WO2010022176A1 - Methods of treatment for skeletal conditons - Google Patents

Abstract

The present invention relates to methods for treatment of a skeletal condition, e.g. osteoporosis, in a subject and/or increasing bone mineral density (BMD) in a subject with a parathyroid hormone (PTH) receptor agonist.

Description

TITLE

[0001] Methods of Treatment for Skeletal Conditions.

CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 61/090,072, filed on August 19, 2008, the entire disclosure of which is incorporated by reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] Not applicable.

BACKGROUND OF THE INVENTION

Field of the invention

[0004] The present invention relates to methods for treatment of a skeletal condition, e.g. osteoporosis, in a subject and/or increasing bone mineral density (BMD) in a subject with a parathyroid hormone (PTH) receptor agonist.

Description of the Related Art

[0005] Recently, evidence has accumulated which clearly demonstrates that parathyroid hormone (PTH), its fragments, e.g. PTH(I -34)_(FORTEO; teriparatide), and other PTH receptor agonists are an effective class of therapeutics for treating skeletal conditions such as osteoporosis. Of particular note, the once daily administration of PTH receptor agonist treatment regimen has been shown to increase lumbar spine bone mineral density (BMD) and increase anti-fracture efficacy in osteoporotic patients. See Finkelstein et al., N Engl J Med 331 : 1618-1623 (1994); Hodsman et al., J Clin Endocrinol Metab 82: 620-28 (1997); Lindsay et al., Lancet 350: 550-555 (1997); Neer et al., N Engl J Med 344: 1434-1441 (2001); Roe et al., Program and Abstracts of the 81st Annual Meeting of the Endocrine Society, p. 59 (1999); Lane et al., J Clin Invest 102: 1627-1633 (1998); U.S. Pat. No. 6,977 ',077; and WO 00/10596.

[0006] Given the known utility of PTH receptor agonists as therapeutics for treating osteoporosis and related skeletal conditions attempts have been made to further improve efficacy through increasing dosage or frequency of administration of the PTH receptor agonists, e.g. more often than once daily administration. However, these efforts have largely failed due to reduced efficacy of the PTH receptor agonist when administered more often than once daily. Unwanted negative effects of these attempts have included reduced bone density, i.e. net bone loss, and/or the development of adverse side effects such as hypercalcemia and cancer. See Winer et al, JCEM 83:3480 (1998); Winer et al, JCEM 88:4214 (2003); Hefti et al., Clinical Science 62:389 (1982); Riond, Clinical Science 85: 223 (1993); Riond et al., Z Ernarungswiss 37: 183 (1998); Dobnig et al., Endocrinology 11 : 138 (1997); Suzuki et al., Journal of Pharmaceutical Sciences 2: 91 (2002); Frolik et al., Bone 33 (2003); Horwitz et al., Journal of Bone and Mineral Research 10:20 (2005); and Locklin et al., Journal of Cellular Biochemistry 89 (2003). A number of possible reasons exist to explain this failure and likely include the known dual nature of PTH. PTH can promote bone formation, and can also stimulate bone resorption (See references above); net bone formation is dependent on treatment regimen. Thus, a need exists for a treatment regimen, i.e. increased frequency of dose per day, where efficacy of the PTH receptor agonists are improved, in comparison to a once daily treatment regimen, and optionally the unwanted negative effects associated with multiple daily administration of PTH receptor agonists can be minimized. The present invention provides for these and other advantages as described below.

SUMMARY OF THE INVENTION

[0007] Disclosed herein is a method for treatment of a skeletal condition in a subject, including: administering to said subject a composition comprising a therapeutically effective amount of a parathyroid hormone (PTH) receptor agonist, wherein the therapeutically effective amount is administered to the subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, wherein the time interval between each daily administration is between about 8 hours and 16 hours, and wherein the administering increases bone mineral density (BMD) in the subject.

[0008] In one embodiment of the method, the skeletal condition can include osteoporosis, a bone fracture, senile osteoporosis, secondary osteoporosis, or an installed orthopaedic device comprising a bone interface. In another embodiment, the subject is a human. In a related embodiment, the subject can include an aged subject, an osteoporotic subject, a post- menopausal woman, a geriatric man, a subject including a bone fracture, a subject including an installed orthopaedic device, or a subject including a secondary osteoporosis. [0009] In another embodiment of the method, the administration is subcutaneous. In another embodiment, the administration can include subcutaneous, oral, intravenous, cutaneous, nasal, intramuscular, or intraperitoneal routes.

[0010] In another embodiment of the method, the therapeutically effective amount is between about 5 and about 30 μg of the PTH receptor agonist. In a related embodiment of the method, the therapeutically effective amount is between about 10 and about 25 μg of the PTH receptor agonist. In a related embodiment, the therapeutically effective amount is about 20 μg of the PTH receptor agonist. In another related embodiment, the therapeutically effective amount is between about 10 and about 200 μg of the PTH receptor agonist, wherein the PTH receptor agonist is PTH(I -84). In another related embodiment, the therapeutically effective amount is about 50 μg of PTH(I -84). In another related embodiment, the therapeutically effective amount is between about 5 and about 80 μg of the PTH receptor agonist, wherein the PTH receptor agonist is Ostabolin-C. In another related embodiment, the therapeutically effective amount is about 30 μg of Ostabolin-C. In another related embodiment, the therapeutically effective amount is between about 10 and about 3000 μg of the PTH receptor agonist, wherein the PTH receptor agonist is a PTHrP analog. [0011] In another embodiment, the time interval between the two administrations is between about 10 hours and 14 hours. In another related embodiment, the time interval between the two administrations is about 12 hours.

[0012] In another embodiment, the PTH receptor agonist includes PTH(l-34), PTH(I- 84), Ostabolin-C, BIM-44058, or a PTHrP analog. In another related embodiment, the PTH receptor agonist includes an isolated PTH receptor agonist peptide. In another related embodiment, the PTH receptor agonist is a recombinant peptide. In another related embodiment, the PTH receptor agonist is a synthetic peptide. In another related embodiment, the PTH receptor agonist includes human PTH(l-34).

[0013] In another embodiment, the administering advantageously affects BMD in the subject more than administration of the composition to the subject one time per day. In another related embodiment, the treatment decreases standard duration of treatment. In another related embodiment, the method further includes determining the BMD of the subject. [0014] Also described herein is a method for treatment of osteoporosis in a human, including: administering to the human a composition including a therapeutically effective amount of PTH(I -34), wherein the therapeutically effective amount is administered to the subject two times per day, wherein each therapeutically effective amount is 20μg, wherein the time interval between each administration of each therapeutically effective amount is 12 hours, and wherein the administering increases BMD in said human.

[0015] Also described herein is a method for reducing the risk of a medical condition in a subject, including treating a skeletal condition in the subject by administering to the subject a composition including a therapeutically effective amount of a PTH receptor agonist, wherein the therapeutically effective amount is administered to the subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, wherein the time interval between each administration is between about 8 hours and 16 hours.

[0016] In one embodiment, the medical condition can include a vertebral bone fracture or a non- vertebral bone fracture.

[0017] Also described herein is a method of advantageously affecting BMD in a subject including a skeletal condition, including: administering to the subject a composition including a therapeutically effective amount of a PTH receptor agonist, wherein the therapeutically effective amount is administered to the subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, and wherein the time interval between each daily administration is between about 8 hours and 16 hours.

[0018] In one embodiment of the above methods, the skeletal condition can include osteoporosis, a bone fracture, senile osteoporosis, secondary osteoporosis, or an installed orthopaedic device comprising a bone interface. In another embodiment, the subject is a human. In a related embodiment, the subject can include an aged subject, an osteoporotic subject, a post-menopausal woman, a geriatric man, a subject including a bone fracture, a subject including an installed orthopaedic device, or a subject including a secondary osteoporosis.

[0019] In another embodiment of the methods, the administration is subcutaneous. In another embodiment, the administration can include subcutaneous, oral, intravenous, cutaneous, nasal, intramuscular, or intraperitoneal routes. [0020] In another embodiment of the methods, the therapeutically effective amount is between about 5 and about 30 μg of the PTH receptor agonist. In a related embodiment of the methods, the therapeutically effective amount is between about 10 and about 25 μg of the PTH receptor agonist. In a related embodiment, the therapeutically effective amount is about 20 μg of the PTH receptor agonist. In another related embodiment, the therapeutically effective amount is between about 10 and about 200 μg of the PTH receptor agonist, wherein the PTH receptor agonist is PTH(I -84). In another related embodiment, the therapeutically effective amount is about 50 μg of PTH(I -84). In another related embodiment, the therapeutically effective amount is between about 5 and about 80 μg of the PTH receptor agonist, wherein the PTH receptor agonist is Ostabolin-C. In another related embodiment, the therapeutically effective amount is about 30 μg of Ostabolin-C. In another related embodiment, the therapeutically effective amount is between about 10 and about 3000 μg of the PTH receptor agonist, wherein the PTH receptor agonist is a PTHrP analog. [0021] In another embodiment, the time interval between the two administrations is between about 10 hours and 14 hours. In another related embodiment, the time interval between the two administrations is about 12 hours.

[0022] In another embodiment, the PTH receptor agonist includes PTH(l-34), PTH(I- 84), Ostabolin-C, BIM-44058, or a PTHrP analog. In another related embodiment, the PTH receptor agonist includes an isolated PTH receptor agonist peptide. In another related embodiment, the PTH receptor agonist is a recombinant peptide. In another related embodiment, the PTH receptor agonist is a synthetic peptide. In another related embodiment, the PTH receptor agonist includes human PTH(l-34).

[0023] In another embodiment, the administering advantageously affects BMD in the subject more than administration of the composition to the subject one time per day. In another related embodiment, the treatment decreases standard duration of treatment. In another related embodiment, the methods further include determining the BMD of the subject.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0024] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where: [0025] Figure 1 shows the bone mineral density (BMD) of the lumbar spine region of

PTH(l-34) treated rats as measured on days -26, 0, 14, 28, and 42 using a PIXImus II scanner.

[0026] Figure 2 shows the bone mineral density (BMD) of the lumbar spine region of

Ostabolin-C (ZT-031) treated rats as measured on days -26, 0, 14, 28, and 42 using a

PIXImus II scanner.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Briefly, and as described in more detail below, described herein are methods for treatment of a skeletal condition, e.g. osteoporosis, methods for accelerating bone growth, and/or methods for increasing bone mineral density (BMD) in a subject with a parathyroid hormone (PTH) receptor agonist. Several features of the current approach should be noted. Twice-daily treatment with a PTH receptor agonist using a specific time interval between doses (e.g., 12 hours) leads to a greater increase in BMD compared to a once-daily regimen for the same total daily dose (e.g., 20 μg of a PTH receptor agonist given twice per day (2x/day) increases BMD more than 40 μg of the PTH receptor agonist given once per day (lx/day)). In addition, the increase in BMD can also occur more rapidly with the 2x daily treatment regimen with the PTH receptor agonist. Advantages of this approach are numerous and can include: superior clinical response rates and efficacy relative to the current treatment lx/day regimen; greater improvement in BMD relative to the current treatment lx/day regimen; a reduced treatment time relative to the current treatment lx/day regimen; and avoidance of severe hypercalcemia and other adverse side effects that can result from multiple daily doses of PTH receptor agonists.

Definitions

[0028] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0029] The term "skeletal condition" means a medical condition in one or more bones in a subject that results in a need for increased bone mineral density.

[0030] The term "PTH" means a parathyroid hormone that is secreted by the parathyroid glands as a protein 84 amino acids in length. See, e.g., GenBank accession number

NM 000315, version NM 000315.2; GI: 39995098 as of August 8, 2008 for the Homo sapiens version of PTH. [0031] The term "PTH receptor agonist" means a molecule that binds to a PTH receptor and activates a PTH receptor.

[0032] The term "PTH(I -34)" means a peptide that consists of the first 34 amino acids of

N-terminal portion of PTH.

[0033] The term "PTH(I -84)" means a peptide that is the full length version of PTH.

[0034] The term "BMD" means bone mineral density and is a measure of the amount of minerals contained in a defined volume of bone.

[0035] The term "PTHrP" means parathyroid hormone-related peptide that is a member of the parathyroid hormone family that regulates endochondral bone development.

[0036] The term "PTHrP(l-34)" means a peptide that consists of the first 34 amino acids of PTHrP.

[0037] The term "Ostabolin-C" means a cyclic 31 amino acid peptide analog fragment of the N-terminal portion of PTH.

[0038] The term "BIM-44058" means an analog of PTHrP: GIu²²'²⁵ , Leu²³'²⁸'³¹ , Aib²⁹ ,

Lys²⁶'³⁰ hPTHrP(l-34). BIM-44058 is also known as BA-058.

[0039] The term "ameliorate" means any therapeutically beneficial result in the treatment of a medical condition, e.g., skeletal disease state, e.g., an osteoporotic disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0040] The term "therapeutically effective amount" is an amount that is effective to ameliorate a symptom of a disease, e.g. ameliorate a skeletal condition. A therapeutically effective amount can be a "prophylactically effective amount" as prophylaxis can be considered therapy.

[0041] As used herein, the terms "treat", "treatment", or "treatment regimen" are used interchangeably and are meant to indicate administering one or more compounds in accordance with the methods of the invention to obtain a desired therapeutic objective.

[0042] The term "modulating" means the effect of a PTH receptor agonist on a PTH receptor.

[0043] The term "enhancing" means that the bone in the treated subject has improved characteristics compared to an untreated subject, or a control subject such as, for example, greater bone strength, mass, and/or density.

[0044] The term "in situ" means processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.

[0045] The term "in vivo" means processes that occur in a living organism. [0046] As used herein, the term "subject" includes mammals. Examples of mammals include, but are not limited to, any member of the Mammalia class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. The term does not denote a particular age or gender.

[0047] The term "mammal" as used herein includes both humans and non-humans and include but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0048] The terms "administering" and "administration" refer to various means of introducing a composition into a cell or into a patient. These means are well known in the art and can include, for example, injection; tablets, pills, capsules, or other solids for oral administration; nasal solutions or sprays; aerosols, inhalants; topical formulations; liposomal forms; and the like.

[0049] The terms "pharmaceutically acceptable" or "pharmacologically acceptable" mean a material which is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing significant undesirable biological effects or interacting in a deleterious manner with components of the composition or formulation in which it is contained.

[0050] The term "peptide" means a polymer of amino acid residues. The term applies to amino acid polymers containing naturally occurring amino acid residues as well as amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid (e.g., non-classical amino acid). The amino acid residues of amino acid polymers are generally linked by covalent peptide bonds but can be linked by any other method known in the art. As used herein, the term encompasses amino acid polymers of any length, including full-length proteins.

[0051] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

Methods of Treatment of Skeletal Conditions in Subjects

[0052] The present invention provides methods for treating or reducing the risk of skeletal conditions and/or increasing BMD in subjects using parathyroid hormone (PTH) receptor agonists. PTH receptor agonists are described in more detail below. A therapeutically effective amount, e.g., about 5-30 μg, e.g., about 20 μg, is administered twice a day e.g. administered twice within a 24 hour time period where the interval between each administration is between about 8 and 16 hours, or e.g., at an interval of about 12 hours. [0053] The PTH receptor agonists are typically formulated (see below) for administering a dose effective for increasing BMD of one or more of a subject's bones and/or for reducing the likelihood and/or severity of medical condition in a subject suffering from a skeletal condition, e.g. osteoporosis. In one aspect, an effective dose provides an improvement in bone structure, density, mass, and/or strength. In another aspect, an effective dose reduces the incidence of vertebral fracture, reduces the incidence of multiple vertebral fractures, reduces the severity of vertebral fracture, and/or reduces the incidence of non- vertebral fracture. In another aspect, a subject receiving a PTH receptor agonist also receives effective doses of calcium and/or vitamin D, which can enhance the effects of the PTH receptor agonist.

[0054] An effective dose of a PTH receptor agonist is typically greater than about 5 μg although, particularly in humans, it can be as large at about 10 to about 30 μg, or larger as is effective to achieve increased BMD, particularly in cortical bone, or to reduce or treat the incidence of fracture. In certain instances, relevant effects of a PTH receptor agonist can be observed at doses less than about 5 μg, or even less than about 1 μg. [0055] Typically, the PTH receptor agonists are administered to a subject at a dosage between 1 and 3,000 μg twice/day, for a period of 0-36 or more months. In alternative embodiments, each dosage can be 5-1,000 μg, between about 5 and 30 μg, between about 10 and 25 μg, or about 20μg. Other dosages of the PTH receptor agonists can include: less than about 5, 5 to 6, 6 to 7, 7 to 8, 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16 to 17, 17 to 18, 18 to 19, 19 to 20, 20 to 21, 21 to 22, 22 to 23, 23 to 24, 24 to 25, 25 to 26, 26 to 27, 27 to 28, 28 to 29, 29 to 30, or about 30 or more micrograms. In one aspect, PTH(l-84) is given at a dose of about 10-200μg. In another aspect, PTH(l-84) is given at a dose of about 50μg. In another aspect, Ostabolin-C is given at a dose of about 5- 80μg. In another aspect, Ostabolin-C is given at a dose of about 30μg. In another aspect, PTHrP analogs are given at a dose of about 10-3000μg. In related aspects of the present invention, less potent analogs of PTH receptor agonists than those noted above are also contemplated for use in the methods of the present invention. [0056] As an example, each dosage of PTH(I -34) can be greater than or equal to about 5μg and less than or equal to about 30μg, between about 10 and 25 μg, or about 20μg. [0057] The increase in bone mass and/or density can be monitored by the assays and methods described herein or others as are known in the art to one of ordinary skill. [0058] One aspect of the invention is administration twice a day. In one aspect, the PTH receptor agonists are administered twice per day at an interval of between about 8 hours and 16 hours. In other embodiments, the PTH receptor agonists are administered twice/day at an interval of between about 10 hours and 14 hours. In yet other embodiments the PTH receptor agonists are administered twice per day at an interval of about 12 hours. In yet other embodiments the PTH receptor agonists are administered twice per day at an interval of about 8 to 9, 9 to 10, 10 to 11, 11 to 12, 12 to 13, 13 to 14, 14 to 15, 15 to 16, 16, or 16 or more hours.

[0059] These PTH receptor agonists can be combined with other art known agents that are known to benefit a subject with a skeletal condition, e.g. vitamin D, calcitonin (e.g., MIACALCIN), bisphosphonates (e.g., Alendronate (Fosamax), Ibandronate (Boniva), Risedronate (Actonel), Zoledronate (Zometa)), denosumab, and/or calcium. [0060] Numerous subjects, e.g., human patients, can benefit from the methods of the present invention for treating a skeletal condition. Subjects can include: aged subjects, osteoporotic subjects, geriatric males, subjects with a fractured bone, postmenopausal females, subjects with any type of internal fixator or orthopaedic device with an interface with bone, subjects with hormonal imbalances that can cause or contribute to a loss of bone density, and subjects treated with any drug that can cause secondary osteoporosis or decreased bone density.

[0061] Typically, the benefits of administration of a PTH receptor agonist persist after a period of administration. The benefits of several months of administration can persist for as much as a year or more, without additional administration.

[0062] Typically, administration of the PTH receptor agonist to a subject twice per day increases BMD in the subject to a level greater than administration of the composition to the subject once per day. In addition, administration of the PTH receptor agonist to a subject twice per day typically decreases the amount of time a subject is treated with the PTH receptor agonist as compared to administration of the PTH receptor agonist to the subject once per day. [0063] The method of treatment can constitute a period of administration of a parathyroid hormone (PTH) receptor agonist, e.g., for a period of time varying between 1-3 months to 15- 18 months to 18-36 months to 36 or more months. The treatment period can be continued until at least the patient BMD is restored to an acceptable level. The period of administration can be less than 1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or more than 18 months. Alternatively, in another embodiment, the method of treatment can constitute a series of administration periods followed by periods of no administration (e.g., sequential periods of three months of administration of a PTH receptor agonist and three months of no administration). The sequential treatment periods can be repeated until the patient BMD is restored (e.g., a T-score < -2.0 or -2.5 below the mean or preferably < -1.0 below the mean). T-score is discussed in greater detail below.

Methods for Assessing Bone in a Subject

[0064] Methods of the invention result in an increase in BMD. Thus, the present invention further provides methods that can be used for assessing one or more bones or the skeleton of a subject before and/or after treatment using the PTH receptor agonists of the invention. The impact of PTH receptor agonist treatment on BMD in a subject by the methods of the invention can be determined, e.g., by examination of bone strength, density, and/or mass after administration of a PTH receptor agonist compared to a control subject or the subject of interest prior to administration of the PTH receptor agonist. Such examination can be performed in situ by using imaging techniques (e.g., X-ray, nuclear magnetic resonance imaging, X-ray tomography, ultrasound, and sound conduction) or stress testing, or ex vivo by standard histological, radiographic, mechanical, or biochemical methods. Modulation of bone density and/or bone mass can be assessed by changes in one or more parameters such as bone mineral density, bone strength, trabecular number, bone size, and bone tissue connectivity. Several other methods for determining BMD are known in the art. For example, BMD measurements can be performed using, e.g., dual energy xray absorptiometry or quantitative computed tomography, and the like, as described in more detail below.

[0065] Several noninvasive techniques are available for estimating skeletal mass and/or bone density. These can include dual-energy x-ray absorptiometry (DEXA), quantitative computed tomography (CT), and ultrasound. Other techniques for estimating skeletal mass or bone density in a subject are well known in the art. [0066] DEXA is a highly accurate x-ray technique that has become the standard for measuring bone density in many medical centers and clinics. DEXA can be used for measurements of any skeletal site, though clinical determinations are usually made based on the lumbar spine and/or hip. Portable DEXA machines have been developed that measure the heel (calcaneus), forearm (radius and ulna), or finger (phalanges), and DEXA can also be used to measure body composition. In the DEXA technique, two x-ray energies are used to estimate the area of mineralized tissue, and the mineral content is divided by the area, which partially corrects for body size. It has become standard practice to relate the results of DEXA to "normal" values using T-scores, which can compare individual results to those in a young population that is matched for race and gender. Alternatively, Z-scores can compare individual results to those of an age-matched population that is also matched for race and gender. Thus, for example, a 60-year-old woman with a Z-score of -1 (1 SD below mean for age) could have a T-score of -2.5 (2.5 SD below mean for a young control group). One example of a DEXA device is a PIXImus II scanner, as used below in Example 1. In addition to DEXA, single-energy x-ray absorptiometry (SXA) can also be used to estimating skeletal mass or bone density.

[0067] CT can be used to measure bone, e.g. the spine, and peripheral CT can be used to measure peripheral bone in e.g. the forearm or tibia. CT has the advantage of studying bone density in subtypes of bone, e.g., trabecular vs. cortical. CT can specifically analyze trabecular bone and can provide a true density (mass of bone per unit volume) measurement. [0068] Ultrasound can be used to measure bone mass by calculating the attenuation of the signal as it passes through bone or the speed with which it traverses the bone. Ultrasound is amenable for use as a screening procedure in, e.g., clinical and/or field settings. [0069] The hip can typically be the site of BMD measurement in most individuals, since it directly assesses bone at an important fracture site. When hip measurements are performed by e.g. DEXA, the spine can be measured at the same time. In younger individuals, such as perimenopausal women, spine measurements can be the most sensitive indicator of bone loss. These and other methods known in the art can be used for measuring the BMD in subjects with skeletal conditions.

Skeletal Conditions

[0070] The present invention relates to skeletal conditions and methods of using the PTH receptor agonists described herein for the treatment of the skeletal conditions and increasing BMD. Skeletal conditions of the present invention can include, e.g., osteoporosis and bone fracture. In addition, a number of skeletal conditions are known in the art which can cause an imbalance in the bone remodeling cycle. Among these are metabolic bone diseases, such as osteoporosis, rickets, osteomalacia, and renal osteodystrophy, which can all result in abnormal or excessive loss of bone mass (osteopenia). Other bone diseases, such as Paget's disease, also cause excessive loss of bone mass at localized sites. These and other diseases and skeletal conditions are discussed in greater detail below.

[0071] Patients suffering from chronic renal (kidney) failure can suffer loss of skeletal bone mass (renal osteodystrophy). In adults, osteodystrophic symptoms often are a significant cause of morbidity. In children, renal failure can result in a failure to grow, due to the failure to maintain and/or to increase bone mass.

[0072] Rickets or Osteomalacia (also known as "soft bones"), is a defect in bone mineralization (e.g., incomplete mineralization), and classically is related to vitamin D (1,25- dihydroxy vitamin D3) deficiency or resistance. The defect can cause compression fractures in bone, and a decrease in bone mass, as well as extended zones of hypertrophy and proliferative cartilage in place of bone tissue. The deficiency can also result from a nutritional deficiency (e.g., rickets in children), malabsorption of vitamin D or calcium, and/or impaired metabolism of the vitamin.

[0073] Bone fractures are a common traumatic injury in all age groups. Approximately 8-10 million bone fractures are reported annually in the United States, with more than 1 million of these requiring hospitalization. The estimated annual cost of treating these fractures exceeds 20 billion dollars. While this is already significant, these numbers are expected to increase due to the aging of the general population. Further, among military personnel, bone fractures are common training and field injuries. Bone fractures, typically located in the arms and legs, are also common battle wounds. Aside from traumatic injury, bone fractures also can be caused by disease, as described above and in more detail below. [0074] Installed orthopaedic devices attached to bone in a subject are another example of a skeletal condition of the present invention. Examples include hip replacement and knee replacement devices. The methods of the present invention can be used, e.g., to strengthen the interface between the device and the bone to which it is attached in the subject. [0075] Osteoporosis is a structural deterioration of the skeleton caused by loss of bone mass resulting from an imbalance in bone formation, bone resorption, or both, such that the resorption dominates the bone formation phase, thereby reducing the weight-bearing capacity of the affected bone. Osteoporosis affects >10 million individuals in the United States alone. In one aspect of osteoporosis, "senile osteoporosis" can occur in geriatric men. In another aspect, osteoporosis also can result from nutritional and endocrine imbalances, hereditary disorders, and a number of malignant transformations. In yet another aspect, "secondary osteoporosis" can result from the use of glucocorticoids, GnRH agonists or antagonists, and other drug therapies.

PTH receptor agonists

[0076] The methods of the invention use PTH receptor agonists to increase BMD in subjects. PTH receptor agonists can include, for example, PTH peptides such as PTH(I -34), Ostabolin-C, BIM-44058, and PTH(l-84), as described in more detail herein. In other aspects of the present invention, one of ordinary skill can combine PTH receptor agonists with siRNAs, microRNAs, or other small regulatory RNAs that target and block the expression of mRNA encoding proteins that inhibit the PTH pathway. [0077] The PTH receptor agonists, or analogs thereof, can increase bone mass and/or density in a subject in need thereof. The PTH receptor agonists can be combined with other art known agents that are known to benefit a subject with a skeletal condition, e.g. vitamin D and/or calcium.

PTH Peptides

[0078] PTH peptides represent one embodiment of the PTH receptor agonists of the present invention. In nature, PTH is initially synthesized as a larger molecule (preproparathyroid hormone, consisting of 115 amino acids), which is then reduced in size by signal peptide cleavage (proparathyroid hormone, 90 amino acids) and then a second prohormone cleavage before secretion as an 84 amino acid peptide. The hydrophobic regions of the preproparathyroid hormone can serve a role in guiding transport of the polypeptide from sites of synthesis on polyribosomes through the endoplasmic reticulum to secretory granules.

[0079] The amino acid sequence of PTH has been characterized in multiple mammalian species and reveals marked conservation in the amino-terminal portion, which is critical for many biologic actions of the molecule. Biological activity is typically associated with the N- terminal portion of PTH. The N-terminal segment of human PTH (hPTH) differs from the N- terminal segment of the bovine (bPTH) and porcine (pPTH) hormones by only three and two amino acid residues, respectively. [0080] In one embodiment, PTH peptide can refer to naturally occurring PTH. In another embodiment, PTH peptide can refer to synthetic PTH. In yet another embodiment, PTH peptide can refer to recombinant PTH. In general, PTH peptides can be obtained by known recombinant or synthetic methods, such as described in U.S. Pat. Nos. 4,086,196 and 5,556,940, each incorporated herein by reference for all purposes, and others as described in more detail below. Furthermore, PTH peptides can include allelic variants, species variants, and conservative amino acid substitution variants of PTH or PTH fragments. PTH peptides can also include full-length PTH, e.g. PTH-(l-84), as well as PTH fragments. Fragments of PTH peptide variants, in amounts giving equivalent or similar biological activity to PTH-(I- 84), can be used in the methods of the invention, if desired by one of ordinary skill in the art. Fragments of PTH typically incorporate at least the amino acid residues of PTH necessary for a biological activity similar to that of intact PTH. Examples of such fragments include: PTH- (1-29), PTH-0-30), PTH-(1-31), PTH-(l-32), PTH-(l-33), PTH-(l-34), PTH(l-38), PTH-(I- 41), PTH-(l-80), PTH-0-81), PTH-(l-82), PTH-(l-83), and PTH-(l-84). In addition, modified, substituted synthetic fragments of the amino-terminal sequence as small as 1-14 residues can be sufficient to activate the major receptor.

[0081] In other embodiments, alternative forms of PTH peptide variants can incorporate from 1 to 5 or more amino acid substitutions that can improve PTH peptide stability and half- life, such as the replacement of methionine residues at positions 8 and/or 18 with leucine or other hydrophobic amino acid that improves PTH peptide stability against oxidation, the replacement of amino acids in the 25-27 region with trypsin-insensitive amino acids such as histidine or other amino acid that improves PTH peptide stability against proteases, and/or the replacement of asparagine at position 16.

[0082] In related embodiments, other suitable forms of PTH peptides can include parathyroid hormone-related peptide (PTHrP), PTHrP(l-34), PTHrP(l-36), and analogs of PTH or PTHrP or their fragments that activate the PTHl receptor. [0083] In other embodiments, PTH peptides can include fragments, variants, and functional analogs of PTH having a 60% or more homologous amino acid sequence with PTH and fragments thereof, e.g. PTH(I -34). The present invention includes pharmaceutical formulations (see below) comprising such PTH peptide variants and functional analogs, carrying modifications like substitutions, deletions, insertions, inversions or cyclisations, but nevertheless having substantially the same biological activities of a full-length PTH peptide. Stability-enhanced variants of PTH peptides are known in the art from, e.g., WO 92/11286 and WO 93/20203, each incorporated herein by reference for all purposes. Cyclized PTH analogs are also contemplated by the present invention as PTH peptides and examples are disclosed in, e.g., WO 98/05683, incorporated herein by reference. PTH peptides can also include amino acid substituted analogs using a PT-(I-11) or PTH-(I -14) backbone. Examples of other PTH peptides of the present invention are described in Shimizu et al., J Biol Chem., 276: 49003-49012 (2001); Shimizu et al., Endocrinology 42: 3068-3074 (2001); Carter and Gardella, Biochim Biophys Acta 1538: 290-304 (2001); Shimizu et al., J Biol Chem., 275: 21836-21843 (2000), each incorporated herein by reference for all purposes. [0084] In other embodiments, PTH peptides useful in the methods of the present invention can include the use of a PTH peptide selected from the group consisting of: (a) full- length parathyroid hormone; (b) biologically active variants of full-length parathyroid hormone; (c) biologically active parathyroid hormone fragments; (d) biologically active variants of parathyroid hormone fragments; (e) biologically active variants having at least 75% homology with PTH; (f) biologically active variants having at least 60% identity with PTH; and (g) biologically active variants encoded by a nucleic acid sequence that hybridizes under stringent conditions to a complementary nucleic acid sequence of PTH or PTH fragments.

[0085] "Homologous amino acid sequence" means an amino acid sequence that differs from an amino acid sequence of a polypeptide, e.g., a PTH peptide that differs by one or more conservative amino acid substitutions, or by one or more non-conservative amino acid substitutions, deletions, or additions located at positions at which they do not destroy the biological activities of the polypeptide. Preferably, such a sequence is at least 75%, preferably 80%, more preferably 85%, more preferably 90%, and most preferably 95% or more homologous to the amino acid sequence. Homologous amino acid sequences can also include sequences that are identical or substantially identical to an amino acid sequence. [0086] By "amino acid sequence substantially identical" is meant a sequence that is at least 60%, preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% or more identical to an amino acid sequence of reference. Preferably, the homologous sequence differs from the reference sequence, if at all, by a majority of conservative amino acid substitutions.

[0087] The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. [0088] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. [0089] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al, infra). [0090] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. MoI. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information web-site. [0091] PTH peptides can be generated wholly or partly by chemical synthesis. The peptides of the invention can be readily prepared according to well-established, standard liquid or solid-phase peptide synthesis methods, general descriptions of which are broadly available (see, for example, in J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, 111. (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, N. Y. (1984); and Applied Biosystems 430A Users Manual, ABI Inc., Foster City, Calif), or they can be prepared in solution, by the liquid phase method or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of a residue by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof. [0092] The PTH peptides can also be obtained by methods well-known in the art for peptide purification and recombinant peptide expression. For recombinant expression of one or more of the peptides, the nucleic acid containing all or a portion of the nucleotide sequence encoding the PTH peptide can be inserted into an appropriate expression vector (i.e., a vector that contains the necessary elements for the transcription and translation of the inserted peptide coding sequence). In one embodiment, the regulatory elements are heterologous (i.e., not the native gene promoter). Alternately, the necessary transcriptional and translational signals can also be supplied by the native promoter for the genes and/or their flanking regions.

[0093] The PTH peptides can also be purified from a natural source. Depending on the source, the PTH peptide can be brought into a solution by breaking the tissue or cells containing it. There are several methods to achieve this, including: repeated freezing and thawing, sonication, homogenization by high pressure or permeabilization by organic solvents. The method of choice typically depends on how fragile the PTH peptide is and how sturdy the cells are. After this extraction process soluble peptide will be in the solvent, and can be separated from cell membranes, DNA, etc. by centrifugation. After the extraction process the peptide of interest can be further purified using methods known in the art including precipitation, differential solubilization, ultracentrifugation, and/or chromatography methods including size exclusion, ion exchange, high pressure liquid, and immunoaffmity.

PTH receptor agonist formulations

[0094] The PTH receptor agonists of the present invention can be formulated for use in a subject to increase BMD in the subject of one or more bones. Formulations of the PTH receptor agonists of the present invention can include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like. Formulations for direct administration, in particular, can include glycerol and other compositions of high viscosity to help maintain the PTH receptor agonist at the desired locus. Biocompatible, e.g. bioresorbable, polymers, including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide, glycolide polymers and lactideiglycolide copolymers, can be useful excipients to control the release of the PTH receptor agonist in vivo. Other potentially useful parenteral delivery systems for these PTH receptor agonists include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration can contain as excipients, for example, lactose, or can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally. Formulations for parenteral administration can also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or cutric acid for vaginal administration. Suppositories for rectal administration can also be prepared by mixing a PTH receptor agonist (alone or in combination another agent) with a non-irritating excipient such as cocoa butter or other compositions that are solid at room temperature and liquid at body temperatures.

[0095] Formulations for topical administration to the skin surface can be prepared by dispersing the molecule capable of releasing a PTH receptor agonist (alone or in combination another agent) with a dermatologically acceptable carrier such as a lotion, cream, ointment, or soap. Particularly useful are carriers capable of forming a film or layer over the skin to localize application and inhibit removal. For topical, administration to internal tissue surfaces, the PTH receptor agonist can be dispersed in a liquid tissue adhesive or other substance known to enhance adsorption to a tissue surface. For example, hydroxypropylcellulose or fibrinogen/thrombin solutions can be used. Alternatively, tissue- coating solutions, such as pectin-containing formulations can be used.

Pharmaceutical compositions of the invention

[0096] Methods for treatment of skeletal conditions are encompassed by the present invention. Said methods of the invention include administering a therapeutically effective amount of a PTH receptor agonist. The compositions of the invention can be formulated in pharmaceutical compositions. These compositions can comprise, in addition to one or more of the PTH receptor agonists, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can depend on the route of administration, e.g. subcutaneous, oral, intravenous, cutaneous, nasal, intramuscular, or intraperitoneal routes. Examples of such and other additives are found in 'Handbook of Pharmaceutical Excipients'; Ed. A.H. Kibbe, 3^rd Ed., American Pharmaceutical Association, USA and Pharmaceutical Press UK, 2000.

[0097] For intravenous, cutaneous, or subcutaneous injection, or injection at the site of affliction, the active ingredient can be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives can be included, as required.

[0098] Pharmaceutical compositions for oral administration can be in tablet, capsule, powder, or liquid form. A tablet can include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions can generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included.

[0099] Whether it is a polypeptide, antibody, nucleic acid, small molecule, or other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is preferably in a "therapeutically effective amount" or "prophylactically effective amount"(as the case can be, although prophylaxis can be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of protein aggregation disease being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980. [00100] A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

EXAMPLES

[00101] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. [00102] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3^rd Ed. (Plenum Press) VoIs A and B(1992).

Example 1: A 42-day subcutaneous pharmacology study to test once vs. twice per day therapy with PTHf 1-34) in aged, ovariectomized Sprague Dawley rats.

[00103] The rats used for the study were ovariectomized (OVX) at 8-9 months old (i.e. greater than 30Og) and then held for at least 70 days post OVX prior to initiating treatment. A control group of rats (sham group) had sham surgery where the ovaries were not removed and another control group (OVX group) had their ovaries removed to induce osteoporosis. Test animals were injected with PTH(I -34) with the following dose regimens: 20μg/kg lX/day, 20μg/kg 2X/day,40μg/kg lX/day, 40μg/kg 2X/day and 80μg/kg lX/day. Sham and OVX control animals received injections of the drug vehicle alone. Injections were made subcutaneous Iy into the scapular region of the rats. The duration between injections given twice per day was 12 hours +/- 1 hour. Animals were treated 5 days per week for the duration of the study. Drug treatment of the rats began on study day 0.

[00104] Bone mineral density (BMD) of the lumbar spine region was measured on study days -26, 0, 14, 28 and 42 using a PIXImus II scanner according to standard procedures described in the PIXImus user manual. Animals were first anesthetized and then scanned. Analysis of the lumbar spine was done by creating a window around last two lumbar vertebrae using a region of interest (ROI) tool of the PIXImus software. Baseline measurements were taken on study day -26 and day 0; drug treatment began on day 0. [00105] Results: Twice per day (2X/day) therapy with PTH(l-34) at doses of 20 and 40μg/kg is more efficacious than once per day therapy of the same doses at increasing lumbar spine BMD (Figure 1 and Table 1). This is also true for total daily dose (e.g. 20μg/kg twice per day is more efficacious than 40μg/kg once per day).

[00106] The rats used for the study are ovariectomized (OVX) at 8-9 months old (i.e. greater than 30Og) and held for at least 70 days post OVX prior to initiating treatment. One control group of rats has sham surgery where the ovaries are not removed. Test animals are injected with PTH(l-84) with the following dose regimens: 20μg/kg lX/day, 20μg/kg 2X/day, 40μg/kg lX/day, 40μg/kg 2X/day, 80μg/kg lX/day, and 80μg/kg 2X/day. When the animals receive 2 daily doses of the PTH receptor agonist, the time interval between doses is 12 hours. Sham animals receive injections of the drug vehicle alone. Injections are made subcutaneously into the scapular region of the rats. Drug treatment of the rats begins on Day 7 of the study.

[00107] BMD of the lumbar spine region is measured on days 0, 7, 14, 21 and 28 using a PIXImus II scanner according to standard procedures described in the PIXImus user manual. Animals are first anesthetized and then scanned. Analysis of the lumbar spine is done by creating a window around last two lumbar vertebrae using a region of interest (ROI) tool of the PIXImus software. Baseline measurements are taken on Day 0; drug treatment begins on Day 7.

[00108] Results: Twice per day therapy with PTH(l-84) at doses of 20, 40 and 80μg/kg is more efficacious than once per day therapy of the same doses at increasing lumbar spine BMD. This is also true for total daily dose (e.g. 20μg/kg twice per day is more efficacious than 40μg/kg once per day).

Example 3: A 42-day subcutaneous pharmacology study to test once vs. twice per day therapy with Ostabolin-C (ZT-031) in aged, ovariectomized Sprague Dawley rats.

[00109] The rats used for the study were ovariectomized (OVX) at 8-9 months old (i.e. greater than 300g) and then held for at least 70 days post OVX prior to initiating treatment. A control group of rats (sham group) had sham surgery where the ovaries were not removed and another control group (OVX group) had their ovaries removed to induce osteoporosis. Test animals were injected with Ostabolin-C (ZT-031) with the following dose regimens: 20μg/kg lX/day, 20μg/kg 2X/day, 40μg/kg lX/day, 40μg/kg 2X/day and 80μg/kg lX/day. Sham and OVX control animals received injections of the drug vehicle alone. Injections were made subcutaneously into the scapular region of the rats. The duration between injections given twice per day was 12 hours +/- 1 hour. Animals were treated 5 days per week for the duration of the study. Drug treatment of the rats began on study day 0. [00110] Bone mineral density (BMD) of the lumbar spine region was measured on study days -26, 0, 14, 28 and 42 using a PIXImus II scanner according to standard procedures described in the PIXImus user manual. Animals were first anesthetized and then scanned. Analysis of the lumbar spine was done by creating a window around last two lumbar vertebrae using a region of interest (ROI) tool of the PIXImus software. Baseline measurements were taken on study day -26 and day 0; drug treatment began on day 0. [00111] Results: Twice per day (2X/day) therapy with Ostabolin-C (ZT-031) at doses of 20 and 40μg/kg is more efficacious than once per day therapy of the same doses at increasing lumbar spine BMD (Figure 2 and Table 1). This is also true for total daily dose (e.g. 20μg/kg twice per day is more efficacious than 40μg/kg once per day).

Example 4: A subcutaneous pharmacology study to test once vs. twice per day therapy with BIM-44058 in aged, ovariectomized Sprague Dawley rats.

[00112] The rats used for the study are ovariectomized (OVX) at 8-9 months old (i.e. greater than 30Og) and held for at least 70 days post OVX prior to initiating treatment. One control group of rats has sham surgery where the ovaries are not removed. Test animals are injected with BIM-44058 with the following dose regimens: 20μg/kg lX/day, 20μg/kg 2X/day, 40μg/kg lX/day, 40μg/kg 2X/day, 80μg/kg lX/day, and 80μg/kg 2X/day. When the animals receive 2 daily doses of the PTH receptor agonist, the time interval between doses is 12 hours. Sham animals receive injections of the drug vehicle alone. Injections are made subcutaneously into the scapular region of the rats. Drug treatment of the rats begins on Day 7 of the study.

[00113] BMD of the lumbar spine region is measured on days 0, 7, 14, 21 and 28 using a PIXImus II scanner according to standard procedures described in the PIXImus user manual. Animals are first anesthetized and then scanned. Analysis of the lumbar spine is done by creating a window around last two lumbar vertebrae using a region of interest (ROI) tool of the PIXImus software. Baseline measurements are taken on Day 0; drug treatment begins on Day 7.

[00114] Results: Twice per day therapy with BIM-44058 at doses of 20, 40 and 80μg/kg is more efficacious than once per day therapy of the same doses at increasing lumbar spine BMD. This is also true for total daily dose (e.g. 20μg/kg twice per day is more efficacious than 40μg/kg once per day).

Example 5: Twice per day therapy with a PTH receptor agonist in humans.

[00115] A randomized, double-blind clinical trial is carried out to determine the outcome of twice daily PTH(I -34). Human patients with osteoporosis are randomly assigned to receive either injectable PTH(l-34) given in two daily doses each of 20μg at an interval of 12 hours or an injectable placebo given in two daily doses at an interval of 12 hours. PTH(l-34) or the injectable placebo is administered by subcutaneous injection by means of a pre filled pen. Patients receive the first two doses of the study drug at the clinical site. They also receive supplementation with calcium carbonate (at a dose of 1000 mg of elemental calcium) and vitamin D (at a dose of 800 IU) to be taken daily throughout the trial. Follow-up evaluations are scheduled at 1, 3, 6, 12, and 18 months. Compliance with the study-drug regimen is assessed by interviewing the patients at each visit and by quantifying the injectable medications that are returned to investigators.

[00116] For bone mineral density determination, areal bone mineral density (in grams per square centimeter) of the lumbar spine and total hip is assessed by dual energy x-ray absorptiometry with the use of densitometers. Quality assurance, cross-calibration adjustment, and data processing are performed. Scan results are withheld from local investigators unless a patient reaches a prespecifϊed safety value of a loss of more than 8% of bone. Lumbar vertebrae that are fractured during the trial are excluded from the calculation of bone mineral density.

[00117] For fracture determination, radiographs of the thoracolumbar spine are obtained at entry, at 18 months, or at early discontinuation, and at unscheduled times if there are new or worsening symptoms suggestive of clinical vertebral fracture. Radiographs are assessed in a blinded fashion by an independent reader for new vertebral fractures. Worsening of a preexisting deformity is not considered a new fracture. Vertebrae are graded individually for compression deformity with the use of semiquantitative criteria. Central adjudication of incident nonvertebral fractures is performed through direct examination of radiographs or evaluation of a radiologist's report.

[00118] For determining markers of bone remodeling, markers of bone formation (intact N-terminal propeptide of type I collagen, bone-specific alkaline phosphatase, and C-terminal propeptide of type I collagen) and bone resorption (C-telopeptide of type I collagen) are measured in serum obtained after an overnight fast in a subgroup of the patients at 1, 6, and 18 months. Frozen serum samples are shipped to a laboratory for analysis and run in batches. [00119] For determining adverse events, data on adverse events occurring or worsening after administration of the first dose of a study drug are collected throughout the study. Adverse events are coded with the use of the Medical Dictionary for Regulatory Activities, version 9.1. In addition to adverse event reports of hypercalcemia and hyperuricemia, total serum calcium concentrations of more than 10.5 mg per deciliter (2.62 mmol per liter) in a sample obtained more than 16 hours after the administration of a study drug; sustained elevated total serum calcium is defined as at least two elevated values at separate study visits is examined. Elevated serum urate is defined as a concentration of more than 9.0 mg per deciliter (535 μmol per liter).

[00120] For statistical analysis, analyses are conducted on data from patients who undergo randomization. For the primary outcome, the change from baseline to the last measurement of bone mineral density at the lumbar spine is examined. Models for continuous variables included fixed effects for the stratification terms and treatment. Analysis of variance is used for continuous variables except for markers of bone turnover, which require nonparametric methods. Categorical variables are compared between study groups with the use of a Cochran-Mantel-Haenszel test stratified according to geographic region or Fisher's exact test. [00121] The effects of treatment on the absolute change in bone mineral density from baseline to 3, 6, 12, and 18 months are assessed with mixed-model repeated measures. Covariates in the models are the treatment assignment, stratification variables, bone mineral density at the lumbar spine at baseline, time of the visit, and interaction between the visit and treatment. These models are used to analyze percent changes. A predefined gatekeeping strategy controls the overall type 1 error for testing of the primary objective and, subsequently, for determining the earliest time at which the increase in bone mineral density at the lumbar spine differs significantly between the study groups. Testing of the remaining secondary outcomes is not adjusted for multiple comparisons, and no interim analyses are conducted. All tests are two-sided.

[00122] PTH(l-34) given twice per day at an interval of 12 hours and a dose of 20μg is shown to be a safe and effective treatment for the treatment of osteoporosis in humans. [00123] While the invention has been particularly shown and described with reference to an embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

[00124] All references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

INFORMAL SEQUENCE LISTING

Claims

1. A method for treatment of a skeletal condition in a subject, comprising: administering to said subject a composition comprising a therapeutically effective amount of a parathyroid hormone (PTH) receptor agonist, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, wherein the time interval between each daily administration is between about 8 hours and 16 hours, and wherein said administering increases bone mineral density (BMD) in said subject.

2. The method of claim 1 , wherein said skeletal condition comprises osteoporosis, a bone fracture, senile osteoporosis, secondary osteoporosis, or an installed orthopaedic device comprising a bone interface.

3. The method of claim 1 , wherein said subject is a human.

4. The method of claim 3, wherein said subject comprises an aged subject, an osteoporotic subject, a post-menopausal woman, a geriatric man, a subject comprising a bone fracture, a subject comprising an installed orthopaedic device, or a subject comprising a secondary osteoporosis..

5. The method of claim 1, wherein said administration is subcutaneous.

6. The method of claim 1, wherein said administration comprises subcutaneous, oral, intravenous, cutaneous, nasal, intramuscular, or intraperitoneal routes.

7. The method of claim 1 , wherein said therapeutically effective amount is between about 5 and about 30 μg of said PTH receptor agonist.

8. The method of claim 1 , wherein said therapeutically effective amount is between about 10 and about 25 μg of said PTH receptor agonist.

9. The method of claim 8, wherein said therapeutically effective amount is about 20 μg of said PTH receptor agonist.

10. The method of claim 1 , wherein said therapeutically effective amount is between about 10 and about 200 μg of said PTH receptor agonist, wherein said PTH receptor agonist is PTH(I -84).

11. The method of claim 10, wherein said therapeutically effective amount is about 50 μg of said PTH receptor agonist.

12. The method of claim 1 , wherein said therapeutically effective amount is between about 5 and about 80 μg of said PTH receptor agonist, wherein said PTH receptor agonist is Ostabolin-C.

13. The method of claim 12, wherein said therapeutically effective amount is about 30 μg of said PTH receptor agonist.

14. The method of claim 1 , wherein said therapeutically effective amount is between about 10 and about 3000 μg of said PTH receptor agonist, wherein said PTH receptor agonist is a PTHrP analog.

15. The method of claim 1 , wherein said time interval between the two administrations is between about 10 hours and 14 hours.

16. The method of claim 15, wherein said time interval between the two administrations is about 12 hours.

17. The method of claim 1, wherein said PTH receptor agonist comprises PTH(l-34), PTH(l-84), Ostabolin-C, BIM-44058, or a PTHrP analog.

18. The method of claim 1, wherein said PTH receptor agonist comprises an isolated PTH receptor agonist peptide.

19. The method of claim 1 , wherein said PTH receptor agonist is a recombinant peptide.

20. The method of claim 1, wherein said PTH receptor agonist is a synthetic peptide.

21. The method of claim 1, wherein said PTH receptor agonist comprises human PTH(I- 34).

22. The method of claim 1 , wherein said administering advantageously affects BMD in said subject more than administration of said composition to said subject one time per day.

23. The method of claim 1 , wherein said treatment decreases standard duration of treatment.

24. The method of claim 1, further comprising determining the BMD of the subject.

25. A method for treatment of osteoporosis in a human, comprising: administering to said human a composition comprising a therapeutically effective amount of PTH(I -34), wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is 20μg, wherein the time interval between each administration of each therapeutically effective amount is 12 hours, and wherein said administering increases BMD in said human.

26. A method for reducing the risk of a medical condition in a subject, comprising treating a skeletal condition in said subject by administering to said subject a composition comprising a therapeutically effective amount of a PTH receptor agonist, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, wherein the time interval between each administration is between about 8 hours and 16 hours.

27. The method of claim 26, wherein said medical condition comprises a vertebral bone fracture or a non- vertebral bone fracture.

28. A method of advantageously affecting BMD in a subject comprising a skeletal condition, comprising: administering to said subject a composition comprising a therapeutically effective amount of a PTH receptor agonist, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, and wherein the time interval between each daily administration is between about 8 hours and 16 hours.

29. The method of any of claims 26-28, wherein said skeletal condition comprises osteoporosis, a bone fracture, senile osteoporosis, secondary osteoporosis, or an installed orthopedic device comprising a bone interface.

30. The method of claim 29, wherein said subject is a human.

31. The method of claim 29, wherein said subject comprises an aged subject, an osteoporotic subject, a post-menopausal woman, a geriatric man, a subject comprising a bone fracture, a subject comprising an installed orthopaedic device, or a subject comprising secondary osteoporosis.

32. The method of any of claims 26-28, wherein said administration is subcutaneous.

33. The method of any of claims 26-28, wherein said therapeutically effective amount is between about 5 and 30 μg of said PTH receptor agonist.

34. The method of any of claims 26-28, wherein said therapeutically effective amount is between about 10 and 25 μg of said PTH receptor agonist.

35. The method of claim 34, wherein said therapeutically effective amount is about 20 μg of said PTH receptor agonist.

36. The method of any of claims 26-28, wherein a time interval between the two administrations is between about 10 hours and 14 hours.

37. The method of claim 36, wherein the time interval between the two administrations is about 12 hours.

38. The method of any of claims 26-28, wherein said PTH receptor agonist comprises PTH(l-34), PTH(l-84), Ostabolin-C, or BIM-44058.

39. The method of any of claims 26-28, wherein said PTH receptor agonist comprises an isolated PTH receptor agonist peptide.

40. The method of any of claims 26-28, wherein said PTH receptor agonist peptide comprises a recombinant peptide.

41. The method of any of claims 26-28, wherein said PTH receptor agonist peptide comprises a synthetic peptide.

42. The method of any of claims 26-28, wherein said PTH receptor agonist comprises PTH(l-34).

43. The method of any of claims 26-28, wherein said administering advantageously affects BMD in said subject more than administration of said composition to said subject one time per day.

44. The method of any of claims 26-28, wherein said treatment decreases standard duration of treatment.

45. The method of any of claims 26-28, further comprising determining the BMD of the subject.

46. The method of any of claims 26-28, wherein said therapeutically effective amount is between about 10 and about 200 μg of said PTH receptor agonist, wherein said PTH receptor agonist is PTH(I -84).

47. The method of claim 46, wherein said therapeutically effective amount is about 50 μg of said PTH receptor agonist.

48. The method of any of claims 26-28, wherein said therapeutically effective amount is between about 5 and about 80 μg of said PTH receptor agonist, wherein said PTH receptor agonist is Ostabolin-C.

49. The method of claim 48, wherein said therapeutically effective amount is about 30 μg of said PTH receptor agonist.

50. The method of any of claims 26-28, wherein said therapeutically effective amount is between about 10 and about 3000 μg of said PTH receptor agonist, wherein said PTH receptor agonist is a PTHrP analog.

51. Use of a composition comprising a therapeutically effective amount of a PTH receptor agonist in the manufacture of a medicament for the treatment of a medical condition in a subject, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, wherein the time interval between each daily administration is between about 8 hours and 16 hours, and wherein said administering increases BMD in said subject.

52. Use of a composition comprising a therapeutically effective amount of PTH(I -34) in the manufacture of a medicament for the treatment of osteoporosis in a subject, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is 20 μg, wherein the time interval between each administration of each therapeutically effective amount is 12 hours, and wherein said administering increases BMD in said human.

53. Use of a composition comprising a therapeutically effective amount of a PTH receptor agonist in the manufacture of a medicament for the treatment of a skeletal condition in a subject, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, wherein the time interval between each administration is between about 8 hours and 16 hours.

54. Use of a composition comprising a therapeutically effective amount of a PTH receptor agonist in the manufacture of a medicament for advantageously affecting BMD in a subject comprising a skeletal condition, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, and wherein the time interval between each daily administration is between about 8 hours and 16 hours.

55. Use of a composition comprising a therapeutically effective amount of a PTH receptor agonist for the treatment of a medical condition in a subject, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, wherein the time interval between each daily administration is between about 8 hours and 16 hours, and wherein said administering increases BMD in said subject.

56. Use of a composition comprising a therapeutically effective amount of PTH(I -34) for the treatment of osteoporosis in a subject, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is 20μg, wherein the time interval between each administration of each therapeutically effective amount is 12 hours, and wherein said administering increases BMD in said human.

57. Use of a composition comprising a therapeutically effective amount of a PTH receptor agonist for the treatment of a skeletal condition in a subject, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, wherein the time interval between each administration is between about 8 hours and 16 hours.

58. Use of a composition comprising a therapeutically effective amount of a PTH receptor agonist for advantageously affecting BMD in a subject comprising a skeletal condition, wherein said therapeutically effective amount is administered to said subject two times per day, wherein each therapeutically effective amount is greater than or equal to about 5μg and less than or equal to about 3000μg, and wherein the time interval between each daily administration is between about 8 hours and 16 hours.