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Recombinant proteins are ever becoming more applicable to the treatment and diagnostics of a wide range of disease states or conditions in modern medicine. The delivery of proteins is hampered by their inherently low bioavailability due to instability to hydrolysis and enzymatic degradation. Formulation challenges include the targeted and controlled release of protein at the site of administration while maintaining protein bioactivity towards its potential target. The individual characteristics of the protein in question will play a large part in the formulation of any carrier vehicle and techniques that may be successful in the case of one biopharmaceutical entity may not apply to any other biopharmaceutical entities. The Poly(ethylene glycols) or PEGs are probably the most successful polymer to date at improving the pharmacokinetics of a wide range of protein molecules and continued interest in improving their delivery profile is a hot topic in research including the elimination of possible anti-PEG IgM stimulation and in situ growth of PEG side chains on recombinant proteins. PLGA and PLA microspheres also have licenced proprietary preparations containing therapeutic proteins but are limited by hydrophobicity and harsh degradation products which may damage protein function, various strategies including the addition of magnesium hydroxide, co-polymerisation and coating with silk fibroin are discussed as methods of addressing these drawbacks. Other strategies reviewed include the poloxamers and their potential influence on improved drug targeting, pH responsive hydrogels for potential delivery of insulin in response to rising glucose concentrations, elastomeric devices which have the potential for sequential protein delivery, the influence of polymer surface topography on targeting and release of protein from a polymeric carrier, cyclodextrins in protein delivery and silk fibroin as a potential carrier for sustained release preparations. While most novel polymer systems reviewed display interesting characteristics and potential advantages in protein delivery it is obvious that PEG is currently the automatic polymer of choice in formulation design and its FDA approved status makes it perhaps the most attractive polymeric carrier available.
In recent years the rise of the biopharmaceutical industry and the trend towards the development of therapeutic protein molecules over chemically synthesised molecules or New Chemical Entities (NCEs) has not only been attributable to the increased ability of protein molecules to specifically target biological pathways in the host but also to the fact that these therapeutic proteins displayed a financial advantage over NCEs with a shorter time to market. Perhaps the most well characterised protein polymer conjugate to date are the Poly(ethylene glycols) or PEG, and many PEGylated therapeutic proteins are currently approved for use in a wide range of indications from Adagen® for the treatment of Severe Combined Immunodeficiency Disease (PEGylated Adenosine deaminase - FDA approval in 1990) to Cimizia® for the treatment of Rheumatoid Arthritis (PEGylated Anti TNF Fab - FDA approval in 2008) with other indications such as Leukaemia, Hepatitis C, Acromegaly and Age Related Macular Degeneration all with licenced PEGylated protein therapies. Two other well defined polymers with licenced drugs on the market are Poly-Lactic Acid or Poly(Lactic co-Glycolic acid) copolymers (PLA/PLGA) microspheres e.g. Lupron® (PLA encapsulated leuporelin acetate) and hyperglycosilated proteins e.g. Aranesp® (hyperglycosilated darbopoetin ¡). The basic rationale for the inclusion of polymeric carriers is to increase the molecular weight and steric bulkiness of the protein with an inert carrier making it less susceptible to degradation and less immunogenic in vivo therefore increasing its therapeutic efficacy ,by way of increased mean residence time and decreasing its side effect profile which usually involves an immune mediated response to high dose protein even when derived from human sources.. The focus of this literature review will be to present to the reader emerging strategies which build on the above principles of therapeutic protein delivery and how these strategies are designed to optimise the release of their conjugated protein molecule be it in a site specific, stimuli responsive or otherwise improved fashion to the technologies currently available under three main headings Poly(ethylene glycols), PLA/PLGA microspheres and Other Polymeric Devices and Strategies.
In general, PEGylation reduces the plasma clearance rate by reducing the metabolic degradation and receptor-mediated uptake of the protein from the systemic circulation. PEGylation also improves the safety profile of the protein by shielding antigenic and immunogenic epitopes. The inert nature of the molecule and the ability to synthesise it with a molecular weight of narrow polydispersity and also to have one functional group made it an ideal choice for protein modification without crosslinking. This has been a constantly developing field since its inception in the 1970s and despite extensive clinical research and the development of clinically useful PEGylated products a greater understanding of its function can yet be achieved. One recent article calls for more in depth studies not directed at the benefit of PEGylation but at elucidating how different methods of PEGylation, namely; new polymer structures and non-covalent methods of PEGylation will improve therapeutic efficacy.
A technique which appears to be of particular interest is the in-situ growth of a PEGmethacrylate (PEGMA) polymer from a recombinant human growth hormone which was functionalised with a polymerisation initiating site. This polymer was grown under aqueous conditions and displayed increased activity when compared to the native protein. The good biocompatibility profile of PEGMA it also displayed increased stability making it a promising candidate for therapeutic applications. The author also highlights the potential benefits which would be seen from the reduced need for aggressive purification methods because of mild conditions of bioconjugation and easier and more versatile ability to conjugate low molecular weight initiators as compared to site-selective large bulky polymer conjugation. A similar study by Gao and Liu et al. demonstrated the first in situ growth of a PEG like polymer from the C terminus of a protein with one method detailed fast and cheap with obvious advantages to industrial applications and another method postulated to be of benefit to the pharmacological profiles of proteins such as interferon ¡, glucagon-like peptide-1, exendin-4 and parathyroid hormone.
In general the conjugation of protein molecules with PEG appears to be clinically beneficial and non-toxic particularly when administered via the parenteral route, there are however reports of increased clearance of PEGylated proteins due to the production of an anti-PEG IgM particularly in the case of liposomal formulations dubbed the ABC (accelerated blood clearance) phenomenon resulting in therapeutic failure. This has been recently attributed to the presence of specific CpG motifs in the PEGylated lipoflex as being causative in the production of anti-PEG IgM due to the lack of anti-PEG IgM production on removal of these motifs.
PLA and PLGA microspheres are have been used to deliver therapeutic proteins highly successfully in a range of disease states. The key factors for effective delivery of protein are desired protein release profile, micro-particle size, micro-pore size, micro-pore coalescence rate, polymer surface area, protein diffusivity, mean molecular weight, non-specific protein adsorption to polymer surface, protein loading, encapsulation efficiency, and bioactivity of the released protein. PLA and PLGA as protein carriers are limited by their hydrophobicity and the acidic nature of degradation products. Both microspheres degrade via an acid catalysed hydrolysis producing carboxylic acids and therefore the acid sensitivity of the selected protein is an important consideration, the addition of magnesium hydroxide has been shown to neutralise the acids leading to improved release kinetics and stability of encapsulated proteins. These issues with stability and release have particularly been highlighted with the mucosal delivery of hepatitis B surface antigen vaccine (HBsAg). PLA encapsulated HBsAg was compared to a tri-block co-polymer (PEG-PLA-PEG) via the nasal mucosa in mice and the conventional aluminium based vaccines received subcutaneously including a booster shot. The findings indicated that the PEG-PLA-PEG co-polymer not only had superior characteristics to the PLA microspheres but yielded a better immune response than was seen with the conventional vaccine (plus booster shot). This paper highlights the limitations of PLA/PLGA microspheres and the harsh environment created during the prolonged release of proteins and its detrimental effect on protein activity. The PEG-PLA-PEG co-polymer also displayed a higher encapsulation efficiency and smaller microparticle size, the author goes on to highlight the potential of these systems as enhanced mucosal vaccine delivery devices and as a potential single shot vaccine for hepatitis B.
Porous systems in PLA/PLGA microencapsulated proteins are of considerable interest currently these are synthesised using a variety of techniques. Newer techniques such as the creation of "open/closed" systems where pores are fabricated, filled with protein in aqueous phase and then re-closed in ethanol vapour to improve drug loading have showed improved release profiles of proteins. It has been proposed that pore opening and closing during degradation events and even during initial incubation of a PLGA polymeric device correlates well with the release rate of biomacromolecules from the microspheres and that temperature and osmotic events are critical in rates of pore opening and closing. This effect however has not been widely investigated with mathematical modelling seeming to be the only investigations performed with benefits thought to be related to the estimation of booster times for single-shot vaccine devices.
Coating the surface of PLGA with various excipients has been used as a strategy to improve the pharmacokinetic profiles of protein release, one such coating of interest is a 1 micron thick coating of a silk fibroin based polymer which has been shown to retard the degradation of the polymer and slow the release of protein from a PLGA microsphere when compared to a traditional PVA [Poly(vinyl alcohol)] coating used in tissue engineering scaffolds offering a potential therapeutic advantage.
Other Polymeric Devices and Strategies
Pluronic block copolymers or poloxamers show considerable potential in the delivery of protein drugs particularly because of their ability to incorporate into membranes. Poloxamer 188 is present in Rebif® (interferon ¢-1-¡) used in the treatment of progressive or relapsing multiple sclerosis. They are triblock co-polymers with hydrophilic, Poly(ethylene oxide), and hydrophobic Poly(propylene oxide) arranged in an A-B-A structure, that is PEO-PPO-PEO and have been cited as promising polymeric carriers for the potential delivery of proteins across the blood brain barrier. These polymers have also been shown to be beneficial in the sensitisation of multi drug resistant tumours to various anticancer agents, enhance drug transport across the intestinal membrane and cause transcriptional activation of gene expression making it one of the most potent drug targeting systems available.
Stimuli Responsive Polymers
Probably one of the most widely studied proteins for stimuli responsive release is insulin, with obvious advantages to therapeutic efficacy if insulin could be released from a device in response to rising glucose levels in the blood. The most promising candidate reviewed is based on enzymatic oxidation of glucose by glucose oxidase yielding gluconic acid. The insulin is contained in a pH responsive hydrogel with glucose oxidase immobilised on its surface. The polymer will swell in response to a lowering in pH (exposure to gluconic acid) becoming more porous thereby releasing insulin in response to higher glucose concentrations. There appears to be difficulty around quantifying an appropriate release of insulin from the polymer especially when attempting to create a hydrogel that will release a reproducible amount of insulin each time even in the presence of declining insulin concentrations this has been highlighted as an area with potential for the development of a nanochip or device to control the amount of released insulin with the responsive polymer triggering the device.
A photo-cross-linked elastomer with cylindrical geometry polymerised from an acrylated star-Poly(¥-caprolactone-co-d,l-lactide) macromonomer investigated for the release of VEGF, IL-2 and IFN-€ displaying zero order release for 60%-80% of the release profile with zero order kinetics with minimal burst. However, in the case of VEGF a significant portion of the loaded protein was rendered inactive leaving around 57% of the original amount as a bioactive protein. This has been cited in later reviews as disadvantageous and that delivery of VEGF at therapeutic concentrations without encountering toxic concentrations of the delivery vehicle has been difficult to realise. More recently the capability of combined and sequential release of proteins from elastomers has been explored. In this study the authors used a Poly(trimethylene carbonate) based photo-cross-linked elastomer, which has the major advantage of a very slow degradation and therefore a lower production of species which may degrade the bioactivity of the embedded protein and released VEGF165 and HGF were highly bioactive over the release period. The same authors in a further study demonstrate angiogenesis following injection of a low viscosity Poly(trimethylene carbonate) elastomer containing VEGF highlighting this formulations potential for the localised delivery of acid sensitive proteins.
Influence of Surface Topography
This is an important factor in protein adsorption with knock on effects on release from polymeric substances and it has been shown that it is not only surface chemistry but also topography which will influence protein adsorption behaviour. This has been of particular interest in the interaction of polymer surfaces with osteoblast-like cells and for its effects on platelet adhesion. In the case of platelets it has been shown that certain nanoscale topographies can result in reduced platelet adhesion and increased fibrinogen adsorption which would be of particular benefit in blood contacting medical devices. The up-regulation of osteoblast like cells activity may be useful in tissue repair and bone re-modelling.
The inclusion of cyclodextrin molecules in the polymeric architecture of supramolecular hydrogels is an area of considerable research in protein drug delivery. Although these materials show some promising characteristics toxicity and biodegradability of these polymers however is an issue and extensive studies would need to be carried out on any potential devices including these molecules in the polymeric structure. ¡-cyclodextrin has been shown to aid gel formation of triblock co-polymers resulting in the formation of a thixotrophic and reversible material with potential for injectable drug delivery.
Silk fibroin matrices have been demonstrated to successfully deliver protein drugs and preserve their potency, it is a slowly biodegradable, biocompatible polymer with excellent mechanical properties and can be manufactured using all-aqueous techniques making it particularly useful for proteins sensitive to harsh manufacturing techniques. The problem with immunogenicity of constituents of the silk matrix namely the sericin proteins can now be easily eliminated by their selective removal while maintaining favourable polymer properties making them promising candidates in the potential delivery of therapeutic proteins.
While investigations of several novel polymeric substances were carried out in this review it is difficult to find a polymer with such wide ranging applications as PEG. Its favourable toxicity profile ease of handling and formulation and the increasing pace of research into new methods of conjugation and structural orientation in conjugation with proteins will probably ensure it remains a market leader in polymeric protein delivery into the future. PLGA and PLA microspheres also look set to be applicable in therapeutic settings possibly with modified characteristics or only in the delivery of acid stable proteins. Poloxamers present a very interesting advantage particularly in the delivery of drugs to the brain and may have potential applications in conditions such as Alzheimer's disease, their influence on intracellular signalling events although heralded as advantageous would have to be examined in detail in specific disease states. Stimuli responsive polymers, although an interesting concept in protein delivery look set to be incorporated into the operation of bioresponsive nanotechnology which may be able to control the release of protein in a more reproducible fashion than relying on the polymeric architecture. Photo-cross-linkable elastomeric devices appear to have significant potential in the delivery of acid sensitive proteins such as VEGF but toxicity issues should not be ignored and further studies in vivo would need to be carried out to rule out possible toxicity at the concentrations needed in these devices. Cyclodextrins also carry toxicity issues which need to be further explored. Details of the influence of polymer topography site specific release appears to be an interesting area with possible applications in targeted systems. Silk from the common silk worm also appears to have ideal characteristics for a range of protein molecules however as it is a naturally occurring compound this may lead to intellectual property rights issues.