Recherche dans les collections de brevets nationales et internationales
Une partie du contenu de cette demande n'est pas disponible pour le moment.
Si cette situation persiste, contactez-nous auObservations et contact
1. (WO1990006124) COMPOSITIONS POUR LE TRAITEMENT DE BLESSURES A CICATRISATION LENTE OU DIFFICILE
Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

FQ-RMITΓATIONS FOR TREATING sτ.nw AND NON-HEALING OUNDS

BACKGROUND OF THE INVENTION
Field of the Invention
This invention relates to compositions for initiating and accelerating the healing of wounds and to methods for purifying them, and, more particularly, to wound-healing compositions which comprise chitin
substantially free of protein.
Slow or non-healing wounds are by their nature difficult to resolve. There are several types of
intractable wounds such as surgical wounds, burns, diabetic ulcerations, stasis ulcerations and decubitus ulcerations. Many skin ulcerations result from a decrease of blood flow to a portion of the skin which keeps the tissue from being properly fed by oxygen and essential nutrients. Long- lasting surgical wounds may result from traumatization of the tissue during surgery in which blood flow is
interrupted.
Ische ic skin ulcers include both decubitus ulcers and ulcers resulting from occlusive, venous and arterial disease. These ulcers can lead to serious infections, are often very slow to heal and remain open and painful for months or years due to inadequate blood supply. In serious cases, the ulcers may lead to infection, amputation and death. Treatments are varied and include drug therapy and arterial reconstructive surgery.
Ulcerations of the skin are a particularly serious problem in the population of para and quadrapalegics, diabetics, long-term bedridden patients and the elderly as the incidence of such wounds is high. Seventy-five percent of the ten thousand new para and quadrapalegics acquire skin ulcerations. Ten percent of all hospital patients develop "bedsores" or stasis ulcerations. The elderly may develop these ulcerations due to ischemia.

Conventional treatments for non- or slow-healing wounds include, but may not be limited to, drug therapy, wound packs and surgery. Unfortunately, often times these modalities are slow and ineffective. Also, costs associated with such treatments can be extremely high and may involve hospitalization.
The compositions of the present invention provide safe and surprisingly effective formulations for initiating and accelerating wound healing. They comprise a
substantially pure form of chitin and may be readily and easily applied as a topical treatment.

Information Disclosure
U.S. Patents Nos. 3,903,268 and 3,911,116 disclose wound healing compositions which contain chitin, partially depolymerized chitin or a chitin derivative. They disclose the use of natural chitin materials as wound dressings, produced by grinding crustacean shells or by using fungal mats which are preferably sterilized as by heat or gas.
Their methods for purifying chitin material beyond
sterilizing it as by gas or heat are basically limited to the use of alternating treatments of NaOH and HC1 at 80 βC.

Increases of 22-25% in wound healing were described through the use of these disclosed chitin compositions in tests involving rats.
U.S. Patent No. 4,572,906 discloses a surgical dressing comprising a blend of gelatin and chitosan for the protection of wounds.
U.S. Patent No. 4,570,629 discloses a wound dressing which comprises a water-soluble linear anionic protein polyelectrolyte component derived from keratin and a second component which may be chitosan.
Wound-healing activities of different chitinaceous substances were evaluated in Balassa and Prudden,
"Applications of Chitin and Chitosan in Wound-Healing
Acceleration," Proc. First Internatl. Conf. on Chitin/ Chitosan 296-305 (1978) where wound-healing activities were evaluated to be from +30% of up to +75%.
None of these references discloses a chitinaceous substance with the degree of purity of those described and claimed herein, nor do any of the prior uses of chitinaceous substances for wound-healing demonstrate the surprisingly high degree of effectiveness of the chitin compositions disclosed herein.

SUMMARY OF THE INVENTION
This invention relates to a chitinaceous wound dressing for the treatment of wounds, particularly non- healing or slow-healing wounds, comprising chitin
substantially free of protein. Preferably, the wound dressings of this invention are powders of 50 mesh size or less and may be used for treating wounds in mammals by applying them directly to a wound surface to be treated.
The wound dressings may be applied in amounts of about 0.01 to about 0.1 g/cm2 of the wound surface to be treated.
Methods for purifying the chitin wound dressings are also contemplated and include the steps of placing crude chitin containing material into an acid bath; placing the chitin material into a heated caustic bath; treating the chitin material with a weak oxidizing agent; and drying the chitin material. Best results are obtained if the chitin material is further sterilized by, for example, using gamma rays and if the chitin material is ground to about a 50 mesh size or less before it is treated with the weak oxidizing agent.
The oxidizing agent is most preferably hydrogen peroxide and the caustic bath solution is most preferably a 1% sodium carbonate solution.

DETAILED DESCRIPTION
This invention relates to a chitin wound dressing material which, due to its substantial purity, results in a surprising increase in the healing capacity of the chitin. The chitin formulations and compositions of this invention approach a 100% healing rate, making these compositions highly superb wound-healing initiators and accelerators. Methods for purifying the chitin and methods for using it in treatment are also described.
Chitin is chemically known as a polysaccharide, poly

(N-acetyl-D-glucosamine) . It forms the cell walls of fungi and the exoskeletons of insects and crustaceans. As used herein, the term "chitin" embraces naturally occurring chitin, synthetic chitin, as well as poly (N-acetyl-D-glucosamine) and its epimer poly (N-acetylgalactosamine) . The N-acetylated partially depolymerized chitin, e.g.
chitotriose, chitobiose, is a substance which retains its polymeric nature but has undergone a reduction in molecular weight (i.e., chain length) as a result of (1) enzymatic action such as by a chitinase enzyme, (2) chemical treatment such as acid hydrolysis *or alkaline treatment, and (3) physical treatment. These materials are known in the art and procedures for their preparation may be found in
"Advances in Carbohydrate Chemistry," Vol. 15, pages 380-384, Academic Press, New York 1960, the disclosure of which is incorporated herein by reference. Thus, the molecular length is in the range from n-1 in which n corresponds to the number of repeating units in chitin to n-0 which is acetylated chitobiose.
The chitin derivatives contemplated are materials such as ethers formed with pharmaceutically-acceptable radicals and esters or salts with pharmaceutically-acceptable acids. Examples of suitable derivatives include hydroxy lower alkyl chitin such as hydroxyethyl chitin, carboxy alkyl chitin such as carboxymethyl chitin, salts of carboxy lower alkyl chitin such as the zinc salt, lower alkyl chitin such as methyl chitin and ethyl chitin, chitin acetate, chitin nitrate, chitin citrate, chitin phosphate, N-acyl derivatives derived from monocarboxylic aliphatic acids such as N-formyl, N-acetyl, N-propionyl, N-caproyl, and the like. It is preferred to use natural chitin as the source for the crude chitin containing material. The naturally occurring chitin from crustaceans is preferred because the source is readily available and because the conformation of the chitin, which is a less compacted matrix in its natural form, allows for enzymatic degradation.
The degree of improvement in wound healing
obtained with the substantially pure chitin of this
invention is greater than that derived from the wound healing chitinaceous materials of the prior art. Further it is surprising that the chitin described herein which is substantially free of protein has such increased wound healing effects.
When chitin is chemically purified from presently available sources, it becomes a superior wound dressing and permits healing of intractable ulcers that might otherwise be untreatable using the wound dressings of the prior art. The purity of the chitin is measured by the absence of proteinaceous contamination and other non-chitin materials. The degree of purity is readily assayed by the Lowry method, J.Biol.Chem. 193:265 (1951), or by polyacrylamide gel electrophoresis. Substantially pure chitin material for the purposes of this invention is that which is substantially free of protein and has detectable levels of protein of less than about 0.1 ug/mg of chitin material, preferably less than about 0.05 ug/mg and most preferably less than about 0.005 ug/mg.
Preparation of Pure Chitin
The means to purify the chitin are non-critical. Chemical hydrolysis of protein or enzymatic degradation are acceptable. Chemical hydrolysis is preferred and involves either extreme basic or acidic conditions such as below about pH 3.0 or above about pH 10.0. Mild oxidation
disrupts the natural matrix of chitin thus allowing
hydrolysis of residue protein. The use of heat will
facilitate the process. Enzymatic degradation using a combination of endo or exo peptidases can be used alone or in combination with chemical hydrolysis. Pronase or Protease K are two commercially available peptidases that are of use in this invention.
To facilitate complete removal of non-chitinaceous material, the chitin is preferably ground into a powder.
The preferred powder is of uniform particulate size wherein the particles average between about 50 to 400 mesh,
preferably to a standard mesh size of 100 or smaller.
The preferred method for preparing a substantially pure chitin material is as follows:
1. Typically the crude chitinaceous material such as crustacean shell is first dried and then placed in a weak acidic bath for 12 hours or more, preferably for 12 to about 18 hours. The particular acid used can be any one of several organic acids such as acetic acid, sulfuric acid, formic acid, lactic acid, ascorbic acid, citric acid or the like. Preferably a 1% hydrochloric acid solution is used. The material is then rinsed with a liquid such as water to bring the pH of the material to about 7.
2. After neutralizing, the material is placed into a heated weak caustic bath for several hours which is heated to between about 180β to about 210βF and allowed to mix, preferably for 4 to 8 hours. This caustic bath can comprise a solution of sodium hydroxide, sodium carbonate or the like, typically at a concentration of 2% to about 4%, preferably at a concentration of about 3% (wt/vol.).
Critical to the preparation of a substantially pure chitin material by this method is that the chitinaceous material is heated first in the acid solution and then in a caustic bath, rather than the reverse.
3. The material is then rinsed again to
neutralize the pH to about 1 , and then it is finely ground to give particles of a 50 mesh size or smaller. The ground material is placed in a weak oxidizing solution for a short period of time, typically about 2 hours. Oxidants which may be used include hydrogen sulfate, sulfuric acid or the like, referably a 3% solution of hydrogen peroxide is used. After oxidation, any remaining trace protein material is removed by placing the material in a second weak caustic solution such as those referred to above, preferably a 1% solution of sodium carbonate which is heated to between about 150 βF to about 250 βF, preferably to about 200 °F for 2 hours to about 6 hours, preferably for about 4 hours. Best results are obtained if the material is again rinsed with water to a pH of about 7 and placed in the caustic bath one more time.
The order of steps is important oxidation allows for easy hydrolysis of protein during the caustic step.
4. The material is then preferably subjected to a solvent such as an alcohol or acetone for about 15 to about 60 minutes, preferably for about 30 minutes. The preferred solvent is an 80% solution of denatured methanol. The material is then filtered to remove the solvent. This solvent bath step may be repeated for best results.
5. The material is then ready for drying which may be accomplished by using a convection or microwave oven or the like. Preferably the material is dried in a
microwave oven for about 10 minutes per 1/2 lb. of material at 30-60% power. It is preferred that the material is further sterilized, for instance, by exposure to about 1.25 rads of gamma rays before packaging or before using as a treatment.
Advantageously between steps 4 and 5 the material is further purified by dissolving the chitin material in concentrated HC1 and then rinsing and filtering the chitin material to bring the chitin material to a pH of about 6.0 to 7.0 by, for example, adding the following steps:
The chitin material is dissolved in concentrated HC1 while slowly adding distilled water to wet all the powder. Typically the material will be dissolved at a rate of about 250 ml of concentrated HC1 per about 25 g of material while adding about 75 to about 125 ml, preferably 100 ml, of water. The resulting solution is stirred for about 10 to 20, preferably 15 minutes. Preferably the solution is then filtered to remove undissolved particles.

The filtrate is then slowly poured into distilled water chilled to about 2'C to about 8βC, preferably to about 5'C, which is kept agitated. The mixture is then filtered and rehydrated with water until the pH of the material is about 6.0 to 7.0, preferably about 7.0.
After reaching neutrality, the cake is washed in ethanol, preferably about 90% ethanol, for about 20 to about 40 minutes, preferably for about 30 minutes and filtered at least once before drying.
Administration of Chitin Wound Dressing
The compositions and pharmaceutical formulations of the present invention may be applied to a wound surface using the same techniques and processes developed for cartilage and other chitinaceous materials, such as through the use of tablets, capsules or pellets of chitin which may be prepared from mixtures of chitin, with well-known
pharmaceutical excipients such as starch, sugar, certain forms of clay or the like. A powder or solution of chitin may also be used to impregnate a surgical gauze or pad which is applied to the wound. However, most preferably the chitin should be allowed to remain in its pure form and should be applied directly to the wound surface as a powder. It is believed that addition of other elements may interfere with the superior healing properties of the pure chitin alone.
The formulation of chitin as described herein may be applied as often as needed, typically it is applied at least in amounts of about 0.01 to about 0.1 g/cm2 preferably 0.05 to about 0.1 g/cm2 of the wound surface to be treated. Further contemplated by this invention are pharmaceutical formulations of the substantially pure chitin contained in amounts of about 1 gram in multiple-use shaker bottles.

Examples

Example 1 Crustacean shells were first mechanically picked clean of meat and extraneous material. The shell was then dried at 180°-200',F through a continuous convection type drier. The dried material was then placed in a 1% HCl acid bath and mixed for 12 hours.
After the acid bath, the material was rinsed with water to bring the material to a pH of 7. The material was then placed in a 3% sodium carbonate caustic bath for four (4) hours. The bath was heated to 180βF and the material was allowed to mix in order to remove protein from the polymer matrix. This material was then rinsed with water to once again bring the material to a pH of 7. This material was then allowed to dry at 150°F.
The resulting purified polymer was then finely ground, using a Bepex grinder (Bepex, Minneapolis, MN) , so that it was capable of passing through a standard 50 mesh screen. The ground material was placed in an oxidizing solution of 3% hydrogen peroxide for 2 hours.
The material was then placed in a weak caustic solution of 1% sodium carbonate and heated to 200 °F for four hours. Then, the material was rinsed with distilled water to a pH of 7 and subjected to a second bath in the caustic solution. After the second caustic treatment, the resulting material was again rinsed with distilled water to a pH of 7.
The rinsed material was then placed in a solvent bath of 80% methanol as a slurry and allowed to mix for 30 minutes. After mixing, the material was filtered for thirty minutes to remove the solvent. This solvent bath procedure was repeated once.
The filtered material was then dried in a
microwave oven for 10 minutes at 50% power and then
sterilized by exposure to 1.25 mrads of gamma rays.

Example 2
An eighty-three year old male suffered for three years from multiple stasis ulcerations of the legs.
Although multiple treatment modalities were attempted.

including surgical placement of skin grafts, no treatment proved effective. The product produced in Example 1 was applied once daily on the ulcerations by shaking the product in a powdered form on to the ulcerated areas at about 0.1 g per cm of affected area. After one week, new skin began to close the wound sites. With continual daily treatment for ten (10) months all ulcerations were entirely cleared and treatment was discontinued.

Example 3
A fifty-three year old male suffered multiple short duration ulcerations of the leg. This patient's family tree is plagued with this problem so it appears that his ulcerations were of a genetic underlying disease state. The ulcerations were treated daily with the powdered product of Example 1 in a manner similar to the treatment of the patient in Example 2. All ulcerations cleared after three weeks of treatment. The ulcerations have not reoccurred more than one year after healing.

Example 4
A seventy-two year old female suffered a large, deep ulceration of the leg due to diabetes. The ulceration was so severe that amputation was considered by her
physician. The product from Example 1 was used daily in treating the ulceration in a manner similar to that used for the patient in Example 2. The ulcerations were treated daily over a six-month period. Healing was evident within two weeks and after six months the ulceration was completely cleared and treatment was discontinued. The ulcer, however, reoccurred three months after initial healing. Therapy was again introduced and the ulcer cleared again within three weeks of treatment.

Example 5
Crustacean shells were first mechanically picked clean of meat and extraneous material. The shell was then dried at I80β-200°F through a continuous convection type drier. The dried material was then placed in a 1% HC1 acid bath and mixed for 12 hours.
After the acid bath, the material was rinsed with water to bring the material to a pH of 7. The material was then placed in a 3% sodium carbonate caustic bath for four (4) hours. The bath was heated to 180*F and the material was allowed to mix in order to remove protein from the polymer matrix. This material was then rinsed with water to once again bring the material to a pH of 7. This material was then allowed to dry at 150 'F.
The resulting purified polymer was then finely ground, using a Bepex grinder (Bepex, Minneapolis, MN) , so that it was capable of passing through a standard 50 mesh screen. The ground material was placed in an oxidizing solution of 3% hydrogen peroxide for 2 hours.
The material was then placed in a weak caustic solution of 1% sodium carbonate and heated to 200 βF for four hours. Then, the material was rinsed with distilled water to a pH of 7 and subjected to a second bath in the caustic solution. After the second caustic treatment, the resulting material was again rinsed with distilled water to a pH of 7.
The rinsed material was then placed in a solvent bath of 80% methanol as a slurry and allowed to mix for 30 minutes. After mixing, the material was filtered for thirty minutes to remove the solvent. This solvent bath procedure was repeated once.
Then, to dissolve each 25 grams of material,
250 ml of concentrated HC1 was added while slowly adding about 100 ml distilled water to wet all of the powder. The solution was stirred for about 15 minutes. The viscous solution was then filtered through closely packed glass wool to remove any undissolved particles.
The filtrate was poured slowly into about 2000 ml of agitating distilled water chilled to about 5°C. A cloudy precipitate appeared after adding the filtrate to the cooled water. The mixture was then vacuum filtered through a Buchner funnel with a #4 Whatman filter to leave a cake which was then rehydrated with about 1000 ml of distilled water. A pH reading was made. The mixture was then
filtered again through a #4 Whatman filter to form a cake. This last step was repeated 4 times until the resulting cake had a pH of about 7.0.
Once the caked material had a neutral pH, it was washed in 1000 ml of 90% ethanol and mixed for 30 minutes. The material was then filtered on #4 Whatman paper and a cake was formed. This step was repeated once.
After the above rinsings the material was dried in a vacuum convection oven at 150*F for 24 hours. The yield was approximately 86%. The dry cake was pulverized for use.
Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, the illustrations and examples should not be construed as a limitation upon the claims.