Compositions and methods for treating collagen-mediated diseases

Abstract

A drug product comprising a combination of highly purified collagenase I and collagenase II from Clostridium histolyticum is disclosed. The drug product includes collagenase I and collagenase II in a ratio of about 1 to 1, with a purity of greater than at least 95%. The invention further disclosed improved fermentation and purification processes for preparing the said drug product.

1 - 55 . (canceled) 
     
     
         56 . A drug product comprising as the sole active ingredients isolated and purified collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, wherein the collagenase I and collagenase II are mixed in a mass ratio of about 1 to 1, and both the collagenase I and collagenase II are at least 95% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         57 . The drug product of  claim 56 , wherein the drug product has one or more of the following characteristics:
 contains less than about 2% by area aggregated protein as determined by reverse phase high performance liquid chromatography;   contains less than about 1% by area of clostripain as determined by reverse phase high performance liquid chromatography;   contains less than about 1% by area of gelatinase as determined by anion exchange chromatography;   contains less than about 1 ug/mg (w/w) of leupeptin;   has a bioburden less than 1 cfu/ml;   contains less than 10 EU/ml of endotoxin.   
     
     
         58 . The drug product of  claim 56 , further comprising a pharmaceutically acceptable carrier or excipient selected from the group consisting of a sugar; a starch; cellulose and its derivatives; powdered tragacanth; malt; gelatin; talc; a glycol; propylene glycol; an ester; ethyl oleate; ethyl laurate; agar; a buffering agent; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; sodium lauryl sulfate; magnesium stearate; a coloring agent; a releasing agent; a coating agent; a perfuming agent; a preservative; an antioxidant; and any combination of the foregoing. 
     
     
         59 . The drug product of  claim 56 , wherein the drug product has a potency of between about 1700 and about 3500 SRC units per milligram of collagenase I and collagenase II. 
     
     
         60 . The drug product of  claim 56 , wherein the drug product has a potency of between about 50,000 and about 90,000 GPA units per milligram of collagenase I and collagenase II. 
     
     
         61 . The drug product of  claim 56 , wherein the collagenase I has a potency of between about 1700 and about 3500 SRC units per milligram of collagenase I, and collagenase II has a potency of between about 79,000 and about 170,000 GPA units per milligram of collagenase II. 
     
     
         62 . The drug product of  claim 56 , wherein and collagenase I and collagenase II both are at least 95% by area pure as determined by reverse phase high performance liquid chromatography, and wherein the drug product has no detectable deterioration of collagenase I or collagenase II for at least 10 days when tested by SDS-PAGE gel electrophoresis. 
     
     
         63 . A pharmaceutical formulation comprising a pharmaceutically acceptable excipient and a drug product comprising as the sole active ingredients isolated and purified collagenase I and collagenase II having the sequence of  Clostridium histolyticum  collagenase I and collagenase II, respectively, wherein the collagenase I and collagenase II have a mass ratio of about 1 to 1 and collagenase I and collagenase II are both at least 95% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         64 . The pharmaceutical formulation of  claim 63 , wherein the drug product is a sterile lyophilized powder and is stored at a temperature of about 5° C. 
     
     
         65 . The pharmaceutical formulation of  claim 63 , wherein the formulation is a lyophilized injectable formulation formulated with Sucrose, Tris and with a pH level of about 8.0. 
     
     
         66 . The pharmaceutical formulation of  claim 63 , wherein the formulation is a lyophilized injectable formulation comprising about 0.9 mg of the said drug product, about 18.5 mg of sucrose and about 1.1 mg of Tris, and wherein the targeting vial fill volume is about 0.9 mL. 
     
     
         67 . The pharmaceutical formulation of  claim 63 , wherein the formulation is a lyophilized injectable formulation comprising about 0.58 mg of the said drug product, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         68 . The pharmaceutical formulation of  claim 63 , wherein the formulation is a sterile lyophilized powder. 
     
     
         69 . The pharmaceutical formulation of  claim 68 , wherein the formulation is a lyophilized injectable composition formulation formulated with sucrose, Tris and with a pH level of about 8.0. 
     
     
         70 . The pharmaceutical formulation of  claim 68 , wherein the formulation is a lyophilized injectable formulation comprising about 0.9 mg of the said drug product, about 18.5 mg of sucrose and about 1.1 mg of Tris, and wherein the targeting vial fill volume is about 0.9 mL. 
     
     
         71 . The pharmaceutical formulation of  claim 63 , wherein the formulation is a lyophilized injectable composition comprising about 0.58 mg of the said drug product, about 12.0 mg of sucrose and about 0.7 mg of Tris. 
     
     
         72 . The pharmaceutical formulation of  claim 63 , wherein collagenase I and collagenase II are both at least 96% by area pure as determined by reverse phase high performance liquid chromatography. 
     
     
         73 . The pharmaceutical formulation of  claim 72 , wherein the formulation is a lyophilized injectable formulation formulated with Sucrose, Tris and with a pH level of about 8.0. 
     
     
         74 . The pharmaceutical formulation of  claim 73 , wherein the formulation is a lyophilized injectable formulation comprising about 0.9 mg of the said drug product, about 18.5 mg of sucrose and about 1.1 mg of Tris, and wherein the targeting vial fill volume is about 0.9 mL. 
     
     
         75 . The pharmaceutical formulation of  claim 73 , wherein the formulation is a lyophilized injectable formulation comprising about 0.58 mg of the said drug product, about 12.0 mg of sucrose and about 0.7 mg of Tris.