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

• US 20040198949A1
• Filed: 04/29/2004
• Published: 10/07/2004
• Est. Priority Date: 06/04/2001
• Status: Active Grant

• Current Assignee: Biocon Limited (Biocon Incorporated)
• Original Assignee: Biocon Limited (Biocon Incorporated)
• Inventors: Radhakrishnan, Balasingam, Ekwuribe, Nnochiri N., Odenbaugh, Amy L., Price, Christopher H., Ansari, Aslam M.
• Granted Patent: US 7,084,114 B2
• Time in Patent Office: Days
• US Class Current: 530/303
• CPC Class Codes: A61K 38/28 Insulins
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• 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 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 5/50 for increasing or potentiating the activity of insulin

Abstract

A mixture of conjugates in which each conjugate in the mixture comprises an insulin drug coupled to an oligomer that includes a polyalkylene glycol moiety is disclosed. The mixture may exhibit higher in vivo activity than a polydispersed mixture of similar conjugates. The mixture may also be more effective at surviving an in vitro model of intestinal digestion than polydispersed mixtures of similar conjugates. The mixture may also result in less inter-subject variability than polydispersed mixtures of similar conjugates.

Claims

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

2. The mixture according to claim 1, wherein the dispersity coefficient is greater than 100,000.

3. The mixture according to claim 1, wherein the polyethylene glycol moiety has at least 2 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 insulin is human insulin and the oligomer is covalently coupled to Lys^B29 of the human insulin and has the formula:

6. The mixture according to claim 1, wherein the mixture has an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of insulin-oligomer conjugates having the same number average molecular weight as the mixture.

7. The mixture according to claim 1, wherein the mixture has an in vitro activity that is greater than the in vitro activity of a polydispersed mixture of insulin-oligomer conjugates having the same number average molecular weight as the mixture.

8. 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 a polydispersed mixture of insulin-oligomer conjugates having the same number average molecular weight as the mixture.

9. The mixture according to claim 1, wherein the mixture has an inter-subject variability that is less than the inter-subject variability of a polydispersed mixture of insulin-oligomer conjugates having the same number average molecular weight as the mixture.

10. The mixture according to claim 1, wherein the oligomer is covalently coupled to an amine function of the insulin.

11. The mixture according to claim 10, wherein the amine function is at Lys^B29 of the insulin.

12. The mixture according to claim 1, wherein the conjugate comprises a first oligomer and a second oligomer.

13. The mixture according to claim 12, wherein the first and the second oligomers are the same.

14. The mixture according to claim 12, wherein the first oligomer is covalently coupled at Lys^B29 of the insulin and the second oligomer is covalently coupled at N-terminal A1 or N-terminal B1 of the insulin.

15. The mixture according to claim 1, wherein the insulin is covalently coupled to the oligomer.

16. The mixture according to claim 1, wherein the insulin is covalently coupled to the oligomer by a hydrolyzable bond.

17. The mixture according to claim 1, wherein the insulin is covalently coupled to the polyethylene glycol moiety of the oligomer.

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

19. The mixture according to claim 18, wherein the insulin is covalently coupled to the lipophilic moiety.

20. The mixture according to claim 18, wherein the polyethylene glycol moiety is covalently coupled to the lipophilic moiety.

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

22. The mixture according to claim 21, wherein the oligomer further comprises a lipophilic moiety covalently coupled to the second polyethylene glycol moiety.

23. The 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.

24. A pharmaceutical composition comprising:

the mixture according to claim 1;

and

a pharmaceutically acceptable carrier.

25. A method of treating insulin deficiency in a subject in need of such treatment, said method comprising:

administering an effective amount of a mixture of conjugates each comprising insulin coupled to an oligomer comprising a polyethylene glycol moiety, wherein the mixture has a dispersity coefficient (DC) greater than 10,000 where

$D<br><br>\text{\hspace{1em}<br><br>}<br><br>C=\frac{{\left(\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i\right)}^2}{\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i^2<br><br>\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i-{\left(\underset{i=1}{\overset{n}{\sum <br><br>}}<br><br>N_i<br><br>M_i\right)}^2}$

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;

to the subject to treat the insulin deficiency.

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

27. The mixture according to claim 26, wherein the polyethylene glycol moiety has at least 2 polyethylene glycol subunits.

28. The mixture according to claim 26, wherein the polyethylene glycol moiety has at least 7 polyethylene glycol subunits.

29. The mixture according to claim 26, wherein at least about 96 percent of the conjugates in the mixture have the same molecular weight.

30. The mixture according to claim 26, wherein the mixture is a monodispersed mixture.

31. The mixture according to claim 26, wherein the mixture is a substantially purely monodispersed mixture.

32. The mixture according to claim 26, wherein at least about 96 percent of the conjugates in the mixture have the same molecular weight and have the same molecular structure.

33. The mixture according to claim 26, wherein the mixture is a purely monodispersed mixture.

34. A substantially monodispersed mixture of conjugates each comprising human insulin covalently coupled at Lys^B29 of the human insulin 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.

35. The substantially monodispersed mixture according to claim 34, wherein the conjugates each comprising human insulin covalently coupled at Lys^B29 of the human insulin to the carboxylic acid moiety of hexanoic 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.

36. A substantially monodispersed mixture of conjugates each comprising insulin coupled to an oligomer that comprises a polyethylene glycol moiety, said mixture having an in vivo activity that is greater than the in vivo activity of a polydispersed mixture of insulin drug-oligomer conjugates having the same number average molecular weight as the substantially monodispersed mixture.

37. The mixture according to claim 36, further having an in vitro activity that is greater than the in vitro activity of the polydispersed mixture of insulin-oligomer conjugates.

38. The mixture according to claim 36, further having an increased resistance to degradation by chymotrypsin when compared to the resistance to degradation by chymotrypsin of the polydispersed mixture of insulin-oligomer conjugates.

39. The mixture according to claim 36, further having an inter-subject variability that is less than the inter-subject variability of the polydispersed mixture of insulin-oligomer conjugates.

40. A mixture of conjugates each comprising insulin coupled to an oligomer that comprises a polyethylene glycol moiety, said mixture having a molecular weight distribution with a standard deviation of less than about 22 Daltons.

41. The mixture according to claim 40, wherein the standard deviation of the molecular weight distribution is less than about 14 Daltons.

42. The mixture according to claim 40, wherein the insulin is human insulin and each oligomer is covalently coupled to Lys^B29 of the human insulin and has the formula:

43. A mixture of conjugates in which each conjugate:

comprises insulin coupled to an oligomer; and

has the same number of polyethylene glycol subunits.

44. The mixture according to claim 43, wherein the insulin is human insulin and each oligomer is covalently coupled to Lys^B29 of the human insulin and has the formula:

45. A mixture of conjugates in which each conjugate is the same and has the formula:

Insulin 

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 insulin.

46. The mixture according to claim 45, wherein the polyalkylene glycol group is a polyethylene glycol moiety.

47. The mixture according to claim 46, wherein the polyethylene glycol moiety has at least 2 polyethylene glycol subunits.

48. The mixture according to claim 46, wherein the polyalkylene glycol moiety is a polyethylene glycol moiety having at least 7 polyethylene glycol subunits.

49. The mixture according to claim 46, wherein:

R is alkyl or alkylene;

R′

is polyethylene glycol having at least 7 polyethylene glycol subunits;

T is alkyl;

j is 1; and

k, m and n are 0.

50. The mixture according to claim 46, wherein:

B is carbonyl;

R is C₅ alkylene;

R′

is polyethylene glycol having 7 polyethylene glycol subunits;

T is methoxy; and

k, m and n are 0.

51. A process for synthesizing a substantially monodispersed mixture of conjugates each conjugate comprising insulin 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—

O⁻

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 an insulin drug; and

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

52. The process according to claim 51, wherein R² is a fatty acid moiety or an ester of a fatty acid moiety.

53. The process according to claim 52, wherein the fatty acid moiety or the ester of a fatty acid moiety comprises an alkyl moiety at least 5 carbon atoms in length.

54. The process according to claim 51, wherein R¹ is a methyl group.

55. The process-according to claim 51, 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).

56. The process according to claim 55, 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—

O⁻

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).

57. The process according to claim 51, 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)

58. The process according to claim 51, 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 insulin.

59. The process according to claim 51, wherein the insulin is human insulin, and wherein the reacting of the substantially monodispersed mixture of activated polymers with a substantially monodispersed mixture of insulin comprises:

reacting the substantially monodispersed mixture of activated polymers with Lys^B29 of the human insulin to provide a substantially monodispersed mixture of monoconjugates each comprising a human insulin coupled to an oligomer that comprises a polyethylene glycol moiety with m+n subunits.