Patent Application
20190119348
This invention relates to parathyroid hormone receptor 1 antagonists or inverse agonists. The invention also relates to compositions of the parathyroid hormone peptides and methods of their use.
Excessive signaling activity of parathyroid hormone receptor 1 (PTHR1) is known to be associated with diseases, such as hypercalcemia, hypophosphatemia, hyperparathyroidism, and Jansen's chondrodysplasia. These diseases can arise from overproduction of either of the two endogenous PTHR1 ligands—PTH, as in primary or secondary hyperparathyroidism (HPT), or PTH-related protein (PTHrP), as in humoral hypercalcemia of malignancy. These diseases are characterized by high levels of blood calcium, excessive urinary excretion of calcium and/or phosphate, and can further be associated with abnormal bones, due to alterations in bone formation/resorption activities mediated by the PTHR1.
Regulation of extracellular calcium concentration is necessary for the normal function of the gastrointestinal, skeletal, neurologic, neuromuscular, and cardiovascular systems. PTH synthesis and release are controlled principally by the serum calcium level; a low level stimulates and a high level suppresses both hormone synthesis and release. PTH, in turn, maintains the serum calcium level by directly or indirectly promoting calcium entry into the blood at three sites of calcium exchange: gut, bone, and kidney. PTH contributes to net gastrointestinal absorption of calcium by favoring the renal synthesis of the active form of vitamin D. PTH promotes calcium resorption from bone indirectly by stimulating differentiation of the bone-resorbing cells, osteoclasts. It also mediates at least three main effects on the kidney: stimulation of tubular calcium reabsorption, enhancement of phosphate clearance, and promotion of an increase in the enzyme that completes synthesis of the active form of vitamin D.
Disruption of calcium homeostasis may produce many clinical conditions (e.g., severe bone disease, anemia, renal impairment, ulcers, myopathy, and neuropathy) and usually results from conditions that produce an alteration in the level of parathyroid hormone. Hypercalcemia is a condition that is characterized by an elevation in the serum calcium level. It is often associated with primary hyperparathyroidism in which an excess of PTH production occurs as a result of a parathyroid gland lesion (e.g., adenoma, hyperplasia, or carcinoma). Another type of hypercalcemia, humoral hypercalcemia of malignancy (HHM), is a common paraneoplastic syndrome. It appears to result in most instances from the production by tumors (e.g., squamous, renal, ovarian, or bladder carcinomas) of a class of protein hormone which shares amino acid homology with PTH. These PTH-related proteins (PTHrP) appear to mimic certain of the renal and skeletal actions of PTH and are believed to interact with the PTH receptor in these tissues.
Antagonist ligands for the parathyroid hormone receptor 1 (PTHR1) can be useful for treating diseases associated with excessive signaling activity at the PTHR1. Some of the antagonist ligands may function as inverse agonists.
There is a need for PTHR1 antagonists and inverse agonists, particularly, those that retain high affinity for PTHR1.
In general, the present invention provides PTHR1 antagonist or inverse agonist peptides. These peptides can be used in a method of treating a condition or a disease of signaling overactivity of PTHR1. The condition or disease may be associated with higher than normal serum levels of calcium, with lower than normal serum levels of phosphate, with higher than normal levels of endogenous PTHR1 agonist(s), or with constitutive activity of PTHR1 mutants.
In one aspect, the invention provides a polypeptide or a pharmaceutically acceptable salt thereof including an N-terminally truncated PTH/PTHrP hybrid peptide or a fragment thereof (e.g., a fragment containing from 24 to 32 amino acid residues of the N-terminally truncated PTH/PTHrP hybrid peptide), where the polypeptide is a PTHR1 antagonist or a PTHR1 inverse agonist.
In some embodiments, the polypeptide is the N-terminally truncated PTH/PTHrP hybrid peptide is of formula (I):
where
X01 is Met, Leu, or Nle;
X02 is Asn, Ala, Val, Asp, Glu, or Gln;
X03 is Leu, Ala, Val, Met, Lys, Ile, Arg, Har, or Trp;
X04 is Gly, Ala, His, Arg, or dTrp;
X05 is Lys, Ala, Leu, Gln, Arg, His, or Trp;
X06 is His, Leu, Arg, Phe, Trp, or Ala;
X07 is Ile or Leu;
X08 is Gln or Asn;
X09 is Asp or Ser;
X10 is Ala, Leu, Met, Glu, Ser, or Phe;
X11 is Ala, Phe, Glu, Ser, Leu, Asn, Trp, or Lys;
X12 is Phe or Trp;
X13 is His, Arg, Leu, Trp, or Lys;
X14 is Lys, His, Ala, Ser, Asn, or Arg; and
X15 is Ile, Cys, or Tyr;
or a fragment thereof containing from 24 to 32 contiguous amino acid residues of formula (I).
In certain embodiments, the polypeptide is a fragment of the N-terminally truncated PTH/PTHrP hybrid peptide containing amino acid residues 1-32 of formula (I). In further embodiments, the polypeptide is a fragment of the N-terminally truncated PTH/PTHrP hybrid peptide containing amino acid residues 3-32 of formula (I). In particular embodiments, the polypeptide is a fragment of the N-terminally truncated PTH/PTHrP hybrid peptide containing amino acid residues 3-33 of formula (I).
In further embodiments, X01 is Met, X04 is Ala, X12 is Phe, and X15 is Ile. In yet further embodiments, X01 is Met, X04 is dTrp, X12 is Trp, and X15 is Ile. In still further embodiments, X01 is Nle, X04 is dTrp, X12 is Trp, and X15 is Tyr. In some embodiments, X01 is Nle, X04 is dTrp, X12 is Trp, and X15 is Cys. In certain embodiments, X01 is Nle, X04 is dTrp, X12 is Trp, and X15 is Ile. In particular embodiments, X01 is Met, X04 is dTrp, X12 is Trp, and X15 is Ile. In some embodiments, X04 is dTrp. In certain embodiments, X12 is Trp.
In other embodiments, the N-terminally truncated PTH/PTHrP hybrid peptide has the amino acid sequence
or a 3-32 or 3-33 fragment thereof.
In yet other embodiments, the polypeptide contains a radionuclide, a polyethylene glycol, or a dye.
In another aspect, the invention provides a pharmaceutical composition containing the polypeptide of the invention and a pharmaceutically acceptable carrier.
In yet another aspect, the invention provides a method of antagonizing or inversely agonizing the activity of parathyroid hormone receptor 1 (PTHR1) in a cell by contacting the cell with the polypeptide of the invention.
In some embodiments, the cell is a human cell.
In a further aspect, the invention provides a method of treating a disease or condition associated with PTHR1 signaling overactivity by administering to the subject an effective amount of the polypeptide of the invention or the pharmaceutical composition of the invention.
In certain embodiments, the disease or condition is hypercalcemia, hypophosphatemia, hyperparathyroidism, or Jansen's chondrodysplasia. In particular embodiments, the administering involves subcutaneous, intravenous, intranasal, transpulmonary, transdermal, transmucosal, or oral administration of the polypeptide or the pharmaceutical composition to the subject.
The invention is also described by the following items.
1. A polypeptide or a pharmaceutically acceptable salt thereof comprising an N-terminally truncated PTH/PTHrP hybrid peptide or a fragment thereof, wherein said polypeptide is a PTHR1 antagonist or a PTHR1 inverse agonist.
2. The polypeptide of item 1, wherein the N-terminally truncated PTH/PTHrP hybrid peptide is of formula (I):
wherein
X01 is Met, Leu, or Nle;
X02 is Asn, Ala, Val, Asp, Glu, or Gln;
X03 is Leu, Ala, Val, Met, Lys, Ile, Arg, Har, or Trp;
X04 is Gly, Ala, His, Arg, or dTrp;
X05 is Lys, Ala, Leu, Gln, Arg, His, or Trp;
X06 is His, Leu, Arg, Phe, Trp, or Ala;
X07 is Ile or Leu;
X08 is Gln or Asn;
X09 is Asp or Ser;
X10 is Ala, Leu, Met, Glu, Ser, or Phe;
X11 is Ala, Phe, Glu, Ser, Leu, Asn, Trp, or Lys;
X12 is Phe or Trp;
X13 is His, Arg, Leu, Trp, or Lys;
X14 is Lys, His, Ala, Ser, Asn, or Arg; and
X15 is Ile, Cys, or Tyr;
or a fragment thereof comprising from 24 to 32 contiguous amino acid residues of formula (I).
3. The polypeptide of item 2, wherein the polypeptide is a fragment of the N-terminally truncated PTH/PTHrP hybrid peptide comprising amino acid residues 1-32 of formula (I).
4. The polypeptide of item 2, wherein the polypeptide is a fragment of the N-terminally truncated PTH/PTHrP hybrid peptide comprising amino acid residues 3-32 of formula (I).
5. The polypeptide of item 2, wherein the polypeptide is a fragment of the N-terminally truncated PTH/PTHrP hybrid peptide comprising amino acid residues 3-33 of formula (I).
6. The polypeptide of any one of items 2 to 5, wherein X01 is Met or Nle.
7. The polypeptide of any one of items 2 to 6, wherein X02 is Asn or Gln.
8. The polypeptide of any one of items 2 to 7, wherein X03 is Leu, Arg, or Har.
9. The polypeptide of any one of items 2 to 8, wherein X05 is Lys.
10. The polypeptide of any one of items 2 to 9, wherein X06 is His or Trp.
11. The polypeptide of any one of items 2 to 10, wherein X10 is Ala, Leu, or Met; X11 is Ala or Phe; X13 is His or Arg; and X14 is Lys or His.
12. The polypeptide of item 11, wherein X10 is Ala, X11 is Ala, X13 is His, and X14 is Lys.
13. The polypeptide of any one of items 2 to 12, wherein X07 is Ile; X08 is Gln; and X09 is Asp.
14. The polypeptide of any one of items 2 to 13, wherein X04 is Gly, Ala, or dTrp.
15. The polypeptide of item 1 or 2, wherein the N-terminally truncated PTH/PTHrP hybrid peptide has the amino acid sequence
or a 3-32 or 3-33 fragment thereof.
16. The polypeptide of any one of items 1 to 15, further comprising a radionuclide, a polyethylene glycol, or a dye.
17. A pharmaceutical composition comprising the polypeptide of any one of items 1 to 16 and a pharmaceutically acceptable carrier.
18. A method of antagonizing the activity of parathyroid hormone receptor 1 (PTHR1) in a cell, the method comprising contacting the cell with the polypeptide of any one of items 1 to 16, wherein, after the contacting, the activity of PTHR1 in the cell is antagonized.
19. A method of inversely agonizing the activity of parathyroid hormone receptor 1 (PTHR1) in a cell, the method comprising contacting the cell with the polypeptide of any one of items 1 to 16, wherein, after the contacting, the activity of PTHR1 in the cell is inversely agonized.
20. The method of item 18 or 19, wherein the cell is a human cell.
21. A method of treating a disease or condition associated with a PTHR1 signaling overactivity, the method comprising administering to the subject an effective amount of the polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17.
22. A method of treating hypercalcemia in a subject, the method comprising administering to the subject an effective amount of the polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17.
23. A method of treating hypophosphatemia in a subject, the method comprising administering to the subject an effective amount of the polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17.
24. A method of treating hyperparathyroidism in a subject, the method comprising administering to the subject an effective amount of the polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17.
25. A method of treating Jansen's chondrodysplasia in a subject, the method comprising administering to the subject an effective amount of the polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17.
26. The method of item 25, wherein the polypeptide is as defined in any one of items 2 to 16, and wherein Xo4 is dTrp.
27. The method of any one of items 21 to 26, wherein the administering comprises subcutaneous, intravenous, intranasal, transpulmonary, transdermal, transmucosal, or oral administration of the polypeptide or the pharmaceutical composition to the subject.
28. The method of any one of items 21 to 27, wherein the subject is a human.
29. The polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17 for treating a disease or condition associated with PTHR1 signaling overactivity in a subject.
30. The polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17 for treating hypophosphatemia in a subject.
31. The polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17 for treating hyperparathyroidism in a subject.
32. The polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17 for treating Jansen's chondrodysplasia in a subject.
33. The polypeptide or the pharmaceutical composition of item 31, wherein the polypeptide is as defined in any one of items 2 to 16, and wherein X04 is dTrp.
34. The pharmaceutical composition of any one of items 29 to 33 formulated for subcutaneous, intravenous, intranasal, transpulmonary, transdermal, transmucosal, or oral administration to the subject.
35. Use of the polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17 in the manufacture of a medicament for treating a disease or condition associated with a PTHR1 signaling overactivity in a subject.
36. Use of the polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17 in the manufacture of a medicament for treating hypercalcemia in a subject.
37. Use of the polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17 in the manufacture of a medicament for treating hypophosphatemia.
38. Use of the polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17 in the manufacture of a medicament for treating hyperparathyroidism in a subject.
39. Use of the polypeptide of any one of items 1 to 16 or the pharmaceutical composition of item 17 in the manufacture of a medicament for treating Jansen's chondrodysplasia.
40. The use of item 39, wherein the polypeptide is as defined in any one of times 2 to 16, and wherein X04 is dTrp.
41. The use any one of items 35 to 40, wherein the medicament is formulated for subcutaneous, intravenous, intranasal, transpulmonary, transdermal, transmucosal, or oral administration to the subject.
The term “dye” is used herein to mean an agent known in the art to be useful in the imaging of biological systems (e.g., a fluorescent dye (e.g., tetramethylrhodamine)).
The term “effective amount,” when used in reference to treating a condition or disease (e.g., hypercalcemia, hypophosphatemia, hyperparathyroidism, or Jansen's chondrodysplasia), refers to an amount of a polypeptide of the invention or a pharmaceutically acceptable salt thereof that treats the condition or disease in a subject.
The term “endogenous agonist” of a parathyroid hormone receptor 1 (PTHR1) is used herein to mean a compound produced by an organism, or a synthetic phenocopy of that compound, i.e., a compound having the same pharmacological activity as the endogenous agonist. For example, the native PTH peptide is (1-84), and PTHrP is ˜(1-140) amino acids; phenocopies of these ligands include PTH(1-34) and PTHrP(1-36), respectively. An endogenous agonist is involved in or modulates the normal physiological activation of the PTHR1. PTHR1 has multiple endogenous agonists (e.g., PTH and PTHrP).
The term “fragment,” when used in reference to an N-terminally truncated PTH/PTHrP hybrid peptide, refers to a portion of the N-terminally truncated PTH/PTHrP hybrid peptide. Thus, a 1-n fragment of formula (I) refers to a polypeptide having a sequence that starts at the first N-terminal amino acid residue in formula (I) and ends at the nth amino acid residue in formula (I). Similarly, a 3-n fragment of formula (I) refers to a polypeptide having a sequence that starts at the third N-terminal amino acid residue in formula (I) and ends at the nth amino acid residue in formula (I).
The term “N-terminally truncated PTH/PTHrP hybrid peptide” is used herein to mean a compound including PTH(X-Y)/PTHrP(Z-37) peptide (e.g., hPTH(X-Y)/hPTHrP(Z-37) peptide), where X is from 2 to 7 (e.g., X is from 5 to 7), Y is from 11 to 18 (e.g., Y is 14), and Z is Y+1, where PTH has a sequence of a 34-amino acid residue-long portion of the parathyroid hormone peptide (e.g., hPTH having a sequence of SEQ ID NO:10 and with the numbering starting at the first N-terminal residue of SEQ ID NO:10 for hPTH), and PTHrP having a sequence of a 37-amino acid residue-long portion of the parathyroid hormone related peptide (e.g., hPTHrP having a sequence of SEQ ID NO:11, and with the numbering starting at the first N-terminal residue of SEQ ID NO:11 for PTHrP). The PTH(5-Y)/PTHrP(Z-37) peptide may be wt-hPTH(5-Y)/wt-hPTHrP(Z-37), which, in some embodiments, includes from 1 to 14 amino acid substitutions in the amino acid sequence of wt-hPTH(X-Y)/wt-hPTHrP(Z-37) peptide. Likewise, PTH(7-Y)/PTHrP(Z-37) peptide may be wt-hPTH(7-Y)/wt-HPTHrP(Z-37), which, in some embodiments, includes from 1 to 14 amino acid substitutions in the amino acid sequence of the wt-hPTH(X-Y)/wt-hPTHrP(Z-37) peptide. PTH(X-Y)/PTHrP(Z-37) may be abbreviated herein as LA-PTH(X-37). Similarly, a fragment of PTH(X-Y)/PTHrP(Z-37), in which the 37th amino acid residue is absent, may be abbreviated herein as LA-PTH(X-36).
The terms “polypeptide” and “peptide” are used interchangeably herein to mean a compound that contains a sequence of amino acids bonded to each other through peptidic bonds. A polypeptide or peptide includes at least 10 amino acids.
The term “PTHR1” is used herein to mean a parathyroid hormone receptor 1 (e.g., a human parathyroid hormone receptor 1 (hPTHR1)). PTHR1 may be wild-type or may be a naturally-occurring mutant PTHR1 which has constitutive activity (e.g., PTHR1 expressed in cells of a subject having Jansen's chondrodysplasia). For example, a naturally-occurring mutant PTHR1 which has constitutive activity can be PTHR1-H223R or PTHR1-T410P.
The term “PTHR1 antagonist” is used herein to mean a polypeptide capable of binding PTHR1, thereby blocking or dampening endogenous agonist-mediated responses without agonizing the signaling activity of PTHR1. The activity of PTHR1 antagonist may be assessed using methods known in the art for assessing antagonist activity or using methods described herein.
The term “PTHR1 inverse agonist” is used herein to mean a polypeptide capable of binding PTHR1 having a constitutive activity and, upon binding, reducing the constitutive activity of PTHR1. The activity of PTHR1 inverse agonist may be assessed using methods known in the art for assessing inverse agonist activity or using methods described herein.
The term “radionuclide” is used herein to mean a radioactive isotope known in the art to be useful in imaging of biological systems.
The term “subject” is used herein to mean a mammal (e.g., a human) diagnosed by a medical practitioner as having a condition or disease, e.g., a disease associated with the PTHR1 signaling overactivity (e.g., hypercalcemia, hypophosphatemia, hyperparathyroidism, or Jansen's chondrodysplasia). Diagnosis may be performed by techniques and methods known in the art. A subject to be treated according to the methods of the invention may have been subjected to standard tests (e.g., tests for serum calcium levels or serum phosphate levels) or may have been identified, without such tests, as one at high risk due to the presence of one or more risk factors (e.g., diseases associated with elevated serum calcium levels (e.g., cancer, tuberculosis, and sarcoidosis) and therapeutic regimens increasing the release of parathyroid hormone (e.g., lithium) or reducing serum phosphate levels (e.g., antacids)).
The terms “treating” or “treatment,” when used herein in reference to a subject, are used herein to mean ameliorating at least one symptom of a condition or disease in a subject having the condition or disease (e.g., a subject diagnosed with hyperparathyroidism, hypercalcemia, hypophosphatemia, or Jansen's chondrodysplasia), as compared with an equivalent untreated control. Such reduction in the symptom (e.g., a reduction in serum calcium levels or an increase in serum phosphate levels) is at least 5% (e.g., at least 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%), as measured in accordance with methods recognized in the art as suitable for assessing the symptom (e.g., serum calcium or phosphate levels).
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. For any term present in the art which is identical to any term expressly defined in this disclosure, the term's definition presented in this disclosure will control in all respects. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods and materials are described herein. Other features and advantages of the invention will be apparent from the following Detailed Description, the drawings, and the claims.
In general, the present invention provides PTHR1 antagonist or inverse agonist peptides. The PTHR1 antagonist or inverse agonist peptides of the invention can be used in a method of treating a condition or a disease of the PTHR1 signaling overactivity (e.g., hypercalcemia, hypophosphatemia, hyperparathyroidism, and Jansen's chondrodysplasia). These diseases are typically associated with higher than normal serum levels of calcium, with lower than normal serum levels of phosphate, with higher than normal levels of endogenous PTHR1 agonist(s), or with constitutive activity of PTHR1 mutants.
Advantageously, the polypeptides of the invention can have higher affinity for PTHR1 in comparison to previously reported PTH(7-34) peptides. This advantageous property may be present despite the absence of N-terminal residues that are believed to contribute to overall binding affinity of PTH(1-34) and PTHrP(1-36). Without wishing to be bound by theory, this advantageous property of the polypeptides of the invention is due to their reduced rate of dissociation from PTHR1. A further advantageous attribute of the polypeptides of the invention can be in their effecting a prolonged reduction in the activity of PTHR1 in comparison to, e.g., an N-terminally truncated PTH (e.g., (7-34)PTH).
The polypeptides of the invention can contain an N-terminally truncated PTH/PTHrP hybrid peptide of formula (I):
wherein
X01 is Met, Leu, or Nle;
X02 is Asn, Ala, Val, Asp, Glu, or Gln;
X03 is Leu, Ala, Val, Met, Lys, Ile, Arg, Har, or Trp;
X04 is Gly, Ala, His, Arg, or dTrp;
X05 is Lys, Ala, Leu, Gln, Arg, His, or Trp;
X06 is His, Leu, Arg, Phe, Trp, or Ala;
X07 is Ile or Leu;
X08 is Gln or Asn;
X09 is Asp or Ser;
X10 is Ala, Leu, Met, Glu, Ser, or Phe;
X11 is Ala, Phe, Glu, Ser, Leu, Asn, Trp, or Lys;
X12 is Phe or Trp;
X13 is His, Arg, Leu, Trp, or Lys;
X14 is Lys, His, Ala, Ser, Asn, or Arg; and
X15 is Ile, Cys, or Tyr;
or a fragment thereof including from 24 to 32 contiguous amino acid residues of formula (I).
In some embodiments, a fragment of a polypeptide of formula (I) is a peptide including from 30 to 32 contiguous amino acid residues. In certain embodiments, a fragment of a polypeptide of formula (I) is a peptide including amino acid residues 1-32 of formula (I), amino acid residues 3-32 of formula (I), or amino acid residues 3-33 of formula (I).
Exemplary PTHR1 antagonist/inverse agonist peptides are listed in Table 1.
In Table 1, dW is dTrp, K′ is Lys conjugated to tetramethyl rhodamine, and C′ stands for Cys conjugated to PEG20. In Table 1, polypeptide LA-PTH(5-36)-1950 is a PTHR1 antagonist, and the remaining polypeptides are antagonists/inverse agonists. In Table 1, polypeptides 1949 and 1952 are duplicates.
The polypeptides of the invention are amenable to production by solution- or solid-phase peptide synthesis and by in-situ synthesis using combination chemistry. The solid phase peptide synthesis technique, in particular, has been successfully applied in the production of human PTH and can be used for the production of these compounds (for guidance, see, e.g., Fairwell et al., Biochem. 22:2691, 1983). Success with producing human PTH on a relatively large scale has been reported in Goud et al., J Bone Min Res 6:781, 1991. The peptide chemical synthesis approach generally entails the use of automated synthesizers and appropriate resin as solid phase, to which the C-terminal amino acid of a desired polypeptide is attached. Extension of the peptide in the N-terminal direction is then achieved by successively coupling a suitably protected form of the next desired amino acid, typically using chemical protocols based on amino-protecting groups (e.g., Fmoc-or Boc-based), until synthesis is complete. Protecting groups are then cleaved from the peptide, usually with concomitant cleavage of the peptide from the resin, and the peptide is then isolated and purified using conventional techniques, such as by reversed phase HPLC using appropriate mobile phase (e.g., acetonitrile as solvent and tri-fluoroacetic acid as ion-pairing agent). Such procedures are generally described in numerous publications and reference may be made, for example, to Stewart and Young, “Solid Phase Peptide Synthesis,” 2nd Edition, Pierce Chemical Company, Rockford, Ill. (1984).
Polypeptides of the invention can also be made recombinantly by any method known in the art. Prokaryotic (e.g., bacterial) and eukaryotic (e.g., yeast and mammalian) expression systems can also be used to produce polypeptides of the invention, particularly, where the polypeptide includes only proteinogenic amino acids.
The polypeptides of the invention can include the dTrp12 modification. The dTrp12-modified polypeptides of the invention, in addition to their antagonist activity, can typically function as PTHR1 inverse agonists.
Further modifications may be included in the polypeptides of the invention (e.g., N-terminal or C-terminal modifications). The polypeptides of the invention typically include amino acids with side chains amenable to modification, for example, through ester or thioester formation (e.g., Ser, Thr, Tyr, Glu, and Asp), amide formation (e.g., Lys, Glu, and Asp), ether formation (e.g., Ser, Thr, Cys), or amine formation (e.g., Lys). For example, the polypeptides of the invention can be modified to include, e.g., a dye (e.g., tetramethylrhodamine (TMR)) or polyethylene glycol (PEG). The inclusion of a dye can permit tracking the polypeptide of the invention in cells or in vivo through the use of fluorescence. The inclusion of a polyethylene glycol (PEG) group can enhance pharmacokinetic properties of the polypeptide of the invention.
Any of the polypeptides of the invention may further include a heterologous sequence (a fusion partner), thus forming a fusion protein. The fusion protein may include a fusion partner such as a purification or detection tag, for example, proteins that may be detected directly or indirectly such as green fluorescent protein, hemagglutinin, or alkaline phosphatase), DNA binding domains (for example, GAL4 or LexA), gene activation domains (for example, GAL4 or VP16), purification tags, or secretion signal peptides (e.g., preprotrypsin signal sequence). In other embodiments the fusion partner may be a tag, such as c-myc, poly histidine, or FLAG. Each fusion partner may contain one or more domains, e.g., a preprotrypsin signal sequence and FLAG tag. In other cases, the fusion partner is an Fc protein (e.g., mouse Fc or human Fc).
The polypeptides disclosed herein may be formulated in a pharmaceutical composition providing an effective amount of the PTHR1 antagonist or inverse agonist to a subject upon administration. The pharmaceutical compositions of the polypeptides disclosed herein can contain an appropriate amount of a suitable carrier or excipient. The pharmaceutical compositions may contain from 0.1% to 95% (w/v) or (w/w) of the PTHR1 antagonist or inverse agonist. The compositions may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal), intranasal, transpulmonary, transdermal, transmucosal, or oral administration. Thus, the composition may be in the form of, e.g., tablets, ampules, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories, enemas, injectables, implants, sprays, or aerosols. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy, 21St edition, 2005, Ed. D. B. Troy, Lippincott Williams & Wilkins, Philadelphia, and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York).
Pharmaceutical compositions may be formulated to release the active compound immediately upon administration or at a predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create substantially constant concentrations of the polypeptides disclosed herein within the body over an extended period of time; (ii) formulations that after a predetermined lag time create substantially constant concentrations of the polypeptides disclosed herein within the body over an extended period of time; (iii) formulations that sustain the action of the polypeptides disclosed herein during a predetermined time period by maintaining a relatively constant, effective level of the polypeptides disclosed herein in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the polypeptides disclosed herein (sawtooth kinetic pattern); (iv) formulations that localize action of the polypeptides disclosed herein, e.g., spatial placement of a controlled release composition adjacent to or in the diseased tissue or organ; (v) formulations that achieve convenience of dosing, e.g., administering the composition once per week or once every two weeks; and (vi) formulations that target the action of the polypeptides disclosed herein by using carriers or chemical derivatives to deliver the compound to a particular target cell type. Administration of the compound in the form of a controlled release formulation is especially preferred for compounds having a narrow absorption window in the gastro-intestinal tract or a relatively short biological half-life.
Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the compound is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the compound in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, molecular complexes, microspheres, nanoparticles, patches, and liposomes.
The composition containing polypeptides described herein may be administered parenterally by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation.
Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), in vials containing several doses and in which a suitable preservative may be added, or in prefilled syringes. The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. In addition to the polypeptides disclosed herein, the composition may include suitable parenterally acceptable carriers and/or excipients. The polypeptides disclosed herein may be incorporated into microspheres, microcapsules, nanoparticles, or liposomes for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents.
As indicated above, the pharmaceutical compositions according to the invention may be in a form suitable for sterile injection. To prepare such a composition, the suitable the polypeptides disclosed herein are dissolved or suspended in a parenterally acceptable liquid vehicle. Among acceptable vehicles and solvents that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, dextrose solution, and isotonic sodium chloride solution. The aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl, or n-propyl p-hydroxybenzoate). In cases where one of the compounds is only sparingly or slightly soluble in water, a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
The polypeptides and the pharmaceutical compositions disclosed herein may be used to treat a condition or a disease of the PTHR1 signaling overactivity (e.g., hypercalcemia, hypophosphatemia, hyperparathyroidism, and Jansen's chondrodysplasia). PTHR1 signaling overactivity may be caused by various factors, such as elevated blood levels of PTH (e.g., hyperparathyroidism) or PTHrP (e.g., humoral hypercalcemia of malignancy).
Some forms of hypercalcemia are related to the interaction of PTHR1 with PTH or PTHrP (e.g., humoral hypercalcemia of malignancy). Hypercalcemia is a condition in which there is an abnormal elevation in serum calcium levels; it is often associated with other diseases, including hyperparathyroidism, osteoporosis, and cancer (e.g., carcinomas of the breast, lung and prostate, epidermoid cancers of the head and neck and of the esophagus, multiple myeloma, and hypernephroma).
Jansen's chondrodysplasia is a rare disease caused by PTHR1 activating mutations (e.g. H223R and T410P) which result in excessive hormone-independent (constitutive) signaling by the receptor itself. Ligands that bind to such constitutively active PTHR1 mutants and suppress its signaling are classified as PTHR1 inverse agonists. Some, but not all, ligands that function as PTHR1 antagonists also function as PTHR1 inverse agonists. Typically, among the polypeptides disclosed herein, those including a dTrp12 modification function as inverse agonists.
In accordance with yet a further aspect of the invention, there is provided a method for treating a disease or condition that is caused by overactivity of PTHR1 in a subject. The method involves administering to the subject an effective amount of the polypeptide of the invention or a pharmaceutically acceptable salt thereof or a fragment thereof or a pharmaceutical composition disclosed herein. The effective amount will typically be sufficient to reduce activation of the PTHR1 of the subject to non-pathological levels, as assessed by the treatment of the subject.
In one embodiment, a subject having a disease or condition that is caused by the constitutive signaling activity of PTHR1 (e.g., Jansen' chondrodysplasia) can be treated using polypeptides of the invention which are PTHR1 inverse agonists. In this embodiment, the PTHR1 inverse agonist polypeptide of the invention may be present as a pharmaceutically acceptable salt thereof or a fragment thereof or in a pharmaceutical composition disclosed herein.
In another embodiment, a subject having a disease or condition that is caused by the non-constitutive signaling overactivity of PTHR1 can be treated using polypeptides of the invention which are PTHR1 antagonists. In this embodiment, the PTHR1 antagonist polypeptide of the invention may be present as a pharmaceutically acceptable salt thereof or a fragment thereof or in a pharmaceutical composition disclosed herein.
To administer the polypeptide of the invention, the appropriate polypeptide of the invention or a pharmaceutically acceptable salt thereof or a fragment thereof can be used in the manufacture of a medicament, generally by being formulated in an appropriate carrier or excipient such as, e.g., physiological saline, and administered through an appropriate route of administration (e.g., parenteral (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal), intranasal, transpulmonary, transdermal, transmucosal, or oral administration). An effective amount of the polypeptide of the invention is typically present in the medicament. For example, typical dosage would be 1 ng to 10 mg of the polypeptide, e.g., per kg body weight, e.g., per day.
The polypeptides disclosed herein may be used to modulate the activity of PTHR1 in a cell. Thus, the present invention features a method of inversely agonizing the activity of PTHR1 in a cell and a method of antagonizing the activity of PTHR1 in a cell. The method may involve contacting the cell with the polypeptide having a desirable activity (e.g., a PTHR1 antagonist or PTHR1 inverse agonist activity). The polypeptide that is a PTHR1 antagonist may be used in this method to antagonizing the signaling activity of PTHR1 in a cell (e.g., by reducing the binding of endogenous agonists to PTHR1). The polypeptide that is a PTHR1 inverse agonist may be used in this method to inversely agonize the constitutive signaling activity of a naturally occurring PTHR1 mutant having constitutive signaling activity (e.g., PTHR1-H223R or PTHR1-T410P). The cell may be in a mammal (e.g., in a subject).
It will be appreciated to those skilled in the art that the invention can be performed within a wide range of equivalent parameters of composition, concentration, modes of administration, and conditions without departing from the spirit or scope of the invention or any embodiment thereof. The following examples are meant to illustrate the invention. They are not meant to limit the invention in any way.
Peptides and Synthesis:
PTH peptides were based on the human PTH(1-34) sequence and were synthesized by the Massachusetts General Hospital Biopolymer Core facility using solid-phase chemistry. Lys13(TMR) derivatives were obtained by post-synthetically attaching a fluorescent tetramethylrhodamine (TMR) group to the epsilon amino function of Lys-13. Pegylated derivatives were obtained by post-synthetically attaching a single PEG group (Mw=20,000) to the side chain thiol of a C-terminal cysteine at position 35.
The 20-kDa thiol-reactive PEG reagent, α-[3-(3-Maleimido-1-oxopropyl)amino]propyl-ω-methoxy, polyoxyethylene, SUNBRIGHT® ME-200MA0B, was obtained from NOF America Corp. (White Plains, N.Y.). The thiol conjugation reaction was performed overnight at room temperature in 100 mM sodium citrate buffer, pH 4.0 containing 1 mM EDTA and 10 mM TCEP reducing agent. Unconjugated PEG reagent was removed from the reaction by cation exchange chromatography using SP-sepharose resin, a linear salt gradient was formed using 20 mM sodium acetate pH 4.0 as buffer A, and the same buffer containing 1 M NaCl was used as buffer B. Elution of the PEG-PTHTMR peptide was monitored by measuring the fractions for TMR absorbance at 543 nm. The peak fractions were pooled and de-salted using a C2tp-reverse-phase cartridge and 75% acetonitrile/0.1% TFA for peptide elution. The eluted sample was then lyophilized, and reconstituted in 10 mM acetic at a final ligand concentration of 12.3 mg/ml (0.5 mM); aliquots of these stock solutions were stored at −80° C. until needed for experiments.
All other non-pegylated PTH peptides were purified by reverse phase HPLC and assessed for quality and purity by analytical HPLC and MALDI mass spectrometry; purity was at least 90%. Peptide concentrations of stock solutions were validated by acid hydrolysis and amino acid analysis.
Other peptides of the invention (e.g., LA-PTH(7-36) or LA-PTH(7-37) (e.g., fragments having 3-32 or 3-33 amino acid residues of SEQ ID NOs: 1-6)) can be prepared using methods known in the art (e.g., the exemplary methods described herein).
Ligand binding to the PTH1 R was assessed in intact GP-2.3 cells or membranes prepared from GP-2.3 cells using a 125I-PTH peptide analog as tracer radioligand and were incubated at room temperature for 90 minutes. Intact cell binding reactions were performed in 96-well plates, and, following incubation, the cells were lysed with 1N NaOH, and the lysate was counted for gamma radiation. For membrane dissociation assays, reactions were assembled in 15 cc tubes, and, at times following membrane addition (t=0), aliquots were withdrawn and filtered through a well of a vacuum filtration 96-well plate, rinsed, and, after washing, the filters were removed and counted for gamma irradiation. Nonspecific binding was determined in reactions containing an excess (5×10−7 M) of unlabeled M-PTH(1-15) or PTH(1-34). Curves were fit to the data using a four-parameter sigmoidal dose-response equation.
cAMP Signaling Properties of the Polypeptides in Cells:
cAMP signaling was assessed in the HEK293-derived or SaOS2-derived cell lines stably expressing the luciferase-based GloSensor™ cAMP reporter (Hattersley et al., Paper presented at: Novel Signaling Mechanisms and Bone Cell Biology 2014; Binkowski et al., Methods in molecular biology. 756:263-271). HEK-293 cells were further transfected to stably express the hPTHR1, hPTHR1-H223R, or hPTHR1-T410P. The cells were seeded into 96-well white plates and were assayed 24 to 48 hours post-confluency. Assays were performed at room temperature in CO2-independent culture media (Life Technologies, Corp., Carlsbad, Calif.) containing 0.1% BSA (CIDB). The cells were pre-loaded with luciferin (0.5 mM in CIDB) for 15 minutes, then PTH peptides were added at varying concentrations. cAMP-dependent luminescence was measured at two-minute intervals using a PerkinElmer Envision plate reader. The time at which maximum luminescence (cps) observed with agonist alone, typically at 10-20 minutes after agonist addition, was used to obtain data from wells containing antagonist with or without agonist, to thus generate antagonist ligand dose-response curves. The resulting cps values were then plotted against ligand concentration using GraphPad Prism 7.0 software and a four-parameter logistics curve fitting equation, which yielded parameters of inhibitory potency (pIC50).
For “wash-out” experiments, the cells pre-loaded with luciferin were treated with media (vehicle) or a test antagonist ligand for 25 minutes; the plate was then removed from the plate reader, the cells were rinsed thrice to remove unbound ligand, and treated with fresh media containing luciferin with or without an agonist ligand. The development of cAMP-dependent luminescence was measured for another 120 minutes.
Wilde-type, ca. 10-week old male C57BL/6J mice were purchased from the Charles River Laboratories (Wilmington, Mass., USA). The origin and method of breeding of Col1-H223R “Jansen's” transgenic mice is described by Calvi et al. (J. Clin. Invest., 107:277-286, 2001). Mice were maintained in facilities operated by the Center for Comparative Research of the Massachusetts General Hospital, and acclimated in the facilities for seven days prior to being used for study. All experimental procedures were approved by the MGH Institutional Animal Care and Use Committee (IACUC). In each study, animals were assigned randomly to treatment groups. Where possible, power calculations established that the number of animals used per study group was sufficient to detect statistically significant differences in intended primary experimental outcomes (i.e., changes in serum Ca and Pi).
Mice were injected IV via the tail vein or subcutaneously with ligands in vehicle (0.05% Tween80; 10 mM citrate; 150 mM NaCl; pH 5.0) to give the intended final ligand dose (e.g. 50 nmol/kg body weight). At times immediately before (t=0) and after injection, tail vein blood was collected and analyzed for blood ionized calcium (Ca2+) measured with a RAPIDLAB 348 analyzer (SIEMENS Healthcare Diagnostic, United Kingdom).
Confocal Microscopy Analysis of PTH-TMR Fluorescence in GP-2.3 cells:
Confocal fluorescence microscopy images were acquired using Olympus FV-1000 MPE confocal system (Center Valley, Pa. 18034), performed by the Photopathology Core facility of the Wellman Center for Photomedicine at the Massachusetts General Hospital. Images were captured with a 40× (LUMPLFL 0.8NA WD 3.3 mm) water-immersion objective, and with 405 nm and 559 nm excitation lasers, at a resolution of 1024×1024.
Ligand internalization properties of PTHTMR analogs were assessed in GP-2.3 cells (HEK-293 with stable transfection of hPTHR1 and GloSensor™); non-specific binding was assessed in GS-22A cells (HEK-293 with stable transfection of GloSensor™; parental to GP-2.3 cells) and was found to be undetectable. The cells were cultured on glass cover-slips in 24-well plates to ˜75% of confluency, then treated with PTHTMR ligand (100 nM) in Hank's balanced salts buffer with 0.1% BSA (HBB) for 15 minutes at room temperature, then rinsed thrice with HBB, fixed with 4% formalin for 5 minutes, mounted with vector-shield containing DAPI on a glass microscope slide, viewed on the microscope, and digitally imaged.
Data were processed using Microsoft Excel® and GraphPad Prism 7.0 software packages and analyzed statistically using Student's t test (two-tailed and unequal variances).
Polypeptides used in this Example are listed in Tables 1 and 2.
In Table 2, dW is dTrp; K′ is Lys conjugated to tetramethyl rhodamine; Ac5c is 1-aminocyclopentane-1-carboxylic acid residue; polypeptides 1894, 1951, 1869, and 1977 are antagonists/inverse agonists; and polypeptides 1923, 1880, 823, and 1962 are agonists; 1894 and 1951 are duplicates.
Other polypeptides of the invention may be used in the assays described herein. For example the fragments of the N-terminally truncated PTH/PTHrP hybrid peptide (e.g., LA-PTH(7-36) or LA-PTH(7-37) (e.g., fragments having 3-32 or 3-33 amino acid residues of SEQ ID NOs: 1-6)) may be tested in the assays described herein.
Pharmacological properties of the ligands were assessed in cells expressing the PTHR1 and the GloSensor™ cAMP reporter. The assays used GP-2.3 cells, derived from HEK293 cells by stable transfection with the hPTHR1 and GloSensor™, as well as SGS-72 cells, which are derived from the human osteosarcoma cell line, Saos-2, in which is endogenously expressed the hPTHR1, by stable transfection with GloSensor™. Binding of representative polypeptides to the PTHR1 was assessed in GP-2.3 cells by radioligand competition methods (
125I-PTH(1-34)
125I-PTHrP(5-36)
Table 3 provides results for radioligand competition binding to GP-2.3 (n=4) cells. The assays were performed in GP-2.3 cells with the two tracer 125I-radioligands shown; values are half-maximal inhibitory concentration (pIC50) and corresponding nanomolar value (below) derived from curve fitting ligand dose-response data. Data are means of four experiments.
In GP-2.3 cells, LA-PTH(5-36)-1950 inhibited the cAMP signal induced by PTH(1-34) (1 nM) ˜five-fold more effectively than did dTrp12-PTH(7-34)-1894 (pIC50s=7.0±0.1 vs. 6.4±0.1 nM), and in SGS-72 cells, it inhibited the cAMP signal induced by PTH(1-34) (0.3 nM) ˜30-fold more effectively (pIC50s=8.6±0.1 vs. 7.2±0.1) (
In washout assays, in which GP-2.3 cells were pre-treated with antagonist at varying doses (e.g., 10 μM, 3.3 μM, 1 μM) for 15 minutes and then rinsed three times prior to application of agonist (e.g., PTH(1-34)-1923 at 1.0 nM (GP-2.3), or 0.3 nM (SGS-72), or M-PTH(1-11)-823 10 nM or isoproterenol-1839 1 μM, as a non-specific control that activates endogenous β2-adrenoreceptors), LA-PTH(5-36)-1950 and dTrp12, Trp23-LA-PTH(5-36)-1949 mediated marked (e.g., >90%) and sustained (e.g. >2 hrs) inhibition of the cAMP response induced by the subsequently applied PTHR1 agonist ligand, whereas dTrp12-PTH(7-34)-1894 (or the equivalent 1951 used in SGS-72 cell experiments) did not diminish the agonist response, as the cAMP levels reached those seen in the vehicle (Veh) pre-treated cells (
indicates data missing or illegible when filed
z,999 concentration of antagonist that inhibited the maximum response to PTH(1-34)-induced in the absence of antagonist (at ˜40 minutes) is reported in units of -log M (pIC50), and the corresponding nanomolar value is shown below each pIC50 value. Data are means of the number of experiments indicated (n). The PTH(1-34)-induced maximum luminescence was ˜80,000 cps and the basal was ˜1,000 cps. TMR indicates Lys13 modified at epsilon amino function with TMR, P vs. inhibition by dW12,Y34-bPTH(7-34).
indicates data missing or illegible when filed
In Table 5, antagonist assays were performed in SGS-72 cells (SaOS2 cells with stable GloSensor™ cAMP reporter) at room temperature. Cells were pre-treated with antagonist at varying concentration for 10 minutes, and then PTH(1-34) or PTHrP(1-36) was added to a concentration of 0.3 nM and peak cAMP-dependent luminescence was recorded and plotted against antagonist ligand concentration. The mean (±sem) concentration of antagonist that inhibited the maximum agonist-induced response observed in the absence of antagonist (at ˜40 minutes) is reported in units of -log M (pIC50), and the corresponding nanomolar value is shown below each pIC50 value. Data are means of the number of seven experiments. The PTH(1-34)-induced maximum luminescence was ˜500,000 cps and the basal was ˜5,000 cps. TMR indicates Lys13 modified at epsilon amino function with TMR. The data are illustrated in
Polypeptide dTrp12, Trp23-LA-PTH(5-36)-1949, which incorporated the Gly12-->dTrp substitution, exhibited inverse agonist properties on the constitutively active PTHR1 mutants (H223R or T410P) stably expressed in HEK293/GloSensor™ cells (GHR-10 and GTP-4 cell lines, respectively) (
The radioiodinated analog, 125I-Nle8, dTrp12, Trp23, Tyr36-LA-PTH(5-36)-1954, dissociated more slowly from the PTHR1 than did either the conventional antagonist radioligand, 125I-Tyr36-PTHrP(5-36)-836, or the N-terminal agonist radioligand 125I-M-PTH(1-15)-779, which corresponds to 125I-Aib1,3, Gln10, hArg11, Ala12, Trp14-PTH(1-15) (
Polypeptides dTrp12, Trp23-LA-PTH(5-36)-1949 and LA-PTH(5-36)-1950 exhibited less residual agonist activity than did the conventional antagonist, dTrp12-PTH(7-34)-1894, as assessed by applying these ligands directly to GP-2.3 cells and assessing cAMP-dependent luminescent responses from GloSensor™ reporter (
Incorporation of tetramethylrhodamine (TMR) at the epsilon amino side chain atom of Lys13 conferred fluorescent properties to LA-PTH antagonist polypeptide, and thus enabled visual demonstration that TMR-LA-PTH(5-36)-1953 effectively retained bound PTHR1 on the cell surface, and thus did not internalize into the cell cytoplasm of GP-2.3 cells at 15 minutes after ligand addition (100 nM), whereas the agonist, TMR-PTH(1-35)-1962 internalized into endosomal vesicles (
Intravenous co-injection of dTrp12, Trp23-LA-PTH(5-36)-1949 into mice with PTH(1-34) inhibited the capacity of the injected PTH(1-34) agonist to induce a calcemic response (
Antagonism at the endogenous hPTHR1 was assessed in SGS-72 cells; cells were pretreated with vehicle or vehicle containing varying doses of antagonist ligand for 15 minutes, then PTH(1-34) or PTHrP(1-36) was added at a concentration of 0.3 nM, and GloSensor™-cAMP responses were measured. The results are summarized in Table 6. IC50s indicate that antagonist concentration at which the response maximum observed in vehicle treated cells was inhibited by 50%. Date are means (±SEM) of the number of separate experiments indicated by n.
To study antagonism at the wild-type PTHR under conditions of excess PTH, as occurs in humans with hyperparathyroidism, we developed a mouse non-genetic, transient model of hyperparathyroidism. Wild-type mice were injected with a PTH agonist analog, PEG-PTH(1-35), which induces hypercalcemia lasting between 24 and 72 hours after a single s.c. injection (see Guo et al., Journal of Bone and Mineral Research, 32:86-98, 2017). Antagonists were injected after 24 hours following the injection of PEG-PTH(1-35) and effects on blood Ca2+ levels were measured. The results are illustrated in
To study antagonism at the wild-type PTHR under conditions of excess PTHrP, as occurs in humans with hypercalcemia of malignancy, we co-injected wild-type mice with PTHrP(1-36) and a test antagonist and measured effects on blood Ca2+ levels. The results are illustrated in
PEG-LA-PTH(5-37)-1978 was tested for antagonism in wild-type mice by co-injection with PTH(1-34). The observed blood Ca2+ levels demonstrated antagonist effect of PEG-LA-PTH(5-37)-1978 (see
The pharmacokinetic effect of pegylation on the antagonist/inverse agonist peptides was assessed by comparing PEG-LA-PTH(5-37)-1978 plasma levels to LA-PTH(5-37)-1975 plasma levels (for an example of similar tests, see Guo et al., Journal of Bone and Mineral Research, 32:86-98, 2017). Mice (9-week old female CD1 strain) were injected with PEG-LA-PTH(5-37)-1978 (30 nmol/kg) or LA-PTH(5-37)-1975 (30 nmol/kg), and TMR fluorescence in plasma was measured. The TMR fluorescence signal was converted to ligand concentration (nM) using a standard curve generated with the same corresponding ligand (Guo et al., Journal of Bone and Mineral Research, 32:86-98, 2017). Data are means±SE; n=3. t1/2=0.9 h for PEG-LA-PTH(5-37)-1978, t1/2 was not determined for LA-PTH(5-37)-1975. The results are illustrated in
These experiments were performed in HEK-293 cells stably transfected to express the GloSensor™ cAMP reporter along with a constitutively active mutant human PTHR1. The HEK-293 cells stably expressing GloSensor™ cAMP reporter along with a constitutively active mutant human PTHR1-H223R are GHR-10 cells. The HEK-293 cells stably expressing GloSensor™ cAMP reporter along with a constitutively active mutant human PTHR1-T410P are GTP-4 cells. The HEK-293 cells stably expressing GloSensor™ cAMP reporter along with a WT-PTHR1 are GP-2.3 cells. GP-2.3 cells were used as control. Cells in 96-well plates were treated with either vehicle or vehicle containing inverse agonist peptide dW12,W23-LA-PTH(7-36)-1990 or Nle8, dW12, W23-LA-PTH(7-36)-1992, or with agonist PTH(1-34)-1923. Effects on basal cAMP signaling over time were assessed during periods of direct ligand addition (
Persistence of the antagonist/inverse agonist effects (prolonged action) as follows. Effects of eight peptides on basal and PTH(1-34)-stimulated cAMP signaling by PTHR-H223R were assessed in GHR-10 cells (HEK-293-derived cells stably expressing glosensor cAMP reporter and PTHR1-H223R). Time courses of changes in cAMP levels were recorded in three consecutive phases:
1) after initial addition of a candidate antagonist/inverse agonist (1×10−6 M-1×10−9 M) to cells pre-loaded for −30 minutes with luciferin (left column);
2) after removal of unbound inverse agonist by extensive wash-out with buffer and replenishment with fresh luciferin-containing buffer (middle column); and
3) after addition of PTH(1-34) (3 nM) to the washed-out cells (right column). Control wells were treated with vehicle. GloSensor™ luminescence, as counts per second (cps), was recorded using a PerkinElmer Envision plate reader. Data are from duplicate wells (means±SEM) of a single experiment representative of two others. The results are illustrated in Table 7 and in
The data in Table 7 were obtained as follows. GHR10 cells were treated with vehicle or vehicle containing the indicated peptide for 24 minutes and cAMP-dependent luminescence was recorded at two-minute intervals. The cells were then washed to remove unbound ligand and media containing fresh luciferin was added, and cAMP luminescence was recorded for another 26 minutes. Percentage reduction was calculated from the area-under-the-curve (AUC) values derived from the time course plots using the equation:
Data are from a representative experiment.
For the studies of inverse agonism at the PTHR1 in vivo, a transgenic mouse model of Jansen's disease, Col1-H223R, was used. In Col1-H223R mice, the human PTHR-H223R mutant allele is expressed specifically via the collagen-type-1a promoter in osteoblastic cells of bone. These mice are described by Calvi et al. (J. Clin. Invest., 107:277-286, 2001), and exhibit a high-bone mass phenotype. To assess the potential capacity of a new inverse agonist analog to correct the bone phenotype, Col1-H223R mice were injected subcutaneously twice daily for 17 days with dW12, W23-LA-PTH(5-36)-1952 (500 nmol/kg); in parallel, control population of Col1-H223R mice was receiving injections with vehicle or peptide L11, dW12, W23, Y36-PTHrP(7-36)-2018 (500 nmol/kg) (
Bone structural parameters were assessed by hematoxylin and eosin (H&E) stain-based histology of tibiae and by micro CT of femurs (
The effect of each regimen on markers of bone-turn-over (Ca, Pi, and CTX) in urine and/or blood and on gene mRNA expression in bone was measured. The results are illustrated in
Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.
Other embodiments are in the claims.
This invention was made with Government support under Grant No. NIH-DK-11794 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/025559 | 3/31/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62317152 | Apr 2016 | US |
