RESUMEN
This work focused on the control of the manufacturing process for a controlled release (CR) pellet product, within a Quality by Design (QbD) framework. The manufacturing process was Wurster coating: firstly layering active pharmaceutical ingredient (API) onto sugar pellet cores and secondly a controlled release (CR) coating. For each of these two steps, development of a Process Analytical Technology (PAT) method is discussed and also a novel application of automated microscopy as the reference method. Ultimately, PAT methods should link to product performance and the two key Critical Quality Attributes (CQAs) for this CR product are assay and release rate, linked to the API and CR coating steps respectively. In this work, the link between near infra-red (NIR) spectra and those attributes was explored by chemometrics over the course of the coating process in a pilot scale industrial environment. Correlations were built between the NIR spectra and coating weight (for API amount), CR coating thickness and dissolution performance. These correlations allow the coating process to be monitored at-line and so better control of the product performance in line with QbD requirements.
Asunto(s)
Microscopía , Preparaciones Farmacéuticas/química , Espectroscopía Infrarroja Corta , Tecnología Farmacéutica/métodos , Automatización de Laboratorios , Celulosa/análogos & derivados , Celulosa/química , Citratos/química , Preparaciones de Acción Retardada , Derivados de la Hipromelosa/química , Cinética , Metilcelulosa/química , Microscopía/normas , Preparaciones Farmacéuticas/normas , Control de Calidad , Solubilidad , Espectroscopía Infrarroja Corta/normas , Tecnología Farmacéutica/normasRESUMEN
This paper describes the use of (1)H nuclear magnetic resonance (NMR) for 3D mapping of the relative density distribution in pharmaceutical tablets manufactured under controlled conditions. The tablets are impregnated with a compatible liquid. The technique involves imaging of the presence of liquid which occupies the open pore space. The method does not require special calibration as the signal is directly proportional to the porosity for the imaging conditions used. The NMR imaging method is validated using uniform density flat faced tablets and also by direct comparison with X-ray computed tomography. The results illustrate (1) the effect of die wall friction on density distribution by compressing round, curved faced tablets using clean and pre-lubricated tooling, (2) the evolution of density distribution during compaction for both clean and pre-lubricated die wall conditions, by imaging tablets compressed to different compaction forces, and (3) the effect of tablet image on density distribution by compressing two complex shape tablets in identical dies to the same average density using punches with different geometries.
Asunto(s)
Densitometría , Imagen por Resonancia Magnética , Comprimidos/química , Celulosa/química , Tamaño de la Partícula , Porosidad , Polvos , Reproducibilidad de los Resultados , Tecnología Farmacéutica/métodos , Tomografía Computarizada por Rayos XRESUMEN
Magnetic Resonance Imaging experiments have been used to investigate the degradation of drug-loaded poly(glycolic lactic acid) (PGLA) 50:50 cylinders. Spatial variation in the rate of degradation throughout the polymer cross-section has been observed non-invasively using quantitative imaging of penetrant concentration, T(2) and self-diffusion coefficient. This spatial variation in the rate of degradation was attributed to the quicker degradation in the inner region of the sample due to autocatalysis by carboxyl end groups generated upon ester bond cleavage.
Asunto(s)
Imagen por Resonancia Magnética/métodos , Poliésteres/química , Agua/química , Catálisis , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Estabilidad de Medicamentos , Glicolatos/química , Ácido Láctico/químicaRESUMEN
X chromosome inactivation in mammals requires the Xist gene, which is exclusively expressed from the inactive X chromosome (Xi). The large heterogeneous Xist nuclear RNA colocalizes with Xi, most likely through nuclear protein interactions. The 5' region of the Xist RNA contains a series of well-conserved tandem repeats known to bind heteronuclear proteins in vitro and to enhance human XIST transcription. We show in an in vitro system that the conserved repeat element located in the 5' region of the mouse Xist gene (Xcr) represses three X-linked genes but has no effect on the autosomal genes Aprt, Ins, and the viral SV40 gene. The repression effect is not mediated by the conserved core sequence (Ccs) of Xcr, but requires the presence of the complete Xcr. This Xcr effect on X-linked genes suggests that Xcr transcript recognizes the genes to be silenced and is involved in the spreading of X inactivation.
Asunto(s)
Ligamiento Genético , ARN no Traducido/genética , Factores de Transcripción/genética , Transcripción Genética/genética , Cromosoma X , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Cartilla de ADN , Glucosafosfato Deshidrogenasa/genética , Hipoxantina Fosforribosiltransferasa/genética , Ratones , Fosfoglicerato Quinasa/genética , ARN Largo no Codificante , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The mouse Xist gene is expressed exclusively from the inactive X chromosome and is involved in the initiation of X inactivation. We previously reported that the -1157/+917 region of the Xist promoter was ubiquitously functional in mammalian cells and that experiments in a transient expression system revealed no trans-acting element responsible for the inactive X specific expression of Xist. In somatic tissues, the 5' end of the silent Xist allele on the active X is known to be fully methylated whereas the expressed allele on the inactive X is unmethylated. In the present study we have used a bisulphite genomic sequencing method to evaluate DNA methylation at all cytosines including CpG dinucleotides within the Xist promoter. We report and confirm that methylation of specific sites plays a key role in Xist gene expression. In vitro DNA methylation of the 5'-region drastically reduced transcriptional activity in transiently transfected fibroblasts. Mobility shift assays showed that methylation does not inhibit Xist promoter activity by preventing the binding of transcription factors and that two distinct nuclear proteins bind in a sequence methyl-CpG-specific manner. Therefore, we suggest that Xist repression involves its promoter methylation and two distinct methylated DNA binding proteins.
Asunto(s)
Islas de CpG/genética , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Regiones Promotoras Genéticas/genética , ARN no Traducido , Factores de Transcripción/genética , Animales , Azacitidina/farmacología , Secuencia de Bases , Línea Celular , Cloranfenicol O-Acetiltransferasa , Citosina , Metilación de ADN/efectos de los fármacos , Femenino , Regulación de la Expresión Génica , Masculino , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/metabolismo , ARN Largo no Codificante , Análisis de Secuencia de ADN , Timina , Cromosoma XRESUMEN
Mutations in the BIGH3 gene on chromosome 5q31 cause four distinct autosomal dominant diseases of the human cornea: granular (Groenouw type I), Reis-Bücklers, lattice type I, and Avellino corneal dystrophies. All four diseases are characterized by both progressive accumulation of corneal deposits and eventual loss of vision. We have identified a specific recurrent missense mutation for each type of dystrophy, in 10 independently ascertained families. Genotype analysis with microsatellite markers surrounding the BIGH3 locus was performed in these 10 families and in 5 families reported previously. The affected haplotype could be determined in 10 of the 15 families and was different in each family. These data indicate that R555W, R124C, and R124H mutations occurred independently in several ethnic groups and that these mutations do not reflect a putative founder effect. Furthermore, this study confirms the specific importance of the R124 and R555 amino acids in the pathogenesis of autosomal dominant corneal dystrophies linked to 5q.
Asunto(s)
Cromosomas Humanos Par 5 , Distrofias Hereditarias de la Córnea/genética , Mutación Puntual , Mapeo Cromosómico , Distrofias Hereditarias de la Córnea/clasificación , Exones , Genes Dominantes , Ligamiento Genético , Marcadores Genéticos , Haplotipos , Humanos , Intrones , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-SimpleRESUMEN
Granular dystrophy Groenouw type I (CDGG1), Reis-Bücklers (CDRB), lattice type I (CDL1) and Avellino (ACD) are four 5q31-linked human autosomal dominant corneal dystrophies. Clinically, they show progressive opacification of the cornea leading to severe visual handicap. The nature of the deposits remains unknown in spite of amyloid aetiology ascribed to the last two. We generated a YAC contig of the linked region and, following cDNA selection, recovered the beta ig-h3 gene. In six affected families we identified missense mutations. All detected mutations occurred at the CpG dinucleotide of two arginine codons: R555W in one CDGG1, R555Q in one CDRB, R124C in two CDL1 and R124H in two ACD families. This suggests, as the last two diseases are characterized by amyloid deposits, that R124 mutated kerato-epithelin (the product of beta ig-h3) forms amyloidogenic intermediates that precipitate in the cornea. Our data establish a common molecular origin for the 5q31-linked corneal dystrophies.