Bart De Spiegeleer

In vitro metabolic stability of obestatin: Kinetics and identification of cleavage products

The in vitro metabolic stability testing on synthetic obestatin peptides from two different species (human hOb and mouse mOb) using HPLC analysis is described. A reversed-phase C(18) column of 300A pore size was used, with a gradient system based on aqueous formic acid and acetonitrile. Electrospray ionization (ESI) ion trap mass spectrometry was used for identification of the chromatographic eluting peptide metabolic products, while UV (DAD) and fluorescence served quantitative purposes. Differences in the metabolic degradation kinetics of hOb and mOb were found in plasma, liver and kidney homogenate, with half-lives ranging between 12.6 and 138.0min. Proteolytic hydrolysis at the N-terminal Phe residue and cleavage at Pro(4)-Phe(5) were found to be two major metabolic pathways, accounting for more than 50% of the metabolic degradation. Several other labile peptide bonds were located. The influence of a standard protease inhibitor cocktail was investigated, as well as the metabolism of iodinated human obestatin in liver homogenate. Our results indicate that the major instability of obestatin peptides, as currently used in biomedical investigations, should be taken into account in the interpretation of the obtained results.

Adsorption of peptides at the sample drying step : Influence of solvent evaporation technique, vial material and solution additive

Although the efficient and careful removal of solvent from samples by centrifugal evaporation or freeze-drying methods is an important step in peptidomics, the recovery of peptides has not yet been fully investigated with these sample drying methods. Moreover, the surface adsorption of the peptides by the container and efforts to reduce this adsorption by organic additives is only scarcely elaborated until now. In this experiment, the recovery of five model peptides, i.e. bovine insulin, mouse obestatin, goserelin, buserelin and leucine-enkephalin was investigated applying dimethylsulfoxide (DMSO), dimethylformamide (DMF), polyethylene glycol 400 (PEG 400), mannitol and n-nonyl-beta-d-glucopyranoside (C(9)-Glu) in function of the two applied solvent evaporation processes (freeze-drying vs. centrifugal evaporation) and vial types, i.e. polypropylene (PP) and glass. Under our experimental conditions, drying resulted in a decreased recovery of the model peptides by 10% on average. Insulin showed the lowest recovery value relative to the other model peptides. For both drying methods, recovery of the model peptides was increased when C(9)-Glu was present. Overall, the use of PP vials is proposed for freeze-drying, while glass vials are found to be more suitable for centrifugal evaporation. The presence of PEG 400 in PP vials caused significantly reduced recoveries for all model peptides using centrifugal evaporation, although this was not observed in glass vials. As a general conclusion, applying C(9)-Glu as an additive along with choosing appropriate vial type (i.e. PP for lyophilization and glass for centrifugal evaporation) can avoid or diminish peptide loss during the evaporation procedure.

Relative response factor determination of β-artemether degradants by a dry heat stress approach

During the stability evaluation of β-artemether containing finished drug products, a consistent and disproportional increase in the UV-peak areas of β-artemether degradation products, when compared to the peak area decline of β-artemether itself, was observed. This suggested that the response factors of the formed β-artemether degradants were significantly higher than β-artemether. Dry heat stressing of β-artemether powder, as a single compound, using different temperatures (125-150 °C), times (10-90 min) and environmental conditions (neutral, KMnO(4) and zinc), resulted in the formation of 17 degradants. The vast majority of degradants seen during the long-term and accelerated ICH stability study of the drug product, were also observed here. The obtained stress results allowed the calculation of the overall average relative response factor (RRF) of β-artemether degradants, i.e. 21.2, whereas the individual RRF values of the 9 most prominent selected degradants ranged from 4.9 to 42.4. Finally, Ames tests were performed on β-artemether as well as a representative stressed sample mixture, experimentally assessing their mutagenic properties. Both were found to be negative, suggesting no mutagenicity problems of the degradants at high concentrations. Our general approach and specific results solve the developmental quality issue of mass balance during stability studies and the related genotoxicity concerns of the key antimalarial drug β-artemether and its degradants.