboxylic acid (9-ACA) in the presence of NADPH, NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. Under the ordinary conditions of the reconstituted system, the catalytic activities (nmol/mim/nmol P450) of cytochrome P450s, 2A1, 2B2, 2C6, 2C11 and 3A2 were 1.53 (1.37 in the presence of cytochrome b₅), 1.20 (2.06), 4.87 (7.75), 18.0 (21.6) and 0.90 (1.17), respectively. Cytochrome P450 2C11 (CYP 2C11) showed the highest catalytic activity of the cytochromes examined. In the reconstituted system using the lipids extracted from microsomes, CYP 3A2 more effectively catalyzed the oxidation of 9-AA to 9-ACA, and its catalytic activity (nmol/min/nmol P450) was 3.33 or 6.61 in the absence or presence of cytochrome b₅, respectively. The antibody against CYP 2C11 inhibited by 90% the hepatic microsomal oxidation of 9-AA MALDO activity in adult male rats, but the activity was not inhibited by antibody against CYP 3A2. These results show that the individual forms of cytochrome P450 have a catalytic activity for the oxidation of 9-AA to 9-ACA, and that CYP 2C11 is the major constitutive catalyst of 9-AA NALDO activity in untreated adult male rat liver.

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This paper reports the role of glutathione (GSH) in the crystalline lens as an antioxidant against the oxidation of lens protein. GSH levels in normal lenses decreased gradually with increasing age, from approximately 5 μmol per g lens (wet weight) to 3 μmol per g lens (wet weight). On the other hand, levels of oxidixzed GSH in the lenses increased until the age of 40. After that, it remained almost constant at the level of approximately 0.9 μmol per g lens. Protein-bound GSH levels in both soluble and insoluble lens proteins dropped noticeably in the 50-year and older age groups, although there were significant differences in levels between both fractions. A decrease of tryptophan and tyrosine residues in lens proteins was proportional to a decrease in GSH levels in the lens as a result of aging. Those residue levels in the cataractous lenses were approximately half those in the normal lens proteins, and GSH levels in such lenses were almost one-tenth that in the normal lens.This study revealed that GSH may play an important role in preventing the oxidation of lens oproteins from various oxidants. Furthermore, it is conceivable that these normal changes in GSH levels in the lenses increase the vulnerability of the lens to senile cataractogenesis.

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For a new drug delivery system of 5-fluorouracil, we prepared prodrugs possessing certain desired properties. The prodrugs, 1-(N-4-chlorophenyl-N-methylcarbamoyl)-5-fluorouracil and 1-(N-2, 4-dichlorophenyl-N-methylcarbamoyl)-5-fluorouracil, contain high serum albumin binding potency and a comparably long half life in the bloodstream in vivo to Tegafur. These two prodrugs are expected to be retained in the bloodstream as a polymeric complex with albumin and to circulate in the body for a long time, like a polymeric prodrug.

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Catalytic RNAs recognize specific sequences of RNA and cleave at a specific site. In this study, we designed hammerhead ribozymes with a thermodynamically stable loop of the sequence 5'C(UUCG)G3' to prevent the aggregation of ribozymes with hammerhead structures. The cleavage activities of these ribozymes were examined using a synthetic pentadecamer with the sequence for the c-Ha-ras mRNA mutated at codon 12 (GG___-U→GU___-U). For in vivo studies, we constructed a plasmid which expressed a highly active ribozyme targeted against the mutated c-Ha-ras mRNA. When this ribozyme-encoding gene and the activated c-Ha-ras gene were cotransfected into NIH3T3 cells, morphologically normal cells were obtained. We also determined that the expression of the c-Ha-ras gene was inhibited in these cells. These results show that ribozymes containing this stable hairpin loop are useful for the rebulation of specific gene expression in vivo.

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Transdermal administration of emedastine was tested in vitro and in vivo. In the diffusion cell method in vitro, emedastine free base was more permeable by transdermal administration than emedastine difumarate. Emedastine had higher permeability in hydrophobic vehicles than in hydrophilic vehicles, and was most permeable in fatty acid monoesters. It was suggested that the change in permeability of emedastine from these vehicles was dependent on the change in its partition from the vehicle to the skin. In studies using rabbits in vivo, emedastine had high permeability from fatty acid monoesters and fatty acid diesters as found in in vitro studies, and bioavailability of the drug after transdermal administration was greater than that after peroral administration. The flux of emedastine in vitro was correlative with the pharmacokinetic parameters in vivo. Consequently, it is clear that transdermal permeability of emedastine is very high and that the drug may be efficacious in the system after administration by these means.

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The transport mechanism of choline was examined using rat renal brush-border membrane vesicles in comparison with tetraethylammonium transport. The stimulatory effect of an outward H⁺ gradient on choline uptake was weak compared with that on tetraehylammonium uptake. [¹⁴C]Tetraethylammonium uptake was cis-inhibited and trans-stimulated by choline, but the effects were less pronounced than those produced by unlabeled tetraethylammonium. [³H]Choline uptake was trans-stimulated by unlabeled choline, but not by tetraethylammonium. An interior-negative membrane potential induced marked stimulation of choline uptake against its concentration gradient (overshoot phenomenon), and the uptake was saturable with an apparent K_m of 0.77 mM. Various compounds such as hemicholinium-3 inhibited the choline uptake by renal brush-border membrane vesicles, but a sulfhydryl reagent did not. These findings suggest that choline can be actively transported by a carrier-mediated system driven by cell interior-negative membrane potential in renal brush-border membrane, and this system may play an important role in the tubular reabsorption of choline.

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The efficacy of percutaneous elcatonin (EC), a hypocalcemic peptide, in the treatment of experimental osteoporosis in rats was evaluated in vivo. Additionally, the effect of the combined use of EC and active vitamin D₃ (1, 25(OH)₂D₃) for the treatment was compared with those of three other groups : 1, 25(OH)₂D₃ alone, estradiol plus 1, 25(OH)₂D₃, and a placebo, and low calcium diet (low Ca). The EC transdermal system and the EC plus 1, 25(OH)₂D₃ system, applied to the rat abdominal skin 6 times for 48 h, significantly increased the ash weight and calcium content of the tibia in the rats, compared with those of placebo group (p<0.05). The EC systems also slightly lowered the alkaline phosphatase activity in plasma of the morbid rats, without a difference in the plasma calcium content. These EC systems were superior to the 1, 25(OH)₂D₃ system and the estradiol plus 1, 25(OH)₂D₃ system in improving osteoporotic parameters. Thus, the EC systems were concluded to be an efficient drug delivery system for Paget's disease and osteoporosis.

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We kinetically examined tissues responsible for the conjugative metabolism (glucuronidation and sulfation) of a component in a crude drug, liquiritigenin (LG; 2, 3-dihydro-7-hydroxy-2-(4-hydroxyphenyl)-(S)-4H-1-benzopyran-4-one) in rats in vivo. LG has been found to form five kinds of conjugates (4'-O-glucuronide (M1), 7-O-glucuronide (M2), 4', 7-O-disulfate (M3), 4'-O-glucuronide-7-O-sulfate (M4) and 7-O-glucuronide-4'-O-sulfate (M5)). Analysis based on metabolite kinetics [K. S. Pang, J. Pharmacokin. Biopharm., 13, 633 (1985)] of the area under the plasma concentration curves (AUC_plasma) and cumulative biliary excretions (Ai_bile) of the ligands after intravenous or hepatic portal venous administration of LG revealed that the liver has the ability to generate all the metabolites. For M1 and M2, the apparent biliary excretion clearance (CL_{bile, app}) obtained by dividing the biliary excretion rate for the metabolite by the plasma concentration of the metabolite decreased with time, confirming that M1 and M2 were formed in the liver. To further analyze the formation rate constants for metabolites in each tissue, we measured the ligand content in several tissues after intravenous administration of LG. By correcting the content of metabolites that were taken up from the plasma, we found that the formation rates per gram of tissue were largest in the liver, except for M3. The metabolic capability of the kidney for M1 and M2 was 15% and 60%, respectively, to that of the liver whereas for M3, the metabolic ability of the kidney was 2.5-fold greater than that of the liver. In contrast, the ability in other tissues was negligible. Considering the weight of each organ in rats, the liver was most responsible for the formation of metabolites, except for M3, where renal conjugation was comparable to hepatic conjugation. The order of formation rate in the liver was M2>M1»M3, M4 and M5, while that in the kidney was M2»M1 and M3. These results were supported by experiments in hepatectomized rats. We could thus quantitatively estimate the formation rate constant for each metabolite in each organ in vivo.

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Doxorubicin, pirarubicin, and FAD-104, but not aclarubicin or MX 2, flattened the morphology of NIH3T3 cells that had been transformed by human H-ras and K-ras. The effect appeared on almost all cells, as early as 2 d following exposure to the antibiotics at concentrations inhibiting cell growth by 50% or more. The morphological alteration accompanied other nornal cell phenotypes, such as the restoration of actin atress fibers, anchorage dependence of cell growth and an increase in nucleoside diphosphate (NDP) kinase activity. NIH3T3 cells transformed by src and other tumor cell lines responded less prominently, if at all.

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The cytotoxicity of monoterpenes, percutaneous absorption enhancers, to cultured human skin cells was investigated in order to quantitatively estimate their skin damage. A neutral red bioassay with epidermal keratinocytes and a contraction test of collagen gel in which dermal fibroblasts were cultured were employed for evaluating the cytotoxicity of terpenes. In the neutral red bioassay, keratinocyte proliferation was inhibited on the addition of terpenes, and cell survival remarkably decreased with an increase in the concentration of terpenes fed into the culture well. When the fibroblasts were cultured in a collagen gel matrix, the lattice of collagen contracted as the cells grew. Therefore, the application of cytotoxic agents brings about an inhibition of collagen gel contraction induced by the fibroblasts. Strong inhibition was observed in the cases of hydrocarbons in terpenes, and the inhibition was dependent on the concentration of these compounds added in the culture medium. The cytotoxicity of terpenes was compared with the skin damage evoked by the application of terpenes in rats in vivo. As a result, it was considered that the skin irritation caused by terpenes was predictable to a certain extent by means of the cytotoxic study of cultured human skin cells.

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To understand the mechanisms responsible for lipopolysaccharide (LPS)-induced enhancement of angiotensinogen synthesis in the liver, studies were carried out in rats with repeated doses of LPS. The administration of sublethal dose (50 μg, i.p.) of LPS to rats resulted in increase in serum levels of tumor necrosis factor (TNF) and interleukin-6 (IL-6), which attained their maximal levels by 1 and 2-4 h, respectively. Serum levels of angiotensinogen and α₂-macroglobulin, a typical acute-phase protein in the rat, were also increased by a primary LPS challenge, and their maximal levels for the formation of TNF and IL-6 were delayed with peaks at 12 and 48 h, respectively. Repeated i.p. administration of LPS (50 μg/d) for 5 consecutive days induced a hyporesponsiveness to its subsequent administration in terms of increasing serum TNF, IL-6 and α₂-macroglobulin. In these LPS-tolerant rats, either LPS-induced elevation of angiotensinogen concentration in serum or angiotensinogen mRNA levels in liver was completely eliminated. Angiotensinogen synthesis in rat hepatoma H4 cells was enhanced in vitro by the addition of sera which had been collected 2 or 4 h after a primary injection of LPS, while not by sera collected from LPS-pretreated rats after a secondary LPS exposure. These results indicate that LPS-induced enhancement of angiotensinogen synthesis in the liver is desensitized in rats after repetitive LPS exposure, presumably by the failure of LPS-induced IL-6 production.

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Studies were performed to characterize the opioid receptors in guinea pig brain using the radiolabeled opioid antagonists, [³H]naloxone and [³H]diprenorphine and the κ-agonist [³H]U-69593. The binding of [³H]U-69593 to guinea pig cerebellar membranes was reduced by NaCl, guanyl-5'-yl-imidodiphosphate (GppNHp) and NaCl + GppNHp, and [³H]naloxone binding to cerebellar membranes was also reduced by NaCl and GppNHp. In the guinea pig cerebral cortex and striatum and the rat cerebellum, [³H]naloxone binding was not affected significantly by GppNHp in the presence or absence of 100nM [D-Ala², N-Me-Phe⁴, Gly⁵-ol]enkephalin (DAMGO) and [D-Ala², D-Leu⁵]enkephalin (DADLE). Guinea pig cerebellar [³H]diprenorphine binding was not affected by NaCl, GppNHp or NaCl + GppNHp. Furthermore, [³H]naloxone binding was reduced after pretreating cerebellar membranes with N-ethylmaleimide (NEM), which also attenuated GppHNp-induced inhibition of cerebellar [³H]naloxone binding. These results suggest that the properties of [³H]naloxone binding in guinea pig cerebellum differ from those in other brain regions and rat cerebellum, and that the interaction of [³H]naloxone and [³H]U-69593, but not [³H]diprenorphine, with guinea pig cerebellar opioid receptors is associated with a G-protein.

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We attempted to develop a fluorescent probe superior to conventional ones (8-anilino-1-naphthalenesulfonate and auramine O) for use in the study of drug-binding sites on α₁-acid glycoprotein (AGP). It was found that quinaldine red (QR) strongly bound to AGP and had an enhanced fluorescence in the presence of AGP at a longer wavelenght, although QR was rarely fluorescent in an aqueous or albumin solution. The binding parameters of QR to AGP were K : 1.3×10⁶M^-1 and n : 0.9, using the fluorometric titration method. The fluorescence of QR in the AGP solution, however, was markedly quenched in the presence of basic drugs, indicating that these drugs competitively displaced QR from its binding site; the results were in good agreement with those in the literature. The good relationship between binding affinities and partition coefficients suggested that hydrophobic forces were involved in the binding of basic drugs to AGP. Moreover, the polarity of the binding site of AGP estimated from the relationship between the emission maximum of QR and Z values was 70, which corresponds to the same Z value of acetonitrile. These results distinguish QR from other conventional AGP probes as a better fluorescent probe by which to understand drug-AGP interaction and the characterization of binding sites on AGP in more detail.

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Two-stage carcinogenesis of mouse skin papillomas induced by 7, 12-dimethylbenz[α]anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA), and mouse pulmonary tumors induced by 4-nitroquinoline-N-oxide (4NQO) and glycerol, were inhibited by digitoxin (1).

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The mitogenicity and lethal toxicity of four synthetic lipid A analogs, glucosamine-4-phosphate with a 7-hydroxy-heptanoyl group (A-166), with a 7-phenyl-heptanoyl group (A-167), or with a 8-(1-phenyl-hexanoyl)-nonanoyl group (A-168), at the C-2 and C-3 positions, and glucosamine-6-phosphate with the same substituents as A-168 at C-2 and C-3 (A-169), were compared. The compound A-166 exhibited no mitogenic activity at various concentrations ranging from 3.13 to 50 μg/ml in the splenocytes of BALB/c mice, but A-167 exhibited weak mitogenic activity at concentrations of 12.5 and 25 μg/ml. A-168 and A-169, as well as A-103, glucosamine-4-phosphate carrying (R)-3-tetradecanoyl-oxytetradecanoyl groups, have remarkable mitogenic activity at concentrations ranging from 12.5 to 100 μg/ml; the activity of A-169 (6-phosphate) was stronger than that of A-168 (4-phosphate). Compound A-167 failed to cause death at doses of 25 and 50 μg/mouse in galactosamine-loaded C57BL/6 mice while A-166 and A-169 were toxic to 2 out of 6 mice at 50 μg/mouse; no deaths were observed at 25 μg/mouse. A-168 showed the highest toxicity of any of the compounds tested at 25 and 50 μg/mouse. The lethal effect of A-103 appeared to be somewhere between that of A-168 and A-169. These findings indicate that lipid A analogs, carrying an aromatic alkyl group as well as a hydroxyacyl group, are mitogenic and lethal when given to mice.

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In an attempt to evaluate its pharmacokinetics, [³H]evodiamine, which is one of the characteristic alkaloids of Evodia fruit was synthesized. The pharmacokinetics of [³H]evodiamine were investigated in rats.In plasma, the main source of radioactivity was a metabolite of d-evodiamine (EM). One hour after oral administration of 200 μg/kg of [³H]evodiamine, the radioactivity level in the plasma was maximal. The radioactivity declined in a biphasic manner with half-life times of 1.6 and 78.4 h. The distribution volume was 560 ml/kg. Radioactivity in tissues was higher in the liver, kidney, heart, lung, and adipose tissue than in plasma, but radioactivity in other tissues it was lower than that in plasma. In all tissues the radioactivity proportionally decreased to the level of that in plasma.At 24 h after administration, 19% and 63% of orally administered radioactivity was excreted in urine and bile, respectively.

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The distribution of fractionated heparin in a primary culture of rat parencymal hepatocytes was investigated optically using the fluorescence labelled drug and confocal imaging system with an inverted fluorescence microscope. The cell-associated fluorescein isothiocyanate (FITC)-fractionated heparin was observed to increase in conjunction with incubation time and also to localize, suggesitng an internalization to cell organella with the exception of the nuclei.

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Significant differences of mutagenic activities between the enantiomers of chiral epoxides, N-nitrosoamines, and halides were exhibited in Ames assay using Salmonella typhimurium TA100.

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The low-sulfated chondroitin 4-sulfate(LSC) chain from human urinary trypsin inhibitor was purified and the structure was characterized. After hyaluronidase SD digestion of LSC, an oligosaccharide which contains the linkage region could be botained. The structure of oligosaccharide was analyzed by HPLC and 500 MHz ¹H-NMR spectroscopy. The analytical results revealed that 4-O-sulfo GalNAc residues were located in the neighborhood of the linkage region.

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