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WO2012111852A1 - Therapeutic agent for vertebral body fracture and method for evaluating same - Google Patents

Therapeutic agent for vertebral body fracture and method for evaluating same Download PDF

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Publication number
WO2012111852A1
WO2012111852A1 PCT/JP2012/054340 JP2012054340W WO2012111852A1 WO 2012111852 A1 WO2012111852 A1 WO 2012111852A1 JP 2012054340 W JP2012054340 W JP 2012054340W WO 2012111852 A1 WO2012111852 A1 WO 2012111852A1
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cha
aib
pthrp
pth
leu
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French (fr)
Japanese (ja)
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美幸 石田
由明 東
善史 原田
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Teijin Pharma Ltd
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Teijin Pharma Ltd
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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
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents

Definitions

  • the present invention relates to a therapeutic agent for vertebral fracture containing a PTH / PTHrP receptor agonist or a substance having an activity of inducing PTH or PTHrP production.
  • the present invention also relates to a novel animal model and a method for evaluating the same that make it possible to evaluate the effect and safety of a therapeutic agent for vertebral fractures for the first time in non-human animals.
  • the vertebral body is the site with the highest fracture frequency (Non-Patent Document 1).
  • the vertebral body is “the main part of the vertebra that is in front of the spinal canal and is distinguished from the vertebral arch” (Stedman Medical Dictionary, 5th edition, Medical View) or “the half that occupies the front of the vertebra "Circular part” (Ojirin, second edition, Sanseido). Therefore, a vertebral body fracture does not include a fracture at the vertebral arch where spinous processes, transverse processes, joint processes, etc. are present, among vertebrae constituting the spine, and has a semi-cylindrical shape. A fracture in the body part.
  • Vertebral fractures may be caused by strong impacts such as accidents or sports, but they are compression fractures of the vertebral body that occur mainly due to increased bone vulnerability and decreased bone strength due to osteoporosis. Yes, it is one of the typical diseases that greatly reduce the patient's QOL (Non-patent Documents 2 and 3).
  • QOL Non-patent Documents 2 and 3
  • a slight external force that usually does not cause a fracture and a weak stress that is about the daily life action are repeatedly applied, resulting in damage to the fine structure of the bone and continuous fracture of the bone structure.
  • Vertebral fractures vary in clinical symptoms, from those without subjective symptoms after injury to those with acute back and back pain. There are no drugs for treating vertebral fractures, and no aggressive drug treatment is available for painful vertebral fractures. Therefore, it is common to treat the patient conservatively for several months by performing external fixation with a resting bed for several weeks while administering an analgesic, and using a device such as a cast, hard or soft corset. At this time, there are many patients who need temporary hospitalization treatment, and the average hospitalization period is reported to be 42.6 days (Non-patent Document 8).
  • Vertebroplasty such as injecting bone cement into fractured vertebral bodies that spread rapidly mainly in the United States since the late 1990s, and kyphoplasty to inflate the balloon to restore the height of the fractured vertebral body and correct kyphosis deformity Vertebral augmentation surgery has been penetrating in Japan (Non-patent Document 9).
  • Non-patent Document 9 Even if external fixation is performed, it is often impossible to maintain good spinal alignment, leaving wedge-shaped deformation of the vertebral body and deformation of the posterior spine.
  • the current situation is that the onset of vertebral body collapse, pseudo-joint, and delayed neuropathy cannot be completely prevented (Non-patent Document 10).
  • a rat femoral closed fracture model which is a general fracture model, is a model in which guillotine is dropped at the center of the femoral shaft and the femur is fractured to evaluate the strength of the femur (Non-patent Document 11).
  • the damage is a cortical bone fracture that is far from the microstructural damage of cancellous bone that occurs in vertebral fractures.
  • the rat femur drilling model in which bone damage is caused by drilling with a dental drill is the same as the femoral closed fracture model, the central part of the femoral shaft, that is, the femoral cortical bone.
  • Non-Patent Documents 12 and 13 It is a damaged model (Non-Patent Documents 12 and 13), and is not a model that can evaluate the damage to the fine structure of cancellous bone caused by vertebral fractures.
  • fracture healing it is known that not only bone formation by osteoblasts but also bone destruction by osteoclasts proceeds.
  • PTH and PTHrP are known to activate osteoclasts.
  • PTH and the like have a function of releasing calcium into the blood by the activity of osteoclasts from bones, and there have been times when it was conventionally considered as a bone destruction factor causing hypercalcemia.
  • Non-patent Document 14 the models used there are fixed bones that are not loaded in an undesired direction by inserting a pin after a fracture has occurred in a long bone (for example, the femur), or flat bones and unweighted bones. It is only the thing which saw the effect in (head skeleton etc.).
  • the vertebral body is the main part of the spine that supports the trunk, and is a bone that is always subjected to a certain amount of weight.
  • Non-patent Document 15 The healing process of a fracture is generally divided into an inflammation phase, a repair phase, and a remodeling phase (Non-patent Document 15).
  • a hematoma is formed at the fracture site, and there are inflammatory cells, from which many factors such as inflammatory cytokines are released.
  • the spinal canal close to the vertebral body is characterized by the passage of the spinal cord, which is important for the control of body movements and sensations, and physiological functions.
  • the disease in which the spinal cord is inflamed for some reason is called myelitis, and its symptoms are abnormal sensory and motor functions when mild, but paralysis of the entire controlled area when severe. Or even death.
  • PTHrP has been shown to be involved in inflammatory responses in several diseases (Non-patent Documents 16-20).
  • Non-patent Document 21 PTH and PTHrP are also known to act on osteoblasts to induce the expression of CXCL1, which is a cell migration factor for neutrophils (Non-patent Document 22). Therefore, when PTH / PTHrP or analogs thereof are administered for the purpose of treating vertebral fractures, it is possible that the inflammation of the fracture site may become more serious, and the inflammation spreads to the spinal cord existing nearby. Possible risk of failure.
  • Prostaglandin E2 acceptor (EP4) selective agronist accelerates bone repair of the felt correlatively-reduced injuries.
  • Ellegaard M Jorgensen NR, Schwartz P. Rockwood and Green's fractures in adults; Seventh Edition. Robert W. Bucholz, James D.B. Heckman, 2009, (Chapter 4,) Lippincott Williams & Wilkins. Int Immunopharmacol.
  • the model can be used to screen for substances that treat vertebral fractures. That is, it becomes a useful animal model for developing a new drug having a therapeutic effect against the damage of the cancellous bone microstructure caused by vertebral fracture.
  • an animal model that can evaluate the damage to the cancellous bone microstructure caused by vertebral fractures could be developed, it would be possible to develop new drugs to treat vertebral fractures Become.
  • the present inventors used a rat, which is a highly versatile animal in drug development, to construct an animal model that causes damage to the fine structure of cancellous bone caused by vertebral fractures, and to evaluate the damage repair process As a drug for treating vertebral body fractures, further, screening is performed using the animal model and the evaluation method, and a substance capable of treating damage to the fine structure of cancellous bone caused by vertebral body fractures is found. Made it possible to develop.
  • An object of the present invention is to provide a therapeutic agent for vertebral fracture, a therapeutic or preventive agent for pain caused by vertebral fracture, by containing a PTH / PTHrP receptor agonist, or a substance having an activity to induce PTH or PTHrP production,
  • the object is to provide a preventive agent for vertebral body compression fracture, or a therapeutic or preventive agent for movement disorders and / or neurological disorders caused by vertebral body fractures.
  • the object of the present invention is also 1) a step of surgically damaging the vertebral bones of a non-human animal, 2) a step of administering a test substance to the non-human animal, and 3) the non-human animal or the same. Measuring the recovery of surgical damage to the vertebral bone of the specimen from which it is derived, thereby providing a method for evaluating a therapeutic agent for vertebral fractures.
  • the present invention is as follows.
  • a vertebral fracture therapeutic agent comprising a PTH / PTHrP receptor agonist.
  • the PTH / PTHrP receptor agonist is parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrP), or a partially active polypeptide thereof, or an analog thereof. Fracture treatment agent.
  • PTH, PTHrP or a partially active polypeptide thereof is PTH (1-84), PTH (1-37), PTH (1-34), PTH (1-31), PTHrP (1-141), Any one of PTHrP (1-139), PTHrP (1-86), PTHrP (1-40), PTHrP (1-37), PTHrP (1-36), and PTHrP (1-34), (2 The therapeutic agent for vertebral fractures according to (1).
  • PTH, PTHrP or a partially active polypeptide thereof is a polypeptide having an amino acid sequence derived from human PTH or human PTHrP.
  • PTH, PTHrP or a partially active polypeptide thereof is a polypeptide in which 1 to 10 amino acids in the amino acid sequence of PTH, PTHrP or a partially active polypeptide thereof are substituted with another amino acid.
  • PTH, PTHrP, or an analog of a partially active polypeptide thereof is PTH (1-84), PTH (1-37), PTH (1-34), PTH (1-31), PTHrP (1-141).
  • the therapeutic agent for vertebral fracture according to (5) which is a polypeptide obtained by substituting 1 to 10 amino acids with another amino acid.
  • An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide in which 1 to 10 amino acids are substituted with another amino acid in the amino acid sequence derived from human PTH or human PTHrP.
  • vertebral body fracture treatment agent according to (6) is also provided.
  • PTH, PTHrP, or a partially active polypeptide thereof, or an analog thereof is a pharmaceutically acceptable salt.
  • An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula: However, A 1 is Ser, Ala, or Dap; A 3 is Ser, Thr, or Aib; A 5 is Leu, Nle, Ile, Cha, ⁇ -Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ; A 7 is Leu, Nle, Ile, Cha, ⁇ -Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ; A 8 is Met, Nva, Leu, Val, Ile, Cha, or Nle; A 11 is Leu, Nle, Ile, Cha, ⁇
  • E 1 is C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12 hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7 -20 hydroxyphenyl alkyl, or with C 11-20 hydroxy naphthyl alkyl Ri; and R 3 is OH, NH 2, C 1-12 alkoxy, or NH-Y-CH 2 -Z, this time, Y is C 1-12 hydrocarbon moiety, Z is H, OH, CO 2 H or CONH 2 ; At least one of A 5 , A 7 , A 8 , A 11 , A 15 , A 18 , A 21 , A 23
  • An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula: [Cha 7,11 ] hPTH (1-34) NH 2 ; [Cha 23 ] hPTH (1-34) NH 2 ; [Cha 24 ] hPTH (1-34) NH 2 ; [Nle 8,18 , Cha 27 ] hPTH (1-34) NH 2 ; [Cha 28 ] hPTH (1-34) NH 2 ; [Cha 31 ] hPTH (1-34) NH 2 ; [Aib 16 ] hPTH (1-34) NH 2 ; [Aib 19 ] hPTH (1-34) NH 2 ; [Aib 34 ] hPTH (1-34) NH 2 ; [Cha 24 , 28 , 31 , Lys 30 ] hPTH (1-34) NH 2 ; [Cha 7,11 , Nle 8,18 , Tyr 34 ] hPTH (1-34)
  • An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula: However, A 1 is Ala, Ser, or Dap; A 3 is Ser or Aib; A 5 is His, Ile, or Cha; A 7 is Leu, Cha, Nle, ⁇ -Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ; A 8 is Leu, Met, or Cha; A 10 is Asp or Asn; A 11 is Lys, Leu, Cha, Phe, or ⁇ -Nal; A 12 is Gly or Aib; A 14 is Ser or His; A 15 is Ile or Cha; A 16 is Gln or Aib; A 17 is Asp or Aib; A 18 is Leu, Aib, or Cha; A 19 is Arg or Aib; A 22 is Phe, Glu, Aib, or Cha; A 23 is Phe, Leu, Lys, or
  • R 1 and R 2 is COE 1 , wherein E 1 is C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20, naphthylalkyl, C 1-12 hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7-20 hydroxyphenylalkyl, or C 11-20 hydroxy naphthyl alkyl There; and R 3 is OH, an NH 2, C 1-12 alkoxy, or NH-Y-CH 2 -Z, this time, Y is C 1-12 hydrocarbon moiety, Z is H, OH, CO 2 H or CONH 2 ; At least one of A 5 , A 7 , A 8 , A 11 , A 15 , A 18 , A 22 , A
  • PTH PTH, analog of PTHrP or their partial active polypeptide, wherein: [Glu 22,25, Leu 23,28,31, Aib 29, Lys 26,30] poly hPTHrP (1-34) NH 2
  • An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula: However, A 1 is Ala, Ser, or Dap; A 3 is Ser or Aib; A 5 represents His, Ile, Acc, or be Cha; A 7 is Leu, Cha, Nle, ⁇ -Nal, Trp, Pal, Acc, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ; A 8 is Leu, Met, Acc, or Cha; A 10 is Asp or Asn; A 11 is Lys, Leu, Cha, Acc, Phe, or ⁇ -Nal; A 12 is Gly, Acc, or Aib; A 14 is Ser or His; A 15 is Ile, Acc, or Cha; A 16 is Gln or Aib; A 17 is Asp or Aib; A 18 is Leu, Aib, Acc, or Cha; A 19 is Arg or Aib; A 22 is Phe
  • R 1 and R 2 Hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7-20 hydroxyphenylalkyl, or C 11-20 hydroxynaphthylalkyl; or only one and one of R 1 and R 2 is COE 1 , wherein E 1 is C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12 hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7 -20 hydroxyphenyl alkyl, or with C 11-20 hydroxy naphthyl alkyl Ri; and R 3 is OH, NH 2, C 1-12 alkoxy, or NH-Y-CH 2 -Z, this time, Y is C 1-12 hydrocarbon moiety, Z is H, OH, CO 2 H or CONH 2 ; A 5, A 7, A 8 , A 11, A 12, A 15, A 18, A 22, A 23, A 24, A 25, A 26, A
  • An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula: [Glu 22 , 25 , Leu 23 , 28 , Lys 26 , 30 , Aib 29 , Ahc 31 ] hPTHrP (1-34) NH 2 ; [Glu 22 , 25 , Ahc 23 , Lys 26 , 30 , Leu 28 , 31 , Aib 29 ] hPTHrP (1-34) NH 2 ; [Glu 22 , 25 , Leu 23 , 28 , 31 , Lys 26 , 30, Ahc 27 , Aib 29 ] hPTHrP (1-34) NH 2 ; [Glu 22 , 25 , 29 , Leu 23 , 28 , 31 , Lys 26 , Ahc 30 ] hPTHrP (1-34) NH 2 ; [Cha 22 , Leu 23 , 28 , 31 , Glu 25 , Lys 26 , 30, Ahc
  • An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula: [Nle 31 ] hPTH (1-34) NH 2 ; [HArg 27 ] hPTH (1-34) NH 2 ; [Dap 1 , Nle 8 , 18 , Tyr 34 ] hPTH (1-34) NH 2 ;
  • An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula: [Glu 22 , 25 , Cha 23 , Lys 26 , Leu 28 , 31 , Aib 29 , Nle 30 ] hPTHrP (1-34) NH 2 ; [Glu 22 , 25 , Cha 23 , Lys 26 , 30 , Leu 28 , 31 , Aib 29 ] hPTHrP (1-34) NH 2 ; [Glu 22,25, Cha 23, Lys 26,30, Aib 29] hPTHrP (1-34) NH 2; [Glu 22 , 25 , Cha 23 , Lys 26 , 30 , Leu 28 , Aib 29 ] hPTHrP (1-34) NH 2 ; [Leu 27 , Aib 29 ] hPTH (1-34) NH 2 ; Alternatively, the therapeutic agent for vertebral fracture according to (1) or (2), which is a pharmaceutically acceptable salt thereof
  • a therapeutic agent for vertebral fracture comprising a substance having an activity of inducing PTH or PTHrP production.
  • the therapeutic agent for vertebral fracture according to (17), wherein the substance having an activity of inducing PTH or PTHrP production is a calcium sensitive receptor antagonist.
  • the therapeutic agent for vertebral fracture according to any one of (1) to (18), wherein the vertebral fracture is a fracture in cancellous bone.
  • a therapeutic or preventive agent for pain caused by vertebral fractures comprising a substance having an inducing activity.
  • a method for evaluating a therapeutic agent for vertebral fracture wherein 1) a step of surgically damaging a vertebral bone of a non-human animal, 2) a step of administering a test substance to the non-human animal, and 3 ) Measuring the recovery from surgical damage to the vertebral bones of the non-human animal or the specimen derived therefrom.
  • the method further includes a step of comparing the result of the measurement of the recovery of the surgical damage to the vertebral bone with the result of the measurement in the normal non-human animal or the target non-human animal or a specimen derived therefrom. ) Evaluation method.
  • the present invention relates to a therapeutic agent for vertebral fracture, a therapeutic or preventive agent for pain caused by vertebral fracture, by containing a PTH / PTHrP receptor agonist, or a substance having an activity of inducing PTH or PTHrP production, It makes it possible to provide a preventive agent for compression fractures, or a therapeutic or preventive agent for movement disorders and / or neurological disorders caused by vertebral fractures.
  • the present invention also includes 1) a surgical damage to the vertebral bones of a non-human animal, 2) a step of administering a test substance to the non-human animal, and 3) the non-human animal or derived therefrom. It is possible to provide a method for evaluating a therapeutic agent for vertebral fractures by including the step of measuring the recovery of the surgical damage to the vertebral bone of the specimen.
  • FIG. 1 is a micro CT image of a damaged vertebral body (a representative example of a 3D image and a 2D image on the model creation date (day 0)).
  • FIG. 2 is a Masson trichrome stained image in the natural restoration process.
  • FIG. 3 is a graph showing the bone volume density of the damaged portion of cancellous bone during the natural repair process.
  • FIG. 4 is a Masson trichrome stained image when hPTH (1-34) or PTHrP analog A is administered for 7 days or 14 days.
  • FIG. 5 is a graph showing the bone volume density in the damaged part of the cancellous bone of the damaged vertebral body when hPTH (1-34) or PTHrP analog A is administered for 7 days or 14 days.
  • FIG. 1 is a micro CT image of a damaged vertebral body (a representative example of a 3D image and a 2D image on the model creation date (day 0)).
  • FIG. 2 is a Masson trichrome stained image in the
  • FIG. 6 is a graph showing bone volume density in cancellous bone of an uninjured vertebral body when hPTH (1-34) or PTHrP analog A is administered for 7 days or 14 days.
  • FIG. 7 is a graph (A) showing the change in body weight over time of a sham-operated rat and a lumbar vertebral body bone injury model rat, and hPTH (1-34) and PTHrP in a vertebral body bone injury model rat. It is the graph (B) which showed the time-dependent body weight change of the rat in the group which administered the analog A, and the group which administered Vehicle.
  • the present inventors have established an animal model that causes damage to the fine structure of cancellous bone caused by vertebral fractures and an evaluation method thereof. With the establishment of an animal model that causes damage to the cancellous bone microstructure caused by vertebral fractures and the evaluation method, it is possible to screen for substances that can treat the damage to the cancellous bone microstructure caused by vertebral fractures. It became. As a result, hPTH (1-34) and PTHrP analog A (SEQ ID NO: 42) were found to be fine in the cancellous bone produced by vertebral fractures in the damaged vertebral body despite the dose not being detected in the undamaged vertebral body. It was effective in treating structural damage.
  • hPTH (1-34) and PTHrP analog A are substances that specifically show a therapeutic effect against damage to the fine structure of cancellous bone caused by vertebral fractures. Therefore, it has been found that a PTH / PTHrP receptor agonist, a substance having an activity of inducing PTH or PTHrP production can be a drug capable of treating cancellous bone microstructure damage caused by vertebral fractures. In addition, a PTH / PTHrP receptor agonist, a substance having an activity of inducing PTH or PTHrP production can treat damage to the fine structure of cancellous bone, so that the bone structure is continuously broken and the vertebral body is crushed. It was found that the progress of vertebral fracture can be stopped.
  • a PTH / PTHrP receptor agonist a substance having an activity of inducing PTH or PTHrP production is a therapeutic or preventive agent for movement disorders and / or neurological disorders caused by vertebral fractures.
  • a PTH / PTHrP receptor agonist, a substance having an activity of inducing PTH or PTHrP production is a revolutionary drug that increases QOL of vertebral fracture patients and improves life prognosis. These drugs for treating new vertebral fractures can not only benefit individual patients, but also reduce inpatients and bedridden patients, reduce medical costs, and contribute significantly to social benefits.
  • PTH / PTHrP receptor agonist The PTH / PTHrP receptor is a type of G protein-coupled receptor, also called PTH1R, PTH1 or PPR, which binds both parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) to the hormone in the cell. It works to transmit signals. That is, it is considered that the function of the PTH / PTHrP receptor is involved in the display of the common function of PTH and PTHrP. In particular, this PTH / PTHrP receptor plays an important role in the action of PTH and PTHrP in calcium metabolism.
  • the therapeutic agent for vertebral fracture in the present invention is characterized by containing a substance that activates the PTH / PTHrP receptor, that is, a PTH / PTHrP receptor agonist.
  • a substance that activates the PTH / PTHrP receptor that is, a PTH / PTHrP receptor agonist.
  • PTH / PTHrP receptor agonists include, but are not limited to, PTH, PTHrP, or partial polypeptides thereof, or analogs thereof, which are described below, and bind to the PTH / PTHrP receptor to signal inside the cell.
  • Any substance capable of generating vertebral body can be used in the therapeutic agent for vertebral fracture of the present invention.
  • PTH / PTHrP receptor agonists other than such PTH, PTHrP, or their partial polypeptides, or analogs thereof include agonist antibodies to PTH / PTHrP receptors, forms that mimic the structure and properties of PTH or PTHrP PTH or PTHrP mimic molecule designed in the above, and low molecular weight compounds obtained by screening using PTH / PTHrP receptor activation as an index.
  • the activation of the PTH / PTHrP receptor can be determined by measuring the binding to the PTH / PTHrP receptor expressed in the cell and the intracellular signal.
  • Measurement of agonist binding to PTH / PTHrP receptor is performed by directly labeling the test substance with a radioactive isotope, fluorescent substance, luminescent substance, or other substance for detection (substance related to biotin-avidin system, tag sequence, etc.) And reacting with a cell expressing the PTH / PTHrP receptor to determine the amount of the label detected for its binding.
  • a standard substance is selected from PTH, PTHrP, or a partial polypeptide thereof, an analog thereof, or a substance that binds to a PTH / PTHrP receptor, and the standard substance is directly a radioisotope.
  • the concentration of cAMP (cyclic adenosine monophosphate) in cells is increased by reacting a test substance with a cell expressing PTH / PTHrP receptor. Can be measured by examining. Measurement of intracellular cAMP concentration can be carried out by a person skilled in the art using information on existing techniques as appropriate, and commercially available kits can also be used. For example, JP-A-11-509201 It can also be implemented by the method described.
  • a test substance is reacted with a cell expressing a PTH / PTHrP receptor to examine an increase in intracellular calcium ion concentration.
  • the increase in intracellular calcium ion concentration can be achieved by using calcium-sensitive fluorescent substances such as Fura-2AM and Fura-4AM and calcium-sensitive luminescent substances such as aequorin. It is possible to implement.
  • it can be measured by examining the activity increase of intracellular phospholipase A2 (PLA2).
  • PTH / PTHrP receptor-expressing cells that use PTH / PTHrP receptor binding or intracellular signals for measurement are cells that endogenously express PTH / PTHrP receptor or PTH / PTHrP artificially expressed Any of the cells can be used.
  • the cells that endogenously express the PTH / PTHrP receptor are cells and cell lines obtained from tissues and cells expressing the PTH / PTHrP receptor in vivo in humans and non-human animals.
  • tissues and cells expressing the PTH / PTHrP receptor include osteoblasts / bone cells and osteoclasts in bones, kidney tubules and collecting duct cells in kidneys, smooth muscles in uterus and small intestine, It refers to cells obtained from glandular tissue, epithelial cells, and other ovarian and liver cells (from the description of PHT1 in IUPHAR DATABASE / http: //www.iufar-db.org/index.jsp/).
  • Examples of cell lines that endogenously express the PTH / PTHrP receptor include, for example, osteoblast cell lines. More specifically, human osteosarcoma cell lines SaOS-2 cells and rat bones And ROS17 / 2.8 cells which are blast-like cells.
  • a cell in which a PTH / PTHrP receptor is artificially expressed is a cell in which a gene encoding a PTH / PTHrP receptor protein is introduced into a host cell by a genetic engineering technique and the PTH / PTHrP receptor protein is expressed.
  • Examples of the PTH / PTHrP receptor protein include a human PTH / PTHrP receptor protein whose amino acid sequence is shown in SEQ ID NO: 1.
  • this human PTH / PTHrP receptor protein In a host cell into which the gene encoding this human PTH / PTHrP receptor protein has been introduced, it is translated as a protein having the amino acid sequence of SEQ ID NO: 1, and then the signal sequence, from the N-terminal to 28 amino acids, is cut off, A mature human PTH / PTHrP receptor protein consisting of the 29th and subsequent amino acid sequences is expressed on the cell surface.
  • the PTH / PTHrP receptor of chimpanzee (amino acid sequence) shown in SEQ ID NO: 2 and the rhesus monkey (Macaca mulatta) whose amino acid sequence is shown in SEQ ID NO: 3 PTH / PTHrP receptor
  • the PTH / PTHrP receptor of orangutan (SEQ ID NO: 4) whose amino acid sequence is shown in SEQ ID NO: 4
  • the PTH / PTHrP receptor of pig (Sus scrofa) whose amino acid sequence is shown in SEQ ID NO: 5 PTHrP receptor, equine PTH / PTHrP receptor whose amino acid sequence is shown in SEQ ID NO: 6, bovine PTH / PTHrP receptor whose amino acid sequence is shown in SEQ ID NO: 7
  • a common marmoset Callithrix jacchus
  • the homology between the human PTH / PThrP receptor and the Pyro / PTHrP of the gray porpoise PTH / PTHrP is a homology that does not allow conservative amino acid substitutions when NCBI's BLASTp method is used in parallel. Identities) and 80% homology to allow conservative amino acid substitutions (Positives). Therefore, the PTH / PTHrP receptor that can be used for the binding of the PTH / PTHrP receptor agonist of the present invention and the measurement of intracellular signal generation includes an amino acid sequence of human PTH / PTHrP receptor and 80% or more homology.
  • PTH / PTHrP receptors other than those described above, which retain the functions of PTH / PTHrP receptors. It is known that PTH / PTHrP such as human binds / acts on the opossum PTH / PTHrP receptor (Jupner H. et al. Endocrinology 134 pp 879 1994). In order to examine that the PTH / PTHrP receptor retains the function of the PTH / PTHrP receptor, as a known PTH / PTHrP receptor agonist such as PTH / PTHrP and its active partial polypeptide or analog thereof, This can be confirmed by examining the binding of substances having activity and the generation of intracellular signals.
  • the host for genetically expressing the PTH / PTHrP receptor is appropriately selected from animal cells (derived from humans and non-human animals), insect cells, yeast cells, bacteria or viruses / bacteriophages. It is preferably an animal cell, more preferably an animal cell line established from an animal cell. Examples of the animal cell line include Cos-7 cell line, LLC-PK1 cell line, HEK293 cell line, Act20 cell line and the like.
  • Animal cell line include Cos-7 cell line, LLC-PK1 cell line, HEK293 cell line, Act20 cell line and the like.
  • PTH, PTHrP or a partially active polypeptide thereof Parathyroid hormone (PTH) is a peptide hormone found in the body of vertebrates more than fish, and is expressed in the form of an inactive preprohormone and is subjected to a process, and usually becomes PTH consisting of 84 amino acids. .
  • PTH consisting of 84 amino acids is also referred to as paratormon, and may be referred to as PTH (1-84) to distinguish it from other active partial polypeptides derived from PTH.
  • PTH used in the present invention is human PTH (1-84) whose amino acid sequence is shown in SEQ ID NO: 13.
  • SEQ ID NO: 14 includes a common marmoset (Callithrix jacchus)
  • SEQ ID NO: 15 includes a giant panda (Ailuropoda melanoleuca)
  • SEQ ID NO: 16 includes Horse (Ecus cavallus), SEQ ID NO: 17 for dogs (Canis lupus familiaris), SEQ ID NO: 18 for bovine (Bos taurus), SEQ ID NO: 19 for cats (Felis catus), SEQ ID NO: 20 for pigs (Sus scrofa)
  • SEQ ID NO: 21 is a rabbit (Oryctolagus cuniculus)
  • SEQ ID NO: 22 is a rat (Rattus norvegicus)
  • SEQ ID NO: 23 is a mouse (Mus muscu).
  • PTH (1-84) used in the present invention is included in PTH (1-84) used in the present invention.
  • PTH partially active polypeptide having a partial amino acid sequence of PTH (1-84).
  • human PTH 1-37) (CP Schmitt et al., Kidney Int., 57, 1484 (2000)) (N-terminal side 1 of SEQ ID NO: 1)
  • human PTH 1-34 (corresponding to the 1st to 34th amino acid sequence on the N-terminal side of SEQ ID NO: 1)
  • human PTH (1-31) J. F. Whitfield and P. Morley, Trends Pharmacol.
  • PTH (1-34) also called teriparatide, is a partially active polypeptide of PTH that was first artificially synthesized and shown to have biological activity equivalent to that of PTH (1-84) (J. T. Potts, J. Endocrinol. 187, 311 (2005)).
  • PTH partially active polypeptides that can be used in the present invention include PTH (1-37) and PTH (1-34) in PTH (1-84) derived from the above-mentioned non-human species.
  • a polypeptide corresponding to PTH (1-31) can be mentioned.
  • PTH peptide consisting of 32 to 33 amino acids derived from other species having an amino acid sequence close to human PTH (1-34)
  • PTH (1-33) of chicken (Gallus gallus) SEQ ID NO: 24
  • Examples include PTH (1-33) (SEQ ID NO: 25) of zebra finch (Taeniopygia guttata) and PTH (1-33) (SEQ ID NO: 26) of Monodelphis domestica, which can also be used in the present invention.
  • PTH PTH / PTHrP such as human binds / acts on the opossum PTH / PTHrP receptor (Jupner H. et al. Endocrinology 134 pp 879 1994).
  • PTHrP Parathyroid hormone-related peptide
  • PTHrP is a peptide hormone found in the living body of vertebrates more than fish and is structurally similar to PTH and acts on calcium metabolism in the same way as PTH.
  • PTHrP is expressed in an inactive preprohormone and undergoes a process, and then becomes PTHrP, usually consisting of 141 or 139 amino acids.
  • PTHrP consisting of 141 amino acids or 139 amino acids may be referred to as PTHrP (1-141) and PTHrP (1-139), respectively, in order to distinguish them from other active partial polypeptides derived from PTH.
  • PTHrP As one example of PTHrP used in the present invention, human PTHrP (1-141) (isoform 1) whose amino acid sequence is shown in SEQ ID NO: 27, or PTHrP (SEQ ID NO: 28) whose amino acid sequence is shown 1-139) (isoform 2). Also, it corresponds to non-human PTHrP (1-141) or PTHrP (1-139)
  • PTH are: Rhesus macaque PTHrP (1-139), SEQ ID NO: 29, Giant Panda (Ailuropoda melanoleuca) PTH (1-141), SEQ ID NO: 31, Bos taurus (SEQ ID NO: 31).
  • human PTHrP (1-141) and rabbit PTHrP (1-141) with NIH's default parameters in the BLAST method, homology (Identity) in which both amino acid sequences match is 90%, amino acids with similar properties When the substitution to was permitted, 91% similarity was shown.
  • human PTHrP (1-141) and 90% of PTHrP (1-141) or PTHrP (1-139) in human or non-human species not specifically described in this specification. Anything exhibiting the above homology is included in PTHrP (1-141) or PTHrP (1-139) used in the present invention.
  • human PTHrP (1-86) E.
  • PTHrP (1-40) E. Lewin et al., Kidney Int., 58, 71 2000) (corresponding to the 1st to 40th amino acid sequences on the N-terminal side of SEQ ID NO: 27)
  • PTHrP (1-37) LJ Suva et al., Science, 237, 893, 1987) (corresponding to the first to 37th amino acid sequence on the N-terminal side of SEQ ID NO: 27)
  • PTHrP (1 -34) BE Kemp et al., Science, 238, 1568 19 7) (corresponding from the N-terminal side 1 of SEQ ID NO: SEQ ID NO: 27 to 34 amino acid sequence) can be exemplified.
  • PTHrP partially active polypeptides examples include PTHrP (1-86) in PTHrP (1-141) or PTHrP (1-139) derived from the above-mentioned non-human species. , PTHrP (1-40), PTHrP (1-37), or a polypeptide corresponding to PTHrP (1-34).
  • PTHrP (1-34) SEQ ID NO: 38 of Platypus (Ornithorhynchus anatinus), zebra finch PTHrP (1-34) (Taeniopygia guttata) (SEQ ID NO: 39), PTHrP (1-34) (Sequence No. 40) of chicken (Gallus gallus), PTHrP (1-34) of Monodelphis domestica SEQ ID NO: 41), and these are also included in the PTH that can be used in the present invention.
  • PTH / PTHrP such as human binds / acts on the opossum PTH / PTHrP receptor (Jupner H. et al. Endocrinology 134 pp 879 1994). Comparing human PTHrP (1-34) and gray porpoise POSSrP (1-34) by the BLAST method, the homology between them is about 77%, and 83% when substitution with an amino acid with similar properties is permitted. The similarity was shown. In addition, as described above, it is known that PTH and PTHrP bind to a common receptor PTH / PTHrP and cause a signal to cells (Jupner H. et al. Science. 254 p1024 1991), and human PTH.
  • PTH, PTHrP, or a partially active polypeptide thereof in human or non-human species not specifically described in this specification may be human PTH (1-34) or human PTHrP (1- 34) any PTH, PTHrP, or a partially active polypeptide thereof having an amino acid sequence having no more than 12 amino acid substitutions relative to the amino acid sequence corresponding to 21 amino acids from the first N-terminal of 34) , PTHrP or a partially active polypeptide thereof.
  • PTH, PTHrP or a partially active polypeptide as described above is produced by extracting from the animal or cell producing the peptide, and a gene encoding the polypeptide is introduced into the host by genetic engineering.
  • PTH, PTHrP or a partially active polypeptide thereof is available from a reagent manufacturer, for example, from Bachem, PTH (1-84) (human) (Cat. No. H-1370), PTH ( 1-37) (human) (Cat. No. H-5974), PTH (1-34) (human) (Cat. No. H-4835), PTH (1-31) (human) (Cat. No. 1). H-2274), PTH (1-31) amide (human) (Cat. No.
  • PTH, PTHrP or a partially active polypeptide thereof used in the present invention has a carboxyl group (COOH) state or amidation (CONH) as its C-terminal structure. 2
  • the PTH, PTHrP or the partially active polypeptide of the present invention is a compound, an amino acid, PTH, PTHrP or a combination thereof at any one of the N-terminus, C-terminus, side chain of amino acid residues, or a combination of two or more thereof.
  • a form to which a protein or peptide other than the partially active polypeptide is added may also be used.
  • the added compound include substances that stabilize when administered in vivo, such as polyethylene glycol (PEG), and compounds having medicinal properties such as bisphosphonates.
  • the state in which an amino acid is added refers to a form in which a methionine residue, acetyl group, pyroglutamic acid, or the like is bonded to the N-terminus, or a proteolytic enzyme prepared as a fusion protein in the production of PTH, PTHrP or a partially active polypeptide thereof Addition of 1 to several amino acids remaining at the N-terminal or C-terminal when cleaving is performed.
  • the protein to be added include proteins involved in stabilization and transportation in vivo such as albumin and immunoglobulin.
  • the peptide to be added include tag sequences (histidine tag, FLAG tag, etc.) used for purification in the production process, other bioactive polypeptides, and the like.
  • a gene for expression that encodes the polypeptide having the structure by genetic engineering can be prepared as a gene expression product using a host cell, a transgenic plant or a non-human transgenic animal into which the gene has been introduced.
  • PTH, PTHrP, or partially active polypeptides thereof is an amino acid sequence of one or more of the amino acid sequences in the above-mentioned PTH, PTHrP, or a partially active polypeptide thereof. In which the activity as a PTH / PTHrP receptor agonist is retained.
  • the PTH / PTHrP described in the above [PTH / PTHrP receptor agonist] is used as a method for examining whether the PTH, PTHrP, or an analog of a partially active polypeptide thereof retains activity as a PTH / PTHrP receptor agonist.
  • Amino acid substitution includes conservative substitution and non-conservative substitution.
  • Conservative substitution refers to substitution between amino acids having similar properties
  • non-conservative substitution refers to other amino acid substitution.
  • amino acid conservative substitutions are more likely to preserve the function of the underlying molecule than non-conservative substitutions. Therefore, it is possible to obtain polypeptides having equivalent effects by conservative substitution of amino acids.
  • One aspect of considering the closeness of amino acid properties in conservative substitution is substitution with a combination of amino acids that can be substituted without significantly affecting the polarity and charge of the amino acid side chain.
  • amino acids having nonpolar side chains are classified into those having aliphatic side chains and those having aromatic side chains. According to these classifications, 20 kinds of amino acids constituting normal natural proteins and peptides are aspartic acid (Asp) and glutamic acid (Glu) are polar / charged / acidic side chain amino acids, arginine (Arg) and lysine (Lys).
  • His histidine
  • glycine Gly
  • Ser serine
  • Thr threonine
  • Asparagine Asn
  • glutamine Gln
  • tyrosine Tyr
  • cysteine Cys
  • Cys is a polar / uncharged side chain amino acid
  • alanine Al
  • valine Val
  • leucine Leu
  • isoleucine Ile
  • methionine Metal
  • Pro proline
  • Amino acids, phenylalanine (Phe) and tryptophan (Trp) are nonpolar / It is classified as an amino acid of the aromatic side chains.
  • Amino acids belonging to the same class are often used for conservative amino acid substitution.
  • Cys may be involved in the formation of intramolecular or intermolecular crosslinks via disulfide bonds, and Pro may affect the secondary structure of proteins / peptides. It may not be handled by replacement.
  • a hydrophobicity index obtained by quantifying hydrophobicity or charge may be used as an index of physicochemical properties of amino acid side chains.
  • the hydrophobic index scores of two kinds of amino acids contained in a natural protein are Arg: ⁇ 10.0, Lys: ⁇ 9.9, Glu and Asp: ⁇ 8.3, Asn: ⁇ 7.1, Gln: -6.0, Ser: -4.3, His and Thr: -3.8, Gly: -2.4, Cys: -2.3, Ala: -1.1, Pro: -0.2, Tyr: 2.5, Val: 4.1, Met: 4.6, Ile: 8.7, Leu and Trp: 9.7, and Phe: 10.
  • Examples of special amino acids not included in normal proteins include ⁇ , ⁇ -diaminopropionic acid (Dap): ⁇ 9.5, ⁇ -aminoisobutyric acid (Aib): 1.1, norleucine (Nle):
  • the hydrophobic index scores such as 9.1, have been calculated (Alessandro et al., PEPTIDES 2002, Proceeding of 27th European Peptide Symposim). In order to perform conservative amino acid substitution, in this hydrophobic index, it is considered that the closer the amino acid score is selected, the closer the hydrophobicity and charge are, and the more likely the structure and function are preserved.
  • Amino acid substitution matrix is a matrix in which sequences with similar structures and functions are collected and compared, and quantified according to the frequency and type of amino acid substitution, and is said to reflect the ease of relative substitution in the evolution process.
  • a typical example is PAM (Point-Accepted-Mutation) (MOO Dayhoff et al., Atlas of Protein Sequence and Structure 5, p345 1978) created by comparing the full-length amino acid sequences of the sequences forming the family.
  • BLOSUM Blocks Substitution Matrix
  • PTH, PTHrP, or an analog of a partially active polypeptide thereof by substitution of amino acids constituting ordinary natural proteins and peptides is a recombinant cell or a trans gene in which a gene encoding the polypeptide is genetically engineered into a host. It can be produced by a method for expression in a transgenic non-human animal or a transgenic plant, or a peptide chemical synthesis method such as a solid phase synthesis method.
  • non-protein amino acids other than the 20 types of amino acids normally contained in natural proteins as described above is also possible It is.
  • non-protein amino acids include D-form amino acids that are engineering isomers.
  • the alpha carbon is an asymmetric carbon atom except for Gly, and takes L and D engineering isomers.
  • the amino acid normally contained in natural protein is a L-form.
  • amino acids in the amino acid sequence of PTH, PTHrP, or a partially active polypeptide thereof are substituted with a D-form amino acid and the activity as a PTH / PTHrP receptor agonist is maintained.
  • amino acids other than the 20 kinds of amino acids may be used as non-protein amino acids, for example, ⁇ - (2-naphthyl) alanine ( ⁇ -Nal), norleucine (Nle), ⁇ , ⁇ -diaminopropionic acid.
  • cyclohexylalanine Cha
  • norvaline Nva
  • 4-amino-phenylalanine Amp
  • 3-pyridinylalanine Amp
  • ⁇ -aminoisobutyric acid Aib
  • 1-amino-1-cyclo- Examples include hexanecarboxylic acid (Ahc).
  • 1-amino-1-cyclopropanecarboxylic acid 1-amino-1-cyclobutanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cycloheptanecarboxylic acid,
  • a non-protein amino acid selected from the group of 1-amino-1-cyclooctanecarboxylic acid and 1-amino-1-cyclononanecarboxylic acid may be represented herein as Acc.
  • those in which the amino acid sequence of PTH, PTHrP, or a partially active polypeptide thereof is substituted with one or more non-protein amino acids and the activity as a PTH / PTHrP receptor agonist is maintained in the present invention.
  • An analog of PTH, PTHrP, or a partially active polypeptide thereof after substitution with these non-protein amino acids can be usually prepared by a known peptide synthesis method such as a solid phase synthesis method.
  • PTH PTHrP
  • an analog of a partially active polypeptide thereof is a peptide that has undergone intramolecular or intermolecular crosslinking.
  • intramolecular cross-linking include cross-linking between Lys at position 26 and Asp at position 30 that are conserved between species in PTH (1-34) and conserved between species in PTHrP (1-34).
  • the analog of PTH, PTHrP, or a partially active polypeptide thereof used in the present invention is an amino acid substitution or amino acid side chain at at least one position of the amino acid sequence of PTH, PTHrP, or a partially active polypeptide thereof.
  • the upper limit of the number of amino acid substitutions is preferably within 12 positions within the range of 21 amino acids from the N-terminus of PTH, PTHrP, or their partially active polypeptides. This is because when comparing the first 21 amino acids of human PTH (1-34) and human PTHrP (1-34), there are 12 amino acid differences, but PTH (1-34) and PTHrP (1- 34) has the same activity as a PTH / PTHrP receptor agonist.
  • the analog of PTH, PTHrP or a partially active polypeptide used in the present invention has a carboxyl group (COOH) state or amidation (CONH) as its C-terminal structure. 2
  • the analog of PTH, PTHrP or a partially active polypeptide thereof according to the present invention can be a compound, amino acid, PTH, PTHrP at the N-terminal, C-terminal, amino acid residue side chain, or a combination of two or more thereof.
  • a form to which a protein or peptide other than an analog of the partially active polypeptide is added may be used.
  • Examples of the added compound include substances that stabilize when administered in vivo, such as polyethylene glycol (PEG), and compounds having medicinal properties such as bisphosphonates.
  • PEG polyethylene glycol
  • the state in which an amino acid is added refers to a form in which a methionine residue, an acetyl group, pyroglutamic acid, or the like is bound to the N-terminus, or after preparation as a fusion protein in the production of an analog of PTH, PTHrP or a partially active polypeptide thereof, Examples include addition of 1 to several amino acids remaining at the N-terminus or C-terminus when cleaved with a proteolytic enzyme.
  • Examples of the protein to be added include proteins involved in stabilization and transportation in vivo such as albumin and immunoglobulin.
  • Examples of the added peptide include tag sequences (histidine tag, FLAG tag, etc.) used for purification in the production process, other bioactive peptides, and the like.
  • tag sequences histidine tag, FLAG tag, etc.
  • JP-A-8-503692 JP-A-62-67099, JP-A-61-57600, JP-A-61-2598, 59-204159 and JP-B-3-14320, JP-A-59-42351, etc.
  • JP-A-59-42351, etc. which are included in analogs of PTH, PTHrP, or partially active polypeptides thereof that can be used in the present invention.
  • PTH PTHrP
  • a partially active polypeptide of PTH represented by the following formula: peptide: However, A 1 Is Ser, Ala, or Dap; A 3 Is Ser, Thr, or Aib; A 5 Is Leu, Nle, Ile, Cha, ⁇ -Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 Is; A 7 Is Leu, Nle, Ile, Cha, ⁇ -Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 Is; A 8 Is Met, Nva, Leu, Val, Ile, Cha, or Nle; A 11 Is Leu, Nle, Ile, Cha, ⁇ -Nal, Trp, Pal, Phe or p-X
  • a polypeptide of the following formula which is an analog of human PTH (1-34) described in JP-T-11-509201 and Japanese Patent No. 4008825 is preferable: [Cha 7, 11 HPTH (1-34) NH 2 ; [Cha 23 HPTH (1-34) NH 2 ; [Cha 24 HPTH (1-34) NH 2 ; [Nle 8, 18 , Cha 27 HPTH (1-34) NH 2 ; [Cha 28 HPTH (1-34) NH 2 ; [Cha 31 HPTH (1-34) NH 2 ; [Aib 16 HPTH (1-34) NH 2 ; [Aib 19 HPTH (1-34) NH 2 ; [Aib 34 HPTH (1-34) NH 2 ; [Cha 24, 28, 31 , Lys 30 HPTH (1-34) NH 2 ; [Cha 7, 11 , Nle 8, 18 , Tyr 34 HPTH (1-34) NH 2 ; [Cha 7, 11 , Nle 8, 18 , Aib 16, 19 , Tyr 34 HPTH (1-34) NH 2
  • an analog of a partially active polypeptide of PTHpP represented by the following formula: Polypeptide: However, A 1 Is Ala, Ser, or Dap; A 3 Is Ser or Aib; A 5 Is His, Ile, or Cha; A 7 Is Leu, Cha, Nle, ⁇ -Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 Is; A 8 Is Leu, Met, or Cha; A 10 Is Asp or Asn; A 11 Is Lys, Leu, Cha, Phe, or ⁇ -Nal; A 12 Is Gly or Aib; A 14 Is Ser or His; A 15 Is Ile or Cha; A 16 Is Gln or Aib; A 17 Is Asp or Aib
  • a polypeptide of the following formula which is an analog of human PTHrP (1-34) described in JP-T-2001-508439 and Japanese Patent No. 3963482: [Glu 22, 25 , Leu 23, 28 , Lys 26, 30 , Aib 29 , Ahc 31 HPTHrP (1-34) NH 2 ; [Glu 22, 25 , Ahc 23 , Lys 26, 30 , Leu 28, 31 , Aib 29 HPTHrP (1-34) NH 2 ; [Glu 22, 25 , Leu 23, 28, 31 , Lys 26, 30 , Ahc 27 , Aib 29 HPTHrP (1-34) NH 2 ; [Glu 22, 25, 29 , Leu 23, 28, 31 , Lys 26 , Ahc 30 HPTHrP (1-34) NH 2 ; [Cha 22 , Leu 23, 28, 31 , Glu 25 , Lys 26, 30 , Ahc 27 , Aib 29 HPTHrP (1-34) NH 2 ; [Glu 22, 25 , Leu 23, 28, 31 ,
  • PTH represented by the following formula Polypeptides that are analogs of partially active polypeptides: [Nle 31 HPTH (1-34) NH 2 ; [HArg 27 HPTH (1-34) NH 2 ; [Dap 1 , Nle 8, 18 , Tyr 34 HPTH (1-34) NH 2 ; Is mentioned. Further, it is an analog of human PTHrP (1-34) or an analog of human PTH (1-34) described in JP-T-2001-508439 and Japanese Patent No.
  • a substance having an activity of inducing PTH or PTHrP production means that the substance itself does not have an agonistic action directly on the PTH / PTHrP receptor, but the gene expression or protein of PTH / PTHrP in tissues or cells in vivo.
  • CaSR calcium sensitive receptor
  • CaSR antagonists include, for example, JTT-305 and MK-5442 (Fukumoto et al., CLINICAL CALCIUM, 21 p89, 2011), NPS2143 (Gowen et al., The Journal of Clinical Investigation, 105 p1595, 2000), SB-423557 (Matheny et al., Bone, 46, p534, 2010), SB-751689 (31th Annual Meeting, American Society for Bone and Mineral Research, oral presentation, presentation number 1051-1130 / poster announcement) ATX914 (American Society for Bone) and Mineral Research, 2010 Annual Meeting, poster presentations, Presentation # SU0372) and the like.
  • the CaSR antagonist examples include compounds disclosed in JP 2010-248183, JP 2005-239611 and JP 2010-159258.
  • the substance having the activity of inducing PTH or PTHrP production is not limited to the above-mentioned substances, and those skilled in the art will know, for example, JP 2010-279372 A about substances having the activity of inducing PTH production. It is possible to obtain the active substance required by the method described in the above, and the CaSR antagonist can be obtained by the method described in Nemeth et al., Journal of Molecular Endocrinology, Vol. 29, p15, 2002, etc. Such substances are also included in the substance having an activity of inducing PTH or PTHrP production in the present invention.
  • the present invention is characterized in that it contains a PTH / PTHrP receptor agonist or a vertebral fracture characterized by containing PTH, PTHrP, or a partially active polypeptide thereof, or an analog thereof. It relates to a therapeutic agent. Furthermore, the present invention relates to a therapeutic agent for vertebral fracture containing a substance having an activity of inducing PTH or PTHrP production.
  • the vertebral body is “the main part of the vertebra that is in front of the spinal canal and is distinguished from the vertebral arch” (Stedman Medical Dictionary, 5th edition, Medical View) or “the half that occupies the front of the vertebra "Circular part” (Ojirin, second edition, Sanseido). Therefore, a vertebral body fracture does not include a fracture at the vertebral arch where spinous processes, transverse processes, joint processes, etc. are present, among vertebrae constituting the spine, and has a semi-cylindrical shape. A fracture in the body part. There are also vertebral fractures caused by strong impacts in accidents and sports, etc., but the social and medical economic problems are vertebral fractures associated with osteoporosis.
  • the main subject for which the therapeutic agent for vertebral fracture of the present invention is used is treatment of vertebral fracture associated with osteoporosis.
  • the vertebra is composed of 7 cervical vertebrae, 12 thoracic vertebrae, 5 lumbar vertebrae, 5 sacral vertebrae (in adults, fused to become sacrum and posterior wall of pelvis), 3-5 tail vertebrae (fused
  • the site of the occurrence of vertebral fractures associated with osteoporosis is concentrated in the thoracic and lumbar vertebrae. Therefore, the rat lumbar vertebral body bone injury model according to the present invention is mainly focused on studies using the lumbar vertebrae. Therefore, the subject to which the therapeutic agent for vertebral fracture of the present invention is used mainly treats vertebral fractures in the thoracic and lumbar vertebrae.
  • the vertebral body As a structural feature of the vertebral body, there is a trabecular structure formed by cancellous bone that is highly developed inside the cortical bone that covers the outside. It is thought that the physical strength of the vertebral body depends greatly on the trabecular structure of the cancellous bone, and the reduction or deformation of the trabecular bone is the cause of the vertebral body compression fracture, which is the final image of the vertebral fracture associated with osteoporosis. It is said to be involved (IONOVICI et al., Romanian Journal of Morphology and Embrology, 50, p79-84, 2009).
  • the subject to which the therapeutic agent for vertebral fracture of the present invention is used mainly treats vertebral fractures in cancellous bone or vertebral fractures that span cortical bone and cancellous bone.
  • the patient using the therapeutic agent for vertebral body fracture in the present invention is a patient in which one or more fractures are already observed in the vertebral body at the start of treatment, and preferably the vertebral body fracture reaches the vertebral body compression fracture.
  • grade 1 change in vertebral body leading edge height, central height and / or trailing edge height
  • grade 1 change in vertebral body leading edge height, central height and / or trailing edge height
  • the area is reduced 10-20%)
  • Diagnosis regarding detection of vertebral fractures and / or reduction in vertebral body height before the start of treatment can be performed by a person skilled in the art using, for example, simple X-ray, CT, MRI, etc.
  • the effect of the therapeutic agent for vertebral fracture in the present invention can be performed using a method such as simple X-ray, CT, MRI, etc.
  • the index of this effect is an index of the fracture line existing before the treatment. Decrease or disappearance of length and / or area, restoration of vertebral body height and area, and the like can be determined as indices. Further, in such diagnosis and treatment effect determination based on the shape of the vertebral body, for example, image analysis described in Japanese Patent No.
  • the present invention also relates to an agent for treating or preventing pain caused by vertebral fractures.
  • the vertebral body compression fracture (one of the vertebral body height / leading edge height / center height / rear edge height is 0.8 or less, or the leading edge When the value of high / rear edge height is 0.75 or less, or when the height of the vertebral body decreases as a whole, or when the height is reduced by 20% or more from the upper and lower vertebral bodies of the judgment vertebral body Vertebral body compression fracture).
  • This vertebral body compression fracture is diagnosed by measurement using simple X-ray, CT, MRI or the like. Therefore, it is possible to determine the effect of the therapeutic agent for vertebral fracture according to the present invention with the effect of preventing the progress to such a compression fracture.
  • the present invention also relates to a preventive agent for vertebral body compression fracture.
  • Symptoms related to vertebral fractures are particularly strong when they are accompanied by movement disorders or neuropathy, or may progress to such movement disorders or neuropathy due to progression of the disease without treatment.
  • the risk is high when severe vertebral body damage is caused by painful vertebral fractures or vertebral body compression fractures. Therefore, it is possible to determine the effect of the therapeutic agent for vertebral fracture of the present invention with the effect of reducing or eliminating such movement disorder and / or neuropathy, or the effect of preventing movement disorder and / or neuropathy.
  • the present invention also relates to a therapeutic or prophylactic agent for movement disorders and / or neurological disorders caused by vertebral fractures.
  • the vertebral fracture treatment agent of the present invention may contain any pharmaceutically acceptable additive.
  • the formulation with pharmaceutically acceptable excipients is published by REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY 20th EDITION Philadelphia University of Science in Wilda It is also possible to carry out by the method described in 1.
  • One form of such a pharmaceutical composition is provided as a solution prepared by dissolving, suspending or emulsifying in a sterile aqueous or oily liquid.
  • solvents include distilled water for injection, physiological saline and the like, and in addition, osmotic pressure regulators (for example, D-glucose, D-sorbitol, D-mannitol, sodium chloride, etc.).
  • solubilizers such as alcohol (eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol), nonionic surfactant (eg polysorbate 80, polyoxyethylene hydrogenated castor oil 50), etc. are used together Sometimes it is done.
  • an oily liquid may be used as the solvent. Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent.
  • a buffer for example, phosphates buffer, acetate buffer
  • a soothing agent for example, benzalkonium chloride, procaine hydrochloride, etc.
  • a stabilizer for example, human serum albumin, Polyethylene glycol, etc.
  • preservatives eg, ascorbic acid, erythorbic acid and their salts
  • coloring agents eg, copper chlorophyll, ⁇ -carotene, red No. 2, blue No.
  • preservatives eg, paraoxybenzoic acid, etc.
  • Esters phenol, benzethonium chloride, benzalkonium chloride, etc.
  • thickeners eg hydroxypropylcellulose, carboxymethylcellulose and their salts
  • stabilizers eg human serum albumin, mannitol, sorbitol, etc.
  • flavoring agents eg Menthol, citrus flavors, etc.
  • solid preparations such as powders, tablets, granules, capsules, pills, suppositories, and lozenges are available. can give.
  • excipients eg, crystalline cellulose, lactose, starch, etc.
  • lubricants eg, magnesium stearate, talc, etc.
  • binding Agents hydroxypropylcellulose, hydroxypropylmethylcellulose, macrogol, etc.
  • disintegrating agents eg, starch, carboxymethylcellulose calcium, etc.
  • additives such as preservatives (for example, benzyl alcohol, chlorobutanol, methyl paraoxybenzoate, propyl paraoxybenzoate, etc.), antioxidants, coloring agents, sweeteners, and the like can be used.
  • Another form is a pharmaceutical composition for mucosa.
  • an adhesive, an adhesion enhancer, and a viscous agent are mainly used as additives for the purpose of imparting adsorptive properties and retention to the mucosa.
  • Thickeners etc. eg mucin, agar, gelatin, pectin, carrageenan, sodium alginate, locust bin gum, xanthan gum, tragacanth gum, gum arabic, chitosan, pullulan, waxy starch, sucralfate, cellulose and derivatives thereof (eg hydroxy In some cases, propylmethylcellulose, polyglycerin fatty acid ester, acrylic acid (meth) acrylate alkyl copolymer or salt thereof, polyglycerin fatty acid ester, and the like may be contained.
  • the vertebral fracture treatment agent can be administered orally or parenterally for the purpose of improving symptoms.
  • dosage forms such as granules, powders, tablets, capsules, solutions, syrups, emulsions or suspensions, and elixirs can be selected.
  • parenteral administration for example, it can be a nasal agent, and a liquid agent, a suspension agent, a solid preparation and the like can be selected.
  • parenteral administration can be an injection, and as the injection, a subcutaneous injection, intravenous injection, infusion, intramuscular injection, intraperitoneal injection or the like can be selected. it can.
  • parenteral administration examples include suppositories, sublingual agents, transdermal agents, transmucosal agents other than nasal agents, and the like. Furthermore, it can also be administered locally to a blood vessel in such a manner that it is contained or applied to a stent or an intravascular embolic agent.
  • the dosage of the pharmaceutical composition varies depending on the patient's age, sex, weight, symptoms, therapeutic effect, administration method, treatment time, type of active ingredient contained in the pharmaceutical composition, etc. Per dose, in the range of 0.001 mg to 500 mg, preferably in the range of 0.005 mg to 200 mg. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be required.
  • the present invention also relates to a method for evaluating a vertebral body fracture therapeutic agent.
  • the evaluation method of the present invention includes 1) a step of surgically damaging a vertebral bone of a non-human animal, 2) a step of administering a test substance to the non-human animal, and 3) the non-human animal or derived therefrom. Morphological, physiological, biochemical or behavioral measurement of recovery of surgical damage to the vertebral bone of the specimen.
  • the results of morphological, physiological, biochemical or behavioral measurements of recovery from surgical damage to vertebral bones are further obtained.
  • the surgical damage given to the vertebral bone by the evaluation method of the present invention is preferably damage that reaches the cancellous bone through the cortical bone of the vertebral bone, and is not particularly limited as long as the damage can be given.
  • a method of damaging by drilling with is preferred. The diameter and depth of a drill or the like and a perforation using the drill can be performed under appropriate conditions using appropriate tools depending on the animal used and the vertebral body used.
  • the perforation diameter is preferably between 0.5 and 2.0 mm, more preferably between 0.5 and 1.5 mm, and even more.
  • the diameter is preferably 0.7 to 0.8 mm.
  • the depth of the perforation may be a depth that reaches the cancellous bone from the cortical bone, but is preferably a depth that reaches the cancellous bone from the cortical bone but does not reach the opposite cortical bone, more preferably 2.5. It is ⁇ 3.0mm deep.
  • the measurement of the healing effect of surgical damage to vertebral bones in the evaluation method of the present invention can be appropriately performed by using a morphological observation method such as simple X-ray, CT, micro CT, or MRI.
  • a method using X-ray micro CT as a method for measuring the healing effect of surgical damage is described as an evaluation method using rats. It is not limited.
  • a method based on histological observation is also possible for measuring the healing effect of surgical damage to vertebral bones in the evaluation method of the present invention.
  • Samples used for histological observation include vertebral body tissues that have undergone surgical damage, other bone tissues, other tissues such as cartilage, muscles, and nerves, and body fluids such as blood and cerebrospinal fluid Is mentioned.
  • a method of collecting tissues by biopsy can be used in addition to collecting tissues by dissection.
  • a sample used for histological observation is subjected to treatment such as fixation, slicing, and / or staining as appropriate, and then subjected to observation.
  • biochemical and / or cytological markers for example, methods for detecting protein or gene expression for markers relating to bone differentiation or metabolism, such as tissue immunochemistry, in-situ hybridization, and PCR are used.
  • a person skilled in the art can appropriately perform histological observation.
  • Methods for measuring the expression of various markers, such as proteins and genes for markers related to bone differentiation and metabolism, can also be carried out by various known methods, and such methods are also included in the histological analysis.
  • Another form of measuring the healing effect of surgical damage to vertebral bones is a measurement by physiological observation, for example, a method by measurement of pain sensation.
  • Methods for measuring pain sensation in non-human animals include, for example, the paw pressure test and von Frey test, which are tests for measuring the degree of mechanical hyperalgesia, and allodynia for measuring the degree of mechanical allodynia.
  • paw pressure test and von Frey test are tests for measuring the degree of mechanical hyperalgesia
  • allodynia for measuring the degree of mechanical allodynia.
  • paw flick test measures the degree of thermal hyperalgesia
  • the evaluation method of the present invention can also evaluate the local or systemic effect on inflammation caused by administration of a test substance.
  • the histological analysis as described above is used to measure the local effect on inflammation, and inflammatory cells such as lymphocytes, monocytes / macrophages, mast cells, neutrophils, eosinophils, eosinophils, For example, the amount of local accumulation of base spheres and the increase / decrease in the amount may be examined.
  • inflammatory factors expressed therein such as tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6
  • TNF tumor necrosis factor
  • IL-1 interleukin 1
  • IL-6 interleukin 6
  • samples such as blood or other body fluids can be used to measure the effects on inflammation throughout the body.
  • Inflammatory cytokine protein or C-reactive protein (CRP) ⁇ 1-antitrypsin, ⁇ 1-antichymotrypsin, ⁇ 1-acid glycoprotein, serum amyloid A, haptoglobin, ceruloplasmin, albumin, transthyretin, transferrin, etc.
  • CRP C-reactive protein
  • ⁇ 1-antitrypsin ⁇ 1-antichymotrypsin
  • ⁇ 1-acid glycoprotein serum amyloid A
  • haptoglobin haptoglobin
  • ceruloplasmin albumin
  • transthyretin transferrin
  • the evaluation method of the present invention can also measure the influence on bone resorption by the administration of the test substance.
  • bone resorption can be measured by measuring bone volume density.
  • Examples of the method for measuring bone volume density include the method using micro CT shown in Examples 3 and 5, and those skilled in the art appropriately select and implement a general method for measuring bone volume density. It is also possible.
  • the evaluation method of the present invention can also evaluate spinal cord inflammation or spinal cord injury caused by administration of the test substance. Histological, physiological, and behavioral methods are used to examine spinal cord inflammation and damage. As a histological method, in addition to the method described in the method based on the histological observation in the measurement of the healing effect of the surgical injury in the measurement of the healing effect, the above-mentioned local inflammation using a sample such as a spinal cord tissue.
  • the non-human animal used in the evaluation method of the present invention is a non-human vertebrate, preferably a non-human primate or a rodent, and a rat is preferable as the rodent animal, such as a mouse. It can be applied to any non-human vertebrates such as rodents, rabbits, dogs and monkeys. Further, as the non-human animal used in the present invention, an osteoporosis model non-human animal can be used, and an example of the osteoporosis model is an ovariectomy model.
  • the vertebral body part used in the evaluation method of the present invention a person skilled in the art selects an appropriate vertebral body part from among cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacral vertebrae and tail vertebrae according to the type of non-human animal used. Although it can be carried out, it is preferably a thoracic and / or lumbar vertebral body, more preferably a method using a lumbar vertebral body.
  • a rat is used as a non-human animal, and in this case, a method using the lumbar vertebrae, particularly the fourth lumbar vertebra and the fifth lumbar vertebra is shown.
  • the vertebral body used is preferably the fourth lumbar vertebra and / or the fifth lumbar vertebra, but is not limited thereto.
  • Administration of the test substance in the evaluation method of the present invention can be carried out by the oral or parenteral route, and those skilled in the art will know the type of non-human animal used, the evaluation site, the physical and / or test substance. In consideration of the chemical properties, it is possible to determine the administration method and administration site, and to prepare a sample for administration containing the test substance corresponding thereto.
  • Example 1 Examination of preparation method of rat lumbar vertebral body bone damage model A new animal model was constructed for the purpose of examining the therapeutic effects of drugs on vertebral fractures in vivo. This model is an in vivo model in which bone damage is caused in the rat lumbar vertebral body and the healing process is observed. Specifically, a hole was drilled in the lumbar spine of a normal rat with a dental drill, and the hole was used as bone damage.
  • Rats were anesthetized with sodium pentobarbital (Kyoritsu Pharmaceutical Co., Ltd.) after a acclimation period of 1 week, shaved around the abdomen with a clipper, and fixed on the operating table on its back. After abdominal laparotomy was performed and the abdominal organs were isolated with sterilized gauze, blood vessels, adipose tissue, etc. around the lumbar spine were carefully detached using a cotton swab. A minute amount of bleeding was stopped by pressing with a cotton swab. The muscle layer around the lumbar spine was torn with tweezers, and a visual field of about 1 cm square was secured for each of the fourth and fifth lumbar vertebrae.
  • a large swab with a diameter of about 1.2 mm and a small swab with a diameter of about 3 mm were used.
  • By gradually exfoliating the tissue with a cotton swab it was possible not only to prevent bleeding but also to minimize damage to the muscle tissue around the vertebral body.
  • a visual field of about 1 cm square is secured for one vertebral body so that the dental drill does not involve the surrounding tissue and minimizes muscular tissue damage.
  • the second step is to make a hole near the center avoiding the anterior longitudinal ligament and blood vessels of the 4th and 5th lumbar vertebrae with a secured visual field.
  • the diameter of the hole is 2 mm (the size of the hole made by Uchida et al. In the femur is about 2-2.5 mm in diameter (Non-patent Documents 12 and 15)), blood vessels cannot be avoided and many blood vessels are damaged. As a result, the bleeding increased, and the rat died without awakening from anesthesia. On the other hand, when the diameter of the hole was 0.4 mm or less, the columnar stable bone damage could not be caused uniformly.
  • FIG. 1 is a model in which a rat lumbar vertebral body damage model was prepared and the vertebral body was photographed using micro CT.
  • a hole having a diameter of about 0.72 mm was formed in the vicinity of the center avoiding the exposed anterior longitudinal ligaments of L4 and L5 and blood vessels.
  • the arrow points to the drilled hole.
  • a bone injury of about 0.72 mm in diameter and 2.5-3.0 mm in depth does not penetrate the vertebral body in the vicinity of the center of the 4th or 5th lumbar vertebra, avoiding the anterior longitudinal ligament and blood vessels.
  • the rat showed a gradual weight gain similar to that of the sham-operated rat that had been awakened from anesthesia and only performed laparotomy.
  • rat lumbar vertebral bone injury model Histological examination of rat lumbar vertebral bone injury model during natural repair process The natural restoration process after bone injury in the rat lumbar vertebral body bone injury model was observed by preparing vertebral body tissue specimens.
  • the model creation date (the day when the hole was made) was set to day 0, and blood was lethal from the abdominal aorta under pentobarbital anesthesia to day 4, day 7, day 14, and day 21, and the lumbar vertebrae were removed.
  • the extracted lumbar vertebra was immersed in 70% ethanol and stored at 4 ° C.
  • Each vertebral body was decalcified by formic acid / sodium citrate and embedded in paraffin (sealed automatic fixed embedding device (Sakura ETP-180BV)). Based on the micro CT image, the damaged part of the cancellous bone was exposed, and the block where the damaged part was exposed was observed.
  • FIG. 2 shows representative examples of MT staining (damaged cancellous bone) at each time point.
  • FIG. 2 shows the MT-stained images of the model production date (day 0), day 4, day 7, day 14, and day 21.
  • 2A was day 0, 2B was day 4, 2C was day 7, 2D was day 14, 2E was day 21, and 2F was a canal bone of a normal vertebral body (sham operated rat) of day 21.
  • the upper part is the damaged part
  • the lower part is the non-injured part
  • the boundary is indicated by an arrow.
  • Example 3 Micro CT imaging and bone volume density (mg / cm) using the image in the natural restoration process of rat lumbar vertebral body bone damage model 3 ) Measurement A microfocus X-ray CT apparatus (Nittsu Elex Co., Ltd.) was used. Using the imaging software ELE SCAN (NX-CP-C80H-IL-021: Nippon Steel ELEX Co., Ltd.), 3D tomographic imaging was performed under imaging conditions of a tube voltage of 50 kV and a tube current of 60 mA.
  • the line where the longitudinal ligament is located from the side of the vertebral body was aligned with the center line, and the vertebral body was fixed so that it did not move during imaging, and 300 images were taken with a width of 0.02 mm.
  • the measured value is the bone volume density (mg / cm 3 ) Calcium carbonate phantom set in 4 stages (0 to 300 mg / cm) 3 CaCO 3 ) Under the same conditions.
  • the CT image was saved as a C3D file and reconstructed using conbeam reconstruction software (ELE SCAN CB3D: Nippon Steel Elex Co., Ltd.) (C3V file creation). Based on the stored C3V file, 300 TIFF files were created and analyzed using this file.
  • Real INTAGE ver2.1 (K.G. TT Co., Ltd.) was used to measure the luminance value.
  • the bone volume density of the damaged part of the cancellous bone of the damaged vertebral body was measured. Specifically, for the 11 Tiff files from 1.90 mm to 2.10 mm, where the line where the posterior longitudinal ligament is located as seen from the side of the vertebral body in the micro CT image taken is 0.40 mm, respectively. 2 The luminance value of the circle was measured. Bone volume density was also measured for cancellous bones of normal vertebral bodies (sham-operated rats).
  • 3 shows the bone volume density of the model creation date (day 0), day 4, day 7, day 14, and day 21.
  • the black circle ( ⁇ ) is the mean bone volume density ⁇ standard deviation of the damaged part of the trabecular bone of the damaged vertebra
  • the square ( ⁇ ) is the mean value of the bone volume density of the cancellous bone of the normal vertebral body (sham operated rat) ⁇ Standard deviation is shown.
  • 1 vertebral body was treated as an example.
  • the average value ⁇ standard deviation of bone volume density immediately after model preparation is 0.0 ⁇ 31.6 mg / cm 3
  • 3 Met is the average value of bone volume density immediately after model preparation.
  • the bone volume density of cancellous bone of normal vertebral bodies is 246.4 ⁇ 64.6 for day 0 and 220.7 ⁇ 50.1 mg / cm for day 21. 3 And showed a substantially constant value within the observation period. Therefore, when a hole was drilled in the lumbar spine with a dental drill, the bone volume density of the hole gradually increased, and it was shown that it recovered to the same level as the normal vertebral body (sham-operated rat) on the 21st day. Moreover, it was shown that the bone damage repair level of the rat lumbar vertebral body bone damage model can be quantified by measuring the bone volume density using micro CT.
  • Example 4 Histological examination when hPTH (1-34) and PTHrP analog A were administered to a rat lumbar vertebral body bone injury model
  • the hPTH (1-34) group, PTHrP analog A and vehicle group were set.
  • As the vehicle administration solution Saline (pH 4.0 ⁇ 0.1) containing 2% rat serum was used.
  • the hPTH (1-34) administration solution and the PTHrP analog A administration solution were prepared to 10 ⁇ g / mL using Vehicle.
  • the first administration was performed 4 hours after the model was prepared, and was administered subcutaneously once a day from the next day.
  • the administration volume was 1 mL / kg.
  • blood was lethal from the abdominal aorta under pentobarbital anesthesia, and the fourth lumbar vertebra, fifth lumbar vertebra (damaged vertebra), third lumbar vertebra, and sixth lumbar vertebra (undamaged vertebral body) were removed.
  • the extracted lumbar vertebra was immersed in 70% ethanol and stored at 4 ° C.
  • Each vertebral body was decalcified with formic acid / sodium citrate and embedded in paraffin (sealed automatic fixed embedding device (Sakura ETP-180BV)).
  • the damaged part of the cancellous bone was exposed, and the block where the damaged part was exposed was observed.
  • Sections (sliding microtome (Sakura IVS-410)) sliced at 3 mm were placed on a slide glass, and after deparalysis, hematoxylin and eosin (HE) staining and Masson trichrome (MT) staining were performed.
  • the prepared tissue specimens were observed using a microscope (Leica DM4000B), a camera (Leica DFC480), and a microscopic photography software (Leica IM500).
  • FIG. 4 shows representative examples of MT staining (damaged cancellous bone) at each time point.
  • FIG. 4 shows an MT-stained image when hPTH (1-34) 10 ⁇ g / kg or PTHrP analog A 10 ⁇ g / kg was subcutaneously administered every day for 7 days or 14 days from the model creation date.
  • 4A is the vehicle group at day7
  • 4B is the hPTH (1-34) group at day7
  • 4C is the PTHrP analog A group at day7
  • 4D is the vehicle group at day14
  • 4E is the hPTH (1-34) at day14 Group
  • 4F shows PTHrP analog A group on day14.
  • the upper part is a damaged part
  • the lower part is an uninjured part
  • the boundary is indicated by an arrow.
  • an increase in fibrous bone and an increase in trabecular bone were observed in the hPTH (1-34) group (FIG. 4B) as compared to the Vehicle group (FIG. 4A).
  • Example 5 Examination of bone volume density at the injured part of cancellous bone of damaged vertebral body when hPTH (1-34) and PTHrP analog A were administered to a rat lumbar vertebral body bone injury model The bone volume density of the damaged part of the trabecular bone of the injured vertebral body when subcutaneously administering hPTH (1-34) 10 ⁇ g / kg or PTHrP analog A 10 ⁇ g / kg for 7 days or 14 days from the model preparation date was measured. The bone volume density was measured using the same method as in Example 3. result Bone volume density (mg / cm) measured using Micro CT images 3 ) was analyzed. In carrying out the analysis, one vertebral body was handled as an example.
  • FIG. 5 shows the bone volume density of the damaged part of cancellous bone when hPTH (1-34) 10 ⁇ g / kg or PTHrP analog A 10 ⁇ g / kg was subcutaneously administered every day for 7 days or 14 days from the model preparation date.
  • the black circle ( ⁇ ) is the mean ⁇ standard deviation of the vehicle group
  • the white circle ( ⁇ ) is the mean ⁇ standard deviation of the hPTH (1-34) group
  • the triangle ( ⁇ ) is the mean ⁇ standard of the PTHrP analog A group Indicates the deviation.
  • Student's t-test was performed between the two groups of the hPTH (1-34) group and the Vehicle group and between the two groups of the PTHrP analog A group and the Vehicle group at each time point.
  • the mean value ⁇ standard deviation of the bone volume density of the damaged part of the cancellous bone was 140.9 ⁇ 58.8 mg / cm in the vehicle group.
  • 3 HPTH (1-34) group is 250.5 ⁇ 78.7 mg / cm 3
  • p 0.0100: Student's t test.
  • bone volume density was shown when PTHrP analog A 10 ⁇ g / kg was subcutaneously administered every day for 7 days or 14 days from the model preparation date (FIG. 5).
  • the mean value ⁇ standard deviation of the bone volume density of the damaged part of the cancellous bone was 91.4 ⁇ 33.8 mg / cm in the Vehicle group.
  • Example 6 Examination of bone volume density of cancellous bone of uninjured vertebral body when hPTH (1-34) and PTHrP analog A were administered to a rat lumbar vertebral body bone injury model 7 days or 14 days from the date of model preparation, the bone volume density of cancellous bone of the non-injured vertebral body was measured when hPTH (1-34) 10 ⁇ g / kg or PTHrP analog A 10 ⁇ g / kg was subcutaneously administered every day. The bone volume density was measured using the same method as in Example 3.
  • the measurement position was set to a position different from the fourth lumbar vertebra and the fifth lumbar vertebra.
  • the third lumbar vertebrae is about 11 Tiff files from 1.70mm to 1.90mm when the line where the posterior longitudinal ligament is located as seen from the side of the vertebral body in the micro CT image taken is 0
  • the 6th lumbar spine is 2.0 mm for each of the 11 Tiff files from 2.10 mm to 2.30 mm when the posterior vertebral body viewed from the side of the vertebral body of the CT image taken is 0. 2
  • the luminance value of the rectangle (not including cortical bone) was measured.
  • Bone volume density (mg / cm) measured using Micro CT images 3 ) was analyzed.
  • one vertebral body was handled as an example. Student's t-test was performed between Day 2 and Day 14 between the hPTH (1-34) group and the Vehicle group, and between the PTHrP analog A group and the vehicle group. The significance level was 5%.
  • the hardware used was Fujitsu Ltd. FMVXD0572 FMV ESPRIMO D3230, and the software used was SAS Institute Japan SAS ver. 8.2 and Arm EXSAS ver. 7.10.
  • the bone volume density of the cancellous bone of the non-injured vertebral bodies was measured.
  • FIG. 6 shows the bone volume density of cancellous bone of the uninjured vertebral body when subcutaneously administering hPTH (1-34) 10 ⁇ g / kg or PTHrP analog A 10 ⁇ g / kg for 7 or 14 days from the model creation date.
  • the black circle ( ⁇ ) is the mean ⁇ standard deviation of the vehicle group
  • the white circle ( ⁇ ) is the mean ⁇ standard deviation of the hPTH (1-34) group
  • the triangle ( ⁇ ) is the mean ⁇ standard of the PTHrP analog A group Indicates the deviation.
  • the mean value ⁇ standard deviation of the bone volume density of cancellous bone was 240.2 ⁇ 59.7 mg / cm in the vehicle group.
  • 3 HPTH (1-34) group is 268.3 ⁇ 30.0 mg / cm 3
  • p 0.2539: Student's t test.
  • bone volume density was shown when PTHrP analog A 10 ⁇ g / kg was subcutaneously administered every day (FIG. 6).
  • the mean value ⁇ standard deviation of the bone volume density of cancellous bone was 214.9 ⁇ 56.6 mg / cm in the vehicle group.
  • the PTHrP analog A group is 233.4 ⁇ 66.6 mg / cm.
  • Example 7 Examination of effects on motor function and body weight change when hPTH (1-34) and PTHrP analog A were administered to a rat lumbar vertebral body bone injury model
  • Administration of hPTH (1-34) and PTHrP analog A exacerbates the inflammatory response in the fracture repair process, or destroys the vertebral body due to excessive bone resorption, resulting in the spinal cord existing in the vicinity of the vertebral body The possibility of being damaged was examined.
  • the spinal injury causes a serious impairment in the lower limb motor function, feeding from the food box attached to the breeding cage is also restricted, resulting in a decrease in food consumption. It is thought that weight loss due to.
  • the production of the rat lumbar vertebral body bone injury model is carried out by the method described in Example 1.
  • the production of the sham-operated rat is carried out by the rat described in Example 1 except for the step of making a hole using a dental drill. It was prepared in the same way as the lumbar spine bone injury model.
  • FIG. 7A shows the results of body weight measurement for each group of rat lumbar vertebral body bone injury model and sham-operated rats. There was no difference in body weight between the lumbar vertebral bone injury model and the sham-operated rats on all the measured days.
  • FIG. 7B shows the change in body weight of each group when hPTH (1-34) 10 ⁇ g / kg or PTHrP analog A 10 ⁇ g / kg or Vehicle was subcutaneously administered for 14 days from the model preparation day. No difference in body weight was observed between the groups treated with hPTH (1-34), PTHrP analog A and vehicle from the start of administration to 14 days. In addition, no visual behavioral abnormality was observed in each animal. Therefore, by administering hPTH (1-34) or PTHrP analog A to a rat lumbar vertebral body bone injury model, there is no effect such as serious spinal cord injury that causes behavioral disorder. confirmed.
  • the present invention relates to a therapeutic agent for vertebral fracture, a therapeutic or preventive agent for pain caused by vertebral fracture, vertebral body by containing a PTH / PTHrP receptor agonist, or a substance having an activity of inducing PTH or PTHrP production. It can be used as a preventive agent for compression fractures, or a therapeutic or prophylactic agent for movement disorders and / or neurological disorders caused by vertebral fractures.
  • the present invention also includes a step of surgically damaging a vertebral bone of a non-human animal, a step of administering a test substance to the non-human animal, and a vertebral bone of a specimen derived from the non-human animal. Including the step of measuring the recovery of surgical damage can be used as an evaluation method for a therapeutic agent for vertebral fractures.

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Abstract

The invention provides a therapeutic agent for vertebral body fracture, a therapeutic or prophylactic agent for pain due to vertebral body fracture, a prophylactic agent for vertebral body compression fracture, or a therapeutic or prophylactic agent for a movement disorder and/or a neurological disorder due to vertebral body fracture, each containing a PTH/PTHrP receptor agonist or a substance having an activity of inducing the production of PTH or PTHrP. Further, the invention can provide a method for evaluating a therapeutic agent for vertebral body fracture, including a step of surgically damaging the bone of the vertebral body in a nonhuman animal, a step of administering a test substance to the nonhuman animal, and a step of measuring recovery from the surgical damage of the bone of the vertebral body in the nonhuman animal or a sample derived from the nonhuman animal.

Description

椎体骨折治療剤及びその評価方法Vertebral fracture treatment and evaluation method thereof

　椎体骨折の治療効果を評価する新規動物モデルを開発し、それによってＰＴＨ／ＰＴＨｒＰ受容体アゴニスト、ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質が椎体骨折の治療効果を有することを見出した。従って、本発明はＰＴＨ／ＰＴＨｒＰ受容体アゴニスト、又はＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有する椎体骨折治療剤に関する。また、本発明は椎体骨折の治療剤の効果及び安全性を非ヒト動物で初めて評価することを可能にした新規動物モデルとその評価方法に関する。 A new animal model for evaluating the therapeutic effect of vertebral fractures was developed, and thereby, it was found that a substance having an activity of inducing PTH / PTHrP receptor agonist, PTH or PTHrP production has a therapeutic effect on vertebral fractures. Therefore, the present invention relates to a therapeutic agent for vertebral fracture containing a PTH / PTHrP receptor agonist or a substance having an activity of inducing PTH or PTHrP production. The present invention also relates to a novel animal model and a method for evaluating the same that make it possible to evaluate the effect and safety of a therapeutic agent for vertebral fractures for the first time in non-human animals.

　椎体は骨折頻度が最も高い部位である（非特許文献１）。椎体とは、「脊椎管の前方にある椎骨の主たる部分で、椎弓と区別される」（ステッドマン医学大辞典、改訂第５版、メジカルビュー社）、又は、「椎骨の前部を占める半円形の部分」（大辞林、第二版、三省堂）と説明される。従って、椎体骨折とは、脊椎を構成する椎骨における骨折のうち、通常は棘突起、横突起や関節突起などが存在する椎弓部分での骨折を含まず、半円柱状の形態を有する椎体部分における骨折のことをいう。
　椎体骨折は、事故やスポーツ等での強い衝撃が原因となる場合もあるが、主に骨粗鬆症による骨の脆弱性の亢進、骨強度の低下を基盤として発生する脊椎椎体の圧迫型骨折であり、患者のＱＯＬを大きく低下させる代表的な疾患の一つである（非特許文献２、３）。骨の脆弱性が亢進すると、通常骨折を生じない程度の軽微な外力、日常生活動作程度の弱いストレスが繰り返し加わることによって、骨の微細構造の損傷が生じ、骨構造が連続的に破断する。骨粗鬆症患者数の増加と共に、脊椎椎体の圧迫型骨折の発生頻度が増加しており、わが国の骨粗鬆症性椎体骨折の有病率は、女性では６０歳代で７．６−１４％、７０歳代で３７−４５％と報告されている（非特許文献４、５、６）。
　椎体骨折患者は他部位の骨折患者よりも生命予後が不良であることが分かっている。例えば、Ｆｒａｃｔｕｒｅ　ｉｎｔｅｒｖｅｎｔｉｏｎ　ｔｒｉａｌ（ＦＩＴ）で得られたデータによると、臨床骨折発生後の死亡リスクは約２倍増加するが、その内訳は、大腿骨近位部骨折は６．７倍増加、椎体骨折は８．６倍増加であった（非特許文献７）。
　椎体骨折は、受傷後に自覚症状がないものから腰背部の急性疼痛が強いものまで臨床症状が多様である。椎体骨折を治療する薬剤はなく、疼痛を伴う椎体の新鮮骨折に対しても、積極的な薬剤治療は行えない。従って、鎮痛剤を投与しながら数週間の安静臥床、更にギプス、硬性又は軟性コルセットなどの装具で外固定法を行い、数カ月間保存的に治療するのが一般的である。この際、一時的な入院加療を必要とする患者も多く、平均的な入院期間は４２．６日と報告されている（非特許文献８）。特に、骨折部位の癒合が遅延した場合、偽関節を呈し頑固な慢性疼痛を生じたり、椎体変形が進行し下肢痛や遅発性神経麻痺を引き起こしたり、脊柱の変形により消化器系、呼吸器系に機能障害を来たしたりするため、患者のＡＤＬやＱＯＬを著しく低下させる。
　重度の椎体骨折に対する治療法として、保存療法にｉｎｓｔｒｕｍｅｎｔａｔｉｏｎを併用した椎体間固定による脊柱再建術がある。１９９０年代後半から米国を中心に急速に広がった骨折椎体に骨セメントを注入する椎体形成術や、バルーンを膨らませ骨折椎体の高さを復元し、後彎変形を矯正するｋｙｐｈｏｐｌａｓｔｙなど、椎体内充填手術（ｖｅｒｔｅｂｒａｌ　ａｕｇｍｅｎｔａｔｉｏｎ　ｓｕｒｇｅｒｙ）が日本国内でも浸透してきている（非特許文献９）。しかしながら、外固定を行っても、椎体の楔状変形、脊椎後彎変形を遺残し良好な脊椎アライメントを維持できないことも多い。さらに保存療法にもかかわらず椎体圧潰や偽関節、遅発性神経障害の発症を完全に防ぎえないのが現状である（非特許文献１０）。
　椎体骨折患者のＱＯＬを高め生命予後を改善するには、椎体骨折を治療する新しい薬剤の開発が必要である。患者個人の利益のみならず、入院患者や寝たきり患者を減らし医療費を削減する社会的利益のためにも、椎体骨折を治療する新しい薬剤の開発は、積極的に取り組むべき課題である。
　椎体骨折を治療する新しい薬剤を開発する上で大きな障害となっていることの一つに、非臨床で使用できる動物モデルがなく、薬剤の効果を非ヒト動物で検証できないことが挙げられる。動物を用いた一般的な骨折モデルはいくつか報告があるが、それらは、長骨骨幹の中央部、すなわち皮質骨の損傷に着目したモデルであり、椎体骨折で生じる海綿骨の微細構造の損傷を評価するモデルではない。例えば、一般的な骨折モデルであるラット大腿骨閉鎖骨折モデルは、その大腿骨骨幹の中央部にギロチンを落として大腿骨を骨折させ、その大腿骨の強度を評価するモデル（非特許文献１１）であるが、その損傷は、皮質骨の骨折であり、椎体骨折で生じる海綿骨の微細構造の損傷とはかけ離れている。また、本発明と同じように、歯科用ドリルで穴をあけることにより骨損傷を生じさせるラット大腿骨ドリリングモデルも、大腿骨閉鎖骨折モデル同様、大腿骨骨幹の中央部、すなわち大腿骨皮質骨を損傷させたモデル（非特許文献１２、１３）であり、椎体骨折で生じる海綿骨の微細構造の損傷を評価できるモデルではない。
　また、骨折治癒の過程においては、骨芽細胞による骨形成のみではなく、破骨細胞による骨破壊の過程も進行することが知られている。ビスフォスフォネートのような破骨細胞の機能を抑制する物質とは異なり、ＰＴＨやＰＴＨｒＰは破骨細胞も活性化することが知られている。事実、ＰＴＨ等は、骨から破骨細胞の活性で血中にカルシウムを放出する作用を持っていることから、従来は高カルシウム血症を引き起こす骨破壊因子と考えられていた時期もある。現在は、骨代謝を高める作用から骨粗鬆症での骨量増加作用に基づく治療剤として利用されている一方、骨折治療にも用いることが期待されている。そのため、いろいろな動物モデルでＰＴＨ等の骨折治癒への作用が検討されている（非特許文献１４）。しかしながら、そこで用いられているモデルは長管骨（例えば大腿骨）で骨折を起こした後にピンなどを挿入することで望ましくない方向に加重がかからないように固定したものや、扁平骨でも非加重骨（頭骸骨など）での効果をみたもののみである。一方、椎体は体幹を支える脊椎の主要部分であり、常にある程度の加重がかかっている骨である。また、臨床的にも実験的にも椎体の構造上、骨折部位をピンなどで固定することは非常に困難である。よって、椎体骨折治療の目的でＰＴＨやＰＴＨｒＰ又はそれらのアナログを投与した場合に、骨折治癒の過程での骨吸収が予想以上に亢進すると、一時的に骨折部位付近の脆弱性が高まる可能性も考えられ、そこに加重がかかることによって、骨折が逆に悪化することや、場合によっては圧壊骨折に至ることで構造的にも機能的にも修復できない状況に至ることも想定される。
　従って、従来から報告されている長骨骨幹の骨折モデルを用いて椎体骨折の薬剤候補となる物質をスクリーニングした場合、大腿骨骨折に最適な物質を見出すことはできても、椎体骨折で生じる海綿骨の微細構造の損傷を治療するのに最適な物質を見出すことは不可能であった。すなわち、非ヒト動物を用いて椎体骨折を評価する方法はなかったため、椎体骨折を治療する薬剤の開発は困難であった。
　骨折の治癒の過程は、一般的に炎症期、修復期、リモデリング期に分けられる（非特許文献１５）。炎症期においては、骨折部位に血腫が形成されるが、その中には炎症系の細胞も存在し、そこから多くの炎症性サイトカインなどの因子が放出される。脊椎の構造をみると、椎体に近接する脊柱管には全身の運動や感覚、また生理作用の制御に重要な脊髄が通っているという特徴がある。この脊髄がなんらかの原因によって炎症を起こした疾患は脊髄炎と呼ばれ、その症状としては軽い場合には感覚や運動機能の異常であるが、重篤な場合にはその支配下の部位全体の麻痺や死に至る場合もある。一方、ＰＴＨｒＰはいくつかの疾患における炎症反応に関与していることが示されている（非特許文献１６−２０）。また、副甲状腺機能亢進症の患者の骨においては肥満細胞（マストセル）が多く存在し、その肥満細胞の集積にＰＴＨが関与しており、ＰＴＨｒＰも同様の作用があると考えらること（非特許文献２１）、ＰＴＨ及びＰＴＨｒＰが骨芽細胞に作用して好中球などに対する細胞遊走因子であるＣＸＣＬ１の発現を誘導するなどの作用も知られている（非特許文献２２）。従って、ＰＴＨ／ＰＴＨｒＰやそれらのアナログなどを椎体骨折の治療目的で投与した場合、骨折部位炎症がさらに重篤になる可能性も考えられ、近傍に存在する脊髄に対しても炎症が広がって障害をおこすおそれが想定される。
　椎体骨折治療の研究においては、上記のように、椎体骨折の有用な動物モデルが存在せず、また重篤な副作用の可能性も考えられていた。
　ＰＴＨの部分活性ポリペプチドは、これまで、上述した長骨骨幹の骨折モデルを用いて、皮質骨の骨折修復促進作用が示されてきた（特許文献１、非特許文献２３、２４）。しかし、椎体骨折で生じる海綿骨の微細構造の損傷に対して治療効果を明確に検出した報告はない。また、特許文献２に示されている化合物が椎体骨折治療剤となる可能性を示した報告がある（特許文献２）が、この報告は、臨床試験で椎体骨折の予防効果を検討しただけで、薬剤の椎体骨折に対する治療効果を明確に検出したものではなく、椎体骨折に対する治療効果を漠然と憶測したものにすぎない。すなわち、本発明のように、椎体骨折の特徴である海綿骨の微細構造の損傷に特化し、その損傷部のみで薬剤の椎体骨折治療効果を明確に示した報告はない。 The vertebral body is the site with the highest fracture frequency (Non-Patent Document 1). The vertebral body is “the main part of the vertebra that is in front of the spinal canal and is distinguished from the vertebral arch” (Stedman Medical Dictionary, 5th edition, Medical View) or “the half that occupies the front of the vertebra "Circular part" (Ojirin, second edition, Sanseido). Therefore, a vertebral body fracture does not include a fracture at the vertebral arch where spinous processes, transverse processes, joint processes, etc. are present, among vertebrae constituting the spine, and has a semi-cylindrical shape. A fracture in the body part.
Vertebral fractures may be caused by strong impacts such as accidents or sports, but they are compression fractures of the vertebral body that occur mainly due to increased bone vulnerability and decreased bone strength due to osteoporosis. Yes, it is one of the typical diseases that greatly reduce the patient's QOL (Non-patent Documents 2 and 3). When the vulnerability of the bone increases, a slight external force that usually does not cause a fracture and a weak stress that is about the daily life action are repeatedly applied, resulting in damage to the fine structure of the bone and continuous fracture of the bone structure. With the increase in the number of osteoporosis patients, the incidence of vertebral body compression-type fractures has increased, and the prevalence of osteoporotic vertebral fractures in Japan is 7.6-14% in women in their 60s, 70 It is reported that it is 37-45% in the age group (nonpatent literature 4, 5, 6).
Vertebral fracture patients have been found to have a poorer prognosis than fracture patients at other sites. For example, according to the data obtained from the Fracture Intervention Trial (FIT), the risk of mortality after the occurrence of a clinical fracture is increased approximately 2-fold, which is 6.7-fold increase in proximal femoral fractures and vertebral bodies The number of fractures increased by 8.6 times (Non-patent Document 7).
Vertebral fractures vary in clinical symptoms, from those without subjective symptoms after injury to those with acute back and back pain. There are no drugs for treating vertebral fractures, and no aggressive drug treatment is available for painful vertebral fractures. Therefore, it is common to treat the patient conservatively for several months by performing external fixation with a resting bed for several weeks while administering an analgesic, and using a device such as a cast, hard or soft corset. At this time, there are many patients who need temporary hospitalization treatment, and the average hospitalization period is reported to be 42.6 days (Non-patent Document 8). In particular, when the fusion of the fracture site is delayed, pseudo-joint and stubborn chronic pain occurs, vertebral body deformation progresses, causing lower limb pain and delayed nerve paralysis, and spinal deformity causes digestive system, respiratory The patient's ADL and QOL are significantly reduced due to the functional disorder of the system.
As a treatment method for severe vertebral body fracture, there is spinal column reconstruction by interbody fusion using instrumentation in combination with preservation therapy. Vertebroplasty such as injecting bone cement into fractured vertebral bodies that spread rapidly mainly in the United States since the late 1990s, and kyphoplasty to inflate the balloon to restore the height of the fractured vertebral body and correct kyphosis deformity Vertebral augmentation surgery has been penetrating in Japan (Non-patent Document 9). However, even if external fixation is performed, it is often impossible to maintain good spinal alignment, leaving wedge-shaped deformation of the vertebral body and deformation of the posterior spine. Furthermore, in spite of conservative therapy, the current situation is that the onset of vertebral body collapse, pseudo-joint, and delayed neuropathy cannot be completely prevented (Non-patent Document 10).
Development of new drugs to treat vertebral fractures is necessary to increase QOL and improve life prognosis of vertebral fracture patients. The development of new drugs to treat vertebral fractures is a challenge that should be actively addressed not only for the benefit of individual patients, but also for the social benefit of reducing inpatients and bedridden patients and reducing medical costs.
One of the major obstacles in developing a new drug for treating vertebral fractures is that there is no animal model that can be used non-clinically, and the effect of the drug cannot be verified in non-human animals. There are several reports of general fracture models using animals, but these models focus on the damage to the central part of the long bone shaft, that is, cortical bone, and the fine structure of cancellous bone that occurs in vertebral fractures. It is not a model for assessing damage. For example, a rat femoral closed fracture model, which is a general fracture model, is a model in which guillotine is dropped at the center of the femoral shaft and the femur is fractured to evaluate the strength of the femur (Non-patent Document 11). However, the damage is a cortical bone fracture that is far from the microstructural damage of cancellous bone that occurs in vertebral fractures. Similarly to the present invention, the rat femur drilling model in which bone damage is caused by drilling with a dental drill is the same as the femoral closed fracture model, the central part of the femoral shaft, that is, the femoral cortical bone. It is a damaged model (Non-Patent Documents 12 and 13), and is not a model that can evaluate the damage to the fine structure of cancellous bone caused by vertebral fractures.
In the process of fracture healing, it is known that not only bone formation by osteoblasts but also bone destruction by osteoclasts proceeds. Unlike substances that suppress osteoclast function such as bisphosphonates, PTH and PTHrP are known to activate osteoclasts. In fact, PTH and the like have a function of releasing calcium into the blood by the activity of osteoclasts from bones, and there have been times when it was conventionally considered as a bone destruction factor causing hypercalcemia. Currently, it is used as a therapeutic agent based on the bone mass increasing action in osteoporosis because of its action to increase bone metabolism, while it is also expected to be used for fracture treatment. Therefore, effects on fracture healing such as PTH have been studied in various animal models (Non-patent Document 14). However, the models used there are fixed bones that are not loaded in an undesired direction by inserting a pin after a fracture has occurred in a long bone (for example, the femur), or flat bones and unweighted bones. It is only the thing which saw the effect in (head skeleton etc.). On the other hand, the vertebral body is the main part of the spine that supports the trunk, and is a bone that is always subjected to a certain amount of weight. Moreover, it is very difficult to fix the fracture site with a pin or the like due to the structure of the vertebral body both clinically and experimentally. Therefore, when PTH, PTHrP, or an analog thereof is administered for the purpose of treating vertebral fractures, if the bone resorption during the fracture healing process is increased more than expected, the vulnerability near the fracture site may temporarily increase. It is also conceivable that the weight is applied to the fracture, and the fracture is worsened, and in some cases, the fracture is fractured, leading to a situation that cannot be repaired structurally or functionally.
Therefore, when a substance that is a drug candidate for vertebral fractures is screened using a long bone fracture model that has been reported in the past, it is possible to find the optimal substance for femoral fractures. It was impossible to find the best material to treat the resulting cancellous bone microstructure damage. That is, since there was no method for evaluating vertebral fractures using non-human animals, it was difficult to develop drugs for treating vertebral fractures.
The healing process of a fracture is generally divided into an inflammation phase, a repair phase, and a remodeling phase (Non-patent Document 15). In the inflammatory phase, a hematoma is formed at the fracture site, and there are inflammatory cells, from which many factors such as inflammatory cytokines are released. Looking at the structure of the spine, the spinal canal close to the vertebral body is characterized by the passage of the spinal cord, which is important for the control of body movements and sensations, and physiological functions. The disease in which the spinal cord is inflamed for some reason is called myelitis, and its symptoms are abnormal sensory and motor functions when mild, but paralysis of the entire controlled area when severe. Or even death. On the other hand, PTHrP has been shown to be involved in inflammatory responses in several diseases (Non-patent Documents 16-20). In addition, there are many mast cells in the bones of patients with hyperparathyroidism, PTH is involved in the accumulation of mast cells, and PTHrP is considered to have the same effect (non- Patent Document 21), PTH and PTHrP are also known to act on osteoblasts to induce the expression of CXCL1, which is a cell migration factor for neutrophils (Non-patent Document 22). Therefore, when PTH / PTHrP or analogs thereof are administered for the purpose of treating vertebral fractures, it is possible that the inflammation of the fracture site may become more serious, and the inflammation spreads to the spinal cord existing nearby. Possible risk of failure.
In the study of vertebral body fracture treatment, as described above, there is no useful animal model of vertebral body fracture, and the possibility of serious side effects has been considered.
The partial active polypeptide of PTH has been shown to promote the repair of cortical bone fracture using the above-described fracture model of the long bone shaft (Patent Document 1, Non-Patent Documents 23 and 24). However, there is no report that clearly detects the therapeutic effect on cancellous bone microstructure damage caused by vertebral fractures. In addition, there is a report showing the possibility that the compound shown in Patent Document 2 can be a therapeutic agent for vertebral fractures (Patent Document 2), but this report examined the prevention effect of vertebral fractures in clinical trials. It is not just a clear detection of the therapeutic effect of the drug on vertebral fractures, but only a vague speculation of the therapeutic effect on vertebral fractures. That is, as in the present invention, there is no report that specifically shows damage to the fine structure of cancellous bone, which is a feature of vertebral fractures, and clearly shows the therapeutic effect of the drug on vertebral fractures only by the damaged part.

特表２００２−５０９８５４Special table 2002-509854 特開２００６−２１９４８５JP 2006-219485

日医大医学誌　２００９．５（２）；１２５−１２９、骨粗鬆症性脊椎椎体骨折、元文　芳和Nihon Medical University Journal, 2009.5 (2); 125-129, osteoporotic vertebral body fracture, original text Yoshikazu Ｂｏｎｅ．１９９６　Ｍａｒ；１８（３　Ｓｕｐｐｌ）：１８５Ｓ−１８９Ｓ．，Ｔｈｅ　ｃｌｉｎｉｃａｌ　ｉｍｐａｃｔ　ｏｆ　ｖｅｒｔｅｂｒａｌ　ｆｒａｃｔｕｒｅｓ：ｑｕａｌｉｔｙ　ｏｆ　ｌｉｆｅ　ｉｎ　ｗｏｍｅｎ　ｗｉｔｈ　ｏｓｔｅｏｐｏｒｏｓｉｓ．，Ｇｏｌｄ　ＤＴ．Bone. 1996 Mar; 18 (3 Suppl): 185S-189S. , The clinical impact of vertebral fractures: quality of life in women with osteoposis. , Gold DT. Ｊ　Ｂｏｎｅ　Ｍｉｎｅｒ　Ｒｅｓ．１９９７　Ａｐｒ；１２（４）：６６３−７５．，Ｃｌｉｎｉｃａｌ　ｇｒａｄｉｎｇ　ｏｆ　ｓｐｉｎａｌ　ｏｓｔｅｏｐｏｒｏｓｉｓ：ｑｕａｌｉｔｙ　ｏｆ　ｌｉｆｅ　ｃｏｍｐｏｎｅｎｔｓ　ａｎｄ　ｓｐｉｎａｌ　ｄｅｆｏｒｍｉｔｙ　ｉｎ　ｗｏｍｅｎ　ｗｉｔｈ　ｃｈｒｏｎｉｃ　ｌｏｗ　ｂａｃｋ　ｐａｉｎ　ａｎｄ　ｗｏｍｅｎ　ｗｉｔｈ　ｖｅｒｔｅｂｒａｌ　ｏｓｔｅｏｐｏｒｏｓｉｓ．，Ｌｅｉｄｉｇ−Ｂｒｕｃｋｎｅｒ　Ｇ，Ｍｉｎｎｅ　ＨＷ，Ｓｃｈｌａｉｃｈ　Ｃ，Ｗａｇｎｅｒ　Ｇ，Ｓｃｈｅｉｄｔ−Ｎａｖｅ　Ｃ，Ｂｒｕｃｋｎｅｒ　Ｔ，Ｇｅｂｅｓｔ　ＨＪ，Ｚｉｅｇｌｅｒ　Ｒ．J Bone Miner Res. 1997 Apr; 12 (4): 663-75. , Clinical grading of spinal osteoporossis: quality of life components and spinal deformity in women with pine, low and low. , Leidig-Bruckner G, Minne HW, Schlaich C, Wagner G, Scheidt-Nave C, Brucker T, Gebest HJ, Ziegler R. 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Ｃａｎｃｅｒ．２００５　Ｍａｙ　１；１０３（９）：１８１０−８．，Ｓｅｒｕｍ　ｐａｒａｔｈｙｒｏｉｄ　ｈｏｒｍｏｎｅ−ｒｅｌａｔｅｄ　ｐｅｐｔｉｄｅ　ｉｓ　ａｓｓｏｃｉａｔｅｄ　ｗｉｔｈ　ｓｙｓｔｅｍｉｃ　ｉｎｆｌａｍｍａｔｉｏｎ　ａｎｄ　ａｄｖｅｒｓｅ　ｐｒｏｇｎｏｓｉｓ　ｉｎ　ｇａｓｔｒｏｅｓｏｐｈａｇｅａｌ　ｃａｒｃｉｎｏｍａ．，Ｄｅａｎｓ　Ｃ，Ｗｉｇｍｏｒｅ　Ｓ，Ｐａｔｅｒｓｏｎ−Ｂｒｏｗｎ　Ｓ，Ｂｌａｃｋ　Ｊ，Ｒｏｓｓ　Ｊ，Ｆｅａｒｏｎ　ＫＣ．Cancer. 2005 May 1; 103 (9): 1810-8. , Serum parathyrido harmonie-related peptide associated is associated with inflation and advertisement promotion in gastroesophageal carcinoma. , Deans C, Wigmore S, Paterson-Brown S, Black J, Ross J, Fearon KC. Ｅｕｒ　Ｒｅｖ　Ｍｅｄ　Ｐｈａｒｍａｃｏｌ　Ｓｃｉ．２０１０　Ｍａｙ；１４（５）：４７１−５．，Ｅｘｐｒｅｓｓｉｏｎ　ｏｆ　ｐａｒａｔｈｙｒｏｉｄ　ｈｏｒｍｏｎｅ−ｒｅｌａｔｅｄ　ｐｒｏｔｅｉｎ　ｉｎ　ｈｕｍａｎ　ｉｎｆｌａｍｅｄ　ｄｅｎｔａｌ　ｐｕｌｐ．，Ｍａｒｉｇｏ　Ｌ，Ｍｉｇｌｉａｃｃｉｏ　Ｓ，Ｍｏｎｅｇｏ　Ｇ，Ｌａ　Ｔｏｒｒｅ　Ｇ，Ｓｏｍｍａ　Ｆ，Ｒａｎｅｌｌｅｔｔｉ　ＦＯ．Eur Rev Med Pharmacol Sci. 2010 May; 14 (5): 471-5. , Expression of parathyrido harmonie-related protein in human inflamed dental pull. , Marigo L, Migliaccio S, Monego G, La Torre G, Soma F, Ranelletti FO. Ｊ　Ｂｏｎｅ　Ｍｉｎｅｒ　Ｒｅｓ．２０１０　Ｊｕｌ；２５（７）：１６３７−４９．，Ｔｈｅ　ｒｏｌｅ　ｏｆ　ｍａｓｔ　ｃｅｌｌｓ　ｉｎ　ｐａｒａｔｈｙｒｏｉｄ　ｂｏｎｅ　ｄｉｓｅａｓｅ．，Ｔｕｒｎｅｒ　ＲＴ，Ｉｗａｎｉｅｃ　ＵＴ，Ｍａｒｌｅｙ　Ｋ，Ｓｉｂｏｎｇａ　ＪＤ．J Bone Miner Res. 2010 Jul; 25 (7): 1637-49. , The role of must cells in parathyroid bone disease. , Turner RT, Iwaniec UT, Marley K, Sibonga JD. ｅｎｄｏｃｒｉｎｏｌｏｇｙ．２００９　Ｍａｙ；１５０（５）：２２４４−５３．Ｅｐｕｂ　２００９　Ｊａｎ　１５．，Ｔｈｅ　ｃｈｅｍｏｋｉｎｅ　Ｃｘｃｌｌ　ｉｓ　ａ　ｎｏｖｅｌ　ｔａｒｇｅｔ　ｇｅｎｅ　ｏｆ　ｐａｒａｔｈｙｒｏｉｄ　ｈｏｒｍｏｎｅ（ＰＴＨ）／ＰＴＨ−ｒｅｌａｔｅｄ　ｐｒｏｔｅｉｎ　ｉｎ　ｃｏｍｍｉｔｔｅｄ　ｏｓｔｅｏｂｌａｓｔｓ．，Ｏｎａｎ　Ｄ，Ａｌｌａｎ　ＥＨ，Ｑｕｉｎｎ　ＪＭ，Ｇｏｏｉ　ＪＨ，Ｐｏｍｐｏｌｏ　Ｓ，Ｓｉｍｓ　ＮＡ，Ｇｉｌｌｅｓｐｉｅ　ＭＴ，Ｍａｒｔｉｎ　ＴＪ．endocrinology. 2009 May; 150 (5): 2244-53. Epub 2009 Jan 15. , The chemokine Cxclli is a novel target gene of parathyroid hormone (PTH) / PTH-related protein in committed osteoblasts. , Onan D, Allan EH, Quinn JM, Gooi JH, Pompolo S, Sims NA, Gillespie MT, Martin TJ. Ｊ　Ｂｏｎｅ　Ｍｉｎｅｒ　Ｒｅｓ．２００２　Ｎｏｖ；１７（１１）：２０３８−４７．，Ｍｅｃｈａｎｉｓｍｓ　ｆｏｒ　ｔｈｅ　ｅｎｈａｎｃｅｍｅｎｔ　ｏｆ　ｆｒａｃｔｕｒｅ　ｈｅａｌｉｎｇ　ｉｎ　ｒａｔｓ　ｔｒｅａｔｅｄ　ｗｉｔｈ　ｉｎｔｅｒｍｉｔｔｅｎｔ　ｌｏｗ−ｄｏｓｅ　ｈｕｍａｎ　ｐａｒａｔｈｙｒｏｉｄ　ｈｏｒｍｏｎｅ（１−３４）．，Ｎａｋａｊｉｍａ　Ａ，Ｓｈｉｍｏｊｉ　Ｎ，Ｓｈｉｏｍｉ　Ｋ，Ｓｈｉｍｉｚｕ　Ｓ，Ｍｏｒｉｙａ　Ｈ，Ｅｉｎｈｏｒｎ　ＴＡ，Ｙａｍａｚａｋｉ　Ｍ．J Bone Miner Res. 2002 Nov; 17 (11): 2038-47. , Mechanisms for the enhancement of fracture healing in rats treated with intermittent low-dose human parathyrido horne (1-34). , Nakajima A, Shimaji N, Shiomi K, Shimizu S, Moriya H, Einhorn TA, Yamazaki M. Ｊ　Ｂｏｎｅ　Ｊｏｉｎｔ　Ｓｕｒｇ　Ａｍ．２００５　Ａｐｒ；８７（４）：７３１−４１．，Ｅｎｈａｎｃｅｍｅｎｔ　ｏｆ　ｅｘｐｅｒｉｍｅｎｔａｌ　ｆｒａｃｔｕｒｅ−ｈｅａｌｉｎｇ　ｂｙ　ｓｙｓｔｅｍｉｃ　ａｄｍｉｎｉｓｔｒａｉｏｎ　ｏｆ　ｒｅｃｏｍｂｉｎａｎｔ　ｈｕｍａｎ　ｐａｒａｔｈｙｒｏｉｄ　ｈｏｒｍｏｎｅ（ＰＴＨ　１−３４）．，Ａｌｋｈｉａｒｙ　ＹＭ，Ｇｅｒｓｔｅｎｆｅｌｄ　ＬＣ，Ｋｒａｌｌ　Ｅ，Ｗｅｓｔｍｏｒｅ　Ｍ，Ｓａｔｏ　Ｍ，Ｍｉｔｌａｋ　ＢＨ，Ｅｉｎｈｏｒｎ　ＴＡ．J Bone Joint Surg Am. 2005 Apr; 87 (4): 731-41. , Enhancement of experimental fracture-healing by system administration of recombinant human parathyroid (PTH 1-34). , Alkhiary YM, Gerstenfeld LC, Kroll E, Westmore M, Sato M, Mitlak BH, Einhorn TA.

　骨粗鬆症性椎体骨折では、骨の脆弱性が亢進すると、通常骨折を生じない程度の軽微な外力、日常生活動作程度の弱いストレスが繰り返し加わることによって、海綿骨の微細構造の損傷が生じ、骨構造が連続的に破断する。しかし、椎体骨折で生じる海綿骨の微細構造の損傷を評価する動物モデルはなく、椎体骨折を治療する物質を非ヒト動物を用いてスクリーニングすることは不可能であった。椎体骨折で生じる海綿骨の微細構造の損傷を評価する動物モデルがこれまで報告されなかったのは、小動物の健康状態に影響を与えず、椎体を損傷させ、その損傷度を評価することが困難であったためである。
　椎体骨折で生じる海綿骨の微細構造の損傷を評価する動物モデルが存在すれば、そのモデルを用いて、椎体骨折を治療する物質をスクリーニングすることができる。すなわち、椎体骨折で生じる海綿骨の微細構造の損傷に対し、治療効果を有する新しい薬剤を開発する有用な動物モデルとなる。これまで椎体骨折を治療するための薬剤はなかったが、椎体骨折で生じる海綿骨の微細構造の損傷を評価する動物モデルを構築できれば、椎体骨折を治療する新しい薬剤の開発が可能となる。また、ＰＴＨ・ＰＴＨｒＰ及びそのアナログによる椎体骨折治療剤を開発する上で、当該モデルを用いることによって、これらの物質の投与によって懸念される炎症反応の増悪や過度の骨吸収などの副作用につながる可能性がないことを確認することも可能になる。
　そこで、本発明者らは、薬剤の開発に当たって汎用性の高い動物であるラットを用い、椎体骨折で生じる海綿骨の微細構造の損傷を生じさせる動物モデルを構築し、その損傷修復過程を評価する評価方法を決定し、さらに、その動物モデルと評価方法を用いて、スクリーニングを行い、椎体骨折で生じる海綿骨の微細構造の損傷を治療できる物質を見出し、椎体骨折を治療する薬剤として開発することを可能とした。
　本発明の目的は、ＰＴＨ／ＰＴＨｒＰ受容体アゴニスト、又はＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することによって、椎体骨折治療剤、椎体骨折に起因する疼痛の治療又は予防剤、椎体圧迫骨折予防剤、又は椎体骨折に起因する運動障害及び／又は神経障害の治療又は予防剤を提供することである。
　また、本発明の目的は、１）非ヒト動物の椎体骨に外科的な損傷を与える工程、２）当該非ヒト動物に被検物質を投与する工程、及び３）当該非ヒト動物又はそれに由来する検体の椎体骨の外科的な損傷の回復を測定する工程、を含むことによって、椎体骨折治療剤の評価方法を提供することである。 In osteoporotic vertebral fractures, when the vulnerability of the bone increases, a slight external force that does not normally cause a fracture and a weak stress that causes daily activities are repeatedly applied, resulting in damage to the fine structure of the cancellous bone. The structure breaks continuously. However, there is no animal model for evaluating cancellous bone microstructure damage caused by vertebral fractures, and it has been impossible to screen non-human animals for substances that treat vertebral fractures. No animal model to evaluate cancellous bone microstructural damage caused by vertebral fractures has been reported so far, without affecting the health of small animals and to assess the degree of damage to the vertebral body. This is because it was difficult.
If there is an animal model that evaluates cancellous bone microstructure damage caused by vertebral fractures, the model can be used to screen for substances that treat vertebral fractures. That is, it becomes a useful animal model for developing a new drug having a therapeutic effect against the damage of the cancellous bone microstructure caused by vertebral fracture. Until now, there was no drug for treating vertebral fractures, but if an animal model that can evaluate the damage to the cancellous bone microstructure caused by vertebral fractures could be developed, it would be possible to develop new drugs to treat vertebral fractures Become. Moreover, when developing a therapeutic agent for vertebral fractures using PTH / PTHrP and analogs thereof, use of the model leads to side effects such as exacerbation of inflammatory reaction and excessive bone resorption that are concerned by administration of these substances. It is also possible to confirm that there is no possibility.
Therefore, the present inventors used a rat, which is a highly versatile animal in drug development, to construct an animal model that causes damage to the fine structure of cancellous bone caused by vertebral fractures, and to evaluate the damage repair process As a drug for treating vertebral body fractures, further, screening is performed using the animal model and the evaluation method, and a substance capable of treating damage to the fine structure of cancellous bone caused by vertebral body fractures is found. Made it possible to develop.
An object of the present invention is to provide a therapeutic agent for vertebral fracture, a therapeutic or preventive agent for pain caused by vertebral fracture, by containing a PTH / PTHrP receptor agonist, or a substance having an activity to induce PTH or PTHrP production, The object is to provide a preventive agent for vertebral body compression fracture, or a therapeutic or preventive agent for movement disorders and / or neurological disorders caused by vertebral body fractures.
The object of the present invention is also 1) a step of surgically damaging the vertebral bones of a non-human animal, 2) a step of administering a test substance to the non-human animal, and 3) the non-human animal or the same. Measuring the recovery of surgical damage to the vertebral bone of the specimen from which it is derived, thereby providing a method for evaluating a therapeutic agent for vertebral fractures.

　本発明は、以下に示すものである。
（１）ＰＴＨ／ＰＴＨｒＰ受容体アゴニストを含有することを特徴とする、椎体骨折治療剤。
（２）ＰＴＨ／ＰＴＨｒＰ受容体アゴニストが、副甲状腺ホルモン（ＰＴＨ）、副甲状腺ホルモン関連ペプチド（ＰＴＨｒＰ）、又はそれらの部分活性ポリペプチド、若しくはそれらのアナログである、（１）に記載の椎体骨折治療剤。
（３）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドが、ＰＴＨ（１−８４）、ＰＴＨ（１−３７）、ＰＴＨ（１−３４）、ＰＴＨ（１−３１）、ＰＴＨｒＰ（１−１４１）、ＰＴＨｒＰ（１−１３９）、ＰＴＨｒＰ（１−８６）、ＰＴＨｒＰ（１−４０）、ＰＴＨｒＰ（１−３７）、ＰＴＨｒＰ（１−３６）、及びＰＴＨｒＰ（１−３４）のいずれかである、（２）に記載の椎体骨折治療剤。
（４）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドが、ヒトＰＴＨ又はヒトＰＴＨｒＰに由来するアミノ酸配列を有するポリペプチドである、（２）又は（３）に記載の椎体骨折治療剤。
（５）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドにおけるアミノ酸配列のうち、１~１０個のアミノ酸を別のアミノ酸に置換したポリペプチドである、（２）に記載の椎体骨折治療剤。
（６）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、ＰＴＨ（１−８４）、ＰＴＨ（１−３７）、ＰＴＨ（１−３４）、ＰＴＨ（１−３１）、ＰＴＨｒＰ（１−１４１）、ＰＴＨｒＰ（１−１３９）、ＰＴＨｒＰ（１−８６）、ＰＴＨｒＰ（１−４０）、ＰＴＨｒＰ（１−３７）、ＰＴＨｒＰ（１−３６）、及びＰＴＨｒＰ（１−３４）のいずれかのアミノ酸配列のうち、１~１０個のアミノ酸を別のアミノ酸に置換したポリペプチドである、（５）に記載の椎体骨折治療剤。
（７）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、ヒトＰＴＨ又はヒトＰＴＨｒＰに由来するアミノ酸配列のうち、１~１０個のアミノ酸を別のアミノ酸に置換したポリペプチドである、（５）又は（６）に記載の椎体骨折治療剤。
（８）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチド、若しくはそれらのアナログが、製薬上許容される塩である、（１）~（７）のいずれかに記載の椎体骨折治療剤。
（９）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが下記式のポリペプチド：

Figure JPOXMLDOC01-appb-C000004
ただし、
Ａ１はＳｅｒ、Ａｌａ、又はＤａｐであり；
Ａ３はＳｅｒ、Ｔｈｒ、又はＡｉｂであり；
Ａ５はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ７はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ８はＭｅｔ、Ｎｖａ、Ｌｅｕ、Ｖａｌ、Ｉｌｅ、Ｃｈａ、又はＮｌｅであり；
Ａ１１はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ１２はＧｌｙ又はＡｉｂであり；
Ａ１５はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ１６はＳｅｒ、Ａｓｎ、Ａｌａ、又はＡｉｂであり；
Ａ１７はＳｅｒ、Ｔｈｒ、又はＡｉｂであり；
Ａ１８はＭｅｔ、Ｎｖａ、Ｌｅｕ、Ｖａｌ、Ｉｌｅ、Ｎｌｅ、Ｃｈａ、又はＡｉｂであり；
Ａ１９はＧｌｕ又はＡｉｂであり；
Ａ２１はＶａｌ、Ｃｈａ、又はＭｅｔであり；
Ａ２３はＴｒｐ又はＣｈａであり；
Ａ２４はＬｅｕ又はＣｈａであり；
Ａ２７はＬｙｓ、Ａｉｂ、Ｌｅｕ、ｈＡｒｇ、Ｇｌｎ、又はＣｈａであり；
Ａ２８はＬｅｕ又はＣｈａであり；
Ａ３０はＡｓｐ又はＬｙｓであり；
Ａ３１はＶａｌ、Ｎｌｅ、若しくはＣｈａである、又は欠損しており；
Ａ３２はＨｉｓである又は欠損しており；
Ａ３３はＡｓｎである又は欠損しており；
Ａ３４はＰｈｅ、Ｔｙｒ、Ａｍｐ、若しくはＡｉｂである、又は欠損しており；
Ｒ１及びＲ２は、それぞれ独立して、Ｈ、Ｃ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；又はＲ１及びＲ２の両方又は一方のみがＣＯＥ１であり、この際、Ｅ１はＣ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；及び
Ｒ３はＯＨ、ＮＨ２、Ｃ１−１２アルコキシ、又はＮＨ−Ｙ−ＣＨ２−Ｚであり、この際、ＹはＣ１−１２炭化水素部分であり、ＺはＨ、ＯＨ、ＣＯ２Ｈ、又はＣＯＮＨ２であり；
Ａ５、Ａ７、Ａ８、Ａ１１、Ａ１５、Ａ１８、Ａ２１、Ａ２３、Ａ２４、Ａ２７、Ａ２８及びＡ３１の少なくとも一つはＣｈａである、又はＡ３、Ａ１２、Ａ１６、Ａ１７、Ａ１８、Ａ１９及びＡ３４の少なくとも一つはＡｉｂである；
又はこれらの製薬上許容できる塩である、（１）又は（２）に記載の椎体骨折治療剤。
（１０）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、下記式のポリペプチド：
［Ｃｈａ７，１１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２３］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｎｌｅ８，１８，Ｃｈａ２７］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２８］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ３１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１６］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１９］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２４，２８，３１，Ｌｙｓ３０］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，Ａｉｂ１６，１９，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，３１，Ａｉｂ１６，１９，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ１１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２８，３１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，Ａｉｂ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ１５］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ａｉｂ１９］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ａｉｂ１６］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１６，１９］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１２］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ３］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ａｉｂ１９，Ｌｙｓ３０］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２４，２８，３１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１７］ｈＰＴＨ（１−３４）ＮＨ２；及び
［Ｃｈａ７，１１，１５］ｈＰＴＨ（１−３４）ＮＨ２、
からなる群より選択されるペプチド、又はこれらの製薬上許容できる塩である、（９）に
記載の椎体骨折治療剤。
（１１）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが下記式のポリペプチド：
Figure JPOXMLDOC01-appb-C000005
 ただし、
Ａ１はＡｌａ、Ｓｅｒ、又はＤａｐであり；
Ａ３はＳｅｒ又はＡｉｂであり；
Ａ５はＨｉｓ、Ｉｌｅ、又はＣｈａであり；
Ａ７はＬｅｕ、Ｃｈａ、Ｎｌｅ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ８はＬｅｕ、Ｍｅｔ、又はＣｈａであり；
Ａ１０はＡｓｐ又はＡｓｎであり；
Ａ１１はＬｙｓ、Ｌｅｕ、Ｃｈａ、Ｐｈｅ、又はβ−Ｎａｌであり；
Ａ１２はＧｌｙ又はＡｉｂであり；
Ａ１４はＳｅｒ又はＨｉｓであり；
Ａ１５はＩｌｅ、又はＣｈａであり；
Ａ１６はＧｌｎ又はＡｉｂであり；
Ａ１７はＡｓｐ又はＡｉｂであり；
Ａ１８はＬｅｕ、Ａｉｂ、又はＣｈａであり；
Ａ１９はＡｒｇ又はＡｉｂであり；
Ａ２２はＰｈｅ、Ｇｌｕ、Ａｉｂ、又はＣｈａであり；
Ａ２３はＰｈｅ、Ｌｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２４はＬｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２５はＨｉｓ、Ａｉｂ、又はＧｌｕであり；
Ａ２６はＨｉｓ、Ａｉｂ、又はＬｙｓであり；
Ａ２７はＬｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２８はＩｌｅ、Ｌｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２９はＡｌａ、Ｇｌｕ、又はＡｉｂであり；
Ａ３０はＧｌｕ、Ｃｈａ、Ａｉｂ、又はＬｙｓであり；
Ａ３１はＩｌｅ、Ｌｅｕ、Ｃｈａ、若しくはＬｙｓである、又は欠損しており；
Ａ３２はＨｉｓである又は欠損しており；
Ａ３３はＴｈｒである又は欠損しており；
Ａ３４はＡｌａである又は欠損しており；
Ｒ１及びＲ２は、それぞれ独立して、Ｈ、Ｃ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；又はＲ１及びＲ２の一方及び一方のみがＣＯＥ１であり、この際、Ｅ１はＣ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０、ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；及び
Ｒ３はＯＨ、ＮＨ２、Ｃ１−１２アルコキシ、又はＮＨ−Ｙ−ＣＨ２−Ｚであり、この際、ＹはＣ１−１２炭化水素部分であり、ＺはＨ、ＯＨ、ＣＯ２Ｈ、又はＣＯＮＨ２であり；
Ａ５、Ａ７、Ａ８、Ａ１１、Ａ１５、Ａ１８、Ａ２２、Ａ２３、Ａ２４、Ａ２７、Ａ２８、Ａ３０、若しくはＡ３１の少なくともひとつはＣｈａである、又はＡ３、Ａ１２、Ａ１６、Ａ１７、Ａ１８、Ａ１９、Ａ２２、Ａ２５、Ａ２６、Ａ２９、Ａ３０、若しくはＡ３４の少なくともひとつはＡｉｂである；
又はこれらの製薬上許容できる塩である、（１）又は（２）に記載の椎体骨折治療剤。
（１２）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、式：［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，３１，Ａｉｂ２９，Ｌｙｓ２６，３０］ｈＰＴＨｒＰ（１−３４）ＮＨ２のポリペプチド又はその製薬上許容できる塩である、（１１）に記載の椎体骨折治療剤。
（１３）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが下記式のポリペプチド：
Figure JPOXMLDOC01-appb-C000006
 ただし、
Ａ１はＡｌａ、Ｓｅｒ、又はＤａｐであり；Ａ３はＳｅｒ又はＡｉｂであり；
Ａ５はＨｉｓ、Ｉｌｅ、Ａｃｃ、又はＣｈａであり；
Ａ７はＬｅｕ、Ｃｈａ、Ｎｌｅ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ａｃｃ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ８はＬｅｕ、Ｍｅｔ、Ａｃｃ、又はＣｈａであり；
Ａ１０はＡｓｐ又はＡｓｎであり；
Ａ１１はＬｙｓ、Ｌｅｕ、Ｃｈａ、Ａｃｃ、Ｐｈｅ、又はβ−Ｎａｌであり；
Ａ１２はＧｌｙ、Ａｃｃ、又はＡｉｂであり；
Ａ１４はＳｅｒ又はＨｉｓであり；
Ａ１５はＩｌｅ、Ａｃｃ、又はＣｈａであり；
Ａ１６はＧｌｎ又はＡｉｂであり；
Ａ１７はＡｓｐ又はＡｉｂであり；
Ａ１８はＬｅｕ、Ａｉｂ、Ａｃｃ、又はＣｈａであり；
Ａ１９はＡｒｇ又はＡｉｂであり；
Ａ２２はＰｈｅ、Ｇｌｕ、Ａｉｂ、Ａｃｃ、又はＣｈａであり；
Ａ２３はＰｈｅ、Ｌｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２４はＬｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２５はＨｉｓ、Ｌｙｓ、Ａｉｂ、Ａｃｃ、又はＧｌｕであり；
Ａ２６はＨｉｓ、Ａｉｂ、Ａｃｃ、又はＬｙｓであり；
Ａ２７はＬｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２８はＩｌｅ、Ｌｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２９はＡｌａ、Ｇｌｕ、Ａｃｃ、又はＡｉｂであり；
Ａ３０はＧｌｕ、Ｌｅｕ、Ｎｌｅ、Ｃｈａ、Ａｉｂ、Ａｃｃ、又はＬｙｓであり；
Ａ３１はＩｌｅ、Ｌｅｕ、Ｃｈａ、Ｌｙｓ若しくはＡｃｃである、又は欠損しており；
Ａ３２はＨｉｓである又は欠損しており；
Ａ３３はＴｈｒである又は欠損しており；
Ａ３４はＡｌａである又は欠損しており；
Ｒ１及びＲ２は、それぞれ独立して、Ｈ、Ｃ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；又はＲ１及びＲ２の一方及び一方のみがＣＯＥ１であり、この際、Ｅ１はＣ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；及び
Ｒ３はＯＨ、ＮＨ２、Ｃ１−１２アルコキシ、又はＮＨ−Ｙ−ＣＨ２−Ｚであり、この際、ＹはＣ１−１２炭化水素部分であり、ＺはＨ、ＯＨ、ＣＯ２Ｈ、又はＣＯＮＨ２であり；
Ａ５、Ａ７、Ａ８、Ａ１１、Ａ１２、Ａ１５、Ａ１８、Ａ２２、Ａ２３、Ａ２４、Ａ２５、Ａ２６、Ａ２７、Ａ２８、Ａ２９、Ａ３０、又はＡ３１の少なくとも一つがＡｃｃである；
又はこれらの製薬上許容できる塩である、（１）又は（２）に記載の椎体骨折治療剤。
（１４）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、下記式のポリペプチド：
［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，Ｌｙｓ２６，３０，Ａｉｂ２９，Ａｈｃ３１］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ａｈｃ２３，Ｌｙｓ２６，３０，Ｌｅｕ２８，３１，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，３１，Ｌｙｓ２６，３０，Ａｈｃ２７，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，２９，Ｌｅｕ２３，２８，３１，Ｌｙｓ２６，Ａｈｃ３０］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｃｈａ２２，Ｌｅｕ２３，２８，３１，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｈｃ２７，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，３１，Ａｈｃ２４，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２９，Ｌｅｕ２３，２８，３１，Ａｉｂ２５，Ｌｙｓ２６，３０，Ａｈｃ２７］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，Ｌｅｕ２３，２８，３１，Ａｉｂ２５，２９，Ｌｙｓ２６，３０，Ａｈｃ２７］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ２２，Ｌｅｕ２３，２８，３１，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｌｅｕ２３，３１，Ｌｙｓ２６，３０，Ａｈｃ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｃｈａ２２，Ａｈｃ２３，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ｌｅｕ２８，３１，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｃｈａ２２，Ｌｅｕ２３，２８，３１，Ａｈｃ２４，２７，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，Ｌｅｕ２３，２８，３１，Ａｈｃ２４，２７，Ｌｙｓ２５，２６，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ１８，２４，２７，Ｇｌｕ２２，Ｃｈａ２３，Ｌｙｓ２５，２６，Ｌｅｕ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，Ｃｈａ２３，Ａｈｃ２４，Ｌｙｓ２５，２６，Ｌｅｕ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ２２，２４，Ｌｅｕ２３，２８，３１，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ２２，２４，Ｌｅｕ２３，２８，Ｌｙｓ２５，２６，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
又はこれらの製薬上許容できる塩である、（１３）に記載の椎体骨折治療剤。
（１５）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、下記式のポリペプチド：
［Ｎｌｅ３１］ｈＰＴＨ（１−３４）ＮＨ２；
［ｈＡｒｇ２７］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｄａｐ１，Ｎｌｅ８，１８，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
又はこれらの製薬上許容できる塩である、（１）又は（２）に記載の椎体骨折治療剤。
（１６）ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、下記式の
ポリペプチド：
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，Ｌｅｕ２８，３１，Ａｉｂ２９，Ｎｌｅ３０］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，３０，Ｌｅｕ２８，３１，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，３０，Ｌｅｕ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｌｅｕ２７，Ａｉｂ２９］ｈＰＴＨ（１−３４）ＮＨ２；
又はこれらの製薬上許容できる塩である、（１）又は（２）に記載の椎体骨折治療剤。
（１７）ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することを特徴とする、椎体骨折治療剤。
（１８）ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質がカルシウム感受性受容体アンタゴニストである、（１７）に記載の椎体骨折治療剤。
（１９）椎体骨折が海綿骨における骨折である、（１）~（１８）のいずれかに記載の椎体骨折治療剤。
（２０）（１）~（１６）のいずれかに記載の、ＰＴＨ、ＰＴＨｒＰ、若しくはそれらの部分活性ポリペプチド、又はそれらのアナログ、若しくは（１７）又は（１８）に記載のＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することを特徴とする、椎体骨折に起因する疼痛の治療又は予防剤。
（２１）（１）~（１６）のいずれかに記載の、ＰＴＨ、ＰＴＨｒＰ、若しくはそれらの部分活性ポリペプチド、又はそれらのアナログ、若しくは（１７）又は（１８）に記載のＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することを特徴とする、椎体圧迫骨折予防剤。
（２２）（１）~（１６）のいずれかに記載の、ＰＴＨ、ＰＴＨｒＰ、若しくはそれらの部分活性ポリペプチド、又はそれらのアナログ、若しくは（１７）又は（１８）に記載のＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することを特徴とする、椎体骨折に起因する運動障害及び／又は神経障害の治療又は予防剤。
（２３）椎体骨折治療剤の評価方法であって、１）非ヒト動物の椎体骨に外科的な損傷を与える工程、２）当該非ヒト動物に被検物質を投与する工程、及び３）当該非ヒト動物又はそれに由来する検体の椎体骨の外科的な損傷の回復を測定する工程、を含むことを特徴とする、評価方法。
（２４）椎体骨の外科的な損傷の回復の測定を行った結果を、さらに正常非ヒト動物若しくは対象非ヒト動物、又はそれに由来する検体における測定の結果と比較する工程を含む、（２３）に記載の評価方法。
（２５）椎体骨の外科的な損傷が、椎体骨の皮質骨を通り海綿骨まで達する損傷であることを特徴とする、（２３）又は（２４）に記載の評価方法。
（２６）椎体骨の外科的な損傷が、ドリルによる穿孔による損傷であることを特徴とする、（２３）~（２５）のいずれかに記載の評価方法。
（２７）椎体骨の外科的な損傷の回復の測定を、検体の組織学的観察、又はＸ線ＣＴを用いた形態観察により行う、（２３）~（２６）のいずれかに記載の評価方法。
（２８）椎体骨の外科的な損傷の回復の測定を、痛覚又は運動機能の測定により行う、（２３）~（２６）のいずれかに記載の評価方法。
（２９）椎体骨の外科的な損傷の回復の計測を、検体の組織化学的解析、骨分化発生マーカーのタンパク質又は遺伝子の発現の解析により行う、（２３）~（２６）のいずれかに記載の評価方法。
（３０）非ヒト動物が、骨粗鬆症のモデル動物である、（２３）~（２９）のいずれかに記載の評価方法。
（３１）骨粗鬆症のモデル動物が、卵巣摘出モデルである、（３０）に記載の評価方法。
（３２）非ヒト動物が、霊長類又は齧歯類の非ヒト動物である、（２３）~（３１）のいずれかに記載の評価方法。
（３３）齧歯類の非ヒト動物が、ラットである、（３２）に記載の評価方法。
（３４）椎体骨が、腰椎の椎体骨である、（２３）~（３３）のいずれかに記載の評価方法。
（３５）腰椎の椎体骨が、第４腰椎及び／又は第５腰椎である、（３４）に記載の評価方法。
（３６）被検物質の投与を、経口又は非経口により行う、（２３）~（３５）のいずれかに記載の評価方法。 The present invention is as follows.
(1) A vertebral fracture therapeutic agent comprising a PTH / PTHrP receptor agonist.
(2) The vertebral body according to (1), wherein the PTH / PTHrP receptor agonist is parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrP), or a partially active polypeptide thereof, or an analog thereof. Fracture treatment agent.
(3) PTH, PTHrP or a partially active polypeptide thereof is PTH (1-84), PTH (1-37), PTH (1-34), PTH (1-31), PTHrP (1-141), Any one of PTHrP (1-139), PTHrP (1-86), PTHrP (1-40), PTHrP (1-37), PTHrP (1-36), and PTHrP (1-34), (2 The therapeutic agent for vertebral fractures according to (1).
(4) The therapeutic agent for vertebral fracture according to (2) or (3), wherein PTH, PTHrP or a partially active polypeptide thereof is a polypeptide having an amino acid sequence derived from human PTH or human PTHrP.
(5) An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide in which 1 to 10 amino acids in the amino acid sequence of PTH, PTHrP or a partially active polypeptide thereof are substituted with another amino acid. The therapeutic agent for vertebral fracture according to (2).
(6) PTH, PTHrP, or an analog of a partially active polypeptide thereof is PTH (1-84), PTH (1-37), PTH (1-34), PTH (1-31), PTHrP (1-141). ), PTHrP (1-139), PTHrP (1-86), PTHrP (1-40), PTHrP (1-37), PTHrP (1-36), and PTHrP (1-34) The therapeutic agent for vertebral fracture according to (5), which is a polypeptide obtained by substituting 1 to 10 amino acids with another amino acid.
(7) An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide in which 1 to 10 amino acids are substituted with another amino acid in the amino acid sequence derived from human PTH or human PTHrP. ) Or vertebral body fracture treatment agent according to (6).
(8) The therapeutic agent for vertebral fracture according to any one of (1) to (7), wherein PTH, PTHrP, or a partially active polypeptide thereof, or an analog thereof is a pharmaceutically acceptable salt.
(9) An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula:
Figure JPOXMLDOC01-appb-C000004
 However,
A 1 is Ser, Ala, or Dap;
A 3 is Ser, Thr, or Aib;
A 5 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 7 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 8 is Met, Nva, Leu, Val, Ile, Cha, or Nle;
A 11 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 12 is Gly or Aib;
A 15 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 16 is Ser, Asn, Ala, or Aib;
A 17 is Ser, Thr, or Aib;
A 18 is Met, Nva, Leu, Val, Ile, Nle, Cha, or Aib;
A 19 is Glu or Aib;
A 21 is Val, Cha, or Met;
A 23 is Trp or Cha;
A 24 is Leu or Cha;
A 27 is Lys, Aib, Leu, hArg, Gln, or Cha;
A 28 is Leu or Cha;
A 30 is Asp or Lys;
A 31 is Val, Nle, or Cha, or is missing;
A 32 is His or missing;
A 33 is Asn or is missing;
A 34 is Phe, Tyr, Amp, or Aib, or is missing;
R 1 and R 2 are each independently H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12. Hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7-20 hydroxyphenylalkyl, or C 11-20 hydroxynaphthylalkyl; or both R 1 and R 2 or only one is COE 1 , wherein E 1 is C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12 hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7 -20 hydroxyphenyl alkyl, or with C 11-20 hydroxy naphthyl alkyl Ri; and R 3 is OH, NH 2, C 1-12 alkoxy, or NH-Y-CH 2 -Z, this time, Y is C 1-12 hydrocarbon moiety, Z is H, OH, CO 2 H or CONH 2 ;
At least one of A 5 , A 7 , A 8 , A 11 , A 15 , A 18 , A 21 , A 23 , A 24 , A 27 , A 28 and A 31 is Cha, or A 3 , A 12 , A 16 , A 17 , A 18 , A 19 and A 34 are Aib;
Alternatively, the therapeutic agent for vertebral fracture according to (1) or (2), which is a pharmaceutically acceptable salt thereof.
(10) An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula:
[Cha 7,11 ] hPTH (1-34) NH 2 ;
[Cha 23 ] hPTH (1-34) NH 2 ;
[Cha 24 ] hPTH (1-34) NH 2 ;
[Nle 8,18 , Cha 27 ] hPTH (1-34) NH 2 ;
[Cha 28 ] hPTH (1-34) NH 2 ;
[Cha 31 ] hPTH (1-34) NH 2 ;
[Aib 16 ] hPTH (1-34) NH 2 ;
[Aib 19 ] hPTH (1-34) NH 2 ;
[Aib 34 ] hPTH (1-34) NH 2 ;
[Cha 24 , 28 , 31 , Lys 30 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Nle 8,18 , Tyr 34 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Nle 8,18 , Aib 16,19 , Tyr 34 ] hPTH (1-34) NH 2 ;
[Cha 7,11, Nle 8,18,31, Aib 16,19, Tyr 34] hPTH (1-34) NH 2;
[Cha 11 ] hPTH (1-34) NH 2 ;
[Cha 28,31 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Nle 8,18 , Aib 34 ] hPTH (1-34) NH 2 ;
[Cha 15 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Aib 19 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Aib 16 ] hPTH (1-34) NH 2 ;
[Aib 16,19 ] hPTH (1-34) NH 2 ;
[Aib 12 ] hPTH (1-34) NH 2 ;
[Aib 3 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Aib 19 , Lys 30 ] hPTH (1-34) NH 2 ;
[Cha 7 ] hPTH (1-34) NH 2 ;
[Cha 24, 28, 31 ] hPTH (1-34) NH 2 ;
[Aib 17 ] hPTH (1-34) NH 2 ; and [Cha 7,11,15 ] hPTH (1-34) NH 2 ,
The therapeutic agent for vertebral fracture according to (9), which is a peptide selected from the group consisting of: or a pharmaceutically acceptable salt thereof.
(11) An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula:
Figure JPOXMLDOC01-appb-C000005
 However,
A 1 is Ala, Ser, or Dap;
A 3 is Ser or Aib;
A 5 is His, Ile, or Cha;
A 7 is Leu, Cha, Nle, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 8 is Leu, Met, or Cha;
A 10 is Asp or Asn;
A 11 is Lys, Leu, Cha, Phe, or β-Nal;
A 12 is Gly or Aib;
A 14 is Ser or His;
A 15 is Ile or Cha;
A 16 is Gln or Aib;
A 17 is Asp or Aib;
A 18 is Leu, Aib, or Cha;
A 19 is Arg or Aib;
A 22 is Phe, Glu, Aib, or Cha;
A 23 is Phe, Leu, Lys, or Cha;
A 24 is Leu, Lys, or Cha;
A 25 is His, Aib, or Glu;
A 26 is His, Aib, or Lys;
A 27 is Leu, Lys, or Cha;
A 28 is Ile, Leu, Lys, or Cha;
A 29 is Ala, Glu, or Aib;
A 30 is Glu, Cha, Aib, or Lys;
A 31 is Ile, Leu, Cha, or Lys, or is missing;
A 32 is His or missing;
A 33 is Thr or missing;
A 34 is Ala or missing;
R 1 and R 2 are each independently H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12. Hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7-20 hydroxyphenylalkyl, or C 11-20 hydroxynaphthylalkyl; or only one and one of R 1 and R 2 is COE 1 , wherein E 1 is C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20, naphthylalkyl, C 1-12 hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7-20 hydroxyphenylalkyl, or C 11-20 hydroxy naphthyl alkyl There; and R 3 is OH, an NH 2, C 1-12 alkoxy, or NH-Y-CH 2 -Z, this time, Y is C 1-12 hydrocarbon moiety, Z is H, OH, CO 2 H or CONH 2 ;
At least one of A 5 , A 7 , A 8 , A 11 , A 15 , A 18 , A 22 , A 23 , A 24 , A 27 , A 28 , A 30 , or A 31 is Cha, or A 3 , A 12 , A 16 , A 17 , A 18 , A 19 , A 22 , A 25 , A 26 , A 29 , A 30 , or A 34 is Aib;
Alternatively, the therapeutic agent for vertebral fracture according to (1) or (2), which is a pharmaceutically acceptable salt thereof.
(12) PTH, analog of PTHrP or their partial active polypeptide, wherein: [Glu 22,25, Leu 23,28,31, Aib 29, Lys 26,30] poly hPTHrP (1-34) NH 2 The therapeutic agent for vertebral fracture according to (11), which is a peptide or a pharmaceutically acceptable salt thereof.
(13) An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula:
Figure JPOXMLDOC01-appb-C000006
 However,
A 1 is Ala, Ser, or Dap; A 3 is Ser or Aib;
A 5 represents His, Ile, Acc, or be Cha;
A 7 is Leu, Cha, Nle, β-Nal, Trp, Pal, Acc, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 8 is Leu, Met, Acc, or Cha;
A 10 is Asp or Asn;
A 11 is Lys, Leu, Cha, Acc, Phe, or β-Nal;
A 12 is Gly, Acc, or Aib;
A 14 is Ser or His;
A 15 is Ile, Acc, or Cha;
A 16 is Gln or Aib;
A 17 is Asp or Aib;
A 18 is Leu, Aib, Acc, or Cha;
A 19 is Arg or Aib;
A 22 is Phe, Glu, Aib, Acc, or Cha;
A 23 is Phe, Leu, Lys, Acc, or Cha;
A 24 is Leu, Lys, Acc, or Cha;
A 25 is His, Lys, Aib, Acc, or Glu;
A 26 is His, Aib, Acc, or Lys;
A 27 is Leu, Lys, Acc, or Cha;
A 28 is Ile, Leu, Lys, Acc, or Cha;
A 29 is Ala, Glu, Acc, or Aib;
A 30 is Glu, Leu, Nle, Cha, Aib, Acc, or Lys;
A 31 is Ile, Leu, Cha, Lys or Acc, or is missing;
A 32 is His or missing;
A 33 is Thr or missing;
A 34 is Ala or missing;
R 1 and R 2 are each independently H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12. Hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7-20 hydroxyphenylalkyl, or C 11-20 hydroxynaphthylalkyl; or only one and one of R 1 and R 2 is COE 1 , wherein E 1 is C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12 hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7 -20 hydroxyphenyl alkyl, or with C 11-20 hydroxy naphthyl alkyl Ri; and R 3 is OH, NH 2, C 1-12 alkoxy, or NH-Y-CH 2 -Z, this time, Y is C 1-12 hydrocarbon moiety, Z is H, OH, CO 2 H or CONH 2 ;
A 5, A 7, A 8 , A 11, A 12, A 15, A 18, A 22, A 23, A 24, A 25, A 26, A 27, A 28, A 29, A 30, or A At least one of 31 is Acc;
Alternatively, the therapeutic agent for vertebral fracture according to (1) or (2), which is a pharmaceutically acceptable salt thereof.
(14) An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula:
[Glu 22 , 25 , Leu 23 , 28 , Lys 26 , 30 , Aib 29 , Ahc 31 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , Ahc 23 , Lys 26 , 30 , Leu 28 , 31 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , Leu 23 , 28 , 31 , Lys 26 , 30, Ahc 27 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , 29 , Leu 23 , 28 , 31 , Lys 26 , Ahc 30 ] hPTHrP (1-34) NH 2 ;
[Cha 22 , Leu 23 , 28 , 31 , Glu 25 , Lys 26 , 30, Ahc 27 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , Leu 23 , 28, 31 , Ahc 24 , Lys 26 , 30 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22,29 , Leu 23,28,31 , Aib 25 , Lys 26,30 , Ahc 27 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , Leu 23 , 28 , 31 , Aib 25 , 29 , Lys 26 , 30, Ahc 27 ] hPTHrP (1-34) NH 2 ;
[Ahc 22 , Leu 23 , 28 , 31 , Glu 25 , Lys 26 , 30 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , Leu 23 , 31 , Lys 26 , 30, Ahc 28 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Cha 22, Ahc 23, Glu 25, Lys 26,30, Leu 28,31, Aib 29] hPTHrP (1-34) NH 2;
[Cha 22 , Leu 23 , 28, 31 , Ahc 24 , 27 , Glu 25 , Lys 26 , 30 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , Leu 23 , 28, 31 , Ahc 24 , 27 , Lys 25 , 26 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Ahc 18 , 24, 27 , Glu 22 , Cha 23 , Lys 25 , 26 , Leu 28 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , Cha 23 , Ahc 24 , Lys 25 , 26 , Leu 28 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Ahc 22 , 24 , Leu 23 , 28 , 31 , Glu 25 , Lys 26 , 30 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Ahc 22 , 24 , Leu 23 , 28 , Lys 25 , 26 , Aib 29 ] hPTHrP (1-34) NH 2 ;
Alternatively, the therapeutic agent for vertebral fracture according to (13), which is a pharmaceutically acceptable salt thereof.
(15) An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula:
[Nle 31 ] hPTH (1-34) NH 2 ;
[HArg 27 ] hPTH (1-34) NH 2 ;
[Dap 1 , Nle 8 , 18 , Tyr 34 ] hPTH (1-34) NH 2 ;
Alternatively, the therapeutic agent for vertebral fracture according to (1) or (2), which is a pharmaceutically acceptable salt thereof.
(16) An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the following formula:
[Glu 22 , 25 , Cha 23 , Lys 26 , Leu 28 , 31 , Aib 29 , Nle 30 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , Cha 23 , Lys 26 , 30 , Leu 28 , 31 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22,25, Cha 23, Lys 26,30, Aib 29] hPTHrP (1-34) NH 2;
[Glu 22 , 25 , Cha 23 , Lys 26 , 30 , Leu 28 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Leu 27 , Aib 29 ] hPTH (1-34) NH 2 ;
Alternatively, the therapeutic agent for vertebral fracture according to (1) or (2), which is a pharmaceutically acceptable salt thereof.
(17) A therapeutic agent for vertebral fracture, comprising a substance having an activity of inducing PTH or PTHrP production.
(18) The therapeutic agent for vertebral fracture according to (17), wherein the substance having an activity of inducing PTH or PTHrP production is a calcium sensitive receptor antagonist.
(19) The therapeutic agent for vertebral fracture according to any one of (1) to (18), wherein the vertebral fracture is a fracture in cancellous bone.
(20) PTH, PTHrP, or a partially active polypeptide thereof, or an analog thereof according to any one of (1) to (16), or PTH or PTHrP production according to (17) or (18) A therapeutic or preventive agent for pain caused by vertebral fractures, comprising a substance having an inducing activity.
(21) PTH, PTHrP, or a partially active polypeptide thereof according to any one of (1) to (16), or an analog thereof, or PTH or PTHrP production according to (17) or (18) An agent for preventing vertebral body compression fracture, comprising a substance having an inducing activity.
(22) PTH, PTHrP, or a partially active polypeptide thereof, or an analog thereof according to any one of (1) to (16), or PTH or PTHrP production according to (17) or (18) A therapeutic or prophylactic agent for a movement disorder and / or a neurological disorder caused by a vertebral fracture, comprising a substance having an inducing activity.
(23) A method for evaluating a therapeutic agent for vertebral fracture, wherein 1) a step of surgically damaging a vertebral bone of a non-human animal, 2) a step of administering a test substance to the non-human animal, and 3 ) Measuring the recovery from surgical damage to the vertebral bones of the non-human animal or the specimen derived therefrom.
(24) The method further includes a step of comparing the result of the measurement of the recovery of the surgical damage to the vertebral bone with the result of the measurement in the normal non-human animal or the target non-human animal or a specimen derived therefrom. ) Evaluation method.
(25) The evaluation method according to (23) or (24), wherein the surgical damage to the vertebral bone is a damage that reaches the cancellous bone through the cortical bone of the vertebral bone.
(26) The evaluation method according to any one of (23) to (25), wherein the surgical damage to the vertebral bone is damage caused by drilling with a drill.
(27) The evaluation according to any one of (23) to (26), wherein measurement of recovery from surgical damage to the vertebral bone is performed by histological observation of a specimen or morphological observation using X-ray CT. Method.
(28) The evaluation method according to any one of (23) to (26), wherein measurement of recovery from surgical damage to vertebral bones is performed by measurement of pain sensation or motor function.
(29) Measurement of recovery from surgical damage to vertebral bones is performed by histochemical analysis of a specimen, or analysis of expression of a protein or gene of a bone differentiation occurrence marker, (23) to (26) The evaluation method described.
(30) The evaluation method according to any one of (23) to (29), wherein the non-human animal is a model animal for osteoporosis.
(31) The evaluation method according to (30), wherein the model animal for osteoporosis is an ovariectomy model.
(32) The evaluation method according to any one of (23) to (31), wherein the non-human animal is a primate or rodent non-human animal.
(33) The evaluation method according to (32), wherein the rodent non-human animal is a rat.
(34) The evaluation method according to any one of (23) to (33), wherein the vertebral bone is a lumbar vertebral bone.
(35) The evaluation method according to (34), wherein the vertebral body bones of the lumbar vertebra are the fourth lumbar vertebra and / or the fifth lumbar vertebra.
(36) The evaluation method according to any one of (23) to (35), wherein the test substance is administered orally or parenterally.

　本発明は、ＰＴＨ／ＰＴＨｒＰ受容体アゴニスト、又はＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することによって、椎体骨折治療剤、椎体骨折に起因する疼痛の治療又は予防剤、椎体圧迫骨折予防剤、又は椎体骨折に起因する運動障害及び／又は神経障害の治療又は予防剤を提供することを可能とする。
　また、本発明は、１）非ヒト動物の椎体骨に外科的な損傷を与える工程、２）当該非ヒト動物に被検物質を投与する工程、及び３）当該非ヒト動物又はそれに由来する検体の椎体骨の外科的な損傷の回復を測定する工程、を含むことによって、椎体骨折治療剤の評価方法を提供することを可能とする。 The present invention relates to a therapeutic agent for vertebral fracture, a therapeutic or preventive agent for pain caused by vertebral fracture, by containing a PTH / PTHrP receptor agonist, or a substance having an activity of inducing PTH or PTHrP production, It makes it possible to provide a preventive agent for compression fractures, or a therapeutic or preventive agent for movement disorders and / or neurological disorders caused by vertebral fractures.
The present invention also includes 1) a surgical damage to the vertebral bones of a non-human animal, 2) a step of administering a test substance to the non-human animal, and 3) the non-human animal or derived therefrom. It is possible to provide a method for evaluating a therapeutic agent for vertebral fractures by including the step of measuring the recovery of the surgical damage to the vertebral bone of the specimen.

　図１は、損傷椎体のｍｉｃｒｏ　ＣＴ画像（モデル作製日（ｄａｙ０）での３Ｄ画像と２Ｄ画像の代表例）である。
　図２は、自然修復過程におけるマッソン・トリクローム染色像である。
　図３は、自然修復過程における海綿骨の損傷部の骨体積密度を示すグラフである。
　図４は、ｈＰＴＨ（１−３４）又はＰＴＨｒＰアナログＡを７日間又は１４日間投与したときのマッソン・トリクローム染色像である。
　図５は、ｈＰＴＨ（１−３４）又はＰＴＨｒＰアナログＡを７日間又は１４日間投与したときの損傷椎体の海綿骨の損傷部における骨体積密度を示すグラフである。
　図６は、ｈＰＴＨ（１−３４）又はＰＴＨｒＰアナログＡを７日間又は１４日間投与したときの非損傷椎体の海綿骨における骨体積密度を示すグラフである。
　図７は、偽手術を行ったラットと腰椎椎体骨損傷モデルのラットの経時的体重変化を示したグラフ（Ａ）、及び椎椎体骨損傷モデルのラットにおいてｈＰＴＨ（１−３４）及びＰＴＨｒＰアナログＡを投与した群とＶｅｈｉｃｌｅを投与した群でのラットの経時的体重変化を示したグラフ（Ｂ）である。 FIG. 1 is a micro CT image of a damaged vertebral body (a representative example of a 3D image and a 2D image on the model creation date (day 0)).
FIG. 2 is a Masson trichrome stained image in the natural restoration process.
FIG. 3 is a graph showing the bone volume density of the damaged portion of cancellous bone during the natural repair process.
FIG. 4 is a Masson trichrome stained image when hPTH (1-34) or PTHrP analog A is administered for 7 days or 14 days.
FIG. 5 is a graph showing the bone volume density in the damaged part of the cancellous bone of the damaged vertebral body when hPTH (1-34) or PTHrP analog A is administered for 7 days or 14 days.
FIG. 6 is a graph showing bone volume density in cancellous bone of an uninjured vertebral body when hPTH (1-34) or PTHrP analog A is administered for 7 days or 14 days.
FIG. 7 is a graph (A) showing the change in body weight over time of a sham-operated rat and a lumbar vertebral body bone injury model rat, and hPTH (1-34) and PTHrP in a vertebral body bone injury model rat. It is the graph (B) which showed the time-dependent body weight change of the rat in the group which administered the analog A, and the group which administered Vehicle.

　本発明者らは、椎体骨折で生じる海綿骨の微細構造の損傷を生じさせる動物モデルとその評価方法を確立した。椎体骨折で生じる海綿骨の微細構造の損傷を生じさせる動物モデルとその評価方法が確立できたことで、椎体骨折で生じる海綿骨の微細構造の損傷を治療できる物質をスクリーニングすることが可能となった。その結果、ｈＰＴＨ（１−３４）とＰＴＨｒＰアナログＡ（配列番号４２）は、非損傷椎体では薬効が検出されない投与量にもかかわらず、損傷椎体では、椎体骨折で生じる海綿骨の微細構造の損傷を治療する効果を発揮した。すなわち、ｈＰＴＨ（１−３４）とＰＴＨｒＰアナログＡは、椎体骨折で生じる海綿骨の微細構造の損傷に対し、特異的に治療効果を示す物質であることを見出した。従って、ＰＴＨ／ＰＴＨｒＰ受容体アゴニスト、ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質は、椎体骨折で生じる海綿骨の微細構造の損傷を治療できる薬剤となることを見出した。
　また、ＰＴＨ／ＰＴＨｒＰ受容体アゴニスト、ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質は、海綿骨の微細構造の損傷を治療できるため、骨構造が連続的に破断し、椎体が圧潰していく椎体骨折の進行を止められることを見出した。
　さらに、ＰＴＨ／ＰＴＨｒＰ受容体アゴニスト、ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質は、椎体骨折に起因する運動障害及び／又は神経障害の治療又は予防剤となることを見出した。
　ＰＴＨ／ＰＴＨｒＰ受容体アゴニスト、ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質は、椎体骨折患者のＱＯＬを高め生命予後を改善する、画期的な薬剤となる。新しい椎体骨折を治療するこれらの薬剤は、患者個人の利益のみならず、入院患者や寝たきり患者を減らし医療費を削減することができ、社会的利益にも大きく貢献できる。
［ＰＴＨ／ＰＴＨｒＰ受容体アゴニスト］
　ＰＴＨ／ＰＴＨｒＰ受容体はＰＴＨ１Ｒ、ＰＴＨ１又はＰＰＲとも呼ばれるＧタンパク質共役型受容体の１種であり、副甲状腺ホルモン（ＰＴＨ）及び副甲状腺ホルモン関連ペプチド（ＰＴＨｒＰ）の両者と結合し細胞内にホルモンのシグナルを伝達する働きをしている。つまり、ＰＴＨ及びＰＴＨｒＰによる共通の機能の発揮には、このＰＴＨ／ＰＴＨｒＰ受容体の働きが関与していると考えられる。特にＰＴＨ及びＰＴＨｒＰのカルシウム代謝における作用においては、このＰＴＨ／ＰＴＨｒＰ受容体が重要な役割を演じている。従って、本発明における椎体骨折治療剤はＰＴＨ／ＰＴＨｒＰ受容体を活性化する物質、つまりＰＴＨ／ＰＴＨｒＰ受容体アゴニストを含有することを特徴とする。
　ＰＴＨ／ＰＴＨｒＰ受容体アゴニストの代表としては、後述のＰＴＨ、ＰＴＨｒＰ、又はそれらの部分ポリペプチド、若しくはそのアナログが挙げられるが、それに限定されず、ＰＴＨ／ＰＴＨｒＰ受容体に結合して細胞内にシグナルを発生させる物質であれば本発明の椎体骨折治療剤に用いることができる。そのようなＰＴＨ、ＰＴＨｒＰ、又はそれらの部分ポリペプチド、若しくはそのアナログ以外のＰＴＨ／ＰＴＨｒＰ受容体アゴニストの例としては、ＰＴＨ／ＰＴＨｒＰ受容体に対するアゴニスト抗体、ＰＴＨ又はＰＴＨｒＰの構造や性状を模倣する形でデザインされたＰＴＨ又はＰＴＨｒＰのミミック分子、またＰＴＨ／ＰＴＨｒＰ受容体の活性化を指標にスクリーニングすることによって得られた低分子化合物などが挙げられる。
　ＰＴＨ／ＰＴＨｒＰ受容体の活性化とは、細胞に発現したＰＴＨ／ＰＴＨｒＰ受容体への結合や細胞内のシグナルを測定することによって判断をすることができる。ＰＴＨ／ＰＴＨｒＰ受容体へのアゴニストの結合の測定は、被検物質に直接放射性同位元素、蛍光物質、発光物質、その他検出用の物質（ビオチン−アビジン系に関連する物質、タグ配列など）で標識を行い、ＰＴＨ／ＰＴＨｒＰ受容体を発現した細胞と反応させてその結合を検出される標識の量を調べることで測定することができる。また、別の方法としてはＰＴＨ、ＰＴＨｒＰ、又はそれらの部分ポリペプチド、若しくはそのアナログやその他びＰＴＨ／ＰＴＨｒＰ受容体の結合する物質の中から標準物質を選定し、その標準物質を直接放射性同位元素、蛍光物質、発光物質、その他検出用の物質（ビオチン−アビジン系に関連する物質、タグ配列など）で標識を行い、被検物質と共存させた形でＰＴＨ／ＰＴＨｒＰ受容体を発現した細胞と反応させて、標準物質のＰＴＨ／ＰＴＨｒＰ受容体への結合が被検物質の存在によって競合的に阻害されたかについてを標準物質の標識の量を調べることによって測定することが可能である。また、ＰＴＨ／ＰＴＨｒＰ受容体へのアゴニストの結合は、ＰＴＨ／ＰＴＨｒＰ受容体を発現した細胞を用いる方法の他、当該細胞から調製された細胞膜を用いることで、当該細胞を用いるのと同様の方法で測定することが可能である。ＰＴＨ／ＰＴＨｒＰ受容体アゴニストの結合による細胞内シグナル発生の測定については、被検物質をＰＴＨ／ＰＴＨｒＰ受容体を発現した細胞と反応させて、細胞内のｃＡＭＰ（環状アデノシン一リン酸）の濃度上昇を調べることによって測定することができる。細胞内のｃＡＭＰ濃度の測定は、当業者であれば適宜既存技術の情報を用いて実施することができ、また市販のキットなども用いることも可能であるが、例えば特表平１１−５０９２０１に記載の方法などでも実施が可能である。また、ＰＴＨ／ＰＴＨｒＰ受容体アゴニストの結合による細胞内シグナル発生を測定する方法としては、被検物質をＰＴＨ／ＰＴＨｒＰ受容体を発現した細胞と反応させて、細胞内のカルシウムイオンの濃度上昇を調べることによって測定することができる。細胞内のカルシウムイオンの濃度上昇は、Ｆｕｒａ−２ＡＭ・Ｆｕｒａ−４ＡＭなどのカルシウム感受性蛍光物質やエクオリンなどのカルシウム感受性発光物質などを用いることで、当業者であれば適宜既存技術の情報を用いて実施することが可能である。また、ＰＴＨ／ＰＴＨｒＰ受容体アゴニストの結合による細胞内シグナル発生を測定する別の方法としては、細胞内のフォスホリパーゼＡ２（ＰＬＡ２）の活性亢進を調べることによって測定することができる。ＰＬＡ２の活性亢進を調べる方法としては、当業者であれば適宜既存技術の情報を用いて実施することが可能であるが、例えばＣａｙｍａｎ　Ｃｈｅｍｉｃａｌ社のｃＰｈｏｓｐｈｏｌｉｐａｓｅ　Ａ２　Ｃｏｌｏｒｉｍｅｔｒｉｃ　Ａｓｓａｙ　Ｋｉｔなどの市販のキットやＭｏｌｅｃｕｌａｒ　Ｐｒｏｂｅｓ社のＲｅｄ／Ｇｒｅｅｎ　ＢＯＤＩＰＹ（Ｒ）　ＰＣ−Ａ２などの市販の試薬などを用いることで調べることが可能である。
　ＰＴＨ／ＰＴＨｒＰ受容体への結合や細胞内のシグナルを測定に用いるＰＴＨ／ＰＴＨｒＰ受容体発現細胞とは、ＰＴＨ／ＰＴＨｒＰ受容体を内在的に発現している細胞又はＰＴＨ／ＰＴＨｒＰを人為的に発現した細胞のいずれでも用いることができる。ＰＴＨ／ＰＴＨｒＰ受容体を内在的に発現している細胞とは、ヒトや非ヒト動物における生体内でＰＴＨ／ＰＴＨｒＰ受容体を発現している組織や細胞から得られた細胞や細胞株であり、ＰＴＨ／ＰＴＨｒＰ受容体を発現している組織や細胞としては例えば骨における骨芽細胞・骨細胞や破骨細胞、腎臓における腎臓の遠位尿細管や集合管の細胞、子宮や小腸における平滑筋・腺組織や上皮の細胞、その他卵巣や肝臓の細胞などから得られた細胞をいう（ＩＵＰＨＡＲ　ＤＡＴＡＢＡＳＥ／ｈｔｔｐ：／／ｗｗｗ．ｉｕｐｈａｒ−ｄｂ．ｏｒｇ／ｉｎｄｅｘ．ｊｓｐ／のＰＨＴ１の説明より）。また、ＰＴＨ／ＰＴＨｒＰ受容体を内在的に発現している細胞株の例としては、例えば骨芽細胞系の細胞株があり、より具体的にはヒト骨肉腫細胞株ＳａＯＳ−２細胞・ラット骨芽細胞様細胞であるＲＯＳ１７／２．８細胞等を挙げることができる。ＰＴＨ／ＰＴＨｒＰ受容体を人為的に発現した細胞とは、ＰＴＨ／ＰＴＨｒＰ受容体タンパク質をコードする遺伝子を遺伝子工学的手法によって宿主細胞の導入し、当該ＰＴＨ／ＰＴＨｒＰ受容体タンパク質を発現させた細胞をいう。ＰＴＨ／ＰＴＨｒＰ受容体タンパク質とは、例えば配列番号１にアミノ酸配列が示されているヒトＰＴＨ／ＰＴＨｒＰ受容体タンパク質が挙げられる。このヒトＰＴＨ／ＰＴＨｒＰ受容体タンパク質をコードする遺伝子が導入された宿主細胞では、配列番号１のアミノ酸配列のタンパク質として翻訳された後、シグナル配列であるＮ末端から２８個までのアミノ酸が切り取られ、２９番目以降のアミノ酸配列からなる成熟配列のヒトＰＴＨ／ＰＴＨｒＰ受容体タンパク質が細胞表面に発現される。またヒトＰＴＨ／ＰＴＨｒＰ受容体タンパク質以外にも、配列番号２にアミノ酸配列が示されているチンパンジー（Ｐａｎ　ｔｒｏｇｌｏｄｙｔｅｓ）のＰＴＨ／ＰＴＨｒＰ受容体、配列番号３にアミノ酸配列が示されているアカゲザル（Ｍａｃａｃａ　ｍｕｌａｔｔａ）のＰＴＨ／ＰＴＨｒＰ受容体、配列番号４にアミノ酸配列が示されているオランウータン（Ｐｏｎｇｏ　ａｂｅｌｉｉ）のＰＴＨ／ＰＴＨｒＰ受容体、配列番号５にアミノ酸配列が示されているブタ（Ｓｕｓ　ｓｃｒｏｆａ）のＰＴＨ／ＰＴＨｒＰ受容体、配列番号６にアミノ酸配列が示されているウマ（Ｅｑｕｕｓ　ｃａｂａｌｌｕｓ）のＰＴＨ／ＰＴＨｒＰ受容体、配列番号７にアミノ酸配列が示されているウシ（Ｂｏｓ　ｔａｕｒｕｓ）のＰＴＨ／ＰＴＨｒＰ受容体、配列番号８にアミノ酸配列が示されているコモンマーモセット（Ｃａｌｌｉｔｈｒｉｘ　ｊａｃｃｈｕｓ）のＰＴＨ／ＰＴＨｒＰ受容体、配列番号９に示されているイヌ（Ｃａｎｉｓ　ｌｕｐｕｓ　ｆａｍｉｌｉａｒｉｓ）のＰＴＨ／ＰＴＨｒＰ受容体、配列番号１０に示されているラット（Ｒａｔｔｕｓ　ｎｏｒｖｅｇｉｃｕｓ）のＰＴＨ／ＰＴＨｒＰ受容体、配列番号１１に示されているマウス（Ｍｕｓ　ｍｕｓｃｕｌｕｓ）のＰＴＨ／ＰＴＨｒＰ受容体、配列番号１２に示されているハイイロジネズミオポッサム（Ｍｏｎｏｄｅｌｐｈｉｓ＿ｄｏｍｅｓｔｉｃａ）のＰＴＨ／ＰＴＨｒＰ受容体等のタンパク質を発現させた細胞も用いることができる。また、ヒトのＰＴＨ／ＰＴｈｒＰ受容体とハイイロジネズミオポッサムのＰＴＨ／ＰＴＨｒＰの間でのホモロジーは、ＮＣＢＩのＢＬＡＳＴｐ法での両者を並列的に並べたところ、保存的なアミノ酸置換を許さない相同性（Ｉｄｅｎｔｉｔｉｅｓ）で８０％、保存的アミノ酸への置換を許容する相同性（Ｐｏｓｉｔｉｖｅｓ）では８６％のホモロジーを示す。そのことから、本発明のＰＴＨ／ＰＴＨｒＰ受容体アゴニストの結合や細胞内シグナル発生の測定に用いることのできるＰＴＨ／ＰＴＨｒＰ受容体には、ヒトＰＴＨ／ＰＴＨｒＰ受容体のアミノ酸配列と８０％以上のホモロジーを有し、ＰＴＨ／ＰＴＨｒＰ受容体の機能を保持している上述以外のＰＴＨ・ＰＴＨｒＰ受容体も含まれる。なお、オポッサムのＰＴＨ／ＰＴＨｒＰ受容体にはヒトなどのＰＴＨ／ＰＴＨｒＰが結合／作用することは公知である（Ｊｕｐｐｎｅｒ　Ｈ．ｅｔ　ａｌ．Ｅｎｄｏｃｒｉｎｏｌｏｇｙ　１３４　ｐｐ８７９　１９９４）。当該ＰＴＨ・ＰＴＨｒＰ受容体がＰＴＨ／ＰＴＨｒＰ受容体の機能を保持していることを調べるためには、ＰＴＨ・ＰＴＨｒＰ及びその活性部分ポリペプチド若しくはそのアナログなどの既知のＰＴＨ／ＰＴＨｒＰ受容体アゴニストとしての活性を有する物質の結合や細胞内シグナル発生を調べることで確認することができる。遺伝子工学的にＰＴＨ／ＰＴＨｒＰ受容体を発現させるための宿主としては、動物細胞（ヒト及び非ヒト動物に由来するもの）・昆虫細胞・酵母細胞・バクテリア又はウイルス／バクテリオファージなどの中から適宜選択して用いることができるが、好ましくは動物細胞であり、より好ましくは動物細胞から樹立された動物細胞株である。当該動物細胞株の一例としては、Ｃｏｓ−７細胞株、ＬＬＣ−ＰＫ１細胞株、ＨＥＫ２９３細胞株又はＡｃｔ２０細胞株などを挙げることができる。
［ＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチド］
　副甲状腺ホルモン（ＰＴＨ）とは、魚類以上の脊椎動物の生体内に認められるペプチドホルモンで、不活性なプレプロホルモンの形で発現されてプロセスを受けた後、通常は８４アミノ酸からなるＰＴＨとなる。８４アミノ酸からなるＰＴＨはパラトルモンとも呼ばれ、また、ＰＴＨに由来する他の活性部分ポリペプチドと区別するためにＰＴＨ（１−８４）と呼ばれることもある。本発明に用いるＰＴＨの一つの例としては、配列番号１３にアミノ酸配列が示されているヒトのＰＴＨ（１−８４）が挙げられる。また、ヒト以外でのＰＴＨ（１−８４）に相当するＰＴＨの例としては、配列番号１４にはコモンマーモセット（Ｃａｌｌｉｔｈｒｉｘ　ｊａｃｃｈｕｓ）、配列番号１５にはジャイアントパンダ（Ａｉｌｕｒｏｐｏｄａ　ｍｅｌａｎｏｌｅｕｃａ）、配列番号１６にはウマ（Ｅｑｕｕｓ　ｃａｂａｌｌｕｓ）、配列番号１７にはイヌ（Ｃａｎｉｓ　ｌｕｐｕｓ　ｆａｍｉｌｉａｒｉｓ）、配列番号１８にはウシ（Ｂｏｓ　ｔａｕｒｕｓ）、配列番号１９にはネコ（Ｆｅｌｉｓ　ｃａｔｕｓ）、配列番号２０にはブタ（Ｓｕｓ　ｓｃｒｏｆａ）、配列番号２１にはウサギ（Ｏｒｙｃｔｏｌａｇｕｓ　ｃｕｎｉｃｕｌｕｓ）、配列番号２２にはラット（Ｒａｔｔｕｓ　ｎｏｒｖｅｇｉｃｕｓ）及び配列番号２３にはマウス（Ｍｕｓ　ｍｕｓｃｕｌｕｓ）のそれぞれの種に由来するＰＴＨ（１−８４）が挙げられ、これらのヒト以外の種に由来するＰＴＨ（１−８４）も本発明に用いることができるが、これらに限定されることはない。
　また、米国のＮａｔｉｏｎａｌ　Ｃｅｔｅｒ　ｆｏｒ　Ｂｉｏｔｅｃｈｎｏｌｏｇｙ　Ｉｎｆｏｒｍａｔｉｎ（ＮＣＢＩ）で提供している配列類似性検索プログラムであるＢＬＡＳＴ（Ｔｈｅ　Ｂａｓｉｃ　Ｌｏｃａｌ　Ａｌｉｇｎｍｅｎｔ　Ｓｅａｒｃｈ　Ｔｏｏｌ）のデフォルトのパラメーター（マトリックス：ＢＬＯＳＵＭ６２、ギャップコスト；Ｅｘｉｔｅｎｃｅ＝１１；Ｅｘｔｅｎｓｉｏｎ＝１）でヒトＰＴＨ（１−８４）とマウスＰＴＨ（１−８４）を比較すると、両者のアミノ酸配列が一致するホモロジー（Ｉｄｅｎｔｉｔｙ）は７１％、性状の近いアミノ酸への置換を許諾した場合には８６％の類似性（Ｓｉｍｉｌａｒｉｔｙ）を示した。従って、この明細書に具体的に配列が記載されていないヒト又はヒト以外の種におけるＰＴＨ（１−８４）であっても、ヒトＰＴＨ（１−８４）と７０％以上のホモロジーを示すものであれば、本発明で用いるＰＴＨ（１−８４）に含まれる。
　また、ＰＴＨ（１−８４）の部分的なアミノ酸配列を有するＰＴＨ部分活性ポリペプチドも本発明で用いることができるＰＴＨに含まれる。ヒトＰＴＨの代表的な部分活性ポリペプチドとしては、ヒトＰＴＨ（１−３７）（Ｃ．Ｐ．Ｓｃｈｍｉｔｔ　ｅｔ　ａｌ．，Ｋｉｄｎｅｙ　Ｉｎｔ．，５７，１４８４（２０００））（配列番号１のＮ末端側１番目から３７番目までのアミノ酸配列に相当）、ヒトＰＴＨ（１−３４）（配列番号１のＮ末端側１番目から３４番目までのアミノ酸配列に相当）、ヒトＰＴＨ（１−３１）（Ｊ．Ｆ．Ｗｈｉｔｆｉｅｌｄ　ａｎｄ　Ｐ．Ｍｏｒｌｅｙ，Ｔｒｅｎｄｓ　Ｐｈａｒｍａｃｏｌ．Ｓｃｉ．，１６，３８２（１９９５））（配列番号１のＮ末端側１番目から３１番目までのアミノ酸配列に相当）が例示できる。特にＰＴＨ（１−３４）はテリパラチドとも呼ばれ、最初に人工的に合成されてＰＴＨ（１−８４）と同等の生物活性を有することが示されたＰＴＨの部分活性ポリペプチドである（Ｊ．Ｔ．Ｐｏｔｔｓ，Ｊ．Ｅｎｄｏｃｒｉｎｏｌ．１８７，３１１（２００５））。また、本発明に用いることができるＰＴＨの部分活性ポリペプチドの例としては、上述のヒト以外の種に由来するＰＴＨ（１−８４）における、ＰＴＨ（１−３７）、ＰＴＨ（１−３４）又はＰＴＨ（１−３１）に相当するポリペプチドが挙げられる。
　それに加えて、ヒトＰＴＨ（１−３４）と近いアミノ酸配列を有する他の種に由来する３２から３３アミノ酸からなるペプチドとして、ニワトリ（Ｇａｌｌｕｓ　ｇａｌｌｕｓ）のＰＴＨ（１−３３）（配列番号２４）、キンカチョウ（Ｔａｅｎｉｏｐｙｇｉａ　ｇｕｔｔａｔａ）のＰＴＨ（１−３３）（配列番号２５）、ハイイロジネズミオポッサム（Ｍｏｎｏｄｅｌｐｈｉｓ　ｄｏｍｅｓｔｉｃａ）のＰＴＨ（１−３３）（配列番号２６）が挙げられ、これらも本発明で用いることができるＰＴＨに含まれる。なお、オポッサムのＰＴＨ／ＰＴＨｒＰ受容体にはヒトなどのＰＴＨ／ＰＴＨｒＰが結合／作用することは公知である（Ｊｕｐｐｎｅｒ　Ｈ．ｅｔ　ａｌ．Ｅｎｄｏｃｒｉｎｏｌｏｇｙ　１３４　ｐｐ８７９　１９９４）。ヒトＰＴＨ（１−３４）とハイイロジネズミオポッサムＰＴＨ（１−３３）をＢＬＡＳＴ法で比較すると両者の間のホモロジーは６３％程度であり、性状の近いアミノ酸への置換を許諾した場合には８２％の類似性を示した。また、ヒトＰＴＨにおいてはＰＴＨ（１−３４）の配列のうち最初の２１アミノ酸がＰＴＨ／ＰＴＨｒＰ受容体への結合及びシグナルの発生に重要であることが公知であるが（Ｇｅｒｄｅｌｌａ　ＦＪ　ｅｔ　ａｌ　Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２７０　ｐｐ６５８４　１９９５、Ｇｅｒｄｅｌｌａ　ＦＪ　ｅｔ　ａｌ　Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２７１　ｐｐ１９８８８，１９９６）、ヒトＰＴＨ（１−３４）に対してハイイロジネズミオポッサムＰＴＨ（１−３３）の間では１２個のアミノ酸配列（性状の近いアミノ酸への置換を許諾した場合には９個）のアミノ酸で置換が起こっていた。
　この明細書に具体的に配列が記載されていないヒト又はヒト以外の種におけるＰＴＨ（１−３４）又はその部分活性ポリペプチドであっても、ヒトＰＴＨ（１−３４）と６３％以上のホモロジーを示すもの、又は１２個以内のアミノ酸置換があるものであれば、本発明で用いるＰＴＨ（１−３４）又はその部分活性ペプチドに含まれる。
　副甲状腺ホルモン関連ペプチド（ＰＴＨｒＰ）とは、魚類以上の脊椎動物の生体内に認められるペプチドホルモンで、ＰＴＨと構造的に類似し、ＰＴＨと同様にカルシウム代謝に作用する。ＰＴＨｒＰは不活性なプレプロホルモンの形で発現されてプロセスを受けた後、通常は１４１アミノ酸又は１３９アミノ酸からなるＰＴＨｒＰとなる。１４１アミノ酸又は１３９アミノ酸からなるＰＴＨｒＰはＰＴＨに由来する他の活性部分ポリペプチドと区別するためにそれぞれＰＴＨｒＰ（１−１４１）、ＰＴＨｒＰ（１−１３９）と呼ばれることもある。本発明に用いるＰＴＨｒＰ一つの例としては、配列番号２７にアミノ酸配列が示されているヒトのＰＴＨｒＰ（１−１４１）（アイソフォーム１）、又は配列番号２８にアミノ酸配列が示されているＰＴＨｒＰ（１−１３９）（アイソフォーム２）が挙げられる。
　また、ヒト以外でのＰＴＨｒＰ（１−１４１）又はＰＴＨｒＰ（１−１３９）に相当す
るＰＴＨの例としては、配列番号２９のアカゲザル（Ｍａｃａｃａ　ｍｕｌａｔｔａ）ＰＴＨｒＰ（１−１３９）、配列番号３０のジャイアントパンダ（Ａｉｌｕｒｏｐｏｄａ　ｍｅｌａｎｏｌｅｕｃａ）ＰＴＨ（１−１４１）、配列番号３１のウシ（Ｂｏｓ　ｔａｕｒｕｓ）のＰＴＨｒＰ（１−１４１）、配列番号３２のブタ（Ｓｕｓ　ｓｃｒｏｆａ）ＰＴＨｒＰ（１−１４１）、配列番号３３のイヌ（Ｃａｎｉｓ　ｌｕｐｕｓ　ｆａｍｉｌｉａｒｉｓ）のＰＴＨｒＰ（１−１４１）、配列番号３４のウマ（Ｅｑｕｕｓ　ｃａｂａｌｌｕｓ）ＰＴＨｒＰ（１−１４１）、配列番号３５のラット（Ｒａｔｔｕｓ　ｎｏｒｖｅｇｉｃｕｓ）ＰＴＨｒＰ（１−１４１）、配列番号３６のマウス（Ｍｕｓ　ｍｕｓｃｕｌｕｓ）ＰＴＨｒＰ（１−１４１）及び配列番号３７のウサギ（Ｏｒｙｃｔｏｌａｇｕｓ　ｃｕｎｉｃｕｌｕｓ）ＰＴＨｒＰ（１−１４１）が挙げられ、これらのヒト以外の種に由来するＰＴＨ（１−８４）も本発明に用いることができるが、これらに限定されることはない。また、ＮＩＨのＢＬＡＳＴ法でのデフォルトのパラメーターでヒトＰＴＨｒＰ（１−１４１）とウサギＰＴＨｒＰ（１−１４１）を比較すると、両者のアミノ酸配列が一致するホモロジー（Ｉｄｅｎｔｉｔｙ）は９０％、性状の近いアミノ酸への置換を許諾した場合には９１％の類似性（Ｓｉｍｉｌａｒｉｔｙ）を示した。
　従って、この明細書に具体的に配列が記載されていないヒト又はヒト以外の種におけるＰＴＨｒＰ（１−１４１）又はＰＴＨｒＰ（１−１３９）であっても、ヒトＰＴＨｒＰ（１−１４１）と９０％以上のホモロジーを示すものであれば、本発明で用いるＰＴＨｒＰ（１−１４１）又はＰＴＨｒＰ（１−１３９）に含まれる。
　ヒトＰＴＨｒＰの代表的な部分活性ポリペプチドとしては、ヒトＰＴＨｒＰ（１−８６）（Ｅ．Ｌｅｗｉｎ　ｅｔ　ａｌ．，Ｋｉｄｎｅｙ　Ｉｎｔ．，５８，７１　２０００）（配列番号２７のＮ末端側１番目から８６番目のアミノ酸配列に相当）、ＰＴＨｒＰ（１−４０）（Ｅ．Ｌｅｗｉｎ　ｅｔ　ａｌ．，Ｋｉｄｎｅｙ　Ｉｎｔ．，５８，７１　２０００）（配列番号配列番号２７のＮ末端側１番目から４０番目のアミノ酸配列に相当）、ＰＴＨｒＰ（１−３７）（Ｌ．Ｊ．Ｓｕｖａ　ｅｔ　ａｌ．，Ｓｃｉｅｎｃｅ，２３７，８９３　１９８７）（配列番号配列番号２７のＮ末端側１番目から３７番目のアミノ酸配列に相当）、ＰＴＨｒＰ（１−３４）（Ｂ．Ｅ．Ｋｅｍｐ　ｅｔ　ａｌ．，Ｓｃｉｅｎｃｅ，２３８，１５６８　１９８７）（配列番号配列番号２７のＮ末端側１番目から３４番目のアミノ酸配列に相当）が例示できる。
　また、本発明に用いることができるＰＴＨｒＰの部分活性ポリペプチドの例としては、上述のヒト以外の種に由来するＰＴＨｒＰ（１−１４１）又はＰＴＨｒＰ（１−１３９）における、ＰＴＨｒＰ（１−８６）、ＰＴＨｒＰ（１−４０）、ＰＴＨｒＰ（１−３７）又はＰＴＨｒＰ（１−３４）に相当するポリペプチドが挙げられる。
　それに加えて、ヒトＰＴＨｒＰ（１−３４）と近いアミノ酸配列を有する他の種に由来する３４アミノ酸からなるポリペプチドとして、カモノハシ（Ｏｒｎｉｔｈｏｒｈｙｎｃｈｕｓ　ａｎａｔｉｎｕｓ）のＰＴＨｒＰ（１−３４）（配列番号３８）、キンカチョウ（Ｔａｅｎｉｏｐｙｇｉａ　ｇｕｔｔａｔａ）のＰＴＨｒＰ（１−３４）（配列番号３９）、ニワトリ（Ｇａｌｌｕｓ　ｇａｌｌｕｓ）のＰＴＨｒＰ（１−３４）（配列番号４０）、ハイイロジネズミオポッサム（Ｍｏｎｏｄｅｌｐｈｉｓ　ｄｏｍｅｓｔｉｃａ）のＰＴＨｒＰ（１−３４）（配列番号４１）が挙げられ、これらも本発明で用いることができるＰＴＨに含まれる。なお、オポッサムのＰＴＨ／ＰＴＨｒＰ受容体にはヒトなどのＰＴＨ／ＰＴＨｒＰが結合／作用することは公知である（Ｊｕｐｐｎｅｒ　Ｈ．ｅｔ　ａｌ．Ｅｎｄｏｃｒｉｎｏｌｏｇｙ　１３４　ｐｐ８７９　１９９４）。ヒトＰＴＨｒＰ（１−３４）とハイイロジネズミオポッサムＰＴＨｒＰ（１−３４）をＢＬＡＳＴ法で比較すると両者の間のホモロジーは７７％程度であり、性状の近いアミノ酸への置換を許諾した場合には８３％の類似性を示した。
　また、前述のようにＰＴＨとＰＴＨｒＰは共通の受容体ＰＴＨ／ＰＴＨｒＰに結合して細胞へのシグナルを惹起することが知られており（Ｊｕｐｐｎｅｒ　Ｈ．ｅｔ　ａｌ．Ｓｃｉｅｎｃｅ．２５４　ｐ１０２４　１９９１）、ヒトＰＴＨ及びヒトＰＴＨｒＰにおいてはＰＴＨ（１−３４）及びＰＴＨｒＰ（１−３４）の配列のうち最初の２１アミノ酸がＰＴＨ／ＰＴＨｒＰ受容体への結合及びシグナルの発生に重要であることも公知である（Ｇｅｒｄｅｌｌａ　ＦＪ　ｅｔ　ａｌ　Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２７０　ｐｐ６５８４　１９９５、Ｇｅｒｄｅｌｌａ　ＦＪ　ｅｔ　ａｌ　Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２７１　ｐｐ１９８８８　１９９６）。ヒトＰＴＨ（１−３４）とヒトＰＴＨｒＰ（１−３４）の最初の２１個のアミノ酸を比較すると、１２個のアミノ酸（性状の近いアミノ酸への置換を許諾した場合には７個）に相違が存在する。従って、この明細書に具体的に配列が記載されていないヒト又はヒト以外の種におけるＰＴＨ、ＰＴＨｒＰ、又はその部分活性ポリペプチドであっても、ヒトＰＴＨ（１−３４）又はヒトＰＴＨｒＰ（１−３４）の最初のＮ末から２１個に相当するアミノ酸配列に対して１２個以内のアミノ酸置換が存在するアミノ酸配列を有するＰＴＨ、ＰＴＨｒＰ、又はその部分活性ポリペプチドであれば、本発明で用いるＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドに含まれる。
　上記のようなＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドは、そのペプチドを産生している動物や細胞から抽出して製造されたもの、そのポリペプチドをコードする遺伝子を遺伝子工学的に宿主に導入した組換え体細胞やトランスジェニック非ヒト動物又はトランスジェニック植物で発現させて製造されたもの、固相合成法などのペプチドの化学合成法によって製造されたものなどのいずれでも使用することができる。また、ＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドは、試薬メーカー等より入手可能なものもあり、例えばＢａｃｈｅｍ社からはＰＴＨ（１−８４）（ｈｕｍａｎ）（Ｃａｔ．Ｎｏ．Ｈ−１３７０）、ＰＴＨ（１−３７）（ｈｕｍａｎ）（Ｃａｔ．Ｎｏ．Ｈ−５９７４）、ＰＴＨ（１−３４）（ｈｕｍａｎ）（Ｃａｔ．Ｎｏ．Ｈ−４８３５）、ＰＴＨ（１−３１）（ｈｕｍａｎ）（Ｃａｔ．Ｎｏ．Ｈ−２２７４）、ＰＴＨ（１−３１）ａｍｉｄｅ（ｈｕｍａｎ）（Ｃａｔ．Ｎｏ．Ｈ−３４０８）、ＰＴＨ−Ｒｅｌａｔｅｄ　Ｐｒｏｔｅｉｎ（１−８６）（ｈｕｍａｎ）（Ｃａｔ．Ｎｏ．Ｈ−９８１５）、ＰＴＨ−Ｒｅｌａｔｅｄ　Ｐｒｏｔｅｉｎ（１−４０）（ｈｕｍａｎ，ｍｏｕｓｅ，ｒａｔ）（Ｃａｔ．Ｎｏ．Ｈ−６８１０）、ＰＴＨ−Ｒｅｌａｔｅｄ　Ｐｒｏｔｅｉｎ（１−３７）（ｈｕｍａｎ，ｍｏｕｓｅ，ｒａｔ）（Ｃａｔ．Ｎｏ．Ｈ−５４９４）、ｐＴＨ−Ｒｅｌａｔｅｄ　Ｐｒｏｔｅｉｎ（１−３４）（ｈｕｍａｎ，ｍｏｕｓｅ，ｒａｔ）（Ｃａｔ．Ｎｏ．Ｈ−６６３０）ＰＴＨ−ＲｅｌａｔｅｄＰｒｏｔｅｉｎ（１−３４）ａｍｉｄｅ（ｈｕｍａｎ，ｍｏｕｓｅ，ｒａｔ）（Ｃａｔ．Ｎｏ．Ｈ−９０９５）などが販売されており、そのようなＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドも使用することが可能である。
　本発明で用いるＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドは、そのＣ末端の構造としてはカルボキシル基（ＣＯＯＨ）の状態又はアミド化（ＣＯＮＨ２）のいずれでも構わない。さらに、本発明のＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドは、そのＮ末端、Ｃ末端又はアミノ酸残基の側鎖の何れか、又は２箇所以上の組合せにおいて、化合物やアミノ酸、ＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチド以外のタンパク質又はペプチドが付加された形態であってもよい。付加される化合物の例としては、ポリエチレングリコール（ＰＥＧ）など生体内に投与した際に安定化するための物質やビスフォスフォネートなどの薬効を有する化合物などが挙げられる。アミノ酸が付加された状態とは、Ｎ末端に、メチオニン残基、アセチル基又はピログルタミン酸等が結合した形態や、ＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドの製造において融合タンパク質として調整した後タンパク質分解酵素で切断を行なった際にＮ末端又はＣ末端に残る１から数個のアミノ酸の付加などが挙げられる。付加されるタンパク質としてはアルブミンや免疫グロブリンなどの生体内での安定化や運搬に関与するタンパク質などが挙げられる。付加されるペプチドについては、製造過程での精製などに用いられるタグ配列（ヒスチジンタグ又はＦＬＡＧタグ等）や他の生体活性ポリペプチドなどが挙げられる。このような、化合物やアミノ酸、ＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチド以外のタンパク質又はペプチドが付加の付加については、公知の方法（ハーマンソン（Ｈｅｒｍａｎｓｏｎ）等　バイオコンジュゲート・テクニックス（Ｂｉｏｃｏｎｊｕｇａｔｅ　ｔｅｃｈｎｉｑｕｅｓ）（ＵＳＡ）１９９６年発行　Ａｃａｄｅｍｉｃ　Ｐｒｅｓｓ）を用いて酵素的又は化学的に会合させて実施することができる。また、ＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドのＮ末端及び／又はＣ末端にアミノ酸やタンパク質・ペプチドが付加された構造の場合には遺伝子工学的に当該構造のポリペプチドをコードする発現用の遺伝子を構築して、その遺伝子が導入された宿主細胞又はトランスジェニック植物又は非ヒトトランスジェニック動物などを用いて遺伝子発現産物として調整することもできる。
［ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログ］
　ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログとは、前記のＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドにおけるアミノ酸配列のうち、一つ以上のアミノ酸配列を人為的に他のアミノ酸又は化合物に置換したもので、ＰＴＨ／ＰＴＨｒＰ受容体アゴニストとしての活性を保持したものをいう。当該ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログがＰＴＨ／ＰＴＨｒＰ受容体アゴニストとしての活性を保持しているかを調べる方法としては、上記［ＰＴＨ／ＰＴＨｒＰ受容体アゴニスト］で記載したＰＴＨ／ＰＴＨｒＰ受容体の活性化を調べる方法と同様に行なうことが可能である。アミノ酸の置換には保存的置換と非保存的な置換があり、保存的置換とは性質が近いアミノ酸同士で置換することをいい、非保存的置換とはそれ以外のアミノ酸置換をいう。一般にアミノ酸の保存的置換の方が非保存的置換より基となる分子の機能が保存される可能性が高いと考えられている。よって、アミノ酸の保存的置換によって、同等の作用を有するポリペプチドを得ることが可能である。
　保存的置換でのアミノ酸の性質の近さを考える一つの観点としては、アミノ酸側鎖の極性や電荷に大きく影響せずに置換できるアミノ酸の組合せでの置換がある。これはアミノ酸側鎖の疎水性や電荷の変化によって二次構造やさらに高次の構造において分子間や分子内での他のアミノ酸側鎖、水分子、他のタンパク質構成要素（金属イオンなど）との相互作用を考慮するうえで、疎水性や電荷の変化が少なければ構造や機能が保存されるであろうとの考えによる。通常の天然タンパク質及びペプチドは２０種類のアミノ酸（何も指定がない場合はＬ体アミノ酸をいう）より構成させているが、それらの側鎖は大きく極性を持つものと極性を持たないもの（非極性）に分類される。極性を持つ側鎖を有するアミノ酸は生理的ｐＨ条件で荷電する側鎖を有するものと荷電を有さないもの（非荷電）に分類される。さらに荷電を有する側鎖を有するものは酸性側鎖と塩基性側鎖に分類される。一方、非極性の側鎖を持つアミノ酸は、脂肪族側鎖を有するものと芳香族側鎖を有するものに分類される。
　これらの分類によると、通常の天然タンパク質及びペプチドを構成する２０種類のアミノ酸は、アスパラギン酸（Ａｓｐ）とグルタミン酸（Ｇｌｕ）は極性／荷電／酸性側鎖のアミノ酸、アルギニン（Ａｒｇ）・リジン（Ｌｙｓ）及びヒスチジン（Ｈｉｓ）は極性／荷電／塩基性側鎖のアミノ酸、グリシン（Ｇｌｙ）・セリン（Ｓｅｒ）・スレオニン（Ｔｈｒ）・アスパラギン（Ａｓｎ）・グルタミン（Ｇｌｎ）・チロシン（Ｔｙｒ）及びシステイン（Ｃｙｓ）は極性／非荷電側鎖のアミノ酸、アラニン（Ａｌａ）・バリン（Ｖａｌ）・ロイシン（Ｌｅｕ）・イソロイシン（Ｉｌｅ）・メチオニン（Ｍｅｔ）及びプロリン（Ｐｒｏ）は非極性／脂肪族側鎖のアミノ酸、フェニルアラニン（Ｐｈｅ）及びトリプトファン（Ｔｒｐ）は非極性／芳香族側鎖のアミノ酸に分類される。それぞれ同じ分類に属するアミノ酸同士は保存的アミノ酸置換に用いられることが多い。但し、Ｃｙｓについてはジスルフィド結合を介して分子内又は分子間での架橋の形成に関与する可能性があり、Ｐｒｏはタンパク質／ペプチドの二次構造に影響する可能性があるので、保存的アミノ酸
置換では扱われない場合もある。
　また、同様にアミノ酸側鎖の物理化学的性質の指標として、疎水性や荷電を数値化した疎水性インデックスを用いる場合がある。疎水性インデックスの例としては、天然タンパク質に含まれる２種類のアミノ酸の疎水性インデックススコアは、Ａｒｇ：−１０．０、Ｌｙｓ：−９．９、Ｇｌｕ及びＡｓｐ：−８．３、Ａｓｎ：−７．１、Ｇｌｎ：−６．０、Ｓｅｒ：−４．３、Ｈｉｓ及びＴｈｒ：−３．８、Ｇｌｙ：−２．４、Ｃｙｓ：−２．３、Ａｌａ：−１．１、Ｐｒｏ：−０．２、Ｔｙｒ：２．５、Ｖａｌ：４．１、Ｍｅｔ：４．６、Ｉｌｅ：８．７、Ｌｅｕ及びＴｒｐ：９．７、Ｐｈｅ：１０とされている。また通常のタンパク質には含まれない特殊なアミノ酸の例としては、α，β−ジアミノプロピオン酸（Ｄａｐ）：−９．５、α−アミノイソ酪酸（Ａｉｂ）：１．１、ノルロイシン（Ｎｌｅ）：９．１など、疎水インデックスのスコアがそれぞれ算定されている（Ａｌｅｓｓａｎｄｒｏ等、ＰＥＰＴＩＤＥＳ　２００２、Ｐｒｏｃｅｅｄｉｎｇ　ｏｆ　２７ｔｈ　Ｅｕｒｏｅａｎ　Ｐｅｐｔｉｄｅ　Ｓｙｍｐｏｓｕｉｍ）。保存的アミノ酸置換を行うには、この疎水インデックスにおいて、そのスコアが近いアミノ酸を選択するほど疎水性や荷電が近く、構造や機能が保存される可能性が高いと考えられる。
　また、別の保存的アミノ酸置換の判断するための他の方法としては、ペプチドやタンパク質のアミノ酸配列のホモロジー比較に用いられるアミノ酸置換行列を用いる方法が挙げられる。アミノ酸置換行列は類似の構造や機能を持つ配列を集めて比較してアミノ酸置換の頻度と種類によって数値化した行列であり、進化過程における相対的な置換のしやすさを反映したものと言われる。その代表的なものとしてはファミリーを形成する配列の全長アミノ酸配列を比較して作成されたＰＡＭ（Ｐｏｉｎｔ−Ａｃｃｅｐｔｅｄ−Ｍｕｔａｔｉｏｎ）（Ｍ．Ｏ．Ｄａｙｈｏｆｆ等，Ａｔｌａｓ　ｏｆ　Ｐｒｏｔｅｉｎ　Ｓｅｑｕｅｎｃｅ　ａｎｄ　Ｓｔｒｕｃｔｕｒｅ　５，ｐ３４５　１９７８）と類似の配列ブロックを集めて比較して作成されたＢＬＯＳＵＭ（Ｂｌｏｃｋｓ　Ｓｕｂｓｔｉｔｕｔｉｏｎ　Ｍａｔｒｉｘ）（Ｓ．Ｈｅｎｉｋｏｆｆ，とＪ．Ｇ．Ｈｅｎｉｋｏｆｆ，Ｐｒｏｃｅｅｄｉｎｇｓ　ｏｆ　ｔｈｅ　Ｎａｔｉｏｎａｌ　Ａｃａｄｅｍｙ　ｏｆ　Ｓｃｉｅｎｃｅｓ　ｏｆ　ｔｈｅ　Ｕｎｉｔｅｄ　Ｓｔａｔｅｓ　ｏｆ　Ａｍｅｒｉｃａ　８９　ｐ１０９１５　１９９２）がある。これらの置換行列では保存的な置換となると考えられるアミノ酸同士の組合せほど大きな値が付けられており、その値を参照して導入するアミノ酸の変異を選択することも可能である。例えば、置換行列の一つであるＢＬＯＳＭ６４についてみると、Ａｌａに対するスコアとして、同じＡｌａであれば４、それに続いてＳｅｒであれば１、Ｃｙｓ・Ｇｌｙ・Ｔｈｒ・Ｖａｌは０となっており、保存的な置換が行なえる可能性が高い。それに対してＡｌａに対してＴｒｐは−３、Ａｓｎ・Ａｓｐ・Ｈｉｓ・Ｐｈｅ・Ｔｙｒは−２と低いスコアであり保存的な置換が行なえる可能性が低いことが分かる。同様に、Ａｒｇに対してＬｙｓが２、Ｇｌｎが１、Ａｓｎ・Ｇｌｕ・Ｈｉｓが０という形で数値が与えられているため、このような置換行列でのスコアを参考にＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログをデザインすることも可能である。
　通常の天然タンパク質及びペプチドを構成するアミノ酸の置換によるＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログは、そのポリペプチドをコードする遺伝子を遺伝子工学的に宿主に導入した組換え体細胞やトランスジェニック非ヒト動物又はトランスジェニック植物で発現させる方法や、固相合成法などのペプチドの化学合成法によって製造することが可能である。
　ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログを作成するためのアミノ酸置換においては上記のように天然タンパク質に通常含まれる２０種類のアミノ酸以外の非タンパク質性アミノ酸と呼ばれるアミノ酸への置換も可能である。非タンパク性アミノ酸の例としては、工学異性体であるＤ体のアミノ酸が挙げられる。天然タンパク質に通常含まれる２０種類のアミノ酸のうち、Ｇｌｙ以外はアルファ炭素が不斉炭素原子となっており、Ｌ体とＤ体の工学異性体を取る。また、天然タンパク質に通常含まれるアミノ酸はＬ体である。したがって、ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアミノ酸配列において一つ以上のアミノ酸をＤ体のアミノ酸に置換して、かつＰＴＨ／ＰＴＨｒＰ受容体アゴニストとしての活性が維持されているものも本発明で用いるＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログに含まれる。また、非タンパク質性アミノ酸として、前記２０種類のアミノ酸以外のアミノ酸が用いられる場合もあり、例えばβ−（２−ナフチル）アラニン（β−Ｎａｌ）、ノルロイシン（Ｎｌｅ）、α，β−ジアミノプロピオン酸（Ｄａｐ）、シクロヘキシルアラニン（Ｃｈａ）、ノルバリン（Ｎｖａ）、４−アミノ−フェニルアラニン（Ａｍｐ）、３−ピリジニルアラニン（Ａｍｐ）、α−アミノイソ酪酸（Ａｉｂ）、１−アミノ−１−シクロ−ヘキサンカルボン酸（Ａｈｃ）などが挙げられる。また、Ａｈｃに加えて、１−アミノ−１−シクロプロパンカルボン酸、１−アミノ−１−シクロブタンカルボン酸、１−アミノ−１−シクロペンタンカルボン酸、１−アミノ−１−シクロヘプタンカルボン酸、１−アミノ−１−シクロオクタンカルボン酸及び１−アミノ−１−シクロノナンカルボン酸の群から選ばれる非タンパク質性アミノ酸をここではＡｃｃとして表記することがある。従って、ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアミノ酸配列において一つ以上の非タンパク質性アミノ酸に置換して、かつＰＴＨ／ＰＴＨｒＰ受容体アゴニストとしての活性が維持されているものも本発明で用いるＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログに含まれる。これらの非タンパク質性アミノ酸への置換を行ったＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログは、通常は固相合成法などの公知のペプチド合成法で作成することができる。
　ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログとしての別の形態としては、分子内又は分子間での架橋が行なわれたペプチドがある。分子内での架橋の一例としては、ＰＴＨ（１−３４）において種間で保存されている２６位のＬｙｓと３０位のＡｓｐの間での架橋やＰＴＨｒＰ（１−３４）において種間で保存されている１３位のＬｙｓと１７位のＡｓｐの間での架橋など、分子内での架橋を行なった環状ペプチドの合成方法やその環状ペプチドがＰＴＨ／ＰＴＨｒＰ受容体アゴニストとしての活性が維持されていることが知られている（Ｂｉｓｅｌｌｏ　Ａ．ｅｔ．ａｌ．，Ｂｉｏｃｈｅｍ　３６　ｐ３２９３　１９９７）。
　本発明で用いるＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログをデザインする場合には、アミノ酸置換による配列の変化が二次構造にできるだけ影響しないようにデザインを行なうことも、構造や機能の保存の上では重要と考えられる。ＰＴＨ（１−３４）及びＰＴＨｒＰ（１−３４）の二次構造を見るとＮ末端側とＣ末端側に２つのα−ヘリックス構造をとることが知られており、それがＰＴＨ／ＰＴＨｒＰ受容体アゴニストとしての活性に重要な働きをしていると考えられている（Ｃｏｈｅｎ　ＥＦ　等，Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．２６６　ｐ１９９７　１９９１）。したがって、ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログをデザインする時に、アミノ酸置換によって、このような二つのα−ヘリックス構造が崩れないようにデザインすることによって、ＰＴＨ／ＰＴＨｒＰ受容体アゴニストとしての活性が保存されたＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログを得ることができる。二次構造を解析する方法としては、ＧＯＲ１（又はＲｏｂｓｏｎ）法（Ｇａｒｎｉｅｒ，ＯｓｇｕｔｈｏｒｐｅとＲｏｂｓｏｎ，Ｊ．Ｍｏｌ．Ｂｉｏｌ．１２０　ｐ９７　１９７８）やＰＲＥＤＥＴＯＲ法（Ｆｒｉｓｈｍａｎ　ＤとＡｒｇｏｓ　Ｐ，Ｐｒｏｔｅｉｎ　Ｅｎｇ　９　ｐ１３３　１９９６）やその他の公知の方法で行なうことができる。
　また、本発明で用いるＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログは、ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアミノ酸配列のうち少なくとも１箇所の位置でのアミノ酸置換又はアミノ酸側鎖の架橋や修飾を伴うが、その数の上限としてはＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのＮ末端から２１個のアミノ酸の範囲では１２箇所以内のアミノ酸置換であることが望ましい。これは、ヒトＰＴＨ（１−３４）とヒトＰＴＨｒＰ（１−３４）の最初の２１個のアミノ酸を比較すると、１２個のアミノ酸に相違があるものの、ＰＴＨ（１−３４）とＰＴＨｒＰ（１−３４）は同様のＰＴＨ／ＰＴＨｒＰ受容体アゴニストとしての活性を有していることによる。
　本発明の用いるＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドのアナログは、そのＣ末端の構造としてはカルボキシル基（ＣＯＯＨ）の状態又はアミド化（ＣＯＮＨ２）のいずれでも構わない。さらに、本発明のＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドのアナログは、そのＮ末端、Ｃ末端又はアミノ酸残基の側鎖の何れか、又は２箇所以上の組合せにおいて、化合物やアミノ酸、ＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドのアナログ以外のタンパク質又はペプチドが付加された形態であってもよい。付加される化合物の例としては、ポリエチレングリコール（ＰＥＧ）など生体内に投与した際に安定化するための物質やビスフォスフォネートなどの薬効を有する化合物などが挙げられる。アミノ酸が付加された状態とは、Ｎ末端に、メチオニン残基、アセチル基又はピログルタミン酸等が結合した形態や、ＰＴＨ、ＰＴＨｒＰ又はその部分活性ポリペプチドのアナログの製造において融合タンパク質として調整した後、タンパク質分解酵素で切断を行なった際にＮ末端又はＣ末端に残る１から数個のアミノ酸の付加などが挙げられる。付加されるタンパク質としてはアルブミンや免疫グロブリンなどの生体内での安定化や運搬に関与するタンパク質などが挙げられる。付加されるペプチドについては、製造過程での精製などに用いられるタグ配列（ヒスチジンタグ又はＦＬＡＧタグ等）や他の生体活性ペプチドなどが挙げられる。
　具体的なＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログの構造については、非常に多くものが公知となっており（例えば特開平９−１５７２９４、特開平５−３２０１９３、特開平５−３２６９６、特表平５−５０９０９８、特表平５−５０５５９４及び特許第３１３５１２２号、特表平８−５０３６９２、特開昭６２−６７０９９、特開昭６１−５７６００、特開昭６１−２４５９８、特開平５９−２０４１５９及び特公平３−１４３２０、特開昭５９−４２３５１等）、それらは本発明で用いることができるＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログに含まれる。
　さらに具体的には特表平１１−５０９２０１に示されているＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログであり、それは下記式で表されるＰＴＨの部分活性ポリペプチドのアナログであるポリペプチド：

Figure JPOXMLDOC01-appb-C000007
ただし、
Ａ１はＳｅｒ、Ａｌａ、又はＤａｐであり；
Ａ３はＳｅｒ、Ｔｈｒ、又はＡｉｂであり；
Ａ５はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ７はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ８はＭｅｔ、Ｎｖａ、Ｌｅｕ、Ｖａｌ、Ｉｌｅ、Ｃｈａ、又はＮｌｅであり；
Ａ１１はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ１２はＧｌｙ又はＡｉｂであり；
Ａ１５はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ１６はＳｅｒ、Ａｓｎ、Ａｌａ、又はＡｉｂであり；
Ａ１７はＳｅｒ、Ｔｈｒ、又はＡｉｂであり；
Ａ１８はＭｅｔ、Ｎｖａ、Ｌｅｕ、Ｖａｌ、Ｉｌｅ、Ｎｌｅ、Ｃｈａ、又はＡｉｂであり；
Ａ１９はＧｌｕ又はＡｉｂであり；
Ａ２１はＶａｌ、Ｃｈａ、又はＭｅｔであり；
Ａ２３はＴｒｐ又はＣｈａであり；
Ａ２４はＬｅｕ又はＣｈａであり；
Ａ２７はＬｙｓ、Ａｉｂ、Ｌｅｕ、ｈＡｒｇ、Ｇｌｎ、又はＣｈａであり；
Ａ２８はＬｅｕ又はＣｈａであり；
Ａ３０はＡｓｐ又はＬｙｓであり；
Ａ３１はＶａｌ、Ｎｌｅ、若しくはＣｈａである、又は欠損しており；
Ａ３２はＨｉｓである又は欠損しており；
Ａ３３はＡｓｎである又は欠損しており；
Ａ３４はＰｈｅ、Ｔｙｒ、Ａｍｐ、若しくはＡｉｂである、又は欠損しており；
Ｒ１及びＲ２は、それぞれ独立して、Ｈ、Ｃ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；又はＲ１及びＲ２の両方又一方のみがＣＯＥ１であり、この際、Ｅ１はＣ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；
及びＲ３はＯＨ、ＮＨ２、Ｃ１−１２アルコキシ、又は−ＮＨ−Ｙ−ＣＨ２−Ｚであり、この際、ＹはＣ１−１２炭化水素部分であり、ＺはＨ、ＯＨ、ＣＯ２Ｈ、又はＣＯＮＨ２であり；
Ａ５、Ａ７、Ａ８、Ａ１１、Ａ１５、Ａ１８、Ａ２１、Ａ２３、Ａ２４、Ａ２７、Ａ２８及びＡ３１の少なくとも一つはＣｈａである、又はＡ３、Ａ１２、Ａ１６、Ａ１７、Ａ１８、Ａ１９及びＡ３４の少なくとも一つはＡｉｂである；ポリペプチドが挙げられる。
　その中でも好ましくは特表平１１−５０９２０１及び日本特許第４００８８２５に記載されているヒトＰＴＨ（１−３４）のアナログである下記式のポリペプチド：
［Ｃｈａ７，１１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２３］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｎｌｅ８，１８，Ｃｈａ２７］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２８］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ３１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１６］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１９］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２４，２８，３１，Ｌｙｓ３０］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，Ａｉｂ１６，１９，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，３１，Ａｉｂ１６，１９，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ１１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２８，３１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，Ａｉｂ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ１５］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ａｉｂ１９］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ａｉｂ１６］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１６，１９］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１２］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ３］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ａｉｂ１９，Ｌｙｓ３０］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２４，２８，３１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１７］ｈＰＴＨ（１−３４）ＮＨ２；及び
［Ｃｈａ７，１１，１５］ｈＰＴＨ（１−３４）ＮＨ２、
からなる群より選択されるポリペプチドである。ここで、［Ｃｈａ７，１１］ｈＰＴＨ（１−３４）ＮＨ２との表記は、ヒトＰＴＨ（１−３４）の７位のＬｅｕと１１位のＬｅｕがＣｈａ（シクロヘキシルアラニン）に置換されており、Ｃ末端がアミド化されていることを示しており、他の同様の表記も同様の意味を示す。
　また、特表平１１−５０９２０１に示されている別のＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログとしての態様としては、下記式で表されるＰＴＨｐＰの部分活性ポリペプチドのアナログであるポリペプチド：
Figure JPOXMLDOC01-appb-C000008
 ただし、
Ａ１はＡｌａ、Ｓｅｒ、又はＤａｐであり；
Ａ３はＳｅｒ又はＡｉｂであり；
Ａ５はＨｉｓ、Ｉｌｅ、又はＣｈａであり；
Ａ７はＬｅｕ、Ｃｈａ、Ｎｌｅ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ８はＬｅｕ、Ｍｅｔ、又はＣｈａであり；
Ａ１０はＡｓｐ又はＡｓｎであり；
Ａ１１はＬｙｓ、Ｌｅｕ、Ｃｈａ、Ｐｈｅ、又はβ−Ｎａｌであり；
Ａ１２はＧｌｙ又はＡｉｂであり；
Ａ１４はＳｅｒ又はＨｉｓであり；
Ａ１５はＩｌｅ、又はＣｈａであり；
Ａ１６はＧｌｎ又はＡｉｂであり；
Ａ１７はＡｓｐ又はＡｉｂであり；
Ａ１８はＬｅｕ、Ａｉｂ、又はＣｈａであり；
Ａ１９はＡｒｇ又はＡｉｂであり；
Ａ２２はＰｈｅ、Ｇｌｕ、Ａｉｂ、又はＣｈａであり；
Ａ２３はＰｈｅ、Ｌｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２４はＬｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２５はＨｉｓ、Ａｉｂ、又はＧｌｕであり；
Ａ２６はＨｉｓ、Ａｉｂ、又はＬｙｓであり；
Ａ２７はＬｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２８はＩｌｅ、Ｌｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２９はＡｌａ、Ｇｌｕ、又はＡｉｂであり；
Ａ３０はＧｌｕ、Ｃｈａ、Ａｉｂ、又はＬｙｓであり；
Ａ３１はＩｌｅ、Ｌｅｕ、Ｃｈａ、若しくはＬｙｓである、又は欠損しており；
Ａ３２はＨｉｓである又は欠損しており；
Ａ３３はＴｈｒである又は欠損しており；
Ａ３４はＡｌａである又は欠損しており；
Ｒ１及びＲ２は、それぞれ独立して、Ｈ、Ｃ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；又はＲ１及びＲ２の両方又は一方のみがＣＯＥ１であり、この際、Ｅ１はＣ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；及びＲ３はＯＨ、ＮＨ２、Ｃ１−１２アルコキシ、又は−ＮＨ−Ｙ−ＣＨ２−Ｚであり、この際、ＹはＣ１−１２炭化水素部分であり、ＺはＨ、ＯＨ、ＣＯ２Ｈ、又はＣＯＮＨ２であり；
Ａ５、Ａ７、Ａ８、Ａ１１、Ａ１５、Ａ１８、Ａ２２、Ａ２３、Ａ２４、Ａ２７、Ａ２８、Ａ３０、若しくはＡ３１の少なくとも一つはＣｈａである、又はＡ３、Ａ１２、Ａ１６、Ａ１７、Ａ１８、Ａ１９、Ａ２２、Ａ２５、Ａ２６、Ａ２９、Ａ３０、若しくはＡ３４の少なくとも一つはＡｉｂである；
ポリペプチドが挙げられる。
　その中でも、特に好ましくはヒトＰＴＨｒＰ（１−３４）のアナログである、式：［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，３１，Ａｉｂ２９，Ｌｙｓ２６，３０］ｈＰＴＨｒＰ（１−３４）ＮＨ２（配列番号４２）で表されるポリペプチドである。
　さらに別のＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログとしての態様としては、特表２００１−５０８４３９に記載されているＰＴＨｒＰの活性部分ペプチドのアナログである下記式：
Figure JPOXMLDOC01-appb-C000009
 ただし、
Ａ１はＡｌａ、Ｓｅｒ、又はＤａｐであり；
Ａ３はＳｅｒ又はＡｉｂであり；
Ａ５はＨｉｓ、Ｉｌｅ、Ａｃｃ、又はＣｈａであり；
Ａ７はＬｅｕ、Ｃｈａ、Ｎｌｅ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ａｃｃ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ８はＬｅｕ、Ｍｅｔ、Ａｃｃ、又はＣｈａであり；
Ａ１０はＡｓｐ又はＡｓｎであり；
Ａ１１はＬｙｓ、Ｌｅｕ、Ｃｈａ、Ａｃｃ、Ｐｈｅ、又はβ−Ｎａｌであり；
Ａ１２はＧｌｙ、Ａｃｃ、又はＡｉｂであり；
Ａ１４はＳｅｒ又はＨｉｓであり；
Ａ１５はＩｌｅ、Ａｃｃ、又はＣｈａであり；
Ａ１６はＧｌｎ又はＡｉｂであり；
Ａ１７はＡｓｐ又はＡｉｂであり；
Ａ１８はＬｅｕ、Ａｉｂ、Ａｃｃ、又はＣｈａであり；
Ａ１９はＡｒｇ又はＡｉｂであり；
Ａ２２はＰｈｅ、Ｇｌｕ、Ａｉｂ、Ａｃｃ、又はＣｈａであり；
Ａ２３はＰｈｅ、Ｌｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２４はＬｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２５はＨｉｓ、Ｌｙｓ、Ａｉｂ、Ａｃｃ、又はＧｌｕであり；
Ａ２６はＨｉｓ、Ａｉｂ、Ａｃｃ、又はＬｙｓであり；
Ａ２７はＬｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２８はＩｌｅ、Ｌｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２９はＡｌａ、Ｇｌｕ、Ａｃｃ、又はＡｉｂであり；
Ａ３０はＧｌｕ、Ｌｅｕ、Ｎｌｅ、Ｃｈａ、Ａｉｂ、Ａｃｃ、又はＬｙｓであり；
Ａ３１はＩｌｅ、Ｌｅｕ、Ｃｈａ、Ｌｙｓ若しくはＡｃｃである、又は欠損しており；
Ａ３２はＨｉｓである又は欠損しており；
Ａ３３はＴｈｒである又は欠損しており；
Ａ３４はＡｌａである又は欠損しており；
Ｒ１及びＲ２は、それぞれ独立して、Ｈ、Ｃ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；又はＲ１及びＲ２の両方又は一方のみがＣＯＥ１であり、この際、Ｅ１はＣ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；及び
Ｒ３はＯＨ、ＮＨ２、Ｃ１−１２アルコキシ、又は−ＮＨ−Ｙ−ＣＨ２−Ｚであり、この際、ＹはＣ１−１２炭化水素部分であり、ＺはＨ、ＯＨ、ＣＯ２Ｈ、又はＣＯＮＨ２であり；
Ａ５、Ａ７、Ａ８、Ａ１１、Ａ１２、Ａ１５、Ａ１８、Ａ２２、Ａ２３、Ａ２４、Ａ２５、Ａ２６、Ａ２７、Ａ２８、Ａ２９、Ａ３０、又はＡ３１の少なくとも一つがＡｃｃである；
ポリペプチドが挙げられる。その中でも特に好ましくは、特表２００１−５０８４３９及び日本特許第３９６３４８２号に記載されているヒトＰＴＨｒＰ（１−３４）のアナログである、下記式のポリペプチド：
［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，Ｌｙｓ２６，３０，Ａｉｂ２９，Ａｈｃ３１］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ａｈｃ２３，Ｌｙｓ２６，３０，Ｌｅｕ２８，３１，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，３１，Ｌｙｓ２６，３０，Ａｈｃ２７，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，２９，Ｌｅｕ２３，２８，３１，Ｌｙｓ２６，Ａｈｃ３０］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｃｈａ２２，Ｌｅｕ２３，２８，３１，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｈｃ２７，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，３１，Ａｈｃ２４，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２９，Ｌｅｕ２３，２８，３１，Ａｉｂ２５，Ｌｙｓ２６，３０，Ａｈｃ２７］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，Ｌｅｕ２３，２８，３１，Ａｉｂ２５，２９，Ｌｙｓ２６，３０，Ａｈｃ２７］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ２２，Ｌｅｕ２３，２８，３１，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｌｅｕ２３，３１，Ｌｙｓ２６，３０，Ａｈｃ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｃｈａ２２，Ａｈｃ２３，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ｌｅｕ２８，３１，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｃｈａ２２，Ｌｅｕ２３，２８，３１，Ａｈｃ２４，２７，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，Ｌｅｕ２３，２８，３１，Ａｈｃ２４，２７，Ｌｙｓ２５，２６，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ１８，２４，２７，Ｇｌｕ２２，Ｃｈａ２３，Ｌｙｓ２５，２６，Ｌｅｕ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，Ｃｈａ２３，Ａｈｃ２４，Ｌｙｓ２５，２６，Ｌｅｕ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ２２，２４，Ｌｅｕ２３，２８，３１，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ２２，２４，Ｌｅｕ２３，２８，Ｌｙｓ２５，２６，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
からなる群より選択されるポリペプチドである。
　さらに、特表平１１−５０９２０１及び日本特許日本特許第４００８８２５に記載されている、別のＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログとしての態様としては、下記式で表されるＰＴＨの部分活性ポリペプチドのアナログであるポリペプチド：
［Ｎｌｅ３１］ｈＰＴＨ（１−３４）ＮＨ２；
［ｈＡｒｇ２７］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｄａｐ１，Ｎｌｅ８，１８，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
が挙げられる。また、さらに特表２００１−５０８４３９及び日本特許第３９６３４８２号に記載されているヒトＰＴＨｒＰ（１−３４）のアナログ又はヒトＰＴＨ（１−３４）のアナログである、式：
［Ｌｅｕ２７，Ａｉｂ２９］ｈＰＴＨ（１−３４）ＮＨ２
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，Ｌｅｕ２８，３１，Ａｉｂ２９，Ｎｌｅ３０］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，３０，Ｌｅｕ２８，３１，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，３０，Ｌｅｕ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
で表されるポリペプチドもＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチドのアナログの例として挙げることができる。
［ＰＴＨ又はＰＴＨｒＰ産生を誘導する物質］
　ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質とは、その物質自体は直接ＰＴＨ／ＰＴＨｒＰ受容体へのアゴニスト作用は有さないが、生体内において組織や細胞でのＰＴＨ／ＰＴＨｒＰの遺伝子発現やタンパク質の産生及び／又は分泌を誘導することによって、全身及び／又は局所でのＰＴＨ／ＰＴＨｒＰの濃度を上昇させることによって、ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分ペプチド、若しくはそのアナログに代表されるＰＴＨ／ＰＴＨｒＰ受容体アゴニストの投与による効果と同様に椎体骨折の治癒効果を有する物質をいう。
　そのようなＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質の例としては、カルシウム感受性受容体（ＣａＳＲ）のアンタゴニストが挙げられる。ＣａＳＲアンタゴニストが投与された動物ではＰＴＨの分泌が亢進することが知られている（Ｇｏｗｅｎ等、Ｔｈｅ　Ｊｏｕｒｎａｌ　ｏｆ　Ｃｌｉｎｉｃａｌ　Ｉｎｖｅｓｔｉｇａｔｉｏｎ、１０５巻　ｐ１５９５、２０００年）。ＣａＳＲアンタゴニストの実例を挙げると、例えばＪＴＴ−３０５やＭＫ−５４４２（福本等、ＣＬＩＮＩＣＡＬ　ＣＡＬＣＩＵＭ、２１巻　ｐ８９、２０１１年）、ＮＰＳ２１４３（Ｇｏｗｅｎ等、Ｔｈｅ　Ｊｏｕｒｎａｌ　ｏｆ　Ｃｌｉｎｉｃａｌ　Ｉｎｖｅｓｔｉｇａｔｉｏｎ、１０５巻　ｐ１５９５、２０００年）、ＳＢ−４２３５５７（Ｍａｔｈｅｎｙ等、Ｂｏｎｅ、４６巻　ｐ５３４、２０１０年）、ＳＢ−７５１６８９（３１ｔｈ　Ａｎｎｕａｌ　Ｍｅｅｔｉｎｇ，Ａｍｅｒｉｃａｎ　Ｓｏｓｉｅｔｙ　ｆｏｒ　Ｂｏｎｅ　ａｎｄ　Ｍｉｎｅｒａｌ　Ｒｅｓｅａｒｃｈ、口頭発表、発表番号１０５１・１１３０／ポスター発表、発表番号ＳＡ０３５０）、ＡＴＸ９１４（Ａｍｅｒｉｃａｎ　Ｓｏｓｉｅｔｙ　ｆｏｒ　Ｂｏｎｅ　ａｎｄ　Ｍｉｎｅｒａｌ　Ｒｅｓｅａｒｃｈ、２０１０　Ａｎｎｕａｌ　Ｍｅｅｔｉｎｇ、ポスター発表、発表番号ＳＵ０３７２）などが挙げられる。またＣａＳＲアンタゴニストの別の例としては、特開２０１０−２４８１８３、特開２００５−２３９６１１や特開２０１０−１５９２５８に開示されている化合物などが挙げられる。但し、ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質としては上述の物質に限定されるものではなく、当業者であれば、ＰＴＨ産生を誘導する活性を有する物質については、例えば特開２０１０−２７９３７２に記載の方法などで求める活性の物質をスクリーニングによって取得することが可能であり、またＣａＳＲアンタゴニストについてはＮｅｍｅｔｈ等、Ｊｏｕｒｎａｌ　ｏｆ　Ｍｏｌｅｃｕｌａｒ　Ｅｎｄｏｃｒｉｎｏｌｏｇｙ、２９巻　ｐ１５、２００２年に記載の方法などで取得することが可能であり、このような物質も本発明におけるＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質に含まれる。
［椎体骨折の治療剤］
　また、本発明はＰＴＨ／ＰＴＨｒＰ受容体アゴニストを含有することを特徴とする、あるいは、ＰＴＨ、ＰＴＨｒＰ、又はそれらの部分活性ポリペプチド、若しくはそれらのアナログを含有することを特徴とする、椎体骨折治療剤に関する。さらに、本発明はＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有する椎体骨折治療剤に関する。
　椎体とは、「脊椎管の前方にある椎骨の主たる部分で、椎弓と区別される」（ステッドマン医学大辞典、改訂第５版、メジカルビュー社）、又は、「椎骨の前部を占める半円形の部分」（大辞林、第二版、三省堂）と説明される。従って、椎体骨折とは、脊椎を構成する椎骨における骨折のうち、通常は棘突起、横突起や関節突起などが存在する椎弓部分での骨折を含まず、半円柱状の形態を有する椎体部分における骨折のことをいう。また、椎体骨折の原因としては、事故やスポーツ等での強い衝撃が原因となる椎体骨折も存在するが、社会的や医療経済的に問題になっているのは骨粗鬆症に伴う椎体骨折であり、これらは必ずしも強い衝撃が受けていない場合でも生じる可能性があるうえに患者の予後に大きく影響するため（元文等、日医大医会誌、５巻　ｐ１２５−１２９、２００９年）、より治療ニーズとしては高いといえる。
　従って、本発明の椎体骨折治療剤が使用される主な対象としては、骨粗鬆症に伴う椎体骨折の治療となる。また、脊椎は７個の頚椎、１２個の胸椎、５個の腰椎、５個の仙椎（成人では融合して仙骨となり骨盤の後壁となる）、３~５個の尾椎（融合して１~数個の尾骨となる）によって形成されるが、骨粗鬆症に伴う椎体骨折の発生部位は胸椎と腰椎に集中している。そのことから、本発明におけるラット腰椎椎体骨損傷モデルは腰椎を用いた検討を中心に行っている。従って、本発明の椎体骨折治療剤が使用される対象としては、主に胸椎及び腰椎における椎体骨折の治療となる。
　椎体の構造的な特徴として、外側を覆う皮質骨の内側に非常に発達した海綿骨によって形成される骨梁構造が存在する。椎体の物理的強度はこの海綿骨による骨梁構造に大きく依存すると考えられており、骨粗鬆症に伴う椎体骨折の最終像である椎体圧迫骨折の発生にはこの骨梁の減少や変形が関与しているといわれる（ＩＯＮＯＶＩＣＩ等、ルーマニアン・ジャーナル・オブ・モーフォロジー・アンド・エンブリオロジー、５０巻　ｐ７９−８４、２００９年）。そのため、海綿骨又は皮質骨と海綿骨にまたがる椎体骨折の治療効果が求められていたが、本発明以前においてはＰＴＨ等の作用としては皮質骨が優位である大腿骨、橈骨や胸骨などにおける骨折治療効果が非臨床又は臨床の研究で示されていたのみであった（Ａｌｋｈｉａｒｙ等、ジャーナル・オブ・ボーン・アンド・ジョイント・サージェリー、８７−Ａ巻　ｐ７３１−７４０、２００５年；Ａｓｐｅｎｂｅｒｇ等、ジャーナル・オブ・ボーン・アンド・ミネラル・リサーチ、２５巻　ｐ４０４−４１４、２０１０年；Ｃｈｉｎｔａｍａｎｅｎｉ等、オステオポローシス・インターナショナル、２１巻　ｐ１０５９−１０６３、２０１０年）。
　また、本発明では、ラット腰椎椎体骨損傷モデルを開発したことにより椎体において皮質骨のみではなく海綿骨の骨折治療効果を解析することを可能にした。従って、本発明の椎体骨折治療剤が使用される対象としては、主に海綿骨における椎体骨折又は皮質骨と海綿骨にまたがる椎体骨折の治療となる。
　また、本発明における椎体骨折治療剤が使用される患者としては、治療開始時にすでに椎体に１箇所以上の骨折が認められる患者であり、好ましくは椎体骨折が椎体圧迫骨折までは至っていない患者であり、より好ましくは椎体に骨折線が認められるが椎体全体の構造にはほぼ変化の無い患者からグレード１（椎体の前縁高、中央高及び／又は後縁高の変化が約２０~２５％減少、ならびに面積が１０~２０％減少）（Ｆｕｋｕｎａｇａ等、ジャーナル・オブ・ボーン・アンド・ミネラル・メタボリズム、２２巻　ｐ１０４−１１０、２００４年）までの患者である。治療開始前の椎体骨折の検出及び／又は椎体高の減少に関する診断は、例えば単純Ｘ線、ＣＴ、ＭＲＩなどの方法で当業者であれば実施することができる（元文等、日医大医会誌、５巻ｐ１２５−１２９、２００９年；Ｍａｊｕｍｄａｒ等、アチーブス・インターナル・メディシン、１６５巻　ｐ９０５−９０９、２００５年；Ｈａｒｖｅｙ等、ザ・ブリティッシュ・ジャーナル・オブ・ラディオロジー、７０巻　ｐ６４５−６４９、１９９７年）。
　また本発明における椎体骨折治療剤の効果についても同様に、単純Ｘ線、ＣＴ、ＭＲＩなどの方法を用いて行なうことが可能であり、この効果の指標としては治療前に存在した骨折線の長さ及び／又は領域の減少又は消失、椎体高や面積の回復などを指標として判定することができる。また、このような椎体の形状に基づく診断や治療効果の判定では、例えば日本特許第３２２９１７９号、日本特許第３２５８２３３号、日本特許特許第４４９５８９１号、日本特許第３８８８９７５号などに記載の画像解析技術を用いて適宜実施することも可能である。
　椎体骨折による症状としては背部及び／又は腰部への疼痛が生じることがある。また、発生した椎体骨折が治療されずに放置された場合、当初は感じられなかった疼痛が発生することもある。従って、そのような疼痛の軽減又は消失、若しくは疼痛の予防効果をもって本発明の椎体骨折治療剤の効果を判定することも可能である。その意味で、本発明はまた、椎体骨折に起因する疼痛の治療又は予防剤に関する。
　一方、椎体骨折が治療されずに進行した場合には椎体圧迫骨折（椎体の中央高／前縁高・中央高／後縁高のいずれかの値が０．８以下、又は前縁高／後縁高の値が０．７５以下の場合、若しくは椎体の高さが全体的に減少する場合、判定椎体の上位・下位椎体より高さが２０％以上減じている場合に椎体圧迫骨折と判定）にまでいたる場合がある。この椎体圧迫骨折の診断は単純Ｘ線、ＣＴ、ＭＲＩなどを用いた測定で行なわれる。従って、そのような圧迫骨折への進展の予防効果をもって本発明の椎体骨折治療剤の効果を判定することも可能である。その意味で、本発明はまた、椎体圧迫骨折の予防剤に関する。
　また、椎体骨折に関連する症状としては、運動障害や神経障害を伴っている場合や治療されずに病態が進行することによってそのような運動障害や神経障害に進展する場合があり、特に強い疼痛を伴う椎体骨折や椎体圧迫骨折で大きな椎体損傷が生じる場合にはリスクが高くなる。従って、そのような運動障害及び／又は神経障害の軽減又は消失、若しくは運動障害及び／又は神経障害の予防効果をもって本発明の椎体骨折治療剤の効果を判定することも可能である。その意味で、本発明はまた、椎体骨折に起因する運動障害及び／又は神経障害の治療又は予防剤に関する。
　本発明の椎体骨折治療剤は、薬学的に許容されるいずれの添加剤を含有してもよい。薬学的に許容される添加剤を用いての製剤は「ＲＥＭＩＮＧＴＯＮ：ＴＨＥ　ＳＣＩＥＮＣＥ　ＡＮＤ　ＰＲＡＣＴＩＣＥ　ＯＦ　ＰＨＡＲＭＡＣＹ　２０ｔｈ　ＥＤＩＴＩＯＮ　フィラデルフィア科学大学（Ｕｎｉｖｅｒｓｉｔｙ　ｏｆ　ｔｈｅ　Ｓｃｉｅｎｃｅｓ　ｉｎ　Ｐｈｉｌａｄｅｌｐｈｉａ）著　Ｗｉｌｌｉａｍｓ　＆　Ｗｉｌｋｉｎｓ社　２０００年１２月１５日発行」に記載の方法で実施することも可能である。このような医薬組成物の一つの形態としては、無菌の水性液若しくは油性液に溶解、懸濁又は乳化することによって調製された液剤として供される。このような溶剤として、水性液としては注射用蒸留水、生理食塩水等が挙げられ、それに加えて浸透圧調節剤（例えば、Ｄ−グルコース、Ｄ−ソルビトール、Ｄ−マンニトール、塩化ナトリウムなど）が添加されたり、適当な溶解補助剤、たとえばアルコール（たとえばエタノール）、ポリアルコール（たとえばプロピレングリコール、ポリエチレングリコール）、非イオン性界面活性剤（たとえばポリソルベート８０、ポリオキシエチレン硬化ヒマシ油５０）などが併用される場合もある。また、溶剤としては油性液が用いられる場合もあり、該油性液の例としてはゴマ油、大豆油などがあげられ、溶解補助剤として安息香酸ベンジル、ベンジルアルコールなどが併用される場合もある。このような液剤においては適宜、緩衝剤（例えば、リン酸塩類緩衝剤、酢酸塩類緩衝剤）、無痛化剤（例えば、塩化ベンザルコニウム、塩酸プロカインなど）、安定剤（例えば、ヒト血清アルブミン、ポリエチレングリコールなど）、保存剤（例えば、アスコルビン酸、エリソルビン酸及びそれらの塩など）着色剤（例えば、銅クロロフィル、β−カロチン、赤色２号、青色１号など）、防腐剤（例えばパラオキシ安息香酸エステル、フェノール、塩化ベンゼトニウム、塩化ベンザルコニウムなど）増粘剤（例えばヒドロキシプロピルセルロース、カルボキシメチルセルロース及びそれらの塩等）、安定化剤（例えば人血清アルブミン、マンニトール、ソルビトールなど）、矯臭剤（例えばメントール、柑橘香料など）の添加剤が用いられる場合がある。また別の医薬組成物（上記いくつか「医薬品組成物」の表現と統一するため）の形態としては、散剤、錠剤、顆粒剤、カプセル剤、丸剤、坐剤、トローチ剤などの固形剤があげられる。経口用製剤の形で投与する固形剤の場合には、添加剤として、賦形剤（例えば、結晶性セルロース、ラクトース、デンプンなど）、滑沢剤（例えば、ステアリン酸マグネシウム、タルクなど）、結合剤（ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、マクロゴールなど）、崩壊剤（例えば、デンプン、カルボキシメチルセルロースカルシウムなど）などが用いられる。また、必要に応じて、防腐剤（例えば、ベンジルアルコール、クロロブタノール、パラオキシ安息香酸メチル、パラオキシ安息香酸プロピルなど）、抗酸化剤、着色剤、甘味剤などの添加剤を用いることができる。さらに別の形態として、粘膜用医薬組成物もあげられ、この製剤においては粘膜への吸着性、滞留性等を付与することを主な目的として添加剤として粘着剤、粘着増強剤、粘稠剤、粘稠化剤等（例えば、ムチン、カンテン、ゼラチン、ペクチン、カラギーナン、アルギン酸ナトリウム、ローカストビンガム、キサンタンガム、トラガントガム、アラビアゴム、キトサン、プルラン、ワキシースターチ、スクラルフェート、セルロース及びその誘導体（例えば、ヒドロキシプロピルメチルセルロース、ポリグリセリン脂肪酸エステル、アクリル酸（メタ）アクリル酸アルキル共重合体又はその塩、ポリグリセリン脂肪酸エステルなど）が含有される場合もある。しかしながら、生体に供与される医薬組成物の形態及び溶剤や添加剤はこれらに限定されるものではなく、当業者であれば適宜選択できる。
　前記椎体骨折治療剤は、症状の改善を目的として、経口又は非経口的に投与することができる。経口投与の場合には、顆粒剤、散剤、錠剤、カプセル剤、液剤、シロップ剤、乳剤又は懸濁剤、エリキシル剤などの剤型を選択することができる。非経口投与の場合には、たとえば経鼻剤とすることができ、液剤、縣濁剤、固形製剤などを選択できる。また別の非経口投与の形態としては、注射剤とすることができ、注射剤としては、皮下注射剤、静脈注射剤、点滴注射剤、筋肉注射剤又は腹腔内注射剤などを選択することができる。またその他の非経口投与に用いる製剤としては、坐剤、舌下剤、経皮剤、経鼻剤以外の経粘膜投与剤なども挙げられる。さらに、ステントや血管内栓塞剤に含有若しくは塗布する態様で、血管内局所投与することもできる。
　前記医薬組成物の投与量は、患者の年齢、性別、体重、症状、治療効果、投与方法、処理時間、又は該医薬組成物に含有される活性成分の種類などにより異なるが、通常成人１人あたり、１回につき０．００１ｍｇから５００ｍｇの範囲で、好ましくは０．００５ｍｇから２００ｍｇの範囲で投与することができる。しかし、投与量は種々の条件により変動するため、上記投与量よりも少ない量で十分な場合もあり、また上記の範囲を超える投与量が必要な場合もある。
［椎体骨折治療剤の評価方法］
　また、本発明は、椎体骨折治療剤の評価方法に関する。
　本発明の評価方法は、１）非ヒト動物の椎体骨に外科的な損傷を与える工程、２）当該非ヒト動物に被検物質を投与する工程、３）当該非ヒト動物又はそれに由来する検体の椎体骨の外科的な損傷の回復について形態学的、生理学的、生化学的又は行動学的な測定をする工程、を含むことを特徴とする。また、本発明の評価方法には好ましくは、椎体骨の外科的な損傷の回復の形態学的、生理学的、生化学的又は行動学的な測定を行った結果を、さらに正常非ヒト動物若しくは又は対照非ヒト動物、又はそれに由来する検体における測定の結果と比較する工程も含まれる。一方、本発明評価方法で椎体骨に与える外科的な損傷については、椎体骨の皮質骨を通り海綿骨まで達する損傷であることが望ましく、損傷を与えることができれば特に限定されないが、ドリルによる穿孔により損傷を与える方法が好ましい。ドリル等及びそれを用いた穿孔の直径及び深さは、用いる動物や用いる椎体の部位によって適宜適切な用具を用いて適切な条件によって実施することが可能である。ラットの腰椎を例にとると歯科用ドリルを用いることが好ましく、その穿孔の直径としては好ましくは０．５~２．０ｍｍの間、より好ましくは０．５~１．５ｍｍの間、さらにより好ましくは０．７~０．８ｍｍの直径である。穿孔の深さは皮質骨から海綿骨に達する深さであればよいが、好ましくは皮質骨から海綿骨に達するが反対側の皮質骨までは達しない深さであり、より好ましくは２．５~３．０ｍｍの深さである。これらの条件は、最も汎用性のある２００~３５０ｇのラットを用いた場合であり、動物の大きさ、椎体の大きさに合わせて、適宜変更したほうが良い。
　本発明の評価方法における椎体骨の外科的な損傷の治癒効果の測定には、単純Ｘ線、ＣＴ若しくはマイクロＣＴ、ＭＲＩなどの形態観察の方法を用いて適宜実施することが可能である。本発明の実施例においては、ラットを用いた評価方法について、その外科的な損傷の治癒効果の測定としてＸ線マイクロＣＴを用いた方法を記載しているが、形態観察の方法としてはこれに限定されるものではない。また、本発明の評価方法における椎体骨の外科的な損傷の治癒効果の測定には、組織学的観察による方法も可能である。組織学的観察に用いる試料としては、外科的損傷を与えた椎体の組織に加えて、周辺の骨組織や軟骨・筋・神経等のその他の組織、また血液や脳脊髄液等の体液などが挙げられる。椎体骨やその周辺の組織の採取には、解剖による組織の採取のほか、バイオプシーによる組織採取の方法を用いることができる。組織学的観察に用いる試料は適宜、固定、薄切、及び／又は染色などの処理を行ったうえで観察に供される。また、生化学的及び／又は細胞学的なマーカー、例えば骨分化や代謝に関するマーカーについてのタンパク質や遺伝子の発現について検出する方法、例えば組織免疫化学やｉｎ・ｓｉｔｕハイブリダイゼーションやＰＣＲなどの手法を用いて、組織学的観察を行うことも、当業者であれば適宜実施することが可能である。また、採取した組織及び／又は体液から分離した細胞や液性成分、又は組織及び／又は細胞や体液から作成した抽出液などを用いて、生化学的なマーカー生化学的及び／又は細胞学的なマーカー、例えば骨分化や代謝に関するマーカーのタンパク質や遺伝子の発現の測定などを行う方法も公知の種々の方法で実施することができ、そのような方法も当該組織学的な解析に含まれる。椎体骨の外科的な損傷の治癒効果の測定を行う別の形態としては生理学的観察による測定であり、その例として痛覚の測定による方法である。非ヒト動物における痛覚の測定方法としては、例えば、機械的刺激性痛覚過敏反応（ｍｅｃｈａｎｉｃａｌ　ｈｙｐｅｒａｌｇｅｓｉａ）の程度を測定するテストであるｐａｗ　ｐｒｅｓｓｕｒｅテストやｖｏｎ　Ｆｒｅｙテスト、機械刺激性アロディニアの程度を測定するアロディニアテスト、温熱性痛覚過敏反応の程度を測定するｐａｗ　ｆｌｉｃｋテストなどが挙げられる（河谷編、痛み研究のアプローチ、ｐ３１−３３、２００６年、真興交易株式会社医書出版部）が、他にも多くの公知の情報があり、当業者であれば用いる動物や評価する部位に応じて適宜その方法を選択して実施することが可能である。
　また、本発明の評価方法では被検物質の投与による局所又は全身での炎症への影響についても評価することが可能である。局所での炎症への影響の測定には、上記のような組織学的な解析が用いられ、炎症性細胞、例えばリンパ球、単球／マクロファージ、肥満細胞、好中球、好酸球、好塩基球などの局所への集積の量や量の増減を調べることなどが挙げられる。また、組織又はそこから分離された細胞もしくはそれらからの抽出液を用いて、そこで発現している炎症性の因子、例えば腫瘍壊死因子（ＴＮＦ）、インターロイキン１（ＩＬ−１）やインターロイキン６（ＩＬ−６）などの炎症性サイトカインのタンパク質又はｍＲＮＡの量や量の増減を調べることでも測定することが可能である。全身での炎症への影響の測定には、血液又はその他の体液などの試料を用いて赤血球沈降速度や白血球数と白血球分画などの一般的な炎症反応のマーカーに加えて、上記のような炎症性サイトカインタンパク質、又はＣ反応性タンパク質（ＣＲＰ）、α１−アンチトリプシン、α１−アンチキモトリプシン、α１−酸性糖蛋白、血清アミロイドＡ、ハプトグロビン、セルロプラスミン、アルブミン、トランスサイレチン、トランスフェリンなど量や量の増減を調べる方法なども挙げられる。その他、局所又は全身での炎症に関する測定は、当業者であれば適宜方法を選択して実施することが可能である。
　さらに、本発明の評価方法では被検物質の投与による骨吸収への影響も測定することができる。骨吸収の測定は上記の外科的な損傷の治癒効果の測定における形態観察の方法が用いられることに加えて、骨体積密度の測定なども用いることができる。骨体積密度の測定方法は、例えば実施例３及び５に示されているマイクロＣＴを用いた方法などが例示できるが、当業者は適宜一般的な骨体積密度の測定方法を選択して実施することも可能である。その他にも、骨吸収への影響を評価する方法として血液や尿などの試料における骨吸収マーカーを用いる方法があり、そのような骨吸収マーカーとしては、例えばデオキシピリジノリン、Ｉ型コラーゲン架橋Ｎ−テロペプチド、Ｉ型コラーゲン架橋Ｃ−テロペプチド、酒石酸抵抗性酸フォスファターゼなどが挙げられるが、これに限定されるものではない。
　さらに、本発明の評価方法では被検物質の投与による脊髄の炎症又はそれに伴う脊髄の損傷についても評価することが可能である。脊髄の炎症や損傷について調べるためには、組織学的方法、生理学的方法や行動学的な方法などが用いられる。組織学的な方法としては、治癒効果の測定における外科的な損傷の治癒効果の測定における組織学的観察による方法に記載した方法に加えて、脊髄組織などの試料を用いて上記局所での炎症への影響の測定に記載した方法などを用いて実施することが可能である。生理学的な方法としては上記の外科的な損傷の治癒効果の測定での生理学的観察と同様の方法を用いることができる。また行動学的な方法としては、Ｂａｓｓｏ，Ｂｅａｔｔｉｅ，ａｎｄ　Ｂｒｅｓｎａｈａｎ（ＢＢＢ）ｓｃａｌｅ、立ち上がり行動やトレッドミルなどによる方法が挙げられる。さらに簡易的には脊髄の損傷にともない下肢に運動障害などが起こった場合は飼育ケージ内での摂餌行動にも支障をきたすことから、摂餌量の測定や体重の変化の測定などでも脊髄の損傷を評価することができる。その他、脊髄の炎症や損傷に関する測定は、当業者であれば適宜方法を選択して実施することが可能である。
　本発明における評価方法で用いる非ヒト動物は非ヒト脊椎動物であり、好ましくはヒト以外の霊長類又は齧歯類であり、当該齧歯類の動物としてはラットが好ましいものとして例示でき、マウスなどげっ歯類、ウサギ、イヌ、サルなど、いずれの非ヒト脊椎動物にも応用できる。また、本発明で用いる非ヒト動物は、骨粗鬆症モデルの非ヒト動物を用いることも可能であり、当該骨粗鬆症モデルとしては卵巣摘出モデルが例示できる。本発明における評価方法で用いる椎体骨の部位は、当業者であれば用いる非ヒト動物の種類によって頚椎・胸椎・腰椎・仙椎・尾椎のうちから適切な椎体の部位を選択して実施することが可能であるが、好ましくは胸椎及び／又は腰椎の椎体であり、より好ましくは腰椎の椎体を用いる方法である。本発明における実施例においては、非ヒト動物としてラットを用いており、この場合には腰椎、特に腰椎のうちの第４腰椎と第５腰椎を用いた方法が示されている。従って、用いる椎体の部位としては第４腰椎及び／又は第５腰椎がより好ましいが、それに限定されるわけではない。
　本発明における評価方法における被検物質の投与は、経口又は非経口による経路で実施することができ、当業者であれば用いる非ヒト動物の種類や評価部位、被検物質の物理的及び／又は化学的性質を勘案して、投与方法や投与部位を判断し、それに対応した被検物質を含有する投与用の試料を調整することが可能である。 The present inventors have established an animal model that causes damage to the fine structure of cancellous bone caused by vertebral fractures and an evaluation method thereof. With the establishment of an animal model that causes damage to the cancellous bone microstructure caused by vertebral fractures and the evaluation method, it is possible to screen for substances that can treat the damage to the cancellous bone microstructure caused by vertebral fractures. It became. As a result, hPTH (1-34) and PTHrP analog A (SEQ ID NO: 42) were found to be fine in the cancellous bone produced by vertebral fractures in the damaged vertebral body despite the dose not being detected in the undamaged vertebral body. It was effective in treating structural damage. That is, it was found that hPTH (1-34) and PTHrP analog A are substances that specifically show a therapeutic effect against damage to the fine structure of cancellous bone caused by vertebral fractures. Therefore, it has been found that a PTH / PTHrP receptor agonist, a substance having an activity of inducing PTH or PTHrP production can be a drug capable of treating cancellous bone microstructure damage caused by vertebral fractures.
In addition, a PTH / PTHrP receptor agonist, a substance having an activity of inducing PTH or PTHrP production can treat damage to the fine structure of cancellous bone, so that the bone structure is continuously broken and the vertebral body is crushed. It was found that the progress of vertebral fracture can be stopped.
Furthermore, it has been found that a PTH / PTHrP receptor agonist, a substance having an activity of inducing PTH or PTHrP production is a therapeutic or preventive agent for movement disorders and / or neurological disorders caused by vertebral fractures.
A PTH / PTHrP receptor agonist, a substance having an activity of inducing PTH or PTHrP production is a revolutionary drug that increases QOL of vertebral fracture patients and improves life prognosis. These drugs for treating new vertebral fractures can not only benefit individual patients, but also reduce inpatients and bedridden patients, reduce medical costs, and contribute significantly to social benefits.
[PTH / PTHrP receptor agonist]
The PTH / PTHrP receptor is a type of G protein-coupled receptor, also called PTH1R, PTH1 or PPR, which binds both parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP) to the hormone in the cell. It works to transmit signals. That is, it is considered that the function of the PTH / PTHrP receptor is involved in the display of the common function of PTH and PTHrP. In particular, this PTH / PTHrP receptor plays an important role in the action of PTH and PTHrP in calcium metabolism. Therefore, the therapeutic agent for vertebral fracture in the present invention is characterized by containing a substance that activates the PTH / PTHrP receptor, that is, a PTH / PTHrP receptor agonist.
Representative examples of PTH / PTHrP receptor agonists include, but are not limited to, PTH, PTHrP, or partial polypeptides thereof, or analogs thereof, which are described below, and bind to the PTH / PTHrP receptor to signal inside the cell. Any substance capable of generating vertebral body can be used in the therapeutic agent for vertebral fracture of the present invention. Examples of PTH / PTHrP receptor agonists other than such PTH, PTHrP, or their partial polypeptides, or analogs thereof include agonist antibodies to PTH / PTHrP receptors, forms that mimic the structure and properties of PTH or PTHrP PTH or PTHrP mimic molecule designed in the above, and low molecular weight compounds obtained by screening using PTH / PTHrP receptor activation as an index.
The activation of the PTH / PTHrP receptor can be determined by measuring the binding to the PTH / PTHrP receptor expressed in the cell and the intracellular signal. Measurement of agonist binding to PTH / PTHrP receptor is performed by directly labeling the test substance with a radioactive isotope, fluorescent substance, luminescent substance, or other substance for detection (substance related to biotin-avidin system, tag sequence, etc.) And reacting with a cell expressing the PTH / PTHrP receptor to determine the amount of the label detected for its binding. As another method, a standard substance is selected from PTH, PTHrP, or a partial polypeptide thereof, an analog thereof, or a substance that binds to a PTH / PTHrP receptor, and the standard substance is directly a radioisotope. Labeled with fluorescent substances, luminescent substances, and other detection substances (substances related to biotin-avidin system, tag sequences, etc.) and coexisting with test substances, and cells expressing PTH / PTHrP receptors By reacting, it is possible to determine whether the binding of the standard substance to the PTH / PTHrP receptor is competitively inhibited by the presence of the test substance by examining the amount of the label of the standard substance. In addition to the method using a cell expressing a PTH / PTHrP receptor, the binding of an agonist to the PTH / PTHrP receptor is similar to the method using the cell by using a cell membrane prepared from the cell. It is possible to measure with. Regarding the measurement of intracellular signal generation by binding of a PTH / PTHrP receptor agonist, the concentration of cAMP (cyclic adenosine monophosphate) in cells is increased by reacting a test substance with a cell expressing PTH / PTHrP receptor. Can be measured by examining. Measurement of intracellular cAMP concentration can be carried out by a person skilled in the art using information on existing techniques as appropriate, and commercially available kits can also be used. For example, JP-A-11-509201 It can also be implemented by the method described. Further, as a method for measuring intracellular signal generation due to binding of a PTH / PTHrP receptor agonist, a test substance is reacted with a cell expressing a PTH / PTHrP receptor to examine an increase in intracellular calcium ion concentration. Can be measured. The increase in intracellular calcium ion concentration can be achieved by using calcium-sensitive fluorescent substances such as Fura-2AM and Fura-4AM and calcium-sensitive luminescent substances such as aequorin. It is possible to implement. In addition, as another method for measuring intracellular signal generation due to the binding of a PTH / PTHrP receptor agonist, it can be measured by examining the activity increase of intracellular phospholipase A2 (PLA2). As a method for investigating the enhancement of PLA2 activity, those skilled in the art can appropriately carry out using information on existing techniques. For example, commercially available kits such as cPhospholipase A2 Colorimetric Assay Kit from Cayman Chemical Co., and Molecular Probes. It is possible to investigate by using commercially available reagents such as Red / Green BODIPY (R) PC-A2 of the company.
PTH / PTHrP receptor-expressing cells that use PTH / PTHrP receptor binding or intracellular signals for measurement are cells that endogenously express PTH / PTHrP receptor or PTH / PTHrP artificially expressed Any of the cells can be used. The cells that endogenously express the PTH / PTHrP receptor are cells and cell lines obtained from tissues and cells expressing the PTH / PTHrP receptor in vivo in humans and non-human animals, Examples of tissues and cells expressing the PTH / PTHrP receptor include osteoblasts / bone cells and osteoclasts in bones, kidney tubules and collecting duct cells in kidneys, smooth muscles in uterus and small intestine, It refers to cells obtained from glandular tissue, epithelial cells, and other ovarian and liver cells (from the description of PHT1 in IUPHAR DATABASE / http: //www.iufar-db.org/index.jsp/). Examples of cell lines that endogenously express the PTH / PTHrP receptor include, for example, osteoblast cell lines. More specifically, human osteosarcoma cell lines SaOS-2 cells and rat bones And ROS17 / 2.8 cells which are blast-like cells. A cell in which a PTH / PTHrP receptor is artificially expressed is a cell in which a gene encoding a PTH / PTHrP receptor protein is introduced into a host cell by a genetic engineering technique and the PTH / PTHrP receptor protein is expressed. Say. Examples of the PTH / PTHrP receptor protein include a human PTH / PTHrP receptor protein whose amino acid sequence is shown in SEQ ID NO: 1. In a host cell into which the gene encoding this human PTH / PTHrP receptor protein has been introduced, it is translated as a protein having the amino acid sequence of SEQ ID NO: 1, and then the signal sequence, from the N-terminal to 28 amino acids, is cut off, A mature human PTH / PTHrP receptor protein consisting of the 29th and subsequent amino acid sequences is expressed on the cell surface. In addition to the human PTH / PTHrP receptor protein, the PTH / PTHrP receptor of chimpanzee (amino acid sequence) shown in SEQ ID NO: 2 and the rhesus monkey (Macaca mulatta) whose amino acid sequence is shown in SEQ ID NO: 3 PTH / PTHrP receptor), the PTH / PTHrP receptor of orangutan (SEQ ID NO: 4) whose amino acid sequence is shown in SEQ ID NO: 4, and the PTH / PTHrP receptor of pig (Sus scrofa) whose amino acid sequence is shown in SEQ ID NO: 5 PTHrP receptor, equine PTH / PTHrP receptor whose amino acid sequence is shown in SEQ ID NO: 6, bovine PTH / PTHrP receptor whose amino acid sequence is shown in SEQ ID NO: 7 A common marmoset (Callithrix jacchus) PTH / PTHrP receptor whose amino acid sequence is shown in SEQ ID NO: 8, a dog (Canis lupus familiaris) PTH / PTHrP receptor shown in SEQ ID NO: 9, The rat (Rattus norvegicus) PTH / PTHrP receptor, the mouse shown in SEQ ID NO: 11 (Mus musculus), the PTH / PTHrP receptor shown in SEQ ID NO: 12, and the Mondelphis_domestica shown in SEQ ID NO: 12 Cells expressing a protein such as PTH / PTHrP receptor can also be used. Furthermore, the homology between the human PTH / PThrP receptor and the Pyro / PTHrP of the gray porpoise PTH / PTHrP is a homology that does not allow conservative amino acid substitutions when NCBI's BLASTp method is used in parallel. Identities) and 80% homology to allow conservative amino acid substitutions (Positives). Therefore, the PTH / PTHrP receptor that can be used for the binding of the PTH / PTHrP receptor agonist of the present invention and the measurement of intracellular signal generation includes an amino acid sequence of human PTH / PTHrP receptor and 80% or more homology. And PTH / PTHrP receptors other than those described above, which retain the functions of PTH / PTHrP receptors. It is known that PTH / PTHrP such as human binds / acts on the opossum PTH / PTHrP receptor (Jupner H. et al. Endocrinology 134 pp 879 1994). In order to examine that the PTH / PTHrP receptor retains the function of the PTH / PTHrP receptor, as a known PTH / PTHrP receptor agonist such as PTH / PTHrP and its active partial polypeptide or analog thereof, This can be confirmed by examining the binding of substances having activity and the generation of intracellular signals. The host for genetically expressing the PTH / PTHrP receptor is appropriately selected from animal cells (derived from humans and non-human animals), insect cells, yeast cells, bacteria or viruses / bacteriophages. It is preferably an animal cell, more preferably an animal cell line established from an animal cell. Examples of the animal cell line include Cos-7 cell line, LLC-PK1 cell line, HEK293 cell line, Act20 cell line and the like.
[PTH, PTHrP or a partially active polypeptide thereof]
Parathyroid hormone (PTH) is a peptide hormone found in the body of vertebrates more than fish, and is expressed in the form of an inactive preprohormone and is subjected to a process, and usually becomes PTH consisting of 84 amino acids. . PTH consisting of 84 amino acids is also referred to as paratormon, and may be referred to as PTH (1-84) to distinguish it from other active partial polypeptides derived from PTH. One example of PTH used in the present invention is human PTH (1-84) whose amino acid sequence is shown in SEQ ID NO: 13. As examples of PTH corresponding to non-human PTH (1-84), SEQ ID NO: 14 includes a common marmoset (Callithrix jacchus), SEQ ID NO: 15 includes a giant panda (Ailuropoda melanoleuca), and SEQ ID NO: 16 includes Horse (Ecus cavallus), SEQ ID NO: 17 for dogs (Canis lupus familiaris), SEQ ID NO: 18 for bovine (Bos taurus), SEQ ID NO: 19 for cats (Felis catus), SEQ ID NO: 20 for pigs (Sus scrofa) SEQ ID NO: 21 is a rabbit (Oryctolagus cuniculus), SEQ ID NO: 22 is a rat (Rattus norvegicus), and SEQ ID NO: 23 is a mouse (Mus muscu). US) PTH (1-84) derived from each species, and PTH (1-84) derived from these non-human species can also be used in the present invention, but is limited to these. There is no.
Also, BLAST (The Basic Local Alignment Search Tool) default parameters (matrix: BLOSUM62, ce ent = exit; Exit cost; Exit; Exc;; Cost cost; Exit; Exc;; Cost cost; Exit; Exc; = 1) When comparing human PTH (1-84) and mouse PTH (1-84), the homology that matches the amino acid sequence of both is 71%. Showed 86% similarity. Therefore, even PTH (1-84) in human or non-human species not specifically described in this specification shows 70% or more homology with human PTH (1-84). If present, it is included in PTH (1-84) used in the present invention.
Also included in the PTH that can be used in the present invention is a PTH partially active polypeptide having a partial amino acid sequence of PTH (1-84). As a representative partial active polypeptide of human PTH, human PTH (1-37) (CP Schmitt et al., Kidney Int., 57, 1484 (2000)) (N-terminal side 1 of SEQ ID NO: 1) To the 37th amino acid sequence), human PTH (1-34) (corresponding to the 1st to 34th amino acid sequence on the N-terminal side of SEQ ID NO: 1), human PTH (1-31) (J. F. Whitfield and P. Morley, Trends Pharmacol. Sci., 16, 382 (1995) (corresponding to the amino acid sequence from the 1st to 31st N-terminal side of SEQ ID NO: 1). In particular, PTH (1-34), also called teriparatide, is a partially active polypeptide of PTH that was first artificially synthesized and shown to have biological activity equivalent to that of PTH (1-84) (J. T. Potts, J. Endocrinol. 187, 311 (2005)). Examples of PTH partially active polypeptides that can be used in the present invention include PTH (1-37) and PTH (1-34) in PTH (1-84) derived from the above-mentioned non-human species. Alternatively, a polypeptide corresponding to PTH (1-31) can be mentioned.
In addition, as a peptide consisting of 32 to 33 amino acids derived from other species having an amino acid sequence close to human PTH (1-34), PTH (1-33) of chicken (Gallus gallus) (SEQ ID NO: 24), Examples include PTH (1-33) (SEQ ID NO: 25) of zebra finch (Taeniopygia guttata) and PTH (1-33) (SEQ ID NO: 26) of Monodelphis domestica, which can also be used in the present invention. Included in PTH. It is known that PTH / PTHrP such as human binds / acts on the opossum PTH / PTHrP receptor (Jupner H. et al. Endocrinology 134 pp 879 1994). Comparing human PTH (1-34) and gray porpoise Poss (1-33) by the BLAST method, the homology between them is about 63%, and 82% when substitution with an amino acid with similar properties is permitted. The similarity was shown. In human PTH, it is known that the first 21 amino acids of the PTH (1-34) sequence are important for binding to the PTH / PTHrP receptor and generation of a signal (Gerdella FJ et al J. Biol. Biol.Chem.270 pp6584 1995, Gerdella FJ et al J.Biol.Chem.271 pp1988, 1996), 12 amino acids among gray porpoise POSS (1-33) against human PTH (1-34) Substitution occurred at amino acids of the sequence (9 when substitution with amino acids with similar properties was permitted).
63% or more homology with human PTH (1-34), even for PTH (1-34) or partially active polypeptides thereof in human or non-human species not specifically described in this specification Or those having 12 or less amino acid substitutions are included in PTH (1-34) or a partially active peptide thereof used in the present invention.
Parathyroid hormone-related peptide (PTHrP) is a peptide hormone found in the living body of vertebrates more than fish and is structurally similar to PTH and acts on calcium metabolism in the same way as PTH. PTHrP is expressed in an inactive preprohormone and undergoes a process, and then becomes PTHrP, usually consisting of 141 or 139 amino acids. PTHrP consisting of 141 amino acids or 139 amino acids may be referred to as PTHrP (1-141) and PTHrP (1-139), respectively, in order to distinguish them from other active partial polypeptides derived from PTH. As one example of PTHrP used in the present invention, human PTHrP (1-141) (isoform 1) whose amino acid sequence is shown in SEQ ID NO: 27, or PTHrP (SEQ ID NO: 28) whose amino acid sequence is shown 1-139) (isoform 2).
Also, it corresponds to non-human PTHrP (1-141) or PTHrP (1-139)
Examples of PTH are: Rhesus macaque PTHrP (1-139), SEQ ID NO: 29, Giant Panda (Ailuropoda melanoleuca) PTH (1-141), SEQ ID NO: 31, Bos taurus (SEQ ID NO: 31). PTHrP (1-141), porcine (Sus scrofa) PTHrP (1-141) of SEQ ID NO: 32, canine (Canis lupus familiaris) PTHrP (1-141) of SEQ ID NO: 34, equine caballus (SEQ ID NO: 34) PTHrP (1-141), rat (Rattus norvegicus) PTHrP (1-141) of SEQ ID NO: 35, mouse (SEQ ID NO: 36) PTHrP (1-14) ) And SEQ ID NO: 37 rabbit (Oryctolagus cuniculus) PTHrP (1-141), and PTH (1-84) derived from these non-human species can also be used in the present invention, but is not limited thereto. Never happen. Moreover, when comparing human PTHrP (1-141) and rabbit PTHrP (1-141) with NIH's default parameters in the BLAST method, homology (Identity) in which both amino acid sequences match is 90%, amino acids with similar properties When the substitution to was permitted, 91% similarity was shown.
Thus, human PTHrP (1-141) and 90% of PTHrP (1-141) or PTHrP (1-139) in human or non-human species not specifically described in this specification. Anything exhibiting the above homology is included in PTHrP (1-141) or PTHrP (1-139) used in the present invention.
As a representative partial active polypeptide of human PTHrP, human PTHrP (1-86) (E. Lewin et al., Kidney Int., 58, 71 2000) (N-terminal side 1st to 86th of SEQ ID NO: 27) PTHrP (1-40) (E. Lewin et al., Kidney Int., 58, 71 2000) (corresponding to the 1st to 40th amino acid sequences on the N-terminal side of SEQ ID NO: 27) ), PTHrP (1-37) (LJ Suva et al., Science, 237, 893, 1987) (corresponding to the first to 37th amino acid sequence on the N-terminal side of SEQ ID NO: 27), PTHrP (1 -34) (BE Kemp et al., Science, 238, 1568 19 7) (corresponding from the N-terminal side 1 of SEQ ID NO: SEQ ID NO: 27 to 34 amino acid sequence) can be exemplified.
Examples of PTHrP partially active polypeptides that can be used in the present invention include PTHrP (1-86) in PTHrP (1-141) or PTHrP (1-139) derived from the above-mentioned non-human species. , PTHrP (1-40), PTHrP (1-37), or a polypeptide corresponding to PTHrP (1-34).
In addition, as a polypeptide consisting of 34 amino acids derived from other species having an amino acid sequence close to human PTHrP (1-34), PTHrP (1-34) (SEQ ID NO: 38) of Platypus (Ornithorhynchus anatinus), zebra finch PTHrP (1-34) (Taeniopygia guttata) (SEQ ID NO: 39), PTHrP (1-34) (Sequence No. 40) of chicken (Gallus gallus), PTHrP (1-34) of Monodelphis domestica SEQ ID NO: 41), and these are also included in the PTH that can be used in the present invention. It is known that PTH / PTHrP such as human binds / acts on the opossum PTH / PTHrP receptor (Jupner H. et al. Endocrinology 134 pp 879 1994). Comparing human PTHrP (1-34) and gray porpoise POSSrP (1-34) by the BLAST method, the homology between them is about 77%, and 83% when substitution with an amino acid with similar properties is permitted. The similarity was shown.
In addition, as described above, it is known that PTH and PTHrP bind to a common receptor PTH / PTHrP and cause a signal to cells (Jupner H. et al. Science. 254 p1024 1991), and human PTH. It is also known that in human PTHrP, the first 21 amino acids of the sequences of PTH (1-34) and PTHrP (1-34) are important for binding to the PTH / PTHrP receptor and generation of signals (Gerdella FJ et al J. Biol. Chem. 270 pp6584 1995, Gerdella FJ et al J. Biol. Chem. 271 pp 1988 1996). Comparing the first 21 amino acids of human PTH (1-34) and human PTHrP (1-34), there is a difference in 12 amino acids (7 if substitution with amino acids with similar properties is permitted) Exists. Therefore, even PTH, PTHrP, or a partially active polypeptide thereof in human or non-human species not specifically described in this specification may be human PTH (1-34) or human PTHrP (1- 34) any PTH, PTHrP, or a partially active polypeptide thereof having an amino acid sequence having no more than 12 amino acid substitutions relative to the amino acid sequence corresponding to 21 amino acids from the first N-terminal of 34) , PTHrP or a partially active polypeptide thereof.
PTH, PTHrP or a partially active polypeptide as described above is produced by extracting from the animal or cell producing the peptide, and a gene encoding the polypeptide is introduced into the host by genetic engineering. Any of those produced by expression in a recombinant cell, transgenic non-human animal or transgenic plant, or those produced by a peptide chemical synthesis method such as a solid phase synthesis method can be used. In addition, PTH, PTHrP or a partially active polypeptide thereof is available from a reagent manufacturer, for example, from Bachem, PTH (1-84) (human) (Cat. No. H-1370), PTH ( 1-37) (human) (Cat. No. H-5974), PTH (1-34) (human) (Cat. No. H-4835), PTH (1-31) (human) (Cat. No. 1). H-2274), PTH (1-31) amide (human) (Cat. No. H-3408), PTH-Related Protein (1-86) (human) (Cat. No. H-9815), PTH-Related Protein (1-40) (human, mouse, rat) (Cat. No. H-6810), P H-Related Protein (1-37) (human, mouse, rat) (Cat. No. H-5494), pTH-Related Protein (1-34) (human, mouse, rat) (Cat. No. H-6630) ) PTH-Related Protein (1-34) amide (human, mouse, rat) (Cat. No. H-9095) and the like, and such PTH, PTHrP or a partially active polypeptide thereof may also be used. Is possible.
PTH, PTHrP or a partially active polypeptide thereof used in the present invention has a carboxyl group (COOH) state or amidation (CONH) as its C-terminal structure.2) Furthermore, the PTH, PTHrP or the partially active polypeptide of the present invention is a compound, an amino acid, PTH, PTHrP or a combination thereof at any one of the N-terminus, C-terminus, side chain of amino acid residues, or a combination of two or more thereof. A form to which a protein or peptide other than the partially active polypeptide is added may also be used. Examples of the added compound include substances that stabilize when administered in vivo, such as polyethylene glycol (PEG), and compounds having medicinal properties such as bisphosphonates. The state in which an amino acid is added refers to a form in which a methionine residue, acetyl group, pyroglutamic acid, or the like is bonded to the N-terminus, or a proteolytic enzyme prepared as a fusion protein in the production of PTH, PTHrP or a partially active polypeptide thereof Addition of 1 to several amino acids remaining at the N-terminal or C-terminal when cleaving is performed. Examples of the protein to be added include proteins involved in stabilization and transportation in vivo such as albumin and immunoglobulin. Examples of the peptide to be added include tag sequences (histidine tag, FLAG tag, etc.) used for purification in the production process, other bioactive polypeptides, and the like. For such addition of compounds or amino acids, proteins or peptides other than PTH, PTHrP or partially active polypeptides thereof, known methods (Hermanson et al., Bioconjugate techniques (Bioconjugate techniques) ( USA) (Academic Press) issued in 1996, which can be carried out by enzymatically or chemically associating. In addition, in the case of a structure in which an amino acid or protein / peptide is added to the N-terminal and / or C-terminal of PTH, PTHrP or a partially active polypeptide thereof, a gene for expression that encodes the polypeptide having the structure by genetic engineering And can be prepared as a gene expression product using a host cell, a transgenic plant or a non-human transgenic animal into which the gene has been introduced.
[Analog of PTH, PTHrP, or partially active polypeptides thereof]
PTH, PTHrP, or an analog of a partially active polypeptide thereof is an amino acid sequence of one or more of the amino acid sequences in the above-mentioned PTH, PTHrP, or a partially active polypeptide thereof. In which the activity as a PTH / PTHrP receptor agonist is retained. As a method for examining whether the PTH, PTHrP, or an analog of a partially active polypeptide thereof retains activity as a PTH / PTHrP receptor agonist, the PTH / PTHrP described in the above [PTH / PTHrP receptor agonist] is used. It can be carried out in the same manner as the method for examining receptor activation. Amino acid substitution includes conservative substitution and non-conservative substitution. Conservative substitution refers to substitution between amino acids having similar properties, and non-conservative substitution refers to other amino acid substitution. In general, it is considered that amino acid conservative substitutions are more likely to preserve the function of the underlying molecule than non-conservative substitutions. Therefore, it is possible to obtain polypeptides having equivalent effects by conservative substitution of amino acids.
One aspect of considering the closeness of amino acid properties in conservative substitution is substitution with a combination of amino acids that can be substituted without significantly affecting the polarity and charge of the amino acid side chain. This is due to changes in the hydrophobicity and charge of the amino acid side chains, and secondary and higher order structures with other amino acid side chains, water molecules, and other protein components (metal ions, etc.) between and within the molecule. This is based on the idea that the structure and function will be preserved if there is little change in hydrophobicity or charge. Normal natural proteins and peptides are composed of 20 kinds of amino acids (L-amino acids when nothing is specified), but their side chains are large and nonpolar (non-polar) Polarity). Amino acids having side chains with polarity are classified into those having side chains that are charged under physiological pH conditions and those that are not charged (uncharged). Further, those having a charged side chain are classified into acidic side chains and basic side chains. On the other hand, amino acids having nonpolar side chains are classified into those having aliphatic side chains and those having aromatic side chains.
According to these classifications, 20 kinds of amino acids constituting normal natural proteins and peptides are aspartic acid (Asp) and glutamic acid (Glu) are polar / charged / acidic side chain amino acids, arginine (Arg) and lysine (Lys). ) And histidine (His) are polar / charged / basic side chain amino acids, glycine (Gly), serine (Ser), threonine (Thr), asparagine (Asn), glutamine (Gln), tyrosine (Tyr) and cysteine ( Cys) is a polar / uncharged side chain amino acid, alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met) and proline (Pro) are nonpolar / aliphatic side chains. Amino acids, phenylalanine (Phe) and tryptophan (Trp) are nonpolar / It is classified as an amino acid of the aromatic side chains. Amino acids belonging to the same class are often used for conservative amino acid substitution. However, Cys may be involved in the formation of intramolecular or intermolecular crosslinks via disulfide bonds, and Pro may affect the secondary structure of proteins / peptides.
It may not be handled by replacement.
Similarly, a hydrophobicity index obtained by quantifying hydrophobicity or charge may be used as an index of physicochemical properties of amino acid side chains. As an example of the hydrophobic index, the hydrophobic index scores of two kinds of amino acids contained in a natural protein are Arg: −10.0, Lys: −9.9, Glu and Asp: −8.3, Asn: − 7.1, Gln: -6.0, Ser: -4.3, His and Thr: -3.8, Gly: -2.4, Cys: -2.3, Ala: -1.1, Pro: -0.2, Tyr: 2.5, Val: 4.1, Met: 4.6, Ile: 8.7, Leu and Trp: 9.7, and Phe: 10. Examples of special amino acids not included in normal proteins include α, β-diaminopropionic acid (Dap): −9.5, α-aminoisobutyric acid (Aib): 1.1, norleucine (Nle): The hydrophobic index scores, such as 9.1, have been calculated (Alessandro et al., PEPTIDES 2002, Proceeding of 27th European Peptide Symposim). In order to perform conservative amino acid substitution, in this hydrophobic index, it is considered that the closer the amino acid score is selected, the closer the hydrophobicity and charge are, and the more likely the structure and function are preserved.
As another method for determining another conservative amino acid substitution, there is a method using an amino acid substitution matrix used for homology comparison of amino acid sequences of peptides and proteins. Amino acid substitution matrix is a matrix in which sequences with similar structures and functions are collected and compared, and quantified according to the frequency and type of amino acid substitution, and is said to reflect the ease of relative substitution in the evolution process. . A typical example is PAM (Point-Accepted-Mutation) (MOO Dayhoff et al., Atlas of Protein Sequence and Structure 5, p345 1978) created by comparing the full-length amino acid sequences of the sequences forming the family. BLOSUM (Blocks Substitution Matrix) (S. Henikoff) and J. G. Henikoff, Processedings of the 19th National AcademyofSciences109 is there. In these substitution matrices, a larger value is given to combinations of amino acids considered to be conservative substitutions, and it is possible to select an amino acid mutation to be introduced with reference to the value. For example, for BLOSM64, one of the permutation matrices, the score for Ala is 4 for the same Ala, 1 for Ser, and 0 for Cys / Gly / Thr / Val. There is a high possibility that a general replacement can be made. On the other hand, Trp is -3 for Asa and Asn, Asp, His, Phe, and Tyr are as low as -2, indicating that the possibility of conservative substitution is low. Similarly, since the numerical values are given in the form of Lys 2, Gln 1, Asn · Glu · His 0 for Arg, PTH, PTHrP, or those with reference to the score in such a permutation matrix It is also possible to design analogs of these partially active polypeptides.
PTH, PTHrP, or an analog of a partially active polypeptide thereof by substitution of amino acids constituting ordinary natural proteins and peptides is a recombinant cell or a trans gene in which a gene encoding the polypeptide is genetically engineered into a host. It can be produced by a method for expression in a transgenic non-human animal or a transgenic plant, or a peptide chemical synthesis method such as a solid phase synthesis method.
In the amino acid substitution for creating analogs of PTH, PTHrP, or partially active polypeptides thereof, substitution to amino acids called non-protein amino acids other than the 20 types of amino acids normally contained in natural proteins as described above is also possible It is. Examples of non-protein amino acids include D-form amino acids that are engineering isomers. Of the 20 types of amino acids normally contained in natural proteins, the alpha carbon is an asymmetric carbon atom except for Gly, and takes L and D engineering isomers. Moreover, the amino acid normally contained in natural protein is a L-form. Accordingly, in the present invention, one in which one or more amino acids in the amino acid sequence of PTH, PTHrP, or a partially active polypeptide thereof are substituted with a D-form amino acid and the activity as a PTH / PTHrP receptor agonist is maintained. Included in the analogs of PTH, PTHrP, or partially active polypeptides thereof used in the invention. In addition, amino acids other than the 20 kinds of amino acids may be used as non-protein amino acids, for example, β- (2-naphthyl) alanine (β-Nal), norleucine (Nle), α, β-diaminopropionic acid. (Dap), cyclohexylalanine (Cha), norvaline (Nva), 4-amino-phenylalanine (Amp), 3-pyridinylalanine (Amp), α-aminoisobutyric acid (Aib), 1-amino-1-cyclo- Examples include hexanecarboxylic acid (Ahc). In addition to Ahc, 1-amino-1-cyclopropanecarboxylic acid, 1-amino-1-cyclobutanecarboxylic acid, 1-amino-1-cyclopentanecarboxylic acid, 1-amino-1-cycloheptanecarboxylic acid, A non-protein amino acid selected from the group of 1-amino-1-cyclooctanecarboxylic acid and 1-amino-1-cyclononanecarboxylic acid may be represented herein as Acc. Accordingly, in the present invention, those in which the amino acid sequence of PTH, PTHrP, or a partially active polypeptide thereof is substituted with one or more non-protein amino acids and the activity as a PTH / PTHrP receptor agonist is maintained in the present invention. Included in the analogs of PTH, PTHrP, or partially active polypeptides thereof used. An analog of PTH, PTHrP, or a partially active polypeptide thereof after substitution with these non-protein amino acids can be usually prepared by a known peptide synthesis method such as a solid phase synthesis method.
Another form of PTH, PTHrP, or an analog of a partially active polypeptide thereof is a peptide that has undergone intramolecular or intermolecular crosslinking. Examples of intramolecular cross-linking include cross-linking between Lys at position 26 and Asp at position 30 that are conserved between species in PTH (1-34) and conserved between species in PTHrP (1-34). A method of synthesizing a cyclic peptide in which intramolecular cross-linking, such as cross-linking between Lys at position 13 and Asp at position 17, and the activity of the cyclic peptide as a PTH / PTHrP receptor agonist are maintained. (Bisello A. et.al., Biochem 36 p3293 1997).
When designing an analog of PTH, PTHrP, or a partially active polypeptide thereof used in the present invention, it is possible to design so that a change in sequence due to amino acid substitution does not affect the secondary structure as much as possible. It is considered important for preservation. The secondary structure of PTH (1-34) and PTHrP (1-34) is known to have two α-helical structures on the N-terminal side and the C-terminal side, which is the PTH / PTHrP receptor. It is considered to play an important role in the activity as an agonist (Cohen EF et al., J. Biol. Chem. 266 p1997 1991). Therefore, when designing analogs of PTH, PTHrP, or partially active polypeptides thereof, by designing such that these two α-helix structures are not disrupted by amino acid substitution, as a PTH / PTHrP receptor agonist PTH, PTHrP, or analogs of partially active polypeptides thereof can be obtained. As a method for analyzing the secondary structure, the GOR1 (or Robson) method (Garnier, Osguthorpe and Robson, J. Mol. Biol. 120 p97 1978) and the PREDETOR method (Frishman D and Argos P, Protein Eng 9 p13319). It can be performed by other known methods.
In addition, the analog of PTH, PTHrP, or a partially active polypeptide thereof used in the present invention is an amino acid substitution or amino acid side chain at at least one position of the amino acid sequence of PTH, PTHrP, or a partially active polypeptide thereof. However, the upper limit of the number of amino acid substitutions is preferably within 12 positions within the range of 21 amino acids from the N-terminus of PTH, PTHrP, or their partially active polypeptides. This is because when comparing the first 21 amino acids of human PTH (1-34) and human PTHrP (1-34), there are 12 amino acid differences, but PTH (1-34) and PTHrP (1- 34) has the same activity as a PTH / PTHrP receptor agonist.
The analog of PTH, PTHrP or a partially active polypeptide used in the present invention has a carboxyl group (COOH) state or amidation (CONH) as its C-terminal structure.2) Furthermore, the analog of PTH, PTHrP or a partially active polypeptide thereof according to the present invention can be a compound, amino acid, PTH, PTHrP at the N-terminal, C-terminal, amino acid residue side chain, or a combination of two or more thereof. Alternatively, a form to which a protein or peptide other than an analog of the partially active polypeptide is added may be used. Examples of the added compound include substances that stabilize when administered in vivo, such as polyethylene glycol (PEG), and compounds having medicinal properties such as bisphosphonates. The state in which an amino acid is added refers to a form in which a methionine residue, an acetyl group, pyroglutamic acid, or the like is bound to the N-terminus, or after preparation as a fusion protein in the production of an analog of PTH, PTHrP or a partially active polypeptide thereof, Examples include addition of 1 to several amino acids remaining at the N-terminus or C-terminus when cleaved with a proteolytic enzyme. Examples of the protein to be added include proteins involved in stabilization and transportation in vivo such as albumin and immunoglobulin. Examples of the added peptide include tag sequences (histidine tag, FLAG tag, etc.) used for purification in the production process, other bioactive peptides, and the like.
There are many known specific structures of analogs of PTH, PTHrP, or partially active polypeptides thereof (for example, JP-A-9-157294, JP-A-5-320193, JP-A-5-32696). JP-A-5-509098, JP-A-5-505594, and Japanese Patent No. 3135122, JP-A-8-503692, JP-A-62-67099, JP-A-61-57600, JP-A-61-2598, 59-204159 and JP-B-3-14320, JP-A-59-42351, etc.), which are included in analogs of PTH, PTHrP, or partially active polypeptides thereof that can be used in the present invention.
More specifically, it is an analog of PTH, PTHrP, or a partially active polypeptide thereof shown in JP-A-11-509201, which is an analog of a partially active polypeptide of PTH represented by the following formula: peptide:
Figure JPOXMLDOC01-appb-C000007
 However,
A1Is Ser, Ala, or Dap;
A3Is Ser, Thr, or Aib;
A5Is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH3Is;
A7Is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH3Is;
A8Is Met, Nva, Leu, Val, Ile, Cha, or Nle;
A11Is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe or p-X-Phe, where X is OH, halogen, or CH3Is;
A12Is Gly or Aib;
A15Is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH3Is;
A16Is Ser, Asn, Ala, or Aib;
A17Is Ser, Thr, or Aib;
A18Is Met, Nva, Leu, Val, Ile, Nle, Cha, or Aib;
A19Is Glu or Aib;
A21Is Val, Cha, or Met;
A23Is Trp or Cha;
A24Is Leu or Cha;
A27Is Lys, Aib, Leu, hArg, Gln, or Cha;
A28Is Leu or Cha;
A30Is Asp or Lys;
A31Is Val, Nle, or Cha, or is missing;
A32Is His or missing;
A33Is Asn or missing;
A34Is Phe, Tyr, Amp, or Aib or is missing;
R1And R2Are independently H, C1-12Alkyl, C2-12Alkenyl, C2-12Alkynyl, C7-20Phenylalkyl, C11-20Naphthylalkyl, C1-12Hydroxyalkyl, C2-12Hydroxyalkenyl, C7-20Hydroxyphenylalkyl or C11-20Hydroxy naphthyl alkyl; or R1And R2Both or only one1In this case, E1Is C1-12Alkyl, C2-12Alkenyl, C2-12Alkynyl, C7-20Phenylalkyl, C11-20Naphthylalkyl, C1-12Hydroxyalkyl, C2-12Hydroxyalkenyl, C7-20Hydroxyphenylalkyl or C11-20Hydroxy naphthyl alkyl;
And R3Is OH, NH2, C1-12Alkoxy or -NH-Y-CH2-Z, where Y is C1-12Is a hydrocarbon moiety, Z is H, OH, CO2H or CONH2Is;
A5, A7, A8, A11, A15, A18, A21, A23, A24, A27, A28And A31At least one of is Cha or A3, A12, A16, A17, A18, A19And A34At least one of which is Aib; including polypeptides.
Among them, a polypeptide of the following formula which is an analog of human PTH (1-34) described in JP-T-11-509201 and Japanese Patent No. 4008825 is preferable:
[Cha7, 11HPTH (1-34) NH2;
[Cha23HPTH (1-34) NH2;
[Cha24HPTH (1-34) NH2;
[Nle8, 18, Cha27HPTH (1-34) NH2;
[Cha28HPTH (1-34) NH2;
[Cha31HPTH (1-34) NH2;
[Aib16HPTH (1-34) NH2;
[Aib19HPTH (1-34) NH2;
[Aib34HPTH (1-34) NH2;
[Cha24, 28, 31, Lys30HPTH (1-34) NH2;
[Cha7, 11, Nle8, 18, Tyr34HPTH (1-34) NH2;
[Cha7, 11, Nle8, 18, Aib16, 19, Tyr34HPTH (1-34) NH2;
[Cha7, 11, Nle8, 18, 31, Aib16, 19, Tyr34HPTH (1-34) NH2;
[Cha11HPTH (1-34) NH2;
[Cha28, 31HPTH (1-34) NH2;
[Cha7, 11, Nle8, 18, Aib34HPTH (1-34) NH2;
[Cha15HPTH (1-34) NH2;
[Cha7, 11, Aib19HPTH (1-34) NH2;
[Cha7, 11, Aib16HPTH (1-34) NH2;
[Aib16, 19HPTH (1-34) NH2;
[Aib12HPTH (1-34) NH2;
[Aib3HPTH (1-34) NH2;
[Cha7, 11, Aib19, Lys30HPTH (1-34) NH2;
[Cha7HPTH (1-34) NH2;
[Cha24, 28, 31HPTH (1-34) NH2;
[Aib17HPTH (1-34) NH2;as well as
[Cha7, 11, 15HPTH (1-34) NH2,
A polypeptide selected from the group consisting of: Here, [Cha7, 11HPTH (1-34) NH2The notation indicates that Leu at position 7 and Leu at position 11 of human PTH (1-34) are substituted with Cha (cyclohexylalanine), and the C-terminus is amidated. Similar notation also has the same meaning.
In addition, as an aspect of another PTH, PTHrP, or an analog of a partially active polypeptide thereof shown in JP-A-11-509201, an analog of a partially active polypeptide of PTHpP represented by the following formula: Polypeptide:
Figure JPOXMLDOC01-appb-C000008
 However,
A1Is Ala, Ser, or Dap;
A3Is Ser or Aib;
A5Is His, Ile, or Cha;
A7Is Leu, Cha, Nle, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH3Is;
A8Is Leu, Met, or Cha;
A10Is Asp or Asn;
A11Is Lys, Leu, Cha, Phe, or β-Nal;
A12Is Gly or Aib;
A14Is Ser or His;
A15Is Ile or Cha;
A16Is Gln or Aib;
A17Is Asp or Aib;
A18Is Leu, Aib, or Cha;
A19Is Arg or Aib;
A22Is Phe, Glu, Aib, or Cha;
A23Is Phe, Leu, Lys, or Cha;
A24Is Leu, Lys, or Cha;
A25Is His, Aib, or Glu;
A26Is His, Aib, or Lys;
A27Is Leu, Lys, or Cha;
A28Is Ile, Leu, Lys, or Cha;
A29Is Ala, Glu, or Aib;
A30Is Glu, Cha, Aib, or Lys;
A31Is Ile, Leu, Cha, or Lys, or is missing;
A32Is His or missing;
A33Is Thr or missing;
A34Is Ala or missing;
R1And R2Are independently H, C1-12Alkyl, C2-12Alkenyl, C2-12Alkynyl, C7-20Phenylalkyl, C11-20Naphthylalkyl, C1-12Hydroxyalkyl, C2-12Hydroxyalkenyl, C7-20Hydroxyphenylalkyl or C11-20Hydroxy naphthyl alkyl; or R1And R2Both or only one1In this case, E1Is C1-12Alkyl, C2-12Alkenyl, C2-12Alkynyl, C7-20Phenylalkyl, C11-20Naphthylalkyl, C1-12Hydroxyalkyl, C2-12Hydroxyalkenyl, C7-20Hydroxyphenylalkyl or C11-20Hydroxynaphthylalkyl; and R3Is OH, NH2, C1-12Alkoxy or -NH-Y-CH2-Z, where Y is C1-12Is a hydrocarbon moiety, Z is H, OH, CO2H or CONH2Is;
A5, A7, A8, A11, A15, A18, A22, A23, A24, A27, A28, A30Or A31At least one of is Cha or A3, A12, A16, A17, A18, A19, A22, A25, A26, A29, A30Or A34At least one of is Aib;
A polypeptide.
Among them, particularly preferred is an analog of human PTHrP (1-34), which has the formula: [Glu22, 25, Leu23, 28, 31, Aib29, Lys26, 30HPTHrP (1-34) NH2A polypeptide represented by (SEQ ID NO: 42).
Further, as an aspect as another analog of PTH, PTHrP, or a partially active polypeptide thereof, the following formula, which is an analog of an active partial peptide of PTHrP described in JP-T-2001-508439:
Figure JPOXMLDOC01-appb-C000009
 However,
A1Is Ala, Ser, or Dap;
A3Is Ser or Aib;
A5Is His, Ile, Acc, or Cha;
A7Is Leu, Cha, Nle, β-Nal, Trp, Pal, Acc, Phe, or p-X-Phe, where X is OH, halogen, or CH3Is;
A8Is Leu, Met, Acc, or Cha;
A10Is Asp or Asn;
A11Is Lys, Leu, Cha, Acc, Phe, or β-Nal;
A12Is Gly, Acc, or Aib;
A14Is Ser or His;
A15Is Ile, Acc, or Cha;
A16Is Gln or Aib;
A17Is Asp or Aib;
A18Is Leu, Aib, Acc, or Cha;
A19Is Arg or Aib;
A22Is Phe, Glu, Aib, Acc, or Cha;
A23Is Phe, Leu, Lys, Acc, or Cha;
A24Is Leu, Lys, Acc, or Cha;
A25Is His, Lys, Aib, Acc, or Glu;
A26Is His, Aib, Acc, or Lys;
A27Is Leu, Lys, Acc, or Cha;
A28Is Ile, Leu, Lys, Acc, or Cha;
A29Is Ala, Glu, Acc, or Aib;
A30Is Glu, Leu, Nle, Cha, Aib, Acc, or Lys;
A31Is Ile, Leu, Cha, Lys or Acc, or is missing;
A32Is His or missing;
A33Is Thr or missing;
A34Is Ala or missing;
R1And R2Are independently H, C1-12Alkyl, C2-12Alkenyl, C2-12Alkynyl, C7-20Phenylalkyl, C11-20Naphthylalkyl, C1-12Hydroxyalkyl, C2-12Hydroxyalkenyl, C7-20Hydroxyphenylalkyl or C11-20Hydroxy naphthyl alkyl; or R1And R2Both or only one1In this case, E1Is C1-12Alkyl, C2-12Alkenyl, C2-12Alkynyl, C7-20Phenylalkyl, C11-20Naphthylalkyl, C1-12Hydroxyalkyl, C2-12Hydroxyalkenyl, C7-20Hydroxyphenylalkyl or C11-20Hydroxy naphthylalkyl; and
R3Is OH, NH2, C1-12Alkoxy or -NH-Y-CH2-Z, where Y is C1-12Is a hydrocarbon moiety, Z is H, OH, CO2H or CONH2Is;
A5, A7, A8, A11, A12, A15, A18, A22, A23, A24, A25, A26, A27, A28, A29, A30Or A31At least one of is Acc;
A polypeptide. Among them, particularly preferred is a polypeptide of the following formula, which is an analog of human PTHrP (1-34) described in JP-T-2001-508439 and Japanese Patent No. 3963482:
[Glu22, 25, Leu23, 28, Lys26, 30, Aib29, Ahc31HPTHrP (1-34) NH2;
[Glu22, 25, Ahc23, Lys26, 30, Leu28, 31, Aib29HPTHrP (1-34) NH2;
[Glu22, 25, Leu23, 28, 31, Lys26, 30, Ahc27, Aib29HPTHrP (1-34) NH2;
[Glu22, 25, 29, Leu23, 28, 31, Lys26, Ahc30HPTHrP (1-34) NH2;
[Cha22, Leu23, 28, 31, Glu25, Lys26, 30, Ahc27, Aib29HPTHrP (1-34) NH2;
[Glu22, 25, Leu23, 28, 31, Ahc24, Lys26, 30, Aib29HPTHrP (1-34) NH2;
[Glu22, 29, Leu23, 28, 31, Aib25, Lys26, 30, Ahc27HPTHrP (1-34) NH2;
[Glu22, Leu23, 28, 31, Aib25, 29, Lys26, 30, Ahc27HPTHrP (1-34) NH2;
[Ahc22, Leu23, 28, 31, Glu25, Lys26, 30, Aib29HPTHrP (1-34) NH2;
[Glu22, 25, Leu23, 31, Lys26, 30, Ahc28, Aib29HPTHrP (1-34) NH2;
[Cha22, Ahc23, Glu25, Lys26, 30, Leu28, 31, Aib29HPTHrP (1-34) NH2;
[Cha22, Leu23, 28, 31, Ahc24, 27, Glu25, Lys26, 30, Aib29HPTHrP (1-34) NH2;
[Glu22, Leu23, 28, 31, Ahc24, 27, Lys25, 26, Aib29HPTHrP (1-34) NH2;
[Ahc18, 24, 27, Glu22, Cha23, Lys25, 26, Leu28, Aib29HPTHrP (1-34) NH2;
[Glu22, Cha23, Ahc24, Lys25, 26, Leu28, Aib29HPTHrP (1-34) NH2;
[Ahc22, 24, Leu23, 28, 31, Glu25, Lys26, 30, Aib29HPTHrP (1-34) NH2;
[Ahc22, 24, Leu23, 28, Lys25, 26, Aib29HPTHrP (1-34) NH2;
A polypeptide selected from the group consisting of:
Furthermore, as an aspect as another PTH, PTHrP, or an analog of those partially active polypeptides described in JP-T-11-509201 and Japanese Patent No. 4008825, PTH represented by the following formula Polypeptides that are analogs of partially active polypeptides:
[Nle31HPTH (1-34) NH2;
[HArg27HPTH (1-34) NH2;
[Dap1, Nle8, 18, Tyr34HPTH (1-34) NH2;
Is mentioned. Further, it is an analog of human PTHrP (1-34) or an analog of human PTH (1-34) described in JP-T-2001-508439 and Japanese Patent No. 3963482,
[Leu27, Aib29] hPTH (1-34) NH2
[Glu22, 25, Cha23, Lys26, Leu28, 31, Aib29, Nle30HPTHrP (1-34) NH2;
[Glu22, 25, Cha23, Lys26, 30, Leu28, 31, Aib29HPTHrP (1-34) NH2;
[Glu22, 25, Cha23, Lys26, 30, Aib29HPTHrP (1-34) NH2;
[Glu22, 25, Cha23, Lys26, 30, Leu28, Aib29HPTHrP (1-34) NH2;
Can also be mentioned as examples of analogs of PTH, PTHrP, or partially active polypeptides thereof.
[Substances that induce PTH or PTHrP production]
A substance having an activity of inducing PTH or PTHrP production means that the substance itself does not have an agonistic action directly on the PTH / PTHrP receptor, but the gene expression or protein of PTH / PTHrP in tissues or cells in vivo. PTH / PTHrP represented by PTH, PTHrP, or a partial peptide thereof, or an analog thereof by increasing the concentration of PTH / PTHrP systemically and / or locally by inducing the production and / or secretion of It refers to a substance having a healing effect on vertebral fractures as well as the effect of administration of a receptor agonist.
Examples of substances having an activity of inducing such PTH or PTHrP production include calcium sensitive receptor (CaSR) antagonists. It is known that secretion of PTH is enhanced in animals administered with a CaSR antagonist (Gowen et al., The Journal of Clinical Investigation, Vol. 105, p1595, 2000). Examples of CaSR antagonists include, for example, JTT-305 and MK-5442 (Fukumoto et al., CLINICAL CALCIUM, 21 p89, 2011), NPS2143 (Gowen et al., The Journal of Clinical Investigation, 105 p1595, 2000), SB-423557 (Matheny et al., Bone, 46, p534, 2010), SB-751689 (31th Annual Meeting, American Society for Bone and Mineral Research, oral presentation, presentation number 1051-1130 / poster announcement) ATX914 (American Society for Bone) and Mineral Research, 2010 Annual Meeting, poster presentations, Presentation # SU0372) and the like. Other examples of the CaSR antagonist include compounds disclosed in JP 2010-248183, JP 2005-239611 and JP 2010-159258. However, the substance having the activity of inducing PTH or PTHrP production is not limited to the above-mentioned substances, and those skilled in the art will know, for example, JP 2010-279372 A about substances having the activity of inducing PTH production. It is possible to obtain the active substance required by the method described in the above, and the CaSR antagonist can be obtained by the method described in Nemeth et al., Journal of Molecular Endocrinology, Vol. 29, p15, 2002, etc. Such substances are also included in the substance having an activity of inducing PTH or PTHrP production in the present invention.
[Treatment for vertebral fractures]
Further, the present invention is characterized in that it contains a PTH / PTHrP receptor agonist or a vertebral fracture characterized by containing PTH, PTHrP, or a partially active polypeptide thereof, or an analog thereof. It relates to a therapeutic agent. Furthermore, the present invention relates to a therapeutic agent for vertebral fracture containing a substance having an activity of inducing PTH or PTHrP production.
The vertebral body is “the main part of the vertebra that is in front of the spinal canal and is distinguished from the vertebral arch” (Stedman Medical Dictionary, 5th edition, Medical View) or “the half that occupies the front of the vertebra "Circular part" (Ojirin, second edition, Sanseido). Therefore, a vertebral body fracture does not include a fracture at the vertebral arch where spinous processes, transverse processes, joint processes, etc. are present, among vertebrae constituting the spine, and has a semi-cylindrical shape. A fracture in the body part. There are also vertebral fractures caused by strong impacts in accidents and sports, etc., but the social and medical economic problems are vertebral fractures associated with osteoporosis. These may not occur even if they are not subjected to a strong impact, and have a great influence on the prognosis of the patient (original sentence, etc., Journal of Nihon Medical College, Vol. 5, p125-129, 2009). It can be said that the treatment needs are high.
Therefore, the main subject for which the therapeutic agent for vertebral fracture of the present invention is used is treatment of vertebral fracture associated with osteoporosis. The vertebra is composed of 7 cervical vertebrae, 12 thoracic vertebrae, 5 lumbar vertebrae, 5 sacral vertebrae (in adults, fused to become sacrum and posterior wall of pelvis), 3-5 tail vertebrae (fused The site of the occurrence of vertebral fractures associated with osteoporosis is concentrated in the thoracic and lumbar vertebrae. Therefore, the rat lumbar vertebral body bone injury model according to the present invention is mainly focused on studies using the lumbar vertebrae. Therefore, the subject to which the therapeutic agent for vertebral fracture of the present invention is used mainly treats vertebral fractures in the thoracic and lumbar vertebrae.
As a structural feature of the vertebral body, there is a trabecular structure formed by cancellous bone that is highly developed inside the cortical bone that covers the outside. It is thought that the physical strength of the vertebral body depends greatly on the trabecular structure of the cancellous bone, and the reduction or deformation of the trabecular bone is the cause of the vertebral body compression fracture, which is the final image of the vertebral fracture associated with osteoporosis. It is said to be involved (IONOVICI et al., Romanian Journal of Morphology and Embrology, 50, p79-84, 2009). Therefore, the therapeutic effect of vertebral fractures that span cancellous bone or cortical bone and cancellous bone has been sought, but prior to the present invention, in the femur, rib, sternum, etc. where cortical bone is dominant as the action of PTH etc. Fracture treatment effects were only shown in non-clinical or clinical studies (Alkhiary et al., Journal of Bone and Joint Surgery, Vol. 87-A, p731-740, 2005; Aspenberg et al., Journal of Bone and Mineral Research, 25, p404-414, 2010; Chinamaneni et al., Osteoporosis International, 21, p1059-1063, 2010).
Further, in the present invention, by developing a rat lumbar vertebral body bone damage model, it was possible to analyze the fracture treatment effect of not only cortical bone but also cancellous bone in the vertebral body. Therefore, the subject to which the therapeutic agent for vertebral fracture of the present invention is used mainly treats vertebral fractures in cancellous bone or vertebral fractures that span cortical bone and cancellous bone.
The patient using the therapeutic agent for vertebral body fracture in the present invention is a patient in which one or more fractures are already observed in the vertebral body at the start of treatment, and preferably the vertebral body fracture reaches the vertebral body compression fracture. From patients with fracture lines in the vertebral body but with almost no change in the structure of the entire vertebral body, grade 1 (change in vertebral body leading edge height, central height and / or trailing edge height) Is about 20-25% decrease, and the area is reduced 10-20%) (Fukunaga et al., Journal of Bone and Mineral Metabolism, Vol. 22, p104-110, 2004). Diagnosis regarding detection of vertebral fractures and / or reduction in vertebral body height before the start of treatment can be performed by a person skilled in the art using, for example, simple X-ray, CT, MRI, etc. Medical Journal, 5 p125-129, 2009; Majumdar et al., Achieves Internal Medicine, 165 p905-909, 2005; Harvey et al., The British Journal of Radiology, 70 p645-649 1997).
Similarly, the effect of the therapeutic agent for vertebral fracture in the present invention can be performed using a method such as simple X-ray, CT, MRI, etc. The index of this effect is an index of the fracture line existing before the treatment. Decrease or disappearance of length and / or area, restoration of vertebral body height and area, and the like can be determined as indices. Further, in such diagnosis and treatment effect determination based on the shape of the vertebral body, for example, image analysis described in Japanese Patent No. 3229179, Japanese Patent No. 3258233, Japanese Patent No. 4495891, Japanese Patent No. 3888975, etc. It is also possible to implement appropriately using a technique.
Symptoms caused by vertebral fractures may cause pain in the back and / or lower back. In addition, when the vertebral fracture that has occurred is left untreated, pain that was not initially felt may occur. Therefore, it is also possible to determine the effect of the therapeutic agent for vertebral fracture according to the present invention with the effect of reducing or eliminating such pain or preventing pain. In that sense, the present invention also relates to an agent for treating or preventing pain caused by vertebral fractures.
On the other hand, if the vertebral fracture progresses without treatment, the vertebral body compression fracture (one of the vertebral body height / leading edge height / center height / rear edge height is 0.8 or less, or the leading edge When the value of high / rear edge height is 0.75 or less, or when the height of the vertebral body decreases as a whole, or when the height is reduced by 20% or more from the upper and lower vertebral bodies of the judgment vertebral body Vertebral body compression fracture). This vertebral body compression fracture is diagnosed by measurement using simple X-ray, CT, MRI or the like. Therefore, it is possible to determine the effect of the therapeutic agent for vertebral fracture according to the present invention with the effect of preventing the progress to such a compression fracture. In that sense, the present invention also relates to a preventive agent for vertebral body compression fracture.
Symptoms related to vertebral fractures are particularly strong when they are accompanied by movement disorders or neuropathy, or may progress to such movement disorders or neuropathy due to progression of the disease without treatment. The risk is high when severe vertebral body damage is caused by painful vertebral fractures or vertebral body compression fractures. Therefore, it is possible to determine the effect of the therapeutic agent for vertebral fracture of the present invention with the effect of reducing or eliminating such movement disorder and / or neuropathy, or the effect of preventing movement disorder and / or neuropathy. In that sense, the present invention also relates to a therapeutic or prophylactic agent for movement disorders and / or neurological disorders caused by vertebral fractures.
The vertebral fracture treatment agent of the present invention may contain any pharmaceutically acceptable additive. The formulation with pharmaceutically acceptable excipients is published by REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY 20th EDITION Philadelphia University of Science in Wilda It is also possible to carry out by the method described in 1. One form of such a pharmaceutical composition is provided as a solution prepared by dissolving, suspending or emulsifying in a sterile aqueous or oily liquid. Examples of such solvents include distilled water for injection, physiological saline and the like, and in addition, osmotic pressure regulators (for example, D-glucose, D-sorbitol, D-mannitol, sodium chloride, etc.). Appropriate solubilizers such as alcohol (eg ethanol), polyalcohol (eg propylene glycol, polyethylene glycol), nonionic surfactant (eg polysorbate 80, polyoxyethylene hydrogenated castor oil 50), etc. are used together Sometimes it is done. In addition, an oily liquid may be used as the solvent. Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. In such a solution, a buffer (for example, phosphates buffer, acetate buffer), a soothing agent (for example, benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (for example, human serum albumin, Polyethylene glycol, etc.), preservatives (eg, ascorbic acid, erythorbic acid and their salts), coloring agents (eg, copper chlorophyll, β-carotene, red No. 2, blue No. 1, etc.), preservatives (eg, paraoxybenzoic acid, etc.) Esters, phenol, benzethonium chloride, benzalkonium chloride, etc.) thickeners (eg hydroxypropylcellulose, carboxymethylcellulose and their salts), stabilizers (eg human serum albumin, mannitol, sorbitol, etc.), flavoring agents (eg Menthol, citrus flavors, etc.) There is a case. As another form of the pharmaceutical composition (in order to unify with the expression of “several“ pharmaceutical compositions ”above), solid preparations such as powders, tablets, granules, capsules, pills, suppositories, and lozenges are available. can give. In the case of solid preparations administered in the form of oral preparations, excipients (eg, crystalline cellulose, lactose, starch, etc.), lubricants (eg, magnesium stearate, talc, etc.), binding Agents (hydroxypropylcellulose, hydroxypropylmethylcellulose, macrogol, etc.), disintegrating agents (eg, starch, carboxymethylcellulose calcium, etc.) are used. If necessary, additives such as preservatives (for example, benzyl alcohol, chlorobutanol, methyl paraoxybenzoate, propyl paraoxybenzoate, etc.), antioxidants, coloring agents, sweeteners, and the like can be used. Another form is a pharmaceutical composition for mucosa. In this preparation, an adhesive, an adhesion enhancer, and a viscous agent are mainly used as additives for the purpose of imparting adsorptive properties and retention to the mucosa. , Thickeners etc. (eg mucin, agar, gelatin, pectin, carrageenan, sodium alginate, locust bin gum, xanthan gum, tragacanth gum, gum arabic, chitosan, pullulan, waxy starch, sucralfate, cellulose and derivatives thereof (eg hydroxy In some cases, propylmethylcellulose, polyglycerin fatty acid ester, acrylic acid (meth) acrylate alkyl copolymer or salt thereof, polyglycerin fatty acid ester, and the like may be contained. Solvents and additives are limited to these. Not intended to be, and can be appropriately selected by those skilled in the art.
The vertebral fracture treatment agent can be administered orally or parenterally for the purpose of improving symptoms. For oral administration, dosage forms such as granules, powders, tablets, capsules, solutions, syrups, emulsions or suspensions, and elixirs can be selected. In the case of parenteral administration, for example, it can be a nasal agent, and a liquid agent, a suspension agent, a solid preparation and the like can be selected. Another form of parenteral administration can be an injection, and as the injection, a subcutaneous injection, intravenous injection, infusion, intramuscular injection, intraperitoneal injection or the like can be selected. it can. Other preparations used for parenteral administration include suppositories, sublingual agents, transdermal agents, transmucosal agents other than nasal agents, and the like. Furthermore, it can also be administered locally to a blood vessel in such a manner that it is contained or applied to a stent or an intravascular embolic agent.
The dosage of the pharmaceutical composition varies depending on the patient's age, sex, weight, symptoms, therapeutic effect, administration method, treatment time, type of active ingredient contained in the pharmaceutical composition, etc. Per dose, in the range of 0.001 mg to 500 mg, preferably in the range of 0.005 mg to 200 mg. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be required.
[Evaluation method for vertebral fracture treatment]
The present invention also relates to a method for evaluating a vertebral body fracture therapeutic agent.
The evaluation method of the present invention includes 1) a step of surgically damaging a vertebral bone of a non-human animal, 2) a step of administering a test substance to the non-human animal, and 3) the non-human animal or derived therefrom. Morphological, physiological, biochemical or behavioral measurement of recovery of surgical damage to the vertebral bone of the specimen. In the evaluation method of the present invention, preferably, the results of morphological, physiological, biochemical or behavioral measurements of recovery from surgical damage to vertebral bones are further obtained. Alternatively, a step of comparing with the result of the measurement in a control non-human animal or a specimen derived therefrom is also included. On the other hand, the surgical damage given to the vertebral bone by the evaluation method of the present invention is preferably damage that reaches the cancellous bone through the cortical bone of the vertebral bone, and is not particularly limited as long as the damage can be given. A method of damaging by drilling with is preferred. The diameter and depth of a drill or the like and a perforation using the drill can be performed under appropriate conditions using appropriate tools depending on the animal used and the vertebral body used. Taking a rat lumbar spine as an example, it is preferable to use a dental drill, and the perforation diameter is preferably between 0.5 and 2.0 mm, more preferably between 0.5 and 1.5 mm, and even more. The diameter is preferably 0.7 to 0.8 mm. The depth of the perforation may be a depth that reaches the cancellous bone from the cortical bone, but is preferably a depth that reaches the cancellous bone from the cortical bone but does not reach the opposite cortical bone, more preferably 2.5. It is ~ 3.0mm deep. These conditions are when the most versatile 200-350 g rat is used, and it is better to change it appropriately according to the size of the animal and the size of the vertebral body.
The measurement of the healing effect of surgical damage to vertebral bones in the evaluation method of the present invention can be appropriately performed by using a morphological observation method such as simple X-ray, CT, micro CT, or MRI. In the examples of the present invention, a method using X-ray micro CT as a method for measuring the healing effect of surgical damage is described as an evaluation method using rats. It is not limited. In addition, a method based on histological observation is also possible for measuring the healing effect of surgical damage to vertebral bones in the evaluation method of the present invention. Samples used for histological observation include vertebral body tissues that have undergone surgical damage, other bone tissues, other tissues such as cartilage, muscles, and nerves, and body fluids such as blood and cerebrospinal fluid Is mentioned. In order to collect the vertebral bones and the surrounding tissues, a method of collecting tissues by biopsy can be used in addition to collecting tissues by dissection. A sample used for histological observation is subjected to treatment such as fixation, slicing, and / or staining as appropriate, and then subjected to observation. In addition, biochemical and / or cytological markers, for example, methods for detecting protein or gene expression for markers relating to bone differentiation or metabolism, such as tissue immunochemistry, in-situ hybridization, and PCR are used. A person skilled in the art can appropriately perform histological observation. In addition, biochemical markers biochemical and / or cytologically using cells or humoral components separated from collected tissues and / or body fluids, or extracts prepared from tissues and / or cells or body fluids, etc. Methods for measuring the expression of various markers, such as proteins and genes for markers related to bone differentiation and metabolism, can also be carried out by various known methods, and such methods are also included in the histological analysis. Another form of measuring the healing effect of surgical damage to vertebral bones is a measurement by physiological observation, for example, a method by measurement of pain sensation. Methods for measuring pain sensation in non-human animals include, for example, the paw pressure test and von Frey test, which are tests for measuring the degree of mechanical hyperalgesia, and allodynia for measuring the degree of mechanical allodynia. There are many other tests, such as the paw flick test that measures the degree of thermal hyperalgesia (Kawatani, Pain Research Approach, p31-33, 2006, Shinko Trading Co., Ltd.). There is known information, and those skilled in the art can appropriately select and implement the method according to the animal to be used and the site to be evaluated.
In addition, the evaluation method of the present invention can also evaluate the local or systemic effect on inflammation caused by administration of a test substance. The histological analysis as described above is used to measure the local effect on inflammation, and inflammatory cells such as lymphocytes, monocytes / macrophages, mast cells, neutrophils, eosinophils, eosinophils, For example, the amount of local accumulation of base spheres and the increase / decrease in the amount may be examined. In addition, using tissues or cells isolated therefrom or extracts from them, inflammatory factors expressed therein, such as tumor necrosis factor (TNF), interleukin 1 (IL-1) and interleukin 6 It can also be measured by examining the amount of protein or mRNA of inflammatory cytokines such as (IL-6) and the increase or decrease of the amount. In addition to general inflammatory reaction markers such as erythrocyte sedimentation rate, leukocyte count and leukocyte fraction, samples such as blood or other body fluids can be used to measure the effects on inflammation throughout the body. Inflammatory cytokine protein or C-reactive protein (CRP), α1-antitrypsin, α1-antichymotrypsin, α1-acid glycoprotein, serum amyloid A, haptoglobin, ceruloplasmin, albumin, transthyretin, transferrin, etc. There is also a method to check increase / decrease. In addition, the measurement regarding local or systemic inflammation can be performed by a person skilled in the art by appropriately selecting a method.
Furthermore, the evaluation method of the present invention can also measure the influence on bone resorption by the administration of the test substance. In addition to the above-described morphological observation method for measuring the healing effect of surgical damage, bone resorption can be measured by measuring bone volume density. Examples of the method for measuring bone volume density include the method using micro CT shown in Examples 3 and 5, and those skilled in the art appropriately select and implement a general method for measuring bone volume density. It is also possible. In addition, there is a method of using a bone resorption marker in a sample such as blood or urine as a method for evaluating the influence on bone resorption. Examples of such a bone resorption marker include deoxypyridinoline, type I collagen cross-linking N Examples include, but are not limited to, telopeptides, type I collagen cross-linked C-telopeptides, tartrate-resistant acid phosphatase, and the like.
Furthermore, the evaluation method of the present invention can also evaluate spinal cord inflammation or spinal cord injury caused by administration of the test substance. Histological, physiological, and behavioral methods are used to examine spinal cord inflammation and damage. As a histological method, in addition to the method described in the method based on the histological observation in the measurement of the healing effect of the surgical injury in the measurement of the healing effect, the above-mentioned local inflammation using a sample such as a spinal cord tissue. It is possible to carry out using the method described in the measurement of the influence on the environment. As a physiological method, the same method as the physiological observation in the measurement of the healing effect of the above-mentioned surgical injury can be used. Examples of behavioral methods include Basso, Beattie, and Bresnahan (BBB) scale, standing-up behavior, and a treadmill. More simply, if movement disorders occur in the lower limbs due to spinal cord injury, the feeding behavior in the cage will also be hindered. Can be evaluated for damage. In addition, measurement of spinal cord inflammation and damage can be performed by a person skilled in the art by appropriately selecting a method.
The non-human animal used in the evaluation method of the present invention is a non-human vertebrate, preferably a non-human primate or a rodent, and a rat is preferable as the rodent animal, such as a mouse. It can be applied to any non-human vertebrates such as rodents, rabbits, dogs and monkeys. Further, as the non-human animal used in the present invention, an osteoporosis model non-human animal can be used, and an example of the osteoporosis model is an ovariectomy model. As for the vertebral body part used in the evaluation method of the present invention, a person skilled in the art selects an appropriate vertebral body part from among cervical vertebrae, thoracic vertebrae, lumbar vertebrae, sacral vertebrae and tail vertebrae according to the type of non-human animal used. Although it can be carried out, it is preferably a thoracic and / or lumbar vertebral body, more preferably a method using a lumbar vertebral body. In the embodiment of the present invention, a rat is used as a non-human animal, and in this case, a method using the lumbar vertebrae, particularly the fourth lumbar vertebra and the fifth lumbar vertebra is shown. Accordingly, the vertebral body used is preferably the fourth lumbar vertebra and / or the fifth lumbar vertebra, but is not limited thereto.
Administration of the test substance in the evaluation method of the present invention can be carried out by the oral or parenteral route, and those skilled in the art will know the type of non-human animal used, the evaluation site, the physical and / or test substance. In consideration of the chemical properties, it is possible to determine the administration method and administration site, and to prepare a sample for administration containing the test substance corresponding thereto.

　以下、実施例をもって本発明をさらに詳しく説明するが、これらの実施例は本発明を制限するものではない。
　動物（雄性Ｓｐｒａｇｕｅ−Ｄａｗｌｅｙラット、手術時８週齢）は、１日１２時間（６：００~１８：００）の人工照明下で、温度２４±２℃、湿度５５±１５％の条件で飼育を行った。飲水条件は、次亜塩素酸消毒した井水（自給・深井戸）の自動給水による自由摂取、摂餌条件は、ＣＥ−２（日本クレア、マウス、ラット用）の自由摂取とした。実験は帝人ファーマ株式会社生物医学総合研究所動物実験委員会の許可のもとに行われた。動物の取り扱いは「生物医学総合研究所　動物実験規則」、処分は「生物医学総合研究所　動物の安楽死に関する規則」に、それぞれ準じた。
［実施例１］ラット腰椎椎体骨損傷モデルの作製方法の検討
　椎体骨折に対する薬剤の治療効果をｉｎ　ｖｉｖｏで検討する目的で、新しい動物モデルを構築した。本モデルは、ラット腰椎椎体に骨損傷を生じさせ、その治癒過程を観察するｉｎ　ｖｉｖｏモデルである。具体的には、正常ラットの腰椎に歯科用ドリルで穴をあけ、その穴を骨損傷とした。
　ラットは１週間の馴化期間の後、ペントバルビタールナトリウム（共立製薬株式会社）により麻酔し、腹部周囲をバリカンで剃毛したのち、手術台に仰向けに固定した。正中線にそって開腹し、腹部の臓器を滅菌ガーゼで隔離した後、腰椎周囲の血管、脂肪組織等は、綿棒を用いて丁寧に剥離した。微量の出血は、綿棒で押さえることで止血した。腰椎周囲の筋層をピンセットで裂き、第４腰椎及び第５腰椎それぞれに対し、約１ｃｍ四方の視野を確保した。露出した第４腰椎及び第５腰椎の前縦靱帯及び血管を避けた中央付近に、歯科用ドリルを用いて穴をあけた。その後、隔離した臓器を正常な位置に戻し、抗生物質を滴下し、縫合糸で切創を縫合した。縫合部はステープラーで留め、ヨードチンキで消毒した。麻酔から覚醒後、飼育ケージに戻し、飼育した。
結果
　本モデルの作製には、２つの工程が重要であった。１工程目は、開腹後の腰椎周囲の血管、脂肪組織等の剥離である。剥離するためには、一般的に用いられている大小２種類の綿棒を用いた。大の綿棒は直径約１．２ｍｍ、小の綿棒は直径約３ｍｍのものを使用した。綿棒に組織を絡ませながら徐々に剥離することで、出血を防ぐだけでなく、椎体周囲の筋組織へのダメージを最小限に抑えることができた。また、歯科用ドリルを椎体に近づける時、歯科用ドリルが周囲組織を巻き込まないようにし、かつ最小限の筋組織障害にとどめるために、１椎体に対し、約１ｃｍ四方の視野を確保した。
　２工程目は、確保した視野で、第４腰椎及び第５腰椎の前縦靱帯及び血管を避けた中央付近に、穴をあけることである。穴の直径を２ｍｍ（Ｕｃｈｉｄａらが大腿骨に作製した穴の大きさは直径約２−２．５ｍｍ（非特許文献１２、１５））にすると、血管を避けることができず多くの血管を傷つけてしまうため、出血多量となり、ラットは麻酔から覚醒することなく死亡した。逆に、穴の直径を０．４ｍｍ以下にすると、円柱状の安定した骨損傷を均等に生じさせることができなかった。従って、ラットが術後死亡することなく生存し、その自然修復過程が２１日間という短期間で、さらにｍｉｃｒｏ　ＣＴを用いた評価ができる骨損傷の大きさとして、直径約０．７２ｍｍ、深さはそれぞれの椎体の大きさに合わせ椎体を貫通しない２．５−３．０ｍｍ程度という条件を決定した。
　代表例として、モデル作製日の損傷椎体（六をあけた椎体）のｍｉｃｒｏ　ＣＴ画像（３Ｄ画像と２Ｄ画像）を示した（図１）。図１は、ラット腰椎椎体損傷モデルを作製し、ｍｉｃｒｏ　ＣＴを用いてその椎体を撮影したものである。具体的には、歯科用ドリルを用い、露出したＬ４及びＬ５の前縦靱帯及び血管を避けた中央付近に直径０．７２ｍｍ程度の穴をあけた。矢印は、あけた穴を指し示す。図のように、第４腰椎又は第５腰椎の前縦靱帯及び血管を避けた中央付近に、直径約０．７２ｍｍ、深さは椎体を貫通しない２．５−３．０ｍｍの骨損傷を作製した。この穴の大きさであれば、ラットは麻酔から覚醒後、開腹のみ実施した偽手術ラットと同様の緩やかな体重増加を示した。また、外観による一般症状の観察では、異常所見はなく、術後２１日間、良好な健康状態を保ちながら飼育することができた。実施例２、３に示すように、術後２１日では、正常椎体と同程度の骨構造の回復を確認できたことから、本モデルを椎体骨折に対する薬剤の治療効果をｉｎ　ｖｉｖｏで検討できる新しいモデルとして、ラット腰椎椎体骨損傷モデルと命名した。
［実施例２］ラット腰椎椎体骨損傷モデルの自然修復過程における組織学的検討
　ラット腰椎椎体骨損傷モデルの骨損傷後の自然修復過程を、椎体の組織標本を作製することにより観察した。モデル作製日（穴をあけた日）をｄａｙ０とし、ｄａｙ４、ｄａｙ７、ｄａｙ１４、ｄａｙ２１に、ペントバルビタール麻酔下で腹部大動脈から放血致死させ、腰椎を摘出した。摘出した腰椎は、７０％エタノールに浸け、４℃で保存した。それぞれの椎体は、蟻酸・クエン酸ナトリウム脱灰し、パラフィン包埋した（密閉式自動固定包埋装置（サクラＥＴＰ−１８０ＢＶ））。ｍｉｃｒｏ　ＣＴ画像を元に海綿骨の損傷部を面出しし、損傷部を面出しできたブロックを観察した。３ｍｍで薄切した切片（滑走式ミクロトーム（サクラＩＶＳ−４１０））をスライドガラスにのせ、脱パラ後、それぞれ、ヘマトキシリン・エオジン（ＨＥ）染色、マッソン・トリクローム（ＭＴ）染色を実施した。作製した組織標本は、顕微鏡（Ｌｅｉｃａ　ＤＭ４０００Ｂ）、カメラ（Ｌｅｉｃａ　ＤＦＣ４８０）、顕微鏡写真撮影ソフト（Ｌｅｉｃａ　ＩＭ５００）を用いて観察した。
結果
　モデル作製日（穴をあけた日）をｄａｙ０とし、ｄａｙ４、ｄａｙ７、ｄａｙ１４、ｄａｙ２１のＨＥ染色像及びＭＴ染色像を観察した。図２に、それぞれの時点でのＭＴ染色の代表例（海綿骨の損傷部）を示した。図２は、モデル作製日（ｄａｙ０）、ｄａｙ４、ｄａｙ７、ｄａｙ１４、ｄａｙ２１のＭＴ染色像を示した。２Ａはｄａｙ０、２Ｂはｄａｙ４、２Ｃはｄａｙ７、２Ｄはｄａｙ１４、２Ｅはｄａｙ２１、さらに、２Ｆはｄａｙ２１の正常椎体（偽手術ラット）の海綿骨を示した。それぞれ、損傷椎体に関しては、上部は損傷部、下部は非損傷部であり、境界を矢印で指し示した。
　モデル作製直後（ｄａｙ０）では、損傷部での骨髄細胞の漏出が観察された（図２Ａ）。ｄａｙ４（図２Ｂ）では、骨折治癒の初期ステージである血腫と肉芽組織の混在と少量の線維性骨が観察された。ｄａｙ７（図２Ｃ）では、血腫は組織化して消失し、線維性骨が増加し、周囲と明らかに区別できる骨梁が認められた。ｄａｙ１４（図２Ｄ）では、骨梁はより増加し、肉芽組織や線維性骨はほとんど認められなかった。ｄａｙ２１（図２Ｅ）では、損傷部は完全に修復し、偽手術ラット（図２Ｆ）と同様の組織像が観察された。
［実施例３］ラット腰椎椎体骨損傷モデルの自然修復過程における、ｍｉｃｒｏ　ＣＴ撮影とその撮影画像を用いた骨体積密度（ｍｇ／ｃｍ３）の計測
　マイクロフォーカスＸ線ＣＴ装置（株式会社日鉄エレックス）を用いた。撮影ソフトＥＬＥ　ＳＣＡＮ（ＮＸ−ＣＰ−Ｃ８０Ｈ−ＩＬ−０２１：株式会社日鉄エレックス）を用い、管電圧５０ｋＶ、管電流６０ｍＡの撮影条件で、３Ｄ断層像撮影した。椎体側面から見た後縦靱帯が位置するラインを中心線に合わせ、撮影中椎体が動かないように固定し、０．０２ｍｍ幅で３００枚撮影した。また、測定値を骨体積密度（ｍｇ／ｃｍ３）に変換するために、４段階に設定した炭酸カルシウムファントム（０~３００ｍｇ／ｃｍ３　ＣａＣＯ３）を同条件で撮影した。ＣＴ画像は、Ｃ３Ｄファイルで保存し、コンビーム再構成ソフトウェア（ＥＬＥ　ＳＣＡＮ　ＣＢ３Ｄ：株式会社日鉄エレックス）を用いて再構成した（Ｃ３Ｖファイル作成）。保存したＣ３Ｖファイルを元に、３００枚のＴＩＦＦファイルを作成し、このファイルを用いて解析を行った。解析には、Ｒｅａｌ　ＩＮＴＡＧＥ　ｖｅｒ２．１（株式会社ケイ・ジー・ティー）を用い、輝度値の計測を行った。
　損傷椎体の海綿骨の損傷部の骨体積密度を計測した。具体的には、撮影したｍｉｃｒｏ　ＣＴ画像の椎体側面から見た後縦靱帯が位置するラインを０としたときの、１．９０ｍｍから２．１０ｍｍの１１枚のＴｉｆｆファイルについて、それぞれ０．４０ｍｍ２の円の輝度値を測定した。
　正常椎体（偽手術ラット）の海綿骨についても骨体積密度を計測した。具体的には、撮影したｍｉｃｒｏ　ＣＴ画像の椎体側面から見た後縦靱帯が位置するラインを０としたときの、１．９０ｍｍから２．１０ｍｍの１１枚のＴｉｆｆファイルについて、それぞれ２．０ｍｍ２の長方形（皮質骨を含まない）の輝度値を測定した。
　測定した輝度値は、同条件で測定した炭酸カルシウムファントムを元に、骨体積密度（ｍｇ／ｃｍ３）に変換した。
結果
　ｍｉｃｒｏ　ＣＴを用いて、海綿骨の損傷部の骨体積密度を計測した。モデル作製日（穴をあけた日）をｄａｙ０とし、ｄａｙ４、ｄａｙ７、ｄａｙ１４、ｄａｙ２１の骨体積密度を示した（図３）。図３は、モデル作製日（ｄａｙ０）、ｄａｙ４、ｄａｙ７、ｄａｙ１４、ｄａｙ２１の骨体積密度を示す。黒丸（●）は、損傷椎体の海綿骨の損傷部の骨体積密度の平均値±標準偏差、四角（□）は、正常椎体（偽手術ラット）の海綿骨の骨体積密度の平均値±標準偏差を示す。
　１椎体を１例として取り扱った。モデル作製直後の骨体積密度の平均値±標準偏差は、０．０±３１．６ｍｇ／ｃｍ３であり、ｄａｙ４、ｄａｙ７、ｄａｙ１４、ｄａｙ２１では、それぞれ、３３．５±３９．５、９２．４±２９．８、１００．２±４５．４、２６３．２±５０．１ｍｇ／ｃｍ３であった。一方、正常椎体（偽手術ラット）の海綿骨の骨体積密度は、ｄａｙ０では２４６．４±６４．６、ｄａｙ２１では２２０．７±５０．１ｍｇ／ｃｍ３であり、観察期間内において、ほぼ一定の値を示した。
　従って、腰椎に歯科用ドリルで穴をあけたとき、その穴の骨体積密度は緩やかに増加し、２１日目で正常椎体（偽手術ラット）と同程度まで回復することが示された。また、ｍｉｃｒｏ　ＣＴを用いて骨体積密度を測定することで、ラット腰椎椎体骨損傷モデルの骨損傷修復レベルを定量できることが示された。
［実施例４］ラット腰椎椎体骨損傷モデルにｈＰＴＨ（１−３４）及びＰＴＨｒＰアナログＡを投与したときの組織学的検討
　モデル作製日から７日間又は１４日間、ｈＰＴＨ（１−３４）（Ｂａｃｈｅｍ社）又はＰＴＨｒＰアナログＡ（Ｉｐｓｅｎ社）（配列番号４２）を投与したときの椎体組織標本を観察した。ｈＰＴＨ（１−３４）群、ＰＴＨｒＰアナログＡ及びＶｅｈｉｃｌｅ群を設定した。Ｖｅｈｉｃｌｅ投与液は、２％ラット血清を含むＳａｌｉｎｅ（ｐＨ４．０±０．１）を用いた。ｈＰＴＨ（１−３４）投与液、ＰＴＨｒＰアナログＡ投与液は、Ｖｅｈｉｃｌｅを用いて１０μｇ／ｍＬに調製した。初回投与は、モデル作製４時間後に実施し、次の日からは１日１回皮下投与した。投与容量は１ｍＬ／ｋｇとした。最終投与２４時間後にペントバルビタール麻酔下で腹部大動脈から放血致死させ、第４腰椎、第５腰椎（損傷椎体）と第３腰椎、第６腰椎（非損傷椎体）を摘出した。摘出した腰椎は、７０％エタノールに浸け、４℃で保存した。
　それぞれの椎体は、蟻酸・クエン酸ナトリウム脱灰し、パラフィン包埋した（密閉式自動固定包埋装置（サクラＥＴＰ−１８０ＢＶ））。ｍｉｃｒｏ　ＣＴ画像を元に海綿骨の損傷部を面出しし、損傷部を面出しできたブロックを観察した。３ｍｍで薄切した切片（滑走式ミクロトーム（サクラＩＶＳ−４１０））をスライドガラスにのせ、脱パラ後、それぞれ、ヘマトキシリン・エオジン（ＨＥ）染色、マッソン・トリクローム（ＭＴ）染色を実施した。作製した組織標本は、顕微鏡（Ｌｅｉｃａ　ＤＭ４０００Ｂ）、カメラ（Ｌｅｉｃａ　ＤＦＣ４８０）、顕微鏡写真撮影ソフト（Ｌｅｉｃａ　ＩＭ５００）を用いて観察した。
結果
　モデル作製日から７日間又は１４日間、ｈＰＴＨ（１−３４）１０μｇ／ｋｇ又はＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを連日皮下投与したときのＨＥ染色像及びＭＴ染色像を観察した。図４に、それぞれの時点でのＭＴ染色の代表例（海綿骨の損傷部）を示した。図４は、モデル作製日から７日間又は１４日間、ｈＰＴＨ（１−３４）１０μｇ／ｋｇ又はＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを連日皮下投与したときのＭＴ染色像を示す。４Ａはｄａｙ７でのｖｅｈｉｃｌｅ群、４Ｂはｄａｙ７でのｈＰＴＨ（１−３４）群、４Ｃはｄａｙ７でのＰＴＨｒＰアナログＡ群、４Ｄはｄａｙ１４でのｖｅｈｉｃｌｅ群、４Ｅはｄａｙ１４でのｈＰＴＨ（１−３４）群、４Ｆはｄａｙ１４でのＰＴＨｒＰアナログＡ群を示す。それぞれ、上部は損傷部、下部は非損傷部であり、境界を矢印で指し示す。
　投与７日目において、ｈＰＴＨ（１−３４）群（図４Ｂ）では、Ｖｅｈｉｃｌｅ群（図４Ａ）と比較して、線維性骨の増加、骨梁の増加傾向が観察された。投与１４日目においては、両群ともに肉芽組織や線維性骨はほとんど認められず、形態学的な差は観察されなかったが、ｈＰＴＨ（１−３４）群（図４Ｅ）では、Ｖｅｈｉｃｌｅ群（図４Ｄ）と比較して、より多くの骨梁が観察された。
　同様に、投与７日目において、ＰＴＨｒＰアナログＡ群（図４Ｃ）では、Ｖｅｈｉｃｌｅ群（図４Ａ）と比較して、線維性骨の増加、骨梁の増加傾向が観察された。投与１４日目においては、両群ともに肉芽組織や線維性骨はほとんど認められず、形態学的な差は観察されなかったが、ＰＴＨｒＰアナログＡ群（図４Ｆ）では、Ｖｅｈｉｃｌｅ群（図４Ｄ）と比較して、より多くの骨梁が観察された。ｈＰＴＨ（１−３４）群とＰＴＨｒＰアナログＡ群の間に、著明な差は観察されなかった。
　また、ＨＥ染色での組織像について、モデル作製日から７日間又は１４日間を観察した。ｈＰＴＨ（１−３４）１０μｇ／ｋｇ又はＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを投与した群をＶｅｈｉｃｌｅ投与群と比較した結果、損傷部及び非損傷部での炎症性細胞の明らかな集積は認められず、形態学的な差はなかった。
［実施例５］ラット腰椎椎体骨損傷モデルにｈＰＴＨ（１−３４）及びＰＴＨｒＰアナログＡを投与したときの損傷椎体の海綿骨の損傷部における骨体積密度の検討
　モデル作製日から７日間又は１４日間、ｈＰＴＨ（１−３４）　１０μｇ／ｋｇ又はＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを連日皮下投与したときの損傷椎体の海綿骨の損傷部の骨体積密度を計測した。骨体積密度は実施例３と同様の方法を用いて測定した。
結果
　Ｍｉｃｒｏ　ＣＴ撮影画像を用いて計測した骨体積密度（ｍｇ／ｃｍ３）に対して、解析を実施した。解析を実施するにあたり、１椎体を１例として取り扱った。
　Ｄａｙ７、ｄａｙ１４でのｈＰＴＨ（１−３４）群とＶｅｈｉｃｌｅ群の２群間、ＰＴＨｒＰアナログＡ群とｖｅｈｉｃｌｅ群の２群間に対し、それぞれＳｔｕｄｅｎｔのｔ検定を実施した。有意水準は５％とした。
　ハードウェアは、富士通株式会社　ＦＭＶＸＤ０５７２　ＦＭＶ　ＥＳＰＲＩＭＯ　Ｄ３２３０を使用し、ソフトウェアは、株式会社ＳＡＳ　インスティチュートジャパン　ＳＡＳ　ｖｅｒ８．２、株式会社アーム　ＥＸＳＡＳ　ｖｅｒ７．１０を使用した。
　ｍｉｃｒｏ　ＣＴを用いて、損傷椎体の海綿骨の損傷部の骨体積密度を計測した。モデル作製日から７日間又は１４日間、ｈＰＴＨ（１−３４）　１０μｇ／ｋｇを連日皮下投与したときの骨体積密度を示した（図５）。図５は、モデル作製日から７日間又は１４日間、ｈＰＴＨ（１−３４）　１０μｇ／ｋｇ又はＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを連日皮下投与したときの海綿骨の損傷部の骨体積密度を示す。黒丸（●）は、ｖｅｈｉｃｌｅ群の平均値±標準偏差、白丸（○）は、ｈＰＴＨ（１−３４）群の平均値±標準偏差、三角（△）は、ＰＴＨｒＰアナログＡ群の平均値±標準偏差を示す。各時点でのｈＰＴＨ（１−３４）群とＶｅｈｉｃｌｅ群の２群間、ＰＴＨｒＰアナログＡ群とＶｅｈｉｃｌｅ群の２群間に対し、それぞれＳｔｕｄｅｎｔのｔ検定を実施したところ、投与１４日目において、Ｖｅｈｉｃｌｅ群とｈＰＴＨ（１−３４）群の２群間、Ｖｅｈｉｃｌｅ群とＰＴＨｒＰアナログＡ群の２群間に統計学的に有意な差が認められた（ｐ＝０．０１００、ｐ＝０．００２５）。
　投与７日目において、海綿骨の損傷部の骨体積密度の平均値±標準偏差は、Ｖｅｈｉｃｌｅ群は９１．４±３３．８ｍｇ／ｃｍ３、ｈＰＴＨ（１−３４）群は１５８．０±７７．５ｍｇ／ｃｍ３であり、２群間に統計学的に有意な差は認められなかった（ｐ＝０．０７４３：Ｓｔｕｄｅｎｔのｔ検定）。投与１４日目において、海綿骨の損傷部の骨体積密度の平均値±標準偏差は、Ｖｅｈｉｃｌｅ群は１４０．９±５８．８ｍｇ／ｃｍ３、ｈＰＴＨ（１−３４）群は２５０．５±７８．７ｍｇ／ｃｍ３であり、２群間に統計学的に有意な差が認められた（ｐ＝０．０１００：Ｓｔｕｄｅｎｔのｔ検定）。
　同様に、モデル作製日から７日間又は１４日間、ＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを連日皮下投与したときの骨体積密度を示した（図５）。投与７日目において、海綿骨の損傷部の骨体積密度の平均値±標準偏差は、Ｖｅｈｉｃｌｅ群は９１．４±３３．８ｍｇ／ｃｍ３、ＰＴＨｒＰアナログＡ群は１４８．５±５６．２ｍｇ／ｃｍ３であり、２群間に統計学的に有意な差は認められなかった（ｐ＝０．０５９０：Ｓｔｕｄｅｎｔのｔ検定）。投与１４日目において、海綿骨の損傷部の骨体積密度の平均値±標準偏差は、Ｖｅｈｉｃｌｅ群は１４０．９±５８．８ｍｇ／ｃｍ３、ＰＴＨｒＰアナログＡ群は２３７．０±４０．４ｍｇ／ｃｍ３であり、２群間に統計学的に有意な差が認められた（ｐ＝０．００２５：Ｓｔｕｄｅｎｔのｔ検定）。従って、椎体の海綿骨の損傷部において、ｈＰＴＨ（１−３４）、ＰＴＨｒＰアナログＡともに、１４日間投与することで有意な骨体積密度増加作用を示すことが分かった。
　また、モデル作製日から７日間、ｈＰＴＨ（１−３４）１０μｇ／ｋｇ又はＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを投与した群においても、有為な差は見られないものの、Ｖｅｈｉｃｌｅ投与群と比較して高い骨体積密度を示したことから、ｈＰＴＨ（１−３４）、ＰＴＨｒＰアナログＡともに、損傷部における過度の骨吸収は起こっていないことが示された。
［実施例６］ラット腰椎椎体骨損傷モデルにｈＰＴＨ（１−３４）及びＰＴＨｒＰアナログＡを投与したときの非損傷椎体の海綿骨の骨体積密度の検討
　モデル作製日から７日間又は１４日間、ｈＰＴＨ（１−３４）　１０μｇ／ｋｇ又はＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを連日皮下投与したときの非損傷椎体の海綿骨の骨体積密度を計測した。骨体積密度は、実施例３と同様の方法を用いて測定した。ただし、非損傷椎体（第３腰椎、第６腰椎）の大きさを考慮し、測定位置を第４腰椎、第５腰椎と異なる位置に設定した。具体的には、第３腰椎は、撮影したｍｉｃｒｏ　ＣＴ画像の椎体側面から見た後縦靱帯が位置するラインを０としたときの、１．７０ｍｍから１．９０ｍｍの１１枚のＴｉｆｆファイルについて、第６腰椎は、撮影したＣＴ画像の椎体側面から見た椎体後方を０としたときの、２．１０ｍｍから２．３０ｍｍの１１枚のＴｉｆｆファイルについて、それぞれ２．０ｍｍ２の長方形（皮質骨を含まない）の輝度値を測定した。
結果
　Ｍｉｃｒｏ　ＣＴ撮影画像を用いて計測した骨体積密度（ｍｇ／ｃｍ３）に対して、解析を実施した。解析を実施するにあたり、１椎体を１例として取り扱った。
　Ｄａｙ７、ｄａｙ１４でのｈＰＴＨ（１−３４）群とＶｅｈｉｃｌｅ群の２群間、ＰＴＨｒＰアナログＡ群とｖｅｈｉｃｌｅ群の２群間に対し、それぞれＳｔｕｄｅｎｔのｔ検定を実施した。有意水準は５％とした。
　ハードウェアは、富士通株式会社　ＦＭＶＸＤ０５７２　ＦＭＶ　ＥＳＰＲＩＭＯ　Ｄ３２３０を使用し、ソフトウェアは、株式会社ＳＡＳ　インスティチュートジャパン　ＳＡＳ　ｖｅｒ８．２、株式会社アーム　ＥＸＳＡＳ　ｖｅｒ７．１０を使用した。
　非損傷椎体（第３腰椎、第６腰椎）の海綿骨の骨体積密度を計測した。モデル作製日から７日間と１４日間、ｈＰＴＨ（１−３４）　１０μｇ／ｋｇを連日皮下投与したときの骨体積密度を示した（図６）。図６は、モデル作製日から７日間又は１４日間、ｈＰＴＨ（１−３４）　１０μｇ／ｋｇ又はＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを連日皮下投与したときの非損傷椎体の海綿骨の骨体積密度を示す。黒丸（●）は、ｖｅｈｉｃｌｅ群の平均値±標準偏差、白丸（○）は、ｈＰＴＨ（１−３４）群の平均値±標準偏差、三角（△）は、ＰＴＨｒＰアナログＡ群の平均値±標準偏差を示す。各時点でのｈＰＴＨ（１−３４）群とＶｅｈｉｃｌｅ群の２群間、ＰＴＨｒＰアナログＡ群とＶｅｈｉｃｌｅ群の２群間に対し、それぞれＳｔｕｄｅｎｔのｔ検定を実施したところ、ｄａｙ７、ｄａｙ１４ともに統計学的に有意な差は認められなかった（ｄａｙ７：ｐ＝０．９９８１、ｐ＝０．５５９１、ｄａｙ１４：ｐ＝０．２５３９、ｐ＝０．１９１９）。投与７日目において、海綿骨の骨体積密度の平均値±標準偏差は、Ｖｅｈｉｃｌｅ群は２１４．９±５６．６ｍｇ／ｃｍ３、ｈＰＴＨ（１−３４）群は２１５．０±４２．１ｍｇ／ｃｍ３であり、２群間に統計学的に有意な差は認められなかった（ｐ＝０．９９８１：Ｓｔｕｄｅｎｔのｔ検定）。投与１４日目において、海綿骨の骨体積密度の平均値±標準偏差は、Ｖｅｈｉｃｌｅ群は２４０．２±５９．７ｍｇ／ｃｍ３、ｈＰＴＨ（１−３４）群は２６８．３±３０．０ｍｇ／ｃｍ３であり、２群間に統計学的に有意な差は認められなかった（ｐ＝０．２５３９：Ｓｔｕｄｅｎｔのｔ検定）。
　同様に、ＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを連日皮下投与したときの骨体積密度を示した（図６）。投与７日目において、海綿骨の骨体積密度の平均値±標準偏差は、Ｖｅｈｉｃｌｅ群は２１４．９±５６．６ｍｇ／ｃｍ３、ＰＴＨｒＰアナログＡ群は２３３．４±６６．６ｍｇ／ｃｍ３であり、２群間に統計学的に有意な差は認められなかった（ｐ＝０．５５９１：Ｓｔｕｄｅｎｔのｔ検定）。投与１４日目において、海綿骨の骨体積密度の平均値±標準偏差は、Ｖｅｈｉｃｌｅ群は２４０．２±５９．７ｍｇ／ｃｍ３、ＰＴＨｒＰアナログＡ群は２７８．０±５０．１ｍｇ／ｃｍ３であり、２群間に統計学的に有意な差は認められなかった（ｐ＝０．１９１９：Ｓｔｕｄｅｎｔのｔ検定）。
　従って、ｈＰＴＨ（１−３４）、ＰＴＨｒＰアナログＡを１４日間投与しても、損傷させていない椎体の海綿骨の骨体積密度は変化しないことが分かった。以上の結果より、損傷椎体の海綿骨の損傷部で認められたｈＰＴＨ（１−３４）、ＰＴＨｒＰアナログＡの骨体積密度の増加作用は、損傷部位特異的であることが分かった。
　上記のように、ラット腰椎椎体骨損傷モデルに、ＰＴＨ（１−３４）やＰＴＨｒＰアナログＡを投与すると、非損傷椎体の海綿骨の骨体積密度は変化しない投与量でも、損傷椎体の海綿骨の損傷部の骨体積密度は有意に増加することを見出した。従って、ＰＴＨ（１−３４）やＰＴＨｒＰアナログＡは、椎体骨折で生じる海綿骨の微細構造の損傷を損傷部位特異的に治療する作用を持つことが分かった。
［実施例７］ラット腰椎椎体骨損傷モデルにｈＰＴＨ（１−３４）及びＰＴＨｒＰアナログＡを投与したときの運動機能及び体重変化への影響の検討
　ｈＰＴＨ（１−３４）及びＰＴＨｒＰアナログＡを投与することで、骨折修復過程の炎症反応が増悪すること、又は過度の骨吸収によって椎体が破壊されることにより、椎体の近傍に存在する脊髄が損傷をうける可能性を検討した。一般的に、脊椎の損傷によって下肢での運動機能に重篤な障害が生じた場合には、飼育ケージに取り付けられた餌箱からの摂餌行動も制限されるため、摂餌量が減ることによる体重減少が起こると考えられる。
　ラット腰椎椎体骨損傷モデルの作製については実施例１に記載の方法で実施し、偽手術ラットの作製については、歯科用ドリルを用いて穴をあける工程以外は、実施例１に記載したラット腰椎椎体骨損傷モデルと同じ方法で作製した。
　まず、本発明のラット腰椎椎体骨損傷モデル作成での運動機能及び体重変化への影響を検討した。ラット腰椎椎体骨損傷モデル及び偽手術ラットの各群について、手術を行った日をＤａｙ０として、各Ｄａｙ４、７、１４、２１日に体重の測定を実施した。また、行動異常については体重測定時に歩行及び立ち上がり等の行動に不自然さがないかを目視で観察した。続いて、ラット腰椎椎体骨損傷モデルを用いてモデル作製日から１４日間、ｈＰＴＨ（１−３４）　１０μｇ／ｋｇ又はＰＴＨｒＰアナログＡ　１０μｇ／ｋｇを連日皮下投与したときの体重変化について検討した。投与開始日であるモデル作成日をＤａｙ０とし、Ｄａｙ１４までの体重を連日測定した。また、行動異常については上記のように目視での観察により行った。
結果
　ラット腰椎椎体骨損傷モデル及び偽手術ラットの各群についての体重測定の結果を図７Ａに示す。測定した日の全てにおいて、ラット腰椎椎体骨損傷モデルと偽手術ラットの各群での体重の差は認められなかった。また、各動物における目視での行動異常も認められなかった。このことから、本発明のラット腰椎椎体骨損傷モデルではモデル作製による運動機能への影響は認めらない。
　ｈＰＴＨ（１−３４）　１０μｇ／ｋｇ又はＰＴＨｒＰアナログＡ　１０μｇ／ｋｇもしくはＶｅｈｉｃｌｅをモデル作製日から１４日間連日皮下投与したときの各群の体重変化を図７Ｂに示す。投与開始から１４日までの間、ｈＰＴＨ（１−３４）、ＰＴＨｒＰアナログＡ及びＶｅｈｉｃｌｅ投与の各群の間で、体重の差は認められなかった。また、各動物における目視での行動異常も認められなかった。このことから、ラット腰椎椎体骨損傷モデルにｈＰＴＨ（１−３４）又はＰＴＨｒＰアナログＡを投与することで、行動障害を起こすような重篤な脊髄への損傷などの作用は起こっていないことが確認された。 Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the present invention.
Animals (male Sprague-Dawley rats, 8 weeks old at the time of operation) are kept under artificial lighting conditions of 12 hours a day (6: 00-18: 00) at a temperature of 24 ± 2 ° C. and a humidity of 55 ± 15%. Went. The drinking water was free intake by automatically supplying well water (self-contained / deep well) sterilized with hypochlorous acid, and the feeding condition was free intake of CE-2 (Japan Claire, for mice and rats). The experiment was conducted with the permission of the Animal Experiment Committee, Teijin Pharma Limited. The handling of animals was in accordance with the “Laboratory for Animal Experiments” and the disposal was in accordance with the “Rule for Animal Euthanasia”.
[Example 1] Examination of preparation method of rat lumbar vertebral body bone damage model
A new animal model was constructed for the purpose of examining the therapeutic effects of drugs on vertebral fractures in vivo. This model is an in vivo model in which bone damage is caused in the rat lumbar vertebral body and the healing process is observed. Specifically, a hole was drilled in the lumbar spine of a normal rat with a dental drill, and the hole was used as bone damage.
Rats were anesthetized with sodium pentobarbital (Kyoritsu Pharmaceutical Co., Ltd.) after a acclimation period of 1 week, shaved around the abdomen with a clipper, and fixed on the operating table on its back. After abdominal laparotomy was performed and the abdominal organs were isolated with sterilized gauze, blood vessels, adipose tissue, etc. around the lumbar spine were carefully detached using a cotton swab. A minute amount of bleeding was stopped by pressing with a cotton swab. The muscle layer around the lumbar spine was torn with tweezers, and a visual field of about 1 cm square was secured for each of the fourth and fifth lumbar vertebrae. A hole was drilled using a dental drill near the center of the exposed fourth lumbar vertebra and fifth lumbar vertebra, avoiding the anterior longitudinal ligament and blood vessels. Thereafter, the isolated organ was returned to a normal position, antibiotics were dropped, and the incision was sutured with a suture. The sutured part was fastened with a stapler and disinfected with iodine tincture. After awakening from anesthesia, the animal was returned to the cage and raised.
result
Two steps were important for the production of this model. The first step is detachment of blood vessels, adipose tissue and the like around the lumbar spine after laparotomy. In order to peel off, two kinds of large and small cotton swabs generally used were used. A large swab with a diameter of about 1.2 mm and a small swab with a diameter of about 3 mm were used. By gradually exfoliating the tissue with a cotton swab, it was possible not only to prevent bleeding but also to minimize damage to the muscle tissue around the vertebral body. In addition, when the dental drill is brought close to the vertebral body, a visual field of about 1 cm square is secured for one vertebral body so that the dental drill does not involve the surrounding tissue and minimizes muscular tissue damage. .
The second step is to make a hole near the center avoiding the anterior longitudinal ligament and blood vessels of the 4th and 5th lumbar vertebrae with a secured visual field. If the diameter of the hole is 2 mm (the size of the hole made by Uchida et al. In the femur is about 2-2.5 mm in diameter (Non-patent Documents 12 and 15)), blood vessels cannot be avoided and many blood vessels are damaged. As a result, the bleeding increased, and the rat died without awakening from anesthesia. On the other hand, when the diameter of the hole was 0.4 mm or less, the columnar stable bone damage could not be caused uniformly. Therefore, the rat survives without dying after surgery, its natural repair process is as short as 21 days, and the bone damage that can be evaluated using micro CT is about 0.72 mm in diameter and depth is The condition of about 2.5-3.0 mm not penetrating the vertebral body was determined according to the size of each vertebral body.
As a representative example, a micro CT image (3D image and 2D image) of an injured vertebral body (sliced vertebral body) on the model preparation date is shown (FIG. 1). FIG. 1 is a model in which a rat lumbar vertebral body damage model was prepared and the vertebral body was photographed using micro CT. Specifically, using a dental drill, a hole having a diameter of about 0.72 mm was formed in the vicinity of the center avoiding the exposed anterior longitudinal ligaments of L4 and L5 and blood vessels. The arrow points to the drilled hole. As shown in the figure, a bone injury of about 0.72 mm in diameter and 2.5-3.0 mm in depth does not penetrate the vertebral body in the vicinity of the center of the 4th or 5th lumbar vertebra, avoiding the anterior longitudinal ligament and blood vessels. Produced. With this hole size, the rat showed a gradual weight gain similar to that of the sham-operated rat that had been awakened from anesthesia and only performed laparotomy. Moreover, in the observation of general symptoms by appearance, there were no abnormal findings, and it was possible to raise the animals while maintaining good health for 21 days after the operation. As shown in Examples 2 and 3, on the 21st day after the operation, the recovery of the bone structure to the same extent as that of the normal vertebral body was confirmed. Therefore, the therapeutic effect of the drug on the vertebral fracture was examined in vivo in this model. As a new model that can be used, it was named rat lumbar vertebral bone injury model.
[Example 2] Histological examination of rat lumbar vertebral bone injury model during natural repair process
The natural restoration process after bone injury in the rat lumbar vertebral body bone injury model was observed by preparing vertebral body tissue specimens. The model creation date (the day when the hole was made) was set to day 0, and blood was lethal from the abdominal aorta under pentobarbital anesthesia to day 4, day 7, day 14, and day 21, and the lumbar vertebrae were removed. The extracted lumbar vertebra was immersed in 70% ethanol and stored at 4 ° C. Each vertebral body was decalcified by formic acid / sodium citrate and embedded in paraffin (sealed automatic fixed embedding device (Sakura ETP-180BV)). Based on the micro CT image, the damaged part of the cancellous bone was exposed, and the block where the damaged part was exposed was observed. Sections (sliding microtome (Sakura IVS-410)) sliced at 3 mm were placed on a slide glass, and after deparalysis, hematoxylin and eosin (HE) staining and Masson trichrome (MT) staining were performed. The prepared tissue specimens were observed using a microscope (Leica DM4000B), a camera (Leica DFC480), and a microscopic photography software (Leica IM500).
result
The model production date (the day when the hole was made) was set to day 0, and the HE-stained image and the MT-stained image of day 4, day 7, day 14, and day 21 were observed. FIG. 2 shows representative examples of MT staining (damaged cancellous bone) at each time point. FIG. 2 shows the MT-stained images of the model production date (day 0), day 4, day 7, day 14, and day 21. 2A was day 0, 2B was day 4, 2C was day 7, 2D was day 14, 2E was day 21, and 2F was a canal bone of a normal vertebral body (sham operated rat) of day 21. Regarding the damaged vertebral bodies, the upper part is the damaged part, the lower part is the non-injured part, and the boundary is indicated by an arrow.
Immediately after model preparation (day 0), leakage of bone marrow cells at the damaged part was observed (FIG. 2A). In day 4 (FIG. 2B), a mixture of hematoma and granulation tissue, which is the initial stage of fracture healing, and a small amount of fibrous bone were observed. In day 7 (FIG. 2C), the hematoma organized and disappeared, fibrous bone increased, and trabecular bone that was clearly distinguishable from the surroundings was observed. In day 14 (FIG. 2D), trabecular bone increased further, and granulation tissue and fibrous bone were hardly observed. In day 21 (FIG. 2E), the damaged part was completely repaired, and a tissue image similar to that in the sham-operated rat (FIG. 2F) was observed.
[Example 3] Micro CT imaging and bone volume density (mg / cm) using the image in the natural restoration process of rat lumbar vertebral body bone damage model3) Measurement
A microfocus X-ray CT apparatus (Nittsu Elex Co., Ltd.) was used. Using the imaging software ELE SCAN (NX-CP-C80H-IL-021: Nippon Steel ELEX Co., Ltd.), 3D tomographic imaging was performed under imaging conditions of a tube voltage of 50 kV and a tube current of 60 mA. The line where the longitudinal ligament is located from the side of the vertebral body was aligned with the center line, and the vertebral body was fixed so that it did not move during imaging, and 300 images were taken with a width of 0.02 mm. In addition, the measured value is the bone volume density (mg / cm3) Calcium carbonate phantom set in 4 stages (0 to 300 mg / cm)3CaCO3) Under the same conditions. The CT image was saved as a C3D file and reconstructed using conbeam reconstruction software (ELE SCAN CB3D: Nippon Steel Elex Co., Ltd.) (C3V file creation). Based on the stored C3V file, 300 TIFF files were created and analyzed using this file. For the analysis, Real INTAGE ver2.1 (K.G. TT Co., Ltd.) was used to measure the luminance value.
The bone volume density of the damaged part of the cancellous bone of the damaged vertebral body was measured. Specifically, for the 11 Tiff files from 1.90 mm to 2.10 mm, where the line where the posterior longitudinal ligament is located as seen from the side of the vertebral body in the micro CT image taken is 0.40 mm, respectively.2The luminance value of the circle was measured.
Bone volume density was also measured for cancellous bones of normal vertebral bodies (sham-operated rats). Specifically, for each of 11 Tiff files from 1.90 mm to 2.10 mm when the line where the posterior longitudinal ligament is located as viewed from the side of the vertebral body of the micro CT image taken is 0 mm, 2.0 mm each.2The luminance value of the rectangle (not including cortical bone) was measured.
Measured luminance value is bone volume density (mg / cm based on calcium carbonate phantom measured under the same conditions.3).
result
The bone volume density of the damaged part of cancellous bone was measured using micro CT. The model production date (the day when the hole was made) was set to day 0, and the bone volume densities of day 4, day 7, day 14, and day 21 were shown (FIG. 3). FIG. 3 shows the bone volume density of the model creation date (day 0), day 4, day 7, day 14, and day 21. The black circle (●) is the mean bone volume density ± standard deviation of the damaged part of the trabecular bone of the damaged vertebra, and the square (□) is the mean value of the bone volume density of the cancellous bone of the normal vertebral body (sham operated rat) ± Standard deviation is shown.
1 vertebral body was treated as an example. The average value ± standard deviation of bone volume density immediately after model preparation is 0.0 ± 31.6 mg / cm3For day 4, day 7, day 14, and day 21, 33.5 ± 39.5, 92.4 ± 29.8, 100.2 ± 45.4, 263.2 ± 50.1 mg / cm, respectively.3Met. On the other hand, the bone volume density of cancellous bone of normal vertebral bodies (sham-operated rats) is 246.4 ± 64.6 for day 0 and 220.7 ± 50.1 mg / cm for day 21.3And showed a substantially constant value within the observation period.
Therefore, when a hole was drilled in the lumbar spine with a dental drill, the bone volume density of the hole gradually increased, and it was shown that it recovered to the same level as the normal vertebral body (sham-operated rat) on the 21st day. Moreover, it was shown that the bone damage repair level of the rat lumbar vertebral body bone damage model can be quantified by measuring the bone volume density using micro CT.
[Example 4] Histological examination when hPTH (1-34) and PTHrP analog A were administered to a rat lumbar vertebral body bone injury model
The vertebral body tissue specimens when hPTH (1-34) (Bachem) or PTHrP analog A (Ipsen) (SEQ ID NO: 42) was administered for 7 days or 14 days from the date of model preparation were observed. The hPTH (1-34) group, PTHrP analog A and vehicle group were set. As the vehicle administration solution, Saline (pH 4.0 ± 0.1) containing 2% rat serum was used. The hPTH (1-34) administration solution and the PTHrP analog A administration solution were prepared to 10 μg / mL using Vehicle. The first administration was performed 4 hours after the model was prepared, and was administered subcutaneously once a day from the next day. The administration volume was 1 mL / kg. 24 hours after the final administration, blood was lethal from the abdominal aorta under pentobarbital anesthesia, and the fourth lumbar vertebra, fifth lumbar vertebra (damaged vertebra), third lumbar vertebra, and sixth lumbar vertebra (undamaged vertebral body) were removed. The extracted lumbar vertebra was immersed in 70% ethanol and stored at 4 ° C.
Each vertebral body was decalcified with formic acid / sodium citrate and embedded in paraffin (sealed automatic fixed embedding device (Sakura ETP-180BV)). Based on the micro CT image, the damaged part of the cancellous bone was exposed, and the block where the damaged part was exposed was observed. Sections (sliding microtome (Sakura IVS-410)) sliced at 3 mm were placed on a slide glass, and after deparalysis, hematoxylin and eosin (HE) staining and Masson trichrome (MT) staining were performed. The prepared tissue specimens were observed using a microscope (Leica DM4000B), a camera (Leica DFC480), and a microscopic photography software (Leica IM500).
result
The HE-stained image and MT-stained image were observed for 7 or 14 days from the date of model preparation when hPTH (1-34) 10 μg / kg or PTHrP analog A 10 μg / kg was subcutaneously administered every day. FIG. 4 shows representative examples of MT staining (damaged cancellous bone) at each time point. FIG. 4 shows an MT-stained image when hPTH (1-34) 10 μg / kg or PTHrP analog A 10 μg / kg was subcutaneously administered every day for 7 days or 14 days from the model creation date. 4A is the vehicle group at day7, 4B is the hPTH (1-34) group at day7, 4C is the PTHrP analog A group at day7, 4D is the vehicle group at day14, 4E is the hPTH (1-34) at day14 Group, 4F shows PTHrP analog A group on day14. In each case, the upper part is a damaged part, the lower part is an uninjured part, and the boundary is indicated by an arrow.
On day 7 after administration, an increase in fibrous bone and an increase in trabecular bone were observed in the hPTH (1-34) group (FIG. 4B) as compared to the Vehicle group (FIG. 4A). On the 14th day after administration, granulation tissue and fibrous bone were hardly observed in both groups, and no morphological difference was observed. In the hPTH (1-34) group (FIG. 4E), the Vehicle group ( More trabecular bone was observed compared to FIG. 4D).
Similarly, on day 7 after administration, an increase in fibrous bone and trabecular bone were observed in the PTHrP analog A group (FIG. 4C) compared to the Vehicle group (FIG. 4A). On day 14 of administration, granulation tissue and fibrous bone were hardly observed in both groups, and no morphological difference was observed. In the PTHrP analog A group (FIG. 4F), the vehicle group (FIG. 4D). More trabeculae were observed compared to. No significant difference was observed between the hPTH (1-34) group and the PTHrP analog A group.
In addition, the tissue images obtained by HE staining were observed for 7 days or 14 days from the date of model preparation. As a result of comparing the group administered with 10 μg / kg of hPTH (1-34) or 10 μg / kg of PTHrP analog A, there was no obvious accumulation of inflammatory cells in the injured and uninjured areas. There was no academic difference.
[Example 5] Examination of bone volume density at the injured part of cancellous bone of damaged vertebral body when hPTH (1-34) and PTHrP analog A were administered to a rat lumbar vertebral body bone injury model
The bone volume density of the damaged part of the trabecular bone of the injured vertebral body when subcutaneously administering hPTH (1-34) 10 μg / kg or PTHrP analog A 10 μg / kg for 7 days or 14 days from the model preparation date was measured. The bone volume density was measured using the same method as in Example 3.
result
Bone volume density (mg / cm) measured using Micro CT images3) Was analyzed. In carrying out the analysis, one vertebral body was handled as an example.
Student's t-test was performed between Day 2 and Day 14 between the hPTH (1-34) group and the Vehicle group, and between the PTHrP analog A group and the vehicle group. The significance level was 5%.
The hardware used was Fujitsu Ltd. FMVXD0572 FMV ESPRIMO D3230, and the software used was SAS Institute Japan SAS ver. 8.2 and Arm EXSAS ver. 7.10.
The bone volume density of the damaged part of the cancellous bone of the damaged vertebral body was measured using micro CT. The bone volume density when hPTH (1-34) 10 μg / kg was subcutaneously administered every day for 7 days or 14 days from the model preparation date was shown (FIG. 5). FIG. 5 shows the bone volume density of the damaged part of cancellous bone when hPTH (1-34) 10 μg / kg or PTHrP analog A 10 μg / kg was subcutaneously administered every day for 7 days or 14 days from the model preparation date. The black circle (●) is the mean ± standard deviation of the vehicle group, the white circle (◯) is the mean ± standard deviation of the hPTH (1-34) group, and the triangle (Δ) is the mean ± standard of the PTHrP analog A group Indicates the deviation. Student's t-test was performed between the two groups of the hPTH (1-34) group and the Vehicle group and between the two groups of the PTHrP analog A group and the Vehicle group at each time point. Statistically significant differences were observed between the two groups, the group and the hPTH (1-34) group, and between the Vehicle group and the PTHrP analog A group (p = 0.0100, p = 0.0025). .
On the 7th day after administration, the mean ± standard deviation of the bone volume density of the damaged part of the cancellous bone is 91.4 ± 33.8 mg / cm in the vehicle group.3, HPTH (1-34) group is 158.0 ± 77.5 mg / cm3There was no statistically significant difference between the two groups (p = 0.0743: Student's t test). On the 14th day after administration, the mean value ± standard deviation of the bone volume density of the damaged part of the cancellous bone was 140.9 ± 58.8 mg / cm in the vehicle group.3HPTH (1-34) group is 250.5 ± 78.7 mg / cm3There was a statistically significant difference between the two groups (p = 0.0100: Student's t test).
Similarly, bone volume density was shown when PTHrP analog A 10 μg / kg was subcutaneously administered every day for 7 days or 14 days from the model preparation date (FIG. 5). On the seventh day after administration, the mean value ± standard deviation of the bone volume density of the damaged part of the cancellous bone was 91.4 ± 33.8 mg / cm in the Vehicle group.3PTHrP analog A group is 148.5 ± 56.2 mg / cm3There was no statistically significant difference between the two groups (p = 0.0590: Student's t test). On the 14th day after administration, the mean value ± standard deviation of the bone volume density of the damaged part of the cancellous bone was 140.9 ± 58.8 mg / cm in the vehicle group.3PTHrP analog A group is 237.0 ± 40.4 mg / cm3There was a statistically significant difference between the two groups (p = 0.0025: Student's t test). Therefore, it was found that both hPTH (1-34) and PTHrP analog A show a significant increase in bone volume density when administered for 14 days in the vertebral cancellous lesion.
In addition, in the group administered with 7 μg / kg of hPTH (1-34) or 10 μg / kg of PTHrP analog A for 7 days from the model creation date, although no significant difference is seen, it is higher than that in the vehicle administration group. Since the bone volume density was shown, it was shown that neither hPTH (1-34) nor PTHrP analog A caused excessive bone resorption at the damaged part.
[Example 6] Examination of bone volume density of cancellous bone of uninjured vertebral body when hPTH (1-34) and PTHrP analog A were administered to a rat lumbar vertebral body bone injury model
7 days or 14 days from the date of model preparation, the bone volume density of cancellous bone of the non-injured vertebral body was measured when hPTH (1-34) 10 μg / kg or PTHrP analog A 10 μg / kg was subcutaneously administered every day. The bone volume density was measured using the same method as in Example 3. However, in consideration of the size of the non-injured vertebral bodies (third lumbar vertebra, sixth lumbar vertebra), the measurement position was set to a position different from the fourth lumbar vertebra and the fifth lumbar vertebra. Specifically, the third lumbar vertebrae is about 11 Tiff files from 1.70mm to 1.90mm when the line where the posterior longitudinal ligament is located as seen from the side of the vertebral body in the micro CT image taken is 0 The 6th lumbar spine is 2.0 mm for each of the 11 Tiff files from 2.10 mm to 2.30 mm when the posterior vertebral body viewed from the side of the vertebral body of the CT image taken is 0.2The luminance value of the rectangle (not including cortical bone) was measured.
result
Bone volume density (mg / cm) measured using Micro CT images3) Was analyzed. In carrying out the analysis, one vertebral body was handled as an example.
Student's t-test was performed between Day 2 and Day 14 between the hPTH (1-34) group and the Vehicle group, and between the PTHrP analog A group and the vehicle group. The significance level was 5%.
The hardware used was Fujitsu Ltd. FMVXD0572 FMV ESPRIMO D3230, and the software used was SAS Institute Japan SAS ver. 8.2 and Arm EXSAS ver. 7.10.
The bone volume density of the cancellous bone of the non-injured vertebral bodies (third lumbar vertebra, sixth lumbar vertebra) was measured. Bone volume density was shown when hPTH (1-34) 10 μg / kg was subcutaneously administered every day for 7 days and 14 days from the date of model preparation (FIG. 6). FIG. 6 shows the bone volume density of cancellous bone of the uninjured vertebral body when subcutaneously administering hPTH (1-34) 10 μg / kg or PTHrP analog A 10 μg / kg for 7 or 14 days from the model creation date. . The black circle (●) is the mean ± standard deviation of the vehicle group, the white circle (◯) is the mean ± standard deviation of the hPTH (1-34) group, and the triangle (Δ) is the mean ± standard of the PTHrP analog A group Indicates the deviation. A Student's t-test was performed between the two groups of the hPTH (1-34) group and the Vehicle group and between the two groups of the PTHrP analog A group and the Vehicle group at each time point. No significant difference was observed (day 7: p = 0.9981, p = 0.5591, day 14: p = 0.2539, p = 0.1919). On day 7 of administration, the mean value ± standard deviation of the bone volume density of cancellous bone was 214.9 ± 56.6 mg / cm in the vehicle group.3HPTH (1-34) group is 215.0 ± 42.1 mg / cm3There was no statistically significant difference between the two groups (p = 0.9981: Student's t test). On the 14th day after administration, the mean value ± standard deviation of the bone volume density of cancellous bone was 240.2 ± 59.7 mg / cm in the vehicle group.3HPTH (1-34) group is 268.3 ± 30.0 mg / cm3There was no statistically significant difference between the two groups (p = 0.2539: Student's t test).
Similarly, bone volume density was shown when PTHrP analog A 10 μg / kg was subcutaneously administered every day (FIG. 6). On day 7 of administration, the mean value ± standard deviation of the bone volume density of cancellous bone was 214.9 ± 56.6 mg / cm in the vehicle group.3The PTHrP analog A group is 233.4 ± 66.6 mg / cm.3There was no statistically significant difference between the two groups (p = 0.5591: Student's t test). On the 14th day after administration, the mean value ± standard deviation of the bone volume density of cancellous bone was 240.2 ± 59.7 mg / cm in the vehicle group.3PTHrP analog A group is 278.0 ± 50.1 mg / cm3There was no statistically significant difference between the two groups (p = 0.1919: Student's t test).
Therefore, it was found that even when hPTH (1-34) or PTHrP analog A was administered for 14 days, the bone volume density of the cancellous bone of the vertebral body that was not damaged did not change. From the above results, it was found that the bone volume density increasing action of hPTH (1-34) and PTHrP analog A observed in the damaged part of cancellous bone of the damaged vertebral body is specific to the damaged site.
As described above, when PTH (1-34) or PTHrP analog A is administered to a rat lumbar vertebral body bone injury model, the bone volume density of the cancellous bone of the uninjured vertebral body is not changed. It was found that the bone volume density of the damaged part of cancellous bone increased significantly. Therefore, it was found that PTH (1-34) and PTHrP analog A have an action of treating damage to the fine structure of cancellous bone caused by vertebral fractures in a site specific to the damaged site.
[Example 7] Examination of effects on motor function and body weight change when hPTH (1-34) and PTHrP analog A were administered to a rat lumbar vertebral body bone injury model
Administration of hPTH (1-34) and PTHrP analog A exacerbates the inflammatory response in the fracture repair process, or destroys the vertebral body due to excessive bone resorption, resulting in the spinal cord existing in the vicinity of the vertebral body The possibility of being damaged was examined. In general, if the spinal injury causes a serious impairment in the lower limb motor function, feeding from the food box attached to the breeding cage is also restricted, resulting in a decrease in food consumption. It is thought that weight loss due to.
The production of the rat lumbar vertebral body bone injury model is carried out by the method described in Example 1. The production of the sham-operated rat is carried out by the rat described in Example 1 except for the step of making a hole using a dental drill. It was prepared in the same way as the lumbar spine bone injury model.
First, the influence on the motor function and weight change in the creation of the rat lumbar vertebral body bone injury model of the present invention was examined. For each group of rat lumbar vertebral body bone injury model and sham-operated rats, the day of surgery was set as Day 0, and the body weight was measured on Days 4, 7, 14, and 21. Regarding behavioral abnormalities, it was visually observed whether there were any unnatural behaviors such as walking and standing when measuring body weight. Subsequently, using the rat lumbar vertebral body bone damage model, changes in body weight were examined when hPTH (1-34) 10 μg / kg or PTHrP analog A 10 μg / kg was subcutaneously administered daily for 14 days from the model preparation date. The model creation date, which is the administration start date, was set to Day 0, and the body weight up to Day 14 was measured every day. In addition, behavioral abnormalities were visually observed as described above.
result
FIG. 7A shows the results of body weight measurement for each group of rat lumbar vertebral body bone injury model and sham-operated rats. There was no difference in body weight between the lumbar vertebral bone injury model and the sham-operated rats on all the measured days. In addition, no visual behavioral abnormality was observed in each animal. Therefore, in the rat lumbar vertebral body bone injury model of the present invention, the influence on the motor function by the model production is not recognized.
FIG. 7B shows the change in body weight of each group when hPTH (1-34) 10 μg / kg or PTHrP analog A 10 μg / kg or Vehicle was subcutaneously administered for 14 days from the model preparation day. No difference in body weight was observed between the groups treated with hPTH (1-34), PTHrP analog A and vehicle from the start of administration to 14 days. In addition, no visual behavioral abnormality was observed in each animal. Therefore, by administering hPTH (1-34) or PTHrP analog A to a rat lumbar vertebral body bone injury model, there is no effect such as serious spinal cord injury that causes behavioral disorder. confirmed.

　本発明は、ＰＴＨ／ＰＴＨｒＰ受容体アゴニスト、又はＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することによって、椎体骨折治療剤、椎体骨折に起因する疼痛の治療又は予防剤、椎体圧迫骨折予防剤、又は椎体骨折に起因する運動障害及び／又は神経障害の治療又は予防剤として使用できる。また、本発明は、非ヒト動物の椎体骨に外科的な損傷を与える工程、当該非ヒト動物に被検物質を投与する工程、及び当該非ヒト動物又はそれに由来する検体の椎体骨の外科的な損傷の回復を測定する工程、を含むことによって、椎体骨折治療剤の評価方法として使用できる。
The present invention relates to a therapeutic agent for vertebral fracture, a therapeutic or preventive agent for pain caused by vertebral fracture, vertebral body by containing a PTH / PTHrP receptor agonist, or a substance having an activity of inducing PTH or PTHrP production. It can be used as a preventive agent for compression fractures, or a therapeutic or prophylactic agent for movement disorders and / or neurological disorders caused by vertebral fractures. The present invention also includes a step of surgically damaging a vertebral bone of a non-human animal, a step of administering a test substance to the non-human animal, and a vertebral bone of a specimen derived from the non-human animal. Including the step of measuring the recovery of surgical damage can be used as an evaluation method for a therapeutic agent for vertebral fractures.

Claims (36)

　ＰＴＨ／ＰＴＨｒＰ受容体アゴニストを含有することを特徴とする、椎体骨折治療剤。 A therapeutic agent for vertebral fracture, comprising a PTH / PTHrP receptor agonist. 　ＰＴＨ／ＰＴＨｒＰ受容体アゴニストが、副甲状腺ホルモン（ＰＴＨ）、副甲状腺ホルモン関連ペプチド（ＰＴＨｒＰ）、又はそれらの部分活性ポリペプチド、若しくはそれらのアナログである、請求項１に記載の椎体骨折治療剤。 The vertebral fracture therapeutic agent according to claim 1, wherein the PTH / PTHrP receptor agonist is parathyroid hormone (PTH), parathyroid hormone-related peptide (PTHrP), or a partially active polypeptide thereof, or an analog thereof. . 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドが、ＰＴＨ（１−８４）、ＰＴＨ（１−３７）、ＰＴＨ（１−３４）、ＰＴＨ（１−３１）、ＰＴＨｒＰ（１−１４１）、ＰＴＨｒＰ（１−１３９）、ＰＴＨｒＰ（１−８６）、ＰＴＨｒＰ（１−４０）、ＰＴＨｒＰ（１−３７）、ＰＴＨｒＰ（１−３６）、及びＰＴＨｒＰ（１−３４）のいずれかである、請求項２に記載の椎体骨折治療剤。 PTH, PTHrP or a partially active polypeptide thereof is PTH (1-84), PTH (1-37), PTH (1-34), PTH (1-31), PTHrP (1-141), PTHrP (1 -139), PTHrP (1-86), PTHrP (1-40), PTHrP (1-37), PTHrP (1-36), and PTHrP (1-34). For treating vertebral fractures. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドが、ヒトＰＴＨ又はヒトＰＴＨｒＰに由来するアミノ酸配列を有するポリペプチドである、請求項２又は３に記載の椎体骨折治療剤。 The therapeutic agent for vertebral fracture according to claim 2 or 3, wherein the PTH, PTHrP or a partially active polypeptide thereof is a polypeptide having an amino acid sequence derived from human PTH or human PTHrP. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドにおけるアミノ酸配列のうち、１~１０個のアミノ酸を別のアミノ酸に置換したポリペプチドである、請求項２に記載の椎体骨折治療剤。 The analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide in which 1 to 10 amino acids in the amino acid sequence of PTH, PTHrP or a partially active polypeptide thereof are substituted with another amino acid. The therapeutic agent for vertebral fracture according to 2. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、ＰＴＨ（１−８４）、ＰＴＨ（１−３７）、ＰＴＨ（１−３４）、ＰＴＨ（１−３１）、ＰＴＨｒＰ（１−１４１）、ＰＴＨｒＰ（１−１３９）、ＰＴＨｒＰ（１−８６）、ＰＴＨｒＰ（１−４０）、ＰＴＨｒＰ（１−３７）、ＰＴＨｒＰ（１−３６）、及びＰＴＨｒＰ（１−３４）のいずれかのアミノ酸配列のうち、１~１０個のアミノ酸を別のアミノ酸に置換したポリペプチドである、請求項５に記載の椎体骨折治療剤。 PTH, PTHrP or analogs of their partially active polypeptides are PTH (1-84), PTH (1-37), PTH (1-34), PTH (1-31), PTHrP (1-141), PTHrP. (1-139), PTHrP (1-86), PTHrP (1-40), PTHrP (1-37), PTHrP (1-36), and any one of the amino acid sequences of PTHrP (1-34), The therapeutic agent for vertebral fracture according to claim 5, which is a polypeptide in which 1 to 10 amino acids are substituted with another amino acid. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、ヒトＰＴＨ又はヒトＰＴＨｒＰに由来するアミノ酸配列のうち、１~１０個のアミノ酸を別のアミノ酸に置換したポリペプチドである、請求項５又は６に記載の椎体骨折治療剤。 The analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide in which 1 to 10 amino acids are substituted with another amino acid in the amino acid sequence derived from human PTH or human PTHrP. The therapeutic agent for vertebral fracture described in 1. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチド、若しくはそれらのアナログが、製薬上許容される塩である、請求項１~７のいずれかに記載の椎体骨折治療剤。 The therapeutic agent for vertebral fracture according to any one of claims 1 to 7, wherein PTH, PTHrP, or a partially active polypeptide thereof, or an analog thereof is a pharmaceutically acceptable salt. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが下記式のポリペプチド：
Figure JPOXMLDOC01-appb-C000001
 ただし、
Ａ１はＳｅｒ、Ａｌａ、又はＤａｐであり；
Ａ３はＳｅｒ、Ｔｈｒ、又はＡｉｂであり；
Ａ５はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ７はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ８はＭｅｔ、Ｎｖａ、Ｌｅｕ、Ｖａｌ、Ｉｌｅ、Ｃｈａ、又はＮｌｅであり；
Ａ１１はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ１２はＧｌｙ又はＡｉｂであり；
Ａ１５はＬｅｕ、Ｎｌｅ、Ｉｌｅ、Ｃｈａ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ１６はＳｅｒ、Ａｓｎ、Ａｌａ、又はＡｉｂであり；
Ａ１７はＳｅｒ、Ｔｈｒ、又はＡｉｂであり；
Ａ１８はＭｅｔ、Ｎｖａ、Ｌｅｕ、Ｖａｌ、Ｉｌｅ、Ｎｌｅ、Ｃｈａ、又はＡｉｂであり；
Ａ１９はＧｌｕ又はＡｉｂであり；
Ａ２１はＶａｌ、Ｃｈａ、又はＭｅｔであり；
Ａ２３はＴｒｐ又はＣｈａであり；
Ａ２４はＬｅｕ又はＣｈａであり；
Ａ２７はＬｙｓ、Ａｉｂ、Ｌｅｕ、ｈＡｒｇ、Ｇｌｎ、又はＣｈａであり；
Ａ２８はＬｅｕ又はＣｈａであり；
Ａ３０はＡｓｐ又はＬｙｓであり；
Ａ３１はＶａｌ、Ｎｌｅ、若しくはＣｈａである、又は欠損しており；
Ａ３２はＨｉｓである又は欠損しており；
Ａ３３はＡｓｎである又は欠損しており；
Ａ３４はＰｈｅ、Ｔｙｒ、Ａｍｐ、若しくはＡｉｂである、又は欠損しており；
Ｒ１及びＲ２は、それぞれ独立して、Ｈ、Ｃ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；又はＲ１及びＲ２の両方又は一方のみがＣＯＥ１であり、この際、Ｅ１はＣ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；及び
Ｒ３はＯＨ、ＮＨ２、Ｃ１−１２アルコキシ、又はＮＨ−Ｙ−ＣＨ２−Ｚであり、この際、ＹはＣ１−１２炭化水素部分であり、ＺはＨ、ＯＨ、ＣＯ２Ｈ、又はＣＯＮＨ２であり；
Ａ５、Ａ７、Ａ８、Ａ１１、Ａ１５、Ａ１８、Ａ２１、Ａ２３、Ａ２４、Ａ２７、Ａ２８及びＡ３１の少なくとも一つはＣｈａである、又はＡ３、Ａ１２、Ａ１６、Ａ１７、Ａ１８、Ａ１９及びＡ３４の少なくとも一つはＡｉｂである；又はこれらの製薬上許容できる塩である、請求項１又は２に記載の椎体骨折治療剤。 An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the formula:
Figure JPOXMLDOC01-appb-C000001
 However,
A 1 is Ser, Ala, or Dap;
A 3 is Ser, Thr, or Aib;
A 5 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 7 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 8 is Met, Nva, Leu, Val, Ile, Cha, or Nle;
A 11 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 12 is Gly or Aib;
A 15 is Leu, Nle, Ile, Cha, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 16 is Ser, Asn, Ala, or Aib;
A 17 is Ser, Thr, or Aib;
A 18 is Met, Nva, Leu, Val, Ile, Nle, Cha, or Aib;
A 19 is Glu or Aib;
A 21 is Val, Cha, or Met;
A 23 is Trp or Cha;
A 24 is Leu or Cha;
A 27 is Lys, Aib, Leu, hArg, Gln, or Cha;
A 28 is Leu or Cha;
A 30 is Asp or Lys;
A 31 is Val, Nle, or Cha, or is missing;
A 32 is His or missing;
A 33 is Asn or is missing;
A 34 is Phe, Tyr, Amp, or Aib, or is missing;
R 1 and R 2 are each independently H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12. Hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7-20 hydroxyphenylalkyl, or C 11-20 hydroxynaphthylalkyl; or both R 1 and R 2 or only one is COE 1 , wherein E 1 is C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12 hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7 -20 hydroxyphenyl alkyl, or with C 11-20 hydroxy naphthyl alkyl Ri; and R 3 is OH, NH 2, C 1-12 alkoxy, or NH-Y-CH 2 -Z, this time, Y is C 1-12 hydrocarbon moiety, Z is H, OH, CO 2 H or CONH 2 ;
At least one of A 5 , A 7 , A 8 , A 11 , A 15 , A 18 , A 21 , A 23 , A 24 , A 27 , A 28 and A 31 is Cha, or A 3 , A 12 , at least one of a 16, a 17, a 18 , a 19 and a 34 is a Aib; a, or a pharmaceutically acceptable salt, vertebral fracture treatment agent according to claim 1 or 2. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、下記式のポリペプチド：
［Ｃｈａ７，１１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２３］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｎｌｅ８，１８，Ｃｈａ２７］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２８］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ３１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１６］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１９］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２４，２８，３１，Ｌｙｓ３０］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，Ａｉｂ１６，１９，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，３１，Ａｉｂ１６，１９，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ１１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２８，３１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ｎｌｅ８，１８，Ａｉｂ３４］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ１５］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ａｉｂ１９］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ａｉｂ１６］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１６，１９］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１２］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ３］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７，１１，Ａｉｂ１９，Ｌｙｓ３０］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ７］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｃｈａ２４，２８，３１］ｈＰＴＨ（１−３４）ＮＨ２；
［Ａｉｂ１７］ｈＰＴＨ（１−３４）ＮＨ２；及び
［Ｃｈａ７，１１，１５］ｈＰＴＨ（１−３４）ＮＨ２、
からなる群より選択されるペプチド、又はこれらの製薬上許容できる塩である、請求項９に記載の椎体骨折治療剤。 An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the formula:
[Cha 7,11 ] hPTH (1-34) NH 2 ;
[Cha 23 ] hPTH (1-34) NH 2 ;
[Cha 24 ] hPTH (1-34) NH 2 ;
[Nle 8,18 , Cha 27 ] hPTH (1-34) NH 2 ;
[Cha 28 ] hPTH (1-34) NH 2 ;
[Cha 31 ] hPTH (1-34) NH 2 ;
[Aib 16 ] hPTH (1-34) NH 2 ;
[Aib 19 ] hPTH (1-34) NH 2 ;
[Aib 34 ] hPTH (1-34) NH 2 ;
[Cha 24 , 28 , 31 , Lys 30 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Nle 8,18 , Tyr 34 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Nle 8,18 , Aib 16,19 , Tyr 34 ] hPTH (1-34) NH 2 ;
[Cha 7,11, Nle 8,18,31, Aib 16,19, Tyr 34] hPTH (1-34) NH 2;
[Cha 11 ] hPTH (1-34) NH 2 ;
[Cha 28,31 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Nle 8,18 , Aib 34 ] hPTH (1-34) NH 2 ;
[Cha 15 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Aib 19 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Aib 16 ] hPTH (1-34) NH 2 ;
[Aib 16,19 ] hPTH (1-34) NH 2 ;
[Aib 12 ] hPTH (1-34) NH 2 ;
[Aib 3 ] hPTH (1-34) NH 2 ;
[Cha 7,11 , Aib 19 , Lys 30 ] hPTH (1-34) NH 2 ;
[Cha 7 ] hPTH (1-34) NH 2 ;
[Cha 24, 28, 31 ] hPTH (1-34) NH 2 ;
[Aib 17 ] hPTH (1-34) NH 2 ; and [Cha 7,11,15 ] hPTH (1-34) NH 2 ,
The therapeutic agent for vertebral fracture according to claim 9, which is a peptide selected from the group consisting of: or a pharmaceutically acceptable salt thereof. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが下記式のポリペプチド：
Figure JPOXMLDOC01-appb-C000002
 ただし、
Ａ１はＡｌａ、Ｓｅｒ、又はＤａｐであり；
Ａ３はＳｅｒ又はＡｉｂであり；
Ａ５はＨｉｓ、Ｉｌｅ、又はＣｈａであり；
Ａ７はＬｅｕ、Ｃｈａ、Ｎｌｅ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ８はＬｅｕ、Ｍｅｔ、又はＣｈａであり；
Ａ１０はＡｓｐ又はＡｓｎであり；
Ａ１１はＬｙｓ、Ｌｅｕ、Ｃｈａ、Ｐｈｅ、又はβ−Ｎａｌであり；
Ａ１２はＧｌｙ又はＡｉｂであり；
Ａ１４はＳｅｒ又はＨｉｓであり；
Ａ１５はＩｌｅ、又はＣｈａであり；
Ａ１６はＧｌｎ又はＡｉｂであり；
Ａ１７はＡｓｐ又はＡｉｂであり；
Ａ１８はＬｅｕ、Ａｉｂ、又はＣｈａであり；
Ａ１９はＡｒｇ又はＡｉｂであり；
Ａ２２はＰｈｅ、Ｇｌｕ、Ａｉｂ、又はＣｈａであり；
Ａ２３はＰｈｅ、Ｌｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２４はＬｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２５はＨｉｓ、Ａｉｂ、又はＧｌｕであり；
Ａ２６はＨｉｓ、Ａｉｂ、又はＬｙｓであり；
Ａ２７はＬｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２８はＩｌｅ、Ｌｅｕ、Ｌｙｓ、又はＣｈａであり；
Ａ２９はＡｌａ、Ｇｌｕ、又はＡｉｂであり；
Ａ３０はＧｌｕ、Ｃｈａ、Ａｉｂ、又はＬｙｓであり；
Ａ３１はＩｌｅ、Ｌｅｕ、Ｃｈａ、若しくはＬｙｓである、又は欠損しており；
Ａ３２はＨｉｓである又は欠損しており；
Ａ３３はＴｈｒである又は欠損しており；
Ａ３４はＡｌａである又は欠損しており；
Ｒ１及びＲ２は、それぞれ独立して、Ｈ、Ｃ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；又はＲ１及びＲ２の一方及び一方のみがＣＯＥ１であり、この際、Ｅ１はＣ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；及び
Ｒ３はＯＨ、ＮＨ２、Ｃ１−１２アルコキシ、又はＮＨ−Ｙ−ＣＨ２−Ｚであり、この際、ＹはＣ１−１２炭化水素部分であり、ＺはＨ、ＯＨ、ＣＯ２Ｈ、又はＣＯＮＨ２であり；
Ａ５、Ａ７、Ａ８、Ａ１１、Ａ１５、Ａ１８、Ａ２２、Ａ２３、Ａ２４、Ａ２７、Ａ２８、Ａ３０、若しくはＡ３１の少なくともひとつはＣｈａである、又はＡ３、Ａ１２、Ａ１６、Ａ１７、Ａ１８、Ａ１９、Ａ２２、Ａ２５、Ａ２６、Ａ２９、Ａ３０、若しくはＡ３４の少なくともひとつはＡｉｂである；
又はこれらの製薬上許容できる塩である、請求項１又は２に記載の椎体骨折治療剤。 An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the formula:
Figure JPOXMLDOC01-appb-C000002
 However,
A 1 is Ala, Ser, or Dap;
A 3 is Ser or Aib;
A 5 is His, Ile, or Cha;
A 7 is Leu, Cha, Nle, β-Nal, Trp, Pal, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 8 is Leu, Met, or Cha;
A 10 is Asp or Asn;
A 11 is Lys, Leu, Cha, Phe, or β-Nal;
A 12 is Gly or Aib;
A 14 is Ser or His;
A 15 is Ile or Cha;
A 16 is Gln or Aib;
A 17 is Asp or Aib;
A 18 is Leu, Aib, or Cha;
A 19 is Arg or Aib;
A 22 is Phe, Glu, Aib, or Cha;
A 23 is Phe, Leu, Lys, or Cha;
A 24 is Leu, Lys, or Cha;
A 25 is His, Aib, or Glu;
A 26 is His, Aib, or Lys;
A 27 is Leu, Lys, or Cha;
A 28 is Ile, Leu, Lys, or Cha;
A 29 is Ala, Glu, or Aib;
A 30 is Glu, Cha, Aib, or Lys;
A 31 is Ile, Leu, Cha, or Lys, or is missing;
A 32 is His or missing;
A 33 is Thr or missing;
A 34 is Ala or missing;
R 1 and R 2 are each independently H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12. Hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7-20 hydroxyphenylalkyl, or C 11-20 hydroxynaphthylalkyl; or only one and one of R 1 and R 2 is COE 1 , wherein E 1 is C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12 hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7 -20 hydroxyphenyl alkyl, or with C 11-20 hydroxy naphthyl alkyl Ri; and R 3 is OH, NH 2, C 1-12 alkoxy, or NH-Y-CH 2 -Z, this time, Y is C 1-12 hydrocarbon moiety, Z is H, OH, CO 2 H or CONH 2 ;
At least one of A 5 , A 7 , A 8 , A 11 , A 15 , A 18 , A 22 , A 23 , A 24 , A 27 , A 28 , A 30 , or A 31 is Cha, or A 3 , A 12 , A 16 , A 17 , A 18 , A 19 , A 22 , A 25 , A 26 , A 29 , A 30 , or A 34 is Aib;
Or the vertebral body fracture therapeutic agent of Claim 1 or 2 which is these pharmaceutically acceptable salt. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、式：［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，３１，Ａｉｂ２９，Ｌｙｓ２６，３０］ｈＰＴＨｒＰ（１−３４）ＮＨ２のポリペプチド又はその製薬上許容できる塩である、請求項１１に記載の椎体骨折治療剤。 PTH, analogs PTHrP or their partial active polypeptide, wherein: [Glu 22,25, Leu 23,28,31, Aib 29, Lys 26,30] hPTHrP (1-34) polypeptide NH 2 or The therapeutic agent for vertebral fracture according to claim 11, which is a pharmaceutically acceptable salt. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが下記式のポリペプチド：
Figure JPOXMLDOC01-appb-C000003
 ただし、
Ａ１はＡｌａ、Ｓｅｒ、又はＤａｐであり；Ａ３はＳｅｒ又はＡｉｂであり；
Ａ５はＨｉｓ、Ｉｌｅ、Ａｃｃ、又はＣｈａであり；
Ａ７はＬｅｕ、Ｃｈａ、Ｎｌｅ、β−Ｎａｌ、Ｔｒｐ、Ｐａｌ、Ａｃｃ、Ｐｈｅ、又はｐ−Ｘ−Ｐｈｅであり、この際、ＸはＯＨ、ハロゲン、又はＣＨ３であり；
Ａ８はＬｅｕ、Ｍｅｔ、Ａｃｃ、又はＣｈａであり；
Ａ１０はＡｓｐ又はＡｓｎであり；
Ａ１１はＬｙｓ、Ｌｅｕ、Ｃｈａ、Ａｃｃ、Ｐｈｅ、又はβ−Ｎａｌであり；
Ａ１２はＧｌｙ、Ａｃｃ、又はＡｉｂであり；
Ａ１４はＳｅｒ又はＨｉｓであり；
Ａ１５はＩｌｅ、Ａｃｃ、又はＣｈａであり；
Ａ１６はＧｌｎ又はＡｉｂであり；
Ａ１７はＡｓｐ又はＡｉｂであり；
Ａ１８はＬｅｕ、Ａｉｂ、Ａｃｃ、又はＣｈａであり；
Ａ１９はＡｒｇ又はＡｉｂであり；
Ａ２２はＰｈｅ、Ｇｌｕ、Ａｉｂ、Ａｃｃ、又はＣｈａであり；
Ａ２３はＰｈｅ、Ｌｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２４はＬｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２５はＨｉｓ、Ｌｙｓ、Ａｉｂ、Ａｃｃ、又はＧｌｕであり；
Ａ２６はＨｉｓ、Ａｉｂ、Ａｃｃ、又はＬｙｓであり；
Ａ２７はＬｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２８はＩｌｅ、Ｌｅｕ、Ｌｙｓ、Ａｃｃ、又はＣｈａであり；
Ａ２９はＡｌａ、Ｇｌｕ、Ａｃｃ、又はＡｉｂであり；
Ａ３０はＧｌｕ、Ｌｅｕ、Ｎｌｅ、Ｃｈａ、Ａｉｂ、Ａｃｃ、又はＬｙｓであり；
Ａ３１はＩｌｅ、Ｌｅｕ、Ｃｈａ、Ｌｙｓ若しくはＡｃｃである、又は欠損しており；
Ａ３２はＨｉｓである又は欠損しており；
Ａ３３はＴｈｒである又は欠損しており；
Ａ３４はＡｌａである又は欠損しており；
Ｒ１及びＲ２は、それぞれ独立して、Ｈ、Ｃ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；又はＲ１及びＲ２の一方及び一方のみがＣＯＥ１であり、この際、Ｅ１はＣ１−１２アルキル、Ｃ２−１２アルケニル、Ｃ２−１２アルキニル、Ｃ７−２０フェニルアルキル、Ｃ１１−２０ナフチルアルキル、Ｃ１−１２ヒドロキシアルキル、Ｃ２−１２ヒドロキシアルケニル、Ｃ７−２０ヒドロキシフェニルアルキル、若しくはＣ１１−２０ヒドロキシナフチルアルキルであり；及び
Ｒ３はＯＨ、ＮＨ２、Ｃ１−１２アルコキシ、又はＮＨ−Ｙ−ＣＨ２−Ｚであり、この際、ＹはＣ１−１２炭化水素部分であり、ＺはＨ、ＯＨ、ＣＯ２Ｈ、又はＣＯＮＨ２であり；
Ａ５、Ａ７、Ａ８、Ａ１１、Ａ１２、Ａ１５、Ａ１８、Ａ２２、Ａ２３、Ａ２４、Ａ２５、Ａ２６、Ａ２７、Ａ２８、Ａ２９、Ａ３０、又はＡ３１の少なくとも一つがＡｃｃである；
又はこれらの製薬上許容できる塩である、請求項１又は２に記載の椎体骨折治療剤。 An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the formula:
Figure JPOXMLDOC01-appb-C000003
 However,
A 1 is Ala, Ser, or Dap; A 3 is Ser or Aib;
A 5 represents His, Ile, Acc, or be Cha;
A 7 is Leu, Cha, Nle, β-Nal, Trp, Pal, Acc, Phe, or p-X-Phe, where X is OH, halogen, or CH 3 ;
A 8 is Leu, Met, Acc, or Cha;
A 10 is Asp or Asn;
A 11 is Lys, Leu, Cha, Acc, Phe, or β-Nal;
A 12 is Gly, Acc, or Aib;
A 14 is Ser or His;
A 15 is Ile, Acc, or Cha;
A 16 is Gln or Aib;
A 17 is Asp or Aib;
A 18 is Leu, Aib, Acc, or Cha;
A 19 is Arg or Aib;
A 22 is Phe, Glu, Aib, Acc, or Cha;
A 23 is Phe, Leu, Lys, Acc, or Cha;
A 24 is Leu, Lys, Acc, or Cha;
A 25 is His, Lys, Aib, Acc, or Glu;
A 26 is His, Aib, Acc, or Lys;
A 27 is Leu, Lys, Acc, or Cha;
A 28 is Ile, Leu, Lys, Acc, or Cha;
A 29 is Ala, Glu, Acc, or Aib;
A 30 is Glu, Leu, Nle, Cha, Aib, Acc, or Lys;
A 31 is Ile, Leu, Cha, Lys or Acc, or is missing;
A 32 is His or missing;
A 33 is Thr or missing;
A 34 is Ala or missing;
R 1 and R 2 are each independently H, C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12. Hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7-20 hydroxyphenylalkyl, or C 11-20 hydroxynaphthylalkyl; or only one and one of R 1 and R 2 is COE 1 , wherein E 1 is C 1-12 alkyl, C 2-12 alkenyl, C 2-12 alkynyl, C 7-20 phenylalkyl, C 11-20 naphthylalkyl, C 1-12 hydroxyalkyl, C 2-12 hydroxyalkenyl, C 7 -20 hydroxyphenyl alkyl, or with C 11-20 hydroxy naphthyl alkyl Ri; and R 3 is OH, NH 2, C 1-12 alkoxy, or NH-Y-CH 2 -Z, this time, Y is C 1-12 hydrocarbon moiety, Z is H, OH, CO 2 H or CONH 2 ;
A 5, A 7, A 8 , A 11, A 12, A 15, A 18, A 22, A 23, A 24, A 25, A 26, A 27, A 28, A 29, A 30, or A At least one of 31 is Acc;
Or the vertebral body fracture therapeutic agent of Claim 1 or 2 which is these pharmaceutically acceptable salt. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、下記式のポリペプチド：
［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，Ｌｙｓ２６，３０，Ａｉｂ２９，Ａｈｃ３１］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ａｈｃ２３，Ｌｙｓ２６，３０，Ｌｅｕ２８，３１，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，３１，Ｌｙｓ２６，３０，Ａｈｃ２７，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，２９，Ｌｅｕ２３，２８，３１，Ｌｙｓ２６，Ａｈｃ３０］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｃｈａ２２，Ｌｅｕ２３，２８，３１，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｈｃ２７，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｌｅｕ２３，２８，３１，Ａｈｃ２４，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２９，Ｌｅｕ２３，２８，３１，Ａｉｂ２５，Ｌｙｓ２６，３０，Ａｈｃ２７］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，Ｌｅｕ２３，２８，３１，Ａｉｂ２５，２９，Ｌｙｓ２６，３０，Ａｈｃ２７］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ２２，Ｌｅｕ２３，２８，３１，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｌｅｕ２３，３１，Ｌｙｓ２６，３０，Ａｈｃ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｃｈａ２２，Ａｈｃ２３，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ｌｅｕ２８，３１，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｃｈａ２２，Ｌｅｕ２３，２８，３１，Ａｈｃ２４，２７，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，Ｌｅｕ２３，２８，３１，Ａｈｃ２４，２７，Ｌｙｓ２５，２６，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ１８，２４，２７，Ｇｌｕ２２，Ｃｈａ２３，Ｌｙｓ２５，２６，Ｌｅｕ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，Ｃｈａ２３，Ａｈｃ２４，Ｌｙｓ２５，２６，Ｌｅｕ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ２２，２４，Ｌｅｕ２３，２８，３１，Ｇｌｕ２５，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ａｈｃ２２，２４，Ｌｅｕ２３，２８，Ｌｙｓ２５，２６，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
又はこれらの製薬上許容できる塩である、請求項１３に記載の椎体骨折治療剤。 An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the formula:
[Glu 22 , 25 , Leu 23 , 28 , Lys 26 , 30 , Aib 29 , Ahc 31 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , Ahc 23 , Lys 26 , 30 , Leu 28 , 31 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , Leu 23 , 28 , 31 , Lys 26 , 30, Ahc 27 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , 29 , Leu 23 , 28 , 31 , Lys 26 , Ahc 30 ] hPTHrP (1-34) NH 2 ;
[Cha 22 , Leu 23 , 28 , 31 , Glu 25 , Lys 26 , 30, Ahc 27 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , Leu 23 , 28, 31 , Ahc 24 , Lys 26 , 30 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22,29 , Leu 23,28,31 , Aib 25 , Lys 26,30 , Ahc 27 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , Leu 23 , 28 , 31 , Aib 25 , 29 , Lys 26 , 30, Ahc 27 ] hPTHrP (1-34) NH 2 ;
[Ahc 22 , Leu 23 , 28 , 31 , Glu 25 , Lys 26 , 30 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , Leu 23 , 31 , Lys 26 , 30, Ahc 28 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Cha 22, Ahc 23, Glu 25, Lys 26,30, Leu 28,31, Aib 29] hPTHrP (1-34) NH 2;
[Cha 22 , Leu 23 , 28, 31 , Ahc 24 , 27 , Glu 25 , Lys 26 , 30 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , Leu 23 , 28, 31 , Ahc 24 , 27 , Lys 25 , 26 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Ahc 18 , 24, 27 , Glu 22 , Cha 23 , Lys 25 , 26 , Leu 28 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , Cha 23 , Ahc 24 , Lys 25 , 26 , Leu 28 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Ahc 22 , 24 , Leu 23 , 28 , 31 , Glu 25 , Lys 26 , 30 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Ahc 22 , 24 , Leu 23 , 28 , Lys 25 , 26 , Aib 29 ] hPTHrP (1-34) NH 2 ;
Alternatively, the therapeutic agent for vertebral fracture according to claim 13, which is a pharmaceutically acceptable salt thereof. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、下記式のポリペプチド：
［Ｎｌｅ３１］ｈＰＴＨ（１−３４）ＮＨ２；
［ｈＡｒｇ２７］ｈＰＴＨ（１−３４）ＮＨ２；
［Ｄａｐ１，Ｎｌｅ８，１８，Ｔｙｒ３４］ｈＰＴＨ（１−３４）ＮＨ２；
又はこれらの製薬上許容できる塩である、請求項１又は２に記載の椎体骨折治療剤。 An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the formula:
[Nle 31 ] hPTH (1-34) NH 2 ;
[HArg 27 ] hPTH (1-34) NH 2 ;
[Dap 1 , Nle 8 , 18 , Tyr 34 ] hPTH (1-34) NH 2 ;
Or the vertebral body fracture therapeutic agent of Claim 1 or 2 which is these pharmaceutically acceptable salt. 　ＰＴＨ、ＰＴＨｒＰ又はそれらの部分活性ポリペプチドのアナログが、下記式のポリペプチド：
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，Ｌｅｕ２８，３１，Ａｉｂ２９，Ｎｌｅ３０］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，３０，Ｌｅｕ２８，３１，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，３０，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｇｌｕ２２，２５，Ｃｈａ２３，Ｌｙｓ２６，３０，Ｌｅｕ２８，Ａｉｂ２９］ｈＰＴＨｒＰ（１−３４）ＮＨ２；
［Ｌｅｕ２７，Ａｉｂ２９］ｈＰＴＨ（１−３４）ＮＨ２；
又はこれらの製薬上許容できる塩である、請求項１又は２に記載の椎体骨折治療剤。 An analog of PTH, PTHrP or a partially active polypeptide thereof is a polypeptide of the formula:
[Glu 22 , 25 , Cha 23 , Lys 26 , Leu 28 , 31 , Aib 29 , Nle 30 ] hPTHrP (1-34) NH 2 ;
[Glu 22 , 25 , Cha 23 , Lys 26 , 30 , Leu 28 , 31 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Glu 22,25, Cha 23, Lys 26,30, Aib 29] hPTHrP (1-34) NH 2;
[Glu 22 , 25 , Cha 23 , Lys 26 , 30 , Leu 28 , Aib 29 ] hPTHrP (1-34) NH 2 ;
[Leu 27 , Aib 29 ] hPTH (1-34) NH 2 ;
Or the vertebral body fracture therapeutic agent of Claim 1 or 2 which is these pharmaceutically acceptable salt. 　ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することを特徴とする、椎体骨折治療剤。 A therapeutic agent for vertebral fracture, comprising a substance having an activity of inducing PTH or PTHrP production. 　ＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質がカルシウム感受性受容体アンタゴニストである、請求項１７に記載の椎体骨折治療剤。 The therapeutic agent for vertebral fracture according to claim 17, wherein the substance having an activity of inducing PTH or PTHrP production is a calcium sensitive receptor antagonist. 　椎体骨折が海綿骨における骨折である、請求項１~１８のいずれかに記載の椎体骨折治療剤。 The therapeutic agent for vertebral fracture according to any one of claims 1 to 18, wherein the vertebral fracture is a cancellous fracture. 　請求項１~１６のいずれかに記載の、ＰＴＨ、ＰＴＨｒＰ、若しくはそれらの部分活性ポリペプチド、又はそれらのアナログ、若しくは請求項１７又は１８に記載のＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することを特徴とする、椎体骨折に起因する疼痛の治療又は予防剤。 The PTH, PTHrP, or a partially active polypeptide thereof, or an analog thereof according to any one of claims 1 to 16, or a substance having an activity of inducing PTH or PTHrP production according to claim 17 or 18. A therapeutic or prophylactic agent for pain caused by vertebral fractures, comprising: 　請求項１~１６のいずれかに記載の、ＰＴＨ、ＰＴＨｒＰ、若しくはそれらの部分活性ポリペプチド、又はそれらのアナログ、若しくは請求項１７又は１８に記載のＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することを特徴とする、椎体圧迫骨折予防剤。 The PTH, PTHrP, or a partially active polypeptide thereof, or an analog thereof according to any one of claims 1 to 16, or a substance having an activity of inducing PTH or PTHrP production according to claim 17 or 18. A vertebral body compression fracture preventing agent, comprising: 　請求項１~１６のいずれかに記載の、ＰＴＨ、ＰＴＨｒＰ、若しくはそれらの部分活性ポリペプチド、又はそれらのアナログ、若しくは請求項１７又は１８に記載のＰＴＨ又はＰＴＨｒＰ産生を誘導する活性を有する物質を含有することを特徴とする、椎体骨折に起因する運動障害及び／又は神経障害の治療又は予防剤。 The PTH, PTHrP, or a partially active polypeptide thereof, or an analog thereof according to any one of claims 1 to 16, or a substance having an activity of inducing PTH or PTHrP production according to claim 17 or 18. A therapeutic or prophylactic agent for dyskinesia and / or neuropathy caused by vertebral fracture, comprising: 　椎体骨折治療剤の評価方法であって、１）非ヒト動物の椎体骨に外科的な損傷を与える工程、２）当該非ヒト動物に被検物質を投与する工程、及び３）当該非ヒト動物又はそれに由来する検体の椎体骨の外科的な損傷の回復を測定する工程、を含むことを特徴とする、評価方法。 A method for evaluating a therapeutic agent for vertebral fracture, comprising 1) a step of surgically damaging a vertebral bone of a non-human animal, 2) a step of administering a test substance to the non-human animal, and 3) the non-human body Measuring the recovery of surgical damage to the vertebral bones of a human animal or a specimen derived therefrom. 　椎体骨の外科的な損傷の回復の測定を行った結果を、さらに正常非ヒト動物若しくは対象非ヒト動物、又はそれに由来する検体における測定の結果と比較する工程を含む、請求項２３に記載の評価方法。 24. The method according to claim 23, further comprising the step of comparing the result of measurement of recovery of vertebral bone surgical damage with the result of measurement in a normal non-human animal or a target non-human animal or a sample derived therefrom. Evaluation method. 　椎体骨の外科的な損傷が、椎体骨の皮質骨を通り海綿骨まで達する損傷であることを特徴とする、請求項２３又は２４に記載の評価方法。 The evaluation method according to claim 23 or 24, wherein the surgical damage to the vertebral bone is damage reaching the cancellous bone through the cortical bone of the vertebral bone. 　椎体骨の外科的な損傷が、ドリルによる穿孔による損傷であることを特徴とする、請求項２３~２５のいずれかに記載の評価方法。 The evaluation method according to any one of claims 23 to 25, wherein the surgical damage to the vertebral bone is damage caused by drilling with a drill. 　椎体骨の外科的な損傷の回復の測定を、検体の組織学的観察、又はＸ線ＣＴを用いた形態観察により行う、請求項２３~２６のいずれかに記載の評価方法。 27. The evaluation method according to claim 23, wherein the measurement of recovery from surgical damage to the vertebral bone is performed by histological observation of a specimen or morphological observation using X-ray CT. 　椎体骨の外科的な損傷の回復の測定を、痛覚又は運動機能の測定により行う、請求項２３~２６のいずれかに記載の評価方法。 27. The evaluation method according to any one of claims 23 to 26, wherein the measurement of recovery from surgical damage to the vertebral bone is performed by measuring pain sensation or motor function. 　椎体骨の外科的な損傷の回復の計測を、検体の組織化学的解析、骨分化発生マーカーのタンパク質又は遺伝子の発現の解析により行う、請求項２３~２６のいずれかに記載の評価方法。 27. The evaluation method according to any one of claims 23 to 26, wherein the measurement of recovery from surgical damage to the vertebral bone is performed by histochemical analysis of a specimen and analysis of protein or gene expression of a bone differentiation occurrence marker. 　非ヒト動物が、骨粗鬆症のモデル動物である、請求項２３~２９のいずれかに記載の評価方法。 30. The evaluation method according to claim 23, wherein the non-human animal is an osteoporosis model animal. 　骨粗鬆症のモデル動物が、卵巣摘出モデルである、請求項３０に記載の評価方法。 The evaluation method according to claim 30, wherein the osteoporosis model animal is an ovariectomy model. 　非ヒト動物が、霊長類又は齧歯類の非ヒト動物である、請求項２３~３１のいずれかに記載の評価方法。 32. The evaluation method according to claim 23, wherein the non-human animal is a primate or rodent non-human animal. 　齧歯類の非ヒト動物が、ラットである、請求項３２に記載の評価方法。 The evaluation method according to claim 32, wherein the rodent non-human animal is a rat. 　椎体骨が、腰椎の椎体骨である、請求項２３~３３のいずれかに記載の評価方法。 The evaluation method according to any one of claims 23 to 33, wherein the vertebral bone is a lumbar vertebral bone. 　腰椎の椎体骨が、第４腰椎及び／又は第５腰椎である、請求項３４に記載の評価方法。 The evaluation method according to claim 34, wherein the vertebral body bones of the lumbar vertebra are the fourth lumbar vertebra and / or the fifth lumbar vertebra. 　被検物質の投与を、経口又は非経口により行う、請求項２３~３５のいずれかに記載の評価方法。 36. The evaluation method according to claim 23, wherein the test substance is administered orally or parenterally.
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