Non-Canonical Insulins and Their Uses

• US 20150353619A1
• Filed: 06/10/2015
• Published: 12/10/2015
• Est. Priority Date: 06/10/2014
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Tirrell, David A., Lieblich, Seth, Fang, Katherine Y., Zisser, Howard C.
• Granted Patent: US 10,640,546 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 3/04 Anorexiants; Antiobesity agents
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C07K 14/62 Insulins
• CPC Class Codes: C07K 2319/21 containing a His-tag

Abstract

In some aspects, the invention relates to an insulin derivative, comprising a cyclic amino acid at position B28 or B29, wherein the cyclic amino acid is not L-proline. In some aspects, the invention relates to a method of making said insulin derivative. In some aspects, the invention relates to a pharmaceutical composition comprising said insulin derivative. In some aspects the invention relates to a method of treating a disease or condition in a subject comprising administering to the subject a composition comprising said insulin derivative.

Claims

1. An insulin derivative, comprising a cyclic amino acid at position B28 or B29, wherein the cyclic amino acid is not L-proline.

2. The insulin derivative of claim 1, wherein the cyclic amino acid is an α

amino acid or a β

amino acid.

3. The insulin derivative of claim 1, wherein the cyclic amino acid is an L amino acid or a D amino acid.

4. The insulin derivative of claim 1, wherein the cyclic amino acid comprises a heterocycle comprising the amide nitrogen, α

carbon, and β

carbon of the cyclic amino acid.

5. The insulin derivative of claim 1, wherein the cyclic amino acid has a neutral, positive, or negative charge at physiological pH.

6. The insulin derivative of claim 1, wherein the cyclic amino acid comprises a 3-membered ring, a 4-membered ring, a 5-membered ring, or a 6-membered ring.

7. The insulin derivative of claim 1, wherein the cyclic amino acid is a fluoroproline analog, a hydroxyproline analog, or a proline ring analog.

8. The insulin derivative of claim 6, wherein the cyclic amino acid is 3-hydroxyproline, 4-hydroxyproline, aziridine-2-carboxylic acid, azetidine-2-carboxylic acid, piperidine-2-carboxylic acid, 3-carboxy-morpholine, 3-carboxy-thiamorpholine, 4-oxaproline, pyroglutamic acid, 1,3-oxazolidine-4-carboxylic acid, 1,3-thiazolidine-4-carboxylic acid, 3-thiaproline, 4-thiaproline, 3-selenoproline, 4-selenoproline, 4-ketoproline, 3,4-dehydroproline, 4-aminoproline, 4-fluoroproline, 4,4-difluoroproline, 4-chloroproline, 4,4-dichloroproline, 4-bromoproline, 4,4-dibromoproline, 4-methylproline, 4-ethylproline, 4-cyclohexyl-proline, 3-phenylproline, 4-phenylproline, 3,4-phenylproline, 4-azidoproline, 4-carboxy-proline, α

-methylproline, α

-ethylproline, α

-propylproline, α

-allylproline, α

-benzylproline, α

-(4-fluorobenzyl)-proline, α

-(2-chlorobenzyl)-proline, α

-(3-chlorobenzyl)-proline, α

-(2-bromobenzyl)-proline, α

-(4-bromobenzyl)-proline, α

-(4-methylbenzyl)-proline, α

-(diphenylmethyl)-proline, α

-(naphthylmethyl)-proline, D-proline, or β

₃-homoproline.

9. The insulin derivative of claim 8, wherein the cyclic amino acid is (2S, 4S)-4-fluoro-L-proline, (2S, 4R)-4-fluoro-L-proline, (2S)-3,4-dehydro-L-proline, (2S, 4S)-4-hydroxy-L-proline, (2S, 4R)-4-hydroxy-L-proline, (2S,4S)-4-azido-L-proline, (2S)-4,4-difluoro-L-proline, (2S)-azetidine-2-carboxylic acid, (2S)-piperidine-2-carboxylic acid, or (4R)-1,3-thiazolidine-4-carboxylic acid.

10. The insulin derivative of claim 9, wherein the cyclic amino acid is cis-4-fluoroproline, trans-4-fluoroproline, cis-4-hydroxyproline, or trans-4-hydroxyproline.

11. The insulin derivative of claim 7, wherein the cyclic amino acid is a substituted cis-4-(1H-1,2,3,-triazol-1-yl)-proline or a substituted trans-4-(1H-1,2,3,-triazol-1-yl)-proline.

12. The insulin derivative of claim 1, wherein the insulin A chain comprises the sequence set forth in SEQ ID NO:

1.

13. The insulin derivative of claim 1, wherein the insulin B chain comprises the sequence set forth in SEQ ID NO:

2 or SEQ ID NO;

32, wherein X₁ corresponds to position B28 and X₂ corresponds to position B29.

14. The insulin derivative of claim 1, wherein:

the amino acid at position B28 is proline, lysine, aspartate, or glutamate;

or

the amino acid at position B29 is proline, lysine, aspartate, or glutamate.

15. The insulin derivative of claim 13, wherein:

the insulin B chain comprises the sequence set forth in SEQ ID NO;

32; and

the amino acid at position B29 is lysine, wherein the lysine is conjugated to a substituted or unsubstituted alkyl group or acyl group.

16. The insulin derivative of claim 15, wherein the lysine is conjugated to hexadecanedioic acid via a γ

-L-glutamyl linker.

17. A method of making the insulin derivative of claim 1, comprising incubating cells comprising an insulin gene in a medium comprising the cyclic amino acid.

18. A method of making the insulin derivative of claim 1, comprising synthesizing the insulin B chain.

19. A method of making the insulin derivative of claim 1, comprising coupling a molecule comprising the cyclic amino acid to a peptide comprising either the insulin A chain, the insulin B chain, or both.

20. A pharmaceutical composition, comprising the insulin derivative of claim 1.

21. The pharmaceutical composition of claim 20, wherein the composition is formulated for subcutaneous injection.

22. The pharmaceutical composition of claim 20, further comprising a second insulin derivative, wherein the second insulin derivative does not comprise the cyclic amino acid.

23. The pharmaceutical composition of claim 22, wherein the second insulin derivative is insulin comprising L-proline at the insulin B chain 28 position and lysine at the insulin B chain 29 position.

24. A method of treating hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, metabolic syndrome X, or dyslipidemia in a subject, comprising administering to the subject the pharmaceutical composition derivative of claim 20.