RESUMEN
The gentle preparation and the functionalization potential of self-emulsifying drug delivery systems (SEDDS) make them an interesting formulation strategy for oral administration of peptide and protein (p/p) drugs. A series of Kolliphor® RH40 (RH40) and Labrasol® (LAB)-based SEDDS containing either long-chain (LC) or medium-chain (MC) glycerides were formulated and characterized with regard to their rheological behavior, as well as the size distribution and zeta potential of the generated emulsions. Insulin, in order to be incorporated in SEDDS, was complexed with soybean phosphatidylcholine. The ability of different SEDDS to protect the incorporated insulin against enzymatic hydrolysis was evaluated by an in vitro model simulating the intestinal proteolysis. SEDDS were incubated in simulated intestinal fluids in the presence of α-Chymotrypsin (α-CT), and HPLC was used to quantify the remaining insulin. Principal component analysis (PCA) was applied to identify the relations between different excipients and properties of SEDDS that describe the SEDDS protective effect on insulin during in vitro proteolysis. The RH40-SEDDS behaved Newtonian in the presence of ethanol (EtOH) and non-Newtonian in the absence of EtOH, which generated emulsion with droplets between 30 to 300 nm. The LAB-SEDDS always behaved Newtonian and generated polydisperse emulsions with broad size distribution (190-4000 nm). During the in vitro proteolysis, insulin can be effectively protected against α-CT (> 60% remaining insulin after 60 min in vitro proteolysis). According to PCA analysis, insulin was better protected in MC-SEDDS compared to LC-SEDDS, and better in LAB-SEDDS compared to RH40-SEDDS. Monoacyl phosphatidylcholine and Capmul® MCM C8 were recognized as excipients favored for SEDDS protection on insulin. However, SEDDS viscosity and the addition of EtOH in SEDDS played insignificant roles on the remaining insulin after in vitro proteolysis. In summary, an in vitro proteolysis model with increased physiological relevance was applied to enable the optimal design of SEDDS for oral p/p drug delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Emulsiones/química , Técnicas In Vitro , Insulina/administración & dosificación , Administración Oral , Quimotripsina/metabolismo , Hidrólisis , Intestinos , ProteolisisRESUMEN
Oral drug delivery is an attractive noninvasive alternative to injectables. However, oral delivery of biopharmaceuticals is highly challenging due to low stability during transit in the gastrointestinal tract (GIT), resulting in low systemic bioavailability. Thus, novel formulation strategies are essential to overcome this challenge. An interesting approach is increasing retention in the GIT by utilizing mucoadhesive biomaterials as excipients. Here, we explored the potential of the GRAS excipient sucrose acetate isobutyrate (SAIB) to obtain mucoadhesion in vivo. Mucoadhesive properties of a 90% SAIB/10% EtOH (w/w) drug delivery system (DDS) were assessed using a biosimilar mucus model and evaluation of rheological behavior after immersion in biosimilar intestinal fluid. To ease readability of this manuscript, we will refer to this as SAIB DDS. The effect of SAIB DDS on cell viability and epithelial membrane integrity was tested in vitro prior to in vivo studies that were conducted using SPECT/CT imaging in rats. When combining SAIB DDS with biosimilar mucus, increased viscosity was observed due to secondary interactions between biosimilar mucus and sucrose ester predicting considerable mucoadhesion. Mucoadhesion was confirmed in vivo, as radiolabeled insulin entrapped in SAIB DDS, remained in the small intestine for up to 22 h after administration. Moreover, the integrity of the system was investigated using the dynamic gastric model under conditions simulating the chemical composition of stomach fluid and physical shear stress in the antrum under fasted conditions. In conclusion, SAIB is an interesting and safe biomaterial to promote high mucoadhesion in the GIT after oral administration.
Asunto(s)
Productos Biológicos/administración & dosificación , Excipientes/farmacología , Insulina/administración & dosificación , Sacarosa/análogos & derivados , Adhesivos Tisulares/farmacología , Animales , Células CACO-2 , Supervivencia Celular/efectos de los fármacos , Liberación de Fármacos , Ácido Gástrico/química , Humanos , Masculino , Moco/química , Organización y Administración , Ratas Endogámicas F344 , Reología , Sacarosa/farmacologíaRESUMEN
To face the challenges of oral delivery of peptide and protein (P/P) drugs, self-emulsifying drug delivery systems (SEDDSs) containing monoacyl phosphatidylcholine (MAPC), Labrasol (LAB) and medium-chain (MC) monoglycerides as permeation enhancers (PEs) were evaluated for their effect on intestinal absorption of insulin. In this study, insulin was complexed with phosphatidylcholine (SPC) to form an insulin-SPC complex (ins-SPC) with increased lipophilicity. The following three SEDDSs: MCT(MAPC) (MC triglycerides and MAPC included), MCT(RH40) (MC triglycerides and Kolliphor® RH40 included) and LCT(MAPC) (long-chain triglycerides and MAPC included) were loading with ins-SPC (4% or 8% w/w of SPC). Three SEDDSs generated emulsions with droplet sizes between 50 and 470â¯nm and with zeta potentials between -5 to -25â¯mV in a simulated intestinal medium. Mucus-secreting Caco-2/HT29-MTX-E12 co-culture and Caco-2 monolayers were used as in vitro cell transport models to investigate insulin permeability. In comparison to insulin HBSS solution, MCT(MAPC) significantly increased the insulin permeability across co-culture and Caco-2 monolayers (2.0-2.5â¯×â¯10-7â¯cm/s). In an intra-jejunal (i.j.) instillation model in rats, MCT(RH40) significantly decreased the rat blood glucose after 0.5â¯h by 17.0⯱â¯2.5% and for MCT(MAPC), it was 23.6⯱â¯10.6%. Furthermore, a lipase inhibitor orlistat was incorporated into MCT(MAPC) to evaluate the effect of lipid digestion on insulin absorption. Results indicated that the incorporation of orlistat did not significantly alter the in vivo insulin absorption. Overall, the SEDDS MCT(MAPC) composed of natural PEs (MAPC and MC glycerides) and synthetic PE (LAB) significantly increased the intestinal absorption of insulin upon i.j. instillation. Although it is not possible to conclude if a single PE is dominating the intestinal absorption of insulin, MCT(MAPC) seems to have the potential for oral insulin delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos , Excipientes/química , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Animales , Células CACO-2 , Técnicas de Cocultivo , Emulsiones , Glicéridos/química , Células HT29 , Humanos , Hipoglucemiantes/farmacocinética , Insulina/farmacocinética , Absorción Intestinal , Yeyuno/metabolismo , Masculino , Modelos Biológicos , Monoglicéridos/química , Orlistat/administración & dosificación , Orlistat/farmacología , Tamaño de la Partícula , Permeabilidad , Fosfatidilcolinas/química , Ratas , Ratas Sprague-DawleyRESUMEN
Group B Streptococcus (GBS) is a leading cause of serious bacterial neonatal infections worldwide, which provides an unmet medical need for a globally effective vaccine. The recombinant GBS fusion antigen GBS-NN contains the N-terminal regions of the GBS Rib and Alpha C proteins. It shows promising immunogenicity eliciting protective immunity in mice and encouraging results in early human clinical trials. Understanding the physical stability of GBS-NN containing conformational B-cell epitopes is crucial to ensure optimal vaccine stability, efficacy, and safety. We initially discovered that GBS-NN is prone to form higher-order structures at elevated temperatures. We therefore investigated the self-assembly behavior of GBS-NN and characterized the higher-order conformational structures as a function of temperature. In the native state, GBS-NN exists as a monomer and has a secondary structure containing α-helix and ß-sheet. Langmuir studies demonstrated that the native protein is highly surface-active and forms a monolayer film at the air-water interface because of its amphipathic properties. The conformational stability of GBS-NN was measured as a function of temperature. GBS-NN has an unusual thermal behavior with a phase transition of approximately 61 °C, which is not accompanied by any major changes in the secondary structure. However, the antigen showed irreversible self-assembly as a function of temperature into higher-order structures with a hydrodynamic diameter of approximately 100 nm. Cryo-transmission electron microscopy analyses demonstrated that these self-assemblies consist of vesicular, ring-like structures with a hollow aqueous interior. Therefore, GBS-NN is a physically stable monomeric protein but is prone to temperature-induced self-assembly above 61 °C.
Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos de Superficie/inmunología , Proteínas Bacterianas/inmunología , Proteínas de la Membrana/inmunología , Infecciones Estreptocócicas/prevención & control , Vacunas Estreptocócicas/inmunología , Streptococcus agalactiae/inmunología , Antígenos Bacterianos/química , Antígenos Bacterianos/aislamiento & purificación , Antígenos de Superficie/química , Antígenos de Superficie/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Epítopos de Linfocito B/química , Epítopos de Linfocito B/inmunología , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/aislamiento & purificación , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Vacunas Estreptocócicas/química , Temperatura , Vacunas Conjugadas/química , Vacunas Conjugadas/inmunologíaRESUMEN
The objective of this study was to investigate the influence of compaction on the conformation of trypsin, its transition temperature (Tm ) of unfolding, and its folding reversibility after thermal denaturation. Plain trypsin was compacted at 40-382 MPa. Pressure-induced changes in the trypsin conformation and the extent of their reversibility were determined using solid- and liquid-state IR spectroscopy together with principal component analysis and an area overlap approach. Trypsin enzymatic activity was determined by a photometric assay. Liquid-state differential scanning calorimetry was performed to determine the Tm as well as the folding reversibility after thermal denaturation of the reconstituted samples. It was found that compacted samples showed reduced activity accompanied by an altered secondary structure. Conformational changes that occur in the solid state were partially reversible upon tablet reconstitution. Aqueous-state IR spectroscopy combined with partial least squares was shown to be a powerful tool to follow irreversible structural changes and evaluate sample bioactivity. Besides its conformation, the thermal stability of trypsin was altered as a result of the applied compaction pressure, indicated by a reduced folding reversibility. In conclusion, this study reveals that tableting can have a negative impact on the biological quality of protein APIs.
Asunto(s)
Proteínas/química , Comprimidos/síntesis química , Tripsina/química , Rastreo Diferencial de Calorimetría/métodos , Calor , Presión , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , TermodinámicaRESUMEN
Oral drug delivery is a preferred route because of good patient compliance. However, most peptide/ protein drugs are delivered via parenteral routes because of the absorption barriers in the gastrointestinal (GI) tract such as enzymatic degradation by proteases and low permeability acrossthe biological membranes. To overcome these barriers, different formulation strategies for oral delivery of biomacromolecules have been proposed, including lipid based formulations and polymer-based particulate drug delivery systems (DDS). The aim of this review is to summarize the existing knowledge about oral delivery of peptide/protein drugs and to provide an overview of formulationand characterization strategies. For a better understanding of the challenges in oral delivery of peptide/protein drugs, the composition of GI fluids and the digestion processes of different kinds of excipients in the GI tract are summarized. Additionally, the paper provides an overview of recent studies on characterization of solid drug carriers for peptide/protein drugs, drug distribution in particles, drug release and stability in simulated GI fluids, as well as the absorption of peptide/protein drugs in cell-based models. The use of biorelevant media when applicable can increase the knowledge about the quality of DDS for oral protein delivery. Hopefully, the knowledge provided in this review will aid the establishment of improved biorelevant models capable of forecasting the performance of particulate DDS for oral peptide/protein delivery.
Asunto(s)
Sistemas de Liberación de Medicamentos , Péptidos/administración & dosificación , Péptidos/química , Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/química , Proteínas/administración & dosificación , Proteínas/química , Administración Oral , Disponibilidad Biológica , Absorción Gastrointestinal/efectos de los fármacos , Humanos , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Insulin suffers from poor oral bioavailability, but lipid-based drug delivery systems (DDS) may constitute promising tools for improving this. Loading of protein drugs into lipid matrices may, however, be challenging, and different formulation approaches must be taken to achieve sufficient loading and preservation of native structure. The aim of the present study was to characterize insulin after complexation with biocompatible surfactants to improve loading into lipid-based DDS. Insulin-surfactant complexes were prepared by freeze-drying with distearyldimethylammonium bromide or soybean phospholipid as complexing surfactant and dimethyl sulfoxide (DMSO) as solvent. Significant change in secondary structure of insulin freeze dried from DMSO was observed using Fourier transform infrared spectroscopy. Changes were quantitatively smaller in the presence of surfactants, demonstrating both a stabilizing effect of surfactants, but also a nonnative secondary structure in the solid state. Finally, circular dichroism analysis of rehydrated complexes showed that the processing did not irreversibly alter the secondary structure of insulin. In short, the present study demonstrates changes in the secondary structure of insulin after freeze-drying from DMSO, constituting a potential generic issue with this technique for protein processing. In the specific case of insulin, the changes were found to be reversible, explaining the success of this strategy in previous studies.
Asunto(s)
Portadores de Fármacos/química , Hipoglucemiantes/administración & dosificación , Insulina/administración & dosificación , Lípidos/química , Tensoactivos/química , Dimetilsulfóxido/química , Liofilización , Hipoglucemiantes/química , Insulina/química , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Triglyceride lipase from Thermomyces lanuginosus (TlL) has been reported to be resistant to denaturation by sodium dodecyl sulfate (SDS). We have found that at neutral pH, structural integrity is strongly dependent on ionic strength. In 10mM phosphate buffer and SDS, the lipase exhibits a far-UV CD spectrum similar to other proteins denatured in this surfactant while the near-UV CD spectrum shows a complete loss of tertiary structure, observations supported by steady state fluorescence spectroscopy. However, when increasing the ionic strength by the addition of NaCl, the lipase was rendered resistant towards SDS denaturation, as observed by all techniques employed. The effect of salt on the critical micelle concentration (CMC) of SDS was observed to correlate with the effect on the degree of SDS-induced denaturation. This finding is compatible with the notion that the concentration of SDS monomers is a crucial factor for SDS-lipase interactions. The presented results are important for the understanding and improvement of protein stability in surfactant systems.
Asunto(s)
Ascomicetos/enzimología , Proteínas Fúngicas/química , Lipasa/química , Dodecil Sulfato de Sodio/farmacología , Dominio Catalítico , Dicroismo Circular , Polarización de Fluorescencia , Proteínas Fúngicas/metabolismo , Lipasa/metabolismo , Concentración Osmolar , Desnaturalización Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Espectrometría de Fluorescencia , Tensoactivos/farmacologíaRESUMEN
In the pharmaceutical industry, protein drugs are modified by, for instance, glycosylation in order to obtain protein drugs with improved delivery profiles and/or increased stability. The effect of glycosylation on protein adsorption behaviour is one of the stability aspects that must be evaluated during development of glycosylated protein drug products. We have studied the effect of glycosylation on the adsorption behaviour of Thermomyces lanuginosus lipase to hydrophobic and hydrophilic surfaces using total internal reflection fluorescence, surface plasmon resonance, far-UV circular dichroism and fluorescence. Three glyco-variants were used, namely the mono-glycosylated wildtype T. lanuginosus lipase, a non-glycosylated variant and a penta-glycosylated variant, the latter two containing one and nine amino acid substitutions, respectively. All the glycosylations were N-linked and contained no charged sugar residues. Glycosylation did not affect the adsorption of wildtype T. lanuginosus lipase to the hydrophobic surfaces. The number of molecules adsorbing per unit surface area, the structural changes occurring upon adsorption, and the orientation upon adsorption were found to be unaffected by the varying glycosylation. However, the interaction with a hydrophilic surface was different between the three glyco-variants. The penta-glycosylated T. lanuginosus lipase adsorbed, in contrast to the two other glyco-variants. In conclusion, adsorption of T. lanuginosus lipase to hydrophobic surfaces was not affected by N-linked glycosylation. Only penta-glycosylated T. lanuginosus lipase adsorbed to the hydrophilic surface, apparently due to its increased net charge of +3 caused by amino acid substitutions in the primary sequence.
Asunto(s)
Ascomicetos/enzimología , Enzimas Inmovilizadas/química , Proteínas Fúngicas/química , Lipasa/química , Adsorción , Sustitución de Aminoácidos , Dicroismo Circular , Estabilidad de Enzimas , Glicosilación , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Peso Molecular , Proteínas Mutantes/química , Compuestos de Organosilicio/química , Estructura Secundaria de Proteína , Cuarzo/química , Proteínas Recombinantes/química , Silanos/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Resonancia por Plasmón de Superficie , Propiedades de SuperficieRESUMEN
The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP by the structurally closely related dUTPase. This pathway provides 70-80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP deaminase is regulated by dTTP, which increases the substrate concentration for half-maximal activity and the cooperativity of dCTP saturation. Likewise, increasing concentrations of dCTP increase the cooperativity of dTTP inhibition. Previous structural studies showed that the complexes of inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type enzyme with dUTP bound were all highly similar and characterized by having an ordered C-terminal. When comparing with a new structure in which dTTP is bound to the active site of E138A, the region between Val120 and His125 was found to be in a new conformation. This and the previous conformation were mutually exclusive within the trimer. Also, the dCTP complex of the inactive H121A was found to have residues 120-125 in this new conformation, indicating that it renders the enzyme inactive. The C-terminal fold was found to be disordered for both new complexes. We suggest that the cooperative kinetics are imposed by a dTTP-dependent lag of product formation observed in presteady-state kinetics. This lag may be derived from a slow equilibration between an inactive and an active conformation of dCTP deaminase represented by the dTTP complex and the dUTP/dCTP complex, respectively. The dCTP deaminase then resembles a simple concerted system subjected to effector binding, but without the use of an allosteric site.
Asunto(s)
Proteínas de Escherichia coli/química , Nucleótido Desaminasas/química , Nucleótidos de Timina/química , Algoritmos , Regulación Alostérica , Sitio Alostérico , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión/genética , Catálisis , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Nucleótidos de Timina/metabolismoRESUMEN
dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg(115) and Glu(138), were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg(2+), and the mutant enzyme also with the substrate dCTP and Mg(2+). The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg(2+) that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.