Human amylin analog polypeptides and methods of use

1. An isolated polypeptide, comprising the amino acid sequence of SEQ ID NO: 203:

X 1 CNTX 5 TCATX 10 RLANX 15 X 16 X 17 X 18 SSNNFGPILPPTKVGSETY-(OH/NH 2 ) (SEQ ID NO: 203), wherein:

X 1 is k or K;

X 5 is S;

X 10 is Q or S;

X 15 is E or F;

X 16 is L;

X 17 is H, V, and Q; and

X 18 is K, H, or R;

wherein:

each K independently represents an L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

each k independently represents a D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer; and

the two cysteine residues of X 1 CNTX 5 TC (SEQ ID NO: 308) are optionally further bound by a disulfide bridge.

2. The isolated polypeptide of claim 1 , comprising the amino acid sequence of SEQ ID NO:209:

X 1 CNTSTCATX 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSETY-(OH/NH 2 ) (SEQ ID NO:209), or a pharmaceutically acceptable salt thereof, wherein:

X 1 is K or k;

X 10 is Q or S;

X 16 is E or F;

X 16 is L; and

X 17 is H, V or Q;

each K independently represents an L-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer;

each k independently represents a D-lysine optionally covalently bound to a lipophilic substituent, optionally via a spacer; and

wherein the two cysteine residues of X 1 CNTSTC (SEQ ID NO: 318) are optionally further bound by a disulfide bridge.

3. The isolated polypeptide of claim 1 , comprising the amino acid sequence of:

(SEQ ID NO: 130)

KC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH 2 ),

or a pharmaceutically acceptable salt thereof,

wherein the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

4. The isolated polypeptide of claim 1 , comprising the amino acid sequence of:

(SEQ ID NO: 64)

K*((γGlu) 2 (CO(CH 2 ) 18 CO 2 H))C*NTSTC*ATQRLANELHKSSNNF

GPILPPTKVGSETY-(NH 2 ),

or a pharmaceutically acceptable salt thereof,

wherein:

K* represents an L-lysine covalently bound to (γGlu) 2 (CO(CH 2 ) 18 CO 2 H); and

the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

5. The isolated polypeptide of claim 1 , comprising the amino acid sequence of:

(SEQ ID NO: 65)

K*((γGlu) 2 (CO(CH 2 ) 16 CO 2 H))C*NTSTC*ATQRLANELHKSSN

NFGPILPPTKVGSETY-(NH 2 ),

or a pharmaceutically acceptable salt thereof,

wherein:

K* represents an L-lysine covalently bound to (γGlu) 2 (CO(CH 2 ) 16 CO 2 H); and

the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

6. The isolated polypeptide of claim 1 , selected from the group consisting of:

(SEQ ID NO: 131)

KC*NTSTC*ATQRLANFLQKSSNNFGPILPPTKVGSETY-(NH 2 ),

or a pharmaceutically acceptable salt thereof,

wherein the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

7. The isolated polypeptide of claim 1 , selected from the group consisting of:

(SEQ ID NO: 109)

K*(γGlu-CO(CH 2 ) 16 CO 2 H)C*NTSTC*ATSRLANFLQKSSNNF G

PILPPTKVGSETY-NH 2 ,

or a pharmaceutically acceptable salt thereof,

wherein:

K* represents an L-lysine covalently bound to γGlu-CO(CH 2 ) 16 CO 2 H; and

the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

8. The isolated polypeptide of claim 1 , comprising an amino acid sequence selected from the group consisting of any of SEQ ID NOS: 55, 64, 65, 109, 112-120, 126, 130, 131, and 143, or a pharmaceutically acceptable salt thereof.

9. The isolated polypeptide of claim 1 , or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent, and optionally comprising a spacer.

10. The isolated polypeptide of claim 9 , or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula VI:

—(Y1) n1 —(V) r —(Y2) n2 —CO—(CH 2 ) m —Z  Formula VI

wherein

Z is —CH 3 or —CO 2 H;

m is from 4 to 24;

Y1 is selected from the group consisting of γGlu, Asp, and Gly;

Y2 is selected from the group consisting of γGlu, Asp, and Gly;

V is —[COCH 2 (O(CH 2 ) 2 ) t OCH 2 NH]—, and t is from 1 to 8;

r is from 1 to 8;

n1 is from 0 to 10; and

n2 is from 0 to 10.

11. The isolated polypeptide of claim 9 , or a pharmaceutically acceptable salt thereof, further comprising a lipophilic substituent and a spacer of Formula III:

-(γGlu) n -CO—(CH 2 ) m —Z (“(γGlu) n ” disclosed as SEQ ID NO: 311)  Formula III

wherein

Z is —CH 3 or —CO 2 H;

m is from 4 to 24; and

n is from 1 to 10.

12. The isolated polypeptide of claim 10 , or a pharmaceutically acceptable salt thereof, wherein the lipophilic substituent, —CO—(CH 2 ) m —Z, is linked to the ε-amino group of a lysine of the isolated polypeptide via the spacer, —(Y1) n1 —(V) r —(Y2) n2 —, which spacer forms a bridge between the amino group of the disclosed polypeptide and the CO— group of the lipophilic substituent.

13. The isolated polypeptide of claim 11 , or a pharmaceutically acceptable salt thereof, wherein the lipophilic substituent, —CO—(CH 2 ) m —Z, is linked to the ε-amino group of a lysine of the isolated polypeptide via the spacer, -(γGlu) n - (“(γGlu) n ” disclosed as SEQ ID NO: 311), which spacer forms a bridge between the amino group of the disclosed polypeptide and the CO— group of the lipophilic substituent.

14. The isolated polypeptide of claim 9 , or a pharmaceutically acceptable salt thereof, wherein:

the lipophilic substituent is —CO—(CH 2 ) m —CO 2 H; and

m is from 14 to 20.

15. The isolated polypeptide of claim 14 , or a pharmaceutically acceptable salt thereof, wherein the spacer is γGlu or 2(γGlu).

16. A pharmaceutical composition comprising the isolated polypeptide of claim 1 or a pharmaceutically acceptable salt thereof.

17. The pharmaceutical composition of claim 16 , wherein the composition further comprises an insulinotropic polypeptide.

18. The isolated peptide of claim 1 , or a pharmaceutically acceptable salt thereof, wherein X 17 is H or Q.

19. The isolated peptide of claim 2 , or a pharmaceutically acceptable salt thereof, wherein X 17 is H or Q.

20. The isolated polypeptide of claim 1 , consisting of the amino acid sequence of:

(SEQ ID NO: 130)

KC*NTSTC*ATQRLANELHKSSNNFGPILPPTKVGSETY-(NH 2 ),

or a pharmaceutically acceptable salt thereof,

wherein the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

21. The isolated polypeptide of claim 1 , consisting of the amino acid sequence of:

(SEQ ID NO: 64)

K*((γGlu) 2 (CO(CH 2 ) 18 CO 2 H))C*NTSTC*ATQRLANELHKSSNN 

FGPILPPTKVGSETY-(NH 2 ),

or a pharmaceutically acceptable salt thereof,

wherein:

K* represents an L-lysine covalently bound to (γGlu) 2 (CO(CH 2 ) 18 CO 2 H); and

the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

22. The isolated polypeptide of claim 1 , comprising the amino acid sequence of:

(SEQ ID NO: 65)

K*((γGlu) 2 (CO(CH 2 ) 16 CO 2 H))C*NTSTC*ATQRLANELHKSSNN 

FGPILPPTKVGSETY-(NH 2 ),

or a pharmaceutically acceptable salt thereof,

wherein:

K* represents an L-lysine covalently bound to (γGlu) 2 (CO(CH 2 ) 16 CO 2 H); and

the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

23. The isolated polypeptide of claim 1 , selected from the group consisting of:

(SEQ ID NO: 131)

KC*NTSTC*ATQRLANFLQKSSNNFGPILPPTKVGSETY-(NH 2 ),

or a pharmaceutically acceptable salt thereof,

wherein the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

24. The isolated polypeptide of claim 1 , selected from the group consisting of:

(SEQ ID NO: 109)

K*(γGlu-CO(CH 2 ) 16 CO 2 H)C*NTSTC*ATSRLANFLQKSSNNF 

GPILPPTKVGSETY-NH 2 ,

or a pharmaceutically acceptable salt thereof,

wherein:

K* represents an L-lysine covalently bound to γGlu-CO(CH 2 ) 16 CO 2 H; and

the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

25. The pharmaceutical composition of claim 16 , wherein said pharmaceutical composition is formulated for injection.

26. The pharmaceutical composition of claim 16 , wherein said pharmaceutical composition is formulated for oral administration.

27. A pharmaceutical composition comprising the isolated polypeptide of claim 20 or a pharmaceutically acceptable salt thereof.

28. A pharmaceutical composition comprising the isolated polypeptide of claim 21 or a pharmaceutically acceptable salt thereof.

29. A pharmaceutical composition comprising the isolated polypeptide of claim 22 or a pharmaceutically acceptable salt thereof.

30. A pharmaceutical composition comprising the isolated polypeptide of claim 23 or a pharmaceutically acceptable salt thereof.

31. A pharmaceutical composition comprising the isolated polypeptide of claim 24 or a pharmaceutically acceptable salt thereof.

32. An isolated polypeptide, comprising the amino acid sequence of SEQ ID NO: 211:

(SEQ ID NO: 211)

X 1 CNTSTCATX 10 RLANX 15 X 16 X 17 KSSNNFGPILPPTKVGSX 35 TY-

(OH/NH 2 ),

wherein:

X 1 is K or k;

X 10 is Q or S;

X 15 is E or F;

X 16 is L;

X 17 is H, V or Q; and

X 35 is E;

wherein:

each K independently represents an L-lysine optionally covalently bound to a protecting group or a spacer optionally bound to a protecting group;

each k independently represents a D-lysine optionally covalently bound to a protecting group or a spacer optionally bound to a protecting group; and

the two cysteine residues of X 1 CNTSTC (SEQ ID NO: 309) are optionally further bound by a disulfide bridge.

33. The isolated peptide of claim 32 , selected from the group consisting of:

(SEQ ID NO. 155)

KC*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-NH2;

and

(SEQ ID NO. 156)

K*(Fmoc)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-

NH 2 ,

or a pharmaceutically acceptable salt thereof,

wherein:

each K* represents an L-lysine covalently bound to a protecting group; and

the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

34. The isolated peptide of claim 32 , selected from the group consisting of:

(SEQ ID NO. 157)

K*(γGlu)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVGSETY-

NH 2 ;

(SEQ ID NO. 158)

K*(γGlu-acetyl)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTK

VGSETY-NH 2 ;

(SEQ ID NO. 159)

K*(γGlu-trityl)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTK

VGSETY-NH 2 ;

and

(SEQ ID NO. 160)

K*(γGlu-tert-butyl)C*NTSTC*ATSRLANFLQKSSNNFGPIL

PPTKVGSETY-NH 2 ,

or a pharmaceutically acceptable salt thereof,

wherein:

each K* independently represents an L-lysine covalently bound to a spacer or a spacer bound to a protecting group; and

the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.

35. The isolated peptide of claim 32 , selected from the group consisting of:

(SEQ ID NO. 161)

K*(γGlu-γGlu)C*NTSTC*ATSRLANFLQKSSNNFGPILPPTKVG

SETY-NH 2 ;

(SEQ ID NO. 162)

K*(γGlu-γGlu-acetyl)C*NTSTC*ATSRLANFLQKSSNNFGPI

LPPTKVGSETY-NH 2 ;

(SEQ ID NO. 163)

K*(γGlu-γGlu-trityl)C*NTSTC*ATSRLANFLQKSSNNFGPI

LPPTKVGSETY-NH 2 ;

and

(SEQ ID NO. 164)

K*(γGlu-γGlu-tert-butyl)C*NTSTC*ATSRLANFLQKSSNN

FGPILPPTKVGSETY-NH 2 ,

or a pharmaceutically acceptable salt thereof,

wherein:

each K* independently represents an L-lysine covalently bound to a spacer or a spacer bound to a protecting group; and

the two cysteine residues denoted C* at positions 2 and 7 are further bound by a disulfide bridge.