RESUMO
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.
RESUMO
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.
Assuntos
Química Farmacêutica/métodos , Descoberta de Drogas/métodos , Preparações Farmacêuticas/química , Administração Oral , Animais , Relação Dose-Resposta a Droga , Humanos , Absorção pela Mucosa Oral , Preparações Farmacêuticas/administração & dosagem , SolubilidadeRESUMO
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.
Assuntos
Anticorpos Monoclonais/química , Di-Hidroxifenilalanina/análise , Estabilidade de Medicamentos , Estabilidade Proteica , Anticorpos Monoclonais/farmacocinética , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Di-Hidroxifenilalanina/química , Combinação de Medicamentos , Espectrometria de Massas/métodos , Oxirredução , Conformação ProteicaRESUMO
One of the most common functional groups encountered in drug molecules is the amide, and the most common degradation pathway for amides is base-mediated hydrolysis to its constituent amine and carboxylic acid. Herein, we report for the first time, a base-mediated oxidative degradation pathway of secondary amides to primary amides. This transformation also represents a novel synthetic methodology, reported for the first time in this work, in transforming secondary amides to primary amides without using any oxidative reagents. The introduction of this mechanism into the pharmaceutical literature is important given that the mechanism and required reactants are present to carry out the chemistry in dosage forms.
Assuntos
Amidas , Preparações Farmacêuticas , Oxirredução , Estresse Oxidativo , FenolRESUMO
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.
Assuntos
Oligonucleotídeos/química , Polietilenoimina/análise , Aerossóis/química , Cromatografia Líquida de Alta Pressão/instrumentação , Desenho de Equipamento , Raios UltravioletaRESUMO
Pharmaceutical scientists are often asked to assess the impact of modifications to the illumination in the manufacturing and product packaging environment on product quality. To assess the impact of switching light sources, four model compounds were exposed to standard fluorescent light, LED, and "yellow light" and the extent of drug photodegradation was determined. Photodegradation under LED light is generally reduced compared to fluorescent light and is often predictable if the UV-Vis absorption spectrum of the active pharmaceutical ingredient (API) and the spectral power distribution emitted by the various light sources overlap. However, lack of noticeable spectral overlap does not ensure absence of API photodegradation and may require additional assessment for selection of appropriate lighting conditions. A detailed evaluation of the API and solid formulation absorbance was performed to assess degradation risk for Compound A and Vitamin D3 when exposed to LED light. The light budget was established for Compound A, spanning all stages of the manufacturing process, under different illumination conditions to enable a complex supply chain. The results also demonstrate that while LEDs used in manufacturing areas are generally "better" compared to fluorescent lights, they are not replacing yellow lights for compounds sensitive to visible light.
Assuntos
Luz , Preparações Farmacêuticas , Estabilidade de Medicamentos , Iluminação , FotóliseRESUMO
Cleaning verification (CV) is a critical step in the pharmaceutical manufacturing process to eliminate or reduce unacceptable contamination of a product as a result of insufficiently cleaned equipment surfaces. The main concern is cross contamination with active pharmaceutical ingredients (APIs) from previous runs that may impact patient safety. Current conventional approaches involve rather tedious sample preparation and analytical methods with relative lengthy analysis time. Potent APIs possessing low acceptable daily intake (ADI) values require analytical methods for CV with very low detection limits to confirm that these APIs are below their acceptance limits prior to the next manufacturing process. In this work, a novel end to end CV workflow was developed, which includes the automated sample and calibration solution preparation as well as high throughput analysis by ultra-high-performance liquid chromatography (UHPLC) coupled with single quadrupole mass spectrometry in multiple injection chromatography and selected ion monitoring mode (MIC-MS-SIM). The method was validated using ten model compounds. Acceptable specificity, linearity (R2 > 0.997) and single digit ng/mL LOQ and LOD were achieved for all model compounds. This approach was also successfully applied to the analysis of 22 internal CV samples from an internal program.
Assuntos
Contaminação de Medicamentos , Preparações Farmacêuticas , Cromatografia Líquida de Alta Pressão , Contaminação de Medicamentos/prevenção & controle , Humanos , Espectrometria de Massas , Fluxo de TrabalhoRESUMO
Novel treatment routes are emerging for an array of diseases and afflictions. Complex dosage forms, based on active pharmaceutical ingredients (APIs) with previously undesirable physicochemical characteristics, are becoming mainstream and actively pursued in various pipeline initiatives. To fundamentally understand how constituents in these dosage forms interact on a molecular level, analytical methods need to be developed that encompass selectivity and sensitivity requirements previously reserved for a myriad of in vitro techniques. The knowledge of precise chemical interactions between drugs and excipients in a dosage form can streamline formulation development and process screening capabilities through the identification of properties that influence rates and mechanisms of drug release in a cost-effective manner, relative to long-term in vivo studies. Through this work, a noncompendial in vitro release (IVR) method was developed that distinguished the presence of individual components in a complex crystalline nanosuspension environment. Doravirine was formulated as a series of long-acting injectable nanosuspensions with assorted excipients, using low- and high-energy wet media milling methods. IVR behavior of all formulation components were monitored using a robust continuous flow-through (CFT) dissolution setup (USP-4 apparatus) with on-line 1H NMR end-analysis (flow-NMR). Results from this investigation led to a better understanding of formulation parameter influences on nanosuspension stability, surface chemistry, and dissolution behavior. Flow-NMR can be applied to a broad range of dosage forms in which specific molecular interactions from the solution microenvironment require further insight to enhance product development capabilities.
Assuntos
Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Injeções , Espectroscopia de Ressonância Magnética/métodos , Nanopartículas/administração & dosagem , Suspensões/administração & dosagem , Suspensões/farmacocinética , Química Farmacêutica/instrumentação , Estabilidade de Medicamentos , Excipientes/química , Técnicas In Vitro/métodos , Nanopartículas/química , Tamanho da Partícula , Piridonas/química , Solubilidade , Triazóis/químicaRESUMO
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.
Assuntos
Cromatografia de Fase Reversa , Dodecilsulfato de Sódio/química , Soluções Tampão , Concentração de Íons de Hidrogênio , Tensoativos/químicaRESUMO
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.
Assuntos
Estabilidade de Medicamentos , Iluminação/instrumentação , Preparações Farmacêuticas/química , Fotólise/efeitos da radiação , Semicondutores , Anticorpos Monoclonais/química , Anticorpos Monoclonais/efeitos da radiação , Produtos Biológicos/química , Produtos Biológicos/efeitos da radiação , Arquitetura de Instituições de Saúde/instrumentação , Arquitetura de Instituições de Saúde/legislação & jurisprudência , Arquitetura de Instituições de Saúde/normas , Iluminação/legislação & jurisprudência , Iluminação/normas , Preparações Farmacêuticas/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
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 (H2O2 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.
Assuntos
Química Farmacêutica/métodos , Composição de Medicamentos/normas , Química Farmacêutica/normas , Cromatografia Líquida de Alta Pressão , Armazenamento de Medicamentos/normas , Peróxido de Hidrogênio/química , Indicadores e Reagentes/química , Oxidantes/química , OxirreduçãoRESUMO
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.
Assuntos
Cromatografia Líquida/métodos , Solventes/química , Estabilidade de Medicamentos , Fenômenos FísicosRESUMO
As science evolves, the need for more efficient and innovative knowledge transfer capabilities becomes evident. Advances in drug discovery and delivery sciences have directly impacted the pharmaceutical industry, though the added complexities have not shortened the development process. These added complexities also make it difficult for scientists to rapidly and effectively transfer knowledge to offset the lengthened drug development timelines. While webcams, camera phones, and iPads have been explored as potential new methods of real-time information sharing, the non-"hands-free" nature and lack of viewer and observer point-of-view render them unsuitable for the R&D laboratory or manufacturing setting. As an alternative solution, the Microsoft HoloLens mixed-reality headset was evaluated as a more efficient, hands-free method of knowledge transfer and information sharing. After completing a traditional method transfer between 3 R&D sites (Rahway, NJ; West Point, PA and Schnachen, Switzerland), a retrospective analysis of efficiency gain was performed through the comparison of a mock method transfer between NJ and PA sites using the HoloLens. The results demonstrated a minimum 10-fold gain in efficiency, weighing in from a savings in time, cost, and the ability to have real-time data analysis and discussion. In addition, other use cases were evaluated involving vendor and contract research/manufacturing organizations.
Assuntos
Descoberta de Drogas/métodos , Indústria Farmacêutica/métodos , Humanos , Preparações Farmacêuticas/química , Estudos Retrospectivos , Estatística como Assunto/métodos , Tecnologia Farmacêutica/métodosRESUMO
In an ongoing effort to explore the use of orexin receptor antagonists for the treatment of insomnia, dual orexin receptor antagonists (DORAs) were structurally modified, resulting in compounds selective for the OX2R subtype and culminating in the discovery of 23, a highly potent, OX2R-selective molecule that exhibited a promising in vivo profile. Further structural modification led to an unexpected restoration of OX1R antagonism. Herein, these changes are discussed and a rationale for selectivity based on computational modeling is proposed.
Assuntos
Antagonistas dos Receptores de Orexina/farmacologia , Orexinas/antagonistas & inibidores , Animais , Eletroencefalografia , Eletromiografia , Estrutura Molecular , Antagonistas dos Receptores de Orexina/química , RatosRESUMO
Metal ions play an important role in oxidative drug degradation. One of the most ubiquitous metal ion impurities in excipients and buffers is Fe(III). In the field of oxidative drug degradation chemistry, the role of Fe(III) has been primarily discussed in terms of its effect in reaction with trace hydroperoxide impurities. However, the role of Fe(III) acting as a direct oxidant of drug molecules, which could operate in the absence of any hydroperoxide impurities, is less common. This work focuses on Fe(III)-induced oxidation of some aromatic drug molecules/drug fragments containing benzylic C-H bonds in the absence of initiating peroxides. Alcohol and ketone degradates are formed at the benzylic carbon atom. The formation of a π-stabilized cation radical is postulated as the key intermediate for the downstream oxidation. Implications are briefly discussed.
Assuntos
Compostos de Benzil/química , Carbono/química , Cloretos/química , Compostos Férricos/química , Peróxidos/química , Compostos de Benzil/metabolismo , Carbono/metabolismo , Cloretos/metabolismo , Compostos Férricos/metabolismo , Ferro/química , Ferro/metabolismo , Oxirredução/efeitos dos fármacos , Peróxidos/metabolismoRESUMO
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.
Assuntos
Nanopartículas/química , Pirrolidinas/química , Compostos de Vinila/química , Química Farmacêutica/métodos , Interações Hidrofóbicas e Hidrofílicas , Tamanho da Partícula , Solubilidade , Soluções/química , Tensoativos/químicaRESUMO
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.
Assuntos
Química Farmacêutica/métodos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Absorção Intestinal , Animais , Área Sob a Curva , Cães , Descoberta de Drogas , Haplorrinos , Humanos , Farmacocinética , Ratos , Solubilidade , Tecnologia Farmacêutica/métodosRESUMO
The ability to define compound solubility targets that are predictive of good oral absorption at high dose preclinical studies (≥ 100 mg compound/kg animal) is of use in drug discovery and development. Two different approaches to identify these targets in preclinical formulations are evaluated herein. The first approach is the use of solubility values from in vitro formulation dilutions using biorelevant parameters for rats. These dilution/solubility results are applied to the maximum absorbable dose (MAD) model to predict compound exposure (AUC) from oral doses and allow the fraction of dose absorbed (F(abs)) calculation. The results from 26 such in vitro evaluations are compared to in vivo studies and discussed. The second approach is the analysis of in vivo AUC proportionality between 10 and 100 mg/kg doses for 28 compounds where only the compound solubility in neat formulation is known. Both assessments suggest similar threshold targets to remove solubility as an absorption limitation for any given compound. Specifically, compound solubility should be >2 mg/mL in aqueous surfactants and >15 mg/mL in cosolvent (PEG400) or pH-adjusted aqueous formulations. The results are a starting place for formulation rule-of-thumb solubility targets applied in discovery and development settings.
Assuntos
Química Farmacêutica/métodos , Preparações Farmacêuticas/química , Administração Oral , Animais , Preparações Farmacêuticas/metabolismo , Polietilenoglicóis/química , Ratos , SolubilidadeRESUMO
Polysorbate 80 is used in the pharmaceutical industry as an additive to enhance the solubility of non-polar compounds in formulation design and during dissolution analysis. In this paper we present the spectroscopic and chromatographic characteristics for a series of commercially available sources of this non-ionic surfactant. The large UV/vis absorbance and broad chromatographic elution of Polysorbate 80 often makes it difficult to accurately quantitate pharmaceutically active compounds in solutions where the surfactant is present. Boundary conditions have been established where analytical interferences can be avoided in spectrophotometric analysis by choice of analysis wavelength and solution concentrations. Chromatographic method development is also presented enabling the removal of Polysorbate interference in instances where spectroscopic interference is too great.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Polissorbatos/química , Espectrofotometria Ultravioleta/métodos , Tensoativos/químicaRESUMO
Medium chain length (C8, C10) mono-, di- and triacylglycerols (MCGs) are used in pharmaceutical formulation development of poorly soluble compounds, as a means to increase their oral bioavailability. The ratios of C8 and C10 fatty acid components along with the ratio of monoglycerides, diglycerides and triglycerides can significantly impact overall solubilizing characteristics both in vitro and in vivo. Existing literature methods either do not have the desired selectivity or simplicity to adequately characterize these MCGs. A reversed-phase HPLC method has been developed utilizing a Waters Symmetry C18 (150 mm x 4.6 mm, 3.5 microm particle size) column with a gradient of acetonitrile and water. The effluent was monitored using a UV detector at 220 nm and relative response factors were determined for all components with available standards to allow for accurate quantitation. The RP-HPLC method was optimized for selectivity, sensitivity and efficiency and was successfully applied to the characterization of commercial samples yielding superior sensitivity and ease of preparation compared to existing compendial methods.