Long-acting parathyroid hormone

Abstract

The present invention provides long-acting parathyroid hormone peptides (PTHs) that remain in serum of a subject for a period of time that is far beyond natural hormones. Long-acting PTHs are conjugated to the C3 position of non-hormonal vitamin D by a discrete length backbone that facilitates its purification, detection, solubility and efficacy on PTH receptors (PTHRs). The PTH may be conjugated to a non-hormonal vitamin D via a 36-membered polyethylene glycol moiety (PTH-PEG36-VitD). The invention also provides an optimized preparation method and a preparation. Compared with a non-conjugated PTH peptide, the PTH-PEG36-VitD has the advantages that the serum half-life period is greatly prolonged, and the bioavailability of the PTH-PEG36-VitD is greatly improved. The PTH-PEG36-VitD can also significantly increase serum calcium, reduce urinary calcium and reduce serum phosphate.

Description

[0001] Cross - Reference to Related Applications

[0002] This invention was made with government support under Grant Numbers R43DK107231 and R44DK107231 awarded by the National Institutes of Health. The government has certain rights in the invention. Field of the Invention

[0003] The present invention provides long-acting parathyroid hormone peptides (PTH) that remain in the serum of a subject for a time period far exceeding that of the native hormone. The long-acting PTH is conjugated at the 3-position of a non-hormonal vitamin D by a discrete length of backbone that facilitates its purification, detection, solubility, and efficacy against the parathyroid hormone receptor (PTHR). In some embodiments, the PTH can be conjugated to the non-hormonal vitamin D by a 36-mer polyethylene glycol moiety (PTH-PEG36-VitD). In other embodiments, the non-hormonal vitamin D lacks a hydroxyl group at the 1-position. In other embodiments, optimized methods of preparation and formulations are provided. PTH-PEG36-VitD has greatly improved serum half-life and bioavailability when compared to unconjugated PTH peptides. In animal models, PTH-PEG36-VitD also significantly increases serum calcium, decreases urinary calcium, and decreases serum phosphate. Background of the Invention

[0005] The present invention provides improved PTH-vitamin D conjugates, which are used in pharmaceutical compositions for treating conditions such as hypoparathyroidism and related conditions. Vitamin D plays a role in calcium, phosphate, and bone homeostasis. Vitamin D is a class of fat-soluble secosteroids. There are several forms (vitamers) of vitamin D. Two major forms are vitamin D2 or ergocalciferol and vitamin D3 or cholecalciferol. Vitamin D without a subscript refers to vitamin D2, D3, or other forms known in the art. In humans, vitamin D can be ingested as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2). The primary source of vitamin D for most people is sunlight. Once vitamin D is produced in the skin or ingested, it needs to be activated through a series of hydroxylation steps, first being converted to 25-hydroxyvitamin D (25(OH)D3) in the liver and then to 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in the kidney. 1α,25(OH)2D3 is the active "hormone" form of vitamin D because it binds to the vitamin D receptor (VDR). 25(OH)D3 is the "non-hormone" form of vitamin D and is the major circulating form in the human body. It binds to the vitamin D binding protein (DBP or VDBP). It is only converted to the hormone form as needed. Examples of non-hormone forms of vitamin D are non-hormone forms of vitamin D lacking the 1α-hydroxy group.

[0006] Parathyroid hormone (PTH)—parathormone or parathyrin is secreted by the chief cells of the parathyroid glands. It is a polypeptide containing 84 amino acids (SEQ ID NO:2). It is normally present in serum at a concentration of 10 - 65 pg / mL. However, the N-terminal 34 amino acids retain full biological activity for the G protein-coupled receptor parathyroid hormone receptor 1 (PTH1R), which is mainly expressed in the kidney and bone (Mosekilde et al., Endocrinology 129:421 - 428, (1991)). (Tay et al., Principles of Bone Biology (Academic Press, 4th Edition, edited by Bilzekian et al.), Chapter 69, 1633 - 1642, 2020); (Mosekilde et al., Endocrinology 129:421 - 428 (1991)). The foregoing is incorporated by reference in its entirety.

[0007] PTH plays a role in mineral homeostasis through at least three mechanisms: (1) PTH stimulates the kidney to convert 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D, which in turn increases intestinal calcium and phosphate absorption, (2) PTH enhances calcium reabsorption at the distal convoluted tubule and collecting duct of the kidney, and (3) PTH increases serum calcium through osteocyte / osteoblast-mediated bone resorption. This releases calcium, as well as magnesium and phosphate, into the circulation (Shoback, N. Engl. J. Med. 359:391-403 (2008)). PTH also regulates serum phosphate levels through bone resorption, vitamin D-mediated intestinal absorption, and renal phosphate excretion (Bilezikian et al., J. Bone Miner. Res. 26(10):2317-2337 (2011); Sikjaer, Rejnmark and Mosekilde, Curr. Drug Saf. 6(2):89-99 (2011)). The foregoing is incorporated by reference in its entirety.

[0008] Although bone resorption and intestinal absorption of phosphate lead to an increase in serum phosphate, the increased renal excretion removes phosphate from the serum at a higher rate, resulting in an overall net decrease in serum phosphate due to PTH (Shoback, N. Engl. J. Med. 359:391-403 (2008)). However, PTH has a very short serum half-life of approximately 5 minutes in humans in the case of intravenous injection (Papapoulos et al., Clinical Endocrinology 7:211-225 (1977)). Due to slow absorption into the circulation, the apparent half-life is longer (1.5 - 2.5 h) after subcutaneous injection (Schweiter et al., Clin. Pharmacol. Ther. 61:360-76 (1997); Rejnmark et al., Scientifica (Cairo) 2014:765629 (2014); Sikjaer et al., J. Bone Miner. Res. 28(10):2232-2243 (2013)). However, this is too short to be used as an effective therapeutic sex hormone replacement. The foregoing is incorporated by reference in its entirety.

[0009] Hypoparathyroidism is a low level of PTH in the blood, which is most commonly due to damage or removal of the parathyroid glands during thyroid surgery, immune system-related damage, genetic or other rare causes. It causes low levels of calcium in the blood, often leading to muscle cramps and twitches or tetany (involuntary muscle contractions) and several other symptoms. Calcium replacements or vitamin D can improve the symptoms but increase the risk of kidney stones and chronic kidney disease. See, e.g., Winer KK et al., J. Clin. Endocrinol. Metab. 97(2):391-399 (2012), which is incorporated by reference in its entirety.

[0010] Hypoparathyroidism can occur when an individual is born without parathyroid glands (usually due to a chromosomal deletion), when the glands are damaged or removed during a surgical procedure (such as thyroidectomy for thyroid cancer or goiter), or when the organ is damaged due to an autoimmune reaction, iron accumulation, magnesium deficiency, or other idiopathic or genetic causes (Bilezikian et al., J. Bone Miner. Res. 26(10):2317-37 (2011); Rubin F1000Research. 9(Faculty Rev):766 (2020)). PTH deficiency leads to low serum calcium (hypocalcemia), high serum phosphate (hyperphosphatemia), high urinary calcium (hypercalciuria) due to reduced renal reabsorption, and increased bone density, which is characterized by increased cortical and trabecular bone density (Shoback et al., J Clin Endocrinol Metab. 101(6):2300-12 (2016)). If untreated, hypoparathyroidism affects almost every organ system in the body. These physiological changes (hypocalcemia, hypercalciuria, hyperphosphatemia, and near absence of PTH), alone or in combination with each other, will lead to an increased risk of neuromuscular symptoms (persistent muscle spasms, paresthesia, tetany, seizures, arrhythmias), ischemic heart disease, kidney stones and nephrocalcinosis, and vertebral fractures (despite increased bone mineral density measurements) (Underbjerg et al., J. Bone Miner. Res. 28(11):2277-2285 (2013); Cipriani et al., J. Clin. Endocrinol. Metab. 106(5):1303-1311 (2021)). In addition, patients with hypoparathyroidism suffer from mood and cognitive disorders such as anxiety, depression, memory problems, and general "brain fog", which are attributed to PTH deficiency (Aggarwal et al., Eur. J. Endocrinol. 168:895-903 (2013)). Importantly, patients with poorly controlled hypoparathyroidism report a significantly reduced quality of life, with 75% of patients reporting that their disease affects their ability to work, and 63% of patients reporting a negative impact on their family relationships (Siggelkow et al., Clin. Endocrinol. 92(2):159-168 (2020)). The foregoing is incorporated by reference in its entirety.

[0011] PTH can have net anabolic or catabolic effects on bone based on the duration of PTH exposure, which is regulated by differential stimulation of osteoblasts and osteoclasts (Frolick et al., Bone 33:372-379 (2003)). Intermittent PTH exposure has anabolic effects and is the basis for (PTH1-34), a therapy for increasing bone mineral density (BMD) in patients with osteoporosis (Neer et al., N. Engl. J. Med., 344(19):1434-1441 (2001)). PTH strongly increases bone deposition (mainly trabecular bone) without resorption, leading to an increase in BMD by preferentially stimulating osteoblasts and osteoblast precursor cells that express the PTH1R, but does not activate osteoclasts that do not express this receptor (Silva et al., J. Endocrinol. Invest. 34(10):801-810 (2011); Kousteni and Bilezikian, Principles of Bone Biology, 3rd ed., Academic Press Inc., 639-656 (2008)). This direct stimulation of osteoblasts and precursor cells reduces osteoblast apoptosis and increases osteoblastogenesis, resulting in an increase in osteoblast number and activity (Kim et al., J. Bone Miner. Res. 27(10):2075-2084 (2012); Bellido et al., J. Biol. Chem. 278(50):50259-50272 (2003)). The foregoing is incorporated by reference in its entirety.

[0012] Patients with hypoparathyroidism have low physiological PTH exposure and exhibit low bone turnover due to reduced and direct osteoblast activity stimulated by PTH1R and reduced osteoclast activation mediated by osteoblasts and osteocytes through RANKL expression (Rubin et al., J. Bone Miner. Res. 23(12):2018-2024 (2008)). This reduction in bone remodeling results in higher BMD in patients with hypoparathyroidism (Bilezikian et al., J. Bone Miner. Res. 26(10):2317-2337 (2011)). Sustained PTH exposure that is more similar to physiological exposure (by insulin pump, multiple daily dosing regimens, or long-acting PTH) will lead to normalization of bone turnover and a decrease in BMD in patients with hypoparathyroidism (Winer et al., J. Pediatr. 203:391-9 (2018)). The foregoing is incorporated by reference in its entirety.

[0013] PTH replacement therapy results in normal serum calcium in patients (Winer et al., J. Clin. Endocrinol. Metab. 83(10):3480-3486 (1998)). However, exogenously administered PTH is rapidly cleared from the body, making it difficult to provide hypoparathyroid patients with physiological PTH concentrations, which need to be maintained within a narrow concentration window to avoid hypocalcemia or hypercalcemia (Tay et al., Br. J. Clin. Pharmacol. 84:252-267 (2018)). In fact, only subcutaneous pump delivery provides a method closest to physiological replacement of PTH compared to once-daily or twice-daily injections (Winer et al., J. Clin. Endocrinol. Metab. 97(2):391-399 (2012)). This is because the plasma half-life of PTH in humans is less than 15 minutes when administered intravenously or is estimated to be 2-3 hours when administered subcutaneously due to slower absorption kinetics (Papapoulos et al., Clin. Endocrinol. 7:211-225 (1977); Sikjaer et al., J. Bone Miner. Res. 28(10):2232-43 (2013)). The foregoing is incorporated by reference in its entirety.

[0014] Current standard of care for hypoparathyroidism consists of calcium and active vitamin D supplementation. Patients manage their symptoms on their own throughout the day, with some patients taking up to 10 pills a day. On average, symptoms last 13 h + / - 9 h per day, and more severe disease even more impacts the patient's life (Hadker et al., Endocr. Pract. 20(7):671-679 (2014), incorporated by reference in its entirety). While supplementation is aimed at controlling the symptoms of hypocalcemia, it worsens the kidney condition, fails to improve bone structure, and does not help with mood and cognitive disorders. An ideal therapy for hypoparathyroidism would mimic endogenous PTH levels, which remain stable throughout the day, to reverse hypocalcemia, hypercalciuria, and hyperphosphatemia.

[0015] Non-hormonal vitamin D forms have a greatly reduced affinity for the VDR and a greatly increased affinity for DBP. DBP is the major transport protein for vitamin D metabolites. Its concentration in plasma is 6 - 7 μM and it has been detected in all fluid compartments. The DBP concentration exceeds the physiological vitamin D metabolite concentration. DBP is important for the transport of vitamin D from the skin into the circulation and across cell membranes into the cytoplasm where vitamin D is activated to the hormonal form. The non-hormonal vitamin D has a significantly higher affinity for DBP than the hormonal form. In contrast, the hormonal form has a significantly higher affinity for the VDR than the non-hormonal form.

[0016] Vitamin D and vitamin D analogs have been approved for the treatment of osteoporosis and secondary hyperparathyroidism. Vitamin D has also been shown to inhibit proliferation and induce differentiation in normal as well as cancer cells. The vitamin D levels required for this activity result in severe toxicity in the form of hypercalcemia. Analogs of vitamin D have been approved for the treatment of psoriasis and other analogs are currently being tested for cancer treatment. Many analogs that have been found to have reduced calcemic effects contain side-chain modifications. These modifications do not have a large effect on VDR binding and thus show equal or even improved efficacy in cell-based proliferation assays. However, many of these modifications have been shown to reduce binding to DBP and thus reduce the half-life in the bloodstream.

[0017] Vitamin D forms that are known to bind to DBP or VDR with higher affinity. (See Haddad, J.G., J. Steroid Biochem. Molec. Biol. Vol. 53, Nos. 1 - 6: 579 - 582 (1995); Norman et al., J. Steroid Biochem. & Mol. Biol. 76: 49 - 59, 51 (2001).) Norman taught the differences between the hormonal and non-hormonal forms of vitamin D. Norman first explained the basis for this teaching: "Compared to other steroid hormones, vitamin D and all of its metabolites... are generally conformationally flexible..." This results in "a wide range of molecular shapes that can be used for binding to receptors involved in 1a,25(OH)2D3-mediated biological responses... as well as for binding to DBP..." (Norman at page 50.) This conformational flexibility is shown in Norman at Figure 2 the place. The vitamin D conformations selected for DBP (non-hormonal) or VDR (hormonal) are shown in Table 1 in Norman. The binding affinities of a large number of vitamin D metabolites for DBP or VDR are provided in Tables 2 and Figure 6At. Norman further states on page 51: "The preferred ligand for DBP is 25(OH)D3, while the preferred ligand for VDR" nuc "is 1a,25(OH)2D3. Thus, 25(OH)D3 binds to DBP 668 times more tightly than 1a,25(OH)2D3. In contrast, 1a,25(OH)2D3 binds to VDR" nuc "668 times more tightly than 25(OH)D3." The foregoing is incorporated by reference in its entirety.

[0018] Adding polyethylene glycol or (PEG) is a known method of increasing the half-life of some compounds by reducing renal clearance, decreasing aggregation, and reducing potential unwanted immune recognition (Jain, Crit. Rev. Ther. Drug Carrier Syst. 25:403 - 447 (2008)). PEG is typically used in fairly large sizes (20 - 40 kDa) to maximize the half-life in circulation. This can be achieved by using a single large PEG attached to the compound or multiple smaller PEGs. (Clark et al. J. Biol. Chem. 271:21969 - 21977 (1996); Fishburn, J. Pharm. Sci. 97:4167 - 4183 (2008)). The foregoing is incorporated by reference in its entirety. Summary of the Invention

[0020] The present invention provides a long-acting parathyroid hormone peptide (PTH) that remains in the serum of a subject for a period of time that is significantly longer than the natural hormone. The long-acting PTH is conjugated at the carbon 3 position of a non-hormonal vitamin D by a discrete length of backbone that facilitates its purification, detection, solubility, and efficacy against the parathyroid hormone receptor (PTHR).

[0021] Accordingly, the present invention provides a pharmaceutical composition comprising a parathyroid hormone peptide (PTH) (PTH conjugate) conjugated to a non-hormonal vitamin D moiety via a backbone, respectively, and a pharmaceutical excipient, wherein the PTH conjugate has a substantially uniform size as measured by mass spectrometry, and wherein when measured at an absorbance at 280 nm using an extinction coefficient of 18,600 M -1 cm -1 the PTH conjugate has a solubility of at least about 0.550 mM in phosphate-buffered saline (PBS). In some embodiments, the PTH conjugate comprises a backbone that is a 36-subunit polyethylene glycol backbone (PTH-PEG36-VitD).

[0022] In some embodiments of the present invention, PTH-PEG36-VitD comprises the following structure:

[0023] H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Cys(succinimidyl-propionamido-PEG36-propionyl-aminopropyl-25-hydroxy-vitamin D)-OH;

[0024] or

[0025] H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Cys(succinimidyl-propionamido-PEG36-propionyl-aminopropyl-25-hydroxy-vitamin D)-NH2.

[0026] In some embodiments of the present invention, the PTH conjugate has substantially the same activity against the PTH receptor (PTHR) as the equivalent unconjugated PTH peptide.

[0027] In some embodiments of the present invention, the PTH conjugate is isolated as a carbonate. In other embodiments, the PTH conjugate is isolated as an acetate. In some embodiments of the present invention, when measured at an absorbance of 28,600 M -1 cm -1 extinction coefficient at 280 nm, the PTH conjugate has a solubility of at least about 9.67 mM in PBS. In other embodiments, the pharmaceutical composition is formulated at a pH of about 4.0 to about 5.5. In a preferred embodiment, the pharmaceutical composition is formulated at a pH of about 5.5. In other embodiments, the pharmaceutical composition is formulated with mannitol. In other embodiments, the pharmaceutical composition is formulated with polysorbate 80 (PS80).

[0028] In some embodiments of the present invention, PTH-PEG36-VitD is formulated as an acetate, wherein the pharmaceutical composition is formulated at a pH of about 5.5, and wherein the pharmaceutical composition comprises mannitol and PS80. In a preferred embodiment, PTH-PEG36-VitD is formulated at a concentration of 0.4 mg / ml in about 10 mM sodium acetate buffer at pH about 5.5, about 4.5% mannitol, and about 0.25% polysorbate 80.

[0029] In some embodiments, PTH-PEG36-VitD is formulated as the acetate salt at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mM or higher. In other embodiments, PTH-PEG36-VitD is formulated at a pH of about 3.5 - 3.9, 4.0 - 4.5, 4.6 - 5.0, 5.1 - 5.5, or 5.6 - 6.0. In other embodiments, PTH-PEG36-VitD is formulated with mannitol at a concentration between about 2.5% and 5%, and more preferably between about 3.0 - 4.5%. In other embodiments, PTH-PEG36-VitD is formulated with PS80 at a concentration of about 0%, 0.1%, 0.2%, 0.25%, 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, or 3.0%. In other embodiments, PTH-PEG36-VitD is formulated with PS80 at a concentration between about 0% and 0.25%.

[0030] In some embodiments of the present invention, the PTH conjugate is formulated with methionine at a concentration between about 0 and 40 mM. In other embodiments, the PTH conjugate is formulated with methionine at a concentration of about 0, 5, 10, 20, or 40 mM. In a preferred embodiment, the PTH conjugate is formulated with methionine at a concentration of about 40 mM.

[0031] In some embodiments of the present invention, the pharmaceutical composition is formulated for subcutaneous delivery, intramuscular delivery, intravenous delivery, controlled release delivery, transdermal delivery, parenteral delivery, or via an implanted reservoir. In a preferred embodiment, the pharmaceutical composition is formulated for subcutaneous delivery.

[0032] In some embodiments of the present invention, the PTH conjugate has a serum half-life in rats of 7 to 15 hours. In other embodiments, the PTH conjugate has a subcutaneous bioavailability in rats of at least about 10 - 13%. In other embodiments, the PTH conjugate has a serum half-life in cynomolgus monkeys of about 24 to 32 hours. In other embodiments, the PTH conjugate has a subcutaneous bioavailability in cynomolgus monkeys of about 45 - 54%.

[0033] In some embodiments of the present invention, a daily dose of about 6 μg / kg of the PTH conjugate, when measured over a 24-hour period after injection, increased the serum calcium level in TPTx rats by an average of at least about 17% compared to the 0-dose level. In other embodiments, a daily dose of about 60 μg / kg of the PTH conjugate, when measured over a 24-hour period after injection, increased the serum calcium level in TPTx rats by an average of at least about 37% compared to the 0-dose level. In other embodiments, a dose of about 2 μg / kg of the PTH conjugate administered every other day, when measured 12 hours after the fifth injection, increased the serum calcium level in cynomolgus monkeys by at least about 0.9 mg / dl compared to the 0-dose level. In other embodiments, a dose of about 100 μg / kg of the PTH conjugate administered every other day, when measured 12 hours after the fifth injection, increased the serum calcium level in cynomolgus monkeys by at least about 6.1 mg / dl compared to the 0-dose level. In other embodiments, a dose of about 10 μg / kg of the PTH conjugate reduced the urinary calcium to serum calcium level ratio in cynomolgus monkeys to equal to or less than about 1.8. In other embodiments, a dose of about 100 μg / kg of the PTH conjugate reduced the urinary calcium to serum calcium level ratio in cynomolgus monkeys to equal to or less than about 1.2.

[0034] In some embodiments of the present invention, when a PTH conjugate of about 6 μg / kg is administered subcutaneously daily, after about 12 days, the PTH conjugate caused a reduction in the serum phosphate level in TPTx rats of at least about 14%. In other embodiments, when a PTH conjugate of about 60 μg / kg is administered subcutaneously daily, after about 12 days, the PTH conjugate caused a reduction in the serum phosphate level in TPTx rats of at least about 39%. In other embodiments, when a PTH conjugate of about 7.0 μg / kg is administered subcutaneously every other day, after about 21 days, the PTH conjugate caused a reduction in the serum phosphate level in cynomolgus monkeys of at least about 1.0 mg / dl. In other embodiments, when a PTH conjugate of about 20 μg / kg is administered subcutaneously every other day, after about 11 days, the PTH conjugate caused a reduction in the serum phosphate level in cynomolgus monkeys of at least about 1.8 mg / dl.

[0035] In some embodiments of the present invention, the PTH conjugate binds to vitamin D binding protein (VDBP) with a dissociation constant of about 5.2 μM.

[0036] The present invention provides a method for treating a subject suffering from a disorder selected from the list consisting of: hypoparathyroidism, hypocalcemia, hyperphosphatemia, hypercalciuria, osteoporosis, fracture repair, osteomalacia, osteomalacia and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, osteogenesis imperfecta, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, fractures, hair loss, chemotherapy-induced hair loss, thrombocytopenia, autoimmune polyendocrine syndrome type 1, DiGeorge syndrome, CHARGE syndrome, Kenny-Caffey type 1, Kenny-Caffey type 2, hereditary deafness and renal dysplasia (HDR), autosomal dominant hypocalcemia type 1 (ADH1), autosomal dominant hypocalcemia type 2 (ADH2), and ADH1 with Bartter's type 5, comprising the step of administering to the subject a pharmaceutical composition of the present invention. In some embodiments, the administering step is achieved by subcutaneous delivery, intramuscular delivery, intravenous delivery, controlled release delivery, transdermal delivery, parenteral delivery, or via an implantable reservoir. In a preferred embodiment, the administration is by the subcutaneous route. In other embodiments, the pharmaceutical composition is administered at a dose of about 2, 7, 10, or 20 μg / kg of the subject's body weight.

[0037] In some embodiments of the present invention, the dose is administered approximately daily. In other embodiments, the dose is administered approximately every other day.

[0038] The present invention provides the pharmaceutical composition of the present invention for treating hypoparathyroidism, hypocalcemia, hyperphosphatemia, hypercalciuria, osteoporosis, fracture repair, osteomalacia, osteomalacia and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, osteogenesis imperfecta, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, fractures, hair loss, chemotherapy-induced hair loss, thrombocytopenia, autoimmune polyendocrine syndrome type 1, DiGeorge syndrome, CHARGE syndrome, Kenny-Caffey type 1, Kenny-Caffey type 2, hereditary deafness and renal dysplasia (HDR), autosomal dominant hypocalcemia type 1 (ADH1), autosomal dominant hypocalcemia type 2 (ADH2), or ADH1 with Bartter's type 5. In other embodiments, the present invention provides a medicament comprising the pharmaceutical composition of the present invention.

[0039] The present invention provides a method of treating a subject having a disorder selected from the group consisting of hypoparathyroidism, hypocalcemia, hyperphosphatemia, hypercalciuria, osteoporosis, fracture repair, osteomalacia, osteomalacia and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, osteogenesis imperfecta, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia of malignancy, osteopenia, periodontal disease, fractures, hair loss, chemotherapy-induced hair loss, thrombocytopenia, autoimmune polyendocrine syndrome type 1, DiGeorge syndrome, CHARGE syndrome, Kenny-Caffey type 1, Kenny-Caffey type 2, hereditary deafness and renal dysplasia (HDR), autosomal dominant hypocalcemia type 1 (ADH1), autosomal dominant hypocalcemia type 2 (ADH2), and ADH1 with Bartter type 5, comprising administering to the subject a pharmaceutical composition comprising a plurality of parathyroid hormone (PTH) peptides (PTH conjugates) each conjugated to a non-hormonal vitamin D through a backbone and a pharmaceutical excipient, wherein each of the plurality of PTH peptides is conjugated to a backbone having a substantially uniform size as measured by mass spectrometry, wherein the method comprises the step of subcutaneously administering repeated doses of the pharmaceutical composition, and wherein the repeated doses result in a maximum to minimum concentration ratio (Cmax / Cmin ratio) of the PTH conjugate in a serum sample from the subject that is equal to or less than about 8.0.

[0040] In some embodiments, the Cmax / Cmin ratio is equal to or less than about 5.37. In preferred embodiments, the Cmax / Cmin ratio is equal to or less than about 4.59. In more preferred embodiments, the Cmax / Cmin ratio is equal to or less than about 3.36. In more preferred embodiments, the Cmax / Cmin ratio is equal to or less than about 2.38. In more preferred embodiments, the Cmax / Cmin ratio is equal to or less than about 1.83. In most preferred embodiments, the Cmax / Cmin ratio is equal to or less than about 1.79.

[0041] In some embodiments of the present invention, the PTH conjugate has a serum half-life of 7 to 15 hours. In other embodiments, the PTH conjugate has a subcutaneous bioavailability of at least about 10 - 13%. In other embodiments, the PTH conjugate has a serum half-life of about 24 to 32 hours. In other embodiments, the PTH conjugate has a subcutaneous bioavailability of about 45 - 54%.

[0042] In some embodiments, a dose of about 2.0 μg / kg of the PTH conjugate administered every other day increased the serum calcium level by at least about 0.9 mg / dl compared to the 0-dose level when measured 12 hours after the injection on day 5. In other embodiments, a dose of about 100 μg / kg of the PTH conjugate administered every other day increased the serum calcium level by at least about 6.1 mg / dl compared to the 0-dose level when measured 12 hours after the injection on day 5.

[0043] In some embodiments, a dose of about 10 μg / kg of the PTH conjugate reduced the ratio of urinary calcium to serum calcium level to equal to or less than about 1.8. In other embodiments, a dose of about 100 μg / kg of the PTH conjugate reduced the ratio of urinary calcium to serum calcium level to equal to or less than about 1.2.

[0044] In some embodiments of the present invention, when about 6 μg / kg of the PTH conjugate is administered subcutaneously daily, after about 12 days, the PTH conjugate causes the serum phosphate level to decrease by at least about 14%. In other embodiments, when about 60 μg / kg of the PTH conjugate is administered subcutaneously daily, after about 12 days, the PTH conjugate causes the serum phosphate level to decrease by at least about 39%.

[0045] The present invention provides a kit comprising the pharmaceutical composition of the present invention and instructions for its use for treating a disorder in a patient. In some embodiments, the disorder is hypoparathyroidism.

[0046] In some embodiments of the present invention, PTH-PEG36-VitD comprises the following structure:

[0047]

[0048] In other embodiments of the present invention, PTH-PEG36-VitD comprises the following structure:

[0049]

[0050] The present invention provides a method for preparing the pharmaceutical composition of the present invention, comprising conjugating a PTH peptide, a backbone, and a non-hormonal vitamin D moiety to form a PTH conjugate, wherein the conjugation step occurs at a pH less than about 7.4. In some embodiments, the PTH peptide comprises the amino acid sequence of SEQ ID NO:3. In other embodiments, the conjugation step occurs at a pH of about 6.0. In other embodiments, the method further comprises a purification step of the conjugate at a pH less than about 8.0. In a preferred embodiment, the purification step is at a pH of about 5.5.

[0051] The present invention provides a pharmaceutical carrier comprising Formula I:

[0052] B——(L) a ——S—(M) b ——C

[0053] I

[0054] Wherein:

[0055] B is a targeting group, which is vitamin D unhydroxylated at the carbon 1 position and conjugated with (L)a at the carbon 3 position;

[0056] S is a backbone moiety, which contains polyethylene glycol composed of 36 repeating ethylene glycol units;

[0057] C is a maleimide group;

[0058] (L) a is a linker, which contains (CH2)3NHC(O)CH2; and

[0059] (M) b is a linker, which contains HNC(O)(CH2)2.

[0060] In some embodiments of the present invention, the above drug carrier comprises a compound having Formula VI:

[0061]

[0062] The present invention provides a method for preparing a pharmaceutical composition comprising parathyroid hormone (PTH) and the above drug carrier, comprising the following steps:

[0063] 1) Purifying the compound having Formula VI;

[0064] 2) Conjugating the compound having Formula VI with PTH;

[0065] In some embodiments, before the conjugation step, the purified compound having Formula VI is not separated from the purification solvent. In other embodiments, the purification step is achieved by high performance liquid chromatography (HPLC). In other embodiments, the conjugate is separated as a carbonate. In other embodiments, the conjugate is separated as an acetate.

[0066] In some of the above embodiments of the present invention, PTH comprises the amino acid sequence of SEQ ID NO:3.

[0067] The present invention provides a method for treating a human suffering from a disorder selected from the group consisting of: hypoparathyroidism, hypocalcemia, hyperphosphatemia, hypercalciuria, osteoporosis, fracture repair, osteomalacia, osteomalacia and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, osteogenesis imperfecta, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, fractures, hair loss, chemotherapy-induced hair loss, thrombocytopenia, autoimmune polyendocrine syndrome type 1, DiGeorge syndrome, CHARGE syndrome, Kenny-Caffey type 1, Kenny-Caffey type 2, hereditary deafness and renal dysplasia (HDR), autosomal dominant hypocalcemia type 1 (ADH1), autosomal dominant hypocalcemia type 2 (ADH2), and ADH1 with Bartter type 5, comprising the step of administering to the human a pharmaceutical composition as described herein. In some embodiments, the pharmaceutical composition is EXT608. In a preferred embodiment, the disorder is hypoparathyroidism.

[0068] In some embodiments, the administering step is effected by subcutaneous delivery, intramuscular delivery, intravenous delivery, controlled release delivery, transdermal delivery, parenteral delivery, or by an implantable reservoir. In a preferred embodiment, the administration is by the subcutaneous route.

[0069] In some embodiments, the pharmaceutical composition is administered at a dose of about 2, 7, 10 or 20 μg / kg of the subject's body weight. In other embodiments, the dose is about 36, 108 or 324 μg. In other embodiments, the dose is administered about daily, about every other day, about every three days, about every four days, about every four days, about every five days, about every six days, about every seven days, about every eight days, or about every nine days.

[0070] In some embodiments, the dose results in a Cmax between about 0.49 and 5.0 ng / ml. In a preferred embodiment, the dose results in a Cmax of about 5.0 ng / ml.

[0071] In some embodiments, the dose results in a Tmax between about 3.3 and 5.3 hours. In a preferred embodiment, the dose results in a Tmax of about 2.7 hours.

[0072] In some embodiments, the dose results in an AUC between about 5.9 and 118 (ng / ml)*h. In a preferred embodiment, the dose results in an AUC of about 118 (ng / ml)*h.

[0073] In some embodiments, the dose results in a Tlast between about 24 and 168 hours. In a preferred embodiment, the dose results in a Tlast of about 168 hours. In a preferred embodiment, the dose results in a Cmax of about 5.0 ng / ml, a Tmax of about 2.7 hours, an AUC of about 118 (ng / ml)*h, and a Tlast of about 168 hours.

[0074] In some embodiments, the dose is about 324 μg SC and results in a T 1 / 2 of about 90 hours. In other embodiments, the dose is about 324 μg SC and results in an AUC inf of about 151 (ng / ml)*h. In other embodiments, the dose is about 324 μg SC and results in a clearance divided by bioavailability (CL / F) of about 2.2 L / h. In other embodiments, the dose is about 324 μg SC and results in an apparent volume of distribution divided by bioavailability during the terminal phase (Vz / F) of about 280 L. In other embodiments, the dose is about 324 μg SC and results in an average residence time (MRT) of about 105 hours. In a preferred embodiment, the dose is about 324 μg SC and results in a T 1 / 2 of about 90 hours, an AUC inf of about 151 (ng / ml)*h, a CL / F of about 2.2 L / h, a Vz / F of about 280 L, and an MRT of about 105 hours.

[0075] In some embodiments of the invention, the pharmaceutical composition raises the serum calcium level for about 24 hours after administration. In other embodiments, the pharmaceutical composition reduces the level of endogenous PTH(1-84). In other embodiments, the pharmaceutical composition does not result in a significant increase in urinary calcium when measured about 36 hours after administration.

[0076] The invention provides a pharmaceutical composition as disclosed herein, wherein the PTH conjugate is formulated with methionine at a concentration between about 0 and 40 mM. In some embodiments, the PTH conjugate is formulated with methionine at a concentration of about 0, 5, 10, 20, or 40 mM. In a preferred embodiment, the PTH conjugate is formulated with methionine at a concentration of about 40 mM.

[0077] The present invention provides a pharmaceutical composition as disclosed herein, wherein after shaking the pharmaceutical composition for 308 hours, when measured at a wavelength of 600 nm, the PTH conjugate shows an optical density (OD) of the precipitate separated from the solution of about 0.411 or lower. In some embodiments, after shaking the pharmaceutical composition for 308 hours, when measured at a wavelength of 600 nm, the PTH conjugate shows an optical density (OD) of the precipitate separated from the solution of about 0.242 or lower. In a preferred embodiment, after shaking the pharmaceutical composition for 308 hours, when measured at a wavelength of 600 nm, the PTH conjugate shows an optical density (OD) of the precipitate separated from the solution of about 0.015 or lower.

[0078] The present invention provides a pharmaceutical composition as disclosed herein, wherein the purity of the PTH conjugate is reduced by about 7.7% or less after incubation at 4 °C for 69 hours. In some embodiments, the purity of the PTH conjugate is reduced by about 14.9% or less after incubation at room temperature for 13 days. Brief Description of the Drawings

[0080] Figure 1 : Method for synthesizing EXT608.

[0081] Figure 2 : UPLC analysis of EXT601.

[0082] Figure 3 : UPLC analysis of EXT608.

[0083] Figure 4 : MALDI-TOF mass spectrometry analysis of EXT601.

[0084] Figure 5 : MALDI-TOF mass spectrometry analysis of EXT608.

[0085] Figure 6 : Cell-based PTHR1 activity assay comparing PTH(1-34), EXT601, EXT607, and EXT608.

[0086] Figure 7 : Kinetic binding analysis of vitamin D binding protein (DBP) to immobilized EXT607 as a function of DBP concentration (0 - 75 μM) by biolayer interferometry (BLI). For each concentration of DBP, both binding association and dissociation data are shown, as well as the calculated fits generated using the ForteBio Octet data analysis software. Inset: Plot of steady-state BLI shift as a function of DBP concentration. The binding equilibrium constant (K) of 8.1 μM was obtained from the calculated steady-state curve fit + 2.5 μMD )。

[0087] Figure 8 : Kinetic binding analysis of vitamin D-binding protein (DBP) to immobilized EXT608 by biolayer interferometry (BLI) as a function of DBP concentration (0 - 75 μM). For each concentration of DBP, both binding association and dissociation data, as well as the calculated fits generated using the ForteBio Octet data analysis software, are shown. Inset: Plot of steady-state BLI shift as a function of DBP concentration. A binding equilibrium constant (K + 1.1 μM was obtained from the calculated steady-state curve fits D )。

[0088] Figure 9 : Cell-based PTHR1 activity assays comparing EXT606, EXT608, EXT611, EXT615, EXT616, and EXT617.

[0089] Figure 10 : Pharmacokinetics of subcutaneous administration of EXT601 (157 μg / kg) in rats. Error bars indicate standard deviation (n = 3).

[0090] Figure 11 : Pharmacokinetics of intravenous (iv, dashed line) and subcutaneous (sc, solid line) administration of EXT607 in rats. Error bars indicate standard deviation (n = 3).

[0091] Figure 12 : Pharmacokinetics of intravenous (iv, dashed line) and subcutaneous (sc, solid line) administration of EXT607 in cynomolgus monkeys. Error bars indicate standard deviation (n = 3).

[0092] Figure 13 : Repeated-dose subcutaneous pharmacokinetics of EXT608 in rats measured after the last dose administration following 21 days of daily dosing (14.2 and 70 μg / kg) or 90 days of daily dosing (1, 3, and 10 μg / kg). Error bars indicate standard deviation (n = 3).

[0093] Figure 14 : Repeated-dose subcutaneous pharmacokinetics of EXT608 in cynomolgus monkeys measured after the last dose administration following 21 days of dosing every other day (1.4, 7, and 20 μg / kg) or 89 days of dosing every other day (0.7 and 2 μg / kg). Error bars indicate standard deviation (n = 3).

[0094] Figure 15: Serum calcium levels in cynomolgus monkeys starting on day 5 after subcutaneous administration of 0, 10, 30, or 100 μg / kg of EXT607 every other day. Animals were dosed at t = 0 on days 1, 3, and 5, with the administration on day 5 indicated by a vertical arrow on the graph. Urine was collected starting on day 8 (72 h on the time axis), as indicated by the horizontal arrow.

[0095] Figure 16 : Serum calcium levels in cynomolgus monkeys on days 1, 11, and 21 after subcutaneous administration of 0, 1.4, 7, or 20 μg / kg of EXT608 every other day. Error bars indicate standard deviation (n = 4). p-values compared to the 0 μg / kg group (vehicle) were calculated using the Student t-test (* < 0.05, # < 0.01, & < 0.001).

[0096] Figure 17 : Serum phosphate levels in cynomolgus monkeys on days 1, 11, and 21 after subcutaneous administration of 0, 1.4, 7, or 20 μg / kg of EXT608 every other day. Error bars indicate standard deviation (n = 4). p-values compared to the 0 μg / kg group (vehicle) were calculated using the Student t-test (* < 0.05, # < 0.01, & < 0.001).

[0097] Figure 18 : Serum calcium levels in cynomolgus monkeys on days 1, 45, and 89 after subcutaneous administration of 0, 0.2, 0.7, or 2 μg / kg of EXT608 every other day. p-values compared to the 0 μg / kg group (vehicle) were calculated using the Student t-test (* < 0.05, # < 0.01, & < 0.001).

[0098] Figure 19 : Serum calcium levels in TPTx rats on days 1, 12, and 27 after daily subcutaneous administration of 0 (vehicle), 1, 3, or 10 nmol / kg of EXT607. Serum calcium values were normalized to the group that underwent sham surgery and received vehicle (sham vehicle). Error bars indicate standard deviation (n = 5). p-values compared to the 0 μg / kg group (vehicle) were calculated using the Student t-test (* < 0.05, # < 0.01, & < 0.001).

[0099] Figure 20 : Serum calcium levels in TPTx rats on days 1, 12, and 27 after daily subcutaneous administration of 10 nmol / kg of PTH(1-34) and PTH(1-84), compared to those from Figure 19The vehicle and sham-operated vehicle control groups were compared. Serum calcium values were normalized according to the sham-operated vehicle group. Error bars indicate standard deviation (n = 5). The p-values compared to the 0 μg / kg group (vehicle) were calculated using Student's t-test (* < 0.05, # < 0.01).

[0100] Figure 21 : Serum phosphate levels in TPTx rats on days 1, 12, and 27 after daily subcutaneous administration of 0 (vehicle), 1, 3, or 10 nmol / kg of EXT607. Serum phosphate values were normalized according to the group that underwent sham surgery and received vehicle (sham-operated vehicle). Error bars indicate standard deviation (n = 5). The p-values compared to the 0 μg / kg group (vehicle) were calculated using Student's t-test (* < 0.05, # < 0.01, & < 0.001).

[0101] Figure 22 : Serum phosphate levels in TPTx rats on days 1, 12, and 27 after daily subcutaneous administration of 10 nmol / kg of PTH(1-34) and PTH(1-84), compared to Figure 21 the vehicle and sham-operated vehicle control groups. Serum phosphate values were normalized according to the sham-operated vehicle group. Error bars indicate standard deviation (n = 5). The p-values compared to the 0 μg / kg group (vehicle) were calculated using Student's t-test (* < 0.05, # < 0.01).

[0102] Figure 23A : The pharmacokinetics of a single subcutaneous dose of EXT608 showed a substantial improvement in serum half-life in humans. Error bars indicate standard error of the mean (n = 3).

[0103] Figure 23B : Changes in albumin-corrected serum calcium levels in healthy human participants after a single subcutaneous dose of 0, 36, 108, 216, or 324 μg of EXT608. The baseline value of serum calcium for each individual was calculated by averaging the -24 h and -1 h measurements. Error bars indicate standard error of the mean (n = 3).

[0104] Figure 23C : Changes in serum endogenous PTH(1-84) levels in healthy human participants after a single subcutaneous dose of 0, 36, 108, 216, or 324 μg of EXT608. For each individual, the -1 h measurement was used as the baseline value. Error bars indicate standard error of the mean (n = 3). Detailed Description of the Invention

[0106] The present invention provides long-acting parathyroid hormone peptides (PTHs) that remain in the serum of a subject for a period of time that far exceeds that of the native hormone. They are intended to replace the endogenous PTH that is missing in patients with hypoparathyroidism and related conditions. The long-acting PTHs are conjugated to the 3-position of a non-hormonal vitamin D through a backbone of discrete length, which facilitates its purification, detection, solubility, and efficacy against the PTH receptor (PTHR). The PTH is conjugated to the non-hormonal vitamin D through a 36-mer polyethylene glycol moiety (PTH-PEG36-VitD). The present invention also provides optimized preparation methods and formulations. PTH-PEG36-VitD has greatly improved serum half-life and bioavailability when compared to non-conjugated PTH peptides. PTH-PEG36-VitD also significantly increases serum calcium, decreases urinary calcium, and decreases serum phosphate.

[0107] The present invention provides a conjugate of a carrier and a drug, which comprises a targeting group, and the targeting group is a non-hormonal vitamin D, a vitamin D analogue, or a vitamin D metabolite. Examples include vitamin D-based molecules that are unhydroxylated at the 1-position of carbon (C1). The carrier is linked to the therapeutic compound at the 3-position of carbon (C3). While not wishing to be bound by theory, it is believed that the hormonal forms of vitamin D are not suitable for the carriers described herein because they may be toxic due to inducing hypercalcemia. In addition, because the hormonal forms bind to the vitamin D receptor in cells, they may inappropriately target the conjugate of the carrier and the drug to unwanted cells or tissues. In contrast, the non-hormonal vitamin D forms bind to the vitamin D binding protein (DBP or VDBP) and remain in circulation for a longer time.

[0108] In some embodiments, the present invention provides EXT607 and EXT608 (described below). They can be used for long-acting PTH replacement therapy and for the preparation of pharmaceutical compositions. PTH is a naturally occurring hormone produced by the parathyroid gland and is normally present in serum at a concentration of 10 - 65 pg / mL (Aloia et al., Endocr. Pract. 12(2):137 - 144 (2006)). Hypoparathyroidism is a rare disease characterized by PTH deficiency and can occur when an individual is born without parathyroid glands (usually due to chromosomal deletion). It can also be caused by damage to the gland during a surgical procedure (such as thyroidectomy), when the organ is damaged due to an autoimmune reaction, iron accumulation, magnesium deficiency, or other idiopathic causes (Bilezikian et al., J. Bone Miner. Res. 26(10):2317 - 2337 (2011)). PTH deficiency leads to hypocalcemia, hypercalciuria, and hyperphosphatemia, which if untreated, can cause persistent muscle spasms, paresthesia, seizures, and arrhythmias (Shoback, N. Engl. J. Med. 359:391 - 403 (2008)). In addition, patients with hypoparathyroidism suffer from cognitive impairments such as anxiety, depression, memory problems, and general "brain fog" (Aggarwal et al., Eur. J. Endocrin. 168:895 - 903 (2013)).

[0109] EXT607 and EXT608 are designed to extend the serum half-life of PTH and achieve pharmacokinetic properties more similar to physiological PTH levels. Vitamin D is conjugated to modify PTH to produce EXT607 and EXT608. The form of 25-hydroxycalciferol [25(OH)-vitamin D] is linked to the C-terminus of PTH(1 - 34) via a small molecular weight polyethylene glycol (PEG) spacer moiety (<2 kDa). The form of 25(OH)-vitamin D used is an inactive "storage" form of vitamin D that is not active on the vitamin D receptor (VDR); thus, vitamin D conjugated to the peptide is unlikely to interfere with normal vitamin D metabolic pathways (Lips, Prog. Biophys. Mol. Biol. 92:4 - 8 (2006)). Since the modification is very small (<2.4 kDa), it does not affect the potency of PTH(1 - 34), as expected for peptides with larger modifications (such as 20 kDa PEG). EXT607 / EXT608 shows extended pharmacokinetics after subcutaneous administration, with a terminal half-life of 8 - 15 hours in rats and 24 - 32 hours in non-human primates (NHPs).

[0110] In some embodiments, EXT607 and EXT608 are maintained in the circulation for an extended period by interacting with VDBP. The half-life of EXT607 and EXT608 is significantly improved compared to PTH(1-34). The half-life of PTH(1-34) in rats increased from 11 minutes to 8 - 15 hours after vitamin D conjugation, and the half-life in NHP is 24 - 32 hours.

[0111] The carrier molecule is linked to PTH using the chemical methods described herein, those described in WO2013172967 and WO2016065042 (incorporated herein by reference in their entirety), or other chemical methods known in the art. The carrier improves the potency, absorption, bioavailability, circulating half-life, or pharmacokinetic properties of the therapeutic compound. In certain embodiments, the carrier also comprises a substance described herein as a "scaffold" that serves, inter alia, as a non-releasable "spacer" between the targeting group and the therapeutic compound. In other embodiments, the carrier lacks a scaffold.

[0112] The carrier is intended for use in humans and animals. The carrier serves the purpose of improving the pharmacokinetic properties of a biological or chemical entity conjugated, linked, or fused to the carrier. This occurs through the interaction of the targeting group with DBP. DBP can rapidly and efficiently actively transport the molecule from the site of administration to the circulating plasma, thereby reducing drug exposure to degradative enzymes. By binding to DBP, the carrier also improves the circulating half-life of the drug. This increases the potency and therapeutic efficacy of the drug by preventing renal filtration and other elimination processes.

[0113] In describing and claiming one or more embodiments of the invention, the following terms will be used according to the definitions described below.

[0114] The term "absorption" is the movement of a drug into the bloodstream. The drug needs to be introduced via some route of administration (e.g., oral, topical or transdermal, subcutaneous, intramuscular, or intravenous) or in a specific dosage form (such as a tablet, patch, capsule, or liquid).

[0115] An "antagonist" refers to a molecule that is capable of neutralizing, blocking, inhibiting, eliminating, reducing, or interfering with the activity of a specific or designated protein, including its binding to one or more receptors in the case of a ligand, or its binding to one or more ligands in the case of a receptor. Antagonists include antibodies and their antigen-binding fragments, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmaceuticals and their metabolites, transcriptional and translational control sequences, etc. Antagonists also include small molecule inhibitors of proteins, hormones, or other bioactive molecules. An antagonist can be a fusion protein, receptor molecule, antisense molecule, aptamer, ribozyme, or derivative that specifically binds to a protein, hormone, or other bioactive molecule and thereby sequesters its binding to its target.

[0116] "Antibody" (Ab) and "immunoglobulin" (Ig) refer to glycoproteins with similar structural features. While antibodies show binding specificity for a particular antigen, immunoglobulins include both antibodies and other antibody-like molecules that typically lack antigen specificity. For example, the latter polypeptides are produced at low levels by the lymphatic system and at increased levels by myelomas.

[0117] "Aptamer" is a nucleic acid-based compound that has been selected to bind to a specific target. Examples of aptamer-based therapeutic compounds can be found in WO07 / 035922, which is incorporated herein by reference in its entirety.

[0118] The term "bioavailability" refers to the fraction of the administered dose of an unchanged drug that reaches the systemic circulation, which is one of the major pharmacokinetic properties of a drug. When a drug is administered intravenously, its bioavailability is 100%. When a drug is administered by other routes (such as orally), its bioavailability is usually reduced (due to incomplete absorption and first-pass metabolism) or can vary among patients. Bioavailability is an important parameter in pharmacokinetics to consider when calculating the dose for non-intravenous administration routes.

[0119] "Carrier" is a compound that can be conjugated, fused, coupled, or formulated with a therapeutic compound to improve the absorption, half-life, bioavailability, pharmacokinetic, or pharmacodynamic properties of the drug. They include targeting groups, coupling groups, and an optional backbone moiety. In some embodiments, the carrier can transport the therapeutic compound from the subcutaneous injection site into the circulation and transport the therapeutic compound in the circulation for an extended period of time.

[0120] "Effective amount" refers to the amount of a therapeutic compound that, at the desired dose and for the desired period of time, effectively achieves the desired therapeutic or prophylactic outcome. The "therapeutically effective amount" of a therapeutic compound can vary depending on factors such as the disease state, age, sex, and weight of the individual. The therapeutically effective amount can be measured, for example, by increased survival rate, more rapid recovery, or improvement, alleviation, or elimination of symptoms, or other acceptable biomarkers or surrogate markers. The therapeutically effective amount is also the amount at which any toxic or harmful effects of the therapeutic compound are outweighed by the therapeutic beneficial effects. "Prophylactically effective amount" refers to the amount of a therapeutic compound that, at the desired dose and for the desired period of time, effectively achieves the desired prophylactic outcome. Usually but not necessarily, since prophylactic doses are used in a subject before or at an earlier stage of a disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0121] "Half-life" is a scientific term known in the art and refers to the amount of time elapsed when half of the amount of a test molecule is no longer detected. The in vivo half-life refers to the amount of time elapsed when half of the test molecule in the circulating serum or tissue of a human or animal is no longer detectable.

[0122] "Hormone" is a biological or chemical messenger that communicates between one cell (or group of cells) and another cell. As described herein, the hormones used in the present invention can be peptides, steroids, pheromones, interleukins, lymphokines, cytokines, or members of other hormone classes known in the art.

[0123] "Homolog" is a bioactive molecule that is similar to a reference molecule at the nucleotide sequence, peptide sequence, functional, or structural level. Homologs can include sequence derivatives that share a certain percentage identity with the reference sequence. Thus, in one embodiment, the homologous or derived sequence shares at least 70% sequence identity. In a preferred embodiment, the homologous or derived sequence shares at least 80% or 85% sequence identity. In a more preferred embodiment, the homologous or derived sequence shares at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity. Homologous or derived nucleic acid sequences can also be defined by their ability to remain bound to the reference nucleic acid sequence under high stringency hybridization conditions. Homologs that have structural or functional similarity to the reference molecule can be chemical derivatives of the reference molecule. Methods for detecting, generating, and screening for structural and functional homologs and derivatives are known in the art.

[0124] "Hybridization" generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below its melting temperature. The higher the degree of homology desired between the probe and the hybridizable sequence, the higher the relative temperature that can be used. Thus, it can be seen that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are less stringent. For additional details and explanations of hybridization reaction stringency, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995), which is incorporated herein by reference in its entirety.

[0125] "Individual", "subject", or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (e.g., mice, hamsters, guinea pigs, and rats). In certain embodiments, the mammal is a human. A "control subject" is a healthy subject who has not been diagnosed with the disease, dysfunction, or disorder identified in the individual, subject, or patient. The control subject does not have any signs or symptoms associated with the disease, dysfunction, or disorder.

[0126] A "drug" is an active pharmaceutical agent prepared for the treatment of a disease, disorder, or condition.

[0127] A "Morpholino" is a synthetic molecule that is a non-natural variant of natural nucleic acids utilizing phosphorodiamidate linkages, described in U.S. Patent No. 8,076,476, which is incorporated herein by reference in its entirety.

[0128] A "nucleic acid" is any one of a group of macromolecules—DNA, RNA, or variants thereof—that carry genetic information that can direct cellular functions. Nucleic acids can have enzyme-like activity (e.g., ribozymes) or can be used to inhibit gene expression in a subject (e.g., RNAi). The nucleic acids used in the present invention described herein can be single-stranded, double-stranded, linear, or circular. The present invention also includes the use of nucleic acid variants, including but not limited to aptamers, PNAs, Morpholinos, or other non-natural nucleic acid variants. For example, nucleic acids useful in the present invention are described in U.S. Patent No. 8,076,476, which is incorporated herein by reference in its entirety.

[0129] "Patient response" or "response" can be evaluated using any endpoint that indicates benefit to the patient, including but not limited to: (1) inhibition of disease progression to some extent, including stabilization, slowing, and complete cessation; (2) reduction in the number of disease episodes and / or symptoms; (3) inhibition (i.e., reduction, slowing, or complete cessation) of infiltration of diseased cells into adjacent peripheral organs and / or tissues; (4) inhibition (i.e., reduction, slowing, or complete cessation) of disease spread; (5) reduction of autoimmune conditions; (6) favorable changes in the expression of biomarkers associated with the disorder; (7) alleviation to some extent of one or more symptoms associated with the disorder; (8) increase in the length of disease-free manifestation after treatment; or (9) reduction in mortality at a given time point after treatment.

[0130] As used herein, the term "peptide" refers to any peptide comprising two or more amino acids. The term peptide includes short peptides (e.g., peptides comprising 2 - 14 amino acids), medium-length peptides (15 - 50), or long-chain peptides (e.g., polypeptides or proteins). The terms peptide, medium-length peptide, and protein may be used interchangeably herein. As used herein, the term "peptide" is interpreted to mean a polymer composed of amino acid residues, related naturally occurring structural variants, and their synthetic non-naturally occurring analogs, linked by peptide bonds, related naturally occurring structural variants, and their synthetic non-naturally occurring analogs. Synthetic peptides can be synthesized, for example, using an automated peptide synthesizer. Peptides can also be synthesized by other means, such as by cells, bacteria, yeast, or other living organisms. Peptides can contain amino acids other than the 20 genetically encoded amino acids. Peptides include peptides modified by natural processes such as processing and other post-translational modifications, as well as peptides modified by chemical modification techniques. Such modifications are fully described in basic textbooks and more detailed monographs and are well known to those skilled in the art. Modifications can occur anywhere in the peptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini.

[0131] As used herein, "pharmaceutically acceptable carrier" or "therapeutically effective carrier" is an aqueous or non-aqueous (solid) carrier, such as an alcohol carrier or an oil carrier, or a mixture thereof, and can contain surfactants, emollients, lubricants, stabilizers, dyes, fragrances, preservatives, acids or bases for adjusting pH, solvents, emulsifiers, gelling agents, humectants, stabilizers, wetting agents, time release agents, humectants, or other components commonly included in a particular form of pharmaceutical composition. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiological buffered saline, or other solvents or vehicles such as glycols, glycerol, and oils such as olive oil or injectable organic esters. Pharmaceutically acceptable carriers can contain physiologically acceptable compounds that serve, for example, to stabilize or increase the absorption of a particular inhibitor, such as carbohydrates (e.g., glucose, sucrose, or dextran), antioxidants (e.g., ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or other stabilizers or excipients.

[0132] The term "pharmacokinetics" is defined as the time course of absorption, distribution, metabolism, and excretion of a therapeutic compound. Improved "pharmacokinetic properties" are defined as: improving one or more pharmacokinetic properties of a particular therapeutic compound as needed. Examples include, but are not limited to: reducing elimination by metabolism or secretion, increasing drug absorption, increasing half-life, and / or increasing bioavailability.

[0133] "PNA" refers to peptide nucleic acid having a chemical structure similar to DNA or RNA. Peptide bonds are used to link nucleotides or nucleosides together.

[0134] A "scaffold" is a molecule to which other molecules can be covalently or non-covalently linked or formulated. The scaffolds of the present invention can act as a "spacer" between a targeting group and a drug. A spacer is a molecular entity that provides a physical distance between two different molecular entities. The scaffold can also contain a reactive "linker" or can have beneficial therapeutic properties other than the drug. A linker is a site of attachment from one molecular entity to another. Thus, the scaffolds of the present invention can be, for example, PEG, serum albumin, thioredoxin, immunoglobulins, modified groups containing reactive linkers, water-soluble polymers, or therapeutic compounds. The scaffolds and linkers of the present invention are stable (i.e., non-releasable). Non-releasable linkers have more stable chemical bonds than releasable linkers to allow the linked molecular entities to remain linked in vivo. However, in certain embodiments, they can be "releasable" under specific conditions. Releasable linkers have inherent instability and allow the release of the linked molecules over time under certain conditions.

[0135] The "stringency" of a hybridization reaction can be readily determined by one of ordinary skill in the art and is typically calculated empirically based on probe length, wash temperature, and salt concentration. Generally, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures.

[0136] As defined herein, "stringent conditions" or "high stringency conditions" can be determined by: (1) washing with low ionic strength and high temperature, such as 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) using a denaturing agent such as formamide during hybridization, such as 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / pH 6.5 50 mM sodium phosphate buffer with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) hybridizing overnight in a solution containing 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μl / ml), 0.1% SDS, and 10% dextran sulfate at 42°C, followed by washing in 0.2x SSC (sodium chloride / sodium citrate) at 42°C for 10 minutes and then a 10-minute high stringency wash at 55°C with 0.1x SSC containing EDTA.

[0137] "Therapeutic compound" generally refers to small molecules, chemical entities, nucleic acids, nucleic acid derivatives, peptides, peptide derivatives, naturally occurring proteins, non-naturally occurring proteins, glycoproteins and steroids, which are administered to a subject to treat a disease or dysfunction or otherwise affect the health of the individual. As used herein, the term "therapeutic compound" has essentially the same meaning as the term "drug" or "therapeutic agent". The therapeutic compound of the present invention is PTH protein and its derivatives.

[0138] As used herein, "treatment" refers to clinical intervention that attempts to change the natural course of the treated individual or cell, and can be performed before or during the clinical pathological process. The desired effects of treatment include preventing the occurrence or recurrence of the disease or its state or symptoms, alleviating the state or symptoms of the disease, reducing any direct or indirect pathological consequences of the disease, reducing the rate of disease progression, improving or alleviating the disease state, and achieving relief or improved prognosis. In some embodiments, the methods and compositions of the present invention can be used to attempt to delay the development of a disease or disorder.

[0139] "Vitamin" is a recognized term in the art and is defined as fat-soluble or water-soluble organic substances essential in trace amounts for normal growth and activity of the body and obtained naturally from plant and animal foods or from supplements.

[0140] "Vitamin D" is a group of fat-soluble, open-ring steroids. There are several forms (vitamin isoenols) of vitamin D. The two main forms are vitamin D2 or ergocalciferol and vitamin D3 or cholecalciferol. Vitamin D without a subscript refers to vitamin D2, D3, or other forms known in the art. In humans, vitamin D can be ingested as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2). In addition, humans can synthesize it from cholesterol when sunlight exposure is sufficient. Cholecalciferol can be modified in the liver or in vitro to 25-hydroxycholecalciferol ("25-hydroxyvitamin D"). In the kidney or in vitro, 25-hydroxyvitamin D can be modified to different hormonal forms of 1,25-hydroxyvitamin D.

[0141] "Vitamin D binding protein", "DBP", or "VDBP" is a naturally occurring circulating serum protein present in all mammals, which is active particularly in binding vitamin D and its analogs and transporting them to sites in the liver and kidneys where the vitamin is modified to its active form, and which in humans retains the various forms of vitamin D in circulation for an average of 30 days. The DBP protein sequence is disclosed in SEQ ID NO:4, and an exemplary nucleic acid sequence encoding the DBP protein sequence is disclosed in SEQ ID NO:5. DBP has multiple naturally occurring isoforms. Exemplary isoforms are available in public sequence databases (e.g., accession numbers NM_001204306.1, NM_001204307.1, NM_000583.3, BC036003.1, M12654.1, X03178.1, AK223458, P_001191235.1, NP_000574.2, AAA61704.1, AAD13872.1, NP_001191236.1, AAA19662.2, I54269, P02774.1, EAX05645.1, AAH57228.1, AAA52173.1, AAB29423.1, AAD14249.1, AAD14250.1, and BAD97178.1).

[0142] The present invention contemplates non-hormonal vitamin D conjugates that bind DBP or functional DBP variants and homologs, said variants and homologs containing conservative or non-conservative amino acid substitutions that substantially retain DBP activity. DBP binding molecules or functional DBP variants can be identified using known techniques and characterized using known methods (Bouillon et al., J Bone Miner Res. 6(10):1051-7(1991), Teegarden et al., Anal. Biochemistry 199(2):293-299(1991), McLeod et al., J Biol Chem. 264(2):1260-7(1989), Revelle et al., J Steroid Biochem. 22:469-474(1985)). The foregoing references are incorporated herein by reference in their entirety.

[0143] The term "water-soluble" refers to a moiety that has a detectable degree of solubility in water. Methods for detecting and / or quantifying water solubility are well known in the art. Exemplary water-soluble polymers include peptides, sugars, polyethers, polyamines, polycarboxylic acids, and the like.

[0144] The present invention provides effective routes for the administration of proteins, peptides, other biological agents, nucleic acids, and small molecule drugs. The present invention also provides effective drug administration routes by transdermal, oral, parenteral, subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-synovial, intra-sternal, intrathecal, intralesional, intracranial injection, infusion, inhalation, ophthalmic, topical, rectal, nasal, buccal, sublingual, vaginal, or implantable reservoir means.

[0145] In addition, the invention described herein provides compositions and methods for maintaining the target binding activity (i.e., pharmacodynamics (PD)) of a therapeutic compound. It also provides compositions and methods for improving the pharmacokinetic (PK) properties of a therapeutic compound as described herein. The present invention also provides compositions and methods for improving drug absorption properties as compared to drug absorption properties of drugs using the same or different administration routes but without the invention described herein. The present invention also provides compositions and methods for improving drug bioavailability properties as compared to drug bioavailability properties of drugs using the same or different administration routes but without the carrier described herein. The present invention also provides compositions and methods for improving drug half-life properties as compared to drug half-life properties of drugs using the same or different administration routes but without the invention described herein.

[0146] The present invention also provides alternative drug administration routes that are more cost-effective or more favorable to the patient when compared to drugs without the invention described herein.

[0147] The non-hormonal vitamin D carriers disclosed herein can improve the absorption, half-life, bioavailability, or pharmacokinetic properties of the attached PTH. Without wishing to be bound by theory, the carrier has the property of binding to native DBP in vivo. DBP can transport the carrier-drug complex from the site of administration to the circulating serum. The vitamin D-DBP interaction can retain the therapeutic compound in the circulation for an extended period of time. This can prevent its excretion from the body and increase the exposure of the therapeutic compound in the body to achieve a more sustained therapeutic effect. In addition, a smaller dose of the drug may be required when conjugated to the carrier as compared to the unmodified form.

[0148] The therapeutic compound carrier conjugates of the present invention generally have about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 targeting groups individually linked to the therapeutic compound. The structures of each targeting group linked to the therapeutic compound can be the same or different. In a preferred embodiment, one or more targeting groups are stably or non-releasably linked to the therapeutic compound at the N-terminus, C-terminus or other part of the therapeutic protein. For example, the therapeutic compound carrier conjugate can comprise a targeting group linked to the N-terminus and an additional targeting group linked to a lysine residue. In another embodiment, the therapeutic compound carrier conjugate has a targeting group linked to the therapeutic protein by a modification such as a sugar residue that is part of a glycosylation site, or at an acylation site of a peptide, or linked to a phosphorylation site, or other natural or non-natural modifications familiar to those skilled in the art. Linkage sites using combinations of the above sites are also contemplated. A preferred embodiment of the present invention includes a targeting group linked to the therapeutic compound at a specific site on the therapeutic compound. In another preferred embodiment, the linkage site on the protein can be cysteine, lysine, N-terminus or C-terminus.

[0149] In another embodiment, the backbone is a pharmaceutically acceptable carrier. In a preferred embodiment, the backbone is polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, peptide, serum albumin, thioredoxin, immunoglobulin, amino acid, nucleic acid, glycan, a modified group containing a reactive linker, a water-soluble polymer, a small carbon chain linker or another therapeutic moiety.

[0150] The length of the backbone of the present invention is discrete to facilitate preparation, solubility, detection and drug efficacy. In one embodiment, the soluble backbone portion has a certain detectable degree of solubility in an aqueous or non-aqueous solution. Methods for detecting and / or quantifying water solubility are well known in the art. Exemplary water-soluble polymers include peptides, sugars, polyethers, polyamines, polycarboxylic acids, etc.

[0151] Peptides can have a mixed sequence or consist of a single amino acid, such as polylysine. Exemplary polysaccharides are polysialic acids. Exemplary polyethers are polyethylene glycols, such as m-PEG. Polyethyleneimine is an exemplary polyamine, and polyacrylic acid is a representative polycarboxylic acid. The polymer backbone of the water-soluble polymer can be polyethylene glycol (i.e., PEG). However, it should be understood that other related polymers are also suitable for use in the practice of the present invention, and the use of the term PEG or polyethylene glycol is intended to be inclusive and not restrictive in this regard. The term PEG includes polyethylene glycol in any of its forms, including alkoxy PEG, bifunctional PEG, multi-arm PEG, forked PEG, branched PEG, pendent PEG (i.e., PEG or a related polymer having one or more functional groups side-attached to the polymer backbone), or PEG having a degradable bond therein. The polymer backbone can be linear or branched.

[0152] Branched polymer backbones are well known in the art. Generally, a branched polymer has a central branched core portion and a plurality of linear polymer chains connected to the central branched core. PEG is commonly used in a branched form, which can be prepared by adding ethylene oxide to various polyols such as glycerol, pentaerythritol, and sorbitol. The central branched portion can also be derived from various amino acids such as lysine. Branched polyethylene glycol can be represented in general form as R(-PEG-OH)m, where R represents the core portion (such as glycerol or pentaerythritol), and m represents the number of arms. Multi-arm PEG molecules (such as those described in U.S. Patent No. 5,932,462, which is incorporated herein by reference in its entirety) can also be used as the polymer backbone.

[0153] Many other polymers are also suitable for the present invention. Non-peptide and water-soluble polymer backbones having from 2 to about 300 termini are particularly useful in the present invention. Examples of suitable polymers include, but are not limited to, other polyalkylene glycols (such as polypropylene glycol (“PPG”)), copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohols, polyvinylpyrrolidone, polylysine, polyethyleneimine, polyhydroxypropylmethacrylamide, poly-α-hydroxy acids, polyvinyl alcohol, polyphosphazenes, polyoxazolines, poly-N-acrylmorpholine (such as described in U.S. Patent No. 5,629,384, which is incorporated herein by reference in its entirety), and copolymers, terpolymers, and mixtures thereof. Although the molecular weight of each chain of the polymer backbone can vary, it generally ranges from about 100 Da to about 100,000 Da.

[0154] In other embodiments, the backbone moiety can be a peptide, serum albumin, thioredoxin, immunoglobulin, amino acid, nucleic acid, glycan, a modified group containing a reactive linker, a water-soluble polymer, a small carbon chain linker, or another therapeutic compound. In one embodiment, the backbone moiety is non-toxic to humans and animals. In another embodiment, the backbone is an endogenous serum protein. In another embodiment, the backbone moiety is a water-soluble polymer. In another embodiment, the backbone is a non-naturally occurring polymer. In another embodiment, the backbone is a naturally occurring moiety that is modified by covalent attachment to another moiety (e.g., PEG, polypropylene glycol, polyaspartic acid, biomolecule, therapeutic moiety, or diagnostic moiety). The backbones and linkers of the present invention are stable (i.e., non-releasable). However, in certain embodiments, they can be "releasable" under specific conditions.

[0155] Conjugation of hydrophilic polymers such as PEG is known in the art. In its most common form, PEG is a linear polymer that is capped with hydroxyl groups at each end: HO—CH2CH2O--(CH2CH2O)x--CH2CH2—OH, where x generally ranges from about 3 to about 4000. In a preferred embodiment, PEG has a substantially uniform molecular weight distribution. In another preferred embodiment, PEG is a linear polymer. In another preferred embodiment, PEG is a branched polymer.

[0156] Many end-functionalized or branched derivatives and various sizes are known in the art and commercially available. For example, conjugation of PEG or polyethylene oxide (also known as PEO) can be performed using the compositions and methods described herein and in U.S. Patent Nos. 7,803,777 (Defrees et al.) and 4,179,337 (Davis et al.), each of which is incorporated herein by reference in its entirety.

[0157] In some embodiments, the therapeutic compound is paired with a smaller backbone moiety or a larger backbone moiety. It is contemplated that the therapeutic compound can be paired with a backbone moiety from 1 Da to 10 kDa. In some embodiments, a backbone having a molecular weight approximately equal to that of the small therapeutic compound results in an effective conjugate. Improvements in efficacy can be obtained by empirically adjusting the backbone size further. Without wishing to be bound by theory, the pharmacokinetic properties and efficacy of the conjugate can be enhanced when the backbone (optionally in combination with a linker) is large enough to eliminate potential steric hindrance of the drug by binding to DBP, and vice versa. Thus, the therapeutic compound is conjugated such that its active region is exposed and available for functional activity, and the carrier is capable of binding to DBP. Additional embodiments provide non-releasable linkages that prolong the circulation of the therapeutic agent. In some small peptide embodiments, such as PTH, the backbone can be selected to be approximately equal to the molecular weight of the therapeutic agent.

[0158] In a preferred embodiment, the conjugation of the therapeutic compound retains some or substantially all of its activity after conjugation. The active region of a given therapeutic agent can be known in the art or determined empirically. In other embodiments, the conjugate has therapeutic activity while remaining attached to the carrier. This embodiment can maximize the time in circulation and its efficacy.

[0159] The backbone of the present invention can have a molecular weight of, for example, 100 Daltons (Da.), 500 Da., 1000 Da., 2000 Da., 5000 Da., 10,000 Da., 15,000 Da., 20,000 Da., 30,000 Da., 40,000 Da., or 60,000 Da. In one embodiment of the present invention, a "small" backbone can be between about 100 Da. and 20,000 Da. In another embodiment, a "large" backbone can be greater than about 20,000 Da. to about 200,000 Da. In a preferred embodiment, the backbone moiety is between about 100 Da. and 200,000 Da. In a more preferred embodiment, the backbone is between about 100 Da to 20,000 Da., 200 Da. to 15,000 Da., 300 Da. to 10,000 Da., 400 Da. to 9,000 Da., 500 Da. to 5,000 Da., 600 Da. to 2,000 Da., 1000 Da. to 200,000 Da., 20,00Da. and 200,000 Da., 100,000 to 200,000 Da., 5000 Da. to 100,000 Da., 10,000 Da. to 80,000 Da., 20,000 Da. to 60,000 Da., or 20,000 Da to 40,000 Da. The size of the backbone can be varied to maximize the absorption, bioavailability, circulating half-life, or efficacy of the conjugated therapeutic compound.

[0160] Another component of the carrier molecule preferably includes a coupling group for covalently linking the drug to the backbone or carrier. The coupling groups of the present invention include amine-reactive groups, thiol-reactive groups, maleimide groups, thiol groups, aldehyde groups, N-hydroxysuccinimide (NHS)-ester groups, haloacetyl groups, iodoacetyl groups, bromoacetyl groups, succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) groups, sulfo-SMCC groups, carbodiimide groups, and bifunctional crosslinkers (such as NHS-maleimide), combinations thereof, or other coupling groups familiar to those skilled in the art. The coupling groups of the present invention can facilitate thiol bonding, amide bonding, oxime bonding, hydrazone bonding, thiazolidinone bonding, or utilize cycloaddition reactions (also known as click chemistry) to conjugate the carrier with the therapeutic compound. In another embodiment, the composition preferably includes a combination of one or more therapeutic compounds linked to the coupling group of the backbone molecule. The linker of the present invention can be between about 40 and 100 daltons. In a preferred embodiment, the linker can be between about 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 daltons. The linker can also be modified to affect the stability or releasability of the linkage between the carrier and the therapeutic compound.

[0161] It is known to those skilled in the art that the NHS group can be used to conjugate with native peptides and proteins without engineering the linkage site. The NHS group allows for linkage with most proteins and peptides containing amino acids with amine groups (such as lysine residues). Utilizing the NHS group allows for flexibility in the carrier conjugation site because the protein structure and reaction time can affect the linkage site and the number of carrier molecules conjugated with the therapeutic compound. For example, by controlling the molar ratio of the NHS carrier to the therapeutic compound, those skilled in the art can have some control over the number of carrier molecules linked to the therapeutic compound, thereby allowing more than one carrier to conjugate with a given therapeutic compound (if desired).

[0162] Conjugation of the carrier with the therapeutic compound is achieved by mixing solutions of the molecules in a specific molar ratio using a compatible solution, buffer, or solvent. For example, molar ratios of about 1:1, 2:1, 4:1, 5:1, 10:1, 20:1, 25:1, 50:1, 100:1, 1000:1, or about 1:2, 1:4, 1:5, 1:10, 1:20, 1:25, 1:50, 1:100, or 1:1000 of the carrier to the therapeutic compound can be used. By varying the ratio, this can result in different numbers of individual carriers being linked to the therapeutic compound or can assist in the selection of specific linkage sites. The conjugation of the carrier is also pH, buffer, salt, and temperature-dependent, and changing these parameters among the parameters can affect the linkage site, the number of carriers linked, and the reaction rate. For example, by selecting a pH for the reaction of pH 6 or below pH 6, it can assist in selectively conjugating the aldehyde form of the carrier to the N-terminus of a therapeutic protein or peptide.

[0163] In addition, to retain substantially the same activity of the therapeutic compound, conjugation with the carrier will be at a site on the molecule that does not interfere with the therapeutic function. For proteins, conjugation may be to the amino terminus, carboxyl terminus, or internal reactive amino acids. For nucleic acids, conjugation may be to the 5'-end, 3'-end, or internal nucleotides, nucleosides, or their derivatives. In one embodiment, the carrier is conjugated to a nucleotide or nucleoside prior to incorporation into the polynucleotide molecule.

[0164] The present invention provides a pharmaceutical carrier comprising Formula I:

[0165] B——(L) a ——S—(M) b ——C

[0166] I

[0167] Wherein:

[0168] B is a targeting group, which is a non-hormonal vitamin D, its analog or metabolite, conjugated with (L) a conjugated;

[0169] S is a backbone moiety comprising polyethylene glycol, polylysine, polyethyleneimine, polypropylene glycol, peptide, serum albumin, thioredoxin, immunoglobulin, amino acid, nucleic acid, glycan, a modified group containing a reactive linker, polylactic acid, a water-soluble polymer, a small carbon chain linker, or another therapeutic moiety;

[0170] C is an amine-reactive group, a thiol-reactive group, a maleimide group, a thiol group, a disulfide group, an aldehyde group, an NHS-ester group, a 4-nitrophenyl ester, an acyl imidazole, a haloacetyl group, an iodoacetyl group, a bromoacetyl group, an SMCC group, a sulfo-SMCC group, a carbodiimide group, and a bifunctional crosslinker (such as NHS-maleimide) or a combination thereof;

[0171] (L) a and (M) b are linkers independently selected from -(CH2) n -, -C(O)NH-, -HNC(O)-, -C(O)O-, -OC(O)-, -O-, -S-S-, -S-, -S(O)-, -S(O)2-, and -NH-;

[0172] a is an integer from 0 to 4; and

[0173] b is an integer from 0 to 4; and

[0174] n is an integer from 0 to 3.

[0175] In a preferred embodiment, the present invention provides a carrier comprising a carrier of formula I:

[0176] B—(L) a —S—(M) b ——C

[0177] I

[0178] wherein:

[0179] B is a targeting group selected from vitamin D, vitamin D analogs, vitamin D-related metabolites, analogs of vitamin D-related metabolites, or small carbon-based molecules that bind DBP;

[0180] S is a backbone moiety comprising polyethylene glycol, polylysine, polypropylene glycol, a peptide, serum albumin, an amino acid, a nucleic acid, a glycan, polylactic acid, a water-soluble polymer, or a small carbon chain linker;

[0181] C is a maleimide group, a thiol group, a disulfide group, an aldehyde group, an NHS-ester group, an iodoacetyl group, or a bromoacetyl group;

[0182] (L) a and (M) b are linkers independently selected from -(CH2) n -, -C(O)NH-, -HNC(O)-, -C(O)O-, -OC(O)-, -O-, -S-S-, -S-, -S(O)-, -S(O)2-, and -NH-;

[0183] a is an integer from 0 to 4; and

[0184] b is an integer from 0 to 4; and

[0185] n is an integer from 0 to 3.

[0186] In a more preferred embodiment, the present invention provides a carrier comprising a carrier of formula I:

[0187] B——(L) a ——S—(M) b ——C

[0188] I

[0189] Wherein:

[0190] B is a targeting group selected from vitamin D, vitamin D analogs or vitamin D-related metabolites;

[0191] S is a backbone moiety comprising polyethylene glycol, polylysine or polypropylene glycol;

[0192] C is a maleimide group, disulfide group, aldehyde group, NHS-ester group or iodoacetyl group;

[0193] (L) a and (M) b are linkers independently selected from -(CH2) n -, -C(O)NH-, -HNC(O)-, -C(O)O-, -OC(O)-, -O-, -S-S-, -S-, -S(O)-, -S(O)2- and -NH-;

[0194] a is an integer from 0 to 4; and

[0195] b is an integer from 0 to 4; and

[0196] n is an integer from 0 to 3.

[0197] In some preferred embodiments, the present invention provides carriers comprising carriers of formula IIa, IIb and IIc:

[0198]

[0199] Wherein:

[0200] B is a targeting group selected from vitamin D, vitamin D analogs or vitamin D-related metabolites;

[0201] S is a backbone moiety comprising polyethylene glycol or polypropylene glycol; and

[0202] C is a maleimide group, disulfide group, aldehyde group, NHS-ester group or iodoacetyl group;

[0203] L 1 is -(CH2) n -;

[0204] L 3 is -(CH2) o -;

[0205] (M) b is a linker independently selected from -(CH2) n -, -C(O)NH-, -HNC(O)-, -C(O)O-, -OC(O)-, -O-, -S-S-, -S-, -S(O)-, -S(O)2- and -NH-;

[0206] b is an integer from 0 - 4; and

[0207] n is 3; and

[0208] o is 1.

[0209] In WO13 / 172967 (which is incorporated herein by reference), conjugation at the carbon 25 (C25) position of 25-hydroxy-vitamin D3 is exemplified. In WO2016 / 065042, conjugation at the carbon 3 (C3) position of 25-hydroxy-vitamin D3 is exemplified.

[0210] In certain most preferred embodiments of formula IIa, B is represented by formula III, S is polyethylene glycol and (M) b -C is represented by formula IVa.

[0211]

[0212] In certain most preferred embodiments of formula IIb, B is represented by formula III, S is polyethylene glycol and (M) b -C is represented by formula IVb.

[0213]

[0214] In certain most preferred embodiments of formula IIc, B is represented by formula III, S is polyethylene glycol and (M) b -C is represented by formula IVc.

[0215]

[0216] In certain most preferred embodiments, S is between about 100 Da and 200,000 Da. In other most preferred embodiments, the backbone moiety is between about 100 Da and 20,000 Da, 200 Da and 15,000 Da, 300 Da and 10,000 Da, 400 Da and 9,000 Da, 500 Da and 5,000 Da, 600 Da and 2,000 Da, 1,000 Da and 200,000 Da, 5,000 Da and 100,000 Da, 10,000 Da and 80,000 Da, 20,000 Da and 60,000 Da, or 20,000 Da and 40,000 Da.

[0217] In a specific embodiment, the present invention provides a carrier represented by Formula V.

[0218]

[0219] In Formula V, x - 1 refers to the fact that after PEG is conjugated with x number of polymers in this reaction, the terminal PEG monomer is converted to an aldehyde, and thus the final formula is x - 1. In another specific embodiment, the present invention provides a carrier represented by Formula VI.

[0220]

[0221] In Formula VI, x refers to the number of repeating ethylene glycol units. In another specific embodiment, the present invention provides a carrier represented by Formula VII.

[0222]

[0223] In certain embodiments, the present invention provides a method for preparing a carrier of Formula I:

[0224] B—(L) a ——S—(M) b ——C

[0225] I

[0226] comprising the steps of: in the presence of an amide coupling agent, reacting a compound of Formula Ia:

[0227] B-L 1 -NH2

[0228] Ia

[0229] with a compound of Formula Ib:

[0230] HOOC——L 3 ——S——(M)b——C

[0231] Ib,

[0232] wherein B, S, C, and L 1 , L 3 and (M) b are as defined above, and L 2 is -C(O)NH-.

[0233] Those skilled in the art will recognize that the compounds of formula Ia can be used as the free base or in a suitable salt form. Suitable salt forms include, but are not limited to, trifluoroacetic acid (TFA), hydrochloric acid (HCl), hydrobromic acid (HBr), methanesulfonic acid (MsOH), (trifluoromethanesulfonic acid) TfOH, and acetic acid (AcOH).

[0234] Any suitable amide coupling agent can be used to form the compounds of formula I. Suitable amide coupling agents include, but are not limited to, 2-chloromethylpyridinium iodide, [benzotriazol-1-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate] (BOP), benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU), and propanephosphonic anhydride (T3P). In certain embodiments, the amide coupling agent is used alone. In certain embodiments, the amide coupling agent is used in combination with a co-reagent (such as 1-hydroxybenzotriazole (HOBT) or N,N-dimethylpyridin-4-amine (DMAP)). In certain embodiments, the amide coupling agent is used in combination with a base (such as triethylamine or diisopropylethylamine). In certain embodiments, the amide coupling agent is used in combination with both a co-reagent (such as HOBT or DMAP) and a base (such as triethylamine or diisopropylethylamine). Those skilled in the art will recognize that co-reagents other than HOBT or DMAP can be used. In addition, those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine can be used.

[0235] Those skilled in the art will recognize that any suitable leaving group can be coupled to the carboxylic acid of formula Ib in the presence of a suitable coupling agent to form the active ester of formula Ic:

[0236]

[0237] Wherein R is a suitable leaving group, including but not limited to imidazole, HOBT, NHS, and 4-nitrophenol. Suitable coupling agents include but are not limited to 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU, and T3P.

[0238] In some embodiments, the present invention provides a method for preparing a carrier of formula I:

[0239] B——(L) a ——S—(M) b ——C

[0240] I

[0241] Comprising the steps of: reacting a compound of formula Ia:

[0242] B-L 1 -NH2

[0243] Ia

[0244] with a compound of formula Ic:

[0245] ROOC—L 3 ——S——(M) b ——C

[0246] Ic

[0247] Wherein B, S, C, R, and L 1 , L 3 and (M) b are as defined above, and L 2 is -C(O)NH-.

[0248] Those skilled in the art will recognize that the compound of formula Ia can be used as a free base or in a suitable salt form. Suitable salt forms include but are not limited to TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0249] In certain embodiments, the amide coupling is carried out with a base (such as triethylamine or diisopropylethylamine). Those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine can be used.

[0250] In certain other embodiments, the present invention provides a method for preparing a carrier of formula IIa:

[0251]

[0252] Comprising the steps of: in the presence of an amide coupling agent, reacting a compound of formula Ia:

[0253] B-L 1 -NH2

[0254] Ia

[0255] React with a compound of formula Id:

[0256] HOOC—L 3 ——S——(M) b ——CH2OH

[0257] Id

[0258] To form a compound of formula Ie; and

[0259]

[0260] Oxidize the primary alcohol of formula Ie to the aldehyde of formula IIa;

[0261]

[0262] Wherein B, S, L 1 、L 3 、(M) b 、b, n and o are as defined above, and L 2 is -C(O)NH-, and C is an aldehyde group.

[0263] Any suitable oxidizing agent can be used to form the compound of formula IIa. Suitable oxidizing agents include, but are not limited to, Collins reagent, pyridinium dichromate (PDC), pyridinium chlorochromate (PCC), oxalyl chloride / DMSO (Swern oxidation), SO3-pyridine / DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane, tetrapropylammonium perruthenate / N-methylmorpholine-N-oxide (TPAP / NMO), and 2,2,6,6-tetramethylpiperidin-1-yloxy / sodium hypochlorite (TEMPO / NaOCl).

[0264] Those skilled in the art will recognize that the compound of formula Ia can be used as a free base or in a suitable salt form. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0265] Any suitable amide coupling agent can be used to form the compound of formula Ie. Suitable amide coupling agents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU, and T3P. In certain embodiments, the amide coupling agent is used alone. In certain embodiments, the amide coupling agent is used in combination with an auxiliary reagent (such as HOBT or DMAP). In certain embodiments, the amide coupling agent is used in combination with a base (such as triethylamine or diisopropylethylamine). In certain embodiments, the amide coupling agent is used in combination with both an auxiliary reagent (such as HOBT or DMAP) and a base (such as triethylamine or diisopropylethylamine). Those skilled in the art will recognize that auxiliary reagents other than HOBT or DMAP can be used. Additionally, those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine can be used.

[0266] In certain embodiments, any suitable leaving group can be coupled with the carboxylic acid of formula Id in the presence of a suitable coupling agent to form the active ester of formula If:

[0267]

[0268] wherein R is a suitable leaving group, including but not limited to imidazole, HOBT, 2,3,5,6-tetrafluorophenol (TFP), NHS, and 4-nitrophenol. Suitable coupling agents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU, and T3P.

[0269] In some embodiments, the present invention provides a method for preparing a carrier of formula Ie:

[0270]

[0271] comprising the steps of: reacting a compound of formula Ia;

[0272] B—L 1 -NH2

[0273] Ia

[0274] with a compound of formula If; and

[0275] ROOC—L 3 ——S——(M) b ——CH2OH

[0276] If

[0277] oxidizing the primary alcohol of formula Ie to the aldehyde of formula IIa;

[0278]

[0279] wherein B, S, C, R, and L 1 , L 3 and (M) b are as defined above, and L 2 is -C(O)NH-.

[0280] Those skilled in the art will recognize that the compounds of formula Ia can be used as the free base or in a suitable salt form. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0281] In certain embodiments, the amide coupling is carried out with a base such as triethylamine or diisopropylethylamine. Those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine can be used.

[0282] Any suitable oxidizing agent can be used to form the compounds of formula IIa. Suitable oxidizing agents include, but are not limited to, Collins reagent, PDC, PCC, oxalyl chloride / DMSO (Swern oxidation), SO3-pyridine / DMSO (Parikh-Doering oxidation), Dess-Martin periodinane, TPAP / NMO, and TEMPO / NaOCl.

[0283] In certain other embodiments, the present invention provides a method for preparing a carrier of formula IIc:

[0284]

[0285] comprising the steps of: reacting a compound of formula Ia:

[0286] B-L 1 -NH2

[0287] Ia

[0288] with a compound of formula Ig:

[0289] ROOC-S——(M) b ——COOH

[0290] Ig

[0291] to form a compound of formula Ih; and

[0292]

[0293] converting the carboxylic acid of formula Ih to the active ester of formula IIc;

[0294]

[0295] wherein B, S, C, R, L 1 , (M) b, b, n, and o are as defined above, and L 2 is -C(O)NH-.

[0296] Those skilled in the art will recognize that the compounds of formula Ia can be used as the free base or in the form of a suitable salt. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0297] Any suitable leaving group can be coupled with the carboxylic acid of formula Ih in the presence of a suitable coupling agent to form the active ester of formula IIc. Suitable leaving groups include, but are not limited to, imidazole, HOBT, 2,3,5,6-tetrafluorophenol (TFP), NHS, and 4-nitrophenol. Suitable coupling agents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU, and T3P.

[0298] In some embodiments, the active ester of formula IIc is formed from the carboxylic acid of formula Ih using a combination of a suitable leaving group and a coupling agent.

[0299] In some embodiments, the active ester of formula IIc is formed from the carboxylic acid of formula Ih using a single reagent that generates the leaving group and also causes the coupling reaction. Such reagents include, but are not limited to, 1,1'-carbonyldiimidazole, N,N'-disuccinimidyl carbonate, 4-nitrophenyl trifluoroacetate, and HBTU. In some embodiments, the single reagent is used alone. In other embodiments, the single reagent is used in combination with an acyl transfer catalyst. Such acyl transfer catalysts include, but are not limited to, DMAP and pyridine. Those skilled in the art will recognize that additional acyl transfer catalysts can be used.

[0300] In a specific embodiment, the present invention provides a method for preparing a carrier represented by formula V:

[0301]

[0302] comprising the steps of: reacting a compound of formula Va:

[0303]

[0304] with a compound of formula Vb:

[0305]

[0306] to form a compound of formula Vc;

[0307]

[0308] reducing the nitrile group to form an amine of formula Vd;

[0309]

[0310] React a compound of formula Vd with a compound of formula Ve;

[0311]

[0312] to form a compound of formula Vf

[0313]

[0314] Oxidize the primary alcohol of formula Vf to form the aldehyde of formula V.

[0315]

[0316] Here, x refers to the number of repeating ethylene glycol units, and x - 1 refers to the fact that after PEG is conjugated with x number of polymers in this reaction, the terminal PEG monomer is converted to an aldehyde, so the final formula is x - 1. In some embodiments, the reaction of the compound of formula Vb with the compound of formula Va is facilitated by adding Triton B. Those skilled in the art will recognize that other reagents can be used to facilitate the nucleophilic addition to acrylonitrile.

[0317] In some embodiments, the reduction of the nitrile of formula Vc to the amine of formula Vd is carried out using AlCl3 / lithium aluminum hydride (LAH). Those skilled in the art will recognize that other reducing agents can be used, including sodium, H2 / Pd, H2 / Raney nickel, and diborane.

[0318] Those skilled in the art will recognize that the compound of formula Vd can be used as a free base or in a suitable salt form. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0319] In certain embodiments, a base (such as triethylamine or diisopropylethylamine) is used to facilitate the coupling of the NHS - ester of formula Ve with the amine of formula Vd. Those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine can be used.

[0320] Any suitable oxidizing agent can be used to form the compound of formula V. Suitable oxidizing agents include, but are not limited to, Collins reagent, PDC, PCC, oxalyl chloride / DMSO (Swern oxidation), SO3 - pyridine / DMSO (Parikh - Doering oxidation), Dess - Martin periodinane, TPAP / NMO, and TEMPO / NaOCl.

[0321] In another specific embodiment, the present invention provides a method for preparing a carrier represented by formula VI:

[0322]

[0323] comprising the following steps: in the presence of an amide coupling agent, reacting a compound of formula Vd:

[0324]

[0325] with a compound of formula VIa:

[0326]

[0327] wherein x represents the number of repeating ethylene glycol units. Those skilled in the art will recognize that the compound of formula Vd can be used as a free base or in the form of a suitable salt. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0328] In a preferred embodiment of formulas VI, Vd, and VIa, x = 36.

[0329] Any suitable amide coupling agent can be used to form the compound of formula VI. Suitable amide coupling agents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU, and T3P. In certain embodiments, the amide coupling agent is used alone. In certain embodiments, the amide coupling agent is used in combination with a co-reagent (such as HOBT or DMAP). In certain embodiments, the amide coupling agent is used in combination with a base (such as triethylamine or diisopropylethylamine). In certain embodiments, the amide coupling agent is used in combination with both a co-reagent (such as HOBT or DMAP) and a base (such as triethylamine or diisopropylethylamine). Those skilled in the art will recognize that co-reagents other than HOBT or DMAP can be used. Additionally, those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine can be used.

[0330] In another specific embodiment, the present invention provides a method for preparing a carrier represented by formula VII:

[0331]

[0332] comprising the following steps: reacting a compound of formula Vd:

[0333]

[0334] with a compound of formula VIIa:

[0335]

[0336] to form a compound of formula VIIb; and

[0337]

[0338] Convert the carboxylic acid of formula VIIb to the active ester of formula VII;

[0339]

[0340] Those skilled in the art will recognize that the compound of formula Vd can be used as a free base or in a suitable salt form. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0341] In certain embodiments, a base (such as triethylamine or diisopropylethylamine) is used to facilitate the coupling of the NHS-ester of formula VIIa with the amine of formula Va. Those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine can be used.

[0342] NHS can be coupled with the carboxylic acid of formula VIIb in the presence of a suitable coupling agent to form the active ester of formula VII. Suitable coupling agents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU, and T3P.

[0343] In some embodiments, a combination of NHS and a coupling agent is used to form the active ester of formula VII from the carboxylic acid of formula VIIb.

[0344] In some embodiments, a single reagent that generates a leaving group and also causes a coupling reaction is used to form the active ester of formula VII from the carboxylic acid of formula VIIb. Such reagents include, but are not limited to, N,N'-disuccinimidyl carbonate. In some embodiments, the single reagent is used alone. In other embodiments, the reagent is used with an acyl transfer catalyst. Such acyl transfer catalysts include, but are not limited to, DMAP and pyridine. Those skilled in the art will recognize that additional acyl transfer catalysts can be used.

[0345] Those skilled in the art will recognize that there are other methods for conjugating a linker and a backbone to the C3 or C25 position of vitamin D derivatives and analogs. For example, the C3 hydroxyl group can be acylated by various groups, as practiced by: N. Kobayashi, K. Ueda, J. Kitahori, and K. Shimada, Steroids, 57, 488 - 493 (1992); J. G. Haddad et al., Biochemistry, 31, 7174 - 7181 (1992); A. Kutner, R. P. Link, H. K. Schnoes, H. F. DeLuca, Bioorg. Chem., 14, 134 - 147 (1986); and R. Ray, S. A. Holick, N. Hanafin, and M. F. Holick, Biochemistry, 25, 4729 - 4733 (1986). The foregoing references are incorporated by reference in their entirety. Those skilled in the art will recognize that these chemical methods can be modified to synthesize compounds of Formula I:

[0346] B——(L) a ——S—(M) b ——C

[0347] I

[0348] wherein B, S, C, (L) a and (M) b are defined as above.

[0349] If desired, gel filtration chromatography and / or ion exchange chromatography can be used to separate therapeutic compound - carrier conjugates having different molecular weights. Gel filtration chromatography can be used to separate different therapeutic compound - carrier conjugates (e.g., monomers, dimers, trimers, etc., where "monomer" indicates one molecule of the targeting group per therapeutic compound, "dimer" indicates two targeting groups linked to the therapeutic compound, etc.) based on their different molecular weights (where the differences essentially correspond to the average molecular weight of the targeting group).

[0350] Gel filtration columns suitable for performing this type of separation include Superdex and Sephadex columns available from Amersham Biosciences (Piscataway, N.J.). The choice of the specific column will depend on the desired separation range. Elution is typically carried out using a suitable buffer, such as phosphate, acetate, etc. The collected fractions can be analyzed by a variety of different methods, e.g., (i) optical density (OD) at 280 nm for protein content, (ii) bovine serum albumin (BSA) protein assay, and (iii) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE).

[0351] The separation of the therapeutic compound carrier conjugate can also be carried out by reverse-phase chromatography using a reverse-phase high-performance liquid chromatography (RP-HPLC) C18 column (Amersham Biosciences or Vydac), or by ion-exchange chromatography using an ion-exchange column (e.g., a DEAE- or CM-Sepharose ion-exchange column available from Amersham Biosciences). The resulting purified composition is preferably substantially free of the therapeutic compound conjugated to non-targeting groups. In addition, the composition is preferably substantially free of all other non-covalently linked targeting groups.

[0352] As described herein, the carrier of the present invention can be a non-hormonal 25-hydroxyvitamin D or an analogue thereof having a coupling group at the 3'-carbon. As used herein, "25-hydroxyvitamin D analogue" includes both naturally occurring forms of vitamin D metabolites and other chemically modified forms. The carrier of the present invention does not include the active (i.e., hormonal) form of vitamin D (which typically has a hydroxyl group at the 1-carbon). These compounds are based on the vitamin D structure and retain some of the functions of vitamin D (i.e., they interact with DBP), although with different affinities. The following list exemplifies forms of vitamin D analogues known in the art. However, they may be hormonal or have a C1 hydroxyl group. Only their chemical nature as vitamin D analogues is provided here, not their functional hormonal properties: OCT is a chemically synthesized form of 1,25(OH)2D3 having an oxygen atom at the 22-position in the side chain (Abe et al., FEBS Lett. 226:58-62 (1987)); Gemini vitamin D analogue - 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteriomethyl-4,4,4-trideuteriobutyl)-23-yne-26,27-hexafluoro-cholecalciferol (BXL0124) (So et al., Mol Pharmacol. 79(3):360-7 (2011)); paricalcitol is a vitamin D2-derived sterol lacking the carbon 19 methylene group found in all natural vitamin D metabolites (Slatopolsky et al., Am J. Kidney Dis. 26:852 (1995)); doxercalciferol (1α-hydroxyvitamin D2), like alfacalcidol (1α-hydroxyvitamin D3), is a prodrug that is hydroxylated in the liver to 1α,25(OH)2D2. However, unlike alfacalcidol, doxercalciferol is also 24-hydroxylated to produce 1α,24(S)-(OH)2D2 (Knutson et al., Biochem Pharmacol 53:829 (1997)); dihydrotachysterol 2 (DHT2), which is hydroxylated in vivo to 25(OH)DHT2, 1,25(OH)2DHT2 (McIntyre et al., Kidney Int. 55:500 (1999)), ED-71 and eldecalcitol. See also Erben and Musculoskel, Neuron Interact. 2(1):59-69 (2001) and Steddon et al., Nephrol. Dial. Transplant. 16(10):1965-1967 (2001). The foregoing references are incorporated by reference in their entirety.

[0353] In another embodiment, the carrier further comprises a pharmaceutically acceptable backbone moiety covalently linked to the targeting group and the therapeutic compound. The backbone moiety of the carrier of the present invention does not necessarily participate but can contribute to the function of the therapeutic compound or improve its pharmacokinetic properties. The backbone of the present invention substantially does not interfere with the binding of the targeting group to DBP. Similarly, the backbone of the present invention substantially does not interfere with the structure or function of the therapeutic compound. The length of the backbone moiety depends on the nature of the targeting group and the therapeutic compound. Those skilled in the art will recognize that, based on the known distances between various bonds, various combinations of atoms provide variable length molecules (Morrison and Boyd, Organic Chemistry, 3rd Edition, Allyn and Bacon, Inc., Boston, Mass. (1977), incorporated herein by reference). Other backbones contemplated by the present invention include peptide linkers, protein linkers (such as human serum albumin or immunoglobulin family proteins or fragments thereof), nucleic acid linkers, small carbon chain linkers, carbon linkers interspersed with oxygen or nitrogen, or combinations thereof. In a preferred embodiment, the linker is non-releasable or stable.

[0354] PTH is a therapeutic peptide. The term "peptide" is used interchangeably herein with the term "protein" and is intended to include a string of amino acids. The amino acids in the peptides of the present invention can be naturally occurring or non-naturally occurring. The peptides of the present invention can be chemically synthesized or biosynthesized and can include cysteine-rich peptides, cyclic peptides, stapled peptides, peptides comprising D- or L-amino acids and mixtures thereof, peptidomimetics, peptide nucleic acids (PNAs), and combinations thereof. Also contemplated within the scope of the embodiments described herein are branched or cyclic therapeutic peptides with or without branches. Cyclic, branched, and branched cyclic peptides are produced by natural post-translational processes and are also prepared by suitable synthetic methods. In some embodiments, any of the peptide products described herein include the above peptide analogs, which are then covalently linked to an alkyl glycoside surfactant moiety.

[0355] Other embodiments include therapeutic peptide chains composed of natural and non-natural amino acids or analogs of natural amino acids. As used herein, peptide and / or protein "analogs" include non-natural amino acids based on natural amino acids, such as tyrosine analogs, which include para-substituted tyrosine, ortho-substituted tyrosine, and meta-substituted tyrosine, where the substituents on tyrosine include acetyl groups, benzoyl groups, amino groups, hydrazines, hydroxylamines, thiol groups, carboxyl groups, methyl groups, isopropyl groups, C2-C20 straight or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl groups, polyether groups, halogens, nitro groups, etc.

[0356] Additional embodiments include therapeutic peptide chains having modified amino acids. Examples include acylated amino acids at the ε-position of lysine, amino acids having fatty acids (such as octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, 3-phenylpropanoic acid, etc.) or amino acids having saturated or unsaturated alkyl chains. (Zhang, L. and Bulaj, G. (2012) Curr Med Chem 19:1602-1618, which is incorporated herein by reference in its entirety).

[0357] The present invention also contemplates therapeutic peptide chains comprising natural and unnatural amino acids or analogs of natural amino acids. In some embodiments, a peptide or protein “analog” comprises unnatural amino acids based on natural amino acids, such as tyrosine analogs, which include para-substituted tyrosine, ortho-substituted tyrosine, and meta-substituted tyrosine, wherein the substituents on tyrosine include acetyl groups, benzoyl groups, amino groups, hydrazines, hydroxylamines, thiol groups, carboxyl groups, methyl groups, isopropyl groups, C2-C20 straight or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl groups, polyether groups, halogens, nitro groups, etc. Examples of Tyr analogs include 2,4-dimethyltyrosine (Dmt), 2,4-diethyltyrosine, O-4-allyltyrosine, 4-propyltyrosine, Ca-methyltyrosine, etc. Examples of lysine analogs include ornithine (Orn), homolysine, Ca-methyllysine (CMeLys), etc. Examples of phenylalanine analogs include, but are not limited to, meta-substituted phenylalanine, wherein the substituents include methoxy groups, C1-C20 alkyl groups, such as methyl groups, allyl groups, acetyl groups, etc. Specific examples include, but are not limited to, 2,4,6-trimethyl-L-phenylalanine (Tmp), O-methyltyrosine, 3-(2-naphthyl)alanine (Nal(2)), 3-(1-naphthyl)alanine (Nal(1)), 3-methylphenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), fluorophenylalanine, isopropylphenylalanine, p-azidophenylalanine, p-acylphenylalanine, p-benzoylphenylalanine, p-iodophenylalanine, p-bromophenylalanine, p-aminophenylalanine, and isopropylphenylalanine, etc.

[0358] Also contemplated within the scope of the embodiments are therapeutic peptide chains containing non-standard or non-natural amino acids known in the art, such as C-α-disubstituted amino acids, such as Aib, Ca-diethylglycine (Deg), aminocyclopentane-1-carboxylic acid (Ac4c), aminocyclopentane-1-carboxylic acid (Ac5c), etc. Such amino acids often result in restricted structures, usually favoring an α-helical structure (Kaul, R. and Balaram, P. (1999) Bioorg Med Chem 7:105-117, which is incorporated herein by reference in its entirety). Additional examples of such non-natural amino acids useful for analog design are homoarginine (Har), etc. In some cases, substitution of the reduced amide bond results in improved protection from enzymatic degradation or altered receptor binding. For example, introduction of the Tic-Phe dipeptide unit with a reduced amide bond between the residues (referred to as Tic-F[CH2-NH]^-Phe) reduces enzymatic degradation.

[0359] In some embodiments, modifications at the amino or carboxyl terminus can optionally be introduced into the peptides or proteins of the present application (Nestor, J.J., Jr. (2009) Current Medicinal Chemistry 16:4399-4418). For example, the peptides or proteins of the present application can be truncated or acylated at the N-terminus (Gourlet, P. et al. (1998) Eur J Pharmacol 354:105-111, Gozes, I. and Furman, S. (2003) Curr Pharm Des 9:483-494, the contents of which are incorporated herein by reference in their entirety). Other modifications to the N-terminus of the peptide or protein, such as deletion or introduction of D-amino acids (such as D-Phe), result in potent and long-acting agonists or antagonists when substituted with modifications described herein, such as long-chain alkyl glycosides.

[0360] Accordingly, the present invention provides therapeutic compound analogs, wherein the natural therapeutic compound is modified by: acetylation, acylation, pegylation, ADP-ribosylation, amidation, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of a covalent cross-link formed by cysteine, formation of pyroglutamic acid, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid linkage, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, transfer RNA-mediated addition of an amino acid to a protein (such as arginylation) and ubiquitination. See, e.g., (Nestor, J.J., Jr. (2007) Comprehensive Medicinal Chemistry II 2:573-601, Nestor, J.J., Jr. (2009) Current Medicinal Chemistry 16:4399-4418, Uy, R. and Wold, F. (1977) Science 198:890-6, Seifter, S. and Englard, S. (1990) Methods Enzymol 182:626-646, Rattan, S.I. et al. (1992) Ann NY Acad Sci 663:48-62). The foregoing references are incorporated by reference in their entirety.

[0361] Glycosylated therapeutic peptides can be prepared using conventional Fmoc chemistry and solid-phase peptide synthesis techniques (e.g., on resin), where the desired protected sugar amino acids are prepared prior to peptide synthesis and then introduced at the desired positions in the peptide chain during peptide synthesis. Thus, therapeutic peptide-polymer conjugates can be conjugated in vitro. Glycosylation can occur prior to deprotection. The preparation of amino acid glycosides is described in U.S. Patent No. 5,767,254, WO 2005 / 097158, and Doores, K. et al., Chem. Commun., 1401-1403, 2006, which are incorporated herein by reference in their entirety. For example, α- and β-selective glycosylation of serine and threonine residues with Schiff base intermediates is carried out using the Koenigs-Knorr reaction and Lemieux's in situ anomerization method. The Schiff base glycosides are then deprotected using mild acidic conditions or hydrogenolysis reactions. Compositions comprising glycosylated therapeutic peptide conjugates are prepared by stepwise solid-phase peptide synthesis, which involves stepwise contacting of the growing peptide chain with protected amino acids, where at least one protected amino acid is glycosylated and then conjugated with a water-soluble polymer. Such compositions can have a purity of at least 95%, at least 97%, or at least 98% of a single species of glycosylated and conjugated therapeutic peptide.

[0362] Monosaccharides that can be introduced at one or more amino acid residues of the therapeutic peptides defined and / or disclosed herein include glucose (dextrose), fructose, galactose, and ribose. Additional monosaccharides suitable for use include glyceraldehyde, dihydroxyacetone, erythrose, threose, erythrulose, arabinose, lyxose, xylose, ribulose, xylulose, allose, altrose, mannose, N-acetylneuraminic acid, fucose, N-acetylgalactosamine, and N-acetylglucosamine, among others. Glycosides such as monosaccharides, disaccharides, and trisaccharides for modifying one or more amino acid residues of the therapeutic peptides defined and / or disclosed herein include sucrose, lactose, maltose, trehalose, melibiose, and cellobiose, among others. Trisaccharides include acarbose, raffinose, and melezitose.

[0363] In additional embodiments of the invention, the therapeutic compounds defined and / or disclosed herein can be chemically conjugated with biotin. The biotin / therapeutic compound can then bind to avidin.

[0364] The presence or concentration of PTH can be measured using an antibody. The term antibody is intended to include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab fragments), single-chain antibodies, bispecific or multispecific antibodies, Llama antibodies, nano-bodies, diabodies, affibodies, Fv, Fab, F(ab')2, Fab', scFv, scFv-Fc, etc. Antibody fusion proteins such as Ig chimeras are also included within the term.

[0365] An antibody that specifically binds to an antigen has a high affinity for that antigen. Antibody affinity can be measured by the dissociation constant (Kd). In certain embodiments, the antibodies provided herein have the following dissociation constants (Kd): equal to or less than about 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM (e.g., 10 -7 M or less, 10 -7 M to 10 -13 M, 10 -8 M to 10 -13 M or 10 -9 M to 10 -13 M).

[0366] Some aspects of vector assembly utilize chemical methods well known in the art. For example, vitamin E-PEG is prepared by Eastman Chemical, and biotin-PEG is prepared by many PEG producers such as Enzon, Nektar, and NOF Corporation. Methods for preparing PEG molecules with some vitamins and other therapeutic compounds attached thereto follow these methods and other chemical methods known in the art. For example, the conjugation of PEG to an oligonucleotide or related molecule occurs when PEG2-N-hydroxysuccinimide ester is coupled to the oligonucleotide via a 5' amine moiety. Several coupling methods are considered and include, for example, the coupling of NHS to an amine group (such as a lysine residue on a peptide), the coupling of maleimide to a thiol group (such as on a cysteine residue), the coupling of iodoacetyl to a thiol group, the coupling of pyridyldisulfide to a thiol group, the use of hydrazide for coupling to a carbohydrate group, the use of aldehyde for coupling to the N-terminus, or the coupling of tetrafluorophenyl ester known to react with primary or secondary amines. Other possible chemical coupling methods are known to those skilled in the art and may be substituted. For example, the conjugation using the coupling groups of the present invention can be carried out using the compositions and methods described in International Publication No. WO93 / 012145. See also U.S. Patent No. 7,803,777. The foregoing is incorporated herein by reference in its entirety.

[0367] Exemplary pharmaceutical formulations of the present invention include aqueous solutions, organic solutions, powder formulations, solid formulations, and mixed-phase formulations.

[0368] The pharmaceutical composition of the present invention comprises any compound of the present invention and its pharmaceutically acceptable salts together with any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical composition of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffering substances (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts), colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polypropylene-block polymers, polyethylene glycol and lanolin.

[0369] Pharmaceutically acceptable salts retain the desired biological activity of the therapeutic composition without toxic side effects. Examples of such salts are (a) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc. / and salts formed with organic acids such as, for example, acetic acid, trifluoroacetic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, etc.; (b) base addition salts or complexes formed with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc.; or base addition salts or complexes formed with organic cations formed from N,N'-dibenzylethylenediamine or ethylenediamine; or (c) a combination of (a) and (b), such as zinc tannate, etc.

[0370] The pharmaceutical composition of the present invention can be administered by subcutaneous, transdermal, oral, parenteral, inhalation, ophthalmic, topical, rectal, nasal, buccal (including sublingual), vaginal or implantable reservoir routes. The pharmaceutical composition of the present invention can contain any conventional, non-toxic, pharmaceutically acceptable carrier, adjuvant or vehicle. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0371] In some embodiments, pharmaceutical compositions are also contemplated that comprise as an active ingredient a therapeutic compound described herein or a pharmaceutically acceptable salt thereof in a mixture with a pharmaceutically acceptable non-toxic component. As described above, such compositions can be prepared for parenteral administration, particularly in the form of a liquid solution or suspension; for oral or buccal administration, particularly in the form of tablets or capsules; for intranasal administration, particularly in the form of a powder, nasal drops, evaporative solution or aerosol; for inhalation, particularly in the form of a liquid solution or dry powder with a broadly defined excipient; for transdermal administration, particularly in the form of a skin patch or microneedle patch; and for rectal or vaginal administration, particularly in the form of a suppository.

[0372] The compositions can be conveniently administered in unit dosage form and can be prepared by any methods well known in the pharmaceutical art, for example, as described in Remington’s Pharmaceutical Sciences, 17th Edition, Mack Publishing Co., Easton, PA (1985), which is incorporated herein by reference in its entirety. Preparations for parenteral administration may contain as excipients sterile water or saline, alkylene glycols such as propylene glycol, polyalkylene glycols such as polyethylene glycol, sugars, oils of vegetable origin, hydrogenated napthalene, serum albumin or other nanoparticles (such as those used in Abraxane TM , American Pharmaceutical Partners, Inc. Schaumburg, IL). For oral administration, the preparations can be enhanced by the addition of bile salts or acyl carnitine. Preparations for intranasal administration can be solid or a solution in an evaporative solvent such as hydrofluoroalkane and can contain excipients for stabilization such as sugars, surfactants, submicron anhydrous α-lactose or dextran, or can be an aqueous solution or oil solution for use in the form of nasal drops or metered sprays. For buccal administration, typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, etc.

[0373] Delivery of a modified therapeutic compound described herein to a subject over an extended period of time (e.g., a period lasting from one week to one year) can be achieved by a single administration of a controlled release system containing a sufficient amount of the active ingredient for the desired release period. For this purpose, various controlled release systems can be utilized, such as monolithic or reservoir-type microcapsules, depot implants, polymeric hydrogels, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, iontophoresis devices and alternative injectable dosage forms. Localization at the site where the active ingredient is desired to be delivered is an additional feature of some controlled release devices, which can prove beneficial for the treatment of certain disorders.

[0374] In certain embodiments for transdermal administration, delivery across the skin barrier can be enhanced using electrodes (e.g., iontophoresis), electroporation, or application of short high-voltage electrical pulses to the skin, radiofrequency, ultrasound (e.g., sonophoresis), microprojection (e.g., microneedles), jet injectors, thermal ablation, magnetophoresis, lasers, velocity, or optomechanical waves. The drug can be contained in a drug-in-adhesive, multilayer drug-in-adhesive, reservoir, matrix, or vapor patch, or a patchless technology can be utilized. Delivery across the skin barrier can also be enhanced using encapsulation, skin lipid fluidizers, or hollow or solid microstructured transdermal systems (MTS, such as those manufactured by 3M), jet injectors. Additives to formulations that assist therapeutic compounds through the skin include prodrugs, chemicals, surfactants, cell-penetrating peptides, permeation enhancers, encapsulation technologies, enzymes, enzyme inhibitors, gels, nanoparticles, and peptide or protein chaperones.

[0375] One form of controlled-release formulation contains a therapeutic compound or its salt dispersed or encapsulated in a slowly degradable, non-toxic, non-antigenic polymer such as co-poly(lactic / glycolic acid), as described in the pioneering work of Kent et al. in U.S. Patent No. 4,675,189, which is incorporated herein by reference. The compound or its salt can also be formulated in cholesterol or other lipid matrix pellets or silicone rubber matrix implants. Additional slow-release, depot implants, or injection formulations will be apparent to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker Inc., New York, 1978; and Controlled Release of Biologically Active Agents, RW Baker, John Wiley & Sons, New York, 1987. The foregoing is incorporated by reference in its entirety.

[0376] Another form of controlled-release formulation includes a solution of a biodegradable polymer such as co-poly(lactic / glycolic acid) or a block copolymer of lactic acid and PEG, in a biocompatible solvent, which is injected subcutaneously or intramuscularly to achieve a depot formulation. Mixing the therapeutic compounds described herein with such polymer formulations is suitable for achieving formulations with a very long duration of action.

[0377] When formulated for nasal administration, absorption through the nasal mucosa can be further enhanced by surfactants such as, for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycochenodeoxycholic acid, cyclodextrin, etc., in an amount ranging between about 0.1 to 15 weight percent, between about 0.5 to 4 weight percent, or in the range of about 2 weight percent. Another class of absorption enhancers that have shown better efficacy and reduced irritation are alkyl maltosides, such as tetradecyl maltoside (Arnold, JJ et al., 2004, J Pharm Sci 93:2205-13; Ahsan, F et al., 2001, Pharm Res 18:1742-46) and references therein, all of which are hereby incorporated by reference.

[0378] The pharmaceutical composition can be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oily suspension. The suspension can be formulated using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents according to techniques known in the art. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, non-volatile oils are conventionally used as solvents or suspending media. For this purpose, any mild non-volatile oil can be used, including synthetic mono- or di-glycerides of fatty acids. Fatty acids (such as oleic acid and its glyceride derivatives) can be used in the preparation of injectables, and natural pharmaceutically acceptable oils (such as olive oil or castor oil, especially in their polyoxylated forms) can also be used in the preparation of injectables. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersing agents, such as Ph.Helv or similar alcohols.

[0379] The pharmaceutical composition of the present invention can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricants, such as magnesium stearate, are usually also added. For oral administration in the form of capsules, useful diluents include lactose and dry corn starch. When an aqueous suspension is orally administered, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents can be added.

[0380] The pharmaceutical compositions of the present invention can also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients which are solid at room temperature but liquid at rectal temperature and will thus melt in the rectum to release the active ingredient. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

[0381] Local administration of the pharmaceutical compositions of the present invention is particularly useful when the desired treatment involves an area or organ that can be readily accessed by topical application. For topical application to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for topical application of the compounds of the present invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compounds, emulsifying wax, and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. The pharmaceutical compositions of the present invention can also be locally administered to the lower intestine by rectal suppository formulations or in suitable enema formulations. Transdermal patches are also included in the present invention.

[0382] The pharmaceutical compositions of the present invention can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0383] When formulated for administration by inhalation, a variety of formulations offer advantages. Adsorbing the therapeutic compound onto a solid that is readily dispersible, such as diketopiperazine (e.g., Technosphere particles (Pfutzner, A and Forst, T, 2005, Expert Opin Drug Deliv 2:1097 - 1106) or similar structures provides formulations that result in rapid initial uptake of the therapeutic compound. Lyophilized powders, particularly glassy particles, containing the therapeutic compound and excipients can be used for delivery to the lungs with good bioavailability.

[0384] For example, see (Inhaled Insulin, Pfizer, Inc. and Aventis Pharmaceuticals Inc.) and (Inhaled Insulin, Mannkind, Corp.).

[0385] Dose levels of the active ingredient compound between about 0.1 to about 300 μg / kg body weight / day, preferably between 0.5 to about 50 μg / kg body weight / day can be used for the prevention and treatment of diseases. Such administration can be used as a chronic or acute therapy. The amount of the drug that can be combined with a carrier to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. Usual formulations will contain from about 0.01% to about 1% of the active compound (w / w). Preferably, such formulations contain from about 0.02% to about 1% of the active compound (w / w).

[0386] When the condition of the patient improves, a maintenance dose of the compound, composition or combination of the present invention can be administered if needed. Subsequently, the dosage or the frequency of administration or both can be reduced to the level at which the improved condition is maintained, depending on the change in symptoms, and treatment should be discontinued when the symptoms have been alleviated to the desired level. However, the patient may request intermittent treatment on a long-term basis at any recurrence of the symptoms of the disease.

[0387] As will be appreciated by those skilled in the art, lower or higher doses than those described above may be required. The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound used, age, body weight, general health status, gender, diet, time of administration, rate of excretion, drug combination, severity and course of the infection, the patient's disposition to the infection and the judgment of the treating physician.

[0388] The carrier-drug conjugates described herein offer advantages to drug manufacturers and patients over unmodified drugs. Specifically, the carrier-drug conjugates or formulations will be more potent, longer-lasting, and require smaller and less frequent dosing. This translates into reduced healthcare costs for the patient and a more convenient drug administration schedule for the patient. Carrier-drug conjugates can also provide subcutaneous or transdermal administration routes as alternatives to intravenous injection. These routes can be self-administered by the patient and thus improve patient compliance.

[0389] In another aspect of the present invention, the level of DBP can be increased as part of a carrier-drug therapy. It has been reported that estrogen can increase the level of DBP (Speeckaert et al., Clinica Chimica Acta 371:33). It is contemplated herein that the level of DBP can be increased by administering estrogen to more effectively deliver the carrier-drug conjugate.

[0390] In another aspect of the present invention, it is contemplated that the carrier can be used for transdermal delivery of drugs. Since DBP usually transports UV-activated vitamin D in the vicinity of the skin surface, the use of a transdermal delivery system with a carrier becomes feasible.

[0391] In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the invention in any way. Specifically, the compositions and methods disclosed herein work with all non-hormonal forms of vitamin D, including its homologues, analogs, and metabolites. This includes vitamin D as used in the following examples. Example

[0392] Example 1: Preparation of PTH coupled to non-hormonal vitamin D at position C3

[0393] PTH(1-34) was synthesized with a C-terminal cysteine ​​residue and a discrete 1.6 kDa PEG using a maleimide group to generate a thioether bond (PTH-C-PEG 1.6K -(3)-VitD). See WO2016 / 065042, which is incorporated herein by reference in its entirety.

[0394] The modified PTH(1-34) compounds were prepared using the following structures:

[0395]

[0396] EXT601 was prepared as described in WO2016 / 065042, which is incorporated herein by reference in its entirety. EXT607 was prepared using the same method described below for EXT608, except that R on EXT607 was OH, while R on EXT608 was NH2. R=NH2 is superior to R=OH in preventing impurity formation during solid phase synthesis of peptides in which the C-terminal amino acid is cysteine.

[0397] The general method for the synthesis of EXT608 is shown in Figure 1 The conversion of 25OH-vitamin D (25-hydroxycholecalciferol, compound 1) to "VitD-NH2" (compound 3) was carried out in two steps, namely by functionalizing the 3'-hydroxyl group of vitamin D with acrylonitrile and then reducing it with lithium aluminum hydride (Ray et al., 1991). The resulting amine (3) was then reacted with heterobifunctional TFP-PEG obtained from Quanta BioDesign, Ltd. (Plain City, OH, USA, catalog number 10555). 36Coupling of the tetrafluorophenyl (TFP) ester of the maleimide linker (4) to obtain VitD-PEG-maleimide (5). The PTH(1-34)-cys-NH2 peptide was synthesized by solid-phase peptide technology. It was then fully deprotected and separated from the resin to generate the crude PTH(1-34)-cys-NH2 peptide. The crude peptide was purified by preparative HPLC, and the purified PTH(1-34)-cys-NH2 was separated by lyophilization. The final synthetic step - conjugation of the purified peptide with VitD-PEG-maleimide was carried out in a solution-phase reaction. The fully assembled EXT608 was then purified using preparative reverse-phase HPLC to remove unreacted PTH(1-34)-cys-NH2, VitD-PEG-maleimide, and other impurities. This allowed for the final isolation of EXT608 upon drying by lyophilization. The detailed steps are as follows:

[0398] VitD-PEG 36 Synthesis of -maleimide (Compound 5), Method 1. VitD-NH2 (Compound 3) was prepared and described as "Compound Vd" in WO2016 / 065042, which is hereby incorporated by reference in its entirety.

[0399]

[0400] Under nitrogen, at 4 °C, 2,6-dimethylpyridine (2.5 equivalents) was added dropwise to a mixture of Compound 3 (1 equivalent) dissolved in anhydrous dichloromethane, and the mixture was stirred for 15 min. Compound 4 (Quanta BioDesign, Plain City, OH, USA, catalog number 10555, 0.8 equivalent) dissolved in 30 mL of dichloromethane was slowly added to the mixture by syringe. The resulting reaction mixture was stirred at room temperature under an inert atmosphere and monitored by thin-layer chromatography (TLC). After 4 hours, the reaction mixture was diluted with dichloromethane and washed with 10% aqueous citric acid, saturated aqueous sodium bicarbonate, and brine. The organic layer was dried over anhydrous Na2SO4, filtered, and concentrated at 30 °C to give a crude product. The crude mixture was purified by silica gel Gold ISCO flash liquid chromatography. The column was eluted with 1-10% MeOH / chloroform (1% ammonium hydroxide). The fractions containing the purified product were combined and evaporated in vacuo to give Compound 5 as a pale yellow solid (R f 0.3 silica gel, dichloromethane solution of 10% methanol), with a yield of 62% and a purity of 68% as measured by HPLC / TLC.

[0401] VitD-PEG 36 Synthesis of -maleimide (Compound 5), Method 2. In an attempt to improve Compound 5 (Mal-PEG 36-VitD), it was found that VitD-PEG prepared by Method 1 36 -maleimide contained impurities formed during lyophilization. Although the exact structures of these impurities were not determined, NMR analysis indicated that the double bonds of maleimide and vitamin D participated in unwanted reactions during lyophilization. Therefore, the crude reaction mixtures of Compounds 3 and 4 prepared by Method 1 were purified by preparative HPLC using a water / acetonitrile mobile phase. The pooled fractions were directly used for the conjugation reaction with PTH(1-34)-cys-NH2. Since Compound 5 was not isolated as a solid, the concentration of Compound 5 was obtained by calculating the concentration using optical absorbance measurements with the extinction coefficient. The purity of Compound 5 using this method was 94.1%.

[0402] Peptide synthesis. PTH(1-34)-cys-NH2 was prepared using the Fmoc (9-fluorenylmethoxycarbonyl) solid-phase peptide synthesis (SPPS) strategy. Ramage resin (tricyclic amide linker) was used during the preparation because it is particularly acid-sensitive and thus very suitable for the preparation of fully protected peptide amides by Fmoc chemistry. With this resin, the C-terminal cysteine-amide (R = NH2) was formed, which is less prone to unwanted side reactions than the C-terminal cysteine-acid (R = OH). The peptide was separated from the resin by treating the peptide resin with the strong acid trifluoroacetic acid (TFA), accompanied by cleavage of the side-chain protecting groups. Scavengers [1,2-ethanedithiol (EDT), triisopropylsilane (TIS), and water] were added to capture the reactive cations during cleavage and avoid alkylation of the side-chain functional groups, resulting in a higher-quality crude peptide. Ammonium iodide (NH4I) was added to the cleavage mixture because the peptide sequence contains a thioether structure. Finally, ascorbic acid was used to neutralize NH4I. These reagents were used to ensure the complete reduction of potentially present methionine sulfoxide in the product. After the cleavage operation, the crude peptide was precipitated using cooled isopropyl ether (IPE) and filtered using a sintered glass funnel. The resulting mass was washed with IPE and dried in a vacuum oven at room temperature.

[0403] Peptide Purification and Lyophilization: The crude product was purified by two-dimensional preparative HPLC on a reverse-phase column, eluted with an acetonitrile (ACN) gradient, and UV detected at 230 nm. For the first preparative HPLC step, a phosphoric acid (H3PO4) buffer system was used as the mobile phase. The C8 reverse-phase resin was the stationary phase. Individual collected fractions were analyzed by ultra-performance liquid chromatography (UPLC, Waters Corporation, Milford, MA) and pooled according to the purity acceptance criteria. Fractions with ≥90% purity were identified as the main pool. The pooled main fractions were diluted with water to reduce their ACN concentration and further processed in the second preparative HPLC step. A trifluoroacetic acid (TFA) buffer system was used for the second purification step by preparative HPLC. The C8 reverse-phase resin was the stationary phase. Individual collected fractions were analyzed by UPLC and pooled according to the purity acceptance criteria. Fractions with ≥95% purity were identified as the main pool. The main product pool from the TFA purification step was filtered through a 0.45 μm membrane filter and lyophilized.

[0404] EXT608 Conjugation Reaction for Preparing Carbonate (Method A): The VitD-PEG-maleimide conjugate reagent (Compound 5) prepared using Method 1 was dissolved in dimethyl sulfoxide (DMSO) and diluted with a Tris buffer solution containing ethylenediaminetetraacetic acid (EDTA). The lyophilized peptide from the above step was dissolved in Tris buffer (pH 7.4). These solutions were mixed together and the reaction was monitored using analytical HPLC. The reaction was stopped by adding a 2% acetic acid solution and it was loaded onto a YMC C8 column equilibrated with an aqueous solution of 10 mM ammonium bicarbonate (NH4HCO3, pH 8) buffer. Product elution was achieved using a gradient of 10 mM NH4HCO3 buffer and acetonitrile, and all fractions meeting the established criteria were collected and pooled together. The purified solution was filtered through a 0.45 μm membrane filtration system. After the purification stage was completed, the solution was lyophilized to obtain EXT608 as the carbonate. The final product was analyzed by UPLC and LC-MS and was determined to have a purity of 80.05%. The major impurity (7.5%) had a molecular weight 18 mass units (M + 18) higher than EXT608 and was assumed to be due to the hydrolysis ring-opening of the thiosuccinimide ring (formed by the addition of cysteine to maleimide). Thus, in some embodiments, the peptide / VitD-PEG-Mal coupling and purification were carried out at a lower pH to prevent thiosuccinimide hydrolysis during reaction, purification, and lyophilization (see below section). However, the ring-opening did not change the activity of the compound.

[0405] EXT608 Conjugation Reaction for Preparing Acetate (Method B): The VitD-PEG-maleimide conjugate reagent (Compound 5) was prepared using Method 2, and the resulting pooled HPLC-purified fractions were added to PTH(1-34)-cys-amide dissolved in 0.5 M ammonium acetate buffer at pH = 6.0. The conjugated peptide solution was purified on a reverse-phase column by preparative HPLC using a pH 5.5 NH4OAc system with UV detection at 220 nm. After elution with an ACN gradient, individual collected fractions were analyzed by UPLC and pooled according to the purity acceptance criteria (≥90%). The main pool was combined and diluted with purified water for lyophilization. The final product was analyzed by UPLC and determined to have a purity of 90.8% with undetectable levels of the M+18 impurity. Thus, lowering the reaction pH from 7.4 in Method A to 6.0 and the purification pH from 8.0 in Method A to 5.5 eliminated the major impurity hypothesized to be generated by thiosuccinimide hydrolysis.

[0406] Example 2: Discrete PEG Linkers are Superior to Polydisperse PEG Linkers

[0407] To provide uniformity for patient administration, maximize preparation yields, and improve the ability to monitor vitamin D PTH conjugates and related impurities using techniques such as UPLC, mass spectrometry, and LC-MS during preparation and treatment, it is important to use discrete PEG chain lengths.

[0408] EXT601 and EXT608 were analyzed by UPLC. The following UPLC method was developed for analyzing vitamin D-modified PTH compounds. An ACQUITY UPLC Peptide CSH C18 column (2.1 x 150 mm, 1.7 μm, 130 Å; Waters Acquity, Milford, MA, catalog number 186006938) was used with a column temperature of 40 °C, a flow rate of 0.4 ml / min, an injection volume of 3 μl, and a detection wavelength of 220 nm. Mobile phase A consisted of 80% water / 20% ACN / 0.1% TFA, and mobile phase B consisted of 20% water / 80% ACN / 0.085% TFA. The following gradient of mobile phase A (%) and mobile phase B (%) was used:

[0409] Time (min) ％A ％B Initial 70 30 15.0 70 30 25.0 58 42 30.0 50 50 36.0 20 80 40.0 20 80

[0410] The UPLC trace of EXT601 is shown in Figure 2In it, the polydisperse length of the PEG linker results in a very wide UPLC peak with a width at the baseline of approximately 3 minutes. Any impurities are masked by the broad EXT601 peak and the fact that for any change in PEG length, any peptide-related or vitamin D-related impurities will exist as multiple peaks. In contrast, the UPLC trace of EXT608 containing a single length PEG linker (x = 36) Figure 3 ) shows narrow peaks with a width at the baseline of approximately 0.5 minutes. The impurities present are well separated from the EXT608 peak. This aids in the further characterization, separation, and determination of the impurities. Additionally, the discrete yields of the therapeutic VitD-PEG 36 -PTH provide important properties for more effectively purifying, quantifying, and monitoring the molecule during preparation and patient treatment.

[0411] Analysis of EXT601 and EXT608 by MALDI-TOF mass spectrometry: EXT601 and EXT608 were analyzed in positive ion mode by MALDI-TOF (matrix-assisted laser desorption / ionization-time of flight) mass spectrometry. The MALDI-TOF analysis of EXT601 is shown in Figure 4 . The polydisperse length of the PEG linker results in a very wide peak, and the distinguishable signals above the baseline are between approximately 6400 - 7650 g / mol. This corresponds to a PEG x linker with x = 34 to 62 repeating units. The average molecular weight is approximately 6880 g / mol, corresponding to a PEG 45 linker. The MALDI-TOF analysis of EXT608 is shown in Figure 5 . Compared to EXT601, EXT608 has a single peak with a molecular weight of 6478 g / mol. For both compounds, the signal at approximately 4215 g / mol is caused by the fragmentation of the intact parent compound during ionization in the mass spectrometer (i.e., in-source fragmentation). Compared to EXT601, EXT608 has a higher signal-to-noise ratio and a higher relative abundance compared to the in-source generated fragments.

[0412] Example 3: PTH(1-34), EXT601, EXT607, and EXT608 equivalently activate the parathyroid hormone receptor 1 (PTHR1).

[0413] Using cells expressing the Gq-coupled receptor A mammalian cell line (DiscoverX, Fremont, CA, USA, now part of Eurofins Discovery, catalog number 86 - 0030P - 2212AG) was used to measure the activity of EXT607 and EXT608 against human parathyroid receptor 1 (PTHR1, also known as PTH1R) compared to PTH(1 - 34) in agonist mode by a cell - based calcium flux assay. EXT607 and EXT608 were diluted to 100 μM in phosphate - buffered saline. The PTH(1 - 34) control was provided by DiscoverX. Cells expressing human PTHR1 were treated repeatedly with 10 concentrations of the test articles, with the highest concentration being 1 μM. After exposure to a calcium - sensitive dye loaded into the cells, intracellular calcium release was measured on a FLIPR Tetra for 2 minutes. The percentage of efficacy was calculated as follows:

[0414] Percentage of efficacy = 100% x (average RFU of test sample - average RFU of vehicle control)

[0415] / (average MAX RFU of control ligand - average RFU of vehicle control)

[0416] The EC50 (half - maximal effective concentration) value was determined from a plot of percentage of efficacy versus agonist concentration ( Figure 6 ). For PTH(1 - 34), the EC50 value was 33 nM, for EXT607 it was 21 nM, and for EXT608 it was 22 nM. As determined in WO2016 / 065042 (incorporated herein by reference in its entirety), for EXT601, the EC50 value was 14 nM. Within the experimental error of the assay, the compounds were equivalent. Thus, conjugation of the vitamin D moiety to PTH(1 - 34) does not interfere with PTHR1 activation. EXT601, EXT607, and EXT608 have approximately equal activity against PTHR1.

[0417] Example 4: EXT607 and EXT608 bind to vitamin D - binding protein (DBP) with equivalent affinities.

[0418] EXT607 and EXT608 were biotinylated using the EZ-Link Sulfo-NHS Biotin No-Weigh Kit (Thermo Fisher Scientific, Waltham, MA, catalog number PIA39256). 1 mmol of biotin was reacted with 1 mmol of conjugate for 1 hour at room temperature. Unbound biotin was separated from the biotinylated conjugate using a PD-10 desalting column (GE Lifesciences / Cytiva, Marlborough, MA, catalog number 17085101). The biotinylation ratio was quantified using the Pierce Biotin Quantification Kit (Thermo Fisher Scientific, Waltham, MA, catalog number PI28005). DBP was purchased from Athens Research and Technology (Athens, GA, catalog number 16-16-070307).

[0419] Affinity measurements were collected on Octet Red96 using pre-hydrated dip and read streptavidin (SA) biosensors (ForteBio, Fremont, CA, catalog number 18-5021). Reagents were diluted into phosphate buffered saline (PBS) with 0.02% Tween 20 in a black 96-well plate. Biotinylated EXT607 or EXT608 was prepared at a biotin concentration of 50 μg / mL and loaded onto the SA tip for 2 minutes. Nonspecific binding of DBP to the SA biosensor was measured using a negative load control containing only buffer. DBP titrations ranging from 0 to 75 μM were prepared. DBP was associated with the loaded tip for 5 minutes and dissociated from the buffer solution for 10 minutes. Binding association and dissociation curves and steady-state curve fitting were generated using ForteBio Octet data analysis software ( Figure 7 and 8 ), and the binding equilibrium constant (KD) was obtained from the steady-state data. Both EXT607 and EXT608 had a maximum response of 1.79 nm displacement. The KD of EXT607 for DBP D 8.1μM+2.5μM. K of EXT608 D It is 5.2μM+1.1μM.

[0420] Example 5: PEG36 is the optimal chain length

[0421] Optimization of PEG chain length is important to maximize the solubility and PTHR1 receptor activity of the vitamin D-PTH conjugate.

[0422] Synthesis of EXT611 (PTH-cys-PEG12-VitD) and EXT606 (PTH-cys-PEG24-VitD): React 1 molar equivalent of Mal-PEG12-NHS (Quanta BioDesign #10284) or Mal-PEG24-TFP (Quanta BioDesign #10554) with 1.1 equivalents of VitD-NH2 (Compound 3) in DMSO at room temperature for 30 minutes, where NHS is the amine-reactive N-hydroxysuccinimide ester. Quench the reaction by adding an equal volume of 0.1 M ammonium acetate at pH = 6.8. Add PTH(1-34)-cys-NH2 (1.0 equivalent, in water) and MES at 0.5 M pH = 6.1 until the final concentration is 50 mM, and allow the reaction to proceed at room temperature for 30 minutes. Purify the reaction by HPLC using an Xselect CSH Phenyl Hexyl column with a mobile phase consisting of 0.1 M ammonium acetate at pH = 5 and acetonitrile. Then lyophilize it to produce the acetate form of the compound. Alternatively, purify the compound using 0.1 M ammonium carbonate at pH = 8 as the aqueous component of the mobile phase to produce the carbonate form of the compound.

[0423] Synthesis of EXT615 (PTH-PEG5kDa-VitD), EXT616 (PTH-PEG10kDa-VitD) and EXT617 (PTH-PEG20kDa-VitD): 1 molar equivalent of Mal-PEG5kDa-NHS (Nanosoft Biotechnology, Lewisville, NC, catalog number 2597-5000, "Mal-PEG5000-SCM"), Mal-PEG10kDa-NHS (Nanosoft Biotechnology, catalog number 2597-10K, "Mal-PEG10K-SCM") or Mal-PEG20kDa-NHS (Advanced BioChemicals, Lawrenceville, GA, catalog number HEP0405, "SC-PEG-MAL, 20k") dissolved in DMF (5kDa and 10kDa) or MeCN (20kDa) was reacted with 1.1 equivalents of VitD-NH2 (Compound 5) in DMSO at room temperature for 30 minutes. The reaction was quenched by adding an equal volume of 0.1 M ammonium acetate at pH = 6.8. The Mal-PEG-VitD intermediate was purified by HPLC using an Xselect CSH phenylhexyl column with a mobile phase consisting of 0.1 M ammonium acetate at pH = 5 and acetonitrile, lyophilized, and dissolved in DMF (5kDa) or MeCN (10kDa and 20kDa). PTH(1-34)-cys-NH2 (1.0 equivalent, in water) and MES at 0.5 M pH = 6.1 were added until a final concentration of 50 mM, and the reaction was allowed to proceed at room temperature for 30 minutes. The PTH-PEG-VitD product was purified by HPLC as above and lyophilized to yield the acetate or carbonate form of the compound.

[0424] Effect of PEG length on PTHR1 receptor activity: The above compounds and EXT608 (all in acetate form) were dissolved in PBS and the concentration was determined using an extinction coefficient of 18,600 M-1 cm-1 at 280 nm. The concentration of each compound was adjusted to 100 μM and submitted to DiscoverX (Fremont, CA, part of Eurofins Scientific), and tested in agonist mode using a calcium mobilization assay in assays with mammalian cells expressing the human PTH1 receptor (PTHR1) as described in Example 3 above. Cells expressing human PTHR1 were treated repeatedly with 10 concentrations of the test article, with the highest concentration being 1 μM, and the EC50 value was determined from the plot of percentage efficacy versus agonist concentration ( Figure 9 ) as shown in Table 1:

[0425] Table 1. Relationship between PTHR1 EC50 value and PEG length.

[0426]

[0427] In the range of x = 12 to ~227 (528 to 10,000 Da.), the EC50 value is independent of the PEGx linker length. However, the PEG20kDa linker (EXT617) shows an approximately 7-fold higher EC50 value (221.3 nM). Without being bound by theory, this increase in the agonist concentration required to achieve 50% maximum efficacy may be due to the large size of the 20kDa PEG linker hindering the binding of the much smaller conjugated PTH peptide (~4 kDa) to PTHR1.

[0428] PEG length and counterion affect solubility: The solubility of the acetate and carbonate forms of each compound was determined as follows. For EXT611, EXT606, and EXT608, phosphate buffered saline (PBS) was added to each compound to form a saturated solution. The undissolved compound was removed by centrifugation. The concentration of the dissolved compound was determined by measuring the absorbance at 280 nm using an extinction coefficient of 18,600 M -1 cm -1 . For EXT615, EXT616, and EXT617, the solubility was actually limited by the high viscosity imparted by the large PEG moiety. Pharmaceutical formulations with high viscosity are difficult to handle and require large needle hole sizes, which result in increased pain at the injection site. PBS was gradually added to EXT615, EXT616, and EXT617 until a solution was obtained that could be successfully pipetted through the tip of a pipette with a hole of approximately 1 mm diameter (enlarged by removing the end with a razor blade). The concentration of the dissolved compound was determined by measuring the absorbance at 280 nm using an extinction coefficient of 18,600 M -1 cm -1 . Surprisingly, for each compound, the acetate has a higher solubility than the carbonate (Table 2). For EXT608, the solubility of the acetate is 17.6 times that of the carbonate. The maximum solubility (9.67 mM) was achieved in the case of EXT608 acetate. Increasing the PEG length leads to an increase in solubility, but this is ultimately limited by the high viscosity semi-solid colloidal solution obtained with ≥5kDa PEG linkers.

[0429] Table 2. Relationship between PBS solubility and PEG length and counterion

[0430]

[0431] Example 6: Single-dose pharmacokinetics of EXT601 in male rats:

[0432] In the single-dose pharmacokinetic study, EXT601 was formulated in PBS and male rats (n = 3) were administered subcutaneously at a volume of 1 ml / kg and a dose of 157 μg / kg. Blood was collected via jugular vein cannulation at 0.5, 1, 2, 4, 6, 8, 24, 32, 48, and 56 hours and processed into plasma. The plasma level of EXT601 was determined using the Immutopics high-sensitivity human parathyroid hormone PTH(1-34) ELISA kit (Catalog No.: 60-3900, Quidel Corporation, San Diego, CA). The relationship between the group-averaged EXT601 level and time is shown in Figure 10 as follows.

[0433] Pharmacokinetic parameters were determined by non-compartmental analysis using Kinetica software (Thermo Fisher Scientific, Waltham, MA) based on the data of plasma concentration versus time. As described in Table 3, EXT601 reached a C max of 64.4 ng / ml at a T max of 2 hours. EXT601 had an elimination half-life of 13.5 hours and a low clearance rate of 20.1 ml / h / kg.

[0434] Table 3: Pharmacokinetic parameters of a single subcutaneous dose of EXT601 in rats

[0435] PK Parameters (unit) Results <![CDATA[t 1 / 2 (h)]]> 13.5 <![CDATA[T max (h)]]> 2.0 <![CDATA[C max (ng / mL)]]> 64.4 <![CDATA[AUC last (h·ng / mL)]]> 665 <![CDATA[AUC inf (h·ng / mL)]]> 700 Cl (mL / h / kg) 20.1 <![CDATA[V Z (mL / kg)]]> 4,376 MRT (h) 16.5

[0436] t 1 / 2 , terminal half-life; Tmax, time to the maximum observed concentration; Cmax, maximum observed concentration; AUC last , area under the concentration-time curve from time = 0 to the time point of the last measurable concentration; AUC inf , area under the concentration-time curve from time = 0 to infinity; Cl, clearance rate; V Z , apparent volume of distribution during the terminal phase; MRT, mean residence time.

[0437] Example 7: Single-dose pharmacokinetics of EXT607 in male rats:

[0438] In the single-dose pharmacokinetic study, EXT607 was formulated in PBS and administered to male rat groups (n = 3) at a volume of 1 ml / kg as follows: Group 1, 100 μg / kg intravenously; Group 2, 30 μg / kg subcutaneously; Group 3, 100 μg / kg subcutaneously; Group 4, 300 μg / kg subcutaneously. Blood (0.25 mL) was collected via jugular vein cannulation, transferred to tubes containing K2EDTA, and kept on wet ice until processed into plasma. Blood samples were collected at 0 (before dosing), 0.083 (IV only), 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, and 72 h post-dose. Plasma levels of EXT607 were determined at Extend Biosciences using the Immutopics high-sensitivity human parathyroid hormone PTH(1-34) ELISA kit (Catalog No.: 60-3900, Quidel Corporation, San Diego, CA). The relationship between the group mean EXT607 levels and time is shown in Figure 11 as follows.

[0439] Pharmacokinetic parameters were determined from the plasma concentration-versus-time data (Table 4) using Version 6.3 (Certara L.P. (Pharsight), St. Louis, MO) by non-compartmental analysis with uniform weighting. The concentration-versus-time data were analyzed using an IV bolus or extravascular administration model. The optimal fit Lambda z range was used. When compared to intravenous injection, EXT607 had a subcutaneous bioavailability (%F) of 10 - 13% based on AUC in male rats. C max was reached within 0.5 to 3 h after SC administration of 30, 100, or 300 μg / kg, and then EXT607 was slowly eliminated with a clearance of 22.9 to 25.9 ml / h / kg, a half-life of 7 to 15 h, and an average residence time of 13.6 to 15.9 h.

[0440] Table 4: Pharmacokinetic parameters of single-dose EXT607 in rats

[0441]

[0442] IV, intravenous; SC, subcutaneous; N / A, not applicable; t 1 / 2 , terminal half-life; Tmax, time of maximum observed concentration; C0, calculated initial concentration; Cmax, maximum observed concentration; AUC last , area under the concentration-time curve from time = 0 to the time point of the last measurable concentration; AUC inf, area under the concentration-time curve from time = 0 to infinity; Cl, clearance; V Z , apparent volume of distribution during the terminal period; MRT, mean residence time.

[0443] Example 8: Single-Dose Pharmacokinetics of EXT607 in Male Monkeys:

[0444] In a single-dose pharmacokinetic study, EXT607 was formulated in PBS and administered to groups (n = 3) of male cynomolgus monkeys, each weighing approximately 4 kg, at a volume of 1 ml / kg as follows: Group 1, 20 μg / kg intravenously; Group 2, 7 μg / kg subcutaneously; Group 3, 20 μg / kg subcutaneously; Group 4, 70 μg / kg subcutaneously. Blood (1.0 mL) was collected via jugular vein cannulation, transferred to tubes containing K2EDTA, and kept on wet ice until processed into plasma. Blood samples were collected at 0 (before dose), 0.083 (IV only), 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60, and 72 h after dose. Plasma levels of EXT607 were determined using the Immutopics high-sensitivity human parathyroid hormone PTH(1-34) ELISA kit (Quidel Corp., San Diego, CA Catalog No. 60-3900). The relationship between the group-averaged EXT607 levels and time is shown in Figure 12 .

[0445] Using Version 6.3 (Certara L.P. (Pharsight), St. Louis, MO), pharmacokinetic parameters were determined from plasma concentration-versus-time data (Table 5) by non-compartmental analysis with uniform weighting. Concentration-versus-time data were analyzed using an IV bolus or extravascular administration model. The best-fit Lambda z range was used. EXT607 had an elimination t 1 / 2 . When delivered subcutaneously, the bioavailability was 45 - 54%, and the clearance was low (6.63 - 7.28 ml / h / kg).

[0446] Dose linearity was observed for AUC (dose adjustment ratio 0.95:0.85:1.00), but not for Cmax (0.28:0.55:1.00). This is because the dose affects the subcutaneous absorption rate and thus affects Cmax. At low doses, the subcutaneous absorption rate is slower than at high doses, resulting in a lower Cmax and an overall flatter PK curve. The slower subcutaneous absorption rate at low doses is also reflected in a higher mean residence time (MRT), which was 29.46 h at low doses, 23.26 h at medium doses, and 17.51 h at high doses.

[0447] Table 5: Pharmacokinetic Parameters of Single-Dose EXT607 in Cynomolgus Monkeys

[0448]

[0449] Example 9: Repeated-Dose Pharmacokinetics of EXT608 in Male and Female Rats

[0450] 21 - day Experiment: EXT608 was formulated in 20 mM sodium acetate buffer at pH = 5.5 with 0.8% sodium chloride. In the repeated-dose toxicokinetics study, a group of 18 rats (9 males + 9 females) was dosed subcutaneously daily with 14.2 μg / kg or 70 μg / kg of EXT608 at a volume of 0.5 ml / kg. On day 21, blood samples (∼0.5 ml) were collected at t = 0 (pre-dose), 0.5, 1, 3, 8, 24, 32, 48, and 72 h, with each time point collected from one of three groups of 3 male and 3 female animals. Each blood sample was collected from the jugular vein into a tube containing K2EDTA and inverted manually several times. Blood samples were kept on wet ice awaiting centrifugation. Samples were centrifuged within 1 h of collection (∼2700 g, ∼10 min, ∼5 °C). Plasma was recovered and evenly divided into replicate aliquots and frozen.

[0451] 90 - day Experiment: EXT608 was formulated in 20 mM sodium acetate buffer at pH = 5.5 with 0.8% sodium chloride and 0.1% polysorbate 80. In the repeated-dose toxicokinetics study, a group of 18 rats (9 males + 9 females) was dosed subcutaneously daily with 1, 3, or 10 μg / kg of EXT608 at a volume of 0.5 ml / kg. On day 90, blood samples (∼0.5 ml) were collected at t = 0 (pre-dose), 0.5, 1, 3, 8, 24, 32, 48, and 72 h, with each time point collected from one of three groups of 3 male and 3 female animals. Plasma was prepared as described above for the 21-day experiment.

[0452] Pharmacokinetic Analysis: Plasma levels of EXT608 were determined using the Immutopics high-sensitivity human parathyroid hormone PTH(1-34) ELISA kit (Quidel Corporation, San Diego, CA catalog number 60-3900). The relationship between the group-averaged EXT608 levels and time after the last dose administration following 21 days of daily dosing (14.2 and 70 μg / kg) or 90 days of daily dosing (1, 3, and 10 μg / kg) is shown in Figure 13Surprisingly, the PK curves vary with the dose level. Higher doses show more prominent peaks, while lower doses have flatter curves and less distinct peaks.

[0453] Using Version 6.4 (Certara L.P. (Pharsight), Princeton, NJ) determined the pharmacokinetic parameters from plasma concentration-time data (Table 6) by a non-compartmental extravascular administration model with uniform weighting. The pharmacokinetic analysis was performed on data collected from t = 0 to 72 h after the last administered dose. The t 1 / 2 values ranged from 10.4 to 13.8 h. To quantify the dose-dependent changes in the observed PK curves, the C max and C min values were determined for the time period between 0 - 24 h (which is the normal dosing cycle). The C max / C min ratio represents the peak-to-trough ratio of the EXT608 concentration observed during the dosing cycle. This ratio decreased from 30 at the highest dose (70 μg / kg) to 8.0 at 14.2 μg / kg, 4.6 at 10 μg / kg, and 1.8 at 3 μg / kg and 1 μg / kg. Without being bound by theory, the low peak-to-trough ratio observed at low doses may be due to the slower rate of absorption of EXT608 from the subcutaneous space into the circulation. A low peak-to-trough ratio is desirable in many therapeutic applications, including parathyroid hormone replacement therapy. It allows for quantitative administration at levels close to the ideal effective dose, without being too high (where toxicity may be observed) or too low (where efficacy is reduced).

[0454] Table 6: PK parameters of EXT608 after repeated daily SC dosing in rats

[0455]

[0456] t 1 / 2 , terminal half-life; T max , time to maximum observed concentration; AUC 0-24h , area under the concentration-time curve from time = 0 to 24 h; AUC last , area under the concentration-time curve from time = 0 to the time point of the last measurable concentration; C max , maximum observed concentration; C min , minimum observed concentration from time = 0 to 24 h; C max / C min (0 - 24 h), the ratio of C max / C min .

[0457] Example 10: Repeated Dose Pharmacokinetics of EXT608 in Male and Female Cynomolgus Monkeys

[0458] 21 - day Experiment: EXT608 was formulated in 20 mM sodium acetate buffer at pH = 5.5 with 0.8% sodium chloride. In the repeated dose toxicokinetics study, a group of 4 cynomolgus monkeys (2 males + 2 females, each weighing approximately 2.5 kg) was dosed subcutaneously every other day with 1.4, 7 or 20 μg / kg of EXT608 at a volume of 0.05 ml / kg. On the last day of dosing - day 21, blood samples (~1.0 ml) were collected at t = 0 (pre-dose), 0.5, 1, 3, 6, 12, 24, 48 and 72 h. Each blood sample was collected from the saphenous vein into a tube containing K2EDTA and centrifuged within 1 hour of collection (~2700 g, ~10 min, ~5 °C). Plasma was recovered and evenly divided into duplicate aliquots and frozen.

[0459] 90 - day Experiment: EXT608 was formulated in 10 mM sodium acetate buffer at pH = 5.5 with 0.8% sodium chloride and 0.1% polysorbate 80. In the repeated dose toxicokinetics study, a group of 6 cynomolgus monkeys (3 males + 3 females) was dosed subcutaneously every other day with 0.7 or 2 μg / kg of EXT608 at a volume of 0.05 ml / kg. On the last day of dosing - day 89, blood samples (~1.0 ml) were collected at t = 0 (pre-dose), 0.5, 1, 3, 6, 12, 24, 48 and 72 h. Plasma was prepared as described above for the 21-day experiment.

[0460] Pharmacokinetic Analysis : Plasma levels of EXT608 were determined using the Immutopics high sensitivity human parathyroid hormone PTH(1-34) ELISA kit (Quidel Corporation, San Diego, CA, catalog number 60-3900). The relationship between the mean EXT608 levels in the groups after the last dose administration following dosing every other day for 21 days (20, 7 and 1.4 μg / kg) or 89 days (2 and 0.7 μg / kg) and time is shown in Figure 14 Figure. Surprisingly, the PK curves varied with the dose level. Higher doses showed more prominent peaks, while lower doses had flatter curves and less distinct peaks.

[0461] Using Version 6.4 (Certara L.P. (Pharsight), Princeton, NJ) determined the pharmacokinetic parameters from plasma concentration-versus-time data (Table 7) by a noncompartmental extravascular administration model with uniform weighting. The pharmacokinetic analysis was performed on data from individual animals collected from t = 0 to 72 h after the last administered dose. The elimination t 1 / 2 values ranged from 34.7 to 51.0 h. Note that the t 1 / 2 values were calculated from data of less than three half-lives. To quantify the dose-dependent changes in the observed PK curves, the C max and C min values were determined for the time period between 0 - 48 h (which is the normal dosing period). The C max / C min ratio represents the peak-to-trough ratio of the EXT608 concentration observed during the dosing period. This ratio decreased from 19.3 at the highest dose (20 μg / kg) to 14.2 at 7 μg / kg, 5.4 at 2 μg / kg, 3.4 at 1.4 μg / kg, and 2.4 at 0.7 μg / kg. Without being bound by theory, the low peak-to-trough ratio observed at low doses may be due to the slower rate of absorption of EXT608 from the subcutaneous space into the circulation. A low peak-to-trough ratio is desirable in many therapeutic applications, including parathyroid hormone replacement therapy. This is because it allows dosing at levels close to the ideal effective dose, without being too high (where toxicity is observed) or too low (where efficacy is reduced).

[0462] Table 7: PK parameters of EXT608 after repeated every-other-day SC dosing in monkeys

[0463]

[0464]

[0465] t 1 / 2 , terminal half-life; T max , time of maximum observed concentration; AUC 0-48h , area under the concentration-time curve from time = 0 to 48 h; AUC last , area under the concentration-time curve from time = 0 to the time point of the last measurable concentration; C max , maximum observed concentration; C min , minimum observed concentration from time = 0 to 48 h; C max / C min (0 - 48 h), ratio of C max / C min , NE, not estimated.

[0466] Example 11: Effects of EXT607 and EXT608 on Serum Calcium, Serum Phosphate, and Urinary Calcium in Male and Female Cynomolgus Monkeys

[0467] 5 - day / 8 - day Experiment: EXT607 was formulated in phosphate buffered saline at pH = 7.4. In a repeated dose toxicokinetics study, a group of 4 cynomolgus monkeys (2 males + 2 females, each weighing 2.5 - 3.7 kg) was dosed subcutaneously every other day at 1 ml / kg volume with 0, 10, 30, or 100 μg / kg of EXT607 on Days 1, 3, and 5. On Day 5, blood samples (~1.0 ml) were collected at t = 0 (pre-dose), 0.5, 1, 3, 6, 12, 24, 48, and 72 h. Each blood sample was collected from the cephalic or saphenous vein into a serum separator tube and centrifuged within 1 h of collection (~2700 g, ~10 min, ~5 °C). Serum was recovered and analyzed for calcium and phosphate by colorimetric assay. EXT607 induced a dose-dependent increase in serum calcium levels, which persisted up to 48 h at the highest dose ( Figure 15 ). The change in serum calcium levels for each time point was calculated by subtracting the baseline calcium level (mean of the 0 μg / kg group for the same time point). The maximum change in calcium over the 72 h period was determined for each animal, and the group mean and standard deviation were calculated (Table 8A). No effect on serum phosphorus was observed.

[0468] Table 8A: Dose-Dependent Changes in Serum Calcium Levels after Injection of EXT607 on Day 5 in Cynomolgus Monkeys

[0469]

[0470] On Days - 3 and 8, urine was collected at room temperature in trays placed under the cages. "Day - 3" refers to the time point before dosing, i.e., three days before the first dose on Day 1. Urinary calcium levels were measured using an Advia 1800 clinical chemistry system (Siemens Medical Solutions USA, Malvern, PA). Urinary calcium levels remained unchanged or decreased in the case of EXT607 treatment (Table 8B). The urine collection on Day 8 corresponded to the serum samples collected 72 h after injection on Day 5. Thus, EXT607 increased serum calcium levels without increasing urinary calcium, as reflected by a lower urinary calcium:serum calcium ratio in the case of EXT607 administration. This is beneficial as high urinary calcium can lead to deposition in the kidney and loss of kidney function.

[0471] Table 8B: Dose-Dependent Effects of EXT607 on Serum Calcium and Urinary Calcium

[0472]

[0473] 21 - day Experiment: EXT608 was formulated in 20 mM sodium acetate buffer, pH = 5.5 with 0.8% sodium chloride. In the repeated-dose toxicokinetics study, a group of 4 cynomolgus monkeys (2 males + 2 females, each weighing approximately 2.5 kg) was dosed subcutaneously every other day starting on Day 1 with 1.4, 7 or 20 μg / kg of EXT608 at a volume of 0.05 ml / kg. On Days 1, 11 and 21, blood samples (∼0.5 ml) were collected at t = 0 (pre-dose), 0.5, 1, 3, 6, 12, 24, 48 and 72 h (Day 21 only). Each blood sample was collected from the saphenous vein into serum separator tubes and centrifuged within 1 h of collection (∼2700 g, ∼10 min, ∼5 °C). Serum was recovered and calcium and phosphate were analyzed using the Advia 1800 Clinical Chemistry System (Siemens Medical Solutions USA, Malvern, PA).

[0474] EXT608 induced a dose-dependent increase in serum calcium levels that lasted up to 24 h( Figure 16 ). Serum phosphate levels did not change significantly on Day 1, but by Day 11, the high dose (20 μg / kg) resulted in a decrease in serum phosphate, and by Day 21, both the high and medium doses (7 μg / kg) decreased serum phosphate for at least 72 h after dosing( Figure 17 ). Changes in serum calcium values for each animal were calculated by subtracting the background level of the 0 μg / kg dosing group from each time point, as described above. The maximum change observed for each animal was used to determine the group mean and standard deviation (Table 8C). Changes in serum phosphate values for each group were calculated by subtracting the background level of the 0 μg / kg dosing group from each time point, as described above. Since the decrease in phosphate levels persisted throughout the sampling period (48 or 72 h), the group mean was determined by averaging the values from each time point within the sampling period (Table 8C). On Day -5 (pre-dose) and Day 24, urine was collected at room temperature in trays placed under the cages. At these time points, neither serum calcium nor urinary calcium levels were elevated.

[0475] Table 8C: Dose-dependent changes in serum calcium (Ca) and phosphate (P i ) in cynomolgus monkeys after 21 days of dosing every other day with EXT608.

[0476]

[0477] 90 - day Experiment:EXT608 was formulated in 10 mM sodium acetate buffer, pH = 5.5, with 0.8% sodium chloride and 0.1% polysorbate 80. In a repeated-dose toxicokinetics study, a group of 6 cynomolgus monkeys (3 males + 3 females) was dosed subcutaneously every other day starting on Day 1 with 0.7 or 2 μg / kg of EXT608 at a volume of 0.05 ml / kg. On Days 1, 45, and 89, blood samples (∼0.5 ml) were collected at t = 0 (pre-dose), 0.5, 1, 3, 6, 12, 24, 48, and 72 h (Day 89 only). Each blood sample was collected from the saphenous vein into a serum separator tube and centrifuged (∼2700 g, ∼10 min, ∼5 °C) within 1 h of collection. Serum was recovered and calcium and phosphate were analyzed using an Advia 1800 clinical chemistry system (Siemens Medical Solutions USA, Malvern, PA). EXT608 induced a dose-dependent increase in serum calcium levels that lasted up to 12 h( Figure 18 ). The change in serum calcium values for each animal was calculated by subtracting the background level of the 0 μg / kg dosed group from each time point as described above. The maximum change observed for each animal was used to determine the group mean and standard deviation as reported in Table 8D. At these EXT608 doses, there was no significant change in serum phosphate levels. On Day -9 (pre-dose) and Day 90, urine was collected at room temperature in trays placed under the cages. At these time points, neither serum calcium nor urinary calcium levels were elevated.

[0478] Table 8D: Dose-dependent change in serum calcium levels after 89 days of dosing EXT608 every other day in cynomolgus monkeys.

[0479]

[0480] Example 12: EXT607 is effective in rats undergoing thyroparathyroidectomy (TPTx)

[0481] A 28-day pharmacodynamics study of EXT607 in TPTx rats. Rats undergoing thyroparathyroidectomy (TPTx) are a model for hypoparathyroidism. To generate TPTx rats, the thyroid and parathyroid glands were surgically removed and the rats were provided with L-thyroxine to compensate for the loss of their thyroid. Due to the absence of PTH, TPTx rats have lower levels of serum calcium and higher levels of serum phosphate. Two weeks after surgery, the animals were dosed subcutaneously (SC) once daily (QD) for 28 days with wild-type PTH(1-34), PTH(1-84), or EXT607 as outlined in Table 9A:

[0482] Table 9A: Group composition for the 28-day TPTx study

[0483]

[0484] Each group consisted of 10 female rats, divided into two subgroups of 5 animals for blood sampling purposes. On days 1, 12, and 27, blood (~0.2 ml) was collected into serum separator tubes at t = 0 (before dose), 2, 6, 10, and 24 h. Samples were processed into serum by standard methods and serum total calcium and phosphate were analyzed.

[0485] Compared to the TPTx control, EXT607 caused an immediate dose-dependent increase in serum calcium that persisted throughout the 24-h dosing period ( Figure 19 ). Calcium levels at 3 and 10 nmol / kg (20 and 60 μg / kg) were at or slightly below the normal calcium level range for healthy animals. At the 1 nmol / kg (6 μg / kg) dose, calcium levels were statistically increased at all time points compared to the TPTx control, but the calcium levels remained below the normal range for healthy animals. The increase in calcium observed with the administration of PTH(1-34) or PTH(1-84) was minimal and did not reach statistical significance except for two isolated time points with PTH(1-84) ( Figure 20 ). For each time point, the percentage increase in serum calcium relative to the TPTx vehicle control group (group 2) was determined and the daily mean values were calculated (Table 9B). Over the three days of sampling (days 1, 12, and 27), EXT608 at 1, 3, and 10 nmol / kg increased serum calcium by an average of 17%, 28%, and 37%, respectively. In contrast, 10 nmol / kg of PTH(1-34) and PTH(1-84) increased by 9%.

[0486] Table 9B: Percentage increase in serum calcium relative to TPTx / vehicle.

[0487]

[0488] Starting on day 12, a significant decrease in serum phosphate was observed in all dose levels of EXT607 and persisted for the remainder of the study ( Figure 21 ). Changes were observed in a dose-dependent manner, with values generally comparable to sham control at 3 and 10 nmol / kg. After treatment with 10 nmol / kg of PTH(1-84), a slight decrease in phosphate levels was observed starting on day 12 post-dose and persisted for the remainder of the study ( Figure 22)。An occasional decrease in serum phosphate was observed in the case of 10 nmol / kg of PTH(1-34). The magnitude of the decrease in serum phosphate in the cases of PTH(1-34) and PTH(1-84) was much smaller than that of EXT607. At each time point, the percentage decrease in serum phosphate relative to the TPTx vehicle control group (Group 2) was determined, and the daily average value was calculated (Table 9C). Taking the average of all time points on Days 12 and 27, EXT608 at 1, 3, and 10 nmol / kg decreased serum phosphate by 15%, 27%, and 38%, respectively, while 10 nmol / kg of PTH(1-34) and PTH(1-84) decreased serum phosphate by 11% and 17%, respectively.

[0489] Table 9C: Percentage decrease in serum phosphate relative to TPTx / vehicle.

[0490]

[0491] Example 13: Improved formulation of EXT608

[0492] Relationship between stability and pH. The optimal pH for maximizing the stability of EXT608 was determined as follows. EXT608 formulations at a concentration of 1 mg / ml were prepared in 50 mM sodium acetate buffer at pH = 4.5 and 5.5 and in phosphate buffer containing 10 mg / ml mannitol at pH 6.5 and 8.0, and incubated at -20 °C, 2 - 8 °C, and 25 °C. After 10 weeks, the concentration and purity of the samples were analyzed by reverse-phase HPLC (Table 10). The stability of EXT608 was highest at pH 4.5 and 5.5 and gradually decreased as the pH increased to 6.5 and 8.0.

[0493] Table 10: Relationship between the concentration and purity % of EXT608 solution after 10 weeks, pH, and incubation temperature.

[0494]

[0495] To further study the effect of pH on the stability of EXT608, formulations of 0.4 mg / mL in acetate buffer at 10 mM pH = 4.0, pH = 4.5, pH = 5.0, pH = 5.5 containing 0.9% saline were prepared by first dissolving the EXT608 API peptide in 10 mM acetic acid, then adjusting the pH with sodium hydroxide, and adjusting the tonicity with saline. The liquid formulations were filled into screw-cap vials at 1 mL per vial. All formulations were incubated at -20 °C, 2 - 8 °C, 25 °C, and the concentration and purity % were tested by reverse-phase HPLC at 6 months (Table 11). The stability of EXT608 was consistent in the pH range of 4.0 to 5.5.

[0496] Table 11: Relationship between the concentration and purity % of EXT608 solution after 6 months and pH and incubation temperature.

[0497]

[0498] Relationship between stability and excipients. During the process of preparing a formulation of EXT608 in 10 mM sodium acetate at pH = 5.5 with 0.8% saline as an isotonic agent added as an excipient, it was found that EXT608 had a tendency to precipitate. Further studies determined that precipitation might be triggered by agitation, such as that occurring during the normal mixing, pumping, and filtration steps during formulation and filling into sterile vials. Polysorbate 80 was investigated as an additional excipient to reduce precipitation. Concentrations of polysorbate 80 from 0.03% to 0.4% were tested, and it was found to reduce precipitation caused by agitation of EXT608 solution in 96-well plates using a rotary shaker platform.

[0499] Further tests were conducted to replace the ionic isotonic agent (saline) with a non-ionic isotonic agent. EXT608 solution formulated with 4.5% mannitol instead of 0.8% saline was less prone to precipitation due to agitation or loss due to non-specific absorption to surfaces. Thus, the formulation with mannitol brought the osmotic pressure of the formulation to the desired range between 275 - 295 mOsm / kg to match the osmotic pressure of human plasma and fluids. This would reduce injection site pain while avoiding increased precipitation in saline.

[0500] An EXT608 formulation was prepared at a concentration of 0.4 mg / ml in 10 mM sodium acetate buffer (pH = 5.5), 4.5% mannitol, and 0.25% polysorbate 80, and then filled into sterile vials.

[0501] Example 14: Single-dose pharmacokinetics and pharmacodynamics of EXT608 in humans

[0502] EXT608 greatly improved the serum half-life in humans in a dose-dependent manner. A sterile EXT608 formulation was prepared at a concentration of 0.4 mg / ml in 10 mM sodium acetate buffer at pH = 5.5, 4.5% mannitol, and 0.25% polysorbate 80, and filled into sterile vials. Healthy human participants were administered EXT608 by subcutaneous injection into the abdomen at doses of 0, 36, 108, 216, or 324 micrograms (n = 3).

[0503] Pharmacokinetics:Four ml of blood samples were collected at -24, -1, 0.5, 1, 2, 4, 8, 12, 18, 24, 36, 48, and 72 hours relative to dosing and on days 5, 7, 14, 21, and 28 after dosing. They were processed into K2EDTA plasma. The plasma levels of EXT608 were determined using the Immutopics High Sensitivity Human Parathyroid Hormone PTH(1-34) ELISA Kit (Catalog No.: 60-3900, Quidel Corporation, San Diego, CA) with the following modifications to the manufacturer's protocol: 1) Prior to analysis, native PTH(1-84) was depleted from plasma samples (100 μl) using goat anti-PTH(39-84) antibody (Catalog No.: 21-3010, Quidel Corporation, San Diego, CA), 2) depleted plasma (75 μl) and 75 μl of 1:1 PBS + 0.05% Tween 20:LowCross buffer (Catalog No.: 100500, Boca Scientific, Dedham, MA) were added to the ELISA plate, and 3) a calibration curve was established using EXT608 reference samples. The relationship between the group-averaged EXT608 levels and time is shown in Figure 23A . The pharmacokinetic parameters for each individual were calculated using Kinetica software (ThermoFisher Scientific, Waltham, MA), and the parameter means and standard deviations are provided in Table 12. For the lower doses (36, 108, and 216 μg), the accurate elimination half-life (t 1 / 2 ) could not be determined because the time points at which the EXT608 concentration was above the lower limit of quantification were limited. For the high dose (324 μg), t 1 / 2 was calculated to be 90 ± 13 h; this parameter and other parameters are provided in Table 13.

[0504] Table 12: PK parameters of EXT608 in humans after a single SC dose

[0505]

[0506] a t last = 48 h (n = 1) or 72 h (n = 2). SC, subcutaneous; Cmax, maximum observed concentration; Tmax, time of maximum observed concentration; AUC last , area under the concentration-time curve from time = 0 to the time point of the last measurable concentration (t last ).

[0507] Table 13: PK parameters of EXT608 in humans after a single 324 μg SC dose

[0508]

[0509] t 1 / 2 , terminal half-life; AUC inf , area under the concentration-time curve from time = 0 to infinity; Cl / F, clearance (Cl) divided by bioavailability (F); V Z / F, apparent volume of distribution (V Z ) divided by bioavailability (F); MRT, mean residence time.

[0510] Pharmacodynamics: Blood samples were collected at -24, -1, 0.5, 1, 2, 4, 8, 12, 18, 24, 36, 48, and 72 hours relative to dosing and on days 5, 7, 14, 21, and 28 after dosing. They were processed to serum and submitted to Quest Diagnostics for standard blood chemistry analysis, including total calcium and albumin. The albumin-corrected total serum calcium levels are shown in Figure 23B . EXT608 increased serum calcium levels in a dose-dependent manner and was able to maintain the elevated calcium levels for 24 h. Blood samples (1.5 ml) were collected at -1, 4, 24, 48, and 72 hours relative to dosing and on days 5, 7, 14, 21, and 28 after dosing. They were processed to EDTA plasma and submitted to Quest Diagnostics for a complete PTH(1-84) analysis (test number #35202). In healthy humans, under conditions of elevated serum calcium, the body will attempt to restore normal calcium levels through multiple mechanisms, including inhibition of endogenous PTH(1-84). EXT608 decreased endogenous PTH(1-84) levels in a dose-dependent manner and was able to maintain the decreased PTH(1-84) levels for approximately 3 - 4 days ( Figure 23C ).

[0511] When measured at approximately 36 hours after administration, EXT608 did not cause a significant increase in urinary calcium at any of the test dose levels (data not shown).

[0512] Example 15: Further improved formulation of EXT608

[0513] Effect of buffer composition and pH on the precipitation tendency of EXT608. In Example 13, it was shown that EXT608 is prone to precipitation, which may be triggered by agitation, such as that occurring during the conventional mixing, pumping, and filtration steps during formulation and filling into sterile vials. It was also shown that the precipitation tendency was reduced by replacing the ionic isotonic agent (saline) with a non-ionic isotonic agent (mannitol) and by including polysorbate 80 (PS80) in the formulation. In this example, further improvement in reducing the precipitation tendency was achieved by changing the buffer composition and pH.

[0514] Analysis 1 : A solution of EXT608 (1 mg / ml) was prepared in 10 mM buffer with 4.5% mannitol. As indicated in Table 14A, acetate, citrate, and histidine buffers with pH values between 4.0 and 5.5 were analyzed. The solution was added to a sealed 96-well plate and shaken at 800 rpm on a rotary shaking platform. Absorbance measurements at 600 nm (OD600) were performed after 24 and 120 h as a measure of light scattering caused by precipitation. After shaking for 24 h, the buffer containing citrate showed a large amount of precipitation, while the buffers containing acetate and histidine showed minimal precipitation.

[0515] Table 14A: Relationship between OD600 absorbance values of EXT608 solution and buffer composition after shaking for 24 and 120 h. All formulations contained the indicated 10 mM buffer and 4.5% mannitol.

[0516]

[0517] Analysis 2 : A solution of EXT608 (1 mg / ml) was prepared in 10 mM buffer with 4.5% mannitol. As indicated in Table 14B, acetate buffer at pH = 4.0, acetate buffer at pH = 5.5, and histidine buffer at pH = 4.0 were tested. PS80 (Super Refined Polysorbate 80 - LQ-(MH)SR48833, Croda, Inc., Plainsboro, NJ) was added to the formulations at 0, 0.05, 0.1, or 0.25% (v / v). Over a period of 308 h, OD600 measurements were performed as in Experiment 1. During the 308 h analysis duration, the EXT608 solution with acetate buffer at pH = 4 did not show any precipitation regardless of the PS80 concentration. In contrast, in the absence of PS80, the solution with acetate buffer at pH = 5.5 had precipitated within 20 h. The introduction of 0.05, 0.10, or 0.25% PS80 delayed the onset of precipitation to 140 h, where some minimal precipitation (OD < 0.1) was observed within 52 h for the 0.10% PS80 sample. In the absence of PS80, the solution with histidine buffer at pH = 4.0 had precipitated within 28 h. The introduction of PS80 delayed the onset of precipitation to 140 h, where high precipitation was observed for the 0.05 and 0.10% PS80 samples, and minimal precipitation was observed for the 0.25% PS80 sample. Thus, acetate buffer at pH = 4.0 is the preferred buffer for preventing precipitation caused by agitation.

[0518] Table 14B: Relationship between OD600 values and shaking time of EXT608 solutions with different buffer compositions, pH, and PS80 concentrations. All formulations contained 10 mM of the indicated buffer and 4.5% mannitol.

[0519]

[0520] Analysis 3 : Solutions of EXT608 (1 mg / ml) were prepared in 10 mM acetate buffer at pH = 4.0, 4.5, 5.0, or 5.5 with 4.5% mannitol. Polysorbate 80 (Ultra-pure Polysorbate 80 - LQ - (MH) SR48833, Croda, Inc., Plainsboro, NJ) was added to the formulations at 0, 0.05, 0.1, or 0.25% (v / v). OD600 measurements were made as in Analysis 1 over the course of 162 h in Table 14C. Regardless of the PS80 concentration, the EXT608 solution with acetate buffer at pH = 4 showed no precipitation over the 162 h duration of the experiment. The solution with acetate buffer at pH = 4.5 showed minimal precipitation (OD < 0.1) within 42 h in the absence of PS80, within 90 h in the presence of 0.05% or 0.10% PS80, and showed little or no precipitation within 162 h in the presence of 0.25% PS80. The solution with acetate buffer at pH = 5.0 showed substantial precipitation within 42 h in the presence of 0 or 0.05% PS80, within 66 h in the presence of 0.1% PS80, and within 90 h in the presence of 0.25% PS80. The solution with acetate buffer at pH = 5.5 showed substantial precipitation within 18 h in the presence of 0% PS80, within 42 h in the presence of 0.05 or 0.1% PS80, and within 66 h in the presence of 0.25% PS80. Thus, the tendency of EXT608 to precipitate in acetate buffer decreases with decreasing pH in the range of 5.5, 5.0, 4.5 to 4.0, where pH 4.0 is optimal for preventing precipitation caused by agitation.

[0521] Table 14C: Relationship between OD600 values of EXT608 solutions and acetate buffer pH and PS80 concentration. All formulations contained 10 mM acetate buffer and 4.5% mannitol.

[0522]

[0523] Effect of formulation composition and pH on the long-term storage stability of EXT608 solution. An improved UPLC method was developed that can better separate impurities from the main EXT608 peak than previous methods (e.g., the UPLC method used to generate the data in Tables 10 and 11 (Example 13)). By providing excellent impurity resolution and quantification, this improved method can better determine the ideal formulation composition and pH to minimize the rate of EXT608 degradation and thereby increase the shelf life. Examples of impurities now resolved by the improved UPLC method include those generated by the oxidation of methionine 8 and / or methionine 18 residues of the PTH1-34 component of EXT608. Oxidation of methionine is known to be catalyzed by peroxides such as hydrogen peroxide or peroxide impurities that may be present in polysorbate 80.

[0524] The improved UPLC method uses a Waters Acquity Peptide CSH C18 column (2.1 x 150 mm, 1.7 μm, Part number: 186006938), with a flow rate between 0.35 - 0.40 ml / min, a column temperature of 40 °C, and a detection wavelength of 220 nm. Mobile phase A consists of 80% water, 20% acetonitrile, and 0.1% trifluoroacetic acid. Mobile phase B consists of 20% water, 80% acetonitrile, and 0.1% trifluoroacetic acid. The gradient conditions are provided in Table 15A.

[0525] Table 15A: UPLC gradient conditions.

[0526]

[0527] To determine whether the addition of free methionine to the formulation reduces the rate of EXT608 degradation, EXT608 was prepared at 0.4 mg / ml in 10 mM sodium acetate buffer at pH 5.5 with 4.5% mannitol and 0.25% PS80 with 0, 5, 10, 20, or 40 mM methionine. Samples were placed in Eppendorf tubes and stored at 4 °C. Free methionine was added to scavenge peroxides and any other oxidizing substances that could otherwise oxidize methionine residues of EXT608. In this analysis, an aged solution of PS80 with more peroxide impurities was used. Aliquots of the EXT608 formulation were taken periodically over a 41-day period, and the purity of EXT608 was determined by UPLC (Table 15B). The addition of methionine to the formulation at concentrations from 5 to 40 mM helped maintain the purity of EXT608.

[0528] Table 15B: Relationship between the purity (%) of EXT608 and methionine concentration upon incubation at 4 °C.

[0529]

[0530] To determine how histidine buffer compares to acetate in terms of affecting the rate of EXT608 degradation, EXT608 was prepared at 0.4 mg / ml in 10 mM sodium acetate buffer at pH 5.5 or 10 mM histidine buffer at pH 5.5. 4.5% mannitol was added to each preparation, and as indicated in Table 15C, 0 or 0.25% PS80 and 0 or 40 mM methionine were added. The samples were placed in Eppendorf tubes and stored at room temperature, where aliquots were taken at days 0, 5, and 11 for UPLC determination of EXT608 purity. For each condition, the acetate buffer showed a slower rate of EXT608 degradation than the histidine buffer. The addition of 40 mM methionine slowed the rate of EXT608 degradation in the acetate buffer but not in the histidine buffer.

[0531] Table 15C: Purity (%) of EXT608 in pH 5.5 acetate vs. histidine buffer incubated at room temperature.

[0532]

[0533] To show how citrate buffer compares to acetate in terms of affecting the rate of EXT608 degradation, EXT608 was prepared at 0.4 mg / ml in 10 mM sodium acetate buffer at pH 5.5 or 10 mM sodium citrate buffer at pH 5.5. 4.5% mannitol was added to each preparation, and as indicated in Table 15D, 0 or 0.25% PS80 and 0, 2.5, 10, or 40 mM methionine were added. The samples were placed in Eppendorf tubes and stored at 4 °C, where aliquots were taken over the course of 134 days for UPLC determination of EXT608 purity. EXT608 showed similar degradation rates in acetate and citrate buffers, with citrate being slightly favored in the case of 0 or 2.5 mM methionine and acetate being slightly favored in the case of 10 or 40 mM methionine. The preparation with the lowest rate of EXT608 degradation was 10 mM acetate at pH 5.5, 4.5% mannitol, 0.25% PS80, and 40 mM methionine.

[0534] Table 15D: Purity (%) of EXT608 in pH 5.5 acetate vs. citrate buffer incubated at 4 °C.

[0535]

[0536] To determine how buffer pH affects the rate of EXT608 degradation, EXT608 was prepared at 1 mg / ml in 10 mM sodium acetate buffer at pH 4.0, 10 mM sodium acetate buffer at pH 5.5, or 10 mM histidine buffer at pH 4.0. As indicated in Table 15E, 0 or 0.25% PS80, 0, 10 or 40 mM methionine, and 4.5, 4.1 or 3.0% mannitol (to keep the osmotic pressure of the formulation constant) were added to each formulation. Samples were placed in 2 ml screw-cap glass vials and stored at 4 °C, where aliquots were removed over the course of 69 days for UPLC determination of EXT608 purity. For each test formulation, the acetate buffer at pH 4.0 was superior to the acetate buffer at pH 5.5. In formulations without PS80, the rate of EXT608 degradation in the acetate buffer at pH 4.0 was similar to the rate of EXT608 degradation in the histidine buffer at pH 4.0. However, in formulations containing 0.25% PS80 and 0, 10 or 40 mM methionine, the acetate at pH 4.0 was superior to the histidine at pH 4.0. The formulation with the lowest rate of EXT608 degradation was 10 mM sodium acetate at pH 4.0 and 4.5% mannitol. The formulation with the second lowest rate of EXT608 degradation was 10 mM sodium acetate at pH 4.0, 3.0% mannitol, 0.25% PS80, and 40 mM methionine.

[0537] Table 15E: Purity (%) of EXT608 in acetate at pH 4.0 vs. acetate at pH 5.5 vs. histidine buffer at pH 4.0 upon incubation at 4 °C.

[0538]

[0539] To further determine how buffer pH affects the rate of EXT608 degradation, EXT608 was prepared at 1 mg / ml in 10 mM sodium acetate buffer at pH 4.0, 4.5, 5.0, or 5.5. As indicated in Table 15F, 0 or 0.25% PS80, 0 or 40 mM methionine, and 4.5 or 3.0% mannitol (to keep the osmotic pressure of the formulation constant) were added to each formulation. Samples were placed in 2 ml screw-cap glass vials and stored at room temperature, where aliquots were removed over the course of 13 days for UPLC determination of EXT608 purity. In the absence of methionine, the rate of EXT608 degradation in the presence of 0.25% PS80 was faster relative to 0% PS80 at all tested pHs; in the presence of 40 mM methionine, the rate of EXT608 degradation in the presence of 0.25% PS80 was similar to or slower than the rate of EXT608 degradation in the presence of 0% PS80. For each condition, the general trend was that the rate of EXT608 degradation at pH 4.0 < / ≈ pH 4.5 < pH 5.0 < pH 5.5.

[0540] Table 15F: Purity (%) of EXT608 in acetate buffer at pH 4.0, 4.5, 5.0, or 5.5 upon incubation at room temperature.

[0541]

[0542] Exemplary sequences

[0543] SEQ ID NO:1 (PTH(1 - 34))

[0544] SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF

[0545] SEQ ID NO:2 (PTH(1 - 84))

[0546] SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPL APRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ

[0547] SEQ ID NO:3 (PTH - C)

[0548] SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFC

[0549] SEQ ID NO:4 (Vitamin D - binding protein (DBP))

[0550] MKRVLVLLLAVAFGHALERGRDYEKNKVCKEFSHLGKEDFTSLSLVLYSRKFPSGTFEQVSQFVKEVVSFTEACCAEGADPDCYDTRTSAFSAKSCESNSPFPVHPGTAECCTKEGFERKLCMAALKHQPQEFPTYVEPTNDEICEAFRKDPKEYANQFMWEYSTNYGQAPLSLLVSYTKSYLSMVGSCCTSASPTVCFLKERLQLKHLSLLTTLSNRVCSQYAAYGEKKSRLSNLIKLAQKVPTADLEDVLPLAEDITNILSKCCESASEDCMAKELPEHTVKLCDNLSTKNSKFEDCCQEKTAMDVFVCTYFMPAAQLPELPDVELPTNKDVCDPGNTKVMDKYTFELSRRTHLPEVFLSKVLEPTLKSLGECCDVEDSTTCFNAKGPLLKKELSSFIDKGQELCADYSENTFTEYKKKLAERLKAKLPDATPTELAKLVNKHSDFASNCCSINSPPLYCDSEIDAELKNIL

[0551] SEQ ID NO:5 (Vitamin D Binding Protein (DBP))

[0552]

[0553] All publications and patent documents disclosed or mentioned in this text are incorporated by reference in their entirety. The foregoing description is provided for illustrative and descriptive purposes only. This description is not intended to limit the invention to the precise form disclosed. The scope of the invention is intended to be defined by the appended claims herein.

Claims

1. A pharmaceutical composition comprising a parathyroid hormone peptide (PTH) (PTH conjugate) each conjugated to a non-hormonal vitamin D moiety via a backbone and a pharmaceutical excipient, wherein the PTH conjugate has a substantially uniform size as measured by mass spectrometry, and wherein the PTH conjugate has a solubility of at least about 0.550 mM in phosphate buffered saline (PBS) when measured at an absorbance of 280 nm using an extinction coefficient of 18,600 M 1 cm -1 .

2. The pharmaceutical composition according to claim 1, wherein the PTH conjugate comprises a backbone, and the backbone is a 36-subunit polyethylene glycol backbone (PTH-PEG36-VitD).

3. The pharmaceutical composition according to claim 2, wherein the PTH-PEG36-VitD comprises the following structure: H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Cys(succinimidyl-propionamido-PEG36-propionyl-aminopropyl-25-hydroxy-vitamin D)-OH; or H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Cys(succinimidyl-propionamido-PEG36-propionyl-aminopropyl-25-hydroxy-vitamin D)-NH2.

4. The pharmaceutical composition according to claim 2, wherein the PTH-PEG36-VitD comprises the following structure: H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-Cys(succinimidyl-propionamido-PEG36-propionyl-aminopropyl-25-hydroxy-vitamin D)-NH2.

5. The pharmaceutical composition according to any one of claims 1-4, wherein the activity of the PTH conjugate on the PTH receptor (PTHR) is substantially the same as that of the equivalent unconjugated PTH peptide.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the PTH conjugate is isolated as a carbonate.

7. The pharmaceutical composition according to any one of claims 1-5, wherein the PTH conjugate is isolated as an acetate.

8. The pharmaceutical composition according to claim 7, wherein when measured at an absorbance at 280 nm using an extinction coefficient of 18,600 M 1 cm -1 , the PTH conjugate has a solubility of at least about 9.67 mM in PBS.

9. The pharmaceutical composition according to any one of claims 1-8, wherein the pharmaceutical composition is formulated at a pH of about 4.0 to about 5.

5.

10. The pharmaceutical composition according to claim 9, wherein the pharmaceutical composition is formulated at a pH of about 5.

5.

11. The pharmaceutical composition according to any one of claims 1-7, wherein the pharmaceutical composition is formulated with mannitol.

12. The pharmaceutical composition according to any one of claims 1-11, wherein the pharmaceutical composition is formulated together with polysorbate 80 (PS80).

13. The pharmaceutical composition according to claim 5, wherein the PTH-PEG36-VitD is formulated as an acetate, wherein the pharmaceutical composition is formulated at a pH of about 5.5, and wherein the pharmaceutical composition comprises mannitol and PS80.

14. The pharmaceutical composition according to claim 13, wherein the PTH-PEG36-VitD is formulated at a concentration of 0.4 mg / ml in about 10 mM sodium acetate buffer at pH about 5.5, about 4.5% mannitol and about 0.25% polysorbate 80.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein the pharmaceutical composition is formulated for subcutaneous delivery, intramuscular delivery, intravenous delivery, controlled release delivery, transdermal delivery, parenteral delivery or via an implantable reservoir.

16. The pharmaceutical composition according to claim 15, wherein the pharmaceutical composition is formulated for subcutaneous delivery.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the PTH conjugate has a serum half-life of 7 to 15 hours in rats.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the PTH conjugate has a subcutaneous bioavailability of at least about 10-13% in rats.

19. The pharmaceutical composition according to any one of claims 1 to 18, wherein the PTH conjugate has a serum half-life of about 24 to 32 hours in cynomolgus monkeys.

20. The pharmaceutical composition according to any one of claims 1 to 17, wherein the PTH conjugate has a subcutaneous bioavailability of about 45-54% in cynomolgus monkeys.

21. The pharmaceutical composition according to any one of claims 1 to 20, wherein a daily dose of about 6 μg / kg of the PTH conjugate, when measured during the 24 hours after injection, increases the serum calcium level in TPTx rats by an average of at least about 17% compared to the 0-dose level.

22. The pharmaceutical composition according to any one of claims 1 to 20, wherein a daily dose of about 60 μg / kg of the PTH conjugate, when measured during the 24 hours after injection, increases the serum calcium level in TPTx rats by an average of at least about 37% compared to the 0-dose level.

23. The pharmaceutical composition according to any one of claims 1 to 20, wherein a dose of about 2 μg / kg of the PTH conjugate administered every other day, when measured 12 hours after the 5th day injection, increases the serum calcium level in cynomolgus monkeys by at least about 0.9 mg / dl compared to the 0-dose level.

24. The pharmaceutical composition according to any one of claims 1 to 20, wherein a dose of about 100 μg / kg of the PTH conjugate administered every other day, when measured 12 hours after the 5th day injection, increases the serum calcium level in cynomolgus monkeys by at least about 6.1 mg / dl compared to the 0-dose level.

25. The pharmaceutical composition according to any one of claims 1 to 21, wherein a dose of about 10 μg / kg of the PTH conjugate reduces the ratio of urinary calcium to serum calcium levels in cynomolgus monkeys to equal to or lower than about 1.

8.

26. The pharmaceutical composition according to any one of claims 1 - 21, wherein a dose of about 100 μg / kg of the PTH conjugate reduces the ratio of urinary calcium to serum calcium levels in cynomolgus monkeys to equal to or lower than about 1.

2.

27. The pharmaceutical composition according to any one of claims 1 to 21, wherein when the PTH conjugate is administered subcutaneously at a daily dose of about 6 μg / kg, after about 12 days, the PTH conjugate causes a reduction in the serum phosphate level of TPTx rats by at least about 14%.

28. The pharmaceutical composition according to any one of claims 1 to 21, wherein when the PTH conjugate is administered subcutaneously at a daily dose of about 60 μg / kg, after about 12 days, the PTH conjugate causes a reduction in the serum phosphate level of TPTx rats by at least about 39%.

29. The pharmaceutical composition according to any one of claims 1 to 21, wherein when the PTH conjugate is administered subcutaneously at a dose of about 7.0 μg / kg every other day, after about 21 days, the PTH conjugate causes a reduction in the serum phosphate level of cynomolgus monkeys by at least about 1.0 mg / dl.

30. The pharmaceutical composition according to any one of claims 1 to 21, wherein when the PTH conjugate is administered subcutaneously at a dose of about 20 μg / kg every other day, after about 11 days, the PTH conjugate causes a reduction in the serum phosphate level of cynomolgus monkeys by at least about 1.8 mg / dl.

31. The pharmaceutical composition according to any one of claims 1 - 30, wherein the PTH conjugate binds to vitamin D - binding protein (VDBP) with a dissociation constant of about 5.2 μM.

32. A method of treating a subject suffering from a disorder selected from the group consisting of: hypoparathyroidism, hypocalcemia, hyperphosphatemia, hypercalciuria, osteoporosis, fracture repair, osteomalacia, osteomalacia and osteoporosis in patients with hypophosphatasia, steroid - induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, osteogenesis imperfecta, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, fractures, hair loss, chemotherapy - induced hair loss, thrombocytopenia, autoimmune polyendocrine syndrome type 1, DiGeorge syndrome, CHARGE syndrome, Kenny - Caffey type 1, Kenny - Caffey type 2, hereditary deafness and renal dysplasia (HDR), autosomal - dominant hypocalcemia type 1 (ADH1), autosomal - dominant hypocalcemia type 2 (ADH2), and ADH1 with Bartter type 5, the method comprising the step of administering to the subject the pharmaceutical composition according to any one of claims 1 to 31.

33. The method according to claim 32, wherein the administration step is achieved by subcutaneous delivery, intramuscular delivery, intravenous delivery, controlled release delivery, transdermal delivery, parenteral delivery, or by an implantable reservoir.

34. The method according to claim 33, wherein the administration is by the subcutaneous route.

35. The method according to any one of claims 32 - 34, wherein the pharmaceutical composition is administered at a dose of about 2, 7, 10, or 20 μg / kg of the body weight of the subject.

36. The method according to any one of claims 32 - 34, wherein the dose is administered approximately daily.

37. The method according to any one of claims 32 - 34, wherein the dose is administered approximately every other day.

38. The pharmaceutical composition according to any one of claims 1 to 31, for the treatment of hypoparathyroidism, hypocalcemia, hyperphosphatemia, hypercalciuria, osteoporosis, fracture repair, osteomalacia, osteomalacia and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, osteogenesis imperfecta, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, fractures, hair loss, chemotherapy-induced hair loss, thrombocytopenia, autoimmune polyendocrine syndrome type 1, DiGeorge syndrome, CHARGE syndrome, Kenny-Caffey type 1, Kenny-Caffey type 2, hereditary deafness and renal dysplasia (HDR), autosomal dominant hypocalcemia type 1 (ADH1), autosomal dominant hypocalcemia type 2 (ADH2), or ADH1 with Bartter type 5.

39. A medicament comprising the pharmaceutical composition according to any one of claims 1 - 31.

40. A method of treating a disorder selected from the group consisting of hypoparathyroidism, hypocalcemia, hyperphosphatemia, hypercalciuria, osteoporosis, fracture repair, osteomalacia, osteomalacia and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, osteogenesis imperfecta, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, fractures, hair loss, chemotherapy-induced hair loss, thrombocytopenia, autoimmune polyendocrine syndrome type 1, DiGeorge syndrome, CHARGE syndrome, Kenny-Caffey type 1, Kenny-Caffey type 2, hereditary deafness and renal dysplasia (HDR), autosomal dominant hypocalcemia type 1 (ADH1), autosomal dominant hypocalcemia type 2 (ADH2), and ADH1 with Bartter type 5, the method comprising administering to the subject a pharmaceutical composition comprising a plurality of parathyroid hormone (PTH) peptides (PTH conjugates) each conjugated to a non-hormonal vitamin D through a backbone and a pharmaceutical excipient, wherein each of the plurality of PTH peptides is conjugated to a backbone having a substantially uniform size as measured by mass spectrometry, wherein the method comprises the step of subcutaneously administering repeated doses of the pharmaceutical composition, and wherein the repeated doses result in a maximum to minimum concentration ratio (Cmax / Cmin ratio) of the PTH conjugate in a serum sample from the subject equal to or less than about 8.

0.

41. The method of claim 40, wherein the Cmax / Cmin ratio is equal to or less than about 5.

37.

42. The method of claim 40, wherein the Cmax / Cmin ratio is equal to or less than about 4.

59.

43. The method of claim 40, wherein the Cmax / Cmin ratio is equal to or less than about 3.

36.

44. The method of claim 40, wherein the Cmax / Cmin ratio is equal to or less than about 2.

38.

45. The method of claim 40, wherein the Cmax / Cmin ratio is equal to or less than about 1.

83.

46. The method of claim 40, wherein the Cmax / Cmin ratio is equal to or less than about 1.

79.

47. The method according to any one of claims 40-46, wherein the PTH conjugate has a serum half-life of 7 to 15 hours.

48. The method according to any one of claims 40-47, wherein the PTH conjugate has a subcutaneous bioavailability of at least about 10-13%.

49. The method according to any one of claims 40-47, wherein the PTH conjugate has a serum half-life of about 24 to 32 hours.

50. The method according to any one of claims 40-49, wherein the PTH conjugate has a subcutaneous bioavailability of about 45-54%.

51. The method according to any one of claims 40 to 49, wherein the PTH conjugate at a dose of about 2.0 μg / kg administered every other day, when measured 12 hours after the injection on day 5, increases the serum calcium level by at least about 0.9 mg / dl compared to the 0-dose level.

52. The method according to any one of claims 40 to 49, wherein the PTH conjugate at a dose of about 100 μg / kg administered every other day, when measured 12 hours after the injection on day 5, increases the serum calcium level by at least about 6.1 mg / dl compared to the 0-dose level.

53. The method according to any one of claims 40 to 50, wherein the PTH conjugate at a dose of about 10 μg / kg reduces the ratio of urinary calcium to serum calcium level to equal to or lower than about 1.

8.

54. The method according to any one of claims 40 to 50, wherein the PTH conjugate at a dose of about 100 μg / kg reduces the ratio of urinary calcium to serum calcium level to equal to or lower than about 1.

2.

55. The method according to any one of claims 40 to 50, wherein when the PTH conjugate at about 6 μg / kg is administered subcutaneously daily, after about 12 days, the PTH conjugate causes the serum phosphate level to decrease by at least about 14%.

56. The method according to any one of claims 40 to 50, wherein when the PTH conjugate at about 60 μg / kg is administered subcutaneously daily, after about 12 days, the PTH conjugate causes the serum phosphate level to decrease by at least about 39%.

57. A kit comprising the pharmaceutical composition according to any one of claims 1 - 31 and instructions for its use in treating a patient's condition.

58. The kit according to claim 57, wherein the condition is hypoparathyroidism.

59. The pharmaceutical composition according to claim 3, wherein the PTH-PEG36-VitD comprises the following structure:

60. The pharmaceutical composition according to any one of claims 3 or 4, wherein the PTH-PEG36-VitD comprises the following structure:

61. A method for preparing the pharmaceutical composition according to any one of claims 1 - 31, comprising conjugating the PTH peptide, the backbone, and the non-hormonal vitamin D moiety to form the PTH conjugate, wherein the conjugation step occurs at a pH less than about 7.

4.

62. The method according to claim 61, wherein the PTH peptide comprises the amino acid sequence of SEQ ID NO:

3.

63. The method according to any one of claims 61 - 62, wherein the conjugation step occurs at a pH of about 6.

0.

64. The method according to any one of claims 61 - 63, further comprising a purification step of the conjugate at a pH less than about 8.

0.

65. The method according to claim 64, wherein the purification step is at a pH of about 5.

5.

66. A drug carrier comprising Formula I: B-(L) a -S-(M) b -C I Wherein: B is a targeting group, which is vitamin D that is not hydroxylated at the 1-carbon position and is conjugated with (L) at the 3-carbon position a conjugated; S is a backbone moiety comprising polyethylene glycol consisting of 36 repeating ethylene glycol units; C is a maleimide group; (L) a is a linker that includes (CH2)3NHC(O)CH2; and (M) b is a linker that includes HNC(O)(CH2)2.

67. The pharmaceutical carrier according to claim 66, which comprises a compound having formula VI:

68. A method for preparing a pharmaceutical composition comprising parathyroid hormone (PTH) and the pharmaceutical carrier according to claim 67, comprising the following steps: 1) Purifying the compound having formula VI; 2) Conjugating the compound having formula VI with the PTH.

69. The method according to claim 8, wherein before the conjugating step, the purified compound having formula VI is not removed from the purification solvent.

70. The method according to any one of claims 68 or 69, wherein the purifying step is achieved by high performance liquid chromatography (HPLC).

71. The method according to any one of claims 68 - 70, wherein the conjugate is separated as a carbonate.

72. The method according to any one of claims 68 - 70, wherein the conjugate is separated as an acetate.

73. The method according to any one of claims 68 - 72, wherein the PTH comprises the amino acid sequence of SEQ ID NO:

3.

74. A method for treating a human being suffering from a disorder selected from the group consisting of: hypoparathyroidism, hypocalcemia, hyperphosphatemia, hypercalciuria, osteoporosis, fracture repair, osteomalacia, osteomalacia and osteoporosis in patients with hypophosphatasia, steroid-induced osteoporosis, male osteoporosis, arthritis, osteoarthritis, osteogenesis imperfecta, fibrous dysplasia, rheumatoid arthritis, Paget's disease, humoral hypercalcemia associated with malignancy, osteopenia, periodontal disease, fractures, hair loss, chemotherapy-induced hair loss, thrombocytopenia, autoimmune polyendocrine syndrome type 1, DiGeorge syndrome, CHARGE syndrome, Kenny-Caffey type 1, Kenny-Caffey type 2, hereditary deafness and renal dysplasia (HDR), autosomal dominant hypocalcemia type 1 (ADH1), autosomal dominant hypocalcemia type 2 (ADH2) and ADH1 with Bartter type 5, the method comprising the step of administering to the human being a pharmaceutical composition according to any one of claims 1 to 31.

75. The method according to claim 74, wherein the disorder is hypoparathyroidism.

76. The method according to claim 74, wherein the pharmaceutical composition is EXT608.

77. The method according to any one of claims 74 or 76, wherein the administering step is achieved by subcutaneous delivery, intramuscular delivery, intravenous delivery, controlled release delivery, transdermal delivery, parenteral delivery or by an implantable reservoir.

78. The method according to claim 77, wherein the administering is by the subcutaneous route.

79. The method according to any one of claims 74 - 78, wherein the pharmaceutical composition is administered at a dose of about 2, 7, 10 or 20 μg / kg of the subject's body weight.

80. The method according to any one of claims 74 - 78, wherein the pharmaceutical composition is administered at a dose of about 36, 108 or 324 μg.

81. The method according to any one of claims 74 - 80, wherein the dose is administered approximately daily.

82. The method according to any one of claims 74 - 80, wherein the dose is administered approximately every other day.

83. The method according to any one of claims 74 - 80, wherein the dose is administered approximately every three days.

84. The method according to any one of claims 74 - 80, wherein the dose is administered approximately every four days.

85. The method according to any one of claims 74 - 80, wherein the dose is administered approximately every five days.

86. The method according to any one of claims 74 - 80, wherein the dose is administered approximately every six days.

87. The method according to any one of claims 74 - 80, wherein the dose is administered approximately every seven days.

88. The method according to any one of claims 74 - 80, wherein the dose is administered approximately every eight days.

89. The method according to any one of claims 74 - 80, wherein the dose is administered approximately every nine days.

90. The method according to any one of claims 74 - 89, wherein the dose results in a Cmax between approximately 0.49 and 5.0 ng / ml.

91. The method according to any one of claims 74 - 89, wherein the dose results in a Cmax of approximately 5.0 ng / ml.

92. The method according to any one of claims 74 - 89, wherein the dose results in a Tmax between approximately 3.3 and 5.3 hours.

93. The method according to any one of claims 74 - 89, wherein the dose results in a Tmax of approximately 2.7 hours.

94. The method according to any one of claims 74 - 89, wherein the dose results in an AUC between approximately 5.9 and 118 (ng / ml)*h.

95. The method according to any one of claims 74 - 89, wherein the dose results in an AUC of approximately 118 (ng / ml)*h.

96. The method according to any one of claims 74 - 89, wherein the dose results in a Tlast between approximately 24 and 168 hours.

97. The method according to any one of claims 74 - 89, wherein the dose results in a Tlast of approximately 168 hours.

98. The method according to any one of claims 74 - 89, wherein the dose results in a Cmax of approximately 5.0 ng / ml, a Tmax of approximately 2.7 hours, an AUC of approximately 118 (ng / ml)*h, and a Tlast of approximately 168 hours. The method according to any one of claims 74-89, wherein the dose is about 324 μg SC and results in a T 1 / 2 of about 90 hours. The method according to any one of claims 74 - 89, wherein the dose is about 324 μg SC and results in an AUC inf of about 151 (ng / ml)*h.

101. The method according to any one of claims 74 - 89, wherein the dose is approximately 324 μg SC and results in a clearance divided by bioavailability (CL / F) of approximately 2.2 L / h.

102. The method according to any one of claims 74 - 89, wherein the dose is approximately 324 μg SC and results in an apparent volume of distribution divided by bioavailability during the terminal phase (Vz / F) of approximately 280 L.

103. The method according to any one of claims 74 - 89, wherein the dose is approximately 324 μg SC and results in an average residence time (MRT) of approximately 105 hours. The method according to any one of claims 74-89, wherein the dose is about 324 μg SC and results in T 1 / 2 being about 90 hours, AUC inf being about 151 (ng / ml)*h, CL / F being about 2.2 L / h, Vz / F being about 280 L and MRT being about 105 hours.

105. The method according to any one of claims 74 - 104, wherein the pharmaceutical composition raises the serum calcium level for about 24 hours after administration.

106. The method according to any one of claims 74 - 105, wherein the pharmaceutical composition reduces the level of endogenous PTH(1 - 84).

107. The method according to any one of claims 74 - 106, wherein the pharmaceutical composition does not cause a significant increase in urinary calcium when measured about 36 hours after administration.

108. The pharmaceutical composition according to any one of claims 1 - 14, wherein the PTH conjugate is formulated with methionine at a concentration between about 0 and 40 mM.

109. The pharmaceutical composition according to claim 108, wherein the PTH conjugate is formulated with methionine at a concentration of about 0, 5, 10, 20, or 40 mM.

110. The pharmaceutical composition according to claim 109, wherein the PTH conjugate is formulated with methionine at a concentration of about 40 mM.

111. The pharmaceutical composition according to any one of claims 1 - 14 and 108 - 110, wherein after shaking the pharmaceutical composition for 308 hours, when measured at a wavelength of 600 nm, the PTH conjugate shows an optical density (OD) of the precipitate separated from the solution of about 0.411 or lower.

112. The pharmaceutical composition according to claim 111, wherein after shaking the pharmaceutical composition for 308 hours, when measured at a wavelength of 600 nm, the PTH conjugate shows an optical density (OD) of the precipitate separated from the solution of about 0.242 or lower.

113. The pharmaceutical composition according to claim 112, wherein after shaking the pharmaceutical composition for 308 hours, when measured at a wavelength of 600 nm, the PTH conjugate shows an optical density (OD) of the precipitate separated from the solution of about 0.015 or lower.

114. The pharmaceutical composition according to any one of claims 1 - 14 and 108 - 113, wherein the purity of the PTH conjugate is reduced by about 7.7% or less after incubation at 4°C for 69 hours.

115. The pharmaceutical composition according to any one of claims 1 - 14 and 108 - 113, wherein the purity of the PTH conjugate is reduced by about 14.9% or less after incubation at room temperature for 13 days.

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