COMPOSITIONS AND METHODS TO PROMOTE WOUND HEALING

Abstract

The present disclosure describes compositions and methods to promote wound healing. The compositions and methods include an interleukin-1 beta (IL-1β) receptor antagonist (IL-1Ra), such as anakinra. The IL-1Ra can be administered topically to a chronic wound including a diabetic ulcer.

Claims

1. A topical composition comprising a therapeutic agent consisting of: a therapeutic protein consisting of the sequence provided in SEQ ID NO: 26 (anakinra); and a platelet-derived growth factor (PDGF) or another growth factor, in a therapeutically effective amount.

2. The topical composition of claim 1, wherein the composition is a gel, ointment, paste, lotion, powder, spray, or cream.

3. The topical composition of claim 1, further comprising a thickening agent.

4. The topical composition of claim 3, wherein the thickening agent is carboxymethylcellulose sodium.

5. The topical composition of claim 1, further comprising a surfactant.

6. The topical composition of claim 5, wherein the surfactant is a co-polymer having the formula ##STR00002## wherein x is 2 to 130, y is 15 to 70, and z is 2 to 130.

7. The topical composition of claim 1, further comprising a buffer.

8. The topical composition of claim 7, wherein the buffer comprises Phosphate Buffered Saline (PBS).

9. The topical composition of claim 1, formulated as an additive to a wound dressing.

10. The topical composition of claim 9, wherein the wound dressing comprises an adhesive bandage.

11. The topical composition of claim 1, wherein the PDGF is Becaplermin (Regnarex™)

12. The topical composition of claim 1, which is formulated to promote healing of a chronic wound in a diabetic subject.

13. The tropical composition of claim 1, wherein the growth factor is a transforming growth factor (TGF).

Description

EXAMPLES

[0096] Example 1. The corneal injury model was utilized in both diabetic and normal rats for 2 days. IL-1β, one of the key genes in the inflammation cascade (pro-inflammasome regulation), was elevated significantly in normal tissue (healing epithelium). This elevation generally requires an increase in IL-1Ra, which is shown in healthy (normal) tissue in the rat experiment. In diabetes, IL-1β expression is increased; however, IL1Ra is not increased or elevated. In short, the IL-1Ra is needed to check inflammation, but remains unchecked in diabetes. Inflammation is a major cause of complications in diabetic patients and can be due to a lack of IL-1Ra. Without being bound by theory, the increased chronic inflammation caused by unchecked IL-1β results in delayed epithelialization and inadequate granulation in the tissues of diabetic patients, leading to a number of complications with ulcerations and infections.

[0097] In experiments, topical application of recombinant mouse IL-1Ra accelerates epithelial wound closure in vivo both in mice and rat type 1 diabetic wound models. Topical application of recombinant mouse IL-1Ra accelerates epithelial wound closure in vivo in both mice and rat type 1 diabetic wound models.

[0098] Example 2. Pluronic® F-127 formulation: One gram of carboxymethylcellulose sodium (1%, w/w, final concentration) was dissolved in 50 ml water with constant agitation. The solution was then cooled to 5-10° C. and 25-30 g Pluronic® F-127 was added while agitating, 10 ml 10x PBS was also added, bringing the total volume to 100 ml. The gel was stored at 4° C. until a clear solution was formed, and the gel was autoclaved (120° C., 20 min). The solution is cooled to 4° C., 67 μl anakinra injection solution (100 mg in 0.67 mL, or 150 mg/ml) will be added to 10 ml solution and stored at 4° C. all the time giving 100 μg/ml. Preventives can be used. REGRANEX® and Anakinra, REGRANEX® 10 ml may be mixed with 6.7 μl anakinra injection solution to yield 100 μg/ml Anakinra.

[0099] Example 3. Subjects with diabetes mellitus (DM) often develop corneal complications and delayed wound healing.

[0100] Animals and Induction of Diabetes. All investigations will conform to the regulations of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, the National Institutes of Health.

[0101] Streptozotocin (STZ) induction of type 1 DM in Sprague-Dawley (SD) rats. Male Sprague-Dawley (SD) rats will be purchased. These rats will be divided into two groups. Half will undergo induction of type I DM with an intraperitoneal injection of 55 mg/kg of streptozotocin in ice-cold 0.01 M citrate buffer (pH 4.5), with the controls will be injected with citrate buffer alone. A second dose of STZ will be injected after 4 to 5 days in animals with serum glucose levels less than 200 mg/dL. This regimen will produce insulin-deficient DM in 100% of the animals. The animals injected with citrate buffer will be normoglycemic. Type 2 Goto-Kakizaki (GK) rats will be maintained under standard conditions. C57BL/6 mice will be induced to develop type 1 DM according a Low-Dose STZ Induction Protocol (mouse). Particularly, for mice, 50 mg/kg will be injected constitutively for 5 days.

[0102] Glucose levels and body weight will be monitored weekly. Animals with blood sugar levels higher than 400 mg/dL (STZ-SD rats), 220 mg/dL (GK), and 350 mg/dL (STD-mice) will be considered diabetic and will be used, with age-matched animals as the control, at 8 weeks post STZ treatment for SD rats, 6 months old for GK rats and 10 week post STZ for mice. These are times when epithelial wound closure is significantly delayed and many pathologies can be observed in DM animals.

[0103] Corneal Epithelial Debridement Wound. Anesthetized rats and mice will be first demarcated with a trephine in the central cornea (5-mm circular wound for rats and 2 mm for mice). Bacitracin ophthalmic ointment will be applied to the cornea after surgery to prevent infection. At 42 hours (rats) or 24 hours (mice) post wounding (hours post wounding: “hpw”), the same size trephine will be used to mark the original wound.

[0104] Subconjunctival Injection of IL-1Ra Polypeptides. For mice, 5 μl of IL-1Ra solution will be injected into the subconjunctival space at 1 site at the superior part of the cornea and for rats, 20 μl at 2 sites, 10 μl each at the superior and inferior quadrants. IL-1Ra will be injected 4-6 hours and/or 24 hours prior to wounding.

[0105] Results will show that IL-1Ra will accelerate delayed epithelial wound closure in DM corneas.

[0106] The Experiment will be repeated with various IL-1Ra (e.g. Anakinra) and REGRANEX® combination therapies. The combination therapies results will show acceleration of delayed epithelial wound closure in DM corneas.

[0107] Example 4. A number of experiments are conducted to further define the role of up-regulation of IL-1Ra (e.g., Anakinra, SEQ ID NOs: 1-26) and the beneficial combination of IL-1Ra with a platelet-derived growth factor (e.g., REGRANEX®) to promote wound healing, promote re-epithelialization, reduce the occurrence and/or severity of ulcers, preserve nerve function and/or integrity, and promote eye health and/or maintenance of vision. In the experiments, each experiment having relevant control conditions, each of the listed compounds is administered as part of a composition (including as a direct therapeutic and/or as part of a genetic therapy as described herein). The experiments assess the function of IL-1Ra as an IL-1 (both IL-1α, and more importantly, IL-1β) neutralizer to suppress inflammation and inflammation caused tissue damage. Particular experiments will follow a procedure described elsewhere herein and/or can include use of the following animal models of chronic wounds: rabbit ear ischemia; pig flap ischemia; rat magnet ischemia reperfusion and/or pig wound infection. With the objective end points for each treatment outcome, the compositions are shown to cause significantly significant improvements as follows:

TABLE-US-00002 Compound Treatment Outcome Objective End Point Anakinra Promotes wound healing Time to wound closure or establishment of a biological barrier Promotes re-epithelialization Establishment of a biological barrier or Superficial Punctate Keratitis (death of small groups of cells on the surface of the cornea that can be examined by a slit lamp) Reduces occurrence and/or severity Slit lamp microscopy for surface of ulcers (ir)regularity Preserves nerve function integrity Corneal (or skin) sensitivity. Preserves nerve integrity Confocal microscopy to determine the nerve fiber health (length, diameters and branches) Promotes eye health Epithelial integrity, ocular surface regularity and/or normal tear secretion. Maintains vision Visual scores as determined by an ophthalmologist Anakinra Promotes wound healing Time to wound closure or formulated for establishment of a biological barrier topical Promotes re-epithelialization Establishment of a biological barrier administration or Superficial Punctate Keratitis Reduces occurrence and/or severity Slit lamp microscopy for surface of ulcers (ir)regularity Preserves nerve function integrity Corneal (or skin) sensitivity. Preserves nerve integrity Confocal microscopy to determine the nerve fiber health (length, diameters and branches) Promotes eye health Epithelial integrity, ocular surface regularity and/or normal tear secretion. Maintains vision Visual scores as determined by an ophthalmologist Anakinra Promotes wound healing Time to wound closure or formulated as establishment of a biological barrier a gel Promotes re-epithelialization Establishment of a biological barrier or Superficial Punctate Keratitis Reduces occurrence and/or severity Slit lamp microscopy for surface of ulcers (ir)regularity Preserves nerve function integrity Corneal (or skin) sensitivity. Preserves nerve integrity Confocal microscopy to determine the nerve fiber health (length, diameters and branches) Promotes eye health Epithelial integrity, ocular surface regularity and/or normal tear secretion. Maintains vision clear and transparent cornea, no fluorescence staining under a slit lamp microscope Anakinra in Promotes wound healing Time to wound closure or combination establishment of a biological barrier with Promotes re-epithelialization Establishment of a biological barrier REGRANEX ® or Superficial Punctate Keratitis Reduces occurrence and/or severity Slit lamp microscopy for surface of ulcers (ir)regularity Preserves nerve function integrity Corneal (or skin) sensitivity. Preserves nerve integrity Confocal microscopy to determine the nerve fiber health (length, diameters and branches) Promotes eye health Epithelial integrity, ocular surface regularity and/or normal tear secretion. Maintains vision Visual scores as determined by an ophthalmologist SEQ ID NOs: Promotes wound healing Time to wound closure or 1, 2, 3, 4, 5, 6, establishment of a biological barrier 7, 8, 9, 10, 11, Promotes re-epithelialization Establishment of a biological barrier 12, 13, 14, 15, or Superficial Punctate Keratitis 16, 17, 18, 19, Reduces occurrence and/or severity Slit lamp microscopy for surface 20, 21, 22, 23, of ulcers (ir)regularity 24, 25 and 26 Preserves nerve function integrity Corneal (or skin) sensitivity. Preserves nerve integrity Confocal microscopy to determine the nerve fiber health (length, diameters and branches) Promotes eye health Epithelial integrity, ocular surface regularity and/or normal tear secretion. Maintains vision Visual scores as determined by an ophthalmologist SEQ ID NOs: Promotes wound healing Time to wound closure or 1, 2, 3, 4, 5, 6, establishment of a biological barrier 7, 8, 9, 10, 11, Promotes re-epithelialization Establishment of a biological barrier 12, 13, 14, 15, or Superficial Punctate Keratitis 16, 17, 18, 19, Reduces occurrence and/or severity Slit lamp microscopy for surface 20, 21, 22, 23, of ulcers (ir)regularity 24, 25 and 26 Preserves nerve function integrity Corneal (or skin) sensitivity. formulated for Preserves nerve integrity Confocal microscopy to determine topical the nerve fiber health (length, administration diameters and branches) and/or in Promotes eye health Epithelial integrity, ocular surface combination regularity and/or normal tear with secretion. REGRANEX ® Maintains vision Visual scores as determined by an ophthalmologist SEQ ID NO: 27 Promotes wound healing Time to wound closure or establishment of a biological barrier Promotes re-epithelialization Establishment of a biological barrier or Superficial Punctate Keratitis Reduces occurrence and/or severity Slit lamp microscopy for surface of ulcers, (ir)regularity Preserves nerve function integrity Corneal (or skin) sensitivity. Preserves nerve integrity Confocal microscopy to determine the nerve fiber health (length, diameters and branches) Promotes eye health Epithelial integrity, ocular surface regularity and/or normal tear secretion. Maintains vision Visual scores as determined by an ophthalmologist

[0108] The tested compounds will also suppress inflammation by neutralizing IL-1β activity.

[0109] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. Thus, the terms “include” or “including” should be interpreted to recite: “comprise, consist of, or consist essentially of.” As used herein, the transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of” limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment. As used herein, a material effect would cause a statistically-significant reduction in an embodiment's ability to promote wound healing in a chronic wound of a diabetic subject.

[0110] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to within a range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17% of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value; ±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the stated value; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% of the stated value; or ±1% of the stated value.

[0111] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0112] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0113] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0114] Certain embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0115] Furthermore, numerous references have been made to patents, printed publications, journal articles and other written text throughout this specification (referenced materials herein). Each of the referenced materials are individually incorporated herein by reference in their entirety for their referenced teaching.

[0116] In closing, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

[0117] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with any drawings and/or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

[0118] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary, 3.sup.rd Edition or a dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).