The challenge of formulating high concentration therapeutic peptides

Therapeutic peptides offer significant opportunities to treat serious and debilitating diseases, with several purported advantages over proteins, such as a simpler characterization process and a reduced regulatory burden. However, many peptides are known to be susceptible to aggregation, and surprisingly little work has been published on designing formulation principles for these therapeutic agents. In this paper the focus will be on the challenges encountered in rational development of a stabilization strategy for two model peptides formulated at high concentration (\u3e50 mg/ml). The first model peptide studied was carbetocin, a 9-amino acid long analogue of oxytocin with improved chemical and enzymatic stability. At high concentrations (70 mg/mL, ca 70 mM) carbetocin is sensitive to interface-induced stress, causing the formation of large amorphous aggregates. The addition of uncharged or charged surfactants surprisingly did not stabilize against this interface-induced stress; instead, positively and negatively charged surfactants steered the aggregation pathway towards the formation of amyloid-like fibrils [1]. The addition of very low concentrations of the uncharged surfactant Span 60 appeared to result in the formation of aggregates with a refractive index close to that of water, creating a false impression of stabilization. For carbetocin, only the removal of the air-water interface by fully filling the vials resulted in a significant stabilization against aggregation [1]. The second model peptide was a 33-amino acid negatively charged peptide which formed amyloid-like fibrils when exposed to heat stress (40 degrees C) at 50 mg/mL concentration. The limited impact of shaking indicated this fibrillation was primarily induced through peptide-peptide interactions in the bulk solution. Peptide-peptide interactions were therefore determined using Taylor Dispersion Analysis. The latter method suggested significant repulsive interactions for this peptide formulation in aqueous solution. Addition of salt (NaCl) or the amino acids Arg or Glu at 150 mM shielded these repulsive interactions. In contrast, addition of 250 mM of the amino acids Val or Ile had a very limited shielding effect. However, none of the added excipients appeared to stabilize the peptide against aggregation, although infrared spectroscopic analysis indicated that the aggregates in the NaCl, Arg and Glu containing solutions consisted of native-like peptides. The aggregates formed in the presence of Val and Ile, or in the absence of any excipients, contained a significant amount of intermolecular beta-sheet [2]. Our results illustrate the challenges in protecting high concentration peptides against aggregation and indicates a strong dependence of the aggregation pathway on the excipients added and their potential interaction with the peptide. A novel formulation toolbox may therefore be required for peptides. The high structural flexibility introduces a significant complicating factor in understanding their interactions with their local environment. [1] Høgstedt UB, Østergaard J, Weiss T, Sjögren H, van de Weert M. Manipulating aggregation behavior of the uncharged peptide carbetocin. Journal of Pharmaceutical Sciences. 2018 Mar;107(3):838-847. [2] Høgstedt UB. Formulation of concentrated peptide solutions – physical stability challenges and the impact of peptide-peptide interactions. PhD thesis University of Copenhagen, 2018

On the purported "backbone fluorescence" in protein three-dimensional fluorescence spectra

In this study, several proteins (albumin, lysozyme, insulin) and model compounds (Trp, Tyr, homopolypeptides) were used to demonstrate the origin of the fluorescence observed upon their excitation at 220–230 nm. In the last 10 years we have observed a worrying increase in the number of articles claiming that this fluorescence originates from the protein backbone, contrary to the established knowledge that UV protein emission is due to aromatic amino acids only. Overall, our data clearly demonstrate that the observed emission upon excitation at 220–230 nm is due to the excitation of Tyr and/or Trp, with subsequent emission from the lowest excited state (i.e. the same as obtained with 280 nm excitation) in agreement with Kasha's rule. Therefore, this fluorescence peak does not provide any information on backbone conformation, but simply reports on the local environment around the aromatic side chains, just as any traditional protein emission spectrum. The many papers in reputable journals erroneously reporting this peak assignment, contradicting 5 decades of prior knowledge, have led to the creation of a new dogma, where many authors and reviewers now take the purported backbone fluorescence as an established fact. We hope the current paper helps counter this new situation and leads to a reassessment of those papers that make this erroneous claim

Rhamnogalacturonan-I based microcapsules for targeted drug release

Drug targeting to the colon via the oral administration route for local treatment of e.g. inflammatory bowel disease and colonic cancer has several advantages such as needle-free administration and low infection risk. A new source for delivery is plant-polysaccharide based delivery platforms such as Rhamnogalacturonan-I (RG-I). In the gastro-intestinal tract the RG-I is only degraded by the action of the colonic microflora. For assessment of potential drug delivery properties, RG-I based microcapsules (~1 μm in diameter) were prepared by an interfacial poly-addition reaction. The cross-linked capsules were loaded with a fluorescent dye (model drug). The capsules showed negligible and very little in vitro release when subjected to media simulating gastric and intestinal fluids, respectively. However, upon exposure to a cocktail of commercial RG-I cleaving enzymes, ~ 9 times higher release was observed, demonstrating that the capsules can be opened by enzymatic degradation. The combined results suggest a potential platform for targeted drug delivery in the terminal gastro-intestinal tract

Interfacial Complexes between a Protein and Lipophilic Ions at an Oil-Water Interface

The interaction between an intact protein and two lipophilic ions at an oil-water interface has been investigated using cyclic voltammetry, impedance based techniques and a newly developed method in which the biphasic oil-water system is analyzed by biphasic electrospray ionization mass spectrometry (BESI-MS), using a dualchannel electrospray emitter. It is found that the protein forms interfacial complexes with the lipophilic ions and that it specifically requires the presence of the oil-water interface to be formed under the experimental conditions. Furthermore, impedance based techniques and BESI-MS with a common ion to polarize the interface indicated that the Galvani potential difference across the oil-water interface significantly influences the interfacial complexation degree. The ability to investigate protein-ligand complexes formed at polarized liquid-liquid interfaces is thus a new analytical method for assessing potential dependent interfacial complexation using a structure elucidating detection principle

Population-based impact of COVID-19 on incidence, treatment, and survival of patients with pancreatic cancer

Background: The COVID-19 pandemic has put substantial strain on the healthcare system of which the effects are only partly elucidated. This study aimed to investigate the impact on pancreatic cancer care. Methods: All patients diagnosed with pancreatic cancer between 2017 and 2020 were selected from the Netherlands Cancer Registry. Patients diagnosed and/or treated in 2020 were compared to 2017–2019. Monthly incidence was calculated. Patient, tumor and treatment characteristics were analyzed and compared using Chi-squared tests. Survival data was analyzed using Kaplan–Meier and Log-rank tests. Results: In total, 11019 patients were assessed. The incidence in quarter (Q)2 of 2020 was comparable with that in Q2 of 2017–2019 (p = 0.804). However, the incidence increased in Q4 of 2020 (p = 0.031), mainly due to a higher incidence of metastatic disease (p = 0.010). Baseline characteristics, surgical resection (15% vs 16%; p = 0.466) and palliative systemic therapy rates (23% vs 24%; p = 0.183) were comparable. In 2020, more surgically treated patients received (neo)adjuvant treatment compared to 2017–2019 (73% vs 67%; p = 0.041). Median overall survival was comparable (3.8 vs 3.8 months; p = 0.065). Conclusion: This nationwide study found a minor impact of the COVID-19 pandemic on pancreatic cancer care and outcome. The Dutch health care system was apparently able to maintain essential care for patients with pancreatic cancer

A Helical Structural Nucleus Is the Primary Elongating Unit of Insulin Amyloid Fibrils

Although amyloid fibrillation is generally believed to be a nucleation-dependent process, the nuclei are largely structurally uncharacterized. This is in part due to the inherent experimental challenge associated with structural descriptions of individual components in a dynamic multi-component equilibrium. There are indications that oligomeric aggregated precursors of fibrillation, and not mature fibrils, are the main cause of cytotoxicity in amyloid disease. This further emphasizes the importance of characterizing early fibrillation events. Here we present a kinetic x-ray solution scattering study of insulin fibrillation, revealing three major components: insulin monomers, mature fibrils, and an oligomeric species. Low-resolution three-dimensional structures are determined for the fibril repeating unit and for the oligomer, the latter being a helical unit composed of five to six insulin monomers. This helical oligomer is likely to be a structural nucleus, which accumulates above the supercritical concentration used in our experiments. The growth rate of the fibrils is proportional to the amount of the helical oligomer present in solution, suggesting that these oligomers elongate the fibrils. Hence, the structural nucleus and elongating unit in insulin amyloid fibrillation may be the same structural component above supercritical concentrations. A novel elongation pathway of insulin amyloid fibrils is proposed, based on the shape and size of the fibrillation precursor. The distinct helical oligomer described in this study defines a conceptually new basis of structure-based drug design against amyloid diseases