Cell-specific targeting systems for drugs and genes have been developed by using glycosylated macromolecule as a vehicle that can be selectively recognized by carbohydrate receptors. Pharmacokinetic analyses of the tissue distribution of glycosylated proteins came to the conclusion that the surface density of the sugar moiety on the protein derivative largely determines the binding affinity for the receptors and plasma lectin. Many glycosylated delivery systems have been developed and their usefulness investigated in various settings. Galactosylated polymers, when properly designed, were found to be effective in delivering prostaglandin E₁ and other low-molecular-weight drugs selectively to hepatocytes. In addition, glycosylated superoxide dismutase and catalase were successfully developed with minimal loss of enzymatic activity. A simultaneous targeting of these two enzymes to liver nonparenchymal cells significantly prevented hepatic ischemia/reperfusion injury. On the other hand, galactosylated catalase, a derivative selectively delivered to hepatocytes, effectively inhibited hepatic metastasis of colon carcinoma cells in mice. Finally, hepatocyte-targeted in vivo gene transfer was achieved by synthesizing a multi-functional carrier molecule, which condenses plasmid DNA, delivering DNA to hepatocytes through recognition by asialoglycoprotein receptors, and releasing DNA from endosomes/lysosomes into cytoplasm.

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Two lignin-degrading basidiomycetes, Stereum hirsutum and Heterobasidium insulare, were used to degrade bisphenol A (BPA) in culture, and the estrogenic activity of the degradation products was examined using MCF-7 cell proliferation assays (E-screen) and analysis of pS2 mRNA expression in MCF 7 cells. Both S. hirsutum and H. insulare showed high resistance to BPA 100 ppm, and their mycelial growth was fully completed within 8 d of incubation at 30 °C. It took 7 to 14 d to achieve complete degradation (ca. 99%) of BPA by both fungi. MCF-7 cells proliferated actively at a BPA concentration of 10⁻⁵ M. However, cell line proliferation was significantly inhibited when the cells were incubated in BPA culture media containing S. hirsutum and H. insulare. Similar results were obtained regarding pS2 mRNA expression. The pS2 mRNA expression levels decreased by 1.5-fold in supernatant from BPA treated with S. hirsutum and H. insulare compared with those treated with BPA alone.

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The specificities of antisera against digoxin C-3′ or C-3″ hemisuccinate–bovine serum albumin (BSA) conjugate were assessed by cross-reactivity studies with digoxin metabolites by radioimmunoassay (RIA) using the homologous and the site heterologous tritium-labeled antigens. One of the tracers used was digoxin 3′-hemisuccinyl-[³H]-leucine; the other was digoxin 3″-hemisuccinyl-[³H]-leucine, which had been prepared from digoxin 3″-hemisuccinate. When the tracer with [³H]-leucine at the C-3′ position was used, antisera (I-1, I-3) elicited by digoxin 3′-hemisuccinate–BSA conjugate showed the following cross-reactivity: digoxigenin bisdigitoxoside (0.34%, 76%), digoxigenin monodigitoxoside (0.11%, 65%), digoxigenin (0.02%, 26%) and dihydrodigoxin (9.4%, 1.2%). However, when using the homologous antigen, antiserum (I-1) was highly specific against the digitoxose chain. When the site heterologous antigen, digoxin 3″-hemisuccinyl-[³H]-leucine was combined, this antiserum showed high cross-reactivity to digoxin degradation products. This digoxin RIA using antiserum (I-1) with the homologous antigen measures unmetabolized digoxin. On the other hand, the RIA system using antiserum (I-3) with the homologous antigen had cross-reactivity with the metabolites in accordance with their relative cardio-activities, so this system would be useful in therapeutic drug monitoring of digoxin.

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The degradation pathways of Amadori compounds in vivo have not been fully understood because of the lack of suitable techniques although the compounds are considered to be key intermediates in glycation, which contributes to the development of pathologies associated with various chronic and age-related diseases. A new approach using ¹³C labeling and NMR spectroscopy has thus been described to obtain more insight into the degradation pathways. A ¹³C-labeled model Amadori compound, [1-¹³C]N^ε-(1-deoxy-D-fructos-1-yl)hippuryl-lysine, was synthesized to investigate the degradation pathways of the sugar moiety. The labeled compound was then incubated under aerobic and physiologic conditions, followed by analysis using ¹³C-NMR spectroscopy to obtain the degradation profile. Consequently, after 28-d incubation at least nine ¹³C signals due to ¹³C-labeled products were observed with those due to unlabeled hippuryl-lysine. These labeled products included not only carboxymethylated hippuryl-lysine as the major product and α- and β-glucose but also acetate and formate. These experiments demonstrate the potential of using a ¹³C label and NMR spectroscopy in that the technique provides the comprehensive profiling of the degradation products containing the labeled position in spite of their chemical structures.

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Microvessels are composed of endothelial cells that cover the luminal surface and pericytes that wrap around and along endothelial cells. In the present study, we investigated the regulation of fibrinolysis in cultured human brain microvascular pericytes and endothelial cells after exposure to thrombin. It was found that the regulation in pericytes is different from that in endothelial cells. Specifically, thrombin increases the secretion of fibrinolytic proteins (tissue- and urokinase-type plasminogen activators and plasminogen activator inhibitor-1), resulting in an enhancement of plasminogen activator activity in endothelial cells, whereas the proteinase increases the secretion of only plasminogen activator inhibitor-1 by activation of proteinase-activated receptor-1 and induces suppression of plasminogen activator activity in pericytes. The present data suggest that thrombin regulation of fibrinolytic activity in endothelial cells and pericytes may be involved in the removal of intravascular thrombi and the maintenance of hemostasis, respectively, in human brain microvessels.

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Recently, the pharmaceutical industry has employed the high-throughput method for the evaluation of cytochrome P450 (CYP) inhibition, using a combination of the heterologously expressed enzyme and a fluoregenic substrate. When buprenorphine (BN), a potent mixed agonist-antagonist analgesic, was evaluated by this method, it exhibited potent inhibition of CYP2D6 with an IC₅₀ value of 0.25 μM in recombinant CYP2D6-expressing insect cell microsomes (rCYP2D6 microsomes). In contrast, the IC₅₀ value was 22.7 μM in human liver microsomes (HLM) using a classical method. Although the substrate concentrations in each study were set to near the K_m values, there was a large discrepancy in IC₅₀ values. When we investigated the effect of nonspecific binding to microsomes on the inhibitory potency, with a view to clarifying this discrepancy, the unbound fraction in microsomes (fu,_mic) was 0.06—0.21 and 0.99 in HLM and rCYP2D6 microsomes, respectively. The corrected IC₅₀ value (1.74 μM) using free BN concentrations was much smaller than the uncorrected value. On the other hand, it was observed that the concentration of BN in HLM decreased rapidly due to metabolism by CYP3A4 while that in rCYP2D6 microsomes decreased only slightly. We then investigated the effect of incubation time on the inhibitory potency, since the rapid elimination of BN in HLM could have been a cause of the discrepancy. The IC₅₀ value for BN was observed to decrease slightly from 22.7 to 17.1 μM, following the shortening of the incubation time from 10 to 2 min in HLM. We conclude that nonspecific binding to microsomes of the inhibitor could affect the inhibitory potency against CYP2D6. If this factor is considered, a more precise estimate of the risk of adverse drug interaction could be achieved.

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We have recently demonstrated the inverse correlation between transglutaminase (TGase) activity and DNA synthesis in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) at 1 d after partial hepatectomy. In order to elucidate a role of polyamines as substrates of TGase in the differential growth capacities between PPH and PVH from regenerating liver, we investigated the zonal differences in alteration of ornithine decarboxylase (ODC) activity and polyamines. In two subpopulations, the inverse correlation between DNA synthesis and ε-(γ-glutamyl) lysine (Gln-Lys) cross-linking catalyzed by TGase was demonstrated at 1 d after partial hepatectomy. ODC activity in PPH significantly increased with a peak at 1 d after partial hepatectomy, whereas did not in PVH. Protein-binding SPD in PPH also transiently increased with a peak at 1 d after partial hepatectomy, but did not in PVH. These results suggest that at 1 d after partial hepatectomy, in PPH, the inhibition of Gln-Lys cross-linking by the formation of N-γ-glutamyl SPD leads to the increase of DNA synthesis, whereas in PVH, enhanced formation of Gln-Lys cross-linking leads to the lower DNA synthesis.

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CKD-732 [6-O-(4-dimethylaminoethoxy) cinnamoyl fumagillol hemioxalate] is a new fumagillin anticancer drug that belongs to an angiogenesis inhibitor. Its effect on the central nervous system (CNS), general behavior, cardiovascular-respiratory system and the other organ systems were studied. CKD-732 was intravenously administered with the dosages of 10, 30, 40 or 50 mg/kg and the highest dosage of 50 mg/kg prolonged the hexobarbital-induced sleep time. CKD-732 at the dosage of 50 mg/kg, also, caused the decrease of body temperature from 15 to 120 min after the administration, which was recovered at 240 min. In the study of the effects on gastric secretion, CKD-732 induced the increase of pH and decrease of total acidity. However, CKD-732 showed no effect on general behavior, spontaneous locomotor activity, motor coordination, analgesia, convulsion, mean arterial pressure, and cardiac functions except for heart rate of isolated rat heart, respiration, isolated smooth muscle, intestinal charcoal transport and renal function. Based on the results, we suggested that CKD-732 is safe general pharmacologically at clinical supposed dose (1.75 mg/kg) and demonstrated to have much better safety than other fumagillin derivatives.

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The effects of the chloroform extract of Maytenus obtufisolia MART. roots on locomotor activity, catalepsy test, amphetamine-induced toxicity and active-avoidance test were studied. The results revealed that the extract caused a decrease in spontaneous activity and induced catalepsy in mice up to 240 min. The extract significantly protected the mice against amphetamine-induced toxicity and decreased the conditioned response in rats, in a dose-related manner. The results suggest that the chloroform extract of Maytenus obtusifolia MART. possesses neuroleptic-like properties.

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Dracorhodin perchlorate, an anthocyanin red pigment, induces human melanoma A375-S2 cell death through the apoptotic pathway. Caspase-3, -8, -9, and -10 inhibitors partially reversed the cell death induced by dracorhodin perchlorate. Caspase-3 and -8 were activated, followed by the degradation of caspase-3 substrates, the inhibitor of caspase-activated DNase, and poly-(ADP-ribose) polymerase. Dracorhodin perchlorate upregulated the expression ratio of Bax/Bcl-2 and significantly increased the expression of p53 and p21^WAF1 proteins. The cell death was partially reduced by the mitogen-activated protein kinase c-JUN NH₂-terminal protein kinase (JNK MAPK) inhibitor (SP600125) and p38 MAPK inhibitor (SB 203580), while the MEK inhibitor (PD98059) augmented cell death; the drug induced sustained phosphorylation of JNK and p38 MAPK. Moreover, the Fas agonistic antibody CH-11 has a synergistic effect with dracorhodin perchlorate. The phoshatidylinositol 3-kinase (PI3-K) family inhibitor wortmanin and tyrosine kinase inhibitor genistein rescued the viability loss induced by dracohodin perchlorate. Taken together, dracorhodin perchlorate induces apoptosis in A375-S2 cells via accumulation of p53, alters the Bax/Bcl-2 ratio, and activates caspases and p38/JNK MAPKs.

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Gumiganghwaltang (GMGHT) is an Oriental herbal prescription, which has been commonly used to treat a cold and inflammatory diseases in Korea. However, the mechanism of GMGHT is not clear. In this study, we investigated the anti-inflammatory mechanism of GMGHT in mouse peritoneal macrophages. GMGHT exerted an anti-inflammatory action through inhibiting lipopolysaccaride (LPS)-induced tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in mouse peritoneal macrophages. The maximal inhibition rate of TNF-α, and IL-6 production by GMGHT (1 mg/ml) was 52.31±2.8% and 56.31±3.1%, respectively. In the inflammatory process, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) increased in peritoneal macrophages. GMGHT decreased the protein level of COX-2 and iNOS in LPS-stimulated mouse peritoneal macrophages. In addition, GMGHT inhibited nuclear factor-κB activation and IκB-α degradation. Our study suggests that an important molecular mechanism by GMKHT reduce inflammation, which might explain its beneficial effect in the regulation of inflammatory reactions.

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We studied the effect of Lo Han Kuo (Siraitia grosvenori Swingle) on histamine-induced nasal rubbing and compound 48/80-induced skin scratching behavior in ICR mice. An extract and glycoside (a complex of sweet components) of Lo Han Kuo were used in the study. Both the extract and glycoside caused no significant effect on nasal rubbing or scratching behavior, even at a dose of 1000 mg/kg when administered in a single dose. However, the effect of Lo Han Kuo became clear after repeated administration, and 300 and 1000 mg/kg of both extract and glycoside significantly inhibited nasal rubbing and skin scratching behavior after consecutive treatment for 4 weeks. Both the extract and glycoside inhibited the histamine release induced by compound 48/80 at concentrations of 300 and 1000 μg/ml. From these results, it is assumed that the inhibition of nasal rubbing and skin scratching behavior induced by Lo Han Kuo occurs through a mast cell-dependent mechanism.

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We have previously reported that the N^ε-(carboxymethyl)lysine (CML) adduct, a major structure of an advanced glycation end product, facilitates proliferation of CD34⁺ endothelial progenitor cells budded from cultured choroidal explants and produces immature vessel-like structures in fibrin gel. The CML adduct is accumulated and facilitates immature neovascularization in cultured choroidal explants of streptozotocin (STZ)-induced diabetic rat. The CML-enhanced neovascularization activity is associated with the actions of tumor necrosis factor (TNF) α, vascular endothelial growth factor and platelet-derived growth factor released from the choroidal explant (Kobayashi et al., Biol. Pharm. Bull., 27, 1382—1387 (2004); 27, 1565—1571 (2004)). The present study was investigated an inhibitory effect of a dihydropyridine calcium antagonist nifedipine on TNF α-induced choroidal neovascularization in the STZ-diabetic rat. TNF α (1—100 ng/ml) increased neovascularization of cultured choroidal explants in the age-matched normal rat but did not increase it in the diabetic rat. Anti-TNF α antibody (1 : 1000) decreased the neovascularization in the diabetic rat but not in the normal rat. Nifedipine (1 μM) inhibited TNF α-induced neovascularization of the normal choroidal explant in a non-competitive manner. Nifedipine (1 μM) also inhibited the diabetic state-induced neovascularization and its inhibitory action was reversed by TNF α (1—10 ng/ml). In conclusion, STZ-diabetic state facilitated choroidal neovascularization through the release of TNF α. Nifedipine inhibited the action of TNF α probably by blocking voltage-dependent Ca²⁺ channels in the endothelial progenitor cells of the diabetic choroid.

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Microsomal triglyceride transfer protein (MTP) is essential for the synthesis of both chylomicron in the intestine and very low-density lipoprotein in the liver. An MTP inhibitor, (2S)-2-cyclopentyl-2-[4-[(2,4-dimethyl-9H-pyrido[2,3-b]indol-9-yl)methyl]phenyl]-N-[(1S)-2-hydroxy-1-phenylethyl]ethanamide (implitapide), has been shown to suppress atherosclerosis in apolipoprotein E knockout (apoE KO) mice. To elucidate the antiatherosclerotic mechanisms of implitapide in the mice, we examined the effects on plasma lipid levels, triglyceride (TG) elevation after oral fat loading, and development of atherosclerosis in apoE KO mice fed a Western-type diet. Implitapide at a dosage of approximately 3.2 mg/kg/day significantly reduced both total cholesterol and TG levels during the 8-week treatment period. In addition, implitapide significantly inhibited the increase in plasma TG levels after oral olive oil loading tests conducted after 4 weeks of treatment. After the treatment, implitapide significantly suppressed the atherosclerotic lesion area by 83% compared with a control group. These results provide direct evidence that the antiatherosclerotic effects of implitapide in apoE KO mice are associated with the inhibition of postprandial TG elevation, in addition to the reduction of both plasma total cholesterol and TG levels.

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The cytotoxicity of flavonoids, including apigenin, eriodictyol, 3-hydroxyflavone, kaempherol, luteolin, naringenin, quercetin, rutin, and taxifolin, toward cultured human normal cells, i.e., human lung embryonic fibroblasts (TIG-1) and human umbilical vein endothelial (HUVE) cells, was examined. When these normal human cells were incubated with each flavonoid in culture medium for 24 h, some of the flavonoids showed considerable cytotoxicity at relatively high concentrations and in a dose-dependent manner. 3-Hydroxyflavone, luteolin, and apigenin were more toxic toward TIG-1 cells than the other flavonoids, and luteolin, 3-hydroxyflavone, and quercetin were more toxic toward HUVE cells. HUVE cells were more vulnerable to flavonoid cytotoxicity than TIG-1 cells. Using 2′,7′-dichlorofluorescin diacetate (DCF-DA), the intracellular reactive oxygen species (ROS) level of flavonoid-treated TIG-1 cells was examined. The ROS level increased significantly in the presence of the flavone apigenin or luteolin or the flavonol 3-hydroxyflavone, quercetin, or kaempherol. These results suggest that these flavones and flavonols exert cytotoxicity through increasing intracellular ROS levels. Further, the incorporation of apigenin, 3-hydroxyflavone, luteolin, and quercetin, which are more toxic, into TIG-1 cells during 24-h incubation was examined. These flavonoids were incorporated into them and the order of their incorporation efficiency was similar to that of their cytotoxicity. In conclusion, some flavonoids are cytotoxic at higher concentrations toward human normal cells. Further, it is suggested that they are incorporated into cells, increase intracellular ROS levels, and then exert cytotoxicity.

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In addition to regulation of normal cell functions, hepatocyte growth factor (HGF) has also been shown to be involved in malignant cell transformation and in growth, invasion and metastasis in cancer cells. Inhibitors of HGF production have a potential for interfering with malignant cell transformation and progression of tumors. We found that tryptanthrin, one of the major compounds extracted from the medicinal plant Polygonum tinctorium, which is known for its antitumor activity, strongly inhibited HGF production stimulated by various HGF inducers in human dermal fibroblasts. HGF production induced by phorbol 12-myristate 13-acetate (PMA) was potently inhibited by tryptanthrin without any appreciable cytotoxic effect. Tryptanthrin also inhibited HGF production induced by epidermal growth factor (EGF) and platelet-derived growth factor. Moreover, proliferation of the fibroblasts induced by the two growth factors was potently suppressed by tryptanthrin to the level of proliferation of unstimulated fibroblasts. However, tryptanthrin did not inhibit HGF production induced by the protein kinase A-activating agents cholera toxin and 8-bromo-cAMP. These effects of tryptanthrin were different from the effects of transforming growth factor β1 and dexamethasone, both of which inhibit HGF production induced by all the above inducers. Upregulations of HGF gene expression by PMA and EGF were also inhibited by tryptanthrin. Activation of the mitogen-activated protein kinase (MAPK) signaling pathway is crucial for PMA-induced HGF production, but tryptanthrin did not attenuate phosphorylation of MAPK induced by PMA. These results indicate that tryptanthrin potently inhibited induction of HGF production probably through events downstream of MAPK activation.

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We investigated the effects of chronic administration of fruit extract (Citrus unshiu MARC) on the endothelial dysfunction seen in aortae from streptozotocin (STZ)-induced diabetic rats. A ten-week administration of this fruit extract preserved acetylcholine (ACh)-induced endothelium-dependent relaxation, but not sodium nitroprusside (SNP)-induced endothelium-independent relaxation, in the diabetic aorta. In age-matched control rats, chronic administration of the fruit extract had no influence on the ACh- or SNP-induced aortic relaxation. The increased total cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride levels seen in STZ-induced diabetic rats were not normalized by fruit-extract treatment. These results suggest that Citrus unshiu MARC extract preserves endothelial function in the aorta in STZ-induced diabetic rats without lowering plasma cholesterol. This beneficial effect may be due to this extract protecting of nitric oxide against inactivation by oxygen free radicals.

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In this study, we investigated the pharmacological effect of humulus lupulus (hops) on gastric juice volume and acidity using a rat pylorus-ligated model. In an intraorally administered experiment, hops clearly increased gastric juice volume without affecting acidity. On the other hand, hops had no influence on gastric juice volume when it was intragastrically administered. A cholinergic agonist, carbachol, increased gastric juice volume without affecting acidity, whereas histamine increased gastric juice volume and acidity. The increase of gastric juice volume induced by carbachol was completely inhibited by atropine. On the other hand, atropine did not inhibit the increase in gastric juice volume induced by histamine. The increase in gastric juice volume induced by hops was completely inhibited by atropine. These results suggested that the increase in gastric juice volume induced by intraorally administered hops could be mediated by the cholinergic nervous system.

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To elucidate the biological significance of the P-site inhibitor of adenylate cyclase, the effect of 2,5-dideoxyadenosine (DDA) on cellular levels of adenine compounds in PC12 cells was studied. The addition of DDA and deoxyadenosine (deoxyAdo), P-site inhibitors of adenylate cyclase, as well as the addition of adenosine (Ado) to the incubation medium containing glucose as the sole nutrient significantly enhanced cellular ATP levels in PC12 cells. N⁶-Methyladenosine and N⁶-cyclohexyladenosine did not augment the ATP levels. The ATP level-enhancing effect of DDA was further enhanced by Ado. After pretreatment of PC12 cells with theophylline, DDA-induced ATP enhancement was potentiated by theophylline but the effect of Ado was suppressed. cAMP levels in PC12 cells were markedly reduced by DDA but the levels were not changed by Ado. These results suggest for the first time that P-site inhibitors of adenylate cyclase may stimulate ATP synthesis via glycolysis by decreasing cAMP levels and the mode of action of the ATP level-enhancing effect of DDA may be different from that of Ado.

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The editorial committee has noticed that this publication as a Note in Biological and Pharmaceutical Bulletin contains data sets identical to those presented in papers, already published in another journal, from the same laboratory with the same corresponding author. Due to a violation of the editorial policies of the journal, this Note has been retracted by the committee. The Editorial Committee of the Pharmaceutical Society of Japan (April 15, 2014)

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The chemopreventive effect of ethanol extract of Indigofera aspalathoides (EIA) on N-nitrosodiethylamine (DEN, 200 mg/kg)-induced experimental liver tumor was investigated in male Wistar rats. Oral administration of ethanol extract of Indigofera aspalathoides (250 mg/kg) effectively suppressed liver tumor induced with DEN as revealed by decrease in the levels of extend of serum glutamate pyruvate transaminase (SGPT), serum glutamate oxaloacetate transaminase (SGOT), alkaline phosphatase (ALP), total bilirubin, gamma glutamate transpeptidase (GGTP), lipid peroxidase (LPO), glutathione peroxidase (Gpx) and glutathione S-transferase (GST) with a concomitant increase in enzymatic antioxidant (superoxide dismutase and catalase) levels when compared to those in liver tumor bearing rats. The histopathological changes of liver sample were compared with respective control. Our results show a significant chemopreventive effect of EIA against DEN induced liver tumor.

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The effects of the ATP-sensitive potassium (K_ATP) channel opener YM099, and the angiotensin-converting enzyme (ACE) inhibitor captopril, on the progression of renal disease in rats with surgical renal mass reduction (RMR) were evaluated. Rats were subtotal (5/6) nephrectomized by resection of the renal poles. After 2 weeks of RMR, rats were randomized to three groups and treated for 6 weeks: no treatment (n=9); YM099 at a dose of 0.3 mg/kg by daily oral administration (n=9); or captopril at a dose of 50 mg/kg by daily oral administration (n=9). Sham-operated rats were used as normal animals (n=9). In RMR rats with no treatment, proteinuria progressively developed. At 8 weeks after RMR, renal function as assessed by plasma creatinine (Pcr) and blood urea nitrogen (BUN) was impaired. Pharmacological activation of K_ATP channel opening by YM099 showed no beneficial effect on proteinuria and renal functional parameters. On the other hand, pharmacological ACE inhibition by captopril significantly attenuated proteinuria, and tended to inhibit the increases in Pcr and BUN; however, these effects were not statistically significant. The presents study indicates that YM099 exhibits no renoprotection with antiproteinuric effect in rats with progressive renal disease. These findings suggest that activation of K_ATP channel opening may play no role in the retardation of progressive renal disease.

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Omeprazole, a proton pump inhibitor (PPI), is widely used in treatment of peptic ulcer, gastro esophageal reflux disease and eradication of Helicobacter pylori. PPIs inhibit final gastric acid secretion stage by blocking H⁺/K⁺-ATPase. But the mechanism except for gastric antisecretory effect has not understood clearly. So, we examined the effects of omeprazole on the levels of gastrointestinal peptides (somatostatin, motilin, gastrin, vasoactive intestinal peptide (VIP), substance P (SP) and calcitonin gene-related peptide (CGRP)) in plasma from healthy subjects. After a single oral administration of omeprazole, the plasma omeprazole concentration was highest at 120 min. Omeprazole caused a significant increase of plasma somatostatin-immunoreactive substance (IS) levels at 60—240 min and plasma motilin-IS levels at 120—180 min, compared with a placebo group, respectively. The physiological release of plasma gastrin-IS was reduced by the administration of omeprazole at 60 min, but the medicine did not alter the levels of VIP-, CGRP- and SP-IS. These results suggested that the pharmacological effects of omeprazole on regulation of gastrointestinal function are closely related to changes of somatostatin-, motilin- and gastrin-IS levels in human plasma.

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We examined the effect of coffee on conjugation reactions in the human colon carcinoma cell line, Caco-2. After supplementing Caco-2 cultures with both 1-naphthol (200 μM) and various concentrations of coffee, the accumulation of 1-naphthyl sulfate and glucuronide in the growth medium was determined by analytical HPLC over a 24-h period. A strong reduction in sulfo-conjugation (<50% of the control value) was observed in cells treated with coffee (IC₅₀=4.3%), but no effect on glucuronic acid conjugation (glucuronidation) was observed. Coffee was also found to inhibit sulfotransferase (SULT) activity towards 1-naphthol in vitro to a similar extent (IC₅₀=5.1%) as in intact Caco-2 cells, but exhibited no effect upon UDP-glucuronosyl transferase (UGT) activity in vitro. PCR analyses showed no significant changes in the expression of either SULT genes (SULT1A1 and SULT1A3) or UGT genes (UGT1A1 and UGT1A6) following treatment with coffee solutions of up to 5% in concentration. These results suggest that the consumption of coffee can modify sulfo-conjugation reactions within intestinal epithelial cells, which may possibly affect the bioavailability of therapeutic drugs and the toxicity of environmental chemicals.

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The effects of soft-shell turtle (Trionyx sinensis) powder (SST) on the proximal tibiae of ovariectomized (OVX) rats were investigated using peripheral quantitative computed tomography (pQCT) and examination of serum biochemical markers. Considering the relationship between the antioxidative property and antiosteoporotic activity, the synergistic effects of a mixture of SST and essential oil of the microalgae Haematococcus pluvialis (OHP) with strong antioxidant activity were also examined. Oral administration of SST (100, 200 mg/kg) or a mixture of SST (100, 200 mg/kg) and OHP (13, 26 mg/kg) three times weekly prevented the decrease in bone mineral content (BMC) in total bone, BMC and bone mineral density (BMD) in cortical bone, and bone strength indices induced by ovariectomy in a dose-dependent manner without uterine side effects. However, OHP alone showed no significant effects.

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Kamisyoyosan (KSS) and Tokisyakuyakusan (TSS) are widely used herbal formulas in Japanese traditional kampo medicine to relieve the symptoms occurred in climacteric disturbance. Since Japanese physicians frequently prescribe these formulas combined with etizolam, one of benzodiazepine anxiolytics, we evaluated the pharmacokinetic interaction between KSS or TSS and etizolam, and in vitro inhibitory effect of KSS and TSS on rat cytochrome P450 (CYP) 3A activity in rat microsomes, to obtain drug information to prevent from disadvantage or adverse effects by their combined therapy. In in vitro experiment, KSS and TSS inhibited CYP3A activity comparable to grapefruit juice. However in in vivo experiments, oral administration of KSS did not influence the plasma concentration profile of etizolam. The maximum concentration (C_max) of etizolam was significantly reduced when TSS was co-administered at 20 times amount of human daily dosage. Since the double of human daily dose of TSS did not suppress the absorption of etizolam, TSS would not influence the pharmacokinetics of etizolam at the usual clinical dosage. Since both KSS and TSS did not influence the metabolism of etizolam, the result of in vitro experiment would not reflect to that of in vivo experiment or in clinic. The combination of etizolam with KSS or TSS at usual dosage would not cause drug interaction.

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The resources of wild Ephedra plants in the Xinjiang Uygur Autonomous Region were surveyed. Ephedra plants mainly grow on the fringes of the Taklimakan Desert and Gureban-tonggute Desert. We found six genotypes of Ephedra przewalskii growing widely in Xinjiang. Three genotypes of Ephedra intermedia were limited to the northern and eastern parts, and Ephedra regeliana scattered in the northern part of Xinjiang. These Ephedra specimens were analyzed for DNA sequences of nuclear ribosomal DNA, internal transcribed spacers 1 and 2, chloroplastic DNA, trnL intron and trnL-trnF intergenic spacer. Intraspecific variation of the nucleotide sequence in E. przewalskii was found in different habitats. Norephedrine, ephedrine, pseudoephedrine, and methylephedrine contents of the specimens were determined. Although Ephedra intermedia of all three genotypes contained ephedrine alkaloids, ephedrine alkaloids were not detected in E. regeliana and E. przewalskii.

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We previously reported the neurotrophic effects of talaumidin (1) from Aristolochia arcuata MASTERS. In the present study, we compared the neurotrophic and neuroprotective effects of six other 2,5-diaryl-3,4-dimethyltetrahydrofuran neolignans isolated from the same plant, veraguensin (2), galgravin (3), aristolignin (4), nectandrin A (5), isonectandrin B (6), and nectandrin B (7), with compound 1 in primary cultured rat neurons. Compounds 3—7 promoted neuronal survival and neurite outgrowth, among which compounds 6 and 7 showed neurotrophic activity comparable with that of 1. Furthermore, compounds 1—7 protected hippocampal neurons against amyloid β peptide (Aβ_25—35)-induced cytotoxicity, while compounds 1 and 4—7 protected against neuronal death from 1-methyl-4-phenylpyridinium ion (MPP⁺)-induced toxicity in cultured rat hippocampal neurons.

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In this study, we examined whether the Kampo formulas Oren-gedoku-to (OGT, Huanglian-jie-du-tang in Chinese) and Keishi-bukuryo-gan-ryo (KBG, Gui-zhi-fu-ling-wan in Chinese) could prevent the progression of atherosclerosis in cholesterol-fed rabbit, an animal model for hypercholesterolemia in vivo. Twenty-four male Japanese white rabbits (2 kg body weight) were divided into four groups. The control group was fed standard rabbit chow containing 1% cholesterol, the OGT group was fed standard rabbit chow containing 1% cholesterol and 1% OGT, the KBG group was fed standard rabbit chow containing 1% cholesterol and 1% KBG, and the vitamin E group was fed standard rabbit chow containing 1% cholesterol and vitamin E (450 mg/1000 g). All four groups were kept on these diets for 8 weeks. At the end of the experiments, the percentage of surface area of the total thoracic aorta with visible plaque was significantly reduced in the OGT and KBG groups. The serum thiobarbituric acid reactive substances of the vitamin E group showed a significantly low value compared with the control group, whereas the serum lipid peroxide levels of the OGT and KBG groups were considerably lower than that of the control groups as well as that of the vitamin E group. Furthermore, the urinary 8-hydroxydeoxyguanosine levels of the OGT and KBG groups were considerably lower than that of the vitamin E group. These results suggest that OGT and KBG prevent the progression of atheromatous plaque by creating a sounder antioxidant defense system than vitamin E.

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Ethanol extracts of lyophilized vegetables were tested for inhibition of human lanosterol synthase (hOSC) in order to find the compounds to suppress cholesterol biosynthesis. Of 130 samples tested, twelve samples showed significant inhibition. Among them, Colocasia esculenta (taro) showed the highest inhibition (55% inhibition at 300 μg/ml). Examination of activity variation among eight taro cultivars indicated that “Aichi-wase” and “Yatsugashira” had the most potent activity for hOSC inhibition. In order to identify the active constituent of taro, ethanol extracts of “Aichi-wase” were partitioned with hexane and aqueous methanol, and fractionated by silica gel column chromatography. Inhibitory activity was concentrated in two major active fractions. Further purification of these fractions by preparative HPLC gave three monogalactosyldiacylglycerols and five digalactosyldiacylglycerols as active compounds that showed 28 to 67% inhibitory activities at the concentration 300 μg/ml.

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Two plastoquinones were isolated from the methanolic extract of the brown alga Sargassum micracanthum, and these were identified as a known 2-geranylgeranyl-6-methylbenzoquinone and its hydroquinone, respectively, based on spectroscopic analysis. The absolute configuration of the secondary hydroxyl group was determined by the modified Mosher's method using the new chromene derivative converted from plastoquinones. One of the plastoquinones and the chromene exhibited significant antioxidant activities, such as an inhibitory effect on lipid peroxidation and a radical scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH). The benzoquinone-type compound and the chromene derivative were found to have potent antiviral activity against human cytomegalovirus (HCMV).

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The effects of inhaling the vapor of Lavandula burnatii super-derived essential oil and one of the main components of lavender oil, linalool on plasma adrenocorticotropic hormone (ACTH), catecholamine and gonadotropin levels in menopausal model rats under ether-inhalation were studied. The increased plasma ACTH levels induced by ether-inhalation tended to decrease by pre-inhalation of Lavandula burnetii super and linalool vapor was induced the decrease of ACTH level. The decrease in adrenaline, noradrenaline and dopamine levels induced by ether-inhalation tended to recover, especially, the dopamine level significantly recovered to the normal level by the inhalation of Lavandula burnetii super and linalool vapor. However, the increased plasma gonadotropin levels in ovariectomized retired female rats (menopausal model rats) was significantly decreased by the inhalation of linalool. These results suggest that lavender oil or one of the main components, linalool may contribute to relieving tension and may be applicable to the treatment of menopausal disorders in human beings.

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In the course of isolating agents preventing sepsis from the EtOAc extract of the roots of Angelica dahurica, four known furanocoumarins, isoimperatorin (1), oxypeucedanin (2), (±)-byakangelicin (3), and (+)-oxypeucedanin hydrate (4), were isolated as active compounds based on the in vivo assay model of sepsis induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN). Among them, 3 showed the highest survival rate (100% with a dose of 30 mg/kg versus 20% for the control experiment) and decreased the plasma levels of tumor necrosis factor-α and alanine aminotransferase in mice adminstered LPS/D-GalN.

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The aim of this study was to characterize a newly developed clonidine transdermal patch, KBD-transdermal therapeutic system (TTS), for the treatment of attention deficit hyperactivity disorder in children. In vitro release, penetration, and in vivo pharmacokinetics in rabbits were investigated. The smaller size of KBD-TTS (2.5 mg/2.5 cm²) showed a similar in vitro penetration to those of Catapres-TTS (2.5 mg/3.5 cm², a clonidine transdermal patch used for the treatment of hypertension, Alza Corporation, U.S.A.). The transdermal penetration rate of clonidine was mainly controlled by the ethylene vinylacetate membrane used in the patch. The skin layer may be only a minor rate-limiting barrier after the topical skin layer at the dosing site is saturated with penetrating clonidine in the initial phase (0 to 12 h). A sensitive liquid chromatography-mass spectrometry method for the quantification of clonidine in rabbit plasma was developed using solid-phase extraction and gradient elution on LC combined with the selected-ion monitoring (SIM) mode. A single dose of clonidine transdermal patch (KBD-TTS) or Catapres-TTS was transdermally administered to rabbits (n=6 each) and removed after 168 h. The average half-life, T_max, C_max and C_ss values of clonidine in rabbits following administration of KBD-TTS were 19.27±4.68 h, 52.56±25.77 h, 27.39±9.03 ng/ml, and 25.82±9.34 ng/ml, similar to those of Catapres-TTS, respectively. The clonidine plasma concentration of KBD-TTS reached a steady state at 24 h through 168 h. The in vitro release rate of the clonidine from KBD-TTS significantly correlated with the in vivo absorption rate (p<0.001).

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The intestinal expression of the CYP3A subfamily was investigated in female rats, and the intestinal metabolism of two CYP3A substrates, testosterone and rifabutin, was examined and compared between males and females. CYP3A1/23 and CYP3A2 intestinal expression was barely detected in male and female rats. Although CYP3A9 was predominantly expressed in the female rat liver, its expression in the intestine was not different between the two sexes. The rate of testosterone 6β-hydroxylation in the female intestine was similar to that for males. Rifabutin was also metabolized at similar rates in both intestines, although the metabolic rate was greater in the female liver. These results indicate that the intestinal drug metabolizing activity of the CYP3A subfamily is similar between males and females, and that CYP3A9 is involved in the intestinal metabolism of CYP3A substrates in both sexes.

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In this study, the in vivo effects of cyclosporin A (CsA) and ketoconazole (KCZ), which are used as inhibitors of P-glycoprotein (Pgp) and cytochrome P450 (CYP) 3A, respectively, on the pharmacokinetics of rhodamine 123 (Rho123), nelfinavir (NFV) and erythromycin (EM) were evaluated in rats. The biliary excretion clearance (Cl_bile) of a known Pgp substrate, Rho123, after intravenous pretreatment with CsA or KCZ (0—20 mg/kg after i.v. administration) showed maximum reduction by 85.6 or 54.1%, respectively, suggesting that the inhibitory potency of KCZ is about half that of Pgp in the liver. Without pretreatment with CsA or KCZ, the clearance ratio of Cl_bile relative to the total body clearances of Rho123, NFV and EM was 10.5, 0.07 and 31.1%, respectively. After CsA pretreatment, these ratios decreased markedly in a manner dependent on the dose of CsA, while after CZ pretreatment the clearance ratios of NFV and EM increased significantly in a manner dependent on the dose of KCZ. However, in the liver, the contribution of Pgp to the changes in the pharmacokinetic parameters of Rho123, NFV and EM after intravenous administration was much less than that of CYP3A. The portal levels of Rho123 and EM but not NFV after intra-loop administration in the presence of 10 μM CsA in the jejunal loop increased significantly, while in the presence of 25 μM KCZ in the jejunal loop, the portal levels of those substrates showed no notable change as compared to the control levels. In conclusion, KCZ had dual potency to inhibit CYP3A and Pgp, and its inhibitory potency for Pgp was half that of CsA in the rat liver. In addition, metabolism via CYP3A contributed more significantly to the clearance of these substrates that did excretion via Pgp in the liver. In the small intestine, the contribution of Pgp is a more important factor in determining the oral bioavailability of EM than metabolism via CYP enzymes. The elimination of NFV is mainly dependent on liver metabolism via CYP3A, and the Pgp efflux mechanism in the liver and intestine did not contribute as importantly to the oral bioavailability of NFV under in vivo conditions, although NFV has been demonstrated to be a substrate of Pgp under in vitro conditions.

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The uptake and metabolism profiles of ginsenoside Rh2 and its aglycon protopanaxadiol (ppd) were studied in the human epithelial Caco-2 cell line. High-performance liquid chromatography-mass spectrometry was applied to determine Rh2 and its aglycon ppd concentration in the cells at different pH, temperature, concentration levels and in the presence or absence of inhibitors. Rh2 uptake was time and concentration dependent, and its uptake rates were reduced by metabolic inhibitors and influenced by low temperature, thus indicating that the absorption process was energy-dependent. Drug uptake was maximal when the extracellular pH was 7.0 for Rh2 and 8.0 for ppd. Rh2 kinetic analysis showed that a non-saturable component (K_d 0.17 nmol·h⁻¹·mg⁻¹ protein) and an active transport system with a K_m of 3.95 μmol·l⁻¹ and a V_max of 4.78 nmol·h⁻¹·mg⁻¹protein were responsible for the drug uptake. Kinetic analysis of ppd showed a non-saturable component (K_d 0.78 nmol·h⁻¹·mg⁻¹ protein). It was suggested that active extrusion of P-glycoprotein and drug degradation in the intestine may influence Rh2 bioavailability.

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Liposomes are an important colloidal carrier system for controlled drug delivery. However some highly hydrophilic small molecules are difficult to entrap into liposomes and store stably, resulting in poor encapsulation efficiency and fast leakage. In the present work, fluorescein sodium (FS) was used as a model drug that was loaded into chitosan nanoparticles and then encapsulated into liposomes by reverse-phase evaporation (RPV). The encapsulation efficiency, particle size, zeta potential, release in vitro and pharmacokinetics in rats were determined in order to characterize the novel drug delivery system. The entrapment efficiency was above 80% in nanoparticles (Np) and 95% in liposomes encapsulating the nanoparticles (Lip-Np). The Lip-Np was composed of soybean phospholipids, cholesterol and chitosan, which the average diameter was 202.6 nm and zeta potential was −34.8 mV. The release rate of fluorescein sodium from Lip-Np was slower than from Np and liposomes. FS in Lip-Np administered to rats exhibited prolonged circulation and higher bioavailability than FS in Np. The results indicated that liposomal release kinetics can be controlled by encapsulating nanoparticles and thus solid-cored liposomes can be used as a potential drug delivery system.

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The color of mammalian skin is determined by many factors, for which visible ones are the degree and distribution of melanin pigmentation. Because tyrosinase, (polyphenol oxidase) is the key enzyme for melanin biosynthesis, the use of various tyrosinase inhibitors is a common practice for whitening purpose in cosmetics. In the present study, the inhibition of tyrosinase by 4,4′-dihydroxybiphenyl (44′-BP) was investigated. In addition to tyrosinase inhibiting activity, melanin biosynthesis was assessed in B16F10 melanoma cells (B16 cells). The results showed that 44′-BP exhibits a strong anti-tyrosinase activity with IC₅₀=1.91 μM. The kinetic analysis of tyrosinase inhibition revealed that 44′-BP acts a competitive inhibitor (K_i=4.0×10⁻⁴ M at 2.5 μM and K_i=2.1×10⁻⁵ M at 5 μM). Furthermore, data on melanin biosynthesis indicated that the amount of melanin was clearly suppressed by 44′-BP.

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Oxatomide is an antiallergic drug used for the treatment of diseases mediated by type I allergy. Recently, terfenadine and astemizole, which have antiallergic actions similar to those of oxatomide, showed side effects on the cardiovascular system. This might be because concomitant drugs such as itraconazole inhibit cytochrome P450 3A4 (CYP3A4), the enzyme responsible for the degradation of terfenadine and astemizole, and thus the blood concentrations of the drugs are abnormally increased. In another article of this issue, we have reported that oxatomide is metabolized by CYP2D6-Val and CYP3A4, and simultaneously inhibits the metabolism of the model substrates for these enzymes. In this study, we performed the kinetic analysis of oxatomide metabolism using microsomes prepared from human liver, and found that the K_m and V_max values were 26.1 μM and 1254.4 pmol/mg protein/min, respectively. Ketoconazole, one of the representative inhibitors for CYP3A4, potently inhibited the metabolism of oxatomide, but other well-known CYP inhibitors did not show significant inhibition. These results suggest that the metabolism of oxatomide is principally catalyzed by CYP3A4. Furthermore, oxatomide inhibited the metabolism of (±) bufuralol and testosterone, model substrates for CYP2D6 and CYP3A4, respectively, in a dose-dependent manner with the K_i values of 57.4 and 24.3 μM, respectively. These observations, together with the finding that the putative highest concentration of oxatomide in blood was ≅40 ng/ml (≅93 nM) at 4 h after each dosage during consecutive 6-d administration, encouraged us to conclude that oxatomide won't inhibit CYP2D6 or CYP3A4 at clinical doses.

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Immunomodulating activity of glycopeptidolipids (GPL), separated from different serovars of Mycobacterium avium complex (MAC), on macrophage functions was compared. When peripheral blood mononuclear cells (PBMC), from healthy donors showing strongly positive reactions to mycobacterial purified protein derivatives (PPD), were incubated with heat-killed Staphylococcus aureus coated with GPL serovar 4, phagocytosis of monocytes increased in dose-dependent manner. However, coating with GPL serovar 9 did not show any effects. After phagocytosis of heat-killed S. aureus, the phagosome-lysosome (P-L) fusion in monocytes was inhibited dose-dependently by coating of S. aureus with GPL serovar 4, but not serovar 9. These results indicate that GPL serovar 4 facilitates invasion of MAC into monocytes and renders resistance to bactericidal reactions due to the inhibition of P-L fusion. Regarding accessory function of macrophages in proliferative responses of T cells, the addition of GPL serovar 4 to cultures resulted in significant inhibition of anti-CD3 monoclonal antibody (mAb)-induced proliferation, whereas both serovar GPLs did not cause reduction of cell viability. Furthermore, the PPD-specific T cell proliferative response was downregulated markedly by GPL serovar 4, but weakly suppressed by GPL serovar 9. These results indicated that the immunomodulating activity of GPL on macrophage functions is serovar-dependent.

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The cultivations of the Candida albicans NIH A-207 strain (A-strain) for 5 d at 27 and 37 °C in 500 mM galactose-added yeast nitrogen base medium (YNB-Gal) decreased the growth of blastoconidia and the pH in the cultures, with dry weights of 56 and 47% and with pHs of 2.41 and 2.47, compared with the dry weight of 100% and pH of 5.63 for a standard cultivation of 2 d at 27 °C in the yeast extract-added Sabouraud liquid medium (YSLM). The cells obtained by cultivations at 27 and 37 °C in the YNB-Gal clearly decreased the agglutination against serum factors 4, 5, and 6 in the commercially available kit ‘Candida Check’, especially at 37 °C, in contrast to those obtained by the standard cultivation. It was also revealed by ¹H-NMR analysis that both the mannans obtained from cultures at 27 and 37 °C in the YNB-Gal had drastically lost a phosphate group and a β-1,2-linked mannopyranose unit, and increased the non-reducing terminal α-1,3-linked mannopyranose unit, especially at 37 °C.

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Utilizing starch-containing alginate beads, a novel drug delivery system (DDS) was developed. With the starch inside, the composite bead could be dried in its original bead shape and handled in the dried state. By employing alginate multi-coating strategy on the starch-alginate beads, detained or controlled release was efficiently achieved and successfully demonstrated for a model peptide drug, L-phenylalanine. The initial latent time and release rate of the drug inside the beads were able to be controlled simply by varying the number of multi-coatings. While the latent time for the initial release was negligible for non-coated starch-alginate beads, the latent times of beads coated one, two, and four times increased to 15, 30, and 70 min, respectively. Furthermore, the alginate component of the composite beads could adsorb and remove heavy metals such as lead from the body. These multifunctional beads combined with the novel coating process will greatly benefit alginate gel-based DDS.

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