Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone-induced toxicities in rats: comparative study with other mitochondrial uncouplers (2,4-dinitrophenol, OPC-163493 and tolcapone).

FCCP (carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone) is known to inhibit oxidative phosphorylation as a protonophore, dissipating the proton gradient across the inner mitochondrial membrane. To understand the toxicity of FCCP, 3-day, 2- and 4-week repeated oral dose studies were performed in male rats. In the 3-day and 2-week repeated dose toxicity studies, observations included salivation, increased body temperature, and dead and moribund animals. Increased liver weight was observed in conjunction with hydropic degeneration and centrilobular necrosis of hepatocytes. In addition, pathological changes were observed in the pancreas, testis, epididymal duct, stomach and parotid gland. Electron microscopic examination revealed mitochondrial pleomorphism in the hepatocytes. Swelling of mitochondria was observed in the alpha cells and beta cells of the pancreas. Dilatation of rough endoplasmic reticulum, Golgi bodies and loss of secretory granules were also noted in the beta cells of the pancreas. FCCP was also compared with three other mUncouplers (DNP, OPC-163493 and tolcapone) with regard to in vitro mitochondrial uncoupling (mUncoupling) activities. FCCP produced the peak ΔOCR (oxygen consumption rate) at the lowest concentration (0.4 µM), followed by OPC-163493, tolcapone, and DNP, based on peak values in ascending order of concentration (2.5, 10, and 50 µM, respectively). Considering the relationship between the mUncoupling activity and toxicity profile of the four mUncouplers, there is no parallel relationship between the in vitro mUncoupling activity and the degree of in vivo toxicity. These findings may contribute to the efficient development of new mitochondrial uncoupler candidates. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00189-x.

Preclinical safety profile of a liver-localized mitochondrial uncoupler: OPC-163493.

Mitochondrial uncouplers (mUncouplers) are known to exhibit a variety of toxic effects in animals. Here we report a safety profile of an mUncoupler, OPC-163493, recently synthesized at Otsuka Pharmaceutical Co, Ltd, and its development as a therapeutic agent for treating diabetes. To understand the acute and subchronic toxicity of OPC-163493, single and repeated oral dose studies in rats, dogs, and monkeys were performed. In the rat studies, rigor mortis and increased body temperatures were observed in the high dose group. Focal necrosis, fatty change, and granular eosinophilic cytoplasm of the hepatocytes were also observed in the high dose group. In the dog studies, gastrointestinal manifestations were observed with decreased body weight and decreased food consumption in the high dose group. Necrotizing arteritis was observed in multiple organs as well as meningitis with hemorrhage in the brain. In the monkey studies, vomiting, decreased food consumption, and decreased locomotor activity were observed in the high dose group. Degeneration of the proximal convoluted tubules and the straight tubular epithelium, regeneration of the proximal tubular epithelium, and degeneration of the collecting tubular epithelium were observed. The target organs of OPC-163493 were liver, blood vessels, and kidney in rats, dogs, and monkeys, respectively. In rats, dogs, and monkeys, safety ratios were 100:1, 13:1, and 20:1, respectively, in terms of total exposure (AUC24h). These safety ratios showed clear separation between exposure to OPC-163493 in animals at NOAEL and the exposure at the effective dose in ZDF rats. This information should contribute to the drug development of new and effective mUncoupler candidates.

New Approach to Drug Discovery of a Safe Mitochondrial Uncoupler: OPC-163493.

We serendipitously found a mitochondrial uncoupler (mUncoupler), compound 1, in the process of screening for inhibitors of a gene product related to calorie restriction (CR) and longevity. Compound 1 has a unique 4-cyano-1,2,3-triazole structure which is different from any known mUncoupler and ameliorated HbA1c in Zucker diabetic fatty (ZDF) rats. However, its administration at high doses was not tolerated in an acute toxicity test in rats. We therefore tried to optimize cyanotriazole compound 1 and convert it into an agent that could be safely administered to patients with diabetes mellitus (DM) or metabolic disorders. Considering pharmacokinetic (PK) profiles, especially organ distribution targeting the liver and avoiding the brain, as well as acute toxicities and pharmacological effects of the derivatives, various conversions and substitutions at the 5-position on the cyanotriazole ring were carried out. These optimizing processes improved PK profiles and effectiveness, and acute toxicities became negligible even at high doses. We finally succeeded in developing an optimized compound, OPC-163493, as a liver-localized/targeted mUncoupler.

Antidiabetic and cardiovascular beneficial effects of a liver-localized mitochondrial uncoupler.

Inducing mitochondrial uncoupling (mUncoupling) is an attractive therapeutic strategy for treating metabolic diseases because it leads to calorie-wasting by reducing the efficiency of oxidative phosphorylation (OXPHOS) in mitochondria. Here we report a safe mUncoupler, OPC-163493, which has unique pharmacokinetic characteristics. OPC-163493 shows a good bioavailability upon oral administration and primarily distributed to specific organs: the liver and kidneys, avoiding systemic toxicities. It exhibits insulin-independent antidiabetic effects in multiple animal models of type I and type II diabetes and antisteatotic effects in fatty liver models. These beneficial effects can be explained by the improvement of glucose metabolism and enhancement of energy expenditure by OPC-163493 in the liver. Moreover, OPC-163493 treatment lowered blood pressure, extended survival, and improved renal function in the rat model of stroke/hypertension, possibly by enhancing NO bioavailability in blood vessels and reducing mitochondrial ROS production. OPC-163493 is a liver-localized/targeted mUncoupler that ameliorates various complications of diabetes.

Establishment and characterization of a novel method for evaluating gluconeogenesis using hepatic cell lines, H4IIE and HepG2.

The liver gluconeogenic pathway is recognized as a target for treating diabetes mellitus. In this study, we attempted to establish a new method to evaluate gluconeogenesis using rat H4IIE hepatoma cells. High-density preculture and exposure to hypertonic solutions, which are known to upregulate the expression of gluconeogenic genes, enhanced glucose release (GR) promoted by gluconeogenic substrates (GS: 1mM pyruvate and 10mM lactate). Our method was also applicable to the human hepatoma HepG2 cells. Measurement of glycogen content in HepG2 cells revealed that GR was compensated by glycogenolysis in the basal state and was generated by gluconeogenesis in the presence of GS. The optimized conditions increased the expression of gluconeogenic genes in HepG2 cells. Insulin and metformin dose-dependently inhibited GR and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) increased it. These results suggest that the present method is useful to evaluate the effects of nutrients, hormones and hypoglycemic agents on hepatic gluconeogenesis.

Characterization of STZ-Induced Type 2 Diabetes in Zucker Fatty Rats.

The Zucker fatty (ZF) rat is a disease model of obesity and metabolic syndrome, such as hyperlipidemia and insulin resistance, resulting from hyperphagia owing to the loss of function of the leptin receptor, but it rarely develops hyperglycemia. We examined the effects of different doses of streptozotocin (STZ). A low dosage of STZ (30 mg/kg body weight, i.p.) elevated blood glucose levels in ZF rats up to 300 mg/dl within a week, and to nearly 500 mg/dl by 5 weeks after injection of STZ. Besides hyperglycemia, STZ-treated ZF (STZ-ZF) rats retained metabolic syndrome features such as hyperlipidemia and hyperinsulinemia. The stimulated insulin secretion in response to orally-loaded glucose disappeared completely in STZ-ZF rats. Although there were no significant differences in the morphology of pancreatic islets between vehicle-treated ZF (Cont-ZF) and STZ-ZF rats, the insulin content was markedly decreased in STZ-ZF rats. The hepatic gene expression for gluconeogenic enzymes was upregulated in STZ-ZF rats compared with Cont-ZF rats. Metformin lowered the blood glucose levels of STZ-ZF rats in a dose-dependent manner. These results suggest that STZ-ZF rats are useful for studies of T2DM and for the evaluation of the efficacy of anti-diabetic drugs.

Mutated G-protein-coupled receptor GPR10 is responsible for the hyperphagia/dyslipidaemia/obesity locus of Dmo1 in the OLETF rat.

1. We have confirmed the Diabetes Mellitus OLETF type I (Dmo1) effect on hyperphagia, dyslipidaemia and obesity in the Otsuka Long-Evans Tokushima Fatty (OLETF) strain. The critical interval was narrowed down to 570 kb between D1Got258 to p162CA1 by segregation analyses using congenic lines. 2. Within the critical 570 kb region of the Dmo1 locus, we identified the G-protein-coupled receptor gene GPR10 as the causative gene mutated in the OLETF strain. The ATG translation initiation codon of GPR10 is changed into ATA in this strain and, so, is unavailable for the initiation of translation. 3. The GPR10 protein has a cognate ligand, namely prolactin-releasing peptide (PrRP). Centrally administered PrRP suppressed the food intake of congenic rats that have a Brown Norway derived Dmo1 region (i.e. with wild-type GPR10), but did not suppress that of the OLETF strain, indicating that GPR10 is without function and could explain hyperphagia in the OLETF strain. 4. Moreover, when restricted in food volume to the same level consumed by the congenic strain, OLETF rats showed few differences in the parameters of dyslipidaemia and obesity compared with congenic strains. 5. Taken together, these results demonstrate that the mutated GPR10 receptor is responsible for the hyperphagia leading to obesity and dyslipidaemia in the obese diabetic strain rat.