MODIFIED COLLAGEN
The present invention relates to a modified collagen obtainable by providing isolated collagen; freezing the isolated collagen; dehydrating the frozen collagen; and maturing the dehydrated collagen. Also disclosed are methods of preparing the modified collagen and uses thereof.
1 .- 26 . (canceled)
27 . A composition comprising matured collagen, wherein the matured collagen comprises collagen stored at a temperature of at least about 40° C. for a period of at least one week or at a temperature of about 2-8° C. for about 1-3 years.
28 . The composition of claim 27 , wherein the matured collagen comprises the collagen stored at a temperature of about 40° C. to about 65° C. for a period of at least one week.
29 . The composition of claim 27 , wherein the matured collagen comprises the collagen stored at a temperature of at least 65° C. for a period of at least one week.
30 . The composition of claim 27 , wherein the matured collagen comprises the collagen stored at a temperature of about 2-8° C. for about 1-3 years
31 . The composition of claim 27 , further comprising gentamicin, bupivacaine or a pharmaceutically acceptable salt thereof.
32 . The composition of claim 31 , wherein the composition comprises bupivacaine HCl.
33 . The composition of claim 31 , wherein the composition comprises about 25% w/w to about 57% w/w bupivacaine HCl.
34 . The composition of claim 31 , wherein the composition exhibits a release profile substantially corresponding to the following pattern:
less than about 60% of the bupivacaine is released in about 45 minutes;
less than about 70% of the bupivacaine is released in about 60 minutes;
less than about 90% of the bupivacaine is released at about 120 minutes; and
when dissolution tested using a Dissolution Apparatus Type II in 500 mL of PBS buffer (pH 7.4, bath temperature 37° C.) and stirred at 50 rpm for 24 hours.
35 . The composition of claim 27 , wherein a 0.9% dispersion of the composition has a viscosity of less than 500 cP at a temperature of 40° C. when tested for viscosity by a method comprising:
preparing a 0.9% dispersion from the composition; and
measuring the viscosity of the dispersion using a Brookfield Digital Rheometer DV-III at a constant shear rate of 15 s −1 at a temperature of 40° C.
36 . The composition of claim 27 , wherein the composition absorbs about 60 mg of water when tested for water uptake by:
weighing a 1.5 cm×4 cm sample of the composition in a dry state to obtain a dry weight;
inserting the sample in water for injection for about 10 minutes;
weighing the sample after 10 minutes of insertion in water to obtain a wet weight; and
wherein the difference between the wet weight and dry weight is the amount of water absorbed.
37 . The composition of claim 27 , wherein the composition swells to a thickness of less than 60 μm when tested for swelling by:
measuring the thickness of a 1.5 cm×4 cm sample of the composition in dry state;
inserting the sample in water for injection for about 10 minutes;
measuring the thickness of the sample after 10 minutes of insertion in water to obtain thickness in a wet state; and
wherein swelling is the difference between the thickness in the wet state and dry state.
38 . The composition of claim 27 , wherein the composition degrades in about 25 minutes when tested for degradation by a method comprising:
(a) placing a 4.5 cm×4.5 cm sample of the composition in 15 mL of 0.2N phosphate buffer (pH 7.4 with CaCl 2 );
(b) adding 0.5 mL of 10 mg/mL Collagenase Type IA-S to the solution in step (a);
(c) agitating the solution of step (b) using a shaking water bath at 37° C. at 120 rpm for 60 minutes; and
(d) measuring the degradation of the sample every 5 minutes throughout step (c).
39 . The composition of claim 27 , wherein about 50% of the composition is degraded in about 15 to 20 minutes upon when tested for degradation by a method comprising:
(a) placing a 3.1 cm×3.1 cm sample of the composition in 15 mL of 0.2N phosphate buffer (pH 7.4 with CaCl 2 );
(b) adding 100 μl of 10 mg/mL Collagenase Type IA-S to the solution in step (a); and
(c) measuring the degradation of the sample after 5, 10, 15, 25, 40, 60, and 90 minutes.
40 . A composition comprising matured collagen prepared by a method comprising the steps of:
(a) providing isolated collagen;
(b) freezing the isolated collagen;
(c) dehydrating the frozen collagen; and
(d) maturing the dehydrated collagen, wherein the maturing step comprises storing the dehydrated collagen at a temperature of at least 40° C. for at least one week or at a temperature of about 2-8° C. for about 1-3 years.
41 . The composition of claim 40 , wherein step (a) comprises providing an isolated collagen dispersion having a concentration of about 3-30% (w/w) collagen particles.
42 . The composition of claim 40 , wherein step (d) comprises storing the dehydrated collagen at a temperature of about 40° C. to about 65° C. for a period of at least one week.
43 . The composition of claim 40 , wherein the maturing step comprises storing the dehydrated collagen at a temperature of at least 65° C. for a period of at least one week.
44 . The composition of claim 40 , wherein the matured collagen comprises the collagen stored at a temperature of about 2-8° C. for about 1-3 years
45 . The composition of claim 40 , further comprising gentamicin, bupivacaine or a pharmaceutically acceptable salt thereof.
46 . The composition of claim 45 , wherein the composition comprises bupivacaine HCl.
47 . The composition of claim 46 , wherein the composition comprises about 25% w/w to about 57% w/w of bupivacaine HCl.
48 . The drug delivery composition of claim 45 , wherein the composition exhibits a release profile substantially corresponding to the following pattern:
less than about 60% of the bupivacaine is released in about 40 minutes;
less than about 70% of the bupivacaine is released in about 60 minutes;
less than about 90% of the bupivacaine is released at about 120 minutes; and
when dissolution tested using a Dissolution Apparatus Type II in 500 mL of PBS buffer (pH 7.4, bath temperature 37° C.) and stirred at 50 rpm for 24 hours.
49 . The composition of claim 40 , further comprising:
(e) preparing an aqueous dispersion of the matured collagen of step (d);
(f) degrading the aqueous dispersion; and
(g) dehydrating the aqueous dispersion.
50 . The composition of claim 49 , wherein the dehydrated collagen of step (c) is milled before step (d).
51 . The composition of claim 49 , wherein step (d) comprises storing the dehydrated collagen at a temperature of at least 40° C. for a period of at least four weeks; or comprises storing the dehydrated collagen at a temperature of at least 65° C. for a period of at least one week.
52 . The composition of claim 49 , wherein a 0.9% dispersion of the composition has a viscosity of less than 500 cP at a temperature of 40° C. when tested for viscosity by a method comprising:
preparing a 0.9% dispersion from the composition; and
measuring the viscosity of the dispersion using a Brookfield Digital Rheometer DV-III at a constant shear rate of 15 s −1 at a temperature of 40° C.
53 . The composition of claim 49 , wherein the composition absorbs about 60 mg of water when tested for water uptake by:
weighing a 1.5 cm×4 cm sample of the composition in a dry state to obtain a dry weight;
inserting the sample in water for injection for about 10 minutes;
weighing the sample after 10 minutes of insertion in water to obtain a wet weight; and
wherein the difference between the wet weight and dry weight is the amount of water absorbed.
54 . The composition of claim 49 , wherein the composition swells to a thickness of less than 60 μm when tested for swelling by:
measuring the thickness of a 1.5 cm×4 cm sample of the composition in dry state;
inserting the sample in water for injection for about 10 minutes;
measuring the thickness of the sample after 10 minutes of insertion in water to obtain thickness in a wet state; and
wherein swelling is the difference between the thickness in the wet state and dry state.
55 . The composition of claim 49 , wherein the composition degrades in about 25 minutes when tested for degradation by a method comprising:
(a) placing a 4.5 cm×4.5 cm sample of the composition in 15 mL of 0.2N phosphate buffer (pH 7.4 with CaCl 2 );
(b) adding 0.5 mL of 10 mg/mL Collagenase Type IA-S to the solution in step (a);
(c) agitating the solution of step (b) using a shaking water bath at 37° C. at 120 rpm for 60 minutes; and
(d) measuring the degradation of the sample every 5 minutes throughout step (c).
56 . The composition of claim 49 , wherein about 50% of the composition is degraded in about 15 to 20 minutes upon when tested for degradation by a method comprising:
(a) placing a 3.1 cm×3.1 cm sample of the composition in 15 mL of 0.2N phosphate buffer (pH 7.4 with CaCl 2 );
(b) adding 100 μl of 10 mg/mL Collagenase Type IA-S to the solution in step (a); and
(c) measuring the degradation of the sample after 5, 10, 15, 25, 40, 60, and 90 minutes.