Patent Application
20220023206
The present invention relates to hydrogel compositions that are useful for therapeutic application in the eye. The present invention further relates to method for preparing these hydrogel compositions and their use for therapeutic applications, especially in the inhibition of ocular scarring.
In 2018 the WHO reported corneal opacity to be a leading cause of blindness globally. Corneal infection caused by conditions such as microbial keratitis result in disorganization of collagen and extracellular matrix to form a scar. Treatment often requires resolution of the infection with steroids and antibiotics. Unresolved corneal opacities may lead to the need of a surgical corneal transplant. Although attempts are made to control corneal scarring through aggressive control of infection/inflammation, there has been little success for the use of potent anti-scarring treatments. One limitation of current eye drop treatments is the low viscosity or weak gelling materials, which do not significantly enhance the retention time of the drug.
Corneal opacity is a leading cause of sight impairment worldwide with an estimated 27.9 million people globally being bilaterally or unilaterally affected[1]. Such opacity is typically derived from alteration of the complex, optically clear, corneal tissue structure, vital for refraction of light onto the retina, and subsequent neuro-visual processing. Commonly, corneal scarring results from ocular infections from a range of pathogens including bacteria, parasites, fungi, viruses and protozoa. In the developed world, devastating corneal infections are most commonly associated with prolonged contact lens wear and/or poor lens hygiene[2-4]; with Pseudomonas aeruginosa being a prominent causative organism. In cases of gram-negative infections, e.g. Pseudomonas, the structural integrity of the cornea becomes compromised through multiple virulence factors, whereby the microbes invade epithelial cells, resulting in activation of numerous inflammatory pathways. Subsequent production of cytokines from epithelial, stromal and intraepithelial inflammatory cells, neovascularization, cellular alterations and degradative stromal processes[5] lead to dysregulated tissue remodeling and disruption of the intricately arranged collagen fibrils[6] leading to the loss of optical transparency, impairment of light refraction and loss of sight.
Following injury that breaches the epithelium and Bowman's layer involving the corneal stroma, an orchestrated wound healing response involving corneal epithelium, stroma and nerves, lacrimal glands and tear film occurs to restore corneal structure and function and maintain the ocular integrity[7]. As part of the corneal wound healing response, the epithelium starts to regenerate in response to stem cell proliferation from the limbal niche almost immediately after the epithelium is injured[8] and keratocytes (transparent cells that function to maintain collagen and ECM turnover) proximal to the wounding site undergo apoptosis (induced by cytokines released from damaged epithelial cells). Proliferation and migration of residual keratocytes peripheral to the injury-site can be detected 12 to 24 hours after injury[9]. The keratocytic response includes production of proteoglycans and synthesis of collagen fibers. These fibers are phenotypically larger than the naive fiber and, due to the water-retention capacity of the proteoglycans, do not assume an ordered regular architecture leading to corneal opacity. The movement of bone-marrow derived precursor cells and circulatory mediators from the from the limbal region activate, transform and differentiate a subset of keratocytes to cell types with fibroblast and myofibroblast characteristics via TGFβ and PDGF activation[10, 9, 11]. TGFβ released from epithelial cells accesses stromal cells through the damaged Bowman's layer to initiate myofibroblast differentiation[12,13]. Myofibroblasts release cytokines that further attract inflammatory cells and ECM deposition (e.g. collagen, fibronectin) to facilitate the fibroblast migration as part of the stromal remodeling phase[14]. Reparative processes lead to improved collagen fiber orientation, contraction of proteoglycan, apoptosis of the stromal myofibroblasts and repopulation of keratocytes enable structural and functional recovery, but with residual stromal opacity. If this is present in the visual axis, sight is impaired. If the corneal epithelial barrier is not restored, stromal metabolism becomes dysregulated leading to keratolysis, degradation of corneal tissue, further disorganization of corneal fibril arrangements, and eventual corneal perforation. This is partly due to the sustained release of TGFβ leading to persistent myofibroblasts preventing stromal re-population of keratocytes[15, 16] Unlike other tissues (e.g. skin), where a persistent ulcer or scar might be tolerated, in the cornea this can have devastating functional effects of permanent corneal scarring with visual disability or loss of eye.
Currently, the standard of clinical care for patients infected with bacterial keratitis focuses initially on sterilizing the infected eye, by eye drop administration of intensive broad-spectrum antibiotics, followed by the addition of topical corticosteroids to reduce inflammation[8, 9]. This is followed by strategies to limit scar formation ranging from intensive lubrication (to reduce biomechanical trauma of the eyelids abrading the wound bed during blinking), to the use of systemic pharmacological agents (sub-antimicrobial dose of tetracyclines for matrix metallo-proteinase inhibition[10]) or supplements (vitamin C used as anti-oxidants and free radical scavengers[11]) in an attempt to promote tissue remodelling. Unfortunately, although effective at sterilizing the eye, the patient is often left with a high degree of corneal hazing which, if it compromises the visual axis, causes loss of visual acuity. Surgical interventions to treat unresponsive and large corneal defects include either application of amniotic membrane as a biologically active bandage releasing anti-inflammatory and anti-fibrotic factors to enhance re-epithelialization and wound healing during acute injury[12-14], or in established cases of visually significant central corneal scars, excision of the scarred tissue and replacement with donor cornea. Reproducibility and repeatability of the clinical outcomes of amnion grafting and corneal transplantation are fraught with risks of failure and rejection[15-18].
If the fibrotic response to injury and infection could be attenuated, it will maximize optical clarity and preserve visual function, and may remove the need for surgical intervention and transplantation. Such an innovation would have the potential to prevent permanent sight-loss in many millions of individuals. As discussed above, fibrosis is driven by raised levels of TGβ-1 activity and so it may be possible to prevent fibrosis using a TGFβ antagonist. Decorin is a naturally occurring pleiotropic anti-fibrotic small leucine-rich proteoglycan that is naturally present at high levels bound to collagen in the corneal stroma22 and which, when released, tightly regulates TGFβ activity by binding the growth factor and sequestering it within the ECM[19]. Decorin regulates cell proliferation, survival and differentiation by modulating numerous growth factors[20-24] including TGF-β as well as directly interfering with collagen fibrillogenesis[25-28].
Decorin is responsible for regulating collagen fibril spacing and ECM to enable corneal transparency and has previously been shown to inhibit scar formation and neovascularization in the cornea[35]. Mutations in decorin are associated with corneal opacities and visual abnormalities associated with congenital stromal dystrophy[40]. Hyperactivity of TGβ3 in corneal fibrosis may overcome the ability of endogenous decorin to maintain homeostasis and there is good evidence that over-expressing decorin in other tissues is able to reduce levels of fibrosis in vivo[41-43].
Human recombinant (hr)Decorin is now available in GMP form and it has been shown is functional in minimizing fibrosis in the brain and spinal cord[29-31]. To date, there has been no reported efficacy of soluble decorin being applied to the surface of the eye for treatment in vivo. One of the possible reasons for this maybe the relatively rapid clearance of eye drops (in the range of minutes[32, 33]), owing to their relatively low viscosities, from the surface of the cornea at early time points, meaning that any efficacy of decorin would be limited.
Previous reports (in particular WO 2017/013414) have suggested that fluid gel hydrogels may be used to deliver the anti-fibrotic agent decorin to the eye in order to reduce scarring. These reports have highlighted the presence of collagen in such compositions as being of vital importance in their effectiveness.
The role attributed to collagen in such compositions has been two-fold: both improving the ability of decorin to binding fibrotic growth factors (such as TGF-β) and contributing to the clearance of these factors once bound.
In a first aspect of the invention there is provided a shear-thinning ocular hydrogel composition suitable for application to the eye, the composition comprising:
In a further aspect of the invention there is provided an ocular hydrogel composition suitable for application to the eye, wherein the ocular hydrogel composition comprises, consist essentially of, or consists of, a shear-thinning hydrogel composition comprising decorin as defined herein.
In a further aspect the present invention provides a method of making a shear-thinning ocular hydrogel composition as defined herein, the method comprising the steps of:
Suitably, the decorin is added to the mixture in the form of an aqueous decorin solution.
In a further aspect the present invention provides a method of making a shear-thinning ocular hydrogel composition as defined herein, the method comprising the steps of:
In a further aspect the present invention provides a shear-thinning ocular hydrogel composition obtainable by, obtained by, or directly obtained by, any of the preparatory methods defined herein.
In a further aspect the present invention provides a shear-thinning ocular hydrogel composition as defined herein for use in therapy.
In a further aspect the present invention provides a shear-thinning ocular hydrogel composition as defined herein for ocular administration.
In a further aspect the present invention provides a shear-thinning ocular hydrogel composition as defined herein for use in the inhibition of scarring.
In a further aspect the present invention provides a shear-thinning ocular hydrogel composition as defined herein for use in the treatment of microbial keratitis.
In a further aspect, the invention provides an ocular composition in accordance with the invention for use as a medicament. Examples of suitable medical uses of the ocular compositions of the invention are described further below. Suitably, the compositions of the invention may be used as medicaments for administration to a surface of the eye.
In a suitable embodiment of the invention, a composition in accordance with the invention is for use in the inhibition of scarring in the eye. Suitably the compositions of the invention may be used as medicaments for inhibition of scarring associated with keratitis, such as microbial keratitis. Suitably the compositions of the invention may be used in the treatment of microbial keratitis to inhibit scarring.
The ocular hydrogel compositions of the invention comprise the anti-fibrotic ECM molecule decorin. An ocular hydrogel composition in accordance with any of the aspects described herein may suitably contain no other biologically-derived agents, in particular no other biologically-derived agents from human or other animal sources. Suitably, an ocular hydrogel composition in accordance with the invention may comprise a polysaccharide microgel-forming polymer, but no protein components capable of gel formation. For example, compositions of the invention may not comprise a protein other than decorin.
In particular, an ocular hydrogel composition of the invention may contain no ECM components other than decorin. Thus an ocular hydrogel composition of the invention may not comprise collagen or fibrin. Indeed, the current invention is based upon the inventors' surprising finding that anti-scarring ocular hydrogel compositions comprising the anti-fibrotic agent decorin can be improved by the exclusion of further ECM components, and particularly from the absence of collagen and fibrin from such compositions. This is in direct contrast to the reports of the prior art, which have indicated that collagen, and optionally fibrin, play vital roles in the ability of these compositions to inhibit scarring.
As set out in WO 2017/013414, the presence of collagen in hydrogel compositions comprising decorin present in such compositions plays a pivotal role in the presentation of decorin in a manner in which decorin's antagonism of TGF-β is optimised.
Furthermore, it is suggested in WO 2017/013414 that when bound to the decorin-collagen complex, TGF-β and other bound factors are more effectively sequestered so that they cannot contribute to fibrosis, inflammation, or angiogenesis. The sequestered factors are then removed from the eye surface as the fluid hydrogel is slowly blinked away.
In view of the above, it will be appreciated that the skilled person would not be motivated to consider the use of ocular hydrogel compositions lacking collagen/fibrin, since to do so would be understood to lose many of the mechanisms by which the compositions of the prior art are stated to achieve their therapeutic activity. Failure to incorporate collagen (or fibrin) would prevent the optimal presentation of decorin for anti-fibrotic activity, negate the ability of a decorin/collagen combination to absorb and remove TGF-β more effectively than decorin alone, and prevent sequestration of fibrotic growth factors by this combination that leads to their subsequent clearance from the eye.
However, against expectations, the inventors have found that use of an ocular hydrogel composition comprising decorin, but lacking collagen or fibrin (or indeed any other ECM), is highly effective in inhibiting scarring of the cornea, such as that associated with microbial keratitis. In fact, the use of shear-thinning ocular hydrogel compositions from which additional ECM components (such as collagen and/or fibrin) are absent offers a number of advantages that are not available in respect of the compositions of the prior art.
These advantages arise with respect to the biological effects of the ocular hydrogel compositions of the invention, their material properties, and the ways in they are manufactured.
As shown in the results set out in the Examples section, the inventors have demonstrated that ocular hydrogel compositions of the invention that comprise decorin, but do not contain collagen or fibrin, are able to effectively inhibit scarring associated with microbial keratitis. Furthermore, the lack of ECM constituents able to interact with and “present” decorin, rather than hindering therapeutic effectiveness, actually confers advantages on the compositions of the invention.
Many ECM components, including collagen and fibrin, incorporate motifs that allow them to bind to other biologically active molecules. It is this property that underpins prior suggestions regarding the use of collagen within compositions in order to present decorin in its native, and therefore more biologically active, context. The favoured microgel particle-forming polymers of the present invention, such as gellan, lack motifs of this sort. Accordingly, it will be recognised they are not able to function in the manner ascribed to collagen and/or fibrin in the prior art, by binding to decorin and maintaining this agent in the conformation in which it is found in vivo.
Binding motifs on ECM components also have important roles in binding to cellular or soluble biological effector molecules. Such molecules provide signals or other biological cues to cells within the host, and the disruption of their signalling may influence the host's response. While some of the alterations of the biological pathways (such as the binding and inhibition of fibrotic growth factors by decorin) have a beneficial therapeutic effect, this is not always the case. Other biological factors can have adverse impacts, causing sensitisation or inflammation at sites where they are provided. By excluding collagen and/or fibrin (or indeed ECM components other than decorin), the ocular hydrogel compositions of the invention are not subject to such undesirable biological effects. Thus, where further biologically-derived agents are not found in the ocular hydrogel compositions of the invention the capacity for adverse responses to such agents on the part of recipients of the compositions is reduced. The use of polysaccharide microgel-forming polymers (and the absence of protein gel-forming polymers) is a suitable approach by which unwanted binding motifs may be excluded from the ocular hydrogel compositions of the invention.
ECM components, such as collagen and/or fibrin, are examples of “biologically-derived” agents, biomaterials that are typically obtained by extraction from naturally occurring sources. These sources, which may be human or other animal, provide naturally occurring proteins which have undergone “correct” processing to their biologically relevant forms (a result that is difficult to achieve by recombinant approaches). However, biologically-derived agents can be subject to significant variation in respect of the sources from which they are derived. These can be “inter-source” variations (e.g. differences between products obtained from different individuals) or “intra-source” variations (e.g. differences in products obtained from the same individual at different times). Intra-source variations may be exacerbated by factors such as health status or medication. Accordingly, a further advantage conferred by the ocular hydrogel compositions of the invention, as compared to those of the prior art, is that, by utilising gel materials (such as gellan) that are not obtained by extraction from human or animal sources they avoid such variation in their properties.
The properties of the fluid gels employed in the compositions of the invention allow for the retention of biologically active decorin at the surface of the cornea, thereby improving the availability of the decorin, and hence its capacity to inhibit scar formation. This is achieved without the need for collagen associated or complexed with the decorin. Instead, these benefits arise as a result of the material properties of the shear-thinning ocular hydrogel compositions.
The properties of the fluid gels employed in the compositions of the invention are such that the decorin is held within the ocular hydrogel composition at the eye's surface. The hydrogel provides a protective layer over damaged sites, such as infections associated with microbial keratitis, contributing to the development of a therapeutic healing milieu.
However, the shear-thinning ocular hydrogel compositions of the invention do not only contribute to the inhibition of scarring through protection of the damaged area. The inventors have found that the material properties of the shear-thinning ocular hydrogel are such that a semi-solid to liquid transition occurs when they are exposed to shear forces consistent with those generated by blinking (i.e. the forces that result from the interaction of the compositions with a recipient's eyelids during blinking). This liquefaction is believed to cause pulsatile release of anti-fibrotic decorin with each blink. After blinking the composition returns to its semi-solid form, effectively storing the remaining decorin in a depot. The regular and controlled release of decorin in this manner establishes highly favourable conditions in which scar-free healing of the ocular surface is able to occur.
It will be appreciated that the shear-thinning hydrogels used in the ocular compositions of the present invention, rely upon an ability to undergo repeated transitions between liquid and semi-solid transitions in order to achieve this effect. Such fluid gels may be termed “self-healing”. However, the gelation mechanism for fibrin is irreversible. Accordingly, the incorporation of fibrin in embodiments of the gels described in the prior art results in compositions that once “broken” are unable to “heal”.
Collagen (another ECM component disclosed as a “carrier” for decorin in the prior) art is also unsuitable for the formation of fluid gels. In this case, the mixing required for formation of such gels causes collagen to fail to gel appropriately during the manufacturing process.
Thus, prior art gels based on collagen and/or fibrin lack the ability to generate regular “pulses” of decorin during their residency in the eye, and so are not able to confer the benefits provided by the ocular hydrogel compositions of the invention.
As mentioned above, the ocular hydrogel compositions of the present invention also offer benefits in terms of the way in which they are manufactured that are not provided for by the compositions of the prior art. The shear-thinning ocular hydrogel compositions of the invention offer benefits on the basis of their reproducibility of manufacture, their ease of manufacture, and the supply chain involved in manufacturing and distributing the product.
Reproducibility of manufacture is of vital importance in compositions for medical use. The ability to achieve a consistent product with little variation between batches is essential in order to allow effective prescribing. As explained further above, the material properties of the shear-thinning ocular hydrogel compositions of the invention are responsible for the manner in which they release therapeutic decorin to the site where scarring is to be inhibited. Thus, it is necessary to be able to manufacture compositions with reproducible material properties, in order to be able to provide compositions that achieve dosing that is consistent between batches.
The incorporation of biologically-derived materials, such as collagen or fibrin, in the compositions of the prior art introduces a number of significant hurdles to the production of reproducible medicaments. Fibrin must be converted from soluble fibrinogen, and this typically involves the use of the enzyme thrombin. Maintaining a consistent degree of enzymatic activity from one batch to another poses a significant problem. Similar difficulties arise as a result of the capacity for variation between batches of fibrinogen, thrombin's substrate. Variations may lead to spontaneous gelation of fibrin, or at least significant differences between the properties of different batches of the materials produced. These differences may give rise to unacceptable variations in the decorin release profiles of the compositions produced.
The role that the enzymatic conversion of fibrinogen to fibrin plays in the production of fibrin gels also poses difficulties in manufacture. Producing compositions of uniform texture and rheological properties proves problematic, as processing in the manner normally employed to manufacture shear-thinning gels yields a “lumpy” product. This may be associated with an unacceptable level of discomfort in those to whose eyes it is administered.
Ensuing continuity and consistency of supply of biologically-sourced materials (whether fibrinogen or thrombin) also introduces difficulties in the supply of manufacturing materials to be used, and the distribution of the compositions produced. Enzymes and their substrates can be sensitive to the way in which they are handled (since variations in temperature, or the like, may significantly impact upon activity), and biological gels of this sort typically require refrigerated (or frozen) storage after manufacture. Use of biological material, such as collagen or fibrin, is also associated with the risk of unwanted contaminants, such as infections agents, being introduced into the products.
In view of the above, it can be seen that the inventors' development of the shear-thinning ocular hydrogel compositions of the invention, from which collagen or fibrin are substantially or entirely absent, represent a considerable technical departure from the products taught in the prior art. However, this departure confers notable unexpected advantages that are not made available on following earlier teachings. The properties, manufacture and uses of the ocular hydrogel compositions are all considered further below.
Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:
The term “hydrogel” is used herein to refer to a gel formed from a hydrophilic polymer dispersed within an aqueous vehicle.
The term “aqueous vehicle” is used herein to refer to water or water-based fluid (e.g. a buffer such as, for example, phosphate buffered saline or a physiological fluid such as, for example, serum).
The term “microgel” is used herein to refer to a microscopic particle of gel formed from a network of microscopic filaments of polymer.
The term “shear-thinning” is used herein to define the hydrogel compositions of the present invention. This terminology is well understood in the art and refers to hydrogel compositions that have a viscosity that reduces when a shear force is applied to the hydrogel. The shear-thinning hydrogel compositions of the invention possess a “resting” viscosity (in the absence of any applied shear force), and a lower viscosity when a shear force is applied. This property of hydrogel compositions enables them to flow and be administered to the body when a shear force is applied (for example, by applying a force to a tube or dispenser containing the hydrogel composition of the invention). Once applied under the application of shear, and the applied shear force is removed, the viscosity of hydrogel composition increases. Typically, the hydrogel compositions of the present invention will have a viscosity of below 1 Pa·s when subjected to a shear force to administer the hydrogel composition. At viscosities below 1 Pa·s, the hydrogel composition will be capable of flowing. The resting viscosity will typically be above 1 Pa·s, for example greater than 2 Pa·s, greater than 3 Pa·s, or greater than 4 Pa·s.
It is to be appreciated that references to “treating” or “treatment” include prophylaxis as well as the alleviation of established symptoms of a condition. “Treating” or “treatment” of a state, disorder or condition therefore includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e., arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.
A “therapeutically effective amount” means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated. Further considerations regarding therapeutically effective amounts of ocular hydrogel compositions of the invention, or of decorin or other agents to be incorporated in such ocular hydrogel compositions, are considered in more detail elsewhere in the specification.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
The present invention is directed to shear-thinning ocular hydrogel compositions, and, unless context requires otherwise, all references within the present disclosure to “hydrogels”, “compositions”, or “hydrogel compositions” should be taken as relating to “shear-thinning ocular hydrogel compositions”.
In a first aspect of the invention there is provided a shear-thinning ocular hydrogel composition comprising:
The ocular hydrogel compositions of the present invention are shear-thinning, meaning that the viscosity of the composition reduces when the hydrogel is exposed to shear. This property enables the hydrogels to reduce in viscosity and flow when a shear force is applied, thereby enabling them to be dispensed and administered, for example from an eye dropper to tube, by applying a shear force (e.g. by squeezing the sides of the eye dropper or tube). Once administered and the shear force applied to the hydrogel diminishes, the viscosity of the hydrogel increases to form a thicker gel capable of residing at the point of administration for a prolonged period.
Typically, the hydrogel compositions of the present invention will have a viscosity of below 1 Pa·s when subjected to a shear force to administer the hydrogel composition. At viscosities below 1 Pa·s, the hydrogel composition will be capable of flowing. The resting viscosity will typically be above 1 Pa·s, for example greater than 2 Pa·s, greater than 3 Pa·s, or greater than 4 Pa·s.
The microgel particle-forming polymer may be any polymer that is capable of forming microgel particles in the aqueous vehicle. The microgel particles formed by the microgel particle-forming polymer may have any suitable morphology (e.g. they may be linear filaments or regular or irregular shaped particles) and/or particle size. The formation of microgel particles, as opposed to a macrogel structure, facilitates the desired shear-thinning characteristics. Without wishing to be bound by any particular theory, it is postulated that, in the absence of shear or at low levels of shear, the microgel particles are bound together, substantially impeding the bulk flow of the hydrogel. However, upon the application of a shear force, the interactions between adjacent microgel particles are overcome, and the viscosity decreases, thereby enabling the hydrogel composition to flow. Once the applied shear force is removed, then the interactions between adjacent microgel particles can reform such that the viscosity increases again and the ability to flow readily is impeded.
In an embodiment, the shear-thinning hydrogel compositions of the present invention do not comprise collagen and/or fibrin.
Suitably the hydrogel composition comprises 0.1 to 5.0 wt. % of the microgel particle-forming polymer. In an embodiment, the hydrogel composition comprises 0.1 to 3.5 wt. % of the microgel particle-forming polymer. In an embodiment, the hydrogel composition comprises 0.5 to 2.5 wt. % of the microgel particle-forming polymer. In an embodiment, the hydrogel composition comprises 0.8 to 1.8 wt. % of microgel particle-forming polymer. In a further embodiment, the hydrogel composition comprises 0.8 to 1.0 wt. % (e.g. 0.9 wt. %) of a microgel particle-forming polymer.
Suitably the microgel particles are formed from one or more polysaccharide microgel particle-forming polymers. Suitably the microgel particles are not formed from decorin.
Suitably, the microgel particle-forming polymer is one or more polysaccharide microgel particle-forming polymers. In an embodiment, the microgel particle-forming polymer is selected from one or more of the following groups: gellans, alginates, carrageenans, agarose. In a particular embodiment, the microgel particle-forming polymer is selected from one or more of the following groups: gellans, alginates or carrageenans. In a more particular embodiment, the microgel particle-forming polymer is selected from gellan or alginate. In yet another embodiment, the microgel particle-forming polymer is gellan. In yet another embodiment, the microgel particle-forming polymer is an alginate.
In an alternative embodiment, the microgel particle-forming polymer is gelatin.
The ocular hydrogel compositions of the invention are transparent or translucent. In a particular embodiment, the ocular hydrogel composition is transparent.
In an embodiment, the hydrogel composition is transparent or translucent and the microgel particle-forming polymer is selected from gellans, alginates and/or carrageenans. In a further embodiment, the hydrogel composition is transparent and the microgel particle-forming polymer is selected from gellans, alginates and/or carrageenans. In a particular embodiment, the hydrogel composition is transparent and the microgel particle-forming polymer is gellan or alginate. In a further embodiment, the hydrogel composition is transparent and the microgel particle-forming polymer is gellan.
Gellan (also referred to gellan gum) is a water-soluble anionic polysaccharide produced by the bacterium Sphingomonas elodea. It is commercially available in a low acyl form under the trade name Kelco gel (Kelco gel CG LA, Azelis, UK).
The hydrogel composition comprises 5 to 100 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent. The metal ion salt may be added to the composition as a component, but it may also be present in other components of the composition, e.g. components such as buffers (e.g. phosphate buffered saline) or any physiological fluids present in the composition, such as, for example, serum.
Suitably, the hydrogel composition comprises 5 to 40 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent. In an embodiment, the hydrogel composition comprises 5 to 30 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent. In another embodiment, the hydrogel composition comprises 5 to 20 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent. In yet another embodiment, the hydrogel composition comprises 5 to 15 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent. In yet another embodiment, the hydrogel composition comprises 8 to 12 mM (e.g. 10 mM) of a monovalent and/or polyvalent metal ion salt as a cross-linking agent.
In a particular embodiment of the invention, the microgel particle-forming polymer is gellan and the composition comprises 0.5 to 40 mM, 5 to 15 mM, 8 to 12 mM or 10 mM of a monovalent metal ion salt (e.g. NaCl) as a cross-linking agent.
In a further embodiment of the invention, the microgel particle-forming polymer is alginate and the composition comprises 0.5 to 40 mM, 5 to 15 mM, 8 to 12 mM or 10 mM of a polyvalent metal ion salt (e.g. a Ca2+ salt) as a cross-linking agent.
Suitably, the hydrogel composition has a pH within the range of 6 to 8. In an embodiment, the hydrogel composition has a pH within the range of 6.5 to 8. In a further embodiment, the hydrogel composition has a pH within the range of 7 to 7.5 (e.g. pH 7.4).
Suitably, the hydrogel composition of the present invention has a resting viscosity (i.e. a viscosity at zero shear) of 1 Pa·s or greater (e.g. 1 Pa·s to 200 Pa·s or 1 Pa·s to 100 Pa·s). More suitably, the resting viscosity will be 2 Pa·s or greater (e.g. 2 Pa·s to 200 Pa·s or 2 Pa·s to 100 Pa·s), 3 Pa·s or greater (e.g. 3 Pa·s to 200 Pa·s or 3 Pa·s to 100 Pa·s), 4 Pa·s or greater (e.g. 4 Pa·s to 200 Pa·s or 4 Pa·s to 100 Pa·s), or 5 Pa·s or greater (e.g. 5 Pa·s to 200 Pa·s or 5 Pa·s to 100 Pa·s).
The viscosity reduces when the hydrogel composition is subjected to a shear force. Suitably, the viscosity reduces to a value below the resting viscosity at which the gel can flow and be administered. Typically, the viscosity will reduce to a value of less than 1 Pa·s when a shear force is applied.
In an embodiment, the hydrogel composition has a resting viscosity of 1 Pa·s or greater (e.g. 1 Pa·s to 200 Pa·s or 1 Pa·s to 100 Pa·s) and when subject to a shear force, the viscosity reduces to below 1 Pa·s.
In another embodiment, the hydrogel composition has a resting viscosity of 2 Pa·s or greater (e.g. 2 Pa·s to 200 Pa·s or 2 Pa·s to 100 Pa·s) and when subject to a shear force, the viscosity reduces to below 2 Pa·s (for example, to below 1 Pa·s).
In another embodiment, the hydrogel composition has a resting viscosity of 3 Pa·s or greater (e.g. 3 Pa·s to 200 Pa·s or 3 Pa·s to 100 Pa·s) and when subject to a shear force, the viscosity reduces to below 3 Pa·s (for example, to below 1 Pa·s).
In another embodiment, the hydrogel composition has a resting viscosity of 4 Pa·s or greater (e.g. 4 Pa·s to 200 Pa·s or 4 Pa·s to 100 Pa·s) and when subject to a shear force, the viscosity reduces to below 4 Pa·s (for example, to below 1 Pa·s).
In another embodiment, the hydrogel composition has a resting viscosity of 5 Pa·s or greater (e.g. 5 Pa·s to 200 Pa·s or 5 Pa·s to 100 Pa·s) and when subject to a shear force, the viscosity reduces to below 5 Pa·s (for example, to below 1 Pa·s).
For the avoidance of doubt, all viscosity values quoted herein are quoted at a normal ambient temperature of 20° C. The viscosity of hydrogel compositions of the present invention can be determined using standard techniques well known in the art. For example, viscosity profiles can be obtained using an AR-G2 (TA Instruments, UK) rheometer equipped with sandblasted parallel plates (40 mm, 1 mm gap height) at 20° C.
Suitably, the hydrogel has an elastic modulus of 5 Pa to 40 Pa at zero shear.
The elastic modulus of the hydrogels of the present invention can be determined by techniques well known in the art.
Particular embodiments of the invention include those in which the shear-thinning ocular hydrogel composition comprises decorin and:
(1) 0.1 to 5.0 wt. % (e.g. 0.1 to 3.5 wt. % or 0.1 to 2.5 wt. %) of a microgel particle-forming polymer (e.g. gellan);
As previously discussed, the shear-thinning ocular hydrogel compositions of the invention comprise the pharmaceutically active therapeutic agent decorin. In certain embodiments of the invention, the hydrogel composition may comprise one or more further pharmacologically active agents. Any suitable pharmacologically active agent may be present. For example, the hydrogel composition may comprise one or more further pharmacologically active agents selected from the group consisting of: a further anti-fibrotic agent; an anti-infective agent; and an anti-inflammatory agent.
In an embodiment, the ocular hydrogel composition comprises decorin in an amount of from 0.1 to 1.0 mg/ml; 0.1 to 0.5 mg/ml; 0.1 to 0.4 mg/ml; or 0.2 to 0.3 mg/ml.
In a further embodiment, the ocular hydrogel composition comprises decorin in an amount of from 0.1 to 1.0 mg/ml; 0.1 to 0.5 mg/ml; 0.1 to 0.4 mg/ml; or 0.2 to 0.3 mg/ml, in any one of the hydrogel compositions defined in paragraphs (1) to (14) above.
The ocular hydrogel composition may comprise any suitable amount of a further pharmacologically active agent. For example, the hydrogel composition may comprise 0.01 to 50 wt. % of a further pharmacologically active agent.
In an embodiment of a composition of the invention comprising an anti-infective agent, such as the antibiotic gentamicin, this may be present in an amount of from 1 to 5 mg/ml. For example, an anti-infective agent, such as gentamicin, may be present in an amount of from 1 to 4 mg/ml, from 1 to 3 mg/ml, or from 1 to 2 mg/ml. An anti-infective agent, such as gentamicin, may be present in an amount of from 2 to 4 mg/ml, or from 2.5 to 3.5 mg/ml.
In an embodiment of a composition of the invention comprising an anti-inflammatory agent, such as the steroid prednisolone, this may be present in an amount of from 0.5 to 250 mg/ml. Suitably, an anti-inflammatory agent such as prednisolone may be present in an amount of from 1.25 to 170 mg/ml, for example from 1.25 to 50 mg/ml, or from 1.25 to 10 mg/ml.
The present invention further provides a method of making a shear-thinning ocular hydrogel composition as defined herein, the method comprising the steps of:
Suitably, step a) is performed by heating the microgel particle-forming polymer and aqueous vehicle to a temperature above the gelling temperature for the microgel particle-forming polymer. For example, in embodiments where the microgel particle-forming polymer is gellan, the gellan/aqueous vehicle mixture may be heated to 60 to 90° C. (e.g. 70° C.) in order to dissolve the gellan polymer.
It will be appreciated that the amount of polymer dissolved will depend on the amount of polymer required in the hydrogel composition (i.e. it will be within the limits defined hereinbefore for the hydrogel composition).
In step b), the solution formed in step a) is suitably maintained at a temperature above the gelation temperature for the microgel particle-forming polymer and is mixed with an aqueous solution of a monovalent or polyvalent metal ion salt. Suitably, in step b), the solution from step a) is continuously agitated before, during and/or after the addition of the solution of the monovalent or polyvalent metal ion salt. For example, the mixture may be mixed at a rate of 50 to 2000 revolutions per minute (rpm) to ensure thorough mixing. In an embodiment, a mixing rate of 300 to 900 rpm or 500 to 800 rpm may be used. A person skilled in the art will appreciate that the mixing rate and mixing apparatus can be varied to provide a desired level of shear/agitation.
In an embodiment, where the microgel particle-forming polymer is gellan, the gellan/aqueous vehicle solution from step a) may be cooled to a temperature of, for example, 35 to 50° C. (e.g. 40° C.) prior to mixture with a monovalent cation solution.
It will be appreciated that the amount of monovalent or polyvalent metal ion salt solution added will depend on the amount of metal ion salt required in the final hydrogel composition (i.e. it will be within the limits defined hereinbefore for the hydrogel composition).
In step c), the mixture from step b) is cooled to a temperature below the gelation temperature for the microgel particle-forming polymer such that microgel particles form in the hydrogel composition. Suitably, the mixture from step b) is cooled gradually with constant mixing. In an embodiment, the mixture from step b) is cooled at a constant cooling rate with continuous agitation/shear applied. The cooling under agitation/shear may continue until the mixture reaches ambient temperature (e.g. 20° C.), at which point the final hydrogel composition may be collected and stored, for example under refrigeration conditions.
The cooling rate used in step c) and the amount of shear/agitation applied can be varied. For example, a cooling rate of 0.2 to 4° C./min, 0.5 to 3° C./min, 0.5 to 2° C./min, 0.5 to 1.5° C./min, or 1° C./min may be used. The amount of shear applied may be, for example, 50 to 2000 rpm, 300 to 900 rpm, or 400 to 500 (e.g. 450) rpm. Any suitable equipment may be used to provide the required agitation/shear. In the accompanying examples, a rotational rheometer (AR-G2, TA Instruments, UK) equipped with cup and vane geometry (cup: 35 mm diameter, vane: 28 mm diameter) is used to provide the required shear.
Decorin may be added to the mixture in step b) or step c) of the method. Suitably, the decorin is added during step c) at a point where the mixture is above the gelling temperature for the microgel particle-forming polymer. Most suitably, the mixture from step b) is cooled to a temperature above the gelling temperature for the microgel particle-forming polymer, the decorin is added and thoroughly mixed into the mixture, and the mixture is then further cooled to a temperature below the gelling temperature for the microgel particle-forming polymer.
Suitably decorin is added to the mixture in either step b) or step c) in the form of an aqueous decorin solution.
In addition to decorin, a further pharmacologically active agent may be added during step b) or step c) (at a temperature above the gelling temperature of the microgel particle-forming polymer).
In a further aspect the present invention provides a method of making a shear-thinning ocular hydrogel composition as defined herein, the method comprising the steps of:
In the above aspect of the invention, the process is the same as the previous process defined above except that the microgel-particle forming polymer is dissolved directly in an aqueous vehicle comprising 0.5 to 100 mM of a monovalent and/or polyvalent metal ion salt as a cross-linking agent. The conditions and variable for steps a), b) and c) described above apply equally to this variant of the process.
Decorin may be added to the mixture in step a) or step b) of the method. Suitably, the decorin is added during step b) at a point where the mixture is above the gelling temperature for the microgel particle-forming polymer. Most suitably, the mixture from step a) is cooled to a temperature above the gelling temperature for the microgel particle-forming polymer, the decorin is added and thoroughly mixed into the mixture, and the mixture is then further cooled to a temperature below the gelling temperature for the microgel particle-forming polymer.
Suitably decorin is added to the mixture in either step a) or step b) in the form of an aqueous decorin solution.
In addition to decorin, a further pharmacologically active agent may be added during step a) or step b) (at a temperature above the gelling temperature of the microgel particle-forming polymer).
In a further aspect the present invention provides a shear-thinning gel composition obtainable by, obtained by, or directly obtained by, any of the preparatory methods defined herein.
An aspect of the invention provides compositions of the invention for use as a medicament. Compositions of the invention are suitable for medical use in the inhibition of scarring (as set out in a further aspect of the invention); as well as the prevention and/or treatment of infection; and the prevention and/or treatment of inflammation. Compositions to be employed in such medical uses may comprise, as required, an active agent selected from the group consisting of: an anti-fibrotic agent; an anti-infective agent; and an anti-inflammatory agent.
As already referred to above, compositions of the invention are also suitable for use in methods of medical treatment. For example, compositions of the invention may be used in methods selected from the group consisting of: methods for the inhibition of scarring; methods for the prevention and/or treatment of infection; and methods for the prevention and/or treatment of inflammation. In practicing such methods, a composition of the invention may be administered, as required, to a subject in need of inhibition of scarring; a subject in need of prevention and/or treatment of infection; or a subject in need of prevention and/or treatment of inflammation.
Suitably, compositions of the invention may be used in methods for the inhibition of scarring in a subject that has microbial keratitis. Such use may also prevent and/or treat infection causing the microbial keratitis. Such use may also prevent and/or treat inflammation associated with microbial keratitis.
As above, compositions to be employed in such methods of treatment may comprise, as required, an active agent selected from the group consisting of: a further anti-fibrotic agent (other than decorin); an anti-infective agent; and an anti-inflammatory agent.
Except for where the context requires otherwise, considerations set out in the present disclosure with respect to medical uses of the compositions of the invention should also be taken as applicable to methods of treatment utilising the compositions of the invention. Similarly, considerations set out in the present disclosure with respect to methods of treatment utilising the compositions of the invention should also be taken as applicable to medical uses of the compositions of the invention.
Inhibition of Scarring
It is recognised that scarring results in deleterious effects in many clinical contexts. For example, scarring of the eye may be associated with loss of sight, and risk of blindness.
It will be appreciated that “inhibition of scarring” encompasses both partial inhibition of scarring and complete inhibition of scarring. Suitable values relating to the extent to which scarring may be inhibited in accordance with the invention are described further below.
Suitably an ocular composition of the invention for use in the inhibition of scarring may comprise gellan. As discussed further above, ocular compositions of the invention comprising gellan and decorin offer surprising benefits in the inhibition of scarring, as compared to prior art compositions. In particular the ocular hydrogel compositions of the invention that incorporate shear-thinning gellan hydrogels offer notable benefits as compared to those compositions of the prior art that employ ECM materials, such as collagen and/or fibrin.
Scarring in the eye, of the sort that may be inhibited by the medical use of compositions of the invention, includes scarring of the cornea, scarring of the retina, scarring of the ocular surface, and scarring in and around the optic nerve. Whilst the compositions of the invention are suitable for topical use, it will be appreciated that agents administered topically may have an effect on the internal anatomy. Thus, compositions administered to the surface of the eye may be effective in inhibiting intraocular scarring. Suitably scarring to be inhibited using the compositions or methods of the invention may include: scarring associated with infection, such as microbial keratitis; scarring associated with accidental injuries; and scarring associated with surgical injuries.
The ocular hydrogel compositions of the invention have particular utility in inhibiting scarring associated with keratitis. Keratitis may arise as a result of infection, for example microbial infection, viral infection, parasitic infection, or fungal infection. The compositions and methods of the invention have shown particular utility in the inhibition of scarring associated with microbial keratitis.
Keratitis may also arise as a result of injury, or of disorders including autoimmune diseases such as rheumatoid arthritis or Sjogren's syndrome. The compositions and methods of the invention may also be used in inhibiting scarring associated with keratitis occurring as a result of these causes.
Suitably, compositions of the invention may comprise a further pharmacologically active agent, such as, a further anti-fibrotic agent; an anti-infective agent; an anti-inflammatory agent
Scarring in the eye that may be inhibited by the medical use of compositions of the invention may also include scarring associated with surgery, such as surgery for the treatment of glaucoma (for example by the insertion of stents); and surgical procedures such as LASIK or LASEK surgery, and scarring associated with accidental injuries.
The skilled person will be aware of many suitable methodologies that allow the identification and quantification of scarring in the eye. These methodologies may also be used to identify inhibition of scarring in the eye. Thus, they may be used to illustrate the effective medical use of the compositions of the invention, to identify therapeutically effective doses of decorin, and also in the identification and/or selection of further pharmacologically active agents to be incorporated in the compositions of the invention.
The skilled person will be aware that there are many parameters by which the inhibition of scarring in the eye can be assessed. Examples of these are discussed further in the Examples. Some of these, such as induction of myofibroblast or ECM components, are also common to body sites outside the eye, while others are specific to the eye.
For example, scarring in the eye may be indicated by an increase in corneal opacity. Such an increase in corneal opacity may be demonstrated by an increase in the area of the cornea that is opaque. Thus, inhibition of scarring may be indicated by a reduction in corneal opacity as compared to a suitable control. Such a decrease in corneal opacity may be demonstrated by a decrease in the area of the cornea that is opaque.
The ability of compositions of the invention, comprising the anti-fibrotic agent decorin, to reduce corneal opacity, and to maintain such a reduction over time, is demonstrated in the data set out in the Examples.
Similarly, scarring of the eye may be indicated by an increase in the presence of myofibroblasts. Thus, inhibition of scarring may be indicated by a reduction in myofibroblast numbers as compared to a suitable control.
Myofibroblasts develop at the site of injuries and are associated with progression of the scarring response. They can be characterised by their expression of α-smooth muscle actin (α-sma). Myofibroblasts can have a number of adverse effects on scar formation, including causing contractions within the healed area. An increase in myofibroblasts associated with scarring may be demonstrated by an increase in α-smooth muscle actin expression. A reduction in myofibroblast numbers of this sort may be demonstrated by a decrease in α-smooth muscle actin expression. The compositions of the invention are able to inhibit α-sma expression as assessed in vitro and in vivo, thus demonstrating their ability to inhibit scarring
As discussed further in the Examples, compositions of the invention are able to inhibit myofibroblast differentiation in vivo, and are also able to maintain this reduced differentiation over time in an experimental model of microbial keratitis.
Myofibroblast differentiation may be increased in response to the action of TGF-β1, a fibrotic growth factor that causes induction of α-sma expression. The Examples set out details of in vitro studies (in human dermal fibroblasts), which illustrate the ability of compositions of the invention to block this increase in α-sma expression. This indicates that the decorin incorporated in the ocular hydrogel compositions of the invention is able to effectively block the activity of fibrotic growth factors (such as TGF-β) despite the absence of collagen and/or fibrin in the exemplary compositions used.
Fibrosis is also associated with the expression and deposition of ECM constituents at a site of injury. The amount of ECM deposited may be increased in scarring, and the arrangement of the ECM may be different from that found in undamaged comparator tissue. The data presented in the Examples illustrate that treatment using compositions of the invention gives rise to tissues in which the arrangement of ECM components more closely resembles that of unwounded tissue, thus illustrating the utility of these compositions in the inhibition of scarring.
The ocular compositions of the invention comprising anti-fibrotic decorin, may be able to achieve an inhibition of fibrosis in the eye by at least 5% as compared to a suitable control agent. For example, a suitable anti-fibrotic agent may be able to achieve an inhibition of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as compared to a suitable control agent. An anti-fibrotic agent suitable for incorporation in a composition of the invention may be able to achieve substantially total inhibition of scarring as compared to a suitable control agent.
By the same token, the medical use of compositions of the invention, or methods of treatment using such compositions, to inhibit scarring in the eye may achieve an inhibition of at least 5% as compared to a suitable control. For example, such medical uses or methods of treatment may achieve an inhibition of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, as compared to a suitable control. The medical uses or methods of treatment of the invention may achieve substantially total inhibition of scarring as compared to a suitable control.
The selection of a suitable control will be readily determined by one of skill in the art. Merely by way of example, a suitable control for assessment of the ability of a composition of the invention to inhibit scarring in the eye may be provided by the recognised standard of care, or an experimental proxy thereof.
Values and other considerations here presented in the context of scarring of the eye generally may, except for where the context requires otherwise, all be applicable specifically to scarring of the cornea.
Active Agents Suitable for Incorporation in the Compositions of the Invention
Compositions of the invention intended for medical use, or use in methods of treatment, may comprise a further active agent, in addition to the anti-fibrotic agent decorin. A suitable further active agent may be selected with reference to the intended medical use. However, for illustration, a suitable further active agent may be selected from the group consisting of: a further anti-fibrotic agent; an anti-infective agent; and an anti-inflammatory agent.
For the avoidance of doubt, a composition of the invention may suitable comprise more than one active agent. In cases where the composition comprises more than one active agent, this may be more than one active agent within a particular class of active agents (e.g. two or more anti-fibrotic agents), or a combination of agents selected from two or more different classes (e.g. anti-fibrotic decorin and an anti-infective agent, or an anti-fibrotic decorin and an anti-inflammatory agent).
Examples of further anti-fibrotic agents that may be incorporated in compositions of the invention are discussed in more detail below.
Merely by way of example, an anti-infective agent suitable for incorporation as an active agent in a composition of the invention may be an anti-microbial agent. For example, such an anti-viral agent, an anti-fungal agent, or anti-helminth agent. In the case of an anti-microbial agent, a suitable anti-infective agent may be an antibiotic, such as gentamicin. Many other suitable examples of antimicrobial agents that can be incorporated in compositions of the invention, including further antibiotics, will be well known to those skilled in the art.
A composition of the invention comprising an anti-infective agent may be used in methods for the prevention and/or treatment of infection. Accordingly, it will be appreciated that such a composition may be administered to a subject in need of prevention and/or treatment of infection. A subject in need of such prevention and/or treatment may be one that has microbial keratitis.
An anti-inflammatory agent for incorporation as an active agent in a composition of the invention may be selected from the group consisting of: a steroid, such as a corticosteroid (for example prednisolone); a non-steroidal anti-inflammatory drug (NSAID), such COX-1 and/or COX-2 enzyme inhibitor; an anti-histamine, such as an H1 receptor antagonist; interleukin-10; pirfenidone; an immunomodulatory agent; and a heparin-like agent.
A composition of the invention comprising an anti-inflammatory agent may be used in methods for the prevention and/or treatment of inflammation. Accordingly, such a composition may be administered to a subject in need of prevention and/or treatment of inflammation. Suitably, the subject may be one having or at risk of developing chronic inflammation or acute inflammation. Merely by way of example, inflammation may be caused by microbial keratitis.
Suitably a composition of the invention may comprise decorin for use in combination with the anti-infective agent gentamicin, and the anti-inflammatory agent prednisolone. A composition of this sort may comprise decorin, prednisolone and gentamicin. Such compositions of the invention are suitable for use in the inhibition of scarring associated with microbial keratitis, as illustrated by the data set out in the Examples.
A composition of the invention incorporates the anti-fibrotic agent decorin in a therapeutically effective amount. Therapeutically effective amounts of anti-fibrotic agents, such as decorin, are discussed in further detail below. A composition of the invention may also comprise a further active agent in a therapeutically effective amount.
A therapeutically effective amount of decorin or a further active agent will be able to achieve a desired clinical outcome either in a single administration, or as part of a course of treatment comprising multiple incidences of administration. The skilled person will be well aware of suitable protocols and procedures for the calculation of therapeutically effective amounts of active agents of various sorts.
Suitably an active agent may be incorporated in a composition of the invention at a concentration of between 0.1 ng/mL and 10 mg/mL. For example, an active agent may be incorporated in a composition of the invention at a concentration of between 1 ng/mL and 5 mg/mL, between 10 ng/mL and 2.5 mg/mL, or between 20 ng/mL and 1 mg/mL, between about 0.1 μg/mL and 0.5 μg/mL, suitably about 0.24 μg/mL.
Anti-Fibrotic Agents
Anti-fibrotic agents are agents that are able to bring about an inhibition of scarring in a body site to which they are provided. The inhibition of scarring is considered more generally elsewhere in the specification.
Decorin, incorporated in the ocular hydrogel compositions of the invention, is an example of an anti-fibrotic agent, and many other anti-fibrotic agents are known to those skilled in the art. Accordingly, the skilled person will be readily able to identify anti-fibrotic agents that may beneficially be incorporated in compositions of the invention for use in the inhibition of scarring. The following provides a non-exclusive list of examples of anti-fibrotic agents suitable for such uses.
Suitable anti-fibrotic agents may be selected from the group consisting of: anti-fibrotic extracellular matrix (ECM) components; anti-fibrotic growth factors (which for purposes of the present disclosure should be taken as also encompassing anti-fibrotic cytokines, chemokines, and the like); and inhibitors of fibrotic agents, such as function blocking antibodies.
Antibodies are useful in disrupting certain cellular activities by binding to cell signalling agents and thereby blocking functions caused by the agents' activity. Examples of such activities that may be blocked include: cell proliferation, cell migration, protease production, apoptosis and anoikis. Merely by way of example, suitable blocking antibodies may be able to bind one or more of the following groups of cell signalling agents: ECM components, growth factors, cytokines, chemokines or matrikines.
Decorin is an example of an anti-fibrotic ECM component. The decorin may be human decorin. Suitably the decorin may be human recombinant decorin. An example of a human recombinant decorin that may be incorporated in the compositions of the invention is that produced and sold by Catalent Pharma Solutions, Inc., under the name Galacorin™.
Decorin for incorporation in a composition of the invention may be a full-length naturally occurring version of this proteoglycan. Alternatively, compositions of the invention may employ anti-fibrotic fragments or anti-fibrotic variants of naturally occurring decorin.
Naturally occurring decorin is a proteoglycan. The proteoglycan (comprising both the core protein and glycosaminoglycan chains), or its fragments, may be used in the hydrogel compositions of the invention. However, the inventors have demonstrated that the core protein alone (without glycosaminoglycan chains) is sufficient to inhibit scarring in the eye. Accordingly, references to decorin (or fragments or variants thereof), in the present specification may alternatively be construed as directed to the core protein without glycosaminoglycan chains. The inventors believe that it is the core protein of decorin that serves to bind to fibrotic growth factors (such as TGF-β), and to block their biological function.
A suitable anti-fibrotic fragment of decorin may comprise up to 50% of the full-length, naturally occurring molecule, up to 75% of the full-length, naturally occurring molecule, or up to 90% of the full-length, naturally occurring molecule. A suitable anti-fibrotic fragment of decorin may comprise the TGF-β-binding portion of decorin.
An anti-fibrotic variant of decorin will differ from the naturally occurring proteoglycan by the presence of one or more mutations in the amino acid sequence of the core protein. These mutations may give rise to additions, deletions, or substitutions of one or more amino acid residues present in the core protein. Merely by way of example, a suitable anti-fibrotic variant of decorin suitable for incorporation in the compositions of the invention may comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, or at least 20 mutations as compared to the amino acid sequence of the naturally occurring core protein.
Except for where the context requires otherwise, references herein to decorin, in connection with the incorporation of this agent in the compositions of the invention, should also be taken as encompassing the use of anti-fibrotic fragments or anti-fibrotic variants of decorin.
In a suitable embodiment, decorin constitutes the only ECM component present in a composition of the invention. Suitably the decorin may be the only anti-fibrotic active agent incorporated in an ocular hydrogel composition in accordance with the invention.
Anti-fibrotic growth factors suitable for incorporation in compositions of the invention include those selected from the group consisting of: transforming growth factor-β3, platelet derived growth factor AA, insulin-like growth factor-1, epidermal growth factor, fibroblast growth factors (FGF) 2, FGF7, FGF10, FGF22, vascular endothelial growth factor A, keratinocyte growth factor, and hepatocyte growth factor.
Inhibitors of fibrotic agents represent suitable examples of further anti-fibrotic agents that may be incorporated in the compositions of the invention. Examples of such inhibitors include agents that bind to, and thereby block, the activity of a fibrotic agent. Examples of such inhibitors include function blocking antibodies or soluble fragments of cell receptors by which the fibrotic agent induces cell signalling. Other examples of such inhibitors include agents that prevent expression of the fibrotic agent. Examples of these sorts of inhibitors include those selected from a group consisting of: anti-sense oligonucleotides, and interfering RNA sequences.
A composition of the invention suitable for use in the inhibition of scarring will incorporate an anti-fibrotic agent in a therapeutically effective amount. Such a therapeutically effective amount will be able to inhibit scarring either in a single administration, or as part of a course of treatment comprising multiple incidences of administration. Details of how inhibition of scarring may be assessed, and so how a therapeutically effective amount may be calculated or recognised, are considered above.
Merely by way of example, the composition of the invention may comprise decorin at a concentration of between 0.1 ng/mL and 10 mg/mL, between 1 ng/mL and 5 mg/mL, between 10 ng/mL and 2.5 mg/mL, between 20 ng/mL and 1 mg/mL, between about 0.1 μg/mL and 0.5 μg/mL, suitably about 0.24 μg/mL.
Topical Administration and Topical Compositions
The compositions of the invention are suitable for topical administration to the eye. For the avoidance of doubt, in the context of the present disclosure, “topical administration” is taken to relate to direct administration of the composition to a surface of the eye. A composition of the invention suitable for such topical administration may be referred to as a topical ocular composition of the invention.
Topical compositions of the invention may be for administration to sites of infection or injury on the surface of the eye including, but not limited to: infections, abrasions, incisions, excisions, burns, and puncture wounds. Suitably topical compositions of the invention may be for administration to the cornea.
It will be appreciated that topical compositions may be formulated in manners conventional for use in such contexts. For example, a suitable topical composition may be formulated such that it does not induce irritation or inflammation of an infected or injured area to which it is administered.
The inventors have provided a novel eye drop system for the sustained delivery of a potent anti-scarring molecule (hrDecorin). The novelty of this eye drop lies in the method of structuring during manufacture, which creates a material that can transition between solid and liquid states, allowing retention in a dynamic environment being slowly removed through blinking. In a murine model of Pseudomonas keratitis, applying the eye drop resulted in reductions of corneal opacity within 16 days. More remarkably, the addition of hrDecorin resulted in scarless restoration and corneal integrity, as shown by complete re-epithelialization and reductions in αSMA, fibronectin and laminin. This drug delivery system is an ideal non-invasive anti-fibrotic treatment for patients with microbial keratitis, potentially without recourse to surgery saving the sight of many in the developing world, where corneal transplantation may not be available.
The present inventors have provided report a new class of eye drop material that allows for prolonged retention of a therapeutic on the surface of the eye, while being gradually cleared through the blinking process. The material is formed through the shearing of a gellan-based hydrogel, a material that is currently used in dilute form to thicken eye drops (e.g. Timoptol) during the gelation process. The application of shear prevents the formation of a continuous polymeric network and results in the formation of interacting particles that can exhibit spherical and ribbon-like morphology. Following shear-processing, these particles interact and form a continuous structure when the solution is at rest. When shear is applied (such as when extruded through an eye dropper), however, the continuous network of particles is disturbed and the material liquefies. Subsequent removal of the shear force results in an immediate healing. The solid-liquid-solid transitions that this material is able to undergo means that it conforms perfectly to the ocular surface and is removed gradually by the eyelid blinking dynamics. Importantly, gellan gum is optically transparent and so the material can continue to transmit light following application, causing minimal disruption to the patient.
A fluid-gel eye drop has been developed which can be loaded with decorin, to provide localized drug delivery and retention at the surface of the eye. The material combines structured gellan gum with the proteoglycan, decorin. Additionally, in conjunction to high optical clarity, the FDA approved polymer (FDA reference number 172.665) coupled with clinical grade hrDecorin, provides a rapid route to translation into the clinic. As such, this study investigated the effects of fluid gel, with and without hrDecorin, on corneal opacity, wound healing and fibrosis within a well-established murine model of Pseudomonas keratitis, as a precursor to clinical application for the management of severe bacterial infection.
Processing of the fluid gel involves passing a polymer solution, gellan, through a jacketed pin-stirrer, where it experiences high levels of shear whilst being forced (thermally) through its sol-gel transition (
The unique properties of fluid gels are such that they exhibit pseudo-solid properties at rest, but can be made to flow under force. Here, increasing the shear force exerted on the system, results in non-Newtonian, shear thinning behavior, typical of highly flocculated or concentrated polymer dispersions/solutions[37] (
The gellan-based eye drop system was formulated for drug delivery with the candidate anti-fibrotic agent, hrDecorin, used for our studies. The rate of release of hrDecorin from the eye drop system was almost linear over time (
Using a well-established model of bacterial keratitis[41], anaesthetized mice (n=6 per group) were challenged with P. aeruginosa (105 CFU) upon the surface of the damaged cornea. A therapeutic protocol to treat the infection based upon the standard treatment for bacterial keratitis patients was developed. After 12 hours of P. aeruginosa incubation to establish the corneal infection, eyes were treated with a 2-hourly regime of Gentamicin (1.5%) over a 12-hour period to sterilize the infection (confirmed by swab cultures).
Following the sterilization phase, 2 days after the initial inoculation, single 5 μl gellan eye drops were administered every 4 hours between 8 am and 8 μm for a further 13 days covering the treatment groups of 1) Gentamicin and Prednisolone (G.P); 2) Gentamicin, Prednisolone and fluid gel (G.P.FG) and; 3) Gentamicin, Prednisolone and hrDecorin fluid gel (G.P.DecFG) (Table 1).
Images of the cornea were taken at intervals throughout the 16-day experiment to measure changes in corneal opacity (
Effects of the Fluid Gel Eye Drops with hrDecorin on Corneal Re-Epithelialization
Epithelial stratification/maturation together with stromal thickness were chosen as outcome measures to assess corneal re-epithelialization, and to observe thickening of the stroma from edema and cellular infiltrates (as markers of infection). Pseudomonas infection severely disrupted the corneal structure, with an averaged increased corneal thickness of 218.7±24 μm after the infection on day 2 compared to naïve corneal thickness values of 129.3±10.7 μm. The infected corneas at day 2 had thinner epithelial layers compared to normal intact controls (19.2±2.1 μm vs 35.5±1.7 μm;
Immunoreactivity (IR) was used to assess the degree of fibrosis, as a ratio of pixel intensity above a baseline obtained from intact corneas (referred to here on as the threshold). In the naïve intact cornea, there were very low levels of αSMA immunoreactivity (IR) in the corneal stroma indicating the presence of few myofibroblasts (
Stromal ECM levels generated by the myofibroblasts were studied using fibronectin and laminin IR (
Improving ocular retention is key to increasing both bio-efficiency and therapeutic response to topical therapies, as turnover of the pre-corneal tear film (ca. 20% per minute[42]) results in rapid elimination of aqueous drugs, reducing titers delivered to the target tissue site. As such, many ocular conditions are currently treated via intensive topical therapies delivered through the day and night, or invasive methods disliked by many patients, including periocular or intravitreal injections to target intraocular pathology. In more severe cases where drugs are ineffective, surgery may be required to treat or remove the resultant corneal scar, increasing the risk of morbidity and increasing the duration of patient discomfort following treatment. The structured or “fluid-gel” formed from gellan provides a pivotal advance since it enables the sustained delivery of molecules such as hrDecorin capable of preventing scarring and obviating the need for invasive surgical repair strategies. The major advantage of the gellan fluid-gel is its capacity to transition between solid and liquid states as it passes through the applicator and solidifies on the surface of the cornea. This unique set of properties originates from the microstructure of the material, which consisted of ribbons and particles that weakly interact with one another at zero shear. These interactions are broken by the application of shear and reform following its removal. In this way, the material may then be gradually cleared from the ocular surface through the natural blinking mechanism. The development of a weak-elastic structure when applied to the surface of the cornea, results in the formation of a transparent and resorbable bandage, with the benefits of eye drops (in application) and hydrogel lens (sustained release), without the drawbacks of either. Indeed, the fluid-gel alone seems to provide a micro-environment conducive to wound healing, with a reduction in corneal opacity and markers of scar formation even without decorin addition. Importantly, the fluid gel does not interfere with hrDecorin's biological activity, as shown by the collagen fibrillogenesis data. As such, the system provides an excellent candidate technology for the clinical setting, with improved drug administration compliance across numerous patient cohorts.
The mouse model of P. aeruginosa keratitis provides a robust, clinically relevant means of evaluating the anti-scarring capacity of the hrDecorin loaded fluid gel against the current standard of care for pseudomonas infection (Gentamicin and Prednisolone)[43]. Once the infection is established, P. aeruginosa invades the corneal epithelial cells disrupting the natural healing responses with transformation of corneal fibroblast into corneal myofibroblasts leading to a fibrogenic microenvironment[44]. Topical administration of the eye drops either with or without the hrDecorin resulted in reduced levels of corneal opacity after 7 and 10 days of eye drop treatments, with the addition of hrDecorin displaying an evident further advantage. The effects of the fluid gel only treatment were not expected as the initial in vitro studies demonstrated that this carrier appeared inert. The therapeutic efficacy of fluid gel alone may be due to the formation of a permissive microenvironment in the damaged cornea, where the occlusive effect of the gel ribbons (that entwine to form a barrier around the wound) provided a therapeutic bandage that prevent biomechanical trauma caused by blinking over the ulcerated eye. It may also have sequestered Prednisolone and Gentamicin within its structure, enhancing retention of the therapeutic substances to the ocular surface, thereby improving bioavailability similar to the prosthetic replacement of the ecosystem (PROSE™ device) but with the added advantage of being resorbable. Such reductions in corneal opacity would benefit patients in terms of preservation of sight[58]. An important aspect to the healing phase encompasses restoration of a stratified non-keratinized epithelium. Together with the tear film, an apical mucosa (composed of lipid, mucins and aqueous layers) provides nutrition and lubrication to the ocular surface and is fundamental to first-line of defense to the eye. hrDecorin treated eyes exhibited the most improved restoration to normal anatomy, with a reduction in stromal edema, thickness and extracellular matrix deposition, coupled with improved epithelial morphology. The reduction in fibrotic markers by hrDecorin has been previously demonstrated across numerous animal models; modulating a range of growth factors (e.g. VEGF, IGF-1, EGF, PDGF) and their receptors, in particular TGFβ signaling via SMAD 2 and 3 pathways, preventing differentiation of corneal fibroblasts. Additionally, it's regulation of matrix metalloproteinase (MMPs) and tissue inhibitors of metalloproteinase (TIMP) results in fibrolysis and attenuated scar formation[29, 46-48].
The intrinsic ability of hrDecorin to aid healing, and in particular reduce scarring, has been enhanced by introducing the fluid gel carrier, improving retention time on the ocular surface. Given the severe extent of injury in this mouse model, we suggest that the endogenous levels of decorin may not have been sufficient to neutralize the TGFβ hyperactivity and subsequent fibrotic cascade to prevent corneal scarring. It is also possible that the endogenous decorin, located at the ocular surface within the tear film, within the cornea and the aqueous humour, is bound and not freely available to sequester the active TGFβ (
A unique feature of our formulations, was their ability to enhance the rate of re-epithelialization compared to standard care. This is central to the limitation of ocular damage as persistent epithelial defects lead to dysfunctional corneal metabolism, stromal melts and perforation. The addition of hrDecorin in the fluid gel eye drop formulation reduced ECM accumulation more than the standard of care and the fluid gel alone and, this was associated with further reductions in opacity (compared with earlier time points) in this model. This suggests our hrDecorin fluid gel eye drop provided sufficient doses to prevent fibrosis and promote wound resolution in this model or altered the chemical structure of the fluid gel to improve the bandaging effect. The benefits of this fluid gel formulation have been clearly demonstrated in vivo, observed both physically, with a reduction in corneal opacity, and pharmacologically, relating to diminished fibrotic markers. However, due to legislative constraints, the data generated within this study was limited to a 16 day time point. However, it would be of interest to examine later time points in future studies.
The effects of the fluid gel alone on the damaged corneal surface suggests an influence over the endogenous growth factors, an effect that is enhanced by the addition of hrDecorin. The fluid-gel may aid corneal healing through several mechanisms: firstly, the unique viscoelastic properties of the fluid gel acts as a liquid that self-structures upon the ocular surface to form a semi-solid occlusive therapeutic dressing for unperturbed healing to take place; secondly, helical domains formed during the gelation of the fluid gel may provide a mimetic scaffold for endogenous decorin to bind, sequestering key growth factors e.g. TGFβ and/or exogenously delivered hrDecorin; thirdly, the fluid gel matrix, comprising primarily of water (99.1%), creates a gradient driven diffusion of cytokines away from the wound site, again resulting in a restoration of the natural equilibria needed to prevent fibrosis.
The inventors have seen two different responses in the presence of the fluid gel and the Decorin fluid gel, and in future studies it will be important to tease apart the mechanism for each. We expect that the fluid gel alone is providing a protective barrier whilst potentially influencing inflammatory cell and fibroblast behavior in a manner that we do not yet understand. Importantly, the fluid gels are facilitating regeneration of the corneal epithelium leading to wound closure. We are hypothesizing that the fluid gel is affecting the limbal epithelium stem cell niche by promoting proliferation and differentiation, which may be dysregulated in the disease situation as well as providing a therapeutic bandage to aide stromal repair. However, in this study we did not have a hyaluronate or a carboxymethylcellulose only group to interrogate whether these ocular lubricant devices have a similar effect. Furthermore, the multifaceted actions of decorin such as inhibition of inflammation and angiogenesis and regulation of autophagy, in addition to sequestering TGFβ within the context of an ocular wound healing environment, need to be investigated further before translation into the clinic.
In conclusion, the inventors have demonstrated that a novel eye drop technology can be used to provide sustained delivery of anti-fibrotic drugs like hrDecorin topically to the cornea in a clinically relevant murine model of fibrosis associated with bacterial keratitis. The eye drop enabled the hrDecorin to remain in contact with the surface of the eye for long enough and at sufficient titers to significantly reduce corneal scarring. Furthermore, this study has demonstrated that the unloaded fluid gel also possesses healing effects in its own right, suggested to arise through its intrinsic material microstructure and subsequent properties. Not only do the material properties of the eye drop enhance anti-scarring drug retention times, but the user-friendly nature of the drops would be welcomed by patients, providing a simple treatment to prevent the scarring pathology that is prevalent after corneal infection. Having demonstrated successful reduction in corneal opacity and a reduction in markers commonly indicative of the scarring process, when compared to the current standard of care, this technology presents an ideal treatment option for patients with microbial keratitis, reducing the occurrence of visually significant corneal opacity and potentially eradicating the need for corrective surgical intervention. Given that transplant availability and the facilities for surgical intervention are often not available in the developing world, we believe that this technology could, in the future, help to save the sight of many patients.
The aim of this study was to explore the use of a novel fluid gel to deliver decorin to the ocular surface in order to reduce corneal opacity and scarring post-bacterial keratitis. The study was split into three evaluation stages: (i) material properties relating to ease of eye drop application, (ii) in vitro assessment of bioactivity of the formulated hrDecorin and (iii) anti-scarring efficacy of the fluid gel with/without hrDecorin in vivo, using a mouse model of Pseudomonas keratitis (once the eyes were sterilized after infection) in comparison to the current standard of care. The sample size (n=6 per experimental group) was based on the resource equation as the effect size was unknown. All analyses were performed by observers masked to experimental groupings and mice were randomly assigned to both treatment and control groups.
Fluid Gel (FG) and hrDecorin Fluid Gel (DecFG) Production
Fluid gels were produced by first dissolving low acyl gellan gum (Kelco gel CG LA, Azelis, UK) in deionized water. Gellan powder was added to deionized water at ambient temperature in the correct ratio to result in a 1% (w/v) solution. The sol was heated to 70° C. under agitation, on a hotplate equipped with a magnetic stirrer, until all the polymer had dissolved. Once dissolved, gellan sol was added to the cup of a rotational rheometer (AR-G2, TA Instruments, UK) equipped with cup and vane geometry (cup: 35 mm diameter, vane: 28 mm diameter). The system was then cooled to 40° C. hrDecorin (Galacorin™ Catalent, USA) in PBS (4.76 mg/ml) and aqueous sodium chloride (0.2 M) was then added to result in final concentrations of 0.9% (w/v) gellan, 0.24 mg/ml hrDecorin and 10 mM NaCl. Following this, the mixture was cooled at a rate of 1° C./min under shear (450/s) to a final temperature of 20° C. The sample was then removed and stored at 4° C. until further use. In the case of fluid gels without hrDecorin, ratios were adjusted so that the final eye drop had a composition of 0.9% (w/v) gellan, 10 mM NaCl.
Microscopy: For transmission microscopy samples were first diluted using polyethylene glycol 400 (PEG400) at a ratio of 1:4 (eye drop to PEG400). Following this, samples were analyzed using an Olympus FV3000. Images were processed using ImageJ (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, Md., USA).
For scanning electron microscopy samples were first prepared for lyophilizing by diluting gellan in deionized water in the same manner as for transmission microscopy to a ratio of 1:9. Samples were then rapidly frozen using liquid nitrogen and placed in a freeze drier overnight to leave a powder. Dried sample was then attached to a carbon stub and analyzed using a SEM.
Rheology: Viscosity profiles were obtained using an AR-G2 (TA Instruments, UK) rheometer equipped with sandblasted parallel plates (40 mm, 1 mm gap height) at 20° C. An equilibrium of 2 minutes was used to ensure constant test temperature. Following this, time dependent ramps up and down were applied ranging from 0.1 to 600/s (3 minutes sweep times). Recovery profiles were obtained using the same apparatus, under single frequency. The sample underwent rejuvenation by shearing at 600/s for 10 s. Following this, storage and loss (G′, G″ respectively) were monitored at 1 Hz, 0.5% strain. The cross over point was used as the point at which the sample started to act like a viscoelastic solid.
hrDecorin Release from the Fluid Gel
Levels of hrDecorin release from the gel were determined cumulatively, by placing 1 ml of the fluid gel containing hrDecorin in a 6 well plate. Then 2 ml of DMEM was placed over the sample and the plates were incubated at 37° C. At each time point, the media was removed for measurement of hrDecorin, and replaced with fresh media. Decorin release was quantified using an ELISA specific for human Decorin (R&D systems, Minneapolis, USA) in accordance with the manufacturer's protocol.
In Vitro hrDecorin Bioactivity Assays
Collagen fibrillogenesis: For the dose response curves, 75 μl of PBS was added to each well of a 96 well plate kept on ice. Varying hrDecorin doses were prepared by adding 400 μg/ml of hrDecorin to the first well and subsequently serial diluting (2-fold dilution) across the plate. Following dilution, a further 150 μl of PBS buffer was added to each well. Then, 75 μl of collagen type I (rat tail; Corning, UK) (800 μg/ml) was added to each well and incubated for 2 hours at 37° C. Subsequent absorbance readings were taken using a 405 nm plate reader. Each assay consisted of duplicate blank controls, and triplicate standard dilutions followed by triplicate sample dilutions. Kinetics of fibril formation were determined using a similar setup as the dose response, without serial dilution; incubating the samples within the plate reader, and taking data points every 2 minutes.
The treatment administration regimes for the in vivo Pseudomonas model are shown in
P. aeruginosa PAO1 strain was cultured in high salt LB (10 g of tryptone, 5 g of yeast extract, and 11.7 g of NaCl per L, supplemented with 10 mM MgCl2 and 0.5 mM CaCl2) at 37° C. for 18 hours. Sub-cultures were derived at an optic density (OD) of 0.2 (OD 650 nm approx. 1×108 CFU/ml). P. aeruginosa were washed (×3) in PBS, centrifuged at 300 rpm for 5 minutes and re-suspended in PBS at a density of 1×105 CFU/2.5 μl. C57BL/6 mice (Jackson Laboratory, CA, USA) were housed in pathogen-free conditions, given free access to food and water and were maintained according to the ARRIVE guidelines, the ARVO statement for the use of animals in ophthalmic and vision research and also adhered to guidelines set out by the University of California, Irvine. For inoculation, mice were anaesthetized and one corneal epithelium was abraded with 3×1 mm parallel scratches using a 26 G needle and inoculated with 2.5 μl P. aeruginosa (1×105 CFU) (strain PAO1)64,65. Mice remained sedated for 2 hours post-inoculation to permit penetration of the infection into the eye, and placed in recovery. After 24 hours, conscious mice were treated with 5 μl of Gentamicin (1.5%, QEHB Pharmacy, Birmingham, UK) every 2 hours for a 12-hour period, to sterilize the infection. After a further 12 hours, mice were administered eye drops (5 μl of each compound) every 4 hours between 8 am and 8 μm for a further 13 days depending on their treatment group: (1) Gentamicin+Prednisolone (0.5%, QEHB Pharmacy), (2) Gentamicin+Prednisolone+Fluid gel, or (3) Gentamicin+Prednisolone+Fluid gel with hrDecorin. Mice were examined for corneal opacification, ulceration and perforation. En-face 24-bit color photographs of the cornea were captured with a SPOT RTKE camera (Diagnostic Instruments) connected to a Leica MZF III stereo Microscope. Mice were euthanized by cervical dislocation under anesthetic at 16 days and eyes enucleated and placed in 4% PFA in PBS for processing for immunohistochemistry.
Two masked independent clinical ophthalmologists analyzed all photographs in the same randomized order (the order was provided by an independent statistician). The area of opacification was delineated and measured in mm2±SEM using ImageJ. Definitions of corneal opacification, adequate and inadequate images were agreed upon prior to commencement of image analysis by the observers. The randomized order dictated that there should be no time-trend in the measured areas. The limits of agreement between observers were assessed using the Bland-Altman method[62] on ninety-nine paired opacity measurements. Differences in measurement were approximately normally distributed. The Bland-Altman analysis revealed that one assessor was likely to assess sizes to be slightly smaller than the other but the mean difference was not statistically different from zero (two-sided p-value=0.29). A small number of replicates of measurements within assessor were conducted but not enough to formally test the limits of agreement within observer.
Enucleated eyes for IHC were post-fixed by immersion in 4% PFA in PBS overnight at 4° C. before cryoprotection using increasing concentrations of sucrose in PBS (10%, 20%, and 30%; Sigma) for 24 hours each at 4° C. Eyes were then embedded in optimal cutting temperature (OCT) embedding medium (Thermo Shandon, Runcorn, UK) in peel-away mold containers (Agar Scientific, Essex, UK) and later sectioned in the parasagittal plane at −22° C. using a cryostat microtome (Bright, Huntingdon, UK) at a thickness of 15 μm, and placed onto Superfrost slides (Fisher Scientific, USA). Central sections (in the optic nerve plane) were used for all IHC studies and stored at −80° C. Frozen sections were left to thaw for 30 minutes before 3×5 minute washings in PBS followed by a 20 minute permeabilization with 0.1% Triton X-100 (Sigma). Non-specific antibody binding sites in tissue sections were blocked for 30 minutes using 0.5% BSA, 0.3% Tween-20 (all from Sigma), and 15% normal goat serum (Vector Laboratories, Peterborough, UK) in PBS before incubating overnight in 4° C. in primary antibody (αSMA, Laminin and fibronectin; 1:200; all from Sigma) again followed by washing 3×5 minutes, and incubating for 1 hour at room temperature with a secondary antibody (Goat anti-mouse Alexa Fluor 488 1:500, Goat anti-mouse Alexa Fluor 594 1:500, Molecular Probes, Paisley, UK). Sections were then washed for 3×5 minutes and mounted in Vectorshield mounting medium containing DAPI (Vector Laboratories). Control tissue sections incubated with secondary antibody alone were all negatively stained.
After IHC, sections were imaged on a Zeiss Axioscanner fluorescent microscope (Axio Scan.Z1, Carl Zeiss Ltd.) at ×20 using the same exposure times for each antibody. IHC staining was quantified by measuring pixel intensity according to the methods previously described61. Briefly, the region of interest used for quantitation of ECM IR was defined by a region of interest which was same prescribed size for all eyes/treatments within the stroma. Each stroma had a total of 30 individual intensity measurements (regions of interest) taken to cover the whole area. ECM deposition was quantified within these defined regions of interests and the percentage of IR pixels above a standardized background threshold from intact corneas was calculated using ImageJ. For each antibody, the threshold level of brightness in the area of the stroma was set using intact untreated corneas to define the reference level for test group analysis. Images were assigned randomized file names to ensure masking of treatment groups for the assessor.
All statistical analyses were performed using SPSS 20 (IBM, Chicago, Ill., USA). Normal distribution tests were carried out to determine the most appropriate statistical analysis to compare treatments. Statistical significance was determined at P<0.05. For opacity measurements, corneal width, epithelial thickness, αSMA, Fibronectin and Laminin data were analyzed using ANOVA with Tukey post-hoc tests. For DAPI measurements of epithelial cell layer numbers, as the data were not normally distributed, a Kruskal-Wallis test was used.
Optimal eye drop viscosity was sought via two main methods: rheological characterisation of current, commercial eye drops/ointments, and consultation with ophthalmic clinicians. Characterisation of the commercial eye products highlighted a large range of viscosities across both eye drops and eye ointments used to medicate conditions such as dry eye; where optimally long retention times are required. Viscosities were collected and compared at 1 s-1 (chosen as a value within initial stages of shear thinning, so as to avoid artefacts for the apparatus) (Table 2 and
In the case both paraffin and carbomer based eye products a warning is given within the instructions to inform the patient that blurring and discomfort could arise due to the drops. As such, the outer limits of the formulation viscosities have been founded based on the values obtained for these products:
In all cases, with all formulations tested, these values have not been exceeded. Thus, all formulations prepared with the gellan as the biopolymer used for gelation could be used within these limits. However, a more optimal formulation, in terms of viscosity, was narrowed upon with aid of clinical advice.
Having made a panel of different formulations, clinicians were asked to manipulate the product and rate them in respect to plausible eye drop products. From this data, it was found that the eye drops in the viscosity range:
were more easily applied, with good retention, with the optimal drop:
Furthermore, the system should exhibit shear thinning behaviour.
Elasticity at rest plays a large role in the use of the product to be retained and deliver actives in a controlled way. It is believed that the ability of the microgel suspension to create a weak elastic network at rest drives the high retention times related to the product. Again, the limits are based upon the characterisation of commercially available eye drops and ointments (
Again, similar to the viscosity, for all formulations tested, the values obtained for current products were not exceeded. Thus, all formulations prepared with the gellan as the biopolymer used for gelation could be used within these limits.
However, when analysed by the clinicians this was narrowed to
with the optimal formulation ranging between
pH
Due to the chemical composition of biopolymers, and varying chemical moieties along their individual backbones, they have varying natural pH's. The pH of products coming into contact with the ocular surface is important, as many chemical injuries are formed in the range of pH<4 and pH>10, with normal physiology close to 7.11±1.5. Therefore, eye drops are formulated in between this range (4-10), with some products dropping as low as pH 3.5 (proparacaine hydrochloride solution)′. Therefore, based on this data from literature the eye drop formulations should have a pH within the range of:
3.5 to 8.6
However, delivery of many actives including proteins requires the formulation to be neutral. In these cases, PBS (phosphate buffered saline) can be added to the eye drop, restricting the pH to a neutral acidity. Therefore, in the formulation has been narrowed to a pH:
Ultimately the material properties of the formulations are governed by the concentration of initial polymer in the product. Therefore, the upper and lower material properties were used to evaluate the material formulations, providing upper and lower limits for polymer concentrations. As all systems exhibited shear thinning behaviours limits were solely based on fulfilling the criteria for both viscosity (at 1 s−1) and elastic behaviour at rest. Thus, the maximum range of:
0.1 to 5.0 wt. % (e.g. 0.1 to 3.5 wt. % or 0.5 to 2.5%) (w/v)
has been set for eye drop formulations, with values within those found for commercially available products. This have been narrowed to fall within the clinician's advice to:
0.5 to 1.5% (w/v)
with the optimised formulation consisting of:
0.9% (w/v)
Data obtained from characterisation of the gellan formulations showed that salt content did not influence the viscosities of the systems, however, it did have an effect on the elastic response of the gel at rest. Again, none of the systems formulated exceeded the upper and lower limits set by the commercial products as such upper and lower concentrations have been defines as:
However, the mechanical spectra showed that at higher salt concentrations, a marked reduction in the elastic network was formed when deformed outside the linear viscoelastic region. This suggests that lower salt concentrations result in more plastic behaviour, which is believed would be more comfortable for the patient. Therefore, the narrowed limits for the formulation have been adjusted to;
The addition of PBS can be used to manipulate the pH of the system. In these cases, 5% v/v (5% was determined as the amount added with the therapeutic decorin, so further studies have not been conducted in this area) is added which will affect the level of salt within the system. The concentration of mono-valent ions within the PBS has been calculated and tabulated in Table 8.
Therefore, the range for the cross-linker has been altered (Table 9), reducing the lower limit as the ion content in the PBS is sufficient to drive the gelation process.
The thermal processing within the manufacture are key to formation of a gel. Typically, the thermal parameters are divided into two sections: the processing temperatures, and the rate of cooling.
The inlet and outlet, are key to make sure the polymer is in a sol prior to processing and exits at a temperature below the gelling transition. Initially the inlet temperature was set as close to the gelling temperature as possible, due to the protein active denaturing at higher temperatures. Therefore, this was set to 40° C. However, this is not necessary, the key aspect to the inlet temperature is to keep it above the gelling temperature, to prevent early gelation and blockages. The function of the outlet temperature is to ensure ordering/structuring of the polymer has completed prior to storage. This prevents aggregation during stage and heterogeneous suspensions forming. As such, for gellan this temperature has been defined as 20° C., allowing the polymer to pass though the gelation process. The exit temperature is therefore controlled by the jacket of the mill, which is set to provide sufficient cooling during the process. This can be changed resulting in various cooling rates.
The cooling rate during the sol-gel transition is known to be very important in regard to the final material properties; as higher cooling rates result in rapid formation of structures and weaker overall moduli. This was observed for the microgel suspensions, however, only at higher polymer concentrations. It was observed that for the optimal eye drop formulation, no changes in material properties were observed suggesting that a large range of parameters could be used:
Whereas at 1.8% w/v polymer it was more dependent on elastic structure required.
Shear rate during processing showed very similar results to cooling rate, with the optimised polymer concentration un-effected by the processing shear. Again, the higher concentrations showed a dependency. Therefore, for the optimised formulation a very broad range of shear could be applied:
NaCl (0.2 M) was prepared through the addition of dry crystals (1.16 g) to deionised water (100 ml) using a volumetric flask. The NaCl was then allowed to dissolve using an inverting technique to aid the process. Once fully dissolved the solution was kept at ambient conditions until further use.
Gellan sols were prepared by dissolving powdered polymer into water/NaCl solution at varying ratios so that the final concentrations post-processing were equal to 0.5, 0.9, 1.35, 1.8 and 2.35% (w/v). In brief, gellan powder was weighed out (2.5, 4.5, 6.75, 9.0 and 11.75 g) and added to 450 ml of deionised water. The mixture was allowed to heat to 95° C. under agitation, allowing the polymer to dissolve. Once fully dissolved, 25 ml of NaCl stock solution (0.2 M) was added to the solution resulting in a 10 mM concentration post-processing. The sol was then allowed to reach thermal equilibrium at 95° C. before processing.
MS were prepared using a jacketed pin mill set to 20° C. Gellan sols were pumped using a peristaltic pump into the pin mill at 3 ml/min so that it entered the processing chamber at 40° C. Prior to entry using a syringe and syringe pump, water was pumped into the gellan stream (at a rate of 0.16 ml/min) so that they impinged, diluting the gellan sol to the final concentrations (0.5, 0.9, 1.35, 1.8 and 2.35% (w/v), 10 mM NaCl). The mixture was then cooled under shear (500 rpm or 1000 rpm) as it passed through the milling unit. On exiting, at 20° C., the gel was packaged and stored at 4° C. until further testing.
A rheometer (TA, AR-G2) equipped with a sandblasted parallel plate (40 mm diameter, 1 mm gap height) was used to test all samples, at 20° C. results are shown in
Amplitude sweeps were obtained in strain controlled mode over a range of 0.1 to 100.0%. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20° C. prior to testing. Measurements were obtained at 1 Hz in a logarithmic fashion.
Viscosity profiles for the samples were obtained using a continuous ramp. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20° C. prior to testing. Increasing shear was applied to the sample in rate controlled mode, between 0.1 and 600 over a 3-minute ramp, with data points obtained in a logarithmic fashion.
Small deformation rheology: see
Large deformation rheology: see
The effects of polymer concentration can be observed for both their elastic nature and viscosity, with both properties, showing the same trend; increasing until a plateau is reached at concentrations above 1.8% (w/v) (
Elasticity (storage modulus, G′) and viscosity of the various suspensions were compared to data collected for current eye drops/ointments, across a range of materials: Paraffin, carbomer and biopolymer based systems (
Yielding behaviours of the suspensions are also very important, especially within retention mechanisms for delivery, as rapidly yielding systems result in fast clearance. Vice versa, where systems do not yield at all, materials are not readily eliminated from the body. The linear viscoelastic region (LVR) is a good indication of the yielding behaviours of the suspensions, as systems leave this linear region, the weak inter-particle interactions begin to break down and the systems flows. The length of the LVR was observed to be a function of the polymer concentration, showing an inverse relationship to gellan content (
Following yielding shear thinning behaviour is also vital for both application and elimination, allowing the suspensions to easily flow upon liquefaction. Shear thinning was observed across all systems, irrespective of polymer concentration (
In summary, it was shown that polymer concentration played a key role in the resulting material properties of the gellan microgel suspensions. Material characteristics such as elasticity, represented by the materials intrinsic G′ values, and viscosity were found to be a function of the polymer concentration, increasing until a plateau was formed at 1.8% (w/v). Effectively this meant that all systems were suitable for application within the ocular environment or injection, comparing closely with already commercially available products and demonstrating strong shear thinning behaviours needed for extrusion through small orifices. Furthermore, by comparing to commercial products and through conversations with independent clinicians, a polymer range between 0.5 and 1.35% (w/v) was narrowed down with 0.9% (w/v) proving optimal for the final formulation.
NaCl (0.1, 0.2, 0.4 and 0.8 M) were prepared through the addition of dry crystals (0.58, 1.16, 2.32 and 4.64 g) to deionised water (100 ml) using a volumetric flask. The NaCl was then allowed to dissolve using an inverting technique to aid the process. Once fully dissolved the solutions were kept at ambient conditions until further use.
Gellan solutions were prepared by dissolving powdered polymer into water/NaCl solution so that the final concentrations post-processing were equal to 0.9% and 1.8% (w/v). In brief, gellan powder was weighed out (4.5 g, 9.0 g) and added to 450 ml of deionised water. The mixture was allowed to heat to 95° C. under agitation, allowing the polymer to dissolve. Once fully dissolved, 25 ml of NaCl stock solution (either 0.1, 0.2, 0.4 and 0.8 M) was added to the solution resulting in a 5, 10, 20 or 40 mM concentration post-processing. The sol was then allowed to reach thermal equilibrium at 95° C. before processing.
MS were prepared using a jacketed pin mill set to 20° C. Gellan sols were pumped using a peristaltic pump into the pin mill at 3 ml/min so that it entered the processing chamber at 40° C. Prior to entry, using a syringe and syringe pump, water was pumped into the gellan stream (at a rate of 0.16 ml/min) so that they impinged, diluting the gellan sol to the final concentrations (0.9% and 1.8% (w/v); 5, 10, 20 or 40 mM NaCl). The mixture was then cooled under shear (1000 rpm) as it passed through the milling unit. On exiting, at 20° C., the gel was packaged and stored at 4° C. until further testing.
A rheometer (TA, AR-G2) equipped with a sandblasted parallel plate (40 mm diameter, 1 mm gap height) was used to test all samples, at 20° C.
Amplitude sweeps were obtained in strain controlled mode over a range of 0.1 to 100.0%. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20° C. prior to testing. Measurements were obtained at 1 Hz in a logarithmic fashion.
Viscosity profiles for the samples were obtained using a continuous ramp. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20° C. prior to testing. Increasing shear was applied to the sample in rate controlled mode, between 0.1 and 600 s−1 over a 3-minute ramp, with data points obtained in a logarithmic fashion.
Small deformation rheology—see
Large deformation rheology—see
Mechanistically, salts play a vital role in the gelation of many polymers, including gellan. Salt type, particularly valency (mono, di, tri etc.) are key to the resultant gel properties; typically, increasing the valency increases the gel strength, as more bridges are formed between polymers. However, in the case of gellan, di-valent ions, e.g. Ca2+, results in clouding (increased turbidity) of the resultant gel. As such mono-valent ions such as Na+ can be used to strengthen the junction sites between helices, forming the 3-dimentional gel structure. Therefore, resultant gel strength is a function of concentration of salt, also termed cross-linker, added. The effects of cross-linker concentration on formation and resultant microgel suspensions (“fluid gels”) formed through sheared gelation can be clearly seen in
The effects of salt concentration on the viscosity of the eye drop was also tested. Little changes were observed across all systems (
In summary, the addition of salt to the biopolymer system resulted in manipulation over the strength of the final product. Increasing salt concentrations ultimately increased the number of crosslinks in the system and final material elastic behaviour. Additionally, such effects were not seen to have drastic changes in viscosity, however, at lower polymer concentrations too much cross-linking could lead to the formation of heterogeneous suspensions and poor stability. Probing the elastic structure using strain sweeps allowed the yielding behaviour of the suspensions to be analysed, highlighting higher strain dependencies at 40 mM formulations. The reduction in plastic nature is expected to arise in discomfort for the patient in ocular applications, thus, the upper limit for cross-linker is suggested to be 20 mM.
NaCl (0.2 M) was prepared through the addition of dry crystals (1.16 g) to deionised water (100 ml) using a volumetric flask. The NaCl was then allowed to dissolve using an inverting technique to aid the process. Once fully dissolved the solutions were kept at ambient conditions until further use.
Gellan solutions were prepared by dissolving powdered polymer into water/NaCl solution so that the final concentrations post-processing were equal to 0.9% and 1.8% (w/v). In brief, gellan powder was weighed out (4.5 g, 9.0 g) and added to 475 ml of deionised water. The mixture was allowed to heat to 95° C. under agitation, allowing the polymer to dissolve. Once fully dissolved, 25 ml of NaCl stock solution (0.2 M) was added to the gellan sol, resulting in a 10 mM final concentration. The sol was then allowed to reach thermal equilibrium at 95° C. before processing.
MS were prepared using a jacketed pin mill, whereby the jacket temperature and the residence time within the mill were altered to result in cooling rates of 1, 3 and 6° C.min−1. As an example: the jacket was set to 5° C. and with a flow rate of 20 mlmin−1, the temperature of the fluid at the inlet was 46 and outlet 16, the residence time at this rate was 5 minutes, thus the cooling rate was equal to 6° C.min−1. On exiting, the gel was packaged and stored at 4° C. until further testing.
A rheometer (TA, AR-G2) equipped with a sandblasted parallel plate (40 mm diameter, 1 mm gap height) was used to test all samples, at 20° C.
Amplitude sweeps were obtained in strain controlled mode over a range of 0.1 to 100.0%. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20° C. prior to testing. Measurements were obtained at 1 Hz in a logarithmic fashion.
Viscosity profiles for the samples were obtained using a continuous ramp. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20° C. prior to testing. Increasing shear was applied to the sample in rate controlled mode, between 0.1 and 600 s−1 over a 3-minute ramp, with data points obtained in a logarithmic fashion.
Small deformation rheology: See
Large deformation rheology: See
Cooling plays a key role in the formation of gellan hydrogels, forcing the polymers through a random coil to helix transition. The effects of cooling rate on the formation of fluid gels was studied to evaluate related changes in material response. It was observed that at the lower polymer concentration (0.9% (w/v)) the cooling rate had little effect on both the degree of elasticity within the system and overall viscosity. However, at higher concentrations (1.8% (w/v)), the cooling rate has a much more pronounced effect on the elastic modulus (G′) (
The data obtained suggests an extra degree of control over the material properties at higher polymer concentrations. Being able to engineer specific elastic properties to the system without changing the overall viscosity. This is important within delivery systems to various areas of the body, allowing a semi-solid like structure to be placed in situ that provides a barrier or prolonged retention. Furthermore, the ability to retain the same viscosity means that even though it acts more solid like at rest, the system remains injectable.
The effects of cooling rate were found to be dependent on the polymer concentration, with the optimised eye drop formulation found to be independent on the cooling rate applied. However, at higher concentrations, the elastic structure can be finely tuned, without effecting the viscosity profiles. As such the degree of solidity can be manipulated when the system is at rest, but remains flowable (syringable) at larger deformation.
NaCl (0.2 M) was prepared through the addition of dry crystals (1.16 g) to deionised water (100 ml) using a volumetric flask. The NaCl was then allowed to dissolve using an inverting technique to aid the process. Once fully dissolved the solutions were kept at ambient conditions until further use.
Gellan solutions were prepared by dissolving powdered polymer into water/NaCl solution so that the final concentrations post-processing were equal to 0.9% and 1.8% (w/v). In brief, gellan powder was weighed out (4.5 g, 9.0 g) and added to 450 ml of deionised water. The mixture was allowed to heat to 95° C. under agitation, allowing the polymer to dissolve. Once fully dissolved, 25 ml of NaCl stock solution (0.2 M) was added to the solution resulting in a 10 mM concentration post-processing. The sol was then allowed to reach thermal equilibrium at 95° C. before processing.
MS were prepared using a jacketed pin mill set to 20° C. Gellan sols were pumped using a peristaltic pump into the pin mill at 3 ml/min so that it entered the processing chamber at 40° C. Prior to entry using a syringe and syringe pump, water was pumped into the gellan stream (at a rate of 0.16 ml/min) so that they impinged, diluting the gellan sol to the final concentrations (0.9 and 1.8% (w/v), 10 mM NaCl). The mixture was then cooled under shear (100, 500, 1000 and 2000 rpm) as it passed through the milling unit. On exiting, at 20° C., the gel was packaged and stored at 4° C. until further testing.
A rheometer (TA, AR-G2) equipped with a sandblasted parallel plate (40 mm diameter, 1 mm gap height) was used to test all samples, at 20° C.
Amplitude sweeps were obtained in strain controlled mode over a range of 0.1 to 100.0%. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20° C. prior to testing. Measurements were obtained at 1 Hz in a logarithmic fashion.
Viscosity profiles for the samples were obtained using a continuous ramp. Samples were loaded into the instrument and upper geometry lowered. Once trimmed, the sample was left to equilibrate at 20° C. prior to testing. Increasing shear was applied to the sample in rate controlled mode, between 0.1 and 600 s−1 over a 3-minute ramp, with data points obtained in a logarithmic fashion.
Small deformation rheology: See
Large deformation rheology: See
The degree of shear applied throughout the sol-gel transition of the gellan biopolymer was studied at two concentrations, 0.9% (w/v) and 1.8% (w/v). At the lower polymer concentration both degree of elasticity, as defined by G′, and viscosity was independent of the degree of shear experienced throughout the gelling profile. In all cases, the resultant material exhibited shear thinning over large deformations and solid-like behaviours at rest, however, the magnitude of such observations did not change (
In summary, changing the mixing speed during processing does not play a large role in the resultant material properties of the microgel suspensions with low polymer concentrations. As such, a wide range of processing shears can be applied without altering the final properties of the eye drop formulation. However, for the higher concentrations, used for “cream-like” spreadable systems, sheared processing plays more of an important role in the degree of solid-like behaviour at rest. In these cases, the degree of elasticity can be manipulated for the intended use.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.
The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.
Analyses of Contact Lens-Associated Microbial Keratitis. Optometry and Vision Science. 2014; 91(1):47-53.
Applications: Springer Netherlands; 2015.
Ophthalmology. 2014; 28(3):198-202.
2011; 11(2):110-28.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1820021.2 | Dec 2018 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2019/053479 | 12/9/2019 | WO | 00 |
