[In vivo integrated safety assessment of the cardiovascular system in drug development].

Drug-induced cardiotoxicity still remains a major cause of concern, and non-clinical integrated risk assessments from both functional and structural alterations in the cardiovascular system are strongly required in the creation of drugs with superior safety profiles. Although systemic blood pressure, heart rate, and electrocardiogram are the main items in safety pharmacology studies, direct cardiac function assessments such as cardiac output and ventricular contractility, mentioned in ICH S7A guideline, are also desirable. General toxicology studies are important to detect structural changes through clinical pathology and histopathological examination, and translational biomarkers and metabolomics analysis with high extrapolation to humans also provide useful insights. In this paper, we will introduce our basic research to investigate the cardiac effects of milrinone, a cAMP phosphodiesterase III inhibitor in cynomolgus monkeys, and share the importance of comprehensive risk assessment in non-clinical in vivo studies.

Trastuzumab-Induced Negative Chronotropic and Lusitropic Effects in Cynomolgus Monkeys.

ABSTRACT: Treatment with trastuzumab, an antihuman epidermal growth factor receptor type 2 humanized monoclonal antibody, has been associated with heart failure in certain patients with cancer; however, the mechanism underlying trastuzumab-induced cardiac dysfunction remains unclear. This study was conducted to clarify the cardiac effects of trastuzumab in cynomolgus monkeys, which are commonly used as cross-reactive species in preclinical safety evaluation. Monkeys were treated with trastuzumab weekly for 1 month (5 doses in total). At first and fifth doses for pressure-volume loop analysis, trastuzumab at 20 mg·kg-1·10 min-1, equivalent to the human therapeutic dose, was administered intravenously to isoflurane-anesthetized animals, followed by 60 mg·kg-1·10 min-1 at a 30-minute interval. The other doses were fixed at 80 mg·kg-1·10 min-1 under unanesthetized conditions. After the first dose, reduced heart rate, decreases in maximal rate of fall of left ventricular pressure, and prolonged time constant for isovolumic relaxation, which are predictors of drug-induced changes in lusitropy, were observed at 20 and 60 mg·kg-1. The changes after the fifth dose were comparable with those after the first dose, indicating trastuzumab did not show exacerbation of cardiac function during the 1-month trial. No significant changes in slope of preload recruitable stroke work, which is a load-independent inotropic parameter, were observed at either dose. In conclusion, trastuzumab-induced little inotropic effect but induced negative chronotropic or lusitropic effects in monkeys, which might be associated with impaired left ventricular diastolic function.

Time for a Fully Integrated Nonclinical-Clinical Risk Assessment to Streamline QT Prolongation Liability Determinations: A Pharma Industry Perspective.

Defining an appropriate and efficient assessment of drug-induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc-prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14-based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B-based "double-negative" nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high-dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double-negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

Comprehensive analysis of cardiac function, blood biomarkers and histopathology for milrinone-induced cardiotoxicity in cynomolgus monkeys.

The objective of this study was to elucidate the underlying cardiotoxic mechanism of milrinone, a cAMP phosphodiesterase 3 inhibitor, by evaluating cardiac functions, blood biomarkers including cardiac troponin I (cTnI), microRNAs (miR-1, miR-133a and miR-499a) and various endogenous metabolites, and histopathology in conscious cynomolgus monkeys. Milrinone at doses of 0, 3 and 30 mg/kg were orally administered to monkeys (n = 3-4/group), and the endpoints were evaluated 1 to 24 h post-dosing. Milrinone caused myocardial injuries characterized by myocardial degeneration/necrosis, cell infiltration and hemorrhage 24 h after drug administration. Cardiac functional analysis revealed that milrinone dose-dependently increased the maximum upstroke velocity of the left ventricular pressure and heart rate, and decreased the QA interval and systemic blood pressure 1-4 h post-dosing, being associated with pharmacological action of the drug. In the blood biomarker analysis, only plasma cTnI was dose-dependently increased 4-7 h after drug administration, suggesting that cTnI is the most sensitive biomarker for early detection of milrinone-induced myocardial injuries. In the metabolomics analysis, high dose of milrinone induced transient changes in lipid metabolism, amino acid utilization and oxidative stress, together with the pharmacological action of increased cAMP and lipolysis 1 h post-dosing before the myocardial injuries were manifested by increased cTnI levels. Taken together, milrinone showed acute positive inotropic and multiple metabolic changes including excessive pharmacological actions, resulting in myocardial injuries. Furthermore, a comprehensive analysis of cardiac functions, blood biomarkers and histopathology can provide more appropriate information for overall assessment of preclinical cardiovascular safety.

Ultrasonographic measurement of the renal resistive index in the cynomolgus monkey (Macaca fascicularis) under conscious and ketamine-immobilized conditions.

Measurement of the renal resistive index (RRI) is one of the standard diagnostic procedures for assessing kidney disability clinically. This method is expected to be used for the same purpose in many kinds of animals, including monkeys utilized in conventional toxicology studies. To establish a practical RRI measurement procedure in cynomolgus monkeys (Macaca fascicularis), RRI was measured by ultrasonography in the spine position in conscious and ketamine-immobilized monkeys. The RRI of conscious monkeys and ketamine-immobilized monkeys could be measured consistently without excessive abdominal or thoracic movement. Consequently, the variability of the RRI in conscious monkeys was comparable to that in ketamine-anesthetized monkeys. No sex difference in RRI was noted between the two conditions. The mean values and SD of the RRI of 48 healthy monkeys (n=24/sex) were 0.55 ± 0.07 and 0.50 ± 0.05, under conscious and ketamine-immobilized conditions, respectively. The RRI of ketamine-immobilized monkeys was significantly lower than that of conscious monkeys, correlating with the decreased blood pressure and heart rate. In a monkey model of cisplatin-induced acute renal injury, which was characterized histopathologically by minimal to mild renal tubular necrosis and regeneration, the RRI was increased beyond the cut off value (mean + 2SD, 0.68) associated with the progression of renal pathogenesis. The present results suggest that ultrasonographic measurement of the RRI in conscious monkeys would be a useful tool in conventional toxicology studies evaluating drug-induced renal injury.

Exposure-response analysis of drug-induced QT interval prolongation in telemetered monkeys for translational prediction to human.

INTRODUCTION: The preclinical in vivo assay for QT prolongation is critical for predicting torsadogenic risk, but still difficult to extrapolate to humans. This study ran preclinical tests in cynomolgus monkeys on seven QT reference drugs containing the drugs used in the IQ-CSRC clinical trial and applied exposure-response (ER) analysis to the data to investigate the potential for translational information on the QT effect. METHODS: In each of six participating facilities in the J-ICET project, telemetered monkeys were monitored for 24 h following administration of vehicle or 3 doses of test drugs, and pharmacokinetic profiles at the same doses were evaluated separately. An individual rate-corrected QT interval (QTca) was derived and the vehicle-adjusted change in QTca from baseline (∆∆QTca) was calculated. Then the relationship of concentration to QT effect was evaluated by ER analysis. RESULTS: For QT-positive drugs in the IQ-CSRC study (dofetilide, dolasetron, moxifloxacin, ondansetron, and quinine) and levofloxacin, the slope of the total concentration-QTca effect was significantly positive, and the QT-prolonging effect, taken as the upper bound of the confidence interval for predicted ∆∆QTca, was confirmed to exceed 10 ms. The ER slope of the negative drug levocetirizine was not significantly positive and the QTca effect was below 10 ms at observed peak exposure. DISCUSSION: Preclinical QT assessment in cynomolgus monkeys combined with ER analysis could identify the small QT effect induced by several QT drugs consistently with the outcomes in humans. Thus, the ER method should be regarded as useful for translational prediction of QT effects in humans.

Negative lusitropic property of nifekalant identified using ventricular pressure-volume loop analyses in anesthetized monkeys.

The present study was conducted to clarify multiple cardiohemodynamic and electrophysiological properties including inotropic/lusitropic effects of nifekalant, a class III antiarrhythmic drug, in an isoflurane-anesthetized monkey. Nifekalant was administered intravenously at the therapeutic dose of 0.3 mg/kg over 10 min to male cynomolgus monkeys (n=4), followed by higher dose of 1 (n=3) or 3 mg/kg (n=1) that was limited due to arrythmogenicity. Left ventricular (LV) pressure-volume (PV) analysis revealed that the 0.3 mg/kg dose of nifekalant induced a negative lusitropic effect, recognized as a decrease in maximal rate of reduction in LV pressure and a prolonged isovolumic relaxation time. Nifekalant also decreased heart rate and increased LV end-diastolic pressure, but had no effects on the other cardiohemodynamic parameters examined. Electrophysiological analysis showed nifekalant at 0.3 mg/kg prolonged QT/QTc intervals with no evidence of arrhythmia. Higher doses of nifekalant induced ventricular arrhythmia in 3 out of 4 animals, in which both the short-term and long-term variability of the QT interval increased just before the occurrence of arrhythmia. In conclusion, a therapeutic dose of nifekalant had no effect on inotropic activity or cardiac compliance, whereas it showed negative lusitropic properties and QT/QTc prolongation in isoflurane-anesthetized monkeys. In addition, higher doses of nifekalant showed remarkable QT/QTc prolongation leading to arrhythmogenicity, which showed good accordance with clinical findings. Caution should be paid to negative lusitropic properties as well as arrhythmogenisity for the safe use of nifekalant.

DS-8500a, an Orally Available G Protein-Coupled Receptor 119 Agonist, Upregulates Glucagon-Like Peptide-1 and Enhances Glucose-Dependent Insulin Secretion and Improves Glucose Homeostasis in Type 2 Diabetic Rats.

G protein-coupled receptor 119 (GPR119) has been shown to be highly expressed in small intestinal L-cells and pancreatic ß-cells and mediates intracellular cAMP concentration, glucagon-like peptide (GLP-1) secretion, and glucose-stimulated insulin secretion (GSIS). This study examined the pharmacological effects of 4-(5-{(1R)-1-[4-(cyclopropylcarbonyl) phenoxy]propyl}-1,2,4-oxadiazol-3-yl)-2-fluoro-N-[(2R)-1-hydroxypropan-2-yl]benzamide (DS-8500a), a novel, orally available, selective GPR119 agonist. In in vitro studies, DS-8500a increased intracellular cAMP in a concentration-dependent manner in human, rat, and mouse GPR119-expressing Chinese hamster ovary (CHO)-K1 cells, with EC50 values of 51.5, 98.4, and 108.1 nmol/l, respectively. DS-8500a had no effect on intracellular cAMP in pcDNA3.1/CHO-K1 cells. In in vivo studies, DS-8500a augmented plasma GLP-1 concentration in Zucker fatty (ZF) rats, and enhanced GSIS and did not change plasma glucose concentration in fasted Sprague-Dawley (SD) rats. A single dose of DS-8500a showed dose-dependent glucose-lowering effects at oral glucose tolerance test (OGTT) in ZF rats. In a repeat-dosing study, DS-8500a had statistically significant glucose-lowering effects at OGTT performed at the 1st day and after 2 weeks of treatment in neonatal streptozotocin-treated (nSTZ) rats, and the efficacy levels of DS-8500a in each test were greater than those of GSK1292263 or MBX-2982, which had been clinically tested previously as GPR119 agonists. Through pharmacokinetics and pharmacodynamics assessment, the high intrinsic activity of DS-8500a was suggested to be one of the reasons for the greater glucose lowering effect in the nSTZ rats. DS-8500a is a useful compound among GPR119 agonists that can maximize the potential benefit of GPR119 in type 2 diabetes.

Inotropic Effects of Nicorandil on Cardiac Contractility Assessed by Left Ventricular Pressure-Volume Relationship Analyses in Anesthetized Monkeys.

Nicorandil is a representative antianginal drug that has dual properties of a nitrate and adenosine triphosphate-sensitive potassium (KATP) channel agonist; however, its effects on integrated cardiac function have not been fully understood. This study was conducted to clarify the functional, hemodynamic, and electrophysiological effects of nicorandil using ventricular pressure-volume loop analysis in isoflurane-anesthetized monkeys. Nicorandil was given intravenously at therapeutic doses of 0.2 and 2 mg/kg over 10 minutes to cynomolgus monkeys (n = 5) with a pause of 10 minutes between the 2 doses. Nicorandil at 0.2 mg/kg caused decreases in systemic blood pressure and left ventricular end-diastolic pressure by its vasodilating action. Nicorandil at 2 mg/kg also exhibited positive inotropic action demonstrated by increased slopes of preload recruitable stroke work relationship, which is a load-independent inotropic parameter. In load-dependent inotropic parameters, positive inotropy of nicorandil was also indicated by the shortened QA interval and increased contractility index; however, significant changes were not observed in the maximal upstroke velocity of left ventricular pressure. Moreover, reflex tachycardia accompanied by shortening of QT/QTc intervals was observed. Overall, the isoflurane-anesthetized monkey model with pressure-volume loop analysis revealed cardiac variables of nicorandil, including a positive inotropy contributable to cardiac performance in addition to its vasodilatory effect. These findings provide useful information when considering the prescription of nicorandil in patients.

Usefulness of simultaneous and sequential monitoring of glucose level and electrocardiogram in monkeys treated with gatifloxacin under conscious and nonrestricted conditions.

Drug-induced cardiac electrophysiological abnormalities accompanied by hypoglycemia or hyperglycemia increase the risk for life-threatening arrhythmia. To assess the drug-induced cardiotoxic potential associated with extraordinary blood glucose (GLU) levels, the effect of gatifloxacin (GFLX) which was frequently associated with GLU abnormality and QT/QTc prolongations in the clinic on blood GLU and electrocardiogram (ECG) parameters was investigated in cynomolgus monkeys (n=4) given GFLX orally in an ascending dose regimen (10, 30, 60 and 100 mg/kg). Simultaneous and sequential GLU and ECG monitoring with a continuous GLU monitoring system and Holter ECG, respectively, were conducted for 24 h under free-moving conditions. Consequently, GFLX at 30 and 60 mg/kg dose-dependently induced a transient decrease in GLU without any ECG abnormality 2-4 h postdose. Highest dose of 100 mg/kg caused severe hypoglycemia with a mean GLU of <30 mg/dL, accompanied by remarkable QT/QTc prolongations by 20-30% in all animals. In contrast, hyperglycemia without QT/QTc prolongations was noted 24 h after dosing in one animal. A close correlation between GLU and QTc values was observed in animals treated with 100 mg/kg, suggesting that GFLX-induced hypoglycemia enhanced QT/QTc prolongations. Furthermore, the 24-h sequential GLU monitoring data clearly distinguished between GFLX-induced GLU abnormality and physiological GLU changes influenced by feeding throughout the day. In conclusion, the combined assessment of continuous GLU and ECG monitoring is valuable in predicting the drug-induced cardio-electrophysiological risk associated with both GLU and ECG abnormalities.

Promising approach for the preclinical assessment of cardiac risks using left ventricular pressure-volume loop analyses in anesthetized monkeys.

INTRODUCTION: Load-independent cardiac parameters obtained from the ventricular pressure-volume relationship are recognized as gold standard indexes for evaluating cardiac inotropy. In this study, for better analyses of cardiac risks, load-independent pressure-volume loop parameters were assessed in addition to load-dependent inotropic, hemodynamic and electrocardiographic changes in isoflurane-anesthetized monkeys. METHODS: The animals were given milrinone (a PDE 3 inhibitor), metoprolol (a ß-blocker), or dl-sotalol (a ß+IKr blocker) intravenously over 10min at two dose levels including clinically relevant doses (n=5/drug). RESULTS: Milrinone and metoprolol produced positive and negative inotropy, respectively. These effects were detected as changes in the slope of the preload-recruitable stroke work, which is a load-independent inotropic parameter. However, dl-sotalol did not alter the slope of the preload-recruitable stroke work. That means dl-sotalol produced no inotropy, although it decreased load-dependent inotropic parameters, including maximal upstroke velocity of left ventricular pressure, attributable to decreased heart rate and blood pressure. Other typical pharmacological effects of the compounds tested were also detected. Both ß-blockers produced PR prolongation, decreased left ventricular end-systolic pressure, increased left ventricular end-diastolic pressure, and increased maximal descending velocity of left ventricular pressure and time constant for isovolumic relaxation. dl-Sotalol also prolonged heart-rate-corrected QT interval. Milrinone induced reflex tachycardia, PR shortening, and decreased left ventricular end-diastolic pressure. DISCUSSION: The overall assessment by not only load-dependent inotropic parameters but also load-independent parameters obtained from the ventricular pressure-volume loop analysis using monkeys can provide further appropriate information for the assessment of drug-induced cardiac risks.

Enhanced Immunostimulating Activity of Lactobacilli-Mimicking Materials by Controlling Size.

The design and synthesis of materials capable of activating the immune system in a safe manner is of great interest in immunology and related fields. Lactobacilli activate the innate immune system of a host when acting as probiotics. Here, we constructed lactobacilli-mimicking materials in which polysaccharide-peptidoglycan complexes (PS-PGs) derived from lactobacilli were covalently conjugated to the surfaces of polymeric microparticles with a wide variety of sizes, ranging from 200 nm to 3 µm. The artificial lactobacilli successfully stimulated macrophages without cytotoxicity. Importantly, we found that the size of artificial lactobacilli strongly influenced their immunostimulating activities, and that artificial lactobacilli of 1 µm exhibited 10-fold higher activity than natural lactobacilli. One major advantage of the artificial lactobacilli is facile control of size, which cannot be changed in natural lactobacilli. These findings provide new insights into the design of materials for immunology as well as the molecular biology of lactobacillus.

Synthesis and immunestimulating activity of lactobacilli-originated polysaccharide-polymeric microparticle conjugates.

The design and synthesis of biomaterials capable of activating the immune system are of interest in immunology-related fields because of their ability to tune up the immune defenses of the host. Lactobacilli are a major constituent of normal human indigenous flora, and some specific strains are known to activate the immune system of the host as probiotics. In this study, we first fabricated novel biohybrid materials in which lactobacilli (L. casei strain Shirota, LcS)-originated polysaccharide-peptidoglycan complexes (PS-PGs) are conjugated with polymeric microparticles (MPs). PS-PGs conjugated onto polymeric MPs surfaces bound its specific antibody, suggesting that PS-PGs kept their original molecular recognition ability. The PS-PGs-based hybrid MPs with an appropriate density of conjugated PS-PGs effectively induced high levels of IL-12 production from macrophages without cytotoxicity. These results suggest that LcS-originated PS-PGs could be available bio-originated materials for developing novel biomaterials capable of activating the immune system in a safe manner.

Oral administration of Bifidobacterium breve attenuates UV-induced barrier perturbation and oxidative stress in hairless mice skin.

Recent studies have shown that some probiotics affect not only the gut but also the skin. However, the effects of probiotics on ultraviolet (UV)-induced skin damage are poorly understood. In this study, we aim to examine whether oral administration of live Bifidobacterium breve strain Yakult (BBY), a typical probiotic, can attenuate skin barrier perturbation caused by UV and reactive oxygen species (ROS) in hairless mice. The mice were orally supplemented with a vehicle only or BBY once a day for nine successive days. Mouse dorsal skin was irradiated with UV from days 6 to 9. The day after the final irradiation, the transepidermal water loss (TEWL), stratum corneum hydration, and oxidation-related factors of the skin were evaluated. We elucidated that BBY prevented the UV-induced increase in TEWL and decrease in stratum corneum hydration. In addition, BBY significantly suppressed the UV-induced increase in hydrogen peroxide levels, oxidation of proteins and lipids, and xanthine oxidase activity in the skin. Conversely, antioxidant capacity did not change regardless of whether BBY was administered or not. In parameters we evaluated, there was a positive correlation between the increase in TEWL and the oxidation levels of proteins and lipids. Our results suggest that oral administration of BBY attenuates UV-induced barrier perturbation and oxidative stress of the skin, and this antioxidative effect is not attributed to enhancement of antioxidant capacity but to the prevention of ROS generation.

Synthesis and optimization of novel (3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as orally active renin inhibitors.

We report synthesis and optimization of a series of (3S,5R)-5-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)piperidine-3-carboxamides as renin inhibitors. Chemical modification of P1', P2' and P3 portions led to a promising 3,5-disubstituted piperidine 32o showing high renin inhibitory activity and favorable oral exposure in both rats and cynomolgus monkeys with acceptable CYP and hERG current inhibition. Compound 32o exhibited a significant blood pressure lowering effect by oral administration in two hypertensive animal models, double transgenic rats and furosemide pretreated cynomolgus monkeys.

Lead optimization of 5-amino-6-(2,2-dimethyl-5-oxo-4-phenylpiperazin-1-yl)-4-hydroxyhexanamides to reduce a cardiac safety issue: discovery of DS-8108b, an orally active renin inhibitor.

With the aim to address an undesired cardiac issue observed with our related compound in the recently disclosed novel series of renin inhibitors, further chemical modifications of this series were performed. Extensive structure-activity relationships studies as well as in vivo cardiac studies using the electrophysiology rat model led to the discovery of clinical candidate trans-adamantan-1-ol analogue 56 (DS-8108b) as a potent renin inhibitor with reduced potential cardiac risk. Oral administration of single doses of 3 and 10 mg/kg of 56 in cynomolgus monkeys pre-treated with furosemide led to significant reduction of mean arterial blood pressure for more than 12 h.

Photoprotective effects of Bifidobacterium breve supplementation against skin damage induced by ultraviolet irradiation in hairless mice.

BACKGROUND: Probiotics have been considered to affect not only the gut but also the skin. This study aimed at examining whether oral administration of live Bifidobacterium breve strain Yakult (BBY), a typical probiotic, could exert photoprotective effects in hairless mouse skin. METHODS: BBY cell suspensions and fermented milk containing BBY (BBYM) were orally administered to hairless mice for 9 and 14 days, respectively. Mice were irradiated with ultraviolet (UV) light daily for the last four consecutive days. Twenty-four hours after the final irradiation, skin elasticity, appearance, elastase activity and interleukin (IL)-1ß levels were evaluated in the dorsal skin. RESULTS: BBY and BBYM significantly prevented UV-induced deleterious changes in skin elasticity and appearance. BBY suppressed the increases in both elastase activity and IL-1ß levels in the skin. There was a significant negative correlation between elastase activity and the ratio of elastic recovery to total deformation and a significant positive correlation between elastase activity and the area ratio of furrows, independent of UV irradiation or BBY administration. CONCLUSION: Our findings suggest that oral administration of probiotic BBY has the potential to prevent UV-induced skin damage, supporting the hypothesis that probiotics are beneficial not only to the intestine but also to the skin.

Atrial fibrillation-associated remodeling does not promote atrial thrombus formation in canine models.

BACKGROUND: The most important complication of atrial fibrillation (AF) is thromboembolic stroke. Although AF-related remodeling is considered important in atrial thrombogenesis, its role never has been directly tested. This study assessed effects of AF-related remodeling on the atrial thrombogenic milieu by using radiofrequency ablation (RFA) to create a quantifiable prothrombotic nidus. METHODS AND RESULTS: We studied normal control dogs (control, n=16) and 3 canine AF-models: (1) atrial tachycardia remodeling (ATR; n=16) induced by atrial tachypacing (400 bpm for 1 week, with atrioventricular block and ventricular pacing at 80 bpm); (2) congestive heart failure (CHF; n=14) attributable to ventricular tachypacing (240 bpm for 2 weeks); and (3) chronic AF (CAF; n=8) induced by atrial tachypacing (35±3 days) without atrioventricular block. CAF dogs had AF for 13±1 days until euthanization. After remodeling was established, RFA lesions were created in both atria. Half the ATR and CHF dogs were subjected to atrial tachypacing during 7-day post-RFA follow-up. Electrophysiological and echocardiographic studies were performed before RFA and 7 days after RFA, and then hearts were removed and atrial thrombi were quantified by histomorphometry. Burst-pacing-induced AF duration was significantly greater in ATR, CHF, and CAF groups versus control group. The atrial effective refractory period shortened in ATR and CAF groups. Left atrial diameter was significantly larger with CHF, but not with ATR. Neither total thrombus volume nor thrombus volume per lesion differed significantly among groups. Table.Properties of Ablation Lesions and Atrial Thrombi Experimental GroupControl (n=16)ATR (n=16)CHF (n=14)CAF (n=8)N of ablation lesions per dog6.9±0.36.6±0.27.2±0.26.9±0.4Ablation lesion area, mm(2)53.1±3.558.3±4.857.7±4.944.3±3.7Ablation lesion depth, mm5.2±0.25.1±0.35.3±0.25.2±0.2Ablation lesion volume, mm(3)205.2±17.8211.6±17.6231.5±29.0176.8±22.2N of thrombi per dog5.4±0.44.7±0.35.6±0.46.5±0.4Presence of thrombus, %80±572±577±695±3Mean thrombus volume in both atria, mm(3)20.8±3.414.9±2.212.2±2.622.5±5.6Mean thrombus volume in left atria, mm(3)8.2±1.54.0±0.95.5±1.68.1±3.3Mean thrombus volume in right atria, mm(3)30.1±5.422.7±4.317.9±4.132.8±8.3Total thrombus volume in both atria, mm(3)140.5±21.399.7±16.886.1±17.5131.1±22.7Total thrombus volume in left atria, mm(3)22.8±5.311.8±3.317.0±3.723.3±6.4Total thrombus volume in right atria, mm(3)117.7±21.587.8±17.269.1±16.1107.8±23.3Thrombus volume normalized to ablation lesion area in both atria, mm(3)/mm(2)0.5±0.10.4±0.11.5±1.10.8±0.3Thrombus volume normalized to ablation lesion volume in both atria0.2±0.10.1±0.00.5±0.40.3±0.1 ATR indicates atrial tachycardia remodeling; CAF, chronic atrial fibrillation; and CHF, congestive heart failure. There were no statistically significant differences for any groups vs control group for any of these variables studied. CONCLUSIONS: None of the AF substrates tested, including sustained atrial tachycardia/AF itself, enhanced post-RFA atrial thrombus formation. Indices of electrical and structural remodeling did not predict post-RFA thrombogenic potential. Contrary to widely held but previously untested notions, we were unable to demonstrate prothrombotic effects of AF-related remodeling.

Discovery of DS-8108b, a Novel Orally Bioavailable Renin Inhibitor.

A novel orally bioavailable renin inhibitor, DS-8108b (5), showing potent renin inhibitory activity and excellent in vivo efficacy is described. We report herein the synthesis and pharmacological effects of 5 including renin inhibitory activity in vitro, suppressive effects of ex vivo plasma renin activity (PRA) in cynomolgus monkey, pharmacokinetic data, and blood pressure-lowering effects in an animal model. Compound 5 demonstrated inhibitory activities toward human renin (IC50 = 0.9 nM) and human and monkey PRA (IC50 = 1.9 and 6.3 nM, respectively). Oral administration of single doses of 3 and 10 mg/kg of 5 in cynomolgus monkey on pretreatment with furosemide led to dose-dependent significant reductions in ex vivo PRA and sustained lowering of mean arterial blood pressure for more than 12 h.

Effects of fermentation conditions and soybean peptide supplementation on hyaluronic acid production by Streptococcus thermophilus strain YIT 2084 in milk.

To increase the hyaluronic acid (HA) yield from Streptococcus thermophilus YIT 2084, fermentation conditions (pH, temperature, agitation, aeration) were optimized in milk-based medium, and the effects of supplemental soybean peptides, which have different molecular weight distributions, were determined. HA production was enhanced to approximately 100 mg/l at pH 6.8 and 33-40 degrees C. Agitation speed and aeration rate slightly affected HA production. Soybean peptides including those of high molecular weight (approximately 27 to 130 kDa) further increased HA production to 208 mg/l under the optimal condition (pH 6.8, 35 degrees C, 100 rpm), which was 20-fold greater than non-optimal condition. HA production was no longer related to the specific growth rate. The HA produced under the optimal condition included a large amount of high-molecular-weight fraction of 100 to 2000 kDa, compared with under the basal condition without optimization.