In eukaryotic cells, pre-mRNA molecules contain multiple intron sequences that are removed by splicing reactions. Truncated ftz pre-mRNA containing one intron and two exons, which mimics RNA under the post-transcriptional splicing, was synthesized and labeled with a fluorescent dye in vitro and then injected to the nucleus of Cos7 cell. The injected pre-mRNAs accumulated in 'speckles' in an intron-dependent manner and were spliced and exported to the cytoplasm with a half-time of about 10 min. Dissociation of pre-mRNAs in speckles exhibited rapid diffusion and slow dissociation of about 100 s. The slow dissociation required metabolic energy of ATP. Some pre-mRNAs shuttled between speckles and nucleoplasm, suggesting that pre-mRNAs repeatedly associated with and dissociated from speckles until introns were removed. These results suggest that speckles function as a checkpoint for whether or not mRNAs are appropriately processed. Next, mature mRNAs of truncated β-globin were synthesized, fluorescently labeled in vitro, and injected to the nucleus. The trajectories of single mRNA molecules in the nucleus were visualized using video-rate confocal microscopy. Approximately half the mRNAs moved by Brownian motion in the nucleoplasm, except the nucleoli, with an apparent diffusion coefficient of 0.2 μm2/s, about 1/150 of that in water. The remaining mRNAs were stationary with an average residence time of about 30 s. These results indicate that mRNAs are transported to nuclear pores by Brownian motion. Finally, intrinsic c-fos mRNA was fluorescently labeled with Cy3-2'O-methyl oligo RNA probes and its concentration was measured by fluorescence correlation spectroscopy.
In vivo redox reaction is involved in processes of oxidative diseases. The redox imaging technique is important to diagnose redox-induced diseases and to assess cure effects of pharmaceutical drugs. A group of nitroxyl radicals is sensitive to redox reactions and we have investigated mechanisms of oxidative diseases, including diabetes, ischemia reperfusion injuries and gastric ulcer. ESR technique has been utilized in analysis of free radicals, which is generated through imbalance of in vivo redox status. We have been developing magnetic resonance approaches for imaging free radicals/redox status in living animals. Overhauser enhanced MRI (OMRI) is a new technique for imaging in vivo redox status in animals via Overhauser effect. We have developed nanometer-scale imaging and simultaneous assessment of redox processes by using OMRI with 14N- and 15N- labeled nitroxyl probes with different distribution properties. We also developed a home-built OMRI imager based on an electromagnet for L-band ESRI. This OMRI technique with dual probes may become a powerful tool to clarify mechanisms of disease and to monitor pharmaceutical therapy.
Molecular imaging by PET/SPECT with radiopharmaceuticals enables noninvasively quantitative evaluation of physiological function, gene expression, pharmacokinetics of proteins and peptides and distribution of receptors with high sensitivity. Together with recent development of imaging equipments, molecular imaging by PET/SPECT is expected to contribute to elucidation of physiological and pathological functions, medical sciences and clinical diagnoses. Molecular imaging with radiopharmaceuticals started from diagnosis of cancer with 18F-2-fluoro-2-deoxyglucose ([18F]FDG). Currently, [18F]FDG is commonly used in the field of clinical diagnosis, because it can provide qualitative information on malignancy and metastasis of tumor. Since its achievement, much effort has been devoted to the development of radiopharmaceuticals that bind or interact with the in vivo biomarkers. For example, a number of radiopharmaceuticals based on proteins and peptides with high binding affinities to various biomarkers have been applied for the diagnosis of tumor, arteriosclerosis, thrombus and so on. Furthermore, Alzheimer's disease is also a major target for diagnosis by PET/SPECT imaging. The development of low-molecular-weight radiolabeled probes for the quantitation of β-amyloid plaques and neurofibrillary tangles in Alzheimer's brains is a topic of current PET/SPECT imaging studies. Here, some recent progress and development of radiopharmaceuticals for PET/SPECT imaging will be reviewed.
One of the most challenging researches in current biology and medicinal chemistry is to understand how individual cellular molecules interact together in living cells. To visualize such molecules, genetically-encoded reporters have been used widely. The most common reporters are firefly luciferase, renilla luciferase, green fluorescent protein (GFP) and its variants with various spectral properties. In this review, novel design of split GFP and split luciferase is described. The principle is based on reconstitution of the split-reporter fragments when they are brought together into close proximity. The reconstitution methods are used for screening organelle-localized proteins, imaging dynamics of nuclear proteins and mRNAs in living cells, and visualizing protease activities in living animals. These methods are generally applicable for imaging of complex cellular processes and evaluating chemical effects in living cells and animals.
Molecular imaging technology such as positron emission tomography (PET) and magnetic resonance imaging (MRI) are known as powerful tools for clinical diagnosis in neurology, oncology and so on. As applications to new drug research and development, there are three methodologies which are PK (Pharmacokinetics study), PD (Pharmacodynamic study), and efficacy study. When we use these methodologies for the drug research, we must consider construction of technological environment (tracer, animal model, imaging analysis software, and clinical database) and regulatory environment for GMP (Good Manufacturing Practice) and GCP (Good Clinical Practice) level. Additionally, concept of microdosing and exploratory clinical study was proposed in western countries and the guidance on microdosing study was also announced by Health, Labor and Welfare Ministry on Jun. 3rd 2008. However they may be still in learning phase, we must meet with complexity, high cost, and indigestion. To promote molecular imaging technology into the drug research, integration of the scientists between academia and industry is important because it needs much type of the advanced technologies and skills.
Immunotoxic effects of heavy metals, as a typical environmental agent, and their mechanisms are reviewed based on our findings on autoimmune response induced by exposure to cadmium (Cd) as CdCl2. Adverse immune effects of chemicals, defined as immunotoxicity, have been used as a sensitive biomarker for assessing health effects of environmental chemicals. My initial research focused on renal toxicity of heavy metals was developed to elucidate characteristics and mechanisms for immune-mediated nephritis induced by heavy metals. In our studies the most interesting finding was autoantibody production enhanced by the oral exposure to Cd at environmental levels. It was observed simultaneously with enhancement of non-specific antibody production and suppression of primed-antigen specific antibody production. Immunostimulation including induction of autoantibodies was found to be the primary immunotoxic effect of Cd, because of the dose-sensitivity, and to be associated with polyclonal B cell activation (PBA). Further mechanism studies on the PBA induced by Cd in vitro showed that it was mediated by T cells, via cytokines, dominantly Type-2 cytokines, and recognition of MHC-II antigens of cell surface. The similarity among PBAs induced by inorganic salts of Cd, mercury and lead suggests that it would be the common effect among the metals to be involved in their pathogenesis of nephritis. Finally possible health significance of chemical-induced PBA is discussed associated with an increasing trend of autoimmune diseases in industrialized countries.
This review provides an overview of the chemistry of hypervalent organohalogans, organoiodanes and organobromanes, developed recently in Tokushima. Vinylic SN2 reactions, cyclopentene syntheses via 1,5 C-H insertion of alkylidene carbenes, reductive Claisen rearrangements, oxidations with peroxy-λ3-iodanes, iodobenzene-catalyzed oxidations, oxidative cleavage of double bonds, oxidative coupling of alcohols with alkynes, Michael additions, and transylidations are involved. These reactions mostly rely on the hyperleaving group ability of aryl-λ3-iodanyl, -bromanyl, and -chloranyl groups.
In Japan, the initial dose of 2.5 mg/3 d is recommended in the package insert of the fentanyl patch preparation to substitute for oral morphine in the dose range of 45-135 mg/d (90 mg/d at the midpoint), while a higher dose is recommended in other countries. To validate the recommended dose of this drug in Japan, we investigated how long the initial recommended dose of the fentanyl patch could control the pain of cancer patients after the switch from other opioids. The dose of the fentanyl patch was increased on the 20th day after the switch from prior opioids at a lower dose than the midpoint of the indicated range, while it was increased on the 3rd day after the switch from the higher dose of prior opioids. Regression analysis showed that the efficacy ratio of the fentanyl patch : oral morphine=80 : 1, suggesting that oral morphine of 25-75 mg/d should be substituted for by the fentanyl patch preparation at a dose of 2.5 mg/3 d.
A single time point isothermal drug stability experiments at constant humidity is introduced. In the new method, kinetic parameters related to both moisture and temperature were obtained by a single pair of experiments: these related to moisture by one with a group of testing humidities and a fixed temperature, those related to temperature by the other with a group of testing temperatures and a constant humidity. By a simulation, the estimates for the kinetic parameters (Ea, m, A) obtained by the proposed method and the reported programmed humidifying and heating method were statistically evaluated and were compared with those obtained by the isothermal measurements at constant humidity. Results indicated that under the same experimental conditions, the estimates obtained by the proposed method were significantly more precise than those obtained by the reported programmed humidifying and heating method. The estimates obtained by the isothermal method at constant humidity were somewhat more precise than those obtained by the proposed method. However, the experimental period needed by the isothermal method at constant humidity was greatly longer than that needed by the proposed method. The stability of dicloxacillin sodium, as a solid state model, was investigated by the single time point isothermal drug stability experiments at constant humidity. The results indicated that the kinetic parameters obtained by the proposed method were comparable to those from the reported.
In this study, we aimed to determine an index of anti-MRSA drugs for long-term treatment. We examined adult patients to whom the anti-MRSA drugs arbekacin sulfate (ABK), vancomycin hydrochloride (VCM), and teicoplanine (TEIC) had been administered in the St. Marianna University School of Medicine, Yokohama City Seibu Hospital, for 1, year. The number of patients treated for≧14 days was 22 (31%) among 71 patients. Immunosuppressive agent positivity (p=0.07), albumin (ALB) level of ≦2.5 g/dl (p=0.03), and C-reactive protein (CRP) level of ≧10 mg/dl (p=0.01),%STAB ≧15% (p=0.11), and the period until the blood drug level is measured (p=0.06) were analyzed with respect to the differences between both groups by univariate analysis. An ALB level of ≦2.5 g/dl was a significant factor as determined by multivariate logistic analysis (p=0.04). It is thought that it is necessary to base long-term administration on necessity in patients with low albumin levels in blood, to consider the appropriate treatment and nutrition management together, and to consider the appropriate treatment depending on the early stage of the blood drug level, and to promote the proper use of drugs.
We evaluated the stability of temozolomide, an alkylating agent, in solutions after opening the capsule. First, we established an analytical method for determination of temozolomide concentration by HPLC. The calibration curve for temozolomide was linear between 0.5-20 μg/ml (r=0.999). We then evaluated the stability of temozolomide in each buffer solution (pH 2-9) for 30 min. Temozolomide was decomposed pH-dependently between pH 7 and 9, and completely decomposed at pH 9. Temozolomide in several drinking water samples and beverages was decomposed according to their pH values. We also examined the time-dependent degradation of temozolomide in different pH solutions. Temozolomide started to decompose at 5 min in alkaline and neutral solutions, whereas 90% of temozolomide remained intact in acidic solution at 60 min. These results indicate that the stability of temozolomide after opening the capsule is affected by pH of solvents, and temozolomide is almost stable in acidic solutions.
Oxaliplatin, established as a therapeutic standard globally for advanced/recurrent colorectal cancer, was approved in Japan in April 2005. With this approval the FOLFOX and FOLFIRI regimens are often selected now as 1st or 2nd line treatment for advanced/recurrent colorectal cancer. Patients receiving these regimens needed to be hospitalized, because the total treatment period was as long as 48 hours. However, the patient who hoped for staying at home has become possible to spend more time at home by using a portable disposable infusion pump (SUREFUSER® A) for continuous intravenous infusion of 5FU. The duration of continuous 5FU infusion is set at an average of 46 hours, however, large variations are observed in the duration of infusion. Due to limitation of time of a patient, there was a case that finished injection on the way. On the contrary, there was a case that finished in a much shorter time than the pre-designated 46 hours. In an attempt to resolve this problem, we analyzed the relation of the total volume of the medicinal solution in SUREFUSER® A and the duration of infusion by regression analysis. The results revealed that it might be possible to bring the total infusion time to close to 48 hours by finding the most suitable volume for continuous 5FU infusion over 46 hours.
When a fluticasone propionate (FP) diskhaler is used to administer inhaled corticosteroid, it has been reported that there is considerable drug residue remaining in the diskhaler after use. The internal structure of the diskhaler is complex, and it is possible that sufficient cleaning of the device is not achieved using the attached brush. In this study, the diskhaler cleaning method was examined using a patient questionnaire. In response to the question on cleaning, 56.3% of patients responded “Having done”, and 66.7% responded to the question on the frequency of the cleaning, “When I use it”. Furthermore, cleaning by a healthy volunteer was examined using Rotadisk® for inhalation practice. When the group that did not perform cleaning was compared with the group that performed cleaning with the brush, the amount of the lactose adhesion was significantly lower in the cleaning group. When the no-cleaning group was compared with the group that shook off the excess residue from the tray and the main body of the diskhaler, the group that shook off the diskhaler components showed a significantly lower amount of lactose adhesion. It was confirmed that drug residue were able to accumulate, and the shaking off method appeared to have an effect equal to that of cleaning with the brush. It seems that providing patients with guidance not only about the method of inhaling with the diskhaler but also about cleaning of the device is an important area of pharmacy patient management.
A reservoir type transdermal patch for delivery of ketorolac, a potent analgesic agent was studied. The low permeability of skin is the rate-limiting step for delivery of most of the drugs. Studies were carried out to investigate the effect of permeation enhancers on the in vitro permeation of ketorolac across rat skin. The reservoir type transdermal patch was fabricated and the core was filled with gel system of a non ionic polymer HPMC (hydroxypropyl methyl cellulose) formulated in PBS (phosphate buffer saline) solution of pH of 5.4 along with isopropyl alcohol at 25% w/w concentration. Various permeation enhancers' viz. dimethyl sulphoxide, d-limonene, eucalyptus oil and transcutol (diethylene glycol monoethyl ether) were incorporated into the gel system. Permeation enhancement of ketorolac with different enhancers followed the order eucalyptus oil> transcutol> DMSO> d-limonene. Cyclic terpene containing eucalyptus oil was found to be the most promising chemical permeation enhancer for transdermal delivery of ketorolac. The increase in concentration of eucalyptus oil further enhanced drug permeation with maximum flux being achieved at 10% w/w of 66.38 μg/cm2/h. Further enhancement of permeation rate of ketorolac across skin was attained by application of abrading gel containing crushed apricot seed onto the skin. There was 5.16 times enhancement and flux of 93.10 μg/cm2/h was attained. A reservoir type transdermal patch for delivery of ketorolac thus appears to be feasible of delivering ketorolac across skin.
To apply human mesenchymal stem cells (hMSC) to regenerative medicines, it is necessary to multiply hMSC in vitro in a short period. In addition, it is desirable that the medium which is used for the hMSC multiplication is not supplemented with the serum, because the addition of the serum has risks of infection. In this study, we cultured hMSC with three kinds of medium used for multiplying hMSC (DMEM, MSCGM, STK2) and compared hMSC proliferation in each medium. As a result, it was confirmed that hMSC proliferation was significantly higher in STK2 medium which is a novel serum-free medium developed for hMSC multiplication. Moreover, we compared the hMSC proliferation in these media under the environment that assumed bone reproduction. When we cultured hMSC in each medium with hydroxyapatite (HAp), the proliferative inhibition by HAp depended on the additive amount, and the degree of the proliferative inhibition was different among the media but the lowest inhibitory effect was observed in STK2 medium.