The Role of Cyclodextrins against Interface-Induced Denaturation in Pharmaceutical Formulations: A Molecular Dynamics Approach

Abstract

Protein-based pharmaceutical products are subject to a variety of environmental stressors, during both production and shelf-life. In order to preserve their structure, and, therefore, functionality, it is necessary to use excipients as stabilizing agents. Among the eligible stabilizers, cyclodextrins (CDs) have recently gained interest in the scientific community thanks to their properties. Here, a computational approach is proposed to clarify the role of β-cyclodextrin (βCD) and 2-hydroxypropyl-β-cyclodextrin (HPβCD) against granulocyte colony-stimulating (GCSF) factor denaturation at the air–water and ice–water interfaces, and also in bulk water at 300 or 260 K. Both traditional molecular dynamics (MD) simulations and enhanced sampling techniques (metadynamics, MetaD) are used to shed light on the underlying molecular mechanisms. Bulk simulations revealed that CDs were preferentially included within the surface hydration layer of GCSF, and even included some peptide residues in their hydrophobic cavity. HPβCD was able to stabilize the protein against surface-induced denaturation in proximity of the air–water interface, while βCD had a destabilizing effect. No remarkable conformational changes of GCSF, or noticeable effect of the CDs, were instead observed at the ice surface. GCSF seemed less stable at low temperature (260 K), which may be attributed to cold-denaturation effects. In this case, CDs did not significantly improve conformational stability. In general, the conformationally altered regions of GCSF seemed not to depend on the presence of excipients that only modulated the extent of destabilization with either a positive or a negative effect.

Investigation of the pH-dependent aggregation mechanisms of GCSF using low resolution protein characterization techniques and advanced molecular dynamics simulations

“…The structure of GCSF is available from X-ray (1CD9 [13] , 1RHG [10] , and 2D9Q [25] ) and NMR (1GNC [26] ) studies, of which 1CD9 has been widely used as the GCSF model structure in various MD simulation studies [27] , [28] , [29] . The initial structure of GCSF for the conventional molecular dynamics simulation (cMD) study was prepared using PDB entry 1CD9 (solved at pH 7.5) [13] .…”

Force Field Parameterization for the Description of the Interactions between Hydroxypropyl-β-Cyclodextrin and Proteins

“…The interaction was favorable, and quite intense, also for most apolar side chains, with the only exclusion of alanine, that showed a value of γ i sc close to zero. The ability of HPβCD to interact with both polar and apolar side chains is not surprising considering the amphiphilic nature of this excipient, which was already the subject of both experimental and computational investigation. ,, …”

“…The ability of HPβCD to interact with both polar and apolar side chains is not surprising considering the amphiphilic nature of this excipient, which was already the subject of both experimental and computational investigation. 13,14,16 The interaction of HPβCD with aromatic side chains (especially tryptophan, phenylalanine, and tyrosine) was particularly pronounced. The preferential interaction of HPβCD with aromatic groups has been observed experimentally and is well documented in the literature.…”

“…HPβCD is more soluble than its unfunctionalized counterpart (>300 mM at ambient temperature) and displays amphiphilic properties. For instance, it was found to be surface-active, as such mitigating surface-induced aggregation of proteins. − …”

Exploration of high dimensional free energy landscapes by a combination of temperature‐accelerated sliced sampling and parallel biasing

“…This problem was addressed to some extent by selectively grouping CVs 51 . The method has been applied to a wide spectrum of complex chemical and biological problems; See references 52–60; see also references 39, 61. Several other attempts to improve the sampling using a MTD‐like bias have also been proposed lately 25,49,62–64 …”