Synthesis of insulin derivatives

• US 6,323,311 B1
• Filed: 09/22/1999
• Issued: 11/27/2001
• Est. Priority Date: 09/22/1999
• Status: Expired due to Term

• Current Assignee: University of Utah Research Foundation (State of Utah)
• Original Assignee: University of Utah Research Foundation (State of Utah)
• Inventors: Kim, Sung Wan, Liu, Feng, Baudys, Miroslav
• Primary Examiner(s): Russel, Jeffrey E.
• Application Number:
  US09/400,942
• Time in Patent Office: 797 Days
• Field of Search: 530/303, 530/304, 530/305, 514/3.4
• US Class Current: 530/303
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• 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: C07K 14/62 Insulins
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

A method for the “one-pot” synthesis of insulin derivatives wherein insulin is modified at the α-amino group of the PheB1 residue is described. The method comprises protecting the α-amino group of the GlyA1 residue and the ε-amino group of the LysB29 residue by reaction of insulin with a cyclic anhydride of a dicarboxylic acid in the presence of a tertiary amine. The protected insulin is then reacted with an activated hydrophilic compound, preferably an activated polyethylene glycol, resulting in a conjugate of the hydrophilic compound coupled to the PheB1 residue of insulin. The protecting groups are then removed from the conjugate under mild acidic conditions, and the resulting insulin derivative can be purified by conventional methods. Monosubstituted insulin derivatives wherein polyethylene glycol or derivatives thereof or glycosides are coupled to the PheB1 residue of insulin are also described.

Claims

1. A method for the synthesis of an insulin derivative having a hydrophilic compound coupled to the PheB1 amino group comprising:

(a) coupling an acyl protective group to the GlyA1 and LysB29 amino groups of insulin comprising reacting insulin with a cyclic anhydride of a dicarboxylic acid in the presence of a tertiary amine thereby obtaining Gly-N<sup>α

A1</sup>, LysN<sup>ε

B29</sup>-disubstituted insulin;

(b) reacting the Gly-N<sup>α

A1</sup>, LysN<sup>ε

B29</sup>-disubstituted insulin with an activated hydrophilic compound thereby covalently bonding the hydrophilic compound to the PheB1 amino group;

(c) quantitatively hydrolyzing the acyl protective group from the GlyA1 and LysB29 residues, thereby obtaining the insulin derivative having the hydrophilic compound coupled to the PheB1 amino group.

2. The method of claim 1 wherein said cyclic anhydride of a dicarboxylic acid is a member selected from the group consisting of maleic anhydride, citraconic anhydride, phthalic anhydride, exo-cis-3,6-endoxo-Δ

⁴-tetrahydrophthalic anhydride, and mixtures thereof.

3. The method of claim 2 wherein said cyclic anhydride of a dicarboxylic acid is maleic anhydride.

4. The method of claim 2 wherein said cyclic anhydride of a dicarboxylic acid is citraconic anhydride.

5. The method of claim 2 wherein said cyclic anhydride of a dicarboxylic acid is phthalic anhydride.

6. The method of claim 2 wherein said cyclic anhydride of a dicarboxylic acid is exo-cis-3,6-endoxo-Δ

⁴-tetrahydrohthalic anhydride.

7. The method of claim 1 wherein said tertiary amine is a member selected from the group consisting of triethylamine and N-methylmorpholine.

8. The method of claim 7 wherein said tertiary amine is triethylamine.

9. The method of claim 1 wherein said activated hydrophilic compound is a derivative of polyethylene glycol represented by the formula:

10. The method of claim 1 wherein said quantitatively hydrolyzing the acyl protective group from the GlyA1 and LysB29 residues comprises mild acid treatment.

11. The method of claim 10 wherein said mild acid treatment comprises treatment with dilute acetic acid.

12. The method of claim 11 wherein said treatment with dilute acetic acid comprises treatment with a medium comprising 1 M acetic acid and 7 M urea.

13. An insulin derivative represented by the formula:

14. The insulin derivative of claim 13 wherein PEG is represented by the formula:

15. The insulin derivative of claim 13 wherein PEG is a branched derivative of polyethylene glycol or alkoxy derivative thereof.

16. An insulin derivative represented by the formula:

17. The insulin derivative of claim 16 wherein PEG is a dendrimeric PEG based on a three-fold consecutive bifurcation.

18. The insulin derivative of claim 17 wherein PEG is represented by the formula:

wherein R₁ is —

OH or —

NH—

(CH₂)_m—

COOH;

m is an integer from 1 to 10;

wherein the carbonyl group is linked to R₁ or —

NH—

Y₁ or —

NH—

Y₂; and

p and r are integers from 0 to about 5;

Y₁ and Y₂ are —

(CH₂)_k—

(O—

CH₂CH₂)_n—

CO—

or —

(CH₂)_k—

(O—

CH₂CH₂)_n—

O—

CO—

wherein n is an integer from 0 to about 400 and can be the same or different in Y₁ and Y₂, and k is an integer from 1 to about 6 and can be the same or different in Y₁ and Y₂ and where the carbonyl group of Y₁ and Y₂ is linked to —

NH—

which is linked to —

(CH₂)_p or —

(CH₂)_r of X, and the other end of Y₁ and Y₂ is linked to —

NH—

group which is linked to carbonyl group of X.

19. The insulin derivative of claim 16 wherein said organic spacer is a member selected from the group consisting of

wherein r is an integer from 1 to 6 and p is an integer from 1 to 8;

and

wherein r is an integer from 1 to 6 and p is an integer from 1 to 8.

20. The insulin derivative of claim 16 wherein PEG is represented by the formula: