WO2008016404A2

WO2008016404A2 - Parathyroid hormone analogues and uses thereof

Info

Publication number: WO2008016404A2
Application number: PCT/US2007/010720
Authority: WO
Inventors: Paul Morley; Martin Stogniew; Brian Macdonald; Gene Scott Merutka; Nagesh R. Palepu
Original assignee: Zelos Therapeutics, Inc.
Priority date: 2006-07-31
Filing date: 2007-05-02
Publication date: 2008-02-07
Also published as: CA2659846A1; JP2010501476A; EP2056862A2; WO2008016404A3

Abstract

The present invention is directed to novel methods of treating a subject with a bone deficit disorder. The methods generally include administering to a subject in need thereof a pharmaceutically acceptable formulation comprising a parathyroid hormone (PTH) peptide analogue in a daily dose sufficient to result in an effective pharmacokinetic profile and maintained adenylate cyclase activity, while simultaneously reducing undesirable side effects.

Description

PARATHYROIDHORMONE ANALOGUES ANDMETHODS OFUSE

FIELD OF THE INVENTION

This application relates to methods and compositions of treating a subject with a bone deficit disorder. The methods generally include administering to a subject in need thereof a pharmaceutically acceptable formulation comprising a parathyroid hormone (PTH) peptide or analog in a dose sufficient to result in an effective pharmacokinetic profile, while simultaneously reducing undesirable side effects. Additionally, the dose can be optimized based on the weight, body surface area, body mass index (BMI), lean body mass or other body characteristic of the patient to be treated. This dose optimization approach applies to PTH peptides and analogs, as well as to other therapeutics as described herein.

BACKGROUND OF THE INVENTION

Bone remodeling, or turnover, consists of two opposing activities: the breakdown (resorption) of old bone by osteoclasts, and the formation of new bone by osteoblasts. Loss of bone mass occurs as part of the natural aging process. Calcium is constantly being added to and taken away from bone. When calcium is taken away faster than it is added, the bones become lighter, less dense, and more porous. This makes the bones weaker and increases their risk of fracture.

Bones naturally become thinner (called osteopenia) as people grow older, because existing bone is broken down faster than new bone is made. As this occurs, the bones lose minerals, heaviness (mass), and structure, making them weaker and more fragile. With further bone loss, osteopenia develops into osteoporosis. Accordingly, the thicker a person's bones are, the longer it takes to develop osteoporosis. Although osteoporosis can occur in men, it is most common in women older than age 65.

Osteoporosis often results in spontaneous fractures of load-bearing bones and the physical and mental deterioration characteristic of immobilizing injuries. In particular, postmenopausal osteoporosis is caused by the disappearance of estrogens which triggers an acceleration of bone turnover with an increased imbalance between resorption of old bone and formation of new bone. Instead of bone mass remaining stable, bone loss results because osteoclasts, the cells that destroy old bone (resorption of bones), outperform osteoblasts, the cells that build new bone (formation of bones). This accelerated bone loss due to resorption without adequate compensation by bone formation results in gradual thinning, increased porosity, and depletion of load-bearing bones.

End stage renal disease is invariably associated with bone disease, known as renal osteodystrophy (ROD). ROD may exist in a high turnover form characterized by high circulating levels of parathyroid hormone (PTH) and overactive bone tissue, often with osteitis fibrosa cystica. The low turnover form of the disease, also known as adynamic bone disease, is characterized by normal or low circulating levels of PTH. Histologically, the bone surfaces are quiescent with little or no cellular activity and osteomalacia may also be present. The incidence of the condition is increased with advanced age, presence of corticosteroid therapy, presence of calcimimetic therapy, calcium containing phosphate binders and high doses of Vitamin D sterols. However, adynamic bone disease is currently difficult to treat without leading to an unacceptable increase in serum calcium. Accordingly, there is a continuous unmet need for effective therapy.

Among the remedies for osteoporosis (which have historically involved increase in dietary calcium, estrogen therapy, and increased doses of vitamin D), human parathyroid hormone (hPTH) treatments are used to build bones to compensate for the bone loss due to osteoporosis. Parathyroid hormone is produced by the parathyroid gland and is involved in the control of calcium levels in blood. It is a hypercalcemic hormone, elevating blood calcium levels. PTH is a polypeptide and synthetic polypeptides may be prepared using the method disclosed by Erickson and Merrifϊeld, The Proteins, Neurath et al., Eds., Academic Press, New York, 1976, page 257, preferably as modified by the method of Hodges et al., Peptide Research, 1, 19 (1988) or by Atherton, E. and Sheppard, R. C, Solid Phase Peptide Synthesis, IRL Press, Oxford, 1989. When serum calcium is reduced to below a "normal" level, the parathyroid gland releases PTH and resorption of bone calcium and increased absorption of calcium from the intestine, as well as renal reabsorption of calcium, occur. An antagonist of PTH is calcitonin, which acts to reduce the level of circulating calcium. Although high levels of PTH can remove calcium from the bone, intermittent low doses can actually promote bone growth. For example, the native hPTH-(l-84) and its fragment hPTH-(l-34) (as sold under the tradename FORTEO^® by Eli Lilly and Co.) have been shown to be useful in the treatment of osteoporosis. The native hPTH-(l-84) and the hPTH-(l-34) fragment, however, suffer a drawback that while they promote bone formation, they simultaneously activate bone resorption. As a consequence hPTH-(l-34) is effective in reducing the fracture frequency of trabecular bone (which make up the bones of the axial skeleton, and include the rib cage, the back bones and the skull, and vertebrate bone), but its fracture reduction efficacy on cortical bone (which serves to protect against torsional loads and includes, for example, the hip and wrists) is considerably less.

There remains a need for therapeutic approaches employing suitable PTH analogues to restore bones and increase bone mineral density in both trabecular and cortical bones in patients with osteoporosis or other bone degenerative/deficit disorders. There further remains a need for therapeutic approaches employing suitable PTH analogues to restore bones and increase bone mineral density and formation without stimulating bone resorption, and without significantly increasing the levels of serum calcium in patients with osteoporosis or other bone degenerative disorders.

SUMMARY OF THE INVENTION

The present invention provides pharmaceutical compositions and formulations containing suitable PTH peptides or analogs thereof for use in methods directed to treating subjects suffering from various bone degenerative or bone deficit disorders. The PTH peptides or analogs described herein induce bone formation in both trabecular and cortical bones, thereby increasing bone mineral density and restoring bones. Unexpectedly, the PTH peptides or analogs described herein induce bone formation while causing less bone resorption than previously known PTH analogs, and also demonstrate lower incidences of and severity in hypercalcemia.

The PTH peptides or analogs disclosed herein, when administered within the specified dosage ranges, are effective in reversing the effects of osteoporosis on cortical bones in animals. Righting the imbalance between resorption of old cortical bone and formation of new cortical bone, these PTH peptides or analogs have been shown to reverse the effects of osteoporosis on bone. Thus, the methods described herein promote cortical bone growth in animals without significantly increasing cortical bone porosity.

These PTH peptides or analogs also promote recovery from bone injuries. Therefore, administration of the specified dosages of the PTH peptides or analogs of the present invention restore osteoporotic cortical bones and promote bone healing in various circumstances, such as in the treatment of fractures.

In one aspect, the invention provides a method for the treatment of osteoporosis, for treating a bone fracture, for inducing bone formation in trabecular and cortical bones, for treating or preventing renal osteodystrophy (ROD) and related disorders, comprising administering to a subject in need thereof a pharmaceutically acceptable formulation comprising a parathyroid hormone (PTH) peptide or analog, wherein said PTH peptide analogue has a reduced phospholipase-C activity and maintains adenylate cyclase activity, wherein the dosage administered results in an effective pharmacokinetic profile and effective bioactivity.

Another embodiment provides the use of the PTH peptides or analogs of the present invention for treating osteoporosis, for treating or preventing a bone fracture, for inducing bone formation in trabecular and cortical bones, for treating or preventing renal osteodystrophy (ROD) and related disorders, or for any other therapeutic use of PTH, wherein calcium monitoring is not required.

Another embodiment provides the use of the PTH peptides of the present invention for treating osteoporosis, for treating or preventing a bone fracture, for inducing bone formation in trabecular and cortical bones, for treating or preventing renal osteodystrophy (ROD) and related disorders, or for any other therapeutic use of PTH, wherein a warning regarding osteosarcoma formation is not required and wherein administration of the PTH peptides of the present invention may lead to lower incidences of osteosarcoma as compared to administration of Forteo.

In another embodiment, the invention provides a pharmaceutical formulation for subcutaneous administration comprising a unit dosage form of a therapeutically effective amount of an aqueous formulation of a parathyroid hormone (PTH) peptide or analog in a daily dosage range of 2 to 100 μg, wherein said PTH peptide or analog has reduced phospholipase-C activity and maintains adenylate cyclase activity and has an effective pharmacokinetic profile and effective bioactivity; and a pharmaceutically acceptable excipient, diluent, or carrier, or combinations thereof. Other therapeutically effective amounts within the pharmaceutical formulation include a daily dosage range of from 0.5 to 50 μg of a formulation stabilized with propylene glycol and/or ethanol for sub-cutaneous delivery or a daily dosage range of 100 to 3,000 μg for inhalation delivery, and weekly doses at 3-7 times the daily doses. Other embodiments include any dosage with any route of administration which results in an effective pharmacokinetic profile and effective bioactivity.

Another embodiment of the invention is a kit for treating a bone deficit disorder comprising, in one or more containers, a therapeutically effective amount of the above- described pharmaceutical composition contained in a device, and a label or packaging insert containing instructions for use.

PTH analogues optionally include less than the first 34 amino acids at the N-terminal end. The PTH peptide analogues of the present invention, when compared to full-length PTH peptides or other PTH peptide analogues which are 34 amino acid residues or longer, trigger less than full activation of phospholipase-C, less bone resorption, and less incidences or lower severity of hypercalcemia, while still maintaining increases in bone mineral density (BMD) at a variety of sites within the body.

Specific embodiments of PTH peptide analogues of the present invention include the following: PTH-(I -31)NH₂, Ostabolin; PTH-(I -3O)NH₂; PTH-(I -29)NH₂; PTH-(I -28)NH₂; Leu²⁷PTH-(l-31)NH₂; Leu²⁷PTH-(l-30)NH₂; LeU²⁷PTH-(I -29)NH₂; Leu²⁷cyclo(22-26)PTH- (1-3I)NH₂ Ostabolin-C™; Leu²⁷cyclo(22-26)PTH-(1-34)NH₂; Leu²⁷cyclo(Lys²⁶-Asp³⁰)PTH- (1-34)NH₂; Cyclo(Lys²⁷-Asp³⁰)PTH-(l-34)NH₂; Leu²⁷cyclo(22-26)PTH-( 1-31)NH₂; Ala²⁷ or NIe²⁷ or Tyr²⁷ or lie²⁷ cyclo(22-26)PTH-( 1-31)NH₂; Leu²⁷cyclo(22-26)PTH-(1-32)NH₂; Leu²⁷cyclo(22-26)PTH-( 1-31)OH; Leu²⁷cyclo(26-30)PTH-(1 -31)NH₂;

Cys²²Cys²⁶Leu²⁷cyclo(22-26)PTH-( 1 -31 )NH₂; Cys²²Cys²⁶Leu²⁷cyclo(26-30)PTH-( 1 -31 )NH₂ ; Cyclo(27-30)PTH-(l-31)NH₂; Leu²⁷cyclo(22-26)PTH-(1 -3O)NH₂; Cyclo(22-26)PTH-(1- 3I)NH₂; Cyclo(22-26)PTH-(1-30)NH₂; Leu²⁷cyclo(22-26)PTH-(1-29)NH₂; Leu²⁷cyclo(22- 26)PTH-(1-28)NH₂; Glu¹⁷,Leu²⁷cyclo(13-17)(22-26)PTH-(l-28)NH₂; and Glu¹⁷,Leu²⁷cyclo(13-l 7)(22-26)PTH-(l-31)NH₂.

Other embodiments of the invention include compositions and methods for treating a bone deficit disorder, while reducing side effects associated with the administration of a parathyroid hormone, comprising administering to a subject with a bone deficit disorder a pharmaceutically acceptable formulation comprising a parathyroid hormone (PTH) peptide analogue in a daily dose sufficient to result in an effective pharmacokinetic profile and maintained adenylate cyclase activity, while simultaneously reducing undesirable side effects. Optionally, the PTH peptide analogue can result in reduced phospholipase-C activity.

In another embodiment, the effective pharmacokinetic profile can be achieved by a variety of routes of administration with a variety of different formulations and comprises a pharmacokinetic parameter selected from the group consisting of: a) a half-life of said PTH peptide analogue of between 2 minutes and 60 minutes; b) a duration of exposure to said PTH peptide analogue of between 30 minutes and 4 hours; c) a T_n^x of said PTH peptide analogue of between 2 minutes and 30 minutes; and d) a C_max of said PTH peptide analogue of between 10 and 400 pg/ml. In additional embodiments, the half-life is between 5-30 minutes, the duration of exposure is between one and two hours, the T_n^x is between 15-30 minutes, and the C_max is between 50-200 pg/ml. This pharmacokinetic profile can be achieved with any route of administration known to those skilled in the art, including oral, topical, transdermal, nasal, pulmonary, transpercutaneous (wherein the skin has been broken either by mechanical or energy means), rectal, buccal, vaginal, via an implanted reservoir, or parenteral. Parenteral includes subcutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion techniques. More preferably, the route of administration is subcutaneous, transcutaneous, intranasal, transdermal, oral, or inhalation administration.

In further embodiments, the undesirable side effects that are reduced are selected from the group consisting of bone resorption, feeling cold, fatigue, loose stool, feeling hot, lower abdominal pain, injection site reaction, arthralgia, injection site hemorrhage, pharyngolaryngeal pain, muscle cramps, and abdominal pain. More specifically, the undesirable side effects that are reduced are selected from the group consisting of hypercalcemia, increase in mean serum calcium level, headache, nausea, back pain, dizziness, and extremity pain.

The PTH peptides of the present invention can also be administered at a variety of doses. Effective dosages can vary according to the type of formulation of PTH peptides or analogs administered as well as the route of administration. One skilled in the art can adjust the dosage by changing the route of administration or formulation, so that the dosage administered would result in a similar pharmacokinetic or biological profile as would result from the preferred dosage ranges described herein. Exemplary dosages include a daily dose of 2 to 100 μg for subcutaneous delivery of an aqueous formulation, a daily dose of 0.5 to 50 μg for subcutaneous delivery of a formulation stabilized with propylene glycol and/or ethanol, a daily dose of 100 to 3,000 μg for inhalation delivery, and weekly doses at 3-7 times the daily doses. Other suitable dosages include any dosage with any route of administration that results in a bioavailability or pharmacokinetic profile similar to those yielded by the above-described dosage ranges.

Preferred dosages for subcutaneous delivery of an aqueous formulation include dosages between 5-9 μg, 10-19 μg, 20-30 μg, 31-40 μg, 42-45 μg, 46-50 μg, and more specifically at 5 μg, 7.5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, or 50 μg. Most preferred doses for subcutaneous delivery of an aqueous formulation include either 7.5, 15, 30, or 45 μg . The dosage can also be calculated based on the size of the patient. The μg dosages can be normalized for patient characteristics such as height, weight, body surface area, BMI, lean body mass, etc., by converting the μg to μg/kg, or μg/m2, or μg /ml. The PTH peptides of the invention can also be administered at a daily dose of between 0.20 and 0.90 μg/kg, more preferably between 0.30 and 0.70 μg/kg, and more preferably between 0.46 and 0.69 μg/kg.

Additional embodiments of the invention include sequential therapy. One embodiment of such a treatment regimen starts treatment with a high dose of suitable PTH peptides or analogs and then after a period of time which could be 1-12 months but preferably 3-9 months and most preferably 4-8 months converts to a lower dose which maintains bone formation at a lower level but does not allow stimulation of bone resorption. Sequential therapy could also start treatment with a low dose and then convert to a high dose. Such sequential therapy can be administered in all doses disclosed herein.

One suitable dosage regimen includes administering an aqueous formulation of a PTH peptide or analog by subcutaneous administration in a first daily dose of from 35 μg to 100 μg, and then after the termination of the first period of time administering for a second period of time a second dose of from 2 μg to 35 μg of a PTH peptide analog. Another suitable dosage regimen includes administering an aqueous formulation of a PTH peptide or analog by subcutaneous administration in a first daily dose of from 2 μg to 35 μg, and then after the termination of the first period of time administering for a second period of time a second dose of from 35 μg to 100 μg of a PTH peptide analog. Additionally, the PTH peptides of the present invention can be administered by inhalation at a first and second daily dose of between 100 μg -2,000 μg, or at a first and second weekly dose of 3-7 times greater than the daily dose. In similar ways, dosages for sequential therapy can be calculated for inhalation administration or for formulations stabilized with propylene glycol or ethanol, or for any other formulations administered by any routes known in the art.

Another embodiment of the present invention is the administration of a dose to a patient based on that patient's weight, height, body surface area, BMI, or other patient characteristic and/or presentation of symptoms. This weight cut off method provides a method for determining a therapeutically effective dosage while maintaining a low incidence of side effects for a patient based upon their weight, body surface area, or BMI. By providing different doses to patients based on their body weight or mass, the amount of exposure of drug to a variety of patients is made more level. The choice of dosage based on weight, body surface area, or BMI of the patient improves the benefit to risk profile of the present peptides by improving the overall efficacy and proportion of patients who respond to the dosage while reducing the side effects of a dose that results in high exposure for an individual. All osteoporosis therapeutics with a predominant action to stimulate bone formation may be administered in a manner where the dosage is based on the patient's weight, height, body surface area, BMI, or other body type characteristic of the patient. Examples of such therapeutics within the scope of the present invention include the anti-sclerostin Mab, inhibitors of negative regulators of the Wnt signaling pathways, and activin receptor agonists. Additionally dosage based on patient weight, body surface area, or BMI is effective for all therapeutics whose bone formation effect is mediated by the action of PTH on its receptor, including PTH, full-length (1-84) and fragments thereof, PTH analogs, PTHrP, and PTHrP analogs. Specific PTH peptides which are effective with dosage based on patient weight, body surface area, or BMI include, but not limited to, full length PTH 1-84, PTH 1-34, PTH- (1-3I)NH₂, Ostabolin; PTH-(I -3O)NH₂; PTH-(I -29)NH₂; PTH-(I -28)NH₂; LeU²⁷PTH-(I- 3I)NH₂; Leu²⁷PTH-(l-30)NH₂; LeU²⁷PTH-(I -29)NH₂; Leu²⁷cyclo(22-26)PTH-(1-31)NH₂ Ostabolin-C™; Leu²⁷cyclo(22-26)PTH-(1-34)NH₂; Leu²⁷cyclo(Lys²⁶-Asp³⁰)PTH-(l -34)NH₂; Cyclo(Lys²⁷-Asp³⁰)PTH-(l-34)NH₂; Leu²⁷cyclo(22-26)PTH-( 1-31)NH₂; Ala²⁷ or NIe²⁷ or Tyr²⁷ or He²⁷ cyclo(22-26)PTH-( 1-31)NH₂; Leu²⁷cyclo(22-26)PTH-(1-32)NH₂; Leu²⁷cyclo(22-26)PTH-(l-31)OH; Leu²⁷cyclo(26-30)PTH-(l-31)NH₂;

Cys²²Cys²⁶Leu²⁷cyclo(22-26)PTH-(l-31)NH₂; Cys²²Cys²⁶Leu²⁷cyclo(26-30)PTH-(l-31)NH₂; Cyclo(27-30)PTH-(l-31)NH₂; Leu²⁷cyclo(22-26)PTH-(l-30)NH₂; Cyclo(22-26)PTH-(l- 3I)NH₂; Cyclo(22-26)PTH-(1-30)NH₂; Leu²⁷cyclo(22-26)PTH-(1-29)NH₂; Leu²⁷cyclo(22- 26)PTH-(1-28)NH₂; Glu^π,Leu²⁷cyclo(13-17)(22-26)PTH-(l-28)NH₂; and Glu¹⁷,Leu²⁷cyclo( 13- 17)(22-26)PTH-(1 -31)NH₂.

Additionally, suitable examples of therapeutics which can be administered based on the weight, body surface area, or BMI of the patient include any drugs which have a narrow therapeutic window, more specifically hormone therapies. Specific therapeutics also include calcium receptor antagonists which stimulate endogenous PTH production, such as those that act as agonists of the PTH receptor, including PTH, full-length and fragments thereof, PTH analogs, PTHrP and analogs thereof. The administration of a dosage based upon the weight, body surface area, or BMI of a patient can be used in a variety of indications, including osteoporosis, fracture repair, renal bone disease, corticosteroid-induced osteoporosis, transplant, and the induction of bone formation in trabecular and cortical bone.

This invention also pertains to specific formulations of Ostabolin-C, including an inhalation powder, and stabilized formulations which have an increased bioavailability. One such stabilized formulation includes Ostabolin-C, 40% ethanol and 60% water, with a pH between 6.0 and 8.0, wherein the formulation is stable for at least 2 years at 5°C. Another formulation includes Ostabolin-C, 40% propylene glycol and 60% water, with a pH between 6.0 and 8.0, wherein the formulation is stable for at least 2 years at 5°C. These formulations may also include, singularly or in combination, methionine, lipoic acid, sucrose and NaCl.

BRIEF DESCRIPTION OF THE DRAWINGS

For Figures 1-17, if not stated otherwise, the measurements following administration of Ostabolin-C were made after a 15 week course of subcutaneous daily administration of the stated dose, and the changes were measured as compared to baseline. As used herein, baseline is the patient's individual measurement prior to receiving any treatment.

Fig. 1 is a bar graph showing the percentage change in lumbar spine bone mineral density (BMD) in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l-31)-NH₂.

Fig. 2 is a graph showing the percentage change in lumbar spine bone mineral density (BMD) in patients with moderate osteoporosis receiving the pharmaceutical formulation containing hPTH-(l-34) teriparatide, Forteo®.

Fig. 3 is a bar graph showing the percentage change in total hip bone mineral density (BMD) in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l -31)-NH₂.

Fig. 4 is a bar graph showing the percentage change in femoral neck bone mineral density (BMD) in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l-31)-NH₂.

Fig. 5 is a bar graph showing the percentage change in trochanter bone mineral density (BMD) in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l-31)-NH₂.

Fig. 6 is a bar graph showing the percentage change in distal radius bone mineral density (BMD) in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-( 1-31)-NH₂. Fig. 7 is a bar graph showing the percentage change in mid-shaft radius bone mineral density (BMD) in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l-31)-NH₂.

Fig. 8 is a bar graph showing the percentage change in the bone formation marker amino terminal pro-peptide of type I pro-collagen (PlNP) in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]- PTH-(1-31)-NH₂.

Fig. 9 is a bar graph showing the percentage change in the bone formation marker osteocalcin in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l-31)-NH₂.

Fig. 10 is a bar graph showing the percentage change in the bone formation marker bone-specific alkaline phosphatase (BSAP) in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l -31)-NH₂.

Fig. 11 is a bar graph showing the percentage change in the bone resorption marker N-telopeptide (NTx) in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l -31)-NH₂.

Fig. 12 is a bar graph showing the percentage change in the bone resorption marker C- terminal telopeptide (CTx) in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l-31)-NH₂.

Fig. 13 is a graph showing the percentage change in the bone formation and bone resorption markers in patients with moderate osteoporosis receiving the pharmaceutical formulation containing rhPTH-(l-34), teriparatide, Forteo®^®.

Fig. 14 is a bar graph showing the percentage of abnormal serum calcium levels in patients with moderate osteoporosis receiving a pharmaceutical formulation containing [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l -31 )-NH₂.

Fig. 15 is a slide showing the Forteo data derived from Deal et al, (2005) J. Bone Min. Res. 20, p. 1905-1991.

Fig. 16 is a slide showing the effectiveness of Ostabolin-C and Forteo. Fig. 17 is a slide showing the effectiveness of Ostabolin-C and Forteo.

Fig. 18 is a graph showing the Phase I pharmacokinetics of Ostabolin-C at different doses.

Fig. 19 is a graph showing the Ostabolin- pharmacokinetics in female rats at different doses.

Fig. 20 is a graph showing the Ostabolin- pharmacokinetics in monkeys in a 6-week subcutaneous study.

Fig. 21 is a graph showing data for the administration of Ostabolin-C at 30 ug, including effects at the lumbar spine, mid-shaft radius, hypercalcemia, and the anabolic window.

Fig. 22 is a graph showing data for the administration of Ostabolin-C at 45 ug, including effects at the lumbar spine, mid-shaft radius, hypercalcemia, and the anabolic window.

Fig. 23 is a graph showing data for the % change from baseline for lumbar spine BMD with increasing Ostabolin-C exposure at 4 months.

Fig. 24 is a linear regression analysis assessing the full range of exposures of Ostabolin-C and the % change from baseline for lumbar spine BMD following exposure to increasing Ostabolin-C exposure at 4 months and 12 month, as compared to placebo.

Fig. 25 is a graph showing the effects of a 45 ug dose of Ostabolin-C on lumbar-spine BMD, s a % change from baseline, at 4 months and 12 months.

Fig. 26 is a linear regression analysis assessing the full range of exposures of Ostabolin-C and the % change from baseline for PlNP and CTx following exposure to increasing Ostabolin-C exposure at 4 months and 12 months, as compared to placebo.

Fig. 27 is a linear regression analysis assessing the full range of exposures of Ostabolin-C and the % change from baseline for total hip BMD following exposure to increasing Ostabolin-C exposure at 4 months and 12 months, as compared to placebo.

Fig. 28 is a linear regression analysis assessing the full range of exposures of Ostabolin-C and the % change from baseline for serum calcium levels following exposure to increasing Ostabolin-C exposure at 4 months and 12 months, as compared to placebos. Fig. 29 is a linear regression analysis assessing the fiill range of exposures of Ostabolin-C and the % change from baseline for femoral neck BMD following exposure to increasing Ostabolin-C exposure at 4 months, as compared to placebo.

Fig. 30 is a linear regression analysis assessing the full range of exposures of Ostabolin-C and the % change from baseline for mid-shaft radius BMD following exposure to increasing Ostabolin-C exposure at 12 months, as compared to placebo.

Fig. 31 is a graph of the incidences of hypercalcemia (at least one episode) following administration of Ostabolin-C at a variety of doses.

Fig. 32 is a graph of the incidences of hypercalcemia (only 1 episode compared to > 1 episode) following administration of Ostabolin-C at a variety of doses.

Fig. 33 is a graph of the exposure range for Ostabolin-C administered at doses of 7.5, 20, 30, and 45 ug.

Fig. 34 is a graph of the exposure range for Ostabolin-C administered at doses of 7.5, 20, 30, and 45 ug, overlaid with the impact of dose optimization on exposure.

Fig. 35 is a graph of the exposure range for Ostabolin-C administered to women and men at 30 ug. The women are represented by the solid line and the men are represented by the dashed line. The mean weight of the men was 82 kg and the mean weight of the women was 64 kg.

Fig. 36 is a graph of the exposure range for Ostabolin-C administered to men and women using a weight cutoff of 68 kg. The women are represented by the solid line and the men are represented by the dashed line. This graph illustrates that with this weight cutoff the men and women receive approximately the same exposure.

Fig. 37 is a graph illustrating the incidences of hypercalcemia at 4 months, applying a weight cutoff. The graph illustrates that with a weight cutoff of approximately 68 kg, the % of hypercalcemia seen is less than 15%.

Fig. 38 is a graph illustrating the incidences of hypercalcemia at 12 months, applying a weight cutoff. Fig. 39 is a graph illustrating more than 1 incidence of hypercalcemia at 4 months, applying a weight cutoff.

Fig. 40 is a graph illustrating more than 1 incidence of hypercalcemia at 12 months, applying a weight cutoff.

Fig. 41 is a graph illustrating the effect of a change in weight cutoff on bone formation.

Fig. 42 is a graph illustrating the effect of a change in weight cutoff on bone resorption.

Fig. 43 is a graph illustrating the effect of a change in weight cutoff on bone formation/resorption ratio.

Fig. 44 is a graph illustrating the incidences of lumbar spine BMD at 4 months, applying a weight cutoff.

Fig. 45 is a graph illustrating the incidences of lumbar spine BMD at 12 months, applying a weight cutoff.

Fig. 46 is a graph illustrating the incidences of lumbar spine BMD > 3% responders at 4 months, applying a weight cutoff.

Fig. 47 is a graph illustrating the dissociative effect of weight cut-off by comparing the hypercalcemia and lumbar spine BMD responder rate.

Fig. 48 is a graph illustrating the dissociation of BMD responders by comparing the site- specific effect of a weight cutoff change.

Fig. 49 is a graph illustrating the comparative effect of weight cutoff, by comparing the effect of a weight cutoff on CTx and hypercalcemia.

Fig. 50 is a graph illustrating the comparative effect of weight cutoff, by comparing the effect of a weight cutoff on CTx and serum calcium.

Fig. 51 is a graph illustrating the comparative effect of weight cutoff, by comparing the effect of a weight cutoff on serum calcium and lumbar spine BMD.

Fig. 52 is a graph illustrating the incidences of headache and nausea at 4 months, applying a weight cutoff. Fig. 53 is a graph illustrating the incidences of total hip BMD at 4 months, applying a weight cutoff.

Fig. 54 is a graph illustrating the incidences of total hip BMD at 12 months, applying a weight cutoff.

Fig. 55 is a graph illustrating the biological AUC (0-4) levels achieved with OCIP, at day 1 following Ostabolin-C inhaled administration.

Fig. 56 is a graph illustrating the biological Cmax levels achieved with OCIP, at day 1 following Ostabolin-C inhaled administration.

Fig. 57 is a graph illustrating the biological Tmax levels achieved with OCIP, at day 1 following Ostabolin-C inhaled administration.

Fig. 58 is a graph illustrating the biological Cmax levels achieved with OCEP, at days 1 and 7 following Ostabolin-C inhaled administration.

Fig. 59 is a graph illustrating the biological AUC (0-4) levels achieved with OCIP, at days 1 and 7 following Ostabolin-C inhaled administration.

Fig. 60 is a graph illustrating the day 1 average blood levels of Ostabolin-C in pg/ml.

Fig. 61 is a graph illustrating the urinary cAMP levels following Ostabolin-C inhaled administration.

Fig. 62 is a graph illustrating the urinary cAMP levels following Ostabolin-C inhaled administration.

Fig. 63 is a graph illustrating the relationship between the increase in cyclic AMP levels and the increase in AUC (0-2) levels following Ostabolin-C inhaled administration.

Fig. 64 is a graph illustrating the relationship between the increase in cyclic AMP levels and the increase in Cmax levels following Ostabolin-C inhaled administration.

Fig. 65 is a graph illustrating the mean % change in PlNP following Ostabolin-C inhaled administration. Fig. 66 is a graph illustrating the mean % change in Osteocalcin following Ostabolin-C inhaled administration.

Fig. 67 is a graph illustrating the relationship between the increase in PlNP levels and the increase in AUC (0-2) levels following Ostabolin-C inhaled administration

Fig. 68 is a graph illustrating the mean % change in CTx following Ostabolin-C inhaled administration.

Fig. 69 is a graph illustrating the mean heart rates following Ostabolin-C inhaled administration.

Fig. 70 is a graph illustrating the mean heart rates following Ostabolin-C inhaled administration

Fig. 71 is a graph illustrating the biological AUC (0-tz) levels achieved with various Ostabolin-C formulations, following subcutaneous administration.

Fig. 72 is a graph illustrating the biological Cmax levels achieved with various Ostabolin-C formulations, following subcutaneous administration.

Fig. 73 is a graph illustrating the biological AUC (0-tz) levels achieved with various Ostabolin-C formulations, following intramuscular and intravenous administration.

Fig. 74 is a graph illustrating the biological Cmax levels achieved with various Ostabolin-C formulations, following intramuscular and intravenous administration.

Fig. 75 is a graph illustrating the plasma concentrations of various Ostabolin-C formulations in female rats following intravenous administration.

Fig. 76 is a graph illustrating the plasma concentrations of various Ostabolin-C formulations in female rats following subcutaneous administration.

Fig. 77 is a graph illustrating the plasma concentrations of various Ostabolin-C formulations in female rats following intramuscular administration

Fig. 78 is a graph illustrating the effects of a dose cutoff with Ostabolin-C as compared to Forteo. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides pharmaceutical compositions and formulations containing suitable PTH peptide analogues for use in methods directed to treating subjects suffering from various bone degenerative or bone deficit disorders. The PTH peptide analogue compounds described herein induce bone formation in both trabecular and cortical bones, thereby increasing bone mineral density and restoring bones. Unexpectedly, the PTH peptide analogues described herein induce bone formation while causing less bone resorption than previously known PTH analogues, and also demonstrate lower incidences and severity of hypercalcemia. Additionally, the PTH peptides of the present invention provide a shorter duration PK profile, which allows for maintaining efficacy while reducing side effects.

The present invention also provides dose optimization as a way to reduce the side effects associated with prior art PTHs. The peptides of the present invention can be administered in varying doses to patients based on the particular patient's weight, height, size, body surface area, or BMI and/or presentation of symptoms. This weight, body surface area, or BMI cut off method provides a method for determining a therapeutically effective dosage while maintaining a low incidence of side effects for a patient based upon their weight, body surface area, or BMI. A dosage that results in high exposure for a particular patient will increase the chance of side effects, including hypercalcemia. Additionally, lack of efficacy may be observed in certain patients, because the dose for that particular patient was too low in a situation for a patient of a certain weight, body surface area, or BMI who could have tolerated a higher dosage of the therapeutic agent.

Transient exposure to PTH receptor agonists causes a bone formation response whereas continuous exposure to some PTH receptor agonists causes a predominant bone resorption effect. Even with transient exposure to PTH receptor agonists, as defined by conventional subcutaneous injection, some stimulation of bone resorption still occurs and this is associated with deleterious clinical effects including hypercalcemia and increased cortical porosity. Modifications to drug delivery that decrease the duration of exposure to PTH receptor agonists, regardless of the interval between doses and the route of administration of the dose, will improve the therapeutic window for PTH receptor agonists by reducing the level of stimulation of bone resorption for a given dose while maintaining or increasing the level of bone formation.

The present invention relates to PTH analogs, including Ostabolin-C and related analogs disclosed herein, which have a shorter duration PK profile than conventional PTHs. Such PTHs generate an increased therapeutic window by reducing the level of stimulation of bone resorption for a given dose equivalent while maintaining or enhancing the stimulation of bone formation.

The present invention also relates to reducing the undesirable side effects associated with the administration of PTH analogues, because of the shorter duration PK profile. Undesirable side effects which can be reduced include bone resorption, feeling cold, fatigue, loose stool, feeling hot, lower abdominal pain, injection site reaction, arthralgia, injection site hemorrhage, pharyngolaryngeal pain, muscle cramps, and abdominal pain. More specifically, undesirable side effects which can be reduced include hypercalcemia, increase in mean serum calcium level, headache, nausea, back pain, dizziness, and extremity pain.

The invention relates to a method for increasing bone toughness and/or stiffness, and/or reducing incidence of fracture in a subject by administering a parathyroid hormone. The method can be employed to increase stiffness and/or toughness at a site of a potential trauma or at a site of an actual trauma. Trauma generally includes fracture, surgical trauma, joint replacement, orthopedic procedures, and the like. Increasing bone toughness and/or stiffness generally includes increasing mineral density of cortical bone, increasing strength of bone, increasing resistance to loading, and the like. Reducing incidence of fracture generally includes reducing the likelihood or actual incidence of fracture for a subject compared to an untreated control population.

The present invention includes a method for increasing the toughness and/or stiffness of bone, including trabecular and cortical bone, and/or reducing the incidence and/or severity of fracture by administering a parathyroid hormone analogue as described herein. More particularly, the invention relates to a method for increasing toughness or stiffness of bone at a site of a potential or actual trauma. Increasing toughness and/or stiffness of bone can be manifested in numerous ways known to those of skill in the art, such as increasing bone mineral density, increasing bone mineral content, increasing work to failure, and the like. In one embodiment, the method of the invention reduces the incidence or severity of vertebral and/or non- vertebral fractures. The method of the invention can be used to decrease the risk of such fractures or for treating such fractures. In particular, the method of the invention can reduce the incidence of vertebral and/or non-vertebral fracture, reduce the severity of vertebral fracture, reduce the incidence of multiple vertebral fracture, improve bone quality, and the like.

The inventors have discovered that PTH peptide analogues that have a reduced phospholipase-C activity, and which maintain adenylate cyclase activity, are surprisingly useful for inducing bone formation in both trabecular and cortical bones, and causing less bone resorption than previous PTH analogues at dosages of about 2 to about 100 μg/day, without significantly increasing levels of serum calcium. The methods provided by this invention are generally practiced by administering to an animal in need thereof a dose of a PTH compound in the amount of about 2 to about 100 μg/day, or weekly at 3 to 7 times greater than the daily dose to induce bone formation and cause less bone resorption and lower incidences of hypercalcemia as compared to the administration of PTH analogues 34 amino acid residues in length or longer. Additionally, the PTH peptides of the present invention can be administered by inhalation at a daily dose of between 100 μg -2,000 μg.

The PTH peptide analogues, either alone or in combination with other bone enhancing agents, of the present invention can be used to treat any mammal, including humans and animals, suffering from a disease, symptom, or condition related to bone deficiency. In an embodiment of the invention, the subject in need of enhanced bone formation is a human patient such as a man or a woman. In a preferred embodiment, the patient is a postmenopausal woman.

Definitions

The following definitions are provided to assist the reader. Unless otherwise defined, all terms of art, notations and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the chemical and medical arts. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over the definition of the term as generally understood in the art.

As used herein, the "PTH peptide analogues" of the present invention are preferably, but not exclusively, non-naturally occurring and may be obtained either recombinantly or by peptide synthesis. The PTH analogues of the present invention include full length PTH (1- 84), 1-31 and 1-34 fragments, and other fragments or variants of fragments of human, rat, porcine, or bovine PTH that have human PTH activity as determined in the ovarectomized rat model of osteoporosis (Kimmel et al., Endocrinology, 1993, 32(4): 1577). Human PTH activity includes the ability of the PTH to increase trabecular and/or cortical bone growth. The PTH analogues of the present invention increase AC activity when administered to a PTH receptor containing or expressing cell in culture, such as an osteoblast or an osteoclast. The PTH analogues of the present invention have certain additional functional activities, as defined below.

As used herein, a PTH peptide analogue that has a "reduced phospholipase-C activity" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less than full activation of phospholipase-C, as compared to the full- length PTH peptide or other PTH peptide analogues which are at least 34 amino acid residues in length.

As used herein, a PTH peptide analogue that leads to "reduced bone resorption" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less bone resorption, as compared to the full-length PTH peptide or other PTH peptide analogues which are at least 34 amino acid residues in length.

As used herein, a PTH peptide analogue that leads to "reduced hypercalcemia levels" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less incidences of hypercalcemia, or lower severity of hypercalcemia, as compared to the full-length PTH peptide or other PTH peptide analogues which are at least 34 amino acid residues in length.

As used herein, "treating" or "treatment of a condition or subject refers to taking steps to obtain beneficial or desired results, including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more disease, symptom, or condition related to bone deficiency. Generally, such bone deficit disease, symptoms, and conditions are treated by inducing bone formation as measured by an increase in bone mineral density ("BMD"). For example, symptoms of osteoporosis include back pain, loss of height and stooped posture, a curved backbone (dowager's hump), or fractures that may occur with a minor injury (especially of the hip, spine, or wrist). Symptoms of Paget' s disease most commonly include bone pain. Other symptoms can include: headaches and hearing loss, neck pain, pressure on nerves, increased head size or bending of spine, hip pain, damage to cartilage of joints (which may lead to arthritis), and Barrel-shaped chest. Symptoms of osteoarthritis can include joint pain and aching, limited range of motion and instability, radiographic evidence of the erosion of the articular cartilage, joint space narrowing, sclerosis of the subchondral bone, and osteophytes (spurs). Symptoms for rheumatoid arthritis include painful, swollen, tender, stiff joints on both sides of the body (symmetrical), especially the hands, wrists, elbows, feet, knees, or neck. Rheumatoid nodules (bumps) ranging in size from a pea to a mothball develop in nearly one-third of people who have rheumatoid arthritis. These nodules usually form over pressure points in the body such as the elbows, knuckles, spine, and lower leg bones.

As used herein, "reduction" of a symptom or symptoms (and grammatical equivalents of this phrase) means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).

As used herein, "administering" or "administration of a drug or pharmaceutical composition or formulation described herein to a subject (and grammatical equivalents of this phrase) includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is administering the drug to the patient.

A variety of administration routes can be used in accordance with the present invention. An effective amount of the peptide described herein can be administered parenterally, orally, by inhalation, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir.

In a preferred embodiment of the invention, an effective amount of the peptide described herein can be administered parenterally. The term "parenteral" as used herein includes transdermal, transcutaneous, subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrasternal, needle-free injection, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. More preferably, the route of administration is subcutaneous administration.

As used herein, a "therapeutically effective amount" of a drug or pharmaceutical composition or formulation, or agent, described herein is an amount of a drug or agent that, when administered to a subject with a disease or condition, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one or more manifestations of the disease or condition in the 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. Thus, a therapeutically effective amount may be administered in one or more administrations.

As used herein, a "prophylactically effective amount" of a drug or pharmaceutical composition or formulation, or agent, described herein is an amount of a drug or agent that, when administered to a subject, will have the intended prophylactic effect, e.g., preventing or delaying the onset (or reoccurrence) of disease or symptoms, or reducing the likelihood of the onset (or reoccurrence) of disease or symptoms. The full prophylactic effect does not necessarily occur by administration of one dose and may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations.

Administration of a bone enhancing agent "in combination with" a drug or pharmaceutical composition or formulation described herein includes parallel administration (i.e., administration of both the drug and the agents to the subject over a period-of time, coadministration (in which both the drug and agents are administered at approximately the same time, e.g., within about a few minutes to a few hours of one another), and co-formulation (in which both the drug and agents are combined or compounded into a single dosage form suitable for oral or parenteral administration).

A "subject" is a mammal, preferably a human, but can also be an animal in need of veterinary treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like).

It should be noted that reference to numeric ranges throughout this specification is intended to encompass all numbers falling within the disclosed ranges. Thus, for example, the recitation of the range of about 1% to about 50% includes 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%, 10%, 12%, 14%, 16%, 20%, 25%, 30%, 35%, 40%, 45%, and 50%, as well as, for example, 21.3%, 7.9%, and 44.5%.

Additional active ingredients can be included in the present compositions. Choices are not limited, but may be chosen for a desired combined therapeutic effect. For example, active ingredients that may be added for a complementary therapeutic effect include, but are not limited to, vitamin D and analogs, estrogen, calcitonin, bisphosphonates, and mixtures thereof. A particularly desirable choice is calcitonin.

Bone Disorders and Diseases

Bone Deficits

In one aspect, the subject in need has a bone deficit, which means that they will have less bone than desirable or that the bone will be less dense or strong than desired. A bone deficit may be localized, such as that caused by a bone fracture or systemic, such as that caused by osteoporosis. Bone deficits may result from a bone remodelling disorder whereby the balance between bone formation and bone resorption is shifted, resulting in a bone deficit.

Examples of such bone remodelling disorders include, for example, osteoporosis, Paget's disease, renal osteodystrophy, renal rickets, osteoarthritis, rheumatoid arthritis, achondroplasia, osteochodrytis, hyperparathyroidism, osteogenesis imperfecta, congenital hypophosphatasia, fribromatous lesions, fibrous displasia, multiple myeloma, abnormal bone turnover, osteolytic bone disease and periodontal disease. Bone remodelling disorders includes metabolic bone diseases which are characterized by disturbances in the organic matrix, bone mineralization, bone remodelling, endocrine, nutritional and other factors which regulate skeletal and mineral homeostasis. Such disorders may be hereditary or acquired and generally are systemic, affecting the entire skeletal system.

Thus, in one aspect the human subject may have a bone remodelling disorder. Bone remodelling as used herein refers to the process whereby old bone is being removed and new bone is being formed by a continuous turnover of bone matrix and mineral that involves bone resorption by osteoclasts and bone formation by osteoblasts.

Osteoporosis is a common bone remodelling disorder characterized by a decrease in bone density of normally mineralized bone, resulting in thinning of trabeculae and increased porosity of bone cortices. The skeletal fragility caused by osteoporosis predisposes sufferers to bone pain and an increased incidence of fractures. Progressive bone loss in this condition may result in a loss of up to 50% of the initial skeletal mass. Primary osteoporosis includes idiopathic osteoporosis which occurs in children or young adults with normal gonadal function, Type I osteoporosis, also described as post-menopausal osteoporosis, and Type II osteoporosis, senile osteoporosis, occurs mainly in those persons older than 70 years of age. Causes of secondary osteoporosis may be endocrine (e.g., glucocorticoid excess, hyperparathyroidism, hypoganodism), drug induced (e.g. corticosteroid, heparin, tobacco) and miscellaneous (e.g., chronic renal failure, hepatic disease and malabsorbtion syndrome osteoporosis).

The phrase "at risk of developing a bone deficit", as used herein, is intended to embrace subjects having a higher than average predisposition towards developing a bone deficit. As an example, those susceptible towards osteoporosis include post-menopausal women, elderly males (e.g., those over the age of 65) and those being treated with drugs known to cause osteoporosis as a side-effect (e.g., steroid-induced osteoporosis). Certain factors are well known in the art which may be used to identify those at risk of developing a bone deficit due to bone remodelling disorders like osteoporosis. Risk factors for osteoporosis are known in the art and include hypogonadal conditions in men and women, irrespective of age, conditions, diseases or drugs that induce hypogonadism, nutritional factors associated with osteoporosis (low calcium or vitamin D being the most common), smoking, alcohol, drugs associated with bone loss (such as glucocorticoids, thyroxine, heparin, lithium, anticonvulsants etc.), loss of eyesight that predisposes to falls, space travel, immobilization, chronic hospitalization or bed rest, and other systemic diseases that have been linked to increased risk of osteoporosis.

Indications of the presence of osteoporosis are known in the art and include radiological evidence of at least one vertebral compression fracture, low bone mass (typically at least 1 standard deviation below mean young normal values), and/or atraumatic fractures. Other important factors include family history, life style, estrogen or androgen deficiency and negative calcium balance. Postmenopausal women are particularly at risk of developing osteoporosis. Hereinafter, references to treatment of bone diseases are intended to include management and/or prophylaxis except where the context demands otherwise.

Bone Trauma

The method of the invention is of benefit to a subject that may suffer or have suffered trauma to one or more bones. The method can benefit mammalian subjects, such as humans, horses, dogs, and cats, in particular, humans. Bone trauma can be a problem for racing horses and dogs, and also for household pets. A human can suffer any of a variety of bone traumas due, for example, to accident, medical intervention, disease, or disorder. In the young, bone trauma is likely due to fracture, medical intervention to repair a fracture, or the repair of joints or connective tissue damaged, for example, through athletics. Other types of bone trauma, such as those from osteoporosis, degenerative bone disease (such as arthritis or osteoarthritis), hip replacement, or secondary conditions associated with therapy for other systemic conditions (e.g., glucocorticoid osteoporosis, burns or organ transplantation) are found most often in older people.

Osteoporosis can lead, for example, to vertebral and/or non-vertebral fractures. Vertebral fractures are those involving the spinal column and non- vertebral fractures refers to any fracture not involving the spinal column. Non- vertebral fractures are more common than fractures of the vertebrae — an estimated 850,000 non-vertebral compared with 700,000 vertebral fractures occur annually in the United States. Non-vertebral fractures include more than 300,000 hip and 250,000 wrist fractures, in addition to 300,000 fractures at other non- vertebral sites. Other examples of non- vertebral fractures include a hip fracture, a fracture of a distal forearm, a fracture of a proximal humerus, a fracture of a wrist, a fracture of a radius, a fracture of an ankle, a fracture of an humerus, a fracture of a rib, a fracture of a foot, a fracture of a pelvis, or a combination of these. The method of the invention can be used to decrease the risk of such fractures or for treating such fractures. The risk of fracture is diminished and the healing of a fracture is aided by increasing the strength and/or stiffness of bone, for example, in the hip, the spine or both. A typical woman at risk for osteoporosis is a postmenopausal woman or a premenopausal, hypogonadal woman. A preferred subject is a postmenopausal woman, and is independent of concurrent hormone replacement therapy (HRT), estrogen or equivalent therapy, or antiresorptive therapy. The method of invention can benefit a subject at any stage of osteoporosis, but especially in the early and advanced stages.

The present invention provides a method, in particular, effective to prevent or reduce the incidence of fractures in a subject with or at risk of progressing to osteoporosis. For example, the present invention can reduce the incidence of vertebral and/or non- vertebral fracture, reduce the severity of vertebral fracture, reduce the incidence of multiple vertebral fracture, improve bone quality, and the like. In another embodiment, the method of the present invention can benefit patients with low bone mass or prior fracture who are at risk for future multiple skeletal fractures, such as patients in which spinal osteoporosis may be progressing rapidly.

Other subjects can also be at risk of or suffer bone trauma and can benefit from the method of the invention. For example, a wide variety of subjects at risk of one or more of the fractures identified above, can anticipate surgery resulting in bone trauma, or may undergo an orthopedic procedure that manipulates a bone at a skeletal site of abnormally low bone mass or poor bone structure, or deficient in mineral. For example, recovery of function after a surgery such as a joint replacement (e.g. knee or hip) or spine bracing, or other procedures that immobilize a bone or skeleton can improve due to the method of the invention. The method of the invention can also aid recovery from orthopedic procedures that manipulate a bone at a site of abnormally low bone mass or poor bone structure, which procedures include surgical division of bone, including osteotomies, joint replacement where loss of bone structure requires restructuring with acetabulum shelf creation and prevention of prosthesis drift, for example. Other suitable subjects for practice of the present invention include those suffering from hypoparathyroidism or kyphosis, who can undergo trauma related to, or caused by, hypoparathyroidism or progression of kyphosis.

Bone Toughness and Stiffness

The method of the invention reduces the risk of trauma or aids recovery from trauma by increasing bone toughness, stiffness or both. Generally toughness or stiffness of bone results from mass and strength of cortical and trabecular (cancellous) bone. The method of the invention can provide levels of bone toughness, stiffness, mass, and/or strength within or above the range of the normal population. Preferably the invention provides increased levels relative to the levels resulting from trauma or giving rise to risk of trauma. Increasing toughness, stiffness, or both decreases risk or probability of fracture compared to an untreated control population.

Certain characteristics of bone when increased provide increased bone toughness and/or stiffness. Such characteristics include bone mineral density (BMD), bone mineral content (BMC), activation frequency or bone formation rate, trabecular number, trabecular thickness, trabecular and other connectivity, periosteal and endocortical bone formation, cortical porosity, cross sectional bone area and bone mass, resistance to loading, and/or work to failure. An increase in one or more of these characteristics is a preferred outcome of the method of the invention.

Certain characteristics of bone, such as marrow space and elastic modulus when decreased provide increased toughness and/or stiffness of bone. Younger (tougher and stiffer) bone has crystallites that are generally smaller than crystallites of older bone. Thus, generally reducing the size of bone crystallites increases toughness and stiffness of bone, and can reduce incidence of fracture. In addition, maturing the crystallites of a bone can provide additional desirable characteristics to the bone, including increased toughness and stiffness of bone and/or can reduced incidence of fracture. A decrease in one or more of these characteristics can be a preferred outcome of the method of the invention!.

The method of the invention is effective for increasing the toughness and/or stiffness of any of several bones. For example, the present method can increase the toughness and/or stiffness of bones including a hip bone, such as an ilium, a leg bone, such as a femur, a bone from the spine, such as a vertebra, or a bone from an arm, such as a distal forearm bone or a proximal humerus. This increase in toughness and/or stiffness can be found throughout the bone, or localized to certain portions of the bone. For example, toughness and/or stiffness of a femur can be increased by increasing the toughness and/or stiffness of a femur neck or a femur trochanter. Toughness and/or stiffness of a hip can be increased by increasing the toughness and/or stiffness of an iliac crest or iliac spine. Toughness and/or stiffness of a vertebra can be increased by increasing the toughness and/or stiffness of a pedicle, lamina, or body. Advantageously, the effect is on vertebra in certain portions of the spine, such as cervical, thoracic, lumbar, sacral, and/or coccygeal vertebrae. Preferably the effect is on one or more mid-thoracic and/or upper lumbar vertebrae. The increase in toughness and/or stiffness can be found in each of the types of bone, or predominantly in one type of the bone. Types of bone include spongy (cancellous, trabecular, or lamellar) bone and compact (cortical or dense) bone and the fracture callus. The method of the invention preferably increases toughness and/or stiffness through its effects on cancellous and cortical bone, or on cortical bone alone. Trabecular bone, bone to which connective tissue is attached can also be toughened and/or stiffened by the present method. For example, it is advantageous to provide additional toughness at a site of attachment for a ligament, a tendon, and/or a muscle.

In another aspect of the invention, increasing toughness or stiffness can reduce incidence of fracture. In this aspect, increasing toughness or stiffness can include reducing incidence of vertebral fracture, reducing incidence of severe fracture, reducing incidence of moderate fracture, reducing incidence of non-vertebral fracture, reducing incidence of multiple fracture, or a combination thereof.

The methods of the invention may also be used to enhance bone formation in conditions where a bone deficit is caused by factors other than bone remodelling disorders. Such bone deficits include fractures, bone trauma, conditions associated with post-traumatic bone surgery (e.g., bone grafts or bone fusions), post-prosthetic joint surgery, post plastic bone surgery, post dental surgery, bone chemotherapy, and bone radiotherapy. Fractures include all types of microscopic and macroscopic fractures. Examples of fractures and/or injuries include avulsion fracture, comminuted fracture, non-union fracture, transverse fracture, oblique fracture, spiral fracture, segmental fracture, a segmental gap, displaced fracture, impacted fracture, greenstick fracture, torus fracture, fatigue fracture, intra-articular fracture (epiphyseal fracture), closed fracture (simple fracture), open fracture (compound fracture), a bone void, and occult fracture in any bones of the subject.

As previously mentioned, a wide variety of bone diseases may be treated in accordance with the present invention, for example all those bone diseases connected with the bone-remodelling cycle. Examples of such diseases include all forms of osteoporosis, osteomalacia and rickets. Osteoporosis, especially of the post-menopausal, male, post- transplant, and steroid-induced types, is of particular note. In addition, PTH peptide analogues find use as bone promotion agents, and as anabolic bone agents. Such uses form another aspect of the present invention.

Parathyroid Hormone Analogues As active ingredient, the pharmaceutically acceptable composition or solution described herein may incorporate full-length PTH (1-84), 1-31 and 1-34 fragments, and other fragments, or variants of fragments, including substitutions, deletions, or insertions, of human PTH, or of rat, porcine or bovine PTH that have human PTH activity as determined in the ovarectomized rat model of osteoporosis reported by Kimmel et al., Endocrinology, 1993, 32(4): 1577. Human PTH activity includes the ability of the PTH to increase trabecular and/or cortical bone growth. The PTH analogues of the present invention increase AC activity when administered to a PTH receptor containing or expressing cell in culture, such as an osteoblast or osteoclast. The PTH analogues used in the present invention are naturally or non- naturally occurring and desirably incorporate less than the first 34 N-terminal residues of PTH.

PTH operates through activation of two second messenger systems, G_s-protein activated adenylyl cyclase (AC) and G_q-protein activated phospholipase C. The latter results in a stimulation of membrane-bound protein kinase Cs (PKC) activity. The PKC activity has been shown to require PTH residues 29 to 32 (Jouishomme et al (1994) J. Bone Mineral Res. 9, (1179-1189). It has been established that the increase in bone growth, i.e. that effect which is useful in the treatment of osteoporosis, is coupled to the ability of the peptide sequence to increase AC activity.

The native PTH sequence, and its truncated 1-34 form, has been shown to have all of these activities. The hPTH-(l-34) sequence is:

Ser VaI Ser GIu He GIn Leu Met His Asn Leu GIy Lys His Leu Asn Ser Met GIu Arg VaI GIu Trp Leu Arg Lys Lys Leu GLn Asp VaI His Asn Phe-OH (SEQ ID NO:1)

AC activity has been shown to require the first few N-terminal residues of the molecule. Thus, in accordance with this embodiment of the invention, it is possible to remove those biological activities associated with the PKC activity by deleting a selected terminal portion of the hPTH-(l-34) molecule. In one embodiment, these shortened analogues are desirably in the form of carboxyl terminal amides. One feature of the invention therefore comprises variants of the human parathyroid analogues PTH(I -25)-NH₂, PTH(I -26)-NH₂, PTH(I -27)-NH₂, PTH(l-28)-NH₂, PTH(I -29)-NH₂, PTH(I -3O)-NH₂, and PTH(I -31)-NH₂.

According to another feature of the PTH analogues to be used in the present invention, it has surprisingly been found that replacing LyS27 with a Leu in the native hPTH sequence results in a higher activity for AC stimulation. This analogue also exhibits its maximum activity when in the form of the carboxyl terminal amide. Thus, another feature of the invention comprises the use of PTH analogues including all sequences from [LeU₂₇J-PTH- (l-25)-NH₂ to [LeU₂₇ ]-PTH-( 1-31)-NH₂.

According to another feature of the present invention, lactams of the PTH analogues are formed, for example, by cyclisation involving the coupling of the side-chains of Glu22 and Lys26, or of the side-chains Lys26 and Asp30, in which Lys27 may be replaced by a Leu or by various other hydrophobic residues, and which has either a C-terminal free amide ending, or has a C-terminal free carboxyl ending. Such substitutions include ornithine, citrulline, alpha-aminobutyric acid, or any linear or branched alpha-amino aliphatic acid, having 2-10 carbons in the side chain, any such analogue having a polar or charged group at the terminus of the aliphatic chain. Example of polar or charged groups include amino, carboxyl, acetamido, guanido and ureido. He, norleucine, Met, and ornithine are expected to be the most active.

The PTH analogues of the present invention may thus feature the formation of a lactam, for example, between either residues Glu22 and Lys26, Ly26 and Asp30, or Glu22 and Lys27. The substitution of Leu for the Lys27 results in a more hydrophobic residue on the hydrophobic face of the amphiphilic helix. This resulted in increased adenylyl cyclase stimulating activity in the PTH receptor containing rat osteosarcoma (ROS) cell line. It will be appreciated by those skilled in the art that other such substitutions would likely result in analogues with the same or increased activities. These hydrophobic substitutions include residues such as Met or norleucine. The combined effect of substitution and either lactam formation is expected to stabilize the alpha-helix and increase bioactivity, and to protect this region of the molecule from proteolytic degradation. The presence of the amide at the C- terminus is further expected to protect the peptide against exoproteolytic degradation (Leslie, F. M. and Goldstein, A. (1982) Neuropeptides 2, 185-196).

In one preferred embodiment of the invention, the peptide used in the disclosed method is PTH(1-31)-NH2 with the following sequence:

Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg- Val-Glu-Trp-Leu-Arg-Lys-Xaa-Leu-Gln-Asp-Val-NH₂ (SEQ ID NO: 2).

Xaa is selected from the group consisting of Lys, Leu, He, NIe and Met. In a preferred embodiment, Xaa is Lys (SEQ ID NO: 3). This embodiment is also referred to as OSTABOLIN. In another preferred embodiment of the invention, the peptide used in the disclosed method is cyclo(22-26)PTH-(l-31)-NH2, cyclized in the form of a lactam between GIu²² and Lys²⁶ with the following sequence:

Ser-Val-Ser-Glu-He-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg- Val-Glu-Trp-Leu-Arg-Lys-Xaa-Leu-Gln-Asp- VaI-Y (SEQ ID NO: 4),

Xaa is selected from the group consisting of Leu, He, NIe and Met and Y is NH₂ or OH. When Xaa is Leu and Y is NH₂ (SEQ ID NO: 5), the PTH is also referred to as OSTABOLIN-C™.

The PTH analogues to be used in the present invention can thus be cyclized or linear, and can be optionally amidated at the C-terminus. Alternatives in the form of PTH variants incorporate from 1 to 5 amino acid substitutions that improve PTH 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 stability against oxidation and 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 stability against protease. Other suitable forms of PTH include PTHrP, PTHrP(l-34), PTHrP(l-36) and analogs of PTH or PTHrP that activate the PTHl receptor. These forms of PTH are embraced by the term "parathyroid hormone analogues" as used generically herein. The hormones may be obtained by known recombinant or synthetic methods, such as described in U.S. Pat. Nos. 4,086,196; 5,556,940; 5,955,425; 6,541,450; 6,316, 410; and 6,110,892, incorporated herein by reference.

Specific embodiments of PTH peptide analogues of the present invention include the following: PTH-(I -31)NH₂, Ostabolin; PTH-(I -3O)NH₂; PTH-(I -29)NH₂; PTH-(I -28)NH₂; Leu²⁷PTH-(l-31)NH₂; LeU²⁷PTH-(I -3O)NH₂; LeU²⁷PTH-(I -29)NH₂; Leu²⁷cyclo(22-26)PTH- (1-3I)NH₂ Ostabolin-C™; Leu²⁷cyclo(22-26)PTH-(1-34)NH₂; Leu²⁷cyclo(Lys26- Asp30)PTH-(l-34)NH₂; Cyclo(Lys27-Asp30)PTH-(l -34)NH₂; Leu²⁷cyclo(22-26)PTH-(1 - 3I)NH₂; Ala²⁷ or NIe²⁷ or Tyr²⁷ or He²⁷ cyclo(22-26)PTH-(l-31)NH₂; Leu²⁷cyclo(22- 26)PTH-(l-32)NH₂; Leu²⁷cyclo(22-26)PTH-(l-31)OH; Leu²⁷cyclo(26-30)PTH-(l-31)NH₂; Cys²²Cys²⁶Leu²⁷cyclo(22-26)PTH-(l -31)NH₂; Cys²²Cys²⁶Leu²⁷cyclo(26-30)PTH-( 1 -31 )NH₂; Cyclo(27-30)PTH-(l-31)NH₂; Leu²⁷cyclo(22-26)PTH-(l-30)NH₂; Cyclo(22-26)PTH-(l- 3I)NH₂; Cyclo(22-26)PTH-(1-30)NH₂; Leu²⁷cyclo(22-26)PTH-(1-29)NH₂; Leu²⁷cyclo(22- 26)PTH-(1-28)NH₂; Glu¹⁷,Leu²⁷cyclo(13-17)(22-26)PTH-(l-28)NH₂; and Glu^l7,Leu²⁷cyclo(l 3- 17)(22-26)PTH-(l -31)NH₂.

Generally, preferred embodiments of PTH peptide analogues include those that when administered result in reduced phospholipase-C activity, reduced bone resorption, and reduced hypercalcemia levels. As defined in the Definitions section herein, "reduced phospholipase-C activity" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less than full activation of phospholipase-C, as compared to the full-length PTH peptide or other PTH peptide analogues which are at least 34 amino acid residues in length; "reduced bone resorption" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less bone resorption, as compared to the full-length PTH peptide or other PTH peptide analogues which are at least 34 amino acid residues in length, and "reduced hypercalcemia levels" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less incidences of hypercalcemia, or lower severity of hypercalcemia, as compared to the full-length PTH peptide or other PTH peptide analogues which are at least 34 amino acid residues in length. The preferred PTH analogues administered in the methods described herein include [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l-31)-NH₂, such as advanced by Zelos Therapeutics, Inc. under the tradename OSTABOLIN-C™ and [Leu²⁷] PTH-(I -31)-NH₂- In another embodiment of the invention, [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l -3O)-NH₂ is used in the methods described herein. In another embodiment, the hormone can be the linear analogue PTH(I -31), which can have a free carboxyl ending, or be amidated, at the C-terminus. In yet another embodiment, the hormone can be PTH(I -30) , which can have a free carboxyl ending, or be amidated, at the C-terminus; or [Leu²⁷]-PTH(l-30)- NH₂. Suitable stabilized solutions of these and other PTH analogues that can be employed in the present methods are described in U.S. Patent Nos. 5,556,940; 5,955,425; 6,541,450; 6,316, 410; and 6,110,892 incorporated herein by reference.

Methods of the Invention and Agents Useful Therein

The methods provided by this invention are generally practiced by administering to an animal in need thereof a daily or weekly dose of a PTH compound in an amount effective to induce bone formation and inhibit or reduce bone loss or resorption.

One aspect of the present invention provides a method for treating osteoporosis by administering to a subject in need thereof a pharmaceutically acceptable formulation comprising a PTH peptide analogue in a daily subcutaneous dose of an aqueous formulation of 2 μg to 100 μg or a weekly dose of from 14 μg to 700 μg , wherein the PTH peptide analogue has a reduced phospholipase-C activity but maintains adenylate cyclase activity. Exemplary dosages include a daily dose of 0.5 to 50 μg for subcutaneous delivery of a formulation stabilized with propylene glycol and/or ethanol, a daily dose of 100 to 3,000 μg for inhalation delivery, and weekly doses at 3-7 times the daily doses. Other suitable dosages include any dosage with any route of administration that results in a bioavailability or pharmacokinetic profile similar to those yielded by the above-described dosage ranges. In one embodiment, the subject is a human man or woman. In a preferred embodiment the woman is post-menopausal.

In another embodiment, the osteoporosis can be selected from the group consisting of advanced-stage osteoporosis, hypogonadal osteoporosis, spinal osteoporosis, transplant- induced osteoporosis, and steroid-induced osteoporosis.

Bone enhancing agents known in the art to increase bone formation, bone density or bone mineralisation, or to prevent bone resorption may be used in the methods and pharmaceutical compositions of the invention. Those of ordinary skill in the bone formation art also recognize that suitable bone enhancing agents include, for example, natural or synthetic hormones, such as selective estrogen receptor modulators (SERMs), estrogens, androgens, calcitonin, prostaglandins and parathormone; growth factors, such as platelet- derived growth factor, insulin-like growth factor, transforming growth factor, epidermal growth factor, connective tissue growth factor and fibroblast growth factor; vitamins, particularly vitamin D; minerals, such as calcium, aluminum, strontium, lanthanides (such as lanthanum (HI) compounds as described and used in U.S. Patent No. 7,078,059, incorporated herein by reference) and fluoride; isoflavones, such as ipriflavone; statin drugs, including pravastatin, fluvastatin, simvastatin, lovastatin and atorvastatin; agonsists or antagonist of receptors on the surface of osteoblasts and osteoclasts, including parathormone receptors, estrogen receptors and prostaglandin receptors; bisphosphonate and anabolic bone agents. In one embodiment, vitamin D, calcium, or both are concurrently administered with the pharmaceutical formulations of the present invention.

Generally, preferred embodiments of PTH peptide analogues include those that when administered result in reduced phospholipase-C activity, reduced ability to stimulate bone resorption, and reduced hypercalcemia levels. As defined in the Definitions section herein, "reduced phospholipase-C activity" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less than full activation of phospholipase-C, as compared to the full-length PTH peptide or other PTH peptide analogues; "reduced bone resorption" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less bone resorption, as compared to the full-length PTH peptide or other PTH peptide analogues, and "reduced hypercalcemia levels" refers to a PTH peptide analogue that has been truncated or modified Ln some manner so as to trigger less incidences of hypercalcemia, or lower severity of hypercalcemia, as compared to the full-length PTH peptide or other PTH peptide analogues.

The preferred PTH analogues administered in the methods described herein include [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l-31)-NH₂, such as advanced by Zelos Therapeutics, Inc. under the tradename OSTABOLIN-C™ and PTH-(I -31)-NH₂, such as advanced by Zelos Therapeutics, Inc. under the tradename OSTABOLIN™. In another embodiment of the invention, [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l-30)-NH₂ is used in the methods described herein. In another embodiment, the hormone can be the linear analogue PTH(I -31), which can have a free carboxyl ending, or be amidated, at the C-terminus. In yet another embodiment, the hormone can be PTH(I -30) , which can have a free carboxyl ending, or be amidated, at the C-terminus; or [Leu²⁷]-PTH(l-30)- NH₂. Suitable stabilized solutions of these and other PTH analogues that can be employed in the present methods are described in U.S. Patent Nos. 5,556,940; 5,955,425; 6,541,450; 6,316, 410; and 6,110,892 incorporated herein by reference.

The pharmaceutical compositions and formulations described herein, and in the doses and routes of administration described in detail below, further operate to induce bone formation by stimulating osteoblast differentiation in trabecular and cortical bone while simultaneously reducing the incidence of hypercalcemia (i.e., higher than normal levels of calcium in the blood).

In another aspect of the invention, methods for treating a bone fracture in a subject are provided. A preferred method can include administering to a subject in need thereof a pharmaceutically acceptable formulation of a PTH peptide analogue in a daily dose of 30 μg for three months, wherein the peptide analogue has reduced phospholipase-C activity and maintains adenylate cyclase activity, and wherein the PTH peptide analogue induces bone formation. In another embodiment, the method can include administering to a subject in need thereof a pharmaceutically acceptable formulation of a PTH peptide analogue in a daily subcutaneous dose of 30 μg - 45 μg for 1, 2, or 3 months, wherein the peptide analogue has reduced phospholipase-C activity and maintains adenylate cyclase activity, and wherein the PTH peptide analogue induces bone formation.

The pharmaceutical formulations described herein can be used to accelerate the healing of a fracture in any bone of the subject's skeleton. In preferred embodiments, the pharmaceutical formulations of the present invention are used to heal fractures of the hip, forearm, humerus, wrist, radius, ankle, rib, femur, tibia, and foot. The fractures can be of multiple types as discussed above, and healing can simultaneously occur in a plurality of bones that may be fractured.

In another aspect, the invention provides methods for inducing bone formation in trabecular and cortical bones, as measured by an increase in BMD by administering to a subject in need thereof a daily dose of a pharmaceutically acceptable formulation of a PTH peptide analogue, wherein the peptide analogue has reduced phospholipase-C activity and maintains adenylate cyclase activity.

In preferred embodiments, the pharmaceutical formulations can be used to induce bone formation at the spine, skull, ribs, hips, ankle, and wrists, although any bone of the subject's skeleton can be induced to form bone. In another embodiment, following administration of the PTH pharmaceutical formulations of the present invention, the incidences in the patient population in which the level of serum calcium is above normal is less than the those seen with administration of prior art PTH peptides.

In yet another aspect, the present invention provides methods of treating or preventing' renal osteodystrophy (ROD) and related disorders by administering to a subject in need thereof a daily dose of a pharmaceutically acceptable formulation of a PTH peptide analogue, wherein the peptide analogue has reduced phospholipase-C activity and maintains adenylate cyclase activity.

In an embodiment, ROD related disorders are osteitis fibrosa cystica and adynamic bone disease.

Unexpected Results

The pharmaceutical compositions and formulations described herein, and in the doses and routes of administration described in detail below, operate to induce bone formation by stimulating osteoblast differentiation in trabecular and cortical bone while simultaneously reducing or inhibiting osteoclast differentiation, and thus, bone resorption. PTH analogues less than 34 amino acids in length are preferred, because these truncated forms maintain the positive effects of increased bone formation, while minimizing the negative effects of increased bone resorption. Minimizing the bone resorption also leads to less cortical porosity.

Administration of the PTH analogues of the present invention at a variety of doses has led to unexpected and superior results when compared to administration of prior PTH analogues. When administered over a course of four months, the PTH analogues of the present invention have been shown to have a similar or greater effect on the increase in BMD of lumbar spine, hip, femoral neck, and trochanter as compared to prior art PTH analogues which are at least 34 amino acid residues in length given over at least a course of a year. For results of prior art PTH analogues, see Neer, N. Eng. J. Med, VoI 344, No. 19, May 2001, p. 1434-1441. These unexpected results are described in detail in the Examples and the Figures.

Administration of the peptides of the present invention also has a positive effect on cortical bone, specifically the wrist (the distal and mid-shaft radius, Figs 6 and 7). Historically, PTH has been known to increase bone resorption, which increases cortical porosity, thus making it difficult for PTH to increase BMD in cortical bone. The dosages and formulations of the present invention have a positive effect on cortical bone growth as compared to both placebo and to teriparatide, Forteo®. This is an unprecedented finding, demonstrating a statistically significant difference from placebo for 3 active doses.

Administration of the PTHs of the present invention also have unexpected results on bone formation and bone resorption markers. The bone formation markers include PlNP, osteocalcin, and BSAP and the bone resorption markers include NTx and CTx. As compared to placebo, the bone formation markers have a greater % change when Ostabolin-C™ is administered at 15, 30, and 45 μg. Figs 8-10. There is a robust effect in the increase in the bone formation markers when Ostabolin-C™ is administered at 30 and 45 μg. The bone resorption markers in Figures 11-13 demonstrate that although there is some increase in bone resorption following the administration of Ostabolin-C™, this increase is less than that which follows administration of the prior art teriparatide, Forteo® PTH. Neer et al., 2001.

Administration of the PTH peptides of the present invention has also been shown to unexpectedly result in a much lower incidence and severity of hypercalcemia as compared to PTHs known in the art. Hypercalcemia for a patient being administered the PTH peptides means the occurrence of at least one serum calcium value for the patient above the upper limit of normal (2.64 mmol/L; 10.6 mg/dL). Neer et al., 2001.

Administration of Forteo^® resulted in an increased level of incidences of hypercalcemia as compared to placebo. FDA approval of Forteo^® was based on the results of treatment of 1637 postmenopausal women (with prior vertebral fractures) with 20 or 40 μg/day of Forteo^® for an average of 19 months. See Forteo^® package insert, incorporated by reference in its entirety, and Neer. While the medication was generally well-tolerated, hypercalcemia was seen at least once in 11% of the 20 μg group subjects and in 28% of the 40 μg group subjects as compared with 2% in the placebo group. In contrast, the administration of low doses of the PTH peptides of the present invention (7.5, 15, and 30 μg) resulted in only a negligible increase in the incidences of hypercalcemia as compared to placebo. As an example, hypercalcemia was seen at least once in 5% of the placebo group and in 5% of the group being administered 15 μg doses, resulting in no net increase of hypercalcemia.

Accordingly, administration of the PTH analogues of the present invention at a variety of doses leads to following unexpected results: 1) similar or greater effect on the increase in BMD of lumbar spine, hip, femoral neck, and trochanter when given over a course of only four months as compared to prior art PTH analogues given over a course of at least a year; 2) increase in BMD on cortical bone, specifically the wrist (the distal and mid-shaft radius), whereas prior art PTH peptides have resulted in decease in BMD of cortical bone; and 3) lower amount of incidences and severity of hypercalcemia as compared to prior art PTH peptides.

The PTH peptides of the present invention offer substantial improvements over currently available therapy , as they are an anabolic agent that lead to much lower incidences and severity of hypercalcemia. Based on preclinical and clinical experience to date, the present PTH peptides are a safe and highly effective anabolic agent for treating osteoporosis, without inducing hypercalcemia. Due to its reduced impact on bone resorption, the present PTH peptides also have an improved clinical profile with respect to its effects on bone quality.

The decrease in bone resorption can be measured by a reduction in the level of bone resorptive markers. Although biochemical markers of bone turnover cannot reveal how much bone is present in the skeleton at any given time, and thus, cannot be used to diagnosis osteoporosis or to tell how severe the disease may be, biochemical markers can be used in conjunction with the pharmaceutical compositions and formulations of the present invention to (1) predict bone loss in peri- and post-menopausal women and to (2) monitor the skeletal response to treatment. Unlike bone mineral density (BMD) measurements, biochemical markers are able to detect acute changes in bone turnover. While BMD tests typically detect bone density changes in years, markers are able to detect changes in bone metabolism in weeks or months. Bone turnover can be assessed via the measurement of various biochemical markers. There are two basic types of markers: markers of bone formation and markers of bone resorption. Additionally, these markers can be categorized into two groups: markers that measure substances released by osteoblasts and osteoclasts and markers that measure substances produced during the formation or breakdown of collagen, a primary protein found in bone. As bone remodeling occurs, these substances are released into the blood and, eventually, excreted in the urine. Many biochemical markers can be detected and measured in both the blood (serum) and urine.

The most commonly used assays for bone formation are serum tests of bone-specific alkaline phosphatase (BSAP), osteocalcin and procollagen peptides, proteins produced by osteoblasts and released into the bloodstream during bone formation. Bone resorption markers typically measure the breakdown of products of collagen, the major protein of bone. These include pyridinoline, deoxypyridinoline, urinary deoxypyridinoline (urinary DPD), N- telopeptides (NTX) and C-telopeptides (CTX) of Type I collagen crosslinks.

Earlier assays, such as total alkaline phosphatase and hydroxyproline, are still used in monitoring such metabolic bone diseases as Paget's disease. However, these tests are not sensitive enough to be used in monitoring the more subtle bone remodeling changes that tend to occur in osteoporosis, as levels tend to be within normal limits in individuals with the disease.

An additional unexpected result is the lack of occurrence of osteosarcoma formation with long term administration of the PTH peptides of the present invention. In its packaging, the prior art Forteo® includes a warning label that Forteo® caused an increase in incidence of osteosarcoma in rats. The label warns that Forteo® should not be prescribed for patients who are at increased baseline risk for osteosarcoma. In contrast, the risk of osteosarcoma occurrence with the long term use of the PTH peptides of the present invention is minimal. The present PTH peptides may have no, or less, incidence of osteosarcoma based on a different sequence and different signaling as compared to PTH (1-34). The phospholipase-C and downstream protein kinase C activity, which are minimized with administration of the PTH peptides of the present invention, may be involved in ostoeoblast growth.

Another unexpected result with the PTH peptides of the present invention is the lack of need to monitor serum calcium levels in patients taking these peptides for possible occurrences of hypercalcemia. Serum calcium levels in patients taking the prior art Forteo® is monitored through samples of blood and/or urine during the course of treatment- The Forteo® package insert warns that administration of Forteo® may "exacerbate hypercalcemia." Use of Forteo® is not recommended for patients with high amounts of calcium in their blood (hypercalcemia), bone cancer or other bone disorders. In contrast, administration of the PTH peptides of the present invention leads to lower incidences of hypercalcemia, as compared to administration of Forteo®. Accordingly, calcium monitoring may not be required with administration of the PTH peptides of the present invention. Another unexpected result with the peptides of the present invention is the ability to tailor the dose administered to a patient based on that patient's weight, body surface area, or BMI and presentation of symptoms. The weight, body surface area, or BMI cut off method provides a method for determining a therapeutically effective dosage while maintaining a low incidence of side effects for a patient based upon their weight, body surface area, or BMI. A dosage that results in high exposure for a particular patient will increase the chance of side effects, including hypercalcemia. Additionally, lack of efficacy may be observed in certain patients, especially men, because the dose for that particular patient was too low in a situation for a patient of a certain weight, body surface area, or BMI who could have tolerated a higher dosage of the therapeutic agent.

AU osteoporosis therapeutics with a predominant action to stimulate bone formation may be administered in a manner where the dosage is based on the weight, body surface area, or BMI of the patient. Examples of such therapeutics within the scope of the present invention include the anti-sclerostin Mab, inhibitors of negative regulators of the Wnt signaling pathways, and activin receptor agonists. Additionally dosage based on patient weight, body surface area, or BMI is effective for all therapeutics whose bone formation effect is mediated by the action of PTH on its receptor, including PTH, full-length and fragments thereof, PTH analogs, PTHrP, and PTHrP analogs. Specific PTH peptides which are effective with dosage based on patient weight, body surface area, or BMI include, but not limited to, full length PTH 1-84, PTH 1-34, PTH-(I -31)NH₂, Ostabolin; PTH-(I -3O)NH₂; PTH-(1-29)NH₂; PTH-(1-28)NH₂; Leu²⁷PTH-(l-31)NH₂; LeU²⁷PTH-(I -3O)NH₂; LeU²⁷PTH- (1-29)NH₂; Leu²⁷cyclo(22-26)PTH-( 1-31)NH₂ Ostabolin-C™; Leu²⁷cyclo(22-26)PTH-(1- 34)NH₂; Leu²⁷cyclo(Lys²⁶-Asp³⁰)PTH-(l-34)NH₂; Cyclo(Lys²⁷-Asp³⁰)PTH-(l-34)NH₂; Leu²⁷cyclo(22-26)PTH-(l-31)NH₂; Ala²⁷ or NIe²⁷ or Tyr²⁷ or He²⁷ cyclo(22-26)PTH-(l- 3I)NH₂; Leu²⁷cyclo(22-26)PTH-(l-32)NH₂; Leu²⁷cyclo(22-26)PTH-(l-31)OH; Leu²⁷cyclo(26-30)PTH-(l-31)NH₂; Cys²²Cys²⁶Leu²⁷cyclo(22-26)PTH-(l-31)NH₂; Cys²²Cys²⁶Leu²⁷cyclo(26-30)PTH-(l-31)NH₂; Cyclo(27-30)PTH-(l-31)NH₂; Leu²⁷cyclo(22- 26)PTH-(l-30)NH₂; Cyclo(22-26)PTH-(l-31)NH₂; Cyclo(22-26)PTH-(l-30)NH₂; Leu²⁷cyclo(22-26)PTH-(l-29)NH₂; Leu²⁷cyclo(22-26)PTH-(l-28)NH₂; Glu¹⁷,Leu²⁷cyclo(13- 17)(22-26)PTH-(l -28)NH₂ ; and Glu¹⁷,Leu²⁷cyclo( 13-17)(22-26)PTH-(l -31 )NH₂.

Additionally, suitable examples of therapeutics which can be administered based on the weight, body surface area, or BMI of the patient include calcium receptor antagonists which stimulate endogenous PTH production, such as those that act as agonists of the PTH receptor, including PTH, full-length and fragments thereof, PTH analogs, PTHrP and analogs thereof. The administration of a dosage based upon the weight, body surface area, or BMI of a patient can be used in a variety of indications, including osteoporosis, fracture repair, renal bone disease, corticosteroid-induced osteoporosis, transplant, and the induction of bone formation in trabecular and cortical bone.

By evaluating the efficacy of a range of doses of the peptides of the present invention, the low doses can be eliminated to reduce the number of non-responders and the high doses, that normally raise safety concerns, can also be eliminated. The choice of multiple effective dosages improves the benefit to risk profile of the present peptides by improving the overall efficacy and proportion of patients who respond to the dosage while reducing the side effects of a dose that results in high exposure for an individual.

Improved PK Profile

Administration of the PTH analogs of the present invention has led to a PK profile of shorter duration (sharper peak) as compared to previously known PK profiles. This shorter PK profile results in maintaining the positive effects of PTH treatment, while simultaneously reducing side effects. Suitable PK parameters within the scope of the present invention include a half life of the PTH peptide analogue of between 2 minutes and 60 minutes; a duration of exposure to the PTH peptide analogue of between 30 minutes and 4 hours; a Tmax of the PTH peptide analogue of between 2 minutes and 30 minutes; and a Cmax of the PTH peptide analogue of between 10 and 400 pg/ml. More preferred PK ranges include a half-life of between 15-30 minutes, a duration of exposure between one and 2 hours, a Tmax of between 15-30 minutes, and a Cmax of between 50-200 pg/ml.

Details of the pharmacokinetics within the scope of the present invention are shown in the figures, both for human and animal administration. For the PTH administered to humans, Ostabolin-C was administered in a liquid formulation with a buffer, a polyol, and a stabilizer, with a pH of between 3 and 5. Alternative embodiments of PTHs can also be administered which result in a similar pharmacokinetic profile. For the PTH administered to animals, Ostabolin-C was formulated in acidified saline and adjusted with phosphates to a pH of 7.2 +/- 0.4. The pharmacokinetics using the above-described Ostabolin-C PTH formulation is shown in figures 18-20.

Pharmaceutical Compositions/Formulations, Dosing, and Administration

A range of PTH peptide analogue compounds can be used in the methods and compositions of the present invention. Generally, preferred embodiments of PTH peptide analogues include those that when administered result in reduced phospholipase-C activity, reduced bone resorption, and reduced hypercalcemia levels. As defined in the Definitions section herein, "reduced phospholipase-C activity" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less than full activation of phospholipase-C, as compared to the full-length PTH peptide or other PTH peptide analogues which are at least 34 amino acid residues; "reduced bone resorption" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less bone resorption, as compared to the full-length PTH peptide or other PTH peptide analogues which are at least 34 amino acid residues, and "reduced hypercalcemia levels" refers to a PTH peptide analogue that has been truncated or modified in some manner so as to trigger less incidences of hypercalcemia, or lower severity of hypercalcemia, as compared to the full- length PTH peptide or other PTH peptide analogues which are at least 34 amino acid residues.

The preferred PTH analogues administered in the methods described herein include [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l-31)-NH₂, such as advanced by Zelos Therapeutics, Inc. under the tradename OSTABOLIN-C™ and PTH-(I -31)-NH₂, such as advanced by Zelos Therapeutics, Inc. under the tradename OSTABOLIN™. In another embodiment of the invention, [Leu²⁷]cyclo[Glu²²-Lys²⁶]-PTH-(l -3O)-NH₂ is used in the methods described herein. In another embodiment, the hormone can be the linear analogue PTH(I -31), which can have a free carboxyl ending, or be amidated, at the C-terminus. In yet another embodiment, the hormone can be PTH(I -30) , which can have a free carboxyl ending, or be amidated, at the C-terminus; or [Leu²⁷]-PTH(l-30)- NH₂. Suitable stabilized solutions of the PTH peptide analogues that can be employed in the present methods are described in U.S. Patent Nos. 5,556,940; 5,955,425; 6,541,450; 6,316, 410; and 6,110,892 incorporated herein by reference.

Dosages

An effective amount of a PTH peptide analogue for use in the present invention is an amount that will provide the desired benefit or therapeutic effect upon administration according to the prescribed regimen. Effective dosages can vary according to the type of formulation of PTH peptides or analogs administered as well as the route of administration. One skilled in the art can adjust the dosage by changing the route of administration or formulation, so that the dosage administered would result in a similar pharmacokinetic or biological profile as result from the preferred dosage ranges described herein. Exemplary dosages include a daily dose of 2 to 100 μg for subcutaneous delivery of an aqueous formulation, a daily dose of 0.5 to 50 μg for subcutaneous delivery of a formulation stabilized with propylene glycol and/or ethanol, a daily dose of 100 to 3,000 μg for inhalation delivery, and weekly doses at 3-7 times the daily doses. Other suitable dosages include any dosage with any route of administration that results in a bioavailability or pharmacokinetic profile similar to those yielded by the above-described dosage ranges.

Nonlimiting examples of an effective amount of PTH analog administered subcutaneously in an aqueous formulation may range from about 2 μg/day to about 100 μg/day, preferably from about 5 μg/day to about 45 μg/day, more preferably from about 7.5 μg/day to about 20 μg/day, more preferably from about 20 μg/day to about 30 μg/day, more preferably from about 30 μg/day to about 45 μg/day, and more preferably 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μg/day. Additional preferred dosages include dosages of 5, 6, 7, 7.5, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 μg/day. Additional examples of an effective amount of PTH analogue administered subcutaneously in an aqueous formulation may range from about 14 μg/week to about 420 μg/week, preferably from about 35 μg/week to about 280 μg/week, more preferably from about 70 μg/week to about 140 μg/week, more preferably from about 140 μg/week to about 210 μg/week, and more preferably 35, 70, 105, 140, 175, 205, or 245 μg/week. The dosages can be administered every day, every two days, every three days, every four days, every five days, every six days, or every seven days (once/week). These dosages can also be adjusted to correct for bioavailability. The doses can also be measured in mmol, taking into account the molecular weight of the PTH peptides used.

The dosage can also be calculated based on the size of the patient. The μg dosages can be normalized for patient characteristics such as height, weight, body surface area, BMI, lean body mass, etc., by converting the μg to μg/kg, μg/m2, or μg /ml, or other suitable conversions known in the art. In suitable embodiments, dosages can also be administered subcutaneously on a μg/kg basis. This calculation is performed as follows using 30 μg and 45 μg as exemplary doses. Based on the assumption that the average human subject weighs about 65 kg, the 30 μg and 45 μg doses are converted to 0.46 μg/kg and 0.69 μg/kg. To convert μg to μg/kg, the μg dose is divided by 65 kg, to give a μg/kg dose (dose/weight = μg/kg). For a dose of 30 μg, 30 μg per 65 kg average human weight gives a μg/kg dose of about 0.46 μg/kg. For 45 μg, 45 μg per 65 kg average human weight gives a μg/kg dose of about 0.69 μg/kg. Dosages within the scope of the present invention for subcutaneous delivery of an aqueous formulation include from 0.20-0.90 μg/kg, more preferably 0.30-0.70 μg/kg, and still more preferably 0.46-0.69 μg/kg. Dosages within the preferred ranges maximize the effectiveness of PTH therapy while simultaneously reducing side effects.

For inhalation therapy, the PTH peptide or analogue can be administered at doses between 100 μg and 3,000 μg per day. More specifically, the PTH peptide or analogue can be administered at doses of 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 700 μg, 800 μg, 900 μg, 1000 μg, 1100 μg, 1200 μg, 1300 μg, 1400 μg, 1500 μg, 1600 μg, 1700 μg, 1800 μg, 1900 μg, 2000 μg, 2100 μg, 2200 μg, 2300 μg, 2400 μg, 2500 μg, 2600 μg, 2700 μg, 2800 μg, 2900 μg, or 3000 μg per day. Inhalation therapy can also be administered weekly at 3 to 7 times greater than the daily dose.

A Phase I clinical study was undertaken using Ostabolin-C™ Inhalation Powder (OCIP) to establish an MTD in post-menopausal women, to compare its PK profile with Phase II sub-cutaneous doses, and to evaluate biological activity with cAMP and biomarkers of bone turnover. Preferred doses of 300 μg and 800 μg resulted in a PK profile similar to subcutaneous administration. This pk profile included a half life of the PTH peptide analogue of between 2 minutes and 60 minutes; a duration of exposure to the PTH peptide analogue of between 30 minutes and 4 hours; a Tmax of the PTH peptide analogue of between 2 minutes and 30 minutes; and a Cmax of the PTH peptide analogue of between 10 and 400 pg/ml. More preferred PK ranges include a half-life of between 15-30 minutes, a duration of exposure between one and 2 hours, a Tmax of between 15-30 minutes, and a Cmax of between 50-200 pg/ml. Details of inhalation dosing is shown in Example 14.

Dosages can also be administered in varying amounts based on the presentation of symptoms and the weight, body surface area, or BMI of the subject. Such dose optimization is discussed in detail below.

Dose Optimization

Dose optimization is important for all drugs, especially for those with a narrow therapeutic window. Hormones in general, including PTH and its analogs, are such drugs which have a narrow therapeutic window. Because of this narrow therapeutic window, a standardized single dose for all patients presenting with a variety of symptoms may not always be effective. Our dose optimization approach of two separate doses, 30 μg and 45 μg, for patients with different symptoms and different weights, body surface area, or BMI, addresses this problem.

Optimizing the dose for the administration of PTH analogs to human patients with an appropriate risk/benefit ratio has proven difficult with prior art PTHs. The administration and potential commercialization of PTH analogs has been analyzed by many companies, with the most effective and least toxic dose being difficult to obtain. Two such companies who have studied PTH dosing are Lilly (with Forteo 1-34 hPTH) and NPS (Preos - recombinant full- length human parathyroid hormone, rhPTH 1-84).

Dose optimization of the PTH peptides of the present invention provides a benefit over the single dose currently available with Forteo. Lilly's Forteo has only been approved for a 20 μg dose. Although Lilly studied the possibility of administering doses of both 20 and 40 μg, only the 20 μg dose was approved. The risk/benefit ratio of the 40 μg dose was too great, as hypercalcemia was seen at least once in 28% of the subjects administered 40 μg of Forteo. Accordingly, the 20 μg dose of Forteo (hPTH 1-34) is the only dose available for all symptoms of all patients. There are some patients for whom a 20 μg dose will not give enough benefit. Additionally, in a Summary Basis of Approval for Forteo, the FDA indicated that the correct dose for men should be 30 μg. Our dose optimization approach of two distinct dosages, 30 μg and 45 μg, will overcome both the risks of Forteo's 40 μg dose and the lack of efficacy in certain patients with Forteo's 20 μg dose.

Additionally, NPS has studied a variety of doses for its recombinant full-length human parathyroid hormone, rhPTH 1-84, Preos. In Phase 2 clinical trials, NPS analyzed does of 50, 75, and 100 μg/day. In Phase 3 clinical trials, the dose administered was limited to a single dose of 100 μg. This dose has not yet been approved due to high incidence levels of hypercalcemia.

The presently described dose/weight cutoff will help avoid the prior art problems of the risks of PTH administration of a single dose outweighing the benefits achieved. The dosages can thus be administered based on the weight, body surface area, or BMI of a subject. As shown in the data below, the greatest benefits, with the least side effects, are obtained when Ostabolin-C is administered at a dose of about 30 μg for a patient below the weight cutoff and at a dose of about 45 μg for a patient above the weight cutoff. These same benefits of dose/weight cutoff occur with PTH analogs described herein in addition to Ostabolin-C, and other therapeutics, including the anti-sclerostin Mab, inhibitors of negative regulators of the Wnt signaling pathways, activin receptor agonists, therapeutics whose bone formation effect is mediated by the action of PTH on its receptor, including PTH, full-length and fragments thereof, PTH analogs, PTHrP, and PTHrP analogs, and calcium receptor antagonists which stimulate endogenous PTH production, such as those that act as agonists of the PTH receptor, including PTH, full-length and fragments thereof, PTH analogs, PTHrP and analogs thereof. The 4 month and 12 month Phase π data from the Ostabolin-C sub-cutaneous program, detailed in the Examples herein, indicate that a daily dose of 30-45 μg has an appropriate risk to benefit profile for the treatment of osteoporosis. The 30 μg dose demonstrates clinically beneficial increases in lumbar spine BMD with a low incidence of hypercalcemia and reduced incidence of side effects. The 45 μg dose causes larger increases in lumbar spine BMD and also produces an increase in BMD of the hip. It is therefore of interest to explore whether dose optimization could capture some or all of the upside efficacy benefits of the high dose without incurring the increased side effects that are also observed at that dose. This analysis is described below.

At the 30 μg dose, lumbar spine BMD increases, mid-radius BMD increases, the percent incidences of hypercalcemia is relatively low, and the increase in bone formation versus bone resorption is great, as demonstrated by the anabolic window. The advantages of this 30 μg dose include early and large increase in bone formation with evidence of reduced bone resorption relative to Forteo. The PTHs within the scope of the present invention which have a shorter duration PK profile, provide a benefit similar to that seen with Forteo in the positive BMD change at lumbar spine and hip, but the present invention reduces the side effects with a reduced potential for cortical bone loss. The present invention also exhibits a decreased propensity to cause hypercalcemia. Data for the administration of 30 and 45 μg doses of Ostabolin-C is shown in figures 21 and 22 .

Administration of PTH analogs at 45 μg dosages also provides additional benefits. These same benefits occur with PTH analogs described herein in addition to Ostabolin-C. This 45 μg dose has superior efficacy, but with the emergence of side effects. Administration of a 45 μg dose of Ostabolin-C results in strong early BMD change at hip or lumbar spine allied with powerful bone formation effects. This positive effect is also combined with some evidence of bone resorption stimulation and hypercalcemia. These results are shown in figure 22.

Based on the effects demonstrated above with dosages of 30 and 45 μg, it is evident that dose optimization is important in order to obtain the benefits while simultaneously minimizing adverse effects. One method of optimizing dose is to administer a dose which results in a shorter PK profile. Suitable PK parameters within the scope of the present invention include a half life of the PTH peptide analogue of between 2 minutes and 60 minutes; a duration of exposure to the PTH peptide analogue of between 30 minutes and 4 hours; a Tmax of the PTH peptide analogue of between 2 minutes and 30 minutes; and a

Cmax of the PTH peptide analogue of between 10 and 400 pg/ml. More preferred PK ranges include a half-life of between 15-30 minutes, a duration of exposure between one and two hours, a Tmax of between 15-30 minutes, and a Cmax of between 50-200 pg/ml.

Another method of optimizing dose is to administer different dosages, depending on the weight, height, body surface area, or BMI of the subject. As shown by the data herein, the high dose of 45 μg has efficacy greater than previously seen with Forteo, particularly at the hip. But this high dose also presents a risk factor of hypercalcemia for certain patients. The low dose of 30 μg has efficacy at least as good as has been seen with Forteo, and does not present a risk factor for hypercalcemia. Neither of the 30 or 45 μg doses are optimal for all patients. The dose optimization presented herein provides a dosing regimen that combines the positive features of both the 30 and 45 μg doses, namely superior efficacy with low risks of adverse effects. This dosing regimen provides for different doses based on the patient's body characteristics, including weight, height, BMI, lean body mass, or body surface area.

In this way, the potential for non response in a subject is mitigated by avoidance of low dose in large individuals and the potential of adverse effects in a subject is mitigated by avoidance of high doses in small individuals. Dosages within the scope of the present invention include administering 45 μg to a subject weighing 50 kg or more, more specifically 65 kg or more, more specifically 68 kg or more, more specifically 70 kg or more and administering 30 μg to a subject weighing less than 90 kg, more specifically less than 75 kg, more specifically less than 68 kg, more specifically less than 65 kg, more specifically less than 59 kg, and more specifically less than 50 kg.

This dose/weight cutoff optimization provides the ability to tailor the dose administered to a patient based on that patient's weight, body surface area, or BMI and presentation of symptoms. The weight cut off method provides a method for determining a therapeutically effective dosage while maintaining a low incidence of side effects for a patient based upon their weight, body surface area, or BMI. A dosage that results in high exposure for a particular patient will increase the chance of side effects, including hypercalcemia. Additionally, lack of efficacy may be observed in certain patients, because the dose for that particular patient was too low in a situation for a patient of a certain weight, body surface area, or BMI who could have tolerated a higher dosage of the therapeutic agent.

By evaluating the efficacy of a ranges of doses, the low doses can be eliminated to reduce the number of non-responders and the high doses, that normally raise safety concerns, can also be eliminated. The weight, body surface area, or BMI cutoff limits the dosage to be administered to a patient to either the high or low dose. The choice of multiple effective dosages improves the benefit to risk profile of the present peptides by improving the overall efficacy and proportion of patients who respond to the dosage while reducing the side effects of a dose that results in high exposure for an individual.

To explore the dosing range between 30 and 45 μg/day, response to Ostabolin-C was analyzed based on individual exposure to drug, with the doses converted to μg/kg (daily dose divided by weight in kg x 100 (for avoidance of decimals)). All patients who received active treatment were converted to an individual dose exposure and then regrouped into ascending exposure dose cohorts of μg/kg (0-15, 16-25, 26-35, 36-45, 46-55, >56 μg/kg). The mean changes for primary and secondary endpoints after 4 months of treatment in these new dose exposure cohorts were calculated. A linear regression analysis enabled the effect of exposure to Ostabolin-C to be assessed across the full range of dose exposures. The group means are presented as a linear regression analysis with 95% CI for continuous variables. The data are shown in the figures for effects on the lumbar spine BMD (figures 23-24), femoral neck BMD (figure 29), forearm midshaft radius (figure 30), total hip (figure 27, 53- 54), serum calcium (figure 28), hypercalcemia (figures 31-32), and bone formation and bone resorption markers (figure 26). The results indicate that all of the biomarker and BMD endpoints and serum calcium (all continuous variables) change in a linear pattern across the entire dose range tested in this study.

Based on the assumption that the average human subject weighs about 65 kg, the 30 μg and 45 μg doses were converted to 0.46 μg/kg and 0.69 μg/kg. To convert μg to μg/kg, the μg dose is divided by 65 kg, to give a μg/kg dose (dose/weight = μg/kg). For a dose of 30 μg, 30 μg per 65 kg average human weight gives a μg/kg dose of about 0.46 μg/kg. For 45 μg, 45 μg per 65 kg average human weight gives a μg/kg dose of about 0.69 μg/kg. These doses are demonstrated in the figures. Dosages within the scope of the present invention include from 0.20-0.90 μg/kg, more preferably 0.30-0.70 μg/kg, and still more preferably 0.46-0.69 μg/kg. Dosages within the preferred ranges maximize the effectiveness of PTH therapy while simultaneously reducing side effects.

One method of reducing the range of dose exposures in a study population is to utilize two dose strengths with a single weight cutoff (i.e. all patients who weigh less than the cutoff receive the low dose (30 μg), whereas all those above the weight cutoff receive the high dose (45 μg). The impact of this dose optimization strategy on Ostabolin-C exposure is illustrated in figures 33-36.

The effect of a weight cutoff at 68 kg reduces the spread of exposures in the 30 and 45 μg dose groups. This is shown in detail in the examples. Comparison of modeled data for three different BMD parameters illustrates that progressively higher dose exposures are required to affect total hip and femoral neck BMD compared to lumbar spine BMD. This will enable therapy to be tailored to the individual needs of the patient. For lumbar spine BMD, the proposed weight cutoff is 68kg, so that patients with lumbar spine BMD who weigh less than 68 kg would be administered the 30 μg dose and patients with lumbar spine BMD weighing 68 kg or more would be administered the 45 μg dose. For hip and femoral neck, the proposed cutoff weight is lower than 68 kg, since higher doses are required to have a similar positive effect.

Plasma Concentration - AUC

The dose may also be selected to provide an effective plasma concentration of PTH analogue or other osteoporosis therapeutic. Examples of an effective maximum plasma concentration of peptide concentration may range from about 10 pg/mL to about 400 pg/mL, preferably from about 20 pg/mL to about 300 pg/mL; from about 50 pg/mL to about 280 pg/mL; from about 80 pg/mL to about 250 pg/mL; from about 100 pg/mL to about 150 pg/mL. Other suitable dosage ranges for maximum plasma concentration of PTH peptide analogues include 20-40 pg/mL, 40-60 pg/mL, 60-80 pg/mL, 80-100 pg/mL, 100-120 pg/mL, 120-140 pg/mL, 140-160 pg/mL, 160-180 pg/mL, 180-200 pg/mL, 200-230 pg/mL, 230-260 pg/mL, 260-300 pg/mL, 300-350 pg/mL, and 350-400 pg/mL.

In another specific embodiment of the invention, the peptide is administered in an effective amount that results in the value for area under the curve (herein referred to as "AUC") in the plasma peptide concentration versus time curve in the range of 5 pg-h/mL — 400 pg-h/mL. More preferably, the range of AUC is between 10 pg-h/mL — 350 pg-h/mL. More preferably, AUC is in the range of 20 pg-h/mL - 300 pg h/mL. More preferably, AUC is in the range of 50 pg h/mL- 250 pg-h/mL. More preferably, AUC is in the range of 70 pg-h/mL — 200 pg-h/mL. More preferably, AUC is in the range of 90 pg-h/mL — 150 pg-h/mL. Even more preferably, AUC is in the range of 95 pg h/mL - 125 pg h/mL. Other suitable range for AUC is 5 pg h/mL - 20 pg h/mL, 20 pg h/mL - 50 pg h/mL, 50 pg h/mL - 70 pg h/mL, 70 pg h/mL - 90 pg h/mL, 90 pg h/mL - 100 pg h/mL, 100 pg-h/mL - 110 pg-h/mL, 110 pg h/mL - 120 pg h/mL, 120 pg-h/mL - 130 pg h/mL, 130 pg h/mL - 150 pg h/mL, 150 pg h/mL - 175 pg h/mL, 175 pg-h/mL - 200 pg h/mL, 200 pg h/mL -225 pg h/mL, 225 pgh/mL - 250 pg h/mL, 250 pg-h/mL -275 pg h/mL, 275 pg h/mL - 300 pg h/mL, 300 -350 pg h/mL, or 350 pg-h/mL - 400 pg h/mL. Accordingly, in one aspect, the invention provides a pharmaceutical formulation comprising a therapeutically effective amount of a PTH peptide analogue as the active ingredient in a daily dosage range of 2 μg to 60 μg or a weekly dosage range of 14 μg to 420 μg, wherein the PTH peptide analogue has reduced phospholipase-C activity and maintains adenylate cyclase activity, in admixture with a pharmaceutically acceptable excipient, diluent, or carrier, or combinations thereof. Effective dosages can vary according to the type of formulation of PTH peptides or analogs administered as well as the route of administration. One skilled in the art can adjust the dosage by changing the route of administration or formulation, so that the dosage administered would result in a similar pharmacokinetic or biological profile as result from the preferred dosage ranges described herein. Exemplary dosages include a daily dose of 2 to 100 μg for subcutaneous delivery of an aqueous formulation, a daily dose of 0.5 to 50 μg for subcutaneous delivery of a formulation stabilized with propylene glycol and/or ethanol, a daily dose of 100 to 3,000 μg for inhalation delivery, and weekly doses at 3-7 times the daily doses. Other suitable dosages include any dosage with any route of administration that results in a bioavailability or pharmacokinetic profile similar to those yielded by the above-described dosage ranges.

Routes of Administration

Administration of the PTH peptide analogues of the present invention includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug and/or provides a patient with a prescription for a drug is administering the drug to the patient.

A variety of administration routes can be used in accordance with the present invention, including oral, topical, transdermal, nasal, pulmonary, transpercutaneous (wherein the skin has been broken either by mechanical or energy means), rectal, buccal, vaginal, via an implanted reservoir, or parenteral. Parenteral includes subcutaneous, intravenous, intramuscular, intraperitoneal, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. More preferably, the route of administration is subcutaneous, transcutaneous, intranasal, transdermal, oral, or inhalation administration.

Formulations A stabilized solution of a parathyroid hormone can include a stabilizing agent, a buffering agent, a preservative, an antibacterial agent and the like. The stabilizing agent incorporated into the solution or composition includes alcohols, ethanol or a polyol which includes a saccharide, preferably a monosaccharide or disaccharide, e.g., glucose, trehalose, raffϊnose, or sucrose; a sugar alcohol such as, for example, mannitol, sorbitol or inositol, and a polyhydric alcohol such as glycerine or propylene glycol or mixtures thereof. A preferred polyol is mannitol or propylene glycol. The concentration of polyol may range from about 1 to about 20 wt-%, preferably about 3 to 10 wt-% of the total solution.

The buffering agent employed in the solution or composition of the present invention may be any acid or salt combination which is pharmaceutically acceptable. Useful buffering systems are, for example, acetate, tartrate or citrate sources. Preferred buffer systems are acetate or tartrate sources, most preferred is an acetate source. The concentration of buffer may be in the range of about 2 mM to about 500 mM, preferably about 2 mM to 100 mM.

The stabilized solution or composition of the present invention may also include a parenterally acceptable preservative. Such preservatives include, for example, cresols, benzyl alcohol, phenol, benzalkonium chloride, benzethonium chloride, chlorobutanol, phenylethyl alcohol, methyl paraben, propyl paraben, thimerosal and phenylmercuric nitrate and acetate. A preferred preservative is m-cresol or benzyl alcohol; most preferred is m-cresol. The amount of preservative employed may range from about 0.1 to about 2 wt-%, preferably about 0.3 to about 1.0 wt-% of the total solution.

The parathyroid hormone compositions can, if desired, be provided in a powder form containing not more than 2% water by weight, that results from the freeze- drying of a sterile, aqueous hormone solution prepared by mixing the selected parathyroid hormone, a buffering agent and a stabilizing agent as above described. Especially useful as a buffering agent when preparing lyophilized powders is a tartrate source. Particularly useful stabilizing agents include glycine, sucrose, trehalose and raffϊnose.

Ready to use formulations containing hPTH, or more specifically, Ostabolin-C, are not stable at room temperature and must be stored under refrigerated conditions (2-8⁰C). Since hPTH undergoes hydrolysis, oxidation and deamidation in aqueous media, it is difficult to develop a solution formulation for room temperature storage. Although, the formulation is stable at 5 ⁰C, it is preferred that the formulation is stable at about pH 7.5, as the pH is closer to physiological pH. Studies have indicated that an Ostabolin-C solution is less stable at pH 7.5 compared to the ready-to-use formulation. Oxidation and deamidation both occur and takes place above pH 7.0. As such, a 100% aqueous formulation above pH 7 under refrigerated conditions may not be feasible. Hence mixtures of ethano I/water or propylene/water systems were used with the antioxidants methionine or lipoic acid to evaluate the stability of the formulations of this invention.

Another additive to help maintain the stability of an hPTH formulation is Methionine. Methionine has been shown to be a potential antioxidant and improve hPTH stability. Additionally, polyols have the potential to stabilize peptide and protein formulations and sucrose concentrations up to IM at pH 5.5 have been found to reduce the rate of both deamidation and oxidation of hPTH. hi addition, parathyroid hormones formulated with typical buffers and excipients employed in the art to stabilize and solubilize proteins for parenteral administration. Buffers also have an effect on stability. Previous models showed that for pHs above 7, TRIS buffer had a much lower deamidation rate constant than a corresponding phosphate buffer. Adding NaCl also has a positive effect of the formulation because of its physiological ionic strength. Art recognized pharmaceutical carriers and their formulations are described in Martin, "Remington's Pharmaceutical Sciences," 15th Ed.; Mack Publishing Co., Easton (1975). More details of stabilizing additives to a hPTH formulation are shown in Examples 15 and 16.

The PTH peptide analogue may also be formulated into a composition suitable for administration by any convenient route, e.g., orally (including sublingually), topically, transdermally (including percutaneous absorption of the composition through the skin, such as by patches, ointments, creams, gels, salves and the like), intranasally, rectally or inhaled as a dry powder, aerosol, or mist, for pulmonary delivery.

Such forms of the compounds of the invention may be administered by conventional means for creating aerosols or administering dry powder medications using devices such as for example, metered dose inhalers, nasal sprayers, dry powder inhaler, jet nebulizers, or ultrasonic nebulizers. Such devices optionally may include a mouthpiece fitted around an orifice. It should be understood, however, that the invention embraces all forms of administration which make the PTH peptide analogues systemically or locally available.

In addition to the usual meaning of administering the formulations described herein to any part, tissue or organ whose primary function is gas exchange with the external environment, for purposes of the present invention, "pulmonary" is also meant to include a tissue or cavity that is contingent to the respiratory tract, in particular, the sinuses. For pulmonary administration, an aerosol formulation containing the active agent, a manual pump spray, nebulizer or pressurized metered-dose inhaler as well as dry powder formulations are contemplated. Suitable formulations of this type can also include other agents, such as antistatic agents, to maintain the disclosed compounds as effective aerosols.

A drug delivery device for delivering aerosols comprises a suitable aerosol canister with a metering valve containing a pharmaceutical aerosol formulation as described and an actuator housing adapted to hold the canister and allow for drug delivery. The canister in the drug delivery device has a head space representing greater than about 15% of the total volume of the canister. Often, the polymer intended for pulmonary administration is dissolved, suspended or emulsified in a mixture of a solvent, surfactant and propellant. The mixture is maintained under pressure in a canister that has been sealed with a metering valve.

Orally administrable compositions may, if desired, contain one or more physiologically compatible carriers and/or excipients and may be solid or liquid. Intranasal administration to the subject includes administering a therapeutically effective amount of the PTH peptide analogue to the mucous membranes of the nasal passage or nasal cavity of the subject. Pharmaceutical compositions for nasal administration can include, for example, nasal spray, nasal drops, suspensions, gels, ointments, creams, or powders.

Pharmaceutically acceptable compositions of the peptide described herein can be used according to the method of the present invention. The pharmaceutical compositions described herein can optionally include one or more pharmaceutically acceptable excipients. Such pharmaceutically acceptable excipients are well known in the art and include, for example, salts (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica and magnesium trisilicate), surfactant(s), water-soluble polymers (such as polyvinyl pyrrolidone, cellulose based substances, polyethylene glycol, polyethylene glycol 400, polyacrylates, sodium carboxymethylcellulose, waxes and polyethylene-polyoxypropylene-block polymers), preservatives, antimicrobials, antioxidants, cryo-protectants, wetting agents, viscosity agents, tonicity modifying agents, levigating agents, absorption enhancers, penetration enhancers, pH modifying agents, muco-adhesive agents, coloring agents, flavoring agents, diluting agents, emulsifying agents, suspending agents, solvents, co-solvents, buffers (such as phosphates, glycine, sorbic acid, potassium sorbate and partial glyceride mixtures of saturated vegetable fatty acids), serum proteins (such as human serum albumin), ion exchangers, tacopherol polyethylene glycol 1000 succinate (TPGS) mygly oil, labrosol, labrofac, ethanol, fillers such as sugars, including lactose sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP), and combinations of these excipients. If desired, disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Further examples of such carriers or excipients include but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Examples of surfactants suitable for use in the formulations of the present invention include, but are not limited to, cholic acid and salts of cholic acid, deoxycholic acid and salts of deoxycholic acid, taurocholic acid and salts of taurocholic acid, polyvinylpyrrolidone, PEG compounds such as cocamines, glyceryl stearates, glyceryl oleates, hydrogenated lanolins, lanolins, laurates and oleates, sorbitan laurates, sorbitan palmitates, sorbitan stearates, quatemium surfactants, sodium sulfates, glyceryl compounds, palmitic acid and its derivatives and oleic acid and its derivatives.

The excipient included within the pharmaceutical compositions of the invention is chosen based on the expected route of administration of the composition in therapeutic applications. Accordingly, compositions designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example, with an inert diluent or with an edible carrier. The compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the pharmaceutical compositions of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums and the like.

Solid dosage forms, such as tablets, pills and capsules, may also contain one or more binding agents, filling agents, suspending agents, disintegrating agents, lubricants, sweetening agents, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art. Examples of filling agents are lactose monohydrate, lactose anhydrous, and various starches. Examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, and silicifϊzed microcrystalline cellulose (SMCC). Suitable lubricants, including agents that act on the flowability of the powder to be compressed, are colloidal silicon dioxide, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel. Examples of sweeteners are any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and accsulfame K. Examples of flavoring agents are bubble gum flavor, fruit flavors, and the like. Examples of preservatives are potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quarternary compounds such as benzalkonium chloride. Suitable diluents include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and/or mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, lactose such as lactose monohydrate, lactose anhydrous, dibasic calcium phosphate, mannitol, starch, sorbitol, sucrose and glucose. Suitable disintegrants include corn starch, potato starch, and modified starches, crosspovidone, sodium starch glycolate, and mixtures thereof. Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts. Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the acid component of the effervescent couple may be present.

Various other materials may be present as coatings or to modify the physical form of the dosage unit. For instance, tablets may be coated with shellac, sugar or both. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propyl parabens as preservatives, a dye and a flavoring such as cherry or orange flavor, and the like.

The compositions may take any convenient form including, for example, tablets, coated tablets, capsules, lozenges, aqueous or oily suspensions, solutions, emulsions, syrups, elixirs and dry products suitable for reconstitution with water or another suitable liquid vehicle before use. The compositions may advantageously be prepared in dosage unit form. Tablets and capsules according to the invention may, if desired, contain conventional ingredients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth or polyvinyl-pyrollidone; fillers, for example lactose, sugar, maize-starch, calcium phosphate, sorbitol or glycine; lubricants, for example magnesium stearate, talc, polyethylene glycol or silica; disintegrants, for example potato starch; or acceptable wetting agents such as sodium lauryl sulphate. Tablets may be coated according to methods well known in the art.

Liquid compositions may contain conventional additives such as suspending agents, for example sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxymethylcellulose, carboxymethylcellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate or acacia; nonaqueous vehicles, which may include edible oils, for example vegetable oils such as arachis oil, almond oil, fractionated coconut oil, fish-liver oils, oily esters such as polysorbate 80, propylene glycol, or ethyl alcohol; and preservatives, for example methyl or propyl p- hydroxybenzoates or sorbic acid. Liquid compositions may conveniently be encapsulated in, for example, gelatin to give a product in dosage unit form.

Formulations for oral delivery may be formulated in a delayed release formulation such that the PTH peptide analogue is delivered to the large intestine. Delayed release formulations are well known in the art and include for example, delayed release capsules or time pills, osmotic delivery capsules etc.

Compositions for parenteral administration may be formulated using an injectable liquid carrier such as sterile pyrogen-free water, sterile peroxide-free ethyl oleate, dehydrated alcohol or propylene glycol or a dehydrated alcohol/propylene glycol mixture, and may be injected intravenously, intraperitoneally, subcutaneously or intramuscularly. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Compositions for rectal administration may be formulated using a conventional suppository base such as cocoa butter or another glyceride.

Compositions for topical administration include ointments, creams, gels, lotions, shampoos, paints, powders (including spray powders), pessaries, tampons, sprays, dips, aerosols, pour-ons and drops. The active ingredient may, for example, be formulated in a hydrophilic or hydrophobic base as appropriate.

It may be advantageous to incorporate an antioxidant, for example ascorbic acid, butylated hydroxyanisole or hydroquinone in the compositions of the invention to enhance their storage life. The pharmacokinetic profiles of various formulations containing Ostabolin-C are detailed in Example 17. Dosing Regimen

Administration in this invention may consist of one or more cycles; during these cycles one or more periods of osteoclastic and osteoblastic activity will occur, as well as one or more periods when there is neither osteoclastic nor osteoblastic activity. Alternatively, administration may be conducted in an uninterrupted regimen; such a regimen may be a long term regimen, e.g., a permanent regimen.

It will be understood that the dosages of compositions and the duration of administration according to the invention will vary depending on the requirements of the particular subject. The precise dosage regime will be determined by the attending physician or veterinary surgeon who will, inter alia, consider factors such as body weight, age and symptoms (if any). The compositions may if desired incorporate one or more further active ingredients.

During the dosing regimen, the hormone can be administered regularly {e.g., once or more each day or week), intermittently (e.g., irregularly during a day or week), or cyclically (e.g., regularly for a period of days or weeks followed by a period without administration). Regular administration can include once daily, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days (once/week). Preferably PTH is administered once daily for 1-7 days for a period ranging from 3 months for up to 3 years in osteoporotic patients, hi additional embodiments, PTH is administered for no less than 8 days. The present invention also encompasses embodiments wherein PTH is administered on a weekly basis.

Preferably, cyclic administration includes administering a parathyroid hormone for at least 2 bone remodeling cycles and withdrawing parathyroid hormone for at least 1 bone remodeling cycle. Another preferred regime of cyclic administration includes administering the parathyroid hormone for at least about 12 to about 24 months and withdrawing parathyroid hormone for at least 6 months. Typically, the benefits of administration of a parathyroid hormone persist after a period of administration. The benefits of several months of administration can persist for as much as a year or two, or more, without additional administration.

If desired, the PTH peptide analogue compound may be administered simultaneously or sequentially with other active ingredients, e.g., bone enhancing agents. These active ingredients may, for example include other medicaments or compositions capable of interacting with the bone remodelling cycle and/or which are of use in fracture repair. Such medicaments or compositions may, for example, be those of use in the treatment of osteoarthritis or osteoporosis as discussed above.

In yet a further aspect, the invention provides a method of treatment or prevention of bone-related diseases, in particular osteoporosis, which comprises administering to a mammal, including humans, in need of such treatment (a) an effective amount of PTH peptide analogues during a period of approximately 6 to 24 months; and (b) after the administration of PTH has been terminated, an effective amount of a bone resorption inhibitor during a period of approximately 12 to 36 months. The bone resorption inhibitor can be a bisphosphonate, e.g. alendronate; or a substance with estrogen-like effect, e.g. estrogen; or a selective estrogen receptor modulator, e.g. raloxifene, tamoxifene, droloxifene, toremifene, idoxifene, or levormeloxifene; or a calcitonin-like substance, e.g. calcitonin; or a vitamin D analog; or a calcium salt.

As discussed above, high dose Ostabolin-C and other PTH analogs of the present invention give a marked early bone formation and BMD response but are associated with stimulated bone resorption that have the potential to decrease the rate of improvement in bone strength by reducing the level of bone mineralization and by increasing cortical porosity. Lower doses of Ostabolin-C and other PTH analogs also cause increases in bone formation at a lower level but are free of bone resorption stimulating activity. In order to obtain the early benefits of the higher dose therapy while simultaneously minimizing side effects, a suitable treatment regimen within the present invention is sequential therapy. One embodiment of such a treatment regimen starts treatment with a high dose of Ostabolin-C or suitable PTH analogs and then after a period of time which could be 1-12 months but preferably 3-9 months and most preferably 4-8 months converts to a lower dose which maintains bone formation at a lower level but does not allow stimulation of bone resorption. Sequential therapy could also start treatment with a low dose and then convert to a high dose. Such a dosing regimen should be superior to high dose and low dose therapy because it will allow continued bone formation and the full maturation of the early bone formation caused by the high dose treatment without degradation by stimulated bone resorption. It will also be superior to high dose therapy in that the incidence of safety and tolerability adverse events will be reduced. Such sequential therapy will thus be an effective therapy while simultaneously minimizing side effects.

Such sequential therapy can be administered in all doses disclosed herein. One suitable dosage regimen includes administering a first daily dose subcutaneously of an aqueous formulation in a dosage range of from 35 μg to 100 μg of a PTH peptide analogue to said human, and then after the termination of the first period of time administering for a second period of time a second dose of from 2 μg to 35 μg of a PTH peptide analogue to said human.

For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA.

Kits

The present invention also encompasses a kit including the present pharmaceutical compositions and to be used with the methods of the present invention. The kit can contain a vial, for example, which contains a formulation of the present invention and suitable carriers, either dried or in liquid form. The kit further includes instructions in the form of a label on the vial and/or in the form of an insert included in a box in which the vial is packaged, for the use and administration of the compounds. The instructions can also be printed on the box in which the vial is packaged. The instructions contain information such as sufficient dosage and administration information so as to allow a worker in the field to administer the drug. It is anticipated that a worker in the field encompasses any doctor, nurse, or technician who might administer the drug, or a patient who might self-administer the pharmaceutical composition.

In one embodiment the kit contains a medication delivery pen that houses a cartridge assembly containing a vial or cartridge that has the capability of holding about a 60 day supply of daily doses of the pharmaceutical compositions described herein. In additional embodiments, the pen has the capability of holding a 1, 2, 3, 4, 5, 6, 7, or 8 week supply of daily doses of the pharmaceutical compositions described herein. In preferred embodiments, the pen has the capability of holding a 2 or 4 week supply of daily doses of the pharmaceutical compositions described herein. Such a device provides ease of use for self- administration of the pharmaceutical compositions described herein.

In a further embodiment, the cartridge can contain a liquid dosage of the pharmaceutical composition, or a lyophilized dosage, which is reconstituted by the user prior to injection. Those of skill in the pharmaceutical arts will recognize that medication delivery pens, cartridge assemblies for holding a liquid or lyophilized pharmaceutical dosage formulation for same, and methods of lyophilizing and sealing an injectable composition are known in the art, as evidenced by U.S. Patent Nos. 5,334,162; 6,053,893; and 6,648,859 the teachings of which are incorporated herein by reference. The examples which follow are illustrative of the invention and are not intended to be limiting.

EXAMPLE 1 Synthesis and Purification of [Leu^lcyclolGIu²² -Lys^2δ]-hPTH-(l-31)-

NH₂

This peptide was synthesized and purified as described in U.S. Patent No. 5,955,425, the teachings of which are incorporated herein by reference, with Lys-Alloc and GIu-OAl 1 substituted at position 26 and 22, respectively. After the addition of Fmoc-Ser¹⁷, the peptide- resin was removed from the column to a reaction vial (Minivial, Applied Science), suspended in 1.7 ml of a solution of tetrakis(triphenylphosphine)palladium(0) (0.24 mmol), 5% acetic acid and 2.5% N-methylmorpholine (NMM) in dichloromethane (DCM) under argon, then shaken at 20° C for 6 hr to remove the allyl and alloc protecting groups (Sole, N. A. et al (1993) In Peptides: Chemistry, Structure, and Biology, Smith, J. And Hodges, R. (Eds), ESCOM pp. 93-94, incorporated herein by reference). The peptide resin was then washed with 0.5% diethyldithiocarbamate (DEDT), 0.5% NMM in DMF (50 ml), followed by DMF (50 ml) and DCM (50 ml). The peptide (0.06 mmol) was cyclized by shaking with 0.06 mmol of l-hydroxy-7-azabenzotriazole (HOAt)/0.12 mmol NMM in 2 ml DMF for 14 h at 20° C (Carpino, L. A. (1993) J. Am. Chem. Soc. 115, 4397-4398). The peptide-resin was filtered, then washed once with DMF, repacked into the column, and washed with DMF until bubbles were removed from the suspension. The remaining synthesis was carried out as the linear counterpart above except that the N-terminal Fmoc group was not removed. The Fmoc- peptide was cleaved from the resin with reagent K as described above. The HPLC was carried out as the linear counterpart above, with the Fmoc group removed prior to the final HPLC.

Other suitable stabilized solutions of the PTH peptide analogues that can be employed in the present methods can be synthesized and purified as described in U.S. Patent Nos. 5,556,940; 5,955,425; 6,541,450; 6,316, 410; and 6,110,892 the teachings of which are incorporated herein by reference.

EXAMPLE 2 [Leu²⁷]cyclo[Glu²² -Lys²⁶]-hPTH-(l-31)-NH₂ Promotes Growth in Both Trabecular and Cortical Bones in a Monkey Model

The peptide [Leu²⁷]cyclo[Glu²² -Lys²⁶]-hPTH-(l-31)-NH₂ Ostabolin-C ™was administered daily by subcutaneous injection to gonad-intact cynomolgus monkeys (4/sex/gτoup) at dose levels of 0, 2, 10 and 25 μg/kg for 52 weeks. Monkeys were 30 to 40 months of age (2.3-3.5 kg) at treatment start. Tibiae were retained for histomorphometry following labeling with calcein green 15 and 5 days prior to euthanasia. Bone mass, as measured by DXA (dual-energy x-ray absorptiometry) and QCT (quantitative computed tomography), was increased at the lumbar spine, femur and tibia. Changes in vertebral BMD (bone mineral density) translated into significant increases in bone strength. The peptide [Leu²⁷]cyclo[Glu²² -Lys²⁶]-hPTH-(l -31)-NH₂ substantially increased osseous accretion in the cancellous and endocortical bone compartments of the proximal tibia at all doses. Tibial cancellous bone volume increased by more than 50% in all the peptide [Leu²⁷]cyclo[Glu²² - Lys²⁶]-hPTH-(l -31)-NH₂ treated groups compared to controls and in the tibial mid-diaphysis, increases in cortical width and relative cortical area with concurrent decreases in medullary area were observed. Only minor increases in cortical porosity were observed at the two highest dose levels. The increase in bone mass appeared to be related to increases in bone formation and decreases in bone resorption as measured by a significant reduction in osteoclast surface. Increases in indices of bone formation were associated with decreases in indices of bone resorption (decreased bone resorption markers, decreased osteoclast surface area, minimal cortical porosity), consistent with the uncoupling of these events. This combination of anabolic and anti-catabolic actions may have significant therapeutic value in the treatment of osteoporosis.

Example 3 - Pre-Clinical Cortical Porosity Data

Comparative data regarding increase in cortical bone porosity in monkey subjects using Ostabolin C at a variety of doses and using the prior art PTHs 1-34 is shown below.

Example 4 - Pre-Clinical Toxicity Data

The below table demonstrates that the prior art PTH, 1-34, teriparatide, Forteo®, is more nephrotoxic than Ostabolin-C™, the difference possibly being linked to the different hypercalcemia states. As shown below, PTH-(I -34) induces a mineralizing nephropathy in monkeys and possibly rats. A NoAEL was not established for the monkey. Ostabolin-C™ was nephrotoxic only in monkeys and a NoAEL was established. Ostabolin-C™ is at least 4- fold safer than PTH-(l-34).

<0.5μg/kg

EXAMPLE 5 Clinical Study of OSTABOLIN-C™

A four month Phase II clinical study was undertaken to investigate the safety, tolerability and efficacy of Ostabolin-C™ in post-menopausal women with low bone mineral density (BMD). Comparative data from this study demonstrates that the use of Ostabolin- C™ has many advantages over the current therapy, use of 1-34 PTH, teriparatide, Forteo®. The clinical protocol is a 16-week phase II randomized, double-blind, placebo-controlled, parallel group, dose finding study to investigate the safety, tolerability and efficacy of Ostabolin-C™ in post-menopausal women with low bone mineral density (BMD). In this study, 261 patients underwent four months of daily dosing of placebo and four active groups. The active groups included daily administration of Ostabolin-C™ in doses of 7.5, 15, 30, and 45 μg. Ostabolin-C™ is formulated as a clear, colorless liquid provided in pre-filled syringes and injected subcutaneously (SC). Subjects self-administer SC 0.1 mL injections of their assigned dose of Ostabolin-C™ 7.5, 15, 30, and 45 μg or placebo daily for 16 weeks in rotating quadrants of the abdomen. The subjects were post-menopausal women (for at least 5 years) with moderate osteoporosis.

The key endpoints for the study include change in mean BMD at the lumbar spine, as assessed by dual energy x-ray absorptiometry (DEXA), and measured by change from the Baseline visit. The Baseline visit is the first visit of the patient, before undergoing any treatment. Secondary efficacy endpoints include the following, as measured by change from Baseline visit:

DEXA: Bone formation and resorption markers:

• Mean femoral neck BMD • Serum osteocalcin

• Mean trochanter BMD • Serum amino terminal pro-peptide of

• Mean total hip BMD type 1 pro-collagen (PlNP)

• Mean radial BMD (distal and • Bone specific alkaline phosphatase midshaft) (BSAP)

• Bone mineral content (BMC) • Serum C-telopeptide (CTx)

• Bone area • Serum N-telopeptide (NTx) Other measurements:

• Lateral thoracic, lumbar spine and left antero-posterior hip radiographs

• Height

EXAMPLE 6 Clinical Results - Effects of Low Dose (7.5, 15, and 30 μg) Ostabolin-C™ Administration of a daily dosage of 7.5, 15, and 30 μg of Ostabolin-C™ as described above in Example 5 demonstrates robust bone anabolic effects at multiple sites in the body, including the spine, the hip, and the wrist without the concomitant negative effects previously seen with the use of prior art PTHs. The unprecedented BMD increases at the mid-radius and the lower incidence and severity of hypercalcemia make these highly attractive doses.

As shown in Figure 1, administration of 7.5, 15, and 30 μg daily dosages of Ostabolin-C™ over a course of 15 weeks results in an increase in lumbar spine BMD. Figures 3, 4, and 5 demonstrate mild BMD increase in hip, femoral neck, and trochanter BMD following administration of Ostabolin-C™ for 15 weeks.

Figures 6 and 7 demonstrate that daily administration of 7.5, 15, and 30 μg of Ostabolin-C™ has an unexpectedly positive effect on cortical bone, specifically the wrist (the distal and mid-shaft radius). There were statistically significant effects at the mid-radius at daily dosages of 7.5, 25, and 30 μg with no negative effect of bone resorption. Historically, PTH has been known to increase bone resorption, which leads to increased cortical porosity, and decreased BMD in radius cortical bone. Neer et al., 2001. As described in Neer, the administration of prior art Forteo® PTH 1-34 led to a decrease in BMD (increased cortical porosity) in the distal and mid-shaft radius as compared to placebo. In contrast, the dosages and formulation of the present invention, namely administration of 7.5, 15, and 30 μg Ostabolin-C™, actually increases cortical BMD in the distal and mid-shaft radius as compared to both placebo and to teriparatide, Forteo®. This is an unprecedented finding, demonstrating a statistically significant difference from placebo for 3 active doses (7.5, 15, and 30 μg).. Figures 8-13 demonstrate the effect which the PTHs of the present invention have on bone formation and bone resorption markers. The bone formation markers include PlNP, osteocalcin, and BSAP and the bone resorption markers include NTx and CTx. As compared to placebo, the bone formation markers have a greater % change when Ostabolin- C™ is administered at 15 and 30 μg.

The bone resorption markers in Figures 11-13 demonstrate that although there is some increase in bone resorption following the administration of Ostabolin-C™, this increase is less than that which follows administration of the prior art teriparatide, Forteo® PTH.

Daily dosages of 7.5, 15, and 30 μg Ostabolin-C™ has also been shown to have a much lower incidence of hypercalcemia as compared to PTHs known in the art. Figure 14 demonstrates that there was no notable difference from placebo on the per cent of abnormal serum calcium for doses of Ostabolin-C™ up to and including 30 μg. In comparison, teriparatide, Forteo® is shown to have a much higher effect at similar doses. For patients receiving Forteo®, hypercalcemia was seen at least once in 11% of the 20 μg group subjects and in 28% of the 40 μg group subjects, as compared with 2% in the placebo group. Neer et al., 2001. The administration of low doses of the PTH peptides of the present invention (7.5, 15, and 30 μg) resulted in no significant increase in the incidences of hypercalcemia as compared to placebo. Hypercalcemia was seen at least once in 5% of the placebo group and in the group being administered 30 μg doses, resulting in no net increase. This is in comparison to the 11% seen with Forteo® administered at 20 μg.

Accordingly, the above results demonstrate that administration of Ostabolin-C™ at 7.5, 15, and 30 μg daily dosages provides many advantages over the administration of Forteo® at 20 μg. The unexpected results include increased cortical BMD in the distal and mid-shaft radius as compared to placebo, less bone resorption than prior art PTH, and lower incidence and severity of hypercalcemia, while maintaining anabolic bone growth as measured by increased BMD at a variety of sites, including spine and hip.

EXAMPLE 7 Pre-Clinical Results - Effects of High Dose (45 μg) Ostabolin-C™

Administration of a daily dosage of 45 μg of Ostabolin-C™ has demonstrated an unprecedented ability to build bone at different sites, including the spine and hip, with early onset of effect in combination with only a mild hypercalcemia signal. This is an improvement over the prior art teriparatide, Forteo® 1-34 PTH.

Figures 1 and 2 demonstrate that administration of 45 μg Ostabolin-C™ leads to an increase in BMD in the lumbar spine. Figure 2 shows the increase in lumbar spine BMD with administration of 20 and 40 μg Forteo®.

Figures 3, 4, and 5 and the table below demonstrate that a daily dosage of 45 μg Ostabolin-C™ has a positive effect on bone formation at the hip, femoral neck, and trochanter. This is an unprecedented finding, demonstrating a statistically significant and clinically meaningful benefit at 45 μg at 15 weeks. The table below demonstrates the change in hip, femoral neck, and trochanter BMD, comparing the administration of teriparatide, Forteo® (20 μg) over a course of at least 12 months versus Ostabolin-C™ (45 μg) at 15 weeks. As shown below, for hip and trochanter, administration of 45 μg Ostabolin-C™ achieved results in 15 weeks similar to the results obtained with administration of Forteo over a course of at least 12 months. Regarding femoral neck, Ostabolin-C™ shows a much greater increase in BMD in a shorter period of time.

Figures 8-13 demonstrate the effect which the PTHs of the present invention have on bone formation and bone resorption markers. The bone formation markers include PlNP, osteocalcin, and BSAP and the bone resorption markers include NTx and CTx. As compared to placebo, the bone formation markers have a greater % change when Ostabolin-C™ is administered at 45 μg. There is a robust effect in the increase in the bone formation markers when Ostabolin-C™ is administered at 45 μg. The bone resorption markers in Figures 11-13 demonstrate that although there is some increase in bone resorption following the administration of Ostabolin-C™, this increase is less than that which follows administration of the prior art teriparatide, Forteo® PTH.

Accordingly, the above results demonstrate that administration of Ostabolin-C™ at 45 μg daily dosages provides many advantages over the administration of rhPTH 1-34 teriparatide, Forteo® at 20 and 40 μg . The unexpected results include increased BMD in the spine and hip, with less bone resorption and lower incidences of hypercalcemia than prior art PTH.

EXAMPLE 8 Pharmacokinetic (PK) Evaluation of Ostabolin-C

The objective of this portion of the study was to evaluate the pharmacokinetics of Ostabolin-C under steady state conditions when given subcutaneously (sc) once a day to postmenopausal female subjects with low bone mineral density.

This study was a Phase π, multicenter, randomized, double-blind, placebo-controlled, parallel group dose-finding study in post-menopausal female subjects. After Screening procedures and a 2- week placebo run-in phase, subjects were to be dosed once a day for 16 weeks with either Placebo or Ostabolin-C (7.5, 15, 30 or 45 μg). A subset of subjects from all treatment groups had blood collected for measurements of Ostabolin-C in order to determine PK parameters and compare them to prior studies.

The full study duration of the study was 22 weeks, which included a 6- week screening period involving a 2-week placebo run-in and then 16 weeks of treatment. The subset of subjects for this component of the study was treated the same as all other subjects with the exception of the additional blood collections at baseline and Week 12.

DATA HANDLING AND PK Analyses

All of the values from the Placebo subject except one (Baseline 2 hour time-point) were below the assay level of detection (i.e., 10 pg/ml). With very limited exceptions, all values from Placebo subjects in prior trials have also been below the levels of detection. Thus, this one value was considered to be a laboratory error and PK parameters for this placebo subject were not calculated.

No samples were obtained for Pre-dose at either Baseline or Week 12. In all prior studies, pre-treatment values have been below the levels of detection and 24-hour time points at doses of 40 ug and below have been below the levels of detection. Thus, no observable values were anticipated and for calculating PK the pre-treatment values for Baseline visit were set to zero.

For pre-dose at Week 12, it was also anticipated that the values would be below the levels of detection based on prior studies and that the 24-hour post dose value would verify this. Only two 24-hour time-point values were above the level of detection; i.e. 24 hours post dosing at Baseline for Subject 030-003 and 24 hours post dosing at Week 12 for Subject 030- 0004. The values of both of these 24-hour time-points were marginally above the assay level of detection. Also, both of these subjects had values below the levels of detection for both the 4 hour and 6 hour time-points after dosing on the prior day. Thus, these values are most likely artifacts and not real values. No valid 24 hour sample for subject 032-0001 at Week 12 was obtained. However, the 6 hour time point after dosing at Week 12 was below the levels of detection, and thus the value for 24-hours was assumed to be also below the level of detection to estimate the AUC(0-24) value. Thus, for calculating PK parameters, the Pre- dose values for Week 12 were also set to zero.

The pharmacokinetic parameters that were estimated at Baseline and Week 12 are as follows: - The area under the drug concentration-time curve from time zero to time 4 hours

- The area under the drug concentration-time curve from time zero to time 24 hours

- The maximum observed drug concentration (Cm_3x)

- The time of the maximum drug concentration (T max)

Since so few subjects were included in this subset of subjects and the time points used for collections were limited, additional PK parameters were not calculated.

AUC values were estimated by a simple summation of trapezoidal areas from each time period. Data from each dose group were summarized using simple statistics on an Excel® spreadsheet; i.e., average (AVG) and Standard Deviation (STD). It should be noted that particularly with the lower doses and associated low blood levels and at late time points, those values just above versus just below the assay limits of detection can have a disproportionate impact to AUC calculations. This adds to that variability of the calculated numbers.

PK VALUES

The following table summarizes the estimated PK parameters. BASELINE PK PARAMETERS

Since there are so few subjects that actually participated in this part of the study and since the values for Cmax, Tmax, and AUC appeared to be very similar for both Baseline and Week 12, the values for all times were averaged to obtain another estimate of these parameters; see table below. Similar PK values for Baseline and Day 7 when steady state kinetics should have been have reached equilibrium have been seen in two previous Phase 1 studies involving this dose range.

WEEK 12 PK PARAMETERS

*Note —no valid 24 hour sample for subject 032-0001 was obtained but since the 6 hour time point was below the levels of detection, the value for 24-hours was assumed to be also below the level of detection to estimate the A UC(0-24) value.

ESTIMATED PK PARAMETERS - AVERAGE OF BASELINE AND WEEK 12 DATA

*Note: N= number of subjects; data from both Baseline and Week 12 combined, each subject had two values for each parameter.

Since Tmax seems to be dose independent in this study as well as in previous studies, the Tmax from all doses in this study determined at Baseline and Week 12 were averaged to obtain an overall estimated value of 0.34 hours with a STD of 0.21 hrs. Discussion

The very limited numbers of subjects involved in this study limit the statistical confidence in the conclusions drawn from the data in this study. However, the data are basically consistent with prior studies.

As seen in prior studies, there was no evidence of accumulation. The PK parameters after 12 weeks of dosing were very similar to those on Day 1 at Baseline.

The Tmax was independent of dose and the overall average form all doses and times was 0.34 hours (srd = 0.21 hrs).

The Cmax and the AUC values increased with dose. There is a rough dose relationship with Cmax and AUVC values in the averaged data.

EXAMPLE 9 Treatment Of Renal Osteodystrophy

End stage renal disease is invariably associated with bone disease, known as renal osteodystrophy (ROD) (for account of pathogenesis see Primer on Metabolic Bone Diseases and Disorders of Mineral Metabolism Chapter 74). ROD may exist in a high turnover form characterized by high circulating levels of PTH (secondary hyperparathyroidism) and overactive bone tissue. This condition is frequently associated with bone pain, muscle weakness, extraskeletal calcification and deformities and growth retardation in children. Reduction in PTH levels is considered necessary to treat these problems. The low turnover form of the disease, also known as adynamic bone disease, is characterized by normal or low circulating levels of PTH and is increasing in incidence due to the increasing use of therapies to effectively control secondary hyperparathyroidism such as Vitamin D sterols, calcium based phosphate binding agents and calcimimetic drμgs. Histologically the bone surfaces are quiescent with little or no osteoblast cellular activity. Clinical consequences of this histological state include increased risk of fractures and growth retardation in prepubertal children.

Adynamic bone disease is currently difficult to treat. The use of parathyroid hormone is contraindicated since reducing parathyroid hormone levels is one of the important goals of the therapies that lead to adynamic disease. Hypercalcemia is a frequent complication of current therapeutic strategies and this would be exacerbated by the use of exogenous PTH. Restoration of normal levels of bone formation activity is therefore difficult to achieve in this setting and there is an unmet need for effective therapy. Agonists of the PTH receptor, exemplified by cyclized or linear PTH (1-31) analogs but also including other cyclic and linear analogs of smaller size and analogs of PTHrP have been shown to increase bone formation but do not have the propensity to stimulate bone resorption that is seen with other PTH fragments and with the naturally occurring hormone. PTH receptor agonists of this type may be able to stimulate osteoblastic function and bone formation and thus effectively treat adynamic bone disease without exacerbation of the risk of hypercalcemia. The use of low doses of these agents may be particularly effective in prevention and treatment of adynamic bone disease to provide restoration of normal osteoblast activity with minimal bone resorption stimulating activity. Specific treatment scenarios in which PTH receptor agonists of this type are used in combination with calcimimetic drμgs, Vitamin D sterols or other agents known to increase the occurrence and/or severity of adynamic bone disease to prevent this occurrence or exacerbation could be created.

PTH receptor agonists could be used in dialysis patients at increased risk of developing adynamic bone disease to prevent the occurrence of adynamic bone disease. PTH receptor agonists of the type described above could also be used to treat patients with osteoporosis and renal disease who have a particularly high risk of fracture due to adynamic bone disease.

Example 10 Rat Oncogenicity Study

Prior art PTHs cause osteosarcomas in animals if administered over a course of two years. The PTH peptides of the present invention, including Ostabolin-C™ and PTH 1-30, are administered subcutaneously to rats for 104 weeks at doses of 0.5, 5, 30, and 50 μg/kg/day. The test article is administered subcutaneously. Analysis of the incidence and morphology of tumours following administration may demonstrate that administration of the PTH peptides of the present invention over the course of two years may lead to lower incidences of osteosarcomas as compared to administration of a similar duration of prior art PTH peptides. This difference could be due to the different amino acids sequences and/or to the different signalling pathways activated by the PTH molecules.

EXAMPLE 11 COMPARISON OF OSTABOLIN-C AND FORTEO

The below table illustrates a comparison of Ostabolin-C with Forteo data derived from Deal et al., (2005) J. Bone Min. Res. 20, p. 1905-1991. As shown below, bone resorption stimulation with 30 μg Ostabolin-C is approximately 50% of the expected effect of 20 μg Forteo despite similar effects on LS-BMD. The effect of 30 μg Ostabolin-C on serum calcium and incidence of hypercalcemia are both diminished. The effect of 45 μg Ostabolin- C on bone formation and BMD is greater than the effect of 20 μg Forteo despite similar effects on bone resorption and calcium. Both 30 μg and 45 μg Ostabolin-C doses have an improved therapeutic window compared to Forteo. These results are also represented in Figures 15, 16, and 17.

EXAMPLE 12- ANALYSIS OF SIDE EFFECTS WITH

DOSE ADMINISTERED BASED ON PATIENT WEIGHT

To confirm that μg/kg exposure is a valid way of analyzing the effect of Ostabolin-C, the cumulative response and side effects profiles were assessed. The cumulative response was analyzed by looking at the proportion of patients who achieved a >3% change in lumbar spine BMD with increasing Ostabolin-C exposure. This analysis illustrates that assessing the effect of Ostabolin-C exposure on an individual patient basis follows a similar pattern to the linear regression analysis from dose groups created on the basis of increasing μg/kg exposure (see figure 23-25 on lumbar spine). Similar trends were also demonstrated for side effect profiles as is illustrated in a cumulative incidence analysis, shown in figures 31, 32, for headache, nausea and hypercalcemia. Increasing exposure was generally associated with a higher incidence of side effects and hypercalcemia.

1 Data from subset with LS-BMD T score <-2.5.

2 Inferred from graphed data in Deal et at. (2005) J. Bone Min. Res. 20, p. 1905-1991. The linear progression of efficacy eπdpoints coupled with the association of higher side effect rates with increased exposure raised the possibility that if the exposure to Ostabolin-C could be maintained between narrower limits than would be produced by administration of a single daily dose to the entire cohort, the observed clinical profile might be superior. Such a strategy could reduce low efficacy responses by eliminating low exposure and also reduce unwanted or excessive responses by eliminating high exposure.

EXAMPLE 13 DOSE OPTIMIZATION -

TWO DOSE STRENGTHS WITH WEIGHT CUT OFF

An analysis of the four month data from the Phase II Trial of the sub-cutaneous Formulation of Ostabolin-C as described in Example 5 demonstrates that a high dose of Ostabolin-C has efficacy greater than Forteo, particularly at the hip, but also presents with some hypercalcemia. The data from Example 5 also demonstrate that the low dose of Ostabolin-C has efficacy similar to Forteo, and without hypercalcemia. Based on these results, we conducted an analysis to identify a dose that combines the favorable clinical features of both Phase II high and low doses, resulting in a dose that both has superior efficacy, and is well tolerated.

In contrast to the actual single dose exposure distribution observed in the table shown below, the effect of a weight cutoff at 68 kg reduces the spread of exposures in the 30 and 45 μg dose groups.

Exposure Ranges for Different Doses

Treatment

Group N Mean Minimum Maximum

Control 59 0.00 0.000 0.00

7.5 μg 49 0.12 0.07 0.16

15 μg 48 0.25 0.17 0.33

30 μg 51 0.48 0.30 0.69

45 μg 54 0.71 0.48 0.99 The effect of the dose optimization strategy was evaluated by performing subgroup analyses using actual data from the 30 and 45 μg cohorts of the study (i.e. creation of a new subgroup by combining all patients below the weight cutoff from the 30 μg dose group with all patients above the weight cutoff from the 45 μg dose group, shown in the table below).

A variety of new subgroups were created out of the patients who participated in the Phase II clinical trial. The groups from Phase II are shown above, where each group received a single dose, either 7.5 μg, 15 μg, 30 μg, 45 μg, or placebo. The 30 μg and 45 μg dose groups were re-grouped, according to the weight of the patient, and over 100 new sub-groups were created. This approach of creating new sub-groups out of dosages which are relatively close to each other, is a novel way of conducting Phase II studies.

For the 105 patients who were originally given 30 or 45 μg, new groups were created by utilizing multiple weight cut-offs in increments of 0.5 kg. For example, one new group had the weight cut-off at 59 kg, meaning that every patient from the original low dose (30 μg) group who weighed less than 59 kg and every patient who weighed more than 59 kg from the original high dose (45 μg) group formed a new cohort. This new cohort represents a dose/weight cut-off of 59 kg, a group in which patients weighing less than 59 kg receive the low dose (30 μg) and patients weighing more than 59 kg receive the high dose (45 μg). Another example can be illustrated using a weight cutoff of 68 kg. Such a group would include all those patients who weigh less than 68 kg and who originally received the low dose (30 μg) as well as all those patients who weigh more than 68 kg and who originally received the high dose (45 μg). By using this method, over 100 separate cohorts were created by altering the dose/weight cut-off in increments of 0.5 kg. A summary of the data generated by these new dose/weight cut-off cohorts, using 68 kg and 59 kg as the dose/weight cut-off, is shown in the table below.

The table above illustrates the actual clinical profile obtained for two dose/weight cutoffs (59 kg which produces a higher exposure profile since more patients receive the higher dose and 68 kg in which more patients receive the lower dose). The profile described in the table indicates that these two cutoffs produce an intermediate clinical profile between 30 and 45 μg. One problem with simple subgroup analysis is that endpoints with a small number of events can skew the observed profile because of large changes in incidence when one of these events moves from one side of the weight cutoff to the other. Therefore, the dose/weight cutoff methodology was applied systematically to the data from the 30 and 45 μg dose groups starting at the lowest weight patient in the study (corresponds to the 45 μg profile because everyone receives the high dose) and increasing the cutoff by 0.5 kg increments through 100 kg (corresponding to the 30 μg profile because everyone receives the low dose). The multiple cutoff profiles thus obtained when graphed can then smooth out the effect of individual events as illustrated for the effect on hypercalcemia. The effect of weight cutoff on clinical profile can then be modeled to eliminate the skewing effects of individual data points as illustrated in figure 37-40. This approach has been applied to primary and secondary endpoints in the study.

Several interesting and unexpected conclusions can be drawn from this analysis. The first is that change in biomarkers (both of formation and resorption) is highly sensitive to change in weight cutoff between 60 and 70 kg, as shown in figures 41-43, 49-50.

Although these changes appear coincident, the ratio of PlNP to CTx also displays a marked shift in this cutoff range (see figures 41-43, 49-50), reflecting the already established bone formation (PlNP) effect at lower doses. The change in incidence of hypercalcemia and in serum calcium also show large increases in this cutoff range, further strengthening the link between the stimulation of bone resorption and the emergence of hypercalcemia in the clinical profile.

By comparing the modeled curves for different clinical variables it is possible to establish whether or not Ostabolin-C will differentially affect clinical parameters if a specific weight cutoff is applied to the dosing regimen. A differential effect can be inferred if the sigmoidal plots of two different clinical variables are not overlapping, as noted with change in incidence of hypercalcemia and lumbar spine BMD responder rate and between change in serum calcium and change in lumbar spine BMD, as shown in figures 44-47, 51. These comparisons illustrate that a cutoff at approximately 68 kg will provide maximal BMD benefits with minimal impact on serum calcium metabolism.

The selection of a definition for BMD responder is different for different BMD sites because the magnitude of change at each site is also different. Different definitions of BMD 'response' have been evaluated based on a separate analysis that demonstrated that actively treated patients had greater BMD changes than placebo at every levels of BMD change (figures 46-48).

Therefore response definitions for the lumbar spine, femoral neck and total hip BMD were selected that produced approximately equal numbers of responders for each BMD rate (>3% for lumbar spine and >0% for total hip and femoral neck BMD). Comparison of modeled data for three different BMD parameters illustrates that progressively higher dose exposures are required to affect total hip and femoral neck BMD compared to lumbar spine BMD. This will enable therapy to be tailored to the individual needs of the patient (see figure 48).

An important benefit of this modeling is that it is possible to project a clinical profile for a specific weight cutoff7dose exposure window with a high degree of accuracy since the effect of individual events is eliminated. The anticipated hypercalcemia and lumbar spine BMD profile for a 68 kg cutoff is illustrated in the table below, which compares the modeled hypercalcemia and lumbar spine BMD changes and the actual Phase II data for the 68 kg weight cutoff. It will also be possible to model the effects of even narrower exposure windows by using multiple dose cutoffs and different doses.

The analysis of the effects of μg/kg exposure to Ostabolin-C has yielded a simple but powerful approach to dose optimization which will provide superior efficacy responses while minimizing the emergence of adverse effects. It is anticipated that this dosing approach will provide additional flexibility to the use of PTH analogs and a superior clinical profile for the treatment of osteoporosis in males and females.

These dose optimization benefits illustrated above for Ostabolin-C will also apply to other therapeutics, including the anti-sclerostin Mab, inhibitors of negative regulators of the Wnt signaling pathways, activin receptor agonists, therapeutics whose bone formation effect is mediated by the action of PTH on its receptor, including PTH, full-length and fragments thereof, PTH analogs, PTHrP, and PTHrP analogs, and calcium receptor antagonists which stimulate endogenous PTH production, such as those that act as agonists of the PTH receptor, including PTH, full-length and fragments thereof, PTH analogs, PTHrP and analogs thereof.

EXAMPLE 14 OSTABOLIN-C -,T 'M ^M INHALATION POWDER PHASE I CLINICAL TRIAL

A Phase I clinical study was undertaken using Ostabolin-C™ Inhalation Powder (OCIP) to establish an MTD in post-menopausal women, to compare its PK profile with Phase II sub-cutaneous doses, and to evaluate biological activity with cAMP and biomarkers of bone turnover. The OCIP was administered using the Nektar T-326 dry powder inhaler (DPI), which is well-accepted by osteoporosis patients in focus groups. An ascending dose tolerance was used. Each cohort was randomized and included 6 active patients and 2 placebo patients. The patients were post menopausal healthy females that were older than 40 years of age and had no known history of osteoporosis or other bone disease.

The primary measurements were taken at clinical laboratories where patient surveys were taken and the patient could describe any adverse events. Additionally the patients' vital signs were taken with Holter monitoring of EKGs and Spirometry. Pharmacokinetics, such as Ostabolin-C blood levels, PK parameters after a single dose and after 7 days of daily dosing were also measured. Pharmacodynamics were also monitored for changes in bone markers including PlNP, Osteocalcin, NTx, CTx, and urine cyclic AMP.

The overall design of the trial is as follows:

The table below shows the dosing summary.

Dosing Summary

The chart below demonstrates that administration of OCIP results in PK parameters similar to those achieved when Ostabolin-C is administered sub-cutaneously.

*AUC and Cmax values normalized to indicated doses

OCIP used 0.3 mg to 1.0 mg (4%) values; SC Used 30 & 45 ug values

**Tmax and Tl/2 dose independent - averaged for all subjects

The chart below shows that PK parameters at Day 1 and Day 7 administration of OCIP are very similar.

*AUC and Cmax values normalized to 0.6 mg dose

OCIP used 0.3 mg to 0.6 mg (4%) values

^•• Tmax and Tl/2 dose independent — averaged for all subjects

Figures 55-60 illustrate that the pharmacokinetic parameters achieved with OCIP administration are similar to those achieved with sub-cutaneous administration. They also demonstrate that the OCIP PK profile is dose proportional. These figures include the following measurements: therapeutic AUC levels, therapeutic Cmax levels, overall cmax and t_max , the C_πra_x, AUC (0-4) values for the OCIP 4% formulation, and that the pk profiles of the 4% ocip formulation.

Generally, the PK trial identified that the Phase II trial would utilize the 4% formulation and the therapeutic range would be dose proportional and there would be single and repeat dosing with no accumulation.

The data in Figures 55-60 demonstrate that lung delivery of Ostabolin-C is biologically active, since cAMP stimulation can be maintained with repeat dosing and bone formation biomarkers increase with delivery to the lung. Additionally, day 1 PK parameters are predictive of cAMP change. The figures also show the urinary cAMP increases within therapeutic ranges on day 1 , that there is a consistent cAMP response with repeat dosing at days 1 , 7 and 28 respectively, that the cAMP generation correlates with AUC and C_max on day 1.

The figures also demonstrate that the levels of PlNP increased from 25 up to 100% by day 28 as compared to baseline and that the levels of osteocalcin increased from 25 up to 100% by day 28 as compared to baseline. The figures also show that the increase in PlNP correlates with AUC. The figures also show that the OCEP administration had no effect on bone resorption markers by showing the percent change in CTx. Accordingly, the data demonstrates that there is a robust urinary cAMP and bone biomarker response with the administration of OCIP. There is therefore a high likelihood of Phase II efficacy comparable with subcutaneous administration, especially since OCIP cAMP dose response exceeds subcutaneous response, the biomarker response correlates with cAMP responses, and the biomarker responses are consistent and clinically relevant.

Adverse Events Profile

The preliminary results of the subjects to whom OCIP was administered as described above showed that the AE profile was consistent with PTH class effects, in that greater than 95% were mild and there were no pulmonary or cardiovascular AEs. Moreover, there were no serious adverse effects, no effects on spirometry parameters or vital signs. The table below shows the distribution of adverse events.

The summary of adverse events in the table below shows that the vast majority (>95%) of adverse events were mild. No adverse events were rated as severe.

The table below shows a summary of adverse events experienced, including headaches, nausea, and vomiting.

Overall the OCIP is Phase II ready and with an appropriate, transient PK profile with acceptable variability and with a biological activity predictive of therapeutic benefit that established a therapeutic window. Moreover, there is a comparability with the SC formulation with a high probability of a late phase success.

Example 15: Formulations

Formulation screening studies on Ostabolin-C solution to develop a stable formulation are detailed. Previous formulations experience oxidation and deamidation at a pH above 7.0. Mixtures of ethanol/water or propylene/water systems with the antioxidants methionine or lipoic acid were evaluated and their stability was accessed.

Two ethanol/water formulations with methionine were examined. The first set of experiments examined the stability of Ostabolin-C solution in the mixture of 40% ethanol/60% water (hPTH #1) above pH 7.0. One mg/ml methionine was included in the formulation to control oxidation. The drug and methionine were dissolved in water and pH was adjusted to 7 with 0.1N NaOH and then ethanol was added to obtain the target ratio of ethanol and water. Ostabolin-C showed excellent stability in 40% ethanol/60% water system. The stability data of hPTH#l are presented in the Table below.

Solution Stability of Ostabolin C in 40% Ethanol/Water with 1 mg/mL of methionine at pH 7 (Batch hPTH#l)

Ostabolin-C showed excellent stability in the 40% ethanol /60% water system. The degradation peaks eluting at RRT of 0.56 to 0.59 and 0.62 to 0.65 are two oxidative degradation peaks. They are present in the initial sample and did not change significantly during the stability study under all conditions with the exception of the 106-day sample stored at 40⁰C. These data suggest that ethanol is stabilizing the formulation. The only other degradant observed (RRT 0.90) is a hydrolysis product which is increasing at 15, 45, and 106 days storage at 40⁰C. The hydrolysis product was not observed at 45 days/25 ⁰C suggesting that the formulation is robust and one could project a shelf life of 2 year under refrigeration conditions.

The other approach was to examine the use of a diol (propylene glycol) to further stabilize the structure of the peptide and thus enhance stability. Additionally, the antioxidant lipoic acid was included. Sample solutions were prepared by dispersing lipoic acid in water, adjusting the pH to 8.0 and then adding drug. The solution pH was adjusted to pH 7.5 and propylene glycol added to obtain a final solution of 60% propylene glycol/40% water. The stability data is presented in the Table below.

The stability of Ostabolin-C in the above solvent system is outstanding. No significant degradant peaks were found at 25 °C and 5 ⁰C storage samples after 106 days. The only degradation we observed is the hydrolysis product at 40⁰C. Some potency loss was observed in 25 ⁰C storage sample as well as the 5 ⁰C sample after 106 days. However no degradation was observed and this loss of potency may be attributable to adherence of drug to the vials. Both hPTH#l and hPTH#2 showed excellent stability and both formulations will be stable for two years under refrigeration storage.

Example 16: Stability Enhancement

Stability studies conducted to data indicate that it is possible to obtain a refrigerator- stable formulation of Ostabolin-C either with a propylene glycol/water or ethanol/water mixture. The addition of other potential stability modifiers is determined. It is well know that hPTHs are susceptible to oxidation during storage. Methionine has been shown to be a potential antioxidant and improve hPTH stability. Additionally, it is well known that polyols have the potential to stabilize peptide and protein formulations. Previously the effect of sucrose on the stability of hPTH (1-34) was examined. Sucrose concentrations up to IM at pH 5.5 were found to reduce the rate of both deamidation and oxidation of hPTH (1-34).

Buffer type could also have an effect on stability. The intent was to examine these solutions at a more physiological relevant pH (pH 7.5). Previously for model compounds it was shown that for pHs above 7, TRIS buffer had a much lower deamidation rate constant than a corresponding phosphate buffer. Lastly, the effect of the addition of 9 mg/ml NaCl was examined as this more represents^~the physiological ionic strength. The stability data on Ostabolin-C solution formulations in the presence of methionine, Tris buffer, sucrose and NaCl in the solvent system consisting of 60% propylene glycol and 40% water at pH 7.5 is examined

Description of Formulations:

Form #1: 250 μg/ml of Ostabolin-C in 0.05M Tris buffer in 60% propylene glycol and 40% water with pH adjusted to 7.5 with HCl

Form #2: 250 μg/ml of Ostabolin-C in 0.05M Tris buffer in 60% propylene glycol and 40% water and 5 mg/ml of methionine with pH adjusted to 7.5 with HCl

Form #3: 250 μg/ml of Ostabolin-C in 0.05M Tris buffer in 60% propylene glycol and 40% water, 5 mg/ml methionine and 200 mg/ml sucrose with pH adjusted to 7.5 with HCl Form #4: 250 μg/ml of Ostabolin-C in 0.05M Tris buffer in 60% propylene glycol and 40% water, 5 mg/ml methionine, 200 mg/ml sucrose and 9 mg/ml of NaCl with pH adjusted to 7.5 with HCl

In a one liter volumetric flask, 600 ml of propylene glycol was added. The contents of the flask were made up to one liter by adding Milli Q water. The resulting solution is 60% propylene glycol and 40% water. Using this as a stock solution, each batch of formulation was prepared by adding and dissolving the inactive ingredients and adjusting the pH of the resultant solution to about 8.0 prior to adding drug. The drug was added and the pH was readjusted to 7.5 with 0. IN HCl.

All test batches were placed at 40?C, 25°C, and 5°C for stability testing. From earlier stability studies, it was observed that the principal degradation peaks of Ostabolin-C are three oxidation peaks OXl (RRT -0.59), OX2 (RRT -0.65), OX3 (RRT -0.7), two hydrolysis peak (RRT ~ 0.90 (HYDl) and 0.98 (HYD2)), three late eluting peaks (RRT between 1.01 to 1.25; DEGl , DEG2 and DEG3) and additional peaks (RRT > 1.4, eluting after the 45 minutes during the gradient wash out period; identified as gradient eluting peaks, GEP(#). The number in parenthesis denotes the number of peaks that are eluting with the gradient change. The degradation peaks observed during the stability analyses will be presented using the above defined nomenclature.

The drug appears to also adhere to the glass and hence 100% of drug is not recovered. Loss in potency is observed with time without any increase in the degradation peak area percent. Four weeks stability of the aforementioned formulations at 40⁰C and 25°C has been complete and the data summarized in the Tables below.

Stability of Ostabolin-C Formulations #1-4 at 40^°C/4 Weeks Storage

Stabilit of Ostabolin-C Formulations #1-4 at 25 C/4 Weeks Storage

ND: Not detected

All four tested formulations showed HYDl ranging 5.3 to 8.3 area percent upon storage 40⁰C for 4 weeks. Hydrolysis degradation is lowest in the formulation containing sucrose and sodium chloride (Form #4) suggesting that the presence of sodium chloride retards or slows down hydrolytic degradation. However, potency of Ostabolin ranges from 65 to 72 for the same storage period, suggesting variable recovery of drug from formulation to formulation. As shown in the Tables above, all formulations showed very little degradation on storage at 25°C for 4 weeks. Hydrolytic degradation is still lowest in the formulation that contains both sucrose and NaCl (Form#4). Other formulations had much higher levels of hydrolytic degradation (about 1 area %) suggesting that the hydrolytic degradation affected by the presence of sodium chloride or the ionic strength of the reaction medium.

With regard to the oxidative degradation, formulations #1 -4 showed a small percent of oxidative degradation despite the presence of high level (5 mg/ml) of the anti oxidizing agent methionine. However, the addition of antioxidizing agent, methionine in the formulation slowed down the oxidative degradation by 50% (Form#l vs Form #2-4). Interestingly, it was not found that oxidative degradation for the first three weeks in the formulations that contained antioxidizing agent. However, the stability analyses of 4 weeks data of 40⁰C samples indicated that all formulation showed the presence of all three oxidative degradants.

Stability analyses of 25°C/4 weeks sample showed oxidative degradation in formulations #1-2. Additionally, only two oxidative degradation peaks were observed. In addition to hydrolytic and oxidative degradations, it was also observed late eluting peak presented as DEGl in the tables. The area percent of this degradation peak varies from 1 to 2% at 40⁰C over a storage period of four weeks, and wasn't observed at 25⁰C under the same condition.

Stability Data of Ostabolin-C Formulations #1-4

In addition to the above experiments, the stability of Ostabolin in water without pH adjustment either without or with the addition of sucrose (200 mg/rnl) and sodium chloride (9 mg/ml), formulations #A and #B, respectively was conducted. The measured pH of these formulations is about 5.6. One week stability data at either 40⁰C or 25°C indicated severe degradation for the water control sample (Form #A). Major degradation products are oxidative degradants and other degradation products that are eluting before the oxidative degradants. Unlike the stability at pH 7.5 system, there were no peaks observed at RRT >1.0. The stability of the same water formulation in the presence of sucrose and sodium chloride (Form #B) has improved significantly. Oxidative degradants levels are significantly lowered compared to Form #A. However, it was observed several late eluting peaks. The stability data are summarized in the Table below.

Stabilit Data of Control Formulations

These data as a whole indicate that propylene glycol/water mixtures significantly enhance the solution stability of Ostabolin-C (Form#A vs. Form #1). Additions of both sucrose and NaCl confer additional stability (Form #1 vs. Form #4 and Form #A vs. Form

#B).

Example 17: Pharmacokinetic Profile of Above Formulations

The following plasma pharmacokinetics profile of various formulations of Ostabolin- C, were studied and compared; subcutaneous, intramuscular and intravenous administrations (clinical formulation) and also subcutaneous administrations (new formulations) with subcutaneous administrations (pre-clinical formulation) to rats. The following shows the details of the formulations used:

Formulations were prepared once only as follows: The bulk freeze-dried peptide was dissolved in 0.01 M acetic acid (supplied by Fisher Scientific, Loughborough, UK) in a 1:1 ratio, re-aliquotted into dosing vials and deep frozen (approximately -7O⁰C). The vials were freeze-dried at <-20°C and stored frozen until required. The freeze-dried aliquots for injection were re-constituted with water for injection (supplied by Animalcare Ltd, York, UK). The peptide was dissolved in an appropriate volume of purified water to an approximate concentration of 2 to 3 mg/mL. Phosphate buffered saline (pH 7.4) was added to give the final required concentration (at approximately pH 7.2). The capped vial(s) were mixed thoroughly to ensure the peptide was fully dissolved. Aseptic techniques and glass vials were used throughout dose preparation. The dose was not sterile filtered. Clinical formulation (50 mg/mL mannitol, 0.166 mg/mL sodium acetate trihydrate, 0.4 mg/mL glacial acetic acid, pH 4.5). The calculated amounts of mannitol, sodium acetate trihydrate and glacial acetic acid were weighed and then dissolved in the correct amount of water for injection. The test article was weighed, added to the solution and stirred to dissolve. The pH was adjusted to 4.5 with 0.1N NaOH or HCl (supplied by Covance, Harrogate) depending on initial pH. Water was added to obtain the required volume. New formulation 1 (40% propylene glycol)

The calculated amount of lipoic acid was weighed and transferred to a suitable container and then dispersed with water for injection. The pH was adjusted to 7.7 with IN NaOH (supplied by BDH Laboratory Supplies, a division of Merck Ltd, Poole, UK). The test article was weighed, added and stirred to dissolve. The pH was adjusted to 7.5 with IN NaOH. Propylene glycol was added to obtain the correct volume. The recorded final pH was

6.6. New formulation 2 (40% ethanol in water pH 7.5)

The calculated amount of DL-Methionine was weighed and transferred to a suitable container and dispersed in water for injection. The test article was weighed and added to the solution.

The pH was adjusted to 7.5 with 0.1N NaOH. Ethanol was added to make the required volume.

The following dose levels were selected:

Main Study

Number of animals in group

Group Dose level

Description Dose Route Number Main study (μg/lcg) Female

1 Treated-] SC (interscapular) 200 12

2 Treated-2 SC (interscapular) 200 12

3 Treated-3 SC (interscapular) 200 12

4 Treated-4 SC (interscapular) 200 12

5 Control IM (calf muscle) 0 12

6 Treated-5 IM (calf muscle) 200 12

7 Treated-6 IV 200 12

Treated 1 = Standard formulation (acidified saline)

Treated 2 = Clinical formulation (50 mg/mL mannitol, 0.166 mg/mL sodium acetate trihydrate, 0.4 mg/mL glacial acetic acid, pH 4.5)

Treated 3 = New formulation 1 (40% propylene glycol)

Treated 4 = New formulation 2 (40% ethanol in water pH 7.5)

Control = Clinical formulation (vehicle)

Treated 5 = Clinical formulation

Treated 6 = Clinical formulation

Repeat Study

Group Number of animals in group ose Route Dose level Number Description D Repeat study

(μg/kg) Female

8 Control-2 IM (calf muscle) 0 12 9 Treated-7 IM (calf muscle) 200 12 10 Treated-8 IV 200 12

Control 2 = Clinical formulation (vehicle) Treated 7 = Clinical formulation Treated 8 = Clinical formulation

The relative bioavailability of Ostabolin-C in the test formulations compared to the standard formulation was markedly higher, being 1.5-, 27.1- and 37.7 fold higher for the clinical, new 2 and new 1 formulations, respectively. The relative bioavailability of Ostabolin-C administered intramuscularly as the clinical formulation was markedly higher than all the SC administered formulations being approximately 200-fold higher than the standard formulation and 5-fold higher than new formulation 1.

Ostabolin-C was not quantifiable in the plasma samples from the intramuscular (IM) control animals (Group 8). Following a single approximate four minute IV infusion of Ostabolin-C at 200 μg/kg to female rats, maximum quantifiable plasma concentrations of Ostabolin-C occurred at a tmax of 5 minutes post the end of the infusion. However, the plasma concentrations at 2 minutes post the end of the infusion were not quantified, being >610000 pg/mL, and suggest that the true Cmax occurred at this earlier time point.

Following single SC administration of Ostabolin-C as four different formulations (standard, clinical, new formulations 1 and 2), plasma drug concentrations increased rapidly to reach maximum levels at the initial blood sampling time of 5 minutes post dose for all formulations. Single EM administration of the clinical formulation resulted in a slightly later tmax of 10 minutes post dose.

The total plasma clearance (CL) of Ostabolin-C was 45.4 mL/min/kg and is similar to hepatic blood flow. The volume of distributions (Vz and Vss) were similar being 0.471 and 0.479 L/kg, respectively, and suggested extensive distribution of Ostabolin-C.

The toxicokinetic parameters for Ostabolin-C following IV administration are presented below:

Treatment 6 = IV Clinical formulation

The pharmacokinetic parameters for Ostabolin-C following SC and IM administration are resented below:

Treatment 1 = SC Standard formulation (acidified saline)

Treatment 2 = SC Clinical formulation (50 mg/mL mannitol, 0.166 mg/mL sodium acetate tihydrate, 0.4 mg/mL glacial acetic acid, pH 4.5)

Treatment 3 = SC New formulation 1 (40% propylene glycol)

Treatment 4 = SC New formulation 2 (40% ethanol in water pH 7.5)

Treatment 5 = IM Clinical formulation

At necropsy, redness or red area was recorded in the subcutaneous injection site of some animals dosed with new formulation 2 (Group 4), which generally correlated with findings seen microscopically. Microscopic findings in animals treated by the subcutaneous dose route were generally infrequent and of a minor nature. The microscopic finding of congestion/haemorrhage was consistent with minor mechanical damage at the time of injection. A minor level of myositis/myopathy was recorded in most animals treated by the intramuscular dose route (control & clinical formulation, Groups 5 & 6); this was consistent with low-grade mechanical damage due to injection and is not considered to be test article related.

Following single SC administration of Ostabolin-C as four different formulations (standard, clinical, new formulations 1 and 2), plasma drug concentrations increased rapidly to reach maximum levels at the initial blood sampling time of 5 minutes post dose for all formulations. Single IM administration of the clinical formulation resulted in a slightly later tmax of 10 minutes post dose.

After attainment of Cmax, plasma concentrations of Ostabolin-C appeared to decline in a generally bi phasic manner after both SC and IM administration. The terminal elimination half-life was only able to be tentatively defined for the clinical formulation and new formulation 2 after SC administration and was 42.4 and 9.1 minutes, respectively. The determination of 11/2 for the clinical formulation is not considered to be robust as it was determined over less than one half-life in duration.

The apparent clearance (CL/F) and volume of distribution (VzJF), calculated where possible for the formulations administered subcutaneously, were formulation dependent being 3850 and 36600 mL/min/kg and 50.6 and 2240 L/kg, respectively, for the new formulation 2 and clinical formulation.

Absolute bioavailability of each SC formulation was very low (<2%) being lowest for the standard formulation (Fabs = 0.04%) and highest for the new formulation 1 (Fabs = 1.6%). The remaining SC formulations (new formulation 2 and clinical formulation) both had a Fabs of approximately 1%. In contrast, the absolute bioavailability of the IM formulation was much greater being 8.7%.

The relative bioavailability of Ostabolin-C in the test formulations compared to the standard formulation was markedly higher, being 1.5-, 27.1- and 37.7 fold higher for the clinical, new 2 and new 1 formulations, respectively. The relative bioavailability of Ostabolin-C administered intramuscularly as the clinical formulation was markedly higher than all the SC administered formulations being approximately 200-fold higher than the standard formulation and 5-fold higher than new formulation 1. The plasma concentrations for Ostabolin-C following IV administration are shown in Figure 75 (treatment 6), and for Ostabolin-C sub-cutaneously and intramuscularly are shown in Figures 76 and 77 (treatments 1-5). This figures show the increased bioavailability with the new formulations.

The AUC0-tz and Cmax of Ostabolin-C after SC administration of the standard, clinical, new 1 and new 2 formulations, as well as the AUC0-tz and Cmax of Ostabolin-C after IM and IV administration of the clinical formulation are shown in figures 70-74.

Overall, following single dose administration of 200 μg/kg Ostabolin-C to female rats by SC, IM and IV dose routes, maximum plasma levels of Ostabolin-C were rapidly reached by 5 minutes after SC administration of all formulations. Tmax occurred at 10 minutes after IM dosing of the clinical formulation. The terminal elimination half life, where calculable, ranged from 7.2 to 42.4 minutes.

Absolute bioavailability of Ostabolin-C was very low (<2%) after SC administration being lowest for the standard formulation (Fabs = 0.04%) and highest for the new formulation 1 (Fabs = 1.6%). Absolute bioavailability following IM dosing was markedly higher being 8.7%.

While this invention has been particularly shown and described with references to preferred embodiment thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

What is claimed is:

1. A method of treating a bone deficit disorder in a subject while reducing side effects associated with the administration of a parathyroid hormone, comprising administering to the subject a parathyroid hormone peptide analog or other peptide with a narrow therapeutic window in an effective dose for the subject, wherein the effective dose for the subject is determined based on the weight of the subject, and wherein there are multiple effective dosages for subjects presenting with different weights, and further wherein the effective dose is calculated based on a predetermined weight cutoff.

2. The method of claim 1, wherein the PTH peptide analogue is selected from the group consisting of PTH 1-84, PTH 1-34, PTH-(1-31)NH₂; PTH-(I -3 O)NH₂; PTH-(1-29)NH₂; PTH-(I -28)NH₂; Leu²⁷PTH-(l-31)NH₂; LeU²⁷PTH-(I -3O)NH₂; Leu²⁷PTH-(l-29)NH₂; Leu²⁷cyclo(22-26)PTH-(1-31)NH₂ Ostabolin-C™; Leu²⁷cyclo(22-26)PTH-(l-34)NH₂; Leu²⁷cyclo(Lys²⁶-Asp³⁰)PTH-(l-34)NH₂; Cyclo(Lys²⁷-Asp³⁰)PTH-(l-34)NH₂; Leu²⁷cyclo(22-26)PTH-(1-31)NH₂; Ala²⁷ or NIe²⁷ or Tyr²⁷ or lie²⁷ cyclo(22-26)PTH-(l-31)NH₂; Leu²⁷cyclo(22-26)PTH-(1- 32)NH₂; Leu²⁷cyclo(22-26)PTH-(l-31)OH; Leu²⁷cyclo(26-30)PTH-(l-31)NH₂; Cys²²Cys²⁶Leu²⁷cyclo(22-26)PTH-(l-31)NH₂; Cys²²Cys²⁶Leu²⁷cyclo(26-30)PTH- (1-3I)NH₂; Cyclo(27-30)PTH-(l-31)NH₂; Leu²⁷cyclo(22-26)PTH-(1-30)NH₂; Cyclo(22-26)PTH-(1 -31 )NH₂; Cyclo(22-26)PTH-(1 -3O)NH₂; Leu²⁷cyclo(22- 26)PTH-(l-29)NH2; Leu²⁷cyclo(22-26)PTH-(l-28)NH₂; Glu¹⁷,Leu²⁷cyclo(13-

17)(22-26)PTH-(l -28)NH₂; and Glu¹⁷,Leu²⁷cyclo( 13-17)(22-26)PTH-(l -31 )NH₂.

3. The method of claim 2, wherein the PTH peptide analogue is selected from the group consisting of: PTH-(I -31) peptide analogues, and PTH-(l-30) peptide analogues.

4. The method of claim 3, wherein the PTH peptide analogue is Ostabolin-C™.

5. The method of claim 1, wherein the peptide with a narrow therapeutic window is selected from the group consisting of anti-sclerostin Mab, inhibitors of negative regulators of the Wnt signaling pathways, activin receptor agonists, hormones, calcium receptor antagonists which stimulate endogenous PTH production.

6. The method of claim 1, wherein the dose is either a high dose or low dose.

7. The method of claim 6, wherein the low dose is in the range of 5-30 μg.

8. The method of claim 7, wherein the low dose is 30 μg

9. The method of claim 6, wherein the high dose is in the range of 45-60 μg.

10. The method of claim 9, wherein the high dose is 45 μg.

11. The method of claim 6, wherein if the patient weighs less than the weight cutoff the patient is administered the low dose.

12. The method of claim 6, wherein if the patient weighs more than the weight cutoff the patient is administered the high dose.

13. The method of claim 1, wherein the single weight cutoff point is between 60-70 kg.

14. The method of claim 13, wherein the single weight cutoff point is 68 kg.

15. The method of claim 1, wherein the undesirable side effects that are reduced are selected from the group consisting of bone resorption, hypercalcemia, increase in mean serum calcium level, headache, nausea, back pain, dizziness, extremity pain, feeling cold, fatigue, loose stool, feeling hot, lower abdominal pain, injection site reaction, arthralgia, injection site hemorrhage, pharyngolaryngeal pain, muscle cramps, and abdominal pain.

16. The method of claim 1, wherein the PTH peptide analogue is administered at a daily dose of between 0.25 and 0.75 μg/kg.

17. The method of claim 16, wherein the PTH peptide analogue is administered at a daily dose of between 0.30 and 0.50 μg/kg.

18. The method of claim 1, wherein the administration is oral, topical, pulmonary, transdermal, intranasal, transpercutaneous, parenteral injection or subcutaneous injection.

19. The method of claim 18, wherein the administration is pulmonary.

20. The method of claim 19, wherein the PTH peptide analogue is administered as a daily inhalation dose of between 100 μg and 2,000 μg.

21. The method of claim 20, wherein the PTH peptide analogue is administered as a daily inhalation dose of between 300 μg and 800 μg.

22. The method of claim 20, wherein the PTH peptide analogue is administered as a weekly inhalation dose 3 to 7 times greater than the daily dose.

23. The method of claim 19, wherein the effective pharmacokinetic profile comprises a pharmacokinetic parameter selected from the group consisting of: a) a half-life of said PTH peptide analogue of between 2 minutes and 60 minutes; b) a duration of exposure to said PTH peptide analogue of between 30 minutes and 4 hours; c) a T_max of said PTH peptide analogue of between 2 minutes and 30 minutes; and d) a C_max of said PTH peptide analogue of between 10 and 400 pg/ml.

24. The method of claim 19, wherein the effective pharmacokinetic profile comprises a half-life of said PTH peptide analogue between 2 minutes and 60 minutes.

25. The method of claim 24, wherein the half-life is between 15-30 minutes.

26. The method of claim 19, wherein the effective pharmacokinetic profile comprises a duration of exposure to said PTH peptide analogue between 30 minutes and 4 hours.

27. The method of claim 26, wherein the duration of exposure is between one and two hours.

28. The method of claim 19, wherein the effective pharmacokinetic profile comprises a Tmax of said PTH peptide analogue between 2 minutes and 30 minutes.

29. The method of claim 28, wherein the Tmax is between 15-30 minutes.

30. The method of claim 19, wherein the effective pharmacokinetic profile comprises a C_max of said PTH peptide analogue between 10 and 400 pg/ml.

31. The method of claim 30, wherein the C_max is between 50-200 pg/ml.

32. A pharmaceutical composition comprising a unit dosage form of a therapeutically effective amount of a parathyroid hormone (PTH) peptide analogue in a dosage determined by a patient's weight and presentation of symptoms, and a pharmaceutically acceptable excipient, diluent, or carrier, or combinations thereof, wherein the pharmaceutical composition administered results in an effective pharmacokinetic profile and maintained adenylate cyclase activity, while simultaneously reducing undesirable side effects associated with the administration of a PTH.

33. The pharmaceutical composition of claim 32, wherein if the patient weighs more than a predetermined weight cutoff, the patient is administered a high dose, and if the patient weighs less than a predetermined weight cutoff the patient is administered a lose dose.

34. The pharmaceutical composition of claim 33, wherein the weight cutoff is between 60-80 kg.

35. The pharmaceutical composition of claim 32, wherein the weight cutoff is 68 kg.

36. The pharmaceutical composition of claim 34, wherein the low dose is between 5- 30 ug.

37. The pharmaceutical composition of claim 33, wherein the low dose is 30 ug.

38. The pharmaceutical composition of claim 35, wherein the high dose is between 45- 60 ug.

39. The pharmaceutical composition of claim 38, wherein the high dose is 45 ug

40. An aqueous pharmaceutical solution, comprising Ostabolin-C in a concentration of about 100-500 ug/ml; which comprises ethanol and water; wherein said solution has a pH of between 6.0-8.0, and wherein the solution is sterile and ready for parenteral administration to a human patient.

41. The solution of claim 40, comprising 40% ethanol and 60% water.

42. The solution of claim 40, further comprising a buffer and a stabilizing agent selected from the group consisting of methionine, lipoic acid, sucrose and NaCL, and mixtures thereof.

43. An aqueous pharmaceutical solution, comprising Ostabolin-C in a concentration of about 100-500 ug/ml; which has an increased bioavailability, and which further comprises propylene and water; wherein said solution has a pH of between 6.0-8.0, and wherein the solution is sterile and ready for parenteral administration to a human patient.

44. The solution of claim 43, further comprising a buffer and a stabilizing agent selected from the group consisting of methionine, lipoic acid, sucrose and NaCL, and mixtures thereof.

45. The solution of claim 44, wherein the buffer is Tris.

46. The solution of claim 45, wherein the concentration of the buffer is in the range of about 2 mM to 100 mM.

47. The solution of claim 43, wherein the concentration of the buffer is about 5 mM.

48. The pharmaceutical solution of claim 40 or 44, wherein said Ostabolin-C concentration is 250 ug/ml.