The recent development of nanotechnology has facilitated a dramatic reduction in the size of materials. Nanomaterials are nanometer-sized materials with specific physicochemical properties that are different from those of the bulk material of the same composition. Such properties make them very attractive for cosmetic and medical applications. However, nanoparticles can act on living cells or bodies at the nano-level resulting in biologically undesirable as well as desirable effects. Thus, reduction in particle size from the micro- to nano-scale not only provides benefits to diverse scientific fields but also poses potential risks to the environment and to human health. Although significant resources are aimed at exploiting the desirable properties of nanoparticles for applications in medicine or cosmetics there are only limited attempts to evaluate potentially undesirable effects in vivo. Thus, there is a pressing need for a careful consideration of the benefits and side effects to the use of nanoparticles in medicine and cosmetics. In recent years, the majority of toxic biological response induced by nanomaterials (Nanotoxicity) has focused on cell culture systems. However, data from these studies will require verification from in vivo experiments using animals. An understanding of Toxicokinetics (the relationship between the physical properties of the nanomaterials and their in vivo behavior) would provide a basis for evaluating undesirable effects. Moreover, toxicoproteomics may identify predictive bio-markers for examining nanotoxicity. In this review article, we describe the assumptions and challenges in the field of nanotoxicity and describe advances for studying nanotoxicity of nanosilicas using toxicokinetics/toxicoproteomics both in vivo and in vitro.
It is considered that the materials with new properties may lead to novel biological effects or unknown adverse health effects. To gather proper hazard information, it is important to develop both experimental protocols and detection/measurement methods for nanomaterials in the body, in parallel. Since 2005, we are running research projects to develop methods to monitor health risk effects for the assessment of manufactured nanomaterials funded by the Ministry of Health, Labour and Welfare. For the experimental protocols, these projects focus on the development of 1) in vitro experimental systems, 2) in vivo experimental systems (mainly focusing on long-term health implication, especially carcinogenesis), and 3) proper inhalation system. Firstly, fullerene (C60), titanium dioxide and multi-walled carbon nanotube were chosen to be tested because of their high production volume. Safety issues for new materials such as nanoparticles is a new paradigm. The key is that the full scale exposure to the public has not been started yet. Therefore, there is a good chance that information from hazard identification studies can be directly fed back to the product development plan. Manufacturers can produce safer products without risking themselves waiting for the toxicology studies to be finished after their products are widely marketed.
Adenovirus vectors (Ad) have been frequently used for cancer gene therapy research because of their high gene transduction efficiency. However, systemic administration of conventional Ad can lead to the acute accumulation of virus particles and transgene expression in the liver, which may cause severe hepatotoxicity. For these reasons, clinical application of Ad for systemic administration has been limited, although intratumor administration of Ad has shown marked antitumor effects. Therefore, to promote the application of Ad in systemic cancer gene therapy, especially against the distant metastatic cancer, a novel Ad with marked accumulation in tumors and minimal hepatic distribution is needed. From this perspective, bioconjugation with polyethylene glycol (PEGylation) to Ad surface is a promising strategy, and we are trying to develop cancer targeted Ad by PEGylation approach. Through our study, we particularly clarified that PEGylated Ad (PEG-Ad) with optimized PEG modification ratio exhibited the enhanced distribution and gene expression in tumor tissue via systemic injection, which was based on the enhanced permeability and retention (EPR) effect. Moreover, PEG-Ad encoding therapeutic gene demonstrated not only stronger tumor-suppressive activity but also fewer hepatotoxic side effects compared with conventional Ad. In addition, we further attempted the active targeting using targeting ligand on the tip of PEG. We revealed that PEG-Ad with transferrin as a tumor targeting ligand could transduce more efficiently into tumor cells, which express transferrin receptor, compared with conventional PEG-Ad. In this symposium, I will present our approach for development of cancer targeted Ad by PEGylation.
Recently several systems including viral and non-viral carriers that can be used to transfer foreign genetic material into cells have been developed with the aim of enhancing gene transfer in vivo. Non-viral vectors are relatively easy to produce in clinically relevant quantities, and associated with fewer safety concerns. Furthermore, synthetic non-viral vectors provide flexibility in formulation design and can be tailored to interact efficiency with DNA cargo and the specific route of vector injection, and can enhance delivery to specific tissues or cells through incorporation of a targeting ligand. Applying cell-specific targeting technology to liposomes would improve in vivo gene transfection efficacy and reduce any unexpected side-effects. Among the various receptors, asialoglycoprotein receptors and mannose receptors are the most promising for gene targeting since they exhibit high affinity and are rapidly internalized. Receptor-mediated delivery systems are able to introduce foreign DNA into specific cell types in vivo. Our group succeeded in the development of glycosylated cationic liposomes for cell-selective targeting of plasmid DNA, siRNA, and NFκB decoy based on the optimization of physicochemical properties of glycosylated liposome complex.
Properties of gene delivery vehicles, including gene transfer efficiencies and toxicities, are a key parameter for successful gene therapy. Among various types of gene delivery vehicles that have been developed so far, adenovirus (Ad) vectors have promising potentials as a vector for gene therapy because they can easily be grown to high titers and can efficiently deliver genes to both dividing and non-dividing cells. However, recent studies demonstrated some drawbacks of conventional Ad vectors, which are composed of subgroup C Ad serotype 5 (Ad5). First, Ad5 vectors poorly transduce cells lacking the primary receptor for Ad5, coxsackievirus and adenovirus receptor (CAR). Second, majority of adults have neutralizing antibodies to Ad5. In order to overcome these drawbacks, we developed a novel Ad vector which is fully composed of subgroup B Ad serotype 35 (Ad35). Ad35 vectors can infect a variety of human cells because the primary receptor for Ad35, CD46, is ubiquitously expressed in human cells. Furthermore, Ad35 vectors efficiently transduce in the presence of anti-Ad5 antibodies, and seroprevalence of Ad35 in adults is much lower than that of Ad5. In the current review, I introduce our recent work on development and evaluation of Ad35 vectors, and I also discuss the potential of Ad35 vectors as gene delivery vehicles.
Legionella pneumophila is a facultative intracellular pathogen which replicates within macrophages and monocytes and finally cause a severe pneumonia known as Legionnaires' disease. An important hallmark of the pathogenesis of this bacterium is their ability to manipulate host cell processes, creating a specified replicative niche within host cells. An L. pneumophila-containing phagosome (LCP) is allowed to associate sequentially with smooth vesicles, mitochondria, and the rough endoplasmic reticulum (RER) to form a compartment called a replicative phagosome. LCPs are biologically characterized by delayed acidification and a low tendency to fuse with lysosomes. The establishment of these specialized phagosomes is mediated by the Icm/Dot Type IV secretion system, which is essential for the intracellular growth of L. pneumophila. L. pneumophila utilizes the Icm/Dot system to inject bacterial effector molecules into the host cell cytosol to survive and replicate in the intracellular compartment through modulation of phagosome biogenesis. This review focuses on our studies on specific aspects of L. pneumophila infection to host cells and bacterial factors which regulates its intracellular growth. We found several characteristic phenomena leading to L. pneumophila infection, which is dependent on LCP formation: active bacterial protein synthesis in L. pneumophila within macrophages, specific exclusion of actin-binding protein p57/Coronin-1 from LCP, and suppression of reactive oxygen species (ROS) production by macrophages upon infection with L. pneumophila. Furthermore, we identified a novel bacterial factor, PmiA, which is involved in multiplication within both protozoa and macrophages. Our recent study has begun to reveal that the biological function of PmiA is closely associated with that of the Icm/Dot type IV secretion system.
The Tokyo Metropolitan Government in Japan enacted an “Ordinance concerning the abuse prevention of the psychoactive drugs” in April 2006 that prohibited the manufacture, cultivation, sales, possession, use, etc., of these drugs. Therefore, we constructed a reproducible, simple, and small-scale determination method of the psychoactive drugs for the re-uptake and the release of monoamines (dopamine, serotonin and norepinephrine), and the activation of [35S]guanosine-5′-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins (G proteins). These assays were then applied to study the effects of different kinds (phenethylamine derivatives, tryptamine derivatives, and piperazine derivatives) of non-medically used psychoactive drugs on monoamine re-uptake and release, and G-protein binding. The results suggested that some drugs strongly act on the central nerve system to the same extent as the drugs. This assay system was able to designate psychoactive drugs as prohibited substances in accordance with criteria set forth by the Tokyo Metropolitan government.
It has been demonstrated that HMG-CoA reductase inhibitors effectively decrease low density lipoprotein and total cholesterol levels, and presently, HMG-CoA reductase inhibitors are most widely used in hyperlipidemia treatment. On the other hand, it has been demonstrated that fibrate agents decrease triglyceride levels more effectively compared to HMG-CoA reductase inhibitors. A cost-effectiveness study comparing fenofibrate, a fibrate agent, and atorvastatin was therefore conducted in hypertriglyceridemia patients. Referring to an analytical method published in the UK, the percentage of patients received fenofibrate and atorvastatin treatments at each dose level was estimated from prescription records at the medical institutions investigated. Changes in the total cholesterol and triglyceride values after the drug administration were investigated examining published reports. Based on the said data, the treatment effectiveness was measured by the percentage of patients who achieved the target lipid levels. The treatment costs were estimated based on the number of patients investigated and reimbursement prices of the drugs. The incremental cost-effectiveness ratio of fenofibrate in decreasing triglyceride levels was dominant over atorvastatin. The incremental cost-effectiveness ratio of atorvastatin in decreasing low density lipoprotein cholesterol levels was JPY 69911. This provides a model for choosing drug treatments that reflects clinical practices at medical institutions by substituting figures for individual cases.
To develop a new mucoadhesive film containing an analgesic combining clinical efficacy and patient comfort, we prepared and evaluated a two-layered film consisting of an adhesive layer containing indomethacin (IM) as the active ingredient and carboxyvinyl polymer (CP) as a bonding agent and a nonadhesive layer containing polyethylene glycol (PEG) to improve film texture. In in vitro and in vivo adhesive tests, the optimal concentration of CP that could be applied to the mucous membrane was 0.2% or 0.3%. Stability testing determined that the optimal storage conditions and expiration period were 4°C without shade and 4 weeks, respectively. The film was clinically evaluated in patients with oral pain. IM at concentrations of 0.5% and 1% provided optimum analgesic effects, and the effects were the greatest in the 1% IM group. The addition of PEG to the nonadhesive layer reduced the number of patients experiencing discomfort at the site where the film was applied. Therefore this film formulation may be useful for local analgesic application due to its low dose requirement, moderate adhesion, and comfortable texture.
A new perfumed dihydro-γ-pyrone, 2-phenyl-5,6-dihydro-4H-pyrane-4-one (12) and seven components of C6-C5 phenylpentanone derivatives have been isolated from the neutral essential oil parts in cultured mycelium of Mycoleptodonoides aitchisonii. These compounds were elucidated on the basis of spectral data and syntheses. The derivatives are odorant components. The perfumed components in cultured mycelium were maximized amount at 7 days.
To investigate the relation between tendency of feverishness and patient profile in pegylated interferon (peginterferon) and ribavirin combination therapy, we performed a retrospective survey of the medical charts of patients with chronic hepatitis C that have been on interferon therapy for the past two years. The sample was 36 patients (18 males, 18 females, aged 30 to 67 years, average of 57.5 years) who were hospitalized for the introduction of peginterferon-alpha 2b and ribavirin combination therapy and did not use NSAIDs before the administration of interferon. We examined body temperature and laboratory values (AST, ALT, WBC, Hb, PLT, BUN, Cr, HCV RNA level, and HCV genotype) for one week from the initiation of peginterferon administration to one week after the administration. Tendency of feverishness was classified into two groups according to the time to reach maximum body temperature. The early fever-developing group reached maximum body temperature within 12 hours of the administration, and the slow fever-developing group reached maximum body temperature after 12 hours post administration. The early fever-developing group had a significantly higher maximum body temperature (early group, 38.5°C±0.55; slow group, 37.8°C±0.55), more persistent fever (early group, 71.0 h±31.3; slow group, 36.5 h±41.8), and a higher percentage of females (early group, 80.0%; slow group, 28.6%). Moreover, females and elderly patients developed significantly higher fever (temperature difference 0.78°C) early (8.5 h) after peginterferon administration. These results indicate the need for careful observation of females and elderly patients in the early stage of an initial treatment of chronic hepatitis C with peginterferon.
We investigated the optimal blood concentration of voriconazole (VRCZ) based on trough blood concentrations (C) and serological test values in patients. With regard to the regulation of VRCZ dosage (D) to maintain optimal blood concentrations, we investigated the relationship between C and D. Twenty-three patients were enrolled in the present study, and at 28 point data were analyzed retrospectively. When the β-D-glucan or the Aspergillus antigen were decreased below the standard set values, it was evaluated as effective. The adverse events were evaluated using the Common Terminology Criteria for Adverse Events ver. 3.0. We found a significant difference (p<0.05) in the average trough blood concentration between patients in whom VRCZ was effective and those in whom VRCZ was ineffective (8.21±2.19 μg/ml vs. 1.01±0.86 μg/ml), and patients presenting with adverse events and those with no adverse events (7.64±2.84 μg/ml vs. 1.49±1.79 μg/ml). There was no significant relationship between C and D (C: D, y=0.018−2.186, r2=0.349). Improved efficacy and more adverse events thus occurred with higher blood concentrations. The careful regulation of the dosage must be performed while measuring blood concentration and observing for adverse events. The implementation of therapeutic drug monitoring for VRCZ is a valuable tool for achieving effective fungal infection therapy and should be aggressively investigated in future studies.
To determine the “home pharmacy” activities including preventive medicine in community pharmacies and their regional differences, we conducted two questionnaire surveys of pharmacies belonging to the pharmacists' association in four areas, two metropolitan areas (Kita-tama area and Minato-ku in Tokyo) and two rural areas (Ueda-shi in Nagano and Aira-gun in Kagoshima) in 1998 and 2007. The questionnaire consisted of 42 questions including the scale and characteristics of the pharmacy, the offering of information to patients and information collection from patients, and activities related to home care medicine, environmental sanitation, and healthcare. Based on 14 factors in the questionnaire, an index of “Community Medicine Contributed by Home Pharmacies” was evaluated to represent the extent of activity including preventive medicine in pharmacy. The median of the indexes in the four areas rose in 2007, and was the highest in Ueda-shi. However, the increase in the index was found to result from increases in activity related to clinical medicine such as the use of “Medication Notebooks” and the circulation of “Pharmaceutical Instructions” and did not result from increased activity related to preventive medicine. Factors to promote preventive medicine activity are discussed based on the data from Aira-gun where the greatest home care medicine activity occurred and from Ueda-shi where the greatest environmental sanitation and healthcare activities occurred.
TLC and HPLC were used to identify possible chemical markers for evaluating the quality of the crude drug “Pogostemoni herba” (aerial part of Pogostemon cablin), which is a component of Kampo medicines. In addition to the reported patchouli alcohol and 2-hydroxy-6-methyl-3-(4-methylpentanoyl)-4-pyrone, three phenylethanoids were isolated from this plant material for the first time: acteoside, isoacteoside, and crenatoside. The usefulness of these compounds as indicators of the crude commercial drug under various TLC conditions was examined, and patchouli alcohol was found to give a definite spot with a reproducible Rf value. Therefore, we propose TLC of the methanol (MeOH) extract using patchouli alcohol as a marker as a convenient method for identifying the crude drug Pogostemoni herba.
Ehime University Hospital has been conducting a four-week practical hospital training course, consisting of 18 programs, for undergraduate students. The course includes experience-oriented programs, such as drug counseling training using case-based learning procedures (CBL practice), evidence based medicine (EBM practice) based on the provision of drug information, and training to avoid adverse drug reactions (pre-avoid practice). A previous study based on a questionnaire survey showed that experience-oriented programs enhanced students' understanding of the hospital practical training, while also increasing their satisfaction. In this trial, written examinations were given to 24 students to evaluate their clinical knowledge and problem-solving abilities at the start and the end of the practical hospital training course to evaluate the educational effects of this curriculum objectively. The problem-solving abilities were examined using a test of a case analysis, which required the students to answer multiple clinical problems or proposals. The examination scores on the clinical knowledge at the end of the practical hospital training course had significantly increased by 9.5% (p<0.01) in comparison to that at the start. In addition, the accuracy rate regarding their problem-solving abilities markedly increased by 28.8% (p<0.001). Moreover, the number of answers also significantly increased by about 1.5-fold (p<0.001). These results suggested that the experience-oriented programs for hospital practical training increased the clinical knowledge and the problem-solving abilities of these students.
Pentabenzensulfonate (QPBS), a potential prodrug for quercetin, was designed and synthesized in high yield. It possesses better physical properties such as solubility, lipid/water partition coefficient, LogP, and hydrolysis kinetics than its original form. The LogP value (2.04) and the half-life of the hydrolysis value (3.85 h) show that oral bioavailability is improved evidently compared with that of quercetin. These results indicate that QPBS can be considered as a potential prodrug for quercetin.
We investigated mRNA expression of c-myc and chromosome aberrations at the c-myc locus in the same passage number of human mesenchymal stem cells (hMSCs). To understand the sensitivity of mRNA expression and the induction of chromosome aberrations, we first tested them in hMSC and cancer cell lines (HeLa S3, HOS, and OUMS-27). The c-myc mRNA expressions in HeLa S3 and OUMS-27 were significantly higher than those in hMSC, but then those in HOS were not. On the other hand, c-myc aberrant cells detected by fluorescence in situ hybridization in HeLa S3, HOS, and OUMS-27 were significantly higher than that in hMSC. Both analyses were performed in hMSCs derived from five donors for the culture period of 50 days. In hMSCs from one donor, the frequency of c-myc aberrant cells significantly increased at 20 and 50 days respectively, and each mRNA expressions had a tendency to increase, but there is no significant change among 3, 20 and 50 days. In hMSCs from the others, both endpoints did not change for 50 days. For safe use of somatic stem cells in the regenerative medicine, the investigation of characteristic change of them during the in vitro culture is important. In the present study, we showed the mRNA expressions and chromosome aberrations of hMSCs in in vitro culture as the first step for establishing of safety evaluation of tissue engineered medical devices using normal hMSCs.