Influence of injection volume and solvent strength on spilantholchromatography using RP fused-core amide stationary phase

For high-throughput purposes, newly developed Fused-Core HPLC stationary phases (HALO® columns) have attracted the interest of the chromatographic community. Due to their small particle size (2.7 μm) and unique particle technology with 0.5 μm porous shell fused to a solid core particle (1.7 µm diameter), these columns create fast and high performance separations. For our skin research, in vitro FDC experiments are performed, resulting in large sample numbers of low concentrated bio-active in physiologic receptor media (e.g. PBS). Spilanthol, present in Spilanthes acmella as most prominent N-alkylamide and having promising transdermal activity [1, 2], could be analyzed in reversed-phase mode on the HALO® amide stationary phase with methanol/H2O (70/30, V/V) acified with 1% formic acid as mobile phase. The influence of the injection volume and the sample solvent composition on six chromatographic characteristics characteristics (retention time, area, height, theoretical plate, symmetry factor and limit of detection) was investigated. Applying different injection volumes (2–100 µl), the chromatographic responses were obtained from a Spilanthes extract, dissolved in a purely aqueous (PBS) as well as methanol-based (70/30, V/V) sample solvent. Our results show that the chromatographic characteristics are highly dependent on the injection volume and solvent strength

Characterisation of modified pharmaceutical proteins: the somatropin case

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin [1]. 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 a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. This yields 9 possible labelling sites for somatropin: 38, 41, 70, 115, 140, 145, 158, 168 and 172. As site-specific labelling is necessary to avoid active region interactions, characterisation of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and Staphylococcus aureus V-8 proteases. The resulting peptides were then monitored using HPLC-MSn, allowing the investigation of the exact position of amino acid modifications

The moving boundaries in starting materials: from small molecules to biopharma and ATMPs

Several guidelines (e.g. ICH) define starting materials for a medicinal drug substance. While a consensus approach for defining the starting material in the synthesis of small molecule API is currently more or less being agreed upon due to the straightforward production process and well characterized API structure, this is not so as the drug nature complicates, e.g. biopharma and Advanced Therapy Medicinal Products (ATMPs). Although some general guidelines are present, Regulatory Authorities and pharmaceutical manufacturers should establish clear approaches for starting material definitions for these complicated APIs, as this will speed up the development and approval process for the benefit of the patient

Exploring the chemical-functional space of cell-penetrating peptides

Cell penetrating peptides (CPPs) are an increasingly growing part of fundamental and applied peptide research. Using their capacity to cross cell barriers, they have already been successfully applied as carriers for problematic cargos like DNA, (si)RNA, proteins and other peptides, (poly/oligo) saccharides and small molecules. Several hundreds of CPPs, showing different properties and activities, are already reported in the literature. To clarify the different types of actions in cell-penetrating behaviour, a database of more than 200 peptides was build, covering the CPPs described over the last five years and for which quantitative data were available. Seen the wide range of techniques, cell lines, peptide concentrations and other operational parameters used to quantify the cellular uptake, a unified response for cellular uptake was firstly defined based upon a concentration corrected standardized response relative to the concentration corrected response of Penetratin, a well known and characterized CPP. In this way a “meta-analytical” comparison of the cellular uptake of different CPPs is established, which was currently hardly needed. Therefore a chemical space was developed using more than 3000 descriptors, calculated from the optimized 3D-structure of the CPPs. By combining these descriptors and the unified responses, clusters of peptides are obtained from which model CPPs can be rationally selected and QSPRs and mechanisms of action established

Adsorption of cyclic depsipeptide mycotoxins to glass

During analytical processes, adsorption of peptides, which is believed to mostly be due to non-covalent interactions and depending upon the experimental conditions, cannot only lead to significant loss of the analyte, but also to increased analytical variability. This undesirable aspect, however, has been given scant attention [1-2]. Some cyclic depsipeptides are considered as mycotoxins, i.e. beauvericin (BEA) and enniatins (ENNs). To quantitatively evaluate this transdermal behaviour, ex-vivo in-vitro Franz Diffusion Cell (FDC) experiments are performed. The adsorption of the cyclic depsipeptide analytes to the FDC glass wall, of which the quality differs from analytical volumetric glassware, was not yet investigated. Therefore, BEA and ENNs were solubilised in six different solvent mixtures (i.e. different concentrations of ACN and EtOH in H2O) and exposed to FDC glass (in duplicate). After 24h equilibration at 25°C, an aliquot of each solution was assayed with a newly developed and validated UHPLC-MS/MS method in multiple reaction monitoring (MRM) mode. Responses were analysed using loglinear models to obtain recoveries. These were fitted using generalised estimating equations with unstructured covariance to account for correlation within the duplicates [3]. QQ-plots confirmed the normality of the raw residuals in these models. Bonferroni corrected p-values and 95% confidence intervals were determined to account for multiplicity in the analysis of each compound separately. Our results showed that at an EtOH or ACN concentration of ≥ 50% there is no statistical or biomedical significant adsorption effect. At lower levels of organic solvents, however, a significant adsorption effect cannot be excluded, i.e. up to approximately 50% adsorption at 10% ACN. References: [1] Herath, H.M.D.R.; Kim, R.-R.; Cabot, P.J.; Shaw, P.N.; Hewavitharana, A.K. Inaccuracies in the quantification of peptides – A case study using β-endorphin assay. LCGC North America, 2013, 31(1): 58-61. [2] Pezeshki, A.; Vergote, V.; Van Dorpe, S.; Baert, B.; Burvenich, C.; Popkov, A.; De Spiegeleer, B. Adsorption of peptides at the sample drying step: influence of solvent evaporation technique, vial material and solution additive. Journal of Pharmaceutical and Biomedical Analysis, 2009, 49: 607-612. [3] Liang, K-Y.; Zeger, S.L. Longitudinal data analysis using generalized linear models. Biometrics, 1986, 78(1): 13-22

Development of geriatric medicines: to GIP or not to GIP?

The elderly population represents a heterogeneous group of patients, which show co-morbidities and is often poly-medicated. Although geriatric patients form the main users of medicines, this group is underrepresented in clinical trials. Safe and efficacious use of drugs by this group is often not ensured. However, since these elderly population will numerically expand in the coming years, this patient group can no longer be neglected during the development and approval stage of medicines. We evaluated the current regulatory status for the geriatric medicines. Next, the views of the different stakeholders in this field will be analyzed. Finally, points of attention and suggestions concerning this regulatory discussion will be emphasized, like i.a. the approach of personalized medicine, the age and disease-related change in blood-brain barrier permeability and use of predictive models. Thus, by analogy with the paediatric investigation plan (PIP), coming to the conclusion whether to GIP or not to GIP

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