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Volume 127, Issue 1
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Symposium Reviews
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Norimichi NAKAHATAArticle type: Foreword
2007 Volume 127 Issue 1 Pages 1-2
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
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Norimichi NAKAHATA, Masaki SAITOArticle type: Review
2007 Volume 127 Issue 1 Pages 3-14
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSG protein-coupled receptors (GPCRs) are seven transmembrane receptors with an N-terminus in the extracellular region and C-terminus in the intracellular region. When an agonist binds to a GPCR, a signal is transduced into a cell through the activation of trimeric G proteins. Recently, it has been shown that the activities of GPCRs are regulated by multiple mechanisms. One of the mechanisms is regulation through the binding proteins to the carboxy (C)-terminus of GPCRs. In the present study, the binding partners for the C-terminus of the parathyroid hormone receptor (PTHR) and thromboxane A2 receptor (TP) were searched for using yeast two-hybrid screening, and the functions of these proteins were investigated. We identified t-complex testis expressed-1 (Tctex-1) and 4.1G as associated proteins for the PTHR. Tctex-1 is one of the light chains of cytoplasmic dynein, which is a motor protein across microtubles. We found that Tctex-1 was involved in agonist-induced internalization of the PTHR. 4.1G, a cytoskeletal protein, facilitated the cell surface localization of the PTHR and augmented PHTR-mediated signal transduction. TPs consists of two splicing variants, TPα and TPβ. As a result of yeast two-hybrid screening, two proteasomal proteins, proteasome activator PA28γ and proteasome subunit α7, were identified as direct interacting proteins for TPβ. TPβ has a tendency to be retained in the intracellular compartment, probably due to its binding to proteasomes. We also demonstrated that TPα and TPβ formed heterodimers, and the signal transduction through TPα was reduced by the formation of heterodimers. In conclusion, the proteins bound to GPCRs may regulate the intracellular traffic of GPCRs.
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Hiroyuki FUKUIArticle type: Review
2007 Volume 127 Issue 1 Pages 15-25
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSHistamine H1 receptors are down-regulated as one step in receptor desensitization. Five phosphorylation sites of the H1 receptor seem to play a key role in receptor down-regulation. In contrast, an increase in the H1 receptor expression level following its mRNA elevation was found in the nasal mucosa in hypersensitivity model rats. Up-regulation of the H1 receptor was induced by the direct stimulation of the H1 receptor. H1 receptor up-regulation was suppressed by pretreatment with antiallergic agents.
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Satoko OHKUBO, Norimichi NAKAHATAArticle type: Review
2007 Volume 127 Issue 1 Pages 27-40
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSLipid rafts and caveolae are microdomains in the cell membranes, which contain cholesterol, glycolipids, and sphingomyelin. While caveolae are relatively stable because caveolin, an integral protein, supports the structure, lipid rafts are considered to be unstable, being dynamically produced and degraded. Recent studies have reported that lipid rafts contain many signaling molecules, such as glycosylphosphatidylinositol-anchored proteins, acylated proteins, G-protein-coupled receptors (GPCRs), trimeric and small G-proteins and their effectors, suggesting that the lipid rafts have an important role in receptor-mediated signal transduction. Therefore drugs that modify the composition of lipid rafts might influence the efficacy of cellular signal transduction. In this review, we demonstrate the role of lipid rafts in GPCR-G-protein signaling and also present our recent results showing that the wasp toxin mastoparan modifies Gq/11-mediated phospholipase C activation through the interaction with gangliosides in lipid rafts.
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Norihiro KOBAYASHI, Junichi GOTOArticle type: Foreword
2007 Volume 127 Issue 1 Pages 41-42
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
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Kunihiko ITOHArticle type: Review
2007 Volume 127 Issue 1 Pages 43-53
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSPhage display has been utilized for making recombinant antibody fragments (Fab or single chain Fv) of human, mouse, or other origins. After construction of an antibody combinatorial library, antigen-specific recombinant antibody fragments can be easily isolated by biopanning of the phage library displaying antibody fragment fused with viral coat protein III against antigen proteins, antigen-expressing live cells, or fixed cells. Using this technique, a variety of human recombinant antibody fragments can be retrieved from bone marrow cells, lymph node cells, or peripheral blood cells of patients with infectious diseases, autoimmune diseases, and cancer. To develop diagnostically and therapeutically useful human antibody medicines, we should first select recombinant antibody fragments not only with antigen-binding activity but also with bioactivity such as virus or toxin neutralization, or tumor-specific cytotoxicity. To achieve this goal, several steps in antibody phage display may be improved: 1) a larger library should be constructed for possible isolation of minor populations present in the repertoire; 2) the biopanning procedure should be improved for isolation of antibody fragments reactive with immunologically minor epitopes; 3) the screening procedure should be based on the measurement of the bioactivity as well as the antigen-binding activity; 4) if necessary, the affinity and specificity of selected antibody fragments should be improved. In this review, I discuss how to isolate clinically useful recombinant antibody fragments efficiently using a phage display system introducing our achievements.
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Norihiro KOBAYASHI, Yoshinori KATO, Hiroyuki OYAMA, Junichi GOTOArticle type: Review
2007 Volume 127 Issue 1 Pages 55-69
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSThe trace characterization of physiologically active substances with low molecular weight (e.g., steroids, catecholamines, prostaglandins, and oligopeptides), which are classified as “haptens”, is an important subject in clinical analysis, and competitive immunoassays have conventionally been used for this purpose. However, the subfemtomole-range determination of haptens is very difficult, as the sensitivity of competitive immunoassays is essentially limited by the affinity of the anti-hapten antibodies that barely reaches the range of 1011 (l/mol) as the affinity constant (Ka). Although a noncompetitive “immunometric assay” format, the two-site immunometric assay (sandwich immunoassay), enables even subattomole-range measurements of macromolecules such as proteins, this principle can not be directly applied to haptens, as their low molecular mass prohibits simultaneous binding by two antibody molecules. To overcome such limitations, we are required either to create artificial antibodies showing ultrahigh affinity to haptens by protein engineering of antibody molecules (“antibody engineering”) or establishment of novel immunometric assay formats applicable to haptens. This review surveys the background and recent approach for subfemtomole-range determination of haptens using novel immunometric assay methods. Our studies for the development of hapten immunometric assays are also described.
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Hiroshi UEDAArticle type: Review
2007 Volume 127 Issue 1 Pages 71-80
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSProteins having multiple epitopes can be usually measured with sandwich ELISA employing two kinds of antibodies, which permits high sensitivity as well as a wide working range of more than three orders of magnitude. On the other hand, so-called monovalent antigens with MW less than 1000 are not susceptible to sandwich assays due to their small size and have almost always been measured in competitive assays. However, while a competitive assay needs only one antibody due to the principle of ratiometric measurement, optimization of the reaction conditions is necessary to attain suitable sensitivity and working range, which are often inferior to those of sandwich assays. As an alternative immunoassay for small antigens, here we propose a noncompetitive “open sandwich” immunoassay, which is based on the principle of stabilization of the antibody variable region Fv upon binding with antigen. Using ELISA to detect labeled VH fragments bound to immobilized VL in the presence of sample in microplate wells, various small molecules with MW around 200—300 can be measured with a superior detection limit and working range compared with those achieved with the corresponding competitive assays. The results indicate a common antigen recognition mode of anti-hapten antibody and wide applicability of the assay to the sensitive and handy analysis of low molecular-weight substances in areas such as environmental analysis and clinical diagnostics.
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Kunihiro OHTA, Hidetaka SEOArticle type: Review
2007 Volume 127 Issue 1 Pages 81-89
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSMonoclonal antibodies (MAbs) have been utilized as research tools, as diagnostic reagents, and for antibody medicine. The preparation of MAbs involves laborious processes and normally takes months. Here we describe an ex vivo B cell-based antibody display system called the ADLib (Autonomously Diversifying Library) system, which enables us to select chicken B cell clones producing antibody against antigens of interest in a couple of weeks. The ADLib system is applicable to self- or highly conserved antigens, polysaccharide chains, peptides, and haptens.
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Ikuo FUJIIArticle type: Review
2007 Volume 127 Issue 1 Pages 91-99
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSAdvances in methods for conformational prediction, structural analysis and site-directed mutagenesis of proteins and peptides have contributed to the understanding of their structure and function. However, with the exception of a few successes, the generation of practical functional molecules solely by rational design remains a difficult challenge. The aim of our study is to investigate molecular design relying on evolutionary processes, called as “directed evolution”, to generate a novel class of biofunctional molecules. This evolutionary approach consists of three steps; 1) constructions of protein/peptide libraries based on structural information, 2) expressions of the libraries on phage particles, and 3) selections with investigator-imposed selective pressures. In this work, we study on directed evolution with antibody libraries. We have succeeded in generating highly active catalytic antibodies in phage-displayed antibody (Fab) libraries. To evolve catalytic antibodies toward higher catalytic activity, we have mimicked an enzyme-evolutional process, in which an enzyme has evolved their ability to use binding energies for catalysis by increasing the affinity for the transition state of a reaction and decreasing the affinity for the ground state. Thus, phage-displayed libraries derived from an original catalytic antiboby were selected against a newly-devised TSA, which was programmed to optimize the differential affinity for the transition state relative to the ground state, to provide variants with improved reaction rates (kcat). The in vitro evolution has great potential for generating novel catalysts as well as for providing opportunities to examine the evolutionary dynamics of enzymes.
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Noriaki HIRAYAMAArticle type: Foreword
2007 Volume 127 Issue 1 Pages 101-102
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
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Hiroki SHIRAI, Masato KOBORIArticle type: Review
2007 Volume 127 Issue 1 Pages 103-112
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSWe describe the prospects of bioinformatics for drug discovery and discuss the current status, problems, and future direction of the interface between bioinformatics and docking studies. We also describe our recent work on sequence and structure analysis using the guanidino-modifying enzymes superfamily as a good example.
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Noriaki HIRAYAMAArticle type: Review
2007 Volume 127 Issue 1 Pages 113-122
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSThe effective integration of detailed structural information with computational chemistry, medicinal chemistry, and informatics transforms the dream of virtual screening into reality. One of the most important technologies essential for virtual screening is an effective docking method to find molecules that efficaciously interact with their target molecules. Since an efficient docking method can be a powerful tool for virtual screening, many different approaches to solving docking problems have been proposed. Docking problems have not yet been solved and none of the currently available programs are perfect in predicting all possible scenarios. Despite the limits and imperfections of the methodology, currently available docking methods are very useful for drug discovery. The basic principles and limits of docking methods together with matters for attention in applying the methods are described in this paper.
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Takatsugu HIROKAWAArticle type: Review
2007 Volume 127 Issue 1 Pages 123-131
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSProtein structure-based molecular design using the computational techniques of protein structure prediction, ligand docking, and virtual screening is an integral part of drug discovery for limiting the application of the structure-based approach to target proteins such as G-protein-coupled receptors (GPCRs). GPCRs play an important role in living organisms and are of major interest to the pharmaceutical industry. However, structural data on ligand-binding forms for GPCRs from experiments to elucidate structural templates for docking simulations are lacking due to the difficulties associated with crystallization and crystallography. Therefore structural prediction of GPCRs in the ligand-bound state using computational methods has been introduced, but the prediction of ligand conformation onto target GPCRs is still constructed manually by human experts. We developed a molecular modeling technique for the prediction of ligand-receptor binding using comparative ligand-binding analysis (CoLBA) that not only considers interaction energy but also the similarity of interaction profiles among ligands. The advantage of CoLBA is that it can facilitate intuitive and flexible screening based on docking results when protein structures with low resolution (or theoretical models) are targeted. We applied CoLBA to ligand-binding prediction in several GPCRs. The predicted ligand-binding models were evaluated in site-directed mutagenesis experiments in collaborative research, and the enrichment rate of activated ligands was compared with random compounds in virtual screening simulations. We propose that CoLBA can be applied in large-scale modeling of ligand-receptor complexes and virtual screening for GPCRs.
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Tomoaki HISHIDAArticle type: Foreword
2007 Volume 127 Issue 1 Pages 133-134
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
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Yoshikazu JOHMURAArticle type: Review
2007 Volume 127 Issue 1 Pages 135-142
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSThe mid- and late stages of adipocyte differentiation are known to be regulated by transcription factors such as peroxisome proliferator-activated receptor (PPAR)γ and CCAAT-box/enhancer binder protein (C/EBP) families. However, events in the early stage of adipocyte differentiation remain largely unknown. To gain insights into the molecular mechanisms underlying the beginning of adipocyte differentiation, we have isolated 102 genes, which are induced at the beginning of the differentiation of mouse 3T3-L1 preadipocytes, using the polymerase chain reaction (PCR)-subtraction method. Of these, 46 appear to be unknown genes. Since rapid amplification of cDNA end (RACE), cDNA library screening, and a genome database search have revealed that two of these genes are novel, we have named them factor for adipocyte differentiation (fad) 24 and fad158. The database research of amino acid sequences revealed that fad24 has a basic leucine zipper motif and an NOC domain, and fad158 has four transmembrane domains and eight leucine-rich repeats. The expression of fad24 and fad158 transiently increased after the addition of adipogenic inducers [insulin, dexamethasone, 3-isobutyl-1-methylxanthine, fetal bovine serum (FBS)]. RNAi-mediated knockdown of fad24 or antisense fad158 inhibited adipogenesis of 3T3-L1 preadipocytes and decreased expressions of PPARγ and C/EBPα. Furthermore, the constitutive overexpression of fad24 or fad158 in the mouse fibroblast cell line NIH-3T3 resulted in adipocyte conversion when stimulated with adipogenic inducers and PPARγ ligand BRL49653. Moreover, it was found that FAD24 localizes in the nucleus, especially within nuclear speckles and nucleolus, and FAD158 localizes to the endoplasmic reticulum (ER). Taken together, fad24 and fad158 appear to regulate adipocyte differentiation by activating the PPARγ pathway.
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Taira WADAArticle type: Review
2007 Volume 127 Issue 1 Pages 143-151
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSObesity, which results from adipose differentiation and adipocyte hypertrophy, is a primary risk factor of these life-style-diseases. Obesity, is primary risk factor of these life-style-diseases, results from adipose differentiation and adipocyte hypertrophy. Adipose differentiation is regulated by several transcriptional factors, and we have focused here on the roles played by endothelial PAS domain protein1 (EPAS1) in adipogenesis. EPAS1 was identified as a factor responsible for hypoxia responses, such as angiogenesis, here we demonstrated that EPAS1 is highly induced during adipose differentiation in vivo and in vitro. We then analyzed EPAS1 promoter activity during adipose differentiation in 3T3-L1 cells. We showed that the sequence -478/-445 is responsible for the up-regulation of EPAS1 expression during adipose differentiation and that the activity of this region is controlled by Sp1 and Sp3. To examine whether EPAS1 exerts an influence on adipogenesis, we overexpressed dominant negative form of EPAS1 in 3T3-L1 cells. The expression of EPAS1 (1-485) allowed cells to accumulate only a minimum amount of lipid droplets. Therefore, induction of EPAS1 expression is necessary for execution of adipose differentiation program. The mechanism involves the direct transcriptional regulation of Glut1, Glut4 and IRS3 genes by EPAS1. These results also confirmed that the protein level of EPAS1 was increased by insulin stimulation in adipocytes. Taken together, this result also indicated that EPAS1 plays a role in the part of insulin action. Therefore, these results suggest that the quantitative and functional alteration of EPAS1 are involved in metabolic syndrome occurrence.
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Makoto ARAKI, Yuka NOZAKI, Kiyoto MOTOJIMAArticle type: Review
2007 Volume 127 Issue 1 Pages 153-162
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSPyruvate dehydrogenase kinase 4 (PDK4) phosphorylates and inactivates the pyruvate dehydrogenase complex to respond to physiologic conditions. This response switches the energy source from glucose to fatty acids to maintain blood glucose levels. Transcription of the PDK4 gene is activated by fasting or by the administration of a peroxisome proliferator-activated receptor α (PPARα) ligand in a tissue-specific manner. However, the two mechanisms to induce PDK4 mRNA as well as the relationship between the two have not been studied in detail. In this study, we show that the two mechanisms are independent, at least in the mouse skeletal muscle, and that estrogen-related receptor α (ERRα) is directly involved in the PPARα-independent transcriptional activation of the PDK4 gene with peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) as a specific partner. The latter conclusion is based on the following evidence: 1) Deletion and point mutation analyses of the cloned mouse PDK4 gene promoter sequence identified an exact possible ERRα-binding motif as the PGC-1α responsive element. 2) The overexpression of ERRα by cotransfection enhanced, and the knocking down of it by specific shRNAs diminished, the PGC-1α-dependent activation. 3) Specific binding of ERRα to the identified PGC-1α-responsive sequence of the mouse PDK4 promoter was confirmed in the electrophoresis mobility shift assay using anti-ERRα antibodies. These results suggest that PGC-1α plays an essential role not only in regulating the amounts of energy creating enzymes, but also at the step of metabolic switching with unevenly distributed tissue transcription factors such as ERRα in the skeletal muscle, thus harmonizing tissue-specific functions and energy metabolism.
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Norimasa TAKAHASHI, Masashi MORITA, Tsuneo IMANAKAArticle type: Review
2007 Volume 127 Issue 1 Pages 163-172
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSAdrenoleukodystrophy (ALD) is an inherited disorder characterized by progressive demyelination of the central nervous system and adrenal dysfunction. The biochemical characterization is based on the accumulation of pathgnomonic amounts of saturated very long-chain fatty acid (VLCFA; C>22) in all tissues, including the brain white matter, adrenal glands, and skin fibroblasts, of the patients. The accumulation of VLCFA in ALD is linked to a mutation in the ALD (ABCD1) gene, an ABC subfamily D member. The ALD gene product, so-called ALDP (ABCD1), is thought to be involved in the transport of VLCFA or VLCFA-CoA into the peroxisomes. ALDP is a half-sized peroxisomal ABC protein and it has 745 amino acids in humans. ALDP is thought to be synthesized on free polysomes, posttranslationally transported to peroxisomes, and inserted into the membranes. During this process, ALDP interacts with Pex19p, a chaperone-like protein for intracellular trafficking of peroxisomal membrane protein (PMP), the complex targets Pex3p on the peroxisomal membranes, and ALDP is inserted into the membranes. After integration into the membranes, ALDP is thought to form mainly homodimers. Here, we chose nine arbitrary mutations of human ALDP with naturally occurring missense mutations and examined the intracellular behavior of their ALDPs. We found that mutant ALDP (S606L, R617H, and H667D) was degraded together with wild-type ALDP by proteasomes. These results suggest that the complex of mutant and wild-type ALDP is recognized as misfolded proteins and degraded by the protein quality control system associated with proteasomes. Further, we found fragmentation of mutant ALDP (R104C) on peroxisomes and it was not inhibited by proteasomes inhibitors, suggesting that an additional protease(s) is also involved in the quality control of mutant ALDP. In addition, mutation of ALDP (Y174C) suggests that a loop between transmembrane domains 2 and 3 is important for the targeting of ALDP to peroxisomes.
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Regular Articles
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Ilhan ÖZTEKIN, Sümeyye GÖKDOGAN, Deniz S. ÖZTEKIN, ...Article type: Regular Article
2007 Volume 127 Issue 1 Pages 173-182
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSIt is not clear how levels of serum lipids and glucose and plasma osmolality change during propofol infusion in the pre- and postoperative period of coronary artery bypass graft surgery (CABG). This prospective, randomized, controlled trial evaluated changes in these parameters during propofol or midazolam infusion during and in the early postoperative period following surgery. Twenty patients undergoing CABG were randomized preoperatively into two groups: 10 patients received propofol (induction 1.5 mg/kg, maintenance 1.5 mg kg-1 h-1) and 10 patients received midazolam (induction 0.5 mg/kg, maintenance 0.1 mg kg-1 h-1). Both groups also received fentanyl (induction 20 μg/kg, maintenance 10 μg kg-1). Serum lipids, glucose, and plasma osmolality were measured preinduction, precardiopulmonary bypass, at the end of cardiopulmonary bypass, at the end of surgery, and 4 and 24 h postoperatively. In the propofol group, we observed a significant increase in triglycerides and very low-density lipoprotein levels 4 h postoperatively. In the midazolam group, we observed a significant decrease in low-density lipoprotein, cholesterol at the end of cardiopulmonary bypass, end of surgery, and 4 and 24 h postoperatively and significant increase in osmolality at the end of cardiovascular bypass. Changes in glucose levels did not differ significantly different between the two groups. In patients with normal serum lipids, glucose, and plasma osmolality undergoing CABG, propofol infusion for maintenance anesthesia is not associated with dangerous changes in serum lipids, glucose, and plasma osmolality compared with midazolam. A propofol infusion technique for maintenance of anesthesia for cardiac surgery where serum lipids and glucose may be of concern could be recommended as an alternative to midazolam.
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Chunjie WU, Hongyi QI, Wenwen CHEN, Chunyan HUANG, Cheng SU, Wenmin LI ...Article type: Regular Article
2007 Volume 127 Issue 1 Pages 183-191
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSThe purpose of this study was to develop a pH-triggered in situ gelling vehicle for ophthalmic delivery of puerarin. The effect of hydroxypropyl-β-cyclodextrin (HP-β-CD) on the aqueous solubility and in vitro corneal permeation of puerarin was also investigated. The puerarin solubility increased linearly and proportionally to the HP-β-CD concentrations and 5% (w/v) HP-β-CD enhanced its ocular permeability significantly. Carbopol® 980NF was used as the gelling agent in combination with HPMC (Methocel E4M) which acted as a viscosity-enhancing agent. The optimum concentrations of Carbopol® 980NF and HPMC E4M for the in situ gel-forming delivery systems were 0.1% (w/v) and 0.4% (w/v), respectively. When these two vehicles were combined, an in situ gel that had the appropriate gel strength and gelling capacity under physiological condition could be obtained. This combined solution could flow freely under non- physiological condition and showed the character of pseudoplastic fluid under both conditions. Both in vitro release studies and in vivo pharmacokinetics studies indicated that the combined polymer systems performed better in retaining puerarin than puerarin eye drops did. These results demonstrate that the Carbopol® 980NF/HPMC E4M can be a viable alternative to conventional puerarin eye drops to enhance ocular bioavailability and patient compliance.
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Mohammad Galal El-BARDICY, Hayam Mahmoud LOTFY, Mohammad Abdalla El-SA ...2007 Volume 127 Issue 1 Pages 193-199
Published: 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSA high performance liquid chromatographic method was developed and validated for determination of meclophenoxate hydrochloride (I) in the presence of its degradation product (p-chlorophenoxy acetic acid) (II). Separation of (I) from (II) was performed using a ZORBAX ODS column with a mobile phase consisting of 0.2% triethylamine in 0.01 M ammonium carbonate: acetonitrile (70:30 v/v). The method showed high sensitivity with good linearity over the concentration range of 50 to 400 μg/ml. The method was successfully applied to the analysis of a pharmaceutical formulation containing (I) with excellent recovery. A kinetics investigation of the alkaline hydrolysis of (I) was carried out in sodium hydroxide solutions of 1, 1.5 and 2 N by monitoring the parent compound itself. The reaction order of (I) followed pseudo-first order kinetics. The activation energy could be estimated from the Arrhenius plot and it was found to be 12.331 kcal/mole.
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Mohammad Galal El-BARDICY, Hayam Mahmoud LOTFY, Mohammad Abdalla El-SA ...Article type: Regular Article
2007 Volume 127 Issue 1 Pages 201-208
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSThe construction and electrochemical response characteristics of polyvinyl chloride (PVC) membrane sensors for the determination of meclophenoxate hydrochloride (I) and pyritinol dihydrochloride (II) in presence of their degradation products are described. The sensors are based on the use of the ion-association complexes of (I) and (II) cation with sodium tetraphenyl borate and ammonium reineckate counteranions as ion-exchange sites in the PVC matrix. In addition β-cyclodextrin (β-CD) membranes were used in the determination of I and II. These ion pairs and β-CD were then incorporated as electroactive species with ortho nitrophenyl octyl ether (oNPOE) as a plasticizer. Three PVC sensors were fabricated for each drug, i.e. meclophenoxate tetraphenyl borate (meclo-TPB), meclophenoxate reineckate (meclo-RNC) and meclophenoxate β-cyclodextrin (meclo-β-CD), and the same was done for pyritinol (pyrit-TPB), (pyrit-RNC) and (pyrit-β-CD). They showed near Nernestian responses for meclophenoxate over the concentration range 10-5—10-2 with slopes of 52.73, 51.64 and 54.05 per concentration decade with average recoveries of 99.92±1.077, 99.96±0.502 and 100.03±0.763 for meclo-TPB, meclo-RNC and meclo-β-CD respectively. Pyritinol also showed near Nernestian responses over the concentration range of 3.162×10-6—3.162×10-4 for pyrit-TPB and pyrit-RNC, and 10-6—3.162×10-4 for pyrit-β-CD with slopes of 30.60, 31.10 and 32.89 per concentration decade and average recoveries of 99.99±0.827, 100.00±0.775 and 99.99±0.680 for pyrit-TPB, pyrit-RNC and pyrit-β-CD respectively. The sensors were used successfully for the determination of I and II in laboratory prepared mixtures with their degradation products, in pharmaceutical dosage forms and in plasma.
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Note
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Toshiro NIWA, Sachiko YAMAMOTO, Miho SAITO, Toshifumi SHIRAGA, Akira T ...2007 Volume 127 Issue 1 Pages 209-216
Published: 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSThe effects of cyclosporine and tacrolimus on cytochrome P450 (CYP) 1A2-mediated 7-ethoxyresorufin O-deethylation, CYP2C9-mediated tolbutamide hydroxylation, CYP2C19-mediated S-mephenytoin 4′-hydroxylation, CYP2D6-mediated debrisoquine 4-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, CYP3A4-mediated nifedipine oxidation, and CYP3A4-mediated testosterone 6β-hydroxylation activities in human liver microsomes were compared. Cyclosporine and tacrolimus, at concentrations of 0.2 or 2 μM, neither inhibited nor stimulated any of the metabolic activities except for those of CYP3A4. On the other hand, cyclosporine and tacrolimus competitively inhibited CYP3A4-mediated nifedipine oxidation activity, with inhibition constants (Ki) of 1.42 and 0.36 μM, respectively. In addition, 20 μM cyclosporine inhibited CYP2C19 and CYP2D6 activities by 29% and 30%, respectively. These results suggest that tacrolimus would not cause clinically significant interactions with other drugs, which are metabolized by CYPs, via the inhibition of hepatic metabolism and that the reason why cyclosporine, but not tacrolimus, has a pharmacokinetic inhibitory effect might be that the dosage and/or the unbound concentrations around its metabolic enzymes are higher than those of tacrolimus, rather than the differences in the inhibition potential. Obvious substrate-dependent effects on CYP3A4-inhibition potential were not observed.
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Article
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Masao UCHIBAYASHIArticle type: Article
2007 Volume 127 Issue 1 Pages 217-224
Published: January 01, 2007
Released on J-STAGE: January 01, 2007
JOURNAL FREE ACCESSA biography of Leo Sternbach, an inventor of benzodiazepine tranquillizers, is presented. It consists of (1) a societal desire for lifestyle pills, (2) Leo's birth in 1908 and youth, (3) education, (4) in Vienna, (5) in Zurich, (6) at Hoffmann-La Roche, Basel, (7) to the New World, (8) at Roche, Nutley NJ, (9) invention of the new drugs, (10) revolution of people's lifestyle, and (11) reward, retirement and obituary in 2005. This paper may be the first comprehensive biography of this remarkable chemist written in Japanese.
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