The mechanism of vesicle-to-micelle or micelle-to-vesicle transition was studied in order to control sizes and fluidities of vesicles during periods of preparation. Dependence of particle sizes measured by quasi-elastic light scattering, turbidities, fluidity parameters monitored by ESR spectroscopy, and morphological changes of mixed aggregates of egg yolk phosphatidylcholine (EPC) and a detergent (octylglucoside (OG) or sodium cholate (Na-chol)) on detergent concentration provided a model of vesicle destruction. It possessed three phase transition points, and proceeded in a stepwise fashion: vesicles, small particles containing large amounts of detergents (SUV*), intermediate structures, and mixed micelles. Vesicle formation on removal of detergents from micelles proceeded oppositely. Micelle-vesicle transition mechanism was common to all detergents examined. The feature of the mechanism was the presence of SUV*. Next, SUV* was prepared by adding appropriate amount of a detergent to small unilamellar vesicles obtained by sonication. Time-dependent size growth of the SUV* was remarkable in the case of OG-containing SUV*, but was insignificant in the case of Na-chol-containing SUV*, suggesting the size determining step to be the stage of the SUV*. The tendency to produce large or small vesicles from micelles was related to the absence or presence, respectively, of a net charge in the detergent molecule. The fluidities of EPC micelles containing small amounts of a detergent possessing a steroidal structure (e.g., Na-chol or CHAPS) were significantly smaller than the corresponding values of a detergent without a steroidal structure (e.g., OG), suggesting a method of control of orderliness of hydrocarbon chains in EPC vesicles.
A vesicle is a compartment composed of lipid bilayer of amphiphilic molecules. The vesicle is applied to carriers of drugs, cosmetics and functional food ingredients in industries. Vesicles are also applied as a model for artificial cell membrane and expected as micro- and nano-reactors. They are generally prepared by the hydration of dry lipid film, but there is no method to prepare vesicles of a controlled size and high entrapment yield of hydrophilic materials inside them. In this article, a microchannel (MC) emulsification method was applied to prepare vesicles aimed at controlling the size and improving the entrapment yield. Firstly, monodisperse water-in-oil (W/O) emulsions were prepared by the MC emulsification method. In this process, hydrophilic materials to be entrapped were contained inside the water droplets of the emulsions. Keeping the water droplets frozen, the emulsifier was replaced by a bilayer-forming lipid mixture, and then the oil phase was evaporated. After hydration of lipid layers surrounding the water droplets, vesicles were formed. We call this preparation “lipid-coated ice droplet hydration method”. The final sizes of the prepared vesicles were comparable to the original emulsion droplet sizes. This means that the size of vesicles can be controlled by controlling the size of original water droplets of the W/O emulsions. Furthermore, calcein as a hydrophilic fluorescent marker and biopolymers, such as enzyme and polysaccharide, were entrapped into the internal water phases of vesicles. The method proposed in this study enables the formation of vesicles with a controlled size and high entrapment yields, potentially useful for expanding the application fields of vesicles as biocompatible carriers and micro- and nano-reactors for biochemical reactions.
The interaction of apolipoprotein A-I (apoA-I) with transmembrane ATP-binding cassette transporter A1 (ABCA1) is a crucial step for high-density lipoprotein (HDL) formation, however, its molecular mechanism is less well understood. Here, we used apoA-I and its model peptide, Ac-18A-NH2, to investigate their interaction with mixed membranes of phosphatidylcholine (PC) with phosphatidylethanolamine (PE) or sphingomyelin (SM). It was shown that PE, possessing the negative spontaneous curvature, increased both the degree of hydration at the membrane interface and the acyl chain order, and that Ac-18A-NH2 had opposite effects to PE, since the α-helix formation at the membrane surface induced positive curvature strain. In addition, increased PE in PC/PE large unilamellar vesicles (LUVs) enhanced the peptide's binding. Although SM significantly lowered the peptide binding capacity, the peptide's binding to PC/SM LUV led to membrane disruption. The interaction with PC/SM LUVs was then investigated using ApoA-I. The spontaneous rHDL formation from pure PC LUV proceeded very slowly at 37°C, but SM-rich PC/SM LUVs, which are in a gel/liquid-disordered (Ld) phase at this temperature, were rapidly solubilized to form rHDL by apoA-I. The addition of cholesterol decreased the rate of the rHDL formation and induced the selective extraction of lipids by apoA-I, which preferably extracted lipids of Ld phase rather than lipids of liquid-ordered (Lo) phase. These results suggest that heterogeneous interface of the mixed membranes facilitates the insertion of apoA-I and induces Ld phase-selective lipid extraction to form rHDL, and are compatible with recent cell works on the apoA-I-dependent HDL generation.
Biosurfactants (BS) are functional amphiphilic compounds produced by a variety of microorganisms. They show unique properties (e.g. mild production conditions, lower toxicity, and environmental compatibility) compared to chemically synthesized counterparts. The numerous advantages of BS have prompted applications not only in the food, cosmetic, and pharmaceutical industries but in energy and environmental technologies as well. Mannosylerythritol lipids (MELs) are one of the most promising BS known, and are produced at yields of over 100 g/l from vegetable oils by yeast strains belonging to the genus Pseudozyma. MELs exhibit excellent surface-active and self-assembling properties leading to the formation of different lyotropic liquid crystals such as sponge (L3), bicontinuous cubic (V2) and lamella (Lα) phases. They also show versatile biochemical actions, including antitumor and differentiation-inducing activities against human leukemia cells, rat pheochromocytoma cells and mouse melanoma cells. MELs also display high binding affinity toward different immunoglobulins and lectins, indicating great potentials as new affinity ligands for the glycoproteins. More significantly, the cationic liposomes bearing MELs increase dramatically the efficiency of gene transfection into mammalian cells via membrane fusion processes. The yeast BS should thus be novel nanobiomaterials, and broaden their applications in various advanced technologies.
Carbohydrate chains in glycoconjugates play important roles in various life phenomena, and there are numerous types of recognition system for carbohydrate chains due to carbohydrate-lectin interactions/carbohydrate-carbohydrate interactions in all higher life forms. It has been proposed that macromolecular polysaccharides isolated from plants, marine organisms, or fungi cross-interact with known and unknown recognition systems in mammals to express their pharmacological activities. Therefore the elucidation of carbohydrate structures related to the activities and functions of these polysaccharide molecules will lead us to utilize the related information in the development of novel carbohydrate-based drugs and functional foods for human health care. Peyer's patches present in the upper intestinal tract play important roles as inductive sites for both protective IgA production and immune tolerance induction in mucosal and systemic immune systems. Dysfunction of the immunocompetent cells of Peyer's patches is thought to induce allergic/autoimmune diseases and down-regulation of the protective system against infectious agents on mucosal sites. We have isolated several Peyer's patch cell-modulating polysaccharides from medicinal herbs used in traditional Japanese herbal remedies, and they have been assumed to comprise the responsible carbohydrate chains with oligosaccharide sizes for expression of modulating activity. Accumulation of knowledge on the structures and functions of these responsible carbohydrate chains in polysaccharide molecules is believed to be important for the development of methodology for logically factitious regulation of functions of immunocompetent cells in Peyer's patches. This review deals with recent results of our study on the structural clarification of responsible carbohydrate chains in modulating polysaccharides against functions of immunocompetent cells in Peyer's patches.
Proteoglycan contains glycosmainoglycans, which are endogenous sulfated polysaccharides, in the molecule. The metabolism of proteoglycans regulates cell behavior and cellular events. It is possible that exogenous polysaccharide-related molecules exhibit their biological activities by two mechanisms. One is the interaction with cells and the other is the interaction with growth factors/cytokines that regulate proteoglycans. In this review, we describe sodium spirulan, a sulfated polysaccharide obtained from a hot-water extract of the blue-green alga Spirulina platensis, as an exogenous polysaccharide that stimulates the release of proteoglycans from vascular endothelial cells. Factors that regulate endothelial proteoglycan metabolism are also being described as possible target molecules of exogenous polysaccharides. Further research is required to obtain exogenous polysaccharide-related molecules that exhibit useful biological activities through controlling endothelial proteoglycan metabolism for protection against vascular lesions such as atheroslcerosis.
Recently, the development of antiviral agents with novel mechanisms of action has been required since many types of infectious disease have become a serious problem in our society. In the present study, we isolated a novel acidic polysaccharide, nostoflan (NSF), from a terrestrial blue-green alga, Nostoc flagelliforme, and examined its structure and antiviral activity. The sugar composition and methylation analyses of NSF revealed that it is mainly composed of (→4)- D-Glcp-(1→, →6,4)-D-Glcp-(1→, →4)-D-Galp-(1→, →4)-D-Xylp-(1→, D-GlcAp-(1→, D-Manp-(1→) with a ratio of ca. 1:1:1:1:0.8:0.2. Oligosaccharide analysis after partial acid hydrolysis of NSF revealed that this polysaccharide might be mainly composed of the sugar sequences of (→4)-β-D-Glcp-(1→4)-D-Xylp-(1 and→4)-[β-D-GlcAp-(1→6)-]-β-D-Glcp-(1→4)-D-Galp-(1→). NSF showed potent antiviral activities against several enveloped viruses including herpes simplex virus type 1, type 2 (HSV-1, HSV-2), human cytomegalovirus, and influenza A virus (IFV). NSF selectively inhibited the attachment of HSV-1 to host cells but not its penetration phase. In an experimental animal study where IFV-infected mice received NSF intranasally, the mortality of mice was significantly decreased. Neutralizing titers in sera of mice treated with NSF were higher than in those treated with oseltamivir. From these results, NSF was found to be a novel polysaccharide that shows antiviral activity in vitro and in vivo in spite of a nonsulfated polysaccharide.
The Japanese animal protection law was amended in 2005 to include the 3Rs principle in animal experiments. According to this new law, the Ministry of Education, Culture, Sports, Science and Technology, the Ministry of Health, Labor and Welfare, and the Ministry of Agriculture, Forestry and Fisheries developed announced several guidelines in 2006. These guidelines indicated responsibility of the president of each research institute conducting animal experiments to meet obligating of the animal experiment committee (AEC) and the education to be provided to scientists. About half a year after this notification, I conducted a survey on how these guidelines were put into practice in the pharmaceutical colleges and universities. I received 29 answers from 24 institutes. It seemed that every institute was following, the guidelines, however, there were many institutes where the details were inadequate. For example, questions on the existence of alternative methods and degree of distress and pain were not asked in some questionnaires sent to the AEC. Education on proper conduct of animal experiments (3Rs, methods to evaluate and decrease distress and pain, and methods of euthanasia) was not conducted in many institutes. Further improvement seems necessary.
Systematic modern animal experimentation was established by Bernard Claude who wrote “An Introduction to the Study of Experimental Medicine” in 1865. At this point, the public was already asking that the pain and distress of experimental animals be reduced. For this, scientists, William Russell and Rex Burch in 1959 proposed the principles of alternatives to animal experimentation, the “3Rs”. Since that time, animal welfare advocates have promoted the 3Rs concept in biomedical research communities. However, cruel animal experiments have continued and there are reports of radical extremists showing their opposition by invasion, arson, theft and even bombing of institutions involved, resulting in killing of the animals. SHAC, one extremist group believed to be animal welfare activitists was recognized as a terrorist group after the 9.11 tragedy in USA and the government viewed their activities very seriously. In 2001, British animal extremists invaded Japanese universities and stole laboratory resources; one individual was arrested and sentenced to prison for three years; Japanese who assisted in the incident were arrested and one was sentenced for one year. In 2006, SHAC USA members were prosecuted and sentenced for up to 6 years for their terrorism activities including arson. We need to consider the background of these activities which are financially supported by animal welfare advocates. The way we, as scientists who conduct such experiments can respond is by promoting alternatives to this experimentation. In Japan, the animal welfare law was revised in 2005 stressing the importance of 3Rs in scientific activities with animals. The promotion of 3Rs should be strengthened in the pharmaceutical community.
In November 2005, the Japanese Center for the Validation of Alternative Methods (JaCVAM) was established as a part of the Division of Pharmacology at the National Center for Biological Safety and Research affiliated with Japan's National Institute of Health Sciences (NIHS). JaCVAM facilitates the validation, peer-review, and international harmonization of alternative to animals testing. Key objectives of JaCVAM are: 1) facilitate 3R's*, prioritizing Reduction and Replacement, and 2) to ensure new test methods are validated, peer reviewed, officially accepted by the regulatory agencies, and made internationally compatible. In this paper, JaCVAM's current activities and future directions are shown in the validation and peer review of alternatives to testing for skin irritation, eye irritation, phototoxicity, skin sensitization, acute toxicity, genotoxicity and endocrine disruptor screening. * 3R's for animal testing (Reduction, Refinement, Replacement)
Reactive oxygen species (ROS) are associated with oxidative stress-mediated alterations under pathophysiological conditions, and particularly brain ischemia, brain tumor, and neurodegenerative diseases. Electron spin resonance (ESR) is recognized as one of the most powerful techniques available for the detection of ROS in tissues and cells. We previously developed an in vitro ESR-based technique for the detection of free radical reactions in biological systems. In addition, significant advances in the field of in vivo ESR techniques over recent years have now made it possible to visualize the distribution and metabolism of oxidative stress, and the degree of tissue oxygenation in vivo. Nitroxyl radicals are very useful as exogenous spin probes for measuring free radical distribution, oxygen concentration, and redox metabolism by in vivo ESR in biological systems, using a combination of these ESR methods collectively focused on animal models of disease such as spontaneously hypertensive rat (SHR) or stroke-prone SHR (SHRSP) for the assessment of antioxidant property of drugs. Our results suggest that ESR could be applied to the assessment of antioxidant property on oxidative stress in target organs, especially brain, using animal disease models, SHR or SHRSP. After screening drugs for antioxidant property using such as in vitro or in vivo ESR assessment, we'll be able to develop and find novel antioxidant drugs for ROS-induced brain disease in the near future.
Currently, the European Centre for the Validation of Alternative Methods in the EU appears to be at the forefront of the development of alternative methods for developmental toxicity test (reproductive/developmental toxicity test). Why is it difficult to develop alternative methods for developmental toxicity test in comparison with other toxicity tests? In developmental toxicity test, chemical substances first enter the bloodstream and then reach the placenta via metabolism in the liver and other organs. After further metabolism in the placenta, chemical substances finally reach the fetus, where they affect fetal development. The difference in the in vivo route of chemical substances is an important reason for the difficulty in the establishment of new methods for developmental toxicologic test in comparison with general toxicity tests. According to the EU, the use of “in silico” techniques for developmental toxicity test may be difficult, and I agree with this. The in silico technique is basically a method for prediction of toxicologic effects from existing data, and cannot predict new effects, because data obtained by developmental toxicologic test are too complex. Three techniques are now being examined to overcome the difficulty in changing the method of developmental toxicologic test: the technique utilizing embryonic stem cells; micromass culture technique; and the whole embryonic culture technique. In this symposium, the current status of developmental toxicity tests and the three techniques being examined in the EU are introduced, and opinions on future progress are presented.
A novel elementary osmotic pump tablet was developed. The system uses the core of drug-resin complexes (DRCs) loaded with propranolol hydrochloride (PNH) for time-controlled delivery. In traditional osmotic pump tablets (OPTs), the lag time was always minimized. However, in the DRCs osmotic pump tablet (DRCOPT), the lag time was increased to achieve the time-controlled delivery. The quantity of osmotic agent in the core and channeling agent in the coating solution as well as weight gain were confirmed to be essential for the release behavior. A spherical symmetric design was applied to the optimization of the DRCOPT. The optimal formulation mainly consisted of DRC 100 mg, polyethyleneoxide (N80) 182 mg, and NaCl 30 mg. The ratio of cellulose acetate (CA)/polyethylene glycol 4000 was 15:3 (w/w) in coating solution, and the weight gain was 8%. The release behavior of the optimal DRCOPT was evaluated in media with different pH, rotation speeds, and ionic strength. It was found to generate a 2-h lag time, to deliver PNH at a rate of zero order from 2 h to 14 h in the medium of NaCl 0.15 mol/l, and the cumulative release at 24 h was 94%. Drug relee was independent of pH and rotation speed, but was proportional to ionic strength. In summary, the lag time could be used in therapeutic regimens with the characteristics of chronotherapy because of the lag time and provides a new concept for the development of osmotic pumps.
We tried to clarify the applicability of “utility” for the evaluation of patient's QOL with gastric cancer after chemotherapy and attempted to compare differences in QOL after treatment with the oral antitumor agent TS-1 or with a conventional injectable combination. Three items, moving activity, pain, and gastrointestinal symptoms, were employed as indicators of patient QOL, and then the assessment of utility was compared based on the expected outcomes that 9 pharmacists working on a ward, 9 nurses working on a neurosurgery ward, and 9 nurses working on a gastrointestinal surgery ward estimated directly using the three methods of standard gamble, time trade-off, and rating scale according to predictive scenarios based on each scenario. The QOL of patients who received the two different types of chemotherapy were also compared as the average utilities from the direct estimation depending on patient conditions as used for chart review. Furthermore, the average utilities were compared with the utility of the mapping method, which can be estimated by applying a utility-converting table defined in the EQ-5D survey. The average utility from each practitioner using the direct estimation revealed that the assessed utility from nurses working on a neurosurgery ward was higher than those of the pharmacists. The average utility obtained using the standard gamble method was higher than those using the rating scale and time trade-off methods. The average utility in the TS-1 therapy group was 0.84-0.94, and that in the conventional injectable therapy group was 0.52-0.79 (p<0.05). The result suggests that utility is applicable for estimation of gastric cancer patient QOL after chemotherapy, and that TS-1 therapy is superior to the traditional injectable combination therapy.
Since patients who used brand-name and generic ketoprofen (KP) tapes dispensed in the Fukuoka City Pharmaceutical Association Pharmacy complained of a difference in feeling between the two products, we conducted a questionnaire survey in patients using KP tapes to determine the usability of brand-name and generic tapes. Patients receiving the brand-name KP tape (product A) and/or a generic KP tape (product B) in our pharmacy were interviewed concerning 20 items including 1) dosage regimen, 2) outer package, 3) liner, 4) the tape itself, and 5) condition of application sites. A significant difference in usability between products A and B was observed in 4 of the 20 items evaluated, i.e., 1) easiness of opening of the outer pouch, 2) easiness of removing the liner film, 3) condition of the application site after removal of the tape, and 4) relief of pain in the target lesion after application. As suggested by patients who had complained of a difference in usability between products A and B, the participants in the survey tended to prefer the brand-name KP tape to the generic product. The findings of the survey indicate that when a brand-name product is switched to generic products, pharmacists should evaluate the usability of such products carefully to select those that will ensure proper drug use by patients.
Erigeron breviscapus (Vant.) Hand-mazz (EB), Erigeron multiradiatus (Lindl.) Benth (EM), and Aster brachytrichus Franch (AB), confused under the vernacular name “meiduoluomi” by native people and traditional healers, have been used for the treatment of meningitis, polyneuritis, hepatitis, adenolymphitis, and enteronitis in traditional Tibetan medicine. In this study, the antiinflammatory activity of methanol extracts of all three plants was investigated in the xylene-induced ear edema model, carrageenan-induced paw edema model, and cotton pellet-induced granuloma model. It was found that the methanolic extracts of both EB and EM had strong inhibitory effects on the acute phase of inflammation in carrageenan-induced paw edema in rats. On the other hand, the methanolic extract of EM showed stronger effects than those of EB in xylene-induced ear edema. In the chronic test, the methanolic extracts of EB and EM resulted in a significant reduction in granuloma weight in rats. In addition, myeloperoxidase (MPO) activity was strongly reduced in the EB-treated and EM-treated groups, which indicated that EB and EM can inhibit certain inflammatory modulator factors that cause neutrophil aggregation in inflamed tissue, e.g., nuclear factor-κB. However, the methanolic extracts of AB had no antiinflammatory effects in the tested models and MPO assay. The similar effects of EM and EB in tested models provided some scientific basis for the traditional usage of meiduoluomi in inflammatory disease. However, the results also suggest that further study is needed to investigate the antiinflammatory profile of AB and provide a scientific basis for the use of AB in inflammatory diseases.
A method for the rapid determination of 11 medical components found in health foods for weight loss using liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been developed. HPLC separation is performed on an ODS column with the gradient elution method. The mobile phase consists of two solvents. Solvent A is water and solvent B is methanol/acetonitrile (1 : 1), and both contain 0.1% formic acid and 5 mmol/l of ammonium acetate. Each medical component is analyzed with multiple-reaction monitoring (MRM) in both negative and positive modes through electrospray ionization (ESI). The recovery rates of the 11 medical components added to commercially available health foods were 46.3-114% and each coefficient of variation was 13.7% or less. It was confirmed that this method is applicable to the urgent analysis of health foods that have caused damage to health.
Many generic drugs have been released to decrease medical expenses, but some problems have been reported with regard to bioavailability and safety. In this study, we compared three once-a-day controlled-release preparations of nifedipine by the dissolution test (one branded and two generic preparations). Although the two generic drugs were equivalent to the branded drug according to the criteria listed in the Japanese “Guideline for Bioequivalence Studies of Generic Products”, there was still a possibility of problems arising. For example, side effects could be caused by a rapid increase in the blood level of nifedipine with one generic drug, while bioavailability might be inadequate with the other due to its small area under the concentration vs. time curve. When each drug was prescribed at a dosage of 20 mg once daily for two weeks, the difference in the copayment for the patient was only 10 yen. Accordingly, it is important for doctors and pharmacists to carefully consider whether such a slight difference in price is really a benefit for the patient.
To help pharmacy students develop applied knowledge concerning clinical diseases and the ability to communicate with patients (and medical staff), we have introduced a new improved program for first and second year pharmaceutical students. This new program involves a 10-20 minute presentation of a clinical disease and clinical case by the students after regular lectures. Our new program may be useful for a 6-year pharmacy education in order to produce pharmacy students who have: 1) wide clinical knowledge, 2) a better basis for understanding advanced subjects such as pharmacology, drug therapeutics and pathologic physiology that are taken in the upper grades, and 3) practical training at medical institutions. In addition, a pamphlet produced as part of the student presentation become reference data when students in the following year study the same topic or teachers of other professional subjects attempt a similar program.