RU2846264C1 - Pth prodrugs - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: invention relates to PTH prodrugs, pharmaceutical compositions containing such PTH prodrugs and uses thereof. Presented is a conjugate of formula 31 or a pharmaceutically acceptable salt thereof, wherein PTH(1-34) is a parathyroid hormone having the sequence SEQ ID NO: 51; N^αS1 is the alpha-amino group of the N-terminal serine of SEQ ID NO: 51.

EFFECT: conjugate or its pharmaceutical composition can be used for treating hypoparathyroidism.

6 cl, 35 ex

Description

The present invention relates to PTH prodrugs, pharmaceutical compositions containing such PTH prodrugs and uses thereof.

Hypoparathyroidism is a rare endocrine disorder characterized by low serum calcium and inappropriately low (deficient) levels of circulating parathyroid hormone, most commonly in adults following thyroid surgery. Standard treatment includes activated vitamin D analogues and calcium supplements, which increase absorption and serum calcium and phosphorus levels and serum levels by abnormally increasing urinary calcium excretion. Hypoparathyroidism is the only major endocrine disorder today where the hormone deficiency is not treated at all by replacing the missing hormone (PTH).

The prevalence of hypoparathyroidism has recently been systematically studied in Denmark, where a total of over 2000 patients were identified with a prevalence of ~24/100,000, of whom only a minority (2/100,000) had non-surgical hypoparathyroidism. These estimates are consistent with recent data from the USA showing a prevalence of similar magnitude in patients with chronic hypoparathyroidism.

Endogenous PTH is synthesized and secreted by the parathyroid glands and is the major endocrine hormone regulating systemic calcium and phosphorus homeostasis. The physiological actions of PTH include the release of calcium and phosphorus from bone, the accumulation of calcium but not phosphorus in the kidneys by increasing renal tubular calcium reabsorption but decreasing renal tubular phosphate reabsorption, and the stimulation of renal production of active vitamin D (1,25(OH))2vitamin D3), which in turn enhances intestinal absorption of calcium and phosphorus. Without the renal actions of PTH to conserve calcium and excrete phosphorus, conventional therapy with vitamin D analogues and calcium supplementation can lead to renal failure or treatment failure due to progressive nephrocalcinosis, as well as ectopic calcifications (in the basal ganglia, lenses of the eye, and vasculature) due to chronically increased calcium x phosphorus product, which precipitates as calcium phosphate crystals when this product is maintained elevated for long periods of time.

Recently, NatparaT, PTH(1-84) was approved by the FDA for the treatment of hypoparathyroidism. Historically, ForteoT PTH(1-34) was also used as a daily injection for hypoparathyroidism once, twice, or three times daily, although it was not approved for this indication.

When PTH is delivered intermittently, such as by daily or multiple daily injections of PTH(1-84) or PTH(1-34), it acts as an anabolic agent on bone, preferentially activating osteoblasts over osteoclasts. This anabolic effect of intermittent PTH exposure is distinct from the bone catabolism that can occur with continuous PTH exposure. The anabolic potential of intermittent administration of PTH agonists has been successfully exploited in the treatment of osteoporosis, where bone turnover is typically high and bone mineral density (BMD) is low, whereas the reverse situation occurs in hypoparathyroidism.

The major complication of hypoparathyroidism is hypercalciuria due to the lack of PTH-dependent calcium reabsorption in the distal renal tubules. Hypercalciuria is associated with an increased risk of nephrocalcinosis, nephrolithiasis, and renal failure. According to the FDA review of Natpara, daily PTH injections did not provide adequate control of urinary calcium excretion due to the short half-life of this PTH agonist in the body.

In addition, non-physiological PTH levels may be associated with hypercalcemia and hypocalcemia. Treatment with Natpara did not reduce the incidence of these complications compared with placebo. This may be partly explained by the unfavorable PK of the Natpara product. For example, administration of currently approved doses of Natpara results in significant supraphysiological PTH levels with a Cmax of 300 pg/mL that returns to baseline within 12 hours. As a result, patients are overtreated in the initial post-dose phase and undertreated in the pre-dose phase.

Hypocalcemia is associated with numerous symptoms, some of which can be life-threatening, including: seizures; paresthesia; cognitive impairment; loss of consciousness with seizures (grand mal seizure); impaired kidney function; cardiac arrhythmia and syncope, and even heart failure.

As such, there is a high unmet need for a more physiological PTH therapy that provides sustained PTH exposure, thereby alleviating symptoms associated with hypocalcemia, hypercalciuria, and hyperphosphatasemia without causing hypercalcemia.

PTH replacement therapy would be more physiological if delivered via continuous infusion, such as with an insulin pump. This was demonstrated by Winer et al. (J Pediatr, 2014, 165 (3), 556-563), where pump delivery simultaneously normalized bone turnover markers and urinary and serum mineral levels, whereas intermittent injection delivery did not.

The normal range of PTH is 15-50 pg/mL, and it is important to recognize that intermittent administration of a PTH agonist does not constitute physiological replacement therapy. PTH polypeptides have inherently short circulating half-lives due to rapid hepatic metabolism. Rapid clearance of the drug from the body prevents sufficient spectrum of drug activity over the dosing interval despite initial supraphysiological drug levels.

Several approaches have been taken to create a longer acting version of PTH, including encapsulating PTH in PLGA microparticles and permanently conjugating the PTH molecule to synthetic or peptide polymers. Kostenuik et al. (J Bone Miner Res, 2007, 22 (10), 1534-1547) described a PTH-Fc fusion protein with a longer half-life than PTH(1-34) and conducted studies in ovariectomized osteopenic rats and mice to determine whether intermittent (once to twice weekly) injections of PTH-Fc would increase bone mass, density, and strength despite prolonged exposure to PTH. A PTH-derived molecule with a long blood half-life has been shown to provide anabolic effects on tubular and cancellous bone comparable to daily PTH administration, but at a reduced dosing frequency. Another approach was proposed by Ponnapakkam et al. (Drug Discov Today, 2014, 19(3), 204.208), in which a PTH-collagen-binding domain fusion polypeptide induced sustained (up to 12 months) increases in bone mineral density in normal male mice after a single dose.

Patients with hypoparathyroidism typically exhibit abnormally low bone turnover rates resulting in increased bone mineral density, and therefore the anabolic effects of PTH should be avoided in the treatment of hypoparathyroidism, and optimal treatment should normalize their bone turnover rates but not increase them above the normal range, as has been demonstrated with daily treatment with PTH(1-34) and PTH(1-84).

Overall, there is a need for more effective treatments for PTH.

Therefore, the object of the present invention is to at least partially overcome the above-described disadvantages.

This problem is solved by means of a PTH prodrug or its pharmaceutically acceptable salt, where the prodrug has the formula (Ia) or (Ib)

(Ia)

(Ib),

Where

-D represents the PTH component;

-L1- is a reversible linker moiety of the prodrug, linked to the PTH moiety of -D via the PTH functional group;

-L2- represents a simple chemical bond or spacer moiety;

-Z is a water-soluble carrier component;

x is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16; and

y is an integer chosen from the group consisting of 1, 2, 3, 4, and 5.

It has been unexpectedly found that the PTH prodrugs of the present invention exhibit low residual prodrug activity and provide a prolonged release of PTH. As a result, administration of the PTH prodrugs of the present invention results in a simultaneous normalization of serum calcium and a decrease in serum phosphate, and thus an increased serum calcium to serum phosphate ratio compared to treatment with PTH1-84, the current standard of care. At the same time, no adverse effects on bone resorption and bone formation markers and overall bone health were observed in a corresponding animal model for humans when physiological doses were administered. At the same time, no adverse effects on bone resorption and bone formation markers and overall bone health were observed in a corresponding animal model for humans when physiological doses were administered.

Surprisingly, it was found that such PTH prodrugs are able to achieve a stable plasma PTH profile that provides physiological serum and urinary calcium levels or even lower than normal urinary calcium levels.

In the context of the present invention, the terms used have the following meanings.

As used herein, the term "PTH" refers to all PTH polypeptides, preferably of a mammalian species, more preferably of a human and mammalian species, more preferably of a human and murine species, as well as variants, analogs, orthologs, homologs and derivatives and fragments thereof, which are characterized by increasing serum calcium and renal phosphorus excretion and decreasing serum phosphorus and renal calcium excretion. The term "PTH" also refers to all PTHrP polypeptides, such as the polypeptide of SEQ ID NO: 121, which binds to and activates the common PTH/PTHrP1 receptor. Preferably, the term "PTH" refers to the PTH polypeptide of SEQ ID NO: 51, as well as variants, homologs and derivatives thereof that exhibit substantially the same biological activity, i.e. increasing serum calcium and renal phosphorus excretion and decreasing serum phosphorus and renal calcium excretion.

Preferably, the term “PTH” refers to the following polypeptide sequences:

SEQ ID NO:1 (PTH 1-84)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ

SEQ ID NO:2 (PTH 1-83)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKS

SEQ ID NO:3 (PTH 1-82)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAK

SEQ ID NO:4 (PTH 1-81)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKA

SEQ ID NO:5 (PTH 1-80)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTK

SEQ ID NO:6 (PTH 1-79)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLT

SEQ ID NO:7 (PTH 1-78)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVL

SEQ ID NO:8 (PTH 1-77)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNV

SEQ ID NO:9 (PTH 1-76)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVN

SEQ ID NO:10 (PTH 1-75)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADV

SEQ ID NO:11 (PTH 1-74)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKAD

SEQ ID NO:12 (PTH 1-73)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKA

SEQ ID NO:13 (PTH 1-72)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADK

SEQ ID NO:14 (PTH 1-71)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEAD

SEQ ID NO:15 (PTH 1-70)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEA

SEQ ID NO:16 (PTH 1-69)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGE

SEQ ID NO:17 (PTH 1-68)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLG

SEQ ID NO:18 (PTH 1-67)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSL

SEQ ID NO:19 (PTH 1-66)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKS

SEQ ID NO:20 (PTH 1-65)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEK

SEQ ID NO:21 (PTH 1-64)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHE

SEQ ID NO:22 (PTH 1-63)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESH

SEQ ID NO:23 (PTH 1-62)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVES

SEQ ID NO:24 (PTH 1-61)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVE

SEQ ID NO:25 (PTH 1-60)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLV

SEQ ID NO:26 (PTH 1-59)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVL

SEQ ID NO:27 (PTH 1-58)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNV

SEQ ID NO:28 (PTH 1-57)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDN

SEQ ID NO:29 (PTH 1-56)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKED

SEQ ID NO:30 (PTH 1-55)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKE

SEQ ID NO:31 (PTH 1-54)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKK

SEQ ID NO:32 (PTH 1-53)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRK

SEQ ID NO:33 (PTH 1-52)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPR

SEQ ID NO:34 (PTH 1-51)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRP

SEQ ID NO:35 (PTH 1-50)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR

SEQ ID NO:36 (PTH 1-49)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQ

SEQ ID NO:37 (PTH 1-48)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGS

SEQ ID NO:38 (PTH 1-47)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAG

SEQ ID NO:39 (PTH 1-46)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDA

SEQ ID NO:40 (PTH 1-45)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRD

SEQ ID NO:41 (PTH 1-44)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPR

SEQ ID NO:42 (PTH 1-43)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAP

SEQ ID NO:43 (PTH 1-42)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLA

SEQ ID NO:44 (PTH 1-41)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPL

SEQ ID NO:45 (PTH 1-40)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAP

SEQ ID NO:46 (PTH 1-39)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGA

SEQ ID NO:47 (PTH 1-38)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALG

SEQ ID NO:48 (PTH 1-37)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVAL

SEQ ID NO:49 (PTH 1-36)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVA

SEQ ID NO:50 (PTH 1-35)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFV

SEQ ID NO:51 (PTH 1-34)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF

SEQ ID NO:52 (PTH 1-33)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHN

SEQ ID NO:53 (PTH 1-32)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH

SEQ ID NO:54 (PTH 1-31)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDV

SEQ ID NO:55 (PTH 1-30)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQD

SEQ ID NO:56 (PTH 1-29)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQ

SEQ ID NO:57 (PTH 1-28)

SVSEIQLMHNLGKHLNSMERVEWLRKKL

SEQ ID NO:58 (PTH 1-27)

SVSEIQLMHNLGKHLNSMERVEWLRKK

SEQ ID NO:59 (PTH 1-26)

SVSEIQLMHNLGKHLNSMERVEWLRK

SEQ ID NO:60 (PTH 1-25)

SVSEIQLMHNLGKHLNSMERVEWLR

SEQ ID NO:61 (amidated PTH 1-84)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ; where the C-terminus is amidated

SEQ ID NO:62 (amidated PTH 1-83)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKS; where the C-terminus is amidated

SEQ ID NO:63 (amidated PTH 1-82)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAK; where the C-terminus is amidated

SEQ ID NO:64 (amidated PTH 1-81)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKA; where the C-terminus is amidated

SEQ ID NO:65 (amidated PTH 1-80)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTK; where the C-terminus is amidated

SEQ ID NO:66 (amidated PTH 1-79)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLT; where the C-terminus is amidated

SEQ ID NO:67 (amidated PTH 1-78)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVL; where the C-terminus is amidated

SEQ ID NO:68 (amidated PTH 1-77)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNV; where the C-terminus is amidated

SEQ ID NO:69 (amidated PTH 1-76)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVN; where the C-terminus is amidated

SEQ ID NO:70 (amidated PTH 1-75)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADV; where the C-terminus is amidated

SEQ ID NO:71 (amidated PTH 1-74)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKAD; where the C-terminus is amidated

SEQ ID NO:72 (amidated PTH 1-73)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKA; where the C-terminus is amidated

SEQ ID NO:73 (amidated PTH 1-72)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADK; where the C-terminus is amidated

SEQ ID NO:74 (amidated PTH 1-71)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEAD; where the C-terminus is amidated

SEQ ID NO:75 (amidated PTH 1-70)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEA; where the C-terminus is amidated

SEQ ID NO:76 (amidated PTH 1-69)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGE; where the C-terminus is amidated

SEQ ID NO:77 (amidated PTH 1-68)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLG; where the C-terminus is amidated

SEQ ID NO:78 (amidated PTH 1-67)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSL; where the C-terminus is amidated

SEQ ID NO:79 (amidated PTH 1-66)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKS; where the C-terminus is amidated

SEQ ID NO:80 (amidated PTH 1-65)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEK; where the C-terminus is amidated

SEQ ID NO:81 (amidated PTH 1-64)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHE; where the C-terminus is amidated

SEQ ID NO:82 (amidated PTH 1-63)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESH; where the C-terminus is amidated

SEQ ID NO:83 (amidated PTH 1-62)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVES; where the C-terminus is amidated

SEQ ID NO:84 (amidated PTH 1-61)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVE; where the C-terminus is amidated

SEQ ID NO:85 (amidated PTH 1-60)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLV; where the C-terminus is amidated

SEQ ID NO:86 (amidated PTH 1-59)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVL; where the C-terminus is amidated

SEQ ID NO:87 (amidated PTH 1-58)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNV; where the C-terminus is amidated

SEQ ID NO:88 (amidated PTH 1-57)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDN; where the C-terminus is amidated

SEQ ID NO:89 (amidated PTH 1-56)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKED; where the C-terminus is amidated

SEQ ID NO:90 (amidated PTH 1-55)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKE; where the C-terminus is amidated

SEQ ID NO:91 (amidated PTH 1-54)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKK; where the C-terminus is amidated

SEQ ID NO:92 (amidated PTH 1-53)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRK; where the C-terminus is amidated

SEQ ID NO:93 (amidated PTH 1-52)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPR; where the C-terminus is amidated

SEQ ID NO:94 (amidated PTH 1-51)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRP; where the C-terminus is amidated

SEQ ID NO:95 (amidated PTH 1-50)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQR; where the C-terminus is amidated

SEQ ID NO:96 (amidated PTH 1-49)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQ; where the C-terminus is amidated

SEQ ID NO:97 (amidated PTH 1-48)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGS; where the C-terminus is amidated

SEQ ID NO:98 (amidated PTH 1-47)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAG; where the C-terminus is amidated

SEQ ID NO:99 (amidated PTH 1-46)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDA; where the C-terminus is amidated

SEQ ID NO:100 (amidated PTH 1-45)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRD; where the C-terminus is amidated

SEQ ID NO:101 (amidated PTH 1-44)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPR; where the C-terminus is amidated

SEQ ID NO:102 (amidated PTH 1-43)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAP; where the C-terminus is amidated

SEQ ID NO:103 (amidated PTH 1-42)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLA; where the C-terminus is amidated

SEQ ID NO:104 (amidated PTH 1-41)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPL; where the C-terminus is amidated

SEQ ID NO:105 (amidated PTH 1-40)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAP; where the C-terminus is amidated

SEQ ID NO:106 (amidated PTH 1-39)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGA; where the C-terminus is amidated

SEQ ID NO:107 (amidated PTH 1-38)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALG; where the C-terminus is amidated

SEQ ID NO:108 (amidated PTH 1-37)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVAL; where the C-terminus is amidated

SEQ ID NO:109 (amidated PTH 1-36)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVA; where the C-terminus is amidated

SEQ ID NO:110 (amidated PTH 1-35)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFV; where the C-terminus is amidated

SEQ ID NO:111 (amidated PTH 1-34)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF; where the C-terminus is amidated

SEQ ID NO:112 (amidated PTH 1-33)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHN; where the C-terminus is amidated

SEQ ID NO:113 (amidated PTH 1-32)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVH; where the C-terminus is amidated

SEQ ID NO:114 (amidated PTH 1-31)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQDV; where the C-terminus is amidated

SEQ ID NO:115 (amidated PTH 1-30)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQD; where the C-terminus is amidated

SEQ ID NO:116 (amidated PTH 1-29)

SVSEIQLMHNLGKHLNSMERVEWLRKKLQ; where the C-terminus is amidated

SEQ ID NO:117 (amidated PTH 1-28)

SVSEIQLMHNLGKHLNSMERVEWLRKKL; where the C-terminus is amidated

SEQ ID NO:118 (amidated PTH 1-27)

SVSEIQLMHNLGKHLNSMERVEWLRKK; where the C-terminus is amidated

SEQ ID NO:119 (amidated PTH 1-26)

SVSEIQLMHNLGKHLNSMERVEWLRK; where the C-terminus is amidated

SEQ ID NO:120 (amidated PTH 1-25)

SVSEIQLMHNLGKHLNSMERVEWLR; where the C-terminus is amidated

SEQ ID NO:121 (PTHrP)

AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTAEIRATSEVSPNSKPSPNTKNHPVRFGSDDEGRYLTQETNKVETYKEQPLKTPGKKKKGKPGKRKEQEKKKRRTRSAWLDSGVTGSGLEGDHLSDTSTTSLELDSRRH

More preferably, the term “PTH” refers to the sequence of SEQ ID:NO 47, 48, 49, 50, 51, 52, 53, 54, 55, 107, 108, 109, 110, 111, 112, 113, 114 and 115. Even more preferably, the term “PTH” refers to the sequence of SEQ ID:NO 50, 51, 52, 110, 111 and 112. In a particularly preferred embodiment of the present invention, the term “PTH” refers to the sequence of SEQ ID NO:51.

As used herein, the term "variant PTH polypeptide" refers to a polypeptide from the same species that differs from a reference PTH or PTHrP polypeptide. Preferably, such a reference polypeptide is the PTH polypeptide sequence of SEQ ID NO:51. In general, the differences are limited such that the amino acid sequences of the reference polypeptide and the variant are generally very similar and, in many regions, identical. Preferably, variant PTH polypeptides are at least 70%, 80%, 90%, or 95% identical to a reference PTH or PTHrP polypeptide, preferably the PTH polypeptide of SEQ ID NO:51. By a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence, it is meant that the amino acid sequence of the polypeptide is identical to the reference sequence, except that the polypeptide sequence may include up to five amino acid changes for every 100 amino acids of the reference amino acid sequence. These substitutions in the reference sequence may occur at the amino (N-terminal) or carboxy terminal (C-terminal) positions of the reference amino acid sequence, or at any location between these terminal positions, distributed either individually among residues in the reference sequence or as one or more contiguous groups in the reference sequence. The reference sequence may be either the entire amino acid sequence of the reference sequence or any fragment refined as described herein. Preferably, the reference sequence is the sequence of SEQ ID NO:51.

Such PTH polypeptide variants may be naturally occurring variants, such as naturally occurring allelic variants encoded by one of several alternative forms of PTH or PTHrP occupying a given locus on a chromosome or organism, or isoforms encoded by naturally occurring splice variants derived from a single primary transcript. Alternatively, a PTH polypeptide variant may be a variant that is not known to occur in nature and that can be produced by mutagenesis techniques known in the art.

It is known in the art that one or more amino acids may be deleted from the N-terminus or C-terminus of a bioactive polypeptide without substantial loss of biological function. Such N- and/or C-terminal deletions are also encompassed by the term PTH polypeptide variant.

It is also known to those skilled in the art that certain amino acid sequences of PTH or PTHrP polypeptides can be altered without significantly affecting the structure or function of the polypeptide. Such mutants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art to have little effect on activity. For example, guidance on how to make phenotypically silent amino acid substitutions is provided in Bowie et al. (1990), Science 247:1306-1310, which is incorporated herein by reference in its entirety, where the authors indicate that there are two general approaches to studying the tolerance of an amino acid sequence to change.

The term PTH polypeptide also encompasses all PTH and PTHrP polypeptides encoded by PTH and PTHrP analogs, orthologs, and/or species homologs. Those skilled in the art will also appreciate that PTHrP and PTHrP analogs are associated with activation of a common PTH/PTHrP1 receptor, so the term PTH polypeptide also encompasses all PTHrP analogs. As used herein, the term "PTH analog" refers to PTH and PTHrP from different and unrelated organisms that perform the same functions in each organism, but that are not derived from a generic structure that the organisms' ancestors had. Instead, the analogous PTH and PTHrP arose separately and then evolved to perform the same or similar functions. In other words, the analogous polypeptides PTH and PTHrP are polypeptides with completely different amino acid sequences, but which have the same biological activity, namely, increasing serum calcium and renal phosphorus excretion, and decreasing serum phosphorus and renal calcium excretion.

As used in this application, the term “PTH ortholog” refers to a PTH and PTHrP from two different species whose sequences are related to each other through a common homologous PTH in the ancestral species, but which have become distinct from each other during evolution.

As used in the present application, the term "PTH homologue" refers to PTH and PTHrP from different organisms that perform the same function in each organism and that are derived from an ancestral structure that the ancestors of the organisms had in common. In other words, homologues of PTH polypeptides are polypeptides with completely similar amino acid sequences that have the same biological activity, namely, increasing serum calcium and renal phosphorus excretion, and decreasing serum phosphorus and renal calcium excretion. Preferably, homologues of a PTH polypeptide can be defined as polypeptides having at least 40%, 50%, 60%, 70%, 80%, 90% or 95% identity with a reference PTH or PTHrP polypeptide, preferably the PTH polypeptide of SEQ ID NO:51.

Thus, the PTH polypeptide according to the present invention can be, for example: (i) a polypeptide in which at least one of the amino acid residues has as a substitute a conserved or non-conserved amino acid residue, preferably a conserved amino acid residue, and such substituted amino acid residue may or may not be encoded by the genetic code; and/or (ii) a polypeptide in which at least one of the amino acid residues includes a substituent group; and/or (iii) a polypeptide wherein the PTH polypeptide is fused to another compound, such as a compound for increasing the half-life of the polypeptide (e.g. polyethylene glycol); and/or (iv) a polypeptide in which additional amino acids are fused to the PTH polypeptide, such as a polypeptide or leader or secretory sequence of an IgG Fc fusion region or a sequence that is used to purify the above-mentioned form of the polypeptide, or a precursor protein sequence.

As used herein, the term “PTH polypeptide fragment” refers to any peptide comprising a consecutive span of a portion of the amino acid sequence of a PTH or PTHrP polypeptide, preferably a polypeptide of the sequence SEQ ID NO:51.

More particularly, a PTH polypeptide fragment comprises at least 6, such as at least 8, at least 10 or at least 17 consecutive amino acids of a PTH or PTHrP polypeptide, more preferably a polypeptide of the sequence SEQ ID NO:51. A PTH polypeptide fragment may be further described as a subspecies of PTH or PTHrP polypeptides comprising at least 6 amino acids, wherein "at least 6" is defined as an integer between 6 and an integer denoting the C-terminal amino acid of a PTH or PTHrP polypeptide, preferably a polypeptide of the sequence SEQ ID No:51. Also included are species of PTH or PTHrP polypeptide fragments of at least 6 amino acids in length, as described above, which are further specified in terms of their N-terminal and C-terminal positions. Also encompassed by the term "PTH polypeptide fragment" as separate species are all fragments of a PTH or PTHrP polypeptide of at least 6 amino acids in length, as described above, which may be particularly specified by their N-terminal and C-terminal position. That is, each combination of N-terminal and C-terminal position that a component of at least 6 consecutive amino acid residues in length may occupy, on any given amino acid sequence of a PTH or PTHrP polypeptide, preferably a PTH polypeptide of the sequence SEQ ID:NO51, is included in the present invention.

The term "PTH" also includes poly(amino acid) conjugates that have a sequence as described above, but having a backbone that contains both amide and non-amide linkages, such as ester linkages, such as depsipeptides. Depsipeptides are chains of amino acid residues in which the backbone contains both amide (peptide) and ester linkages. Accordingly, the term "side chain" as used herein refers to either a moiety attached to the alpha carbon atom of an amino acid moiety if the amino acid moiety is linked via amine linkages, such as in polypeptides, or to any carbon atom-containing moiety attached to the backbone of a poly(amino acid) conjugate, such as in the case of depsipeptides. Preferably, the term "PTH" refers to polypeptides having a backbone formed via amide (peptide) linkages.

Since the term PTH includes the above-described variants, analogs, orthologs, homologs, derivatives and fragments of PTH and PTHrP, all references to specific positions in the reference sequence also include equivalent positions in the variants, analogs, orthologs, homologs, derivatives and fragments of the PTH or PTHrP moiety, even if not specifically stated.

As used in the present application, the term "random coil" refers to a peptide or protein adopting/having/forming, preferably having, a conformation that is substantially free of defined secondary and tertiary structure, as determined by circular dichroism spectroscopy performed in an aqueous buffer at ambient temperature and pH 7.4. Preferably, the ambient temperature is about 20°C, i.e. between 18°C and 22°C, the most preferred ambient temperature is 20°C.

As used in this application, the term "pharmaceutical composition" refers to a composition comprising one or more active ingredients, such as a drug or prodrug, in this application in particular PTH prodrugs according to the present invention and optionally one or more excipients, as well as any product that directly or indirectly results from the combination, complexation or aggregation of any two or more ingredients of the composition, or the dissociation of one or more ingredients, or other types of reactions or interactions of one or more ingredients. Accordingly, the pharmaceutical composition according to the present invention encompasses any composition obtained by mixing one or more PTH prodrugs according to the present invention and optionally a pharmaceutically acceptable excipient.

As used herein, the term “liquid composition” refers to a mixture comprising a water-soluble PTH prodrug and one or more solvents, such as water.

The term "suspension composition" refers to a mixture comprising a water-insoluble PTH prodrug, wherein for example the carrier Z' is a hydrogel, and one or more solvents, such as water. Due to the water-insoluble polymer, the polymeric prodrug cannot be dissolved and the prodrug is in particulate form.

As used in the present application, the term "dry composition" means that the pharmaceutical composition is provided in dry form. Suitable drying methods are spray drying and lyophilization, i.e. freeze drying. Such a dry prodrug composition has a residual water content of maximum 10%, preferably less than 5% and more preferably less than 2%, as determined by the Karl Fischer method. Preferably, the pharmaceutical composition according to the present invention is dried by lyophilization.

The term "drug" as used in this application refers to a substance used to treat, alleviate, prevent or diagnose a disease or otherwise improve physical or mental health. If a drug is conjugated to another moiety, the moiety of the resulting product that is derived from the drug is referred to as the "biologically active moiety". The PTH prodrug of the present invention comprises a PTH moiety that is released from the PTH prodrug in the form of the PTH drug.

As used herein, the term "prodrug" refers to a conjugate comprising a biologically active moiety reversibly and covalently linked to a specialized protecting group through a reversible linker moiety of the prodrug, which is a linker moiety comprising a reversible bond to the biologically active moiety, and wherein the specialized protecting group alters or reduces undesirable properties in the parent molecule. This also includes enhancements of desirable properties of the drug and suppression of undesirable properties. The specialized non-toxic protecting group is referred to as the "carrier". The prodrug releases the reversibly and covalently linked biologically active moiety in the form of its corresponding drug. In other words, a prodrug is a conjugate comprising a biologically active moiety that is reversibly and covalently conjugated to a carrier moiety through a reversible linker moiety of the prodrug, wherein the covalent or reversible conjugation of the carrier to the reversible linker moiety of the prodrug is either direct or through a spacer. Such a conjugate releases the previously conjugated biologically active moiety in the form of free drug.

The terms “biodegradable linkage” or “reversible linkage” mean a linkage that is hydrolytically degradable, i.e. cleavable, in the absence of enzymes under physiological conditions (aqueous buffer at pH 7.4, 37°C) with a half-life in the range of one hour to two months, preferably one hour to one month. Accordingly, a stable linkage is a linkage having a half-life under physiological conditions (aqueous buffer at pH 7.4, 37°C) of more than two months.

Accordingly, a "reversible linker moiety of a prodrug" is a moiety that is covalently conjugated to a biologically active moiety, such as PTH, via a reversible bond and is also covalently conjugated to a carrier moiety, such as -Z or Z', wherein the covalent conjugation to said carrier moiety is either direct or via a spacer moiety, such as -L2-. Preferably, the bond between -Z or Z' and L2- is a stable bond.

As used in this application, the term "traceless prodrug linker" means a reversible prodrug linker that, when cleaved, releases the drug in its free form. As used in this application, the term "free form" of a drug means the drug in its unmodified, pharmacologically active form.

As used herein, the term "excipient" refers to a diluent, adjuvant, or carrier with which a therapeutic agent, such as a drug or prodrug, is administered. Such a pharmaceutical excipient may be a sterile liquid such as water and oils, including liquids of petroleum, animal, vegetable, or synthetic origin, including, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water is a preferred excipient when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred vehicles when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably used as liquid excipients for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry milk, glycerin, propylene glycol, water, ethanol, and the like. The composition may optionally also contain small amounts of wetting or emulsifying agents, pH buffering agents such as, for example, acetate, succinate, tris, carbonate, phosphate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, MES (2-(N-morpholine)ethanesulfonic acid) or may contain detergents such as Tween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids such as, for example, glycine, lysine or histidine. Such pharmaceutical compositions may be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release preparations and the like. The pharmaceutical composition may be in the form of a suppository, with conventional binders and excipients such as triglycerides. The preparation for oral administration may contain standard excipients such as pharmaceutical grades mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Such compositions contain a therapeutically effective amount of the drug or biologically active component, together with a suitable amount of excipient, so as to obtain a form suitable for administration to a patient. The composition must be appropriate for the route of administration.

As used in this application, the term "reagent" means a chemical compound that contains at least one functional group for reaction with a functional group of another chemical compound or a drug. It is understood that a drug containing a functional group (such as a primary or secondary amine or a hydroxyl functional group) is also a reagent.

As used in this application, the term "moiety" means a portion of a molecule that is missing one or more atoms compared to the corresponding reactant. If, for example, a reactant of the formula "H-X-H" reacts with another reactant and becomes part of the reaction product, the corresponding moiety of the reaction product has the structure "H-X-" or "-X-", where each "-" denotes attachment to another moiety. Accordingly, the biologically active moiety is released from the prodrug as a drug.

It is understood that if a sequence or chemical structure of a group of atoms is provided where the group of atoms is attached to two constituents or interrupts a constituent, said sequence or structure may be attached to the two constituents in any orientation unless otherwise specified. For example, the moiety “-C(O)N(R12)-” may be attached to the two constituents or interrupts a constituent as either “-C(O)N(R)-” or as “-N(R)C(O)-”. Similarly, the moiety

can be joined to two components or can interrupt a component either as , or as .

As used in this application, the term “functional group” means a group of atoms that can react with other groups of atoms. Functional groups include, but are not limited to, the following groups: carboxylic acid (-(C=O)OH), primary and secondary amine ( _-NH2 , -NH-), maleimide, thiol (-SH), sulfonic acid (-(O=S=O)OH), carbonate, carbamate (-O(C=O)N<), hydroxyl (-OH), aldehyde (-(C=O)H), ketone (-(C=O)-), hydrazine (>NN<), isocyanate, isothiocyanate, phosphoric acid (-O(P=O)OHOH), phosphonic acid (-O(P=O)OHH), haloacetyl, alkyl halide, acryloyl, aryl fluoride, hydroxylamine, disulfide, sulfonamides, sulfuric acid, vinyl sulfone, vinyl ketone, diazoalkane, oxirane, and aziridine.

In case the prodrugs according to the present invention contain one or more acidic or basic groups, the present invention also includes their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically acceptable salts. Thus, the prodrugs according to the present invention containing acidic groups can be used according to the present invention, for example, in the form of alkali metal salts, alkaline earth metal salts or ammonium salts. More specific examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or ammonium salts or with organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. The prodrugs according to the present invention containing one or more basic groups, i.e. groups that can be protonated, can be present and used according to the present invention in the form of their addition salts with inorganic or organic acids. Examples of suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivaloic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfamic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid and other acids known to those skilled in the art. Those skilled in the art are familiar with the transformation of a basic group into a cation, such as alkylation of an amine group to produce a positively charged ammonium group and the corresponding counterion of the salt. If the prodrugs of the present invention simultaneously contain acidic and basic groups, the present invention also includes, in addition to the mentioned salt forms, internal salts or betaines (zwitterions). The corresponding salts can be prepared by conventional methods known to those skilled in the art, for example by contacting these prodrugs with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange with other salts. The present invention also includes all salts of the prodrugs of the present invention which, due to low physiological compatibility, are not suitable directly for use in pharmaceutical products, but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.

The term "pharmaceutically acceptable" means a substance that does not cause harm when administered to a patient and is preferably approved by a regulatory authority such as EMEA (Europe) and/or FDA (USA) and/or any other national regulatory authority for use in animals, preferably humans.

As used in the present application, the term “about” in combination with a numerical value is used to indicate a range in the range and including the numerical value plus or minus no more than 10% of the stated numerical value, more preferably no more than 8% of the stated value, even more preferably no more than 5% of the stated value, and most preferably no more than 2% of the stated value. For example, the phrase “about 200” is used to indicate a range in the range and including 200 +/- 10%, i.e. in the range and including 180 to 220; preferably 200 +/- 8%, i.e. in the range and including 184 to 216; even more preferably in the range and including 200 +/- 5%, i.e. in the range and including 190 to 210; and most preferably 200 +/- 2%, i.e. in the range and including 196 - 204. It is clear that the percentage given as “about 20%” does not mean “20% +/- 10%”, i.e. in the range and including 10 - 30%, but “about 20%” means in the range and including 18 - 22%, i.e. plus and minus 10% of the numerical value, which is 20.

As used in the present application, the term "polymer" means a molecule comprising repeating structural units, i.e. monomers linked by chemical bonds in a linear, ring, branched, cross-linked or dendrimeric manner or a combination thereof, which may be of synthetic or biological origin or a combination of both. It is understood that the polymer may also comprise one or more other chemical groups and/or moiety/moieties, such as, for example, one or more functional groups. Preferably, the soluble polymer has a molecular weight of at least 0.5 kDa, such as a molecular weight of at least 1 kDa, a molecular weight of at least 2 kDa, a molecular weight of at least 3 kDa, or a molecular weight of at least 5 kDa. If the polymer is soluble, it preferably has a molecular weight of at most 1000 kDa, such as at most 750 kDa, such as at most 500 kDa, such as at most 300 kDa, such as at most 200 kDa, such as at most 100 kDa. It is understood that for insoluble polymers, such as hydrogels, there are no significant ranges of molecular weight. It is also understood that a protein is a polymer in which amino acids are repeating structural units, even though the side chains of each amino acid may be different.

As used in this application, the term "polymeric" means a reactant or constituent comprising one or more polymers or a polymeric constituent/constituents. The polymeric reactant or constituent may optionally also comprise one or more other constituents, which are preferably selected from the group consisting of:

C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl; And

links selected from a group containing

Where

dotted lines indicate the addition of a component or reagent to the remainder, and

-R and Ra are independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

One skilled in the art understands that the polymerization products obtained from a polymerization reaction do not all have the same molecular weight, but rather have a molecular weight distribution. Therefore, molecular weight ranges, molecular weights, ranges of the number of monomers in a polymer, and the number of monomers in a polymer, as used in this application, refer to the number average molecular weight and the average number of monomers, i.e., the arithmetic mean of the molecular weight of the polymer or polymer fragment and the arithmetic mean of the number of monomers of the polymer or polymer fragment.

Accordingly, in a polymeric constituent comprising “x” monomer units, any whole number given for “x” therefore corresponds to the arithmetic average number of monomers. Any range of whole numbers given for “x” provides the range of whole numbers in which the arithmetic average number of monomers lies. An integer for “x” given as “about x” means that the arithmetic average numbers of monomers lie in the range of whole numbers x +/- 10%, preferably x +/- 8%, more preferably x +/- 5%, and most preferably x +/- 2%.

As used in this application, the term “number average molecular weight” means the simple arithmetic mean of the molecular weights of the individual polymers.

As used in this application, the term "water-soluble" with reference to a carrier means that when such carrier is part of the PTH prodrug of the present invention, at least 1 g of PTH prodrug containing such water-soluble carrier can be dissolved in one liter of water at 20°C to form a homogeneous solution. Accordingly, the term "water-insoluble" with reference to a carrier means that when such carrier is part of the PTH prodrug of the present invention, less than 1 g of PTH prodrug containing such water-soluble carrier can be dissolved in one liter of water at 20°C to form a homogeneous solution.

As used in this application, the term "hydrogel" means a hydrophilic or amphiphilic polymer network composed of homopolymers or copolymers that is insoluble due to the presence of hydrogen bonds, ionic interactions, and/or covalent chemical cross-links. The cross-links provide the network structure and physical integrity.

As used in this application, the term "thermogel" means a compound that is a low viscosity liquid or solution having a viscosity of less than 500 cP at 25°C at a shear rate of about 0.1/s at a low temperature, the low temperature being in the range of about 0°C to about 10°C, but which is a compound with a higher viscosity of less than 10,000 cP at 25°C at a shear rate of about 0.1/s at a higher temperature, the higher temperature being in the range of about 30°C to about 40°C, for example at a temperature of about 37°C.

As used in this application, the term “PEG-based” with respect to a moiety or reactant means that said moiety or reactant contains PEG. Preferably, the PEG-based moiety or reactant comprises at least 10% (w/w) PEG, such as at least 20% (w/w) PEG, such as at least 30% (w/w) PEG, such as at least 40% (w/w) PEG, such as at least 50% (w/w), such as at least 60 (w/w) PEG, such as at least 70% (w/w) PEG, such as at least 80% (w/w) PEG, such as at least 90% (w/w) PEG, such as at least 95%. The remaining weight percent of the PEG-based constituent or reactant is comprised of other constituents, preferably selected from the following constituents and bonds:

C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl; and

links selected from a group containing

Where

dotted lines indicate the addition of a component or reagent to the remainder, and

-R and Ra are independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

As used in this application, the term "PEG-based comprising at least X% PEG" with respect to a moiety or reactant means that said moiety or reactant comprises at least X% (w/w) of ethylene glycol units (-CH2CH2O-), wherein the ethylene glycol units may be arranged in a block, alternating, or random arrangement in the moiety or reactant, and preferably all of the ethylene glycol units of said moiety or reactant are present in a single block; the remaining weight percent of the PEG-based moiety or reactant is comprised of other moieties, preferably selected from the following moieties and bonds:

C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, phenyl, naphthyl, indenyl, indanyl and tetralinyl; and

links selected from a group containing

Where

dotted lines indicate the addition of a component or reagent to the remaining part, and

-R and Ra are independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

The term “hyaluronic acid based, containing at least X% hyaluronic acid” is used accordingly.

The term “substituted” as used in this application means that one or more hydrogen atoms of a molecule or moiety are replaced by another atom or group of atoms, which are designated as a “substituent.”

Preferably, the one or more additional optional substituents are independently selected from the group consisting of halogen, -CN, -COORx1, -ORx1, -C(O)Rx1, -C(O)N(Rx1Rx1a), -S(O)2N(Rx1Rx1a), -S(O)N(Rx1Rx1a), -S(O)2Rx1, -S(O)Rx1, -N(Rx1)S(O)2N(Rx1aRx1b), -SRx1, -N(Rx1Rx1a), -NO2, -OC(O)Rx1, -N(Rx1)C(O)Rx1a, -N(Rx1)S(O)2Rx1a, -N(Rx1)S(O)Rx1a, -N(Rx1)C(O)ORx1a, -N(Rx1)C(O)N(Rx1aRx1b), -OC(O)N(Rx1Rx1a), -T0, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl; wherein -T0, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally substituted with one or more -Rx2, which are the same or different, and wherein the C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T0-, -C(O)O-, -O-, -C(O)-, -C(O)N(Rx3)-, -S(O)2N(Rx3)-, -S(O)N(Rx3)-, -S(O)2-, -S(O)-, -N(Rx3)S(O)2N(Rx3a)-, -S-, -N(Rx3)-, -OC(ORx3)(Rx3a)-, -N(Rx3)C(O)N(Rx3a)- and -OC(O)N(Rx3)-;

-Rx1, -Rx1a, -Rx1b are independently selected from the group consisting of -H, -T0, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl; wherein -T0, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally substituted with one or more substituents -Rx2, which are the same or different, and wherein C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T0-, -C(O)O-, -O-, -C(O)-, -C(O)N(Rx3)-, -S(O)2N(Rx3)-, -S(O)N(Rx3)-; -S(O)2-, -S(O)-, -N(Rx3)S(O)2N(Rx3a)-, -S-, -N(Rx3)-, -OC(ORx3)(Rx3a)-, -N(Rx3)C(O)N(Rx3a)- and -OC(O)N(Rx3)-;

each T0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl; wherein each T0 is independently optionally substituted with one or more -Rx2 substituents that are the same or different;

each -Rx2 is independently selected from the group consisting of halogen, -CN, oxo (=O), -COORx4, -ORx4, -C(O)Rx4, -C(O)N(Rx4Rx4a), -S(O)2N(Rx4Rx4a), -S(O)N(Rx4Rx4a), -S(O)2Rx4, -S(O)Rx4, -N(Rx4)S(O)2N(Rx4aRx4b), -SRx4, -N(Rx4Rx4a), -NO2, -OC(O)Rx4, -N(Rx4)C(O)Rx4a, -N(Rx4)S(O)2Rx4a, -N(Rx4)S(O)Rx4a, -N(Rx4)C(O)ORx4a, -N(Rx4)C(O)N(Rx4aRx4b), -OC(O)N(Rx4Rx4a) and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogen substituents which are the same or different;

each -Rx3, -Rx3a, -Rx4, -Rx4a, -Rx4b is independently selected from the group consisting of -H and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogen substituents that are the same or different.

More preferably, one or more additional optional substituents are independently selected from the group consisting of halogen, -CN, -COORx1, -ORx1, -C(O)Rx1, -C(O)N(Rx1Rx1a), -S(O)2N(Rx1Rx1a), -S(O)N(Rx1Rx1a), -S(O)2Rx1, -S(O)Rx1, -N(Rx1)S(O)2N(Rx1aRx1b), -SRx1, -N(Rx1Rx1a), -NO2, -OC(O)Rx1, -N(Rx1)C(O)Rx1a, -N(Rx1)S(O)2Rx1a, -N(Rx1)S(O)Rx1a, -N(Rx1)C(O)ORx1a, -N(Rx1)C(O)N(Rx1aRx1b), -OC(O)N(Rx1Rx1a), -T0, C1-10 alkyl, C2-10 alkenyl and C2-10 alkynyl; wherein -T0, C1-10 alkyl, C2-10 alkenyl and C2-10 alkynyl are optionally substituted with one or more -Rx2, which are the same or different, and wherein the C1-10 alkyl, C2-10 alkenyl and C2-10 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T0-, -C(O)O-, -O-, -C(O)-, -C(O)N(Rx3)-, -S(O)2N(Rx3)-, -S(O)N(Rx3)-, -S(O)2-, -S(O)-, -N(Rx3)S(O)2N(Rx3a)-, -S-, -N(Rx3)-, -OC(ORx3)(Rx3a)-, -N(Rx3)C(O)N(Rx3a)- and -OC(O)N(Rx3)-;

each -Rx1, -Rx1a, -Rx1b, -Rx3, -Rx3a is independently selected from the group consisting of H, halogen, C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl;

each T0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl; wherein each T0 is independently optionally substituted with one or more -Rx2, which are the same or different;

each -Rx2 is independently selected from the group consisting of halogen, -CN, oxo (=O), -COORx4, -ORx4, -C(O)Rx4, -C(O)N(Rx4Rx4a), -S(O)2N(Rx4Rx4a), -S(O)N(Rx4Rx4a), -S(O)2Rx4, -S(O)Rx4, -N(Rx4)S(O)2N(Rx4aRx4b), -SRx4, -N(Rx4Rx4a), -NO2, -OC(O)Rx4, -N(Rx4)C(O)Rx4a, -N(Rx4)S(O)2Rx4a, -N(Rx4)S(O)Rx4a, -N(Rx4)C(O)ORx4a, -N(Rx4)C(O)N(Rx4aRx4b), -OC(O)N(Rx4Rx4a), and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogens which are the same or different;

each -Rx4, -Rx4a, -Rx4b is independently selected from the group consisting of H, halogen, C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl;

Even more preferably, one or more additional optional substituents are independently selected from the group consisting of halogen, -CN, -COORx1, -ORx1, -C(O)Rx1, -C(O)N(Rx1Rx1a), -S(O)2N(Rx1Rx1a), -S(O)N(Rx1Rx1a), -S(O)2Rx1, -S(O)Rx1, -N(Rx1)S(O)2N(Rx1aRx1b), -SRx1, -N(Rx1Rx1a), -NO2, -OC(O)Rx1, -N(Rx1)C(O)Rx1a, -N(Rx1)S(O)2Rx1a, -N(Rx1)S(O)Rx1a, -N(Rx1)C(O)ORx1a, -N(Rx1)C(O)N(Rx1aRx1b), -OC(O)N(Rx1Rx1a), -T0, C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl; wherein -T0, C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl are optionally substituted with one or more -Rx2, which are the same or different, and wherein the C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl are optionally interrupted by one or more groups selected from the group consisting of -T0-, -C(O)O-, -O-, -C(O)-, -C(O)N(Rx3)-, -S(O)2N(Rx3)-, -S(O)N(Rx3)-, -S(O)2-, -S(O)-, -N(Rx3)S(O)2N(Rx3a)-, -S-, -N(Rx3)-, -OC(ORx3)(Rx3a)-, -N(Rx3)C(O)N(Rx3a)-, and -OC(O)N(Rx3)-;

each -Rx1, -Rx1a, -Rx1b, -Rx2, -Rx3, -Rx3a is independently selected from the group consisting of H, halogen, C1-6 alkyl, C2-6 alkenyl and C2-6 alkynyl;

each T0 is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl; wherein each T0 is independently optionally substituted with one or more -Rx2, which are the same or different.

Preferably, a maximum of 6 -H atoms of the optionally substituted molecule are independently substituted with a substituent, for example, 5 -H atoms are independently substituted with a substituent, 4 -H atoms are independently substituted with a substituent, 3 -H atoms are independently substituted with a substituent, 2 -H atoms are independently substituted with a substituent, or 1 -H atom is substituted with a substituent.

The term “interrupted” means that the moiety is inserted between two carbon atoms or, if the insertion is at one end of the constituent, between a carbon atom or heteroatom and a hydrogen atom, preferably between a carbon atom and a hydrogen atom.

The term “spacer” refers to any moiety that is suitable for connecting two moieties. Preferably, the spacer is selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(Ry1)-, -S(O)2N(Ry1)-, -S(O)N(Ry1)-, -S(O)2-, -S(O)-, -N(Ry1)S(O)2N(Ry1a)-, -S-, -N(Ry1)-, -OC(ORy1)(Ry1a)-, -N(Ry1)C(O)N(Ry1a)-, -OC(O)N(Ry1)-, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl; wherein -T-, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally substituted with one or more -Ry2, which are the same or different, and wherein the C1-50 alkyl, C2-50 alkenyl and C2-50 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(Ry3)-, -S(O)2N(Ry3)-, -S(O)N(Ry3)-, -S(O)2-, -S(O)-, -N(Ry3)S(O)2N(Ry3a)-, -S-, -N(Ry3)-, -OC(ORy3)(Ry3a)-, -N(Ry3)C(O)N(Ry3a)-, and -OC(O)N(Ry3)-;

-Ry1 and -Ry1a are independently selected from the group consisting of H, -T, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl; wherein -T, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally substituted with one or more -Ry2 that are the same or different, and wherein the C1-50 alkyl, C2-50 alkenyl and C2-50 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(Ry4)-,
-S(O)2N(Ry4)-, -S(O)N(Ry4)-, -S(O)2-, -S(O)-, -N(Ry4)S(O)2N(Ry4a)-, -S-, -N(Ry4)-, -OC(ORy4)(Ry4a)-, -N(Ry4)C(O)N(Ry4a)-, and -OC(O)N(Ry4)-;

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl; wherein each T is independently optionally substituted with one or more -Ry2 that are the same or different;

each -Ry2 is independently selected from the group consisting of halogen, -CN, oxo (=O), -COORy5, -ORy5, -C(O)Ry5, -C(O)N(Ry5Ry5a), -S(O)2N(Ry5Ry5a), -S(O)N(Ry5Ry5a), -S(O)2Ry5, -S(O)Ry5, -N(Ry5)S(O)2N(Ry5aRy5b), -SRy5, -N(Ry5Ry5a), -NO2, -OC(O)Ry5, -N(Ry5)C(O)Ry5a, -N(Ry5)S(O)2Ry5a, -N(Ry5)S(O)Ry5a, -N(Ry5)C(O)ORy5a, -N(Ry5)C(O)N(Ry5aRy5b), -OC(O)N(Ry5Ry5a), and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogens which are the same or different; and

each -Ry3, -Ry3a, -Ry4, -Ry4a, -Ry5, -Ry5a and Ry5b is independently selected from the group consisting of H, and C1-6 alkyl, wherein C1-6 alkyl is optionally substituted with one or more halogens that are the same or different.

Even more preferably, the spacer is selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(Ry1)-, -S(O)2N(Ry1)-, -S(O)N(Ry1)-, -S(O)2-, -S(O)-, -N(Ry1)S(O)2N(Ry1a)-, -S-, -N(Ry1)-, -OC(ORy1)(Ry1a)-, -N(Ry1)C(O)N(Ry1a)-, -OC(O)N(Ry1)-, C1-50 alkyl, C2-50 alkenyl, and C2-50 alkynyl; wherein -T-, C1-20 alkyl, C2-20 alkenyl and C2-20 alkynyl are optionally substituted with one or more -Ry2, which are the same or different, and wherein the C1-20 alkyl, C2-20 alkenyl and C2-20 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(Ry3)-, -S(O)2N(Ry3)-, -S(O)N(Ry3)-, -S(O)2-, -S(O)-, -N(Ry3)S(O)2N(Ry3a)-, -S-, -N(Ry3)-, -OC(ORy3)(Ry3a)-, -N(Ry3)C(O)N(Ry3a)-, and -OC(O)N(Ry3)-;

-Ry1 and -Ry1a are independently selected from the group consisting of H, -T, C1-10 alkyl, C2-10 alkenyl and C2-10 alkynyl; wherein -T, C1-10 alkyl, C2-10 alkenyl and C2-10 alkynyl are optionally substituted with one or more -Ry2, which are the same or different, and wherein C1-10 alkyl, C2-10 alkenyl and C2-10 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(Ry4)-, -S(O)2N(Ry4)-, -S(O)N(Ry4)-, -S(O)2-, -S(O)-, -N(Ry4)S(O)2N(Ry4a)-, -S-, -N(Ry4)-, -OC(ORy4)(Ry4a)-, -N(Ry4)C(O)N(Ry4a)- and -OC(O)N(Ry4)-;

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl; wherein each T is independently optionally substituted with one or more -Ry2 that are the same or different;

-Ry2 is selected from the group consisting of halogen, -CN, oxo (=O), -COORy5, -ORy5, -C(O)Ry5, -C(O)N(Ry5Ry5a), -S(O)2N(Ry5Ry5a), -S(O)N(Ry5Ry5a), -S(O)2Ry5, -S(O)Ry5, -N(Ry5)S(O)2N(Ry5aRy5b), -SRy5, -N(Ry5Ry5a), -NO2, -OC(O)Ry5, -N(Ry5)C(O)Ry5a, -N(Ry5)S(O)2Ry5a, -N(Ry5)S(O)Ry5a, -N(Ry5)C(O)ORy5a, -N(Ry5)C(O)N(Ry5aRy5b), -OC(O)N(Ry5Ry5a), and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogens which are the same or different; and

each -Ry3, -Ry3a, -Ry4, -Ry4a, -Ry5, -Ry5a and Ry5b is independently of each other selected from the group consisting of H, and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogens that are the same or different.

Even more preferably, the spacer is selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(Ry1)-, -S(O)2N(Ry1)-, -S(O)N(Ry1)-, -S(O)2-, -S(O)-, -N(Ry1)S(O)2N(Ry1a)-, -S-, -N(Ry1)-, -OC(ORy1)(Ry1a)-, -N(Ry1)C(O)N(Ry1a)-, -OC(O)N(Ry1)-, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl; wherein -T-, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally substituted with one or more -Ry2, which are the same or different, and wherein the C1-50 alkyl, C2-50 alkenyl and C2-50 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(Ry3)-, -S(O)2N(Ry3)-, -S(O)N(Ry3)-, -S(O)2-, -S(O)-, -N(Ry3)S(O)2N(Ry3a)-, -S-, -N(Ry3)-, -OC(ORy3)(Ry3a)-, -N(Ry3)C(O)N(Ry3a)-, and -OC(O)N(Ry3)-;

-Ry1 and -Ry1a are independently selected from the group consisting of H, -T, C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl;

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl;

each -Ry2 is independently selected from the group consisting of halogen and C1-6 alkyl; and

each -Ry3, -Ry3a, -Ry4, -Ry4a, -Ry5, -Ry5a and Ry5b is independently selected from the group consisting of -H and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogens that are the same or different.

As used herein, the term “C1-4 alkyl” by itself or in combination means a straight or branched alkyl moiety having from 1 to 4 carbon atoms. When present at the end of a molecule, examples of straight or branched C1-4 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. When two constituent molecules are linked by C1-4 alkyl, examples of such C1-4 alkyl groups include -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)-, -C(CH3)2-. Each hydrogen of the C1-4 alkyl carbon may optionally be substituted with a substituent as defined above. Optionally, the C1-4 alkyl may be interrupted by one or more moieties as defined below.

As used herein, the term “C1-6 alkyl” by itself or in combination means a straight or branched alkyl moiety having from 1 to 6 carbon atoms. When present at the end of a molecule, examples of straight or branched C1-6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methylbutyl, 2,2-dimethylpropyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl and 3,3-dimethylpropyl. When two constituent molecules are linked by a C1-6 alkyl group, examples of such C1-6 alkyl groups are -CH2-, -CH2-CH2-, -CH(CH3)-, -CH2-CH2-CH2-, -CH(C2H5)- and C(CH3)2-. Each hydrogen atom on a C1-6 carbon atom may optionally be substituted with a substituent as defined above. Optionally, the C1-6 alkyl may be interrupted by one or more moieties as defined below.

Accordingly, “C1-10 alkyl,” “C1-20 alkyl,” or “C1-50 alkyl” means an alkyl chain having from 1 to 10, from 1 to 20, or from 1 to 50 carbon atoms, respectively, wherein each hydrogen atom at a C1-10, C1-20, or C1-50 carbon atom may optionally be substituted with a substituent as defined above. Optionally, a C1-10 or C1-50 alkyl may be interrupted by one or more moieties as defined below.

As used herein, the term “C2-6 alkenyl” by itself or in combination means a straight or branched hydrocarbon moiety containing at least one carbon-carbon double bond having from 2 to 6 carbon atoms. When present at the end of a molecule, examples are CH=CH2, -CH=CH-CH3, -CH2-CH=CH2, -CH=CHCH2-CH3 and -CH=CH-CH=CH2. When two molecular moieties are linked by a C2-6 alkenyl group, then an example of such a C2-6 alkenyl is -CH=CH-. Each hydrogen atom of a C2-6 alkenyl moiety may optionally be substituted with a substituent as defined above. Optionally, a C2-6 alkenyl may be interrupted by one or more moieties as defined below.

Accordingly, the term “C2-10 alkenyl,” “C2-20 alkenyl,” or “C2-50 alkenyl,” by itself or in combination, means a straight-chain or branched hydrocarbon moiety containing at least one carbon-carbon double bond having from 2 to 10, from 2 to 20, or from 2 to 50 carbon atoms. Each hydrogen atom of a C2-10 alkenyl, C2-20 alkenyl, or C2-50 alkenyl group may optionally be substituted with a substituent as defined above. Optionally, a C2-10 alkenyl, C2-20 alkenyl, or C2-50 alkenyl may be interrupted by one or more moieties as defined below.

As used herein, the term “C2-6 alkynyl” by itself or in combination means a straight or branched hydrocarbon moiety containing at least one carbon-carbon triple bond having from 2 to 6 carbon atoms. When present at the end of the molecule, examples are -CєCH, -CH2-Cє CH, CH2-CH2-Cє CH and CH2-Cє C-CH3. When two molecular constituents are linked by an alkynyl group, then an example is -Cє C-. Each hydrogen atom of the C2-6 alkynyl group may optionally be substituted with a substituent as defined above. Optionally, one or more double bonds may be present. Optionally, the C2-6 alkynyl may be interrupted by one or more moieties as defined below.

Accordingly, as used herein, the term “C2-10 alkynyl,” “C2-20 alkynyl,” and “C2-50 alkynyl” by itself or in combination means a straight or branched hydrocarbon moiety containing at least one carbon-carbon triple bond having from 2 to 10, from 2 to 20, or from 2 to 50 carbon atoms, respectively. Each hydrogen atom of a C2-10 alkynyl, C2-20 alkynyl, or C2-50 alkynyl group may optionally be substituted with a substituent as defined above. Optionally, one or more double bonds may be present. Optionally, a C2-10 alkynyl, C2-20 alkynyl, or C2-50 alkynyl may be interrupted by one or more moieties as defined below.

As mentioned above, the C1-4 alkyl, C1-6 alkyl, C1-10 alkyl, C1-20 alkyl, C1-50 alkyl, C2-6 alkenyl, C2-10 alkenyl, C2-20 alkenyl, C2-50 alkenyl, C2-6 alkynyl, C2-10 alkynyl, C2-20 alkenyl or C2-50 alkynyl may optionally be interrupted by one or more moieties, which are preferably selected from the group consisting of

Where

dotted lines represent addition to the remainder of the constituent or reactant; and

-R and Ra are independently selected from the group consisting of -H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

As used herein, the term "C3-10 cycloalkyl" means a cyclic alkyl chain having from 3 to 10 carbon atoms, which may be saturated or unsaturated, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl or cyclodecyl. Each hydrogen atom of the C3-10 cycloalkyl carbon may be substituted with a substituent as defined above. The term "C3-10 cycloalkyl" also includes bridged bicycles such as norbornane or norbornene.

The term “8- to 30-membered carbopolycyclyl” or “8- to 30-membered carbopolycycle” means a cyclic moiety of two or more rings with 8 to 30 ring atoms, wherein two adjacent rings share at least one ring atom, and which may contain up to a maximum number of double bonds (an aromatic or non-aromatic ring that is fully saturated, partially unsaturated, or unsaturated). Preferably, 8- to 30-membered carbopolycyclyl means a cyclic moiety of two, three, four, or five rings, more preferably two, three, or four rings.

As used herein, the term "3- to 10-membered heterocyclyl" or "3- to 10-membered heterocycle" means a ring with 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms that may contain up to the maximum number of double bonds (an aromatic or non-aromatic ring that is fully saturated, partially unsaturated or unsaturated), wherein at least one ring atom to four ring atoms are substituted with a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-), and wherein the ring is bonded to the remainder of the molecule through a carbon or nitrogen atom. Examples of 3-10 membered heterocycles include, but are not limited to, aziridine, oxirane, thirane, azirine, oxirene, thiirene, azetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline, isoxazole, isoxazoline, thiazole, thiazoline, isothiazole, isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran, tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran, imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine, piperidine, morpholine, tetrazole, triazole, triazolidine, tetrazolidine, diazepane, azepine or homopiperazine. Each hydrogen atom of a 3- to 10-membered heterocyclyl or 3- to 10-membered heterocyclic group may be substituted with a substituent as defined below.

As used herein, the term "8-11 membered heterobicyclyl" or "8-11 membered heterobicycle" means a heterocyclic moiety of two rings with 8 to 11 ring atoms, wherein at least one ring atom is shared between the two rings, and which contains up to the maximum number of double bonds (an aromatic or non-aromatic ring that is fully saturated, partially unsaturated, or unsaturated), wherein at least one ring atom up to six ring atoms are substituted with a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen, and nitrogen (including =N(O)-), and wherein the ring is bonded to the remainder of the molecule through a carbon or nitrogen atom. Examples of an 8- to 11-membered heterobicycle are indole, indoline, benzofuran, benzothiophene, benzoxazole, benzisoxazole, benzothiazole, benzisothiazole, benzimidazole, benzimidazole, benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline, dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline, decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline, benzazepine, purine or pteridine. The term 8- to 11-membered heterobicycle also includes spiro structures of two rings such as 1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles such as 8-azabicyclo[3.2.1]octane. Each hydrogen atom of an 8- to 11-membered heterobicyclyl or 8- to 11-membered heterobicycle may be substituted with a substituent as defined below.

Similarly, the term “8- to 30-membered heteropolycyclyl” or “8- to 30-membered heteropolycycle” means a heterocyclic moiety of more than two rings with 8 to 30 ring atoms, preferably three, four or five rings, wherein two adjacent rings share at least one ring atom and which may contain up to a maximum number of double bonds (an aromatic or non-aromatic ring that is fully saturated, partially unsaturated or unsaturated), wherein at least one ring atom to 10 ring atoms are substituted with a heteroatom selected from the group consisting of sulfur (including -S(O)-, -S(O)2-), oxygen and nitrogen (including =N(O)-), and wherein the ring is bonded to the remainder of the molecule through a carbon or nitrogen atom.

It is understood that the phrase “the Rx/Ry pair joins together with the atom to which they are attached to form a C3-10 cycloalkyl or a 3-10 membered heterocyclyl” in relation to the constituent structure

means that Rx and Ry form the following structure:

,

where R is C3-10 cycloalkyl or 3-10 membered heterocyclyl.

It is also clear that the phrase “the Rx/Ry pair combines with the atom to which they are attached to form a ring A” in relation to the constituent structure

means that Rx and Ry form the following structure:

.

As used in this application, the term “terminal alkyne” means a moiety

.

As used in this application, "halogen" means fluorine, chlorine, bromine or iodine. In general, it is preferred that the halogen is fluorine or chlorine.

In general, the term “comprises” or “containing” also encompasses “consists of” or “consisting of.”

It is clear that in formula (Ia) and (Ib) -D is connected to -L1- by means of a covalent or reversible bond.

Another object of the present invention is a prodrug of PTH or a pharmaceutically acceptable salt thereof comprising a D-L conjugate, wherein

-D represents the PTH component; and

-L contains a reversible prodrug linker moiety -L1-, which is a -L1- moiety linked to a PTH moiety -D via a PTH functional group;

where -L1- is substituted by -L2-Z’ and is optionally further substituted; where

-L2- represents a single chemical bond or spacer moiety; and

-Z’ is a water-soluble carrier component.

It is clear that the set of components -L2-L1-D is connected to a water-insoluble carrier -Z’, and that the bond between -D and L1- is covalent and reversible.

Preferably, -D has the sequence of SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114 or SEQ ID NO:115. More preferably, -D has the sequence of SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:110, SEQ ID NO:111 or SEQ ID NO:112.

In one embodiment of the present invention, -D has the sequence SEQ ID NO:50.

In another embodiment of the present invention, -D has the sequence SEQ ID NO:52.

In another embodiment of the present invention, -D has the sequence SEQ ID NO:110.

In another embodiment of the present invention, -D has the sequence SEQ ID NO:111.

In another embodiment of the present invention, -D has the sequence SEQ ID NO:112.

Most preferably, -D has the sequence SEQ ID NO:51.

The -L1- moiety is either conjugated to a side chain functional group of an amino acid residue -D, to an N-terminal amine functional group or to a C-terminal carboxyl functional group of -D, or to a nitrogen atom of the polypeptide backbone -D. Attachment to either the N-terminus or the C-terminus can be either directly via the corresponding amine or carboxyl functional group, respectively, or indirectly, when the spacer moiety is first conjugated to the amine or carboxyl functional group to which the -L1- spacer moiety is conjugated.

Preferably, the amino acid residue of PTH to which -L1- is conjugated comprises a functional group selected from the group consisting of carboxylic acid, primary and secondary amine, maleimide, thiol, sulfonic acid, carbonate, carbamate, hydroxyl, aldehyde, ketone, hydrazine, isocyanate, isothiocyanate, phosphoric acid, phosphonic acid, haloacetyl, alkyl halide, acryloyl, aryl fluoride, hydroxylamine, sulfate, disulfide, vinyl sulfone, vinyl ketone, diazoalkane, oxirane, guanidine and aziridine. Even more preferably, the amino acid residue of PTH to which -L1- is conjugated comprises a functional group selected from the group consisting of hydroxyl, primary and secondary amine and guanidine. Even more preferably, the amino acid residue of PTH to which -L1- is conjugated comprises a primary or secondary amine functional group. Most preferably, the amino acid residue of PTH to which -L1- is conjugated comprises a primary amine functional group.

If the -L1- moiety is conjugated to a functional group of the side chain of an amino acid residue of PTH, said amino acid residue is selected from the group consisting of proteogenic amino acid residues and non-proteogenic amino acid residues.

In one embodiment of the present invention, -L1- is conjugated to a functional group of the side chain of a non-proteogenic amino acid residue of PTH. It is understood that such a non-proteogenic amino acid is not found in the sequence of natural PTH or its fragments, and that it can only be present in variants, analogs, orthologs, homologs and derivatives of PTH.

In another embodiment of the present invention, -L1- is conjugated to a functional group of the side chain of a proteogenic acid residue in PTH. Preferably, said amino acid is selected from the group consisting of consisting of histidine, lysine, tryptophan, serine, threonine, tyrosine, aspartic acid, glutamic acid and arginine. Even more preferably, said amino acid is selected from the group consisting of consisting of lysine, aspartic acid, arginine and serine. Even more preferably, said amino acid is selected from the group consisting of consisting of lysine, arginine and serine.

In one embodiment of the present invention, -L1- is conjugated to the histidine side chain functional group of PTH.

In another embodiment of the present invention, -L1- is conjugated to a lysine side chain functional group of PTH.

In another embodiment of the present invention, -L1- is conjugated to a functional group of the side chain of tryptophan PTH.

In another embodiment of the present invention, -L1- is conjugated to the serine side chain functional group of PTH.

In another embodiment of the present invention, -L1- is conjugated to the threonine side chain functional group of PTH.

In another embodiment of the present invention, -L1- is conjugated to a tyrosine side chain functional group of PTH.

In another embodiment of the present invention, -L1- is conjugated to the aspartic acid side chain functional group of PTH.

In another embodiment of the present invention, -L1- is conjugated to the glutamic acid side chain functional group of PTH.

In another embodiment of the present invention, -L1- is conjugated to the arginine side chain functional group of PTH.

It is clear that not every component of PTH can contain all of these amino acid residues.

In a preferred embodiment of the present invention, -L1- is conjugated to the N-terminal amine functional group of PTH, either directly via the corresponding amine functional group, or indirectly, when a spacer moiety is first conjugated to the amine functional group to which the spacer moiety -L1- is conjugated. Even more preferably, -L1- is directly conjugated to the N-terminal amine functional group of PTH, preferably PTH 1-34, i.e. PTH having the sequence SEQ ID NO:51.

Surprisingly, it was found that the N-terminal attachment of L1 is preferred, i.e., the attachment of L1 to the N-terminus of PTH, since such an attachment site was found to protect the N-terminus, which is essential for the activity of PTH. Furthermore, it was surprisingly found that the major metabolite formed from the PTH prodrug with N-terminal attachment of L1 is PTH 1-33, i.e., the 33 N-terminal amino acids of PTH, the metabolite of which is known to be active.

In another embodiment of the present invention, -L1- is conjugated to the C-terminal functional group of PTH either directly via the corresponding carboxyl functional group, or indirectly when the spacer moiety is first conjugated to the carboxyl functional group to which the spacer moiety -L1- is conjugated.

Most preferably, L1- is directly conjugated to the N-terminal amine functional group of PTH.

The -L1- moiety may be bonded to -D via any type of bond, provided that it is reversible. Preferably, -L1- is bonded to -D via a bond selected from the group consisting of an amide, an ester, a carbamate, an acetal, an aminal, an imine, an oxime, a hydrazone, a disulfide, and an acylguanidine. Even more preferably, -L1- is bonded to -D via a bond selected from the group consisting of an amide, an ester, a carbamate, and an acylguanidine. It is understood that some of these bonds are not reversible in themselves, but in the present invention, the adjacent groups contained in L1 make these bonds reversible.

In one embodiment of the present invention, -L1- is linked to -D via an ether linkage.

In another embodiment of the present invention, -L1- is linked to -D via a carbamate linkage.

In another embodiment of the present invention, -L1- is linked to -D via an acylguanidine linkage.

In a preferred embodiment of the present invention, -L1- is linked to -D via an amide bond.

The -L1- moiety is a reversible prodrug linker from which the drug, i.e. PTH, is released in its free form, i.e. it is a traceless prodrug linker. Suitable prodrug linkers are known in the art, such as reversible prodrug linker moieties disclosed in WO 2005/099768 A2, WO 2006/136586 A2, WO 2011/089216 A1 and WO 2013/024053 A1, which are incorporated herein by reference.

In another embodiment of the present invention, -L1- is a reversible prodrug linker as described in WO 2011/012722 A1, WO 2011/089214 A1, WO 2011/089215 A1, WO 2013/024052 A1 and WO 2013/160340 A1, which are incorporated herein by reference.

A particularly preferred moiety -L1- is disclosed in WO 2009/095479 A2. Accordingly, in a preferred embodiment of the present invention, the moiety -L1- has the formula (II):

,

where the dotted line represents the attachment of a -D moiety to a nitrogen, hydroxyl or thiol atom, which is the PTH moiety;

-X- is -C(R4R4a)-; -N(R4)-; -O-; -C(R4R4a)-C(R5R5a)-; -C(R5R5a)-C(R4R4a)-; -C(R4R4a)-N(R6)-; -N(R6)-C(R4R4a)-; -C(R4R4a)-O-; -O-C(R4R4a)-; or C(R7R7a)-;

X1 is C; or S(O);

-X2- represents -C(R8R8a)-; or C(R8R8a)-C(R9R9a)-;

=X3 represents =O; =S; or =N-CN;

-R1, -R1a, -R2, -R2a, -R4, -R4a, -R5, -R5a, -R6, -R8, -R8a, -R9, -R9a are independently selected from the group consisting of -H; and C1-6 alkyl;

-R3, -R3a are independently selected from the group consisting of -H; and C1-6 alkyl, provided that if one or both of -R3, -R3a are other than hydrogen, they are bonded to N to which they are attached through an SP3-hybridized carbon atom;

-R7 is -N(R10R10a); or -NR10-(C=O)-R11;

-R7a, -R10, -R10a, -R11 independently of one another represent -H; or C1-6 alkyl;

optionally, one or more of the pairs -R1a/-R4a, -R1a/-R5a, -R1a/-R7a, -R4a/-R5a, -R8a/-R9a form a chemical bond;

optionally, one or more of the pairs -R1/-R1a, -R2/-R2a, -R4/-R4a, -R5/-R5a, -R8/-R8a, -R9/-R9a join together with the atom to which they are attached to form a C3-10 cycloalkyl; or a 3-10 membered heterocyclyl;

optionally, one or more of the pairs -R1/-R4, -R1/-R5, -R1/-R6, -R1/-R7a, -R4/-R5, -R4/-R6, -R8/-R9, -R2/-R3 join together with the atoms to which they are attached to form ring A;

optionally, R3/R3a join together with the nitrogen atom to which they are attached to form a 3-10 membered heterocycle;

A is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C3-10 cycloalkyl; 3-10 membered heterocyclyl; and 8-11 membered heterobicyclyl; and

where -L1- has as a substituent -L2-Z or -L2-Z’, and where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (II) is not substituted by -L2-Z or -L2-Z’ or a substituent;

Where

-L2- represents a simple chemical bond or spacer;

-Z is a water-soluble carrier; and

-Z’ is a water-insoluble carrier.

Preferably, -L1- of formula (II) has as a substituent one constituent -L2-Z or -L2-Z’.

In one embodiment of the present invention, -L1- of formula (II) is further unsubstituted.

It is clear that if -R3/-R3a of formula (II) are joined together with the nitrogen atom to which they are attached to form a 3- to 10-membered heterocycle, only such 3- to 10-membered heterocycles can be formed in which the atoms directly attached to the nitrogen atom are SP3-hybridized carbon atoms. In other words, such a 3- to 10-membered heterocycle formed by -R3/-R3a together with the nitrogen atom to which they are attached has the following structure:

,

Where

the dotted line indicates the addition to the residue -L1-;

the ring contains from 3 to 10 atoms, including at least one nitrogen atom; and

R# and R## represent an SP3-hybridized carbon atom.

It is also clear that the 3-10 membered heterocycle can be further substituted.

Exemplary embodiments of suitable 3- to 10-membered heterocycles formed by -R3/-R3a of formula (II) together with the nitrogen atom to which they are attached are the following:

Where

the dotted line represents attachment to the remainder of the molecule; and

-R is selected from the group consisting of -H and C1-6 alkyl.

-L1- of formula (II) may optionally be further substituted. In general, any substituent may be used as long as the cleavage principle is not affected, i.e. the hydrogen marked with an asterisk in formula (II) is not replaced and the nitrogen of the constituent

formula (II) remains part of a primary, secondary or tertiary amine, i.e. -R3 and R3a independently of each other represent -H or are linked to -N< through an SP3-hybridized carbon atom.

In one embodiment of the present invention, -R1 or R1a of formula (II) is substituted with -L2-Z or L2-Z'. In another embodiment of the present invention, -R2 or R2a of formula (II) is substituted with -L2-Z or L2-Z'. In another embodiment of the present invention, -R3 or R3a of formula (II) is substituted with -L2-Z or L2-Z'. In another embodiment of the present invention, -R4 of formula (II) is substituted with -L2-Z or L2-Z'. In another embodiment of the present invention, -R5 or R5a of formula (II) is substituted with -L2-Z or L2-Z'. In another embodiment of the present invention, -R6 of formula (II) is substituted with -L2-Z or L2-Z'. In another embodiment of the present invention, -R7 or R7a of formula (II) is substituted with -L2-Z or L2-Z'. In another embodiment of the present invention, -R8 or R8a of formula (II) is substituted with -L2-Z or L2-Z'. In another embodiment of the present invention, -R9 or R9a of formula (II) is substituted with -L2-Z or L2-Z'. In another embodiment of the present invention, -R10 is substituted with -L2-Z or -L2-Z'. In another embodiment of the present invention, -R11 is substituted with -L2-Z or -L2-Z'.

Preferably, -X- of formula (II) is selected from the group consisting of -C(R4R4a)-, -N(R4)- and -C(R7R7a)-.

In one embodiment of the present invention, -X- of formula (II) is -C(R4R4a)-.

In one preferred embodiment of the present invention, X of formula (II) is -C(R7R7a)-.

Preferably, -R7 of formula (II) is -NR10-(C=O)-R11.

Preferably, -R7a of formula (II) is selected from H, methyl and ethyl. Most preferably, -R7a of formula (II) is -H.

Preferably, -R10 is selected from H, methyl and ethyl. Most preferably, -R10 is methyl.

Preferably, -R11 is selected from H, methyl and ethyl. Most preferably, -R11 is -H.

Preferably, -R11 is substituted with -L2-Z or -L2-Z’.

In another preferred embodiment of the present invention, -X- of formula (II) is -N(R4)-.

Preferably, -R4 is selected from the group consisting of H, methyl and ethyl. Preferably, -R4 is -H.

Preferably, X1 of formula (II) is C.

Preferably, =X3 of formula (II) represents =O.

Preferably, -X2- of formula (II) is -C(R8R8a)-.

Preferably, -R8 and -R8a of formula (II) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R8 and -R8a of formula (II) is -H. Even more preferably, both -R8 and -R8a of formula (II) are -H.

Preferably, -R1 and -R1a of formula (II) are independently selected from the group consisting of H, methyl and ethyl.

In one preferred embodiment of the present invention, at least one of -R1 and -R1a of formula (II) is -H, more preferably both -R1 and -R1a of formula (II) are -H.

In another preferred embodiment of the present invention, at least one of -R1 and -R1a of formula (II) is methyl, more preferably both -R1 and R1a of formula (II) are -methyl.

Preferably, -R2 and R2a of formula (II) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R2 and R2a of formula (II) is -H. Even more preferably, both -R2 and R2a of formula (II) are -H.

Preferably, -R3 and R3a of formula (II) are independently selected from the group consisting of H, methyl, ethyl, propyl and butyl.

In one preferred embodiment of the present invention, at least one of -R3 and -R3a of formula (II) is methyl, more preferably -R3 of formula (II) is methyl and R3a of formula (II) is -H.

In another preferred embodiment of the present invention, -R3 and -R3a of formula (II) are both -H.

Preferably, -D is linked to -L1- via a nitrogen atom by forming an amide bond.

In one preferred embodiment of the present invention, the constituent -L1- has the formula (IIa-i):

(IIa-i),

Where

where the dotted line represents the addition of the D moiety, which is the PTH moiety, to the nitrogen atom by forming an amide bond;

-R1, -R1a, -R2, -R2a, -R3, -R3a, -R7, -R7a and -X2- have the meanings as defined in formula (II); and

where -L1- is substituted by -L2-Z or L2-Z’, and where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIa-i) is not substituted by -L2-Z or -L2-Z’ or a substituent.

Preferably, -L1- of formula (IIa-i) is substituted by one moiety -L2-Z or -L2-Z’.

Preferably, the -L1- moiety of formula (IIa-i) is not further substituted.

Preferably, -R1 and R1a of formula (IIa-i) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R1 and -R1a of formula (IIa-i) is -H. Even more preferably, both -R1 and -R1a of formula (IIa-i) are -H.

Preferably, -R7 of formula (IIa-i) is -NR10-(C=O)-R11.

Preferably, -R7a of formula (II-i) is selected from H, methyl and ethyl. Most preferably, -R7a of formula (II-i) is -H.

Preferably, -R10 of formula (IIa-i) is selected from H, methyl and ethyl. Most preferably, -R10 of formula (IIa-i) is methyl.

Preferably, -R11 of formula (IIa-i) is selected from H, methyl and ethyl. Most preferably, -R11 of formula (IIa-i) is -H.

Preferably, -R11 of formula (IIa-i) is substituted with -L2-Z or L2-Z’.

Preferably, -X2- of formula (IIa-i) is -C(R8R8a)-.

Preferably, -R8 and -R8a of formula (IIa-i) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R8 and -R8a of formula (IIa-i) is -H. Even more preferably, both -R8 and -R8a of formula (IIa-i) are -H.

Preferably, -R2 and -R2a of formula (IIa-i) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R2 and -R2a of formula (IIa-i) is -H. Even more preferably, both -R2 and -R2a of formula (IIa-i) are -H.

Preferably, -R3 and -R3a of formula (IIa-i) are independently selected from the group consisting of H, methyl, ethyl, propyl and butyl. Even more preferably, at least one of -R3 and -R3a of formula (IIa-i) is methyl.

Preferably, -R3 of formula (IIa-i) is -H, and -R3a of formula (IIa-i) is methyl.

More preferably, the component -L1- has the formula (IIa-ii):

(IIa-ii),

where the dotted line represents the addition of the D moiety, which is the PTH moiety, to the nitrogen atom by forming an amide bond;

-R2, -R2a, -R10, -R11 and -X2- have the meanings as defined in formula (II); and

where -L1- is substituted by -L2-Z or L2-Z’ and where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIa-ii) is not substituted by -L2-Z or -L2-Z’ or a substituent.

Preferably, -L1- of formula (IIa-ii) is substituted by one moiety -L2-Z or -L2-Z’.

Preferably, the -L1- moiety of formula (IIa-ii) is not further substituted.

Preferably, -X2- of formula (IIa-ii) is -C(R8R8a)-.

Preferably, -R8 and -R8a of formula (IIa-ii) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R8 and R8a of formula (IIa-ii) is -H. Even more preferably, both -R8 and R8a of formula (IIa-ii) are -H.

Preferably, -R3 and R3a of formula (IIa-ii) are independently selected from the group consisting of H, methyl, ethyl, propyl and butyl. Even more preferably, at least one of -R3 and R3a of formula (IIa-ii) is methyl.

Preferably, -R3 of formula (IIa-ii) is -H, and R3a of formula (IIa-ii) is methyl.

Preferably, -R10 of formula (IIa-ii) is selected from H, methyl and ethyl. Most preferably, -R10 of formula (IIa-ii) is methyl.

Preferably, -R11 of formula (IIa-ii) is selected from H, methyl and ethyl. Most preferably, -R11 of formula (IIa-ii) is -H.

Preferably, -R11 of formula (IIa-ii) is substituted with -L2-Z or L2-Z’.

In an even more preferred embodiment of the present invention, the constituent -L1- has the formula (IIa-ii’):

(IIa-ii'),

Where

where the dotted line represents the addition of the D moiety, which is the PTH moiety, to the nitrogen atom by forming an amide bond;

the dotted line marked with an asterisk indicates attachment to -L2-;

-R3, -R3a, -R10 and X2- have the meanings as defined in formula (II); and

where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIa-ii’) is not substituted by a substituent.

Preferably, the -L1- moiety of formula (IIa-ii’) is not further substituted.

Preferably, -X2- of formula (IIa-ii’) is -C(R8R8a)-.

Preferably, -R8 and R8a of formula (IIa-ii') are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R8 and R8a of formula (IIa-ii') is -H. Even more preferably, both -R8 and R8a of formula (IIa-ii') are -H.

Preferably, -R3 and R3a of formula (IIa-ii') are independently selected from the group consisting of H, methyl, ethyl, propyl and butyl. Even more preferably, at least one of -R3 and R3a of formula (IIa-ii') is methyl.

Preferably, -R3 of formula (IIa-ii’) is -H, and R3a of formula (IIa-ii’) is methyl.

Preferably, -R10 of formula (IIa-ii') is selected from H, methyl and ethyl. Most preferably, -R10 of formula (IIa-ii') is methyl.

Even more preferably, the -L1- component has the formula (IIa-iii):

(IIa-iii),

where the dotted line represents the addition to the nitrogen atom of the D moiety, which is the PTH moiety, by forming an amide bond; and

where -L1- is substituted by -L2-Z or -L2-Z’, and where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIa-iii) is not substituted by -L2-Z or -L2-Z’ or a substituent.

Preferably, -L1- of formula (IIa-iii) is substituted by one moiety -L2-Z or -L2-Z’.

Preferably, the -L1- moiety of formula (IIa-iii) is not further substituted.

The most preferred component -L1- has the formula (IIa-iii’):

(IIa-iii'),

Where

where the dotted line represents the addition of the D moiety, which is the PTH moiety, to the nitrogen atom by forming an amide bond;

the dotted line marked with an asterisk indicates attachment to -L2-;

-R2, -R2a, -R3, -R3a and X2- have the meanings as defined in formula (II); and

where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIa-iii’) is not substituted by a substituent.

Preferably, the -L1- moiety of formula (IIa-iii’) is not further substituted.

In another preferred embodiment of the present invention, the constituent -L1- has the formula (IIb-i)

(IIb-i),

Where

where the dotted line represents the addition of the D moiety, which is the PTH moiety, to the nitrogen atom by forming an amide bond;

-R1, -R1a, -R2, -R2a, -R3, -R3a, -R4 and X2- have the meanings as defined in formula (II); and

where -L1- is substituted by -L2-Z or -L2-Z’, and where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIb-i) is not substituted by -L2-Z or -L2-Z’ or a substituent.

Preferably, -L1- of formula (IIb-i) is substituted by one moiety -L2-Z or -L2-Z’.

Preferably, the -L1- moiety of formula (IIb-i) is not further substituted.

Preferably, -R1 and R1a of formula (IIb-i) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R1 and R1a of formula (IIb-i) is methyl. Even more preferably, both -R1 and R1a of formula (IIb-i) are -methyl.

Preferably, -R4 of formula (IIb-i) is selected from the group consisting of H, methyl and ethyl. More preferably, -R4 of formula (IIb-i) is -H.

Preferably, -X2- of formula (IIb-i) is -C(R8R8a)-.

Preferably, -R8 and R8a of formula (IIb-i) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R8 and R8a of formula (IIb-i) is -H. Even more preferably, both -R8 and R8a of formula (IIb-i) are -H.

Preferably, -R2 and R2a of formula (IIb-i) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R2 and R2a of formula (IIb-i) is -H. Even more preferably, both -R2 and R2a of formula (IIb-i) are -H.

Preferably, -R3 and R3a of formula (IIb-i) are independently selected from the group consisting of H, methyl, ethyl, propyl and butyl. Even more preferably, at least one of -R3 and R3a of formula (IIb-i) is -H. Even more preferably, both -R3 and R3a of formula (IIb-i) are -H.

More preferably, the component -L1- has the formula (IIb-ii):

(IIb-ii),

where the dotted line represents the addition of the D moiety, which is the PTH moiety, to the nitrogen atom by forming an amide bond;

-R2, -R2a, -R3, -R3a and X2- have the meanings as defined in formula (II); and

where -L1- is substituted by -L2-Z or -L2-Z’, and where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIb-ii) is not substituted by -L2-Z or -L2-Z’ or a substituent.

Preferably, -L1- of formula (IIb-ii) is substituted by one moiety -L2-Z or -L2-Z’.

Preferably, the -L1- moiety of formula (IIb-ii) is not further substituted.

Preferably, -X2- of formula (IIb-ii) is -C(R8R8a)-.

Preferably, -R8 and R8a of formula (IIb-ii) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R8 and R8a of formula (IIb-ii) is -H. Even more preferably, both -R8 and R8a of formula (IIb-ii) are -H.

Preferably, -R2 and R2a of formula (IIb-ii) are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R2 and R2a of formula (IIb-ii) is -H. Even more preferably, both -R2 and R2a of formula (IIb-ii) are -H.

Preferably, -R3 and R3a of formula (IIb-ii) are independently selected from the group consisting of H, methyl, ethyl, propyl and butyl. Even more preferably, at least one of -R3 and R3a of formula (IIb-ii) is -H. Even more preferably, both -R3 and R3a of formula (IIb-ii) are -H.

Even more preferably, the -L1- component has the formula (IIb-ii’):

(IIb-ii'),

Where

where the dotted line represents the addition of the D moiety, which is the PTH moiety, to the nitrogen atom by forming an amide bond;

-R2, -R2a, -R3, -R3a and X2- have the meanings as defined in formula (II); and

where -L1- is substituted by -L2-Z or L2-Z’, and where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIb-ii’) is not substituted by -L2-Z or -L2-Z’ or a substituent.

Preferably, the -L1- moiety of formula (IIb-ii’) is not further substituted.

Preferably, -X2- of formula (IIb-ii’) is -C(R8R8a)-.

Preferably, -R8 and R8a of formula (IIb-ii') are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R8 and R8a of formula (IIb-ii') is -H. Even more preferably, both -R8 and R8a of formula (IIb-ii') are -H.

Preferably, -R2 and R2a of formula (IIb-ii') are independently selected from the group consisting of H, methyl and ethyl. More preferably, at least one of -R2 and R2a of formula (IIb-ii') is -H. Even more preferably, both -R2 and R2a of formula (IIb-ii') are -H.

Preferably, -R3 and R3a of formula (IIb-ii') are independently selected from the group consisting of H, methyl, ethyl, propyl and butyl. Even more preferably, at least one of -R3 and R3a of formula (IIb-ii') is -H. Even more preferably, both -R3 and R3a of formula (IIb-ii') are -H.

Even more preferably, the -L1- component has the formula (IIb-iii):

(IIb-iii),

Where

where the dotted line represents the addition to the nitrogen atom of the D moiety, which is the PTH moiety, by forming an amide bond; and

where -L1- is substituted by -L2-Z or L2-Z’, and where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIb-iii) is not substituted by -L2-Z or L2-Z’ or a substituent.

Preferably, -L1- of formula (IIb-iii) is substituted by one moiety -L2-Z or L2-Z’.

Preferably, the -L1- moiety of formula (IIb-iii) is not further substituted.

The most preferred component -L1- has the formula (IIb-iii’):

(IIb-iii'),

Where

where the dotted line represents the addition of the D moiety, which is the PTH moiety, to the nitrogen atom by forming an amide bond;

-R2, -R2a, -R3, -R3a and X2- have the meanings as defined in formula (II); and

where -L1- is substituted by -L2-Z or L2-Z’, and where -L1- is optionally further substituted, provided that the hydrogen marked with an asterisk in formula (IIb-iii’) is not substituted by -L2-Z or L2-Z’ or a substituent.

Preferably, the -L1- moiety of formula (IIb-iii’) is not further substituted.

Another preferred moiety -L1- is disclosed in unpublished European patent application 14180004, which has a corresponding international application with application number PCT/EP2015/067929. Accordingly, in another preferred embodiment of the present invention, the moiety -L1- has formula (III):

(III),

Where

the dotted line shows the addition to the primary or secondary amine or hydroxyl of the D moiety by forming an amide or ester bond, respectively;

-R1, -R1a, -R2, -R2a, -R3 and R3a are independently selected from the group consisting of -H, -C(R8R8aR8b), -C(=O)R8, -Cє N, -C(=NR8)R8a, -CR8(=CR8aR8b), -Cє CR8 and T;

-R4, -R5 and R5a are independently selected from the group consisting of -H, -C(R9R9aR9b) and T;

a1 and a2 independently represent 0 or 1;

-R6, -R6a, -R7, -R7a, -R8, -R8a, -R8b, -R9, -R9a, -R9b are each independently selected from the group consisting of -H, halogen, -CN, -COOR10, -OR10, -C(O)R10, -C(O)N(R10R10a), -S(O)2N(R10R10a), -S(O)N(R10R10a), -S(O)2R10, -S(O)R10, -N(R10)S(O)2N(R10aR10b), -SR10, -N(R10R10a), -NO2, -OC(O)R10, -N(R10)C(O)R10a, -N(R10)S(O)2R10a,
-N(R10)S(O)R10a, -N(R10)C(O)OR10a, -N(R10)C(O)N(R10aR10b), -OC(O)N(R10R10a), -T, C1-20 alkyl, C2-20 alkenyl and C2-20 alkynyl; wherein -T, C1-20 alkyl, C2-20 alkenyl and C2-20 alkynyl are optionally substituted with one or more substituents -R11, which are the same or different, and wherein C1-20 alkyl, C2-20 alkenyl and C2-20 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(R12)-, -S(O)2N(R12)-, -S(O)N(R12)-, -S(O)2-, -S(O)-, -N(R12)S(O)2N(R12a)-, -S-, -N(R12)-, -OC(OR12)(R12a)-, -N(R12)C(O)N(R12a)- and -OC(O)N(R12)-;

each -R10, -R10a, -R10b is independently selected from the group consisting of -H, -T, C1-20 alkyl, C2-20 alkenyl, and C2-20 alkynyl; wherein -T, C1-20 alkyl, C2-20 alkenyl and C2-20 alkynyl are optionally substituted with one or more substituents -R11, which are the same or different, and wherein C1-20 alkyl, C2-20 alkenyl and C2-20 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(R12)-, -S(O)2N(R12)-, -S(O)N(R12)-, -S(O)2-, -S(O)-, -N(R12)S(O)2N(R12a)-, -S-, -N(R12)-, -OC(OR12)(R12a)-, -N(R12)C(O)N(R12a)- and -OC(O)N(R12)-;

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, and 8-11 membered heterobicyclyl; wherein each T is independently optionally substituted with one or more -R11 substituents that are the same or different;

each -R11 is independently selected from halogen, -CN, oxo (=O), -COOR13, -OR13, -C(O)R13, -C(O)N(R13R13a), -S(O)2N(R13R13a), -S(O)N(R13R13a), -S(O)2R13, -S(O)R13, -N(R13)S(O)2N(R13aR13b), -SR13, -N(R13R13a), -NO2, -OC(O)R13, -N(R13)C(O)R13a, -N(R13)S(O)2R13a, -N(R13)S(O)R13a, -N(R13)C(O)OR13a, -N(R13)C(O)N(R13aR13b), -OC(O)N(R13R13a) and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogen substituents which are the same or different;

each -R12, -R12a, -R13, -R13a, -R13b is independently selected from the group consisting of -H and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogen substituents that are the same or different;

optionally, one or more of the pairs -R1/-R1a, -R2/-R2a, -R3/-R3a, -R6/-R6a, -R7/-R7a join together with the atom to which they are attached to form a C3-10 cycloalkyl or a 3-10 membered heterocyclyl;

optionally, one or more of the pairs -R1/-R2, -R1/-R3, -R1/-R4, -R1/-R5, -R1/-R6, -R1/-R7, -R2/-R3, -R2/-R4, -R2/-R5, -R2/-R6, -R2/-R7, -R3/-R4, -R3/-R5, -R3/-R6, -R3/-R7, -R4/-R5, -R4/-R6, -R4/-R7, -R5/-R6, -R5/-R7, -R6/-R7 join together with the atoms to which they are attached to form ring A;

A is selected from the group consisting of phenyl; naphthyl; indenyl; indanyl; tetralinyl; C3-10 cycloalkyl; 3-10 membered heterocyclyl; and 8-11 membered heterobicyclyl;

where -L1- has as a substituent -L2-Z or L2-Z’, and where -L1- is optionally further substituted;

Where

-L2- represents a simple chemical bond or spacer;

-Z is a water-soluble carrier; and

-Z’ is a water-insoluble carrier.

Optional other substituents of the -L1- moiety of formula (III) are preferably as described above.

Preferably, -L1- of formula (III) has as a substituent one constituent -L2-Z or L2-Z’.

In one embodiment of the present invention, -L1- of formula (III) is further unsubstituted.

Further preferred embodiments for -L1- are disclosed in EP1536334B1, WO2009/009712A1, WO2008/034122A1, WO2009/143412A2, WO2011/082368A2 and US8618124B2, which are incorporated herein by reference in their entirety.

Further preferred embodiments for -L1- are disclosed in US8946405B2 and US8754190B2, which are incorporated herein by reference in their entirety. Accordingly, the preferred moiety -L1- has formula (IV):

(IV),

Where

the dotted line shows the attachment to -D, which is the PTH moiety, and where the attachment is via a functional group of the -D moiety selected from the group consisting of -OH, -SH and NH2;

m is 0 or 1;

at least one or both of -R1 and R2 is/are independently selected from the group consisting of -CN, -NO2, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted alkenyl, optionally substituted alkynyl, -C(O)R3, -S(O)R3, -S(O)2R3 and -SR4,

one and only one of -R1 and R2 is selected from the group consisting of -H, optionally substituted alkyl, optionally substituted arylalkyl, and optionally substituted heteroarylalkyl;

-R3 is selected from the group consisting of -H, optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, optionally substituted heteroarylalkyl, -OR9, and N(R9)2;

-R4 is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

each -R5 is independently selected from the group consisting of -H, optionally substituted alkyl, optionally substituted alkenylalkyl, optionally substituted alkynylalkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

-R9 is selected from the group consisting of -H and optionally substituted alkyl;

-Y- is absent and -X- is -O- or S-; or

-Y- is -N(Q)CH2-, and X- is -O-;

Q is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted arylalkyl, optionally substituted heteroaryl, and optionally substituted heteroarylalkyl;

optionally, -R1 and R2 can join to form a 3-8 membered ring; and

optionally, both -R9 together with the nitrogen atom to which they are attached form a heterocyclic ring;

where -L1- has as a substituent -L2-Z or L2-Z’, and where -L1- is optionally further substituted;

Where

-L2- represents a simple chemical bond or spacer;

-Z is a water-soluble carrier; and

-Z’ is a water-insoluble carrier.

Only in the context of formula (IV) the terms used have the following meanings:

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

The term “alkoxy” includes alkyl groups bonded to oxygen, including methoxy, ethoxy, isopropoxy, cyclopropoxy, cyclobutoxy and the like.

The term "alkenyl" includes non-aromatic unsaturated hydrocarbons with carbon-carbon double bonds.

The term "alkynyl" includes non-aromatic unsaturated hydrocarbons with carbon-carbon triple bonds.

The term "aryl" includes aromatic hydrocarbon groups having from 6 to 18 carbon atoms, preferably from 6 to 10 carbon atoms, including groups such as phenyl, naphthyl and anthracenyl. The term "heteroaryl" includes aromatic rings containing from 3 to 15 carbon atoms containing at least one N, O or S atom, preferably from 3 to 7 carbon atoms containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isooxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl and the like.

In some cases, alkenyl, alkynyl, aryl, or heteroaryl moieties may be attached to the remainder of the molecule through an alkylene bond. Under these circumstances, the substituent will be referred to as an alkenylalkyl, alkynylalkyl, arylalkyl, or heteroarylalkyl, indicating that the alkylene moiety is between the alkenyl, alkynyl, aryl, or heteroaryl and the molecule to which the alkenyl, alkynyl, aryl, or heteroaryl is bonded.

The term "halogen" includes bromine, fluorine, chlorine and iodine.

The term “heterocyclic ring” refers to a 4- to 8-membered aromatic or non-aromatic ring containing 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 listed above for the term “heteroaryl.”

When the 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 in any group, including those above, include halo, nitro, cyano, OR, -SR, -NR2, -OCOR, -NRCOR, -COOR, -CONR2, -SOR, -SO2R, -SONR2, -SO2NR2, where 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.

Preferably, -L1- of formula (IV) has as a substituent one constituent -L2-Z or L2-Z’.

A further preferred embodiment -L1- is disclosed in WO2013/036857A1, which is incorporated herein by reference in its entirety. Accordingly, the preferred moiety -L1- has formula (V):

(V),

Where

the dotted line shows the attachment to -D, which represents

PTH component, and where the attachment is via the amine functional group of the -D component;

-R1 is selected from the group consisting of optionally substituted C1-C6 linear, branched, or cyclic alkyl; optionally substituted aryl; optionally substituted heteroaryl; alkoxy; and NR52;

-R2 is selected from the group consisting of -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

-R3 is selected from the group consisting of -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

-R4 is selected from the group consisting of -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl;

each -R5 is independently selected from the group consisting of -H; optionally substituted C1-C6 alkyl; optionally substituted aryl; and optionally substituted heteroaryl; or two -R5 taken together may be cycloalkyl or cycloheteroalkyl;

where -L1- has as a substituent -L2-Z or L2-Z’, and where -L1- is optionally further substituted;

Where

-L2- represents a simple chemical bond or spacer;

-Z is a water-soluble carrier; and

-Z’ is a water-insoluble carrier.

Only in the context of formula (V) the terms used have the following meanings:

“Alkyl,” “alkenyl,” and “alkynyl” include linear, branched, or cyclic hydrocarbon groups having from 1 to 8 carbon atoms or 1 to 6 carbon atoms or 1 to 4 carbon atoms, 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, they contain 1 to 6 carbon atoms.

“Aryl” includes aromatic hydrocarbon groups having from 6 to 18 carbon atoms, preferably 6-10 carbon atoms, including groups such as phenyl, naphthyl and anthracene. “Heteroaryl” includes aromatic rings containing 3-15 carbon atoms containing at least one N, O or S atom, preferably 3-7 carbon atoms containing at least one N, O or S atom, including groups such as pyrrolyl, pyridyl, pyrimidinyl, imidazolyl, oxazolyl, isooxazolyl, thiazolyl, isothiazolyl, quinolyl, indolyl, indenyl and the like.

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. The substituents may in general 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; ketone; sulfone; sulfonamide; aryl including phenyl, naphthyl and anthracenyl; heteroaryl including 5-membered heteroaryls including pyrrole, imidazole, furan, thiophene, oxazole, thiazole, isoxazole, isothiazole, thiadiazole, triazole, oxadiazole and tetrazole, 6-membered heteroaryls including pyridine, pyrimidine, pyrazine, and fused heteroaryls including benzofuran, benzothiophene, benzoxazole, benzimidazole, indole, benzothiazole, benzisoxazole and benzisothiazole.

Preferably, -L1- of formula (V) has as a substituent one constituent -L2-Z or L2-Z’.

Another preferred embodiment -L1- is disclosed in US7585837B2, which is incorporated herein by reference in its entirety. Accordingly, the preferred moiety -L1- has formula (VI):

(VI),

Where

the dotted line shows the attachment to -D, which is the PTH moiety, and where the attachment is via the amine functional group of the -D moiety;

R1 and R2 are independently selected from the group consisting of hydrogen, alkyl, alkoxy, alkoxyalkyl, aryl, alkaryl, aralkyl, halogen, nitro, -SO3H, -SO2NHR5, amino, ammonium, carboxyl, PO3H2 and OPO3H2;

R3, R4 and R5 are independently selected from the group consisting of hydrogen, alkyl and aryl;

where -L1- has as a substituent -L2-Z or L2-Z’, and where -L1- is optionally further substituted;

Where

-L2- represents a simple chemical bond or spacer;

-Z is a water-soluble carrier; and

-Z’ is a water-insoluble carrier.

Suitable substituents for formula (VI) are alkyl (such as C1-6 alkyl), alkenyl (such as C2-6 alkenyl), alkynyl (such as C2-6 alkynyl), aryl (such as phenyl), heteroalkyl, heteroalkenyl, heteroalkynyl, heteroaryl (such as an aromatic 4-7 membered heterocycle) or halogen moieties.

Only in the context of formula (VI) the terms used have the following meanings:

The terms “alkyl”, “alkoxy”, “alkoxyalkyl”, “aryl”, “alkaryl” and “aralkyl” mean alkyl radicals containing 1-8, preferably 1-4 carbon atoms, for example methyl, ethyl, propyl, isopropyl and butyl, and aryl radicals containing 6-10 carbon atoms, for example phenyl and naphthyl. The term “halogen” includes bromine, fluorine, chlorine and iodine.

Preferably, -L1- of formula (VI) has one -L2-Z or L2-Z’ moiety as a substituent.

Another preferred embodiment -L1- is disclosed in WO2002/089789A1, which is incorporated herein by reference in its entirety. Accordingly, the preferred moiety -L1- has formula (VII):

*

(VII),

Where

the dotted line shows the attachment to -D, which is the PTH moiety, and where the attachment is via the amine functional group of the -D moiety;

L1 is a bifunctional linking group,

Y1 and Y2 independently represent O, S or NR7;

R2, R3, R4, R5, R6 and R7 are independently selected from the group consisting of hydrogen, C1-6 alkyl, C3-12 branched alkyl, C3-8 cycloalkyl, C1-6 substituted alkyl, C3-8 substituted cycloalkyl, aryl, substituted aryl, aralkyl, C1-6 heteroalkyl, substituted C1-6 heteroalkyl, C1-6 alkoxy, phenoxy and C1-6 heteroalkoxy;

Ar is a moiety which, when included in formula (VII), forms a multi-substituted aromatic hydrocarbon or a multi-substituted heterocyclic group;

X represents a chemical bond or moiety that is actively transferred into the target cell, a hydrophobic moiety, or a combination of both,

y is 0 or 1;

where -L1- has as a substituent -L2-Z or L2-Z’, and where -L1- is optionally further substituted;

Where

-L2- represents a simple chemical bond or spacer;

-Z is a water-soluble carrier; and

-Z’ is a water-insoluble carrier.

Only in the context of formula (VII) the terms used have the following meanings:

The term “alkyl” should be understood to include, for example, straight-chain, branched, substituted C1-12 alkyls including alkoxy, C3-8 cycloalkyls or substituted cycloalkyls, etc.

The term “substitution” is understood to include the addition or replacement of one or more atoms contained in a functional group, or the combination with one or more different atoms.

Substituted alkyls include carboxyalkyls, aminoalkyls, dialkylamines, hydroxyalkyls, and mercaptoalkyls; substituted cycloalkyls include moieties such as 4-chlorocyclohexyl; aryls include moieties such as naphthyl; substituted aryls include moieties such as 3-bromophenyl; aralkyls include moieties such as toluyl; heteroalkyls include moieties such as ethylthiophene; substituted heteroalkyls include moieties such as 3-methoxythiophene; alkoxy includes moieties such as methoxy; and phenoxy includes moieties such as 3-nitrophenoxy. Halo should be understood to include fluorine, chlorine, iodine, and bromine.

Preferably, -L1- of formula (VII) has as a substituent one constituent -L2-Z or L2-Z’.

In another preferred embodiment of the present invention, -L1- comprises a substructure of formula (VIII)

(VIII),

Where

the dotted line marked with an asterisk represents the addition of the D component, which is the PTH component, to the nitrogen atom by forming an amide bond;

unlabeled dotted lines show attachment to the remainder of -L1-; and

where -L1- has as a substituent -L2-Z or L2-Z’, and where -L1- is optionally further substituted;

Where

-L2- represents a simple chemical bond or spacer;

-Z is a water-soluble carrier; and

-Z’ is a water-insoluble carrier.

Preferably, -L1- of formula (VIII) has one -L2-Z or L2-Z’ moiety as a substituent.

In one embodiment of the present invention, -L1- of formula (VIII) is further unsubstituted.

In another preferred embodiment of the present invention, -L1- comprises a substructure of formula (IX)

(IX),

Where

the dotted line marked with an asterisk represents the addition of the D component, which is the PTH component, to the nitrogen atom by forming a carbamate bond;

unlabeled dotted lines show attachment to the remainder of -L1-; and

where -L1- has as a substituent -L2-Z or L2-Z’, and where -L1- is optionally further substituted;

Where

-L2- represents a simple chemical bond or spacer;

-Z is a water-soluble carrier; and

-Z’ is a water-insoluble carrier.

Preferably, -L1- of formula (IX) has as a substituent one constituent -L2-Z or L2-Z’.

In one embodiment of the present invention, -L1- of formula (IX) is further unsubstituted.

In one embodiment of the present invention, -L1- of formula (IX) is further unsubstituted.

In the prodrugs of the present invention, -L2- is a chemical linkage or spacer moiety.

In one embodiment of the present invention, -L2- is a chemical bond.

In another embodiment of the present invention, -L2- is a spacer moiety.

When -L2- is other than a simple chemical bond, -L2- is preferably selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(Ry1)-, -S(O)2N(Ry1)-, -S(O)N(Ry1)-, -S(O)2-, -S(O)-, -N(Ry1)S(O)2N(Ry1a)-, -S-, -N(Ry1)-, -OC(ORy1)(Ry1a)-, -N(Ry1)C(O)N(Ry1a)-, -OC(O)N(Ry1)-, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl; wherein -T-, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally substituted with one or more -Ry2, which are the same or different, and wherein the C1-50 alkyl, C2-50 alkenyl and C2-50 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(Ry3)-, -S(O)2N(Ry3)-, -S(O)N(Ry3)-, -S(O)2-, -S(O)-, -N(Ry3)S(O)2N(Ry3a)-, -S-, -N(Ry3)-, -OC(ORy3)(Ry3a)-, -N(Ry3)C(O)N(Ry3a)-, and OC(O)N(Ry3)-;

-Ry1 and Ry1a are independently selected from the group consisting of H, -T, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl; wherein -T, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally substituted with one or more -Ry2, which are the same or different, and wherein the C1-50 alkyl, C2-50 alkenyl and C2-50 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(Ry4)-, -S(O)2N(Ry4)-, -S(O)N(Ry4)-, -S(O)2-, -S(O)-, -N(Ry4)S(O)2N(Ry4a)-, -S-, -N(Ry4)-, -OC(ORy4)(Ry4a)-, -N(Ry4)C(O)N(Ry4a)-, and OC(O)N(Ry4)-;

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl; wherein each T is independently optionally substituted with one or more -Ry2 that are the same or different;

each -Ry2 is independently selected from the group consisting of halogen, -CN, oxo (=O), -COORy5, -ORy5, -C(O)Ry5, -C(O)N(Ry5Ry5a), -S(O)2N(Ry5Ry5a), -S(O)N(Ry5Ry5a), -S(O)2Ry5, -S(O)Ry5, -N(Ry5)S(O)2N(Ry5aRy5b), -SRy5, -N(Ry5Ry5a), -NO2, -OC(O)Ry5, -N(Ry5)C(O)Ry5a, -N(Ry5)S(O)2Ry5a, -N(Ry5)S(O)Ry5a, -N(Ry5)C(O)ORy5a, -N(Ry5)C(O)N(Ry5aRy5b), -OC(O)N(Ry5Ry5a), and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogens which are the same or different; and

each -Ry3, -Ry3a, -Ry4, -Ry4a, -Ry5, -Ry5a and Ry5b is independently selected from the group consisting of -H, and C1-6 alkyl, wherein C1-6 alkyl is optionally substituted with one or more halogens that are the same or different.

When -L2- is other than a simple chemical bond, -L2- is even more preferably selected from -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(Ry1)-, -S(O)2N(Ry1)-, -S(O)N(Ry1)-, -S(O)2-, -S(O)-, -N(Ry1)S(O)2N(Ry1a)-, -S-, -N(Ry1)-, -OC(ORy1)(Ry1a)-, -N(Ry1)C(O)N(Ry1a)-, -OC(O)N(Ry1)-, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl; wherein -T-, C1-20 alkyl, C2-20 alkenyl, and C2-20 alkynyl are optionally substituted with one or more -Ry2, which are the same or different, and wherein the C1-20 alkyl, C2-20 alkenyl, and C2-20 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(Ry3)-, -S(O)2N(Ry3)-, -S(O)N(Ry3)-, -S(O)2-, -S(O)-, -N(Ry3)S(O)2N(Ry3a)-, -S-, -N(Ry3)-, -OC(ORy3)(Ry3a)-, -N(Ry3)C(O)N(Ry3a)-, and OC(O)N(Ry3)-;

-Ry1 and Ry1a are independently selected from the group consisting of H, -T, C1-10 alkyl, C2-10 alkenyl and C2-10 alkynyl; wherein -T, C1-10 alkyl, C2-10 alkenyl and C2-10 alkynyl are optionally substituted with one or more -Ry2, which are the same or different, and wherein the C1-10 alkyl, C2-10 alkenyl and C2-10 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(Ry4)-, -S(O)2N(Ry4)-, -S(O)N(Ry4)-, -S(O)2-, -S(O)-, -N(Ry4)S(O)2N(Ry4a)-, -S-, -N(Ry4)-, -OC(ORy4)(Ry4a)-, -N(Ry4)C(O)N(Ry4a)-, and OC(O)N(Ry4)-;

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl; wherein each T is independently optionally substituted with one or more -Ry2 that are the same or different;

-Ry2 is selected from the group consisting of halogen, -CN, oxo (=O), -COORy5, -ORy5, -C(O)Ry5, -C(O)N(Ry5Ry5a), -S(O)2N(Ry5Ry5a), -S(O)N(Ry5Ry5a), -S(O)2Ry5, -S(O)Ry5, -N(Ry5)S(O)2N(Ry5aRy5b), -SRy5, -N(Ry5Ry5a), -NO2, -OC(O)Ry5, -N(Ry5)C(O)Ry5a, -N(Ry5)S(O)2Ry5a, -N(Ry5)S(O)Ry5a, -N(Ry5)C(O)ORy5a, -N(Ry5)C(O)N(Ry5aRy5b), -OC(O)N(Ry5Ry5a), and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogens which are the same or different; and

each -Ry3, -Ry3a, -Ry4, -Ry4a, -Ry5, -Ry5a and Ry5b is independently of each other selected from the group consisting of H, and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogens that are the same or different.

When -L2- is other than a single chemical bond, -L2- is even more preferably selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(Ry1)-, -S(O)2N(Ry1)-, -S(O)N(Ry1)-, -S(O)2-, -S(O)-, -N(Ry1)S(O)2N(Ry1a)-, -S-, -N(Ry1)-, -OC(ORy1)(Ry1a)-, -N(Ry1)C(O)N(Ry1a)-, -OC(O)N(Ry1)-, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl; wherein -T-, C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally substituted with one or more -Ry2, which are the same or different, and wherein the C1-50 alkyl, C2-50 alkenyl and C2-50 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(Ry3)-, -S(O)2N(Ry3)-, -S(O)N(Ry3)-, -S(O)2-, -S(O)-, -N(Ry3)S(O)2N(Ry3a)-, -S-, -N(Ry3)-, -OC(ORy3)(Ry3a)-, -N(Ry3)C(O)N(Ry3a)-, and OC(O)N(Ry3)-;

-Ry1 and Ry1a are independently selected from the group consisting of H, -T, C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl;

each T is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl;

each -Ry2 is independently selected from the group consisting of halogen and C1-6 alkyl; and

each -Ry3, -Ry3a, -Ry4, -Ry4a, -Ry5, -Ry5a and Ry5b is independently of each other selected from the group consisting of H, and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogens that are the same or different.

Even more preferably, -L2- is a C1-20 alkyl chain which is optionally interrupted by one or more groups independently selected from -O-, -T- and -C(O)N(Ry1)-; and wherein the C1-20 alkyl chain is optionally substituted with one or more groups independently selected from -OH, -T and C(O)N(Ry6Ry6a); wherein -Ry1, -Ry6, -Ry6a are independently selected from the group consisting of H and C1-4 alkyl, and wherein T is selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10-membered heterocyclyl, 8-11-membered heterobicyclyl, 8-30-membered carbopolycyclyl, and 8-30-membered heteropolycyclyl.

Preferably, -L2- has a molecular weight in the range of 14 g/mol to 750 g/mol.

Preferably, -L2- comprises a constituent selected from

, , , , , , , , , , , , , , , , , , ,

, , And ;

Where

the dotted line denotes attachment to the rest of -L2-, -L1-, -Z and/or -Z', respectively; and

-R and Ra are independently selected from the group consisting of H, methyl, ethyl, propyl, butyl, pentyl and hexyl.

In one preferred embodiment of the present invention, L2- has a chain length of from 1 to 20 atoms.

As used in this application, the term “chain length” with respect to a moiety -L2- refers to the number of -L2- atoms present in the shortest link between -L1- and Z.

Preferably, -L2- has the formula (i)

(i),

Where

the dotted line marked with an asterisk indicates attachment to -L1-;

The unmarked dotted line indicates attachment to -Z or Z';

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

wherein the constituent of formula (i) is optionally further substituted.

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 of the present invention, n of formula (i) is 4. In another embodiment of the present invention, n of formula (i) is 5. In another embodiment of the present invention, n of formula (i) is 6.

In one preferred embodiment of the present invention, the component -L1-L2- is selected from the group consisting of (IIca-i),

(IIca-ii) and

(IIca-iii);

Where

the unmarked dotted line represents the addition to the nitrogen atom of the D moiety, which is the PTH moiety, by forming an amide bond; and

The dotted line marked with an asterisk indicates an attachment to -Z or -Z’.

In one preferred embodiment of the present invention, the component -L1-L2- is selected from the group consisting of

(IIcb-i),

(IIcb-ii) and

(IIcb-iii);

Where

the unmarked dotted line represents the addition to the nitrogen atom of the D moiety, which is the PTH moiety, by forming an amide bond; and

The dotted line marked with an asterisk indicates attachment to -Z or -Z’.

In a preferred embodiment of the present invention, the -L1-L2- constituent has the formula (IIca-ii).

In another preferred embodiment of the present invention, the -L1-L2- moiety has the formula (IIcb-iii).

Preferably, the PTH prodrug according to the present invention has formula (Ia) with x = 1.

The -Z carrier comprises a C8-24 alkyl or polymer. Preferably, -Z comprises a polymer, preferably a polymer selected from the group consisting of 2-methacryloyloxyethyl phosphoylcholines, 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(ethylene glycols), poly(ethylene oxides), poly(ethyl phosphates), poly(ethyl oxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl oxazolines), poly(hydroxymethacrylates), poly(hydroxypropyl methacrylamides), poly(hydroxypropyl methacrylates), poly(hydroxypropyl oxazolines), poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic acid copolymers), poly(methacrylamides), poly(methacrylates), poly(methyl oxazolines), poly(organophosphazenes), poly(orthoesters), poly(oxazolines), poly(propylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinylamines), poly(vinyl methyl ethers), poly(vinylpyrrolidones), silicones, celluloses, carbomethylcelluloses, hydroxypropyl methylcelluloses, chitins, chitosans, dextrans, dextrins, gelatins, hyaluronic acids and derivatives, functionalized hyaluronic acids, mannans, pectins, rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethyl starches and other polymers based on carbohydrates, xylans and their copolymers.

Preferably, -Z has a molecular weight in the range of 5 to 200 kDa. Even more preferably, -Z has a molecular weight in the range of 8 to 100 kDa, even more preferably in the range of 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 of the present invention, -Z has a molecular weight of about 40 kDa.

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

In one embodiment of the present invention, -Z is polysarcosine.

In another preferred embodiment of the present invention, -Z comprises poly(N-methylglycine).

In a particularly preferred embodiment of the present invention, -Z comprises a random coil protein moiety.

In one preferred embodiment of the present invention, -Z comprises one random coil protein moiety.

In another preferred embodiment of the present invention, -Z comprises two random coil protein constituents.

In another preferred embodiment of the present invention, -Z comprises three random coil protein constituents.

In another preferred embodiment of the present invention, -Z comprises four random coil protein constituents.

In another preferred embodiment of the present invention, -Z comprises five random coil protein constituents.

In another preferred embodiment of the present invention, -Z comprises six random coil protein constituents.

In another preferred embodiment of the present invention, -Z comprises seven random coil protein constituents.

In another preferred embodiment of the present invention, -Z comprises eight random coil protein constituents.

Preferably, the random coil protein constituent comprises at least 25 amino acid residues and at most 2000 amino acids. Even more preferably, the random coil protein constituent comprises at least 30 amino acid residues and at most 1500 amino acid residues. Even more preferably, the random coil protein constituent comprises at least 50 amino acid residues and at most 500 amino acid residues.

In a preferred embodiment of the present invention, -Z comprises a random coil protein moiety, wherein 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 the random coil protein moiety are proline residues. Preferably, the random coil protein moiety is as described in WO 2011/144756 A1, which is incorporated herein 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 is incorporated herein by reference. A moiety comprising such a protein with a random helix structure containing alanine and proline is referred to as “PA” or a “PA moiety”.

Accordingly, -Z contains a PA component.

In an equally preferred embodiment of the present invention, -Z comprises a random coil protein moiety in 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 the random coil protein moiety are proline residues. Preferably, the random coil protein moiety is described in WO 2008/155134 A1, which is incorporated herein 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 described in WO 2008/155134 A1, which is incorporated herein by reference. The moiety comprising a protein with a random helix structure containing alanine, serine and proline is referred to as “PAS” or “PAS moiety”.

Accordingly, -Z contains a PAS component.

In an equally preferred embodiment of the present invention, -Z comprises a random coil protein moiety in 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 reverse coil protein moiety are selected from alanine, glycine and proline. The moiety comprising a random coil protein comprising alanine, glycine and proline is referred to as "PAG" or "PAG moiety".

Accordingly, -Z contains a PAG component.

In an equally preferred embodiment of the present invention, -Z comprises a random coil protein moiety in 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 reverse coil protein moiety are selected from proline and glycine. The moiety comprising the random coil protein containing proline and glycine is referred to as "PG" or "PG moiety".

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

[(Gly)p-Pro-(Gly)q]r (a-0);

Where

p is selected from the group consisting of 0, 1, 2, 3, 4, and 5;

q is selected from the group consisting of 0, 1, 2, 3, 4, and 5;

r is an integer in the range and inclusive from 10 to 1000;

provided that at least one of p and q is at least 1;

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

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

Even more preferably, the PG moiety comprises the sequence SEQ ID:NO 122: GGPGGPGPGGPGPGPGGPGGPG

Even more preferably, the PG moiety comprises the sequence SEQ ID:NO 97 of formula (a-0-a)

(GGPGGPGPGGPGGPGPGGPG)v (a-0-a),

Where

v is an integer in the range 1 to 50, inclusive.

Accordingly, -Z contains a PG component.

In an equally preferred embodiment of the present invention, -Z comprises a moiety of a protein with a random coil structure, in 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 protein with a reverse coil structure are selected from alanine, glycine, serine, threonine, glutamate and proline. Preferably, the moiety of the protein with a random coil structure is as described in WO 2010/091122 A1, which is incorporated herein 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 described in WO2010/091122A1, which is incorporated herein by reference. The moiety comprising a protein with a random helix structure comprising alanine, glycine, serine, threonine, glutamate and proline is referred to as “XTEN” or “XTEN moiety” along with its designation in WO 2010/091122 A1.

Accordingly, -Z contains an XTEN component.

In another preferred embodiment of the present invention, Z comprises a fatty acid derivative. Preferred fatty acid derivatives are those described in WO 2005/027978 A2 and WO 2014/060512 A1, which are incorporated herein by reference.

In another preferred embodiment of the present invention, Z is a hyaluronic acid-based polymer.

In one embodiment of the present invention, Z is a carrier as disclosed in WO 2012/02047 A1, which is incorporated herein by reference.

In another embodiment of the present invention, Z is a carrier as disclosed in WO 2013/024048 A1, which is incorporated herein by reference.

In another preferred embodiment of the present invention, -Z is a PEG-based polymer, such as a linear, branched or multi-armed PEG-based polymer.

In one embodiment of the present invention, -Z is a linear PEG-based polymer.

In another embodiment of the present invention, -Z is a multi-arm PEG-based polymer. Preferably, -Z is a multi-arm PEG-based polymer having at least 4 PEG-based arms.

Preferably, such a PEG-based polymer with multiple -Z branches is connected to a plurality of -L2-L1-D moieties, wherein each -L2-L1-D moiety is preferably connected to the end of a branch, preferably to the end of a branch. Preferably, such a PEG-based polymer with multiple -Z branches is connected to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 -L2-L1-D moieties. Even more preferably, such a multi-armed -Z PEG-based polymer is linked to 2, 3, 4, 6 or 8 -L2-L1- D moieties. Even more preferably, such a multi-armed -Z PEG-based polymer is linked to 2, 4 or 6 -L2-L1- D moieties, even more preferably, such a multi-armed -Z PEG-based polymer is linked to 4 or 6 -L2-L1- D moieties, and most preferably, such a multi-armed -Z PEG-based polymer is linked to 4 -L2-L1- D moieties.

Preferably, such a multi-arm PEG-based polymer Z is a multi-arm PEG derivative, such as described in detail for the products listed in JenKem Technology, USA (available at http://www.jenkemusa.com/Pages/PEGProducts.aspx on December 18, 2014), such as a 4-arm PEG derivative, in particular a 4-arm PEG derivative comprising a pentaerythritol core, an eight-arm PEG derivative comprising a hexaglycerol core, and an eight-arm PEG derivative comprising a tripentaerythritol core. More preferably, the water-soluble PEG-based carrier -Z comprises a moiety selected from:

A 4-arm PEG amine containing a pentaerythritol core:

with n in the range from 20 to 500;

An eight-armed PEG amine containing a hexaglycerol core:

with n in the range from 20 to 500; and

R = hexaglycerol or tripentaerythritol core structure; and

A six-armed PEG amine containing a sorbitol or dipentaerythritol core:

with n in the range from 20 to 500; and

R = containing a core of sorbitol or dipentaerythritol;

and where the dotted lines represent attachment to the rest of the PTH prodrug.

In a preferred embodiment of the present invention, -Z is a branched PEG-based polymer. In one embodiment of the present invention, -Z is a branched PEG-based polymer having one, two, three, four, five or six branch points. Preferably, -Z is a branched PEG-based polymer having one, two or three branch points. In one embodiment of the present invention, -Z is a branched PEG-based polymer having one branch point. In another embodiment of the present invention, -Z is a branched PEG-based polymer having two branch points. In another embodiment of the present invention, -Z is a branched PEG-based polymer having three branch points.

The branching point is preferably selected from the group consisting of -N<, -CH< and >C<.

Preferably, such branched -Z moiety has a molecular weight of at least 10 kDa.

In one embodiment of the present invention, such a branched -Z moiety has a molecular weight in the range of and including 10 kDa to 500 kDa, more preferably in the range of and including 10 kDa to 250 kDa, even more preferably in the range of and including 10 kDa to 150 kDa, even more preferably in the range of and including 12 kDa to 100 kDa, and most preferably in the range of and including 15 kDa to 80 kDa.

Preferably, such branched moiety -Z has a molecular weight in the range and including 10 kDa - 80 kDa. In one embodiment of the present invention, the molecular weight is about 10 kDa. In another embodiment of the present invention, the molecular weight of such branched moiety -Z is about 20 kDa. In another embodiment of the present invention, the molecular weight of such branched moiety -Z is about 30 kDa. In another embodiment of the present invention, the molecular weight of such branched moiety -Z is about 40 kDa. In another embodiment of the present invention, the molecular weight of such branched moiety -Z is about 50 kDa. In another embodiment of the present invention, the molecular weight of such branched moiety -Z is about 60 kDa. In another embodiment of the present invention, the molecular weight of such branched moiety -Z is about 70 kDa. In another embodiment of the present invention, the molecular weight of such branched moiety -Z is about 80 kDa. Most preferably, such branched moiety -Z has a molecular weight of about 40 kDa.

Preferably, -Z or Z' contains a constituent

.

In an equally preferred embodiment of the present invention, Z comprises an amide bond.

Preferably -Z contains a constituent of formula (a)

(a),

Where

the dotted line shows the attachment to -L2- or to the remaining part of -Z;

BPa is a branch point selected from the group consisting of -N<, -CR<, and >C<;

-R is selected from the group consisting of -H and C1-6 alkyl;

a is 0 if BPa is -N< or CR<, and n is 1 if BPa is >C<;

-Sa-, -Sa’-, -Sa’’- and Sa’’’- independently of each other represent a chemical bond or are selected from the group consisting of C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl; wherein the C1-50 alkyl, C2-50 alkenyl and C2-50 alkynyl are optionally substituted with one or more substituents -R1, which are the same or different, and wherein the C1-50 alkyl, C2-50 alkenyl and C2-50 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(R2)-, -S(O)2N(R2)-, -S(O)N(R2)-, -S(O)2-, -S(O)-, -N(R2)S(O)2N(R2a)-, -S-, -N(R2)-, -OC(OR2)(R2a)-, -N(R2)C(O)N(R2a)- and -OC(O)N(R2)-;

each -T- is independently selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, tetralinyl, C3-10 cycloalkyl, 3-10 membered heterocyclyl, 8-11 membered heterobicyclyl, 8-30 membered carbopolycyclyl, and 8-30 membered heteropolycyclyl; wherein each -T- is independently optionally substituted with one or more substituents -R1 that are the same or different;

each -R1 is independently selected from the group consisting of halogen, -CN, oxo (=O), -COOR3, -OR3, -C(O)R3, -C(O)N(R3R3a), -S(O)2N(R3R3a), -S(O)N(R3R3a), -S(O)2R3, -S(O)R3, -N(R3)S(O)2N(R3aR3b), -SR3, -N(R3R3a), -NO2, -OC(O)R3, -N(R3)C(O)R3a, -N(R3)S(O)2R3a, -N(R3)S(O)R3a, -N(R3)C(O)OR3a, -N(R3)C(O)N(R3aR3b), -OC(O)N(R3R3a) and C1-6 alkyl; wherein C1-6 alkyl is optionally substituted with one or more halogen substituents which are the same or different;

each -R2, -R2a, -R3, -R3a and R3b is independently selected from the group consisting of -H and C1-6 alkyl, wherein C1-6 alkyl is optionally substituted with one or more halogen substituents that are the same or different; and

-Pa’, -Pa’’ and Pa’’’ independently represent the polymer component.

In one embodiment of the present invention, BPa of formula (a) is -N<.

In another embodiment of the present invention, BPa of formula (a) is >C<.

In a preferred embodiment of the present invention, BPa of formula (a) is -CR<. Preferably, -R is -H. Accordingly, a of formula (a) is 0.

In one embodiment of the present invention, -Sa- of formula (a) is a chemical bond.

In another embodiment of the present invention, -Sa- of formula (a) is selected from the group consisting of C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl, wherein the C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl are optionally interrupted by one or more chemical groups selected from the group consisting of -T-, -C(O)O-, -O-, -C(O)-, -C(O)N(R4)-, -S(O)2N(R4)-, -S(O)N(R4)-, -S(O)2-, -S(O)-, -N(R4)S(O)2N(R4a)-, -S-, -N(R4)-, -OC(OR4)(R4a)-, -N(R4)C(O)N(R4a)-, and OC(O)N(R4)-; where -T- is 3-10 membered heterocyclyl; and R4 and R4a are independently selected from the group consisting of H, methyl, ethyl, propyl, and butyl.

Preferably, -Sa- of formula (a) is selected from the group consisting of C1-10 alkyl that is interrupted by one or more chemical groups selected from the group consisting of -T-, -C(O)N(R4)- and O-.

In one embodiment of the present invention, -Sa'- of formula (a) is a chemical bond.

In another embodiment of the present invention, -Sa’- of formula (a) is selected from the group consisting of C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl, wherein the C1-10 alkyl, C2-10 alkenyl, and C2-10 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(R4)-, -S(O)2N(R4)-, -S(O)N(R4)-, -S(O)2-, -S(O)-, -N(R4)S(O)2N(R4a)-, -S-, -N(R4)-, -OC(OR4)(R4a)-, -N(R4)C(O)N(R4a)-, and OC(O)N(R4)-; where -R4 and R4a are independently selected from the group consisting of H, methyl, ethyl, propyl and butyl. Preferably -Sa'- 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(R4)-.

In one embodiment of the present invention, -Sa’’- of formula (a) is a chemical bond.

In another embodiment of the present invention, -Sa’’- of formula (a) is selected from the group consisting of C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl, wherein the C1-10 alkyl, C2-10 alkenyl, and C2-10 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(R4)-, -S(O)2N(R4)-, -S(O)N(R4)-, -S(O)2-, -S(O)-, -N(R4)S(O)2N(R4a)-, -S-, -N(R4)-, -OC(OR4)(R4a)-, -N(R4)C(O)N(R4a)-, and OC(O)N(R4)-; where -R4 and R4a are independently selected from the group consisting of H, methyl, ethyl, propyl and butyl. Preferably -Sa’’- 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(R4)-.

In one embodiment of the present invention, -Sa’’’- of formula (a) is a chemical bond.

In another embodiment of the present invention, -Sa’’’- of formula (a) is selected from the group consisting of C1-10 alkyl, C2-10 alkenyl, and C2-10 alkynyl, wherein the C1-10 alkyl, C2-10 alkenyl, and C2-10 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(R4)-, -S(O)2N(R4)-, -S(O)N(R4)-, -S(O)2-, -S(O)-, -N(R4)S(O)2N(R4a)-, -S-, -N(R4)-, -OC(OR4)(R4a)-, -N(R4)C(O)N(R4a)-, and OC(O)N(R4)-; where -R4 and R4a are independently selected from the group consisting of H, methyl, ethyl, propyl and butyl. Preferably -Sa’’’- 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(R4)-.

Preferably, -Pa', -Pa'' and Pa''' of formula (a) independently comprise a polymer selected from the group consisting of 2-methacryloyloxyethylphosphoylcholines, 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(ethylene glycols), poly(ethylene oxides), poly(ethyl phosphates), poly(ethyl oxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl oxazolines), poly(hydroxymethacrylates), poly(hydroxypropyl methacrylamides), poly(hydroxypropyl methacrylates), poly(hydroxypropyl oxazolines), poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic acid copolymers), poly(methacrylamides), poly(methacrylates), poly(methyl oxazolines), poly(organophosphazenes), poly(orthoesters), poly(oxazolines), poly(propylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinylamines), poly(vinyl methyl ethers), 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 their copolymers.

More preferably, -Pa', -Pa'' and Pa''' of formula (a) independently comprise a PEG-based moiety. Even more preferably, -Pa', -Pa'' and Pa''' of formula (a) independently comprise a PEG-based 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.

Preferably, -Pa’, -Pa’’ and Pa’’’ of formula (a) independently have a molecular weight in the range and including 5 kDa - 50 kDa, more preferably have a molecular weight in the range and including 5 kDa - 40 kDa, even more preferably in the range and including 7.5 kDa - 35 kDa, even more preferably in the range of and including 7.5 - 30 kDa, even more preferably in the range and including 10 - 30 kDa.

In one embodiment of the present invention, -Pa', -Pa'' and Pa''' of formula (a) have a molecular weight of about 5 kDa.

In another embodiment of the present invention, -Pa', -Pa'' and Pa''' of formula (a) have a molecular weight of about 7.5 kDa.

In another embodiment of the present invention, -Pa', -Pa'' and Pa''' of formula (a) have a molecular weight of about 10 kDa.

In another embodiment of the present invention, -Pa', -Pa'' and Pa''' of formula (a) have a molecular weight of about 12.5 kDa.

In another embodiment of the present invention, -Pa', -Pa'' and Pa''' of formula (a) have a molecular weight of about 15 kDa.

In another embodiment of the present invention, -Pa', -Pa'' and Pa''' of formula (a) have a molecular weight of about 20 kDa.

In one embodiment of the present invention, -Z comprises one moiety of formula (a).

In another embodiment of the present invention, -Z comprises two components of formula (a).

In another embodiment of the present invention, -Z comprises three components of formula (a).

Preferably, -Z is a constituent of formula (a).

More preferably, -Z contains a constituent of formula (b)

(b),

Where

the dotted line shows the attachment to -L2- or to the remainder of -Z; and

m and p independently of each other represent an integer in the range and inclusive from 150 to 1000; preferably an integer in the range and inclusive from 150 to 500; more preferably an integer in the range and inclusive from 200 to 500; and most preferably an integer in the range and inclusive from 400 to 500.

Preferably, m and p of formula (b) represent the same integer.

Most preferably, m and p of formula (b) are equal to about 450.

Preferably, -Z is a constituent of formula (b).

The carrier -Z’ is a water-insoluble polymer, even more preferably a hydrogel. Preferably, such a hydrogel comprises a polymer selected from the group consisting of 2-methacryloyloxyethyl phosphoylcholines, 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(ethylene glycols), poly(ethylene oxides), poly(ethyl phosphates), poly(ethyl oxazolines), poly(glycolic acids), poly(hydroxyethyl acrylates), poly(hydroxyethyl oxazolines), poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides), poly(hydroxypropyl methacrylates), poly(hydroxypropyl oxazolines), poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolic acid copolymers), poly(methacrylamides), poly(methacrylates), poly(methyl oxazolines), poly(organophosphazenes), poly(orthoesters), poly(oxazolines), poly(propylene glycols), poly(siloxanes), poly(urethanes), poly(vinyl alcohols), poly(vinylamines), poly(vinyl methyl ethers), 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 their copolymers.

If the carrier -Z' is a hydrogel, it is preferably a hydrogel containing PEG or hyaluronic acid. Most preferably, such a hydrogel contains PEG.

Even more preferably, the carrier -Z' is a hydrogel as disclosed in WO 2006/003014 A2, WO 2011/012715 A1 or WO 2014/056926 A1, which are incorporated herein by reference in their entirety.

In another embodiment of the present invention, -Z' is a polymer network formed by physical aggregation of polymer chains, wherein the physical aggregation is preferably caused by hydrogen bonding, crystallization, helicalization, or complexation. In one embodiment of the present invention, such a polymer network is a thermogel polymer.

Preferably, the total mass of the PTH prodrug according to the present invention is 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. Preferably, the total mass of the PTH prodrug according to the present invention is at most 250 kDa, such as at most 200 kDa, 180 kDa, 150 kDa or 100 kDa.

In a preferred embodiment of the present invention, the PTH prodrug of the present invention has formula (IIe-i):

(IIe-i),

Where

the unmarked dotted line represents the addition to the nitrogen atom of the D moiety, which is the PTH moiety, by forming an amide bond; and

the dotted line marked with an asterisk means joining to the component

Where

m and p independently represent an integer in the range and inclusive from 400 to 500.

Preferably, -D is attached to the PTH prodrug of formula (IIe-i) via the N-terminal amine functional group of the PTH moiety.

In another preferred embodiment of the present invention, the PTH prodrug of the present invention has the formula (IIf-i):

(IIf-i),

Where

the unmarked dotted line represents the addition to the nitrogen atom of the D moiety, which is the PTH moiety, by forming an amide bond; and

the dotted line marked with an asterisk means joining to the component

Where

m and p independently represent an integer in the range and inclusive from 400 to 500.

Preferably, -D is attached to the PTH prodrug of formula (IIf-i) via the N-terminal amine functional group of the PTH moiety.

In a preferred embodiment of the present invention, the residual activity of the PTH prodrug of the present invention 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%.

As used herein, the term “residual activity” refers to the activity exhibited by a PTH prodrug of the present invention, wherein the PTH moiety is bound to a carrier, relative to the activity exhibited by the corresponding free PTH. In this context, the term "activity" refers to binding to activate the PTH/PTHrP1 receptor, resulting in activation of adenylate cyclase to form cAMP, phospholipase C to form 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 requires a time during which a certain amount of PTH will be released from the PTH prodrug of the present invention, and that such released PTH will distort the results measured for the PTH prodrug. Therefore, in practice, it is common practice to test the residual activity of the prodrug with a conjugate in which the drug moiety, in this case PTH, is irreversibly, i.e. stably bound to a carrier that resembles as closely as possible the structure of the PTH prodrug for which the residual activity is to be measured.

A suitable assay for measuring the activity of PTH and the residual activity of the PTH prodrug according to the present invention, preferably in the form of a stable analogue, is, for example, the measurement of cAMP production from HEK293 cells overexpressing the PTH receptor/PTHrP1 (Hohenstein et al., Journal of Pharmaceutical and Biomedical Analysis, September 2014, 98: 345-350) or a cell-based assay for detecting the release of cyclic AMP, detected by homogeneous time-resolved fluorescence (HTRF) or ELISA, which has been validated according to ICHQ2(R1) (http://www.criver.com/files/pdfs/bps/bp_r_in_vitro_bioassays.aspx).

Surprisingly, it was found that the use of N-terminal attachment of L1 and the use of a branched PEG carrier for Z, i.e. 2x 20 kDa PEG, resulted in particularly low residual activity. Reduced residual activity is desirable as it reduces side effects.

It has been surprisingly found that the PTH prodrugs of the present invention are able to achieve a stable plasma PTH profile that provides physiological serum and urinary calcium levels or even reduced urinary calcium levels.

Preferably, after subcutaneous administration, the pharmacokinetic profile of the PTH prodrug of the present invention exhibits a peak-to-trough ratio of less than 4 within one injection interval.

As used herein, the term “injection interval” refers to the time between two successive administrations of a pharmaceutical composition according to the present invention.

As used herein, the term “peak to trough ratio” refers to the ratio between the highest plasma concentration and the lowest plasma concentration of PTH released from a PTH prodrug of the present invention during the time period between two consecutive administrations to a non-human primate, preferably a cynomolgus monkey.

The period of time between two successive subcutaneous administrations, i.e. the administration interval, is preferably at least 24 hours, such as 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, every 84 hours, 96 hours, 108 hours, 120 hours, 132 hours, 144 hours, 156 hours, one week, two weeks, three weeks or four weeks.

In one embodiment of the present invention, the time period between two consecutive subcutaneous administrations is 24 hours.

In another embodiment of the present invention, the time period between two consecutive subcutaneous administrations is 48 hours.

In another embodiment of the present invention, the time period between two consecutive subcutaneous administrations is 72 hours.

In another embodiment of the present invention, the time period between two consecutive subcutaneous administrations is 96 hours.

In another embodiment of the present invention, the time period between two consecutive subcutaneous administrations is 120 hours.

In another embodiment of the present invention, the time period between two consecutive subcutaneous administrations is 144 hours.

In another embodiment of the present invention, the time period between two consecutive subcutaneous administrations is one week.

The peak to trough ratio measured for each administration interval is less than 4, preferably less than 3.8, more preferably less than 3.6, even more preferably less than 3.4, even more preferably less than 3.2, even more preferably less than 3, even more preferably less than 2.8, even more preferably less than 2.6, even more preferably less than 2.4, even more preferably less than 2.2 and most preferably less than 2.

Another object of the present invention is a pharmaceutical composition comprising at least one PTH prodrug according to the present invention and at least one excipient.

Preferably, the pharmaceutical composition comprising at least one PTH prodrug according to the present invention has a pH value in the range of and including pH 3 to pH 8. More preferably, the pharmaceutical composition has a pH value in the range of and including pH 4 to pH 6. Most preferably, the pharmaceutical composition has a pH value in the range of and including pH 4 to pH 5.

In one embodiment of the present invention, the pharmaceutical composition comprising at least one PTH prodrug of the present invention and at least one excipient is a liquid composition or a suspension composition. It is understood that the pharmaceutical composition is a suspension composition if the PTH prodrug of the present invention comprises a water-insoluble carrier -Z'.

In another embodiment of the present invention, the pharmaceutical composition comprising at least one PTH prodrug of the present invention and at least one excipient is a dry composition.

Such a liquid pharmaceutical composition, a pharmaceutical composition in the form of a suspension or a dry pharmaceutical composition contains at least one excipient. Excipients used in parenteral compositions can be categorized as follows, for example, buffering agents, isotonicity modifiers, preservatives, stabilizers, anti-adsorption agents, anti-oxidation agents, thickeners/viscosity increasing agents or other auxiliary agents. However, in some cases, one excipient may have dual or triple functions. Preferably, at least one excipient contained in the pharmaceutical composition according to the present invention is selected from the group consisting of the following:

Buffering agents: physiologically acceptable buffers to maintain the pH value in the desired range such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulfate, nitrate, chloride, pyruvate; acid neutralizers such as Mg(OH)2 or ZnCO3 can also be used;

Isotonicity modifiers: to minimize pain that may result from cell damage due to osmotic pressure differences when injecting slow-release agents; glycerol and sodium chloride are examples; effective concentrations can be determined by osmometry using a permissible osmolality of 285-315 mOsmol/kg for serum;

Preservatives and/or antimicrobials: Multidose parenteral formulations require the addition of preservatives at sufficient concentration to minimize the risk of contamination of the patient upon injection, and appropriate regulatory requirements should be 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;

Stabilizers: Stabilization is achieved by enhancing protein stabilizing forces, destabilizing the denatured state or directly binding excipients to the protein; stabilizers can 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 sulfate, 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 can be used;

Anti-adsorption agents: Generally ionic or non-ionic surfactants or other proteins or soluble polymers are used to coat or competitively adsorb to the inner surface of the formulation container. Examples include poloxamer (Pluronic F-68), PEG dodecyl ether (Brij 35), polysorbate 20 and 80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA, and gelatins; the concentration and type of excipient selected depends on the effect to be avoided, but typically a monolayer of surfactant is formed at the interface just above the CMC value;

Oxidation protectants: Antioxidants such as ascorbic acid, ectoine, methionine, glutathione, monothioglycerol, morin, polyethyleneimine (PEI), propyl gallate and vitamin E; chelating agents such as citric acid, EDTA, hexaphosphate and thioglycolic acid may also be used;

Thickening agents or viscosity increasing agents: in the case of suspensions, they slow down the settling of particles in the vial and syringe and are used to facilitate mixing and resuspension of particles and to facilitate injection of the suspension (i.e. with low force on the syringe plunger); Suitable thickeners or viscosity enhancers are, for example, carbomer thickeners such as Carbopol 940, Carbopol Ultrez 10, cellulose derivatives such as hydroxypropyl methylcellulose (hypromellose, HPMC) or diethylaminoethylcellulose (DEAE or DEAE-C), colloidal magnesium silicate (Veegum) or sodium silicate, hydroxyapatite gel, tricalcium phosphate gel, xanthans, carrageenans such as 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 form a poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) triblock (e.g. PluronicT), a polyether-polyester copolymer such as polyethyleneglycol terephthalate/polybutylene terephthalate copolymer, sucrose acetate isobutyrate (SAIB), dextran or its derivatives, combinations of dextrans and PEG, polydimethylsiloxane, collagen, chitosan, polyvinyl alcohol (PVA) and derivatives, polyalkylimides, poly(acrylamide-co-diallyldimethylammonium (DADMA)), polyvinylpyrrolidone (PVP), glycosaminoglycans (GAGs) such as dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronan, ABA triblock or AB block copolymers consisting of hydrophobic A-blocks such as polylactide (PLA) or poly(lactide-co-glycolide) (PLGA) and hydrophilic B-blocks such as polyethyleneglycol (PEG) or polyvinylpyrrolidone; such block copolymers, like the above-mentioned poloxamers, can exhibit reversible thermal gelation (a liquid state at room temperature for ease of administration and a gel state above the sol-gel transition temperature at body temperature after injection);

Lyophilizing or diffusing agent: modifies the permeability of connective tissue by hydrolyzing extracellular matrix components in the interstitial space, such as, but not limited to, hyaluronic acid, a polysaccharide found in the intercellular space of connective tissue; a lyophilizing agent, such as, but not limited to, hyaluronidase, temporarily reduces the viscosity of the extracellular matrix and promotes diffusion of injected drugs; and

Other auxiliaries: such as wetting agents, viscosity modifiers, antibiotics, hyaluronidase; acids and bases such as hydrochloric acid and sodium hydroxide are auxiliaries necessary to adjust the pH value during production.

The pharmaceutical composition comprising at least one PTH prodrug can be administered to a patient by various routes of administration, such as local, enteral or parenteral administration, as well as by external application, injection or infusion methods, including intra-articular, periarticular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, intracapsular, intraorbital, intravitreal, intratympanic, intravesical, intracardiac, transtracheal, subcuticular, subcapsular, subarachnoid, intraspinal, intraventricular, intrasternal injections or infusions, intranasal, oral, transpulmonary and transdermal administration, direct delivery to the brain via an implanted device allowing delivery of the invention or the like to brain tissue or brain fluids (e.g., an Ommaya reservoir), direct intracerebroventricular injection or infusion, injection or infusion into the brain or areas associated with the brain, injection into the suprachoroidal space, retro-orbital injection or ocular instillation. Preferably, the pharmaceutical composition containing at least one PTH prodrug is administered by subcutaneous injection.

Subcutaneous injection is preferably given with a syringe and needle or with a syringe pen, even more preferably with a syringe pen.

Another object of the present invention is the use of a PTH prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing at least one PTH prodrug according to the present invention as a medicine.

Another object of the present invention is a PTH prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one PTH prodrug according to the present invention for use in the treatment of a disease that can be treated with PTH.

Preferably, said disease is selected from the group consisting of hypoparathyroidism, hyperphosphatasemia, osteoporosis, fracture healing, bone softening, bone softening and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, brittle bone syndrome, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, bone fracture, alopecia, chemotherapy-induced alopecia and thrombocytopenia. Most preferably, said disease is hypoparathyroidism.

In one embodiment of the present invention, the patient undergoing the method of treatment according to the present invention is a mammalian patient, preferably a human.

Another object of the present invention is the use of a PTH prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing at least one PTH prodrug according to the present invention for the preparation of a medicament for the treatment of a disease that can be treated with PTH.

Preferably, said disease is selected from the group consisting of hypoparathyroidism, hyperphosphatasemia, osteoporosis, fracture healing, bone softening, bone softening and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, brittle bone syndrome, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, bone fracture, alopecia, chemotherapy-induced alopecia and thrombocytopenia. Most preferably, said disease is hypoparathyroidism.

In one embodiment of the present invention, a disease to be treated with a PTH prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising at least one PTH prodrug according to the present invention occurs in a mammalian patient, preferably a human patient.

Another object of the present invention is a method for treating, controlling, slowing down or preventing in a mammalian patient, preferably a human, in need of treatment of one or more diseases that can be treated with PTH, comprising the step of administering to said patient in need thereof a therapeutically effective amount of a PTH prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition containing a PTH prodrug according to the present invention.

Preferably, one or more diseases that can be treated with PTH are selected from the group consisting of hypoparathyroidism, hyperphosphatasemia, osteoporosis, fracture healing, bone softening, bone softening and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, brittle bone syndrome, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, bone fracture, alopecia, chemotherapy-induced alopecia and thrombocytopenia. Most preferably, said disease is hypoparathyroidism.

Another object of the present invention is a method of administering a PTH prodrug, a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the present invention, wherein the method comprises the step of administering the PTH prodrug, a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the present invention by topical, enteral or parenteral administration and by external application methods, injection or infusion, including intra-articular, periarticular, intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, intracapsular, intraorbital, intravitreal, intratympanic, intravesical, intracardiac, transtracheal, subcuticular, subcapsular, subarachnoid, intraspinal, intraventricular, intrasternal injections or infusions, intranasal, oral, transpulmonary and transdermal administration, direct delivery to the brain through an implanted device allowing delivery of the invention or the like into brain tissue or brain fluids (eg, Ommaya reservoir), direct intracerebroventricular injection or infusion, injection or infusion into the brain or areas associated with the brain, injection into the suprachoroidal space, retro-orbital injection or ocular instillation, preferably by subcutaneous injection.

In a preferred embodiment of the present invention, the present invention relates to a PTH prodrug or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to the present invention, for use in the treatment of hypoparathyroidism by subcutaneous injection.

Examples

Substances and Methods

PTH(1-34) with a protected side chain (SEQ ID NO:51) on TCP resin having a Boc protecting group at the N-terminus and a Lys26 side chain with an ivDde protecting group (synthesized according to the Fmoc strategy) was obtained from CASLO ApS, Kongens Lyngby, Denmark and Peptide Specialty Laboratories GmbH, Heidelberg, Germany.

PTH(1-34) with a protected side chain on a TCP resin having a Fmoc protecting group at the N-terminus (synthesized according to the Fmoc strategy) was obtained from CASLO ApS, Kongens Lyngby, Denmark and Peptide Specialty Laboratories GmbH, Heidelberg, Germany.

PEG 2x20 kDa-maleimide, Sunbright GL2-400MA and PEG 2x10 kDa-maleimide, Sunbright GL2-200MA were purchased from NOF Europe N.V., Grobbendonk, Belgium. S-Trityl-6-mercaptohexanoic acid was purchased from Polypeptide, Strasbourg, France. HATU was purchased from Merck Biosciences GmbH, Schwalbach/Ts, Germany. Fmoc-N-Me-Asp(OBn)-OH was purchased 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 otherwise noted.

Compound 11a (examples 11-15) was synthesized according to the procedure described in example WO29095479A2, example 1.

Syringes equipped with a polyethylene frit (MultiSynTech GmbH, Witten, Germany) were used as reaction vessels or at the washing stage of peptide resins.

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

General procedure for Fmoc deprotection of protected PTH on resin: the resin was pre-swollen in DMF for 30 min and the solvent was removed. 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 x 10 min. Fresh DMF/piperidine/DBU solution was used for each step. Finally, the resin was washed with DMF (10x), DCM (10x) and dried under vacuum.

Purification by RP-HPLC:

Preparative RP-HPLC was performed using a Waters 600 controller and 2487 Dual Absorbance detector equipped with the following columns: Waters XBridgeTM BEH300 Prep C18 5 μm, 150 x 10 mm, flow rate 6 mL/min, or Waters XBridgeTM BEH300 Prep C18 10 μm, 150 x 30 mm, flow rate 40 mL/min. Linear gradients of solvent system A (water containing 0.1% TFA v/v or 0.01% conc. HCl v/v) and solvent system B (acetonitrile containing 0.1% TFA v/v or 0.01% conc. HCl v/v) were used. HPLC fractions containing product were pooled and lyophilized unless otherwise noted.

Flash chromatography

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

Ion exchange chromatography:

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

Size exclusion chromatography:

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

For in vitro release kinetic studies of compound 31, pH 7.40 buffer (100 mM phosphate, 10 mM L-methionine, 3 mM EDTA, 0.05% Tween-20) was used instead of 0.1% AcOH as the mobile phase.

Analytical methods

Analytical ultra-high performance LC (UPLC)-MS was performed on a Waters Acquity system equipped with a Waters BEH300 C18 column (particle size 2.1 x 50 mm, particle size 1.7 μm, 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 Thermo Scientific LTQ Orbitrap Discovery mass spectrometer or coupled to a Waters Microsass ZQ.

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

Quantitative determination of total plasma PTH(1-34) concentrations:

Total plasma PTH(1-34) concentrations were determined by quantifying the signature peptide near the N-terminus (sequence: IQLMHNLGK) and the C-terminal signature peptide (sequence: LQDVHNF) after protein precipitation in plasma, followed by sequential digestion with endoproteinase Lys-C (origin: Lysobacter enzymogenes) and endoproteinase Glu-C (origin: Staphylococcus aureus V8) of the supernatant. Then, reverse phase liquid chromatography analysis and mass spectrometry detection (RP-HPLC-MS) were performed.

Calibration standards of PTH(1-34) conjugate in blank plasma were prepared as follows: PTH(1-34) conjugate composition was pre-diluted with buffer composition to aqueous standard solutions ranging from 5 to 300 μg/mL PTH(1-34) equiv (concentration range 1) and from 0.5 μg/mL to 100 μg/mL PTH(1-34) equiv (concentration range 2), respectively. Each aqueous standard solution was then diluted 1:100 with thawed heparin plasma to yield compound concentration ranges from 50 to 3000 ng/mL PTH(1-34) eq (dilution with rat plasma in concentration range 1) and 5 to 1000 ng/mL PTH(1-34) eq (dilution with macaque plasma in concentration range 2).

These solutions were used to obtain a calibration curve. The calibration curves were obtained 1/x2 for both signature peptides. For quality control, three samples were obtained, independently of the calibration standard solutions, respectively. Concentrations at the low end (3-5 times the concentration of the corresponding LLOQ), the middle range (0.05 - 0.1 times the concentration of the corresponding ULOQ) and at the high end (0.5-0.8 times the concentration of the corresponding ULOQ).

Sample preparation volumes can be varied depending on the target signal response after sample preparation. The protein precipitation workup procedure is described here for the analysis of plasma samples that occur in simian species. Protein precipitation was performed by adding 200 μl pre-chilled (5-10°C) methanol to 100 μl of plasma sample. 180 μl of the supernatant was transferred to a new well plate and evaporated to dryness (under a gentle stream of nitrogen at 45°C). 50 μl of reducing solvent (50 mM Tris, 0.5 mM _CaCl2 buffer, adjusted to pH 8.0) was used to solubilize the residue. Proteolytic digestion was performed as follows:

20 μg Lys-C (Part Number 125-05061, Wako Chemicals GmbH, Neuss, Germany) were dissolved in 80 μl 10 mM acetic acid. 3 μl of the Lys-C solution were added to each well and the samples were incubated for 15 h at 37°C. Then, 10 μg Glu-C (Part Number V1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 μl water, added to each well and incubation was continued for 1.5 h at 37°C. After incubation, the samples were acidified with 2 μl water/formic acid 4:6 (v/v) and 10 μl were injected into the UPLC-MS system.

LC-MS analysis was performed using an Agilent 1290 UPLC coupled to an Agilent 6460 TripleQuad mass spectrometer via an ESI probe. Chromatography was performed on a Waters Acquity BEH300 C18 analytical column (1.7 μm particle size, the column dimensions used were 50 x 2.1 mm for the analysis of samples originating from rat species or 100 x 2.1 mm for the analysis of samples originating from monkey species) with a prefilter at a flow rate of 0.30 ml/min (T = 60 °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 was used as mobile phase B.

The gradient system for the analysis of samples derived from rat plasma included a linear increase from 0.1% B to 40% B over 7 min. The gradient system for the analysis of samples derived from monkey plasma included a linear increase from 8.0% B to 11.0% B over 6 min, followed by a linear increase to 26% B over 4 min. Mass analysis was performed in the multiple reaction monitoring (MRM) mode, monitoring the transitions of m/z 437.2 to 131.0 and m/z 352.3 to 463.0.

Alternatively, quantitative determination of total plasma PTH(1-34) concentrations was performed according to the following procedure:

Total plasma PTH(1-34) concentrations were determined by quantifying the signature peptide near the N-terminus (sequence: IQLMHNLGK) and the C-terminal signature peptide (sequence: LQDVHNF) after protein precipitation in plasma, followed by sequential digestion with Endoproteinase Lys-C (origin: Lysobacter enzymogenes) and Endoproteinase Glu-C (origin: Staphylococcus aureus V8) of the supernatant. Then, reverse phase liquid chromatography analysis and mass spectrometry detection (RP-HPLC-MS) were performed.

Standards for calibration of PTH(1-34) conjugate in blank heparinized plasma were prepared in the concentration range from 50 to 3000 ng/mL PTH(1-34) eq (diluted in rat plasma) and from 1 to 1000 ng/mL PTH(1-34) eq (diluted in monkey plasma).

These solutions were used to generate the calibration curve. For quality control, three samples were prepared independently of the calibration standard solutions, respectively. Concentrations at the low end (3-5 times the concentration of the corresponding LLOQ), mid-range (0.05 - 0.1 times the concentration of the corresponding ULOQ) and high end (0.5-0.8 times the concentration of the corresponding ULOQ).

Sample preparation volumes can be varied depending on the target signal response after sample preparation. The protein precipitation workup procedure is described here for the analysis of plasma samples that occur in simian species. Protein precipitation was performed by adding 200 μl pre-chilled (5-10°C) methanol to 50 μl of plasma sample. 60 μl of the supernatant was transferred to a new well plate and evaporated to dryness (under a gentle stream of nitrogen at 45°C). To solubilize the residue, 60 μl of reducing solvent (50 mM Tris, 0.5 mM CaCl2 buffer, adjusted to pH 8.0) was used. Proteolytic digestion was performed as follows:

20 μg Lys-C (Part Number 125-05061, Wako Chemicals GmbH, Neuss, Germany) were dissolved in 80 μl 10 mM acetic acid. 3 μl of the Lys-C solution were added to each well and the samples were incubated for 15 h at 37°C. Then, 10 μg Glu-C (Part Number V1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 μl water, added to each well and incubation was continued for 1.5 h at 37°C. After incubation, the samples were acidified with 2 μl water/formic acid 4:6 (v/v) and 10 μl were injected into the UPLC-MS system.

Chromatography was performed on a Waters Acquity BEH300 C18 analytical column (particle size 1.7 μm, column dimensions 50x2.1 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 was used as mobile phase B.

Quantitative determination of plasma PEG concentrations:

Plasma total PEG concentrations were determined by quantification of the polymeric portion of PTH(1-34) conjugates after plasma protein precipitation and enzymatic digestion of the supernatant. Size exclusion chromatography analysis and mass spectrometry detection (SEC-MS) followed.

Calibration standards of PTH(1-34) conjugate in blank heparinized macaque plasma were prepared over a concentration range of 50 to 1200 ng/mL PEG equivalents.

These solutions were used to generate a quadratic calibration curve. The calibration curves were scaled up 1/x. For quality control, three samples were prepared independently of the calibration standards, respectively. The concentrations were at the low end (2-4-fold LLOQ concentration), mid-range (0.1-0.2-fold ULOQ concentration) and high end (0.8-fold ULOQ concentration). Protein precipitation was performed by adding 200 µl pre-cooled (5-10°C) methanol to 100 µl plasma sample. 180 µl of the supernatant were transferred to a new well plate and evaporated to dryness (under a gentle stream of nitrogen at 45°C). 50 µl of reducing solvent (50 mM Tris, 0.5 mM CaCl2 buffer, adjusted to pH 8.0) were used to solubilize the residue. Proteolytic digestion was performed as follows: 20 μg Lys-C (serial number 125-05061, Wako Chemicals GmbH, Neuss, Germany) were dissolved in 80 μl 10 mM acetic acid. 3 μl of the Lys-C solution were added to each well and the samples were incubated for 15 h at 37°C. Then, 10 μg Glu-C (serial number V1651, Promega GmbH, Mannheim, Germany) were dissolved in 25 μl water, added to each well and incubation was continued for 1.5 h at 37°C. After incubation, the samples were acidified with 2 μl water/formic acid 4:6 (v/v) and 10 μl were injected into the UPLC-MS system.

LC-MS analysis was performed using an Agilent 1290 UPLC coupled to an Agilent 6460 TripleQuad mass spectrometer via an ESI probe. The isolation of the polymer precursor ion was achieved by applying high voltage fragmentation in the ion source (200-300 V) at the MS interface. Chromatography was performed on a TOSOH TSK Gel SuperAW3000 analytical column (4.0 μm particle size, 150 x 6.0 mm column dimensions) at a flow rate of 0.50 mL/min (T = 60 °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 was used as mobile phase B. The chromatographic setup for sample analysis involves isocratic elution with 50% B for 8 minutes.

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

Because of the reversible nature of the -L1- to -D addition, measurements of PTH receptor activity were performed using stable analogs of the PTH prodrugs of the present invention, i.e., they were prepared using structures similar to the PTH prodrugs of the present invention, which instead of a reversible -Z to -D addition have a stable addition.

This was necessary because the PTH prodrugs of the present invention could release PTH during the course of the experiment, and said release of PTH would influence the result.

Example 1

Synthesis of linker reagent 1f

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

To a solution of N-methyl-N-Boc-ethylenediamine (2 g, 11.48 mmol) and NaCNBH3 (819 mg, 12.63 mmol) in MeOH (20 mL) was added 2,4,6-trimethoxybenzaldehyde (2.08 g, 10.61 mmol) in portions. The mixture was stirred at room temperature for 90 min, acidified with 3 M HCl (4 mL) and stirred for another 15 min. The reaction mixture was added to saturated NaHCO3 (200 mL) and extracted 5x with DCM. The combined organic phases were dried over Na2SO4 and the solvents were evaporated in vacuo. The resulting N-methyl-N-Boc-N'-Tmob-ethylenediamine 1a was dried under high vacuum and used in the next step without further processing.

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

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

To a solution of 1a (2 g, 5.65 mmol) in CH2Cl2 (24 mL) COMU (4.84 g, 11.3 mmol), N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4.52 mmol) and 2,4,6-collidine (2.65 mL, 20.34 mmol) were added. The reaction mixture was stirred for 3 h at room temperature, diluted with CH2Cl2 (250 mL) and washed 3x with 0.1 M H2SO4 (100 mL) and 3x with brine (100 mL). The aqueous phases were re-extracted with CH2Cl2 (100 mL). The combined organic phases were dried over Na2SO4, filtered and the residue was concentrated to a volume of 24 mL. Compound 1b was purified using flash chromatography.

Yield: 5.31 g (148%, 6.66 mmol)

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

To a solution of 1b (5.31 g, max 4.52 mmol, designated as 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 room temperature, diluted with DCM (400 mL) and washed 3x with 0.1 M H2SO4 (150 mL) and 3x with brine (150 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na2SO4 and filtered. Compound 1c was isolated by evaporation of the solvent and used in the next step without further purification.

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

Compound 1c (5.31 g, 4.52 mmol, crude) was dissolved in acetonitrile (26 mL) and COMU (3.87 g, 9.04 mmol), 6-tritylmercaptocaproic 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 room temperature, diluted with DCM (400 mL) and washed 3x with 0.1 M H2SO4 (100 mL) and 3x with brine (100 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na2SO4, filtered and 1d was isolated by evaporation of the solvent. Product 1d was purified using flash chromatography.

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

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

To a solution of 1d (2.63 g, 2.78 mmol) in i-PrOH (33 mL) and H2O (11 mL) was added LiOH (267 mg, 11.12 mmol) and the reaction mixture was stirred for 70 min at room temperature. The mixture was diluted with DCM (200 mL) and washed 3x with 0.1 M H2SO4 (50 mL) and 3x with brine (50 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na2SO4, filtered and compound 1e was isolated by evaporation of the solvent. Compound 1e was purified using flash chromatography.

Yield: 2.1 g (88%)

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

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-hydroxysuccinimide (114 mg, 0.99 mmol) was added and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was filtered, the solvent was removed in vacuo and the residue was dissolved in 90% acetonitrile plus 0.1% TFA (3.4 mL). The crude mixture was purified by RP-HPLC. The 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 by evaporation of the solvent.

Yield: 154 mg (81%)

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

Example 2

Synthesis of linker reagent 2g

4-Methoxytriphenylmethyl chloride (3.00 g, 9.71 mmol) was dissolved in DCM (20 mL) and added dropwise with 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 room temperature, after which it was diluted with diethyl ether (300 mL), washed 3x with brine/0.1 M NaOH 30/1 (v/v) and once with brine. The organic phase was dried over Na2SO4, and compound 2b was isolated by evaporation of the solvent.

Yield: 3.18 g (98%)

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 room temperature. The solution was diluted with diethyl ether (250 mL), washed 3 x with brine/0.1 M NaOH 30/1 (v/v) and once with brine. The organic phase was dried over Na2SO4 and the solvent was removed in vacuo. 2c was purified using flash chromatography.

Yield: 5.69 g (85%)

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

Compound 2c (3.19 g, 4.53 mmol) was dissolved in anhydrous THF (50 mL), 1 M BH3 THF solution in THF (8.5 mL, 8.5 mmol) was added, and the mixture was stirred for 16 h at room temperature. Another 1 M BH3 THF solution in THF (14 mL, 14.0 mmol) was added, and the mixture was stirred for another 16 h at room temperature. Methanol (8.5 mL) and N,N´-dimethylethylenediamine (3.00 mL, 27.9 mmol) were added, and the mixture was heated under reflux for 3 h. The mixture was allowed to cool, and ethyl acetate (300 mL) was added. The solution was washed with 2x aqueous Na2CO3 and 2x aqueous NaHCO3. The organic phase was dried over Na2SO4 and the solvent was removed in vacuo to give compound 2d.

Yield: 3.22 g (103%)

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

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 room temperature. The solution was concentrated in vacuo and purified using flash chromatography to afford product 2e.

Yield: 2.09 g (74%)

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

Compound 2e (5.01 g, 6.34 mmol) was dissolved in acetonitrile (80 mL). 0.4 M aqueous HCl (80 mL) and then acetonitrile (20 mL) were added and the mixture was stirred for 1 h at room temperature. The pH was adjusted to pH 5.5 by adding aqueous 5 M NaOH. The organic solvent was removed in vacuo and the remaining aqueous solution was extracted 4x with DCM. The combined organic phases were dried over Na2SO4 and the solvent was removed in vacuo to give product 2f.

Yield: 4.77 g (95%)

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

Compound 2f (5.27 g, 6.65 mmol) was dissolved in DCM (30 mL) and added to a solution of p-nitrophenyl fluoroformate (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 room temperature. The solution was concentrated in vacuo and purified using flash chromatography to afford product 2g.

Yield: 4.04 g (89%)

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

Example 3

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

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

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

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

Example 4

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

PTH(1-34) with protected side chain on TCP resin having Boc group at N-terminus and Lys26 side chain with ivDde group was subjected to ivDde deprotection according to the procedure described in Materials and Methods. A solution of 6-tritylmercaptohexanoic acid (107 mg, 273 μmol), PyBOP (141 mg, 273 μmol) and DIPEA (95 μL, 545 μmol) in DMF (3 mL) was added to 0.80 g (90.9 μmol) of resin. The suspension was shaken for 1 h at room temperature. The resin was washed 10x with DMF, 10x with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of the protecting groups were achieved by adding 6 ml of a 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole cleavage cocktail and shaking the suspension for 1 h at room temperature. Crude compound 4 was precipitated in pre-chilled diethyl ether (-18°C). The residue was dissolved in ACN/water and purified by RP-HPLC. The product fractions were lyophilized.

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

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

Example 5

Synthesis of temporary conjugate S1 PTH(1-34)

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

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

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

Example 6

Synthesis of temporary conjugate S1 PTH(1-34) 6

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

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

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

Example 7

Synthesis of temporary conjugate S1 PTH(1-34) 7

Side chain protected PTH(1-34) on TCP resin having an Fmoc protecting group at the N-terminus was subjected to Fmoc deprotection according to the procedure described in Materials and Methods. A solution of Fmoc-Ser(Trt)-OH (117 mg, 205 μmol), PyBOP (108 mg, 207 μmol) and DIPEA (53 μL, 305 μmol) in DMF (2 mL) was added to 0.50 g (51 μmol) of resin. The suspension was shaken for 1 h at room temperature. The resin was washed 10x with DMF, 10x with DCM and dried in vacuo. Fmoc deprotection was performed as described above. A solution of 2 g (144 mg, 211 μmol) and DIPEA (53 μl, 305 μmol) in DMF (1.8 ml) was added to the resin. The suspension was shaken for 7 h at room temperature. The resin was washed 10x with DMF, 10x with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of the protecting groups were achieved by adding 6 ml of a 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole cleavage cocktail and shaking the suspension for 1 h at room temperature. Crude compound 7 was precipitated in pre-cooled diethyl ether (-18 °C). The residue was dissolved in ACN/water and purified by RP-HPLC. The product fractions were lyophilized.

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

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

Example 8

Synthesis of temporary conjugate S1 PTH(1-34) 8

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

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

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

Example 9

Synthesis of temporary conjugate S1 PTH(1-34) 9

Side chain protected PTH(1-34) on TCP resin having an Fmoc protecting group at the N-terminus was subjected to Fmoc deprotection according to the procedure described in Materials and Methods. A solution of compound 1e (182 mg, 213 μmol), PyBOP (111 mg, 213 μmol), and DIPEA (93 μL, 532 μmol) in DMF (5 mL) was added to 2.00 g (107 μmol) of resin. The suspension was shaken for 16 h at room temperature. The resin was washed 10x with DMF, 10x with DCM, and dried in vacuo. Cleavage of the peptide from the resin and removal of the protecting groups were achieved by adding 20 mL of a 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole cleavage cocktail and shaking the suspension for 1 h at room temperature. Crude compound 9 was precipitated in pre-chilled diethyl ether (-18°C). The residue was dissolved in ACN/water and purified by RP-HPLC. The product fractions were lyophilized.

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

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

Example 10

Synthesis of temporary conjugate K26 PTH(1-34) 10

PTH(1-34) with protected side chain on TCP resin having Boc group at N-terminus and Lys26 side chain with ivDde group was subjected to ivDde deprotection according to the procedure described in Materials and Methods. A solution of compound 1f (867 mg, 910 μmol) and DIPEA (0.24 mL, 1.36 mmol) in DMF (5 mL) was added to 1.91 g (227 μmol) of resin. The suspension was shaken for 1 h at room temperature. The resin was washed 10x with DMF, 10x with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of the protecting groups were achieved by adding 20 mL of a 100/3/3/2/1 (v/w/v/v/v) TFA/DTT/TES/water/thioanisole cleavage cocktail and shaking the suspension for 1 h at room temperature. Crude compound 10 was precipitated in pre-chilled diethyl ether (-18°C). The residue was dissolved in ACN/water and purified by RP-HPLC. The product fractions were lyophilized.

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

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

Example 11

Synthesis of low molecular weight transient conjugate S1 PEG 11b

0.15 mL of 0.5 M NaH2PO4 buffer (pH 7.4) was added to 0.5 mL of a 20 mg/mL solution of thiol 5 (10 mg, 1.84 μmol) in 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v). The solution was incubated at room temperature for 10 min, after which 238 μL of a 10 mg/mL solution of maleimide 11a (2.4 mg, 2.21 μmol) in 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v) were added. The solution was incubated for 20 min at room temperature. 10 μL of TFA was added and the mixture was purified by RP-HPLC. The product fractions were lyophilized to yield compound 11b.

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

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

Example 12

Synthesis of low molecular weight temporary conjugate S1 PEG 12

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

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

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

Example 13

Synthesis of low molecular weight transient conjugate S1 PEG 13

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

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

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

Example 14

Synthesis of low molecular weight transient conjugate S1 PEG 14

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

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

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

Example 15

Synthesis of low molecular weight temporary conjugate K26 PEG 15

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

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

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

Example 16

Synthesis of permanent conjugate 2x20 kDa S1 PEG 16

772 μL of a solution containing thiol 3 (19.4 mg/mL, 15 mg, 3.54 μmol) and 2.5 mg/mL Boc-L-Met in 1/1 (v/v) acetonitrile/water containing 0.1% TFA (v/v) were added to 1.87 mL of a solution containing PEG 2x20 kDa - maleimide (Sunbright GL2-400MA, 187 mg, 4.32 μmol) and 2.5 mg/mL Boc-L-Met in water containing 0.1% TFA (v/v). 0.5 M NaH2PO4 buffer (0.66 mL, pH 7.0) was added and the mixture was stirred for 30 min at room temperature. 10 μl of 270 mg/ml 2-mercaptoethanol in water were added. The mixture was stirred for 5 min at room temperature and 0.33 ml of 1 M HCl was 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 fractions containing the solution were lyophilized.

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

Example 17

Synthesis of permanent conjugate 2x20 kDa K26 PEG 17

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

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

Example 18

Synthesis of a temporary 2x20 kDa S1 PEG 18 conjugate

Conjugate 18 was prepared as described for 16 by reaction of thiol 5 (37 mg, 8.40 μmol) and PEG 2x20 kDa-maleimide (Sunbright GL2-400MA, 445 mg, 9.24 μmol). The reaction was quenched by addition of 50 μl TFA without addition of 2-mercaptoethanol. Conjugate 18 was purified by IEX followed by SEC for desalting. Fractions containing the solution were lyophilized.

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

Example 19

Synthesis of a temporary 2x20 kDa S1 PEG 19 conjugate

Conjugate 19 was prepared as described for 16 by reaction of thiol 7 (27 mg, 6.14 μmol) and PEG 2x20 kDa-maleimide (Sunbright GL2-400MA, 325 mg, 7.50 μmol).

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

Example 20

Synthesis of a temporary 2x20 kDa S1 PEG 20 conjugate

Conjugate 20 was prepared as described for 16 by reaction of thiol 9 (38 mg, 8.59 μmol) and PEG 2x20 kDa-maleimide (Sunbright GL2-400MA, 455 mg, 9.45 μmol). The reaction was quenched by addition of 50 μl TFA without addition of 2-mercaptoethanol. Conjugate 20 was purified by IEX followed by SEC for desalting. Fractions containing the solution were lyophilized.

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

Example 21

Synthesis of a temporary 2x20 kDa K26 PEG 21 conjugate

Conjugate 21 was prepared as described for 16 by reaction of thiol 10 (34 mg, 7.58 μmol) and PEG 2x20 kDa-maleimide (Sunbright GL2-400MA, 401 mg, 9.26 μmol).

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

Example 22

In vitro release kinetics of a transient low molecular weight PEG conjugate

Conjugates 11b, 12, 13, 14, and 15 were dissolved in pH 7.4 phosphate buffer (60 mM NaH2PO4, 3 mM EDTA, 0.01% Tween-20, adjusted to pH 7.4 with NaOH) containing 0.05 mg/mL pentafluorophenol as an internal standard at a concentration of approximately 1 mg conjugate/mL. The solutions were sterile filtered and incubated at 37°C. Aliquots were taken at time points and analyzed by RP-HPLC and ESI-MS. The fraction of released PTH at a time point was calculated from the ratio of the UV peak areas of released PTH and PEG conjugate. The % PTH released was plotted against incubation time. Curve fitting software was used to calculate the corresponding half-life times.

Results:

For conjugate 11b, a half-life of 3.2 days was obtained.

For conjugate 12, a half-life of 8.7 days was obtained.

For conjugate 13, a half-life of 10.8 days was obtained.

For conjugate 14, a half-life of 25.3 days was obtained.

For conjugate 15, a half-life of 6.9 days was obtained.

Example 23

In vitro release kinetics of transient 2x20 kDa PEG conjugates

Conjugates 18, 19, 20, b 21 were dissolved in pH 7.4 phosphate buffer (60 mM NaH2PO4, 3 mM EDTA, 0.01% Tween-20, adjusted to pH 7.4 with NaOH) containing 0.08 mg/mL pentafluorophenol as an internal standard at a concentration of about 5 mg conjugate/mL. The solutions were sterile filtered and incubated at 37°C. Aliquots were taken at time points and analyzed by RP-HPLC. The fraction of released PTH at a time point was calculated from the ratio of the UV peak areas of released PTH and PEG conjugate. The % PTH released was plotted against incubation time. Curve fitting software was used to calculate the corresponding half-lives.

Results:

For conjugate 18, a half-life of 2.8 days was obtained.

For conjugate 19, a half-life of 13.4 days was obtained.

For conjugate 20, a half-life of 1.3 days was obtained.

For conjugate 21, a half-life of 7.1 days was obtained.

Example 24

PTH receptor activity of permanent 2x20 kDa PEG conjugates 16 and 17 in a cell-based assay

The residual PTH activity of permanently PEGylated conjugates 16 and 17 was quantified by measuring cAMP production from HEK293 cells overexpressing 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 a reference standard.

Results:

For conjugate 16, a receptor activity of 0.12% was found relative to the reference PTH(1-34).

For conjugate 17, a receptor activity of 0.11% was found relative to the reference PTH(1-34).

The results demonstrate effective reduction of receptor activity in permanent 2x20 kDa PEG conjugates 16 and 17. It can be concluded that similar conjugates with PTH transiently linked to Ser1 or Lys26 (e.g. 18 and 21) are suitable PTH prodrugs providing low residual receptor activity. Direct analysis of transient conjugates in a cell-based assay is not possible due to cleavage of the linker under the assay conditions. The released PTH will affect the assay result.

Example 25

Pharmacokinetic study of permanent 2x20 kDa conjugates of PEG 16 and 17 in rats

Male Wistar rats (6 weeks, 230-260 g) received either a single intravenous (2 groups, n = 3 animals each) or subcutaneous (2 groups, n = 3 animals each) administration of 16 or 17 at doses of 29 μg/rat PTHeq PTH eq and 31 μg/rat PTH eq, respectively. Blood samples were collected up to 168 h post-dose and plasma prepared. Plasma PTH(1-34) concentrations were determined by quantifying the N-terminal signature peptide (sequence: IQLMHNLGK) and C-terminal signature peptide (sequence: LQDVHNF) after LysC and GluC digestion as described in Substances and Methods.

Results: Doses were well tolerated with no apparent discomfort during or after administration. No adverse dose reactions were observed throughout the study. Following intravenous administration of 16 and 17, tmax for total PTH(1-34) occurred at 15 min (the earliest time point analyzed), followed by a slow decline in total PTH(1-34) with a half-life of approximately 13 h and 11 h, respectively. Following subcutaneous injection, total PTH(1-34) peaked at tmax of 24 h for 16 and 17, followed by a slow decline in total PTH(1-34) with a half-life of approximately 1.5 days for both conjugates. Bioavailability was approximately 40% and 60%, respectively. Similar PK curves were obtained for the N- and C-terminal signature peptide up to 168 h post-dose, indicating the presence of intact PTH(1-34) in the conjugate.

The favorable long PKs and stability of PTH in the conjugates indicate the suitability of permanent 2x20 kDa PEG model compounds, as there is a slow release of PTH prodrugs after subcutaneous injection. It can be concluded that similar conjugates with transiently linked Ser1 (e.g., 18) or Lys26 PTH are suitable PTH prodrugs providing sustained levels of released bioactive PTH.

Example 26

Pharmacokinetic study of a transient 2x20 kDa S1 PEG 19 conjugate in cynomolgus monkeys

Naïve male cynomolgus macaques (2-4 years, 3.7-5.4 kg) received a single subcutaneous administration (n = 3 animals) of conjugate 19 at a dose of 70 μg/kg PTH eq. Blood samples were collected up to 504 h post-dose and plasma was prepared. Total plasma PTH(1-34) concentrations were determined by quantifying the N-terminal signature peptide (sequence: IQLMHNLGK) and C-terminal signature peptide (sequence: LQDVHNF) following LysC and GluC cleavage as described in the Materials and Methods section. PEG concentrations were determined using the method described in the Materials and Methods section.

Results: Dosing was well tolerated with no apparent discomfort during dosing. One animal showed visible discomfort at 72 h post-dose but recovered within a few days. No adverse reactions to dosing were observed throughout the study. Total PTH(1-34) concentrations peaked at tmax of 24 h, followed by a slow decline in total PTH(1-34) with a half-life of approximately 2.5 days for the N-terminal signature peptide and 0.9 days for the C-terminal signature peptide. PEG concentrations peaked at tmax of 24 h, followed by a slow decline in PEG concentrations with a half-life of 3.5 days.

It can be concluded that conjugate 19 is a suitable prodrug for prolonged delivery of PTH.

Example 27

Pharmacokinetic study of a transient 2x20 kDa S1 PEG 18 conjugate in cynomolgus monkeys

Naïve cynomolgus macaques (2-3 yr, 2.5-4 kg) received daily subcutaneous administration (n = 2 animals - 1 male/1 female) of conjugate 18 at dose levels of 0.2, 0.5, and 1 μg/kg PTH eq for 28 days. Blood samples were collected for up to 28 days (on days 1, 13, and 27, samples were collected predose, 2 h, 4 h, 8 h, 12 h, and 24 h postdose) and plasma was obtained. Plasma PTH(1-34) concentrations were determined by quantifying the N-terminal signature peptide (sequence: IQLMHNLGK) and C-terminal signature peptide (sequence: LQDVHNF) after LysC and GluC cleavage as described in the Substances and Methods section.

Results: All doses were administered uneventfully. No adverse dose reactions were observed throughout the study. Dose linearity was observed across the three groups. Dose stacking was observed from day 1 to day 13 and day 27. Total PTH(1-34) concentrations were determined using an N-terminal signature peptide (sequence: IQLMHNLGK) at steady state (through day 27).

Low peak-to-trough ratios of total PTH(1-34) for all dose groups below 3 were observed after daily subcutaneous administration at steady state in cynomolgus monkeys. Since free peptide concentrations at steady state correlate with total PTH(1-34) concentrations, the peak-to-trough ratio for free peptide is below 4 in cynomolgus monkeys.

Example 28

Pharmacodynamic effects in thyroid-parathyroidectomized (TPTx) rats in a 28-day study with daily subcutaneous injections of conjugate 18 or PTH(1-84)

This study was conducted to test and compare the effects of daily subcutaneous injection of compound 18 and PTH(1-84), the current safety standard, in an animal disease model relevant to hypoparathyroidism (HP) treatment research. Rats subjected to thyroparathyroidectomy (TPTx) by blunt dissection fail to produce parathyroid hormone, PTH, the master regulator of calcium homeostasis. Consequently, TPTx rats develop hypocalcemia and hyperphosphatasemia, characteristic of HP. Seventeen-week-old female SD TPTx rats (n = 9/group) were dosed subcutaneously for 28 days with compound 18 (5 μg PTH eq/kg/day, 1.2 nmol/kg/day, in 10 mM succinic acid, 46 g/L mannitol, pH 4.0), PTH(1-84) (70 μg PTH eq/kg/day, 7.3 nmol/kg/day, in 10 mM citrate, mannitol 39.0 g/L, pH 5.0), or vehicle. Additionally, one group of sham-operated rats (n = 9), representing normal physiological background controls, also received vehicle. Serum calcium (sCa) and phosphorus (sP) levels in animals were measured pre- and post-dose on days 1, 6, 12, and 27. In addition, bone turnover markers (P1NP and CTx) were measured and bone quality was assessed by ex vivo pQCT.

Results: Mean sCa in TPTx rats predosed on day 1 was 8.3 mg/dL compared to 10.9 mg/dL in sham-operated controls. sP values were 8.7 mg/dL and 5.9 mg/dL, respectively. Compound 18 was administered daily at 1.2 nmol/kg, increasing sCa to near-normal levels while decreasing sP over several days of administration. On day 12 (day 5 at steady state with compound 18), sCa had stabilized at normal levels (10.7 mg/dL) in this group of animals (compound 18/sham control ratio = 1.01) in contrast to the hypocalcemic level (8.1 mg/dL) measured in PTH(1-84)-treated rats (PTH(1-84)/sham control ratio = 0.76). Furthermore, 24-hour urinary Ca excretion on day 12 was comparable between compound 18-treated animals and sham controls. Bone mineral density (BMD) and bone mineral content (BMC) were increased in TPTx controls, as observed in HP patients. Compound 18 treatment decreased BMD, BMC and area in parallel with an increase in CTx compared to sham-operated and TPTx vehicle-treated animals. A significant increase in trabecular BMD was observed in PTH(1-84)-treated animals compared to both control groups.

It was concluded that compound 18, at a dose even less than 20% of the molar equivalent of the PTH(1-84) dose tested here, was able to maintain sCa at a level comparable to that of sham-operated controls (representing normal levels here) for 24 hours. In contrast, PTH(1-84) at 7.3 nmol/kg/day did not result in an increase in sCa compared to vehicle-injected TPTx rats. However, minimal decreases in sP were observed in PTH(1-84)-treated animals, confirming the effects and responses to PTH(1-84) in rats. After 28 days of treatment with compound 18, trabecular and cortical BMD in the vertebrae were in the normal range, whereas an anabolic effect was observed for PTH(1-84) on trabecular and cortical bone in the vertebrae.

Example 29

Synthesis of linker reagent 29h

To a solution of compound 29a (250 g, 294 mmol, 1 equiv) in dichloromethane (1 L) was added a solution of Na2CO3 (187 g, 1.8 mol, 6 equiv) in H2O (1 L). The reaction solution was stirred at 15-30 °C for 0.5 h. TLC (DCM/MeOH = 10:1, Rf = 0.5) showed that the starting material was completely consumed. The organic layer was separated and the aqueous phase was extracted with dichloromethane (1 L). The organic layers were combined and washed with brine (800 mL), then dried with anhydrous Na2SO4, filtered and concentrated in vacuo to give compound 29b as a yellow oil.

Yield: 200 g, 272 mmol, 93%

Four reactions were carried out in parallel.

To a solution of compound 29b (50 g, 68.1 mmol, 1 equiv) and Fmoc-5-aminovaleric acid (25.4 g, 74.9 mmol, 1.1 equiv), DIPEA (61.6 g, 477 mmol, 83.3 mL, 7 equiv) in acetonitrile (500 mL) was added dropwise T3P 50% [EtOAc] (130 g, 204 mmol, 122 mL, 3 equiv) at 15-30 °C over 1 h. After addition, the reaction mixture was stirred at 15-30 °C for 18 h. TLC (Petroleum ether/Ethyl acetate = 1:1, Rf = 0.5) showed that the starting material was completely consumed. The four reactions were combined for study. The mixture was diluted with water (3 L), then adjusted to pH = 3~4 with 0.5 N HCl. The mixture was extracted with EtOAc (3 L), then the aqueous phase was extracted with EtOAc (2 L). The organic layers were combined and washed with brine (1 L), then dried with anhydrous Na2SO4, filtered and concentrated in vacuo to give the crude product as a yellow oil. The crude product was purified by column chromatography on silica gel using petroleum ether/ethyl acetate to give compound 29c as a yellow solid.

Yield: 220 g, 199 mmol, 73%

Four reactions were carried out in parallel.

Piperidine (47.3 g, 555 mmol, 55 mL, 10.7 equiv) was added to a solution of compound 29c (55 g, 52 mmol, 1 equiv) in dichloromethane (275 mL). The reaction solution was stirred for 3 h at 15-30 °C. TLC (Petroleum ether/Ethyl acetate = 1:1, Rf = 0) showed that the starting material was completely consumed. The four reactions were combined and the mixture was diluted with water (800 mL) and dichloromethane (800 L), then adjusted to pH = 3~4 with 0.5 N HCl solution. The organic layer was separated and the aqueous phase was extracted with dichloromethane (800 mL). The organic layers were combined and washed with brine (1 L), then dried with anhydrous Na2SO4, filtered and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography on silica gel using DCM/MeOH to give compound 29d as a white solid.

Yield: 140 g, 168 mmol, 81%

Four reactions were carried out in parallel.

To a solution of compound 29d (30 g, 36 mmol, 1 equiv) in THF (300 mL) were added (1,3-dioxo-1,3-dihydro-2H-isoindolyl-2-yl)acetaldehyde (6.8 g, 36 mmol, 1 equiv) and NaBH(OAc)3 (15.3 g, 72 mmol, 2 equiv) in one portion. After addition, the reaction mixture was stirred at 15-30 °C for 18 h. TLC (DCM/MeOH = 10:1, Rf = 0.4) showed that the starting material was completely consumed. The four reactions were combined and the mixture was diluted with water (2 L) and EtOAc (1.5 L). The organic layer was separated and the aqueous phase was extracted with EtOAc (1 L). The organic layers were combined and washed with brine (1 L), then dried over anhydrous Na2SO4, filtered and concentrated in vacuo to afford compound 29e as a yellow oil.

Yield: 164g, crude compound

Three reactions were carried out in parallel.

To a solution of compound 29e (50 g, 49.7 mmol, 1 equiv) in DCM (150 mL) were added Et3N (25.1 g, 248 mmol, 34.4 mL, 5 equiv) and Boc2O (21.7 g, 99.4 mmol, 22.8 mL, 2 equiv). After addition, the reaction mixture was stirred at 15-30 °C for 12 h. TLC (Petroleum ether/Ethyl acetate = 1:1, Rf = 0.4) showed that the starting material was completely consumed. The three reactions were combined and the mixture was diluted with water (800 mL), then adjusted to pH = 3~4 with 0.5 N HCl solution. The organic layer was separated and the aqueous phase was extracted with dichloromethane (800 mL). The organic layers were combined and washed with brine (800 mL), then dried with anhydrous Na2SO4, filtered and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography on silica gel using petroleum ether/ethyl acetate to give compound 29f as a yellow solid.

Yield: 80 g, 72.3 mmol, 48.5%

Three reactions were carried out in parallel.

To a solution of compound 29f (25 g, 22.6 mmol, 1 equiv) in DCM (125 mL) and EtOH (300 mL) was added NH2NH2.H2O (28.9 g, 565 mmol, 28 mL, 98% purity, 25 equiv) in one portion. After the addition, the reaction mixture was stirred at 15-30 °C for 18 h. TLC (Petroleum ether/Ethyl acetate = 1:1, Rf = 0.03) showed that the starting material was completely consumed. The three reactions were combined and the mixture was diluted with water (1 L) and dichloromethane (800 mL), then adjusted to pH = 3~4 with 0.5 N HCl solution. The organic layer was separated and the aqueous phase was extracted with dichloromethane (500 mL). The organic layers were combined and washed with brine (800 mL), then dried with anhydrous Na2SO4, filtered and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography on silica gel using DCM/MeOH to give compound 29g as a yellow oil.

Yield: 45 g, 46.1 mmol, 68%

Four reactions were carried out in parallel.

To a solution of compound 29g (11 g, 11.3 mmol, 1.0 equiv) in THF (100 mL) were added Et3N (3.4 g, 33.8 mmol, 4.7 mL, 3.0 equiv) and 4-nitrophenyl carboxychloridate (2.5 g, 12.4 mmol, 1.1 equiv). After addition, the reaction mixture was stirred at 15-30 °C for 18 h. TLC (Petroleum ether/Ethyl acetate = 1:1, Rf = 0.4) showed that the starting material was completely consumed. The four reactions were combined and the mixture was diluted with water (800 mL) and EtOAc (800 mL), then adjusted to pH = 3~4 with 0.5 N HCl solution. The organic layer was separated and the aqueous phase was extracted with EtOAc (500 mL). The organic layers were combined and washed with brine (800 mL), then dried with anhydrous Na2SO4, filtered and concentrated in vacuo to give the crude product. The crude product was purified by column chromatography on silica gel using petroleum ether:ethyl acetate to give compound 29h as a thick pale yellow oil.

Yield: 29 g, 25.4 mmol, 56%

Example 30

Synthesis of temporary conjugate S1 PTH(1-34) 30

Side chain protected PTH(1-34) on TCP resin having an Fmoc protecting group at the N-terminus was subjected to Fmoc deprotection according to the procedure described in the Materials and Methods section. A solution of Fmoc-Ser(Trt)-OH (997 mg, 1.75 mmol), PyBOP (911 mg, 1.75 mmol), and DIPEA (305 μL, 1.75 mmol) in DMF (5 mL) was added to 5.0 g (0.58 mmol) of resin. The suspension was shaken overnight at room temperature. The resin was washed 10x with DMF and Fmoc deprotection was performed as described above. A solution of compound 29h (2.66 g, 2.33 mmol) and DIPEA (611 μL, 3.50 mmol) in DMF (5 mL) was added to the resin. The suspension was shaken overnight at room temperature. The resin was washed 10x with DMF, 10x with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of the protecting groups were achieved by adding 30 mL of a 100/3/3/2/1 (v/w/v/v/v) TFA/EDT/TES/water/thioanisole cleavage cocktail and shaking the suspension for 1 h at room temperature. Crude compound 30 was precipitated in pre-cooled diethyl ether (-18 °C). The residue was dissolved in ACN/water and purified by RP-HPLC. The product fractions were lyophilized.

Yield: 168 mg (5%), 30*9 TFA

MS: m/z 1155.92 = [M+4H]4+, (calculated monoisotopic mass for [M+4H]4+ = 1155.85).

Example 31

Synthesis of a temporary 4x10 kDa S1 PEG 31 conjugate

2.3 mL of a solution containing compound 30 (13 mg/mL, 30 mg, 5.31 μmol) in 8/2 (v/v) water/ethanol containing 0.1% TFA (v/v) and 10 mM methionine were added to 3.4 mL of a solution containing PEG 2x10 kDa - maleimide (Sunbright GL2-200MA, 342 mg, 15.9 μmol) in the same solvent. 0.5 M NaH2PO4 buffer (0.8 mL, pH 7.0) was added and the mixture was stirred for 30 min at room temperature. 20 μL of TFA was added and the mixture was stored at 4°C overnight. Conjugate 31 was purified by IEX followed by RP-HPLC using a linear gradient of solvent system A (water containing 0.2% AcOH v/v) and solvent system B (acetonitrile containing 0.2% AcOH v/v). Fractions containing the solution were lyophilized.

Yield: 161 mg (3.55 μmol, 67%) of conjugate 31*9 AcOH

Example 32

In vitro release kinetics of a transient 4x10 kDa PEG 31 conjugate

Conjugate 31 (11 mg) was dissolved in 1 v/v % acetic acid in water (1.8 mL) at 0.5 mg PTH eq/mL. Buffer exchange to pH 7.4 phosphate buffer (100 mM NaH2PO4, 10 mM L-methionine, 3 mM EDTA, 0.05 % Tween-20, adjusted to pH 7.4 with NaOH) was performed by SEC chromatography. The eluate was further diluted with phosphate buffer to achieve a concentration of 0.1 mg PTH eq/mL. The resulting solution was sterile filtered and incubated at 37°C. Aliquots were taken at time points and analyzed by RP-HPLC. The fraction of released PTH at a time was calculated from the ratio of the UV peak areas of released PTH and PEG conjugate. The % PTH released was plotted against incubation time. Curve fitting software was used to calculate the corresponding half-lives.

Results:

For conjugate 31, a half-life of 14.5 days was obtained.

Example 33

Pharmacodynamic effects in cynomolgus monkeys in a single dose PK/PD study using compound 18

Compound 18 was administered at 1 μg/kg to male cynomolgus monkeys (N = 3) in a single subcutaneous PK/PD study assessing serum calcium (sCa) levels and urinary calcium excretion for 96 hours post-dose.

Results: Following administration of compound 18 at 1 μg/kg to cynomolgus monkeys, sCa levels remained within the normal range for 96 hours post-dose, and a clear downward trend in urinary calcium levels was observed within the first 24 hours.

Conclusion: At a dose maintaining sCa in the normocalcemic range, a concomitant reduction in urinary Ca excretion was observed, and therefore compound 18 addresses a key unmet medical need in patients with HP.

Example 34

Pharmacokinetic study of a transient 2x20 kDa S1 PEG 18 conjugate in cynomolgus monkeys

Naïve cynomolgus macaques (2-3.5 years, 2-5 kg) (3-5 males/3-5 females) received daily subcutaneous administration of compound 18 at dose levels of 0.2, 0.5, and 1.5 μg PTH/kg. Blood samples were collected at; day 1: pre-dose, 4 h, 8 h, 12 h, 18 h, and 24 h post-dose, day 8: pre-dose, day 14: pre-dose, 8 h, and 12 h, and day 28: 3 h, 6 h, 8 h, 12 h, 18 h, 24 h, 72 h, 168 h, and 336 h) and plasma was obtained. Total plasma PTH concentrations were determined by quantifying the N-terminal signature peptide (sequence: IQLMHNLGK) after LysC and GluC cleavage as described in the Substances and Methods section.

Results: Systemic exposure, expressed as Cmax and AUC, increased proportionally with dose. Systemic exposure to total PTH, expressed as AUC, accumulated approximately 3-fold from day 1 to day 28.

Low peak-to-trough ratios of total PTH for all dose groups were observed after daily subcutaneous administration to cynomolgus monkeys at day 28 (steady state was observed from day 8).

Example 35

Pharmacokinetic study of a transient 2x20 kDa S1 PEG 18 conjugate in Sprague-Dawley rats

Sprague-Dawley Crl:CD(SD) rats (dosing started at 8 weeks of age) received daily subcutaneous administration of compound 18 at dose levels of 10, 30, and 60 μg PTH/kg for 28 days. The TK group, containing 9 males and 9 females per dose group, was divided into 3 subgroups with 3 rats per subgroup. Blood samples were collected through day 28 from 3 rats per sex, per sampling time point. Samples were collected on day 1: pre-dose, 4 h, 8 h, 12 h, 18 h, and 24 h post-dose and on day 28: 3 h, 6 h, 8 h, 12 h, 18 h, 24 h, and 336 h, and plasma was prepared. Total plasma PTH concentrations were determined by quantifying the N-terminal signature peptide (sequence: IQLMHNLGK) after LysC and GluC cleavage as described in the Substances and Methods section.

Results: Systemic exposure, expressed as mean Cmax and AUC, increased proportionally with dose. Systemic exposure to total PTH, expressed as mean AUC, accumulated 3- to 6-fold from day 1 to day 28. Systemic exposure in female rats was approximately 2-fold higher than in males.

Low peak-to-trough ratios of total PTH for all dose groups were observed after daily subcutaneous administration to Sprague-Dawley rats at day 28 (steady state observed from day 8).

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-oxoethylideneaminooxy)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 - - dimethylaminopyridine

DMF - N,N-dimethylformamide

DMSO - - dimethyl sulfoxide

DTT - dithiothreitol

EDTA - ethylenediaminetetraacetic acid

eq - stoichiometric equivalent

ESI-MS - Electrospray Ionization Mass Spectrometry

Et - ethyl

Fmoc - 9-fluorenylmethyloxycarbonyl

Glu-C - Glu-C endoproteinase

h - hour

HATU - O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate

HP - hypoparathyroidism

HPLC - High Performance Liquid Chromatography

ivDde - 4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl

LC - liquid chromatography

LTQ - Linear Quadrupole Trap

Lys-C - endoproteinase Lys-C

LLOQ - lower limit of quantitative indicator

Mal - 3-maleimidopropyl

Me - methyl

MeOH - methanol

min - minutes

Mmt - monomethoxytrityl

MS - mass spectrometry

m/z - mass to charge ratio

OtBu - tert-butyloxy

PEG - poly(ethylene glycol)

pH - Potency of Hydrogens

PK - pharmacokinetics

Pr - saw cut

PTH - parathyroid hormone

PyBOP - benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate

Q-TOF - quadrupole time-of-flight

RP-HPLC - reversed-phase high-performance liquid chromatography

rt - room temperature

sCa - serum calcium

SIM - monitoring of selected ions

SEC - size exclusion chromatography

sc - subcutaneously

sP - serum phosphate

t1/2 - half-life

TCP - trityl chloride polystyrene

TES - triethylsilane

TFA - trifluoroacetic acid

THF - tetrahydrofuran

TK - toxicokinetic

Tmob - 2,4,6-trimethoxybenzyl

TPTx - thyroparathyroidectomy

Trt - triphenylmethyl, trityl

ULOQ - Upper Limit of Quantity

UPLC - Ultra High Performance Liquid Chromatography

UV - ultraviolet

ZQ – single quadrupole.

--->

SEQUENCE LIST

<110> Ascendis Pharma A/S

<120> PTH PRODRUGS

<130>CPX70238PC

<160> 122

<170> PatentIn version 3.5

<210> 1

<211> 84

<212> PRT

<213> Homo sapiens

<400> 1

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys

65 70 75 80

Ala Lys Ser Gln

<210> 2

<211> 83

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-83

<400> 2

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys

65 70 75 80

Ala Lys Ser

<210> 3

<211> 82

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-82

<400> 3

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys

65 70 75 80

Ala Lys

<210> 4

<211> 81

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-81

<400> 4

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys

65 70 75 80

Ala

<210> 5

<211> 80

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-80

<400> 5

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys

65 70 75 80

<210> 6

<211> 79

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-79

<400> 6

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr

65 70 75

<210> 7

<211> 78

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-78

<400> 7

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu

65 70 75

<210> 8

<211> 77

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-77

<400> 8

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val

65 70 75

<210> 9

<211> 76

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-76

<400> 9

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn

65 70 75

<210> 10

<211> 75

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-75

<400> 10

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val

65 70 75

<210> 11

<211> 74

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-74

<400> 11

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp

65 70

<210> 12

<211> 73

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-73

<400> 12

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala

65 70

<210> 13

<211> 72

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-72

<400> 13

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys

65 70

<210> 14

<211> 71

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-71

<400> 14

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp

65 70

<210> 15

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-70

<400> 15

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala

65 70

<210> 16

<211> 69

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-69

<400> 16

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu

65

<210> 17

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-68

<400> 17

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly

65

<210> 18

<211> 67

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-67

<400> 18

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu

65

<210> 19

<211> 66

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-66

<400> 19

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser

65

<210> 20

<211> 65

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-65

<400> 20

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys

65

<210> 21

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-64

<400> 21

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

<210> 22

<211> 63

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-63

<400> 22

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His

50 55 60

<210> 23

<211> 62

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-62

<400> 23

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser

50 55 60

<210> 24

<211> 61

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-61

<400> 24

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu

50 55 60

<210> 25

<211> 60

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-60

<400> 25

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val

50 55 60

<210> 26

<211> 59

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-59

<400> 26

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu

50 55

<210> 27

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-58

<400> 27

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val

50 55

<210> 28

<211> 57

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-57

<400> 28

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn

50 55

<210> 29

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-56

<400> 29

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp

50 55

<210> 30

<211> 55

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-55

<400> 30

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu

50 55

<210> 31

<211> 54

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-54

<400> 31

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys

50

<210> 32

<211> 53

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-53

<400> 32

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys

50

<210> 33

<211> 52

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-52

<400> 33

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg

50

<210> 34

<211> 51

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-51

<400> 34

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro

50

<210> 35

<211> 50

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-50

<400> 35

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg

50

<210> 36

<211> 49

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-49

<400> 36

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln

<210> 37

<211> 48

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-48

<400> 37

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

<210> 38

<211> 47

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-47

<400> 38

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly

35 40 45

<210> 39

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-46

<400> 39

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala

35 40 45

<210> 40

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-45

<400> 40

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp

35 40 45

<210> 41

<211> 44

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-44

<400> 41

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg

35 40

<210> 42

<211> 43

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-43

<400> 42

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro

35 40

<210> 43

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-42

<400> 43

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala

35 40

<210> 44

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> human PTH-41

<400> 44

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu

35 40

<210> 45

<211> 40

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-40

<400> 45

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro

35 40

<210> 46

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-39

<400> 46

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala

35

<210> 47

<211> 38

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-38

<400> 47

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly

35

<210> 48

<211> 37

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-37

<400> 48

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu

35

<210> 49

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-36

<400> 49

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala

35

<210> 50

<211> 35

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-35

<400> 50

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val

35

<210> 51

<211> 34

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-34

<400> 51

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe

<210> 52

<211> 33

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-33

<400> 52

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn

<210> 53

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-32

<400> 53

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

<210> 54

<211> 31

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-31

<400> 54

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val

20 25 30

<210> 55

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-30

<400> 55

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp

20 25 30

<210> 56

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-29

<400> 56

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln

20 25

<210> 57

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-28

<400> 57

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu

20 25

<210> 58

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-27

<400> 58

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys

20 25

<210> 59

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-26

<400> 59

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys

20 25

<210> 60

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> human PTH 1-25

<400> 60

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg

20 25

<210> 61

<211> 84

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-84

<220>

<221>MOD_RES

<222> (84)..(84)

<223> amidation

<400> 61

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys

65 70 75 80

Ala Lys Ser Gln

<210> 62

<211> 83

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-83

<220>

<221>MOD_RES

<222> (83)..(83)

<223> amidation

<400> 62

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys

65 70 75 80

Ala Lys Ser

<210> 63

<211> 82

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-82

<220>

<221>MOD_RES

<222> (82)..(82)

<223> amidation

<400> 63

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys

65 70 75 80

Ala Lys

<210> 64

<211> 81

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-81

<220>

<221>MOD_RES

<222> (81)..(81)

<223> amidation

<400> 64

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys

65 70 75 80

Ala

<210> 65

<211> 80

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-80

<220>

<221>MOD_RES

<222> (80)..(80)

<223> amidation

<400> 65

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys

65 70 75 80

<210> 66

<211> 79

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-79

<220>

<221>MOD_RES

<222> (79)..(79)

<223> amidation

<400> 66

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr

65 70 75

<210> 67

<211> 78

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-78

<220>

<221>MOD_RES

<222> (78)..(78)

<223> amidation

<400> 67

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu

65 70 75

<210> 68

<211> 77

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-77

<220>

<221>MOD_RES

<222> (77)..(77)

<223> amidation

<400> 68

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val

65 70 75

<210> 69

<211> 76

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-76

<220>

<221>MOD_RES

<222> (76)..(76)

<223> amidation

<400> 69

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn

65 70 75

<210> 70

<211> 75

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-75

<220>

<221>MOD_RES

<222> (75)..(75)

<223> amidation

<400> 70

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val

65 70 75

<210> 71

<211> 74

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-74

<220>

<221>MOD_RES

<222> (74)..(74)

<223> amidation

<400> 71

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp

65 70

<210> 72

<211> 73

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-73

<220>

<221>MOD_RES

<222> (73)..(73)

<223> amidation

<400> 72

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys Ala

65 70

<210> 73

<211> 72

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-72

<220>

<221>MOD_RES

<222> (72)..(72)

<223> amidation

<400> 73

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp Lys

65 70

<210> 74

<211> 71

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-71

<220>

<221>MOD_RES

<222> (71)..(71)

<223> amidation

<400> 74

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala Asp

65 70

<210> 75

<211> 70

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-70

<220>

<221>MOD_RES

<222> (70)..(70)

<223> amidation

<400> 75

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu Ala

65 70

<210> 76

<211> 69

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-69

<220>

<221>MOD_RES

<222> (69)..(69)

<223> amidation

<400> 76

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly Glu

65

<210> 77

<211> 68

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-68

<220>

<221>MOD_RES

<222> (68)..(68)

<223> amidation

<400> 77

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu Gly

65

<210> 78

<211> 67

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-67

<220>

<221>MOD_RES

<222> (67)..(67)

<223> amidation

<400> 78

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser Leu

65

<210> 79

<211> 66

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-66

<220>

<221>MOD_RES

<222> (66)..(66)

<223> amidation

<400> 79

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys Ser

65

<210> 80

<211> 65

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-65

<220>

<221>MOD_RES

<222> (65)..(65)

<223> amidation

<400> 80

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

Lys

65

<210> 81

<211> 64

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-64

<220>

<221>MOD_RES

<222> (64)..(64)

<223> amidation

<400> 81

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu

50 55 60

<210> 82

<211> 63

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-63

<220>

<221>MOD_RES

<222> (63)..(63)

<223> amidation

<400> 82

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His

50 55 60

<210> 83

<211> 62

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-62

<220>

<221>MOD_RES

<222> (62)..(62)

<223> amidation

<400> 83

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser

50 55 60

<210> 84

<211> 61

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-61

<220>

<221>MOD_RES

<222> (61)..(61)

<223> amidation

<400> 84

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu

50 55 60

<210> 85

<211> 60

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-60

<220>

<221>MOD_RES

<222> (60)..(60)

<223> ACETYLATION

<400> 85

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val

50 55 60

<210> 86

<211> 59

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-59

<220>

<221>MOD_RES

<222> (59)..(59)

<223> amidation

<400> 86

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu

50 55

<210> 87

<211> 58

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-58

<220>

<221>MOD_RES

<222> (58)..(58)

<223> amidation

<400> 87

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn Val

50 55

<210> 88

<211> 57

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-57

<220>

<221>MOD_RES

<222> (57)..(57)

<223> amidation

<400> 88

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp Asn

50 55

<210> 89

<211> 56

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-56

<220>

<221>MOD_RES

<222> (56)..(56)

<223> amidation

<400> 89

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu Asp

50 55

<210> 90

<211> 55

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-55

<220>

<221>MOD_RES

<222> (55)..(55)

<223> amidation

<400> 90

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys Glu

50 55

<210> 91

<211> 54

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-54

<220>

<221>MOD_RES

<222> (54)..(54)

<223> amidation

<400> 91

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys Lys

50

<210> 92

<211> 53

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-53

<220>

<221>MOD_RES

<222> (53)..(53)

<223> amidation

<400> 92

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg Lys

50

<210> 93

<211> 52

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-52

<220>

<221>MOD_RES

<222> (52)..(52)

<223> amidation

<400> 93

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro Arg

50

<210> 94

<211> 51

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-51

<220>

<221>MOD_RES

<222> (51)..(51)

<223> amidation

<400> 94

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg Pro

50

<210> 95

<211> 50

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-50

<220>

<221>MOD_RES

<222> (50)..(50)

<223> amidation

<400> 95

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln Arg

50

<210> 96

<211> 49

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-49

<220>

<221>MOD_RES

<222> (49)..(49)

<223> amidation

<400> 96

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

Gln

<210> 97

<211> 48

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-48

<220>

<221>MOD_RES

<222> (48)..(48)

<223> amidation

<400> 97

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser

35 40 45

<210> 98

<211> 47

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-47

<220>

<221>MOD_RES

<222> (47)..(47)

<223> amidation

<400> 98

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly

35 40 45

<210> 99

<211> 46

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-46

<220>

<221>MOD_RES

<222> (46)..(46)

<223> amidation

<400> 99

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala

35 40 45

<210> 100

<211> 45

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-45

<220>

<221>MOD_RES

<222> (45)..(45)

<223> amidation

<400> 100

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp

35 40 45

<210> 101

<211> 44

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-44

<220>

<221>MOD_RES

<222> (44)..(44)

<223> amidation

<400> 101

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg

35 40

<210> 102

<211> 43

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-43

<220>

<221>MOD_RES

<222> (43)..(43)

<223> amidation

<400> 102

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro

35 40

<210> 103

<211> 42

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-42

<220>

<221>MOD_RES

<222> (42)..(42)

<223> amidation

<400> 103

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu Ala

35 40

<210> 104

<211> 41

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-41

<220>

<221>MOD_RES

<222> (41)..(41)

<223> amidation

<400> 104

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro Leu

35 40

<210> 105

<211> 40

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-40

<220>

<221>MOD_RES

<222> (40)..(40)

<223> amidation

<400> 105

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala Pro

35 40

<210> 106

<211> 39

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-39

<220>

<221>MOD_RES

<222> (39)..(39)

<223> amidation

<400> 106

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly Ala

35

<210> 107

<211> 38

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-38

<220>

<221>MOD_RES

<222> (38)..(38)

<223> amidation

<400> 107

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu Gly

35

<210> 108

<211> 37

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-37

<220>

<221>MOD_RES

<222> (37)..(37)

<223> amidation

<400> 108

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala Leu

35

<210> 109

<211> 36

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-36

<220>

<221>MOD_RES

<222> (36)..(36)

<223> amidation

<400> 109

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val Ala

35

<210> 110

<211> 35

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-35

<220>

<221>MOD_RES

<222> (35)..(35)

<223> amidation

<400> 110

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe Val

35

<210> 111

<211> 34

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-34

<220>

<221>MOD_RES

<222> (34)..(34)

<223> amidation

<400> 111

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn Phe

<210> 112

<211> 33

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-33

<220>

<221>MOD_RES

<222> (33)..(33)

<223> amidation

<400> 112

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

Asn

<210> 113

<211> 32

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-32

<220>

<221>MOD_RES

<222> (32)..(32)

<223> amidation

<400> 113

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His

20 25 30

<210> 114

<211> 31

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-31

<220>

<221>MOD_RES

<222> (31)..(31)

<223> amidation

<400> 114

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val

20 25 30

<210> 115

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-30

<220>

<221>MOD_RES

<222> (30)..(30)

<223> amidation

<400> 115

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp

20 25 30

<210> 116

<211> 29

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-29

<220>

<221>MOD_RES

<222> (29)..(29)

<223> amidation

<400> 116

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln

20 25

<210> 117

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-28

<220>

<221>MOD_RES

<222> (28)..(28)

<223> amidation

<400> 117

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu

20 25

<210> 118

<211> 27

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-27

<220>

<221>MOD_RES

<222> (27)..(27)

<223> amidation

<400> 118

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys

20 25

<210> 119

<211> 26

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-26

<220>

<221>MOD_RES

<222> (26)..(26)

<223> amidation

<400> 119

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg Lys

20 25

<210> 120

<211> 25

<212> PRT

<213> artificial sequence

<220>

<223> amidated human PTH 1-25

<220>

<221>MOD_RES

<222> (25)..(25)

<223> amidation

<400> 120

Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn

1 5 10 15

Ser Met Glu Arg Val Glu Trp Leu Arg

20 25

<210> 121

<211> 141

<212> PRT

<213> Homo sapiens

<400> 121

Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln

1 5 10 15

Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu Ile His

20 25 30

Thr Ala Glu Ile Arg Ala Thr Ser Glu Val Ser Pro Asn Ser Lys Pro

35 40 45

Ser Pro Asn Thr Lys Asn His Pro Val Arg Phe Gly Ser Asp Asp Glu

50 55 60

Gly Arg Tyr Leu Thr Gln Glu Thr Asn Lys Val Glu Thr Tyr Lys Glu

65 70 75 80

Gln Pro Leu Lys Thr Pro Gly Lys Lys Lys Lys Gly Lys Pro Gly Lys

85 90 95

Arg Lys Glu Gln Glu Lys Lys Lys Arg Arg Thr Arg Ser Ala Trp Leu

100 105 110

Asp Ser Gly Val Thr Gly Ser Gly Leu Glu Gly Asp His Leu Ser Asp

115 120 125

Thr Ser Thr Thr Ser Leu Glu Leu Asp Ser Arg Arg His

130 135 140

<210> 122

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> artificial random spiral

<400> 122

Gly Gly Pro Gly Gly Pro Gly Pro Gly Gly Pro Gly Gly Pro Gly Pro

1 5 10 15

Gly Gly Pro Gly

20

<---

Claims (15)

1. Conjugate of formula 31 or its pharmaceutically acceptable salt, moreover PTH(1-34) is a parathyroid hormone having the sequence SEQ ID NO: 51; N ^αS1 is the alpha-amino group of the N-terminal serine of SEQ ID NO: 51; and 2x10 kDa PEG is a fragment of formula (b) where the dotted line indicates the point of attachment to the -S- link in formula 31; and each of m and p is independently an integer such that each polyethyleneglycol arm has a molecular mass of about 10 kDa. 2. A pharmaceutical composition for treating hypoparathyroidism, comprising an effective amount of the conjugate according to claim 1 or a pharmaceutically acceptable salt thereof and at least one excipient. 3. A method for treating hypoparathyroidism, comprising administering to a subject in need thereof a pharmaceutically effective amount of a conjugate or a pharmaceutically acceptable salt thereof according to claim 1 or a pharmaceutical composition according to claim 2. 4. The method according to claim 3, wherein the conjugate or its pharmaceutically acceptable salt or pharmaceutical composition is administered by injection. 5. The method according to claim 3 or 4, wherein the conjugate or its pharmaceutically acceptable salt or pharmaceutical composition is administered by subcutaneous injection. 6. The method according to item 5, wherein the subcutaneous injection is carried out using a syringe pen.