RELATED APPLICATIONS
This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application Nos. 62/374,532, filed August 12, 2016, 62/407,863, filed October 13, 2016, and 62/467,697, filed March 6, 2017, which applications are incorporated herein by reference.
GOVERNMENT SUPPORT
This invention was made with government support under Grant No. 1152561 awarded by National Science Foundation and Grant No. Al 51-061-0107 awarded by the United States Army. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Inflammation describes the cooperative response of afflicted cells to harmful stimuli (e.g., infection) or local tissue injury in attempt to restore homeostasis. Exposure of resident cells to these aberrant conditions initiates intracellular signaling cascades that result in the production and secretion of inflammatory mediators. Localized deposition of these

inflammatory entities serves to recruit immune cells (e.g., neutrophils) from the interstitium and vasculature to the site of injury or insult. Successful removal of the stimulus is followed by tissue repair, which introduces new immune cell types (e.g., macrophages) and signaling intermediaries and concludes the acute inflammatory response (Medzhitov, Nature, 2008.

454(7203): 428-435). However, if tissue homeostasis is not achieved within this timespan, a chronic inflammatory response ensues, whereby additional immune cells are introduced to the site of injury in attempt to contain it. Nevertheless, chronic inflammation can permanently undermine the healthy tissue state. Dysregulated signaling pathways that result from chronic inflammation have been implicated in a multitude of diseases, including dry eye, autoimmune disorders, cardiovascular disease, and cancer.

While the instigating cause of the immune response can be foreign to the host, disruption of localized tissue homeostasis due to aberrant cell signaling can also generate concentration gradients of signaling molecules that drive immune cell recruitment and response. For example, disruptions in tear film composition at the apical surface of the eye results in the increased production of proinflammatory cytokines that stimulate the immune cascade both acutely and

chronically (Luo et al., Eye & Contact Lens, 2005. 31(5): 186-193). This condition, known as keratoconjunctivitis sicca or dry eye syndrome (DES), persists due to a constant inflammatory stimulus that translates into altered cellular mechanical stress (via cell shrinkage) and gene expression (Brocker et al., Biomolecular Concepts, 2012. 3(4): 345-364). This further lends to the production of cytokines, which act on the local microenvironment and recruit mediator cell types of the acute inflammatory response. In turn, migratory neutrophils secrete additional proinflammatory morphogens that alter ocular limbal vascular permeability and thereby permit influx of activated T cells to the irritated eye surface, transitioning to a chronic inflammatory state (Baudouin, Survey of Ophthalmology, 2001. 45(2): S211-220).

One specific example of such a stimulus occurs with tear film fluid hyperosmolarity, which is caused by accelerated tear evaporation or tear gland hyposecretion. If the hyperosmotic stimulus is not addressed by the actions of immune cell mediators, homeostasis is not achieved and destruction of the ocular surface and tear glands evolves over time through dysregulated tissue remodeling mechanisms of the ocular surface. This cascade can lead to the increased production of matrix metalloproteinase 9 (MMP-9) that degrades the ocular surface in a runaway feed-forward mechanism of tissue remodeling.

Approaches to mitigate the inflammatory response typically target the production of proinflammatory signaling molecules. These include the use of glucocorticoid steroids, which function to decrease production of proinflammatory proteins while simultaneously increasing production of anti-inflammatory proteins within a recipient cell (Rhen et al., The New England Journal of Medicine, 2005. 353(16): 1711-1723). However, the effects of glucocorticoid signaling are potent and not confined to immune cell signaling, with impacts on metabolic and fluid homeostasis, neuronal function, and fetal development. Therefore, glucocorticoid signaling is heavily regulated and generally restricted to chronic hyperactive immune system disorders. Conversely, non-steroidal anti-inflammatory drugs (NSAIDs), which include aspirin, ibuprofen, and naproxen, function to inhibit cyclooxygenase (COX) enzyme activity, which precedes prostaglandin production that is heavily increased in inflamed cells (Ricciotti et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 2011. 31(5): 986-1000). NSAIDs are effective combatants of the inflammatory process, but are typically administered systemically and inhibit the functions of COX enzymes elsewhere in the body, which can contribute to stomach ulcerations and renal dysfunction. Given the off-target side effects of the above-mentioned therapeutic strategies, the anti-inflammatory agent ideally should be localized to the injured or infected tissue (e.g., skin, or eye surface).

The application of targeted anti-inflammatory therapies offers promise to attenuate the immune cell response with minimal side effects. For example, the development of antagonist antibodies against pro-inflammatory mediators (e.g., chemokines) has been employed for inflammatory diseases with promising efficacy (Skov et al., Journal of Immunology, 2008.

181(1): 669-679). However, the production cost of these proteins is significant and variability in antibody production may influence therapeutic efficacy. Alternatively, pharmacological inhibitors of signaling pathways upstream of chemokine production and/or secretion would be theoretically ideal, since they would eliminate recruitment of immune cell types involved in the acute and eventual chronic inflammatory response. Among these theoretical targets would be the nuclear factor-kappa B ( F-κΒ) transcription factor family, which is heavily implicated in the production of acute pro-inflammatory morphogens (Hayden et al., Cell Research, 2011. 21(2): 223-244). Natively, NF-κΒ subunits reside in the cytoplasm and are prevented from nuclear translocation by the masking of protein residues that target delivery to this region.

However, upon stimulation, the inhibitory protein is quickly degraded, thereby allowing translocation and DNA binding of NF-κΒ proteins and subsequent gene transcription.

A number of natural and synthetic inhibitors of NF-κΒ exist. Among the former is silk fibroin, which is a dimer composed of heavy and light protein chains (390 kD and 26 kD, respectively) isolated from the silkworm cocoon (reviewed by Altman et al., Biomaterials, 2003. 24(3): 401-416). These globular proteins assemble into a fibrillar architecture by the disulfide linkage of light and heavy chains and exhibit remarkable homogeneity in β-sheet secondary structure. Fibroin has been shown to inhibit transcription and upstream activation (i.e., via inhibition of protein kinases) of NF-κΒ protein subunits (Chon et al., International Journal of Molecular Medicine, 2012. 30(5): 1203-1210). Furthermore, hydrolyzed peptide fragments of fibroin have been shown to inhibit transcription of proinflammatory molecules that are classically under control of NF-κΒ (Kim et al., J. Neurosurg., 2011. 114(2): 485-90; J.

Microbiol. Biotechnol., 2012. 22(4): 494-500). However, the use of silk fibroin has not resulted in effective treatments for inflammatory conditions and wounds.

Furthermore, eye disease and injury remain persistent and serious concerns to the general world population. Ocular disease and trauma pose an immediate threat to normal vision by extending throughout the healing process and risking permanent disability or blindness from prolonged infection, chronic inflammation, and scar formation. As such, there is an immediate need for therapies to reduce inflammation and accelerate healing of the injured or inflamed ocular tissue.

SUMMARY

The invention provides a modified silk fibroin protein for therapeutic applications such as reducing inflammation as well as promoting wound healing and tissue regeneration. The modified protein has been shown to support corneal epithelial cell attachment and proliferation. The silk-derived protein (SDP) described herein is a fibroin-derived protein composition that has reduced beta-sheet activity, resulting in a highly-soluble and aqueous-stable material. SDP can be readily incorporated into solution-based product formulations at high concentrations.

Another advantage is that SDP has a high level of miscibility with other dissolved ingredients, such as those typically included in an ophthalmic formulation. One specific use of SDP is its inclusion in ophthalmic formulations as a novel protein component to enhance solution-wetting characteristics on the ocular surface. The SDP can be fractionated and it was surprisingly discovered that low molecular weight fractions of SDP have enhanced anti-inflammatory properties.

The invention therefore provides a fibroin-derived protein composition that possesses enhanced stability in an aqueous solution, wherein the primary amino acid sequences of the fibroin-derived protein composition differ from native fibroin by at least 4% with respect to the absolute values of the combined differences in amino acid content of serine, glycine, and alanine; cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains of fibroin are reduced or eliminated; a plurality of peptide chains in the protein composition terminate in amide (-C(=0)NEi2) groups; the composition has a serine content that is reduced by greater than 25% compared to native fibroin protein, wherein the serine content is at least about 5%; and wherein the average molecular weight of the fibroin-derived protein composition is less than 40 kDa and greater than 2 kDa.

In some embodiments, greater than 50% of the protein chains of the protein composition have a molecular weight within the range of 10 kDa to 60 kDa. In various embodiments, the protein composition does not gel upon sonication of an aqueous solution of the protein composition at concentrations of up to 10% w/w.

The protein composition can have less than 8% serine, less than 7% serine, or less than 6% serine amino acid residues. The protein composition can have greater than 46% glycine amino acids, greater than 46.5% glycine amino acids. The protein can have greater than 30% alanine amino acids, or greater than 30.5% alanine amino acids.

The protein composition can completely re-dissolves in water after being dried to a thin film. Beta-sheet protein structures are minimal or absent in aqueous solution. The protein

composition can maintain an optical absorbance in aqueous solution of less than 0.25 at 550 nm after at least five seconds of sonication.

The invention also provides an ophthalmic formulation comprising the protein composition described herein, and water, and optionally one or more of a buffering medium, a salt, a stabilizer, a preservative, and a lubricant.

The invention further provides a method for reducing inflammation comprising administering a fibroin-derived protein composition to inflamed tissue; wherein the primary amino acid sequences of the fibroin-derived protein composition differ from native fibroin by at least 4% with respect to the absolute value of the combined differences in amino acid content of serine, glycine, and alanine; cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains of fibroin are reduced or eliminated; a plurality of peptide chains in the protein composition terminate in amide (-C(=0) H2) groups; the composition has a serine content that is reduced by greater than 25% compared to native fibroin protein, and wherein the serine content is at least about 5%; and wherein the average molecular weight of the fibroin-derived protein composition is less than 60 kDa and greater than 2 kDa; thereby reducing transcription factor signaling within cell nuclei of the tissue, thereby reducing the inflammation. The average molecular weight of the fibroin-derived protein composition can also be less than 55 kDa, and/or greater than about 5 kDa, greater than 10 kDa, greater than 15 kDa, or greater than 20 kDa.

The administration to inflamed tissue can reduce transcription of one or more of the inflammatory genes TNF-a, MMP-9, IL-Ιβ, and IL-6. The reduction can be as much as 20%, 40%), 50%), or 60%) compared to in absence of the protein composition. The administration can be to the cornea and the administration can reduce the presence of MMP-9 in the cornea. The administration can be to the eye and the administration reduces inflammation on the ocular surface, for example, as determined by ELISA measurement of proinflammatory markers in the tear film. The the reduction in inflammation can be accompanied by an increase in cell migration rates at the point of inflammation, for example, an increase in cell proliferation, as determined by an MTT assay.

The protein composition can have an average molecular weight less than 40 kDa, or less than 35 kDa. The fibroin-derived protein composition can be dissolved in an ophthalmic formulation comprising one or more of a buffering medium, a salt, a stabilizer, a preservative, and a lubricant.

The inflammation can be inflammation caused by an ocular condition, wherein the ocular condition is dry eye syndrome, corneal ulcer, corneal erosion, corneal abrasion, corneal degeneration, corneal perforation, corneal scarring, epithelial defect, keratoconjunctivitis, idiopathic uveitis, corneal transplantation, age-related macular degeneration, diabetic eye, blepharitis, glaucoma, ocular hypertension, post-operative eye pain and inflammation, posterior segment neovascularization, proliferative vitreoretinopathy, cytomegalovirus retinitis, endophthalmitis, choroidal neovascular membrane, vascular occlusive disease, allergic eye disease, tumor, retinitis pigmentosa, eye infection, scleritis, ptosis, miosis, eye pain, mydriasis, neuralgia, cicatrizing ocular surface disease, ocular infection, inflammatory ocular disease, ocular surface disease, corneal disease, retinal disease, ocular manifestations of systemic diseases, hereditary eye condition, ocular tumor, increased intraocular pressure, herpetic infection, ptyrigium or scleral tumor, wound sustained to ocular surface, post-photorefractive keratotomy eye pain and inflammation, thermal or chemical burn to the cornea, scleral wound, or keratoconus and conjunctival wound. In one embodiment, the inflammation is caused by dry eye syndrome.

The invention further provides for the use of a fibroin-derived protein composition described herein for treating inflammation, wherein the primary amino acid sequences of the fibroin-derived protein composition differ from native fibroin by at least 4% with respect to the absolute value of the combined differences in amino acid content of serine, glycine, and alanine; cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains of fibroin are reduced or eliminated; a plurality of peptide chains in the protein composition terminate in amide (-C(=0) H2) groups; the composition has a serine content that is reduced by greater than 25% compared to native fibroin protein, and wherein the serine content is at least about 5%; and wherein the average molecular weight of the fibroin-derived protein composition is less than 60 kDa and greater than 10 kDa. The protein composition can have an average molecular weight less than 35 kDa. The composition can be a composition for the treatment of dry eye syndrome.

Accordingly, SDP compositions are provided herein that possess enhanced stability in aqueous solutions in which the primary amino acid sequence of native fibroin is modified from native silk fibroin, wherein cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains reduced or eliminated; wherein the composition has a serine content that is reduced by greater than 40% compared to native fibroin protein; and wherein the average molecular weight of the SDP is less than about 60 kDa.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the specification and are included to further demonstrate certain embodiments or various aspects of the invention. In some instances, embodiments of the invention can be best understood by referring to the accompanying

drawings in combination with the detailed description presented herein. The description and accompanying drawings may highlight a certain specific example, or a certain aspect of the invention. However, one skilled in the art will understand that portions of the example or aspect may be used in combination with other examples or aspects of the invention.

Figure 1A-D. p65 protein immunostaining (white) of hCLE cultures for F-κΒ activation. (A) Negative control cultures treated with PBS showed cytosolic p65 staining indicating native NF-κΒ inactivity. (B) Positive control cultures treated with PBS containing 1 ng/mL TNF-a demonstrated punctate p65 nuclear staining indicating protein translocation and hence a high level of NF-κΒ activation. (C and D) Culture treated with PBS, TNF-a, and 0.1% SDP or 1% SDP demonstrated a dose-dependent reduction in nuclear p65 staining indicating higher SDP concentrations inhibit NF-κΒ activation to a greater extent, respectively. (Scale bars = 20 um).

Figure 2. Summary qPCR results of relative fold gene expression for TNF-a and MMP-9 for hCLE cultures treated with PBS, PBS plus 0.5% SDP, PBS plus 1 ng/mL TNF-a cytokine, and PBS plus 1 ng/mL TNF-a cytokine plus 0.5% SDP. TNF-a and MMP-9 are known genetic markers of NF-κΒ activation. Cultures stimulated with TNF-a and treated with 0.5% SDP were found to have a 6-fold reduction in gene expression relative to TNF-a cytokine stimulated controls (Δ p < 0.01 compared to PBS for respective GOI; Θ p < 0.01 vs. SDP for respective GOI; and # p < 0.05 vs. indicated groups; n = 3).

Figure 3A-E. (A) Representative cross-section image of corneal tissue obtained from native rabbits immunostained for MMP-9. (B-D) Representative immunohistochemical images of corneal cross-sections obtained from rabbits harvested 72-hours post-surgery for the various treatment groups. MMP-9 staining decreased for both SDP treated groups (C and D) when compared to PBS- treated animals (B). (Scale bar = 50 μπι). (E) Summary graph of measured staining intensity (fluorescence intensity) of MMP-9 in corneas treated with PBS, 0.5% SDP, or 2% SDP (* p < 0.01 vs. Control; # p < 0.01 compared to 0.5% SDP, n = 3).

Figure 4. qPCR results of relative fold gene expression of IL-Ιβ and IL-6 for rabbit corneas treated with PBS, PBS plus 0.5% SDP, and PBS plus 2% SDP over a 72-hour period following surgical denudement of the epithelial surface. IL-Ιβ and IL-6 are known genetic markers of inflammation within the corneal tissue environment. Expression of both markers was significantly reduced in the presence of SDP treatment (* p < 0.01 vs. PBS for each GOI; n = 6).

Figure 5. Summary graph of H2O2 levels measured by electron paramagnetic resonance (EPR) spectroscopy in the presence of defined concentrations of dissolved proteins (PASF, SDP or SDP-4). H2O2 (20 μΜ) was incubated in the absence (Control) or presence of PASF, SDP, or SDP-4 (each at 0.5%, 1%, or 5%), and then introduced to a H2O2 -specific spin probe. EPR signal generated by the oxidized spin probe for each sample was measured and normalized to control samples (i.e., lacking protein). PASF increased EPR signal amplitude with increasing protein concentration. In contrast, SDP evoked a concentration-dependent reduction in EPR signal amplitude, demonstrating that SDP proteins scavenge H2O2. H2O2 scavenging was even more robust in the presence of SDP-4 proteins. Error bars are represented as S.D., N=3.

Figure 6. SDS-PAGE lanes 2-5 represent the respective molecular weight (MW) distributions of SEC-fractionated SDP populations for which biological impact was evaluated (SDP-1, SDP-2, SDP-3, SDP-4, and SDP). Lane 6 illustrates the non-fractionated SDP distribution from which fractions were derived. MW standards are shown in lane 1.

Figure 7. Representative images from in vitro wound healing assays demonstrate that cell growth and migration into the cell-free region (wound), outlined in white, is significantly accelerated in the presence of 5-mg/mL SDP-3 or SDP-4.

Figure 8. Summary bar graph illustrating percent wound closure at indicated time points during the scratch wound assay (*p< 0.05 vs Control), (#p< 0.05 vs SDP-1, n=3), (†p< 0.05 vs SDP-2, n=3).

Figure 9. MTT analysis of epithelial cell viability in hCLE cultures treated with 5-mg/mL of fractionated SDPs or (saline buffer) control. Treatment with SDP-3 and SDP-4 significant increased cell proliferation relative to control cells. Treatment with SDP-1 or SDP-2 did not change cell proliferation relative to controls (* p<0.05 vs. Control, n=3; # p<0.05 vs. SDP-1, n=3;† p<0.05 vs. SDP-2, n=3).

Figure 10. qPCR summary of TNF-a, MMP-9, and Interleukins -1α/β, -6, and -8 transcription in hCLE cells untreated (native) or stimulated with TNF-a to initiate inflammatory signaling, and treated with 1 mg/mL of fractionated SDP. Treatment with SDP-3 and SDP-4 significantly decreased transcription of the defined inflammatory genes, relative to control cells treated with PBS. († p<0.05 vs Native, n=3; * p<0.05 vs Control, n=3).

Figure 11. ELISA analysis of TNF-a cytokine secretion by hCLE cells untreated (native) or stimulated with TNF-a to initiate inflammatory signaling, and treated with 1 mg/mL of SDP fractions. Treatment with SDP-3 and SDP-4 significantly decreased secretion of the pro-inflammatory cytokine TNF-a, while SDP-2 significantly increased secretion, relative to control cells treated with PBS. († p<0.05 vs Native, n=3), (* p<0.05 vs Control, n=3).

Figure 12. Summary of Transwell migration assay demonstrating that treatment with TNF-a significantly increased HL-60 inflammatory cell migration relative to untreated (native) cultures. Addition of SDP-4 (1 mg/mL) resulted in a significant reduction of TNF-a driven HL-60 cell migration († p<0.05 vs Control, n=3; * p<0.05 vs Native, n=3).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides protein compositions derived from SDP for treating

inflammation and for treating wounds. Evidence supports that proteins isolated from the silkworm cocoon stimulate growth of corneal cells and alter expression of genes implicated in wound healing and inflammation (Figures 1-5). The protein compositions described herein also possess enhanced solubility and stability in aqueous solutions. Methods of making protein compositions include modifying the primary amino acid sequence of native fibroin such that cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains are reduced or eliminated. Additionally, the serine content of the protein composition is reduced by greater than 40% compared to native fibroin protein, and the average weight molecular weight of the proteins is less than about 60 kDa. In some cases, protein compositions described herein include or be derived from the protein compositions described in U.S. Patent No. 9,394,355, the entire disclosure of which is hereby incorporated by reference into this specification. Lower average molecular weight fractions can also be isolated to provide compositions with enhanced antiinflammatory activity by virtue of their enhanced ability to reduce the expression of proinflammatory genes compared to larger molecular weight fractions or the SDP composition in its entirety.

Discrete SDP subpopulations further enhance healing and reduce inflammation in the body, particularly in corneal tissue. Selected SDP fractions have been shown to enhance the effective potency of SDP on cell migration response and inflammation. The SDP fractions were prepared by extracting Bombyx mori silkworm cocoons fibers in 0.3% sodium carbonate at 95 °C, and then fibroin fiber was dissolved in 54% LiBr solution. The dissolved solution was autoclaved, coarse filtered, and then purified by diafiltration. The material was then filtered through a nominal polypropylene filter to produce a final SDP solution. The SDP solution was then separated by molecular weight (MW) through the use of one of two methods depending on the specific experiment. In the first method, centrifugation using molecular weight cutoff filters was utilized to separate out SDP protein fractions by molecular weight cutoff (MWCO) size. For example, SDP can be centrifuged at 5000 χ g until samples are reduced to 10% of starting volume (e.g., 15 mL initial volume concentrated to 1.5 mL, for certain experiments described herein). Proteins sieved through the filter are less than the molecular MWCO of a particular filter; the retained proteins are generally of equal or greater molecular weight. In a second method, sample SDP fractions can also be isolated by size exclusion chromatography (SEC) to produce discrete protein sub-populations, or fractions. Four fractions of decreasing average molecular weight were produced and are referred to as SDP-1, SDP-2, SDP-3, and SDP -4

(Figure 6)

The two smallest molecular weight SDP fractions, SDP-3 and SDP-4, significantly reduce inflammation and enhance wound healing of hCLE cultures in vitro through increased cell migration and proliferation effects (Figure 7-12). These SDP fractions inhibit inflammatory signaling, which can further enhance wound healing and improve long-term patient outcomes. The protein fractions derived from SDP can therefore be used for treating inflammation and related conditions. One specific therapeutic application is in the treatment of dry eye disease, which is known to be an inflammatory related disease that is driven, in part, by the F-KB signaling pathway, which is inhibited by SDP. In another specific therapeutic application, SDP may be utilized to treat post-surgical injuries to induce enhanced healing outcomes by reducing inflammation and/or increasing cell proliferation and/or migration, such as those injuries produced during refractive eye surgery or cataract removal, and/or accidental injuries where the corneal epithelium is compromised.

DEFINITIONS

The following definitions are included to provide a clear and consistent understanding of the specification and claims. As used herein, the recited terms have the following meanings. All other terms and phrases used in this specification have their ordinary meanings as one of skill in the art would understand. Such ordinary meanings may be obtained by reference to technical dictionaries, such as Haw ley 's Condensed Chemical Dictionary 14th Edition, by R.J. Lewis, John Wiley & Sons, New York, N.Y., 2001.

References in the specification to "one embodiment", "an embodiment", etc., indicate that the embodiment described may include a particular aspect, feature, structure, moiety, or characteristic, but not every embodiment necessarily includes that aspect, feature, structure, moiety, or characteristic. Moreover, such phrases may, but do not necessarily, refer to the same embodiment referred to in other portions of the specification. Further, when a particular aspect, feature, structure, moiety, or characteristic is described in connection with an embodiment, it is within the knowledge of one skilled in the art to affect or connect such aspect, feature, structure, moiety, or characteristic with other embodiments, whether or not explicitly described.

The singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a component" includes a plurality of such components, so that a component X includes a plurality of components X. It is further noted that the claims may be drafted to exclude an optional element. As such, this statement is intended to serve as antecedent basis for the use of exclusive terminology, such as "solely," "only," "other than", and the like, in connection with any element described herein, and/or the recitation of claim elements or use of "negative" limitations.

The term "and/or" means any one of the items, any combination of the items, or all of the items with which this term is associated. The phrases "one or more" and "at least one" are readily understood by one of skill in the art, particularly when read in context of its usage. For example, the phrase can mean one, two, three, four, five, six, ten, 100, or any upper limit approximately 10, 100, or 1000 times higher than a recited lower limit.

The term "about" can refer to a variation of ± 5%, ± 10%, ± 20%, or ± 25% of the value specified. For example, "about 50" percent can in some embodiments carry a variation from 45 to 55 percent. For integer ranges, the term "about" can include one or two integers greater than and/or less than a recited integer at each end of the range. Unless indicated otherwise herein, the term "about" is intended to include values, e.g., weight percentages, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, element, the composition, or the embodiment. The term about can also modify the end-points of a recited range as discuss above in this paragraph.

As will be understood by the skilled artisan, all numbers, including those expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, are approximations and are understood as being optionally modified in all instances by the term "about." These values can vary depending upon the desired properties sought to be obtained by those skilled in the art utilizing the teachings of the descriptions herein. It is also understood that such values inherently contain variability necessarily resulting from the standard deviations found in their respective testing measurements.

As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges recited herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof, as well as the individual values making up the range, particularly integer values. A recited range (e.g., weight percentages or carbon groups) includes each specific value, integer, decimal, or identity within the range. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, or tenths. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art, all language such as "up to", "at least", "greater than", "less than", "more than", "or more", and the like, include the number recited and such terms refer to ranges that can be subsequently broken down into subranges as discussed above. In the same manner, all ratios recited herein also include all sub-ratios falling within the broader ratio. Accordingly, specific values recited for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for radicals and substituents.

One skilled in the art will also readily recognize that where members are grouped together in a common manner, such as in a Markush group, an invention encompasses not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group. Additionally, for all purposes, an invention encompasses not only the main group, but also the main group absent one or more of the group members. An invention therefore envisages the explicit exclusion of any one or more of members of a recited group. Accordingly, provisos may apply to any of the disclosed categories or embodiments whereby any one or more of the recited elements, species, or embodiments, may be excluded from such categories or embodiments, for example, for use in an explicit negative limitation.

The term "contacting" refers to the act of touching, making contact, or of bringing to immediate or close proximity, including at the cellular or molecular level, for example, to bring about a physiological reaction, a chemical reaction, or a physical change, e.g., in a solution, in a reaction mixture, in vitro, or in vivo.

For a therapeutic application, an "effective amount" refers to an amount effective to treat a disease, disorder, and/or condition, or to bring about a recited effect. For example, an effective amount can be an amount effective to reduce the progression or severity of the condition or symptoms being treated. Determination of a therapeutically effective amount is within the capacity of persons skilled in the art. The term "effective amount" is intended to include an amount of a composition described herein, or an amount of a combination of peptides described herein, e.g., that is effective to treat or prevent a disease or disorder, or to treat the symptoms of the disease or disorder, in a host. Thus, an "effective amount" generally means an amount that provides the desired effect.

Fibroin is a protein derived from the silkworm cocoon (e.g., Bombyx mori). Fibroin includes a heavy chain that is about 350-400 kDa in molecular weight and a light chain that is about 24-27 kDa in molecular weight, wherein the heavy and light chains are linked together by a disulfide bond. The primary sequences of the heavy and light chains are known in the art. The fibroin protein chains possess hydrophilic N and C terminal domains, and alternating blocks of hydrophobic/hydrophilic amino acid sequences allowing for a mixture of steric and electrostatic

interactions with surrounding molecules in solution. At low concentration dilutions (1% or less) the fibroin protein molecule is known to take on an extended protein chain form and not immediately aggregate in solution. The fibroin protein is highly miscible with hydrating molecules such as HA, PEG, glycerin, and CMC, has been found to be highly biocompatible, and integrates or degrades naturally within the body through enzymatic action. Native fibroin, or also referred to herein as prior art silk fibroin (PASF), is known in the art and has been described by, for example, Daithankar et al. {Indian J. Biotechnol. 2005, 4, 115-121) and International Publication No. WO 2014/145002 (Kluge et al.).

The terms "silk-derived protein" (SDP) and "fibroin-derived protein" are used interchangeably herein. These materials are prepared by the processes described herein involving heat, pressure, and a high concentration of a heavy salt solution. Therefore 'silk-derived' and 'fibroin-derived' refer to the starting material of the process that structurally modifies the silk fibroin protein to arrive at a protein composition (SDP) with the structural, chemical and physical properties described herein. The SDP compositions possess enhanced solubility and stability in an aqueous solution. The SDP may be derived from silkworm silk (e.g., Bombyx mori), spider silk, or genetically engineered silk.

As used herein, the terms "molecular weight" and "average molecular weight" refer to weight average molecular weight determined by standard Sodium Dodecyl Sulfate

Polyacrylamide Gel Electrophoresis (SDS-PAGE) electrophoresis methods undertaken with a NuPAGE™ 4% - 12% Bis-Tris protein gel (ThermoFisher Scientific, Inc.) in combination analysis with ImageJ software (National Institutes of Health). ImageJ is used to determine the relative amount of protein of a given molecular weight in a sample. The software accomplishes this by translating the staining on the gel (i.e., the amount of protein) into a quantitative signal intensity. The user then compares this signal to a standard (or ladder) consisting of species of known molecular weights. The amount of signal between each marker on the ladder is divided by the whole signal. The cumulative summation of each protein sub-population, also referred to herein as fractions and interchangeably also referred to as fragments, allows the user to determine the median molecular weight, which is referred to herein as the average molecular weight. In practice, electrophoresis gels are stained, and then scanned into greyscale images, which are converted into histograms using ImageJ. Total pixel intensity within each gel lane is quantified by ImageJ (i.e., total area under the histogram), and subsequently fractionated into populations demarcated by protein molecular weight standards also stained on the gel. The histogram pixel area between any two molecular weight standards is divided by the total histogram area of the protein, thereby providing the fraction of total protein that falls within these molecular weights. Analysis by other methods may provide different values that account for certain peptides that are not accounted for by SDS-PAGE methods. For example, FIPLC can be used to analyze the average molecular weights, which method provides values that are typically about 10-30%, lower than determined by SDS-PAGE (increasing differences as molecular weights decrease).

PREPARATION OF SDP COMPOSITIONS

SDP compositions described herein can possess enhanced stability compared to native fibroin in aqueous solutions. The enhanced stability achieved by the SDP compositions provided herein, which is also referred herein as a SDP, allow the material to remain in solution significantly longer than the native / PASF proteins (referred to herein as PASF). Enhanced stability of the SDP materials provided herein also allow for the preparation of SDP solutions of high concentration without aggregation, precipitation, or gelation. In commercial applications such as eye drops or applications requiring protein to be soluble in solution, enhanced stability can provide suitably lengthy shelf life and increased quality of the product by reducing protein aggregation. Potential aggregation of protein in solution can negatively impact a product's desired performance for a particular application. This is especially true for eye drop

formulations given that aggregates could cause abrasive damage to the ocular surface. The ability to concentrate the SDP to high constitutions in solution (over 50% w/v or > 500 mg/mL) is significantly advantageous for inventorying a useful working solution that can be used as-is or diluted for any number of applications. Examples of such applications are the use of SDP as an ingredient in ophthalmic formulations, such as those provided herein, as a protein supplement or additive.

Transforming the primary amino acid sequences of the native fibroin protein into the SDP material may enhance its stability in aqueous solutions by decreasing the susceptibility of the molecules to aggregate. Aggregation eventually leads to gel formation. In the

transformation of the native fibroin, both serine and cysteine amino acids are cleaved in the presence of high heat and dehydrating conditions. Similarly, Patchornik et al. (J. Am. Chem. Soc. 1964, 86, 1206) demonstrated that a dehydroalanine (DHA) intermediate is formed from serine and cysteine in solution. The amino acid degradation is further driven when in the presence of a strong dehydrating solvent system, such as the 50-55%) w/v LiBr solution as described herein, in which a hydride shift takes place to induce removal of water. The degradation reaction can take place in the presence of hydroxide ions (e.g., pH 7.5 to pH 11), which further drives cleavage of the DHA intermediate. This cleavage forms an amide, a

pyruvoyl peptide, and LiBr. One viable chemical mechanism is outlined in Scheme 1 for a serine amino acid, which scheme is also applicable for cysteine amino acids. Chemical alteration of the serine and cysteine amino acids of the PASF protein into a DHA intermediate with further hydrolytic cleavage leads to enhanced solution stability of the SDP products.

Scheme 1. Schematic of an underlying chemical reaction for serine and cysteine degradation.

Degradation is driven by the production of a DHA intermediate that is formed from a hydride shift reaction in the presence of a dehydrating high salt concentration environment. Degradation of DHA is then accomplished through an SN2 reaction within the basic solvent environment.

This cleavage reaction discussed above can significantly affect macromolecular properties of the resulting peptides, which results in an aqueous solution of stabilized SDP material. The initial protein aggregation of fibroin is believed to be instigated by interactions of the native fibroin heavy and light chains at the cysteine amino acids as described by Greving et al. (Biomacromolecules 2012, 13(3): 676-682). The cysteine amino acids within the fibroin light and heavy protein chains interact with one another through disulfide linkages. These disulfide bridges participate in fibroin protein aggregation and gel network flocculation.

Without the native fibroin light chain present, the proteins are significantly less susceptible to aggregation. Therefore, the process described herein can effectively reduce\ the native fibroin light chain's ability to form disulfide bonds by reducing cysteine content and thus reducing or eliminating disulfide bond-forming capability. Through this mechanism, the transformative process described herein functionally stabilizes the resulting SDP in solution by reducing or eliminating the ability to form cysteine-derived aggregations.

In addition to aggregation-inducing disulfide bridges, the susceptibility of the silk fibroin to further aggregate into flocculated structure is also driven by the protein's amino acid chemistry as described by Mayen et al. {Biophysical Chemistry 2015, 197: 10-17). Molecular modeling of silk fibroin serine, alanine, and glycine amino acid sequences have shown that the presence of serine enhances initial protein-to-protein interaction through a greater propensity to create hydrogen bonding between adjacent fibroin protein chain moieties. The models demonstrate that reduced serine and increased alanine and glycine decrease the initial propensity for protein aggregation. The molecular modeling observations indicate that by altering the native amino acid chemistry of the fibroin protein a material could be generated that would have higher stability in aqueous solution.

One strategy to accomplish enhanced stability is to eliminate charged functional groups, such as hydroxyls, from the protein. Due to the relatively high electronegativity of hydroxyl groups, this chemistry can drive both hydrogen bonding with available hydrogen atoms and nonspecific charge interactions with positively charged amino acid groups. Almost 12% of the native fibroin protein's content is composed of serine, which bears a hydroxyl functional group. Therefore, by reducing the availability of hydroxyl groups that facilitate hydrogen bonding, the overall protein stability in solution may be enhanced. The process described herein effectively reduces the amount of serine content and increases the relative alanine and glycine content, which eliminates the number of available hydroxyl groups available to create hydrogen bonds. Through this mechanism the process described herein functionally stabilizes the resulting SDP in solution extended periods of time (e.g., at least several [6-8] months, and/or for more than 1.5 years; extended studies are ongoing, indicating that stability may be maintained for more than 2 years, or more than 3 years).

In addition to the reduction of cysteine and serine moieties, solvent charge interaction is important for stabilizing a protein solution. After initial protein flocculation, the gelation process is believed to continue to drive closer associations among the native fibroin heavy chains, which leads to both intra- and inter-molecular beta-sheet formation among hydrophobic blocks of the heavy chains. Once significant beta-sheet formation occurs, the fibroin solution transitions to a gel. As the solution transitions to a gel, and the fibroin becomes insoluble and is no longer useful as a solution-based product. To prevent gelation, the pH of the SDP solution can be raised to high alkalinity to enhance stability, for example over a pH of 7.5. As a result, the increased pH produces additional free hydroxyl groups that form a valence shield around the SDP molecules in solution. The formed valence shield acts to produce a zeta potential that stabilizes the protein by reducing protein-protein interactions derived from hydrogen bonding or non-specific charged and/or hydrophobic interactions. The fibroin-transformation process

functionally stabilizes processed SDP in solution through this mechanism and others. The SDP can be derived from Bombyx mori silkworm fibroin or other fibroin from the Bombyx genus or other silk proteins.

SDP material can be prepared by the following process.

1. Silk cocoons are prepared by removing pupae material and pre-rinsing in warm water.

2. Native fibroin protein fibers are extracted from the gum-like sericin proteins by washing the cocoons in water at high water temperature, typically 95 °C or more, at alkaline pH.

3. The extracted fibroin fibers are dried and then dissolved using a solvent system that neutralizes hydrogen bonding between the beta-sheets; a 54% LiBr aqueous solution of 20% w/v silk fibroin protein is effective for this neutralization step.

4. The fibroin protein dissolved in LiBr solution is processed in an autoclave

environment (-121 °C [-250 °F], at -15-17 PSI pressure, for approximately 30 minutes at temperature).

5. The heat-processed fibroin protein and LiBr solution are then dialyzed to remove lithium and bromide ions from the solution. At this point in the process the material has been chemically transformed to SDP.

6. The dialyzed SDP is then filtered to remove any non-dissolved aggregates and contaminating bioburden.

The SDP solution is produced using a distinctly different process than the process used for current silk fibroin solution production. Notably, the autoclaving of the silk fibroin protein while it is combined with LiBr in solution initiates chemical transitions to produce the stabilized SDP material. The fibroin protein is dissolved in LiBr solution, which neutralizes hydrogen bonding and electrostatic interactions of the solubilized native fibroin protein. This leaves the protein without specific secondary structure confirmations in solution. As a result, the thermodynamic energy required to hydrolyze covalent bonding within the fibroin protein chain is at its lowest energy requirements to initiate hydrolytic cleavage.

In one embodiment, the temperature is set to 121 °C for 30 minutes at 15-17 PSI autoclave conditions. However, in various embodiments, the processing conditions may be modified to stabilize the SDP material to varying degrees. In other embodiments, additional protein solubilization agents can be used in the process, including other or additional halide salts such as calcium chloride and sodium thiocyanate, organic agents such as urea, guanidine hydrochloride, and 1,1, 1,3,3,3-hexafluoroisopropanol, additional strong ionic liquid solution additives such as calcium nitrate and l-butyl-3-methylimidazolium chloride, or a combination thereof.

SDP COMPOSITIONS

Protein composition described herein can be derived from silk fibroin and possess enhanced solubility and stability in aqueous solutions. The compositions can be used to treat and reduce inflammation. In one embodiment, the SDP and/or fractions thereof have primary amino acid sequences that differ from native fibroin by at least 4% (via summation of the absolute values of the differences) with respect to the combined amino acid content of serine, glycine, and alanine. A plurality of the protein fragments of SDP can terminate in amide (-C(=0) H2) groups. SDP can have a serine content that is reduced by greater than 40% compared to native fibroin, wherein the serine content is at least about 5%. The cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains of fibroin may be reduced or eliminated. The SDP compositions provided herein possess enhanced stability in an aqueous solution. In certain embodiments, at least 75 percent of the protein fragments have a molecular weight of less than about 60 kDa and act as an anti-inflammatory that also promotes cell migration and proliferation in the tissue to close the wound. The composition may comprise less than 8.5% serine amino acid residues. In some embodiments, the average molecular weight of the SDP is less than 55 kDa.

In some cases, protein compositions provided herein are prepared by a process comprising heating an aqueous fibroin solution at an elevated pressure. The aqueous fibroin solution includes lithium bromide at a concentration of at least 8M. The aqueous fibroin solution is heated to at least about 105 °C (221 °F) under a pressure of at least about 10 PSI for at least about 20 minutes, to provide the protein composition. As a result of these processing conditions, the polypeptides of the protein composition comprise less than 8.5% serine amino acid residues, and a plurality of the protein fragments terminate in amide
groups.

In some cases, protein compositions provided herein are prepared by a process comprising heating an aqueous fibroin solution at an elevated pressure, wherein the aqueous fibroin solution comprises lithium bromide at a concentration of 9-10M, and wherein the aqueous fibroin solution is heated to a temperature in the range of about 115 °C (239 °F) to about 125 °C (257 °F), under a pressure of about 15 PSI to about 20 PSI for at least about 20 minutes; to provide the protein composition. The protein composition can include less than 6.5% serine amino acid residues.

SDP compositions provided herein can possess enhanced stability in aqueous solution, wherein: the primary amino acid sequences of the SDP composition differs from native fibroin by at least 4% with respect to the combined (absolute value) difference in serine, glycine, and alanine content (SDP vs. PASF); cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains are reduced or eliminated; and the composition has a serine content that is reduced by greater than 25% compared to native fibroin protein. The average molecular weight of the SDP composition can be less than about 60 kDa and greater than about 2 kDa, or greater than about 10 kDa, as determined by the MWCO of the dialyzing membrane and SDS-PAGE analysis.

In some cases, SDP compositions provided herein possess enhanced stability in aqueous solution, wherein: the primary amino acid sequences of the SDP composition differs from native fibroin by at least 6% with respect to the combined difference in serine, glycine, and alanine content; cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains are reduced or eliminated; and the composition has a serine content that is reduced by greater than 40% compared to native fibroin protein. The average molecular weight of the SDP composition can be less than about 55 kDa and greater than about 10 kDa, as determined by the MWCO of the dialyzing membrane and SDS-PAGE analysis.

In some cases, SDP compositions provided herein possess enhanced stability in aqueous solutions, wherein: the primary amino acid sequences of the SDP composition is modified from native silk fibroin; cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains are reduced or eliminated; the average molecular weight of the SDP composition is less than about 60 kDa and greater than about 10 kDa; and a 5% w/w aqueous solution of the SDP composition maintains an optical absorbance at 550 nm of less than 0.25 for at least two hours after five seconds of sonication.

In some cases, SDP compositions provided herein possess enhanced stability in aqueous solutions, wherein: the primary amino acid sequences of the SDP composition is modified from native silk fibroin such that they differ from native fibroin by at least 5% with respect to the combined (absolute value) difference in serine, glycine, and alanine content. In some embodiments, the difference of is at least 6%, 8%, 10%, 12% or 14% compared to native fibroin. Cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains are reduced or eliminated; the average molecular weight of the SDP composition is less than about 60 kDa and greater than about 15 kDa; and the SDP composition maintains an optical absorbance at 550 nm of less than 0.2 for at least two hours after five seconds of sonication.

In some cases, SDP compositions provided herein can be isolated and/or purified as a dry powder or film, for example, by dialysis and/or filtration. Alternatively, SDP compositions provided herein can be isolated and/or purified as a stable aqueous solution, which can be modified for use as a therapeutic formulation, such as an ophthalmic formulation.

In various embodiments, the amino acid compositions of the SDP found in protein

compositions provided herein can differ from the amino acid composition of native fibroin by at least 4%, by at least 4.5%, by at least 5%, or by at least 5.5%, or by at least 6%, with respect to the content of serine, glycine, and alanine combined.

In some cases, protein compositions described herein have a serine content that is reduced by greater than 25%, by greater than 30%, by greater than 35%, by greater than 40%, or by greater than 45%, compared to the serine content of native fibroin protein.

The average molecular weight of SDP compositions provided herein can be less than about 80 kDa, less than about 70 kDa, less than about 60 kDa, or less than about 55 kDa, or the composition has an average molecular weight of about 50-60 kDa, or about 51-55 kDa. In various embodiments, the average molecular weight of the SDP composition can be greater than about 2 kDa, greater than about 10 kDa, greater than about 15 kDa, greater than about 20 kDa, greater than about 25 kDa, greater than about 30 kDa, greater than about 35 kDa, greater than about 40 kDa, or greater than about 50 kDa. Accordingly, the (weight average) average molecular weight of SDP compositions provided herein can be about 5 kDa to about 80 kDa, about 10 kDa to about 65 kDa, about 15 kDa to about 60 kDa, about 15 kDa to about 60 kDa, about 20 kDa to about 65 kDa, about 20 kDa to about 55 kDa. In various embodiments, the average molecular weight of the SDP composition is about 45 kDa to about 65 kDa, about 45 kDa to about 60 kDa, about 50 kDa to about 65 kDa, or about 50 kDa to about 60 kDa.

The SDP protein compositions can be soluble in water at 40% w/w without any precipitation observable by ocular inspection.

In some embodiments, protein compositions provided herein comprise less than 8% serine amino acid residues. In some cases, protein compositions provided herein comprise less than 7.5%) serine amino acid residues, less than 7% serine amino acid residues, less than 6.5% serine amino acid residues, or less than 6% serine amino acid residues. The serine content of the peptide compositions is generally at least about 4%, or at least about 5%, or about 4-5%.

In some embodiments, protein compositions provided herein comprise greater than 46.5%) glycine amino acids, relative to the total amino acid content of the protein composition. In some cases, protein compositions provided herein comprise greater than 47% glycine amino acids, greater than 47.5% glycine amino acids, or greater than 48% glycine amino acids.

In some embodiments, protein compositions provided herein comprise greater than 30% alanine amino acids, relative to the total amino acid content of the protein composition. In some cases, protein compositions provided herein comprise greater than 30.5% alanine, greater than 31% alanine, or greater than 31.5% alanine.

In some embodiments, protein compositions provided herein can completely re-dissolve

after being dried to a thin film. In various embodiments, protein compositions provided herein can lack beta-sheet protein structure in aqueous solution. The protein composition can maintain an optical absorbance in aqueous solution of less than 0.25 at 550 nm after at least five seconds of soni cation.

In some embodiments, protein compositions provided herein can be in combination with water. In some cases, protein compositions provided herein can completely dissolve in water at a concentration of 10% w/w, or even greater concentrations such as 15% w/w, 20% w/w, 25% w/w, 30%) w/w, 35%) w/w, or 40% w/w. In some embodiments, protein compositions provided herein can be isolated and purified, for example, by dialysis, filtration, or a combination thereof.

In various embodiments, protein compositions provided herein can enhance the spreading of an aqueous solution comprising the protein composition and ophthalmic formulation components, for example, compared to the spreading of a corresponding

composition that does not include the protein composition. This enhanced spreading can result in an increase in surface area of the aqueous solution by greater than twofold, or greater than threefold.

In various embodiments, the SDP protein compositions do not form a gel at

concentrations up to 20% w/v, up to 30% w/v, or up to 40% w/v in water. In some

embodiments, SDP compositions provided herein can have glycine-alanine-glycine-alanine (GAGA) (SEQ ID NO: 1) segments of amino acids that comprise at least about 47.5% of the amino acids of the SDP composition. In some cases, SDP compositions provided herein can also have GAGA (SEQ ID NO: 1) segments of amino acids that comprise at least about 48%, at least about 48.5%, at least about 49%, at least about 49.5%, or at least about 50%, of the amino acids of the protein composition.

In various embodiments, SDP compositions provided herein can have glycine-alanine (GA) segments of amino acids that comprise at least about 59% of the amino acids of the SDP composition. In some cases, SDP compositions provided herein can also have GA segments of amino acids that comprise at least about 59.5%, at least about 60%, at least about 60.5%, at least about 61%), or at least about 61.5%, of the amino acids of the protein composition.

Protein compositions provided herein can be prepared by a process comprising heating an aqueous fibroin solution at an elevated pressure, wherein the aqueous fibroin solution comprises lithium bromide at a concentration of at least 8M, and wherein the aqueous fibroin solution is heated to at least about 105 °C (221 °F) under a pressure of at least about 10 PSI for at least about 20 minutes; to provide the protein composition, wherein the protein composition comprises less than 8.5% serine amino acid residues. Therefore, methods of preparing a SDP

composition are also provided herein. Methods of preparing a SDP composition provided herein can include one or more of the process steps described herein.

In some cases, methods of preparing provided herein can use lithium bromide having a concentration between about 8.0M and about 11M. In some embodiments, the concentration of lithium bromide is about 9M to about 10M, or about 9.5M to about 10M.

In some embodiments, the aqueous fibroin solution that contains lithium bromide is heated to at least about 107 °C (225 °F), at least about 110 °C (230 °F), at least about 113 °C (235 °F), at least about 115 °C (239 °F), or at least about 120 °C (248 °F).

In some embodiments, the aqueous fibroin solution that contains lithium bromide is heated under a pressure of at least about 12 PSI, at least about 14 PSI, at least about 15 PSI, or at least about 16 PSI, up to about 18 PSI, or up to about 20 PSI.

In some embodiments, the aqueous fibroin solution that contains lithium bromide is heated for at least about 20 minutes, at least about 30 minutes, at least about 45 minutes, or at least about 1 hour, up to several (e.g., 12-24) hours.

In some embodiments, the protein composition can be dissolved in water at 40% w/w without observable precipitation.

In some embodiments, the fibroin has been separated from sericin.

In some embodiments, lithium bromide has been removed from the protein composition to provide a purified protein composition. In various embodiments, the protein composition has been isolated and purified, for example, by dialysis, filtration, or a combination thereof.

In additional embodiments, the protein composition has properties as described above, and amino acid compositions as described above regarding serine, glycine, and alanine content.

In various embodiments, the protein composition re-dissolves after drying as a thin film, a property not found with native fibroin. The protein composition can lack beta-sheet protein structure in solution. The protein composition can maintain an optical absorbance in solution of less than 0.25 at 550 nm after at least five seconds of sonication.

In one specific embodiment, the invention provides a protein composition prepared by a process comprising heating an aqueous fibroin solution at an elevated pressure, wherein the aqueous fibroin solution comprises lithium bromide at a concentration of 9-10M, and wherein the aqueous fibroin solution is heated to a temperature in the range of about 115 °C (239 °F) to about 125 °C (257 °F), under a pressure of about 15 PSI to about 20 PSI for at least about 30 minutes; to provide the protein composition, wherein the protein composition comprises less than 6.5% serine amino acid residues, and the protein composition has an aqueous viscosity of less than 10 cP as a 15% w/w solution in water.

SDP compositions are chemically distinct from native silk fibroin protein as a result of the preparation process, resulting in changes in amino acid content and the formation of terminal amide groups. The resulting SDP has enhanced solubility and stability in aqueous solution. The SDP can be used in a method for forming, for example, ophthalmic formulations with a protein composition described herein, for example, an aqueous solution of the protein composition. The solution can include about 0.01% to about 92% w/v SDP. The solution can be about 8%> to about 99.9% w/v water.

In some embodiments, processes are provided that induces hydrolysis, amino acid degradation, or a combination thereof, of fibroin protein such that the average molecular weight of the protein is reduced from about 100-200 kDa for silk fibroin produced using prior art methods to about 30-90 kDa, or about 30-50 kDa, for the SDP material described herein. The resulting polypeptides can be a random assortment of peptides of various molecular weights averaging to the ranges recited herein.

In addition, the amino acid chemistry can be altered by reducing cysteine content to non-detectable levels by standard assay procedures. For example, the serine content can be reduced by over 50%> from the levels found in the native fibroin, which can result in increases of overall alanine and glycine content by 5% (relative amino acid content), as determined by standard assay procedures. The SDP material can have a serine content of less than about 8%> relative amino acid content, or a serine amino acid content of less than about 6% relative amino acid content. The SDP material can have a glycine content above about 46.5%>, and/or an alanine content above about 30%> or above about 30.5%. The SDP material can have no detectable cysteine content, for example, as determined by HPLC analysis of the hydrolyzed polypeptide of the protein composition. The SDP material can form 90% less, 95% less, or 98% less beta-sheet secondary protein structures as compared to native silk fibroin protein, for example, as determined by the FTIR analysis.

Stability Evaluations. The stability of a protein solution can be evaluated a number of different ways. One suitable evaluation is the Lawrence Stability Test described below in Example 1 below. Another suitable evaluation is the application of sonication to a protein solution, followed by optical absorbance analysis to confirm continued optical clarity (and lack of aggregation, beta-sheet formation, and/or gelation). Standard sonication, or alternatively ultrasonication (sound frequencies greater than 20 kHz), can be used to test the stability of an SDP solution. Solutions of SDP are stable after subjecting to sonication. The SDP composition maintains an optical absorbance at 550 nm of less than 0.25 for at least two hours after five seconds of sonication. For example, a 5% w/w solution of the protein composition can maintain an optical absorbance of less than 0.1 at 550 nm after five seconds of sonication at -20 kHz, the standard conditions used for the sonication described herein. In various embodiments, SDP composition aqueous solutions do not gel upon sonication at concentrations of up to 10% w/w. In further embodiments, SDP composition aqueous solutions do not gel upon ultrasoni cation at concentrations of up to 15% w/w, up to 20% w/w, up to 25% w/w, up to 30% w/w, up to 35% w/w, or up to 40% w/w.

Low viscosity. As a result of its preparation process and the resulting changes in the chemical structures of its peptide chains, SDP has a lower viscosity than native silk fibroin (PASF). As a 5% w/w solution in water (at 25.6 °C), native silk fibroin has a viscosity of about 5.8 cP, whereas under the same conditions, SDP has a viscosity of about 1.8 cP, and SDP -4 has a viscosity of about 2.7 cP. SDP maintains a low viscosity compared to PASF at higher concentrations as well. The SDP composition can have an aqueous viscosity of less than 5 cP, or less than 4 cP, as a 10% w/w solution in water. In various embodiments, SDP remains in solution up to a viscosity of at least 9.8 cP. SDP also has an aqueous viscosity of less than 10 cP as a 15%) w/w solution in water. SDP can also have an aqueous viscosity of less than 10 cP as a 24%) w/w solution in water.

The process described herein provides a protein composition where the fibroin light chain protein is not discernable after processing, as well when the sample is run using standard Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) electrophoresis methods undertaken with a NuPAGE™ 4%-12% Bis-Tris protein gel (ThermoFisher Scientific, Inc.). For example, in one embodiment, the SDP material can have the fibroin light chain over 50%) removed when compared to native silk fibroin protein. Furthermore, the resulting SDP material forms minimal to no beta-sheet protein secondary structure post-processing, while silk fibroin solution produced using prior art methods forms significant amounts of beta-sheet secondary structure. In one embodiment, the SDP material can be prepared by processing silk fibroin fibers under autoclave or autoclave-like conditions (i.e., approximately 120 °C and 14-18 PSI) in the presence of a 40-60%> w/v lithium bromide (LiBr) solution.

SDP COMPOSITION FRACTIONS

Silk Technologies, Ltd. has developed the silk-derived protein (SDP) product that can be readily incorporated into ophthalmic product formulations for reducing inflammation and enhancing the wound healing process. The SDP product can be separated into smaller protein fractions or sub-populations based on molecular weight to enhance the anti-inflammatory and wound healing properties. SDP protein sub-populations, also referred to as fractions or

fragments, can be separated by any suitable and effective method, for example, by size exclusion chromatography or membrane dialysis. For example, the fractions can be separated in to 2-4 different groups based on decreasing average molecular weights. Example 6 describes one method for preparing four different fractions that have the same overall amino acid content and terminal amide content but different average molecular weights. It was surprisingly discovered that the different fractions also possess different biological properties, for example, for reducing inflammation in the body and in various tissues as a result of differences in cellular uptake of the different fractions.

This disclosure therefore provides methods of reducing inflammation and/or enhancing wound healing using SDP, including low average molecular weight fractions of SDR Also described are compositions for reducing inflammation in the treatment of ocular conditions, such as, but not limited to, dry eye disease, and/or injury, including corneal wounds. The treatments can include the administration of a formulation that includes SDP, or a low molecular weight SDP sub-population. In certain embodiments, the invention provides methods for treating a disease state and/or wound comprising administering to a subject in need thereof a composition comprising low molecular weight SDP (e.g., SDP-3 or SDP -4).

The methods can include applying a composition of SDP fractions to diseased or injured tissue. The protein fractions can have primary amino acid sequences that differ (via summation of absolute value differences) from native fibroin by at least 4% with respect to the combined amino acid content of serine, glycine, and alanine. A plurality of the protein fragments can terminate in amide (-C(=0)NEi2) groups. Compositions provided herein may have a serine content that is reduced by greater than 40% compared to native fibroin, wherein the serine content is at least about 5%. The cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains of fibroin may be reduced or eliminated. In some embodiments, at least 75 percent of the protein fragments have a molecular weight of less than about 100 kDa. Such compositions reduce inflammation, and promote cell migration and/or proliferation in the tissue to treat the disease state and/or enhance closure of the wound. The SDP compositions possess enhanced solubility and stability in an aqueous solution.

SDP composition fractions can have an average molecular weight between about 2 kDa and 60 kDa. In one embodiment, a low molecular weight fraction having an average molecular weight of 25-38 kDa, of 32-35 kDa, or about 34 kDa ± 5%, is isolated, which fraction is referred to herein as SDP-4.

In some embodiments, at least 60 percent of the protein fragments have a molecular weight of less than about 60 kDa, or less than about 55 kDa, to promote cell migration and

proliferation in the tissue to close the wound. In another embodiment, at least 90 percent of the protein fragments have a molecular weight of less than about 100 kDa and promote cell migration and proliferation in the tissue to close the wound.

In some embodiments, at least 80 percent of the protein fragments have a molecular weight between about 10 kDa and 85 kDa. In some embodiments, at least 50 percent of the protein fragments have a molecular weight between about 20 kDa and 60 kDa. In some embodiments, at least 85 percent of the protein fragments have a molecular weight of greater than about 10 kDa. In some embodiments, at least 90 percent of the protein fragments have a molecular weight of greater than about 5 kDa.

In certain embodiments, the invention provides an SDP composition comprising low molecular weight SDP and a pharmaceutically acceptable carrier. The low molecular weight SDP can have an average molecular weight of less than 60 kDa. In some embodiments, the low molecular weight SDP is less than 40 kDa and the fraction reduces inflammation and/or enhances cell migration and/or proliferation.

In one embodiment, the low molecular weight SDP, for example, SDP -4, is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater of the total SDP in a composition. In some embodiments, the composition does not comprise high molecular weight SDP, for example, the sample has an average molecular weight of less than about 35 kDa.

In one embodiment, the SDP-4 fraction has an average molecular weight of 33-35 kDa, as determined by SDS-PAGE / ImageJ analysis, as previously described above, and a pH 8.1-8.3, an osmolarity of about 23 mOsm, and a viscosity of about 1.5-3 cP at 25 °C, each as a 50 mg/mL solution in water.

Various compositions can be prepared to include low molecular weight protein fragments or high molecular weight protein fragments or combinations thereof. Low molecular weight protein fragments can reduce inflammation and/or enhance cell migration and/or proliferation on a diseased tissue surface and/or wound. Low molecular weight protein fragments are also useful in treating inflamed tissue surfaces due to an active disease state and/or the presence of a wound or wounds. In some cases, it may be useful to apply a composition of low molecular weight protein fragments to enhance the wound healing process. These cases may include wounds acquired on the battlefield during war, surgical wounds of a person who desires faster healing, for example, of an infection or for pain relief. The wound healing process is enhanced by increasing cell numbers, reducing inflammatory molecules, such as MMP-9, and/or increasing epithelial cell proliferation.

High molecular weight protein fragments may increase cell adhesion to the basement

membrane or aid in basement membrane formation. In some cases, it may be useful to apply a composition of high molecular weight protein fragments for chronic wounds or wounds that fester or wounds that have difficulty healing up, such as diabetic ulcers or skin burns. Whereas low molecular weight protein fragments may be involved in wound closure rate, high molecular weight protein fragments may be involved in wound closure quality. In some cases, it may be used to apply a composition of carefully selected amounts of low molecular weight protein fragments and high molecular weight protein fragments for optimal wound healing rate and quality. The wound healing process is enhanced by increasing structural proteins, such focal adhesion kinases (FAK) and/or tight junctions between cells, such as zonula occluden (ZO-1) structures.

Low average molecular weight fractions such as SDP-4 possess certain properties making the fraction distinct from SDP and higher molecular weight fractions. For example, SDP cellular uptake is dependent on molecular weight of the peptide chains. SDP peptide molecules smaller than about 60 kDa in size are readily absorbed by cells in culture, and more specifically human corneal limbal epithelial (hCLE) cells. SDP molecules larger than about 60 kDa in size are mostly excluded from being absorbed by the cell cultures. It is also important to note that SDP molecules do not co-localize with lysosomal-associated membrane protein 1 (LAMP-1), which is a marker for the lysosomal endocytotic degradation pathway. As a result, the SDP molecules appear to associate with a non-specified cellular membrane receptor, in which molecules of less than about 60 kDa are then absorbed by the hCLE cells. More importantly, because the SDP molecules are not absorbed through the lysosomal degradation pathway they are bioavailable and able to elicit biological activity.

SDP FORMULATIONS

The SDP compositions and sub-fractions described herein can be formulated with water and/or a pharmaceutical carrier. The pharmaceutical carrier can be, for example, phosphate buffered saline, a film, a fiber, a foam, a hydrogel, a protein or polymer matrix, a three-dimensional scaffold, a microparticle, a nanoparticle, a polymer, or a mat. In some

embodiments, the protein fragments may be attached to a substrate such as a corneal transplant, a wound dressing, a contact lens, a tissue, a tissue-graft, or a degradable material. In a specific embodiment, the carrier is phosphate buffered saline, for example, in an ocular formulation.

In some embodiments, ophthalmic compositions are provided for the treatment of dry eye syndrome in a human or mammal. Compositions provided herein can be an aqueous solution that includes an amount of SDP effective for treating dry eye syndrome. For example, the effective amount of the SDP in the aqueous solution can be about 0.01% by weight to about 80%) by weight SDP. In other embodiments, the aqueous solution can include SDP at about 0.1%) by weight to about 10%> by weight, or about 0.5% by weight to about 2% by weight. In certain specific embodiments, the ophthalmic composition can include about 0.05%> w/v SDP, about 0.1% w/v SDP, about 0.2% w/v SDP, about 0.25% w/v SDP, about 0.5% w/v SDP, about 0.75% w/v SDP, about 1% w/v SDP, about 1.5% w/v SDP, about 2% w/v SDP, about 2.5% w/v SDP, about 5% w/v SDP, about 8% w/v SDP, or about 10% w/v SDP.

In various embodiments, the ophthalmic formulation can include additional components in the aqueous solution, such as a demulcent agent, a buffering agent, and/or a stabilizing agent. The demulcent agent can be, for example, hyaluronic acid (HA), hydroxyethyl cellulose, hydroxypropyl methylcellulose, dextran, gelatin, a polyol, carboxymethyl cellulose (CMC), polyethylene glycol, propylene glycol (PG), hypromellose, glycerin, polysorbate 80, polyvinyl alcohol, or povidone. The demulcent agent can be present, for example, at about 0.01%> by weight to about 10%> by weight, or at about 0.2% by weight to about 2% by weight. In one specific embodiment, the demulcent agent is HA. In various embodiments, the HA can be present at about 0.2% by weight of the formulation.

The buffering or stabilizing agent of an ophthalmic formulation can be phosphate buffered saline, borate buffered saline, citrate buffer saline, sodium chloride, calcium chloride, magnesium chloride, potassium chloride, sodium bicarbonate, zinc chloride, hydrochloric acid, sodium hydroxide, edetate disodium, or a combination thereof.

An ophthalmic formulation can further include an effective amount of an antimicrobial preservative. The antimicrobial preservative can be, for example, sodium perborate, polyquaterium-1 (e.g., Poly quad® preservative), benzalkonium (BAK) chloride, sodium chlorite, brimonidine, brimonidine purite, polexitonium, or a combination thereof.

An ophthalmic formulation can also include an effective amount of a vasoconstrictor, an anti-histamine, or a combination thereof. The vasoconstrictor or antihistamine can be naphazoline hydrochloride, ephedrine hydrochloride, phenylephrine hydrochloride,

tetrahydrozoline hydrochloride, pheniramine maleate, or a combination thereof.

In one embodiment, an ophthalmic formulation can include an effective amount of SDP as described herein in combination with water and one or more ophthalmic components. The ophthalmic components can be, for example, a) polyvinyl alcohol; b) PEG and hyaluronic acid; c) PEG and propylene glycol, d) CMC and glycerin; e) propylene glycol and glycerin; f) glycerin, hypromellose, and PEG; or a combination of any one or more of the preceding components. The ophthalmic formulation can include one or more inactive ingredients such as

HP -guar, borate, calcium chloride, magnesium chloride, potassium chloride, zinc chloride, and the like. The ophthalmic formulation can also include one or more ophthalmic preservatives such as sodium chlorite (Purite® preservative (NaClC ), polyquad, BAK, EDTA, sorbic acid, benzyl alcohol, and the like. Ophthalmic components, inactive ingredients, and preservatives can be included at about 0.1% to about 5% w/v, such as about 0.25%, 0.3%, 0.4%, 0.5%, 1%, 2%), 2.5%), or 5%), or a range in between any two of the aforementioned values.

What is claimed is:
1. A fibroin-derived protein composition that possesses enhanced stability in an aqueous solution, wherein:
the primary amino acid sequences of the fibroin-derived protein composition differ from native fibroin by at least 4% with respect to the absolute values of the combined differences in amino acid content of serine, glycine, and alanine;

cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains of fibroin are reduced or eliminated;

a plurality of peptide chains in the protein composition terminate in amide (-C(=0)NEi2) groups;

the composition has a serine content that is reduced by greater than 25% compared to native fibroin protein, wherein the serine content is at least about 5%; and

wherein the average molecular weight of the fibroin-derived protein composition is less than 40 kDa and greater than 2 kDa.

2. The protein composition of claim 1 wherein greater than 50% of the protein chains of the protein composition have a molecular weight within the range of 10 kDa to 60 kDa.

3. The protein composition of claim 1 wherein the protein composition does not gel upon sonication of an aqueous solution of the protein composition at concentrations of up to 10% w/w.

4. The protein composition of claim 1 wherein the protein composition comprises less than 8%) serine amino acid residues.

5. The protein composition of claim 1 wherein the protein composition comprises greater than 46.5%) glycine amino acids.

6. The protein composition of claim 1 wherein the protein composition comprises greater than 30.5%) alanine amino acids.

7. The protein composition of claim 1 wherein the protein composition completely re-dissolves in water after being dried to a thin film.

8. The protein composition of claim 1 wherein the protein composition lacks beta-sheet protein structure in aqueous solution.

9. The protein composition of claim 1 wherein the protein composition maintains an optical absorbance in aqueous solution of less than 0.25 at 550 nm after at least five seconds of soni cation.

10. An ophthalmic formulation comprising the protein composition of any one of claims 1-9 and water, and optionally one or more of a buffering medium, a salt, a stabilizer, a preservative, and a lubricant.

11. A method for reducing inflammation comprising administering a fibroin-derived protein composition to inflamed tissue;

wherein the primary amino acid sequences of the fibroin-derived protein composition differ from native fibroin by at least 4% with respect to the absolute value of the combined differences in amino acid content of serine, glycine, and alanine;

cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains of fibroin are reduced or eliminated;

a plurality of peptide chains in the protein composition terminate in amide (-C(=0)NEi2) groups;

the composition has a serine content that is reduced by greater than 25% compared to native fibroin protein, and wherein the serine content is at least about 5%; and

wherein the average molecular weight of the fibroin-derived protein composition is less than 60 kDa and greater than 10 kDa;

thereby reducing transcription factor signaling within cell nuclei of the tissue, thereby reducing the inflammation.

12. The method of claim 11 wherein the administration to inflamed tissue reduces transcription of one or more of the inflammatory genes T F-α, MMP-9, IL-Ιβ, and IL-6.

13. The method of claim 11 wherein the administration is to the cornea and the

administration reduces the presence of MMP-9 in the cornea.

14. The method of claim 11 wherein the administration is to the eye and the administration reduces inflammation on the ocular surface.

15. The method of claim 11 wherein the reduction in inflammation is accompanied by increased cell migration rates at the point of inflammation.

16. The method of claim 11 wherein the protein composition has an average molecular weight less than 40 kDa.

17. The method of claim 11 wherein the protein composition has an average molecular weight less than 35 kDa.

18. The method of claim 11 wherein the fibroin-derived protein composition is dissolved in an ophthalmic formulation comprising one or more of a buffering medium, a salt, a stabilizer, a preservative, and a lubricant.

19. The method of claim 11 wherein the inflammation is caused by an ocular condition, wherein the ocular condition is dry eye syndrome, corneal ulcer, corneal erosion, corneal abrasion, corneal degeneration, corneal perforation, corneal scarring, epithelial defect, keratoconjunctivitis, idiopathic uveitis, corneal transplantation, age-related macular

degeneration, diabetic eye, blepharitis, glaucoma, ocular hypertension, post-operative eye pain and inflammation, posterior segment neovascularization, proliferative vitreoretinopathy, cytomegalovirus retinitis, endophthalmitis, choroidal neovascular membrane, vascular occlusive disease, allergic eye disease, tumor, retinitis pigmentosa, eye infection, scleritis, ptosis, miosis, eye pain, mydriasis, neuralgia, cicatrizing ocular surface disease, ocular infection, inflammatory ocular disease, ocular surface disease, corneal disease, retinal disease, ocular manifestations of systemic diseases, hereditary eye condition, ocular tumor, increased intraocular pressure, herpetic infection, ptyrigium or scleral tumor, wound sustained to ocular surface, post-photorefractive keratotomy eye pain and inflammation, thermal or chemical burn to the cornea, scleral wound, or keratoconus and conjunctival wound.

20. The method of claim 19 wherein the inflammation is caused by dry eye syndrome.

21. Use of a fibroin-derived protein composition for treating inflammation, wherein the primary amino acid sequences of the fibroin-derived protein composition differ from native fibroin by at least 4% with respect to the absolute value of the combined differences in amino acid content of serine, glycine, and alanine; cysteine disulfide bonds between the fibroin heavy and fibroin light protein chains of fibroin are reduced or eliminated; a plurality of peptide chains in the protein composition terminate in amide (-C(=0)NEi2) groups; the composition has a serine content that is reduced by greater than 25% compared to native fibroin protein, and wherein the serine content is at least about 5%; and wherein the average molecular weight of the fibroin-derived protein composition is less than 60 kDa and greater than 10 kDa.

22. The use of claim 21 wherein the protein composition has an average molecular weight less than 35 kDa.

23. The use of claim 22 wherein the composition is a composition for the treatment of dry eye syndrome.