Wound healing compositions

Claim 2

A composition for stimulating wound healing comprising a polypeptide which is isolated from a mammalian fluid or pancreas, comprises 52 amino acids and has a molecular weight of about 5800 in a physiologically acceptable carrier.

Claim 3

A method of accelerating wound healing comprising treating a wound with the composition of claim 1.

Claim 4

The method of claim 2, wherein the wound is treated with the composition in an amount sufficient to provide 25 ng/ml compsition of the polypeptide to the wound.

Claim 5

The composition of claim 1 wherein the carrier is selected from the group consisting of carboxymethyl-cellulose, oil-in-water emulsion and water-in-oil emulsions.

Claim 6

The composition of claim 1 wherein the polypeptide is isolated from a mammalian fluid selected from the group consisting of blood, plasma and serum.

Claim 7

The composition of claim 1 wherein the polypeptide is isolated from the fluid or pancreas of a mammal selected from the group consisting of human, swine, horses, sheep and beef.

Claim 8

The composition of claim 1 wherein the polypeptide is isolated by: (a) isolating as a solid fraction the portion of the mammalian starting material containing alpha and beta globulins, (b) homogenizing the isolated fraction in 40 volumes of 75% ethanol - 0.02 M hydrochlorid acid at 4°C, (c) centrifuging the homogenate and separating the resulting sediment and supernatant, (d) adjusting the pH of the separated supernatant to 8.5 with sodium hydroxide, (e) mixing the pH adjusted supernatant with four times its volume of acetone-ethanol, (f) cooling the resulting mixture to at least -20°C for at least fourteen hours, (g) thereafter separating and drying the resulting precipitate, (h) extracting the dried precipitate with acetic acid, (i) passing the supernatant of the extraction through a molecular sieve column and collecting the eluate with stimulating activity for cellular growth, (j) dissolving the dried precipitate of the eluate in a pH 7 phosphate buffer solution, (k) depositing the resulting buffered solution on an equilibrated cationic exchanger, (1) eluting the cationic exchanger column and collecting the eluate with stimulating activity for cellular growth, and (m) evaporating the solvent from this fraction.

Claim 9

The composition of claim 7 wherein the fraction of mammalian material containing alpha and beta globulins is isolated as a precipitate by adding 9% (v/v) ethanol to a fluid suspension of the material, precipitating protein for 12 hours at -3 0 C and pH 7.2, removing the precipitate by centrifugation, adding ethanol to the clear supernatant fluids to a final concentration of 19%(v/v) and adjusting the pH to 5.85, precipitating protein for 12 hours at -5 0 C, removing the precipitate by centrifugation, adding ethanol to the clear supernatant fluid to a final concentration of 40% (v/v) maintaining a pH of 5.85, precipitating protein for 12 hours at -9°C, and collecting the precipitate by centrifugation.

Claim 10

A composition for stimulating wound healing comprising a glycosteroid and a carrier, said glycosteroid having the structure: wherein R is an oligosaccharide group containing five sugars.

Claim 11

A composition for stimulating wound healing comprising a glycosteroid and a carrier, said glycosteroid having the structure: wherein R is an oligosaccharide group containing five sugars.

Claim 12

A method of accelerating wound healing comprising treating a wound with the composition of claim 9.

Claim 13

The method of claim 10 wherein the wound is treated with the composition in an amount sufficient to provide 1 to 10 p gram/ml composition of the glycosteroid to the wound.

Claim 14

The method of claim 9 wherein the carrier is selected from the group consisting of carboxymethylcellulose, oil-in-water emulsions, water-in-oil emulsions and aqueous solutions.

Claim 15

The composition of claim 9 wherein the glycosteroid is isolated from a mammalian tissue selected from the group consisting of heart, lung, muscle and spleen.

Claim 16

The composition of claim 9 wherein the glycosteroid is isolated from a mammalian fluid selected from the group consisting of blood, plasma and serum.

Claim 17

The composition of claim 9 wherein the glycosteroid is isolated from the fluid or organ of a mammal selected from the group consisting of human, swine, horses, sheep and beef.

Claim 18

The composition of claim 9 wherein the glycosteroid is isolated from mammalian tissue or fluid by: (a) homogenizing the mammalian tissue or fluid with an aqueous solution with 0.2% NaCl; (b) clearing the homogenate from·the cellular debris by centrifugation or filtration; (c) thereafter ultra-filtering the cleared homogenate; (d) concentrating the ultrafiltrate at 40°C until the ionic strength thereof correspond to a 0.8% NaCl solution; (e) binding organic material in the concentrated ultrafiltrate to active carbon; (f) thereafter extracting the active carbon with 10-30% acetic acid; (g) evaporating the solvent from the extract; (h) dissolving the residue in 0.2-2M acetic acid; (i) chromatographing the resulting solution over a molecular sieve column ; (j) thereafter eluting the column with 0.2-2M acetic acid and collecting second fraction with cell preliferating activity. (k) removing the solvent from the collected eluate.

Claim 19

The composition of claim 16 wherein the glycosteroid is further concentrated by: (a) dissolving the dried eluate resulting from step (k) of claim 8 in ethanol-methylene chloride; (b) chromatographing the dissolved eluate over a silica gel column; (c) collecting the fractions of silica gel column eluate which have R f values of 0.4, 0.55 and 0.7; and (d) evaporating the solvent.

Claim 20

A composition for stimulating wound healing comprising a glycosphingolipid and a carrier, said glycosphingolipid having the formula: wherein R is an oligosaccharide containing five sugar units.

Claim 21

A composition for stimulating wound healing comprising a glycosphingolipid and a carrier, said glycosphingolipid having the formula: wherein R is an oligosaccharide containing five sugar units.

Claim 22

A method of accelerating wound healing comprising treating a wound with the composition of claim 18.

Claim 23

The method of claim 19 wherein the wound is treated with the composition in an amount sufficient to provide 5 to 50 µ gram/ml ointment of the glyco- sphingolipid to the wound.

Claim 24

The method of claim 18 wherein the carrier is selected from the group consisting of carbcxy- methylcellulose, oil-in-water emulsions, water-in-oil emulsions and aqueous solutions.

Claim 25

The compositions of claim 18 wherein the glycosphingolipid is isolated from a mammalian tissue selected from the group consisting of heart, lung, muscle and spleen.

Claim 26

The composition of claim 18 wherein the glycosphingolipid is isolated from a mammalian fluid selected from the group consisting of blood, plasma and serum.

Claim 27

The composition of claim 18 wherein the glycosphingolipid is isolated from the fluid or organ of a mammal selected from the group consisting of humans, swine, horses, sheep and beef.

Claim 28

The composition of claim 18 wherein the glycosphingolipid is isolated from mammalian tissue or fluid by: (a) homogenizing the mammalian tissue or fluid with an aqueous solution with 0.2% NaCl; (b) clearing the homogenate from cellular debris by centrifugation or filtration; (c) thereafter ultra-filtering the cleared homogenate; (d) concentrating the ultrafiltrate at 40°C until the ionic strength thereof correspond to a 0.8% NaCl solution; (e) binding organic material in the concentrated ultrafiltrate to active carbon (1% by weight); (f) thereafter extracting the active carbon with 10-30% acetic acid; (g) evaporating the solvent from the extract; (h) dissolving the residue in 0.2-2M acetic acid; (i) chromatographing the resulting solution over a molecular sieve column; (j) thereafter eluting the column with 0.2-2M acetic acid and collecting the first fraction with cell proliferating activity; and (k) removing the solvent from the collected eluates.

Claim 29

The composition of claim 25 wherein the glycosphingolipid is further concentrated by: (a) dissolving the dried eluate resulting from step (k) of claim 8 in acetic acid; (b) adsorbing the dissolved eluate on an equilibrated silica gel column having C-8 chains; (c) thereafter washing the column with a logarithmic gradient of 0.1% acetic acid - 90% alcohol; . (d) collecting the 30% alcohol containing fraction; and (e) evaporating the solvent.