Fibrillation-resistant insulin and insulin analogues
A fibrillation-resistant insulin analogue may be a single-chain insulin analogue or a physiologically acceptable salt thereof, containing an insulin A chain sequence or an analogue thereof and an insulin B chain sequence or an analogue thereof connected by a polypeptide of 4-10 amino acids. The fibrillation-resistant insulin analogue preferably displays less than 1 percent fibrillation with incubation at 37° C. for at least 21 days. A single-chain insulin analogue displays greater in vitro insulin receptor binding than normal insulin while displaying less than or equal binding to IGFR than normal insulin. The fibrillation-resistant insulin may be used to treat a patient using an implantable or external insulin pump, due to its greater fibrillation resistance.
1. An insulin analogue comprising a single chain polypeptide of formula I,
B-C-A (I)
wherein B comprises a polypeptide having the sequence:
(SEQ. ID. NO. 33)
FVNQHLCGSX 2 LVEALYLVCGERGFFYTX 3 X 4 T
where X 2 is D or H, X 3 is P, D or K, and X 4 is K or P,
wherein C is a polypeptide selected from the group consisting of a polypeptide having the sequence GGGPRR (SEQ ID NO: 19) and a polypeptide having the sequence GGPRR (SEQ ID NO: 20),
and wherein A comprises a polypeptide having the sequence:
GIVEQCCX 1 SICSLYQLENYCN,
(SEQ. ID. NO. 32)
where X 1 is T or H.
2. The insulin analogue of claim 1 , wherein the analogue displays less than 1 percent fibrillation with incubation at 37° C. for 90 days.
3. The insulin analogue of claim 1 , wherein A comprises a polypeptide having the sequence GIVEQCCHSICSLYQLENYCN (SEQ ID NO: 29).
4. The insulin analogue of claim 3 , wherein C is a polypeptide having the sequence GGGPRR (SEQ ID NO:19).
5. The insulin analogue of claim 4 , wherein X 3 is D, and X 4 is P.
6. The insulin analogue of claim 5 , wherein X 2 is D.
7. The insulin analogue of claim 5 , wherein X 2 is H.
8. The insulin analogue of claim 1 , wherein C is a polypeptide having the sequence GGGPRR (SEQ ID NO: 19).
9. The insulin analogue of claim 8 , wherein X 3 is D, and X 4 is P.
10. The insulin analogue of claim 9 , wherein X 2 is D.
11. The insulin analogue of claim 9 , wherein X 2 is H.
12. A nucleic acid encoding a polypeptide of formula I,
B-C-A (I)
wherein B comprises a polypeptide having the sequence:
FVNQHLCGSX 2 LVEALYLVCGERGFFYTX 3 X 4 T (SEQ ID NO: 33)
where X 2 is D or H, X 3 is P, D or K, and X 4 is K or P,
wherein C is a polypeptide selected from the group consisting of a polypeptide having the sequence GGGPRR (SEQ ID NO: 19) and a polypeptide having the sequence GGPRR (SEQ ID NO: 20),
and wherein A comprises a polypeptide having the sequence:
GIVEQCCX 1 SICSLYQLENYCN (SEQ ID NO: 32)
where X 1 is T or H.
13. An expression vector comprising the nucleic acid according to claim 12 .
14. A host cell transformed with the expression vector of claim 13 .
15. The nucleic acid of claim 12 , wherein A comprises a polypeptide having the sequence GIVEQCCHSICSLYQLENYCN (SEQ ID NO: 29).
16. The nucleic acid of claim 15 , wherein C is a polypeptide having the sequence GGGPRR (SEQ ID NO:19).
17. The nucleic acid of claim 16 , wherein X 3 is D, and X 4 is P.
18. A method of reducing blood glucose levels of a mammal comprising administering a physiologically effective amount of a single chain insulin analogue or a physiologically acceptable salt thereof to a mammal, wherein the single chain insulin analogue comprises a single chain polypeptide of formula I,
B-C-A (I)
wherein B comprises a polypeptide having the sequence:
FVNQHLCGSX 2 LVEALYLVCGERGFFYTX 3 X 4 T (SEQ ID NO: 33)
where X 2 is D or H, X 3 is P, D or K, and X 4 is K or P,
wherein C is a polypeptide selected from the group consisting of a polypeptide having the sequence GGGPRR (SEQ ID NO: 19) and a polypeptide having the sequence GGPRR (SEQ ID NO: 20),
and wherein A comprises a polypeptide having the sequence:
GIVEQCCX 1 SICSLYQLENYCN (SEQ ID NO: 32)
where X 1 is T or H.
19. The method of claim 18 , wherein X 2 is D.
20. The method of claim 19 , wherein the insulin A chain polypeptide comprises a histidine substitution at position A10.
21. The method of claim 18 , wherein the single chain insulin analogue is a polypeptide having the sequence FVNQHLCGSNLVEALYLVCGERGFFYT NPTGGGPRRGIVEQCCHSICSLYQLENYCN (SEQ ID NO: 26).
22. The method of claim 18 , wherein the mammal is a human.