Dosage regimen for a controlled-release PTH compound

Abstract

The present invention relates to a pharmaceutical composition comprising at least one controlled-release PTH compound or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in the treatment, control, delay or prevention of a condition that can be treated, controlled, delayed or prevented with PTH, pharmaceutical composition comprising at least one controlled-release PTH compound or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in the treatment, control, delay or prevention of a condition that can be treated, controlled, delayed or prevented with PTH, wherein said pharmaceutical composition is administered no more frequently than once every 24 hours with a dosage of the controlled-release PTH compound that corresponds to no more than 70% of the molar equivalent dose of PTH 1-84 administered every 24 hours required to maintain serum calcium within normal levels over said 24 hour period in humans.

Claims

1. A method of treating or controlling hypoparathyroidism in a mammalian patient, comprising the step of administering a pharmaceutical composition comprising at least one controlled-release parathyroid hormone (PTH) compound or a pharmaceutically acceptable salt thereof by subcutaneous injection no more frequently than once every 24 hours with a dosage of the controlled-release PTH compound that corresponds to 20-40% of the molar equivalent dose of PTH 1-84 administered subcutaneously every 24 hours, which is required to maintain a serum albumin-adjusted calcium level in serum of above 8.5 mg/dL over a 24 hour period in humans and wherein the controlled release PTH compound is of formula: ##STR00068## wherein the unmarked dashed line indicates the attachment to an N-terminal amine group of -D, which is a PTH moiety having the amino sequence of SEQ ID NO:51, by an amide bond; and the dashed line marked with the asterisk indicates attachment to a moiety ##STR00069## wherein each of m and p is independently an integer ranging from and including 400 to 500.

2. The method of claim 1, wherein the pharmaceutical composition is administered once every 24 hours.

3. The method of claim 1, wherein the pharmaceutical composition has a pH ranging from and including pH 3 to pH 8.

4. The method of claim 1, wherein the mammalian patient is a human patient.

5. The method of claim 1, wherein the dosage of the controlled-release PTH compound corresponds to no more than 30% of the molar equivalent dose of PTH 1-84.

Description

EXAMPLES

(1) Materials and Methods

(2) Side chain protected PTH(1-34) (SEQ ID NO:51) on TCP resin having Boc protected N-terminus and ivDde protected side chain of Lys26 (synthesized by Fmoc-strategy) was obtained from custom peptide synthesis providers.

(3) Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus (synthesized by Fmoc-strategy) was obtained from custom peptide synthesis providers.

(4) PEG 2×20 kDa maleimide, Sunbright GL2-400MA was purchased from NOF Europe N.V., Grobbendonk, Belgium. S-Trityl-6-mercaptohexanoic acid was purchased from Polypeptide, Strasbourg, France. HATU was obtained from Merck Biosciences GmbH, Schwalbach/Ts, Germany. Fmoc-N-Me-Asp(OBn)-OH was obtained from Peptide International Inc., Louisville, Ky., USA. Fmoc-Aib-OH was purchased from Iris Biotech GmbH, Marktredwitz, Germany. All other chemicals and reagents were purchased from Sigma Aldrich GmbH, Taufkirchen, Germany, unless a different supplier is mentioned.

(5) Compound 11a (examples 11-15) was synthesized following the procedure described in patent W02009/095479A2, example 1.

(6) Syringes equipped with polyethylenene frits (MultiSynTech GmbH, Witten, Germany) were used as reaction vessels or for washing steps of peptide resins.

(7) General procedure for the removal of ivDde protecting group from side chain protected PTH on resin: The resin was pre-swollen in DMF for 30 min and the solvent was discarded. The ivDde group was removed by incubating the resin with DMF/hydrazine hydrate 4/1 (v/v, 2.5 mL/g resin) for 8×15 min. For each step fresh DMF/hydrazine hydrate solution was used. Finally, the resin was washed with DMF (10 x), DCM (10×) and dried in vacuo.

(8) General procedure for the removal of Fmoc protecting group from protected PTH on resin: The resin was pre-swollen in DMF for 30 min and the solvent was discarded. The Fmoc group was removed by incubating the resin with DMF/piperidine/DBU 96/2/2 (v/v/v, 2.5 mL/g resin) for 3×10 min. For each step fresh DMF/piperidine/DBU hsolution was used. Finally, the resin was washed with DMF (10 x), DCM (10 x) and dried in vacuo.

(9) RP-HPLC Purification:

(10) For preparative RP-HPLC a Waters 600 controller and a 2487 Dual Absorbance Detector was used, equipped with the following columns: Waters XBridge™ BEH300 Prep C18 5 μm, 150×10 mm, flow rate 6 mL/min, or Waters XBridge™ BEH300 Prep C18 10 μm, 150×30 mm, flow rate 40 mL/min. Linear gradients of solvent system A (water containing 0.1% TFA v/v) and solvent system B (acetonitrile containing 0.1% TFA v/v) were used. HPLC fractions containing product were pooled and lyophilized if not stated otherwise.

(11) Flash Chromatography:

(12) Flash chromatography purifications were performed on an Isolera One system from Biotage AB, Sweden, using Biotage KP-Sil silica cartridges and n-heptane and ethyl acetate as eluents. Products were detected at 254 nm.

(13) Ion Exchange Chromatography:

(14) Ion exchange chromatography (IEX) was performed using an Amersham Bioscience AEKTAbasic system equipped with a MacroCap SP cation exchanger column (Amersham Bioscience/GE Healthcare). 17 mM acetic acid pH 4.5 (solvent A) and 17 mM acetic acid, 1 M NaCl, pH 4.5 (solvent B) were used as mobile phases.

(15) Size Exclusion Chromatography:

(16) Size exclusion chromatography (SEC) was performed using an Amersham Bioscience AEKTAbasic system equipped with HiPrep 26/10 desalting columns (Amersham Bioscience/GE Healthcare). 0.1% (v/v) acetic acid was used as mobile phase.

(17) Analytical Methods

(18) Analytical ultra-performance LC (UPLC)-MS was performed on a Waters Acquity system equipped with a Waters BEH300 C18 column (2.1×50 mm, 1.7 μm particle size, flow: 0.25 mL/min; solvent A: water containing 0.04% TFA (v/v), solvent B: acetonitrile containing 0.05% TFA (v/v)) coupled to a LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific or coupled to a Waters Micromass ZQ.

(19) Quantitative measurements of serum calcium (sCa), urinary calcium and serum phosporous (sP) were performed on a Roche-Hitachi P800 modular biochemistry instrument.

Example 1

(20) Synthesis of Linker Reagent 1f

(21) Linker reagent 1f was synthesized according to the following scheme:

(22) ##STR00047##

(23) To a solution of N-methyl-N-Boc-ethylenediamine (2 g, 11.48 mmol) and NaCNBH.sub.3 (819 mg, 12.63 mmol) in MeOH (20 mL) was added 2,4,6-trimethoxybenzaldehyde (2.08 g, 10.61 mmol) portion wise. The mixture was stirred at rt for 90 min, acidified with 3 M HCl (4 mL) and stirred further 15 min. The reaction mixture was added to saturated NaHCO.sub.3 solution (200 mL) and extracted 5× with DCM. The combined organic phases were dried over Na.sub.2SO.sub.4 and the solvents were evaporated in vacuo. The resulting N-methyl-N-Boc-N′-Tmob-ethylenediamine 1a was dried in high vacuum and used in the next reaction step without further purification.

(24) Yield: 3.76 g (11.48 mmol, 89% purity, 1a: double Tmob protected product=8:1)

(25) MS: m/z 355.22=[M+H].sup.+, (calculated monoisotopic mass=354.21).

(26) To a solution of 1a (2 g, 5.65 mmol) in DCM (24 mL) COMU (4.84 g, 11.3 mmol), N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4.52 mmol) and 2,4,6-collidine (2.65 mL, 20.34 mmol) were added. The reaction mixture was stirred for 3 h at rt, diluted with DCM (250 mL) and washed 3× with 0.1 M H.sub.2SO.sub.4 (100 mL) and 3× with brine (100 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtrated and the residue concentrated to a volume of 24 mL. 1b was purified using flash chromatography.

(27) Yield: 5.31 g (148%, 6.66 mmol)

(28) MS: m/z 796.38=[M+H].sup.+, (calculated monoisotopic mass=795.37).

(29) To a solution of 1b (5.31 g, max. 4.52 mmol ref. to N-Fmoc-N-Me-Asp(OBn)-OH) in THF (60 mL) DBU (1.8 mL, 3% v/v) was added. The solution was stirred for 12 min at rt, diluted with DCM (400 mL) and washed 3× with 0.1 M H.sub.2SO.sub.4 (150 mL) and 3× with brine (150 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4 and filtrated. 1c was isolated upon evaporation of the solvent and used in the next reaction without further purification.

(30) MS: m/z 574.31=[M+H].sup.+, (calculated monoisotopic mass=573.30). 1c (5.31 g, 4.52 mmol, crude) was dissolved in acetonitrile (26 mL) and COMU (3.87 g, 9.04 mmol), 6-tritylmercaptohexanoic acid (2.12 g, 5.42 mmol) and 2,4,6-collidine (2.35 mL, 18.08 mmol) were added. The reaction mixture was stirred for 4 h at rt, diluted with DCM (400 mL) and washed 3× with 0.1 M H.sub.2SO.sub.4 (100 mL) and 3× with brine (100 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and 1d was isolated upon evaporation of the solvent. Product 1d was purified using flash chromatography.

(31) Yield: 2.63 g (62%, 94% purity)

(32) MS: m/z 856.41=[M+H].sup.+, (calculated monoisotopic mass=855.41).

(33) To a solution of 1d (2.63 g, 2.78 mmol) in i-PrOH (33 mL) and H.sub.2O (11 mL) was added LiOH (267 mg, 11.12 mmol) and the reaction mixture was stirred for 70 min at rt. The mixture was diluted with DCM (200 mL) and washed 3× with 0.1 M H.sub.2SO.sub.4 (50 mL) and 3× with brine (50 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na.sub.2SO.sub.4, filtered and 1e was isolated upon evaporation of the solvent. 1e was purified using flash chromatography.

(34) Yield: 2.1 g (88%)

(35) MS: m/z 878.4=[M+Na].sup.+, (calculated monoisotopic mass=837.40).

(36) To a solution of 1e (170 mg, 0.198 mmol) in anhydrous DCM (4 mL) were added DCC (123 mg, 0.59 mmol), and a catalytic amount of DMAP. After 5 min, N-hydroxy-succinimide (114 mg, 0.99 mmol) was added and the reaction mixture was stirred at rt for 1 h. The reaction mixture was filtered, the solvent was removed in vacuo and the residue was taken up in 90% acetonitrile plus 0.1% TFA (3.4 mL). The crude mixture was purified by RP-HPLC. Product fractions were neutralized with 0.5 M pH 7.4 phosphate buffer and concentrated. The remaining aqueous phase was extracted with DCM and if was isolated upon evaporation of the solvent.

(37) Yield: 154 mg (81%)

(38) MS: m/z 953.4=[M+H].sup.+, (calculated monoisotopic mass=952.43)

Example 2

(39) Synthesis of Linker Reagent 2 g

(40) ##STR00048##

(41) 4-Methoxytriphenylmethyl chloride (3.00 g, 9.71 mmol) was dissolved in DCM (20 mL) and added dropwise under stirring to a solution of ethylenediamine 2a (6.5 mL, 97.3 mmol) in DCM (20 mL). The reaction mixture was stirred for 2 h at rt after which it was diluted with diethyl ether (300 mL), washed 3× with brine/0.1 M NaOH 30/1 (v/v) and once with brine. The organic phase was dried over Na.sub.2SO.sub.4 and 2b was isolated upon evaporation of the solvent.

(42) Yield: 3.18 g (98%)

(43) Mmt protected intermediate 2b (3.18 g, 9.56 mmol) was dissolved in DCM (30 mL). 6-(Tritylthio)-hexanoic acid (4.48 g, 11.5 mmol), PyBOP (5.67 g, 10.9 mmol) and DIPEA (5.0 mL, 28.6 mmol) were added and the mixture was stirred for 30 min at rt. The solution was diuted with diethyl ether (250 mL), washed 3× with brine/0.1 M NaOH 30/1 (v/v) and once with brine. The organic phase was dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo. 2c was purified using flash chromatography.

(44) Yield: 5.69 g (85%)

(45) MS: m/z 705.4=[M+H].sup.+, (calculated monoisotopic mass=704.34).

(46) Compound 2c (3.19 g, 4.53 mmol) was dissolved in abhydrous THF (50 mL), 1 M BH.sub.3.THF solution in THF (8.5 mL, 8.5 mmol) was added and the mixture was stirred for 16 h at rt. More 1 M BH.sub.3.THF solution in THF (14 mL, 14.0 mmol) was added and the mixture was stirred for further 16 h at rt. Methanol (8.5 mL) and N,N′-dimethyl-ethylendiamine (3.00 mL, 27.9 mmol) were added and the mixture was heated under reflux for 3 h. The mixture was allowed to cool down and ethyl acetate (300 mL) was added. The solution was washed 2× with aqueous Na.sub.2CO.sub.3 and 2× with aqueous NaHCO.sub.3. The organic phase was dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo to obtain 2d.

(47) Yield: 3.22 g (103%)

(48) MS: m/z 691.4=[M+H].sup.+, (calculated monoisotopic mass=690.36).

(49) Di-tert-butyl dicarbonate (2.32 g, 10.6 mmol) and DIPEA (3.09 mL, 17.7 mmol) were dissolved in DCM (5 mL) and added to a solution of 2d (2.45 g, 3.55 mmol) in DCM (5 mL). The mixture was stirred for 30 min at rt. The solution was concentrated in vacuo and purified by flash chromatography to obtain product 2e.

(50) Yield: 2.09 g (74%)

(51) MS: m/z 791.4=[M+H].sup.+, (calculated monoisotopic mass=790.42).

(52) Compound 2e (5.01 g, 6.34 mmol) was dissolved in acetonitrile (80 mL). 0.4 M aqueous HCl (80 mL) followed by acetonitrile (20 mL) was added and the mixture was stirred for 1 h at rt. The pH was adjusted to pH 5.5 by addition of aqueous 5 M NaOH. The organic solvent was removed in vacuo and the remaining aqueous solution was extracted 4× with DCM. The combined organic phases were dried over Na.sub.2SO.sub.4 and the solvent was removed in vacuo to obtain product 2f.

(53) Yield: 4.77 g (95%)

(54) MS: m/z 519.3=[M+H].sup.+, (calculated monoisotopic mass=518.30).

(55) Compound 2f (5.27 g, 6.65 mmol) was dissolved in DCM (30 mL) and added to a solution of p-nitrophenyl chloroformate (2.01 g, 9.98 mmol) in DCM (25 mL). 2,4,6-trimethylpyridine (4.38 mL, 33.3 mmol) was added and the solution was stirred for 45 min at rt. The solution was concentrated in vacu and purified by flash chromatography to obtain product 2 g.

(56) Yield: 4.04 g (89%)

(57) MS: m/z 706.32=[M+Na].sup.+, (calculated monoisotopic mass=683.30).

Example 3

(58) Synthesis of Permanent S1 PTH(1-34) Conjugate 3

(59) ##STR00049##

(60) Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of 6-tritylmercaptohexanoic acid (62.5 mg, 160 μmol), PyBOP (80.1 mg, 154 μmol) and DIPEA (53 μL, 306 μmol) in DMF (2 mL) was added to 0.21 g (51 μmol) of the resin. The suspension was agitated for 80 min at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 10 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 3 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried.

(61) Yield: 36 mg (14%), 3*8 TFA

(62) MS: m/z 1062.31=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1062.30).

Example 4

(63) Synthesis of Permanent K26 PTH(1-34) Conjugate 4

(64) ##STR00050##

(65) Side chain protected PTH(1-34) on TCP resin having Boc protected N-terminus and ivDde protected side chain of Lys26 was ivDde deprotected according to the procedure given in Materials and Methods. A solution of 6-tritylmercaptohexanoic acid (107 mg, 273 μmol), PyBOP (141 mg, 273 μmol) and DIPEA (95 μL, 545 μmol) in DMF (3 mL) was added to 0.80 g (90.9 μmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 6 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 4 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried.

(66) Yield: 40 mg (8%), 4*8 TFA

(67) MS: m/z 1062.30=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1062.30).

Example 5

(68) Synthesis of Transient S1 PTH(1-34) Conjugate

(69) ##STR00051##

(70) Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Aib-OH (79 mg, 244 μmol), PyBOP (127 mg, 244 μmol) and DIPEA (64 μL, 365 μmol) in DMF (1.5 mL) was added to 0.60 g (61 μmol) of the resin. The suspension was agitated for 16 h at rt. The resin was washed 10× with DMF and Fmoc-deprotected as described above. A solution of 2 g (167 mg, 244 μmol) and DIPEA (64 μL, 365 μmol) in DMF (1.5 mL) was added to the resin. The suspension was agitated for 24 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 7 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 5 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried.

(71) Yield: 78 mg (24%), 5*9 TFA

(72) MS: m/z 1101.59=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1101.57).

Example 6

(73) Synthesis of Transient S1 PTH(1-34) Conjugate 6

(74) ##STR00052##

(75) Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Ala-OH (32 mg, 102 μmol), PyBOP (53 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) was added to 0.25 g (25 μmol) of the resin. The suspension was shaken for 1 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried under vacuum. Fmoc-deprotection was performed as described above. A solution of 2 g (69 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) was added to the resin. The suspension was agitated for 1.5 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 3 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 6 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried.

(76) Yield: 25 mg (18%), 6*9 TFA

(77) MS: m/z 1098.75=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1098.07).

Example 7

(78) Synthesis of Transient S1 PTH(1-34) Conjugate 7

(79) ##STR00053##

(80) Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Ser(Trt)-OH (117 mg, 205 μmol), PyBOP (108 mg, 207 μmol) and DIPEA (53 μL, 305 μmol) in DMF (2 mL) was added to 0.50 g (51 μmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried under vacuum.

(81) Fmoc-deprotection was performed as described above. A solution of 2 g (144 mg, 211 μmol) and DIPEA (53 μL, 305 μmol) in DMF (1.8 mL) was added to the resin. The suspension was shaken for 7 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 6 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 7 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried.

(82) Yield: 54 mg (20%), 7*9 TFA

(83) MS: m/z 1102.08=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1102.07).

Example 8

(84) Synthesis of Transient S1 PTH(1-34) Conjugate 8

(85) ##STR00054##

(86) Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Leu-OH (36 mg, 102 μmol), PyBOP (53 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) was added to 0.25 g (25 μmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried under vacuum. Fmoc-deprotection was performed as described above. A solution of 2 g (69 mg, 102 μmol) and DIPEA (27 μL, 152 μmol) in DMF (3 mL) was added to the resin. The suspension was agitated for 1.5 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 3 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 8 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried.

(87) Yield: 31 mg (22%), 8*9 TFA

(88) MS: m/z 1109.32=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1108.58).

Example 9

(89) Synthesis of Transient S1 PTH(1-34) Conjugate 9

(90) ##STR00055##

(91) Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of 1e (182 mg, 213 μmol), PyBOP (111 mg, 213 μmol) and DIPEA (93 μL, 532 μmol) in DMF (5 mL) was added to 2.00 g (107 μmol) of the resin. The suspension was agitated for 16 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried under vacuum. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 20 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and agitating the suspension for 1 h at rt. Crude 9 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried.

(92) Yield: 47 mg (8%), 9*9 TFA

(93) MS: m/z 1108.58=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1108.57).

Example 10

(94) Synthesis of Transient K26 PTH(1-34) Conjugate 10

(95) ##STR00056##

(96) Side chain protected PTH(1-34) on TCP resin having Boc protected N-terminus and ivDde protected side chain of Lys26 was ivDde deprotected according to the procedure given in Materials and Methods. A solution of 1f (867 mg, 910 μmol) and DIPEA (0.24 mL, 1.36 mmol) in DMF (5 mL) was added to 1.91 g (227 μmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10× with DMF, 10× with DCM and dried under vacuum. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 20 mL cleavage cocktail 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole and shaking the suspension for 1 h at rt. Crude 10 was precipitated in pre-cooled diethyl ether (−18° C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried.

(97) Yield: 92 mg (7%), 10*9 TFA

(98) MS: m/z 1108.58=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1108.57).

Example 11

(99) Synthesis of Low Molecular Weight Transient 51 PEG Conjugate 11b

(100) ##STR00057##

(101) 0.15 mL of a 0.5 M NaH.sub.2PO.sub.4 buffer (pH 7.4) was added to 0.5 mL of a 20 mg/mL solution of thiol 5 (10 mg, 1.84 μmol) in 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v). The solution was incubated at rt for 10 min after which 238 μL of a 10 mg/mL solution of maleimide 11a (2.4 mg, 2.21 μmol) in 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v) were added. The solution was incubated for 20 min at rt. 10 μL TFA was added and the mixture was purified by RP-HPLC. The product fractions were freeze-dried to obtain 11b.

(102) Yield: 3.1 mg (26%), 11b*9 TFA

(103) MS: m/z 1097.00=[M+4H].sup.4+, (calculated monoisotopic mass for [M+5H].sup.5+=1096.99).

Example 12

(104) Synthesis of Low Molecular Weight Transient S1 PEG Conjugate 12

(105) ##STR00058##

(106) Conjugate 12 was synthesized as described for 11b by using thiol 6 (10 mg, 1.85 μmol) and maleimide 11a (2.4 mg, 2.21 μmol).

(107) Yield: 10 mg (83%), 12*9 TFA

(108) MS: m/z 1094.20=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1094.19).

Example 13

(109) Synthesis of Low Molecular Wight Tansient 51 PEG Cnjugate 13

(110) ##STR00059##

(111) Conjugate 13 was synthesized as described for 11b by using thiol 7 (10 mg, 1.84 μmol) and maleimide 11a (2.4 mg, 2.21 μmol).

(112) Yield: 8 mg (67%), 13*9 TFA

(113) MS: m/z 1097.40=[M+5H].sup.5+, (calculated monoisotopic mass for [M+5H].sup.5+=1097.39).

Example 14

(114) Synthesis of Low Molecular Weight Transient S1 PEG Conjugate 14

(115) ##STR00060##

(116) Conjugate 14 was synthesized as described for 11b by using thiol 8 (10 mg, 1.83 μmol) and maleimide 11a (2.4 mg, 2.21 μmol).

(117) Yield: 4 mg (33%), 14*9 TFA

(118) m/z 1378.01=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1378.00).

Example 15

(119) Synthesis of Low Molecular Weight Transient K26 PEG Conjugate 15

(120) ##STR00061##

(121) Conjugate 15 was synthesized as described for 11b by using thiol 10 (5.2 mg, 0.95 μmol) and maleimide 11a (1.23 mg, 1.14 μmol).

(122) Yield: 2.1 mg (33%), 15*9 TFA

(123) MS: m/z 1102.60=[M+5H].sup.5+, (calculated monoisotopic mass for [M+5H].sup.5+=1102.59).

Example 16

(124) Synthesis of Permanent 2×20 kDa S1 PEG Conjugate 16

(125) ##STR00062##

(126) 772 μL of a solution containing thiol 3 (19.4 mg/mL, 15 mg, 3.54 μmol) and 2.5 mg/mL Boc-L-Met in 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v) were added to 1.87 mL of a solution containing PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 187 mg, 4.32 μmol) and 2.5 mg/mL Boc-L-Met in water containing 0.1% TFA (v/v). 0.5 M NaH.sub.2PO.sub.4 buffer (0.66 mL, pH 7.0) was added and the mixture was stirred for 30 min at rt. 10 μL of a 270 mg/mL solution of 2-mercaptoethanol in water was added. The mixture was stirred for 5 min at rt and 0.33 mL 1 M HCl were added. Conjugate 16 was purified by IEX followed by RP-HPLC using a linear gradient of solvent system A (water containing 0.1% AcOH v/v) and solvent system B (acetonitrile containing 0.1% AcOH v/v). The product containing fractions were freeze-dried.

(127) Yield: 97 mg (2.01 μmol, 57%) conjugate 16*8 AcOH

Example 17

(128) Synthesis of Permanent 2×20 kDa K26 PEG Conjugate 17

(129) ##STR00063##

(130) Conjugate 17 was prepared as described for 16 by reaction of thiol 4 (15 mg, 3.53 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 187 mg, 4.32 μmol).

(131) Yield: 80 mg (1.79 μmol, 51%) conjugate 17*8 AcOH

Example 18

(132) Synthesis of Transient 2×20 kDa 51 PEG Conjugate 18

(133) ##STR00064##

(134) Conjugate 18 was prepared as described for 16 by reaction of thiol 5 (37 mg, 8.40 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 445 mg, 9.24 μmol). The reaction was quenched by addition of 50 μL TFA without prior addition of 2-mercaptoethanol. Conjugate 18 was purified by IEX followed by SEC for desalting. The product containing fractions were freeze-dried.

(135) Yield: 161 mg (3.33 μmol, 40%) conjugate 18*9 AcOH

Example 19

(136) Synthesis of Transient 2×20 kDa 51 PEG Conjugate 19

(137) ##STR00065##

(138) Conjugate 19 was prepared as described for 16 by reaction of thiol 7 (27 mg, 6.14 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 325 mg, 7.50 mol).

(139) Yield: 249 mg (5.16 μmol, 84%) conjugate 19*9 AcOH

Example 20

(140) Synthesis of transient 2×20 kDa S1 PEG Conjugate 20

(141) ##STR00066##

(142) Conjugate 20 was prepared as described for 16 by reaction of thiol 9 (38 mg, 8.59 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 455 mg, 9.45 μmol). The reaction was quenched by addition of 50 μL TFA without prior addition of 2-mercaptoethanol. Conjugate 20 was purified by IEX followed by SEC for desalting. The product containing fractions were freeze-dried.

(143) Yield: 194 mg (4.01 μmol, 47%) conjugate 20*9 AcOH

Example 21

(144) Synthesis of Transient 2×20 kDa K26 PEG Conjugate 21

(145) ##STR00067##

(146) Conjugate 21 was prepared as described for 16 by reaction of thiol 10 (34 mg, 7.58 μmol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 401 mg, 9.26 μmol).

(147) Yield: 256 mg (5.30 μmol, 70%) conjugate 21*9 AcOH

Example 22

(148) Pharmacodynamic Actions in Thyroparathyroidectomised (TPTx) Rats During a 28-Days Study With Daily Subcutaneous Injections With Conjugate 18 or PTH(1-84)

(149) This study was performed in order to test and compare the effect of daily subcutaneous injection of compound 18 and PTH(1-84), the current standard of care, in an animal disease model relevant for investigating treatment of hypoparathyroidism (HP). Rats subjected to thyroparathyroidectomy (TPTx) by blunt dissection are unable to produce parathyroid hormone, PTH, the major regulator of calcium homeostasis. Hence, TPTx rats develop hypocalcemia and hyperphosphatemia characteristic of HP. 17 weeks old female SD TPTx rats (n=9/group) were dosed subcutaneously for 28 days with compound 18 (5 μg PTH eq/kg/d; 1.2 nmol/kg/d, in 10 mM succinic acid, 46 g/L mannitol, pH 4.0), PTH(1-84) (70 μg PTH eq/kg/d; 7.3 nmol/kg/d; in 10 mM citrate, mannitol 39.0 g/L, pH 5.0) or vehicle. Additionally, one group of sham operated rats (n=9) representing normophysiological background control were also given vehicle. Serum calcium (sCa) and phosporous (sP) levels in the animals were measured pre- and post-dose on days 1, 6, 12 and 27. Moreover, bone turnover markers (P1NP and CTx) were measured and bone quality assessed by ex vivo pQCT.

(150) Results: The average sCa in the TPTx rats pre-dosing at day 1 was 8.3 mg/dL compared to 10.9 mg/dL in the sham operated control rats. The sP values were 8.7 mg/dL and 5.9 mg/dL, respectively. Compound 18 given daily at 1.2 nmol/kg elevated sCa to near-normal levels while lowering sP within a few days of administration. At day 12 (day 5 at steady state with compound 18) sCa had stabilised at normal level (10.7 mg/dL) in this group of animals (compound 18/sham-control ratio=1.01) as opposed to the hypocalceamic level (8.1 mg/dL) measured in the PTH(1-84) treated rats (PTH(1-84)/sham-control ratio=0.76). Additionally, the 24-hour urinary Ca excretion at day 12 was comparable between the animals treated with compound 18 and sham-control. Bone mineral density (BMD) and bone mineral content (BMC) were increased in TPTx controls as seen in HP patients. Treatment with Compound 18 decreased BMD, BMC and area in parallel with an increase in CTx compared to sham and vehicle-treated TPTx animals. A significant increase in trabecular BMD was observed in animals dosed with PTH(1-84) compared to both control groups.

(151) It was concluded that compound 18 at a dosage even as low as less than 20% of the molar equivalent of the here tested dose of PTH(1-84) was able to maintain sCa at a level comparable to the sCa level in sham-control animals (here representing normal level) over a 24 hour period. In contrast, PTH(1-84) at a dose of 7.3 nmol/kg/d did not lead to increase in sCa as compared to the levels in the vehicle-injected TPTx rats. However, a minimal decrease in sP was observed in the PTH(1-84) dosed animals confirming exposure and response to PTH(1-84) in the rats. Following the 28-days of treatment with Compound 18, trabecular and cortical BMD in vertebrae were within normal range, whereas an anabolic effect was observed for PTH(1-84) on trabecular and cortical bone in vertebrae. Abbreviations: ACN acetonitrile AcOH acetic acid Aib 2-aminoisobutyric acid BMD bone mineral density Bn benzyl Boc tert-butyloxycarbonyl COMU (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate cAMP cyclic adenosine monophosphate d day p DBU 1,3-diazabicyclo[5.4.0]undecene DCC N,N′-dicyclohexylcarbodiimide DCM dichloromethane DIPEA N,N-diisopropylethylamine DMAP dimethylamino-pyridine DMF N,N-dimethylformamide DMSO dimethylsulfoxide DTT dithiothreitol EDTA ethylenediaminetetraacetic acid eq stoichiometric equivalent ESI-MS electrospray ionization mass spectrometry Et ethyl Fmoc 9-fluorenylmethyloxycarbonyl Glu-C endoproteinase Glu-C h hour HATU O-(7-azabenzotriazole-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate HP hypoparathyroidism HPLC high performance liquid chromatography ivDde 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl LC liquid chromatography LTQ linear trap quadrupole Lys-C endoproteinase Lys-C LLOQ lower limit of quantification Mal 3-maleimido propyl Me methyl MeOH methanol min minutes Mmt monomethoxytrityl MS mass spectrum/mass spectrometry m/z mass-to-charge ratio OtBu tert-butyloxy PEG poly(ethylene glycol) pH potentia Hydrogenii PK pharmacokinetics Pr propyl PTH parathyroid hormone PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Q-TOF quadrupole time-of-flight RP-HPLC reversed-phase high performance liquid chromatography rt room temperature sCa serum calcium SIM single ion monitoring SEC size exclusion chromatography sc subcutaneous sP serum phosphate t.sub.1/2 half life TCP tritylchloride polystyrol TES triethylsilane TFA trifluoroacetic acid THF tetrahydrofuran Tmob 2,4,6-trimethoxybenzyl TPTx thyroparathyroidectomy Trt triphenylmethyl, trityl ULOQ upper limit of quantification UPLC ultra performance liquid chromatography UV ultraviolet ZQ single quadrupole