Product Highlight: Ligand Binding Assays for GLP-1 analogs (Peptide Drugs) in line with FDA Considerations

Background:

Glucagon-like peptide-1 (GLP-1) analogs also known as incretin mimetics are class of drugs indicated for type-2 diabetes mellitus to lower the blood sugar level. The mechanism of the GLP-1 analogs is to prevent the breakdown of GLP-1. This enhances the glucose depended insulin release and suppresses the glucagon release (which reduces hepatic glucose output), and reduces gastric emptying rate (resulting in weight loss from reduced appetite). The other benefit of GLP-1 analogs is that they do not cause hypoglycaemia like other class of anti-diabetic medications and also provide weight reduction benefit.

According to the International Diabetes Federation™ (IDF) Diabetes Atlas 2017, around 425 million patients suffer from diabetes worldwide. As per a World Health Organization (WHO) report 2017, diabetes is one of top three cause of death among the non-communicable diseases. China (114.4 million), India (72.9 million) and U.S. (30.2 million) are top three economies having high prevalence of diabetes.

Introduction:

Pancreas safety has become a subject of much debate concerning dipeptidylpeptidase-4 (DPP-4) inhibitors (DPP-4is) and glucagon-like peptide-1 (GLP-1) receptor agonists (GLP-1RAs). This concern is partly based on the well-described effect of GLP-1 to induce growth of pancreatic Î?2;-cells. Both drug classes increase effective GLP-1 levels, but to a different degree, and have different modes of action; thus, it is important to differentiate between them, particularly when considering mechanistic hypotheses for potential safety concerns or signals. GLP-1RAs mediate their effects directly through the GLP-1R. Although increased levels of GLP-1 and glucose-dependent insulinotropic peptide (GIP) are considered important parts of the mechanism of action of DPP-4is (4), DPP-4 is known to degrade many other hormones. DPP-4is have been shown to increase GLP-1, GIP, and peptide YY. This complicates the understanding of both desired and potentially undesired effects of this class of compounds. Within GLP-1RAs, differences exist in safety-related parameters. One subgroup is the exendin-4 based drugs with exenatide and lixisenatide, which are structurally distinct from human GLP-1. Owing to the low amino acid homology to native human GLP-1, these medications are associated with an increased number of immune reactions that are, however, all of a relatively mild form, that is, mostly antibody development, injection site nodules, and loss of efficacy. The other subgroup is based on human GLP-1 and contains liraglutide, taspoglutide, and larger covalently conjugated molecules such as albiglutide and dulaglutide. Clinical development of taspoglutide was stopped due to severe immune-related side effects, with cases of anaphylactic shock, possibly caused by the formulation. No such findings have been reported with other GLP-1 based analogs.

Semaglutide is a once-weekly GLP-1 analog that is in phase 3 clinical development. Where liraglutide is acylated with a palmitic acid and has an extra amino acid as a spacer between the palmitic acid and the Lys26, where the fatty acid is attached, semaglutide is acylated with a stearic diacid at Lys26 but has a much larger synthetic spacer and is, furthermore, modified for DPP-4 stability in position 8, where the amino acid Î±-aminobutyric acid has been introduced.

KRISHGEN has followed a consistent and successful approach in developing lignd binding assays for exenatide, liraglutide, lixisenatide, semaglutide. The principle used has been to bound the ligand complex by using a secondary antibody to bind the biotinylated peptide-antibody complex to the microtiter plate. Subsequently the detection is done using a streptavidin HRP conjugate. This successful approach has helped to ensure sensitive, consistent detection levels and remove the time consuming step of sample preparation or acid dissociation with a SPE/cleanup step as in LC/MS assays.

These kits have been validated as per ICH Guidelines for Bioassays including for sample matrix interferences and have reported recovery within acceptable limits.

Conclusion:

Successfully developing robust bioanalytical PK assays for peptides requires a combination of skill sets including assay development expertise, familiarity with small and large molecule assay platforms (LC-MS/MS and LBA), creativity to find unique solutions, and perseverance. Standard LC-MS/MS or LBA techniques are not often sufficient in solving the riddles that peptide PK assays present. As the number and types of innovative peptides in preclinical and clinical development continue to grow, bioanalytical laboratories will need to adapt to meet the challenges ahead.

References:

The Human GLP-1 Analogs Liraglutide and Semaglutide: Absence of Histopathological Effects on the Pancreas in Nonhuman Primates. …Carsten F. Gotfredsen, Anne-Marie MÃ¸lck, Inger Thorup, Niels C. Berg Nyborg, Zaki Salanti, Lotte Bjerre Knudsenâ‡‘ and Marianne O. Larsen … Diabetes 2014 Jul; 63(7): 2486-2497. https://doi.org/10.2337/db13-1087