WO2011071480A1 - Hormone parathyroïde de l'ours noir et ses procédés d'utilisation - Google Patents

Hormone parathyroïde de l'ours noir et ses procédés d'utilisation Download PDF

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Publication number
WO2011071480A1
WO2011071480A1 PCT/US2009/066974 US2009066974W WO2011071480A1 WO 2011071480 A1 WO2011071480 A1 WO 2011071480A1 US 2009066974 W US2009066974 W US 2009066974W WO 2011071480 A1 WO2011071480 A1 WO 2011071480A1
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Prior art keywords
bone
pth
seq
polypeptide
amino acid
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PCT/US2009/066974
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English (en)
Inventor
Seth W. Donahue
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Michigan Technological University
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Priority to PCT/US2009/066974 priority Critical patent/WO2011071480A1/fr
Priority to US13/514,362 priority patent/US8987201B2/en
Priority to AU2009356227A priority patent/AU2009356227A1/en
Priority to BR112012013725A priority patent/BR112012013725A2/pt
Priority to JP2012543070A priority patent/JP2013512688A/ja
Priority to CA2782640A priority patent/CA2782640A1/fr
Priority to EP09764429A priority patent/EP2509996A1/fr
Publication of WO2011071480A1 publication Critical patent/WO2011071480A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone (parathormone); Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention provides an isolated polypeptide comprising at least 10 consecutive amino acid residues of SEQ ID NO: 2 wherein the polypeptide comprises at least one of amino acid residues 41 and 52 of SEQ ID NO: 2.
  • isolated polypeptides comprising amino acid residues 1 -84 of SEQ ID NO: 2 are provided.
  • Polynucleotides encoding such polypeptides are also provided.
  • the present invention also provides an isolated polynucleotide comprising SEQ ID NO: 1 .
  • the invention provides a method of increasing cAMP levels in a bone-forming cell comprising contacting the bone-forming cell with an effective amount of at least one polypeptide selected from a polypeptide comprising amino acid residues 1 -34 of SEQ ID NO: 2, a polypeptide comprising amino acid residues 1 -36 of SEQ ID NO: 2, a polypeptide comprising amino acid residues 1-84 of SEQ ID NO: 2, wherein contacting the bone-forming cell with the polypeptide increases cAMP levels in the bone- forming cell.
  • the invention provides a method of reducing apoptosis in a bone-forming cell comprising contacting the bone-forming cell with an effective amount of at least one polypeptide selected from a polypeptide comprising amino acid residues 1 -34 of SEQ ID NO: 2, a polypeptide comprising amino acid residues 1 -36 of SEQ ID NO: 2, a polypeptide comprising amino acid residues 1-84 of SEQ ID NO: 2, wherein contacting the bone-forming cell with the polypeptide reduces apoptosis in the bone- forming cell.
  • the invention provides a method of decreasing the ratio of expression levels of Bax protein to Bcl-2 protein in a bone-forming cell comprising contacting the bone-forming cell with an effective amount of at least one polypeptide selected from a polypeptide comprising amino acid residues 1-34 of SEQ ID NO: 2, a polypeptide comprising amino acid residues 1 -36 of SEQ ID NO: 2, a polypeptide comprising amino acid residues 1-84 of SEQ ID NO: 2, wherein contacting the bone-forming cell with the polypeptide decreases the ratio of expression levels of Bax protein to Bcl-2 protein in the bone-forming cell.
  • the invention provides a method of increasing the expression level of a bone matrix protein, a transcriptional activator, or a transcriptional regulator in a bone-forming cell comprising contacting the bone-forming cell with an effective amount of at least one polypeptide selected from a polypeptide comprising amino acid residues 1 -34 of SEQ ID NO: 2, a polypeptide comprising amino acid residues 1 -36 of SEQ ID NO: 2, a polypeptide comprising amino acid residues 1-84 of SEQ ID NO: 2, wherein contacting the bone-forming cell with the polypeptide increases the expression level of the bone matrix protein, the transcriptional activator, or the transcriptional regulator in the bone- forming cell.
  • the invention provides a method of enhancing bone mineral density, increasing bone mass, decreasing bone loss, or reducing the incidence of bone fractures, or any combination thereof, in a subject, comprising contacting a bone- forming cell in the subject with an effective amount of at least one polypeptide selected from a polypeptide comprising amino acid residues 1 -34 of SEQ ID NO: 2, a polypeptide comprising amino acid residues 1 -36 of SEQ ID NO: 2, a polypeptide comprising amino acid residues 1-84 of SEQ ID NO: 2, wherein contacting the bone-forming cell with the polypeptide enhances bone mineral density, increases bone mass, decreases bone loss, or reduces the incidence of bone fractures in the subject.
  • the invention provides a method of enhancing bone mineral density, increasing bone mass, decreasing bone loss, or reducing the incidence of bone fractures, or any combination thereof, in a subject, comprising contacting a bone- forming cell in the subject with an effective amount of a polypeptide comprising bear parathyroid hormone or a functional fragment thereof, wherein contacting the bone-forming cell with the polypeptide enhances bone mineral density, increases bone mass, decreases bone loss, or reduces the incidence of bone fractures in the subject.
  • the invention provides an isolated polypeptide consisting of amino acid residues 1 -34 of SEQ ID NO: 2. In certain embodiments, the invention provides an isolated polypeptide consisting of amino acid residues 1 -36 of SEQ ID NO: 2. In certain embodiments, the invention provides an isolated polypeptide consisting of amino acid residues 1 -84 of SEQ ID NO: 2.
  • Figure 1 shows ultimate stress, a measure of bone strength, and ash fraction, a measure of bone mineral content, which both increase with age in black bears.
  • PTH serum parathyroid hormone
  • Figure 3 shows normalized serum resorption (ICTP) and formation (PICP and osteocalcin) marker concentrations during the 3 month disuse period.
  • FIG. 4 shows that the amount of PGE2 released by osteoblastic cells was greatest when the cells were treated with serum collected in the post-hibernation period.
  • Figure 5 shows the sequence of the mature black bear PTH protein compared to other known PTH sequences.
  • Figure 8 shows uCT images of a proximal rat tibia (left), 1 .6 mm analysis region of cortical and trabecular bone starting 2 mm distal to the proximal physis (top right), and the trabecular analysis region (bottom right).
  • Figures 9A-9F show pCT images of bones from rats following sham ovariectomy (OVX) (Fig. 9A) or actual OVX followed by six (Fig. 9B) or fourteen (Fig. 9C) weeks of recovery with no PTH treatment, or by fourteen weeks of recovery including eight weeks of treatment with 3 pg/kg (Fig. 9D), 10 ⁇ g/kg (Fig. 9E), or 30 pg/kg (Fig. 9F) black bear PTH 1-34.
  • OVX sham ovariectomy
  • Figure 10 shows a graph of bone volume as a fraction of total volume (BV/TV) for bones from rats having had sham or actual OVX procedures followed by treatment with vehicle (saline) or human or bear PTH1 -34.
  • Figure 1 1 shows a graph of bone mineral apparent density for bones from rats having had sham or actual OVX procedures followed by treatment with vehicle (saline) or human or bear PTH1 -34.
  • Figure 12 shows a graph of trabecular strut number for bones from rats having had sham or actual OVX procedures followed by treatment with vehicle (saline) or human or bear PTH1 -34.
  • Figure 13 shows a graph of trabecular thickness for bones from rats having had sham or actual OVX procedures followed by treatment with vehicle (saline) or human or bear PTH 1-34.
  • Figure 14 shows a graph of trabecular bone mineralization for bones from rats having had sham or actual OVX procedures followed by treatment with vehicle (saline) or human or bear PTH1 -34.
  • Figure 15 shows a graph of tibial cortical bone volume for bones from rats having had sham or actual OVX procedures followed by treatment with vehicle (saline) or human or bear PTH1 -34.
  • Figure 16 shows a graph of tibial cortical porosity for bones from rats having had sham or actual OVX procedures followed by treatment with vehicle (saline) or human or bear PTH 1-34.
  • Figure 17 shows a graph of femoral cortical bone strength for bones from rats having had sham or actual OVX procedures followed by treatment with vehicle (saline) or human or bear PTH1 -34.
  • Figure 18 shows a graph of serum calcium levels of rats having had sham or actual OVX procedures followed by treatment with vehicle (saline) or human or bear PTH 1- 34.
  • Figure 19 shows the relative caspase-3 activity in MC3T3-S4 cells pretreated with 0-100 nM human or bear PTH peptides and then subjected to serum starvation.
  • LS means with standard error bars are presented. Bars with different letters are significantly (p ⁇ 0.05) different from each other.
  • Figure 20 shows ultimate load of the femur in OVX rats treated with vehicle (V), 2.5 nmol/kg hPTH1 -84, or 2.5 nmol/kg bbPTH 1-84.
  • Figure 21 shows normalized ultimate load of the femur in OVX rats treated with vehicle (V), 2.5 nmol/kg hPTH1-84, or 2.5 nmol/kg bbPTH1 -84.
  • Figure 22 shows bone volume fraction in OVX rats treated with vehicle (V), 2.5 nmol/kg hPTH 1 -84, or 2.5 nmol/kg bbPTH1 -84.
  • Figure 23 shows trabecular number in OVX rats treated with vehicle (V), 2.5 nmol/kg hPTH1 -84, or 2.5 nmol/kg bbPTH1 -84.
  • Figure 24 shows trabecular thickness in OVX rats treated with vehicle (V), 2.5 nmol/kg hPTH1 -84, or 2.5 nmol/kg bbPTH1 -84.
  • Figure 25 shows trabecular separation in OVX rats treated with vehicle (V), 2.5 nmol/kg hPTH1 -84, or 2.5 nmol/kg bbPTH1 -84.
  • Figure 26 shows apparent mineral density in OVX rats treated with vehicle (V), 2.5 nmol/kg hPTH1 -84, or 2.5 nmol/kg bbPTH 1-84.
  • Figure 27 shows material mineral density in OVX rats treated with vehicle (V), 2.5 nmol/kg hPTH 1 -84, or 2.5 nmol/kg bbPTH1 -84.
  • Figure 28 shows caspase 3/7 activity in cells cultured in prehibernation, hibernation, and posthibernation bear serum.
  • Figure 29 shows ultimate load of femurs in mice treated with vehicle or various PTH peptides.
  • the deleterious effects of disuse on bone may continue into the remobilization period. Some bone may be recovered during remobilization, but recovery is slow and often incomplete. For example, the rate of bone loss during bedrest is more than three times greater than the rate of bone gain during remobilization, and the recovery of bone lost in spaceflight can be incomplete even after 5 years. When disuse-induced changes in bone can be completely reversed by resumed activity, the remobilization period is often 2 to 3 times longer than the immobilization period. Bone formation decreases and/or bone resorption increases in many situations that reduce mechanical loads on bone. However, both resorption and formation increase during canine forelimb immobilization, yet there is significant bone loss in that case. Likewise, thigh bone turnover occurs in patients with spinal cord injury, which leads to bone loss and increased fracture incidence.
  • black bears do not suffer significant bone loss due either to aging ( Figure 1 ) or, more importantly, to the extended periods of disuse that occur during hibernation.
  • Hibernating black bears have immobilization and active periods that can be approximately equal in length in northern regions.
  • Data on serum markers of bone metabolism suggest that both resorption and formation increase during disuse in bears, with a normal lag time (i.e., reversal period) between resorption and formation, and that the increase in formation remains coupled and balanced with the increase in resorption.
  • Histological data from black bear iliac crest biopsies also show increased resorption and formation during inactivity.
  • bears are unique in that trabecular bone volume, bone mineral density, and bone mineral content do not decrease during hibernation.
  • cortical bone strength and ash fraction increase with age, and porosity decreases with age in black bears, despite annual periods of disuse.
  • Cortical bone porosity is significantly lower in hibernating grizzly bears than in active grizzly bears, and femoral cross-sectional geometry and strength are unaffected by hibernation.
  • Bears have evolved many unique biological mechanisms to survive long periods of immobilization without food. These mechanisms appear to include the recycling of calcium and other products of bone catabolism, since bears increase bone turnover but do not excrete waste during hibernation. In humans, bedrest-induced disuse osteoporosis is caused primarily by increased resorption without a corresponding increase in formation. This results in hypercalcemia and a negative calcium balance brought about by increased urinary and fecal calcium. Since bears do not urinate or defecate during hibernation, it is likely that most of the calcium released from bone by resorption is recycled back into bone via osteoblastic bone formation.
  • Ionized calcium is found to increase by about 23% during hibernation, possibly because of the lag time between resorption and formation.
  • black bear PTH levels are highest when levels of ionized calcium are highest (Example 2, Table 1 ).
  • PTH is the primary regulator of blood calcium levels, and thus plays a role in maintaining homeostatic serum calcium levels in black bears during disuse. Serum PTH levels are positively correlated with the bone formation marker osteocalcin in active and hibernating black bears (Figure 2), and both osteocalcin and PTH increase during hibernation. In addition, black bear PTH concentration is highest when ionized calcium concentration is highest. Since bone resorption increases during hibernation but total serum calcium (tCa) remains unchanged, increased levels of PTH likely cause increased renal reabsorption of calcium, facilitating the recycling of mineral back into the bone with a balanced increase in bone formation.
  • black bear PTH has 9 different amino acid residues out of a total of the 84 amino acid residues of the full-length, mature PTH polypeptide. Also described herein are various methods of use for black bear PTH and functional fragments thereof. It is specifically envisioned that polypeptide subfragments comprising at least 10 consecutive amino acid residues of SEQ ID NO: 2 and including at least one of amino acid residues 41 or 52 can be used to develop antibodies specific for black bear PTH. These antibodies can be used to quantify black bear PTH, e.g. in an ELISA assay.
  • PTH receptors on the surface of bone-forming cells are coupled to cyclic adenosine monophosphate (cAMP)-dependent second-messenger signaling pathways inside the cells. These signaling pathways, in turn, lead to increased expression of genes involved in bone formation such as those encoding type I collagen, osteonectin, and osteopontin. Since the cAMP/protein kinase A pathway is responsible for the majority of PTH-induced increases in histological and serum indices of bone formation, it follows that an increased cAMP response can lead to greater bone formation. A relatively small number of amino acid substitutions in the sequence of a given PTH protein can stimulate greater cyclic adenosine monophosphate (cAMP) production compared to the native form.
  • cAMP cyclic adenosine monophosphate
  • ovariectomized rats demonstrated a 25% greater bone formation response to daily 25 g injections of bovine PTH 1 -34 than to rat PTH 1-34, where rat PTH 1 -34 has 5 amino acid sequence differences compared to bovine PTH 1-34. Injection of bovine PTH 1-34 results in a 37% greater increase in bone volume fraction during treatment.
  • contacting a bone-forming cell with black bear PTH or a functional fragment thereof increases cAMP levels in the bone-forming cell.
  • the bone- forming cell is contacted with a polypeptide comprising amino acid residues 1 -34 or 1 -36 of SEQ ID NO: 2.
  • the bone-forming cell is contacted with a polypeptide comprising SEQ ID NO: 2.
  • contacting a cell includes adding the polypeptide to the culture solution, in the case of in vitro experiments, or administering the polypeptide to a subject using appropriate administration procedures for polypeptide therapeutic agents.
  • Contacting a cell also includes introducing into a subject an exogenous polynucleotide that encodes the desired polypeptide in an expression system so as to synthesize and release the polypeptide in the subject.
  • bone-forming cells includes, but is not limited to, osteoblasts, osteocytes, bone lining cells, chondroblasts, and chondrocytes. Suitably, the bone-forming cell may be in a subject.
  • Bone-forming cells regularly turn over, with most of the cells' death being due to programmed cell death, or apoptosis. Given this regular rate of turnover, any mechanism that decreases apoptosis of bone-forming cells will lead to an increased number of bone- forming cells which presumably will promote bone growth. Thus, in certain embodiments of the present invention, contacting a bone-forming cell with black bear PTH or a functional fragment thereof reduces apoptosis in the bone-forming cell.
  • the bone-forming cell is contacted with a polypeptide comprising amino acid residues 1 -34 or 1-36 of SEQ ID NO: 2. In certain embodiments of the present invention, the bone-forming cell is contacted with a polypeptide comprising SEQ ID NO: 2.
  • C-terminal fragments of endogenous black bear PTH play a role in seasonal bone remodeling processes via binding to CPTHRs (C-terminal PTH receptors).
  • C-terminal fragments of bear PTH may antagonize the calcemic effects of PTH 1 -84 and 1-34 by preventing osteoclastogenesis and possibly by affecting mature osteoclast activity that would normally occur in response to resorptive stimuli such as disuse (Divieti, P. et al., 2002, Endocrinology 143(1 ): 171-6). This may help bears to maintain homeostatic calcium levels throughout hibernation.
  • osteocytes and osteoblasts may be contacted with at least one polypeptide selected from a polypeptide comprising amino acid residues 11 -84 and a polypeptide comprising amino acid residues 7-84 of SEQ ID NO: 2.
  • the protein Bax promotes apoptosis while the Bcl-2 protein protects cells from apoptosis, and a decrease in the expression ratio of Bax to Bcl-2 is indicative of a decrease in apoptosis in the particular cell population.
  • contacting a bone-forming cell with black bear PTH or a functional fragment thereof decreases the ratio of expression levels of Bax protein relative to expression levels of Bcl-2 protein in the bone-forming cell.
  • the bone-forming cell is contacted with a polypeptide comprising amino acid residues 1 -34 or 1 -36 of SEQ ID NO: 2.
  • the bone-forming cell is contacted with a polypeptide comprising SEQ ID NO: 2.
  • Example 1 1 shows that black bear PTH 1 -34 decreases the expression ratio of Bax/Bcl-2 in cultured cells whereas human PTH 1 -34 increases the expression ratio of Bax/Bcl-2 ( Figure 7).
  • black bear PTH 1-34 appears to be more effective at preventing apoptosis than human PTH 1 -34. Without being bound by theory, this difference may be the result of the two amino acid differences between human and black bear PTH 1 -34.
  • the transcriptional activator is Runx2.
  • the transcriptional regulator is c-fos.
  • Exemplary bone matrix proteins include, but are not limited to, osteocalcin, osteopontin, and type I collagen.
  • the bone-forming cell is contacted with a polypeptide comprising amino acid residues 1-34 or 1 -36 of SEQ ID NO: 2.
  • the bone-forming cell is contacted with a polypeptide comprising SEQ ID NO: 2.
  • Exogenous human PTH is used to treat post-menopausal and age-related osteoporosis in humans, but it is not an ideal therapeutic.
  • Only recombinant human PTH 1 - 34 (LY333334, Eli Lilly, Indianapolis IN) is currently approved for clinical use, and only one form of recombinant human PTH 1 -84 is under consideration for approval by the U.S. Food and Drug Administration (ALX1-1 1 , NPS Pharmaceuticals, Parsippany, NJ).
  • ALX1-1 1 NPS Pharmaceuticals, Parsippany, NJ.
  • LY333334 and ALX1 -1 1 can stimulate approximately the same magnitude of bone formation in vivo, their biological actions are not identical.
  • PTH 1 -34 downregulates production of procollagen-1 mRNA, whereas PTH 1 -84 does not (Nasu et al., 1998, Endocr J, 45, 229-34).
  • PTH 1 -84 does not (Nasu et al., 1998, Endocr J, 45, 229-34).
  • the C-terminal portion of human PTH when cleaved from the mature hormone, has important biological functions such as inhibition of bone resorption.
  • the present invention is a method of treating osteroporosis comprising administering an effective amount of black bear PTH or a functional fragment thereof to a subject in need thereof.
  • the osteoporosis is primary or age-related osteoporosis and/or disuse osteoporosis.
  • contacting a bone-forming cell in a subject with black bear PTH or a functional fragment thereof increases bone mineral density, increases bone mass, decreases bone loss or reduces the incidence of bone fracture in the subject.
  • the bone-forming cell is contacted with a polypeptide comprising amino acid residues 1 -34 or 1-36 of SEQ ID NO: 2.
  • the bone-forming cell is contacted with a polypeptide comprising SEQ ID NO: 2.
  • contacting a bone-forming cell in a subject with black bear PTH or a functional fragment thereof increases bone mineral density, increases bone mass, decreases bone loss or reduces the incidence of bone fracture by at least about 5% or at least about 10%.
  • the increase in bone mineral density, increase in bone mass, decrease in bone loss or reduction in the incidence of bone fracture may be at least about 15%, at least about 30%, at least about 50%, at least about 75% or at least about 90%.
  • the increase in bone mineral density, increase in bone mass, decrease in bone loss or reduction in the incidence of bone fracture is determined by measuring the desired characteristic on the same patient before and after treatment by a technique known to one of ordinary skill in the art.
  • bone mineral density can be determined by methods involving taking dual energy x-rays (DEXA) or CT scans of bones in the spinal column, wrist, arm or leg.
  • the subject may suitably be a mammal, including without limitation human, horse, dog, cat, mouse, bear, bovine, pig, or deer.
  • the subject is a post-menopausal human female.
  • the subject may have osteoporosis or may be at risk to develop osteoporosis.
  • Risk factors for developing osteoporosis include: personal history of fracture after age 50; current low bone mass; history of fracture in a first-degree relative; being female; being thin and/or having a small frame; advanced age; a family history of osteoporosis; estrogen deficiency as a result of menopause, especially early or surgically induced; abnormal absence of menstrual periods (amenorrhea); anorexia nervosa; low lifetime calcium intake; vitamin D deficiency; use of certain medications (corticosteroids, chemotherapy, anticonvulsants and others); presence of certain chronic medical conditions, such as those that decrease calcium absorption in the gut such as Crohn's disease; low testosterone levels in men; an inactive lifestyle; current cigarette smoking; excessive use of alcohol; and being Caucasian or Asian; although African Americans and Hispanic Americans are at significant risk as well. Moreover, women can lose up to 20 percent of their bone mass in the five to seven years following menopause, making them more susceptible to osteoporosis.
  • Black bear PTH or functional fragments thereof are also useful as a preventative (rather than a restorative), or prophylactic, measure to combat disuse osteoporosis or to prevent osteoporosis in a subject at risk for developing osteoporosis. Since bears appear to be the only animals that maintain balanced bone remodeling during disuse, black bear PTH or functional fragments thereof are also useful to prevent bone loss during reduced skeletal unloading that occurs, for example, in astronauts during spaceflight and in spinal cord injury patients after injury.
  • Black bear PTH or functional fragments thereof may be administered in combination with calcium and/or vitamin D.
  • "Vitamin D” refers to the entire Vitamin D class of compounds.
  • Black bear PTH or functional fragments thereof may also be administered in combination with other anabolic or antiresporptive active agents.
  • Suitable anabolic active agents include various fragments of human PTH (e.g. 1 -34 and 1 -84), fluoride, GH, insulin-like growth factor I, statins, and PYK2 kinase inhibitors.
  • Suitable antiresorptive active agents include, but are not limited to estrogens, selective estrogen receptor modulators, calictonin, and bisphosphonates.
  • an anabolic functional fragment of black bear PTH (e.g. black bear PTH 1-34) is administered in combination with an antiresorptive active agent.
  • different functional fragments of black bear PTH may be administered in combination with each other. The different functional fragments may be administered concurrently or in any order that is suitable.
  • the calcium, vitamin D, anabolic active agent, and/or antiresporptive active agent may be administered concurrently with black bear PTH or functional fragments thereof or may be administered before or after black bear PTH or functional fragments thereof.
  • the calcium, vitamin D, anabolic active agent, and/or antiresporptive active agent may be in a separate composition or may be in the same pharmaceutical composition as the black bear PTH or functional fragments thereof.
  • Black bear PTH or functional fragments thereof or compositions comprising black bear PTH or functional fragments thereof can be accomplished by any suitable technique.
  • Black bear PTH or functional fragment thereof may be administered by any suitable route including, for example, oral, nasal, rectal, and parenteral routes of administration.
  • parenteral includes but is not limited to subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, and intrathecal administration, such as by injection.
  • administration of a polypeptide includes administration of an exogenous polynucleotide operably connected to a promoter such that the polynucleotide expresses the polypeptide in the subject.
  • Administration of the polypeptide also includes administration of a viral vector comprising a polynucleotide encoding the polypeptide.
  • the viral vector is an adenoviral vector.
  • Black bear PTH or functional fragments thereof, or compositions comprising black bear PTH or functional fragments thereof, can be administered continuously or at discrete time intervals as can be readily determined by a person skilled in the art. An ordinarily skilled clinician can determine a suitable amount of black bear PTH or a functional fragment thereof to be administered to a subject.
  • the effective dose for any particular subject will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; route of administration; the rate of excretion or inactivation of black bear PTH or functional fragments thereof employed; the duration of the treatment; other pharmaceuticals used in combination or coincidental with black bear PTH or functional fragments thereof and like factors well known in the medical arts. For example, it is well within the level of ordinary skill in the art to start doses at levels lower than those required to achieve the desired effect and to gradually increase the dosage until the desired effect is achieved.
  • the dosage of black bear PTH or functional fragments thereof in certain embodiments is in a range of 0.10 pg/kg per day to 40 pg/kg per day. In certain embodiments, the dosage is in a range of 5 pg/kg per day to 20 pg/kg per day. In certain embodiments, the dosage is 10 pg/kg per day. In certain embodiments, the dosage is in a range of 10 pg/day to 400 pg/day per subject. In certain embodiments, the dosage is in a range of 20 pg/day to 40 pg/day per subject. In certain embodiments, the dosage is 30 pg/day per subject.
  • the dosage of antiresorptives is about 5 to about 100 mg per day
  • the dosage of calcium is from about 500 to about 1500 mg per day
  • the dosage of vitamin D is from about 500 to about 1500 mg per day
  • the dosage of anaoblics is from about 0.1 mg to about .5 mg per day or about 10 pg/day to 400 pg/day.
  • the subject is a human.
  • the daily dosages in certain embodiments are given for one week, in certain embodiments for one month, in certain embodiments for three months, in certain embodiments for six months, in certain embodiments for one year, in certain embodiments for one and a half years, in certain embodiments for two years, and in certain embodiments for three years.
  • the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.
  • a suitable delivery device is loaded with the effective daily dose for more than one day, for example, for seven days, fourteen days, twenty-one days, twenty-eight days or the like, and the delivery device is used to repeatedly administer the desired daily single dose or daily multiple doses for the desired total number of days.
  • those of ordinary skill in the art will readily optimize effective doses and co-administration regimens as determined by good medical practice and the clinical condition of the individual subject.
  • compositions containing black bear PTH or functional fragments thereof useful in the methods of the present invention can be formulated according to known methods for preparing pharmaceutically useful compositions.
  • Formulations are described in detail in a number of sources which are well known and readily available to those skilled in the art. For example, Remington's Pharmaceutical Science, by E. W. Martin, describes formulations which can be used in the disclosed methods.
  • the compositions will be formulated such that an effective amount of the black bear PTH or functional fragment thereof is combined with a suitable carrier in order to facilitate effective administration of the composition.
  • compositions used in the present methods can also be in a variety of forms. These include, for example, solid, semi-solid, and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspension, suppositories, injectable and infusible solutions, and sprays. The form will depend on the intended mode of administration and therapeutic application.
  • the compositions also suitably include conventional pharmaceutically acceptable excipients which are known to those skilled in the art.
  • excipients include water for injection, ethanol, dimethyl sulfoxide, glycerol, alumina, starch, glacial acetic acid, sodium acetate, mannitol, metacresol, hydrochloric acid and/or sodium hydroxide to adjust the pH of a composition to a suitable value, and equivalent or otherwise suitable carriers and diluents.
  • pharmaceutical compositions will comprise between about 0.1 % and 99%, and suitably between about 1 and 15% by weight of the total of one or more of the polypeptides of the present invention based on the weight of the total composition including the carrier or diluent.
  • a polynucleotide amplified using PCR so that it is sufficiently and easily distinguishable (on a gel, for example) from the rest of the cellular components is considered “isolated".
  • the nucleic acid molecules, polynucleotides, and polypeptides of the invention may be "substantially pure,” i.e., having the highest degree of purity that can be achieved using purification techniques known in the art.
  • a "functional fragment” refers to any region or portion of a polypeptide or polynucleotide which is a region or portion of a larger polypeptide or polynucleotide, the region or portion having an activity or function attributable to the larger polypeptide or polynucleotide.
  • a functional fragment of human PTH is the 1-34 region of human PTH.
  • Functional fragments of black bear PTH include, but are not limited to, 1 -34 and 1 -36.
  • any numerical range recited herein includes all values from the lower value to the upper value, i.e., all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application. For example, if a concentration range is stated as 1 % to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1 % to 3%, etc., are expressly enumerated in this specification. If a concentration range is "at least 5%,” it is intended that all percentage values up to and including 100% are also expressly enumerated. These are only examples of what is specifically intended.
  • Black bear genomic DNA was used for PCR amplification of PTH, using consensus primers designed based on alignment of eight full-length mammalian PTH sequences available in GenBank including bovine (Bos taurus, AAA30749), cat (Felis catus, Q9GL67), dog (Canis familiaris, P52212), human (Homo sapiens, NP_000306), macaque (Macaca fascicularis, Q9XT35), mouse (Mus musculus, NP_065648), pig (Sus scrofa, NP_999566), and rat (Rattus norvegicus, NP_058740).
  • PCR amplification was performed using 10-15 ng genomic DNA, 100 ⁇ dNTPs, 0.2 ⁇ each primer, and 1 unit REDTaq (Sigma, St. Louis, MO) in 20 pL reaction volume. PCR products were gel-purified using the UltraClean GelSpin Kit (MoBio Carlsbad, CA) and cloned into the pCRII vector using the TA cloning kit (Invitrogen, Carlsbad, CA). DNA sequencing was performed using the DTCS Quick Start kit and the CEQ8000 Genetic Analysis System (Beckman Coulter, Fullerton, CA), following the manufacturer's instructions.
  • Nucleotide sequences were searched against the GenBank protein database using BlastX (Altschul et al., 1997; Nucleic Acids Res., 25, 3389-402) to confirm their putative identity as PTH. Multiple sequence alignment was performed by ClustalW version 1.82 (Chenna et al., 2003; Nucleic Acids Res., 31 , 3497-500). Phylogenetic analysis was carried out using the neighbor-joining (NJ) method implemented in the Molecular Evolutionary Genetics Analysis (MEGA) package version 3.0 (Kumar et al., 2004; Brief Bioinform., 5, 150-63), with the pairwise deletion option for handling alignment gaps, and with the Poisson correction model for distance computation.
  • NJ neighbor-joining
  • MEGA Molecular Evolutionary Genetics Analysis
  • Black bear PTH shares 84-95% sequence similarity with other mammalian PTHs, and is most similar to dog PTH (91 % identity, 95% similarity) ( Figure 5). Interestingly, two amino acid residues, 41 and 52 of the mature hormone, are unique to black bear PTH.
  • Blood samples were drawn from the femoral vein while the bears were anesthetized, and the samples were transported to the laboratory in an ice-packed cooler. Immediately on return to the laboratory, the blood was centrifuged to isolate the serum, which was frozen at -20° F. Blood samples were collected from each bear every 10 days from the first of October through the end of May. Hibernation began in early January and ended in early April. Thus, the collection dates encompassed an active pre- hibernation period, a disuse hibernation period, and an active post-hibernation remobilization period.
  • the crude EDTA solution was diluted 2-fold and passed over a bulk column containing 10 g Sepralyte C18 particles (Analytichem International, Harbor City, CA) previously activated with methanol and equilibrated with 0.1 % trifluoroacetic acid in water (0.1 % TFA). An extensive wash with 0.1 % TFA was followed by 30% methanol/0.1 % TFA until UV absorbance dropped to baseline. Osteocalcin was eluted with 80% methanol/0.1 % TFA. Methanol was evaporated under a stream of air and the remaining solution lyophilized.
  • the resulting dried protein was suspended in 0.05 M Tris buffer, pH 8.0 and applied to a 5 ml Biorad Econo-Q column previously equilibrated with the same buffer.
  • the column was developed with a gradient from 0.1 to 0.6 M NaCI in 0.5 M Tris, pH 8.0.
  • Osteocalcin eluted in a symmetric peak, the last to elute from the column. Identity of this peak as osteocalcin was qualitatively verified by reacting fraction aliquots with diazobenzene sulfonic acid yielding a pink color in those fractions containing osteocalcin, with intensity corresponding to peak height.
  • Both the C18 and the Econo-Q column were new and never exposed to protein from other species. Previous experience with other species suggests the final osteocalcin peak is greater than 99% pure. Concentration of black bear osteocalcin in the final elute was determined with BCA reagents from Pierce Chemical (Rockford, IL).
  • the serum was assayed for PTH, 25-OH D, leptin, IGF-I, and osteocalcin (a bone formation marker) using RIA and ELISA.
  • the bone resorption marker (ICTP) began to increase immediately after the onset of hibernation ( Figure 3). Each data point is the mean value from 5 bears. After 10-20 days, the bone formation markers (osteocalcin and PICP) also increased and appeared to remain coupled to the increased resorption for the duration of hibernation. This is consistent with the 1 -2 week histological "reversal" period between resorption and formation. These remodeling markers showed trends of increased resorption and formation throughout the hibernation period, and formation appeared to remain coupled and balanced with resorption. Mean osteocalcin levels were higher (p ⁇ 0.0001 ) during and after hibernation compared to pre-hibernation (Table 1 ).
  • Serum leptin did not change during hibernation relative to pre-hibernation, but was significantly (p ⁇ 0.004) lower during post-hibernation remobilization (Table 1 ).
  • IGF-1 significantly (p ⁇ 0.0001 ) decreased during hibernation relative to pre-hibernation and reached its highest value during remobilization (Table 1 ).
  • Table 1 Mean Serum Metabolite Concentrations
  • MC-3T3 cells were treated with bear serum and prostaglandin E 2 (PGE 2 ) release was quantified.
  • MC-3T3 cells were grown in alpha minimum essential media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Hyclone , Logan, UT) and 1 % penicillin-streptomycin solution at 37°C in 5% C0 2 for 24 hours. The media was aspirated and replaced with 10 ml of fresh media containing 10% bear serum collected prior to hibernation, during hibernation, or after hibernation.
  • PGE 2 prostaglandin E 2
  • the cells were allowed to grow for an additional 24 hours, and then the media was collected and frozen at -20°C for PGE2 analysis.
  • the cells were removed from the culture dishes using 0.25% trypsin in EDTA, pelleted by centrifugation, and quantified with a trypan blue and hemocytometer.
  • the PGE2 levels were determined using the BiotrakTM PGE 2 competitive enzyme immunoassay (Amersham Biosciences, Piscataway, NJ). The assay was performed in duplicate using 50 ⁇ samples from all experimental media samples. The reaction was halted prior to endpoint determination using 1 M sulfuric acid and read at 450 nm using a microplate reader (VERSAmax, Molecular Devices Corporation, Sunnyvale, CA). The duplicate optical density values were corrected for nonspecific binding and were averaged, and compared to a standard curve to determine the amount of PGE 2 in each well. These values were corrected for total media volume and normalized by the number of cells in the sample. ANOVA was used to compare the normalized PGE 2 between the three serum groups.
  • Example 4 Culture in bear serum decreases the ratio of gene expression of Bax to Bcl-2 during hibernation
  • RNA samples were removed from 4 female black bears between 2004-2005 as described above. Sample dates encompassed a pre-hibernation active period, a hibernation disuse period, and a post-hibernation remobilization period. MC-3T3 osteoblastic cells were cultured for 24 hours in media containing 10% bear serum, after which total RNA was isolated using a BioRad AquaPure RNA Isolation Kit (#732-6370, Bio- Rad Laboratories, Hercules, CA).
  • endogenous PTH and the bone formation marker osteocalcin both increase during hibernation (Donahue et al., 2006; J. Exp. Biol. , 209, 1630-8), it is possible that endogenous bear PTH causes a decrease in osteoblast apoptosis during hibernation, which in turn increases bone formation.
  • Full-length recombinant black bear PTH (residues 1 -84) is produced and its effects on levels of cyclic adenosine monophosphate (cAMP) concentration in bone cell lines (MC-3T3 osteoblastic cells and MLO-Y4 osteocytic cells) are investigated and compared to results obtained using recombinant human PTH 1 -84.
  • Equivalent experiments are conducted using subfragments of black bear and human PTH, the subfragments including amino acid residues 1 -34, 1 -36, 7-84, 1 1-84, and 41 -52 of the full-length (1 -84) mature protein.
  • black bear and human PTH polypeptides are synthesized with solid-phase methods.
  • the cultured bone cells (MC- 3T3 and MLO-Y4) are contacted for 10 or 30 minutes with human or black bear PTH full- length (i.e. amino acid residues 1-84) polypeptide or one of the above-listed subfragments.
  • PTH full- length i.e. amino acid residues 1-84
  • cAMP concentration in the cell is measured using a competitive binding assay as described further below.
  • the lyophilized peptides are reconstituted to 100 uM stock concentrations in 1 mM acetic acid, and diluted to 10 uM working stock concentrations before use.
  • MC-3T3 subclone 14 cells ATCC, CRL-2594
  • MLO-Y4 cells obtained from L.F. Bonewald, University of Missouri, Kansas City, MO
  • MC-3T3 10% fetal bovine serum (FBS)
  • MLO-Y4 5% FBS and 5% bovine calf serum
  • MC-3T3 and MLO-Y4 cells are seeded at appropriate densities (MC-3T3: 50,000 cells/cm 2 , MLO-Y4 15,000 cells/cm 2 ) in 6-well plates. Cells are cultured overnight to reach optimal confluence. The culture media is then aspirated and replaced with media containing either 10% serum + vehicle (1 mM acetic acid) or 10% serum + 100 nM PTH (human or bear 1 -84, or a subfragment thereof). Cells are cultured under these conditions for 10 or 30 minutes (Carter, P. H. et al., 1999, J Biol. Chem. 274(45), 31955-60; Chen, X. et al., 2002, Am. J.
  • Full-length recombinant black bear PTH (residues 1 -84) is produced and its effects on apoptosis in bone cell lines (MC-3T3 osteoblastic cells and MLO-Y4 osteocytic cells) are investigated and are compared to results obtained using recombinant human PTH 1-84. Equivalent experiments are conducted using subfragments of black bear and human PTH, the subfragments including amino acid residues 1 -34, 1 -36, 7-84, 1 1 -84, and 41 -52 of the full-length (1-84) mature protein. For some experiments, black bear and human PTH polypeptides are synthesized with solid-phase methods.
  • the lyophilized peptides are reconstituted to 100 uM stock concentrations in 1 mM acetic acid, and diluted to 10 uM working stock concentrations before use.
  • MC-3T3 subclone 14 cells ATCC, CRL-2594
  • MLO-Y4 cells obtained from L.F. Bonewald, University of Missouri, Kansas City, MO
  • MC-3T3 10% fetal bovine serum (FBS)
  • MLO-Y4 5% FBS and 5% bovine calf serum
  • the PTH polypeptide or vehicle is left in situ during apoptosis induction because the suppression of apoptosis by PTH is self-limiting (Bellido et al., 2003). After 6 hours, cells are trypsinized, centrifuged, resuspended, and counted using a hemocytometer. 50,000 cells are removed from the suspension and placed into lysis buffer. The lysate supernatant (following centrifugation) is removed for analysis and stored at -20° C.
  • Apoptosis is quantified from the lysate supernatant with an ELISA (Cell Death Detection ELISA, #1544675, Roche Applied Science, Indianapolis, IN).
  • ELISA Cell Death Detection ELISA, #1544675, Roche Applied Science, Indianapolis, IN.
  • This assay detects mono- and oligonucleosomes from fragmented cellular DNA in the cytoplasmic fraction of cell lysates, and therefore provides a good measure of the early and middle stages of apoptosis. Briefly, samples are diluted in buffer solution and added to microplate wells coated with an anti-histone mouse monoclonal (clone H1 1 -4) antibody. Lysate supernatant from the vehicle-treated cells serves as a negative control.
  • Optical densities are measured at 405 nm following the addition of a peroxidase-conjugated anti-DNA mouse monoclonal (clone MCA-33) antibody, and the amount of apoptosis in each sample is determined relative to its corresponding negative control. All samples are assayed in duplicate.
  • the lyophilized peptides are reconstituted to 100 uM stock concentrations in 1 mM acetic acid, and diluted to 10 uM working stock concentrations before use.
  • MC-3T3 subclone 14 cells ATCC, CRL-2594
  • MLO-Y4 cells obtained from L.F. Bonewald, University of Missouri, Kansas City, MO
  • MC-3T3 10% fetal bovine serum (FBS)
  • MLO-Y4 5% FBS and 5% bovine calf serum
  • MC-3T3 cells are seeded at a density of 50,000 cells/cm 2
  • MLO-Y4 cells are seeded at a density of 15,000 cells/cm 2 in 6-well plates and cultured overnight to reach optimal confluence.
  • the culture media are aspirated and replaced with media containing either 10% serum + vehicle (1 mM acetic acid) or 10% serum + 100 nM PTH (human or bear 1-84, or a subfragment as listed above).
  • Cells are cultured under these conditions for 1 or 3 hours; these time points correspond with PTH-induced upregulation of c-fos and osteocalcin (Jiang et al., 2004, J. Biol.
  • Primers for all genes of interest are designed using PrimerQuest software (Integrated DNA Technologies, Coralville, IA) and the NCBI gene bank sequences, and the PCR conditions are optimized using RNA from MC-3T3 and MLO-Y4 cells.
  • Real-time PCR is performed using the Mx3000P real-time PCR system (Stratagene, La Jolla, CA).
  • the protocol involves a hot start at 95°C for 10 minutes followed by 40 cycles of 95°C for 30 seconds (denaturation), 69°C for 1 minute (annealing) and 72°C for 1 minute (extension).
  • the exception to this protocol is for c-fos, which has an annealing temperature of 66°C.
  • Each 25 ⁇ reaction contains 1 x AbsoluteTM qPCR SYBR® green mix (ABgene, Rochester, NY), 0.1 ⁇ forward and reverse primer, and 2.5 ng total RNA equivalent cDNA template.
  • Gene expression is determined using the relative standard curve method normalized to the geometric mean of the three housekeeping genes. All samples are measured in duplicate, and any samples with a coefficient of variation (CV) greater than 10% are reanalyzed.
  • CV coefficient of variation
  • Example 8 Comparison of the Effects of Black Bear Serum from Different Seasons on Bone Cell Apoptosis and Gene Expression, and Correlation with Serum Levels of PTH and Osteocalcin
  • the bears are anesthetized with a 2:1 mixture of ketamine (100 mg/ml):xylazine (100-mg/ml); the dosage is 1 cc of the mixture per 45.5 kg of body mass.
  • Blood samples are drawn from the femoral vein while the bears are anesthetized, and the samples are transported to the laboratory in an ice-packed cooler.
  • This assay has previously been validated for bears (Donahue et al., 2006; J. Exp. Biol., 209, 1630-8).
  • the antibody is guinea-pig anti-rat osteocalcin and tracer is 125 l-labeled rat osteocalcin.
  • Aliquots of 100 ⁇ of bear serum are assayed in duplicate for PTH concentration (Donahue et al., 2006; J. Exp. Biol., 209, 1630-8) with an ELISA (Porcine Intact PTH ELISA Kit, #60-3305, Immutopics, Inc., San Clemente, CA).
  • This assay binds the 39-84 region of PTH, and requires the 13-34 region of PTH to colorimetrically report PTH concentration. Thus, it provides a good measure of intact (1 -84) PTH concentration as well as C-terminal subfragments 7-84 and 1 1 -84.
  • This ELISA has been shown to cross-react with bear PTH (Donahue et al., 2006; J. Exp. Biol., 209, 1630-8), and has 100% cross- reactivity with human PTH.
  • samples of culture media containing 10 nM recombinant black bear or human PTH 1-84 are assayed in duplicate. The known concentration of the PTH samples is compared to the measured concentration determined from the assay's standard curve. Any potential difference in cross-reactivity determined from these samples is used as a correction for endogenous black bear PTH concentration in the black bear serum samples.
  • Serum from the hibernation and post-hibernation seasons causes a greater prevention of apoptosis compared to pre-hibernation serum, because PTH is higher during hibernation and post-hibernation than in pre-hibernation serum.
  • Endogenous serum PTH concentrations are inversely related to apoptosis levels, in that higher serum PTH levels correspond to lower rates of apoptosis, i.e. serum PTH concentrations are negatively correlated with apoptosis levels.
  • Black bear PTH either full-length (1 -84) or one of several functional subfragments thereof (1 -34; 1-36; 7-84; 1 1-84; 41 -52), are tested in vivo for anabolic stimulation of bone cells compared to an equivalent human PTH or subfragment.
  • Each of the PTH polypeptides is synthesized and suspended in a pharmaceutically-appropriate carrier for subcutaneous injection.
  • Full-length PTH or a functional fragment thereof, from either black bear or human, is administered to mice at a dose of 40 ⁇ g/kg body weight daily for 7 weeks.
  • Black bear PTH or a functional fragment thereof cause greater increases in bone strength, mass, and mineral content than the equivalent human PTH polypeptide.
  • Total RNA was isolated, and cDNA was generated with reverse transcription.
  • Primers for the bone matrix proteins type I collagen and osteocalcin, the pro-apoptotic protein Bax, the anti-apoptotic protein Bcl-2, and the housekeeping genes Gapdh, ⁇ -actin, and cyclophillin were designed using PrimerQuest software (Integrated DNA Technologies, Coralville, IA).
  • Real-time PCR was performed using the Mx3000P real-time PCR system (Stratagene, LaJolla, CA). All samples were measured in duplicate.
  • Gene expression was determined using the relative standard curve method normalized to the geometric mean of the three housekeeping genes (Gapdh, ⁇ - actin, and cyclophillin). Apoptosis-related genes were analyzed as the expression ratio of Bax / Bcl-2, since a decrease in this ratio is associated with decreased apoptosis in vitro.
  • the current dose of PTH1 -34 for humans is approximately 0.3 pg/kg while a dose as low as 5 pg/kg has been shown to be carcinogenic in rats (Tashjian et al., J. Bone Mineral Res. 23(6): 803-81 1 (2008)).
  • the sham OVX group as well as one group of actual OVX rats were euthanized at six weeks while the remaining animals were euthanized at fourteen weeks following the OVX procedure. Samples were obtained from the euthanized animals for further analysis.
  • FIG. 9A Three-dimensional images of bones were obtained using micro-computed tomography (pCT). The three-dimensional images were then used to quantify bone parameters, as explained below. The measurements were conducted on a 1 .6 mm-thick region that begins at a position 2 mm from the growth plate (Fig. 8). Bone samples were obtained and analyzed from sham (Fig. 9A, "Sham OVX 6 weeks”) and actual (Fig. 9B, "OVX 6 weeks”) OVX rats that were euthanized at six weeks after the procedure, as well as a group of actual OVX rats that were euthanized at fourteen weeks after the procedure (Fig. 9C, "OVX 14 weeks”).
  • the rats in the OVX 14 group were given injections of vehicle (saline) for 8 weeks following the 6 week bone loss period, in parallel with the PTH1-34 injections for eight weeks (Figs. 9D, 9E, and 9F).
  • vehicle saline
  • PTH1-34 injections for eight weeks.
  • the samples shown in Figures 9D, 9E, and 9F were obtained from rats that were injected for eight weeks following the 6 week bone loss period, with 3, 10, or 30 pg/kg black bear PTH 1-34 ("bbPTH”), respectively.
  • FIG. 10 shows bone volume as a fraction of total volume (BV/TV) for the various treatment groups.
  • the baseline level of bone volume fraction in the presence of ovaries (“sham baseline,” light diamond) and the baseline level six weeks after loss of ovaries and in the absence of PTH treatment ("OVX baseline,” dark diamond) are shown at the left, in the column labeled "6 weeks.”
  • Treatment of OVX rats with vehicle (saline) alone for fourteen weeks (“OVX vehicle,” triangle) led to a further decline in bone volume fraction.
  • bone mineral apparent density was determined for each of the samples ( Figure 1 1 ) and is shown as milligrams hydroxyapatite per cubic centimeter ("App.Dn (mgHA ccm)").
  • the bone mineral apparent density was significantly higher (p ⁇ 0.05) for bones of OVX rats treated with 3, 10, or 30 pg/kg hPTH or bbPTH than for bones of OVX rats treated with vehicle (saline) alone.
  • the bone mineral apparent density for OVX rats treated with 30 ⁇ g kg hPTH was significantly higher (p ⁇ 0.05) than OVX rats treated with 30 pg/kg bbPTH.
  • Black bear PTH 1-34 showed a trend of greater trabecular bone mineralization ("M.Dn (mgHA ccm)"; Figure 14) with increasing dose compared to human PTH1 -34.
  • Treatment of OVX rats with either bear (“bbPTH1 -34,” circles) or human (“hPTH1 -34,” squares) PTH1 -34 produced significantly (p ⁇ 0.05) greater trabecular bone mineralization than OVX rats treated with vehicle (saline) alone.
  • Tibial cortical porosity in bones of OVX rats was observed to be significantly (p ⁇ 0.05) decreased when the rats were treated with middle (10 pg/kg) and higher doses (30 pg/kg) of either bear ("bbPTH1-34," circles) or human ("hPTH1-34,” squares) PTH 1-34 relative to bones from OVX rats treated with vehicle (saline) alone ( Figure 16).
  • bbPTH1-34 circles
  • human hPTH1-34
  • An additional benefit that black bear PTH1 -34 may confer over human PTH1 - 34 is lower serum calcium.
  • Hypercalcemia is a side effect of PTH treatment in humans.
  • serum calcium levels were seen to be lower in rats treated with bear PTH1 -34 compared to human PTH 1 -34, although the results are not significant at the p ⁇ 0.05 level.
  • statistical analyses predict that serum calcium concentrations would be significantly (p ⁇ 0.05) lower in black bear PTH 1-34 treated rats if the sample size were doubled.
  • black bear PTH1-34 treatment may reduce the incidence of this side effect.
  • MC3T3 subclone 4 cells (ATCC, CRL-2593) were maintained in standard culture media (89% alpha-minimum essential media, 1 % penicillin/streptomycin, and 10% FBS) at 37°C in 5% C02. Cells were not used beyond passage 30.
  • MC3T3 cells were seeded at a density of 20,000 cells/well in 96-well plates and allowed to attach overnight. Culture media was then aspirated and replaced with 100 ⁇ of culture media containing 10% FBS and either vehicle (PBS) or PTH (10-100 nM) for one hour, after which media was aspirated and cells were washed with PBS to remove residual serum. To induce apoptosis, cells were incubated in control or serum-free media for 6 hours. Replacement media contained 89% alpha-minimum essential media, 1 % penicillin/streptomycin, and either 10% FBS (control media) or 0% FBS (starvation media).
  • mice received subcutaneous injections of calcein at a dosage of 10 mg/kg on days 12 and 3 before death to label mineralizing bone surfaces. Twenty-four hours following the last treatment injection, mice were sacrificed by carbon dioxide asphyxiation. Right femurs were fixed in 10% neutral buffered formalin. Left femurs were wrapped in 0.15M saline soaked gauze and stored at -20 deg C. Left tibias were stored in 70% ethanol at 4 deg C.
  • Trabecular bone architecture and mineralization were evaluated in the left proximal tibial metaphysis (0.7 mm distal to the growth plate) using micro-computed tomography.
  • Tibias from experiment 1 were scanned on a ⁇ 35 scanner (Scanco Medical AG, Basserdorf, Switzerland) at 3.5 ⁇ (high) resolution.
  • Tibias from experiment 2 were scanned on a ⁇ 40 scanner (Scanco Medical AG, Basserdorf, Switzerland) at 7 ⁇ (high) resolution. Bones were scanned in 70% ethanol.
  • Trabecular bone volume fraction (BV/TV, %), trabecular number (Tb.N, mm-1 ), trabecular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, mm), trabecular apparent mineral density (App.Mn.Dn, mgHA/cm3) and trabecular tissue mineral density (Mat.Mn.Dn, mgHA cm3) were computed using the manufacturer's software.
  • the left femoral diaphyses were ashed in a furnace to determine mineral content (ash fraction).
  • Right femoral diaphyses were embedded in methyl methacrylate and sectioned with a diamond saw to expose the midshaft cross-section. Images of the midshaft cross-sections were captured using a digital camera (Spot Insight QE, Diagnostic Instruments Inc., Sterling Heights, Michigan), and Scion Image analysis software (Scion Corporation, Frederick, Maryland) was used to calculate the periosteal area (Ps.Ar), cortical area (Ct.Ar), and endosteal area (Es.Ar) for each sample.
  • Ps.Ar periosteal area
  • Ct.Ar cortical area
  • Es.Ar endosteal area
  • Cortical bone thickness was calculated in 0.1 mm increments for each anatomical quadrant and for the entire cross-section using image analysis software (Bioquant Osteo, Nashville, TN). Thin sections from the midshaft of the right femur were ground to a thickness of 50 ⁇ and mounted on glass slides. Images of calcein labels were captured at 100X magnification and periosteal and endosteal mineral apposition rates (Ps.MAR and Es.MAR, respectively) and mineralizing surfaces (Ps.MS/BS and Es. MS/MS) were quantified (Bioquant) for each femur.
  • Table 1 Trabecular bone architectural properties and mineral density in the proximal tibia of mice from the PTH 1-34 study.
  • Table 2 Trabecular bone architectural properties and mineral density in the proximal
  • Bone periosteal area was highest in vehicle treated mice from the PTH 1-34 study and lowest in vehicle-treated mice from the PTH 1 -84 study (Table 3).
  • Neither cortical bone area nor endosteal area were different between treatment groups (p > 0.136).
  • Cortical bone thickness was greater in mice from the PTH 1-84 study compared to the PTH 1 -34 study, but there were no other differences between treatment groups (Table 3).
  • Table 3 Cortical bone geometrical properties in the femoral midshaft of PTH-treated mice.
  • mice [0160] The hPTH peptides in these studies did not produce an increase in trabecular bone mass compared to vehicle treatment (Tables 1 and 2), likely because of the relatively low peptide dosage administered to the mice.
  • a dosage of 2.5 nmol/kg was used in our studies because it is the lowest peptide dosage shown to increase osteoblast number and decrease osteoblast apoptosis in trabecular bone secondary spongiosa in mice (see Bellido et al. (2003) J Biol Chem 278(50):50259-72), and it is closer to a clinical dosage (20 ⁇ g day, approximately 0.07 nmol/kg for a 70 kg patient) compared to PTH dosages used in other short-term in vivo studies.
  • mice treated with the same dosage of bbPTH 1 -84 showed improvement in trabecular bone properties (Table 2) and cortical bone strength (Figure 29) compared to hPTH 1 -84.
  • Trabecular thickness tended to be elevated in mice treated with bbPTH 1 -84 (Table 2).
  • trabecular bone volume fraction and apparent mineral density were only significantly increased over vehicle treatment by bbPTH 1 -84 (Table 2).
  • Most of the peptide analogs investigated tended to increased femoral ultimate load, but the increase over vehicle treatment was only statistically significant for bbPTH 1 -84 ( Figure 29).
  • Bone volume fraction in the proximal tibia was also greatest in mice treated with bbPTH 1 -84 (Table 1 ), even though bbPTH 1-84 was less anti-apoptotic than the other peptide analogs tested in MC3T3 cells. While the 1-34 fragment of hPTH is sufficient to replicate the anabolic effects of the full hPTH peptide, the fact that bbPTH 1 -84 demonstrated a superior performance in vivo compared to both bbPTH 1-34 and both hPTH peptides suggests that the amino acid changes in bbPTH's C-terminal portion may be involved in receptor binding and receptor activation behavior.
  • Example 15 In vivo effects of human ⁇ 1 -84 and bear PTH1 -84 in ovariectomized rats
  • OVX ovariectomized
  • Nov.-Dec active pre-hibernation
  • 9 hibernation Jan. -March
  • Amr.-May active post-hibernation
  • MC3T3-E1 pre-osteoblasts were seeded in Alpha-Modified Minimum Essential Media ( -MEM) with 10% fetal bovine serum at 10,000 cells per well in a white-walled tissue culture plate and allowed to attach overnight. Media was then aspirated and replaced with 2% seasonal bear serum in -MEM for a 24 hour treatment.
  • -MEM Alpha-Modified Minimum Essential Media

Abstract

L'invention concerne l'hormone parathyroïde (PTH) de l'ours noir et des fragments fonctionnels de celle-ci. L'invention concerne également des procédés d'utilisation de la PTH d'ours noir et des fragments fonctionnels permettant d'accroître l'AMPc dans un ostéoblaste; réduisant l'apoptose dans un ostéoblaste; de réduire le rapport des niveaux d'expression de la protéine Bax par rapport à la protéine Bcl-2 dans un ostéoblaste; d'augmenter le niveau d'expression d'un ou plusieurs parmi une protéine de matrice osseuse, un activateur transcriptionnel ou un régulateur de la transcription dans un ostéoblaste; d'améliorer la teneur minérale de l'os, d'augmenter la masse osseuse, de réduire la perte osseuse ou de réduire l'incidence des fractures osseuse ou n'importe quelle combinaison de ceux-ci, chez un sujet. L'invention concerne également des anticorps dirigés contre l'hormone parathyroïde (PTH) de l'ours noir et des fragments fonctionnels de celle-ci.
PCT/US2009/066974 2009-12-07 2009-12-07 Hormone parathyroïde de l'ours noir et ses procédés d'utilisation WO2011071480A1 (fr)

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PCT/US2009/066974 WO2011071480A1 (fr) 2009-12-07 2009-12-07 Hormone parathyroïde de l'ours noir et ses procédés d'utilisation
US13/514,362 US8987201B2 (en) 2009-12-07 2009-12-07 Black bear parathyroid hormone and methods of using black bear parathyroid hormone
AU2009356227A AU2009356227A1 (en) 2009-12-07 2009-12-07 Black bear parathyroid hormone and methods of using black bear parathyroid hormone
BR112012013725A BR112012013725A2 (pt) 2009-12-07 2009-12-07 paratormônio de urso preto e métodos de usar o paratormônio de uso preto.
JP2012543070A JP2013512688A (ja) 2009-12-07 2009-12-07 クロクマの副甲状腺ホルモン及びクロクマの副甲状腺ホルモンを使用する方法
CA2782640A CA2782640A1 (fr) 2009-12-07 2009-12-07 Hormone parathyroide de l'ours noir et ses procedes d'utilisation
EP09764429A EP2509996A1 (fr) 2009-12-07 2009-12-07 Hormone parathyroïde de l'ours noir et ses procédés d'utilisation

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CN109320600A (zh) * 2018-10-31 2019-02-12 杨德鸿 一种基于蛋白结构域重建的新型pth模拟肽及其应用

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CN109320600A (zh) * 2018-10-31 2019-02-12 杨德鸿 一种基于蛋白结构域重建的新型pth模拟肽及其应用

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US8987201B2 (en) 2015-03-24
US20120244232A1 (en) 2012-09-27
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