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The nasal drug application has drawn much attention as the strategy for the delivery route of many drug modalities such as the poorly absorbed drugs, peptides, nucleic acid, and central nervous system drugs. The absorption of drug after intranasal (IN) application depends on the nasal residence time of applied drug, affected by mucociliary clearance (MC). MC is a decisive factor in the nasal absorption of drug. We describe the establishment of in vitro evaluation system of nasal MC via the moving velocity of a marker particle on nasal mucosa, and the development of the pharmacokinetic model to which in vitro parameters on nasal MC was incorporated to enable the prediction of drug absorption after IN application. Moreover, the pharmacokinetics of norfloxacin after IN application was investigated using MC-modified rats pretreated with MC modulators. Nasal absorption fluctuated due to changes in the nasal residence time of drug in response to changes in MC. The prediction system enables quantitative evaluation of changes in drug absorption associated with MC fluctuations. In addition, for a precise prediction system for drug absorption after IN application from the drug absorption model, the relationships between in vitro drug permeability through Calu-3 layers, in vivo transnasal permeation of drug and in vivo bioavailability after IN application were evaluated. The significant correlations between these parameters were obtained, suggesting that transnasal permeability and drug absorption after IN application can be predicted from in vitro membrane permeability.

Distigmine bromide (distigmine) is a carbamate cholinesterase (ChE) inhibitor, which is mainly used for the treatment of myasthenia gravis. Distigmine is also used in Japan for the treatment for underactive bladder and glaucoma. The effectiveness of distigmine for underactive bladder treatment has been confirmed by many clinical reports, and this effect is thought to be caused by potentiating urinary bladder smooth muscle contraction due to inhibition of acetylcholine degradation during micturition. However, the pharmacological effects of distigmine on urinary bladder smooth muscle have not been well studied. The most distinctive pharmacological feature of distigmine is that it shows long-lasting effects than other ChE inhibitors; however, few studies have investigated the persistence of the enhancing effect of distigmine on the contractile function of urinary bladder smooth muscle. Moreover, this mechanism remains unclear. In this review, we present our findings on the mechanism of the potentiating effect of distigmine on isolated guinea pig urinary bladder smooth muscle contraction. We also discuss the long-lasting potentiating effect of distigmine on urinary bladder motility and the mechanism of these effects using guinea pig urinary bladder smooth muscle in vivo and in vitro. In addition, we present our investigations on the long-lasting mechanism of distigmine using recombinant human acetylcholinesterase.

We previously reported that tolvaptan may influence warfarin pharmacodynamics in vivo; however, the mechanism responsible for this influence was not clear. In this study, we investigated the drug-drug interactions between warfarin and tolvaptan by measuring warfarin blood concentrations in 18 patients who received warfarin therapy and in 24 who received warfarin+tolvaptan therapy. The free warfarin concentrations significantly increased in patients who were also receiving oral tolvaptan (p=0.04). In vitro albumin-binding experiments showed that the free warfarin concentrations significantly increased with the addition of tolvaptan, in a dose-dependent manner, through albumin-binding substitution (approximately 2.5 times). Both clinical and in vitro data showed that tolvaptan increased the unbound warfarin serum concentration. The prothrombin time-international normalized ratio (PT-INR) tended to increase within 2 weeks when tolvaptan was added at clinically used doses (p=0.14). Special attention is warranted in cases with a serum tolvaptan concentration of ≥125 ng/mL (≥7.5 mg/d) for at least 2 weeks following oral tolvaptan administration.

The latest chemical management policies require toxicological evaluation of marketed but untested chemicals. Furthermore, in Europe, for animal welfare reasons sales of cosmetics and raw materials for which animal experiments were conducted were totally banned, in 2013. Responding to these regulatory trends, a strong demand exists to develop new in vitro test methods and to improve in silico prediction models for safety assessments. In recent years, the development of adverse outcome pathways (AOPs) has been actively promoted in the Organisation for Economic Co-operation and Development (OECD). Since it is difficult to replace a particular in vivo animal test with a single in vitro test method or in silico prediction model, integrated approaches to testing and assessment (IATA) have been studied based on AOP information. With regard to skin sensitization, several in vitro test methods that measure key events of AOP have been established, and integrated strategies using in vitro tests have been examined using AOP. Currently, numerous AOPs are under development for a wide range of complex toxicity endpoints in the OECD AOP program. The AOPs are expected to contribute to the development of many accurate in vitro test methods and to establish IATA as well as to evaluate safety in humans of many substances, including household chemicals, food-related chemicals, cosmetics, and pharmaceuticals.

Because the liver is the primary target organ for chemicals and pharmaceuticals, evaluation of these substances' liver toxicity is of critical importance. New evaluation methods without animal testing (i.e., in vitro and/or in silico) are eagerly anticipated, both for animal welfare and for decreasing cost. Also, the importance of mechanistic interpretation of the output derived from non-animal testing has been increasing. Accordingly, we investigated the potential for evaluating liver toxicity by applying the adverse outcome pathway (AOP) concept using gene set enrichment analysis (GSEA) from gene expression (GEx) data. A case study targeting hepatocellular fatty degeneration (HFD) is reported and discussed. We first identified the events detectable in an in vitro system by comparing the GEx data from the rat primary hepatocyte (in vitro) and rat liver (in vivo) treated with a chemical with the ability to induce HFD as one of the phenotypes in a 28-day repeated-dose toxicity test. Then, the scores based on GSEA were calculated after establishing the gene sets for each event leading to HFD. As a result, the mechanistic information leading to HFD was obtained from the score calculated based on the GSEA and the usefulness of the transcriptome-driven evaluation using AOP was demonstrated.

Interactions between carbohydrate-containing glycoproteins, proteoglycans, and glycolipids on the cell surface are important biological stages for the processes of bacterial or viral infection and tumor metastasis. Moreover, supramolecular interaction by macromolecules with two-fold (C 2 ) or three-fold (C 3 ) geometry is one of the common interactions in many important biological responses. To develop new multivalent symmetrical bioactive compounds or leads, we designed and synthesized several new molecules with these geometries and evaluated their bioactivities in an attempt to find new types of bioactive leads that may interfere with the sugar recognition process. We evaluated bioactivities including antibacterial, antiviral, and anticancer activities of targeted molecules in vitro using biological assay systems. Among the synthesized target derivatives examined, some bivalent symmetrical derivatives showed high levels of bioactivities. In this review, the author describes the results of synthesis of oligovalent symmetrical target compounds and some interesting guiding results of evaluation of their biological activities and structure-activity relationships.

Pathological α-synuclein (αSyn) has been shown to retain the ability to propagate as prions in humans and animals. However, the molecular basis underlying the prion-like properties of αSyn remains poorly understood. We examined whether brain tissues from cases of dementia with Lewy bodies (DLB), which contain serine 129 (Ser129)-phosphorylated insoluble aggregates of αSyn, exhibit prion-like seeding activity in vitro using the real-time quaking-induced conversion (RT-QuIC) seeding assay. Brain tissues from cases of diffuse neocortical DLB yielded a 50% seeding dose of 10 7.3 -10 9.8 /g brain. The RT-QuIC assay could discriminate between DLB and other neurological and neurodegenerative disorders, suggesting its potential applicability for differential diagnosis. Insoluble aggregates of αSyn>250 kDa detected only in DLB brain tissues by Western blotting analysis were specifically phosphorylated at Ser129. Therefore, we postulated that Ser129-phosphorylated insoluble aggregates of αSyn have prion-like seeding activity. However, insoluble aggregates of recombinant human αSyn (rSyn) with increased β-sheet structures showed little seeding activity in either the phosphorylated or nonphosphorylated state. In contrast, prefibrillar oligomers of rSyn showed seeding activity both with and without phosphorylation. The findings of the present study suggested that soluble oligomeric αSyn, but not the fully fibrillary form, is a seeding species in vitro.

The molecular basis underlying the conversion of normal prion protein (PrP C ) into abnormal prion protein (PrP Sc ) has not been fully elucidated. The protein-misfolding cyclic amplification (PMCA) technique, which can amplify PrP Sc in vitro with the use of intermittent sonication, mimics the process of in vivo PrP Sc replication. Accumulating evidence suggests that co-factors other than PrP may play a crucial role in the faithful replication of PrP Sc . In conventional PMCA, brain homogenates (BHs) from normal animals are used as the PrP C substrate. Since BHs contain many impurities, it is difficult to identify the co-factors using conventional PMCA. Thus, we developed a modified PMCA system using baculovirus and insect cell-derived recombinant PrP as a substrate (insect cell PMCA; iPMCA). We demonstrated that nucleic acids and glycosaminoglycans (GAGs) such as heparan sulfate (HS) or its analogue heparin (HP) are critical for PrP Sc amplification in iPMCA. Of note, the addition of HS or HP restored the conversion efficiency in iPMCA under nucleic acid-depleted conditions. Moreover, the iPMCA products were infectious and preserved the strain properties of the input seed PrP Sc . These data suggest that not only nucleic acids but also some GAGs play an important role in facilitating faithful replication of prions, at least in vitro.

Tissues of the small intestine are crucial to understanding drug disposition because these tissues regulate the bioavailability of drugs. However, no evaluation system is currently available for precise and comprehensive analysis of intestinal pharmacokinetics. To address this, functional intestinal epithelial cells were generated from human induced pluripotent stem (iPS) cells for use in pharmacokinetic studies. An improved intestinal differentiation method was established by screening a variety of small molecule compounds against cells during differentiation. The mRNA expression levels of intestinal markers, drug transporters, and CYP3A4 were found to increase following treatment with compounds that act as inhibitors of mitogen-activated protein kinase, DNA methyltransferase, and transforming growth factor-β. Therefore, we inferred that these compounds enhanced differentiation into intestinal epithelial cells. The differentiated intestinal epithelial cells in the presence of these compounds possessed drug-metabolizing enzyme activities, such as those of CYPs, UDP-glucuronosyltransferase, and sulfotransferase. In addition, these cells had the ability to induce CYP3A4 in the presence of 1α,25-dihydroxyvitamin D 3 . The differentiated intestinal epithelial cells seeded on cell culture inserts formed loose-tight junctions, similar to those in the human small intestine, rather than Caco-2 cells. The cells exhibited polarity, such as apical and basal sides. We also demonstrated that the uptake and efflux transport activities in the cells occurred via peptide transporter and breast cancer resistance protein, respectively. Taken together, it was suggested that human iPS cell-derived intestinal epithelial cells are pharmacokinetically functional, and represent a promising model system for pharmacokinetic studies of drug candidates.

We present a case of heparin resistance whereby open heart surgery was discontinued. A 53-year-old woman who was diagnosed with ventricular septal defect and atrial septal defect was scheduled for intracardiac repair. However, after intravenous heparin (400 U kg-1) supplementation, the activated clotting time (ACT) increased only to seconds. The operation was discontinued because the addition of heparin( 200 U kg-1) did not show sufficient prolongation of ACT, fully indicative of heparin resistance. Additional antithrombin III concentrate was also ineffective. Postoperative study of the administration of heparin in vitro to the patient's serum showed the probability of transient heparin resistance arising from the stress of surgery.

Tocopheryl phosphoric acid (TPA, a hydrophilic vitamin E derivative) loaded liposome and glycerin containing phospholipid nanoparticles (GPLNP) were prepared using the film rehydration and extrusion method. Nanoparticle formulations were evaluated for size, zeta potential, and in vitro permeation across hairless mouse skin, and 31 P NMR spectral analysis was performed. The prepared formulations were stable for 2 weeks, and their mean nanoparticle size varied between 90 and 140 nm. Although glycerin did not affect the particle size of the empty (no TPA) system, TPA-loading resulted in the reduction of particle size and conferred a negative charge. The 31 P NMR spectral analysis showed that the presence of glycerin in the formulation changed the nanoparticle structure from a bilayer to a nonbilayer. Moreover, it was suggested that TPA molecules interacted with phospholipid by entrapping nanoparticles in the formulations. TPA did not permeate across the hairless mouse skin after 48 h. However, the TPA concentration in the hairless mouse skin after permeation study increased in the nanoparticle systems and the 30% GPLNP formulation was the best formulation for the accelerated TPA permeation in the hairless mouse skin. These results demonstrate that 30% GPLNP improved TPA permeation in the hairless mouse skin model. And it was strongly suggested that glycerin has an important role for changing the structure of nanoparticles and enhancing the skin permeation of TPA.

Proteins such as membrane transporters, enzymes, receptors and channels play key roles in drug absorption, distribution, metabolism, and elimination, and also influence efficacy and the likelihood of adverse reactions. Therefore, if we can quantify the activities of these molecules, it may be possible to predict the behavior of candidate drugs in humans in disease states; such methodology would be extremely helpful for efficient drug development. We have developed an in silico method to select appropriate peptides within amino acid sequences in order to quantify targeted proteins by LC-MS/MS in selected reaction monitoring (SRM) mode. We have applied this method for the quantification of functional proteins in order to validate various in vitro and in vivo models. We found fairly good correlation between protein amounts and the enzymatic activities of microsomal cytochrome P450 (CYP) isoforms and uridine 5'-diphospho-glucuronosyltransferase (UGT) in human liver, as well as between protein amounts and the transport activities of multiple transporters in human lung cells. These results suggest that protein quantification can be useful in predicting activity. We have applied this approach to evaluate the usefulness and limitations of an immortalized human brain capillary endothelial cell line (D3 cells) and a P-glycoprotein humanized (hMDR1) mouse model by comparing the amounts of functional proteins in the models with those in isolated capillaries from human brain. In order to obtain sufficient human tissue specimens for further studies leading to clinical applications, we believe that international collaboration will be crucial.

Growing evidence indicates that serotonergic neurons play a crucial role in brain function and dysfunction, such as in major depressive disorder. However, the complexity of serotonergic projections severely hampers the elucidation of their precise mechanisms. Here we summarize our recent studies on the effects of ketamine and olanzapine, which have been reported to be effective in treatment-resistant depression, on dorsal raphe nucleus serotonergic neurons, using microdialysis, electrophysiology experiments, and slice cultures. Furthermore, we discuss the results of our recent approach using viral vectors, in particular, HIV-1 based lentiviral vectors.

The pluripotent stem cells have a self-renewal ability and can be differentiated into theoretically all of cell types. The induced pluripotent stem (iPS) cells overcame the ethical problems of the human embryonic stem (ES) cell, and enable pathologic analysis of intractable diseases and drug discovery. The in vitro disease model using disease-specific iPS cells enables repeated analyses of human cells without influence of environment factors. Even though autoimmune diseases are polygenic diseases, autoimmune disease-specific iPS cells are thought to be a promising tool for analyzing the pathogenesis of the diseases and drug discovery in future.

Two topical therapeutic agents were approved in Japan from 2015 to 2016, adding new options for onychomycosis therapy in the clinical field. In order to confirm the differences of formulation properties and nail pharmacokinetics between 5% luliconazole solution and 10% efinaconazole solution, drug concentration and antifungal activity in the nail were measured after topical treatment using human nail plates. In the in vitro permeation study, concentration of each drug was measured in the transversely sliced nail after single treatment with the two topical therapeutic agents. The results showed that concentration of luliconazole is higher than that of efinaconazole at all nail layers, differing by 1.7-8.4 times at each measurement point. Next, we examined antifungal activities of each drug in sliced nail after 14-day topical treatment. Mean rates of formation of inhibition zones for 5% luliconazole solution and 10% efinaconazole solution were 71.0% and 12.6%, respectively, and were statistically different. These results show that the two topical therapeutic agents have different properties, and suggest that 5% luliconazole solution has good nail permeation and retention characteristics. Moreover, luliconazole was found to retain enough antifungal activity in the nail plate against Trichophyton spp. after treatment with the topical agent.

Membrane proteins allow a cell to communicate with its environment by relaying a signal or transporting a molecule through the cell membrane. Elucidation of the three-dimensional structure of a membrane protein provides a greater understanding of its function and mechanisms. Ultimately, this knowledge will enlighten researchers on how these proteins can be regulated to elicit a desired cellular response, which could lead to novel therapeutic medicine. Unfortunately, the determination of the high-resolution crystal structures of transmembrane proteins remains a challenge due to their poor solubility and high conformational flexibility. Additives and cocrystallization ligands are being used to address these problems. In vitro selected macrocyclic peptides have recently been successfully employed as cocrystallization ligands. Although originally intended as inhibitors and drug lead molecules, in vitro selected macrocyclic peptides are now showing that their pharmacodynamic properties also allow them to serve as excellent cocrystallization ligands. Structures for macrocyclic peptide-bound transporters, the multidrug and toxic compound extrusion family transporter from Pyrococcus furiosus (PfMATE) and the ABC transporter subfamily B member 1 from Cyanidioschyzon meraloe (CmABCB1), have been elucidated using X-ray crystallography. The cocrystal structures reveal that the macrocyclic peptides improve crystallization by binding in a similar manner as a small molecule or a biologic. The PfMATE-binding macrocyclic peptides MaD3S and MaD5 bind to the surfaces buried in the center channel of the transporters. Although both transporters possess a center channel and substrate-binding pocket, the CmABCB1-binding macrocyclic peptide, aCAP, binds to the outer surface of the transporter in a similar manner to a biologic.

The in vitro metabolism of 2,2',3,4,4', 5,5'-heptachlorobiphenyl (CB180) was examined using liver microsomes of rats, guinea pigs and hamsters. Of liver microsomes from untreated animals, rats and guinea pigs produced one metabolite (M-1) with the activity of 1.2 and 18.1 pmol/hr/mg protein, respectively, but hamsters did not at all. Pretreatment of phenobarbital (PB) resulted in about 32-fold increase in rats, 4-fold increase in guinea pigs and an appearance of M-1 in hamsters (15 pmol/hr/mg protein). In addition, another metabolite (M-2) was formed only by liver microsomes of PB-treated guinea pigs. In contrast, pretreatment of 3-methylcholanthrene showed no metabolite in three animals. By comparison of the GC-MS data of the metabolites with synthesized authentic samples, M-1 and M-2 was determined to be 3'-hydroxy (OH)-CB180 and 4'-OH-2,2',3,4,5,5'-hexachlorobiphenyl (CB141), respectively. These results suggest that 3'-OH-CB180 is a major metabolite and is formed by PB-inducible cytochrome P450 (CYP2B enzymes) in animals and also guinea pigs possess much higher activity to metabolize CB180 than rats and hamsters.

Drug-induced liver injury (DILI) is one of leading causes of attrition during both early and late stages of drug development and postmarketing. DILI is generally classified into the intrinsic and idiosyncratic types. Intrinsic DILI is dose dependent and predictable as exemplified by acetaminophen toxicity. However, the occurrence of idiosyncratic DILI with very low incidence and severe liver damage is difficult to predict because of the complex nature of DILI and poor understanding of its mechanism. In this review, we summarize current knowledge and our accumulated experimental findings on the pathogenic mechanisms of DILI focusing on the reactive metabolites of drugs formed by drug-metabolizing enzymes and immune- and inflammation-related responses. Considering drug metabolism and pharmacokinetics, we have established nonclinical animal models of DILI for 10 types of clinical drug known to cause idiosyncratic DILI in humans. Using animal models, it has been shown that the formation of reactive metabolites and both innate and adaptive immunity are involved in the pathogenesis of drug hepatotoxicity. Based on information on biomarkers obtained from animal models, we developed a cell-based system that predicts the potential DILI risks of drugs. The results of these studies increased our understanding of the mechanisms of DILI and help to predict and prevent idiosyncratic DILI caused by drug candidates.

A generic drug is defined as a drug product that is comparable to a brand name drug in terms of dosage, form, strength, route of administration, quality, performance characteristics, and indicated use. Generic drugs for topical use, in the case of sheet-like products, are required to be the same as the original drug in terms of application area and dosage form. The composition of such generic drug formulations may differ from that of the original product. The adhesive of any pharmaceutically-active tape that directly contacts the skin plays a role in delivering the active ingredient into the skin, and affects the sensation and ease of handling. Therefore, adhesives are an important ingredient in these products. Thus, the aim of this study was to characterize original and generic lidocaine tape products, and to evaluate the adhesive properties of each. The tack force, peel strength and shear adhesion were measured as adhesive properties. In addition, in vitro drug releasing profiles and skin permeation profiles of the products were evaluated. In vivo transdermal absorption was also evaluated to predict the possibility of adverse effects. Adhesive properties differed among the three analyzed products. These differences may have been caused by differences in the adhesives. Drug-releasing profiles and skin permeation profiles also differed among the three products, even though the pharmacokinetics were not significantly different. By obtaining an adequate understanding of the characteristics of original and generic products, we will be able to provide better tailor-made medications for drug therapies for patients.

Background: In the 1990s, the Japanese Society of Allergology (JSA) standardized Japanese cedar pollen allergen vaccines. In the present study, the task force for house dust mite (HDM) allergen standardization of the Committee for Allergens and Immunotherapy of JSA reports the standardization of HDM allergen vaccines in Japan., Methods: In vivo allergenic potency was determined by intradermal testing of 51 Japanese adults with positive serum specific IgE to HDM allergens. In vitro total IgE binding potency was analyzed by the competitive ELISA using a pooled serum, with sera obtained from 10 allergic patients. Concentrations of HDM group 1 (Der 1) and group 2 major allergens in eight HDM allergen extracts were measured by sandwich ELISAs. Correlation between the in vitro total IgE binding potency and major allergen levels was analyzed., Results: We selected a JSA reference HDM extract and determined its in vivo allergenic potency. The in vitro total IgE binding potency significantly correlated with Der 1 content, group 2 allergen content, and their combined amount, indicating that measurement of major allergen contents can be used as a surrogate in vitro assay., Conclusions: The task force determined the in vivo allergenic potency (100000 JAU/ml) and Der 1 content (38.5 μg/ml) of the JSA reference HDM extract, selected the measurement of Der 1 content as the surrogate in vitro assay, and decided that manufacturers can label a HDM allergen extract as having a titer of 100000 JAU/ml if it contains 22.2-66.7 μg/ml of Der 1.

PL cream (combination of lidocaine and procaine) was launched on the market in April 2012 in Japan. We investigated differences in the anesthetic effect by employing two types of base: Carbopol and methylcellulose. Electron microscopy showed a distinct difference in appearance: densely-scattered, fine particles for Carbopol and sparse, large particles for methylcellulose. Accordingly, the extensibility of the cream was significantly greater at 4 and 25 degrees centigrade for methylcellulose, but was greater at 34 degrees centigrade for Carbopol. The steady flow viscosity (1 s(-1)) was greater for the Carbopol than methylcellulose base. The difference in the cutaneous permeability between the two bases increased over time: the methylcellulose base was removed at 90 min after application and, 30 min later, showed a significant difference. These results suggest that the methylcellulose base has a superior anesthetic effect in clinical settings.

Inchinkoto (ICKT) is one of the most commonly used herbal medicines and is a hepatoprotective agent. Among the numerous chemical compounds included in ICKT, geniposide is the most abundant component. After oral intake, geniposide is converted into the active metabolite genipin by intestinal bacteria and absorbed in the portal circulation. The biological properties of ICKT and its major active ingredient genipin have been studied in numerous experiments using cells and animals. ICKT or genipin administration exerts a choleretic effect through upregulation of multidrug resistance-associated protein 2 in hepatocytes. ICKT also exerts antiapoptoic activity by inhibiting the transforming growth factor beta 1- or tumor necrosis factor alpha-dependent signaling pathway. The excessive inflammatory response induced by various forms of hepatic stress is also attenuated by ICKT preadministration. Proinflammatory cytokine-induced upregulation of inducible nitric oxide synthase is strongly attenuated by ICKT in both in vivo and in vitro experiments. Moreover, ICKT enhances antioxidant enzymes in the liver under oxidative stress. These experimental results clearly indicate the effects of ICKT on hepatic stress. To date, however, clinical data on the benefits of ICKT for liver disease are very rare. To extend the clinical applications of ICKT in humans, it is crucial to design and perform a rigorous clinical trial. In this review article, recent evidence relating to the hepatoprotective effects of ICKT in the field of basic and clinical science is summarized and discussed.

A 56-year-old man was referred to our clinic because of left lumbar pain and a left solitary renal tumor (9. 8 cm in diameter) and bilateral pulmonary metastases detected by computed tomographic scan. Pathologic diagnosis following open radical nephrectomy was papillary renal cell carcinoma, G2, pT2aN0M1. Subsequently, the patient was sequentially treated with interleukin-2 (3 months (mo), progressive disease (PD)), interferon-alpha (3 mo, PD), and oral S-1 as a clinical trial (28 mo, PD). Because of skin fibrofolliculomas, pulmonary cysts, and spontaneous pneumothorax history, Birt-Hogg-Dubé (BHD) syndrome was suspected during the treatment course, despite his having no family history of the disease. Subsequent genetic testing revealed a FLCN germline mutation (c. 1285dupC). He was started on molecular-targeting therapies sequentially, i.e., sorafenib (1 mo, PD), sunitinib (4 mo, PD), and everolimus (7 mo, PD). The patient died of progressive disease at 78 mo from the initial nephrectomy and 30 mo from the start of targeted agents. Loss of FLCN function has been shown to result in the upregulation of the PI3K/mTORC1 pathway in both in vitro experiments and in vivo FLCN knockout mice models. Despite its use as the sixth-line systematic treatment, the mTOR inhibitor everolimus exhibited a relatively long-term effect as compared with the previously used tyrosine kinase inhibitors and in contrast to the results in the RECORD-1 clinical trial. This finding may provide insight into the molecular mechanism of BHDassociated renal tumors.

The in vitro metabolism of 2, 2', 3, 4', 5, 5'-hexachlorobiphenyl (hexaCB) (CB146) was examined using liver microsomes of rats, guinea pigs, hamsters and human. Untreated animal livers produced one metabolite (M-2) in rats, three metabolites (M-l, M-2and M-3) in guinea pigs and no metabolite in hamsters. Pretreatment of phenobarbital (PB) resulted in a marked increase of M-1 in three animals and of M-2in guinea pigs. In contrast, pretreatment of 3-methylcholanthrene showed a significant increase of M-3 in guinea pigs and a decrease of M-2in rats. Human liver microsomes prepared from nine Caucasians mainly formed M-2and M-3 at a ratio of 2: 1 and two individuals also formed one more metabolite M-1. The formation of M-1 was significantly correlated with CYP2B6 activity. By comparison of the GC-MS data of three metabolites with synthesized authentic samples, M-1 and M-2were determined to be 3'-hydroxy (OH)-CB146 and 4-OH-CB146, respectively. However, M-3 is unclear at present except the fact that it is OH-hexaCB. These results suggest that 3'-OH-CB146 is formed by PB-inducible cytochrome P450 (CYP2B enzymes) in animal and human livers and 4-OH-CB146 is a major metabolite in rat and human liver.

Hypertension and oxidant stress predispose to the onset and progression of arteriosclerotic diseases. In this study, the components of two kinds of porcine liver hydrolysates (LH-I and LH-II) were analyzed, and the antioxidant effects and angiotensin converting enzyme (ACE)-inhibiting effects of LH-I and LH-II were examined in vitro. Furthermore, the effects of LH-I and LH-II on the blood pressure were examined in spontaneously hypertensive rats (SHR). The results showed that peptides and amino acids accounted for 70% or more of the constituents of both LH-I and LH-II. The results of gel filtration HPLC showed that most of the nitrogen-containing components were peptides or amino acids with molecular weights of 6000 or less. The DPPH radical scavenging activities of LH-I and LH-II were 55.6 and 38.1 μM Trolox Equivalent/g, respectively. The IC(50) values for the ACE-inhibiting activity of LH-I and LH-II were 0.18 and 0.31 mg/mL, respectively. Oral administration of 1 g/rat of LH-I or LH-II to SHR resulted in significant lowering of the blood pressure. These findings indicate that both LH-I and LH-II have antioxidant activity and ACE-inhibiting activity. Moreover, both exerted a blood pressure-lowering effect in SHR. The antioxidant activity and ACE-inhibiting activity of LH-I and LH-II are presumed to be based on the actions of the component peptides.

Nanoparticles are defined as particles whose diameter is 1-100 nm. Many investigations about the toxicology of nanoparticles have been reported recently. The toxicity of nanoparticles has been examined both in vivo and in vitro and many results are being achieved. However, the results of in vivo and in vitro examinations are sometimes different. According to the in vitro examinations, it is suggested that solubility, adsorption ability and surface activity are involved in the cytotoxicity of nanoparticles. On the other hand, in in vivo, clearance is an important factor in the lung toxicity of nanoparticles. In the hazard assessment of nanoparticles, in vivo and in vitro examinations are necessary for an accurate evaluation of their biological influences, including the toxic mechanisms.

Topical dermatological formulations of non-steroidal anti-inflammatory drugs (NSAIDs) are reported to show their pharmacological effect partially through the systemic circulation, and to induce systemic side effects. However, pharmaceutical equivalence and pharmacokinetic bioequivalence between brand-name and generic products are not required. Therefore, we aimed to predict systemic drug exposure from brand-name and nine generic ketoprofen tapes. In vitro release profiles were examined using the paddle-over-disk method, then analyzed by the W. I. Higuchi equation incorporating an initial burst effect. Pharmacokinetic parameters were estimated from observed release profiles and the reported time-plasma concentration profile of the brand-name product. Plasma concentration profiles of generic products were predicted from the observed release profiles and the pharmacokinetic parameters of the brand-name product. In vitro release profiles differed markedly, and estimated release rates for initial burst effect and at 24 hours ranged from 4.20 to 88.75% and from 45.27 to 95.83%, respectively. The predicted plasma concentration profile of each product reflected its release profile, and estimated C(max) ranged from 61.70 to 290.30 ng/mL (0.46- to 2.15-fold vs. brand-name product). Generic products were classified into three types, i.e., systemic exposure comparable with, higher than and lower than that of brand-name product. C(max) was predicted to increase with enhanced skin permeability for all products, but the increase rates differed among products. These results suggest that safety and efficacy differ between brand-name and generic ketoprofen tapes. Healthcare professionals should carefully monitor systemic side effects, especially when switching from brand-name to generic products for which higher systemic exposure is predicted.

Dipeptidyl peptidase-4 (DPP-4) inhibitors were available in Japan since the end of 2009, and incretin-based therapies including glucagon-like peptide-1(GLP-1) mimetics are currently expected to be effective to Japanese patients with type 2 diabetes. Recently, there are a few reports that DPP-4 inhibitors cause severe hypoglycemia when combined with sulfonylureas although no major hypoglycemic episodes were reported in clinical studies. We discuss the mechanism of hypoglycemia by combination therapy, based on our islet studies about ATP-sensitive K+ channel inhibition by sulfonylureas and novel effects and mechanism of incretin.

The combination of anti-glaucoma eye drops is frequently used in clinical treatment, and it is known that the combination can cause corneal damage. Recently, an anti-glaucoma combination eye drops is developed, and the treatment by the combination eye drops is expected to enhance quality of life. However, effects of the combination eye drops on corneal epithelial cell damage have not been clarified. In this study, we investigated the corneal epithelial cell damage of commercially available anti-glaucoma combination eye drops, such as Xalacom® (latanoprost/timolol maleate combination eye drops), Duotrav® (travoprost/timolol maleate combination eye drops) and Cosopt® (dorzolamide hydrochloride/timolol maleate combination eye drops) using the human corneal epithelial cell (HCE-T). The cytotoxicity in Xalacom® was higher than that in Xalatan® (eye drops containing latanoprost) and Timoptol® (eye drops containing timolol maleate), and the benzalkonium chloride (BAC) and timolol maleate were related to cytotoxicity in Xalacom®. The cytotoxicity in Duotrav® and Cosopt® was lower than that in Timoptol®. The Duotrav® is preserved with a non-BAC system (POLYQUAD, polidronium chloride). Therefore, it was suggested that the POLYQUAD related to the low cytotoxicity in Duotrav®. On the other hand, the D-mannitol reduced the cytotoxicity by BAC in this study. This result suggested that the cytotoxicity in Cosopt® was reduced by D-mannitol. The Duotrav® and Cosopt® may be less damaging to the ocular surface of glaucoma patients receiving long-term eye drop therapy in compared with the combination of anti-glaucoma eye drops.

Unlabelled: The response rate of Irinotecan for gastric cancer is reported to be 18. 4%. The rate is improved by combination with 5-FU. However, it remains unclear whether or not the effect of the two drugs is synergy or antagonistic. The purpose of this study is to clarify whether the effect of Irinotecan and 5-FU in gastric cancer is synergy or antagonistic. We performed study using 13 specimens removed surgically and 2 specimen collected from ascites. We performed the Collagen Gel Droplet Embedded Culture Drug Sensitivity Test (CD-DST) with 3 assumptions. In the first assumption, we let 5-FU come in contact with a tumor at a level of 1 mg/mL for 24 hours. The second assumption was SN-38 at a level of 30 mg/mL for 24 hours and the 3rd assumption was SN-38 at a level of 30 mg/mL and 5-FU at a level of 1 mg/mL for 24 hours. If the combination index was more than 1, the combination therapy was judged as synergic; if less than 1, it was considered antagonistic., Results: The inhibition rate of combination therapy was significantly higher than that of monotherapy. The inhibition rate of combination therapy was significantly correlate with that of monotherapy (Irinotecan; r=0.704, p=0.003, 5-FU; r=0.746, p=0.001). The combination index was antagonistic in only 6 of 15 cases. However, it was synergic in all well-differentiated adenocarcinomas (4/4)., Discussion: We conclude that combination therapy is antagonistic in most cases of gastric cancer in vitro. However, it may be synergic in well-differentiated adenocarcinomas.

Background: Increased osmolality of the airway surface fluid due to water loss associated with hyperventilation is considered to be a cause of exercise-induced asthma (EIA)., Aim: We investigated the influence of changes in osmolality on airway epithelial ion transport in guinea pigs., Methods: We explored from measuring open circuit potential difference in challenge with hypertonic saline or mannitol., Results: Diphenylamine-2-carboxylate (DPC), amiloride, and disodium cromoglycate (DSCG) prevented an increase of the potential difference (PD) after exposure of tracheal mucosa to 0.9-10.8% hypertonic saline solutions (HSSs) (p < 0.05, p < 0.01, and p < 0.01, respectively on 1.8%). An increase of the PD was observed after a single dose of 1.8% hypertonic saline solution (HSS) was applied to the mucosa, but no increase was observed after a single exposure to a 585 mOsm/kg aqueous mannitol solution (585AMS). The results remained the same when a Cl-free solution was used as the perfusate. The change of airway epithelial cell thickness was only suppressed significantly by DPC (p < 0.01) after 1.8% HSS challenge., Conclusions: Not only the change of osmolality, but also changes of the Cl- and Na+ concentrations in airway surface fluid seem to have an important influence on the PD. cAMP-dependent Cl- channel may have a role, and the same mechanism may provoke EIA.

Propolis is known to have abundant bioactive constituents and a variety of biological activities. To investigate the effect of Brazilian propolis on insulin resistance, 10-week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a non-insulin-dependent type 2 diabetic model, were treated for 4 weeks with propolis (100 and 300 mg/kg, p.o.) or vehicle (control). Propolis treatment significantly decreased the plasma levels of insulin and insulin resistance index (Homeostasis Model Assessment-Insulin Resistance; HOM-IR), without affecting blood glucose levels and tended to lower systolic blood pressure compared with the control. In isolated and perfused mesenteric vascular beds of OLETF rats, propolis treatment resulted in significant reduction of sympathetic nerve-mediated vasoconstrictor response to periarterial nerve stimulation (PNS) and tended to increase calcitonin gene-related peptide (CGRP) nerve-mediated vasodilator response to PNS compared with in vehicle-treated OLETF rats. However, propolis treatment did not significantly affect the vasoconstrictor and vasodilator response to noradrenaline, CGRP, acetylcholine, and sodium nitroprusside. These results suggest that propolis could be an effective and functional food to prevent development of insulin resistance.

Half-logistic (h-L) function, which is half of the sigmoidal, logistic function with a boundary at the inflection point, curve-fits the isovolumic relaxation left ventricular (LV) pressure curve from the minimum of the first order time derivative of pressure (dP/dt(min)), and the myocardial isometric relaxation tension curve from the minimum of the first order time derivative of tension (dF/dt(min)) superior to the conventional mono-exponential function. Recently, we found that h-L function could curve-fit the other partial curves. The isovolumic LV pressure curve in the excised, cross-circulated canine heart, was divided into four distinct phases with boundaries set at the maximum of dP/dt (dP/dt(max)), peak LV pressure, dP/dt(min), and LV end-diastolic pressure (LVEDP) resulting in the first phase from the point corresponding to QR on the electrocardiogram to dP/dt(max); the second phase from dP/dt(max) to the peak LV pressure; the third phase from the peak LV pressure to dP/dt(min); and the fourth phase from dP/dt(min) to LVEDP. Similarly, the isometric twitch tension curves in the mouse LV and rabbit right ventricular (RV) papillary muscles were divided into four distinct phases with boundaries set at the maximum of dF/dt (dF/dt(max)), peak tension, dF/dt(min), and resting tension resulting in the first phase from the point corresponding to twitch stimulation to dF/dt(max); the second phase from dF/dt(max) to the peak tension; the third phase from the peak tension to dF/dt(min); and the fourth phase from dF/dt(min) to resting tension. The h-L correlation coefficient (r) values for the sequential curves were larger than the m-E r values, respectively, and the h-L residual mean squares (RMS) were smaller than the m-E RMS values, respectively. The h-L time constants are indices which quantify cardiac and myocardial inotropism and lusitropism more accurately. We consider that the h-L approach also applies for evaluation of the isovolumic contraction phase and the isovolumic relaxation phase in the beating hearts.

Many electrophysiological studies have been conducted on the 'limbic system', which is known to play a crucial role in mnemonic, emotional, and olfactory functions. Because of the difficulty in accessing such deep brain structures under in vivo conditions, in vitro brain slice preparations are often used. Recertly, an isolated whole brain preparation in which multi-synaptic circuits and the intracellular activity they generate are well preserved has also become available. In this paper, we described about the studies of the olfactory circuits by using this unique preparation. This experimental approach combines the advantages of the in vivo experimental condition with those of in vitro slice preparations, i.e. an intact synaptic network, excellent mechanical stability, and control over the ionic and biochemical extracellular environment. In particular, it provides easy access to the limbic system and preserves the neuronal network of the entorhinal-hippocampal loop. We used this preparation in combination with optical imaging performed using voltage-sensitive dyes in order to investigate how olfactory information is transferred from the olfactory bulb to the piriform, entorhinal and amygdaloid cortices. We visualized the propagation pattern of the neural activity after olfactory nerve stimulation and found that piriform and entorhinal activities converge in the amygdaloid cortex. This convergence may allow the amygdaloid cortex to integrate olfactory sensation with the information retained or processed in the entorhinal cortex.

Some of the cytokines and growth factors in the aqueous humor activate Rho, and the Rho/ROCK signal transduction participates in signaling pathways via rearrangement of actin cytoskeleton that leads to various cellular reactions. In a previous study, we demonstrated that a selective ROCK inhibitor significantly reduced intraocular pressure, the mechanism of which was attributed to improved outflow. ROCK inhibitors induced the actomyosin assembly, cell adhesive interactions, and the expression of extracellular matrix (ECM) proteins in cultured TM cells. The inhibition of Rho also has been implicated in pathological wound healing by regulating neovascularization, migration, and ECM in scar tissue. In this study, we investigated the role of ROCK inhibitor in regulating human Tenon fibroblast (HTF) activity and postoperative scar formation in a rabbit sclerostomy model. ROCK inhibitor showed decreased aSMA expression in HTF, and prevented enhanced contractility, assembly of actin stress fibers, and myofibroblastic transdifferentiation. In vivo sclerostomy studies showed that the bleb survival was significantly improved in ROCK inhibitor-treated eyes. In another study by us, ROCK inhibitor showed neuroprotective effects against rat retinal ischemia reperfusion injury. Collectively, ROCK inhibitors are thus a potential new strategy for developing medical therapy for glaucomatous dis-