Functional differences of iodinated obestatin peptides: in vitro metabolic stability profile

Peptides are small polymers of 50 amino acids, situated between organic molecules and proteins. Most peptides perform a biochemical-physical function and are therefore considered as potential drugs. In vivo studies, such as blood-brain barrier transport and medical imaging require sensitive analytical techniques, achieved by using radiolabeled tracers. These peptide tracers are synthesised from the reaction with radioactive sodium iodide, incorporating the iodine in the tyrosine or histidine amino acid residue[1]. However, next to enzymatic degradation, it is reported that deiodination occurs in vitro as well as in vivo [2, 3]. This phenomenon should be taken into account since the deiodinated metabolite(s) can cause the biologic activity. Therefore, the in vitro stability of different iodinated mouse obestatin derivatives is characterised in the main metabolic compartments: plasma, liver and kidney. Using LC-UV for quantification, significant differences in the degradation kinetics of the iodinated peptides, arising from both enzymatic proteolysis and deiodination, were found when compared to the native, unmodified peptide. LC-MS/MS analysis demonstrated that the cleavage sites were dependent upon the biological matrix and the location of the amino acid residue incorporating the iodine atom(s). The degrading enzymes were found to target peptide bonds further away from the iodine incorporation, while proteolytic cleavages of nearby peptide bonds were inhibited. Di-iodinated amino acid residue containing peptides were found to be more susceptible to deiodination than the mono-iodinated derivative. References [1] J. Nemeth, G. Oroszi, B. Jakab, M. Magyarlaki, Z. Szilvassy, E. Roth, B. Farkas, Journal of Radioanalytical and Nuclear Chemistry 2002, 251, 129. [2] J. C. Solis-S, P. Villalobos, A. Orozco, C. Valverde-R, Journal of Endocrinology 2004, 181, 385. [3] E. R. Goldberg, L. A. Cohen, Bioorganic Chemistry 1993, 21, 41

Stability indicating method development for low level calcitonin

Salmon calcitonin is a 32 amino acid peptide drug, therapeutically used in Paget’s disease and osteoporosis in post-menopausal women. The assay and degradation of salmon calcitonin formulated at low concentrations (400 ppm m/m) in a bioadhesive nasal powder is examined. Sample preparation was developed and optimized using Plackett-Burman and Onion designs. Best results were obtained by treating the sample with 0.45% (v/v) trifluoroacetic acid at 60°C for 40 minutes, obtaining maximum recovery of 99.63% without yielding any observable degradation. Two HPLC–UV/MS methods were developed and validated as well, showing fitness for their intended use of assay respectively degradation monitoring Pilot stability tests of the bioadhesive powder under different storage conditions showed a temperature dependent decrease in salmon calcitonin assay, with no equivalent increase in degradation products, which can be explained by the chemical interaction between calcitonin and carbomer present in the finished drug product

HPLC-MS characterisation of chelate modified somatropin

Somatropin is a recombinant human growth hormone, consisting of 191 amino acids. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is currently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. The modification of the protein with the chelating agent NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein for diagnostic (e.g. 68Ga) or therapeutic (e.g. 90Y) purposes. The NOTA unit is selectively introduced on a lysine side chain. This yields 9 possible labelling sites for somatropin: FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF We have applied an enzymatic digestion procedure for the characterisation of the modified somatropin, using trypsin, chymotrypsin and Staphylococcus aureus V-8 proteases. The resulting peptides were then monitored using HPLC-MS2, allowing the characterisation of the modified protein