U.S. patent application number 10/839037 was filed with the patent office on 2004-10-28 for modulators of receptors for parathyroid hormone and parathyroid hormone-related protein.
This patent application is currently assigned to Amgen Inc.. Invention is credited to Kostenuik, Paul, Lacey, David Lee, Liu, Chuan-Fa.
Application Number | 20040214996 10/839037 |
Document ID | / |
Family ID | 27498365 |
Filed Date | 2004-10-28 |
United States Patent
Application |
20040214996 |
Kind Code |
A1 |
Kostenuik, Paul ; et
al. |
October 28, 2004 |
Modulators of receptors for parathyroid hormone and parathyroid
hormone-related protein
Abstract
The present invention concerns therapeutic agents that modulate
the activity of PTH and PTHrP. In accordance with the present
invention, modulators of PTH and PTHrP comprise: (a) a PTH/PTHrP
modulating domain; and (b) a vehicle, such as a polymer (e.g., PEG
or dextran) or an Fc domain, which is preferred; wherein the
vehicle is covalently attached to the C-terminus of the PTH/PTHrP
modulating domain. The vehicle and the PTH/PTHrP modulating domain
may be linked through the N-- or C-terminus of the PTH/PTHrP
modulating domain, as described further below. The preferred
vehicle is an Fc domain, and the preferred Fc domain is an IgG Fc
domain. Preferred PTH/PTHrP modulating domains comprise the PTH and
PTHrP-derived amino acid sequences described hereinafter. Other
PTH/PTHrP modulating domains can be generated by phage display,
RNA-peptide screening and the other techniques mentioned herein.
Such peptides typically will be modulators of both PTH activity and
PTHrP activity, although such techniques can be used to generate
peptide sequences that serve as selective modulators (e.g.,
agonists of PTH activity but not PTHrP activity).
Inventors: |
Kostenuik, Paul; (Newbury
Park, CA) ; Liu, Chuan-Fa; (Longmont, CO) ;
Lacey, David Lee; (Newbury Park, CA) |
Correspondence
Address: |
AMGEN INCORPORATED
MAIL STOP 27-4-A
ONE AMGEN CENTER DRIVE
THOUSAND OAKS
CA
91320-1799
US
|
Assignee: |
Amgen Inc.
|
Family ID: |
27498365 |
Appl. No.: |
10/839037 |
Filed: |
May 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10839037 |
May 4, 2004 |
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09843221 |
Apr 26, 2001 |
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6756480 |
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60266673 |
Feb 6, 2001 |
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60214860 |
Jun 28, 2000 |
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60200053 |
Apr 27, 2000 |
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Current U.S.
Class: |
530/391.1 |
Current CPC
Class: |
A61P 35/02 20180101;
A61P 9/00 20180101; A61P 37/06 20180101; A61P 11/00 20180101; A61K
38/00 20130101; A61P 3/10 20180101; A61P 3/14 20180101; A61P 35/00
20180101; A61P 35/04 20180101; A61P 5/22 20180101; A61P 13/12
20180101; A61K 47/61 20170801; A61P 17/14 20180101; A61P 19/10
20180101; A61P 11/06 20180101; A61K 47/60 20170801; C07K 14/635
20130101; A61P 29/00 20180101; A61P 5/46 20180101; A61P 1/16
20180101; A61P 21/00 20180101; A61P 1/02 20180101; A61P 5/06
20180101; A61P 19/02 20180101; A61P 7/06 20180101; A61K 47/6425
20170801; C07K 2319/00 20130101; A61P 19/00 20180101; A61P 19/08
20180101; A61P 5/14 20180101; A61P 5/18 20180101; A61P 5/16
20180101 |
Class at
Publication: |
530/391.1 |
International
Class: |
C07K 016/46 |
Claims
What is claimed is:
1. A composition of matter of the formula
P.sup.1-(L.sup.1).sub.a-F.sup.1 and multimers thereof, wherein:
F.sup.1 is a vehicle and is attached at the C-terminus of
P.sup.1-(L.sup.1).sub.a; P.sup.1 is a PTH/PTHrP modulating domain;
L.sup.1 is a linker; and a is 0 or 1.
2. The composition of matter of claim 1 of the formulae
P.sup.1-F.sup.1.
3. The composition of matter of claim 1, wherein F.sup.1 is an Fc
domain.
4. The composition of matter of claim 1 wherein F.sup.1 is an IgG
Fc domain.
5. The composition of matter of claim 1 wherein F.sup.1 is an IgG1
Fc domain.
6. The composition of matter of claim 1 wherein F.sup.1 comprises
the sequence of SEQ ID NO: 2.
7. The composition of matter of claim 1 wherein the PTH/PTHrP
modulating domain is of the formula
11
X.sup.NHX.sup.10X.sup.11X.sup.12KX.sup.14X.sup.15X.sup.16X.sup.1-
7X.sup.18X.sup.19RX.sup.21 (SEQ ID NO: 3) X.sup.22X.sup.23X.sup.24-
X.sup.25X.sup.26X.sup.27X.sup.28X.sup.C
wherein: X.sup.N is absent or is
X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7,
X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7,
X.sup.1X.sup.2X.sup.3X.sup.4X- .sup.5X.sup.6X.sup.7, or
YX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.- 7; X.sup.1
through X.sup.7, X.sup.10, X.sup.11, X.sup.12, X.sup.14 through
X.sup.28 are each independently amino acid residues; X.sup.C is
absent or is X.sup.29, X.sup.29X.sup.30, X.sup.29X.sup.30X.sup.31,
X.sup.29X.sup.30X.sup.31X.sup.32,
X.sup.29X.sup.30X.sup.31X.sup.32X.sup.3- 3,
X.sup.29X.sup.30X.sup.31X.sup.32X.sup.33X.sup.34,
X.sup.29X.sup.30X.sup.31X.sup.32X.sup.33X.sup.34X.sup.35, or
X.sup.29X.sup.30X.sup.31X.sup.32X.sup.33X.sup.34X.sup.35X.sup.36;
X.sup.29 through X.sup.36 are each independently amino acid
residues.
8. The composition of matter of claim 7, wherein: X.sup.N is
X.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7; X.sup.1 is a
hydrophilic or nonfunctional residue; X.sup.2 is V; X.sup.3 is S;
X.sup.4 is E; X.sup.5 is a nonfunctional or basic residue; X.sup.6
is Q; X.sup.7 is L; X.sup.10 is an acidic or hydrophilic residue;
X.sup.11 is a nonfunctional or basic residue; X.sup.12 is a
nonfunctional residue; X.sup.14 is a basic or hydrophilic residue;
X.sup.15 is a nonfunctional residue; X.sup.16 is a nonfunctional or
hydrophilic residue; X.sup.17 is an acidic, hydrophilic, or
nonfunctional residue; X.sup.18 is a nonfunctional residue;
X.sup.19 is an acidic or basic residue; X.sup.21 is a nonfunctional
or basic residue; X.sup.22 is a hydrophilic, acidic, or aromatic
residue; X.sup.23 is an aromatic or lipophilic residue; X.sup.24 is
a lipophilic residue (L preferred); X.sup.25 is a hydrophilic or
basic residue; X.sup.26 is a hydrophilic or basic residue; X.sup.27
is a lipophilic, basic, or nonfunctional residue; and X.sup.28 is a
lipophilic or nonfunctional residue.
9. The composition of matter of claim 8, wherein: X.sup.C is
X.sup.29X.sup.30X.sup.31X.sup.32X.sup.33X.sup.34; X.sup.29 is a
hydrophilic or nonfunctional residue; X.sup.30 is a hydrophilic or
acidic residue; X.sup.31 is a lipophilic or nonfunctional residue;
X.sup.32 is H; X.sup.33 is a hydrophilic residue; and X.sup.34 is a
nonfunctional or aromatic residue.
10. The composition of matter of claim 8, wherein: X.sup.C is
X.sup.29X.sup.30X.sup.31; X.sup.29 is a hydrophilic or
nonfunctional residue; X.sup.30 is a hydrophilic or acidic residue;
and X.sup.31 is a lipophilic or nonfunctional residue.
11. The composition of matter of claim 8, wherein: X.sup.C is
X.sup.29X.sup.30; X.sup.29 is a hydrophilic or nonfunctional
residue; and X.sup.30 is a hydrophilic or acidic residue.
12. The composition of matter of claim 8, wherein: X.sup.C is
X.sup.29; and; X.sup.29 is a hydrophilic or nonfunctional
residue.
13. The composition of matter of claim 8, wherein X.sup.C is
absent.
14. The composition of matter of claim 8, wherein: X.sup.1 is A, S
or Y; X.sup.5 is H or I; X.sup.10 is N or D; X.sup.11 is L, R, or
K; X.sup.12 is G, F, or W; X.sup.14 is H or S; X.sup.15 is L or I;
X.sup.16 is Q, N, S, or A; X.sup.17 is S, D, or L; X.sup.18 is M,
L, V or Nle; X.sup.19 is E or R; X.sup.21 is V, M, R, or Nle;
X.sup.22 is E or F; X.sup.23 is W or F; X.sup.25 is R or H;
X.sup.26 is K or H; X.sup.27 is K or L; and X.sup.28 is L or I.
15. The composition of matter of claim 14, wherein: X.sup.C is
X.sup.29X.sup.30X.sup.31X.sup.32X.sup.33X.sup.34; X.sup.29 is Q or
A; X.sup.30 is D or E; X.sup.31 is V or I; X.sup.33 is N or T; and
X.sup.34 is A, F or Y.
16. The composition of matter of claim 14, wherein: X.sup.C is
X.sup.29X.sup.30X.sup.31; X.sup.29 is Q or A; X.sup.30 is D or E;
and X.sup.31 is V or I;
17. The composition of matter of claim 14, wherein: X.sup.C is
X.sup.29X.sup.30; X.sup.29 is Q or A; and X.sup.30 is D or E.
18. The composition of matter of claim 14, wherein: X.sup.C is
X.sup.29; and; X.sup.29 is Q or A.
19. The composition of matter of claim 14, wherein X.sup.C is
absent.
20. The composition of matter of claim 1, wherein the PTH/PTHrP
modulating domain is of the formula
12 J.sup.NJ.sup.7J.sup.8HNJ.sup.11J.sup.12KHLJ.sup.16 (SEQ ID NO:
4) SJ.sup.18J.sup.19RJ.sup.21EWLRKKLJ.sup.C
wherein: J.sup.N is absent or is selected from
J.sup.1J.sup.2J.sup.3J.sup.- 4J.sup.5J.sup.6,
J.sup.2J.sup.3J.sup.4J.sup.5J.sup.6, J.sup.3J.sup.4J.sup.5J.sup.6;
J.sup.1 is an amino acid residue; J.sup.2 is an amino acid residue;
J.sup.3 is an amino acid residue; J.sup.4 is an amino acid residue;
J.sup.5 is an amino acid residue; J.sup.6 is an amino acid residue;
J.sup.7 is an amino acid residue; J.sup.8 is an amino acid residue;
J.sup.11 is a nonfunctional or basic residue; J.sup.12 is an amino
acid residue; J.sup.16 is an amino acid residue; J.sup.18 is an
amino acid residue; J.sup.19 is an acidic or basic residue;
J.sup.21 is an amino acid residue; J.sup.C is absent or is
J.sup.29, J.sup.29J.sup.30, J.sup.29J.sup.30J.sup.31,
J.sup.29J.sup.30J.sup.31J.sup- .32,
J.sup.29J.sup.30J.sup.31J.sup.32J.sup.33,
J.sup.29J.sup.30J.sup.31J.s- up.32J.sup.33J.sup.34; and J.sup.29 is
an amino acid residue; J.sup.30 is an amino acid residue; J.sup.31
is an amino acid residue; J.sup.32 is an amino acid residue;
J.sup.33 is an amino acid residue; J.sup.34 is an amino acid
residue.
21. The composition of matter of claim 20, wherein: J.sup.N is
J.sup.1J.sup.2J.sup.3J.sup.4J.sup.5J.sup.6; J.sup.1 is a
nonfunctional or aromatic residue; J.sup.2 is a nonfunctional
residue; J.sup.3 is a hydrophilic residue; J.sup.4 is an acidic
residue; J.sup.5 is a nonfunctional residue; J.sup.6 is a basic
residue; J.sup.7 is a nonfunctional or aromatic residue; J.sup.8 is
a nonfunctional residue; J.sup.11 is a basic or a nonfunctional
residue; J.sup.12 is a nonfunctional or aromatic residue; J.sup.16
is a nonfunctional or hydrophilic residue; J.sup.18 is a
nonfunctional residue; J.sup.19 is an acidic or basic residue; and
J.sup.21 is a nonfunctional residue; J.sup.C is
J.sup.29J.sup.30J.sup.31J.sup.32J.sup.33J.sup.34; J.sup.29 is a
hydrophilic or nonfunctional residue; J.sup.30 is a hydrophilic or
acidic residue; J.sup.31 is a lipophilic or nonfunctional residue;
J.sup.32 is a basic residue; J.sup.33 is an acidic residue; and
J.sup.34 is an aromatic residue.
22. The composition of matter of claim 21, wherein: J.sup.1 is A, S
or Y; J.sup.2 is V; J.sup.3 is S; J.sup.4 is E; J.sup.5 is I;
J.sup.6 is Q; J.sup.7 is L or F; J.sup.8 is M or Nle; J.sup.11 is
L, R, or K; J.sup.12 is G or W; J.sup.16 is N, S, or A; J.sup.18 is
M, Nle, L, or V; J.sup.19 is E or R; J.sup.21 is V, M, or Nle;
J.sup.29 is Q or A; J.sup.30 is D or E; J.sup.31 is V or I;
J.sup.32 is H; J.sup.33 is N; and J.sup.34is F or Y.
23. The composition of matter of claim 20, wherein: J.sup.N is
J.sup.1J.sup.2J.sup.3J.sup.4J.sup.5J.sup.6; J.sup.1 is a
nonfunctional or aromatic residue; J.sup.2 is a nonfunctional
residue; J.sup.3 is a hydrophilic residue; J.sup.4 is an acidic
residue; J.sup.5 is a nonfunctional residue; J.sup.6 is a basic
residue; J.sup.7 is a nonfunctional or aromatic residue; J.sup.8 is
a nonfunctional residue; J.sup.11 is a basic or a nonfunctional
residue; J.sup.12 is a nonfunctional or aromatic residue; J.sup.16
is a nonfunctional or hydrophilic residue; J.sup.18 is a
nonfunctional residue; J.sup.19 is an acidic or basic residue;
J.sup.21 is a nonfunctional residue; J.sup.C is
J.sup.29J.sup.30J.sup.31; J.sup.29 is a hydrophilic or
nonfunctional residue; J.sup.30 is a hydrophilic or acidic residue;
and J.sup.31 is a lipophilic or nonfunctional residue.
24. The composition of matter of claim 23, wherein: J.sup.1 is A, S
or Y; J.sup.2 is V; J.sup.3 is S; J.sup.4is E; J.sup.5 is I;
J.sup.6 is Q; J.sup.7is L or F; J.sup.8 is M or Nle; J.sup.11 is L,
R, or K; J.sup.12 is G or W; J.sup.16 is N, S, or A; J.sup.18 is M,
Nle, L, or V; J.sup.19 is E or R; J.sup.21 is V, M, or Nle;
J.sup.29 is Q or A; J.sup.30 is D or E; and J.sup.31 is V or I.
25. The composition of matter of claim 20, wherein: J.sup.N is
J.sup.1J.sup.2J.sup.3J.sup.4J.sup.5J.sup.6; J.sup.1 is a
nonfunctional or aromatic residue; J.sup.2 is a nonfunctional
residue; J.sup.3 is a hydrophilic residue; J.sup.4 is an acidic
residue; J.sup.5 is a nonfunctional residue; J.sup.6 is a basic
residue; J.sup.7 is a nonfunctional or aromatic residue; J.sup.8 is
a nonfunctional residue; J.sup.11 is a basic or a nonfunctional
residue; J.sup.12 is a nonfunctional or aromatic residue; J.sup.16
is a nonfunctional or hydrophilic residue; J.sup.18 is a
nonfunctional residue; J.sup.19 is an acidic or basic residue;
J.sup.21 is a nonfunctional residue; J.sup.C is J.sup.29J.sup.30;
J.sup.29 is a hydrophilic or nonfunctional residue; and J.sup.30 is
a hydrophilic or acidic residue.
26. The composition of matter of claim 25, wherein: J.sup.1 is A, S
or Y; J.sup.2 is V; J.sup.3 is S; J.sup.4 is E; J.sup.5 is I;
J.sup.6 is Q; J.sup.7 is L or F; J.sup.8 is M or Nle; J.sup.11 is
L, R, or K; J.sup.12 is G or W; J.sup.16 is N, S, or A; J.sup.18 is
M, Nle, L, or V; J.sup.19 is E or R; J.sup.21 is V, M, or Nle;
J.sup.29 is Q or A; and J.sup.30 is D or E.
27. The composition of matter of claim 20, wherein: J.sup.N is
J.sup.1J.sup.2J.sup.3J.sup.4J.sup.5J.sup.6; J.sup.1 is a
nonfunctional or aromatic residue; J.sup.2 is a nonfunctional
residue; J.sup.3 is a hydrophilic residue; J.sup.4 is an acidic
residue; J.sup.5 is a nonfunctional residue; J.sup.6 is a basic
residue; J.sup.7 is a nonfunctional or aromatic residue; J.sup.8 is
a nonfunctional residue; J.sup.11 is a basic or a nonfunctional
residue; J.sup.12 is a nonfunctional or aromatic residue; J.sup.16
is a nonfunctional or hydrophilic residue; J.sup.18 is a
nonfunctional residue; J.sup.19 is an acidic or basic residue;
J.sup.21 is a nonfunctional residue; J.sup.C is J.sup.29; and
J.sup.29 is a hydrophilic or nonfunctional residue.
28. The composition of matter of claim 27, wherein: J.sup.1 is A, S
or Y; J.sup.2 is V; J.sup.3 is S; J.sup.4 is E; J.sup.5 is I;
J.sup.6 is Q; J.sup.7 is L or F; J.sup.8 is M or Nle; J.sup.11 is
L, R, or K; J.sup.12 is G or W; J.sup.16 is N, S, or A; J.sup.18 is
M, Nle, L, or V; J.sup.19 is E or R; J.sup.21 is V, M, or Nle; and
J.sup.29 is Q or A.
29. The composition of matter of claim 20, wherein: J.sup.N is
J.sup.1J.sup.2J.sup.3J.sup.4J.sup.5J.sup.6; J.sup.1 is a
nonfunctional or aromatic residue; J.sup.2 is a nonfunctional
residue; J.sup.3 is a hydrophilic residue; J.sup.4 is an acidic
residue; J.sup.5 is a nonfunctional residue; J.sup.6 is a basic
residue; J.sup.7 is a nonfunctional or aromatic residue; J.sup.8 is
a nonfunctional residue; J.sup.11 is a basic or a nonfunctional
residue; J.sup.12 is a nonfunctional or aromatic residue; J.sup.16
is a nonfunctional or hydrophilic residue; J.sup.18 is a
nonfunctional residue; J.sup.19 is an acidic or basic residue;
J.sup.21 is a nonfunctional residue; and J.sup.C is absent.
30. The composition of matter of claim 29, wherein: J.sup.1 is A, S
or Y; J.sup.2 is V; J.sup.3 is S; J.sup.4 is E; J.sup.5 is I;
J.sup.6 is Q; J.sup.7 is L or F; J.sup.8 is M or Nle; J.sup.11 is
L, R, or K; J.sup.12 is G or W; J.sup.16 is N, S, or A; J.sup.18 is
M, Nle, L, or V; J.sup.19 is E or R; and J.sup.21 is V, M, or
Nle.
31. The composition of matter of claim 20, wherein the PTH/PTHrP
modulating domain is selected from Table 1.
32. The composition of matter of claim 1 wherein the PTH/PTHrP
modulating domain is of the formula
13 O.sup.NLHO.sup.10O.sup.11O.sup.12KSIO.sup.16 (SEQ ID NO: 5)
O.sup.17LRRRFO.sup.23LHHLIO.sup.C
wherein: O.sup.N is absent or is
YO.sup.1O.sup.2O.sup.3O.sup.4O.sup.5O.sup- .6O.sup.7,
O.sup.1O.sup.2O.sup.3O.sup.4O.sup.5O.sup.6O.sup.7,
O.sup.2O.sup.3O.sup.4O.sup.5O.sup.6O.sup.7,
O.sup.3O.sup.4O.sup.5O.sup.6O- .sup.7,
O.sup.4O.sup.5O.sup.6O.sup.7, O.sup.5O.sup.6O.sup.7,
O.sup.6O.sup.7, or O.sup.7; O.sup.1 is an amino acid residue;
O.sup.2 is an amino acid residue; O.sup.3 is an amino acid residue;
O.sup.4 is an amino acid residue; O.sup.5 is an amino acid residue;
O.sup.6 is an amino acid residue; O.sup.7 is an amino acid residue;
O.sup.10 is an amino acid residue; O.sup.11 is an amino acid
residue; O.sup.12 is an amino acid residue; O.sup.16 is an amino
acid residue; O.sup.17 is an amino acid residue; O.sup.23 is an
amino acid residue; O.sup.C is absent or is O.sup.29,
O.sup.29O.sup.30, O.sup.29O.sup.30O.sup.31,
O.sup.29O.sup.30O.sup.31O.sup.32,
O.sup.29O.sup.30O.sup.31O.sup.32O.sup.3- 3,
O.sup.29O.sup.30O.sup.31O.sup.32O.sup.33O.sup.34,
O.sup.29O.sup.30O.sup.31O.sup.32O.sup.33O.sup.34O.sup.35, or
O.sup.29O.sup.30O.sup.31O.sup.32O.sup.33O.sup.34O.sup.35O.sup.36;
and O.sup.29 through O.sup.36 are each independently amino acid
residues.
33. The composition of matter of claim 27, wherein: O.sup.N is
O.sup.7; O.sup.7 is a nonfunctional residue; O.sup.10 is an acidic
or hydrophilic residue; O.sup.11 is a basic or nonfunctional
residue; O.sup.12 is an aromatic or nonfunctional residue; O.sup.15
is a hydrophilic or nonfunctional residue; O.sup.16 is a
hydrophilic residue; O.sup.17 is an acidic or nonfunctional
residue; O.sup.23 is an aromatic residue; and O.sup.C is
absent.
34. The composition of matter of claim 23, wherein: O.sup.N is
O.sup.1O.sup.2O.sup.3O.sup.4O.sup.5O.sup.6O.sup.7; O.sup.1 is a
nonfunctional amino acid residue; O.sup.2 is a nonfunctional amino
acid residue; O.sup.3 is a hydrophilic amino acid residue; O.sup.4
is an acidic amino acid residue; O.sup.5 is a basic or
nonfunctional amino acid residue; O.sup.6 is a hydrophilic amino
acid residue; O.sup.7 is a nonfunctional residue; O.sup.10 is an
acidic or hydrophilic residue; O.sup.11 is a basic or nonfunctional
residue; O.sup.12 is an aromatic or nonfunctional residue; O.sup.15
is a hydrophilic or nonfunctional residue; O.sup.16 is a
hydrophilic residue; and O.sup.17 is an acidic or nonfunctional
residue; and O.sup.23 is an aromatic residue.
35. The composition of matter of claim 34, wherein: O.sup.1 is A;
O.sup.2 is V; O.sup.3 is S; O.sup.4 is E; O.sup.5 is H or I;
O.sup.6 is Q; O.sup.7 is L; O.sup.10 is N or D; O.sup.11 is K or L;
O.sup.12 is G, F, or W; O.sup.15 is I or S; O.sup.16 is Q or N;
O.sup.17 is D or L; O.sup.23 is F or W.
36. The composition of matter of claim 27, wherein the PTH/PTHrP
modulating domain is selected from Table 2.
37. The composition of matter of claim 1, wherein the PTH/PTHrP
modulating domain has the amino acid sequence of TIP39.
38. The composition of matter of claim 1, comprising a sequence
selected from Table 4.
39. A composition of matter, which comprises a peptide selected
from SEQ ID NOS: 17,18,19, and 69.
40. A nucleic acid encoding a composition of matter of claim 1.
41. A nucleic acid encoding a composition of matter of claim 7.
42. A nucleic acid encoding a composition of matter of claim
20.
43. A nucleic acid encoding a composition of matter of claim
32.
44. A nucleic acid encoding a composition of matter of claim
39.
45. An expression vector comprising the DNA of claim 40.
46. An expression vector comprising the DNA of claim 41.
47. An expression vector comprising the DNA of claim 42.
48. An expression vector comprising the DNA of claim 43.
49. An expression vector comprising the DNA of claim 44.
50. A host cell comprising the expression vector of claim 45.
51. A host cell comprising the expression vector of claim 46.
52. A host cell comprising the expression vector of claim 47.
53. A host cell comprising the expression vector of claim 48.
54. A host cell comprising the expression vector of claim 49.
55. The cell of claim 50, wherein the cell is an E. coli cell.
56. The cell of claim 51, wherein the cell is an E. coli cell.
57. The cell of claim 52, wherein the cell is an E. coli cell.
58. The cell of claim 53, wherein the cell is an E. coli cell.
59. A process for preparing an antagonist of the PTH/PTHrP
receptor, which comprises: a) selecting at least one peptide that
binds to the receptor; and b) preparing a pharmacologic agent
comprising at least one Fc domain covalently linked to at least one
amino acid sequence of the selected peptide or peptides.
60. The process of claim 59, wherein the peptide is selected from
the SEQ ID NOS: 3,4, or 5.
61. The process of claim 59, wherein the peptide is selected in a
process comprising screening of a phage display library, an E. coli
display library, a ribosomal library, an RNA-peptide library, or a
chemical peptide library.
62. The process of claim 59, wherein the preparation of the
pharmacologic agent is carried out by: a) preparing a gene
construct comprising a nucleic acid sequence encoding the selected
peptide and a nucleic acid sequence encoding an Fc domain; and b)
expressing the gene construct.
63. The process of claim 59, wherein the gene construct is
expressed in an E. coli cell.
64. The process of claim 59, wherein the selection of the peptide
is carried out by a process comprising: a) preparing a gene
construct comprising a nucleic acid sequence encoding a first
selected peptide and a nucleic acid sequence encoding an Fc domain;
b) conducting a polymerase chain reaction using the gene construct
and mutagenic primers, wherein i) a first mutagenic primer
comprises a nucleic acid sequence complementary to a sequence at or
near the 5' end of a coding strand of the gene construct, and ii) a
second mutagenic primer comprises a nucleic acid sequence
complementary to the 3' end of the noncoding strand of the gene
construct.
65. A method of treating osteopenia, which comprises administering
a PTH agonist and a bone resorption inhibitor, wherein the PTH
agonist comprises a composition of matter of claim 1.
66. A method of treating osteopenia, which comprises administering
a PTH agonist and a bone resorption inhibitor, wherein the PTH
agonist comprises a composition of matter of claim 7.
67. A method of treating osteopenia, which comprises administering
a PTH agonist and a bone resorption inhibitor, wherein the PTH
agonist comprises a composition of matter of claim 20.
68. A method of treating osteopenia, which comprises administering
a PTH agonist and a bone resorption inhibitor, wherein the PTH
agonist comprises a composition of matter of claim 32.
69. A method of treating osteopenia, which comprises administering
a PTH agonist and a bone resorption inhibitor, wherein the PTH
agonist comprises a composition of matter of claim 39.
70. The method of claim 65, wherein the bone resorption inhibitor
is selected from OPG, OPG-L antibody, calcitonin, bisphosphonates,
estrogens, estrogen receptor modulators, and tibolone.
71. The method of claim 66, wherein the bone resorption inhibitor
is selected from OPG, OPG-L antibody, calcitonin, bisphosphonates,
estrogens, estrogen receptor modulators, and tibolone.
72. The method of claim 67, wherein the bone resorption inhibitor
is selected from OPG, OPG-L antibody, calcitonin, bisphosphonates,
estrogens, estrogen receptor modulators, and tibolone.
73. The method of claim 68, wherein the bone resorption inhibitor
is selected from OPG, OPG-L antibody, calcitonin, bisphosphonates,
estrogens, estrogen receptor modulators, and tibolone.
74. The method of claim 69, wherein the bone resorption inhibitor
is selected from OPG, OPG-L antibody, calcitonin, bisphosphonates,
estrogens, estrogen receptor modulators, and tibolone.
75. A method of treating osteopenia, which comprises administering
a composition of matter of claim 1.
76. A method of treating osteopenia, which comprises administering
a composition of matter of claim 7.
77. A method of treating osteopenia, which comprises administering
a composition of matter of claim 20.
78. A method of treating osteopenia, which comprises administering
a composition of matter of claim 32.
79. A method of treating osteopenia, which comprises administering
a composition of matter of claim 39.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/843,221, filed Apr. 26, 2001, which claims the benefit of
U.S. Provisional Application No. 60/266,673, filed Feb. 6, 2001,
U.S. Provisional Application No. 60/214,860, filed Jun. 28, 2000,
and U.S. Provisional Application No. 60/200,053, filed Apr. 27,
2000, which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Parathyroid hormone (PTH) and parathyroid hormone-related
protein (PTHrP) play important physiological roles in calcium
homeostasis and in development, respectively. Calcium concentration
in the blood is tightly regulated, due to the essential role of
calcium in cell metabolism. PTH is an endocrine hormone which is
secreted from the parathyroid gland in response to decreased serum
calcium levels. PTH acts directly to increase bone resorption and
to stimulate renal calcium reabsorption, thus increasing or
preserving circulating calcium stores. PTH also indirectly
increases calcium absorption in the gut by stimulating the renal
hydroxylation of vitamin D.
[0003] Both primary and secondary hyperparathyroidism are
conditions that are associated with excessive levels of circulating
parathyroid hormone. Through the aforementioned pathways, excess
PTH levels can cause hypercalcemia and osteopenia. Bone resorption
inhibitors such as bisphosphonates and OPG can effectively protect
bone and can inhibit the skeleton's contribution to hypercalcemia.
However, the calcemic effects of hyperparathyroidism on the kidney
and gut are not addressed by currently available therapy.
[0004] PTHrP is produced by many cell types, and plays an important
role in regulating skeletal development. Postnatally, the roles for
PTHrP are less clearly defined. Circulating levels of PTHrP are
essentially non-detectable in normal healthy adults. However, many
tumors of diverse embryological origins produce and secrete PTHrP
in quantities sufficient to cause hypercalcemia. In fact, humoral
hypercalcemia of malignancy (HHM) is the most common paraneoplastic
syndrome, which accounts for significant patient morbidity and
mortality.
[0005] Currently, HHM is treated with saline hydration followed by
bone resorption inhibitors such as bisphosphonates. This treatment
regimen typically takes 3-4 days to achieve significant reductions
in serum calcium, and the effects are relatively short-lived (less
than one month). For patients with high circulating levels of
PTHrP, the effects of current treatment options are even less
impressive. Repeated administration of conventional therapies are
usually progressively less effective. These limitations to current
therapy strongly indicate an unmet medical need for rapid,
effective, and long-lasting treatments for HHM.
[0006] A major reason for the limited benefits of current HHM
therapy is the failure to directly inhibit PTHrP, which is very
well established as the principal pathophysiologic factor in HHM.
Bone resorption inhibitors such as bisphosphonates only inhibit
bone resorption, while PTHrP also has significant calcemic effects
on the kidney and the gut. Total neutralization of PTHrP would be
the ideal adjuvant therapeutic approach to treatment of HHM.
[0007] Both PTH and PTHrP interact with PTH-1 receptor, which
accounts for most of their known effects. Mannstadt et al. (1999),
Am. J. Physiol. 277. 5Pt 2. F665-75 (1999). Only PTH interacts with
the newly discovered PTH-2 receptor. Id. PTHrP can be changed to a
PTH-2 receptor agonist, however, by changing two residues to the
residues at those positions in PTH. Gardella et al. (1996), J.
Biol. Chem. 271 (33): 19888-93.
[0008] An N-terminal fragment of PTH has been used as a therapeutic
agent. Intermittently administered native PTH-(1-84) exhibits
osteogenic properties, and it has been recognized for decades that
these properties can be fully realized with the C-terminally
truncated fragment PTH-(1-34). Both peptides bind and activate the
PTH-1 receptor with similar affinities, causing the activation of
adenylate cyclase (AC) as well as phospholipase C (PLC). AC
activation through PTH-1 receptor generates cAMP, while PLC
activation through PTH-1 receptor generates PKC and intracellular
calcium transients. PTH-(1-34) can maximally activate both the AC
and the PLC pathways. It has been demonstrated that the anabolic
effects of PTH-(1-34) require short intermittent (daily) exposures
Dobnig (1998), Endocrinol. 138: 4607-12. In human trials on
postmenopausal women, daily subcutaneous injection of low doses of
PTH(1-34) were shown to result in impressive bone formation in the
spine and femoral neck with significant reduction in incidence of
vertebral fractures. These clinical data reveal PTH as one of the
most efficacious agents tested for osteoporosis.
[0009] Truncated PTH fragments have diminished AC/cAMP activation
and similarly diminished anabolic activity. Rixon et al. (1994), J.
Bone Min. Res. 9: 1179-89; Hilliker et al. (1996), Bone 19:
469-477; Lane et al. (1996), J. Bone Min. Res. 11: 614-25. Such
truncated PTH fragments have this diminished activity (Rixon et al.
(1994); Hilliker et al. (1996); Lane et al. (1996)) even if they
maintain full agonism towards PKC. Rixon et al., (1994). These
observations have led to the proposal that the AC/cAMP pathway is
critical for the bone anabolic properties of PTH, while the PLC/PKC
pathway is dispensable in this regard. Rixon et al., (1994);
Whitfield et al. (1996), Calcified Tissue International 53:
81-7.
[0010] An opposing, but not mutually exclusive, theory suggests
that PLC activation (in addition to AC) might also be an important
property of anabolic PTH fragments. Takasu (1998), Endocrinol. 139:
4293-9. The apparent absence of PLC activation by some anabolic
C-terminally truncated PTH peptides may be an artifact of
insensitive assay methods combined with lower receptor binding.
Takasu (1998). Progressive truncations from the C-terminus of
PTH-(1-34) result in stepwise reductions in binding affinity for
the PTH1R Takasu (1998). PKC activation through PTH-1 receptor
appears to be acutely sensitive to binding affinity and to receptor
density (Guo et al. (1995), Endocrinol 136: 3884-91), whereas cAMP
activation is far less sensitive to these variables. As such,
hPTH-(1-31) has a slightly reduced (1-6 fold) affinity for PTH-1
receptor compared to hPTH-(1-34), while hPTH-(1-30) has a
significantly reduced (10-100 fold) affinity Takasu (1998). Perhaps
due to this decreased PTH-1 receptor affinity, PTH-(1-30) is a weak
and incomplete agonist for PLC activation via the rat PTH-1
receptor.
[0011] Compared to PTH-(1-34), PTH-(1-31) has similar or slightly
reduced anabolic potential (Rixon et al. (1994); Whitfield et al.
(1996), Calcified Tissue International 53: 81-7; Whitfield et al.
(1996), Calcified Tissue International 65: 143-7), binding affinity
for PTH1R, and cAMP induction (Takasu (1998)). PTH-(1-31) also has
slightly reduced PLC activation. Takasu (1998). In healthy humans,
infusion of PTH-(1-31) and PTH-(1-34) had similar stimulatory
effects on plasma and urinary cAMP concentration, but unlike
PTH-(1-34), PTH-(1-31) failed to elevate serum calcium, plasma
1,25(OH)2D3, or urinary N-TX levels. Fraher et al. (1999), J. Clin.
Endocrin. Met. 84: 2739-43. These data suggest that PTH-(1-31) has
diminished capacity to induce bone resorption and to stimulation
vitamin D synthesis, which is a favorable profile for bone anabolic
agents.
[0012] PTH-(1-30) was initially shown to lack anabolic properties
Whitfield et al. (1996), Calcified Tissue International 53: 81-7.
More recently, however, it has been demonstrated that PTH-(1-30) is
anabolic when administered at very high doses (400-2,000 .mu.g/kg,
vs. 80 .mu.g/kg for PTH-(1-34)). The lower potency of PTH-(1-30)
could be predicted by its lower binding affinity for PTH-1
receptor, its diminished cAMP activation, and/or to its greatly
diminished PKC activation. Takasu (1998). It remains to be
determined whether PTH-(1-30) has a similar or even more desirable
reduction in apparent bone resorption activity.
[0013] PTH-(1-28) is the smallest reported fragment to fully
activate cAMP. Neugebauer et al. (1995), Biochem. 34: 8835-42.
However, hPTH-(1-28) was initially reported to have no osteogenic
effects in OVX rats. Miller et al. (1997), J. Bone Min. Res. 12:
S320 (Abstract). Recently, a very high dose of PTH-(1-28) (1,000
.mu.g/kg/day) was shown to be anabolic in OVX rats, whereas 200
.mu.g/kg/day was ineffective. Whitfield et al. (2000), J. Bone Min.
Res. 15: 964-70. The diminished or absent anabolic effects of some
truncated PTH fragments has been attributed to rapid clearance in
vivo. Rixon et al. (1994).
[0014] Recombinant and modified proteins are an emerging class of
therapeutic agents. Useful modifications of protein therapeutic
agents include combination with the "Fc" domain of an antibody and
linkage to polymers such as polyethylene glycol (PEG) and dextran.
Such modifications are discussed in detail in a patent application
entitled, "Modified Peptides as Therapeutic Agents," U.S. Ser. No.
09/428,082, PCT appl. no. WO 99/25044, which is hereby incorporated
by reference in its entirety.
[0015] A much different approach to development of therapeutic
agents is peptide library screening. The interaction of a protein
ligand with its receptor often takes place at a relatively large
interface. However, as demonstrated for human growth hormone and
its receptor, only a few key residues at the interface contribute
to most of the binding energy. Clackson et al. (1995), Science 267:
383-6. The bulk of the protein ligand merely displays the binding
epitopes in the right topology or serves functions unrelated to
binding. Thus, molecules of only "peptide" length (2 to 40 amino
acids) can bind to the receptor protein of a given large protein
ligand. Such peptides may mimic the bioactivity of the large
protein ligand ("peptide agonists") or, through competitive
binding, inhibit the bioactivity of the large protein ligand
("peptide antagonists").
[0016] Phage display peptide libraries have emerged as a powerful
method in identifying such peptide agonists and antagonists. See,
for example, Scott et al. (1990), Science 249: 386; Devlin et al.
(1990), Science 249: 404; U.S. Pat. No. 5,223,409, issued Jun. 29,
1993; U.S. Pat. No. 5,733,731, issued Mar. 31, 1998; U.S. Pat. No.
5,498,530, issued Mar. 12, 1996; U.S. Pat. No. 5,432,018, issued
Jul. 11, 1995; U.S. Pat. No. 5,338,665, issued Aug. 16, 1994; U.S.
Pat. No. 5,922,545, issued Jul. 13, 1999; WO 96/40987, published
Dec. 19, 1996; and WO 98/15833, published Apr. 16, 1998 (each of
which is incorporated by reference in its entirety). In such
libraries, random peptide sequences are displayed by fusion with
coat proteins of filamentous phage. Typically, the displayed
peptides are affinity-eluted against an antibody-immobilized
extracellular domain of a receptor. The retained phages may be
enriched by successive rounds of affinity purification and
repropagation. The best binding peptides may be sequenced to
identify key residues within one or more structurally related
families of peptides. See, e.g., Cwirla et al. (1997), Science 276:
1696-9, in which two distinct families were identified. The peptide
sequences may also suggest which residues may be safely replaced by
alanine scanning or by mutagenesis at the DNA level. Mutagenesis
libraries may be created and screened to further optimize the
sequence of the best binders. Lowman (1997), Ann. Rev. Biophys.
Biomol. Struct. 26: 401-24.
[0017] Structural analysis of protein-protein interaction may also
be used to suggest peptides that mimic the binding activity of
large protein ligands. In such an analysis, the crystal structure
may suggest the identity and relative orientation of critical
residues of the large protein ligand, from which a peptide may be
designed. See, e.g., Takasaki et al. (1997), Nature Biotech. 15:
1266-70. These analytical methods may also be used to investigate
the interaction between a receptor protein and peptides selected by
phage display, which may suggest further modification of the
peptides to increase binding affinity.
[0018] Other methods compete with phage display in peptide
research. A peptide library can be fused to the carboxyl terminus
of the lac repressor and expressed in E. coli. Another E.
coli-based method allows display on the cell's outer membrane by
fusion with a peptidoglycan-associated lipoprotein (PAL).
Hereinafter, these and related methods are collectively referred to
as "E. coli display." In another method, translation of random RNA
is halted prior to ribosome release, resulting in a library of
polypeptides with their associated RNA still attached. Hereinafter,
this and related methods are collectively referred to as "ribosome
display." Other methods employ peptides linked to RNA; for example,
PROfusion technology, Phylos, Inc. See, for example, Roberts &
Szostak (1997), Proc. Natl. Acad. Sci. USA, 94: 12297-303.
Hereinafter, this and related methods are collectively referred to
as "RNA-peptide screening." Chemically derived peptide libraries
have been developed in which peptides are immobilized on stable,
non-biological materials, such as polyethylene rods or
solvent-permeable resins. Another chemically derived peptide
library uses photolithography to scan peptides immobilized on glass
slides. Hereinafter, these and related methods are collectively
referred to as "chemical-peptide screening." Chemical-peptide
screening may be advantageous in that it allows use of D-amino
acids and other unnatural analogues, as well as non-peptide
elements. Both biological and chemical methods are reviewed in
Wells & Lowman (1992), Curr. Opin. Biotechnol. 3: 355-62.
Conceptually, one may discover peptide mimetics of any protein
using phage display, RNA-peptide screening, and the other methods
mentioned above.
SUMMARY OF THE INVENTION
[0019] The present invention concerns therapeutic agents that
modulate the activity of PTH and PTHrP. In accordance with the
present invention, modulators of PTH and PTHrP comprise:
[0020] a) a PTH/PTHrP modulating domain, preferably the amino acid
sequence of PTH/PTHrP modulating domains of PTH and/or PTHrP, or
sequences derived therefrom by phage display, RNA-peptide
screening, or the other techniques mentioned above; and
[0021] b) a vehicle, such as a polymer (e.g., PEG or dextran) or an
Fc domain, which is preferred;
[0022] wherein the vehicle is covalently attached to the carboxyl
terminus of the PTH/PTHrP modulating domain. The preferred vehicle
is an Fc domain, and the preferred Fc domain is an IgG Fc domain.
Preferred PTH/PTHrP modulating domains comprise the PTH and
PTHrP-derived amino acid sequences described hereinafter. Other
PTH/PTHrP modulating domains can be generated by phage display,
RNA-peptide screening and the other techniques mentioned herein.
Such peptides typically will be antagonists of both PTH and PTHrP,
although such techniques can be used to generate peptide sequences
that serve as selective inhibitors (e.g., inhibitors of PTH but not
PTHrP).
[0023] Further in accordance with the present invention is a
process for making PTH and PTHrP modulators, which comprises:
[0024] a) selecting at least one peptide that binds to the PTH-1 or
PTH-2 receptor; and
[0025] b) covalently linking said peptide to a vehicle.
[0026] The preferred vehicle is an Fc domain. Step (a) is
preferably carried out by selection from the peptide sequences in
Tables 1 and 2 hereinafter or from phage display, RNA-peptide
screening, or the other techniques mentioned herein.
[0027] The compounds of this invention may be prepared by standard
synthetic methods, recombinant DNA techniques, or any other methods
of preparing peptides and fusion proteins. Compounds of this
invention that encompass non-peptide portions may be synthesized by
standard organic chemistry reactions, in addition to standard
peptide chemistry reactions when applicable.
[0028] The primary use contemplated for the compounds of this
invention is as therapeutic or prophylactic agents. The
vehicle-linked peptide may have activity comparable to--or even
greater than--the natural ligand mimicked by the peptide.
[0029] The compounds of this invention may be used for therapeutic
or prophylactic purposes by formulating them with appropriate
pharmaceutical carrier materials and administering an effective
amount to a patient, such as a human (or other mammal) in need
thereof. Other related aspects are also included in the instant
invention.
[0030] Of particular interest in the present invention are
molecules comprising PTH/PTHRP modulating domains having a
shortened PTH C-terminal sequence, such as PTH-(1-28) or (1-34).
The prior art shows no anabolic studies using sustained duration
delivery of such C-terminally truncated PTH fragments. Although the
art does not suggest it, molecules comprising smaller fragments
such as PTH-(1-30)-Fc can be anabolic on their own. Despite their
weak agonism towards PLC (see Background of the Invention),
hPTH-(1-30) is nearly as effective at cAMP stimulation as is
hPTH-(1-34). While not wanting to be constrained by theory, the
inventors note that the anabolic properties of PTH fragments may be
selectively related to their cAMP activation, rather than PLC
activation, so that PTH fragments with reduced receptor affinity
will have a favorable anabolic profile. It is possible that
continuous exposure to truncated PTH fragments would have a
different, and more favorable effect on bone compared to continuous
exposure to PTH-(1-34) or PTH-(1-84) that has been demonstrated in
humans by Fraher et al. (1999).
[0031] Numerous additional aspects and advantages of the present
invention will become apparent upon consideration of the figures
and detailed description of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0032] FIG. 1 shows exemplary Fc dimers that may be derived from an
IgG1 antibody. "Fc" in the figure represents any of the Fc variants
within the meaning of "Fc domain" herein. "X.sup.1" and "X.sup.2"
represent peptides or linker-peptide combinations as defined
hereinafter. The specific dimers are as follows:
[0033] A: Single disulfide-bonded dimers. IgG1 antibodies typically
have two disulfide bonds at the hinge region between the constant
and variable domains. The Fc domain in FIG. 1A may be formed by
truncation between the two disulfide bond sites or by substitution
of a cysteinyl residue with an unreactive residue (e.g., alanyl).
In FIG. 1A, the Fc domain is linked at the C-terminus of the
peptide.
[0034] B: Doubly disulfide-bonded dimers. This Fc domain may be
formed by truncation of the parent antibody to retain both
cysteinyl residues in the Fc domain chains or by expression from a
construct including a sequence encoding such an Fc domain. In FIG.
1B, the Fc domain is linked at the C-terminus of the peptide.
[0035] C: Noncovalent dimers. This Fc domain may be formed by
elimination of the cysteinyl residues by either truncation or
substitution. One may desire to eliminate the cysteinyl residues to
avoid impurities formed by reaction of the cysteinyl residue with
cysteinyl residues of other proteins present in the host cell. The
noncovalent bonding of the Fc domains is sufficient to hold
together the dimer. Other dimers may be formed by using Fc domains
derived from different types of antibodies (e.g., IgG2, IgM).
[0036] FIG. 2 shows the structure of additional compounds of the
invention. FIG. 2A shows a single chain molecule and may also
represent the DNA construct for the molecule. FIG. 2B shows a dimer
in which the linker-peptide portion is present on only one chain of
the dimer. FIG. 2C shows a dimer having the peptide portion on both
chains. The dimer of FIG. 2C will form spontaneously in certain
host cells upon expression of a DNA construct encoding the single
chain as shown in FIG. 3. In other host cells, the cells could be
placed in conditions favoring formation of dimers or the dimers can
be formed in vitro.
[0037] FIG. 3 shows exemplary nucleic acid and amino acid sequences
(SEQ ID NOS: 1 and 2, respectively) of human IgG1 Fc that may be
used in this invention.
[0038] FIG. 4 shows the calcemic response of normal mice to
PTH-(1-34) and to PTH-(1-34)-Fc. Mice were challenged with vehicle
(PBS, --X--), or with PTH-(1-34) (open symbols) or with
PTH-(1-34)-Fc (closed symbols). Doses were 156 nmol/kg (circles),
469 nmol/kg (triangles) or 1,560 nmol/kg (squares). Data represent
group means, n=6 mice/group.
[0039] FIG. 5 shows that [Asn10,Leu11]PTHrP-(7-34)-Fc inhibits the
calcemic response of normal mice to PTHrP. Normal male mice were
injected SC with vehicle (PBS, circles) or with human PTHrP-(1-34)
at 0.5 mg/kg (squares). PTHrP-challenged mice were then immediately
injected SC with [Asn10,Leu11]PTHrP-(7-34)-Fc at 10 mg/kg
(triangles) or 30 mg/kg (diamonds). Data represent group means,
with an n of 6 mice/group.
[0040] FIG. 6 shows the effect of [Asn10,Leu11]PTHrP-(7-34)-Fc on
chronic hypercalcemia induced by PTH-(1-34)-Fc. Normal male mice
were challenged once by SC injection with PTH-(1-34)-Fc (30 mg/kg)
(open circles), or with vehicle (PBS, open squares). Some
PTH-(1-34)-Fc-challenged mice were treated once, at the time of
challenge, with [Asn10,Leu11]PTHrP-(7-34)-Fc at 10 (closed
triangle), 30 (closed circle), or 100 mg/kg (closed square). All
doses of [Asn10,Leu11]PTHrP-(7-34)-Fc caused a significant
suppression of PTH-(1-34)-Fc-mediated hypercalcemia. Data represent
means.+-.SEM, n=5 mice/group.
[0041] FIG. 7 shows cAMP accumulation in ROS 17/2.8 rat
osteoblast-like cells. Cultures were treated with the
phosphodiesterase inhibitor IBMX and then challenged for 15 minutes
with various PTH fragments. cAMP was measured by ELISA.
[0042] FIG. 8 shows the effects of single treatments on clinical
chemistry. Peripheral blood was obtained daily for 3 days following
single subcutaneous injections of the indicated compounds. FIG. 8A
shows total serum calcium; FIG. 8B, alkaline phosphatase (AP), a
marker of osteoblast activity; FIG. 8C, tartrate-resistant acid
phosphatase (TRAP), a marker of osteoclast activity, and FIG. 8D,
AP:TRAP ratio, an index of relative osteoblas: osteoclast
activity.
[0043] FIG. 9 shows the effects of PTH constructs on bone mineral
density. Peripheral quantitative computed tomography (pQCT) was
performed on the proximal tibial metaphysis of mice on day 15,
after injections of PTH constructs on day 0, 5 and 10.
[0044] FIG. 10 shows the effect of twice-weekly PTH-(1-34)-Fc
versus daily PTH-(1-34) on tibial, trabecular, and cortical bone
mineral density (BMD). Daily PTH [PTH-(1-34)] was given at 80
.mu.g/kg/day (20 nmol/kg/day).
[0045] FIG. 11 shows the effects of twice-weekly treatment on BMD
and serum calcium in aged ovariectomized (OVX) rats. Eleven months
after OVX, rats were treated twice per week with phosphate-buffered
saline (PBS, vehicle) or with APD (0.5 mg/kg) or with PTH-(1-34)-Fc
(50 nmol/kg). DEXA was performed weekly. Blood was drawn 24 hours
after the second weekly injection, when the calcemic effects of
PTH-Fc are typically maximal.
[0046] FIG. 12 shows the effect of a single subcutaneous injection
of PTH-(1-34)-Fc into OVX cynomologus monkeys. Monkeys were
injected with PTH-(1-34)-Fc at doses of 1-30 .mu.g/kg (n=1/group)
or 100-1000 .mu.g/kg (n=2/group). Serum was analyzed for total
calcium. The dotted line indicates the threshold for hypercalcemia,
based on an elevation of calcium greater than three standard
deviations above the normal mean, on two or more consecutive
timepoints.
DETAILED DESCRIPTION OF THE INVENTION
[0047] Definition of Terms
[0048] The terms used throughout this specification are defined as
follows, unless otherwise limited in specific instances.
[0049] The term "comprising" means that a compound may include
additional amino acids on either or both of the N-- or C-termini of
the given sequence. Of course, these additional amino acids should
not significantly interfere with the activity of the compound.
[0050] The term "acidic residue" refers to amino acid residues in
D- or L-form having sidechains comprising acidic groups. Exemplary
acidic residues include D and E.
[0051] The term "aromatic residue" refers to amino acid residues in
D- or L-form having sidechains comprising aromatic groups.
Exemplary aromatic residues include F, Y, and W.
[0052] The term "basic residue" refers to amino acid residues in D-
or L-form having sidechains comprising basic groups. Exemplary
basic residues include H, K, and R.
[0053] The terms "hydrophilic residue" and "Haa" refer to amino
acid residues in D- or L-form having sidechains comprising at least
one hydrophilic functional group or polar group. Exemplary
hydrophilic residues include C, D, E, H, K, N, Q, R, S, and T.
[0054] The terms "lipophilic residue" and "Laa" refer to amino acid
residues in D- or L-form having sidechains comprising uncharged,
aliphatic or aromatic groups. Exemplary lipophilic sidechains
include F, I, L, M, V, W, and Y. Alanine (A) is amphiphilic--it is
capable of acting as a hydrophilic or lipophilic residue. Alanine,
therefore, is included within the definition of both "lipophilic
residue" and "hydrophilic residue."
[0055] The term "nonfunctional residue" refers to amino acid
residues in D- or L-form having sidechains that lack acidic, basic,
or aromatic groups. Exemplary nonfunctional amino acid residues
include M, G, A, V, I, L and norleucine (Nle).
[0056] The term "vehicle" refers to a molecule that prevents
degradation and/or increases half-life, reduces toxicity, reduces
immunogenicity, or increases biological activity of a therapeutic
protein. Exemplary vehicles include an Fc domain (which is
preferred) as well as a linear polymer (e.g., polyethylene glycol
(PEG), polylysine, dextran, etc.); a branched-chain polymer (see,
for example, U.S. Pat. No. 4,289,872 to Denkenwalter et al., issued
Sep. 15, 1981; U.S. Pat. No. 5,229,490 to Tam, issued Jul. 20,
1993; WO 93/21259 by Frechet et al., published 28 Oct. 1993); a
lipid; a cholesterol group (such as a steroid); a carbohydrate or
oligosaccharide (e.g., dextran); human serum albumin (HSA) and
related molecules; transtheratin (TTR) and related molecules; or
any natural or synthetic protein, polypeptide or peptide that binds
to a salvage receptor. Vehicles are further described
hereinafter.
[0057] The term "native Fc" refers to molecule or sequence
comprising the sequence of a non-antigen-binding fragment resulting
from digestion of whole antibody, whether in monomeric or
multimeric form. The original immunoglobulin source of the native
Fc is preferably of human origin and may be any of the
immunoglobulins, although IgG1 and IgG2 are preferred. Native Fc's
are made up of monomeric polypeptides that may be linked into
dimeric or multimeric forms by covalent (i.e., disulfide bonds) and
non-covalent association. The number of intermolecular disulfide
bonds between monomeric subunits of native Fc molecules ranges from
1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g.,
IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is a
disulfide-bonded dimer resulting from papain digestion of an IgG
(see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The
term "native Fc" as used herein is generic to the monomeric,
dimeric, and multimeric forms.
[0058] The term "Fc variant" refers to a molecule or sequence that
is modified from a native Fc but still comprises a binding site for
the salvage receptor, FcRn. International applications WO 97/34631
(published 25 Sep. 1997) and WO 96/32478 describe exemplary Fc
variants, as well as interaction with the salvage receptor, and are
hereby incorporated by reference in their entirety. Thus, the term
"Fc variant" comprises a molecule or sequence that is humanized
from a non-human native Fc. Furthermore, a native Fc comprises
sites that may be removed because they provide structural features
or biological activity that are not required for the fusion
molecules of the present invention. Thus, the term "Fc variant"
comprises a molecule or sequence that lacks one or more native Fc
sites or residues that affect or are involved in (1) disulfide bond
formation, (2) incompatibility with a selected host cell (3)
N-terminal heterogeneity upon expression in a selected host cell,
(4) glycosylation, (5) interaction with complement, (6) binding to
an Fc receptor other than a salvage receptor, or (7)
antibody-dependent cellular cytotoxicity (ADCC). Fc variants are
described in further detail hereinafter.
[0059] The term "Fc domain" encompasses native Fc and Fc variant
molecules and sequences as defined above. As with Fc variants and
native Fc's, the term "Fc domain" includes molecules in monomeric
or multimeric form, whether digested from whole antibody or
produced by other means.
[0060] The term "multimer" as applied to Fc domains or molecules
comprising Fc domains refers to molecules having two or more
polypeptide chains associated covalently, noncovalently, or by both
covalent and non-covalent interactions. IgG molecules typically
form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers,
dimers, trimers, or tetramers. Multimers may be formed by
exploiting the sequence and resulting activity of the native Ig
source of the Fc or by derivatizing (as defined below) such a
native Fc.
[0061] The term "dimer" as applied to Fc domains or molecules
comprising Fc domains refers to molecules having two polypeptide
chains associated covalently or non-covalently. Thus, exemplary
dimers within the scope of this invention are as shown in FIGS. 1
and 2.
[0062] The terms "derivatizing" and "derivative" or "derivatized"
comprise processes and resulting compounds respectively in which
(1) the compound has a cyclic portion; for example, cross-linking
between cysteinyl residues within the compound; (2) the compound is
cross-linked or has a cross-linking site; for example, the compound
has a cysteinyl residue and thus forms cross-linked dimers in
culture or in vivo; (3) one or more peptidyl linkage is replaced by
a non-peptidyl linkage; (4) the N-terminus is replaced by
--NRR.sup.1, NRC(O)R.sup.1, --NRC(O)OR.sup.1,
--NRS(O).sub.2R.sup.1, --NHC(O)NHR, a succinimide group, or
substituted or unsubstituted benzyloxycarbonyl-NH--, wherein R and
R.sup.1 and the ring substituents are as defined hereinafter; (5)
the C-terminus is replaced by --C(O)R.sup.2 or --NR.sup.3R.sup.4
wherein R.sup.2, R.sup.3 and R.sup.4 are as defined hereinafter;
and (6) compounds in which individual amino acid moieties are
modified through treatment with agents capable of reacting with
selected side chains or terminal residues. Derivatives are further
described hereinafter.
[0063] The term "peptide" refers to molecules of 1 to 85 amino
acids, with molecules of 5 to 34 amino acids preferred. Exemplary
peptides may comprise the PTH/PTHrP modulating domain of a
naturally occurring molecule or comprise randomized sequences.
[0064] The term "randomized" as used to refer to peptide sequences
refers to fully random sequences (e.g., selected by phage display
methods or RNA-peptide screening) and sequences in which one or
more residues of a naturally occurring molecule is replaced by an
amino acid residue not appearing in that position in the naturally
occurring molecule. Exemplary methods for identifying peptide
sequences include phage display, E. coli display, ribosome display,
RNA-peptide screening, chemical screening, and the like.
[0065] The term "PTH/PTHrP modulating domain" refers to any amino
acid sequence that binds to the PTH-1 receptor and/or the PTH-2
receptor and comprises naturally occurring sequences or randomized
sequences. Exemplary PTH/PTHrP modulating domains can be identified
or derived as described in the references listed for Tables 1 and
2, which are hereby incorporated by reference in their
entirety.
[0066] The term "PTH agonist" refers to a molecule that binds to
PTH-1 or PTH-2 receptor and increases or decreases one or more PTH
activity assay parameters as does full-length native human
parathyroid hormone. An exemplary PTH activity assay is disclosed
in Example 1.
[0067] The term "PTH antagonist" refers to a molecule that binds to
PTH-1 or PTH-2 receptor and blocks or prevents the normal effect on
those parameters by full length native human parathyroid hormone.
An exemplary PTH activity assay is disclosed in Example 2.
[0068] The term "bone resorption inhibitor" refers to such
molecules as determined by the assays of Examples 4 and 11 of WO
97/23614:, which is hereby incorporated by reference in its
entirety. Exemplary bone resorption inhibitors include OPG and
OPG-L antibody, which are described in WO 97/23614 and WO98/46751,
respectively, which are hereby incorporated by reference in their
entirety.
[0069] Additionally, physiologically acceptable salts of the
compounds of this invention are also encompassed herein. By
"physiologically acceptable salts" is meant any salts that are
known or later discovered to be pharmaceutically acceptable. Some
specific examples are: acetate; trifluoroacetate; hydrohalides,
such as hydrochloride and hydrobromide; sulfate; citrate; tartrate;
glycolate; and oxalate.
[0070] Structure of Compounds
[0071] In General. PTH and PTHrP receptor binding amino acid
sequences are described in Tables 1 and 2. Other information on PTH
and PTHrP is found in Mannstadt et al. (1999), Am. J. Physiol. 277.
5Pt 2: F665-75; and Gardella (1996), J. Biol. Chem. 271 (33):
19888-93. Each of these references is hereby incorporated by
reference in its entirety.
[0072] From the foregoing sequences, the present inventors
identified in particular preferred sequences derived from PTH and
PTHrP. These sequences can be randomized through the techniques
mentioned above by which one or more amino acids may be changed
while maintaining or even improving the binding affinity of the
peptide.
[0073] In the compositions of matter prepared in accordance with
this invention, the peptide may be attached to the vehicle through
the peptide's C-terminus. Thus, the vehicle-peptide molecules of
this invention may be described by the following formula I:
P.sup.1-(L.sup.1).sub.a-F.sup.1
[0074] and multimers thereof, wherein:
[0075] F.sup.1 is a vehicle (preferably an Fc domain) and is
attached at the C-terminus of P.sup.1-(L.sup.1).sub.a;
[0076] P.sup.1 is a sequence of a PTH/PTHrP modulating domain;
[0077] L.sup.1 is a linker; and
[0078] a is 0 or 1.
[0079] Peptides. Any number of peptides may be used in conjunction
with the present invention. Peptides may comprise part of the
sequence of naturally occurring proteins, may be randomized
sequences derived from the sequence of the naturally occurring
proteins, or may be wholly randomized sequences. Phage display and
RNA-peptide screening, in particular, are useful in generating
peptides for use in the present invention.
[0080] A PTH/PTHrP modulating domain sequence particularly of
interest is of the formula II
1
X.sup.NX.sup.8HX.sup.10X.sup.11X.sup.12KX.sup.14X.sup.15X.sup.16X-
.sup.17X.sup.18X.sup.19 (SEQ ID NO: 3)
RX.sup.21X.sup.22X.sup.23X.sup.24X.sup.25X.sup.26X.sup.27X.sup.28X.sup.C
[0081] wherein:
[0082] X.sup.N is absent or is X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7,
X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.7,
X.sup.1X.sup.2X.sup.3X.sup.4X- .sup.5X.sup.6X.sup.7, or
YX.sup.1X.sup.2X.sup.3X.sup.4X.sup.5X.sup.6X.sup.- 7;
[0083] X.sup.1 is an amino acid residue (nonfunctional, hydrophilic
or aromatic residue preferred; A, S or Y preferred);
[0084] X.sup.2 is an amino acid residue (nonfunctional residue
preferred, V most preferred);
[0085] X.sup.3 is an amino acid residue (hydrophilic residue
preferred, S most preferred);
[0086] X.sup.4 is an amino acid residue (acidic residue preferred,
E most preferred);
[0087] X.sup.5 is an amino acid residue (nonfunctional or basic
residue preferred, H or I most preferred);
[0088] X.sup.6 is an amino acid residue (acidic or hydrophilic
residue preferred, Q or E most preferred);
[0089] X.sup.7 is an amino acid residue (nonfunctional or aromatic
residue preferred, L or F most preferred);
[0090] X.sup.8 is an amino acid residue (nonfunctional residue
preferred, M or Nle most preferred);
[0091] X.sup.10 is an amino acid residue (an acidic or hydrophilic
residue preferred, N or D most preferred);
[0092] X.sup.11 is an amino acid residue (nonfunctional or basic
residue preferred, L, R, or K most preferred);
[0093] X.sup.12 is an amino acid residue (nonfunctional or aromatic
residue preferred, G, F, or W most preferred);
[0094] X.sup.14 is an amino acid residue (basic or hydrophilic
residue preferred, H or S most preferred);
[0095] X.sup.15 is an amino acid residue (nonfunctional residue
preferred, with L or I most preferred);
[0096] X.sup.16 is an amino acid residue (nonfunctional or
hydrophilic residue preferred, Q, N, S, or A most preferred);
[0097] X.sup.17 is an amino acid residue (acidic, hydrophilic, or
nonfunctional residue preferred; S, D, or L most preferred);
[0098] X.sup.18 is an amino acid residue (nonfunctional residue
preferred, M, L, V or Nle most preferred);
[0099] X.sup.19 is an amino acid residue (acidic or basic residue
preferred, E or R most preferred);
[0100] X.sup.21 is an amino acid residue (nonfunctional residue or
basic residue preferred; V, M, R, or Nle most preferred);
[0101] X.sup.22 is an amino acid residue (hydrophilic, acidic, or
aromatic residue preferred, E or F most preferred);
[0102] X.sup.23 is an aromatic or lipophilic residue (W or F
preferred);
[0103] X.sup.24 is a lipophilic residue (L preferred);
[0104] X.sup.25 is an amino acid residue (hydrophilic or basic
residue preferred, R or H most preferred);
[0105] X.sup.26 is an amino acid residue (hydrophilic or basic
residue preferred, K or H most preferred);
[0106] X.sup.27 is an amino acid residue (lipophilic, basic, or
nonfunctional residue preferred, K or L most preferred);
[0107] X.sup.28 is an amino acid residue (lipophilic or
nonfunctional residue preferred, L or I most preferred);
[0108] X.sup.C is absent or is X.sup.29, X.sup.29X.sup.30,
X.sup.29X.sup.30X.sup.31, X.sup.29X.sup.30X.sup.31X.sup.32,
X.sup.29X.sup.30X.sup.31X.sup.32X.sup.33,
X.sup.29X.sup.30X.sup.31X.sup.3- 2X.sup.33X.sup.34,
X.sup.29X.sup.30X.sup.31X.sup.32X.sup.33X.sup.34X.sup.3- 5, or
X.sup.29X.sup.30X.sup.31X.sup.32X.sup.33X.sup.34X.sup.35X.sup.36;
[0109] X.sup.29 is an amino acid residue (hydrophilic or
nonfunctional residue preferred, Q or A most preferred);
[0110] X.sup.30 is an amino acid residue (hydrophilic or acidic
residue preferred, D or E most preferred);
[0111] X.sup.31 is an amino acid residue (lipophilic or
nonfunctional residue preferred, V or I most preferred);
[0112] X.sup.32 is an amino acid residue (basic residue preferred,
H most preferred);
[0113] X.sup.33 is an amino acid residue (hydrophilic residue
preferred, N or T most preferred);
[0114] X.sup.34 is an amino acid residue (nonfunctional or aromatic
residue preferred, A, F or Y most preferred);
[0115] X.sup.35 is an amino acid residue (acidic residue preferred,
E most preferred); and
[0116] X.sup.36 is an amino acid residue (aromatic residue
preferred, Y most preferred).
[0117] A preferred PTH/PTHrP modulating domain sequence formula is
III
2 J.sup.NJ.sup.7J.sup.8HNLJ.sup.12KHLJ.sup.16SJ.sup.18 (SEQ ID NO:
4) J.sup.19RJ.sup.21EWLRKKLJ.sup.C
[0118] wherein:
[0119] J.sup.N is absent or is selected from
J.sup.1J.sup.2J.sup.3J.sup.4J- .sup.5J.sup.6,
J.sup.2J.sup.3J.sup.4J.sup.5J.sup.6,
J.sup.3J.sup.4J.sup.5J.sup.6;
[0120] J.sup.1 is an amino acid residue (nonfunctional,
hydrophilic, or aromatic residue preferred; A, S or Y most
preferred);
[0121] J.sup.2 is an amino acid residue (nonfunctional residue
preferred, V most preferred);
[0122] J.sup.3 is an amino acid residue (hydrophilic residue
preferred, S most preferred);
[0123] J.sup.4 is an amino acid residue (acidic residue preferred,
E most preferred);
[0124] J.sup.5 is an amino acid residue (nonfunctional residue
preferred, I most preferred);
[0125] J.sup.6 is an amino acid residue (basic residue preferred, Q
preferred);
[0126] J.sup.7 is an amino acid residue (nonfunctional or aromatic
residue preferred, L or F most preferred);
[0127] J.sup.8 is an amino acid residue (nonfunctional residue
preferred, M or Nle most preferred);
[0128] J.sup.12 is an amino acid residue (nonfunctional or aromatic
residue preferred, G or W most preferred);
[0129] J.sup.16 is an amino acid residue (nonfunctional or
hydrophilic residue preferred, N, S, or A most preferred);
[0130] J.sup.18 is an amino acid residue (nonfunctional residue
preferred, M, Nle, L, or V most preferred);
[0131] J.sup.19 is an acidic or basic residue (E or R
preferred);
[0132] J.sup.21 is an amino acid residue (nonfunctional residue
preferred, V, M, or Nle most preferred);
[0133] J.sup.C is absent or is J.sup.29, J.sup.29J.sup.30,
J.sup.29J.sup.30J.sup.31, J.sup.29J.sup.30J.sup.31J.sup.32,
J.sup.29J.sup.30J.sup.31J.sup.32J.sup.33, or
J.sup.29J.sup.30J.sup.31J.su- p.32J.sup.33J.sup.34;
[0134] J.sup.29 is an amino acid residue (hydrophilic or
nonfunctional residue preferred, Q or A most preferred);
[0135] J.sup.30 is an amino acid residue (hydrophilic or acidic
residue preferred, D or E most preferred);
[0136] J.sup.31 is an amino acid residue (lipophilic or
nonfunctional residue preferred, V or I most preferred);
[0137] J.sup.32 is an amino acid residue (basic residue preferred,
H most preferred);
[0138] J.sup.33 is an amino acid residue (acidic residue preferred,
N most preferred);
[0139] J.sup.34 is an amino acid residue (aromatic residue
preferred, F or Y most preferred).
[0140] From the formula of SEQ ID NO: 4, peptides appearing in
Table 1 below are most preferred.
[0141] Another preferred PTH/PTHrP modulating domain sequence is
IV
3 O.sup.NLHO.sup.10O.sup.11O.sup.12KSIO.sup.15 (SEQ ID NO: 5)
O.sup.16LRRRFO.sup.23LHHLIO.sup.C
[0142] wherein:
[0143] O.sup.N is absent or is
YO.sup.1O.sup.2O.sup.3O.sup.4O.sup.5O.sup.6- O.sup.7,
O.sup.1O.sup.2O.sup.3O.sup.4O.sup.5O.sup.6O.sup.7,
O.sup.2O.sup.3O.sup.4O.sup.5O.sup.6O.sup.7,
O.sup.3O.sup.4O.sup.5O.sup.6O- .sup.7,
O.sup.4O.sup.5O.sup.6O.sup.7, O.sup.5.sup.6O.sup.7, O.sup.6O.sup.7,
or O.sup.7;
[0144] O.sup.1 is an amino acid residue (nonfunctional residue
preferred, A most preferred);
[0145] O.sup.2 is an amino acid residue (nonfunctional residue
preferred, V most preferred);
[0146] O.sup.3 is an amino acid residue (hydrophilic residue
preferred, S most preferred);
[0147] O.sup.4 is an amino acid residue (acidic residue preferred,
E most preferred);
[0148] O.sup.5 is an amino acid residue (basic or nonfunctional
residue preferred, H or I preferred);
[0149] O.sup.6 is an amino acid residue (hydrophilic residue
preferred, Q most preferred);
[0150] O.sup.7 is an amino acid residue (nonfunctional residue
preferred, L most preferred);
[0151] O.sup.10 is an amino acid residue (acidic or hydrophilic
residue preferred, N or D most preferred);
[0152] O.sup.11 is an amino acid residue (basic or nonfunctional
residue preferred, K or L most preferred);
[0153] O.sup.12 is an amino acid residue (aromatic or nonfunctional
residue preferred, G, F, or W most preferred);
[0154] O.sup.15 is an amino acid residue (hydrophilic or
nonfunctional residue preferred, I or S most preferred);
[0155] O.sup.16 is an amino acid residue (hydrophilic residue
preferred, Q or N most preferred);
[0156] O.sup.17 is an amino acid residue (acidic or nonfunctional
residue preferred, D or L most preferred);
[0157] O.sup.23 is an amino acid residue (aromatic residue
preferred, with F or W most preferred);
[0158] O.sup.C is absent or is O.sup.29, O.sup.29O.sup.30,
O.sup.29O.sup.30O.sup.31, O.sup.29O.sup.30O.sup.31O.sup.32,
O.sup.29O.sup.30O.sup.31O.sup.32O.sup.33,
O.sup.29O.sup.30O.sup.31O.sup.3- 2O.sup.33O.sup.34,
O.sup.29O.sup.30O.sup.31O.sup.32O.sup.33O.sup.34O.sup.3- 5, or
O.sup.29O.sup.30O.sup.31O.sup.32O.sup.33O.sup.34O.sup.35O.sup.36;
and
[0159] O.sup.29 through O.sup.36 are each independently amino acid
residues.
[0160] From the formula of SEQ ID NO: 5, peptides appearing in
Table 2 below are most preferred.
[0161] Exemplary peptide sequences for this invention appear in
Tables 1 and 2 below. These peptides may be prepared as described
in the cited references or in U.S. Pat. Nos. 4,423,037, 4,968,669,
5,001,22, and 6,051,686, each of which is hereby incorporated by
reference in its entirety. Molecules of this invention
incorporating these peptide sequences may be prepared by methods
known in the art. Single letter amino acid abbreviations are used.
Any of these peptides may be linked in tandem (i.e., sequentially),
with or without linkers. Any peptide containing a cysteinyl residue
may be cross-linked with another Cys-containing peptide, either or
both of which may be linked to a vehicle. Any peptide having more
than one Cys residue may form an intrapeptide disulfide bond, as
well. Any of these peptides may be derivatized as described
hereinafter.
4TABLE 1 PTH/PTHrP modulating domains based on PTH SEQ ID
Description Sequence NO: human PTH(1-84).sup.1
SVSEIQLMHNLGKHLNSMERVE 10 WLRKKLQDVHNFVALGAPLAPR
DAGSQRPRKKEDNVLVESHEKS LGEADKADVNVLTKAKSQ rat PTH(1-84).sup.2
AVSEIQLMHNLGKHLASVERMQ 11 WLRKKLQDVHNFVSLGVQMAAR
EGSYQRPTKKEDNVLVDGNSKS LGEGDKADVDVLVKAKSQ human PTH.sup.3(7-84)
LMHNLGKHLNSMERVEWLRKKL 12 QDVHNFVALGAPLAPRDAGSQR
PRKKEDNVLVESHEKSLGEADK ADVNVLTKAKSQ human PTH(1-44).sup.3
SVSEIQLMHNLGKHLNSMERVE 13 WLRKKLQDVHNFVALGAPLAPR human
PTH(1-38).sup.3 SVSEIQLMHNLGKHLNSMERVE 14 WLRKKLQDVHNFVALG human
PTH(2-38).sup.3 VSEIQLMHNLGKHLNSMERVEW 15 LRKKLQDVHNFVALG human
PTH(1-34).sup.4 SVSEIQLMHNLGKHLNSMERVE 16 WLRKKLQDVHNF [Arg11]
human PTH(1-34) SVSEIQLMHNRGKHLNSMERVE 17 WLRKKLQDVHNF [Lys11]
human PTH(1-34) SVSEIQLMHNKGKHLNSMERVE 18 WLRKKLQDVHNF [Arg19]
human PTH(1-34) SVSEIQLMHNLGKHLNSMRRVE 19 WLRKKLQDVHNF [Tyr1] human
PTH(1-34).sup.3 YVSEIQLMHNLGKHLNSMERVE 20 WLRKKLQDVHNF [Leu(8, 18),
Tyr34] SVSEIQLLHNLGKHLNSLERVE 21 human PTH(1-34).sup.3 WLRKKLQDVHNY
bovine PTH(1-34).sup.5 AVSEIQFMHNLGKHLSSMERVE 22 WLRKKLQDVHNF
[Leu(8, 18), Tyr34] AVSEIQFLHNLGKHLSSLERVE 23 bovine (1-34).sup.6
WLRKKLQDVHNY porcine PTH(1-34).sup.3 SVSEIQLMHNLGKHLSSLERVE 24
WLRKKLQDVHNF rat PTH(1-34).sup.3 AVSEIQLMHNLGKHLASVERMQ 25
WLRKKLQDVHNF [Leu(8, 21), Tyr34] rat AVSEIQLLHNLGKHLASVERLQ 26
PTH(1-34).sup.3 WLRKKLQDVHNY human PTH(1-31).sup.7
SVSEIQLMHNLGKHLNSMERVE 27 WLRKKLQDV [Leu27] human PTH(1-31).sup.8
SVSEIQLMHNLGKHLNSMERVE 28 WLRKLLQDV [Leu(8, 18) Tyr34]
SEIQLLHNLGKHLNSLERVEWL 29 PTH(3-34).sup.9 RKKLQDVHNY bovine
PTH(3-34).sup.10 SEIQFMHNLGKHLSSMERVEWL 30 RKKLQDVHNF [Leu(8, 18),
Tyr34] SEIQFLHNLGKHLSSLERVEWL 31 bovine PTH(3-34).sup.11 RKKLQDVHNY
human PTH(7-34).sup.12 LMHNLGKHLNSMERVEWLRKKL 32 QDVHNF [Leu(8, 18)
Tyr34] LLHNLGKHLNSLERVEWLRKKL 33 human PTH(7-34).sup.9 QDVHNY
bovine PTH(7-34).sup.13 FMHNLGKHLSSMERVEWLRKKL 34 QDVHNF [Tyr34]
bovine FMHNLGKHLSSMERVEWLRKKL 35 PTH(7-34).sup.14 QDVHNY [Leu(8,
18), Tyr34] FLHNLGKHLSSLERVEWLRKKL 36 bovine PTH(7-34).sup.15
QDVHNY [Leu(8, 18), Trp12, FLHNLWKHLSSLERVEWLRKKL 37 Tyr34] bovine
QDVHNY PTH(7-34).sup.16 [D-Trp12, Tyr34] FMHNL-D-Trp-KHLSSMERVE 38
bovine PTH(7-34).sup.17 WLRKKLQDVHNY human PTH(1-30)
SVSEIQLMHNLGKHLNSMERVE 39 WLRKKLQD [Arg11] human PTH(1-30)
SVSEIQLMHNRGKHLNSMERVE 40 WLRKKLQD [Lys11] human PTH(1-30)
SVSEIQLMHNKGKHLNSMERVE 41 WLRKKLQD [Arg19] human PTH(1-30)
SVSEIQLMHNLGKHLNSMRRVE 42 WLRKKLQD [Tyr1] human PTH(1-30)
YVSEIQLMHNLGKHLNSMERVE 43 WLRKKLQD [Leu(8, 18)] human
SVSEIQLLHNLGKHLNSLERVE 44 PTH(1-30) WLRKKLQD bovine PTH(1-30)
AVSEIQFMHNLGKHLSSMERVE 45 WLRKKLQD [Leu(8, 18)] bovine
AVSEIQFLHNLGKHLSSLERVE 46 PTH(1-30) WLRKKLQD porcine PTH(1-30)
SVSEIQLMHNLGKHLSSLERVE 47 WLRKKLQD rat PTH(1-30)
AVSEIQLMHNLGKHLASVERMQ 48 WLRKKLQD [Leu(8, 21), Tyr34] rat
AVSEIQLLHNLGKHLASVERLQ 49 PTH(1-30) WLRKKLQD [Leu27] human
PTH(1-30) SVSEIQLMHNLGKHLNSMERVE 50 WLRKLLQD human PTH(1-29)
SVSEIQLMHNLGKHLNSMERVE 51 WLRKKLQ human PTH(1-28)
SVSEIQLMHNLGKHLNSMERVE 52 WLRKKL [Leu(8, 18)] PTH(3-30)
SEIQLLHNLGKHLNSLERVEWL 53 RKKLQD bovine PTH(3-30)
SEIQFMHNLGKHLSSMERVEWL 54 RKKLQD [Leu(8, 18)] bovine
SEIQFLHNLGKHLSSLERVEWL 55 PTH(3-30) RKKLQD human PTH(7-30)
LMHNLGKHLNSMERVEWLRKKL 56 QD [Leu(8, 18)] human
LLHNLGKHLNSLERVEWLRKKL 57 PTH(7-30) QD bovine PTH(7-30)
FMHNLGKHLSSMERVEWLRKKL 58 QD [Leu(8, 18)] bovine
FLHNLGKHLSSLERVEWLRKKL 59 PTH(7-30) QD [Leu(8, 18), Trp12]
FLHNLWKHLSSLERVEWLRKKL 60 bovine PTH(7-30) QD [D-Trp12] bovine
FMHNL-D-Trp-KHLSSMERVE 61 PTH(7-30) WLRKKLQD .sup.1Hendy et al.
(1981), Proc. Natl. Acad. Sci USA 78: 7365; Kimura et al. (1983),
Biochem. Biophys. Res. Commun. 114: 493; Zanelli et al. (1985),
Endocrinology 117: 1962; Wingender et al. (1985), J. Biol. Chem.
264: 4367. .sup.2Heinrich et al. (1984), J. Biol. Chem. 259: 3320.
.sup.3Bachem Catalogue (1999). .sup.4Doppelt et al. (1981), Calcif.
Tissue Int. 33: 649; Podbesek et al. (1983) Endocrinology 112:
1000; Kent et al. (1985), Clin. Sci. 68: 171; McKee and Caulfield
(1989), Peptide Res. 2: 161; Lee and Russell (1989); Biopolymers
28: 1115; Reeve et al. (1990), Br. Med. J. 301: 314; Neugebauer et
al. (1994), Int. J. Peptide Protein Res. 43: 555. .sup.5Nakamura et
al. (1981); Proc. Soc. Exp. Biol. Med. 168: 168; Law et al. (1983),
J. Clin. Endocrinol. Metab. 56: 1335; Wang et al. (1984), Eur. J.
Pharmacol. 97, 209; Sham et al. (1986), Gen. Comp. Endocrinol. 61:
148; Smith et al. (1987), Arch. Biochem. Biophys. 253: 81.
.sup.6Based on Coltrera. (1981), J. Biol. Chem. 256: 10555;
Bergeron et al. (1981), Endocrinology 109: 1552. .sup.7Jouishomme
et al. (1994), J. Bone Miner. Res. 9: 943; Whitfield and Morley;
TIPS 16: 382. .sup.8Barbier et al. (1997), J. Med. Chem. 40: 1373.
.sup.9Based on Schipani et al. (1993), Endocrinology 132: 2157-65.
.sup.10Scharla et al. (1991), Horm. Metab. Res. 23: 66-9; McGowan
et al. (1983), Science 219: 67; Lowik et al. (1985), Cell Calcium
6: 311. .sup.11Based on Jobert et al. (1997), Endocrinology 138:
5282; Schipani et al. (1993); Rosenblatt et al. (1977), J. Biol.
Chem. 252: 5847; Segre et al. (1979), J. Biol. Chem. 254: 6980;
Nussbaum et al. (1980), J. Biol. Chem. 225: 10183; Gray et al.
(1980), Br. J. Pharmac. 76: 259. .sup.12Nissenson et al. (1999),
Endocrinology 140: 1294-1300. .sup.13Jueppner et al. (1996),
Endocrinology. .sup.14Horiuchi et al. (1983), Science 220: 1053.
.sup.15Schipani et al. (1993); Holick et al. (1995), Bone 16: 140S
(abstract 223, Conference, Melbourne, February 1995). .sup.16Based
on Dresner-Pollak et al. (1996), JBMR 11: 1061-5. .sup.17Goldman et
al. (1988), Endocrinology 123: 2597.
[0162]
5TABLE 2 PTH/PTHrP modulating domains based on PTHrP SEQ ID
Description Sequence NO: human PTHrP(1-86).sup.3 AVSEHQLLHDKGKSIQDL
62 RRRFFLHHLIAEIHTAEI RATSEVSPNSKPSPNTKN HPVRFGSDDEGRYLTQET
NKVETYKEQPLKTP human PTHrP(1-34).sup.18 AVSEHQLLHDKGKSIQDL 63
RRRFFLHHLIAEIHTA [Tyr36] human PTHrP(1-36).sup.3 AVSEHQLLHDKGKSIQDL
64 RRRFFLHHLIAEIHTAEY [Ile5, Trp23, Tyr36] human AVSEIQLLHDKGKSIQDL
65 PTHrP(1-36).sup.3 RRRFWLHHLIAEIHTAEY Tyr-human PTHrP(1-34).sup.3
YAVSEHQLLHDKGKSIQD 66 LRRRFFLHHLIAEIHTA [Asn10, Leu11, D-Phe12]
AVSEHQLLHNL-D-Phe- 67 human PTHrP(1-34).sup.19 KSIQDLRRRFFLHHLIAE
IHTA PTHrP(7-34).sup.20 LLHDKGKSIQDLRRRFFL 68 HHLIAEIHTA [Asn10,
Leu11] human LLHNLGKSIQDLRRRFFL 69 PTHrP(7-34) HHLIAEIHTA [Asn16,
Leu17] LLHDKGKSINLLRRRFFL 70 PTHrP(7-34).sup.21 HHLIAEIHTA [Leu11,
D-Trp12] human LLHDL-D-Trp-KSIQDL 71 PTHrP(7-34).sup.22
RRRFFLHHLIAEIHTA [Asn10, Leu11, D-Trp12] LLHNL-D-Trp-KSIQDL 72
PTHrP(7-34).sup.23 RRRFFLHHLIAEIHTA [D-Trp12] PTHrP(8-34)
LHNL-D-Trp-KSIQDLR 73 RRFFLHHLIAEIHTA [D-Phe12] PTHrP(8-34)
LHNL-D-Phe-KSIQDLR 74 RRFFLHHLIAEIHTA [Asn10, Leu11, D-Trp12]
LLHNL-D-Trp-KSIQDL 75 human PTHrP(7-34).sup.20 RRRFFLHHLIAEIHTA
human PTHrP(1-30) AVSEHQLLHDKGKSIQDL 76 RRRFFLHHLIAE [Ile5, Trp23]
human AVSEIQLLHDKGKSIQDL 77 PTHrP(1-30) RRRFWLHHLIAE Tyr-human
PTHrP(1-30) YAVSEHQLLHDKGKSIQD 78 LRRRFFLHHLIAE [Asn10, Leu11,
D-Phe12] AVSEHQLLHNL-D-Phe- 79 human PTHrP(1-30) KSIQDLRRRFFLHHLIAE
PTHrP(7-30) LLHDKGKSIQDLRRRFFL 80 HHLIAE [Asn10, Leu11] human
LLHNLGKSIQDLRRRFFL 81 PTHrP(7-30) HHLIAE [Asn16, Leu17] PTHrP(7-30)
LLHDKGKSINLLRRRFFL 82 HHLIAE [Leu11, D-Trp12] human
LLHDL-D-Trp-KSIQDL 83 PTHrP(7-30) RRRFFLHHLIAE [Asn10, Leu11,
D-Trp12] LLHNL-D-Trp-KSIQDL 84 PTHrP(7-30) RRRFFLHHLIAE [D-Trp12]
PTHrP(8-30) LHNL-D-Trp-KSIQDLR 85 RRFFLHHLIAE [D-Phe12] PTHrP(8-30)
LHNL-D-Phe-KSIQDLR 86 RRFFLHHLIAE [Asn10, Leu11, D-Trp12]
LLHNL-D-Trp-KSIQDL 87 human PTHrP(7-30) RRRFFLHHLIAE [Haa(Laa Laa
Haa Haa)2 Laa SVSEIQLMHNLGKHLNSM 88 22-31] human PTH(1-34).sup.24
ERVELLEKLLEKLHNF [Haa(Laa Laa Haa Haa)2 Laa SVSEIQLMHNLGKHLNSM 89
22-31] human PTH(1-34).sup.24 ERVELLEKLLKKLHNF [Haa(Laa Laa Haa
Haa)2 Laa SVSEIQLMHNLGKHLNSM 90 22-31] human PTH(1-34).sup.25
ERVALAEALAEALHNF [Haa(Laa Laa Haa Haa)2 Laa SVSEIQLMHNLGKHLNSM 91
22-31] human PTH(1-34).sup.26 ERVSLLSSLLSSLHNF [Haa(Laa Laa Haa
Haa)2 Laa SVSEIQLMHNLGKHLNSM 92 22-31] human PTH(1-34).sup.27
ERVAFYDKVAEKLHNF [Haa(Laa Laa Haa Haa).sub.2 Laa LMHNLGKHLNSMERVELL
93 22-31] human PTH(7-34).sup.24 EKLLEKLHNF [Haa(Laa Laa Haa
Haa).sub.2 Laa LMHNLGKHLNSMERVELL 94 22-31] human PTH(7-34).sup.24
EKLLKKLHNF [Haa(Laa Laa Haa Haa)2 Laa LMHNLGKHLNSMERVALA 95 22-31]
human PTH(7-34).sup.25 EALAEALHNF [Haa(Laa Laa Haa Haa)2 Laa
LMHNLGKHLNSMERVSLL 96 22-31] human PTH(7-34).sup.26 SSLLSSLHNF
[Haa(Laa Laa Haa Haa)2 Laa LMHNLGKHLNSMERVAFY 97 22-31] human
PTH(7-34).sup.27 DKVAEKLHNF [Haa(Laa Laa Haa Haa)2 Laa
AVSEHQLLHDKGKSIQDL 98 22-31] human PTHrP(1-34).sup.24
RRRELLEKLLEKLHTA [Haa(Laa Laa Haa Haa)2 Laa AVSEHQLLHDKGKSIQDL 99
22-31] human PTHrP(1-34).sup.24 RRRELLEKLLKKLHTA [Haa(Laa Laa Haa
Haa)2 Laa AVSEHQLLHDKGKSIQDL 100 22-31] human PTHrP(1-34).sup.25
RRRALAEALAEALHTA [Haa(Laa Laa Haa Haa)2 Laa AVSEHQLLHDKGKSIQDL 101
22-31] human PTHrP(1-34).sup.26 RRRSLLSSLLSSLHTA [Haa(Laa Laa Haa
Haa)2 Laa AVSEHQLLHDKGKSIQDL 102 22-31[ human PTHrP(1-34).sup.27
RRRAFYDKVAEKLHTA [Haa(Laa Laa Haa Haa)2 Laa LLHDKGKSIQDLRRRELL 103
22-31] human PTHrP(7-34).sup.28 EKLLEKLHTA [Haa(Laa Laa Haa Haa)2
Laa LLHDKGKSIQDLRRRELL 104 22-31] human PTHrP(7-34).sup.24
EKLLKKLHTA [Haa(Laa Laa Haa Haa)2 Laa LLHDKGKSIQDLRRRALA 105 22-31]
human PTHrP(7-34).sup.25 EALAEALHTA [Haa(Laa Laa Haa Haa)2 Laa
LLHDKGKSIQDLRRRSLL 106 22-31] human PTHrP(7-34).sup.26 SSLLSSLHTA
[Haa(Laa Laa Haa Haa)2 Laa LLHDKGKSIQDLRRRAFY 107 22-31] human
PTHrP(7-34).sup.27 DKVAEKLHTA [Lys11, Lys13; Arg19, Arg21;
AVSEHQLLHDKGKSIQDL 108 Haa(Laa Laa Haa Haa)2 Laa RRRELLEKLLRKLHTA
22-31] human PTHrP(1-34).sup.29 [Lys11, Lys13; Arg19, Arg21;
AVSEHQLLHDKGKSIQDL 109 Haa(Laa Laa Haa Haa)2 Laa RRRELLEKLLEKLHTS
22--31] human PTHrP(1-34).sup.30 [Lys11, Lys13; Arg19, Arg21;
AVSEHQLLHDKGKSIQDL 110 Haa(Laa Laa Haa Haa)2 Laa RRRELLEKLLEKLHTAGR
22-31] human PTHrP(1-34).sup.31 R [Lys11, Lys13; Arg19, Arg21;
AVSEHQLLHDKGKSIQDL 111 Haa(Laa Laa Haa Haa)2 Laa RRRELLEKLLEKLKEL
22-31] human PTHrP(1-34).sup.32 [Lys11, Lys13, Ala19, Arg21,
AVSEHQLLHDKGKSIQDL 112 Haa(Laa Laa Haa Haa).sub.2 Laa
ARRELLEKLLEKLHTA 22-31] human PTHrP(1-34).sup.33 [Lys11, Lys13,
Arg19, Ala21, AVSEHQLLHDKGKSIQDL 113 Haa(Laa Laa Haa Haa).sub.2 Laa
RRAELLEKLLEKLHTA 22-31] human PTHrP(1-34).sup.34 [Leu11, Lys13,
Arg19, Arg21, AVSEAQLLHDLGKSIQDL 114 Haa(Laa Laa Haa Haa).sub.2 Laa
RRRELLEKLLEKLHAL 22-31] human PTHrP(1-34).sup.35 [Lys11, Lys13,
Arg19, Arg21, AVSEHQLLHDKGKSIQDL 115 Haa(Laa Laa Haa Haa).sub.2 Laa
RRRELLERLLERLHTA 22-31] human PTHrP(1-34).sup.36 [Arg11, Arg13,
Arg19, Arg21, AVSEHQLLHDRGRSIQDR 116 Haa(Laa Laa Haa Haa).sub.2 Laa
RRELLERLLERLHTA 22-31] human PTHrP(1-34).sup.37 [Arg11, Lys13,
Arg19, Arg21, AVSEHQLLHDRGKSIQDL 117 Haa(Laa Laa Haa Haa).sub.2 Laa
RRRELLERLLKRLHTA 22-31] human PTHrP(1-34).sup.38 [Arg11, Arg13,
Arg19, Arg21, AVSEHQLLHDRGRSIQDL 118 Haa(Laa Laa Haa Haa).sub.2 Laa
RRRELLERLLKRLHTA 22-31] human PTHrP(1-34).sup.39 Haa(Laa Laa Haa
Haa).sub.2 Laa AVSEHQLLHDKGKSIQDL 119 22-31] human
PTHrP(1-34).sup.40 RRRALAEALAEALHTA Haa(Laa Laa Haa Haa).sub.2 Laa
AVSEHQLLHDKGKSIQDL 120 22-31] human PTHrP(1-34).sup.41
RRRSLLSSLLSSLHTA Haa(Laa Laa Haa Haa).sub.2 Laa AVSEHQLLHDKGKSIQDL
121 22-31] human PTHrP(1-34).sup.42 RRRAFYDKVAEKLHTA Haa(Laa Laa
Haa Haa).sub.2 Laa AVSEIQFMHNLGKHLSSM 122 22-31] human
PTHrP(1-34).sup.43 ERVELLEKLLEKLHNY Haa(Laa Laa Haa Haa).sub.2 Laa
AVSEIQFMHNLGKHLSSM 123 22-31] human PTHrP(1-34).sup.44
RRRELLEKLLEKLHNY [Haa(Laa Laa Haa Haa).sub.2 Laa SVSEIQLMHNLGKHLNSM
124 22-30] human PTH(1-30) ERVELLEKLLEK [Haa(Laa Laa Haa Haa).sub.2
Laa SVSEIQLMHNLGKHLNSM 125 22-30] human PTH(1-30) ERVELLEKLLKK
[Haa(Laa Laa Haa Haa).sub.2 Laa SVSEIQLMHNLGKHLNSM 126 22-30] human
PTH(1-30) ERVALAEALAEA [Haa(Laa Laa Haa Haa).sub.2 Laa
SVSEIQLMHNLGKHLNSM 127 22-30] human PTH(1-30) ERVSLLSSLLSS [Haa(Laa
Laa Haa Haa).sub.2 Laa SVSEIQLMHNLGKHLNSM 128 22-30] human
PTH(1-34).sup.27 ERVAFYDKVAEKLHNF [Haa(Laa Laa Haa Haa).sub.2 Laa
LMHNLGKHLNSMERVELL 129 22-30] human PTH(7-30) EKLLEK [Haa(Laa Laa
Haa Haa).sub.2 Laa LMHNLGKHLNSMERVELL 130 22-30] human PTH(7-30)
EKLLKK [Haa(Laa Laa Haa Haa).sub.2 Laa LMHNLGKHLNSMERVALA 131
22-30] human PTH(7-30) EALAEA [Haa(Laa Laa Haa Haa).sub.2 Laa
LMHNLGKHLNSMERVSLL 132 22-30] human PTH(7-30) SSLLSS [Haa(Laa Laa
Haa Haa).sub.2 Laa LMHNLGKHLNSMERVAFY 133 22-30] human PTH(7-30)
DKVAEK [Haa(Laa Laa Haa Haa).sub.2 Laa AVSEHQLLHDKGKSIQDL 134
22-30] human PTHrP(1-30) RRRELLEKLLEK [Haa(Laa Laa Haa Haa).sub.2
Laa AVSEHQLLHDKGKSIQDL 135 22-30] human PTHrP(1-30) RRRELLEKLLKK
[Haa(Laa Laa Haa Haa).sub.2 Laa AVSEHQLLHDKGKSIQDL 136 22-30] human
PTHrP(1-30) RRRALAEALAEA [Haa(Laa Laa Haa Haa).sub.2 Laa
AVSEHQLLHDKGKSIQDL 137 22-30] human PTHrP(1-30) RRRSLLSSLLSS
[Haa(Laa Laa Haa Haa).sub.2 Laa AVSEHQLLHDKGKSIQDL 138 22-30] human
PTHrP(1-30) RRRAFYDKVAEK [Haa(Laa Laa Haa Haa).sub.2 Laa
LLHDKGKSIQDLRRRELL 139 22-30] human PTHrP(7-30) EKLLEK [Haa(Laa Laa
Haa Haa).sub.2 Laa LLHDKGKSIQDLRRRELL 140 22-30] human PTHrP(7-30)
EKLLKK [Haa(Laa Laa Haa Haa).sub.2 Laa LLHDKGKSIQDLRRRALA 141
22-30] human PTHrP(7-30) EALAEA [Haa(Laa Laa Haa Haa).sub.2 Laa
LLHDKGKSIQDLRRRSLL 142 22-30] human PTHrP(7-30) SSLLSS [Haa(Laa Laa
Haa Haa).sub.2 Laa LLHDKGKSIQDLRRHAFY 143 22-30] human PTHrP(7-30)
DKVAEK [Lys11, Lys13; Arg19, Arg21; AVSEHQLLHDKGKSIQDL 144 Haa(Laa
Laa Haa Haa).sub.2 Laa RRRELLEKLLRK 22-30] human PTHrP(1-30)
[Lys11, Lys13; Arg19, Arg21; AVSEHQLLHDKGKSIQDL 145 Haa(Laa Laa Haa
Haa).sub.2 Laa RRRELLEKLLEK 22-30] human PTHrP(1-30) ]Lys11, Lys13;
Arg19, Arg21; AVSEHQLLHDKGKSIQDL 146 Haa(Laa Laa Haa Haa).sub.2 Laa
RRRELLEKLLEKLHT 22-30] human PTHrP(1-30) [Lys11, Lys13; Arg19,
Arg21; AVSEHQLLHDKGKSIQDL 147 Haa(Laa Laa Haa Haa).sub.2 Laa
RRRELLEKLLEK 22-30] human PTHrP(1-30) [Lys11, Lys13, Ala19, Arg21,
AVSEHQLLHDKGKSIQDL 148 Haa(Laa Laa Haa Haa).sub.2 Laa ARRELLEKLLEK
22-30] human PTHrP(1-30) [Lys11, Lys13, Arg19, Ala21,
AVSEHQLLHDKGKSIQDL 149 Haa(Laa Laa Haa Haa).sub.2 Laa RRAELLEKLLEK
22-30] human PTHrP(1-30) [Leu11, Lys13, Arg19, Arg21,
AVSEAQLLHDLGKSIQDL 150 Haa(Laa Laa Haa Haa).sub.2 Laa RRRELLEKLLEK
22-30] human PTHrP(1-30) [Lys11, Lys13, Arg19, Arg21,
AVSEHQLLHDKGKSIQDL 151 Haa(Laa Laa Haa Haa).sub.2 Laa RRRELLERLLER
22-30] human PTHrP(1-30) [Arg11, Arg13, Arg19, Arg21,
AVSEHQLLHDRGRSIQDR 152 Haa(Laa Laa Haa Haa).sub.2 Laa RRELLERLLER
22-30] human PTHrP(1-30) [Arg11, Lys13, Arg19, Arg21,
AVSEHQLLHDRGKSIQDL 153 Haa(Laa Laa Haa Haa).sub.2 Laa RRRELLERLLKR
22-30] human PTHrP(1-30) [Arg11, Arg13, Arg19, Arg21,
AVSEHQLLHDRGRSIQDL 154 Haa(Laa Laa Haa Haa).sub.2 Laa RRRELLERLLKR
22-30] human PTHrP(1-30) Haa(Laa Laa Haa Haa).sub.2 Laa
AVSEHQLLHDKGKSIQDL 155 22-30] human PTHrP(1-30) RRREALAEALAEA
Haa(Laa Laa Haa Haa).sub.2 Laa AVSEHQLLHDKGKSIQDL 156 22-30] human
PTHrP(1-30) RRRSLLSSLLSS Haa(Laa Laa Haa Haa).sub.2 Laa
AVSEHQLLHDKGKSIQDL 157 22-30] human PTHrP(1-30) RRRAFYDKVAEK
Haa(Laa Laa Haa Haa).sub.2 Laa AVSEIQFMHNLGKHLSSM 158 22-30] human
PTHrP(1-30) ERVELLEKLLEK Haa(Laa Laa Haa Haa).sub.2 Laa
AVSEIQFMHNLGKHLSSM 159 22-30] human PTHrP(1-30) RRRELLEKLLEK
.sup.18Moseley et al. (1987), Proc. Natl. Acad. Sci. USA 84: 5048;
Suva et al. (1987), Science 237: 893; Kemp et al. (1987), Science
238: 1568; Paspaliaris et al. (1995), Bone 16: 141S (abstract 225,
Conference, Melbourne 1995). .sup.19Based on JP 07316195, May 25,
1994 (Nippon Kayaku). .sup.20Nagasaki et al. (1989), Biochem.
Biophys. Res. Commun. 158: 1036; Nutt et al.; Endocrinology 127,
491 (1990). .sup.21Williams et al. (1998), J. Reproduction &
Fertility 112: 59-67. .sup.22Gardella et al. (1996), Endocrinol.
137: 3936-41; Fukayama et al. (1998), Am. J. Physiol. 274:
E297-E303. .sup.23Li et al. (1996), Endocrinology.
.sup.24Incorporating SEQ ID NO: 26 from U.S. Pat. No. 6,051,686.
.sup.25Incorporating SEQ ID NO: 28 from U.S. Pat. No. 6,051,686.
.sup.26Incorporating SEQ ID NO: 29 from U.S. Pat. No. 6,051,686.
.sup.27Incorporating SEQ ID NO: 30 from U.S. Pat. No. 6,051,686.
.sup.28Incorporating SEQ ID NO: 26 from U.S. Pat. No. 6,051,686
.sup.29Incorporating SEQ ID NO: 5 from U.S. Pat. No. 6,051,686.
.sup.30Based on SEQ ID NOS: 8, 9 from U.S. Pat. No. 6,051,686
.sup.31Incorporating SEQ ID NO: 10 from U.S. Pat. No. 6,051,686
.sup.32Incorporating SEQ ID NO: 11 from U.S. Pat. No. 6,051,686
.sup.33Incorporating SEQ ID NO: 12 from U.S. Pat. No. 6,051,686
.sup.34Incorporating SEQ ID NO: 12 from U.S. Pat. No. 6,051,686
.sup.35Incorporating SEQ ID NO: 14 from U.S. Pat. No. 6,051,686
.sup.36Incorporating SEQ ID NO: 15 from U.S. Pat. No. 6,051,686
.sup.37Incorporating SEQ ID NO: 16 from U.S. Pat. No. 6,051,686
.sup.38Incorporating SEQ ID NO: 17 and 18 from U.S. Pat. No.
6,051,686 .sup.39Incorporating SEQ ID NO: 19 from U.S. Pat. No.
6,051,686 .sup.40Incorporating SEQ ID NO: 20 from U.S. Pat. No.
6,051,686 .sup.41Incorporating SEQ ID NO: 21 from U.S. Pat. No.
6,051,686 .sup.42Incorporating SEQ ID NO: 22 from U.S. Pat. No.
6,051,686 .sup.43Modified from SEQ ID NO: 23 from U.S. Pat. No.
6,051,686 .sup.44Modified from SEQ ID NO: 24 from U.S. Pat. No.
6,051,686
[0163] Another useful PTH/PTHrP modulating domain has the sequence
of the peptide known as TIP39:
6 SLALADDAAFRERARLLAAL (SEQ ID NO: 160) ERRHWLNSYMHKLLVLDAP
[0164] TIP39 is described by Usdin et al. (1999), Nature Neurosci.
2(11): 941-3; Usdin et al. (1996), Endocrinology 137(10): 4285-97;
Usdin et al. (1995), J. Biol. Chem. 270(26): 15455-8; Usdin et al.
(1999), Endocrinol. 140(7): 3363-71.
[0165] Additional useful PTH/PTHrP modulating domain sequences may
result from conservative and/or non-conservative modifications of
the amino acid sequences of SEQ ID NOS: 3, 4, 5, TIP39, or the
sequences listed in Tables 1 and 2.
[0166] Conservative modifications will produce peptides having
functional and chemical characteristics similar to those of the PTH
or PTHrP peptide from which such modifications are made. In
contrast, substantial modifications in the functional and/or
chemical characteristics of the peptides may be accomplished by
selecting substitutions in the amino acid sequence that differ
significantly in their effect on maintaining (a) the structure of
the molecular backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the size
of the molecule.
[0167] For example, a "conservative amino acid substitution" may
involve a substitution of a native amino acid residue with a
nonnative residue such that there is little or no effect on the
polarity or charge of the amino acid residue at that position.
Furthermore, any native residue in the polypeptide may also be
substituted with alanine, as has been previously described for
"alanine scanning mutagenesis" (see, for example, MacLennan et al.,
1998, Acta Physiol. Scand. Suppl. 643:55-67; Sasaki et al., 1998,
Adv. Biophys. 35:1-24, which discuss alanine scanning
mutagenesis).
[0168] Desired amino acid substitutions (whether conservative or
non-conservative) can be determined by those skilled in the art at
the time such substitutions are desired. For example, amino acid
substitutions can be used to identify important residues of the
peptide sequence, or to increase or decrease the affinity of the
peptide or vehicle-peptide molecules (see preceding formulae)
described herein. Exemplary amino acid substitutions are set forth
in Table 3.
7TABLE 3 Amino Acid Substitutions Original Exemplary Preferred
Residues Substitutions Substitutions Ala Val, Leu, Ile Val Arg Lys,
Gln, Asn Lys Asn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn
Glu Asp Asp Gly Pro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu,
Val, Met, Ala, Leu Phe, Norleucine Leu Norleucine, Ile, Val, Ile
Met, Ala, Phe Lys Arg, 1,4 Diamino- Arg butyric Acid, Gln, Asn Met
Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr Leu Pro Ala Gly Ser
Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp, Phe, Thr,
Ser Phe Val Ile, Met, Leu, Phe, Leu Ala, Norleucine
[0169] In certain embodiments, conservative amino acid
substitutions also encompass non-naturally occurring amino acid
residues which are typically incorporated by chemical peptide
synthesis rather than by synthesis in biological systems.
[0170] As noted in the foregoing section "Definition of Terms,"
naturally occurring residues may be divided into classes based on
common sidechain properties that may be useful for modifications of
sequence. For example, non-conservative substitutions may involve
the exchange of a member of one of these classes for a member from
another class. Such substituted residues may be introduced into
regions of the peptide that are homologous with non-human
orthologs, or into the non-homologous regions of the molecule. In
addition, one may also make modifications using P or G for the
purpose of influencing chain orientation.
[0171] In making such modifications, the hydropathic index of amino
acids may be considered. Each amino acid has been assigned a
hydropathic index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0172] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
understood in the art. Kyte et al., J. Mol. Biol., 157: 105-131
(1982). It is known that certain amino acids may be substituted for
other amino acids having a similar hydropathic index or score and
still retain a similar biological activity. In making changes based
upon the hydropathic index, the substitution of amino acids whose
hydropathic indices are within .+-.2 is preferred, those which are
within .+-.1 are particularly preferred, and those within .+-.0.5
are even more particularly preferred.
[0173] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. The greatest local average hydrophilicity of a
protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.,
with a biological property of the protein.
[0174] The following hydrophilicity values have been assigned to
amino acid residues: arginine (+3.0); lysine (+3.0); aspartate
(+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine
(+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline
(-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0);
methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine
(-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
In making changes based upon similar hydrophilicity values, the
substitution of amino acids whose hydrophilicity values are within
.+-.2 is preferred, those which are within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred. One may also identify epitopes from primary amino acid
sequences on the basis of hydrophilicity. These regions are also
referred to as "epitopic core regions."
[0175] A skilled artisan will be able to determine suitable
variants of the polypeptide as set forth in the foregoing sequences
using well known techniques. For identifying suitable areas of the
molecule that may be changed without destroying activity, one
skilled in the art may target areas not believed to be important
for activity. For example, when similar polypeptides with similar
activities from the same species or from other species are known,
one skilled in the art may compare the amino acid sequence of a
peptide to similar peptides. With such a comparison, one can
identify residues and portions of the molecules that are conserved
among similar polypeptides. It will be appreciated that changes in
areas of a peptide that are not conserved relative to such similar
peptides would be less likely to adversely affect the biological
activity and/or structure of the peptide. One skilled in the art
would also know that, even in relatively conserved regions, one may
substitute chemically similar amino acids for the naturally
occurring residues while retaining activity (conservative amino
acid residue substitutions). Therefore, even areas that may be
important for biological activity or for structure may be subject
to conservative amino acid substitutions without destroying the
biological activity or without adversely affecting the peptide
structure.
[0176] Additionally, one skilled in the art can review
structure-function studies identifying residues in similar peptides
that are important for activity or structure. In view of such a
comparison, one can predict the importance of amino acid residues
in a peptide that correspond to amino acid residues that are
important for activity or structure in similar peptides. One
skilled in the art may opt for chemically similar amino acid
substitutions for such predicted important amino acid residues of
the peptides.
[0177] One skilled in the art can also analyze the
three-dimensional structure and amino acid sequence in relation to
that structure in similar polypeptides. In view of that
information, one skilled in the art may predict the alignment of
amino acid residues of a peptide with respect to its three
dimensional structure. One skilled in the art may choose not to
make radical changes to amino acid residues predicted to be on the
surface of the protein, since such residues may be involved in
important interactions with other molecules. Moreover, one skilled
in the art may generate test variants containing a single amino
acid substitution at each desired amino acid residue. The variants
can then be screened using activity assays know to those skilled in
the art. Such data could be used to gather information about
suitable variants. For example, if one discovered that a change to
a particular amino acid residue resulted in destroyed, undesirably
reduced, or unsuitable activity, variants with such a change would
be avoided. In other words, based on information gathered from such
routine experiments, one skilled in the art can readily determine
the amino acids where further substitutions should be avoided
either alone or in combination with other mutations.
[0178] A number of scientific publications have been devoted to the
prediction of secondary structure. See Moult J., Curr. Op. in
Biotech., 7(4): 422-427 (1996), Chou et al., Biochemistry 13(2):
222-245 (1974); Chou et al., Biochemistry, 113(2): 211-222 (1974);
Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol., 47: 45-148
(1978); Chou et al., Ann. Rev. Biochem., 47: 251-276 and Chou et
al., Biophys. J., 26: 367-384 (1979). Moreover, computer programs
are currently available to assist with predicting secondary
structure. One method of predicting secondary structure is based
upon homology modeling. For example, two polypeptides or proteins
which have a sequence identity of greater than 30%, or similarity
greater than 40% often have similar structural topologies. The
recent growth of the protein structural data base (PDB) has
provided enhanced predictability of secondary structure, including
the potential number of folds within a polypeptide's or protein's
structure. See Holm et al., Nucl. Acid. Res., 27(1): 244-247
(1999). It has been suggested (Brenner et al., Curr. Op. Struct.
Biol., 7(3): 369-376 (1997)) that there are a limited number of
folds in a given polypeptide or protein and that once a critical
number of structures have been resolved, structural prediction will
gain dramatically in accuracy.
[0179] Additional methods of predicting secondary structure include
"threading" (Jones, D., Curr. Opin. Struct. Biol., 7(3): 377-87
(1997); Sippl et al., Structure, 4(1): 15-9 (1996)), "profile
analysis" (Bowie et al., Science, 253: 164-170 (1991); Gribskov et
al., Meth. Enzym., 183: 146-159 (1990); Gribskov et al., Proc. Nat.
Acad. Sci., 84(13): 4355-8 (1987)), and "evolutionary linkage" (See
Home, supra, and Brenner, supra).
[0180] Vehicles. This invention requires the presence of at least
one vehicle (F.sup.1) attached to a peptide through the C-terminus
or a sidechain of one of the amino acid residues. Multiple vehicles
may also be used; e.g., an Fc at the C-terminus and a PEG group at
a sidechain.
[0181] An Fc domain is the preferred vehicle. The Fc domain may be
fused to the C terminus of the peptides.
[0182] As noted above, Fc variants are suitable vehicles within the
scope of this invention. A native Fc may be extensively modified to
form an Fc variant in accordance with this invention, provided
binding to the salvage receptor is maintained; see, for example WO
97/34631 and WO 96/32478. In such Fc variants, one may remove one
or more sites of a native Fc that provide structural features or
functional activity not required by the fusion molecules of this
invention. One may remove these sites by, for example, substituting
or deleting residues, inserting residues into the site, or
truncating portions containing the site. The inserted or
substituted residues may also be altered amino acids, such as
peptidomimetics or D-amino acids. Fc variants may be desirable for
a number of reasons, several of which are described below.
Exemplary Fc variants include molecules and sequences in which:
[0183] 1. Sites involved in disulfide bond formation are removed.
Such removal may avoid reaction with other cysteine-containing
proteins present in the host cell used to produce the molecules of
the invention. For this purpose, the cysteine-containing segment at
the N-terminus may be truncated or cysteine residues may be deleted
or substituted with other amino acids (e.g., alanyl, seryl). In
particular, one may truncate the N-terminal 20-amino acid segment
of SEQ ID NO: 2 or delete or substitute the cysteine residues at
positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are
removed, the single chain Fc domains can still form a dimeric Fc
domain that is held together non-covalently.
[0184] 2. A native Fc is modified to make it more compatible with a
selected host cell. For example, one may remove the PA sequence
near the N-terminus of a typical native Fc, which may be recognized
by a digestive enzyme in E. coli such as proline iminopeptidase.
One may also add an N-terminal methionine residue, especially when
the molecule is expressed recombinantly in a bacterial cell such as
E. coli. The Fc domain of SEQ ID NO: 2 is one such Fc variant.
[0185] 3. A portion of the N-terminus of a native Fc is removed to
prevent N-terminal heterogeneity when expressed in a selected host
cell. For this purpose, one may delete any of the first 20 amino
acid residues at the N-terminus, particularly those at positions 1,
2, 3, 4 and 5.
[0186] 4. One or more glycosylation sites are removed. Residues
that are typically glycosylated (e.g., asparagine) may confer
cytolytic response. Such residues may be deleted or substituted
with unglycosylated residues (e.g., alanine).
[0187] 5. Sites involved in interaction with complement, such as
the C1q binding site, are removed. For example, one may delete or
substitute the EKK sequence of human IgG1. Complement recruitment
may not be advantageous for the molecules of this invention and so
may be avoided with such an Fc variant.
[0188] 6. Sites are removed that affect binding to Fc receptors
other than a salvage receptor. A native Fc may have sites for
interaction with certain white blood cells that are not required
for the fusion molecules of the present invention and so may be
removed.
[0189] 7. The ADCC site is removed. ADCC sites are known in the
art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with
regard to ADCC sites in IgG1. These sites, as well, are not
required for the fusion molecules of the present invention and so
may be removed.
[0190] 8. When the native Fc is derived from a non-human antibody,
the native Fc may be humanized. Typically, to humanize a native Fc,
one will substitute selected residues in the non-human native Fc
with residues that are normally found in human native Fc.
Techniques for antibody humanization are well known in the art.
[0191] Preferred Fc variants include the following. In SEQ ID NO: 2
(FIG. 4) the leucine at position 15 may be substituted with
glutamate; the glutamate at position 99, with alanine; and the
lysines at positions 101 and 103, with alanines. In addition, one
or more tyrosine residues can be replaced by phenyalanine
residues.
[0192] An alternative vehicle would be a protein, polypeptide,
peptide, antibody, antibody fragment, or small molecule (e.g., a
peptidomimetic compound) capable of binding to a salvage receptor.
For example, one could use as a vehicle a polypeptide as described
in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al.
Peptides could also be selected by phage display or RNA-peptide
screening for binding to the FcRn salvage receptor. Such salvage
receptor-binding compounds are also included within the meaning of
"vehicle" and are within the scope of this invention. Such vehicles
should be selected for increased half-life (e.g., by avoiding
sequences recognized by proteases) and decreased immunogenicity
(e.g., by favoring non-immunogenic sequences, as discovered in
antibody humanization).
[0193] As noted above, polymer vehicles may also be used for
F.sup.1 and F.sup.2. Various means for attaching chemical moieties
useful as vehicles are currently available, see, e.g., Patent
Cooperation Treaty ("PCT") International Publication No. WO
96/11953, entitled "N-Terminally Chemically Modified Protein
Compositions and Methods," herein incorporated by reference in its
entirety. This PCT publication discloses, among other things, the
selective attachment of water soluble polymers to the N-terminus of
proteins.
[0194] A preferred polymer vehicle is polyethylene glycol (PEG).
The PEG group may be of any convenient molecular weight and may be
linear or branched. The average molecular weight of the PEG will
preferably range from about 2 kiloDalton ("kD") to about 100 kD,
more preferably from about 5 kD to about 50 kD, most preferably
from about 5 kD to about 10 kD. The PEG groups will generally be
attached to the compounds of the invention via acylation or
reductive alkylation through a reactive group on the PEG moiety
(e.g., an aldehyde, amino, thiol, or ester group) to a reactive
group on the inventive compound (e.g., an aldehyde, amino, or ester
group).
[0195] A useful strategy for the PEGylation of synthetic peptides
consists of combining, through forming a conjugate linkage in
solution, a peptide and a PEG moiety, each bearing a special
functionality that is mutually reactive toward the other. The
peptides can be easily prepared with conventional solid phase
synthesis. The peptides are "preactivated" with an appropriate
functional group at a specific site. The precursors are purified
and fully characterized prior to reacting with the PEG moiety.
Ligation of the peptide with PEG usually takes place in aqueous
phase and can be easily monitored by reverse phase analytical HPLC.
The PEGylated peptides can be easily purified by preparative HPLC
and characterized by analytical HPLC, amino acid analysis and laser
desorption mass spectrometry.
[0196] Polysaccharide polymers are another type of water soluble
polymer which may be used for protein modification. Dextrans are
polysaccharide polymers comprised of individual subunits of glucose
predominantly linked by .alpha.1-6 linkages. The dextran itself is
available in many molecular 25 weight ranges, and is readily
available in molecular weights from about 1 kD to about 70 kD.
Dextran is a suitable water soluble polymer for use in the present
invention as a vehicle by itself or in combination with another
vehicle (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309.
The use of dextran conjugated to therapeutic or diagnostic
immunoglobulins has been reported; see, for example, European
Patent Publication No. 0 315 456, which is hereby incorporated by
reference in its entirety. Dextran of about 1 kD to about 20 kD is
preferred when dextran is used as a vehicle in accordance with the
present invention.
[0197] Linkers. Any "linker" group is optional. When present, its
chemical structure is not critical, since it serves primarily as a
spacer. The linker is preferably made up of amino acids linked
together by peptide bonds. Thus, in preferred embodiments, the
linker is made up of from 1 to 20 amino acids linked by peptide
bonds, wherein the amino acids are selected from the 20 naturally
occurring amino acids. Some of these amino acids may be
glycosylated, as is well understood by those in the art. In a more
preferred embodiment, the 1 to 20 amino acids are selected from
glycine, alanine, proline, asparagine, glutamine, and lysine. Even
more preferably, a linker is made up of a majority of amino acids
that are sterically unhindered, such as glycine and alanine. Thus,
preferred linkers are polyglycines (particularly (Gly).sub.4,
(Gly).sub.5), poly(Gly-Ala), and polyalanines. Other specific
examples of linkers are:
8 (Gly).sub.3Lys(Gly).sub.4; (SEQ ID NO: 6)
(Gly).sub.3AsnGlySer(Gly).sub.2; (SEQ ID NO: 7)
(Gly).sub.3Cys(Gly).sub.4; and (SEQ ID NO: 8) GlyProAsnGlyGly. (SEQ
ID NO: 9)
[0198] To explain the above nomenclature, for example,
(Gly).sub.3Lys(Gly).sub.4 means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly.
Combinations of Gly and Ala are also preferred. The linkers shown
here are exemplary; linkers within the scope of this invention may
be much longer and may include other residues.
[0199] Non-peptide linkers are also possible. For example, alkyl
linkers such as --NH--(CH.sub.2).sub.5--C(O)--, wherein s=2-20
could be used. These alkyl linkers may further be substituted by
any non-sterically hindering group such as lower alkyl (e.g.,
C.sub.1-C.sub.6) lower acyl, halogen (e.g., Cl, Br), CN, NH.sub.2,
phenyl, etc. An exemplary non-peptide linker is a PEG linker, VI
1
[0200] wherein n is such that the linker has a molecular weight of
100 to 5000 kD, preferably 100 to 500 kD. The peptide linkers may
be altered to form derivatives in the same manner as described
above.
[0201] Derivatives. The inventors also contemplate derivatizing the
peptide and/or vehicle portion of the compounds. Such derivatives
may improve the solubility, absorption, biological half life, and
the like of the compounds. The moieties may alternatively eliminate
or attenuate any undesirable side-effect of the compounds and the
like. Exemplary derivatives include compounds in which:
[0202] 1. The compound or some portion thereof is cyclic. For
example, the peptide portion may be modified to contain two or more
Cys residues (e.g., in the linker), which could cyclize by
disulfide bond formation.
[0203] 2. The compound is cross-linked or is rendered capable of
cross-linking between molecules. For example, the peptide portion
may be modified 20 to contain one Cys residue and thereby be able
to form an intermolecular disulfide bond with a like molecule. The
compound may also be cross-linked through its C-terminus, as in the
molecule shown below. V 2
[0204] 3. One or more peptidyl [--C(O)NR--] linkages (bonds) is
replaced by a non-peptidyl linkage. Exemplary non-peptidyl linkages
are --CH.sub.2-carbamate [--CH.sub.2--OC(O)NR--], phosphonate,
--CH.sub.2-sulfonamide [--CH.sub.2--S(O).sub.2NR--], urea
[--NHC(O)NH--], --CH.sub.2-secondary amine, and alkylated peptide
[--C(O)NR.sup.6-- wherein R.sup.6 is lower alkyl].
[0205] 4. The N-terminus is derivatized. Typically, the N-terminus
may be acylated or modified to a substituted amine. Exemplary
N-terminal derivative groups include --NRR.sup.1 (other than
--NH.sub.2), --NRC(O)R.sup.1, --NRC(O)OR.sup.1,
--NRS(O).sub.2R.sup.1, --NHC(O)NHR.sup.1, succinimide, or
benzyloxycarbonyl-NH-- (CBZ-NH--), wherein R and R.sup.1 are each
independently hydrogen or lower alkyl and wherein the phenyl ring
may be substituted with 1 to 3 substituents selected from the group
consisting of C.sub.1-C.sub.4 alkyl, C.sub.1-C.sub.4 alkoxy,
chloro, and bromo.
[0206] 5. The free C-terminus is derivatized. Typically, the
C-terminus is esterified or amidated. Exemplary C-terminal
derivative groups include, for example, --C(O)R.sup.2 wherein
R.sup.2 is lower alkoxy or --NR.sup.3R.sup.4 wherein R.sup.3 and
R.sup.4 are independently hydrogen or C.sub.1-C.sub.8 alkyl
(preferably C.sub.1-C.sub.4 alkyl).
[0207] 6. A disulfide bond is replaced with another, preferably
more stable, cross-linking moiety (e.g., an alkylene). See, e.g.,
Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al.
(1993) Thirteenth Am. Pep. Symp., 357-9.
[0208] 7. One or more individual amino acid residues is modified.
Various derivatizing agents are known to react specifically with
selected sidechains or terminal residues, as described in detail
below.
[0209] Lysinyl residues and amino terminal residues may be reacted
with succinic or other carboxylic acid anhydrides, which reverse
the charge of the lysinyl residues. Other suitable reagents for
derivatizing alpha-amino-containing residues include imidoesters
such as methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;
2,4 pentanedione; and transaminase-catalyzed reaction with
glyoxylate.
[0210] Arginyl residues may be modified by reaction with any one or
combination of several conventional reagents, including
phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and
ninhydrin. Derivatization of arginyl residues requires that the
reaction be performed in alkaline conditions because of the high
pKa of the guanidine functional group. Furthermore, these reagents
may react with the groups of lysine as well as the arginine
epsilon-amino group.
[0211] Specific modification of tyrosyl residues has been studied
extensively, with particular interest in introducing spectral
labels into tyrosyl residues by reaction with aromatic diazonium
compounds or tetranitromethane. Most commonly, N-acetylimidizole
and tetranitromethane are used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively.
[0212] Carboxyl sidechain groups (aspartyl or glutamyl) may be
selectively modified by reaction with carbodiimides
(R'--N.dbd.C.dbd.N--R') such as
1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,
aspartyl and glutamyl residues may be converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0213] Glutaminyl and asparaginyl residues may be deamidated to the
corresponding glutamyl and aspartyl residues. Alternatively, these
residues are deamidated under mildly acidic conditions. Either form
of these residues falls within the scope of this invention.
[0214] Cysteinyl residues can be replaced by amino acid residues or
other moieties either to eliminate disulfide bonding or,
conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al.
(1996), J. Med. Chem. 39: 3814-9.
[0215] Derivatization with bifunctional agents is useful for
cross-linking the peptides or their functional derivatives to a
water-insoluble support matrix or to other macromolecular vehicles.
Commonly used cross-linking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides
such as bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and
4,330,440 are employed for protein immobilization.
[0216] Carbohydrate (oligosaccharide) groups may conveniently be
attached to sites that are known to be glycosylation sites in
proteins. Generally, O-linked oligosaccharides are attached to
serine (Ser) or threonine (Thr) residues while N-linked
oligosaccharides are attached to asparagine (Asn) residues when
they are part of the sequence Asn-X-Ser/Thr, where X can be any
amino acid except proline. X is preferably one of the 19 naturally
occurring amino acids other than proline. The structures of
N-linked and O-linked oligosaccharides and the sugar residues found
in each type are different. One type of sugar that is commonly
found on both is N-acetylneuraminic acid (referred to as sialic
acid). Sialic acid is usually the terminal residue of both N-linked
and O-linked oligosaccharides and, by virtue of its negative
charge, may confer acidic properties to the glycosylated compound.
Such site(s) may be incorporated in the linker of the compounds of
this invention and are preferably glycosylated by a cell during
recombinant production of the polypeptide compounds (e.g., in
mammalian cells such as CHO, BHK, COS). However, such sites may
further be glycosylated by synthetic or semi-synthetic procedures
known in the art.
[0217] Other possible modifications include hydroxylation of
proline and lysine, phosphorylation of hydroxyl groups of seryl or
threonyl residues, oxidation of the sulfur atom in Cys, methylation
of the alpha-amino groups of lysine, arginine, and histidine side
chains. Creighton, Proteins: Structure and Molecule Properties (W.
H. Freeman & Co., San Francisco), pp. 79-86 (1983).
[0218] Compounds of the present invention may be changed at the DNA
level, as well. The DNA sequence of any portion of the compound may
be changed to codons more compatible with the chosen host cell. For
E. coli, which is the preferred host cell, optimized codons are
known in the art. Codons may be substituted to eliminate
restriction sites or to include silent restriction sites, which may
aid in processing of the DNA in the selected host cell. The
vehicle, linker and peptide DNA sequences may be modified to
include any of the foregoing sequence changes.
[0219] Methods of Making
[0220] The compounds of this invention largely may be made in
transformed host cells using recombinant DNA techniques. To do so,
a recombinant DNA molecule coding for the peptide is prepared.
Methods of preparing such DNA molecules are well known in the art.
For instance, sequences coding for the peptides could be excised
from DNA using suitable restriction enzymes. Alternatively, the DNA
molecule could be synthesized using chemical synthesis techniques,
such as the phosphoramidate method. Also, a combination of these
techniques could be used.
[0221] The invention also includes a vector capable of expressing
the peptides in an appropriate host. The vector comprises the DNA
molecule that codes for the peptides operatively linked to
appropriate expression control sequences. Methods of effecting this
operative linking, either before or after the DNA molecule is
inserted into the vector, are well known. Expression control
sequences include promoters, activators, enhancers, operators,
ribosomal binding sites, start signals, stop signals, cap signals,
polyadenylation signals, and other signals involved with the
control of transcription or translation.
[0222] The resulting vector having the DNA molecule thereon is used
to transform an appropriate host. This transformation may be
performed using methods well known in the art.
[0223] Any of a large number of available and well-known host cells
may be used in the practice of this invention. The selection of a
particular host is dependent upon a number of factors recognized by
the art. These include, for example, compatibility with the chosen
expression vector, toxicity of the peptides encoded by the DNA
molecule, rate of transformation, ease of recovery of the peptides,
expression characteristics, bio-safety and costs. A balance of
these factors must be struck with the understanding that not all
hosts may be equally effective for the expression of a particular
DNA sequence. Within these general guidelines, useful microbial
hosts include bacteria (such as E. coli sp.), yeast (such as
Saccharomyces sp.) and other fungi, insects, plants, mammalian
(including human) cells in culture, or other hosts known in the
art.
[0224] Next, the transformed host is cultured and purified. Host
cells may be cultured under conventional fermentation conditions so
that the desired compounds are expressed. Such fermentation
conditions are well known in the art. Finally, the peptides are
purified from culture by methods well known in the art.
[0225] The compounds may also be made by synthetic methods. For
example, solid phase synthesis techniques may be used. Suitable
techniques are well known in the art, and include those described
in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis
and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149;
Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young
(1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763;
Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson
et al. (1976), The Proteins (3rd ed.) 2: 257-527. Solid phase
synthesis is the preferred technique of making individual peptides
since it is the most cost-effective method of making small
peptides.
[0226] Compounds that contain derivatized peptides or which contain
non-peptide groups may be synthesized by well-known organic
chemistry techniques.
[0227] Uses of the Compounds
[0228] The compounds of this invention have pharmacologic activity
resulting from their interaction with PTH-1 receptor or PTH-2
receptor. Mannstadt et al. (1999), Am. J. Physiol. 277. 5Pt 2.
F665-75. PTH and agonists thereof increase bone resorption,
increase renal calcium reabsorption, decrease epidermal
proliferation, and decrease hair growth. Holick et al. (1994) Proc.
Natl. Sci. USA 91 (17): 8014-6; Schilli et al. (1997), J. Invest.
Dermatol. 108(6): 928-32. Thus, antagonists of PTH-1 receptor
and/or PTH-2 receptor are useful in treating:
[0229] primary and secondary hyperparathyroidism;
[0230] hypercalcemia, including hypercalcemia resulting from solid
tumors (breast, lung and kidney) and hematologic malignacies
(multiple myeloma, lymphoma and leukemia); idiopathic
hypercalcemia, and hypercalcemia associated with hyperthyroidism
and renal function disorders;
[0231] tumor metastases, particularly metastases to bone, and
particularly related to breast and prostate cancer;
[0232] cachexia and anorexia, particularly as associated with
cancer;
[0233] osteopenia that is related to or aggravated by aberrant PTH
receptor signaling, including various forms of osteoporosis, such
as:
[0234] primary osteoporosis;
[0235] post-menopausal and age-related osteoporosis;
[0236] endocrine osteoporosis (hyperthyroidism,
hyperparathyroidism, Cushing's syndrome, and acromegaly);
[0237] hereditary and congenital forms of osteoporosis (e.g.,
osteogenesis imperfecta, homocystinuria, Menkes' syndrome, and
Riley-Day syndrome);
[0238] osteoporosis due to immobilization of extremities;
[0239] osteoporosis secondary to other disorders, such as
hemochromatosis, hyperprolactinemia, anorexia nervosa,
thyrotoxicosis, diabetes mellitus, celiac disease, inflammatory
bowel disease, primary biliary cirrhosis, rheumatoid arthritis,
ankylosing spondylitis, multiple myeloma, lymphoproliferative
diseases, and systemic mastocytosis;
[0240] osteoporosis secondary to surgery (e.g., gastrectomy) or to
drug therapy, such as chemotherapy, anticonvulsant therapy,
immunosuppressive therapy, and anticoagulant therapy;
[0241] osteoporosis secondary to glucocorticosteroid treatment for
such diseases as rheumatoid arthritis (RA), systemic lupus
erythematosus (SLE), asthma, temporal arteritis, vasculitis,
chronic obstructive pulmonary disease, polymyalgia rheumatica,
polymyositis, chronic interstitial lung disease;
[0242] osteoporosis secondary to glucocorticosteroid and/or
immunomodulatory treatment to prevent organ rejection following
organ transplant such as kidney, liver, lung, heart
transplants;
[0243] osteoporosis due to submission to microgravity, such as
observed during space travel;
[0244] osteoporosis associated with malignant disease, such as
breast cancer, prostate cancer;
[0245] Paget's disease of bone (osteitis deformans) in adults and
juveniles;
[0246] osteomyelitis, or an infectious lesion in bone, leading to
bone loss;
[0247] osteopenia following surgery, induced by steroid
administration, and associated with disorders of the small and
large intestine and with chronic hepatic and renal diseases.
[0248] Osteonecrosis, or bone cell death, associated with traumatic
injury or nontraumatic necrosis associated with Gaucher's disease,
sickle cell anemia, systemic lupus erythematosus, rheumatoid
arthritis, periodontal disease, osteolytic metastasis, and other
conditions;
[0249] alopecia (deficient hair growth or partial or complete hair
loss), including androgenic alopecia (male pattern baldness), toxic
alopecia, alopecia senilis, alopecia areata, alopecia pelada, and
trichotillomania;
[0250] and the like.
[0251] There are other conditions wherein a patient would benefit
from the activity of PTH or PTHrP. For those indications, PTH
receptor agonists are useful as a therapeutic treatment. In
particular, such indications include fracture repair (including
healing of non-union fractures), osteopenia, including various
forms of osteoporosis, such as:
[0252] primary osteoporosis;
[0253] post-menopausal and age-related osteoporosis;
[0254] endocrine osteoporosis (hyperthyroidism, Cushing's syndrome,
and acromegaly);
[0255] hereditary and congenital forms of osteoporosis (e.g.,
osteogenesis imperfecta, homocystinuria, Menkes' syndrome, and
Riley-Day syndrome);
[0256] osteoporosis due to immobilization of extremities;
[0257] osteoporosis secondary to other disorders, such as
hemochromatosis, hyperprolactinemia, anorexia nervosa,
thyrotoxicosis, diabetes mellitus, celiac disease, inflammatory
bowel disease, primary biliary cirrhosis, rheumatoid arthritis,
ankylosing spondylitis, multiple myeloma, lymphoproliferative
diseases, and systemic mastocytosis;
[0258] osteoporosis secondary to surgery (e.g., gastrectomy) or to
drug therapy, such as chemotherapy, anticonvulsant therapy,
immunosuppressive therapy, and anticoagulant therapy;
[0259] osteoporosis secondary to glucocorticosteroid treatment for
diseases such as RA, SLE, asthma, temporal arteritis, vasculitis,
chronic obstructive pulmonary disease, polymyalgia rheumatica,
polymyositis, chronic interstitial lung disease;
[0260] osteoporosis secondary to glucocorticosteroid and/or
immunomodulatory treatment to prevent organ rejection following
organ transplant such as kidney, liver, lung, heart
transplants;
[0261] osteoporosis due to submission to microgravity, such as
observed during space travel;
[0262] osteoporosis associated with malignant disease, such as
breast cancer, prostate cancer;
[0263] PTH agonists with extended half-life (e.g., those linked to
Fc domains) may be used with an inhibitor of bone resorption.
Inhibitors of bone resorption include OPG and OPG derivatives,
OPG-L (RANKL) antibody, calcitonin (e.g., Miacalcin.RTM.,
Calcimar.RTM.), bisphosphonates (e.g., APD, alendronate,
risedronate, etidronate, pamidronate, tiludronate, clodronate,
neridronate, ibandronate, zoledronate), estrogens (e.g.,
Premarin.RTM., Estraderm.RTM., Prempro.RTM., Alora.RTM.,
Climara.RTM., Vivelle.RTM., Estratab.RTM. Ogen.RTM.), selective
estrogen receptor modulators (e.g., raloxifene, droloxifene,
lasofoxifene), tibolone, and the like. Exemplary bone resorption
inhibitors are described in WO98/46751 and WO97/23614, which are
hereby incorporated by reference in their entirety.
[0264] The compounds of this invention may be appropriate as a
monotherapy for the treatment of Osteoporosis, and it is possible
that the addition of an antiresorptive agent to PTH-Fc treatment
will increase both their efficacy and therapeutic window. Both PTH
and PTH-Fc cause an increase in both bone formation and bone
resorption. The ability of antiresorptives to block the osteoclast
response could limit the hypercalcemic effects of PTH-Fc and could
also increase bone mas
[0265] Pharmaceutical Compositions
[0266] In General. The present invention also provides methods of
using pharmaceutical compositions of the inventive compounds. Such
pharmaceutical compositions may be for administration for
injection, or for oral, pulmonary, nasal, transdermal or other
forms of administration. In general, the invention encompasses
pharmaceutical compositions comprising effective amounts of a
compound of the invention together with pharmaceutically acceptable
diluents, preservatives, solubilizers, emulsifiers, adjuvants
and/or carriers. Such compositions include diluents of various
buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic
strength; additives such as detergents and solubilizing agents
(e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic
acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl
alcohol) and bulking substances (e.g., lactose, mannitol);
incorporation of the material into particulate preparations of
polymeric compounds such as polylactic acid, polyglycolic acid,
etc. or into liposomes. Hyaluronic acid may also be used, and this
may have the effect of promoting sustained duration in the
circulation. Such compositions may influence the physical state,
stability, rate of in vivo release, and rate of in vivo clearance
of the present proteins and derivatives. See, e.g., Remington's
Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co.,
Easton, Pa. 18042) pages 1435-1712 which are herein incorporated by
reference in their entirety. The compositions may be prepared in
liquid form, or may be in dried powder, such as lyophilized form.
Implantable sustained release formulations are also contemplated,
as are transdermal formulations.
[0267] Twice weekly dosing of the compounds of this invention is
superior to daily injection of PTH (1-34) for increasing osteoblast
number, bone volume, and bone mineral density in rodents. In adult
mice, twice weekly dosing with PTH-(1-34)-Fc caused greater
increases in bone density and bone volume compared to daily
PTH-(1-34). (See FIG. 10.) In an aged OVX rat model of
osteoporosis, twice weekly dosing was able to reverse more than 50%
of the bone loss induced by one year of estrogen ablation. The
effect seen in the aged rat model was even greater when combined
with a bisphosphonate (APD). In rats, a single SC injection of
PTH-(1-34)-Fc (340 nmol/kg) caused a hypercalcemic response which
persisted for 72 hours (FIG. 8). This duration is concordant with
the rate of clearance of PTH-(1-34)-Fc from the serum, and is
consistent with an optimal twice-weekly dosing regimen in rats.
[0268] The optimal dosing of primates may be less frequent compared
to rats or mice. Weekly (or less frequent) dosing may be optimal in
primates, based on the observation that the hypercalcemic response
of OVX cynomolgus monkeys to a single subcutaneous injection of
PTH-(1-34)-Fc (10-34 nmol/kg) persisted for about 168 hours (FIG.
11). This observation suggests that a single subcutaneous dose of
PTH-(1-34)-Fc in primates is cleared within about 1 week, which
could also represent the maximum dosing frequency required for
anabolic effects.
[0269] Oral dosage forms. Contemplated for use herein are oral
solid dosage forms, which are described generally in Chapter 89 of
Remington's Pharmaceutical Sciences (1990), 18th Ed., Mack
Publishing Co. Easton Pa. 18042, which is herein incorporated by
reference in its entirety. Solid dosage forms include tablets,
capsules, pills, troches or lozenges, cachets or pellets. Also,
liposomal or proteinoid encapsulation may be used to formulate the
present compositions (as, for example, proteinoid microspheres
reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation may
be used and the liposomes may be derivatized with various polymers
(e.g., U.S. Pat. No. 5,013,556). A description of possible solid
dosage forms for the therapeutic is given in Chapter 10 of
Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker
and C. T. Rhodes, herein incorporated by reference in its entirety.
In general, the formulation will include the inventive compound,
and inert ingredients which allow for protection against the
stomach environment, and release of the biologically active
material in the intestine.
[0270] Also specifically contemplated are oral dosage forms of the
above inventive compounds. If necessary, the compounds may be
chemically modified so that oral delivery is efficacious.
Generally, the chemical modification contemplated is the attachment
of at least one moiety to the compound molecule itself, where said
moiety permits (a) inhibition of proteolysis; and (b) uptake into
the blood stream from the stomach or intestine. Also desired is the
increase in overall stability of the compound and increase in
circulation time in the body. Moieties useful as covalently
attached vehicles in this invention may also be used for this
purpose. Examples of such moieties include: PEG, copolymers of
ethylene glycol and propylene glycol, carboxymethyl cellulose,
dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
See, for example, Abuchowski and Davis, Soluble Polymer-Enzyme
Adducts Enzymes as Drugs (1981), Hocenberg and Roberts, eds.,
Wiley-Interscience, New York, N.Y., pp. 367-83; Newmark, et al.
(1982), J. Appl. Biochem. 4:185-9. Other polymers that could be
used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for
pharmaceutical usage, as indicated above, are PEG moieties.
[0271] For oral delivery dosage forms, it is also possible to use a
salt of a modified aliphatic amino acid, such as sodium
N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to
enhance absorption of the therapeutic compounds of this invention.
The clinical efficacy of a heparin formulation using SNAC has been
demonstrated in a Phase II trial conducted by Emisphere
Technologies. See U.S. Pat. No. 5,792,451, "Oral drug delivery
composition and methods".
[0272] The compounds of this invention can be included in the
formulation as fine multiparticulates in the form of granules or
pellets of particle size about 1 mm. The formulation of the
material for capsule administration could also be as a powder,
lightly compressed plugs or even as tablets. The therapeutic could
be prepared by compression.
[0273] Colorants and flavoring agents may all be included. For
example, the protein (or derivative) may be formulated (such as by
liposome or microsphere encapsulation) and then further contained
within an edible product, such as a refrigerated beverage
containing colorants and flavoring agents.
[0274] One may dilute or increase the volume of the compound of the
invention with an inert material. These diluents could include
carbohydrates, especially mannitol, .alpha.-lactose, anhydrous
lactose, cellulose, sucrose, modified dextrans and starch. Certain
inorganic salts may also be used as fillers including calcium
triphosphate, magnesium carbonate and sodium chloride. Some
commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500,
Emcompress and Avicell.
[0275] Disintegrants may be included in the formulation of the
therapeutic into a solid dosage form. Materials used as
disintegrants include but are not limited to starch including the
commercial disintegrant based on starch, Explotab. Sodium starch
glycolate, Amberlite, sodium carboxymethylcellulose,
ultramylopectin, sodium alginate, gelatin, orange peel, acid
carboxymethyl cellulose, natural sponge and bentonite may all be
used. Another form of the disintegrants are the insoluble cationic
exchange resins. Powdered gums may be used as disintegrants and as
binders and these can include powdered gums such as agar, Karaya or
tragacanth. Alginic acid and its sodium salt are also useful as
disintegrants.
[0276] Binders may be used to hold the therapeutic agent together
to form a hard tablet and include materials from natural products
such as acacia, tragacanth, starch and gelatin. Others include
methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl
cellulose (CMC). Polyvinyl pyrrolidone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in
alcoholic solutions to granulate the therapeutic.
[0277] An antifrictional agent may be included in the formulation
of the therapeutic to prevent sticking during the formulation
process. Lubricants may be used as a layer between the therapeutic
and the die wall, and these can include but are not limited to;
stearic acid including its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and
waxes. Soluble lubricants may also be used such as sodium lauryl
sulfate, magnesium lauryl sulfate, polyethylene glycol of various
molecular weights, Carbowax 4000 and 6000.
[0278] Glidants that might improve the flow properties of the drug
during formulation and to aid rearrangement during compression
might be added. The glidants may include starch, talc, pyrogenic
silica and hydrated silicoaluminate.
[0279] To aid dissolution of the compound of this invention into
the aqueous environment a surfactant might be added as a wetting
agent. Surfactants may include anionic detergents such as sodium
lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium
sulfonate. Cationic detergents might be used and could include
benzalkonium chloride or benzethonium chloride. The list of
potential nonionic detergents that could be included in the
formulation as surfactants are lauromacrogol 400, polyoxyl 40
stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60,
glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty
acid ester, methyl cellulose and carboxymethyl cellulose. These
surfactants could be present in the formulation of the protein or
derivative either alone or as a mixture in different ratios.
[0280] Additives may also be included in the formulation to enhance
uptake of the compound. Additives potentially having this property
are for instance the fatty acids oleic acid, linoleic acid and
linolenic acid.
[0281] Controlled release formulation may be desirable. The
compound of this invention could be incorporated into an inert
matrix which permits release by either diffusion or leaching
mechanisms e.g., gums. Slowly degenerating matrices may also be
incorporated into the formulation, e.g., alginates,
polysaccharides. Another form of a controlled release of the
compounds of this invention is by a method based on the Oros
therapeutic system (Alza Corp.), i.e., the drug is enclosed in a
semipermeable membrane which allows water to enter and push drug
out through a single small opening due to osmotic effects. Some
enteric coatings also have a delayed release effect.
[0282] Other coatings may be used for the formulation. These
include a variety of sugars which could be applied in a coating
pan. The therapeutic agent could also be given in a film coated
tablet and the materials used in this instance are divided into 2
groups. The first are the nonenteric materials and include methyl
cellulose, ethyl cellulose, hydroxyethyl cellulose,
methylhydroxy-ethyl cellulose, hydroxypropyl cellulose,
hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,
providone and the polyethylene glycols. The second group consists
of the enteric materials that are commonly esters of phthalic
acid.
[0283] A mix of materials might be used to provide the optimum film
coating. Film coating may be carried out in a pan coater or in a
fluidized bed or by compression coating.
[0284] Pulmonary delivery forms. Also contemplated herein is
pulmonary delivery of the present protein (or derivatives thereof).
The protein (or derivative) is delivered to the lungs of a mammal
while inhaling and traverses across the lung epithelial lining to
the blood stream. (Other reports of this include Adjei et al.,
Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J.
Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet et al.
(1989), J. Cardiovasc. Pharmacol. 13 (suppl.5): s.143-146
(endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12
(.alpha.1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84:
1145-6 (.alpha.1-proteinase); Oswein et al. (March 1990),
"Aerosolization of Proteins", Proc. Symp. Resp. Drug Delivery II,
Keystone, Colorado (recombinant human growth hormone); Debs et al.
(1988), J. Immunol. 140: 3482-8 (interferon-.gamma. and tumor
necrosis factor .alpha.) and Platz et al., U.S. Pat. No. 5,284,656
(granulocyte colony stimulating factor).
[0285] Contemplated for use in the practice of this invention are a
wide range of mechanical devices designed for pulmonary delivery of
therapeutic products, including but not limited to nebulizers,
metered dose inhalers, and powder inhalers, all of which are
familiar to those skilled in the art. Some specific examples of
commercially available devices suitable for the practice of this
invention are the Ultravent nebulizer, manufactured by
Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer,
manufactured by Marquest Medical Products, Englewood, Colo.; the
Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research
Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured
by Fisons Corp., Bedford, Mass.
[0286] All such devices require the use of formulations suitable
for the dispensing of the inventive compound. Typically, each
formulation is specific to the type of device employed and may
involve the use of an appropriate propellant material, in addition
to diluents, adjuvants and/or carriers useful in therapy.
[0287] The inventive compound should most advantageously be
prepared in particulate form with an average particle size of less
than 10 .mu.m (or microns), most preferably 0.5 to 5 .mu.m, for
most effective delivery to the distal lung.
[0288] Pharmaceutically acceptable carriers include carbohydrates
such as trehalose, mannitol, xylitol, sucrose, lactose, and
sorbitol. Other ingredients for use in formulations may include
DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be
used. PEG may be used (even apart from its use in derivatizing the
protein or analog). Dextrans, such as cyclodextran, may be used.
Bile salts and other related enhancers may be used. Cellulose and
cellulose derivatives may be used. Amino acids may be used, such as
use in a buffer formulation.
[0289] Also, the use of liposomes, microcapsules or microspheres,
inclusion complexes, or other types of carriers is
contemplated.
[0290] Formulations suitable for use with a nebulizer, either jet
or ultrasonic, will typically comprise the inventive compound
dissolved in water at a concentration of about 0.1 to 25 mg of
biologically active protein per mL of solution. The formulation may
also include a buffer and a simple sugar (e.g., for protein
stabilization and regulation of osmotic pressure). The nebulizer
formulation may also contain a surfactant, to reduce or prevent
surface induced aggregation of the protein caused by atomization of
the solution in forming the aerosol.
[0291] Formulations for use with a metered-dose inhaler device will
generally comprise a finely divided powder containing the inventive
compound suspended in a propellant with the aid of a surfactant.
The propellant may be any conventional material employed for this
purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a
hydrofluorocarbon, or a hydrocarbon, including
trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or
combinations thereof. Suitable surfactants include sorbitan
trioleate and soya lecithin. Oleic acid may also be useful as a
surfactant.
[0292] Formulations for dispensing from a powder inhaler device
will comprise a finely divided dry powder containing the inventive
compound and may also include a bulking agent, such as lactose,
sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which
facilitate dispersal of the powder from the device, e.g., 50 to 90%
by weight of the formulation.
[0293] Nasal delivery forms. Nasal delivery of the inventive
compound is also contemplated. Nasal delivery allows the passage of
the protein to the blood stream directly after administering the
therapeutic product to the nose, without the necessity for
deposition of the product in the lung. Formulations for nasal
delivery include those with dextran or cyclodextran. Delivery via
transport across other mucous membranes is also contemplated.
[0294] Dosages. The dosage regimen involved in a method for
treating the above-described conditions will be determined by the
attending physician, considering various factors which modify the
action of drugs, e.g. the age, condition, body weight, sex and diet
of the patient, the severity of any infection, time of
administration and other clinical factors. Generally, the daily
regimen should be in the range of 0.1-1000 micrograms of the
inventive compound per kilogram of body weight, preferably 0.1-150
micrograms per kilogram.
[0295] Specific Preferred Embodiments
[0296] The inventors have determined preferred structures for the
preferred peptides listed in Table 4 below. The symbol "A" may be
any of the linkers described herein or may simply represent a
normal peptide bond (i.e., so that no linker is present). Tandem
repeats and linkers are shown separated by dashes for clarity.
9TABLE 4 Preferred embodiments Peptide SEQ Sequence/structure
description ID NO: SVSEIQLMHNLGKHLNSMERVEWL PTH(1-34) 161
RKKLQDVHNF-.LAMBDA.-F.sup.1 SVSEIQLMHNRGKHLNSMERVEWL (L11R) 162
RKKLQDVHNF-.LAMBDA.-F.sup.1 PTH(1-34) SVSEIQLMHNKGKHLNSMERVEWL
(L11K) 163 RKKLQDVHNF-.LAMBDA.-F.sup.1 PTH(1-34)
SVSEIQLMHNLGKHLNSMRRVEWL (E19R) 164 RKKLQDVHNF-.LAMBDA.-F.sup.1
PTH(1-34) SVSEIQLMHNLGKHLNSMERVEWL PTH(1-31) 165
RKKLQDV-.LAMBDA.-F.sup.1 SVSEIQLMHNLGKHLNSMERVEWL PTH(1-30) 166
RKKLQD-.LAMBDA.-F.sup.1 F.sup.1-.LAMBDA.-SVSEIQLMHNLGKHLNSME
PTH(1-29) 167 RVEWLRKKLQ F.sup.1-.LAMBDA.-SVSEIQLMHNLGKHLNSME
PTH(1-28) 168 RVEWLRKKL LLHNLGKSIQDLRRRFFLHHLIAE (D10N, K11L) 169
IHTA-.LAMBDA.-F.sup.1 PTHrP(7-34) SLALADDAAFRERARLLAALERRH TIP39
170 WLNSYMHKLLVLDAP-.LAMBDA.-F.sup.1
[0297] "F.sup.1" is an Fc domain as defined previously herein. In
addition to those listed in Table 4, the inventors further
contemplate heterodimers in which each strand of an Fc dimer is
linked to a different peptide sequence; for example, a molecule in
which one strand can be described by SEQ ID NO: 166, the other by
SEQ ID NO: 170 or an Fc linked with any of the sequences in Tables
1 and 2.
[0298] All of the compounds of this invention can be prepared by
methods described in PCT appl. no. WO 99/25044.
[0299] The invention will now be further described by the following
working examples, which are illustrative rather than limiting.
EXAMPLE 1
Bioactivity of an Fc-conjugated PTH/PTHrP Receptor (PTH-R1) Agonist
[PTH-(1-34)-Fc]
[0300] Introduction
[0301] Parathyroid hormone [PTH-(1-34) or native PTH-(1-84)] causes
increased bone formation and increased bone mass when injected
daily. This anabolic response was previously thought to require
brief exposure to PTH, which is facilitated by the short half-life
(less than 1 h) of PTH. Clinically, the anabolic effect of PTH
therapy requires daily SC injection, which is a significant barrier
to the widespread use of PTH. Less frequent injections of PTH would
be clinically desirable and could be achieved by increasing the in
vivo half-life of PTH. Short-term (intermittent) exposure to PTH
(<1 h/day) stimulates osteoblastic bone formation, while
long-term (continuous) exposure (>2 h/day) stimulates
osteoclastic bone resorption (Dobnig et al, Endocrinology 138:
4607, 1998). The art suggests that PTH with an extended half-life
on its own may increase bone resorption and lead to hypercalcemia.
However, it should be possible to prevent PTH-induced osteoclast
activity with bone resorption inhibitors. Osteoprotegerin (OPG) may
be well suited for this purpose. A single treatment of rats, mice
or humans with OPG-Fc causes sustained inhibition of bone
resorption, by essentially eradicating the osteoclast population.
Co-administration of a potent bone resorption inhibitor, like OPG,
may provide greater effect. This regimen would theoretically permit
the unopposed stimulation of bone formation by PTH, leading to
increased bone mass. It is likely that other bone resorption
inhibitors, including bisphosphonates or estrogen, would also
inhibit PTH-induced bone resorption and could therefore be used in
combination with a long-acting PTH molecule. Towards this goal, we
have cloned, expressed and purified human PTH-(1-34)-Fc. Fc
conjugation of proteins causes a significant increase in their
circulating half life, which may permit injections of PTH-(1-34)-Fc
on a schedule similar to or identical to that of OPG-Fc. The
benefits of this invention include less frequent injections of PTH,
from the current standard of once per day to as infrequently as
once per quarter.
[0302] Materials, Methods, and Results
[0303] Hypercalcemia Assay
[0304] We tested the potency and duration of effect of
PTH-(1-34)-Fc in a murine hypercalcemia model. Briefly, mice were
injected once SC with varying doses of PTH-(1-34) or PTH-(1-34)-Fc,
and peripheral blood was collected from the retroorbital sinus for
determination of blood ionized calcium. The half-life and the
potency of PTH-(1-34)-Fc was greater than that of PTH-(1-34), as
evidenced by the sustained hypercalcemic response of mice to the
former agent (FIG. 4). Hypercalcemia induced by PTH-(1-34)
persisted for 6-24 h, while equimolar doses of PTH-(1-34)-Fc caused
more sustained hypercalcemia (48-72 h). This duration of response
is consistent with greater half-life of the PTH-(1-34)-Fc construct
vs. PTH-(1-34). The potency of PTH-(1-34)-Fc was also significantly
greater than that of PTH-(1-34) (FIG. 4). The highest dose of
PTH-(1-34)-Fc caused a greater increase in peak ionized calcium
levels compared with an equimolar dose of PTH-(1-34). Analysis of
the area under the curve (AUC) demonstrated that at the highest
dose employed, PTH-(1-34)-Fc caused a 2.6-fold greater
hypercalcemic response than did equimolar doses of PTH-(1-34).
[0305] Anabolic Assay
[0306] Having demonstrated the superior pharmacology and half-life
of PTH-(1-34)-Fc over PTH-(1-34), we conducted a pilot study to
determine whether PTH-(1-34)-Fc co-treatment with OPG-Fc would
increase bone mass. Briefly, 6-month-old male Sprague Dawley (SD)
rats were divided into groups of 6. Baseline bone mineral density
(BMD) was determined in the third lumbar vertebra (L3) of all rats
by dual-energy X-ray absorptiometry (DEXA) (Day 0). Rats were then
treated according to the following schedule:
[0307] Group 1: Vehicle controls (PBS, injected SC, Days 1, 3, and
5)
[0308] Group 2: OPG-Fc, single SC injection (1 mg/kg) on Day 1
[0309] Group 3: PTH-(1-34), SC injections on Days 1, 3, and 5, at
20 nMoles PTH/kg/injection. This represents an optimal anabolic PTH
regimen.
[0310] Group 4: Same as group 3, but with a single OPG-Fc injection
on Day 1.
[0311] Group 5: Single SC injection of PTH-(1-34)-Fc at 60
nMoles/kg, on Day 1. This represents a molar dose which is
equivalent to the total dose of PTH-(1-34) received by group 3, but
in a single injection.
[0312] Group 6: Same as group 5, but with a single OPG-Fc injection
(SC, 1 mg/kg) on Day 1.
[0313] DEXA of the lumbar spine was performed again on Day 7 to
evaluate changes in BMD. BMD in L3 increased modestly with a single
injection of OPG-Fc, or with 3 injections of PTH-(1-34), compared
to PBS-treated rats (FIG. 5). PTH-(1-34)+OPG caused a greater
increase in BMD than either OPG or PTH-(1-34) alone. As a
monotherapy, a single injection of PTH-(1-34)-Fc failed to increase
BMD. However, a single injection of PTH-(1-34)-Fc plus a single
injection of OPG-Fc caused a significant increase in BMD (FIG. 5).
This result provides proof of principle that a PTH construct with
extended circulating half life can be combined with a potent
antiresorptive, like OPG-Fc, to create an anabolic skeletal
response. The anabolic effect of a single treatment with
PTH-(1-34)-Fc plus OPG-Fc was greater than that induced by multiple
injections of PTH-(1-34), with or without OPG-Fc co-treatment. In
conclusion, maximal gains in BMD can be achieved with infrequent
injections of PTH-(1-34)-Fc+OPG-Fc, which is a superior treatment
regimen compared to PTH-(1-34), which must be injected daily or
every second day.
[0314] FIG. 5 shows the effect of PTH-Fc +OPG-Fc on bone mineral
density (BMD) in the third lumbar vertebra (L3). Normal 6 month old
male rats were treated with PTH-Fc or PTH or vehicle by a single SC
injection. Some rats also received a single SC injection of OPG.
BMD was determined 7 days later by DEXA. Data represent
means.+-.SD, n=6 rats/group.
EXAMPLE 2
Bioactivity of an Fc-conjugated PTH/PTHrP Receptor (PTH-R1)
Antagonist ([Asn10,Leu11]PTHrP-(7-34)-Fc)
[0315] Introduction
[0316] Several disease states are associated with increased
circulating levels of PTH or PTHrP. Primary and secondary
hyperparathyroidism (PHPT and SHPT, respectively), are associated
with increased PTH levels, while humoral hypercalcemia of
malignancy (HHM) results in elevated PTHrP levels. Both proteins
signal through the common PTH/PTHrP receptor (PTH-R1) to cause
increases in bone resorption, renal calcium reabsorption, and renal
biosynthesis of vitamin D. While bone resorption inhibitors have
variable success in inhibiting osteoclastic bone resorption in
these disease states, no therapy currently mitigates the intestinal
and renal influence of PTH or PTHrP excess on calcemia. Agents
which directly antagonize PTH or PTHrP signaling are therefore
likely to have greater efficacy compared to resorption
inhibitors.
[0317] The most studied antagonists of PTH-R1 signaling are based
on amino terminal truncations. PTH-(7-34) peptides are fairly
effective PTH-R1 antagonists with very mild agonist activity.
Compared to PTH-(7-34), PTHrP-(7-34) peptide has greater affinity
for PTH-R1 and as such is a more potent antagonist. However,
PTHrP-(7-34) also has greater (but still mild) agonist activity
compared to PTH-(7-34) (McKee (1990), Endocrinol. 127: 76). The
optimal antagonist may combine the weaker agonism of PTH-(7-34)
with the stronger antagonism of PTHrP-(7-34). Nutt et al (1990),
Endocrinol. 127: 491, demonstrates that substituting Asn10 and
Leu11 of PTH into the PTHrP sequence (replacing Asp10 and Lys11)
results in a peptide ([Asn10,Leu11]PTHrP-(7-34)-Fc) with virtually
no agonist activity but with very potent antagonist activity.
[0318] Like native PTH, all peptide-based PTH-R1 antagonists share
the property of very short circulating half-lives (<1 h).
Furthermore, the amino-terminal truncations which are required to
remove receptor agonism, also significantly reduce the affinity of
these peptides for PTH-R1. These properties limit the clinical
potential of conventional peptide antagonists. Fc-conjugation of
amino-terminally truncated PTH- or PTHrP peptides should
significantly increase their circulating half life, such that
continuous antagonism of PTH-R1 might be achieved with sufficient
exposure to these Fc-antagonists.
[0319] Materials, Methods and Results
[0320] We have cloned, expressed and purified
[Asn10,Leu11]PTHrP-(7-34)-Fc- . We tested the ability of this
compound to antagonize both acute and chronic hypercalcemia
responses in mice. PTHrP-(1-34) was used as a calcemic agent to
evaluate the acute effects of [Asn10,Leu11]PTHrP-(7-34)- -Fc.
Because PTHrP is the principal mediator of HHM, this study also
represents a model for hypercalcemia-inducing tumors. Briefly,
blood ionized calcium (BIC) was measured at baseline, and mice were
then challenged with vehicle (PBS) or with PTHrP-(1-34) (0.5 mg/kg)
by SC injection. Mice were then treated once SC with varying doses
of [Asn10,Leu11]PTHrP-(7-34)-Fc, or with vehicle (PBS). In
vehicle-treated mice challenged with PTHrP-(1-34), a transient
hypercalcemic response was observed. The peak calcemic response
occurred at 3 h post challenge, and persisted until at least 6 h
post challenge. [Asn10,Leu11]PTHrP-(7-34)-Fc at 10 mg/kg caused a
more rapid normalization of PTHrP-induced hypercalcemia compared to
vehicle treatment. A dose of 30 mg/kg completely blocked the
calcemic response to PTHrP-(1-34) (FIG. 6).
[0321] In order to test the ability of [Asn10,Leu11]PTHrP-(7-34)-Fc
to antagonize more chronic hypercalcemia, we used PTH-(1-34)-Fc as
a long-acting calcemic agent. This study also represents a model
for primary and secondary hyperparathyroidism, diseases which are
characterized by persistent elevation of PTH levels. In
vehicle-treated mice, a single SC injection of PTH-(1-34)-Fc (30
mg/kg) caused a robust hypercalcemic response in normal mice,
reaching a level of 2.75 mg/dl at 24 h post challenge (vs. normal
control value of 1.35). A single SC injection of
[Asn10,Leu11]PTHrP-(7-34)-Fc at 10-100 mg/kg caused a significant
decrease in the peak hypercalcemic response to PTH-(1-34)-Fc at 24
h (FIG. 6).
[0322] In conclusion, we have demonstrated antagonistic activity of
[Asn10,Leu11]PTHrP-(7-34)-Fc, in both acute and chronic animal
models of hypercalcemia. These models employed calcemic agents
based on both PTH and on PTHrP sequences. These data suggest that
[Asn10,Leu11]PTHrP-(7-34)- -Fc, as well as other Fc-conjugated
PTH-R1 antagonists, may be effective treatment options for
hyperparathyroidism, HHM, and other diseases associated with
aberrant PTH-R1 signaling.
EXAMPLE 3
Osteogenic Properties of Fc-Conjugated and Native C-Terminally
Truncated PTH Fragments
[0323] A. cAMP Assays
[0324] We tested the relative ability of PTH-Fc constructs to
stimulate cAMP accumulation in rat osteoblast-like ROS 17/2.8
cells. Cultures were treated with the phosphodiesterase inhibitor
IBMX to promote the accumulation of cAMP. Cultures were then
challenged for 15 minutes with either vehicle (PBS), or various PTH
constructs. Dose-dependent cAMP accumulation was demonstrated for
all fragments. Non-Fc-conjugated PTH-(1-34) was slightly more
potent than PTH-(1-31)-Fc and PTH-(1-30)-Fc (FIG. 7). These data
demonstrate that Fc-conjugated PTH fragments maintain the ability
to activate the AC pathway in osteoblasts.
[0325] B. Mouse Bioassay
[0326] We then tested the effects of PTH-(1-34), PTH-(1-34)-Fc,
PTH-(1-31)-Fc and PTH-(1-30)-Fc in mice. Four week old male mice
were injected on days 0, 5, and 10 with vehicle or with PTH
fragments, by SC injection. Peripheral blood was obtained for
clinical chemistry at 24, 48, and 72 h. Mice were killed on day 15,
vertebrae, tibiae and femurs were harvested for histology and one
tibia was collected for bone density measurements (peripheral
quantitative computed tomography, pQCT). Clinical chemistry
endpoints included total serum calcium, serum alkaline phosphatase
(AP, a marker of osteoblast activity), and serum tartrate-resistant
acid phosphatase (TRAP, a marker of osteoclast activity). For each
animal, the ratio of AP:TRAP was calculated as an index of relative
osteoblast activity compared to osteoclast activity. A higher
AP:TRAP ratio would indicate a potentially more anabolic agent. The
relatively high doses (15-fold greater than optimal anabolic doses)
were selected base on previous studies which demonstrated
significant changes in clinical chemistry endpoints. It was
anticipated that lower doses might be required to demonstrate
anabolic effects on bone density, and that antiresorptive
co-treatment might also be required to achieve anabolic
responses.
[0327] The clinical chemistry results are demonstrated in FIG. 8.
Serum calcium was not significantly different at 24, 48, or 72 h
after injection of 300 nmoles/kg (1.2 mg/kg) of PTH-(1-34). This
result is consistent with the short half-life of the non-Fc
conjugated peptide, which normally causes a transient (12 h)
increase in serum calcium. In contrast, an equimolar dose of
PTH-(1-34)-Fc caused a dramatic and sustained increase in serum
calcium, which peaked at 24 h. PTH-(1-31)-Fc was a more potent
calcemic agent, while PTH-(1-30)-Fc was the least calcemic of the 3
Fc peptides (FIG. 8A). Serum AP (osteoblast marker) was unchanged
by PTH-(1-34) administration, but was significantly elevated by 300
nmoles/kg of PTH-(1-34)-Fc and by PTH-(1-31)-Fc at 72 h.
PTH-(1-30)-Fc demonstrated the greatest elevation of AP, which
peaked 72 h after injection of 1,000 nmoles/kg (FIG. 8B). Serum
TRAP (osteoclast marker) was not significantly changed by
PTH-(1-34), PTH-(1-34)-Fc, or PTH-(1-30)-Fc, but was dramatically
increased by PTH-(1-31)-Fc (FIG. 8C). The calculated AP:TRAP ratios
were unchanged by PTH-(1-34), and were increased over time by
PTH-(1-34)-Fc. The low dose of PTH-(1-31)-Fc (100 nmoles/kg)
increased AP:TRAP, while the high dose (1,000 nmoles/kg) decreased
AP:TRAP. The greatest increase in AP:TRAP was realized with
PTH-(1-30)-Fc (1,000 nmoles/kg) (FIG. 8D).
[0328] The effects of the various PTH constructs on bone mineral
density (proximal tibial metaphysis) are demonstrated in FIG. 9. At
the end of the 15-day study, PTH-(1-34) (300 nmoles/kg) was
observed to have a modest (non-significant) anabolic effect when
injected on day 0, day 5 and day 10. PTH-(1-34)-Fc (300 nmoles/kg)
had no effect on bone density, nor did PTH-(1-31)-Fc at 100
nmoles/kg. Higher doses of PTH-(1-31)-Fc (300-1,000 nmoles/kg)
caused significant hypercalcemia-related toxicity, and bones were
not harvested from these animals for pQCT. PTH-(1-30)-Fc caused the
greatest increase in bone density. There was an apparent reverse
dose-response, where PTH-(1-30)-Fc at 100 nmoles/kg had the
greatest effect and at 1,000 nmoles/kg had the least effect,
although at all doses BMD was greater than in controls (FIG. 9).
The reverse dose-response was consistent with the notion that doses
employed (chosen for clinical chemistry endpoints) were 5-50 fold
higher than the optimal anabolic doses. Low doses of PTH (or
PTH-Fc) which fail to significantly increase serum calcium are
optimal for anabolic effects. See Hock, J. M. (1992), J. Bone Min.
Res. 7:65-72. In the current study, the treatment regimen with the
greatest anabolic effect (PTH-(1-30)-Fc at 100 nmoles/kg) was also
the only PTH-Fc treatment which failed to significantly increase
serum calcium (FIG. 8A).
[0329] These data demonstrate the potential anabolic effects of
C-terminally truncated PTH-Fc peptides. The longer half-life
conferred by Fc conjugation, combined with the selective
stimulation of AC/cAMP by C-terminal truncations, may explain the
anabolic effect in the absence of a potent bone resorption
inhibitor. It is expected that stepwise C-terminal truncation of
PTH-(1-30)-Fc will reveal shorter fragments which maintain or
exceed the anabolic profile of PTH-(1-30)-Fc. These fragments may
be more selective at stimulating osteoblasts, and may be less
calcemic, thus providing a wider therapeutic window for anabolic
therapy.
EXAMPLE 4
PTH-Fc Treatment as a Monotherapy
[0330] The efficacy of PTH-(1-34)-Fc as a monotherapy was
demonstrated in adult mice. Briefly, male BDF1 mice (4 months of
age) were treated twice per week by subcutaneous injection with
various doses of PTH-(1-34)-Fc or with vehicle (PBS). Other mice
were treated daily with SC injections of PTH-(1-34) at a dose of 80
.mu.g/kg/day (20 nmol/kg/day), a treatment regimen which is optimal
for increasing bone mass in rodents (M. Gunness-Hey and J. M. Hock,
Metab. Bone Dis. & Rel. Res. 5:177-181, 1984). After 3 weeks,
mice were sacrificed and tibiae were analyzed for bone mineral
density (BMD) via pQCT (FIG. 10).
[0331] Total tibial BMD and cancelled BMD were both significantly
increased by daily PTH-(1-34) injections compared to
vehicle-treated controls (FIG. 1, two-way ANOVA, p<0.05).
Twice-weekly injections of PTH-(1-34)-Fc caused dose-dependent
increases in both total and cancellous BMD which, at the two
highest doses (50 and 150 nmol/kg), were significantly greater than
the effects of either vehicle or daily PTH-(1-34). Cortical BMD in
the tibia was not significantly enhanced by daily PTH-(1-34)
treatments. Twice-weekly PTH-(1-34)-Fc caused a dose-dependent
increase in cortical BMD which at the highest dose was signficantly
greater than that observed in mice treated with vehicle or with
daily PTH-(1-34) (p<0.05).
[0332] Twice-weekly PTH-(1-34)-Fc also effectively increased BMD as
a monotherapy in aged ovariectomized (OVX) rats. Sprague Dawley
rats were OVX'd at 3 months of age and allowed to lose bone for 11
months. Other rats were sham-operated and treated twice per week
with vehicle (PBS). OVX rats were treated twice per week with SC
injections of either vehicle or the bisphosphonate APD
(pamidronate, 0.5 mg/kg), or with PTH-(1-34)-Fc (50 nmol/kg) or
with APD+PTH-(1-34)-Fc. BMD was determined weekly via dual energy
X-ray absorptiometry (DEXA). Rats were sacrificed after 4 weeks of
treatment. At the start of treatment, OVX rats had significant
reductions in BMD at all skeletal sites analyzed, compared to
vehicle-treated OVX rats (FIG. 11, p<0.05, 2-way ANOVA). APD
alone did not significantly increase BMD at any skeletal site
compared to vehicle-treated OVX rats. PTH-(1-34)-Fc alone caused a
significant increase in BMD at the femoral metaphysis after 4 weeks
of treatment (p<0.05). Treatment of OVX rats with PTH+ABD was
associated with an earlier significant increase in BMD at this site
(3 weeks). The combination of APD+PTH-(1-34)-Fc also caused
significant BMD increases at the lumbar vertebrae and at the
femoral metaphysis (p<0.05). PTH-(1-34)-Fc alone caused a mild
and transient hypercalcemic response which resolved spontaneously
after day 10 despite continued treatments. The co-administration of
APD completely blocked the calcemic effect of PTH-(1-34)-Fc.
[0333] These data suggest that PTH-(1-34)-Fc is an effective
anabolic agent when used as a monotherapy in both adult mice and
aged OVX rats. We have also demonstrated that the addition of an
antiresorptive agent (APD) to PTH-(1-34)-Fc was associated with
similar or more rapid increases in BMD in aged OVX rats.
Co-administration of APD also blocked the transient hypercalcemic
response produced by PTH-(1-34)-Fc, which suggests that the
therapeutic index of PTH-(1-34)-Fc could be significantly improved
by co-administering an effective antiresorptive agent.
[0334] The invention now being fully described, it will be apparent
to one of ordinary skill in the art that many changes and
modifications can be made thereto, without departing from the
spirit and scope of the invention as set forth herein.
10 Abbreviations AC adenylate cyclase AP alkaline phosphatase BMD
bone mineral density cAMP cyclic adenosine monophosphate DEXA
dual-energy X-ray absorptiometry HHM humoral hypercalcemia of
malignancy OPG osteoprotegerin OVX ovariectomized PBS
phosphate-buffered saline pQCT peripheral quantitative computed
tomography PTH parathyroid hormone PTHrP parathyroid
hormone-related protein TRAP tartrate-resistant acid
phosphatase
[0335]
Sequence CWU 1
1
170 1 684 DNA Homo sapiens CDS (1)..(684) 1 atg gac aaa act cac aca
tgt cca cct tgt cca gct ccg gaa ctc ctg 48 Met Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 1 5 10 15 ggg gga ccg tca
gtc ttc ctc ttc ccc cca aaa ccc aag gac acc ctc 96 Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 20 25 30 atg atc
tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc 144 Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 35 40 45
cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag 192
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50
55 60 gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac agc
acg 240 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr 65 70 75 80 tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac
tgg ctg aat 288 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn 85 90 95 ggc aag gag tac aag tgc aag gtc tcc aac aaa
gcc ctc cca gcc ccc 336 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro 100 105 110 atc gag aaa acc atc tcc aaa gcc aaa
ggg cag ccc cga gaa cca cag 384 Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln 115 120 125 gtg tac acc ctg ccc cca tcc
cgg gat gag ctg acc aag aac cag gtc 432 Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val 130 135 140 agc ctg acc tgc ctg
gtc aaa ggc ttc tat ccc agc gac atc gcc gtg 480 Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 145 150 155 160 gag tgg
gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 528 Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175
ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag ctc acc 576
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180
185 190 gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc tcc
gtg 624 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val 195 200 205 atg cat gag gct ctg cac aac cac tac acg cag aag agc
ctc tcc ctg 672 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu 210 215 220 tct ccg ggt aaa 684 Ser Pro Gly Lys 225 2
228 PRT Homo sapiens 2 Met Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu Leu 1 5 10 15 Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 20 25 30 Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 35 40 45 His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60 Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 65 70 75 80 Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 85 90
95 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
100 105 110 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln 115 120 125 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val 130 135 140 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val 145 150 155 160 Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro 165 170 175 Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 180 185 190 Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205 Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215
220 Ser Pro Gly Lys 225 3 21 PRT Artificial Sequence PTH/PTHrP 3
Xaa His Xaa Xaa Xaa Lys Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Xaa Xaa 1 5
10 15 Xaa Xaa Xaa Xaa Xaa 20 4 22 PRT Artificial Sequence PTH/PTHrP
4 Xaa Xaa His Asn Leu Xaa Lys His Leu Xaa Ser Xaa Xaa Arg Xaa Glu 1
5 10 15 Trp Leu Arg Lys Lys Leu 20 5 21 PRT Artificial Sequence
PTH/PTHrP 5 Leu His Xaa Xaa Xaa Lys Ser Ile Xaa Xaa Leu Arg Arg Arg
Phe Xaa 1 5 10 15 Leu His His Leu Ile 20 6 8 PRT Artificial
Sequence Preferred linker 6 Gly Gly Gly Lys Gly Gly Gly Gly 1 5 7 8
PRT Artificial Sequence Preferred linker 7 Gly Gly Gly Asn Gly Ser
Gly Gly 1 5 8 8 PRT Artificial Sequence Preferred linker 8 Gly Gly
Gly Cys Gly Gly Gly Gly 1 5 9 5 PRT Artificial Sequence Preferred
linker 9 Gly Pro Asn Gly Gly 1 5 10 84 PRT Homo sapiens 10 Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20
25 30 Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly
Ser 35 40 45 Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu
Ser His Glu 50 55 60 Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val
Asn Val Leu Thr Lys 65 70 75 80 Ala Lys Ser Gln 11 84 PRT Rattus
rattus 11 Ala Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His
Leu Ala 1 5 10 15 Ser Val Glu Arg Met Gln Trp Leu Arg Lys Lys Leu
Gln Asp Val His 20 25 30 Asn Phe Val Ser Leu Gly Val Gln Met Ala
Ala Arg Glu Gly Ser Tyr 35 40 45 Gln Arg Pro Thr Lys Lys Glu Asp
Asn Val Leu Val Asp Gly Asn Ser 50 55 60 Lys Ser Leu Gly Glu Gly
Asp Lys Ala Asp Val Asp Val Leu Val Lys 65 70 75 80 Ala Lys Ser Gln
12 78 PRT Homo sapiens 12 Leu Met His Asn Leu Gly Lys His Leu Asn
Ser Met Glu Arg Val Glu 1 5 10 15 Trp Leu Arg Lys Lys Leu Gln Asp
Val His Asn Phe Val Ala Leu Gly 20 25 30 Ala Pro Leu Ala Pro Arg
Asp Ala Gly Ser Gln Arg Pro Arg Lys Lys 35 40 45 Glu Asp Asn Val
Leu Val Glu Ser His Glu Lys Ser Leu Gly Glu Ala 50 55 60 Asp Lys
Ala Asp Val Asn Val Leu Thr Lys Ala Lys Ser Gln 65 70 75 13 44 PRT
Homo sapiens 13 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys
His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys
Leu Gln Asp Val His 20 25 30 Asn Phe Val Ala Leu Gly Ala Pro Leu
Ala Pro Arg 35 40 14 38 PRT Homo sapiens 14 Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe
Val Ala Leu Gly 35 15 37 PRT Homo sapiens 15 Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn Ser 1 5 10 15 Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn 20 25 30 Phe
Val Ala Leu Gly 35 16 34 PRT Homo sapiens 16 Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu
Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn
Phe 17 34 PRT Artificial Sequence modified human PTH 17 Ser Val Ser
Glu Ile Gln Leu Met His Asn Arg Gly Lys His Leu Asn 1 5 10 15 Ser
Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25
30 Asn Phe 18 34 PRT Artificial Sequence modified human PTH 18 Ser
Val Ser Glu Ile Gln Leu Met His Asn Lys Gly Lys His Leu Asn 1 5 10
15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30 Asn Phe 19 34 PRT Artificial Sequence modified human PTH
19 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15 Ser Met Arg Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
Val His 20 25 30 Asn Phe 20 34 PRT Artificial Sequence modified
human PTH 20 Tyr Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys
His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys
Leu Gln Asp Val His 20 25 30 Asn Phe 21 34 PRT Artificial Sequence
modified human PTH 21 Ser Val Ser Glu Ile Gln Leu Leu His Asn Leu
Gly Lys His Leu Asn 1 5 10 15 Ser Leu Glu Arg Val Glu Trp Leu Arg
Lys Lys Leu Gln Asp Val His 20 25 30 Asn Tyr 22 34 PRT Artificial
Sequence bovine 22 Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly
Lys His Leu Ser 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys
Lys Leu Gln Asp Val His 20 25 30 Asn Phe 23 34 PRT Artificial
Sequence modified bovine PTH 23 Ala Val Ser Glu Ile Gln Phe Leu His
Asn Leu Gly Lys His Leu Ser 1 5 10 15 Ser Leu Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Tyr 24 34 PRT
Artificial Sequence porcine 24 Ser Val Ser Glu Ile Gln Leu Met His
Asn Leu Gly Lys His Leu Ser 1 5 10 15 Ser Leu Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe 25 34 PRT
Rattus rattus 25 Ala Val Ser Glu Ile Gln Leu Met His Asn Leu Gly
Lys His Leu Ala 1 5 10 15 Ser Val Glu Arg Met Gln Trp Leu Arg Lys
Lys Leu Gln Asp Val His 20 25 30 Asn Phe 26 34 PRT Artificial
Sequence modified rat PTH 26 Ala Val Ser Glu Ile Gln Leu Leu His
Asn Leu Gly Lys His Leu Ala 1 5 10 15 Ser Val Glu Arg Leu Gln Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Tyr 27 31 PRT Homo
sapiens 27 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His
Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu
Gln Asp Val 20 25 30 28 31 PRT Artificial Sequence modified human
PTH 28 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu
Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Leu Leu Gln
Asp Val 20 25 30 29 32 PRT Artificial Sequence PTH 29 Ser Glu Ile
Gln Leu Leu His Asn Leu Gly Lys His Leu Asn Ser Leu 1 5 10 15 Glu
Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Tyr 20 25
30 30 32 PRT Artificial Sequence bovine 30 Ser Glu Ile Gln Phe Met
His Asn Leu Gly Lys His Leu Ser Ser Met 1 5 10 15 Glu Arg Val Glu
Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe 20 25 30 31 32 PRT
Artificial Sequence modified bovine PTH 31 Ser Glu Ile Gln Phe Leu
His Asn Leu Gly Lys His Leu Ser Ser Leu 1 5 10 15 Glu Arg Val Glu
Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Tyr 20 25 30 32 28 PRT
Homo sapiens 32 Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu
Arg Val Glu 1 5 10 15 Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn
Phe 20 25 33 28 PRT Artificial Sequence modified human PTH 33 Leu
Leu His Asn Leu Gly Lys His Leu Asn Ser Leu Glu Arg Val Glu 1 5 10
15 Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Tyr 20 25 34 28 PRT
Artificial Sequence bovine PTH 34 Phe Met His Asn Leu Gly Lys His
Leu Ser Ser Met Glu Arg Val Glu 1 5 10 15 Trp Leu Arg Lys Lys Leu
Gln Asp Val His Asn Phe 20 25 35 28 PRT Artificial Sequence
modified bovine PTH 35 Phe Met His Asn Leu Gly Lys His Leu Ser Ser
Met Glu Arg Val Glu 1 5 10 15 Trp Leu Arg Lys Lys Leu Gln Asp Val
His Asn Tyr 20 25 36 28 PRT Artificial Sequence modified bovine PTH
36 Phe Leu His Asn Leu Gly Lys His Leu Ser Ser Leu Glu Arg Val Glu
1 5 10 15 Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Tyr 20 25 37
5 PRT Artificial Sequence modified bovine PTH 37 Cys Asn Gly Arg
Cys 1 5 38 27 PRT Artificial Sequence modified bovine PTH 38 Phe
Met His Asn Leu Lys His Leu Ser Ser Met Glu Arg Val Glu Trp 1 5 10
15 Leu Arg Lys Lys Leu Gln Asp Val His Asn Tyr 20 25 39 30 PRT Homo
sapiens 39 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His
Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu
Gln Asp 20 25 30 40 30 PRT Artificial Sequence modified human PTH
40 Ser Val Ser Glu Ile Gln Leu Met His Asn Arg Gly Lys His Leu Asn
1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30 41 30 PRT Artificial Sequence modified human PTH 41 Ser
Val Ser Glu Ile Gln Leu Met His Asn Lys Gly Lys His Leu Asn 1 5 10
15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp 20 25 30
42 30 PRT Artificial Sequence modified human PTH 42 Ser Val Ser Glu
Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met
Arg Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp 20 25 30 43 30 PRT
Artificial Sequence modified human PTH 43 Tyr Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp 20 25 30 44 30 PRT
Artificial Sequence modified human PTH 44 Ser Val Ser Glu Ile Gln
Leu Leu His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Leu Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp 20 25 30 45 30 PRT
Artificial Sequence bovine 45 Ala Val Ser Glu Ile Gln Phe Met His
Asn Leu Gly Lys His Leu Ser 1 5 10 15 Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp 20 25 30 46 30 PRT Artificial Sequence
modified bovine PTH 46 Ala Val Ser Glu Ile Gln Phe Leu His Asn Leu
Gly Lys His Leu Ser 1 5 10 15 Ser Leu Glu Arg Val Glu Trp Leu Arg
Lys Lys Leu Gln Asp 20 25 30 47 30 PRT Artificial Sequence porcine
PTH 47 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu
Ser 1 5 10 15 Ser Leu Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln
Asp 20 25 30 48 30 PRT Artificial Sequence rat PTH 48 Ala Val Ser
Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Ala 1 5 10 15 Ser
Val Glu Arg Met Gln Trp Leu Arg Lys Lys Leu Gln Asp 20 25 30 49 30
PRT Artificial Sequence modified rat PTH 49 Ala Val Ser Glu Ile Gln
Leu Leu His Asn Leu Gly Lys His Leu Ala 1 5 10 15 Ser Val Glu Arg
Leu Gln Trp Leu Arg Lys Lys Leu Gln Asp 20 25 30 50 30 PRT
Artificial Sequence modified human PTH 50 Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Leu Leu Gln Asp 20 25 30 51 29 PRT Homo
sapiens 51 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His
Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu
Gln 20
25 52 28 PRT Homo sapiens 52 Ser Val Ser Glu Ile Gln Leu Met His
Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu 20 25 53 28 PRT Artificial Sequence modified
PTH 53 Ser Glu Ile Gln Leu Leu His Asn Leu Gly Lys His Leu Asn Ser
Leu 1 5 10 15 Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp 20 25
54 28 PRT Artificial Sequence bovine 54 Ser Glu Ile Gln Phe Met His
Asn Leu Gly Lys His Leu Ser Ser Met 1 5 10 15 Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp 20 25 55 28 PRT Artificial Sequence
modified bovine PTH 55 Ser Glu Ile Gln Phe Leu His Asn Leu Gly Lys
His Leu Ser Ser Leu 1 5 10 15 Glu Arg Val Glu Trp Leu Arg Lys Lys
Leu Gln Asp 20 25 56 24 PRT Homo sapiens 56 Leu Met His Asn Leu Gly
Lys His Leu Asn Ser Met Glu Arg Val Glu 1 5 10 15 Trp Leu Arg Lys
Lys Leu Gln Asp 20 57 24 PRT Artificial Sequence modified human PTH
57 Leu Leu His Asn Leu Gly Lys His Leu Asn Ser Leu Glu Arg Val Glu
1 5 10 15 Trp Leu Arg Lys Lys Leu Gln Asp 20 58 24 PRT Artificial
Sequence bovine 58 Phe Met His Asn Leu Gly Lys His Leu Ser Ser Met
Glu Arg Val Glu 1 5 10 15 Trp Leu Arg Lys Lys Leu Gln Asp 20 59 24
PRT Artificial Sequence modified bovine PTH 59 Phe Leu His Asn Leu
Gly Lys His Leu Ser Ser Leu Glu Arg Val Glu 1 5 10 15 Trp Leu Arg
Lys Lys Leu Gln Asp 20 60 24 PRT Artificial Sequence modified
bovine PTH 60 Phe Leu His Asn Leu Trp Lys His Leu Ser Ser Leu Glu
Arg Val Glu 1 5 10 15 Trp Leu Arg Lys Lys Leu Gln Asp 20 61 24 PRT
Artificial Sequence modified bovine PTH 61 Phe Met His Asn Leu Lys
Trp His Leu Ser Ser Met Glu Arg Val Glu 1 5 10 15 Trp Leu Arg Lys
Lys Leu Gln Asp 20 62 86 PRT Homo sapiens 62 Ala Val Ser Glu His
Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg
Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His 20 25 30 Thr
Ala Glu Ile Arg Ala Thr Ser Glu Val Ser Pro Asn Ser Lys Pro 35 40
45 Ser Pro Asn Thr Lys Asn His Pro Val Arg Phe Gly Ser Asp Asp Glu
50 55 60 Gly Arg Tyr Leu Thr Gln Glu Thr Asn Lys Val Glu Thr Tyr
Lys Glu 65 70 75 80 Gln Pro Leu Lys Thr Pro 85 63 34 PRT Homo
sapiens 63 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser
Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile
Ala Glu Ile His 20 25 30 Thr Ala 64 36 PRT Artificial Sequence
modified human PTHrP 64 Ala Val Ser Glu His Gln Leu Leu His Asp Lys
Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Phe Phe Leu His
His Leu Ile Ala Glu Ile His 20 25 30 Thr Ala Glu Tyr 35 65 36 PRT
Artificial Sequence modified human PTHrP 65 Ala Val Ser Glu Ile Gln
Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg
Arg Phe Trp Leu His His Leu Ile Ala Glu Ile His 20 25 30 Thr Ala
Glu Tyr 35 66 35 PRT Artificial Sequence modified human PTHrP 66
Tyr Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile 1 5
10 15 Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu
Ile 20 25 30 His Thr Ala 35 67 33 PRT Artificial Sequence modified
human PTHrP 67 Ala Val Ser Glu His Gln Leu Leu His Asn Leu Lys Ser
Ile Gln Asp 1 5 10 15 Leu Arg Arg Arg Phe Phe Leu His His Leu Ile
Ala Glu Ile His Thr 20 25 30 Ala 68 28 PRT Homo sapiens 68 Leu Leu
His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe 1 5 10 15
Phe Leu His His Leu Ile Ala Glu Ile His Thr Ala 20 25 69 28 PRT
Artificial Sequence modified human PTHrP 69 Leu Leu His Asn Leu Gly
Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe 1 5 10 15 Phe Leu His His
Leu Ile Ala Glu Ile His Thr Ala 20 25 70 28 PRT Artificial Sequence
modified PTHrP 70 Leu Leu His Asp Lys Gly Lys Ser Ile Asn Leu Leu
Arg Arg Arg Phe 1 5 10 15 Phe Leu His His Leu Ile Ala Glu Ile His
Thr Ala 20 25 71 28 PRT Artificial Sequence modified human PTHrP 71
Leu Leu His Asp Leu Trp Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe 1 5
10 15 Phe Leu His His Leu Ile Ala Glu Ile His Thr Ala 20 25 72 28
PRT Artificial Sequence modified PTHrP 72 Leu Leu His Asn Leu Trp
Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe 1 5 10 15 Phe Leu His His
Leu Ile Ala Glu Ile His Thr Ala 20 25 73 27 PRT Artificial Sequence
modified PTHrP 73 Leu His Asn Leu Trp Lys Ser Ile Gln Asp Leu Arg
Arg Arg Phe Phe 1 5 10 15 Leu His His Leu Ile Ala Glu Ile His Thr
Ala 20 25 74 27 PRT Artificial Sequence modified PTHrP 74 Leu His
Asn Leu Phe Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe Phe 1 5 10 15
Leu His His Leu Ile Ala Glu Ile His Thr Ala 20 25 75 28 PRT
Artificial Sequence modified human PTHrP 75 Leu Leu His Asn Leu Trp
Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe 1 5 10 15 Phe Leu His His
Leu Ile Ala Glu Ile His Thr Ala 20 25 76 30 PRT Homo sapiens 76 Ala
Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10
15 Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu 20 25 30
77 30 PRT Artificial Sequence modified human PTHrP 77 Ala Val Ser
Glu Ile Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp
Leu Arg Arg Arg Phe Trp Leu His His Leu Ile Ala Glu 20 25 30 78 31
PRT Artificial Sequence human PTHrP with non-human N-terminal
peptide 78 Tyr Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys
Ser Ile 1 5 10 15 Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu
Ile Ala Glu 20 25 30 79 30 PRT Artificial Sequence modified human
PTHrP 79 Ala Val Ser Glu His Gln Leu Leu His Asn Leu Phe Lys Ser
Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile
Ala Glu 20 25 30 80 24 PRT Homo sapiens 80 Leu Leu His Asp Lys Gly
Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe 1 5 10 15 Phe Leu His His
Leu Ile Ala Glu 20 81 24 PRT Artificial Sequence modified human
PTHrP 81 Leu Leu His Asn Leu Gly Lys Ser Ile Gln Asp Leu Arg Arg
Arg Phe 1 5 10 15 Phe Leu His His Leu Ile Ala Glu 20 82 24 PRT
Artificial Sequence modified PTHrP 82 Leu Leu His Asp Lys Gly Lys
Ser Ile Asn Leu Leu Arg Arg Arg Phe 1 5 10 15 Phe Leu His His Leu
Ile Ala Glu 20 83 23 PRT Artificial Sequence modified human PTHrP
83 Leu Leu His Asp Leu Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe Phe
1 5 10 15 Leu His His Leu Ile Ala Glu 20 84 23 PRT Artificial
Sequence modified PTHrP 84 Leu Leu His Asn Leu Lys Ser Ile Gln Asp
Leu Arg Arg Arg Phe Phe 1 5 10 15 Leu His His Leu Ile Ala Glu 20 85
23 PRT Artificial Sequence modified PTHrP 85 Leu His Asn Leu Trp
Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe Phe 1 5 10 15 Leu His His
Leu Ile Ala Glu 20 86 23 PRT Artificial Sequence modified PTHrP 86
Leu His Asn Leu Phe Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe Phe 1 5
10 15 Leu His His Leu Ile Ala Glu 20 87 24 PRT Artificial Sequence
modified human PTHrP 87 Leu Leu His Asn Leu Trp Lys Ser Ile Gln Asp
Leu Arg Arg Arg Phe 1 5 10 15 Phe Leu His His Leu Ile Ala Glu 20 88
34 PRT Artificial Sequence modified human PTH 88 Ser Val Ser Glu
Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met
Glu Arg Val Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu His 20 25 30
Asn Phe 89 34 PRT Artificial Sequence modified human PTH 89 Ser Val
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15
Ser Met Glu Arg Val Glu Leu Leu Glu Lys Leu Leu Lys Lys Leu His 20
25 30 Asn Phe 90 34 PRT Artificial Sequence modified human PTH 90
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5
10 15 Ser Met Glu Arg Val Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu
His 20 25 30 Asn Phe 91 34 PRT Artificial Sequence modified human
PTH 91 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu
Asn 1 5 10 15 Ser Met Glu Arg Val Ser Leu Leu Ser Ser Leu Leu Ser
Ser Leu His 20 25 30 Asn Phe 92 34 PRT Artificial Sequence modified
human PTH 92 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys
His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Ala Phe Tyr Asp Lys Val
Ala Glu Lys Leu His 20 25 30 Asn Phe 93 28 PRT Artificial Sequence
modified human PTH 93 Leu Met His Asn Leu Gly Lys His Leu Asn Ser
Met Glu Arg Val Glu 1 5 10 15 Leu Leu Glu Lys Leu Leu Glu Lys Leu
His Asn Phe 20 25 94 28 PRT Artificial Sequence modified human PTH
94 Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu
1 5 10 15 Leu Leu Glu Lys Leu Leu Lys Lys Leu His Asn Phe 20 25 95
28 PRT Artificial Sequence modified human PTH 95 Leu Met His Asn
Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Ala 1 5 10 15 Leu Ala
Glu Ala Leu Ala Glu Ala Leu His Asn Phe 20 25 96 28 PRT Artificial
Sequence modified human PTH 96 Leu Met His Asn Leu Gly Lys His Leu
Asn Ser Met Glu Arg Val Ser 1 5 10 15 Leu Leu Ser Ser Leu Leu Ser
Ser Leu His Asn Phe 20 25 97 28 PRT Artificial Sequence modified
human PTH 97 Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu
Arg Val Ala 1 5 10 15 Phe Tyr Asp Lys Val Ala Glu Lys Leu His Asn
Phe 20 25 98 34 PRT Artificial Sequence modified human PTHrP 98 Ala
Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10
15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu His
20 25 30 Thr Ala 99 34 PRT Artificial Sequence modified human PTHrP
99 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln
1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Lys Lys
Leu His 20 25 30 Thr Ala 100 34 PRT Artificial Sequence modified
human PTHrP 100 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys
Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Ala Leu Ala Glu Ala Leu
Ala Glu Ala Leu His 20 25 30 Thr Ala 101 34 PRT Artificial Sequence
modified human PTHrP 101 Ala Val Ser Glu His Gln Leu Leu His Asp
Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Ser Leu Leu
Ser Ser Leu Leu Ser Ser Leu His 20 25 30 Thr Ala 102 34 PRT
Artificial Sequence modified human PTHrP 102 Ala Val Ser Glu His
Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg
Arg Arg Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu His 20 25 30 Thr
Ala 103 28 PRT Artificial Sequence modified human PTHrP 103 Leu Leu
His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg Glu 1 5 10 15
Leu Leu Glu Lys Leu Leu Glu Lys Leu His Thr Ala 20 25 104 28 PRT
Artificial Sequence modified human PTHrP 104 Leu Leu His Asp Lys
Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg Glu 1 5 10 15 Leu Leu Glu
Lys Leu Leu Lys Lys Leu His Thr Ala 20 25 105 28 PRT Artificial
Sequence modified human PTHrP 105 Leu Leu His Asp Lys Gly Lys Ser
Ile Gln Asp Leu Arg Arg Arg Ala 1 5 10 15 Leu Ala Glu Ala Leu Ala
Glu Ala Leu His Thr Ala 20 25 106 28 PRT Artificial Sequence
modified human PTHrP 106 Leu Leu His Asp Lys Gly Lys Ser Ile Gln
Asp Leu Arg Arg Arg Ser 1 5 10 15 Leu Leu Ser Ser Leu Leu Ser Ser
Leu His Thr Ala 20 25 107 28 PRT Artificial Sequence modified human
PTHrP 107 Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg Arg
Arg Ala 1 5 10 15 Phe Tyr Asp Lys Val Ala Glu Lys Leu His Thr Ala
20 25 108 34 PRT Artificial Sequence modified human PTHrP 108 Ala
Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10
15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Arg Lys Leu His
20 25 30 Thr Ala 109 34 PRT Artificial Sequence modified human
PTHrP 109 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser
Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu
Glu Lys Leu His 20 25 30 Thr Ser 110 37 PRT Artificial Sequence
modified human PTHrP 110 Ala Val Ser Glu His Gln Leu Leu His Asp
Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu
Glu Lys Leu Leu Glu Lys Leu His 20 25 30 Thr Ala Gly Arg Arg 35 111
34 PRT Artificial Sequence modified human PTHrP 111 Ala Val Ser Glu
His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu
Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu Lys 20 25 30
Glu Leu 112 34 PRT Artificial Sequence modified human PTHrP 112 Ala
Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10
15 Asp Leu Ala Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu His
20 25 30 Thr Ala 113 34 PRT Artificial Sequence modified human
PTHrP 113 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser
Ile Gln 1 5 10 15 Asp Leu Arg Arg Ala Glu Leu Leu Glu Lys Leu Leu
Glu Lys Leu His 20 25 30 Thr Ala 114 34 PRT Artificial Sequence
modified human PTHrP 114 Ala Val Ser Glu Ala Gln Leu Leu His Asp
Leu Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu
Glu Lys Leu Leu Glu Lys Leu His 20 25 30 Ala Leu 115 34 PRT
Artificial Sequence modified human PTHrP 115 Ala Val Ser Glu His
Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg
Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg Leu His 20 25 30 Thr
Ala 116 33 PRT Artificial Sequence modified human PTHrP 116 Ala Val
Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile Gln 1 5 10 15
Asp Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg Leu His Thr 20
25 30 Ala 117 34 PRT Artificial Sequence modified human PTHrP 117
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Lys Ser Ile Gln 1 5
10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Lys Arg Leu
His 20 25 30 Thr Ala 118 34 PRT Artificial Sequence modified
human PTHrP 118 Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg
Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu
Leu Lys Arg Leu His 20 25 30 Thr Ala 119 34 PRT Artificial Sequence
modified human PTHrP 119 Ala Val Ser Glu His Gln Leu Leu His Asp
Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Ala Leu Ala
Glu Ala Leu Ala Glu Ala Leu His 20 25 30 Thr Ala 120 34 PRT
Artificial Sequence modified human PTHrP 120 Ala Val Ser Glu His
Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg
Arg Arg Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu His 20 25 30 Thr
Ala 121 34 PRT Artificial Sequence modified human PTHrP 121 Ala Val
Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15
Asp Leu Arg Arg Arg Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu His 20
25 30 Thr Ala 122 34 PRT Artificial Sequence modified human PTHrP
122 Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu Ser
1 5 10 15 Ser Met Glu Arg Val Glu Leu Leu Glu Lys Leu Leu Glu Lys
Leu His 20 25 30 Asn Tyr 123 34 PRT Artificial Sequence modified
human PTHrP 123 Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys
His Leu Ser 1 5 10 15 Ser Met Arg Arg Arg Glu Leu Leu Glu Lys Leu
Leu Glu Lys Leu His 20 25 30 Asn Tyr 124 30 PRT Artificial Sequence
modified human PTH 124 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu
Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Leu Leu Glu
Lys Leu Leu Glu Lys 20 25 30 125 30 PRT Artificial Sequence
modified human PTH 125 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu
Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Leu Leu Glu
Lys Leu Leu Lys Lys 20 25 30 126 30 PRT Artificial Sequence
modified human PTH 126 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu
Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Ala Leu Ala Glu
Ala Leu Ala Glu Ala 20 25 30 127 30 PRT Artificial Sequence
modified human PTH 127 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu
Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Ser Leu Leu Ser
Ser Leu Leu Ser Ser 20 25 30 128 34 PRT Artificial Sequence
modified human PTH 128 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu
Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Ala Phe Tyr Asp
Lys Val Ala Glu Lys Leu His 20 25 30 Asn Phe 129 24 PRT Artificial
Sequence modified human PTH 129 Leu Met His Asn Leu Gly Lys His Leu
Asn Ser Met Glu Arg Val Glu 1 5 10 15 Leu Leu Glu Lys Leu Leu Glu
Lys 20 130 24 PRT Artificial Sequence modified human PTH 130 Leu
Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Glu 1 5 10
15 Leu Leu Glu Lys Leu Leu Lys Lys 20 131 24 PRT Artificial
Sequence modified human PTH 131 Leu Met His Asn Leu Gly Lys His Leu
Asn Ser Met Glu Arg Val Ala 1 5 10 15 Leu Ala Glu Ala Leu Ala Glu
Ala 20 132 24 PRT Artificial Sequence modified human PTH 132 Leu
Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu Arg Val Ser 1 5 10
15 Leu Leu Ser Ser Leu Leu Ser Ser 20 133 24 PRT Artificial
Sequence modified human PTH 133 Leu Met His Asn Leu Gly Lys His Leu
Asn Ser Met Glu Arg Val Ala 1 5 10 15 Phe Tyr Asp Lys Val Ala Glu
Lys 20 134 30 PRT Artificial Sequence modified human PTHrP 134 Ala
Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10
15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys 20 25 30
135 30 PRT Artificial Sequence modified human PTHrP 135 Ala Val Ser
Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp
Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Lys Lys 20 25 30 136 30
PRT Artificial Sequence modified human PTHrP 136 Ala Val Ser Glu
His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu
Arg Arg Arg Ala Leu Ala Glu Ala Leu Ala Glu Ala 20 25 30 137 30 PRT
Artificial Sequence modified human PTHrP 137 Ala Val Ser Glu His
Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg
Arg Arg Ser Leu Leu Ser Ser Leu Leu Ser Ser 20 25 30 138 30 PRT
Artificial Sequence modified human PTHrP 138 Ala Val Ser Glu His
Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg
Arg Arg Ala Phe Tyr Asp Lys Val Ala Glu Lys 20 25 30 139 24 PRT
Artificial Sequence modified human PTHrP 139 Leu Leu His Asp Lys
Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg Glu 1 5 10 15 Leu Leu Glu
Lys Leu Leu Glu Lys 20 140 24 PRT Artificial Sequence modified
human PTHrP 140 Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg
Arg Arg Glu 1 5 10 15 Leu Leu Glu Lys Leu Leu Lys Lys 20 141 24 PRT
Artificial Sequence modified human PTHrP 141 Leu Leu His Asp Lys
Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg Ala 1 5 10 15 Leu Ala Glu
Ala Leu Ala Glu Ala 20 142 24 PRT Artificial Sequence modified
human PTHrP 142 Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg
Arg Arg Ser 1 5 10 15 Leu Leu Ser Ser Leu Leu Ser Ser 20 143 24 PRT
Artificial Sequence modified human PTHrP 143 Leu Leu His Asp Lys
Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg Ala 1 5 10 15 Phe Tyr Asp
Lys Val Ala Glu Lys 20 144 30 PRT Artificial Sequence modified
human PTHrP 144 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys
Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu
Leu Arg Lys 20 25 30 145 30 PRT Artificial Sequence modified human
PTHrP 145 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser
Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu
Glu Lys 20 25 30 146 33 PRT Artificial Sequence modified human
PTHrP 146 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser
Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu
Glu Lys Leu His 20 25 30 Thr 147 30 PRT Artificial Sequence
modified human PTHrP 147 Ala Val Ser Glu His Gln Leu Leu His Asp
Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu
Glu Lys Leu Leu Glu Lys 20 25 30 148 30 PRT Artificial Sequence
modified human PTHrP 148 Ala Val Ser Glu His Gln Leu Leu His Asp
Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Ala Arg Arg Glu Leu Leu
Glu Lys Leu Leu Glu Lys 20 25 30 149 30 PRT Artificial Sequence
modified human PTHrP 149 Ala Val Ser Glu His Gln Leu Leu His Asp
Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Ala Glu Leu Leu
Glu Lys Leu Leu Glu Lys 20 25 30 150 30 PRT Artificial Sequence
modified human PTHrP 150 Ala Val Ser Glu Ala Gln Leu Leu His Asp
Leu Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu
Glu Lys Leu Leu Glu Lys 20 25 30 151 30 PRT Artificial Sequence
modified human PTHrP 151 Ala Val Ser Glu His Gln Leu Leu His Asp
Lys Gly Lys Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu
Glu Arg Leu Leu Glu Arg 20 25 30 152 29 PRT Artificial Sequence
modified human PTHrP 152 Ala Val Ser Glu His Gln Leu Leu His Asp
Arg Gly Arg Ser Ile Gln 1 5 10 15 Asp Arg Arg Arg Glu Leu Leu Glu
Arg Leu Leu Glu Arg 20 25 153 30 PRT Artificial Sequence modified
human PTHrP 153 Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Lys
Ser Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu
Leu Lys Arg 20 25 30 154 30 PRT Artificial Sequence modified human
PTHrP 154 Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser
Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu
Lys Arg 20 25 30 155 30 PRT Artificial Sequence modified human
PTHrP 155 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser
Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Ala Leu Ala Glu Ala Leu Ala
Glu Ala 20 25 30 156 30 PRT Artificial Sequence modified human
PTHrP 156 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser
Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Ser Leu Leu Ser Ser Leu Leu
Ser Ser 20 25 30 157 30 PRT Artificial Sequence modified human
PTHrP 157 Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser
Ile Gln 1 5 10 15 Asp Leu Arg Arg Arg Ala Phe Tyr Asp Lys Val Ala
Glu Lys 20 25 30 158 30 PRT Artificial Sequence modified human
PTHrP 158 Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His
Leu Ser 1 5 10 15 Ser Met Glu Arg Val Glu Leu Leu Glu Lys Leu Leu
Glu Lys 20 25 30 159 30 PRT Artificial Sequence modified human
PTHrP 159 Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His
Leu Ser 1 5 10 15 Ser Met Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu
Glu Lys 20 25 30 160 39 PRT Artificial Sequence TIP39 160 Ser Leu
Ala Leu Ala Asp Asp Ala Ala Phe Arg Glu Arg Ala Arg Leu 1 5 10 15
Leu Ala Ala Leu Glu Arg Arg His Trp Leu Asn Ser Tyr Met His Lys 20
25 30 Leu Leu Val Leu Asp Ala Pro 35 161 34 PRT Artificial Sequence
Preferred embodiments - PTH 161 Ser Val Ser Glu Ile Gln Leu Met His
Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp
Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe 162 34 PRT
Artificial Sequence Preferred embodiments - PTH 162 Ser Val Ser Glu
Ile Gln Leu Met His Asn Arg Gly Lys His Leu Asn 1 5 10 15 Ser Met
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30
Asn Phe 163 34 PRT Artificial Sequence Preferred embodiments - PTH
163 Ser Val Ser Glu Ile Gln Leu Met His Asn Lys Gly Lys His Leu Asn
1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
Val His 20 25 30 Asn Phe 164 34 PRT Artificial Sequence Preferred
embodiments - PTH 164 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu
Gly Lys His Leu Asn 1 5 10 15 Ser Met Arg Arg Val Glu Trp Leu Arg
Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe 165 31 PRT Artificial
Sequence Preferred embodiments - PTH 165 Ser Val Ser Glu Ile Gln
Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg
Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val 20 25 30 166 30 PRT
Artificial Sequence Preferred embodiments - PTH 166 Ser Val Ser Glu
Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp 20 25 30 167 29 PRT
Artificial Sequence Preferred embodiments - PTH 167 Ser Val Ser Glu
Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln 20 25 168 28 PRT
Artificial Sequence Preferred embodiments - PTH 168 Ser Val Ser Glu
Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu 20 25 169 28 PRT Artificial
Sequence Preferred embodiments - PTHrP 169 Leu Leu His Asn Leu Gly
Lys Ser Ile Gln Asp Leu Arg Arg Arg Phe 1 5 10 15 Phe Leu His His
Leu Ile Ala Glu Ile His Thr Ala 20 25 170 39 PRT Artificial
Sequence Preferred embodiments - TIP39 170 Ser Leu Ala Leu Ala Asp
Asp Ala Ala Phe Arg Glu Arg Ala Arg Leu 1 5 10 15 Leu Ala Ala Leu
Glu Arg Arg His Trp Leu Asn Ser Tyr Met His Lys 20 25 30 Leu Leu
Val Leu Asp Ala Pro 35
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