As a first step to characterize the unknown functional properties of the human mitochondrial ADP/ATP carrier (AAC), we tried to express human type 1 AAC (hAAC1) in Saccharomyces cerevisiae. Expression of hAAC1 in yeast mitochondrial membrane was very low, although its transcript level was high. Its expression was improved greatly by replacement of its N-terminal region with the corresponding region of yeast type 2 AAC (yAAC2), as observed with the bovine type 1 AAC (bAAC1). This chimeric hAAC1 showed similar ADP transport activity to that of chimeric bAAC1, corresponding to the transport activity of bAAC1 in bovine heart mitochondria. These results suggested that the N-terminal region of yAAC2 is important for expression of the mammalian carriers in yeast mitochondria. Using the present expression system, studies on the functional properties of the human AAC isoforms in relation to their structures are now possible.
We have reported fatty alcohol synthesis accompanied by chain elongation in liver peroxisomes (Biochim. Biophys. Acta, 1346, 38 (1997)). In the present experiment, we studied what kind of acyl-CoA(s) destined to be utilized as primer for fatty alcohol synthesis accumulate(s) during peroxisomal β-oxidation. Peroxisomes were prepared from rat liver treated with clofibrate, a peroxisome proliferator, and incubated with [U-14C]palmitate, in order to investigate acyl-CoAs after β-oxidation. At 1 mM concentration, MgATP activated β-oxidation, but inhibited β-oxidation at concentrations higher than 1 mM. After incubation of peroxisomes with palmitate, various acyl-CoAs were formed. Among medium-chain labelled acyl-CoAs, octanoyl-CoA was mainly detected. There results suggest that octanoyl-CoA accumulates during β-oxidation of palmitate. When peroxisomes were incubated with [9,10-3H]palmitate and [9,10-3H]stearate, among medium-chain acyl-CoAs, octanoyl-CoA and decanoyl-CoA were primarily detected, respectively, suggesting the occurrence of at least 4 cycles of β-oxidation of both fatty acids by peroxisomes.
α-Thujaplicin, a minor component of Thujopsis dolabrata SIEB. Et ZUCC. Var. hondai MAKINO, which was synthesized, showed the antibacterial activity, phytogrowth-inhibitory effect, inhibition of carboxypeptidase A and cytotoxic effect. Antibacterial activity of α-thujaplicin on Enterococcus faecalis IFO-12965 [minimum inhibitory concentration (MIC): 1.56 μg/ml] was higher than that of gentamicin (MIC: 6.25 μg/ml) used as a positive control. Inhibitory activity of α-thujaplicin on carboxypeptidase A [50% inhibitory concentration (IC50): 3.24×10-5M] was higher than that of 1,10-phenanthroline used as a positive control. α-Thujaplicin showed germination inhibition toward the seed of Echinochloa utilis Ohwi et Yabuno even at the low concentration of 10 ppm and its growth inhibitory effect was stronger than that of sodium 2,4-dichlorophenoxyacetate used as a standard. α-Thujaplicin at 1.25 μg/ml inhibited cell growth of human stomach cancer KATO-III by 86%, and Ehrlich’s ascites carcinoma by 87%, respectively. This compound even at the low concentration of 0.32 μg/ml also inhibited cell growth of the former by 66%, and the latter by 75%, respectively. The acute toxicity of α-thujaplicin [50% lethal dose (LD50) value: 256 mg/kg] in mice was as strong as those of β-dolabrin (LD50 value: 232 mg/kg) and γ-thujaplicin (LD50 value: 277 mg/kg).
Haloperidol, an antipsychotic, was investigated in cells overexpressing P-glycoprotein to detemine whether it was a clinically effective drug to reverse for reversing multidrug resistance (MDR) mediated by P-glycoprotein. A nontoxic concentration of haloperidol (1—30 μM) enhanced the cytotoxic effects of vinblastine (VBL) concentration-dependently in VBL-resistant human leukemia (K562/VBL) cells, but had no effect in the parent cells. Haloperidol also enhanced the cytotoxicities of epirubicin, doxorubicin and actinomycin D in the K562/VBL cells, but not those of idarubicin or cisplatin; this enhancement was less than that of the VBL toxicity in the VBL-resistant tumor line. Haloperidol increased the intracellular accumulation of VBL in the K562/VBL cells, and the binding of [3H]-azidopine to the cell-surface protein, P-glycoprotein, was inhibited by haloperidol in a concentration-dependent manner. Haloperidol was less potent than verapamil. Thus, haloperidol appeared to potentiate anticancer agents through the reversal of MDR by competitively inhibiting drug-binding to P-glyco-protein. In contrast, the main metabolite of haloperidol, dihydrohaloperidol, without antipsychotic activity, had less of an effect. Therefore, haloperidol might be useful in reversing drug-resistance.
The effects of 2-chloro-3-(4-acetophenyl)-amino-1,4-naphthoquinone (NQ301), an antithrombotic agent, on aggregation, binding of fibrinogen to glycoprotein (GP)Iib/IIIa complex and intracellular signals were investigated using human platelets. NQ301 significantly inhibited the collagen-, thrombin-, arachidonic acid-, thapsigargin- and calcium ionophore A23187-induced aggregation of washed human platelets with IC50 values of 13.0±0.1, 11.2±0.5, 21.0±0.9, 3.8±0.1 and 46.2±0.8 μM, respectively. NQ301 also significantly inhibited FITC-conjugated fibrinogen binding to human platelet surface GPIIb/IIIa complex, but failed to inhibit the fibrinogen binding to purified GPIIb/IIIa complex. These data demonstrate that NQ301 inhibits platelet aggregation by suppression of the intracellular pathway, rather than by direct inhibition of fibrinogen-GPIIb/IIIa complex binding. NQ301 significantly inhibited the increase of cytosolic Ca2+ concentration and ATP secretion, and also significantly increased platelet cAMP levels in the activated platelets. These results suggest that the antiplatelet activity of NQ301 may be mediated by inhibition of cytosolic Ca2+ mobilization, enhancement of cAMP production and inhibition of ATP secretion in activated platelets.
Secretory diarrhea occurs when the balance between intestinal absorption and secretion is disturbed by excessive secretion caused by enterotoxins produced by the pathogen. Wood creosote has long been used as a traditional antidiarrheal remedy. The goal of our study was to extend our knowledge about the antisecretory action of wood creosote against Escherichia coli enterotoxin-induced secretion in the small intestine and colon. Experiments were performed in mucosal sheets of rat jejunum and colon which were stripped of the external muscle layers to eliminate interactions with smooth muscle activity and local blood flow. Mucosal sheets were placed in modified Ussing chambers and hypersecretory conditions were induced by heat-labile (LT) or heat-stable (STa) E. coli enterotoxins added cumulatively (0.01—10 μg/ml) to the mucosal bathing solution. Intestinal secretion was monitored electrophysiologically as transmucosal short circuit current (Isc). LT induced a concentration-dependent increase in Isc in the rat jejunum, with no effect in the colon. In contrast, STa induced a significant increase in colonic Isc, without causing any change in Isc across the jejunum. In separate experiments the effects of increasing concentrations of wood creosote (0.1—50 μg/ml), added to the mucosal or serosal bathing solution, were examined against the secretory responses induced by LT or STa. In the small intestine the antisecretory activity of wood creosote against LT-induced secretion was more potent following serosal application, whereas in the colon wood creosote inhibited STa-induced secretion with equal potency following either serosal or mucosal addition. In summary, our findings demonstrate that wood creosote possesses antidiarrheal activity suppressing E. coli enterotoxin-induced secretion in both the small intestine and colon.
Several studies have suggested that high blood pressure is associated with the risk of bone loss. Since various antihypertensive drugs are in wide use for the treatment of hypertension, it is important to investigate the influences of these drugs on bone metabolism. Osteoblasts play a pivotal role in the regulation of bone formation. During differentiation, they sequentially express type I collagen, alkaline phosphatase (ALP), other bone matrix proteins, and finally undergo mineral deposition. In this study, we examined the effects of various antihypertensive drugs on the function of osteoblast using clonal MC3T3-E1 cells. Drugs examined include dihydropyridine-type calcium channel blockers (benidipine, amlodipine, and nifedipine), angiotensin-converting enzyme (ACE) inhibitors (captopril, lisinopril, and enalapril), and angiotensin II receptor type1 (AT1) antagonists (TCV-116 and KW-3433). None of the ACE inhibitors or AT1 antagonists affected ALP activity or cellular DNA content significantly. In contrast, benidipine, amlodipine, and nifedipine increased ALP activity when used in amounts 1 pM, 100 nM, and 100 nM, respectively. Benidipine blocked calcium influx through the L-type voltage dependent calcium channel of MC3T3-E1 more potently than amlodipine or nifedipine. These calcium channel blockers did not change collagen accumulation. Benidipine significantly increased in vitro mineralization at a concentration of 1 nM and higher, while amlodipine did so at 1 μM and nifedipine did not. Comparison of the effective concentration of each calcium channel blocker in our study with the reported maximum serum concentration of each drug suggests that benidipine, but not amlodipine or nifedipine, promotes mineral deposition in human.
The gastric mucus is an important factor in the gastric mucosal protection from acid, pepsin and various reagents (alcohol, aspirin, etc.). MUC5AC is the mucin secreted from surface mucous cells, and belongs to the gel-forming mucin. We examined the regulation of rat MUC5AC (rMUC5AC) mRNA by glucocorticoid in vivo and in vitro, comparing it with that of pepsinogen (Pg) mRNA. By adrenal gland resection, rMUC5AC and Pg mRNA levels and Pg content in rats significantly decreased to 70%, 46% and 42% of those in the sham operated controls, respectively. With the treatment of hydrocortisone (1, 5 and 50 mg/kg), Pg mRNA level and Pg content in adrenalectomized rats was restored. On the other hand, the rMUC5AC mRNA level exceeded the control with 1 or 5 mg/kg injections of hydrocortisone, but drastically decreased to 18% of sham operation levels with it (50 mg/kg). Similar results were obtained in normal rats with treatment of hydrocortisone (50 mg/kg). Mucus and DNA content of cultured rat gastric epithelial cells were not affected by hydrocortisone, but rMUC5AC mRNA level was significantly decreased in a dose-dependent manner. From the in vivo and in vitro results, at least a physiological concentration of glucocorticoid was necessary in the expression of rMUC5AC mRNA. However, high dose of hydrocortisone directly suppressed the expression of rMUC5AC mRNA. These results suggested that hydrocortisone might directly cause the suppression and indirectly the enhancement of the mucin biosynthesis.
The enhancing effect of α-monoisostearyl glyseryl ether (GE-IS) on the percutaneous penetration of indomethacin (IM) from test solutions in propylene glycol (PG) was investigated using the excised abdominal skin of rats in vitro. The percutaneous penetration of IM into diffusion cells was significantly increased in the presence of 0.2% or 1% (w/w) GE-IS compared with enhancer-free PG solution. Permeation parameters of IM, such as lag time and permeability coefficient, revealed that GE-IS significantly augmented the percutaneous penetration of IM from PG. These results strongly suggested that GE-IS functions as a penetration enhancer of IM through rat skin. To elucidate the mode of action of GE-IS as a penetration enhancer, the solubility of IM in the test solution and the percutaneous penetration of IM through damaged skin from which the stratum corneum had been stripped were investigated. The results suggested that GE-IS acts directly on the stratum corneum and alters the permeability of the skin.
Interleukin-6 (IL-6) is known as a proinflammatory cytokine involved in immune response, inflammation, and hematopoiesis. Inhibitory effects of anti-inflammatory drugs on IL-6 bioactivity using IL-6-dependent hybridoma have been evaluated. Three out of 16 nonsteroidal anti-inflammatory drugs (NSAIDs) showed IC50 values of less than 100 μM, which were in the order of oxyphenylbutazone hydrate (IC50=7.5 μM)>meclofenamic acid sodium salt (31.9 μM)>sulindac (74.9 μM). Steroidal anti-inflammatory drugs (SAIDs) exhibited significant inhibitory effects at 100 μM on the IL-6 bioactivity, and their inhibitory potencies were in the order of budesonide (IC50=2.2 μM)>hydrocortisone 21-hemisuccinate (6.7 μM), prednisolone (7.5 μM), betamethasone (10.9 μM)>dexamethasone (18.9 μM) and triamcinolone acetonide (24.1 μM). The results would provide an additional mechanism by which anti-inflammatory drugs display their anti-inflammatory and immunosuppressive effects at higher concentrations.
The DNA binding properties of three novel extended diphenylfuran bisamidines (1—3) possessing different dicationic terminal side chains were studied. The ultrafiltration assay showed that bisamidines 1—3 have significant affinity for DNA. The DNA-binding data for bisamidines 1—3 using homopolymers poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC), indicated that these compounds show moderate specificity for AT base pairs. We studied the cytotoxicity effects of bisamidines 1—3, Hoechst 33258 and DAPI (4',6-diamidino-2-phenylindole) in cultured breast cancer MCF-7 cells. The bisamidines 1—3 showed comparable antitumour activity to Hoechst 33258, but were substantially more cytotoxic compared to DAPI. These data show that in broad terms the cytotoxic potency of bisamidines 1—3 in cultured breast cancer MCF-7 cells decreases with the size of the alkyl group substituent (cyclopropyl>isopropyl>cyclopentyl), in accord with their increases in DNA affinity, as shown by the binding constant values.
Shikunshito-Kamiho (SKTK) is a traditional Chinese medicine composed of eight crude drugs (Ginseng Radix, Hoelen, Atractylodis Rhizoma, Glycyrrhizae Radix, Prunellae Spica, Ostreae Testa, Laminaria Thallus, Sargassum). We investigated the effects of SKTK on pH, ammonia, fecal enzymes such as β-glucuronidase, tryptophanase, urease, and formation aberrant crypt foci in the colon carcinogenesis model induced by 1, 2-dimethylhydrazine (DMH). Water extract of SKTK was administered orally for 5 weeks to DMH-treated mice as 0.5% and 1.5% of the diet. β-Glucuronidase, pH and tryptophanase were significantly inhibited after treatment of 0.5% and 1.5% SKTK, while urease was significantly reduced only during and after treatment of 1.5% SKTK as compared with control data. However, the ammonia concentration wasn’t different in SKTK treated groups from control group. The incidence number of aberrant crypts foci (ACF) and aberrant crypts/focus in colon was significantly decreased by 0.5% and 1.5% SKTK mixed diets compared with that in rats treated with DMH alone. These results suggest that SKTK exterts anticarcinogenic activity on experimental murine colorectal cancer.
Extract of Gymnema sylvestre leaves was administered to rats receiving either a high fat diet or normal fat diet for 10 weeks to investigate its influence on plasma and liver lipids and on visceral fat accumulation. In addition, its effect was compared with those of chitosan and the influence of combined use of these two substances was also evaluated. Within the high fat diet groups, the extract suppressed body weight gain and accumulation of liver lipids to the same extent as chitosan and the combined use. In addition, intraperitoneal fat and fat drop vacuoles on the epithelium of renal tubules, noted in the high fat diet group, were scattered by administration of the extract with the same results as for chitosan and combined use. Within the normal fat diet groups, plasma triglyceride levels decreased by administration of the extract, with similar results as chitosan and combined use. Concerning plasma total cholesterol, there was no decreasing effects with the extract, as found with chitosan and combined use. However, the effect of chitosan on plasma total cholesterol tended to be enhanced when used in combination with the extract. In addition, long-term administration of the extract did not show any influence on hematological and blood chemical parameters.
“The extract of shikon” (SK) and shikonin play important roles in the development of granulomatous tissue formation. To reveal the augmenting effect of SK or shikonin on vascular endothelial growth factor (VEGF) production and neovascularization, we investigated murine granulomatous tissue induced by SK and shikonin, comparing them to pouches in which trehalose 6,6'-dimycolate (TDM) was injected. The development of granulomatous tissue formation was evaluated by the wet weight of pouch walls. At day 5 and 7 after SK and shikonin injection, prominent granulomatous tissue formation was detected. Histological observations on the development of granulomatous tissue showed that the pouch was formed in the submuscular connective tissue and necrotic tissue directly facing the cavity and granulomatous tissue developed in the connective tissue. At day 1, VEGF-positive neutrophils accumulated in the pouch wall. Granulomatous tissue formation and neovascularization by injection of SK or shikonin was not more prominent than TDM. However, the present results indicate that SK and shikonin induce neovascularization in granulomatous tissue.
Hydrolyzable tannins were found to be the active cytotoxic constituents of three Greek Cytinus taxa: Cytinus ruber, Cytinus hypocistis subsp. hypocistis and Cytinus hypocistis subsp. orientalis. The cytotoxic activity was evaluated against a broad spectrum of cancer cell lines. The structure of the active compounds was investigated with NMR and electrospray-MS/MS techniques.
In the present study, we assayed the antioxidant properties of Ginkgo biloba (Gb) extract on rats submitted to 21 d of chronic hypoxia. Doses of 25 and 50 mg/kg were examined. Oxygenated free radical production measured by the chemiluminescence technique was significantly decreased in treated rats compared to control rats placed in similar experimental conditions, and this effect was more significant at the 50 mg/kg dose. On the other hand, no antioxidant enzyme activities of the drug were observed towards red blood cells. These results suggest that ginkgo biloba extract has a free radical scavenging action. These antioxidant properties could explain the beneficial hematological properties of Gb extract.
Extract of Gymnema sylvestre R. Br leaves (GE) was orally administered once a day to rats fed a high fat diet or normal fat diet for 3 weeks to investigate its influence on lipid metabolism. As a result, GE did not influence body weight gain or feed intake in both diet groups during the experimental period. The apparent fat digestibility was significantly decreased by GE in both diet groups for the last 2 weeks of the experimental period, though not the apparent protein digestibility. In addition, the excretion of neutral sterols and acid steroids into feces was increased by GE in both diet groups. Furthermore, GE decreased the total cholesterol and triglyceride levels in serum. On the other hand, blood lecithin-cholesterol acyltransferase (LCAT) activity was increased by GE. More-over, it was suggested that GE influenced cecal fermentation and that propionic acid and acetic acid contents in cecum were significantly increased by GE. Consequently, it was suggested that GE improved serum cholesterol and triglyceride levels through influence over a wide range of lipid metabolism in rats.
Monodesmosides which were obtained from the partial degradation of hederagenin bisdesmosides exhibited significant antifungal effect against Microsporum canis, Coccidioides immitis, Trichophyton mentagrophytes, Cryptococcus neoformans, and Candida albicans at the minimal inhibitory concentrations of 6.25—25 μm/ml. The hederagenin glycosides were isolated from the leaves of Kalopanax pictum var. chinense.
We examined the effects of 75 kinds of natural compounds, such as alkaloids, phenylpropanoids, flavonoids, steroids and terpenoids on the in vitro migration and proliferation of colon 26-L5 cells, in comparison with anti-cancer drugs used for chemotherapy. Twenty-three of the 75 compounds inhibited markedly tumor cell migration. Among the 23 compounds, evodiamine showed the most potent and selective inhibitory activity on tumor cell migration with an IC50 value of 1.25 μg/ml, which was about 20 times lower than that for tumor cell proliferation. The migratory inhibition reached about 70% at 10 μg/ml of evodiamine. On the other hand, most of anti-cancer drugs tested, except for paclitaxel, had little effect on tumor cell migration at the concentrations strongly inhibiting tumor cell proliferation. Paclitaxel suppressed tumor cell migration in a concentration-dependent manner and achieved about 70% inhibition at 10 μg/ml with a marginal effect of cell proliferation. These results suggest that evodiamine and paclitaxel may be regarded as leading compounds for anti-metastatic agents acting through the inhibition of tumor cell migration without cytotoxicity.
This study was designed to develop an oral dosage form of elcatonin (EC), a hypocalcemic peptide. The EC absorption was estimated by the reduction in plasma calcium concentrations. When EC was orally coadministered with nitroso-N-acetyl-D, L-penicillamine (SNAP, 4.0 mg) and 0.02% Carbopol solution or with taurocholate (20 mM) and 0.02% Carbopol solution, the lowering effect was increased compared with that after EC alone, but the F values (0.32 and 0.30%) were extremely small. The oral administration of the mucoadhesive emulsion, which was prepared by coating the W/O/W emulsion with 0.1% Carbopol, enhanced the calcium lowering effect, with the F value of 0.43%. The strong mucoadhesion of the mucoadhesive emulsion to the gastrointestinal mucosa was observed. A capsule containing EC (500 μg), taurocholate (6 mg) and lyophilized Carbopol (3.5 mg) administered orally gave a sustained but comparatively small calcium lowering effect. In the in vitro enzymatic hydrolysis experiment, EC was more rapidly hydrolyzed in the intestinal fluid than in the mucosal extract. The combination of 20 mM taurocholate with 0.02% Carbopol showed the greatest inhibitory effect in both fluid and extract. These data indicated that EC was effectively absorbed through the intestinal wall, but the peptide was dominantly degraded by proteolytic enzymes in the GI tract. These results will offer a potential approach to the oral delivery of EC.
The objective of this study was to investigate the pharmacokinetics and in vivo anti-tumor effect of recombinant human tumor necrosis factor-α (rHuTNF-α) encapsulated in poly(methoxypolyethyleneglycol cyanoacrylate-co-n-hexadecyl cyanoacrylate) (PEG-PHDCA) nanoparticles. Our experimental results showed that PEG-PHDCA nanoparticles could extend the half-life of rHuTNF-α to 7.42 h and obviously change the protein biodistribution in tissues, and in particular, increase accumulation of rHuTNF-α in tumor. Compared with PHDCA nanoparticles and free rHuTNF-α, PEG-PHDCA nanoparticles loaded with rHuTNF-α showed higher anti-tumor potency at the same dose, which might be related to its higher accumulation in tumor tissues and longer plasma circulation time. Therefore, PEG-PHDCA nanoparticles could be an effective carrier for rHuTNF-α.
The aim of the present work was to investigate and assess the merit of PEGylated recombinant human tumor necrosis factor-α (rHuTNF-α) following our previous work. The rHuTNF-α was modified using activated polyethylene glycol (PEG), N-succinimidyl succinnate monomethoxy polyethylene glycol (SS-PEG). The pharmacokinetics and anti-tumor effect were investigated. The experimental results showed that PEGylated rHuTNF-α could obviously alter in vivo behavioral characteristics of rHuTNF-α. Among the synthesized PEG-rHuTNF-αs with different PEG molecules, PEG20000-rHuTNF-α demonstrated the longest circulating half-life (24.8 h) which was about 50 times longer than that of rHuTNF-α (28.8 min). In addition, there was much more PEG20000-rHuTNF-α distributed into tumor tissues than other PEG-rHuTNF-αs or rHuTNF-α with time, and PEG20000-rHuTNF-α also showed the highest anti-tumor potency. These results indicated that PEG20000-rHuTNF-α was a useful long circulating molecule with selective localization in tumor tissues and enhanced anti-tumor activity of rHuTNF-α.
The objective of this study was to investigate the relationship between drug lipophilicity and the transdermal absorption processes in the iontophoretic delivery in vivo. Anodal iontophoresis of β-blockers as model drugs having different lipophilicity (atenolol, pindolol, metoprolol, acebutolol, oxprenolol and propranolol) was performed with rats (electrical current, 0.625 mA/cm2; application period, 90 min), and the drug concentrations in skin, cutaneous vein and systemic vein were determined. Increasing the lipophilicity of β-blockers caused a greater absorption into the skin. Exceptionally, it was found that pindolol had high skin absorption, irrespective of its hydrophilic nature. Further, the drug transfer rate from skin to cutaneous vein (RSC) was evaluated from the arterio-venous plasma concentration difference of drug in the skin. Normalized RSC by skin concentration showed a negative correlation with the logarithm of n-octanol/buffer partition coefficient (Log P, pH 7.4), suggesting the partitioning between stratum corneum and viable epidermis was a primary process to determine the transfer properties of β-blockers to local blood circulation. Pindolol exhibited both high skin absorption and high transfer from skin to cutaneous vein. These characteristics of pindolol could be explained by the chemical structure, molecular size and hydrophilicity. These findings for pindolol should be valuable for the optimal design of drug candidates for iontophoretic transdermal delivery.
In this study, we identified the metabolites and the CYP forms that are specifically involved in emetine O-demethylation in human liver microsomes, and cleared the inhibitory potential of cephaeline and emetine on the activity of the major drug-metabolizing CYP enzymes. Incubation of emetine with human liver microsomes yielded three metabolites identified by using HPLC by comparison of the retention time with the authentic sample of cephaeline, 9-O-demethylemetine and 10-O-demethylemetine. CYP3A4 and CYP2D6 were able to metabolize emetine to cephaeline and 9-O-demethylemetine, and CYP3A4 also participated in metabolizing emetine to 10-O-demethylemetine. Cephaeline and emetine inhibited probe substrates metabolism. IC50 for cephaeline against CYP2D6 and CYP3A4 were 121 and 1000 μM, respectively. For the emetine, CYP2D6 and CYP3A4 were 80 and 480 μM, respectively. Inhibition constants (Ki) for both compounds on the CYP2D6 and CYP3A4 activities were determined by graphic analysis of Dixon plots at various concentrations. The obtained Ki values of cephaeline for CYP2D6 and CYP3A4 were 54 and 355 μM, respectively, and the values of emetine were 43 and 232 μM, respectively. We concluded that these in vitro inhibitions of cephaeline and emetine would hardly increase plasma concentrations of co-administered drugs in clinical therapy.
The methodology to distinguish the patients showing considerable fluctuation of the whole blood concentration of cyclosporin A (CYA) was investigated from a viewpoint of laboratory test values. First, we retrospectively examined the CYA trough blood concentrations monitored continuously. The patients were classified into three groups by the fluctuation of CYA trough blood concentrations during the examination period (Cmax/Cmin): Group 1(Cmax/Cmin=100—200%; n=21), Group 2 (Cmax/Cmin=200—300%; n=25), and Group 3 (Cmax/Cmin=more than 300%; n=32). In the laboratory tests examined, the serum triglyceride concentrations were considerably different among the groups, and it was the highest in Group 3. Next, to elucidate the effect of serum triglyceride concentration on the CYA blood concentration, in vivo pharmacokinetic studies after single intravenous or repetitive oral administration of CYA were conducted in the model rats with pseudo-hypertriglyceridemia, hypocythemia, and acute renal failure. Only in pseudo-hypertriglyceridemia rats, the CYA blood concentration after a single intravenous injection was significantly higher than that in normal rats because of the restriction of CYA distribution to the extravascular tissues. On the other hand, the increase in the serum triglyceride concentration did not affect the fluctuation of CYA trough blood concentration after repetitive oral administration. Taken together, the fluctuation of CYA trough blood concentrations observed in the clinical situation could be due to the fluctuation of serum triglyceride concentration, and the patients with such fluctuation of serum triglyceride concentrations might also be distinguishable by the higher concentration of serum triglyceride in laboratory tests.
Endothelin-1 (ET-1) has been reported to be expressed in human epidermis at both the gene and protein levels. ET-1 plays a pivotal role in ultraviolet B (UVB)-induced pigmentation due to its accentuated secretion after UVB irradiation and its function as a mitogen and as a melanogen for human melanocytes. We have recently found that endothelin-converting enzyme (ECE)-1α plays a constitutive role in the secretion of ET-1 by human keratinocytes and that an extract of Sanguisorba officinalis L. inhibits ECE activity in human endothelial cells, which predominantly express ECE-1α. In this report, to clarify the potential use of this botanical extract as a whitening agent, we examined whether this extract inhibits UVB-induced pigmentation in vivo. When this extract was applied to human keratinocytes after UVB irradiation, secretion of ET-1 by those cells was reduced, and this was accompanied by a concomitant increase in the secretion of inactive precursor Big endothelin-1. When hairless mice were exposed to UVB light and were treated with the extract, it suppressed the induction of ET-1 in the UVB-irradiated epidermis. In the course of UVB-induced pigmentation of brownish guinea pig skin, this extract significantly diminished pigmentation in UVB-exposed areas. These findings indicate that ECE-1α in keratinocytes plays a pivotal role in the induction of pigmentation following UVB irradiation and that an extract of S. officinalis, which inhibits ET-1 production in human keratinocytes, is a good ingredient for a whitening agent.
The effects of two saline cathartics (sodium chloride and sodium citrate) on the adsorptive capacity of activated charcoal (AC) for rifampicin were studied. Solutions of rifampicin alone and rifampicin with 7.5 mg/ml cathartic solution were vortex-mixed for 30 s with different quantities of AC. These were incubated for 30 min at 37°C and analyzed for free rifampicin spectrophotometrically at 320 nm. The addition of sodium citrate significantly increased (p<0.05) the adsorptive capacity of AC for rifampicin with a resulting decrease in B-50 values at both the therapeutic and simulated toxic doses. Sodium chloride addition reduced the binding of rifampicin to AC at the toxic doses. The adsorption of rifampicin to activated charcoal, both alone and with the two saline cathartics, obeyed quantity-dependent kinetics. AC may be co-administered with sodium citrate in the management of rifampicin overdose.
Glycosidases play a pivotal role in processing of various glycoproteins and glycolipids. It is well known that glycosidases are also involved in a variety of degenerative metabolic disorders such as cancer and AIDS. In order to develop potent α-glucosidase inhibitors, we first screened 2,2-diphenyl-1-picrylhydrazyl hydrate as a candidate. 2,2-Diphenyl-1-picrylhydrazyl hydrate was found to inhibit α- and β-glucosidases as well as α- and β-mannosidases. It was also shown to be a non-competitive inhibitor of yeast α-glucosidase with a Ki value of 1.1×10-6M. Taken together, we anticipate that 2,2-diphenyl-1-picrylhydrazyl hydrate may be a potent inhibitor for some incurable metabolic disorders including AIDS.
Uridine analogue binding sites, the so-called uridine receptor, were observed in the experiments on specific [3H]N3-phenacyluridine binding to bovine synaptic membranes using two isomers, N3-(S)-(+)- and N3-(R)-(−)-α-hydroxy-β-phenethyluridine, as ligands. The potent hypnotic, N3-(S)-(+)-α-hydroxy-β-phenethyluridine, but not the (R)-isomer, strongly inhibited [3H]N3-phenacyluridine binding. The racemate had inhibitory activity intermediate between that of the two α-hydroxy-β-phenethyluridines ((R)- or (S)-isomers). The inhibitory constants of these compounds were determined. The Ki values of N3-phenacyluridine, α-hydroxy-β-phenethyluridine (racemate), N3-(R)-(−)-, and N3-(S)-(+)-α-hydroxy-β-phenethyluridine were 0.65, 397.4, 1908, and 10.2 nM, respectively. The present results indicate the existence of uridine receptors in the central nervous system in relation to their hypnotic activities reported previously.