Exploring the Brainpeps database

Since the discovery that peptides can cross the blood-brain barrier (BBB), doors have been opened to new therapeutics for CNS diseases and pain management. Recently, we have constructed the Brainpeps database (brainpeps.ugent.be) to give an overview of the available BBB transport data of peptides, which are scattered in the literature [1]. One possible application of the Brainpeps database is the study of structure-property relationships (QSPRs). Before peptides can be used as drugs, their impurity profile needs to be examined as part of the International Conference on Harmonization (ICH) risk assessment of peptide drugs. Compared to small molecules, no in-silico predictive programs are available for toxicity screening of the different peptide impurities towards passing the BBB. To predict the BBB-behaviour of peptides as well as their impurities, we explored the Brainpeps database. During this presentation, the first results of the modelling experiments are presented. Our starting hypothesis is that the interactions of peptides at the blood-brain barrier are comparable with those of peptides in HPLC systems. Therefore, we determined the retention characteristics on different fused-core HPLC systems of a set of model peptides selected from the Brainpeps database and explored the relationship between the chromatographic characteristics and their BBB-influx properties [2]. In conclusion, using the Brainpeps database and experimental HPLC data, a first step towards in-silico profiling of peptides, including their impurities, at the blood-brain barrier level is taken. More chromatographic analyses of BBB peptides and harmonization on testing the BBB transport of peptides are future challenges to validate and unify this model. References [1] Van Dorpe S., Bronselaer A., Nielandt J., Stalmans S., Wynendaele E., Audenaert K., Van de Wiele C., Burvenich C., Peremans K., Hsuchou H., De Tré G., De Spiegeleer B. Brainpeps: the blood-brain barrier peptide database. Brain Struct Funct (2012), DOI: 10.1007/s00429-011-0375-0. [2] D’Hondt M., Van Dorpe S., Gevaert B., Wynendaele E., Stalmans S., Peremans K., Burvenich C., De Spiegeleer B. Fused-core RP-HPLC modelling of peptides. Journal of Pharmaceutical Analysis (2012), Accepted for publication

Dry heat stress stability of model peptide: buserelin

Buserelin, an gonadotropin hormone releasing agonist peptide implemented in the treatment of prostate cancer, was subjected in its dry powder state to short-term heat exposure to evaluated its potential application in hot melt extrusion processing. Five different temperature and time conditions, ranging from 150°C to 180°C and 10 to 160 min, respectively, were evaluated (4 time points per temperature). Each experimental condition was performed in duplicate. A stability indicating UPLC-DAD (220 nm) method was used both for buserelin assay as degradant quantification. Linear regression, assuming first order degradation kinetics, was employed to calculate the Arrhenius activation energy (Ea) and frequency factor (A). During dry heat stressing, a change from solid to molten state was observed at 165°C. This change is also reflected in the Arrhenius regression curve, which is of sigmoidal nature. Given this change in state, two Arrhenius Ea were calculated, i.e. 217 and 87 kJ/mol for solid and molten state respectively. Three different degradant groups were observed using HPLC ESI/iontrap-MS: [M+H+] 453 amu, [M+H+] 902 amu and busereline diastereoisomers. The apparent degradation mechanisms included hydrolysis, dissociation of the tertiary butyl group on Ser6 and isomerization. Further structure elucidation is ongoing using nanoUPLC ESI/Orbitrap-MS

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

Fused-core HPLC method development implemented in a short-term stability study of Triple IT solution

For the majority of children with acute lymphoblastic leukemia (ALL), treatment consists in part of a triple intrathecal (Triple IT) therapy, i.e. a combination of cytarabine (CB), methotrexate (MTX) and methylprednisolone sodium succinate (MPSS) [1]. This combination product is prepared ex-tempore. However, no in-use shelf-life under defined storage conditions has yet been established. During stability studies, a large number of samples are generated, thus creating the need for a fast, accurate and selective analytical method. In this study, a fused-core HPLC method was developed. This hybrid technology, consisting of a 0.5 µm thick porous shell fused to a 1.7 µm inert core, enables faster chromatographic separation with sufficiently high resolution. During method development, both stressed and unstressed solutions containing both single Triple IT components and the mixture thereof, were analyzed using different linear gradient times, ranging from 5 to 30 min. The mobile phase composition was fixed (A: 0.1% glacial acid in H2O; B: 0.1% glacial acid in ACN), starting with A:B (90:10, V/V) and ending with A:B (10:90, V/V). Method selectivity was evaluated based on the observed peaks in stressed CB, MTX and MPSS solutions, i.e. incubation at 40°C and 80°C. A balance between fast separation and sufficient resolution between the Triple IT components and related degradants, was found by setting the gradient time at 15 min. The Triple IT related degradation peaks were chromatographically separated from the remaining Triple IT components. Moreover, selectivity was supported by a peak purity analysis on the observed peaks. Linearity was demonstrated (R² > 0.999) for the three Triple IT components. Repeatability was evaluated by triplicate injections of 100% reference assay: relative standard deviation varied between 0.155% (MPSS), 0.464% (CB) and 1.352% (MTX) [2]. References [1] A. Ruggiero, V. Conter, M. Milani, E. Biagi, I. Lazzareschi, P. Sparano, R. Riccardi. Intrathecal chemotherapy with antineoplastic agents in children. Paediatric drugs 3(4) (2001) 237-246. [2] M. D’Hondt, E. Vangheluwe, S. Van Dorpe, J. Boonen, T. Bauters, B. Pelfrene, J. Vandenbroucke, H. Robays, B. De Spiegeleer. Stability of ex-tempore prepared Triple intrathecal solution consisting of cytarabine, methotrexate and methylprednisolone sodium succinate. American Journal of Health-System Pharmacy, submitted for publication