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.

A monkey model of acetaminophen-induced hepatotoxicity; phenotypic similarity to human.

Species-specific differences in the hepatotoxicity of acetaminophen (APAP) have been shown. To establish a monkey model of APAP-induced hepatotoxicity, which has not been previously reported, APAP at doses up to 2,000 mg/kg was administered orally to fasting male and female cynomolgus monkeys (n = 3-5/group) pretreated intravenously with or without 300 mg/kg of the glutathione biosynthesis inhibitor, L-buthionine-(S,R)-sulfoximine (BSO). In all the animals, APAP at 2,000 mg/kg with BSO but not without BSO induced hepatotoxicity, which was characterized histopathologically by centrilobular necrosis and vacuolation of hepatocytes. Plasma levels of APAP and its reactive metabolite N-acethyl-p-benzoquinone imine (NAPQI) increased 4 to 7 hr after the APAP treatment. The mean Cmax level of APAP at 2,000 mg/kg with BSO was approximately 200 µg/mL, which was comparable to high-risk cutoff value of the Rumack-Matthew nomogram. Interestingly, plasma alanine aminotransferase (ALT) did not change until 7 hr and increased 24 hr or later after the APAP treatment, indicating that this phenotypic outcome was similar to that in humans. In addition, circulating liver-specific miR-122 and miR-192 levels also increased 24 hr or later compared with ALT, suggesting that circulating miR-122 and miR-192 may serve as potential biomarkers to detect hepatotoxicity in cynomolgus monkeys. These results suggest that the hepatotoxicity induced by APAP in the monkey model shown here was translatable to humans in terms of toxicokinetics and its toxic nature, and this model would be useful to investigate mechanisms of drug-induced liver injury and also potential translational biomarkers in humans.

MicroRNA profiles in a monkey testicular injury model induced by testicular hyperthermia.

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.

MicroRNA profiling in ethylene glycol monomethyl ether-induced monkey testicular toxicity model.

To establish and characterize ethylene glycol monomethyl ether (EGME)-induced testicular toxicity model in cynomolgus monkeys, EGME at 0 or 300 mg/kg was administered orally to sexually mature male cynomolgus monkeys (n = 3/group) for 4 consecutive days. Circulating and testicular microRNA (miRNA) profiles in this model were investigated using miRNA microarray or real-time quantitative reverse transcription-PCR methods. EGME at 300 mg/kg induced testicular toxicity in all the monkeys, which was characterized histopathologically by decreases in pachytene spermatocytes and round spermatids, without any severe changes in general conditions or clinical pathology. In microarray analysis, 16 down-regulated and 347 up-regulated miRNAs were detected in the testis, and 326 down-regulated but no up-regulated miRNAs were detected in plasma. Interestingly, miR-1228 and miR-2861 were identified as abundant miRNAs in plasma and the testis of control animals, associated presumably with apoptosis and cell differentiation, respectively, and were prominently increased in the testis of EGME-treated animals, reflecting the recovery from EGME-induced testicular damages via stimulating cell proliferation and differentiation of sperm. Furthermore, down-regulation of miR-34b-5p and miR-449a, which are enriched in meiotic cells like pachytene spermatocytes, was obvious in the testis, suggesting that these spermatogenic cells were damaged by the EGME treatment. In conclusion, EGME-induced testicular toxicity in cynomolgus monkeys was shown, and this model would be useful for investigating the mechanism of EGME-induced testicular toxicity and identifying testicular biomarkers. Additionally, testicular miR-34b-5p and miR-449a were suggested to be involved in damage of pachytene spermatocytes.

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.