Long-acting parathyroid hormone

Abstract

The present invention provides a long-acting parathyroid hormone peptide (PTH) that is retained in a subject's serum for a period significantly longer than that of the native hormone. Long-acting PTH is conjugated at carbon 3 of non-hormonal vitamin D via a modestly long scaffold that facilitates its purification, detection, solubility, and efficacy at the PTH receptor (PTHR). PTH may also be conjugated to non-hormonal vitamin D via a 36-mer poly(ethylene glycol) moiety (PTH-PEG36-VitD). The present invention also provides optimized manufacturing methods and formulations. PTH-PEG36-VitD exhibits significantly improved serum half-life and bioavailability compared to unconjugated PTH peptide. PTH-PEG36-VitD also significantly increases serum calcium, reduces urinary calcium, and reduces serum phosphate.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This invention was made with government support under grants R43DK107231 and R44DK107231 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] FIELD OF THE INVENTION The present invention provides long-acting parathyroid hormone peptides (PTH) that are retained in a subject's serum for periods significantly longer than the native hormone. Long-acting PTH is conjugated to the carbon-3 position of non-hormonal vitamin D via a modestly long scaffold that facilitates its purification, detection, solubility, and efficacy at the PTH receptor (PTHR). In some embodiments, PTH may be conjugated to non-hormonal vitamin D via a 36-mer poly(ethylene glycol) moiety (PTH-PEG36-VitD). In other embodiments, the non-hormonal vitamin D lacks a hydroxyl group at carbon-1. In other embodiments, optimized manufacturing methods and formulations are provided. PTH-PEG36-VitD exhibits significantly improved serum half-life and bioavailability compared to unconjugated PTH peptide. In animal models, PTH-PEG36-VitD also significantly increases serum calcium, reduces urinary calcium, and reduces serum phosphate. [Background technology]

[0003] The present invention provides improved PTH-vitamin D conjugates for use in pharmacological 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 group of fat-soluble secosteroids. Vitamin D exists in several forms (vitamers). The 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 taken orally as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2). For most people, sunlight is the primary source of vitamin D. Once manufactured in the skin or ingested, vitamin D must be activated by a series of hydroxylation steps, first to 25-hydroxyvitamin D (25(OH)D3) in the liver and then to 1,25-dihydroxyvitamin D3 (1α,25(OH)2D3) in the kidneys. 1α,25(OH)2D3 is the active "hormonal" form of vitamin D because it binds to the vitamin D receptor (VDR). 25(OH)D3 is the "non-hormonal" form of vitamin D and is the major circulating form in the human body. It binds to vitamin D-binding protein (DBP or VDBP). It is converted to its hormonal form only when needed. Examples of non-hormonal vitamin D forms are those lacking the 1α-hydroxyl group.

[0004] Parathyroid hormone (PTH), also known as parathyroid hormone or parathyroid hormone, is secreted from the chief cells of the parathyroid gland. It is a polypeptide containing 84 amino acids (SEQ ID NO: 2). It is normally present in serum at concentrations of 10-65 pg / mL. However, the N-terminal 34 amino acids retain full biological activity at the G protein-coupled receptor parathyroid hormone receptor 1 (PTH1R), which is expressed primarily in the kidney and bone (Mosekilde et al., Endocrinology 129:421-428, (1991)). (Tay et al., Principles of Bone Biology (Academic Press, 4 th Edition, Bilzekian et al. editors), Chapter 69, 1633-1642, 2020; (Mosekilde et al., Endocrinology 129:421-428 (1991)), which are incorporated by reference in their entirety.

[0005] PTH plays a role in mineral homeostasis through at least three mechanisms: (1) PTH stimulates the conversion of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D in the kidney, which then increases calcium and phosphate absorption in the intestinal tract; (2) PTH enhances calcium resorption in the distal renal tubule and collecting duct; and (3) PTH increases serum calcium through osteocyte / osteoblast-mediated bone resorption, which 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)), all of which are incorporated by reference in their entireties.

[0006] While bone resorption and intestinal phosphate absorption increase serum phosphate, increased renal excretion removes phosphate from serum at a faster rate, resulting in an overall net decrease in serum phosphate with PTH (Shoback, N. Engl. J. Med. 359:391-403(2008)). However, intravenously administered PTH has a very short serum half-life of approximately 5 minutes in humans (Papapoulos et al., Clinical Endocrinology 7:211-225(1977)). The apparent half-life after subcutaneous injection is long (1.5-2.5 h) due to slow absorption into the circulation (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 hormone replacement. The above is incorporated by reference in its entirety.

[0007] Hypoparathyroidism is a condition in which the level of PTH in the blood is low, most commonly caused by damage or removal of the parathyroid glands during thyroid surgery, immune system-related damage, genetic inheritance, or other rare causes. Low blood calcium levels can often cause muscle spasms and muscle cramps or tetany (involuntary muscle contractions), as well as several other symptoms. Calcium supplementation or vitamin D can alleviate symptoms, but may increase the risk of kidney stones and chronic kidney disease. See, for example, Winer KK, et al. J. Clin. Endocrinol. Metab. 97(2):391-399(2012) (incorporated by reference in its entirety).

[0008] Hypoparathyroidism can occur when an individual is born without parathyroid glands (usually due to a chromosomal deletion), when the parathyroid glands are damaged or removed during surgery (e.g., thyroidectomy due to thyroid cancer or goiter), or when the organs are damaged by an autoimmune response, 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)). Deficiency of PTH leads to decreased serum calcium levels (hypocalcemia), elevated serum phosphate levels (hyperphosphatemia), elevated urinary calcium levels due to decreased renal resorption (hypercalciuria), and increased bone mineral density characterized by increased cortical and trabecular bone density (Shoback et al., J Clin Endocrinol Metab. 101(6):2300-12(2016)). If left untreated, hypoparathyroidism affects virtually every organ system in the body. These physiological changes (hypocalcemia, hypercalciuria, hyperphosphatemia, and near-absence of PTH), alone or in combination with each other, can lead to neuromuscular symptoms (persistent muscle spasms, dysesthesias, tetany, seizures, cardiac arrhythmias), ischemic heart disease, kidney stones and nephrocalcinosis, and an increased risk of 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)). Additionally, patients with hypoparathyroidism suffer from emotional and cognitive disorders, including anxiety, depression, memory impairment, and various "brain fog" symptoms, which are thought to result from a deficiency of PTH (Aggarwal et al., Eur. J. Endocrinol. 168:895-903(2013)).Importantly, patients with inadequately controlled hypoparathyroidism report a significantly reduced quality of life, with 75% reporting that their disease affects their ability to work and 63% reporting a negative effect on family relationships (Siggelkow et al., Clin. Endocrinol. 92(2):159-168(2020)). The above is incorporated by reference in its entirety.

[0009] PTH can have purely anabolic or catabolic effects on bone, depending on the duration of PTH exposure, which is regulated by different stimuli for osteoblasts and osteoclasts (Frolick et al., Bone 33:372-379 (2003)). Intermittent PTH exposure has anabolic effects and is the basis for the treatment Forteo® (PTH1-34), which increases bone mineral density (BMD) in osteoporotic patients (Neer et al., N. Engl. J. Med., 344(19):1434-1441 (2001)). PTH acutely increases bone deposition (primarily cancellous bone) without resorption, resulting in increased bone mineral density by preferentially stimulating osteoblasts and osteoblast precursor cells. These osteoblasts and osteoblast precursor cells express PTH1R, but do not express the receptor unless osteoclasts are activated (Silva et al., J. Endocrinol. Invest. 34(10):801-810(2011); Kousteni and Bilezikian, Principles of Bone Biology, 3rd Edition, Academic Press Inc., 639-656(2008)). Direct stimulation of these osteoblasts and precursor cells reduces osteoblast apoptosis and increases osteoblastogenesis, leading to increased osteoblast numbers 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 above is incorporated by reference in its entirety.

[0010] Patients with hypoparathyroidism have low physiological PTH exposure and exhibit reduced bone turnover. This is due to reduced acute and direct osteoblast activation via PTH1R stimulation and reduced osteoclast activation via osteoblast- and osteocyte-mediated RANKL expression (Rubin et al., J. Bone Miner. Res. 23(12):2018-2024(2008)). This reduced bone remodeling results in increased BMD in hypoparathyroid patients (Bilezikian et al., J. Bone Miner. Res. 26(10):2317-2337(2011)). Continuous PTH exposure (via insulin-type pumps, multiple daily dosing regimens, or long-acting PTH), which more closely mimics physiological exposure, results in normalization of bone turnover and reduction of BMD in patients with hypoparathyroidism (Winer et al., J. Pediatr. 203:391-9(2018)). The above is incorporated by reference in its entirety.

[0011] PTH replacement therapy can achieve normal calcium levels in patients (Winer et al., J. Clin. Endocrinol. Metab. 83(10):3480-3486(1998)). However, exogenously administered PTH is rapidly cleared from the body. Therefore, it is difficult to provide hypoparathyroid patients with physiological PTH concentrations, which must remain within a narrow concentration window to avoid either hypocalcemia or hypercalcemia (Tay et al., Br. J. Clin. Pharmacol. 84:252-267(2018)). In fact, compared with once- or twice-daily injections, only subcutaneous pump delivery provided the closest approach to physiological replacement of PTH (Winer et al., J. Clin. Endocrinol. Metab. 97(2):391-399(2012)). This is because the plasma half-life of PTH in humans is estimated to be less than 15 minutes when administered intravenously, or 2-3 hours when administered subcutaneously due to slow absorption kinetics (Papapoulos et al., Clin. Endocrinol. 7:211-225(1977); Sikjaer et al., J. Bone Miner. Res. 28(10):2232-43(2013)), all of which are incorporated by reference in their entireties.

[0012] The current standard of care for hypoparathyroidism consists of calcium and active vitamin D supplementation. Patients must self-manage their symptoms throughout the day, with some taking up to 10 tablets per day. On average, symptoms last 13 ± 9 hours per day, and more severe disease has a greater impact on patients' lives (Hadker et al., Endocr. Pract. 20(7):671-679 (2014), incorporated by reference in its entirety). While supplementation aims to suppress the symptoms of hypocalcemia, it worsens renal function, fails to improve bone structure, and fails to improve emotional and cognitive impairment. The ideal treatment for hypoparathyroidism would mimic endogenous levels of PTH, which are maintained throughout the day, in order to improve hypocalcemia, hypercalciuria, and hyperphosphatemia.

[0013] Non-hormonal vitamin D forms have significantly reduced affinity for the VDR and significantly increased affinity for DBP. DBP is the primary transporter of vitamin D metabolites. Its plasma concentration is 6-7 μM and is detected in all body fluid compartments. DBP concentrations exceed those of physiological vitamin D metabolites. DBP is important for transporting vitamin D from the skin into the circulation and across cell membranes into the cytoplasm, where it is activated to its hormonal form. The affinity of non-hormonal vitamin D for DBP is significantly higher than that of hormonal forms. In contrast, the affinity of hormonal forms for the VDR is significantly higher than that of non-hormonal forms.

[0014] Vitamin D and vitamin D analogs have been approved for the treatment of osteoporosis and secondary hyperparathyroidism. Vitamin D has been shown to inhibit proliferation and induce differentiation in both normal and cancer cells. The levels of vitamin D required for this activity cause severe toxicity in the form of hypercalcemia. Vitamin D analogs have been approved for the treatment of psoriasis, and others are currently being tested for the treatment of cancer. Many of the analogs found to have reduced calcemic effects contain side-chain modifications. These modifications do not significantly affect VDR binding and therefore exhibit comparable or even greater efficacy in cell-based proliferation assays. However, many of these modifications have been shown to reduce binding to DBP, thereby shortening their half-life in the bloodstream.

[0015] Forms of vitamin D are known that bind with higher affinity to DBP or VDR. (See Haddad, JG, J. Steroid Biochem. Molec. Biol. Vol. 53, No. 1-6:579-582 (1995); Norman et al., J. Steroid Biochem. & Mol. Biol. 76:49-59, 51 (2001)). Norman teaches the difference between hormonal and non-hormonal forms of vitamin D. Norman first explains the basis for this teaching: "Vitamin D, and all of its metabolites, are conformationally very flexible compared to other steroid hormones..." This results in "a wide range of molecular shapes available for binding to receptors involved in 1a,25(OH)2D3-mediated biological responses, as well as binding to DBP..." (Norman, 50). This conformational flexibility is demonstrated in Norman's Figure 2. Vitamin D conformations that select for DBP (non-hormonal) or VDR (hormonal) are presented in Norman's Table 1. The binding affinities of a number of vitamin D metabolites to DBP or VDR are shown in Norman's Table 2 and Figure 6. Norman further states on page 51: "The preferred ligand for DBP is 25(OH)D3, and for VDRnuc The preferred ligand for VDR is 1a,25(OH)2D3. Thus, 25(OH)D3 binds to DBP 668 times more strongly than 1a,25(OH)2D3. In contrast, 1a,25(OH)2D3 binds to VDR 668 times more strongly than 25(OH)D3. nuc , which is incorporated by reference in its entirety.

[0016] The addition of poly(ethylene glycol) or (PEG) is a known method for extending the half-life of some compounds by decreasing renal clearance, reducing aggregation, and potentially lowering undesirable immune recognition (Jain, Crit. Rev. Ther. Drug Carrier Syst. 25:403-447 (2008)). PEG is typically used at a fairly large size (20-40 kDa) to maximize circulatory half-life. This can be achieved by using either a single large PEG or multiple small PEGs attached to the compound (Clark et al. J. Biol. Chem. 271:21969-21977 (1996); Fishburn, J. Pharm. Sci. 97:4167-4183 (2008)). The above is incorporated by reference in its entirety. Summary of the Invention

[0017] The present invention provides a long-acting parathyroid hormone peptide (PTH) that is retained in the serum of a subject for a period significantly longer than the native hormone. The long-acting PTH is conjugated to the carbon-3 position of non-hormonal vitamin D via a modest-length scaffold that facilitates its purification, detection, solubility, and efficacy at the PTH receptor (PTHR).

[0018] Accordingly, the present invention provides pharmaceutical compositions comprising parathyroid hormone peptides (PTH), each conjugated via a scaffold to a non-hormonal vitamin D moiety (PTH conjugate) and a pharmaceutical excipient, wherein the PTH conjugates are substantially uniform in size and have an absorbance of 18,600 M at 280 nm as measured by mass spectrometry. -1 cm -1 The PTH conjugate has a solubility in phosphate buffered saline (PBS) of at least about 0.550 mM as measured using an extinction coefficient of 1000 .mu.m. In some embodiments, the PTH conjugate comprises a scaffold, wherein the scaffold is a 36-subunit poly(ethylene glycol) scaffold (PTH-PEG36-VitD).

[0019] In some embodiments of the invention, PTH-PEG36-VitD has 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(succinimido-propionylamino-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(succinimido-propionylamino-PEG-propionyl-aminopropyl-25-hydroxy-vitamin D)-NH

[0020] In some embodiments of the invention, the PTH conjugates have approximately the same activity at the PTH receptor (PTHR) as the equivalent unconjugated PTH peptide.

[0021] In some embodiments of the invention, the PTH conjugate is isolated as a carbonate salt. In other embodiments, the PTH conjugate is isolated as an acetate salt. In some embodiments of the invention, the PTH conjugate has an absorbance of 18,600 M at 280 nm. -1 cm -1 The pharmaceutical composition has a solubility of at least about 9.67 mM in PBS, as measured using an extinction coefficient of 0.05%. In another embodiment, 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 another embodiment, the pharmaceutical composition is formulated with mannitol. In another embodiment, the pharmaceutical composition is formulated with polysorbate 80 (PS80).

[0022] In some embodiments of the invention, PTH-PEG36-Vit D is formulated as an acetate salt, the pharmaceutical composition is formulated at a pH of about 5.5, and the pharmaceutical composition comprises mannitol and PS 80. In a preferred embodiment, PTH-PEG36-Vit D is formulated at a concentration of 0.4 mg / ml in about 10 mM sodium acetate buffer, about 4.5% mannitol, and about 0.25% polysorbate 80 at a pH of about 5.5.

[0023] In some embodiments, PTH-PEG36-VitD is formulated as an acetate salt at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 mM or greater. 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 of about 2.5%-5%, more preferably 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 of about 0% to 0.25%.

[0024] In some embodiments of the invention, the PTH conjugate is formulated with a methionine concentration of about 0-40 mM. In other embodiments, the PTH conjugate is formulated with a methionine concentration of about 0, 5, 10, 20, or 40 mM. In a preferred embodiment, the PTH conjugate is formulated with a methionine concentration of about 40 mM.

[0025] In some embodiments of the 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.

[0026] In some embodiments of the present invention, the PTH conjugate has a serum half-life of 7-15 hours in rats. In other embodiments, the PTH conjugate has a subcutaneous bioavailability of at least about 10-13% in rats. In other embodiments, the PTH conjugate has a serum half-life of about 24-32 hours in cynomolgus monkeys. In other embodiments, the PTH conjugate has a subcutaneous bioavailability of about 45-54% in cynomolgus monkeys.

[0027] In some embodiments of the invention, daily administration of a PTH conjugate at a dose of about 6 μg / kg results in an average increase in serum calcium levels in TPTx rats of at least about 17% above the zero dose level when measured within 24 hours after injection. In other embodiments, daily administration of a PTH conjugate at a dose of about 60 μg / kg results in an average increase in serum calcium levels in TPTx rats of at least about 37% above the zero dose level when measured within 24 hours after injection. In other embodiments, administration of a PTH conjugate at a dose of about 2 μg / kg every other day results in an increase in serum calcium levels in cynomolgus monkeys of at least about 0.9 mg / dl above the zero dose level when measured 12 hours after injection on day 5. In other embodiments, administration of a PTH conjugate at a dose of about 100 μg / kg every other day results in an increase in serum calcium levels in cynomolgus monkeys of at least about 6.1 mg / dl above the zero dose level when measured 12 hours after injection on day 5. In another embodiment, a dose of about 10 μg / kg of the PTH conjugate reduces the ratio of urinary calcium levels to serum calcium levels in cynomolgus monkeys to about 1.8 or less, hi another embodiment, a dose of about 100 μg / kg of the PTH conjugate reduces the ratio of urinary calcium levels to serum calcium levels in cynomolgus monkeys to about 1.2 or less.

[0028] In some embodiments of the invention, when the PTH conjugate is administered subcutaneously at about 6 μg / kg daily, it reduces serum phosphate levels in TPTx rats by at least about 14% after about 12 days. In other embodiments, when the PTH conjugate is administered subcutaneously at about 60 μg / kg daily, it reduces serum phosphate levels in TPTx rats by at least about 39% after about 12 days. In other embodiments, when the PTH conjugate is administered subcutaneously at about 7.0 μg / kg every other day, it reduces serum phosphate levels in cynomolgus monkeys by at least about 1.0 mg / dl after about 21 days. In other embodiments, when the PTH conjugate is administered subcutaneously at about 20 μg / kg every other day, it reduces serum phosphate levels in cynomolgus monkeys by at least about 1.8 mg / dl after about 11 days.

[0029] In some embodiments of the invention, the PTH conjugate binds to vitamin D binding protein (VDBP) with a dissociation constant of about 5.2 μM.

[0030] The present invention provides a method of treating a subject suffering from a condition selected from the list comprising 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, alopecia, chemotherapy-induced alopecia, thrombocytopenia, autoimmune polyglandular 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 subject a pharmaceutical composition of the present invention. In some embodiments, the administering step is accomplished via subcutaneous delivery, intramuscular delivery, intravenous delivery, controlled release delivery, transdermal delivery, parenteral delivery, or via an implanted reservoir. In preferred embodiments, administration is via 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.

[0031] In some embodiments of the invention, a dose is administered about every day, hi other embodiments, a dose is administered about every other day.

[0032] The present invention provides a pharmaceutical composition of the present invention for use in treating hypoparathyroidism, hypocalcemia, hyperphosphatemia, hypercalciuria, osteoporosis, fracture repair, osteomalacia, osteoporosis 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, alopecia, chemotherapy-induced alopecia, thrombocytopenia, polyglandular autoimmune 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. In another embodiment, the present invention provides a medicament comprising the pharmaceutical composition of the present invention.

[0033] 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, alopecia, chemotherapy-induced alopecia, thrombocytopenia, polyglandular autoimmune 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 A with Bartter type 5 The present invention provides a method of treating a subject suffering from a condition selected from the group consisting of DH1, the method comprising administering to the subject a pharmaceutical composition, the pharmaceutical composition comprising a plurality of parathyroid hormone peptides (PTH), each of which is conjugated via a scaffold to a non-hormonal vitamin D (PTH conjugate) and a pharmaceutical excipient, wherein each PTH peptide in the plurality is conjugated to a scaffold of approximately uniform size as measured by mass spectrometry, and the method comprises subcutaneously administering repeated doses of the pharmaceutical composition, wherein the repeated doses result in a ratio of maximum concentration to minimum concentration of the PTH conjugate in a serum sample from the subject (Cmax / Cmin ratio) of about 8.0 or less.

[0034] In some embodiments, the Cmax / Cmin ratio is about 5.37 or less. In preferred embodiments, the Cmax / Cmin ratio is about 4.59 or less. In more preferred embodiments, the Cmax / Cmin ratio is about 3.36 or less. In more preferred embodiments, the Cmax / Cmin ratio is about 2.38 or less. In more preferred embodiments, the Cmax / Cmin ratio is about 1.83 or less. In most preferred embodiments, the Cmax / Cmin ratio is about 1.79 or less.

[0035] In some embodiments of the invention, the PTH conjugate has a serum half-life of 7-15 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 24-32 hours. In other embodiments, the PTH conjugate has a subcutaneous bioavailability of about 45-54%.

[0036] In some embodiments, administration of the PTH conjugate at a dose of about 2.0 μg / kg every other day results in an increase in serum calcium levels of at least about 0.9 mg / dl over the zero dose level when measured 12 hours post-injection on day 5. In other embodiments, administration of the PTH conjugate at a dose of about 100 μg / kg every other day results in an increase in serum calcium levels of at least about 6.1 mg / dl over the zero dose level when measured 12 hours post-injection on day 5.

[0037] In some embodiments, a dose of about 10 μg / kg of the PTH conjugate reduces the ratio of urinary calcium levels to serum calcium levels to about 1.8 or less, and in other embodiments, a dose of about 100 μg / kg of the PTH conjugate reduces the ratio of urinary calcium levels to serum calcium levels to about 1.2 or less.

[0038] In some embodiments of the invention, when the PTH conjugate is administered subcutaneously at about 6 μg / kg daily, it reduces serum phosphate levels by at least about 14% after about 12 days. In other embodiments, when the PTH conjugate is administered subcutaneously at about 60 μg / kg daily, it reduces serum phosphate levels by at least about 39% after about 12 days.

[0039] The invention provides kits comprising the pharmaceutical composition of the invention and instructions for its use to treat a condition in a patient. In some embodiments, the condition is hypoparathyroidism.

[0040] In some embodiments of the invention, PTH-PEG36-VitD has the following structure: [ka] Includes.

[0041] In another embodiment of the invention, PTH-PEG36-VitD has the following structure: [ka] Includes.

[0042] The present invention provides a method for producing a pharmaceutical composition of the present invention, comprising conjugating a PTH peptide, a scaffold, and a non-hormonal vitamin D moiety to form a PTH conjugate, wherein the conjugating step is carried out at a pH of 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 conjugating step is carried out at a pH of about 6.0. In other embodiments, the method further comprises purifying the conjugate at a pH of less than about 8.0. In a preferred embodiment, the purification step is at a pH of about 5.5.

[0043] The present invention provides a pharmaceutical carrier comprising Formula I, [ka] During the ceremony, B is a targeting group that is vitamin D that is not hydroxylated at carbon 1 and is conjugated to (L)a at carbon 3; S is a scaffold moiety, comprising poly(ethylene glycol) consisting of 36 repeating ethylene glycol units; C is a maleimide group, (L) a is a linker containing (CH2)3NHC(O)CH2, (M) b is a linker containing HNC(O)(CH2)2.

[0044] In some embodiments of the present invention, the pharmaceutical carrier comprises a compound having Formula VI. [ka]

[0045] The present invention provides a method for producing a pharmaceutical composition comprising parathyroid peptide (PTH) and the pharmaceutical carrier described above, comprising the steps of: 1) purifying the compound having formula VI; 2) conjugating a compound having formula VI to PTH; The present invention provides a method comprising:

[0046] In some embodiments, the purified compound having Formula VI is not removed from the purification solvent prior to the conjugation step. In other embodiments, the purification step is accomplished by high pressure liquid chromatography (HPLC). In other embodiments, the conjugate is isolated as a carbonate salt. In other embodiments, the conjugate is isolated as an acetate salt.

[0047] In some embodiments of the invention described above, the PTH comprises the amino acid sequence of SEQ ID NO:3.

[0048] 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, alopecia, chemotherapy-induced alopecia, thrombocytopenia, autoimmune polyglandular syndrome The present invention provides a method of treating a human suffering from a condition selected from the group consisting of Group 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 human a pharmaceutical composition as described herein. In some embodiments, the pharmaceutical composition is EXT608. In a preferred embodiment, the condition is hypoparathyroidism.

[0049] In some embodiments, the administering step is accomplished via subcutaneous delivery, via intramuscular delivery, via intravenous delivery, via controlled release delivery, via transdermal delivery, via parenteral delivery, or via an implanted reservoir. In a preferred embodiment, administration is via the subcutaneous route.

[0050] 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 every day, about every other day, about every third day, about every fourth day, about every fourth day, about every fifth day, about every sixth day, about every seventh day, about every eighth day, or about every ninth day.

[0051] In some embodiments, the dose provides a Cmax of about 0.49-5.0 ng / ml. In one preferred embodiment, the dose provides a Cmax of about 5.0 ng / ml.

[0052] In some embodiments, the dose provides a Tmax of about 3.3 to 5.3 hours. In one preferred embodiment, the dose provides a Tmax of about 2.7 hours.

[0053] In some embodiments, the dose provides an AUC of about 5.9 to 118 (ng / ml)*h. In a preferred embodiment, the dose provides an AUC of about 118 (ng / ml)*h.

[0054] In some embodiments, the dose provides a Tlast of about 24 to 168 hours. In a preferred embodiment, the dose provides a Tlast of about 168 hours. In one preferred embodiment, the dose provides 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.

[0055] In some embodiments, the dose is about 324 μg SC and the T 1 / 2 In other embodiments, the dose is about 324 μg SC, resulting in an AUC of about 151 (ng / ml)*h. inf In another embodiment, the dose is about 324 μg SC, resulting in a clearance divided by bioavailability (CL / F) of about 2.2 L / h. In another embodiment, the dose is about 324 μg SC, resulting in an apparent volume of distribution during the terminal phase divided by bioavailability (Vz / F) of about 280 L. In another embodiment, the dose is about 324 μg SC, resulting in a mean retention time (MRT) of about 105 hours. In a preferred embodiment, the dose is about 324 μg SC, resulting in a T of about 90 hours. 1 / 2 , AUC of approximately 151 (ng / ml)*h inf , resulting in a CL / F of about 2.2 L / h, a Vz / F of about 280 L, and an MRT of about 105 hours.

[0056] In some embodiments of the present invention, the pharmaceutical composition increases serum calcium levels for about 24 hours after administration. In other embodiments, the pharmaceutical composition reduces endogenous PTH(1-84) levels. In other embodiments, the pharmaceutical composition does not cause a significant increase in urinary calcium when measured about 36 hours after administration.

[0057] The present invention provides pharmaceutical compositions as disclosed herein, wherein the PTH conjugate is formulated with a methionine concentration of about 0-40 mM. In some embodiments, the PTH conjugate is formulated with a methionine concentration of about 0, 5, 10, 20, or 40 mM. In a preferred embodiment, the PTH conjugate is formulated with a methionine concentration of about 40 mM.

[0058] The present invention provides pharmaceutical compositions as disclosed herein, wherein the PTH conjugate exhibits precipitation from a solution of about 0.411 or less optical density (OD) when measured at a wavelength of 600 nm after shaking the pharmaceutical composition for 308 hours. In some embodiments, the PTH conjugate exhibits precipitation from a solution of about 0.242 or less optical density (OD) when measured at a wavelength of 600 nm after shaking the pharmaceutical composition for 308 hours. In a preferred embodiment, the PTH conjugate exhibits precipitation from a solution of about 0.015 or less optical density (OD) when measured at a wavelength of 600 nm after shaking the pharmaceutical composition for 308 hours.

[0059] The present invention provides pharmaceutical compositions as disclosed herein, wherein the purity of the PTH conjugate decreases by no more than about 7.7% after 69 hours of incubation at 4° C. In some embodiments, the purity of the PTH conjugate decreases by no more than about 14.9% after 13 days of incubation at room temperature. [Brief explanation of the drawings]

[0060] [Figure 1] How to synthesize EXT608. [Figure 2] UPLC analysis of EXT601. [Figure 3] UPLC analysis of EXT608. [Figure 4] MALDI-TOF mass spectrometry analysis of EXT601. [Figure 5] MALDI-TOF mass spectrometry analysis of EXT608. [Figure 6] Cell-based PTHR1 activity assay comparing PTH(1-34), EXT601, EXT607, and EXT608. [Figure 7] Kinetic binding analysis of vitamin D binding protein (DBP) to immobilized EXT607 by biolayer interferometry (BLI) as a function of DBP concentration (0-75 mM). For each DBP concentration, both binding association and dissociation data are shown, along with a calculated fit generated using ForteBio Octet Data Analysis Software. Inset: Plot of steady-state BLI shift as a function of DBP concentration. The calculated steady-state curve fit yielded a binding equilibrium constant (KD) of 8.1 μM + 2.5 μM. [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 mM). For each DBP concentration, both binding association and dissociation data are shown, along with a calculated fit generated using ForteBio Octet Data Analysis Software. Inset: Plot of steady-state BLI shift as a function of DBP concentration. The calculated steady-state curve fit yielded a binding equilibrium constant (KD) of 5.2 μM + 1.1 μM. [Figure 9] Cell-based PTHR1 activity assay comparing EXT606, EXT608, EXT611, EXT615, EXT616 and EXT617. [Figure 10] Pharmacokinetics of EXT601 (157 mg / kg) administered subcutaneously to rats. Error bars indicate standard deviation (n=3). [Figure 11]Pharmacokinetics of EXT607 administered intravenously (iv, dashed line) and subcutaneously (sc, solid line) to rats. Error bars indicate standard deviation (n=3). [Figure 12] Pharmacokinetics of EXT607 administered intravenously (iv, dashed line) and subcutaneously (sc, solid line) to cynomolgus monkeys. Error bars indicate standard deviation (n=3). [Figure 13] Repeat-dose subcutaneous pharmacokinetics of EXT608 in rats measured after the last dose following either 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). [Figure 14] Repeat-dose subcutaneous pharmacokinetics of EXT608 in cynomolgus monkeys measured after the last dose following either 21 days of every-other-day dosing (1.4, 7, and 20 μg / kg) or 89 days of every-other-day dosing (0.7 and 2 μg / kg). Error bars indicate standard deviation (n=3). [Figure 15] Serum calcium levels in cynomolgus monkeys following alternate-day subcutaneous dosing of EXT607 at 0, 10, 30, or 100 mg / kg beginning on day 5. Animals were dosed at t=0 on days 1, 3, and 5, with the day 5 dose indicated by the vertical arrow on the graph. Urine was collected beginning on day 8 (72 h on the time axis), as indicated by the horizontal arrow. [Figure 16] Serum calcium levels in cynomolgus monkeys on days 1, 11, and 21 after subcutaneous administration of EXT608 at 0, 1.4, 7, or 20 mg / kg every other day. Error bars indicate standard deviation (n=4). p values ​​relative to the 0 mg / kg group (vehicle) were calculated using Student's t-test (*<0.05, #<0.01, &<0.001). [Figure 17] Serum phosphate levels in cynomolgus monkeys on days 1, 11, and 21 after alternate-day subcutaneous administration of EXT608 at 0, 1.4, 7, or 20 mg / kg. Error bars indicate standard deviation (n=4). p values ​​relative to the 0 mg / kg group (vehicle) were calculated using Student's t-test (*<0.05, #<0.01, &<0.001). [Figure 18]Serum calcium levels in cynomolgus monkeys on days 1, 45, and 89 after alternate-day subcutaneous administration of EXT608 at 0, 0.2, 0.7, or 2 mg / kg. p values ​​relative to the 0 mg / kg group (vehicle) were calculated using Student's t-test (*<0.05, #<0.01, &<0.001). [Figure 19] Serum calcium levels in TPTx rats on days 1, 12, and 27 after daily subcutaneous administration of EXT607 at 0 (vehicle), 1, 3, or 10 nmol / kg. Serum calcium levels were normalized to the group that underwent sham surgery and received vehicle (sham vehicle). Error bars indicate standard deviation (n=5). p values ​​relative to the 0 mg / kg group (vehicle) were calculated using Student's t-test (*<0.05, #<0.01, &<0.001). [Figure 20] Serum calcium levels in TPTx rats on days 1, 12, and 27 after daily subcutaneous administration of 10 nmol / kg PTH(1-34) and PTH(1-84) were compared with the vehicle and sham vehicle control groups in Figure 19. Serum calcium values ​​were normalized to the sham vehicle group. Error bars indicate standard deviation (n=5). p values ​​relative to the 0 mg / kg group (vehicle) were calculated using Student's t-test (*<0.05, #<0.01). [Figure 21] Serum phosphate levels in TPTx rats on days 1, 12, and 27 after daily subcutaneous administration of EXT607 at 0 (vehicle), 1, 3, or 10 nmol / kg. Serum phosphate values ​​were normalized to a group that underwent sham surgery and received vehicle (sham vehicle). Error bars indicate standard deviation (n=5). p values ​​relative to the 0 mg / kg group (vehicle) were calculated using Student's t-test (*<0.05, #<0.01, &<0.001). [Figure 22]Serum phosphate levels in TPTx rats on days 1, 12, and 27 after daily subcutaneous administration of 10 nmol / kg PTH(1-34) and PTH(1-84) were compared with vehicle and sham vehicle control groups in Figure 21. Serum phosphate values ​​were normalized to the sham vehicle group. Error bars indicate standard deviation (n=5). p values ​​relative to the 0 mg / kg group (vehicle) were calculated using Student's t-test (*<0.05, #<0.01). [Figure 23A] Pharmacokinetics of a single subcutaneous dose of EXT608 demonstrates significantly improved serum half-life in humans. Error bars represent standard error of the mean (n=3). [Figure 23B] Changes in albumin-adjusted serum calcium levels in healthy human participants after a single subcutaneous dose of EXT608 at 0, 36, 108, 216, or 324 mg. Baseline serum calcium values ​​were calculated by averaging each individual's -24h and -1h measurements. Error bars indicate the standard error of the mean (n=3). [Figure 23C] Changes in serum endogenous PTH(1-84) levels in healthy human participants after a single subcutaneous dose of EXT608 at 0, 36, 108, 216, or 324 mg. For each individual, the -1 h measurement was used as the baseline value. Error bars indicate the standard error of the mean (n=3). Detailed Description of the Invention

[0061] The present invention provides long-acting parathyroid hormone peptides (PTH) that are retained in the serum of subjects for periods significantly longer than the natural hormone. These are intended to replace the endogenous PTH that is deficient in patients with hypoparathyroidism and related conditions. Long-acting PTH is conjugated to the carbon-3 position of non-hormonal vitamin D via a modestly long scaffold that facilitates its purification, detection, solubility, and efficacy at the PTH receptor (PTHR). PTH is conjugated to non-hormonal vitamin D via a 36-mer poly(ethylene glycol) moiety (PTH-PEG36-VitD). The present invention also provides optimized manufacturing methods and formulations. PTH-PEG36-VitD exhibits significantly improved serum half-life and bioavailability compared to unconjugated PTH peptide. PTH-PEG36-VitD also significantly increases serum calcium, reduces urinary calcium, and reduces serum phosphate.

[0062] The present invention provides carrier-drug conjugates containing a targeting group that is non-hormonal vitamin D, a vitamin D analog, or a vitamin D metabolite. Examples include vitamin D-based molecules that are not hydroxylated at carbon 1 (C1). The carrier is linked to a therapeutic compound at carbon 3 (C3). Without wishing to be bound by theory, it is believed that hormonal forms of vitamin D are not suitable for the carriers described herein because they can be toxic due to the induction of hypercalcemia. Furthermore, hormonal forms bind intracellular vitamin D receptors, potentially inappropriately targeting the carrier-drug conjugate to undesirable cells or tissues. In contrast, non-hormonal forms of vitamin D bind vitamin D binding proteins (DBP or VDBP) and remain in circulation longer.

[0063] In some embodiments, the present invention provides EXT607 and EXT608 (described below), which are useful 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 concentrations of 10-65 pg / mL (Aloia et al., Endocr. Pract. 12(2):137-144(2006)). Hypoparathyroidism is a rare disorder characterized by PTH deficiency and can occur when individuals are born without parathyroid glands (usually due to a chromosomal defect). It can also occur when the parathyroid glands are damaged during surgery (e.g., thyroidectomy), or when organs are damaged due to an autoimmune response, iron accumulation, magnesium deficiency, or other idiopathic causes (Bilezikian et al., J. Bone Miner. Res. 26(10):2317-2337(2011)). PTH deficiency causes hypocalcemia, hypercalciuria, hyperphosphatemia, and, if left untreated, persistent muscle spasms, paresthesia, seizures, and irregular heartbeat (Shoback, N. Engl. J. Med. 359:391-403(2008)). Additionally, patients with hypoparathyroidism suffer from cognitive impairments, including anxiety, depression, memory impairment, and various forms of "brain fog" (Aggarwal et al., Eur. J. Endocrin. 168:895-903(2013)).

[0064] EXT607 and EXT608 were designed to extend the serum half-life of PTH and achieve a pharmacokinetic profile that more closely resembles physiological PTH levels. EXT607 and EXT608 were created by modifying PTH using a vitamin D conjugate. A version of 25-hydroxycholecalciferol [25(OH)-vitamin D] was attached to the C-terminus of PTH(1-34) via a low-molecular-weight poly(ethylene glycol) (PEG) spacer moiety (<2 kDa). The 25(OH)-vitamin D version used is an inactive "storage" form of vitamin D that has no activity at the vitamin D receptor (VDR). Therefore, peptide-conjugated vitamin D is unlikely to interfere with normal vitamin D metabolic pathways (Lips, Prog. Biophy. Mol. Biol. 92:4-8 (2006)). Because the modifications are very small (<2.4 kDa), they do not affect the potency of PTH(1-34), as would be expected for peptides with larger modifications, such as 20 kDa PEG. EXT607 / EXT608 exhibit prolonged pharmacokinetics after subcutaneous administration, with terminal half-lives of 8-15 hours in rats and 24-32 hours in NHPs.

[0065] In some embodiments, EXT607 and EXT608 are retained in the circulation for extended periods through their interaction 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 increases from 11 minutes to 8-15 hours after vitamin D conjugation, while in NHPs the half-life is 24-32 hours.

[0066] The carrier molecule is attached to PTH using chemical reactions described herein, those described in WO2013172967 and WO2016065042 (incorporated herein by reference in their entireties), or other known chemical reactions in the art. The carrier improves the efficacy, absorption, bioavailability, circulatory half-life, or pharmacokinetic properties of the therapeutic compound. In certain embodiments, the carrier further comprises what is described herein as a "scaffold," which functions, among other things, as a non-releasable "spacer" between the targeting group and the therapeutic compound. In other embodiments, the carrier lacks a scaffold.

[0067] The carriers are designed to be suitable for both human and veterinary use. The carriers serve the purpose of improving the pharmacokinetic properties of the biological or chemical entities coupled, conjugated, or fused to the carrier. This occurs through the interaction of the targeting group with DBP. DBP actively transports molecules from the administration site into circulating plasma quickly and efficiently, thereby reducing the drug's exposure to degradative enzymes. By binding to DBP, the carriers also improve the drug's circulatory half-life. This increases the drug's potency and therapeutic effectiveness by preventing renal filtration and other excretion processes.

[0068] In describing and claiming one or more embodiments of the present invention, the following terminology will be used in accordance with the definitions set out below.

[0069] The term "absorption" refers to the movement of a drug into the bloodstream. The drug must be introduced via some route of administration (e.g., oral, topical or transdermal, subcutaneous, intramuscular, or intravenous) or in a particular dosage form such as a tablet, patch, capsule, or liquid.

[0070] "Antagonist" refers to a molecule capable of neutralizing, blocking, inhibiting, neutralizing, reducing, or preventing the activity of a particular or designated protein, including, in the case of a ligand, binding to one or more receptors, or, in the case of a receptor, binding to one or more ligands. Antagonists include antibodies and antigen-binding fragments thereof, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and their metabolites, transcriptional and translational control sequences, and the like. Antagonists also include small molecule inhibitors of proteins, hormones, or other bioactive molecules. Antagonists may be fusion proteins, receptor molecules, antisense molecules, aptamers, ribozymes, or derivatives that specifically bind to proteins, hormones, or other bioactive molecules, thereby blocking their binding to the target.

[0071] "Antibody" (Ab) and "immunoglobulin" (Ig) refer to glycoproteins having similar structural characteristics. While antibodies exhibit binding specificity to a specific antigen, immunoglobulins include both antibodies and other antibody-like molecules which generally lack antigen specificity. The latter type of polypeptide is produced at low levels by the lymph system, for example, and at increased levels by myelomas.

[0072] An "aptamer" is a nucleic acid-based compound that is selected to bind to a specific target. Examples of aptamer-based therapeutic compounds are described in WO07 / 035922, the contents of which are incorporated herein by reference in their entirety.

[0073] The term "bioavailability" refers to the fraction of an administered dose that reaches the systemic circulation unchanged and is one of the key pharmacokinetic properties of a drug. When a drug is administered intravenously, its bioavailability is 100%. When a drug is administered via other routes (such as orally), its bioavailability is generally reduced (due to incomplete absorption and first-pass metabolism) or may vary from patient to patient. Bioavailability is an important pharmacokinetic parameter that is considered when calculating dosages for non-intravenous routes of administration.

[0074] "Carriers" are compounds that can be conjugated, fused, coupled, or formulated with a therapeutic compound to improve drug absorption, half-life, bioavailability, pharmacokinetic, or pharmacodynamic properties. These include targeting groups, coupling groups, and optionally, scaffold moieties. In some embodiments, carriers not only carry the therapeutic compound from a subcutaneous injection site into the circulatory system, but may also carry the therapeutic compound within the circulatory system for extended periods of time.

[0075] An "effective amount" refers to an amount of a therapeutic compound effective (at dosages and for periods of time necessary) to achieve a desired therapeutic or preventative result. A "therapeutically effective amount" of a therapeutic compound may vary depending on factors such as an individual's medical condition, age, sex, and weight. A therapeutically effective amount may be measured, for example, by improved survival, faster recovery, or alleviation, improvement, or elimination of symptoms, or other accepted biomarkers or surrogate markers. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the therapeutic compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount of a therapeutic compound effective (at dosages and for periods of time necessary) to achieve a desired preventative result. Typically, a prophylactic dose is used in subjects before or at an earlier stage of disease, so the prophylactically effective amount will be less than the therapeutically effective amount, but this is not necessarily the case.

[0076] "Half-life" is a scientific term known in the art and refers to the time that elapses until half of the amount of a test molecule is no longer detectable. In vivo half-life refers to the time that elapses until half of the test molecule is no longer detectable in the circulating serum or tissues of a human or animal.

[0077] A "hormone" is a biological or chemical messenger that communicates between one cell (or group of cells) and another. As described herein, hormones for use in the present invention may be peptides, steroids, pheromones, interleukins, lymphokines, cytokines, or members of other hormone classes known in the art.

[0078] A "homolog" is a biologically active molecule similar to a reference molecule at the nucleotide sequence, peptide sequence, function, or structure level. Homologs may include sequence derivatives that share a specific percent identity with the reference sequence. Thus, in one embodiment, a homologous or derived sequence shares at least 70 percent sequence identity. In a preferred embodiment, a homologous or derived sequence shares at least 80 percent or 85 percent sequence identity. In a more preferred embodiment, a homologous or derived sequence shares at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity. A homologous or derived nucleic acid sequence may be defined by its ability to remain bound to a reference nucleic acid sequence under high stringency hybridization conditions. A homolog that shares structural or functional similarity with a reference molecule may also be a chemical derivative of the reference molecule. Methods for detecting, generating, and screening structural and functional homologs and derivatives are known in the art.

[0079] "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 desired degree of homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, higher relative temperatures tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation regarding stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers (1995), the contents of which are incorporated herein by reference in their entirety.

[0080] An "individual," "subject," or "patient" is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including human primates 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" refers to a healthy subject who has not been diagnosed with the disease, dysfunction, or condition identified in the individual, subject, or patient. A control subject does not suffer from any signs or symptoms associated with the disease, dysfunction, or condition.

[0081] A "drug" is an active substance manufactured for the treatment of a disease, disorder, or condition.

[0082] "Morpholinos" are synthetic molecules that are non-natural variants of natural nucleic acids that utilize phosphorodiamidate linkages and are described in U.S. Pat. No. 8,076,476, the contents of which are incorporated herein by reference in their entirety.

[0083] A "nucleic acid" is any group of polymers, either DNA, RNA, or variants thereof, that carry genetic information capable of directing cellular function. Nucleic acids may have enzymatic activity (e.g., ribozymes) or may be used to suppress gene expression in a subject (e.g., RNAi). Nucleic acids used in the inventions described herein may be single-stranded, double-stranded, linear, or circular. The present invention further incorporates the use of nucleic acid variants, including, but not limited to, aptamers, PNAs, morpholinos, or other non-natural variants of nucleic acids. By way of example, nucleic acids useful in the present invention are described in U.S. Patent No. 8,076,476, the contents of which are incorporated herein by reference in their entirety.

[0084] "Patient response" or "response" can be assessed 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 disease cell infiltration into adjacent peripheral organs and / or tissues; (4) inhibition (i.e., reduction, slowing, or complete cessation) of disease spread; (5) reduction in autoimmune pathology; (6) favorable change in expression of biomarkers associated with the disorder; (7) alleviation to some extent of one or more symptoms associated with the disorder; (8) an increase in the symptom-free period following treatment; or (9) a reduction in mortality at a given time point following treatment.

[0085] As used herein, the term "peptide" refers to any peptide containing two or more amino acids. The term peptide includes short peptides (e.g., peptides containing 2-14 amino acids), medium-length peptides (15-50 amino acids), 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 intended to mean a polymer composed of amino acid residues linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. 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 organisms. Peptides may contain amino acids other than the 20 genetically encoded amino acids. Peptides include those modified both by natural processes (e.g., processing and other post-translational modifications) as well as by chemical modification techniques. Such modifications are fully described in basic texts and, more specifically, in monographs, and are well known to those skilled in the art. Modifications can occur anywhere in a peptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.

[0086] As used herein, a "pharmaceutically acceptable carrier" or "therapeutically effective carrier" can be aqueous or non-aqueous (solid), such as alcoholic or oily, or a mixture thereof, and can contain surfactants, emollients, lubricants, stabilizers, dyes, fragrances, preservatives, acids or bases for adjusting pH, solvents, emulsifiers, gelling agents, moisturizers, stabilizers, humectants, sustained-release agents, moisturizers, or other ingredients commonly included in certain forms of pharmaceutical compositions. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically 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, for example, physiologically acceptable compounds that act to stabilize or increase the absorption of the specific inhibitor, such as sugar chains (such as glucose, sucrose, or dextran), antioxidants (such as ascorbic acid or glutathione), chelating agents, low molecular weight proteins, or other stabilizers or excipients.

[0087] 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 of the pharmacokinetic properties as desired for a particular therapeutic compound. Examples include, but are not limited to, decreased metabolic or secretory excretion, increased drug absorption, increased half-life, and / or increased bioavailability.

[0088] "PNA" refers to peptide nucleic acid, which has a chemical structure similar to DNA or RNA, using peptide bonds to link nucleotides or nucleosides together.

[0089] A "scaffold" is a molecule to which other molecules can be covalently or non-covalently attached 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 physical distance between two different molecular entities. A scaffold may also contain a reactive "linker" or may have beneficial therapeutic properties in addition to a drug. A linker is a site of attachment from one molecular entity to another. Thus, a scaffold of the present invention may be, for example, PEG, serum albumin, thioredoxin, immunoglobulins, reactive linker-containing modifying groups, water-soluble polymers, or therapeutic compounds. The scaffolds and linkers of the present invention are stable (i.e., non-releasable). Non-releasable linkers provide a more stable chemical bond than releasable linkers, and the attached molecular entity remains attached in vivo. However, in certain embodiments, they may be "releasable" under specific conditions. Releasable linkers have inherent instability, allowing the attached molecule to be released over time and under specific conditions.

[0090] "Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures.

[0091] As defined herein, "stringent conditions" or "high stringency conditions" are defined as: (1) those employing low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C; (2) those employing denaturing agents such as formamide during hybridization, e.g., 50% (v / v) formamide with 0.1% bovine serum albumin at 42°C; (3) overnight hybridization in 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, with a 10-minute wash at 42°C in 0.2x SSC (sodium chloride / sodium citrate) followed by a 10-minute high-stringency wash at 55°C consisting of 0.1x SSC containing EDTA.

[0092] "Therapeutic compounds" generally refer to small molecules, chemical entities, nucleic acids, nucleic acid derivatives, peptides, peptide derivatives, natural proteins, non-natural proteins, glycoproteins, and steroids that are administered to a subject to treat a disease or dysfunction or otherwise affect the health of an individual. As used herein, the term "therapeutic compound" has essentially the same meaning as the term "drug" or "therapeutic agent." The therapeutic compounds of the present invention are PTH protein and its derivatives.

[0093] As used herein, "treatment" refers to a clinical intervention that seeks to alter the natural course of the individual or cell being treated and can be performed before or during the course of a clinical condition. Desirable effects of treatment include preventing the occurrence or recurrence of a disease or condition or symptoms thereof, alleviating the disease condition or symptoms, reducing any direct or indirect pathological consequences of a disease, slowing the rate of disease progression, reducing or palliating the condition, and achieving remission or improved prognosis. In some embodiments, the methods and compositions of the invention are useful in attempting to delay the onset of a disease or disorder.

[0094] "Vitamin" is an art-recognized term and is defined as a fat-soluble or water-soluble organic substance essential in trace amounts for the normal growth and function of the body and obtained naturally from plant and animal foods or supplements.

[0095] "Vitamin D" is a group of fat-soluble secosteroids. Vitamin D exists in several forms (vitamers). The 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 taken orally as cholecalciferol (vitamin D3) or ergocalciferol (vitamin D2). Additionally, humans can synthesize it from cholesterol with sufficient sun exposure. Cholecalciferol may be modified in the liver or in vitro to 25-hydroxycholecalciferol ("25-hydroxyvitamin D"). 25-hydroxyvitamin D can be modified in the kidney or in vitro to the unique hormonal form of 1,25-hydroxyvitamin D.

[0096] "Vitamin D binding protein," "DBP," or "VDBP" is a naturally occurring circulating serum protein found in all mammals that, among other activities, can bind and transport vitamin D and its analogs to sites in the liver and kidney, where the vitamin is modified into its active form, maintaining various forms of vitamin D in the circulation for an average of 30 days in humans. 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).

[0097] The present invention contemplates non-hormonal vitamin D conjugates that bind DBP or functional DBP variants, and homologs containing conservative or non-conservative amino acid substitutions that substantially retain DBP activity. DBP-binding molecules or functional DBP variants may 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 above references are incorporated herein by reference in their entirety.

[0098] The term "water-soluble" refers to a moiety that has some 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, saccharides, poly(ethers), poly(amines), poly(carboxylic acids), and the like.

[0099] The present invention provides effective routes for the administration of proteins, peptides, other biologics, nucleic acids, and small molecule drugs. The present invention further provides effective routes of drug administration via transdermal, oral, parenteral, subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, intracranial injection, infusion, inhalation, ocular, topical, rectal, nasal, buccal, sublingual, vaginal, or implanted reservoir modes.

[0100] Additionally, the inventions described herein provide compositions and methods for maintaining the target binding activity, i.e., pharmacodynamics (PD), of a therapeutic compound. Additionally, compositions and methods for improving the pharmacokinetic (PK) profile of a therapeutic compound as described herein are also provided. The invention further provides compositions and methods for improving a drug absorption profile compared to the drug absorption profile of a drug using the same or a different route of administration but without the inventions described herein. The invention further provides compositions and methods for improving a drug bioavailability profile compared to the drug bioavailability profile of a drug using the same or a different route of administration but without the carriers described herein. The invention further provides compositions and methods for improving a drug half-life profile compared to the drug half-life profile of a drug using the same or a different route of administration but without the inventions described herein.

[0101] The present invention also provides an alternative route of drug administration that may be more cost effective or preferable for patients compared to drugs without the invention described herein.

[0102] The non-hormonal vitamin D carriers disclosed herein may improve the absorption, half-life, bioavailability, or pharmacokinetic properties of the conjugated PTH. Without wishing to be bound by theory, the carriers have the property of binding to natural DBP in the body. DBP may transport the carrier-drug complex from the administration site into the circulating serum. The vitamin D-DBP interaction may allow the therapeutic compound to remain in the circulation for a prolonged period of time. This prevents excretion from the body and increases the exposure of the therapeutic compound in the body, achieving a longer-lasting therapeutic effect. Furthermore, when conjugated to a carrier, a lower dose of the drug may be required compared to the unmodified form.

[0103] The therapeutic compound-carrier conjugates of the present invention typically have about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targeting groups individually attached to the therapeutic compound. The structures of each targeting group attached to the therapeutic compound can be the same or different. In a preferred embodiment, one or more targeting groups are stably or non-releasably attached to the therapeutic compound at the N-terminus, C-terminus, or other portion of the therapeutic protein. For example, a therapeutic compound-carrier conjugate may include a targeting group attached to the N-terminus and an additional targeting group attached to a lysine residue. In another embodiment, a therapeutic compound-carrier conjugate has a targeting group attached to the therapeutic protein via a modification such as a sugar residue, as part of a glycosylation site, or on an acylation site of the peptide, or attached to a phosphorylation site, or other natural or non-natural modification known to those skilled in the art. Attachment sites using a combination of the above sites are also contemplated. A preferred embodiment of the present invention includes a targeting group attached to a therapeutic compound at a specific site on the therapeutic compound. In another preferred embodiment, the attachment site on the protein may be a cysteine, a lysine, the N-terminus or the C-terminus.

[0104] In another embodiment, the scaffold is a pharmaceutically acceptable carrier. In a preferred embodiment, the scaffold is poly(ethylene glycol), polylysine, polyethyleneimine, poly(propylene glycol), a peptide, serum albumin, thioredoxin, an immunoglobulin, an amino acid, a nucleic acid, a glycan, a modifying group containing a reactive linker, a water-soluble polymer, a small carbon chain linker, or an additional therapeutic moiety.

[0105] The scaffolds of the present invention are of discrete lengths, facilitating manufacturing, solubility, detection, and pharmaceutical efficacy. In one embodiment, the soluble scaffold portion has some detectable solubility in aqueous or non-aqueous solutions. Methods for detecting and / or quantifying water solubility are well known in the art. Exemplary water-soluble polymers include peptides, saccharides, poly(ethers), poly(amines), poly(carboxylic acids), and the like.

[0106] Peptides may have mixed sequences or may be composed of a single amino acid, such as poly(lysine). An exemplary polysaccharide is poly(sialic acid). An exemplary poly(ether) is poly(ethylene glycol), e.g., m-PEG. Poly(ethyleneimine) is an exemplary polyamine, and poly(acrylic) acid is a representative poly(carboxylic acid). The polymer backbone of the water-soluble polymer can be poly(ethylene glycol) (i.e., PEG). However, it should be understood that other related polymers are suitable for use in the practice of the present invention, and the use of the terms PEG or poly(ethylene glycol) is intended to be inclusive rather than exclusive in this regard. The term PEG includes poly(ethylene glycol) in any of its forms, including alkoxy PEG, bifunctional PEG, multiarm PEG, forked PEG, branched PEG, pendant PEG (i.e., PEG or related polymers having one or more functional groups pendant to the polymer backbone), or PEG with degradable linkages. The polymer backbone can be linear or branched.

[0107] Branched polymer backbones are generally known in the art. Typically, branched polymers have a central branch core portion and multiple linear polymer chains connected to the central branch core. PEG is commonly used in branched forms, which can be prepared by adding ethylene oxide to various polyols, such as glycerol, pentaerythritol, and sorbitol. The central branch portion can also be derived from several amino acids, such as lysine. Branched poly(ethylene glycol) can be represented in the general form as R(-PEG-OH) m, where R represents a core portion, such as glycerol or pentaerythritol, and m represents the number of arms. Multi-armed PEG molecules, such as those described in U.S. Pat. No. 5,932,462 (the contents of which are incorporated herein by reference in their entirety), can also be used as polymer backbones.

[0108] Many other polymers are also suitable for the present invention. Nonpeptidic, 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 poly(alkylene glycols) such as poly(propylene glycol) ("PPG"), copolymers of ethylene glycol and propylene glycol, poly(oxyethylated polyols), poly(olefinic alcohols), polyvinylpyrrolidone), polylysine, polyethyleneimine, poly(hydroxypropylmethacrylamide), poly(α-hydroxy acids), poly(vinyl alcohols), polyphosphazenes, polyoxazolines, poly(N-acryloylmorpholines) (examples of which include those described in U.S. Pat. No. 5,629,384, the contents of which are incorporated herein by reference in their entirety), and copolymers, terpolymers, and mixtures thereof. The molecular weight of each chain of the polymer backbone can vary but typically ranges from about 100 Da to about 100,000 Da.

[0109] In other embodiments, the scaffold moiety may be a peptide, serum albumin, thioredoxin, immunoglobulin, amino acid, nucleic acid, glycan, modifying group containing a reactive linker, water-soluble polymer, small carbon chain linker, or additional therapeutic compound. In one embodiment, the scaffold moiety is non-toxic to humans and animals. In another embodiment, the scaffold is an endogenous serum protein. In another embodiment, the scaffold moiety is a water-soluble polymer. In another embodiment, the scaffold is a non-natural polymer. In another embodiment, the scaffold is a natural moiety modified by covalent attachment to an additional moiety (e.g., PEG, poly(propylene glycol), poly(aspartic acid), biomolecule, therapeutic moiety, or diagnostic moiety). The scaffolds and linkers of the present invention are stable (i.e., non-releasable). However, in certain embodiments, they may be "releasable" under specific conditions.

[0110] Conjugates of hydrophilic polymers such as PEG are known in the art. In its most common form, PEG is a linear polymer terminated at each end with a hydroxyl group, i.e., HO-CH2CHO--(CH2CHO)x--CH2CH2-OH, where x typically ranges from about 3 to about 4000. In a preferred embodiment, PEG has an essentially homodisperse molecular weight distribution. In another preferred embodiment, PEG is a linear polymer. In another preferred embodiment, PEG is a branched polymer.

[0111] Many terminally functionalized or branched derivatives, and a variety of sizes, are known in the art and commercially available. By way of example, conjugation of PEG or poly(ethylene oxide) (also known as PEO) may be carried out using the compositions and methods described herein, as well as 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.

[0112] In some embodiments, therapeutic compounds are paired with smaller or larger scaffold moieties. It is contemplated that therapeutic compounds can be paired with scaffold moieties ranging from 1 Da to 10 kDa. In some embodiments, scaffolds approximately equal to the molecular weight of small therapeutic compounds result in effective carrier-drug conjugates. Further empirical tuning of the scaffold size may result in improved efficacy. Without wishing to be bound by theory, if the scaffold (optionally in combination with a linker) is large enough to eliminate potential steric hindrance of the drug due to DBP binding, the pharmacokinetic properties and efficacy of the conjugate may be enhanced, and vice versa. Thus, upon conjugation of the therapeutic compound, its active region becomes exposed and available for functional activity, and the carrier becomes capable of binding DBP. Additional embodiments provide non-releasable attachments that prolong the circulation of the therapeutic agent. In some small peptide embodiments, such as PTH, the scaffold may be selected to approximately equal the molecular weight of the therapeutic agent.

[0113] In preferred embodiments, the conjugate of a therapeutic compound retains some or substantially all of its activity after conjugation. The active region of a given therapeutic agent is known in the art or can be empirically determined. In other embodiments, the conjugate remains therapeutically active while attached to the carrier. This embodiment may maximize circulation time as well as efficacy.

[0114] For example, scaffolds of the present invention can have a molecular weight of 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" scaffold may be between about 100 Da and 20,000 Da. In another embodiment, a "large" scaffold may be greater than about 20,000 Da, up to about 200,000 Da. In a preferred embodiment, the scaffold portion is between about 100 Da and 200,000 Da. In more preferred embodiments, the scaffold is 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, 20,00 Da to 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 scaffold may be varied to maximize the absorption, bioavailability, circulatory half-life or efficacy of the conjugated therapeutic compound.

[0115] Another component of the carrier molecule preferably includes a coupling group used to covalently attach a drug to the scaffold or carrier. 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 cross-linkers such as NHS-maleimide, combinations thereof, or other coupling groups familiar to those skilled in the art. The coupling groups of the present invention facilitate thiol, amide, oxime, hydrazone, and thiazolidinone linkages, or utilize cycloaddition reactions, also known as click chemistry, to attach the carrier to a therapeutic compound. In another embodiment, the composition preferably includes a combination of one or more therapeutic compounds attached to the coupling group of the scaffold molecule. The linkers of the present invention may be in the range of approximately 40 to 100 daltons. In preferred embodiments, the linker may be in the range of about 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 daltons. The linker may be modified to affect the stability or releasability of the linkage between the carrier and the therapeutic compound.

[0116] Those skilled in the art know that NHS groups are useful for coupling to natural peptides and proteins without the need for manipulation at the attachment site. NHS groups allow attachment to most proteins and peptides containing amino acids with amine groups, such as lysine residues. The use of NHS groups provides flexibility in the carrier conjugation site, as the protein structure and reaction time can affect the attachment site and number of carrier molecules conjugated to the therapeutic compound. Illustratively, by controlling the molar ratio of NHS carrier to therapeutic compound, those skilled in the art have some control over the number of carrier molecules attached to the therapeutic compound, thus allowing for conjugation of more than one carrier to a given therapeutic compound, if desired.

[0117] Conjugation of a carrier to a therapeutic compound is achieved by mixing a solution of the molecules together at a specific molar ratio using a compatible solution, buffer, or solvent. For example, a molar ratio of carrier to therapeutic compound 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 can be used. Varying the ratio can vary the number of distinct carriers attached to a therapeutic compound or can help select specific attachment sites. Attachment of carriers is also dependent on pH, buffer, salt, and temperature; varying these parameters, among others, can affect attachment sites, the number of carriers attached, and reaction kinetics. For example, selecting a reaction pH of pH 6 or less may be useful for selectively conjugating an aldehyde version of the carrier to the N-terminus of a therapeutic protein or peptide.

[0118] Furthermore, to maintain substantially the same activity of the therapeutic compound, conjugation to the carrier is performed at a site on the molecule that does not interfere with the therapeutic function. In the case of proteins, conjugation to the amino-terminus, carboxy-terminus, or internal reactive amino acid may be required. In the case of nucleic acids, conjugation to the 5'-terminus, 3'-terminus, or internal nucleotide, nucleoside, or derivative thereof may be required. In one embodiment, the carrier is conjugated to the nucleotide or nucleoside before being incorporated into the polynucleotide molecule.

[0119] The present invention provides a pharmaceutical carrier comprising Formula I, [ka] During the ceremony, B is (L) a a targeting group that is non-hormonal vitamin D, an analog thereof, or a metabolite thereof conjugated to S is a scaffold moiety and comprises poly(ethylene glycol), polylysine, polyethyleneimine, poly(propylene glycol), peptides, serum albumin, thioredoxin, immunoglobulins, amino acids, nucleic acids, glycans, modifying groups containing reactive linkers, polylactic acid, water soluble polymers, small carbon chain linkers, or additional therapeutic moieties; C is a bifunctional cross-linker such as 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 acylimidazole, a haloacetyl group, an iodoacetyl group, a bromoacetyl group, an SMCC group, a sulfo-SMCC group, a carbodiimide group, and an NHS-maleimide, or a combination thereof; (L )a and (M) b is -(CH2) n are linkers independently selected from -, -C(O)NH-, -HNC(O)-, -C(O)O-, -OC(O)-, -O-, -SS-, -S-, -S(O)-, -S(O)2- and -NH-; a is an integer from 0 to 4, b is an integer from 0 to 4, n is an integer from 0 to 3.

[0120] In a preferred embodiment, the present invention provides a carrier comprising a carrier of formula I: [ka] During the ceremony, B is a targeting group selected from vitamin D, a vitamin D analog, a vitamin D-related metabolite, an analog of a vitamin D-related metabolite, or a small carbon-based molecule to which DBP is attached; S is a scaffold moiety, and includes poly(ethylene glycol), polylysine, poly(propylene glycol), peptide, serum albumin, amino acid, nucleic acid, glycan, polylactic acid, water-soluble polymer, or small carbon chain linker; 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; (L) a and (M) b is -(CH2) n are linkers independently selected from -, -C(O)NH-, -HNC(O)-, -C(O)O-, -OC(O)-, -O-, -SS-, -S-, -S(O)-, -S(O)2- and -NH-; a is an integer from 0 to 4, b is an integer from 0 to 4, n is an integer from 0 to 3.

[0121] In a more preferred embodiment, the present invention provides a carrier comprising a carrier of formula I: [ka] During the ceremony, B is a targeting group selected from vitamin D, a vitamin D analog, or a vitamin D-related metabolite; S is a scaffold moiety and comprises poly(ethylene glycol), polylysine, or poly(propylene glycol); C is a maleimide group, a disulfide group, an aldehyde group, an NHS-ester group, or an iodoacetyl group; (L) a and (M) b is -(CH2) n are linkers independently selected from -, -C(O)NH-, -HNC(O)-, -C(O)O-, -OC(O)-, -O-, -SS-, -S-, -S(O)-, -S(O)2- and -NH-; a is an integer from 0 to 4, b is an integer from 0 to 4, n is an integer from 0 to 3.

[0122] In some preferred embodiments, the present invention provides supports comprising supports represented by Formula IIa, Formula IIb, and Formula IIc, [ka] During the ceremony, B is a targeting group selected from vitamin D, a vitamin D analog, or a vitamin D-related metabolite; S is a scaffold moiety and comprises poly(ethylene glycol) or poly(propylene glycol); C is a maleimide group, a disulfide group, an aldehyde group, an NHS-ester group, or an iodoacetyl group; L 1 is -(CH2) n - and L 3 is -(CH2) o - and (M) b is -(CH2) n are linkers independently selected from -, -C(O)NH-, -HNC(O)-, -C(O)O-, -OC(O)-, -O-, -SS-, -S-, -S(O)-, -S(O)2- and -NH-; b is an integer from 0 to 4, n is 3, o is 1.

[0123] WO13 / 172967 (incorporated herein by reference) exemplifies conjugates at carbon 25 (C25) of 25-hydroxy-vitamin D3. WO2016 / 065042 exemplifies conjugates at carbon 3 (C3) of 25-hydroxy-vitamin D3.

[0124] In certain most preferred embodiments of Formula IIa, B is represented by Formula III, S is poly(ethylene glycol), and (M) b -C is represented by formula IVa. [ka]

[0125] In certain most preferred embodiments of Formula IIb, B is represented by Formula III, S is poly(ethylene glycol), and (M) b-C is represented by formula IVb. [ka]

[0126] In certain most preferred embodiments of Formula IIc, B is represented by Formula III, S is poly(ethylene glycol), and (M) b -C is represented by formula IVc. [ka]

[0127] In certain most preferred embodiments, S is between about 100 Da and 200,000 Da. In other most preferred embodiments, the scaffold portion 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, 1000 Da and 200,000 Da, 5000 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.

[0128] In a specific embodiment, the present invention provides a carrier represented by formula V: [ka]

[0129] In Formula V, x-1 refers to the fact that when PEG is conjugated to x number of polymers in this reaction, the terminal PEG monomer is converted to an aldehyde, resulting in x-1 in the final formulation. In another specific embodiment, the present invention provides a carrier represented by Formula VI: [ka]

[0130] 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: [ka]

[0131] In certain embodiments, the present invention provides a method of producing a carrier of formula I, [ka] The method comprises reacting a compound of formula Ia with [ka] a compound of formula Ib; [ka] reacting in the presence of an amide coupling agent, B, S, C and L 1 , L 3 , and (M) b is defined as above, and L 2 is -C(O)NH-.

[0132] Those skilled in the art will recognize that the compounds of Formula Ia can be used either as the free base or as a suitable salt form, including, but not limited to, trifluoroacetic acid (TFA), hydrochloric acid (HCl), hydrobromic acid (HBr), methanesulfonic acid (MsOH), (trifluoromethanesulfonic acid) TfOH, and acetic acid (AcOH).

[0133] Any suitable amide coupling agent may be used to form the compound of Formula I. Suitable amide coupling agents include 2-chloromethylpyridinium iodide, [benzotriazol-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate] (BOP), benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and 1-[bis(dimethylamino)methyl]-2-(2-methyl-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU). Amide coupling agents include, but are not limited to, [amino)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-tetramethylaminium 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 conjunction with a co-reagent, examples of which include 1-hydroxybenzotriazole (HOBT) or N,N-dimethylpyridin-4-amine (DMAP). In certain embodiments, the amide coupling agent is used in conjunction with a base, such as triethylamine or diisopropylethylamine. In certain embodiments, the amide coupling agent is used with both a co-reagent, such as HOBT or DMAP, and a base, such as triethylamine or diisopropylethylamine. One skilled in the art will recognize that co-reagents other than HOBT or DMAP may also be used. Furthermore, one skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine may also be used.

[0134] Those skilled in the art will recognize that any suitable leaving group may be coupled with a carboxylic acid of formula Ib in the presence of a suitable coupling agent to form an activated ester of formula Ic. [ka] where R is a suitable leaving group, including, but not limited to, imidazole, HOBT, NHS, and 4-nitrophenol. Suitable coupling reagents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU, and T3P. In some embodiments, the present invention provides a method for producing a carrier of formula I. [ka] The method comprises reacting a compound of formula Ia with [ka] with a compound of formula Ic [ka] B, S, C, R and L 1 , L 3 , and (M) b is defined as above, and L 2 is -C(O)NH-.

[0135] Those skilled in the art will recognize that the compounds of Formula Ia can be used either as the free base or as a suitable salt form, including, but not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0136] In certain embodiments, the amide coupling is carried out using a base such as triethylamine or diisopropylethylamine. Those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine may also be used.

[0137] In another particular embodiment, the present invention provides a method for producing a carrier of formula IIa. [ka] The method comprises reacting a compound of formula Ia with [ka] a compound of formula Id, [ka] reacting in the presence of an amide coupling agent to form a compound of formula Ie; [ka]

[0138] oxidizing the primary alcohol of formula Ie to an aldehyde of formula IIa; [ka] B, S, L 1 , L 3 , (M) b , b, n, and o are defined as above, and L 2 is —C(O)NH—, where C is an aldehyde group.

[0139] Any suitable oxidizing agent may be used to form the compound of Formula IIa, including, but 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-yl)oxyl / sodium hypochlorite (TEMPO / NaOCl).

[0140] Those skilled in the art will recognize that the compounds of Formula Ia can be used either as the free base or as a suitable salt form, including, but not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0141] Any suitable amide coupling agent may 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 with a co-reagent such as HOBT or DMAP. In certain embodiments, the amide coupling agent is used with a base such as triethylamine or diisopropylethylamine. In certain embodiments, the amide coupling agent is used 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 may also be used. Furthermore, those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine may also be used.

[0142] In certain embodiments, any suitable leaving group can be coupled with a carboxylic acid of formula Id in the presence of a suitable coupling reagent to form an activated ester of formula If. [ka] where 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 reagents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU, and T3P.

[0143] In some embodiments, the present invention provides methods for producing a carrier of formula Ie. [ka] The method comprises reacting a compound of formula Ia with [ka] reacting with a compound of formula If; [ka]

[0144] oxidizing the primary alcohol of formula Ie to an aldehyde of formula IIa; [ka] B, S, C, R and L 1 , L 3 , and (M) b is defined as above, and L 2 is -C(O)NH-.

[0145] Those skilled in the art will recognize that the compounds of Formula Ia can be used either as the free base or as a suitable salt form, including, but not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0146] In certain embodiments, the amide coupling is carried out using a base such as triethylamine or diisopropylethylamine. Those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine may also be used.

[0147] Any suitable oxidizing agent may be used to form the compound of Formula IIa, including, but not limited to, Collins' reagent, PDC, PCC, oxalyl chloride / DMSO (Swern oxidation), SO3-pyridine / DMSO (Parikh-Doehring oxidation), Dess-Martin periodinane, TPAP / NMO, and TEMPO / NaOCl.

[0148] In another particular embodiment, the present invention provides a method for producing a carrier of formula IIc. [ka] The method comprises reacting a compound of formula Ia with [ka] reacting with a compound of formula Ig [ka] forming a compound of formula Ih; [ka] converting the carboxylic acid of formula Ih to an activated ester of formula IIc, [ka] B, S, C, R, L 1, (M) b , b, n, and o are defined as above, and L 2 is -C(O)NH-.

[0149] Those skilled in the art will recognize that the compounds of Formula Ia can be used either as the free base or as a suitable salt form, including, but not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0150] Any suitable leaving group can be coupled with a carboxylic acid of formula Ih in the presence of a suitable coupling reagent to form an activated 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 reagents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU, and T3P.

[0151] In some embodiments, an activated ester of formula IIc is formed from a carboxylic acid of formula Ih using a suitable leaving group and coupling reagent combination.

[0152] In some embodiments, an activated ester of Formula IIc is formed from a carboxylic acid of Formula Ih using a single reagent that both generates a leaving group and effects 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, a single reagent is used alone. In other embodiments, a single reagent is used in conjunction 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 may also be used.

[0153] In a specific embodiment, the present invention provides a method for producing a carrier of formula V: [ka] The method comprises reacting a compound of formula Va with [ka] reacting with a compound of formula Vb [ka] forming a compound of formula Vc; [ka] reducing the nitrile group to form an amine of formula Vd; [ka] reacting a compound of formula Vd with a compound of formula Ve, [ka] forming a compound of formula Vf; [ka] and oxidizing the primary alcohol of formula Vf to form an aldehyde of formula V. [ka]

[0154] where x refers to the number of repeating ethylene glycol units, and x-1 refers to the fact that conjugating PEG to x number of polymers in this reaction converts the terminal PEG monomer to an aldehyde, resulting in x-1 in the final formulation. In some embodiments, the reaction of a compound of Formula Vb with a compound of Formula Va is facilitated by the addition of Triton B. One of skill in the art will recognize that other reagents may be used to facilitate the nucleophilic addition to acrylonitrile.

[0155] In some embodiments, the reduction of the nitrile of formula Vc to the amine of formula Vd is carried out using AlCl / lithium aluminum hydride (LAH). One skilled in the art will recognize that other reducing reagents may be used, including sodium, H / Pd, H / Raney nickel, and diborane.

[0156] Those skilled in the art will recognize that the compound of formula Vd can be used either as the free base or as a suitable salt form, including, but not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0157] In certain embodiments, a base such as triethylamine or diisopropylethylamine is used to facilitate the coupling of an NHS ester of formula Ve with an amine of formula Vd. One skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine may also be used.

[0158] Any suitable oxidizing agent may 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-Doehring oxidation), Dess-Martin periodinane, TPAP / NMO, and TEMPO / NaOCl.

[0159] In another specific embodiment, the present invention provides a method for producing a carrier of formula VI. [ka] The method comprises reacting a compound of formula Vd with [ka] with a compound of formula VIa in the presence of an amide coupling agent. [ka]

[0160] where x refers to the number of repeating ethylene glycol units. One skilled in the art will recognize that the compound of formula Vd can be used either as a free base or as a suitable salt form. Suitable salt forms include, but are not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0161] In a preferred embodiment of Formula VI, Formula Vd, and Formula VIa, x=36.

[0162] Any suitable amide coupling agent may 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 with a co-reagent such as HOBT or DMAP. In certain embodiments, the amide coupling agent is used with a base such as triethylamine or diisopropylethylamine. In certain embodiments, the amide coupling agent is used 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 may also be used. Furthermore, those skilled in the art will recognize that bases other than triethylamine or diisopropylethylamine may also be used.

[0163] In another specific embodiment, the present invention provides a method for producing a carrier of formula VII. [ka] The method comprises reacting a compound of formula Vd with [ka] by reacting with a compound of formula VIIa [ka] forming a compound of formula VIIb; [ka] and converting the carboxylic acid of formula VIIb to an activated ester of formula VII. [ka]

[0164] Those skilled in the art will recognize that the compound of formula Vd can be used either as the free base or as a suitable salt form, including, but not limited to, TFA, HCl, HBr, MsOH, TfOH, and AcOH.

[0165] 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 may also be used.

[0166] NHS can be coupled with a carboxylic acid of formula VIIb in the presence of a suitable coupling reagent to form an activated ester of formula VII. Suitable coupling reagents include, but are not limited to, 2-chloromethylpyridinium iodide, BOP, PyBOP, HBTU, HATU, DCC, EDCI, TBTU, and T3P.

[0167] In some embodiments, the activated ester of formula VII is formed from the carboxylic acid of formula VIIb using a combination of NHS and a coupling reagent.

[0168] In some embodiments, an activated ester of Formula VII is formed from a carboxylic acid of Formula VIIb using a single reagent that both generates a leaving group and effects the coupling reaction. Such a reagent includes, but is not limited to, N,N'-disuccinimidyl carbonate. In some embodiments, a single reagent is used alone. In other embodiments, the reagent is used in conjunction 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 may also be used.

[0169] Those skilled in the art will recognize that there are other methods for conjugating linkers and scaffolds to the C3 or C25 position of vitamin D derivatives and analogs. For example, the C3 hydroxyl group can be acylated with various groups, as described in N. Kobayashi, K. Ueda, J. Kitahori, and K. Shimada, Steroids, 57, 488-493 (1992); JG Haddad, et al., Biochemistry, 31, 7174-7181 (1992); A. Kutner, RP Link, HK Schnoes, HF DeLuca, Bioorg. Chem., 14, 134-147 (1986); and R. Ray, SA Holick, N. Hanafin, and MF Holick, Biochemistry, 25, 4729-4733 (1986). The above references are incorporated by reference in their entirety. Those skilled in the art will recognize that these chemical reactions can be modified to synthesize compounds of Formula I. [ka] where B, S, C, (L) a and (M) b is defined as above.

[0170] If desired, therapeutic compound-carrier conjugates having different molecular weights can be isolated using gel filtration chromatography and / or ion exchange chromatography. Gel filtration chromatography can be used to fractionate various therapeutic compound-carrier conjugates (e.g., monomers, dimers, trimers, etc., where "monomer" refers to one targeting group molecule per therapeutic compound, "dimer" refers to two targeting groups attached to a therapeutic compound, etc.) based on their different molecular weights (where the difference essentially corresponds to the average molecular weight of the targeting groups).

[0171] Suitable gel filtration columns for carrying out this type of separation include Superdex and Sephadex columns available from Amersham Biosciences (Piscataway, NJ). The choice of a particular column depends on the desired fractionation range desired. Elution is generally carried out using a suitable buffer, such as phosphate, acetate, etc. Collected fractions may be analyzed by several different methods, examples of which include (i) optical density at 280 nm (OD) for protein content, (ii) bovine serum albumin (BSA) protein analysis, and (iii) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE).

[0172] Separation of the therapeutic compound-carrier conjugates 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-Sepharose or CM-Sepharose ion-exchange column available from Amersham Biosciences). The resulting purified composition is preferably substantially free of non-targeting group-conjugated therapeutic compounds. Furthermore, the composition is preferably substantially free of all other non-covalently bound targeting groups.

[0173] As described herein, the carrier of the present invention may be non-hormonal 25-hydroxyvitamin D or an analog thereof having a coupling group at the 3' carbon. As used herein, "25-hydroxyvitamin D analog" includes both naturally occurring metabolite forms of vitamin D as well as other chemically modified forms. The carrier of the present invention does not include the active (i.e., hormonal) form of vitamin D (typically having a hydroxyl group at carbon 1). These compounds are based on the structure of vitamin D and retain partial vitamin D function (i.e., interacting with DBP), albeit with different affinities. The following list is illustrative of vitamin D analog forms known in the art, although they may be hormonal or have a C1 hydroxyl group. These are presented solely for their chemical properties as vitamin D analogs, not their functional hormonal properties. OCT is a chemically synthesized version of 1,25(OH)2D3, which has an oxygen atom at position 22 of the side chain (Abe et al., FEBS Lett. 226:58-62 (1987)). The gemini vitamin D analog is 1α,25-dihydroxy-20R-21(3-hydroxy-3-deuteromethyl-4,4,4-trideuterobutyl)-23-ine-26,27-hexafluoro-cholecalciferol (BXL0124) (So et al., Mol Pharmacol. 79(3):360-7(2011)). Paricalcitol is a vitamin D2-derived sterol that lacks the 19-carbon 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) 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 above references are incorporated by reference in their entireties.

[0174] In another embodiment, the carrier further comprises a pharmaceutically acceptable scaffold moiety covalently bonded to the targeting group and the therapeutic compound. The scaffold moiety of the carrier of the present invention is not necessarily involved in the function of the therapeutic compound, but may contribute to the function of the therapeutic compound or improve the pharmacokinetic properties of the therapeutic compound. The scaffold of the present invention does not substantially interfere with the binding of the targeting group to the DBP. Similarly, the scaffold of the present invention does not substantially interfere with the structure or function of the therapeutic compound. The length of the scaffold moiety depends on the properties of the targeting group and the therapeutic compound. Those skilled in the art will recognize that various atom combinations will provide molecules of variable length based on the known distances between the various bonds (Morrison and Boyd, Organic Chemistry, 3rd Ed., Allyn and Bacon, Inc., Boston, Mass. (1977), incorporated herein by reference). Other scaffolds 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, oxygen- or nitrogen-interspersed carbon linkers, or combinations thereof. In preferred embodiments, the linkers are non-releasable or stable.

[0175] PTH is a therapeutic peptide. The term "peptide," used interchangeably herein with the term "protein," is intended to include a string of amino acids. The amino acids in the peptides of the present invention may be natural or non-natural. The peptides of the present invention may be chemically or biologically synthesized and may include cysteine-rich peptides, cyclic peptides, stapled peptides, peptides containing D- or L-amino acids and mixtures thereof, peptidomimetics, peptide-nucleic acids (PNAs), and combinations thereof. Branched or cyclic therapeutic peptides, with or without branching, are also contemplated within the scope of the embodiments described herein. Cyclic peptides, branched peptides, and branched cyclic peptides can be obtained by post-translational natural processes or produced by suitable synthetic methods. In some embodiments, any peptide product described herein includes the above-described peptide analogs, which are then covalently attached to an alkyl glycoside surfactant moiety.

[0176] Other embodiments include therapeutic peptide chains composed of natural and unnatural amino acids, or analogs of natural amino acids. As used herein, "analogs" of peptides and / or proteins include unnatural amino acids based on natural amino acids, examples of which include tyrosine analogs (including para-substituted tyrosines, ortho-substituted tyrosines, and meta-substituted tyrosines), and substituents on tyrosine include acetyl groups, benzoyl groups, amino groups, hydrazine, hydroxylamine, thiol groups, carboxy groups, methyl groups, isopropyl groups, C2-C20 straight or branched hydrocarbons, saturated or unsaturated hydrocarbons, O-methyl groups, polyether groups, halogens, nitro groups, etc.

[0177] Additional embodiments include therapeutic peptide chains with modified amino acids. Examples include amino acids acylated at the epsilon position of lysine, amino acids with fatty acids (such as octanoic acid, decanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, 3-phenylpropanoic acid, etc.), or amino acids with saturated or unsaturated alkyl chains. (Zhang, L. and Bulaj, G. (2012) Curr Med Chem 19:1602-1618, the contents of which are incorporated herein by reference in their entirety.)

[0178] The present invention further contemplates therapeutic peptide chains comprising natural and unnatural amino acids, or analogs of natural amino acids. In some embodiments, peptide or protein "analogs" include unnatural amino acids based on natural amino acids, examples of which include tyrosine analogs (including para-substituted tyrosines, ortho-substituted tyrosines, and meta-substituted tyrosines), where substituents on tyrosine include acetyl groups, benzoyl groups, amino groups, hydrazine, hydroxylamine, thiol groups, carboxy 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-dimethyl-tyrosine (Dmt), 2,4-diethyl-tyrosine, O-4-allyl-tyrosine, 4-propyl-tyrosine, Ca-methyl-tyrosine, etc. Examples of lysine analogs include ornithine (Orn), homo-lysine, Ca-methyl-lysine (CMeLys), etc. Examples of phenylalanine analogs include, but are not limited to, meta-substituted phenylalanines, where 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-methyl-tyrosine, 3-(2-naphthyl)alanine (Nal(2)), 3-(1-naphthyl)alanine (Nal(1)), 3-methyl-phenylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), fluorinated phenylalanine, isopropyl-phenylalanine, p-azido-phenylalanine, p-acyl-phenylalanine, p-benzoyl-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-phenylalanine, and isopropyl-phenylalanine.

[0179] Also contemplated within the scope of the embodiments are therapeutic peptide chains containing non-standard or unnatural amino acids known in the art, such as C-alpha disubstituted amino acids (Aib, Ca-diethylglycine (Deg), aminocyclopentane-l-carboxylic acid (Ac4c), aminocyclopentane-l-carboxylic acid (Ac5c), etc.). Such amino acids often induce constrained structures, often biasing toward an α-helical structure (Kaul, R. and Balaram, P. (1999) Bioorg Med Chem 7:105-117, the contents of which are incorporated herein by reference in their entirety). A further example of such an unnatural amino acid useful for analog design is homo-arginine (Har). In some cases, replacing reduced amide bonds improves protection from enzymatic destruction or alters receptor binding. Illustratively, incorporation of a Tic-Phe dipeptide unit (designated Tic-F[CH2-NH]^-Phe) with a reduced amide bond between residues reduces enzymatic degradation.

[0180] In some embodiments, amino- or carboxyl-terminal modifications may be optionally introduced into the peptides or proteins (Nestor, JJ, Jr. (2009) Current Medicinal Chemistry 16:4399-4418). For example, the peptides or proteins may 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 peptides or proteins, such as the deletion or incorporation of a D-amino acid such as D-Phe, when substituted with modifications described herein, such as long-chain alkyl glycosides, result in potent, long-acting agonists or antagonists.

[0181] Thus, the present invention provides therapeutic compound analogs, where the native therapeutic compound has been modified by acetylation, acylation, PEGylation, ADP-ribosylation, amidation, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cysteine ​​cross-links, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation, selenoylation, sulfation, addition of amino acids to proteins (such as arginylation) via transfer RNA, and ubiquitination. See, e.g., (Nestor, JJ, Jr. (2007) Comprehensive Medicinal Chemistry II 2:573-601; Nestor, JJ, 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 above references are incorporated by reference in their entireties.

[0182] Glycosylated therapeutic peptides may be prepared using conventional Fmoc chemistry and solid-phase peptide synthesis techniques (e.g., on resin), in which the desired protected sugar amino acid is prepared prior to peptide synthesis and then introduced into the peptide chain at the desired position during peptide synthesis. Thus, therapeutic peptide-polymer conjugates may be conjugated in vitro. Glycosylation may occur before deprotection. Preparation of amino acid glycosides is described in U.S. Pat. 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 entireties. For example, alpha- and beta-selective glycosylation of serine and threonine residues is carried out using the König-Knorr reaction and the Lemieux in situ anomerization method using a Schiff base intermediate. Deprotection of the Schiff base glycoside is then carried out using mildly acidic conditions or hydrogenolysis. Compositions comprising glycosylated therapeutic peptide conjugates are produced by stepwise solid-phase peptide synthesis, which involves contacting a growing peptide chain with protected amino acids in a stepwise manner, glycosylation of at least one of the protected amino acids, followed by water-soluble polymer conjugation. 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.

[0183] Monosaccharides that can be introduced into 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, as well as others. Glycosides (such as monosaccharides, disaccharides, and trisaccharides used in modifying therapeutic peptides), i.e., 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.

[0184] In a further embodiment of the present invention, a therapeutic compound as defined and / or disclosed herein may be chemically coupled to biotin, which can then bind to avidin.

[0185] The presence or concentration of PTH may be measured using antibodies. 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, nanobodies, diabodies, affibodies, Fv, Fab, F(ab')2, Fab', scFv, scFv-Fc, etc. The term also includes antibody fusion proteins such as Ig chimeras.

[0186] An antibody that specifically binds to an antigen has a high affinity for that antigen. The affinity of an antibody may be measured by the dissociation constant (Kd). In certain embodiments, the antibodies provided herein have a dissociation constant of 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~10 -13 M, 10-8 M~10 -13 M or 10 -9 M~10 -13 It has a dissociation constant (Kd) of less than or equal to 1 M.

[0187] Some embodiments of carrier assembly utilize chemical methods well known in the art. For example, vitamin E-PEG is manufactured by Eastman Chemical, and biotin-PEG is manufactured by a number of PEG manufacturers, including Enzon, Nektar, and NOF Corporation. Methods for producing PEG molecules linked to several vitamins and other therapeutic compounds follow these and other chemical methods known in the art. Attachment of PEG to oligonucleotides or related molecules occurs, for example, as a PEG2-N-hydroxysuccinimide ester coupled to the oligonucleotide via a 5' amine moiety. Several coupling methods are contemplated, including, for example, NHS coupling to amine groups (e.g., lysine residues on peptides), maleimide coupling to sulfhydryl groups (e.g., on cysteine ​​residues), iodoacetyl coupling to sulfhydryl groups, pyridyldithiol coupling to sulfhydryl groups, hydrazides for coupling to carbohydrate groups, aldehydes for coupling to the N-terminus, or tetrafluorophenyl ester coupling, which is known to react with primary or secondary amines. Other possible chemical coupling methods are known to those skilled in the art and can be substituted. By way of example, conjugation using the coupling groups of the present invention may be carried out using the compositions and methods described in International Publication No. WO 93 / 012145. See also U.S. Patent No. 7,803,777, the entire contents of which are incorporated herein by reference.

[0188] Exemplary drug formulations of the present invention include aqueous solutions, organic solutions, powder formulations, solid formulations, and mixed phase formulations.

[0189] The pharmaceutical composition of the present invention comprises any of the compounds of the present invention and pharmaceutically acceptable salts thereof, and 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), buffer substances (such as phosphoric acid), 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 silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0190] Pharmaceutically acceptable salts retain the desired biological activity of the therapeutic composition without toxic side effects. Examples of such salts include (a) acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.) and organic acids (e.g., 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 (e.g., zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, etc.) or organic cations formed from N,N'-dibenzylethylenediamine or ethylenediamine, or (c) combinations of (a) and (b), such as zinc tannate.

[0191] The pharmaceutical compositions of the present invention may be administered subcutaneously, transdermally, orally, parenterally, by inhalation, ophthalmically, topically, rectally, nasally, buccally (including sublingually), vaginally, or via an implanted reservoir. The pharmaceutical compositions of the present invention may contain any conventional, non-toxic, pharmaceutically acceptable carrier, adjuvant, or vehicle. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0192] In some embodiments, pharmaceutical compositions comprising the therapeutic compound or its pharmaceutically acceptable salt as described herein in combination with pharmaceutically acceptable non-toxic components are also contemplated as active ingredients.As mentioned above, such compositions can be prepared for parenteral administration, particularly in the form of 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 powder, nasal drops, evaporation solution or aerosol, for inhalation, particularly in the form of liquid solution or dry powder containing excipients in the broad sense, for transdermal administration, particularly in the form of skin patch or microneedle patch, and for rectal or vaginal administration, particularly in the form of suppository.

[0193] The compositions may be conveniently administered in unit dosage form and prepared by any method known in the pharmaceutical arts (e.g., as described in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, PA (1985), the contents of which are incorporated herein by reference in their entirety). Formulations for parenteral administration may contain excipients such as sterile water or salts of alkylene glycols (e.g., propylene glycol), polyalkylene glycols (e.g., polyethylene glycol), sugars, vegetable oils, hydrogenated naphthalenes, serum albumin or other nanoparticles (such as those used in Abraxane™, American Pharmaceutical Partners, Inc., Schaumburg, Illinois). For oral administration, the formulation may be enriched by the addition of bile salts or acylcarnitines. Formulations for nasal administration may be solids or solutions in evaporative solvents (such as hydrofluorocarbons), may contain stabilizing excipients (e.g., sugars, surfactants, submicron anhydrous α-lactose or dextran), or may be aqueous or oily solutions for use in the form of nasal drops or metered-dose sprays. For buccal administration, typical excipients include sugars, calcium stearate, magnesium stearate, pregelatinized starch, and the like.

[0194] Delivery of the modified therapeutic compounds described herein to a subject over an extended period, such as a period of one week to one year, can be achieved by administering a single dose of a controlled-release system containing sufficient active ingredient for the desired release period. A variety of controlled-release systems can be used for this purpose, including monolithic or reservoir-type microcapsules, depot implants, polymer hydrogels, osmotic pumps, vesicles, micelles, liposomes, transdermal patches, iontophoresis devices, and alternative injectable dosage forms. Localization of the active ingredient at the desired site of delivery is an additional feature of some controlled-release devices and may prove beneficial in the treatment of certain disorders.

[0195] In certain embodiments of transdermal administration, delivery across the skin barrier is enhanced using electrodes (e.g., iontophoresis), electroporation or application of brief high-voltage electrical pulses to the skin, radiofrequency, ultrasound (e.g., sonophoresis), microprojection (such as microneedles), jet injectors, thermal ablation, magnetophoresis, laser, velocity, or photomechanical waves. The drug can be contained in a single-layer drug-dissolving adhesive patch, a multi-layer drug-dissolving adhesive patch, a reservoir patch, a matrix patch, or a vapor patch, or patchless technology can be utilized. Delivery through the skin barrier can also be enhanced using encapsulation, skin lipid fluidizers, hollow or solid microstructured transdermal systems (such as MTS, manufactured by 3M), or jet injectors. Additives to formulations to aid in the passage of therapeutic compounds through the skin include prodrugs, chemicals, surfactants, cell membrane-penetrating peptides, penetration enhancers, encapsulation techniques, enzymes, enzyme inhibitors, gels, nanoparticles, and peptide or protein chaperones.

[0196] One form of controlled-release formulation contains a therapeutic compound or its salt dispersed or encapsulated in a slowly degrading, non-toxic, non-antigenic polymer such as copoly(lactic / glycolic acid) (described in the pioneering work of Kent et al., U.S. Pat. No. 4,675,189, incorporated herein by reference). These compounds or their salts may also be formulated into cholesterol or other lipid matrix pellets, or silastomer matrix implants. Additional sustained-release depot implant or injectable formulations will be apparent to the skilled artisan. See, e.g., *Sustained and Controlled Release Drug Delivery Systems*, *J.R. Robinson*, ed., *Marcel Dekker Inc., *New York, 1978*; and *Controlled Release of Biologically Active Agents*, *RW Baker*, *John Wiley & Sons, *New York, 1987*. The above are incorporated by reference in their entireties.

[0197] A further form of controlled-release formulation comprises a solution of a biodegradable polymer (such as copoly(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.

[0198] When formulated for nasal administration, absorption through the nasal mucosa may be further enhanced by surfactants, examples of which include, for example, glycocholic acid, cholic acid, taurocholic acid, ethocholic acid, deoxycholic acid, chenodeoxycholic acid, dehydrocholic acid, glycodeoxycholic acid, cyclodextrin, and the like, in amounts ranging from about 0.1 to 15 weight percent, about 0.5 to 4 weight percent, or about 2 weight percent. An additional class of absorption enhancers that has been reported to exhibit greater efficacy with reduced irritation is the alkyl maltoside class, an example of which is 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.

[0199] The pharmaceutical compositions may be in the form of a sterile injectable preparation (e.g., a sterile injectable aqueous or oleaginous suspension). This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80, etc.) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., as a solution in 1,3-butanediol). Acceptable vehicles and solvents that may be employed include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile fixed oils are conventionally employed as solvents or suspending media. For this purpose, any bland fixed oil may be employed, including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, particularly in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph. Helv or a similar alcohol.

[0200] The pharmaceutical composition of the present invention may be orally administered in any orally acceptable dosage form, including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. For tablets for oral use, commonly used carriers include lactose and corn starch. Lubricating agents such as magnesium stearate are also commonly added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. For oral administration of aqueous suspensions, the active ingredient is combined with emulsifying and suspending agents. Optionally, some sweetening agents and / or flavoring agents and / or coloring agents may be added.

[0201] The pharmaceutical composition of the present invention may also be administered in the form of suppositories for rectal administration.These compositions can be prepared by mixing the compound of the present invention with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the active ingredient.Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycol.

[0202] Topical administration of the pharmaceutical compositions of the present invention is particularly useful when the desired treatment involves areas or organs easily accessible by topical application. For topical application to the skin, the pharmaceutical composition should be formulated in a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for topical administration 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 in 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 may also be applied topically to the lower intestinal tract via a rectal suppository formulation or in a suitable enema formulation. Topically transdermal patches are also included in the present invention.

[0203] The pharmaceutical compositions of the invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation, and may be prepared as solutions in saline employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0204] When formulated for delivery by inhalation, several formulations offer advantages. Adsorption of a therapeutic compound to an easily dispersible solid, such as a diketopiperazine (e.g., Technosphere particles (Pfutzner, A. and Forst, T., 2005, Expert Opin Drug Deliv. 2:1097-1106) or similar structure), results in a formulation that provides rapid initial absorption of the therapeutic compound. Lyophilized powders, particularly glassy particles, containing a therapeutic compound and excipients are useful for pulmonary delivery with good bioavailability. See, e.g., Exubera® (inhaled insulin, Pfizer, Inc. and Aventis Pharmaceuticals Inc.) and Afrezza® (inhaled insulin, Mannkind, Corp.).

[0205] Dosage levels of about 0.1 to about 300 mg / kg body weight per day, preferably 0.5 to about 50 mg / kg body weight per day, of the active ingredient compound are useful for the prevention and treatment of disease. Such administration can be used as chronic or acute therapy. The amount of drug that may be combined with the carriers to produce a single dosage form will vary depending on the host treated and the particular mode of administration. Typical preparations contain about 0.01% to about 1% (w / w) of the active compound. Preferably, such preparations contain about 0.02% to about 1% (w / w) of the active compound.

[0206] Once the patient's condition has improved, a maintenance dose of the compound, composition or combination of the present invention may be administered as needed. Thereafter, when the symptoms have been alleviated to a desired level, the dosage or frequency of administration, or both, may be reduced as a function of the symptoms to a level at which the improved condition is maintained, and treatment should be discontinued. However, if any symptoms of the disease recur, the patient may require long-term intermittent treatment.

[0207] As the skilled artisan will appreciate, lower or higher doses than those set forth above may be necessary. The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, severity and course of the infection, the pharmacokinetics of the patient's response to the infection, and the judgment of the treating physician.

[0208] The carrier-drug conjugates described herein offer advantages to drug manufacturers and patients over unmodified drugs. Specifically, the carrier-drug conjugates or formulations are more potent, longer-lasting, and require smaller and less frequent doses. This translates to reduced healthcare costs and more convenient drug administration schedules for patients. The carrier-drug conjugates can also provide subcutaneous or transdermal administration routes as an alternative to intravenous injection. These routes allow patients to self-administer, improving patient compliance.

[0209] In yet another embodiment of the present invention, DBP levels can be increased as part of carrier-drug therapy. It has been reported that estrogen can increase DBP levels (Speeckaert et al., Clinica Chimica Acta 371:33). It is envisioned herein that DBP levels can be increased by administering estrogen to more effectively deliver the carrier-drug conjugate.

[0210] In yet another aspect of the present invention, it is contemplated that a carrier can be used to deliver the drug transdermally. DBPs typically transport UV-activated vitamin D close to the surface of the skin, allowing the use of a transdermal delivery system with a carrier.

[0211] In order to more fully understand the invention described herein, the following examples are provided. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the invention in any manner. In particular, the compositions and methods disclosed herein work with all non-hormonal forms of vitamin D, including its homologs, analogs, and metabolites. This includes vitamin D as used in the following examples. [Example]

[0212] Example 1: Preparation of PTH coupled to non-hormonal vitamin D at the C3 position PTH(1-34) bearing a C-terminal cysteine ​​residue and a separate 1.6 kDa PEG was synthesized using a maleimide group to create a thioether bond (PTH-C-PEG). 1.6K -(3)-VitD). See WO2016 / 065042, the contents of which are incorporated herein by reference in their entirety.

[0213] The modified PTH(1-34) compound was made with the following structure: [ka]

[0214] EXT601 was prepared as described in WO2016 / 065042, the contents of which are incorporated herein by reference in their entirety. EXT607 was prepared using the same method as described below for EXT608, except that R in EXT607 is OH, whereas R in EXT608 is NH. R=NH is more advantageous than R=OH to prevent impurity formation during solid-phase synthesis of peptides whose C-terminal amino acid is cysteine.

[0215] The overall 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) is accomplished in two steps by functionalizing the 3'-hydroxyl group of vitamin D with acrylonitrile, followed by reduction with lithium aluminum hydride (Ray, et al. 1991). The resulting amine (3) was then catalyzed by the heterobifunctional TFP-PEG (obtained from Quanta BioDesign, Ltd.). 36 The PTH(1-34)-cys-NH2 peptide was then coupled to the tetrafluorophenyl (TFP) ester of the α-maleimide linker (4) (Plain City, OH, USA, Catalog No. 10555) to yield VitD-PEG-maleimide (5). The PTH(1-34)-cys-NH2 peptide was synthesized using solid-phase peptide techniques. It was then fully deprotected and removed from the resin to yield crude PTH(1-34)-cys-NH2 peptide. The crude peptide was purified by preparative HPLC, and the purified PTH(1-34)-cys-NH2 was isolated by lyophilization. The final synthetic step, conjugation of the purified peptide and 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 process is as follows:

[0216] VitD-PEG 36 -Synthesis of maleimide (compound 5), method 1. VitD-NH2 (compound 3) is prepared and described as "compound Vd" in WO2016 / 065042, the contents of which are incorporated herein by reference in their entirety. [ka]

[0217] To a mixture of compound 3 (1 eq) dissolved in anhydrous dichloromethane under nitrogen at 4 °C, 2,6-lutidine (2.5 eq) was added dropwise and stirred for 15 min. Compound 4 (Quanta BioDesign, Plain City, OH, USA, Cat. No. 10555, 0.8 eq) dissolved in 30 mL of dichloromethane was slowly added to this mixture via syringe. The resulting reaction mixture was stirred at room temperature under an inert atmosphere and monitored by thin layer chromatography (TLC). After 4 h, 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 the crude product. The crude mixture was purified by silica gel gold ISCO flash chromatography. The column was eluted with 1–10% MeOH / chloroform (1% ammonium hydroxide). Fractions containing the purified product were combined and evaporated under vacuum to give a pale yellow solid (R f Compound 5 was obtained in 62% yield with 68% purity by HPLC / TLC (0.3 silica gel, 10% methanol in dichloromethane).

[0218] VitD-PEG 36 Synthesis of -maleimide (compound 5), method 2. Compound 5 (Mal-PEG 36 In an effort to improve the purity of VitD-PEG produced by Method 1, 36 We found that the -maleimide contained impurities formed during lyophilization. Although the exact structures of these impurities have not been determined, NMR analysis indicated that the double bonds of the maleimide and vitamin D were involved in undesired reactions during lyophilization. Therefore, the crude reaction mixture of compounds 3 and 4 prepared by Method 1 was purified by preparative HPLC using a water / acetonitrile mobile phase instead. The pooled fractions were used directly in the conjugation reaction with PTH(1-34)-cys-NH2. Because compound 5 was not isolated as a solid, quantification of compound 5 was obtained by optical absorption measurements, using the extinction coefficient to calculate the concentration. The purity of compound 5 using this method was 94.1%.

[0219] Peptide synthesis. PTH(1-34)-cys-NH2 was produced using the Fmoc (9-fluorenylmethoxycarbonyl) solid-phase peptide synthesis (SPPS) strategy. Ramage resin (a tricyclic amide linker) was used in the production process because it is particularly acid-sensitive and therefore suitable for preparing fully protected peptide amides using Fmoc chemistry. This resin formed a C-terminal cysteine ​​amide (R = NH2), which is less prone to undesired side reactions than a C-terminal cysteic acid (R = OH). Treating the peptide resin with the strong acid trifluoroacetic acid (TFA) simultaneously cleaved the side-chain protecting groups and separated the peptide from the resin. Scavenger agents [1,2-ethanedithiol (EDT), triisopropylsilane (TIS), and water] were added to capture reactive cations during cleavage, avoiding alkylation of side-chain functional groups and resulting in higher-quality crude peptides. Because the peptide sequence contains a thioether structure, ammonium iodide (NH4I) was added to the cleavage cocktail. Finally, ascorbic acid was used to neutralize the NHI. These reagents were used to ensure complete reduction of methionine sulfoxide potentially present in the product. After the cleavage procedure, the crude peptide was precipitated using chilled isopropyl ether (IPE) and filtered using a fritted glass funnel. The resulting cake was washed with IPE and dried in a vacuum oven at room temperature.

[0220] Peptide purification and lyophilization: The crude product is purified by two-dimensional preparative HPLC on a reversed-phase column with acetonitrile (ACN) gradient elution and UV detection at 230 nm. In the first preparative HPLC step, phosphoric acid (HPO 4)A buffer system was used as the mobile phase. C8 reversed-phase resin was used as the stationary phase. The collected individual fractions were analyzed by ultra-performance liquid chromatography (UPLC, Waters Corporation, Milford, MA) and pooled according to purity acceptance criteria. Fractions with a purity of ≥90% were identified as the main pool. The pooled main fractions were diluted with water to reduce the ACN concentration and further processed in a second preparative HPLC step. The second purification step by preparative HPLC used a trifluoroacetic acid (TFA) buffer system. C8 reversed-phase resin was used as the stationary phase. The collected individual fractions were analyzed by UPLC and pooled according to purity acceptance criteria. Fractions with a purity of ≥95% 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.

[0221] EXT608 conjugation reaction to produce 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 solution of Tris buffer containing ethylenediaminetetraacetic acid (EDTA). The lyophilized peptide from the previous 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 2% acetic acid solution and loaded onto a YMC C8 column equilibrated with 10 mM ammonium bicarbonate (NH4HCO3, pH 8) buffer in water. Elution of the product was achieved using a gradient of 10 mM NH4HCO3 buffer and acetonitrile. All fractions meeting the established criteria were collected and pooled together. The purified solution was filtered through a 0.45 μm membrane filtration system. Upon completion of the purification step, the solution was lyophilized to obtain EXT608 as the carbonate salt. The final product was analyzed by UPLC and LC-MS and determined to have a purity of 80.05%. The major impurity (7.5%), with a molecular weight 18 mass units higher than EXT608 (M+18), is hypothesized to be due to hydrolytic ring-opening of the thiosuccinimide ring (formed by the addition of cysteine ​​to the maleimide). Therefore, in some embodiments, the peptide / VitD-PEG-Mal coupling and purification are performed at a lower pH to prevent thiosuccinimide hydrolysis during the reaction, purification, and lyophilization (see section below). However, ring-opening does not alter the activity of the compound.

[0222] EXT608 Conjugation Reaction to Produce Acetate Salt (Method B): 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, pH 6.0. The conjugated peptide solution was purified by preparative HPLC with a pH 5.5 NHOAc system using a reversed-phase column and UV detection at 220 nm. After elution with an ACN gradient, the collected individual fractions were analyzed by UPLC and pooled according to 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. Therefore, 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 a major impurity hypothesized to arise from hydrolysis of thiosuccinimide.

[0223] Example 2: Discrete PEG linkers are advantageous over polydisperse PEG linkers The use of distinct PEG chain lengths was important to ensure uniformity of patient dosing, maximize manufacturing yield, and enhance the ability to monitor the vitamin D PTH conjugates and associated impurities during manufacturing and treatment processes using techniques such as UPLC, mass spectrometry, and LC-MS.

[0224] Analysis of EXT601 and EXT608 by UPLC. The following UPLC method was developed for the analysis of vitamin D-modified PTH compounds. An ACQUITY UPLC Peptide CSH C18 column (2.1 x 150 mm, 1.7 μm, 130A, 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 ml, 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 (%A) and mobile phase B (%B) was used: [Table 1]

[0225] The UPLC trace for EXT601 is shown in Figure 2. The polydisperse lengths of the PEG linkers resulted in a very broad UPLC peak with a baseline width of approximately 3 minutes. Any impurities were obscured by the broad EXT601 peak and the fact that any peptide or vitamin D-related impurities were present as multiple peaks for each PEG length variation. In contrast, the UPLC trace for EXT608 (Figure 3), which contains a single-length PEG linker (x=36), showed a narrow peak with a baseline width of approximately 0.5 minutes. The impurities present were well resolved from the EXT608 peak. This facilitated further characterization, separation, and identification of the impurities. Furthermore, the therapeutic VitD-PEG 36 The modest yield of -PTH provides an important attribute for more efficient purification, quantification and monitoring of the molecule during manufacturing and patient treatment.

[0226] Analysis of EXT601 and EXT608 by MALDI-TOF mass spectrometry: EXT601 and EXT608 were analyzed by MALDI-TOF (matrix-assisted laser desorption / ionization time-of-flight) mass spectrometry in the positive ion mode. The MALDI-TOF analysis of EXT601 is shown in Figure 4. The polydisperse length of the PEG linker resulted in a very broad peak with distinguishable signals above the baseline between approximately 6400 and 7650 g / mol. This is due to the PEGs with repeating units x = 34 to 62. x The average molecular weight was approximately 6880 g / mol, and the PEG 45 The MALDI-TOF analysis of EXT608 is shown in Figure 5. In contrast to EXT601, EXT608 exhibited a single peak with a molecular weight of 6478 g / mol. For both compounds, the signal at approximately 4215 g / mol results from fragmentation of the intact parent compound during ionization in the mass spectrometer (i.e., in-source fragmentation). Compared to EXT601, EXT608 exhibited a higher signal-to-background ratio and a higher relative abundance compared to the in-source-generated fragments.

[0227] Example 3: PTH(1-34), EXT601, EXT607 and EXT608 equally activate parathyroid hormone receptor 1 (PTHR1). The activity of EXT607 and EXT608 relative to PTH(1-34) on human parathyroid receptor 1 (PTHR1, also known as PTH1R) was measured in agonist mode by a cell-based calcium flux assay using the PathHunter® mammalian cell line (DiscoverX, Fremont, CA, USA, now part of Eurofins Discovery, catalog number 86-0030P-2212AG) expressing a Gq-coupled receptor. EXT607 and EXT608 were diluted to 100 μM in phosphate-buffered saline. A PTH(1-34) control was provided by DiscoverX. Cells expressing human PTHR1 were treated in duplicate with 10 concentrations of test substance, with a top concentration of 1 μM. Intracellular calcium release was measured 2 minutes after exposure using a FLIPR Tetra with a calcium-sensitive dye loaded into the cells. The percent activation efficacy was calculated as follows:

number

[0228] EC50 (half-maximal effective concentration) values ​​were determined from plots of percent efficacy versus agonist concentration (Figure 6). EC50 values ​​were 33 nM for PTH(1-34), 21 nM for EXT607, and 22 nM for EXT608. The EC50 value for EXT601 was 14 nM, as determined in WO2016 / 065042, the contents of which are incorporated herein by reference in their entirety. The compounds exhibited comparable potency within the experimental error of the assay. Thus, conjugation of a vitamin D moiety to PTH(1-34) does not interfere with PTHR1 activation. EXT601, EXT607, and EXT608 exhibited approximately comparable activity at PTHR1.

[0229] Example 4: EXT607 and EXT608 bind to vitamin D binding protein (DBP) with equal affinity. EXT607 and EXT608 were biotinylated using the EZ-Link Sulfo-NHS Biotin No-Weigh Kit (Thermo Fisher Scientific, Waltham, MA, Catalog No. 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 No. 17085101). The biotinylation rate was quantified using the Pierce Biotin Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, Catalog No. PI28005). DBP was purchased from Athens Research and Technology (Athens, GA, Catalog No. 16-16-070307).

[0230] Affinity measurements were collected with Octet Red 96 using prehydrated dip-and-read streptavidin (SA) biosensors (ForteBio, Fremont, CA, catalog number 18-5021). The reagent was diluted in phosphate-buffered saline (PBS) containing 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 chip for 2 minutes. A negative loading control of buffer only was used to measure nonspecific binding of DBP to the SA biosensor. A DBP titration ranging from 0 to 75 μM was prepared. DBP was allowed to associate with the loaded chip for 5 minutes and then dissociated in buffer solution for 10 minutes. The binding association and dissociation curves, as well as the steady-state curve fits, were generated using ForteBio Octet data analysis software (Figures 7 and 8), and the binding equilibrium constants (KD) were obtained from the steady-state data. Both EXT607 and EXT608 had a maximum response of 1.79 nm shift. The KD of EXT607 relative to DBP was D is 8.1μM + 2.5μM. KD is 5.2μM + 1.1μM.

[0231] Example 5: PEG36 is the optimal chain length Optimizing the PEG chain length is important to maximize the solubility and PTHR1 receptor activity of the vitamin D-PTH conjugate.

[0232] Synthesis of EXT611 (PTH-cys-PEG12-VitD) and EXT606 (PTH-cys-PEG24-VitD): One molar equivalent of Mal-PEG12-NHS (Quanta BioDesign #10284) or Mal-PEG24-TFP (Quanta BioDesign #10554) was reacted with 1.1 equivalents of VitD-NH2 (compound 3) in DMSO at room temperature for 30 minutes, where NHS is an amine-reactive N-hydroxysuccinimide ester. The reaction was quenched by adding an equal volume of 0.1 M ammonium acetate pH = 6.8. PTH(1-34)-cys-NH2 (1.0 equivalent in water) was added to a final concentration of 50 mM with 0.5 M MES pH = 6.1, and the reaction was allowed to proceed at room temperature for 30 minutes. The reaction was purified by HPLC using an Xselect CSH phenylhexyl column with a mobile phase consisting of 0.1 M ammonium acetate pH = 5 and acetonitrile. It was then lyophilized to obtain the acetate salt form of the compound. Alternatively, the compound was purified using 0.1 M ammonium carbonate pH=8 as the aqueous component of the mobile phase to obtain the carbonate salt form of the compound.

[0233] Synthesis of EXT615 (PTH-PEG5kDa-VitD), EXT616 (PTH-PEG10kDa-VitD), and EXT617 (PTH-PEG20kDa-VitD): Mal-PEG5kDa-NHS (Nanosoft Biotechnology, Louisville, NC, Catalog No. 2597-5000, "Mal-PEG5000-SCM"), Mal-PEG10kDa-NHS (Nanosoft Biotechnology, Catalog No. 2597-10K, "Mal-PEG10K-SCM"), or Mal-PEG20kDa-NHS (Advanced Biotechnology, Catalog No. 2597-10K, "Mal-PEG10K-SCM") dissolved in DMF (5kDa and 10kDa) or MeCN (20kDa) was used. One molar equivalent of "SC-PEG-MAL, 20kDa" (BioChemicals, Lawrenceville, GA, catalog number HEP0405) was reacted with 1.1 equivalents of VitD-NH2 (compound 5) in DMSO for 30 minutes at room temperature. The reaction was quenched by adding an equal volume of 0.1 M ammonium acetate 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 pH 5 and acetonitrile, lyophilized, and dissolved in DMF (5 kDa) or MeCN (10 kDa and 20 kDa). PTH(1-34)-cys-NH2 (1.0 equivalent in water) was added to a final concentration of 50 mM with 0.5 M MES pH 6.1, and the reaction was allowed to proceed for 30 minutes at room temperature. The PTH-PEG-VitD product was purified by HPLC as described above and lyophilized to yield either the acetate or carbonate form of the compound.

[0234] Effect of PEG length on PTHR1 receptor activity: The compounds listed above were dissolved in PBS with EXT608 (all acetate salts), and concentrations were determined using an extinction coefficient of 18,600 M1cm-1 at 280 nm. Each compound was adjusted to 100 μM and submitted to DiscoverX (a part of Eurofins Scientific, Fremont, CA) for testing in an assay with mammalian cells expressing the human PTH1 receptor (PTHR1) using a calcium mobilization assay in agonist mode, as described above in Example 3. PTHR1-expressing cells were treated in duplicate with 10 concentrations of test article, with a top concentration of 1 μM. EC50 values ​​were determined from a plot of percent efficacy as a function of agonist concentration (Figure 9), as shown in Table 1. [Table 2]

[0235] The EC50 values ​​were independent of the PEG linker length throughout the range of x = 12 to approximately 227 (528 to 10,000 Da). However, the PEG 20 kDa linker (EXT617) exhibited an approximately 7-fold higher EC50 value (221.3 nM). Without wishing to be bound by theory, this increase in agonist concentration required to achieve 50% maximal efficacy may have been due to the larger size of the 20 kDa PEG linker blocking binding of the much smaller conjugated PTH peptide (approximately 4 kDa) to PTHR1.

[0236] 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, each compound was added to phosphate buffered saline (PBS) to form a saturated solution. Undissolved compound was removed by centrifugation. The concentration of dissolved compound was determined to be 18,600 M. -1 cm -1The solubility of EXT615, EXT616, and EXT617 was determined by measuring absorbance at 280 nm using an extinction coefficient of 1000 M. For EXT615, EXT616, and EXT617, solubility was practically limited by the high viscosity imparted by the large PEG moiety. Highly viscous drug formulations are difficult to manipulate and require a large needle hole size, which can cause 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 a pipette tip with a diameter of approximately 1 mm (enlarged by cutting the end with a razor blade). The concentration of the dissolved compound was determined to be 18,600 M. -1 cm -1 The solubility of each compound was determined by measuring absorbance at 280 nm using an extinction coefficient of 0.05. Surprisingly, the acetate salt solubility was higher than that of the carbonate salt for each compound (Table 2). In the case of EXT608, the acetate salt was 17.6 times more soluble than the carbonate salt. Maximum solubility was achieved with EXT608 acetate (9.67 mM). Increasing PEG length increases solubility, but this is ultimately limited by the highly viscous, semi-solid, gelatinous solution obtained with PEG linkers ≥ 5 kDa. [Table 3]

[0237] Example 6: Single-dose pharmacokinetics of EXT601 in male rats: In a single-dose pharmacokinetic study, EXT601 was formulated in PBS and administered subcutaneously to male rats (n=3) at a volume of 1 ml / kg and a dose of 157 μg / kg. Blood was collected via a jugular vein cannula at 0.5, 1, 2, 4, 6, 8, 24, 32, 48, and 56 hours and processed to plasma. Plasma levels of EXT601 were measured using an Immutopics high-sensitivity human parathyroid hormone PTH(1-34) ELISA kit (catalog number: 60-3900, Quidel Corporation, San Diego, CA). Group mean EXT601 levels as a function of time are shown in Figure 10.

[0238] Pharmacokinetic parameters were determined from plasma concentration versus time data by non-compartmental analysis using Kinetica software (Thermo Fisher Scientific, Waltham, MA). EXT601 exhibited a 2-hour T max C of 64.4ng / ml max EXT601 has an elimination half-life of 13.5 hours and a low clearance of 20.1 ml / h / kg. [Table 4]

[0239] Example 7: Single-dose pharmacokinetics of EXT607 in male rats: In a single-dose pharmacokinetic study, EXT607 was formulated in PBS and administered to groups of male rats (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; and Group 4, 300 μg / kg subcutaneously. Blood (0.25 mL) was collected via the jugular vein cannula, transferred to tubes containing K2EDTA, and stored on wet ice until plasma processing. Blood samples were collected at 0 (pre-dose), 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 measured at Extend Biosciences using an Immutopics high-sensitivity human parathyroid hormone PTH(1-34) ELISA kit (catalog number: 60-3900, Quidel Corporation, San Diego, CA). Group mean EXT607 levels as a function of time are shown in Figure 11.

[0240] Pharmacokinetic parameters were determined from the plasma concentration versus time data (Table 4) by noncompartmental analysis with uniform weighting using Phoenix® WinNonlin® version 6.3 (Certara LP (Pharsight), St. Louis, MO). Concentration versus time data were analyzed using an IV bolus or extravascular administration model. A best-fit lambda z range was used. EXT607 had a subcutaneous bioavailability (%F) of 10-13% in male rats based on AUC compared with intravenous injection. Following SC administration of 30, 100, or 300 μg / kg, C was observed within 0.5-3 hours. max , and then EXT607 was slowly excreted with a clearance of 22.9–25.9 ml / h / kg, a half-life of 7–15 h, and a mean retention time of 13.6–15.9 h. [Table 5]

[0241] Example 8: Single-dose pharmacokinetics of EXT607 in male monkeys: In a single-dose pharmacokinetic study, EXT607 was formulated in PBS and administered at a volume of 1 ml / kg to groups (n=3) of male cynomolgus monkeys weighing approximately 4 kg each: Group 1, 20 μg / kg intravenously; Group 2, 7 μg / kg subcutaneously; Group 3, 20 μg / kg subcutaneously; and Group 4, 70 μg / kg subcutaneously. Blood (1.0 mL) was collected via a jugular vein cannula, transferred to tubes containing K2EDTA, and stored on wet ice until plasma processing. Blood samples were collected at 0 (pre-dose), 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 measured using Immutopics' highly sensitive human parathyroid hormone PTH(1-34) ELISA kit (Quidel Corp., San Diego, CA, catalog number 60-3900). Group mean EXT607 levels as a function of time are shown in Figure 12.

[0242] Pharmacokinetic parameters were determined from the plasma concentration versus time data (Table 5) by noncompartmental analysis with uniform weighting using Phoenix® WinNonlin® version 6.3 (Certara LP (Pharsight), St. Louis, MO). Concentration versus time data were analyzed using an IV bolus or extravascular administration model. A best fit lambda z range was used. EXT607 exhibited a t of 24 to 32 hours of elimination. 1 / 2 When delivered subcutaneously, bioavailability is 45-54% and clearance is low (6.63-7.28 ml / h / kg).

[0243] Dose-linearity was observed for AUC (dose-adjusted ratio 0.95:0.85:1.00), but not for Cmax (0.28:0.55:1.00). This is because dose affects the subcutaneous absorption rate and therefore Cmax. At low doses, the subcutaneous absorption rate is slower than at high doses, resulting in a lower Cmax and a flatter overall PK profile. The slower subcutaneous absorption rate at low doses is also reflected in the increased mean retention time (MRT), which was 29.46 h at the low dose, 23.26 h at the mid dose, and 17.51 ​​h at the high dose. [Table 6]

[0244] Example 9: Repeat-dose pharmacokinetics of EXT608 in male and female rats 21-day study: EXT608 was formulated in 20 mM sodium acetate pH 5.5 buffer containing 0.8% sodium chloride. In a repeated-dose toxicokinetic study, groups of 18 rats (9 males and 9 females) received daily subcutaneous doses of either 14.2 μg / kg or 70 μg / kg EXT608 at a volume of 0.5 ml / kg. On day 21, blood samples (approximately 0.5 ml) were collected at t = 0 (pre-dose), 0.5, 1, 3, 8, 24, 32, 48, and 72 h from one of three cohorts of three male and three female animals per time point. Each blood sample was collected from the jugular vein into a tube containing K2EDTA and manually inverted several times. Blood samples were stored on wet ice pending centrifugation. Samples were centrifuged (approximately 2700 g, approximately 10 minutes, approximately 5°C) within 1 hour of collection. Plasma was collected, divided equally into duplicate aliquots, and frozen.

[0245] 90-day study: EXT608 was formulated in 20 mM sodium acetate pH=5.5 buffer containing 0.8% sodium chloride and 0.1% polysorbate 80. In a repeated-dose toxicokinetic study, groups of 18 rats (9 males + 9 females) received daily subcutaneous doses of EXT608 at 0.5 ml / kg at either 1, 3, or 10 μg / kg. On day 90, blood samples (approximately 0.5 ml) were collected at t=0 (pre-dose), 0.5, 1, 3, 8, 24, 32, 48, and 72 h from one of three cohorts of three male and three female animals at each time point. Plasma was prepared as described above for the 21-day study.

[0246] Pharmacokinetic Analysis: Plasma levels of EXT608 were measured using an Immutopics high-sensitivity human parathyroid hormone PTH(1-34) ELISA kit (Quidel Corporation, San Diego, CA, catalog number 60-3900). Group mean EXT608 levels as a function of time for the last dose administration after either 21 days of daily dosing (14.2 and 70 μg / kg) or 90 days of daily dosing (1, 3, and 10 μg / kg) are shown in Figure 13. Surprisingly, the PK profile varied depending on the dose level. Higher doses showed a more pronounced peak, while lower doses had a flatter profile with a less distinct peak.

[0247] Pharmacokinetic parameters were determined from the plasma concentration versus time data (Table 6) using Phoenix® WinNonlin® version 6.4 (Certara LP (Pharsight), Princeton, NJ) by a non-compartmental extravascular administration model with uniform weighting. Pharmacokinetic analysis was performed on data collected from t = 0 to 72 h after the last dose was administered. The t values ​​for elimination were 1 / 2 The C ranged from 10.4 to 13.8 h. To quantify the dose-dependent changes observed in the PK profile, the C was measured during the usual dosing period of 0 to 24 h. max Value and C min The value was determined. max / C minThe ratio represents the peak-to-trough ratio of EXT608 concentrations observed during the dosing period. This ratio decreased from 30 at the highest dose (70 mg / kg) to 8.0 at 14.2 mg / kg, 4.6 at 10 mg / kg, and 1.8 at 3 mg / kg and 1 mg / kg. Without being bound by theory, the low peak-to-trough ratio observed at lower doses may be the result of a slower rate of absorption of EXT608 from the subcutaneous space into the circulation. For many therapeutic applications, including parathyroid hormone replacement therapy, a low peak-to-trough ratio is desirable. This allows dosing at levels closer to the ideal effective dose, without being too high, so that toxicity may be observed, or too low, so that efficacy is reduced. [Table 7]

[0248] Example 10: Repeat-dose pharmacokinetics of EXT608 in male and female cynomolgus monkeys 21-day study: EXT608 was formulated in 20 mM sodium acetate pH=5.5 buffer containing 0.8% sodium chloride. In a repeat-dose toxicokinetic study, groups of four cynomolgus monkeys (two males and two females, each weighing approximately 2.5 kg) received either 1.4, 7, or 20 μg / kg of EXT608 subcutaneously every other day at a volume of 0.05 ml / kg. On the final day of dosing, day 21, blood samples (approximately 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 tubes containing K2EDTA and centrifuged (approximately 2700 g, approximately 10 minutes, approximately 5°C) within 1 hour of collection. Plasma was collected, divided equally into duplicate aliquots, and frozen.

[0249] 90-day study: EXT608 was formulated in 10 mM sodium acetate pH=5.5 buffer containing 0.8% sodium chloride and 0.1% polysorbate 80. In a repeated-dose toxicokinetic study, groups of six cynomolgus monkeys (3 males + 3 females) received either 0.7 or 2 μg / kg of EXT608 subcutaneously every other day at a volume of 0.05 ml / kg. Blood samples (approximately 1.0 ml) were collected at t=0 (pre-dose), 0.5, 1, 3, 6, 12, 24, 48, and 72 h on Day 89, the final day of dosing. Plasma was prepared as described above for the 21-day study.

[0250] Pharmacokinetic Analysis: Plasma levels of EXT608 were measured using an Immutopics high-sensitivity human parathyroid hormone PTH(1-34) ELISA kit (Quidel Corporation, San Diego, CA, catalog number 60-3900). Group mean EXT608 levels as a function of time for the last dose administration after either 21 days of every-other-day dosing (20, 7, and 1.4 μg / kg) or 89 days of every-other-day dosing (2 and 0.7 μg / kg) are shown in Figure 14. Surprisingly, the PK profile varied depending on dose level. Higher doses showed a more pronounced peak, while lower doses had a flatter profile with a less distinct peak.

[0251] Pharmacokinetic parameters were determined from the plasma concentration versus time data (Table 7) using Phoenix® WinNonlin® version 6.4 (Certara LP (Pharsight), Princeton, NJ) with a non-compartmental extravascular administration model with uniform weighting. Pharmacokinetic analysis was performed on data for individual animals collected from t = 0 to 72 h after the last dose was administered. The t 1 / 2 The range of t was 34.7 to 51.0 h. 1 / 2 The values ​​were calculated from data from less than three half-lives. To quantify the dose-dependent changes observed in the PK profile, the C values ​​were calculated for the period 0–48 h, which is the usual dosing period. maxValue and C min The value was determined. max / C min The ratio represents the peak-to-trough ratio of EXT608 concentrations observed during the dosing period. This ratio decreased from 19.3 at the highest dose (20 mg / kg) to 14.2 at 7 mg / kg, 5.4 at 2 mg / kg, 3.4 at 1.4 mg / kg, and 2.4 at 0.7 mg / kg. Without being bound by theory, the low peak-to-trough ratio observed at lower doses may be the result of a slower rate of absorption of EXT608 from the subcutaneous space into the circulation. For many therapeutic applications, including parathyroid hormone replacement therapy, a low peak-to-trough ratio is desirable because it allows dosing at levels closer to the ideal effective dose, without being too high to cause observed toxicity or too low to reduce efficacy. [Table 8]

[0252] Example 11: Effects of EXT607 and EXT608 on serum calcium, serum phosphate, and urinary calcium in male and female cynomolgus monkeys 5-day / 8-day study: EXT607 was formulated in phosphate-buffered saline (PBS) at pH 7.4. In a repeated-dose toxicokinetic study, groups of four cynomolgus monkeys (2 males and 2 females, each weighing 2.5-3.7 kg) received EXT607 at 0, 10, 30, or 100 μg / kg subcutaneously at 1 ml / kg every other day on days 1, 3, and 5. On day 5, blood samples (approximately 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 serum separator tubes and centrifuged (approximately 2700 g, approximately 10 minutes, approximately 5°C) within 1 hour of collection. Serum was collected and analyzed for calcium and phosphate by colorimetric analysis. EXT607 induced a dose-dependent increase in serum calcium levels, which persisted for up to 48 h at the highest dose (Figure 15). The change in serum calcium levels was calculated for each time point by subtracting the baseline calcium level (mean of the 0 mg / 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. [Table 9]

[0253] On days 3 and 8, urine was collected at room temperature via a pan placed under the cage. "Day -3" refers to the pre-dose time point, 3 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 the same or decreased with EXT607 treatment (Table 8B). The urine collection on day 8 corresponds to the serum sample collected 72 h after injection on day 5. Thus, EXT607 increased serum calcium levels without increasing urinary calcium, as reflected by the decreased urinary:serum calcium ratio with EXT607 administration. This is beneficial because high urinary calcium levels can lead to kidney deposition and loss of kidney function. [Table 10]

[0254] 21-day study: EXT608 was formulated in 20 mM sodium acetate pH=5.5 buffer containing 0.8% sodium chloride. In a repeat-dose toxicokinetic study, groups of four cynomolgus monkeys (2 males and 2 females, each weighing approximately 2.5 kg) received either 1.4, 7, or 20 μg / kg of EXT608 subcutaneously in a volume of 0.05 ml / kg every other day, starting on Day 1. Blood samples (approximately 0.5 ml) were collected on Days 1, 11, and 21 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 (approximately 2700 g, approximately 10 minutes, approximately 5°C) within 1 hour of collection. Serum was collected and analyzed for calcium and phosphate using an Advia1800 clinical chemistry system (Siemens Medical Solutions USA, Malvern, PA).

[0255] EXT608 induced a dose-dependent increase in serum calcium levels that persisted for up to 24 h (Figure 16). Serum phosphate levels were not significantly changed on day 1, but by day 11, the high dose (20 μg / kg) caused a decrease in serum phosphate, and by day 21, both the high and medium doses (7 μg / kg) reduced serum phosphate, with a sustained decrease lasting for at least 72 h after dosing (Figure 17). Changes in serum calcium levels were calculated for each animal by subtracting the background level of the 0 mg / kg group from each time point, as described above. The maximum change observed for each animal was used to determine group means and standard deviations (Table 8C). Changes in serum phosphate levels were calculated for each group by subtracting the background level of the 0 mg / kg group from each time point, as described above. Because the decrease in phosphate levels persisted throughout the entire sampling period (48 h or 72 h), group means were determined by averaging the values ​​for each time point within the sampling period (Table 8C). Urine was collected at room temperature in a pan placed under the cage on day -5 (before dosing) and day 24. Neither serum nor urinary calcium levels were elevated at these time points. [Table 11]

[0256] 90-day study: EXT608 was formulated in 10 mM sodium acetate pH=5.5 buffer containing 0.8% sodium chloride and 0.1% polysorbate 80. In a repeated-dose toxicokinetic study, groups of six cynomolgus monkeys (3 males + 3 females) received either 0.7 or 2 μg / kg of EXT608 subcutaneously in a volume of 0.05 ml / kg every other day starting on Day 1. Blood samples (approximately 0.5 ml) were collected on Days 1, 45, and 89 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 serum separator tubes and centrifuged (approximately 2700 g, approximately 10 minutes, approximately 5°C) within 1 hour of collection. Serum was collected and analyzed for calcium and phosphate using an Advia 1800 clinical chemistry system (Siemens Medical Solutions USA, Malvern, PA). EXT608 induced a dose-dependent increase in serum calcium levels, which persisted for up to 12 h (Figure 18). Changes in serum calcium levels were calculated for each animal by subtracting the background level of the 0 mg / kg dose group from each time point, as described above. The maximum change observed in each animal was used to determine group means and standard deviations, as reported in Table 8D. Serum phosphate levels did not change significantly at these EXT608 doses. Urine was collected at room temperature via a pan placed under the cage on days -9 (pre-dose) and 90. Neither serum nor urinary calcium levels were elevated at these time points. [Table 12]

[0257] Example 12: EXT607 is effective in thyroparathyroidectomized (TPTx) rats A 28-day pharmacodynamic study of EXT607 in TPTx rats. Thyroparathyroidectomy (TPTx) rats are a model of hypoparathyroidism. To generate TPTx rats, the thyroid and parathyroid glands were surgically removed, and the rats were given L-thyroxine to compensate for the loss of thyroid gland. Due to the absence of PTH, TPTx rats had low serum calcium levels and high serum phosphate levels. Two weeks after surgery, animals received subcutaneous (SC) administration of wild-type PTH(1-34), PTH(1-84), or EXT607, as summarized in Table 9A, once daily (QD) for 28 days. [Table 13]

[0258] Each group consisted of 10 female rats, divided into two cohorts of 5 rats for blood sampling purposes. Blood (approximately 0.2 ml) was collected into serum separator tubes at t = 0 (pre-dose), 2, 6, 10, and 24 h on days 1, 12, and 27. Samples were processed to serum using standard methods and analyzed for serum total calcium and phosphate.

[0259] EXT607 caused an immediate, dose-dependent increase in serum calcium compared to the TPTx control group, and the increase was sustained throughout the 24-h dosing period (Figure 19). Calcium levels at 3 and 10 nmol / kg (20 and 60 μg / kg) were within or slightly below the normal range for calcium levels in healthy animals. At a dose of 1 nmol / kg (6 μg / kg), calcium levels were statistically increased compared to the TPTx control group at various time points, but the calcium levels remained below the normal range for healthy animals. The calcium increases observed with PTH(1-34) or PTH(1-84) administration were minimal and did not reach statistical significance except at two isolated time points for PTH(1-84) (Figure 20). For each time point, the percent increase in serum calcium compared to the TPTx vehicle control group (Group 2) was measured, and daily means were calculated (Table 9B). Over the 3-day sampling period (days 1, 12, and 27), EXT608 increased serum calcium by an average of 17, 28, and 37% at 1, 3, and 10 nmol / kg, respectively, compared to a 9% increase with PTH(1-34) and PTH(1-84) at 10 nmol / kg.

[0260] [Table 14]

[0261] A significant decrease in serum phosphate was observed with EXT607 at all dose levels beginning on day 12 and continuing through the remainder of the study (Figure 21). The changes were observed in a dose-dependent manner, with values ​​generally comparable to sham controls at 3 nmol / kg and 10 nmol / kg. A slight decrease in phosphate levels was observed after treatment with PTH(1-84) at 10 nmol / kg beginning on day 12 post-dose and continuing through the remainder of the study (Figure 22). Occasional decreases in serum phosphate were observed with PTH(1-34) at 10 nmol / kg. The decreases in serum phosphate with PTH(1-34) and PTH(1-84) were significantly smaller in magnitude than with EXT607. At each time point, the percent decrease in serum phosphate compared to the TPTx vehicle control group (Group 2) was measured, and daily means were calculated (Table 9C). Averaging across all time points on days 12 and 27, EXT608 reduced serum phosphate by 15, 27, and 38% at 1, 3, and 10 nmol / kg, respectively, compared with 11% and 17% reductions in PTH(1-34) and PTH(1-84), respectively, at 10 nmol / kg.

[0262] [Table 15]

[0263] Example 13: Improved formulation of EXT608 Stability as a Function of pH. The optimal pH for maximizing EXT608 stability was determined as follows. EXT608 formulations at 1 mg / ml were prepared in 50 mM sodium acetate buffer (pH 4.5 and 5.5) and phosphate buffer (pH 6.5 and 8.0) containing 10 mg / ml mannitol and incubated at -20°C, 2-8°C, and 25°C. After 10 weeks, samples were analyzed by reverse-phase HPLC to determine concentration and purity (Table 10). EXT608 stability was highest at pH 4.5 and 5.5 and gradually decreased as the pH increased to 6.5 and 8.0. [Table 16]

[0264] To further investigate the effect of pH on EXT608 stability, 0.4 mg / mL formulations were prepared in 10 mM acetate buffers containing 0.9% saline at pH 4.0, 4.5, 5.0, and 5.5 by dissolving the EXT608 API peptide in 10 mM acetic acid after first adjusting the pH with sodium hydroxide and the tonicity with saline. This liquid formulation was filled in 1 mL aliquots into screw-cap vials. All formulations were incubated at -20°C, 2-8°C, and 25°C and tested for concentration and % purity by reverse-phase HPLC at 6 months (Table 11). EXT608 stability was consistent across the pH range of 4.0 to 5.5. [Table 17]

[0265] Stability as a Function of Excipients. During the process of preparing formulations of EXT608 in 10 mM sodium acetate, pH 5.5, with the excipient 0.8% saline as an isotonicity agent, it was discovered that EXT608 had a tendency to precipitate. Further investigation revealed that precipitation could be caused by agitation (such as that encountered during the normal mixing, pumping, and filtering steps during formulation and filling into sterile vials). Polysorbate 80 was investigated as an additional excipient to reduce precipitation. Concentrations of 0.03% to 0.4% polysorbate 80 were tested and found to reduce precipitation caused by agitation of EXT608 solutions in 96-well plates using a rotary shaking platform.

[0266] Further testing was performed by replacing the ionic isotonic agent (saline) with a nonionic isotonic agent. EXT608 solutions formulated with 4.5% mannitol instead of 0.8% saline were less susceptible to precipitation caused by agitation or loss due to nonspecific absorption to surfaces. Thus, formulations containing mannitol increased the formulation's osmolality to a desirable range of 275–295 mOsm / kg, matching the osmolality of human plasma and body fluids. This reduced pain at the injection site and avoided increased precipitation in saline.

[0267] The 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.

[0268] Example 14: Single-dose pharmacokinetics and pharmacodynamics of EXT608 in humans EXT608 significantly improves serum half-life in humans in a dose-dependent manner. Sterile EXT608 formulations were 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 filled into sterile vials. Healthy human participants received EXT608 by subcutaneous injection in the abdomen at doses of 0, 36, 108, 216, or 324 micrograms (n=3).

[0269] Pharmacokinetics: Four-ml blood samples were collected at -24, -1, 0.5, 1, 2, 4, 8, 12, 18, 24, 36, 48, and 72 hours after dosing, as well as on days 5, 7, 14, 21, and 28. They were processed to K2EDTA plasma. Plasma levels of EXT608 were measured using the Immutopics High Sensitivity Human Parathyroid Hormone PTH(1-34) ELISA Kit (Cat. No. 60-3900, Quidel Corporation, San Diego, CA) according to the manufacturer's protocol with the following modifications. Prior to analysis, 100 ml of plasma samples were depleted of native PTH(1-84) using a goat anti-PTH(39-84) antibody (catalog number: 21-3010, Quidel Corporation, San Diego, CA). 2) 75 ml of depleted plasma was added to the ELISA plate along with 75 ml of 1:1 PBS + 0.05% Tween 20: LowCross buffer (catalog number: 100500, Boca Scientific, Dedham, MA). 3) A calibration curve was established using an EXT608 reference sample. Group mean EXT608 levels as a function of time are shown in Figure 23A. Pharmacokinetic parameters were calculated for each individual using Kinetica software (ThermoFisher Scientific, Waltham, MA). The mean and standard deviation of the parameters are shown in Table 12. For the low doses (36, 108, and 216 mg), the exact elimination half-life (t 1 / 2 ) could not be determined. At the high dose (324 mg), t 1 / 2 was calculated to be 90±13 h. This value and other parameters are shown in Table 13. [Table 18] [Table 19]

[0270] Pharmacodynamics: Blood samples were collected at -24, -1, 0.5, 1, 2, 4, 8, 12, 18, 24, 36, 48, and 72 hours after dosing, as well as on days 5, 7, 14, 21, and 28. These were processed to serum and submitted to Quest Diagnostics for standard blood chemistry analysis, including total calcium and albumin. Albumin-adjusted total serum calcium levels are shown in Figure 23B. EXT608 increased serum calcium levels in a dose-dependent manner and was able to maintain these elevated calcium levels for 24 hours. Blood samples (1.5 ml) were collected at -1, 4, 24, 48, and 72 hours after dosing, as well as on days 5, 7, 14, 21, and 28. These were processed to EDTA plasma and submitted to Quest Diagnostics for intact PTH(1-84) analysis (study code number 35202). In healthy humans, under conditions of elevated serum calcium levels, the body attempts to restore normal calcium levels through various mechanisms, including suppression of endogenous PTH(1-84). EXT608 reduced endogenous PTH(1-84) levels in a dose-dependent manner and maintained the reduced PTH(1-84) levels for approximately 3–4 days (Figure 23C).

[0271] EXT608 did not cause a significant increase in urinary calcium at any dose level tested when measured approximately 36 hours after administration (data not shown).

[0272] Example 15: Additional and improved formulations of EXT608 Effect of Buffer Composition and pH on the Propensity for Precipitation of EXT608. Example 13 demonstrated that EXT608 is prone to precipitation, which can be caused by agitation (such as that occurring during the normal mixing, pumping, and filtering steps involved in formulation and filling into sterile vials). It was also demonstrated that replacing an ionic isotonic agent (saline) with a non-ionic isotonic agent (mannitol) and including polysorbate 80 (PS80) in the formulation reduced the tendency for precipitation. In this example, modifications to the buffer composition and pH are made to further reduce the tendency for precipitation.

[0273] Assay 1: A solution of EXT608 (1 mg / ml) was prepared in 10 mM buffer containing 4.5% mannitol. Acetate, citrate, and histidine buffers with pH values ​​ranging from 4.0 to 5.5 were analyzed, as shown in Table 14A. The solutions were added to a sealed 96-well plate and shaken at 800 rpm on a rotary shaking platform. Absorbance measurements at 600 nm (OD600) were taken after 24 h and 120 h as a measure of light scattering caused by precipitation. After 24 h of shaking, buffers containing citrate showed extensive precipitation, while buffers containing acetate and histidine showed minimal precipitation. [Table 20]

[0274] Analysis 2: Solutions of EXT608 (1 mg / ml) were prepared in 10 mM buffer containing 4.5% mannitol. Acetate pH = 4.0, acetate pH = 5.5, and histidine pH = 4.0 buffers were tested as shown in Table 14B. PS80 (Highly Purified Polysorbate 80-LQ-(MH)SR48833, Croda, Inc., Plainsboro, NJ) was added to the formulation at 0, 0.05, 0.1, or 0.25% (v / v). OD600 measurements were taken over a 308 h period, as in Experiment 1. EXT608 solutions containing acetate pH = 4 buffer did not exhibit any precipitation, regardless of PS80 concentration, over the 308 h analysis period. In contrast, solutions containing acetate pH = 5.5 buffer without PS80 exhibited precipitation by 20 h. The inclusion of 0.05, 0.10, or 0.25% PS80 delayed the onset of precipitation until 140 h, with some minimal precipitation (OD<0.1) observed by 52 h in the 0.10% PS80 sample. Solutions containing histidine pH=4.0 buffer in the absence of PS80 precipitated by 28 h. The inclusion of PS80 delayed the onset of precipitation until 140 h, with a high degree of precipitation observed in the 0.05% and 0.10% PS80 samples and minimal precipitation observed in the 0.25% PS80 sample. Therefore, acetic acid pH=4.0 is the preferred buffer to prevent precipitation induced by agitation. [Table 21-1] [Table 21-2]

[0275] Assay 3: Solutions of EXT608 (1 mg / ml) were prepared in 10 mM acetate buffer containing 4.5% mannitol at pH 4.0, 4.5, 5.0, or 5.5. PS80 (Highly Purified Polysorbate 80-LQ-(MH)SR48833, Croda, Inc., Plainsboro, NJ) was added to the formulation at 0, 0.05, 0.1, or 0.25% (v / v). OD600 measurements were performed over a 162 h period as in Assay 1 (Table 14C). EXT608 solutions containing acetate pH 4 buffer did not exhibit any precipitation over the 162 h experimental period, regardless of PS80 concentration. Solutions containing acetate pH = 4.5 buffer showed minimal (OD < 0.1) precipitation by 42 h in the absence of PS80, minimal precipitation by 90 h with 0.05% or 0.10% PS80, and little or no precipitation by 162 h with 0.25% PS80. Solutions containing acetate pH = 5.0 buffer showed extensive precipitation by 42 h with 0 or 0.05% PS80, by 66 h with 0.1% PS80, and by 90 h with 0.25% PS80. Solutions containing acetate pH = 5.5 buffer showed extensive precipitation by 18 h with 0% PS80, by 42 h with 0.05 or 0.1% PS80, and by 66 h with 0.25% PS80. Thus, the tendency of EXT608 to precipitate in acetate buffer decreased as the pH decreased across the range of 5.5, 5.0, 4.5, and 4.0, with pH 4.0 being optimal for preventing precipitation induced by agitation. [Table 22-1] [Table 22-2]

[0276] Effect of Formulation Composition and pH on Long-Term Storage Stability of EXT608 Solutions. An improved UPLC method was developed that provides better separation of 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)). This improved method provides superior resolution and quantification of impurities, allowing for better determination of the ideal formulation composition and pH for minimizing the rate of EXT608 degradation and thereby extending shelf life. Examples of impurities resolved here by the improved UPLC method include impurities resulting from oxidation of the methionine 8 and / or methionine 18 residue(s) of the PTH1-34 component of EXT608. Peroxides, such as hydrogen peroxide, or peroxide impurities that may be present in polysorbate 80, are known to catalyze the oxidation of methionine.

[0277] The improved UPLC method used a Waters Acquity Peptide CSH C18 column (2.1 x 150 mm, 1.7 mm, 130 Å, part number: 186006938) with a flow rate of 0.35-0.40 mL / min, a column temperature of 40 °C, and a detection wavelength of 220 nm. Mobile phase A consisted of 80% water, 20% acetonitrile, and 0.1% trifluoroacetic acid. Mobile phase B consisted of 20% water, 80% acetonitrile, and 0.1% trifluoroacetic acid. The gradient conditions are shown in Table 15A. [Table 23]

[0278] To determine whether free methionine added to the formulation reduced the degradation rate of EXT608, EXT608 was prepared at 0.4 mg / ml in 10 mM sodium acetate pH 5.5 buffer containing 4.5% mannitol and 0.25% PS80, and either 0, 5, 10, 20, or 40 mM methionine was added. Samples were placed in Eppendorf tubes and stored at 4°C. Free methionine was added to remove peroxides and any other oxidizing species that could potentially oxidize the methionine residues in EXT608. For this analysis, an aged solution of PS80, which has a higher concentration of peroxide impurities, was used. Aliquots of the EXT608 formulation were taken periodically over a 41-day period, and the purity of EXT608 was measured by UPLC (Table 15B). The addition of methionine to the formulation at concentrations ranging from 5 to 40 mM helped maintain the purity of EXT608. [Table 24]

[0279] To determine how histidine buffer affects the degradation rate of EXT608 compared to acetate buffer, EXT608 was formulated at 0.4 mg / ml in 10 mM sodium acetate pH 5.5 buffer or 10 mM histidine pH 5.5 buffer. Each formulation was supplemented with 4.5% mannitol, either 0% or 0.25% PS80, and either 0 mM or 40 mM methionine, as shown in Table 15C. Samples were placed in Eppendorf tubes and stored at room temperature. Aliquots were removed on days 0, 5, and 11 for UPLC analysis of EXT608 purity. Acetate buffer demonstrated a slower rate of EXT608 degradation than histidine buffer in each condition. The addition of 40 mM methionine slowed the rate of EXT608 degradation in acetate buffer but not in histidine buffer. [Table 25]

[0280] To demonstrate how citrate buffer affects the degradation rate of EXT608 compared to acetate buffer, EXT608 was formulated at 0.4 mg / ml in 10 mM sodium acetate pH 5.5 buffer or 10 mM sodium citrate pH 5.5 buffer. Each formulation was supplemented with 4.5% mannitol, either 0% or 0.25% PS80, and either 0, 2.5, 10, or 40 mM methionine, as shown in Table 15D. Samples were placed in Eppendorf tubes and stored at 4°C, and aliquots were removed for UPLC determination of EXT608 purity over a 134-day period. EXT608 exhibited similar degradation rates in acetate versus citrate buffer, with citrate buffer slightly preferred at 0 or 2.5 mM methionine and acetate buffer slightly preferred at 10 or 40 mM methionine. The formulation with the lowest rate of degradation of EXT608 was 10 mM acetic acid pH 5.5, 4.5% mannitol, 0.25% PS80 and 40 mM methionine. [Table 26]

[0281] To determine how buffer pH affects the degradation rate of EXT608, EXT608 was formulated at 1 mg / ml in either 10 mM sodium acetate pH 4.0 buffer, 10 mM sodium acetate pH 5.5 buffer, or 10 mM histidine pH 4.0 buffer. As shown in Table 15E, each formulation was supplemented with either 0 or 0.25% PS80, 0, 10, or 40 mM methionine, and 4.5, 4.1, or 3.0% mannitol (to maintain constant formulation osmolality). Samples were placed in 2 ml screw-top glass vials and stored at 4°C. Aliquots were removed for UPLC analysis of EXT608 purity over a 69-day period. For each formulation tested, acetate pH 4.0 buffer outperformed acetate pH 5.5 buffer. In formulations without PS80, the degradation rate of EXT608 in acetate pH 4.0 buffer was similar to that in histidine pH 4.0 buffer. However, for formulations containing 0.25% PS80 and either 0, 10, or 40 mM methionine, acetate pH 4.0 was superior to histidine pH 4.0. The formulation with the lowest rate of EXT608 degradation was 10 mM sodium acetate pH 4.0 and 4.5% mannitol. The formulation with the next lowest rate of EXT608 degradation was 10 mM sodium acetate pH 4.0, 3.0% mannitol, 0.25% PS80, and 40 mM methionine.

[0282] [Table 27]

[0283] To further determine how buffer pH affects the degradation rate of EXT608, EXT608 was prepared at 1 mg / ml in either 10 mM sodium acetate buffer at pH 4.0, 4.5, 5.0, or 5.5. As shown in Table 15F, either 0 or 0.25% of PS80, either 0 or 40 mM of methionine, and either 4.5 or 3.0% of mannitol were added to each formulation (to keep the osmotic pressure of the formulation constant). Samples were placed in 2 ml screw-top glass vials, stored at room temperature, and aliquots were taken out to measure the purity of EXT608 by UPLC over a 13-day period. When methionine was absent, the degradation rate of EXT608 was faster with 0.25% PS80 compared to 0% PS80 at all tested pHs. When 40 mM methionine was added, the degradation rate of EXT608 with 0.25% PS80 was equal to or slower than that with 0% PS80. For each condition, the overall trend of the degradation rate of EXT608 was Degradation rate at pH 4.0 <≒ Degradation rate at pH 4.5 < Degradation rate at pH 5.0 < Degradation rate at pH 5.5.

Table 28

[0284] Exemplary sequences SEQ ID NO: 1 (PTH(1 - 34)) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF SEQ ID NO: 2 (PTH(1 - 84)) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ SEQ ID NO: 3 (PTH - C) SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFC[[ID=2..3]] SEQ ID NO: 4 (Vitamin D - binding protein (DBP)) MKRVLVLLLAVAFGHALERGRDYEKNKVCKEFSHLGKEDFTSLSLVLYSRKFPSGTFEQVSQFVKEVVSFTEACCAEGADPCYDTRTSAFSAKSCESNSPFPVHPGTAECCTKEGFE RKLCMAALKHQPQEFPTYVEPTNDEICEAFRKDPKEYANQFMWEYSTNYGQAPLSLLVSYTKSYLSMVGSCCTSASPTVCFLKERLQLKHLSLLTTLSNRVCSQYAAYGEKKSRLSNLI KLAQKVPTADLEDVLPLAEDITNILSKCCESASEDCMAKELPEHTVKLCDNLSTKNSKFEDCCQEKTAMDVFVCTYFMPAAQLPELPDVELPTNKDVCDPGNTKVMDKYTFELSRRTH LPEVFLSKVLEPTLKSLGECCDVEDSTTCFNAKGPLLKKELSSFIDKGQELCADYSENTFTEYKKKLAERLKAKLPDATPTELAKLVNKHSDFASNCCSINSPPLYCDSEIDAELKNIL SEQ ID NO: 5 (Vitamin D Binding Protein (DBP))

[0285] All publications and patent documents disclosed or referenced herein are incorporated by reference in their entirety. The foregoing description has been presented for purposes of illustration and description only. It is not intended to limit the invention to the precise form disclosed. It is intended that the scope of the invention be defined by the claims appended hereto.

Claims

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

2. 2. The pharmaceutical composition of claim 1, wherein the PTH conjugate comprises a scaffold, the scaffold being a 36-subunit poly(ethylene glycol) scaffold (PTH-PEG36-VitD).

3. The PTH-PEG36-VitD has 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(succinimido-propionylamino-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(succinimido-propionylamino-PEG-propionyl-aminopropyl-25-hydroxy-vitamin D)-NH 3. The pharmaceutical composition of claim 2, comprising:

4. The PTH-PEG36-VitD has 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(succinimido-propionylamino-PEG-propionyl-aminopropyl-25-hydroxy-vitamin D)-NH 3. The pharmaceutical composition of claim 2, comprising:

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the PTH conjugate has approximately the same activity at the PTH receptor (PTHR) as the equivalent unconjugated PTH peptide.

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

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

8. The PTH conjugate had an absorbance of 18,600 M at 280 nm. １ cm －１ 8. The pharmaceutical composition of claim 7, having a solubility of at least about 9.67 mM in PBS as measured using an extinction coefficient of

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

5.

10. 10. The pharmaceutical composition of 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 to 7, wherein the pharmaceutical composition is formulated with mannitol.

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

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

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

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

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

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

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

19. 19. The pharmaceutical composition of 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. 18. The pharmaceutical composition of any one of claims 1 to 17, wherein the PTH conjugate has a subcutaneous bioavailability of about 45-54% in cynomolgus monkeys.

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

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

23. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein administration of the PTH conjugate at a dose of about 2 μg / kg every other day results in an increase in serum calcium levels in cynomolgus monkeys of at least about 0.9 mg / dl over the zero dose level when measured 12 hours post-injection on the fifth day.

24. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein administration of the PTH conjugate at a dose of about 100 μg / kg every other day results in an increase in serum calcium levels in cynomolgus monkeys of at least about 6.1 mg / dl over the zero dose level when measured 12 hours post-injection on the fifth day.

25. 22. The pharmaceutical composition of 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 level to serum calcium level to about 1.8 or less in cynomolgus monkeys.

26. 22. The pharmaceutical composition of any one of claims 1 to 21, wherein a dose of about 100 μg / kg of the PTH conjugate reduces the ratio of urinary calcium level to serum calcium level to about 1.2 or less in cynomolgus monkeys.

27. 22. The pharmaceutical composition of any one of claims 1 to 21, wherein the PTH conjugate reduces serum phosphate levels by at least about 14% in TPTx rats after about 12 days when administered subcutaneously at about 6 μg / kg daily.

28. 22. The pharmaceutical composition of any one of claims 1 to 21, wherein the PTH conjugate reduces serum phosphate levels by at least about 39% in TPTx rats after about 12 days when administered subcutaneously daily at about 60 μg / kg.

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

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

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

32. 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, alopecia, chemotherapy-induced alopecia, thrombocytopenia, polyglandular autoimmune disease 32. A method of treating a subject suffering from a condition selected from the group consisting of: 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 said subject a pharmaceutical composition according to any one of claims 1 to 31.

33. 33. The method of claim 32, wherein the administering step is accomplished via subcutaneous delivery, via intramuscular delivery, via intravenous delivery, via controlled release delivery, via transdermal delivery, via parenteral delivery, or via an implanted reservoir.

34. 34. The method of claim 33, wherein the administration is via a subcutaneous route.

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

36. 35. The method of any one of claims 32 to 34, wherein the dose is administered about daily.

37. 35. The method of any one of claims 32 to 34, wherein the doses are administered approximately every other day.

38. 32. The pharmaceutical composition of any one of claims 1 to 31 for use in 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, alopecia, chemotherapy-induced alopecia, thrombocytopenia, autoimmune polyglandular 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 to 31.

40. 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, alopecia, chemotherapy-induced alopecia, thrombocytopenia, polyglandular autoimmune 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 ADH with Bartter type 5 1. A method of treating a condition selected from the group consisting of (a) and (b), comprising administering to the subject a pharmaceutical composition comprising a plurality of parathyroid hormone peptides (PTH), each of which is conjugated via a scaffold to a non-hormonal vitamin D (a PTH conjugate) and a pharmaceutical excipient, wherein each PTH peptide in the plurality is conjugated to a scaffold of approximately uniform size as measured by mass spectrometry, the method comprising subcutaneously administering repeated doses of the pharmaceutical composition, wherein the repeated doses result in a ratio of maximum concentration to minimum concentration of the PTH conjugate (Cmax / Cmin ratio) of about 8.0 or less in a serum sample from the subject.

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

37.

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

59.

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

36.

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

38.

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

83.

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

79.

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

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

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

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

51. 50. The method of any one of claims 40-49, wherein administering the PTH conjugate at a dose of about 2.0 μg / kg every other day results in an increase in serum calcium levels of at least about 0.9 mg / dl over the zero dose level when measured 12 hours post-injection on the fifth day.

52. 50. The method of any one of claims 40-49, wherein administering the PTH conjugate at a dose of about 100 μg / kg every other day results in an increase in serum calcium levels of at least about 6.1 mg / dl over the zero dose level when measured 12 hours post-injection on the fifth day.

53. 51. The method of any one of claims 40-50, wherein a dose of about 10 μg / kg of the PTH conjugate reduces the ratio of urinary calcium levels to serum calcium levels to about 1.8 or less.

54. 51. The method of any one of claims 40-50, wherein a dose of about 100 μg / kg of the PTH conjugate reduces the ratio of urinary calcium levels to serum calcium levels to about 1.2 or less.

55. 51. The method of any one of claims 40-50, wherein the PTH conjugate reduces serum phosphate levels by at least about 14% after about 12 days when administered subcutaneously at about 6 μg / kg daily.

56. 51. The method of any one of claims 40-50, wherein the PTH conjugate reduces serum phosphate levels by at least about 39% after about 12 days when administered subcutaneously at about 60 μg / kg daily.

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

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

59. The PTH-PEG36-VitD has the following structure: 【Chemical 1】 4. The pharmaceutical composition of claim 3, comprising:

60. The PTH-PEG36-VitD has the following structure: 【Chemistry 2】 5. The pharmaceutical composition of claim 3, comprising:

61. 32. A method of making the pharmaceutical composition of any one of claims 1 to 31, comprising conjugating the PTH peptide, the scaffold, and the non-hormonal vitamin D moiety to form the PTH conjugate, wherein the conjugating step is performed at a pH of less than about 7.

4.

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

3.

63. 63. The method of any one of claims 61 to 62, wherein the conjugating step is carried out at a pH of about 6.

0.

64. 64. The method of any one of claims 61 to 63, further comprising purifying the conjugate at a pH below about 8.

0.

65. 65. The method of claim 64, wherein the pH is about 5.5 in the purification step.

66. Formula I: 【Chemistry 3】 1. A pharmaceutical carrier comprising: B is a targeting group that is vitamin D that is not hydroxylated at carbon 1 and (L) at carbon 3. ａ It is conjugated to S is a scaffold moiety and comprises poly(ethylene glycol) consisting of 36 repeating ethylene glycol units; C is a maleimide group; (L) ａ is (CH ２ ) ３ NHC(O)CH ２ is a linker comprising (M) ｂ is HNC(O)(CH ２ ) ２ The pharmaceutical carrier is a linker comprising:

67. Formula VI: 【Chemistry 4】 67. The pharmaceutical carrier of claim 66, comprising a compound having the formula:

68. 68. A method for preparing a pharmaceutical composition comprising parathyroid peptide (PTH) and a pharmaceutical carrier according to claim 67, comprising: 1) purifying the compound having formula VI; 2) conjugating the compound having formula VI to the PTH; The method comprising:

69. 9. The method of claim 8, wherein the purified compound having Formula VI is not removed from a purification solvent prior to the conjugation step.

70. 70. The method of any one of claims 68 or 69, wherein the purification step is accomplished by high pressure liquid chromatography (HPLC).

71. 71. The method of any one of claims 68 to 70, wherein the conjugate is isolated as a carbonate salt.

72. 71. The method of any one of claims 68 to 70, wherein the conjugate is isolated as an acetate salt.

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

3.

74. 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, alopecia, chemotherapy-induced alopecia, thrombocytopenia, polyglandular autoimmune disease 32. A method of treating a human suffering from a condition selected from the group consisting of: 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 said human a pharmaceutical composition according to any one of claims 1 to 31.

75. 75. The method of claim 74, wherein the condition is hypoparathyroidism.

76. 75. The method of claim 74, wherein the pharmaceutical composition is EXT608.

77. 77. The method of any one of claims 74 or 76, wherein the administering step is accomplished via subcutaneous delivery, via intramuscular delivery, via intravenous delivery, via controlled release delivery, via transdermal delivery, via parenteral delivery, or via an implanted reservoir.

78. 78. The method of claim 77, wherein the administration is via a subcutaneous route.

79. 79. The method of any one of claims 74 to 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. 79. The method of any one of claims 74 to 78, wherein the pharmaceutical composition is administered at a dose of about 36, 108 or 324 μg.

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

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

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

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

85. 81. The method of any one of claims 74-80, wherein the dose is administered about every 5 days.

86. 81. The method of any one of claims 74-80, wherein the dose is administered about every 6 days.

87. 81. The method of any one of claims 74-80, wherein the dose is administered about every 7 days.

88. 81. The method of any one of claims 74-80, wherein the dose is administered about every 8 days.

89. 81. The method of any one of claims 74-80, wherein the dose is administered about every 9 days.

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

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

92. 90. The method of any one of claims 74-89, wherein the dose results in a Tmax of about 3.3 to 5.3 hours.

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

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

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

96. 90. The method of any one of claims 74-89, wherein the dose results in a Tlast of about 24 to 168 hours.

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

98. 90. The method of any one of claims 74-89, wherein 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.

99. The dose was about 324 μg SC, with a T of about 90 hours １／２ The method of any one of claims 74 to 89, wherein

100. The dose was about 324 μg SC, with an AUC of about 151 (ng / ml)*h ｉｎｆ The method of any one of claims 74 to 89, wherein

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

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

103. 90. The method of any one of claims 74-89, wherein the dose is about 324 μg SC and results in a mean retention time (MRT) of about 105 hours.

104. The dose was about 324 μg SC, with a T of about 90 hours １／２ , AUC of about 151 (ng / ml)*h ｉｎｆ 90. The method of any one of claims 74 to 89, wherein the method results in a CL / F of about 2.2 L / h, a Vz / F of about 280 L, and an MRT of about 105 hours.

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

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

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

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

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

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

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

112. 112. The pharmaceutical composition of claim 111, wherein the PTH conjugate exhibits precipitation from a solution having an optical density (OD) of about 0.242 or less when measured at a wavelength of 600 nm after shaking the pharmaceutical composition for 308 hours.

113. 113. The pharmaceutical composition of claim 112, wherein the PTH conjugate exhibits precipitation from a solution with an optical density (OD) of about 0.015 or less when measured at a wavelength of 600 nm after shaking the pharmaceutical composition for 308 hours.

114. 114. The pharmaceutical composition of any one of claims 1-14 and 108-113, wherein the purity of the PTH conjugate decreases by no more than about 7.7% after incubation at 4°C for 69 hours.

115. 114. The pharmaceutical composition of any one of claims 1-14 and 108-113, wherein the purity of the PTH conjugate decreases by no more than about 14.9% after incubation at room temperature for 13 days.

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