In the central nervous system (CNS), the synapse is a specialized junctional complex by which axons and dendrites emerging from different neuron intercommunicates. Changes in the efficiency of synaptic transmission are important for a number of aspects of neural function. Much has been learned about the activity-dependent synaptic modifications that are thought to underlie memory storage, but the mechanism by which these modifications are stored remains unclear. Thus, it is important to find and characterize “memory molecules,” and “memory apparatus or memory forming apparatus.” A good candidate for the storage mechanism is Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). CaM kinase II is one of the most prominent protein kinases, present in essentially every tissue but most concentrated in the brain. Neuronal CaM kinase II regulates important neuronal functions, including neurotransmitter synthesis, neurotransmitter release, modulation of ion channel activity, cellular transport, cell morphology and neurite extension, synaptic plasticity, learning and memory, and gene expression. Studies concerning this kinase open a door of the molecular basis of nerve function, especially learning and memory, and indicate one direction for the studies in the field of neuroscience. This review presents molecular structure, properties and functions of CaM kinase II, as a major component of neuron, which are mainly developed in our laboratory.
NF-E2-related factor-2 (Nrf2), a basic leucine zipper transcription factor, is involved in the expression of numerous detoxifying and antioxidant genes via the antioxidant response element (ARE). Keap1, a cytoplasmic protein, sequesters Nrf2 in the cytoplasm under normal conditions. Various stimuli, including electrophiles and oxidative stress, liberate Nrf2 from Keap1, allowing Nrf2 to translocate into the nucleus and to bind to the ARE. Recently, there is increasing evidence that compounds that stimulate the activation of the Nrf2-ARE pathway may become useful therapeutic drugs for neurodegenerative diseases associated with oxidative stress. Apomorphine (Apo), a dopamine D1/D2 receptor agonist, is used for clinical therapy of Parkinson's disease. On the other hand, Apo is a potent radical scavenger and has protective effects on oxidative stress-induced cell death. We previously reported that pretreatment of human neuroblastoma SH-SY5Y cells with Apo enhanced the protective effects. In addition, we have recently demonstrated that Apo stimulates the translocation of Nrf2 into the nucleus and the transactivation of the ARE. Our findings suggest that not only the function as a radical scavenger, but also the function as an Nrf2-ARE pathway activator may be involved in the neuroprotective effects of Apo on oxidative stress-induced neuronal cell death. In this review, our recent studies on the mechanism underlying Apo-induced neuroprotection are summarized.
A new method for consecutive α- and β-activation of propiolates toward electrophiles has been developed, which is mediated by suitable tertiary amines (e.g., DABCO) involving intramolecular silyl migration as a key step. Methyl 3-trimethylsilylpropiolate was reacted with aromatic aldehyde in the presence of DABCO in refluxing benzene to give a highly functionalized olefin product, in which new carbon-carbon bonds were formed at both α- and β-positions of the starting propiolate. On the other hand, when using aliphatic aldehyde the reaction course was dramatically changed to afford a propargyl TMS ether as a sole product. However, we suppose that these reactions have a common reaction pathway partly, including ammonium ylide-alkylidene carbene equilibrium, and that the former products arise from the ylide form and the latter from the carbene form. These domino reactions were successfully applied for an intramolecular version by use of substrates having both formyl group and TMS-propiolate structure derived from salicylaldehyde, leading to a new formylcoumarin-forming reaction.
Vitamin D receptor antagonist has attracted significant level of interests because of its potential utility in the treatment of Paget's disease, which is known as the most flagrant example of disordered bone remodeling and the second most common bone disease after osteoporosis in Anglo-Saxons. Recent studies on Paget's disease suggested a specific increase in osteoclasts sensitivity to the differentiation activity of active vitamin D3 as the principal mechanism for abnormal bone formation. We set out to conduct a structure-activity relationship study on the first VDR antagonists of TEI-9647 and TEI-9648 (25-dehydro-1α-hydroxyvitamin D3-26,23-lactone) toward improved VDR antagonistic activity. Given that both potent agonists and antagonists must have high affinity for the VDR, we hoped that our accumulated knowledge in VDR agonists would help us identify potent antagonists. First, 2α-modified TEI-9647 analogs were synthesized, and then, 24-substitution was next investigated to stabilize its lactone structure under the physiological conditions. Finally, 2α-modified 24-methyl-, 24,24-dimethyl-25-dehydro-1α-hydroxyvitamin D3-26,23-lactone analogs were synthesized. It was found that 2α,24,24-trimethyl-TEI-9647 was found to possess approximately 90-fold improved antagonistic activity (IC50 0.093 nM) over the original TEI-9647 (IC50 8.3 nM).
Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors and commonly play an important role in the regulation of lipid homeostasis. Although three PPAR subtypes, α, δ and γ show a relatively close amino acid sequence homology, the functions of each PPAR are distinct. For example, PPARα and PPARδ induce lipid oxidation, while PPARγ activates lipid storage and adipogenesis. To analyze the detail functions of human PPARs, we previously established tetracycline-regulated human hepatoblastoma cell lines that can be induced to express each human PPAR subtype. The expression of each PPAR subtype in established cell line was tightly controlled by the concentration of doxycycline. DNA microarray analyses using these cell lines were performed with or without adding ligand and provided the important information on the PPAR target genes. Furthermore, we analyzed the 5′-flanking region of the human adipose differentiation-related protein (adrp) gene that responded to all subtypes of PPARs, and determined the functional PPRE of the human adrp gene. Here we discuss the usefulness of these cell lines.
Spinosin is the major effective single constituent in the traditional Chinese herb Semen Ziziphi Spinosae, which is used for sedation and hypnosis. For the further use of spinosin in treating insomnia, the pharmacokinetics and tissue distribution of spinosin after intravenous administration to rats was investigated. An HPLC method with an ODS column (250 mm×4.6 mm, i.d.) and a mobile phase of acetonitrile-water-acetic acid (23:77:1) was used for the determination of spinosin in the plasma and tissues of rats. Vanillin was used as an internal standard, and spinosin was detected at 334 nm. The calibration curve of spinosin in plasma showed good linearity over the concentration range of 1-300 μg/ml, and the quantitation of limit of plasma was 1 μg/ml. The linear range of concentrations of spinosin in the heart, spleen, stomach, lung, testis, brain, and intestine was 0.1-40 μg/ml and the quantitation limit was 0.1 μg/ml. The linear range of concentrations of spinosin in the liver and kidney was 1-150 μg/ml, and the quantitation limit was 1 μg/ml. The correlation coefficients of all calibration curves were between 0.9939 and 0.9980. The intra and interrun precision for all samples was less than ≤11.0%. The time-concentration curve of spinosin after the intravenous administration of a single dose of 20 mg/kg to rats corresponded to the two-compartment model. The main pharmacokinetic parameters T0.5α, T0.5β, CLs, AUC0-T, and Vc were 6.66 min, 51.5 min, 1.42 l·min-1, 2.83 mg·min·ml-1, and 14.0 l·kg-1, respectively. At 20 min, a concentration peak occurred in liver and brain tissues. The highest level of spinosin occurred in the liver, followed by the spleen and kidney. The lowest level of spinosin appeared in the testis, followed by the brain. Spinosin was not detected in smooth and skeletal muscle. After intravenous administration, the drug was distributed extensively and transferred quickly in rats in vivo.
The comparison study was performed with 3 kinds of Lipo PGE1 (5 μg/ml) preparations (Formulation A, B, and C), which are now used in clinical. Under alkali condition, Lipo PGE1 (5 μg/ml) preparations in combination with physiological solution containing calcium ion were susceptible to stop dropping because of the formation of aggregates. There was a difference of feasibility to form aggregates among these preparations. The percentage of PGE1 in the LM (lipid microspheres) was 68.8% (Formulation A) when determined by filtration with the pore size of 0.1 μm, and the respective value (%) of Formulation B and Formulation C was 43.0% and 13.9%. This indicates that the latter formulations were significantly susceptible to leak from the LM. PGE1 can induce an extensive irritation. The potency of irritation was the most in Formulation C. This seems similar with the result of LM retention of PGE1. PGE1 increased the blood flow. Formulation A reached the peak by 2.27 fold, which was significantly higher than Formulation C and PGE1 alone (PGE1-cyclodextrin, PGE1-CD). The peak was also significantly higher in Formulation B than that of PGE1-CD. The AUC value of blood flow rate showed a significant increase in Formulation A and Formulation B as compared to that of PGE1-CD. Drug retention in the LM can be a determinant factor for drug distribution and pharmacological effect. This study indicates that there can be some differences among Lipo PGE1 preparations, which have the same drug dose.
Mouth washing after inhalation of corticosteroids is effective for prevention of local adverse effects. We determined the amounts of drug residues remaining on the oropharyngeal mucosa following inhalation of budesonide (BUD) via a Turbuhaler (BUD-TH) (100 μg). Further, we studied the effects of mouth washing on the removal of drug residues by quantification of BUD in expectorated wash solution using an HPLC method. The amount of BUD recovered after gargling and rinsing for 5 s each was 19.4±9.4 μg, as compared to 23.8±13.6 μg after rinsing alone for 10 s and 18.3±8.9 μg after gargling alone for 10 s, though the differences were not significant. Our results indicated that about 20% of the dose was remaining on the oropharyngeal mucosa after inhalation. In a comparison of washing times, the amounts of BUD recovered were 26.3±3.2 μg after gargling and rinsing for 3 s each, and 19.4±9.3 μg after those for 5 s each. As for the effect of lag time before beginning mouth washing, the ratio of BUD recovered following mouth washing with a lag time of 1 min was 73.2%, while it was reduced to 27.8% after 10 min, as compared to immediate mouth washing following administration. Our results suggest that the amount of BUD removed by mouth washing is associated with the lag time between inhalation and mouth washing, however, not with the duration of mouth washing. We concluded that immediate mouth washing after inhalation is most useful for the removal of drugs following BUD-TH administration.
Coenzyme Q10 (CoQ10) is widely consumed as a food supplement because of its recognition as an important nutrient in supporting human health. Absorption of compounds from the gastrointestinal tract is one of the important determinants of oral bioavailability. However, the absorption of dietary CoQ10 is slow and limited due to its hydrophobicity and large molecular weight. The absorption of orally applied compounds can be enhanced by interactions with food or food components. Thus, we investigated the effect of food intake on the absorption of CoQ10 after oral supplementation. In this study, we demonstrated that food intake enhanced the intestinal absorption of CoQ10. In order to improve intestinal absorption of CoQ10 after oral supplementation, we developed an emulsion formulation. Intestinal absorption of CoQ10 after administration of the emulsion formulation was also enhanced by food intake. Moreover, the peak concentration and the extent of absorption after administration of the emulsion formulation were greater than those after administration of a suspension formulation. It is possible that administration of CoQ10 in an emulsion formulation enhances the pharmacological effects of CoQ10.
Prednisolone is a safe antiinflammatory agent for the treatment of inflammatory diseases. To improve the aqueous solubility of the drug and dissolution rate, the complexation of prednisolone with skimmed milk was studied. A physical mixture and solid dispersion of prednisolone with skimmed milk were prepared. The lyophilization method was used to prepare the solid dispersion. Detection of inclusion complexes was performed in the solid state using differential scanning calorimetry (DSC), powder X-ray diffractometry, and scanning electron microscopy. The diffractogram of the complex differed from that of the physical mixture, where the characteristic peaks of prednisolone, particularly at 23.9°, 44.6°, and 72.2° (2 θ), nearly disappeared, indicating the formation of a true inclusion complex. These observations were in accordance with the results of the DSC analysis. Disappearance of the specific DSC peaks of the drug in the DSC curve of the solid dispersion showed that the drug interacts with the carrier.
Sedative effect of the aqueous extract of Centranthus longiflorus ssp. longiflorus (Cle-1) on intact and adrenalectomized rats was investigated using a thiopental sleeping test to clarify the relationship of this effect on adrenal gland hormones, particularly glucocorticoids. Adrenal gland hormones were found to play an important role in inhibiting the sedative effect of the investigated drugs. It is clear, however, that these hormones are not glucocorticoids. Glucocorticoids were not responsible for shortening the sleep period.
The construction and electrochemical response characteristics of polyvinyl chloride (PVC) membrane sensors for determination of tiapride in presence of its degradation products are described. The sensors are based on the ion association complexes of tiapride cation with sodium tetraphenyl borate (Tia-TPB) [sensor 1]) or ammonium reineckate (Tia-R) [sensor 2] counter anions as ion exchange sites in PVC matrix. The performance characteristics, sensitivity and selectivity of these electrodes in presence of tiapride degradation products were evaluated according to IUPAC recommendations. It reveals a fast, stable and linear response for tiapride over the concentration range 10-5-10-2 M with cationic slopes of 28.997 and 30.580 mV per concentration decade with sensors 1 and 2, respectively. These sensors exhibit fast response time (20-30 s), low quantitation limit (4.5×10-6 and 3.6×10-6, respectively), and good stability (6-8 weeks). The direct potentiometric determination of tiapride hydrochloride using the proposed sensors gave average recoveries of 99.95±0.678 and 99.92±1.157 for sensors 1 and 2, respectively. The sensors are used for determination of tiapride hydrochloride, in pure form, in presence of its degradation products in tablets, and in plasma. Validation of the method shows suitability of the proposed sensors for use in the quality control assessment of tiapride hydrochloride and for routine analysis as stability indicating method. The developed method was found to be simple, accurate and precise when compared with a reference company spectrophotometric method.
The ethyl acetate, chloroform extracts, and the two flavonoids kaempferol 1 and kaempferol-3-O-glucoside 2 isolated from whole plants of Thesium chinense Turcz were investigated for their antiinflammatory and analgesic activities. For the antiinflammatory activity, carrageenan-induced hind paw edema and xylene-induced mouse ear edema models, and for the analgesic activity, the acetic acid-induced abdominal constriction test was used. The ethyl acetate extract and two flavonoids showed significant (p<0.05 and p<0.01) and dose-dependent antiinflammatory and analgesic activity. The chloroform extract was inactive in the assay.
The aim of the current study was to design an oral sustained release matrix tablet of metformin HCl and to optimize the drug release profile using response surface methodology. Tablets were prepared by non-aqueous wet granulation method using HPMC K 15M as matrix forming polymer. A central composite design for 2 factors at 3 levels each was employed to systematically optimize drug release profile. HPMC K 15M (X1) and PVP K 30 (X2) were taken as the independent variables. The dependent variables selected were % of drug released in 1 hr (rel1 hr), % of drug released in 8 hrs (rel8 hrs) and time to 50% drug release (t50%). Contour plots were drawn, and optimum formulations were selected by feasibility and grid searches. The formulated tablets followed Higuchi drug release kinetics and diffusion was the dominant mechanism of drug release, resulting in regulated and complete release within 8 hrs. The polymer (HPMC K 15M) and binder (PVP K 30) had significant effect on the drug release from the tablets (p<0.05). Polynomial mathematical models, generated for various response variables using multiple linear regression analysis, were found to be statistically significant (p<0.05). Validation of optimization study, performed using 8 confirmatory runs, indicated very high degree of prognostic ability of response surface methodology, with mean percentage error (±S.D.) 0.0437±0.3285. Besides unraveling the effect of the 2 factors on the in vitro drug release, the study helped in finding the optimum formulation with sustained drug release.
Gambir (asen'yaku in Japanese), an aqueous extract of leaves and young twigs of Uncaria gambir Roxb., has traditionally been used as a treatment for diarrhea and dysentery as an internal medicine and for sore throat as a gargle. Although it is a pharmacopoeic medicine in Japan, the quantitative evaluation of its constituents has not yet been adopted in the Japanese pharmacopoeia. We analyzed polyphenolic constituents in 31 gambir and related products to establish evaluation methods, since gambir contains large amounts of polyphenolic constituents. The total flavan contents in the samples revealed using the vanillin-HCl estimation method ranged from 24-79%. Reversed-phase high performance liquid chromatography (RP-HPLC) analysis indicated that catechin was the most abundant constituent in each sample, with contents in the range of 7-76%. The catechin contents in the gambir products between the first and third quartiles were 28-54%. Thus, the lower limit of the catechin content in gambir products can be set at around 20% for quality management. Fifteen tested samples were subjected to HPLC analysis to show the presence of epicatechin (1.5% on average) and the dimeric compounds procyanidin B1, procyanidin B3, and gambiriin A1 (ca. 1% each). The molecular weight distributions of polymeric flavans in the gambir products were analyzed by gel permeation chromatography (GPC) and showed that the average degree of polymerization for each sample was 3 to 7. These results indicate that the combination of the vanillin-HCl method, RP-HPLC analysis, and GPC analysis gives valuable information for evaluating the polyphenolic profiles of gambir products.
We performed a retrospective study to examine the preventive effects of newquinolones for endogenous infection in patients receiving various allogeneic hematopoietic stem cell transplantation including bone marrow transplantation (BMT), peripheral blood stem cell transplantation (PBSCT), and cord blood transplantation (CBT). Forty-nine patients were enrolled. Ciprofloxacin or norfloxacin was orally administered for intestinal sterilization from day -14 until engraftment. As a result, the period from transplantation until engraftment was significantly longer in CBT group than in BMT group. The febrile index (the ratio of the febrile (≥38.0°C) period during neutropenia (≤500 cells/mm3) and C-reactive protein (CRP)-positive index (the ratio of CRP-positive (≥2.0 mg/dl) period during neutropenia) were comparable among the three groups. In addition, no gram-negative bacteria in stool was isolated in the three groups; that is, an endogenous infection of gram-negative bacteria, a potential pathogen, was well controlled by newquinolones. We should be careful when interpreting the results of this small study; however, newquinolones are clinically effective for endogenous infection of gram-negative bacteria in patients receiving not only BMT, but also PBSCT and CBT.
Evidence has recently been accumulating that a sirolimus-eluting stent (DES) is superior to a bare-metal stent (BMS) in preventing restenosis after percutaneous coronary intervention (PCI), and an increasing number of Japanese hospitals have been adopting DES. We conducted a retrospective study to identify clinical factors that influence the risk of restenosis after PCI, including stent types, by analyzing the data of 49 continuous patients who received PCI and follow-up coronary angiography in Hiratsuka City Hospital between March, 2004 and March, 2005. Age, sex, body mass index, smoking, complications, clinical diagnosis before PCI, the site and number of stenoses, implanted stent type (BMS or DES), the number of stents used, maximum inflating pressure and withdrawal of ticlopidine due to its adverse drug reactions were chosen as potential factors that may influence the risk of restenosis, and the correlation between these factors and restenosis was tested by Student's t-test or chi-square test. Coronary restenosis developed in 10 out of 49 patients, and factors having significant correlation with restenosis were age (73±7 in the restenosis group (R) and 64±12 in the non-restenosis group (N) (p<0.05)) and the type of stent (DES used in only one of 10 cases in R whereas in 24 of 39 in N (p<0.001)). Multivariate analysis showed older age (odds ratio (OR): 1.200 (95% CI: 1.038-2.823)) and the use of DES are independent predictors for restenosis (OR: 0.015 (95%CI: 0.001-0.249)). Our study further supports the efficacy of DES in PCI, but its long-term outcome is yet to be confirmed.