text:
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 or through a sidechain at any residue from residue 14 through the C-terminal residue. 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 PEG. 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).
1. A composition of matter of the formula
and multimers thereof, wherein:
F1 is a vehicle and is attached at the C-terminus of P1-(L1)a or through a sidechain at any residue from residue 14 through the C-terminal residue;
P1 is a PTH/PTHrP modulating domain;
L1 is a linker; and
a is 0 or 1.
2. The composition of matter of
3. The composition of matter of
4. The composition of matter of
XN is absent or is X3X4X5X6X7, X2X3X4X5X6X7, X1X2X3X4X5X6X7, or YX1X2X3X4X5X6X7;
X1 through X7, X10, X11, X12, X14 through X28 are each independently amino acid residues;
XC is absent or is X29, X29X30 X29X30X31, X29X30X31X32, X29X30X31X32X33, X29X30X31X32X33X34, X29X30X31X32X33X34X35, or X29X30X31X32X33X34X35X36.
X29 through X36 are each independently amino acid residues;
provided that one or more of X14 through X36 is a cysteine residue.
5. The composition of matter of
XN is X1X2X3X4X5X6X7;
X1 is a hydrophilic or nonfunctional residue;
X2 is V;
X3is S;
X4 is E;
X5 is a nonfunctional or basic residue;
X6 is Q;
X7 is L;
X10 is an acidic or hydrophilic residue;
X11 is a nonfunctional or basic residue;
X12 is a nonfunctional residue;
X14 is a basic or hydrophilic residue;
X15 is a nonfunctional residue;
X16 is a nonfunctional or hydrophilic residue;
X17 is an acidic, hydrophilic, or nonfunctional residue;
X18 is a nonfunctional residue;
X19 is an acidic or basic residue;
X21 is a nonfunctional or basic residue;
X22 is a hydrophilic, acidic, or aromatic residue;
X23 is an aromatic or lipophilic residue;
X24 is a lipophilic residue (L preferred);
X25 is a hydrophilic or basic residue;
X26 is a hydrophilic or basic residue;
X27 is a lipophilic, basic, or nonfunctional residue; and
X28 is a lipophilic or nonfunctional residue.
6. The composition of matter of
XC is X29X30X31X32X33X34;
X29 is a hydrophilic or nonfunctional residue;
X30 is a hydrophilic or acidic residue;
X31 is a lipophilic or nonfunctional residue;
X32 is H;
X33 is a cysteine or a hydrophilic residue; and
X34 is a nonfunctional or aromatic residue.
7. The composition of matter of
XC is X29X30X31;
X29 is a hydrophilic or nonfunctional residue;
X30 is a hydrophilic or acidic residue; and
X31 is a lipophilic or nonfunctional residue.
8. The composition of matter of
XC is X29X30;
X29 is a hydrophilic or nonfunctional residue; and
X30 is a hydrophilic or acidic residue.
9. The composition of matter of
XC is X29; and
X29 is a hydrophilic or nonfunctional residue.
10. The composition of
11. The composition of matter of
12. The composition of matter of
X1 is A, S or Y;
X5 is H or I;
X10 is N or D;
X11 is L, R, or K;
X12 is G, F, or W;
X14 is H or S;
X15 is L or I;
X16 is Q, N, S, or A;
X17 is S, D, or L;
X18 is M, L, V or Nle;
X19 is E or R;
X21 is V, M, R, or Nle;
X22 is E or F;
X23 is W or F;
X25 is R or H;
X26 is K or H;
X27 is C; and
X28 is L or I.
13. The composition of matter of
XC is X29X30X31X32X33X34;
X29 is Q or A;
X30 is D or E;
X31 is V or I;
X33 is C;
X34 is N or T; and
X35is A, F or Y.
14. The composition of matter of
XC is X29X30X31;
X29 is Q or A;
X30 is D or E; and
X31 is V or I;
15. The composition of matter of
XC is X29X30;
X29 is Q or A; and
X30 is D or E.
16. The composition of matter of
XC is X29; and;
X29 is Q or A.
17. The composition of matter of
18. The composition of matter of
JN is absent or is selected from J1J2J3J4J5J6, J2J3J4J5J6, J3J4J5J6;
J1 is an amino acid residue;
J2 is an amino acid residue;
J3 is an amino acid residue;
J4 is an amino acid residue;
J5 is an amino acid residue;
J6 is an amino acid residue;
J7 is an amino acid residue;
J8 is an amino acid residue;
J11 is a nonfunctional or basic residue;
J12 is an amino acid residue;
J16 is an amino acid residue;
J18 is an amino acid residue;
J19 is an acidic or basic residue;
J21 is an amino acid residue;
JC is absent or is J29, J29J30, J29J30J31, J29J30J31J32, J29J30J31J32J33, J29J30J31J32J33J34; and
J29 is an amino acid residue;
J30 is an amino acid residue;
J31 is an amino acid residue;
J32 is an amino acid residue;
J33 is an amino acid residue;
J34 is an amino acid residue;
provided that one or more of J14 through the C-terminal residue is a cysteine residue.
19. The composition of matter of
JN is J1J2J3J4J5J6;
J1 is a nonfunctional or aromatic residue;
J2 is a nonfunctional residue;
J3 is a hydrophilic residue;
J4 is an acidic residue;
J5 is a nonfunctional residue;
J6 is a basic residue;
J7 is a nonfunctional or aromatic residue;
J8 is a nonfunctional residue;
J11 is a basic or a nonfunctional residue;
J12 is a nonfunctional or aromatic residue;
J16 is a nonfunctional or hydrophilic residue;
J18 is a nonfunctional residue;
J19 is an acidic or basic residue; and
J21 is a nonfunctional residue;
JC is J29J30J31J32J33J34;
J29 is a hydrophilic or nonfunctional residue;
J30 is a hydrophilic or acidic residue;
J31 is a lipophilic or nonfunctional residue;
J32 is a basic residue;
J33 is an acidic residue; and
J34 is an aromatic residue.
20. The composition of matter of
J1 is A, S or Y;
J2 is V;
J3 is S;
J4 is E;
J5 is I;
J6 is Q;
J7 is L or F;
J8 is M or Nle;
J11 is L, R, or K;
J12 is G or W;
J16 is N, S, or A;
J18 is M, Nle, L, or V;
J19 is E or R;
J21 is V, M, or Nle;
J is Q or A;
J30 is D or E;
J31 is V or I;
J32 is H;
J33 is N; and
J34 is F or Y.
21. The composition of matter of
JN is J1J2J3J4J5J6;
J1 is a nonfunctional or aromatic residue;
J2 is a nonfunctional residue;
J3 is a hydrophilic residue;
J4 is an acidic residue;
J5 is a nonfunctional residue;
J6 is a basic residue;
J7 is a nonfunctional or aromatic residue;
J8 is a nonfunctional residue;
J11 is a basic or a nonfunctional residue;
J12 is a nonfunctional or aromatic residue;
J16 is a nonfunctional or hydrophilic residue;
J18 is a nonfunctional residue;
J19 is an acidic or basic residue;
J21 is a nonfunctional residue;
JC is J29J30J31;
J29 is a hydrophilic or nonfunctional residue;
J30 is a hydrophilic or acidic residue; and
J31 is a lipophilic or nonfunctional residue.
22. The composition of matter of
J1 is A, S or Y;
J2 is V;
J3 is S;
J4 is E;
J5 is I;
J6 is Q;
J7 is L or F;
J8 is M or Nle;
J11 is L, R, or K;
J12 is G or W;
J16 is N, S, or A;
J18 is M, Nle, L, or V;
J19 is E or R;
J21 is V, M, or Nle;
J29 is Q or A;
J30 is D or E; and
J31 is V or I.
23. The composition of matter of
JN is J1J2J3J4J5J6;
J1 is a nonfunctional or aromatic residue;
J2 is a nonfunctional residue;
J3 is a hydrophilic residue;
J4 is an acidic residue;
J5 is a nonfunctional residue;
J6 is a basic residue;
J7 is a nonfunctional or aromatic residue;
J8is a nonfunctional residue;
J11 is a basic or a nonfunctional residue;
J12 is a nonfunctional or aromatic residue;
J16 is a nonfunctional or hydrophilic residue;
J18 is a nonfunctional residue;
J19 is an acidic or basic residue;
J21 is a nonfunctional residue;
JC is J29J30;
J29 is a hydrophilic or nonfunctional residue; and
J30 is a hydrophilic or acidic residue.
24. The composition of matter of
J is A, S or Y;
J2 is V;
J3 is S;
J4 is E;
J5 is I;
J6 is Q;
J7 is L or F;
J8 is M or Nle;
J11 is L, R, or K;
J12 is G or W;
J16 is N, S, or A;
J18 is M, Nle, L, or V;
J19 is E or R;
J21 is V, M, or Nle;
J29 is Q or A; and
J30 is D or E.
25. The composition of matter of
JN is J1J2J3J4J5J6;
J1 is a nonfunctional or aromatic residue;
J2 is a nonfunctional residue;
J3 is a hydrophilic residue;
J4 is an acidic residue;
J5 is a nonfunctional residue;
J6 is a basic residue;
J7 is a nonfunctional or aromatic residue;
J8 is a nonfunctional residue;
J11 is a basic or a nonfunctional residue;
J12 is a nonfunctional or aromatic residue;
J16 is a nonfunctional or hydrophilic residue;
J18 is a nonfunctional residue;
J19 is an acidic or basic residue;
J21 is a nonfunctional residue;
JC is J29; and
J29 is a hydrophilic or nonfunctional residue.
26. The composition of matter of
J1 is A, S or Y;
J2 is V;
J3 is S;
J4 is E;
J5 is I;
J6 is Q;
J7 is L or F;
J8is M or Nle;
J11 is L, R, or K;
J12 is G or W;
J16 is N, S, or A;
J18 is M, Nle, L, or V;
J19 is E or R;
J21 is V, M, or Nle; and
J29 is Q or A.
27. The composition of matter of
JN is J1J2J3J4J5J6;
J1 is a nonfunctional or aromatic residue;
J2 is a nonfunctional residue;
J3 is a hydrophilic residue;
J4 is an acidic residue;
J5 is a nonfunctional residue;
J6 is a basic residue;
J7 is a nonfunctional or aromatic residue;
J8 is a nonfunctional residue;
J11 is a basic or a nonfunctional residue;
J12 is a nonfunctional or aromatic residue;
J16 is a nonfunctional or hydrophilic residue;
J18 is a nonfunctional residue;
J19 is an acidic or basic residue;
J21 is a nonfunctional residue; and
JC is absent.
28. The composition of matter of
J1 is A, S or Y;
J2 is V;
J3 is S;
J4 is E;
J5 is I;
J6 is Q;
J7 is L or F;
J8 is M or Nle;
J11 is L, R, or K;
J12 is G or W;
J16 is N, S, or A;
J18 is M, Nle, L, or V;
J19 is E or R; and
J21 is V, M, or Nle.
29. The composition of matter of
30. The composition of matter of
ON is absent or is YO1O2O3O4O5O6O7, O1O2O3O4O5O6O7, O2O3O4O5O6O7, O3O4O5O6O7, O4O5O6O7, O5O6O7, O6O7, or O7;
O1 is an amino acid residue;
O2 is an amino acid residue;
O3 is an amino acid residue;
O4 is an amino acid residue;
O5 is an amino acid residue;
O6 is an amino acid residue;
O7 is an amino acid residue;
O10 is an amino acid residue;
O11 is an amino acid residue;
O12 is an amino acid residue;
O16 is an amino acid residue;
O17 is an amino acid residue;
O23 is an amino acid residue;
OC is absent or is O29, O29O30, O29O30O31, O29O30O31O32, O29O30O31O32O33, O29O30O31O32O33O34, O29O30O31O32O33O34O35, or O29O30O31O32O33O34O35O36; and
O29 through O36 are each independently amino acid residues;
provided that one or more of O14 through the C-terminal residue is a cysteine residue.
31. The composition of matter of
ON is O7;
O7 is a nonfunctional residue;
O10 is an acidic or hydrophilic residue;
O11 is a basic or nonfunctional residue;
O12 is an aromatic or nonfunctional residue;
O15 is a hydrophilic or nonfunctional residue;
O16 is a hydrophilic residue;
O17 is an acidic or nonfunctional residue;
O23 is an aromatic residue; and
OC is absent.
32. The composition of matter of
ON is O1O2O3O4O5O6O7;
O1 is a nonfunctional amino acid residue;
O2 is a nonfunctional amino acid residue;
O3 is a hydrophilic amino acid residue;
O4 is an acidic amino acid residue;
O5 is a basic or nonfunctional amino acid residue;
O6 is a hydrophilic amino acid residue;
O7 is a nonfunctional residue;
O10 is an acidic or hydrophilic residue;
O11 is a basic or nonfunctional residue;
O12 is an aromatic or nonfunctional residue;
O15 is a hydrophilic or nonfunctional residue;
O16 is a hydrophilic residue; and
O17 is an acidic or nonfunctional residue; and
O23 is an aromatic residue.
33. The composition of matter of
O1 is A;
O2 is V;
O3 is S;
O4 is E;
O5 is H or I;
O6 is Q;
O7 is L;
O10 is N or D;
O11 is K or L;
O12 is G, F, or W;
O15 is I or S;
O16 is Q or N;
O17 is D or L;
O23 is F or W.
34. The composition of matter of
35. The composition of matter of
36. The composition of matter of
37. A composition of matter, which comprises a peptide selected from Table 1B.
38. 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
39. 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
40. 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
41. 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
42. 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
43. The method of
44. The method of
45. The method of
46. The method of
47. The method of
48. A method of treating osteopenia, which comprises administering a composition of matter of
49. A method of treating osteopenia, which comprises administering a composition of matter of
50. A method of treating osteopenia, which comprises administering a composition of matter of
51. A method of treating osteopenia, which comprises administering a composition of matter of
52. A method of treating osteopenia, which comprises administering a composition of matter of
This application is a continuation-in-part of U.S. 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.
This invention relates to parathyroid hormone PTH), parathyroid hormone-related protein (PTHrP) and modulators of PTH and PTHrP receptors. This invention also relates to proteins modified for extended half-life and, in particular, to proteins modified with polyethylene glycol.
PTH and 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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 μg/kg, vs. 80 μg/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.
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 μg/kg/day) was shown to be anabolic in OVX rats, whereas 200 μg/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).
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, WO 00/24782, which is hereby incorporated by reference in its entirety.
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�).
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.
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.
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.
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:
Further in accordance with the present invention is a process for making PTH and PTHrP modulators, which comprises:
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.
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.
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.
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).
Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.
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
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
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).
Definition of Terms
The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances.
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.
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.
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.
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.
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.
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.�
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).
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; 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.
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.
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 September 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.
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.
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.
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
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 �NRR1, NRC(O)R1, �NRC(O)OR1, �NRS(O)2R1, �NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH�, wherein R and R1 and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by �C(O)R2 or �NR3R4 wherein R2, R3 and R4 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.
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.
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.
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 1A and 2, which are hereby incorporated by reference in their entirety.
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.
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.
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 WO 98/46751, respectively, which are hereby incorporated by reference in their entirety.
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.
Structure of Compounds
In General.
Exemplary PTH and PTHrP receptor binding amino acid sequences are described in Tables 1A, 1B, 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.
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.
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:
F1 is a vehicle (preferably a PEG molecule) and is attached at the C-terminus of P1-(L1)a or through a sidechain at any residue from residue 14 through the C-terminal residue;
P1 is a sequence of a PTH/PTHrP modulating domain;
L1 is a linker; and
a is 0 or 1.
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.
A PTH/PTHrP modulating domain sequence particularly of interest is of the formula II
XN is absent or is X3X4X5X6X7, X2X3X4X5X6X7, X1X2X3X4X5X6X7, or YX1X2X3X4X5X6X7;
X1 is an amino acid residue (nonfunctional, hydrophilic or aromatic residue preferred; A, S or Y preferred);
X2 is an amino acid residue (nonfunctional residue preferred, V most preferred);
X3 is an amino acid residue (hydrophilic residue preferred, S most preferred);
X4 is an amino acid residue (acidic residue preferred, E most preferred);
X5 is an amino acid residue (nonfunctional or basic residue preferred, H or I most preferred);
X6 is an amino acid residue (acidic or hydrophilic residue preferred, Q or E most preferred);
X7 is an amino acid residue (nonfunctional or aromatic residue preferred, L or F most preferred);
X8 is an amino acid residue (nonfunctional residue preferred, M or Nle most preferred);
X10 is an amino acid residue (an acidic or hydrophilic residue preferred, N or D most preferred);
X11 is an amino acid residue (nonfunctional or basic residue preferred, L, R, or K most preferred);
X12 is an amino acid residue (nonfunctional or aromatic residue preferred, G, F, or W most preferred);
X14 is an amino acid residue (basic or hydrophilic residue preferred, H or S most preferred);
X15 is an amino acid residue (nonfunctional residue preferred, with L or I most preferred);
X16 is an amino acid residue (nonfunctional or hydrophilic residue preferred, Q, N, S, or A most preferred);
X17 is an amino acid residue (acidic, hydrophilic, or nonfunctional residue preferred; S, D, or L most preferred);
X18 is an amino acid residue (nonfunctional residue preferred, M, L, V or Nle most preferred);
X19 is an amino acid residue (acidic or basic residue preferred, E or R most preferred);
X21 is an amino acid residue (nonfunctional residue or basic residue preferred; V, M, R, or Nle most preferred);
X22 is an amino acid residue (hydrophilic, acidic, or aromatic residue preferred, E or F most preferred);
X23 is an aromatic or lipophilic residue (W or F preferred);
X24 is a lipophilic residue (L preferred);
X25 is an amino acid residue (hydrophilic or basic residue preferred, R or H most preferred);
X26 is an amino acid residue (hydrophilic or basic residue preferred, K or H most preferred);
X27 is an amino acid residue (lipophilic, basic, or nonfunctional residue preferred, K or L most preferred);
X28 is an amino acid residue (lipophilic or nonfunctional residue preferred, L or I most preferred);
XC is absent or is X29, X29X30, X29X30X31, X29X30X31X32, X29X30X31X32X33, X29X30X31X32, X33X34, X29X30X31X32X33X34X35, or X29X30X31X32X33X34X35X36;
X29 is an amino acid residue (hydrophilic or nonfunctional residue preferred, Q or A most preferred);
X30 is an amino acid residue (hydrophilic or acidic residue preferred, D or E most preferred);
X31 is an amino acid residue (lipophilic or nonfunctional residue preferred, V or I most preferred);
X32 is an amino acid residue (basic residue preferred, H most preferred);
X33 is an amino acid residue (hydrophilic residue preferred, N or T most preferred);
X34 is an amino acid residue (nonfunctional or aromatic residue preferred, A, F or Y most preferred);
X35 is an amino acid residue (acidic residue preferred, E most preferred);
X36 is an amino acid residue (aromatic residue preferred, Y most preferred);
provided that one or more of X14 through X36 is a cysteine residue.
A preferred PTH/PTHrP modulating domain sequence formula is III
JN is absent or is selected from J1J2J3J4J5J6, J2J3J4J5J6, J3J4J5J6;
J1 is an amino acid residue (nonfunctional, hydrophilic, or aromatic residue preferred; A, S or Y most preferred);
J2 is an amino acid residue (nonfunctional residue preferred, V most preferred);
J3 is an amino acid residue (hydrophilic residue preferred, S most preferred);
J4 is an amino acid residue (acidic residue preferred, E most preferred);
J5 is an amino acid residue (nonfunctional residue preferred, I most preferred);
J6 is an amino acid residue (basic residue preferred, Q preferred);
J7 is an amino acid residue (nonfunctional or aromatic residue preferred, L or F most preferred);
J8 is an amino acid residue (nonfunctional residue preferred, M or Nle most preferred);
J12 is an amino acid residue (nonfunctional or aromatic residue preferred, G or W most preferred);
J16 is an amino acid residue (nonfunctional or hydrophilic residue preferred, N, S, or A most preferred);
J18 is an amino acid residue (nonfunctional residue preferred, M, Nle, L, or V most preferred);
J19 is an acidic or basic residue (E or R preferred);
J21 is an amino acid residue (nonfunctional residue preferred, V, M, or Nle most preferred);
JC is absent or is J29, J29J30, J29J30J31, J29J30J31J32, J29J30J31J32J33, or J29J30J31J32J33J34;
J29 is an amino acid residue (hydrophilic or nonfunctional residue preferred, Q or A most preferred);
J30 is an amino acid residue (hydrophilic or acidic residue preferred, D or E most preferred);
J31 is an amino acid residue (lipophilic or nonfunctional residue preferred, V or I most preferred);
J32 is an amino acid residue (basic residue preferred, H most preferred);
J33 is an amino acid residue (acidic residue preferred, N most preferred);
J34 is an amino acid residue (aromatic residue preferred, F or Y most preferred);
provided that one or more of J14 through the C-terminal residue is a cysteine residue.
From the formula of SEQ ID NO: 4, peptides appearing in Table 1A or 1B below are most preferred.
Another preferred PTH/PTHrP modulating domain sequence is IV
ON is absent or is YO1O2O3O4O5O6O7, O1O2O3O4O5O6O7, O2O3O4O5O6O7, O3O4O5O6O7, O4O5O6O7, O5O6O7, O6O7, or O7;
O1 is an amino acid residue (nonfunctional residue preferred, A most preferred);
O2 is an amino acid residue (nonfunctional residue preferred, V most preferred);
O3 is an amino acid residue (hydrophilic residue preferred, S most preferred);
O4 is an amino acid residue (acidic residue preferred, E most preferred);
O5 is an amino acid residue (basic or nonfunctional residue preferred, H or I preferred);
O6 is an amino acid residue (hydrophilic residue preferred, Q most preferred);
O7 is an amino acid residue (nonfunctional residue preferred, L most preferred);
O10 is an amino acid residue (acidic or hydrophilic residue preferred, N or D most preferred);
O11 is an amino acid residue (basic or nonfunctional residue preferred, K or L most preferred);
O12 is an amino acid residue (aromatic or nonfunctional residue preferred, G, F, or W most preferred);
O15 is an amino acid residue (hydrophilic or nonfunctional residue preferred, I or S most preferred);
O16 is an amino acid residue (hydrophilic residue preferred, Q or N most preferred);
O17 is an amino acid residue (acidic or nonfunctional residue preferred, D or L most preferred);
O23 is an amino acid residue (aromatic residue preferred, with F or W most preferred);
OC is absent or is O29, O29O30, O29O30O31, O29O30O31O32, O29O30O31O32O33, O29O30O31O32O33O34, O29O30O31O32O33O34O35, or O29O30O31O32O33O34O35O36; and
O29 through O36 are each independently amino acid residues; provided that one or more of O14 through the C-terminal residue is a cysteine residue.
From the formula of SEQ ID NO: 5, peptides appearing in Table 2 below are most preferred.
Exemplary peptide sequences for this invention appear in Tables 1A, 1B 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, or as described hereinafter. Molecules of this invention incorporating these peptide sequences may be prepared by methods known in the art or as described hereinafter. 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.
Another useful PTH/PTHrP modulating domain has the sequence of the peptide known as TIIP39:
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 1A, 1B, and 2. In particular, useful PTH/PTHrP modulating domains further comprise molecules in which any residue at position 14 through the C-terminal amino acid of any sequence in Tables 1A and 2 is substituted with a cysteine residue to provide an attachment site for a polymer (PEG preferred). Cysteine substitutions at position 27 through the C-terminal peptide are preferred.
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.
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).
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.
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.
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.
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).
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.
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.
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.�
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.
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.
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.
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.
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).
This invention requires the presence of at least one vehicle (F1) 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.
Fc domain.
An Fc domain is the preferred vehicle. The Fc domain may be fused to the C terminus of the peptides.
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:
Preferred Fc variants include the following. In SEQ ID NO: 2 (
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).
Polymer Vehicles.
As noted above, polymer vehicles may also be used for F1 and F2. 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.
A preferred polymer vehicle is polyethylene glycol (PEG). The chemical modification of therapeutic proteins with polyethylene glycol (PEG) has been broadly applied to improve the in vivo efficacy of protein drugs. See �Protein Conjugates� chapter in Harris, et al., American Chemical Society pp. 118-216 (1997). PEGylation achieves this effect by extending the drug's circulating half-life, increasing its solubility and in some cases, reducing the drug's toxicity and immunogenicity.
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. The PEG group may also be attached to more than one therapeutic molecule; for example, in a peptide-PEG-peptide configuration. Two PEG molecules may also be attached to multiple sites or to a single site of a therapeutic molecule; for example, two PEG molecules attached to a cysteine sidechain through a linker. The average molecular weight of the PEG will preferably range from about 2 kiloDalton (�kD�) to about 100 kDa, more preferably from about 5 kDa to about 50 kDa, most preferably from about 5 kDa, 20 kDa, or 30 kDa. In the present invention, linear monomethoxy PEGmaleimides of molecular weights in the range of 5-30 kDa are preferred, with 20-30 kDa polymers most preferred. Also preferred is a 40 kDa branched PEG-maleimide, comprised of two 20 kDa polymer �arms� joined through a linker at the peptide attachment site. Another preferred embodiment employs an 8 kDa bis-functional PEG-(maleimide)2 which can be used to generate a peptide-PEG-peptide configuration.
A variety of conjugation chemistries have been investigated for coupling PEG non-specifically to proteins. Zalipsky, Advanced Drug Delivery Reviews 16:157-182 (1995). The PEG groups can 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).
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.
Solid phase synthesis is also useful to prepare molecules PEGylated through an available amino group (e.g., a lysine sidechain) using an orthogonal protection strategy. In such synthesis, the peptide is synthesized with removable protecting groups (e.g., Dde for lysine sidechains) attached to the amino groups and any other reactive groups that are not selected for PEGylation. The synthesized peptide can then undergo a reaction resulting in PEGylation of the unprotected amino group. The protecting groups on the other reactive groups can then be removed by conventional means. This technique is especially useful for PEGylation of one of the lysine residues of the PTH fragments mentioned herein. For PTH(1-34), for example, the sidechain of one of the lysine residues at positions 13, 26, or 27 is left unprotected while the others comprise a Dde protecting group. The Dde groups are selectively removed using 2% hydrazine in water for 5 to 30 minutes at room temperature. One may use this technique to prepare a preferred molecule that is PEGylated through the sidechain of the lysine residue at position 27.
Solid phase synthesis techniques may also be employed to prepare molecules having a PEG moiety at the C-terminus. The molecule may comprise a PEG moiety linking it to a resin used for solid phase synthesis. The synthesized molecule may then be cleaved from the resin such that the PEG moiety is retained with the peptide.
Site-directed approaches may be useful to maximize retention of biological activity while minimizing conjugate heterogeneity. Site-directed PEGylation is typically achieved through a combination of recombinant protein techniques and selective conjugation chemistries. First, site-directed mutagenesis is used to introduce unique amino acids with reactive functional groups into the polypeptide sequence at positions predicted to have minimal impact on protein activity Goodson, et al, Bio/Technology 8:343-346 (1990) and Tsutsumi, et al., Proc. Natl. Acad. Sci. 97:8548-8553 (2000). Cysteine is the preferred residue for engineering directed conjugation sites because it is relatively scarce in proteins and the thiol sidechain is among the most reactive of the protein nucleophiles. Next, the mutagenic nucleic acid is introduced into a vector, which then is used to transfect a host cell (e.g., E. coli, which is preferred), and the peptide is expressed and isolated from the host cell. For the PEG portion of the molecule, an activated monofunctional PEG polymer is prepared or obtained commercially. Although there are a wide variety of activated PEG polymers available which react specifically with cysteine thiols, such as PEG-maleimide, -vinylsulfone, -iodoacetamide, -orthopyridyl-disulphide and -epoxides, among others, the PEG-maleimides are by far the most commonly used for conjugation. Zalipsky et al., pp. 347-370 (1992). Finally, the cysteine-containing protein and activated PEG are combined under appropriate reaction conditions to promote formation of a PEG-protein conjugate which is subsequently purified and characterized. PEG-maleimides are preferred PEGylating reagents, but PEGylation may also be achieved with PEG-vinylsulfones, PEG-orthopyridyl-disulphides and PEG-iodoacetamides or any other activated PEG that is selective for cysteine thiols.
Although cysteine residues are convenient substrates for PEGylation chemistry, as noted above, the PTH-(1-34) peptide and other peptides useful in this invention (e.g., see Tables 1A and 2) contains no natural Cys residues for PEG conjugation. Cys mutations can be introduced at any position, but structure-activity data suggests that C-terminal domain insertions or substitutions are preferred. In one preferred embodiment, cysteine residues were introduced into PTH-(1-34) by substituting Cys for Lysine at position 27, and/or by inserting Cys between histidine and asparagine at position 33.
Polysaccharide polymers are another type of water soluble polymer which may be used for protein modification and may be prepared by techniques generally as described above. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular 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.
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)4, (Gly)5), poly(Gly-Ala), and polyalanines. Other specific examples of linkers are:
Non-peptide linkers are also possible. For example, alkyl linkers such as �NH�(CH2)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., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary non-peptide linker is a PEG linker, VI
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:
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.
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.
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.
Carboxyl sidechain groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R′�N═C═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.
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.
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.
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.
Carbohydrate (oligosaccharide) groups may conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, 0-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.
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).
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.
Methods of Making
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.
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.
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.
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.
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.
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.
Compounds that contain derivatized peptides or which contain non-peptide groups may be synthesized by well-known organic chemistry techniques.
Uses of the Compounds
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:
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:
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
Pharmaceutical Compositions
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.
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
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 (
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.
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, NY,, 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.
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�.
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.
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.
One may dilute or increase the volume of the compound of the invention with an inert material. These diluents could include carbohydrates, especially mannitol, α-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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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 (α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84: 1145-6 (α1-proteinase); Oswein et al. (March 1990), �Aerosolization of Proteins�, Proc. Symp. Resp. Drug Delivery II, Keystone, Colo. (recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140: 3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).
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, Col.; 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.
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.
The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 μm (or microns), most preferably 0.5 to 5 μm, for most effective delivery to the distal lung.
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.
Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
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.
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 chlorofluorocarbons 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.
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.
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.
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.
Specific Preferred Embodiments
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.
All of the compounds of this invention can be prepared by methods described in (WO 00/24782).
The invention will now be further described by the following working examples, which are illustrative rather than limiting.
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.
Materials, Methods, and Results
Hypercalcemia Assay
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 (
Anabolic Assay
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:
Group 1:
Vehicle controls (PBS, injected SC, Days 1, 3, and 5)
Group 2:
OPG-Fc, single SC injection (1 mg/kg) on Day 1
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.
Group 4:
Same as group 3, but with a single OPG-Fc injection on Day 1.
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.
Group 6:
Same as group 5, but with a single OPG-Fc injection (SC, 1 mg/kg) on Day 1.
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 OPGFc, or with 3 injections of PTH-(1-34), compared to PBS-treated rats (
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.
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.
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.
Materials, Methods and Results
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) (
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 (
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.
A. cAMP Assays
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 (
B. Mouse Bioassay
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.
The clinical chemistry results are demonstrated in
The effects of the various PTH constructs on bone mineral density (proximal tibial metaphysis) are demonstrated in
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.
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 μg/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 (
Total tibial BMD and cancelled BMD were both significantly increased by daily PTH-(1-34) injections compared to vehicle-treated controls (
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 (
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.
Cys mutations were introduced into PTH(1-34) to provide PEGylation sites. The initial cysteine positions were selected based on structure/function activity data suggesting that the C-terminal domain may be most tolerant to PEGylation. These analogs represent a substitution mutant K27C, an insertion mutant at C33 and the dual mutant combining both cysteine positions:
Additional cysteine analogs of PTH (1-34) have also been prepared (see Table 1B).
Chemical Reagents.
All Fmoc-amino acids and Fmoc-Phe-Wang resin was obtained from Midwest Bio-Tech. (Fishers, Ind.). HOBt/DMF and DCC/NMP were purchased from Q Bio-Gene (Carlsbad, Calif.). HBTU was purchased from either Q Bio-Gene (Carlsbad, Calif.) or NovaBiochem (San Diego, Calif.) Bulk solvents: ACN, MeOH, DCM, DMF, and NMP were purchased from EM Science (Gibbstown, N.J.). NEM and piperidine were obtained from Aldrich (Milwaukee, Wis.). Phenol, and TIS were purchased from Sigma (St. Louis, Mo.). TFA was purchased from either PE Applied Biosystems (Foster City, Calif.) or Chem-Impex (Wood Dale, Ill.). All reagents for amino acid analysis were purchased from PE Applied Biosystems (Foster City, Calif.). All chemicals were of the highest grade possible.
Solid-Phase Peptide Synthesis of PTH(1-34) and Analogues.
All peptides were prepared by solid phase synthesis using the Fmoc/t-butyl-based methodology with Fmoc-Phe-Wang or Fmoc-Cyc(Trt)-Wang resin as a solid support. The sidechain protection scheme is as follows: Arg(Pbf), Asn(Trt), Asp(OtBu), Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc), Ser(tBu), and Trp(Boc). A PE Applied Biosystems (Foster City, CA) 431 instrument or a Rainin Symphony Multiplex Peptide Synthesizer (Protein Technologies Inc, Woburn, Mass.) with the manufacturer's standard Fmoc program was utilized for solid phase synthesis peptide chain assembly at either 0.05, 0.10, or 0.20 mmol scale. Each coupling consisted of the following modules: (i) removal of the α-amino Fmoc protection by piperidine in NMP (ABI instrument) or NEM in DMF (Rainin instrument); (ii) activation with either HBTU or DCC followed by (iii) single 60-minute coupling of the HOBt ester of the Fmoc amino acid (20, 10, or 5 equivalents) in NMP or DMF; (iv) resin washes.
Upon completion of the automated synthesis, the peptide-resin was dried in vacuo and subjected to acidolytic deprotection and cleavage (TFA:H2O:TIS:EDT:thioanisol (85:5:5:2.5:2.5, 10 ml, 4 hr, 20� C.). After cleavage and deprotection, the resin/peptide suspension was filtered, peptide-filtrate was precipitated using cold diethyl ether (40 ml), pelleted by centrifugation, washed with cold diethyl ether (2x , 40 ml), and dried in vacuo or by speedvac.
The precipitated crude peptides were analyzed by HPLC-MS for crude purity and expected molecular ion. Analytical HPLC was carried out on a Vydac (Hesperia, Calif.) 214TPTM C18 column (300 Å pore size, 5m particle size, 0.46�25 cm). The conditions for this and all subsequent primary analytical RP-HPLC were linear gradients of 5-50% ACN in 0.1% aqueous TFA over 25 minutes, and a flow rate of 1.0 ml/min, unless otherwise noted. The effluent was monitored with a PDA detector from which the 220 nm absorbance profile was extracted.
Electrospray Mass Spectrometry.
Mass spectra of all peptides were obtained with an PE-Sciex API-I (Thornhill, ON) single quadrupole electrospray mass spectrometer equipped with a nebulizer-assisted electrospray source. Samples of preparative HPLC fractions were introduced by means of an Alltech (Deerfield, Ill.) 426 HPLC pump and Perkin Elmer (Norwalk, Conn.) Series 200 autosampler, using a 0.40 ml/min isocratic gradient of 1 mM NH4OAc in ACN:H20 (50:50). The mass spectrometer was scanned from mass to charge (m/z) 300 to 2400 for the characterization of the purified peptide. The acquisition time was 0.20 minutes, and 10 scans were used for each spectrum. Masses were calculated from the m/z ratios from the observed protonation states of the peptide using Sciex (Thornhill, ON) MacSpec� software. Theoretical masses were calculated using Sciex (Thornhill, ON) MacBioSpec� software
Secondary RP-HPLC Analysis.
All peptides were subjected to two additional analytical RP-HPLC protocols to ensure peak homogeneity. Samples were analyzed on a YMC (Wilmington, Calif.) ODS-AQ C18 column (2.0�50 mm). The conditions for this chromatography were a linear gradient of 5 to 50% ACN in 0.1% aqueous TFA over 45 minutes, and a flow rate of 0.4 ml/min. Samples were also analyzed on a Vydac (Hesperia, Calif.) 218TPTM C18 column (300 Å pore size, 5 μm particle size, 0.46�25 cm). The conditions for this analysis were a linear gradient of 5 to 20% ACN in 0.1% aqueous TFA over 10 minutes, followed by 20 to 50% ACN in 0.1% aqueous TFA over 40 minutes, and a flow rate of 1 ml/min. All secondary analytical RP-HPLC were monitored with a PDA detector from which the 220 nm absorbance result was extracted.
Amino Acid Analysis.
Qualitative amino acid analyses were performed on purified peptides. All analyses were performed on an PE Applied Biosystems (Foster City, Calif.) 420A derivatizer with a 130A separation system. The peptides were manually hydrolyzed in vacuo using 6 N HCl at 150� C. for 60 minutes and then applied (approximately 1 μg) to the amino acid analyzer for derivatization as the PTC derivative. The amino acid mixture was then separated by RP-HPLC on a Brownlee PTC C18 column (2.1�220 mm) with a linear gradient of 2% to 11% ACN in H20 over 4 minutes, 11% to 27% ACN in H20 over 6 minutes, and 27% to 47% ACN in H2O over 10 minutes. Both solvents were buffered with NaOAc to pH 5.4, and the flow rate was 0.3 ml/min. Quantitative amino acid analyses were performed in triplicate, and composition was determined by comparison of each amino acid peak area in the peptide to the area of a known amount of amino acid standard.
The PTH (1-34) cysteine analogs were PEGylated with a variety of PEG-maleimides, as shown in Reaction Scheme 1 below.
The PEGylation reactions were carried out in 20 mM sodium phosphate, 5 mM EDTA, pH 6.5 with PTH (1-34) peptide concentrations from 2-10 mg/ml and PEG:peptide stoichiometry of 0.5 to 5 fold molar excess. Although reactant concentrations may exceed these limits, the preferred conditions are 5 mg/ml peptide with an equimolar PEG-maleimide concentration. Reaction times may range from 15 minutes to overnight at room temperature or 4 degrees C., with 2 hours at room temperature preferred. Optionally, the reaction may be stopped with the addition of excess mercaptan, such as β-mercaptoethanol. It is further anticipated that PTH (1-34) PEGylation can be achieved by coupling in non-aqueous solvents or through orthogonal approaches during peptide synthesis. The PEG-PTH (1-34) conjugates were then purified by aqueous phase cation exchange chromatography (
A list of PEGylated PTH peptides appears in Table 5 below
In vitro Characterization by cAMP Assay.
PEG-PTH molecules were screened in vitro for evidence of PTH receptor (PTHR1) activation by monitoring production of cAMP in murine MC3T3-E1 osteoblast cultures. PTH-(1-34) peptide consistently demonstrates the greatest potency in this assay, while PTH-(1-34)-Fc is a full agonist with slightly reduced potency. The mutant peptide analogs used for PEGylation (C27, C33 and C27/33) had potencies in the cAMP assay that were similar to each other and to PTH-(1-34)-Fc (
In vivo Characterization:
Hypercalcemia assays. PEGPTH molecules were also screened for evidence of sustained PTHR1 activation in young male BDF1 mice. Briefly, mice were injected subcutaneously with PEG-PTH or control constructs and blood ionized calcium (BIC) levels were monitored for up to 120 hours. Increases in BIC reflect increased bone resorption, which is a pharmacologic response to PTH treatment. PTH-(1-34) peptide causes a transient hypercalcemic response in mice which ends within about 6 hours of injection. In contrast, the hypercalcemia induced by equimolar injections with PTH-(1-34)-Fc lasts for approximately 72 hours (
Other PEG-PTH constructs caused hypercalcemic responses which were similar to those observed with equimolar doses of PTH-(1-34)-Fc, including:
The C27-30K construct caused the greatest hypercalcemic response in this assay, and this peptide and C33-30K were then tested in a dose-response hypercalcemia assay. At equimolar doses, C27-30K and C33-30K PEG-PTH molecules were 3-10 fold more potent than PTH-(1-34)-Fc in hypercalcemia assays (
In vivo Characterization:
Bone anabolic assays. PEG-PTH molecules were also tested for bone anabolic activity in adult male BDF1 mice. The doses were chosen based on the relative potencies of C27-30K PEG-PTH and PTH-(1-34)-Fc in mouse hypercalcemia assays. PTH-(1-34)-Fc at 150 nmol/kg caused a significant increase in tibial trabecular bone mineral density (BMD) when injected twice-weekly for 4 weeks. C27-30K PEG-PTH was similarly anabolic at 3-10 fold lower doses than PTH-(1-34)-Fc when injected twice-weekly (
Discussion and Conclusions.
These data indicate the feasibility of using PEGylation to increase the circulating half-life of PTH peptides. The longer half-life conferred by PEGylation permits infrequent subcutaneous dosing in rodents to obtain bone anabolic effects. The pharmacologic effects of PEG-PTH are similar to those observed with PTH-Fc constructs, with respect to CAMP activation in vitro as well as hypercalcemic and bone anabolic effects in vivo. These similarities in pharmacology suggest that increasing the half-life of PTH peptides, regardless of the means, could represent an important therapeutic advance. Neither Fc nor PEG are absolutely required to realize the therapeutic benefit of a long-acting PTH molecule. These data would also predict that a slow-release formulation of PTH peptides such as those described in Tables 1A, 1B and 2 would achieve the same desired benefit of less frequent injections.
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.
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