Investigating the delivery of antimicrobial proteins and aminoglycoside antibiotics to the airways

Biopharmaceuticals are finding wide applications in the management of diverse disease conditions. Pulmonary delivery of proteins may constitute an effective and efficient non-invasive alternative to parenteral delivery, which is currently the main route of administration of biopharmaceutical drugs. A particular area, in which pulmonary delivery of peptides and proteins may find ready application, is in the local delivery of antimicrobial peptides and proteins to the airway, a measure that could potentially bring about improvements to currently available antipseudomonal therapies. This thesis has therefore sought to develop inhalable antimicrobial proteins in combination with antibiotics that have particularly good antimicrobial activity against Pseudomonas aeruginosa infections in the respiratory tract of people with cystic fibrosis (CF). Through process optimisation, a suitable spray drying method was developed and used for the preparation of active, inhalable dry powder formulations of the antimicrobial protein, lactoferrin, and aminoglycosides (tobramycin and gentamicin). The physicochemical properties, aerosolisation performance and the antibacterial properties of the various spray-dried formulations were assessed. In addition, a relevant in vitro cellular model was employed to investigate the potential cytotoxic and pro-inflammatory effects of the various formulations on four bronchial human epithelial cells together with their effectiveness at reducing bacterial colonies when administered on to biofilm co-cultured on the epithelial cells. It was found that following spray drying the particles obtained were mostly spherical, amorphous and possessed suitable aerosolisation characteristics. The various spray-dried antimicrobial proteins (lactoferrin or apo lactoferrin) and co-spray dried combinations of the proteins and aminoglycosides were found to exhibit bactericidal activity against planktonic and biofilms of P. aeruginosa. In general, the spray drying process was found not to significantly affect the antimicrobial activities of the protein. Treatment of the different bronchial epithelial cell lines with the antimicrobial formulations showed that the various formulations were non-toxic and that the co-spray dried combinations significantly reduced established P. aeruginosa biofilms on the four bronchial epithelial cells. Overall, the results from this thesis demonstrates that spray drying could potentially be employed to prepare inhalable antimicrobial agents comprised of proteins and antibiotics. These new combinations of proteins and aminoglycosides has promising applications in the management of P. aeruginosa in the airway of cystic fibrosis patients.

Playing catch-up with Escherichia coli:using yeast to increase success rates in recombinant protein production experiments

Several host systems are available for the production of recombinant proteins, ranging from Escherichia coli to mammalian cell-lines. This article highlights the benefits of using yeast, especially for more challenging targets such as membrane proteins. On account of the wide range of molecular, genetic, and microbiological tools available, use of the well-studied model organism, Saccharomyces cerevisiae, provides many opportunities to optimize the functional yields of a target protein. Despite this wealth of resources, it is surprisingly under-used. In contrast, Pichia pastoris, a relative new-comer as a host organism, is already becoming a popular choice, particularly because of the ease with which high biomass (and hence recombinant protein) yields can be achieved. In the last few years, advances have been made in understanding how a yeast cell responds to the stress of producing a recombinant protein and how this information can be used to identify improved host strains in order to increase functional yields. Given these advantages, and their industrial importance in the production of biopharmaceuticals, I argue that S. cerevisiae and P. pastoris should be considered at an early stage in any serious strategy to produce proteins.

Composite cell support membranes based on collagen and polycaprolactone for tissue engineering of skin

The preparation and characterisation of collagen:PCL composites for manufacture of tissue engineered skin substitutes and models are reported. Films having collagen:PCL (w/w) ratios of 1:4, 1:8 and 1:20 were prepared by impregnation of lyophilised collagen mats by PCL solutions followed by solvent evaporation. In vitro assays of collagen release and residual collagen content revealed an expected inverse relationship between the collagen release rate and the content of synthetic polymer in the composite that may be exploited for controlled presentation and release of biopharmaceuticals such as growth factors. DSC analysis revealed the characteristic melting point of PCL at around 60°C and a tendency for the collagen component, at high loading, to impede crystallinity development within the PCL phase. The preparation of fibroblast/composite constructs was investigated using cell culture as a first stage in mimicking the dermal/epidermal structure of skin. Fibroblasts were found to attach and proliferate on all the composites investigated reaching a maximum of 2×105/cm2 on 1:20 collagen:PCL materials at day 8 with cell numbers declining thereafter. Keratinocyte growth rates were similar on all types of collagen:PCL materials investigated reaching a maximum of 6.6×104/cm2 at day 6. The results revealed that composite films of collagen and PCL are favourable substrates for growth of fibroblasts and keratinocytes and may find utility for skin repair. © 2003 Elsevier Ltd. All rights reserved.

The RGBarrier assay: the parallel study of gene regulatory element performance at defined chromosomal locations

Vertebrate genomes are organised into a variety of nuclear environments and chromatin states that have profound effects on the regulation of gene transcription. This variation presents a major challenge to the expression of transgenes for experimental research, genetic therapies and the production of biopharmaceuticals. The majority of transgenes succumb to transcriptional silencing by their chromosomal environment when they are randomly integrated into the genome, a phenomenon known as chromosomal position effect (CPE). It is not always feasible to target transgene integration to transcriptionally permissive “safe harbour” loci that favour transgene expression, so there remains an unmet need to identify gene regulatory elements that can be added to transgenes which protect them against CPE. Dominant regulatory elements (DREs) with chromatin barrier (or boundary) activity have been shown to protect transgenes from CPE. The HS4 element from the chicken beta-globin locus and the A2UCOE element from a human housekeeping gene locus have been shown to function as DRE barriers in a wide variety of cell types and species. Despite rapid advances in the profiling of transcription factor binding, chromatin states and chromosomal looping interactions, progress towards functionally validating the many candidate barrier elements in vertebrates has been very slow. This is largely due to the lack of a tractable and efficient assay for chromatin barrier activity. In this study, I have developed the RGBarrier assay system to test the chromatin barrier activity of candidate DREs at pre-defined isogenic loci in human cells. The RGBarrier assay consists in a Flp-based RMCE reaction for the integration of an expression construct, carrying candidate DREs, in a pre-characterised chromosomal location. The RGBarrier system involves the tracking of red, green and blue fluorescent proteins by flow cytometry to monitor on-target versus off-target integration and transgene expression. The analysis of the reporter (GFP) expression for several weeks gives a measure of the protective ability of each candidate elements from chromosomal silencing. This assay can be scaled up to test tens of new putative barrier elements in the same chromosomal context in parallel. The defined chromosomal contexts of the RGBarrier assays will allow for detailed mechanistic studies of chromosomal silencing and DRE barrier element action. Understanding these mechanisms will be of paramount importance for the design of specific solutions for overcoming chromosomal silencing in specific transgenic applications.

Revisión del uso de los medicamentos biosimilares vs. biológicos: implicaciones para la salud en Colombia.

Este documento tiene como objetivo describir las implicaciones para la salud con el uso de medicamentos biosimilares en comparación con los medicamentos biológicos en Colombia. Así mismo, describir el contexto normativo acerca del uso de medicamentos biosimilares, las recomendaciones y lineamientos sobre seguridad y efectividad del uso de medicamentos Biosimilares y Biológicos, partiendo de sus diferencias biomoleculares. Para esto, se desarrolló una revisión documental electrónica y manual de la literatura en bases de datos, revistas y libros limitada a términos MeSH. La selección de los artículos incluyo documentos completos publicados en revistas indexadas de los últimos 10 años, en español e inglés; la información recolectada se organizó para la construcción del presente documento. Concluyendo, se encontró que las patentes de muchos medicamentos biológicos han vencido o están próximas a caducar y varios biosimilares están desarrollándose y comercializándose incluso en países sin regulaciones estrictas. Los biosimilares nunca podrán ser iguales al original por su complejidad molecular, por ello debemos integrarlos a los sistemas de farmacovigilancia mejorando trazabilidad e identificando su origen mientras se establecen denominaciones comunes distinguibles. La evidencia actual sugiere que la regulación de medicamentos biosimilares debe ser evaluada y armonizada en todo el mundo.