Transposons are mobile genetic elements that move between or within vectors and chromosomes. For the transposition, an enzyme called transposase recognizes transposon-specific terminal inverted repeat sequences (IRs) located on both ends of transposons, and remove them from their original sites and, integrates them into other sites. Because of this feature, transposons containing genes of interest between their two IRs are able to carry the genes from vectors to chromosomes. Transposons are promising systems for chromosomal integration because they can not only integrate exogenous genes efficiently, but also be transfected to a variety of cells or organs using a range of transfection methods. In this review, we focused on the therapeutic application of transposons. A few transposons can integrate transgenes into mammalian chromosomes. They have been used in preclinical studies of gene therapy and cell therapy. In addition, they have recently been used for generation of induced pluripotent stem cells. Transposon-based integrative vector systems have two components. One is the transposon containing transgenes, and the other is the expression cassette of the transposase. Both viral and non-viral vectors have been used to deliver these two components to mammalian cells or organs, and sustained transgene expression has been achieved. Transposon-mediated sustained transgene expression has also produced therapeutic effect in disease models of hereditary and chronic diseases. Although transposon-based integrative vector systems have problems, such as insertional mutagenesis, studies to overcome these problems have been progressing, and these vector systems will become indispensable tools to cure refractory diseases.
Liposomes modified with polyethylene glycol (PEG) can stably exist in the bloodstream because the PEG on the liposomes attracts a water shell to the liposomal surface. Since these liposomes are long circulating nanocarriers, they are used as drug and gene delivery tools. Repeat injection of PEGylated liposomes, however, is known to induce the accelerated blood clearance (ABC) phenomenon. In the ABC phenomenon, PEGylated liposomes that are injected subsequent to the first injection are cleared rapidly from the bloodstream and accumulate in the liver, resulting in loss of their long-circulating characteristics. The induction of ABC phenomenon is related to the production of anti-PEG IgM from splenic B cells. To elucidate the mechanism of the phenomenon, we firstly examined the relationship between the induction of ABC phenomenon and the concentration of PEGylated liposomes, and observed that the high dose of those did not induce the phenomenon. Next, we investigated whether polymeric micelles trigger ABC phenomenon or not. Finally, the size-dependency of ABC phenomenon was investigated by use of variously sized PEGylated liposomes and polymeric micelles having PEG chains. Our data suggest that the initiation of ABC phenomenon would be size-dependent, and particles smaller than 30 nm did not induce ABC phenomenon. We anticipate that the elucidation of the ABC phenomenon will be helpful for the development of DDS formulations.
Transdermal Drug Delivery Systems (TDDS), where active drugs must be absorbed into the systemic circulation after penetrating the skin barrier, were first launched in 1979, and about 10 TDDS containing different kinds of drugs were developed during the initial decade. Interestingly, a developmental rush has come again in the present century. Various penetration-enhancing approaches to improve drug permeation of the skin (stratum corneum) have been attempted. These approaches are of two types: chemical and physical. Examples of the chemical approach are enhancers such as alcohol, monoterpenes and fatty acid esters, as well as chemical modification of prodrugs. In contrast, physical approaches include the use of electrical-, thermal- and mechanical-energy, as well as microneedles, needle-free injectors or electroporation to completely or partially evade the barrier function in the stratum corneum. The chemical approaches are mainly effective in increasing the skin permeation of low-molecular chemicals, whereas physical means are effective for these chemicals but also high-molecules like peptides, proteins and nucleotides (DNA or RNA). Marked development has been observed in these physical means in the past decade. In addition, recent developments in tissue engineering technologies enables the use of cultured skin containing keratinocytes and fibroblasts as a TDDS. An effective “cell delivery system” may be a reality in the near future. This paper will look back on the 30-year history of TDDS and evaluate the feasibility of a new generation of these systems.
Carious and periodontal disease is strongly associated with pulmonary infections. Aspiration pneumonia often develops lung abscess and/or empyema, and sometimes leads to death in elderly patients. It is often repeatedly seen in most of elderly patients, which leads to general weakness, prolonged bed rest, and several complications. There are two pathophysiological factors for aspiration pneumonia. One is due to odontogenic infections: aspirated oral microorganisms reach pulmonary alveoli, grow, and develop their pathogenicity. The other is host factors: alcoholism, diabetes, or bedridden status reduces cough reflex, airway clearance, and functions of phagocytes. The prevention of aspiration pneumonia is significant from medical, social, and economical viewpoints, although the main management of pneumonia is antimicrobial chemotherapy. “Oral care” has recently been of interest as a control means for odontogenic infections and aspiration pneumonia. A dental hygienist and speech therapists in our hospital have implemented active intervention in oral care of patients with risk of aspiration pneumonia, which has made considerable achievements.
What we medical coordinators aim at is “an advance intervention” that prevents disease and hardships in life through preventive health counseling. It is true that these interventions focus on improving health conditions. “Mind and skills” are the pillars of medical coordination. Medical coordinators are concerned about and sympathize with clients. Advance intervention enables clients and families to make satisfactory decisions. Currently, time limitations prevent full implementation of advance interventions. We intend in the future to promote health literacy so that any community member can perform advance interventions when needed, with our support and under our supervision. Here I introduce two cases where I as a medical coordinator, on the client's behalf, have supported clients' and families' decision making and reduced their hardships, especially economic burdens. In these cases, the client's life was threatened because medical staff did not understand the client's real intention and because the client did not know how to inform the medical staff of their situation. How can we bridge the gap between clients and medical staff? How can we find out our clients' real needs? How can clients achieve satisfactory decision making? I want to consider these things with you at the symposium.
The growth of the dietary supplement in Japanese market suggests that the patient's need for assistance with self-care will also continue to grow. Patients' burgeoning acceptance and use of alternative therapies is another indication that patients seeking more from the health care system. The questionnaire showed that the most of them are expecting the pharmacists to provide their knowledge of dietary supplements. However, only limited amount of information is available. We founded the group Alterna in 2003, that compose the pharmacists working in community pharmacies accompanied with those in pharmaceutical universities. We have published the journal named “Alterna” that includes the information of dietary supplements, and it attained Vol. 9 in 2008. In the past studies, we evaluated the content and solubility of coenzyme Q10 dietary supplements in Japanese markets, some of which showed poor solubility. In others, we had taken up the information about vitamin and minerals, tea catechins, DHA and EPA, cooking oils to reduce body fat, collagen, etc. Findings in these studies present the opportunities for the pharmacists to provide the significant positive impact on health care outcomes and costs to patients.
In recent years, appropriate medication and guarantees of safety are being sought not only by medical circles but also by the world of sport. Under normal circumstances, sport should be wholesome in both mind and body, but “doping” by the misuse and abuse of drugs and such is developing into a social issue. This is not just a result of the deliberate behavior of a certain number of people; many cases include use due to a lack of knowledge of drugs and doping, although eventually the sanctions received are the same. Doping tends to be perceived as the problem of just a section of elite athletes, but since the introduction of doping control at the National Athletic Meet 2003, anti-doping measures continue to be a problem close at hand. In 2004, the World Anti-Doping Code came into effect and subsequently not just the world of sport but various national governments became deeply involved with anti-doping. Anti-doping guidelines in Japan were formulated by the Ministry of Education, Culture, Sports, Science and Technology in 2007, stipulating that doctors and pharmacists should be proactive in anti-doping activities. With the aim of eradicating doping, it was deemed that pharmacists can intervene by providing support regarding such issues as drug enlightenment, consultation; the supply of drug information; database production; and therapeutic use exemption. It can be considered that pharmacists can sufficiently use their knowledge and experience gained in these fields, and that such knowledge could lead to more appropriate drug use in sport.
Even though they have not been diagnosed with a recognized disease, many people have or are at risk of contracting debilitating conditions. They can be referred to as being in the “ill-health zone.” For example, many bedridden elderly develop pressure ulcers. The prevention and treatment of pressure ulcers should focus on two main factors: the role of pressure in the development of circulatory disorders; and increased dermal pH. In preventing the development of circulatory disorders resulting in pressure ulcers, using an air or polyurethane mattress is helpful. However, changing the mattress has little effect if the position of the bedridden person is not also changed regularly. To avoid an increase in dermal pH, caregivers should apply moisture-repellent cream and/or oil to the sacral region after careful cleansing. It is important that such preventive measures and treatment be performed daily, and caregivers should be educated on this need and subsequently monitored. Pharmacists have a role in caring for those in the ill-health zone.
Together with xanthine oxidase, aldehyde oxidase (AO) is a major member of a relatively small family of molybdenum hydroxylases. Both enzymes are homodimers with a subunit molecular weight of about 150 kDa and exhibit catalytic activity only as a dimer. An AO subunit contains a molybdopterin cofactor, an FAD and two different 2Fe-2S redox centers. The enzyme catalyzes oxidation of a wide range of endogenous and exogenous aldehydes and N-heterocyclic aromatic compounds. N-heterocycle-containing drugs such as methotrexate, 6-mercaptopurine, cinchona alkaloids and famciclovir are oxidized by this enzyme. Marked species differences have been well documented for the AO-catalyzed metabolism of drugs including methotrexate and famciclovir. In addition, a large rat strain variation has also been demonstrated in the oxidation activity of benzaldehyde and methotrexate. Marked differences in species, large differences in rat strains and individual differences in AO activities in some rat strains have been reported. However, little has been elucidated about any related molecular biological mechanisms. We examined the mechanism of individual variations and strain difference of rat AO using the technology of molecular biology. Our recent studies regarding the inter- and intra-difference of AO activities in rats are described.
Three-dimensional structure of a molecule and its alteration is an important issue, and it is essential problem for controlling the function of a molecule such as a molecular switch or device. In most cases, molecular switches have relatively high activation energy for interconversion between alternative structures, however in some biological systems, conformational preference plays an important role in regulation of bioactivity. We have interested in conformational alteration of aromatic amides, which have interesting features about conformation. Most of secondary aromatic amides such as benzanilide tend to prefer trans conformation, whereas N-methylation makes its conformation cis-favored. Recently we found several aromatic amides altered the preferred conformation depending on the external stimuli. Thus, N-phenyl-N-quinonyl type of amides changed their preferred conformation according to redox state of quinonyl moiety. N-Methyl pyridylamides showed conformational alteration according to solvent acceptor ability or addition of acid. N-Methylated pyridylamide oligomer also showed unique conformational folding and unfolding. These properties of the aromatic amides can be applied to stimuli-responsive molecular switches and functional molecules.
Chemicals act on biological molecules and affect their functions. DNA is one of the most important targets, damaging of which could lead to diverse diseases including cancer. The mode of action of chemicals to DNA contains chemical reaction and protein factor-mediated modulation of the function. In this review, these actions are described in view of effects of chemicals on DNA. First, oxidative damage of DNA is described in several cases of chemicals focusing on its mechanisms involving metals such as copper. We have demonstrated: DNA binding of copper ions prior to reduction-oxidation reaction is crucial for the damaging, probably due to the proximal attack of reactive oxygen species; reduction of the bound copper induces a conformational change of DNA strand through rearrangement of copper-coordination geometry; RNA, another nucleic acid, is more liable to oxidative damage than DNA. Impact of RNA damage on oxidative stress-related diseases is discussed. Second, a group of chemicals called endocrine disruptors is described. Phthalate esters are ubiquitous endocrine disruptors of which mechanisms are still elusive. Here, we present our research performed for elucidation of the active metabolite and molecular target. Novel candidates of active metabolite are suggested. Finally, toxicological activity dynamics are described, showing several chemicals exert toxic potential by structural alteration in the environment, metabolism, or both. These imply gene-environment interactions that would underlie various diseases induced by environmental chemicals.
An attempt was made to prepare FITC-labeled-lactoferrin (LF-FTC)-loaded microparticles, durable under gastrointestinal conditions, first by the combination of alginate/calcium complexation and emulsification-evaporation and next by treatment with chitosan solution. The obtained microparticles were examined for particle characteristics, in vitro release profiles and physical stability in solutions of pH 1.2 and 6.8. The obtained chitosan-coated alginate/calcium complex microparticles (Ch/Al/Ca-MP) showed almost uniform size of 1-2 μm and a spherical shape with a non-smooth surface. The content and recovery of LF-FTC in Ch/Al/Ca-MP fell as the concentration of chitosan solution used in chitosan coating increased. The release rate of LF-FTC was faster in Ch/Al/Ca-MP prepared with more chitosan at pH 1.2 and 6.8. Ch/Al/Ca-MP coated with 0.25 and 0.5% (w/v) chitosan solution showed good gradual release characteristics in vitro. Furthermore, they were durable at pH 1.2 and 6.8, though swelling and softening of the microparticles occurred at pH 6.8. It is suggested that alginate/calcium complex microparticles coated with 0.25-0.5% (w/v) chitosan solution would be useful for the intestinal delivery of LF.
Dissolution testing is a core performance test in pharmaceutical development and quality control. Generally, the HPLC method uses the analysis of dissolution testing. In this study, we attempted to improve the dissolution test by using microdialysis methods. We also investigated the comparison of the conventional HPLC dissolution method (batch-sampling method) and the improved dissolution test (microdialysis method). Histamine H2-receptor antagonist cimetidine tablets (200 mg), which are used clinically and of which there are also some generic examples, were selected for this comparison, and the dissolution behavior of the tablets by the two methods were found to be similar. On the other hand, standard deviation in the microdialysis method was lower than that of the batch-sampling method. In addition, the microdialysis method can omit many steps such as the filtration, collection and replenishment of sample solutions, and is also able to accomplish continuous sampling of sample solutions. These findings provide significant information that can be used in the pharmaceutical development and quality control of original and generic products.
The effect of tacrolimus (FK506) on peptic ulcer was evaluated using pyloric ligation (PL) model in rats. Tacrolimus was administered orally at different doses (1, 2 and 3 mg/kg) and it showed a gastric ulcer healing effect in a dose dependent manner. Gastric volume, total and free acidity and ulcerative index parameters were reduced in the tacrolimus treated rats as compared to pyloric ligated rats. The higher dose (3 mg/kg) treated group produced significant results similar to that of the ranitidine (50 mg/kg) treated group. Pretreatment with tacrolimus also produced significant (p<0.05) reduction in TBARS, total calcium, TNF-α, IL-8 and MPO whereas it showed an increase in GSH level at higher dose. The anti-secretory and anti-ulcerative effect of tacrolimus may be due to immunosuppressive actions by inhibition of calcineurin and the oxidative pathway. It can be concluded that tacrolimus can play an important role in the treatment of peptic ulcer disorder to improve the quality of life.
Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are circulating hormones secreted predominantly in patients with hypertension or congestive heart failure. To obtain background data on plasma ANP and BNP levels in rats, we investigated the circadian rhythms and effects of anesthesia on these peptides. To determine the circadian rhythms, plasma samples from thirty rats were collected by non-anesthesia (decapitation) at six time points every fourth hour. To determine the effects of anesthesia, plasma samples from thirty-two rats were collected under diethyl ether, pentobarbital or urethane anesthesia. The plasma ANP and BNP levels were determined using a radioimmunoassay. The plasma ANP levels were high from the evening to early morning, while the plasma BNP levels were relatively low at 2:30 AM. The difference in the BNP levels was statistically significant. The plasma BNP levels were relatively high when the rats were anesthetized using urethane. These results suggest that blood collection should be performed between 10:30 AM to 2:30 PM to determine plasma ANP and BNP. The use of pentobarbital is also recommended for toxicological studies in rats.
The present study was aimed at developing a soft chewable dosage form for calcium carbonate for nutraceutical application. Two different types of the formulations viz., sugar based and sugar free soft chews were prepared. The effect of various ingredients on the different organoleptic characteristics (grittiness, sweetness, hardness and mouthfeel) and the emulsion stability of the dosage form were checked and evaluated on the basis of an in-house numerical scale on healthy human volunteers. The study revealed that the type of emulsifying agent, heating temperature, particle size of the drug, ratio and quantity of sugars were found to have significant impact on the organoleptic characteristics of the dosage form. The study also indicates that the proper selection of packaging material is important in order to maintain the long term integrity of the formulation.
A simple, sensitive, selective and reproducible reversed-phase HPLC method was developed for the determination of sophoricoside in rat plasma after intravenous administration. Naringin was successfully used as internal standard (IS) for calibration. The chromatographic separation was accomplished on a reversed-phase C18 column using acetonitrile-methanol-0.08% phosphoric acid (8:29:63, v/v/v) as mobile phase with a flow rate of 1.0 ml/min, with UV detection at 260 nm. Plasma samples were injected into the HPLC system after precipitating protein directly by methanol. Good linearity was achieved in the range of 0.0240~48.0 μg/ml (R2=0.9989). The limit of detection (LOD) and limit of quantification (LOQ) of this method were 0.0075 μg/ml and 0.0240 μg/ml, respectively. The absolute recoveries of sophoricoside from plasma were 95.8%, 93.2%, 98.0% at concentrations of 0.0240, 1.92, 15.0 μg/ml. The intra-day and inter-day variabilities were 3.39%~5.78% and 2.17%~4.72%, respectively. The developed method was successfully applied to the pharmacokinetic study of sophoricoside after intravenous administration of 2.5, 10 and 20 mg/kg in rats.
We examined a simple screening method for judging the complex formation between a drug and aluminum(III) on a spot plate. As few drug had color reaction by basing on the binary complex formation of drug-aluminum(III), the ternary complex formation of drug-aluminum(III)-dye was studied this time using 50 kinds of drugs. The dyes used were Chromazurol S and Erythrosin. As a result, in the drug that the complex formation with aluminum(III) was assumed, a remarkable coloration difference was recognized in comparison with the blank prepared under the same conditions. The proposed simple screening method should be very useful for judging instantly the complex formation between a drug and aluminum(III).
The goal of our investigation was to develop oral solution of coenzyme Q10 (CoQ10) by using self-emulsifying systems. CoQ10 is practically insoluble in water. Therefore, it is difficult to develop oral solution of CoQ10. To solubilize CoQ10 and to develop it as oral solution, self-emulsifying systems consisted of oil, surfactant and co-surfactant were studied and using the self-emulsifying system chosen, oral solution was developed. After the evaluation of solubilizing abilities of various oils and surfactants, the self-emulsifying system consisted of acetylated monoglyceride (AM) as oil, polyoxyethylene (20) sorbitan monolaurate (P2SM) as surfactant and propylene glycol laurate (PGL) as co-surfactant, was chosen because the emulsions prepared by that system showed spontaneous emulsification and transparent appearance. All of these components could be used for the development of functional foods. Finally, a CoQ10 solution with concentrations of 100 mg/70 ml and 100 mg/100 ml could successfully be developed by the addition of water to one milliliter of self-emulsifying system consisted of AM (10%), P2SM (70%) and PGL (20%) along with 100 mg of CoQ10. This oral solution formulation was stable for more than one month.
This study was aimed to propose a novel dosing schedule of docetaxel based on α1-acid glycoprotein (AGP)as an index. For this purpose, we performed Monte Carlo simulation using a population pharmacokinetic/pharmacodynamic (PPK/PPD) model, which we previously developed to estimate the ANC Nadir distribution after docetaxel administration. AGP values, which were incorporated in PPK/PPD, were sampled from normal distributions (S.D. 44, range from 19 to 259), as various mean levels of 125, 150, 175 and 200 (mg/dl). Monte Carlo simulation was conducted using docetaxel doses of 40, 50 and 60 (mg/m2) for each AGP distribution. Simulation was performed 200 times, and distributions of ANC Nadir median were obtained from simulations. We accepted a dose when 20 percentile of the distribution of ANC Nadir median was greater than 500 (counts/μl), in order to avoid the grade 4 neutropenia. From the results of simulations, 40, 50, 60 and 60 doses (mg/m2) were recommended for 125, 150, 175, and 200 AGP mean (mg/dl) respectively. Secondly, to evaluate this dosing schedule, we adopted these recommended doses to 16 patients whose ANC Nadir observed is lesser than 500, and simulated the ANC Nadir. The number of patients whose simulated time below ANC=500 was higher than 6 days decreased from 8 to 2, implying that this dosing schedule might be effective to avoid neutropenia induced by docetaxel. In conclusion, we proposed a novel dosing schedule of docetaxel using AGP as an index, which might be effective to avoid neutropenia induced by docetaxel.