WO2015057836A2 - Hormone parathyroïdienne anabolique osseuse et analogues de protéine apparentée à l'hormone parathyroïdienne - Google Patents

Hormone parathyroïdienne anabolique osseuse et analogues de protéine apparentée à l'hormone parathyroïdienne Download PDF

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WO2015057836A2
WO2015057836A2 PCT/US2014/060689 US2014060689W WO2015057836A2 WO 2015057836 A2 WO2015057836 A2 WO 2015057836A2 US 2014060689 W US2014060689 W US 2014060689W WO 2015057836 A2 WO2015057836 A2 WO 2015057836A2
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pth
bone
pthrp
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WO2015057836A3 (fr
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Hesham TAWFEEK
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The Trustees Of Columbia University In The City Of New York
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis

Definitions

  • Bone is a dynamic organ that undergoes a complex and well-coordinated process of remodeling. This process involves continuous removal of bone or bone resorption by the osteoclast cells (multinucleated cells of hematopoietic origin), followed by synthesis of new bone matrix and subsequent mineralization or bone formation by the osteoblast cells (cells of mesenchymal origin). Remodeling is important both for maintaining a healthy skeleton by repairing strain-induced micro-damages and calcium homeostasis. The balance between bone formation and resorption determines the net outcome on bone mass. When bone resorption exceeds bone formation, diseases of bone loss manifest such as in postmenopausal or glucocorticoid- induced osteoporosis.
  • osteoporosis is a common world-wide bone disorder and a major public health threat for 44 million Americans over the age of 50, 68% of them being women.
  • BMD bone mineral density
  • strength will lead to prevention of these fractures.
  • various treatment options have become available, mostly anti-resorptive agents such as bisphosphonates, estrogens, calcitonin, and antibodies (Denosumab).
  • the parathyroid hormone (PTH) is an 84-amino acid peptide hormone that is an important regulator of bone remodeling through calcium and phosphate homeostasis.
  • PTH is secreted by the parathyroid glands in response to low blood calcium, and its primary function is to act on the kidney, bone and intestine to regulate calcium and phosphate homeostasis.
  • PTH maintains blood calcium through the stimulation of bone resorption, vitamin D hydroxylation, and renal calcium reabsorption.
  • PTH also acts on osteoblasts through its cognate PTH/parathyroid hormone-related peptide (PTHrP) receptor (PTH/PTHrP receptor or PTHR1) to stimulate new bone formation (1).
  • the amino terminal human PTH(l-34) fragment which is functionally similar to the full length human PTH(l-84), is the only osteo-anabolic agent so far that is approved by the US FDA for treatment of osteoporosis and patients on glucocorticoid therapy who have a high fracture risk.
  • PTH(l-34) is sold as Teriparatide or Forteo.
  • PTH(l-34) needs to be self-injected daily for 2 years resulting in compliance and cost concerns, especially compared with other forms of treatment, and resistance to PTH(l-34) develops after 2 years of treatment with no further increase in BMD or bone mass.
  • PTH is not a pure anabolic agent, it still has some bone resorptive activity and may cause hypercalcemia and hypercalciuria and other undesired side effects. All these factors limit the widespread use of PTH(l-34) and necessitate optimization of human PTH(l-34). Therefore there is a need for further study of PTH and analogs or fragments thereof for treatment of bone diseases.
  • FIG. 1 Human PTH (2-34) is a potent bone anabolic agent.
  • FIG. 2 The human PTH (2-32) is another potent bone anabolic agent.
  • FIG. 3 Removal of carboxy-terminal Region 29-34 of human PTH(l-34) reduces PTH bone anabolic effects.
  • FIG. 4 The human PTH (2-34) does not stimulate osteoclast formation as compared to PTH (1-28) and PTH (1-34).
  • Primary osteoblasts were co-cultured with BM macrophages at 10,000 and 2,000 cells, respectively, per well of 48-well plate. Next day, co-culture was treated with vehicle or the indicated PTH analogs. Every 48h, half of the medium was replaced with fresh medium and analogs. After 6 days of culture, cells were fixed and then stained for TRAP. Osteoclasts were considered when a multinucleated (>3 nuclei) TRAP positive cell was found. Each condition was performed in triplicates. The data in the graph are the means + SD.
  • FIG. 5 The human PTH (2-34) and PTH (2-32) do not stimulate osteoclast formation as compared to PTH (1-28) and PTH (1-34).
  • Primary osteoblasts were co-cultured with BM macrophages at 10,000 and 2,000 cells, respectively, per well of 48-well plate. Next day, co- culture was treated with vehicle or the indicated PTH analogs. Every 48h, half of the medium was replaced with fresh medium and analogs. After 6 days of culture, cells were fixed and then stained for TRAP. Images show effect of treatment on osteoclast formation. Osteoclasts are multinucleated (>3 nuclei) TRAP positive cells.
  • FIG. ⁇ -gal Enzyme Complementation Assay showing that the human PTH (2-34) and the human PTH (2-32) stimulation of ⁇ -arrestin/PTHRl interaction was much weaker than human PTH (1-34) and human PTH (1-28).
  • Chinese Hamster Ovary (CHO) cell line expressing PTHR1 and ⁇ -arrestin each tagged with a complementary fragment of ⁇ -gal were treated with vehicle or the indicated analog dose for 60 min.
  • Light generation after adding enzyme substrate was measured as an indication of ⁇ -gal enzyme fragment complementation and ⁇ -arrestin / PTHR1 interaction.
  • Dose for PTH (1-34) ranged from 0.0001-10 nM.
  • PTH 0.001-1000 nM.
  • PTH (2-34) and PTH (2-32) ranged from 10-1000 nM.
  • V is vehicle treated. Each condition was performed in triplicates and the results are the means + SD.
  • FIG. 7 Shows a diagram representing the putative cellular/molecular mechanisms of PTH and its interaction with PTHR1.
  • FIG. 8 The human PTH (2-33) is another potent bone anabolic agent.
  • FIG. 9 The human PTH (2-33) is as potent as Human PTH (1-34) in increasing bone formation.
  • FIG. 10 The human PTH (2-34) is much weaker than Human PTH (1-34) in increasing bone resorption in vivo.
  • Bone disease means a bone disease, disorder or a condition that is either primarily or secondarily associated with bone loss, a therapy that causes or is associated with bone loss, failure of bone to regenerate, osteoporosis, osteopenia, bone fractures, delayed fracture healing, non-union fracture, demineralization of bone (low bone mineral density), osteomyelitis, osteonecrosis, subclinical low bone density or decreased bone strength, tooth extraction tooth socket damage, inflammatory bone loss, bone marrow depression, bone marrow transplantation, peri-tooth implant bone damage, any infectious lesion affecting bone density/mass, chronic periodontitis, bone implants, and bone grafts. Also include are traumatic injury or non-traumatic necrosis associated with e.g., Gaucher' s disease, sickle cell anemia, systemic lupus erythematosus and other conditions.
  • Bone Loss includes for the purpose of this disclosure lower than normal bone mineral density (BMD), lower than normal bone mass, lower than normal bone volume and combinations of these; an increase in the level of bone resorption above normal resorption levels and a decrease in the level of bone formation below normal levels are also indications of bone loss.
  • BMD bone mineral density
  • Ctx carboxy terminal telopeptide
  • Anabolic Bone Markers indicate that bone is growing. These markers include amino terminal propeptide of type I collagen (PINP) that rises in the serum when there is anabolic bone development indicating that bones are forming/growing/regenerating.
  • PINP amino terminal propeptide of type I collagen
  • Bone density and “bone mineral density (BMD)” and “bone mass” are used interchangeably herein to refer to the amount of mineral matter per square centimeter of bones.
  • BMD is used in clinical medicine as an indirect indicator of osteoporosis and fracture risk.
  • Bone density as used here is an indicia of bone growth and bone regeneration that is used to determine the efficacy of treatment and therapeutic dose. While there are many different types of BMD tests, all are non-invasive. Most tests differ in which bones are measured to determine the BMD result. Bone density can be measured in vivo for example using PlXImus bone densitometer. Other tests include:
  • Dual-energy X-ray absorptiometry DXA or DEXA
  • Quantitative computed tomography QCT
  • DPA Dual photon absorptiometry
  • DXR Digital X-ray radiogrammetry
  • SEXA Single energy X-ray absorptiometry
  • Bone Mass/bone density is an important determinant of osteoporotic fracture risk.
  • the bone mass of a given part of the skeleton is directly dependent upon both its volume or size and the density of the mineralized tissue contained within the periosteal envelope.
  • the techniques of single- 1 and dual-energy photon or X-ray absorptiometry measure the so-called 'areal' or 'surface' bone mineral density (BMD), a variable which has been shown to be directly related to bone strength.
  • BMD bone mineral density
  • Bone mass, bone volume and bone density are used here as an indicia of bone growth and bone regeneration that is used to determine the efficacy of treatment and therapeutic dose.
  • Bone volume is measured herein in mice (and in non-human animals) as the bone volume fraction (BV/TV) using MicroCT, which method is not used on living humans.
  • Dual- energy X-ray absorptiometry or DEXA is currently the most widely used, but ultrasound has been described as a more cost-effective approach to measure bone density.
  • the test works by measuring a specific bone or bones, usually the spine, total hip, proximal femur and wrist. The density of these bones is then compared with an average index based on age, sex, and size (hereafter normal bone density or control bone density). The resulting comparison is used to determine risk for fractures and the stage of osteoporosis in an individual, or in this case efficacy of treatment.
  • Average bone mineral density BMC / W [g/cm2] ;
  • t-score the number of standard deviations above or below the mean for a healthy 30 year old adult of the same sex and ethnicity as the patient (a t-score of above -1 is indicative of a normal range, between -1 and -2.5 is indicative of early bone loss, and below -2.5 is indicative of osteoporosis)
  • Bone formation, bone growth, bone regeneration and bone regrowth are synonyms for any form of anabolic bone activity, including mineralization of bone.
  • Osteoporosis is a progressive bone disease that is characterized by a decrease in bone mass and density and that leads to an increased risk of fracture.
  • the bone mineral density (BMD) is reduced, bone microarchitecture deteriorates, and the amount and variety of proteins in bone are altered.
  • Osteoporosis is defined by the World Health Organization (WHO) as a bone mineral density of 2.5 standard deviations or more below the mean peak bone mass (average of young, healthy adults).
  • WHO World Health Organization
  • preventing refers to reducing or delaying the onset of the disease in a subject who is at high risk of developing the disease.
  • treating a disorder described herein including but not limited to bone disease, alopecia, enhancing fracture healing, improving the outcome of bone marrow transplantation as used herein includes prophylaxis, amelioration, mitigation, reducing the severity of one or more symptoms or eliminating the disease once it has been diagnosed.
  • the term “treating” includes stopping or reversing the progression of a disease.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a condition or disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a condition or disease and/or adverse effect attributable to the condition or disease.
  • terapéuticaally effective amount means an amount that achieves the intended therapeutic effect of reducing bone loss by increasing bone mass and bone density in a subject.
  • the full therapeutic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses.
  • a therapeutically effective amount may be administered in one or more administrations per day for successive days.
  • a series of doses administered over the course of a period of time is referred to herein as a "regimen.”
  • animal means any animal for example mammals, including, but not limited to humans, primates, dogs, cattle, cows, horses, kangaroos, pigs, sheep, goats, cats, rabbits, rodents, and transgenic non-human animals, and the like, which are to be the recipient of a particular treatment.
  • animal means any animal for example mammals, including, but not limited to humans, primates, dogs, cattle, cows, horses, kangaroos, pigs, sheep, goats, cats, rabbits, rodents, and transgenic non-human animals, and the like, which are to be the recipient of a particular treatment.
  • the terms “animal” “subject” and “patient” are used interchangeably herein in reference to a human subject or a rodent.
  • the preferred animal, patient, or subject is a human.
  • PTHrP PTH related peptide
  • PTHrP (2-31), PTHrP (2-32), PTHrP (2-33), PTHrP (2-34) will behave similar to PTH fragments.
  • PTH(l-34) the FDA-approved drug for treating osteoporosis, PTH(2-32), and (2-34), and most likely (2-33) and (2-31), neither increase bone resorption nor carry the risk of developing hypercalcemia or hypercalcuria.
  • the structure of these fragments is shorter than PTH (1-34), the fragments are expected to have less or minimal side effects compared to PTH (1-34).
  • PTH bone anabolic fragments PTH(2-34), PTH(2-33), PTH(2-32) and PTH(2-31), as well as PTHrP (2-32), PTHrP (2-33) or PTHrP (2-34), of the invention, (herein the enumerated PTH analogs, PTH peptides, or PTH fragments, which include cyclic, pegylated and fused versions thereof) can thus be formulated to prolong the anabolic effects of PTH and reduce the frequency of administration without the risk of drug-induced bone resorption or hypercalcemia.
  • Certain embodiments of the invention are directed to the therapeutic use of PTH(2-34), PTH(2-33), PTH(2-32) PTH(2-31), PTHrP (2-32), PTHrP (2-33) and PTHrP (2-34) to treat or prevent bone diseases associated with bone loss.
  • Bone loss is indicated, inter alia, by reduced bone density, reduced bone mass, reduced bone volume and a reduction in anabolic bone markers such as PINP as defined herein.
  • Other embodiments include pharmaceutical formulations of PTH(2-34), PTH(2-33), PTH(2-32) PTH(2-31), PTHrP (2-32), PTHrP (2-33) and PTHrP (2-34) for administration to animals.
  • the enumerated PTH fragments are administered as slow release or long acting formulations that are achieved for example by depot preparations or by attaching the enumerated PTH fragments to a collagen binding domain (54-58) or an FC antibody fragment.
  • the enumerated PTH fragments can also be fused or bound to a hydroxyapatite (HA) binding protein or peptides such as acidic oligopeptide (L-Asp or L- Glu) which targets PTH to bone. Reducing the frequency of PTH administration is important both for lowering the cost of PTH( ⁇ 1000 US dollars/month) and ensuring patient compliance and acceptance of PTH treatment option.
  • the fragments are modified by PEGylation to enhance further their bone anabolic and therapeutic properties (Patent # US7820179 B2 and US20050148763).
  • the enumerated PTH fragments can be made into a cyclic amino acid analog of parathyroid hormone (PTH) similar to one that is in development by Zelos Therapeutics Inc (ZT-031 ) for the treatment of osteoporosis and other bone-related disorders, which drug is in phase 3 clinical trials.
  • the cyclic analog may be 31-33 amino acids.
  • Osteoblasts which build bone, and osteoclasts, which resorb bone, are the key cellular modulators of bone remodeling and metabolism.
  • the PTH(l-34) fragment directly stimulates osteoblasts by increasing osteoblast number and activity, promoting the deposition of new bone matrix and accelerating the rate of mineralization.
  • PTH(l-34) indirectly stimulates bone resorption by increasing the recruitment, differentiation, and activity of osteoclasts.
  • Osteoclasts lack PTH receptors but respond to factors, including receptor activator of nuclear factor kB ligand (RANKL) and osteoprotegrin (OPG) that are secreted by osteoblasts in response to PTH(l-34).
  • RNKL nuclear factor kB ligand
  • OPG osteoprotegrin
  • iPTH has complex effects on cortical bone, including increases in cortical porosity and cortical thickness that result in neutral to positive effects on cortical strength. (2-11). This striking difference between the effects of iPTH and cPTH on bone mass reflects complex interactions between the osteoblast cells, osteoclasts, osteocytes and T cells, however, osteoblast cells and signaling through the PTHR1 receptor on osteoblasts remain central for PTH effects on bone formation and resorption. (19-21). [0034] It is conceivable that transient (as caused by iPTH) versus prolonged activation (as caused by cPTH) of some signaling pathways has a different outcome.
  • G-protein GTP-binding protein
  • GPCR GTP-binding protein-coupled receptor
  • PTHR1 activation stimulates multiple downstream pathways and events such as Gs/adenylate cyclase (AC)/cAMP/protein kinase A (PKA), Gql l/ phospholipase C (PLC)/diacylglycerol (DAG)-inositol-triphosphate (IP3)-Ca /protein kinase C (PKC), phospholipase D and Src/p-arrestin/MAPK pathways and PTHR1 phosphorylation and endocytosis.
  • PAG Gql l/ phospholipase C
  • DAG diacylglycerol
  • IP3 phospholipase D
  • Src/p-arrestin/MAPK phospholipase D
  • PTHR1 phosphorylation and endocytosis phosphorylation and end
  • Human PTH(2-34) efficiently increases bone volume while the human PTH(3-34) exhibits no effect.
  • the human PTH(2-32) is another potent bone anabolic agent.
  • the human PTH(2-34) and PTH (2-32) do not stimulate osteoclast formation as compared to PTH(l-28) and PTH(l-34).
  • ⁇ -gal Enzyme Complementation Assay (hereafter the " ⁇ -gal " assay) is a new technology that measures PTHRl interaction with the ⁇ -arrestin, an adapter molecule that mediates PTHRl internalization and signaling. Specifically a PathHunter express PTHRl Chinese Hamster Ovary- Kl (CHO-K1) ⁇ -arrestin GPCR Assay (Discoverx, Fremont, CA) was used. This technology is based on the ⁇ -gal enzyme fragment complementation. The assay is extremely sensitive and detects interactions at less than pico molar PTH concentrations. The assay can therefore be used as a platform for PTH drug discovery and screening.
  • PTH(2-34) and PTH(2-32) ranged from 10-1000 nM. V is vehicle treated. Each condition was performed in triplicates and the results are the means + SD. The ⁇ -gal assay showed that the human PTH(2-34) and the human PTH(2-32) stimulation of ⁇ -arrestin/PTHRl interaction was much weaker than stimulation with human PTH(l-34) or human PTH(l-28) (FIG 6).
  • Certain embodiments of the invention are directed to methods for treating bone diseases associated with low bone mass by administering one or more enumerated PTH analogs to an individual in need of such treatment in a therapeutically effective amount that increases bone density/mass or the level of anabolic bone markers.
  • These indicia of bone anabolism can be determined using routine methods in the art that are set forth herein.
  • Preferred formulations enhance uptake of the PTH fragments by bone.
  • Other embodiments are directed to slow-release or long acting formulations.
  • Bone loss can be due to loss of bone density or reduced bone mass (used interchangeably herein) or by loss of bone volume, although volume is difficult to measure in a living human.
  • Means for measuring loss of bone density/mass include conventional radiography that is useful, both by itself and in conjunction with CT or MRI, which also detect fractures.
  • radiography is relatively insensitive to detection of early disease and requires a substantial amount of bone loss (about 30%) to be apparent on X-ray images.
  • Bone loss is typically measured as a loss of bone density using dual-energy X-ray absorptiometry (DEXA). Other means for measuring bone density are described above.
  • Anabolic bone chemical biomarkers are also a useful tool in detecting bone formation/growth/regeneration, thus enabling one to monitor the efficacy of PTH fragment treatment in reversing bone loss.
  • Anabolic bone markers in the serum that indicate the level of bone formation can be measured by ELISA and used as early indicators of efficacy that can be detected before bone density changes may appear.
  • serum Osteocalcin (OCN) and the anabolic marker aminoterminal propeptide of type I collagen (PINP) can be monitored, with an increase in post-treatment levels compared to pre-treatment levels of these markers being an indication of PTH fragment are effective in stimulating bone formation/growth/regeneration as a means for treating diseases associated with bone loss.
  • serum OCN and PINP are monitored to determine efficacy of PTH fragment therapy.
  • monitoring bone resorption factors provides an indication of the state of the bone physiology and they can be contrasted with the level of anabolic bone markers.
  • a common catabolic marker that indicates bone resorption is carboxy (C) terminal telopeptide (Ctx). Ctx has been used to monitor the progress of Rheumatoid Arthritis and osteoarthritis (OA), for example.
  • the extent of PTH fragment therapy can be monitored by both the level of anabolic and catabolic or together known as bone turnover markers, such that if the increase in anabolic markers is greater than the increase in catabolic markers, there is net bone growth or bone gain.
  • Bone diseases that can be treated by administering one or more of the enumerated PTH fragments defined above include any bone disease, disorder or a condition that is associated with bone loss, including failure of bone to regenerate, osteoporosis, osteopenia, osteonecrosis, bone fractures, demineralization of bone (low bone mineral density), osteomyelitis, osteonecrosis, subclinical low bone density or decreased bone strength.
  • the enumerated PTH fragments will promote peri-implant bone healing, and in an embodiment can be administered together with zoledronic acid adsorbed onto the implant surface, In chronic periodontitis PTH fragments can cause greater resolution of alveolar bone defects and accelerate osseous wound healing in the oral cavity.
  • the enumerated PTH fragments can be used where there is a high fracture risk or history of fracture, or a loose implant, The enumerated PTH fragments can slow down vertebral degeneration by enhancing bone formation.
  • osteoporosis a disease that is also associated with a high incidence of fractures.
  • PTH(l-34) is an FDA-approved drug for treating osteoporosis, however it retains some catabolic function that promotes bone resorption, and can cause hypercalcemia and hypercalcuria.
  • the enumerated PTH fragments retain anabolic activity but have minimal levels of catabolic activity.
  • the enumerated PTH fragments are administered individually to avoid competition for binding to PTHR1 receptor. However, routine experimentation will determine which combinations of PTH fragments can be administered together with optimal efficacy.
  • the enumerated PTH fragments can be used in combination with other anti-resorptive therapies as has been done with PTH (1-34), or with other drugs that stimulate bone formation and regrowth.
  • a therapeutic dose for the enumerated PTH fragments is similar to that established for PTH(l-34).
  • the current PTH (1-34) dose approved by the US FDA for Forteo® administered for treating osteoporosis is 20 ⁇ g/day subcutaneously, an amount that can vary from about 20.0 ⁇ g/ day to about 40.0 ⁇ g/ /day. However, many factors affect the dose, and thus a broad range of between about 1 ⁇ g/day to about 500.0 ⁇ g/ day is possible.
  • a therapeutic dose/amount of one of the enumerated PTH fragments is from about 1 ⁇ g/day to 500 ⁇ g/day.
  • Fusion proteins of (PTH) (1-33) joined/bound to the collagen binding domain of ColH (CBD) have been administered as long acting formulations in amounts up to 320 g/kg on a weekly or monthly basis.
  • Routes of administration, frequency of administration and pharmaceutical formulation type, pharmacokinetcis and individual subject physiology are just some of the factors that affect dosage.
  • Osteopenia and subtherapeutic bone mass or bone density can also be treated similarly to osteoporosis, although perhaps with lower doses or less frequent administration, as can be determined by routine experimentation.
  • PTH has been established as a stimulator of hematopoietic stem cell expansion (34-36) and is presently in clinical trials testing its action on improving the outcome of bone marrow (BM) transplantation. Therefore certain other embodiments are directed to methods of treating a subject undergoing bone marrow transplantation (either before or after or before and after the transplant) with one or more of the enumerated PTH fragments. Other embodiments are directed to methods for treating a subject having inflammatory arthritis with one or more of the enumerated PTH fragments.
  • Osteoporosis can be defined as a systemic skeletal disease characterized by low bone mass, microarchitecture deterioration of bone tissue, increased bone fragility and susceptibility to fracture.
  • Osteoporosis may be caused by, or associated with, various factors. Being female, particularly a post-menopausal female, having a low body weight, and leading a sedentary lifestyle are all risk factors for osteoporosis (loss of bone mineral density, leading to fracture risk).
  • Persons having any of the following profiles may be candidates for treatment with an enumerated PTH fragment: a post-menopausal woman and not taking estrogen or other hormone replacement therapy; a person with a personal or maternal history of hip fracture or smoking; a post-menopausal woman who is tall (over 5 feet 7 inches) or thin (less than 125 pounds); a man with clinical conditions associated with bone loss; a person using medications that are known to cause bone loss (including corticosteroids such as PrednisoneTM, various anti-seizure medications such as DilantinTM and certain barbiturates, or high-dose thyroid replacement drugs); a person having type 1 diabetes, liver disease, kidney disease or a family history of osteoporosis; a person having high bone turnover (e.g., excessive collagen in urine samples); a person with a thyroid condition, such as hyperthyroidism; a person who has experienced a fracture after only mild trauma; a person who has had x-ray evidence of vertebral fracture or other signs of osteo
  • Bone loss resulting from cancer therapy is widely recognized and termed cancer therapy induced bone loss (CTIBL) and can be treated with these PTH fragments.
  • CTIBL cancer therapy induced bone loss
  • PTH (1-34) is contraindicated in bone metastasis, bone cancers or other metabolic bone diseases.
  • bone diseases as candidates for treatment further include osteomyelitis, osteopenia, osteonecrosis, and any infectious lesion in bone that leads to bone loss.
  • Osteonecrosis, or bone cell death is associated with traumatic injury or non-traumatic necrosis associated with e.g., Gaucher' s disease, sickle cell anemia, systemic lupus erythematosus and other conditions.
  • the enumerated PTH fragments may be useful in treatment of periodontal bone loss, and bone fracture healing.
  • the enumerated PTH fragments may be useful in patients that are diagnosed with subclinical low bone density, as a protective measure against the development of osteoporosis.
  • methods and compositions of the present invention may find medical utility in the healing of bone fractures and cartilage defects including open fracture reduction and fixation of artificial joints. It has been reported that local or systemic PTH does help cartilage regeneration and prevent further degeneration in animal osteo-arthritis by Livne et al 1989, Chang et al 2009, Sampson et al 2011, Eswaramoorthy et al 2012).
  • One route of administration in addition to systemic administration is implantation of slow-release preparations of one or more of the enumerated PTH fragments. De novo bone formation induced by the enumerated PTH fragments will contribute to the repair of congenital, trauma-induced, or oncologic resection induced craniofacial defects.
  • Other methods are contemplated that are useful for identifying subjects having bone disease that causes bone loss, measured as a loss of bone mass or bone density, or subjects at risk of developing such a disease.
  • pre-treatment levels of bone density or bone mass or both are made and compared to control levels in a normal subject. If the level of bone density/mass of the subject having the bone diseases is significantly lower than the level of bone density of the control, then the subject is administered a therapeutically effective amount of an enumerated PTH fragment such as PTH(2-34), PTH(2-33), PTH(2-32) and/or PTH(2-31).
  • Post-treatment levels of bone density/mass can be determined at multiple times after treatment begins to monitor and adjust the therapeutic dose to determine the subject's response to treatment. If the post-treatment bone density/mass of the subject is significantly higher than the pretreatment level, then treatment is continued until the subject bone density level reaches the control level of bone density.
  • a subject that responds to therapy may be put on a maintenance dose of PTH fragments in order to prevent deterioration.
  • a maintenance dose may be lower than the dose used to raise the bone mass to control levels and it can be determined by monitoring the subject's response to treatment.
  • the enumerated PTH fragments can also be administered to treat osteoarthritis, Rheumatoid arthritis (Fukata S, et al., 2004 Arthritis Rheum 50:4060-4069 AND Redlich K et al 2004 Am J Pathol AND Schett G et al, 2006 Ann N Y Acad Sci 1068:477-488 AND Migliore A, 2012 Eur Rev Med Pharmacol Sci), or to treat or prevent alopecia, preferably conjugated to a collagen binding domain.
  • a composition comprising one or more of the enumerated PTH fragments is administered in a subject in need at a dosage and frequency to increase bone density or bone formation markers and/or decrease bone resorption markers.
  • the subject in need has suffered bone loss and/or is at an elevated risk of bone fractures.
  • the increase in bone density serves to reduce the risk of bone fractures.
  • the subject in need exhibits symptoms of osteoporosis.
  • a method of reducing the risk of bone fractures that involves administering one or more of the enumerated PTH fragments.
  • the PTH fragments are administered according to a regimen that increases bone density or bone formation markers, and which optionally do not increase or decrease bone resorption markers.
  • Regulatory approval of PTH (1-34) involved the demonstration of reduced risk of fracture in patients diagnosed with osteoporosis. However, demonstration of reduced risk of fracture requires extended monitoring times (1-2 years). Changes in bone formation markers and bone density can occur much sooner (approx. 3 months), which allows one to determine therapeutic efficacy earlier in clinical studies.
  • compositions or formulations and administration are provided.
  • a "therapeutically effective amount" of an enumerated PTH fragment will vary depending on such factors as weight, diet, the subject's general health, concurrent medication the particular mode of administration and the pharmacokinetics of the formulation. Because these factors and their relationship to determining a therapeutically effective amount are well known in the medical arts, the determination of therapeutically effective dose is within the ambit of the skilled person.
  • administering is initiated at lower dosage levels, with the dosage level being increased until the desired therapeutic result is achieved for a particular individual being treated.
  • a therapeutic dose can be similar to that established for PTH(l-34) for treating osteoporosis, which for humans is 20-40 ⁇ g/day. This dose can vary widely from between about 5.0 ⁇ g/ day to about 500.0 ⁇ g/ day depending on the factors described above.
  • compositions When administered other than by injection, such as oral, rectal, transdermal, nasal or pulmonary, the pharmaceutical compositions can be prepared in conventional forms.
  • Oral administration may be in liquid formulation or as tablets or capsules. Rectal administration may be as suppositories.
  • Nasal and pulmonary administration can be as liquid or powder.
  • the appropriate mode of administration will vary inter alia depending upon factors known in the art, including the acuteness and severity of the condition being treated, the frequency of administration and the dosage required. Any mode of administration that produces desired therapeutic effect without unacceptable adverse effects is relevant in practicing this invention.
  • the enumerated PTH fragments are typically administered intermittently.
  • Preferred intermittent administration schedules include daily, every second day, every third day, twice per week, every fourth day, every fifth day, every sixth day, and once per week.
  • the enumerated PTH fragments of the present invention can be formulated in various ways including as salts or derivatives thereof, for storage or administration depending on the particular need of the subject and the bone disease being treated.
  • the enumerated PTH analogs were chemically synthesized (95% pure) and therefore have residue Trifluoroacetic acid (TFA). Almost all peptide drugs are in the TFA salt form. Typically peptide drugs for clinical trials are in the acetate salt form.
  • lyophilized peptides in 0.9% NaCl + 100 mM Acetic acid were stored at -20° C until they were used.
  • compositions comprising the enumerated PTH fragments (e.g., PTH(2-34), PTH(2-33), PTH(2-32) and PTH(2-31)), can be formulated in any suitable amount, vehicle or pharmaceutically acceptable carrier for delivery. It is within the invention to provide a pharmaceutical composition, wherein one or more of the enumerated PTH fragments, are present in an amount effective to increase bone mass in a subject to whom they are administered (a therapeutically effective amount).
  • compositions of the present invention may be formulated and used as tablets, capsules, or elixirs for oral or buccal administration; for use in vaginal or rectal administration particularly in semisolid forms such as creams and suppositories. They may also be formulated in sterile solutions and suspensions for injection, inhalation or pulmonary administration in the form of powders, nasal drops or aerosols.
  • the enumerated PTH fragment(s) are formulated with a carrier that is pharmaceutically acceptable and is appropriate for delivery by the chosen route of administration.
  • Suitable pharmaceutically acceptable carriers are those used conventionally with peptide-based drugs, such as diluents, excipients and the like.
  • Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition, 1995).
  • compositions suitable for parenteral administration conveniently comprise a sterile aqueous preparations of one or more of the enumerated fragments that are preferably isotonic with the blood of the recipient.
  • This aqueous preparation may be formulated according to known methods using those suitable dispersing or wetting agents and suspending agents.
  • the injectable aqueous formulation for Forteo® which contains human PTH(l-34), is exemplary of a suitable pharmaceutical formulation for embodiments of the present invention for PTH fragments.
  • the FDA approved dose for Forteo is 20 ⁇ g subcutaneously once a day, but it is also administered up to 40 ⁇ g /day.
  • a therapeutic dose/amount of one of the enumerated PTH fragments is from about 1 ⁇ g /day to 500 ⁇ g /day.
  • Fusion proteins of (PTH) (1-33) joined/bound to the collagen binding domain of ColH (CBD) have been administered as long acting formulations in amounts up to 320 ⁇ g/kg on a weekly or monthly basis.
  • Administer as a An example of an aqueous formulation is a solution in 1,3-butane diol. Water, Ringer's solution, and isotonic sodium chloride solution are exemplary acceptable diluents.
  • Sterile, fixed oils may be employed as a solvent or suspending medium.
  • Bland fixed oils, including synthetic mono or diglycerides, and fatty acids, such as oleic acid, may also be used.
  • Formulations for vaginal or rectal administration may contain as excipients, for example, polyalkylene glycols, vaseline, cocoa butter, and the like.
  • Formulations for inhalation administration may be solid and contain as excipients, for example, lactose or may be aqueous or oily solutions for administration in the form of nasal drops.
  • excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like.
  • Preparations of the invention may optionally comprise pharmaceutically acceptable salts, buffering agents, preservatives and excipients. Examples of these salts are those of mineral or organic acids, e.g. of hydrochloric, acetic or methanesulfonic acid. Also salts as alkaline metal or alkaline earth salts, such as sodium or magnesium salts of the carboxylic acid group, are conceivable.
  • the present enumerated PTH fragments may be administered in a vehicle, such as distilled water or in saline, phosphate buffered saline, 5% dextrose solutions or oils.
  • a vehicle such as distilled water or in saline, phosphate buffered saline, 5% dextrose solutions or oils.
  • the solubility of the enumerated PTH fragment may be enhanced, if desired, by incorporating a solubility enhancer, such as detergents and emulsifiers.
  • the enumerated PTH fragments may be utilized in the form of a sterile-filled vial or ampoule containing a pharmacologically effective amount of the enumerated PTH fragment(s), in either unit dose or multi-dose amounts.
  • the vial or ampoule may contain the enumerated PTH fragment and the desired carrier, as an administration ready formulation.
  • the vial or ampoule may contain the enumerated PTH fragment in a form, such as a lyophilized form, suitable for reconstitution in a suitable carrier, such as sterile water or phosphate-buffered saline.
  • Suitable buffering agents are systems of acetic acid (1-2% w/v), citric acid (1-3% w/v); boric acid (0.5-2.5% w/v), and phosphoric acid (0.8-2% w/v).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).
  • the pharmaceutical compositions are formulated for administration by infusion, parenteral administration, or by injection, for example subcutaneous, intraperitoneal or intravenous injection, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered to physiologically tolerable pH.
  • Formulation for intramuscular administration may be based on solutions or suspensions in plant oil, e.g. canola oil, corn oil or soy bean oil. These oil based formulations may be stabilized by antioxidants e.g. BHA (butylated hydroxianisole) and BHT (butylated hydroxytoluene).
  • compositions of the present invention may be placed within containers, or kits, along with packaging material which provides instructions regarding the use of such pharmaceutical compositions.
  • instructions will include a tangible expression describing the reagent concentration, as well as within certain embodiments, relative amounts of excipient ingredients or diluents (e.g., water, saline, or PBS) which may be necessary to reconstitute the pharmaceutical composition.
  • PTH and its related peptide PTHrP have been crystalized for studying PTH/PTHR1 interaction (Eli-Lilly) (Jin et al 2000 JBC).
  • delayed absorption of a parenterally administered drug form can be accomplished by dissolving or suspending the drug in an oil vehicle.
  • PTH(l-34) has been fused to the Fc fragment of human IgGl to increase the half-life of PTH.
  • Skeletal anabolism was examined in mice and rats treated once or twice per week with this PTH-Fc- fusion protein and resulted in significant increases in bone volume, density, and strength in osteopenic ovariectomized mice and rats.
  • Kostenuik, P., et al. Infrequent Delivery of a Long-Acting PTH-Fc Fusion Protein Has Potent Anabolic Effects on Cortical and Cancellous Bone, J Bone and Miner Res, Vol. 22, No. 10, Pages 1534-1546 (June 18, 2007).
  • PTH(2-33) has been fused to a collagen binding domain (PTH-CBD) to extend its activity, and has shown an anabolic bone effect with monthly dosing.
  • PTH-CBD collagen binding domain
  • the duration of action of such a compound was tested with different routes of administration.
  • Normal young C57BL/6J mice received a single intraperitoneal injection of PTH-CBD (320 ⁇ g/kg), which caused sustained increases in bone mineral density by >10 % for 1 year in normal mice, regardless of the route of administration. See, Ponnapakkam, T., et ah, , Calcif Tissue Int (2012) 91: 196-203.
  • the enumerated PTH fragments are made into fusion proteins to make them long acting, as is described in Ponnapakkam, T., et al., Calcif Tissue Int (2011) 88: 511-520.
  • the fusion proteins comprised parathyroid hormone (PTH) (1-33) joined/bound to the collagen binding domain of ColH (CBD).
  • PTH-PKD-CBD Two fusion proteins were synthesized, linking the carboxy terminus of the PTH fragment either directly to the amino terminal of the CBD or to the CBD through an adjacent ColH domain.
  • PTH-PKD-CBD Two fusion proteins were synthesized, linking the carboxy terminus of the PTH fragment either directly to the amino terminal of the CBD or to the CBD through an adjacent ColH domain.
  • Both PTH-CBD and PTH-PKD-CBD had biological activity and both fusion peptides bound to type 1 collagen in flow-through assays.
  • the PTH-CBD was concentrated in the bone and skin, tissues with abundant collagen and blood flow. These fusion proteins were administered at a dose of 320 ⁇ g/kg on a weekly or monthly basis.
  • the enumerated PTH fragments are cyclic amino acid analogs of the respective PTH peptide; or are pegylated.
  • the modifications taught herein can be applied not only to the PTH analogs taught herein (e.g.,PTH(2- 31), PTH (2-32), PTH (2-33), PTH (2-34), PTHrP (2-31), PTHrP (2-32), PTHrP (2-33) or PTHrP (2-34)), but may be applied to human or non-human PTH (1-34), PTH (1-38), PTH (1-84) or any other fragment, or PTHrP (1-34), PTHrP (1-36) or any other PTHrP fragment.
  • the cyclic analogs are 31-33 amino acids in length.
  • the 2-34C and 2-33C cyclic analogs are 33 and 32 amino acids, respectively.
  • a dosage form a may contain a pharmaceutically acceptable non-toxic salt of the enumerated PTH fragments which has a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, or with an organic cation formed from e.g., ⁇ , ⁇ '-dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b) e.
  • a polybasic acid such as phosphoric acid, sulfuric acid, citric
  • the enumerated PTH fragments of the present invention or, preferably, a relatively insoluble salt such as those just described, may be formulated in a gel, for example, an aluminum monostearate gel with, e.g. sesame oil, suitable for injection.
  • Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like.
  • Another type of slow release depot formulation for injection would contain the enumerated fragment dispersed for encapsulated in a slow degrading, non-toxic, non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer, for example as described in U.S. Pat. No. 3,773,919.
  • injectable depot forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polyactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include polyorthoesters and polyanhidrides.
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissue.
  • the compounds or, preferably, relatively insoluble salts such as those described above may also be formulated in cholesterol matrix silastic pellets, particularly for use in animals.
  • Aqueous carriers or vehicles can also be supplemented for use as injectables with an amount of gelatin that serves to depot the enumerated PTH fragment at or near the site of injection, for its slow release to the desired site of action.
  • Alternative gelling agents such as hyaluronic acid, may also be useful as depot agents.
  • Micro-CT Computerized Tomography
  • BS trabecular bone surface
  • Tb.Th trabecular thickness
  • Tb.N trabecular number
  • cortical thickness Co.Th: ⁇
  • cortical area Co.S mm2
  • the dynamic indices of bone formation such as the bone formation rate/bone surface, the mineralizing surface/bone surface and the mineral apposition rate can be measured.
  • the Micro-CT analyses are performed using a SCANCO Micro-CT Scanner instrument.
  • Osteoblast and osteoclast cell proliferation and apoptosis can be measured in vivo in non- human animals to monitor bone resorption using 5-bromo-2'-deoxyuridine (BrdU) and Tunnel staining, respectively, as described previously.
  • BadU 5-bromo-2'-deoxyuridine
  • Tunnel staining respectively, as described previously.
  • Calvi LM et al., 2001
  • Activated parathyroid hormone/parathyroid hormone-related protein receptor in osteoblastic cells differentially affects cortical and trabecular bone. J Clin Invest 107:277-286; Jilka RL, et al., 1998
  • Osteoblast programmed cell death (apoptosis) modulation by growth factors and cytokines.
  • the biochemical analysis of bone turnover markers can be measured by ELISA and includes indices of bone formation (osteocalcin or OCN, amino-terminal propeptide of type I collagen or P1NP, and BSAP) and bone resorption (CTX1 and TRAP5b) in the serum.
  • PTH fragments are expected to increase the levels of bone formation and not to affect or to slightly increase resorption markers.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference.
  • One embodiment of the present invention is related to an analog of the human PTH (1- 34) with unique clinically-relevant bone anabolic properties. Disclosed is the structure of the polypeptide, chemical methods for synthesizing and producing the polypeptide, the method and the dose of the polypeptide used for demonstrating the bone anabolic effect in wild type mice. Furthermore, disclosed is the method that demonstrates the inability of this polypeptide to stimulate osteoclast cell formation in vitro.
  • the polypeptide human PTH (2-34) efficiently increases bone mass.
  • Results The results of the micro-CT analysis demonstrate that the human PTH fragment (2-34) polypeptide maintained a great bone anabolic effect compared to the human PTH (1-34). See Fig 1.
  • the present invention is related to another related synthetic fragment of the human parathyroid hormone (PTH) (1-34) with unique clinically-relevant bone anabolic properties.
  • PTH human parathyroid hormone
  • the invention discloses the structure of this polypeptide, chemical methods for synthesizing and producing the polypeptide, the method and the dose of the polypeptide used for demonstrating the bone anabolic effect in wild type mice. Furthermore, the invention discloses the method that demonstrates the failure of this polypeptide to stimulate osteoclast cell formation in vitro.
  • the polypeptide, human PTH (2-32) human increases bone mass.
  • Results The results of the micro-CT analysis demonstrate that the human PTH fragment (2-32) polypeptide still exhibits a bone anabolic effect compared to the human PTH (1-34). See Fig 2.
  • the present invention is related to the human parathyroid hormone (PTH) (1-34) amino acid sequences that have a PTH bone anabolic effect, human PTH (1-28) is a carboxy terminal- truncated human PTH polypeptide fragment that lost most of the bone anabolic activity is the.
  • PTH parathyroid hormone
  • the polypeptide human PTH (1-28) is much less potent than the human PTH (1-34) in increasing bone mass.
  • Results The results of the micro-CT analysis demonstrate that the human PTH fragment (1-28) polypeptide is much less bone anabolic agent than the human PTH fragment (1-34). See Fig 3. [0113] Conclusion: Removal of sequences 29-34 from human PTH (1-34) reduces the bone anabolic effect of human PTH (1-34).
  • the polypeptides human PTH (2-34) and human PTH (2-32) do not stimulate osteoclast formation compared to human PTH (1-34) and human PTH (1-28).
  • BM bone marrow
  • RBC lysis buffer was then neutralized by adding 10X volume of phosphate buffered saline (PBS) and cells were spun in a table top centrifuge for 10 min at 1500 rpm at room temperature. Supernatant was discarded and the cells were resuspended in medium and cultured in a 10 cm dish plate in 10 ml full medium.
  • the medium consisted of alpha modification of minimum essential eagle medium (a-MEM) containing 10% fetal bovine serum, 10% penicillin/streptomycin and 50 ug/ml ascorbic acid. Next day, cells in suspension were removed and the medium was replaced.
  • a-MEM minimum essential eagle medium
  • adherent cells osteoblasts progenitors and BM macrophages
  • adherent cells were rinsed twice with PBS.
  • Adherent primary osteoblasts were separated from macrophages by differential trypsinization. Osteoblasts were lifted from the plate after 5 min of incubation at 37 C with 2 mis of 0.05% trypsin. Trypsin was inhibited by adding 3X volume of a-MEM medium and spinning in a table top centrifuge for 5 min at 1500 rpm. Remaining adherent cells are macrophages which were lifted from the plate by incubation with trypsin for 20 min and then gentle scraping of cells.
  • the co- culture was treated continuously with vehicle or 10 and 100 nM of human PTH (1-34), the human PTH (2-34), the human PTH (2-32) or the human PTH (1-28) for 6 days.
  • Half of the medium was replaced every 48-72h with fresh medium and reagents.
  • Osteoclast formation was measured by counting the multinucleated (at least 3 nuclei) Tartrate-resistant acid phosphatase (TRAP) stain positive cells.
  • Certain embodiments are directed to the novel PTH fragments, human PTH (2-34) and (2-32), which are bone anabolic agents that do not stimulate osteoclast formation that leads to . undesired bone resorptive properties. It is believed that PTH (2-33) will also exhibit similar properties. Furthermore, the human PTH (1-28) is a more bone catabolic than an anabolic agent.
  • the polypeptides human PTH (2-34) fails to stimulate ⁇ -arrestin interaction with PTHR1 as compared to the human PTH (1-34) and the human PTH (1-28).
  • Methods We used PathHunter express PTHR1 CHO-K1 ⁇ -arrestin GPCR Assay from Discoverx (Catalog Number 93-0315E2). The assay is based on a ⁇ -gal Enzyme Complementation. Chinese Hamster Ovary (CHO) cell line expressing PTHR1 and ⁇ -arrestin each tagged with a complementary fragment of ⁇ -gal were treated with vehicle or the indicated analog dose in Fig 6 for 60 min. Light generation after adding enzyme substrate was measured as an indication of ⁇ -gal enzyme fragment complementation and ⁇ -arrestin / PTHR1 interaction. The dose for PTH (1-34) ranged from 0.0001-10 nM. The dose for PTH (2-34) ranged from 10- 1000 nM.
  • Position 1 deletion results in loss of ⁇ -arrestin /PTHR1 interaction and possibly attenuation of ⁇ -arrestin-mediated signaling.
  • Lack of ⁇ -arrestin /PTHR1 interaction in response to PTH (2-34) may account for lack of osteoclast formation capabilities for PTH (2-34) and (2-32).
  • ⁇ -arrestin pathway may, therefore, be a potential bone catabolic pathway.
  • the human PTH (2-33) is another potent bone anabolic agent.
  • Results The results of the micro-CT analysis demonstrate that the human PTH fragment (2-33) polypeptide exhibits a bone anabolic effect compared to the human PTH (1-34). See Fig. 8.
  • the human PTH (2-33) is as potent as Human PTH (1-34) in increasing bone formation.
  • Results The results of the osteocalcin level analysis demonstrate that the proposed human PTH (2-33) polypeptide exhibits a similar bone formation effect to that of the human PTH (1-34). See Fig 9.
  • the human PTH (2-34) is much weaker than Human PTH (1-34) in increasing bone resorption in vivo. [0128] Goal: To examine the bone resorption potency of human PTH (2-34).
  • Results The results of the CTX sera levels analysis demonstrates that the proposed human PTH (2-34) polypeptide exhibits decreased bone resorption compared to the human PTH (1-34). See Fig 10.
  • Amino acid (AA) sequence Val - Ser - Glu - He - Gin - Leu - Met - His - Asn - Leu - Gly - Lys - His - Leu - Asn - Ser - Met - Glu - Arg - Val - Glu - Trp - Leu - Arg - Lys - Lys - Leu - Gin - Asp - Val - His - Asn - Phe- Gly- He- Asn- Ser- Asn- Leu- Ser- Gly- Lys- Phe- Lys- Ala- Asp- Lvs-NH2
  • the 33 AA sequence that is not underlined is AA sequence 2-34 of human PTH.
  • the 4 AA underlined sequence is a short flexible linker.
  • the 10 AA underlined sequence is a collagen binding domain sequence for bone targeting.
  • the 32 AA sequence that is not underlined is AA sequence 2-33 of human PTH.
  • the 4 AA underlined sequence is a short flexible linker.
  • the 10 AA underlined sequence is a collagen binding domain sequence for bone targeting. Modifications of Human PTH (2-34) and PTH (2-33) analogs for enhancing activity and prolonging the action:
  • the enumerated PTH analogs can be made via chemical synthesis methods or genetic recombinant/expression techniques (cell-based or cell-free systems) known in the art.
  • cells such as bacteria or yeast cells can be transfected with an expression vector having a sequence encoding the enumerated PTH analog.
  • the cells expressing the analog are cultured so that they transcribe and translate the PTH analog, which can then be obtained from the cell and cleaned, typically by lysing the cells and subjecting the lysate to purification.
  • PEG polyethylene glycol group
  • the peptides were chemically synthesized using Fmoc (9-fluorenylmethoxy carbonyl) chemistry.
  • the peptide chains were synthesized from the carboxyl terminus to the Asn amino terminus onto resin.
  • the resin was incubated with dichloromethane (DCM) for 30 minutes and then washed with dimethylformamide (DMF) three times.
  • DCM dichloromethane
  • DMF dimethylformamide
  • Fmoc-protecting groups at the amino terminus were deprotected with an alkaline buffer and then washed with DMF three times to remove the deprotection buffer.
  • the second amino acid was Fmoc-His(Trt)-OH coupled to the first amino acid and then DMF cleaned.
  • the Fmoc-Lys(Dde)-OH was used for the 26th amino acid.
  • the Boc-Val-OH was used for the last amino acid.
  • Dde was removed and Lys was coupled with Fmoc-mini-PEG-OH.
  • the peptide was ninhydrin tested and the coupling and washing steps repeated until the crude peptide was fully synthesized.
  • the peptide was cleaved from the resin.
  • the crude peptides were then diethyl ether precipitated, drained and washed.
  • the peptides were isolated and purified by high-performance liquid chromatography (HPLC). The purity and molecular weight of the respective peptides were confirmed by mass spectrometry.
  • Peptide cyclization was achieved by forming a disulfide bond or lactam bridge between Glu21 and Lys25 using The Dde/ODmab Strategy as follows:
  • the peptides were chemically synthesized using Fmoc (9-fluorenylmethoxy carbonyl) chemistry.
  • the peptide chains were synthesized from the carboxyl terminus to the Asn amino terminus onto resin.
  • the resin was incubated with dichloromethane (DCM) for 30 minutes and then washed with dimethylformamide (DMF) three times.
  • DCM dichloromethane
  • DMF dimethylformamide
  • Fmoc-protecting groups at the amino terminus were deprotected with an alkaline buffer and then washed with DMF three times to remove the deprotection buffer.
  • the second amino acid was Fmoc-His(Trt)-OH coupled to the first amino acid and then DMF cleaned.
  • the Fmoc-Lys(Dde)-OH was used for the 25th amino acid.
  • the Fmoc-Glu(odmab)-OH was used for the 21st amino acid.
  • the Boc-Val-OH was used for the last amino acid. After the last amino acid Val, Dde and Odmab were removed to form cyclization.
  • the peptide was ninhydrin tested and the coupling and washing steps repeated until the crude peptide was fully synthesized. After the final Fmoc group was removed, the peptide was cleaved from the resin. The crude peptides were then diethyl ether precipitated, drained and washed. The peptides were isolated and purified by high-performance liquid chromatography (HPLC). The purity and molecular weight of the respective peptides were confirmed by mass spectrometry.
  • Osteoporos Int 1 162- 170.

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Abstract

La présente invention concerne une nouvelle hormone parathyroïdienne ainsi que des analogues de protéine apparentée à l'hormone parathyroïdienne. L'invention concerne également des méthodes d'utilisation d'un ou de plusieurs desdits analogues en vue de traiter un sujet souffrant d'une maladie osseuse associée à une perte osseuse. L'invention concerne en outre d'autres méthodes de traitement d'états pathologiques à l'aide d'un ou de plusieurs desdits analogues, notamment des méthodes de traitement de l'alopécie, de l'arthrite rhumatoïde, et de lésions/d'infections dérivant en une perte osseuse. L'invention concerne encore en outre de nouvelles formulations comprenant un ou plusieurs analogues.
PCT/US2014/060689 2013-10-15 2014-10-15 Hormone parathyroïdienne anabolique osseuse et analogues de protéine apparentée à l'hormone parathyroïdienne WO2015057836A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10406202B2 (en) 2014-10-22 2019-09-10 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US10420819B2 (en) 2014-10-22 2019-09-24 Extend Biosciences, Inc. Insulin vitamin D conjugates
WO2020160118A1 (fr) 2019-01-29 2020-08-06 Shire-Nps Pharmaceuticals, Inc. Variantes d'hormone parathyroïdienne
US20220293274A1 (en) * 2019-03-21 2022-09-15 Assistance Publique - Hopitaux De Paris Method of predicting whether a kidney transplant recipient is at risk of having allograft loss

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996007416A1 (fr) * 1994-09-09 1996-03-14 The Procter & Gamble Company Oestrogenes et hormone parathyroidienne utiles pour traiter l'osteoporose
US20040214996A1 (en) * 2000-04-27 2004-10-28 Amgen Inc. Modulators of receptors for parathyroid hormone and parathyroid hormone-related protein
US20070299009A1 (en) * 1995-07-13 2007-12-27 Dong Zheng X Analogs of parathyroid hormone
WO2010022176A1 (fr) * 2008-08-19 2010-02-25 Ferring International Center S.A. Méthodes pour traiter des maladies du squelette

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996007416A1 (fr) * 1994-09-09 1996-03-14 The Procter & Gamble Company Oestrogenes et hormone parathyroidienne utiles pour traiter l'osteoporose
US20070299009A1 (en) * 1995-07-13 2007-12-27 Dong Zheng X Analogs of parathyroid hormone
US20040214996A1 (en) * 2000-04-27 2004-10-28 Amgen Inc. Modulators of receptors for parathyroid hormone and parathyroid hormone-related protein
WO2010022176A1 (fr) * 2008-08-19 2010-02-25 Ferring International Center S.A. Méthodes pour traiter des maladies du squelette

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US10420819B2 (en) 2014-10-22 2019-09-24 Extend Biosciences, Inc. Insulin vitamin D conjugates
US10702574B2 (en) 2014-10-22 2020-07-07 Extend Biosciences, Inc. Therapeutic vitamin D conjugates
US11116816B2 (en) 2014-10-22 2021-09-14 Extend Biosciences, Inc. Therapeutic vitamin d conjugates
WO2020160118A1 (fr) 2019-01-29 2020-08-06 Shire-Nps Pharmaceuticals, Inc. Variantes d'hormone parathyroïdienne
CN113840625A (zh) * 2019-01-29 2021-12-24 夏尔-Nps医药品有限公司 甲状旁腺激素变体
EP3917577A4 (fr) * 2019-01-29 2023-07-12 Takeda Pharmaceuticals U.S.A., Inc. Variantes d'hormone parathyroïdienne
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