PTH COMPOUNDS WITH LOW PEAK-TO-TROUGH RATIOS

Abstract

The present invention relates to a pharmaceutical composition comprising a PTH compound, wherein after subcutaneous administration the pharmacokinetic profile of the PTH compound exhibits a peak to trough ratio of less than 4 within one injection interval.

Claims

1. A pharmaceutical composition comprising a controlled-release PTH compound, wherein after subcutaneous administration the pharmacokinetic profile of the PTH compound exhibits a peak to trough ratio of less than 4 within one injection interval.

2. The pharmaceutical composition of claim 1, wherein the time period between two consecutive subcutaneous administrations is one week.

3. The pharmaceutical composition of claim 1, wherein the subcutaneous administration is via subcutaneous injection.

4. The pharmaceutical composition of claim 1, wherein the subcutaneous administration occurs with a pen device.

5. The pharmaceutical composition of claim 1, wherein the peak to trough ratio is less than 3.

6. The pharmaceutical composition of claim 1, wherein the administration is to a non-human primate.

7. The pharmaceutical composition of claim 6, wherein the non-human primate is a cynomolgus monkey.

8. The pharmaceutical composition of claim 1, wherein the administration is to a human.

9. The pharmaceutical composition of claim 1, wherein the controlled-release PTH compound is water-insoluble.

10. The pharmaceutical composition of claim 9, wherein the water-insoluble controlled-release PTH compound is selected from the group consisting of crystals, nanoparticles, microparticles, nanospheres and microspheres.

11. The pharmaceutical composition of claim 9, wherein the water-insoluble controlled-release PTH compound is a microparticle comprising at least one PTH molecule or PTH moiety.

12. The pharmaceutical composition of claim 9, wherein the water-insoluble controlled-release PTH compound is a nanosphere comprising at least one PTH molecule or PTH moiety.

13. The pharmaceutical composition of claim 9, wherein the water-insoluble controlled-release PTH compound is a microsphere comprising at least one PTH molecule or PTH moiety.

14. The pharmaceutical composition of claim 9, wherein the water-insoluble controlled-release PTH compound comprises at least one PTH moiety covalently and reversibly conjugated to a water-insoluble polymer.

15. The pharmaceutical composition of claim 9, wherein the water-insoluble polymer comprises a polymer selected from the group consisting of 2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides), poly(aspartamides), poly(butyric acids), poly(glycolic acids), polybutylene terephthalates, poly(caprolactones), poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters), poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides), poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines), poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides), poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines), poly(iminocarbonates), poly(lacticacids), poly(lactic-co-glycolic acids), poly(methacrylamides), poly(methacrylates), poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters), poly(oxazolines), poly(propylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinyl amines), poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses, carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins, hyaluronic acids and derivatives, functionalized hyaluronic acids, mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl starches and other carbohydrate-based polymers, xylans, and copolymers thereof.

16. The pharmaceutical composition of claim 9, wherein the water-insoluble controlled-release PTH compound is a compound comprising a conjugate D-L, wherein -D is a PTH moiety; and -L comprises a reversible prodrug linker moiety -L.sup.1-, which moiety -L.sup.1- is connected to the PTH moiety -D through a functional group of PTH; wherein -L.sup.1- is substituted with -L.sup.2-Z and is optionally further substituted; wherein -L.sup.2- is a single chemical bond or a spacer moiety; and Z is a water-insoluble carrier moiety to which a multitude of moieties -L.sup.2-L.sup.1-D is connected.

17. The pharmaceutical composition of claim 16, wherein -D has the sequence of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 or SEQ ID NO:115.

18. The pharmaceutical composition of claim 16, wherein -D has the sequence of SEQ ID NO:51.

19. The pharmaceutical composition of claim 16, wherein -L.sup.1- is conjugated to a functional group of the side chain of an amino acid residue of -D, to the N-terminal amine functional group or to the C-terminal carboxyl functional group of -D or to a nitrogen atom in the backbone polypeptide chain of -D.

20. The pharmaceutical composition of claim 16, wherein -L.sup.1- is conjugated to a functional group of the side chain of a proteinogenic amino acid residue of PTH selected from the group consisting of histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid and arginine.

21. The pharmaceutical composition of claim 16, wherein -L.sup.1- is conjugated to a functional group of the side chain of a lysine residue of PTH.

22. The pharmaceutical composition of claim 16, wherein -L.sup.1- is conjugated to a functional group of the side chain of a serine residue of PTH.

23. The pharmaceutical composition of claim 16, wherein -L.sup.1- is conjugated to the N-terminal amine functional group of PTH.

24. The pharmaceutical composition of claim 16, wherein -L.sup.1- is conjugated to the C-terminal functional group of PTH.

25. The pharmaceutical composition of claim 16, wherein L.sup.1- is connected to -D through a linkage selected from the group consisting of amide, ester, carbamate, acetal, aminal, imine, oxime, hydrazone, disulfide and acylguanidine.

26. The pharmaceutical composition of claim 16, wherein -L.sup.1- is a reversible prodrug linker from which PTH is released in its free form.

27. The pharmaceutical composition of claim 16, wherein -L.sup.1- is of formula (IV): ##STR00068## wherein the dashed line indicates attachment to -D and wherein attachment is through a functional group of -D selected from the group consisting of OH, SH and NH.sub.2; m is 0 or 1; at least one or both of R and R.sup.2 is/are independently of each other selected from the group consisting of CN, NO.sub.2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, C(O)R.sup.3, S(O)R.sup.3, S(O).sub.2R.sup.3, and SR.sup.4, one and only one of R.sup.1 and R.sup.2 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted arylalkyl, and optionally substituted heteroarylalkyl; R.sup.3 is selected from the group consisting of H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, OR.sup.9 and N(R.sup.9).sub.2; R.sup.4 is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl; each R.sup.5 is independently selected from the group consisting of H, optionally substituted alkyl, optionally substituted alkenylalkyl, optionally substituted alkynylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl and optionally substituted heteroarylalkyl; R.sup.9 is selected from the group consisting of H and optionally substituted alkyl; Y is absent and X is O or S; or Y is N(Q)CH.sub.2 and X is O; Q is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl and optionally substituted heteroarylalkyl; optionally, R.sup.1 and R.sup.2 may be joined to form a 3 to 8-membered ring; and optionally, both R.sup.9 together with the nitrogen to which they are attached form a heterocyclic ring; wherein -L.sup.1- is substituted with -L.sup.2-Z and wherein -L.sup.1- is optionally further substituted.

28. The pharmaceutical composition of claim 16, wherein -L.sup.1- of formula (IV) is substituted with one moiety -L.sup.2-Z.

29. The pharmaceutical composition of claim 16, wherein -L.sup.2- is a chemical bond.

30. The pharmaceutical composition of claim 16, wherein -L.sup.2- is a spacer moiety.

31. The pharmaceutical composition of claim 16, wherein -L.sup.2- is preferably selected from the group consisting of -T-, C(O)O, O, C(O), C(O)N(R.sup.y1), S(O).sub.2N(R.sup.y1), S(O)N(R.sup.y1), S(O).sub.2, S(O), N(R.sup.y1)S(O).sub.2N(R.sup.y1a), S, N(R.sup.y1), OC(OR.sup.y1)(R.sup.y1a), N(R.sup.y1)C(O)N(R.sup.y1a), OC(O)N(R.sup.y1), C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T-, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one or more R.sup.y2, which are the same or different and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, C(O)O, O, C(O), C(O)N(R.sup.y3), S(O).sub.2N(R.sup.y3), S(O)N(R.sup.y3), S(O).sub.2, S(O), N(R.sup.y3)S(O).sub.2N(R.sup.y3a), S, N(R.sup.y3), OC(OR.sup.y3)(R.sup.y3a), N(R.sup.y3)C(O)N(R.sup.y3a), and OC(O)N(R.sup.y3); R.sup.y1 and R.sup.y1a are independently of each other selected from the group consisting of H, -T, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; wherein -T, C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally substituted with one or more R.sup.2, which are the same or different, and wherein C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T-, C(O)O, O, C(O), C(O)N(R.sup.y4), S(O).sub.2N(R.sup.y4), S(O)N(R.sup.y4), S(O).sub.2, S(O), N(R.sup.y4)S(O).sub.2N(R.sup.y4a), S, N(R.sup.y4), OC(OR.sup.y4)(R.sup.y4a), N(R.sup.y4)C(O)N(R.sup.y4a), and OC(O)N(R.sup.y4); each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl; wherein each T is independently optionally substituted with one or more R.sup.2, which are the same or different; each R.sup.y2 is independently selected from the group consisting of halogen, CN, oxo (O), COOR.sup.y5, OR.sup.y5, C(O)R.sup.y5, C(O)N(R.sup.y5R.sup.y5a), S(O).sub.2N(R.sup.y5R.sup.y5a), S(O)N(R.sup.y5R.sup.y5a), S(O).sub.2R.sup.5, S(O)R.sup.y5, N(R.sup.y5)S(O).sub.2N(R.sup.y5aR.sup.y5b), SR.sup.5, N(R.sup.y5R.sup.y5a), NO.sub.2, OC(O)R.sup.y5, N(R.sup.y5)C(O)R.sup.y5a, N(R.sup.y5)S(O).sub.2R.sup.y5a, N(R.sup.y5)S(O)R.sup.y5a, N(R.sup.y5)C(O)OR.sup.y5a, N(R.sup.y5)C(O)N(R.sup.y5aR.sup.y5b), OC(O)N(R.sup.y5R.sup.y5a), and C.sub.1-6 alkyl; wherein C.sub.1-6 alkyl is optionally substituted with one or more halogen, which are the same or different; and each R.sup.y3, R.sup.y3a, R.sup.4, R.sup.y4a, R.sup.y5, R.sup.y5a and R.sup.y5b is independently selected from the group consisting of H, and C1.6 alkyl, wherein C1.6 alkyl is optionally substituted with one or more halogen, which are the same or different.

32. The pharmaceutical composition of claim 16, wherein -L.sup.2- is a C.sub.1-20 alkyl chain, which is optionally interrupted by one or more groups independently selected from O, -T- and C(O)N(R.sup.y1); and which C.sub.1-20 alkyl chain is optionally substituted with one or more groups independently selected from OH, -T and C(O)N(R.sup.y6R.sup.y6a); wherein R.sup.y1, R.sup.y6, R.sup.y6a are independently selected from the group consisting of H and C.sub.1-4 alkyl and wherein T is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C.sub.3-10 cycloalkyl, 3- to 10-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8- to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl.

33. The pharmaceutical composition of claim 16, wherein -L.sup.2- has a molecular weight in the range of from 14 g/mol to 750 g/mol.

34. The pharmaceutical composition of claim 16, wherein -L.sup.2- has a chain length of 1 to 20 atoms.

35. The pharmaceutical composition of claim 16, wherein Z is a hydrogel.

36. The pharmaceutical composition of claim 35, wherein the hydrogel comprises a polymer selected from the group consisting of 2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides), poly(aspartamides), poly(butyric acids), poly(glycolic acids), polybutylene terephthalates, poly(caprolactones), poly(carbonates), poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters), poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides), poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl-oxazolines), poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides), poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines), poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic acids), poly(methacrylamides), poly(methacrylates), poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters), poly(oxazolines), poly(propylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinyl amines), poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses, carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins, hyaluronic acids and derivatives, functionalized hyaluronic acids, mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl starches and other carbohydrate-based polymers, xylans, and copolymers thereof.

37. The pharmaceutical composition of claim 35, wherein the hydrogel comprises PEG or hyaluronic acid.

38. The pharmaceutical composition of claim 35, wherein the hydrogel comprises PEG.

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

40. A method of treating, controlling, delaying or preventing in a mammalian patient in need of the treatment of one or more diseases which can be treated with PTH, comprising the step of administering to said patient in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 1.

41. The method of claim 40, wherein the disease is selected from the group consisting of hypoparathyroidism, hyperphosphatemia, osteoporosis, fracture repair, osteomalacia, osteomalacia and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, osteogenesis imperfect, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, bone fracture, alopecia, chemotherapy-induced alopecia, and thrombocytopenia.

42. The method of claim 40, wherein the condition is hypoparathyroidism.

Description

EXAMPLES

[0670] Materials and Methods

[0671] 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.

[0672] 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.

[0673] PEG 220 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.

[0674] Compound 11a (examples 11-15) was synthesized following the procedure described in patent WO29095479A2, example 1.

[0675] Syringes equipped with polyethylenene frits (MultiSynTech GmbH, Witten, Germany) were used as reaction vessels or for washing steps of peptide resins.

[0676] 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 815 min. For each step fresh DMF/hydrazine hydrate solution was used. Finally, the resin was washed with DMF (10), DCM (10) and dried in vacuo.

[0677] 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 310 min. For each step fresh DMF/piperidine/DBU solution was used. Finally, the resin was washed with DMF (10 x), DCM (10 x) and dried in vacuo.

[0678] RP-HPLC Purification:

[0679] 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 m, 15010 mm, flow rate 6 mL/min, or Waters XBridge BEH300 Prep C18 10 m, 15030 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.

[0680] Flash Chromatography:

[0681] 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.

[0682] Ion Exchange Chromatography:

[0683] 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.

[0684] Size Exclusion Chromatography:

[0685] 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.

[0686] Analytical Methods

[0687] Analytical ultra-performance LC (UPLC)-MS was performed on a Waters Acquity system equipped with a Waters BEH300 C18 column (2.150 mm, 1.7 m 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.

[0688] Quantification of Plasma Total PTH(1-34) Concentrations:

[0689] Plasma total PTH(1-34) concentrations were determined by quantification of a signature peptide close to the N-terminus (sequence: IQLMHNLGK) and a C-terminal signature peptide (sequence: LQDVHNF) after plasma protein precipitation, followed by sequential digestion with Endoproteinase Lys-C(origin: Lysobacter enzymogenes) and Endoproteinase Glu-C(origin: Staphylococcus aureus V8) of the supernatant. Subsequently, analysis by reversed phase liquid chromatography and detection by mass spectrometry (RP-HPLC-MS) was performed.

[0690] Calibration standards of PTH(1-34) conjugate in blank heparin or EDTA plasma were prepared in concentration ranges from 1 to 1000 ng/mL PTH(1-34) eq (dilution with rat plasma) and 1 to 1000 ng/mL PTH(1-34) eq (dilution with monkey plasma).

[0691] These solutions were used for the generation of a calibration curve. For quality control, three samples independent from the calibration standard solutions were prepared accordingly. Concentrations at the lower end (3-5 fold concentration of the respective LLOQ), the middle range (0.05-0.1 fold concentration of the respective ULOQ) and the upper end (0.5-0.8 fold concentration of the respective ULOQ).

[0692] Sample preparation volumes can be altered depending on the targeted signal response after sample preparation. Processing procedure of the protein precipitation is described here for the analysis of plasma samples originated in rat species. Protein precipitation was carried out first by addition of 100 L of internal standard solution (625 ng/mL of deuterated conjugate) and then by addition of 400 L of acetonitrile to 50 L of the plasma sample. 2 times 150 L of the supernatant were transferred into a new well-plate and evaporated to dryness (under a gentle nitrogen stream at 50 C.). 50 L of reconstitution solvent (50 mM Tris 0.5 mM CaCl.sub.2 buffer, adjusted to pH 8.0) were used to dissolve the residue. Proteolytic digestion was performed as follows:

[0693] 20 g of Lys-C(order number 125-05061, Wako Chemicals GmbH, Neuss, Germany) were dissolved in 80 L of 10 mM acetic acid. 3 L of the Lys-C solution were added to each cavity and samples incubated for 15 hours at 37 C. Afterwards 10 g of Glu-C(order number V1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 L water, and 1.5 L of the Glu-C solution added to each cavity and incubation continued for 1.5 hours at 37 C. After incubation samples were acidified with 2 L water/formic acid 4:6 (v/v) and 10 L were injected into the UPLC-MS system.

[0694] Chromatography was performed on a Waters Acquity BEH300 C18 analytical column (1.7 m particle size; column dimensions 502.1 mm). Water (UPLC grade) containing 0.1% formic acid (v/v) was used as mobile phase A and acetonitrile (UPLC grade) with 0.1% formic acid as mobile phase B.

[0695] Mass analysis was performed on an AB Sciex 6500.sup.+ QTrap in multiple reaction monitoring (MRM) mode, monitoring the transition m/z 352.0 to 462.1 (signature peptide IQLMHNLGK), 355.4 to 467.1 (signature peptide IQLMHNLGK, internal standard), 436.9 to 631.4 (signature peptide LQDVHNF), and 441.9 to 631.4 (signature peptide LQDVHNF, internal standard).

[0696] Quantification of Plasma PEG Concentrations:

[0697] Plasma total PEG concentrations were determined by quantification of the polymeric part of PTH(1-34) conjugates after plasma protein precipitation and enzymatic digestion of the supernatant. Analysis by size exclusion chromatography and detection by mass spectrometry (SEC-MS) followed.

[0698] Calibration standards of PTH(1-34) conjugate in blank heparinized monkey plasma were prepared in concentration ranges from 50 to 4500 ng/mL PEG equivalents.

[0699] These solutions were used for the generation of a quadratic calibration curve. Calibration curves were weighted 1/x. For quality control, three samples independent from the calibration standard solutions were prepared accordingly. Concentrations at the lower end (2-4 fold concentration of the LLOQ), the middle range (0.1-0.2 fold concentration of the ULOQ) and the upper end (0.8 fold concentration of the ULOQ). Protein precipitation was carried out by addition of 200 L of precooled (5-10 C.) methanol to 100 L of the plasma sample. 180 L of the supernatant were transferred into a new well-plate and evaporated to dryness (under a gentle nitrogen stream at 45 C). 50 L of reconstitution solvent (50 mM Tris 0.5 mM CaCl.sub.2 buffer, adjusted to pH 8.0) were used to dissolve the residue. Proteolytic digestion was performed as follows: 20 g of Lys-C(order number 125-05061, Wako Chemicals GmbH, Neuss, Germany) were dissolved in 80 L of 10 mM acetic acid. 3 L of the Lys-C solution were added to each cavity and samples incubated for 15 hours at 37 C. Afterwards 10 g of Glu-C(order number V1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 L water, and 1.5 L of the Glu-C solution added to each cavity and incubation continued for 1.5 hours at 37 C. After incubation samples were acidified with 2 L water/formic acid 4:6 (v/v) and 5 L were injected into the SEC-MS system.

[0700] SEC-MS analysis was carried out by using an Agilent 1290 UPLC coupled to an Agilent 6460 TripleQuad mass spectrometer via an ESI probe. Acquisition of a distinct precursor ion of the polymer was achieved by applying high voltage in-source fragmentation (200-300V) at the MS interface. Chromatography was performed on a TOSOH TSK Gel SuperAW3000 analytical column (4.0 m particle size; column dimensions 1506.0 mm) at a flow rate of 0.50 mL/min (T=65 C). Water (UPLC grade) containing 0.1% formic acid (v/v) was used as mobile phase A and acetonitrile (UPLC grade) with 0.1% formic acid as mobile phase B. The chromatographic setup for sample analysis comprises an isocratic elution of 50% B over 8 minutes.

[0701] Mass analysis was performed in single reaction monitoring (SRM) mode, monitoring the transition m/z 133.1 to 45.1.

[0702] Quantification of Plasma Free PTH Concentrations:

[0703] Free PTH concentrations in acidified plasma were determined as the sum of peptide PTH(1-34) and peptide PTH(1-33) after plasma protein precipitation, followed by solid phase extraction. Subsequently, analysis using liquid chromatography separation and detection by mass spectrometry (LC-MS) was performed.

[0704] Calibration standards of PTH(1-34) and PTH(1-33) in blank acidified EDTA rat plasma were prepared in concentrations ranging from 5.00 to 500 g/mL in acidified plasma for each analyte. The corresponding concentration range is 7.00 to 700 g/mL for both analytes in neat plasma, as plasma is acidified as volume ratio plasma:0.5 M citrate buffer pH 4=1:0.4 v/v.

[0705] Standard solutions were used for the generation of a calibration curve. For quality control, three samples were prepared independent of the calibration standard solutions at 15.0, 150 and 400 g/mL in acidified plasma.

[0706] Protein precipitation was carried out by addition of 150 L of cold acetonitrile to 150 L of the plasma sample after addition of 50.0 L of cold internal standard solution, followed by centrifugation. The supernatant was decanted into a new polypropylene tube and 900 L of cold water was added. After another centrifugation step, the tubes were kept in ice-water until loading on the SPE column.

[0707] Solid phase extraction: the HLB elution columns were conditioned with 200 L methanol followed by 200 L water. The columns were loaded 3 times with 420 L of the diluted samples by applying positive pressure. The SPE-columns were washed with 200 L of methanol:water 5:95 v/v. The samples were eluted with 40.0 L SPE elution solvent (Aceonitrile:water:Trifluoroactic acid 60:40:1 v/v/v), followed by 40.0 L of water. The elution solvent was left standing on the columns for 2 minutes, after which very gentle pressure was applied for elution.

[0708] Separation between metabolites and interfering endogenous compounds was achieved by LC-MS using an Xselect CSH C18 column (2.1100 mm, 2.5 m) at 50 C. and using 0.2% Formic acid and 0.5% dimethyl sulfoxide in water as mobile phase A, 0.5% dimethyl sulfoxide in acetonitrile:methanol (75:25, v/v) as mobile phase B, and operating at a gradient with a flow rate of 0.500 mL/min.

[0709] A triple 6500 quadrupole mass spectrometer equipped with a turbo ion spray source was used for detection in positive ion mode. For PTH(1-34), PTH(1-33) and PTH(1-33) (Leu-d.sub.10).sub.3, quantification was done by counting 5 times the same SRM transition.

[0710] Quantification is based on multiple reaction monitoring (MRM) of the transitions of m/z:

687.3-787.3 for PTH(1-34)

662.8-757.9 for PTH(1-33)

[0711] 692.3-793.3 for PTH(1-34) (Leu-d.sub.10).sub.3
667.8-763.9 for PTH(1-33) (Leu-d.sub.10).sub.3

[0712] A linear calibration curve with a 1/x.sup.2 weighing factor was used for both analytes.

[0713] Concentrations of free PTH were determined in acidified plasma, as a means to stabilize the analytes. Acidified plasma was prepared by diluting rat EDTA plasma 1.4 times (in case of blank, zero, calibration and QC samples) or rat whole blood (in case of study samples) 1.2 times. Assuming approximately 50% (v/v) of whole blood being plasma, the neat plasma concentrations are approximately 1.4 times higher than the reported concentrations in acidified plasma. Free PTH concentrations are calculated as sum of Free PTH (1-34) and Free PTH (1-33) in PTH(1-34) equivalents).

[0714] Due to the reversible nature of the attachment of -L.sup.1- to -D, measurements for PTH receptor activity were made using stable analogs of the PTH prodrugs of the present invention, i.e. they were made using similar structures to those of the PTH prodrugs of the present invention which instead of a reversible attachment of Z to -D have a stable attachment.

[0715] This was necessary, because the PTH prodrugs of the present invention would release PTH in the course of the experiment and said released PTH would have influenced the result.

Example 1

[0716] Synthesis of Linker Reagent 1f

[0717] Linker reagent 1f was synthesized according to the following scheme:

##STR00047##

[0718] 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.

[0719] Yield: 3.76 g (11.48 mmol, 89% purity, 1a: double Tmob protected product=8:1)

[0720] MS: m/z 355.22=[M+H].sup.+, (calculated monoisotopic mass=354.21).

[0721] 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.

[0722] Yield: 5.31 g (148%, 6.66 mmol)

[0723] MS: m/z 796.38=[M+H].sup.+, (calculated monoisotopic mass=795.37).

[0724] 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.

[0725] MS: m/z 574.31=[M+H].sup.+, (calculated monoisotopic mass=573.30).

[0726] 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 id was isolated upon evaporation of the solvent. Product 1d was purified using flash chromatography.

[0727] Yield: 2.63 g (62%, 94% purity)

[0728] MS: m/z 856.41=[M+H].sup.+, (calculated monoisotopic mass=855.41).

[0729] 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.

[0730] Yield: 2.1 g (88%)

[0731] MS: m/z 878.4=[M+Na].sup.+, (calculated monoisotopic mass=837.40).

[0732] 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.

[0733] Yield: 154 mg (81%)

[0734] MS: m/z 953.4=[M+H].sup.+, (calculated monoisotopic mass=952.43).

Example 2

[0735] Synthesis of Linker Reagent 2g

##STR00048##

[0736] 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.

[0737] Yield: 3.18 g (98%)

[0738] 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 DTPEA (5.0 mL, 28.6 mmol) were added and the mixture was stirred for 30 min at rt. The solution was diluted 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.

[0739] Yield: 5.69 g (85%)

[0740] MS: m/z 705.4=[M+H].sup.+, (calculated monoisotopic mass=704.34).

[0741] Compound 2c (3.19 g, 4.53 mmol) was dissolved in anhydrous 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.

[0742] Yield: 3.22 g (103%)

[0743] MS: m/z 691.4=[M+H].sup.+, (calculated monoisotopic mass=690.36).

[0744] 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.

[0745] Yield: 2.09 g (74%)

[0746] MS: m/z 791.4=[M+H].sup.+, (calculated monoisotopic mass=790.42).

[0747] 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.

[0748] Yield: 4.77 g (95%)

[0749] MS: m/z 519.3=[M+H].sup.+, (calculated monoisotopic mass=518.30).

[0750] 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 2g.

[0751] Yield: 4.04 g (89%)

[0752] MS: m/z 706.32=[M+Na]+, (calculated monoisotopic mass=683.30).

Example 3

[0753] Synthesis of Permanent S1 PTH(1-34) Conjugate 3

##STR00049##

[0754] 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 mol), PyBOP (80.1 mg, 154 mol) and DIPEA (53 L, 306 mol) in DMF (2 mL) was added to 0.21 g (51 mol) 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.

[0755] Yield: 36 mg (14%), 3*8 TFA

[0756] MS: m/z 1062.31=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1062.30).

Example 4

[0757] Synthesis of Permanent K26 PTH(1-34) Conjugate 4

##STR00050##

[0758] 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 mol), PyBOP (141 mg, 273 mol) and DIPEA (95 L, 545 mol) in DMF (3 mL) was added to 0.80 g (90.9 mol) 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.

[0759] Yield: 40 mg (8%), 4*8 TFA

[0760] MS: m/z 1062.30=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1062.30).

Example 5

[0761] Synthesis of Transient S1 PTH(1-34) Conjugate

##STR00051##

[0762] 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 mol), PyBOP (127 mg, 244 mol) and DIPEA (64 L, 365 mol) in DMF (1.5 mL) was added to 0.60 g (61 mol) 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 2g (167 mg, 244 mol) and DIPEA (64 L, 365 mol) 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.

[0763] Yield: 78 ng (24%), 5*9 TFA

[0764] MS: m/z 1101.59=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1101.57).

Example 6

[0765] Synthesis of Transient S1 PTH(1-34) Conjugate 6

##STR00052##

[0766] 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 mol), PyBOP (53 mg, 102 mol) and DIPEA (27 L, 152 mol) in DMF (3 mL) was added to 0.25 g (25 mol) 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 2g (69 mg, 102 mol) and DIPEA (27 L, 152 mol) 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 pr-cooled diethyl ether (18 C.). The precipitate was dissolved in ACN/water and purified by RP-HPLC. The product fractions were freeze-dried.

[0767] Yield: 25 mg (18%), 6*9 TFA

[0768] MS: m/z 1098.75=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1098.07).

Example 7

[0769] Synthesis of Transient S1 PTH(1-34) Conjugate 7

##STR00053##

[0770] 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 mol), PyBOP (108 mg, 207 mol) and DIPEA (53 L, 305 mol) in DMF (2 mL) was added to 0.50 g (51 mol) 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 2g (144 mg, 211 mol) and DIPEA (53 L, 305 mol) 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.

[0771] Yield: 54 mg (20%), 7*9 TFA

[0772] MS: m/z 1102.08=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1102.07).

Example 8

[0773] Synthesis of Transient S1 PTH(1-34) Conjugate 8

##STR00054##

[0774] 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 mol), PyBOP (53 mg, 102 mol) and DIPEA (27 L, 152 mol) in DMF (3 mL) was added to 0.25 g (25 mol) 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 2g (69 mg, 102 mol) and DIPEA (27 L, 152 mol) 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.

[0775] Yield: 31 mg (22%), 8*9 TFA

[0776] MS: m/z 1109.32=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1108.58).

Example 9

[0777] Synthesis of Transient S1 PTH(1-34) Conjugate 9

##STR00055##

[0778] 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 mol), PyBOP (111 mg, 213 mol) and DIPEA (93 L, 532 mol) in DMF (5 mL) was added to 2.00 g (107 mol) 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.

[0779] Yield: 47 mg (8%), 9*9 TFA

[0780] MS: m/z 1108.58=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1108.57).

Example 10

[0781] Synthesis of Transient K26 PTH(1-34) Conjugate 10

##STR00056##

[0782] 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 if (867 mg, 910 mol) and DIPEA (0.24 mL, 1.36 mmol) in DMF (5 mL) was added to 1.91 g (227 mol) 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.

[0783] Yield: 92 mg (7%), 10*9 TFA

[0784] MS: m/z 1108.58=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1108.57).

Example 11

[0785] Synthesis of Low Molecular Weight Transient S1 PEG Conjugate 11b

##STR00057##

[0786] 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 mol) 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 L of a 10 mg/mL solution of maleimide 11a (2.4 mg, 2.21 mol) 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 L TFA was added and the mixture was purified by RP-HPLC. The product fractions were freeze-dried to obtain 1b.

[0787] Yield: 3.1 mg (26%), 11b*9 TFA

[0788] MS: m/z 1097.00=[M+4H].sup.4+, (calculated monoisotopic mass for [M+5H].sup.5+=1096.99).

Example 12

[0789] Synthesis of Low Molecular Weight Transient S1 PEG Conjugate 12

##STR00058##

[0790] Conjugate 12 was synthesized as described for 11b by using thiol 6 (10 mg, 1.85 mol) and maleimide 11a (2.4 mg, 2.21 mol).

[0791] Yield: 10 mg (83%), 12*9 TFA

[0792] MS: m/z 1094.20=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1094.19).

Example 13

[0793] Synthesis of Low Molecular Weight Transient S1 PEG Conjugate 13

##STR00059##

[0794] Conjugate 13 was synthesized as described for 11b by using thiol 7 (10 mg, 1.84 mol) and maleimide 11a (2.4 mg, 2.21 mol).

[0795] Yield: 8 mg (67%), 13*9 TFA

[0796] MS: m/z 1097.40=[M+5H].sup.5+, (calculated monoisotopic mass for [M+5H].sup.5+=1097.39).

Example 14

[0797] Synthesis of Low Molecular Weight Transient S1 PEG Conjugate 14

##STR00060##

[0798] Conjugate 14 was synthesized as described for 11b by using thiol 8 (10 mg, 1.83 mol) and maleimide 11a (2.4 mg, 2.21 mol).

[0799] Yield: 4 mg (33%), 14*9 TFA

[0800] MS: m/z 1378.01=[M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+=1378.00).

Example 15

[0801] Synthesis of Low Molecular Weight Transient K26 PEG Conjugate 15

##STR00061##

[0802] Conjugate 15 was synthesized as described for 11b by using thiol 10 (5.2 mg, 0.95 mol) and maleimide 11a (1.23 mg, 1.14 mol).

[0803] Yield: 2.1 mg (33%), 15*9 TFA

[0804] MS: m/z 1102.60=[M+5H].sup.5+, (calculated monoisotopic mass for [M+5H].sup.5+=1102.59).

Example 16

[0805] Synthesis of Permanent 220 kDa S1 PEG Conjugate 16

##STR00062##

[0806] 772 L of a solution containing thiol 3 (19.4 mg/mL, 15 mg, 3.54 mol) 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 220 kDa maleimide (Sunbright GL2-400MA, 187 mg, 4.32 mol) 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 L 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.

[0807] Yield: 97 mg (2.01 mol, 57%) conjugate 16*8 AcOH

Example 17

[0808] Synthesis of Permanent 220 kDa K26 PEG Conjugate 17

##STR00063##

[0809] Conjugate 17 was prepared as described for 16 by reaction of thiol 4 (15 mg, 3.53 mol) and PEG 220 kDa maleimide (Sunbright GL2-400MA, 187 mg, 4.32 mol).

[0810] Yield: 80 mg (1.79 mol, 51%) conjugate 17*8 AcOH

Example 18

[0811] Synthesis of Transient 220 kDa S1 PEG Conjugate 18

##STR00064##

[0812] Conjugate 18 was prepared as described for 16 by reaction of thiol 5 (37 mg, 8.40 mol) and PEG 220 kDa maleimide (Sunbright GL2-400MA, 445 mg, 9.24 mol). The reaction was quenched by addition of 50 L 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.

[0813] Yield: 161 mg (3.33 mol, 40%) conjugate 18*9 AcOH

Example 19

[0814] Synthesis of Transient 220 kDa S1 PEG Conjugate 19

##STR00065##

[0815] Conjugate 19 was prepared as described for 16 by reaction of thiol 7 (27 mg, 6.14 mol) and PEG 220 kDa maleimide (Sunbright GL2-400MA, 325 mg, 7.50 mol).

[0816] Yield: 249 mg (5.16 mol, 84%) conjugate 19*9 AcOH

Example 20

[0817] Synthesis of Transient 220 kDa S1 PEG Conjugate 20

##STR00066##

[0818] Conjugate 20 was prepared as described for 16 by reaction of thiol 9 (38 mg, 8.59 mol) and PEG 220 kDa maleimide (Sunbright GL2-400MA, 455 mg, 9.45 mol). The reaction was quenched by addition of 50 L 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.

[0819] Yield: 194 mg (4.01 mol, 47%) conjugate 20*9 AcOH

Example 21

[0820] Synthesis of Transient 220 kDa K26 PEG Conjugate 21

##STR00067##

[0821] Conjugate 21 was prepared as described for 16 by reaction of thiol 10 (34 mg, 7.58 mol) and PEG 220 kDa maleimide (Sunbright GL2-400MA, 401 mg, 9.26 mol).

[0822] Yield: 256 mg (5.30 mol, 70%) conjugate 21*9 AcOH

Example 22

[0823] In vitro release kinetics of transient low molecular weight PEG conjugates Conjugates 11b, 12, 13, 14, and 15 were dissolved in pH 7.4 phosphate buffer (60 mM NaH.sub.2PO.sub.4, 3 mM EDTA, 0.01% Tween-20, adjusted to pH 7.4 by NaOH) containing 0.05 mg/mL pentafluorophenol as internal standard at a concentration of approximately 1 mg conjugate/mL. The solutions were filtered sterile and incubated at 37 C. At time points, aliquots were withdrawn and analysed by RP-HPLC and ESI-MS. The fraction of released PTH at a particular time point was calculated from the ratio of UV peak areas of liberated PTH and PEG conjugate. The % released PTH was plotted against incubation time. Curve-fitting software was applied to calculate the corresponding half times of release.

[0824] Results:

[0825] For conjugate 11b a release half life time of 3.2 d was obtained.

[0826] For conjugate 12 a release half life time of 8.7 d was obtained.

[0827] For conjugate 13 a release half life time of 10.8 d was obtained.

[0828] For conjugate 14 a release half life time of 25.3 d was obtained.

[0829] For conjugate 15 a release half life time of 6.9 d was obtained.

Example 23

[0830] In Vitro Release Kinetics of Transient 220 kDa PEG Conjugates

[0831] Conjugates 18, 19, 20, and 21 were dissolved in pH 7.4 phosphate buffer (60 mM NaH.sub.2PO.sub.4, 3 mM EDTA, 0.01% Tween-20, adjusted to pH 7.4 by NaOH) containing 0.08 mg/mL pentafluorophenol as internal standard at a concentration of approximately 5 mg conjugate/mL. The solutions were filtered sterile and incubated at 37 C. At time points, aliquots were withdrawn and analysed by RP-HPLC. The fraction of released PTH at a particular time point was calculated from the ratio of UV peak areas of liberated PTH and PEG conjugate. The % released PTH was plotted against incubation time. Curve-fitting software was applied to calculate the corresponding half times of release.

[0832] Results:

[0833] For conjugate 18 a release half life time of 2.8 d was obtained.

[0834] For conjugate 19 a release half life time of 13.4 d was obtained.

[0835] For conjugate 20 a release half life time of 1.3 d was obtained

[0836] For conjugate 21 a release half life time of 7.1 d was obtained

Example 24

[0837] PTH Receptor Activity of Permanent 220 kDa PEG Conjugates 16 and 17 in Cell Based Assay

[0838] The residual PTH activity of permanently PEGylated conjugates 16 and 17 was quantified by measuring cAMP production from HEK293 cells over-expressing the PTH/PTHrP1 receptor (Hohenstein A, Hebell M, Zikry H, El Ghazaly M, Mueller F, Rohde, J. Development and validation of a novel cell-based assay for potency determination of human parathyroid honnone (PTH), Journal of Pharnaceutical and Biomedical Analysis September 2014, 98: 345-350). PTH(1-34) from NIBSC (National Institute for Biological Standards and Control, UK) was used as reference standard.

[0839] Results:

[0840] For conjugate 16 a receptor activity of 0.12% was found relative to PTH(1-34) reference

[0841] For conjugate 17 a receptor activity of 0.11% was found relative to PTH(1-34) reference

[0842] The results indicate an effective lowering of receptor activity in the permanent 220 kDa PEG conjugates 16 and 17. It can be concluded that similar conjugates with transiently Ser1 or Lys26 linked PTH (like e.g. 18 and 21) are suitable PTH prodrugs providing low residual receptor activity. Direct analysis of transient conjugates in the cell assay is not possible due to linker cleavage under the assay conditions. The released PTH would influence the assay result.

Example 25

[0843] Pharmacokinetic Study of Permanent 220 kDa PEG Conjugates 16 and 17 in Rats

[0844] Male Wistar rats (6 weeks, 230-260 g) received either a single intravenous (2 groups, n=3 animals each) or a single subcutaneous (2 groups, n=3 animals each) administration of 16 or 17 at doses of 29 g/rat PTH.sub.eq and 31 g/rat PTH.sub.eq respectively. Blood samples were collected up to 168 h post dose, and plasma was generated. Plasma PTH(I-34) concentrations were determined by quantification of the N-terminal signature peptide (sequence: IQLMHNLGK) and the C-terminal signature peptide (sequence: LQDVHNF) after LysC and GluC digestion as described in Materials and Methods.

[0845] Results: Dose administrations were well tolerated with no visible signs of discomfort during administration and following administration. No dose site reactions were observed any time throughout the study. After intraveneous injection of 16 and 17 the total PTH(1-34) t.sub.max was observed at 15 min (earliest time point analyzed), followed by a slow decay in total PTH(1-34) content with a half life time of approx. 13 h and 11 h respectively. After subcutaneous injection the total PTH(1-34) concentration peaked at a tax of 24 h for both 16 and 17, followed by a slow decay in total PTH(1-34) content with half life times of approx. 1.5 days for both conjugates. The bioavailability was approx. 40% and 60% respectively. Similar PK curves were obtained for the N- and the C-terminal signature peptide up to 168 h post dose, indicating the presence of intact PTH(1-34) in the conjugate.

[0846] The favourable long lasting PK and the stability of PTH in the conjugates indicate the suitability of the permanent 220 kDa PEG model compounds as slow releasing PTH prodrugs after subcutaneous injection. It can be concluded that similar conjugates with transiently Ser (like e.g. 18) or Lys26 linked PTH are suitable PTH prodrugs providing long lasting levels of released bioactive PTH.

Example 26

[0847] Pharmacokinetic Study of Transient 220 kDa S1 PEG Conjugate 19 in Cynomolgus Monkeys

[0848] Male non nave cynomolgus monkeys (2-4 years, 3.7-5.4 kg) received a single subcutaneous (n=3 animals) administration of 19 at a dose of 70 g/kg PTH.sub.eq. Blood samples were collected up to 504 h post dose, and plasma was generated. Total plasma PTH(1-34) concentrations were determined by quantification of the N-terminal signature peptide (sequence: IQLMHNLGK) and the C-terminal signature peptide (sequence: LQDVHNF) after LysC and GluC digestion as described in Materials and Methods. The PEG concentrations were determined using the method described in Materials and Methods.

[0849] Results: Dose administrations were well tolerated with no visible signs of discomfort during administration. One animal showed showed visible signs of discomfort 72 h post dose, but recovered the days after. No dose site reactions were observed any time throughout the study. The total PTH(1-34) concentration peaked at a t.sub.max of 24 h, followed by a slow decay in total PTH(1-34) content with a half life time of approx. 2.5 d for the N-terminal signature peptide and 0.9 d for the C-terminal signature peptide. The PEG concentration peaked at t.sub.max of 24 h, followed by a slow decay in PEG concentration with a half life time of 3.5 d.

[0850] It can be concluded that conjugate 19 is a suitable prodrug for sustained delivery of PTH.

Example 27

[0851] Pharmacokinetic Study of Transient 220 kDa S1 PEG Conjugate 18 in Cynomolgus Monkeys

[0852] Non nave cynomolgus monkeys (2-3 years, 2.5-4 kg) received daily subcutaneous (n=2 animals-1 male/1 female) administration of 18 at dose levels of 0.2, 0.5, and 1 g/kg PTH.sub.eq for 28 days. Blood samples were collected up to 28 days (at days 1, 13, and, 27 samples were collected at pre-dose, 2 h, 4 h, 8 h, 12 h, and 24 h post-dose) and plasma was generated. Plasma PTH(1-34) concentrations were determined by quantification of the N-terminal signature peptide (sequence: IQLMHNLGK) and the C-terminal signature peptide (sequence: LQDVHNF) after LysC and GluC digestion as described in Materials and Methods.

[0853] Results: All dose administrations were performed without incident. No dose site reactions were observed any time throughout the study. Dose linearity was observed in the three groups. Dose stacking was observed from day 1 compared with day 13 and day 27. Total PTH(1-34) concentrations were quantified via the N-terminal signature peptide (sequence: IQLMHNLGK) at steady state (during day 27).

[0854] A low peak-to-trough ratio of total PTH(1-34) for all dose groups of below 3 was observed after daily subcutaneous application at steady state in cynomolgus monkeys. As free peptide concentrations at steady state are correlated to total PTH(1-34) concentration, the peak-to-trough ratio for the free peptide is below 4 in cynomolgus monkeys.

Example 28

[0855] Pharmacokinetic Study of Transient 220 kDa S1 PEG Conjugate 18 in Cynomolgus Monkeys

[0856] Nave cynomolgus monkeys (2-3.5 years, 2-5 kg) (3-5 males/3-5 females) received daily subcutaneous administrations of 18 at dose levels of 0.2, 0.5 and 1.5 g PTH/kg. Blood samples were collected at; Day 1: pre-dose, 4 h, 8 h, 12 h, 18 h, and 24 h post-dose, at Day 8: pre-dose, at Day 14: predose, 8 h, and 12 h and at Day 28: 3 h, 6 h, 8 h, 12 h, 18 h, 24 h, 72 h, 168 h, and 336 h) and plasma was generated. Total PTH plasma concentrations were determined by quantification of the N-terminal signature peptide (sequence: IQLMHNLGK) after LysC and GluC digestion as presented earlier in Materials and Methods.

[0857] Results: Systemic exposure expressed as C.sub.max and AUC increased in an approximately dose proportional manner. Systemic exposure of Total PTH expressed as AUC accumulated approximately 3-fold from Day 1 to Day 28.

[0858] A low mean peak-to-trough ratio of Total PTH for all dose groups of 1.5 was observed after daily subcutaneous administration in cynomolgus monkeys at Day 28 (steady state observed from Day 8). This low mean peak-to-trough ratio of Total PTH can also be translated to Free PTH.

Example 29

[0859] Pharmacokinetic Study of Transient 220 kDa S1 PEG Conjugate 18 in Sprague-Dawley Rats

[0860] Sprague-Dawley Crl:CD(SD) rats (initiation of dosing at 8 weeks of age) received daily subcutaneous administrations of 18 at dose levels of 10, 30 and 60 g PTH/kg for 28 days. A TK group containing of 9 males and 9 females per dose group was divided into 3 subgroup with 3 rats per subgroup. Blood samples were collected up to 28 days with 3 rats per sex, per sampling time point. Samples were collected at Day 1: pre-dose, 4 h, 8 h, 12 h, 18 h, and 24 h post-dose, and at Day 28: 3 h, 6 h, 8 h, 12 h, 18 h, 24 h, and 336 h and plasma was generated. The total PTH plasma concentrations were determined by quantification of the N-terminal signature peptide (sequence: IQLMHNLGK) after LysC and GluC digestion as presented earlier in Materials and Methods.

[0861] The free PTH plasma concentrations were determined by quantification as the sum of PTH(1-34) and PTH(1-33) by LC-MS/MS as presented earlier in Materials and Methods.

[0862] Results: Systemic exposure of total PTH and free PTH expressed as mean C.sub.max and AUC increased in an approximately dose proportional manner. Systemic exposure of total PTH expressed as mean AUC accumulated 3-6 fold from day 1 to day 28 and free PTH expressed as mean AUC accumulated 2-3 fold from day 1 to day 28. Systemic exposure of total PTH in the female rat was approximately 2-fold higher than in males. Systemic exposure of free PTH was slightly higher in the female rat than in males.

[0863] A low mean peak-to-trough ratio of total PTH for all dose groups of 1.2 was observed after daily subcutaneous administration in Sprague-Dawley rats at day 28 (steady state observed from Day 8). A low mean peak-to-trough ratio of free PTH in the range 1.5-2.4 was observed after daily subcutaneous administration in Sprague-Dawley rats at day 28.

ABBREVIATIONS

[0864] ACN acetonitrile [0865] AcOH acetic acid [0866] Aib 2-aminoisobutyric acid [0867] BMD bone mineral density [0868] Bn benzyl [0869] Boc tert-butyloxycarbonyl [0870] COMU (1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate [0871] cAMP cyclic adenosine monophosphate [0872] d day [0873] DBU 1,3-diazabicyclo[5.4.0]undecene [0874] DCC N,N-dicyclohexylcarbodiimide [0875] DCM dichloromethane [0876] DIPEA N,N-diisopropylethylamine [0877] DMAP dimethylamino-pyridine [0878] DMF N,N-dimethylformamide [0879] DMSO dimethylsulfoxide [0880] DTT dithiothreitol [0881] EDTA ethylenediaminetetraacetic acid [0882] eq stoichiometric equivalent [0883] ESI-MS electrospray ionization mass spectrometry [0884] Et ethyl [0885] Fmoc 9-fluorenylmethyloxycarbonyl [0886] Glu-C Endoproteinase Glu-C [0887] h hour [0888] HATU O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate [0889] HPLC high performance liquid chromatography [0890] ivDde 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl [0891] LC liquid chromatography [0892] LTQ linear trap quadrupole [0893] Lys-C endoproteinase Lys-C [0894] LLOQ lower limit of quantification [0895] Mal 3-maleimido propyl [0896] Me methyl [0897] MeOH methanol [0898] min minutes [0899] Mint monomethoxytrityl [0900] MS mass spectrum/mass spectrometry [0901] m/z mass-to-charge ratio [0902] OtBu tert-butyloxy [0903] PEG poly(ethylene glycol) [0904] pH potentia Hydrogenii [0905] PK pharmacokinetics [0906] Pr propyl [0907] PTH parathyroid hormone [0908] PyBOP benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate [0909] Q-TOF quadrupole time-of-flight [0910] RP-HPLC reversed-phase high performance liquid chromatography [0911] rt room temperature [0912] SIM single ion monitoring [0913] SEC size exclusion chromatography [0914] sc subcutaneous [0915] t.sub.1/2 half life [0916] TCP tritylchloride polystyrol [0917] TES triethylsilane [0918] TFA trifluoroacetic acid [0919] THF tetrahydrofuran [0920] Tmob 2,4,6-trimethoxybenzyl [0921] Trt triphenylmethyl, trityl [0922] ULOQ upper limit of quantification [0923] UPLC ultra performance liquid chromatography [0924] UV ultraviolet [0925] ZQ single quadrupole