Amorphous solid dispersions (ASDs) have been increasingly used to maximize human exposures from poorly soluble drug candidates. One well-studied advantage of ASDs is the increased amorphous drug ...solubility compared to crystalline forms. This provides more rapid dissolution rates. An additional advantage of ASDs is that the dissolution process of the ASD particle may also rapidly transform much of the drug present in the ASD particle to small (<1 μm) amorphous drug nanoparticles which will have fast dissolution rates. This work examines the mechanism by which this nanoparticle formation occurs by studying an ASD consisting of 70–80% copovidone, 20% anacetrapib (a low solubility lipophilic drug), and 0–10% TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate, a surfactant). Nanoparticle formation is found to derive from a rapid amorphous drug domain formation within the ASD particle, driven by copovidone dissolution from the particle. The role of surfactant in the ASD particle is to prevent an otherwise rapid, local drug domain aggregation event, which we term “hydrophobic capture”. Surfactant thus allows the amorphous drug domains to escape hydrophobic capture and diffuse to bulk solution, where they are reported as nanoparticles. This view of surfactant and nanoparticle formation is compared to the prevailing view in the literature. The work here clarifies the different roles that surfactant might play in increasing nanoparticle yields and extending the useful drug loading ranges in copovidone-based ASDs.
Small molecule developability challenges have been well documented over the last two decades. One of these critical developability parameters is aqueous solubility. In general, more soluble compounds ...have improved oral absorption. While enabling formulation technologies exist to improve bioperformance for low solubility compounds, these are often more complex, expensive, and challenging to scale up. Therefore, to avoid these development issues, medicinal chemists need tools to rapidly profile and improve the physicochemical properties of molecules during discovery. Dose number (Do) is a simple metric to predict whether a compound will be reasonably absorbed based on solubility at an expected clinical dose and represents a valuable parameter to the medicinal chemist defining a clinical candidate. The goal of this mini-Perspective is to present the background of the Do equation and how it can be effectively used to rapidly predict oral absorption potential for molecules in the discovery space.
Forced degradation is a method of studying the stability of pharmaceuticals in order to design stable formulations and predict drug product shelf life. Traditional methods of reaction and analysis ...usually take multiple days, and include LC‐UV and LC‐MS product analysis. In this study, the reaction/analysis sequence was accelerated to be completed within minutes using Leidenfrost droplets as reactors (acceleration factor: 23–188) and nanoelectrospray ionization MS analysis. The Leidenfrost droplets underwent the same reactions as seen in traditional bulk solution experiments for three chemical degradations studied. This combined method of accelerated reaction and analysis has the potential to be extended to forced degradation of other pharmaceuticals and to drug formulations. Control of reaction rate and yield is achieved by manipulating droplet size, levitation time and whether or not make‐up solvent is added. Evidence is provided that interfacial effects contribute to rate acceleration.
Traditional forced degradation of drug substances and drug products can take days to weeks but accelerated reaction/analysis of active pharmaceutical ingredients was achieved within minutes using Leidenfrost droplets and MS analysis. Lifetimes and sizes of the droplets and degree of solvent evaporation control reaction rate and product yield. Interfacial effects increase reaction rates in small droplets.
Linear polyethylenimines are polycationic excipients that have found many pharmaceutical applications, including as a delivery vehicle for gene therapy through formation of polyplexes with ...oligonucleotides. Accurate quantitation of linear polyethylenimines in both starting solution and formulation containing oligonucleotide/polyethylenimine polyplexes is critical. Existing methods using spectroscopy, matrix‐assisted laser desorption/ionization mass spectrometry time‐of‐flight, or nuclear magnetic resonance are either complex or suffer from low selectivity. Here, the development and performance of a simple analytical method is described whereby linear polyethylenimines are resolved by ultra‐high‐performance liquid chromatography and quantified using either a charged aerosol detector or an ultraviolet detector. For formulated oligonucleotide/polyethylenimine polyplexes, sample preparation through decomplexation/digestion by trifluoroacetic acid was necessary to eliminate separation interference. The method can be used not only to support formulation development but also to monitor the synthesis/purification and characterization of linear polyethylenimines.
•Interaction of SDS in sample solution with basic analytes in RPLC was explored.•Micelle concentration and strength found to be the origin of the peak splitting.•Modifications to pH or instrument ...configuration can affect the amount of splitting.
In the process of dissolution method development for Merck proprietary compound A, a basic analyte, abnormal chromatographic behavior involving peak splitting and retention time shifting in the presence of sodium dodecyl sulfate (SDS) in the sample solution was observed. A mechanistic study was conducted and the level and type of surfactant, along with the pKa of the analyte, were determined to be the critical variables in the degree of effect seen. Chromatographically, the effect was further impacted by the injection volume used, the pH and identity of the mobile phase buffer and the amount of system volume between the autosampler and the column. A simple resolution using a basic mobile phase pH was identified to be an effective way to eliminate abnormal chromatographic behavior and produce robust and reproducible analysis.
Macrocyclic peptides show promise in targeting high-value therapeutically relevant binding sites due to their high affinity and specificity. However, their clinical application is often hindered by ...low membrane permeability, which limits their effectiveness against intracellular targets. Previous studies focused on peptide conformations in various solvents, leaving a gap in understanding their interactions with and translocation through lipid bilayers. Addressing this, our study explores the membrane interactions of stapled peptides, a subclass of macrocyclic peptides, using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. We conducted ssNMR measurements on ATSP-7041M, a prototypical stapled peptide, to understand its interaction with lipid membranes, leading to an MD-informed model for peptide membrane permeation. Our findings reveal that ATSP-7041M adopts a stable α-helical structure upon membrane binding, facilitated by a cation-π interaction between its phenylalanine side chain and the lipid headgroup. This interaction makes the membrane-bound state energetically favorable, facilitating membrane affinity and insertion. The bound peptide displayed asymmetric insertion depths, with the C-terminus penetrating deeper (approximately 9 Å) than the N-terminus (approximately 4.3 Å) relative to the lipid headgroups. Contrary to expectations, peptide dynamics was not hindered by membrane binding and exhibited rapid motions similar to cell-penetrating peptides. These dynamic interactions and peptide-lipid affinity appear to be crucial for membrane permeation. MD simulations indicated a thermodynamically stable transmembrane conformation of ATSP-7041M, reducing the energy barrier for translocation. Our study offers an in silico view of ATSP-7041M's translocation from the extracellular to the intracellular region, highlighting the significance of peptide-lipid interactions and dynamics in enabling peptide transit through membranes.Macrocyclic peptides show promise in targeting high-value therapeutically relevant binding sites due to their high affinity and specificity. However, their clinical application is often hindered by low membrane permeability, which limits their effectiveness against intracellular targets. Previous studies focused on peptide conformations in various solvents, leaving a gap in understanding their interactions with and translocation through lipid bilayers. Addressing this, our study explores the membrane interactions of stapled peptides, a subclass of macrocyclic peptides, using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. We conducted ssNMR measurements on ATSP-7041M, a prototypical stapled peptide, to understand its interaction with lipid membranes, leading to an MD-informed model for peptide membrane permeation. Our findings reveal that ATSP-7041M adopts a stable α-helical structure upon membrane binding, facilitated by a cation-π interaction between its phenylalanine side chain and the lipid headgroup. This interaction makes the membrane-bound state energetically favorable, facilitating membrane affinity and insertion. The bound peptide displayed asymmetric insertion depths, with the C-terminus penetrating deeper (approximately 9 Å) than the N-terminus (approximately 4.3 Å) relative to the lipid headgroups. Contrary to expectations, peptide dynamics was not hindered by membrane binding and exhibited rapid motions similar to cell-penetrating peptides. These dynamic interactions and peptide-lipid affinity appear to be crucial for membrane permeation. MD simulations indicated a thermodynamically stable transmembrane conformation of ATSP-7041M, reducing the energy barrier for translocation. Our study offers an in silico view of ATSP-7041M's translocation from the extracellular to the intracellular region, highlighting the significance of peptide-lipid interactions and dynamics in enabling peptide transit through membranes.
This work demonstrates the use of a fluorescent probe to screen protein conformational changes in mixtures of monoclonal antibodies and determine the region of where such changes may originate ...through a footprinting mass spectrometry approach. The oxidative stress of mixtures of two different immunoglobulins (IgG1, IgG2, or IgG4) performed in the presence of 2,2′-azobis(2-amidinopropane dihydrochloride) results in sequence-specific tyrosine oxidation reactions depending on the time of incubation of the IgG molecules and the nature of the excipients present in the formulation. The combination of a fluorescence assay, based on the detection of 3,4-dihydroxyphenylalanine (DOPA) and mass spectrometry analyses, permits the identification of protein conformation changes. In a mixture of IgG2 and IgG4, a destabilization of IgG4 in the presence of IgG2 is observed. The destabilized region involves the Fab region of IgG4 between Tyr63 and Tyr81 and potentially multiple regions of IgG2.
The ability to produce and isolate relatively pure amounts of relevant degradation products is key to several aspects of drug product development: (a) aid in the unambiguous structural identification ...of such degradation products, fulfilling regulatory requirements to develop safe formulations (International Conference on Harmonization Q3B and M7); (b) pursue as appropriate safety evaluations with such material, such as chronic toxicology or Ames testing; (c) for a specified degradation product in a late-stage regulatory filing, use pure and well-characterized material as the analytical standard. Producing such materials is often a resource- and time-intensive activity, either relying on the isolation of slowly formed degradation products from stressed drug product or by re-purposing the drug substance synthetic route. This problem is exacerbated if the material of interest is an oxidative degradation product, because typical oxidative stressing (H
O
and radical initiators) tends to produce a myriad of irrelevant species beyond a certain stress threshold, greatly complicating attempts for isolating the relevant degradation product. In this article, we present reagents and methods that may allow the rapid and selective enrichment of active pharmaceutical ingredient with the desired oxidative degradation product, which can then be isolated and used for purposes described above.
This article describes how the increased use of energy-efficient solid-state light sources (e.g., light-emitting diode LED-based illumination) in hospitals, pharmacies, and at home can help alleviate ...concerns of photodegradation for pharmaceuticals. LED light sources, unlike fluorescent ones, do not have spurious spectral contributions <400 nm. Because photostability is primarily evaluated in the International Council of Harmonization Q1B tests with older fluorescent bulb standards (International Organization for Standardization 10977), the amount of photodegradation observed can over-predict what happens in reality, as products are increasingly being stored and used in environments fitted with LED bulbs. Because photodegradation is premised on light absorption by a compound of interest (or a photosensitizer), one can use the overlap between the spectral distribution of a light source and the absorption spectra of a given compound to estimate if photodegradation is a possibility. Based on the absorption spectra of a sample of 150 pharmaceutical compounds in development, only 15% would meet the required overlap to be a candidate to undergo direct photodegradation in the presence of LED lights, against a baseline of 55% of compounds that would, when considering regular fluorescent lights. Biological drug products such as peptides and monoclonal antibodies are also expected to benefit from the use of more efficient solid-state lighting.
In the drug discovery setting, the ability to rapidly identify drug absorption risk in preclinical species at high doses from easily measured physical properties is desired. This is due to the large ...number of molecules being evaluated and their high attrition rate, which make resource-intensive in vitro and in silico evaluation unattractive. High-dose in vivo data from rat, dog, and monkey are analyzed here, using a preclinical dose number (PDo) concept based on the dose number described by Amidon and other authors (Pharm. Res., 1993, 10, 264–270). PDo, as described in this article, is simply calculated as dose (mg/kg) divided by compound solubility in FaSSIF (mg/mL) and approximates the volume of biorelevant media per kilogram of animal that would be needed to fully dissolve the dose. High PDo values were found to be predictive of difficulty in achieving drug exposure (AUC)–dose proportionality in in vivo studies, as could be expected; however, this work analyzes a large data set (>900 data points) and provides quantitative guidance to identify drug absorption risk in preclinical species based on a single solubility measurement commonly carried out in drug discovery. Above the PDo values defined, >50% of all in vivo studies exhibited poor AUC–dose proportionality in rat, dog, and monkey, and these values can be utilized as general guidelines in discovery and early development to rapidly assess risk of solubility-limited absorption for a given compound. A preclinical dose number generated by biorelevant dilutions of formulated compounds (formulated PDo) was also evaluated and defines solubility targets predictive of suitable AUC–dose proportionality in formulation development efforts. Application of these guidelines can serve to efficiently identify compounds in discovery that are likely to present extreme challenges with respect to solubility-limited absorption in preclinical species as well as reduce the testing of poor formulations in vivo, which is a key ethical and resource matter.
