Angiogenin-1 (p15, an angiogenesis inducer with RNase activity) and lactogenin-like protein (p17) isolated from partially purified bovine lactoferrin (bLF) preparations were characterized as glycyrrhizin (GL)-binding proteins (gbPs). As expected, bLF-affinity column chromatography confirmed these two gbPs to be bLF-binding proteins. These two purified gbPs exhibited RNase activities when incubated with poly(C) as a substrate. Both GL and glycyrrhetinic acid (GA) at 100 μM significantly inhibited RNase activities of these two gbPs, both of which functioned as phosphate acceptors of C-kinase in vitro. Phosphorylation of p15 and p17 by C-kinase was inhibited by GA in a dose-dependent manner with the 50% inhibition dose (ID50) of approx. 10 μM, whereas GL required a relatively high dose (300 μM) to inhibit significantly it. A GA derivative (oGA, ID50=approx. 0.3 μM) was found to be a potent inhibitor of the C-kinase-mediated phosphorylation of these two gbPs in vitro. In addition, a possible physiological significance of C-kinase on the physiological interaction between bLF and two bLF-binding proteins (p15 and p17) is noted.
Aromatization of 16α-hydroxyandrostenedione (16α-OH AD) with aromatase in human placental microsomes was studied by gas chromatography-mass spectrometry (GC-MS) using [2,4,6,6,9α, 16β, 17α-2H7]estriol as an internal standard. 16α-OH AD was incubated with the microsomes in the presence of NADPH in air. The metabolite was extracted with ethyl acetate and treated with NaBH4. The reduced product, estriol, was isolated by Sep-Pak C18 cartridge and then analyzed as the tris(trimethylsilyl)ether by a GC-MS (EI mode). The production of estriol was dependent upon protein concentration and incubation time. Apparent Km and Vmax values of the microsomal aromatase for 16α-OH AD were 568 nM and 25.5 pmol/min/mg protein, respectively. In this assay, aromatase activity, estriol formation, could be determined at a level as low as 1 pmol/min/mg protein. Aromatase inhibitors, 4-hydroxy- and 6-oxo-androstenediones, prevented the estriol formation in a competititve manner with 25 and 30 nM of apparent Ki values, respectively.
A selective and sensitive HPLC measurement of 3',5'-cyclic nucleotide phosphodiesterase (PDE) activity in human platelets using (3,4-dimethoxyphenyl)glyoxal (DMPG) as a fluorogenic reagent for guanine and its nucleosides and nucleotides is described. cGMP, a substrate for PDE, and GMP, which was produced by the enzyme reaction, are selectively converted by the reaction with DMPG to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. Human platelet PDE activity was measured and the inhibitory effects of several compounds were investigated.
We prepared anti-plasma hyaluronan binding protein (PHBP) mouse monoclonal antibodies and studied the fragmentation profile of PHBP with them. PHBP is present in human plasma as a single polypeptide chain (70 kDa). During the purification, PHBP partially fragmentated into the 50-kDa N-terminal fragment and the 27-kDa C-terminal fragment. After the incubation of the purified PHBP, the 70-kDa precursor form was completely cleaved to the 50- and 27-kDa fragments, followed by the 50-kDa to the 26-kDa, and the 27-kDa to the 17-kDa plus the 8-kDa fragments, respectively. Because the purified PHBP contained no other detectable proteins and PHBP has a typical serine protease domain, we concluded that the fragmentation of PHBP was caused by own serine protease activity. PHBP cleaved the C-terminal side of Arg in the peptide effectively and that of Lys weakly. The results of the pre-incubation experiments of PHBP suggested that the single-chain form of PHBP is a precursor, the two-subunit structure is an active form and the three- or four-chain structure is an inactive form of a serine protease.
Nitric oxide (NO) induces apoptotic cell death and cAMP has a significantly protective effect on NO-induced cytotoxicity in human osteoblasts, MG-63 cells. Treatment with S-nitroso-N-acetylpenicillamine (SNAP)(0.6 mM) resulted in genomic DNA fragmentation, characteristic of apoptosis. However, concomitant incubation of the cells with either DBcAMP or forskolin markedly inhibited SNAP-induced apoptosis in a dose-dependent manner. Furthermore, pretreatment of MG-63 cells with H-89 or KT5720, which is known to inhibit cAMP-dependent protein kinase (PKA), abolished the protective effect of DBcAMP and forskolin on SNAP-induced apoptosis. In this study, we explored the involvement of caspases in the regulatory mechanism of SNAP-induced apoptosis by cAMP. Our data show that DBcAMP or forskolin blocked SNAP-induced caspase-3-like cysteine protease activation and that H-89, a PKA inhibitor, reversed the cAMP-induced regulatory effect of caspase-3 like protease. Consistent with the results, cAMP inhibited the proteolytic cleavage of caspase-3, -6, -9 and cytochrome c release to cytoplasm. The inhibition of caspase-3 activation did not block SNAP-induced cytochrome c release to cytoplasm, suggesting that caspase-3 activation may occur downstream of cytochrome c release. In summary, these findings show that the exposure of MG-63 cells to cAMP analogs renders them more resistant to NO-induced damage and suggests the presence of regulatory mechanisms of the cell death pathway by cAMP in which caspase-3, -6, and -9 and cytochrome c release serves to mediate NO-induced apoptosis.
Nitric oxide (NO), an endothelium-dependent relaxing factor, regulates relaxation, proliferation, and migration of smooth muscle cells (SMCs) and most likely attenuates developing vascular disease such as atherosclerosis. We investigated whether or not NO is associated with regulation of aortic elasticity. S-Nitrosoglutathione (GSNO), a NO donor, stimulated tropoelastin synthesis in cultured SMCs during both the quiescent and proliferating phases. The stimulation of tropoelastin synthesis was dose-dependent within 1—100 nM. Maximum stimulation was detected by treatment with 100 nM GSNO for 24 h. 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP), an exogenous cyclic GMP analog, also upregulated tropoelastin synthesis. Tropoelastin and lysyl oxidase mRNA expression, as assessed by Northern blot analysis, was also stimulated by GSNO. Administration of KT5823, a cyclic GMP-dependent protein kinase inhibitor, inhibited the GSNO-induced tropoelastin synthesis. These results indicate that the stimulatory effects of GSNO are due to cyclic GMP dependent protein kinase (PKG) activation by NO. In conclusion, NO seems to enhance aortic elasticity via tropoelastin and lysyl oxidase upregulation.
Arylsulfate sulfotransferase (ASST) transfers a sulfate group from a phenolic sulfate ester to a phenolic acceptor substrate. In the present study, the gene encoding ASST was cloned from a genomic library of Salmonella typhimurium. The gene was subcloned into the vector pKF3 and was sequenced. A recombinant clone harboring the gene was directly identified using a fluorescent assay. Sequencing revealed two contiguous open reading frames (ORFs) on the same strand. Based on amino acid sequence homology, ORF1 and ORF2 are designated as astA and dsbA, respectively. The deduced amino acid sequence of astA from S. typhimurium was highly similar to those of the Enterobacter amnigenus, Klebsiella, and Campylobacter jejuni ASSTs, encoded by the astA genes. However, an ASST activity assay revealed a different acceptor specificity. Using p-nitrophenyl sulfate (PNS) as a donor substrate, phenol is the best acceptor substrate, followed by α-naphthol, resorcinol, tyramine, acetaminophen, and tyrosine.
Effect of exogenously added water-soluble antioxidants on the mouse macrophage lectin-like receptor activity for oxidized erythrocytes was investigated. A monolayer of thioglycollate-induced mouse peritoneal macrophages was preincubated with each of the antioxidants at 37°C for 1 h, and the binding for mouse erythrocytes oxidized with ADP-chelated Fe(III) was examined. The binding was decreased by preincubation of macrophages with ascorbic acid-related compounds including ascorbic acid, erythorbic acid and dehydroascorbic acid in a dose-dependent fashion at relatively high concentrations above 10 μM. The binding was similarly decreased by preincubation of macrophages with catechin compounds including epicatechin, epigallocatechin, epicatechin gallate and epigallocatechin gallate in a dose-dependent fashion at 0.01-100 μM. The binding was more effectively decreased by preincubation of macrophages with thiol-related compounds including glutathione, oxidized glutathione, glutathione isopropyl ester and N-acetylcysteine in a dose dependent fashion at relatively low doses below 1 μM. These results showed that water-soluble antioxidants especially glutathione and its derivatives reduced the ability of macrophages to bind oxidized erythrocytes, suggesting that the activity of lectin-like receptors of macrophages for oxidized erythrocytes was regulated by oxidative mechanisms.
We examined the levels of arginine amidase secreted from isolated rabbit arteries treated with dermatan and dextran sulfates, and the relation between the secretion of arginine amidase activity and the concentration of dermatan sulfate added to these arteries. The results showed that while dextran sulfate tended to accelerate the release of arginine amidase activity from the isolated rabbit ear artery, the induction was not significant. There was a significant increase in the level of arginine amidase released from the lower portion of isolated rabbit aorta (p<0.05), but no significant change in the upper portion of the aorta. In contrast, the addition of dermatan sulfate significantly increased the level of arginine amidase activity released from the isolated rabbit ear artery and the upper and lower portions of the aorta (p<0.05). Linear dose-response relationships were observed between the level of arginine amidase activity released from the isolated rabbit ear artery and aorta and the concentration of dermatan sulfate added.
A sterol fraction composed of campesterol (7.6%), stigmasterol (28.4%) and β-sitosterol (61.1%) was obtained by activity-guided fractionation of the acetone extract of Sideritis foetens CLEM. This sterol fraction showed anti-infammatory activity in vivo murine models of inflammation. It decreased carrageenan paw oedema in mice after oral administration of 30 and 60 mg/kg and inhibited mouse ear oedema induced by 12-O-tetradecanoylphorbol acetate (TPA) after topical application. Quantitation of the neutrophil specific marker myeloperoxidase (MPO) demonstrated that its topical anti-inflammatory activity was associated with reduction in neutrophil infiltration into inflamed tissues. Non-cytotoxic concentrations of the sterol fraction inhibited leukocyte granular enzyme release (β-glucuronidase) and superoxide generation. However, it did not shown any significant inhibitory effect on histamine release from mast cells. In vitro modulatory activity towards the classical pathway of the complement system shown by this fraction would correlate with the anti-inflammatory profile shown in vivo.
Doxorubicin (DOX) is an anthracyclie drug widely used in chemotherapy for cancer patients, but it often gives rise to multidrug resistance in cancer cells. The purpose of this work was to study the effect of hydrogen peroxide in DOX-sensitive mouse P388/S leukemia cells and in the DOX-resistant cell line. Hydrogen peroxide induced a significant increase in dose- and time-response cell death in cultured P388/S cells. The degree of cell death in P388/DOX cells induced by hydrogen peroxide was less than that in P388/S cells treated with hydrogen peroxide. Parent cells exposed to 3 mM of hydrogen peroxide showed a loss of mitochondrial membrane potential correlated with cell death. Hydrogen peroxide at a concentration greater than 0.3 mM increased the intracellular Ca2+ of P388/S cells dose-dependently; however, no change following addition of hydrogen peroxide (0.3—1 mM) was observed in the resistant cells. Hydrogen peroxide (0.1 and 1 mM) treatment also induced the production of intracellular ROS in P388/S cells, while no such increase was produced by this substance in P388/DOX cells. Resistant cells also showed a significant level of glutathione (GSH) compared with the parent cells. In addition, N-acetyl-L-cysteine and reduced GSH antioxidants abolished death of P388/S cells caused by hydrogen peroxide. Therefore, it is believed that the reduced effect of oxidative stress towards the resistant cells may be related to an increase in intracellular GSH level.
2-[(4-Cyanophenyl)amino]-3-chloro-1,4-naphthalenedione (NQ-Y15) is a dual action drug which acts as a thromboxane A2 (TXA2) synthase inhibitor and TXA2/PGH2 receptor antagonist. In the present study, we examined the effects of NQ-Y15 on Ca2+ mobilization, which is the common event in various types of platelet activation, in arachidonic acid (AA)-stimulated rat platelets. The elevation of cytosolic Ca2+ concentration ([Ca2+]i) induced by AA was inhibited by NQ-Y15 in a concentration-dependent manner. This inhibition-effect of NQ-Y15 was found to be based on the suppression of the rise in [Ca2+]i by the inhibition of both Ca2+ release from internal stores and Ca2+ influx from the extracellular space. Our successive trial was focused on the role of cyclic AMP (cAMP) in the action of NQ-Y15, because cAMP was reported to be increased by dual action drugs such as picotamide and to inhibit the increase in [Ca2+]i. NQ-Y15 was confirmed to increase cAMP in AA-stimulated rat platelets. These results suggested that NQ-Y15 might inhibit the rise in [Ca2+]i in AA-treated rat platelets by increasing cAMP, which is involved in the inhibition of platelet activation.
We, previously demonstrated that one shot administration of glycyrrhizin (Grz) reduced the postprandial blood glucose rise, using Std ddY mice. Subsequently, we evaluated the effects of long-term Grz treatment (2.7, 4.1 g/kg diet) on diabetic symptoms using genetically non-insulin dependent diabetic model mice (KK-Ay). Male KK-Ay mice were divided into 3 groups: the control group, 0.27% Grz diet (2.7 g of Grz/kg diet) group and 0.41% Grz diet (4.1 g of Grz/kg diet) group. The elevation of blood glucose concentration was almost entirely suppressed in mice fed the 0.41% Grz diet 7 weeks after the beginning of test feeding, although it was not suppressed in mice fed the control diet or the 0.27% Grz diet. Water intake in the control and 0.27% Grz diet groups increased gradually, whereas, this was not true in the 0.41% Grz diet group. Grz treatment significantly lowered blood insulin level. Throughout the experiment, Grz did not affect the food intake or body weight among the three groups. The mice fed the 0.41% Grz diet also improved their tolerance to oral glucose loading 9 weeks after the beginning of test feeding. This study shows that Grz has an antidiabetic effect in noninsulin-dependent diabetes model mice.
The effect of two different stress stimuli, restraint stress and footshock stress, on small intestinal motility was evaluated by a more reliable method with improvement of the previous method using a radionuclide, 51Cr. The small intestinal transit was significantly inhibited by restraint stress, but not by footshock stress, although plasma corticosterone levels were significantly elevated to the same extent by restraint stress and footshock stress. These results suggest that restraint stress and footshock stress stimuli influence small intestinal motility via different mechanisms, but the reason for the difference is unclear. This experimental system using 51Cr seems to be useful for the elucidation of mechanisms for restraint stress-induced dysfunction of small intestinal motility be cause of its excellent quantitative evaluation of small intestinal transit.
Ex vivo muscarinic receptor binding of oxybutynin and propiverine, the most commonly used anticholinergic agents for the treatment in patients with urinary incontinence, was investigated in rat tissues. The oral administration of oxybutynin (50.8 and 127 μmol/kg) caused a significant increase in the apparent dissociation constant (Kd) for specific (−)-[3H]QNB binding in the rat bladder, prostate, submaxillary gland, heart and cerebral cortex, compared with each of the control values. Also, in the submaxillary gland of these rats, there was a reduction in the maximal number of binding sites (Bmax) for (−)-[3H]QNB binding. Similarly, oral administration of propiverine at doses of 74.3-297 μmol/kg brought about a significant increase in the Kd values for (−)-[3H]QNB binding in rat tissues including the bladder, and greater increase in Kd values was seen in the rat prostate, heart and submaxillary gland. On the other hand, oral administration of propiverine, unlike oxybutynin, resulted in very little reduction in the Bmax valules for (−)-[3H]QNB binding in the submaxillary gland. In conclusion, the present study has shown that oxybutynin and propiverine, after oral administration, bind significantly to muscarinic receptors in tissues such as the bladder, which is the target organ for the treatment of urinary incontinence, and that oxybutynin appears to exhibit long-term binding to muscarinic receptors in the salivary gland.
We examined the effects of paeoniflorin on adenosine A1 receptor-mediated memory disturbance in the mouse passive avoidance test and inhibition of long-term potentiation (LTP) in the rat hippocampal CA1 region. The pretraining administration of the selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) significantly impaired the retention performance determined 24 h after the training test. The intraperitoneal injections of paeoniflorin and the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) significantly attenuated the deficit in retention performance caused by CPA. The in vitro studies revealed that adenosine (1 and 10 μM) dose dependently reduced both the population spike (PS) amplitudes and the tetanic stimulation-induced LTP in the hippocampus. DPCPX, at the concentration (0.1 μM) that had no effect on PS amplitudes or LTP induction, significantly reversed the suppressive effects of adenosine on both indices. Paeoniflorin also dose dependently reversed 10 μM adenosine-induced suppression of LTP but had no effect on PS reduced by adenosine. These results suggest that paeoniflorin ameliorates memory disruption mediated by adenosine A1 receptor and that modulation of adenosine-mediated inhibition of LTP in the hippocampus is implicated in its beneficial effect on learning and memory impairment in rodents.
In the present study, we investigated the effects of TA-993 and its metabolite MB3 on platelet activation in vitro. TA-993 and MB3 concentration-dependently inhibited platelet aggregation and ATP release induced by collagen in human platelets. Thromboxane (Tx) A2 formation, as determined by the production of TxB2, and the increase in intracellular Ca2+ concentration ([Ca2+]i) were also suppressed by TA-993 and MB3. TA-993 and MB3 did not inhibit TxA2 formation caused by arachidonic acid. These results suggest that the inhibition of platelet activation by TA-993 and MB3 is partly mediated by an inhibition of TxA2 formation at a step prior to cyclooxygenase. Furthermore, TA-993 and MB3 inhibited U-46619-induced platelet aggregation without blockade of the increase in [Ca2+]i, suggesting that they are likely to exert some additional effects on the intracellular events induced by Ca2+.
Plasma high-density lipoprotein cholesterol (HDL-C) concentrations are regulated by plasma cholesteryl ester transfer protein (CETP) in humans. The aim of this study was to ascertain the relationship between plasma HDL-C and plasma CETP activities in mouse, rat, dog, hamster, rabbit and monkey. In this study, the plasma HDL-C levels were highest in dogs and lowest in rabbits among the six species. Plasma CETP activities were higher in hamsters, rabbits and monkeys compared to mice, rats and dogs. The present study shows that there are species differences in HDL-C and CETP activity in six species of healthy experimental animals, with the six species being separated into two types. The first type showed a high HDL-C/TC ratio with low or absent CETP activity, and included mouse, rat and dog, whereas the second type showed a low HDL-C/TC ratio and high CETP activity, and included hamster, rabbit and monkey. The present study also shows that there is a strong relationship between plasma HDL-C levels and CETP activity in high CETP activity animals and that the relationship between the HDL-C/TC ratio and CETP activity is an important factor in all animals, regardless of CETP activity level.
The effect of 4-(4-chlorobenzyl)pyridine (4-CBP) on rat hepatic microsomal cytochrome P450 (P450) and its molecular species (CYP2B1, 2E1, 3A2, 2C11, and 2C12) and on drug-metabolizing enzyme activities were examined in vivo and in vitro. Treatment of rats with 4-CBP resulted in the induction of P450 and drug-metabolizing enzymes in a dose-dependent manner, but it was markedly inhibitory at higher dose levels. Immunoblot analyses revealed that 4-CBP induces both CYP2B1 and 2E1; however, both were decreased by increasing the dose of 4-CBP. The in vitro inhibitory experiment revealed that 4-CBP strongly inhibited benzphetamine N-demethylase activity, but not dimethylnitrosamine N-demethylase activity. The present findings provide information on the induction and inhibition effect of chlorinated benzylpyridine on hepatic microsomal P450s and drug-metabolizing enzymes in vivo and in vitro.
Concerning arsenic-induced tumorigenesis, an animal model must be developed for understanding the mechanism of human carcinogenesis by arsenics. To determine whether orally administered dimethylarsinic acid (DMA) promotes and causes the progression of skin tumorigenesis, an animal protocol by topical application of dimethylbenz(a)anthracene (DMBA) with or without UVB, a tumor promoter, in hairless mice was used. The administration of DMA by the oral route promoted not only the formation of papillomas induced by DMBA alone but also the formation of malignant tumors induced by way of the formation of atypical keratoses by treatment with DMBA and UVB. A phenomenon, the progression of keratoses→atypical keratoses→squamous cell carcinomas (SCCs), observed in the present study may resemble the development of tumors in arsenic-exposed humans. We also discussed the involvement of a reactive oxygen species (ROS), e.g., the dimethylarsenic peroxy radical [(CH3)2AsOO·], produced during the metabolic processing of DMA, in skin and in multi-organ tumorigenesis.
The cytotoxicity of reactive oxygen species and related agents toward cultured rat adrenal medullary phenochromocytoma PC12 cells was examined. These species and agents include hydrogen peroxide, linoleic acid hydroperoxide(LOOH), tert-butyl hydroperoxide, paraquat, 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), 2,2'-azobis(2,4-dimethylvaleronitrile)(AMVN), and a hypoxanthine-xanthine oxidase system. The respective 50% lethal concentrations (LC50) for undifferentiated and differentiated PC12 cells were found to be 275 and 165 μM of hydrogen peroxide, 58.3 and 35.3 μM of LOOH, 536 and 212 μM of tert-butyl hydroperoxide, 42.5 and 26.5 mM of paraquat, 79.5 and 74.5 mM of AAPH, 412 and 300 μM of AMVN, and 37.2 and 16.6 mU·ml-1 xanthine oxidase activity of the hypoxanthine-xanthine oxidase system. These results show that the differentiated cells were more susceptible to these oxidative agents than the undifferentiated cells. The glutathione peroxidase activity level of the undifferentiated cells was 2—3 times higher than the differentiated cells, the catalase activity level also tended to be higher, the superoxide dismutase activity level was higher on a per-protein-quantity basis but lower on a per-cell-number basis, and the total and reduced glutathione concentration levels were considerably higher. The enhanced susceptibility of the differentiated cells may result from decreases in the activity of glutathione peroxidase and the concentration of its substrate, reduced glutathione (GSH). Further, the preincubation of PC12 cells with α-tocopherol or L-buthionine-(R,S)-sulfoximine(BSO) lowered or enhanced their cytotoxicities, respectively.
We established a simple method for the purification of human plasma kallikrein (PK) by affinity chromatography and characterized it by analytical reverse phase-HPLC and Time of Flight Mass Spectroscopy (TOF-MS). The affinity resin (PKSI-Toyopearl) was synthesized using a selective synthetic inhibitor of plasma kallikrein (PKSI-527) as a ligand. The resin was found to have the highest efficiency in PK purification when the coupling ratio of PKSI-527 per resin was 9—14 μmol/g. PK was purified 466-fold with a yield of 83% from acetone-activated human plasma by affinity chromatography. The purity of PK thus obtained was confirmed by reverse phase-HPLC with a linear gradient of acetonitrile. The molecular weight of the purified PK was determined to be 86, 151 by TOF-MS.
The 95% EtOH extract of the seeds of Alpinia blepharocalyx (Zingiberaceae) showed significant antiproliferative activity towards human HT-1080 fibrosarcoma and murine colon 26-L5 carcinoma cells. Chemical investigation of the extract led to the isolation of forty-four new (1—44) and one known (45) diarylheptanoids, eleven phenolic compounds (46—56) together with β-sitosterol glucoside (57). Almost all the isolated compounds showed significant antiproliferative activity in a concentration-dependent manner. Among the compounds, epicalyxin F (17) exhibited the most potent activity against the proliferation of colon 26-L5 carcinoma cells with an ED50 value of 0.89 μM, while calyxin B (2) exhibited the most potent activity against human HT-1080 fibrosarcoma cells with an ED50 value of 0.69 μM. Moreover, calyxins B (2) and K (11), epicalyxins F (17), I (20) and K (22), 6-hydroxycalyxin F (25), blepharocalyxin B (27) and mixtures of 7 and epicalyxin G (18) and of calyxin J (10) and epicalyxin J (21) possessed more potent activity than a clinically used anticancer drug, 5-fluorouracil, towards HT-1080 fibrosarcoma cells. Analysis of the structure activity relationship suggested that the position of the attachment of a chalcone or a flavanone moiety does not affect the activity, although their presence in association causes a substantial enhancement of the antiproliferative activity. Moreover, the conjugated double bond of the chalcone moiety and the phenolic hydroxyl group potentiate the antiproliferative activity of the compounds.
The metabolic pathway of chiisanoside isolated from leaves of Acanthopanax divaricatus var. albeofructus (Araliaceae) by human intestinal bacteria and by the protein fraction of leaves of this plant were investigated, and the cytotoxic and anti-rotaviral activities of chiisanoside and its metabolite, chiisanogenin, were assayed. Chiisanogenin was produced as a main metabolite, when chiisanoside were incubated for 15 h with human intestinal bacteria. This metabolic pathway proceeded more potently with the protein fraction than with human intestinal bacteria. The in vitro cytotoxicity of chiisanogenin was superior to that of chiisanoside. H+/K+ ATPase was more potently inhibited by chiisanogenin than by chiisanoside. However, the anti-rotaviral activity of chiisanoside was more potent than that of chiisanogenin.
The diethyl ether extract of Anemarrhenae Rhizoma (rhizomes of Anemarrhena asphodeloides BUNGE) showed testosterone 5α-reductase inhibitory activity. Two major constituents, cis-hinokiresinol (1) and 2,6,4'-trihydroxy-4-methoxybenzophenone (2) were identified as the active principles. The inhibitory activity of 1 was superior to that of ethinylestradiol, but that of 2 was weak.
The effects of 4-hydroxyantipyrine (4-OH), a major metabolite of antipyrine, and its 4-O-sulfate (4-S) on the pharmacokinetics of antipyrine were investigated in rats. Plasma elimination of intravenously administered antipyrine was significantly decelerated under a steady-state concentration of 4-OH but not under that of 4-S. Tissue-to-plasma concentration ratio (Kp) of antipyrine under its steady-state concentration was significantly increased in the brain and heart by the concomitant use of 4-OH, while similar use of 4-S had no effect. The enhancement of the blood-brain barrier (BBB) permeability of antipyrine caused by the concomitant use of 4-OH was believed to be concerned with the increase of the Kp value of antipyrine in the brain. These results suggested that 4-OH could be used as a biodistribution promoter.
Using the Walker 256 model for carcinosarcoma-bearing rats, we intravenously administered 5 polysaccharide carriers with various molecular weights (MWs) and electric charges and tested for their plasma and tissue distribution. Two carriers, carboxymethylated-D-manno-D-glucan (CMMG) and CMdextran (CMDex), showed higher plasma AUC than the other carriers tested, namely, CMchitin (CMCh), N-desulfated N-acetylated heparin (DSH), and hyaluronic acid (HA). This was consistently found to be true over the range of MWs tested. For CMDex, the maximum value of plasma AUC was obtained when the MW exceeded 150 kDa. As for the anionic charge, CMDex (110—180 kDa) with a degree of substitution (DS) of the CM groups ranging from 0.2 to 0.6, showed maximum plasma AUC values. Twenty-four hours after administration, the concentration of CMDex (180—250 kDa; DS: 0.6—1.2) in tumors was more than 3% of dose/g—approximately 10-fold higher than those observed with CMCh, DSH and HA. Doxorubicin (DXR) was bound to these carriers via a peptide spacer, GlyGlyPheGly (GGFG), to give carrier-GGFG-DXR conjugates (DXR content: 4.2—7.0 (w/w)%), and the antitumor effects of these conjugates were tested with Walker 256 carcinosarcoma-bearing rats by monitoring the tumor weights after a single intravenous injection. Compared with free DXR, CMDex-GGFG-DXR and CMMG-GGFG-DXR conjugates significantly suppressed tumor growth, while the CMCh-GGFG-DXR, DSH-GGFG-DXR, and HA-GGFG-DXR conjugates in a similar comparison showed weak tumor growth inhibition. These findings suggest that the antitumor effect of the carrier-DXR conjugates was related to the extent with which the carriers accumulated in the tumors.
Isoniazid (INH) is metabolized by polymorphic N-acetyltransferase2 (NAT2). In the present study, the relationship between the NAT2 genotype and the INH acetylator phenotype was examined in Japanese tuberculous patients and compared with healthy subjects. Subjects were classified according to the genotyping into NAT2*5B (allele4), NAT2*6A (allele3) and NAT2*7B (allele2), using the PCR-RFLP method. Twelve healthy subjects and 7 tuberculous patients participated in the INH acetylator phenotyping study, in which each subject was administered an oral dose of INH, followed by urine sampling for 24 h. Urinary concentrations of INH and N-acetylisoniazid (AcINH) were measured by the HPLC method. The urinary recoveries of INH (% of dose) in healthy subjects in relation to NAT2 genotyping were as follows: 6.4±2.2 in the homozygotes for the wild-type allele, 10.7±2.2 in the compound heterozygotes for the mutant allele, and 38.6±6.4 in the homozygotes for the mutant allele. In the patients study, the findings in the corresponding three groups were 4.0±1.7, 8.8 and 18.3±9.3. Although no significant difference was found because of the lower systemic exposure of INH in patients compared with healthy subjects, there were differences in the disposition kinetics of INH between subjects with and without mutations in the NAT2 gene, and these findings were observed not only in healthy subjects but also in patients who had comedicated drugs and hepatic dysfunctions. The findings indicated that the metabolism of INH by NAT2 is clearly impaired in subjects with mutations in the NAT2 gene, and thus genotyping for three NAT2 point mutations was adequate to predict the metabolism of INH in Japanese tuberculous patients as well as healthy subjects. This NAT2 genotyping could become a useful alternative to TDM for INH.
Among several classes of antidepressants, tricyclic antidepressants are known to prolong QTc intervals (QT interval corrected by heart rate) in electrocardiograms, while selective serotonin uptake inhibitors (SSRI) are considered to be devoid of arrhythmogenicity. In this study, we aimed to compare the arrhythmogenic potencies of imipramine (IMI), a typical tricyclic antidepressant, and fluvoxamine (FLV), an SSRI, at therapeutic and supratherapeutic concentrations using guinea pigs in vivo. Guinea pigs were anesthetized, and IMI (10 and 20 mg/kg/h) or FLV (20 mg/kg/h) was intravenously administered for 90 minutes to obtain the time-courses of drug concentrations in plasma and the changes in the QTc intervals during and after the drug administration. IMI induced distinct QTc prolongation in a dose-dependent manner, while FLV prolonged QTc intervals only slightly. A pharmacokinetic-pharmacodynamic analysis revealed that the potency for QTc prolongation of IMI was 1.7-fold higher than that of FLV. Taking the therapeutic concentration into account, the clinical risk of FLV for QTc prolongation was suggested to be 5-fold lower than that of IMI. Therefore, this SSRI agent was suggested to be safer than the tricyclic antidepressant for patients with cardiac risk factors, including arrhythmia, or for those taking other arrhythmogenic drugs concomitantly.
Acquired resistance to chemotherapy is a major problem during cancer treatment. One mechanism for drug resistance is overexpression of the MDR (multidrug resistance) 1 gene encoding the transmembrane efflux pump, P-glycoprotein (P-gp). Calcium channel blockers such as verapamil, nifedipine and nicardipine have been shown to reverse cellular drug resistance by inhibiting P-gp drug efflux. This study evaluated whether a new calcium channel blocker, lomerizine, influenced doxorubicin (Dox) cytotoxicity and P-gp activity in a P-gp-expressing cell line compared to a non-expressing subline. Verapamil, and even more markedly, lomerizine, increased cellular uptake of calcein transported by P-gp in a P-gp-expressing erythroleukemia cell line, K562-Dox. Ten μM of lomerizine reduced the IC50 of doxorubicin in the K562-Dox from 60000 ng/ml to 800 ng/ml, whereas the IC50 of doxorubicin in the K562 subline was only marginally affected by these drugs. Lomerizine showed greater reduction in P-gp efflux than verapamil at an equimolar concentration. These results suggest that lomerizine has the clinical potential to reverse tumor MDR involving the efflux protein P-gp.
This study was designed to clarify the percutaneous penetration of bupranolol (BP), a β-adrenoceptor antagonist, through rabbit skin and to compare the in vitro penetration with the in vivo absorption. BP penetrated across the skin slowly in the absence of enhancers in vitro. Isopropyl myristate and N-methyl-2-pyrrolidone enhanced the in vitro penetration, with a 3.6 times higher flux compared with that without enhancers. However, in the in vivo percutaneous absorption, the maximal penetration was obtained with the formulation added d-limonene, with a 3.0 times higher area under the concentration-time curve (AUC) than that for the formulation without enhancers. The plasma levels of BP determined, however, were extremely lower than the theoretical plasma steady-state concentrations predicted. The plasma levels of BP after application of these formulations were maintained in the range of 7—22 ng/ml for 30 h, of which concentrations were above the therapeutically effective concentration (1.5—4 ng/ml). Therefore, the transdermal systems will offer an efficient drug delivery system for the treatment of angina pectoris and tachycardia.
A traditional Chinese herbal medicine, Quzhu Tang (QZT) was studied for its in vitro antioxidant activity and the effect on cerebral oxidative damage after forebrain ischemia followed by reperfusion in rats. The QZT decoction was shown to have strong hydroxyl radical (·OH) scavenging activity (approx. 0.1 mM as Trolox equivalent) when determined by ESR using DMPO as a spin trap reagent and H2O2/UV as the ·OH source. When the QZT decoction was injected into rat duodenum 2 h before cerebral ischemia, the oxidative brain damage after 45 min reperfusion was strongly inhibited in terms of two biochemical indications, thiobarbituric acid reactive substance formation and the loss of glutathione peroxidase. Since the QZT formula consists of 4 herbal constituents (Rhizoma atractylodis, Poria, Radix notoginseng and Radix astragali), each of the component herbs and their combinations were also examined for their protective effects on the cerebral ischemia/reperfusion injury and the effects were compared with their in vitro antioxidant potential. Although some of the incomplete formulas showed as strong antioxidant activities as complete QZT in vitro, only the complete QZT formula was effective in preventing cerebral oxidative injury in rats, and other preparations showed limited activity in vivo.
Procyanidolic oligomers (leucocyanidines, LCs) extracted from grape seeds (Vitis vinifera) are known to have antioxidant and antimutagenic activities, and a protective effect against cardiovascular disease. In the present study we examined the influence of LCs on the activities of phase I enzymes and conjugation enzymes and on antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase. Administration of LCs (25, 50, and 100 mg/kg. p.o. for 7 d) markedly decreased the activities of NADPH-cytochrome P450 reductase, P4501A1, P4501A2, and P4503A4, but significantly increased the activities of glutathione S-transferase and phenolsulfotransferase in rat liver. However, the activities of antioxidant enzymes were not affected by LC administration. The inhibition of P450s and increases in phase II enzyme activities indicate a role for LCs as a chemopreventive agent against toxic or carcinogenic metabolites of P450 isozymes.