Application of in vitro models in the catabolism studies of therapeutic peptides
Thesis event information
Date and time of the thesis defence
Place of the thesis defence
IT116, University of Oulu
Topic of the dissertation
Application of in vitro models in the catabolism studies of therapeutic peptides
Doctoral candidate
Master of Science Juha Jyrkäs
Faculty and unit
University of Oulu Graduate School, Faculty of Technology, Sustainable Chemistry
Subject of study
Chemistry
Opponent
Associate Professor Tiina Sikanen, University of Helsinki
Custos
Docent Ari Tolonen, University of Oulu
Research on the in vitro catabolism of therapeutic peptides revealed a novel mechanism
A study conducted at the University of Oulu has discovered a mechanism that may have implications for how peptide drugs are studied in the future. Peptide drugs have been used in medicine for over a century, but they have long been overshadowed by small molecule drugs, mainly because peptide drugs cannot traditionally be administered orally and are therefore not as widely and easily used. However, GLP-1 analogues, originally developed for the treatment of diabetes, have gained popularity as weight loss drugs, which has also increased the interest to utilize other peptide drugs for various indications. The aim of the study was therefore to investigate the usefulness of different in vitro models for studying the catabolism of peptide drugs.
Four different peptide drugs were studied in models derived from human and rat tissues, and the samples formed were analyzed using liquid chromatography-mass spectrometry. During the study, it was found that the cofactor NADPH affected the catabolite profiles of one linear peptide, leuprorelin, in a previously unreported manner: the addition of NADPH appeared to activate the enzymes that degrade leuprorelin, resulting in an increase in the number of catabolites. NADPH acts as an electron source for various enzymes, such as CYP enzymes that commonly break down small-molecule drugs, but peptidases, enzymes responsible for the degradation of both therapeutic and endogenous peptides, do not normally utilize NADPH in the process. Therefore, the goal of the study was to determine the mechanism behind this activating phenomenon. Further studies revealed that the effect of NADPH on peptidase activity was indirect: peptidases often contain thiol groups that can form a disulfide bridge, which affects the activity of peptidases. Cells contain enzymes that reduce disulfide bridges to free thiol groups, utilizing the electron pair donated by NADPH. These enzymes, known as oxidoreductases, have been known for decades, but this study was the first to report their effect on the degradation of peptide drugs in in vitro models.
Finally, animal studies conducted during the investigation revealed that the leuprorelin catabolite profiles formed in the presence of NADPH did not correspond as well to the catabolite profiles formed in animals as those without NADPH. However, the target of leuprorelin is outside the cell, and linear peptides are generally unable to cross the cell membrane, so the effect of this mechanism may be greater and more significant for peptide drugs that can. Further research is needed to clarify the potential impact of this mechanism on the correlation between the catabolite profiles and clearance of other peptide drugs between in vitro models and in vivo.
Four different peptide drugs were studied in models derived from human and rat tissues, and the samples formed were analyzed using liquid chromatography-mass spectrometry. During the study, it was found that the cofactor NADPH affected the catabolite profiles of one linear peptide, leuprorelin, in a previously unreported manner: the addition of NADPH appeared to activate the enzymes that degrade leuprorelin, resulting in an increase in the number of catabolites. NADPH acts as an electron source for various enzymes, such as CYP enzymes that commonly break down small-molecule drugs, but peptidases, enzymes responsible for the degradation of both therapeutic and endogenous peptides, do not normally utilize NADPH in the process. Therefore, the goal of the study was to determine the mechanism behind this activating phenomenon. Further studies revealed that the effect of NADPH on peptidase activity was indirect: peptidases often contain thiol groups that can form a disulfide bridge, which affects the activity of peptidases. Cells contain enzymes that reduce disulfide bridges to free thiol groups, utilizing the electron pair donated by NADPH. These enzymes, known as oxidoreductases, have been known for decades, but this study was the first to report their effect on the degradation of peptide drugs in in vitro models.
Finally, animal studies conducted during the investigation revealed that the leuprorelin catabolite profiles formed in the presence of NADPH did not correspond as well to the catabolite profiles formed in animals as those without NADPH. However, the target of leuprorelin is outside the cell, and linear peptides are generally unable to cross the cell membrane, so the effect of this mechanism may be greater and more significant for peptide drugs that can. Further research is needed to clarify the potential impact of this mechanism on the correlation between the catabolite profiles and clearance of other peptide drugs between in vitro models and in vivo.
Created 2.11.2025 | Updated 3.11.2025