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 method of treating a human patient having hypoparathyroidism comprising subcutaneously administering to the subject a PTH compound, wherein the pharmacokinetic profile of the PTH compound exhibits a peak to trough ratio of less than 4 within one administration interval at a steady state.

2. The method composition of claim 1, wherein the administration interval is at least 24 hours.

3. The method of claim 1, wherein the administration interval is 24 hours.

4. The method of claim 1, wherein the administration interval is one week.

5. The method of claim 1, wherein the subcutaneous administration is via subcutaneous injection.

6. The method of claim 1, wherein the subcutaneous administration occurs with a pen device.

7. The method of claim 1, wherein the peak to trough ratio is less than 3.

8. (canceled)

9. (canceled)

10. The method of claim 1, wherein the PTH compound is a stable PTH compound.

11. The method of claim 10, wherein the PTH moiety is either directly or through a spacer moiety covalently conjugated through a stable linkage to a water-soluble polymeric moiety.

12. The method of claim 10, wherein the PTH moiety is either directly or through a spacer moiety covalently conjugated through a stable linkage to an albumin-binding moiety.

13. The method of claim 1, wherein the PTH compound is a controlled-release PTH compound.

14. The method of claim 13, wherein the controlled-release PTH compound is water-insoluble.

15. The method of claim 13, wherein the controlled-release PTH compound is water-soluble.

16. (canceled)

17. The method of claim 1, wherein the PTH compound is administered as a pharmaceutical composition having a pH ranging from and including pH 3 to pH 8.

18. The method of of claim 18, wherein the pharmaceutical composition has a pH ranging from and including pH 4 to pH 5.

19. The method of claim 1, wherein the PTH compound is a water-soluble controlled-release PTH compound of formula (Ia) or a pharmaceutically acceptable salt thereof ##STR00126## wherein —D is a PTH moiety, which has the sequence of SEQ ID NO:51; —L.sup.2—L.sup.1— has the formula: ##STR00127## wherein the unmarked dashed line indicates the attachment to a nitrogen of —D by an amide bond; and the dashed line marked with an asterisk indicates attachment to —Z; —Z comprises a polyethylene glycol polymer of about 40 kDa; x is 1; and the PTH moiety is released with a release half-life of at least 12 hours.

20. The method of claim 19, wherein —L.sup.2—L.sup.1 is attached to the N-terminal amine functional group of —D.

21. The method of claim 20, wherein —Z comprises a branched polyethylene glycol polymer.

22. The method of claim 21, wherein —Z has one branching point.

Description

[0413] The term “alkyl” as used herein includes linear, branched or cyclic saturated hydrocarbon groups of 1 to 8 carbons, or in some embodiments 1 to 6 or 1 to 4 carbon atoms.

[0414] The term “alkoxy” includes alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy, and similar.

[0415] The term “alkenyl” includes non-aromatic unsaturated hydrocarbons with carbon-carbon double bonds.

[0416] The term “alkynyl” includes non-aromatic unsaturated hydrocarbons with carbon-carbon triple bonds.

[0417] The term “aryl” includes aromatic hydrocarbon groups of 6 to 18 carbons, preferably 6 to 10 carbons, including groups such as phenyl, naphthyl, and anthracenyl. The term “heteroaryl” includes aromatic rings comprising 3 to 15 carbons containing at least one N, O or S atom, preferably 3 to 7 carbons containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl, and similar.

[0418] In some instance, alkenyl, alkynyl, aryl or heteroaryl moieties may be coupled to the remainder of the molecule through an alkylene linkage. Under those circumstances, the substituent will be referred to as alkenylalkyl, alkynylalkyl, arylalkyl or heteroarylalkyl, indicating that an alkylene moiety is between the alkenyl, alkynyl, aryl or heteroaryl moiety and the molecule to which the alkenyl, alkynyl, aryl or heteroaryl is coupled.

[0419] The term “halogen” includes bromo, fluoro, chloro and iodo.

[0420] The term “heterocyclic ring” refers to a 4 to 8 membered aromatic or non-aromatic ring comprising 3 to 7 carbon atoms and at least one N, O, or S atom. Examples are piperidinyl, piperazinyl, tetrahydropyranyl, pyrrolidine, and tetrahydrofuranyl, as well as the exemplary groups provided for the term “heteroaryl” above.

[0421] When a ring system is optionally substituted, suitable substituents are selected from the group consisting of alkyl, alkenyl, alkynyl, or an additional ring, each optionally further substituted. Optional substituents on any group, including the above, include halo, nitro, cyano, —OR, —SR, —NR.sub.2, —OCOR, —NRCOR, —COOR, —CONR.sub.2, —SOR, —SO.sub.2R, —SONR.sub.2, —SO.sub.2N R.sub.2, wherein each R is independently alkyl, alkenyl, alkynyl, aryl or heteroaryl, or two R groups taken together with the atoms to which they are attached form a ring.

[0422] Preferably —L.sup.1— of formula (IV) is substituted with one moiety —L.sup.2—Z or —L.sup.2—Z′.

[0423] An additional preferred embodiment for —L.sup.1— is disclosed in WO2013/036857A1, which is herewith incorporated by reference in its entirety. Accordingly, a preferred moiety —L.sup.1— is of formula (V):

##STR00060##

wherein [0424] the dashed line indicates attachment to —D which is a PTH moiety and wherein attachment is through an amine functional group of —D; [0425] —R.sup.1 is selected from the group consisting of optionally substituted C.sub.1-C.sub.6 linear, branched, or cyclic alkyl; optionally substituted aryl; optionally substituted heteroaryl; alkoxy; and —NR.sup.5.sub.2; [0426] —R.sup.2 is selected from the group consisting of —H; optionally substituted C.sub.1-C.sub.6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl; [0427] —R.sup.3 is selected from the group consisting of —H; optionally substituted C.sub.1-C.sub.6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl; [0428] —R.sup.4 is selected from the group consisting of —H; optionally substituted C.sub.1-C.sub.6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl; [0429] each —R.sup.5 is independently of each other selected from the group consisting of —H; optionally substituted C.sub.1-C.sub.6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl; or when taken together two —R.sup.5 can be cycloalkyl or cyclohetero alkyl; [0430] wherein —L.sup.1— is substituted with —L.sup.2—Z or —L.sup.2—Z′ and wherein —L.sup.1— is optionally further substituted; [0431] wherein —L.sup.2— is a single chemical bond or a spacer; [0432] —Z is a water-soluble carrier; and [0433] —Z′ is a water-insoluble carrier.

[0434] Only in the context of formula (V) the terms used have the following meaning:

[0435] “Alkyl”, “alkenyl”, and “alkynyl” include linear, branched or cyclic hydrocarbon groups of 1-8 carbons or 1-6 carbons or 1-4 carbons wherein alkyl is a saturated hydrocarbon, alkenyl includes one or more carbon-carbon double bonds and alkynyl includes one or more carbon-carbon triple bonds. Unless otherwise specified these contain 1-6 C.

[0436] “Aryl” includes aromatic hydrocarbon groups of 6-18 carbons, preferably 6-10 carbons, including groups such as phenyl, naphthyl, and anthracene “Heteroaryl” includes aromatic rings comprising 3-15 carbons containing at least one N, O or S atom, preferably 3-7 carbons containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isoxazolyl, thiszolyl, isothiazolyl, quinolyl, indolyl, indenyl, and similar.

[0437] The term “substituted” means an alkyl, alkenyl, alkynyl, aryl, or heteroaryl group comprising one or more substituent groups in place of one or more hydrogen atoms. Substituents may generally be selected from halogen including F, Cl, Br, and I; lower alkyl including linear, branched, and cyclic; lower haloalkyl including fluoroalkyl, chloroalkyl, bromoalkyl, and iodoalkyl; OH; lower alkoxy including linear, branched, and cyclic; SH; lower alkylthio including linear, branched and cyclic; amino, alkylamino, dialkylamino, silyl including alkylsilyl, alkoxysilyl, and arylsilyl; nitro; cyano; carbonyl; carboxylic acid, carboxylic ester, carboxylic amide, aminocarbonyl; aminoacyl; carbamate; urea; thiocarbamate; thiourea; ketne; sulfone; sulfonamide; aryl including phenyl, naphthyl, and anthracenyl; heteroaryl including 5-member heteroaryls including as pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole, and tetrazole, 6-member heteroaryls including pyridine, pyrimidine, pyrazine, and fused heteroaryls including benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole, and benzisothiazole.

[0438] Preferably —L.sup.1— of formula (V) is substituted with one moiety —L.sup.2—Z or —L.sup.2—Z′.

[0439] A further preferred embodiment for —L.sup.1— is disclosed in US7585837B2, which is herewith incorporated by reference in its entirety. Accordingly, a preferred moiety —L.sup.1— is of formula (VI):

##STR00061##

wherein [0440] the dashed line indicates attachment to —D which is a PTH moiety and wherein attachment is through an amine functional group of —D; [0441] R.sup.1 and R.sup.2 are independently selected from the group consisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, —SO.sub.3H, —SO.sub.2NHR.sup.5, amino, ammonium, carboxyl, PO.sub.3H.sub.2, and OPO.sub.3H.sub.2; [0442] R.sup.3, R.sup.4, and R.sup.5 are independently selected from the group consisting of hydrogen, alkyl, and aryl; [0443] wherein —L.sup.1— is substituted with —L.sup.2—Z or —L.sup.2—Z′ and wherein —L.sup.1— is optionally further substituted; [0444] wherein —L.sup.2— is a single chemical bond or a spacer; [0445] —Z is a water-soluble carrier; and [0446] —Z′ is a water-insoluble carrier.

[0447] Suitable substituents for formulas (VI) are alkyl (such as C.sub.1-6 alkyl), alkenyl (such as C.sub.2-6 alkenyl), alkynyl (such as C.sub.2-6 alkynyl), aryl (such as phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl (such as aromatic 4 to 7 membered heterocycle) or halogen moieties.

[0448] Only in the context of formula (VI) the terms used have the following meaning:

[0449] The terms “alkyl”, “alkoxy”, “alkoxyalkyl”, “aryl”, “alkaryl” and “aralkyl” mean alkyl radicals of 1-8, preferably 1-4 carbon atoms, e.g. methyl, ethyl, propyl, isopropyl and butyl, and aryl radicals of 6-10 carbon atoms, e.g. phenyl and naphthyl. The term “halogen” includes bromo, fluoro, chloro and iodo.

[0450] Preferably —L.sup.1— of formula (VI) is substituted with one moiety —L.sup.2—Z or —L.sup.2—Z′.

[0451] A further preferred embodiment for —L.sup.1— is disclosed in WO2002/089789A1, which is herewith incorporated by reference in its entirety. Accordingly, a preferred moiety —L.sup.1— is of formula (VII):

##STR00062##

wherein [0452] the dashed line indicates attachment to —D which is a PTH moiety and wherein attachment is through an amine functional group of —D; [0453] L.sub.1 is a bifunctional linking group, [0454] Y.sub.1 and Y.sub.2 are independently O, S or NR.sup.7; [0455] R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6 and R.sup.7 are independently selected from the group consisting of hydrogen, C.sub.1-6 alkyls, C.sub.3-12 branched alkyls, C.sub.3-8 cycloalkyls, C.sub.1-6 substituted alkyls, C.sub.3-8 substituted cycloalkyls, aryls, substituted aryls, aralkyls, C.sub.1-6 heteroalkyls, substituted C.sub.1-6 heteroalkyls, C.sub.1-6 alkoxy, phenoxy, and C.sub.1-6 heteroalkoxy; [0456] Ar is a moiety which when included in formula (VII) forms a multisubstituted aromatic hydrocarbon or a multi-substituted heterocyclic group; [0457] X is a chemical bond or a moiety that is actively transported into a target cell, a hydrophobic moiety, or a combination thereof, [0458] y is 0 or 1; [0459] wherein —L.sup.1— is substituted with —L.sup.2—Z or —L.sup.2—Z′ and wherein —L.sup.1— is optionally further substituted; [0460] wherein —L.sup.2— is a single chemical bond or a spacer; [0461] —Z is a water-soluble carrier; and [0462] —Z′ is a water-insoluble carrier.

[0463] Only in the context of formula (VII) the terms used have the following meaning:

[0464] The term “alkyl” shall be understood to include, e.g. straight, branched, substituted C.sub.1-12 alkyls, including alkoxy, C.sub.3-8 cycloalkyls or substituted cycloalkyls, etc.

[0465] The term “substituted” shall be understood to include adding or replacing one or more atoms contained within a functional group or compounds with one or more different atoms.

[0466] Substituted alkyls include carboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls and mercaptoalkyls; substtued cycloalkyls include moieties such as 4-chlorocyclohexyl; aryls include moieties such as napthyl; substituted aryls include moieties such as 3-bromo-phenyl; aralkyls include moieties such as toluyl; hctcroalkyls include moieties such as cthylthiophcnc; substituted heteroalkyls include moieties such as 3-methoxythiophone; alkoxy includes moieities such as methoxy; and phenoxy includes moieties such as 3-nitrophenoxy. Halo-shall be understood to include fluoro, chloro, iodo and bromo.

[0467] Preferably —L.sup.1— of formula (VII) is substituted with one moiety —L.sup.2—Z or —L.sup.2—Z′.

[0468] In another preferred embodiment —L.sup.1— comprises a substructure of formula (VIII)

##STR00063##

wherein [0469] the dashed line marked with the asterisk indicates attachment to a nitrogen of —D which is a PTH moiety by forming an amide bond; [0470] the unmarked dashed lines indicate attachment to the remainder of —L.sup.1—; and [0471] wherein —L.sup.1— is substituted with —L.sup.2—Z or —L.sup.2—Z′ and wherein —L.sup.1— is optionally further substituted; [0472] wherein —L.sup.2— is a single chemical bond or a spacer; [0473] —Z is a water-soluble carrier; and [0474] —Z′ is a water-insoluble carrier.

[0475] Preferably —L.sup.1— of formula (VIII) is substituted with one moiety —L.sup.2—Z or —L.sup.2—Z′.

[0476] In one embodiment —L— of formula (VIII) is not further substituted.

[0477] In another preferred embodiment —L.sup.1— comprises a substructure of formula (IX)

##STR00064##

wherein [0478] the dashed line marked with the asterisk indicates attachment to a nitrogen of —D which is a PTH moiety by forming a carbamate bond; [0479] the unmarked dashed lines indicate attachment to the remainder of —L.sup.1—; and [0480] wherein —L.sup.1— is substituted with —L.sup.2—Z or —L.sup.2—Z′ and wherein —L.sup.1— is optionally further substituted; [0481] wherein —L.sup.2— is a single chemical bond or a spacer; [0482] —Z is a water-soluble carrier; and [0483] —Z′ is a water-insoluble carrier.

[0484] Preferably —L.sup.1— of formula (IX) is substituted with one moiety —L.sup.2—Z or —L.sup.2—Z′.

[0485] In one embodiment —L.sup.1— of formula (IX) is not further substituted.

[0486] In the prodrugs of the present invention —L.sup.2— is a chemical bond or a spacer moiety.

[0487] In one embodiment —L.sup.2— is a chemical bond.

[0488] In another embodiment —L.sup.2— is a spacer moiety.

[0489] When —L.sup.2— is other than a single chemical bond, —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.yl)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)—;

[0490] —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.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 arc 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.y1)—, —OC(OR.sup.y4)(R.sup.y4a)—, —N(R.sup.y4)C(O)N(R.sup.y4a)—, and —OC(O)N(R.sup.y4)—; [0491] 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.y2, which are the same or different; [0492] 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.y5, —S(O)R.sup.y5, —N(R.sup.y5)S(O).sub.2N(R.sup.y5aR.sup.y5b), —SR.sup.y5, —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 [0493] each —R.sup.y3, —R.sup.y3a, —R.sup.y4, —R.sup.y4a, —R.sup.y5, —R.sup.y5a and —R.sup.y5b is independently selected from the group consisting of —H, 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.

[0494] When —L.sup.2— is other than a single chemical bond, —L.sup.2— is even more preferably selected from —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.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 alkynyl are optionally substituted with one or more —R.sup.y2, which are the same or different and wherein C.sub.1-20 alkyl, C.sub.2-20 alkenyl, and C.sub.2-20 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)—; [0495] —R.sup.y1 and —R.sup.y1a are independently of each other selected from the group consisting of —H, —T, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl; wherein —T, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl arc optionally substituted with one or more —R.sup.y2, which arc the same or different, and wherein C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 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.y1)—, —OC(OR.sup.y4)(R.sup.y4a)—, —N(R.sup.y4)C(O)N(R.sup.y4a)—, and —OC(O)N(R.sup.y4)—; [0496] 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.y2, which are the same or different; [0497] —R.sup.y2 is 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.y5, —S(O)R.sup.y5, —N(R.sup.y5)S(O).sub.2N(R.sup.y5aR.sup.y5b), —SR.sup.y5, —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 [0498] each —R.sup.y3, —R.sup.y3a, —R.sup.y4, —R.sup.y4a, —R.sup.y5, —R.sup.y5a and —R.sup.y5b is independently of each other selected from the group consisting of —H, 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.

[0499] When —L.sup.2— is other than a single chemical bond, —L.sup.2— is even more 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)—; [0500] —R.sup.y1 and —R.sup.y1a arc independently selected from the group consisting of —H, —T, C.sub.1-10 alkyl, C.sub.2-10 alkenyl, and C.sub.2-10 alkynyl; [0501] 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; [0502] each —R.sup.y2 is independently selected from the group consisting of halogen, and C.sub.1-6 alkyl; and [0503] each —R.sup.y3, —R.sup.y3a, —R.sup.y4, —R.sup.y4a, —R.sup.y5, —R.sup.y5a and —R.sup.y5b is independently of each other selected from the group consisting of —H, 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.

[0504] Even more preferably, —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 1 0-membered heterocyclyl, 8- to 11-membered heterobicyclyl, 8-to 30-membered carbopolycyclyl, and 8- to 30-membered heteropolycyclyl.

[0505] Preferably, —L.sup.2— has a molecular weight in the range of from 14 g/mol to 750 g/mol.

[0506] Preferably, —L.sup.2— comprises a moiety selected from

##STR00065##

##STR00066##

##STR00067##

##STR00068##

##STR00069##

##STR00070##

##STR00071##

##STR00072##

##STR00073##

##STR00074##

##STR00075##

##STR00076##

##STR00077##

##STR00078##

##STR00079##

##STR00080##

##STR00081##

##STR00082##

##STR00083##

##STR00084##

##STR00085##

##STR00086##

wherein [0507] dashed lines indicate attachment to the rest of —L.sup.2—, —L.sup.1—, —Z and/or —Z′, respectively; and [0508] —R and —R.sup.a are independently of each other selected from the group consisting of —H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

[0509] In one preferred embodiment —L.sup.2— has a chain lengths of 1 to 20 atoms.

[0510] As used herein the term “chain length” with regard to the moiety —L.sup.2— refers to the number of atoms of —L.sup.2— present in the shortest connection between —L.sup.1— and —Z.

[0511] Preferably, —L.sup.2— is of formula (i)

##STR00087##

wherein [0512] the dashed line marked with the asterisk indicates attachment to —L.sup.1—; [0513] the unmarked dashed line indicates attachment to —Z or —Z′; [0514] n is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18; and [0515] wherein the moiety of formula (i) is optionally further substituted.

[0516] Preferably, n of formula (i) is selected from the group consisting of 3, 4, 5, 6, 7, 8, and 9. Even more preferably n of formula (i) is 4, 5, 6, or 7. In one embodiment n of formula (i) is 4. In another embodiment n of formula (i) is 5. In another embodiment n of formula (i) is 6.

[0517] In one preferred embodiment the moiety —L1—L2— is selected from the group consisting of

##STR00088##

##STR00089##

##STR00090##

wherein [0518] the unmarked dashed line indicates the attachment to a nitrogen of —D which is a PTH moiety by forming an amide bond; and [0519] the dashed line marked with the asterisk indicates attachment to —Z or —Z′.

[0520] In another preferred embodiment the moiety —L.sup.1—L.sup.2— is selected from the group consisting of

##STR00091##

##STR00092##

##STR00093##

wherein [0521] the unmarked dashed line indicates the attachment to a nitrogen of —D which is a PTH moiety by forming an amide bond; and [0522] the dashed line marked with the asterisk indicates attachment to —Z or —Z′.

[0523] In a preferred embodiment the moiety —L.sup.1—L.sup.2— is of formula (IIca-ii).

[0524] In another preferred embodiment the moiety —L.sup.1—L.sup.2— is of formula (IIcb-iii).

[0525] Preferably, the controlled-release PTH compound of the present invention is of formula (Ia) with x = 1.

[0526] The carrier -Z comprises a C.sub.8-24 alkyl or a polymer. Preferably, -Z comprises a polymer, preferably a polymer selected from the group consisting of 2-methacryloyl-oxyethyl phosphoyl cholins, poly(acrylic acids), poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers, poly(amidcs), poly(amidoamincs), poly(amino acids), poly(anhydridcs), 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.

[0527] Preferably, —Z has a molecular weight ranging from 5 to 200 kDa. Even more preferably, —Z has a molecular weight ranging from 8 to 100 kDa, even more preferably ranging from 10 to 80 kDa, even more preferably from 12 to 60, even more preferably from 15 to 40 and most preferably —Z has a molecular weight of about 20 kDa. In another equally preferred embodiment —Z has a molecular weight of about 40 kDa.

[0528] In one embodiment such water-soluble carrier —Z comprises a protein. Preferred proteins are selected from the group consisting of carboxyl-terminal polypeptide of the chorionic gonadotropin as described in US 2012/0035101 A1 which are herewith incorporated by reference; albumin; XTEN sequences as described in WO 2011123813 A2 which are herewith incorporated by reference; proline/alanine random coil sequences as described in WO 2011/144756 A1 which are herewith incorporated by reference; proline/alanine/serine random coil sequences as described in WO 2008/155134 A1 and WO 2013/024049 A1 which are herewith incorporated by reference; and Fc fusion proteins.

[0529] In one embodiment —Z is a polysarcosine.

[0530] In another preferred embodiment —Z comprises a poly(N-mcthylglycinc).

[0531] In a particularly preferred embodiment —Z comprises a random coil protein moiety.

[0532] In one preferred embodiment —Z comprises one random coil protein moiety.

[0533] In another preferred embodiment —Z comprises two random coil proteins moieties.

[0534] In another preferred embodiment —Z comprises three random coil proteins moieties.

[0535] In another preferred embodiment —Z comprises four random coil proteins moieties.

[0536] In another preferred embodiment —Z comprises five random coil proteins moieties.

[0537] In another preferred embodiment —Z comprises six random coil proteins moieties.

[0538] In another preferred embodiment —Z comprises seven random coil proteins moieties.

[0539] In another preferred embodiment —Z comprises eight random coil proteins moieties.

[0540] Preferably such random coil protein moiety comprises at least 25 amino acid residues and at most 2000 amino acids. Even more preferably such random coil protein moiety comprises at least 30 amino acid residues and at most 1500 amino acid residues. Even more preferably such random coil protein moiety comprises at least 50 amino acid residues and at most 500 amino acid residues.

[0541] In a preferred embodiment, —Z comprises a random coil protein moiety of which at least 80%, preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% of the total number of amino acids forming said random coil protein moiety are selected from alanine and proline. Even more preferably, at least 10%, but less than 75%, preferably less than 65%, of the total number of amino acid residues of such random coil protein moiety are proline residues. Preferably, such random coil protein moiety is as described in WO 2011/144756 A1 which is hereby incorporated by reference in its entirety. Even more preferably —Z comprises at least one moiety selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:51 and SEQ ID NO:61 as disclosed in WO2011/144756 which are hereby incorporated by reference. A moiety comprising such random coil protein comprising alanine and proline will be referred to as “PA” or “PA moiety”.

[0542] Accordingly, —Z comprises a PA moiety.

[0543] In an equally preferred embodiment, —Z comprises a random coil protein moiety of which at least 80%, preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% of the total number of amino acids forming said random coil protein moiety are selected from alanine, serine and proline. Even more preferably, at least 4%, but less than 40% of the total number of amino acid residues of such random coil protein moiety are proline residues. Preferably, such random coil protein moiety is as described in WO 2008/155134 A1 which is hereby incorporated by reference in its entirety. Even more preferably —Z comprises at least one moiety selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:56 as disclosed in WO 2008/155134 A1, which are hereby incorporated by reference. A moiety comprising such random coil protein moiety comprising alanine, serine and proline will be referred to as “PAS” or “PAS moiety”.

[0544] Accordingly, —Z comprises a PAS moiety.

[0545] In an equally preferred embodiment, —Z comprises a random coil protein moiety of which at least 80%, preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% of the total number of amino acids forming said random coil protein moiety are selected from alanine, glycine and proline. A moiety comprising such random coil protein moiety comprising alanine, glycine and proline will be referred to as “PAG” or “PAG moiety”.

[0546] Accordingly, —Z comprises a PAG moiety.

[0547] In an equally preferred embodiment, —Z comprises a random coil protein moiety of which at least 80%, preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% of the total number of amino acids forming said random coil protein moiety are selected from proline and glycine. A moiety comprising such random coil protein moiety comprising proline and glycine will be referred to as “PG” or “PG moiety”.

[0548] Preferably, such PG moiety comprises a moiety of formula (a-0)

##STR00094##

wherein [0549] p is selected from the group consisting of 0, 1, 2, 3, 4 and 5; [0550] q is selected from the group consisting of 0, 1, 2, 3, 4 and 5; [0551] r is an integer ranging from and including 10 to 1000; [0552] provided that at least one of p and q is at least 1;

[0553] Preferably, p of formula (a-0) is selected from the group consisting of 1, 2 and 3.

[0554] Preferably, q of formula (a-0) is selected from 0, 1 and 2.

[0555] Even more preferably the PG moiety comprises the sequence of SEQ ID NO:122: GGPGGPGPGGPGGPGPGGPG

[0556] Even more preferably, the PG moiety comprises the sequence of formula (a-0-a) (GGPGGPGPGGPGGPGPGGPG).sub.v (a-0-a), wherein v is an integer ranging from and including 1 to 50.

[0557] It is understood that the sequence of formula (a-0-a) comprises v replicates of the sequence of SEQ ID NO:122.

[0558] Accordingly, —Z comprises a PG moiety.

[0559] In an equally preferred embodiment, —Z comprises a random coil protein moiety of which at least 80%, preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% of the total number of amino acids forming said random coil protein moiety are selected from alanine, glycine, serine, threonine, glutamate and proline. Preferably, such random coil protein moiety is as described in WO 2010/091122 A1 which is hereby incorporated by reference. Even more preferably —Z comprises at least one moiety selected from the group consisting of SEQ ID NO:182, SEQ ID NO:183, SEQ ID NO:184; SEQ ID NO:185, SEQ ID NO:186, SEQ ID NO:187, SEQ ID NO:188, SEQ ID NO:189, SEQ ID NO:190, SEQ ID NO:191, SEQ ID NO:192, SEQ ID NO:193, SEQ ID NO:194, SEQ ID NO:195, SEQ ID NO:196, SEQ ID NO:197, SEQ ID NO:198, SEQ ID NO:199, SEQ ID NO:200, SEQ ID NO:201, SEQ ID NO:202, SEQ ID NO:203, SEQ ID NO:204, SEQ ID NO:205, SEQ ID NO:206, SEQ ID NO:207, SEQ ID NO:208, SEQ ID NO:209, SEQ ID NO:210, SEQ ID NO:211, SEQ ID NO:212, SEQ ID NO:213, SEQ ID NO:214, SEQ ID NO:215, SEQ ID NO:216, SEQ ID NO:217, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:221, SEQ ID NO:759, SEQ ID NO:760, SEQ ID NO:761, SEQ ID NO:762, SEQ ID NO:763, SEQ ID NO:764, SEQ ID NO:765, SEQ ID NO:766, SEQ ID NO:767, SEQ ID NO:768, SEQ ID NO:769, SEQ ID NO:770, SEQ ID NO:771, SEQ ID NO:772, SEQ ID NO:773, SEQ ID NO:774, SEQ ID NO:775, SEQ ID NO:776, SEQ ID NO:777, SEQ ID NO:778, SEQ ID NO:779, SEQ ID NO:1715, SEQ ID NO:1716, SEQ ID NO:1718, SEQ ID NO:1719, SEQ ID NO:1720, SEQ ID NO:1721 and SEQ ID NO:1722 as disclosed in WO2010/091122A1, which are hereby incorporated by reference. A moiety comprising such random coil protein moiety comprising alanine, glycine, serine, threonine, glutamate and proline will be referred to as “XTEN” or “XTEN moiety” in line with its designation in WO 2010/091122 A1.

[0560] Accordingly, —Z comprises an XTEN moiety.

[0561] In another preferred embodiment, —Z comprises a fatty acid derivate. Preferred fatty acid derivatives are those disclosed in WO 2005/027978 A2 and WO 2014/060512 A1 which are herewith incorporated by reference.

[0562] In another preferred embodiment —Z is a hyaluronic acid-based polymer.

[0563] In one embodiment —Z is a carrier as disclosed in WO 2012/02047 A1 which is herewith incorporated by reference.

[0564] In another embodiment —Z is a carrier as disclosed in WO 2013/024048 A1 which is herewith incorporated by reference.

[0565] In another preferred embodiment —Z is a PEG-based polymer, such as a linear, branched or multi-arm PEG-based polymer.

[0566] In one embodiment —Z is a linear PEG-based polymer.

[0567] In another embodiment —Z is a multi-arm PEG-based polymer. Preferably, —Z is a multi-arm PEG-based polymer having at least 4 PEG-based arms.

[0568] Preferably, such multi-arm PEG-based polymer —Z is connected to a multitude of moieties —L.sup.2—L.sup.1—D, wherein each moiety —L.sup.2—L.sup.1—D is preferably connected to the end of an arm, preferably to the end of an arm. Preferably such multi-arm PEG-based polymer —Z is connected to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 moieties —L.sup.2—L.sup.1—D. Even more preferably such multi-arm PEG-based polymer —Z is connected to 2, 3, 4, 6 or 8 moieties —L.sup.2—L.sup.1—D. Even more preferably such multi-arm PEG-based polymer —Z is connected to 2, 4 or 6 moieties —L.sup.2—L.sup.1—D, even more preferably such multi-arm PEG-based polymer —Z is connected to 4 or 6 moieties —L.sup.2—L.sup.1—D, and most preferably such multi-arm PEG-based polymer —Z is connected to 4 moieties —L.sup.2—L.sup.1—D.

[0569] Preferably, such multi-arm PEG-based polymer -Z is a multi-arm PEG derivative as, for instance, detailed in the products list of JenKem Technology, USA (accessed by download from http://www.jenkemusa.com/Pages/PEGProducts.aspx on Dec. 18, 2014), such as a 4-arm-PEG derivative, in particular a 4-arm-PEG comprising a pentaerythritol core, an 8-arm-PEG derivative comprising a hexaglycerin core, and an 8-arm-PEG derivative comprising a tripentaerythritol core. More preferably, the water-soluble PEG-based carrier -Z comprises a moiety selected from: [0570] a 4-arm PEG Amine comprising a pentaerythritol core: with n ranging from 20 to 500; [0571] an 8-arm PEG Amine comprising a hcxaglyccrin core: with n ranging from 20 to 500; and [0572] R = hexaglycerin or tripentaerythritol core structure; and [0573] a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core: with n ranging from 20 to 500; and [0574] R = comprising a sorbitol or dipentaerythritol core; [0575] and wherein dashed lines indicate attachment to the rest of the PTH prodrug.

[0576] In a preferred embodiment —Z is a branched PEG-based polymer. In one embodiment —Z is a branched PEG-based polymer having one, two, three, four, five or six branching points. Preferably, —Z is a branched PEG-based polymer having one, two or three branching points. In one embodiment —Z is a branched PEG-based polymer having one branching point. In another embodiment —Z is a branched PEG-based polymer having two branching points. In another embodiment —Z is a branched PEG-based polymer having three branching points.

[0577] A branching point is preferably selected from the group consisting of —N<, —CH< and >C<.

[0578] Preferably, such branched PEG-based moiety —Z has a molecular weight of at least 10 kDa.

[0579] In one embodiment such branched moiety —Z has a molecular weight ranging from and including 10 kDa to 500 kDa, more preferably ranging from and including 10 kDa to 250 Da, even more preferably ranging from and including 10 kDa to 150 kDa, even more preferably ranging from and including 12 kDa to 100 kDa and most preferably ranging from and including 15 kDa to 80 kDa.

[0580] Preferably, such branched moiety —Z has a molecular weight ranging from and including 10 kDa to 80 kDa. In one embodiment the molecular weight is about 10 kDa. In another embodiment the molecular weight of such branched moiety —Z is about 20 kDa. In another embodiment the molecular weight of such branched moiety —Z is about 30 kDa. In another embodiment the molecular weight of such a branched moiety —Z is about 40 kDa. In another embodiment the molecular weight of such a branched moiety —Z is about 50 kDa. In another embodiment the molecular weight of such a branched moiety —Z is about 60 kDa. In another embodiment the molecular weight of such a branched moiety —Z is about 70 kDa. In another embodiment the molecular weight of such a branched moiety —Z is about 80 kDa. Most preferably, such branched moiety —Z has a molecular weight of about 40 kDa.

[0581] Preferably, —Z or —Z′ comprises a moiety

##STR00098##

In an equally preferred embodiment —Z comprises an amide bond.

[0582] Preferably —Z comprises a moiety of formula (a)

##STR00099##

wherein [0583] the dashed line indicates attachment to —L.sup.2— or to the remainder of —Z; [0584] BP.sup.a is a branching point selected from the group consisting of —N<, —CR< and >C<; [0585] —R is selected from the group consisting of —H and C.sub.1-6 alkyl; [0586] a is 0 if BP.sup.a is —N< or —CR< and n is 1 if BP.sup.a is >C<; [0587] —S.sup.a—, —S.sup.a′—, —S.sup.a″— and —S.sup.a‴— are independently of each other a chemical bond or are selected from the group consisting of C.sub.1-50 alkyl, C.sub.2-50 alkenyl, and C.sub.2-50 alkynyl; [0588] wherein 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.1, 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.2)—, —S(O).sub.2N(R.sup.2)—, —S(O)N(R.sup.2)—, —S(O).sub.2—, —S(O)—, —N(R.sup.2)S(O).sub.2N(R.sup.2a)—, —S—, —N(R.sup.2)—, —OC(OR.sup.2)(R.sup.2a)—, —N(R.sup.2)C(O)N(R.sup.2a)—, and —OC(O)N(R.sup.2)—; [0589] 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.1, which are the same or different; [0590] each —R.sup.1 is independently selected from the group consisting of halogen, —CN, oxo (═O), —COOR.sup.3, —OR.sup.3, —C(O)R.sup.3, —C(O)N(R.sup.3R.sup.3a), —S(O).sub.2N(R.sup.3R.sup.3a), —S(O)N(R.sup.3R.sup.3a), —S(O).sub.2R.sup.3, —S(O)R.sup.3, —N(R.sup.3)S(O).sub.2N(R.sup.3aR.sup.3b), —SR.sup.3, —N(R.sup.3R.sup.3a), —NO.sub.2, —OC(O)R.sup.3, —N(R.sup.3)C(O)R.sup.3a, —N(R.sub.3)S(O).sub.2R.sup.3a, —N(R.sub.3)S(O)R.sup.3a, —N(R.sup.3)C(O)OR.sup.3a, —N(R.sup.3)C(O)N(R.sup.3aR.sup.3b), —OC(O)N(R.sup.3R.sup.3a), 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; [0591] each —R.sup.2, —R.sup.2a, —R.sup.3, —R.sup.3a and —R.sup.3b is independently selected from the group consisting of —H, 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 —P.sup.a′ , —P.sup.a″ and —P.sup.a‴ are independently a polymeric moiety.

[0592] In one embodiment BP.sup.a of formula (a) is —N<.

[0593] In another embodiment BP.sup.a of formula (a) is >C<.

[0594] In a preferred embodiment BP.sup.a of formula (a) is —CR<. Preferably, —R is —H. Accordingly, a of formula (a) is 0.

[0595] In one embodiment —S.sup.a— of formula (a) is a chemical bond.

[0596] In another embodiment —S.sup.a— of formula (a) is selected from the group consisting of C.sub.1-10 alkyl, C.sub.2-10 alkenyl and C.sub.2-10 alkynyl, which C.sub.1-10 alkyl, C.sub.2-10 alkenyl and C.sub.2-10 alkynyl arc optionally interrupted by one or more chemical groups selected from the group consisting of —T—, —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.4)—, —S(O).sub.2N(R.sup.4)—, —S(O)N(R.sup.4)—, —S(O).sub.2—, —S(O)—, —N(R.sup.4)S(O).sub.2N(R.sup.4a)—, —S—, —N(R.sup.4)—, —OC(OR.sup.4)(R.sup.4a)—, —N(R.sup.4)C(O)N(R.sup.4a)—, and —OC(O)N(R.sup.4)—; wherein —T— is a 3- to 10-membered heterocyclyl; and —R.sup.4 and —R.sup.4a are independently selected from the group consisting of —H, methyl, ethyl, propyl and butyl.

[0597] Preferably —S.sup.a— of formula (a) is selected from the group consisting of C.sub.1-10 alkyl which is interrupted by one or more chemical groups selected from the group consisting of —T—, —C(O)N(R.sup.4)— and —O—.

[0598] In one embodiment —S.sup.a′— of formula (a) is a chemical bond.

[0599] In another embodiment —S.sup.a′— of formula (a) is selected from the group consisting of C.sub.1-10 alkyl, C.sub.2-10 alkenyl and C.sub.2-10 alkynyl, which C.sub.1-10 alkyl, C.sub.2-10 alkenyl and C.sub.2-10 alkynyl are optionally interrupted by one or more chemical groups selected from the group consisting of —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.4)—, —S(O).sub.2N(R.sup.4)—, —S(O)N(R.sup.4)—, —S(O).sub.2—, —S(O)—, —N(R.sup.4)S(O).sub.2N(R.sup.4a)—, —S—, —N(R.sup.4)—, —OC(OR.sup.4)(R.sup.4a)—, —N(R.sup.4)C(O)N(R.sup.4a)—, and —OC(O)N(R.sup.4)—; wherein —R.sup.4 and —R.sup.4a are independently selected from the group consisting of —H, methyl, ethyl, propyl and butyl. Preferably —S.sup.a′— of formula (a) is selected from the group consisting of methyl, ethyl, propyl, butyl, which are optionally interrupted by one or more chemical groups selected from the group consisting of —O—, —C(O)— and —C(O)N(R.sup.4)—.

[0600] In one embodiment —S.sup.a″— of formula (a) is a chemical bond.

[0601] In another embodiment —S.sup.a″— of formula (a) is selected from the group consisting of C.sub.1-10 alkyl, C.sub.2-10 alkenyl and C.sub.2-10 alkynyl, which C.sub.1-10 alkyl, C.sub.2-1O alkenyl and C.sub.2-10 alkynyl are optionally interrupted by one or more chemical groups selected from the group consisting of —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.4)—, —S(O).sub.2N(R.sup.4)—, —S(O)N(R.sup.4)—,—S(O).sub.2—, —S(O)—, —N(R.sup.4)S(O).sub.2N(R.sup.4a)—, —S—, —N(R.sup.4)—, —OC(OR.sup.4)(R.sup.4a)—, —N(R.sup.4)C(O)N(R.sup.4a)—, and —OC(O)N(R.sup.4)—; wherein —R.sup.4 and —R.sup.4a are independently selected from the group consisting of —H, methyl, ethyl, propyl and butyl. Preferably —S.sup.a″—of formula (a) is selected from the group consisting of methyl, ethyl, propyl, butyl, which are optionally interrupted by one or more chemical groups selected from the group consisting of —O—, —C(O)— and —C(O)N(R.sup.4)—.

[0602] In one embodiment —S.sup.a‴—of formula (a) is a chemical bond.

[0603] In another embodiment —S.sup.a‴— of formula (a) is selected from the group consisting of C.sub.1-10 alkyl, C.sub.2-10 alkenyl and C.sub.2-10 alkynyl, which C.sub.1-10 alkyl, C.sub.2-1O alkenyl and C.sub.2-10 alkynyl are optionally interrupted by one or more chemical groups selected from the group consisting of —C(O)O—, —O—, —C(O)—, —C(O)N(R.sup.4)—, —S(O).sub.2N(R.sup.4)—, —S(O)N(R.sup.4)—,—S(O).sub.2—, —S(O)—, —N(R.sup.4)S(O).sub.2N(R.sup.4a)—, —S—, —N(R.sup.4)—, —OC(OR.sup.4)(R.sup.4a)—, —N(R.sup.4)C(O)N(R.sup.4a)—, and —OC(O)N(R.sup.4)—; wherein —R.sup.4 and —R.sup.4a are independently selected from the group consisting of —H, methyl, ethyl, propyl and butyl. Preferably —S.sup.a‴— of formula (a) is selected from the group consisting of methyl, ethyl, propyl, butyl, which are optionally interrupted by one or more chemical groups selected from the group consisting of —O—, —C(O)— and —C(O)N(R.sup.4)—.

[0604] Preferably, —P.sup.a′, —P.sup.a″ and —P.sup.a‴ of formula (a) independently comprise 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.

[0605] More preferably, —P.sup.a′, —P.sup.a″ and —P.sup.a‴ of formula (a) independently comprise a PEG-based moiety. Even more preferably, —P.sup.a′, —P.sup.a″ and —P.sup.a‴ of formula (a) independently comprise a PEG-bascd moiety comprising at least 20% PEG, even more preferably at least 30%, even more preferably at least 40% PEG, even more preferably at least 50% PEG, even more preferably at least 60% PEG, even more preferably at least 70% PEG, even more preferably at least 80% PEG and most preferably at least 90% PEG.

[0606] Preferably, —P.sup.a′, —P.sup.a″ and —P.sup.a‴ of formula (a) independently have a molecular weight ranging from and including 5 kDa to 50 kDa, more preferably have a molecular weight ranging from and including 5 kDa to 40 kDa, even more preferably ranging from and including 7.5 kDa to 35 kDa, even more preferably ranging from and 7.5 to 30 kDa, even more preferably ranging from and including 10 to 30 kDa.

[0607] In one embodiment —P.sup.a′, —P.sup.a″ and —P.sup.a‴ of formula (a) have a molecular weight of about 5 kDa.

[0608] In another embodiment —P.sup.a′, —P.sup.a″ and —P.sup.a‴ of formula (a) have a molecular weight of about 7.5 kDa.

[0609] In another embodiment —P.sup.a′, —P.sup.a″ and —P.sup.a‴ of formula (a) have a molecular weight of about 10 kDa.

[0610] In another embodiment —P.sup.a′, —P.sup.a″ and —P.sup.a‴ of formula (a) have a molecular weight of about 12.5 kDa.

[0611] In another embodiment —P.sup.a′, —P.sup.a″ and —P.sup.a‴ of formula (a) have a molecular weight of about 15 kDa.

[0612] In another embodiment —P.sup.a′, —P.sup.a″ and —P.sup.a‴ of formula (a) have a molecular weight of about 20 kDa.

[0613] In one embodiment —Z comprises one moiety of formula (a).

[0614] In another embodiment —Z comprises two moieties of formula (a).

[0615] In another embodiment —Z comprises three moieties of formula (a).

[0616] Preferably, —Z is a moiety of formula (a).

[0617] More preferably, —Z comprises a moiety of formula (b)

##STR00100##

wherein [0618] the dashed line indicates attachment to —L.sup.2— or to the remainder of —Z; and [0619] m and p are independently of each other an integer ranging from and including 150 to 1000; preferably an integer ranging from and including 150 to 500; more preferably an integer ranging from and including 200 to 500; and most preferably an integer ranging from and including 400 to 500.

[0620] Preferably, m and p of formula (b) are the same integer.

[0621] Most preferably m and p of formula (b) are about 450.

[0622] Preferably, —Z is a moiety of formula (b).

[0623] The carrier —Z′ is a water-insoluble polymer, even more preferably a hydrogel. Preferably, such 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(vinylmcthylcthcrs), poly(vinylpyrrolidoncs), silicones, celluloses, carbomcthyl 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.

[0624] If the carrier —Z′ is a hydrogel, it is preferably a hydrogel comprising PEG or hyaluronic acid. Most preferably such hydrogel comprises PEG.

[0625] Even more preferably, the carrier —Z′ is a hydrogel as described in WO 2006/003014 A2, WO 2011/012715 A1 or WO 2014/056926 A1, which are herewith incorporated by reference in their entirety.

[0626] In another embodiment —Z′ is a polymer network formed through the physical aggregation of polymer chains, which physical aggregation is preferably caused by hydrogen bonds, crystallization, helix formation or complexation. In one embodiment such polymer network is a thermogelling polymer.

[0627] If the controlled-release PTH compound of the present invention is a prodrug, its total mass is preferably at least 10 kDa, such as at least 12 kDa, such as at least 15 kDa, such as at least 20 kDa or such as at least 30 kDa. If the controlled-release PTH compound is a water-soluble prodrug, its total mass preferably is at most 250 kDa, such as at most 200 kDa, 180 kDa, 150 kDa or 100 kDa. It is understood that no meaningful upper molecular weight limit can be provided in case the controlled-release PTH compound is water-insoluble.

[0628] In one preferred embodiment the controlled-release PTH compound is of formula (IIe-i):

##STR00101##

wherein [0629] the unmarked dashed line indicates the attachment to a nitrogen of —D which is a PTH moiety by forming an amide bond; and [0630] the dashed line marked with the asterisk indicates attachment to a moiety [0631] wherein m and p are independently an integer ranging from and including 400 to 500.

[0632] Preferably, —D is attached to the PTH prodrug of formula (IIe-i) through the N-terminal amine functional group of the PTH moiety.

[0633] In another preferred embodiment the PTH prodrug of the present invention is of formula (IIf-i):

##STR00103##

wherein [0634] the unmarked dashed line indicates the attachment to a nitrogen of —D which is a PTH moiety by forming an amide bond; and [0635] the dashed line marked with the asterisk indicates attachment to a moiety [0636] wherein m and p are independently an integer ranging from and including 400 to 500.

[0637] Preferably, —D is attached to the PTH prodrug of formula (IIf-i) through the N-terminal amine functional group of the PTH moiety.

[0638] In a preferred embodiment the residual activity of the controlled-release PTH in the form of a PTH prodrug is less than 10%, more preferably less than 1%, even more preferably less than 0.1%, even more preferably less than 0.01%, even more preferably less than 0.001% and most preferably less than 0.0001%.

[0639] As used herein the term “residual activity” refers to the activity exhibited by the PTH prodrug with the PTH moiety bound to a carrier in relation to the activity exhibited by the corresponding free PTH. In this context the term “activity” refers to binding to an activation domain of the PTH/PTHrP1 receptor resulting in activation of adenylate cyclase to generate cAMP, phospholipase C to generate intracellular calcium, or osteoblastic expression of RANKL (which binds to RANK (Receptor Activator of Nuclear Factor kB) on osteoclasts. It is understood that measuring the residual activity of the PTH prodrug of the present invention takes time during which a certain amount of PTH may be released from the PTH prodrug of the present invention and that such released PTH will distort the results measured for the PTH prodrug. It is thus accepted practice to test the residual activity of a prodrug with a conjugate in which the drug moiety, in this case PTH, is non-reversibly, i.e. stably, bound to a carrier, which as closely as possible resembles the structure of the PTH prodrug for which residual activity is to be measured.

[0640] Preferably, the pharmaceutical composition of the present invention has a pH ranging from and including pH 3 to pH 8. More preferably, the pharmaceutical composition has a pH ranging from and including pH 4 to pH 6. Most preferably, the pharmaceutical composition has a pH ranging from and including pH 4 to pH 5.

[0641] In one embodiment the pharmaceutical composition of the present invention is a liquid or suspension formulation. It is understood that the pharmaceutical composition is a suspension formulation if the controlled-release PTH compound of the present invention is water-insoluble.

[0642] In another embodiment the pharmaceutical composition of the present invention is a dry formulation which is reconstituted before administration to a patient.

[0643] Such liquid, suspension, dry or reconstituted pharmaceutical composition comprises at least one excipient. Excipients used in parenteral formulations may be categorized as, for example, buffering agents, isotonicity modifiers, preservatives, stabilizers, anti-adsorption agents, oxidation protection agents, viscosifiers/viscosity enhancing agents, or other auxiliary agents. However, in some cases, one excipient may have dual or triple functions. Preferably, the at least one excipient comprised in the pharmaceutical composition of the present invention is selected from the group consisting of [0644] (i) Buffering agents: physiologically tolerated buffers to maintain pH in a desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulphate, nitrate, chloride, pyruvate; antacids such as Mg(OH).sub.2 or ZnCO.sub.3 may be also used; [0645] (ii) Isotonicity modifiers: to minimize pain that can result from cell damage due to osmotic pressure differences at the injection depot; glycerin and sodium chloride are examples; effective concentrations can be determined by osmometry using an assumed osmolality of 285-315 mOsmol/kg for serum; [0646] (iii) Preservatives and/or antimicrobials: multidose parenteral formulations require the addition of preservatives at a sufficient concentration to minimize risk of patients becoming infected upon injection and corresponding regulatory requirements have been established; typical preservatives include m-cresol, phenol, methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosol, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and benzalkonium chloride; [0647] (iv) Stabilizers: Stabilisation is achieved by strengthening of the protein-stabilising forces, by destabilisation of the denatured state, or by direct binding of excipients to the protein; stabilizers may be amino acids such as alanine, arginine, aspartic acid, glycine, histidine, lysine, proline, sugars such as glucose, sucrose, trehalose, polyols such as glycerol, mannitol, sorbitol, salts such as potassium phosphate, sodium sulphate, chelating agents such as EDTA, hexaphosphate, ligands such as divalent metal ions (zinc, calcium, etc.), other salts or organic molecules such as phenolic derivatives; in addition, oligomers or polymers such as cyclodextrins, dextran, dendrimers, PEG or PVP or protamine or HSA may be used; [0648] (v) Anti-adsorption agents: Mainly ionic or non-ionic surfactants or other proteins or soluble polymers are used to coat or adsorb competitively to the inner surface of the formulation’s container; e.g., poloxamer (Pluronic F-68), PEG dodecyl ether (Brij 35), polysorbate 20 and 80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA and gelatins; chosen concentration and type of excipient depends on the effect to be avoided but typically a monolayer of surfactant is formed at the interface just above the CMC value; [0649] (vi) Oxidation protection agents: antioxidants such as ascorbic acid, ectoine, methionine, glutathione, monothioglycerol, morin, polyethylenimine (PEI), propyl gallate, and vitamin E; chelating agents such as citric acid, EDTA, hexaphosphate, and thioglycolic acid may also be used; [0650] (vii) Viscosifiers or viscosity enhancers: in case of a suspension retard settling of the particles in the vial and syringe and are used in order to facilitate mixing and resuspension of the particles and to make the suspension easier to inject (i.e., low force on the syringe plunger); suitable viscosifiers or viscosity enhancers are, for example, carbomer viscosifiers like Carbopol 940, Carbopol Ultrez 10, cellulose derivatives like hydroxypropylmethylcellulose (hypromellose, HPMC) or diethylaminoethyl cellulose (DEAE or DEAE-C), colloidal magnesium silicate (Veegum) or sodium silicate, hydroxyapatite gel, tricalcium phosphate gel, xanthans, carrageenans like Satia gum UTC 30, aliphatic poly(hydroxy acids), such as poly(D,L- or L-lactic acid) (PLA) and poly(glycolic acid) (PGA) and their copolymers (PLGA), terpolymers of D,L-lactide, glycolide and caprolactone, poloxamers, hydrophilic poly(oxyethylene) blocks and hydrophobic poly(oxypropylene) blocks to make up a triblock of poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) (e.g. Pluronic®), polyetherester copolymer, such as a polyethylene glycol terephthalate/polybutylene terephthalate copolymer, sucrose acetate isobutyrate (SAIB), dextran or derivatives thereof, combinations of dextrans and PEG, polydimethylsiloxane, collagen, chitosan, polyvinyl alcohol (PVA) and derivatives, polyalkylimides, poly (acrylamide-co-diallyldimethyl ammonium (DADMA)), polyvinylpyrrolidone (PVP), glycosaminoglycans (GAGs) such as dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronan, ABA triblock or AB block copolymers composed of hydrophobic A-blocks, such as polylactide (PLA) or poly(lactide-co-glycolide) (PLGA), and hydrophilic B-blocks, such as polyethylene glycol (PEG) or polyvinyl pyrrolidone; such block copolymers as well as the abovementioned poloxamers may exhibit reverse thermal gelation behavior (fluid state at room temperature to facilitate administration and gel state above sol-gel transition temperature at body temperature after injection); [0651] (viii) Spreading or diffusing agent: modifies the permeability of connective tissue through the hydrolysis of components of the extracellular matrix in the intrastitial space such as but not limited to hyaluronic acid, a polysaccharide found in the intercellular space of connective tissue; a spreading agent such as but not limited to hyaluronidase temporarily decreases the viscosity of the extracellular matrix and promotes diffusion of injected drugs; and [0652] (ix) Other auxiliary agents: such as wetting agents, viscosity modifiers, antibiotics, hyaluronidase; acids and bases such as hydrochloric acid and sodium hydroxide are auxiliary agents necessary for pH adjustment during manufacture.

[0653] A further aspect of the present invention is a method of treating, controlling, delaying or preventing in a mammalian patient, preferably a human patient, one or more conditions which can be treated, controlled, delayed or prevented with PTH, comprising the step of administering a pharmaceutical composition comprising at least one controlled-release PTH compound once every 24 hours in a dosage of no more than 70% of the molar equivalent dosage of PTH 1-84 required to maintain serum calcium above 8.5 mg/dL over a 24 hour period.

[0654] Preferred embodiments of the controlled-release PTH compound are as described above.

[0655] Preferably, the condition that can be treated, controlled, delayed or prevented with PTH 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. More preferably, the condition that can be treated, controlled, delayed or prevented with PTH is selected from the group consisting of hypoparathyroidism, hyperphosphatemia, fracture repair, arthritis, osteoarthritis, rheumatoid arthritis, osteopenia, periodontal disease, bone fracture, alopecia, chemotherapy-induced alopecia, and thrombocytopenia.

[0656] Most preferably said condition is hypoparathyroidism.

[0657] Preferably, the patient undergoing the method of treatment of the present invention is a mammalian patient, preferably a human patient.

EXAMPLES

Materials and Methods

[0658] 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.

[0659] 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.

[0660] PEG 2x20 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.

[0661] Compound 11a (examples 11-15) was synthesized following the procedure described in patent WO29095479A2, example 1.

[0662] Syringes equipped with polyethylenene frits (MultiSynTech GmbH, Witten, Germany) were used as reaction vessels or for washing steps of peptide resins.

[0663] 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 x) and dried in vacuo.

[0664] 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.

RP-HPLC Purification

[0665] 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 .Math.m, 150 × 10 mm, flow rate 6 mL/min, or Waters XBridge™ BEH300 Prep C18 10 .Math.m, 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.

Flash Chromatography

[0666] 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.

Ion Exchange Chromatography

[0667] 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.

Size Exclusion Chromatography

[0668] 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.

Analytical Methods

[0669] 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 .Math.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.

Quantification of Plasma Total PTH(1-34) Concentrations

[0670] 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.

[0671] 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).

[0672] 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).

[0673] 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 .Math.L of internal standard solution (625 ng/mL of deuterated conjugate) and then by addition of 400 .Math.L of acetonitrile to 50 .Math.L of the plasma sample. 2 times 150 .Math.L of the supernatant were transferred into a new well-plate and evaporated to dryness (under a gentle nitrogen stream at 50° C.). 50 .Math.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:

[0674] 20 .Math.g of Lys-C (order number 125-05061, Wako Chemicals GmbH, Neuss, Germany) were dissolved in 80 .Math.L of 10 mM acetic acid. 3 .Math.L of the Lys-C solution were added to each cavity and samples incubated for 15 hours at 37° C. Afterwards 10 .Math.g of Glu-C (order number V1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 .Math.L water, and 1.5 .Math.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 .Math.L water/formic acid 4:6 (v/v) and 10 .Math.L were injected into the UPLC-MS system.

[0675] Chromatography was performed on a Waters Acquity BEH300 C18 analytical column (1.7 .Math.m particle size; column dimensions 50 × 2.1 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.

[0676] 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).

Quantification of Plasma PEG Concentrations

[0677] 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.

[0678] 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.

[0679] 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 .Math.L of precooled (5-10° C.) methanol to 100 .Math.L of the plasma sample. 180 .Math.L of the supernatant were transferred into a new well-plate and evaporated to dryness (under a gentle nitrogen stream at 45° C.). 50 .Math.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 .Math.g of Lys-C (order number 125-05061, Wako Chemicals GmbH, Neuss, Germany) were dissolved in 80 .Math.L of 10 mM acetic acid. 3 .Math.L of the Lys-C solution were added to each cavity and samples incubated for 15 hours at 37° C. Afterwards 10 .Math.g of Glu-C (order number V1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 .Math.L water, and 1.5 .Math.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 .Math.L water/formic acid 4:6 (v/v) and 5 .Math.L were injected into the SEC-MS system.

[0680] 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 (200-300 V) at the MS interface. Chromatography was performed on a TOSOH TSK Gel SuperAW3000 analytical column (4.0 .Math.m particle size; column dimensions 150 × 6.0 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.

[0681] Mass analysis was performed in single reaction monitoring (SRM) mode, monitoring the transition m/z 133.1 to 45.1.

Quantification of Plasma Free PTH Concentrations

[0682] 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.

[0683] 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 pg/mL in acidified plasma for each analyte. The corresponding concentration range is 7.00 to 700 pg/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.

[0684] 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 pg/mL in acidified plasma.

[0685] Protein precipitation was carried out by addition of 150 .Math.L of cold acetonitrile to 150 .Math.L of the plasma sample after addition of 50.0 .Math.L of cold internal standard solution, followed by centrifugation. The supernatant was decanted into a new ploypropylene tube and 900 .Math.L of cold water was added. After another centrifugation step, the tubes were kept in ice-water until loading on the SPE column.

[0686] Solid phase extraction: the HLB .Math.elation columns were conditioned with 200 .Math.L methanol followed by 200 .Math.L water. The columns were loaded 3 times with 420 .Math.L of the diluted samples by applying positive pressure. The SPE-columns were washed with 200 .Math.L of methanol:water 5:95 v/v. The samples were eluted with 40.0 .Math.L SPE elution solvent (Aceonitrile:water:Trifluoroactic acid 60:40:1 v/v/v), followed by 40.0 .Math.L of water. The elution solvent was left standing on the columns for 2 minutes, after which very gentle pressure was applied for elution.

[0687] Separation between metabolites and interfering endogenous compounds was achieved by LC-MS using an Xselect CSH C18 column (2.1×100 mm, 2.5 .Math.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.

[0688] 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.

[0689] Quantification is based on multiple reaction monitoring (MRM) of the transitions of m/z: [0690] 687.3-787.3 for PTH(1-34) [0691] 662.8-757.9 for PTH(1-33) [0692] 692.3-793.3 for PTH(1-34) (Leu-d.sub.10).sub.3 [0693] 667.8-763.9 for PTH(1-33) (Leu-d.sub.10).sub.3

[0694] A linear calibration curve with a 1/x.sup.2 weighing factor was used for both analytes.

[0695] 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).

[0696] 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.

[0697] 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

Synthesis of Linker Reagent 1f

[0698] Linker reagent 1f was synthesized according to the following scheme:

##STR00105##

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.

TABLE-US-00122 Yield: 3.76 g (11.48 mmol, 89 % purity, 1a: double Tmob protected product = 8 :1) MS: m/z 355.22 = [M+H].sup.+, (calculated monoisotopic mass = 354.21).

[0699] To a solution of 1a (2 g, 5.65 mmol) in DCM (24 mL) COMU (4.84 g, 11.3 mmol), N-Fmoe-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.

TABLE-US-00123 Yield: 5.31 g (148 %, 6.66 mmol) MS: m/z 796.38 = [M+H].sup.+, (calculated monoisotopic mass = 795.37).

[0700] 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.

TABLE-US-00124 MS: m/z 574.31 = [M+H].sup.+, (calculated monoisotopic mass = 573.30).

[0701] 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.

TABLE-US-00125 Yield: 2.63 g (62 %, 94 % purity) MS: m/z 856.41 = [M+H].sup.+, (calculated monoisotopic mass = 855.41).

[0702] 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.

TABLE-US-00126 Yield: 2.1 g (88 %) MS: m/z 878.4 = [M+Na].sup.+, (calculated monoisotopic mass = 837.40).

[0703] 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 1f was isolated upon evaporation of the solvent.

TABLE-US-00127 Yield: 154 mg (81%) MS: m/z 953.4 = [M+H].sup.+, (calculated monoisotopic mass = 952.43).

Example 2

[0704] Synthesis of linker reagent 2g

##STR00106##

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.

TABLE-US-00128 Yield: 3.18 g (98%)

[0705] 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.

TABLE-US-00129 Yield: 5.69 g (85 %) MS: m/z 705.4 = [M+H].sup.+, (calculated monoisotopic mass = 704.34).

[0706] 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.

TABLE-US-00130 Yield: 3.22 g (103 %) MS: m/z 691.4 = [M+H].sup.+, (calculated monoisotopic mass = 690.36).

[0707] 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.

TABLE-US-00131 Yield: 2.09 g (74 %) MS: m/z 791.4 = [M+H].sup.+, (calculated monoisotopic mass = 790.42).

[0708] 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.

TABLE-US-00132 Yield: 4.77 g (95 %) MS: m/z 519.3 = [M+H].sup.+, (calculated monoisotopic mass = 518.30).

[0709] 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.

TABLE-US-00133 Yield: 4.04 g (89 %) MS: m/z 706.32 = [M+Na].sup.+, (calculated monoisotopic mass = 683.30).

Example 3

[0710] Synthesis of permanent S1 PTH(1-34) conjugate 3

##STR00107##

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

TABLE-US-00134 Yield: 36 mg (14 %), 3*8 TFA MS: m/z 1062.31 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+ = 1062.30).

Example 4

[0711] Synthesis of permanent K26 PTH(1-34) conjugate 4

##STR00108##

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

TABLE-US-00135 Yield: 40 mg (8 %), 4*8 TFA

TABLE-US-00136 MS: m/z 1062.30 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+ = 1062.30).

Example 5

[0712] Synthesis of transient S1 PTH(1-34) conjugate

##STR00109##

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 .Math.mol), PyBOP (127 mg, 244 .Math.mol) and DIPEA (64 .Math.L, 365 .Math.mol) in DMF (1.5 mL) was added to 0.60 g (61 .Math.mol) of the resin. The suspension was agitated for 16 h at rt. The resin was washed 10 x with DMF and Fmoc-deprotected as described above. A solution of 2g (167 mg, 244 .Math.mol) and DIPEA (64 .Math.L, 365 .Math.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.

TABLE-US-00137 Yield: 78 mg (24 %), 5*9 TFA MS: m/z 1101.59 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+ = 1101.57).

Example 6

[0713] Synthesis of transient S1 PTH(1-34) conjugate 6

##STR00110##

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

TABLE-US-00138 Yield: 25 mg (18 %), 6*9 TFA MS: m/z 1098.75 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+ = 1098.07).

Example 7

[0714] Synthesis of transient S1 PTH(1-34) conjugate 7

##STR00111##

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

TABLE-US-00139 Yield: 54 mg (20 %), 7*9 TFA

TABLE-US-00140 MS: m/z 1102.08 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+ = 1102.07).

Example 8

[0715] Synthesis of transient S1 PTH(1-34) conjugate 8

##STR00112##

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

TABLE-US-00141 Yield: 31 mg (22 %), 8*9 TFA MS: m/z 1109.32 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+ = 1108.58).

Example 9

[0716] Synthesis of transient S1 PTH(1-34) conjugate 9

##STR00113##

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

TABLE-US-00142 Yield: 47 mg (8 %), 9*9 TFA MS: m/z 1108.58 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+ = 1108.57).

Example 10

[0717] Synthesis of transient K26 PTH(1-34) conjugate 10

##STR00114##

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 .Math.mol) and DIPEA (0.24 mL, 1.36 mmol) in DMF (5 mL) was added to 1.91 g (227 .Math.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 prc-coolcd diethyl ether (-18° C.). The precipitate was dissolved in ACN/watcr and purified by RP-HPLC. The product fractions were freeze-dried.

TABLE-US-00143 Yield: 92 mg (7 %), 10*9 TFA MS: m/z 1108.58 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+ = 1108.57).

Example 11

[0718] Synthesis of low molecular weight transient S1 PEG conjugate 11b

##STR00115##

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

TABLE-US-00144 Yield: 3.1 mg (26 %), 11b.sup.∗9 TFA MS: m/z 1097.00 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+5H].sup.5+ = 1096.99).

Example 12

[0719] Synthesis of low molecular weight transient S1 PEG conjugate 12

##STR00116##

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

TABLE-US-00145 Yield: 10 mg (83 %), 12*9 TFA MS: m/z 1094.20 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+ = 1094.19).

Example 13

[0720] Synthesis of low molecular weight transient S1 PEG conjugate 13

##STR00117##

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

TABLE-US-00146 Yield: 8 mg (67 %), 13*9 TFA MS: m/z 1097.40 = [M+5H].sup.5+, (calculated monoisotopic mass for [M+5H].sup.5+ = 1097.39).

Example 14

[0721] Synthesis of low molecular weight transient S1 PEG conjugate 14

##STR00118##

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

TABLE-US-00147 Yield: 4 mg (33 %), 14*9 TFA MS: m/z 1378.01 = [M+4H].sup.4+, (calculated monoisotopic mass for [M+4H].sup.4+ = 1378.00).

Example 15

[0722] Synthesis of low molecular weight transient K26 PEG conjugate 15

##STR00119##

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

TABLE-US-00148 Yield: 2.1 mg (33 %), 15*9 TFA MS: m/z 1102.60 = [M+5H].sup.5+, (calculated monoisotopic mass for [M+5H].sup.5+ = 1102.59).

Example 16

[0723] Synthesis of permanent 2x20 kDa S1 PEG conjugate 16

##STR00120##

772 .Math.L of a solution containing thiol 3 (19.4 mg/mL, 15 mg, 3.54 .Math.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 2x20 kDa maleimide (Sunbright GL2-400MA, 187 mg, 4.32 .Math.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 .Math.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.

[0724] Yield: 97 mg (2.01 .Math.mol, 57 %) conjugate 16*8 AcOH

Example 17

[0725] Synthesis of permanent 2x20 kDa K26 PEG conjugate 17

##STR00121##

Conjugate 17 was prepared as described for 16 by reaction of thiol 4 (15 mg, 3.53 .Math.mol) and PEG 2x20 kDa maleimide (Sunbright GL2-400MA, 187 mg, 4.32 .Math.mol).

[0726] Yield: 80 mg (1.79 .Math.mol, 51 %) conjugate 17*8 AcOH

Example 18

[0727] Synthesis of transient 2×20 kDa S1 PEG conjugate 18

##STR00122##

Conjugate 18 was prepared as described for 16 by reaction of thiol 5 (37 mg, 8.40 .Math.mol) and PEG 2x20 kDa maleimide (Sunbright GL2-400MA, 445 mg, 9.24 .Math.mol). The reaction was quenched by addition of 50 .Math.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.

[0728] Yield: 161 mg (3.33 .Math.mol, 40 %) conjugate 18*9 AcOH

Example 19

[0729] Synthesis of transient 2×20 kDa S1 PEG conjugate 19

##STR00123##

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

TABLE-US-00149 Yield: 249 mg (5.16 .Math.mol, 84 %) conjugate 19*9 AcOH

Example 20

[0730] Synthesis of transient 2×20 kDa S1 PEG conjugate 20

##STR00124##

Conjugate 20 was prepared as described for 16 by reaction of thiol 9 (38 mg, 8.59 .Math.mol) and PEG 2×20 kDa maleimide (Sunbright GL2-400MA, 455 mg, 9.45 .Math.mol). The reaction was quenched by addition of 50 .Math.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.

TABLE-US-00150 Yield: 194 mg (4.01 .Math.mol, 47 %) conjugate 20*9 AcOH

Example 21

[0731] Synthesis of transient 2×20 kDa K26 PEG conjugate 21

##STR00125##

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

TABLE-US-00151 Yield: 256 mg (5.30 .Math.mol, 70 %) conjugate 21*9 AcOH

Example 22

In Vitro Release Kinetics of Transient Low Molecular Weight PEG Conjugates

[0732] 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.

[0733] Results: [0734] For conjugate 11b a release half life time of 3.2 d was obtained. [0735] For conjugate 12 a release half life time of 8.7 d was obtained. [0736] For conjugate 13 a release half life time of 10.8 d was obtained. [0737] For conjugate 14 a release half life time of 25.3 d was obtained. [0738] For conjugate 15 a release half life time of 6.9 d was obtained.

Example 23

In Vitro Release Kinetics of Transient 2×20 kDa PEG Conjugates

[0739] 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.

[0740] Results: [0741] For conjugate 18 a release half life time of 2.8 d was obtained. [0742] For conjugate 19 a release half life time of 13.4 d was obtained. [0743] For conjugate 20 a release half life time of 1.3 d was obtained [0744] For conjugate 21 a release half life time of 7.1 d was obtained

Example 24

PTH Receptor Activity of Permanent 2x20 kDa PEG Conjugates 16 and 17 in Cell Based Assay

[0745] 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 hormone (PTH), Journal of Pharmaceutical 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.

[0746] Results: [0747] For conjugate 16 a receptor activity of 0.12 % was found relative to PTH(1-34) reference [0748] For conjugate 17 a receptor activity of 0.11 % was found relative to PTH(1-34) reference

[0749] The results indicate an effective lowering of receptor activity in the permanent 2x20 kDa PEG conjugates 16 and 17. It can be concluded that similar conjugates with transiently Serl 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

Pharmacokinetic Study of Permanent 2×20 kDa PEG Conjugates 16 and 17 in Rats

[0750] 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 .Math.g/rat PTH.sub.eq and 31 .Math.g/rat PTH.sub.eq respectively. Blood samples were collected up to 168 h post dose, and plasma was generated. Plasma PTH(1-34) concentrations were determined by quantification of the N-tcrminal signature peptide (sequence: IQLMHNLGK) and the C-terminal signature peptide (sequence: LQDVHNF) after LysC and GluC digestion as described in Materials and Methods.

[0751] 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 t.sub.max 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.

[0752] The favourable long lasting PK and the stability of PTH in the conjugates indicate the suitability of the permanent 2×20 kDa PEG model compounds as slow releasing PTH prodrugs after subcutaneous injection. It can be concluded that similar conjugates with transiently Serl (like e.g. 18) or Lys26 linked PTH are suitable PTH prodrugs providing long lasting levels of released bioactive PTH.

Example 26

Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate 19 in Cynomolgus Monkeys

[0753] Male non naive 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 .Math.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.

[0754] 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.

[0755] It can be concluded that conjugate 19 is a suitable prodrug for sustained delivery of PTH.

Example 27

Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate 18 in Cynomolgus Monkeys

[0756] Non naive 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 .Math.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.

[0757] 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).

[0758] 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

Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate 18 in Cynomolgus Monkeys

[0759] Naïve 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 .Math.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, 8h, 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.

[0760] 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.

[0761] 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

Pharmacokinetic Study of Transient 2×20 kDa S1 PEG Conjugate 18 in Sprague-Dawley Rats

[0762] 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 .Math.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.

[0763] 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.

[0764] 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.

[0765] 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.

TABLE-US-00152 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 DBU 1,3-diazabicyclo[5.4.0]undecene DCC N,N′-dicyclohexylcarbodiimide DCM dichloromethane DIPEA N,N-diisopropylethylamine DMAP dimcthylamino-pyridinc 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 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 SIM single ion monitoring SEC size exclusion chromatography sc subcutaneous t.sub.½ half life TCP tritylchloride polystyrol TES triethylsilane TFA trifluoroacetic acid THF tetrahydrofuran Tmob 2,4,6-trimcthoxybcnzyl Trt triphenylmethyl, trityl ULOQ upper limit of quantification UPLC ultra performance liquid chromatography UV ultraviolet ZQ single quadrupole