This study was conducted to examine any changes caused by feed restriction in dogs to contribute to safety evaluation in toxicity studies. Two male 7-month-old beagle dogs/group were fed 300 (control), 150 (50% of control), or 70 g/animal of diet daily (23% of control) for 4 weeks. Effects of feed restriction, except for clinical signs, were noted depending on the feed dosage in almost all examinations. The principal outcomes were: decreased body weight and water consumption, ECG changes (decreased heart rate and prolonged QTc), and hematopoietic and lymphopoietic suppression (decreased reticulocyte ratio or white blood cell count in hematology, decreased nucleated cell count in bone marrow, decreased erythroid parameters in myelography, and hypocellularity of bone marrow and thymic atrophy in histopathology). In addition, some changes were noted in urinalysis (decreased urine volume and sodium and potassium excretion), blood chemistry (decreased ALP and inorganic phosphorus and increased creatinine), organ weights, and gastric histopathology. These results provide important reference data for distinguishing the primary effects of test compounds from secondary effects of decreased food consumption in toxicity studies in beagle dogs.

The importance of cancer-associated fibroblasts (CAFs) in cancer biology has been recently highlighted owing to their critical roles in cancer growth, progression, metastasis, and therapeutic resistance. We have previously established a primary culture of breast cancer cells, which showed epithelial-mesenchymal transition and cancer stem cell-like properties. In this study, we found that the primary culture also showed CAF-like properties. For example, hypoxia inducible factor 1α (HIF1A) and its downstream genes, nuclear factor-kappa B2 (NF-κB2) and BCL2/adenovirus E1B 19 kd-interacting protein 3 (BNIP3), and many enzymes involved in glycolysis, such as GAPDH, LDH, PGAM1, and PKM2, were highly overexpressed in the primary culture. Moreover, media conditioned with the primary culture cells enhanced the growth of breast cancer cells. Similar to previous CAF studies, this enhancement suggested to be occurred through fibroblast growth factor signaling. This MCKH primary culture cell, which showed simultaneous expression of tumorigenic and CAF properties, offers a unique experimental system for studying the biology of CAFs.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are used for cardiac safety assessment but have limitations for the evaluation of drug-induced contractility. Threedimensional (3D) cardiac tissues are similar to native tissue and valuable for the assessment of contractility. However, a longer time and specialized equipment are required to generate 3D tissues. We previously developed a simple method to generate 3D tissue in a short period by coating the cell surfaces with extracellular matrix proteins. We hypothesized that this 3D cardiac tissue could be used for simultaneous evaluation of drug-induced repolarization and contractility. In the present work, we examined the effects of several compounds with different mechanisms of action by cell motion imaging. Consequently, human ether-ago-go-related gene (HERG) channel blockers with high arrhythmogenic risk caused prolongation of contraction-relaxation duration and arrhythmia-like waveforms. Positive inotropic drugs, which increase intracellular Ca 2+ levels or myocardial Ca 2+ sensitivity, caused an increase in maximum contraction speed (MCS) or average deformation distance (ADD) (ouabain, 138% for MCS at 300 nM; pimobendane, 132% for ADD at 3 mM). For negative inotropic drugs, verapamil reduced both MCS and ADD (61% at 100 nM). Thus, this 3D cardiac tissue detected the expected effects of various cardiovascular drugs, suggesting its usefulness for cardiotoxicity evaluation.

Acotiamide hydrochloride hydrate (acotiamide-HH) has been newly developed as an indication for functional dyspepsia, which is characterized by digestive symptoms such as postprandial fullness, abdominal bloating, or early satiation, and is now being prescribed in Japan. As part of a safety assessment, 2-year long-term carcinogenicity studies using rats and mice were conducted. In the mouse carcinogenicity study, no evidence of carcinogenicity was obtained, even in the high-dose-treated group (up to 2000 mg/kg/day). In the rat carcinogenicity study, acotiamide-HH was administered at 200, 600, and 2000 mg/kg/day. Detailed histopathological examination revealed that the incidence of endometrial adenocarcinoma significantly increased in the 600 mg/kg/day treated group. There was no trend of this incidence and no accompanying increase in pre-neoplastic lesions or related histological changes in the genital tissues, suggesting the absence of abnormalities in the sexual endocrine system. Results of genotoxicity and reproductive/developmental studies showed that acotiamide-HH is a non-genotoxic substance and did not affect sexual balance. Acotiamide-HH did not induce an estrogen-dominant hormonal imbalance that could cause the incidence of uterine cancer and did not have initiation activity. Therefore, the proliferation of endometrial adenocarcinoma in this middle dose group in the rat carcinogenesis study was considered an accidental event of naturally occurring tumors. However, the incidence of endometrial adenocarcinoma in this group deviated from the background data collected in the same laboratory during the study period. Therefore, it is considered necessary to conduct another pre-clinical study in order to obtain data that would dispel any concerns of safety.