Subcutaneous (SC) injection is the most common administration route for peptide therapeutics. Catabolism at the injection site can be a specific and major degradation pathway for many SC administered peptides. In some cases, it can significantly affect pharmacokinetics, particularly bioavailability, and have detrimental effects on the efficacy of the drug. This work describes a liquid chromatography-high resolution mass spectrometry based in vitro assay to assess peptide metabolism in the SC tissue (SCiMetPep assay). The SCiMetPep assay was developed using human, Sprague-Dawley rat and Göttingen minipig SC tissue homogenate supernatant, and allows for both determination of degradation rate (half-life) of the parent peptide and identification of metabolites generated from enzymatic proteolysis. The assay was developed and validated using known peptides including human insulin and four GLP-1 analogues (lixisenatide, exenatide, liraglutide and semaglutide). Different experimental parameters were evaluated in order to optimize the homogenization process of the SC tissue and the peptide incubation conditions. In vitro metabolism of these peptides was in good agreement with in vivo data reported in the literature. Finally, when SCiMetPep assay was applied on a series of structurally related peptides, a fairly good correlation was found between SC metabolic stability and bioavailability, suggesting that catabolism at the injection site can have a major role in the absorption, distribution, metabolism, and excretion (ADME) of peptide therapeutics. The SCiMetPep showed the ability to identify analogs with improved SC metabolic stability and hence higher bioavailability. The assay can be used in the early phases of drug discovery to identify peptide metabolic soft spots at the injection site and guide the peptide drug discovery process.
Keywords: Bioavailability; HRMS; In vitro; Metabolism; Peptide; Subcutaneous.
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