Amylin peptides

• US 5,367,052 A
• Filed: 05/01/1989
• Issued: 11/22/1994
• Est. Priority Date: 04/27/1987
• Status: Expired due to Term

• Current Assignee: Amylin Corporation
• Original Assignee: Amylin Pharmaceuticals Incorporated (Bristol-Myers Squibb Company)
• Inventors: Willis, Antony C., Cooper, Garth J. S.
• Primary Examiner(s): LEE, LESTER
• Application Number:
  US07/346,624
• Time in Patent Office: 2,031 Days
• Field of Search: 424/85.8, 530/307, 530/324, 514/12
• US Class Current: 530/307
• 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: C07K 14/575 Hormones derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695

Abstract

A biologically active peptide associated with diabetes and designated herein "amylin" and processes for preparing it and assaying for it and for Type 2 diabetes are disclosed. The invention includes peptides having the amino acid sequence KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY, substantially homologous sequences of amino acids, proamylin, as well as biologically active subfragments and conservative mutations. The peptide may be prepared from diabetic pancreata by solubilization of amyloid and isolation of the peptide by gel filtration and reverse phase chromotography. Amylin may also be synthesized, or it may be produced by recombinant DNA techniques using the disclosed nucleic acid sequences.

Claims

1. An amylin peptide having thirty-seven amino acids, in which the C-terminal tyrosine of said peptide is carboxyamidated, said peptide contains an intramolecular linkage between cysteine resides at positions 2 and 7, and wherein said peptide is capable of reducing insulin-induced incorporation of glucose into glycogen in isolated rat soleus muscle.

5. An amylin peptide as recited in claim 1 or 2 which is at least about 50% pure.

6. An amylin peptide as recited in claim 1 or 2 which is at least about 80% pure.

7. An amylin peptide as recited in claim 1 or 2 which is at least about 90% pure.

8. An amylin peptide as recited in claim 1 or 2 which is at least about 95% pure.

9. An amylin peptide as recited in claim 1 or 2 which is at least about 99% pure.

11. The amylin peptide of claim 5 wherein said amylin peptide is an amylin peptide having thirty-seven amino acids, in which the C-terminal tyrosine of said peptide is carboxyamidated, said peptide contains an intramolecular linkage between the cysteine residues at positions 2 and 7, and wherein said peptide is capable of reducing insulin-induced incorporation of glucose into glycogen in isolated rat soleus muscle.

12. The amylin peptide of claim 6 wherein said amylin peptide is an amylin peptide having thirty-seven amino acids, in which the C-terminal tyrosine of said peptide is carboxyamidated, said peptide contains an intramolecular linkage between the cysteine residues at positions 2 and 7, and wherein said peptide is capable of reducing insulin-induced incorporation of glucose into glycogen in isolated rat soleus muscle.

13. The amylin peptide of claim 7 wherein said amylin peptide is an amylin peptide havng thirty-seven amino acids, in which the C-terminal tyrosine of said peptide is carboxyamidated, said peptide contains an intramolecular linkage between the cysteine residues at positions 2 and 7, and wherein said peptide is cabable of reducing insulin-induced incorporation of glucose into glycogen in isolated rat soleus muscle.

14. The amylin peptide of claim 8 wherein said amylin peptide is an amylin peptide having thirty-seven amino acids, in which the C-terminal tyrosine of said peptide is carboxyamidated, said peptide contains an intramolecular linkage between the cysteine residues at positions 2 and 7, and wherein said peptide is capable of reducing insulin-induced incorporation of glucose into glycogen in isolated rat soleus muscle.

15. The amylin peptide of claim 9 wherein said amylin peptide is an amylin peptide having thirty-seven amino acids, in which the C-terminal tyrosine of said peptide is carboxyamidated, said peptide contains an intramolecular linkage between the cysteine residues at positions 2 and 7, and wherein said peptide is capable of reducing insulin-induced incorporation of glucose into glycogen in isolated rat soleus muscle.

17. The amylin peptide of claim 5 wherein said amylin peptide is the amylin peptide KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY which contains an intramolecular linkage between the cysteine residues at positions 2 and 7, and wherein the C-terminal tyrosine is carboxyamidated.

18. The amylin peptide of claim 6 wherein said amylin peptide is the amylin peptide KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY which contains an intramolecular linkage between the cysteine residues at positions 2 and 7, wherein the C-terminal tyrosine is carboxyamidated.

19. The amylin peptide of claim 7 wherein said amylin peptide is the amylin peptide KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY which contains an intramolecular linkage between the cysteine residues at positions 2 and 7, wherein the C-terminal tyrosine is carboxyamidated.

20. The amylin peptide of claim 8 wherein said amylin peptide is the amylin peptide KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY which contains an intramolecular linkage between the cysteine residues at positions 2 and 7, wherein the C-terminal tyrosine is carboxyamidated.

21. The amylin peptide of claim 9 wherein said amylin peptide is the amylin peptide KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY which contains an intramolecular linkage between the cysteine residues at positions 2 and 7, wherein the C-terminal tyrosine is carboxyamidated.

2. The amylin peptide KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY which contains an intramolecular linkage between the cysteine residues at positions 2 and 7, and wherein the C-terminal tyrosine is carboxyamidated.

3. A process for the preparation of an amylin peptide as recited in claim 2, comprising the steps of (a) preparing solubilized amyloid by use of formic acid in conjunction with ultrasound, (b) subjecting the amyloid material from step (a) to normal phase high performance liquid chromatography gel filtration using a mobile phase comprising aqueous guanidine and sodium phosphate, and (c) subjecting the amyloid material from step (b) to high performance liquid chromatography using a mobile phase comprising triflouracetic acid and elution by acetonitrile.

4. A substantially pure amylin peptide prepared by the process of claim 3.

10. The amylin peptide of claim 4 wherein said amylin peptide is an amylin peptide having thirty-seven amino acids, in which the C-terminal tyrosine of said peptide is carboxyamidated. said peptide contains an intramolecular linkage between the cysteine residues at positions 2 and 7, and wherein said peptide is capable of reducing insulin-induced incorporation of glucose into glycogen in isolated rat soleus muscle.

16. The amylin peptide of claim 4 wherein said amylin peptide is the amylin peptide KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY which contains an intramolecular linkage between the cysteine residues at positions 2 and 7, and wherein the C-terminal tyrosine is carboxyamidated.