CA2325371C - Method of increasing bone toughness and stiffness and reducing fractures - Google Patents

Abstract

The invention relates to a method for increasing the toughness and/or stiffness of bone and/or reducing the likelihood and/or severity of bone fracture by administering a parathyroid hormone. The method can be employed to increase toughness or stiffness of bone at a site of a potential or actual trauma, such as the hip or spine of a person at ris k of or suffering from osteoporosis. The method of the invention can reduce the incidence of vertebral fracure, reduce the incidence of multiple vertebral fractures, reduce the severity of vertebral fracture, and/or reduce the incidence of non-vertebral fracture.

Description

METHOD OF INCREASING BONE TOUGHNESS AND STIFFNESS AND REDUCING FRACTURES
TECHNICAL FIELD
This invention relates to methods for increasing the toughness and/or stiffness of bone and/or reducing the likelihood and/or severity of bone fracture by administering a parathyroid hormone. More particularly, the invention relates a method for increasing toughness or stiffness of bone at a site of a potential or actual trauma, such as the hip or spine of a person at risk of or suffering from osteoporosis.
1o More particularly, the invention relates to a method of reducing the incidence of vertebral fracture, reducing the incidence of multiple vertebral fractures, reducing the severity of vertebral fracture, and/or reducing the incidence of non-vertebral fracture.
is BACKGROUND OF THE INVENTION
Existing agents such as estrogen, bisphosphonates, fluoride, or calcitonin can prevent bone loss and induce a 3 - 5% increase of bone mass by refilling the remodeling space, but net bone formation is not significantly stimulated. The retention of bone by inhibition of bone turnover may not be sufficient protection 2o against fracture risk for patients who already have significant bone loss.
Anabolic agents that increase bone strength by stimulating bone formation preferentially may provide better protection against fracture in patients with established osteoporosis.
Parathyroid hormone (PTH) is a secreted, 84 amino acid product of the mammalian parathyroid gland that controls serum calcium levels through its action 25 on various tissues, including bone. The N-terminal 34 amino acids of bovine and human PTH (PTH(1-34)) is deemed biologically equivalent to the full length hormone. Other amino terminal fragments of PTH (including 1-31 and 1-38 for example), or PTHrP (PTH-related peptide/protein) or analogues of either or both, that activate the PTH/PTHrP receptor (PTH1 receptor) have shown similar biologic 3o effects on bone mass, although the magnitude of such effects may vary.
Studies in humans with various forms of PTH have demonstrated an anabolic effect on bone, and have prompted significant interest in its use for the treatment of osteoporosis and related bone disorders. The significant anabolic effects of PTH on bone, including stimulation of bone formation which results in a net gain in bone mass and/or strength, have been demonstrated in many animal models and in humans.
It is commonly believed that PTH administration in humans and in relevant animal models has a negative effect on cortical bone. In fact, naturally occurring increases in endogenous PTH, which occur in the disorder hyperparathyroidism, result in thinning of cortical bone accompanied by an increase in connectivity and mass of trabecular bone. Past studies suggest that when Haversian cortical bone (found in humans and higher mammals) remodels under the influence of PTH, there will be a re-distribution of bone such that cortical bone mass and strength decrease, while trabecular bone increases in mass and strength. For example, in published clinical studies of administering PTH, cortical bone mass decreased after treatment with exogenous PTH and these findings have raised concern that the treatment of PTH will lead to reduced cortical bone mass and strength. One concern raised by such studies is that there would be a loss of total skeletal bone mass due to the loss of cortical bone. This is of high clinical relevance as, in osteoporosis, the greater loss of trabecular bone compared to loss of cortical bone, means that mechanical loading is predominantly borne by the remaining cortical bone. Continued loss of cortical bone would increase the fracture risk. Therefore, it is important that a therapeutic agent for osteoporosis maintain or increase a subjects residual cortical bone.
The effects of PTH on cortical bone have been investigated in nonhuman animals with Haversian remodeling, such as dogs, ferrets, sheep and monkeys, but sample sizes are typically too small for reliable statistical analysis. The impact of the changes induced by PTH treatment on mechanical properties of cortical bone in such animals remains unknown. Published studies of rodents have shown increased cortical bone mass during administration of PTH but a loss of this benefit after withdrawal of PTH. However, rodent cortical bone has a distinctly different structure from Haversian cortical bone, and remodels by surface appositional formation and resorption, rather than by intracortical remodeling of osteons.
Furthermore, technological limitations in biomechanical testing on the relatively short bones of rodents give rise to artifacts of measurement when an agent, such as a PTH, alters bone geometry to thicken the bone. Such artifacts make extrapolation of rat cortical bone responses to those of humans or other animals with osteonal remodeling unreliable. Therefore, the existing data for animals, like humans, undergoing Haversian remodeling indicates that PTH may have an adverse impact on cortical bone, causing net loss of bone mass through depletion of cortical bone.
As a consequence, it has been a popular belief regarding the action of PTH
that patients require concurrent or subsequent treatment with an antiresorptive to minimize loss of bone induced by PTH. In fact, this model has been the basis for several clinical studies in women. For example, three clinical studies have used PTH in post-menopausal women on concurrent therapy with calcitonin or estrogen, or in premenopausal women taking GnRH agonist, Synarel, for endometriosis. The opposing effects of estrogen and PTH on cortical bone turnover make it particularly difficult to observe effects of just PTH during combination therapy with these two agents.
There remains a need for a method for employing a PTH to increase strength and stiffness of bone in humans and other animals exhibiting Haversian remodeling, and for reducing the incidence of fracture of bones in these animals.
Furthermore, there remains a need for a method for increasing the quality and amount of cortical bone.
SUMMARY OF THE INVENTION
The present invention includes a method for increasing the toughness and/or stiffness of bone, preferably cortical bone, and/or reducing the incidence and/or severity of fracture by administering a parathyroid hormone. 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 method can increase toughness or stiffness at a site of a potential trauma, such as a hip or spine of a person with osteoporosis, or at another site having abnormally low bone mass or poor bone structure. The method can also increase bone toughness or stiffness at a site of an actual trauma, such as a fracture, for example, in a hip or vertebra. A preferred subject for the method of the invention is a woman or man at risk for or having osteoporosis, preferably a postmenopausal 1o woman, and is independent of concurrent hormone replacement therapy (HRT), estrogen or equivalent therapy, or antiresorptive therapy. In one embodiment, the patient also receives supplements of calcium and/or vitamin D.
A parathyroid hormone, such as the N-terminal amino acids 1-34 of recombinant human parathyroid hormone, can be administered either cyclically or t5 intermittently. Preferably, cyclic administration includes administering PTH for 2 or more remodeling cycles and withdrawing PTH for one or more remodeling cycles.
Further, according to the method of the invention, the increases in toughness and/or stiffness of a bone can persist for several remodeling cycles, or up to several years, after the last administration of a parathyroid hormone.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A and 1B show that BMD (bone mineral density) and BMC (bone mineral content) in the femoral midshaft (cortical bone) (A) and in the proximal femur (cancellous bone + cortical bone) (B) were significantly greater in PTH-treated animals than controls at both doses.
Figures 2A through 2D show the effects of PTH on mechanical strength and cross sectional moment of inertia (CSMI) in the cortical bone of the femoral midshaft.
Figure 3 illustrates the percent change in DXA measures of whole bone 3o mineral content in control and treatment groups.

i FROM MEACH.ANT & GOULD CA 02325371 20100-09-21 00 9:25/ST. 9:20/N0. 42b060b912 Figures 4A-C illustrate the percCnt change in DX~\ measures of the spine for control and treatment groups in thr: lumbar vertebrae 2-4 for bone area (A), bone mineral content (B), and bone mineral density (C).
Figures ~A and SB illustrate the increase in bone mass (A) and bone strength (B) in lumbar vertebrae of primates treated with a parathyroid hormone.
Figures 6A and fiB illustrate the increase in strength of femur neck (A) and the constant strength of humerus mid-diaphysis (B) in primates treated with a parathyroid hormone.
Figure 7 illustrates activation ofbone formation rates on endosteal and v o periosteal surfaces of the midshaf3 humerus.
Figure 8 illustr-atcs the histogram analysis of the shift in bone voxel densities in lumbar vertebra, resulting from PTH treatment wmpared to control. Note the increase in density in cortical bone compartment after withdrawal of PTH
treatment.
Figure 9 illustrates increases in lumbar spine BMD through 23 months of 15 treatment of patients with either 20 y PTH or 40 pg~day PTH, compared to placebo treated controls.
Figure 10 illustrates increases in femur and hip neck 13M.U through 24 months of treatment orpatients with either 20 ~.g/ day PTfI or 40 ~g/day pTH, compared to placebo treated contmls.
DETAILED D.h:SCIFtIPTION
The invention rotates 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 tougluiess 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, mcre~ssing resistance to loading, and the like. Reducing incidence of fracture 3U generally includes reducing the likelihood or actual incidence of fracture for a subject compared to an untreated control population.

WO 00/10596 6 PCT/L'S99;18961 As used herein, ultimate force refers to maximum force that a bone specimen sustains; stiffness refers to the slope of the linear portion of a load-deformation cun~e; and work to failure refers to the area under the load-deformation cun~e before failure. Each of these can be measured and calculated by methods standard in the 5 bone study art. These parameters are structural properties that depend on intrinsic material properties and geometry, and can be determined as described in Turner CH, Burr DB 1993 "Basic biomechanical measurements of bone: a tutorial." Bone 14:595-608. Ultimate force, stiffness, and work to failure can be normalized to obtain intrinsic material properties such as to ultimate stress, elastic modulus, and toughness, which are independent of size and shape. As used herein, ultimate stress refers to maximum stress that a specimen can sustain; elastic modulus refers to material intrinsic stiffness; and toughness refers to resistance to fracture per unit volume. Each of these can be determined by methods known in the art. Id. Femoral bone strength, as referred to herein, can be measured 15 at the femur neck, or at the midshaft typically using three-point or four-point bending at the latter site.
Bone Trauma The method of the invention is of benefit to a subject that may suffer or have 20 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 25 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 3o transplantation) are found most often in older people.
Preferred subjects include a human, preferably a woman, at risk for or suffering from osteoporosis. Risk factors for osteoporosis are known in the art and 6 ~ PCT/US99/18961 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 1o deviation below mean young normal values), andlor atraumatic fractures.
Osteoporosis can lead, for example, to vertebral and/or non-vertebral fractures. 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 2o 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 WO 0(1110596 g PCT/US99/18961 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, trabecular, and 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 3o 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 ~o 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 2o increased by increasing the toughness and/or stiffness of a femur neck or a femur trochantera. 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 WO 00/10596 1~ Pt'1'/US99ii 8961 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 lineament.
a tendon, andlor 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.
to Parathyroid Hormone As active ingredient, the composition or solution may incorporate the full length, 84 amino acid form of parathyroid hormone, particularly the human form, hPTH (1-84), obtained either recombinantly, by peptide synthesis or by extraction 15 from human fluid. See, for example, U.S. Pat. No. 5,208,041, The amino acid sequence for hPTH (1-84) is reported by Kimura et al.
in Biochem. Biophys. Res. Comm., 114(2):493.
The composition or solution may also incorporate as active ingredient fragments or variants of fragments of human PTH or of rat, porcine or bovine PTH
20 that have human PTH activity as determined in the ovariectomized rat model of osteoporosis reported by Kimmel et al., Endocrinology, 1993, 32(4):1577.
The parathyroid hornone fragments desirably incorporate at least the first 28 N-terminal residues, such as PTH(1-28), PTH(1-31), PTH(1-34), PTH(1-37), PTH(1-38) and PTH(1-41). Alternatives in the form of PTH variants incorporate 25 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.
3o Other suitable forms of PTH include.PTHrP, PTHrP(1-34), PTHrP(1-36) and analogs of PTH or PTHrP that activate the PTH1 receptor. These forms of PTH
are embraced by the term "parathyroid hormone" as used generically herein. The WO 00/10596 11 PCT/l!S99!18961 hormones may be obtained by known recombinant or synthetic methods. such as described in U.S. Pat. Nos. 4,086,196 and S,SS6,940 .
The preferred hormone is human PTH( 1-34), also known as teriparatidc.
5 Stabilized solutions of human PTH( 1-34), such as recombinant human PTH( 1-34) (rhPTH(1-34), that can be employed in the present method are described in PCT application W099/29337.
Crystalline forms ofhuman PTH(1-34) that can be employed in the present method are described in PCT application W099/31137.
Administering Parathyroid Hormone A parathyroid hormone can typically be administered parenteraIiy, preferably by subcutaneous injection, by methods and in formulations well known in the art.
Stabilized formulations of human PTH(1-34) that can advantageously be employed in the present method are described in PCT application W099/29337.
This patent application also describes numerous other formulations for storage and administration of parathyroid hormone. A
stabilized solution of a parathyroid hormone can include a stabilizing agent, a 2o buffering agent, a preservative, and the like.
The stabilizing agent incorporated into the solution or composition includes a polyol which includes a saccharide, preferably a monosaccharide or disaccharide, e.g., glucose, trehalose, raffinose, or sucrose; a sugar alcohol such as, for example, mannitol, sorbitol or inositol, and a polyhydric alcohol such as glycerine or 25 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 3o and capable of maintaining the aqueous solution at a pH range of 3 to 7, preferably 3-6. 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 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, benzalkoniurn 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 l0 1.0 wt-% of the total solution.
Thus, the stabilized teriparatide solution can contain mannitol, acetate and m-cresol with a predicted shelf life of over 15 months at 5°C.
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 raffinose.
2o In addition, parathyroid hormone can be formulated with typical buffers and excipients employed in the art to stabilize and solubilize proteins for parenteral administration. Art recognized pharmaceutical carriers and their formulations are described in Martin, "Remington's Pharmaceutical Sciences," 1 Sth Ed.; Mack Publishing Co., Easton ( 1975). A parathyroid hormone can also be delivered via the lungs, mouth, nose, by suppository, or by oral formulations.
The parathyroid hormone is formulated for administering a dose effective for increasing toughness and/or stiffness of one or more of a subject's bones and/or for reducing the likelihood andlor severity of bone fracture. Preferably, an effective dose provides an improvement in cortical bone structure, mass, and/or strength.
3o Preferably, an effective dose reduces the incidence of vertebral fracture, reduces the incidence of multiple vertebral fractures, reduces the severity of vertebral fracture, and/or reduces the incidence of non-vertebral fracture. Preferably, a subject receiving parathyroid hormone also receives effective doses of calcium and vitamin D, which can enhance the effects of the hormone. An effective dose of parathyroid hormone is typically greater than about 5 p,g/kg/day although, particularly in humans, it can be as large at about 10 to about 40 ~.g/kg/day, or larger as is effective to achieve increased toughness or stiffness, particularly in cortical bone, or to reduce the incidence of fracture. A subject suffering from hypoparathyroidism can require additional or higher doses of a parathyroid hormone; such a subject also requires replacement therapy with the hormone. Doses required far replacement therapy in hypoparathyroidism are known in the art. In certain instances, relevant effects of to PTH can be observed at doses less than about S pg/kglday, or even less than about 1 p,g/kglday.
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).
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. Preferably, cyclic administration includes administering a parathyroid hormone for at least 2 remodeling cycles and withdrawing parathyroid hormone for at least 1 remodeling cycle. Another preferred regime of cyclic administration includes administering the parathyroid hormone for 2o 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.
Uses of Formulations of a Parathryoid Hormone 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 which contains a formulation of the present invention and 3o suitable corners, 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.
The present invention also relates to a pharmaceutical composition including a formulation of one or more parathyroid hormones, such as human PTH(1-84) or human PTH(1-34), and that is suitable for parenteral administration. According to the invention, a formulation of one or more parathyroid hormones, such as human to PTH(1-84) or human PTH(1-34), can be used for manufacturing a composition or medicament suitable for administration by parenteral administration. The invention also relates to methods for manufacturing compositions including a formulation of one or more parathyroid hormones, such as human PTH(1-84) or human PTH(1-34), in a form that is suitable for parenteral administration. For example, a liquid or solid formulation can be manufactured in several ways, using conventional techniques. A
liquid formulation can be manufactured by dissolving the one or parathyroid hormones, such as human PTH(1-84) or human PTH(1-34), in a suitable solvent, such as water, at an appropriate pH, including buffers or other excipients, for example to form one of the stabilized solutions described hereinabove.
The examples which follow are illustrative of the invention and are not intended to be limiting.
EXAMPLES
Example 1- - Increased Bone Strength and Density Upon Administration of rhPTH(1-34) to Rabbits Experimental Procedures Female intact New Zealand white rabbits (HRP Inc. Denver, Pa.), one of the smallest animals that form osteons by intracortical remodeling, approximately 3o months old and weighing 3.25-3.75 kg, were sorted by mean group body weight into 3 groups of 6 animals each. Two experimental groups received biosynthetic PTH(1-34) at doses of 10 or 40 ug/ml/kg/day. The control group was given 1.0 ml/kglday of acidified 0.9M saline containing 2% heat-inactivated rabbit sera. PTH( 1-3=ll or vehicle were injected by once daily subcutaneous injections on 5 days a week for 140 days. Rabbits were fed rabbit lab chow containing 0.~% Ca and 0.41°roP, and given water ad libitum.
5 The selection of doses was based on a series of preliminary studies showing that (1) after a single injection of PTH(1-34) at 100 pg/hg, serum calcium increased and failed to return to baseline by 24 hours, whereas after a single dose of 50 pg/kg, serum calcium returned to baseline within 24 hours, (2) repeated injections of pg/kg PTH(1-34) resulted in transient rise in serum calcium with return to baseline to values in 6-24 hours, and (3) PTH(1-34) at s S ug/l:g did not alter histomorphometry of bone surfaces.
A set of double alizarin labels (Sigma, St. Louis) was given i.m. at 20 mg/hg on days 55 and 63, and a set of double calcein labels (Sigma, St. Louis) was given s.c. at 5 mglkg on days 1 S and 7, prior to sacrifice. Rabbits were anesthetized by IS CO, in a random order sequence, approximately 3-6 hours after the last injection, to obtain blood by cardiac puncture and then killed with sodium pentobarbital (100 mg/kg), injected i.p.. The right humerus, both femora, lumbar vertebrae (L3-LS) and the right tibia were removed.
2o Blood chemistry Serum calcium, phosphate, alkaline phosphatase, creatinine and urea nitrogen were measured by computerized multichannel serum analysis.
HistomorQhometry 25 Histomorphometric measurements were done on cortical bone of tibial midshaft and on cancellous bone of L3. After sacrifice, these bones were removed from each animal and fixed in 10% neutral buffered formalin for 24 hours. The tissues were dehydrated in a graded series of alcohols (70-100%, 2 changes per grade, each for 4 hours under vacuum). The specimens were then placed in xylene, 3o and infiltrated with methylmethacrylate under vacuum at 20 psi on 2 hours/step and 24 hours infiltration schedule in a Shandon Hypercenter automatic processor (Shandon Lipshaw, Pittsburgh, PA). The specimens were embedded in 2% DDK-* Trademark plast with 0.2% initiator (Delaware Diamond Knives, Vi'ilmington, DE). Cross-sections of tibia were cut at 80 ~m using a diamond wire saw (Delaware Diamond Knives, Ine., DE) and stained with Goldner's trichrome. Unstained cross sections approximately 80 pm thick were processed for dynamic histomorphometry of 5 fluorochrome labels. Sagittal sections of L3 were cut on Reichert-Jung 2050 microtome (Magee Scientific Ine., Dexter, MI) at S~m and stained with McNeal's tetraehrome, or left unstained for dynamic histomorphometry.
Histomorphometry was done at 1 SOx magnification, using a IvTikon fluorescence microscope (OptiphotNikon, Tokyo, Japan) and a semi-automatic digitizing system (Bioquani IV, R&M Biometrics, Nashville, TN). Bone formation and resorption in the periosteal, endocorticaI and intracortical envelopes were measured across the entire cross-sectional area of the mid-diaphyseal sections of the tibia. Measurements on cancellous bone were made within a 6 mm--area in the center of the lumbar vertebra, 0.5 mm from the margin of the surrounding cortical shell. The nomenclature was in accordance with the ASBMR Committee on histomorphometric nomenclature (Parfitt AM, Drezner MK, Florieux FH, Kanis JA, Malluche H, Meunier PJ, Ott SM, Recker RR 1987 "Bone histomorphometry:
standardization of nomenclature, symbols, and units. Report of the ASBMR
Histomorphometry Nomenclature Committee". J. Bone Miner. Res. 2:595-610.) Dynamic parameters were measured based on the calcein label.
* Trademark Bone mass measurements The mid-shaft of femora and the fourth lumbar vertebra in 50%
ethanol/saline were scanned in cross-section by quantitated computer tomography (QCT or pQCT) employing a 960A pQCT and analyzed using Dichte software version 5.1 (Norland/Stratec, Ft. Atkinson, WI). Whole tissue parameters were measured including volumetric bone mineral density (vBMD, mg/cm'), cross-sectional area (X-Area, mmz), and bone mineral content (BMC, mg), using voxel dimensions of 148 x 148 x 1200 um. Volume can be calculated by multiplying X-Area by the slice thickness of 1.2 mm. The entire femoral neck of excised femora in to a bath of 50% ethanol, saline were scanned using a peripheral dual energy absorptiometry (pDEXA, Norland/Stratec). Specifically, apparent bone mineral density (aBMD, g/cm2), projected area (cm2) and bone mineral content (BMC, g) were measured using scan steps of 0.5 x 1.0 mm and threshold of 0.04.
Biomechanical testing Bone mechanical properties were measured in the right femoral midshaft and the body of L5. Bones were resected, cleaned of connective tissue, wrapped in gauze soaked in isotonic saline and frozen at -20°C until testing.
Prior to testing, specimens were thawed for 1-2 hours at room temperature. All specimens were 2o tested to failure in a circulating water bath at 37°C using an MTS
810 servohydraulic testing machine (MTS Corp., Minneapolis, MN). Load-deformation curves were recorded using the HP 7090A measurement plotting system (Hewlett Packard, Camas, WA). Ultimate force (maximum force that specimens sustain), stiffness (the slope of the linear portion of the load-deformation curve) and work to failure (area under the load-deformation curve before failure) were measured using a digitizer system (Jandel Scientific, Corte Madera, CA). These parameters are structural properties which depend on intrinsic material properties and geometry. Turner CH, Burr DB 1993 "Basic biomechanical measurements of bone: a tutorial." Bone 14:595-608. The data were normalized to obtain intrinsic material properties such as 3o ultimate stress (maximum stress that specimens sustain), elastic modulus (material intrinsic stiffness), and toughness (resistance to fracture per unit volume) which are independent of size and shape. Id.

i WO 00/10596 t fi PCT/l,'S99I18961 Femoral bone strength was measured at the midshaft using three-point bending. The femur was positioned on a fixture with the anterior side facin;
toward the loader. Load was applied on the mid-point between two supports that were ~~
mm apart. The load cell was displaced at the rate of 1 mmisec until failure occurred.
To normalize the data obtained from the load-deformation curve, bending ultimate stress was calculated from ultimate force by of FuLr/8I (1) where a~ is bending fracture stress, Fu is the ultimate force, L is the length between supports, r is the radius in anterior-posterior direction, and I is the moment of inertia.
1 o Id. The value for the moment of inertia was calculated, assuming that the femoral cross-sections were elliptical.
Average cortical thickness was calculated from thiclatess measurements made in each of four quadrants of the femoral cross-section with a pair of digital calipers, accurate to 0.01 mm with a precision of t 0.005 mm (Mitutoyo, Japan).
Elastic modulus of the femur (Et) was calculated using the following equation:
E~{stiffness)*(L'/48I) (2) Toughness of the femur (Toughnessf) was also calculated using the following equation:
2o Toughnessf 3*(work to failure) *r'-/LI (3) For the mechanical testing of fifth lumbar vertebra (LS), both end plates of the vertebral body were cut parallel using a Buehler Isomet slow speed saw (Buehler LTD, Evanston, IL). After resection of the posterior processes, mechanical strength of L5 was measured in compression. The compressive load was applied in stroke control, with a cross-head speed of lmm/sec through a pivoting platen to correct for nonparallel alignment of the faces of the vertebral body. To normalize the data obtained from the load-deformation curve, and to evaluate intrinsic material properties that are independent of bone geometry, ultimate stress was calculated as the ultimate force divided by the gross cross-sectional area.
Cross-sectional area (CSA) was calculated by CSA=nab/4 (4) *.Trademark where a and b are the width in the anterior-posterior and medial-lateral directions, respectively).
Elastic modulus of the vertebra (E~) was calculated by E~=(stiffness)/(CSA/h) (S) where h is the cranio-caudal height of vertebral body.
Toughness of the vertebra (Toughness) was calculated by Toughness=(work to failure)n(CSA*h) (6) Acoustic microscopy i0 500-pm thick cross-sections were cut from the mid-diaphysis of the right humerus using a diamond wire saw. Precise thickness of each specimen was measured using a micrometer (Mitutoyo, Japan) at a resolution of 1 wm.
Acoustic velocity measurements were made using a scanning acoustic microscope (UH3, Olympus, Japan) by the method described previously by Hasegawa K, Turner CH, I5 Recker RR, Wu E, Burr DB 1995 "Elastic properties of osteoporotic bone measured by scanning acoustic microscopy". Bone 16:85-90. Using this technique, detailed intrinsic mechanical properties at a selected focal point can be measured. A
50 MHz transducer (V-390, Panametrics, Waltham, MA) was used to generate acoustic waves in pulse-echo mode. The 50 MHz lens produced an acoustic beam, 2o approximately 60 pm in diameter. Specimens were fixed to the bottom of a chamber filled with water at constant temperature {22°C). Delay time between acoustic waves reflected from the top of the specimens and those reflected from the bottom of the specimens was measured using a digital oscillosocope (TDS 620, Tekronix, Beaverton, OR). Delay times were measured at five different locations such that 25 each site was more than 300 pm from each other in the anterior cortex of the humerus. Acoustic velocity was calculated as twice the thickness of specimens, divided by the average delay time. Wet weight (Ww) and submerged weight (Ws) in 100% ethyl alcohol were measured using a balance (AJ100, Mettler Instrument Corp., Heightstown, NJ). Wet density (p) was calculated using Archimedes's 30 principle:
p={Wwl(Ww-Ws)} *pETOH (7) where pETOH is the density of alcohol (0.789 g/cm3). Assuming the acoustic wave pathway in bone as homogenous, the elastic coefficient (C) representing the intrinsic stiffness of the specimens is calculated:
C-p*v2 {8) where p is wet density and v is acoustic velocity.
Statistical analysis Bartlett analysis was used to check homogeneity of variance. When variance was homogeneous, one-way ANOVA with Fisher's PLSD tests for post-hoc 1o comparison was applied. When variance was not homogeneous, Kruskal-Wallis non-parametric analysis of variance was applied, with post-hoc analysis using Mann-Whitney's U-tests. Statistical significance was ascribed at p<0.05. Results are presented as mean ~ SEM.
Results Body weight and biochemistry Rabbits treated with vehicle PTH(1-34) at 10 mg/kg/day exhibited minor increments in body weight over 140 days. Rabbits given PTH(1-34) at 40 ug/kg/day exhibited a small decrement of 51 g in body weight, representing a 1.4 ~ 1.6%
loss 2o in body weight during the experiment (Table 1). Serum measures were within the normal physiological response for rabbits, although small increases in serum calcium and urea nitrogen were observed. Serum alkaline phosphatase increased by 2-fold at the higher PTH{1-34) dose (Table 2).
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Histomorphometry Bone formation on periosteal (Ps.MSBS) and endocortical (Ec.MSBS) surfaces of the tibial midshaft increased in PTH(1-34) treated groups (Table 3).
Ps.MSBS in the higher dose group was significantly greater than in the other 2 groups (p<0.001) and Ec.MSBS in the higher dose group was significantly greater than in the control group (p<0.05). Consistent with the increase in serum alkaline phosphatase, bone formation rates on each surface (Ps.BFRBS and Ec.BFRBS) were significantly greater in the higher dose group than the other 2 groups (p<0.05).
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Intracortically, the number of resorption sites (Rs.N/Ct.Ar) in rabbits given PTH(1-34) at 40 pglkg/day was significantly greater by 7-fold, than in the other 2 groups (p<0.05) (Table 4). The number of labeled osteons (L.On.N/Ct.Ar) in rabbits given PTH(1-34) at 40 pglkg/day also significantly increased compared to the other 2 groups (p<0.41 vs the control group, p<0.05 vs 10 pglkg/day group). MAR was significantly greater in both treatment groups than in the control group {p<0.01), but there was no significant difference between the PTH-treated groups. Bone formation rate (BFRBV) and activation frequency (Ac.F) increased (p<0.05 and p<0.01, respectively) at both doses.
2o Although bone area (B.Ar) increased at both doses, a significant difference was only found between the higher dose group and the control group (p<0.01).
Marrow area (Ma.Ar) decreased after treatment, but was not significantly different among the three groups. However, cortical area (Ct.Ar) in the higher dose group was significantly greater than the other 2 groups (p<0.0001 vs the control group, p<0.05 vs the lower dose group). Ct.Ar in the lower dose group was also significantly higher than the control (p<0.05). Similar results were found in %Ct.Ar.
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WO 00/10596 2~ PCT/US99/18961 In cancellous bone, most of the formation parameters (OSBS, Ob.SBS, OV/TV, and MSBS) increased with PTH(1-34) treatment (Table 5). Those in rabbits given PTH(1-34) at 40 ~tg/kg/day were significantly greater than in the other 2 groups (p<0.01 vs both the control group and 10 uglkg/day group in all parameters). Bone formation rate (BFR/BS) also significantly increased in rabbits given PTH(1-34) at 40 pg/kg/day compared to the other 2 groups (p<0.0001 vs both the control and 10 ~.glkg/day groups). Although resorption (ESBS and Oc.SBS) increased in both PTH(1-34) treated groups, only eroded surface (ESBS) in the higher dose group was significantly greater than the control group (p<0.001}.
There 1o were no differences in osteoid thickness (O.Th) among the three groups.
Despite the evidence for accelerated bone turnover, fractional bone volume (BV/TV) did not change after PTH(1-34} treatment. Tunneling resorption and peritrabecular fibrosis were not observed in any of the groups.
Bone mass measurements vBMD and BMC in the midshaft of the femur assessed by pQCT in 40 p.g/kg/day group were significantly higher than in the other 2 groups (p<0.001 in vBMD and p<0.0001 in BMC vs the control group, p<0.05 in vBMD and p<0.01 in BMC vs the lower dose group) (Fig. 1A). vBMD and BMC in 10 uglkg/day group were also significantly higher than in the control group (p<0.05 in both vBMD
and BMC). Although bone area of the midshaft of the femur also increased dose-dependently, it significantly increased only in 40 pg/kglday group (p<0.05).
aBMD and BMC in the proximal femur, measured by dual X-ray absorbtiometry (DXA or pDXA), increased dose-dependently. Significant differences were present in both aBMD and BMC between the control group and 10 pg/kg/day group (p<0.05) as well as between the control group and 40 pglkg/day group (p<0.001) (Fig. 1B). No significant differences were found in bone area among the three groups.
Overall, Figure 1 shows that BMD (bone mineral density) and BMC (bone 3o mineral content) in the femoral midshaft (cortical bone) (A) and in the proximal femur (cancellous bone + cortical bone) (B) were significantly greater in PTH-treated animals than controls at both doses. Cortical bone area at the femoral midshaft in rabbits treated at the higher dose was significantly greater than controls.
No significant differences were found between groups in bone area of the proximal femur. Data are expressed as mean ~ SEM. *P<0.05 compared with the control.
tP<0.05 compared with PTH 10 ~glkg/day.
There were no significant differences in vBMD, BMC or bone area of the lumbar vertebra (L4) assessed by pQCT, among the three groups.
Biomechanical testing Structural properties of the midshaft of the femur, such as ultimate force, to stiffness and work to failure increased dose-dependently (Fig. 2). Figure 2 shows the effects of PTH on mechanical strength and cross sectional moment of inertia (CSMI) in the cortical bone of the femoral midshaft. Structural mechanical properties (open bars) and CSMI increased significantly in the higher dose group, while stiffness also increased significantly in the lower dose group. Of the intrinsic 15 material properties (dark bars), only elastic modulus increased significantly in the lower dose group when compared to controls. Elastic modulus in the higher dose group was significantly decreased when compared to the lower dose group. In Figure 2: data are expressed as mean ~ SEM; * indicates P<0.05 compared with the control; and ~ indicates P<0.05 compared with 10 ~g/kg/day.
2o In this study and the results shown in Figure 2, all parameters were significantly higher in rabbits given PTH(1-34) at 40 ~glkg/day than in the control group (p<0.01 for ultimate force and work to failure, p<0.05 for stiffness).
Stiffness in the lower dose group was also significantly higher than in the control group (p<0.05). Of the intrinsic material properties, elastic modulus was significantly less 25 in rabbits given 40 ug/kg/day than those given 10 ug/kglday {p<0.01).
In the lumbar vertebral body, no significant differences were found in mechanical properties among the three groups.
Acoustic microscopy 3o There were no significant differences in acoustic velocity or elastic coefficient among the three groups.

Discussion The skeletal response of cortical bone to biosynthetic hPTH(1-34) involved both a direct regulation of material properties and a compensatory regulation of biomechanical properties in the long bones of intact, mature female rabbits.
PTH(1-34) increased bone turnover and cortical porosity and, at the 40 p.g/kg dose, reduced the material elastic modulus of cortical bone. However, the decreased elastic modulus was more than compensated by increased bone apposition on periosteal and endocortical surfaces, resulting in a significant improvement in the structural strength, stiffness and work to failure of cortical bone in rabbits.
to In this study using intact rabbits, cancellous bone volume of the lumbar vertebra did not change after PTH(1-34) treatment despite the evidence for increased bone turnover. Previous use as an osteopenic model, presence of intracortical remodeling, and short remodeling period--together with the rabbit's rapid growth and early skeletal maturation (by 6-9 months), formed the basis for selection of the rabbit 15 as a model in which to test the effects of intermittently administered PTH(1-34).
Rabbits can exhibit a wide variation in serum calcium levels (10-16 mg/dl), but these levels are not directly influenced by the amount of dietary calcium, another advantage of the model. Although transient significant increases of approximately 1 mg/ml were recorded in rabbits treated with PTH(1-34) at 40 pglkg, the actual 2o values were always within the known physiologic range.
In the current study, biosynthetic hPTH(1-34) for 140 days increased bone formation rate intracortically as well as on periosteal and endocortical surfaces.
Intracortical Ac.F increased in the lower dose group by 8X and in the higher dose group by 20X. This led to a 2-fold increase in cortical porosity in the tibia in the 25 lower dose group and a 6-fold increase in the higher dose group. The data from acoustic microscopy shows that elastic properties of the bone material itself in the humerus was not affected, indicating that intrinsic cortical bone quality is normal.
Therefore, the increased porosity must account for the slight reduction in elastic modulus, a material properties measurement that includes the spaces in the cortex.
3o Increased cortical porosity was more than compensated, however, by significantly increased MSBS and BFR/BS on both periosteal and endocortical surfaces in the midshaft of the tibia in the higher dose group, resulting in significantly increased bone area. This would increase the cross-sectional moment of inertia, which is proportional to the bone's bending rigidity, as it did in the femoral midshaft (Fig. 2). The consequence of these changes in shape and material properties was to improve the mechanical strength and stiffness of the femoral diaphysis when compared to controls, thus offsetting the potentially deleterious mechanical effects of increased cortical porosity.
Conclusion In conclusion, the increases in bone turnover and cortical porosity after 1o PTH(1-34) treatment were accompanied by concurrent increases in bone at periosteal and endocortical surfaces. The combination of these phenomena resulted in an enhancement of the toughness ultimate stress, stiffness, and work to failure of the femur.
i5 Example 2 - - Increased Bone Strength and Density Upon Administration of rhPTH(1-34) to Monkeys Experimental Procedures General The live phase of the study used feral, adult (closed growth plates) 2o cynomolgus primates (Macaca fascicularis), weighing 2.77~0.03 kg (mean ~
standard error of the mean [SEM]). Monkeys were held in quarantine for 3 months, then started on a diet containing 0.3% calcium, 0.3% phosphate, and 250 IU
vitamin D3/100 g diet, and given fluoridated water (1 ppm fluoride) ad libitum. The calcium content corresponded to 1734 mg calcium/2000 calories. After 1 month on the diet, 25 animals were sorted into groups of 21 or 22, sham operated or ovariectomized.
Once daily subcutaneous injections of vehicle (sham and ovariectomized controls) or rhPTH(1-34), at 1 ~glkg (PTH1) or 5 ~glkg (PTHS), were started 24 hours after ovariectomy. Animals were treated for either 18 months (PTH1 and PTHS), or for 12 months followed by withdrawal of treatment (PTH1-W and PTHS-W).
3o The study groups were divided as shown in Table 6.

Table 6 - - Study Groups for Primate Study MonkeyMonkeys in s Final Abbreviaat Analyses lion Uutset(n=121) (n=128) Sham ovariectomized, 18 monthsSham 21 21 vehicle Ovariectomized, 18 months OVX 22 20 vehicle Ovariectomized, 18 months PTH1 21 19 i pg rhPTH(1-34~kglday Overiectomized, 12 months PTH1-W21 20 lltg rhPTH(1-34/kg/day, 6 months vehicle Ovariectomized, 18 months PTHS 22 21 S~g rhPTH(1-34ykglday Ovariectomized, 12 months PTHS-W21 20 Sltg rhPTH(1-34)/kg/day, months vehicle Serum and urinary samples were taken 24 hours after vehicle or rhPTH(1-34) injection at 3-month intervals. A sparse sampling design of 5 monkeys in each rhPTH(1-34)-treated group was used for pharmacokinetics, with sampling (spanning 0 to 240 minutes each time) at baseline, 7, 11, and 17 months. At 0 time and at 6-month intervals, total skeleton and spine (L-2 to L-4) bone mass were assessed by dual-energy x-ray absorptiometry (DXA); peripheral quantitative computed x-ray to tomography (pQCT) was used to assess bone mass in the midshaft and distal radii, and the proximal tibia. Iliac biopsies were taken at 6 and 15 months for histomorphometry. All animals were euthanized after 18 months.
Biomechanical tests were done on lumbar vertebrae L-3 to L-4, the femur neck, humerus midshaft, and on a cortical bone specimen machined from the femur diaphysis (measures defined in Table 7). Conventional static and dynamic histomorphometry were done (measures described in Table 11 ) on the humerus midshaft, lumbar vertebra L-2, femoral neck, femur midshaft, the radius midshaft and distal radius. Initial statistical analyses compared all groups to vehicle-treated ovariectomized controls. The data is suitable for additional exploratory analyses to 2o examine dose dependency, effects of withdrawal, interactions between outcomes, and changes in time by methods known to those of skill in the art. All assays were conducted and determined by methods known in the art.
For certain experimental subjects, cortical bone of the humerus was examined by histomorphometry and by polarized Fourier transform infrared microscopy. The Fourier transform infrared microscopy was conducted by an adaptation of known methods for such microscopy.

3D Finite Elernent Modeling Studies These studies determined 3D finite element modeling data on vertebra from monkeys of the study dosed with PTH for 18 months. Excised L-5 vertebra in 50%
ethanol/saline from the ovariectomized (n=7) and PTH (n=7) groups were serially scanned in 500 um steps by quantitative computed tomography (QCT, Norland, Ft.
Atkinson, WI), using 70x70 ~m pixels. Each of the 500 pm cross-sections was analyzed for volumetric bone mineral density (BMD, mg/cc), bone mineral content (BMC, mg), cross-sectional area (X-Area), cancellous bone volume (BV/TV), to trabecular thickness (Tb.Th), and connectivity (node density, strut analysis). Pixels in each serial scan were averaged to create 490x490x500 ~tm voxels. The serial scans were then stacked and a triangular surface mesh generated for each bone using the "marching cubes" algorithm (see e.g. Lorensen and Cline 1987 "Marching cubes, a high resolution 3D surface construction algorithm." Computer Graphics 21, 15 169). A smoothed version of each surface mesh was then used to generate a tetrahedral mesh for 3D finite-element modeling.
Young's modulus for each tetrahedral element was derived from the original voxel densities and material properties from a beam of cortical bone milled from the femoral diaphysis of the monkeys. Each tetrahedral mesh was rotated so that the 20 bottom surface of each vertebra was aligned with a plane. Linear elastic stress analysis was then performed for each L-S model in which a distributed load of was applied to the top surface of the centrum, perpendicular to the bottom plane while the bottom surface was fixed in the direction of loading. The resulting axial strain contours were evaluated, as were the BMD distributions, and compared 25 between PTH and ovariectomized. At this resolution, the density of each voxel is dependent on the extent to which each voxel is filled with bone as opposed to soft tissue.
Results 3o Reports of differences in the text are statistically significant, p<0.05.
All animals gained 4% to 9% of initial body weight during the study independent of treatment.

Serum and Urine Measures Serum estradiol levels at 3 and 18 months were below 5 pg/mL in all ovariectomized monkeys. When measures of calcium homeostatis were compared to s sham controls, ovariectomized controls had lower serum calcium and phosphate and 1,25-dihydroxyvitamin D levels, but did not differ in endogenous PTH, urinary cyclic adenosine monophosphate (CAMP), urinary calcium, urinary creatinine, or serum urea nitrogen measured 24 hours after last injection. Animals treated with rhPTH(1-34) had lower serum phosphate, lower endogenous PTH, and higher 1,25-dihydroxyvitamin D and urinary cAMP compared to ovariectomized. Serum bone formation marker assays showed that ovariectomized monkeys had low serum total alkaline phosphatase (ALP) and osteocalcin compared to shams, and rhPTH(1-34) restored levels back to sham values. Urinary C-telopeptide (CrossLaps) excretion, used as a biochemical marker of bone resorption, was not altered by rhPTH(1-34) compared to ovariectomized controls.
Bone Mass Overall skeletal bone mass, expressed as total body BMC, was increased significantly by PTH(1-34) (Fig. 3). Spine bone mineral density (BMD) remained 2o stable in ovariectomized controls for 18 months, while sham controls gained approximately 5% above baseline (Figs. 4A-4C and SA). rhPTH(1-34) increased spine BMD by 7% to 14% and whole body bone mineral content (BMC, Figure 3) by up to 6% compared to baseline (Figs. 4A-4C and SA). Spine bone mineral content also increased (Fig. 5A). In rhPTH(1-34)-treated primates, the magnitude of these increases was significantly higher than that of ovariectomized controls, and matched (PTH1) or exceeded (PTHS) that of shams. rhPTH(1-34) did not alter BMD of the midshaft or distal radius. The cross-sectional area of the midshaft increased by 7% in the PTHS group. In the proximal tibia, there was no increase in cross-sectional area but rhPTH(1-34) increased BMC and BMD compared to ovariectomized controls. Six months after treatment was withdrawn, BMD and BMC in the spine and femur neck remained higher than ovariectomized controls, with no change in the cortical midshaft of the humerus.

Bone Strength rhPTH(1-34) increased strength (FY) in the vertebrae by up to 43% (Tables 7 and 8, Fig. 5B). rhPTH(1-34) improved strength in the femur neck (f,~ by up to 12%
(Tables 7 and 9, Fig. 6A). rhPTH(1-34) did not alter measures in the cortical diaphysis of the humerus midshaft (Tables 7 and 10), or the material properties of beam specimens machined from the femoral diaphysis (Tables 7 and 9, Fig. 6B) when compared to ovariectomized controls. In animals treated with rhPTH( 1-34) for 12 months and then withdrawn from treatment for 6 months, bone strength measures remained significantly higher than ovariectomized controls (Tables 7-10, Figs. 5B and 6A).
Table 7 Variables Reported for the Third and Fourth Lumbar Vertebrae (L-3 and L-4), Humerus Midshaft, Proximal Femoral Neck, and Femoral Beam Specimens Variable Units Descri lion Lumbar Vertebrae, L-3 and L-4 A mm Cross-sectional area F,. N Yield force is the force at a 0.2% offset S N/mm Slope of the linear portion of the force-displacement curve stiffness M a Yield stress E M a Youn 's modulus Humerus Midshaft t mm Aver a cortical thickness F N Ultimate force is the maximum force the specimen can withstand S N/mm Slope ofthe linear portion ofthe force-displacement curve stiffness here's stiffness mJN N-mm Area under the load-dis lacement curve U=work to failure Proximal Femoral Neck F N Ultimate force is the maximum force the specimen can withstand Femur Dia h sical Beam S cimens a" M a Ultimate stress E G Youn 's modulus a J/m Tou hness E Ultimate strain Table 8 Biomechanical Measures of Strength in the Spine (Lumbar Vertebrae, L-3 and L-4 Combined) of Ovariectomized Primates at 18 Months Variable units Sham OVX ControlPTH 1 PTH PTHS PTHS-W
' 1-W

A mm' 90.5*2.186.7t2.388.3*2.090.9t2_387.3*2.782.8*2.1 * 52 * 94' * 105 * 73 * 77'~ * 59 S /mm 7312 5805 7701 7401 8012 7074 * 319 * 476' * 474 * 452 * 367 f 314 Q~ 19.4 17.3 21.9 21.1 24.6 21.9 M * 0.6 * L0 * 1.3 * 0.8 * 1. t 0.9 sa l'~

E M 650 * 546 * 717 * 659 759 * 698 *
a 32 49 48 * 42 36 41 Abbreviations: OVX=ovariectomized; PTH1=rhPTH(1-34) 1 p.glkg for 18 months;
PTH 1-W= withdrawal for 6 months after treatment with rhPTH( 1-34) 1 pg/kg for months; PTHS=rhPTH(1-34) 5 ltg/kg for 18 months; PTHS-W=withdrawal for 6 months after treatment with rhPTH(1-34) 5 ~.g/kg for 12 months.
aSee Table 4.1 for description of variables bData expressed as mean ~ standard error of the mean (SEM) per group.
°Statistically significant compared to OVX controls (p<0.05).
SStatistically significant compared to sham controls (p<0.05).
Table 9 Biomechanical Measures of Material Properties of Equivalent Size l0 Beam Specimens from Femur Diaphysis, and Biomechanical Measure of Strength of Femur Neck of Ovariectomized Primates at 18 Months Variable -units'Sham OVX ControlPTH1 PTH1-W PTHS PTHS-W

6 M 222 f 21615 22214_ 21416 206 f 20816 a 5 6 E G 17.210.616.410.4 17.1f0.4 16.610.6 15.410.6'15.3f0.6' a a 5.9103 5.810.4 6./10.4 S.St0.4 5.4f0.4 6./10.4 mJlm3 0.035 0.035 0.036 0.034 f 0.034 0.038 t 0.001 f 0.002 t 0.002 0.002 t f 0.002 .002 Proximal _ femur _ neck F ~ 1288 1105 f 1235 t 1258 f 1362 1213 f f 41 53' ~ 45 ~ 52 ~ t 30 42 ~

Abbreviations: OVX=ovariectomized; PTH1=rhPTH(1-34) 1 p.g/kg for 18 months;
15 PTH1-W= withdrawal after treatment with rhPTH(1-34) 1 pg/kg for 12 months;
PTHS=rhPTH(1-34) 5 pglkg for 18 months; PTHS-W=withdrawal after treatment with rhPTH{ 1-34) 5 p.g/kg for 12 months.
aSee Table 4.1 for description of variables bData expressed as mean ~ standard error of the mean (SEM) per group.
20 °Statistically significant compared to OVX controls (p<0.05).
SStatistically significant compared to sham controls {p<0.05).
Table 10 Biomechanical Measures of Cortical Bone of the Midshaft of the 25 Humerus of Ovariectomized Primates at 18 Months Variable units'Sham OVX ControlPTH1 PTHI-W PTHS PTHS-W

mm 1.74 I .63 f 1.68 1.66 f 1.80 f 1.72 t t 0.04 0.03' t 0.04 0.04 0.05 0.03 F 725 f 636 t 26 654 689 f 680 f 15 707 t 26 t 23 ' 24 S /mm 601 t 520 t 26 54 4 573 t 548 t 18 573 f 23 t 20 24 U mJ 1797 1542 t 1641 1751 t 1804 f 1775 f t 85 92 t 84 99 113 Abbreviations: OVXwariectomized; PTH1=rhPTH(1-34) 1 p,glkg for 18 months;
PTHI-W= withdrawal after treatment with rhPTH(1-34) 1 p,g/kg for 12 months;
PTHS=rhPTH(1-34) 5 p,g/kg for 18 months; PTHS-W=withdrawal after treatment 3o with rhPTH(1-34) 5 p.g/kg for 12 months.
aSee Table 4.1 for description of variables bData expressed as mean t standard error of the mean (SEM).
°Statistically significant compared to OVX controls {p<0.05).
gStatistically significant compared to sham controls (p<0.05).
Bone Histomorphometry Although turnover rates were greater in ovariectomized than sham controls, there was no significant loss of bone volume in the iliac crest. As the tetracycline label given at 6 months was not detectable in many animals, only static parameters were measured for this time point. Static and dynamic histomorphometry data at months showed that treatment with rhPTH(1-34) increased cancellous bone area compared to ovariectomized, and increased bone formation without increasing resorption measures above those measured in ovariectomized controls. Bone formation rate was increased progressively by higher doses of rhPTH(1-34).
Although cancellous bone remained increased compared to ovariectomized controls after withdrawal of rhPTH(1-34) following 12 months of treatment, bone formation and resorption reverted to that seen in ovariectomized controls, and bone turnover remained higher than in sham controls. rhPTH(1-34) did not affect mineralization, activation frequency, or remodeling periods. There were no differences in individual bone multicellular unit (BMLJ)-based bone balance between resorption and formation. In summary, rhPTH(1-34) increased cancellous bone by selective stimulation of bone formation.
In the cortical bone of the humerus, where rhPTH(1-34) did not significantly modify BMD or bone strength measures, rhPTH(1-34) stimulated changes in the periosteal, endosteal, and intracortical compartments (Tables 11 and 12).
Although 2o there were no differences in total area or medullary area between groups, rhPTH(1-34) increased cortical area, and the PTHS and PTHS-W groups had significantly more cortical bone, suggestive of increased cross-sectional moment of inertia, a measure of strength. The increase in area could be attributed to increased formation on both periosteal and endosteal surfaces (Fig. 7).
Sham controls and PTHS-W groups had reduced periosteal mineralizing surfaces compared to ovariectomized controls and the other rhPTH(1-34)-treated groups. Endocortical mineralizing surfaces were significantly greater in ovariectomized controls compared to shams and rhPTH(1-34) did not increase above ovariectomized control values. In intracortical remodeling, there were more 3o resorption spaces in ovariectomized animals, and activation frequency was greater in ovariectomized, PTH1, and PTHS groups than in sham controls or either of the withdrawal groups. There were significantly more labeled osteons per unit area in WO 00/10596 3~ PCT/US99/18961 ovariectomized compared to sham controls, and rhPTH(1-34) did not increase these significantly above ovariectomized control values.
Intracortical porosity was greater in ovariectomized compared to shams, but not different between ovariectomized controls and PTH 1. PTHS and PTHS-W
increased porosity above that seen in ovariectomized controls. Data from the rabbit studies suggested the hypothesis that the increase in porosity, accompanied by increased cortical bone, may be a structural response to maintain the biomechanical properties of rhPTH(1-34)-treated bone. There were no differences between ovariectomized and the other groups in formation period, osteoid width, wall width, or osteoid maturation at 18 months.
In summary, there were no differences in turnover rates between ovariectomized controls and either dose of rhPTH(1-34). Sham controls had a lower turnover rate than either ovariectomized controls or rhPTH(1-34)-treated animals.
When rhPTH(1-34) was withdrawn for 6 months, turnover rates decreased significantly, but BMD and biomechanical strength measures remained higher than ovariectomized controls. Normal values for osteoid width and maturation time intracortically for all groups indicates treatment did not cause any defect in the normal timing of the mineralizing process. Normal values for wall width indicate that treatment did not alter the normal balance between resorption and formation at 2o the level of the individual BMU.

Table 11 Histomorphometric Variables for Cortical Bone Measurements of the Humerus Variable' Units I7escri lion Ac.F cles/ Activation ar fre uenc BFRBS.Ec da Bone formation rate endocortical surface referent BFRBS.Ps da Bone formationriosteal surface rate, referent BFR/BV / ear Bone fonnationbone volume referent rate FP da s Formation riod L.On.N/Ct.A#/mm Number of fluorochrome labeled osteons r unit cortical area MAR da Mineral a ition rate intracortical MAR.Ec da Mineral a sition rate, endocortical surface MAR.Ps da Mineral a ition rate riosteal surface MS/BS.Ec % Mineralizing endocortical surface normalized to total endocortical surface MS/BS.Ps % Mineralizing periosteal surface normalized to total periosteal surface O.Wi Osteoidwidth ' Rs.NICt.A #/mm Number of reso lion s aces r unit cortical area W.Wi Osteonal wall width omt da s Osteoid maturation time Po % Porosi the rcenta a of bone area occu ied b s aces B.Ar mm Bore; area, the total area within the eriosteal surface Ct. Ar mm Cortical area, the area of bone within the periosteal surface includes rosities Mc.Ar mm Medull cavi area 'Nomenclature is that recommended in Journal of Bone and Mineral Research, 1987.

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v, o The analysis by histomorphometry and polarized Fourier transform infrared microscopy revealed that administration of PTH improved bone quality by replacing old bone (large crystallites) with young bone (range of sized crystallites, tending to smaller size). Further, upon withdrawal of PTH from monkeys given low doses, 5 there is an additional benefit as the matrix becomes optimally mineralized, and the crystallites mature. The data derived from histomorphometry and Fourier transform infrared microscopy show an unexpected benefit on 'bone quality of the cortical bone as optimal mineralization occurs the mineral phase matures.
1o 3D Finite Element Modeling Studies Examination of the middle 500 pm slice of L-5 showed a 21 % increase in BMD for PTH compared to ovariectomized that was due to a 27% increase in BMC
with no change in the cross sectional area. Analysis of the centrum from PTH
showed a 73% increase in BV/TV that was due to a 30% increase in Tb.Th and 37%
15 greater Tb.N, compared to ovariectomized.. Connectivity analysis for this region showed a 140% greater node density (nodeltissue volume) and 286% greater node-to-node struts for the PTH vertebra.
Histogram distribution analysis of bone voxel densities for PTH showed a decrease in the proportion of low densities (0-355 mg/cc), an increase in middle 2o densities (356-880 mg/cc), with little effect on the high density voxels (887-1200 mg/cc), compared to ovariectomized (Fig. 8). Most striking was the shift to a greater bone voxel density in the cortical bone compartment following withdrawal of treatment for 6 months (Fig. 8).
The proportion of vertebral bone elements (voxels) that fell within a certain 25 range of BMD values was calculated. The BMD ranges chosen were the following:
low BMD, 0-300 mg/cc; medium BMD, 300-700 mg/cc; high BMD, 700-1000 mg/cc; and cortical BMD, >1000 mglcc (Table 13). Compared to ovarectomized controls, PTH treatment significantly decreased the volume of low BMD bone and increased the volume of medium BMD bone. After withdrawal of PTH, there was a 3o decrease in medium BMD bone and an increase in high BMD bone, indicating that medium BMD bone became more dense.

wo oonosv6 ~cTntsvv,nsv61 Table 13. Percentages of LS vertebral volume grouped by BMD values (mean~SEM) Treatment low BMD medium BMD hteh BI~1D cortical 1~:) (,-01 l.ol BhID i.pi ovanectomized 30.41:,?48.711.6 1Q.9*l.? 0.9~U.:

PTH 17.711.6'58.9f 1.9' ~2.8=?.7 0.61U.:

PTH-W 22.4f 49.711 ? 2 7 .0t 0.830.2 1.3' Lh' S
Table 14. BMC at the midlevel of the LS vertebra and vertebral effective strain Treatment BMC lmg) ettecnve strain lustram I

ovartectomized 37.211.6 701164 PTH 47.411.5' 447136' PTH-~1' 44.21 t 539134' .2' *statistically different (p<0.0S by Fisher's PLSD test) The data summarized in Figure 8 show that L-S vertebra from cvnomolgus 1o monkeys treated for 18 months with PTH respond with significant increases in bone mass, trabecular thickness, and trabecular connectivity, with marginal effects on the outer dimensions (X-Area) of the vertebra. Analysis of the distribution of bone elements in L-S showed that the heavily mineralized bone regions change the least with no evidence of bone sclerosis. Rather, it is the porous trabecular bone that 1S responded the most to PTH. The shift in BMD led to a substantial reduction in the axial strain, indicating mechanical improvement. As clearly shown in the histograms of PTH and ovariectomy BMD, PTH converted the low density bone voxels into medium density voxels with no significant effect on the high density voxels.
2o The data summarized in Table 14 show that the BMC through the middle of the vertebrae was significantly increased by PTH treatment and a beneficial effect of PTH remained after 6 months of withdrawal. The average mechanical strain in the vertebra was reduced 36% by PTH treatment and remained 23% below OVX after withdrawal of PTH. This study indicates that withdrawal of PTH treatment for 6 25 months did not lead to resorption of newly formed bone, but instead there was a beneficial redistribution of medium density bone into lower and higher density bone.
This redistribution led to continued strain reduction in the vertebrae and thus improved mechanical function.

Discussion This primate study indicates that PTH, given in the absence of other medicines that might affect bone, benefits both cortical and trabecular bone to increase overall skeletal bone mass. Moreover, withdrawal of PTH did not result in significant loss of benefits associated with PTH treatment over a period of at least 2 remodeling cycles.
Surrogate markers have been used in other trials to indicate activity in bone, and they assumed that changes in value reflect changes in bone mass. Although 1o there are published data on humans and primates to show that both formation and resorption markers increase, consistent with activation of bone turnover, for example, during early menopause or in active disease states, the high turnover is considered to be indicative of bone loss. In adolescence, high turnover during maturation of the human skeleton has been less well studied, but is accompanied by an anabolic gain in bone mass. Such a phenomenon would be totally unexpected in drug therapy of osteoporosis, according to current art. Thus, the increase in bone turnover markers is inconsistent with the known anabolic effect of PTH to increase bone mass and strength, as shown by data in the present study.
The data from this 18 month study on cynomolgus monkeys supports the 2o following unexpected findings:
~ Overall significant increase in total skeletal mass ~ Significant increase in bone mass and strength at the femur neck.
~ No evidence of "steal" from cortical bone to increase trabecular bone.
Increase in bone mass and strength were statistically significant at sites enriched for either cortical bone (femur neck) or trabecular bone (lumbar vertebrae). At purely cortical bone sites (femur mid-diaphysis), there was a trend for PTH to stabilize or slightly increase bone mass and strength, compared to ovariectomized controls.
~ Changes in bone markers in ovariectomized monkeys (and humans) do 3o not reflect the beneficial, anabolic effects of PTH on the skeleton. Use of body fluids from primates in the present study allows development of new and more valid surrogate markers.
~ Retention of the gain in bone mass and strength for at least 2 remodeling cycles after withdrawal of treatment.
This PTH primate study differs from published studies on rhesus and cynomolgus monkeys in that it used a large sample size to provide appropriate statistical power to detect differences that may not have been apparent in previous much smaller studies; controls included both ovariectomized primates (used in published studies) and sham operated, but intact primates. The latter control group to has not been previously reported in this type of study, so that some of the benefits of PTH, and the restoration of certain measures to sham control levels, compared to values for ovariectomized animals were assessed for the f rst time.
Conclusions 15 This 18-month study in mature, feral, ovariectomized (OVX) cynomolgus monkeys, Macaca fascicularis, assured eff cacy and safety in bone following treatment with rhPTH(1-34) for either 12 months followed by withdrawal of treatment for 6 months, or treatment for 18 months. rhPTH{1-34) significantly increased bone mass and strength of the spine and femur neck above ovariectomized 2o controls to levels equivalent to or greater than those of sham controls. In ovariectomized monkeys treated with rhPTH(1-34), measures of calcium homeostatic (serum calcium, phosphate, and 1,25-dihydroxyvitamin D) were restored to that of sham controls. Serum, urine, and histomorphometry measures used to assess bone turnover showed rhPTH(1-34) maintained formation rates 25 equivalent to or higher than those of ovariectomized controls, while biochemical markers of bone resorption remained equivalent to those of shame controls. In all animals treated with rhPTH(1-34) for up to 18 months, pharmacokinetic measures did not change with time, and there was no accumulation of rhPTH( 1-34). There was no evidence of sustained hypercalcemia or kidney pathology after 18 months of 3o treatment. There were no changes in mineralization or remodeling periods.
The net gain in skeletal bone mineral content observed with rhPTH(1-34) may be explained WO 00/10596 PCT/US991189b1 by increased bone formation rate and bone forming surface with little or no effect on bone resorption. There were significant increases in bone mineral content, bone mineral density and biomechanical measures of strength, including toughness and stiffness, at clinically relevant sites such as the spine, femur neck and proximal tibia.
rhPTH(1-34) increased the rate of turnover in cortical bone of midshaft of the humerus and radius, but did not significantly alter bone mass or biomechanical measures of strength compared to either ovariectornized or sham controls.
However, the increase in cortical width and/or cortical bone area is consistent with an increase in cross-sectional moment of inertia, a measure of strength and stiffness.
rhPTH(1-34) had no significant effects on the intrinsic material properties of cortical bone.
Endocortical bone formation was stimulated, thus increasing cortical width and intracortical porosity. It appears that these changes in porosity are responsible for maintaining the elasticity of the bone.
In monkeys, 12 months of treatment with rhPTH(1-34) followed by withdrawal for 6 months was associated with smaller, but still significant, gains in bone mass and strength in the spine and femur neck. Following withdrawal, no significant effects were noted on the cortical bone midshaft of the humerus and radius. Bone markers and histornorphometry showed trends to return to the low turnover values measured in sham controls.
In vivo mechanistic studies in rodents showed that genes associated with anabolic outcomes of rhPTH(1-34) are upregulated within 1 to 6 hours, and the increase in bone forming surfaces can be detected within 24 hours after the first dose in the absence of detectable effects on resorption. rhPTH(1-34) appears to recruit osteoprogenitors in S-phase, and stimulate their differentiation into osteoblasts, thereby rapidly increasing the percent bone forming surfaces. Single or multiple injections of rhPTH(1-34) may be given within a 1-hour period to induce the anabolic effect in bone. However, when the equivalent dose is given in young rats as multiple injections over 6 hours or 8 hours, the anabolic effect was abrogated, suggesting that brief, limited exposure to rhPTH(1-34) is required to induce the anabolic effect.
In summary, rhPTH(1-34) is anabolic on the bone in monkeys and rabbits, increasing bone mass and biomechanical strength measures at clinically relevant sites such as the lumbar spine and femur neck by selective stimulation of bone formation. The increases in bone turnover, endocortical surface formation, and porosity, detected by histomorphometry at cortical sites, did not alter bone mass or 5 biomechanical measures of bone strength, but did increase the cross sectional moment of inertia by increasing cortical bone area and/or width.
These studies demonstrate that administration of parathyroid hormone receptor activators, such as recombinant human PTH(1-34) improve bone quality both during and following treatment. In fact, administering PTH once daily for 1o months, or at the same doses for 12 months followed by a 6 month withdrawal phase, showed marked improvement of the quality of cortical bone of the humerus as analyzed by histomorphometry and polarized Fourier transform infrared (FTIR) microscopy. This analysis revealed that administration of PTH improved bone quality by replacing old bone (large crystallites) with young bone (range of sized is crystallites, tending to smaller size). Thus, administration of PTH can increase cortical bone quality, improve mineralization, and accelerate mineralization and replacement of old bone by new bone.
Further, upon withdrawal of PTH from monkeys given low doses, there is an additional benefit as the matrix becomes more optimally mineralized, and the 2o crystallites mature. That is, at low doses, PTH can have additional benefits during the withdrawal phase of treatment by enhancing mineralization. These data indicate benefits of a finite regime of treatment with PTH followed by a withdrawal period to achieve enhanced benefit. Current definitions of bone quality do not include these aspects of improved mineralization.
25 In earlier studies of a treatment phase of PTH followed by a phase of no treatment, the treatment phase was less than 1 month. The prolonged but finite treatment phase of 18-24 months followed by a period of at least 2 remodeling cycles has not previously been explored. The continued benefit in primates after withdrawal of treatment is in marked contrast to results achieved in rodents upon 3o dosing with PTH. Studies of rats have uniformly shown that bone is rapidly lost following withdrawal of treatment. Gunness-Hey, M. and Hock, J. M. (1989) Bone 10: 447-452; Shen, V. et al. (1993) J. Clin Invest 91: 2479-2487; Shen, V. et al.
(1992) Calcif. Tissue Int. 50: 214-220; and Mosekilde, L. et al. (1997) Bone 20:
429-437.
Such a method of enhancing bone mineralization has not previously been observed and is unexpected, revealing a new method by which PTH strengthens and toughens bone and can prevent fractures. This new method includes enhancing and regulating mineralization, to provide tougher, stiffer, more fracture resistant bone.
Such beneficial effects require more than new matrix formation. These findings indicate that PTH has benefits in patients with immobilized bones or skeletons, or in l0 skeletons deficient in mineral, provided there is also adequate calcium and vitamin D supplementation.
Example 3 - - Increased Bone Strength and Density, and Reduced Fractures Upon Administration of rhPTH(1-34) to Humans IS
Number of Subjects: rhPTH(1-34): 1093 enrolled, 848 finished.
Placebo: 544 enrolled, 447 finished.
Diagnosis and Inclusion Women ages 30 to 85 years, postmenopausal for a minimum of Criteria: 5 years, with a minimum of one moderate or two mild atraumatic vertebral fractures.
Dosage and Administration: Test Product (blinded)_ rhPTH(1-34): 20 pg/day, given subcutaneously rhPTH(1-34): 40 wg/day, given subcutaneously Reference Therapy fblinded~
Placebo study material for injection Duration of Treatment: rhPTH(1-34): 17-23 months (excluding 6-month run-in phase) Placebo: 17-23 months (excluding 6-month run-in phase) Criteria for Evaluation: Spine x-ray; serum biological markers (calcium, bone-specific alkaline phosphatase, procollagen I carboxy-terminal propeptide); urine markers (calcium, N-telopeptide, free deoxypyridinoline); 1,25-dihydroxyvitamin D; bone mineral density: spine, hip, wrist, and total body; height; population pharmacokinetics; bone biopsy (selected study sites).

s EPOM MERCHANT ~ GOULD CA 02325371 'iooo-o9-ii00 9:25/ST. 9;20/N0.4260606912 P

Patient Characteristics Placebo PTH-20 1'T>-I-40 (N=544) (N=541) (N=552) p-value Caucasian 98.9% 98.9% 98.4% 0.672 Age 69.07.0 69.57.1 69.96.8 0.099 Years post menopausal 20.98.5 21.518.7 21.88.2 0.273 Hysterectomized 23.8% 23.1% 21.6% 0.682 Uterus + 0 or 1 ovary 57 51 58 Uterus + 2 ovaries 61 57 51 Unlrnown 11 17 10 Previous osteoporosis drug 14.9% 15.5% 13.0% 0.479 use Baseline spine BMD U.82-fi0.170.820.17 0.820.17 >0.990 Baseline # of vert, fx >0.990 U 54 (10.4%) 45 (8.8%) 54 (10.1%) 1 144 (27.8%)159 (31.1 169 (31.6%) %) 2 128 (24.7%)128 (25.0%)125 (23.4%) 3 75 (14.5%) 67 (13.1%) 81 (15.1%) 4 s9 (11.4%) 49 (9.G%) 45 (8.4%j 28 (5.4%) 31 (6.1%) 21 (3.9%) 6 13 (2.5%) 20 (3.9%) 25 (4.7%) 7 6 (1.2%) 7 (1.4%) 10 (1.9%) ' $ 9 (1.7%) 5 (1.0%) 3 (0.6%) 9 I (0.2%) 0 2 (0.4%) 1 (0.2%) 1 (0.2%) 0 Unspecified 26 29 17 5 Results Data from this clinical trial including a total of 1637 women treated with recombinant human parathyroid hormone (1-34), rhPTH(1-34) 0, 20, or 40 ~,glday, and supplemented with vitamin D and calcium, for 18-24 months, showed results reported in Tables 15-19.
Table 15 illustrates data showing the reduction upon treatment with PTH of the munher and severity of vertebral fractures. Comparing all PTH treated patients with placebo, the overall reduction in number of patients with vertebral fractures was 67%
{p<0.001), with a GS% reduction (p<0.001) at 20 pg/day P'IH compared to placebo, and a 69%
reduction at 40 ~g/day PTH compared to placebo (Table 15j. Comparing all PTIi treated patients with p1 acebo, the overall reduction in number of patients with multiple vertebral fractures was 81 % (p<0.001 ), with a 77% reduction (p<0.001) at 20 p,g/day PTH compared to placebo, and a 86% reduction at 40 p,g/day PTH compared to placebo. Comparing all PTH treated patients with placebo, the overall reduction in number of patients with moderate to severe vertebral fractures was 84% (p<0.001), with a 90% reduction (p<0,001) at 20 ~.g/day PTH compared to placebo, and a 78% reduction at 40 pg/day PTH compared to placebo (Table 15).
Table 15. Effect of treatment with PTH on number and severity of vertebral fractures.
Placebo 20 pg/day PTH 40 ~glday PTH

(n*=448) (n=444) (n=434) Number and 64 22 19 percentage of (14.3%) (5.0%) (4.4%) patients with new vertebral fractures Number and 22 5 3 percentage of (4.9%) (1.1%) (0.7%) patients with 2 or more new vertebral fractures Number and 42 4 9 percentage of (9.4%) (0.9%) (2.1 %) patients with new moderate to severe fractures**

*n = number of patients with both baseline and endpoint x-rays 1o ** Moderate fracture results in more than 25% loss of vertebral height (or an equivalent measure). Severe fracture results in more than 40% loss of vertebral height (or an equivalent measure). Fractures are as defined by Genant et al. (1993) Vertebral fracture assessment using a semiquantitative technique; J. Bone & Min Res 81137-1148.
Table 16 illustrates the effect of treatment with PTH on the number of fractures at various non-vertebral bones throughout the body. The number of fractures apparently decreased at each of the hip, radius, ankle, humerus, ribs, foot, pelvis, and other sites (Table 16). The reduction is statistically significant when 2o viewed as the reduction in the total number of fractures among the PTH
treated patients compared to the placebo treated patients. The reduction is even more significant when considered as the reduction in the total number of fractures of hip, radius, ankle, humerus, ribs, foot, and pelvis among the PTH treated patients compared to the placebo treated patients (Table 16).
Table 16. Effect of treatment with PTH on number of non-vertebral fractures.
p-values Placebo PTH-20 PTH-40 (N=544) (N=541) (N=552)Overall PTH-pbo* 20-pbo 40-pbo Hip 4 2 3 0.718 0.474 0.417 0.690 Radius 13 7 10 0.404 0.236 0.180 0.504 Ankle 4 2 2 0.601 0.313 0.417 0.403 Humerus 5 4 3 0.767 0.534 0.744 0.465 Ribs 10 S 5 0.277 0.109 0.197 0.184 Foot 4 1 4 0.374 0.474 0.181 0.983 Pelvis 3 1 0 0.171 0.076 0.319 0.081 Other 16 14 9 0.338 0.296 0.723 0.146 Total 53 34 32 0.024 0.007 0.036 0.015 Total 41 21 24 0.013 0.003 0.010 0.025 w/o "Other"

*Placebo (pbo) The effect of PTH on bone mineral content (BMC), bone mineral density (BMD), and bone area were determined by dual energy absorptiometry (DEXA), and the results are reported in Tables 17-19. PTH administration caused apparent 1o increases in BMC at the patient's lumbar spine, femur and hip, wrist, and throughout the patient's whole body (Table 17). Treatment with PTH caused significant increases in the patient's BMD at the lumbar spine, femur and hip (Table 18).
The increases at the lumbar spine, femur and hip were statistically significant with p<0.001 (Table 18). Bone area apparently increased upon PTH treatment for the patient's lumbar spine, femur and hip (Table 19). The increases were statistically significant for the lumbar spine and hip neck (Table 19).
The effect of PTH on the whole body the measure of bone quantity and quality, BMC, is particularly significant. This whole body effect indicates that the amount of bone in the patient's body is increasing. PTH does not merely result in 2o moving bone mass from one portion of the patient's body to another.
Instead, treatment with PTH increases the amount and quality of bone in the patient's body.

Figures 9 and 10 illustrate the increases over time in lumbar spine BMD and femurlhip neck BMD, respectively, for PTH treated and placebo control patients.
The patient's lumbar spine BMD increases steadily for at least about 18 months, with no or a less significant increase over the subsequent months. The patient's femur/hip s BMD apparently increases for at least 18 months, and may increase upon further duration of PTH treatment.
Table 17. Effect of PTH on bone mineral content expressed as endpoint % change (SD) from baseline Placebo PTH-20 PTH-40 p-value Lumbar spine 1.60 (6.92)11.85 (8.83)16.62 ( <0.001 11.1 ) Femur/Hip Total -0.38 (5.18)3.50 {6.26) 4.78 (6.70)<0.001 Neck -O.s 1 2.99 (7.26) 5.80 (8.71)<0.001 (7.06) Trochanter 0.98 (14.97)5.68 (15.58)6.53 (15.33)<0.001 Intertrochanter -0.23 (6.28)3.59 (7.32) 4.99 (7.79)<0.001 Ward's triangle 0.01 (14.75)5.36 (14.78)8.86 (17.02)<0.001 Wrist Ultradistal -1.67 (7.44)-0.25 (6.53)-1.88 (7.97)0.184 1/3 radius -1.19 (6.12)-I.37 (4.51)-3.04 (6.09)0.025 Whole body -0.74 (4.76)1.30 (4.48) 2.28 (5.44)<0.001 to Table 18. Effect of PTH on bone mineral density expressed as endpoint % change (SD) from baseline Placebo PTH-20 PTH-40 p-value Lumbar spine 1.13 (5.47) 9.70 (7.41 13.7 (9.69)<0.001 ) Femur/Hip Total -1.01 (4.25)2.58 (4.88)3.60 (5.42)<0.001 Neck -0.69 (5.39)2.79 (5.72)5.06 (6.73)<0.001 Trochanter -0.21 (6.30)3.50 (6.81)4.40 (7.45)<0.001 Intertrochanter-1.29 (4.53)2.62 (5.52)3.98 (5.96)<0.001 Ward's triangle-0.80 (11.73)4.19 (11.93)7.85 (13.24)<0.041 Wrist Ultradistal -1.89 (7.98)-0.05 (7.14)-1.76 (7.20)0.108 1/3 radius -1.22 (3.37)-1.94 (4.07)-3.17 (4.62)0.001 1s Table 19. Effect of PTH on bone area expressed as endpoint % change (SD) from baseline Placebo PTH-20 PTH-40 p-value Lumbar spine 0.46 (2.97)2.52 (3.52)3.34 (3.72)<0.001 Femur/Hip Total 0.54 (3.02)0.84 (3.16)1.05 (2.98)0.144 Neck 0.04 (4.60)0.27 (4.91)0.81 (5.56)0.035 Trochanter 0.95 (12.75)1.99 (12.16)1.92 (11.30)0.197 Intertrochanter 1.01 (5.17)1.01 (4.99)1.01 (4.89)0.964 Ward's triangle 0.44 (7.60)1.13 (7.34)0.99 (8.06)0.309 Wrist Ultradistal 0.25 (6.40)-0.25 (6.00)-0.39 (4.80)0.653 1/3 radius -0.02 (5.73)0.52 (3.40)0.01 (4.42)0.586 In summary, the data presented above indicate that patients treated with PTH
have reduced fractures. Specifically, PTH treatment reduced by more than 66%
the number of patients with prior vertebral fractures who suffered new vertebral fractures. PTH treatment also reduced by more than 78% the number of patients with prior vertebral fractures who suffered new, multiple vertebral fractures.
In 1 o addition, PTH decreased the severity of vertebral fractures, with a significant reduction by 78% in the number of patients with moderate or severe fractures.
Patients receiving PTH benefited from a significant reduction in all non-vertebral fractures (including fractures of hip, radius, wrist, pelvis, foot, humerus, ribs or ankle) with significance at a level of p<0.007. Bone quality increases as well.
Patients with prior fracture benefited from a significant increase in bone mineral content of the hip, spine and total body. This increase indicates that fracture reduction at these sites can occur as early as after 12 months of therapy.
Discussion 2o These data on fractures are the first data on fracture reduction by PTH in humans. These findings demonstrate an improvement in bone quality and bone strength, like the preclinical data reported hereinabove. These results also show benefits in bone quality and strength at non-vertebral sites. The findings of a reduction in the numbers of fractures sustained during the 18-23 month period of treatment has not previously been observed in clinical or preclinical studies.

The question of whether PTH alone increases toughness and strength of bone to improve resistance to fracture has not previously been tested in humans.
The published literature has consistently suggested that PTH must be given in combination with an anti-resorptive or estrogen. Previous, published clinical trials included patient populations too small to determine a significant reduction of fracture. In one study the benefits of PTH alone could not be assessed because there were no placebo controls. In a second study, employing the commonly accepted definition of fracture, no reduction in fracture was observed.
The findings of a reduction of fractures at combined non-vertebral sites is to particularly unexpected in light of the common belief that PTH has negative effects at such sites. Common dogma holds that PTH will increase cortical porosity and therefore weaken bone, especially early in therapy. Further, this dogma asserts that cortical bone sites are at high risk of fracture and that PTH will offer no benefit in fracture reduction at non-vertebral sites. The dogma also holds that PTH alone is t5 unlikely to be efficacious and will require concurrent anti-resorptive therapy to block negative effects on cortical bone. The present data demonstrate the previously unobserved benefits of PTH given to patients receiving vitamin D and calcium supplements. Unexpectedly, PTH strengthens bone to reduce the number of new fractures in a patient at risk for multiple fractures of the spine, at risk for additional 2o non-vertebral fractures, at risk for moderate to severe additional fractures of the spine, and the like.
This clinical study on post-menopausal women showed particular benefits from treating patients with low dose (20 pg/day) since the dose of PTH (which, at high doses, could show side effects in some patients) was reduced, but fracture 25 prevention and fracture reduction was retained, and similar to those noted at the high dose (40 p.g/day). The FT-IR monkey data provide a possible, but not limiting, mechanistic explanation. The monkey study shows that low dose PTH increased crystal formation and accelerated mineralization in cortical bone. In addition, low dose monkeys showed additional benefits after withdrawal, as PTH enhanced 3o mineral content of the bone. The present data demonstrate the novel finding that PTH given at low doses to patients receiving vitamin D and calcium supplements, is effective in preventing both vertebral and non-vertebral fractures. Contrary to popular belief, PTH strengthens bone at non-vertebral sites to prevent new fractures or reduce the severity of fractures, apparently by improving the mineralization and mineral content of the bone.
The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention. All publications and patent applications in this specification to are indicative of the level of ordinary skill in the art to which this invention pertains.

Claims (10)

CLAIMS:

1. Use of a parathyroid hormone consisting of amino acid sequence 1-34 of human parathyroid hormone for the manufacture of a medicament for concurrently reducing the risk of both vertebral and non-vertebral bone fracture in a man or postmenopausal woman at risk of or having osteoporosis, in a daily dose of 20 µg to 40 µg, without concurrent administration of an antiresorptive agent other than vitamin D or calcium.

2. The use according to claim 1 wherein said daily dose is 20 µg.

3. The use according to claim 1 wherein said human subject is at risk or has osteoporosis arising from an age-related hypogonadal condition.

4. The use according to claim 1 wherein said human subject is a postmenopausal woman.

5. The use according to any one of claims 1 to 4 wherein said medicament is used for at least about 12 months up to 3 years.

6. Use of a parathyroid hormone consisting of amino acid sequence 1-34 of human parathyroid hormone for concurrently reducing the risk of both vertebral and non-vertebral bone fracture in a man or postmenopausal woman at risk of or having osteoporosis, in a daily dose of 20 µg to 40 µg, without concurrent administration of an antiresorptive agent other than vitamin D or calcium.

7. The use according to claim 6 wherein said daily dose is 20 µg.

8. The use according to claim 6 wherein said human subject is at risk or has osteoporosis arising from an age-related hypogonadal condition.

9. The use according to claim 6 wherein said human subject is a postmenopausal woman.

10. The use according to any one of claims 6 to 9 wherein said use is for at least about 12 months up to 3 years.