Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/66—Phosphorus compounds
• • 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
  • A61K38/23—Calcitonins
• • 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
  • A61K38/24—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
• • 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 (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

• 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. PTHrP shares many of the biological effects of PTH including binding to a common PTH/PTHrP receptor. Tregear, et. al, Endocrinol. 93:1349 (1983). PTH peptides from many different sources, e.g. human, bovine, rat, chicken, have been characterized. Nissenson. et al., Receptor, 3:193 (1993).
• PTH has been shown to both improve bone mass and quality Dempster, et al., Endocrine Rev., 14:690 (1993); and Riggs, Amer. J. Med. 91 (Suppl 5B):37S (1991).
• the anabolic effect of intermittently administered PTH has been observed in osteoporitic men and women either with or without concurrent antiresorptive therapy. Slovik, et al, J. Bone Miner. Res., 1:377 (1986); Reeve, et al., Br. Med. J., 301:314 (1990); and Hesch, R-D., et al., Calcif. Tissue Int'l, 44:176 (1989).
• the invention features a peptide of the formula (I), wherein
• a preferred embodiment of the immediately foregoing peptide is where A 3 is Ser; A 5 is Ile or Acc; A 7 is Leu, Acc, or Cha; A 8 is Acc, Met, Nva, Leu, Val, Ile, or Nle; A 11 is Leu, Acc, or Cha; A 12 is Acc or Gly; A 15 is Leu, Acc, or Cha; A 16 is Asn or Aib; A 17 is Ser or Aib; A 18 is Acc, Met, or Nle; A 21 is Val or Acc; A 27 is Lys, hArg, Acc, or Cha; A 31 is Val, Leu, Nle, Acc, or Cha; A 32 is His; A 33 is Asn; A 34 is Phe, Tyr, Amp, or Aib; or a pharmaceutically acceptable salt thereof.
• a preferred embodiment of the immediately foregoing peptide, designated Group B, is where A 5 is Ile or Ahc; A 7 is Leu, Ahc, or Cha; A 8 is Ahc, Met, or Nle; A 11 is Leu, Ahc, or Cha; A 12 is Ahc or Gly; A 15 is Leu, Ahc, or Cha; A 18 is Met or Ahc; A 21 is Val or Ahc; A 22 is Glu or Ahc; A 23 is Trp, Ahc, or Cha; A 24 is Leu, Ahc, or Cha; A 27 is Lys, hArg, Ahc, or Cha; A 28 is Leu, Ahc, or Cha; A 29 is Gln, Ahc or Aib; A 31 is Val, Leu, Nle, Ahc, or Cha; R 1 is H; R 2 is H; and R 3 is NH 2 ; or a pharmaceutically acceptable salt thereof.
• a preferred group of peptides of Group B is where at least one of A 7 , A 11 , A 15 , A 23 , A 24 , A 27 , A 28 , or A 31 is Cha.
• Another preferred group of peptides of Group B is where at least one of A 16 , A 17 , A 19 , A 29 , or A 34 is Aib.
• Preferred peptides of formula (I) are [Ahc 7,11 ]hPTH (1-34)NH 2 ; [Ahc 7,11 , Nle 8,18 , Tyr 34 ]hPTH(1-34)NH 2 ; [Ahc 11 ]hPTH(1-34)NH 2 ; [Ahc 7,11,15 ]hPTH(1-34)NH 2 ; [Ahc 7 ]hPTH(1-34)NH 2 ; [Ahc 23 ]hPTH(1-34)NH 2 ; [Ahc 24 ]hPTH(1-34)NH 2 ; [Nle 8,18 , Ahc 27 ]hPTH(1-34)NH 2 ; [Ahc 28 ]hPTH(1-34)NH 2 ; [Ahc 31 ]hPTH(1-34)NH 2 ; [Ahc 24,28,31 ]hPTH(1-34)NH 2 ; [Ahc 24,28,31 , Lys 30 ]hPTH (1-34)NH 2 ; [Ahc 28,31
• the invention also features peptides of the following formulae; [Cha 22,23 , Glu 25 , Lys 26,30 , Leu 28 , Aib 29 ]hPTHrP (1-34)NH 2 ; [Cha 22,23 , Glu 25 , Lys 26,30 , Aib 29 ]hPTHrP(1-34) NH 2 ; [Glu 22,23 , Leu 25,28,31 , Lys 26 , Aib 29 , Nle 30 ]hPTHrP (1-34)NH 2 ; [Glu 22,25 , Leu 23,28,30,31 , Lys 26 , Aib 29 ]hPTHrP (1-34)NH 2 ; [Glu 22,25,29 , Leu 23,28,30,31 , Lys 26 ]hPTHrP (1-34)NH 2 ; [Glu 22,25,29 , Leu 23,28,30,31 , Lys 26 ]hPTHrP (1-34)NH 2 ; [Glu 22,25,29 , Leu 23,
• the invention relates to peptide variants of PTH(1-34) of the following generic formula: wherein
• At least one of A 5 , A 7 , A 8 , A 11 , A 15 , A 18 , A 21 , A 23 , A 24 , A 27 , A 28 , and A 31 is Cha, or at least one of A 3 , A 12 , A 16 , A 17 , A 18 , A 19 , and A34 is Aib; or that (ii) at least A 1 is Dap, A 7 is ⁇ -Nal, Trp, Pal, Phe, or p-X-Phe, A 15 is ⁇ -Nal, Trp, Pal, Phe, or p-X-Phe, A 27 is hArg, or A 31 is Nle; or a pharmaceutically acceptable salt thereof.
• the invention relates to peptide variants of PTH(1-34) of the following formula (II):
• a 5 , A 7 , A 8 , A 11 , A 15 , A 18 , A 21 , A 23 , A 24 , A 27 , A 28 , and A 31 is Cha, or at least one of A 3 , A 12 , A 16 , A 17 , A 18 , A 19 , and A 34 is Aib, and the peptide is not [Aib 12 , Tyr 34 ]hPTH(1-34)NH 2 or a pharmaceutically acceptable salt thereof.
• a preferred group of peptides of formula (II), designated Group (i) is where at least one of A 7 , A 11 , A 15 , A 23 , A 24 , A 27 , A 28 , and A 31 is Cha; or a pharmaceutically acceptable salt thereof.
• a preferred group of peptides of Group (i), designated Group (ii), is where A 3 is Ser; A 5 is Ile; A 7 is Leu or Cha; A 8 is Met, Nva, Leu, Val, Ile, or Nle; A 12 is Leu or Cha; A 12 is Gly; A 15 is Leu or Cha; A 16 is Asn or Aib; A 17 is Ser; A 18 is Met or Nle.
• a 21 is Val; A 27 is Lys, hArg, or Cha; A 32 is His; A 31 is Val, Nle, or Cha; A 33 is Asn; A 34 is Phe, Tyr, Amp, or Aib; R 1 is H; R 2 is H, and R 3 is NH 2 ; or a pharmaceutically acceptable salt thereof.
• a preferred group of peptides of Group (ii), designated Group (iii), is where at least one of A 7 and A 11 is Cha, or a pharmaceutically acceptable salt thereof.
• Preferred peptides of Group (iii) are [Cha 7,11 ]hPTH (1-34)NH 2 , [Cha 7,11 , Nle 8,18 , Tyr 34 ]hPTH(1-34)NH 2 ; [Cha 11 ]hPTH(1-34)NH 2 ; [Cha 7,11,15 ]hPTH(1-34)NH 2 ; and [Cha 7 ]hPTH(1-34)NH 2 ; or a pharmaceutically acceptable salt thereof.
• Another preferred group of peptides of Group (ii), designated Group (iv), is where at least one of A 15 , A 23 , A 24 , A 27 , A 28 , and A 31 is Cha; or a pharmaceutically acceptable salt thereof.
• Preferred peptides of Group (iv) are [Cha 23 ]hPTH(1-34) NH 2 , [Cha 24 ]hPTH(1-34)NH 2 , [Nle 8,18 , Cha 27 ]hPTH (1-34)NH 2 , [Cha 28 ]hPTH(1-34)NH 2 , [Cha 31 ]hPTH(1-34) NH 2 , [Cha 24,28,31 ]hPTH(1-34)NH 2 ; [Cha 24,28,31 , Lys 30 ]hPTH(1-34)NH 2 ; [Cha 28,31 ]hPTH(1-34)NH 2 ; and [Cha 15 ]hPTH(1-34)NH 2 ; or a pharmaceutically acceptable salt thereof.
• Another preferred group of peptides of formula (II), designated Gropu (v), is where at least one of A 3 , A 12 , A 16 , A 17 , A 18 , A 19 , and A 34 is Aib; or a pharmaceutically acceptable salt thereof.
• a preferred group of peptides of Group (v), designated Group (vi), is where A 3 is Ser or Aib; A 5 is Ile, A 7 is Leu or Cha; A 8 is Met, Nva, Leu, Val, Ile, or Nle; A 11 is Leu or Cha; A 15 is Leu or Cha; A 16 is Asn or Aib; A 18 is Met, Aib, or Nle; A 21 is Val; A 27 is Lys, Aib, Leu, hArg, or Cha; A 31 is Val, Nle, or Cha; A 32 is His; A 33 is Asn; A 34 is Phe, Tyr, Amp, or Aib; R 1 is H; R 2 is H, and R 3 is NH 2 ; or a pharmaceutically acceptable salt thereof.
• a preferred group of peptides of Group (vi), designated Group (vii), is where at least one of A 3 , A 12 , A 16 , A 17 , A 19 , and A 34 is Aib; or a pharmaceutically acceptable salt thereof.
• Preferred peptides of Group (vii) are [Aib 16 ]hPTH(1-34) NH 2 , [Aib 19 ]hPTH(1-34)NH 2 [Aib 34 ]hPTH(1-34)NH 2 ; [Aib 16,19 ]hPTH(1-34)NH 2 ; [Aib]hPTH(1-34)NH 2 , [Aib 17 ]hPTH(1-34)NH 2 ; and [Aib 12 ]hPTH(1-34)NH 2 ; or a pharmaceutically acceptable salt thereof.
• Another preferred group of peptides of formula (II), designated Group (viii), is where at least one of A 7 , A 11 , A 15 , A 23 , A 24 , A 27 , A 28 , and A 31 is Cha and at least one of A 3 , A 12 , A 16 , A 17 , A 18 , A 19 , and A 34 is Aib; or a pharmaceutically acceptable salt thereof.
• a preferred group of peptides of Group (viii), designated Group (ix), is where A 3 is Ser or Aib; A 5 is Ile; A 7 is Leu or Cha; A 8 is Met, Nva, Leu, Val, Ile, or Nle; A 11 is Leu or Cha; A 15 is Leu or Cha; A 16 is Asn or Aib; A 18 is Met, Aib, or Nle, A 21 is Val; A 27 is Lys, Aib, Leu, hArg, or Cha; A 31 is Val, Nle, or Cha; A 32 is His; A 33 is Asn; A 34 is Phe, Tyr, Amp, or Aib; R 1 is H, R 2 is H; and R 3 is NH 2 , or a pharmaceutically acceptable salt thereof.
• a preferred group of peptides of Group (ix), designated Group (x), is where at least one of A 7 and A 11 is Cha and at least one of A 16 , A 19 , and A 34 is Aib; or a pharmaceutically acceptable salt thereof.
• Preferred peptides of Group (x) are [Cha 7,11 , Nle 8,18 , Aib 16,19 , Tyr 34 ]hPTH(1-34)NH 2 , [Cha 7,11 , Nle 8,18,31 , Aib 16, 19 , Tyr 34 ]hPTH(1-34)NH 2 , [Cha 7,11 , Aib 19 ]hPTH(1-34) NH 2 ; [Cha 7,11 , Aib 16 ]hPTH(1-34)NH 2 ; [Cha 7,11 , Nle 8,18 , Aib 34 ]hPTH(1-34)NH 2 ; or [Cha 7,11 , Aib 19 , Lys 30 ]hPTH (1-34)NH 2 ; or a pharmaceutically acceptable salt thereof.
• Another preferred group of peptides of Group (ix), designated Group (xi), is where at least one of A 24 , A 28 , and A 31 is Cha and at least one of A 16 and A 17 is Aib; or a pharmaceutically acceptable salt thereof.
• Preferred peptides of Group (xi) are [Cha 28 , Nle 8,18 , Aib 16,19 , Tyr 34 ]hPTH(1-34)NH 2 , and [Cha 28 , Aib 16,19 ]PTH (1-34)NH 2 ; or a pharmaceutically acceptable salt thereof.
• the present invention is directed to a peptide of the formula (III):
• a 1 is Dap
• a 7 is ⁇ -Nal, Trp, Pal, Phe, or p-X-Phe
• a 15 is ⁇ -Nal, Trp, Pal, Phe, or p-X-Phe
• a 27 is hArg, or A 31 is Nle; or a pharmaceutically acceptable salt thereof.
• a preferred group of peptides of formula (III) is where A 1 is Ser, Gly, or Dap; A 3 is Ser or Aib; A 8 is Met, Nva, Leu, Val, Ile, or Nle; A 16 is Asn or Aib; A 18 is Met, Aib, or Nle; A 21 is Val; A 27 is Lys, Aib, Leu, hArg, or Cha; A 31 is Val, Nle, or Cha; A 32 is His; A 33 is Asn; A 34 is Phe, Tyr, Amp, or Aib; R 1 is H; R 2 is H; and R 3 is NH 2 , or a pharmaceutically acceptable salt thereof.
• Preferred peptides of the immediately foregoing peptides are [Nle 31 ]hPTH(1-34)NH 2 , [hArg 27 ]hPTH(1-34)NH 2 , and [Dap 1 , Nle 8,18 , Tyr 34 ]hPTH(1-34)NH 2 ; or a pharmaceutically acceptable salt thereof.
• the present invention is directed to a peptide of the formula (IV):
• a 5 , A 7 , A 8 , A 11 , A 15 , A 18 , A 22 , A 23 , A 24 , A 27 , A 28 , A 30 , or A 31 is Cha, or at least one of A 3 , A 12 , A 16 , A 17 , A 18 , A 19 , A 22 , A 25 , A 26 , A 29 , A 30 , or A 34 is Aib; or a pharmaceutically acceptable salt thereof.
• a preferred group of peptides of formula (IV) is where A 22 is Phe or Cha; A 23 is Phe or Cha; A 25 is His; A 26 is His; A 27 is Leu or Cha; A 28 is Ile or Cha; A 29 is Ala; A 30 is Glu or Lys; A 31 is Ile or Cha; A 32 is His; A 33 is Thr; and A 34 is Ala; or a pharmaceutically acceptable salt thereof.
• Two preferred groups of peptides of the immediately foregoing group of peptides is where at least one of A 7 and A 11 is Cha; or where at least one of A 16 or A 19 is Aib; or a pharmaceutically acceptable salt thereof.
• a 22 is Glu, Aib, or Cha
• a 23 is Leu, Lys, or Cha
• a 25 is Aib or Glu
• a 26 is Aib or Lys
• a 28 is Leu, Lys, or Cha
• a 29 is Glu or Aib
• a 30 is Cha, Aib, or Lys,
• a 31 is Leu, Cha, or Lys
• a 32 is His
• a 33 is Thr
• a 34 is Ala; or a pharmaceutically acceptable salt thereof.
• Two preferred groups of peptides of the immediately foregoing group of peptides is where at least one of A 7 and A 11 is Cha; or where at least one of A 16 or A 19 is Aib; or a pharmaceutically acceptable salt thereof.
• this invention is directed to a peptide of the formula (V):
• a preferred group of peptides of formula (V) is where A 22 is Glu, Aib, Acc, or Cha; A 23 is Leu, Lys, Acc, or Cha; A 25 is Aib or Glu; A 26 is Aib or Lys, A 28 is Leu, Lys, Acc, or Cha; A 29 is Glu or Aib; A 30 is Cha, Aib, Acc, or Lys; A 31 is Leu, Cha, Acc, or Lys; A 32 is His; A 33 is Thr; and A 34 is Ala; or a pharmaceutically acceptable salt thereof.
• Two preferred groups of peptides of the immediately foregoing group of peptides is where at least one of A 7 and A 11 is Cha; or where at least one of A 16 or A 19 is Aib, or a pharmaceutically acceptable salt thereof.
• the present invention is directed to a method of treating osteoporosis in a patient in need thereof, which comprises administering to said patient a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof, as defined hereinabove.
• the present invention is directed to a method of treating osteoporosis in a patient in need thereof, which comprises administering to said patient a combination of a bisphosphonate or calcitonin and a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof, as defined hereinabove.
• the present invention is directed to a pharmaceutical composition
• a pharmaceutical composition comprising a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.
• the present invention is directed to a pharmaceutical composition
• a pharmaceutical composition comprising a compound of formula (I), (II), (III), (IV) or (V) or a pharmaceutically acceptable salt thereof as defined hereinabove, a bisphosphonate or calcitonin and a pharmaceutically acceptable carrier or diluent.
• the present invention is directed to a method of treating osteoporosis in a patient in need thereof, which comprises administering to said patient a peptide of the formula [Glu 22,25 , Leu 23,28,31 , Aib 29 , Lys 26,30 ]hPTHrP (1-34)NH 2 or a pharmaceutically acceptable salt thereof.
• the present invention is directed to a method of treating osteoporosis in a patient in need thereof, which comprises administering to said patient a combination of a bisphosphonate or calcitonin and a peptide of the formula [Glu 22,25 , Leu 23,28,31 , Aib 29 , Lys 26,30 ]hPTHrP (1-34)NH 2 or a pharmaceutically acceptable salt thereof.
• the present invention is directed to a pharmaceutical composition
• a pharmaceutical composition comprising a peptide of the formula [Glu 22,25 , Leu 23,28,31 , Aib 29 , Lys 26,30 ]hPTHrP (1-34)NH 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.
• the present invention is directed to a pharmaceutical composition
• a pharmaceutical composition comprising a peptide of the formula [Glu 22,25 , Leu 23,28,31 , Aib 29 , Lys 26,30 ]hPTHrP (1-34)NH 2 or a pharmaceutically acceptable salt thereof, a bisphosphonate or calcitonin, and a pharmaceutically acceptable carrier or diluent.
• the present invention is directed to a method of treating osteoporosis in a patient in need thereof, which comprises administering to said patient a peptide of the formula (VI):
• a 5 , A 7 , A 8 , A 11 , A 12 , A 15 , A 18 , A 22 , A 23 , A 24 , A 25 , A 26 , A 27 , A 28 , A 29 , A 30 , or A 31 is Acc; or a pharmaceutically acceptable salt thereof.
• the present invention is directed to a method of treating osteoporosis in a patient in need thereof, which comprises administering to said patient a combination of bisphosphonate or calcitonin and a peptide of formula (VI), as defined hereinabove.
• the present Invention is directed to a pharmaceutical composition
• a pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and a peptide of formula (VI), as defined hereinabove.
• the present invention is directed to a pharmaceutical composition
• a pharmaceutical composition comprising a bisphosphonate or calcitonin, a pharmaceutically acceptable carrier or diluent, and a peptide of formula (VI), as defined hereinabove.
• N-terminal amino acid all abbreviations (e.g. Ala or A 1 ) of amino acids in this disclosure stand for the structure of —NH—CH(R)—CO—, wherein R is a side chain of an amino acid (e.g., CH 3 for Ala).
• N-terminal amino acid For the N-terminal amino acid, the abbreviation stands for the structure of ⁇ N—CH(R)—CO—, wherein R is a side chain of an amino acid, ⁇ -Nal, Nle, Dap, Cha, Nva, Amp, Pal, Ahc, and Aib are the abbreviations of the following ⁇ -amino acids; : ⁇ -(2-naphthyl)alanine, norleucine, ⁇ , ⁇ -diaminopropionic acid, cyclohexylalanine, norvaline, 4-amino-phenylalanine, ⁇ -(3-pyridinyl)alanine, 1-amino-1-cyclo-hexanecarboxylic acid, and ⁇ -aminoisobutyric acid, respectively.
• ⁇ -amino acids : ⁇ -(2-naphthyl)alanine, norleucine, ⁇ , ⁇ -diaminoprop
• Acc is an amino acid selected from the group of 1-amino-1-cyclopropanecarboxylic acid. , 1-amino-1-cyclobutanecarboxylic acid; , 1-amino-1-cyclopentanecarboxylic acid; , 1-amino-1-cyclohexanecarboxylic acid; , 1-amino-1-cycloheptanecarboxylic acid, 1-amino-1-cyclooctanecarboxylic acid, and 1-amino-1-cyclononanecarboxylic acid.
• hydroxyalkyl, hydroxyphenyl-alkyl, and hydroxynaphthylalkyl may contain 1-4 hydroxy substituents.
• COE 1 stands for —C ⁇ O.E 1 —C ⁇ O.E 1 .
• Examples of —C ⁇ O.E 1 —C ⁇ O.E 1 include, but are not limited to, acetyl and phenylpropionyl.
• a peptide of this invention is also denoted herein by another format, e.g., [Ahc 7,11 ]hPTH(1-34)NH 2 , with the substituted amino acids from the natural sequence placed between the second set of brackets (e.g., Ahc 7 for Leu 7 , and Ahc 11 for Leu 11 in hPTH).
• the abbreviation hPTH stands for human PTH; hPTHrP for human PTHrP, rPTH for rat PTH, and bPTH for bovine PTH.
• the numbers between the parentheses refer to the number of amino acids present in the peptide (e.g., hPTH(1-34) is amino acids 1 through 34 of the peptide sequence for human PTH).
• hPTH (1-34) The sequences for hPTH (1-34), hPTHrP(1-34), bPTH(1-34), and rPTH(1-34) are listed in Nissenson, et al., Receptor, 3 193 (1993).
• the designation of “NH 2 ” in PTH(1-34)NH 2 indicates that the C-terminus of the peptide is amidated.
• PTH(1-34) on the other hand, has a free acid C-terminus.
• Each of the peptides of the invention is capable of stimulating the growth of bone in a subject (i.e., a mammal such as a human patient).
• a subject i.e., a mammal such as a human patient.
• antiresorptive therapy e.g., bisphosphonate and calcitonin.
• the peptides of this invention can be provided in the form of pharmaceutically acceptable salts.
• such salts include, but are not limited to, those formed with organic acids (e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamoic acid), inorganic acids (e.g., hydrochloric acid, sulfuric acid, or phosphoric acid), and polymeric acids (e.g., tannic acid, carboxymethyl cellulose, polylactic, polyglycolic, or copolymers of polylactic-glycolic acids).
• organic acids e.g., acetic, lactic, maleic, citric, malic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamoic acid
• inorganic acids e.g., hydrochloric acid, sulfuric acid, or
• a therapeutically effective amount of a peptide of this invention and a pharmaceutically acceptable carrier substance e.g., magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can form a micelle
• a pharmaceutically acceptable carrier substance e.g., magnesium carbonate, lactose, or a phospholipid with which the therapeutic compound can form a micelle
• a therapeutic composition e.g., a pill, tablet, capsule, or liquid
• administration e.g., orally, intravenously, transdermally, pulmonarily, vaginally, subcutaneously, nasally, iontophoretically, or by intratracheally
• the pill, tablet, or capsule that is to be administered orally can be coated with a substance for protecting the active composition from the gastric acid or intestinal enzymes in the stomach for a period of time sufficient to allow it to pass undigested into the small intestine.
• the therapeutic composition can also be in the form of a biodegradable or nonbiodegradable sustained release formulation for subcutaneous or intramuscular administration. See, e.g., U.S. Pat. Nos. 3,773,919 and 4,767,628 and PCT Application No WO 94/15587.
• Continuous administration can also be achieved using an implantable or external pump (e.g., INFUSAIDTM pump).
• the administration can also be conducted intermittently, e.g., single daily injection, or continuously at a low dose, e.g., sustained release formulation.
• the dose of a peptide of the present invention for treating the above-mentioned diseases or disorders varies depending upon the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian.
• peptide covered by the above generic formula for use in treating diseases or disorders associated with deficiency in bone growth or the like, e.g. osteoporosis or fractures.
• PTH(1-34) and PTHrP(1-34) have been reported to have two amphophilic alpha helical domains. See, e.g., Barden, et al., Biochem., 32:7126 (1992). The first ′′-helix is formed between amino acid residues 4 through 13, while the second ′′-helix is formed between amino acid residues 21 through 29.
• Some peptides of this invention contain the substitution of Acc for one or more residues within or near these two regions of PTH(1-34) and PTHrP(1-34), e.g., Ahc 7 and Ahc 11 within the first ′′-helix or Ahc 27 and Ahc 28 within the second ′′-helix; or Cha 7 and Cha 11 within the first ⁇ -helix or Cha 27 and Cha 28 within the second ⁇ -helix.
• the peptides of the invention can be prepared by standard solid phase synthesis. See, e.g., Stewart, J. M., et al., Solid Phase Synthesis (Pierce Chemical Co., 2d ed. 1984). The following is a description of how [Glu 22,25 , Leu23,28, Lys26,30, Aib 29 , or Ahc 31 ]hPTH(1-34)NH 2 was prepared.
• Other peptides of the invention can be prepared in an analogous manner by a person of ordinary skill in the art.
• the pH of the aqueous layer was adjusted to about 3 by adding 4N HCl.
• the organic layer was separated.
• the aqueous layer was extracted with ethyl acetate (1 ⁇ 100 ml).
• Two organic layers were combined and washed with water (2 ⁇ 150 ml), dried over anhydrous MgSO 4 , filtered and concentrated to dryness under reduced pressure.
• the residue was recrystallized in ethyl acetate/hexanes, 9.2 g of a pure product was obtained, 29% yield.
• Other protected Acc amino acids can be prepared in an analogous manner by a person or ordinary skill in the art.
• the peptide was synthesized on an Applied Biosystems (Foster City, Calif.) model 430A peptide synthesizer which was modified to do accelerated Boc-chemistry solid phase peptide synthesis. See Schnoize, et al., Int. J. Peptide Protein Res., 90:180 (1992). 4-Methylbenz-hydrylamine (MBHA) resin (Peninsula, Belmont, Calif.) with the substitution of 0.93 mmol/g was used.
• MBHA 4-Methylbenz-hydrylamine
• Boc amino acids (Bachem, Calif., Torrance, Calif.; Nova Biochem., LaJolla, Calif.) was used with the following side chain protection: Boc-Ala-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHex)-OH, Boc-Glu(OcHex)-OH, Boc-His(DNP)-OH, Boc-Val-OH, Boc-Leu-OH, Boc-Gly-OH, Boc-Gln-OH, Boc-Ile-OH, Boc-Lys (2ClZ)-OH, Boc-Ahc-OH, Boc-Thr(Bzl)-OH, Boc-Ser (Bzl)-OH; and Boc-Aib-OH.
• Boc amino acids 2.5 mmol
• HBTU 2.0 mmol
• DIEA 1.0 mL
• Coupling times were 5 min except for the Boc-Aib-OH, and its following residue Boc-Leu-OH, and Boc-Ahc-OH, and its following residue Boc-Lys(2Clz)-OH, wherein the coupling times for these four residues were 2 hrs.
• the resin was treated with a solution of 20% mercaptoethanol/10% DIEA in DMF for 2 ⁇ 30 min. to remove the DNP group on the His side chain.
• the N-terminal Boc group was then removed by treatment with 100% TFA for 2 ⁇ 2 min.
• the partially-deprotected peptide-resin was washed with DMF and DCM and dried under reduced pressure.
• the final cleavage was done by stirring the peptide-resin in 10 mL of HF containing 1 mL of anisole and dithiothreitol (24 mg) at OEC for 75 min. HF was removed by a flow of nitrogen. The residue was washed with ether (6 ⁇ 10 mL) and extracted with 4N HOAc (6 ⁇ 10 mL).
• Boc-His(DNP)-OH, Boc-Asn-GH, Boc-Val-OH, Boc-Leu-OH, Boc-Ser-OH, Boc-Gly-Oh, Boc-Met-OH, Boc-Gln-OH, Boc-Ile-OH, Boc-Lys(2ClZ)-OH, Boc-Ser(Bzl)-OH, and Boc-Trp(Fm)-OH The synthesis was carried out on a 0.14 mmol scale. The Boc groups were removed by treatment with 100% TFA for 2 ⁇ 1 min.
• Boc amino acids (2.5 mmol) were pre-activated with HBTU (2.0 mmol) and DIEA (1.0 mL) in 4 mL of DMF and were coupled without prior neutralization of the peptide-resin TFA salt. Coupling times were 5 min except for the Boc-Aib-OH and the following residue. Boc-Asn(Xan)-OH, wherein the coupling times were 20 min.
• the resin was treated with a solution of 20% mercaptoethanol/10% DIEA in DMF for 2 ⁇ 30 min. to remove the DNP group on the His side chain.
• the N-terminal Boc group was then removed by treatment with 100% TFA for 2 ⁇ 2 min.
• the formyl group on the side chain of Trp was removed by treatment with a solution of 15% ethanolamine/15% water/70% DMF for 2 ⁇ 30 min.
• the partially-deprotected peptide-resin was washed with DMF and DCM and dried under reduced pressure.
• the final cleavage was done by stirring the peptide-resin in 10 mL of HF containing 1 mL of anisole at 0° C. for 75 min. HF was removed by a flow of nitrogen. The residue was washed with ether (6 ⁇ 10 mL) and extracted with 4N HOAc (6 ⁇ 10 mL).
• the pH of the aqueous layer was adjusted to about 3 by adding 4N HCl.
• the organic layer was separated.
• the aqueous layer was extracted with ethyl acetate (1 ⁇ 100 ml).
• Two organic layers were combined and washed with water (2 ⁇ 150 ml), dried over anhydrous MgSO 4 , filtered and concentrated to dryness under reduced pressure.
• the residue was recrystallized in ethyl acetate/hexanes 9.2 g of a pure product was obtained, 29% yield.
• Other protected Acc amino acids can be prepared in an analogous manner by a person or ordinary skill in the art.
• the peptide was synthesized on an Applied Biosystems (Foster City, Calif.) model 430A peptide synthesizer which was modified to do accelerated Boc-chemistry solid phase peptide synthesis. See Schnoize, et al., Int. J. Peptide Protein Res., 90:180 (1992). 4-Methylbenz-hydrylamine (MBHA) resin (Peninsula, Belmont, Calif.) with the substitution of 0.93 mmol/g was used.
• MBHA 4-Methylbenz-hydrylamine
• Boc amino acids (Bachem, Calif., Torrance, Calif.; Nova Biochem., LaJolla, Calif.) was used with the following side chain protection: Boc-Ala-OH, Boc-Arg(Tos)-OH, Boc-Asp(OcHex)-OH, Boc-Glu(OcHex)-OH, Boc-His(DNP)-OH, Boc-Val-OH. Boc-Leu-OH, Boc-Gly-OH, Boc-Gln-OH, Boc-Ile-OH, Boc-Lys (2ClZ)-OH, Boc-Ahc-OH, Boc-Thr(Bzl)-OH, Boc-Ser (Bzl)-OH; and Boc-Aib-OH.
• Boc amino acids 2.5 mmol
• HBTU 2.0 mmol
• DIEA 1.0 mL
• Coupling times were 5 min except for the Boc-Aib-OH, and its following residue Boc-Leu-OH, and Boc-Ahc-OH, and its following residue Boc-Lys(2Clz)-OH, wherein the coupling times for these four residues were 2 hrs.
• the resin was treated with a solution of 20% mercaptoethanol/10% DIEA in DMF for 2 ⁇ 30 min. to remove the DNP group on the His side chain.
• the N-terminal Boc group was then removed by treatment with 100% TFA for 2 ⁇ 2 min.
• the partially-deprotected peptide-resin was washed with DMF and DCM and dried under reduced pressure.
• the final cleavage was done by stirring the peptide-resin in 10 mL of HF containing 1 mL of anisole and dithiothreitol (24 mg) at 0° C. for 75 min.
• HF was removed by a flow of nitrogen.
• the residue was washed with ether (6 ⁇ 10 mL) and extracted with 4N HOAc (6 ⁇ 10 mL).
• the substituents R 1 and R 2 of the above generic formula may be attached to the free amine of the N-terminal amino acid by standard methods known in the art.
• alkyl groups e.g., C 1-12 alkyl
• Hydroxyalkyl groups e.g., C 1-12 hydroxyalkyl
• Acyl groups may be attached by coupling the free acid, e.g., E 1 COOH, to the free amine of the N-terminal amino acid by mixing the completed resin with 3 molar equivalents of both the free acid and diisopropylcarbodiimide in methylene chloride for one hour and cycling the resulting resin through steps (a) to (f) in the above wash program. If the free acid contains a free hydroxy group, e.g., p-hydroxyphenylpropionic acid, then the coupling should be performed with an additional 3 molar equivalents of HOBT.
• peptides of this invention can be prepared in an analogous manner by a person of ordinary skill in the art.
• the peptides of the invention were tested for their ability to bind to the PTH receptor present on the SaOS-2 (human osteosarcoma cells).
• SaOS-2 cells American Type Culture Collection. Rockville, Md.; ATCC #HTB 85
• RPMI 1640 medium Sigma, St. Louis, Mo.
• FBS fetal bovine serum
• 2 mM glutamine at 37EC in a humidified atmosphere of 5% CO 2 in air.
• the medium was changed every three or four days and the cells were subcultured every week by trypsinization.
• SaOS-2 cells were maintained for four days until they had reached confluence.
• the medium was replaced with 5% FBS in RPMI 1640 medium and incubated for 2 hrs at room temperature with 10 ⁇ 10 4 cpm mono- 125 I-[Nle 8,18 , Tyr 34 (3- 125 I)]bPTH(1-34)NH 2 in the presence of a competing peptides of the invention at various concentrations between 10 ⁇ 11 M to 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 binding assay was conducted with various peptides of the invention, and the Kd value (half maximal inhibition of binding of mono- 125 I-[Nle 8,18 , Tyr 34 (3- 125 I)]bPTH(1-34) NH 2 ) for each peptide was calculated.
• any stimulation of the adenylate cyclase was determined by measuring the level of synthesis of cAMP (adenosine 3′,5′-monophosphate) as described previously in Rodan, et al., J. Clin. Invest. 72: 1511 (1983) and Goldman, et al., Endocrinol., 123:1468 (1988).
• cAMP adenosine 3′,5′-monophosphate
• Confluent SAOS-2 cells in 24 wells plates were incubated with 0.5:Ci [ 3 H] adenine (26.9 Ci/mmol, New England Nuclear, Boston, Mass.) in fresh medium at 37EC for 2 hrs, and washed twice with Hank's balanced salt solution (Gibco, Gaithersburg, Md.).
• the cells were treated with 1 mM IBMX [isobutylmethyl-xanthine, Sigma, St. Louis, Mo.] in fresh medium for 15 min, and the peptides of the invention were added to the medium to incubate for 5 min.
• the reaction was stopped by the addition of 1.2 M trichloroacetic acid (TCA) (Sigma, St.
• cAMP was isolated by the two-column chromatographic method (Salmon, et al. 1974, Anal. Biochem. 58, 541). The radioactivity was counted in a scintillation counter (Liquid Scintillation Counter 2200CA, PACKARD, Downers Grove, Ill.).
• the respective EC 50 values (half maximal stimulation of adenylate cyclase) for the tested peptides were calculated and shown in Table I. All tested peptides were found to be potent stimulators of adenylate cyclase activity, which is a biochemical pathway indicative as a proximal signal for osteoblast proliferation (e.g. bone growth).

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