Effects on hepatocellular carcinoma of doxorubicin-loaded immunoliposomes designed to target the VEGFR-2

To maintain a tumour vasculature in proportion of the tumour growth, the endothelial cells proliferate and up-regulate the expression of the VEGF receptor 2 (VEGFR-2), whose expression is restricted to this cell type. This specificity implies that one therapeutically target the tumour endothelium. We investigated the use of immunoliposomes (IL), containing conjugated Fab' fragments of the monoclonal rat anti-VEGFR-2 antibody DC101 (DC101-IL) to cargo doxorubicin to the tumour endothelium. In vitro, fluorescein-labelled IL displayed a 7 fold better binding to VEGFR-2-positive 293T cells in comparison to unspecific liposomes. Balb/C mice were injected subcutaneously with syngeneic hepatocellular carcinoma cells. One set of animals was treated with DC101-IL filled with doxorubicin when the tumours were bigger than 400 mm3. A specific delivery of doxorubicin to endothelial cells of the tumour vessels could be demonstrated by the red fluorescence of doxorubicin with laser scanning microscopy, but neither a delay of tumour growth nor a shrinking of the tumour mass was observed. Yet necrosis in the tumours treated with doxorubicin containing vehicles was larger than in the tumours of the control groups. A second set of animals was treated with DC101-IL filled with doxorubicin when the tumours were smaller than 1 mm3. DC101-IL filled with doxorubicin led to a significant delay in tumour growth up to 7 weeks compared to empty DC101-IL, free doxorubicin, and HEPES/Glucose (HEPES/Glucose vs. DOX-DC101-IL, p = 0.001; unpaired, two-tailed Student's t-test) and to a higher amount of necrotic areas in the tumours (p = 0.053; 1 way ANOVA with 4 groups). These findings suggest that IL designed to bind specifically to VEGFR-2 can be used to deliver doxorubicin to the tumour endothelium and may impair the "angiogenic switch" of the tumours.

Elucidating the IGFBP2 signaling pathway in glioma development and progression

Diffuse gliomas are highly lethal central nervous system malignancies which, unfortunately, are the most common primary brain tumor and also the least responsive to the very few therapeutic modalities currently available to treat them. IGFBP2 is a newly recognized oncogene that is operative in multiple cancer types, including glioma, and shows promise for a targeted therapeutic approach. Elevated IGFBP2 expression is present in high-grade glioma and correlates with poor survival. We have previously demonstrated that IGFBP2 induces glioma development and progression in a spontaneous glioma mouse model, which highlighted its significance and potential for future therapy. However, we did not yet know the key physiological pathways associated with this newly characterized oncogene. We first evaluated human glioma genomics data harnessed from the publicly available Rembrandt source to identify major pathways associated with IGFBP2 expression. Integrin and ILK, among other cell migration and invasion-related pathways, were the most prominently associated. We confirmed that these pathways are regulated by IGFBP2 in glioma cells lines, and demonstrated that 1) IGFBP2 activates integrin α5β1, leading to the activation of key pathways important in glioma; 2) IGFBP2 mediates cell migration pathways through ILK; and 3) IGFBP2 activates NF-kB via an integrin α5 interaction. We then sought to determine whether this was a physiologically active signaling pathway in vivo by assessing its ability to induce glioma progression in the RCAS/tv-a spontaneous glioma mouse model. We found that ILK is a key downstream mediator of IGFBP2 that is required for the induction of glioma progression. Most significantly, a genetic therapeutic approach revealed that perturbation of any point in the pathway thwarted tumor progression, providing strong evidence that targeting the key players could potentially produce a significant benefit for human glioma patients. The elucidation of this signaling pathway is a critical step, since efforts to create a small molecule drug targeting IGFBP2 have so far not been successful, but a number of inhibitors of the other pathway constituents, including ILK, integrin and NF-kB, have been developed.

Nanoinformatics approaches for information extraction and text mining in nanomedical research texts

La nanotecnología es un área de investigación de reciente creación que trata con la manipulación y el control de la materia con dimensiones comprendidas entre 1 y 100 nanómetros. A escala nanométrica, los materiales exhiben fenómenos físicos, químicos y biológicos singulares, muy distintos a los que manifiestan a escala convencional. En medicina, los compuestos miniaturizados a nanoescala y los materiales nanoestructurados ofrecen una mayor eficacia con respecto a las formulaciones químicas tradicionales, así como una mejora en la focalización del medicamento hacia la diana terapéutica, revelando así nuevas propiedades diagnósticas y terapéuticas. A su vez, la complejidad de la información a nivel nano es mucho mayor que en los niveles biológicos convencionales (desde el nivel de población hasta el nivel de célula) y, por tanto, cualquier flujo de trabajo en nanomedicina requiere, de forma inherente, estrategias de gestión de información avanzadas. Desafortunadamente, la informática biomédica todavía no ha proporcionado el marco de trabajo que permita lidiar con estos retos de la información a nivel nano, ni ha adaptado sus métodos y herramientas a este nuevo campo de investigación. En este contexto, la nueva área de la nanoinformática pretende detectar y establecer los vínculos existentes entre la medicina, la nanotecnología y la informática, fomentando así la aplicación de métodos computacionales para resolver las cuestiones y problemas que surgen con la información en la amplia intersección entre la biomedicina y la nanotecnología. Las observaciones expuestas previamente determinan el contexto de esta tesis doctoral, la cual se centra en analizar el dominio de la nanomedicina en profundidad, así como en el desarrollo de estrategias y herramientas para establecer correspondencias entre las distintas disciplinas, fuentes de datos, recursos computacionales y técnicas orientadas a la extracción de información y la minería de textos, con el objetivo final de hacer uso de los datos nanomédicos disponibles. El autor analiza, a través de casos reales, alguna de las tareas de investigación en nanomedicina que requieren o que pueden beneficiarse del uso de métodos y herramientas nanoinformáticas, ilustrando de esta forma los inconvenientes y limitaciones actuales de los enfoques de informática biomédica a la hora de tratar con datos pertenecientes al dominio nanomédico. Se discuten tres escenarios diferentes como ejemplos de actividades que los investigadores realizan mientras llevan a cabo su investigación, comparando los contextos biomédico y nanomédico: i) búsqueda en la Web de fuentes de datos y recursos computacionales que den soporte a su investigación; ii) búsqueda en la literatura científica de resultados experimentales y publicaciones relacionadas con su investigación; iii) búsqueda en registros de ensayos clínicos de resultados clínicos relacionados con su investigación. El desarrollo de estas actividades requiere el uso de herramientas y servicios informáticos, como exploradores Web, bases de datos de referencias bibliográficas indexando la literatura biomédica y registros online de ensayos clínicos, respectivamente. Para cada escenario, este documento proporciona un análisis detallado de los posibles obstáculos que pueden dificultar el desarrollo y el resultado de las diferentes tareas de investigación en cada uno de los dos campos citados (biomedicina y nanomedicina), poniendo especial énfasis en los retos existentes en la investigación nanomédica, campo en el que se han detectado las mayores dificultades. El autor ilustra cómo la aplicación de metodologías provenientes de la informática biomédica a estos escenarios resulta efectiva en el dominio biomédico, mientras que dichas metodologías presentan serias limitaciones cuando son aplicadas al contexto nanomédico. Para abordar dichas limitaciones, el autor propone un enfoque nanoinformático, original, diseñado específicamente para tratar con las características especiales que la información presenta a nivel nano. El enfoque consiste en un análisis en profundidad de la literatura científica y de los registros de ensayos clínicos disponibles para extraer información relevante sobre experimentos y resultados en nanomedicina —patrones textuales, vocabulario en común, descriptores de experimentos, parámetros de caracterización, etc.—, seguido del desarrollo de mecanismos para estructurar y analizar dicha información automáticamente. Este análisis concluye con la generación de un modelo de datos de referencia (gold standard) —un conjunto de datos de entrenamiento y de test anotados manualmente—, el cual ha sido aplicado a la clasificación de registros de ensayos clínicos, permitiendo distinguir automáticamente los estudios centrados en nanodrogas y nanodispositivos de aquellos enfocados a testear productos farmacéuticos tradicionales. El presente trabajo pretende proporcionar los métodos necesarios para organizar, depurar, filtrar y validar parte de los datos nanomédicos existentes en la actualidad a una escala adecuada para la toma de decisiones. Análisis similares para otras tareas de investigación en nanomedicina ayudarían a detectar qué recursos nanoinformáticos se requieren para cumplir los objetivos actuales en el área, así como a generar conjunto de datos de referencia, estructurados y densos en información, a partir de literatura y otros fuentes no estructuradas para poder aplicar nuevos algoritmos e inferir nueva información de valor para la investigación en nanomedicina. ABSTRACT Nanotechnology is a research area of recent development that deals with the manipulation and control of matter with dimensions ranging from 1 to 100 nanometers. At the nanoscale, materials exhibit singular physical, chemical and biological phenomena, very different from those manifested at the conventional scale. In medicine, nanosized compounds and nanostructured materials offer improved drug targeting and efficacy with respect to traditional formulations, and reveal novel diagnostic and therapeutic properties. Nevertheless, the complexity of information at the nano level is much higher than the complexity at the conventional biological levels (from populations to the cell). Thus, any nanomedical research workflow inherently demands advanced information management. Unfortunately, Biomedical Informatics (BMI) has not yet provided the necessary framework to deal with such information challenges, nor adapted its methods and tools to the new research field. In this context, the novel area of nanoinformatics aims to build new bridges between medicine, nanotechnology and informatics, allowing the application of computational methods to solve informational issues at the wide intersection between biomedicine and nanotechnology. The above observations determine the context of this doctoral dissertation, which is focused on analyzing the nanomedical domain in-depth, and developing nanoinformatics strategies and tools to map across disciplines, data sources, computational resources, and information extraction and text mining techniques, for leveraging available nanomedical data. The author analyzes, through real-life case studies, some research tasks in nanomedicine that would require or could benefit from the use of nanoinformatics methods and tools, illustrating present drawbacks and limitations of BMI approaches to deal with data belonging to the nanomedical domain. Three different scenarios, comparing both the biomedical and nanomedical contexts, are discussed as examples of activities that researchers would perform while conducting their research: i) searching over the Web for data sources and computational resources supporting their research; ii) searching the literature for experimental results and publications related to their research, and iii) searching clinical trial registries for clinical results related to their research. The development of these activities will depend on the use of informatics tools and services, such as web browsers, databases of citations and abstracts indexing the biomedical literature, and web-based clinical trial registries, respectively. For each scenario, this document provides a detailed analysis of the potential information barriers that could hamper the successful development of the different research tasks in both fields (biomedicine and nanomedicine), emphasizing the existing challenges for nanomedical research —where the major barriers have been found. The author illustrates how the application of BMI methodologies to these scenarios can be proven successful in the biomedical domain, whilst these methodologies present severe limitations when applied to the nanomedical context. To address such limitations, the author proposes an original nanoinformatics approach specifically designed to deal with the special characteristics of information at the nano level. This approach consists of an in-depth analysis of the scientific literature and available clinical trial registries to extract relevant information about experiments and results in nanomedicine —textual patterns, common vocabulary, experiment descriptors, characterization parameters, etc.—, followed by the development of mechanisms to automatically structure and analyze this information. This analysis resulted in the generation of a gold standard —a manually annotated training or reference set—, which was applied to the automatic classification of clinical trial summaries, distinguishing studies focused on nanodrugs and nanodevices from those aimed at testing traditional pharmaceuticals. The present work aims to provide the necessary methods for organizing, curating and validating existing nanomedical data on a scale suitable for decision-making. Similar analysis for different nanomedical research tasks would help to detect which nanoinformatics resources are required to meet current goals in the field, as well as to generate densely populated and machine-interpretable reference datasets from the literature and other unstructured sources for further testing novel algorithms and inferring new valuable information for nanomedicine.

PLGA microspheres for the delivery of a novel subunit TB vaccine

Biodegradable poly(dl-lactide-co-glycolide) microspheres were prepared using a modified double emulsion solvent evaporation method for the delivery of the subunit tuberculosis vaccine (Ag85B-ESAT-6), a fusion protein of the immunodominant antigens 6-kDa early secretory antigenic target (ESAT-6) and antigen 85B (Ag85B). Addition of the cationic lipid dimethyl dioctadecylammonium bromide (DDA) and the immunostimulatory trehalose 6,6'-dibehenate (TDB), either separately or in combination, was investigated for the effect on particle size and distribution, antigen entrapment efficiency, in vitro release profiles and in vivo performance. Optimised formulation parameters yielded microspheres within the desired sub-10 mu m range (1.50 +/- 0.13 mu m), whilst exhibiting a high antigen entrapment efficiency (95 +/- 1.2%) and prolonged release profiles. Although the microsphere formulations induced a cell-mediated immune response and raised specific antibodies after immunisation, this was inferior to the levels achieved with liposomes composed of the same adjuvants (DDA-TDB), demonstrating that liposomes are more effective vaccine delivery systems compared with microspheres.

Targeted delivery of a novel group of site-directed transglutaminase inhibitors to the liver using liposomes:a new approach for the potential treatment of liver fibrosis

Liver fibrosis and its end-stage disease cirrhosis are a main cause of mortality and morbidity worldwide. Thus far, there is no efficient pharmaceutical intervention for the treatment of liver fibrosis. Liver fibrosis is characterized by excessive accumulation of the extracellular matrix (ECM) proteins. Transglutaminase (TG)-mediated covalent cross-linking has been implicated in the stabilization and accumulation of ECM in a number of fibrotic diseases. Thus, the use of tissue TG2 inhibitors has potential in the treatment of liver fibrosis. Recently, we introduced a novel group of site-directed irreversible specific inhibitors of TGs. Here, we describe the development of a liposome-based drug-delivery system for the site-specific delivery of these TG inhibitors into the liver. By using anionic or neutral-based DSPC liposomes, the TG inhibitor can be successfully incorporated into these liposomes and delivered specifically to the liver. Liposomes can therefore be used as a potential carrier system for site-specific delivery of the TG2 inhibitors into the liver, opening up a potential new avenue for the treatment of liver fibrosis and its end-stage disease cirrhosis.

Liposomes act as stronger sub-unit vaccine adjuvants when compared to microspheres

The ability of liposomes and microspheres to enhance the efficacy of a sub-unit antigen was investigated. Microspheres were optimised by testing a range of surfactants employed in the external aqueous phase of a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation process for the preparation of microspherescomposed of poly(d,l-lactide-co-glycolide) and the immunological adjuvant dimethyl dioctadecyl ammonium bromide (DDA)and then investigated with regard to the physico-chemical and immunological characteristics of the particles produced. The results demonstrate that this parameter can affect the physico-chemical characteristics of these systems and subsequently, has a substantial bearing on the level of immune response achieved, both humoural and cell mediated, when employed for the delivery of the sub-unit tuberculosis vaccine antigen Ag85B-ESAT-6. Moreover, the microsphere preparations investigated failed to initiate immune responses at the levels achieved with an adjuvant DDA-based liposome formulation (DDA-TDB), further substantiating the superior ability of liposomes as vaccine delivery systems.

Liposome-mediated DNA immunisation via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper, we have investigated the application of liposome-entrapped DNA and their cationic lipid composition on such potency after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmoles dioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol) and either the cationic lipid 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP) or cholesteryl 3-N-(dimethyl amino ethyl) carbamate (DC-Chol). This method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded incorporation values of 90-94% of the DNA used. Mixing or rehydration of preformed cationic liposomes with 100 μg plasmid DNA also led to similarly high complexation values (92-94%). In an attempt to establish differences in the nature of DNA association with these various liposome preparations their physico-chemical characteristics were investigated. Studies on vesicle size, zeta potential and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, formulation of liposomal DNA by the dehydration-rehydration generated submicron size liposomes incorporating most of the DNA in a manner that prevents DNA displacement through anion competition. The bilayer composition of these dehydration-rehydration vesicles (DRV(DNA)) can also further influence these physicochemical characteristics with the presence of DOPE within the liposome bilayer resulting in a reduced vesicle zeta potential. Subcutaneous liposome-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 μg liposome-entrapped DNA compared to naked DNA. At all time points measured, mice immunised with naked DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with DNA entrapped in DRV liposomes containing DOTAP compared to the control group and mice immunised with naked DNA. By day 56, all total IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing DC-Chol dropped to significantly lower levels that those measured in mice which received the DOTAP formulation. Similar results were found when the IgG1 and IgG2a subclass responses were determined. These results suggest that, not only can DNA be effectively entrapped within liposomes using the DRV method but that such DRV liposomes containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. © 2003 Taylor & Francis Ltd.

Bioreaction and separation in preparative batch chromatographic columns : the hydrology of lactose to yield glucose,galactose and oligosaccharides

The initial aim of this project was to improve the performance of a chromatographic bioreactor-separator (CBRS). In such a system, a dilute enzyme solution is pumped continuously through a preparative chromatographic column, while pulses of substrate are periodically injected on to the column. Enzymic reaction and separation are therefore performed in a single unit operation. The chromatographic columns used were jacketed glass columns ranging from 1 to 2 metres long with an internal diameter of 1.5 cm. Linking these columns allowed 1, 2, 3 and 4 metre long CBRS systems to be constructed. The hydrolysis of lactose in the presence of β~galactosidase was the reaction of study. From previous work at Aston University, there appeared to be no difficulties in achieving complete lactose hydrolysis in a CBRS. There did, however, appear to be scope for improving the separative performance, so this was adopted as an initial goal. Reducing the particle size of the stationary phase was identified as a way of achieving this improvement. A cation exchange resin was selected which had an average particle size of around half that previously used when studying this reaction. A CBRS system was developed which overcame the operational problems (such as high pressure drop development) associated with use of such a particle size. A significant improvement in separative power was achieved. This was shown by an increase in the number of theoretical plates (N) from about 500 to about 3000 for a 2 metre long CBRS, coupled with higher resolution. A simple experiment with the 1 metre column showed that combined bioreaction and separation was achievable in this system. Having improved the separative performance of the system, the factors affecting enzymic reaction in a CBRS were investigated; including pulse volume and the degree of mixing between enzyme and substrate. The progress of reaction in a CBRS was then studied. This information was related to the interaction of reaction and separation over the reaction zone. The effect of injecting a pulse over a length of time as in CBRS operation was simulated by fed batch experiments. These experiments were performed in parallel with normal batch experiments where the substrate is mixed almost instantly with the enzyme. The batch experiments enabled samples to be taken every minute and revealed that reaction is very rapid. The hydrodynamic characteristics of the two injector configurations used in CBRS construction were studied using Magnetic Resonance Imaging, combined with hydrodynamic calculations. During the optimisation studies, galactooligosaccharides (GOS) were detected as intermediates in the hydrolysis process. GOS are valuable products with potential and existing applications in food manufacture (as nutraceuticals), medicine and drug targeting. The focus of the research was therefore turned to GOS production. A means of controlling reaction to arrest break down of GOS was required. Raising temperature was identified as a possible means of achieving this within a CBRS. Studies were undertaken to optimise the yield of oligosaccharides, culminating in the design, construction and evaluation of a Dithermal Chromatographic Bioreactor-separator.

The role of lipid geometry in designing liposomes for the solubilisation of poorly water soluble drugs

Liposomes are well recognised for their ability to improve the delivery of a range of drugs. More commonly they are applied for the delivery of water-soluble drugs, but given their structural attributes they can also be employed as solubilising agents for low solubility drugs as well as drug targeting agents. To further explore the potential of liposomes as solubilising agents, we have investigated the role of bilayer packaging in promoting drug solubilisation in liposome bilayers. The effect of alkyl chain length and symmetry was investigated to consider if using 'mis-matched' phospholipids could be used to create 'voids' within the bilayers, and enhance bilayer loading capacity. Lipid packing was investigated using Langmuir studies, which demonstrated that increasing the alkyl chain length enhanced lipid packing, with condensed monolayer forming, whilst asymmetric lipids formed less condensed monolayers. However this more open packing did not translate into improved drug loading, with the longer chain, condensed bilayers formed from long-chain, saturated lipids offering higher drug loading capacity. These studies demonstrate that liposomes formulated from longer chain, saturated lipids offer enhanced solubilisation capacity. However the molecular size, rather than lipophilicity, of the drug to be incorporated was also a key factor dominating bilayer incorporation efficiency.

Characterization and optimization of bilosomes for oral vaccine delivery

Oral vaccines offer significant benefits due to the ease of administration, better patient compliance and non-invasive, needle-free administration. However, this route is marred by the harsh gastro intestinal environment which is detrimental to many vaccine formats. To address this, a range of delivery systems have been considered including bilosomes; these are bilayer vesicles constructed from non-ionic surfactants combined with the inclusion of bile salts which can stabilize the vesicles in the gastro intestinal tract by preventing membrane destabilization. The aim of this study was to investigate the effect of formulation parameters on bilosome carriers using Design of Experiments to select an appropriate formulation to assess in vivo. Bilosomes were constructed from monopalmitoylglycerol, cholesterol, dicetyl phosphate and sodium deoxycholate at different blends ratios. The optimized bilosome formulation was identified and the potential of this formulation as an oral vaccine delivery system were assessed in biodistribution and vaccine efficacy studies. Results showed that the larger bilosomes vesicles (~6 µm versus 2 µm in diameter) increased uptake within the Peyer's patches and were able to reduce median temperature differential change and promote a reduction in viral cell load in an influenza challenge study. © 2013 Informa UK, Ltd.

The role of lipid geometry in designing liposomes for the solubilisation of poorly water soluble drugs

Liposomes are well recognised for their ability to improve the delivery of a range of drugs. More commonly they are applied for the delivery of water-soluble drugs, but given their structural attributes, they can also be employed as solubilising agents for low solubility drugs as well as drug targeting agents. To further explore the potential of liposomes as solubilising agents, we have investigated the role of bilayer packaging in promoting drug solubilisation in liposome bilayers. The effect of alkyl chain length and symmetry was investigated to consider if using 'mis-matched' phospholipids could create 'voids' within the bilayers, and enhance bilayer loading capacity. Lipid packing was investigated using Langmuir studies, which demonstrated that increasing the alkyl chain length enhanced lipid packing, with condensed monolayers forming, whilst asymmetric lipids formed less condensed monolayers. However, this more open packing did not translate into improved drug loading, with the longer chain, condensed bilayers formed from long-chain, saturated lipids offering higher drug loading capacity. These studies demonstrate that liposomes formulated from longer chain, saturated lipids offer enhanced solubilisation capacity. However the molecular size, rather than lipophilicity, of the drug to be incorporated was also a key factor dominating bilayer incorporation efficiency. © 2012 Elsevier B.V. All rights reserved.

Enhanced dispersibility and deposition of spray-dried powders for pulmonary gene therapy

Spray-drying represents a viable alternative to freeze-drying for preparing dry powder dispersions for delivering macromolecules to the lung. The dispersibility of spray-dried powders is limited however, and needs to be enhanced to improve lung deposition and subsequent biological activity. In this study, we investigate the utility of leucine as a dry powder dispersibility enhancer when added prior to spray-drying a model non-viral gene therapy formulation (lipid:polycation:pDNA, LPD). Freeze-dried lactose-LPD, spray-dried lactose-LPD and spray-dried leucine-lactose-LPD powders were prepared. Scanning electron microscopy showed that leucine, increased the surface roughness of spray-dried lactose particles. Particle size analysis revealed that leucine-containing spray-dried powders were unimodally dispersed with a mean particle diameter of 3.12 μm. Both gel electrophoresis and in vitro cell (A549) transfection showed that leucine may compromise the integrity and biological functionality of the gene therapy vector. The deposition of the leucine containing powder was however significantly enhanced as evidenced by an increase in gene expression mediated by dry powder collected at lower stages of a multistage liquid impinger (MSLI). Further studies are required to determine the potential of leucine as a ubiquitous dispersibility enhancer for a variety of pulmonary formulations. © 2003 Taylor & Francis Ltd.

Genomic evaluation during permeability of indomethacin and its solid dispersion

Drug resistance was first identified in cancer cells that express proteins known as multidrug resistance proteins that extrude the therapeutic agents out of the cells resulting in alteration of pharmacokinetics, tissue distribution, and pharmacodynamics of drugs. To this end studies were carried out to investigate the role of pharmacological inhibitors and pharmaceutical excipients with a primary focus on P-glycoprotein (P-gp). The aim of this study was to investigate holistic changes in transporter gene expression during permeability upon formulation of indomethacin as solid dispersion. Initial characterization studies of solid dispersion of indomethacin showed that the drug was dispersed within the carrier in amorphous form. Analysis of permeability data across Caco-2 monolayers revealed that drug absorption increased by 4-fold when reformulated as solid dispersion. The last phase of the work involved investigation of gene expression changes of transporter genes during permeability. The results showed that there were significant differences in the expression of both ATP-binding cassette (ABC) transporter genes as well as solute carrier transporter (SLC) genes suggesting that the inclusion of polyethylene glycol as well as changes in molecular form of drug from crystalline to amorphous have a significant bearing on the expression of transporter network genes resulting in differences in drug permeability. © 2011 Informa UK, Ltd.

Receptor activity-modifying proteins; multifunctional G protein-coupled receptor accessory proteins

Receptor activity-modifying proteins (RAMPs) are single pass membrane proteins initially identified by their ability to determine the pharmacology of the calcitonin receptor-like receptor (CLR), a family B G protein-coupled receptor (GPCR). It is now known that RAMPs can interact with a much wider range of GPCRs. This review considers recent developments on the structure of the complexes formed between the extracellular domains (ECDs) of CLR and RAMP1 or RAMP2 as these provide insights as to how the RAMPs direct ligand binding. The range of RAMP interactions is also considered; RAMPs can interact with numerous family B GPCRs as well as examples of family A and family C GPCRs. They influence receptor expression at the cell surface, trafficking, ligand binding and G protein coupling. The GPCR-RAMP interface offers opportunities for drug targeting, illustrated by examples of drugs developed for migraine.

Magnetic nanoformulation of azidothymidine 5’-triphosphate for targeted delivery across the blood–brain barrier

Despite significant advances in highly active antiretroviral therapy (HAART), the prevalence of neuroAIDS remains high. This is mainly attributed to inability of antiretroviral therapy (ART) to cross the blood–brain barrier (BBB), thus resulting in insufficient drug concentration within the brain. Therefore, development of an active drug targeting system is an attractive strategy to increase the efficacy and delivery of ART to the brain. We report herein development of magnetic azidothymidine 5′-triphosphate (AZTTP) liposomal nanoformulation and its ability to transmigrate across an in vitro BBB model by application of an external magnetic field. We hypothesize that this magnetically guided nanoformulation can transverse the BBB by direct transport or via monocyte-mediated transport. Magnetic AZTTP liposomes were prepared using a mixture of phosphatidyl choline and cholesterol. The average size of prepared liposomes was about 150 nm with maximum drug and magnetite loading efficiency of 54.5% and 45.3%, respectively. Further, magnetic AZTTP liposomes were checked for transmigration across an in vitro BBB model using direct or monocyte-mediated transport by application of an external magnetic field. The results show that apparent permeability of magnetic AZTTP liposomes was 3-fold higher than free AZTTP. Also, the magnetic AZTTP liposomes were efficiently taken up by monocytes and these magnetic monocytes showed enhanced transendothelial migration compared to normal/non-magnetic monocytes in presence of an external magnetic field. Thus, we anticipate that the developed magnetic nanoformulation can be used for targeting active nucleotide analog reverse transcriptase inhibitors to the brain by application of an external magnetic force and thereby eliminate the brain HIV reservoir and help to treat neuroAIDS.