WO1995002610A1 - Analogues de la parathormone et peptide apparente a la parathormone: synthese et utilisation pour le traitement de l'osteoporose - Google Patents

Analogues de la parathormone et peptide apparente a la parathormone: synthese et utilisation pour le traitement de l'osteoporose Download PDF

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Publication number
WO1995002610A1
WO1995002610A1 PCT/US1994/000589 US9400589W WO9502610A1 WO 1995002610 A1 WO1995002610 A1 WO 1995002610A1 US 9400589 W US9400589 W US 9400589W WO 9502610 A1 WO9502610 A1 WO 9502610A1
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WIPO (PCT)
Prior art keywords
xaa
leu
lys
glu
arg
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PCT/US1994/000589
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English (en)
Inventor
John L. Krstenansky
John J. Nestor, Jr.
Teresa L. Ho
Brian H. Vickery
Chinh T. Bach
Original Assignee
Syntex (U.S.A.) Inc.
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Priority claimed from PCT/US1993/006465 external-priority patent/WO1994001460A1/fr
Application filed by Syntex (U.S.A.) Inc. filed Critical Syntex (U.S.A.) Inc.
Publication of WO1995002610A1 publication Critical patent/WO1995002610A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to novel analogs of parathyroid hormone and parathyroid hormone related peptide, their synthesis by solid phase and recombinant techniques, and their use for increasing bone mass in mammalian subjects.
  • Osteoporosis is the most common form of metabolic bone disease and may be considered the symptomatic, fracture stage of bone loss
  • osteoporosis Although osteoporosis may occur secondary to a number of underlying diseases, 90% of all cases appear to be idiopathic.
  • Postmenopausal women are particularly at risk for idiopathic osteoporosis (postmeno-pausal or Type I osteoporosis).
  • Another high risk group for idiopathic osteoporosis is the elderly of either sex (senile or Type II osteoporosis).
  • Osteoporosis has also been related to corticosteroid use, immobilization or extended bed rest, alcoholism, diabetes, gonadotoxic chemotherapy, hyperprolactinemia, anorexia nervosa, primary and secondary amenorrhea, and oophorectomy.
  • osteoporosis In the various forms of osteoporosis, bone fractures, which are the result of bone loss that has reached the point of mechanical failure, frequently occur. Postmenopausal osteoporosis is characterized by fractures of the wrist and spine, while femoral neck fractures seem to be the dominant feature of senile osteoporosis.
  • the mechanism by which bone is lost in osteoporotics is believed to involve an imbalance in the process by which the skeleton renews itself.
  • bone remodeling This process has been termed bone remodeling. It occurs in a series of discrete pockets of activity. These pockets appear spontaneously within the bone matrix on a given bone surface as a site of bone resorption.
  • Osteoclasts bone dissolving or resorbing cells
  • Osteoclasts are responsible for the resorption of a portion of bone of generally constant dimension. This resorption process is followed by the appearance of osteoblasts (bone forming cells) which then refill with new bone the cavity left by the osteoclasts.
  • osteoporotics In a healthy adult subject, the rate at which osteoclasts and osteoblasts are formed is such that bone formation and bone resorption are in balance. However, in osteoporotics an imbalance in the bone remodeling process develops which results in bone being lost at a rate faster than it is being made. Although this imbalance occurs to some extent in most individuals as they age, it is much more severe and occurs at a younger age in postmenopausal osteoporotics or following oophorectomy.
  • osteoclasts Daily subcutaneous injections of hPTH(1-34) completely reversed the loss of trabecular bone and resulted in amounts of trabecular bone exceeding that of sham operated controls. The number of osteoblasts increased and the number of osteoclasts decreased.
  • the mammalian parathyroid hormones e.g. human (hPTH), bovine (bPTH), and porcine (pPTH)
  • hPTH human
  • bPTH bovine
  • pPTH porcine
  • Biological activity is associated with the N-terminal portion, with residues (1-34) apparently the minimum required.
  • the N-terminal segment of human PTH differs from the
  • PTH The primary function of PTH is to elicit the adaptive changes that serve to maintain a constant concentration of Ca 2+ in the extracellular fluid.
  • PTH acts on the kidneys to increase tubular reabsorption of Ca 2+ from the urine, as well as stimulating the conversion of calcifediol to calcitriol, which is responsible for absorption of Ca 2+ from the
  • PTH acts on bone to increase the rate of resorption of Ca 2+ and phosphate.
  • PTH stimulates the rate of bone resorption by osteoclasts, increases the rate of differentiation of mesenchymal cells to osteoclasts, and prolongs the half life of these latter cells .
  • With prolonged action of PTH the number of bone forming osteoblasts is also increased; thus, the rate of bone turnover and remodeling is enhanced.
  • individual osteoblasts appear to be less active than normal.
  • PTHrp Parathyroid hormone-related peptide
  • a 140+ amino acid protein and fragments thereof, reproduce the major biological actions of PTH.
  • PTHrp is elaborated by a number of human and animal tumors and other tissues and may play a role in hypercalcemia of malignancy.
  • the sequence of hPTHrp (1-34) is as follows: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile 1 5 10 15
  • sequence homology between hPTH and hPTHrp is largely limited to the 13 N-terminal residues, 8 of which are identical; only 1 of 10 amino acids in the (25-34) receptor binding region of hPTH is conserved in hPTHrp. Conformational similarity may underIle the common activity.
  • This invention provides synthetic polypeptide analogs of parathyroid hormone (PTH) , parathyroid hormone related peptide (PTHrp) , and of the physiologically active truncated homologs and analogs of PTH and PTHrp, and salts thereof, in which amino acid residues (22-31) form an amphipathic cx-helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence:
  • this invention provides analogs of PTH, PTHrp, and the physiologically active truncated analogs and homologs of PTH and
  • amino acid residues (22-31) form an amphipathic ⁇ -helix, the sequence of said residues (22-31) selected from: a) Xaa 1 Xaa 2 Leu Xaa 4 Xaa 5 Leu Xaa 7 Xaa 8 Xaa 9 Xaa 10 wherein
  • Xaa 1 and Xaa 4 are independently Glu, Glu(OCH 3 ), His, or Phe; Xaa 2 is Leu or
  • Xaa 5 is Lys or His
  • Xaa 7 and Xaa 10 are independently Leu or Ile
  • Xaa 8 is
  • Xaa 9 is Lys or Glu (SEQ ID NO:26); b) Xaa 1 Xaa 2 Leu Xaa 4 Arg Leu Leu Xaa 8 Arg Leu wherein
  • Xaa 1 and Xaa 4 are independently Glu, Glu(OCH 3 ), His, or Phe; Xaa 2 is Leu or Phe; Xaa 8 is Glu, Lys, or Lys (COCR-PEGX) and PEGX is a poly- (ethylene glycol methyl ether) radical of molecular
  • this invention provides analogs of PTH, PTHrp, and the physiologically active truncated analogs and homologs of PTH and PTHrp, or salts thereof, of the formula:
  • Xaa 1 is absent or is Ala
  • Xaa 2 is absent or is Val
  • Xaa 3 is absent or is Ser
  • Xaa 4 is absent or is Glu or Glu (OCH-);
  • Xaa 5 is absent or is His or Ala
  • Xaa 6 is absent or is Gln;
  • Xaa 7 is absent or is Leu;
  • Xaa 10 and Xaa 17 are independently Asp or Asp (OCH 3 );
  • Xaa 11 is Lys, Arg, or Leu;
  • Xaa 13 is Lys, Arg, Tyr, Cys, Leu, Cys (CH 2 CONH (CH 2 ) 2 NH (biotinyl) ) , Lys (7-dimethylamino-2 -oxo-2H-l-benxopyran-4 -acetyl) , or Lys (dihydrocinnamoyl);
  • Xaa 20 is Arg or Leu
  • Xaa 19 and Xaa 21 are independently Lys, Ala, or Arg;
  • Xaa 22-31 is selected from (SEQ ID NOS : 26 , 27 , 28 , 29 , or 30); Xaa 32 is His ,
  • Xaa 33 is absent, or is Pro, Thr, Glu, or Ala;
  • Xaa 34 is absent, or is Pro, Arg, Met, Ala, hSer, hSer lactone, Tyr, Leu, or 1 , 4 -diaminobutyryl lactam;
  • Xaa 35 is absent or is Pro, Glu, Ser, Ala, or Gly;
  • Xaa 36 is absent or is Ala, Arg, or Ile;
  • Xaa 37 is absent or is Arg, Trp, or 3- (-2-naphthyl) -L-alanine;
  • Xaa 38 is absent or is Ala or hSer or Xaa 38j)2 is Thr Arg Ser Ala Trp;
  • Term is OR or NR 2 where each R is independently H, (C,-C 4 )alkyl or phenyl (C 1 -C 4 )alkyl; and the pharmaceutically acceptable salts thereof.
  • this invention provides analogs of PTH,
  • amino acid residues (22-31) form an amphipathic ⁇ -helix, the sequence of said residues (22-31) selected from:
  • Xaa is Glu or Arg (SEQ ID NO: 26);
  • Xaa 29 is Glu, Lys, or Lys (COCH-PEGX) and PEGX is a poly- (ethylene glycol methyl ether) radical of molecular weight 100 to 10,000 (SEQ ID NO:27); c) Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu (SEQ ID NO:28);
  • compositions for the prevention or treatment of conditions characterized by decreases in bone mass comprising an effective bone mass increasing amount of a polypeptide analog of parathyroid hormone (PTH) , parathyroid hormone related peptide (PTHrp) , and of the physiologically active truncated homologs and analogs of PTH and
  • amino acid residues (22-31) form an amphipathic ⁇ -helix
  • said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence: Haa (Laa Laa Haa Haa), Laa. in admixture with a pharmaceutically acceptable carrier.
  • processes for preparing pharmaceutical compositions which comprise mixing the above described compounds with a pharmaceutically acceptable carrier.
  • this invention provides methods for treating mammalian conditions characterized by decreases in bone mass which methods comprise administering to a mammal in need thereof an effective bone mass increasing amount of a polypeptide analog of PTH, PTHrp, or of a physiologically active truncated homolog or analog of PTH or PTHrp, or a salt thereof, in which amino acid residues (22-31) form an amphipathic ⁇ -helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence:
  • this invention provides methods for treating mammalian conditions characterized by decreases in bone mass which methods comprise administering to a mammal in need thereof an effective bone mass increasing amount of a polypeptide analog of PTH, PTHrp, or of a
  • amino acid residues (22-31) form an amphipathic ⁇ -helix, the sequence of said residues (22-31) selected from: (SEQ ID NOS: 26, 27, 28, 29, and 30).
  • This invention also includes processes for the solid phase synthesis of polypeptide analogs of PTH, PTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, and salts thereof, in which amino acid residues (22-31) form an amphipathic ⁇ -helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence:
  • Haa (Laa Laa Haa Haa) 2 Laa which processes comprise sequentially coupling protected amino acids on a suitable resin support, removing the side chain and W-protecting groups, and cleaving the polypeptide from the resin.
  • This invention also encompasses processes for the solid phase synthesis of polypeptide analogs of PTH, pTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, and salts thereof, in which amino acid residues (22-31) form an amphipathic ⁇ -helix, the sequence of said residues (22-31) selected from: (SEQ ID NOS: 26, 27, 28, 29, and 30); which processes comprise sequentially coupling protected amino acids on a suitable resin support, removing the side chain and N ⁇ -protecting groups, and cleaving the polypeptide from the resin.
  • amino acid residues (22-31) form an amphipathic ⁇ -helix, said residues (22-31) selected from hydrophilic amino acids (Haa) and lipophilic amino acids (Laa) ordered in the sequence:
  • the invention also encompasses DNA sequences, vectors, and plasmids for the recombinant synthesis of such polypeptide analogs.
  • the invention provides DNA sequences, vectors, and plasmids for the recombinant synthesis of polypeptide analogs of PTH, PTHrp, and of the physiologically active truncated homologs and analogs of PTH and PTHrp, and salts thereof, in which amino acid residues (22-31) form an amphipathic ⁇ -helix, the sequence of said residues (22-31) selected from: (SEQ ID NOS: 26, 27, 28, 29, and 30). BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 presents the DNA sequence and enzyme restriction sites of a synthetic gene coding for a PTHrp (1-34) analog of this invention (hPTH PCR Amplification).
  • Figure 2 outlines the preparation of a plasmid incorporating a
  • Figure 3 outlines the preparation of a plasmid incorporating two copies of a PTHrp(1-34) analog gene (Trp LE 18 hPTHrp (1-34) 2 Construction).
  • Figure 4 outlines the preparation of a plasmid incorporating four copies of a PTHrp(1-34) analog gene (Trp LE 18 hPTHrp (1-34) 4 Construct).
  • Valine Val V The abbreviations represent L-amino acids unless otherwise designated as D- or D,L-. Certain amino acids, both natural and non-natural, are achiral, e.g. glycine. All peptide sequences are presented with the N-terminal amino acid on the left and the C-terminal amino acid on the right.
  • Hydrophilic amino acid refers to an amino acid having at least one hydrophilic functional group in addition to those required for peptide bond formation, such as arginine, asparagine, aspartic acid, glutamic acid, glutamine, histidine, lysine, serine, threonine, and their homologs.
  • Lipophilic amino acid (Laa) refers to an uncharged, aliphatic or aromatic amino acid, such as isoleucine, leucine, methionine,
  • alanine is classified as
  • amphiphilic i.e., capable of acting as either hydrophilic or lipophilic.
  • Physiologically active truncated homolog or analog of PTH or PTHrp refers to a polypeptide having a sequence comprising less than the full complement of amino acids found in PTH or PTHrp which, however, elicits a similar physiological response.
  • the truncated PTH or PTHrp need not be fully homologous with PTH or PTHrp to elicit a similar physiological response.
  • PTH (1-34) and PTHrp (1-34) are preferred, but not exclusive, representatives of this group.
  • Amphipathic ⁇ -helix refers to the secondary structure exhibited by certain polypeptides in which the amino acids assume an ⁇ -helical
  • ⁇ H [ ( ⁇ H N sin ⁇ (N-1) ) 2 + ( ⁇ H N cos ⁇ (N-1) ) 2 ] 1 ⁇ 2 ,
  • the hydrophobic moment may be expressed as the mean hydrophobic moment per residue by dividing ⁇ H by N to obtain ⁇ H >.
  • a value of ⁇ H > at 100° ⁇ 20° of about 0.20 or greater is suggestive of amphipathic helix formation.
  • the ⁇ H > values at 100° for hPTHrp (22-31) and hPTH (22-31) are 0.19 and 0.37, respectively.
  • amphipathic index as a predictor of amphipathicity. They concluded that approximately half of all known ⁇ -helices are amphipathic, and that the dominant frequency is 97.5° rather than 100°, with the number of residues per turn being closer to 3.7 than 3.6. While such refinements are scientifically interesting, the basic approach of Eisenberg, et al. is sufficient to classify a given sequence as amphipathic, particularly when one is designing a sequence ab initio to form an amphipathic
  • a substitute amphipathic ⁇ -helical amino acid sequence may lack homology with the sequence of a given segment of a naturally occurring polypeptide but elicits a similar secondary structure, i. e. an ⁇ -helix having opposing polar and nonpolar faces, in the physiological environment.
  • Replacement of the naturally occurring amino acid sequence with an alternative sequence may beneficially affect the physiological activity, stability, or other properties of the altered parent polypeptide. Guidance as to the design and selection of such sequences is provided in J. L.
  • Haa's are selected from the group of hydrophilic amino acids and the Laa's are selected from the group of lipophilic amino acids, as defined above.
  • residues 1, 4, 5, 8, and 9 are distributed along one face (A) of the helix within about a 140° arc of each other, while residues 2, 3, 6, 7, and 10 occupy an opposing 140° arc on the other face (B) of the helix.
  • all the residues on one face are of the same polarity while all those on the other face are of the opposite polarity, i.e., if face A is all hydrophilic, face B is all lipophilic and vice versa.
  • face A is all hydrophilic
  • face B is all lipophilic and vice versa.
  • Laa (Haa Haa Laa Laa) 2 Haa will also meet the residue distribution criteria and is an equivalent descriptor of the helices of this invention.
  • Alanine may be substituted for either hydrophilic or lipophilic amino acids, since Ala can reside readily on either face of an amphipathic ⁇ -helix, although Ala 10 does not form an amphipathic ⁇ -helix.
  • proline, cysteine, and tyrosine are not used; however, their presence and other random errors in the sequence may be tolerated, e.g. a hydrophilic residue on the lipophilic face, as long as the remaining amino acids in the segment substantially conform to the hydrophilic face - lipophilic face division.
  • sufficiently amphipathic to be a sequence of this invention is to calculate the mean hydrophobic moment, as defined above. If the peak mean moment per residue at 100° ⁇ 20° exceeds about 0 .20 , then the sequence will form an amphipathic helix and is a sequence of this invention.
  • the mean peak hydrophobic moment occurs at 92° and has a value of 0.48.
  • this invention provides analogs of PTH, PTHrp, and the physiologically active truncated analogs and homologs of PTH and PTHrp, or salts thereof, in which amino acid residues (22-31) form an amphipathic ⁇ -helix, the sequence of said residues (22-31) selected from:
  • Xaa 1 and Xaa 4 are independently Glu, Glu(0CH 3 ), His, or Phe;
  • Xaa 2 is Leu or Phe;
  • Xaa 5 is Lys or His;
  • Xaa 7 and Xaa 10 are independently Leu or Ile;
  • Xaa 8 is Ala, Arg, or Glu; and
  • Xaa 9 is Lys or Glu (SEQ ID NO:26);
  • Xaa 1 and Xaa 4 are independently Glu, Glu(OCH 3 ), His, or Phe; Xaa 2 is Leu or Phe; Xaa 8 is Glu, Lys, or Lys (COCH 2 PEGX) and PEGX is a poly- (ethylene glycol methyl ether) radical of molecular
  • this invention provides analogs of PTH, PTHrp, and the physiologically active truncated analogs and homologs of PTH and PTHrp, or salts thereof, of the formula:
  • Xaa 1 is absent or is Ala
  • Xaa 2 is absent or is Val ;
  • Xaa 3 is absent or is Ser ;
  • Xaa 4 is absent or is Glu or Glu (OCH 3 );
  • Xaa 5 is absent or is His or Ala
  • Xaa 6 is absent or is Gln;
  • Xaa 7 is absent or is Leu;
  • Xaa 10 and Xaa 17 are independently Asp or Asp (OCH 3 );
  • Xaa 11 is Lys , Arg, or Leu;
  • Xaa 13 is Lys , Arg, Tyr, Cys , Leu, Cys (CH 2 CONH (CH 2 ) 2 NH (biotinyl) ) , Lys (7 -dimethylamino-2 -oxo-2H- 1 -benxopyran-4 -acetyl) , or Lys (dihydrocinnamoyl);
  • Xaa 20 is Arg or Leu;
  • Xaa 19 and Xaa 21 are independently Lys , Ala, or Arg;
  • Xaa 22"31 is selected from (SEQ ID NOS : 26 , 27 , 28 , 29 , or 30); Xaa 32 is His ,
  • Xaa 33 is absent, or is Pro, Thr, Glu, or Ala;
  • Xaa 34 is absent, or is Pro, Arg, Met, Ala, hSer, hSer lactone, Tyr, Leu, or
  • Xaa 35 is absent or is Pro, Glu, Ser, Ala, or Gly;
  • Xaa 36 is absent or is Ala, Arg, or Ile;
  • Xaa 37 is absent or is Arg, Trp, or 3- (-2-naphthyl) -L-alanine;
  • Xaa 38 is absent or is Ala or hSer or Xaa 3iM2 is Thr Arg Ser Ala Trp;
  • Term is OR or NR- where each R is independently H, (C,-C 4 )alkyl or phenyl (C 1 -C 4 )alkyl; and the pharmaceutically acceptable salts thereof.
  • this invention includes polypeptide analogs of the physiologically active truncated homolog hPTHrp (1-34) , as shown in Formula (I) :
  • Xaa 5 is His or Ala
  • Xaa 22-31 is selected from:
  • Xaa is Glu or Arg (SEQ ID NO:26);
  • Xaa is Glu, Lys, or Lys (COCHjPEGX) and PEGX is a
  • Xaa 32 is His or Lys
  • Xaa 33 is Thr, Glu, or Ala
  • Xaa 34 is Ala, hSer, Tyr, or Leu;
  • a more specific aspect of the invention includes
  • a still more specific aspect of the invention includes those Formula (I) polypeptides wherein Xaa 22-31 is (SEQ ID NO: 26); Xaa 11 and Xaa 13 are both Lys; and Xaa 19 and Xaa 21 are both Arg.
  • polypeptides include, but are not limited to:
  • Another aspect of this invention includes those polypeptides of Formula (I) wherein Xaa 22-31 is (SEQ ID NO:26); Xaa 11 and Xaa 13 are both Lys; and one of Xaa 19 and Xaa 21 is Arg and the other is Ala.
  • Representative poly-peptides of this subgenus include, but are not limited to:
  • this invention includes those polypeptides of
  • Xaa 22-31 is (SEQ ID NO:26); one of Xaa 11 and Xaa 13 is Leu and the other is Lys; and Xaa 19 and Xaa 21 are both Arg.
  • Representative polypeptides of this subgenus include , but are not limited to :
  • this invention includes those polypeptides of Formula (I) wherein Xaa 22-31 is (SEQ ID N0:27) , for which ⁇ H > at 100° exceeds 0.50.
  • a further aspect of this invention includes those Formula (I) polypeptides wherein Xaa 22-31 is (SEQ ID NO:27); Xaa 11 and Xaa 13 are both Lys or both Arg; and Xaa 19 and Xaa 21 are both Arg.
  • Representative polypeptides of this subgenus include, but are not limited to:
  • this invention includes polypeptides of Formula (I) wherein Xaa 22-31 is (SEQ ID NO:28) , for which ⁇ H > at 100° is about 0.25.
  • Representative polypeptides of this subgenus include, but are not limited to:
  • this invention includes polypeptides of Formula (I) wherein Xaa 22-31 is (SEQ ID NO:29) , for which ⁇ H > at 100° is about 0.28.
  • polypeptides of this subgenus include, but are not limited to:
  • this invention includes polypeptides of Formula (I) wherein Xaa 22-31 is (SEQ ID NO:30) , for which ⁇ H > at 100° is about 0.29.
  • Representative polypeptides of this subgenus include, but are not limited to:
  • Still another aspect of this invention includes polypeptide analogs of the physiologically active truncated homolog bPTH(1-34), as shown in
  • Xaa 1 is Ser or Ala
  • Xaa 7 is Leu or Phe ;
  • Xaa 8 is Met or Nle ;
  • Xaa 16 is Asn or Ser
  • Xaa 18 is Leu, Met, or Nle ;
  • Xaa 19 is Glu or Arg
  • Xaa 21 is Val or Arg
  • Xaa 22-31 is selected from (SEQ ID NO : 26 , 27 , 28, 29 , and 30);
  • Xaa 34 is Phe or Tyr
  • Term is OH or NR 2 , where each R is H or (C 1 -C 4 ) alkyl ;
  • polypeptides include, but are not limited to :
  • polypeptides include, but are not limited to:
  • polypeptides of the instant invention may be synthesized by methods such as those set forth in
  • these methods involve the sequential addition of protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid and any reactive side chain group are protected. This protected amino acid is then either attached to an inert solid support, or utilized in solution, and the next amino acid in the sequence, also suitably protected, is added under conditions amenable to formation of the amide linkage . After all the desired amino acids have been linked in the proper sequence, protecting groups and any solid support are removed to afford the crude polypeptide. The polypeptide is desalted and purified, preferably chromatographically, to yield the final product.
  • a preferred method of preparing the analogs of the physiologically active truncated polypeptides, having fewer than about forty amino acids involves solid phase peptide synthesis.
  • this method the ⁇ -amino (N ⁇ ) functions and any reactive side chains are protected by acid- or
  • the protecting group should be stable to the conditions of peptide linkage formation, while being readily removable without affecting the extant polypeptide chain.
  • protecting groups include, but are not limited to t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), o-chlorobenzyloxycarbonyl,
  • Suitable side chain protecting groups include, but are not limited to: acetyl, benzyl (Bzl), benzyloxymethyl (Bom), o-bromobenzyloxycarbonyl, t-butyl,
  • Suitable resin supports are those materials which are inert to the reagents and reaction conditions of the stepwise condensation and deprotection reactions, as well as being insoluble in the media used. Examples of commercially available resins include
  • styrene/divinylbenzene resins modified with a reactive group e.g., chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylated co-poly-(styrene-divinylbenzene), and the like.
  • a reactive group e.g., chloromethylated co-poly-(styrene-divinylbenzene), hydroxymethylated co-poly-(styrene-divinylbenzene), and the like.
  • Benzylated, hydroxymethylated phenylacetamidomethyl (PAM) resin is preferred.
  • C-terminus of the compound is an amide
  • a preferred resin is
  • Attachment to the PAM resin may be accomplished by reaction of the
  • ⁇ T-protected amino acid preferably the Boc-amino acid, as its ammonium, cesium, triethylammonium, 1,5-diazabicyclo-[5.4.0]undec-5-ene,
  • DMF N,N-dimethylformamide
  • the N ⁇ -Boc-amino acid may be attached to the benzhydrylamine resin by means of, for example, an N,N'-diisopropylcarbodiimide
  • DIC /1-hydroxybenzotriazole (HOBt) mediated coupling for from about 2 to about 24 hours, preferably about 2 hours at a temperature of between about 10° and 50°C, preferably 25°C in a solvent such as dichloro-methane or dimethylformamide, preferably dichloromethane.
  • a solvent such as dichloro-methane or dimethylformamide, preferably dichloromethane.
  • each protected amino acid is preferably introduced in approximately 1.5 to 2.5 fold molar excess and the coupling carried out in an inert, nonaqueous, polar solvent such as dichloromethane, DMF, or mixtures thereof, preferably in dichloromethane at ambient temperature.
  • Representative coupling agents are N,N' -dicyclohexylcarbodiimide (DCC) , N,N'-diisopropyl-carbodiimide (DIC) or other carbodiimide, either alone or in the presence of
  • cleavage may be effected by means of aminolysis with an alkylamine or fluoroalkylamine for peptides with an alkylamide C-terminus, or by aminolysis with, for example, ammonia/methanol or ammonia/ethanol for peptides with an unsubstituted amide C-terminus, at a temperature between about -10° and 50°C, preferably about 25°C, for between about 12 and 24 hours, preferably about 18 hours.
  • Peptides with a hydroxy C-terminus may be cleaved by HF or other strongly acidic deprotection regimen or by saponification.
  • the peptide may be removed from the resin by transesterification, e.g., with methanol, followed by aminolysis or saponification.
  • the protected peptide may be purified by silica gel chromatography.
  • the side chain protecting groups may be removed from the peptide by treating the aminolysis product with, for example, anhydrous liquid hydrogen fluoride in the presence of anisole or other carbonium ion scavenger, treatment with hydrogen fluoride/pyridine complex, treatment with tris(trifluoroacetyl) boron and trifluoroacetic acid, by reduction with hydrogen and palladium on carbon or polyvinylpyrrolidone, or by reduction with sodium in liquid ammonia, preferably with liquid hydrogen fluoride and anisole at a temperature between about -10° and +10°C, preferably at about 0°C, for between about 15 minutes and 2 hours, preferably about 1.5 hours.
  • anhydrous liquid hydrogen fluoride in the presence of anisole or other carbonium ion scavenger
  • treatment with hydrogen fluoride/pyridine complex treatment with tris(trifluoroacetyl) boron and trifluoroacetic acid
  • hydrogen and palladium on carbon or polyvinylpyrrolidone
  • the resin cleavage and deprotection steps may be combined in a single step utilizing liquid hydrogen fluoride and anisole as described above.
  • the solution may be desalted (e.g. with BioRad AG-3 ® anion exchange resin) and the peptide purified by a sequence of chromatographic steps employing any or all of the following types: ion exchange on a weakly basic resin in the acetate form; hydrophobic adsorption chromatography on underivatized co-poly (styrene-divinylbenzene), e.g.. Amberlite ® XAD; silica gel adsorption chromatography; ion exchange chromatography on
  • HPLC octyl- or octadecylsilylsilica
  • Another aspect of the present invention relates to processes for preparing polypeptides and pharmaceutically acceptable salts thereof which processes comprise sequentially condensing protected amino acids on a suitable resin support, removing the protecting groups and resin support, and purifying the product, to afford analogs of the physiologically active truncated homologs and analogs of PTH and PTHrp, preferably of PTH(1-34) and PTHrp(1-34), in which the amino acids at positions (22-31) form an amphipathic ⁇ -helical peptide sequence, as defined above.
  • the polypeptides of this invention may be prepared by cloning and expression of a gene encoding for the desired polypeptide.
  • a plasmid containing the desired DNA sequence is prepared and inserted into an appropriate host microorganism, typically a bacteria, such as E. coli, or a yeast, such as Saccharomyces cerevisiae, inducing the host microorganism to produce multiple copies of the plasmid, and so of the cDNA encoding for the polypeptide analogs of this invention.
  • PTH or PTHrp analog is designed with convenient restriction enzyme cleavage sites to facilitate subsequent alterations.
  • Polymerase chain reaction (PCR) as taught by Mullis in U. S. Patent Nos. 4,683,195 and 4,683,202, may be used to amplify the sequence.
  • the amplified synthetic gene may be isolated and ligated to a suitable plasmid, such as a Trp LE plasmid, into which four copies of the gene may be inserted in tandem.
  • a suitable plasmid such as a Trp LE plasmid
  • Trp LE plasmids Preparation of Trp LE plasmids is described in U.S. Patent No. 4,738,921 and European Patent Publication No. 0212532.
  • Trp LE plasmids generally produce 8-10 times more protein than Trp E plasmids.
  • the multi-copy gene may then be expressed in an
  • E. coli or S. cerevisiae.
  • Trp LE 18 Prot (Ile 3 , Pro 5 ) containing the following elements: a pBR322 fragment (EcoRI-BamHI) containing the ampicillin resistant gene and the plasmid origin of replication; an EcoRI-SacII fragment containing the trp promoter and the trpE gene; an HIV protease (Ile 3 , Pro 5 ) gene fragment (SacII-HindIII); a bGRF gene fragment (HindIII-BamHI); and a transcription terminator from E. coli rpoc gene.
  • the HIV protease and bGRF gene fragments are not critical and may be replaced with other coding sequences, if desired.
  • the expressed multimeric fusion proteins accumulate intracellularly into stable inclusion bodies and may be separated by centrifugation from the rest of the cellular protein.
  • the isolated fusion protein is converted to the monomeric PTH or PTHrp analog and may be purified by cation exchange and/or reverse phase HPLC.
  • polypeptides of this invention are useful for the prevention and treatment of a variety of mammalian conditions manifested by loss of bone mass.
  • the compounds of this invention are indicated for the prophylaxis and therapeutic treatment of osteoporosis and osteopenia in humans.
  • the polypeptides of this invention, or salts thereof are administered in amounts between about 0.002 and 1 ⁇ g/kg body weight per day, preferably from about 0.04 to about 0.2 ⁇ g/kg body weight per day.
  • the daily dose of active ingredient is from about 0.1 to about 50 ⁇ gs, preferably from about 2.0 to about 10 ⁇ gs. In other mammals, such as horses, dogs, and cattle, higher doses may be required.
  • This dosage may be delivered in a conventional pharmaceutical composition by a single administration, by multiple applications, or via controlled release, as needed to achieve the most effective results, preferably one or more times daily by injection.
  • the selection of the exact dose and composition and the most appropriate delivery regimen will be influenced by, inter alia, the pharmacological properties of the selected polypeptide, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient.
  • Representative delivery regimens include oral, parenteral (including subcutaneous, intramuscular and intravenous), rectal, buccal (including sublingual), pulmonary, transdermal, and intranasal.
  • salts retain the desired biological activity of the parent polypeptide without toxic side effects.
  • examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalene disulfonic acids,
  • polygalacturonic acid and the like (b) base addition salts formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or with an organic cation formed from N,N'-dibenzylethylenediamine or ethylenediamine; or (c) combinations of (a) and (b), e.g., a zinc tannate salt and the like.
  • polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like
  • organic cation formed from N,N'-dibenzylethylenediamine or ethylenediamine or combinations of (a) and (b), e.g., a zinc tannate salt and the like.
  • a further aspect of the present invention relates to pharmaceutical compositions comprising as an active ingredient a polypeptide of the present invention, or pharmaceutically acceptable salt thereof, in admixture with a pharmaceutically acceptable, non-toxic carrier.
  • compositions may be prepared for parenteral
  • compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA., (1985).
  • Formulations for parenteral administration may contain as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • the formulation can be enhanced by the addition of bile salts or acylcarnitines.
  • Formulations for nasal administration may be solid and may contain excipients, for example, lactose or dextran, or may be aqueous or oily solutions for use in the form of nasal drops or metered spray.
  • excipients include sugars, calcium stearate, magnesium stearate,
  • pregelatinated starch and the like.
  • the absorption across the nasal mucous membrane may be enhanced by surfactant acids, such as for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid,
  • surfactant acids such as for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid,
  • glycodeoxycholic acid cyclodextrins and the like in an amount in the range between about 0.2 and 15 weight percent, preferably between about 0.5 and 4 weight percent, most preferably about 2 weight percent.
  • Delivery of the compounds of the present invention to the subject over prolonged periods of time, for example, for periods of one week to one year, may be accomplished by a single administration of a controlled release system containing sufficient active ingredient for the desired release period.
  • a controlled release system containing sufficient active ingredient for the desired release period.
  • Various controlled release systems such as monolithic or reservoir-type microcapsules, depot implants, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, iontophoretic devices and alternative injectable dosage forms may be utilized for this purpose.
  • Localization at the site to which delivery of the active ingredient is desired is an additional feature of some controlled release devices, which may prove beneficial in the treatment of certain disorders.
  • controlled release formulation contains the polypeptide or its salt dispersed or encapsulated in a slowly degrading, non-toxic, non-antigenic polymer such as copoly (lactic/glycolic) acid, as described in the pioneering work of Kent, Lewis, Sanders, and Tice, U.S. 4,675,189.
  • the compounds or, preferably, their relatively insoluble salts may also be formulated in cholesterol or other lipid matrix pellets, or silastomer matrix implants. Additional slow release, depot implant or injectable formulations will be apparent to the skilled artisan. See, for example,
  • the protected amino acids were obtained from Applied Biosystems Inc. (Foster City, CA) .
  • EXAMPLE I Compound 1 (SEQ ID NO: 7) was prepared on 0.5 mmol scale by the solid phase method on 4-methylbenzhydrylamine resin, using an automated Applied Biosystems Model 430A peptide synthesizer. The ⁇ -amino groups were protected with t-butoxycarbonyl (Boc) . The side chain protecting groups were: benzyl (Bzl) for Asp, Glu, and Ser; tosyl (Tos) for Arg;
  • the completed peptide was cleaved from the resin with simultaneous deprotection of the side chain protecting groups using anhydrous HF (25 mL) in the presence of anisole (2.5 mL) at -10°C for 30 minutes and at 0°C for 60 minutes. After evaporation of the HF in vacuo, the residue was washed with anhydrous ether, and the crude peptide extracted with 10% acetic acid.
  • Lyophilization of the 10% acetic acid extract gave 900 mgs of crude product.
  • the peptide was purified by medium pressure ODS reversed phase column chromatography using a gradient of 22-45% CH 3 CN in 0.1% TFA.
  • the product eluted in three fractions, which were concentrated and lyophilized to give 130 mgs of white solid of >98% purity.
  • AAA Asp, 1.9(2); Glu, 5.6(6); Ser, 1.6(2); His, 2.7(3); Gly, 1.0(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.8(3); Val, 1.0(1); Ile, 0.9(1); Leu, 7.3(8); Lys, 4.0(4) .
  • AAA Asp, 2.1(2); Glu, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly 1.1(1); Thr,
  • AAA Asp, 2.1(2); Gly, 6.1(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 5.0(5); Val, 1.0(1); Ile, 0.9(1); Leu, 7.7(8); Lys, 1.9(2) .
  • AAA Asp, 2.1(2); Glu, 6.1(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.2(1); Thr,
  • AAA Asp, 2.0(2); Glu, 4.8(5); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 6.7(7); Val, 1.0(1); Ile, 1.0(1); Lys,
  • AAA Asp, 2.0(2); Glu, 4.8(5); Ser, 1.8(2); His, 2.7(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 2.0(2); Arg, 3.9(4); Val, 1.0(1); Ile, 1.0(1); Leu, 7.9(8); Lys, 4.0(4) .
  • AAA Asp, 2.0(2); Glu, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly, 2.3(2); Thr, 1.0(1); Ala, 1.9(2); Arg, 5.0(5); Val, 1.2(1); Ile, 1.0(1); Leu, 7.8(8); Lys, 4.3(4). .ompound 10
  • AAA Asp, 2.0(2); Glu, 6.0(6); Ser, 1.8(2); His, 2.0(2); Gly, 1.2(1); Ala, 3.0(3); Arg, 2.8(3); Val, 1.1(1); Ile, 1.0(1); Leu, 9.9(10); Lys, 3.0(3) .
  • AAA Asp, 2.2(2); Glu, 7.7(7); Ser, 1.7(2); His, 2.0(2); Gly, 1.0(1); Ala,
  • AAA Asp, 2.1(2); Glu, 6.5(6); Ser, 2.7(3); His, 3.1(3); Gly, 1.1(1); Ala, 1.0(1); Arg, 1.0(1); Tyr, 0.8(1); Val, 2.0(2); Phe, 1.0(1); Ile,
  • AAA Asp, 2.1(2); Glu, 5.5(5); Ser, 2.6(3); His, 3.1(3); Ala, 1.0(1); Gly, 1.1(1); Arg, 3.2(3); Tyr, 1.0(1); Val, 1.0(1); Phe, 1.0(1); Ile, 1.0(1); Leu, 9.0(9); Lys, 3.0(3) .
  • AAA Asp, 2.1(2); Glu, 4.9(5); Ser, 1.7(2); His, 2.6(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 7.6(7); Arg, 2.8(3); Val, 1.2(1); Ile, 1.0(1); Leu, 6.6(6) ; Lys, 1.9(2) .
  • AAA Asp, 2.1(2); Glu, 6.1(6); Ser, 1.7(2); His, 3.1(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 3.0(3); Arg, 2.1(2); Val, 1.1(1); Ile, 1.0(1); Leu, 8.0(8) ; Lys, 4.4(4) .
  • AAA Asp, 2.1(2); Glu, 6.0(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1); Thr,
  • AAA Asp, 2.1(2); Glu, 2.9(3); Ser, 6.8(7); His, 3.1(3); Gly, 1.2(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Val, 1.0(1); Ile, 1.0(1); Leu, 8.2(8) ; Lys, 2.0(2) .
  • AAA Asp, 2.9(3) , Glu, 3.5(4); Ser, 1.4(2); His, 2.6(3); Gly, 0.9(1); Thr,
  • AAA Asx, 2.1(2); Glx, 5.0(5); Ser, 2.7(3); His, 3.0(3); Gly, 1.0(1); Ala,
  • AAA Asx, 2.0(2); Glx, 4.9(5); Ser, 2.6(3); His, 2.8(3); Gly, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.0(1); Phe, 1.0(1); Ile, 0.9(1); Leu, 8.8(9) ; Lys, 3.4(3) ;
  • AAA Asx, 2.1(2); Glx, 4.9(5); Ser, 2.7(3); His, 2.8(3); Gly, 1.0(1);
  • AAA Asx, 2.1(2); Glx, 6.5(6); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1); Thr,
  • AAA Asx, 2.2(2); Glx, 6.2(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 5.2(5); Val, 1.1(1); Ile, 1.1(1); Leu, 8.4(8) ; Lys, 2.2(2) .
  • AAA Asx, 2.1(2); Glx, 6.2(6); Ser, 1.8(2); His, 2.9(3); Gly, 1.1(1); Thr,
  • AAA Asx, 2.1(2); Glx, 5.9(6); Ser, 1.6(2); His, 2.7(3); Gly, 2.2(2); Thr, 1.0(1); Ala, 1.8(2); Arg, 7.3(7); Val, 0.8(1); Ile, 1.0(1); Leu, 8.1(8) ; Lys, 2.1(2) .
  • AAA Asx, 2.2(2); Glx, 6.1(6); Ser, 1.7(2); His, 3.0(3); Gly, 1.9(2);
  • AAA Asx, 2.0(2); Glx, 5.6(6); Ser, 1.7(2); His, 3.1(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 0.9(1); Arg, 3.0(3); Tyr, 0.9(1); Val, 1.0(1); Ile,
  • AAA Asx, 2.0(2); Glx, 5.5(6); Ser, 1.8(2); His, 3.4(3); Gly, 1.1(1); Thr,
  • AAA Asx+Cys, 3.0(2+1); Glx, 5.6(6); Ser, 1.7(2); His, 3.0(3); Gly,
  • AAA Asx, 2.2(2); Glx, 6.1(6); Ser, 1.8(2); His, 3.8(3); Gly, 1.0(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 1.1(1); Ile, 0.9(1); Leu, 8.3(3); Lys, 3.3(3).
  • AAA Asx, 1.9(2); Glx, 6.3(6); Ser, 1.7(2); His, 3.2(3); Gly, 1.0(1); Thr, 1.1(1); Ala, 2.0(2); Arg, 3.2(3); Val, 1.1(1); Ile, 0.9(1); Leu, 8.2(8); Lys, 4.5(4).
  • AAA Asx, 2.0(2); Glx, 5.7(6); Ser, 1.8(2); His, 3.0(3); Gly, 2.2(2); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.8(3); Val, 1.2(1); Ile, 0.9(1); Leu,
  • AAA Asx, 2.1(2); Glx, 6.3(6); Ser, 1.8(2); His, 3.3(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 1.1(1); Ile, 0.9(1); Leu, 8.0(8); Lys, 4.2(4) .
  • AAA Asx, 2.1(2); Glx, 6.2(6); Ser, 1.4(2); His, 3.0(3); Gly, 1.1(1); Thr, 1.1(1); Ala, 1.7(2); Arg, 3.2(3); Val, 0.6(1); Ile, 0.9(1); Leu, 8.0(8); Lys, 4.1(4).
  • AAA Asx, 2.1(2); Glx, 5.9(6); Ser, 2.0(2); His, 3.1(3); Gly, 0.8(1);
  • hSer 0.8(1); Thr, 1.0(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.3(1); Ile, 1.0(1); Leu, 8.1(8); Lys, 3.8(4).
  • AAA Asx, 2.0(2); Glx, 5.6(6); Ser, 1.7(2); His, 2.9(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.9(3); Val, 1.2(1); Ile, 1.0(1); Leu, 7.7(8); Lys, 4.3(4) .
  • AAA Asx, 2.0(2); Glx, 5.5(6); Ser, 1.8(2); His, 2.9(3); Gly, 1.0(1); Thr, 1.0(1); Ala, 0.9(1); Arg, 2.9(3); Val, 1.2(1); Ile, 0.9(1); Leu,
  • AAA Asx, 2.0(2); Glx, 5.6(6); Ser, 1.9(2); His, 2.9(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 0.9(1); Arg, 2.8(3); Val, 1.2(1); Ile, 1.1(1); Leu,
  • AAA Asx, 2.0(2); Glx, 5.7(6); Ser, 1.8(2); His, 3.0(3); Gly, 1.1(1); Ala,
  • AAA Asx, 2.2(2); Glx, 5.9(6); Ser, 1.9(2); His, 2.1(2); Gly, 1.1(1); Ala, 1.0(1); Arg, 3.0(3); Val, 1.1(1); Ile, 1.0(1); Leu, 7.9(8); Lys, 4.3(4); Pro, 0.9(1).
  • AAA Asx, 2.0(2); Glx, 5.8(6); Ser, 2.8(3); His, 2.8(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 3.7(4); Ile, 0.9(1); Leu, 7.5(8); Lys,
  • AAA Asx, 2.2(2); Glx, 6.0(6); Ser, 2.7(3); His, 3.0(3); Gly, 2.2(2); Thr,
  • AAA Asx, 1.9(2); Glx, 5.6(6); Ser, 2.6(3); His, 3.3(3); Gly, 2.1(2); Thr,
  • AAA Asx, 2.1(2) , Glx, 6.3(6); Ser, 2.8(3); His, 3.2(3); Gly, 2.1(2) , Thr, 2.0(2); Ala, 3.2(3); Arg, 9.9(9); Val, 1.0(1) , Ile, 0.9(1); Leu, 8.6(8); Lys, 1.1(1); Trp, 1.1(1) .
  • Compound 47
  • AAA Asx, 3.0(3); Glx, 2.9(3); Ser, 3.7(4); His, 2.8(3); Gly, 1.1(1); Thr, 0.9(1); Ala, 1.9(2); Arg, 2.0(2); Tyr, 1.0(1); Val, 1.7(2); Phe, 0.9(1); Ile, 0.9(1); Leu+Nle 5.8(2+4); Lys, 3.4(3); Trp, 1.1(1).
  • AAA Asx, 2.3(2); Glx, 6.6(6); Ser, 1.4(2); His, 3.2(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 0.9(1); Met, 1.1(1); Ile,
  • AAA Asx, 2.1(2); Glx, 6.3(6); Ser, 1.7(2); His, 3.3(3); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 2.9(3); Val, 0.9(1); Ile, 0.9(1); Leu, 8.0(8); Lys, 3.8(4) .
  • AAA Asx, 2.2(2); Glx, 5.0(5); Ser, 1.9(2); His, 3.3(3); Gly, 1.0(1); Thr,
  • AAA Asx, 2.2(2); Glx, 4.9(5); Ser, 1.8(2); His, 4.3(4); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.1(3); Val, 1.1(1); Ile, 1.0(1); Leu, 8.1(8); Lys, 3.9(4).
  • AAA Asx, 2.1(2); Glx, 5.9(6); Ser, 1.8(2); His, 4.2(4); Gly, 1.1(1); Thr, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Val, 1.1(1); Ile, 0.9(1); Leu,
  • AAA Asx, 2.2(2); Glx, 4.9(5); Ser, 1.8(2); His, 3.1(3); Gly, 1.1(1); Thr,
  • AAA Asx, 2.2(2); Glx, 7.1(7); Ser, 1.7(2); His, 2.8(3); Gly, 1.0(1); Thr, 1.0(1); Ala, 2.1(2); Arg, 3.1(3); Val, 1.1(1); Ile, 1.7(2); Leu, 7.1(7); Lys, 2.7(3).
  • AAA Asx, 2.1(2); Glx, 5.8(6); Ser, 1.7(2); His, 3.1(3); Gly, 0.9(1);
  • AAA Asx, 2.0(2); Glx, 5.9(6); Ser, 1.7(2); His, 2.9(3); Gly, 0.9(1);
  • AAA Asx, 1.9(2); Glx, 5.9(6); Ser, 1.8(2); His, 3.2(3); Gly, 1.1(1); Ala, 2.0(2); Arg, 3.0(3); Thr, 1.0(1); Val, 1.1(1); Ile, 0.9(1); Leu,
  • AAA Asx, 2.0(2); Glx, 5.5(6); Ser, 2.7(3); His, 3.1(3); Gly, 1.0(1); Ala, 1.8(2); Arg, 4.0(4); Thr, 0.9(1); Val, 0.9(1); Ile, 0.9(1); Leu,
  • AAA Asx, 2.1(2); Glx, 5.5(6); Ser, 2.8(3); His, 2.9(3); Gly, 1.0(1); Ala, 2.0(2); Arg, 4.0(4); Thr, 0.9(1); Val, 1.0(1); Ile, 0.9(1); Leu, 7.5(8); Lys, 4.2(4); Nai, 1.1(1).
  • AAA Asx, 2.0(2); Glx, 5.6(6); Ser, 2.7(3); His, 3.2(3); Gly, 1.0(1); Ala, 3.1(3); Arg, 2.8(3); Thr, 1.0(1); Val, 1.1(1); Ile, 0.9(1); Leu,
  • AAA Asx, 2.2(2); Glx, 6.9(7); Ser, 1.7(2); His, 3.2(3); Gly, 1.1(1); Ala,
  • AAA Asx, 2.0(2); Glx, 6.6(7); Ser, 1.9(2); His, 3.4(3); Gly, 1.1(1); Ala, 2.0(2); Arg, 3.8(4); Thr, 1.0(1); Val, 1.1(1); Ile, 1.7(2); Leu,
  • AAA Asx, 2.2(2); Glx, 6.8(7); Ser, 1.8(2); His, 3.3(3); Gly, 1.0(1); Ala, 2.0(2); Arg, 3.0(3); Thr, 1.0(1); Val, 1.0(1); Ile, 1.8(2); Leu,
  • AAA Asx, 2.0(2); Glx, 6.6(7); Ser, 1.9(2); His, 3.3(3); Gly, 1.1(1); Ala, 2.0(2); Arg, 2.9(3); Thr, 1.0(1); Val, 1.1(1); Ile, 1.0(1); Leu, 8.2(8); Lys, 3.8(4) .
  • AAA Asx, 3.1(3); Glx, 4.8(5); Ser, 2.9(3); His, 2.9(3); Gly, 1.1(1); Ala, 1.1(1); Arg, 2.0(2); Val, 2.7(3); Phe, 1.0(1); Ile, 1.0(1); Leu + Nle 5.9 (4 + 2); Lys, 2.8(3) .
  • AAA Asx, 2.0(2); Glx, 5.7(6); Ser, 1.7(2); His, 2.9(3); Gly, 1.0(1); Thr, 0.9(1); Ala, 1.0(1); Arg, 2.8(3); Ile, 0.9(1); Leu, 7.4(8); Lys,
  • AAA Asx, 2.0(2); Glx, 4.1(4); Ser, 0.9(1); His, 2.1(2); Gly, 1.0(1); Thr,
  • AAA Asx, 2.0(2); Glx, 3.9(4); Ser, 0.9(1); His, 1.9(2); Gly, 1.0(1); Thr, 1.0(1); Ala, 1.0(1); Arg, 2.9(3); Ile, 0.9(1); Leu, 6.8(7); Lys, 4.2(4) .
  • AAA Asx, 2.2(2); Glx, 6.2(6); Ser, 2.8(3); His, 3.1(3); Gly, 2.2(2); Thr, 2.2(2); Ala, 2.2(2); Arg, 10.4(10); Ile, 1.0(1); Leu, 8.0(8); Trp, 1.1(1).
  • AAA Asx, 2.1(2); Glx, 4.1(4); Ser, 1.9(2); His, 2.0(2); Gly, 2.1(2); Thr, 2.0(2); Ala, 2.1(2); Arg, 9.7(10); Ile, 0.9(1); Leu, 7.4(8) .
  • Example I This polypeptide was converted to the homoserine lactone as follows.
  • the purified peptide 160 mgs was dissolved in 44% formic acid (4 mL) .
  • This solution was combined with a premixed solution of cyanogen bromide (700 mgs) and phenol (1.6 mgs) in 44% formic acid (4 mL) at 0°C.
  • the solution was stirred at 0°C for 2 hr and at room temperature for 2 hrs.
  • the formation of the product was monitored by HPLC (Vydac ® C-18, 300 A, 4.6 ⁇ 250 mm, flow of 1.2 mL/min, gradient 25-45% acetonitrile in 0.1% TFA over 10 min) .
  • AAA Asp, 2.1(2); Glu, 6.1(6); Ser, 1.8(2); His, 3.0(3); Thi, 1.1(1); Ala, 1.1(1); Arg, 2.7(3); Val, 1.0(1); Ile, 1.0(1); Leu, 8.2(8); Lys,
  • AAA Asp, 2.1(2); Glu, 6.1(6); Ser, 1.6(2); His, 2.8(3); Gly, 0.97(1); hSer, 0.97(1); Thi, 1.0(1); Ala, 1.0(1); Arg, 2.9(3); Val, 1.0(1); Ile, 1.0(1); Leu, 7.6(8); Lys, 3.9(4).
  • BocAVS (Bzl) E (OBz) H (Bom) QLLHD (OBzl) R(Ts) GR (Ts) S (Bzl) IQD (OBz) - LR(Ts)R(Ts)E(OBz)LLE(OBzl)R(Ts)LLK(Fmoc)R(Ts)LH(Bom)T(Bzl)A- O-PAM was synthesized on a 0.35 mmol scale. All N" groups were protected with t-butoxycarbonyl (Boc); side chain protecting groups were as indicated.
  • the peptide resin was treated with 50 mL of 20% piperidine in dimethylformamide (DMF) at room temperature for 30 minutes to remove the fluorenylmethoxy-carbonyl (Fmoc) protecting group on lysine.
  • the resin was washed successively with DMF, MeOH, CH 2 C1 2 and dried to give 1.6 g partially protected peptide.
  • Example I 100 mgs of Compound 19 were obtained.
  • AAA Asp, 2.1(2); Glu, 5.0(5); Ser, 1.6(2); His, 2.9(3); Gly, 0.9(1); Thr, 1.9(2); Arg, 7.1(7); Val, 1.1(1); Ile, 1.0(1); Leu, 8.0(8); Lys, 0.9(1) .
  • AAA Asp, 2.0(2); Glu, 4.8(5); Ser, 1.6(2); His, 2.6(3); Gly, 1.1(1); Thr, 1.1(1); Arg, 7.3(7); Val, 0.8(1); Ile, 0.9(1); Leu, 8.3(8); Lys, 1.1(1); Ala, 1.8(2).
  • a synthetic gene coding for the hPTHrp (1-34) analog Compound 4 (SEQ ID NO: 9) was designed having the nucleotide sequence and enzyme restriction sites shown in Figure 1.
  • the requisite oligodeoxynucleotides were prepared with a DNA synthesizer (Milligen/Biosearch) using the phosphoramidite process of Sinha, et al., Nucleic Acid Research 12, 4539-4557 (1984).
  • oligonucleotides were located with UV, excised from the gel, desalted over Waters cl8 Sep-pak ® cartridges, and lyophilized.
  • PCR polymerase chain reaction
  • PTHPCR2 CCTCGAAGCT TATGCATCAT TATC (SEQ ID NO: 34) , the entire gene was amplified by PCR.
  • the amplified DNA products were purified by gel
  • hPTHrp (1-34) 1 Analog Gene 200 ng of the amplified DNA was isolated and cut by restriction enzymes HinD III and Sac II. As shown in Figure 2, the DNA was ligated to 2 ⁇ g TrpLE 18 Prot (Ile 3 , Pro 5 ) plasmid previously cleaved with Hind III and Sac II.
  • TrpLE 18 hPTHrp (1-34) 1 containing one copy of the hPTHrp (1-34) analog gene was then transformed into competent E. coli HB 101 cells (CLONTECH, Palo Alto, CA) . Transformants were subjected to PCR analysis to verify insertion. Transformed cell colonies were selected and boiled in 200 ⁇ L of water for 5 minutes; 2 ⁇ L were subjected to PCR with two primers flanking the insert. The PCR product was then analyzed on 1% agarose gel to confirm the presence of one copy of the hPTHrp (1-34) gene insert. TrpLE 18 hPTHrp (1-34) 1 construct was then verified by DNA sequencing on an automated DNA sequencer (Applied Biosystems Model 373A, Foster City, CA) using the vendor's Dye Deoxy Terminator Sequencing kit.
  • Nhe I and Xba I restriction sites are located near the beginning and end of the hPTHrp (1-34) analog gene sequence. These two sites, which recognize different sequences, but produce identical single strand cohesive termini, allow the construction of multiple copy
  • hPTHrp (1-34) genes within the Trp LE 18 vector.
  • Trp LE 18 hPTHrp (1-34) 1 The strategy for constructing repeated hPTHrp (1-34) sequences in tandem is outlined in Figure 3.
  • 5 ⁇ g of plasmid Trp LE 18 hPTHrp (1-34) 1 containing a single copy of the gene was cleaved with Bam HI + Nhe I and Xba I + Bam HI. From each digest, about 300 ng of the fragment containing the hPTHrp (1-34) analog gene was isolated. These two fragments were mixed and ligated to form the Trp LE 18 hPTHrp (1-34) 2 plasmid. This plasmid was used to transform competent E. coli HB 101 cells.
  • TrpLE 18 hPTHrp (1-34) 2 containing two copies of the gene was then confirmed by DNA sequencing.
  • the correct fusion of the two hPTHrp (1-34) genes results in the elimination of Nhe I and Xba I sites at the junction. This makes the remaining Xba I and Nhe I sites flanking the tandem genes unique.
  • Trp LE 18 hPTHrp (1-34) 4 containing four copies of the hPTHrp (1-34) gene was constructed, as shown in Figure 4.
  • the sequence of Trp LE 18 hPTHrp (1-34)4 was found to be correct by DNA sequence analysis.
  • the culture was then grown with vigorous shaking to an A 550 of between 0.6 and 0.8, whereupon 2 mL of a 10 mg/mL solution of indole acrylic acid (IAA) was added. Growth was continued with good aeration for about 16 hr to a final A 550 of about 6 (typically, between 4 and 10).
  • the cells were concentrated by centrifugation and resuspended in 500 mL of 50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA buffer solution (Tris buffer).
  • the suspension was sonicated using a Heat Systems-Ultrasonics, Inc. model 220F sonicator (equipped with a 3/4" horn) operated at 50% of full capacity to avoid overheating.
  • the whole cells were analyzed by SDS-PAGE.
  • the gene products derived from the TrpLE 18 hPTHrp (1-34)4 construct were seen as a major band of the predicted MW of approximately 17,000. This accounts for as much as 10% of the total cellular protein.
  • the cell lysate was centrifuged for 15 min. at about 3600 x g to pellet the Trp LE 18 hPTHrp (1-34) 4 fusion protein; the supernatant was discarded. The pellet was resuspended in 200 mL Tris buffer, (typically 40-80 A 550 /mL).
  • TrpLE 18 hPTHrp (1-34) 4 fusion protein was resuspended by gently stirring in 60 mL of 70% formic acid (about 20 mg/mL total protein; typically material from 1000 A 550 units of cells is dissolved in 3 mL) .
  • a few drops of octanol were added and N 2 bubbled through the solution for 20 minutes before adding 5.5 g CNBr.
  • This reaction was allowed to proceed for 6 hours at 25°C before an equal volume of 50:50 MeOH:Hp was mixed with the sample and subsequently removed by rotary evaporation. After 2 to 4 repetitions of this process, the bulk of the formic acid and CNBr were essentially removed.
  • the sample was then evaporated to dryness, redissolved in 200 mL water and lyophilized for storage.
  • the CNBr cleaved supernatant was dialyzed against 50 mM KH-PO 4 pH 6.5 for 24 hours with multiple changes. During dialysis, pH was maintained at 6.5. After dialysis, the precipitates were removed by high speed
  • Retention time of the homo-Serlactone hPTHrp (1-34), Compound 4 was approximately 2.943 minutes.
  • the purified peptide was approximately 98% pure as determined by mass spectroscopy.
  • EXAMPLE XI The compounds of this invention were evaluated for their effect on bone mass in ovariectomized rats, generally in accord with the procedures of Gunness-Hey and Hock, Metab. Bone Dis. 5:177 181 (1984).
  • the rats were sacrificed and the right femurs excised.
  • the femurs were cut in half and the distal half femurs (DHF) were further separated into trabecular bone (TB) and cortical bone
  • Calcette calcium analyzer and expressed as mean bone Ca in mg/DHF/100 g body weight.
  • the two sample t-test was used to compare OVX and sham groups.
  • Oneway ANOVA was used to compare OVX groups, followed by Fisher's LSD multiple comparison to compare each treatment group to vehicle.
  • Ovariectomy induced substantial total bone loss, primarily from trabecular bone.
  • Total bone calcium was 47 to 54% lower than for shamoperated controls.
  • bPTH(1-34) and hPTHrp (1-34) at 80 ⁇ g/kg/day provided statistically significant increases in total bone calcium for treated OVX rats, ranging from 53 to 95% and 18 to 40%, respectively; however, there was no

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Abstract

L'invention concerne des analogues polypeptidiques synthéthiques de la parathormone (PTH) et des peptides apparentés à la parathormone (PTHrp), ainsi que des homologues et des analogues physiologiquement actifs de formes tronquées de la PTH et de la PTHrp dans lesquels les restes d'acides aminés (22-31) forment une hélice α amphipathique. Ces restes (22-31), choisis parmi des acides aminés hydrophiles (Haa) et des acides aminés lipophiles (Laa), sont disposés suivant la séquence: Haa(Laa Laa Haa Haa)2Laa. Ces composés et leurs sels acceptables sur le plan pharmaceutique sont utiles pour la prophylaxie et le traitement de l'ostéoporose chez les mammifères. L'invention concerne également des procédés pour la production de ces polypeptides utilisant la synthèse en phase solide et des méthodes de recombinaison.
PCT/US1994/000589 1993-07-13 1994-01-21 Analogues de la parathormone et peptide apparente a la parathormone: synthese et utilisation pour le traitement de l'osteoporose WO1995002610A1 (fr)

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PCT/US1993/006465 WO1994001460A1 (fr) 1992-07-14 1993-07-13 Analogues de pth et pthrp, leur synthese et utilisation pour le traitement de l'osteoporose
USPCT/US93/06465 1993-07-13

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WO1996040775A1 (fr) * 1995-06-07 1996-12-19 Syntex (U.S.A.) Inc. Procede de traitement de l'osteopenie induite par les corticosteroides
WO1997007815A2 (fr) * 1995-08-29 1997-03-06 Syntex (U.S.A.) Inc. Compositions pharmaceutiques servant a l'administration nasale de composes utiles pour le traitement de l'osteoporose
EP0822200A1 (fr) * 1996-07-30 1998-02-04 F. Hoffmann-La Roche Ag Procédé de préparation de composés analogues de PTH et PTrP
WO1998004591A1 (fr) * 1996-07-31 1998-02-05 The General Hospital Corporation Nouveaux analogues de la proteine liee a l'hormone parathyroide
US5723577A (en) * 1995-07-13 1998-03-03 Biomeasure Inc. Analogs of parathyroid hormone
WO1999012561A2 (fr) * 1997-09-09 1999-03-18 F. Hoffman-La Roche Ag GUERISON DE FRACTURES AU MOYEN D'ANALOGUES DE PTHrP
US5955574A (en) * 1995-07-13 1999-09-21 Societe De Conseils De Recherches Et D'applications Scientifiques, S.A. Analogs of parathyroid hormone
US5969095A (en) * 1995-07-13 1999-10-19 Biomeasure, Inc. Analogs of parathyroid hormone
WO2003059291A2 (fr) * 2002-01-10 2003-07-24 Osteotrophin Llc Traitement des affections osseuses avec des medicaments anabolisants du squelette
US6921750B2 (en) 1995-07-13 2005-07-26 Societe De Conseils De Recherches Et D'applications Scientifiques, S.A.S. Analogs of parathyroid hormone
US7410948B2 (en) 1995-07-13 2008-08-12 Societe De Conseils De Recherches Et D'applications Scientifiques, Sas Analogs of parathyroid hormone
US7994129B2 (en) 2005-11-10 2011-08-09 Michigan Technological University Methods of using black bear parathyroid hormone
US8987201B2 (en) 2009-12-07 2015-03-24 Michigan Technological University Black bear parathyroid hormone and methods of using black bear parathyroid hormone
US9057727B2 (en) 2007-08-01 2015-06-16 The General Hospital Corporation Screening methods using G-protein coupled receptors and related compositions
US9492508B2 (en) 2010-05-13 2016-11-15 The General Hospital Corporation Parathyroid hormone analogs and uses thereof
EP2957278B1 (fr) 2006-10-03 2017-05-17 Radius Health, Inc. Compositions stables contenant pthrp et leur emploi
CN110114065A (zh) * 2016-11-30 2019-08-09 珀杜研究基金会 通过甲状旁腺激素受体刺激进行的骨折靶向性骨再生
USRE49444E1 (en) 2006-10-03 2023-03-07 Radius Health, Inc. Method of treating osteoporosis comprising administration of PTHrP analog
US11782041B2 (en) 2017-04-28 2023-10-10 Radius Health, Inc. Abaloparatide formulations and methods of testing, storing, modifying, and using same

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WO2005105132A1 (fr) * 2004-05-04 2005-11-10 Metabolic Pharmaceuticals Limited Methodes destinees a prevenir ou traiter des maladies osseuses
CN106146648B (zh) * 2015-03-26 2020-06-12 深圳翰宇药业股份有限公司 一种甲状旁腺激素类似物的合成方法
CN107375910B (zh) * 2017-07-12 2020-03-24 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 PTHrP在制备治疗男性性腺功能低下综合征中的应用

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EP0451867A1 (fr) * 1990-04-12 1991-10-16 Mitsubishi Chemical Corporation Antagonistes d'hormone parathyroide
EP0477885A2 (fr) * 1990-09-28 1992-04-01 Takeda Chemical Industries, Ltd. Dérivées d'hormone parathyroid
WO1993006845A1 (fr) * 1991-10-10 1993-04-15 Pang Peter K T Analogues de l'hormone parathyroidienne substitues au niveau des aminoacides ?25,26,27¿, et leur utilisation dans le traitement de l'osteoporose
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JP2008024709A (ja) * 1995-06-07 2008-02-07 Syntex Usa Inc コルチコステロイド誘導オステオペニアの処置法
WO1996040775A1 (fr) * 1995-06-07 1996-12-19 Syntex (U.S.A.) Inc. Procede de traitement de l'osteopenie induite par les corticosteroides
US7632811B2 (en) 1995-07-13 2009-12-15 Societe De Conseils De Recherches Et D'applications Scientifiques, Sas Analogs of parathyroid hormone
US5723577A (en) * 1995-07-13 1998-03-03 Biomeasure Inc. Analogs of parathyroid hormone
US6921750B2 (en) 1995-07-13 2005-07-26 Societe De Conseils De Recherches Et D'applications Scientifiques, S.A.S. Analogs of parathyroid hormone
USRE40850E1 (en) 1995-07-13 2009-07-14 Societe De Conseils De Recherches Et D'applications Scientifiques, S.A.S. Analogs of parathyroid hormone
US5955574A (en) * 1995-07-13 1999-09-21 Societe De Conseils De Recherches Et D'applications Scientifiques, S.A. Analogs of parathyroid hormone
US5969095A (en) * 1995-07-13 1999-10-19 Biomeasure, Inc. Analogs of parathyroid hormone
US7410948B2 (en) 1995-07-13 2008-08-12 Societe De Conseils De Recherches Et D'applications Scientifiques, Sas Analogs of parathyroid hormone
WO1997007815A2 (fr) * 1995-08-29 1997-03-06 Syntex (U.S.A.) Inc. Compositions pharmaceutiques servant a l'administration nasale de composes utiles pour le traitement de l'osteoporose
WO1997007815A3 (fr) * 1995-08-29 1997-09-18 Syntex Inc Compositions pharmaceutiques servant a l'administration nasale de composes utiles pour le traitement de l'osteoporose
US6849710B1 (en) 1996-07-30 2005-02-01 F. Hoffmann-La Roche Ag Method for the synthesis of analogs of parathyroid hormone and parathyroid hormone related peptide
KR100518982B1 (ko) * 1996-07-30 2006-05-29 에프. 호프만-라 로슈 아게 부갑상선호르몬및부갑상선호르몬과연관된펩티드의유사체의합성방법
EP0822200A1 (fr) * 1996-07-30 1998-02-04 F. Hoffmann-La Roche Ag Procédé de préparation de composés analogues de PTH et PTrP
US6362163B1 (en) 1996-07-31 2002-03-26 The General Hospital Corporation Parathyroid hormone-related peptide analogs
US6147186A (en) * 1996-07-31 2000-11-14 The General Hospital Corporation Parathyroid hormone-related peptide analogs
WO1998004591A1 (fr) * 1996-07-31 1998-02-05 The General Hospital Corporation Nouveaux analogues de la proteine liee a l'hormone parathyroide
AU752925B2 (en) * 1997-09-09 2002-10-03 F. Hoffmann-La Roche Ag Fracture healing using PTHrP analogs
US6583114B2 (en) 1997-09-09 2003-06-24 Roche Palo Alto Llc Fracture healing using pthrp analogs
WO1999012561A3 (fr) * 1997-09-09 1999-05-14 Hoffmann La Roche GUERISON DE FRACTURES AU MOYEN D'ANALOGUES DE PTHrP
WO1999012561A2 (fr) * 1997-09-09 1999-03-18 F. Hoffman-La Roche Ag GUERISON DE FRACTURES AU MOYEN D'ANALOGUES DE PTHrP
KR100679778B1 (ko) * 1997-09-09 2007-02-07 에프. 호프만-라 로슈 아게 PTHrP 유사체를 사용하는 골절 치료법
US7015195B2 (en) 2002-01-10 2006-03-21 Osteotrophin, Llc Treatment of bone disorders with skeletal anabolic drugs
US7384912B2 (en) 2002-01-10 2008-06-10 Osteotrophin, Llc Treatment of bone disorders with skeletal anabolic drugs
WO2003059291A3 (fr) * 2002-01-10 2005-03-24 Osteotrophin Llc Traitement des affections osseuses avec des medicaments anabolisants du squelette
WO2003059291A2 (fr) * 2002-01-10 2003-07-24 Osteotrophin Llc Traitement des affections osseuses avec des medicaments anabolisants du squelette
US7994129B2 (en) 2005-11-10 2011-08-09 Michigan Technological University Methods of using black bear parathyroid hormone
USRE49444E1 (en) 2006-10-03 2023-03-07 Radius Health, Inc. Method of treating osteoporosis comprising administration of PTHrP analog
EP2957278B1 (fr) 2006-10-03 2017-05-17 Radius Health, Inc. Compositions stables contenant pthrp et leur emploi
US9057727B2 (en) 2007-08-01 2015-06-16 The General Hospital Corporation Screening methods using G-protein coupled receptors and related compositions
US8987201B2 (en) 2009-12-07 2015-03-24 Michigan Technological University Black bear parathyroid hormone and methods of using black bear parathyroid hormone
US9492508B2 (en) 2010-05-13 2016-11-15 The General Hospital Corporation Parathyroid hormone analogs and uses thereof
CN110114065A (zh) * 2016-11-30 2019-08-09 珀杜研究基金会 通过甲状旁腺激素受体刺激进行的骨折靶向性骨再生
US11782041B2 (en) 2017-04-28 2023-10-10 Radius Health, Inc. Abaloparatide formulations and methods of testing, storing, modifying, and using same
US11835506B2 (en) 2017-04-28 2023-12-05 Radius Health, Inc. Abaloparatide formulations and methods of testing, storing, modifying, and using same
US11977067B2 (en) 2017-04-28 2024-05-07 Radius Health, Inc. Abaloparatide formulations and methods of testing, storing, modifying, and using same

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