Title:  Keratin-based powders and hydrogel for pharmaceutical applications

United States Patent:  6,544,548

Issued:  April 8, 2003

Inventors:   Siller-Jackson; Arlene J. (Helotes, TX); Van Dyke; Mark E. (Fair Oaks Ranch, TX); Timmons; Scott F. (San Antonio, TX); Blanchard; Cheryl R. (San Antonio, TX); Smith; Robert A. (Jackson, MS)

Assignee:  Keraplast Technologies, Ltd. (San Antonio, TX)

Appl. No.:  638643

Filed:  August 14, 2000

A hydratable, highly absorbent keratin solid fiber or powder capable of absorbing a large weight excess of water may be produced by partially oxidizing hair keratin disulfide bonds to sulfonic acid residues and reacting the sulfonic acid residues with a cation. The neutralized suspension can be filtered, washed, and dried, leaving keratin solid which can be shredded into fibers and further ground into powder. Addition of water to the solid produces a hydrogel. The powder or hydrogel may be useful as an absorbent material, as a therapeutic for skin, or as an excipient. The keratin materials can be incorporated into nonwoven films. The hydrogel can be used as a biocompatible viscoelastic filler for implant applications. Another use for the absorbent keratin and keratin hydrogel is as an excipient in pharmaceutical and cosmetic applications.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes a hydratable solid derived from keratin that is highly absorbent and can form a hydrogel or viscoelastic hydrogel upon the application of water. The keratin solid can include protein having an ionizable pendant group such as sulfonic acid that can be derived from an oxidized protein disulfide linkage. A preferred source of protein is keratin, and particularly preferred is keratin obtained from hair, including human hair. While hair is a preferred source of keratinous material, other keratinous materials are also believed suitable for use in the present invention. Examples of other sources include animal hair, skin, hooves, feathers, beaks, feet and horns. The patient or a human donor are some preferred sources of hair, as hair from these sources is most likely to result in a non-immunogenic product, although animal hair may be acceptable for many individuals for many applications. In one method according to the present invention, hair is provided, preferably clean and unbleached. In another method, the hair is washed with Versa-Clean.TM. (Fisher Scientific, Pittsburgh, Pa.), rinsed with deionized water, and allowed to dry.

A. Preparation of Oxidized Keratin

The hair can be oxidized in peracetic acid or another suitable reagent such as H₂ O₂. One method utilizes between about 1% to 32% peracetic acid, at a temperature between about 0 degrees C. and 100 degrees C. for between 0.5 and 24 hours. In one method, about 1 weight/volume percent peracetic acid is used. One method treats 30 grams of hair with 500 mL of 4% peracetic acid at 4 degrees C. for 24 hours. Another method treats the hair at room temperature for 24 hours. Yet another method treats the hair at about 90 degrees C. for about 10 hours. In a preferred method, the hair is treated by heating the hair in the oxidizing agent for between about 1 and 4 hours at a temperature between about 20 and 100 degrees C. In a more preferred method, the hair is treated by heating the hair in the oxidizing agent for between about 1 and 2 hours at a temperature between about 80 and 100 degrees C. In a most preferred method, the hair is treated by heating the hair in about 2 weight/volume percent oxidizing agent for about 2 hours at a temperature of about 100 degrees C. The oxidation is believed to cleave a significant portion of keratin disulfide bonds forming cysteic acid residues having sulfonic acid groups. The sulfonic acid groups are believed to be hydrophilic in nature and will ionically bond to cations later in the process, forming a salt of the keratin and cation. The partial oxidation is also believed by Applicants to release existing short chain peptides, or form additional short chain peptides, which can remain associated with, or entrained in the keratin structure.

After oxidation, the keratin solid can be recovered from the oxidizing liquid using filtration or other suitable methods such as centrifugation or decantation. The recovered, oxidized solid can be washed with water or alcohol such as methanol or ethanol to remove the excess oxidizing agent. In a preferred embodiment, washing is limited to avoid removing too much of any soluble peptide chains entrained in the keratin.

B. Preparation of Hydratable Keratin

The solid fraction can be suspended in a suitable solvent or solvent mixture. The solvent should be capable of at least suspending the hair or keratin solid and keeping the solid sufficiently swelled for subsequent reaction. The solvent is preferably a non-aqueous solvent, as the presence of water can act to hydrolyze peptide backbone bonds, which can result in an inferior product. The solvent should be able to solubilize the later added base. One group of suitable solvents includes alcohols such as methanol and ethanol. Other solvents such as ether, tetrahydrofuran (THF), acetone, propylene glycol, 1,4-dioxane and glycol ethers may also be suitable as solvents. Small amounts of water will assist in swelling the keratin and may therefore be added to the aforementioned solvents in an amount up to 20 volume percent. The solvent used is preferably volatile to promote evaporation from the final solid product.

The hair or keratin solvent suspension can then have the pH titrated upward to at least about pH 7. Increasing the pH deprotonates the sulfonic acid groups, leaving the sulfonic acids free to exchange with another cation. The pH can be adjusted with a base, preferably having a monovalent cation. Preferred bases include sodium hydroxide and potassium hydroxide.

The pH-adjusted keratin suspension can be heated for a time and temperature sufficient to swell the keratin structure and promote neutralizing of the sulfonic acid sites with the provided cation. In a preferred method, the keratin suspension is boiled between about 0.5 hours and 12 hours. More preferably, the keratin suspension is boiled between about 0.5 hours and 3 hours. In one method, the keratin suspension is boiled for about 1 hour. Boiling for too long a time period leads to a mushy keratin which results from degradation of the peptide backbone. A hydrated keratin product is less preferred due to the greater difficulty of grinding the keratin.

After boiling, the keratin is preferably allowed to continue to react with the provided base cation at lower temperature and with stirring. The lower temperature reaction preferably takes place at a temperature of between about 15 and 30 degrees C. for between about 1 and 24 hours. More preferably, the lower temperature reaction takes place at a temperature of between about 20 and 25 degrees C. for between about 1 and 5 hours. In one method, the keratin suspension is allowed to react with stirring at room temperature for about 5 hours. In certain embodiments the reaction is held at the boiling point of the solvent for about 2 hours.

After reacting at lower temperature, the reacted solid can be separated from the solvent using any suitable method such as filtration. The solid is preferably washed with a solvent such as the same solvent used in the reaction. Washing the keratin removes some of the base, which is preferably removed. The base is preferably removed to make the keratin solid less caustic.

After filtration and washing, the keratin can be dried by a method such as evaporation under vacuum. In one method, the keratin is dried at room temperature under about 5 mm Hg vacuum for about 2 hours. The dried keratin is preferably somewhat brittle, which can result in a better product after grinding. The dried keratin can be shredded into fibers and can further be ground into a powder. The dried keratin can be directly ground into a powder using a mortar and pestle, a ball mill, or other means of breaking down or comminuting the dried keratin into particles. Alternatively, the keratin can be ground or milled in the solvent used for said neutralization step.

One resulting hydratable fiber or powder has been observed to absorb about 10 to 13 times its own weight in water. In one test, fibers having a length of between one quarter and one-half inch were observed to absorb an average of 1300%+/-33% of their weight in water at a temperature of 21.5 degrees C. The fiber has been observed to absorb at least 10 times its own weight in water within about 10 seconds. The powder has been observed to rapidly absorb water as well.

The fibers were also tested for various toxicity parameters and were found to be non-toxic, non-irritating, non-sensitizing, and biocompatible as indicated in Table 1.

 

C. Preparation of Nonwoven Films

1. Nonwoven Film Comprising Hydratable Keratin Fibers

Hydratable keratin fibers may be incorporated into a nonwoven film by admixing with synthetic fibers which serve as a binder. Such a nonwoven film can be formed by mixing synthetic fibers made from, but not limited to, .alpha.-olefins, acrylates, urethanes, acetates, nylons esters, or copolymers thereof with water-absorbent keratin fibers and heat pressing the mixture into a film of desired thickness. The synthetic fibers will serve as a binder for the keratin fibers, while not completely encapsulating them. This morphology provides mechanical integrity to the film, while allowing the keratin fibers to absorb water. The hydrated fibers can also release material which has been shown to be beneficial for repairing damaged epithelial tissue.

Nonwoven films can be prepared by preparing nonwoven webs of a synthetic polymer and then placing a layer of hydratable keratin fibers between two layers of the nonwoven polymer-web. For example, a nonwoven film was produced by first preparing a nonwoven web measuring approximately one half inch thick by 24 inches wide using 9 denier, 38 mm length polypropylene fibers. The web was made using a Rando-Webber, (Fiber Controls, Inc. Gastonia, N.C.) air laying machine operating at 2000 rpm, 12 ft./minute let off speed with a feed rate of 4 ft./minute. A web of approximately 20 feet in length was coated over half of its length on one side with keratin fibers of approximately 2-5 mm in length. The keratin was spread on the web using a hand sifter. The uncoated section of the web was folded back over the coated section to form a laminate of keratin between two layers of polypropylene. The laminate was passed through a Sigrna heated roller press (BF Perkins, Rochester N.Y.). The rolls were oil heated to 160^oC. and a pressure of 350 pounds per linear inch was applied. The surface of the top roller was textured so as to impress a texture in the finished nonwoven film. The laminate was fed through the rollers at approximately 4 ft./minute and the polypropylene softened and pressed such that a film of approximately 3 mm in thickness resulted. This nonwoven film was bound together by the polypropylene, but retained some flexibility. The keratin fibers were at least partially exposed such that the film wetted easily and the keratin became gelatinous upon addition of water.

Nonwoven films can be made by other procedures. For example, if a more open nonwoven is desired, a laminate of keratin and synthetic fibers can be prepared as described above, and this laminate processed by a through air dryer. The through air dryer is capable of heating the laminate but does not apply pressure to the film. In this process, the synthetic fibers can be softened and bound together, thus providing a structural matrix for the keratin fibers. The result is a nonwoven web which retains more of its original, open morphology. Also, films made with synthetic fibers can sometimes be stiff. The example given above resulted in a film resembling burlap. If a softer film is desired, alternative fibers or blends of fibers may be used to produce the nonwoven web. A blend of cotton and polypropylene, for example, would provide a softer, more pliable nonwoven film. Cotton fibers can conveniently be blended into the nonwoven web during the air laying or carding process, prior to coating with keratin fibers. Other natural fibers such as hemp may also be used.

These nonwoven films are produced from a loose, laminated precursor. However, the keratin fibers are exposed to the surface in the final product. Although the exemplified polymeric binder is hydrophobic, the nonwoven film wets easily and readily absorbs water. Once water is applied to the film, the keratin fibers absorb it and swell, thus forming a hydrogel which is entrained in the unswollen binder. This type of film is of utility as a wound dressing because of the capability of absorbing wound exudate and forming a hydrated, gelatinous cover over the wound site. Such a dressing provides a closed, moist environment, conducive to wound healing. Drug actives that are useful in wound healing applications such as antibiotics, anti-inflammatory agents, analgesics and the like may also be bound to the keratin used in producing the nonwoven films. The nonwoven film may be produced using a biocompatible synthetic binder material such as polylactic acid, polyglycolic acid, copolymers thereof, and drug loaded keratin. Such a nonwoven device is useful as a biocompatible implant for controlled and/or sustained delivery of active agents. Due to the water absorbency, these nonwoven films also have utility as components of disposable diapers, feminine hygiene products as well as any other application where a nontoxic film with biocompatibility and absorbency is desired and the healing of damaged skin or other epithelial tissue is deemed beneficial or necessary. These films also have utility as implant materials for the repair of damaged hard or soft tissues, as cell scaffolds and tissue engineering applications.

2. Nonwoven Film Comprising Oxidized Hydratable Keratin Powder

Nonwoven films can also be prepared with oxidized keratin powder. For example, a nonwoven web measuring approximately one half inch thick by 24 inches wide was prepared using 1.7 denier, 38 mm length Fortrel.RTM. polyester fibers supplied from Wellman, Inc. (Johnsonville, S.C.). A blend of 20 wt. % low melt and 80 wt. % high melt fibers was first mixed by hand, then run through a Garnett fine opener, and finally carded. This was done prior to laying the web to provide a homogeneous sample. The web was made using a Rando-Webber (Fiber Controls, Inc, Gastonia, N.C.) air laying machine operating at 2000 rpm, 12 ft./minute let off speed with a feed rate of 4 ft./minute.

The web was mechanically entangled using a hydrobonder from Honeycomb Systems, Div. (Division of Valmet, Inc., Biddeford, Me.). This equipment consists of a screen conveyor and a manifold of high pressure waterjets. The web passes under the water jet manifold and the force of the water forces the fibers through the screen, thereby entangling them. The degree of entanglement can be controlled by the mesh size of the screen conveyor. The excess water was removed using a vacuum stripper manufactured by Evac Corporation, (Spartanburg, S.C.). This process reduced the web's thickness to approximately one eighth inch and resulted in a more tightly entangled web with more structural integrity than one produced using only the air laying technique.

Two rolls of web, 20 feet in length, were prepared using this process and used to make a laminate with hydratable keratin powder. The keratin powder was less than 300 .mu.m in size and was prepared as described for keratin fibers. The laminate was prepared by conveying the two webs from separate spools and spraying the powder onto the bottom web. Powder was sprayed using a GEMA.TM. powder sprayer with an electrostatic spray gun (the electrostatic feature was not used). The gun was operated at 2 psi with a flow of 4.5 m³ /hour through the reservoir and a make-up flow of 1.5 m³ /hour through the gun. The nonwoven laminate was conveyed with a take-up winder operating at 32 ft./minute. The use of a tighter web allowed small keratin particles (length of less than 1 mm) to be used without significant loss. This was especially important during the winding and unwinding operations prior to thermal bonding. The web could also be moistened slightly prior to spraying the keratin in order to promote adhesion.

The nonwoven laminate was passed through a Sigma heated roller press. The rolls were oil heated to 160^oC. and a pressure of 200 to 215 pounds per linear inch was applied. The surface of the top roller was textured so as to impress a texture in the finished nonwoven film. The laminate was fed through the rollers at approximately 15 to 17 ft./minute. This procedure resulted in a nonwoven film of approximately 3 mm in thickness. The surface of the film was smoother than the film described previously and the use of polyester, rather than polypropylene, produced a softer, more pliable film. The keratin powder was at least partially exposed such that the film wetted easily and the keratin became gelatinous upon addition of water.

D. Keratin Delivery Systems

Active agents can be incorporated into a hydratable keratin excipient to form a delivery system. By "active agent" is meant a compound, the delivery of which is the object of the application of the preparation comprising that compound and the keratin excipient of the present invention. Delivery of an active agent is generally desired because of a beneficial and/or desired effect or attribute imparted by the agent upon delivery. Physical classes of active agents that can be incorporated into the keratin excipient of the present invention include, but are not limited to, compounds that may ion exchange with sulfonic acid groups, those compounds that may otherwise be formulated as hydrochlorides, compounds that form an electrostatic association with the keratin excipient, polar agents, polynucleotide agents, and polypeptide and peptide agents. Polypeptide agents include both recombinant and native polypeptides. For example, insulin is a polypeptide agent that may be incorporated into the keratin excipient of the present invention. Polar compounds include, but are not limited to 4-acetaminophenol, aspirin and beta-lactams. Compounds that may otherwise be formulated as hydrochlorides include, but are not limited to phenylpropanolamine and pseudoephedrine.

The active agent may be a pharmaceutical agent. By "pharmaceutically active agent" is meant any compound commonly referred to as a "drug" and its equivalents which include any physiologically or pharmacologically active substance that produces a localized or systemic effect or effects in animals. The term "animal" includes mammals, humans and primates such as domestic, household, sport or farm animals such as sheep, goats, cattle, horses and pigs, laboratory animals such as mice, rats and guinea pigs, fishes, avians, reptiles and zoo animals. Examples of pharmaceutical agents that may be formulated as hydrochlorides, and examples of pharmaceutical agents in general, are found in Remington: The Science and Practice of Pharmacy, (19th ed., ed. A. Gennaro) 1995, The Pharmacological Basis of Therapeutics, by Goodman and Gilman, 6th Ed., 1980, published by the MacMillan Company, London and in The Merck Index, 11th Edition, 1989, published by Merck & Co., Rahway, N.J., herein incorporated by reference in their entirety. A non-exhaustive list that exemplifies some of the classes and types of pharmaceutical agents that may be used in the present invention is provided in Table 2.

 

The active agent may be a cosmetic agent. The term "cosmetic" used in relation to a formulation or product means a formulation or product that qualifies as a cosmetic under the Federal Food, Drug and Cosmetic Act, 21 U.S.C. .sctn.321(i). By "cosmetic agent" is meant an agent that is incorporated in a cosmetic formulation or product and that agent is reported or believed to impart a beneficial and/or desirable effect or attribute upon application of the cosmetic. Cosmetic agents include, but are not limited to, anti-wrinkle agents, such as retinol and alpha-hydroxy acids, polypeptide and peptide agents derived from skin proteins (e.g., keratin, collagen and elastin), sunscreens, humectants, antioxidants, vitamins, tanning agents (both artificial and those that effect melanogensis), and whitening agents. Descriptions of cosmetic agents may be found in International Cosmetic Ingredient Dictionary and Handbook, Cosmetic Toiletries and Fragrance Association, 8^th ed. 2000, and A Consumers Dictionary of Cosmetic Ingredients, Ruth Winter, Three Rivers Printers, 5^th ed, 1999, both herein incorporated by reference.

In some embodiments of the present invention, the keratin excipient may be incorporated in a nonwoven film. In other embodiments of the present invention, the keratin excipient may be incorporated into a drug delivery device. Examples of systems that may be adapted for transdermal use with the compositions described herein described in U.S. Pat. No. 4,816,252; U.S. Pat. No. 5,122,382; U.S. Pat. No. 5,198,223; U.S. Pat. No. 5,023,084; U.S. Pat. No. 4,906,169; U.S. Pat. No. 5,145,682; U.S. Pat. No. 4,624,665; U.S. Pat. No. 4,687,481; U.S. Pat. No. 4,834,978; and U.S. Pat. No. 4,810,499 (all incorporated herein by reference). Examples of systems that may be adapted for inhalation of the compositions described herein are described in U.S. Pat. No. 5,884,620 and U.S. Pat. No. 5,960,792, both incorporated herein by reference.

In some embodiments, the basis of drug delivery is binding an active agent in a keratin matrix for later release by some mechanical, chemical, biochemical or cellular mechanism. Active agents can either be electrostatically bound or physically entrained in the matrix. Electrostatic binding can occur in the form of ionic bond formation (specific, results in tightly bound active agent) and Van der Waal's bond formation (less specific, results in a less tightly bound drug). Entraining an active agent can be most effectively accomplished by providing a processes that incorporates intimate mixing of the agent and the matrix, most notably, solution processing. Multiple combinations of these binding mechanisms are possible, depending on the structure and functionality of the drug molecule.

Electrostatic binding (ionic and Van der Waal's) can be accomplished by performing an ion exchange with oxidized keratin in nonaqueous media. The media needs to solubilize the drug and effectively suspend, and at least slightly swell the keratin. Ionic bonds will form between ionic species, whereas Van der Waal's bonds will form between ionizable functional groups of opposite partial charge.

Entrainment can be accomplished by physical mixing of the active agent and the keratin matrix. The more intimate the mixing, the more likely to result in tightly bound drug. This approach works best for large molecules such as protein therapeutics because larger molecules can entangle more effectively than smaller ones. A most effective approach to promote intimate mixing is to add an active agent and keratin matrix in suspension or solution. This can be accomplished by adding an aqueous solution of active agent to dehydrated keratin absorbent and forming a hydrogel, by adding a keratin hydrogel to dehydrated active agent, or by adding a solution of active agent to a keratin hydrogel. The resulting drug loaded hydrogel can be processed into a dosage for in the gel state or dried and ground into a powder.

The keratin matrix needs to be of a particle size category in dosage forms to be used for inhalation or intravenous (IV) injection. Dry, solid keratin loaded with an active agent as described previously can be processed into a powder of specific particle size using any one of a variety of grinding techniques known to those skilled in the art such as grinding or milling. Classification into a specific particle size range can be performed by sieving. For inhalation applications, particulates of different sizes will reach specific areas of the respiratory system. For example, particles that are greater than 5 microns will reach the nasopharynx. Particles between about 2 and about 5 microns will reach the trachea and bronchus. To reach the alveoli, particles must typically be less than about 1 micron. It has been shown that particles of about 3 microns or larger are deposited in the respiratory tree and become encysted. Keratin particles for drug delivery must reach the alveoli in order to be efficiently absorbed by the bloodstream and therefore must be less than about 1 micron.

For intravenous injections applications, the drug loaded keratin particles need to be small enough to be metabolized in the bloodstream (i.e. absorbed by cells) and also sufficiently small enough so as not to cause blockage of the small capillaries in the circulatory system. The limiting size in this regard is the lesser of the two, namely the mean capillary diameter (in humans, ca. 5 microns).

For both the inhalation and intravenous injection dosage forms, the particles sizes discussed must be the fully hydrated particle size. Depending on the processing parameters of the keratin used and the conditions under which the drug is incorporated into the keratin matrix, hydration capacities will vary. Considering a maximum absorption mass of ca. 20 times, the maximum particle sizes discussed previously would be reduced by ca. 20 times. Specific swelling volumes would depend on the materials used since resulting densities will vary.

In various embodiments, the particle size is less than 0.5 micron, or less than 1.0 micron, or less than 2 microns, or less than 3 microns, or less than 4 microns, or less than 5 microns, or less than 10 microns, or less than 20 microns, or less than 30 microns, or less than 40 microns, or less than 50 microns, or less than 100 microns. In other embodiments the particle size is between about 0.1 micron and about 1 micron, or between about 0.1 micron and about 2 microns, or between about 0.1 micron and about 3 microns, or between about 1 micron and about 3 microns, or between about 1 micron and about 5 microns, or between about 1 micron and about 10 microns.

The active agent delivery system of the present invention offers distinct advantages over conventional drug dosage forms. As with most delivery systems, sustained or controlled release allows the level of an agent to be maintained at a more consistent concentration, thereby allowing larger doses to be administered on a less frequent basis. In the system described here, the chemical and material properties of the keratin determine the properties of the dosage form. For example, loading can be varied by the availability of sulfonic acid binding sites, which can in turn be controlled by the keratin oxidation process. Further, the disintegration and breakdown of the keratin can also be controlled by the relative amount of disulfide crosslinks remaining after the oxidation process. Disintegration and dissolution will effect the release kinetics of the dosage form. For longer term release applications, the release rate can be controlled by the hydrolysis rate of the keratin, which in turn can be controlled by processing and formulation parameters such as oxidant type, oxidation time, and solids content. When incorporated into a nonwoven wound dressing, the bound drugs can be tailored to those most beneficial to wound healing such as, for example, antibiotics, biocides, pain medications and growth factors.

Claim 1 of 48 Claims

What is claimed is:

1. A preparation comprising a cross-linked insoluble keratin excipient associated with a pharmaceutical or cosmestic active agent, where said keratin excipient is chemically modified to contain sulfonate groups.
 

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