WO1996019246A1 - Continuous low-dose administration of parathyroid hormone or its agonist - Google Patents

Continuous low-dose administration of parathyroid hormone or its agonist Download PDF

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Publication number
WO1996019246A1
WO1996019246A1 PCT/US1995/016554 US9516554W WO9619246A1 WO 1996019246 A1 WO1996019246 A1 WO 1996019246A1 US 9516554 W US9516554 W US 9516554W WO 9619246 A1 WO9619246 A1 WO 9619246A1
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Prior art keywords
nle
arg
tyr
phe
bpth
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PCT/US1995/016554
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French (fr)
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Michael Chorev
Michael Rosenblatt
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Beth Israel Hospital Association
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Priority to JP8519949A priority Critical patent/JPH10511095A/en
Priority to AU44728/96A priority patent/AU4472896A/en
Priority to EP95943474A priority patent/EP0800405A4/en
Publication of WO1996019246A1 publication Critical patent/WO1996019246A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575Hormones
    • C07K14/635Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22Hormones
    • A61K38/29Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00Drugs for disorders of the metabolism
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • Parathyroid hormone has been shown to effect a positive bone balance (reviewed in Dempster, D. . et al., Endocrine Rev., 1993, 14, 690-709; Riggs, L. , Amer. J. Med., 1991, 91 (Suppl 5B) , 37S-41S) .
  • the mammalian parathyroid hormone is a polypeptide product of the parathyroid glands.
  • the mature circulating form of the hormone is comprised of 84 amino acid residues.
  • 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
  • PTHi. 34 is a synthetic amino-terminal fragment of PTH 1 _ 8 (Tregear, G.W. , et al., Endocrinology, 1973, 93, 1349-1353; Mosekilde, L. , et al., Endocrinology, 1991, 129, 421-428).
  • the invention features a method of promoting bone formation in a human patient (e.g., a patient who suffers f om osteoporosis) .
  • the method includes the steps of administering (e.g., transmucously, intravenously, transdermally, subcutaneously, via implantation, or via infusion) continuously to the patient PTH (mature form) , PTHrP, or an agonist thereof for a period of at least one month (as long as the life time of the patient, if necessary) at a dosage between 10 and 400 units/24 hrs.
  • PTH and PTHrP include, but are not limited to, human PTH (hPTH) , human PTHrP (hPTHrP) , bovine PTH (bPTH) , bovine PTHrP (bPTHrP) , and rat PTH (rPTH) .
  • An agonist of PTH or PTHrP is a peptide which is a structural analog or fragment (preferably, an N-terminal fragment containing 50 or fewer amino acids) of a naturally occurring PTH or PTHrP and, like PTH and PTHrP, also capable of binding to PTH receptor and, thereby, promoting bone formation.
  • Examples of such an agonist include, but are not limited to, hPTH 1 _ 34 NH 2 , hPTH 1 _ 38 NH 2 , hPTH !
  • NH 2 denotes amidation of the carboxyl group (-CO*OH) of the C-terminal amino acid to form -CO*NH 2 .
  • the following publications disclose the sequences of PTH peptides: The Parathyroids Basic and Clinical Concepts, ed. John P. Bilezikian, 239-258 (Raven Press, NH 1994); Nissenson, R. , et al., Structure & Function of the Receptor for Parathyroid Hormone and Parathyroid Hormone-Releasing Hormone, 3 Receptor 193-202 1993; Bachem California 1993-1994 Catalog (Torrance, CA) ; and Sigma Peptides and Amino Acids 1994 Catalog (St. Louis, MO) .
  • partial PTH agonists can also be used to practice the method of this invention.
  • Examples of partial PTH agonists include, but are not limited to, N- terminal deletion analogs (e.g., [Tyr 34 ]bPTH 3 _ 34 NH 2 ; see U.S. Patent No. 4,771,124 (1988).
  • Preferred ranges of dosages include 10-300 units/24 hrs, 10-200 units/24 hrs, 10-100 units/24 hrs, 100-400 units/24 hrs, 200-400 units/24 hrs, and 300-400 units/24 hrs.
  • PTH, PTHrP, or an agonist thereof is defined by utilizing an in vitro cAMP accumulation assay with human SaOS-2 cells.
  • Human SaOS-2 cells respond upon exposure to PTH, PTHrP, or an agonist thereof with a dose-dependent stimulation of cAMP accumulation.
  • [Nle 8 ' 18 ,Tyr 34 ]hPTH 1 _ 34 NH 2 as the reference standard analog (10,000 units/mg) a dose-response relationship can be generated using standard non-linear regression analysis.
  • the relative potency for various PTH analogs (in units/mg) can be determined from the ratio of the EC 50 of the reference standard analog to that of the PTH analog.
  • EC 50 is defined as the dose that evokes a half maximal response — cAMP accumulation herein.
  • the detailed procedure for handling the cells, setting up the assay, as well as methods for cAMP quantitation, is described in Sistane, E. , et al., Pharmacopeial Forum 20(3), 7509-7520 (1994).
  • an ambulatory e.g., MINIMEDTM 404-SP, MiniMed Technologies, Sylmar, CA; Pharmacia Deltec CADD-MICRO MODELTM S900, Pharmacia Deltec Inc., St. Paul, MN; or Disetronic Medical System's PANOMATTM, Madison, MN
  • an implantable pump e.g., MEDTRONIC SYNCROMEDTM, Medtronic, Inc., Minneapolis, MN
  • MEDTRONIC SYNCROMEDTM Medtronic, Inc., Minneapolis, MN
  • PTH, PTHrP, or an agonist thereof is administered as a sustained release formulation.
  • the formulation may contain a homo- or a co- polymer prepared from lactic acid (D-isomer, L-isomer, or a racemate) , glycolide, glycolic acid, or caprolactone.
  • suitable sustained release formulations can be found in the following publications: U.S. Patent No. 3,773,919 (1973) U.S. Patent No. 5,187,150 (1993) U.S. Patent No. 4,767,628 (1988)
  • the sustained release formulations can be administered parenterally (e.g. subcutaneously, or intravenously) or by inhalation (e.g. using an aerosol delivery system; e.g., see WO93/00951 and WO94/07514) .
  • PTH, PTHrP, or an agonist thereof can be administered transmucously (e.g. nasal, vaginal, rectal) or transdermally (e.g. iontophoretic patch) .
  • a bone resorption inhibiting agent can also be administered during performance of the above-described method.
  • a suitable bone resorption inhibiting agent include, but are not limited to, estrogen, calcitonin, bisphosphonate, tamoxifen, vitamin D, and calcium. Also, see U.S. Patent No. 5,118,667 (1992).
  • the preferred dose and duration for practicing the above-described method 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.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient(s) into association with the carrier which constitutes one or more accessory ingredients.
  • the formulations for tablets or powders are prepared by uniformly and intimately blending the active ingredient (e.g., hPTH) with finely divided solid carriers, and then, if necessary as in the case of tablets, forming the product into the desired shape and size.
  • the invention also features novel PTH or PTHrP agonists of the following formula: ⁇
  • a 2 is Ser or Ala
  • a 7 is Leu or Phe
  • a 12 is Gly, Aib, Ala, or D-Ala;
  • a 16 is Asn, Ser or Ala;
  • a 19 is Glu, Arg, Lys, Asp, Ser, Thr, Gin, Asn, or Ala;
  • a 21 is Val, Met, Arg, Lys, Glu, Asp, Ser, Thr, Gin, Asn, Leu, lie, Nle, Ala, Phe, or p-X-Phe where X is OH, CH 3 , N0 2 , or a halogen;
  • a 22 is Glu, Asp, Phe, p-X-Phe where X is OH, CH 3 , N0 2 or a halogen, Ser, Thr, Gin, Asn, Leu, lie, Nle, Val, Ala, or Met;
  • a 23 is Trp, 1-Nal, or 2-Nal;
  • a 34 is Phe, or p-X-Phe where X is OH, CH 3 , N0 2 , or a halogen;
  • W is OH, C ⁇ _ 12 alkoxy, c 7 - 20 phenylalkoxy, C 11 _ 20 napthylalkoxy, or NR 3 R 4 ; provided that when A 12 is Gly,
  • a ⁇ g is Glu, A 21 is Val, and A 22 is Gin, then A 23 must be 1- Nal or 2-Nal; and each of R 2 , R 2 , R 3 , and R 4 is, independently, H, c ⁇ -i 2 alkyl, C 7 _ 10 phenylalkyl, or CO * E where E is C x _ 12 alkyl, C 2 _ 12 alkenyl, phenyl, naphthyl, or C 7 _ 20 phenylalkyl; or a pharmaceutically acceptable salt thereof.
  • the N-terminus is at the left and the C-terminus at the right in accordance with the conventional representation of a polypeptide chain.
  • R is - CH(CH 3 ) 2 for Val.
  • the amino acid residue is optically active, it is the L-form configuration that is intended unless D-form is expressly designated.
  • Preferred groups of compounds covered by the above formula include (i) those where h- is Ala; A 7 is Phe; and A 16 is Ser; (ii) those where A 19 is Glu or Arg; and A 21 is Val or Arg; and (iii) those where A 22 is p-X-Phe where X is OH, CH 3 , N0 2 , or a halogen.
  • PTH and PTH-like agonist activities of peptide fragments or derivatives of parathyroid hormone or PTHrP can be tested by contacting a concentration range of the test compound with the cells in culture and assessing the stimulation of the PTH/PTHrP receptors.
  • Receptor stimulation is evidenced by the activation of second messenger molecules coupled to the receptors, for example, a stimulation of cyclic AMP accumulation in the cell or an increase in enzymatic activity of protein kinase C, both of which are readily monitored by conventional assays (Jouis Subscribe, H. , et al., Endocrinology, 1992, 130, 53-60; Abou-Samra, A.B. , et al..
  • PTH-like agonist activities of subfragments of PTH have been successfully analyzed by contacting peptides with rat kidney cells in culture and assessing cyclic AMP accumulation (Blind, E. , et al., Clin. Endocrinol., 1993, 101, 150-155) and the stimulation of 1,25- dehydroxyvitamin D 3 production (Janulis, M. , et al., Endocrinology, 1993, 133, 713-719).
  • PTH or PTHrP agonists of the invention were synthesized on an APPLIED BIOSYSTEMSTM 430A Automated Peptide Synthesizer (Applied Biosystems Inc. , Foster City, CA) using version 1.40 of the software for NMP/HOBt Boc-based chemistry.
  • Boc for t-butoxycarbonyl
  • For for formayl cycHx for cyclohexyl
  • Cl-Z for 2- chlorobenzyloxycarbonyl
  • OBzl is O-benzyl
  • BOM for benzyloxymethyl
  • Bzl for benzyl
  • Br-Z for 2-bromo- benzyloxycarbonyl
  • I ⁇ -Tosyl for tosyl at guanidyl site
  • pMBHA-R paramethoxybenzhydrylamine resin.
  • the crude peptides were purified on a WATERS DELTA PREPTM 4000 (Waters, Milford, MA), preparative HPLC system, connected to a PrepPack cartridge of VydacTM C18 300A, 15-20 ⁇ [47 x 300 mm] (Waters, Milford, MA) at a flow rate of 70 mL/min monitored at 220 nm.
  • the analytical HPLC system included the following components: Waters 600E multisolvent delivery system, 490E programmable multiwavelength detector, 717 autosampler and a 747 data module.
  • the samples were analyzed on a VYDACTM C18 218TP5415 (150 x 4.6 mm, 5 ⁇ m) , The Separation Group (Hesperia, CA) at a flow rate of 1 mL/min monitored at 220 nm.
  • the solvent mixtures for both the analytical and preparative HPLC were: A: 0.1% TFA in H 2 0, and B: 0.1% TFA in acetonitrile.
  • the purity of the peptides and their derivatives exceeded 99% as determined from the analytical RP-HPLC.
  • Example 2 SaOS-2 BIO cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37°C in a humidified atmosphere of 5% C0 2 in air. The medium was changed every three or four days, and the cells were subcultured every week by trypsinization.
  • FBS fetal bovine serum
  • SaOs-2 BIO cells were maintained for four days after they had reached confluence.
  • the medium was replaced with 5% FBS RPS/1640 medium and incubated for 2 hrs at room temperature with 10 x 10 4 cpm mono- 125" I- [Nle 8 ' 18 ,Tyr 34 (3-I 125 ) ]bPTH 1 _ 34 NH 2 in the presence or absence of a competing tested PTH agonist.
  • 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. Synthesis of the radiolabelled [Nle 8 ' 18 . Tyr 34 (3-I 125 ) ]bPTH 1 _ 34 NH, was carried out as described in Goldman ME et al., Endocrinology, 1988, 123, 1468-1475.
  • the binding assay was conducted on Analogs I-IV and Analog V (i.e., [Nle 8 , Nle 18 , Tyr 3 ]bPTH 1 _ 34 NH 2 ) .
  • the IC 5 ⁇ 0n '' s (half maximal inhibition of binding of mono- 125 I- ' [Nle 8 ' 18 ,Tyr 34 (3-I 125 )]bPTH 1 _ 34 NH 2 ) for the five tested analogs were calculated and shown in Table IV below:
  • In vivo bone anabolic activities of PTH/PTHrP agonists are tested by administering the peptide or a formulation containing the peptide into intact animals or an experimental animal model of osteopenia.
  • the animal model can be osteoporosis in rats induced by ovariectomy (Hori, M. , et al., Bone Miner., 1988, 3, 193-199; Geral, et al., J. Bone Miner. Res., 1989, 4, Suppl. 1, S303; Liu C-C. & Kalu, D.N., J. Bone Miner. Res., 1990, 5, 973-982; Mosekilde, L.
  • the bone anabolic effects of the compound are determined following 12 to 60 days of treatment by assessing the change in bone mineral density by dual energy x-ray absorptiometry or dry weight of femurs or total ash weight (Hori, H. , et al., Bone Miner., 1988, 3, 193-199; Hefti, E. , et al., Clin. Sci., 1982, 62, 389- 396) .
  • Increase in the rate of bone formation and mineralization are assessed using metabolic labels, e.g. tetracycline (Tam, C.S., et al., Endocrinology, 1982, 110, 506-512).
  • the patients prior to the study are subject to the following: complete health and physical examination, evaluation of the nutritional status (particularly of calcium intake and serum calcium) , full analysis of biomarkers for bone-turnover (Riis, B.J. , Amer. J. Med. 1993, 95 [Suppl 5A] , 17s-21s; Delmas, P.D. , Amer. J.
  • the treatments are extended to twelve months, wherein bone mass measurements and bone biopsy further provide clear indication of bone growth in the population treated with continuous infusion hPTH 1 _ 34 .

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Abstract

A method of promoting bone formation in a human patient, which includes the step of administering continuously to the patient parathyroid hormone or its agonist for a period of at least one month at a dosage between 10 and 400 units/24 hrs. Also disclosed are novel parathyroid hormone agonists.

Description

CONTINUOUS LOW-DOSE ADMINISTRATION OF PARATHYROID HORMONE OR ITS AGONIST
BACKGROUND OF THE INVENTION Parathyroid hormone ("PTH") has been shown to effect a positive bone balance (reviewed in Dempster, D. . et al., Endocrine Rev., 1993, 14, 690-709; Riggs, L. , Amer. J. Med., 1991, 91 (Suppl 5B) , 37S-41S) . The mammalian parathyroid hormone is a polypeptide product of the parathyroid glands. The mature circulating form of the hormone is comprised of 84 amino acid residues. 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
(Schipani E. , et al., Endocrinology, 1993, 132, 2157- 2165; Broadus, A.E., Steward, A.F., Parathyroid hormone- related protein: In: The Parathyroids, Bilezikian, J.P., Levine, M.A. , Marcus R. Eds, Raven Press, NY. 1994, 259- 294).
The general efficacy of daily acute administration of PTH2.8 and P Hj^^j has been demonstrated in young, adult, and aged rats of both sexes (Dempster, D.W. et al., Endocrine Rev., 1993, 14, 690-709; Gunness-Hey, M. , et al., Metab. Bone Dis. & Rel. Res., 1984, 5, 177-181), as well as in other animal models, both normal and osteoporotic (Liu, C.C. et al., J. Bone Miner. Res., 1990, 5, 973-981; Podbesek, R. , et al., Endocrinology, 1983, 112, 1000-1006). The anabolic effect of intermittently administered PTH is also observed in osteoporotic men (Slovik, D.M. , et al., J. Bone Miner. Res., 1986, 1, 377-381) and women (Reeve, J. , et al., Br. Med. J., 1990, 301, 314-318) and with concurrent antiresorptive therapy (Hesch, R-D., et al. Calcif Tissue Int, 1989, 176-180) suggesting that the process is not coupled to active resorption. PTHi.34 is a synthetic amino-terminal fragment of PTH1_8 (Tregear, G.W. , et al., Endocrinology, 1973, 93, 1349-1353; Mosekilde, L. , et al., Endocrinology, 1991, 129, 421-428).
The mechanism for the anabolic effect of intermittent PTH is not entirely clear (Dempster, D.W. et al.. Endocrine Rev., 1993, 14, 690-709). The effect is dose-dependent within a defined dose range. There is increase in net calcium absorption from the intestine, and an increase in both calcium balance and calcium accretion into bone, with a corresponding increase in bone mass. This effect is also evident in the increase in trabecular bone forming surfaces and osteoblast number.
However, contrary to the demonstrated anabolic efficacy of daily subcutaneous injections of PTH, data from animal studies indicate that continuous administration (infusion) of the hormone either had no effect on bone growth or resulted in bone loss in dogs (Podbesek, R. , et al., Endrocrinology, 1983, 112, 1000- 1006; Malluche, H.H. , et al.. Am. J. Physiol., 1982, 242, F197-F202) . Continuous administration of PTH has also been shown to inhibit osteoblast activity in humans. Simon, et al. (Simon, L.S., et al., J. Bone Miner. Res., 1988, 3, 241-246) examined the effects of intravenously infused hPTH1_3 at a dose of 0.55 U/kg/hr (approximately 800 U over 24 hr) continuously for 24 hours in six osteoporotic subjects and two normal subjects. In all cases, serum levels of collagen type I, produced principally by osteoblasts, decreased within 16 hours of infusion, which also correlated with an increase in blood levels of ionized calcium. SUMMARY OF THE INVENTION The invention features a method of promoting bone formation in a human patient (e.g., a patient who suffers f om osteoporosis) . The method includes the steps of administering (e.g., transmucously, intravenously, transdermally, subcutaneously, via implantation, or via infusion) continuously to the patient PTH (mature form) , PTHrP, or an agonist thereof for a period of at least one month (as long as the life time of the patient, if necessary) at a dosage between 10 and 400 units/24 hrs. PTH and PTHrP include, but are not limited to, human PTH (hPTH) , human PTHrP (hPTHrP) , bovine PTH (bPTH) , bovine PTHrP (bPTHrP) , and rat PTH (rPTH) . An agonist of PTH or PTHrP is a peptide which is a structural analog or fragment (preferably, an N-terminal fragment containing 50 or fewer amino acids) of a naturally occurring PTH or PTHrP and, like PTH and PTHrP, also capable of binding to PTH receptor and, thereby, promoting bone formation. Examples of such an agonist include, but are not limited to, hPTH1_34 NH2, hPTH1_38 NH2, hPTH!.^ NH2, hPTH-^ge NH2, [Nle8'18, Tyr34]hPTH1_34 NH2, bPTH1_34 NH2, [Nle8'18,Tyr34]bPTH1_34, [Nle8'18,Phe22,Tyr3 ]hPT^.^ NH2, [Nle8'18,Arg19,Tyr34]hPT^.^ NH2, [Nle8'18,Arg21,Tyr3 ]bPTH1_34 NH2, or [Nle8'18,Arg19'21 , Tyr3 ]bPTH1_34 NH2. The symbol NH2 denotes amidation of the carboxyl group (-CO*OH) of the C-terminal amino acid to form -CO*NH2. The following publications disclose the sequences of PTH peptides: The Parathyroids Basic and Clinical Concepts, ed. John P. Bilezikian, 239-258 (Raven Press, NH 1994); Nissenson, R. , et al., Structure & Function of the Receptor for Parathyroid Hormone and Parathyroid Hormone-Releasing Hormone, 3 Receptor 193-202 1993; Bachem California 1993-1994 Catalog (Torrance, CA) ; and Sigma Peptides and Amino Acids 1994 Catalog (St. Louis, MO) . The following publications disclose the sequences of PTHrP peptides: Yasuda, et al., 264 J. Biol. Chem. 7720-7725 (1989); Schermer, D.T., Journal of Bone & Mineral Research 6:149-155 (1991) and Burtis, W.J., 38(11) Clinical Chemistry 2171-2183 (1992). More examples can be found in the following publications: German Application 4203040 Al (1993); PCT Application 94/01460 (1994); PCT Application 94/02510 (1994); EP Application 477885 A2(1992);
EP Application 561412 Al (1993); PCT Application 93/20203 (1993); U.S. Patent No. 4,771,124 (1988); PCT Application 92/11286 (1992) PCT Application 93/06846 (1993)
PCT Application 92/10515 (1992) U.S. Patent No. 4,656,250 (1987); EP Application 293158 A2 (1988); PCT Application 94/03201 (1994); EP Application 451,867 Al (1991);
US Patent No. 5,229,489 (1993); and PCT Application 92/00753 (1992). Note that partial PTH agonists can also be used to practice the method of this invention. Examples of partial PTH agonists include, but are not limited to, N- terminal deletion analogs (e.g., [Tyr34]bPTH3_34 NH2; see U.S. Patent No. 4,771,124 (1988).
Preferred ranges of dosages include 10-300 units/24 hrs, 10-200 units/24 hrs, 10-100 units/24 hrs, 100-400 units/24 hrs, 200-400 units/24 hrs, and 300-400 units/24 hrs.
One unit of PTH, PTHrP, or an agonist thereof is defined by utilizing an in vitro cAMP accumulation assay with human SaOS-2 cells. Human SaOS-2 cells respond upon exposure to PTH, PTHrP, or an agonist thereof with a dose-dependent stimulation of cAMP accumulation. With [Nle8'18,Tyr34]hPTH1_34 NH2 as the reference standard analog (10,000 units/mg) , a dose-response relationship can be generated using standard non-linear regression analysis. The relative potency for various PTH analogs (in units/mg) can be determined from the ratio of the EC50 of the reference standard analog to that of the PTH analog. EC50 is defined as the dose that evokes a half maximal response — cAMP accumulation herein. The detailed procedure for handling the cells, setting up the assay, as well as methods for cAMP quantitation, is described in Sistane, E. , et al., Pharmacopeial Forum 20(3), 7509-7520 (1994).
If the administration is to be achieved via infusion, an ambulatory (e.g., MINIMED™ 404-SP, MiniMed Technologies, Sylmar, CA; Pharmacia Deltec CADD-MICRO MODEL™ S900, Pharmacia Deltec Inc., St. Paul, MN; or Disetronic Medical System's PANOMAT™, Plymouth, MN) or an implantable pump (e.g., MEDTRONIC SYNCROMED™, Medtronic, Inc., Minneapolis, MN) can be used.
Preferably, PTH, PTHrP, or an agonist thereof is administered as a sustained release formulation. As an example, the formulation may contain a homo- or a co- polymer prepared from lactic acid (D-isomer, L-isomer, or a racemate) , glycolide, glycolic acid, or caprolactone. Examples of suitable sustained release formulations can be found in the following publications: U.S. Patent No. 3,773,919 (1973) U.S. Patent No. 5,187,150 (1993) U.S. Patent No. 4,767,628 (1988)
U.S. Patent No. 4,675,189 (1987) U.S. Patent No. 5,271,945 (1993) U.S. Patent No. 4,917,893 (1990); and U.S. Patent No. 3,887,699 (1975). The sustained release formulations can be administered parenterally (e.g. subcutaneously, or intravenously) or by inhalation (e.g. using an aerosol delivery system; e.g., see WO93/00951 and WO94/07514) . In other embodiments, PTH, PTHrP, or an agonist thereof can be administered transmucously (e.g. nasal, vaginal, rectal) or transdermally (e.g. iontophoretic patch) .
Also, if desired, a bone resorption inhibiting agent can also be administered during performance of the above-described method. Examples of a suitable bone resorption inhibiting agent include, but are not limited to, estrogen, calcitonin, bisphosphonate, tamoxifen, vitamin D, and calcium. Also, see U.S. Patent No. 5,118,667 (1992).
The preferred dose and duration for practicing the above-described method 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. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient(s) into association with the carrier which constitutes one or more accessory ingredients. In general, the formulations for tablets or powders are prepared by uniformly and intimately blending the active ingredient (e.g., hPTH) with finely divided solid carriers, and then, if necessary as in the case of tablets, forming the product into the desired shape and size.
The invention also features novel PTH or PTHrP agonists of the following formula: \
A1-Val-Ser-Glu-Ile-Gln-A7-Nle-His-Asn-Leu-A12- R2
Lys-His-Leu-A16-Ser-Nle-A19-Asn-A21-A22-A23-Leu-Arg- Lys- Lys-Leu-Gln-Asp-Val-His-Asn-A34-W
wherein:
A2 is Ser or Ala; A7 is Leu or Phe;
A12 is Gly, Aib, Ala, or D-Ala; A16 is Asn, Ser or Ala;
A19 is Glu, Arg, Lys, Asp, Ser, Thr, Gin, Asn, or Ala; A21 is Val, Met, Arg, Lys, Glu, Asp, Ser, Thr, Gin, Asn, Leu, lie, Nle, Ala, Phe, or p-X-Phe where X is OH, CH3, N02, or a halogen;
A22 is Glu, Asp, Phe, p-X-Phe where X is OH, CH3, N02 or a halogen, Ser, Thr, Gin, Asn, Leu, lie, Nle, Val, Ala, or Met;
A23 is Trp, 1-Nal, or 2-Nal;
A34 is Phe, or p-X-Phe where X is OH, CH3, N02, or a halogen;
W is OH, Cλ_12 alkoxy, c 7-20 phenylalkoxy, C11_20 napthylalkoxy, or NR3R4; provided that when A12 is Gly,
Aιg is Glu, A21 is Val, and A22 is Gin, then A23 must be 1- Nal or 2-Nal; and each of R2, R2, R3, and R4 is, independently, H, cι-i2 alkyl, C7_10 phenylalkyl, or CO* E where E is Cx_12 alkyl, C2_12 alkenyl, phenyl, naphthyl, or C7_20 phenylalkyl; or a pharmaceutically acceptable salt thereof.
In the above formula, the N-terminus is at the left and the C-terminus at the right in accordance with the conventional representation of a polypeptide chain. Also, The symbol Alf Val, A7, or the like in a peptide sequence stands for an amino acid residue, i.e., =N- CH(R)-CO- when it is at the N-terminus or -NH-CH(R)-C0- when it is not at the N-terminus, where R denotes the side chain of that amino acid residue. Thus, R is - CH(CH3)2 for Val. Also, when the amino acid residue is optically active, it is the L-form configuration that is intended unless D-form is expressly designated. Further, the symbols Aib, 1-Nal, and 2-Nal herein are abbreviations for α-aminoisobutyric acid, 3-(l-naphthyl) alanine, and 3-(2-naphthyl)alanine, respectively.
Preferred groups of compounds covered by the above formula include (i) those where h- is Ala; A7 is Phe; and A16 is Ser; (ii) those where A19 is Glu or Arg; and A21 is Val or Arg; and (iii) those where A22 is p-X-Phe where X is OH, CH3, N02, or a halogen. Below are particularly pre-ferred compounds: [Nle8'18,Arg19,Tyr34]bPTH1_34 NH2, [Nle8'18, Arg21,Tyr34]bPTH1_34 NH2, [Nle8'18,Arg19'21,Tyr34]bPTH1_34 NH2, and [Nle8'18,Phe22,Tyr34]bPTH1_34 NH2. Such compounds, as PTH or PTHrP agonists, can be used to promote bone formation in a manner described above.
Other features and advantages of the present invention will be apparent from the following description of the preferred embodiments, and also from the appending claims.
DESCRIPTION OF PREFERRED EMBODIMENTS The synthesis, selection and use of PTH, PTHrP or agonists thereof which are capable of promoting bone formation are within the ability of a person of ordinary skill in the art. For example, well-known in vitro or in vivo assays can be used to determine the efficacy of various PTH/PTHrP agonists to promote bone formation in human patients. For in vitro binding assays, osteoblast-like cells which are permanent cell lines with osteoblastic characteristics and possess receptors for PTH/PTHrP of either rat or human origin can be used. Suitable osteoblast-like cells include ROS 17/2 (Jouishomme, H. , et al., Endocrinology, 1992, 130, 53-60), UMR 106 (Fujimori, A., et al.. Endocrinology, 1992, 130, 29-60) and the human derived SaOS-2 (Fukuyama, S., et al., Endocrinology, 1992, 131, 1757-1769). The cell lines are available from American Type Culture Collection, Rockville, Maryland, and can be maintained in standard specified growth media. Additionally, transfected human embryonic kidney cells (HEK 293) expressing the human PTC receptor can also be utilized for in vitro binding assays. See Pines, et al., Endocrinology, 1994, 135, 1713-1716. For in vitro functional assays, PTH and PTH-like agonist activities of peptide fragments or derivatives of parathyroid hormone or PTHrP can be tested by contacting a concentration range of the test compound with the cells in culture and assessing the stimulation of the PTH/PTHrP receptors. Receptor stimulation is evidenced by the activation of second messenger molecules coupled to the receptors, for example, a stimulation of cyclic AMP accumulation in the cell or an increase in enzymatic activity of protein kinase C, both of which are readily monitored by conventional assays (Jouishomme, H. , et al., Endocrinology, 1992, 130, 53-60; Abou-Samra, A.B. , et al.. Endocrinology, 1989, 125, 2594-2599; Fujimori, A., et al., Endocrinology, 1991, 128, 3032-3039; Fukayama, S., Et al., Endocrinology, 1994, 134, 1851-1858; Abou- Samra, A.B., et al.. Endocrinology, 1991, 129, 2547-2554; and Pines, et al., Endocrinology, 1994, 135, 1713-1716). Other parameters of PTH action include increase in cytosolic calcium and phosphoinositols, and biosynthesis of collagen, osteocalcin, and alteration in alkaline phosphatase activity.
PTH-like agonist activities of subfragments of PTH have been successfully analyzed by contacting peptides with rat kidney cells in culture and assessing cyclic AMP accumulation (Blind, E. , et al., Clin. Endocrinol., 1993, 101, 150-155) and the stimulation of 1,25- dehydroxyvitamin D3 production (Janulis, M. , et al., Endocrinology, 1993, 133, 713-719).
The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Furthermore, all of the references cited in this disclosure are incorporated by reference.
Example 1
The PTH or PTHrP agonists of the invention were synthesized on an APPLIED BIOSYSTEMS™ 430A Automated Peptide Synthesizer (Applied Biosystems Inc. , Foster City, CA) using version 1.40 of the software for NMP/HOBt Boc-based chemistry. The following side-chain protected amino acid derivatives were used in the course of the synthesis: N-Boc-ArgJ^-TosylJ-OH, N-Boc-Asp(cycHx)-OH, N-Boc-Glu(OBzl)-OH, N-Boc-His(Bom)-OH, N-Boc-Lys(Cl-Z)- OH, N-Boc-Ser(Bzl)-OH, N-Boc-Thr(Bzl)-OH, N-Boc-Tyr(Br- Z)-0H, and N-Boc-Trp(N'-For)-OH.
Cleavage from resin with concomitant removal of the side-chain protecting groups of pMBHA-R-bound peptide was performed by liquid HF in the presence of 10% anisole (20 mL/g resin-bound peptide) 1 hr at 0°C. Low-high HF procedures shall be used for clevage from resin and deprotection for PTH agonists containing Trp. See Tam, et al., J. Am. Chem. Soc, 1983, 105, 6442. The resin- crude peptide mixture was washed with petroleum ether and ether. The dry resin-crude peptide mixture was extracted consecutively with 50% acetic acid and water. The combined washes were lyophilized. The lyophilized crude peptides were subjected to preparative RP-HPLC purification.
The full names for the abbreviations used above are as follows: Boc for t-butoxycarbonyl, For for formayl, cycHx for cyclohexyl, Cl-Z for 2- chlorobenzyloxycarbonyl, OBzl is O-benzyl, BOM for benzyloxymethyl, Bzl for benzyl, Br-Z for 2-bromo- benzyloxycarbonyl, I^-Tosyl for tosyl at guanidyl site, and pMBHA-R for paramethoxybenzhydrylamine resin. The crude peptides were purified on a WATERS DELTA PREP™ 4000 (Waters, Milford, MA), preparative HPLC system, connected to a PrepPack cartridge of Vydac™ C18 300A, 15-20 μ [47 x 300 mm] (Waters, Milford, MA) at a flow rate of 70 mL/min monitored at 220 nm. The analytical HPLC system included the following components: Waters 600E multisolvent delivery system, 490E programmable multiwavelength detector, 717 autosampler and a 747 data module. The samples were analyzed on a VYDAC™ C18 218TP5415 (150 x 4.6 mm, 5 μm) , The Separation Group (Hesperia, CA) at a flow rate of 1 mL/min monitored at 220 nm. The solvent mixtures for both the analytical and preparative HPLC were: A: 0.1% TFA in H20, and B: 0.1% TFA in acetonitrile. The purity of the peptides and their derivatives exceeded 99% as determined from the analytical RP-HPLC.
The following four bovine PTH agonists were synthesized:
[Nle8'18,Phe22,Tyr3 ]bPTH1_34 NH2 (Analog I); [Nle8'18,Arg19,Tyr34]bPTH1_34 NH2 (Analog II); [Nle8'18,Arg21,Tyr34]bPTH1_34 NH2 (Analog III) ; and [Nle8'18,Arg19'21,Tyr34]bPTH1_34 ^ (Analog IVj .
These four analogs were analyzed using HPLC (gradient of 30 min) and the results are shown in Table I below. For each solvent gradient, only the intitial and final percentages of B of the solvent (which consists of both A and B — see above for their compositions) are indicated.
TABLE I
ANALOG RETENTION TIME SOLVENT GRADIENT (min.) (%)
I 20.6 25 - 45
II 14.2 25 - 50
III 13.6 25 - 50
IV 17.1 20 - 50
The results of the amino acid analysis of the four analogs are shown in Table II below, which lists both calculated and found values.
TABLE II
Amino Acid Analog I Analog 11 Analog HI Analog IV j
Calcd. 3.00 3.00 3.00 3.00
Asx Found 3.23 3.09 3.16 3-22
Calcd. 3.00 3.00 3.00 3.00
Ser Found 2.75 234 2.48 2.86
Calcd. 4.00 4.00 5.00 4.00
Glx Found 4.27 4.27 5.35 4.34
Calcd. 1.00 1.00 1.00 1.00
Gly Found 1.03 1.08 1.01 1.07
Calcd. 1.00 1.00 1.00 1.00
Ala Found 1.04 1.03 0.97 0.95
Calcd. 3.00 3.00 2.00 2.00
Val Found 2.80 3.03 1.97 1.68
Calcd. 1.00 1.00 1.00 1.00 lie Found 0.99 .096 0.91 0.81
Calcd. 4.00 4.00 4.00 4.00
Leu Found 4.24 4.32 4.11 4.25
Calcd. 2.00 2.00 2.00 2.00
Nle Found 1.61 1-50 1.94 2.19
Calcd. 1.00 1.00 1.00 1.00
Tyr Found 1.15 1.06 1.01 1.10
Calcd. 2.00 1.00 1.00 1.00
Phe Found 2.06 0.97 1.00 0.93
Calcd. 3.00 3.00 3.00 3.00
His Found 2.85 3.03 2.93 2.64
Calcd. 3.00 3.00 3.00 3.00
Lys Found 3.00 2.93 3.05 2.90
Calcd. 2.00 3.00 3.00 4.00
Arg Found 1.97 3.20 3.11 4.04 Table III demonstrates the FAB (fast-atom bombardment) mass spectrometer analysis of the analogs.
TABLE III
ANALOG MEASURED PREDICTED
I 4106.8 4105.8
II 4114.7 4114.8
III 4146.4 4144.8
IV 4173.5 4172.8
Example 2 SaOS-2 BIO cells were maintained in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37°C in a humidified atmosphere of 5% C02 in air. The medium was changed every three or four days, and the cells were subcultured every week by trypsinization.
SaOs-2 BIO cells were maintained for four days after they had reached confluence. The medium was replaced with 5% FBS RPS/1640 medium and incubated for 2 hrs at room temperature with 10 x 104cpm mono-125"I- [Nle8'18,Tyr34(3-I125) ]bPTH1_34 NH2 in the presence or absence of a competing tested PTH agonist. 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. Synthesis of the radiolabelled [Nle8'18. Tyr34 (3-I125) ]bPTH1_34 NH, was carried out as described in Goldman ME et al., Endocrinology, 1988, 123, 1468-1475.
The binding assay was conducted on Analogs I-IV and Analog V (i.e., [Nle8, Nle18, Tyr3 ]bPTH1_34 NH2) . The IC 5ς0n '' s (half maximal inhibition of binding of mono-125I- '[Nle8'18,Tyr34(3-I125)]bPTH1_34 NH2) for the five tested analogs were calculated and shown in Table IV below:
TABLE IV
ANALOG IC50 (nM)
I 1. 5
II 0. 6
III 0. 3
IV 0. 3
V 1 . 5
Example 3
The adenylate cyclase activity induced by each of Analogs I-V was also measured in SaOS-2 BIO cells as described previously (Rodan et al. 1983 J. Clin. Invest. 72;1511; Goldman et al. 1988 Endocrinology 123, 1468). Confluent SaOS-2 BIO cells in 24 wells plates were incubated 0.5 μ.Ci[3H]adenine (26.9 Ci/mmol, New England Nuclear, Boston, MA) in fresh medium at 37°C for 2 hrs, and washed twice with Hank's solution. The cells were treated with 1 mM IBMX [isobutylmethylxanthine, Sigma, St. Louis, MO] in fresh medium for 15 min, and a tested PTH analog was added to the medium to incubate for 5 min. The reaction was stopped by the addition of 1.2 M TCA followed by sample neutralization with 4 N KOH. Cyclic AMP was isolated by the two-column chromatographic method (see Salomon et al. 1974 Anal. Biochem. 58;541). The radioactivity was counted in a scintillation counter (Liquid scintillation counter 2200CA, PACKARD, Downers Grove, IL) . The EC50 ,s (half maximal stimulation of adenylate cyclase) were calculated for the five PTH analogs and are shown below: TABLE V
ANALOG EC50 (nM)
I 1. 5
II 2 . 0
III 0. 2
IV 0 . 5
V 2 . 0
Example 4
In vivo bone anabolic activities of PTH/PTHrP agonists are tested by administering the peptide or a formulation containing the peptide into intact animals or an experimental animal model of osteopenia. The animal model can be osteoporosis in rats induced by ovariectomy (Hori, M. , et al., Bone Miner., 1988, 3, 193-199; Geral, et al., J. Bone Miner. Res., 1989, 4, Suppl. 1, S303; Liu C-C. & Kalu, D.N., J. Bone Miner. Res., 1990, 5, 973-982; Mosekilde, L. , et al., Endocrinol., 1991, 129, 421-428; Wronski, T.J. , Yen C-F. Bone, 1994, 15, 51-58; Reviewed in Demster D.W. , et al.. Endocrine Rev., 1993, 14, 690- 709) .
The bone anabolic effects of the compound are determined following 12 to 60 days of treatment by assessing the change in bone mineral density by dual energy x-ray absorptiometry or dry weight of femurs or total ash weight (Hori, H. , et al., Bone Miner., 1988, 3, 193-199; Hefti, E. , et al., Clin. Sci., 1982, 62, 389- 396) . Increase in the rate of bone formation and mineralization are assessed using metabolic labels, e.g. tetracycline (Tam, C.S., et al., Endocrinology, 1982, 110, 506-512). Qualitative and quantitative evaluations of changes in trabecular/cortical bone volume and complexity are determined by standard histomorphometric methods (Wronski, T.J. , Yet C-F, Bone, 1994, 15, 51-58; Tam C.S., et al., Endocrinology, 1982, 110, 506-612; Podbesek, R. , et al., Endocrinology 1983, 112, 1000-1006) of bone samples from control (untreated) and treated animals.
Example 5
The anabolic efficacy of the PTH/PTHrP agonists are tested in humans (Review in Dempster D.W. , et al., Endocrine Rev., 1993, 14, 690-709). To determine if continuous administration of hPTHj.^ or its agonist is effective in promoting bone growth in humans, 30 post- menopausal women with established osteoporosis based on bone-density measurements are selected. A double-blind, placebo-controlled, randomized experiment is conducted where two groups of 15 women each are either placed on placebo treatment or continuous infusion of a defined dose of hPTH1_34 (25 units to 400 units/24 hrs) using an infusion pump. The patients prior to the study are subject to the following: complete health and physical examination, evaluation of the nutritional status (particularly of calcium intake and serum calcium) , full analysis of biomarkers for bone-turnover (Riis, B.J. , Amer. J. Med. 1993, 95 [Suppl 5A] , 17s-21s; Delmas, P.D. , Amer. J.
Med., 1951, 91 [Suppl 5B] , 59s-63s) , radiology, bone mass measurements of vertebral and axial sites (Gerant, H.K., et al., Amer. J. Med., 1991, 91 [Suppl 5B] , 49s-53s; Wasnich, R.D., Amer J Med, 1991, 91 [Suppl 5B] , 54s-58s) and bone biopsy, to establish the baseline parameters for each individual. After one and three months of treatment, the patients are reevaluated for changes in serum calcium and biomarkers of bone turnover to determine the outcome of continuous hPTH1_34 administration. When the bio arker analysis suggests an increase in osteoblast activity (e.g., increase in serum alkaline phosphatase and serum osteocalcin) , the treatments are extended to twelve months, wherein bone mass measurements and bone biopsy further provide clear indication of bone growth in the population treated with continuous infusion hPTH1_34.
OTHER EMBODIMENTS From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

Claims

1. A method of promoting bone formation in a human patient, said method comprising administering continuously to said patient PTH, PTHrP, or an agonist thereof for a period of at least one month at a dosage between 10 and 400 units/24 hrs.
2. A method of claim 1, wherein said dosage is 10-300 units/24 hrs.
3. A method of claim 1, wherein said dosage is 10-200 units/24 hrs.
4. A method of claim 1, wherein said dosage is 10-100 units/24 hrs.
5. A method of claim 1, wherein said dosage is 100-400 units/24 hrs.
6. A method of claim 1, wherein said dosage is 200-400 units/24 hrs.
7. A method of claim 1, wherein said dosage is 300-400 units/24 hrs.
8. A method of claim 1, wherein said agonist is hPTHi.34 NH2 or [Nle8'18, Tyr34]hPTH1_34 NH2.
9. A method of claim 1, wherein said agonist is bPTH1_34 NH2, [Nle8'18,Tyr34]bPTH1_34 NH2, [Nle8'18,Phe22, Tyr34]bPTH1.34 NH2, [Nle8'18,Arg19,Tyr34]bPTH1.34 NH2, [Nle8'18,Arg21,Tyr34]bPTH1_34 NH2, or [Nle8'18,Arg19'21 , Tyr3 ]bPTH1_34 NH2.
10. A method of claim 1, wherein infusion is performed to administer said PTH, PTHrP, or an agonist thereof.
11. A method of claim 10, wherein said infusion is achieved with an ambulatory or implantable infusion pump.
12. A method of claim 1, wherein said PTH, PTHrP, or an agonist thereof is administered as a sustained release formulation.
13. A method of claim 12, wherein said sustained release formulation comprising a polymer prepared from lactic acid, caprolactone, glycolide, or glycolic acid.
14. A method of claim 1, further comprising the step of administering to said patient a bone resorption inhibiting agent.
15. A compound having the formula:
\
A1-Val-Ser-Glu-Ile-Gln-A7-Nle-His-Asn-Leu-A12-
/
Lys-His-Leu-A16-Ser-Nle-A19-Asn-A21-A22-A23-Leu-Arg- Lys- Lys-Leu-Gln-Asp-Val-His-Asn-A34-W
wherein: Ax is Ser or Ala; A7 is Leu or Phe; A12 is Gly, Aib, Ala, or D-Ala; A16 is Asn, Ser or Ala; A19 is Glu, Arg, Lys , Asp, Ser, Thr, Gin , Asn, or Ala ;
A21 is Val , Met, Arg, Lys, Glu, Asp, Ser, Thr, Gin, Asn, Leu, lie, Nle, Ala , Phe , or p-X-Phe where X is OH, CH3, N02, or a halogen;
A22 is Glu, Asp, Phe, p-X-Phe where X is OH, CH3 , N02 or a halogen, Ser, Thr, Gin, Asn, Leu, lie, Nle, Val, Ala , or Met;
A23 is Trp, 1-Nal, or 2-Nal; A34 is Phe, or p-X-Phe where X is OH, CH3, N02, or a halogen;
W is OH, _12 alkoxy, C7_20 phenylalkoxy, C^^Q napthylalkoxy, or NR3R4; provided that when A12 is Gly, A19 is Glu, A21 is Val, and A22 is Gin, then A23 must be 1- Nal or 2-Nal; and each of Rl t R2, R3, and R4 is, independently, H, Ci-12 alkyl, c 7-ιo Phenylalkyl, or CO* E where E is Cα_12 alkyl, C2_12 alkenyl, phenyl, naphthyl, or C7_20 phenylalkyl; or a pharmaceutically acceptable salt thereof.
16. A compound of claim 15, wherein: A is Ala;
A7 is Phe; and
A16 is Ser; or a pharmaceutically acceptable salt thereof.
17. A compound of claim 15, wherein: A19 is Glu or Arg; and
A21 is Val or Arg, or a pharmaceutically acceptable salt thereof.
18. A compound of claim 15, wherein:
A22 is p-X-Phe where X is OH, CH3, N02, or a halogen; or a pharmaceutically acceptable salt thereof.
19. A compound of claim 15, wherein said compound is [Nle8'18,Arg19,Tyr34]bPTH1_34 NH2, [Nle8'18,Arg21, Tyr^lbPTHi.^ NH2, or [Nle8'18,Arg19'21,Tyr3 ]bPTH1.34 NH2; or a pharmaceutically acceptable salt thereof.
20. A compound of claim 15, wherein said compound is [Nle8'18,Phe22,Tyr34]bPTH1_34 NH2; or a pharmaceutically acceptable salt thereof.
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998014478A1 (en) * 1996-09-30 1998-04-09 Beth Israel Deaconess Medical Center Continuous low-dose administration of parathyroid hormone or its agonist
WO1998030590A2 (en) * 1997-01-07 1998-07-16 Biomeasure Incorporated Analogs of parathyroid hormone
US5969095A (en) * 1995-07-13 1999-10-19 Biomeasure, Inc. Analogs of parathyroid hormone
US6544949B1 (en) * 1995-07-13 2003-04-08 Societe De Conseils De Recherches Et D'applications Scientifiques, S.A.S. Analogs of parathyroid hormone
WO2003053167A1 (en) 2001-12-11 2003-07-03 Societe Des Produits Nestle S.A. Composition for promotion of bone growth and maintenance of bone health
US7393837B2 (en) 1998-09-28 2008-07-01 Mcgill University Inhibition of PEX in the treatment of metabolic bone diseases
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

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DZ2873A1 (en) * 1998-08-19 2003-12-15 Lilly Co Eli Method for increasing bone hardness and stiffness.

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4656250A (en) * 1983-08-05 1987-04-07 Toyo Jozo Kabushiki Kaisha [Nle8, Nle18, Tyr34 or Phe34 ]-h-PTH peptide derivatives

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4747825A (en) * 1984-06-29 1988-05-31 Ferring Laboratories, Inc. Apparatus and methodology for pulsed administration of growth promoting agents
JP2653255B2 (en) * 1990-02-13 1997-09-17 武田薬品工業株式会社 Long-term sustained release microcapsules
WO1993011786A1 (en) * 1991-12-17 1993-06-24 Procter & Gamble Pharmaceuticals, Inc. Methods for the treatment of osteoporosis using bisphosphonates and parathyroid hormone

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4656250A (en) * 1983-08-05 1987-04-07 Toyo Jozo Kabushiki Kaisha [Nle8, Nle18, Tyr34 or Phe34 ]-h-PTH peptide derivatives

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0800405A4 *

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7632811B2 (en) 1995-07-13 2009-12-15 Societe De Conseils De Recherches Et D'applications Scientifiques, Sas 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
US6544949B1 (en) * 1995-07-13 2003-04-08 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
US6921750B2 (en) 1995-07-13 2005-07-26 Societe De Conseils De Recherches Et D'applications Scientifiques, S.A.S. Analogs of parathyroid hormone
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EP1645566A3 (en) * 1997-01-07 2006-10-11 Societe De Conseils De Recherches Et D'applications Scientifiques S.A.S. Analogs of parathyroid hormone
EP1645566A2 (en) * 1997-01-07 2006-04-12 Societe De Conseils De Recherches Et D'applications Scientifiques S.A.S. Analogs of parathyroid hormone
CZ301497B6 (en) * 1997-01-07 2010-03-24 Societe De Conseils De Recherches Et D´Applications Scientifiques, S.A.S. Peptide variant of fragment of parathyroid hormone and parathyroid hormone-related protein peptides
WO1998030590A3 (en) * 1997-01-07 1998-12-10 Biomeasure Inc Analogs of parathyroid hormone
WO1998030590A2 (en) * 1997-01-07 1998-07-16 Biomeasure Incorporated Analogs of parathyroid hormone
US7393837B2 (en) 1998-09-28 2008-07-01 Mcgill University Inhibition of PEX in the treatment of metabolic bone diseases
EP2135517A1 (en) 2001-12-11 2009-12-23 Société des Produits Nestlé S.A. Composition for promotion of bone growth and maintenance of bone health
WO2003053167A1 (en) 2001-12-11 2003-07-03 Societe Des Produits Nestle S.A. Composition for promotion of bone growth and maintenance of bone health
EP2263480A1 (en) 2001-12-11 2010-12-22 Société des Produits Nestlé S.A. Composition for promotion of bone growth and maintenance of bone health, comprising Mentha extracts
EP2272382A1 (en) 2001-12-11 2011-01-12 Société des Produits Nestlé S.A. Composition for promotion of bone growth and maintenance of bone health, comprising soybean or soybean extracts
EP2286675A1 (en) 2001-12-11 2011-02-23 Société des Produits Nestlé S.A. Composition for promotion of bone growth and maintenance of bone health
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

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AU4472896A (en) 1996-07-10
EP0800405A4 (en) 1998-11-11
JPH10511095A (en) 1998-10-27
EP0800405A1 (en) 1997-10-15
CA2205959A1 (en) 1996-06-27

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