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WO2013025940A1 - Angiogenèse induite par l'alpha-kératose - Google Patents

Angiogenèse induite par l'alpha-kératose Download PDF

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Publication number
WO2013025940A1
WO2013025940A1 PCT/US2012/051212 US2012051212W WO2013025940A1 WO 2013025940 A1 WO2013025940 A1 WO 2013025940A1 US 2012051212 W US2012051212 W US 2012051212W WO 2013025940 A1 WO2013025940 A1 WO 2013025940A1
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composition
keratose
alpha
alpha keratose
recipient
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Luke Burnett
Chad Johnson
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Keranetics LLC
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Keranetics LLC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0031—Rectum, anus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0034—Urogenital system, e.g. vagina, uterus, cervix, penis, scrotum, urethra, bladder; Personal lubricants
    • A61K9/0036—Devices retained in the vagina or cervix for a prolonged period, e.g. intravaginal rings, medicated tampons, medicated diaphragms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/02—Suppositories; Bougies; Bases therefor; Ovules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/12—Aerosols; Foams

Definitions

  • the invention relates to compositions of keratin-based biomaterials and methods for modulating or promoting angiogenesis.
  • Angiogenesis is a physiological process that involves the growth of new blood vessels or capillaries from existing vessels during growth, development, wound healing, and tumorigenesis.
  • Angiogenesis may encompass both vasculogenesis and arteriogenesis.
  • Vasculogenesis describes the formation of vascular structures from circulating or tissue- resident endothelial stem cells (angioblasts), which proliferate into endothelial cells.
  • Vasculogenesis typically relates to the embryonal development of the vascular system.
  • Arteriogenesis is the formation of medium-sized blood vessels.
  • angiogenesis describes the formation of thin-walled endothelium-lined structures with muscular smooth muscle wall and fibrocytes.
  • Angiogenesis is important for the repair of damaged tissues. Thus, there is a great need for compositions that modulate angiogenesis for a number of biological processes.
  • Keratins are a family of proteins found in the hair, skin, and other tissues of vertebrates. Human hair is an important source of keratins because it is inexpensive and readily available. Keratins can be extracted from human hair fibers by oxidation or reduction. These extraction methods typically comprise a two-step process whereby the crosslinked structure of hair keratins is broken down by either oxidation or reduction. In these reactions, the disulfide bonds formed by cystine amino acid residues are either oxidized or reduced, rendering the keratins soluble without disrupting the peptide bonds. After oxidation or reduction, keratins can remain trapped within the hair cuticle and often require a secondary denaturing step to efficiently extract the cortical proteins.
  • Denaturants useful for such extractions include urea, guanidine salts, strong bases (hydroxides), detergents, and combinations thereof.
  • a typical extraction method uses urea at concentrations between 0.1 and 10 M in buffered solutions.
  • Keratin proteins obtained using reductive extractions are referred to as kerateines owing to the reduction of many of the cystine bonds to sulfhydryl moieties.
  • kerateines owing to the reduction of many of the cystine bonds to sulfhydryl moieties.
  • oxidative extraction is used, the resulting keratins are referred to as keratoses.
  • cystines in keratoses have been oxidized to cysteic acids that contain sulfonic acid moieties.
  • Crude extracts of keratins can be further refined into alpha, gamma, and keratin associated protein ("KAP") and/or acidic or basic fractions by a variety of purification methods such as isoelectric precipitation, dialysis, or high performance liquid chromatography (HPLC).
  • KAP keratin associated protein
  • HPLC high performance liquid chromatography
  • the alpha fraction precipitates under acidic conditions.
  • the alpha fraction begins to precipitate at pHs below 6 and is completely precipitated by pH 4.2.
  • the KAP fraction typically co-precipitates with the alpha fraction, thereby producing an alpha KAP mixture.
  • the gamma fraction remains in solution under these conditions, but can be precipitated by addition of polar organic solvents.
  • Such solvents are water-mi scible but do not dissolve keratins.
  • a useful polar organic solvent for this purpose is ethanol. Precipitation of the gamma fraction can be aided by cooling the ethanol and adding the keratin solution dropwise to the ethanol, rather than adding the ethanol to the keratin. Additional fractionation procedures have been described and will be apparent to one having ordinary skill in the art.
  • High molecular weight keratins or alpha keratins, which are predominantly alpha helical, originate from the microfibrillar regions of the hair follicle, and typically range in molecular weight from about 40-85 kDa.
  • Beta keratins have molecular weights of 10-20 kDa and are found in the cuticle region of hair.
  • Low molecular weight keratins, such as the gamma keratins and keratin-associated proteins originate from the matrix regions of the hair follicle, and typically have molecular weights from about 3-30 kDa for KAP and 10-15 kDa for gamma keratins.
  • oxidized keratin preparations (alpha, alpha/KAP, and/or gamma keratose) have molecular weights from about 85 kDa to 100 kDa.
  • Dialysis can be used to separate the alpha/KAP keratose components from the gamma keratose fraction.
  • the alpha/KAP/gamma keratose mixture may be dialyzed in a regenerated cellulose dialysis membrane with a nominal molecular weight cut off (MWCO) of 100 kDa.
  • MWCO molecular weight cut off
  • the gamma fraction which has a molecular weight of 10-15 kDa, permeates through the membrane leaving the alpha KAP fraction in the retentate.
  • the permeate also contains some alpha keratose monomers.
  • the retained dialysate solution is then re-dialyzed in a 5-kDa MWCO dialysis membrane to remove residual oxidant, salts, or contaminants.
  • the majority of the alpha keratose fraction remains in the retentate during the first dialysis step despite that the alpha monomer molecular weight is around 55 kDa. This is likely due to the propensity of the alpha monomers to exist as multimers of greater than 100 kDa under native, non- reducing conditions.
  • the KAP also remain in the retentate/dialysate. Although they have relatively low molecular weights, the KAP complexes with the alpha fraction, does not pass through the dialysis membrane, and must be separated via chromatography.
  • the resulting solutions from dialysis may then be separately lyophilized and ground with a mill into fine particulates that are several hundred micrometers in diameter.
  • the alpha and gamma fractions can then be recombined as dry powders, sterilized via gamma radiation, and reconstituted with aqueous solutions such as water or phosphate buffered saline to produce keratose solutions or hydrogels.
  • the independent alpha, gamma, and KAP fractions can be recombined to form "meta keratins" or "meta keratoses” that have different biochemical properties than the independent fractions or crude starting mixtures.
  • the solution viscosity, gel-forming propensity, biocompatibility, tissue-compatibility, vasodilation, and other biochemical properties of the resulting keratin sub-fractions and meta keratins differ from each other.
  • These sub-fractions of keratin have interesting characteristics such as an ability to promote vascularization and angiogenesis around a wound site and to promote blood flow and perfusion to cells and tissues.
  • the material properties and therapeutic effects of keratose can be controlled.
  • alpha keratose Described herein is alpha keratose.
  • the alpha keratose proteins described herein, and compositions comprising alpha keratose can be used to modulate angiogenesis and vascularization and the use of alpha keratose compositions alone or in combination with tissue scaffolds, to treat recipients in need of such therapy,
  • keratin may be isolated from human hair.
  • the keratin may be oxidized.
  • the keratin may comprise alpha keratose.
  • the keratin may comprise basic alpha-keratose or acidic alpha- keratose.
  • the keratin may comprise alpha keratose KAP.
  • the keratin may comprise meta keratin.
  • the meta keratin may comprise at least one of alpha keratose or alpha keratose/KAP, or combinations thereof.
  • the meta keratin may comprise alpha keratose, alpha keratose/KAP, gamma keratose, KAP, or combinations thereof.
  • the meta keratin may comprise basic alpha-keratose, acidic alpha-keratose, basic gamma-keratose, acidic gamma- keratose, or combinations thereof.
  • compositions comprising alpha keratose may comprise alpha keratose in an amount of about 0.1%, about 0.2%, about 0.5%, about 0.75%, about 1%, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the composition by weight.
  • compositions comprising alpha keratose may be in the form ofhydrogels, gels, membranes, films, scaffolds, sponges, beads, matrices, powders, particulates, absorbents, adsorbents, bandages, dressings, pessaries, saturated tampons, transdermal patches, coatings, creams, lotions, pastes, gels, jelly, mucilages, ointments, foams, sprays, suppositories, aqueous or oily suspensions, emulsions, the like, or combinations thereof.
  • the recipient may be a mammal, such as a human being, or a dog, cat, mouse, rat, or rabbit.
  • the composition comprising alpha keratose can be topically administered.
  • the composition comprising alpha keratose may further comprise drugs, biomaterials, crosslinking agents, antibodies, biological tissues or artificial tissues.
  • composition comprising keratose may be in the form of hydrogels, gels, membranes, films, scaffolds, sponges, beads, matrices, powders, particulates, absorbents, adsorbents, bandages, dressings, transdermal patches, pessaries, saturated tampons, coatings, creams, lotions, pastes, gels, jelly, mucilages, ointments, foams, sprays, suppositories, aqueous or oily suspensions, emulsions, the like, or combinations thereof.
  • the alpha keratose may be administered at a controlled rate.
  • the alpha keratose may be administered to a specific tissue comprising connective tissue (bone, blood, lymph, fibrous tissue), muscle tissue (striated and smooth muscle), nervous tissue, epithelial tissue (skin, organ linings), or endothelial tissue (vascular tissue).
  • the alpha keratose administration may be site-specific and spatially restricted.
  • the composition comprising alpha keratose may comprise alpha keratose, gamma keratose, or combinations thereof.
  • composition comprising alpha keratose may comprise about 0.1%, about 0.2%, about 0.5%, about 0.75%, about 1 %, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the composition by weight.
  • Also described herein are methods for modulating angiogenesis in the periphery of an implantable scaffold or topical dressing comprising administering an effective amount of alpha keratose, in the periphery of an implantable scaffold or topical dressing.
  • compositions for delivering alpha keratose to a recipient in need thereof comprising a composition comprising alpha keratose, may further comprise drugs, biomaterials, crosslinking agents, antibodies, biological tissues or artificial tissues.
  • compositions comprising keratose may be in the form of hydrogels, gels, membranes, films, scaffolds, sponges, beads, matrices, powders, particulates, absorbents, adsorbents, bandages, dressings, transdermal patches, pessaries, saturated tampons, coatings, creams, lotions, pastes, gels, jelly, mucilages, ointments, foams, sprays, suppositories, aqueous or oily suspensions, emulsions, the like, or combinations thereof.
  • Suitable means for delivering include drugs, biomaterials, crosslinking agents, antibodies, biological tissues, artificial tissues, hydrogels, gels, membranes, films, scaffolds, sponges, beads, matrices, powders, particulates, absorbents, adsorbents, bandages, dressings, transdermal patches, pessaries, saturated tampons, coatings, creams, lotions, pastes, gels, jelly, mucilages, ointments, foams, sprays, suppositories, aqueous or oily suspensions, emulsions, the like, or combinations thereof.
  • composition comprising alpha keratose, for the modulation of angiogenesis comprising administering an effective amount of alpha keratose.
  • Also described herein are devices for administering alpha keratose to a recipient in need thereof comprising a composition comprising alpha keratose; and a means for delivering the alpha keratose composition to a recipient in need thereof.
  • containers for storing compositions of alpha keratose for administration to a recipient in need thereof comprising a composition comprising alpha keratose; and a means for storing the composition comprising alpha keratose.
  • kits for administering a alpha keratose composition to a recipient in need thereof comprising at least one container comprising individual premeasured dosages, the containers including at least one composition comprising alpha keratose; wherein the composition comprising alpha keratose comprises one or more of a drug, biomaterial, crosslinking agent, antibodies, biological tissues, artificial tissues, hydrogels, gels, membranes, films, scaffolds, sponges, beads, matrices, powders, particulates, absorbents, adsorbents, and the like, or combinations thereof; optionally, solutions for rehydrating dehydrated compositions; a means for administering a alpha keratose composition to a recipient in need thereof; and instructions describing a method for administering a alpha keratose composition to a recipient in need thereof.
  • FIGURE 1 shows representative fluorescent microangiograph images for the (A) SIS control and (B) SIS + alpha keratose three weeks post-implantation. These fluorescent photomicrographs demonstrated that the alpha keratose (B) released from the SIS disk augments or enhances angiogenesis relative to the non-treated control SIS (A). The scale bar is 100 ⁇ .
  • FIGURE 2 Figure 2 shows hematoxylin and eosin histological staining of (A) SIS control and (B) SIS + alpha keratose three weeks post-implantation. These images show that the alpha keratose (B) released from the SIS disk did not augment or attenuate the remodeling properties of the SIS base material relative to the non-treated control (A).
  • the scale bar is 100 um.
  • angiogenesis encompasses all different types and modifications of arterial or venous vessel growth, including vasculogenesis, arteriogenesis, and vascularization, in general.
  • the downregulation, prevention, suppression, reduction, or cessation of any process related to angiogenesis, as used herein, is referred to as “inhibition” or “inhibiting” angiogenesis.
  • the induction, initiation, or upregulation of angiogenesis are referred to herein as "promoting,” “enhancing,” or “augmenting” angiogenesis.
  • scaffold refers to a structure that can be used as a substrate for seeding cells thereon.
  • the scaffold can be porous or solid.
  • the scaffold can be implantable, bioabsorbable, inert, or removable, depending on the application.
  • Such scaffolds can comprise biomolecules or other polymers.
  • Often scaffolds comprise hydrogels and can be made of the materials described herein for creating hydrogels.
  • dressing refers to a covering for a wound or injury.
  • a dressing may comprise compositions of the invention to modulate angiogenesis at the wound site or in surrounding tissue.
  • implantable refers to the insertion, grafting, or fixation of a foreign material into a living body or tissue.
  • topical application refers to the administration of an agent to the surface of a body, tissue, or opening thereof, such as to skin, wounds, burns, site of surgery, or other injuries.
  • peripheral refers to the area immediately surrounding portion of a surface or area, such as a wound, injury, site of surgery, or site of application of an agent.
  • administered at a controlled rate refers to the release of an agent from a carrier or delivery agent at a specific rate of time.
  • the release rate is dependent on the carrier or delivery agent composition, the surface area of release, the temperature, the absorption rate of the agent, and other chemical, physical, or pharmacological factors.
  • administered to a specific tissue refers to the delivery of an agent from a carrier or delivery agent to one or more specific tissue systems in or on an animal's body.
  • site specific refers to the delivery of an agent at a specific region or area of a body or wound wherein the effect of the agent is relegated to the area of administration.
  • spatialally restricted refers to the limited ability of an agent to affect areas that the agent is in contact with, or within the immediate vicinity (periphery) of the contact region.
  • the promotion of angiogenesis as used herein is site specific and spatially restricted because the inhibition of angiogenesis only occurs at the site of application of the compositions described herein and the angiogenic effects are relegated to the site of administration and the immediate periphery.
  • Keratin refers to keratinous proteins that can be isolated from hair.
  • alpha keratose refers to oxidized alpha keratin, isolated from hair. Keratin may be derived from many sources; a typical source of keratin is human hair. Keratin can also be purified from wool, animal fur, feathers, horns, nails, or other keratinous structures. Keratins from wool also have generally good biocompatibility. See Ito et al., Biocompatibility of denatured wool keratin, Kobunshi Ronbunshu 39(4):249-256 (1982).
  • the hair may be end-cut as one would typically find in a barbershop or salon or purchased through commercial sources.
  • the hair may be cleaned by washing in a warm water solution of mild detergent and freed of surface oils by washing with an organic solvent such as ethanol, ether, or acetone.
  • a typical solvent comprises an alcohol, e.g., ethanol.
  • Keratin may be extracted from a number of sources. Keratin present in hair, wool, fur, feathers, horns, hooves, nails, beaks, scales, and the like are acceptable for the invention In a specific embodiment, human hair is used because of its biocompatibility and low antigenicity in human medical applications.
  • One method for the production of keratoses is by oxidation of keratin with hydrogen peroxide, peracetic acid, or performic acid.
  • the oxidant is peracetic acid.
  • a solution of peracetic acid is used at a concentration range of about 1% to about 10%.
  • a specific concentration used can be a 2% solution of peracetic acid.
  • the oxidant concentrations range from a ratio of about 5: 1 to about 50: 1 weight to weight to the keratin protein source to be extracted.
  • a specific embodiment uses a weight to weight ratio of 25: 1 of a 2% peracetic acid solution.
  • the oxidation temperature is between 0 and 100° Celsius. In a specific embodiment, the oxidation temperature is 37°C. In some embodiments, the oxidation time is between 0.5 and 24 hours. In a specific embodiment, the oxidation time is 12 hours. In some embodiments, mechanical mixing is used to maximize oxidation efficiency.
  • the keratin protein source can be rinsed free of residual oxidant using copious amounts of purified water.
  • the oxidized keratin protein source is washed with water until residual oxidant is removed.
  • the washing step is performed until the washed keratin protein source does not test positive for oxidant.
  • the keratoses may be extracted from the oxidized keratin protein source using an aqueous solution of a denaturing agent.
  • Protein denaturants are well known in the art, including but not limited to, urea, transition metal hydroxides (e.g. sodium and potassium hydroxide), ammonium hydroxide, and tris(hydroxymethyl)aminomethane (Tris, also known as Trizma® base).
  • Tris is used at a ratio of about 5:1 to about 50: 1 weight of protein source, to a Tris solution of a concentration of about 0.01 to 1 . In a specific embodiment, the ratio is 25: 1. In another specific embodiment, Tris is used at a concentration of lOOmM.
  • the extraction temperature is between 0° and 100° C. In a specific embodiment, the extraction temperature is 37° C. In some embodiments, the extraction time is between 0.5 and 24 hours. In a specific embodiment, the extraction time is about 2 hours. Additional yield can be achieved with subsequent extractions with dilute solutions of Tris or purified water. Often, the extraction is performed with mechanical agitation in a mixing tank to ensure a more efficient yield.
  • a high speed centrifugation step may be used.
  • Current methods known in the art generally use a low speed centrifugation (around 4,000 rpm) to clear particulate matter, However, this speed does not create enough force to remove many of the beta keratin protein contaminants present in the extracted protein solution.
  • high speed centrifugation is employed. Speeds in excess of about 5,000 rpm to about 30,000 rpm can be used.
  • the extracted protein solution is spun at about 20,000 rpm to produce a clarified protein solution of solubilized keratin proteins.
  • the high speed centrifugation step is performed at about 4° C.
  • a clarified protein solution is produced by the high speed centrifugation of the soluble keratin protein solution.
  • dialysis is used to separate and/or to concentrate certain protein species present in the sample. Accordingly, the clarified protein solution described herein may be subjected to a dialysis step to fractionate certain protein species.
  • a 100 kDa molecular weight cutoff membrane is employed in the purification of alpha-keratose or alpha-kerateine.
  • a 5 kDa molecular weight cutoff membrane is employed to purify gamma-keratose or gamma kerateine.
  • a common matrix for the dialysis membranes is regenerated cellulose, however, many other membrane preparations suitable for protein purification may be used,
  • the dialysis is performed under conditions that maintain a transmembrane pressure from about 30 to about 70 psi. In a specific embodiment the transmembrane pressure is about 30 to about 40 psi. Further, it is important to minimize the heat buildup developed by the shear stress of pressurized dialysis. Thus, in some embodiments, the dialysis is carried out at a temperature from about 4° C to about 20° C. In a specific embodiment, the dialysis is carried out at about 15° C.
  • the conductivity is adjusted.
  • the conductivity is adjusted down to about or below 0.6 mS. In some instances, the conductivity is adjusted with water.
  • proteins can be freeze-dried (lyophilized) to achieve storage conditions while maintaining protein stability.
  • lyophilization is used to produce a protein cake of purified protein. The lyophilization is used to stabilize the extracted keratin proteins. Methods known in the art such as shell freezing followed by vacuum or bulk freezing and applying high heat tend to degrade proteins.
  • a keratin protein cake comprising keratose alpha or gamma and/or kerateine alpha or gamma is produced by a lyophilization of a clarified keratin protein solution, optionally after dialysis.
  • the clarified protein solution post-dialysis is bulk frozen at about -40° C, and then a vacuum is applied until the containment containing the solution reaches about 250 torr.
  • heat is then applied in a step-wise fashion, bringing the material to about 0° C, then to about 25° C, then to about 37° C, white maintaining 250 torr pressure.
  • the lyophilization process occurs over a 24 hour period.
  • Precise grinding of the lyophilized material aids in the homogeneity of reconstitution and protein stability.
  • Previous methods involve crude grinding methods, including grinding or chopping of the material in a laboratory blender.
  • some embodiments employ a commercial grinding apparatus to machine the material to a homogenous particle size.
  • a pharmaceutical mill is employed.
  • the particle size is about 1000 microns or less in diameter.
  • the ground material is deionized.
  • Hydrogels were prepared for analysis by carefully weighing the appropriate keratin lyophilized powder or powders. The powders were diluted in either sterile phosphobuffer saline or sterile water to generate the described percent mass to volume ratio. These solutions were placed in a 37° C incubator overnight before analysis.
  • Hydrogels comprising the compositions described herein are useful.
  • Natural biomaterials useful for forming hydrogels for use in the compositions may comprise alginate; chitosan; collagen; gelatin; fibrin; hyaluronic acid; silk; or keratins.
  • Synthetic biomaterials useful in the compositions described herein may comprise poly(lactic acid) (i.e., PLA), poly(glycolic acid) (i.e., PGA), poly(dioxanone) (i.e., PDS or PDO), poly(caprolactone), or
  • poly(PCCP-SA anhydride) examples include poly(esters), poly(hydroxy acids), poly(lactones), poly(amides), poly(ester-amides), poly(amino acids), poly(anhydrides), poly(ortho-esters), poly(carbonates), poly(phosphazines), poly(thioesters), polysaccharides and mixtures thereof. Additional hydrogel forming compositions are described in U.S. Patent No. 5,854,382.
  • the keratin sub-fractions such as alpha, gamma, and KAP, have distinct characteristics such as differential effects on vascularization and angiogenesis around wound sites and the promotion of blood flow and perfusion to cells and tissues.
  • the differential properties of the alpha, gamma and KAP keratin sub-fractions, as described above, either alone or in combination, can be used to tailor the chemical and material properties and fine tune the biochemical, medicinal, and therapeutic effects of specific keratin compositions.
  • the useful features of keratins include the ability to recombine purified keratin sub-fractions into "meta keratins" that have controllable physical properties and the angiogenic promoting effects of alpha keratose.
  • Optimizing the molecular weight using meta-keratins allows formulations to be optimized for a variety of properties.
  • the manipulation of the percentage of the components of a meta-keratose hydrogel may affect properties such as viscosity, film strength and elasticity, fiber strength, and hydrolytic susceptibility.
  • a higher percentage of alpha-keratose in the composition leads to decreased hydrolytic susceptibility.
  • lowering the percentage of alpha-keratose in the composition leads to increased hydrolytic susceptibility.
  • crosslinking may improve strength.
  • hydrolysis of a hydrogel may be measured to determine the effective release window for compound of interest.
  • Keratose hydrogel formation may be accomplished by rehydrating sterile or pyrogen- free keratose or meta-keratose powders with saline or other isotonic solution (e.g., lactated Ringer's solution) under aseptic conditions at protein concentrations greater than 5% (w/v).
  • saline or other isotonic solution e.g., lactated Ringer's solution
  • Alpha keratose, or meta keratose solutions can be formulated by rehydrating at concentrations less than 5% (w/v).
  • the product can take the form of a ready -to-inject sterile or pyrogen-free gel, or a fluid that may be used in conjunction with a tissue scaffold or as a coating for an implant.
  • the composition may comprise alpha keratose in an amount of about 0.1 %, about 0.2%, about 0.5%, about 0.75%, about 1 %, about 2%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% by weight.
  • the composition may comprise alpha keratose in an amount of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 5
  • compositions described herein may comprise alpha keratose, gamma keratose, KAP, or combinations thereof.
  • the composition may comprise alpha keratose/KAP and gamma keratose.
  • the weight : weight ratio of alpha keratose/KAP to gamma keratose may be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • the composition may comprise alpha keratose and KAP.
  • the weight : weight ratio of alpha keratose to KAP may be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • the composition may comprise alpha keratose and gamma keratose.
  • the weight : weight ratio of alpha keratose to gamma keratose may be about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.
  • the composition comprises alpha keratose, gamma keratose, and KAP.
  • the weight : weight : weight fraction of alpha keratose to gamma keratose to KAP may be about 1:1:1, 1:1:2, 1:1:3, 1:1:4, 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:2:1, 1:3:1, 1:4:1, 1:5:1, 1:6:1, 1:7:1, 1:8:1, 1:8:1, 1:10:1,2:1:1, 3:1:1,4:1:1, 5:1:1, 6:1:1, 7:1:1, 8:1:1, 8:1:1, or 10:1:1.
  • the composition comprises a mixture of alpha keratose, gamma keratose, and KAP.
  • the composition may comprise about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% alpha keratose by weight.
  • the composition may comprise about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% gamma keratose by weight.
  • the composition may comprise about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% KAP by weight.
  • the composition comprises a mixture comprising alpha keratose.
  • the composition may comprise about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% alpha keratose by weight.
  • compositions comprising alpha keratose.
  • Such compositions may be in the form of powders, particulates, granules, beads, matrices or the like, and can be made in accordance with known techniques such as freeze drying (i.e., lyophilization), spray drying, dehydration, or the like.
  • compositions described herein may be produced by mixing dry powder compositions with aqueous solutions to produce solutions of alpha keratose and/or other keratin(s) solubilized therein.
  • the mixing step can be carried out at any suitable temperature, typically room temperature, and can be carried out by any suitable technique such as stirring, shaking, agitation, sonication, etc. Salts, buffers, excipients, pharmaceutically acceptable carriers, gel-forming agents, disintegrants, coatings, anti-adherents, emulsifiers, detergents, reductants, oxidants, proteins, carbohydrates, or other constituent ingredients that may comprise a dry powder.
  • the alpha keratose and/or other keratin(s) may be suspended in solutions, gels, or provided as dry powders.
  • the compositions may be provided as hydrated gels, films, membranes, etc. or in dehydrated form.
  • Compositions in dehydrated form may be provided with sterile or pyrogen-free water or buffer/electrolyte solutions for rehydrating.
  • compositions comprising aqueous solutions, emulsions, or dry powders intended to be rehydrated with aqueous compositions or emulsions.
  • Such compositions may contain proteins, carbohydrates, salts, buffers, excipients, pharmaceutically acceptable carriers, stabilizers, gel-forming agents, crowding agents, binders, coatings, solubilizers, emulsifiers, detergents, reductants, oxidants, preservatives, sorbents, polar or nonpolar organic solvents, non-aqueous vehicles including edible oils, and aqueous solvents.
  • compositions comprising delivery agents or carriers such as drugs, biomaterials, crosslinking agents, antibodies, biological tissues, artificial tissues, hydrogels, gels, membranes, films, scaffolds, sponges, beads, matrices, powders, absorbents, adsorbents, bandages, dressings, transdermal patches, pessaries, saturated tampons, coatings, creams, lotions, gels, pastes, jelly, mucilages, ointments, foams, sprays, suppositories, aqueous or oily suspensions, or emulsions containing alpha keratose and/or other keratose compositions.
  • delivery agents or carriers such as drugs, biomaterials, crosslinking agents, antibodies, biological tissues, artificial tissues, hydrogels, gels, membranes, films, scaffolds, sponges, beads, matrices, powders, absorbents, adsorbents, bandages, dressings, transdermal patches, pessaries,
  • any substrate, agent, or device that is capable of releasing alpha keratose to living tissue, wound, site of injury, or site of surgery is acceptable for delivering such compositions to a site of application of a recipient.
  • substrates, agents, or delivery devises are useful for retaining and releasing the KAP composition in the vicinity or periphery of a site of administration or at a specific tissues at a controlled release rate.
  • administration of an alpha keratose composition as described herein may be spatially restricted and/or site specific.
  • compositions described herein may be non-pyrogenic or sterile. Keratin solutions may be sterile filtered and processed aseptically, or terminally sterilized using ethylene oxide, e-beam, gamma radiation, or other low temperature methods (i.e., ⁇ 50 °C).
  • compositions described herein may be provided in a precursor solution aseptically packaged in a suitable container.
  • a gel precursor solution can be provided in a glass ampoule ready to use directly or after dilution by the user. A user would break open the ampoule, mix in a compound of interest, and use the solution directly or after dilution.
  • compositions described herein may be provided preformed and aseptically packaged in suitable containers, such as a flexible polymeric bags, plastic packaging, bottles, or foil envelopes or may be provided as a kit of sterile or non-pyrogenic dry powder in one container and sterile aqueous solution in a separate container for mixing just prior to use.
  • suitable containers such as a flexible polymeric bags, plastic packaging, bottles, or foil envelopes
  • the composition When provided pre-formed and packaged in a sterile container the composition preferably has a shelf life of at least 4 or 6 months (up to 2 or 3 years or more) at room temperature, prior to substantial loss of viscosity (e.g., more than 10 or 20 percent) and/or structural integrity of the gel or hydrogel.
  • kits wherein the compositions described herein are provided in a suitable container (e.g. a plastic or glass bottle, sterile ampoule, etc.), optionally packaged in sterile or non-pyrogenic form.
  • the kit may be contained in a hermetically sealed package also in sterile form.
  • the compositions may be provided as powders, liquids, gels, membranes, dressings, creams, and so forth, and may be provided in different volumes.
  • the composition may be provided in a dehydrated from that can be rehydrated using sterile water or solution provided in the kit.
  • the kit can comprise a delivery means or applicator for administering the composition.
  • kit may contain printed instructions for using the kit, including descriptions of the compositions, directions for use of the applicator, and other relevant information.
  • an alpha keratose composition comprising alpha keratose.
  • Various alpha keratose compositions are described herein.
  • the administration of an alpha keratose composition may be done by topical, subcutaneous, intradermal, intraperitoneal, intramuscular, intravenous, intracerebral, epidural, or transdermal administration.
  • the dose may be administered at a specific rate or to a specific tissue.
  • Specific tissues include connective tissue (bone, blood, lymph, fibrous tissue), muscle tissue (striated and smooth muscle), nervous tissue, epithelial tissue (skin, organ linings), or endothelial tissue (vascular tissue) and others.
  • the administration of an alpha keratose composition described herein is site-specific and/or spatially restricted.
  • the alpha keratose composition may be administered at a specific place on a tissue to modulate angiogenesis and thereby spatially restricting the angiogenesis promoting activities of the composition.
  • the alpha keratose compositions described herein are placed or deposited on the periphery of devices to be placed on or within the body of a recipient where modulation of angiogenesis is desired.
  • alpha keratose compositions may be placed on the periphery of implantable scaffolds or topical dressings.
  • mice Twelve adult BALB/c mice were divided into two groups; six mice in each group were subcutaneously implanted with scaffolds consisting of either Small Intestinal Submucosa (SIS; control; Cook Medical Group) or SIS + alpha keratose as follows.
  • SIS Small Intestinal Submucosa
  • control Cook Medical Group
  • SIS + alpha keratose as follows.
  • Four- layered lyophilized SIS samples were cut into 15 mm diameter disks using a die press and sterilized using low temperature ethylene oxide.
  • Nylon filter disks 13 mm
  • mice After three weeks (21 days), all mice were humanely euthanized using an overdose of anesthesia and the diaphragms opened.
  • mice in each group were exsanguinated, flushed with saline, and injected with a fluorescent bead suspension through the left heart ventricle to measure only intact, functional vasculature.
  • the SIS disks were then explanted and imaged using a fluorescent confocal microscope and photographed to determine the furthest point of neo-vascular formation.
  • Two SIS disks from each group were explanted, fixed in formalin, sectioned, stained with hematoxylin and eosin (HE) or Masson's trichrome, imaged, and photographed.
  • HE hematoxylin and eosin

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Abstract

La présente invention concerne des compositions comprenant de l'alpha-kératose et la capacité de telles compositions à induire la vascularisation et l'angiogenèse autour d'un site de plaie, et des procédés pour l'utilisation de telles compositions seules ou en combinaison avec d'autres sous-fractions de kératine et d'échafaudages de tissu non-kératine pour moduler l'angiogenèse.
PCT/US2012/051212 2011-08-17 2012-08-16 Angiogenèse induite par l'alpha-kératose Ceased WO2013025940A1 (fr)

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CN107335088A (zh) * 2016-05-03 2017-11-10 薛培贤 一种可采用自体的毛发制作伤口敷料的方法
CN107335094A (zh) * 2016-05-03 2017-11-10 薛培贤 一种可采用自体指甲制作人体组织修复的材料的方法
WO2019005724A1 (fr) * 2017-06-26 2019-01-03 Virtue Labs, LLC Compositions fongicides et méthodes d'utilisation
US10279045B2 (en) 2011-08-17 2019-05-07 Keranetics Llc Low protein percentage gelling compositions
US10385095B2 (en) 2011-08-17 2019-08-20 Keratin Biosciences, Inc Methods for extracting keratin proteins
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US20080274165A1 (en) * 2006-02-17 2008-11-06 Wake Forest University Health Sciences Wound healing compositions containing keratin biomaterials
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US20110070276A1 (en) * 2002-01-28 2011-03-24 Keraplast Technologies, Ltd. Bioactive keratin peptides
US20070298070A1 (en) * 2006-02-10 2007-12-27 Van Dyke Mark E Nerve regeneration employing keratin biomaterials
US20080274165A1 (en) * 2006-02-17 2008-11-06 Wake Forest University Health Sciences Wound healing compositions containing keratin biomaterials

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US10709789B2 (en) 2011-08-17 2020-07-14 KeraNetics, Inc. Low protein percentage gelling compositions
US10279045B2 (en) 2011-08-17 2019-05-07 Keranetics Llc Low protein percentage gelling compositions
US10385095B2 (en) 2011-08-17 2019-08-20 Keratin Biosciences, Inc Methods for extracting keratin proteins
US11034722B2 (en) 2011-08-17 2021-06-15 KeraNetics, Inc. Methods for extracting keratin proteins
US10792239B2 (en) 2012-11-06 2020-10-06 Kernnetics, Inc. White keratin compositions
CN107335094A (zh) * 2016-05-03 2017-11-10 薛培贤 一种可采用自体指甲制作人体组织修复的材料的方法
CN107335088A (zh) * 2016-05-03 2017-11-10 薛培贤 一种可采用自体的毛发制作伤口敷料的方法
WO2019005724A1 (fr) * 2017-06-26 2019-01-03 Virtue Labs, LLC Compositions fongicides et méthodes d'utilisation
CN111263629A (zh) * 2017-06-26 2020-06-09 德美实验室有限责任公司 化妆品组合物和使用方法
US11534383B2 (en) 2017-06-26 2022-12-27 Virtue Labs, LLC Cosmetic compositions and methods of use
AU2018292381B2 (en) * 2017-06-26 2023-10-26 Virtue Labs, LLC Cosmetic compositions and methods of use
US11883519B2 (en) 2017-06-26 2024-01-30 Virtue Labs, LLC Cosmetic compositions and methods of use
US12208153B2 (en) 2017-06-26 2025-01-28 Virtue Labs, LLC Cosmetic compositions and methods of use

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