Recent studies suggest that chronic local inflammation and cellular stresses are the key steps in organ defects in obesity-related diseases such as atherosclerosis, diabetes, and fatty liver. We have shown that an excess energy state activates sterol regulatory element-binding protein (SREBP)-1c, a master transcription factor for fatty acid synthesis causing the accumulation of lipids leading to fatty liver, insulin resistance, insulin secretion defects, and dyslipidemia and we clarified their molecular mechanisms. Recently, we shifted focus to the quality aspect of accumulated lipids. It has long been known that saturated and poly-unsaturated fatty acids are atherogenic and anti-atherogenic, respectively. Besides desaturation, we found that the chain-length of fatty acids is another important factor. Elovl6 that we have cloned as an SREBP-1 target is a fatty acid elongase that catalises the last step of fatty acid synthesis. Elovl6 KO mice exhibit obesity and fatty liver on a high energy diet, but unexpectedly were immune to insulin resistance (Nat. Med., 13, 2007, Matsuzaka et al.), atherosclerosis (Arterioscler. Thromb. Vasc. Biol., 31, 2011, Saito et al.) and non-alcoholic steatohepatitis (NASH) pathology including liver damage, ROS, and fibrosis (Hepatology, 56, 2012, Matsuzaka et al.). Elovl6 is crucial for protection against lung fibrosis (Nat. Commun., 4, 2013, Sunaga et al.). These data implicate that fatty acid composition is a new therapeutic target for a variety of chronic metabolic diseases. In this symposium review, a novel strategy for the prevention of life-related diseases will be discussed in the standpoint of application of wet-dry fusion strategies for theoretical and medicinal chemistry.
The influenza A virus genome consists of eight-segmented, single-stranded, negative-sense RNAs. Each genomic viral RNA segment (vRNA) encodes different viral proteins that are necessary for efficient virus replication, and forms a ribonucleoprotein complex (RNP) together with viral nucleoproteins and an RNA polymerase complex. Later in infection, progeny virions, which are released from the plasma membrane of the infected cell, must incorporate the eight separate vRNAs to be infectious. However, the mechanism by which the segmented vRNAs are incorporated into each progeny virion remains unclear. To elucidate the genome packaging mechanism of influenza A virus, we examined the architecture of RNPs within progeny virions using several electron microscopic analyses. We demonstrated that each progeny virion incorporates eight RNPs arranged in a specific pattern, in which seven RNPs surround the central one. Such characteristic arrangement is found in all influenza A virus strains tested here, suggesting that the mechanism by which well-organized eight RNPs is incorporated into virion is common to influenza A viruses. In addition, there seem to be physical interactions among the eight RNPs via nucleic acid-like structures, suggesting that there are specific interactions among the eight vRNAs in the form of RNPs. These results indicate that influenza A virion selectively packages a complete set of eight separate vRNAs.
For the prevention and control of infectious viral diseases, vaccines and antiviral drugs targeting viral proteins are of great importance. Amino acid substitutions in viral proteins occasionally cause the emergence of antibody-escape and drug-resistant mutants. With regard to this, we have studied the proteins of several viruses, especially the influenza A virus, by using techniques of computational chemistry and biology such as molecular modeling, molecular docking, and molecular dynamics simulations. Influenza A virus is a zoonotic pathogen that is transmitted from animals to humans. This virus has two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). The HA of influenza viruses plays a key role in the initiation of viral infection. And HA is also the major target of antibodies that neutralize viral infectivity. Some amino acid substitutions in the antigenic epitope on HA could decrease the interaction between HA and antibodies, leading to the generation of antigenic variants with novel antigenic structures of HA. In addition, HA protein seems to be a favorable target for anti-influenza drugs, but effective HA inhibitors have not been developed due to the emergence of drug-resistant viruses with amino acid substitutions on the HA. To understand how amino acid substitutions affect changes in drug susceptibility, we have been computationally analyzing the three-dimensional structures of influenza virus proteins. In this paper, we review the results obtained through our current analysis.
Alzheimer's disease (AD) is the most common senile dementia. One of the pathological characteristics of AD is the appearance of senile plaques composed of amyloid-β (Aβ) depositions. Aβ is generated by consecutive cleavages of Aβ precursor protein (APP) by β- and γ-secretases. The common pathogenesis for familial AD (FAD) is believed to involve misprocessing of APP by γ-secretase, resulting in increased Aβ42 peptide deposition. However, little is known about γ-secretase function in sporadic AD (SAD), which is the major type of AD. This may be because Aβ42 peptide has highly aggregative properties; therefore it is not easy to estimate the quantitative alteration of net Aβ42 in SAD patients. Alcadein is a family of neural type I membrane proteins. Processing of Alcadein by APP α- and γ-secretases results in secretion of non-aggregative peptide, p3-Alc, into CSF and blood. The C-terminuses of Aβ and p3-Alc are altered by FAD-linked genetic mutations in catalytic components of γ-secretase, in association with an increase in minor Aβ and p3-Alc species. Thus p3-Alcs are expected to behave as useful indicators of γ-secretase dysfunction in SAD brain. Quantitative and qualitative analyses of p3-Alcs raise the possibility that γ-secretase dysfunction may exist even in the absence of genetic mutations. p3-Alc peptides may be a novel biomarker for AD and an indicator of γ-secretase dysfunction for drug development.
Functional multilayer thin films have been prepared by layer-by-layer (LbL) deposition for the development of sensors, separators, and drug delivery systems. In particular, glucose-sensitive LbL films have been widely studied for use as glucose sensors and in glucose-triggered drug delivery systems. In this work, I report on glucose-sensitive LbL films that consist of concanavalin A (ConA), phenylboronic acid (PBA), and glucose oxidase (GOx). ConA/glycogen LbL films were prepared by LbL deposition of ConA and glycogen through a lectin-sugar interaction. Similarly, PBA-modified poly(amidoamine) dendrimer/poly(vinyl alcohol) (PVA) LbL films were prepared through cyclic boronate ester bonds. Both types of films decomposed in the presence of glucose, by the competitive binding of glucose, although these LbL films did not show a satisfactory response to millimolar concentrations of glucose under physiological conditions. PBA-modified poly(allylamine hydrochloride) and PVA films were prepared on a GOx-modified quartz slide. The LbL film was stable over a wide pH range, from 3.0 to 9.0, in the absence of glucose. In contrast, the film decomposed upon exposure to 0.1-10 mM glucose solutions for 60 min at pH 7.4. The glucose-induced decomposition of the film can be explained by the scission of the carbon-boron bond of the PBA residues by hydrogen peroxide, which was produced through the GOx-catalyzed oxidation of glucose. These results suggest this multilayer film may be useful for the development of glucose-sensitive drug delivery systems.
Large individual variations in drug efficacy and safety could be explained in part by pharmacokinetics regulated by drug transporters and drug-metabolizing enzymes. However, expression and/or function of these proteins often fluctuate in pathological conditions, causing individual pharmacokinetic variability. To achieve a personalized pharmacotherapy after liver transplantation, our group has been investigating the pharmacokinetics of drugs and factors causing its variation based on molecular biological analysis using rats with liver ischemia-reperfusion (I/R) injury as a model for injuries immediately after liver transplantation. The first finding is that the oral bioavailability of cyclosporine A (CsA), which is an immunosuppressant, was decreased by increased first-pass metabolism due to elevated CYP3A and P-glycoprotein (P-gp) specifically in the upper small intestine after liver I/R. Expression of CYP3A in the small intestine was also elevated through transcriptional regulation by endogenous bile acids, whereas expression and function of intestinal P-gp were increased by post-transcriptional regulation via microRNA-145. Next, the pharmacokinetics of a cationic drug, cimetidine, which is eliminated from the kidney, and the expressional variation of drug transporters in the kidney after liver I/R were examined. Liver I/R decreased tubular secretion of cimetidine, mainly because of decreased expression of rat organic cation transporter 2 in the kidney. These findings provide useful information on the etiology of liver I/R injury and appropriate use of immunosuppressants and drugs eliminated from the kidney after liver transplantation.
A separation-oriented derivatization method combined with LC has been developed for the selective analysis of biogenic and related compounds. In this method, we utilized a specific affinity between perfluoroalkyl-containing compounds, i.e., ‘fluorous’ compounds (fluorophilicity). Our strategy involves the derivatization of target analytes with perfluoroalkyl reagents, followed by selective retention of the derivatives with a perfluoroalkyl-modified stationary phase LC column. The perfluoroalkylated derivatives are strongly retained on the column owing to their fluorophilicity, whereas non-derivatized species, such as sample matrices, are hardly retained. Therefore, utilizing this derivatization method, target analytes can be determined selectively without interference from matrices. This method has been successfully applied to the LC analysis of some biogenic and related compounds in complex biological samples.
Cancer patients often report a decreased quality of life due to cancer-related pain, especially neuropathic pain, which is difficult to manage and often develops resistance to morphine. Thus a supplementary analgesic can play an important role in the treatment of cancer-related pain. Carbamazepine (CBZ), for example, can be effective, but only if the patient can take medication orally because it is limited to oral administration. In this study we investigated the efficacy of a suppository containing CBZ tablet in hospital preparations. We selected a base for the suppository of either polyethylene glycol (P), Witepsol VOSCO® H-15 (H), or Witepsol VOSCO® S-55 (S). Six to eight in vitro and in vivo groups were divided randomly based on route of administration and treatment: intravenous (i.v.), per os (p.o.), and intrarectal administration (i.r.) (composed of CBZ powder and tablet formulations, CBZp and CBZt) prepared in either a base of P, H, or S (CBZp/P, CBZp/H, CBZp/S, CBZt/P, CBZt/H, and CBZt/S). The hardness levels of the CBZt suppository group were significantly lower than the CBZp suppository group. The drug release profiles of the CBZt suppository group were high in order of P, H, and S; there were no significant differences between these groups and the CBZp suppository group. The maximum drug concentration time levels of the CBZt suppository group significantly increased compared with the p.o. group. These two groups had equivalent maximum drug concentration and bioavailability levels. These results suggest that a suppository containing CBZ tablet can be useful for hospital preparations.
In Japan the prevalence of dementia has increased considerably, and pharmacists are involved in addressing these patients' medication-related problems. Here, we determined whether pharmacists' comprehensive assessment of medication profiles could reduce the burden of dementia patients' medication-related problems. In this historical cohort study 120 community pharmacies were randomly selected, and participating pharmacists completed questionnaires concerning comprehensive assessment of patient medication profiles, using a “start” questionnaire for patients prescribed medication prior to or during the study period and a “follow-up” questionnaire for patients who subsequently visited pharmacies for prescriptions. Numbers and details of problems and solutions implemented by pharmacists and identified in the start and follow-up questionnaires were compared. Changes in start and follow-up scores were also compared between patients whose problems were identified by pharmacists (identified group) and those whose problems were not (non-identified group). Data were collected for 349 patients issued medication by 60 pharmacies. The most common medication-related problems identified in the start survey were key person's understanding of donepezil (60 cases) and other dementia treatments (60 cases), and adherence to treatment (53 cases). Solutions implemented by pharmacists included gathering information regarding drug administration and dementia awareness from the key person and providing pharmaceutical counseling and instruction. Subsequently, problems related to understanding of dementia treatment, understanding donepezil, and adherence were resolved by 70.0%, 65.0%, and 58.5%, respectively. Pharmacists' comprehensive assessment of medication profiles could effectively solve dementia patients' medication-related problems.
Despite an increase in the number of reports on medical safety interventions conducted by ward-based pharmacists, only a few reports have classified intervention cases in detail. We classified and compared the types of medical safety intervention conducted by ward-based pharmacists since the introduction of their services. The interventions were classified into: cases that were identified by pharmacists or through asking questions about physicians' prescriptions before dispensing medications (active interventions); and those in which pharmacy technicians could contribute to medical safety by receiving inquiries from patients or healthcare providers (passive interventions). The numbers of the two types of intervention were compared. The number of interventions significantly increased after the introduction of ward-based clinical pharmacy services. Especially, the numbers of cases identified during ward rounds conducted by ward-based pharmacists (active interventions) and those identified by receiving inquiries from physicians or nurses (passive interventions) significantly increased, possibly because the collection of patient information was performed more efficiently by conducting ward rounds, and an environment where physicians and nurses can easily make inquiries to pharmacists was established after increasing the time pharmacists spend on hospital wards. The results demonstrate that the introduction of ward-based clinical pharmacy services has improved communication with patients, facilitated information-sharing among physicians, nurses, and other healthcare providers, contributed to the safer management of pharmaceutical operations, and increased interest of patients.
Long-term clinical training based on a model core curriculum was conducted to nurture highly competent pharmacists in the clinical field. Pharmacists' responsibilities are expanding, and a system has been developed to help pharmacists gain accreditation, identify specialties, and improve their training. However, this system requires research competency. Therefore clinical research should be considered a part of clinical training to encourage high competency among pharmacists. Because the model core curriculum does not include a section on clinical research. Osaka City University Hospital introduced a hands-on clinical research experience program and evaluated its usefulness. A significant improvement in the level of knowledge and awareness of clinical research was seen among students who underwent the clinical research experience program. In addition, the level of student satisfaction was higher. These findings suggest that a clinical research experience program may be useful to nurture a greater awareness of clinical research and knowledge acquisition among pharmacists.