CSAHi study-2: Validation of multi-electrode array systems (MEA60/2100) for prediction of drug-induced proarrhythmia using human iPS cell-derived cardiomyocytes: Assessment of reference compounds and comparison with non-clinical studies and clinical information.

With the aim of reconsidering ICH S7B and E14 guidelines, a new in vitro assay system has been subjected to worldwide validation to establish a better prediction platform for potential drug-induced QT prolongation and the consequent TdP in clinical practice. In Japan, CSAHi HEART team has been working on hiPS-CMs in the MEA (hiPS-CMs/MEA) under a standardized protocol and found no inter-facility or lot-to-lot variability for proarrhythmic risk assessment of 7 reference compounds. In this study, we evaluated the responses of hiPS-CMs/MEA to another 31 reference compounds associated with cardiac toxicities, and gene expression to further clarify the electrophysiological characteristics over the course of culture period. The hiPS-CMs/MEA assay accurately predicted reference compounds potential for arrhythmogenesis, and yielded results that showed better correlation with target concentrations of QTc prolongation or TdP in clinical setting than other current in vitro and in vivo assays. Gene expression analyses revealed consistent profiles in all samples within and among the testing facilities. This report would provide CiPA with informative guidance on the use of the hiPS-CMs/MEA assay, and promote the establishment of a new paradigm, beyond conventional in vitro and in vivo assays for cardiac safety assessment of new drugs.

Syk-dependent signaling pathways in neutrophils and macrophages are indispensable in the pathogenesis of anti-collagen antibody-induced arthritis.

Spleen tyrosine kinase (Syk) is associated with Fcγ receptors (FcγRs) and transmits activation signals through FcγRs in myeloid cells. Thus, application of drugs to inhibit Syk activity can affect the development of immune diseases mediated by autoantibodies, while unexpected systemic effects by the inhibition may be concerned because Syk has multiple physiological functions. We used tamoxifen-inducible systemic conditional Syk knockout (KO) mice to evaluate the role of Syk in the pathogenesis of autoimmune arthritis and to investigate the systemic effects of Syk deletion. In a collagen antibody-induced arthritis model, Syk KO mice were almost completely protected from disease induction and showed significantly attenuated accumulation of neutrophils and macrophages in the joints. Syk-deleted macrophages showed less IL-6 and MCP-1 production upon FcγR ligation and exhibited reduced FcγR-mediated phagocytosis in vitro. Syk-deleted macrophages produce more RANTES upon FcγR ligation, indicating a Syk-independent signaling through the FcγR. We further found that both wild-type and Syk-deleted macrophages induced neutrophil chemotaxis upon FcγR ligation in vitro, and air-pouch model demonstrated that Syk-deleted neutrophils have a potential to infiltrate into local tissues in response to collagen and anti-collagen antibodies. However, Syk-deleted neutrophils exhibited greatly decreased neutrophil extracellular traps formation and FcγR-mediated phagocytosis. Our results indicated that Syk deficiency rendered mice completely unresponsive to immune activation by anti-collagen antibodies with disabling one pathway of FcγR-mediated signaling that was crucial for arthritis induction.

Predictors of early postoperative pneumonia after oncologic surgery with the patients receiving professional oral health care: A prospective, multicentre, cohort study.

This study was a prospective, multicentre, cohort study on 685 patients who had undergone oncologic surgery. The patients were divided into two groups according to the presence or absence of postoperative pneumonia. The two groups were compared with respect to their background, index operation, food eaten, oral condition, contents of oral care and dental treatment, laboratory data, and bacterial flora. All postoperative pneumonias occurred in six cases within four days postoperatively. The multivariable logistic regression analysis showed that preoperative serum C-reactive protein was the strongest predictor of postoperative pneumonia. In addition, decreased postoperative Candida albicans colonies was an effective predictor of postoperative pneumonia. For patients with predictors of postoperative pneumonia, perioperative strategies for its prevention should be considered in addition to professional oral health care. This study was approved by the National Hospital Organization's Central Ethics Review Board and was also approved by the directors of the participating institutions.

Isolation and identification of diglucuronides of some endogenous steroids in dogs.

Diglucuronidation is a novel glucuronidation reaction where the second glucuronosyl moiety is attached at the C2' position of the first glucuronosyl moiety. To examine whether diglucuronidation takes place in endogenous substrates in vivo, control urine and bile samples were collected from male Crl:CD(SD) IGS rats, beagle dogs, and cynomolgus monkeys and analyzed by liquid chromatography-mass spectrometry (LC-MS) after solid phase extraction. Several diglucuronides of C(19) steroids, including M1 (C(31)H(46)O(14)) and M2 (C(31)H(44)O(14)), were detected in the urine and bile of the dogs but not in the excreta of the rats and monkeys. A milligram quantity of M1 was successfully isolated from the pooled dog urine and analyzed by nuclear magnetic resonance (NMR) spectroscopy. M1 was unambiguously identified as epiandrosterone 3-O-diglucuronide by comparing the LC-MS and two-dimensional NMR data of M1 with those of the biosynthesized epiandrosterone 3-O-diglucuronide. M2 was identified as dehydroepiandrosterone 3-O-diglucuronide. According to these findings, the diglucuronidation reaction was proven to be occurring on steroid hormones in vivo in dogs.

[Case of atopic myelitis with acute onset and atypical distribution of spinal cord lesions].

A 23-year-old woman was admitted to our department because of gait disturbance, sensory impairment in the lower limbs, and sphincter disturbance, all of which had been developing within 24 hours before admission. Neurological examination disclosed symmetric muscle weakness, sensory impairment, and diminished tendon reflexes in the lower limbs. The urinary bladder was hypoactive, and the anal tone was reduced. The spinal cord MRI performed on the day of admission revealed swelling of the epiconus. The CSF findings were not remarkable, except for the elevated levels of IgE (8 IU/ml) and MBP (7.8 ng/ml). Besides, there was a marked increase in the serum mite-specific IgE titers. Collectively, we made a diagnosis of atopic myelitis. She was treated with steroid pulse therapy and plasma exchange, which led to a significant amelioration of her neurological manifestations. The repeat MRI carried out on the 21st day of her admission displayed several foci scattered in the lumbar and sacral spinal cord segments, which exhibited high intensity signals on the T2-weighted images. The values of IgE and albumin in the CSF and serum raised the possibility of intrathecal IgE synthesis. We measured her CSF IgE levels at several time points during admission. The temporal profile of her CSF IgE levels was not correlated with that of her neurological disabilities.

Characterization of resistance to bromobenzene-induced hepatotoxicity by microarray.

In our previous study, we demonstrated that the initial hepatic injury caused by bromobenzene (BB) was no longer detected in rats despite subsequent dosing, indicating that the liver acquired resistance to BB-induced hepatotoxicity. In this experiment, microarray analysis was conducted to characterize this resistance. The liver samples for the analysis utilized were obtained from previous experiments where F344 rats were treated intraperitoneally with BB (150 mg/kg). At 24 hr post-dose, hepatic injury was confirmed by monitoring the AST values and then the rats were maintained at the same dosing regimen for an additional 8 days. The gene expression profiles of the BB-treated rat livers were compared with a vehicle-treated group by Affymetrix RG_U34A arrays. As results, a decreased expression level of CYP3A9 and an increased expression level of GST Yc2 and glutathione peroxidase (GPX) were detected. These changes indicated suppression of the phase I reaction and induction of the phase II reaction (glutathione conjugation). Increased expression levels of epoxide hydrolase (EH) and NAD(P)H:quinone oxidoreductase (NQO1) also suggested the involvement of EH- and NQO1-mediated hydrolysis other than glutathione conjugation with resistance in the phase II reaction. Moreover, an increased expression level of abcc3 (multidrug resistance protein 3; Mrp3) was significantly noted. Based on the present findings, it was suggested that Mrp3 in the phase III reaction (drug elimination) contributed to the resistance to BB hepatotoxicity in addition to the suppression of the phase I reaction (metabolic activation) and the induction of the phase II reaction (detoxification). Among them, the factors which contributed most were considered to be the increased GST Yc2 and Mrp3, based on the degree of the gene expression changes.

Resistance to the skeletal muscle injury expressed by repeated treatment with compound A that has HMG-CoA reductase inhibitory activity.

It has been noted that chemical-induced initial insult is sometimes no longer detected in examinations after additional consecutive treatments, suggesting that the target organs acquire resistance to the chemical toxicity. In this study, whether acquired resistance to the skeletal muscle toxicity is observed during repeated treatment of a toxic dose of Compound A that has a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitory activity was examined. F344 male rats (7-weeks old) were given a mixed diet with 0.12% Compound A (corresponding to approximately 100 mg/kg/day) for up to 56 days. Blood samples were obtained from the tail vein periodically during the dosing period, and utilized for the measurement of creatine kinase (CK) as a marker of skeletal muscle injury. In the necropsies on Days 4, 8, 11, 28, 42 and 56, the skeletal muscles from the rectus femoris were removed for histopathology or gene expression analysis. A satellite group was provided to measure the plasma concentrations of Compound A and M1, the active metabolite of Compound A. CK levels increased from Day 9 and reached approximately 30 times those of the controls on Day 12. Histopathology of the skeletal muscle on Day 11 revealed severe necrosis of the muscle fibers. However, in spite of continuous treatments to the damaged rats, the CK levels decreased after that and returned to normal levels on Day 18. No skeletal muscle injury was observed on Days 42 and 56. There were no marked differences in the exposure levels of Compound A and M1 between Days 8 (prior to CK elevation) and 28 (post CK elevation). As for the most significant changes in the gene expression analysis for the skeletal muscle on Days 42 and 56, the probe for IkappaBa, which is known as an inhibitor for nuclear factor-kappaB (NF-kappaB), increased 2-fold compared to the control. Furthermore, an increased probe for CCAAT/enhancer-binding protein (C/EBP) delta, a transcriptional factor, and a decreased probe for cAMP-response element-binding protein (CBP)/p300, a transcriptional coactivator, were also noted significantly on Day 56. These changes in the gene expression analysis suggested suppressed NF-kappaB-mediated transactivation, which was responsible for the protective effects on the muscle injury. Based on the present findings, the resistance to skeletal muscle injury observed in this study may be attributable to the suppressed NF-kappaB-mediated transactivation, but not to the decreased exposure to toxicants.

Natural history of the Nihon (Bhd gene mutant) rat, a novel model for human Birt-Hogg-Dubé syndrome.

In the Nihon rat, an established model of hereditary renal cell carcinoma (RCC), the propensity for tumor development, is inherited as an autosomal dominant trait due to a single germline nucleotide insertion mutation in the rat Bhd ortholog. The Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant disease characterized by fibrofolliculoma, pulmonary cysts, spontaneous pneumothorax, and renal neoplasm. The renal lesions of the Nihon rat are characterized, and extrarenal lesions are also described in this work. The earliest lesion of the RCC was identified as an altered tubule at as early as 3 weeks of age and rapidly progressed through adenoma to carcinoma with the primary cell type being clear/acidophilic where some similarities were evident to RCCs in BHD syndrome. The Nihon rats demonstrate a heterotopic ossification within RCCs and three extrarenal lesions, clear cell hyperplasia/adenoma of the endometrium, clear cell change of the epithelium of striated portions of salivary glands, and cardiac rhabdomyomatosis. This rat model of hereditary RCC provides a useful tool for analyzing the series of events leading to renal tumorigenesis and for studying BHD gene functions.

CSAHi study: Evaluation of multi-electrode array in combination with human iPS cell-derived cardiomyocytes to predict drug-induced QT prolongation and arrhythmia--effects of 7 reference compounds at 10 facilities.

INTRODUCTION: Drug-induced QT prolongation is a major safety issue during drug development because it may lead to lethal ventricular arrhythmias. In this study, we evaluated the utility of multi-electrode arrays (MEA) with human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) to predict drug-induced QT prolongation and arrhythmia. METHODS: Ten facilities evaluated the effects of 7 reference drugs (E-4031, moxifloxacin, flecainide, terfenadine, chromanol 293B, verapamil, and aspirin) using a MED64 MEA system with commercially available hiPS-CMs. Field potential duration (FPD), beat rate, FPD corrected by Fridericia's formula (FPDc), concentration inducing FPDc prolongation by 10% (FPDc10), and incidence of arrhythmia-like waveform were evaluated. RESULTS: The inter-facility variability of absolute values before drug application was similar to the intra-facility variability for FPD, beat rate, and FPDc. The inter-facility variability of FPDc10 for 5 reference drugs ranged from 1.8- to 5.8-fold. At all 10 facilities, E-4031, moxifloxacin, and flecainide prolonged FPDc and induced arrhythmia-like waveforms at concentrations 1.8- to 6.1-fold higher than their FPDc10. Terfenadine prolonged FPDc and induced beating arrest at 8.0 times the FPDc10. The average FPDc10 values for E-4031, moxifloxacin, and terfenadine were comparable to reported plasma concentrations that caused QT prolongation or Torsade de Pointes in humans. Chromanol 293B, a IKs blocker, also prolonged FPDc but did not induce arrhythmia-like waveforms, even at 7.4 times the FPDc10. In contrast, verapamil shortened FPDc and aspirin did not affect FPDc or FP waveforms. DISCUSSION: MEA with hiPS-CMs can be a generalizable method for accurately predicting both QT prolongation and arrhythmogenic liability in humans.

Overexpression of the peroxisome proliferator activated receptor alpha or the human c-Ha-ras transgene is not involved in tumorigenesis induced by di(2-ethylhexyl)phthalate in rasH2 mice.

The mRNA profiles for peroxisome proliferator activated receptor (PPAR) alpha and human c-Ha-ras genes were determined by real-time semi-quantitative polymerase chain reaction analysis of hepatocellular adenomas induced by di(2-ethylhexyl)phthalate (DEHP) in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice). The mRNA levels were essentially equal in hepatocellular adenomas and adjacent non-neoplastic hepatocytes, in spite of a remarkable elevation in the cell proliferation index in tumors determined by anti-Ki-67 immunohistochemistry. From the results, it is concluded that overexpression of PPARalpha or the transgene is not associated with the liver tumorigenesis induced by DEHP in rasH2 mice.

A novel model of continuous depletion of glutathione in mice treated with L-buthionine (S,R)-sulfoximine.

L-buthionine (S,R)-sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis, was administered to mice via drinking water for 14 days in order to establish an animal model with continuously depleted levels of GSH. No toxicity was observed at 20 mM BSO, even though a significant decrease in liver weight was observed at 30 mM BSO. GSH levels in the liver, kidney, brain, lung, heart, spleen, pancreas, small intestine, large intestine, skeletal muscle, plasma and blood cells from mice given 20 mM of BSO were all less than those from the control mice continuously throughout a 24-hr period. The ratios of the GSH levels to that of the control were 46.4% and 16.7% in the liver and kidney, respectively, suggesting a decrease in GSH conjugation activity in vivo by GSH depletion. Liver cytochrome P450 content and UDP-glucuronosyltransferase activity to p-nitrophenol were not influenced by the BSO dosing. To confirm the adequacy of this GSH-depletion model, 0.125 or 0.25% of acetaminophen (APAP) was administered via diet to this model for 14 days. Nine out of the ten mice given both 20 mM BSO and 0.25% APAP died on Day 2, and remarkable necrosis was observed in the hepatocytes and renal tubular epithelium. Moreover, focal necrosis of hepatocytes with proliferation of fibroblasts was observed on Day 15 in some mice coadministered 20 mM BSO and 0.125% APAP. However, no toxicity was observed in mice given APAP alone. Based on these results, a mouse given 20 mM of BSO via drinking water for 14 days was concluded to be an animal model with continuously depleted levels of GSH in various organs without toxicity. This model shows high susceptibility to toxicity induced by chemicals which are metabolized to electrophilic and reactive metabolite(s), such as APAP.

Thirteen-week Intravenous Toxicity Study of a Novel Humanized Anti-Human Death Receptor 5 Monoclonal Antibody, CS-1008, in Cynomolgus Monkeys.

CS-1008, a humanized monoclonal antibody that is agonistic to human death receptor 5, was intravenously administered to cynomolgus monkeys twice a week for 13 weeks at 3 different dose levels (5, 15 and 42 mg/kg) in order to evaluate its potential toxicity. A control group received phosphate buffered saline containing 0.01% polysorbate 80. Each of the 4 groups consisted of 3 male and 3 female cynomolgus monkeys. No animal in any group died during the dosing period. No toxic changes in clinical signs, food consumption, body weight, electrocardiography, ophthalmology, urinalysis, hematology, blood chemistry, gross pathology, organ weights or histopathology were noted in any group during the dosing period. In the toxicokinetic analysis, the values for the maximum concentration of CS-1008 in plasma and the area under the curve generally increased with increasing dose. No clear differences in the toxicokinetic parameters or profiles were observed between the sexes. Development of anti-CS-1008 antibodies was not detected in any sample. The no-observed adverse-effect level (NOAEL) of CS-1008 in cynomolgus monkeys under the conditions of this study was concluded to be 42 mg/kg in both sexes, when administered intravenously twice a week for 13 weeks. This study supports the development of CS-1008 as a therapeutic biopharmaceutical.

Ocular lesions in Watanabe heritable hyperlipidemic rabbits.

Hyperlipidemic ocular lesions are described for Watanabe heritable hyperlipidemic (WHHL) rabbits. Male WHHL rabbits 8 months old exhibited serum hyperlipidemia and ophthalmoscopically yellowish-white lesions along the corneoscleral junction and in the iris. Histopathologically, foamy macrophages aggregated in the stroma of the cornea, iris, and ciliary body were observed. These findings have been interpreted as lipid keratopathy. In addition, multiple clusters of a large number of foamy macrophages occurred throughout the choroid and sclera in association with the blood vessels. The lesions in the choroid and sclera could not be detected ophthalmoscopy, yet were much more prominent than those in the cornea, iris, and ciliary body, suggesting greater involvement and earlier onset of lipidosis at these sites associated with hyperlipidemia in WHHL rabbits.

Mutation and overexpression of the transgene in ethylnitrosourea-induced tumors in mice carrying a human prototype c-Ha-ras gene.

To investigate mechanisms underlying accelerated carcinogenesis in mice carrying a human prototype c-Ha-ras gene (rasH2 mouse), mutations and the expression profile of the transgene were evaluated in 14 tumors induced by a single injection of ethylnitrosourea (ENU), with or without additional beta-estradiol 3-benzoate (EB) treatment. Although no codon 12 mutations were detected, changes in codon 61 were evident in all lung adenocarcinomas, skin squamous cell carcinomas and forestomach squamous cell carcinomas examined. The mRNA levels of the transgene in these lesions were also elevated 1.71- to 4.77-fold, 3.04- to 5.18-fold, and 3.00- to 5.67-fold, respectively, in comparison with those in the normal livers of rasH2 mice. The results obtained in this study suggest that mutations in codon 61 and amplification of the transgene play key roles in the carcinogenesis induced by ENU in rasH2 mice.

Twenty-six-Week carcinogenicity study of chloroform in CB6F1 rasH2-transgenic mice.

The carcinogenic potential of chloroform was evaluated in a short-term carcinogenicity testing system using CB6F1 rasH2-Tg (rasH2-Tg) mice. Chloroform was administered to rasH2-Tg males at doses of 28, 90, or 140 mg/kg and rasH2-Tg females at 24, 90, or 240 mg/kg by oral gavage for 26 weeks. Wild-type (non-Tg) male and female mice received doses of 140 mg/kg and 240 mg/kg, respectively. N-methyl-N-nitrosourea was administered to rasH2-Tg mice by single intraperitoneal injection (75 mg/kg) as a positive control. In both the rasH2-Tg and non-Tg mice, there was no significant increase in the incidence of neoplastic lesions by chloroform treatment. The incidence of hepatocellular foci in the rasH2- and non-Tg females receiving 240 mg/kg was increased. Forestomach tumors and malignant tumors occurred in most of the rasH2-mice in the positive control group. Swelling or vacuolation of hepatocytes, a toxic change induced by chloroform, occurred in both the rasH2-Tg and non-Tg mice. It is concluded that chloroform, a putative human noncarcinogen, did not show evidence of carcinogenic potential in the present study using rasH2-Tg mice. This study suggests that the rasH2-Tg mouse model may not be appropriate for detecting nongenotoxic carcinogens. However, the sensitivity of rasH2-Tg mice to nongenotoxic carcinogens should be assessed with consideration of the results from the other ILSI-HESI project studies.

Molecular mechanism investigation of phenobarbital-induced serum cholesterol elevation in rat livers by microarray analysis.

Phenobarbital (PB) increases serum total cholesterol levels in rodents and humans. To investigate the underlying molecular mechanisms, we performed a microarray analysis on liver of rats treated repeatedly with 100 mg/kg PB, and examined the serum blood chemistry. The serum concentration of non-esterified fatty acids was decreased from day 1 to day 14 except for day 7, and that of cholesterol was increased from day 4 to day 14. The serum concentration of total ketone bodies was increased on day 7, and that of triglycerides was decreased on day 14. Transcript content of glycolytic genes was decreased by PB treatments, while that of lipoprotein lipase was continuously increased, suggesting a notion that repetitive PB treatments impaired glycolysis and stimulated lipolysis in the liver. The hypothesis was examined by using a previously reported flux-balance model. The increase in mRNA content of malic enzyme after the PB treatment agreed well with the flux-balance model result, suggesting the validity of our hypothesis. The findings also suggested that there was an abundance of acetyl-CoA and shortage of glycolytic products after the repeated PB treatments. Although ketogenesis would normally occur under such cellular conditions, it was only weakly observed after the repeated PB treatments, presumably owing to a decrease in HMG-CoA synthase mRNA content. On the other hand, the mRNA content of several cholesterogenic genes was slightly induced by PB treatments. Thus, serum chemistry and microarray results suggested that repeated PB treatments induced cholesterogenesis in rat livers, which may have contributed to the elevation of the serum total cholesterol concentration.