DOSAGE SCHEME FOR CONTROLLED RELEASE OF A PTH CONNECTION

Description

[0001] The present invention relates to a pharmaceutical composition comprising at least one controlled-release PTH compound or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in the treatment of hypoparathyroidism.

[0002] Hypoparathyroidism is a rare endocrine disorder of calcium and phosphate metabolism that most often arises as a result of parathyroid gland damage or removal during surgery of the thyroid gland. Hypoparathyroidism is unusual among endocrine disorders in that it has not been treated, until recently, by replacement with the missing hormone, parathyroid hormone, or PTH. Conventional therapy for hypoparathyroidism involves large doses of vitamin D and oral calcium supplementation, which, although often effective, is associated with marked swings in blood Ca 2+< resulting in hypercalcemia and hypocalcemia, excess urinary calcium excretion, nephrocalcinosis and ectopic calcifications, including vascular, basal ganglia, and lens of eye.

[0003] Calcium is the most abundant mineral in the human body, and its tight regulation is required for many critical biological functions, such as bone mineralization, muscle contraction, nerve conduction, hormone release, and blood coagulation. It is particularly important to maintain calcium concentration as stable as possible, because of the high sensitivity of a variety of cell systems or organs, including the central nervous system, muscle, and exo / endocrine glands, to small variations in Ca 2+< . PTH is a major regulator of calcium homeostasis.

[0004] The inappropriately low PTH level in relation to serum Ca 2+< concentration, characteristic of hypoparathyroidism, leads to decreased renal tubular reabsorption of Ca 2+< and simultaneously, to increased renal tubular reabsorption of phosphate. Thus, the main biochemical abnormalities of hypoparathyroidism are hypocalcemia and hyperphosphatemia. Clinical features of the disease include symptoms of hypocalcemia, such as perioral numbness, paresthesias, and carpal / pedal muscle spasms. Laryngeal spasm, tetany, and seizures are serious and potentially life-threatening complications. Hyperphosphatemia and an elevated calcium x phosphate product contributes to ectopic deposition of insoluble calcium phosphate complexes in soft tissues, including vasculature, brain, kidneys, and other organs.

[0005] Standard therapy of hypoparathyroidism is oral calcium and vitamin D supplementation. The goals of therapy are to a) ameliorate symptoms of hypocalcemia; b) maintain fasting serum calcium within or slightly below to the low-normal range; c) maintain fasting serum phosphorus within the high normal range or only slightly elevated; d) avoid or minimize hypercalciuria; e) maintain a calcium-phosphate product at levels well below the upper limit of normal and f) avoid ectopic calcification of the kidney (stones and nephrocalcinosis) and other soft tissues.

[0006] Several concerns arise with prolonged use of calcium and active vitamin D in large doses, particularly with regard to hypercalciuria, kidney stones, nephrocalcinosis and ectopic soft tissue calcification. In addition, conventional therapy with calcium and active vitamin D does not alleviate quality of life complaints nor does it reverse abnormalities in bone remodeling characteristic of the disease. In short, there is a high need for improved therapies for hypoparathyroidism.

[0007] In 2015, Natpara, PTH(1-84), was approved for once-daily subcutaneous injection as an adjunct to vitamin D and calcium in patients with hypoparathyroidism. Natpara, PTH(1-84), was approved to control hypocalcemia based on a pivotal trial demonstrating that 42 percent of PTH(1-84) treated participants achieved normal blood calcium levels on reduced doses of calcium supplements and active forms of vitamin D, compared to 3 percent of placebo-treated participants. Following a time course in which serum calcium was monitored after injection, 71 percent of patients treated with PTH(1-84) developed hypercalcemia at one or more measurements during a 24-hour period. PTH(1-84) reduced urinary calcium excretion 2-8 hours after injection but over the 24-hour period, urinary calcium excretion did not change. Similarly, urinary phosphate excretion increased only during the first 8 hours after PTH(1-84) injection.

[0008] While this represents an important advance in the treatment of the disease, Natpara has not demonstrated an ability to reduce incidences of hypercalcemia (elevated serum calcium levels), hypocalcemia (low serum calcium), or hypercalciuria (elevated urinary calcium) relative to conventional therapy in treated patients.

[0009] As such, there is a high need for improved PTH based therapies for hypoparathyroidism.

[0010] PTH(1-34), or teriparatide, was approved by the FDA in 2002 for the treatment of osteoporosis. Despite not being approved for this indication, PTH(1-34) has historically been used for treatment of hypoparathyroidism with patients receiving twice- or thrice-daily injections. To facilitate more physiological PTH levels, clinical studies have been conducted with PTH(1-34) administered by pump delivery in comparison with twice-daily injections. Over 6-months, pump delivery produced normal, steady state calcium levels with minimal fluctuation and avoided the rise in serum and urine calcium levels that are evident soon after PTH injection. The marked reduction in urinary calcium excretion when PTH(1-34) is administered by pump may indicate that PTH must be continuously exposed to the renal tubule for the renal calcium-conserving effects to be realized. Pump delivery of PTH(1-34) achieved simultaneous normalization of markers of bone turnover, serum calcium, and urine calcium excretion. These results were achieved with a 65 percent lower daily PTH(1-34) dose and a reduced need for magnesium supplementation compared with the twice daily PTH(1-34) injection regimen.

[0011] However, continuous pump therapy is inconvenient and challenging for patients, and it is an object of the current invention to provide for a more convenient therapeutic option of providing continuous exposure to PTH.

[0012] Long-term daily administration of PTH is associated with a progressive cortical bone loss due to increased bone metabolism. In a 6-year follow-up of patients treated with PTH(1-84) (Rubin, JCEM 2016) bone turnover markers remained greater than pretreatment values, peaking at the early years after PTH(1-84) initiation and declining thereafter but remaining significantly higher than baseline values by year 6. Bone mineral density (BMD) by dual X-ray absorptometry (DXA) was consistent with known site-specific effects of PTH, namely increases in lumbar spine and declines in the distal 1 / 3 radius. The decrease observed at the distal 1 / 3 radius is consistent with the known effects of intermittent PTH to increase cortical porosity and endosteal resorption.

[0013] It is an object of this invention to provide for a method of intermittently administering PTH, with improved control of serum and urine calcium, serum phosphorus, and lower elevation of bone turnover markers than currently applied PTH therapies. Preferably intermittent means with daily intervals, or more preferred with weekly intervals.

[0014] In the preclinical development program of both Forteo, PTH(1-34) and Natpara, PTH(1-84) a dose dependent increase in osteosarcoma rate was observed in rats treated with daily injections of the PTH compound. In the Natpara study, dosing of the high dose rats were discontinued due to excessive deaths in this group, primarily from metastatic osteosarcoma. This is felt to be due to the sensitivity of rats to the anabolic effects of intermittent PTH. In contrast, continuous exposure to PTH is known to lack significant bone anabolic activity. As such is an object of this invention to provide for an intermittent PTH replacement therapy that provides for an infusion-like profile of PTH, resulting in improved symptom control with a lower administered dose. Preferably intermittent means with daily intervals, or more preferred or alternatively with weekly intervals.

[0015] In summary, there is a need for a more convenient and safer treatment of hypoparathyroidism with reduced side-effects.

[0016] It is therefore an object of the present invention to at least partially overcome the shortcomings described above.

[0017] This object is achieved with a pharmaceutical composition as defined in claim 1.

[0018] It was surprisingly found that such controlled-release PTH compound has a higher potency than PTH 1-84, so fewer molar equivalents need to be administered in a single administration to a patient to achieve beneficial serum calcium levels for at least 24 hours which improves efficacy and reduces the risks of side-effects.

[0019] It is understood that PTH 1-84 is the polypeptide with the sequence of SEQ ID NO: 1.

[0020] Within the present invention the terms are used having the meaning as follows.

[0021] As used herein the terms "no more frequently than once every 24 hours" and "at least once every 24 hours" are used synonymously and mean that the time between two consecutive administrations is 24 hours or longer, meaning that there may for example be 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours or one week between two consecutive administrations.

[0022] As used herein the terms "within normal level" and "within the normal range" with regard to serum calcium levels refer to the calcium level ordinarily found in a subject of a given species, sex and age, provided as the range given by the lower limit of normal and the upper limit of normal. In humans, the normal level corresponds to a serum calcium level of above 8.5 mg / dL (albumin-adjusted). In humans the upper limit of normal is below 10.5 mg / dL

[0023] As used herein the term "serum calcium above 8.5 mg / dL" refers to albumin-adjusted calcium concentrations.

[0024] As used herein the term "albumin-adjusted" with regard to calcium levels means that the measured serum calcium level is corrected for calcium bound to albumin according to the following formula: albumin - adjusted serum calcium mg / dL = measured total Ca mg / dL + 0.8 4.0 − serum albumin g / dL

[0025] As used herein the term "non-adjusted" with regard to calcium levels means that the measured total calcium concentration (mg / dL) is not adjusted for albumin-binding of calcium.

[0026] The term "molar equivalent dose" refers to the dose in which the controlled-release PTH compound comprises the same number of PTH molecules or PTH molecules as a particular dose of PTH 1-84 comprises PTH 1-84 molecules. For example, if a controlled-release PTH compound comprises one PTH molecule or PTH moiety per controlled-release PTH compound the molar equivalent dose is 1 controlled-release PTH compound for every 1 molecule of PTH 1-84.

[0027] The term PTH 1-84 refers to the polypeptide sequence SEQ ID NO:1 (PTH 1-84):

[0028] As used herein the term "controlled-release PTH compound" refers to the compound of formula 19.

[0029] "PTH(1-34)" refers to the PTH polypeptide of SEQ ID NO:51: SEQ ID NO:51 (PTH 1-34) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF

[0030] As used herein the term "polypeptide" refers to a peptide comprising up to and including 50 amino acid monomers.

[0031] As used herein the term "pharmaceutical composition" refers to a composition containing one or more active ingredients, such as for example the controlled-release PTH compound, and one or more excipients, as well as any product which results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients of the composition, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients.

[0032] As used herein the term "liquid composition" refers to a mixture comprising water-soluble controlled-release PTH compound and one or more solvents, such as water.

[0033] As used herein, the term "dry composition" means that a pharmaceutical composition is provided in a dry form. Suitable methods for drying are spray-drying and lyophilization, i.e. freeze-drying. Such dry composition of prodrug has a residual water content of a maximum of 10%, preferably less than 5% and more preferably less than 2%, determined according to Karl Fischer. Preferably, the pharmaceutical composition for use of the present invention is dried by lyophilization.

[0034] As used herein, the term "excipient" refers to a diluent, adjuvant, or vehicle with which the therapeutic, such as a drug or prodrug, is administered. Such pharmaceutical excipient can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including peanut oil, soybean oil, mineral oil and sesame oil. Water is a preferred excipient when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred excipients when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed 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, dried skim milk, glycerol, propylene, glycol, water and ethanol. The pharmaceutical composition, if desired, can also contain minor amounts of wetting or emulsifying agents, pH buffering agents, like, for example, acetate, succinate, tris, carbonate, phosphate, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), or can contain detergents, like Tween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids like, for example, glycine, lysine, or histidine. These pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders and sustained-release formulations. The pharmaceutical composition can be formulated as a suppository, with traditional binders and excipients such as triglycerides. Oral formulation can include standard excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions will contain a therapeutically effective amount of the drug or biologically active moiety, together with a suitable amount of excipient so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0035] In case the controlled-release PTH compound for use of the present invention comprise one or more acidic or basic groups, the invention also comprises their corresponding pharmaceutically or toxicologically acceptable salts, in particular their pharmaceutically utilizable salts. Thus, the controlled-release PTH compound for use of the present invention comprising acidic groups can be used according to the invention, for example, as alkali metal salts, alkaline earth metal salts or as ammonium salts. More precise examples of such salts include sodium salts, potassium salts, calcium salts, magnesium salts or salts with ammonia or organic amines such as, for example, ethylamine, ethanolamine, triethanolamine or amino acids. Controlled-release PTH compound for use of the present invention comprising one or more basic groups, i.e. groups which can be protonated, can be present and can be used according to the invention in the form of their addition salts with inorganic or organic acids. Examples for suitable acids include hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuric acid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid, pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelic acid, fumaric acid, maleic acid, malic acid, sulfaminic acid, phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid, citric acid, adipic acid, and other acids known to the person skilled in the art. For the person skilled in the art further methods are known for converting the basic group into a cation like the alkylation of an amine group resulting in a positively-charge ammonium group and an appropriate counterion of the salt. If the controlled-release PTH compound for use of the present invention simultaneously comprise acidic and basic groups, the invention also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). The respective salts can be obtained by customary methods which are known to the person skilled in the art like, for example by contacting these compounds 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 compounds for use of the present invention which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.

[0036] The term "pharmaceutically acceptable" means a substance that does cause harm when administered to a patient and preferably means approved by a regulatory agency, such as the EMA (Europe) and / or the FDA (US) and / or any other national regulatory agency for use in animals, preferably for use in humans.

[0037] In general, the term "comprise" or "comprising" also encompasses "consist of" or "consisting of'.

[0038] It is understood that experimenting with humans is subject to strict rules. Therefore, a more easily accessible test system in the form of an animal model may be needed to determine the dosage of PTH 1-84 required to maintain serum calcium within normal levels in otherwise hypocalcaemic subjects, which in human preferably refers to a serum albumin-adjusted calcium level of above 8.5 mg / dL and below 10.5 mg / dL with an optimal level of 9.5 mg / dL which corresponds to a range of 2.125 to 2.625 nmol / L with an optimum of 2.375 nmol / L. One such animal model are thyroparathyroidectomized (TPTX) rats. Rats subjected to thyroparathyroidectomy are unable to produce parathyroid hormone, PTH, the major regulator of calcium homeostasis, and consequently will develop hypocalcemia.

[0039] However, it is also understood that serum calcium levels may vary between different species, such as between human and rats, so in order to achieve comparable results values have to be adjusted to accommodate these inter-species differences. Therefore, when using such animal models, it is necessary to first determine the species' normal serum calcium levels for a given sex and age. For example, Watchorn (Biochem J. 1933; 27(6): 1875-1878) provides the normal range of serum calcium (not adjusted) of male rats as 10.29 to 13.16 mg / dL and as 9.61 to 14.04 mg / dL for female rats. Preferably, the normal serum calcium range is determined as a serum calcium concentration above the lower range of normal, e.g. as above 9.6 mg / dL (not adjusted) for female rats, even more preferably for female rats aged 13 to 22 weeks. Preferably, the normal serum calcium range is determined as a serum calcium concentration below the upper range of normal, e.g. as below 14 mg / dL (not adjusted) for female rats, even more preferably for female rats aged 13 to 22 weeks.

[0040] Accordingly, when subjecting female rats to thyroparathyroidectomy to obtain TPTX rats, the normal range to target with treatment would be above 9.6 mg / dL (not adjusted) and preferably below 14 mg / dL (not adjusted) in such animals aged 13 to 22 weeks.

[0041] The pharmaceutical composition for use of the present invention is administered no more frequent than once every 24 hours, such as every 24 hours, every 48 hours, every 72 hours, every 96 hours, every 120 hours, every 144 hours, once a week, once every two weeks. Preferably, administration of the pharmaceutical composition of the present invention occurs in multiples of 24 hours, i.e. every N times 24 hours, whereas N is an integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14.

[0042] In one embodiment the pharmaceutical composition for use of the present invention is administered once every 24 hours.

[0043] In another embodiment the pharmaceutical composition for use of the present invention is administered once every 48 hours.

[0044] In another embodiment the pharmaceutical composition for use of the present invention is administered once every 72 hours.

[0045] In another embodiment the pharmaceutical composition for use of the present invention is administered once every 96 hours.

[0046] In another embodiment the pharmaceutical composition for use of the present invention is administered once every 120 hours.

[0047] In another embodiment the pharmaceutical composition for use of the present invention is administered once every 144 hours.

[0048] In another embodiment the pharmaceutical composition for use of the present invention is administered once every week.

[0049] In the present invention PTH 1-84 is administered every 24 hours. This means that if the pharmaceutical composition comprising the controlled-release PTH compound is administered every 24 hours, both the pharmaceutical composition comprising the controlled-release PTH compound and PTH 1-84 are administered with the same frequency. If the pharmaceutical composition comprising the controlled-release PTH compound is administered with intervals longer than 24 hours, PTH 1-84 is administered more than once within the interval between two consecutive administrations of the pharmaceutical composition comprising the controlled-release PTH compound. In such case the total amount of PTH1-84 is calculated from all administrations occurring in said interval between two consecutive administrations of the pharmaceutical composition comprising the controlled-release PTH compound of the present invention to obtain the basis for determining the molar equivalent dose of the controlled-release PTH compound.

[0050] The pharmaceutical composition for use of the present invention is administered by subcutaneous injection.

[0051] It is understood that the mode of administration of the controlled-release PTH compound and the mode of administration of the PTH 1-84 are identical, i.e. the PTH 1-84 is administered by subcutaneous injection and also the controlled-release PTH compound is administered by subcutaneous injection.

[0052] In one embodiment the subcutaneous injection of the pharmaceutical composition for use of the present invention is performed with a syringe. In another embodiment the subcutaneous injection of the pharmaceutical composition for use of the present invention is administered with a pen injector. In another embodiment the subcutaneous injection of the pharmaceutical composition for use of the present invention is administered with an auto injector.

[0053] In the present invention the pharmaceutical composition for use of the present invention is administered no more frequently than every 24 hours with a dosage of the controlled-release PTH compound that corresponds to no more than 40% of the molar equivalent dose of PTH 1-84 required to maintain serum calcium within a normal range, i.e., above 8.5 mg / dL and below 10.5 mg / dL, in humans over a 24 hour period. Preferably, the pharmaceutical composition for use of the present invention is administered with a dosage of the controlled-release PTH compound that corresponds to no more than 35% and most preferably with a dosage of the controlled-release PTH compound that corresponds to no more than 30% of the molar equivalent dose of PTH 1-84 required to maintain serum calcium within the normal range, i.e., above 8.5 mg / dL and below 10.5 mg / dL, in humans over a 24 hour period.

[0054] The controlled-release PTH compound is of formula 19

[0055] Preferably, the pharmaceutical composition comprising the controlled-release PTH compound for use of the present invention has a pH ranging from and including pH 3 to pH 8. More preferably, the pharmaceutical composition has a pH ranging from and including pH 4 to pH 6. Most preferably, the pharmaceutical composition has a pH ranging from and including pH 4 to pH 5.

[0056] In one embodiment the pharmaceutical composition comprising the controlled-release PTH compound for use of the present invention is a liquid formulation.

[0057] In another embodiment the pharmaceutical composition comprising the controlled-release PTH compound for use of the present invention is a dry formulation which is reconstituted before administration to a patient.

[0058] Such liquid, dry or reconstituted pharmaceutical composition comprises at least one excipient. Excipients used in parenteral formulations may be categorized as, for example, buffering agents, isotonicity modifiers, preservatives, stabilizers, anti-adsorption agents, oxidation protection agents, viscosifiers / viscosity enhancing agents, or other auxiliary agents. However, in some cases, one excipient may have dual or triple functions. Preferably, the at least one excipient comprised in the pharmaceutical composition for use of the present invention is selected from the group consisting of (i) Buffering agents: physiologically tolerated buffers to maintain pH in a desired range, such as sodium phosphate, bicarbonate, succinate, histidine, citrate and acetate, sulphate, nitrate, chloride, pyruvate; antacids such as Mg(OH) 2 or ZnCO 3 may be also used; (ii) Isotonicity modifiers: to minimize pain that can result from cell damage due to osmotic pressure differences at the injection depot; glycerin and sodium chloride are examples; effective concentrations can be determined by osmometry using an assumed osmolality of 285-315 mOsmol / kg for serum; (iii) Preservatives and / or antimicrobials: multidose parenteral formulations require the addition of preservatives at a sufficient concentration to minimize risk of patients becoming infected upon injection and corresponding regulatory requirements have been established; typical preservatives include m-cresol, phenol, methylparaben, ethylparaben, propylparaben, butylparaben, chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosol, sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and benzalkonium chloride; (iv) Stabilizers: Stabilisation is achieved by strengthening of the protein-stabilising forces, by destabilisation of the denatured state, or by direct binding of excipients to the protein; stabilizers may be amino acids such as alanine, arginine, aspartic acid, glycine, histidine, lysine, proline, sugars such as glucose, sucrose, trehalose, polyols such as glycerol, mannitol, sorbitol, salts such as potassium phosphate, sodium sulphate, chelating agents such as EDTA, hexaphosphate, ligands such as divalent metal ions (zinc, calcium, etc.), other salts or organic molecules such as phenolic derivatives; in addition, oligomers or polymers such as cyclodextrins, dextran, dendrimers, PEG or PVP or protamine or HSA may be used; (v) Anti-adsorption agents: Mainly ionic or non-ionic surfactants or other proteins or soluble polymers are used to coat or adsorb competitively to the inner surface of the formulation's container; e.g., poloxamer (Pluronic F-68), PEG dodecyl ether (Brij 35), polysorbate 20 and 80, dextran, polyethylene glycol, PEG-polyhistidine, BSA and HSA and gelatins; chosen concentration and type of excipient depends on the effect to be avoided but typically a monolayer of surfactant is formed at the interface just above the CMC value; (vi) Oxidation protection agents: antioxidants such as ascorbic acid, ectoine, methionine, glutathione, monothioglycerol, morin, polyethylenimine (PEI), propyl gallate, and vitamin E; chelating agents such as citric acid, EDTA, hexaphosphate, and thioglycolic acid may also be used; (vii) Viscosifiers or viscosity enhancers: in case of a suspension retard settling of the particles in the vial and syringe and are used in order to facilitate mixing and resuspension of the particles and to make the suspension easier to inject (i.e., low force on the syringe plunger); suitable viscosifiers or viscosity enhancers are, for example, carbomer viscosifiers like Carbopol 940, Carbopol Ultrez 10, cellulose derivatives like hydroxypropylmethylcellulose (hypromellose, HPMC) or diethylaminoethyl cellulose (DEAE or DEAE-C), colloidal magnesium silicate (Veegum) or sodium silicate, hydroxyapatite gel, tricalcium phosphate gel, xanthans, carrageenans like Satia gum UTC 30, aliphatic poly(hydroxy acids), such as poly(D,L- or L-lactic acid) (PLA) and poly(glycolic acid) (PGA) and their copolymers (PLGA), terpolymers of D,L-lactide, glycolide and caprolactone, poloxamers, hydrophilic poly(oxyethylene) blocks and hydrophobic poly(oxypropylene) blocks to make up a triblock of poly(oxyethylene)-poly(oxypropylene)-poly(oxyethylene) (e.g. Pluronic ®< ), polyetherester copolymer, such as a polyethylene glycol terephthalate / polybutylene terephthalate copolymer, sucrose acetate isobutyrate (SAIB), dextran or derivatives thereof, combinations of dextrans and PEG, polydimethylsiloxane, collagen, chitosan, polyvinyl alcohol (PVA) and derivatives, polyalkylimides, poly (acrylamide-co-diallyldimethyl ammonium (DADMA)), polyvinylpyrrolidone (PVP), glycosaminoglycans (GAGs) such as dermatan sulfate, chondroitin sulfate, keratan sulfate, heparin, heparan sulfate, hyaluronan, ABA triblock or AB block copolymers composed of hydrophobic A-blocks, such as polylactide (PLA) or poly(lactide-co-glycolide) (PLGA), and hydrophilic B-blocks, such as polyethylene glycol (PEG) or polyvinyl pyrrolidone; such block copolymers as well as the abovementioned poloxamers may exhibit reverse thermal gelation behavior (fluid state at room temperature to facilitate administration and gel state above sol-gel transition temperature at body temperature after injection); (viii) Spreading or diffusing agent: modifies the permeability of connective tissue through the hydrolysis of components of the extracellular matrix in the intrastitial space such as hyaluronic acid, a polysaccharide found in the intercellular space of connective tissue; a spreading agent such as hyaluronidase temporarily decreases the viscosity of the extracellular matrix and promotes diffusion of injected drugs; and (ix) Other auxiliary agents: such as wetting agents, viscosity modifiers, antibiotics, hyaluronidase; acids and bases such as hydrochloric acid and sodium hydroxide are auxiliary agents necessary for pH adjustment during manufacture.

[0059] The condition that can be treated with the controlled-release PTH compound is hypoparathyroidism.Examples Materials and Methods

[0060] Side chain protected PTH(1-34) (SEQ ID NO:51) on TCP resin having Boc protected N-terminus and ivDde protected side chain of Lys26 (synthesized by Fmoc-strategy) was obtained from custom peptide synthesis providers.

[0061] Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus (synthesized by Fmoc-strategy) was obtained from custom peptide synthesis providers.

[0062] PEG 2x20 kDa maleimide, Sunbright GL2-400MA was purchased from NOF Europe N.V., Grobbendonk, Belgium. S-Trityl-6-mercaptohexanoic acid was purchased from Polypeptide, Strasbourg, France. HATU was obtained from Merck Biosciences GmbH, Schwalbach / Ts, Germany. Fmoc-N-Me-Asp(OBn)-OH was obtained from Peptide International Inc., Louisville, KY, USA. Fmoc-Aib-OH was purchased from Iris Biotech GmbH, Marktredwitz, Germany. All other chemicals and reagents were purchased from Sigma Aldrich GmbH, Taufkirchen, Germany, unless a different supplier is mentioned.

[0063] Compound 11a (examples 11-15) was synthesized following the procedure described in patent WO29095479A2, example 1.

[0064] Syringes equipped with polyethylenene frits (MultiSynTech GmbH, Witten, Germany) were used as reaction vessels or for washing steps of peptide resins.

[0065] General procedure for the removal of ivDde protecting group from side chain protected PTH on resin: The resin was pre-swollen in DMF for 30 min and the solvent was discarded. The ivDde group was removed by incubating the resin with DMF / hydrazine hydrate 4 / 1 (v / v, 2.5 mL / g resin) for 8 x 15 min. For each step fresh DMF / hydrazine hydrate solution was used. Finally, the resin was washed with DMF (10 x), DCM (10 x) and dried in vacuo.

[0066] General procedure for the removal of Fmoc protecting group from protected PTH on resin: The resin was pre-swollen in DMF for 30 min and the solvent was discarded. The Fmoc group was removed by incubating the resin with DMF / piperidine / DBU 96 / 2 / 2 (v / v / v, 2.5 mL / g resin) for 3 x 10 min. For each step fresh DMF / piperidine / DBU hsolution was used. Finally, the resin was washed with DMF (10 x), DCM (10 x) and dried in vacuo.RP-HPLC purification:

[0067] For preparative RP-HPLC a Waters 600 controller and a 2487 Dual Absorbance Detector was used, equipped with the following columns: Waters XBridge ™< BEH300 Prep C18 5 µm, 150 x 10 mm, flow rate 6 mL / min, or Waters XBridge ™< 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) and solvent system B (acetonitrile containing 0.1 % TFA v / v) were used. HPLC fractions containing product were pooled and lyophilized if not stated otherwise.Flash Chromatography:

[0068] Flash chromatography purifications were performed on an Isolera One system from Biotage AB, Sweden, using Biotage KP-Sil silica cartridges and n-heptane and ethyl acetate as eluents. Products were detected at 254 nm.Ion exchange chromatography:

[0069] Ion exchange chromatography (IEX) was performed using an Amersham Bioscience AEKTAbasic system equipped with a MacroCap SP cation exchanger column (Amersham Bioscience / GE Healthcare). 17 mM acetic acid pH 4.5 (solvent A) and 17 mM acetic acid, 1 M NaCl, pH 4.5 (solvent B) were used as mobile phases.Size exclusion chromatography:

[0070] Size exclusion chromatography (SEC) was performed using an Amersham Bioscience AEKTAbasic system equipped with HiPrep 26 / 10 desalting columns (Amersham Bioscience / GE Healthcare). 0.1 % (v / v) acetic acid was used as mobile phase.Analytical methods

[0071] Analytical ultra-performance LC (UPLC)-MS was performed on a Waters Acquity system equipped with a Waters BEH300 C18 column (2.1 x 50 mm, 1.7 µm particle size, flow: 0.25 mL / min; solvent A: water containing 0.04% TFA (v / v), solvent B: acetonitrile containing 0.05% TFA (v / v)) coupled to a LTQ Orbitrap Discovery mass spectrometer from Thermo Scientific or coupled to a Waters Micromass ZQ.

[0072] Quantitative measurements of serum calcium (sCa), urinary calcium and serum phosporous (sP) were performed on a Roche-Hitachi P800 modular biochemistry instrument.Example 1 - not according to the invention Synthesis of linker reagent 1f

[0073] Linker reagent 1f was synthesized according to the following scheme:

[0074] To a solution of N-methyl-N-Boc-ethylenediamine (2 g, 11.48 mmol) and NaCNBH 3 (819 mg, 12.63 mmol) in MeOH (20 mL) was added 2,4,6-trimethoxybenzaldehyde (2.08 g, 10.61 mmol) portion wise. The mixture was stirred at rt for 90 min, acidified with 3 M HCl (4 mL) and stirred further 15 min. The reaction mixture was added to saturated NaHCO 3 solution (200 mL) and extracted 5 x with DCM. The combined organic phases were dried over Na 2 SO 4 and the solvents were evaporated in vacuo. The resulting N-methyl-N-Boc-N'-Tmob-ethylenediamine 1a was dried in high vacuum and used in the next reaction step without further purification. Yield:3.76 g (11.48 mmol, 89 % purity, 1a : double Tmob protected product = 8 :1)MS:m / z 355.22 = [M+H] +< , (calculated monoisotopic mass = 354.21).

[0075] To a solution of 1a (2 g, 5.65 mmol) in DCM (24 mL) COMU (4.84 g, 11.3 mmol), N-Fmoc-N-Me-Asp(OBn)-OH (2.08 g, 4.52 mmol) and 2,4,6-collidine (2.65 mL, 20.34 mmol) were added. The reaction mixture was stirred for 3 h at rt, diluted with DCM (250 mL) and washed 3 x with 0.1 M H 2 SO 4 (100 mL) and 3 x with brine (100 mL). The aqueous phases were re-extracted with DCM(100 mL). The combined organic phases were dried over Na 2 SO 4 , filtrated and the residue concentrated to a volume of 24 mL. 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).

[0076] To a solution of 1b (5.31 g, max. 4.52 mmol ref. to N-Fmoc-N-Me-Asp(OBn)-OH) in THF (60 mL) DBU (1.8 mL, 3 % v / v) was added. The solution was stirred for 12 min at rt, diluted with DCM (400 mL) and washed 3 x with 0.1 M H 2 SO 4 (150 mL) and 3 x with brine (150 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na 2 SO 4 and filtrated. 1c was isolated upon evaporation of the solvent and used in the next reaction without further purification. MS:m / z 574.31 = [M+H] +< , (calculated monoisotopic mass = 573.30).

[0077] 1c (5.31 g, 4.52 mmol, crude) was dissolved in acetonitrile (26 mL) and COMU (3.87 g, 9.04 mmol), 6-tritylmercaptohexanoic acid (2.12 g, 5.42 mmol) and 2,4,6-collidine (2.35 mL, 18.08 mmol) were added. The reaction mixture was stirred for 4 h at rt, diluted with DCM (400 mL) and washed 3 x with 0.1 M H 2 SO 4 (100 mL) and 3 x with brine (100 mL). The aqueous phases were re-extracted with DCM(100 mL). The combined organic phases were dried over Na 2 SO 4 , filtered and 1d was isolated upon 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).

[0078] To a solution of 1d (2.63 g, 2.78 mmol) in i-PrOH (33 mL) and H 2 O (11 mL) was added LiOH (267 mg, 11.12 mmol) and the reaction mixture was stirred for 70 min at rt. The mixture was diluted with DCM (200 mL) and washed 3 x with 0.1 M H 2 SO 4 (50 mL) and 3 x with brine (50 mL). The aqueous phases were re-extracted with DCM (100 mL). The combined organic phases were dried over Na 2 SO 4 , filtered and 1e was isolated upon evaporation of the solvent. 1e was purified using flash chromatography. Yield:2.1 g (88 %)MS:m / z 878.4 = [M+Na] +< , (calculated monoisotopic mass = 837.40).

[0079] To a solution of 1e (170 mg, 0.198 mmol) in anhydrous DCM (4 mL) were added DCC (123 mg, 0.59 mmol), and a catalytic amount of DMAP. After 5 min, N-hydroxy-succinimide (114 mg, 0.99 mmol) was added and the reaction mixture was stirred at rt for 1 h. The reaction mixture was filtered, the solvent was removed in vacuo and the residue was taken up in 90 % acetonitrile plus 0.1 % TFA (3.4 mL). The crude mixture was purified by RP-HPLC. Product fractions were neutralized with 0.5 M pH 7.4 phosphate buffer and concentrated. The remaining aqueous phase was extracted with DCM and 1f was isolated upon evaporation of the solvent. Yield:154 mg (81%)MS:m / z 953.4 = [M+H] +< , (calculated monoisotopic mass = 952.43) Example 2Synthesis of linker reagent 2g

[0080]

[0081] 4-Methoxytriphenylmethyl chloride (3.00 g, 9.71 mmol) was dissolved in DCM (20 mL) and added dropwise under stirring to a solution of ethylenediamine 2a (6.5 mL, 97.3 mmol) in DCM (20 mL). The reaction mixture was stirred for 2 h at rt after which it was diluted with diethyl ether (300 mL), washed 3 x with brine / 0.1 M NaOH 30 / 1 (v / v) and once with brine. The organic phase was dried over Na 2 SO 4 and 2b was isolated upon evaporation of the solvent. Yield:3.18 g (98%)

[0082] Mmt protected intermediate 2b (3.18 g, 9.56 mmol) was dissolved in DCM (30 mL). 6-(Tritylthio)-hexanoic acid (4.48 g, 11.5 mmol), PyBOP (5.67 g, 10.9 mmol) and DIPEA (5.0 mL, 28.6 mmol) were added and the mixture was stirred for 30 min at rt. The solution was diuted with diethyl ether (250 mL), washed 3 x with brine / 0.1 M NaOH 30 / 1 (v / v) and once with brine. The organic phase was dried over Na 2 SO 4 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).

[0083] Compound 2c (3.19 g, 4.53 mmol) was dissolved in abhydrous THF (50 mL), 1 M BH 3 ·THF solution in THF (8.5 mL, 8.5 mmol) was added and the mixture was stirred for 16 h at rt. More 1 M BH 3 ·THF solution in THF (14 mL, 14.0 mmol) was added and the mixture was stirred for further 16 h at rt. Methanol (8.5 mL) and N,N'-dimethyl-ethylendiamine (3.00 mL, 27.9 mmol) were added and the mixture was heated under reflux for 3 h. The mixture was allowed to cool down and ethyl acetate (300 mL) was added. The solution was washed 2 x with aqueous Na 2 CO 3 and 2 x with aqueous NaHCO 3 . The organic phase was dried over Na 2 SO 4 and the solvent was removed in vacuo to obtain 2d. Yield:3.22 g (103 %)MS:m / z 691.4 = [M+H] +< , (calculated monoisotopic mass = 690.36).

[0084] Di-tert-butyl dicarbonate (2.32 g, 10.6 mmol) and DIPEA (3.09 mL, 17.7 mmol) were dissolved in DCM (5 mL) and added to a solution of 2d (2.45 g, 3.55 mmol) in DCM (5 mL). The mixture was stirred for 30 min at rt. The solution was concentrated in vacuo and purified by flash chromatography to obtain product 2e. Yield:2.09 g (74 %)MS:m / z 791.4 = [M+H] +< , (calculated monoisotopic mass = 790.42).

[0085] Compound 2e (5.01 g, 6.34 mmol) was dissolved in acetonitrile (80 mL). 0.4 M aqueous HCl (80 mL) followed by acetonitrile (20 mL) was added and the mixture was stirred for 1 h at rt. The pH was adjusted to pH 5.5 by addition of aqueous 5 M NaOH. The organic solvent was removed in vacuo and the remaining aqueous solution was extracted 4 x with DCM. The combined organic phases were dried over Na 2 SO 4 and the solvent was removed in vacuo to obtain product 2f. Yield:4.77 g (95 %)MS:m / z 519.3 = [M+H] +< , (calculated monoisotopic mass = 518.30).

[0086] Compound 2f (5.27 g, 6.65 mmol) was dissolved in DCM (30 mL) and added to a solution of p-nitrophenyl chloroformate (2.01 g, 9.98 mmol) in DCM (25 mL). 2,4,6-trimethylpyridine (4.38 mL, 33.3 mmol) was added and the solution was stirred for 45 min at rt. The solution was concentrated in vacu and purified by flash chromatography to obtain product 2g. Yield:4.04 g (89 %)MS:m / z 706.32 = [M+Na] +< , (calculated monoisotopic mass = 683.30). Example 3 - not according to the invention Synthesis of permanent S1 PTH(1-34) conjugate 3

[0087]

[0088] Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of 6-tritylmercaptohexanoic acid (62.5 mg, 160 µmol), PyBOP (80.1 mg, 154 µmol) and DIPEA (53 µL, 306 µmol) in DMF (2 mL) was added to 0.21 g (51 µmol) of the resin. The suspension was agitated for 80 min at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 10 mL cleavage cocktail 100 / 3 / 3 / 2 / 1 (v / w / v / v / v) TFA / DTT / TES / water / thioanisole and agitating the suspension for 1 h at rt. Crude 3 was precipitated in pre-cooled diethyl ether (-18 °C). The precipitate was dissolved in ACN / water and purified by RP-HPLC. The product fractions were freeze-dried. Yield:36 mg (14 %), 3 *8 TFAMS:m / z 1062.31 = [M+4H] 4+< , (calculated monoisotopic mass for [M+4H] 4+< = 1062.30). Example 4 - not according to the invention Synthesis of permanent K26 PTH(1-34) conjugate 4

[0089]

[0090] Side chain protected PTH(1-34) on TCP resin having Boc protected N-terminus and ivDde protected side chain of Lys26 was ivDde deprotected according to the procedure given in Materials and Methods. A solution of 6-tritylmercaptohexanoic acid (107 mg, 273 µmol), PyBOP (141 mg, 273 µmol) and DIPEA (95 µL, 545 µmol) in DMF (3 mL) was added to 0.80 g (90.9 µmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 6 mL cleavage cocktail 100 / 3 / 3 / 2 / 1 (v / w / v / v / v) TFA / DTT / TES / water / thioanisole and agitating the suspension for 1 h at rt. Crude 4 was precipitated in pre-cooled diethyl ether (-18 °C). The precipitate was dissolved in ACN / water and purified by RP-HPLC. The product fractions were freeze-dried. Yield:40 mg (8 %), 4 *8 TFAMS:m / z 1062.30 = [M+4H] 4+< , (calculated monoisotopic mass for [M+4H] 4+< = 1062.30). Example 5 - not according to the invention Synthesis of transient S1 PTH(1-34) conjugate

[0091]

[0092] Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Aib-OH (79 mg, 244 µmol),PyBOP (127 mg, 244 µmol) and DIPEA (64 µL, 365 µmol) in DMF (1.5 mL) was added to 0.60 g (61 µmol) of the resin. The suspension was agitated for 16 h at rt. The resin was washed 10 x with DMF and Fmoc-deprotected as described above. A solution of 2g (167 mg, 244 µmol) and DIPEA (64 µL, 365 µmol) in DMF (1.5 mL) was added to the resin. The suspension was agitated for 24 h at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 7 mL cleavage cocktail 100 / 3 / 3 / 2 / 1 (v / w / v / v / v) TFA / DTT / TES / water / thioanisole and agitating the suspension for 1 h at rt. Crude 5 was precipitated in pre-cooled diethyl ether (-18 °C). The precipitate was dissolved in ACN / water and purified by RP-HPLC. The product fractions were freeze-dried. Yield:78 mg (24 %), 5 *9 TFAMS:m / z 1101.59 = [M+4H] 4+< , (calculated monoisotopic mass for [M+4H] 4+< = 1101.57). Example 6 - not according to the invention Synthesis of transient S1 PTH(1-34) conjugate 6

[0093]

[0094] Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Ala-OH (32 mg, 102 µmol),PyBOP (53 mg, 102 µmol) and DIPEA (27 µL, 152 µmol) in DMF (3 mL) was added to 0.25 g (25 µmol) of the resin. The suspension was shaken for 1 h at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried under vacuum. Fmoc-deprotection was performed as described above. A solution of 2g (69 mg, 102 µmol) and DIPEA (27 µL, 152 µmol) in DMF (3 mL) was added to the resin. The suspension was agitated for 1.5 h at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 3 mL cleavage cocktail 100 / 3 / 3 / 2 / 1 (v / w / v / v / v) TFA / DTT / TES / water / thioanisole and agitating the suspension for 1 h at rt. Crude 6 was precipitated in pre-cooled diethyl ether (-18 °C). The precipitate was dissolved in ACN / water and purified by RP-HPLC. The product fractions were freeze-dried. Yield:25 mg (18 %), 6 *9 TFAMS:m / z 1098.75 = [M+4H] 4+< , (calculated monoisotopic mass for [M+4H] 4+< = 1098.07). Example 7 Synthesis of transient S1 PTH(1-34) conjugate 7

[0095]

[0096] Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Ser(Trt)-OH (117 mg, 205 µmol),PyBOP (108 mg, 207 µmol) and DIPEA (53 µL, 305 µmol) in DMF (2 mL) was added to 0.50 g (51 µmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried under vacuum. Fmoc-deprotection was performed as described above. A solution of 2g (144 mg, 211 µmol) and DIPEA (53 µL, 305 µmol) in DMF (1.8 mL) was added to the resin. The suspension was shaken for 7 h at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 6 mL cleavage cocktail 100 / 3 / 3 / 2 / 1 (v / w / v / v / v) TFA / DTT / TES / water / thioanisole and agitating the suspension for 1 h at rt. Crude 7 was precipitated in pre-cooled diethyl ether (-18 °C). The precipitate was dissolved in ACN / water and purified by RP-HPLC. The product fractions were freeze-dried. Yield:54 mg (20 %), 7 *9 TFAMS:m / z 1102.08 = [M+4H] 4+< , (calculated monoisotopic mass for [M+4H] 4+< = 1102.07). Example 8 - not according to the invention Synthesis of transient S1 PTH(1-34) conjugate 8

[0097]

[0098] Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of Fmoc-Leu-OH (36 mg, 102 µmol), PyBOP (53 mg, 102 µmol) and DIPEA (27 µL, 152 µmol) in DMF (3 mL) was added to 0.25 g (25 µmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried under vacuum. Fmoc-deprotection was performed as described above. A solution of 2g (69 mg, 102 µmol) and DIPEA (27 µL, 152 µmol) in DMF (3 mL) was added to the resin. The suspension was agitated for 1.5 h at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried in vacuo. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 3 mL cleavage cocktail 100 / 3 / 3 / 2 / 1 (v / w / v / v / v) TFA / DTT / TES / water / thioanisole and agitating the suspension for 1 h at rt. Crude 8 was precipitated in pre-cooled diethyl ether (-18 °C). The precipitate was dissolved in ACN / water and purified by RP-HPLC. The product fractions were freeze-dried. Yield:31 mg (22 %), 8 *9 TFAMS:m / z 1109.32 = [M+4H] 4+< , (calculated monoisotopic mass for [M+4H] 4+< = 1108.58). Example 9 - not according to the invention Synthesis of transient S1 PTH(1-34) conjugate 9

[0099]

[0100] Side chain protected PTH(1-34) on TCP resin having Fmoc protected N-terminus was Fmoc deprotected according to the procedure given in Materials and Methods. A solution of 1e (182 mg, 213 µmol),PyBOP (111 mg, 213 µmol) and DIPEA (93 µL, 532 µmol) in DMF (5 mL) was added to 2.00 g (107 µmol) of the resin. The suspension was agitated for 16 h at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried under vacuum. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 20 mL cleavage cocktail 100 / 3 / 3 / 2 / 1 (v / w / v / v / v) TFA / DTT / TES / water / thioanisole and agitating the suspension for 1 h at rt. Crude 9 was precipitated in pre-cooled diethyl ether (-18 °C). The precipitate was dissolved in ACN / water and purified by RP-HPLC. The product fractions were freeze-dried. Yield:47 mg (8 %), 9 *9 TFAMS:m / z 1108.58 = [M+4H] 4+< , (calculated monoisotopic mass for [M+4H] 4+< = 1108.57). Example 10 - not according to the invention Synthesis of transient K26 PTH(1-34) conjugate 10

[0101]

[0102] Side chain protected PTH(1-34) on TCP resin having Boc protected N-terminus and ivDde protected side chain of Lys26 was ivDde deprotected according to the procedure given in Materials and Methods. A solution of 1f (867 mg, 910 µmol) and DIPEA (0.24 mL, 1.36 mmol) in DMF (5 mL) was added to 1.91 g (227 µmol) of the resin. The suspension was agitated for 1 h at rt. The resin was washed 10 x with DMF, 10 x with DCM and dried under vacuum. Cleavage of the peptide from the resin and removal of protecting groups was achieved by adding 20 mL cleavage cocktail 100 / 3 / 3 / 2 / 1 (v / w / v / v / v) TFA / DTT / TES / water / thioanisole and shaking the suspension for 1 h at rt. Crude 10 was precipitated in pre-cooled diethyl ether (-18 °C). The precipitate was dissolved in ACN / water and purified by RP-HPLC. The product fractions were freeze-dried. Yield:92 mg (7 %), 10 *9 TFAMS:m / z 1108.58 = [M+4H] 4+< , (calculated monoisotopic mass for [M+4H] 4+< = 1108.57). Example 11 - not according to the invention Synthesis of low molecular weight transient S1 PEG conjugate 11b

[0103]

[0104] 0.15 mL of a 0.5 M NaH 2 PO 4 buffer (pH 7.4) was added to 0.5 mL of a 20 mg / mL solution of thiol 5 (10 mg, 1.84 µmol) in 1 / 1 (v / v) acetonitrile / water containing 0.1 % TFA (v / v). The solution was incubated at rt for 10 min after which 238 µL of a 10 mg / mL solution of maleimide 11a (2.4 mg, 2.21 µmol) in 1 / 1 (v / v) acetonitrile / water containing 0.1 % TFA (v / v) were added. The solution was incubated for 20 min at rt. 10 µL TFA was added and the mixture was purified by RP-HPLC. The product fractions were freeze-dried to obtain 11b. Yield:3.1 mg (26 %), 11b *9 TFAMS:m / z 1097.00 = [M+4H] 4+< , (calculated monoisotopic mass for [M+5H] 5+< = 1096.99). Example 12 - not according to the invention Synthesis of low molecular weight transient S1 PEG conjugate 12

[0105]

[0106] R =

[0107] 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 TFAMS:m / z 1094.20 = [M+4H] 4+< , (calculated monoisotopic mass for [M+4H] 4+< = 1094.19). Example 13 - not according to the invention Synthesis of low molecular weight transient S1 PEG conjugate 13

[0108]

[0109] R =

[0110] 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 TFAMS:m / z 1097.40 = [M+5H] 5+< , (calculated monoisotopic mass for [M+5H] 5+< = 1097.39). Example 14 - not according to the invention Synthesis of low molecular weight transient S1 PEG conjugate 14

[0111]

[0112] R =

[0113] 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 TFAMS:m / z 1378.01 = [M+4H] 4+< , (calculated monoisotopic mass for [M+4H] 4+< = 1378.00). Example 15 - not according to the invention Synthesis of low molecular weight transient K26 PEG conjugate 15

[0114]

[0115] R =

[0116] 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 TFAMS:m / z 1102.60 = [M+5H] 5+< , (calculated monoisotopic mass for [M+5H] 5+< = 1102.59). Example 16 - not according to the invention Synthesis of permanent 2x20 kDa S1 PEG conjugate 16

[0117]

[0118] 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 NaH 2 PO 4 buffer (0.66 mL, pH 7.0) was added and the mixture was stirred for 30 min at rt. 10 µL of a 270 mg / mL solution of 2-mercaptoethanol in water was added. The mixture was stirred for 5 min at rt and 0.33 mL 1 M HCl were added. Conjugate 16 was purified by IEX followed by RP-HPLC using a linear gradient of solvent system A (water containing 0.1 % AcOH v / v) and solvent system B (acetonitrile containing 0.1 % AcOH v / v). The product containing fractions were freeze-dried. Yield:97 mg (2.01 µmol, 57 %) conjugate 16 *8 AcOH Example 17 - not according to the invention Synthesis of permanent 2x20 kDa K26 PEG conjugate 17

[0119]

[0120] 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 %) conjugate 17 *8 AcOH Example 18 - not according to the invention Synthesis of transient 2x20 kDa S1 PEG conjugate 18

[0121]

[0122] 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 prior addition of 2-mercaptoethanol. Conjugate 18 was purified by IEX followed by SEC for desalting. The product containing fractions were freeze-dried. Yield:161 mg (3.33 µmol, 40 %) conjugate 18 *9 AcOH Example 19 Synthesis of transient 2x20 kDa S1 PEG conjugate 19

[0123]

[0124] 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 %) conjugate 19 *9 AcOH Example 20 - not according to the invention Synthesis of transient 2x20 kDa S1 PEG conjugate 20

[0125]

[0126] 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 prior addition of 2-mercaptoethanol. Conjugate 20 was purified by IEX followed by SEC for desalting. The product containing fractions were freeze-dried. Yield:194 mg (4.01 µmol, 47 %) conjugate 20 *9 AcOH Example 21 - not according to the invention Synthesis of transient 2x20 kDa K26 PEG conjugate 21

[0127]

[0128] 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 - not according to the invention Pharmacodynamic actions in thyroparathyroidectomised (TPTx) rats during a 28-days study with daily subcutaneous injections with conjugate 18 or PTH(1-84)

[0129] This study was performed in order to test and compare the effect of daily subcutaneous injection of compound 18 and PTH(1-84), the current standard of care, in an animal disease model relevant for investigating treatment of hypoparathyroidism (HP). Rats subjected to thyroparathyroidectomy (TPTx) by blunt dissection are unable to produce parathyroid hormone, PTH, the major regulator of calcium homeostasis. Hence, TPTx rats develop hypocalcemia and hyperphosphatemia characteristic of HP. 17 weeks old female SD TPTx rats (n = 9 / group) were dosed subcutaneously for 28 days with compound 18 (5 µg PTH eq / kg / d; 1.2 nmol / kg / d, in 10 mM succinic acid, 46 g / L mannitol, pH 4.0), PTH(1-84) (70 µg PTH eq / kg / d; 7.3 nmol / kg / d; in 10 mM citrate, mannitol 39.0 g / L, pH 5.0) or vehicle. Additionally, one group of sham operated rats (n =9) representing normophysiological background control were also given vehicle. Serum calcium (sCa) and phosporous (sP) levels in the animals were measured pre- and post-dose on days 1, 6, 12 and 27. Moreover, bone turnover markers (P1NP and CTx) were measured and bone quality assessed by ex vivo pQCT.

[0130] Results: The average sCa in the TPTx rats pre-dosing at day 1 was 8.3 mg / dL compared to 10.9 mg / dL in the sham operated control rats. The sP values were 8.7 mg / dL and 5.9 mg / dL, respectively. Compound 18 given daily at 1.2 nmol / kg elevated sCa to near-normal levels while lowering sP within a few days of administration. At day 12 (day 5 at steady state with compound 18) sCa had stabilised at normal level (10.7 mg / dL) in this group of animals (compound 18 / sham-control ratio = 1.01) as opposed to the hypocalceamic level (8.1 mg / dL) measured in the PTH(1-84) treated rats (PTH(1-84) / sham-control ratio = 0.76). Additionally, the 24-hour urinary Ca excretion at day 12 was comparable between the animals treated with compound 18 and sham-control. Bone mineral density (BMD) and bone mineral content (BMC) were increased in TPTx controls as seen in HP patients. Treatment with Compound 18 decreased BMD, BMC and area in parallel with an increase in CTx compared to sham and vehicle-treated TPTx animals. A significant increase in trabecular BMD was observed in animals dosed with PTH(1-84) compared to both control groups.

[0131] It was concluded that compound 18 at a dosage even as low as less than 20% of the molar equivalent of the here tested dose of PTH(1-84) was able to maintain sCa at a level comparable to the sCa level in sham-control animals (here representing normal level) over a 24 hour period. In contrast, PTH(1-84) at a dose of 7.3 nmol / kg / d did not lead to increase in sCa as compared to the levels in the vehicle-injected TPTx rats. However, a minimal decrease in sP was observed in the PTH(1-84) dosed animals confirming exposure and response to PTH(1-84) in the rats. Following the 28-days of treatment with Compound 18, trabecular and cortical BMD in vertebrae were within normal range, whereas an anabolic effect was observed for PTH(1-84) on trabecular and cortical bone in vertebrae.Abbreviations:

[0132] ACNacetonitrile AcOHacetic acid Aib2-aminoisobutyric acid BMDbone mineral density Bnbenzyl Boctert-butyloxycarbonyl COMU(1-cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate cAMPcyclic adenosine monophosphate dday DBU1,3-diazabicyclo[5.4.0]undecene DCCN,N'-dicyclohexylcarbodiimide DCMdichloromethane DIPEAN,N-diisopropylethylamine DMAPdimethylamino-pyridine DMFN,N-dimethylformamide DMSOdimethylsulfoxide DTTdithiothreitol EDTAethylenediaminetetraacetic acid eqstoichiometric equivalent ESI-MSelectrospray ionization mass spectrometry Etethyl Fmoc9-fluorenylmethyloxycarbonyl Glu-Cendoproteinase Glu-C hhour HATUO-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HPhypoparathyroidism HPLChigh performance liquid chromatography ivDde4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl LCliquid chromatography LTQlinear trap quadrupole Lys-Cendoproteinase Lys-C LLOQlower limit of quantification Mal3-maleimido propyl Memethyl MeOHmethanol minminutes Mmtmonomethoxytrityl MSmass spectrum / mass spectrometry m / zmass-to-charge ratio OtButert-butyloxy PEGpoly(ethylene glycol) pHpotentia Hydrogenii PKpharmacokinetics Prpropyl PTHparathyroid hormone PyBOPbenzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate Q-TOFquadrupole time-of-flight RP-HPLCreversed-phase high performance liquid chromatography rtroom temperature sCaserum calcium SIMsingle ion monitoring SECsize exclusion chromatography scsubcutaneous sPserum phosphate t 1 / 2 half life TCPtritylchloride polystyrol TEStriethylsilane TFAtrifluoroacetic acid THFtetrahydrofuran Tmob2,4,6-trimethoxybenzyl TPTxthyroparathyroidectomy Trttriphenylmethyl, trityl ULOQupper limit of quantification UPLCultra performance liquid chromatography UVultraviolet ZQsingle quadrupole

Claims

1. A pharmaceutical composition comprising at least one controlled-release PTH compound or a pharmaceutically acceptable salt, hydrate or solvate thereof, for use in the treatment of hypoparathyroidism, wherein said pharmaceutical composition is administered by subcutaneous injection no more frequently than once every 24 hours with a dosage of the controlled-release PTH compound that corresponds to no more than 40% of the molar equivalent dose of PTH 1-84 of SEQ ID NO: 1 administered subcutaneously every 24 hours required to maintain serum calcium within normal levels over said 24 hour period in humans, which normal levels of serum calcium refer to a serum albumin-adjusted calcium level of above 8.5 mg / dL and below 10.5 mg / dL, and wherein the controlled-release PTH is a water-soluble conjugate of formula 19