WO2006033912A2 - Traitement des affections osseuses a l'aide de medicaments anabolisants du squelette - Google Patents

Traitement des affections osseuses a l'aide de medicaments anabolisants du squelette Download PDF

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WO2006033912A2
WO2006033912A2 PCT/US2005/032706 US2005032706W WO2006033912A2 WO 2006033912 A2 WO2006033912 A2 WO 2006033912A2 US 2005032706 W US2005032706 W US 2005032706W WO 2006033912 A2 WO2006033912 A2 WO 2006033912A2
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pthrp
bone
pth
day
patient
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PCT/US2005/032706
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WO2006033912B1 (fr
WO2006033912A3 (fr
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Andrew F. Stewart
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Osteotrophin Llc
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Priority to EP05797693A priority Critical patent/EP1799240A4/fr
Priority to AU2005287139A priority patent/AU2005287139A1/en
Priority to MX2007003185A priority patent/MX2007003185A/es
Priority to CA002580281A priority patent/CA2580281A1/fr
Priority to JP2007532415A priority patent/JP2008513459A/ja
Publication of WO2006033912A2 publication Critical patent/WO2006033912A2/fr
Publication of WO2006033912A3 publication Critical patent/WO2006033912A3/fr
Publication of WO2006033912B1 publication Critical patent/WO2006033912B1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/29—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/12—Drugs for disorders of the metabolism for electrolyte homeostasis
    • A61P3/14—Drugs for disorders of the metabolism for electrolyte homeostasis for calcium homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/18—Drugs for disorders of the endocrine system of the parathyroid hormones

Definitions

  • the present invention relates generally to methods for the prevention and treatment of a variety of mammalian conditions manifested by loss of bone mass, including osteoporosis. More particularly, the present invention relates to methods of using PTHrP, or an analog thereof, for the treatment of metabolic bone disorders that are effective and have an increased safety.
  • bone Throughout adult life, bone continually undergoes remodeling through the interactive cycles of bone formation and resorption (bone turnover). Bone resorption is typically rapid, and is mediated by osteoclasts (bone resorbing cells), formed by mononuclear phagocytic precursor cells at bone remodeling sites. This process then is followed by the appearance of osteoblasts (bone forming cells), which form bone slowly to replace the lost bone. The fact that completion of this process normally leads to balanced replacement and renewal of bone indicates that the molecular signals and events that influence bone remodeling are tightly controlled.
  • the mechanism of bone loss is not well understood, but in practical effect, the disorder arises from an imbalance in the formation of new healthy bone and the resorption of old bone, skewed toward a net loss of bone tissue.
  • This bone loss includes a decrease in both mineral content and protein matrix components of the bone, and leads to an increased fracture rate of the femoral bones and bones in the forearm and vertebrae predominantly. These fractures, in turn, lead to an increase to general morbidity, a marked loss of stature and mobility, and in many cases, an increase in mortality resulting from complications.
  • a number of bone growth disorders are known which cause an imbalance in the bone remodeling cycle.
  • Osteoporosis or porous bone
  • osteoporosis is a disease characterized by low bone mass and structural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures of the hip, spine, and wrist. It is a devastating disease among both postmenopausal women as well as among older men.
  • the costs at the national level for medications and hospitalizations are estimated to be in the $50,000,000 per year range at present and are likely to increase as the US population ages.
  • the mainstays of therapy are oral calcium supplements, vitamin D supplements, and a family of medications termed "anti-resorptives" which reduce osteoclastic bone resorption.
  • estrogens such as conjugated estrogens (Premarin®); selective estrogen receptor modulators (SERMs), such as raloxifene (Evista®); calcitonin (Miacalcin®); and bisphosphonates, such as alendronate (Fosamax®), risedronate (Actonel®), etidronate (Didronel®), pamidronate (Aredia®), tiludronate (Skelid®), or zoledronic acid (Zometa®).
  • Osteoporosis exists, in general, when skeletal mineral losses are in the range of 50% below peak bone mass, which occurs at approximately age 30. Seen from the perspective of correcting the deficit in bone mineral, complete reversal of this 50% loss would require a 100% increase in bone mass. Thus, seen from this perspective, the 2-8% increases in bone mineral density which result from anti-resorptive therapy, while clinically significant and beneficial, leaves very significant room for improvement. Since the use of anti-resorptives to prevent bone loss does not result in new bone production, the ultimate effectiveness of anti-resorptives in quantitative terms is limited. These considerations emphasize the need for the development of pharmaceutical mechanisms to produce new bone.
  • parathyroid hormone is a very effective new member of such a new osteoporosis therapeutic armamentarium.
  • PTH parathyroid hormone
  • PTH was first identified in parathyroid gland extracts in the 1920's. The complete amino acid sequence of PTH was determined ih the 1970's. Because patients with overproduction of parathyroid hormone (i.e., hyperparathyroidism) develop a decline in bone mass (sometimes very severe), PTH has widely been seen as a catabolic skeletal agent over the past century. However, both animal and human studies have now clearly demonstrated that when administered subcutaneously as a single daily dose, (so called "intermittently" - in contrast to the continuous overproduction of PTH which occurs in patients with hyperparathyroidism), PTH can induce marked increases in bone mineral density and bone mass.
  • parathyroid hormone i.e., hyperparathyroidism
  • PTH is very different from the anti-resorptive class of drugs. See, Finkelstein et al, N Engl J Med 331: 1618-1623 (1994); Hodsman et al, JCUn Endocrinol Metab 82: 620-28 (1997); Lindsay et al, Lancet 350: 550-555 (1997); Neer et al, N Engl J Med 344: 1434-1441 (2001); Roe et aU Program and Abstracts of the 81st Annual Meeting of the Endocrine Society, p. 59 (1999); Lane et al, JCUn Invest 102: 1627-1633 (1998).
  • spine bone mineral density was reported to be increased by as much as 30%, when assessed using dual energy x-ray absorptiometry (DXA), and as much as 80% when using quantitative computerized tomography (QCT) of lumbar spine trabecular bone ⁇ see, Roe et al., Program and Abstracts of the 81st Annual Meeting of the Endocrine Society, 59 (1999)).
  • DXA dual energy x-ray absorptiometry
  • QCT quantitative computerized tomography
  • PTH has recently been demonstrated to have significant anti-fracture efficacy, both at the spine and at non-vertebral sites. PTH has been shown to reduce fractures by between 60% and 90% depending on the skeletal site and the definition of fracture. Neer et al, NEnglJMed344: 1434-1441 (2001).
  • PTH appears to be the first member of a new class of anti-osteoporosis drugs, which in contrast to the anti-resorptives, have been termed the skeletal "anabolic" class of osteoporosis drugs, or "anabolics.”
  • PTHrP Parathyroid hormone-related protein
  • PTHrP is the product of a gene distinct from that which encodes PTH.
  • PTHrP shares approximately 60% homology at the amino acid level with PTH in the first 13 amino acids, and then the sequences diverge completely.
  • PTHrP is initially translated as a pro-hormone that then undergoes extensive post-translational processing.
  • PTHrP-(I -36) One of the processed forms, or authentic secretory forms, as identified in the inventor's laboratory, is PTHrP-(I -36). ⁇ u et al, J Biol Chem 271: 24371-24381 (1996). PTHrP-(l-36) binds to the common PTH/PTHrP receptor, also termed the PTH-I receptor, in bone and kidney. Everhart-Caye et al, J Clin Endocrinol Metab, 81: 199-208 (1996); Orloff et al, Endocrinology, 131: 1603-1611 (1992).
  • PTHrP-(l-36) binds to this receptor with equal affinity to PTH, and activates the PKA and PKC signal transduction pathways with equal potency as PTH.
  • PTHrP was originally identified by the inventor (Burtis et al, J Biol Chem 262: 7151-7156 (1987); Stewart et al, Biochem Biophys Res Comm 146: 672-678 (1987)) and others (Strewler et al, J Clin Invest, 80: 1803, (1987); Moseley et al, Proc. Natl. Acad. ScI USA. 84: 5048-5052 (1987)) through its role as the causative agent for the common human paraneoplastic syndrome termed humoral hypercalcemia of malignancy (HHM). Stewart et al, N Engl J Med 303: 1377-1383 (1980).
  • the present invention provides methods for the prevention and treatment of a variety of mammalian conditions manifested by loss of bone mass, including osteoporosis.
  • the invention is based on the surprising observation that the administration of very high doses of a PTHrP, or a related analog, can produce drastic increases in BMD in a very short time period.
  • the period of administration is preferably 15, 18, 21, 24, 30, or 36 months, more preferably 7, 8, 9, 10, 11, or 12 months, and most preferably 1, 2, 3, 4, 5, or 6 months.
  • the high doses of the skeletal anabolic drug do not produce any significant adverse side effects when administered for short periods of time or at intermittant dosing intervals. Accordingly, the methods of the present invention offer greater safety by substantially eliminating or reducing the risk of negative side effects commonly associated with skeletal anabolic drags, such as hypercalcemia, renal failure, hypotension, or the risk of developing osteogenic sarcomas.
  • the rates of increase in BMD achieved with the methods of the present invention are extremely rapid. In one embodiment, three months of treatment with PTHrP-(I -36) yielded rates of increase in BMD that were greater than any rates previously obtained with anti-resorptives and lower doses of PTH for longer periods of administrations.
  • the rates of increase in BMD achieved with the methods of the present invention are preferably at least 1% per month, 1.1% per month or 1.2% per month, more preferably 1.3% per month or 1.4% per month, and most preferably over 1.5% per month or 1.6% per month.
  • the increases in BMD observed are not generally obtained with anti-resorptives for two to three years of administrations.
  • the present invention provides methods for the prevention and treatment of bone disorders using skeletal anabolic drugs that are both safe and effective.
  • the resulting increase in BMD achieved with the methods of the present invention preferably results in T-scores >-2.5, more preferably results in T-scores >-2.0, and most preferably results in T-scores >-1.0. Furthermore, the resulting increase in BMD achieved with the methods of the present invention preferably prevents fractures resulting preferably in at least a 50%, 60%, or 70% reduction in incidence of fractures, more preferably in at least a 75%, 80%, or 85% reduction in incidence of fractures, and most preferably in at least a 90% or 95% reduction in incidence of fractures.
  • the present invention provides methods of increasing bone mass in an animal or a human patient by administering intermittently to the patient PTHrP, or an analog thereof, at a dosage between 5 ⁇ g/day and 50 mg/day or greater.
  • a preferred dose range is 10-45,000 ⁇ g/day.
  • Other preferred dose ranges include 25-40,000 ⁇ g/day, 35-37,500- ⁇ g/day, 50-35,000 ⁇ g/day, 100-30,000 ⁇ g/day, 150-25,000 ⁇ g/day, 200-20,000 ⁇ g/day, 250-15,000 ⁇ g/day, 300-10,000 ⁇ g/day, 350-7,500 ⁇ g/day, 400-3,000 ⁇ g/day, 400-1,500 ⁇ g/day, 400-1,200 ⁇ g/day, 400-900 ⁇ g/day, 400-600 ⁇ g/day, 80-500 ⁇ g/day, 90-500 ⁇ g/day, 100-500 ⁇ g/day, 150-500 ⁇ g/day, 200-500 ⁇ g/day, 250-500 ⁇ g/day, 300-500 ⁇ g/day, 350-500 ⁇ g/day, 400-500 ⁇ g/day, and 450-500 ⁇ g/day.
  • PTHrP-(I -36) is administered at a intermittant dosages between 50 and 10,000 ⁇ g/day.
  • a more preferred dose range is 200-7,500 ⁇ g/day.
  • An even more preferred dose range is 400-5,000 ⁇ g/day.
  • Other preferred dose ranges include 400-1,500 ⁇ g/day, 400-1,200 ⁇ g/day, 400-900 ⁇ g/day, and 400-600 ⁇ g/day (approximately 6.5-18 ⁇ g/kg/day, 6.5-15 ⁇ g/kg/day, 6.5-12 ⁇ g/kg/day, and 6.5-9 ⁇ g/kg/day).
  • the present invention also provides methods for increasing bone density using administration of PTHrP, or analogs thereof, for periods of time longer than previously administered in animals or humans.
  • the present invention provides methods of increasing bone mass in an animal or a human patient by intermittently administering PTHrP, or an analog thereof, for a period of between 1-36 months.
  • the period of administration is preferably 15, 18, 21, 24, 30, or 36 months, more preferably 7, 8, 9, 10, 11, or 12 months, and most preferably 1, 2, 3, 4, 5, or 6 months.
  • the methods of the invention can be employed with a patient afflicted with, or at risk of, a metabolic bone disorder including primary or secondary osteoporosis, osteomalacia, renal osteodystrophy, and other types of skeletal disorders with associated bone loss.
  • a metabolic bone disorder including primary or secondary osteoporosis, osteomalacia, renal osteodystrophy, and other types of skeletal disorders with associated bone loss.
  • the rates of increase in BMD achieved by the methods of the present invention are at least 1.5% per month.
  • the PTHrP, or an analog thereof can be administered to a patient afflicted with a bone fracture, e.g., a compound fracture or a simple fracture.
  • Preferred embodiments include administration of PTHrP -(1-34) at the dosages described above, to patients afflicted with a bone fracture.
  • the PTHrP, or an analog thereof can be administered to a surgical patient to promote bone healing following surgery, for example hip replacement surgery or cardiac surgery, or other invasive procedures that displace or damage bone.
  • Preferred embodiments of this include administration of PTHrP-(I -36) at the dosages described above.
  • Other embodiments include PTHrP, fragments or analogs thereof, administered or applied to the damaged bone in formulations of bone pastes having the dosages described above.
  • PTHrP, or an analog thereof, used in the methods of the present invention can be defined by SEQ ID NO:2; have at least 70% homology with SEQ ID NO:2; or be encoded by a nucleic acid sequence that hybridizes under stringent conditions to a complementary nucleic acid sequence of SEQ ID NO:1.
  • PTHrP analogs that can be used in the methods of this invention include fragments PTHrP-(l-30) through PTHrP-(l-173).
  • PTHrP analogs can also include analogs with a model amphipathic alpha-helical peptide (MAP) sequence substituted in the C-terminal region of hPTHrP(l-34) such as [MAP 1-10] 22-31 hPTHrP-(l-34)NH 2 ).
  • PTHrP analogs can also include peptidomimetics and small molecule drugs having skeletal anabolic agonistic biological activities, as defined herein.
  • PTHrP can be administered by subcutaneous, oral, intravenous, intraperitoneal, intramuscular, topically (on the bone surface e.g., as a paste or solution), buccal, rectal, vaginal, intranasal and ' aerosol administration. Intermittent administration may be by periodic injections once daily, once every two days, once every three days, once weekly, twice weekly, biweekly, twice monthly, and monthly. Alternatively, the use of pulsatile administration of the skeletal anabolic drug by mini-pump can be employed in the methods of the present invention. Slow or extended release matrices having PTHrP, fragments or analogs thereof, are also suitable.
  • the present invention provides methods of increasing bone mass in an animal or a human patient.
  • the method comprises administering between 1.5 and 90 mg of PTHrP, or an analog thereof, intermittently over a period ranging from one week to one month.
  • the method comprises administering between 3 and 180 mg of PTHrP, or an analog thereof, intermittently over a period ranging from one week to two months.
  • the method comprises administering between 4.5 and 270 mg of PTHrP, or an analog thereof, intermittently over a period ranging from one week to three months.
  • the method comprises administering between 9 and 540 mg of PTHrP, or an analog thereof, intermittently over a period ranging from one week to six months.
  • the method comprises administering between 18 and 1080 mg of PTHrP, or an analog thereof, intermittently over a period ranging from one week to one year. In yet another embodiment, the method comprises administering between 36 and 2160 mg of PTHrP, or an analog thereof, intermittently over a period ranging from one week to two years. In yet another embodiment, the method comprises administering between 54 and 3240 mg of PTHrP, or an analog thereof, intermittently over a period ranging from one week to three years. In still even another embodiment, the method comprises administering between 70 and 10,000 mg of PTHrP, or an analog thereof, intermittently over a period ranging from one week to three years.
  • the method comprises administering between 100 and 50,000 mg of PTHrP, or an analog thereof, intermittently over a period ranging from one week to three years.
  • the PTHrP, or analog thereof can be administered at an intermittant dosing interval of twice daily, once daily, once every two days, once every three days, once-weekly, twice-weekly, biweekly, twice-monthly, or monthly.
  • the present invention provides kit for increasing bone mass in an animal or a human patient.
  • the kit comprises between 1.5 and 90 mg of PTHrP, or an analog thereof, and written directions providing instructions for intermittent administration of PTHrP, or an analog thereof, to an animal or a human patient over a period ranging from one week to one month.
  • the kit comprises between 3 and 180 mg of PTHrP, or an analog thereof, and written directions providing instructions for intermittent administration of PTHrP, or an analog thereof, to an animal or a human patient over a period ranging from one week to two months.
  • the kit comprises between 4.5 and 270 mg of PTHrP, or an analog thereof, and written directions providing instructions for intermittent administration of PTHrP, or an analog thereof, to an animal or a human patient over a period ranging from one week to three months.
  • the kit comprises between 9 and 540 mg of PTHrP, or an analog thereof, and written directions providing instructions for intermittent administration of PTHrP, or an analog thereof, to an animal or a human patient over a period ranging from one week to six months.
  • the kit comprises between 18 and 1080 mg of PTHrP, or an analog thereof, and written directions providing instructions for intermittent administration of PTHrP, or an analog thereof, to an animal or a human patient over a period ranging from one week to one year. In yet another embodiment, the kit comprises between 36 and 2160 mg of PTHrP, or an analog thereof, and written directions providing instructions for intermittent administration of PTHrP, or an analog thereof, to an animal or a human patient over a period ranging from one week to two years.
  • the kit comprises between 54 and 3240 mg of PTHrP, or an analog thereof, and written directions providing instructions for intermittent administration of PTHrP, or an analog thereof, to an animal or a human patient over a period ranging from one week to three years.
  • the kit comprises between 70 and 10,000 mg of PTHrP, or an analog thereof, and written directions providing instructions for intermittent administration of PTHrP, or an analog thereof, to an animal or a human patient over a period ranging from one week to three years.
  • the kit comprises between 100 and 50,000 mg of PTHrP, or an analog thereof, and written directions providing instructions for intermittent administration of PTHrP, or an analog thereof, to an animal or a human patient over a period ranging from one week to three years.
  • the methods of the present invention can further comprise the step of co-administering, either simultaneously or sequentially with PTHrP, a bone resorption inhibiting agent.
  • the bone resorption-inhibiting agent can be a bisphosphonate, estrogen, a selective estrogen receptor modulator, a selective androgen receptor modulator, calcitonin, a vitamin D analog, or a calcium salt.
  • the bone resorption-inhibiting agent can also be alendronate, risedronate, etidronate, pamidronate, t ⁇ luc ⁇ ronate, zoledronic acid, raloxifene, tamoxifene, droloxifene, toremifene, idoxifene, levormeloxifene, or conjugated estrogens.
  • the patient receives intermittent administration of the skeletal anabolic drug for a three-month period of time, followed by a three-month period of treatment with a bone resorption-inhibiting agent.
  • the sequential treatment regimen could begin with a treatment period with a bone resorption inhibiting agent followed by a treatment period with the skeletal anabolic drug, that the length of sequential treatment periods can be modified (e.g., 1-18 months), and that the skeletal anabolic drug can be co-administered with the bone resorption inhibiting agent (e.g., sequential treatment period of a skeletal anabolic drug and a bone resorption inhibiting agent followed by a treatment period of a bone resorption inhibiting agent alone).
  • the bone resorption inhibiting agent e.g., sequential treatment period of a skeletal anabolic drug and a bone resorption inhibiting agent followed by a treatment period of a bone resorption inhibiting agent alone.
  • the sequential treatment periods (e.g., three months of the skeletal anabolic drug followed by three month of the bone resorption inhibiting agent) can be repeated until the patient BMD is restored (e.g., a T-score ⁇ -2.0 or -2.5 below the mean).
  • the invention includes a computer system and methods for the design of peptidomimetics and small molecule drugs having skeletal anabolic agonistic or antagonistic biological activities.
  • the system includes a processor, memory, a display or data output means, a data input means, and a computer readable instruction set having at least an algorithm capable or rendering a three-dimensional structure of a skeletal anabolic agent, fragment, or derivative thereof, as well as a receptor for such skeletal anabolic agent.
  • the system comprises a computer aided design (CAD) algorithm capable of rendering a peptidomimetic or small molecule drug based on the active sites of the skeletal anabolic agent or receptor.
  • CAD computer aided design
  • FIG. 1 is a homology alignment of human PTHrP-(l-36) with the corresponding sequence in other species, aligned to maximize amino acid identity, and wherein amino acids that differ from the corresponding amino acid in the human sequence are bolded and amino acids that are conservative amino acid substitution variants of the corresponding amino acids in the human sequence are bolded and underlined.
  • FIG. 2 is a homology alignment of human PTH-(I -34) with the corresponding sequence in other species, aligned to maximize amino acid identity, and wherein amino acids that differ from the corresponding amino acid in the human sequence are bolded and amino acids that are conservative amino acid substitution variants of the corresponding amino acids in the human sequence are bolded and underlined.
  • FIG. 3 is a homology alignment of human TIP-(I -39) with the corresponding sequence in mouse, aligned to maximize amino acid identity, and wherein amino acids that differ from the corresponding amino acid in the human sequence are bolded and amino acids that are conservative amino acid substitution variants of the corresponding amino acids in the human sequence are bolded and underlined.
  • FIG. 5 illustrates changes in bone mineral density as percent changes from baseline, following treatments with PTHrP or a placebo (PBO), as measured at the lumbar spine (L/S), the femoral neck (FN) and the total hip (TH).
  • PBO placebo
  • L/S lumbar spine
  • FN femoral neck
  • TH total hip
  • FIG. 6 illustrates bone turnover markers in the placebo and PTHrP-treated subjects.
  • FIG. 6(a) demonstrates the serum osteocalcin results expressed as change from baseline.
  • FIG. 6(b) indicates the serum N-telopeptide (NTX) values in the two groups.
  • FIG. 6(c) indicates the urinary deoxypyridinolines in the two groups. The results demonstrate that PTHrP stimulates serum osteocalcin, and by inference, bone formation, but not bone resorption.
  • FIG. 7 illustrates serum total calcium (left panel) and ionized calcium (right panel) in the PTHrP and placebo groups. There is no difference in serum total or ionized calcium between the, PTHrP and control groups, and no patient in either group developed hypercalcemia as measured by total or ionized serum calcium.
  • BMD lumbar vertebral bone mass density
  • FIG. 9 are line graphs depicting competition binding studies (Top Panels) of 125 I-[Tyr 36 ]PTHrP-(l-36)NH 2 under equilibrium conditions to human renal cortical membranes (RCM) (Panel A), SaOS-2 membranes (Panel B), and SaOS-2 intact cells (Panel C).
  • RCM renal cortical membranes
  • FIG. 10 are line graphs depicting the stimulation of adenylate cylcase activity in human renal cortical membranes (RCM) (Panel A), SaOS-2 membranes (Panel B), and SaOS-2 intact cells (Panel C) by [Tyr 36 ]PTHrP-(l-36)NH 2 ( ⁇ ), [Nle 8>18 ,Tyr 34 ]hPTH-(l-34) (o),rPTH-(l-34)
  • FIG 11 illustrates a line graph depicting the time course for binding of PTHrP and PTH peptides including 125 I[NIe 8 ' 18 , Tyr 34 ]-hPTH-(l-34)NH 2 to canine renal membranes at 2O 0 C: —o— total binding of radioligand; -- ⁇ -- binding of radioligand in the presence of 10 '6 M unlabeled bPTH-(l-34) (nonspecific binding); --•-- specific binding of radioligand. Points represent the mean ⁇ SEM of triplicate determinations. The SEM was too small to indicate in those points . without error bars. Results are representative of those obtained in three experiments.
  • FIG. 12 is a line graph depicting competition binding studies of 125 I-[NIe 8 ' 18 , Tyr 34 ] hPTH-(l-34)NH 2 to canine renal membranes at 2O 0 C with unlabeled [NIe 8 ' 18 , Tyr 34 ] hPTHh-(l-34)NH 2 (A), bPTH-(l-34) (•), and [Tyr 36 ] PTHrP-(I -36)NH 2 (o). Points represent the mean ⁇ S.E.
  • FIG. 13 is a line graph depicting competition binding studies of 125 I-[Tyr 36 ]PTHrP-(l-36) NH 2 to canine renal membranes at 2O 0 C with unlabeled [Nle 8>I8 ,Tyr 34 ]hPTH-(l-34)NH 2 (A) 5 bPTH-(l-34) (•), [Tyr 36 ]PTHrP-(l-36)NH 2 (o), PTHrP-(49-74) ( ⁇ ) and [Cys 5 ,Trp ⁇ ,Gly 13 ] PTHrP-(5-18) (Pl-PEPTIDE) (D). Points represent the mean ⁇ S.E.
  • FIG. 14 illustrates the change in femoral bone mineral content in the five groups.
  • BMC is shown as a percent change from the sham animals at each time point. Note that there is a progressive increase in femoral bone mineral content in each group of peptide-treated rats, and that the changes are highly significant in statistical terms.
  • FIG. 15 is a series of photomicrographs of the right proximal tibia following 90 days of treatment. A. Sham; B. OVX; C. SDZ-PTH-893; D. rhPTH(l-34); E. hPTHrP(l-36). Following ovariectomy, bone is lost in the proximal tibia.
  • FIG. 16 depicts selected bone histomorphometric changes during the six month period.
  • the key points are that: a) trabecular area, bone formation rate and resorption surface decline with age in the OVX groups; b) all three peptides had markedly positive effects compared to OVX controls on trabecular area and bone formation rate; and, c) despite this marked increase in bone formation rate, bone resorption rates were similar in months 1-6 among the treated and control groups.
  • FIG. 17 illustrates changes in biomechanical strength (load to failure) during the six months of treatment.
  • the key points are that:. a).jtnarked improvements in biomechanical measures occurred in all three groups for each of the three peptides; and b), improvements occurred at both predominantly trabecular and predominantly cortical sites.
  • FIG. 18 illustrates changes in serum calcium and renal calcium content during the six months. Note that rats treated with SDZ-PTH-893 developed moderate hypercalcemia, and marked increases in renal calcium content.
  • bone is continually undergoing remodeling through the interactive cycles of bone formation and resorption (bone turnover). Bone resorption is typically rapid, and is mediated by osteoclasts (bone resorbing cells), formed by mononuclear phagocytic precursor cells at bone remodeling sites. This process then is followed by the appearance of osteoblasts (bone forming cells), which form bone slowly to replace the lost bone.
  • osteoclasts bone resorbing cells
  • the activities of the various cell types that participate in the remodeling process are controlled by interacting systemic ⁇ e.g., hormones, lymphokines, growth factors, vitamins) and local factors ⁇ e.g., cytokines, adhesion molecules, lymphokines and growth factors).
  • cytokines cytokines, adhesion molecules, lymphokines and growth factors
  • the mechanism of bone loss is not well understood but, in practical effect, the disorder arises from an imbalance in the formation of new healthy bone and the resorption of old bone, skewed toward a net loss of bone tissue.
  • This bone loss includes a decrease in both mineral content and protein matrix components of the bone, and leads to an increased fracture rate of the femoral bones and bones in the forearm and vertebrae predominantly. These fractures, in turn, lead to an increase to general morbidity, a marked loss of stature and mobility, and in many cases, an increase in mortality resulting from complications.
  • a number of bone growth disorders are known which cause an imbalance in the bone remodeling cycle.
  • kidney failure Patients suffering from chronic renal (kidney) failure almost universally suffer loss of skeletal bone mass (renal osteodystrophy). While it is known that kidney malfunction causes a calcium and phosphate imbalance in the blood, to date replenishment of calcium and phosphate by dialysis does not significantly inhibit osteodystrophy in patients suffering from chronic renal failure. In adults, osteodystrophic symptoms often are a significant cause of morbidity. In children, renal failure often results in a failure to grow, due to the failure to maintain and/or to increase bone mass.
  • Rickets or Osteomalacia (“soft bones"), is a defect in bone mineralization (e.g., incomplete mineralization), and classically is related to vitamin D (1,25-dihydroxy vitamin D 3 ) deficiency or resistance.
  • the defect can cause compression fractures in bone, and a decrease in bone mass, as well as extended zones of hypertrophy and proliferative cartilage in place of bone tissue.
  • the deficiency may result from a nutritional deficiency (e.g., rickets in children), malabsorption of vitamin D or calcium, and/or impaired metabolism of the vitamin.
  • Hyperparathyroidism (overproduction of the parathyroid hormone) has been known to cause abnormal bone loss since its initial description in the 1920's. In children, hyperparathyroidism can inhibit growth. In adults with hyperparathyroidism, the skeleton integrity is compromised and fractures of the hip, vertebrae, and other sites are common.
  • the parathyroid hormone imbalance typically may result from parathyroid adenomas or parathyroid gland hyperplasia. Secondary hyperparathyroidism may result from a number of disorders such as vitamin D deficiency or prolonged pharmacological use of a glucocorticoid such as cortisone. Secondary hyperparathyroidism and renal osteodystrophy may result from chronic renal failure.
  • osteoclasts are stimulated to resorb bone in response to the excess hormone present.
  • the trabecular and cortical bone may ultimately be resorbed and marrow is replaced with fibrosis, macrophages, and areas of hemorrhage as a consequence of microfractures.
  • This condition occurring in both primary and secondary hyperparathyroidism, is referred to pathologically as osteitis fibrosa cystica.
  • Osteoporosis is a structural deterioration of the skeleton caused by loss of bone mass resulting from an imbalance in bone formation, bone resorption, or both, such that the resorption dominates the bone formation phase, thereby reducing the weight-bearing capacity of the affected bone. Osteoporosis affects >10 million individuals in the United States, but only 10 to 20% are diagnosed and treated.
  • osteoporotic individuals In a healthy adult, the rates at which bone is formed and resorbed are tightly coordinated so as to maintain the renewal of skeletal bone.
  • an imbalance in these bone-remodeling cycles develops which results in both loss of bone mass and in formation of microarchitectural defects in the continuity of the skeleton.
  • These skeletal defects created by perturbation in the remodeling sequence, accumulate and finally reach a point at which the structural integrity of the skeleton is severely compromised and bone fracture is likely.
  • the chief clinical manifestations are vertebral and hip fractures, but all parts of skeleton may be affected. Osteoporosis is defined as a reduction of bone mass (or density) or the presence of a fragility fracture.
  • Osteoporosis is defined operationally by the National Osteoporosis Foundation and World Health Organization as a bone density that falls -2.0 or -2.5 standard deviations (SD) below the mean (also referred to as a T-score of -2.0 or -2.5). Those who fall at the lower end of the young normal range (a T-score of >1 SD below the mean) have low bone density and are considered to be "osteopenic" and be at increased risk of osteoporosis.
  • SD standard deviations
  • osteoporosis Although this imbalance occurs gradually in most. individuals. as they age. (“senile... osteoporosis”), it is much more severe and occurs at a rapid rate in postmenopausal women. In addition, osteoporosis also may result from nutritional and endocrine imbalances, hereditary disorders and a number of malignant transformations.
  • osteoporosis In the United States, as many as 8 million women and 2 million men have osteoporosis (T-score ⁇ -2.5), and an additional 18 million individuals have bone mass levels that put them at increased risk of developing osteoporosis (e.g., bone mass T-score ⁇ -1.0). Osteoporosis occurs more frequently with increasing age, as bone tissue is progressively lost. In women, the loss of ovarian function at menopause (typically after age 50) precipitates rapid bone loss such that most women meet the criteria for osteoporosis by age 70. The epidemiology of fractures follows similar trends as the loss of bone density.
  • Osteoporosis results from bone loss due to normal age-related changes in bone remodeling as well as extrinsic and intrinsic factors that exaggerate this process. These changes may be superimposed on a low peak bone mass. Consequently, the bone remodeling process is fundamental for understanding the pathophysiology of osteoporosis.
  • the skeleton increases in size by linear growth and by apposition of new bone tissue on the outer surfaces of the cortex. This latter process is the phenomenon of remodeling, which also allows the long bones to adapt in shape to the stresses placed upon them. Increased sex hormone production at puberty is required for maximum skeletal maturation, which reaches maximum mass and density in early adulthood. Nutrition and lifestyle also play an important role in growth, though genetic factors are the major determinants of peak skeletal mass and density.
  • This process has three primary functions: (1) to repair microdamage within the skeleton, (2) to maintain skeletal strength, and (3) to supply calcium from the skeleton to maintain serum calcium.
  • Acute demands for calcium involve osteoclast-mediated resorption as well as calcium transport by osteocytes.
  • the activation of remodeling may be induced by microdamage to bone due to excessive or accumulated stress.
  • Bone remodeling is also regulated by several circulating hormones, including estrogens, androgens, vitamin D, and PTH, as well as locally produced growth factors such as IGF-I and -II, transforming growth factor (TGF) ⁇ , PTHrP, ILs, prostaglandins, tumor necrosis factor (TNF), and osteoprotegrin and many others. Additional influences include nutrition (particularly calcium intake) and physical activity level.
  • TGF transforming growth factor
  • PTHrP ILs
  • ILs transforming growth factor
  • TNF tumor necrosis factor
  • osteoprotegrin osteoprotegrin
  • Additional influences include nutrition (particularly calcium intake) and physical activity level.
  • the end result of this remodeling process is that the resorbed bone is replaced by an equal amount of new bone tissue.
  • the mass of the skeleton remains constant after peak bone mass is achieved in adulthood. After age 30 to 45, however, the resorption and formation processes become unbalanced, and resorption exceeds formation.
  • This imbalance may begin at different ages and varies at different skeletal sites; it becomes exaggerated in women after menopause. Excessive bone loss can be due to an increase in osteoclastic activity and/or a decrease in osteoblastic activity. In addition, an increase in remodeling activation frequency can magnify the small imbalance seen at each remodeling unit.
  • DXA dual-energy x-ray absorptiometry
  • SXA single-energy x-ray absorptiometry
  • CT quantitative computed tomography
  • DXA is a highly accurate x-ray technique that has become the standard for measuring bone density in most centers. Though it can be used for measurements of any skeletal site, clinical determinations are usually made of the lumbar spine and hip.
  • Portable DXA machines have been developed that measure the heel (calcaneus), forearm (radius and ulna), or finger (phalanges), and DXA can also be used to measure body composition.
  • two x-ray energies are used to estimate the area of mineralized tissue, and the mineral content is divided by the area, which partially corrects for body size.
  • this correction is only partial since DXA is a two-dimensional scanning technique and cannot estimate the depths or posteroanterior length of the bone.
  • BMD bone mineral density
  • Newer DXA techniques that measure information BMD are currently under evaluation. Bone spurs, which are frequent in osteoarthritis, tend to falsely increase bone density of the spine. Because DXA instrumentation is provided by several different manufacturers, the output varies in absolute terms. Consequently, it has become standard practice to relate the results to "normal" values using T-scores, which compare individual results to those in a young population that is matched for race and gender. Alternatively, Z-scores compare individual results to those of an age-matched population that is also matched for race and gender. Thus, a 60-year-old woman with a Z-score of-1 (1 SD below mean for age) could have a T-score of -2.5 (2.5 SD below mean for a young control group).
  • CT is used primarily to measure the spine, and peripheral CT is used to measure bone in the forearm or tibia.
  • Research into the use of CT for measurement of the hip is ongoing.
  • CT has the added advantage of studying bone density in subtypes of bone, e.g., trabecular vs. cortical.
  • the results obtained from CT are different from all others currently available since this technique specifically analyzes trabecular bone and can provide a true density (mass of bone per unit volume) measurement.
  • CT remains expensive, involves greater radiation exposure, and is less reproducible.
  • Ultrasound is used to measure bone mass by calculating the attenuation of the signal as it passes through bone or the speed with which it traverses the bone. It is unclear whether ultrasound assesses bone quality, but this may be an advantage of the technique. Because of its relatively low cost and mobility, ultrasound is amenable for use as a screening procedure.
  • PTHrP Parathyroid hormone-related peptide
  • a 140+ amino acid protein and fragments thereof, reproduce the major biological actions of PTH.
  • PTHrP is elaborated by a number of human and animal tumors and other tissues and may play a role in hypercalcemia of malignancy.
  • the nucleotide and amino acid sequences of hPTHrP-(l-36) are provided in SEQ DD NOS:1 and 2, respectively.
  • Biological activity is associated with the N-terminal portion.
  • the amino acid sequence of the N-terminal segment of human PTHrP shows great homology with the N-terminal segment of various species, as illustrated in FIG. 1.
  • PTH and PTHrP although distinctive products of different genes, exhibit considerable functional and structural homology and may have evolved from a shared ancestral gene.
  • the structure of the gene for human PTHrP is more complex than that of PTH, containing multiple exons and multiple sites for alternate splicing patterns during formation of the mRNA. Protein products of 141, 139, and 173 amino acids are produced, and other molecular forms may result from tissue-specific cleavage at accessible internal cleavage sites.
  • PTHrP The biologic roles of these various molecular species and the nature of the circulating forms of PTHrP are unclear. It is uncertain whether PTHrP circulates at any significant level in normal human adults; as a paracrine factor, PTHrP may be produced, act, and be destroyed locally within tissues. In adults PTHrP appears to have little influence on calcium homeostasis, except in disease states, when large tumors, especially of the squamous cell type, lead to massive overproduction of the hormone.
  • hPTH and hPTHrP are largely limited to the 13 N-terminal residues, 8 of which are identical; only, l.of 10-amino acids in.the (25-34) receptor- binding region of hPTH is conserved in hPTHrP. Conformational similarity may underlie the common activity. Cohen et al. (J. Biol. Chem. 266: 1997-2004 (1991)) have suggested that much of the sequence of PTH-(l-34) and PTHrP-(I -34), in particular regions (5-18) and (21-34), assumes an ⁇ -helical configuration, while noting that there is some question whether this configuration prevails for the carboxyl terminal end under physiological conditions.
  • thyroid hormone related protein encompasses naturally- occurring PTHrP, as well as synthetic or recombinant PTHrP (rec PTHrP). Further, the term “parathyroid hormone related protein” encompasses allelic variants, species variants, and conservative amino acid substitution variants. The term also encompasses full-length PTHrP- (1-36), as well as PTHrP fragments, including small peptidomimetic molecules having PTHrP- like bioactivity, for example, in the assays described herein.
  • PTHrP the biological activity of PTHrP is associated with the N-terminal portion, with residues (1-30) apparently the minimum required. It will thus be understood that fragments of PTHrP variants, in amounts giving equivalent biological activity to PTHrP-(l-36), can be used in the methods of the invention, if desired. Fragments of PTHrP incorporate at least the amino acid residues of PTHrP necessary for a biological activity similar to that of intact PTHrP-(I -36).
  • fragments include PTHrP-(l-30), PTHrP-(l-31), PTHrP-(l-32), PTHrP-(l-33), PTHrP-(l-34), PTHrP-(l-35), PTHrP-(l-36), ... PTHrP-(I-139), PTHrP-(I-HO), and PTHrP-(l-141).
  • thyroid hormone-related protein also encompasses variants and functional analogues of PTHrP having an homologous amino acid sequence with PTHrP-(I -36).
  • the present invention thus includes pharmaceutical formulations comprising such PTHrP variants and functional analogs, carrying modifications like substitutions, deletions, insertions, inversions or cyclisations, but nevertheless having substantially the biological activities of parathyroid hormone.
  • homologous amino acid sequence means an amino acid sequence that differs from an amino acid sequence shown in SEQ ID NO:2, by one or more conservative amino acid substitutions, or by one or more non-conservative amino acid substitutions, deletions, or additions located at positions at which they do not destroy the biological activities of the polypeptide.
  • Conservative amino acid substitutions typically include substitutions among amino acids of the same class. These classes include, for example, (a) amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; (b) amino acids having basic side chains, such as lysine, arginine, and histidine; (c) amino acids having acidic side chains, such as aspartic acid and glutamic acid; and (d) amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.
  • amino acids having uncharged polar side chains such as asparagine, glutamine, serine, threonine, and tyrosine
  • amino acids having basic side chains such as lysine, arginine, and histidine
  • amino acids having acidic side chains such as aspartic acid and glut
  • homologous amino acid sequences include sequences that are identical or substantially identical to an amino acid sequence as shown in SEQ ID NO:2.
  • amino acid sequence substantially identical is meant a sequence that is at least 60%, preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% identical to an amino acid sequence of reference.
  • the homologous sequence differs from the reference sequence, if at all, by a majority of conservative amino acid substitutions.
  • % homology and % identity are determined by first aligning a candidate PTHrP polypeptide with SEQ ID NO:2, as provided in FIG. 1. Once aligned, the total number of identical amino acids and/or the number of conservative amino acid substitution variants shared between the candidate polypeptide and SEQ ID NO:2 are counted. For the calculation of % identity, the number of identical amino acids between the candidate PTHrP polypeptide and the reference sequence is divided by the total number of amino acids in the reference sequence, and this number is multiplied by 100 to obtain a percentage value.
  • FIG. 1 provides a homology alignment of human PTHrP-(l-36) (SEQ ID NO:2) with the corresponding sequence in other species, aligned to maximize amino acid identity.
  • the amino acids in other species that differ from the corresponding amino acid in the human sequence are bolded and amino acids that are conservative amino acid substitution variants of the corresponding amino acids in the human sequence are bolded and underlined.
  • the values of % identity and % homology are provided.
  • homology can be measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Similar amino acid sequences are aligned to obtain the maximum degree of homology (i.e., identity). To this end, it may be necessary to artificially introduce gaps into the sequence. Once the optimal alignment has been set up, the degree of homology (i.e., identity) is established by recording all of the positions in which the amino acids of both sequences are identical, relative to the total number of positions. Similarity factors include similar size, shape and electrical charge.
  • a similarity score is first calculated as the sum of the aligned pairwise amino acid similarity scores. Insertions and deletions are ignored for the purposes of percent homology and identity. Accordingly, gap penalties are not used in this calculation.
  • the raw score is then normalized by dividing it by the geometric mean of the scores of the candidate compound and the reference sequence. The geometric mean is the square root of the product of these scores. The normalized raw score is the percent homology. Polypeptides having a sequence homologous to one of the sequences shown in SEQ ID
  • NOS: 1 or 2 include naturally-occurring allelic variants, as well as mutants and variants or any other non-naturally-occurring variants that are analogous in terms of bone formation activity, to a polypeptide having a sequence as shown in SEQ ID NO:2.
  • allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does not substantially alter the biological function of the polypeptide.
  • biological function is meant the function of the polypeptide in the cells in which it naturally occurs, even if the function is not necessary for the growth or survival of the cells.
  • the biological function of a porin is to allow the entry into cells of compounds present in the extracellular medium.
  • a polypeptide can have more than one biological function.
  • Allelic variants are very common in nature. Allelic variation may be equally reflected at the polynucleotide level. Polynucleotides, e.g., DNA molecules, encoding allelic variants can easily be retrieved by polymerase chain reaction (PCR) amplification of genomic DNA extracted by conventional methods. This involves the use of synthetic oligonucleotide primers matching upstream and downstream of the 5' and 3' ends of the encoding domain. Suitable primers can be designed according to the nucleotide sequence information provided in SEQ ID NO:1. Typically, a primer can consist of 10-40, preferably 15-25 nucleotides.
  • primers containing C and G nucleotides in a proportion sufficient to ensure efficient hybridization; e.g., an amount of C and G nucleotides of at least 40%, preferably 50% of the total nucleotide amount.
  • Useful homologs that do not naturally occur can be designed using known methods for identifying regions of a PTHrP peptide that are likely to be tolerant of amino acid sequence changes and/or deletions. For example, stability-enhanced or modified variants of PTHrP are known in the art. For example, Vickery et al, (J. Bone Miner.
  • These homologs and other such biologically active peptidomimetic compounds are useful for creating small- molecule agonists or antagonists of PTHrP, PTH, or TIP peptides, as is discussed in Example 6.
  • Polypeptide derivatives that are encoded by polynucleotides of the invention include, e.g., fragments, polypeptides having large internal deletions derived from full-length polypeptides, and fusion proteins.
  • Polypeptide fragments of the invention can be derived from a polypeptide having a sequence homologous to any of the sequences shown in SEQ ID NOS:2-13, to the extent that the fragments retain the desired substantial bone formation properties of the parent polypeptide.
  • a polynucleotide of the invention having a homologous coding sequence, can hybridize, preferably under stringent conditions, to a polynucleotide having a sequence complementary to the nucleotide sequence in SEQ ID NO:1. Hybridization procedures are described in, e.g., Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons Inc.
  • Tm 81.5 + 0.5 x (% G+C) + 1.6 log (positive ion concentration) - 0.6 x (% formamide).
  • hybridization temperature (Th) is approximately 20-40°C, 20-25°C or, preferably, 30-40°C below the calculated Tm.
  • stringent conditions can be achieved, both for pre-hybridizing and hybridizing incubations, (i) within 4-16 hours at 42 0 C, in 6xSSC containing 50% formamide or (ii) within 4-16 hours at 65 0 C in an aqueous 6xSSC solution (1 M NaCl, 0.1 M sodium citrate (pH 7.0)).
  • Tm For polynucleotides containing 30 to 600 nucleotides, the above formula is used and then is corrected by subtracting (600/polynucleotide size in base pairs). Stringency conditions are defined by a Th that is 5 to 1O 0 C below Tm. Hybridization conditions with oligonucleotides shorter than 20-30 bases do not exactly follow the rules set forth above. In such cases, the formula for calculating the Tm is as follows:
  • Tm 4 x (G+C) + 2 (A+T).
  • G+C 3 x (G+C) + 2 (A+T).
  • A+T 2 (A+T).
  • an 18 nucleotide fragment of 50% G+C would have an approximate Tm of 54 0 C.
  • the methods of the present invention includes the use of a PTHrP peptide selected from the group consisting of:
  • biologically active variants having at least 60% identity with SEQ ID NO:2; and (g) biologically active variants encoded by a nucleic acid sequence that hybridizes under stringent conditions to a complementary nucleic acid sequence of SEQ ID NO:1.
  • PTHrP includes, but is not limited to, human PTHrP (hPTHrP), bovine PTHrP (bPTHrP), and rat PTHrP (rPTHrP).
  • An analog of PTHrP is a peptide which is a structural analog or fragment (preferably, anN-terminal fragment containing 50 or fewer amino acids) of a naturally- occurring PTHrP and, like PTHrP, also capable of binding to PTH receptor and stimulating adenylate cyclase activity, thereby promoting bone formation.
  • fragments include, but are not limited to, PTHrP-(l-30), PTHrP-(I -31), PTHrP-(l-32), PTHrP-(l-33), PTHrP-(l-34), PTHrP-(l-35), PTHrP-(l-36), ... PTHrP-(I -139), PTHrP-(l-140), and PTHrP- (1-141).
  • the following publications disclose the sequences of PTHrP peptides: Yasuda et al, J. Biol. Chem. 264: 7720-.7725 (1989); Schermer, J. Bone & Min. Res. 6: 149-155 (1991); and Burtis, Clin.
  • PTHrP exerts important developmental influences on fetal bone development and in adult physiology.
  • a homozygous knockout of the PTHrP gene (or the gene for the PTH receptor) in mice causes a lethal deformity in which animals are born with severe skeletal deformities resembling chondrodysplasia.
  • Many different cell types produce PTHrP, including brain, pancreas, heart, lung, mammary tissue, placenta, endothelial cells, and smooth muscle.
  • PTHrP directs transplacental calcium transfer, and high concentrations of PTHrP are produced in mammary tissue and secreted into milk.
  • Human and bovine milk for example, contain very high concentrations of the hormone; the biologic significance of the latter is unknown.
  • PTHrP may also play a role in uterine contraction and other biologic functions, still being clarified in other tissue sites.
  • PTHrP Because PTHrP shares a significant homology with PTH in the critical amino terminus, it binds to and activates the PTH/PTHrP receptor, with effects very similar to those seen with PTH. However, PTHrP, not PTH, appears to be the predominant physiologic regulator of bone mass, with PTHrP being essential for the development of full bone mass. Demonstrating this, conditional gene knockout strategies, employing mice in which the PTHrP gene was disrupted in osteoblasts prevented the production of PTHrP locally within adult bone, but which had normal PTH levels in adult bone. Absent PTHrP, and these mice developed osteoporosis demonstrating that osteoblast-derived PTHrP exerts anabolic effects in bone by promoting osteoblast function.
  • the 500-amino-acid PTH/PTHrP receptor (also known as the PTHl receptor) belongs to a subfamily of GCPR that includes those for glucagon, secretin, and vasoactive intestinal peptide.
  • the extracellular regions are involved in hormone binding, and the intracellular domains, after hormone activation, bind G protein subunits to transduce hormone signaling into cellular responses through stimulation of second messengers.
  • a second PTH receptor (PTH2 receptor) is expressed in brain, pancreas, and several other tissues. Its amino acid sequence and the pattern of its binding and stimulatory response to PTH and PTHrP differ from those of the PTHl receptor.
  • the PTH/PTHrP receptor responds equivalently to PTH and PTHrP, whereas the PTH2 receptor responds only to PTH.
  • the endogenous ligand of this receptor appears to be tubular infundibular peptide-39 or TIP-39.
  • TIP-39 tubular infundibular peptide
  • the physiological significance of the PTH2 receptor-TIP-39 system remains to be defined. Recently, a 39-amino-acid hypothalamic peptide, tubular infundibular peptide (TIP-39), has been characterized and is a likely natural ligand of the PTH2 receptor.
  • the PTHl and PTH2 receptors can be traced backward in evolutionary time to fish.
  • the zebrafish PTHl and PTH2 receptors exhibit the same selective responses to PTH and PTHrP as do the human PTHl and PTH2 receptors.
  • the evolutionary conservation of structure and function suggests unique biologic roles for these receptors.
  • G proteins of the G s class link the PTH/PTHrP receptor to adenylate cyclase, an enzyme that generates cyclic AMP 5 leading to activation of protein kinase A.
  • Coupling to G proteins of the G q class links hormone action to phospholipase C, an enzyme that generates inositol phosphates (e.g., IP 3 ) and DAG, leading to activation of protein kinase C and intracellular calcium release.
  • Studies using the cloned PTH/PTHrP receptor confirm that it can be coupled to more than one G protein and second-messenger kinase pathway, apparently explaining the multiplicity of pathways stimulated by PTH and PTHrP.
  • Incompletely characterized second- inessenger responses may be independent of phospholipase C or adenylate cyclase stimulation (the latter, however, is the strongest and best characterized second messenger signaling pathway for PTH and PTHrP).
  • the responses in bone include effects on collagen synthesis; increased alkaline phosphatase, ornithine decarboxylase, citrate decarboxylase, and glucose-6-phosphate dehydrogenase activities; DNA, protein, and phospholipid synthesis; calcium and phosphate transport; and local cytokine/growth factor release.
  • these biochemical events lead to an integrated hormonal response in bone turnover and calcium homeostasis.
  • PTHrP provides anabolic effects, similar to those demonstrated by PTHrP, for example, PTH, and TIP.
  • Compositions of PTH and TIP, and their uses, are similar to those for PTHrP disclosed herein.
  • These skeletal anabolic agents, PTH and TIP, or analogs thereof, increase bone mass in a human patient in need thereof, when administered to said patient at a dosage between 10 and 3,000 ⁇ g/day for a period of 1-36 months.
  • the dosage is preferably 10 and 50,000 ⁇ g/day, 20 and 30,000 ⁇ g/day, 35 and 20,000 ⁇ g/day, 40 and 15,000 ⁇ g/day, 45 and 10,000 ⁇ g/day, 50-5,000 ⁇ g/day, more preferably 75-1,500 ⁇ g/day, even more preferably 100-1,200 ⁇ g/day, and most preferably 300-1,000 ⁇ g/day.
  • the period of administration is preferably 12, 15, or 18 months, more preferably 7, 8, 9, 10, or 11 months, and most preferably .1, 2, 3, 4, 5, or 6 months.
  • the increase in bone mass can be monitored by the assays described herein.
  • These skeletal anabolic agents can be combined with PTHrP. They are described below.
  • PTH Peptides PTH is an 84 amino-acid single-chain peptide.
  • the amino acid sequence of PTH has been characterized in multiple mammalian species, revealing marked conservation in the amino- terminal portion, which is critical for many biologic, actions of the molecule... Biological activity is associated with the N-terminal portion, with residues (1-29) apparently the minimum required.
  • the N-terminal segment of human PTH (hPTH) differs from the N-terminal segment of the bovine (bPTH) and porcine (pPTH) hormones by only three and two amino acid residues, respectively.
  • PTH is initially synthesized as a larger molecule (preproparathyroid hormone, consisting of 115 amino acids), which is then reduced in size by signal peptide cleavage (proparathyroid hormone, 90 amino acids) and then a second prohormone cleavage before secretion as an 84 amino acid peptide.
  • preproparathyroid hormone consisting of 115 amino acids
  • signal peptide cleavage can be reduced in size by signal peptide cleavage
  • prohormone cleavage prohormone cleavage before secretion as an 84 amino acid peptide.
  • the hydrophobic regions of the preproparathyroid hormone serve a role in guiding transport of the polypeptide from sites of synthesis on polyribosomes through the endoplasmic reticulum to secretory granules.
  • Modified, substituted synthetic fragments of the amino-terminal sequence as small as 1-14 residues are sufficient to activate the major receptor.
  • Biologic roles for the carboxyl- terminal region of PTH e.g., 35-84 are under investigation; a separate receptor or receptors may exist for this region of the molecule. Fragments shortened or modified at the amino terminus still bind to the PTH receptor but lose the capacity to stimulate biologic responses.
  • the peptide composed of the sequence 7-34 is a competitive inhibitor of active hormone binding to receptors in vitro but is a weak inhibitor in vivo.
  • parathyroid hormone encompasses naturally occurring PTH, as well as synthetic or recombinant PTH (rec PTH). Further, the term “parathyroid hormone” encompasses allelic variants, species variants, and conservative amino acid substitution variants. The term also encompasses full-length PTH-(I -84), as well as PTH fragments. It will thus be understood that fragments of PTH variants, in amounts giving equivalent biological activity to PTH-(l-84), can be used in the methods of the invention, if desired. Fragments of PTH incorporate at least the amino acid residues of PTH necessary for a biological activity similar to that of intact PTH.
  • fragments include: PTH-(I -29), PTH-(I -30), PTH-(I -31), PTH-(l-32), PTH- (1-33), PTH-Q-34), PTH-(l-80), PTH-(1-81), PTH-(l-82), PTH-(l-83), and PTH-(l-84).
  • thyroid hormone also encompasses variants and functional analogs of
  • the present invention thus includes pharmaceutical formulations comprising such PTH variants and functional analogs, carrying modifications like substitutions, deletions, insertions, inversions or cyclisations, but nevertheless having substantially the biological activities of parathyroid hormone.
  • Stability- enhanced variants of PTH are known in the art from, e.g., WO 92/11286 and WO 93/20203, each incorporated herein by reference.
  • Variants of PTH can incorporate, for example, amino acid substitutions that improve PTH stability and half-life, such as the replacement of methionine residues at positions 8 and/or 18, and replacement of asparagine at position 16.
  • Cyclized PTH analogs are disclosed in, e.g., WO 98/05683, incorporated herein by reference.
  • the term "parathyroid hormone” also encompasses amino acid substituted analogs using the PTH-(I-11) or PTH-(1-14) backbone.
  • homology alignment of the reference sequence, human PTH-(l-34) (SEQ ID NO: 15), with the corresponding sequence in other species, aligned to maximize amino acid identity.
  • "Homologous amino acid sequence” means an amino acid sequence that differs from an amino acid sequence shown in SEQ ID NO: 15, by one or more conservative amino acid substitutions, or by one or more non-conservative amino acid substitutions, deletions, or additions located at positions at which they do not destroy the biological activities of the polypeptide.
  • such a sequence is at least 75%, preferably 80%, more preferably 85%, more preferably 90%, and most preferably 95% homologous to the amino acid sequence in SEQ ID NO: 2.
  • Homologous amino acid sequences also include sequences that are identical or substantially identical to an amino acid sequence as shown in SEQ ID NO: 15.
  • amino acid sequence substantially identical is meant a sequence that is at least 60%, preferably 70%, more preferably 80%, more preferably 90%, and ' most preferably 95% identical to an amino acid sequence of reference. Preferably the homologous sequence differs from the reference sequence, if at all, by a majority of conservative amino acid substitutions.
  • PTH peptides useful in the methods of the present invention include the use of a PTH peptide selected from the group consisting of:
  • TIP-39 tubular infundibular peptide
  • tubular infundibular peptide encompasses naturally-occurring TIP, as well as synthetic or recombinant TIP (rec TIP). Further, the term “tubular infundibular peptide” encompasses allelic variants, species variants, and conservative amino acid substitution variants. The term also encompasses full-length TEP-(l-39), as well as TIP fragments. It will thus be understood that fragments of TIP variants, in amounts giving equivalent biological activity to TIP-(l-39), can be used in the methods of the invention, if desired. Fragments of TIP incorporate at least the amino acid residues of TIP necessary for a biological activity similar to that of intact TIP-(l-39). Examples of such fragments are TIP-(l-29), TIP-(l-30), TIP-(1-31), ... TIP-(l-37), TIP-(l-38), and TIP-(l-39).
  • tubular infundibular peptide also encompasses variants and functional analogues of TIP having an homologous amino acid sequence with TIP-(I -39).
  • the present invention thus includes pharmaceutical formulations comprising such TIP variants and functional analogs, carrying modifications like substitutions, deletions, insertions, inversions or cyclisations, but nevertheless having substantially the biological activities of TIP-(I -39).
  • % homology and % identity are determined by first aligning a candidate TIP polypeptide with SEQ ID NO:26, as provided in FIG. 3.
  • "Homologous amino acid sequence” means an amino acid sequence that differs from an amino acid sequence shown in SEQ ID NO: 15, by one or more conservative amino acid substitutions, or by one or more non-conservative amino acid substitutions, deletions, or additions located at positions at which they do not destroy the biological activities of the polypeptide.
  • such a sequence is at least 75%, preferably 80%, more preferably 85%, more preferably 90%, and most preferably 95% homologous to the amino acid sequence in SEQ ID NO: 26.
  • Homologous amino acid sequences also include sequences that are identical or substantially identical to an amino acid sequence as shown in SEQ ID NO: 26.
  • amino acid sequence substantially identical is meant a sequence that is at least 60%, preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% identical to an amino acid sequence of reference.
  • the homologous sequence differs from the reference sequence, if at all, by a majority of conservative amino acid substitutions.
  • the methods of the present invention includes the use of a TIP peptide selected from the group consisting of:
  • compositions of the present invention may be administered intermittently by any route which is compatible with the particular molecules and, when included, with the particular bone resorption inhibiting agent.
  • administration may be oral or parenteral, including subcutaneous, intravenous, inhalation, nasal, and intraperitoneal routes of administration.
  • intermittent administration may be by periodic injections of a bolus of the composition once daily, once every two days, once every three days, once weekly, twice weekly, biweekly, twice monthly, and monthly
  • compositions of the present invention may be provided to an individual by any suitable means, directly (e.g., locally, as by injection, implantation or topical administration to a tissue locus) or systemically (e.g., parenterally or orally).
  • parenterally such as by intravenous, subcutaneous, intramolecular, ophthalmic, intraperitoneal, intramuscular, buccal, rectal, vaginal, intraorbital, intracerebral, intracranial, intraspinal, intraventricular, intrathecal, intracisternal, intracapsular, intranasal or by aerosol administration
  • the composition preferably comprises part of an aqueous or physiologically compatible fluid suspension or solution.
  • the carrier or vehicle is physiologically acceptable so that in addition t ⁇ ' del ⁇ very " ⁇ "f the desired composition to the patient, it does not otherwise adversely affect the patient's electrolyte and/or volume balance.
  • the fluid medium for the agent thus can comprise normal physiologic saline (e.g., 0.9% aqueous NaCl, 0.15 M, pH 7-7.4).
  • the use of pulsatile administration of the skeletal anabolic drug by mirri- pump can be employed in the methods of the present invention.
  • Useful solutions for parenteral administration may be prepared by any of the methods well known in the pharmaceutical art, described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES (Gennaro, A., ed.), Mack Pub., 1990.
  • Formulations of the therapeutic agents of the invention may include, for example, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and the like.
  • Formulations for direct administration in particular, may include glycerol and other compositions of high viscosity to help maintain the agent at the desired locus.
  • Biocompatible, preferably bioresorbable, polymers including, for example, hyaluronic acid, collagen, tricalcium phosphate, polybutyrate, lactide, and glycolide polymers and lactide/glycolide copolymers, may be useful excipients to control the release of the agent in vivo.
  • Other potentially useful parenteral delivery systems for these agents include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation administration contain as excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oily solutions for administration in the form of nasal drops, or as a gel to be applied intranasally.
  • Formulations for parenteral administration may also include glycocholate for buccal administration, methoxysalicylate for rectal administration, or cutric acid for vaginal administration.
  • Suppositories for rectal administration may also be prepared by mixing the PTHrP peptide (alone or in combination with a bone resorption-inhibiting agent) with a non-irritating excipient such as cocoa butter or other compositions that are solid at room temperature and liquid at body temperatures.
  • a non-irritating excipient such as cocoa butter or other compositions that are solid at room temperature and liquid at body temperatures.
  • Formulations for topical administration to the skin surface may be prepared by dispersing the molecule capable of releasing the PTHrP peptide (alone or in combination with a bone resorption-inhibiting agent, or an anabolic agent) with a dermatologically acceptable carrier such as a lotion, cream, ointment or soap. Particularly useful are carriers capable of forming a film or layer over the skin to localize application and inhibit removal.
  • a dermatologically acceptable carrier such as a lotion, cream, ointment or soap.
  • the agent may be dispersed in a liquid or semisolid tissue adhesive or other substance known to enhance adsorption to a tissue surface, e.g., a bone paste.
  • hydroxypropylcellulose or fibrinogen/thrombin solutions may be used to advantage.
  • tissue-coating solutions such as pectin-containing formulations may be used.
  • the method of treatment can constitute a single period of intermittent administration of a skeletal anabolic drug ⁇ e.g., for a period of time varying between 1-3 months to 15-18 months).
  • the period of administration is preferably 12, 15, or 18 months, more preferably 7, 8, 9, 10, or 11 months, and most preferably 1, 2, 3, 4, 5, or 6 months.
  • the method of treatment can constitute a series of administration periods followed by periods of no administration ⁇ e.g., sequential periods of three months of intermittent administration of a skeletal anabolic drug and three months of no drug administration).
  • the sequential treatment periods can be repeated until the patient BMD is restored (e.g., a T-score ⁇ -2.0 or -2.5 below the mean or preferably ⁇ -l .0 below the mean).
  • the method of treatment further includes the step of co-administering, either simultaneously or sequentially to said patient a bone resorption inhibiting agent.
  • the bone resorption-inhibiting agent can be a bisphosphonate, estrogen, a selective estrogen receptor modulator, a selective androgen receptor modulator, calcitonin, a vitamin D analog, or a calcium salt.
  • the bone resorption-inhibiting agent can also be alendronate, risedronate, etidronate, pamidronate, tiludronate, zoledronic acid, raloxifene, tamoxifene, droloxifene, toremifene, idoxifene, levormeloxifene, or conjugated estrogens.
  • the patient receives intermittent administration of the skeletal anabolic drug for a period of time, followed by a period of treatment with a bone resorption inhibiting agent, either alone or in combination with the skeletal anabolic drug.
  • an anabolic agent such as PTHrP is first administered, for example, over a three month period or longer, followed by administration of an antiresorptive agent either alone or in combination with the skeletal anabolic drug, for example, over an additional three month period or longer.
  • reverse administration i.e., giving the antiresorptive agent before administration of the anabolic agent, diminishes the efficacy of the anabolic agent.
  • anabolic agents such as PTHrP should be the primary osteoporosis therapeutics, with antiresorptives used later to maintain and enhance the PTHrP/PTH/TIP effects, and tor example, estrogen or bisphosphonates osteoporosis administered as second line agents after the anabolics.
  • the sequential treatment regimen could begin with a treatment period with a bone resorption inhibiting agent followed by a treatment period with the skeletal anabolic drug, that the length of sequential treatment periods can be modified (e.g., 1-18 months), and that the skeletal anabolic drug can be co-administered with the bone resorption inhibiting agent (e.g., sequential treatment period of a skeletal anabolic drug and a bone resorption inhibiting agent followed by a treatment period of a bone resorption inhibiting agent alone).
  • the bone resorption inhibiting agent e.g., sequential treatment period of a skeletal anabolic drug and a bone resorption inhibiting agent followed by a treatment period of a bone resorption inhibiting agent alone.
  • the sequential treatment periods e.g., three months of the skeletal anabolic drug followed by three month of the bone resorption inhibiting agent
  • the sequential treatment periods can be repeated until the patient BMD is restored (e.g., a T-score ⁇ -2.0 or -2.5 below the mean or preferably ⁇ -l .0 below the mean).
  • Skeletal anabolic agents are commonly believed to demonstrate numerous adverse side effects, and as a result, the dosage and administration of these agents is carefully controlled, and the patient carefully monitored for emergence of unwanted side effects.
  • PTHrP was originally thought to be responsible for most instances of hypercalcemia of malignancy, a syndrome that resembles hyperparathyroidism, with a toxicity profile believed to be similar to or even greater to that of PTH.
  • the toxicity profiles of other skeletal anabolic agents do not appear to be applicable to PTHrP.
  • the findings of the present invention indicate that despite being administered in doses, for example, at least 20 times higher than those considered safe for PTH, PTHrP does not cause significant side effects.
  • intermittent doses of PTHrP of about 50 micrograms to about 400 micrograms given subcutaneously (Q2H for 8 hours after a dose) does not appear to cause hypercalcemia.
  • administration of PTHrP has never been observed to cause hypercalcemia at any dose yet given, such as doses exceeding 450 micrograms and up to 1 milligram are safe, well tolerated by patients and efficacious.
  • individual doses of 3-10 milligrams appear safe, and even up to 50 mg or greater appear well tolerated and are also possible given proper patient monitoring.
  • hypercalcemia defined in the studies described in Example 1 and Example 5 as a serum calcium above 9.9 mg/dl, a very conservative definition of hypercalcemia
  • Neer, et al. defined hypercalcemia as a serum calcium greater than 10.6 mg/dl.
  • PTHrP offers many advantages over PTH as a therapeutic. It is a pure anabolic skeletal agent which is non-hypercalcemic, and has no other adverse effects even when administered in the comparatively higher doses explored to date. Second it appears far more efficacious than PTH in increasing bone mass density. Third, it is more stable than PTH.
  • Suitable osteoblast-like cells include ROS 17/2 (Jouis Subscribe et al, Endocrinology, 130: 53-60 (1992)), UMR 106 (Fujimori et al., Endocrinology, 130: 29-60 (1992)), and the human derived SaOS-2 (Fukuyama et al, Endocrinology, 131: 1757-1769 (1992)).
  • the cell lines are available from American Type Culture Collection, Rockville, Md., and can be maintained in standard specified growth media.
  • transfected human embryonic kidney cells (HEK 293) expressing the human PTHl or PTH2 receptors can also be utilized for in vitro binding assays. See, Pines et al, Endocrinology, 135: 1713-1716 (1994).
  • PTHrP -like analog activities of peptide fragments or derivatives of PTHrP can be tested by contacting a concentration range of the test compound with the cells in culture and assessing the stimulation of the activation of second messenger molecules coupled to the receptors, e.g., the stimulation of cyclic AMP accumulation in the cell or an increase in enzymatic activity of protein kinase C, both of which are readily monitored by conventional assays.
  • Agonist activities of subfragments of PTH have been successfully analyzed by contacting peptides with rat kidney cells in culture and assessing cyclic AMP accumulation (Blind et al, Clin. Endocrinol, 101: 150-155 (1993)) and the stimulation of l,25-dehydroxyvitaminD 3 production (Janulis et al, Endocrinology, 133: 713-719 (1993)).
  • PTH and PTHrP with bone formation activity bind specifically with PTH/PTHrP receptors and produce a dose-dependent stimulation of cAMP accumulation in human renal cortical membranes, in human osteoblast-like osteosarcoma membranes and intact cells (Example 2), and in canine renal cortical membranes (Example 3).
  • [Nle 8 ' 18 ,Tyr 34 ] hPTH-(l-34) NH 2 or hPTHrP-(l-36) as the reference standard analogs, a dose-response relationship can be generated using standard non-linear regression analysis.
  • the relative potency for various PTHrP analogs can be determined from the ratio of the EC 50 of the reference standard analog to that of the PTHrP analog.
  • EC 50 is defined as the dose that evokes a half-maximal response-cAMP accumulation herein.
  • the detailed procedure for handling the cells, setting up the assay, as well as methods for cAMP quantitation, is described in Sistane et al, Pharmacopeial Forum 20: 7509-7520 (1994).
  • candidate PTHrP analogs can be characterized by their abilities to increase trabecular and cortical bone mass in ovariectomized, osteopenic rats, as described in Example 4.
  • Example 5 describes a three-month double blind, prospective, placebo-controlled randomized clinical trial, demonstrating the effectiveness of PTHrP as a skeletal anabolic agent.
  • PTHrP displays minimal side effects, for example, despite the comparatively high doses, no significant increase in hypercalcemia is observed.
  • Example 6 describes a computer system and methods of using the same, for structural based design of peptidomimetics and small molecules having skeletal anabolic biological activity.
  • Parathyroid hormone-related protein is the quintessential skeletal catabolic agent. It was initially discovered as the cause of the common lethal paraneoplastic syndrome, humoral hypercalcemia of malignancy or "HHM". Hypercalcemia occurring among patients with HHM results principally from a striking activation of osteoclastic bone resorption. Thus, PTHrP would seem an unlikely candidate as a skeletal anabolic agent.
  • the purpose of the present study was to determine whether the administration of intermittent high doses of a PTHrP peptide, for a short period of time could produce significant increases in BMD without negative side effects, and that as such, PTHrP might be an effective skeletal anabolic agent in women with postmenopausal osteoporosis.
  • PTH parathyroid hormone
  • Example 1 the study described herein as Example 1 is three month double- blinded, randomized placebo-controlled pilot clinical trial in which PTHrP was compared to placebo treatment.
  • the rate of increase, as well as the absolute increase, observed in lumbar spine bone mineral density with PTHrP are large, and may equal or exceed those reported to date using currently available osteoporosis drugs.
  • PTHrP administered subcutaneously in high doses for only three months appears to be a potent anabolic agent, producing a 4.7% increase in lumbar spine BMD. This compares very favorably to available anti-resorptive drugs for osteoporosis, and PTH. Despite the high doses, PTHrP was well tolerated.
  • hPTHrP-(l-36) Preparation of hPTHrP-(l-36) and placebo for human injection
  • Synthetic hPTHrP-(l-36) was prepared by solid-phase synthesis, as previously described
  • hPTHrP-(l-36) was weighed, dissolved in 10 mM acetic acid, filtered through a sterile 0.2 ⁇ m syringe filter, aseptically aliquoted into 5-600 ⁇ g aliquots in sterile glass vials, aseptically sealed into the vials, frozen at — 8O 0 C, and lyophilized. Placebo vials were prepared in exactly the same manner. The vials were stored at — 8O 0 C.
  • Peptide content was confirmed by amino acid analysis, PTHrP-(l-36) RIA (described below) or PTHrP -(1-36) RNA, and adenylyl cyclase bioassay (described below). Pyrogen testing was performed by limulus amebocyte lysate gel-clot assay method (Associates of Cape Cod, Falmouth, MA), using standard endotoxin from Escherichia coli 0113 as a control. The endotoxin concentration in the vials was below the lower limit of detection ( ⁇ 0.03 endotoxin units/mL). The vials were labeled in coordination with the University of Pittsburgh Medical Center Investigational Pharmacy.
  • the PTHrP-(I -36) from a vial was reconstituted in 1.0 mL 0.9% saline.
  • the mass of hPTHrP (1-36) is 4260.6 Da.
  • the structures of the peptides were confirmed by mass spectroscopy and amino acid analysis. Greater than 99% purity was confirmed by reverse-phase high performance liquid chromatography.
  • hPTHrP-(l-36) The biological potency of hPTHrP-(l-36) was tested using an adenylyl cyclase assay performed in confluent SaOS2 human osteosarcoma cells, using a method previously described in detail (Everhart-Caye et al. JCUn Endocrinol Metab 81: 199-208 (1996); Orloff et al. Endocrinol 131: 1603-1611 (1992); Merendino et al. Science 231: 388-390 (1986)).
  • SaOS2 cells were obtained from American Type Culture Collection, Rockville, MD, and were maintained in McCoy's medium supplemented with 10% FBS, 2 mmol/L L-glutamine, penicillin (50 U/mL), and streptomycin (50 ⁇ g/mL). Cells were plated approximately 10 days before assay in 24-well plates and had been confluent for approximately 7 days previous to assay. The cells were incubated at 25 0 C with isobutylmethylxanthine (500 mmol/L) for 10 min, the peptides added, and the incubation continued at 25°C for another 10 min.
  • McCoy's medium supplemented with 10% FBS, 2 mmol/L L-glutamine, penicillin (50 U/mL), and streptomycin (50 ⁇ g/mL).
  • Duplicates of assay standard or sample (100 ⁇ L) were incubated overnight at 4°C with (100 ⁇ L) of a 1:1500 dilution of S-2 in PlOBT buffer (PBS containing 10% BSA and 0.1 % Triton X-100).
  • Iodinated Tyr 36 of a 1 1500 dilution of S2 in PlOBT buffer (PBS containing 10% BSA and 0.1% Triton X-100).
  • Iodinated Tyr 36 hPTHrP- (l-36)amide (2000-8000 cpm) in PBT buffer was added to the tubes, and the mixture was incubated overnight at 4 0 C. Phase separation was accomplished using dextran-coated charcoal. The sensitivity of the assay is 50 pmol/L.
  • the antiserum recognizes hPTHrP-(l-74), (1-36) and (1-141) with equal affinity, but fails to cross-react with hPTHrP-(37-74) or with hPTH-(l-34) or hPTH-(l-84) (Yang et al, Biochem., 33: 1460-1469 (1994)). Serum and Urine Biochemistries
  • each subject underwent a bone mineral density scan (DXA) of the lumbar spine and hip at the beginning and at the conclusion of the study.
  • Inclusion criteria included a T-score of less than -2.5 at the lumbar spine, " being more than three years postmenopausal, being on estrogen replacement for at least three years, and being in generally excellent health.
  • Exclusion criteria included use in the past of any osteoporosis medication, including bisphosphonates, calcitonin, or selective estrogen receptor modifiers.
  • Current use of medications or agents that might influence calcium or bone metabolism ⁇ e.g., thiazides, non- physiologic doses of thyroid hormone, glucocorticoids, lithium, alcohol, etc. was also an exclusion criterion. All study subjects provided informed consent. The protocol was approved by the University of Pittsburgh Institutional Review Board.
  • the sixteen subjects were randomized to receive three months of treatment with either PTHrP or placebo (identically prepared empty vials containing no PTHrP). Each subject also received 400 IU of vitamin D and 1000 mg of calcium as calcium carbonate per day (Os-CaI, Smith Kline Beecham/Glaxo, King of Prussia, PA), and this was started two weeks before the initiation of PTHrP or placebo treatments. Subjects were taught in the home storage at -20°C, reconstitution and self-injection of PTHrP or placebo.
  • Vials were reconstituted by the study subjects in 1.0 ml of sterile bacteriostatic saline immediately prior to use, to an average dosage of PTHrP of 410.25 ⁇ g per day, or saline placebo, and was self-administered into the abdominal subcutaneous fat. Subjects returned for blood and urine studies at 0, 14, 30, 60 and 90 days of the study. A final bone density study was performed on day 90 of the study.
  • Study subjects were monitored at 0, 2, 4, 8, and 12 weeks for hypercalcemia, rashes, GI complaints, cardiovascular complaints or symptoms, or other non-specific complaints. Subjects were questioned regarding adverse effects at each visit, i.e., 0, 14, 30, 60 and 90 days of the study.
  • Baseline Demographics The baseline demographics in the two groups are shown in Table I. The subjects were of similar age, weight, height, BMI, years since menopause, years on estrogen, calcium intake, and had similar plasma 25 vitamin D concentrations. In the placebo group, two were smokers and one was on a normal replacement dose of thyroid hormone for hypothyroidism. Both groups displayed osteoporosis at the lumbar spine. Study Compliance.
  • the changes in BMD at the lumbar spine over the three months of the study are shown in FIG. 4.
  • the left panel shows the changes in bone mineral density as measured by DXA as percent changes from baseline.
  • the right panel shows the same data as absolute changes in bone mineral density from baseline in gm/cm 2 .
  • the error bars represent SEM. P-values were determined using Student's paired T-test.
  • the changes in BMD expressed as percent change from baseline at the total hip and femoral neck are shown in FIG. 5, and are compared to the changes at the lumbar spine.
  • the light gray bars indicate the placebo group (PBO), and the black bars indicate the experimental group (PTHrP).
  • the L/S data are the same as those presented in FIG. 4 and include the outlier.
  • the error bars indicate SEM, and P-values were determined using Student's paired T-test. There was no significant difference between the PTHrP or PBO groups at either hip site during the study.
  • FIG. 6 illustrates three different bone turnover markers in the placebo and PTHrP -treated subjects.
  • FIG. 6(a) illustrates serum osteocalcin, a marker of bone formation, increased in a statistically significant fashion during the study in the PTHrP-treated subjects but not the placebo controls. Indeed, as illustrated in FIG. 6(a), increases in serum osteocalcin were apparent as early as day 15 (the earliest time period blood samples were obtained).
  • Urinary DPD excretion a second marker of bone resorption, was also unchanged, see, FIG. 6(c).
  • the dotted line indicates the placebo group and the sold line the PTHrP group.
  • FIG. 7 illustrates serum total and ionized serum calcium in the placebo and PTHrP- treated subjects.
  • the dotted line indicates the placebo group and the sold line, the PTHrP group.
  • the error bars indicate SEM, and the P-values were determined using ANOVA for repeated measures.
  • Calcium levels remained normal and constant in both the PTHrP-treated subjects as well as in the placebo controls. No subject developed a significant increase in serum total or ionized calcium.
  • FIG. 8 illustrates a comparison of the anabolic activity of PTHrP with results from selected previously published osteoporosis clinical trials.
  • "Ralox 150” refers to Delmas PD, et al, N Engl J Med 337:1641-7, (1997); “Ralox 120” to Ettinger B, et ah, JAMA 282:637-45, (1999); and “calcitonin” to Chestnut C, et al, Osteoporosis Int 8(suppl 3):13 (1998); “alendro”, “risedro” and “zoledro” refer to studies employing alendronate (Liberman UA, et ah, N Engl J Med 333:1437-43, (1995) and Murphy MG, et ah, J Clin Endocrinol Metab 86:1116-25, (2001), to risedronate (Fogelman I, et ah, J Clin Endocrinol
  • PTH refers to two studies employing parathyroid hormone, Lindsay R, et ah, Lancet 350:550-5 (1997), and Neer RM, et ah, N Engl J Med 344:1434-41 (2001), and “PTHrP” refers to the current study.
  • Estrogen causes similar increments in spine BMD, but a change of 5% requires three years of treatment.
  • the changes observed using some bisphosphonates, including etidronate, alendronate, risedronate, and zoledronate, may equal or exceed 5%, but require far longer than three months, typically one or more years. Indeed, the changes observed compare favorably to, and may possibly exceed those observed in studies reported to date using PTH over a three month period. Viewed from the perspective of available anti-resorptive therapies, the effects of short-term high dose PTHrP are striking.
  • the doses of PTHrP employed in this study were large compared to those used in similar PTH studies. Subjects in this study received 6.56 micrograms/kg/day, which on average was 410.25 micrograms per day in the eight subjects who received PTHrP. This is some 10- to 20- fold larger than doses of hPTH(l-34) (20-40 micrograms/day) commonly employed in osteoporosis studies. Doses of PTH in excess of 20 micrograms/day are associated with hypercalcemia and other adverse effects in humans. It is surprising, therefore, that healthy subjects would tolerate doses of this magnitude without developing hypercalcemia, postural hypotension, nausea, flushing or other adverse effects.
  • the bone turnover marker data ⁇ see, FIG. 6(a), (b), and (c)) suggest that PTHrP may have purely anabolic effects on the skeleton, without the accompanying increase in bone resorption observed using PTH.
  • PTH which displays both formation- and resorption-stimulating properties
  • PTHrP appears to have selective osteoblastic or anabolic effects, without concomitant resorption-stimulating effects.
  • the lack of a resorptive effect is unlikely to be due to concomitant estrogen use since the resorptive response to PTH is not abolished by estrogen.
  • the doses of PTHrP employed in this study were very large. Subjects in this study received an average dosage 410.25 ⁇ g per day in the eight subjects who received PTHrP. This is some 10- to 20-fold larger than doses of hPTH(l-34) (20-40 ⁇ g/day) commonly employed in osteoporosis studies. Doses of PTH in excess of 20 ⁇ g/day are known to be associated with hypercalcemia and other adverse effects. It is surprising, therefore, that healthy subjects would tolerate PTHrP doses of this magnitude without developing hypercalcemia, postural hypotension, nausea, flushing or other adverse effects.
  • PTHrP(l-36) Short-term, very high dose treatment with PTHrP(l-36) causes a remarkable increase in spine BMD.
  • PTHrP appears to have predominantly anabolic effects with little or no resorptive component.
  • the differences between PTH and PTHrP are not likely to reflect differences in receptor interactions or signaling between the two molecules, but likely reside in the differing pharmacokinetic properties of the two molecules following subcutaneous administration.
  • the eight subjects receiving PTHrP demonstrated important increases in lumbar spine BMD, with a mean value of approximately A.I 5%.
  • PTHrP is widely viewed as the quintessential catabolic skeletal hormone responsible for dramatic skeletal mineral losses in patients with HHM.
  • the observation that PTHrP is actually markedly anabolic for the skeleton when administered "intermittently" (e.g., once per day) was not anticipated. This is evidenced by the fact that many investigators and pharmaceutical firms have worked for more than 10 years (and likely as long as 70 years) with PTH in osteoporosis, but none has embraced PTHrP despite its having been in the public domain since its initial description in 1987.
  • the treatment regimen of the present invention for the treatment of osteoporosis has one additional unanticipated and unpredictable strength relating to safety.
  • bPTH-(l-84) was obtained from the National Hormone and Pituitary Program through the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).
  • (Tyr 36 ) chicken (c)PTHrP-(l-36)amide was purchased from Belmont, CA.
  • hPTHrP-(l-141) was provided by Genentech, Inc., So. San Francisco, CA, and transaminated rPTH-(l-34) was provided by Dr. David L. Carnes, Jr. (San Antonio, TX).
  • Radioiodination of hPTHrP-(l-36) was performed using a modification of the lactoperoxidase method as previously described (Orloff et al. J. Biol. Chem., 264: 6097-6103 (1989); Orloff et al. J Bone Min Res 6: 279-287 (1991)). Purification of radioligand was accomplished by reverse-phase HPLC using a 30 cm ⁇ -Bondapak C 18 column " (Waters
  • the radioligand prepared and purified in this manner is composed almost exclusively of the monoiodinated form.
  • the specific activity ranged from 300-450 ⁇ Ci/ ⁇ g at the time of iodination.
  • the radioligand displayed full biological activity in the canine renal adenylate cyclase assay when compared to the unlabeled peptide (Orloff et al. J. Biol. Chem., 264: 6097-6103 (1989)).
  • the human osteoblast-like osteosarcoma cell line SaOS-2 (American Type Culture Collection, Rockville, MD), was maintained in McCoy's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (50 U/ml), and streptomycin (50 ⁇ g/ml). The medium was changed every other day, and studies were performed at 5-7 days post confluence. Cell numbers were determined using a Coulter counter.
  • Highly purified human RCM were prepared using discontinuous sucrose gradient ultracentrifugation as previously described (Orloff et al. J Bone Min Res 6: 279-287 (1991)). All steps were performed in the presence of the following protease inhibitors: aprotinin [10 Kallikrein inhibitor units (KIU/ml], pepstatin (5 ⁇ g/ml), leupeptin (45 ⁇ g/ml), and phenylmethanesulfanylfluoride (10 ⁇ g/ml).
  • KIU/ml Kallikrein inhibitor units
  • pepstatin 5 ⁇ g/ml
  • leupeptin 45 ⁇ g/ml
  • phenylmethanesulfanylfluoride 10 ⁇ g/ml.
  • Normal human kidney cortex was from four separate nephrectomy specimens removed for localized transitional cell carcinoma, renal cell carcinoma, or benign cysts. Renal function in all individuals was normal as assessed by serum creatinine and py
  • SaOS-2 cell membranes were prepared as previously described in detail. Briefly, postconfluent cells in 150 cm 2 flasks were scraped into membrane buffer [10 mMTris HCl (pH 7.5), 0.2 mM MgCl 2 , 0.5 mM EGTA, 1 mM dithiothreitol, leupeptin 45 ⁇ g/ml, pepstatin 5 ⁇ g/ml, aprotinin 10 KIU/ml, and phenylmethane sulfanyl-fluoride 10 ⁇ g/ml] at O 0 C. Cell disruption was achieved by sonification and the suspension was centrifuged at 13,000 x g for 15 min at 4 0 C.
  • the pellet was resuspe ⁇ decl witl ⁇ 'a''D ⁇ utl'Ce' glass homogenizer in membrane buffer (minus dithiothreitol) containing 250 mM sucrose.
  • the suspension was layered onto a cushion of the membrane buffer containing 45% sucrose and centrifuged at 70,000 x g for 30 min at 4 0 C.
  • the membrane fraction layering at the interface was collected, diluted 5-fold with membrane buffer containing 250 mM sucrose, and recentrifuged.
  • the pellet was resuspended in membrane buffer containing 250 mM sucrose, aliquoted, and stored at -7O 0 C. Protein concentrations were determined by the method of Lowry using BSA as standard.
  • the binding assay for SaOS-2 membranes was conducted as for human RCM. Membranes were added to a final concentration of 112.5 ⁇ g/ml and specific binding reached equilibrium by 60 min at 3O 0 C. TB ranged from 15-20% and NSB from 4.0-4.3%.
  • Binding to intact SaOS-2 cells was performed as described (Orloff et al. Am J Physiol 262: E599-E607 (1992)) with the following modifications. Binding studies were conducted at 15°C in the presence of chymostatin (100 ⁇ g/ml) and bacitracin (200 ⁇ g/ml). Specific binding of I25 I-(Tyr 36 )hPTHrP-(l-36)NH 2 reached equilibrium by 150 min at 15°C. The incubation time of 150 min was therefore used for competitive binding studies. Cell viability, as assessed by exclusion of trypan blue, was greater than 95% at the end of a standard incubation. Total binding (TB) ranged from 18-23% of total radioactivity added and non-specific binding (NSB) consistently ranged between 5-7%.
  • Adenylate cyclase-stimulating activity was examined in confluent SAOS-2 cells as previously described for ROS 17/2.8 cells (Merendino et al, Science 231 :388-390, (1986)), with the following modification.
  • the intact cell assay was conducted at 15 0 C, the same conditions employed for binding to intact SaOS-2 cells (vide supra).
  • Time course experiments demonstrated that peak cAMP stimulation for PTHrP and PTH occurred after a 60 min incubation.
  • Dose response curves for each peptide were thus generated using 60 min incubations at 15°C under binding assay conditions. Under these conditions, maximal stimulation varied between 80- and 200-fold above basal activity.
  • Adenylate cyclase activity was examined in human kidney membranes and SaOS-2 cell membranes as previously described in detail for canine renal membranes (Orloff et al, J Biol Chem 264:6097-6103, (1989); Orloff et al J Bone Min Res 6: 279-287 (1991)), with the following modifications: Time course experiments conducted at 3O 0 C demonstrated peak cAMP accumulation at 10 min for human kidney membranes and 30 min for SaOS-2 membranes. Therefore, dose response curves for each peptide were generated at 3O 0 C for 10 min in human kidney and 3O 0 C for 30 min in SaOS-2 membranes.
  • kidney and bone cell membrane adenlyate cyclase assays were performed under binding assay conditions. Results are expresses as percentage of maximal cAMP stimulation in order to compare peptide dose responses from different experiments. Maximal cAMP .stimulation varied from 3- to 8-fold above basal for human RCM, and from 2- to 7-fold for SaOS-2 membranes.
  • IC 50 values for competitive binding experiments and EC 50 values for adenylate cyclase dose response curves were determined from the concentration of peptide yielding 50% of the maximal response. Statistical differences were assessed by paired and unpaired two-tailed Student's t test. Further analysis of competition binding data was carried out with the LIGAND computerized least-squares nonlinear curve-iittmg program (Munson et al. Anal Biochem 107: 220-239 (1980)).
  • bPTH-(l-84) which displayed lower binding affinity, retained its lower relative potency as compared to hPTHrP-(l-36) in the SaOS-2 membrane adenylate cyclase assay, but it was essentially equipotent to hPTHrP-(l-36) in stimulating cAMP production in RCM.
  • SaOS-2 intact cells were also studied (Table III and FIGS. 9C and 10C). In general, the relative affinity and cAMP-stimulating potency of the peptide agonists that were tested closely paralleled the results in RCM and SaOS-2 membranes.
  • hPTHrP-(l-141) had 5-fold greater affinity than hPTHrP-(l-74) in both assays, but it remained less potent than hPTHrP-(l-36).
  • the relative affinity of bPTH-(l-84) was similar to that of hPTHrP-(l-74), but as noted in the preceding paragraph, it did not display the enhanced coupling to adenylate cyclase in SaOS-2 cells or membranes as it had in RCM.
  • the purpose of the present study was to compare the properties of renal receptors for PTH and PTHrP and determine if the two peptides interact with the same receptors.
  • the PTH-related peptide, [Tyr 36 ]PTHrP-(l-36)amide (PTHrP-(I -36)), and [Nle M8 5 Tyr 34 ]hPTH-(l-34)amide (NNT-hPTH-(l-34)) were radioiodinated and used in competition binding studies using canine renal cortical membranes (CRMC) to assess the binding of several PTH and PTHrP analogs.
  • CRMC canine renal cortical membranes
  • the PTH-related peptide (Tyr 36 ) PTHrP-(l-36)amide (PTHrP-(l-36)) was prepared by solid-phase synthesis as previously described (Stewart et al., J. Clin. Invest., 81: 596-600 (1988)).
  • PTHrP-(49-74) and (Cys 5 ,Trp n ,Gly I3 )PTHrP-(5-18) (Pl-peptide) were prepared using similar solid-phase methods.
  • Radio-iodination of the peptides PTHrP (1-36) andNHT-hPTH (1-34) was performed using a modification (Thorell et at, Biochim. Biophys. Acta, 251: 363-369 (1971)) of the lactoperoxidase method (Marchalonis, Biochem. J., 113: 299-305 (1969)).
  • the peptide (10 ⁇ g/10 ⁇ l) was mixed with Na 125 I (1 mCi/10 ⁇ l) (Amersham, Arlington Heights, IL) and lactoperoxidase (2 ⁇ g) (Sigma Chemicals, St. Louis, MO). The reaction was initiated by the ..
  • the eluate was lyophilized and purified by reverse-phase HPLC using a 30 cm u-Bondapak C18 column (Waters Associates). The column was equilibrated with H 2 O containing 0.1% TFA and developed with acetonitrile in 0.1% TFA.
  • I NNT-PTH 1-34
  • the gradient employed was a 60 min linear gradient of 33-43% acetonitrile.
  • 125 I PTHrP-(I -36) elution was accomplished with a 50 min linear 27-34% acetonitrile gradient.
  • Eluted fractions were collected in borosilicate glass tubes (12 x 75mm) containing 30 ⁇ l of 1% BSA and monitored for radioactivity in a gamma spectrometer.
  • HPLC-purified radioligand was subjected to complete enzymatic digestion in 100 ⁇ l of a buffer consisting of Tris-HCl (50 mM) pH 7.5, NaCl (75 mM), and sodium aside (0.005%) (Brown etal, Biochem., 20: 4538-4546 (1981)).
  • a mixture of trypsin (1 ⁇ g/10 ⁇ l), carboxypeptidase Y (1 ⁇ g/10 ⁇ l), leucine aminopeptidase (1 ⁇ g/10 ⁇ l), and pronase E (2 ⁇ g/10 ⁇ l) was added and digestion carried out at 37°C for 24 hours. The reaction was stopped by adding 100 ⁇ l of 0.1% TFA.
  • CRCM canine renal cortical membranes
  • Membranes were purified further by a modification of the procedure described by Segre et al. (J. Biol. Chem., 254: 6980-6986 (1979)).
  • the white pellet described above was centrifuged at 2200xg for 15 mm and the supernatant and upper portion of the resulting double- layered pellet was removed and resuspended in SET buffer. This was centrifuged at 20,000xg for 15 mm and the supernatant discarded.
  • the pellet was then layered onto a discontinuous gradient of sucrose in 0.01 M Tris, 0.001 MNa 2 EDTA (pH 7.5), 6.5 KIU/ml aprotinin, and 50 ⁇ g/ml bacitracin.
  • the gradient consisted of 39% sucrose (2 ml), 37% sucrose (4 ml), and 32% sucrose (2 ml).
  • the membranes were centrifuged at 25,000 rpm (75,000xg) for 90 mm at 4 0 C. Major bands were present at each interface in addition to a pellet at the bottom of the tube.
  • Preliminary studies of the lightest fraction (not entering the sucrose) and the fraction at the 32%-37% interface indicated the highest specific binding and lowest non-specific binding.
  • the lightest fraction demonstrated less degradation of the radioligand in rebinding studies. Therefore, all subsequent experiments were conducted with this fraction except where specifically indicated.
  • a second membrane preparation was performed using the same procedure as above, but in the presence of leupeptin (5 ⁇ g/ml), pepstatin (5 ⁇ g/ml), aprotmin (10 KIU/ml), N-ethylmaleimide (NEM) (1.0 mM), and phenylmethanesulfanyl fluoride (PMSF) (10 ⁇ g/ml) in all steps (Nissenson et ah, Biochem., 26: 1874-1-878 (1987)). Protein was measured by the method of Lowry using BSA as standard.
  • Binding assays were conducted in siliconized 12x75 mm borosilicate glass test tubes at 2O 0 C in a final volume of 0.2 ml.
  • the binding buffer consisted of 50 mM Tris HCl (pH 7.5), 4.2 mM MgCl 2 , 0.3% BSA, 26 mM KCl, approximately 60-8OxIO 3 cpm/tube of radioligand, and, where appropriate, unlabeled peptides.
  • bacitracin was added to a final concentration of 100 ⁇ g/ml for experiments conducted with 125 I NNT-hPTH-(l-34) and 200 ⁇ g/ml for 125 I PTHrP-(l-36).
  • Binding was initiated by adding 50 ⁇ g membrane. At the end of the incubation periods described, 50 ⁇ l triplicate aliquots were layered onto 300 ⁇ l of iced binding buffer containing 1.0% BSA in 500 ⁇ l polypropylene tubes. The tubes were centrifuged at approximately 16,000xg for three min at 4°C in a microcentrifuge. The supernatant was aspirated arid the tip of the tube containing the membrane-associated radioligand was cut off. Radioactivity in both the pellet and supernatant was measured. Total binding (TB) of radioligand varied between 7.2-14.6% of total counts added for
  • Adenylate cyclase-stimulating activity was examined using a guanyl nucleotide-amplified canine renal cortical membrane (CRCM) PTH-sensitive adenylate cyclase assay, performed as previously described in detail (Stewart et al, Proc. Natl. Acad. ScI USA, 80: 1454-1478 (1987)). Briefly, synthetic PTHrP-(l-36) or bPTH-(l-34) was added in duplicate to assay tubes containing crude CRCM, and the conversion of ⁇ -[ 32 P]cAMP to [ 32 P]cAMP at 30°C for 30 min was examined. Results are expressed as the percent increment in adenylate cyclase activity in tubes containing the peptides as compared with tubes containing vehicle only.
  • CRCM canine renal cortical membrane
  • Adenylate cyclase-stimulating activity of both peptides was also examined using highly purified 32% interface membranes. Incubation was carried out under binding conditions at 2O 0 C for 20 min in the presence of the protease inhibitor, bacitracin (200 ⁇ g/ml). All other aspects of this assay were identical to the standard assay.
  • Dissociation constants were determined by Scatchard analysis of the data obtained from competitive binding experiments using radioligand and increasing concentrations of unlabeled ligand.
  • binding affinities Ki
  • IC 50 concentration of unlabeled ligand displacing 50% of specific radioligand binding
  • EBDA computer program
  • the binding affinity constant (K 1 ) for PTHrP-(I -36) was 11.5 nM (Table II, top).
  • K 1 The binding affinity constant for PTHrP-(I -36) was 11.5 nM (Table II, top).
  • 125 I-PTHrP-(I -36) was used as the radioligand, all three synthetic peptides were approximately equipotent in inhibiting binding (FIG. 13).
  • Binding dissociation constants for [Nle 8 ' 18 5 Tyr 34 ]hPTH-l-34)amide, bPTH-(l-34), and PTHrP-(I -36) were 8.5, 10.5, and 14.1 nM, respectively (Table II, top).
  • PTHrP-(49-74) and a synthetic 13-amino acid bio-inactive amino-te ⁇ ninal PTHrP failed to inhibit binding of 125 I-PTHrP -(1-36) to canine renal membranes (FIG. 13).
  • bPTH-(l-34) was substantially more potent than PTHrP-(I -36) in the canine renal cortical adenylate cyclase assay (Table II). This relationship was seen in both the standard assay conditions (30 0 C for 30 min) and under binding assay conditions (20°C for 20 min, with bacitracin). In the standard assay (30 min,
  • bPTH-(l-34) had greater than 6-fold the potency of PTHrP-(I -36) with K n , values of 0.06 and 0.40 nM, respectively.
  • the adenylate cyclase assay was performed under binding conditions which had been demonstrated to result in negligible proteolysis of radioligands. Under conditions identical to the equilibrium binding assay (2O 0 C, 20 min, with bacitracin), adenylate cyclase stimulation by bPTH-(l-34) was 15-fold greater than for PTHrP -(1-36).
  • the K m values under binding assay conditions were 0.13 and 2.00 nM, respectively.
  • PTHrP analogs are evaluated for their effect on bone mass in ovariectomized rats, generally in accord with the procedures of Stewart et al, J. Bone Min Res, 15: 1517-1525 (2000), incorporated by reference herein.
  • three PTHTPTHrP molecules were selected for direct comparison: PTH(l-34), PTHrP(l-36) and the PTH analog, SDZ-PTH-893 (Leu 8 , Asp 10 , Lys 1 ⁇ Ala 16 , GIn 18 , Thr 33 , Ala 34 hPTH(l -34)).
  • Recombinant human PTH(l-34) (rec hPTH(l-34) or LY333334) was prepared as described previously (Hirano et al, J Bone Min Res 14: 536-545 (1999); Frolick et al, J Bone Min Res 14: 163-72 (1999)).
  • PTHrP(I -36) was prepared using solid phase synthesis as described previously (Everhart-Caye et al, JCHn Endocrinol Metab 81: 199-208 (1996); Henry et al, J Clin Endocrinol Metab 82: 900-906 (1997); Plotkin et al, J Clin Endocrinol Metab 83: 2786-2791 (1998)).
  • Protocol The protocol employed is described in schematic form in Table IV. Animals were randomly assigned to 17 groups of 10 as described in the Table. Except for animals in the first group which were sacrificed at five months of age, the remaining animals were observed for one month, and treatment with the various test peptides or vehicle was begun at six months of age. For the peptide-treated animals, the peptide was administered daily, subcutaneously at a dose of 40 ⁇ g/kg/day, in the vehicle described above. For vehicle-treated animals, vehicle alone was administered in an identical fashion.
  • Serum and urine chemistries as described in Table XI were performed using standard auto analysesr methods (Boehringer-Mannheim-Hitachi, Indianapolis, IN). Kidney calcium content was determined following extraction of whole kidneys in 5% trichloroacetic acid, followed by calcium measurement by calcium analyzer (Calcette, Midfield, MA).
  • Bone mass was assessed using bone ash weight as well as DEXA measurements of the radius, femur and whole body.
  • Whole body bone mineral content was determined using a Norland DXA Eclipse densitometer, and results are expressed in mg.
  • Left femur bone mineral density in mg/cm 2 (BMD) 3 bone mineral content in mg (BMC), and cross sectional area in cm 2 (X-area) were determined using a calibrated Hologic QDR 4500A densitometer coupled to Small Animal Regional High Resolution software, as performed by S. Orwoll at Oregon Health Sciences University, Portland OR.
  • Left radius maximal length measurements were performed using Fowler/Sylvac Ultra-Cal III calipers (Newton, MA).
  • Bone histomorphometry was performed on methyl methacrylate embedded sections of the right tibia of each animal following sacrifice as described in Table IV. Animals were labeled using calcein, 30 mg/kg, administered subcutaneously seven and three days prior to sacrifice. Standard histomorphometric measures were performed as shown in Tables IV-VI (Parfitt et al, J Bone Min Res 2: 595-610 (1987)).
  • Bone histomorphometry was performed in order to assess structural features of the skeletal changes as well as changes in bone turnover.
  • trabecular area Tb.Ar
  • FIGS. 15 and 16 trabecular area (Tb.Ar) declined markedly in the OVX control animals and remained depressed throughout the study, as compared to the sham animals.
  • marked increases in trabecular area occurred in all three peptide-treated groups, with the same rank order observed in the bone mass measures: SDZ-PTH>PTH>PTHrP.
  • the increased Tb.Ar in treated animals was principally the result of increased trabecular thickness, which resulted in reduced trabecular separation (see Table V).
  • Bone formation MS/BS
  • FIG. 15 Bone formation
  • FIG. 15 at each time point following initiation of treatment, bone formation parameters were significantly increased in all three peptide-treated animal groups as compared to the age-matched OVX and sham animals (Table VI, FIG. 15).
  • Biomechanical measures improved in all three peptide treated groups (FIG. 17, and see Table IX and X for additional detail).
  • measures of biomechanical strength increased with each of the peptides.
  • ultimate load also increased with all three peptides. The changes were statistically significant and quantitatively large.
  • biomechanical measures at the lumbar spine and femoral neck exceeded those found not only in the OVX controls, but also the sham controls.
  • a cortical bone site Similar findings were observed
  • FIG. 17 In general, the three peptide-treated groups showed augmented or improved biomechanical parameters as compared to both the sham and OVX control groups, and these changes were statistically, quantitatively, and functionally very significant (see Table X for complete details). Body weight increased with increasing age in all groups throughout the study but there were no significant differences among the treated and control . groups._ Animals gained weight at approximately the same rate (see Table XI for details).
  • PTHrP analogs, or other skeletal anabolic agents can be tested using the methods described above.
  • PTHrP analogs, or other skeletal anabolic agents, useful in the methods of the present invention are expected to significantly increase the total bone calcium, trabecular calcium, cortical bone calcium, trabecular thickness, and bone volume over untreated OVX controls.
  • PTHrP, PTH, and TIP peptides may be used to develop peptidomimetics and small molecule drugs, that are useful as agonists and antagonists of these skeletal anabolic agents.
  • a "peptidomimetic" refers to derivatives of the fragments or full length peptides of the skeletal anabolic agents PTHrP, PTH, or TIP, described above, that demonstrate biological activity involving modulation of bone mass, as well as mixtures, pharmaceutical compositions, and compositions comprising the same.
  • a "small molecule drug” refers to a non-naturally occurring low-molecular weight compound, having similar activity.
  • the biological activity of a peptidomimetic or small molecule drug can be agonistic or antagonistic to that of PTHrP, PTH, or TIP, or may include a spectrum of activity, i.e., may be antagonistic to PTH activity and agonistic to PTHrP activity.
  • the biological activity of PTHrP is associated with the N-terminal portion, with residues (1-30) minimally providing the biological activity. Truncated forms of the 39 amino acid tubular infundibular peptide (TIP) are also being assayed for biological activity.
  • Receptors for these agents are also targets for structural-based drug design.
  • the 500-amino-acid PTH/PTHrP receptor also known as the PTHl receptor
  • the extracellular regions are involved in hormone binding, and the intracellular domains, after hormone activation, bind G protein subunits to transduce hormone signaling into cellular responses through stimulation of second messengers. These second messengers likewise provide drug targets.
  • a second PTH receptor (PTH2 receptor) is expressed in brain, pancreas, and several other tissues.
  • compositions modulate, i.e., upregulate or downregulate PTHl or PTH2 receptor activity.
  • a system comprising structural information relating to the atomic coordinates obtained by* x-ray diffraction of a PTHrP, PTH, or TIP peptide, fragment, peptidomimetic or small molecule drug is provided.
  • a PTHrP, PTH, or TIP peptide, fragment, peptidomimetic or small molecule drug is provided.
  • a purified crystalline preparation of a PTHrP, PTH, or TIP peptide, fragment, peptidomimetic or small molecule drug is provided.
  • Structures of PTHl or PTH2 receptors, or a PTHrP, PTH, or TIP peptide, fragment, peptidomimetic or small molecule drug are obtained by x-ray diffraction of crystallized polypeptides, 2-D nuclear magnetic resonance spectroscopy of the same, or by similar methods of obtaining high resolution structures of biological materials.
  • High resolution structures refer to structures solved to greater than 2.8 angstroms, and preferable greater than 2.3 angstroms, and are used to map the active sites of these receptors and their ligands.
  • Structures are determined and interpreted using computer systems described in the art, e.g., having at least a memory bank, a display, a data input means, a processor and an instruction set comprising an algorithm for reading, interpreting and rendering the structural data, all of which are well known in the art, for example see, U.S. Patent No.: 6,273,598 to Keck et ah, entitled, Computer system and methods for producing morphogen analogs of human OP-I, incorporated herein by reference. According to the present invention, such systems may be standalone or networked, i.e., through a packet switched network.
  • CAD Computer aided design
  • Binding studies were conducted at 20 0 C using monoiodinated [Nle 8>18l Tyr 34 ]hPTH-(l-34) amide ( 125 I-PTH) or Tyr 36 ]PTHrP-(l-36)amide ( 125 I-PTHrP) as the radioligand.
  • the IQ values were determined by Scatchard analysis, and the K, values were derived from the IC 50 values.
  • Adenylate cyclase stimulation was evaluated under standard assay conditions, employing partially purified canine renal membranes and 30-min incubations at 30°C. Adenylate cyclase stimulation was also evaluated under binding assay conditions, using highly purified canine renal membranes in the presence of bacitracin (200 ⁇ g/ml) and 20-min incubations at 20 0 C.
  • Baseline OVX 1 66 ⁇ 1" 55 ⁇ 1 34.6 ⁇ 0.2 1.62 ⁇ 0.03 0.289 ⁇ 0.008 0.177 ⁇ 0.002
  • Baseline Sham 2 53 ⁇ 1 52 ⁇ 1 33.6 ⁇ 0.2 1.55 ⁇ 0.03 0.294 ⁇ 0.007 0.190 ⁇ 0.004
  • BMD bone mineral density
  • OVX ovariectomized.
  • WW Wet weight
  • OVX ovariectomized.
  • n number rats per group
  • OVX ovariectomized
  • WW Wet weight
  • OVX ovariectomized.

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Abstract

L'invention concerne des méthodes de prévention et de traitement de diverses maladies touchant les mammifères se manifestant par une perte de la masse osseuse, telles que l'ostéoporose. L'invention concerne des méthodes utilisant PTHrP (protéine liée à l'hormone parathyroïde) ou des analogues de celles-ci, qui sont à la fois efficaces et plus sûrs, dans le traitement des affections osseuses métaboliques.
PCT/US2005/032706 2004-09-16 2005-09-14 Traitement des affections osseuses a l'aide de medicaments anabolisants du squelette WO2006033912A2 (fr)

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MX2007003185A MX2007003185A (es) 2004-09-16 2005-09-14 Tratamiento de trastornos oseos con farmacos anabolicos esqueletales.
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US8568737B2 (en) 2007-08-01 2013-10-29 The General Hospital Corporation Screening methods using G-protein coupled receptors and related compositions
US9492508B2 (en) 2010-05-13 2016-11-15 The General Hospital Corporation Parathyroid hormone analogs and uses thereof

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US7015195B2 (en) * 2002-01-10 2006-03-21 Osteotrophin, Llc Treatment of bone disorders with skeletal anabolic drugs
CA2496618A1 (fr) * 2002-05-23 2003-12-04 Michael Holick Utilisation d'analogues peptidiques d'hormone parathyroide pour le traitement de l'atrophie vaginale

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US8568737B2 (en) 2007-08-01 2013-10-29 The General Hospital Corporation Screening methods using G-protein coupled receptors and related compositions
EP2650009A3 (fr) * 2007-08-01 2014-01-29 The General Hospital Corporation Procédés de criblage utilisant les récepteurs couplés à la protéine G et compositions associées
CN104072606A (zh) * 2007-08-01 2014-10-01 总医院有限公司 利用g蛋白偶联受体和相关组合物的筛选方法
US9057727B2 (en) 2007-08-01 2015-06-16 The General Hospital Corporation Screening methods using G-protein coupled receptors and related compositions
CN104072606B (zh) * 2007-08-01 2017-09-22 总医院有限公司 利用g蛋白偶联受体和相关组合物的筛选方法
US9492508B2 (en) 2010-05-13 2016-11-15 The General Hospital Corporation Parathyroid hormone analogs and uses thereof

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