RESUMEN
OBJECTIVES: To prepare oral controlled-release cilostazol formulations and evaluate their pharmacokinetics and pharmacodynamics in dogs and humans compared with a commercial twice-daily immediate-release formulation (Pletal), thereby showing the potential for the development of an improved once-daily cilostazol formulation. METHODS: Six different controlled-release preparations were formulated using a micronized cilostazol, solubilizer/absorption enhancer and erodible hydrogel. In-vitro drug release profiles were tailored by varying hydrogel viscosity. Pharmacokinetics and pharmacodynamic (antithrombotic) efficacy were evaluated in beagle dog model of arterial thrombosis. Finally, their pharmacokinetics and pharmacodynamics were also evaluated in healthy human volunteers after single and multiple oral administrations. KEY FINDINGS: Hydrogel viscosity-dependent sustained drug release profiles were observed with zero-order release kinetics during 8-12 h. In dogs and humans, compared with Pletal, prolonged drug absorption profiles were observed in the two controlled-release formulations studied. In dogs, the controlled-release formulations showed greater antithrombotic efficacy than twice-daily Pletal. In humans, the antithrombotic efficacy of the selected once-daily cilostazol formulation was equivalent to that of twice-daily Pletal after single and multiple administrations. CONCLUSIONS: The prepared oral controlled-release cilostazol formulation may provide prolonged drug absorption and sufficient therapeutic efficacy, potentially serving as an oral once-daily cilostazol formulation to improve patient compliance.
Asunto(s)
Fibrinolíticos/administración & dosificación , Absorción Intestinal , Tetrazoles/administración & dosificación , Trombosis/tratamiento farmacológico , Administración Oral , Animales , Cilostazol , Preparaciones de Acción Retardada , Modelos Animales de Enfermedad , Perros , Portadores de Fármacos/química , Fibrinolíticos/farmacocinética , Fibrinolíticos/farmacología , Geles , Humanos , Masculino , Solubilidad , Tetrazoles/farmacocinética , Tetrazoles/farmacología , ViscosidadRESUMEN
Large-scale proteomics of three wild relatives of wheat grain (A, B, and D genomes) were analyzed by using multidimensional protein identification technology coupled to liquid chromatography quadruple mass spectrometry. A total of 1568 (peptide match ≥1) and 255 (peptide match ≥2) unique proteins were detected and classified, which represents the most wide-ranging proteomic exploitation to date. The development of standard proteomes exhibiting all of the proteins involved in normal physiology will facilitate the delineation of disease/defense, metabolism, energy metabolism, and protein synthesis. A relative proteome exploration of the expression patterns indicates that proteins are involved in abiotic and biotic stress. Functional category analysis indicates that these differentially expressed proteins are mainly involved in disease/defense (15.38%, 21.26%, and 16.78%), metabolism (8.39%, 12.07%, and 14.09%), energy metabolism (11.19%, 11.49%, and 13.42%), protein synthesis (9.09%, 9.20%, and 8.72%), cell growth and division (9.09%, 4.60%, and 6.04%), cellular organization (4.20%, 5.75%, and 5.37%), development (6.29%, 2.87%, 3.36%), folding and stability (6.29%, 8.62%, and 8.05%), signal transduction (11.19%, 7.47%, and 8.05%), storage protein (4.20%, 1.72%, and 2.01%), transcription (5.59%, 5.17%, and 4.03%), and transport facilitation (1.40%, 1.15%, and 3.36%) in A, B, and D genomes, respectively. Here, we reported genome-specific protein interaction network using Cytoscape software, which provides further insight into the molecular functions and mechanism of biochemical pathways. We provide a promising understanding about the expressed proteins and protein functions. Our approach should be applicable as a marker to assist in breeding or gene transfer for quality and stress research of cultivated wheat.
Asunto(s)
Proteínas de Plantas/análisis , Proteoma/análisis , Proteómica/métodos , Triticum/metabolismo , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Redes Reguladoras de Genes , Genoma de Planta/genética , Espectrometría de Masas , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Proteoma/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Triticum/genéticaRESUMEN
Cockroaches have been recognized as a major cause of asthma. Bla g 4 is one of the most important German cockroach allergens. The aim of this study is to investigate IgE reactivity to the recombinant Bla g 4 (rBla g 4) in the sera of allergic patients and identify linear IgE binding epitope. For protein expression, full-length Bla g 4 (EF202172) was divided into 5 overlapping peptide fragments (E1: aa 1-100, E2: aa 34-77, E3: aa 74-117, E4: aa 114-156, and E5: aa 153-182). The full-length and 5 peptide fragments of Bla g 4 was generated by PCR and over-expressed in E. coli BL21 (DE3). The IgE binding reactivities of the full-length and peptide fragments were measured by ELISA using 32 serum samples of cockroach allergy. The sera of 8 patients (25%) reacted with rBla g 4. Four sera (100%) showed IgE-binding reactivity to full-length and peptide fragment 4, and 2 sera (50%) reacted with peptide fragment 2. One (20%) serum reacted with peptide fragment 3. The results of ELISA using overlapping recombinant fragments indicated that the epitope region was located at amino acid sequences 34-73 and 78-113, and major IgE epitope of Bla g 4 was located at amino acid sequences 118-152 of C-terminal. B-cell epitope analysis of German cockroach allergen Bla g 4 could contribute to the strategic development of more specific and potentially efficacious immunotherapy.
Asunto(s)
Alérgenos/inmunología , Cucarachas/inmunología , Mapeo Epitopo , Hipersensibilidad/inmunología , Inmunoglobulina E/inmunología , Proteínas de Insectos/inmunología , Adolescente , Adulto , Alérgenos/química , Alérgenos/genética , Secuencia de Aminoácidos , Animales , Antígenos de Plantas , Niño , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Alineación de Secuencia , Adulto JovenRESUMEN
BACKGROUND: The allergenicity of allergens could be influenced by amino acid substitutions in B- or T-cell epitope regions. The German cockroach is known to produce potent allergens inducing strong IgE-mediated allergic reactions. This study was performed to investigate sequence variations in major allergens of the German cockroach. METHODS: Reverse transcriptase PCR was used to amplify the cDNA sequences encoding major allergens of the German cockroach (Bla g 1, Bla g 2, Bla g 4, and Bla g 5). RESULTS: The deduced amino acid sequences revealed 38 Bla g 1 variants with 1-7 amino acid substitutions (98.6-99.8% identity), 28 Bla g 2 variants with 1-3 substitutions (99.1-99.7%), 27 Bla g 4 variants with 0-32 substitutions (82.4-100%), and 8 Bla g 5 variants with 1-2 substitutions (99.0-99.5%), respectively. Bla g 1 and Bla g 2 showed sporadic amino acid substitutions despite the divergence in their sequences. Bla g 4 exhibited frequent variations, with clusters of substitutions in residues 29-38, 52-80, and 132-155. Sequence variations in Bla g 4 imply the presence of multiple isoforms and isoallergens, which may in turn have various effects on the IgE-binding capacity and T-cell responsiveness. Only 8 variants were found in Bla g 5, with infrequent amino acid changes of one or two residues. CONCLUSIONS: Analyses of T-cell and IgE-binding epitope regions would clarify the effect of sequence polymorphisms on allergenicity, which in turn will aid in the design of allergen formulations for diagnosis and immunotherapy for cockroach allergies.
Asunto(s)
Alérgenos/genética , Cucarachas/genética , Proteínas de Insectos/genética , Secuencia de Aminoácidos , Animales , Cucarachas/inmunología , ADN Complementario/genética , Femenino , Masculino , Datos de Secuencia Molecular , Polimorfismo Genético , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Bla g 2 is a cockroach allergen of great importance. This study was conducted to identify IgE-binding epitope(s) of Bla g 2 using the recombinant protein technique. Approximately 50% of tested sera showed IgE reactivity to Pichia-expressed Bla g 2 (PrBla g 2) and E. coli-expressed Bla g 2 (ErBla g 2). Only 5.3% of serum samples showed stronger reactivity to PrBla g 2 than ErBla g 2, indicating that serum was reactive to conformational or carbohydrate epitopes. The full-length and 5 peptide fragments of Bla g 2 were produced in E. coli. All fragments showed IgE-binding activity to the cockroach-allergy patients' sera. Specifically, peptide fragments of amino acid residue 1-75 and 146-225 appeared to be important for IgE-binding. The information about the IgE-binding epitope of Bla g 2 can aid in the diagnosis and treatment for cockroach allergies.