In interviews on the traditional herbal medicines of Tupi-Guarany Indians at the herbal market of Asuncion and questionnaire from their users, it was clarified that various useful medicinal plants are available in Paraguay and most of them are generally used without drying. In the search for bioactive substances from those plants, a β-glucuronidase-inhibitory diterpene called scoparic acid A (SA) was isolated from Scoparia dulcis L. together with scoparic acid B, scoparic acid C, and the aphidicolin-like tetracyclic diterpenes scopadulcic acid A (SDA) and scopadulcic acid B (SDB). HPLC analysis of diterpenes in the individual plants of Paraguayan and Asian S. dulcis revealed the presence of three chemotypes based on major component, i.e., SA type, SDB type, and SDX type containing mainly scopadiol and scopadulciol (SDC). SA and SDB were elucidated to be mainly biosynthesized in the leaves via 2-C-methyl-D-erythritol- 4-phosphate pathway, and a leaf organ culture system containing methyl jasmonate 10 μM was found to enhance the production of diterpenes by activation of Ca-signal transduction systems such as calmodulin and protein kinase C. On the other hand, SDB and SDC were found to show multifaceted pharmacological effects such as inhibitory effects on gastric acid excretion, bone resorption, replication of herpes simplex virus type 1 (HSV-1), etc. In addition, SDC was suggested to be applicable to cancer gene therapy using ganciclovir or acyclovir and the HSV-1 thymidine kinase gene called the suicide gene.
This review describes studies on drug discovery using a rational formulation design and innovative, drug delivery systems (DDS) for biomaterials such as therapeutic peptides and nucleotides. The microcapsules of the LH-RH superagonist leuprorelin acetate prepared using the new in-water drying method and biodegradable polymers, such as PLGA and PLA, could achieve a long-term sustained release for 1-6 months thereby facilitating easily treatment of hormone-dependent diseases, prostate cancer, endometriosis, and precocious puberty. This DDS technology showed an improvement in patient QOL and highly promoted the clinical value of the agonist. Moreover, PLGA microcapsules of siRNAs against VEGF, cFLIP, Raf-1, and Int6 have also been developed to treat various cancers and arteriosclerosis obliterans. To develop therapeutic nucleotides, a particle design is created using functional peptides, such as cell penetrating peptides (CPP), nuclear localizing signals (NLS), tight junction reversible openers (AT1002), bombesin, and dynein light chain-associated sequences. siRNA use should lead to a paradigm shift in drug discovery against various diseases. Tat analog with NLS could enhance the potency of a vaginal DNA vaccine. The artificial Tat CPP of STR-CH2R4H2C synthesized in our laboratory could efficiently deliver siRNAs into many types of cells and enhance the therapeutic effects for treating sarcoma, atopic dermatitis, allergic rhinitis, and asthma by intratumor injection and inhalation of the nanoparticles. Tat and AT1002 analogs used to treat atopic dermatitis in mice increased cell membrane permeability to siRelA, a siRNA against a subclass of NF-κB, and exhibited striking therapeutic and preventive effects.
The traditional Oriental medicine and health supplement have been empirically practiced but most of them have not come through objective examination to prove their efficacy. From pharmacological aspect, we have been investigating the medical benefits of traditional Oriental medicines and health supplements as curatives and their varied actions and mechanisms. The study on airway inflammation has shown that even a Kampo preparation, Bakumondo-to, has anti-inflammatory, anti-allergic, immunomodulatory, secretory-modulating and metabolic regulatory actions. The base of all its actions is founded on the restoration of normal molecular and cellular functions through DNA transcriptional regulation. In other previous studies, we showed that a health supplement, royal jelly (RJ) has weak estrogenic activity. RJ competes with 17β-estradiol for binding to the human estrogen receptors α and β, though it is much weaker than diethylstilbestrol in binding affinity. Treatment of MCF-7 cells with RJ enhances proliferation, and concomitant treatment with tamoxifen blocked this effect. A reporter gene assay showed that RJ enhanced transcription of the luciferase gene through the estrogen-responsive element in MCF-7 cells. Furthermore, subcutaneous injection of RJ restored the expression of vascular endothelial growth factor gene in the uteri of ovariectomized rats. We suggest that the diverse pharmacological functions of RJ can be ascribed, in part, to its estrogenic effects. We hypothesize that polyherbal medicines and health supplements, which have multiple actions, may be better than Western medicine of single component to treat various diseases including ‘Mibyou’ (presymptomatic disease). Our findings provide us with a new idea on the nature of disorder and disease-state development which involve complicated mechanisms and will contribute to novel principles to prevent diseases and establish new treatment. Adoption of means of translational research should provide objective background for efficacy and stimulate broader application and usage of traditional medicines and health supplements as curatives of Mibyou.
Studies on trypsin-specific compounds are useful for the design of clinically useful compounds. It is well known that several benzamidine derivatives are potent competitive inhibitors of trypsin and trypsin-like enzymes. Many kinds of Schiff base metal chelate containing either amidine or guanidine have been synthesized and their inhibitory activities against trypsin have been characterized. Recently, the interactions of the Schiff base metal chelate inhibitors with trypsin enzyme have been determined by X-ray crystal structure analysis. The structural information and inhibitory activity data for amidine- and guanidine-containig Schiff base metal chelate inhibitors provide new avenues for designing novel inhibitors against physiologically important trypsin-like serine proteases.
Mass spectrometry-based quantitative analysis and biomarker discovery using metabolomics approach represent one of the major platforms in clinical fields including for the prognosis or diagnosis, assessment of severity and response to therapy in a number of clinical disease states as well as therapeutic drug monitoring (TDM). This review first summarizes our mass spectrometry-based research strategy and some results on relationship between cysteinyl leukotriene (cysLT), thromboxane (TX), 12-hydroxyeicosatetraenoic acid (12-HETE) and other metabolites of arachidonic acid and diseases such as atopic dermatitis, rheumatoid arthritis and diabetes mellitus. For the purpose of evaluating the role of these metabolites of arachidonic acid in disease status, we have developed sensitive determination methods with simple solid-phase extraction and applied in clinical settings. In addition to these endogenous compounds, using mass spectrometry, we have developed actually applicable quantitative methods for TDM. Representative example was a method of TDM for sirolimus, one of the immunosuppressant agents for a recipient of organ transplant, which requires rigorous monitoring of blood level. As we recognized great potential in mass spectrometry during these researches, we have become interested in metabolomics as the non-targeted analysis of metabolites. Now, established strategy for the metabolomics investigation applies to samples from cells, animals and humans to separate groups based on altered patterns of metabolites in biological fluids and to identify metabolites as potential biomarkers discriminating groups. We would be honored if our research using mass spectrometry would contribute to provide useful information in the field of medical pharmacy.
In this review, we have summarized our recent studies on the functionality of ionotropic (iGluR) and metabotropic (mGluR) glutamate receptors expressed by undifferentiated neural progenitor cells (NPC) isolated from embryonic rat and mouse brains. NPC are primitive cells with the self-renewal capacity as well as the multipotentiality to generate different neural lineages including neurons, astrocytes, and oligodendrocytes. Isolated cells were cultured in the presence of growth factors for the formation of round spheres by clustered cells so-called ‘neurospheres’ under floating conditions. Reverse transcription polymerase chain reaction analyses revealed expression of mRNA for particular iGluR and mGluR subtypes in NPC. Moreover, sustained exposure to an agonist for the N-methyl-D-aspartate receptor (NMDAR) not only inhibited the formation of neurospheres but also promoted differentiation of NPC into cells immunoreactive to a neuronal marker protein on immunocytochemistry and western blot analyses. On the other hand, sustained exposure to an agonist for the group III mGluR subtype led to suppression of proliferation activity in these neurospheres along with facilitation of the subsequent differentiation into astrocytes. Accordingly, glutamate could play a pivotal role in the mechanisms underlying proliferation for self-replication, together with determination of the subsequent differentiation fate toward particular progeny lineages through activation of NMDAR and group III mGluR subtypes in NPC.
The cerebral cortex is organized into six cell layers, each of which contains neurons with similar morphology, functions, gene-expression profiles, and projection patterns. These layer-specific neuronal phenotypes are sequentially generated from common cortical progenitor cells in the ventricular zone of dorsal telencephalon. Although recent investigations have clarified important roles of intrinsic factors such as transcription factors and regulators of the cell cycle for the maturation of cortical progenitors, growth factors and neurotrophic factors environmentally supplied by the cerebral cortex are thought to regulate proliferation and neural development and determine neuronal differentiation in the cerebral cortex. In this review, I focus on the function of neurotrophin-family neurotrophic factor, including nerve growth factor, brain-derived neurotrophic factor (BDNF), neurotropin-3 (NT-3) and neurotrophin-4 in the formation of the neuronal layer of the cerebral cortex. Especially, BDNF and NT-3 are expressed in the proliferating cortical progenitors and influence the biological properties of cortical progenitors prior to neurogenesis and play distinct roles in generation of cortical neuronal subtypes.
We have developed a new method of iodocyclization based on reagent-controlled oxidative aromatization. Our strategy takes advantage of the dual nature of iodine as both an iodinating and an oxidizing agent. This approach enabled “product switch” and enhanced the flexibility of the synthetic pathway toward pyrazoles and isoxazoles. In addition, the iodo moiety of the cyclized product could create further diversity. The utility of our methodology was demonstrated in the synthesis of valdecoxib and its 2,5-dihydro analogs.
Skeletal muscle has great regenerative potential that depends on muscle stem cells, called satellite cells. In uninjured muscle, satellite cells reside beneath the basal lamina and are maintained in quiescent and undifferentiated state. This state is considered a requisite for sustaining the satellite cell pool, but the molecular mechanism is still unknown. In our previous study, we developed a novel monoclonal antibody that specifically recognized muscle satellite cells in skeletal muscle. Using this monoclonal antibody, we purified satellite cells and performed genome-wide transcriptome analysis. In these analyses, we found that satellite cells expressed Hesr1/Hey1 and Hesr3/HeyL genes known as down stream target of Notch signaling. Although each single knock out mice did not indicate obvious phenotype in skeletal muscle, Hesr1/Hesr3 double knock out mice showed remarkably decreased number of satellite cells. Intriguingly, dKO satellite cells were not kept in quiescent and differentiated state in adult skeletal muscle. This dysregulated state of satellite cells lead to gradually decreased number of satellite cells and age-dependent regeneration defect. These results indicate that Hesr1/3 is essential for muscle stem cell biology and will facilitate this research field.
Because embryonic stem (ES) cells and induced pluripotent stem (iPS) cells can differentiate into various types of cells in vitro, they are considered as a valuable model to understand the processes involved in the differentiation into functional cells as well as an unlimited source of cells for therapeutic applications. Efficient gene transduction method is one of the powerful tools for the basic researches and for differentiating ES and iPS cells into lineage-committed cells. Recently, we have developed an adenovirus (Ad) vector for efficient transduction into ES and iPS cells. We showed that Ad vectors containing the cytomegalovirus enhancer/β-actin promoter with β-actin intron (CA) promoter or the elongation factor (EF)-1α promoter were the appropriate for the transduction into ES and iPS cells. We also found that enforced expression of a PPARγ gene or a Runx2 gene into mouse ES and iPS cells by an optimized Ad vector markedly augmented the differentiation of adipocytes or osteoblasts, respectively. Thus, a gene transfer technique using an Ad vector could be an advantage for the regulation of stem cell differentiation and could be applied to regenerative medicine based on ES and iPS cells.
Biaryl compounds are versatile building blocks in the synthesis of natural products, pharmaceuticals, agricultural chemicals and π-conjugated organic materials. This review describes a recent progress for the biaryl cross-coupling reaction of heteroaromatic compounds using hypervalent iodine reagent. Our novel biaryl coupling reaction is a unique method for constructing various heteroaromatic biaryls without use of transition metal catalysts. From mechanistic point of view, the coupling reaction was realized through stable iodine intermediate generated from heteroaroamatic compound and iodine (III) reagent.
Chronic kidney disease (CKD) has been increasingly recognized as a major public health problem in the world. Recent studies have showed that CKD is an independent risk factor for the occurrence of cardiovascular disease (CVD). Reactive oxygen species (ROS), generated by reduction-oxidation actions, have been generated by reduction-oxidation actions, recognized as the important chemical mediators that regulate signal transduction in various cells including vascular smooth muscle cells (VSMC) and mesangial cells (MC). It has been showed that increase in ROS generation may relate to a risk for CVD and CKD. In addition, ROS mediate activation of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, p38, and big MAP kinase 1, in various cells leading to change in gene expressions. Control of the oxidative stress and ROS-mediated alterations of signaling molecules including MAP kinases may provide new therapeutic strategy against CKD and CVD. In this review, we summarize mainly our data regarding the pharmacological effects of renin-angiotensin-aldosterone system blockers, bioflavonoids and adiponectin in VSMC and MC. Also we review the data on a possible new class drug against oxidative stress to improve CKD and CVD.
To minimize their occurrence, it is important to gather and analyze data regarding cases of not only medical accidents but also of incidents involving potential harm to patients. In gathering data, we have separated reporting between the details of such incidents and information about their occurrence. We have implemented a system involving a first report to achieve prompt notification and a second report to provide details. An online report input system has been established taking into consideration both ease of input and promptness of information sharing. We discuss the input of the first and second reports in a total of 951 cases over a period of 6 months. From the data regarding the timing of the first report, 307 and 789 cases were reported within 24 h and 48 h, respectively, indicating that the first report was input mostly without delay in accordance with the operational guidelines. On the other hand, it took 14 days to surpass a second report rate of 80%. Cases that took more than 2 weeks to be reported would likely have gone unreported had there not been a first report to indicate and confirm that an incident had even occurred. Investigation is needed, especially for problematic cases, so we assume that discovering important incidents via the first report has been successful. In addition, details of incidents can be input into this system in free-text, yielding information that cannot be acquired with multiple choice input as in standard reporting systems.
Personnel who prepare and administer chemotherapeutic agents have been reported to develop untoward effects. The use of appropriate techniques for preparing these agents is encouraged, and educational training systems that involve the use of a fluorescent or chemiluminescence reagent as placebos have been established to minimize potential exposure to these agents. However, the optimum conditions for the use and visibility of these placebos remain obscure. In this study, our results indicated that the fluorescence intensity of fluorescent reagent decreased when it was used at a concentration greater than 0.01%. Because drops created due to splashes and leaks are extremely small and easily evaporate, it is possible that the fluorescence resulting from such drops readily disappears despite using an anti-evaporation reagent. We also developed a method to evaluate the visibility of the small drop; using this method, we determined the distance at which the drop present on the pin could be seen by the observer. The distance at which the drop was clearly recognized as a pinpoint by using the fluorescence method was almost comparable to that for the chemiluminescence method. In the chemiluminescence method, the drop on the pin was faintly visible as a slightly bright area because of low background when observed at a certain distance that was much greater than that at which the drop was clearly visible; however, such an area was not observed in the fluorescence method. The results of our study will help in the selection of a training method depending on the situation.
At Tohoku Pharmaceutical University, problem-based learning (PBL) tutorials were incorporated into “prescription analysis” and “case analysis” for fifth-year students in 2010 with the following objectives: ① application and confirmation of acquired knowledge and skills, and acquisition of ② communication ability, ③ presentation ability, ④ cooperativeness through groupwork, and ⑤ information collecting ability. In the present study, we conducted a questionnaire survey on a total of 158 fifth-year students in order to investigate the educational benefits of PBL tutorials. The results showed that the above five objectives of PBL tutorials were being achieved, and confirmed the educational benefits expected of PBL tutorials. In contrast, it was found to be necessary to improve the contents of scenarios and lectures, time allocation regarding schedules, the learning environment, the role of tutors, and other matters. In order to maximize the educational benefits of PBL tutorials, it will be necessary in the future to continue to conduct surveys on students and make improvements to the curriculum based on survey results.
δ-Elemene, an antitumor component, is a chemical compound isolated from Curcuma wenyujin, a Chinese traditional herb. We examined whether δ-elemene could inhibit cell growth and cell cycle progression and induce apoptosis in human leukemia HL-60 cells. The results demonstrated that δ-elemene induces significant apoptosis of HL-60 cells, as shown by MTT assay, annexin V (AnV) binding of externalized phosphatidylserine (PS), and the mitochondrial probe JC-1 using flow cytometry. HL-60 cells treated with δ-elemene showed high percentages in the early apoptotic and late apoptoctic/necrotic stages, as well as caspase-3 activation of HL-60 cells. By monitoring the changes in cell cycle profiles, we confirmed that δ-elemene could interfere with the cell cycle in the G2/M phase and induce apoptosis in HL-60 cells in a time-dependent manner. Caspase-3 plays a direct role in proteolytic cleavage of the cellular proteins responsible for progression to apoptosis. Therefore we examined apoptosis in HL-60 cells after exposure to δ-elemene and measured caspase-3 activities with or without Z-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk, a broad-spectrum caspase inhibitor) pretreatment using flow cytometric analysis. The results showed that δ-elemene could induce caspase-3 activation as detected by the decrease in δ-elemene-induced caspase-3 activities after treatment with z-VAD-fmk. In the present study, δ-elemene activated typical caspase-dependent apoptosis in HL-60 cells, as demonstrated by an inhibitory effect of z-VAD-fmk on this cell death. During δ-elemene-induced apoptosis, cytochrome c and apoptosis-inducing factor were released into the cytosol and BAX was translocated from the cytosol to mitochondria. However, these were not prevented by z-VAD-fmk. In conclusion, our study demonstrated that δ-elemene could induce G2/M cell cycle transition and trigger apoptosis through a caspase-3-dependent pathway.
Hemopurification is an effective therapy for acute kidney injury, defined as creatinine clearance less than 30 ml/min, which occurs frequently in the intensive care unit. These critically ill patients often have severe infectious complications and are thus often treated with antibiotics. However, the effect of hemopurification on the pharmacokinetics of antibiotics is not well understood. In this study, we investigated the pharmacokinetics of doripenem (DRPM) in critically ill patients with accompanying renal dysfunction undergoing continuous hemodiafiltration by high-volume filtration/high-flow dialysis (high-flow CHDF) and compared it to the pharmacokinetics of DRPM during conventional CHDF. We studied 8 patients (2 in the high-flow group and 6 in the conventional group) in whom DRPM was administered while performing CHDF for acute kidney injury. DRPM (250 mg) was intravenously infused over 1 h. For the conventional group, CHDF was performed at a blood flow rate (QB) of 100 ml/min, dialysate flow rate (QD) of 500 ml/h, and filtration flow rate (QF) of 300 ml/h. For the high-flow group, CHDF was performed at a blood flow rate (QB) of 100 ml/min, dialysate flow rate (QD) of 1500 ml/h, and filtration flow rate (QF) of 900 ml/h. For both groups, a polysulfonehemofilter with a membrane area of 1.0 m2 was used. Mean half-life, total body clearance, and clearance via hemodiafiltration of DRPM were 2.9 h, 118 ml/min, and 41.9 ml/min, respectively, in the high-flow group, and 7.9 h, 58 ml/min, and 13.5 ml/min in the conventional group. Clearance via hemodiafiltration increased approximately 3-fold by tripling the hemopurification rate. Therefore, CHDF parameters greatly affected DRPM pharmacokinetics in patients receiving CHDF. These results suggest that clearance via hemodiafiltration increases proportionally to the hemopurification rate. Thus, it is reasonable to conclude that DRPM dose must be increased to 1.0-1.5 g/day when performing high-flow CHDF.