Safety Assessment of a Novel Intravenous Diclofenac Sodium Formulation Following 28-Day Repeated Administration in Wistar Rats.

These toxicity studies aimed to assess the safety and tolerability of a novel intravenous diclofenac sodium (37.5 mg/mL) formulation containing povidone K12 (80 mg/mL) as the key excipient in Wistar rats. This formulation was tested at doses of 3, 7, and 15 mg/kg/day and was administered daily for 28 days by intravenous route. Toxicokinetic estimation revealed a dose-proportional increase in plasma exposure to diclofenac. The formulation was well tolerated in males; however, mortality was observed in females (2/15) at the highest dose (15 mg/kg/day). Adverse gastrointestinal events related to NSAIDS and a few other treatment-related effects on clinical and anatomic pathology were noted at the 15 mg/kg/day dose, which normalized at the end of the 2-week recovery period. In addition, the excipient povidone K12 was present in a higher amount than the approved Inactive Ingredient Database (IID) limit in the proposed novel formulation. It was qualified through a separate 28-day repeated dose toxicity study by intravenous route in Wistar rats. Povidone K12 was found to be well tolerated and safe up to a dose of 165 mg/kg/day. No treatment-related adverse effects were observed in this study. In conclusion, repeated administration of a novel intravenous formulation containing diclofenac sodium was found to be safe up to the dose of 7 mg/kg/day in female rats and 15 mg/kg/day in male rats.

Saroglitazar suppresses the hepatocellular carcinoma induced by intraperitoneal injection of diethylnitrosamine in C57BL/6 mice fed on choline deficient, l-amino acid- defined, high-fat diet.

BACKGROUND: Saroglitazar is a novel PPAR-α/γ agonist with predominant PPAR-α activity. In various preclinical models, saroglitazar has been shown to prevent & reverse symptoms of NASH. In view of these observations, and the fact that NASH is a progressive disease leading to HCC, we hypothesized that saroglitazar may prevent the development of HCC in rodents. METHODS: HCC was induced in C57BL/6 mice by a single intraperitoneal injection of 25 mg/kg diethylnitrosamine (DEN) at the age of 4 weeks and then feeding the animal a choline-deficient, L-amino acid- defined, high-fat diet (CDAHFD) for the entire study duration. Eight weeks after initiation of CDAHFD, saroglitazar (1 and 3 mg/kg) treatment was started and continued for another 27 weeks. RESULTS: Saroglitazar treatment significantly reduced the liver injury markers (serum ALT and AST), reversed hepatic steatosis and decreased the levels of pro-inflammatory cytokines like TNF-α in liver. It also resulted in a marked increase in serum adiponectin and osteopontin levels. All disease control animals showed hepatic tumors, which was absent in saroglitazar (3 mg/kg)- treatment group indicating 100% prevention of hepatic tumorigenesis. This is the first study demonstrating a potent PPARα agonist causing suppression of liver tumors in rodents, perhaps due to a strong anti-NASH activity of Saroglitazar that overrides its rodent-specific peroxisome proliferation activity. CONCLUSION: The data reveals potential of saroglitazar for chemoprevention of hepatocellular carcinoma in patients with NAFLD/NASH.

Non-clinical safety evaluation of a novel pharmaceutical salt, rosuvastatin ethanolamine, in Wistar rats.

Rosuvastatin, a second generation 3-Hydroxy-3-Methyl Glutaryl Coenzyme-A reductase inhibitor, is widely used for the management of hypercholesterolemia. Rosuvastatin ethanolamine, developed by Cadila Healthcare Ltd., is a novel, chemically stable, and pharmaceutically acceptable salt, having better physiochemical properties than commercially available Rosuvastatin salt. The objective of the present study is to evaluate safety, tolerability, and toxicokinetic profile of novel salt. Therefore, four weeks repeated dose oral (gavage) toxicity and toxicokinetic study of Rosuvastatin ethanolamine was carried out. The drugs were administered once daily at salt corrected dose of 15, 40, and 100 mg/kg for four weeks. No signs of toxicity were observed during repeated (four weeks) oral administrations of Rosuvastatin ethanolamine in rats up to 40 mg/kg. Single male mortality was observed at 100 mg/kg dose. Microscopy finding in liver was minimal to mild bile ductular proliferation, single cell necrosis, and hepatocellular vacuolation of cytoplasm with associated statistically significant serum elevation of transaminase enzymes; AST, ALT, ALP, and/or liver functional marker; total bilirubin with at ≥40 mg/kg. The systemic exposures (AUC0-24 and Cmax) were not markedly different between males and females, or between the administration periods (except high dose, where exposure on day 28 was approximately 2 to 3 fold higher than that of day 1. In conclusion, Rosuvastatin ethanolamine exhibited toxicities to liver as the target organ at ≥40 mg/kg in this study. These adverse effects with associated exposures should be taken into consideration for the future assessing of potential Rosuvastatin toxicities.

Coagonist of glucagon-like peptide-1 and glucagon receptors ameliorates nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is often associated with obesity and type 2 diabetes. Coagonists of glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) are under clinical investigation for the treatment of obesity and type 2 diabetes. In this study, we have demonstrated the effect of a balanced coagonist in the treatment of NAFLD using mouse models. GLP-1R agonist exendin-4, glucagon, and coagonist (Aib2 C24 chimera2) were administered to C57BL6/J mice, in which NAFLD was induced by carbon tetrachloride (CCl4) treatment after high-fat diet (HFD) feeding, and choline-deficient, L-amino-acid-defined HFD (CDAHFD) feeding. Repeated dose administration of coagonist significantly attenuated liver inflammation and steatosis induced by acute and long-term treatment with CCl4 in HFD-fed mice. Coagonist markedly attenuated the CDAHFD-induced expression of TIMP-1, MMP-9, TNF-α, MCP-1, COL1A1, and α-SMA. It also inhibited progression of hepatic steatosis and fibrosis in mice. Exendin-4 was better than glucagon, but coagonist was most effective in reduction of hepatic inflammation as well as steatosis. Coagonist of GLP-1R and GCGR improved NAFLD in C57BL6/J mice. This effect is mediated by reduction in lipotoxicity and inflammation in liver.

Coagonist of GLP-1 and glucagon decreases liver inflammation and atherosclerosis in dyslipidemic condition.

Dyslipidemia enhances progression of atherosclerosis. Coagonist of GLP-1 and glucagon are under clinical investigation for the treatment of obesity and diabetes. Earlier, we have observed that coagonist reduced circulating and hepatic lipids, independent of its anorexic effects. Here, we investigated the role of coagonist of GLP-1 and glucagon receptors in complications of diet-induced dyslipidemia in hamsters and humanized double transgenic mice. Hamsters fed on high fat high cholesterol diet were treated for 8 weeks with coagonist of GLP-1 and glucagon receptors (75 and 150 µg/kg). Pair-fed control was maintained. Cholesterol fed transgenic mice overexpressing hApoB100 and hCETP with coagonist (300 µg/kg) for 4 weeks. After the completion of treatment, biochemical estimations were done. Coagonist treatment reduced triglycerides in plasma, liver and aorta, plasma cholesterol and hepatic triglyceride secretion rate. Expressions of HMG-CoA reductase and SBREBP-1C were reduced and expressions of LDLR, CYP7A1, ABCA1 and ABCB11 were increased in liver, due to coagonist treatment. Coagonist treatment increased bile flow rate and biliary cholesterol excretion. IL-6 and TNF-α were reduced in plasma and expression of TNF-α, MCP-1, MMP-9 and TIMP-1 decreased in liver. Treatment with coagonist reduced oxidative stress in liver and aorta. Energy expenditure was increased and respiratory quotient was reduced by coagonist treatment. These changes were correlated with reduced hepatic inflammation and lipids in liver and aorta in coagonist treated hamsters. Coagonist treatment also reduced lipids in cholesterol-fed transgenic mice. These changes were independent of glycaemia and anorexia observed after coagonist treatment. Long term treatment with coagonist of GLP-1 and glucagon receptor ameliorated diet-induced dyslipidemia and atherosclerosis by regulating bile homeostasis, liver inflammation and energy expenditure.

Coagonist of GLP-1 and Glucagon Receptor Ameliorates Development of Non-Alcoholic Fatty Liver Disease.

BACKGROUND: Obesity, diabetes and dyslipidemica are the key pathogenic stimulus that enhances progression of Non-Alcoholic Fatty Liver Disease (NAFLD). Coagonist of Glucagon Like- Peptide-1 (GLP-1) Receptor (GLP-1R) and Glucagon Receptor (GCGR) are being evaluated for obesity and diabetes. GLP-1 analogs have shown to reverse diabetes and obesity. Glucagon treatment reduces lipids after acute and chronic treatment. OBJECTIVE: In this study, we have investigated the effect of co-agonist on the prevention of NAFLD induced by long-term feeding of High Fat Diet (HFD). METHOD: We have used HFD to induce NAFLD after chronic feeding in mice. Co-agonist treatment (150 µg.kg-1, s.c.) was initiated with induction of HFD, which was continued for 40 weeks. Body weight, food intake, glucose homeostasis, lipid profile, inflammatory and fibrotic markers were assessed at the end of treatment. RESULTS: Co-agonist treatment prevented body weight gain, glucose intolerance and insulin resistance. Treatment with co-agonist reduced NEFA, increased FGF21 and adiponectin levels. Co-agonist increased glycerol release and energy expenditure, while decreased respiratory quotient. Co-agonist reduced lipids in circulation and liver. Expression of SREBP-1C, SCD-1, ACC and FAS were decreased, while ACOX1 and CPT1 were increased after co-agonist treatment. Inflammatory cytokine TNF-α and IL-6 in plasma and expression of MCP-1, TGF-ß, MMP-9, TNF-α, TIMP-1, α-SMA, and COL1A1 were decreased after co-agonist treatment. Plasma transaminases, hepatic TBARS, hepatic hydroxyproline and relative liver weight were suppressed after co-agonist treatment. Fat accumulation, inflammation and fibrosis were reduced in histological assessment of liver in co-agonist treated animals. CONCLUSION: Co-agonist prevented development of HFD-induced NAFLD by ameliorating obesity, diabetes, inflammation and fibrosis.

Dual PPARα/γ agonist saroglitazar improves liver histopathology and biochemistry in experimental NASH models.

BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are common clinico-pathological conditions that affect millions of patients worldwide. In this study, the efficacy of saroglitazar, a novel PPARα/γ agonist, was assessed in models of NAFLD/NASH. METHODS & RESULTS: HepG2 cells treated with palmitic acid (PA;0.75 mM) showed decreased expression of various antioxidant biomarkers (SOD1, SOD2, glutathione peroxidase and catalase) and increased expression of inflammatory markers (TNFα, IL1ß and IL6). These effects were blocked by saroglitazar, pioglitazone and fenofibrate (all tested at 10µM concentration). Furthermore, these agents reversed PA-mediated changes in mitochondrial dysfunction, ATP production, NFkB phosphorylation and stellate cell activation in HepG2 and HepG2-LX2 Coculture studies. In mice with choline-deficient high-fat diet-induced NASH, saroglitazar reduced hepatic steatosis, inflammation, ballooning and prevented development of fibrosis. It also reduced serum alanine aminotransferase, aspartate aminotransferase and expression of inflammatory and fibrosis biomarkers. In this model, the reduction in the overall NAFLD activity score by saroglitazar (3 mg/kg) was significantly more prominent than pioglitazone (25 mg/kg) and fenofibrate (100 mg/kg). Pioglitazone and fenofibrate did not show any improvement in steatosis, but partially improved inflammation and liver function. Antifibrotic effect of saroglitazar (4 mg/kg) was also observed in carbon tetrachloride-induced fibrosis model. CONCLUSIONS: Saroglitazar, a dual PPARα/γ agonist with predominant PPARα activity, shows an overall improvement in NASH. The effects of saroglitazar appear better than pure PPARα agonist, fenofibrate and PPARγ agonist pioglitazone.

Spontaneous lesions in endocrine glands of experimental Wistar rats and beagle dogs.

A retrospective analysis was undertaken at Zydus Research Centre to understand the incidences of spontaneous lesions in endocrine glands of Wistar rats and beagle dogs. The data from a total of 841 Wistar rats (418 males and 423 females) and 144 beagle dogs (72 males and 72 females) was used from placebo/vehicle treated control group of different non-clinical toxicity studies. The lesions in various endocrine glands were classified according to the species and age of the animals at termination of study. Among the endocrine glands, the highest numbers (types) of spontaneous lesions were observed in adrenal glands followed in descending order by pituitary, thyroid, endocrine pancreas and parathyroid glands in Wistar rats. In beagle dogs, highest numbers (types) of spontaneous lesions were seen in adrenals followed by thyroid, endocrine pancreas, pituitary and parathyroid gland. In adrenal glands of Wistar rats, the incidences of cortical cell vacuolation, hemorrhages and hemangiectasis/peliosis were increased with age. Incidence of peliosis at ∼110 weeks of age was higher in female rats. Among the proliferative lesions in rats, higher incidences of cortical cell hyperplasia was observed followed by medullary hyperplasia, complex pheochromocytoma, cortical cell adenoma and cortical adenocarcinoma. In beagle dogs, the incidences of hemangiectasis and cortical cell vacuolation in adrenal glands were higher in 18-21 months aged dogs in both the sexes as compared to 10-12 months of age. In pituitary gland, the incidences of cystic changes were higher in older rats and dogs and the incidences were more in beagles as compared to rats. In thyroid glands, C-cell (parafollicular cells) hyperplasia/complex was observed more frequently in both the species. Few incidences of cystic changes were observed in parathyroid of 18-21 months aged beagle dogs. In endocrine pancreas, few incidences of islet-cell vacuolation, atrophy and hyperplasia were observed in both the species. The Islet cell hyperplasia was found to be more frequent in male rats at ∼110 weeks of age.

Saroglitazar, a novel PPARα/γ agonist with predominant PPARα activity, shows lipid-lowering and insulin-sensitizing effects in preclinical models.

Saroglitazar is a novel nonthiazolidinediones (TZD) and nonfibric acid derivative designed to act as dual regulator of lipids and glucose metabolism by activating peroxisome proliferator-activated receptors (PPAR). These studies evaluate the efficacy and safety profile of Saroglitazar in preclinical in vitro and in vivo models. The EC50 values of Saroglitazar assessed in HepG2 cells using PPAR transactivation assay for hPPARα and hPPARγ were 0.65 pmol/L and 3 nmol/L, respectively. In db/db mice, 12-day treatment with Saroglitazar (0.01-3 mg/kg per day, orally) caused dose-dependent reductions in serum triglycerides (TG), free fatty acids (FFA), and glucose. The ED50 for these effects was found to be 0.05, 0.19, and 0.19 mg/kg, respectively with highly significant (91%) reduction in serum insulin and AUC-glucose following oral glucose administration (59%) at 1 mg/kg dose. Significant reduction in serum TG (upto 90%) was also observed in Zucker fa/fa rats, Swiss albino mice, and in high fat -high cholesterol (HF-HC)-fed Golden Syrian hamsters. LDL cholesterol was significantly lowered in hApoB100/hCETP double transgenic mice and HF-HC diet fed Golden Syrian Hamsters. Hyperinsulinemic-Euglycemic clamp study in Zucker fa/fa rats demonstrated potent insulin-sensitizing activity. Saroglitazar also showed a significant decrease in SBP (22 mmHg) and increase (62.1%) in serum adiponectin levels in Zucker fa/fa rats. A 90-day repeated dose comparative study in Wistar rats and marmosets confirmed efficacy (TG lowering) potential of Saroglitazar and has indicated low risk of PPAR-associated side effects in humans. Based on efficacy and safety profile, Saroglitazar appears to have good potential as novel therapeutic agent for treatment of dyslipidemia and diabetes.

Effect of repeated freezing and thawing on 18 clinical chemistry analytes in rat serum.

In a preclinical research laboratory, using serum samples that have been frozen and thawed repeatedly is sometimes unavoidable when needing to confirm previous results or perform additional analysis. Here we determined the effects of multiple cycles of refrigeration or freezing and thawing of rat serum at 3 temperature conditions for different storage times on clinical chemistry analytes. Serum samples obtained from adult Wistar rats were stored at 2 to 8 °C and -10 to -20 °C for as long as 72 h and at -70 °C for as long as 30 d. At different time points (24, 48, and 72 h for samples stored at 2 to 8 °C or -10 to -20 °C and 1, 7, and 30 d for samples stored at -70 °C), the samples were brought to room temperature, analyzed, and then stored again at the designated temperature. The results obtained after each storage cycle were compared with those obtained from the initial analysis of fresh samples. Of the 18 serum analytes evaluated, 14 were stable without significant changes, even after 3 freeze-thaw cycles at the tested temperature ranges. Results from this study will help researchers working with rat serum to interpret the biochemical data obtained from serum samples that have been frozen and thawed repeatedly.

Evaluation of Cardioprotective Effect of 3,5,3'-Tri-iodo-L-thyronine in Isoproterenol-Induced Cardiotoxicity.

T(3) (3,5,3'-triiodothyronine) has drawn relatively little attention in relation to cardiovascular (CVS) diseases. The present study was designed to evaluate the cardioprotective action of T(3) in isoproterenol-(ISO-) induced cardiac toxicity. Female Wistar rats were exposed with ISO (100 mg/kg, body weight, subcutaneously) for 2 days at the interval of 24 h followed by T(3) (3 µg/kg, body weight, orally) treatment for 3 days. Positive control rats received only ISO (100 mg/kg, body weight, subcutaneously) for 2 days at the interval of 24 hrs. Control group animals received normal saline as a vehicle. As expected, ISO-induced significant changes were observed in low-density lipoprotein, total cholesterol, ALT, CK-MB to TCK ratio, and prolongation of QT interval in electrocardiogram, which is toward normalization after T(3) treatment. Lower heart weight, upregulation of cardiac myosin heavy chain alpha (MHC-α), and reduced inflammatory cell infiltration, myonecrosis, vacuolar changes, and a trend toward normal cardiac muscle fiber architecture in microscopic examination of cardiac tissue further support the cardioprotective effect of T(3).