Mixtures of calcitonin drug-oligomer conjugates comprising polyalkylene glycol, uses thereof, and methods of making same

• US 20030060606A1
• Filed: 06/04/2001
• Published: 03/27/2003
• Est. Priority Date: 06/04/2001
• Status: Active Grant

• Current Assignee: Biocon Limited (Biocon Incorporated)
• Original Assignee: Nobex Corporation (Biocon Incorporated)
• Inventors: Ekwuribe, Nnochiri N., Odenbaugh, Amy L., Price, Christopher H., Ansari, Aslam M.
• Granted Patent: US 6,713,452 B2
• Time in Patent Office: Days
• US Class Current: 530/399
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: C07K 14/585 Calcitonins

Abstract

A mixture of conjugates in which each conjugate in the mixture comprises a calcitonin drug coupled to an oligomer that includes a polyalkylene glycol moiety is disclosed. The mixture may lower serum calcium levels in a subject by 10, 15 or even 20 percent or more. Moreover, the mixture may be more effective at surviving an in vitro model of intestinal digestion than non-conjugated calcitonin. Furthermore, the mixture may exhibit a higher bioavailability than non-conjugated calcitonin.

Claims

1. A substantially monodispersed mixture of conjugates, each conjugate comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety.

2. The mixture according to claim 1, wherein the polyethylene glycol moiety has at least 2, 3 or 4 polyethylene glycol subunits.

3. The mixture according to claim 1, wherein the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits.

4. The mixture according to claim 1, wherein the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.

5. The mixture according to claim 1, wherein the mixture of conjugates is a monodispersed mixture.

6. The mixture according to claim 1, wherein the mixture of conjugates is a substantially purely monodispersed mixture.

7. The mixture according to claim 1, wherein the mixture of conjugates is a purely monodispersed mixture.

8. The mixture according to claim 1, wherein the mixture has the capability of lowering serum calcium levels by at least 5 percent.

9. The mixture according to claim 1, wherein the mixture has an increased resistance to degradation by chymotrypsin when compared to the resistance to degradation by chymotrypsin of the calcitonin drug which is not coupled to the oligomer.

10. The mixture according to claim 1, wherein the mixture has a bioefficacy that is greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.

11. The mixture according to claim 1, wherein the calcitonin drug is salmon calcitonin.

12. The mixture according to claim 11, wherein the oligomer is covalently coupled to an amine function of the salmon calcitonin.

13. The mixture according to claim 12, wherein the amine function is at Lys¹¹ or Lys¹⁸ of the salmon calcitonin.

14. The mixture according to claim 11, wherein the conjugate comprises a first oligomer and a second oligomer.

15. The mixture according to claim 14, wherein the first oligomer is covalently coupled to Lys¹¹ of the salmon calcitonin and the second oligomer is covalently coupled to Lys¹⁸ of the salmon calcitonin.

16. The mixture according to claim 1, wherein the calcitonin drug is covalently coupled to the oligomer.

17. The mixture according to claim 1, wherein the calcitonin drug is covalently coupled to the oligomer by a hydrolyzable bond.

18. The mixture according to claim 1, wherein the calcitonin drug is covalently coupled to the polyethylene glycol moiety of the oligomer.

19. The mixture according to claim 1, wherein the oligomer further comprises a lipophilic moiety.

20. The mixture according to claim 19, wherein the calcitonin drug is covalently coupled to the lipophilic moiety.

21. The mixture according to claim 20, wherein the polyethylene glycol moiety is covalently coupled to the lipophilic moiety.

22. The mixture according to claim 1, wherein the conjugate comprises a plurality of oligomers.

23. The mixture according to claim 22, wherein each oligomer in the plurality of oligomers has the same molecular structure.

24. The mixture according to claim 1, wherein the oligomer comprises a first polyethylene glycol moiety covalently coupled to the calcitonin drug by a non-hydrolyzable bond and a second polyethylene glycol moiety covalently coupled to the first polyethylene glycol moiety by a hydrolyzable bond.

25. The monodispersed mixture according to claim 24, wherein the oligomer further comprises a lipophilic moiety covalently coupled to the second polyethylene glycol moiety.

26. The monodispersed mixture according to claim 1, wherein the conjugates are each amphiphilically balanced such that each conjugate is aqueously soluble and able to penetrate biological membranes.

27. A pharmaceutical composition comprising:

the mixture according to claim 1;

and

a pharmaceutically acceptable carrier.

28. A substantially monodispersed mixture of conjugates each comprising salmon calcitonin covalently coupled at Lys¹¹ of the salmon calcitonin to the carboxylic acid moiety of a carboxylic acid, which is covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having at least 7 polyethylene glycol subunits, and covalently coupled at Lys¹⁸ of the salmon calcitonin to the carboxylic acid moiety of a carboxylic acid, which is covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having at least 7 polyethylene glycol subunits.

29. The mixture according to claim 28, wherein the conjugates each consist of salmon calcitonin covalently coupled at Lys¹¹ of the salmon calcitonin to the carboxylic acid moiety of octanoic acid, which is covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having 7 polyethylene glycol subunits, and covalently coupled at Lys¹⁸ of the salmon calcitonin to the carboxylic acid moiety of octanoic acid, which is covalently coupled at the end distal to the carboxylic acid moiety to a methyl terminated polyethylene glycol moiety having 7 polyethylene glycol subunits.

30. The mixture according to claim 28, wherein the mixture is a monodispersed mixture.

31. The mixture according to claim 28, wherein the mixture is a substantially purely monodispersed mixture.

32. The mixture according to claim 28, wherein the mixture is a purely monodispersed mixture.

33. A method of treating a bone disorder in a subject in need of such treatment, said method comprising:

administering an effective amount of a substantially monodispersed mixture of conjugates each comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety to the subject to treat the bone disorder.

34. The method according to claim 33, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.

35. The method according to claim 33, wherein the bone disorder is osteoporosis Paget'"'"'s disease, or hypercalcemia.

36. A substantially monodispersed mixture of conjugates each conjugate comprising a calcitonin drug coupled to an oligomer comprising a polyethylene glycol moiety, said mixture capable of lowering serum calcium levels in a subject by at least 5 percent.

37. The mixture according to claim 36, wherein the mixture is capable of lowering serum calcium levels in a subject by at least 10 percent.

38. The mixture according to claim 36, wherein the mixture is capable of lowering serum calcium levels in a subject by at least 20 percent.

39. The mixture according to claim 36, further having an increased resistance to degradation by chymotrypsin or trypsin when compared to the resistance to degradation by chymotrypsin or trypsin of the calcitonin drug which is not coupled to the oligomer.

40. The mixture according to claim 36, further having a bioefficacy higher than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.

41. A mixture of conjugates each conjugate comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety having at least 4 polyethylene glycol subunits, said mixture having a molecular weight distribution with a standard deviation of less than about 22 Daltons.

42. The mixture of conjugates according to claim 41, wherein the standard deviation of the molecular weight distribution is less than about 14 Daltons.

43. The mixture of conjugates according to claim 41, wherein the standard deviation of the molecular weight distribution is less than about 11 Daltons.

44. The mixture of conjugates according to claim 41, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ to a first oligomer and coupled at Lys¹⁸ to a second oligomer, and wherein the first oligomer and the second oligomer each have the formula:

45. The mixture of conjugates according to claim 41, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ to a first oligomer and coupled at Lys¹⁸ to a second oligomer, and wherein the first oligomer and the second oligomer each have the formula:

46. The mixture of conjugates according to claim 41, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ or Lys¹⁸ to an oligomer having the formula:

52. The mixture of conjugates according to claim 41, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ or Lys¹⁸ to an oligomer having the formula:

47. A mixture of conjugates each comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety, wherein the mixture has a dispersity coefficient (DC) greater than 10,000 where

48. The mixture of conjugates according to claim 47, wherein the dispersity coefficient is greater than 100,000.

49. The mixture of conjugates according to claim 47, wherein the dispersity coefficient is greater than 500,000.

50. The mixture of conjugates according to claim 47, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ to a first oligomer and coupled at Lys¹⁸ to a second oligomer, and wherein the first oligomer and the second oligomer each have the formula:

51. The mixture of conjugates according to claim 47, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ to a first oligomer and coupled at Lys¹⁸ to a second oligomer, and wherein the first oligomer and the second oligomer each have the formula:

53. A mixture of conjugates in which each conjugate:

comprises a calcitonin drug coupled to an oligomer; and

has the same number of polyethylene glycol subunits.

54. The mixture of conjugates according to claim 53, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ to a first oligomer and coupled at Lys¹⁸ to a second oligomer, and wherein the first oligomer and the second oligomer each have the formula:

55. The mixture of conjugates according to claim 53, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ to a first oligomer and coupled at Lys¹⁸ to a second oligomer, and wherein the first oligomer and the second oligomer each have the formula:

56. The mixture of conjugates according to claim 53, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ or Lys¹⁸ to an oligomer having the formula:

57. A mixture of conjugates in which each conjugate has the same molecular weight and has the structure of the formula:

Calcitonin Drug

B-L_j-G_k-R-G′

_m-R′

G″

_n-T]_p 

 

(A)

wherein;

B is a bonding moiety;

L is a linker moiety;

G, G′ and

G″

are individually selected spacer moieties;

R is a lipophilic moiety and R′

is a polyalkylene glycol moiety, or R′

is the lipophilic moiety and R is the polyalkylene glycol moiety;

T is a terminating moiety;

j, k, m and n are individually 0 or 1; and

p is an integer from 1 to the number of nucleophilic residues on the calcitonin drug.

58. The mixture of conjugates according to claim 57, wherein the polyalkylene glycol moiety is a polyethylene glycol moiety.

59. The mixture of conjugates according to claim 58, wherein the polyethylene glycol moiety has at least 2, 3 or 4 polyethylene glycol subunits.

60. The mixture of conjugates according to claim 58, wherein the polyethylene glycol moiety has at least 5 or 6 polyethylene glycol subunits.

61. The mixture of conjugates according to claim 58, wherein the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.

62. The mixture of conjugates according to claim 57, wherein:

R is alkyl or alkylene;

R′

is a polyethylene glycol moiety having at least 6 polyethylene glycol subunits;

T is alkyl or alkoxy;

j is 1; and

k, m and n are 0.

63. The mixture of conjugates according to claim 57, wherein:

B is carbonyl;

R is C₇-C₁₆ alkylene;

R′

is a polyethylene glycol moiety having between 6 and 8 polyethylene glycol subunits;

T is methyl or methoxy; and

k, m and n are 0.

64. A process for synthesizing a substantially monodispersed mixture of conjugates each conjugate comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety, said process comprising:

reacting a substantially monodispersed mixture comprising compounds having the structure of Formula I;

R¹(OC₂H₄)_m—

O³¹ X⁺ 

 

(I)

wherein R¹ is H or a lipophilic moiety;

m is from 1 to 25; and

X⁺ is a positive ion,

with a substantially monodispersed mixture comprising compounds having the structure of Formula II;

R²(OC₂H₄)_n—

OMs 

 

(II)

wherein R² is H or a lipophilic moiety; and

n is from 1 to 25, under conditions sufficient to provide a substantially monodispersed mixture comprising polymers having the structure of Formula III;

R²(OC₂H₄)_m+n—

OR¹ 

 

(III);

activating the substantially monodispersed mixture comprising polymers of Formula III to provide a substantially monodispersed mixture of activated polymers capable of reacting with a calcitonin drug; and

reacting the substantially monodispersed mixture of activated polymers with a substantially monodispersed mixture of calcitonin drugs under conditions sufficient to provide a substantially monodispersed mixture of conjugates each comprising a calcitonin drug coupled to an oligomer that comprises a polyethylene glycol moiety with m+n subunits.

65. The process according to claim 64, wherein R² is a fatty acid moiety or an ester of a fatty acid moiety.

66. The process according to claim 65, wherein the fatty acid moiety or the ester of a fatty acid moiety comprises an alkyl moiety at least 5 carbon atoms in length.

67. The process according to claim 64, wherein R¹ is a methyl group.

68. The process according to claim 64, further comprising:

reacting a substantially monodispersed mixture comprising compounds having the structure of Formula V;

R²(OC₂H₄)_n—

OH 

 

(V)

with a methanesulfonyl halide under conditions sufficient to provide a substantially monodispersed mixture comprising compounds having the structure of Formula II;

R²(OC₂H₄)_n—

OMs 

 

(II).

69. The process according to claim 68, further comprising:

reacting a substantially monodispersed mixture comprising compounds having the structure of Formula VI;

R²—

OMs 

 

(VI)

wherein R² is a lipophilic moiety;

with a substantially monodispersed mixture comprising compounds having the structure of Formula VII;

R³(OC₂H₄)_m—

O³¹ X₂⁺ 

 

(VII)

wherein R³ is benzyl, trityl, or THP; and

X₂⁺ is a positive ion;

under conditions sufficient to provide a substantially monodispersed mixture comprising compounds having the structure of Formula VIII;

R³(OC₂H₄)_m—

OR² 

 

(VIII); and

reacting the substantially monodispersed mixture comprising compounds having the structure of Formula VIII under conditions sufficient to provide a substantially monodispersed mixture comprising compounds having the structure of Formula V;

R²(OC₂H₄)_m—

OH 

 

(V).

70. The process according to claim 64, further comprising:

reacting a substantially monodispersed mixture comprising compounds having the structure of Formula IV;

R¹(OC₂H₄)_n—

OH 

 

(IV)

under conditions sufficient to provide a substantially monodispersed mixture comprising compounds having the structure of Formula I;

R¹(OC₂H₄)_n—

O⁻

X⁺ 

 

(I).

71. The process according to claim 64, wherein the activating of the substantially monodispersed mixture comprises reacting the substantially monodispersed mixture of polymers of Formula III with N-hydroxy succinimide to provide an activated polymer capable of reacting with a calcitonin drug.

72. The process according to claim 64, wherein the calcitonin drug is salmon calcitonin, and wherein the reacting of the substantially monodispersed mixture of activated polymers with a substantially monodispersed mixture of salmon calcitonin comprises:

reacting the substantially monodispersed mixture of activated polymers with Lys¹¹ and Lys¹⁸ of the salmon calcitonin to provide a substantially monodispersed mixture of diconjugates each comprising a salmon calcitonin coupled to two oligomers that each comprise a polyethylene glycol moiety with m+n subunits.