Recombinant Adenovirus (Ad) vectors are considered to be a promising gene delivery vehicle of high utility because they are easy to construct, can be produced at high titers, and efficiently transduce various types of cells. Ad vectors commonly used in the world, including clinical trials, are composed of Ad serotype 5 (Ad5), which belongs to subgroup C. In recent years, however, it has become apparent that Ad5 vectors have some drawbacks, such as high seroprevalence of anti-Ad5 antibodies in adults and low transduction efficiencies of Ad5 vectors in cells lacking a primary receptor for Ad5, coxsackievirus and adenovirus receptor (CAR). To overcome these limitations of Ad5 vectors, we have developed a novel type of Ad vector, which is composed of Ad serotype 35 (Ad35), belonging to subgroup B. Ad35 vectors recognize human CD46, not CAR, as a cellular receptor for infection. Human CD46 is expressed in almost all of human cells, leading to a broad tropism of Ad35 vectors to human cells, in contrast, expression of rodent CD46 is limited to the testis. Therefore, in vivo transduction properties of Ad35 vectors are not appropriately evaluated in normal mice. In order to evaluate the in vivo transduction properties of Ad35 vectors, Ad35 vectors were applied to human CD46-transgenic mice and nonhuman primates, which express CD46 in a similar pattern to humans. The data obtained using CD46-transgenic mice and nonhuman primates would provide valuable information towards clinical applications of Ad35 vectors.
AAV vector is derived from nonpathogenic virus and has a number of attractive features as a vector for human gene transfer including safety, broad tissue specificity, and low immunogenicity following gene transfer. Moreover, persistent transgene expression (for years) was demonstrated in multiple animal experiments. For these reasons, applications to a wide spectrum of diseases are expected, and several clinical trials have been conducted. Although it is too early to conclude the outcome, the efficacy of treatment was not sufficiently substantiated in most of the trials despite confirming the safety of the vector. These results are primarily due to low levels of transgene expression. One of the approaches to improve this situation is the use of alternative serotypes of AAV. Traditionally, serotype 2 was considered to be a prototype of AAV, and the majority of studies including human clinical trials have been conducted using this serotype. On the other hand, there are five “classical” serotypes, and several have been additionally discovered from tissues of primates including humans. These serotypes are considered to be valuable resources for vector development to overcome the shortcomings of serotype 2. This review focuses on the difference in expression levels and tissue specificity of various serotype-derived vectors and summarizes current status in the treatment of candidate diseases.
The spatiotemporal distribution of transgenes determines the therapeutic efficacy of in vivo gene transfer. The important parameters of gene transfer are the level, duration, and cell specificity of expression, and the number of transfected cells. Interaction of vectors with blood cells, antigen-presenting cells, serum proteins, and other biological components affects the tissue distribution of vectors and the profile of transgene expression. Although plasmid DNA is less immunogenic than viral vectors, it can induce inflammatory cytokine release, due mainly to the presence of unmethylated CpG dinucleotides (CpG motifs). It was clearly demonstrated that intravenous injection of a plasmid DNA/cationic liposome complex resulted not only in the induction of inflammatory cytokines, but also in the activation of nuclear factor κB (NF-κB) in the lung. Insertion of additional NF-κB-binding sequences into conventional plasmid DNA resulted in a high transgene expression in the lung, suggesting that the biological response to vectors can be used to increase transgene expression. In a marked contrast to this strategy, long-term transgene expression was achieved by reducing the number of the CpG motifs in plasmid DNA. A plasmid encoding murine interferon (IFN)-β or IFN-γ with reduced numbers of CpG motifs was highly effective in inhibiting metastatic tumor growth in mice. These results clearly demonstrate the importance of the regulation of biological responses to plasmid vectors to optimize plasmid-based in vivo gene transfer.
Gene therapy based on ultrasound with microbubbles offers a novel approach for the prevention and treatment of variety of diseases. The major development of gene transfer has importantly contributed to intense investigation of the potential of gene therapy in cancer or cardiovascular medicine. The amazing advances in molecular biology have provided a dramatic improvement of the technology that is necessary to transfer target genes into somatic cells. Gene transfer methods have been surprisingly improved. In fact, some of them (retroviral vectors, adenoviral vectors or liposome based vectors, etc.) have been used in the clinical trials already. But some severe side effects were reported in clinical gene therapy using such viral, so people desire safe and efficient clinical gene therapy. Recently, ultrasound-mediated gene transfer has been reported to augment the transfection efficiency and facilitate local gene expression. Interestingly, gene transfer into the fetal central nervous system was successfully achieved by intrauterine injection with microbubble-enhanced ultrasound. Compared to other viral vectors, there are some theoretical advantages including safety, simplicity of preparation, and local gene transfer. Thus, we focused on the development of gene transfer using naked plasmid DNA with an ultrasound or microbubble-enhanced ultrasound method.
For the development of efficient gene vector, intracellular processes such as cellular uptake, endosomal release and nuclear delivery must be overcome. Viruses have also evolved and have developed sophisticated mechanisms for controlling intracellular trafficking for the efficient delivery of their genomes to nuclei in host cells for symbiosis. In the light of these mechanisms, various kinds of artificial devices have been developed to overcome the intracellular barriers. However, in the majority of studies, variation of the transfection activity before and after the modification of devices was evaluated, and intracellular trafficking remained unclear. Therefore, it is understand to recognize which of the intracellular barrier should be intensively improved to enhance the transfection activity. To clarify the rate-limited process in the current non-viral vector, we compared the intracellular trafficking between adenovirus and LipofectAMINE PLUS. As a result, we found that difference of the transfection efficiency between adenovirus and LipofectAMINE PLUS was dominantly derived from the differences on transcription activity. Therefore it is essential to consider the regulation of the intranuclear events to improve the transfection activity of artificial vector.
This review describes new syntheses of organofluorine compounds taking advantage of the special properties of fluorine compounds as synthones. The main reactions presented are as follows: 1) Trifluoromethylation of aryl or alkyl halides. Application of this reaction for the synthesis of fluorine analogues of nucleic acid bases is discussed. 2) Some syntheses of trifluoromethyl compounds using the Diels-Alder or the 1,3-dipolar reaction, trifluoromethylated carbene, and photoreaction. 3) The Friedel-Crafts reaction of 3,3,3-trifluoropropene, where linear alkylation occurs due to electronic effect of the trifluoromethyl group in good contrast with the Friedel-Crafts reaction of propene. 4) The ene reaction of trifluoromethylated carbonyl compounds, which work as good enophiles. Application of this reaction to the synthesis of trifluoro analogues of terpenes is discussed. 5) The ene reaction of trifluoromethylated imines. 6) Reaction of halothane, which has a highly acidic hydrogen and two halogens adjacent to the trifluoromethyl group and shows interesting reactivities with various bases and metals to give products with unexpected structures. 7) Reaction of 2-bromo-2,2-difluoroacetate with Cu, where the cross-coupling reaction, Michael-type reaction, and radical reaction for different types of difluoroacetates are presented. 8) Reaction of 2-bromo-2,2-difluoroacetate in the presence of Rh catalyst. This reaction provided a new methodology for the introduction of fluoroalkyl substituents to the α-position of α,β-unsaturated ketones. The Rh catalyst solved some difficulties in the introduction of difluoroacetate to carbonyl compounds (Reformatsky reaction). Application of this reaction to imines provided easy access to β,β-difluoro-β-lactams.
Cell proliferation is regulated through a transition between the G0 phase and cell cycle. We isolated a mammalian temperature-sensitive mutant cell line defective in the function from the G0 phase to cell cycle. Senescent human somatic cells fail to enter into the cell cycle from the G0 phase with stimulation by any growth factor. Telomere shortening was found to be a cause of cellular senescence, and reexpression of telomerase immortalized human somatic cells. Immortalized human somatic cells showed normal phenotypes and were useful not only for basic research but also for clinical and applied fields. The importance of p53 and p21 activation/induction i now well accepted in the signal transduction process from telomere shortening to growth arrest, but the precise mechanism is largely unknown as yet. We found that the MAP kinase cascade and histone acetylase have an important role in the signaling process to express p21. Tumor tissues and cells were found to have strong telomerase activity, while most normal somatic human tissues showed very weak or no activity. Telomerase activity was shown to be a good marker for early tumor diagnosis because significant telomerase activity was detected in very early tumors or even in some precancerous tissues compared with adjacent normal tissues. Telomere/telomerase is a candidate target for cancer chemotherapeutics, and an agent that abrogated telomere functions was found to kill tumor cells effectively by inducing apoptosis whereas it showed no effect on the viability of normal cells.
Aging induces a decrease in bone mass, and osteoporosis with its accompanying decrease in bone mass is widely recognized as a major public health problem. Bone loss with increasing age may be due to decreased bone formation and increased bone resorption. Pharmacologic and nutritional factors may prevent bone loss with aging, although chemical compounds in food and plants which act on bone metabolism are poorly understood. We have found that isoflavones (including genistein and daidzein), which are contained in soybeans, have a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption, thereby increasing bone mass. Menaquinone-7, an analogue of vitamin K2 which is abundant in fermented soybeans, has been demonstrated to stimulate osteoblastic bone formation and to inhibit osteoclastic bone resorption. Of various carotenoids, β-cryptoxanthin, which is abundant in Satsuma mandarin (Citrus unchiu MARC), has a stimulatory effect on osteoblastic bone formation and an inhibitory effect on osteoclastic bone resorption. The supplementation of these factors has a preventive effect on bone loss induced by ovariectomy in rats, which are an animal model of osteoporosis, and their intake has been shown to have a stimulatory effect on bone mass in humans. Factors with an anabolic effect on bone metabolism were found in extracts obtained from wasabi leafstalk (Wasabi japonica MATSUM), the marine alga Sargassum horneri, and bee pollen Cistus ladaniferus. Phytocomponent p-hydroxycinnamic acid was also found to have an anabolic effect on bone metabolism. Food chemical factors thus play a role in bone health and may be important in the prevention of bone loss with increasing age.
Sterols are widely and abundantly distributed in nature. It is convenient to utilize them for the preparation of useful compounds such as pharmaceuticals with steroid and secosteroid skeletons. This paper describes the synthesis and structure-activity relationships of naturally occurring active forms of vitamin D analogues, sterols having neurite outgrowth activity, and liver X receptor agonist. The active form of vitamin D4 showed similar biological activities but had higher affinity to the vitamin D-binding protein compared with the corresponding vitamins D2 and D3. This shows that the active form of vitamin D4 is a good candidate for an agent to replace the active forms of vitamins D2 and D3. In the course of screening for low molecular-weight compounds that exhibit neurite outgrowth activity in the culture broth, we found that the natural product dictyosterol showed strong activity. From screening of the analogues, it was found that the double bond between C22 and C23 in the side chain of the sterol is essential for its activity. Ergost-22-ene-1α,3β-diol was found to serve as a stronger liver X receptor agonist than 24(S), 25-epoxycholesterol, which regulates the expression of genes involved in lipid metabolism. Structure-function study showed that the 1α-hydroxyl group, the saturated steroid structure, and the double bond between C22 and C23 are needed to function as a liver X receptor agonist.
Antimicrobial agents sometimes cause the adverse effects of diarrhea and loose stool. Antibiotic-resistant lactic acid bacteria are used to prevent these adverse effects. The bacteria are not resistant to several antimicrobial agents, although the bacterium preparations are sometimes prescribed the antimicrobial agents concomitantly. Therefore this paper reports that the minimal inhibitory concentration of three new antimicrobial agents against antibiotic-resistant lactic acid bacteria were determined using a microdilution method with cation-adjusted Mueller-Hinton broth. Furthermore, we investigated antimicrobial agents that are prescribed concomitantly with antibiotic-resistant lactic acid bacterium preparations or a clostridium butyricum preparation. The bacteria were susceptible to the three new antimicrobial agents. Approximately 50% of the bacterium preparations were prescribed alone, and 30% were prescribed concomitantly with antimicrobial agents that show antimicrobial activity against the bacteria. Consequently, we suggest that pharmacists need to confirm prescriptions and to provide more drug information on antibiotic-resistant lactic acid bacterium preparations.
Because it is well known that drug remains in the fluticasone propionate Diskhaler (FP-DH) following a single inhalation, the following patient information is recommended. “Please inhale more than once or twice if any drug remains in the device after inhalation”. It is believed the inspiratory flow rate of the individual patient has an influence on the amount of arug that remains in the device. If the dosing performance of FP-DH is dependent on inspiratory effort, establishment of a method of inhalation that makes it independent of inspiratory flow rate is important in clinical practice. In the present study, we investigated the influence of various methods of inhalation of drug remaining in the FP-DH. No significant differences were observed regarding the drug remaining in the device among the inhalation times examined (range, 0.5—2.5 s) or the number of inhalations (range, 1—3 times). On the other hand, the amount of drug remaining in the device did decrease by tapping the device before the second inhalation. The results suggest that the amount of drug remaining in the device can be decreased by tapping the device after the first inhalation if the patient's inspiratory flow rate is low.
AIM: The present study was carried out to evaluate the antinociceptive, anti-inflammatory and antipyretic effects of chloroform extract of Solanum nigrum leaves using various animal models. METHODS: The extract was prepared by soaking (1:20; w/v) the air-dried powdered leaves (20 g) in chloroform for 72 hrs followed by evaporation (40°C) under reduced pressure to dryness (1.26 g) and then dissolved (1:50; w/v) in dimethylsulfoxide (DMSO). The supernatant, considered as the stock solution with dose of 200 mg/kg, was diluted using DMSO to 20 and 100 mg/kg, and all doses were administered (s.c.; 10 ml/kg) in mice/rats 30 min prior to tests. RESULTS: The extract exhibited significant (p<0.05) antinociceptive activity when assessed using the abdominal constriction, hot plate and formalin tests. The extract also produced significant (p<0.05) anti-inflammatory and antipyretic activities when assessed using the carrageenan-induced paw edema and brewer's yeast-induced pyrexia tests. Overall, the activities occurred in a dose-independent manner. CONCLUSION: The present study demonstrated that the lipid-soluble extract of S. nigrum leaves possessed antinociceptive, anti-inflammatory and anti-pyretic properties and confirmed the traditional claims.
We evaluated the usefulness of small group discussion using the KJ method (KJ-SGD) in regards to early exposure for first-year pharmaceutical science students. Fourteen students were divided into 3 groups. They were asked to discuss the societal role of pharmaceutical science faculty members and then write the results of their discussion. Thereafter, each of the groups presented the contents of their written results to all of the students. Discussion sessions were held on two separate occasions, the first prior to early exposure and the second after early exposure. After receiving early exposure, the students were asked to revise their previous written results. Further, a questionnaire was given to the students following each discussion session, which revealed that more than 92% understood the KJ-SGD method and most answered that it was useful. In addition, all answered that their perceptions were changed after early exposure. Our findings showed that KJ-SGD was useful and that the students were able to effectively understand the contents following early exposure.
High-performance liquid chromatography was employed to determine the contents of the eight marker components liquiritin, naringin, hesperidin, thymol, imperatorin, honokiol, isoimperatorin, and magnolol in the traditional Chinese medicinal preparation Huoxiang-zhengqi liquid. The separation was performed on a C18 column by stepwise gradient elution with water-methanol-acetonitrile (0.01 min, 68:30:2; 20 min, 60:38:2; 50 min, 34:64:2; 65 min, 34:64:2; 75 min, 28:70:2; 85 min, 68:30:2) as the mobile phase at a flow rate of 1 ml/min, with UV detection at 283 nm. Eight regression equations showed good linear relationships between the peak area ratio of each marker to internal standard and amounts. The recoveries of the markers listed above were 97.4, 98.5, 97.4, 98.6, 97.8, 99.2, 97.0, and 97.5%, respectively. The repeatability and reproducibility (relative standard deviation) of the method were less than 2.2 and 3.0%, respectively.
Our series of studies aimed to examine the possibility of interactions between prescription drugs and over-the-counter (OTC) drugs by monitoring plasma drug concentrations in rats. When a levodopa preparation indicated for patients with Parkinson's disease was administered in combination with Takeda Kampo Ichoyaku K-matsu (A), Taisho Kampo Ichoyaku (B), or Kanebo Kampo Ichoyaku H(C), which are OTC kampo medicines for upset stomach, the plasma levodopa concentration-time curves were shifted downward and the AUC for levodopa was significantly lowered. These results indicate that there may be some interactions between the levodopa preparation and these OTC kampo medicines when ingested together, which leads to a reduction in the bioavailability of levodopa. On the other hand, concomitant administration of the levodopa preparation with Takeda Kampo Ichoyaku A-matsu (D) did not alter any of the pharmacokinetic parameters for levodopa. According to the package inserts for the OTC kampo medicines, A, B and C, but not D, contain metallic additives, such as aluminum silicate and magnesium stearate. In addition, combination with a kampo basis of D (Koshaheiisan-ka-shakuyaku) showed no detectable change in levodopa bioavailability. From these results, it was concluded that metallic additives may play an essential role in generating the drug-interaction between levodopa preparation and OTC kampo medicine for upset stomach.
The present study was carried out to establish the antinociceptive and anti-inflammatory properties of Dicranopteris linearis leaves chloroform extract in experimental animals. The antinociceptive activity was measured using the abdominal constriction, formalin and hot plate tests, while the anti-inflammatory activity was measured using the carrageenan-induced paw edema. The extract, obtained after 72 h soaking of the air-dried leaves in chloroform followed by evaporation under vacuo (40°C) to dryness, was dissolved in dimethyl sulfoxide to the doses of 20, 100 and 200 mg/kg and administered subcutaneously 30 min prior to subjection to the above mentioned assays. The extract, at all doses used, was found to exhibit significant (p<0.05) antinociceptive activity in a dose-dependent manner. However, the significant (p<0.05) anti-inflammatory activity observed occur in a dose-independent manner. As a conclusion, the chloroform extract of D. linearis possesses antinociceptive and anti-inflammatory activity and thus justify its traditional uses by the Malays to treat various ailments.