Experience in validating a method for assessing GLP-1R receptor activation in vitro upon stimulation with tirzepatide

Introduction. Tirzepatide is a molecule capable of controlling blood glucose levels by combining dual agonism of the glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptors. GLP-1 and GIP are incretin hormones involved in the regulation of glycemia and energy metabolism. Their combined agonism enhances the insulin response, reduces glucagon secretion and appetite, making it an effective strategy for the treatment of type 2 diabetes mellitus (T2DM) and obesity. The specific biological activity of such compounds can be evaluated in vitro using cell lines expressing GLP-1R or GIPR.

Aim. To validate a method for determining GLP-1R receptor activation in vitro using a cell line stimulated with tirzepatide in the "Calcium Flux" assay. This approach is necessary for subsequent studies of the comparability of bioequivalent drugs.

Materials and methods. A genetically engineered HTS163L cell line expressing the human GLP-1 receptor was used as the model. Receptor activation was recorded by changes in fluorescence signal caused by an increase in intracellular calcium concentration after incubation of cells with tirzepatide at various concentrations. Method validation was carried out in accordance with the requirements of the State Pharmacopoeia of the Russian Federation, ICH, and EMA. Statistical analysis of the results was performed using MARS software (BMG LABTECH).

Results and discussion. The applied method demonstrated a high level of specificity (placebo signal <2 % of the drug signal), a satisfactory limit of quantification (signal exceeded the blank by 43 %), good linearity (R² = 0.981), accuracy (within 77–119 % of nominal EC₅₀), and precision (CV ≤ 5 % within runs, ≤15 % between runs). All validation parameters met the established acceptance criteria, confirming the correctness of the chosen approach.

Conclusion. The proposed method demonstrates high sensitivity and reproducibility, making it suitable for functional analysis of the biological activity of GLP-1 receptor agonists, including tirzepatide, in in vitro models.

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