absolute-quantification-plasma-microrna-levels-cynomolgus-monkeys

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

Daiichi Sankyo Co., Ltd.

Very late antigen-4 (VLA-4), which is concerned with cell-cell adhesion, plays important roles in development of the heart, and some VLA-4 antagonists cause cardiac anomalies. In this study, we evaluated the teratogenic potential of VLA-4 antagonist derivatives as screening, and investigated the conditions that induce cardiac anomalies. Seventeen compounds were orally administered to pregnant rats throughout the organogenesis period, and fetal examinations were performed. In addition, drug concentrations in the embryos were assayed. As a result, the incidence of ventricular septal defect (VSD) ranged from 0 to 100% depending on the compound. Plasma drug concentrations in the dams were related to increased incidence of VSD; however, these incidences were not increased when the concentration of the compound in the embryos at 24h after dosing was low. It is considered that continuous pharmacological activity in the embryo for more than 24h might disrupt closure of the ventricular septum.

Investigation of the teratogenic potential of VLA-4 antagonist derivatives in rats.

To characterize microRNAs (miRNAs) involved in testicular toxicity in cynomolgus monkeys, miRNA profiles were investigated using next-generation sequencing (NGS), microarray and reverse transcription-quantitative real-time-PCR (RT-qPCR) methods. First, to identify organ-specific miRNAs, we compared the expression levels of miRNAs in the testes to those in representative organs (liver, heart, kidney, lung, spleen and small intestine) obtained from naïve mature male and female monkeys (n = 2/sex) using NGS analysis. Consequently, miR-34c-5p, miR-202-5p, miR-449a and miR-508-3p were identified to be testicular-specific miRNAs in cynomolgus monkeys. Next, we investigated miRNA profiles after testicular-hyperthermia (TH) treatment to determine which miRNAs are involved in testicular injury. In this experiment, mature male monkeys were divided into groups with or without TH-treatment (n = 3/group) by immersion of the testes in a water bath at 43 °C for 30 min for 5 consecutive days. As a result, TH treatment induced testicular injury in all animals, which was characterized by decreased numbers of spermatocytes and spermatids. In a microarray analysis of the testis, 11 up-regulated (>2.0 fold) and 13 down-regulated (<0.5 fold) miRNAs were detected compared with those in the control animals. Interestingly, down-regulated miRNAs included two testicular-specific miRNAs, miR-34c-5p and miR-449a, indicating their potential use as biomarkers for testicular toxicity. Furthermore, RT-qPCR analysis revealed decreased expression levels of testicular miR-34b-5p and miR-34c-5p, which are enriched in meiotic cells, reflecting the decrease in pachytene spermatocytes and spermatids after TH treatment. These results provide valuable insights into the mechanism of testicular toxicity and potential translational biomarkers for testicular toxicity. Copyright © 2016 The Authors. Journal of Applied Toxicology published by John Wiley & Sons Ltd.

Ken Sakurai

Medicinal Safety Research Laboratories

Daiichi Sankyo Co., Ltd.

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

Ken Sakurai

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Daiichi Sankyo Co., Ltd.

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

Medicinal Safety Research Laboratories