Ultrasound is a good tool for theranostics due to have multi-potency both of diagnostics with sonography and therapeutics with high intensity focused ultrasound (HIFU). In addition, microbubbles and nanobubbles are utilized as not only contrast imaging agent but also enhancer of drug and gene delivery by combination of ultrasound. Recently, we developed novel liposomal nanobubbles (Bubble liposomes) which were containing perfluoropropane. Bubble liposomes induced jet stream by low intensity ultrasound exposure and resulted in enhancing permeability of cell membrane. This phenomenon has been utilized as driving force for drug and gene delivery. On the other hand, the combination of Bubble liposomes and high intensity ultrasound induces strong jet stream and increase temperature. This condition can directly damage to tumor cells, we are applying this for cancer therapy. Therefore, their combination has potency for various cancer therapies such as gene therapy, immunotherapy and hyperthermia. In this review, we discuss about cancer therapy by the combination of Bubble liposomes and ultrasound.
Visual images provide an immensely rich source of information about the external world. Eye has characteristic structure sensory cells are arranged along the eye wall, and is filled inside with vitreous body. In recent years, intravitreal injection of anti-vascular endothelial growth factor (VEGF) agent had widely spread, and numerous number of patients who suffered ocular angiogenic disease such as diabetic retinopathy, age-related macular degeneration and retinal vascular occlusion for the disease, were treated and spared the blindness. Vitreous cavity was regarded as reservoir of drug, intravitreal injection is thought a sort of drug delivery. However, with regard to the administration of a selective drug deliver, it has not yet been solved. Our aim is to establish a new method of gene transfer, drug delivery using low-energy ultrasound to the eye, to date, we confirmed drug and gene deliver to the ocular tissue such as cornea, conjunctiva and retina with high efficiency. In addition, tissue damage was minimal. We have also shown that ultrasound irradiation with combination of a microbubbles or bubble liposome could be introduced drug and gene more effectively. Based on these knowledge, we will focus on development of a new device for intraocular ultrasound exposure and potential for therapeutic application of ultrasound to humans retinal disease such as retinal artery obstruction.
We applied a polymeric micelle carrier system for the targeting of a magnetic resonance imaging (MRI) contrast agent. Prepared polymeric micelle MRI contrast agent exhibited a long circulation characteristic in blood, and considerable amount of the contrast agent was found to accumulate in colon 26 solid tumor by the EPR effect. The signal intensities of tumor area showed 2-folds increase in T1-weighted images at 24 h after i.v. injection. To observe enhancement of the EPR effect by Cderiv pretreatment on tumor targeting, we used the contrast agent for the evaluation by means of MRI. Cderiv pretreatment significantly enhanced tumor accumulation of the contrast agent. Interestingly, very high signal intensity in tumor region was found at 24 h after the contrast agent injection in Cderiv pretreated mice. The contrast agent visualized a microenvironmental change in tumor. These results indicate that the contrast agent exhibits potential use for tumor diagnostic agent. To combine with a polymeric micelle carrier system for therapeutic agent, the usage of the combination makes a new concept of “theranostic” for a better cancer treatment.
Cyclodextrins (CyDs) are cyclic oligosaccharides composed of 6, 7, or 8 glucopyranoside units, named α-, β-, or γ-CD, respectively. CyDs consist of a hydrophobic cavity in which hydrophobic molecules are encapsulated to form an inclusion complex. CyDs are widely used in pharmaceutical applications because they function as nanocapsules to improve the stability and solubility of drugs. Recently, CyDs have attracted much attention as for use as components of supramolecular nanostructures that are particularly attractive because of their unique structures. We modified CyDs with phenylboronic acid (PBA), which forms covalent bonds with the diol groups of sugar, and used the resulting PBA-CyDs to prepare supramolecular nanomachines that undergo structural transformation in the presence of a chemical signal in the form of a sugar. PBA-α-CyD formed a supramolecular polymer that showed consecutive intermolecular interactions between PBA and the cavity of another PBA-α-CyD, whereas PBA-β-CyD formed head-to-head dimers in which one PBA moiety was encapsulated in the other. These supramolecular nanostructures disintegrated in the presence of sugars because of the structural change in the PBA moiety and loss of the driving force of the supramolecular assembly. These features of disintegration can be potentially used to prepare a nanomachine that would act as a sugar-responsive insulin release system. Currently, we are studying sugar-responsive nanomachines composed of PEGylated insulin and PBA-γ-CyD.
High accumulation and selective delivery of boron into tumor tissue are the most important requirements to achieve the efficient cell-killing effect of boron neutron capture therapy (BNCT) that relies on the nuclear reaction of two essentially nontoxic species, boron-10 (10B) and thermal neutrons in boron-loaded tissues. Recent development of boron cluster lipids and their liposomal boron delivery system (BDS) are summarized in this article. Boron compounds that have no affinity to tumor can potentially be delivered to tumor tissues by liposomes, therefore, liposomal BDS would be one of the most attractive approaches for efficient BNCT of various cancers. There are two approaches for BDS: encapsulation of boron compounds into liposomes and incorporation of boron-conjugated lipids into the liposomal bilayer. The combination of both approaches has a potential for reduction of the total dose of liposomes without reducing the efficacy of BNCT.
World Health Organization (WHO) indicated that recognition of the responsibility of individuals for their own health and awareness that professional care for minor ailments is often unnecessary were important points of view for “Self-Medication” in “Guidelines for the Regulatory Assessment of Medicinal Products for Use in Self-Medication” published in 2000. In Japan, educational curriculum in each school is formulated by “the Courses of Study”. Education for health at junior and senior high school is mainly carried out in “Health and Physical Education” class. The objectives are to enable students to develop qualities and abilities to appropriately manage and improve their health throughout their lives through understanding of health and safety in personal and social life. This idea is common with self-medication at a basic concept level. The previous Courses of Study were revised in 2008 and 2009. They described about medicine for senior but not junior high school. The interim report of “The Central Council for Education” in 2005 pointed out that understanding about effects/side effects by medicines, and abilities to use them appropriately are one of minimal qualities that all children must acquire. Due to revision of the Pharmaceutical Affairs Law in 2006, the necessity of endeavoring to widespread knowledge and enlighten about proper use of medicines in school education was considered. Since the new Courses of Study in the junior high school shows content that medicines should be used properly, it is thought that educational frame of medicines for children is going into new era in Japan.
Teaching the three health principles and proper use of medicines are the basis of education on medicines. Before seeking prescription drugs, day-to-day health management is important. It is also important to understand that if a minor ailment persists, self-treatment with over-the-counter medications should be attempted. Since medications are double-edged swords, their proper use is the responsibility of patients to minimize the risk and maximize the effectiveness. This awareness should be taught during education on how to use medicines. A better understanding of medicines and fostering awareness through education on medicines will contribute to reductions in healthcare costs and promote the health of patients when they participate in their own care and learn how to self-medicate.
To promote the education on medicines in school, guidance is, as a rule, to be given in the health education of the Subject, “Health and Physical Education” indicated as a curriculum standard in lower and upper secondary school, and in the health guidance, which carry out in Special Activities and Integrated Studies, etc. Guidance is mainly carried by the teacher for health and physical in health education and school nurse teacher (yogo teacher) in health guidance. In health education we have only limited school hours, and generally use the text book. Some teachers feel resistance to teach medicines because of needs on the special knowledge, however teachers should deal with medicines based on curriculum standard. School pharmacist is a member of school, and has a special knowledge for medicines, and he/she can support teachers as a provider of teaching materials, an adviser, and for a guest teacher. It is important for school pharmacist to understand the contents indicated in curriculum standard and to use glossary to be able to understand for children. In the guidance of health, it is not necessary to teach based on curriculum standard, and it can deal with advanced contents on medicines. However it is important to understand for children what are appropriate contents according to the development stage. To use the packages and instructions for medicines provided at home are good materials for children to have interest the medicines in their guidance. The objectives of education on medicines enable children to cultivate practical abilities for the maintenance and improvement of health.
Under new courses of study, medicine-related content has been incorporated into health and physical education at junior high schools, and classes on the “Correct use of medicine” began as part of this content in 2012. Based on the “School Health and Safety Law” implemented in April 2009, health guidance provided by school physicians, pharmacists, and dentists has also been incorporated. This has raised expectations that educational programs concerning the “Correct use of medicine” could be effectively implemented through cooperation between school pharmacists and health and physical education teachers. In order to clarify current knowledge and awareness regarding the “Correct use of medicine” among students, as well as the guidance provided at schools, we conducted a training workshop for teachers at elementary and junior high schools in Gifu City. Based on the results, we developed a “Correct use of medicine” educational program. We then presented this program in a class with 40 third-year junior high school students on January 31, 2012. It consisted of an introduction (7 min), a development portion (35 min), and a conclusion (8 min). After the class, a question and answer session was held with observers (n=11) and a questionnaire survey, which resulted in high evaluations, was conducted on the students. Comments including concern over the large volume of educational content and the need to raise awareness were heard, but the results of the questionnaire survey showed that many students found the class content useful and interesting.
Natural vitamin K is found in two forms: a plant form, phylloquinone (PK) and bacterial forms, menaquinones (MKs). PK is a major form of dietary vitamin K; however, the most prevalent form of vitamin K in animals and humans is menaquinone-4 (MK-4). Despite its high concentrations, the origin of MK-4 is yet to be defined. It is postulated that PK is converted into MK-4 and accumulates in extrahepatic tissues. The molecular mechanisms for these conversion reactions have been unclear. To identify the MK-4 biosynthetic enzyme, we screened the human genome database for prenylation enzyme. We found UbiA prenyltransferase domain containing 1 (UBIAD1), a human homologue of Escherichia coli prenyltransferase menA. The short interfering RNA against the UBIAD1 gene inhibited the conversion of deuterium-labelled vitamin K derivatives into deuterium-labelled-MK-4 (MK-4-d7) in human cells. We confirmed that the UBIAD1 gene encodes an MK-4 biosynthetic enzyme through its expression and conversion of deuterium-labelled vitamin K derivatives into MK-4-d7 in insect cells infected with UBIAD1 baculovirus. UBIAD1 was localized in endoplasmic reticulum. Our results show that UBIAD1 is a human MK-4 biosynthetic enzyme. This identification will permit more effective decisions to be made about vitamin K intake and bone health.
In recent times, glaucoma has become the leading cause of acquired blindness among the Japanese. As visual disorders markedly decrease the quality of life (QOL), it is important to develop new strategies for preventing the onset of and delaying the progression of glaucoma. Glaucoma has long since been recognized as a serious disease caused by increased intraocular pressure and subsequent injury and death of the neuronal retinal cells. Therefore, numerous studies have focused on the mechanisms that damage neuronal cells and on the drugs that possess protective effects in reversing this damage. However, injury to the retinal vasculature has been recently shown in animal models of glaucoma. Hence, thus far, only few papers have been published on retinal circulation in glaucoma. These study results have indicated that retinal circulation is altered in glaucoma and that this vascular abnormality may be the cause of and/or may accelerate retinal degeneration. In this report, we have attempted to elucidate the mechanisms of retinal circulation and explore novel drugs for the treatment of retinal circulation disorders. We have also introduced here our previous research results on retinal circulation. We reported that the drugs that improved retinal circulation, by intravitreal injection, in the rat model of glaucoma also inhibited retinal nerve injury, thereby representing possibilities that they might be novel candidate drugs for glaucoma prevention and treatment.
Pulmonary arterial hypertension (PAH) is a rare, progressive, and fetal disease. The five-year survival rate after diagnosis is ～50%. In Japan, PAH is listed in the Specified Rare and Intractable Diseases. Pulmonary vascular remodeling and sustained pulmonary vasoconstriction are the major causes for the elevated pulmonary vascular resistance (PVR) in PAH. The pathogenic mechanisms involved in the pulmonary vascular abnormalities in PAH remain unclear. Sustained vasoconstriction and vascular remodeling owing to proliferation of pulmonary arterial smooth muscle cells (PASMCs) are key pathogenic events that lead to early morbidity and mortality. These events have been closely linked to Ca2+ mobilization and signaling in PASMCs. An increase in cytosolic Ca2+ concentration ([Ca2+]cyt) in PASMCs is an important stimulus for pulmonary vasoconstriction and cell proliferation which subsequently cause pulmonary vascular wall thickening followed by the increase in PVR. Increased resting [Ca2+]cyt and enhanced Ca2+ influx have been implicated in PASMCs from PAH patients, but precise therapeutic targets to interrupt these signal pathways have not been identified. We recently found that the extracellular Ca2+-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), is upregulated in PASMCs from patients with idiopathic pulmonary arterial hypertension (IPAH). In addition, blockage of the CaSR with an antagonist (NPS2143) prevents the development of pulmonary hypertension and right ventricular hypertrophy in animal models of pulmonary hypertension. The functionally upregulated CaSR in PASMCs is a novel pathogenic mechanism contributing to the augmented Ca2+ signaling and excessive cell proliferation in IPAH. Targeting CaSR in PASMCs may help develop novel therapeutic approach for PAH.
The recent vigorous transnational migration of people and materials reflecting the development of transportation facilities, changes in social structure, and war disasters has increased the global spread of emerging and re-emerging infectious diseases. Vaccine, which is the major fundamental prophylaxis against infectious diseases, has greatly contributed to the maintenance and improvement of human health worldwide. However, the disadvantages of conventional injection systems hamper the speedy mass-vaccination and the global distribution of vaccines. Transcutaneous immunization systems, which are easy-to-use and low-invasive methods of vaccination, have the potential to overcome certain issues associated with injectable vaccinations. In this review, we provide an outline of recent trends in the development of techniques for the transcutaneous delivery of vaccine antigens. We also introduce basic and clinical research involving our transcutaneous immunization systems that incorporate self-dissolving microneedle patch.
Biomarkers are useful tools as indicators/predictors of disease severity and drug responsiveness, and thus, are expected to make drug development more efficient and to accelerate proper use of approved drugs. Many academic achievements on biomarkers have been reported, but only several biomarkers are used in drug development and clinical settings. We first show our results on the pharmacogenomic analysis of the anti-cancer drug irinotecan and of Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). UGT1A1*6 and *28 were significantly associated with altered pharmacokinetics of an irinotecan metabolite, SN-38, and with increased frequency of severe neutropenia. HLA* 58:01 and HLA-B*15:11/HLA-A*31:01 were associated with SJS/TEN by allopurinol and carbamazepine, respectively. Our papers have been cited in the package inserts of irinotecan and allopurinol. In addition to these genomic biomarkers, metabolomic biomarkers, which can reflect the disease phenotype and drug responsiveness, have been exploring for 12 major diseases in Japan, as a part of a multi-omics team with multi-national centers. In animal models of dilated cardiomyopathy and Alzheimer's disease, we found several changes in lipid metabolite levels in the diseased tissues. Moreover, two oxidized fatty acids were correlatively changed in the brain and plasma from Alzheimer's model mice before its onset, and thus, could be candidates for predictive biomarkers. Finally, we propose/discuss several key issues for academic researches on biomarker discovery and development, especially for newly coming researchers in the field of pharmaceutical sciences. We hope that this review would help novel biomarker identification and qualification in Japan.
The goal of pharmaceutical sciences is to deliver effective and safe medicinal products to patients. To achieve this goal, we need to ensure the efficacy, safety and quality of the products. Currently, many attempts are made to utilize human induced pluripotent stem cells (hiPSCs) in regenerative medicine/cell therapy. There are significant obstacles, however, preventing the clinical use of hiPSC-derived products. One of the most obvious safety issues is the presence of residual undifferentiated cells that have tumorigenic potential. Therefore, the assessment and control of the tumorigenicity of hiPSC-derived products is essential in order to prevent tumor development by residual pluripotent stem cells after implantation. We recently examined three in vitro assay methods to detect undifferentiated cells: soft agar colony formation assay, flow cytometry assay and quantitative real-time polymerase chain reaction assay (qRT-PCR). Although the soft agar colony formation assay was unable to detect hiPSCs, the flow cytometry assay using anti-TRA-1-60 antibody detected 0.1% undifferentiated hiPSCs that were spiked in primary retinal pigment epithelial (RPE) cells. Moreover, qRT-PCR with a specific probe and primers was found to detect a trace amount of LIN28 mRNA, which is equivalent to that present in a mixture of a single hiPSC and 5.0×104 RPE cells. Our findings provide highly sensitive and quantitative in vitro assays essential for facilitating safety profiling of hiPSC-derived RPE cells for their clinical use.
Alzheimer's disease (AD) is the most common cause of dementia. AD treatments currently approved are commonly regarded as purely symptomatic. Therefore, development of effective disease modifying treatments is eagerly required. Pathological hallmarks of AD brain include amyloid-β (Aβ) accumulation, neurofibrillary tangle (NFT), synaptic loss, and neurodegeneration. Understanding of AD pathogenesis provides deep insight into the therapeutic approach for AD. In this review, we introduce our current translational researches for new therapeutic strategies against AD. We recently revealed that a key molecule for actin assembly, Wiscott-Aldrich syndrome protein family verprolin-homologous protein (WAVE), is accumulated into NFT in an Aβ pathology-dependent manner. Because synaptic function is maintained by precise regulation of actin assembly, abnormal accumulation of WAVE induced by Aβ pathology may expand the NFT pathology to synaptic dysfunction. These results imply that lowering of brain Aβ at the early stage of AD pathogenesis is a promising therapeutic approach. As the approach for the elimination of brain Aβ, we have studied microglial Aβ phagocytosis and proposed its therapeutic application for AD treatment. We demonstrated that anti-Aβ antibody and galantamine promote microglial Aβ phagocytosis by binding to Fc receptors and nicotinic acetylcholine receptors on microglia, respectively. On the other hand, we demonstrated that the transplantation with microglia effectively eliminate brain Aβ and microglia-like Aβ phagocytic cells were successfully prepared from bone marrow derived cells by the stimulation with macrophage colony-stimulating factor. Thus, we have studied cell therapeutic strategy aim at its clinical application for AD treatment by the activation of endogenous microglia and transplantation of exogenous microglia-like Aβ phagocytic cells.
Toll-like receptor-2 (TLR2) is one of the important innate immune receptors that play an important role in recognizing the pathogens and producing inflammatory cytokines. In this review, we focus on the regulatory mechanisms of expression, function and inflammatory responses of TLR2 during pathogenic infection in innate immune cells. We first showed that nontypeable Haemophilus influenzae (NTHi), an important human pathogen that exacerbates otitis media and chronic obstructive pulmonary diseases (COPD), induces inflammatory responses through activation of NF-κB via two distinct signals, NIK-IKKα/β-IκBα and MKK3/6-p38 pathways. Moreover, TLR2 was greatly up-regulated by NTHi through the positive IKK-IκBα-dependent NF-κB pathway and the negative MKK3/6-p38α/β pathway. Importantly, glucocorticoids synergistically enhance NTHi-induced TLR2 up-regulation likely via a negative cross-talk with the inhibitory p38 MAPK. The results provide novel insights into the role of glucocorticoids in regulating host defense and innate immune responses through TLR2 regulation. We next focused on the pathogenesis of cystic fibrosis (CF), a common lethal inherited disorder characterized by recurrent pulmonary infections and obstruction caused by chronic mucus hypersecretion and inflammation. We demonstrated an increased expression of TLR2, due to an enhanced DNA de-methylation and Sp1-dependent transcriptional activation in CF epithelial cells. Furthermore, a Th17 cytokine IL-17A synergistically increased TLR2 signal through an enhancement of p38 phosphorylation. These studies suggest the importance of TLR2 and IL-17 signals in the pathogenesis of CF. Finally, by focusing on neutrophils and CF airway epithelial cells, we identified curcumin as a potent inhibitor of TLR2-mediated inflammatory responses.
To accurately predict the stability of thiamine nitrate as a model drug in pharmaceutical products under uncontrolled temperature conditions, the average reaction rate constant was determined, taking into account the heat transfer from the atmosphere to the product. The stability tests of thiamine nitrate in the three packages with different heat transfers were performed under non-isothermal conditions. The stability data observed were compared with the predictions based on a newly developed method, showing that the stability was well predicted by the method involving the heat transfer. By contrast, there were some deviations observed from the predicted data, without considering heat transfer in the packages with low heat transfer. The above-mentioned result clearly shows that heat transfer should be considered to ensure accurate prediction of the stability of commercial pharmaceutical products under non-isothermal atmospheres.
Borrelia duttonii, a causative agent of relapsing fever, is transmitted between the distinct microenvironments of the vector tick, Ornithodoros moubata, and a mammalian host. We identified the total and membrane fraction proteins of B. duttonii strain Ly isolated from a patient in Tanzania by using two-dimensional gel electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The analyses of the total and membrane fractions from bacterial cultures incubated at 37°C identified 68 and 15 proteins, respectively. Since spirochaete clearance in mice is associated with an immunoglobulin M (IgM) and immunoglobulin G3 (IgG3)-mediated response, immunoblot analyses were used to identify the proteins reactive with IgM and IgG3 of gerbil serum against B. duttonii strain Ly. The outcome showed that six proteins (antigen p83/100, membrane-associated protein P66 (P66), flagellar filament outer layer protein, hypothetical protein BDU_412, vlp protein gamma subfamily (γ-Vlp) and flagellin (FlaB)) were identified against IgM, and four (antigen p83/100, P66, γ-Vlp and FlaB) of the six proteins also reacted with IgG3. It is believed that these proteins are immunodominant antigens for the host immune response. Some of these immunogenic proteins might be used as molecular diagnostic tools in the study of relapsing fever in Tanzania.