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

• US 6,713,452 B2
• Filed: 06/04/2001
• Issued: 03/30/2004
• Est. Priority Date: 06/04/2001
• Status: Expired due to Fees

• Current Assignee: Biocon Limited (Biocon Incorporated)
• Original Assignee: Nobex Corporation (Biocon Incorporated)
• Inventors: Odenbaugh, Amy L., Ansari, Aslam M., Ekwuribe, Nnochiri N., Price, Christopher H.
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US09/873,777
• Publication Number:
  US 20030060606A1
• Time in Patent Office: 1,030 Days
• Field of Search: 514/2, 514/11, 514/12, 514/21, 530/307, 530/345, 530/410, 528/425, 554/227, 562/587, 568/613, 568/618, 568/622, 568/623
• US Class Current: 514/11.9
• 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, wherein the conjugate comprises a first oligomer and a second oligomer, wherein each oligomer comprises a polyethylene glycol moiety and is coupled to salmon calcitonin and wherein the first oligomer is covalently coupled to an amine function of Lys¹¹ of the salmon calcitonin and the second oligomer is covalently coupled to an amine function of Lys¹⁸ of the salmon calcitonin.

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

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

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

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

6. The mixture according to claim 1, wherein the conjugates have 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.

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

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

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

10. The mixture according to claim 9, wherein the polyethylene glycol moiety is covalently coupled to the lipophilic moiety.

11. The mixture according to claim 1, wherein the first oligomer and the second oligomer have the same molecular structure.

12. The mixture according to claim 1, wherein each conjugate is amphiphilically balanced such that each conjugate is aqueously soluble and able to penetrate biological membranes.

13. A pharmaceutical composition comprising:

the mixture according to claim 1, and

a pharmaceutically acceptable carrier.

14. The mixture according to claim 1, wherein the PEG moiety has from 2-50 PEG subunits.

15. The mixture according to claim 1, wherein the PEG moiety has from 2-7 PEG subunits.

16. The mixture according to claim 1, wherein the oligomer further comprises an alkyl moiety.

17. The mixture according to claim 1, wherein the oligomer further comprises an alkyl moiety having from 1 to 28 carbon atoms.

18. The mixture according to claim 1, wherein the oligomer further comprises an alkyl moiety having from 2 to 12 carbon atoms.

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

20. The mixture according to claim 1, wherein the oligomer further comprises a fatty acid moiety having from 2 to 18 carbon atoms.

21. The mixture according to claim 1, wherein the oligomer further comprises a fatty acid moiety having from 3 to 14 carbon atoms.

22. The mixture according to claim 1, wherein the oligomer further comprises a fatty acid moiety having 4, 5, or 6 carbon atoms.

32. A pharmaceutical composition comprising:

the mixture according to claim 3, and

a pharmaceutically acceptable carrier.

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

administering an effective amount of a pharmaceutical composition comprising a mixture according to claim 1, to the subject to treat the bone disorder.

71. The method according to claim 70, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.

72. The method according to claim 70, wherein the bone disorder is osteoporosis, Paget'"'"'s disease, or hypercalcemia.

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

24. The mixture according to claim 23, wherein the oligomer further comprises a lipophilic moiety covalently coupled to the second polyethylene glycol moiety.

25. The mixture according to claim 23, wherein each conjugate is amphiphilically balanced such that each conjugate is aqueously soluble and able to penetrate biological membranes.

26. The mixture according to claim 23, wherein the mixture of conjugates is a monodispersed mixture.

27. The mixture according to claim 23, wherein the mixture of conjugates is a substantially purely monodispersed mixture.

28. The mixture according to claim 23, wherein the mixture of conjugates is a purely monodispersed mixture.

29. The mixture according to claim 23, wherein the mixture has the capability of lowering serum calcium levels by at least 5 percent.

30. The mixture according to claim 23, wherein the conjugates have 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.

31. The mixture according to claim 23, wherein the mixture has a bioefficacy that is greater than the bioefficacy of the calcitonin drug which is not coupled to the oligomer.

33. The mixture according to claim 23, wherein the PEG moiety has from 2-50 PEG subunits.

34. The mixture according to claim 23, wherein the PEG moiety has from 2-7 PEG subunits.

35. The mixture according to claim 23, wherein the oligomer further comprises an alkyl moiety.

36. The mixture according to claim 23, wherein the oligomer further comprises an alkyl moiety having from 1 to 28 carbon atoms.

37. The mixture according to claim 23, wherein the oligomer further comprises an alkyl moiety having from 2 to 12 carbon atoms.

38. The mixture according to claim 23, wherein the oligomer further comprises a fatty acid moiety.

39. The mixture according to claim 23, wherein the oligomer further comprises a fatty acid moiety having from 2 to 18 carbon atoms.

40. The mixture according to claim 23, wherein the oligomer further comprises a fatty acid moiety having from 3 to 14 carbon atoms.

41. The mixture according to claim 23, wherein the oligomer further comprises a fatty acid moiety having 4, 5, or 6 carbon atoms.

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

administering an effective amount of a pharmaceutical composition comprising a mixture according to claim 23 to the subject to treat the bone disorder.

74. The method according to claim 73, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.

75. The method according to claim 73, wherein the bone disorder is osteoporosis, Paget'"'"'s disease, or hypercalcemia.

42. 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.

43. The mixture according to claim 42, 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.

44. The mixture according to claim 42, wherein the mixture is a monodispersed mixture.

45. The mixture according to claim 42, wherein the mixture is a substantially purely monodispersed mixture.

46. The mixture according to claim 42, wherein the mixture is a purely monodispersed mixture.

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

administering an effective amount of a pharmaceutical composition comprising a mixture according to claim 42 to the subject to treat the bone disorder.

77. The method according to claim 76, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.

78. The method according to claim 76, wherein the bone disorder is osteoporosis, Paget'"'"'s disease, or hypercalcemia.

47. A mixture of conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons, 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:

48. The mixture of conjugates according to claim 47, wherein the standard deviation of the molecular weight distribution is less than about 14 Daltons.

49. The mixture of conjugates according to claim 47, wherein the standard deviation of the molecular weight distribution is less than about 11 Daltons.

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

administering an effective amount of a pharmaceutical composition comprising a mixture according to claim 47 to the subject to treat the bone disorder.

80. The method according to claim 79, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.

81. The method according to claim 79, wherein the bone disorder is osteoporosis, Paget'"'"'s disease, or hypercalcemia.

50. A mixture of conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons, 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 50, wherein the standard deviation of the molecular weight distribution is less than about 14 Daltons.

52. The mixture of conjugates according to claim 50, wherein the standard deviation of the molecular weight distribution is less than about 11 Daltons.

53. A mixture of conjugates having a molecular weight distribution with a standard deviation of less than about 22 Daltons, wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ or Lys¹⁸ to an oligomer having the formula:

54. The mixture of conjugates according to claim 53, wherein the standard deviation of the molecular weight distribution is less than about 14 Daltons.

55. The mixture of conjugates according to claim 53, wherein the standard deviation of the molecular weight distribution is less than about 11 Daltons.

56. A mixture of conjugates having a dispersity coefficient (DC) greater than 10,000 where:

$\mathrm{DC}=\frac{{\left(\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i\right)}^2}{\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i^2<br><br>\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i-{\left(\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i\right)}^2}$

wherein;

n is the number of different molecules in the sample;

N_i is the number of i^th molecules in the sample; and

M_i is the mass of the i^th molecule, and

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;

57. The mixture of conjugates according to claim 56, wherein the dispersity coefficient is greater than 100,000.

58. The mixture of conjugates according to claim 56, wherein the dispersity coefficient is greater than 500,000.

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

administering an effective amount of a pharmaceutical composition comprising a mixture according to claim 56 to the subject to treat the bone disorder.

83. The method according to claim 82, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.

84. The method according to claim 82, wherein the bone disorder is osteoporosis, Paget'"'"'s disease, or hypercalcemia.

59. A mixture of conjugates having a dispersity coefficient (DC) greater than 10,000 where:

$\mathrm{DC}=\frac{{\left(\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i\right)}^2}{\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i^2<br><br>\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i-{\left(\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i\right)}^2}$

wherein;

n is the number of different molecules in the sample;

N_i is the number of i^th molecules in the sample; and

M_i is the mass of the i^th molecule, and

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;

60. The mixture of conjugates according to claim 59, wherein the dispersity coefficient is greater than 100,000.

61. The mixture of conjugates according to claim 59, wherein the dispersity coefficient is greater than 500,000.

62. A mixture of conjugates having a dispersity coefficient (DC) greater than 10,000 where:

$\mathrm{DC}=\frac{{\left(\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i\right)}^2}{\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i^2<br><br>\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i-{\left(\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i\right)}^2}$

wherein;

n is the number of different molecules in the sample;

N_i is the number of i^th molecules in the sample; and

M_i is the mass of the i^th molecule, and

wherein each conjugate in the mixture comprises salmon calcitonin coupled at Lys¹¹ or Lys¹⁸ to an oligomer having the formula;

63. The mixture of conjugates according to claim 62, wherein the dispersity coefficient is greater than 100,000.

64. The mixture of conjugates according to claim 62, wherein the dispersity coefficient is greater than 500,000.

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

administering an effective amount of a pharmaceutical composition comprising a mixture according to claim 62 to the subject to treat the bone disorder.

86. The method according to claim 85, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.

87. The method according to claim 85, wherein the bone disorder is osteoporosis, Paget'"'"'s disease, or hypercalcemia.

65. A mixture of conjugates in which each conjugate 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:

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

administering an effective amount of a pharmaceutical composition comprising a mixture according to claim 65 to the subject to treat the bone disorder.

95. The method according to claim 94, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.

96. The method according to claim 94 wherein the bone disorder is osteoporosis, Paget'"'"'s disease, or hypercalcemia.

66. A mixture of conjugates in which each conjugate 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:

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

administering an effective amount of a pharmaceutical composition comprising a mixture according to claim 66 to the subject to treat the bone disorder.

89. The method according to claim 88, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.

90. The method according to claim 88, wherein the bone disorder is osteoporosis, Paget'"'"'s disease, or hypercalcemia.

67. A mixture of conjugates in which each conjugate comprises salmon calcitonin coupled at Lys¹¹ or Lys¹⁸ to an oligomer having the formula:

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

administering an effective amount of a pharmaceutical composition comprising a mixture according to claim 67 to the subject to treat the bone disorder.

92. The method according to claim 91, wherein the bone disorder is characterized by excessive osteoclastic bone resorption or hypercalcemic serum effects.

93. The method according to claim 91, wherein the bone disorder is osteoporosis, Paget'"'"'s disease, or hypercalcemia.

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

Calcitonin Drug-oligomer where the oligomer has the formula;

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

Calcitonin Drug-oligomer where the oligomer has the formula;

97. 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;

98. The process according to claim 97, wherein R² is a fatty acid moiety or an ester of a fatty acid moiety.

99. The process according to claim 98, wherein the fatty acid moiety or the ester of a fatty acid moiety comprises an alkyl moiety at least 5 carbon atoms in length.

100. The process according to claim 97, wherein R¹ is a methyl group.

101. The process according to claim 97, further comprising:

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

102. The process according to claim 101, further comprising:

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

103. The process according to claim 97, further comprising:

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

104. The process according to claim 97, 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.