Scalable Total Syntheses and Structure-Activity Relationships of Haouamines†A, B, and Their Derivatives as Stable Formate Salts.

Haouamines A, B, and their derivatives were synthesized via Suzuki-Miyaura coupling and three key cyclization reactions as follows: the newly developed palladium(0)-catalyzed arylative cyclization of phenylalanine-derived alkyne-aldehydes with 2-bromoarylboronic acid (an "anti-Wacker"-type cyclization); BF3 ⋅OEt2 -promoted Friedel-Crafts-type cyclization of symmetrical electron-rich aromatic rings adjacent to a tertiary allylic alcohol leading to the indeno-tetrahydropyridine skeleton; and (cyanomethyl)trimethylphosphonium iodide-mediated macrocyclization of amino alcohols to afford aza-paracyclophane precursors. The palladium-catalyzed reduction of mono- and di-triflate intermediates in the later stages enabled the alteration of both the position and number of hydroxyl groups on the C-ring. The instability of haouamine B was dramatically improved by salt formation with formic acid. An unambiguous evaluation of the cytotoxicity of the prepared haouamine derivative formates with and without hydroxyl groups at different positions on the C-ring indicated that the catechol structure in haouamine B produced weak cytotoxicity.

Evaluation of the effects of ontogenetic or maturation functions and chronic heart failure on the model analysis for the dose-response relationship of warfarin in Japanese children.

PURPOSE: We previously demonstrated that the rational pediatric dosage of warfarin can be well-described by a SIZE parameter that includes an allometry exponent of weight. On the other hand, allometry alone is considered to be insufficient to predict drug clearance in neonates and infants. The primary purpose of the present study was to evaluate the effects of incorporation of the maturation process into the analysis model for the dose-response relationship of warfarin in Japanese children. In addition, we evaluated the effect of chronic heart failure (CHF) on the response to warfarin as an independent risk factor for increased anticoagulant effects. METHODS: Thirty-eight patients with stable anticoagulation by warfarin were enrolled. During a mean follow-up period of 4.74 ± 3.51 years, 1092 data points including prothrombin time-international normalized ratio (PT-INR) were obtained. The data were subjected to multiple regression analysis to identify covariates related to the anticoagulant effects. RESULTS: Two different models describing the maturation process did not improve the predictive performance for the dose-response relationship in pediatric patients. In addition to the SIZE-normalized daily dose, the vitamin K epoxide reductase complex 1 (VKORC1) genotype, and concomitant use of bosentan, CHF was identified as a covariate increasing the anticoagulant effects of warfarin to 118%. CONCLUSION: The SIZE parameter was useful even without incorporation of maturation models to describe the response to warfarin in pediatric patients, and our longitudinal follow-up study design with multiple observations was beneficial to detect changes within individual subjects.

The possible clinical impact of risperidone on P-glycoprotein-mediated transport of tacrolimus: A case report and in vitro study.

The authors encountered the case of an 8-fold increase in the concentration/dose (C/D) ratio of tacrolimus (TAC) following the coadministration of voriconazole (VRCZ) in a hematopoietic stem cell transplantation (HSCT) recipient. The interaction observed was much greater than expected and the patient had also been treated with oral risperidone (RSP). It was hypothesized that cytochrome P450 (CYP)3A inhibition of the small intestine by voriconazole and P-glycoprotein (P-gp) inhibition of the small intestine by risperidone exerted a synergistic effect on the bioavailability of tacrolimus. The aim of the present study was to evaluate the effect of risperidone on the P-gp-mediated transport of tacrolimus. The transcellular transport of P-gp substrates was examined in Caco-2 and P-gp-expressing renal epithelial LLC-GA5-COL150 cells. In Caco-2 cells, the apical-basal (A-B) transport of rhodamine123 (Rh123) after a 120 min incubation was increased by 47.1%, whereas that in the B-A direction was decreased by 61.7% in the presence of risperidone (100 µm). These results indicate that risperidone showed an inhibitory effect on the P-gp-mediated transport of Rh123. In LLC-GA5-COL150 cells, the A-B transport of tacrolimus after 120 min incubation was increased by 21.7% in the presence of risperidone (100 µm), whereas that in the B-A direction was decreased by 10.7%. These results suggest that risperidone was at least partly involved in the mechanism of the marked increase in the C/D ratio of tacrolimus. This case report provides new insights into the diversity of drug interactions of tacrolimus triggered by the combination of two concomitant drugs.

The intrinsic microglial clock system regulates interleukin-6 expression.

Similar to neurons, microglia have an intrinsic molecular clock. The master clock oscillator Bmal1 modulates interleukin-6 upregulation in microglial cells exposed to lipopolysaccharide. Bmal1 can play a role in microglial inflammatory responses. We previously demonstrated that gliotransmitter ATP induces transient expression of the clock gene Period1 via P2X7 purinergic receptors in cultured microglia. In this study, we further investigated mechanisms underlying the regulation of pro-inflammatory cytokine production by clock molecules in microglial cells. Several clock gene transcripts exhibited oscillatory diurnal rhythmicity in microglial BV-2 cells. Real-time luciferase monitoring also showed diurnal oscillatory luciferase activity in cultured microglia from Per1::Luciferase transgenic mice. Lipopolysaccharide (LPS) strongly induced the expression of pro-inflammatory cytokines in BV-2 cells, whereas an siRNA targeting Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), a core positive component of the microglial molecular clock, selectively inhibited LPS-induced interleukin-6 (IL-6) expression. In addition, LPS-induced IL-6 expression was attenuated in microglia from Bmal1-deficient mice. This phenotype was recapitulated by pharmacological disruption of oscillatory diurnal rhythmicity using the synthetic Rev-Erb agonist SR9011. Promoter analysis of the Il6 gene revealed that Bmal1 is required for LPS-induced IL-6 expression in microglia. Mice conditionally Bmal1 deficient in cells expressing CD11b, including microglia, exhibited less potent upregulation of Il6 expression following middle cerebral artery occlusion compared with that in control mice, with a significant attenuation of neuronal damage. These results suggest that the intrinsic microglial clock modulates the inflammatory response, including the positive regulation of IL-6 expression in a particular pathological situation in the brain, GLIA 2016. GLIA 2017;65:198-208.

A randomized-controlled trial focusing on socio-economic status for promoting vegetable intake among adults using a web-based nutrition intervention programme: study protocol.

BACKGROUND: Web-based nutritional education programmes appear to be comparable to those delivered face-to-face. However, no existing web-based nutrition education or similar programme has yet been evaluated with consideration of socio-economic status. The objective of a nutritional education programme of promoting vegetable intake designed a randomized controlled trial (RCT) is to evaluate the results of intervention and to determine how socio-economic status influences the programme effects. METHODS/DESIGN: Participants will be randomly sampled individuals (aged 30-59) stratified according national population statistics for sex, age, and household income. Participants were consented to survey participation (n = 1500), and will be randomly divided into intervention and control groups. The intervention period is 5 weeks with one step of diet-related education per week. The main outcome of the programme is dietary behaviour as eating vegetable (350 g per day, five small bowl). To encourage behavioural changes, the programme contents are prepared using behavioural theories and techniques tailored to the assumed group stages of behavioural change. In the first step, we employ the health belief model to encourage a shift from the pre-contemplative to the contemplative phase; in the second and third steps, social cognitive theory is used to encourage transition to the preparatory phase; in the fourth step, social cognitive theory and strengthening social support are used to promote progression to the execution phase; finally, in the fifth step, strengthening social capital and social support are used to promote the shift to the maintenance phase. The baseline, post intervention and follow-up survey was assessed using a self-administered questionnaire. For process evaluation, we use five items relating to programme participation and satisfaction. A follow-up survey of participants will be carried out 3 months after intervention completion. DISCUSSION: The fact that this study is an RCT with an established control group is a strong advantage. Information and communications technology is not limited by time or place. If we could show this web-based nutrition education programmes has a positive effect, it may be an appropriate tool for reaching individuals in lower socio-economic state. TRIAL REGISTRATION: Current Controlled Trials UMIN-ICDR UMIN 000019376 (Registered October 16, 2015).

Reduction in Vegetable Intake Disparities With a Web-Based Nutrition Education Intervention Among Lower-Income Adults in Japan: Randomized Controlled Trial.

BACKGROUND: No existing Web-based nutrition education interventions have been evaluated in light of socioeconomic status just in Japan. OBJECTIVE: The aim was to investigate the effect of a Web-based intervention program on reducing vegetable intake disparities between low- and middle-income Japanese adults. METHODS: In this randomized controlled trial, participants were assessed at three time points-baseline, postintervention (5 weeks later), and a follow-up after 3 months-from October 2015 to March 2016. We collected data via a Japanese online research service company from 8564 adults aged 30 to 59 years. Participants were stratified according to national population statistics for gender and age, and randomly selected. They were then randomly allocated into intervention (n=900) and control (n=600) groups such that both groups contained an equal number of individuals with low and middle income. The intervention program encouraged behavior change using behavioral theories and techniques tailored to their assumed stage of change. The outcome was vegetable intake servings per day (1 serving being approximately 70 g). RESULTS: Out of 900 participants who started, 450 were from the middle income group (of which 386 or 85.7% completed the intervention), and 450 were from the low income group (of which 371 or 82.4% completed). In the intervention group, vegetable intake increased in the low-income participants from baseline to postintervention (0.42 servings, 95% CI 0.11-0.72). A two-way analysis of variance showed that low-income participants had significant main effects of group (η2=0.04, P=.01) and time (η2=0.01, P<.001), and a significant interaction (η2=0.01, P=.009). Middle-income participants also had a significant main effect of time (η2=0.01, P=.006) and a significant interaction (η2=0.01, P=.046). CONCLUSIONS: This Web-based nutritional education intervention could fill the vegetable intake gap between low- and middle-income adults in Japan, and is expected to prevent noncommunicable and lifestyle-related diseases. Further intervention program improvements are necessary to maintain and increase vegetable intake for other groups. TRIAL REGISTRATION: Current Controlled Trials (UMIN-ICDR): UMIN000019376; https://upload.umin.ac.jp/cgi-open-bin/ icdr_e/ctr_view.cgi?recptno=R000022404 (Archived by WebCite at http://www.webcitation.org/6u9wihBZU).

Taste-Masking Effect of Chlorogenic Acid (CGA) on Bitter Drugs Evaluated by Taste Sensor and Surface Plasmon Resonance on the Basis of CGA-Drug Interactions.

The purpose of this study was to evaluate the taste-masking effects of chlorogenic acid (CGA) on bitter drugs using taste sensor measurements and surface plasmon resonance (SPR) analysis of CGA-drug interactions. Six different bitter drugs were used: amlodipine besylate (AMD), diphenhydramine hydrochloride (DPH), donepezil hydrochloride (DNP), rebamipide (RBM), diclofenac sodium (DCF) and etodolac (ETD). Taste sensor outputs were significantly inhibited by the addition of CGA to all drugs. The inhibition ratio of the taste sensor output decreased in the following order DPH>DNP>AMD≈DCF≈RBM≈ETD. The association rate constant (ka) for the interaction between drugs and CGA as evaluated by SPR measurement also decreased in the following order DPH>DNP>AMD>DCF≈ETD≈RBM. It was suggested that basic drugs (AMD, DNP, DPH) associate more easily with CGA than acidic drugs (DCF, RBM, ETD). The inhibition ratios (%) of the taste sensor output of bitter drugs caused by CGA and the association rate constants (ka) between the drugs and CGA were significantly correlated (rs=0.886, p<0.05, Spearman's correlation test). Our findings suggest that the taste-masking effects of CGA are due to its direct association with the drugs. CGA may therefore be a useful taste-masking agent for basic drugs.

Suppression in Bitterness Intensity of Bitter Basic Drug by Chlorogenic Acid.

The purpose of the study was to evaluate suppression of the bitterness intensity of bitter basic drugs by chlorogenic acid (CGA) using the artificial taste sensor and human gustatory sensation testing and to investigate the mechanism underlying bitterness suppression using 1H-NMR. Diphenhydramine hydrocholoride (DPH) was the bitter basic drug used in the study. Quinic acid (QNA) and caffeic acid (CFA) together form CGA. Although all three acids suppressed the bitterness intensity of DPH in a dose-dependent manner as determined by the taste sensor and in gustatory sensation tests, CFA was less effective than either CGA or QNA. Data from 1H-NMR spectroscopic analysis of mixtures of the three acids with DPH suggest that the carboxyl group, which is present in both QNA and CGA but not CFA, interact with the amine group of DPH. This study showed that the bitterness intensity of DPH was suppressed by QNA and CGA through a direct electrostatic interaction with DPH as confirmed in 1H-NMR spectroscopic analysis. CGA and QNA may therefore be useful bitterness-masking agents for the basic drug DPH.

Protective upregulation of activating transcription factor-3 against glutamate neurotoxicity in neuronal cells under ischemia.

This study evaluates the pathological role of the stress sensor activating transcription factor-3 (ATF3) in ischemic neurotoxicity. Upregulation of the transcript and protein for ATF3 was seen 2-10 hr after reperfusion in the ipsilateral cerebral hemisphere of mice with transient middle cerebral artery occlusion for 2 hr. Immunohistochemical analysis confirmed the expression of ATF3 by cells immunoreactive for a neuronal marker in neocortex, hippocampus, and striatum within 2 hr after reperfusion. In murine neocortical neurons previously cultured under ischemic conditions for 2 hr, transient upregulation of both Atf3 and ATF3 expression was similarly found during subsequent culture for 2-24 hr under normoxia. Lentiviral overexpression of ATF3 ameliorated the neurotoxicity of glutamate (Glu) in cultured murine neurons along with a slight but statistically significant inhibition of both Fluo-3 and rhodamine-2 fluorescence increases by N-methyl-D-aspartate. Similarly, transient upregulation was seen in Atf3 and ATF3 expression during the culture for 48 hr in neuronal Neuro2A cells previously cultured under ischemic conditions for 2 hr. Luciferase reporter analysis with ATF3 promoter together with immunoblotting revealed the possible involvement of several transcription factors responsive to extracellular and intracellular stressors in the transactivation of the Atf3 gene in Neuro2A cells. ATF3 could be upregulated to play a role in mechanisms underlying mitigation of the neurotoxicity mediated by the endogenous neurotoxin Glu at an early stage after ischemic signal inputs.

Upregulation of Slc38a1 Gene Along with Promotion of Neurosphere Growth and Subsequent Neuronal Specification in Undifferentiated Neural Progenitor Cells Exposed to Theanine.

We have shown marked promotion of both cluster growth and neuronal specification in pluripotent P19 cells with overexpression of solute carrier 38a1 (Slc38a1), which is responsible for membrane transport of glutamine. In this study, we evaluated pharmacological profiles of the green tea amino acid ingredient theanine, which is a good substrate for glutamine transporters, on proliferation and neuronal specification in neural progenitor cells from embryonic rat neocortex. Sustained exposure to theanine, but not glutamine, accelerated the growth of neurospheres composed of proliferating cells and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reducing activity at concentrations of 1-100 µM in undifferentiated progenitor cells. Such prior exposure to theanine promoted spontaneous and induced commitment to a neuronal lineage with concomitant deteriorated astroglial specification. Selective upregulation was seen in the expression of Slc38a1 in progenitor cells cultured with theanine. Similarly significant increases in cluster growth and MTT reducing activity were found in P19 cells cultured with theanine for 4 days. Luciferase activity was doubled in a manner sensitive to the deletion of promoter regions in P19 cells with a luciferase reporter plasmid of the Slc38a1 promoter after sustained exposure to theanine for 4 days. Overexpression of X-box binding protein-1 led to a marked increase in luciferase activity in P19 cells transfected with the Slc38a1 reporter plasmid. These results suggest that theanine accelerates cellular proliferation and subsequent neuronal specification through a mechanism relevant to upregulation of Slc38a1 gene in undifferentiated neural progenitor cells.

In Vitro Enhancement of Carvedilol Glucuronidation by Amiodarone-Mediated Altered Protein Binding in Incubation Mixture of Human Liver Microsomes with Bovine Serum Albumin.

Carvedilol is mainly metabolized in the liver to O-glucuronide (O-Glu). We previously found that the glucuronidation activity of racemic carvedilol in pooled human liver microsomes (HLM) was increased, R-selectively, in the presence of amiodarone. The aim of this study was to clarify the mechanisms for the enhancing effect of amiodarone on R- and S-carvedilol glucuronidation. We evaluated O-Glu formation of R- and S-carvedilol enantiomers in a reaction mixture of HLM including 0.2% bovine serum albumin (BSA). In the absence of amiodarone, glucuronidation activity of R- and S-carvedilol for 25 min was 0.026, and 0.51 pmol/min/mg protein, and that was increased by 6.15 and 1.60-fold in the presence of 50 µM amiodarone, respectively. On the other hand, in the absence of BSA, or when BSA was replaced with human serum albumin, no enhancing effect of amiodarone on glucuronidation activity was observed, suggesting that BSA played a role in the mechanisms for the enhancement of glucuronidation activity. Unbound fraction of S-carvedilol in the reaction mixture was greater than that of R-carvedilol in the absence of amiodarone. Also, the addition of amiodarone caused a greater increase of unbound fraction of R-carvedilol than that of S-carvedilol. These results suggest that the altered protein binding by amiodarone is a key mechanism for R-selective stimulation of carvedilol glucuronidation.

Association of household income and education with eating behaviors in Japanese adults: a cross-sectional study.

BACKGROUND: Socioeconomic inequalities as social determinants of health are important issues in public health and health promotion. However, the association between socioeconomic status and eating behaviors has been investigated poorly in Japanese adults. To fill this gap, the present study examines the association of eating behaviors with household income and education. METHODS: The sample comprised 3,137 Japanese adults (1,580 men and 1,557 women) aged 30 to 59 years who responded to an Internet-based cross-sectional survey in 2014. Data on the following eating behaviors were collected via self-report: "taking care of one's diet for health," "eating vegetables," "frequency of eating breakfast," "frequency of family breakfasts," "frequency of family dinners," "using the information on nutrition labels," and "conversations with family or friends during meals." Self-reported data on socioeconomic status (household income and education) and demographic variables (gender, age, district of residence, marital status, residence status, and employment status) were also collected. The associations between eating behaviors and household income or education were tested using binomial logistic regression analysis with eating behaviors as dependent variables and household income and education as independent variables. A trend P -value was calculated for three categories of household income (less than 3,000,000 JPY, 3,000,000-7,000,000 JPY, and over 7,000,000 JPY) and education (junior high/high school, 2-year college, and 4-year college/graduate school). RESULTS: Higher household income and education were significantly associated with higher rates of eating vegetables, using the information on nutrition labels, and conversation with family or friends during meals in Japanese men and women. Higher household incomes were significantly associated with lower rates of frequency of family breakfasts in Japanese men and lower rates of frequency of family dinners in Japanese men and women. CONCLUSIONS: Higher socioeconomic status as indicated by household income or education was associated with eating more vegetables and conversation with family or friends during meals in Japanese men and women. Socioeconomic status should be considered in health promotion and diet improvement.

Variations in prebiotic oligosaccharide fermentation by intestinal lactic acid bacteria.

Prebiotic oligosaccharides confer health benefits on the host by modulating the gut microbiota. Intestinal lactic acid bacteria (LAB) are potential targets of prebiotics; however, the metabolism of oligosaccharides by LAB has not been fully characterized. Here, we studied the metabolism of eight oligosaccharides by 19 strains of intestinal LAB. Among the eight oligosaccharides used, 1-kestose, lactosucrose and galactooligosaccharides (GOSs) led to the greatest increases in the numbers of the strains tested. However, mono- and disaccharides accounted for more than half of the GOSs used, and several strains only metabolized the mono- and di-saccharides in GOSs. End product profiles indicated that the amounts of lactate produced were generally consistent with the bacterial growth recorded. Oligosaccharide profiling revealed the interesting metabolic manner in Lactobacillus paracasei strains, which metabolized all oligosaccharides, but left sucrose when cultured with fructooligosaccharides. The present study clearly indicated that the prebiotic potential of each oligosaccharide differs.

Upregulation of Runt-Related Transcription Factor-2 Through CCAAT Enhancer Binding Protein-ß Signaling Pathway in Microglial BV-2 Cells Exposed to ATP.

We have shown constitutive expression of the master regulator of osteoblastogenesis, runt-related transcription factor-2 (Runx2), by microglia cells outside bone. Here, we attempted to evaluate the pathological significance of Runx2 in microglial BV-2 cells exposed to ATP at a high concentration. Marked upregulation of Runx2 transcript and protein expression was seen in cells exposed to 1 mM ATP for a period longer than 30 min without inducing cytotoxicity. The Runx2 upregulation by ATP was prevented by extracellular and intracellular Ca(2+) chelators, while thapsigargin upregulated Runx2 expression alone without affecting the upregulation by ATP. A calmodulin antagonist prevented the upregulation by ATP, with calcineurin inhibitors being ineffective. Although ATP markedly increased nuclear levels of nuclear factor of activated T cell-2 (NFAT2), Runx2 promoter activity was not simulated by the introduction of either NFAT1 or NFAT2, but facilitated by that of CCAAT enhancer binding protein-α (C/EBPα), C/EBPß and nuclear factor (erythroid-derived 2)-like-2 (Nrf2). Exposure to ATP up-regulated C/EBPß and Nrf2, but not C/EBPα, expression, in addition to increasing nuclear levels of respective corresponding proteins. Runx2 upregulation by ATP was deteriorated by knockdown of C/EBPß but not by that of Nrf2, however, while exposure to ATP up-regulated matrix metalloproteinase-13 (Mmp13) expression in a Runx2-dependent manner. Overexpression of Runx2 up-regulated Mmp13 expression with promoted incorporation of fluorescent beads into BV-2 cells without ATP. These results suggest that extracellular ATP up-regulates Runx2 expression through activation of the C/EBPß signaling in a calmodulin-dependent manner to play a pivotal role in phagocytosis in microglial BV-2 cells.

Daily oral intake of theanine prevents the decline of 5-bromo-2'-deoxyuridine incorporation in hippocampal dentate gyrus with concomitant alleviation of behavioral abnormalities in adult mice with severe traumatic stress.

Posttraumatic stress disorder is a long-lasting psychiatric disease with the consequence of hippocampal atrophy in humans exposed to severe fatal stress. We demonstrated a positive correlation between the transient decline of 5-bromo-2'-deoxyuridine (BrdU) incorporation in the hippocampal dentate gyrus (DG) and long-lasting behavioral abnormalities in mice with traumatic stress. Here, we investigated pharmacological properties of theanine on the declined BrdU incorporation and abnormal behaviors in mice with traumatic stress. Prior daily oral administration of theanine at 50-500 mg/kg for 5 days significantly prevented the decline of BrdU incorporation, while theanine significantly prevented the decline in the DG even when administered for 5 days after stress. Consecutive daily administration of theanine significantly inhibited the prolonged immobility in mice with stress in forced swimming test seen 14 days later. Although traumatic stress significantly increased spontaneous locomotor activity over 30 min even when determined 14 days later, the increased total locomotion was significantly ameliorated following the administration of theanine at 50 mg/kg for 14 days after stress. These results suggest that theanine alleviates behavioral abnormalities together with prevention of the transient decline of BrdU incorporation in the hippocampal DG in adult mice with severe traumatic stress.

Accumulation of alkaline earth metals by the green macroalga Bryopsis maxima.

Twenty-five days after the disaster at the Fukushima Daiichi nuclear power plant in 2011, we collected samples of the green macroalga Bryopsis maxima from the Pacific coast of Japan. Bryopsis maxima is a unicellular, multinuclear, siphonous green macroalga. Radiation analysis revealed that B. maxima emitted remarkably high gamma radiation of (131)I, (134)Cs, (137)Cs, and (140)Ba as fission products of (235)U. Interestingly, B. maxima contained naturally occurring radionuclides derived from (226)Ra and (228)Ra. Analysis of element content revealed that B. maxima accumulates many ocean elements, especially high quantities of the alkaline earth metals Sr (15.9 g per dry-kg) and Ba (3.79 g per dry-kg), whereas Ca content (12.5 g per dry-kg) was lower than that of Sr and only 61 % of the mean content of 70 Japanese seaweed species. Time-course analysis determined the rate of radioactive (85)Sr incorporation into thalli to be approximately 0.13 g Sr per dry-kg of thallus per day. Subcellular fractionation of B. maxima cells showed that most of the (85)Sr was localized in the soluble fraction, predominantly in the vacuole or cytosol. Given that (85)Sr radioactivity was permeable through a dialysis membrane, the (85)Sr was considered to be a form of inorganic ion and/or bound with a small molecule. Precipitation analysis with sodium sulfate showed that more than 70% of the Sr did not precipitate as SrSO4, indicating that a proportion of the Sr may bind with small molecules in B. maxima.

Contribution of CYP3A isoforms to dealkylation of PDE5 inhibitors: a comparison between sildenafil N-demethylation and tadalafil demethylenation.

The aim of this study was to characterize the kinetics of metabolite formation of the phosphodiesterase type-5 (PDE5) inhibitors sildenafil and tadalafil by CYP3A4, CYP3A5, and CYP3A7 isoforms. The formations of N-desmethyl sildenafil and desmethylene tadalafil were examined using CYP3A supersomes co-expressing human P450 oxidoreductase and cytochrome b5. Both sildenafil N-demethylation and tadalafil demethylenation were catalyzed by CYP3A4, CYP3A5, and to a lesser extent by CYP3A7. The kinetics of desalkyl metabolite formation of the two drugs were well fitted to the Hill equation; however, the Hill coefficients (n) suggested CYP3A-mediated negative cooperativity. Next, we analyzed the kinetics with a two binding sites model assuming two reaction steps: reaction 1 with high-affinity and low-capacity metabolism and reaction 2 with low-affinity and high-capacity metabolism. The kinetics of desalkyl metabolite formation were also fitted to the two binding sites model. The intrinsic clearance (CLint) values of reactions 1 and 2 for sildenafil N-demethylation were 0.733 and 0.033 µL/min/pmol P450 for CYP3A4, 0.788 and 0.019 µL/min/pmol P450 for CYP3A5, and 0.079 and 0.004 µL/min/pmol P450 for CYP3A7, respectively. The CLint values of reactions 1 and 2 for tadalafil demethylenation were 0.187 and 0.014 µL/min/pmol P450 for CYP3A4, 0.050 and <0.001 µL/min/pmol P450 for CYP3A5, and 0.004 and <0.001 µL/min/pmol P450 for CYP3A7, respectively. These results may provide the basis not only for understanding the metabolic properties of the two PDE5 inhibitors, but also for one possible explanation of the mechanisms of CYP3A-mediated negative cooperativity.

Pharmacological characterization of standard analgesics on oxaliplatin-induced acute cold hypersensitivity in mice.

Oxaliplatin, a platinum-based chemotherapeutic agent, causes an acute peripheral neuropathy triggered by cold in almost all patients during or within hours after its infusion. We recently reported that a single administration of oxaliplatin induced cold hypersensitivity 2 h after the administration in mice. In this study, we examined whether standard analgesics relieve the oxaliplatin-induced acute cold hypersensitivity. Gabapentin, tramadol, mexiletine, and calcium gluconate significantly inhibited and morphine and milnacipran decreased the acute cold hypersensitivity, while diclofenac and amitriptyline had no effects. These results suggest that gabapentin, tramadol, mexiletine, and calcium gluconate are effective against oxaliplatin-induced acute peripheral neuropathy.

Phenotypic screening using waveform analysis of synchronized calcium oscillations in primary cortical cultures.

At present, in vitro phenotypic screening methods are widely used for drug discovery. In the field of epilepsy research, measurements of neuronal activities have been utilized for predicting efficacy of anti-epileptic drugs. Fluorescence measurements of calcium oscillations in neurons are commonly used for measurement of neuronal activities, and some anti-epileptic drugs have been evaluated using this assay technique. However, changes in waveforms were not quantified in previous reports. Here, we have developed a high-throughput screening system containing a new analysis method for quantifying waveforms, and our method has successfully enabled simultaneous measurement of calcium oscillations in a 96-well plate. Features of waveforms were extracted automatically and allowed the characterization of some anti-epileptic drugs using principal component analysis. Moreover, we have shown that trajectories in accordance with the concentrations of compounds in principal component analysis plots were unique to the mechanism of anti-epileptic drugs. We believe that an approach that focuses on the features of calcium oscillations will lead to better understanding of the characteristics of existing anti-epileptic drugs and allow to predict the mechanism of action of novel drug candidates.

Acute cold hypersensitivity characteristically induced by oxaliplatin is caused by the enhanced responsiveness of TRPA1 in mice.

BACKGROUND: Oxaliplatin, a platinum-based chemotherapeutic agent, causes an unusual acute peripheral neuropathy. Oxaliplatin-induced acute peripheral neuropathy appears in almost all patients rapidly after infusion, and is triggered or exacerbated by cold, while its mechanisms are poorly understood. In this study, the involvement of thermosensitive transient receptor potential channels (TRPA1, TRPM8 and TRPV1) in oxaliplatin-induced acute hypersensitivity was investigated in mice. RESULTS: A single intraperitoneal administration of oxaliplatin (1-10 mg/kg) induced cold but not mechanical hypersensitivity within 2 h in a dose-dependent manner. Infusion of the oxaliplatin metabolite, oxalate (1.7 mg/kg), also induced acute cold hypersensitivity, while another platinum-based chemotherapeutic agent, cisplatin (5 mg/kg), or the non-platinum-containing chemotherapeutic agent, paclitaxel (6 mg/kg) failed to induce mechanical or cold hypersensitivity. The oxaliplatin-induced acute cold hypersensitivity was abolished by the TRPA1 antagonist HC-030031 (100 mg/kg) and by TRPA1 deficiency. The nocifensive behaviors evoked by intraplantar injections of allyl-isothiocyanate (AITC; TRPA1 agonist) were significantly enhanced in mice treated for 2 h with oxaliplatin (1-10 mg/kg) in a dose-dependent manner, while capsaicin (TRPV1 agonist)-evoked nocifensive behaviors were not affected. Menthol (TRPM8/TRPA1 agonist)-evoked nocifensive-like behaviors were also enhanced by oxaliplatin pretreatment, which were inhibited by TRPA1 deficiency. Similarly, oxalate enhanced, but neither cisplatin nor paclitaxel affected AITC-evoked nocifensive behaviors. Pretreatment of cultured mouse dorsal root ganglia (DRG) neurons with oxaliplatin (30-300 µM) for 1, 2, or 4 h significantly increased the number of AITC-sensitive neurons in a concentration-dependent manner whereas there was no change in the number of menthol- or capsaicin-sensitive neurons. CONCLUSIONS: Taken together, these results suggest that a brief treatment with oxaliplatin or its metabolite oxalate is sufficient to enhance the responsiveness of TRPA1 but not that of TRPM8 and TRPV1 expressed by DRG neurons, which may contribute to the characteristic acute peripheral neuropathy induced by oxaliplatin.