EP0942744A1 - Treatment of bone fracture - Google Patents

Treatment of bone fracture

Info

Publication number
EP0942744A1
EP0942744A1 EP97946311A EP97946311A EP0942744A1 EP 0942744 A1 EP0942744 A1 EP 0942744A1 EP 97946311 A EP97946311 A EP 97946311A EP 97946311 A EP97946311 A EP 97946311A EP 0942744 A1 EP0942744 A1 EP 0942744A1
Authority
EP
European Patent Office
Prior art keywords
pth
pthrp
antagonist
peptide
administered parenterally
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97946311A
Other languages
German (de)
French (fr)
Inventor
Jacques-Pierre Moreau
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ipsen Pharma SAS
Original Assignee
Societe de Conseils de Recherches et dApplications Scientifiques SCRAS SAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Societe de Conseils de Recherches et dApplications Scientifiques SCRAS SAS filed Critical Societe de Conseils de Recherches et dApplications Scientifiques SCRAS SAS
Publication of EP0942744A1 publication Critical patent/EP0942744A1/en
Withdrawn legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides

Definitions

  • Parathyroid hormone is a polypeptide produced by the parathyroid glands. The mature circulating form of the hormone is comprised of 84 amino acid residues. The biological action of PTH can be reproduced by a peptide fragment of its N-terminus (e.g., amino acid residues 1 through 34) .
  • Parathyroid hormone- related protein (“PTHrP”) is a 139 to 173 amino acid- protein with N-terminal homology to PTH. PTH and PTHrP bind to an identical receptor, the PTH/PTHrP receptor, expressed in kidneys and bone cells (Schipani, E. , et al., Endocrinology 132:2157-2165 (1993)).
  • PTH also binds to the recently cloned PTH2 receptor (Usdin, T.B., et al., J. Biol. Chem., 270:15455-15458 (1995)), while PTHrP has a much lower affinity for this receptor.
  • the binding of PTH or PTHrP to the PTH/PTHrP receptor results in intracellular accumulation of cAMP and inositol triphosphates and increases intracellular free calcium (Abou-Samra, A-B, et al., Proc. Natl. Acad. Sci. USA 89:2732-2736 (1992)).
  • PTH and PTHrP agonists increase Vit D3 conversion in the kidneys, enhance reabsorption of calcium from the renal tubules, and increase calcium absorption from the gut (In: The Parathyroids-Basic and Clinic Concepts, Eds. Bilezikian, J.P., Marcus, R. , Levine, M.A., Raven Press, 1994) .
  • the following invention relates to the novel discovery that a PTH/PTHrP antagonist can stimulate bone growth during fracture.
  • the invention features a method of promoting bone formation at a bone fracture site in a subject (e.g., a mammal such as a human) .
  • the method includes the step of administering (e.g., intravenously, subcutaneously, transmucosally, transdermally, by inhalation, orally, or by release from a sustained release formulation) to the subject a therapeutically effective amount of a PTH/PTHrP antagonist.
  • PTH/PTHrP antagonist will be defined below. A therapeutically effective amount depends upon the condition being treated, the route of administration chosen, and the specific activity of the compound used and ultimately will be decided by the attending physician or veterinarian.
  • the PTH/PTHrP antagonist may be administered locally to the site of the fracture or administered systemically to the subject.
  • the PTH/PTHrP antagonist may also be administered intermittently or continuously to the subject.
  • the PTH/PTHrP antagonist is administered to the subject until the bone fracture has healed.
  • the PTH/PTHrP antagonist may be injected parenterally, e.g., subcutaneously, into the bloodstream of the subject being treated.
  • the route such as intravenous, intramuscular, intraperitoneal, enterally, transdermally, transmucously, parenteral release from a sustained released polymer composition (e.g., a lactic acid polymer or lactic- glycolic acid copolymer microparticle or implant) , profusion, nasal, oral, etc., will vary with the condition being treated and the activity and bioavailability of the PTH/PTHrP antagonist being used.
  • a sustained released polymer composition e.g., a lactic acid polymer or lactic- glycolic acid copolymer microparticle or implant
  • the PTH/PTHrP antagonist While it is possible for the PTH/PTHrP antagonist to be administered as the pure or substantially pure compound, it may also be presented as a pharmaceutical formulation or preparation.
  • the formulations to be used in the present invention, for both humans and animals, comprise any of the PTH/PTHrP antagonists to be described below, together with one or more pharmaceutically acceptable carriers thereof, and optionally other therapeutic ingredients.
  • the carrier must be "acceptable” in the sense of being compatible with the active ingredient(s) of the formulation (e.g., capable of stabilizing peptides) and not deleterious to the subject to be treated.
  • the formulation should not include oxidizing agents or other substances with which certain ingredients (e.g., peptides) are known to be incompatible. Consequently, it is important to carefully select the excipient. pH is another key factor, and it may be necessary to buffer the product under slightly acidic conditions (pH 5 to 6) .
  • formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient(s) into association with the carrier which constitutes one or more accessory ingredients.
  • the formulations for tablets or powders are prepared by uniformly and intimately blending the active ingredient with finely divided solid carriers, and then, if necessary, as in the case of tablets, forming the product into the desired shape and size.
  • Formulations suitable for parenteral (e.g., intravenous) administration conveniently comprise sterile aqueous solutions of the active ingredient(s) .
  • the solutions are isotonic with the blood of the subject to be treated.
  • Such formulations may be conveniently prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering said solution sterile.
  • the formulation may be presented in unit or multi-dose containers, for example, sealed ampoules or vials.
  • Formulations suitable for sustained release parenteral administrations e.g., biodegradable polymer formulations
  • a bone resorption inhibiting agent can also be administered during performance of the above described method.
  • the term “inhibition of bone resorption” refers to prevention of bone loss, especially the inhibition of removal of existing bone either from the mineral phase and/or the organic matrix phase, through direct or indirect alteration of osteoclast formation or metabolism.
  • the term “inhibitor of bone resorption” as used herein refers to agents that prevent bone loss by the direct or indirect alteration of osteoclast formation or metabolism.
  • Suitable bone resorption inhibiting agents includes, but is not limited to, an estrogen, a bisphosphonate (e.g., PamidronateTM, CilodronateTM, IbandoronateTM, ElendronateTM, or RecidronateTM) , sodium fluoride, vitamin D, a calcitonin, and calcium.
  • an estrogen e.g., PamidronateTM, CilodronateTM, IbandoronateTM, ElendronateTM, or RecidronateTM
  • sodium fluoride e.g., sodium fluoride, vitamin D, a calcitonin, and calcium.
  • PTH and PTHrP includes specie homologs of PTH and PTHrP, e.g., human PTH (hPTH) , bovine PTH (bPTH) , rat PTH (rPTH) , human PTHrP (hPTHrP) , and rat PTHrP (rPTHrP) .
  • the sequence of various specie homologs of PTH and PTHrP are recited in Nissenson, et al., Receptor 3:193 (1993).
  • a PTH/PTHrP antagonist is a compound which both binds to the PTH/PTHrP receptor and inhibits PTH/PTHrP stimulated mobilization of calcium and/or PTH/PTHrP stimulated mobilization of adenylate cyclase, i.e., as described in the biological assays described herein.
  • the PTH/PTHrP antagonist is a peptide.
  • Such a peptide may be a homologous (e.g., at least 70 percent homology) fragment of PTH (a "PTH peptide antagonist") or PTHrP (a "PTHrP peptide antagonist”) .
  • the first four to the first seven amino acids of the N-terminus of the homologous PTH/PTHrP may be deleted.
  • antagonists include hPTH(7- 34)NH 2 , bPTH(7-34)NH 2 , hPTHrP(7-34)NH 2 , [Nle 8 ' 18 , Tyr 3 ]hPTH(7-34)NH 2 , [Nle 8 ' 18 , Tyr 34 ]bPTH(7-34)NH 2 , [Nle 8 ' 18 , D-Trp 12 , Tyr 34 ]bPTH(7-34)NH 2 , and [Leu 11 , D- Trp 12 ]bPTH(7-34)NH 2 , (where the number in parentheses corresponding to the amino acid of the natural peptide which are present in the peptide, the symbol NH 2 denotes the amidation of the carboxyl group of the C-terminal amino acid to form -CONH 2 , and the numbered amino acids in the brackets refer
  • the PTH/PTHrP antagonist is a non-peptide (e.g., a peptidomimetic).
  • Human osteogenic sarcoma SaOS-2 cells (ATCC No. HTB85) , expressing the PTH/PTHrP receptor, were maintained in RPM1 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37sc in a humidified atmosphere of 5% C0 2 in air. The medium was changed every three or four days, and the cells were subcultured every week by trypsinization.
  • RPM1 1640 medium Sigma, St. Louis, MO
  • FBS fetal bovine serum
  • 2 mM glutamine 2 mM glutamine
  • SaOS 2 cells were maintained for four days until they had reached confluence.
  • the medium was replaced with 5% FBS in RPS1 1640 medium and incubated for 2 hrs at room temperature with 10 x 10 4 cpm [Nle 8 ' 18 , Tyr 34 (3- I 125 ) ]bPTH 1 _ 34 NH 2 in the presence of a competing test PTH/PTHrP antagonist at varying concentrations (i.e., 10 " n ito 10 ⁇ 4 m) .
  • the cells were washed four times with ice-cold PBS and lysed with 0.1 M NaOH, and the radioactivity associated with the cells was counted in a scintillation counter.
  • the IC 50 value (half maximal inhibition of binding of [Nle 8 ' 18 , Tyr 34 (3- 125 l) ]bPTH(l- 34)NH 2 can be calculated for each peptide.
  • Synthesis of the radiolabelled [Nle 8 ' 18 , Tyr 34 (3-I 125 ) ]bPTH 1 _ 34 NH 2 was carried out as described in Goldman, ME, et al., Endocrinology, 1988, 123, 1468-1475. Cells such as the human kidney 293 cells transfected with the PTH/PTHrP receptor may also be used as described in Pines, et al., Endocrinology 135:1713 (1994).
  • test PTH/PTHrP antagonists to inhibit adenylate cyclase release in response to PTH/PTHrP stimulation was also measured in SaOS-2 cells. More specifically, an inhibition of the adenylate cyclase was determined by measuring the level of synthesis of cAMP (adenosine 3' ,5'-monophosphate) . Confluent SaOS-2 cells in 24 wells plates are incubated with 0.5 ⁇ Ci[ 3 H]adenine (26.9 Ci/mmol, New England Nuclear,
  • test PTH/PTHrP antagonists to inhibit calcium mobilization in response to PTH/PTHrP stimulation can also be measured in SaOS-2 cells.
  • Confluent SaOS-2 BIO cells in 24 wells plates are incubated in 0.3 percent EDTA/phosphate buffered saline solution at 25sc for 2 hrs, and washed twice with Hank's solution.
  • the cells are resuspended in Hank's buffered saline solution for loading of the fluorescent Ca 2+ indicator Fura-2AM.
  • the cells are incubated with 2 mM fluorescent Ca 2+ indicator Fura-2AM (Molecular Probes,
  • the IC 50 's (half maximal concentration for the inhibition of calcium mobilization) can be calculated for the PTH/PTHrP antagonists.
  • Cells, such as human kidney 293 cells, which are transfected with the PTH/PTHrP receptor may also be used.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Endocrinology (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention features a method of promoting bone formation at a bone fracture site in a subject. The method includes the step of administering to the subject a therapeutically effective amount of a PTH/PTHrP antagonist.

Description

TREATMENT OF BONE FRACTURE
Background of the Invention Parathyroid hormone ("PTH") is a polypeptide produced by the parathyroid glands. The mature circulating form of the hormone is comprised of 84 amino acid residues. The biological action of PTH can be reproduced by a peptide fragment of its N-terminus (e.g., amino acid residues 1 through 34) . Parathyroid hormone- related protein ("PTHrP") is a 139 to 173 amino acid- protein with N-terminal homology to PTH. PTH and PTHrP bind to an identical receptor, the PTH/PTHrP receptor, expressed in kidneys and bone cells (Schipani, E. , et al., Endocrinology 132:2157-2165 (1993)). In contrast, PTH also binds to the recently cloned PTH2 receptor (Usdin, T.B., et al., J. Biol. Chem., 270:15455-15458 (1995)), while PTHrP has a much lower affinity for this receptor. The binding of PTH or PTHrP to the PTH/PTHrP receptor results in intracellular accumulation of cAMP and inositol triphosphates and increases intracellular free calcium (Abou-Samra, A-B, et al., Proc. Natl. Acad. Sci. USA 89:2732-2736 (1992)).
PTH and PTHrP agonists increase Vit D3 conversion in the kidneys, enhance reabsorption of calcium from the renal tubules, and increase calcium absorption from the gut (In: The Parathyroids-Basic and Clinic Concepts, Eds. Bilezikian, J.P., Marcus, R. , Levine, M.A., Raven Press, 1994) . PTH and PTHrP agonists administered intermittently to animals and man augment bone mass, prevent bone loss in osteopenic states, and build new bone in established osteopenia (Dempster, D.W., et al., Endocrine Rev. 14:690-709 (1993); and Leaffer, D. , et al., Endocrinology 3624-3631 (1995)). The following invention relates to the novel discovery that a PTH/PTHrP antagonist can stimulate bone growth during fracture.
Summary of the Invention The invention features a method of promoting bone formation at a bone fracture site in a subject (e.g., a mammal such as a human) . The method includes the step of administering (e.g., intravenously, subcutaneously, transmucosally, transdermally, by inhalation, orally, or by release from a sustained release formulation) to the subject a therapeutically effective amount of a PTH/PTHrP antagonist.
Definition of "PTH/PTHrP antagonist" will be defined below. A therapeutically effective amount depends upon the condition being treated, the route of administration chosen, and the specific activity of the compound used and ultimately will be decided by the attending physician or veterinarian.
The PTH/PTHrP antagonist may be administered locally to the site of the fracture or administered systemically to the subject. The PTH/PTHrP antagonist may also be administered intermittently or continuously to the subject. In one embodiment, the PTH/PTHrP antagonist is administered to the subject until the bone fracture has healed. The PTH/PTHrP antagonist may be injected parenterally, e.g., subcutaneously, into the bloodstream of the subject being treated. However, it will be readily appreciated by those skilled in the art that the route, such as intravenous, intramuscular, intraperitoneal, enterally, transdermally, transmucously, parenteral release from a sustained released polymer composition (e.g., a lactic acid polymer or lactic- glycolic acid copolymer microparticle or implant) , profusion, nasal, oral, etc., will vary with the condition being treated and the activity and bioavailability of the PTH/PTHrP antagonist being used.
While it is possible for the PTH/PTHrP antagonist to be administered as the pure or substantially pure compound, it may also be presented as a pharmaceutical formulation or preparation. The formulations to be used in the present invention, for both humans and animals, comprise any of the PTH/PTHrP antagonists to be described below, together with one or more pharmaceutically acceptable carriers thereof, and optionally other therapeutic ingredients.
The carrier must be "acceptable" in the sense of being compatible with the active ingredient(s) of the formulation (e.g., capable of stabilizing peptides) and not deleterious to the subject to be treated. Desirably, the formulation should not include oxidizing agents or other substances with which certain ingredients (e.g., peptides) are known to be incompatible. Consequently, it is important to carefully select the excipient. pH is another key factor, and it may be necessary to buffer the product under slightly acidic conditions (pH 5 to 6) .
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient(s) into association with the carrier which constitutes one or more accessory ingredients.
In general, the formulations for tablets or powders are prepared by uniformly and intimately blending the active ingredient with finely divided solid carriers, and then, if necessary, as in the case of tablets, forming the product into the desired shape and size.
Formulations suitable for parenteral (e.g., intravenous) administration, on the other hand, conveniently comprise sterile aqueous solutions of the active ingredient(s) . Preferably, the solutions are isotonic with the blood of the subject to be treated. Such formulations may be conveniently prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering said solution sterile. The formulation may be presented in unit or multi-dose containers, for example, sealed ampoules or vials. Formulations suitable for sustained release parenteral administrations (e.g., biodegradable polymer formulations) are also well known in the art. See, e.g., U.S. Patent Nos. 3,773,919 and 4,767,628 and PCT Publication No. WO 94/15587.
Also, if desired, a bone resorption inhibiting agent can also be administered during performance of the above described method. The term "inhibition of bone resorption" refers to prevention of bone loss, especially the inhibition of removal of existing bone either from the mineral phase and/or the organic matrix phase, through direct or indirect alteration of osteoclast formation or metabolism. Thus, the term "inhibitor of bone resorption" as used herein refers to agents that prevent bone loss by the direct or indirect alteration of osteoclast formation or metabolism. An example of suitable bone resorption inhibiting agents includes, but is not limited to, an estrogen, a bisphosphonate (e.g., Pamidronate™, Cilodronate™, Ibandoronate™, Elendronate™, or Recidronate™) , sodium fluoride, vitamin D, a calcitonin, and calcium.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.
Detailed Description of the Invention It is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Also, all publications, patent applications, patents, and other references mentioned herein are incorporated by reference.
PTH/PTHrP Antagonist
The term PTH and PTHrP includes specie homologs of PTH and PTHrP, e.g., human PTH (hPTH) , bovine PTH (bPTH) , rat PTH (rPTH) , human PTHrP (hPTHrP) , and rat PTHrP (rPTHrP) . The sequence of various specie homologs of PTH and PTHrP are recited in Nissenson, et al., Receptor 3:193 (1993). What is meant by a PTH/PTHrP antagonist is a compound which both binds to the PTH/PTHrP receptor and inhibits PTH/PTHrP stimulated mobilization of calcium and/or PTH/PTHrP stimulated mobilization of adenylate cyclase, i.e., as described in the biological assays described herein. In one embodiment, the PTH/PTHrP antagonist is a peptide. Such a peptide may be a homologous (e.g., at least 70 percent homology) fragment of PTH (a "PTH peptide antagonist") or PTHrP (a "PTHrP peptide antagonist") . The first four to the first seven amino acids of the N-terminus of the homologous PTH/PTHrP may be deleted. Examples of such antagonists include hPTH(7- 34)NH2, bPTH(7-34)NH2, hPTHrP(7-34)NH2, [Nle8' 18, Tyr3 ]hPTH(7-34)NH2, [Nle8' 18, Tyr34]bPTH(7-34)NH2, [Nle8' 18, D-Trp12, Tyr34]bPTH(7-34)NH2, and [Leu11, D- Trp12]bPTH(7-34)NH2, (where the number in parentheses corresponding to the amino acid of the natural peptide which are present in the peptide, the symbol NH2 denotes the amidation of the carboxyl group of the C-terminal amino acid to form -CONH2, and the numbered amino acids in the brackets refer to substitutions to the natural sequence at the recited number position) . Examples of such antagonists are recited in the following patent applications: U.S. Patent Nos. 5,093,233; 4,968,669; 5,001,223; 4,771,124; 4,423,037; 5,087,562; and 5,114,843 and European Patent Application Nos. 341,963; 293,160; 293,158; 293,130; 293,159; and 341,961. In another embodiment, the PTH/PTHrP antagonist is a non-peptide (e.g., a peptidomimetic).
Synthesis of PTH/PTHrP Antagonists
The methods for synthesizing PTH/PTHrP antagonists are well documented and are within the ability of a person of ordinary skill in the art. Synthesis of short amino acid sequences is well established in the peptide art. For example, U.S. Patent No. 5,093,233 describes the synthesis of PTH antagonists while U.S. Patent No. 5,114,843 describes the synthesis of PTHrP antagonists.
PTH/PTHrP Antagonist Binding Assay
Human osteogenic sarcoma SaOS-2 cells (ATCC No. HTB85) , expressing the PTH/PTHrP receptor, were maintained in RPM1 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37sc in a humidified atmosphere of 5% C02 in air. The medium was changed every three or four days, and the cells were subcultured every week by trypsinization.
SaOS2 cells were maintained for four days until they had reached confluence. The medium was replaced with 5% FBS in RPS1 1640 medium and incubated for 2 hrs at room temperature with 10 x 104 cpm [Nle8'18, Tyr34(3- I125) ]bPTH1_34 NH2 in the presence of a competing test PTH/PTHrP antagonist at varying concentrations (i.e., 10" n ito 10~4m) . The cells were washed four times with ice-cold PBS and lysed with 0.1 M NaOH, and the radioactivity associated with the cells was counted in a scintillation counter. The IC50 value (half maximal inhibition of binding of [Nle8'18, Tyr34(3-125l) ]bPTH(l- 34)NH2 can be calculated for each peptide. Synthesis of the radiolabelled [Nle8'18, Tyr34 (3-I125) ]bPTH1_34NH2 was carried out as described in Goldman, ME, et al., Endocrinology, 1988, 123, 1468-1475. Cells such as the human kidney 293 cells transfected with the PTH/PTHrP receptor may also be used as described in Pines, et al., Endocrinology 135:1713 (1994).
Adenylate Cyclase Inhibition Assay
The ability of the test PTH/PTHrP antagonists to inhibit adenylate cyclase release in response to PTH/PTHrP stimulation was also measured in SaOS-2 cells. More specifically, an inhibition of the adenylate cyclase was determined by measuring the level of synthesis of cAMP (adenosine 3' ,5'-monophosphate) . Confluent SaOS-2 cells in 24 wells plates are incubated with 0.5 μCi[3H]adenine (26.9 Ci/mmol, New England Nuclear,
Boston, MA) in fresh medium at 372C for 2 hrs, and washed twice with Hank's balanced salt solution (Gibco, Gaithersberg, MD) . The cells are treated with 1 mM IBMX (isobutylmethylxanthine, Sigma, St. Louis, MO) in fresh medium for 15 min. The test PTH/PTHrP antagonist, at varying concentrations, followed by PTH, PTHrP, or a PTH or PTHrP agonist (e.g., hPTH(l-34)NH2 or [Nle8'18,Tyr34]bPTH(l-34) NH2) at a concentration approximate to its EC50 are added to the medium and incubated for 5 min. The reaction is stopped by the addition of 1.2 M trichloroacetic acid (Sigma) followed by sample neutralization with 4 N KOH. Cyclic AMP is isolated by the two-column chromatographic method. See Salomon, et al., Anal. Biochem. 58:541 (1974). The radioactivity is counted in a scintillation counter (Liquid scintillation counter 2200CA, PACKARD, Downers Grove, IL) . The IC50's (half maximal concentration for the inhibition of adenylate cyclase release ) can be calculated for each of the test PTH/PTHrP antagonists. Cells, such as human kidney 293 cells, which are transfected with the PTH/PTHrP receptor, may also be used as described in Pines, et al., Endocrinology 35:1713 (1994) .
Calcium Inhibition Assay
The ability of the test PTH/PTHrP antagonists to inhibit calcium mobilization in response to PTH/PTHrP stimulation can also be measured in SaOS-2 cells. Confluent SaOS-2 BIO cells in 24 wells plates are incubated in 0.3 percent EDTA/phosphate buffered saline solution at 25sc for 2 hrs, and washed twice with Hank's solution. The cells are resuspended in Hank's buffered saline solution for loading of the fluorescent Ca2+ indicator Fura-2AM. The cells are incubated with 2 mM fluorescent Ca2+ indicator Fura-2AM (Molecular Probes,
Eugene, OR) in fresh medium for 30 min at 25sc. Unloaded Fura-2AM is removed by centrifugation twice with Hank's - buffered saline solution, and the final cell suspensions is transformed to a spectrofluorometer (Hitachi F-2000, Japan) , equipped with a magnetic stirring mechanism and a temperature regulated cuvette holder after equilibration to 372C, the test PTH/PTHrP antagonist, at various concentrations, followed by PTH, PTHrP, or a PTH or PTHrP agonist (at a concentration approximate to its EC50) are added to the medium and incubated for 5 min. The IC50's (half maximal concentration for the inhibition of calcium mobilization) can be calculated for the PTH/PTHrP antagonists. Cells, such as human kidney 293 cells, which are transfected with the PTH/PTHrP receptor may also be used.
Other Embodiments The foregoing description has been limited to specific embodiments of this invention. It will be apparent, however, that variations and modifications may be made to the invention, with the attainment of some or all of the advantages of the invention. Such embodiments are also within the scope of the following claims.
What is claimed is:

Claims

Claims
1. A method of promoting bone formation at a bone fracture site in a subject, said method comprising the step of administering to said patient a therapeutically effective amount of a PTH/PTHrP antagonist.
2. A method of claim 1, wherein said PTH antagonist or said PTHrP antagonist is administered locally to said fracture site.
3. A method of claim 1, wherein said PTH/PTHrP antagonist is administered systemically to said patient.
4. A method of claim 1, wherein said PTH/PTHrP antagonist is a PTH peptide antagonist.
5. A method of claim 1, wherein said PTH/PTHrP antagonist is a PTHrP peptide antagonist.
6. A method of claim 2, wherein said PTH/PTHrP antagonist is a PTH peptide antagonist.
7. A method of claim 2, wherein said PTH/PTHrP antagonist is a PTHrP peptide antagonist.
8. A method of claim 3, wherein said PTH/PTHrP antagonist is a PTH peptide antagonist.
9. A method of claim 3, wherein said PTH/PTHrP antagonist is a PTHrP peptide antagonist.
10. A method of claim 1, wherein said PTH/PTHrP antagonist is administered parenterally.
11. A method of claim 2, wherein said PTH/PTHrP antagonist is administered parenterally.
12. A method of claim 3 , wherein said PTH/PTHrP antagonist is administered parenterally.
13. A method of claim 4 , wherein said PTH/PTHrP antagonist is administered parenterally.
14. A method of claim 5, wherein said PTH/PTHrP antagonist is administered parenterally.
15. A method of claim 6, wherein said PTH/PTHrP antagonist is administered parenterally.
16. A method of claim 7, wherein said PTH/PTHrP antagonist is administered parenterally.
17. A method of claim 8, wherein said PTH/PTHrP antagonist is administered parenterally.
18. A method of claim 9, wherein said PTH/PTHrP antagonist is administered parenterally.
EP97946311A 1996-12-05 1997-10-28 Treatment of bone fracture Withdrawn EP0942744A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US76085396A 1996-12-05 1996-12-05
US760853 1996-12-05
PCT/US1997/019370 WO1998024471A1 (en) 1996-12-05 1997-10-28 Treatment of bone fracture

Publications (1)

Publication Number Publication Date
EP0942744A1 true EP0942744A1 (en) 1999-09-22

Family

ID=25060364

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97946311A Withdrawn EP0942744A1 (en) 1996-12-05 1997-10-28 Treatment of bone fracture

Country Status (4)

Country Link
EP (1) EP0942744A1 (en)
AU (1) AU5150798A (en)
CA (1) CA2273843A1 (en)
WO (1) WO1998024471A1 (en)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5114843A (en) * 1988-05-09 1992-05-19 Merck & Co., Inc. Humoral hypercalcemic factor antagonists
US5093233A (en) * 1990-04-25 1992-03-03 Merck & Co., Inc. Antagonists with position 13 modification
TW273513B (en) * 1993-10-27 1996-04-01 Chugai Pharmaceutical Co Ltd

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9824471A1 *

Also Published As

Publication number Publication date
AU5150798A (en) 1998-06-29
CA2273843A1 (en) 1998-06-11
WO1998024471A1 (en) 1998-06-11

Similar Documents

Publication Publication Date Title
US5747456A (en) Continuous low-dose administration of parathyroid hormone or its agonist
EP0917537B1 (en) Novel parathyroid hormone-related peptide analogs
US7632811B2 (en) Analogs of parathyroid hormone
US7410948B2 (en) Analogs of parathyroid hormone
EP0948541B1 (en) Analogs of PTHrP
NICKOLS et al. Binding of parathyroid hormone and parathyroid hormone-related protein to vascular smooth muscle of rabbit renal microvessels
US9492508B2 (en) Parathyroid hormone analogs and uses thereof
US6541450B1 (en) Parathyroid hormone analogues for the treatment of osteoporosis
JP4837888B2 (en) Conformationally restricted parathyroid hormone (PTH) analog
CZ291382B6 (en) Peptide derivatives influencing growth hormone release, pharmaceutical composition containing them and their use
Malluche et al. Influence of the parathyroid glands on bone metabolism
Östenson et al. Effects of porcine diazepam-binding inhibitor on insulin and glucagon secretion in vitro from the rat endocrine pancreas
JPH10511095A (en) Low dose continuous administration of parathyroid hormone or its agonist
EP0942744A1 (en) Treatment of bone fracture
CACABELOS et al. Time-and dose-dependent responses of brain histamine to intracerebroventricular and intraperitoneal administrations of growth hormone-releasing factor (GRF1-44)
RABBANIS et al. Influence of the Amino-Terminus on in Vitro and in Vivo Biological Activity of Synthetic Parathyroid Hormone-Like Peptides of Malignancy
EP1352912A1 (en) Parathyroid hormone analogues for the treatment of osteoporosis
CZ20003999A3 (en) PTH analog, method for selective binding to receptor, method of inducing agonistic and antagonistic response, pharmaceutical preparation and therapy method

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19990621

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20010502