PL191239B1 - Analog of parathyroid hormone peptide - Google Patents

Abstract

Peptides, parathyroid hormone analogs of the formulas: [Cha22,23, Glu25, Lys26,30, Leu28, Aib29] hPTHrP (1-34) NH2; [Cha22.23, Glu25, Lys26.30, Aib29] hPTHrP (1-34) NH2; [Glu22.25, Leu23.28.31, Lys26, Aib29, Nle30] hPTHrP (1-34) NH2; [Glu22.25, Leu23.28.30.31, Lys26, Aib29] hPTHrP (1-34) NH2; [Glu22,25,29, Leu23, 28, 30, 31, Lys26] hPTHrP (1-34) NH2; [Glu22.25.29, Leu23.28.31, Lys26, Nle30] hPTHrP (1-34) NH2; [Ser1, Ile5, Met8, Asn10, Leu11.23.28.31, His14, Cha15, Glu22.25, Lys26.30, Aib29] hPTHrP (1-34) NH2; [Glu22.25, Cha23, Lys26, Leu28.31, Aib29, Nle30] hPTHrP (1-34) NH2; [Cha22.23, Glu25, Lys26.30, Leu28.31 'Aib29] hPTHrP (1- 34) NH2; [Cha22, Leu23,28,31, Glu25,29, Lys26, Nle30] hPTHrP (1-34) NH2; [Cha7,11,15] hPTHrP (1-34) NH2; [Cha7,8,15] hPTHrP (1-34) NH2; [Glu22, Cha23, Aib25.29, Lys26.30, Leu28.31] hPTHrP (1-34) NH2; [Glu22, Cha23, Aib25.29, Lys26, Leu28] hPTHrP (1-34) NH2; [Glu22, Leu23.28, Aib25.29, Lys26] hPTHrP (1-34) NH2; [Aib29] hPTHrP (1-34) NH2; [Glu22.25, Cha23, Lys26, Leu28.31, Aib29, Nle30] hPTHrP (1-34) NH2; [Glu22.25, Cha23, Lys26.30, Aib29, Leu31] hPTHrP (1-34) NH2; [Glu22.25, Leu23.28.31, Lys26, Aib29.30] hPTHrP (1-34) NH2; [Glu22.25, Leu23.28.31, Lys26, Aib29] hPTHrP (1-34) NH2; [Glu22.25, Leu23.28.31, Aib26.29, Lys30] hPTHrP- (1-34) NH2; [Glu22.25, Cha23, Lys26.30, Leu28.31, Aib29] hPTHrP (1-34) NH2; [Glu22.25, Cha23, Lys26.30, Aib29] hPTHrP (1-34) NH2; [Glu22.25, Cha23, Lys26.30, Leu28, Aib29] hPTHrP (1-34) NH2; [Leu27, Aib29] hPTH (1-34) NH2; or a pharmaceutically acceptable salt thereof.

Description

Description of the invention

The invention relates to peptides, parathyroid hormone analogs.

Parathyroid hormone ("PTH") is a polypeptide produced by the parathyroid glands. The mature, circulating form of the hormone comprises 84 amino acid residues. The biological activity of PTH can be recreated using its N-terminal peptide fragment (e.g., amino acid residues 1 to 34). The parathyroid hormone-related protein (parathyroid hormone) is a protein between 139 and 173 amino acids in length with the N-terminus homologous to PTH. PTHrP exhibits many of the same biological effects as PTH, including binding to a receptor common to PTH / PTHrP. Tregear, et al., Endocrinol., 93: 1349 (1983). A variety of PTH peptides derived from various organisms, e.g., human, cattle, rat, chickens, have been characterized. Nissenson, et al., Receptor, 3: 193 (1993).

PTH has been shown to improve both bone mass and quality. Dempster, et al., Endocrine Rev., 14: 690 (1993); and Riggs, Amer. J. Med., 91 (Suppl. 5B): 37S (1991). An anabolic effect in men and women suffering from osteoporosis was observed with intermittent therapy with and without concomitant 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 relates to peptides having the following formulas: [Cha ^22,23 , Glu

Aib ²⁹ ] hPTHrP (1-34) NH2; [Cha ^22.23 , Glu ²⁵ , Lys ^26.30 , Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu ^22.25

Aib ²⁹ , Nle ³⁰ ] hPTHrP (1-34) NH2; [Glu ^22.25 , Leu ^23.28,30.31 , Lys ²⁶ , Aib ²⁹ ] hPTHrP (1-34) NH2 22.25.29 23.28.31

Leu

Underworld

Leu [Cha

23,28,30,31

Asn ¹

Lys

Leu [Glu ² ] hPTHrP (1-34) NH2; [Glu ² His ¹⁴ , Cha ¹⁵ , Glu ² , Lys ^26.30 , Leu ²⁸ _Leu ^23.28.31 , Lys ²⁶ 22.25.29

Aib ²

Leu

Nle 23,28,31] hPTHrP (1-34) NH2

Glu

25.29

Lys [Cha ^7,8,15 ] hPTHrP (1-34) NH2; [Glu ²² , Cha Aib ^25.29 , Lys ²⁶ , [Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu

Leu ²⁸ ] hPTHrP (1-34) NH [Cha

Nle

Aib

Lys

22.23

Leu

26.30

Lys ²⁶ , Nle ³⁰ ] hPTHrP (1-34) NH2

22.25 [Glu [Ser

How many ⁵

Aib ²⁹ ] hPTHrP (1-34) NH ₂ ; [Glu ^22.25 , Cha ²³ , Lys

26.30

28.31

Glu ²⁵ , Lys ^26.30 , Leu ^28.31 'Aib ²⁹ ] hPTHrP (1-34) NH2] hPTHrP (1-34) NH2 Lys ^26.30 , Leu ^28.31 ] hPTHrP (1-34) NH2

23.28

25.29

Cha ² [Glu ²

Lys ² Leu ²

34) NH2; [Glu [Cha ^7,11,15 ] hPTHrP (1-34) NH ₂ [Glu ²² , Cha ²³

Lys ²⁶ ] hPTHrP (1-34) NH ₂ ; [Glu ²² , Leu ^23.28 , Aib ^22.25 Cha ²³ , Lys ²⁶ , Leu ^28.31 , Aib ²⁹ , ^22.25 , Leu ^23.28.31 , Lys ²⁶ ,

Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu ^22.25 , Leu ^23.28.31 , Aib ^26.29 , Lys ^26.30 , Leu ^28.31 , Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu ^22.25 ,

Aib ²⁹ , Leu ³¹ ] hPTHrP (1-34) NH2; [Glu ²

22.25

Lys

Cha

Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu

22.25

Cha ²³ , Lys

26.30

Leu

Nle ³⁰ ] hPTHrP (1-34) NH ₂ ; [Glu ^22.25

Aib ^29.30 ] hPTHrP (1-34) NH ₂ 30

Lys ³⁰ ] hPTHrP (1 Cha ²³ , Lys ^26.30

Aib ²⁹ ] hPTHrP (1-34) NH2; or [Leu ²⁷

Aib ²⁹ ] hPTH (1-34) NH2; or a pharmaceutically acceptable salt thereof.

With the exception of the N-terminal amino acids, all amino acid abbreviations (e.g., Ala) in the present disclosure have the structure -NH-CH (R) -CO-, where R is an amino acid side chain (e.g., CH3 for Ala). For N-terminal amino acids, the abbreviations indicate the structure = N-CH (R) -CO-, where R is the amino acid side chain. β-Nal, Nle, Dap, Cha, Nva, Amp, Pal, Ahc, and Aib are abbreviations of the following α-amino acids, respectively: e- (2-naphthyl) alanine, norleucine, α, β-diaminopropionic acid, cyclohexylalanine, norvaline , 4-amino-phenylalanine, e- (3-pyridinyl) alanine, 1-amino-1-cyclohexane carboxylic acid, and α-aminoisobutyric acid. Acc is an amino acid selected from the group consisting of 1-amino-1-cyclopropane carboxylic acid;

1-amino-1-cyclobutane carboxylic acid; 1-amino-1-cyclopentanecarboxylic acid; 1-amino-1-cyclohexane carboxylic acid; 1-amino-1-cycloheptanecarboxylic acid; 1-amino-1-cyclooctanecarboxylic acid; and 1-amino-1-cyclononanecarboxylic acid.

In the above formula, the hydroxyalkyl, hydroxyphenylalkyl, and hydroxynaphthylalkyl may include 1-4 hydroxy substituents. Also, COE ₁ means -C = O ^ E ₁ . examples of -C = O 4 E ₁ include, but are not limited to, acetyl and phenylpropionyl.

The peptides according to the invention have the format, e.g. [Ahc ^7.11 ] hPT (1-34) NH2, with the amino acids in parentheses substituted for the amino acids of the natural sequence (e.g. Ahc ⁷ for Leu ⁷ , and Ahc ¹¹ for Leu ¹¹ in hPTH). The abbreviation hPTH stands for human PTH, hPTHrP stands for human PTHrP, rPTH stands for rat PTH, and bPTH stands for bovine PTH. The numbers in parentheses refer to the number of amino acids present in the peptide (e.g., hPTH (1-34) represents amino acids 1 to 34 of the human PTH peptide sequence). The sequence for hPTH (1-34), hPTHrP (1-34), bPTH (1-34), and rPTH (1-34) is provided in Nissenson, et al., Receptor, 3: 193 (1993). The designation "NH2" in PT (1-34) NH2 means that the C-terminus of the peptide is amidated. On the other hand, PTH (1-34) has a free end C.

PL 191 239 B1

The peptide of the invention is capable of promoting bone growth in a subject (i.e. a mammal such as a human patient). Thus, in the treatment of osteoporosis and bone fractures, it is useful when administered alone or concurrently with anti-resorptive treatment, e.g., bisphosphonates and calcitonin.

The peptides of the invention may be provided in the form of pharmaceutically acceptable salts. Examples of such salts include, but are not limited to, those prepared with organic acids (e.g., acetic, lactic, malic, citric, maleic, ascorbic, succinic, benzoic, methanesulfonic, toluenesulfonic, or pamic acids), inorganic acids (e.g., hydrochloric, sulfuric, or phosphoric). ), and polymeric acids (e.g., tannic, carboxymethylcellulose, polylactic, polyglycolic or polylactic glycolic acid copolymers).

A therapeutically effective amount of a peptide of the invention and a pharmaceutically acceptable carrier (e.g., magnesium carbonate, lactose or a phospholipid with which the active compound may take the form of a micelle) together form a therapeutic composition (e.g., pill, tablet, capsule or liquid) for administration (e.g., orally, intravenously, transdermally, pulmonary, vaginally, subcutaneously, intranasally, iontophoretically or intraventricularly). The pills, tablets or capsules for oral administration may be coated with substances to protect the active composition from stomach acid or intestinal enzymes in the stomach for a period of time sufficient to permit undigested passage to the small intestine. The therapeutic composition can also be in the form of a biodegradable or non-biodegradable extended release formulation for subcutaneous or intramuscular administration. See U.S. Patent Nos. 3,773,919 and 4,767,628 and PCT Application No. WO 94/15587. Continuous delivery can also be achieved using implantable or external pumps (eg, an INFUSAID ™ pump). Administration can also be performed intermittently, e.g. by single daily injections, or continuously at low doses, e.g. in a sustained release formulation.

The dose of the peptide for the treatment of the above diseases and disorders will vary with the mode of administration, the age and weight of the subject, and the condition of the subject being treated, and will always be determined by the attending physician or veterinarian.

The peptide according to the invention also finds use in the treatment of diseases or disorders related to insufficient bone growth etc., e.g. osteoporosis or fractures.

Other features and advantages of the invention will become apparent from the following detailed description and claims.

Detailed description of the invention

Based on the present description, the invention can be fully utilized. The following detailed examples are intended to illustrate only and not to limit the remainder of the description in any way. Further, all the publications mentioned are incorporated by reference.

Building

PTH (1-34) and PTHrP (1-34) have been reported to include two amphophilic alpha-helical domains. See, e.g., Barden et al., Biochem. 32: 7126 (1992). The first α helix is formed by amino acid residues 4 to 13, while the second α helix is formed by amino acids 21 to 29. Some peptides of the invention are substituted with a different amino acid at one or more residues inside or near these two PTH regions (1 -34) and PTHrP (1-34) (in the first α helix or in the second α helix).

Synthesis

The peptides of the invention can be made by standard solid phase synthesis. See, e.g., Stewart JM et al., Solid Phase Synthesis (Pierce Chemical Co., 2nd Ed. 1984). The peptides of the invention were also synthesized and purified according to procedures well known in the art in a manner analogous to that shown below for the peptide of formula [Glu ^22.25 , Leu ^23.28 , Lys ^26.30 , Aib ²⁹ , Ahc ³¹ ] hPTH (1-34) NH2. Other peptides according to the invention can be produced by a person skilled in the art analogously.

1- [N-tert-butoxycarbonylamino] -1-cyclohexanecarboxylic acid (Boc-Ahc-OH) was synthesized as follows:

19.1 g (0.133 mol) of 1-amino-1-cyclohexanecarboxylic acid (Acros Organics, Fisher Scientific, Pittsburgh, PA) was dissolved in 200 ml of dioxin and 100 ml of water. Thereto was added 67 mg of 2 N NaOH. The solution was cooled in an ice-water bath. To this solution was added 32.0 g (0.147 mol) of di-tert-butyl dicarbonate. The reaction mixture was stirred overnight at room temperature. Then the dioxin was removed under reduced pressure. 200 ml was added to the remaining solution

Of ethyl acetate. The mixture was cooled in an ice-water bath. 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 x 100 ml). The two organic layers were combined and washed with water (2 x 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 pure product was obtained with a yield of 29%. Other protected Acc amino acids can be produced by a person skilled in the art in an analogous manner.

The peptide was synthesized on an Applied Biosystems (Foster City, CA) model 430A peptide synthesizer, modified for solid phase synthesis by the Boc accelerated method. See, Schnoize et al., Int. J. Peptide Protein Res., 90: 180 (1992). 4-Methylbenzylhydrylamine (MBHA) resin (Peninsula, Belmont, CA) was used with a 0.93 mmol / g substitution. The amino acids used were Boc (Bachem, CA, Torrance, CA; Nova Biochem., LaJolla, CA) with the following side chains protected with groups: Boc-Ala-OH, Boc-Arg (Tos) -OH, Boc-Asp (OcHex) -OH , Boc-Glu (OcHex) -OH, BocHis (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. The synthesis was performed on a 0.14 mmol scale. Boc groups were removed by treatment with 100% TFA 2 x 1 min. The Boc amino acids (2.5 mmol) were pre-activated with HBTU (2.0 mmol) and DIEA (1.0 ml) in 4 ml DMF and coupled without prior neutralization of the peptide-resin TFA salt. The coupling times were 5 minutes, with the exception of Boc-Aib-OH and its Boc-Leu-OH residues, and Boc-Ahc-OH and its Boc-Lys (2Clz) -OH residues, when the coupling time for these residues was 2 hours .

At the end of peptide chain linkage, the resin was treated with a solution of 20% mercaptoethanol / 10% DIEA in DMF 2 x 30 minutes to remove DNP groups on the His side of the chain. The N-terminal Boc group was removed by treatment with 100% TFA for 2x2 minutes. The partially deprotected peptide-resin complex was washed with DMF and DCM then dried under vacuum. Final cleavage was performed by mixing the peptide-resin in 10 ml of HF containing 1 ml of anisole and dithiothreitol (24 mg) at 0 ° C for 75 minutes. HF was removed under a stream of nitrogen. The residue was washed with ether (6 x 10 ml) and extracted with 4 N HOAc (6 x 10 ml).

The peptide mixture in the aqueous extract was purified by reverse phase preparative high pressure liquid chromatography (HPLC) using a Vydac ™ C18 reverse phase column (Nest Group, Southborough, MA). The column was eluted with a linear gradient (10% to 45% solution B over 130 minutes) at a flow rate of 10 ml / min (solution A = 0.1% aqueous TFA; solution B = acetonitrile containing 0.1% TFA). Fractions were collected and checked by analytical HPLC. Pure product containing fractions were combined and lyophilized to dryness. 85 mg of a white solid were obtained. Based on analytical HPLC analysis, the purity was> 99%. Mass spectrometry analysis gave a molecular weight of 3,972.4 (consistent with the calculated molecular weight of 3,972.7).

The full names of the abbreviations used herein are as follows: Boc, t-butylcarbonyl; HF, hydrogen fluoride; Fm, formate; Xan, xanthyl; Bzl, benzyl; Tos, tosyl; DNP, 2,4-dinitrophenol; DMF, dimethylformamide; DCM, dichloromethane; HBTU, 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate; DIEA, diisopropylethylamine; HOAc, acetic acid; TFA, trifluoroacetic acid; 2 Cl 2, 2-chlorobenzyloxycarbonyl and OcHex, O-cyclohexyl.

Substituents such as, for example, alkyl groups, e.g. C1-12 alkyl groups, may be attached to the free amine of the N-terminal amino acid by standard methods, using reductive alkylation. Hydroxyalkyl groups, e.g., C1-12 hydroxyalkyl groups, can be attached using reductive alkylation in which the free hydroxyl groups are protected with a t-butyl ester. Acyl groups, e.g., COE1, can be attached by binding the free acid, e.g., E1COOH, to the free amine of the N-terminal amino acid, by mixing the terminated resin with 3 molar equivalents of the free acid and diisopropylcarbodiimide in methylene chloride for one hour, and carrying the resulting resin through steps (a ) to (f) of the rinsing program above. If the free acid contains free hydroxyl groups, e.g. p-hydroxyphenylpropionic acid, the coupling should be carried out with an additional 3 molar equivalents of HOBT.

Other peptides according to the invention can be produced by a person skilled in the art analogously.

Functional tests

A. PTH receptor binding

The peptides of the invention were tested for their ability to bind to the PTH receptor present on SaOS-2 cells (human bone sarcoma). SaOS-2 cells (American Type Culture Collection, Rockville, Maryland, USA; ATCC #HBT 85) were maintained in RPMI 1640 medium (Sigma, St. Louis, MO)

PL 191 239 B1 with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37 ° C in a humidified atmosphere with 5% CO2 in air. The medium was changed every three to four days and cells were cloned weekly by trypsinization.

SaOS-2 cells were maintained for four days until they were confluent growth. Medium was replaced with RPMI 1640 medium with 5% FBS and incubated for 2 hours at room temperature with 10x10 ⁴ cpm mono- ¹²⁵ I- [Nle ^8,18, Tyr ³⁴ (3- ¹²⁵ I)] bPTH (1-34) NH2 in the presence of competing of peptides according to the invention at various concentrations between 10 ^-11 M and 10 ^-4 M. The cells were washed three times with ice-cold PBS and lysed in 0.1 M NaOH, after which the cell-bound radioactivity was measured in a scintillation counter. The synthesis of mono- ¹²⁵ I- [Nle ^8,18, Tyr ³⁴ (3- ¹²⁵ I)] bPTH (1-34) NH2 was carried out as described in Goldman et al., Endocrinol., 123: 1468 (1988).

The binding assay was performed for the various peptides of the invention and for each peptide was calculated Kd value (half maximal inhibition of binding of mono- ¹²⁵ I- [Nle ^8,18, Tyr ³⁴ (3- ¹²⁵ I)] bPTH (1-34) NH2).

As shown in Table I, all tested peptides show high affinity for the PTH receptor on SaOS-2 cells.

T a b e l a I

Peptide Kd (μΜ) EC50 (nM) [Glu ^22.25 , Leu ^23.28 , Lys ^26.30 , Aib ²⁹ , Ahc ³¹ ] hPTHrP (1-34) NH2; 0.200 3.7 [Glu ^{22 '25,} Ahc ^23, Lys ^26,30, Leu ^28,31, Aib ^29] hPTHrP (1-34) NH2; 0.070 3.9 [Glu ^22.25 , Leu ^23.28.31 , Lys ^26.30 , Ahc ²⁷ , Aib ²⁹ ] hPTHrP (1-34) NH2; 0.230 3.0 [Glu ^22.25.29 , Leu ^23.28.31 , Lys ²⁶ , Ahc ³⁰ ] hPTHrP (1-34) NH2; 0.230 7 [Cha ^22, Leu ^23,28,31, Glu ^25, Lys ^26,30, Ahc ^27, Aib ^29] hPTHrP (1-34) NH2; 0.060 2.0 [Glu ^22.25 , Leu ^23.28.31 , Ahc ²⁴ , Lys ^26.30 , Aib ²⁹ ] hPTHrP (1-34) NH2; 0.006 0.5 [Glu ^22.29 , Leu ^23.28.31 , Aib ²⁵ , Lys ^26.30 , Ahc ²⁷ ] hPTHrP (1-34) NH2; 5 [Glu ²² , Leu ^23.28.31 , Aib ^25.29 , Lys ^26.30 , Ahc ²⁷ ] hPTHrP (1-34) NH2; 2 [Ahc ²² , Leu ^23.28.31 , Glu ²⁵ , Lys ^26.30 , Aib ²⁹ ] hPTHrP (1-34) NH2 0.3 [Glu ^22.25 , Leu ^23.31 , Lys ^26.30 , Ahc ²⁸ , Aib ²⁹ ] hPTHrP (1-34) NH2 0.5 [Cha ²² , Ahc ²³ , Glu ²⁵ , Lys ^26.30 , Leu ^28.31 , Aib ²⁹ ] hPTHrP (1-34) NH2 0.4

B. Stimulation of adenylate cyclase activity

The ability of the peptides of the invention to elicit a biological response in SaOS-2 cells was measured. Specifically, adenylate cyclase induction was tested by measuring the level of cAMP (adenosine-3 ', 5'-monophosphate) synthesis as previously described in Rodan et al., J. Clin. Invest.

72: 1511 (1983) and Goldman et al., Endocrinol., 123: 1468 (1988). Confluent SaOS-2 cells in plates

24-wells were incubated with 0.5 μθ [ ³ H] -adenine (26.9 Ci / mmol, New England Nuclear, Boston, MA) in fresh medium at 37 ° C for 2 hours and washed twice with Hank's buffered salt solution (Gibco, Gaithersburg, MD). Cells were treated with 1 mM IBMX (Isobutylmethylxanthine, Sigma, St. Louis, MO) in fresh medium for 15 minutes, then peptides of the invention were added to the medium and incubated for 5 minutes. The reaction was stopped by the addition of 1.2 M trichloroacetic acid (TCA) (Sigma, St. Louis, MO) and then the sample was neutralized with 4 N KOH. cAMP was isolated by two column chromatography (Salmon et al., 1974, Anal. Biochem. 58, 541). Radioactivity was counted in a scintillation counter (Liquid Scintillation Counter 2200CA, Packard, Dovners Grove, IL).

The corresponding EC50 values (half maximal adenylate cyclase stimulation) for the test peptides were calculated as described in Table I. All tested peptides were found to be potent stimulants of adenylate cyclase activity, which is an indicator of the biochemical pathway of the proximal osteoblast proliferation signal (e.g. bone growth). .

PL 191 239 B1

Other executions

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention which is defined within the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the claims.

Claims (1)

Patent claim Peptides, parathyroid hormone analogs of the formulas: [Cha ^22.23 , Glu Aib ²⁹ ] hPTHrP (1-34) NH2; [Cha ^22.23 , Glu ²⁵ , Lys ^26.30 , Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu ^22.25 , Leu _Lys ^26.30 , Leu ^{28 31} , Lys ²⁶ Leu ²³ , ²⁸ , ³⁰ , ³¹ , Lys ²⁶ , Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu ^22.25.29 Leu ^23.28.30.31 , Lys ²⁶ ] hPTHrP (1-34) NH2; [Glu ^22.25.29 , Leu ^23.28.31 , Lys ²⁶ , Nle ³⁰ ] hPTHrP (1-34) NH2; [Ser ¹ , Ile ⁵ Met ⁸ , Asn ¹⁰ , Leu ^11,23,28,31 , His ¹⁴ , Cha ¹⁵ , Glu ^22.25 , Lys ^26.30 , Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu ^22.25 , Cha ²³ , Lys ²⁶ Aib ²⁹ , Nle ³⁰ ] hPTHrP (1-34) NH ₂ ; [Glu ^22.25 Leu ^28.31 , Aib ²⁹ , Nle ³⁰ ] hPTHrP (1-34) NH ₂ ; [Cha ^22,23 Glu ²⁵ , Lys 26.30 Leu ^28.31 'Aib ²⁹ ] hPTHrP (1-34) NH ₂ ; [Cha ^22, Leu ^23,28,31, Glu ^25,29, Lys ^26, Nle ^30] hPTHrP (1-34) NH2; [Cha ^7,11,15 ] hPTHrP (1-34) NH2; [Cha ^7,8,15 ] hPTHrP (1-34) NH2; [Glu ²² , Cha ²³ , Aib ^25.29 , Lys ^26.30 , Leu ^28.31 ] hPTHrP (1-34) NH2; [Glu ²² , Cha ²³ Aib ^25.29 , Lys ²⁶ , Leu ²⁸ ] hPTHrP (1-34) NH2; [Glu ²² , Leu 23.28 Aib 25.29 Lys ²⁶ ] hPTHrP (1-34) NH2; Nle ³⁰ ] hPTHrP (1-34) NH ₂ ; [Glu ^22.25 [Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu ^22.25 , Cha ²³ , Lys ²⁶ , Leu ^28.31 , Aib Cha ²³ , Lys ^26.30 , Aib ²⁹ , Leu ³¹ ] hPTHrP (1-34) NH2; [Glu ^22.25 , Leu ^23.28.31 , Lys ²⁶ , Aib ^29.30 ] hPTHrP (1-34) NH ₂ ; [Glu ^22.25 , Leu ^23.28.31 , Lys ²⁶ , Aib ²⁹ ] hPTHrP (1-34) NH ₂ ; [Glu ^22.25 , Leu ^23.28.31 , Aib ^26.29 , 34) NH ₂ ; [Glu ^22.25 , Cha ²³ , Lys ^26.30 , Leu ^28.31 , Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu ^22.25 , Aib ²⁹ ] hPTHrP (1-34) NH2; [Glu ^22.25 , Cha ²³ , Lys ^26.30 , Leu ²⁸ , Aib ²⁹ ] hPTHrP (1-34) NH2; [Leu ²⁷ , Aib ²⁹ ] hPTH (1Lys ³⁰ ] hPTHrP (1Cha ²³ , Lys ^26.30 34) NH2; or a pharmaceutically acceptable salt thereof. Publishing Department of the Polish Patent Office. Circulation 50 copies. Price PLN 2.00.