Hepatic in vitro metabolism of peptides; comparison of human liver S9, hepatocytes and Upcyte Hepatocytes with cyclosporine A, leuprorelin, desmopressin and cetrorelix as model compounds

Abstract The number of approved peptide therapeutics has increased significantly in recent years. Peptide therapeutics have many advances over small molecule drugs, such as higher affinity to target and lower toxicity profiles. Although peptide-like drugs are mainly metabolized/catabolized in the body for smaller peptides and amino acids, metabolite identification still has an essential part of in their development, especially if their structure contains modified amino acids, and also to identify the metabolic soft spots enabling modification to more stable sequence. The use of human derived in vitro systems is an important tool when investigating metabolism of peptide drugs, and comparison of results by various hepatic systems was investigated here. Peptides were incubated in several different in vitro human liver-derived subcellular and cellular incubation systems, i.e. liver S9 fraction, suspended cryo-preserved human primary hepatocytes and plated Upcyte hepatocytes. Samples were collected at different time points and analysed by UPLC/HR-MS-method developed for the purpose. Both substrate disappearance and metabolite formation were monitored, and the systems were compared. S9 fraction formed the highest number of metabolites for leuprorelin and cetrorelix, while for desmopressin and cyclosporin, primary hepatocytes and liver S9 produced similar metabolite profiles. Interestingly, not only cyclosporin, but also leuprorelin and cetrorelix showed metabolites whose formation was CYP (NADPH) dependent in liver S9. For leuprorelin and cetrorelix, the metabolites that showed NADPH dependency with liver S9, were not detected with hepatocytes, even though for leuprorelin these reactions played a major role in liver S9. The hydrolytic metabolic reactions were very similar between liver S9 and hepatocytes, i.e. the metabolite profiles in hepatocytes matched better with liver S9 profiles without NADPH, which may be caused by cell uptake rate limitation with hepatocytes, or then hydrolytic processes are more stressed in peptide metabolism with hepatocytes, in comparison to CYP-mediated processes.