05 Jun Peptide Metabolism: Identification of metabolite structures of GLP-1 receptor agonists in different in-vitro systems using high resolution mass spectrometry

Andreas Brink(1), Alessandra Piranha(1,2), Yves Siegrist(1), Aynur Ekicilier(1), Nicola Thum(1,3), Fabien Fontaine(4), Ismael Zamora(4), Marcel Gubler(1), Silke Simon(1), Nicole Kratochwil(1), Simone Schadt(1)
1. Drug Disposition & Safety, Pharmaceutical Sciences, Pharma Research and Early Development, Roche Innovation Center Basel F. Hoffmann-La Roche Ltd., Grenzacherstrasse 124, 4070 Basel, Switzerland; 2. Royal Melbourne Institute of Technology, Melbourne, Australia; 3.School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland; 4. Lead Molecular design, S.L., Spain

Introduction Peptide drugs are an important class of therapeutics under investigation in various pharmaceutical companies. Assessment of peptide stability in vitro, the identification of cleavage sites and structure elucidation of degradation products are important tasks of drug metabolism scientists. However, most in vitro systems established to investigate metabolism of small molecules (e.g. microsomes) are not relevant for peptides because most peptides show low cell membrane permeability and are subject to hydrolysis by enzymes expressed on epithelial cell surfaces. In addition to relevant in vitro systems, appropriate mass spectrometry approaches and tailored software tools are required due to the higher molecular weight, presence of multiple-charge stages upon electrospray ionization and increasing molecular complexity (modified amino acids, cyclisation etc.) of peptide drug candidates. Methods Glucagon-Like Peptide-1, (GLP-1) and three analogs (taspoglutide, liraglutide, exenatide) were incubated with the human recombinantly expressed enzymes dipeptidyl-peptidase-IV (DPP-4) and neutral endopeptidase (NEP) as well as with various cellular systems, namely primary and immortalized human umbilical vein endothelial cells (HUVEC cells), TERT1-immortalized renal proximal tubule epithelial cells (RPTECs-TERT1 cells) and human hepatocytes. Samples were analyzed up to 24 hours using a Q Exactive™ Hybrid Quadrupole-Orbitrap Mass Spectrometer in data dependent scan mode. The mass inclusion list set-up (up to z = 5) and the post-acquisition data analyses were performed using the recently introduced peptide mode integrated in Mass-MetaSite (MMS 3.2.0). MMS extracted metabolite peaks and interpreted MS/MS fragmentation to provide structural proposals. The results were reviewed using WebMetabase (version 3.1.4). Preliminary Data The peptide mode of the Mass-Metasite software was successfully applied to process full scan HRMS data to detect and identify metabolites of 4 model peptides. MMS proposed definitive metabolite structures to the identified metabolite peaks based on the interpretation of MS2 fragmentation data. Based on these metabolite structures, peptide bond cleavage sites could be demonstrated. WebMetabase was able to sort and match metabolites based on retention time and MS2 fragmentation across the different in vitro experiments resulting in an efficient workflow to compile results for comparison of different in vitro systems with regard to metabolites formed. The results showed that GLP-1 was metabolized rapidly in the presence of DPP-4 (t1/2 ≈ 7 min). The main metabolite identified by MMS (< 2 ppm) resulted from N-terminal cleavage after amino acid 8 (Ala), corresponding to GLP-1 (9-37). This observation was in line with reports from literature. The same metabolite increased with time in incubates with primary HUVEC, immortalized HUVEC and hepatocytes indicative of functional DPP-4 activity in these cell lines. Turnover in presence of DPP-4 was as well seen for taspoglutide and liraglutide, however at a slower rate compared to GLP-1. Analog to GLP-1 the cleavage sites were assigned after amino acid 8 at the N-terminus resulting in liraglutide(9-37) and taspoglutide(9-37). Taspoglutide in presence of NEP was initially cleaved between the amino acids Ser18-Tyr19 and Tyr19-Leu20 position (analog for liraglutide). Further peptidic cleavage lead to shorter peptides mainly seen in human hepatocytes. For taspoglutide and liraglutide, mostly the same metabolites were seen in HUVEC cells when compared with the isolated enzyme systems NEP and DPP-4 alone. Preliminary data suggest that no significant qualitative difference was observed between primary and immortalized HUVEC cells for degradation products of GLP-1 and its structural analogs. In conclusion this approach might be used for peptide metabolism investigations. Novel Aspect New software-aided approach to analyze HRMS data to investigate stability and cleavage sites of peptides in different in vitro systems.