Evaluation of reproductive safety of glucokinase activator zygk1, a potential therapeutic agent for diabetes

Abstract

Diabetes, especially type 2 diabetes, is a consequence of insulin resistance that occurs due to impairment of the glucose metabolism, resulting in decreased absorption and use of glucose in tissues like liver as well as muscle. Hyperglycaemia, typically observed in type 2 diabetes is linked with reduced capacity of pancreas to secrete insulin hormone in response to glucose and decreased glycolysis or glycogenesis in liver. Type 2 diabetes is a progressive disease, which is difficult to treat since the therapeutic effect of the medications decreases over time, and the therapies are also linked with side effects mainly concerning cardiovascular and metabolic systems. Hence, new drug discoveries for treating diabetes are required to satisfy the unmet need in therapy. Diabetes during pregnancy, or gestational diabetes has very high prevalence, with limited therapeutic options like insulin, metformin, and sulfonylureas. Many of the oral antidiabetic agents reach the fetus through placenta and may show teratogenic potential or embryo lethal fatality during preclinical toxicity studies. It is therefore necessary to conduct toxicity studies related to embryo fetal development to support the development in humans for any antidiabetic agent. These kinds of research are necessary to check unexpected side effects on the reproductive system and organogenesis, where doses more than the human proposed therapeutic doses need to be checked for evaluating the possible effects of the investigational drug on pregnancy, fetal viability and development. Glucokinase (GK) is an enzyme that causes the phosphorylation of glucose to glucose-6-phosphate in the presence of ATP, for both glycogen synthesis and glycolysis. GK is mainly expressed in liver and pancreas, with a lesser role in intestine, muscles, and brains, and hence has a significant role as a possible target for treating type 2 diabetes mellitus. In beta cells of pancreas, glucokinase enzyme acts as the sensor of glucose concentration and accordingly secretes insulin, whereas in liver, it causes glucose-metabolism. Loss of function mutations of glucokinase lead to maturity-onset diabetes of the young type 2 and gestational diabetes indicating the incidence of diabetes in case of decreased glucokinase activity. On the other hand, individuals with gain of function glucokinase mutations demonstrate hypoglycaemia caused by increased insulin secretion. The increased insulin secretion in gain of function glucokinase mutations suggest that a small molecule glucokinase activators (GKAs) may show significant antidiabetic activity by increasing glucose-stimulated insulin release and by increasing glucose metabolism in liver. Many small molecule compounds that are allosteric modulators of glucokinase have been synthesised and studied in preclinical and clinical studies for antidiabetic activity. In this study, I have evaluated the activity of a novel glucokinase activator ZYGK1, 3-[4-5-Methyl-1,3,4oxadiazol-2-yl]phenoxy]-5-[[(3S)-1-methyl-2-oxo-3 pyrrolidinyl] oxy]-N-2-thiazolylbenzamide using various preclinical models of diabetes. I have also evaluated the toxicity of this compound in rats. My study indicates that ZYGK1, a novel glucokinase activator can stimulate glucokinase enzyme in the liver as well as pancreas that translate into therapeutically significant antihyperglycemic effect in glucose intolerant type 2 diabetes models. The overall effect seems to be devoid of any derangement in body weight or lipid levels indicating that ZYGK1 can be a safe and effective option for treating of type 2 diabetes. Based on its mechanism, ZYGK1 appears to combine the effect of two first line antidiabetic agents, namely sulfonylurea that works in pancreas and metformin, which works in liver. Further, since ZYGK1 appears to reverse the desensitization induced by sulfonylurea therapy, it appears to be a suitable therapy for a wide range of patients besides the ones that develop resistance to sulfonylurea therapy. It is crucial to understand the pathological responses of healthy animals to evident and consistent hypoglycaemia while evaluating the risk of findings to human. This helps in translating the results from animal studies to human clinical settings. In this study, I have investigated the effects of repeated dosing of ZYGK1 and observed that it caused persistent hypoglycaemia in healthy male as well as female rats. This condition caused structural changes in peripheral nerves, which appear to be due to hypoglycaemia or hyperinsulinemia. These changes were observed at very high doses and were found to be reversible after cessation of treatment. Hypoglycaemia-associated changes appear to be attributed to the exaggerated pharmacology of ZYGK1, as they are evident only at high doses. The study highlights the need for understanding these effects to interpret results from pre clinical testing of new glucokinase activators. Since hypoglycaemia of mother during gestation can lead to adverse effects on pregnancy and embryo-fetal development, we further conducted the study to understand the effects of ZYGK1 on embryo-fetal developmental toxicity in rats to evaluate reproductive safety. In this study, ZYGK1 was administered at doses (60 mg/kg and 120 mg/kg) to pregnant rats. The findings indicated that though ZYGK1 induced significant decrease in blood glucose in pregnant rats, however, it did not cause major malformation and hence it can be considered as non-teratogenic in Wistar rats. Blood glucose levels decreased in all ZYGK1-treated pregnant rats at initiation of treatment i.e., gestation day 6 as well as completion of treatment i.e., gestation day 15. In contrast to the similar glucose levels at the start of the treatment (start of dosing on Day 6 of pregnancy), the treatment showed severe hypoglycemia on Day 15 of pregnancy. Despite these effects, there were only nonsignificant compensatory increases in food consumption, which decreased after cessation of treatment on Day 15 of pregnancy. The sustenance of this metabolic adaptation was also observed in other glucokinase activator treatments in toxicity studies, indicating the reversibility of the toxicity. After the fetal examination post caesarean section, the body weight of fetuses was significantly reduced in both 60 and 120 mg/kg group, and the crown-rump length was also decreased at 120 mg/kg dose of ZYGK1. However, no changes in ophthalmic tissues were found. Eye abnormalities were reported to be caused by maternal hypoglycaemia induced by insulin injections as well as by small molecule glucokinase activator in toxicity studies. Though the ophthalmic abnormalities were not found in ZYGK1-treated rats, it appears that the decrease in fetal body weight as well as nonsignificant increase in implantation loss or resorption could be the effect of maternal hypoglycemia, and not the direct effect of ZYGK1. In conclusion, oral administration of ZYGK1 treatment was well tolerated, without any mortality in male and female Wistar rats, with partially reversible peripheral nerve changes that can be attributed to the sustained hypoglycaemia. For embryo fetal toxicity study, the pregnant rats did not show any toxicity up to the dose of 120 mg/kg. The toxicities were also not evident in gestation period including external, visceral and skeletal examinations in this study. The correlation between the extent of hypoglycaemia and fetal anomalies in higher species like rabbits can give novel insights for linking embryo-fetal toxicity with maternal hypoglycaemia. Therefore, it may be concluded that ZYGK1 is non-teratogenic and can be considered as a potential therapeutic agent for treating gestational diabetes.