With recent development of the nanotechnology, nanomaterials have been successfully employed in various industrial applications such as medicine and cosmetics. Nanomaterials show the useful properties such as electronic reactivity and the tissue permeability that were not provided by micromaterials. Thus, nanomaterials are expected as innovative materials for the development of medicine and cosmetics. However, these innovative properties may show unknown biological responses that could not been detected by the conventional toxicity assay. For industrial development and affluent society establishment that enjoyed only a benefit of nanomaterials, it is urgent to gather information of the properties and the biological effects, and to establish the standard safety evaluation method of nanomaterials. So, we are analyzing association among property, biodistribution and biological effects of nanomaterials to search for the safety biomarker (functional-, molecular- and biochemical-biomarker) using nanosilicas (nSP) as a standard nanomaterial. Because nSP shows high uniform dispersibility and is already used in medicine, cosmetics and food additive, the results of this study are useful to extrapolate it to other nanomaterials and to make practicable as safety biomarker. In this report, we show the latest knowledge about the linkage information among property, biodistribution and biological effects of nSP by toxicokinetics and toxicoproteomics, and the search study of safety biomarker based on these basic information.

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  To clarify the whole picture of epidermal growth factor (EGF) signaling pathway, we identified proteins from the EGF-stimulated A431 cells by anti-phospho-tyrosine antibody column chromatography. Over 150 proteins were detected including previously unidentified proteins as well as well-studied proteins. Among these proteins, we picked up four proteins that had not been known in EGF signaling pathway and analyzed their functions. We report the functions of these proteins in this article. 1) CFBP interacts with CD2AP family proteins and functions as a key component in downregulation of EGF receptor protein level following EGF stimulation. 2) Ymer is found to be phosphorylated and ubiquitinated upon EGF stimulation, and functions as a regulator for the downregulation and endocytosis of EGF receptor. 3) CLPABP binds to mitochondria-specific phospholipids, cardiolipin, through its PH domain, and its complex includes various proteins related to mRNA metabolism. 4) GAREM is associated with Grb2 and Shp2. Each association affects the ERK activity. Finally, we discuss the possibilities that these proteins can be used as a novel biomarker protein for cancer and other diseases.

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  Tumor blood vessels are essential for tumor growth. Therefore, these blood vessels are potential targets for anti-cancer therapy. The purpose of this study is to develop anti-tumor endothelial cell (TEC) antibodies for delivering anti-cancer agents or drugs. To achieve this goal, we utilized the phage antibody display library method to create monoclonal antibodies in vitro. Accordingly, we developed anti-TEC antibodies from an single chain Fv fragment (scFv) phage display library prepared using the Fv genes amplified from the mRNAs isolated from the TEC-immunized mice. The size of the phage antibody library prepared from the mRNA of the TEC-immunized mice was approximately 1.3×10⁷ CFU. To select and enrich for the phages displaying the anti-TEC antibodies, cell panning was performed first using the TEC followed by subtractive panning using the normal endothelial cell. After five cycles of panning, the affinity of bound phage clones increased approximately 10 000 folds. Subsequently, clones isolated from the post-panning output library were tested for their antigen-specificity by ELISA and western blotting. One of the scFv phage clones showing antigen-specificity recognized only TEC in vitro, and when injected into the Colon26 bearing mice, this clone accumulated more on the tumor tissue than the wild type phage. These results suggest that the isolated an antibody and this clone's target molecule could be potentially useful for novel anti-tumor therapies.

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  Molecular biomarkers are keys to the development of new diagnostic protocols and therapies. Recently, significant research effort has been devoted to the development of these biomarkers using various approaches. Perhaps the most promising approach is disease proteomics. This method involves analyzing and identifying changes in the expression pattern at the protein level in the diseased condition (disease-related proteins) by using two-dimensional differential gel electrophoresis analysis (2D-DIGE). In the case of disease proteomics, hundreds of candidate disease-related proteins can be identified at a time. Therefore, how to pick the really valuable proteins up from a number of candidate drug targets is the most important issue to be solved worldwide. Here, we introduce a novel approach, termed “antibody proteomics”, which addresses this issue. Using antibody proteomics it is possible to identify a variety of disease-related proteins by 2D-DIGE and simultaneously prepare monoclonal antibodies to these proteins by using a phage antibody library. The advantage of this technology is that the target proteins are identified in a high-throughput manner. Our approach relies on the fact that tissue microarray analysis can evaluate the relationship between disease-related proteins and disease progression, based on clinical and pathological information. In this review, we discussed the development and application of antibody proteomics and gave an overview of future work.

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  Conformational transition of monomeric amyloid β-peptide (Aβ) to a self-associated β-sheet structure is considered to be an initial step in the development of Alzheimer's disease. Several lines of evidence suggest that physiologically abundant lipid membranes and metal ions are involved in this step. We have demonstrated that Aβ binds to the phosphatidylcholine membrane in the lamellar gel phase but not in the liquid crystalline phase by using fluorescence and circular dichroism spectroscopy. The membrane-bound Aβ molecule takes α-helical or β-sheet structure depending on the temperature. Tightly packed phosphatidylcholine membranes appear to serve as a platform for non-electrostatic binding and self-association of Aβ. We have also examined Zn(II) and Cu(II) binding modes of Aβ by Raman spectroscopy. The Raman spectra demonstrate that three histidine residues in the N-terminal region of Aβ provide primary metal binding sites. Zn(II) binds to the N_τ atom of histidine and the peptide aggregates through intermolecular His-Zn-His bridges. In contrast, Cu(II) ion is chelated by the N_π atom of histidine and deprotonated main-chain amide nitrogens to form a soluble complex. Our findings on the conformational regulation of Aβ may help in better understanding the molecular basis for the development of Alzheimer's disease.

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  We have proposed that a nucleation-dependent polymerization model could explain the general mechanisms of amyloid fibril formation in vitro. Based on this model, we systematically demonstrated that several classes of organic compounds (e.g., wine-related polyphenols, non-steroidal anti-inflammatory drugs) not only inhibit the formation of Aβ amyloid fibrils from Aβ and their extension, but also destabilize Aβ amyloid fibrils dose-dependently in vitro. We found significant positive correlations of the effective concentrations (EC₅₀) of these compounds ranging from 10 nM to 10 μM, for the formation and destabilization of Aβ amyloid fibrils. We next investigated the anti-amyloidogenic effects of five flavonoids on Aβ amyloid fibrils in vitro. Oxidized flavonoids generally inhibited fibril formation significantly more potently than fresh compounds. By surface plasmon resonance (SPR) analysis, distinct association and dissociation reactions of myricetin (Myr) to Aβ amyloid fibrils were observed, in contrast to the very weak binding to the Aβ monomer. A significant decrease in the rate of fibril extension was observed when>0.5 μM of Myr was injected into the SPR experimental system. These findings suggest that flavonoids, especially Myr exert an anti-amyloidogenic effect in vitro by preferentially and reversibly binding to the amyloid fibril structure of fibrils, rather than to Aβ monomers. This working model should prove useful not only for the rational development of preventives and therapeutics for Alzheimer's disease and other human amyloidosis, but also for understanding the basic mode of action of amyloid imaging compounds.

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  The conversion of soluble, nontoxic amyloid β-peptide (Aβ) to aggregated, toxic Aβ rich in β-sheet structures by seeded polymerization is considered to be the key step in the development of Alzheimer's disease. Accumulated evidence suggests that lipid rafts (microdomains) in membranes mainly composed of sphingolipids (gangliosides and sphingomyelin) and cholesterol play a pivotal role in this process. Our model membrane studies revealed the following mechanism of Aβ aggregation in membranes. Soluble Aβ with unordered structures specifically binds to raft-like membranes containing a ganglioside cluster, the formation of which is facilitated by cholesterol. The membrane-bound Aβ forms an α-helix-rich structure at lower densities. At higher densities, Aβ undergoes a conformational transition to a β-sheet-rich structure that can serve as a seed for amyloid fibril formation. This model was confirmed at a cellar level using rat pheochromocytoma PC12 cells. Amyloid fibrils formed in lipid rafts were cytotoxic, whereas fibrils formed in solution were almost nontoxic and had different morphologies. Thus, the role of membranes in Aβ fibrillization is not merely the acceleration of Aβ aggregation but the generation of toxic fibrils. Furthermore, nordihydroguaiaretic acid was found to effectively inhibit the ganglioside-induced amyloidogenesis by preventing the binding of Aβ to the membrane.

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  Alzheimer's disease (AD) has become a major health burden to society. However, no fundamentally therapeutic drugs for AD have been developed. Increasing evidence suggests that the elevation of β-amyloid (Aβ) peptides in the brain is central to AD pathogenesis. Recently, in the course of our survey of substances having anti-dementia activity from natural resources, we have successfully found nobiletin, a polymethoxylated flavone contained in AURANTII NOBILIS PERICARPIUM which is a component of traditional Chinese medicines. In this review, we describe the beneficial effects of nobiletin on memory impairment and Aβ pathology in a transgenic mouse model introduced human “Swedish” and “London” mutant amyloid precursor protein. We also note the possible molecular mechanism underlying the protective action against Aβ-induced memory impairment provided by our studies using cultured hippocampal neurons. Namely, daily administration of nobiletin for four months rescued the memory impairment in fear conditioning, and decreased hippocampal Aβ deposit in the transgenic mice as analyzed by immunohistochemistry. PKA-dependent signaling and membrane trafficking of AMPA receptor subunit, GluR1, which are known to be required for long-term potentiation (LTP), have been demonstrated to be inhibited by a sublethal concentration of Aβ in cultured hippocampal neurons. Our in vitro studies evidently showed that a sublethal concentration of Aβ actually inhibited glutamate-induced increases in both PKA substrates phosphorylation and GluR1 membrane trafficking in cultured hippocampal neurons, whereas nobiletin reversed the Aβ-induced inhibition of such biochemical processes. The natural compound with these unique actions has thus potential to become a novel drug for fundamental treatment of AD.

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  Currently, there are five anti-Alzheimer's disease drugs approved. These are tacrine, donepezil, rivastigmine, galantamine, and memantine. The mechanism of the first four drugs is acetylcholinesterase inhibition, while memantine is an NMDA-receptor antagonist. However, these drugs do not cure Alzheimer's, but are only symptomatic treatments. Therefore, a cure for Alzheimer's disease is truly needed. Alzheimer's disease is a progressive neurodegenerative disease characterized by cognitive deficits. The cause of the disease is not well understood, but research indicates that the aggregation of β-amyloid is the fundamental cause. This theory suggests that β-amyloid aggregation causes neurotoxicity. Therefore, development of the next anti-Alzheimer's disease drug is based on the β-amyloid theory. We are now studying natural products, such as mulberry leaf extracts and curcumin derivatives, as potential cure for Alzheimer's disease. In this report, we describe some data about these natural products and derivatives.

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  We have developed two cell culture systems for use in pharmaceutical research using nano-biotechnology. First, we developed a double layered co-culture system using cell sheet technology, and showed that in a layered co-culture system with HepG2 and bovine endothelial cells, the expression levels of various cytochrome P450 (CYP) genes were significantly increased compared to monolayer cultured HepG2 cells. In the layered HepG2 co-culture, expression of the CYP2C and CYP3A family genes was induced by phenobarbital treatment. We also detected CYP3A4 enzyme induction using this co-culture system. Next, we developed sensor cells. Living cells maintain homeostasis by responding quickly and with great sensitivity to changes in the external environment. Consequently, sensors using cells as active elements are thought to be able to perform analyses faster and with more sensitivity than previous methods. We have modified mammalian cells using genetic engineering techniques to develop next-generation cell sensors that can visually represent specific reactions. We successfully produced devices using sensor cells that can process a variety of specimens using Micro-Electro-Mechanical System (MEMS), Nano-Electro-Mechanical System (NEMS), and other nano/micro processing technologies. These systems may serve as a useful model for in vitro pharmacological studies on the coordinated regulation of metabolism and cytotoxicity. In this review, we introduce our research and describe recent trends in this field.

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  Cultured hepatocytes are expected to be used for drug screening and bioartificial liver. Since hepatocytes lose their functions very rapidly in vitro, many attempts have been made to maintain their viability and functions. First, we want to introduce the surface modification of culture substrate using a starburst dendrimer. Addition of fructose to the terminal of the dendrimer was shown to be effective in maintianing hepatocyte function. As the second topic, we will show results of the use of a three-dimensional carrier for hepatocyte cultivation. Hepatocytes and bone marrow stromal cells were cocultured in silane beads, and packed into a radial flow-type bioreactor. The perfusion culture showed the effectiveness of bone marrow stromal cells for the maintenance of hepatocyte function. The next topic will be the trial of adenoviral gene transfer into hepatocytes. Thioredoxin gene was chosen because the products play important roles in redox control and antiapoptosis. The introduction of the gene could inhibit apoptosis and maintain the hepatocyte viability. Finally, we want to introduce the results on differentiation of stem cells into hepatocytes, because it is very difficult to obtain sufficient number of human hepatocytes. Human mesenchymal stem cells were cultured in the presence of several protein factors and the hepatocyte-specific marker was expressed after 2 weeks of induction culture. The use of human stem cells could be an important strategy for the support of a drug development system.

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  Limitation of conventional human Ether-a-go-go Related Gene (hERG) assay and QT prolongation testing for accurate prediction of Torsades de Pointes (TdP) by compounds showed us the necessity of a new approach to evaluate global cardiac safety. As one of the advanced applications of an on-chip cellomics system, on-chip cardiomyocyte cell network-based re-entry model assay has the potential to measure the TdP probability as a pre-clinical test for cardiac safety. This system also can estimate the heart pressure, Na, K, and Ca ion channel conditions using a single cell-based optical/electrical measurement system. In this presentation, we present the system setup and then its possible application for drug discovery and toxicology.

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  Sensors have been developed to determine the concentration of specific compounds in situ. They are already widely employed as a practical technological tool in the clinical and healthcare fields. Recently, another concept of biosensing has been receiving attention: biosensing for the evaluation of molecular potency. The author described the idea as qualified analysis. The development of this novel concept has been supported by the development of related technologies, such as electrochemistry, molecular interface science, molecular design, molecular biology (genetic engineering), and cellular/tissual engineering. This study addresses this new concept of biosensing and its application to the evaluation of the potency of chemicals in biological systems, in the field of cellular/tissual engineering. Cellular biosensing will provide valuable information for both pharmaceutical research and chemical safety, and be applicable in drug discovery in vitro as a screening tool.

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  The white of an egg, rendered opaque by boiling, can be converted into a thin, transparent and rigid material like glass by evaporating the moisture. This phenomenon is known as the vitrification of heat-denatured proteins. We applied vitrification technology to a collagen gel and converted it into a rigid glass-like material. We attempted to rehydrate the glass-like material and succeeded in preparing a novel stable state of collagen gel that was a thin and transparent membrane with excellent gel strength and protein permeability. We called it “collagen vitrigel” because it was produced from the vitrification process of a traditional hydrogel. Further, a framework-embedded collagen vitrigel membrane that can be easily turned inside out with tweezers was prepared by inserting a nylon membrane ring in the collagen sol prior to the gelation, thereby allowing the membrane to function as a removable cell culture substratum. Different types of anchorage-dependent cells could be cultured on both surfaces of the substratum by the manipulation of two-dimensional cultures, and consequently a three-dimensional crosstalk model with paracrine effects from each cell type was reconstructed. Also, the collagen vitrigel membrane containing a bioactive molecule provided a drug delivery system (DDS) with sustainable release. In this review, we summarize the recent progress of applied studies using the collagen vitrigel membrane as follows: a corneal model for eye irritant and permeability tests, a skin model for sensitization test, a renal glomerular model for evaluating blood filtration, an endometrial model for developing a new treatment and a DDS of hepatocyte growth factor for improving liver disorder.

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  In January 2007, Daini Okamoto General Hospital introduced a new system in which pharmacists, together with physicians and nurses, address patients with cardiopulmonary arrest on arrival and inpatients with de novo cardiopulmonary arrest. Over the past 2 years since the introduction of the system, the role of pharmacists in cardiopulmonary resuscitation (CPR) at the hospital has become increasingly established. Pharmacists prepare drugs for CPR, measure intervals during drug administration, and check the heart rhythm, produce records, pass drugs to physicians and nurses, and serve as CPR staff. CPR involves a large number of processes, and requires rapid responses. The participation of pharmacists in time management and drug administration, playing specific roles, has promoted role-sharing among physicians, nurses, and pharmacists, as well as the establishment of a “resuscitation team” in the hospital. Emergency medicine is in a difficult situation. We believe that our efforts have helped pharmacists contribute to emergency care and provide high-quality CPR.

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  Life-support (particularly, advanced life-support) training is not included in pharmacist education; however, the life-support should be mastered since a pharmacist is a medical professional. We consider it to be important to master other skills before the life-support practicing, because a pharmacist does not check a patient to assess their clinical condition and administer drugs (suppository, intravenous injection etc.) The pharmacist prepares medicines, but does not administer medicines to treat the patient. Furthermore, the pharmacist is not interested in the vital signs of the patient receiving the medicines (the pharmacist has not identified the patient has complaint from changes in vital signs), which is why pharmacists can not develop themselves as medical professionals. Based on this observation, life-support training should be considered. In other words, to foster pharmacists with high clinical competence, pharmacy students should receive life-support training after training in drug administration and vital sign checks in a bedside training room. Drug administration using a pharmacy system versatile-type training model and pharmacy training model, vital signs check and auscultation using a physical assessment model and a cardiac disease disorder simulator in our bedside practice are useful for advanced life-support using a high-performance care simulator (monitoring vital signs, adrenalin administration and oxygen inhalation for ventricular fibrillation (VF). These training skills can improve the clinical competence of pharmacy students.

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  Emergency medicine is an interdisciplinary area that covers medical care in an emergency room, trauma center and intensive care unit. It also provides prehospital emergency medical service and disaster medicine. Pharmacists are expected to play major roles as a member of the emergency medical team. The roles of pharmacist include inventory management, formulary management, administration guidance, medication delivery, identification of a tablet, analysis of intoxicating substance, and therapeutic drug monitoring. During disaster response, accompanying disaster relief team to provide supervision for stock and prescription of medications could also be an important mission of a pharmacist. As a member of the emergency medical team, knowledge and skills for effective communication is essential to discuss patients' condition and therapeutic strategy with other healthcare providers. Basic life support and first-aid ability are mandatory to all the medical professionals who take care of patients. Safety and security management at the emergency or disaster scene is another important requirement. We hope more pharmacists will join the emergency and disaster medicine and contribute to extend a hand to the sufferers.

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  We commenced to estimate the economic impact of salmeterol/fluticasone combination (SFC) therapy compared to fluticasone propionate (FP) therapy for asthma control in Japanese patients. A Markov model with five health states, developed by Price in 2002, was used. 1-week transition probabilities among status of asthma management were obtained from literature and epidemiological data from public data base. Direct cost for treatment was estimated from Japan medical fee schedule. Cost and effectiveness were not discounted due to 12-week simulation by the model. Univariate sensitivity analyses were undertaken to examine the main variables affecting cost-effectiveness. Probabilistic analysis was also undertaken to discuss statistical argument and to provide information for decision-making. In this analysis, the model was run over a 12-week period of time using transition probabilities. The results showed that treatment with SFC resulted in a higher proportion of totally controlled weeks per patient than treatment with FP (65.0 vs. 49.5%; incremental effectiveness by 15.5%), and lower mean direct asthma management costs (¥168 702 vs. ¥227 820). Probabilistic sensitivity analysis, conducted to assess robustness of the above base case result, showed that in the 95% of cases SFC was dominant (more effective and less costly) to FP. It suggested that SFC will be the most cost-effective therapy for asthma control. It would, however, be required to further evaluate cost-effectiveness of SFC in long-term observation.

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  Recently, it is required that community pharmacists participate in palliative home care. In this study, we designed educational activities for palliative home care to local residents as a new approach performed by community pharmacists. In addition, we proposed this approach to community pharmacists and discussed its roles to promote palliative home care in the future. We designed and held an educational seminar on palliative home care focusing on safe use of opioids in local residents. After this seminar, we conducted a questionnaire survey of the participants. Then, we made a proposition of this new approach by presentation of a seminar to community pharmacists (members of “Heartfulcare INC”). After the proposition, a questionnaire survey of the seminar on palliative home care was performed. In total, 79 people participated in the educational seminar. Most (87.3%) participants thought that it was informative. Furthermore, about 40% of participants answered “misunderstanding of opioids” or “anxiety regarding side effects of opioids” were removed following the seminar. All participating pharmacists evaluated the seminar as useful. The educational activity in the present study seemed effective to local residents on their misunderstanding of opioids. Furthermore, this approach was appreciated as an important role of community pharmacists in the palliative home care not only by local residents but also by pharmacists themselves. Therefore it seems important to continue such activities so as to improve palliative home care in the future.

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  We established a method for estimating pediatric doses of drugs metabolized by cytochrome P450 (CYP) isozymes, using the free fraction of drug in plasma (fu), serum protein level (P), liver volume (LV), and CYP activity (Vmax/Km) as indices of physiological and biochemical development in children up to 15 years old. This method allows the child/adult dose ratio (D_C/D_A)=child/adult oral clearance ratio (CL_(PO)C/CL_(PO)A) of drugs mainly metabolized in the liver to be estimated by the following equation:
\\[ \\frac{D_C}{D_A} \\cong \\left( \\frac{1}{fu_A + (1 - fu_A) \\frac{P_C}{P_A}} \\ ight) \\cdot \\left( \\frac{BSA_C}{BSA_A} \\ ight)^{1.176} \\cdot \\left( \\frac{Vmax_C/Km_C}{Vmax_A/Km_A} \\ ight) \\]
Major metabolism of drugs was ascribed to CYP1A2 for theophylline and caffeine, and CYP1A2 and CYP2D6 for propranolol and mexiletine. For theophylline and caffeine, CL_(PO)C/CL_(PO)A calculated from the child/adult body surface area ratio (BSA ratio) and the value calculated by our method were compared, using CL_(PO)C/CL_(PO)A calculated from the clearance ratio based on population pharmacokinetics (PPK ratio) as a reference. For all drugs, pediatric doses calculated from the Crawford equation and our equation were compared, with predetermined doses as the reference. For theophylline and caffeine, the relative accuracy of our method was significantly higher than that of BSA-based estimation when the PPK ratio was used for reference. For theophylline, caffeine, and propranolol, the relative accuracy of our method was significantly higher than that of BSA-based estimation when predetermined doses were used for reference. These findings indicate the validity of our method which considers the physiological and biochemical development (i.e., fu, P, LV, and CYP activity) for pediatric dose estimation.

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  We formerly reported the most suitable volumes of medicinal solution for continuous 5FU infusion over 46 hours in the FOLFOX6 and FOLFIRI regimens, respectively, by analyzing the relation of the total volume of the medicinal solution in a portable disposable infusion pump (SUREFUSER^®A) and the duration of infusion using a regression analysis. A total infusion time of about 48 hours was obtained. As the ambient temperature increased, however, we noticed that the continuous 5FU infusion finished earlier than anticipated. Using an additional analysis, we found that not only the medicinal solution's original coefficient of viscosity, but also the ambient temperature influenced the duration of infusion. Here, we report that the speed of continuous 5FU infusion increases as the coefficient of viscosity decreases in response to increases in ambient temperature. Thus, the composition of medicinal solutions and the ambient temperature must be considered to ensure a correct duration of continuous infusion.

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