Non-aqueous silicone elastomer gels as a vaginal microbicide delivery system for the HIV-1 entry inhibitor maraviroc

Aqueous semi-solid polymeric gels, such as those based on hydroxyethylcellulose (HEC) and polyacrylic acid (e.g. Carbopol®), have a long history of use in vaginal drug delivery. However, despite their ubiquity, they often provide sub-optimal clinical performance, due to poor mucosal retention and limited solubility for poorly water-soluble actives. These issues are particularly pertinent for vaginal HIV microbicides, since many lead candidates are poorly water-soluble and where a major goal is the development of a coitally independent, once daily gel product. In this study, we report the use of a non-aqueous silicone elastomer gel for vaginal delivery of the HIV-1 entry inhibitor maraviroc. In vitro rheological, syringeability and retention studies demonstrated enhanced performance for silicone gels compared with a conventional aqueous HEC gel, while testing of the gels in the slug model confirmed a lack of mucosal irritancy. Pharmacokinetic studies following single dose vaginal administration of a maraviroc silicone gel in rhesus macaques showed higher and sustained MVC levels in vaginal fluid, vaginal tissue and plasma compared with a HEC gel containing the same maraviroc loading. The results demonstrate that non-aqueous silicone gels have potential as a formulation platform for coitally independent vaginal HIV microbicides.

Increased potential of a cationic liposome-based delivery system:enhancing stability and sustained immunological activity in pre-clinical development

The combination of dimethyl dioctadecyl ammonium bromide (DDA) and the synthetic cord factor trehalose dibehenate (TDB) with Ag85B-ESAT-6 (H1 fusion protein) has been found to promote strong protective immune responses against Mycobacterium tuberculosis. The development of a vaccine formulation that is able to facilitate the requirements of sterility, stability and generation of a vaccine product with acceptable composition, shelf-life and safety profile may necessitate selected alterations in vaccine formulation. This study describes the implementation of a sterilisation protocol and the use of selected lyoprotective agents in order to fulfil these requirements. Concomitantly, close analysis of any alteration in physico-chemical characteristics and parameters of immunogenicity have been examined for this promising DDA liposome-based tuberculosis vaccine. The study addresses the extensive guidelines on parameters for non-clinical assessment, suitable for liposomal vaccines and other vaccine delivery systems issued by the World Health Organisation (WHO) and the European Medicines Agency (EMEA). Physical and chemical stability was observed following alteration in formulations to include novel cryoprotectants and radiation sterilisation. Immunogenicity was maintained following these alterations and even improved by modification with lysine as the cryoprotective agent for sterilised formulations. Taken together, these results outline the successful alteration to a liposomal vaccine, representing improved formulations by rational modification, whilst maintaining biological activity.

Towards an improved ocular drug delivery system

The ultimate aim of this project was to design new biomaterials which will improve the efficiency of ocular drug delivery systems. Initially, it was necessary to review the information available on the nature of the tear fluid and its relationship with the eye. An extensive survey of the relevant literature was made. There is a common belief in the literature that the ocular glycoprotein, mucin, plays an important role in tear film stability, and furthermore, that it exists as an adherent layer covering the corneal surface. If this belief is true, the muco-corneal interaction provides the ideal basis for the development of sustained release drug delivery. Preliminary investigations were made to assess the ability of mucin to adhere to polymer surfaces. The intention was to develop a synthetic model which would mimic the supposed corneal/mucin interaction. Analytical procedures included the use of microscopy (phase contrast and fluorescence), fluorophotometry, and mucin-staining dyes. Additionally, the physical properties of tears and tear models were assessed under conditions mimicking those of the preocular environment, using rheological and tensiometric techniques. The wetting abilities of these tear models and opthalmic formulations were also investigated. Tissue culture techniques were employed to enable the surface properties of the corneal surface to be studied by means of cultured corneal cells. The results of these investigations enabled the calculation of interfacial and surface characteristics of tears, tear models, and the corneal surface. Over all, this work cast doubt on the accepted relationship of mucin with the cornea. A corneal surface model was designed, on the basis of the information obtained during this project, which would possess similar surface chemical properties (i.e. would be biomimetic) to the more complex original. This model, together with the information gained on the properties of tears and solutions intended for ocular instillation, could be valuable in the design of drug formulations with enhanced ocular retention times. Furthermore, the model itself may form the basis for the design of an effective drug-carrier.

Subunit vaccines distearoylphosphatidylcholine-based liposomes entrapping antigen offer a neutral alternative to dimethyldioctadecylammonium-based cationic liposomes as an adjuvant delivery system

The adjuvanticity of liposomes can be directed through formulation to develop a safe yet potent vaccine candidate. With the addition of the cationic lipid dimethyldioctadecylammonium bromide (DDA) to stable neutral distearoylphosphatidylcholine (DSPC):cholesterol (Chol) liposomes, vesicle size reduces while protein entrapment increases. The addition of the immunomodulator, trehalose 6,6-dibehenate (TDB) to either the neutral or cationic liposomes did not affect the physiochemical characteristics of these liposome vesicles. However, the protective immune response, as indicated by the amount of IFN-? production, increases considerably when TDB is present. High levels of IFN-? were observed for cationic liposomes; however, there was a marked reduction in IFN-? release over time. Conversely, for neutral liposomes containing TDB, although the initial amount of IFN-? was slightly lower than the cationic equivalent, the overall protective immune responses of these neutral liposomes were effectively maintained over time, generating good levels of protection. To that end, although the addition of DSPC and Chol reduced the protective immunity of DDA:TDB liposomes, relatively high protection was observed for the neutral counterpart, DSPC:Chol:TDB, which may offer an effective neutral alternative to the DDA:TDB cationic system, especially for the delivery of either zwitterionic (neutral) or cationic molecules or antigens.

Polycaprolactone fibres as a potential delivery system for collagen to support bone regeneration

Poly(e-caprolactone) (PCL) is biocompatible, non-immunogenic and non-toxic, and slowly degrades, allowing sufficient time for tissue regeneration. PCL has the potential for application in bone and cartilage repair as it may provide the essential structure required for bone regeneration, however, an ideal scaffold system is still undeveloped. PCL fibres were prepared using the gravity spinning technique, in which collagen was either incorporated into or coated onto the 'as-spun' fibres, in order to develop novel biodegradable polymer fibres which will effectively deliver collagen and support the attachment and proliferation of human osteoblast (HOB) cells for bone regeneration. The physical and mechanical characteristics and cell fibre interactions were analysed. The PCL fibres were found to be highly flexible and inclusion of collagen did not alter the mechanical properties of PCL fibres. Overall, HOB cells were shown to effectively adhere and proliferate on all fibre platforms tested, although proliferation rates were enhanced by surface coating PCL fibres with collagen compared to PCL fibres incorporating collagen and PCL-only fibres. These findings highlight the potential of using gravity spun PCL fibres as a delivery platform for extracellular matrix proteins, such as collagen, in order to enhance cell adherence and proliferation for tissue repair.

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.

Drug delivery strategies for the treatment of Helicobacter pylori infections

Helicobacter pylori is one of the most common pathogenic bacterial infections, colonising an estimated half of all humans. It is associated with the development of serious gastroduodenal disease - including peptic ulcers, gastric lymphoma and acute chronic gastritis. Current recommended regimes are not wholly effective and patient compliance, side-effects and bacterial resistance can be problematic. Drug delivery to the site of residence in the gastric mucosa may improve efficacy of the current and emerging treatments. Gastric retentive delivery systems potentially allow increased penetration of the mucus layer and therefore increased drug concentration at the site of action. Proposed gastric retentive systems for the enhancement of local drug delivery include floating systems, expandable or swellable systems and bioadhesive systems. Generally, problems with these formulations are lack of specificity, limited to mucus turnover or failure to persist in the stomach. Gastric mucoadhesive systems are hailed as a promising technology to address this issue, penetrating the mucus layer and prolonging activity at the mucus-epithelial interface. This review appraises gastroretentive delivery strategies specifically with regard to their application as a delivery system to target Helicobacter. As drug-resistant strains emerge, the development of a vaccine to eradicate and prevent reinfection is an attractive proposition. Proposed prophylactic and therapeutic vaccines have been delivered using a number of mucosal routes using viral and non-viral vectors. The delivery form, inclusion of adjuvants, and delivery regime will influence the immune response generated. © 2005 Bentham Science Publishers Ltd.

Reducible disulfide-based non-viral gene delivery systems

The design and synthesis of safe efficient non-viral vectors for gene delivery has attracted significant attention in recent years due primarily to the severe side-effect profile reported with the use of their viral counterparts. Previous experiments have revealed that the strong interaction between the carriers and nucleic acid may well hinder the release of the gene from the complex in the cytosol adversely affecting transfection efficiency. However, incorporating reducible disulfide bonds within the delivery systems themselves which are then cleaved in the glutathione-rich intracellular environment may help in solving this puzzle. This review focuses on recent development of these reducible carriers. The biological rationale and approaches to the synthesis of reducible vectors are discussed in detail. The in vitro and in vivo evaluations of reducible carriers are also summarized and it is evident that they offer a promising approach in non-viral gene delivery system design.

Development and evaluation of a novel intranasal spray for the delivery of amantadine

The aim of this study was to develop and characterize an intranasal delivery system for amantadine hydrochloride (AMT). Optimal formulations consisted of a thermosensitive polymer Pluronic® 127 and either carboxymethyl cellulose or chitosan which demonstrated gel transition at nasal cavity temperatures (34 ± 1°C). Rheologically, the loss tangent (Tan δ) confirmed a 3-stage gelation phenomena at 34 ± 1°C and non-Newtonian behavior. Storage of optimized formulation carboxymethyl cellulose and optimal formulation chitosan at 4°C for 8 weeks resulted in repeatable release profiles at 34°C when sampled, with a Fickian mechanism earlier on but moving toward anomalous transport by week 8. Polymers (Pluronic® 127, carboxymethyl cellulose, and chitosan) demonstrated no significant cellular toxicity to human nasal epithelial cells up to 4 mg/mL and up to 1 mM for AMT (IC50: 4.5 ± 0.05 mM). Optimized formulation carboxymethyl cellulose and optimal formulation chitosan demonstrated slower release across an in vitro human nasal airway model (43%-44% vs 79 ± 4.58% for AMT). Using a human nasal cast model, deposition into the olfactory regions (potential nose-to-brain) was demonstrated on nozzle insertion (5 mm), whereas tilting of the head forward (15°) resulted in greater deposition in the bulk of the nasal cavity.

Gene delivery using cationic liposomes

The development of cationic liposomes for gene delivery has been ongoing for almost 20 years; however, despite extensive efforts to develop a successful therapeutic agent, there has been limited progress towards generating an effective pharmaceutical product. Since the introduction of N-(1-[2,3-dioley-loxy]propyl)-N,N,N-trimethylammonium chloride, an immense number of different cationic lipids have been synthesised and used to formulate cationic liposome - DNA complexes. Structural modification of the cationic lipids and the addition of components within the delivery system that can facilitate the fusion, cellular uptake and targeting of liposome - DNA complexes have all been used in a bid to enhance their transfection efficiency. Unfortunately, the overall impact of these improvements is still nominal, with the vast majority of clinical trials (∼ 85%) continuing to rely on more potent viral delivery of DNA despite their associated toxicity issues. Key characteristics of the most effective cationic liposomes for the delivery of plasmid DNA (from a consensus of the literature) is identified here and the problems of converting these attributes into an effective pharmaceutical product are outlined. © 2006 Informa UK Ltd.

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.

The fabrication of biodegradable nanospheres from novel hydroxalkanoates for the delivery of actives of pharmaceutical and agricultural interest

This work describes the fabrication of nanospheres from a range of novel polyhydroxyalkanoates supplied by Monsanto, St Louis, Missouri, USA for the delivery of selected actives of both pharmaceutical and agricultural interest. Initial evaluation of established microsphere and nanosphere fabrication techniques resulted in the adoption and optimisation of a double sonication solvent evaporation method involving the synperonic surfactant F68. Nanospheres could be consistently generated with this method. Studies on the incorporation and release of the surrogate protein Bovine Serum Albumin V demonstrated that BSA could be loaded with between 10-40% w/w BSA without nanosphere destabilisation. BSA release from nanospheres into Hanks Balanced Salts Solution, pH 7.4, could be monitored for up to 28 days at 37°C. The incorporation and release of the Monsanto actives - the insecticide Admire® ({ 1-[(6-chloro-3-pyridinyl)methyIJ-N-nitro-2-imidazolidinimine}) and the plant growth hormone potassium salt Gibberellic acid (GA3K) from physico-chemically characterised polymer nanospheres was monitored for up to 37 days and 28 days respectively, at both 4°C and 23°C. Release data was subsequently fitted to established kinetic models to elaborate the possible mechanisms of release of actives from the nanospheres. The exposure of unloaded nanospheres to a range of physiological media and rural rainwater has been used to investigate the role polymer biodegradation by enzymatic and chemical means might play in the in vivo release of actives and agricultural applications. The potential environmental biodegradation of Monsanto polymers has been investigated using a composting study (International Standard ISO/FDIS 14855) in which the ultimate aerobic biodegradation of the polymers has been monitored by the analysis of evolved carbon dioxide. These studies demonstrated the potential of the polymers for use in the environment, for example as a pesticide delivery system.

Development and formulation of wax-based transdermal drug delivery systems

Topical and transdermal formulations are promising platforms for the delivery of drugs. A unit dose topical or transdermal drug delivery system that optimises the solubility of drugs within the vehicle provides a novel dosage form for efficacious delivery that also offers a simple manufacture technique is desirable. This study used Witepsol® H15 wax as a abase for the delivery system. One aspect of this project involved determination of the solubility of ibuprofen, flurbiprofen and naproxen in the was using microscopy, Higuchi release kinetics, HyperDSC and mathematical modelling techniques. Correlations between the results obtained via these techniques were noted with additional merits such as provision of valuable information on drug release kinetics and possible interactions between the drug and excipients. A second aspect of this project involved the incorporation of additional excipients: Tween 20 (T), Carbopol®971 (C) and menthol (M) to the wax formulation. On in vitro permeation through porcine skin, the preferred formulations were: ibuprofen (5% w/w) within Witepsol®H15 + 1% w/w T; flurbiprofen (10% w/w) within Witepsol®H15 + 1% w/w T; naproxen (5% w/w) within Witepsol®H15 + 1% w/w T + 1% C and sodium diclofenac (10% w/w) within Witepsol®H15 + 1% w/w T + 1% w/w T + 1% w/w C + 5% w/w M. Unit dose transdermal tablets containing ibuprofen and diclofenac were produced with improved flux compared to marketed products; Voltarol Emugel® demonstrated flux of 1.68x10-3 cm/h compared to 123 x 10-3 cm/h for the optimised product as detailed above; Ibugel Forte® demonstrated a permeation coefficient value of 7.65 x 10-3 cm/h compared to 8.69 x 10-3 cm/h for the optimised product as described above.

Oral delivery strategies for the controlled release of cimetidine

It is advantageous to develop controlled release dosage forms utilising site-specific delivery or gastric retention for those drugs with frequent or high dosing regimes. Cimetidine is a potent and selective H2 -reception antagonist used in the treatment of various gastrointestinal disorders and localisation in the upper gastrointestinal tract could significantly improve the drug absorption. Three strategies were undertaken to prepare controlled release systems for the delivery of cimetidine to the GI tract. Firstly, increasing the contact time of the dosage form with the mucus layer which coats the gastrointestinal tract, may lead to increased gastric residence times. Mucoadhesive microspheres, by forming a gel-like structure in contact with the mucus, should prolong the contact between the delivery system and the mucus layer, and should have the potential for releasing the drug in sustained and controlled manner. Gelatin microspheres were prepared, optimised and characterised for their physicochemical properties. Crosslinking concentration, particle size and cimetidine loading influenced drug release profiles. Particle size was influenced by surfactant concentration and stirring speed. Mucoadheisve polymers such as alginates, chitosans, carbopols and polycarbophil were incorporated into the microspheres using different strategies. The mucoadhesion of the microspheres was determined using in vitro surface adsorption and ex vivo rat intestine models. The surface-modification strategy resulted in highest levels of microsphere adhesion, with chitosan, carbopols and polycarbophil as the most successful candidates for improvement of adhesion, with over 70% of the microspheres retained ex vivo. Specific targeting agent UEA I lectin was conjugated to the surface of gelatin microspheres, which enhanced the adhesion of the microspheres. Alginate raft systems containing antacids have been used extensively in the treatment of gastro-oesophageal disease and protection of the oesophageal mucosa from acid reflux by forming a viscous raft layer on the surface of the stomach content, and could be an effective delivery system for controlled release of cimetidine.

Evaluation of biodegradable polymer microspheres and an anionic dendrimer for the potential delivery of antisense oligonucleotides

Antisense oligonucleotides (AODNs) can selectively inhibit individual gene expression by binding specifically to rnRNA. The over-expression of the epidermal growth factor receptor (EGFR) has been observed in human breast and glioblastoma tumours and therefore AODNs designed to target the EGFR would be a logical approach to treat such tumours. However, poor pharmacokinetic/pharmacodynamic and cellular uptake properties of AODNs have limited their potential to become successful therapeutic agents. Biodegradable polymeric poly (lactide-co-glycolide) (P(LA-GA)) and dendrimer delivery systems may allow us to overcome these problems. The use of combination therapy of AODNs and cytotoxic agents such as 5-fluorouracil (5-FU) in biodegradable polymeric formulations may further improve therapeutic efficacy. AODN and 5-FU were either co-entrapped in a single microsphere formulation or individually entrapped in two separate microsphere formulations (double emulsion method) and release profiles determined in vitro. The release rates (biphasic) of the two agents were significantly slower when co-entrapped as a single microsphere formulation compared to those obtained with the separate formulations. Sustained release over 35 days was observed in both types of formulation. Naked and microsphere-loaded AODN and 5-FU (in separate formulations) were tested on an A431 vulval carcinoma cell line. Combining naked or encapsulated drugs produced a greater reduction in viable cell number as compared with either agent alone. However, controls and Western blotting indicated that non-sequence specific cytotoxic effects were responsible for the differences in viable cell number. The uptake properties of an anionic dendrimer based on a pentaerythritol structure covalently linked to AODNs (targeting the EGFR) have been characterised. The cellular uptake of AODN linked to the dendrimer was up to 3.5-fold higher in A431 cells as compared to naked AODN. Mechanistic studies suggested that receptor-mediated and adsorptive (binding protein-mediated) endocytosis were the predominant uptake mechanisms for the dendrimer-AODN. RNase H cleavage assay suggested that the dendrimer-AODN was able to bind and cleave the target site. A reduction of 20%, 28% and 45% in EGFR expression was observed with 0.05μM, 0.1μM and 0.5μM dendrimer-AODN treatments respectively with a reduction in viable cell number. These results indicated that the dendrimer delivery system may reduce viable cell number by an antisense specific mechanism.