Evaluation of New-Modelled Recombinant Human Insulin (rh-Insulin) Analog Expressed in E. coli Using Radioiodination Technique Followed by In Vivo Biodistribution in Diabetes-Induced Mice

Abstract

Biologists have significantly improved various techniques for confirming the physiological and pharmacological activity of new proteins produced by recombinant DNA technology, such as Western blotting, ELISA, and flow cytometry. Although these methods are costly and comparatively low in efficiency, our study focuses on developing a real-time approach to investigate the physiological activity of our new recombinant human insulin (rh-Insulin), which is expressed in Escherichia coli. An in vivo biodistribution study of radioiodinated rh-Insulin (¹²⁵I-rh-Insulin) was conducted in diabetic-induced mice, exploiting the capability of tyrosine residues in protein molecules to undergo electrophilic substitution of hydrogen atoms with traceable ¹²⁵I atoms. We studied many factors to optimize the conditions for the iodination reaction, including the amount of substrate, the amount of chloramine-T, pH, temperature, and reaction time. A high radiochemical yield of 99.01 ± 0.2% was achieved. The in vivo step involved the administration of ¹²⁵I-rh-Insulin intravenously (I.V.) in previously induced diabetic mice to study the pharmacokinetics of the new insulin analog. Results show a homogeneous distribution of insulin molecules throughout the body organs, correlating with organ mass, size, and functionality, with no accumulation in distinct organs. The clearance of insulin from the body occurs via both renal and hepatic routes due to the aqueous nature of insulin. Additionally, a parallel experiment was conducted on diabetic mice using only rh-Insulin, resulting in a significant reduction in glucose levels in the mice's blood, thereby exploring the physiological activity of insulin and confirming the ability of our new construct to lower blood glucose levels in diabetic mice. Consequently, this method appears to be much more rapid and effective for the evaluation of biological molecules in vivo using radioactive tracing techniques.