Polyacetylenic alcohols such as panaxytriol, panaxynol and panaxydol isolated from the roots of Panax ginseng C. A. MEYER have antiproliferative activity against various cultured tumor cells. Anti-panaxytriol antibody was obtained by immunizing rabbits with panaxytriol-bovine serum albumin conjugates. Although the antibody reactivity was directed mainly toward panaxytriol, there was a slight cross-reactivity with other polyacetylenic compounds. The antibody was, therefore, used for screening a large number of crude drugs for polyacetylenic compounds such as panaxytriol. Methanol-extracts from 31 crude drugs were examined. Significant reactivity was observed in 15 methanol-extracts from Aralieaceae, Compositae and Umbelliferae as reported by other investigators. Three out of the 15 crude drugs were selected for determination of the potent cross-reactive compounds. Four kinds of cross-reactive compounds were isolated by silica gel column chromatography, monitoring each fraction using the enzyme-linked immunosorbent assay (ELISA). Among them, panaxynol and heptadeca-1, 8-diene-4, 6-diyne-3, 10-diol were identified from Saposhnikoviae Radix. Falcarindiol was newly identified from Peucedani Radix. A new polyacetylenic alcohol, 9, 10-epoxy-16-hydroxy-octadeca-17-ene-12, 14-diyne-1-al, was also isolated from Foeniculi Fructus. All these polyacetylenic alcohols inhibited the growth of a human gastric adenocarcinoma cell line, MK-1 cells, in a dose-dependent manner. These results indicate that the antibody against panaxytriol is an effective tool for"screening"antiproliferative polyacetylenic compounds.

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To investigate the effect of ethanol or colchicine on the intracellular proteolytic processing of lysosomal β-glucuronidase, which is considered to occur in the Golgi complex in the intracellular sorting pathway, three rat liver Golgi subfractions, GF-1, GF-2, and GF-3, were isolated from ethanol-or colchicine-treated rats, and the electrophoretic patterns of the extracted Golgi β-glucuronidase on polyacrylamide gel were examined. The isolated Golgi subfractions from the drug-treated rats gave a better yield of fraction than that from the control rats. The enzymatic characterization of these three subfractions showed no significant contamination by other subcellular structures such as plasma membranes, microsomes, or lysosomes, and no inhibitory effect of the drugs was observed. On the other hand, suppressed galactosyltransferase activity, a marker enzyme of the Golgi complex, was detected in the colchicine-treated rats. The electrophoretic pattern of Golgi β-glucuronidase on polyacrylamide gel revealed one major band which moved to the same position as the lysosomal enzyme type in the control rats. In contrast, in the ethanol-and colchicine-treated rats, Golgi β-glucuronidase was found to have two major bands stained for enzyme activity resulting from a mixture of microsomal-and lysosomal-type enzymes. These results suggested that the post-translational modification step, during conversion from a microsomal-type enzyme to a lysosomal-type enzyme, was apparently inhibited. Taken together, these findings indicated that ethanol or colchicine administration to rats caused an inhibitory effect on the intracellular post-translational modification of Golgi β-glucuronidase destined for targeting to the lysosomes.

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To investigate the intracellular processing event for lysosomal cathespin L, we examined the effect of leupeptin, a non-covalent cysteine proteinase inhibitor, on the intracellular processing kinetics of cathepsin L as analyzed by pulse-chase experiments in vivo with [³⁵S] methionine in primary cultures of rat hepatocytes. This revealed that cathepsin L was initially synthesized as a proenzyme of molecular weight 39 kDa and the proenzyme was subsequently processed to the mature form of the enzyme, 30 and 25 kDa. In the leupeptin-treated cells, the proteolytic conversion of cellular procathepsin L, of molecular weight 39 kDa, to the mature enzyme was significantly inhibited and considerable amounts of proenzyme were found in the cell after 8 h chase periods. Furthermore, the subcellular fractionation experiment demonstrated that the intracellular processing of procathepsin L in the high density lysosomal fraction was significantly inhibited and that considerable amounts of the procathepsin L form were still observed in the dense lysosomal fraction after a 2 h chase period. These results suggest that leupeptin treatment caused significant inhibition of the intracellular maturation of cathepsin L. These findings show that cysteine proteinase plays an important role in the intracellular proteolytic processing and activation of lysosomal cathepsin L in vivo and that this processing event occurs within the lysosomes.

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We investigated the effects of selective β₁ adrenoceptor stimulation on oxygen tension (pO₂) in the myocardium of anesthetized dogs. A β₁-selective full agonist, T-0509 (0.01-0.05μg/kg, i. v.), caused positive inotropic and chronotropic effects, and increased left circumflex blood flow, although it did not change arterial blood pressure. These effects were inhibited by bisoprolol (10μg/kg), but not by ICI 118551 (30μg/kg). Under control conditions, subepicardial pO₂ (pO_2epi) and subendocardial pO₂ (pO_2endo) were approximately 33 and 27 mmHg, respectively. T-0509 (0.05μg/kg) decreased pO_2epi in all cases, with a mean decrease of 2.6±0.5mmHg, and this was significantly inhibited by bisoprolol. T-0509 caused an increase (7 out of 10 dogs) or a slight decrease (3 out of 10) in the pO_2endo; the mean increment was 2.0±1.3mmHg (n=10). Isoproterenol (0.01-0.05μg/kg, i. v.) exerted positive inotropic and chronotropic effects that were sensitive to bisoprolol, and a hypotensive effect that was sensitive to ICI 118551. Isoproterenol caused an increase in blood flow that was sensitive to ICI 118551. Isoproterenol (0.05μg/kg) decreased pO_2epi in all cases, with a mean decrease of 2.7±0.5mmHg, which was significantly inhibited by bisoprolol. Isoproterenol caused an increase (5 out of 10) or a slight decrease (5 out of 10) in the pO_2endo; the mean increment was 1.1±1.2mmHg (n=10). These results suggest that, while coronary arteries dilate during selective stimulation of β₁-adrenoceptors or nonselective stimulation of β-adrenoceptors, the oxygen supply is insufflcient in pO_2epi due to a marked increase in oxygen demand. However, pO_2endo does not readily decrease under these conditions.

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We studied nephrotoxicity following a single injection of cisplatin (CDDP) at low dose (1mg/kg) in two groups of rats aged 52 weeks (adult, A group) and 9 weeks (young, Y group). Renal platinum (Pt) was detectable in both groups 3 h after the CDDP injection, and, from 6 h to 3 d after injection, its level in the A group was higher than that in the Y group. Compared with the levels in age-matched normal rats (non-treated rats examined at time zero), the plasma urea nitrogen and creatinine levels in the A group were significantly increased, beginning 3 d after CDDP injection, while those in the Y group showed little change for 10 d after injection. Beginning 3 d after CDDP injection, the level of renal metallothionein in the Y group increased, while that in the A group decreased remarkably. The renal tissue levels of the heavy metals Zn, Cu, Mn showed similar patterns. There were no significant changes in the renal lipid peroxide (LPO) level in either the A and Y group at any time measured after CDDP injection compared with the value in the respective age-matched untreated group. Morphological evaluation demonstrated degeneration of the proximal tubules in the A group 3d after CDDP injection. These results suggested that the renal disorders observed following CDDP injection in the A group were caused by mechanisms other than LPO, such as decreased tissue metabolic function associated with aging.

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Methanolic extract (CM-ext) from tubers of Corydalis turtschaninovii forma yanhusuo has been screened for activity in experimental models of types I-IV allergy. In type I allergic models, CM-ext at doses of 200, 500mg/kg, p. o. inhibited 48-h homologous passive cutaneous anaphylaxis (PCA) in rats which is related to IgE, and 4-h heterologous PCA in guinea pigs which is related to IgG. The inhibition of CM-ext on 48-h PCA was also recognized in adrenalectomized rats. CM-ext exhibited the inhibitory effect on formation of IgE antibody in BALB/c mice. In type II allergic model, it was found that CM-ext inhibits reversed cutaneous anaphylaxis (RCA). In type III allergic model, CM-ext showed the inhibitory effect on direct passive arthus reaction (DPAR) in rats. Furthermore, in type IV allergic model, CM-ext had the inhibitory effects on induction phase and effector phase in picryl chloride-induced contact dermatitis (PC-CD). It also showed therapeutic action on PC-CD. These results indicated that CM-ext not only inhibits antibody-mediated allergic reactions but also influences cell-mediated allergic reactions and should be recognized as a potent material for allergic reactions, although the mechanisms and active principles of CM-ext have not yet been completely determined.

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This study was designed to clarify the relationship between the properties of propiverine and skin penetration, and to compare the in vitro penetration characteristics of propiverine and terodiline through rat skin. Propiverine in both hydrochloride and free forms penetrated across the skin extremely slowly, with a 2.6 times higher flux in the hydrochloride than that in the free base, in the absence of enhancers. Various enhancers failed to enhance the penetration of propiverine hydrochloride, whereas the same agents slightly increased the flux of the free form, these being due to the slow release rate of the free form from the gel formulations, an extremely high lipophilicity (log P_oct/water >4.97), much less solubility (0.141 mg/ml) and a large partition capacity of the drug to skin components. Terodiline in both forms was able to rapidly penetrate through the skin, even in the absence of enhancers, with 20.2 and 9.8 times higher fluxes respectively, than the corresponding forms of propiverine. The high penetration characteristics of terodiline would be due to a suitable lipophilicity, low binding property as well as the structural masking from the binding to the epidermal components. Propiverine hydrochloride penetrated through the stratum corneum 4.4 times and viable skin 3.1 times higher than through full-thickness skin, while the fluxes of terodiline through the stratum corneum and viable skin were similar to each other, with high penetration rates for each form.

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Intestinal absorption characteristics of azetirelin, a new thyrotropin-releasing hormone (TRH) analogue, were studied in rats by means of in situ closed loop and in vitro everted sac experiments. Plasma concentrations of azetirelin obtained in the in situ closed loop experiments were not significantly different among the intestinal segments. Area under the plasma concentration-time curve (AUC) of azetirelin following administration into the duodenal loop increased in proportion to the dose. The serosal to mucosal concentration ratio of the analogue in the everted sac experiment was constant over the mucosal drug concentration range of 0.01-10 mM. There was no directional difference in the transfer rate of azetirelin across the everted and non-everted sacs of the duodenum. Furthermore, its transport across the duodenum was not influenced by low incubation temperature (25°C), addition of dipeptide (Gly-Gly), or pretreatment of the mucosal surface with 2, 4-dinitrophenol, while that of TRH was inhibited under these conditions. These results suggest that the intestinal absorption mechanism of azetirelin is different from that of TRH, and that azetirelin is predominantly transported via a passive diffusion.

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The effect of temperature on the local hepatic disposition of BOF-4272 [(±)-8-(3-methoxy-4-phenylsulfinyl-phenyl) pyrazolo [1, 5-a]-1, 3, 5-triazine-4-olate], a new drug used to treat hyperuricemia, was investigated by means of a perfusion experiment following the pulse input into the portal vein of rat, in which the temperature of the perfusate was changed from 37°C down to 4°C. The same perfusion experiment was also attempted using bovine serum albumin (BSA) as a reference substance to compare with the hepatic disposition of BOF-4272. The elution time profiles of BOF-4272 and BSA from the liver into the hepatic vein were evaluated by moment analysis. The recovery ratio (F_H) and the mean transit time (t^^-_H) of BOF-4272 were 22.8±3.3% and 0.111±0.008 min at 37°C, respectively. Both F_H and t^^-_H significantly increased with the decrease in the temperature of the perfusate, 3 times and 2 times greater at 4°C than at 37°C, respectively. The F_H and t^^-_H of BSA were 98.3±4.5% and 0.129±0.013min at 37°C, respectively. These parameters of BSA were independent of temperature, while those of BOF-4272 showed a definite dependency on temperature. A new estimation method for the elimination rate constant (k_e) and the partition ratio (k') in the dispersion model was developed by rearranging the theoretical equations of F_H and t^^-_H. The index for the elimination (k_e) of BOF-4272 decreased with the decrease in temperature, while the index for the distribution (k') increased with the decrease in temperature. This result shows that the metabolism (or the biliary excretion) decreased and the distribution increased with a decrease in temperature, indicating that the hepatic metabolizing pathway which is presumably temperature-dependent is blocked, and the blocked portion of BOF-4272 thus returns back to the perfusate at the low temperature.

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Studies on controlled release dosage forms were conducted by using waxy materials for a newly developed anti-allergy drug, 6-methyl-N-(1H-tetrazol-5-yl)-2-pyridinecarboxamide (TA-5707F), which is not maintained at an effective level in blood for a long duration. Four kinds of tablets were prepared by changing the amount of hydrogenated oil (K3 wax) and polyethyleneglycol-6000 in the tablets. Then, they were administered orally to beagle dogs, and the TA-5707F concentration in the plasma was determined by a HPLC method. The pharmacokinetic parameters were estimated and compared with the results of the in vitro dissolution test determined by the JP paddle method and by the disintegration method. The linearity between the in vivo mean dissolution time (MDT) and in vitro MDT was good in both in vitro dissolution methods. However, the MDTs obtained by the disintegration method were one-third of the in vivo MDT, while those obtained by the paddle methods were 3 times higher. This suggests that both the diffusion of TA-5707F through the waxy matrix and the erosion of the wax matrix caused by the gastrointestinal (GI) tract mobility contributed to the in vivo dissolution mechanism. The blood levels were very low when the tablet was administered after giving food. The prolongation of resident time in the stomach and the low solubility of TA-5707F in an acidic medium seemed to be related to the phenomena. By the depression of GI motility using propantheline bromide, the blood levels could be markedly prolonged and the area under the plasma concentration-time curve (AUC) increased 1.3 times.

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The concentrations of dopamine, 3, 4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in rat striatum were increased after the i. v. administration of chlorpromazine (CPZ). Assuming that the enhancement of dopamine concentration in the striatum after CPZ administration is caused by the release of dopamine from the dopamine neuronal terminals, the relationship between the enhancement of dopamine concentration in the striatum and CPZ concentration in the cerebrospinal fluid (CSF) and the striatum were analyzed using the sigmoid E_max model. The enhancement of dopamine concentration in the striatum could be described quantitatively by this model. The time courses of DOPAC and HVA concentration in the striatum after CPZ administration were analyzed using the dopamine metabolism model, which has an apparent first-order clearance from dopamine to DOPAC and HVA, and also using the Michaelis-Menten type elimination kinetics of DOPAC and HVA. The values of the metabolism parameters for DOPAC and HVA were fixed to the estimated values of the L-dopa study. The calculated values of DOPAC and HVA concentrations in the striatum were greater than those of the observed data. The elimination parameters for DOPAC and HVA were reestimated by the nonlinear least squares method. The time courses of DOPAC and HVA concentration in the striatum could be described using these reestimated elimination parameters. These results indicated that the turnover rate of dopamine and dopamine metabolites, DOPAC and HVA in the striatum after CPZ administration is different from that after L-dopa administration. Thus, analysis using the dopamine metabolism model is a useful method not only for the quantitative explanation of the relationship between the CPZ concentration and the enlargement of dopamine concentration in the striatum, but also for the examination of the effect of CPZ on the turnover rate of dopamine and dopamine metabolites, DOPAC and HVA in the striatum.

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The anti-human immunodeficiency virus (HIV) activity of polyoxometalates of representative structural families, such as Keggin, lacunary Keggin, trivacant Keggin, Keggin sandwich, Wells-Dawson and Wells-Dawson sandwich, was determined using two strains of HIV type 1 (HIV-1_HTLV-IIIBand HIV-1_SF-2H). The compounds were preferably selected to cover both polyoxotungstates and polyoxomolybdates in each structural family. In general, polyoxotungstates of Keggin, lacumary Keggin, trivacant Keggin, Keggin sandwich, Wells-Dawson and Wells-Dawson sandwich structures showed anti-HIV-1_HTLV-IIIBactivity, whereas most compounds not included in these structural categories were inactive. Among the compounds with a potent anti-HIV-1_HTLV-IIIBactivity, those of Keggin and its closely related structural families (lacunary Keggin, trivacant Keggin and Keggin sandwich) inhibited the cytopathogenicity and syncytium formation caused by HIV-1_SF-2Hto a much higher extent compared with HIV-1_HTLV-IIIB-related ones. The difference between the spectra of anti-HIV-1_HTLV-IIIBactivity and the specificity for HIV-1_SF-2Hmight result from differential structural requirements in these functions.

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To clarify whether α₁-adrenergic receptors contribute to cardiac hypertrophy, we examined α₁-adrenergic receptor densities in the right and left ventricles of 5-and 20-week-old spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) using [³H] prazosin. Also, we analyzed a pharmacologically distinct subtype of α₁-adrenergic receptors by pretreatment with chloroethylclonidine. The ratio of right ventricular weight to body weight as well as left ventricular weight to body weight was higher in SHR than in WKY at 5 weeks of age. At 20 weeks of age, only the left ventricular weight to body weight ratio was higher in SHR. At 5 weeks of age, the right and left ventricular α₁-adrenergic receptor densities in SHR were greater than those in WKY (LV 1760 vs. 1275, RV 2543 vs. 1521 fmol/mg protein). At 20 weeks of age, only right ventricular α₁-adrenergic receptor density remained higher in SHR (1425 vs. 885 fmol/mg protein). The percentage of pharmacologically distinct α_1A receptor was higher in the right ventricle of SHR than in that of WKY at 5 weeks of age (25% vs. 18%). There were no significant differences in the dissociation constants among the α₁-adrenergic receptor assays. These findings suggest that an increase in α₁-adrenergic receptors might be involved in cardiac hypertrophy in the early phase of hypertension in SHR.

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The antioxidant effects in the liver and kidney obtained from rats fed diets containing 3% green or black tea leaf powder, which were prepared from the same lot tea leaves, were studied using the tissue slice-antioxidant evaluation method with two lipid peroxidation inducers. After 50 d on the diets, liver slices prepared from green and black tea-supplemented rats showed significant inhibitory effects against tert-butyl hydroperoxide-induced lipid peroxidation. These effects, however, were not proportional to the amounts of (-)-epicatechins and antioxidant vitamins in the tea leaves. In the kidney, the antioxidant effect was observed only in the green tea-fed group. A similar antioxidant effect on the kidney was observed after oral administration of a major tea polyphenol, (-)-epigallocatechin gallate (50 mg/kg body weight for. 7d). Liver slices from black tea-fed rats also inhibited bromotrichloromethane-induced lipid peroxidation. These results demonstrated that dietary green and black tea had antioxidant effects on tissue lipid peroxidation ex vivo.

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Regulation of the activity of lanosterol 14α-demethylase (cytochrome P-450_14DM) by dietary cholesterol was studied in rats. In male rats fed a 3% cholesterol diet for 1 and 4 weeks, the activity of 24, 25-dihydrolanosterol 14α-demethylase was decreased in the liver. The cytochrome P-450_14DM protein content detected by immunoblotting was also decreased by cholesterol feeding. These results demonstrate that dietary cholesterol acts as a repressive factor for lanosterol 14α-demethylase. Further, the activity was enhanced by preincubation with phosphatase of the enzyme solution from both the control and cholesterol fed rats at the same rate. This result suggests that regulation of the activity involves phosphorylation of this enzyme.

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Coumestrol, a representative phytoestrogen, inhibited bone resorption-stimulating agent-induced bone resorption, whereas it did not inhibit basal bone resorption of cultured fetal rat limb bone. Coumestrol increased the calcium content of 9-d-old chick embryonic femurs in organ culture, indicating that this phytoestrogen stimulated bone-mineralizing activity. Therefore, coumestrol is a unique substance in that it inhibits bone resorption and, at the same time, stimulates bone mineralization. Coumestrol exhibited less estrogenic activity than 17β-estradiol as estimated by the increase in uterine weight of ovariectomized rats. As coumestrol has less estrogenic activity than 17β-estradiol, it may prove to be a more potent drug against bone diseases including osteoporosis.

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We investigated the antihypertensive effect of sesamin, a lignan from sesame oil, using deoxycorticosterone acetate (DOCA)-salt hypertensive rats. The animals were unilaterally nephrectomized, and then separated into a sham-operated group (sham group) and a DOCA-salt-treated group. The latter was further separated into a normal diet group (control group) and a sesamin-containing diet group (sesamin group). The systolic blood pressure of control group progressively increased in comparison with that of sham group. This DOCA-salt-induced hypertension was markedly suppressed by feeding a sesamin-containing diet. Systolic blood pressure after 5 weeks was 130.6±1.9mmHg in the sham group, 198.1±7.3mmHg in the control group and 152.5±8.4mmHg in the sesamin group, respectively. The treatment with DOCA and salt for 5 weeks significantly increased the weight of the left ventricle plus the septum. However, this increase was signiflcantly suppressed in the sesamin group. When the degree of vascular hypertrophy of the aorta and superior mesenteric artery was histochemically evaluated, there were significant increases in wall thickness, wall area and the wall-to-lumen ratio in the control group, compared with the sham. Sesamin feeding ameliorated the development of DOCA-salt-induced vascular hypertrophy in both the aorta and mesenteric artery. These findings strongly suggest that sesamin is useful as a prophylactic treatment in the development of hypertension and cardiovascular hypertrophy.

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The effect of the vesicle size on in vivo release of daunorubicin, an anthracycline anticancer drug, from liposomes into the circulation was studied in rats. The liposomes were prepared from hydrogenated egg phosphatidylcholine (HEPC) or egg phosphatidylcholine (EPC), cholesterol and dicetylphosphate at a molar ratio of 5 : 4 : 1, and their mean vesicle sizes were adjusted to about 50 and 100 nm. The drug retained in the liposomes and the drug that leaked were separated from plasma by gel filtration. On the assumption that the lipid content does not change, the drug release from each liposome preparation was estimated from the latency (%) calculated from the drug/lipid concentration ratio of the liposome preparation. From HEPC-liposomes with a diameter of 50 nm, the drug was released gradually after intravenous administration, and the cumulative percent release of the drug reached 40% after 240 min. However, from EPC-liposomes with the same size, 50% of the drug was released within 5 min, and more than 90% of the drug was released within 60 min. From HEPC-liposomes with a diameter of 100 nm, no drug release was observed for 240 min after administration. These findings indicate that the vesicle size and the lipid composition of liposome preparation affect the in vivo drug release.

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The objective of this study is to investigate the metabolism of the antitumor drug, irinotecan (CPT-11), to its active metabolite, SN-38, in tumor tissue. Using Walker 256 carcinoma, we prepared a tissue-isolated tumor model : tumor preparation was continuously perfused with Krebs-Henseleit bicarbonate buffer containing 4% bovine serum albumin (BSA) and CPT-11 (10μg/ml), and the concentration of SN-38 in the perfusate was monitored using HPLC. The concentration of SN-38 in the perfusate was gradually increased to a level of 9.69ng/ml 60 min after the start of perfusion. As a control, an aliquot of the perfusate was separately incubated ; however, no significant increase in SN-38 levels was observed. At the end of the perfusion, a part of the tumor tissue was homogenized and the level of SN-38 was determined ; the levels in tumor tissue were 2.2-4.5 times higher than in the perfusate. From above results, CPT-11 was found to be metabolized to its active metabolite, SN-38, in tumor tissue-a desirable feature of an antitumor prodrug.

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The stereoisomeric inversion of (25R)-and (25S)-3α, 7α, 12α-trihydroxy-5β-cholestanoic acid (THCA) in rat liver peroxisome was studied. After incubation of an isomer of THCA-CoA thioester with a peroxisomal fraction, 5β-cholestanoic acids were extracted and optical antipodes were separated and determined by LC/APCI-MS. The transformation of (24E)-3α, 7α, 12α-trihydroxy-5β-cholest-24-en-26-oic acid (Δ²⁴-THCA) formed by acyl-CoA oxidase was also analyzed by GC/NICI-MS. Rapid enzymatic epimerization from either direction was observed prior to biotransformation into (24E)-Δ²⁴-THCA.

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