RESUMO
PURPOSE: To mechanistically study and model the effect of lipids, either from food or self-emulsifying drug delivery systems (SEDDS), on drug transport in the intestinal lumen. METHODS: Simultaneous lipid digestion, dissolution/release, and drug partitioning were experimentally studied and modeled for two dosing scenarios: solid drug with a food-associated lipid (soybean oil) and drug solubilized in amodel SEDDS (soybean oil and Tween 80 at 1:1 ratio). Rate constants for digestion, permeability of emulsion droplets, and partition coefficients in micellar and oil phases were measured, and used to numerically solve the developed model. RESULTS: Strong influence of lipid digestion on drug release from SEDDS and solid drug dissolution into food-associated lipid emulsion was observed and predicted by the developed model. Ninety minutes after introduction of SEDDS, there was 9% and 70% drug release in the absence and presence of digestion, respectively. However, overall drug dissolution in the presence of food-associated lipids occurred over a longer period than without digestion. CONCLUSION: A systems-based mechanistic model incorporating simultaneous dynamic processes occurring upon dosing of drug with lipids enabled prediction of aqueous drug concentration profile. This model, once incorporated with a pharmacokinetic model considering processes of drug absorption and drug lymphatic transport in the presence of lipids, could be highly useful for quantitative prediction of impact of lipids on bioavailability of drugs.
Assuntos
Portadores de Fármacos/metabolismo , Emulsões/metabolismo , Preparações Farmacêuticas/administração & dosagem , Óleo de Soja/metabolismo , Simulação por Computador , Digestão , Ingestão de Alimentos , Humanos , Lipólise , Modelos Biológicos , Preparações Farmacêuticas/química , Farmacocinética , SolubilidadeRESUMO
Alpha-synuclein (alpha-Syn) is an abundant brain protein whose mutations have been linked to early-onset Parkinson's disease (PD). We recently demonstrated, by means of a single-molecule force spectroscopy (SMFS) methodology, that the conformational equilibrium of monomeric wild-type (WT) alpha-Syn shifts toward beta-containing structures in several unrelated conditions linked to PD pathogenicity. Herein, we follow the same methodology previously employed for WT alpha-Syn to characterize the conformational heterogeneity of pathological alpha-Syn mutants A30P, A53T, and E46K. Contrary to the bulk ensemble-averaged spectroscopies so far employed to this end by different authors, our single-molecule methodology monitored marked differences in the conformational behaviors of the mutants with respect to the WT sequence. We found that all the mutants have a much higher propensity than the WT to adopt a monomeric compact conformation that is compatible with the acquiring of beta structure. Mutants A30P and A53T show a similar conformational equilibrium that is significantly different from that of E46K. Another class of conformations, stabilized by mechanically weak interactions (MWI), shows a higher variety in the mutants than in the WT protein. In the A30P mutant these interactions are relatively stronger, and therefore the corresponding conformations are possibly more structured. The more structured and globular conformations of the mutants can explain their higher propensity to aggregate with respect to the WT.
Assuntos
Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutação/genética , alfa-Sinucleína/química , alfa-Sinucleína/genética , Fenômenos Biomecânicos , Escherichia coli/genética , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/genética , Proteínas Mutantes Quiméricas/metabolismo , Proteínas Mutantes/metabolismo , Conformação Proteica , Desnaturação Proteica , Análise Espectral , alfa-Sinucleína/metabolismoRESUMO
BACKGROUND: The link between mucus plugs and airflow obstruction has not been established in chronic severe asthma, and the role of eosinophils and their products in mucus plug formation is unknown. METHODS: In clinical studies, we developed and applied a bronchopulmonary segment-based scoring system to quantify mucus plugs on multidetector computed tomography (MDCT) lung scans from 146 subjects with asthma and 22 controls, and analyzed relationships among mucus plug scores, forced expiratory volume in 1 second (FEV1), and airway eosinophils. Additionally, we used airway mucus gel models to explore whether oxidants generated by eosinophil peroxidase (EPO) oxidize cysteine thiol groups to promote mucus plug formation. RESULTS: Mucus plugs occurred in at least 1 of 20 lung segments in 58% of subjects with asthma and in only 4.5% of controls, and the plugs in subjects with asthma persisted in the same segment for years. A high mucus score (plugs in ≥ 4 segments) occurred in 67% of subjects with asthma with FEV1 of less than 60% of predicted volume, 19% with FEV1 of 60%-80%, and 6% with FEV1 greater than 80% (P < 0.001) and was associated with marked increases in sputum eosinophils and EPO. EPO catalyzed oxidation of thiocyanate and bromide by H2O2 to generate oxidants that crosslink cysteine thiol groups and stiffen thiolated hydrogels. CONCLUSION: Mucus plugs are a plausible mechanism of chronic airflow obstruction in severe asthma, and EPO-generated oxidants may mediate mucus plug formation. We propose an approach for quantifying airway mucus plugging using MDCT lung scans and suggest that treating mucus plugs may improve airflow in chronic severe asthma. TRIAL REGISTRATION: Clinicaltrials.gov NCT01718197, NCT01606826, NCT01750411, NCT01761058, NCT01761630, NCT01759186, NCT01716494, and NCT01760915. FUNDING: NIH grants P01 HL107201, R01 HL080414, U10 HL109146, U10 HL109164, U10 HL109172, U10 HL109086, U10 HL109250, U10 HL109168, U10 HL109257, U10 HL109152, and P01 HL107202 and National Center for Advancing Translational Sciences grants UL1TR0000427, UL1TR000448, and KL2TR000428.
Assuntos
Asma/patologia , Eosinofilia/patologia , Muco/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Adulto , Asma/complicações , Estudos de Casos e Controles , Cisteína/química , Elasticidade , Peroxidase de Eosinófilo/metabolismo , Eosinofilia/complicações , Feminino , Volume Expiratório Forçado , Humanos , Hidrogéis , Masculino , Pessoa de Meia-Idade , Tomografia Computadorizada Multidetectores , Oxidantes/química , Compostos de Sulfidrila/química , Tomografia Computadorizada por Raios XRESUMO
HYPOTHESIS: Bile micelles are thought to mediate intestinal absorption, in part by providing a phase into which compounds can partition. Solubilizing capacity of bile micelles is enhanced during the digestion of fat rich food. We hypothesized that the intestinal digestion of triglycerides causes an increase in volume of micelles that can be quantitatively monitored over the course of digestion using small-angle neutron scattering (SANS), and that SANS can enable evaluation of the contribution of each of the components present during digestion to the size of micelles. EXPERIMENTS: SANS was used to characterize the size and shape of micelles present prior to and during the in vitro simulated intestinal digestion of a model food-associated lipid, triolein. FINDINGS: Pre-lipolysis mixtures of a bile salt and phospholipid simulating bile concentrations in fed conditions were organized in micelles with an average volume of 40 nm3. During lipolysis, the micelle volume increased 2.5-fold over a 2-h digestion period due to growth in one direction as a result of insertion of monoglycerides and fatty acids. These efforts represent a basis for quantitative mechanistic understanding of changes in solubilizing capacity of the intestinal milieu upon ingestion of a fat-rich meal.
RESUMO
Lipids, either derived from food or used as drug delivery agents, can have a significant effect on orally delivered drug dissolution, absorption, and bioavailability. Despite numerous studies about fat-rich food/drug interactions, there is still an incomplete understanding of the influence of ingested lipids on oral bioavailability, and a lack of a general in vitro model that is able to predict a priori the in vivo performance of drug-lipid systems. In order to determine the impact of lipid and lipid digestion on drug dissolution and absorption, the choice of a bio-relevant in vitro model is the first and crucial point. A suitable in vitro model should include a medium that mimics as much as possible the gastrointestinal (GI) tract contents after food intake. The goal of this review is to provide an updated overview of (i) in vivo measurements examining stomach and intestine contents in fasted and fed states and (ii) the wide variety of bio-relevant media used in dissolution testing and in vitro lipid digestion studies. These approaches are compared and discussed in light of their capability to model physiological and physicochemical properties of GI tract contents in the fasted and fed states, and in particular when the lipid digestion process occurs.