RH-Insulin (Sunshine Lake)

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.

Insulin might be associated with changes in infant gastrointestinal microbiota. The objective of this randomized controlled trial was to assess the efficacy of two doses of recombinant human(rh) enteral insulin administration compared to placebo in intestinal microbiota.

19 preterm patients were recruited at the NICU of La Paz University Hospital (Madrid, Spain). Subjects received 2000 µIU of rh enteral insulin/ml(n = 8), 400 µIU of rh enteral insulin/ml(n = 6) or placebo(n = 5) for 28 days administered once per day. Extracted DNA from fecal samples collected at the beginning and end of treatment were analyzed. The 16S rRNA V4 region was amplified and sequenced in a Miseq(Illumina®) sequencer using 2 × 250 bp paired end. Resulting reads were filtered and analyzed using Qiime2 software. Metabolic activity was assessed by GC.

Gestational age and birth weight did not differ between groups. At the phylum level, both insulin treated groups increased the relative abundance of Bacillota, while Pseudomonadota decreased. No change was observed in infants receiving placebo. At the genus level, insulin at both doses showed enriching effects on Clostridium. We found a significant increase in concentrations of fecal propionate in both rh insulin treated groups.

Rh insulin may modify neonatal intestinal microbiota and SCFAs in preterm infants.

Decrease of Pseudomonadota (former Proteobacteria phylum) and increase of Bacillota (former Firmicutes phylum) obtained in this study are the changes observed previously in low-risk infants for NEC. The administration of recombinant enteral insulin may modify the microbiota of preterm new-borns and SCFAs. Modulation of the microbiota may be a mechanism whereby insulin contributes to neonatal intestinal maturation and/or protection.