Comprehensive toxicological evaluation of novel anti-hypertensive small molecule KSD179019

Abstract

The most common factor for mortality and morbidity worldwide is hypertension. Unfortunately, 10% of hypertensive patients do not respond to the treatment regimen that is currently prescribed; this condition is referred to as resistant hypertension. Since our lab reseach focusing on the function of Secretin and the Secretin receptor (SCTR) in the water homeostasis for ten years, our team has discovered SCTR to be a novel target for anti-hypertensive medications and Small molecule KSD179019 as a Positive Allosteric Modulator (PAM) for SCTR that has recently been identified as having anti-hypertensive effects in hypertensive animal models. A novel drug molecule's safety must be taken very seriously. As a result, we pursued to establish the maximum tolerated level of dose for KSD179019 and examine its possible toxicity. A sequence of in vitro toxicity tests were designed to assess the mutagenicity of S. Typhimurium and E. coli strains, which is been in practice for more than 4 decades to detect mutagenicity of chemicals. Also, the cell toxicity of HepG2 and hek 293 cell lines. The outcome of in vitro study data showed that there was no cell toxicity observed on HepG2 and Hek293 cell lines up to 1 mM concentration of KSD179019 and no toxic mutagenic effect of the KSD179019 was observed up to 5000 µg/ml on TA98, TA 100, TA 1535, TA1537 strains of S. Typhimurium and E. coli WP2 [pKM101], coli WP2 uvrA. As these findings were encouraging, we carry on to conduct in vivo toxicity study following to the OECD guideline. Following the OECD test recommendations 426, an acute toxicity study for 14 days of KSD179019 was carried out. The dosage of 2000 mg/kg was administered with KSD179019 suspension on both female and male C56/BL/6J mice (n = 6). The sign of acute toxicity was observed by carefully monitoring the parameters such as abnormalities in eyes, changes in body weight, skin and fur, aberrant behaviour and mortality for respective 14 days. No death was observed for the dosage of 2000 mg/kg at the end of 14 days. As no death was observed for the dosage of 2000 mg/kg, we increased the dose to 5000 mg/kg (limit test) to determine the lethal dose (LD50). There was still no death caused by KSD179019 during the limit test. Then, we moved to perform 90 days sub-chronic study following the OECD test guidelines 408 with three different dose groups of KSD179019 (1000,700, 500 mg/kg) through oral gavage administration in each group of both sexes (n=10). Further 180 days chronic toxicity study was conducted with the doses of 500, 350, 250 with doubled number of mice as per the OECD guidelines 452. After the dose period in both sub chronic and chronic study, all the animals including the control were sacrificed and detained urine analysis for the parameters like pH, ketones, specific gravity, bilirubin, glucose, blood, protein, nitrate, urobilinogen, and leukocytes. The blood collected from mice was used to analyse the biochemical tests to assess the kidney and liver functions, thyroid gland function, lipid profile and haematological analysis. / The data from invitro showed no toxic effect of KSD179019 on cells and bacteria. From 14 days acute toxicity study, KSD179019 was grouped under drug category 5 (OCED 423), where the Lethal death is above 5000 mg/kg. From the 90 days sub-chronic toxicity study KSD179019 did not appear to cause considerable toxicity up to 500 mg/kg and selected as reference dose for chronic study. The dose of 350 mg/kg b.w. of KSD179019, as determined by the findings of tests on chronic toxicity, was not harmful and was designated as the dose with no-observed-adverse-effect-level (NOAEL), which will be useful in future dose selection in human clinical investigations.