Endothelin (ET) has been implicated in the pathogenesis of several cardiovascular disorders because of its powerful vasoconstrictor and growth-promoting properties. The ET family consists of three isoforms, ET-1, ET-2 and ET-3. ET-1 appears to be the predominant member of the family generated by vascular endothelial cells. In view of the multiple cardiovascular actions of ET-1, there has been much interest in its contribution to the pathophysiology of hypertension and arteriosclerosis. We have been investigating the roles of ETA and ETB receptors in ET-1-related cardiovascular diseases using subtype-selective ET receptor antagonists and ETB receptor-deficient animals. Our studies have demonstrated that ET-1 overproduction and ETA-mediated ET-1 actions seem to play a crucial role in the development of several types of hypertensive and post-ischemic diseases. On the other hand, ET-1 biosynthesis and release are regulated at the transcriptional level, and various endogenous substances are known to stimulate ET-1 gene expression by DNA binding of transcription factors. We and others have recently demonstrated that nuclear factor-κB (NF-κB), a transcription factor with a pivotal role in inducing genes involved in immune, inflammatory and stress responses, is responsible for endothelial ET-1 production. In in vivo studies, agents that can inhibit the NF-κB activation improved the development of ET-1-related cardiovascular diseases. Thus, NF-κB inhibition may be a pertinent treatment for ET-1 related diseases.
Recent clinical studies have indicated the utility of mineralocorticoid receptor (MR) antagonists in cardiovascular and renal injuries. Chronic treatment with aldosterone/salt resulted in severe cardiac and renal injuries in rats. Further studies showed that the aldosterone-induced organ injuries were associated with increases in expression of NADPH oxidase components and reactive oxygen species (ROS) levels. Treatment with a selective MR antagonist, eplerenone, prevented the elevation of ROS levels and ameliorated organ injuries. In vitro studies also showed that MR is highly expressed in cultured vascular smooth muscle cells, glomerular mesangial cells and renal fibroblasts. In these cells, aldosterone-induced cell injuries were associated with increases in NADPH oxidase activity and superoxide generation. Further, the aldosterone-dependent cell injuries were markedly attenuated by treatment with eplerenone. These accumulating data support the notion that the aldosterone/MR is involved in the pathogenesis of cardiovascular and renal injuries through NADPH oxidase-dependent ROS production.
Although the pro-inflammatory and pro-fibrotic actions of aldosterone on the vasculature have been reported, the effects and molecular mechanisms of aldosterone on endothelial function are yet to be determined. We investigated how aldosterone regulates endothelial nitric oxide synthase (eNOS) function in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated for 16 hrs with 10-7 mol/l of aldosterone. The concentration of reactive oxygen species (ROS) was estimated by measuring DCF chemiluminescence. Signal transduction was estimated by Western immunoblots. Realtime RT-PCR was performed to measure expression of transcripts of endogenous GTP cyclohydrolase-1 (GCH1) and components of NAD(P)H oxidase. In order to eliminate the possible effect of the glucocorticoid receptor (GR), and to emphasize the role of mineralocorticoid receptor (MR), we used GR siRNA and knocked down GR expression in several experiments. NO output was estimated by intracellular cGMP concentration. ROS production increased significantly in aldosterone-treated HUVEC, but was abolished by pre-treatment with eplerenone. Transcripts of p47phox were increased by aldosterone treatment. Vascular endothelial growth factor (VEGF)-induced eNOS Ser 1177 but not Akt Ser 473 phosphorylation levels were reduced significantly by pretreatment with aldosterone. Pretreatment with either eplerenone or okadaic acid restored phosphorylation levels of eNOS Ser 1177 in aldosterone-treated cells, suggesting that protein phosphatase (PP) 2A was upregulated by aldosterone via MR. The decrease in NO output caused by aldosterone pretreatment was reversed significantly by either 5,6,7,8-tetrahydrobiopterin (BH4), GCH1 overexpression, or p47phox knockdown. These results suggest that aldosterone inhibits eNOS function through bimodal mechanisms of BH4 deficiency and PP2A activation.
The nitric oxide (NO) synthases (NOSs) system consists of three different isoforms, including neuronal (nNOS), inducible (iNOS), and endothelial NOSs (eNOS). The roles of NO in vivo have been extensively investigated in pharmacological studies with NOS inhibitors and in studies with mice lacking each NOS isoform. However, in the pharmacological studies, the specificity of NOS inhibitors continues to be an issue of debate, while in the studies with mice lacking each NOS isoform, compensatory mechanism by other NOSs appears to be involved. Thus, the ultimate roles of endogenous NO in our body still remain to be fully elucidated. To address this important issue, we have successfully developed mice in which all three NOS genes are completely disrupted. NOS expression and activities were totally absent in the triply n/i/eNOS-/- mice before and after treatment with lipopolysaccharide. While the triply n/i/eNOS-/- mice were viable, their survival and fertility rates were markedly reduced as compared with wild-type mice. The first noticeable phenotypes were polyuria, polydipsia, and renal unresponsiveness to vasopressin, characteristics consistent with nephrogenic diabetes insipidus. We subsequently observed that in those mice, arteriosclerosis is spontaneously developed with a clustering of cardiovascular risk factors. These results provide the first evidence that genetic disruption of all three NOSs causes a variety of cardiovascular diseases in mice in vivo, demonstrating the critical role of the endogenous NOSs system in maintaining cardiovascular homeostasis.
Angiotensin II (Ang II) signaling is mediated by two receptor subtypes, type 1 (AT1) and type 2 (AT2). The activation of AT1 receptors is responsible for the development of Ang II-dependent hypertension, whereas the activation of AT2 receptor is thought to play a counter-regulatory protective role in the regulation of blood pressure that opposes the AT1 receptor-mediated vasoconstriction. However, the precise mechanisms by which increased numbers of AT2 receptors counterbalance the AT1-mediated actions of Ang II are unknown. We have demonstrated that the abdominal aortic banding in mice and rats and the 2-kidney, 1-clip Goldblatt model of hypertension in mice induces up-regulation of AT2 receptors in the pressure-overloaded thoracic aorta. In these hypertensive animals, the AT1-receptor antagonists but not calcium antagonist abolish up-regulation of the aortic AT2 receptor as well as blood pressure elevation, suggesting that the pressure-overload up-regulates the aortic AT2 receptor by Ang II via the activation of AT1 receptor. Ang II binding to up-regulated AT2 receptors induces vasodilation in these aortas through bradykinin B2-receptor-mediated phosphorylation of endothelial nitric oxide synthase (eNOS) at Ser633 and Ser1177 via a protein kinase A-dependent signaling pathway, resulting in sustained production of nitric oxide. These studies provide evidence that the vascular AT2 receptor is up-regulated in the course of hypertension through the activation of AT1 receptor, thereby activating a vasodilatory pathway in vessels through the AT2 receptor via the bradykinin/nitric oxide/cGMP. This issue is important because the antihypertensive effect of AT1-receptor blockers is, at least in part, dependent on AT2-receptor activation.
The Myxomycetes (true slime molds) are an unusual group of primitive organisms that may be assigned to one of the lowest classes of eukaryotes. As their fruit bodies are very small and it is very difficult to collect much quantity, few studies have been made on the chemistry of myxomycetes. We studied spore germination experiments of hundreds of field-collected myxomycetes collected in Japan, and succeeded in laboratory culture of plasmodia of several myxomycetes in a practical scale for natural products chemistry studies. Pyrroloiminoquinones, polyene yellow pigments, and a peptide lactone were isolated from cultured plasmodia of myxomycetes, while new naphthoquinone pigments, cycloanthranilylprolines, tyrosine-kinase inhibitory bisindoles, a cytotoxic triterpenoid aldehyde lactone, a dibenzofuran glycoside, and sterols possessing an unprecedented 2,6-dioxabicyclo[2.2.2] octan-3-one ring system, were also isolated from field-collected fruit bodies of myxomycetes.
Two new catalysts, ruthenium hydride with a nitrogen-containing heterocyclic carbene (A) and an organopalladium catalyst supported on a sulfur-terminated semi-conductor, gallium arsenide (001) (B) were discovered. Both catalysts are environmentally benign, because A can yield indole derivatives with good atom economy, and B can catalyze the Mizoroki-Heck reaction more than 10 times with only trace amounts of leached palladium (ppb level). Substituted 1,2-dihydroquinoline, indole and 3-methylene-2,3-dihydroindole were also prepared selectively from the common starting material, N-allyl-o-vinylaniline, and catalyst by slight modification of the reaction conditions. These procedures address an important issue in diversity-oriented synthesis. These methods utility were demonstrated by application to biologically active natural products synthesis.
Development of new synthetic reactions that feature a tandem process triggered by Brook rearrangement, a C-to-O 1,2-anionic shift of a silyl group, will be discussed. A basic motif for the strategy is the generation of an α-siloxy carbanion by the reaction of acylsilanes with ketone enolates and then trapping the anions by intra- and inter-molecular electrophiles. For example, the reaction of benzoyltrimethylsilane with lithium enolates of methyl ketones produced 1,2-cyclopropanediols via Brook rearrangement of the initial 1,2-adduct and subsequent internal aldol reaction. This concept was applied to the synthesis of five- and seven-membered carbocycles using the reaction of acryloylsilanes with enolates of alkyl and alkenyl methyl ketones, respectively. Furthermore, we found that the use of enolate of 2-cycloheptenone instead of the enolates of alkenyl methyl ketone as the four-carbon unit in the [3+4] annulation produces bicyclo[3.3.2]- decenone derivatives, in which the two-atom internal tether could be cleaved to give the cis-3,4,8-trisubstituted cyclooctenone enol silyl ethers stereoselectively. The α-siloxy carbanions can be also generated by an γ-anion-induced ring cleavage of α,β-epoxysilanes. Thus, O-silyl cyanohydrins of β-silyl-α,β-epoxyaldehyde can function as a highly functionalized homoenolate equivalent via a tandem sequence involving base-promoted ring opening, Brook rearrangement, and alkylation at the allylic position. Based on these results, we developed several new synthetically useful reactions in which three methods for the generation of a carbanion at the γ-position, i.e., deprotonation, reaction of acylsilanes with a nucleophile followed by Brook rearrangement, and a conjugate addition of a nucleophile to an enoate system bearing an epoxysilane moiety at the α-position, were used.
Drug delivery systems (DDS) using liposomes as drug carriers for targeting to macrophages have been developed for the treatment of diseases that macrophages are related to their progress. Initially, DDS for the treatment of atherosclerosis are described. The influence of particle size on the drug delivery to atherosclerotic lesions that macrophages are richly present and antiatherosclerotic effects following intravenous administration of liposomes containing dexamethasone (DXM-liposomes) was investigated in atherogenic mice. Both the drug delivery efficacy of DXM-liposomes (particle size, 200 nm) to atherosclerotic lesions and their antiatherosclerotic effects were greater than those of 70 and 500 nm. These results indicate that there is an optimal particle size for drug delivery to atherosclerotic lesions. DDS for the treatment of respiratory infections are then described. The influence of particle size and surface mannosylation on the drug delivery to alveolar macrophages (AMs) and antibacterial effects following pulmonary administration of liposomes containing ciprofloxacin (CPFX-liposomes) was investigated in rats. The drug delivery efficacy of CPFX-liposomes to AMs was particle size-dependent over the range 100-1000 nm and then became constant at over 1000 nm. These results indicate that the most effective size is 1000 nm. Both the drug delivery efficacy of mannosylated CPFX-liposomes (particle size, 1000 nm) to AMs and their antibacterial effects were significantly greater than those of unmodified CPFX-liposomes. These results indicate that the surface mannosylation is useful method for drug delivery to AMs. This review provides useful information to help in the development of novel pharmaceutical formulations aimed at drug targeting to macrophages.
Cellular slime molds are thought to be excellent model organisms for the study of cell and developmental biology because of their simple pattern of development. However, there have been few reports on secondary metabolites of them. We have focused on the utility of cellular slime molds as novel resources for natural product chemistry, and have studied the diversity of secondary metabolites produced by them as well as their physiological and pharmacological activities. We have recently isolated many novel compounds from the fruiting bodies of various species of Dictyostelium cellular slime molds. Total syntheses and biological evaluation of these compounds have been carried out. It was shown that dictyopyrones and dictyomedins may regulate Dictyostelium development. Amino sugar derivatives such as furanodictines and dictyoglucosamines induced neuronal differentiation of rat PC-12 cells. In addition, brefelamide inhibited the cellular proliferation of 1321N1 human astrocytoma cells. These results show that cellular slime molds are promising sources in natural product chemistry.
Clinical pharmacists now have many jobs in a hospital, including dispensing services, drug information, drug counseling for patients, therapeutic drug monitoring (TDM), and clinical research coordinating. These are important and essential jobs for a hospital pharmacist in his drug profession. During the performance of pharmaceutical services for patients and physicians, clinical pharmacists have an opportunity to change the medicine of a prescription. In this review, we outline the new developments in TDM and pharmacoeconomics achieved by pharmaceutical care at Hiroshima University Hospital. Pharmaceutical care is the professional practice in which the pharmacist takes charge of the needs of a patient related to his/her drugs. One of the aims was to investigate drug induced interaction in pharmacotherapy, and another was evaluation of the clinical efficacy of Bayesian analysis in detoxication treatments of overdoses using TDM. Pharmacoeconomics is an important aspect of the practice of pharmaceutical care. This review investigated the daily cost of ophthalmic solutions used in treating glaucoma and allergic conjunctivitis in Japan. It also summarizes the recent practice of pharmaceutical care in pharmacotherapy and pharmacoeconomics.
We investigated whether the deleterious side effects of chemotherapeutic agents on the physiologic functions of fish could be modulated by lactoferrin (LF). Goldfish, weighing about 25 g, were treated intramuscularly with methotrexate (MTX: 2.5 mg/kg body weight) and fluorouracil (FU: 15 or 50 mg/kg body weight) three times every other day. In control fish fed a commercial diet, MTX induced severe immunosuppression, increased the number of total bacteria and Enterobacteriaceae in the intestinal tract, and caused intestinal damage such as lowered and thickened mucosa and thinned muscularis externa, with moderate renal dysfunction. A few fish treated with MTX died. In fish injected with FU or FU plus MTX, the side effects were slightly less in comparison with those in the MTX group. Pretreatment with LF (oral administration at 200 mg/kg body weight/day) for 3 weeks reduced the deleterious side effects of MTX and FU. One intraperitoneal injection of LF (200 mg/kg body weight) immediately after the first MTX injection also reduced the side effects. These results show that LF reduces the physiologic dysfunction of fish treated with chemotherapeutic agents.
Various nutritional supplements have become available in recent years. However, health problems resulting from the misuse of these supplements are on the rise, and have been attributed to a lack of knowledge among consumers. In addition, a survey of university students revealed that approximately 20% of students erroneously considered nutritionally balanced supplements as substitutes for meals. Given this background, we conducted a questionnaire survey of first- and fourth-year students at the Faculty of Pharmaceutical Sciences at Kobe Gakuin University with the objective of elucidating factors such as the awareness of supplements among pharmacy students and whether these students had a superior understanding of supplements compared to the general student population. Awareness of supplements among students was determined in terms of the degrees of emphasis on meals and supplements in nutritional intake. The proportion of students who essentially believed that “nutritionally balanced supplements can be used as substitutes for meals” did not significantly differ between pharmacy students and the general student population. In addition, only 30% of students had an accurate understanding of supplements. Following graduation, pharmacy students may become pharmacists and thus be responsible for providing directions regarding usage of supplements. These findings suggest that in order to nurture professional pharmacists, it is necessary to first implement practical nutrition education and consumer education to promote healthier dietary habits among the students themselves.
A novel two-step release system for the traditional Chinese medicine compound Danshen was developed by combining an effervescent osmotic pump tablet (EOPT) and a pulsed-released tablet (PT) of compound Danshen into one hard capsule. The EOPT of Danshen was prepared with sodium chloride, mannitol, hydroxypropylmethylcellulose (HPMC), and sodium bicarbonate as osmotic agents. The osmotic pressure from EOPT was greatly enhanced by carbon dioxide generated from the reaction between sodium bicarbonate and acidic components from Danshen. It was shown that the tested Danshen components could be completely released from the prepared EOPT following a zero-order release for up to 12 h. The PT of compound Danshen was a three-layer coated tablet composed of organic acid and osmotic agents. Eudragit RL, HPMC and the mixture of EC and Eudragit RS, RL were the major constituents of the separation layer, swelling layer and controlling release membrane, respectively. The swelling test of the PT indicated that swelling is a prerequisite for drug release from this PT device. In addition, the swelling behavior further suggested the drug release mechanism of PT involves diffusion, the osmotic pumping effect, and organic acid-induced effect, among which the osmotic pumping effect was the most important. Moreover, there was no significant difference among the five active constituents in their release profiles from the final combined two-step release system of compound Danshen.
A simple and sensitive high performance liquid chromatography method with UV detection was described for the determination of colchicine (COL) in mouse plasma. After single-step deproteinization by acetonitrile using berberine hydrochloride as an internal standard (I.S.), solutes were separated on a Diamonsil C18 column (250 mm×4.6 mm I.D., 5 μm particle size) (Dikma), using acetonitrile-0.15% phosphoric acid solution (27:73, v/v) as mobile phase (flow-rate 1.0 ml/min); wavelength of the UV detector was set at 350 nm. No interference from any endogenous substances was observed during the elution of COL and internal standard (I.S., berberine hydrochloride). The retention times for COL and I.S. were 11.23 min and 8.82 min, respectively. The limit of quantification was evaluated to be 1.5 ng/ ml and the limit of detection was 0.5 ng/ml. The method was used in the study of pharmacokinetics of COL after intravenous injection (i.v.) and intraperitoneal injection (i.p.). The result indicated that COL disappears from the plasma according to a three compartment open model.
Osteoporosis is a common adverse reaction induced by glucocorticoid treatment. Bisphosphonate, vitamin D3 (VD3) or vitamin K2 (VK2) is recommended as first or second choice of drug for treatment of glucocorticoid-induced osteoporosis. In the present study, the treatment effect of risedronate against glucocorticoid-induced osteoporosis in rheumatoid arthritic patients was compared with that of alfacalcidol. Twelve patients were randomized to receive either risedronate (2.5 mg) or alfacalcidol (0.5 μg) daily for 48 weeks. Each patient also received 800 mg of calcium supplementation (800 mg/day) daily. Bone mineral density (BMD) and the biochemical markers of bone turnover were measured before (baseline) and 12, 24, and 48 weeks after treatment with risedronate or alfacalcidol, and the percentage changes in these parameters from baseline were compared.The BMD values 12, 24 and 48 weeks after treatment with risedronate increased by 3.9%, 4.1% and 5.2%, respectively, which were significantly higher than those after treatment with alfacalcidol (2.8%, 2.1% and 2.5%, respectively). Urinary excretion of N-telopeptides of type I collagen and deoxypyridinoline after risedronate treatment were more significantly decreased than that after alfacalcidol treatment. The present findings at least suggest that risedronate is more useful for the prevention and treatment of glucocorticoid-induced osteoporosis in patients with rheumatoid arthritis than alfacalcidol, although the number of patients studied was small.
RGD conjugation liposomes (RGD-liposomes) were evaluated for brain-targeting drug delivery. The flow cytometric in vitro study demonstrated that RGD-liposomes could bind to monocytes and neutrophils effectively. Ferulic acid (4-hydroxy-3-methoxycinnamic, FA) was loaded into liposomes. Rats were subjected to intrastriatal microinjections of 100 units of human recombinant IL-1β to produce brain inflammation and caudal vein injection of three formulations (FA solution, FA liposome and RGD-coated FA liposome). Animals were sacrificed 15, 30, 60 and 120 min after administration to study the body distribution of the FA in the three formulations. HPLC was used to determine the concentration of FA in vivo with salicylic acid as internal standard. The results of body distribution indicated that RGD-coated liposomes could be mediated into the brain with a 6-fold FA concentration compared to FA solution and 3-fold in comparison to uncoated liposome. Brain targeted delivery was achieved and a reduction in dosage might be allowed.
Bauhinia variegata (Leguminosae) commonly known as Kachnar, is widely used in Ayurveda as tonic to the liver. The present work was carried out to assess the potential of Bauhinia variegata bark as hepatoprotective agent. The hepatoprotective activity was investigated in carbon tetrachloride (CCl4) intoxicated Sprague-Dawley rats. Bauhinia variegata alcoholic Stem Bark Extract (SBE) at different doses (100 and 200 mg/kg) were administered orally to male Sprague-Dawley rats weighing between 100-120 g. The effect of SBE on the serum marker enzymes, viz., AST, ALT, ALP and GGT and liver protein and lipids were assessed. The extract exhibited significant hepatoprotective activity. Hence, B. variegata appears to be a promising hepatoprotective agent.
We examined the effects of Hachimi-jio-gan (HJ) on the small intestinal function in streptozotocin (STZ)-induced diabetic rats. The rats had free access to pellets containing 1% HJ extract powder for 4 weeks after STZ administration. The intestinal disaccharidase (sucrase and maltase) activity was elevated in STZ-treated rats compared with control rats, whereas it was significantly reduced by HJ administration. This suggested that HJ suppresses or delays monosaccharide production in the small intestinal epithelium. In addition, the intestinal mucosal weights and DNA contents that were significantly increased in the STZ-treated rats were restrained to the control level by HJ treatment. Simultaneously, we examined the changes in the plasma levels of glucagon-like peptide 2 (GLP-2), which is a trophic factor specific for the intestine. The plasma GLP-2 levels significantly increased in the STZ-treated rats, whereas HJ decreased the plasma GLP-2 levels. Thus intestinal mucosal weights and DNA contents correlated with plasma GLP-2 levels in diabetes-associated bowel growth. These results suggest that HJ may normalize or suppress the small intestinal disaccharidase activity and the epithelial cell proliferation mediated by GLP-2 in the animal model rats.
In this study, a survey was conducted to determine the rate of drug-dispensing errors with the use of medicine bags printed with photographs of prescribed medicines (hereafter “medicine bag”) for a 6-week period from June 20 to July 31, 2005. During this period, 393928 prescriptions were filled in 127 medical facilities that use the medicine bag. The efficacy of the medicine bag in the prevention of drug-dispensing errors was investigated. A total of 6550 (1.66%) drug-dispensing errors were identified: 70.6% were identified at the inspecting stage; 27.4% at the providing medicine and information stage; and 2% after the medication was dispensed. The drug-dispensing errors identified in the inspecting and providing stages included a) using the wrong contents, b) dispensing the wrong drugs, c) missing drugs, d) calculation errors, e) weighing/measuring errors, and f) others. No significant difference was observed in the error rates; thus it was assumed that the type of error was not dependent on the stage at which dispensing errors was discovered. However, it was found that approximately 25% of errors at the providing stage were discovered as a result of the medicine bag. Errors of types a), b), and c) were often discovered because the photograph was printed on the medicine bag. Therefore it was assumed that the photographs contributed to the discovery of drug-dispensing errors.