RNA interference (RNAi) is a posttranscriptional gene-silencing event in which short double-stranded RNA (siRNA) degrades target mRNA. Because of its potent and highly specific gene-silencing effect, RNAi is expected to be used in the treatment of various diseases. Cancer is one of the major targets of RNAi-based therapy, because silencing oncogenes or other genes contributing to tumor progression can be target genes for RNAi. The delivery of RNAi effector to target cells is one of the key factors determining therapeutic efficacy, because gene silencing is limited to cells reached by RNAi effectors. Tumor cell lines stably expressing reporter genes were confirmed to be effective in sensitively and quantitatively evaluating RNAi effects in tumor cells in vitro and in vivo. Quantitative analyses of the gene-silencing effect revealed that short-hairpin RNA expressing plasmid DNA (pshRNA) has more durable effects than siRNA. Intratumoral injection of RNAi effectors was effective in suppressing target gene expression in tumor cells, and silencing of β-catenin or hypoxia-inducible factor-1α (HIF-1α) significantly inhibited tumor growth. RNAi effectors were successfully delivered to tumor cells colonizing the liver through the vascular route. We found that tumor-bearing liver showed elevated HIF-1α expression in the cells, and the silencing of the expression in normal liver cells is also effective in inhibiting metastatic tumor growth. These results indicate the possibility of RNAi-based cancer therapy.
Peritoneal dissemination is one of the most common causes of metastasis from malignancies in the abdominal cavity. However, the treatment of peritoneal dissemination is difficult; patients receiving normal chemotherapy have a 0-1% chance of surviving for 5 years. Milky spots in the greater omentum are considered to facilitate the adhesion and invasion of abdominal free cancer cells, and subsequently lymph node metastasis occurs. Since immune cells such as macrophages and lymphocytes are present in the greater omentum and lymph nodes, the activation of immune cells would be a promising strategy for treatment. Single-stranded oligonucleotides containing CpG dinucleotides (CpG DNA) are recognized by Toll-like receptor-9 on antigen-presenting cells such as macrophages to stimulate Th-1-type immune responses. However, a delivery system for CpG DNA to immune cells is essential to develop effective therapy against peritoneal dissemination. Here we review the pathophysiologic basis of peritoneal dissemination and introduce our approach that employs cationic liposomes as a carrier for CpG DNA as a new approach in the treatment of peritoneal dissemination.
The development of nonviral vectors for the efficient and safe delivery to cells has long been awaited to facilitate gene therapy. Recently, many nonviral vectors modified with cationic lipids, cationic polymers, etc. have been reported. However, those nonviral vectors with cationic materials require improved stability, longer duration of gene expression, and reduced cytotoxicity. We successfully prepared mucoadhesive poly (lactide-co-glycolide) nanospheres (PLGA NS) by modifying the nanoparticulate surface with chitosan to improve mucosal peptide absorption after oral and pulmonary administration. Furthermore, we found that nucleic acid, which was not dispersed in the organic solvent, could be dispersed by forming a complex with cationic lipid. Using this phenomenon, polynucleic acids for gene therapy (plasmid DNA, antisense oligonucleotide, small interfering RNA, etc.) can be encapsulated into the matrix of the polymer particles with the emulsion solvent diffusion method. The advantages of this preparation method are its simple process and avoidance of an ultrasonication process for submicronization of particles. The resultant nanospheres show better cellular uptake and different gene therapeutic effects compared with conventional vectors due to their improved adherence to cells and sustained release of polynucleic acid in the cells. In conclusion, chitosan-coated PLGA NS can possibly be applied in nonviral vectors for gene therapy.
For successful cancer gene therapy via intravenous administration, it is essential to optimize the stability of carriers in the systemic circulation and the cellular association after the accumulation of the carrier in tumor tissue. However, a dilemma exists regarding the use of poly(ethylene glycol) (PEG), which is useful for conferring stability in the systemic circulation, but is undesirable for the cellular uptake and subsequent processes. We report the development of a PEG-peptide-lipid ternary conjugate (PPD). In this strategy, PEG is removed from the carriers via cleavage by a matrix metalloproteinase (MMP), which is specifically expressed in tumor tissues. An in vitro study revealed that the PPD-modified gene carrier (multifunctional envelope-type nano device, MEND) exhibited pDNA expression activity that was dependent on the MMP expression level in the host cells. In vivo studies further revealed that the PPD was potent in stabilizing MEND in the systemic circulation and facilitating tumor accumulation. Moreover, the intravenous administration of PPD or PEG/PPD dually modified MEND resulted in the stimulation of pDNA expression in tumor tissue, as compared with a conventional PEG-modified MEND. Thus MEND modified with PPD is a promising device with the potential to make in vivo cancer gene therapy achievable.
Lupine alkaloids have been studied from the viewpoints of biosynthesis, biotechnology, chemotaxonomy, and biological activity, on the basis of the chemical investigation of the leguminous plants of the 28 species belonging to the 9 genera, which mainly grow in Japan. The results obtained have been comprehensively reviewed by authors. This review describes the stereochemistry of lupine alkaloids and focuses on the conformational flexibility of nitrogen-fused systems such as quinolizidine and indolizidine, syntheses of new unusual types of alkaloids from known lupine alkaloids, and pharmacological activity of lupine alkaloids, especially κ-opioid receptor- mediated antinociceptive effects of matrine-type lupine alkaloids.
Homocysteine, a sulfur amino acid, is an intermediate metabolite of methionine. In 1969, McCully reported autopsy evidence of extensive arterial thrombosis and atherosclerosis in children with elevated plasma homocysteine concentrations and homocystinuria. On the basis of this observation, he proposed that elevated plasma homocysteine (hyperhomocysteinemia) can cause atherosclerotic vascular disease. Hyperhomocysteinemia is now well established as an independent risk factor for atherosclerotic vascular disease. Mild hyperhomocysteinemia is quite prevalent in the general population. It can be caused by genetic defects in the enzymes involved in homocysteine metabolism or nutritional deficiencies in vitamin cofactors, certain medications or renal disease. An increase of 5 μmol per liter in the plasma homocysteine concentration raises the risk of coronary artery disease by as much as an increase of 20 mg per deciliter in the cholesterol concentration. In this article, we review the biochemical, experimental and clinical studies on hyperhomocysteinemia, with emphasis on the metabolism and pharmacokinetics of homocysteine.
In order to quality control for medicinal plants such as Aconitum charmicaelii, Rhemannia glutinosa, Atractyrodes lancea, Pinellia ternata, Panax species and Gentiana scabra were clonally micropropagated by tip tissue culture and embryogenetic techniques. Monoclonal antibodies against the bioactive compounds contained in the crude drugs were prepared and set up the ELISA as a high sensitive and quick determination method. A newly developed eastern blotting methodology can stain typically the only antigen molecule and the related compounds having a same aglycone. Knockout extract can be prepared by using an immunoaffinity column conjugated with monoclonal antibody, and its importance has been discussed. Single chain fragment-variable gene against solamargine was cloned and transformed to a host plant, Solanum chasianum resulting in the increase of antigen molecule as 2.5 to 3 times. Three biosynthetic enzymes regarding marihuana compounds, THCA-, CBDA-, CBCA-synthases were isolated. THCAsynthase was cloned, over expressed and confirmed its characteristics including FAD combining enzyme, and finally determined the structure by X-ray analysis. The distribution of THCAsynthase was also investigated using its and GFP hybrid gene. We found new functions for saffron like improving learning and memory and LTP for blocking by ethanol. A folk medicine in Taiwan, Anoectochilus formosanus was propagated in vitro and investigated opening new pharmacological activities in lipid methabolism.
This review starts with an introduction of derivative spectrophotometry followed by a description on the construction of a personal computer-assisted derivative spectrophotometric (DS) system. An acquisition system for inputting digitalized absorption spectra into personal computers and a BASIC program for calculating derivative spectra were developed. Then, applications of the system to drug analyses that are difficult with traditional absorption methods are described. Following this, studies on the interactions of drugs with biological macromolecules by the DS and NMR methods were discussed. An 1H NMR study elucidated that the small unilamellar vesicle (SUV) has a single membrane made of a phosphatidylcholine bilayer, and that chlorpromazine interacts with both the outer and inner layers. 13C NMR revealed a reduction of the dissociation constants of phenothiazine drugs due to their interaction with SUV. The partition coefficients of phenothiazine, benzodiazepine and steroid drugs in an SUV-water system and the effects of cholesterol or amino lipids content on these partition coefficients were examined by the DS method. The binding constants of phenothiazine drugs to bovine serum albumin (BSA) and the influence of Na+, K+, Cl-, Br-, and I- on these binding constants were determined by DS. It was found that I-, Br-, Cl- reduce the binding constants in this order, and that Na+ and K+ have no effect. A 19F NMR study revealed that triflupromazine binds to BSA and human serum albumin in two regions including Site II with different populations, and that a nonsteroidal anti-inflammatory drug, niflumic acid, binds Sites Ia and Ib.
This review focuses on two pharmacologic approaches to the functional evaluation of new target molecules for drug discovery. One is the development of a novel specific antagonist of the Na+-Ca++ exchanger (NCX) SEA0400. The other is a comprehensive analysis of the functions of pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide ligand for G protein-coupled receptors. NCX is the one of the last target molecules regulating the cellular Ca++ concentration. There was no efficient way to address the pathophysiologic roles of NCX until a specific antagonist, 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400), was developed. Our recent studies using SEA0400 clearly showed the possible roles of NCX in several pathologic states of cardiovascular and nervous tissues. In our second approach including gene-targeting methods, we found new, unexpected roles of PACAP in higher brain functions, such as psychomotor, cognition, photoentrainment, and nociception. Based on these experimental findings, a genetic association study in schizophrenia patients revealed that the single-nucleotide polymorphisms of the PACAP gene are significantly associated with the hypofunction of the hippocampus. Regarding the peripheral roles of PACAP, we found that PACAP is involved not only in the regulation of insulin secretion in pancreatic islets, but also in the regulation of islet turnover. In subsequent phenotypic analysis of PACAP transgenic mice, we identified novel candidate genes that probably have promising functional roles.
This review introduces a new concept “Programmed Packaging” to develop a non-viral gene delivery system. Based on this concept, multifunctional envelope type nano devices (MEND) were developed for in vitro, in situ and in vivo conditions. A quantitative study to identify a rate limiting step in intracellular trafficking was also shown between viral and non-viral vectors, which indicated an important role of controlled intranuclear disposition for development a safe and efficient non-viral gene delivery system. This review will provide a future direction of non-viral gene delivery system.
Structural biology teaches us about the fundamental nature of biological molecules. Drug design is the most immediate medical application of structural biology. Therefore our studies have been focused on structural and functional studies of human disease-related proteins and proteins essential for the growth and development of pathogenic organisms. The present paper describes 1) structural biological studies of human autocrine motility factor, 2) structural biological studies of human ribonuclease L, and 3) structural biological studies of Plasmodium falciparum S-adenosyl- L-homocysteine hydrolase.
For efficient gene delivery to the nucleus, nonviral vectors need to overcome several barriers such as the plasma membrane, endosomal membrane, and nuclear membrane. To overcome these obstacles, it is necessary to equip the delivery system with various functional devices. However, it is difficult to package all such functional devices into a single system to exert each of their functions at the appropriate time and at the correct location. Thus our group proposed a new packaging concept, “programmed packaging.” A multifunctional envelope-type nano device (MEND) was developed for use as an efficient nonviral system for the delivery of plasmid DNA (pDNA), oligodeoxynucleotide (ODN), and siRNA using octaarginine (R8) as an internalizing ligand based on the programmed packaging. The R8-modified MEND (R8-MEND) encapsulating pDNA showed significantly high transfection activity comparable to that of adenovirus, and the uptake pathway of R8-MEND was macropinocytosis, which can avoid lysosomal degradation. R8-MEND successfully delivered gene to hair follicles after in vivo topical application to mouse skin. Moreover, R8-MEND encapsulating anti-luciferase ODN using protamine showed a 90% antisense effect, and R8-MEND encapsulating siRNA condensed with stearylated R8 significantly silenced luciferase activity. Our group thus succeeded in the development of R8-MEND based on programmed packaging, and MEND is a promising new delivery system for pDNA and functional nucleic acids.
Photoaffinity labeling enables the direct probing of a target protein through a covalent bond between a ligand and its binding protein. We used carbene-generating phenyldiazirine as a photophore because practical examinations had shown that the phenyldiazirine functioned as the powerful barb on the hook. This review describes improvements of synthetic strategies of the photoaffinity ligands bearing diazirine. First, we dramatically improved the direct formylation of phenyldiazirine, which was a practical diazirine source, to obtain a versatile diazirine unit. Second, we established “the photoreactive unit technique” for a one-step introduction of phenyldiazirine into peptides, proteins, DNAs, and sugars. Since the photoreactive units can be easily integrated into physiological ligands by chemoselective reaction, the biochemists, who are not familiar with organic synthesis, can prepare the photoaffinity ligands using their interested ligands. Our improvements would promote the utilization of phenyldiazirine for analyzing biological interfaces, and extend the potential of photoaffinity labeling as a sensitive means of rapidly elucidating protein structures and proteomic profiling.
The present study was aimed at investigating the effects of Anwala churna (Emblica officinalis GAERTN.), an Ayurvedic preparation, on memory in rats. Anwala churna was administered orally in three doses (50, 100 and 200 mg/kg) for 15 days to different groups of young and aged rats. The elevated plus-maze and Hebb-Williams maze served as exteroceptive behavioral models for testing memory. Diazepam-, scopolamine-, and ageing induced amnesia served as the interoceptive behavioral models. Anwala churna (50, 100, and 200 mg/kg, p.o.) produced a dose-dependent improvement in memory scores of young and aged rats. Furthermore, it reversed the amnesia induced by scopolamine (0.4 mg/kg, i.p.) and diazepam (1 mg/kg, i.p.). Based on these results, Anwala churna may prove to be a useful remedy for the management of Alzheimer's disease due to its multifarious beneficial effects such as memory improvement and reversal of memory deficits.
The aim of this work was to study the pharmacokinetics of piperaquine in healthy volunteers. Healthy volunteers received piperaquine and tablets of Artekin by oral administration. The plasma samples were analyzed for piperaquine by liquid-liquid extraction and determined by HPLC-UV. The results demonstrated that the plasma drug concentration-time curves of single and multiple dose of piperaquine were fitted to a two-compartment open model. The pharmacokinetics parameters of piperaquine alone in a single dose were: t1/2(β)=(317.2-/+126.6)h, AUC0→∞=(44293-/+12636)h×ng/ml, Vd=(9490.9-/+2161.9)ml/kg, and Cl=(22.83-/+9.83)ml/h/kg. In Artekin in a single dose these parameters were: t1/2(β)=(302.8-/+180.7)h, AUC0→∞=(46419-/+13670)h×ng/ml, Vd=(10188.6-/+3520.3)ml/kg, and Cl=(25.48-/+10.89)ml/h/kg, while in Artekin in multiple doses they were: t1/2(β)=(298.9-/+101.9)h, AUC0→∞=(227692-/+56294)h×ng/ml, Vd=(5031.5-/+1097.8)ml/kg, Cl=(11.91-/+3.046)ml/h/kg, respectively. The absorption and distribution of piperaquine were quick while the elimination was quite slow. There were significant differences in the pharmacokinetics parameters of piperaquine in Artekin between a single dose and multiple doses (p<0.001), suggesting that piperaquine might accumulate in vivo and that attention should be given to its possible adverse drug reactions in clinical treatment.
Cortex Lycii Radicis (CLR) has been used as a traditional Oriental medicine as an antipyretic and to treat pneumonia, night-sweats, cough, hematemesis, inflammation, and diabetes mellitus for centuries. This study aimed to determine the effects of CLR on alloxan-induced diabetic mice and its mechanisms. Based on thin-layer chromatography (TLC) assay, the main compounds of CLR include an organic acid, flavone, alkaloid, polysaccharide, anthraquinone, and saponin. The mice were divided into four groups: normal control (NC), diabetes control (DC), diabetes+high-dose CLR (200 mg kg-1), and diabetes+low-dose CLR (100 mg kg-1). The diabetic mice were administered CLR daily for 28 days. The CLR treatment resulted in significant decreases in fasting blood glucose, total cholesterol, and triglycerides. CLR also showed a tendency to improve body weight gain in diabetic mice. Furthermore, the serum insulin level of each group was assayed, and the DC group had a lower serum insulin level than the NC group. Insulin levels were dose dependently raised in the CLR-treated groups compared with the DC group. According to single-cell gel electrophoresis and LD50 analysis, CLR was nontoxic to the animals. The results indicate that CLR alleviates the blood glucose and lipid increases associated with diabetes and improves the abnormal glucose metabolism and increases insulin secretion by restoring impaired pancrease βcells in alloxan-induced diabetic mice. The results suggest that CLR has hypoglycemic potential and could be useful in diabetes therapy.
The estimation of paracetamol and orphenadrine citrate in a multicomponent pharmaceutical dosage form by spectrophotometric method has been reported. Because of highly interference in the spectra and the presence of non-linearity caused by the analyte concentrations which deviate from Beer and Lambert's law, partial least-squares (PLS) and artificial neural networks (ANN) techniques were used for the calibration. A validation set of spiked samples was employed for testing the accuracy and precision of the methods. Reasonably good recoveries were obtained with PLS for paracetamol and the use of an ANN allowed the estimation of orphenadrine citrate, a minor component which could not be adequately modeled by PLS. Three production batches of a commercial sample were analysed, and there was statistically no significant difference (P<0.05) between the results with the proposed method and those obtain with the official comparative method.
The minimental state examination (MMSE) is a widely used, standardized method to assess cognitive function including movement-related disorders with high reliability. We studied the relationship between MMSE scores and the ability to take oral medications correctly (ingestion compliance) in 70 elderly inpatients (mean age 71.3±7.0 years). Patients with abnormal glucose tolerance as determined by an HbA1c level of 5.8% or greater including diabetes showed a trend of lower MMSE scores compared with patients with normal glucose tolerance, and the scores were negatively correlated with HbA1c, age, and systolic blood pressure (P<0.05). Self-management in taking oral medications was very difficult in 4 patients whose MMSE scores were 21 points or less. Thus ingestion supervisions by nurses were required in these patients. Furthermore, 9 of 12 noncompliant patients had MMSE scores ranging from 22 to 26 points. We instructed these patients to take medications in a one-dose package as a useful tool to improve compliance. The MMSE score was 27 or higher in 44 of 54 compliant patients, and 10 patients had scores ranging from 21 to 26. The sensitivity and specificity for noncompliance at an MMSE score cut-off point of 26 were 75.0% and 81.5%, respectively. In conclusion, it is necessary to coordinate ingestion methods matched to each patient according to their abilities to comply with medication schedules. They should be preevaluated with the MMSE to improve ingestion compliance. The MMSE is a recommended test in hospitalized elderly patients for the assessment of the ability to take medications safely.
In vitro transcorneal permeation of diclofenac from oil drops was studied using freshly excised goat cornea.The maximum apparent corneal permeability coefficient (Papp) was obtained with 0.2% (w/v) diclofenac drops in sesame oil followed by safflower oil, while formulation in castor oil provided minimal Papp. The addition of benzyl alcohol, a preservative, in oil drops, increased the Papp value of diclofenac. Partition experiments indicated increased partitioning of diclofenac in the aqueous phase in the presence of benzyl alcohol, and the same could be responsible for the benzyl alcohol-induced increase in Papp. The solubility of diclofenac was higher in castor, arachis, and sunflower oil. But drug permeation from 0.5-1.0% (w/v) diclofenac drops in castor oil or 0.5% (w/v) drops in arachis /sunflower oil was less than that observed with 0.2% (w/v) drops in sesame oil. Thus diclofenac 0.2% (w/v) drops in sesame oil containing 0.5% (v/v) benzyl alcohol provides maximum Papp. The formulation increased corneal hydration indicating corneal damage. Since corneal hydration is less than 83% the damage appears to be reversible. The saturation solubility of diclofenac in sesame oil at 4°C is 0.33% (w/v). Hence diclofenac 0.2% (w/v) solution in sesame oil will not precipitate at 4°C and therefore the chances of crystallization of diclofenac from the formulation due to climatic change leading to physical instability appear to be remote.
The secondary and adverse effects when biguanides, alpha-glycosidase inhibitor or thiazolidine derivative was used with sulphonylurea agent (SU) as compared with those with SU alone in Type 2 diabetes patients by using Systematic Review. Two-agent concurrent treatment groups, taken from studies in which subjects were assigned to a group given only a sulfonylurea agent and a group given a sulfonylurea agent with the other glycemic control agent (combination of a sulfonylurea agent and a biguanide agent (I), combination of a sulfonylurea agent and an α-glucosidase inhibitor (II), and combination of a sulfonylurea agent and thiazolidinedione (III)), were studied in a randomized controlled trial. The secondary efficacy outcome measures were total cholesterol (TC), triglyceride (TG), HDL-C, LDL-C, and change in body weight. The incidence of hypoglycemia, feeling of fullness, diarrhea, liver dysfunction, and edema was investigated as a safety outcome measure, and the clinical significance of concurrent treatment with a sulfonylurea agent in addition to the other glycemic control agent was investigated. With respect to (II), an antidiabetic effect was showed. As for (III), it had the disadvantage of increased body weight. Furthermore, increase of HDL-C levels, in particular, was observed. The improving effect of (III) on serum lipids may be clinically effective for considering the pathologic condition of diabetes, which is often complicated by hyperlipidemia.
The search for newer non-steroidal antiinflammatory drugs (NSAIDs) and the importance of oxadiazoles as antiinflammatory agents prompted us to undertake the synthesis of some novel oxadiazole and related analogues with unreported antiinflammatory activities. The antiinflammatory potential of the compounds was investigated using the carrageenan-induced rat paw edema method and cotton pellet-induced granuloma method. Some compounds demonstrated marked antiinflammatory activities. The antiinflammatory activity of oxadiazoles at doses of 100 mg/kg was shown by their ability to provide 28-55%, 21-36%, and 27-49% protection against carrageenan-induced rat paw edema, moist cotton pellet-induced, and dry cotton pellet-induced granuloma, respectively. On the other hand, the antiinflammatory properties of oxadiazolines at doses of 100 mg/kg was reflected by their ability to provide 15-47%, 22-39%, and 23-47% protection against carrageenan-induced rat paw edema, moist cotton pellet-induced, and dry cotton pellet-induced granuloma, respectively. Structure-activity relationships among synthesized compounds were also studied.