The 1990s has seen the arrival of many protein therapeutic drugs. In response to the increased availability and use of these macromolecular pharmacological agents, the FDA is moving its review of these products from the """"""""biologicals"""""""" category into the """"""""drugs"""""""" category. As such, increased emphasis on physiological disposition would be expected. Yet, suitable methods for studying protein drug disposition do not exist. Radioisotopes, bioassays, and immunoassays all present problems. In contrast, a new experimental approach developed in this laboratory promises more definitive results, especially in the area of metabolism. This approach uses uniformly stable-isotope labeled proteins as test species, separates biological fluids with HPLC, and quantifies stable- isotope enriched chromatographic peaks with Chemical Reaction Interface Mass Spectrometry (HPLC/CRIMS). Uniform labeling ensures that all metabolites of the protein can be detected. Such proteins can be generated by growing a suitable cell line in stable-isotope labeled medium. The three protein disposition studies proposed are: (1) the angiotensinogen cascade and the effect of a protease inhibitor on that cascade; (2) the intracellular proteolysis of fibroblast growth factor-1 (FGF-1); and (3) the kinetics and metabolism of insulin connecting peptide (C-peptide). The purpose of the first study is to demonstrate how the uniformly labeled protein + HPLC/CRIMS analysis provides a comprehensive survey of the consequences of an inhibitor of angiotensin converting enzyme (a dipeptidyl carboxypeptidase). In the FGF-1 experiments, the goals are to generate an unambiguous profile of the intracellular metabolites produced in NIH 3T3 cells, evaluate which of these peptide metabolites contribute to the activity of FGF-1, and identify the active species using electrospray mass spectrometry. Recently, C-peptide was shown to lessen the vascular and neurological consequences in experimentally-diabetic rats. Because C-peptide has long been believed to be inactive, little information exists about its kinetics, and nothing is known about its metabolic fate. Overall, this work will show he capabilities of a new strategy by which a more accurate understanding of the fundamental pharmacology of an increasingly important class of drugs can be gained.
| Lecchi, Paolo; Gupte, Anand R; Perez, Ricardo E et al. (2003) Size-exclusion chromatography in multidimensional separation schemes for proteome analysis. J Biochem Biophys Methods 56:141-52 |
| Abramson, F P (2001) The use of stable isotopes in drug metabolism studies. Semin Perinatol 25:133-8 |
