CNS delivery of antisense oligodeoxynucleotides using biodegradable microspheres

Antisense oligodeoxynucleotides can selectively inhibit gene expression provided they are delivered to their target site successfully for a sufficient duration. Biodegradable microspheres have previously been developed for the potential systemic delivery of antisense oligodeoxynucleotides and offer an excellent strategy for central administration of antisense oligodeoxynucleotides, providing a sustained-release delivery system. Biodegradable microspheres were formulated to entrap antisense oligodeoxynucleotides for stereotaxic implantation into site-specific regions of the rat brain.Release profiles of antisense oligodeoxynucleotides from biodegradable microspheres over 56 days that were triphasic were observed with high molecular weight polymers. Antisense oligodeoxynucleotides loaded into microspheres (1-10μm) had a five-fold increase in cellular association with glial and neuronal cells compared to the naked molecule, which was partially due to a greater cellular accumulation as observed by a slower efflux profile. In vivo distribution studies of antisense oligodeoxynucleotides demonstrated that the use of microspheres provided a sustained-release over more than 2 days compared to 12 hours of the naked molecule. Efficacy of antisense oligodeoxynucleotides was demonstrated during locomotor activity investigations, which significantly reduced cocaine-induced locomotor activity, where no efficacy was demonstrated with microspheres, possibly attributed to antisense loading and measurements being taken during a lag phase of antisense oligodeoxynucleotide release. Biodegradable microspheres can be delivered site-specifically into the brain and provide sustained-release of antisense oligodeoxynucleotides, offering the potential of in vivo efficacy in these reagents in the brain.

Biodegradable polymers for the sustained release of antisense nucleic acids

Antisense oligodeoxynucleotides can selectively inhibit individual gene expression provided they remain stable at the target site for a sufficient period of time. Thus, the efficacy of antisense oligodeoxynucleotides may be improved by employing a sustained release delivery system which would protect from degradation by nucleases whilst delivering the nucleic acid in a controlled manner to the site of action. Biodegradable polymer films and micro spheres were evaluated as delivery devices for the oligodeoxynucleotides and ribozymes. Polymers such as polylactide, polyglycolide, polyhydroxybutyrate and polyhydroxyvalerate were used due to their biocompatability and non toxic degradation products. Release profiles of antisense nucleic acids from films over 28 days was biphasic, characterised by an initial burst release during the first 48 hours followed by a more sustained release. Release from films of longer antisense nucleic acids was slower compared to shorter nucleic acids. Backbone type also affected release, although to a lesser extent than length. Total release of the nucleic acids is dependent upon polymer degradation, no degradation of the polymer films was evident over the 28 day period, due to the high molecular weight and crystallinity of the polymers required to make solvent cast films. Backbone length and type did not affect release from microspheres, release was generally faster than from films, due to the increased surface area, and low molecular weight polymers which showed signs of degradation over the release period, resulting in a triphasic release profile. An increase in release was observed when sphere size and polymer molecular weight were decreased. The polymer entrapped phosphodiester oligodeoxynucleotides and ribozymes had enhanced stability compared to free oligodeoxynucleotides and ribozymes when incubated in serum. The released nucleic acids were still capable of hybridising to their target sequence, indicating that the fabrication processes did not adversely effect the properties of the antisense nucleic acids. Oligodeoxynucleotides loaded in 2μm spheres had a 10 fold increase in macrophage association compared to free oligodeoxynucleotides. Fluorescent microscopy indicates that the polymer entrapped oligodeoxynucleotide is concentrated inside the cell, whereas free oligodeoxynucleotides are concentrated at the cell membrane. Biodegradable polymers can reduce the limitations of antisense therapy and thus offer a potential therapeutic advantage.

A study in radiopaque polymeric materials

The problems associated with x-ray-transparent denture base are defined and conventional approaches to their solution are assessed. Consideration of elemental absorption parameters leads to the postulation that atoms such as zinc, and bromine, may be effective radiopacifiers over at least part of the clinical x-ray spectrum. These elements had hitherto been considered too light to be effective. Investigation of copolymers of methylmethacrylate and p-bromostyrene revealed no deleterious effects arising from the aromatically brominated monomer (aliphatic bromination caused UV destabilisation). For effective x-ray absorption a higher level of bromination would be necessary, but the expense of suitable compounds made further study unjustifiable. Incorporation of zinc atoms into the polymer was accomplished by copolymerisation of zinc acrylate with methylmethacrylate in solution. At high zinc levels this produced a powder copolymer convenient for addition to dental polymers in the dough moulding process. The resulting mouldings showed increasing brittleness at high loadings of copolymer. Fracture was shown to be through the powder particles rather than around them, indicating the source of weakness to be in the internal structure of the copolymer. The copolymer was expected to be cross-linked through divalent zinc ions and its insolubility and infusibility supported this. Cleavage of the ionic cross links with formic acid produced a zinc-free linear copolymer of high molecular weight. Addition of low concentrations of acrylic acid to the dough moulding monomer appeared to 'labilise' the cross links producing a more homogeneous moulding with adequate wet strength. Toxicologically the zinc-containing materials are satisfactory and though zinc is extracted at a measurable rate in an aqueous system, this is very small and should be acceptable over the life of a denture. In other respects the composite is quite satisfactory and though a marketable product is not claimed the system is considered worthy of further study.

The behaviour of polymer quenchants

The internationally accepted Wolfson Heat Treatment Centre Engineering Group test was used to evaluate the cooling characteristics of the most popular commercial polymer quenchants: polyalkylene glycols, polyvinylpyrrolidones and polyacrylates. Prototype solutions containing poly(ethyloxazoline) were also examined. Each class of polymer was capable of providing a wide range of cooling rates depending on the product formulation, concentration, temperature, agitation, ageing and contamination. Cooling rates for synthetic quenchants were generally intermediate between those of water and oil. Control techniques, drag-out losses and response to quenching in terms of hardness and residual stress for a plain carbon steel, were also considered. A laboratory scale method for providing a controllable level of forced convection was developed. Test reproducibility was improved by positioning the preheated Wolfson probe 25mm above the geometric centre of a 25mm diameter orifice through which the quenchant was pumped at a velocity of 0.5m/s. On examination, all polymer quenchants were found to operate by the same fundamental mechanism associated with their viscosity and ability to form an insulating polymer-rich-film. The nature of this film, which formed at the vapour/liquid interface during boiling, was dependent on the polymer's solubility characteristics. High molecular weight polymers and high concentration solutions produced thicker, more stable insulating films. Agitation produced thinner more uniform films. Higher molecular weight polymers were more susceptible to degradation, and increased cooling rates, with usage. Polyvinylpyrrolidones can be cross-linked resulting in erratic performance, whilst the anionic character of polyacrylates can lead to control problems. Volatile contaminants tend to decrease the rate of cooling and salts to increase it. Drag-out increases upon raising the molecular weight of the polymer and its solution viscosity. Kinematic viscosity measurements are more effective than refractometer readings for concentration control, although a quench test is the most satisfactory process control method.

Reduced metal transferrin binding in neurological diseases

By employing G75 gel-filtration chromotography, it has been demonstrated that human plasma gallium speciation (and by implication, Al speciation) is bimodal. Normally, gallium was predominantly bound to a high molecular weight fraction which was presumably transferrin. Literature reviews and experimental work throughout this thesis provided evidence to support this idea. An aluminium-transferrin species was assumed to be relatively non-toxic and a protective function for this complex has been suggested. A second, low molecular weight species of gallium was observed and its identity has been suggested to be citrate. The results of this thesis support the concept citrate was a gallium binding ligand present in the plasma, but there was another species (tentatively identified as phosphate) which bound gallium to a much greater degree than did citrate in the majority of samples studied. The consequence of a low molecular weight species of aluminium is the possibility that this leads to a more rapid, uncontrolled deposition of the metal in the brain compared to a transferrin mediated mechanism. Plasma speciation studies in Alzheimer's disease, Parkinson's disease, Down's syndrome, and neonates has revealed an altered ratio of the two gallium species found in control subjects. In all groups there was an increase in the potentially more neurotoxic low molecular weight species. These observations have led to a suggested mechanism of accumulation of metals in the brain, which is known to occur in the first three groups. Possible pathogenic mechanisms are described. The results can also offer an explanation to the reported increased sensitivity to the toxic effects of aluminium in the neonate. Speciation studies on normal plasma has shown the balance between high and low molecular weight species of gallium to be influenced by many physiological factors. There appears to be a fine equilibrium between both species which can be altered without any great difficulty. Therefore, in the diseased groups studied, it is possible that there are subtle biochemical changes within the circulatory system to affect the equilibrium which results in an increased low molecular weight species of aluminium. Furthermore, it has been demonstrated that there is a group of normal controls with no clinical signs of Alzheimer's or Parkinson's disease which have reduced transferrin binding. This indicates there is a population of healthy people who are at risk to the development of either disease.

Amino acid, peptide and drug transport across monolayers of human intestinal (CAC0-2) cells in vitro

The properties of Caco-2 monolayers were compared on aluminium oxide and nitrocellulose permeable-supports. On nitrocellulose, Caco-2 cells displayed a higher rate of taurocholic acid transport than those cultured on aluminium oxide inserts. In addition, Caco-2 cells grown on these two inserts were not comparable with respect to cell morphology, cell numbers and transepithelial electrical resistance. The low adsorption potential of the aluminium oxide inserts, particularly for high molecular weight or lipophilic ligands, offers a distinct advantage over nitrocellulose inserts for drug transport studies. The carrier-mediated uptake and transport of the imino acid (L-proline) and the acidic amino acids (L-aspartate and L-glutamate) have been studied. At pH7.4, L-proline uptake is mediated via an A-system carrier. Elevated uptake and transport under acidic conditions occurs by activation of a distinct carrier population. Acidic amino acid transport is mediated via a X-AG system. The flux of baclofen, CGP40116 andCGP40117 across Caco-2 monolayers was described by passive transport. The transport of three peptides, thyrotrophin-releasing hormone, SQ29852 and cyclosporin were investigated. Thyrotrophin-releasing hormone transport acrossCaco-2 monolayers was characterised by a minor saturable (carrier-mediated,approximately 25%) pathway, superimposed onto a major non-saturable (diffusional)pathway. SQ29852 uptake into Caco-2 monolayers is described by a major saturable mechanism (Km = 0.91 mM) superimposed onto a minor passive component.However, the initial-rate of SQ29852 transport is consistent with a passive transepithelial transport mechanism. These data highlight the possibility that itsbasolateral efflux is severely retarded such that the passive paracellular transportdictates the overall transepithelial transport characteristics. In addition, modelsuitable for investigating the transepithelial transport of cyclosporin A has been developed. A modification of the conventional Caco-2 model has been developed which has a calcium-free Ap donor-solution and a Bl receiver-solution containing the minimumcalcium concentration required to maintain monolayer integrity (100 μM). The influence of calcium and magnesium on the absorption of [14C]pamidronate was evaluated by comparing its transport across the conventional and minimum calciumCaco-2 models. Ap calcium and magnesium ions retard the Ap-to-Bl flux of pamidronate across Caco-2 monolayers. The effect of self-emulsifying oleic acid-Tween 80 formulations on Caco-2monolayer integrity has been investigated. Oleic acid-Tween 80 (1 0:1) formulations produced a dose-dependent disruption of Caco-2 monolayer integrity. This disruption was related to the oleic acid content of the formulation.

The application of counter immunoelectrophoresis (CIE) in ocular protein studies. Part II:kinin activity in the lens wearing eye

The kinin family are a group of bioactive peptides that are closely involved in the modulation of vascular inflammation and local injury. We have demonstrated here, for the first time, a link between kinin activity and contact lens wear. Protein extracts from daily and extended wear etafilcon A, Group IV, Acuvue lenses (Vistakon), were analysed by counter immunoelectrophoresis. In this way, kinin activity associated with contact lens wear was detected. High molecular weight kininogen was used as the marker protein. In contrast, no kinin activity was detected in the non-lens wearing normal eye. © 2002 British Contact Lens Association. Published by Elsevier Science Ltd. All rights reserved.

The detection of kinin activity in contact lens wear

Many factors can be, and have been, attributed to the appearance of complications in lens wear, but the greatest is associated with deposition. Reduced acuity, irritation and inflammatory responses are often referred to as adverse reactions arising as a result of deposition. In this study, particular attention was paid to the potential role of adsorbed proteins in activating, mediating and/or stimulating a host immune response, i.e., the hypothesis that the adsorption of certain proteins from the tears and ocular surfaces may actively affect successful lens wear. In particular, the purpose of this study was to investigate the presence of a group of proteins previously undiscovered in the ocular environment. The intention was to target a family of proteins/glycoproteins that have become prominent recently in a variety of inflammatory responses and disorders at many other mucosal associated sites around the body, e.g. in nasal rhinitis and in joint inflammation. The protein cascade in question is the kinin family of inflammatory mediators. The aim was to investigate their presence in the ocular environment, specifically in relation to contact lens wear, and consequently assess the implications of their discovery. High molecular weight kininogen (HMWK), with its central role in kinin responses, was investigated initially as the marker of kinin activity, with subsequent members examined thereafter.

Direct observation of morphological development during the spin-coating of polystyrene-poly(methyl methacrylate) polymer blends

We present results of the direct observation, in real-space, of the phase separation of high molecular weight polystyrene and poly(methyl methacrylate) from ortho-xylene using our newly developed technique of high speed stroboscopic interference microscopy. Taking a fixed concentration (3 wt % in o-xylene) at a fixed composition (1:4 by weight) and by varying the rotational rate during the spin-coating process, we are able to observe the formation of a range of phase separated bicontinuous morphologies of differing length-scales. Importantly, we are able to show that the mechanism by which the final phase separated structure is formed is through domain coarsening when rich in solvent, before vitrification occurs and fixes the phase separated structure. The ability to directly observe morphological development offers a route toward controlling the length-scale of the final morphology through process control and in situ feedback, from a single stock solution. © 2013 Wiley Periodicals, Inc.

An assessment of the performance of high density polyethylene copolymers for natural gas distribution pipes

The total thermoplastics pipe market in west Europe is estimated at 900,000 metric tonnes for 1977 and is projected to grow to some 1.3 million tonnes of predominantly PVC and polyolefins pipe by 1985. By that time, polyethylene for gas distribution pipe and fittings will represent some 30% of the total polyethylene pipe market. The performance characteristics of a high density polyethylene are significantly influenced by both molecular weight and type of comonomer; the major influences being in the long-term hoop stress resistance and the environmental stress cracking resistance. Minor amounts of hexene-1 are more effective than comonomers lower in the homologous series, although there is some sacrifice of density related properties. A synergistic improvement is obtained by combining molecular weight increase with copolymerisation. The Long-term design strength of polyethylene copolymers can be determined from hoop stress measurement at elevated temperatures and by means of a separation factor of approximate value 22, extrapolation can be made to room temperature performance for a water environment. A polyethylene of black composition has a sufficiently improved performance over yellow pigmented pipe to cast doubts on the validity of internationally specifying yellow coded pipe for gas distribution service. The chemical environment (condensate formation) that can exist in natural gas distribution networks has a deleterious effect on the pipe performance the reduction amounting to at least two decades in log time. Desorption of such condensate is very slow and the influence of the more aggressive aromatic components is to lead to premature stress cracking. For natural gas distribution purposes, the design stress rating should be 39 Kg/cm2 for polyethylenes in the molecular weight range of 150 - 200,000 and 55 Kg/cm2 for higher molecular weight materials.