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WO2005001075A1 - Compositions rdp58 et procedes de modulation de l'activite des osteoclastes et des osteoblastes - Google Patents

Compositions rdp58 et procedes de modulation de l'activite des osteoclastes et des osteoblastes Download PDF

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Publication number
WO2005001075A1
WO2005001075A1 PCT/US2004/015490 US2004015490W WO2005001075A1 WO 2005001075 A1 WO2005001075 A1 WO 2005001075A1 US 2004015490 W US2004015490 W US 2004015490W WO 2005001075 A1 WO2005001075 A1 WO 2005001075A1
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Prior art keywords
osteoblast
rdp58
osteoclast
activity
bone
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PCT/US2004/015490
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English (en)
Inventor
Timothy Fong
Mirella E. Lazarov
Ajith Welihinda
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Sangstat Medical Corporation
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Publication of WO2005001075A1 publication Critical patent/WO2005001075A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the invention concerns the physiological processes of bone deposition and bone resorption.
  • the invention relates to osteoclast development and function, osteoblast development and function, leukocyte infiltration, and the regulated production of various osteoclastogenic and osteoblastogenic factors.
  • BACKGROUND [003] The formation and maintenance of living bone requires coordinated regulation of the opposing processes of bone deposition and bone resorption.
  • Bone deposition and bone resorption are mainly effected by the activities of osteoblasts and osteoclasts, respectively.
  • Osteoblasts are derivable from mesenchymal stem cells, and mediate synthesis of the organic matrix of bone.
  • Osteoclasts are multinuclear phagocytic cells obtainable from bone marrow-derived monocyte/macrophage precursor cells, and mediate dissolution of the bone matrix and solubilization of bone salts.
  • TNF ⁇ tumor necrosis factor a
  • IL-1 interleukin-1
  • IL-4 interleukin-4
  • IL-6 interleukin-6
  • LIF leukemia inhibitory factor
  • M-CSF macrophage colony stimulating factor
  • Osteoblasts and osteoblast precursors produce RANKL, a ligand for the cell surface molecule RANK, a TNF receptor superfamily member that is highly expressed in osteoclast precursors.
  • RANKL is also known as OPG-ligand (OPG-L), osteoclast differentiation factor (ODF), and TNF-related activation induced cytokine (TRANCE).
  • OPG-L OPG-ligand
  • ODF osteoclast differentiation factor
  • TRANCE TNF-related activation induced cytokine
  • RANKL has been shown to promote osteoclast differentiation and activation, and has also been shown to inhibit osteoclast apoptosis. Osteoclasts are absent in RANKL nullizygous mice, and mutant animals exhibit severe osteopetrosis and defects in tooth eruption. RANK nullizygous mice also lack osteoclasts and exhibit severe osteopetrosis. In addition, a soluble RANK-Fc fusion protein that effectively blocks RANKL-induced RANK activation can cause osteopetrosis.
  • Osteoblasts also produce a RANK antagonist, osteoprotegerin (OPG), which binds to RANKL, inhibiting RANK activation and osteoclast formation.
  • OPG osteoprotegerin
  • OPG is synthesized as a 401 amino acid precursor protein, which includes a 21 amino acid propeptide that is cleaved to yield the mature form of OPG.
  • OPG like RANK, belongs to the TNF receptor superfamily, but does not have a transmembrane or cytoplasmic domain, and is secreted as a soluble protein.
  • OPG nullizygous mice exhibit a marked increase in osteoclast numbers and bone resorption, and have severe osteoporosis, underscoring the importance of RANK-induced signals in osteoclast development and the regulation of bone resorption.
  • the dysfunction or dysregulation of osteoblasts and/or osteoclasts can also shift the deposition:resorption balance and result in conditions or diseases characterized by excessive or insufficient bone mass.
  • Increased osteoclast activity and/or decreased osteoblast activity can result in increased bone resorption, and is associated with osteopenia disorders (bone loss disorders).
  • decreased osteoclast activity and/or increased osteoblast activity can result in decreased bone resorption, and is associated with osteopetrosis and its characteristic increase in bone density.
  • Periodontitis has been reported to afflict 20-30% of all adults in the industrialized world, and is one of the leading causes of tooth loss in adults. Periodontitis results from a bacterial infection that spreads from the gums to the ligaments and bones that support the teeth, causing loss of bone and loosening of teeth. The bacterial infection promotes infiltration by polymorphonuclear monocytes and T cells, as well as the production of osteoclastogenic factors and an increase in osteoclast activity. Further, the oral bacteria that cause periodontal disease have also been linked to a number of systemic diseases, including infective endocarditis, stroke, atheroscleoris, myocardial infarction, and preterm low birth weight.
  • Bone metastases are often osteolytic, causing bone destruction which can result in pathologic fractures. Additionally, much of the pain associated with many types of cancer is thought to be due to bone metastases. Bone metastases are commonly seen in patients with breast, prostate, thyroid, bladder, lung and renal cancer, as well as patients with malignant melanoma and other forms of cancer. Among the most common are breast cancer and prostate cancer. Additionally these lesions can cause nerve root or spinal cord compression. Current treatment regimens, including local radiation, bisphosphonates, or trastuzumab can be helpful but are not curative.
  • Osteoclastogenesis has been implicated as a mechanism by which malignant tumors accomplish destruction of bone. These metastatic tumors may cause activation of osteoclasts, thereby leading to bone destruction. Agents capable of inhibiting tumor-promoted osteoclastogenesis and resultant tumor-induced osteolysis are highly desirable.
  • the invention provides compositions and methods for modulating osteoclast and osteoblast activity.
  • the "RDP58 compositions” disclosed herein are potent modulators of osteoclast and osteoblast activity, and methods of using RDP58 compositions to decrease osteoclast activity and increase osteoblast activity are provided. Additionally, these RDP58 compositions are capable of inhibiting (i) polymorphonuclear cell infiltration into the vicinity of bone, (ii) the production of a variety of osteoclastogenic factors, and (iii) the production of matrix metalloproteinases and the destruction of tissue. These RDP58 compositions are useful for modulating bone formation, maintenance, and repair.
  • the invention provides compositions and methods for decreasing osteoclast activity in a cell population comprising at least one osteoclast or osteoclast precursor cell.
  • the methods involve contacting the osteoclast or osteoclast precursor cell with an RDP58 composition, whereby contact with the RDP58 composition decreases osteoclast activity.
  • methods for decreasing osteoclast-mediated bone resorption involve contacting osteoclasts or osteoclast precursor cells with an RDP58 composition, whereby the RDP58 composition decreases osteoclast-mediated bone resorption by decreasing the osteoclast activity of the osteoclasts or decreasing the differentiation of osteoclast precursor cells.
  • the invention provides methods for decreasing osteoclast differentiation induced by lipopolysaccharide (LPS), TNFcr, or RANKL.
  • the methods involve contacting osteoclast precursor cells with an RDP58 composition, whereby the RDP58 composition decreases the signaling activity of LPS, TNF ⁇ , or RANKL therein.
  • the invention provides methods for decreasing the bone resorption induced by LPS, TNF ⁇ , or RANKL.
  • the methods involve contacting an osteoclast with an RDP58 composition, whereby the RDP58 composition decreases the signaling activity of LPS, TNF ⁇ , or RANKL therein.
  • the invention provides compositions and methods for decreasing TRAF activity in osteoclasts and osteoclast precursor cells.
  • TRAF activity may be, for example, attendant activation of a TNF receptor superfamily member such as RANK.
  • the invention provides methods for decreasing one or more osteoclast activities mediated by TRAF activity, which involve contacting an osteoclast or osteoclast precursor cell with an RDP58 composition, whereby the RDP58 composition decreases TRAF activity therein.
  • the TRAF activity is preferably binding activity, as directed at a binding partner of TRAF, and/or kinase activity as directed at a substrate of TRAF.
  • methods for decreasing TRAF6, TRAF2, and TRAF5 activity are provided.
  • the invention provides compositions and methods for decreasing IRAK activity in osteoclasts and osteoclast precursor cells.
  • IRAK activity may be, for example, attendant activation of a TNF receptor superfamily member such as RANK.
  • the invention provides methods for decreasing one or more osteoclast activities mediated by IRAK activity, which involve contacting an osteoclast or osteoclast precursor cell with an RDP58 composition, whereby the RDP58 composition decreases IRAK activity therein.
  • the IRAK activity is preferably binding activity, as directed at a binding partner of IRAK, preferably TRAF, and/or kinase activity as directed at a substrate of IRAK.
  • the invention provides compositions and methods for decreasing MyD88 activity in osteoclasts and osteoclast precursors.
  • MyD88 activity may be, for example, attendant activation of a TNF receptor superfamily member such as RANK.
  • the invention provides methods for decreasing one or more osteoclast activities mediated by MyD88 activity, which involve contacting an osteoclast or osteoclast precursor cell with an RDP58 composition, whereby the RDP58 composition decreases MyD88 activity therein.
  • the MyD88 activity is preferably binding activity, as directed at a binding partner of MyD88, preferably TRAF.
  • the invention provides compositions and methods for decreasing MyD88/IRAK/TRAF complex activity in osteoclasts and osteoclast precursors.
  • MyD88/IRAK/TRAF complex activity may be, for example, attendant activation of a TNF receptor superfamily member such as RANK.
  • the invention provides methods for decreasing one or more osteoclast activities mediated by MyD88/IRAK TRAF complex activity, which involve contacting an osteoclast or osteoclast precursor cell with an RDP58 composition, whereby the RDP58 composition decreases MyD88/IRAK/TRAF complex activity therein.
  • MyD88/IRAK/TRAF complex activity may be inhibited, for example, by inhibiting formation of the complex.
  • the invention provides compositions and methods for increasing osteoblast activity in a cell population comprising at least one osteoblast or osteoblast precursor cell.
  • the methods involve contacting the osteoblast or osteoblast precursor cell with an RDP58 composition, whereby contact with the RDP58 composition increases osteoblast activity.
  • methods for increasing osteoblast-mediated bone deposition involve contacting osteoblasts or osteoblast precursor cells with an RDP58 composition, whereby the RDP58 composition increases osteoblast-mediated bone deposition by increasing osteoblast activity of the osteoblasts or increasing the differentiation of osteoblast precursor cells.
  • the invention provides compositions and methods for decreasing integrin signaling in osteoblasts and osteoblast precursor cells.
  • the invention provides methods for increasing one or more osteoblast activities inhibited by integrin signaling, which involve contacting an osteoblast or osteoblast precursor cell with an RDP58 composition, whereby the RDP58 composition decreases integrin signaling therein.
  • the integrin signaling is preferably signal transduction leading to the activation of the transcription factors AP1 and NF- ⁇ B in osteoblasts and osteoblast precursor cells.
  • the integrin signaling is ⁇ lll integrin signaling.
  • the invention provides compositions and methods for decreasing AP1 and NF- KB activity in osteoblasts and osteoblast precursor cells.
  • the AP1 and NF- ⁇ B activity may be, for example, attendant integrin signaling.
  • the invention provides methods for increasing one or more osteoblast activities inhibited by AP1 or NF- ⁇ B activity, which involve contacting an osteoblast or osteoblast precursor cell with an RDP58 composition, whereby the RDP58 composition decreases AP1 and NF- ⁇ B activity therein.
  • the AP1 and NF- ⁇ B activity is preferably DNA-binding activity, as directed at AP1 and NF- ⁇ B recognition elements, respectively, and/or transcription activating activity as directed from AP1 and NF- ⁇ B recognition elements, respectively.
  • the invention provides compositions and methods for coordinately increasing osteoblast activity and decreasing osteoclast activity in a cell population comprising at least one osteoblast or osteoblast precursor cell and at least one osteoclast or osteoclast precursor cell.
  • the methods involve (i) contacting the osteoblast or osteoblast precursor cell with an RDP58 composition, whereby contact with the RDP58 composition increases osteoblast activity, and (ii) contacting the osteoclast or osteoclast precursor cell with an RDP58 composition, whereby contact with the RDP58 composition decreases osteoclast activity.
  • the invention provides compositions and methods for the treatment of bone loss disorders.
  • Bone loss disorders include conditions and diseases wherein the inhibition of bone loss and/or the promotion of bone formation is desirable.
  • conditions and diseases include osteoporosis, osteomyelitis, Paget's disease, periodontitis, hypercalcemia, osteonecrosis, osteosarcoma, osteolyic metastases, familial expansile osteolysis, prosthetic loosening, periprostetic osteolysis, and cleiodocranial dysplasia (CCD).
  • the methods involve administering an RDP58 composition to a patient having a bone loss disorder.
  • methods for decreasing osteoclast activity and increasing osteoblast activity in a patient having a bone loss disorder using an RDP58 composition are also provided.
  • methods for decreasing matrix metailoproteinase production in an patient having a bone loss disorder using an RDP58 composition are also provided.
  • the invention provides methods for treating periodontitis. Also provided are methods for inhibiting polymorphonuclear cell infiltration into the vicinity of the alveolar bone, and inhibiting the production of a variety of osteoclastogenic cytokines in the vicinity of the alveolar bone, in patients having periodontitis. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in the vicinity of the alveolar bone in patients having periodontitis. Also provided are methods for decreasing matrix metailoproteinase production, preferably MMP2 or MMP9 production, in the vicinity of the alveolar bone in patients having periodontitis. The methods involve providing an RDP58 composition to the vicinity of the alveolar bone of a patient having periodontitis.
  • the invention provides methods for treating a variety of osteoporosis disorders, such as primary osteoporosis, endocrine osteoporosis (hyperthyroidism, hyperparathyroidism, Cushing's syndrome, and acromegaly), hereditary and congenital forms of osteoporosis (osteogenesis imperfecta, homocystinuria, Menkes' syndrome, and Rile-Day syndrome) and osteoporosis due to immobilization of extremities. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having an osteoporosis disorder. Also provided are methods for decreasing matrix metailoproteinase production, preferably MMP2 or MMP9 production, in patients having an osteoporosis disorder. The methods involve administering an RDP58 composition to a patient having an osteoporosis disorder.
  • osteoporosis disorders such as primary osteoporosis, endocrine osteoporosis (hyperthyroidism, hyperparathyroidism, Cushing's
  • the invention provides methods for treating osteomyelitis, or an infectious lesion in bone leading to bone loss. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having osteomyelitis, or an infectious lesion in bone leading to bone loss. Also provided are methods for decreasing matrix metailoproteinase production in patients having osteomyelitis. The methods involve administering an RDP58 composition to a patient having osteomyelitis, or an infectious lesion in bone leading to bone loss.
  • the invention provides methods for treating osteosarcoma. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having osteosarcoma. Also provided are methods for decreasing matrix metailoproteinase production in patients having osteosarcoma. Also provided are methods for increasing cell differentiation and decreasing cell proliferation in bone tissue of patients having osteosarcoma. The methods involve administering an RDP58 composition to a patient having osteosarcoma.
  • the invention provides methods for treating hypercalcemia, such as resulting from solid tumors (breast, lung and kidney) or hematologic malignacies (multiple myeloma, lymphoma and leukemia), idiopathic hypercalcemia, and hypercalcemia associated with hyperthrybidism and renal function disorders. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having hypercalcemia. Also provided are methods for decreasing matrix metailoproteinase production in patients having hypercalcemia. The methods involve administering an RDP58 composition to a patient having hypercalcemia.
  • the invention provides methods for treating osteonecrosis, or bone cell death, associated with traumatic injury or nontraumatic necrosis associated with Gaucher's disease, sickle cell anemia, systemic lupus erythematosus and other conditions. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having osteonecrosis. Also provided are methods for decreasing matrix metailoproteinase production in patients having osteonecrosis. The methods involve administering an RDP58 composition to a patient having osteonecrosis.
  • the invention provides methods for inhibiting bone loss attendant rheumatoid arthritis. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having rheumatoid arthritis. Also provided are methods for decreasing matrix metailoproteinase production in patients having rheumatoid arthritis. The methods involve administering an RDP58 composition to a patient having rheumatoid arthritis.
  • the invention provides methods for treating periprosthetic osteolysis. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having periprosthetic osteolysis. Also provided are methods for decreasing matrix metailoproteinase production in patients having periprosthetic osteolysis. The methods involve administering an RDP58 composition to a patient having periprosthetic osteolysis.
  • the invention provides methods for treating bone loss due to osteolytic metastasis. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having osteolytic metastasis. Also provided are methods for decreasing matrix metailoproteinase production in patients having osteolytic metastasis. The methods involve administering an RDP58 composition to a patient having osteolytic metastasis.
  • the invention provides methods for treating familial expansile osteolysis. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having familial expansile osteolysis. Also provided are methods for decreasing matrix metailoproteinase production in patients having familial expansile osteolysis. The methods involve administering an RDP58 composition to a patient having familial expansile osteolysis.
  • the invention provides methods for treating Paget's disease (osteitis deformans). Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having Paget's disease. Also provided are methods for decreasing matrix metailoproteinase production in patients having Paget's disease. The methods involve administering an RDP58 composition to a patient having Paget's disease. [0040] In another preferred embodiment, the invention provides methods for treating CCD. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having CCD. Also provided are methods for decreasing matrix metailoproteinase production in patients having CCD. The methods involve administering an RDP58 composition to a patient having CCD.
  • Paget's disease osteoitis deformans
  • the invention provides methods for decreasing prosthetic loosening. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having a prosthesis. Also provided are methods for decreasing matrix metailoproteinase production in patients having a prosthesis. The methods involve administering an RDP58 composition to a patient having a prosthesis.
  • the invention also provides compositions and methods for stimulating bone regeneration.
  • the bone regeneration may be following reconstruction of bone defects in cranio-maxillofacial surgery, or following an implant into bone, for example a dental implant, bone supporting implant, or prosthesis.
  • the bone regeneration may also be following a bone fracture.
  • the methods involve administering an RDP58 composition to a patient in need of bone regeneration.
  • RDP58 compositions increase the activity of osteoblasts while decreasing the activity of osteoclasts.
  • RDP58 compositions are able to decrease osteoclast differentiation and bone resorption, including the osteoclast differentiation and bone resorption promoted by a variety of agents.
  • RDP58 compositions are also able to increase osteoblast differentiation and bone deposition, including the osteoblast differentiation and bone deposition promoted by a variety of agents.
  • osteoclast differentiation is meant the formation of a cell having at least one osteoclast activity from a cell which lacks the activity but is of the osteoclast lineage, and is therefore referred to as an "osteoclast precursor”.
  • osteoclast precursor includes cells that give rise to osteoclasts without proliferation, as well as cells that go through one or more rounds of cell division to provide cells that give rise to osteoclasts without proliferation.
  • osteoblast differentiation is meant the formation of a cell having at least one osteoblast activity from a cell which lacks the activity but is of the osteoblast lineage, and is therefore referred to as an "osteoblast precursor”.
  • osteoblast precursor includes cells that give rise to osteoblasts without proliferation, as well as cells that go through one or more rounds of cell division to provide cells that give rise to osteoblasts without proliferation.
  • Osteoclast activity includes but is not limited to the ability to polarize on bone or dentate; the ability to form a polarized ruffled membrane on bone or dentate; the ability to form a polarized ruffled membrane rich in an electrogenic protein pump on bone or dentate; the ability to form matrix attachment or sealing zones with bone or dentate, which attachment structures are rich in filamentous actin (forming plasma membrane protrusions known as podosomes), largely devoid of organelles and are normally organized as a ring surrounding a ruffled membrane rich in an electrogenic protein pump; the ability to achieve a pH of about 4.5 in a portion of the extracellular environment of bone or dentate; the ability to mobilize bone or dentate mineral; the ability to release cathepsin extracellularly into a portion of the extracellular environment of bone or dentate; the ability to achieve a pH of about 4.5 in a portion of the extracellular environment of bone or dentate and release cathepsin extracellularly into this environment where catheps
  • “decreasing osteoclast activity” is meant decreasing partially or completely one or more osteoclast activities.
  • “Decreasing osteoclast activity” also includes inhibiting osteoclast differentiation (i.e., inhibiting osteoclastogenesis), whereby a precursor cell is kept from differentiating and obtaining the ability to exert an osteoclast activity.
  • “Decreasing osteoclast activity” also includes inhibiting osteoclast or osteoclast precursor cell survival, whereby an osteoclast or osteoclast precursor cell undergoes cell death that it would not undergo in the absence of an RDP58 composition.
  • “Decreasing osteoclast activity” also includes promoting osteoclast or osteoclast precursor cell death, whereby an osteoclast or osteoclast precursor cell undergoes cell death that it would not undergo in the absence of an RDP58 composition.
  • Osteoblast activity includes but is not limited to expression of the transcription factor Cbfa-1 ; expression and/or secretion of osteocalcin; expression and presentation and/or secretion of RANKL; expression and presentation and/or secretion of OPG; expression and presentation and/or secretion of M-CSF (an activity high in osteoblast precursors); Cbfa-1 mediated expression of genes having Cbfa-1 binding sites in their regulatory regions; the ability to participate in endochronal ossification; the ability to promote osteoclast development, activation or survival (an activity high in osteoblast precursors); the ability to synthesize collagen type I protein and deposit collagen type I protein extracellularly; the ability to synthesize the components of osteoid and deposit osteoid extracellularly.
  • osteoclast activation is meant the promotion or induction of osteoclast activity.
  • increasing osteoblast activity is meant increasing the level of one or more extant osteoblast activities, or inducing one or more osteoblast activities where no such activity was extant prior to induction.
  • Increasing osteoblast activity also includes promoting osteoblast differentiation (i.e., promoting osteoblastogenesis), whereby a precursor cell is induced to differentiate and obtain the ability to exert an osteoblast activity.
  • Increasing osteoblast activity also includes promoting osteoblast or osteoblast precursor cell survival, whereby an osteoblast or osteoblast precursor cell does not undergo cell death that it would undergo in the absence of an RDP58 composition.
  • Increasing osteoblast activity also includes inhibiting osteoblast or osteoblast precursor cell death, whereby an osteoblast or osteoblast precursor cell does not undergo cell death that it would undergo in the absence of an RDP58 composition.
  • the invention provides compositions and methods for decreasing osteoclast activity in a cell population comprising at least one osteoclast or osteoclast precursor cell.
  • the methods involve contacting the osteoclast or osteoclast precursor cell with an RDP58 composition, whereby contact with an RDP58 composition decreases osteoclast activity.
  • the invention provides compositions and methods for increasing osteoblast activity in a cell population comprising at least one osteoblast or osteoblast precursor cell.
  • the methods involve contacting the osteoblast or osteoblast precursor cell with an RDP58 composition, whereby contact with an RDP58 composition increases osteoblast activity.
  • the invention provides compositions and methods for coordinately increasing osteoblast activity and decreasing osteoclast activity in a cell population comprising at least one osteoblast or osteoblast precursor cell and at least one osteoclast or osteoclast precursor cell.
  • the methods involve (i) contacting the osteoblast or osteoblast precursor cell with an RDP58 composition, whereby contact with an RDP58 composition increases osteoblast activity, and (ii) contacting the osteoclast or osteoclast precursor cell with the RDP58 composition, whereby contact with the RDP58 composition decreases osteoclast activity.
  • Cell populations may be in vivo or in vitro populations.
  • Preferred cell populations are in vivo cell populations that include osteoclasts and osteoblasts, as well as mesenchymal stem cells and bone marrow-derived monocyte/macrophage precursor cells, which are precursors of osteoblasts and osteoclasts, respectively.
  • the cell population is in the vicinity of the alveolar bone. In another embodiment, the cell population is in the vicinity of a bone fracture.
  • the reduction of osteoclast activity effected by an RDP58 composition is an inhibition of osteoclast precursor cell differentiation induced by LPS, TNF ⁇ , or RANKL.
  • the methods involve contacting osteoclast precursor cells with an RDP58 composition, whereby the RDP58 composition decreases the signaling activity of LPS, TNF-r, or RANKL therein.
  • the reduction of osteoclast activity effected by an RDP58 composition is a reduction of the bone resorption induced by LPS, TNF ⁇ , or RANKL.
  • the methods involve contacting an osteoclast with an RDP58 composition, whereby the RDP58 composition decreases the signaling activity of LPS, TNF ⁇ , or RANKL therein.
  • contacting with an RDP58 composition is meant providing the RDP58 composition to the cell in such a manner and in such an amount as to effect physical contact between the RDP58 composition and the cell.
  • the cell population is an In vivo population, and the method involves providing an RDP58 composition to the vicinity of osteoblasts, osteoblast precursor cells, osteoclasts, or osteoclast precursor cells in vivo.
  • Providing an RDP58 composition to the vicinity of means providing an RDP58 composition within an effective distance of the reference site.
  • Effective distance means a distance within which the RDP58 composition can exert a bioactivity, particularly the ability to increase osteoblast activity, or decrease osteoclast activity at the reference site. Providing can be done, for example, by local delivery, oral delivery, systemic delivery, etc.
  • the RDP58 composition need not be directly delivered within the effective distance of the reference site to be "provided to the vicinity" of the reference site. The effective distance will vary with the nature of the RDP58 composition, the amount and formulation of the RDP58 composition used, and the nature of the tissue, but will be readily determined with standardizing experiments.
  • the invention provides compositions and methods for modulating signal transduction in osteoclasts, osteoclast precursor cells, osteoblasts, and osteoblast precursor cells.
  • the methods involve contacting osteoclasts, osteoclast precursor cells, osteoblasts, or osteoblast precursor cells with an RDP58 composition.
  • the methods may be used to decrease osteoclast activity and/or increase osteoblast activity.
  • the invention provides methods for inhibiting integrin signaling in osteoblast and osteoblast precursor cells.
  • the methods involve contacting osteoblasts or osteoblast precursor cells with an RDP58 composition.
  • the methods may be used to promote the differentiation of osteoblast precursor cells into osteoblasts, and to increase osteoblast activity.
  • the integrin signaling pathways that are inhibited by the RDP58 composition activate NF- ⁇ B and/or AP1 in osteoblasts or osteoblast precursor cells in the absence of the RDP58 composition.
  • the integrin signaling pathway is preferably ⁇ lll integrin signaling pathway.
  • the invention provides methods for inhibiting NF- ⁇ B and AP1 activity in osteoblast and osteoblast precursor cells.
  • the methods involve contacting osteoblasts or osteoblast precursor cells with an RDP58 composition.
  • the methods may be used to promote the differentiation of osteoblast precursor cells into osteoblasts, and to increase osteoblast activity.
  • the invention provides methods for inhibiting the signaling activity of members of the TNF receptor superfamily in osteoclasts and osteoclast precursor cells.
  • the methods involve contacting osteoclasts or osteoclast precursor cells with an RDP58 composition.
  • the methods may be used to inhibit the differentiation of osteoclast precursor cells into osteoclasts, and to decrease osteoclast activity.
  • the invention provides methods for inhibiting TRAF activity in osteoclasts and osteoclast precursor cells.
  • the TRAF is TRAF6 which lies downstream of RANK.
  • the TRAF activity is preferably binding activity, as directed at a binding partner of TRAF, and/or kinase activity as directed at a substrate of TRAF.
  • the invention provides methods for inhibiting IRAK activity in osteoclasts and osteoclast precursor cells.
  • IRAK lies downstream of RANK.
  • the IRAK activity is preferably binding activity, as directed at a binding partner of IRAK, preferably TRAF, and/or kinase activity as directed at a substrate of IRAK.
  • the invention provides methods for inhibiting MyD88 activity in osteoclasts and osteoclast precursor cells.
  • MyD88 lies downstream of RANK.
  • the MyD88 activity is preferably binding activity, as directed at a binding partner of MyD88, preferably TRAF6.
  • the invention provides methods for inhibiting formation of a MyD88/IRAK TRAF complex in osteoclasts and osteoclast precursors.
  • the complex lies downstream of RANK.
  • the invention provides compositions and methods for the treatment of bone loss disorders.
  • Bone loss disorders include conditions and diseases wherein the inhibition of bone loss and/or the promotion of bone formation is desirable.
  • conditions and diseases include osteoporosis, osteomyelitis, Paget's disease, periodontitis, hypercalcemia, osteonecrosis, osteosarcoma, osteolyic metastases, familial expansile osteolysis, prosthetic loosening, periprostetic osteolysis, and cleiodocranial dysplasia (CCD).
  • the methods involve administering a therapeutic amount of an RDP58 composition to a patient having a bone loss disorder.
  • methods for decreasing osteoclast activity and increasing osteoblast activity in patients having a bone loss disorder using an RDP58 composition are also provided.
  • methods for decreasing matrix metailoproteinase production in patents having a bone loss disorder using an RDP58 composition are also provided.
  • an RDP58 composition may be used alone or in conjunction with other factors for the treatment of bone disorders.
  • an RDP58 composition is used in combination with other osteoclast inhibitors, such as OPG and RANK-based proteins designed to inhibit RANK signaling, such as RANK-Fc, function-blocking anti-RANK antibodies, function-blocking anti-RANKL antibodies, and other function-blocking RANK-binding or RANKL-binding moieties.
  • a therapeutically effective amount of a factor which stimulates bone formation may be used in conjunction with an RDP58 composition.
  • Such bone formation factors include but are not limited to the bone morphogenic factors designated BMP-1 through BMP-12, TGF- ⁇ and TGF-,ff family members, FGF-1 to FGF-10, interleukin-1 inhibitors, TNF ⁇ inhibitors, parathyroid hormone and analogs thereof, parathyroid related protein and analogs thereof, E series prostaglandins, bisphosphonates (such as alendronate and others), vitamin D3, dexamethasone, and bone-enhancing minerals such as fluoride and calcium.
  • BMP-1 through BMP-12 TGF- ⁇ and TGF-,ff family members
  • FGF-1 to FGF-10 include interleukin-1 inhibitors, TNF ⁇ inhibitors, parathyroid hormone and analogs thereof, parathyroid related protein and analogs thereof, E series prostaglandins, bisphosphonates (such as alendronate and others), vitamin D3, dexamethasone, and bone-enhancing minerals such as fluoride and calcium.
  • the invention is directed to the use of an RDP58 composition in the treatment of periodontitis.
  • RDP58 compositions have at least three additional abilities that add to their potency as agents for the treatment of periodontitis: first, RDP58 compositions have the ability to inhibit polymorphonuclear cell infiltration into the vicinity of the alveolar bone, which infiltration is normally associated with periodontitis; second, RDP58 compositions have the ability to inhibit the production of a variety of osteoclastogenic factors in the vicinity of the alveolar bone, which production is normally associated with periodontitis; and third, RDP58 compositions have the ability to inhibit the production of matrix metalloproteinases, for example, MMP9, in the vicinity of the alveolar bone, and inhibit tissue destruction thereby.
  • matrix metalloproteinases for example, MMP9
  • the invention provides RDP58 compositions and methods for the inhibition of leukocyte infiltration into the vicinity of the alveolar bone, which infiltration is a characteristic of periodontitis.
  • an RDP58 composition may be used to reduce monocyte, macrophage, and neutrophil occupation of the extracellular space in the vicinity of the alveolar bone.
  • the invention provides RDP58 compositions and methods for the inhibition of the production of a variety of osteoclastogenic factors in the vicinity of the alveolar bone, including TNF ⁇ , TNF , IL-1 , IL-4, IL-6, and LIF.
  • the invention provides methods for treating a variety of osteoporosis disorders, such as primary osteoporosis, endocrine osteoporosis (hyperthyroidism, hyperparathyroidism, Cushing's syndrome, and acromegaly), hereditary and congenital forms of osteoporosis (osteogenesis imperfecta, homocystinuria, Menkes' syndrome, and Rile-Day syndrome) and osteoporosis due to immobilization of extremities.
  • methods for decreasing osteoclast activity and increasing osteoblast activity in patients having an osteoporosis disorder are also provided.
  • methods for decreasing MMP production in patients having an osteoporosis disorder The methods involve administering an RDP58 composition to a patient having an osteoporosis disorder.
  • the invention provides methods for treating osteomyelitis, or an infectious lesion in bone leading to bone loss. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having osteomyelitis, or an infectious lesion in bone leading to bone loss. Also provided are methods for decreasing matrix metailoproteinase activity in patients having osteomyelitis. The methods involve administering an RDP58 composition to a patient having osteomyelitis, or an infectious lesion in bone leading to bone loss.
  • the invention provides methods for treating hypercalcemia, such as resulting from solid tumors (breast, lung and kidney) or hematologic malignacies (multiple myeloma, lymphoma and leukemia), idiopathic hypercalcemia, and hypercalcemia associated with hyperthryoidism and renal function disorders. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having hypercalcemia. Also provided are methods for decreasing matrix metailoproteinase production in patients having hypercalcemia. The methods involve administering an RDP58 composition to a patient having hypercalcemia.
  • the invention provides methods for treating osteonecrosis, or bone cell death, associated with traumatic injury or nontraumatic necrosis associated with Gaucher's disease, sickle cell anemia, systemic lupus erythematosus and other conditions. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having osteonecrosis. Also provided are methods for decreasing matrix metailoproteinase production in patients having osteonecrosis. The methods involve administering an RDP58 composition to a patient having osteonecrosis. [0079] In another preferred embodiment, the invention provides methods for inhibiting bone loss attendant rheumatoid arthritis.
  • the invention provides methods for treating periprosthetic osteolysis. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having periprosthetic osteolysis. Also provided are methods for decreasing matrix metailoproteinase production in patients having periprosthetic osteolysis. The methods involve administering an RDP58 composition to a patient having periprosthetic osteolysis.
  • the invention provides methods for treating osteosarcoma. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having osteosarcoma. Also provided are methods for decreasing matrix metailoproteinase production in patients having osteosarcoma. Also provided are methods for increasing cell differentiation and decreasing cell proliferation in bone tissue of patients having osteosarcoma. The methods involve administering an RDP58 composition to a patient having osteosarcoma.
  • the invention provides methods for treating bone loss due to osteolytic metastasis. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having osteolytic metastasis. Also provided are methods for decreasing matrix metailoproteinase production in patients having osteolytic metastasis. The methods involve administering an RDP58 composition to a patient having osteolytic metastasis.
  • the invention provides methods for treating familial expansile osteolysis. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having familial expansile osteolysis. Also provided are methods for decreasing matrix metailoproteinase production in patients having familial expansile osteolysis. The methods involve administering an RDP58 composition to a patient having familial expansile osteolysis.
  • the invention provides methods for treating Paget's disease (osteitis deformans). Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having Paget's disease. Also provided are methods for decreasing matrix metailoproteinase production in patients having Paget's disease. The methods involve administering an RDP58 composition to a patient having Paget's disease.
  • Paget's disease osteoitis deformans
  • methods for decreasing osteoclast activity and increasing osteoblast activity in patients having Paget's disease are also provided.
  • methods for decreasing matrix metailoproteinase production in patients having Paget's disease The methods involve administering an RDP58 composition to a patient having Paget's disease.
  • the invention provides methods for treating CCD. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having CCD. Also provided are methods for decreasing matrix metailoproteinase production in patients having CCD. The methods involve administering an RDP58 composition to a patient having CCD. [0086] In another preferred embodiment, the invention provides methods for decreasing prosthetic loosening. Also provided are methods for decreasing osteoclast activity and increasing osteoblast activity in patients having a prosthesis. Also provided are methods for decreasing matrix metailoproteinase production in patients having a prosthesis. The methods involve administering an RDP58 composition to a patient having a prosthesis.
  • the invention provides compositions and methods for promoting bone regeneration.
  • the methods involve administering an RDP58 composition to a patient in need of bone regeneration.
  • the bone regeneration may follow reconstruction of bone defects in cranio-maxillofacial surgery, or follow introduction of an implant into bone, for example a dental implant, bone supporting implant, or prosthesis.
  • methods for augmenting the repair of bone fractures involve administering a therapeutic amount of an RDP58 composition to a patient having a bone fracture.
  • the bones concerned and the extent of the fracture may vary.
  • an RDP58 composition is provided to the vicinity of the bone defect, bone fracture, implant, or prosthesis, where osteoblasts and/or osteoblast precursor cells are present.
  • an RDP58 composition is administered in combination with one or more bone regeneration-promoting cofactors.
  • Preferred bone deposition-promoting cofactors include bone morphogenic factors designated BMP-1 through BMP-12, TGF- ? and TGF- ?
  • FGF-1 to FGF-10 interleukin-1 inhibitors, TNF ⁇ inhibitors, parathyroid hormone and analogs thereof, parathyroid related protein and analogs thereof, E series prostaglandins, bisphosphonates (such as alendronate and others), vitamin D3, dexamethasone, and bone-enhancing minerals such as fluoride and calcium.
  • Provided to the vicinity of the bone defect means providing an RDP58 composition within an effective distance of the bone defect.
  • Effective distance means a distance within which the RDP58 composition can exert a bioactivity, particularly the ability to increase osteoblast activity, or decrease osteoclast activity at the bone defect site.
  • Providing can be done, for example, by local delivery, oral delivery, systemic delivery, etc.
  • the RDP58 composition need not be directly delivered within the effective distance of the bone defect to be "provided to the vicinity" of the bone defect.
  • RDP58 composition formulations in a paste or gel form for local application are highly preferred. See for example, US Patent No. 6,716,883, and US Patent No. 6,620,406.
  • RDP58 composition formulations preferably adhere to the tooth surface, thereby precluding the need for dressings or adhesives.
  • an RDP58 composition is used as an adjunctive therapy to scaling and root planing.
  • an RDP58 composition is combined with carriers conventionally used to formulate a paste, including thickening agents such as methylcellulose or hydroxypropyl methylcellulose, humectants and surfactants, as described in more detail in EP 568 160 and U.S. Pat. No. 5,496,541 , the contents of which are incorporated herein by reference.
  • an RDP58 composition may be admixed with gel carriers such as gelatin, polyethylene glycol, guar gum or combinations thereof.
  • gel carriers such as gelatin, polyethylene glycol, guar gum or combinations thereof.
  • Structurant compounds are also normally present in a gel, examples of which include polyoxyethylene-polyoxypropylenecopolymers.
  • Such structurants are generally present in amounts ranging from about 18 to about 25% by weight of the composition.
  • Toothpaste or gel forms of RDP58 compositions may further comprise an abrasive.
  • suitable abrasives include water-insoluble alkali or alkaline earth metal salts of metaphosphate, calcium carbonate, aluminate and silicate.
  • the amount of such abrasive generally contained in a toothpaste or gel ranges from about 5 to about 80% by weight of the composition.
  • an RDP58 composition may be combined with conventionally used carriers including one or more natural or synthetic elastomers, optionally supplemented with one or more solvents, plasticizers or fillers.
  • Natural elastomers suitable for use include substances of vegetable origin such as chicle, jelutong, gutta percha, guayule and crown gum.
  • Examples of synthetic elastomers include butadiene-styrene copolymers, isobutylene- isoprenecopolymers, polyethylene, polyisobutylene, polyvinylacetateand combinations thereof.
  • the elastomer generally comprises from about 14% to about 50% by weight of the composition.
  • Solvent may additionally be added to soften the elastomer component.
  • Suitable solvents include methyl, glycerol or pentaerythritol esters of rosins or modified rosins, such as hydrogenated, dimerized or polymerized rosins as well as terpene resins such as polyterpene.
  • Specific examples of such solvents include pentaerythritol ester of wood rosin, glycerol ester of partially dimerized or polymerized rosin, glycerol ester of tall oil rosin or wood rosin, and partially hydrogenated methyl ester of rosin.
  • Such solvents may be used in an amount ranging from about 10% to about 75% of the composition.
  • Plasticizers, softeners or emulsifiers may also be included in the gum composition in an amount of up to about 30% by weight of the composition.
  • these components include lanolin, lecithin, glyceryl monostearate, stearic acid, sodium stearate, postassium stearate, glyceryl triacetate, triacetin and glycerin, as well as natural waxes, petroleum waxes, paraffin waxes and microcrystalline waxes to improve texture and consistency.
  • a therapeutically effective amount of an RDP58 composition is administered to the oral cavity of the individual for a suitable period of time.
  • therapeutically effective amount means an amount of an RDP58 composition sufficient to treat periodontal disease without causing intolerable side effects.
  • Precise dosages of the composition appropriate for use to treat an individual are established in appropriately controlled clinical trials. As will be appreciated, the appropriate dosage will vary with the administrable form of the RDP58 composition.
  • RDP58 compositions suitable for use in the methods disclosed herein will generally comprise at least one peptide, polypeptide or oligopeptide capable of decreasing osteoclast or increasing osteoblast activity.
  • peptides selected from the family of RDP58 peptides described in PCT Publication WO 98/46633, which are characterized therein as being capable of inhibiting the cytotoxic activity of lymphocytic cells, inhibiting the production of inflammatory cytokines and inflammatory responses associated with those cytokines, inhibiting the activity of heme-containing enzymes and delaying the onset of autoimmune disease in a mammal at risk of developing such a disease.
  • peptides also have the ability to modulate a variety of biochemical pathways and decrease osteoclast activity and increase osteoblast activity.
  • Suitable peptides for use in the compositions and methods provided herein have a variety of characteristics, and may be identified in a number of ways.
  • Peptides may be identified by their ability to inhibit osteoclast activity.
  • putative peptides may be screened by incubation with osteoclast precursor cells under conditions known to promote osteoclast differentiation, such as in the presence of M-CSF-1 and RANKL.
  • Means for measuring osteoclast differentiation, such as TRAP assays are well known in the art. See, for example, assays described in U.S. Patent No. 6,271 ,349.
  • the ability of a putative peptides to modulate LPS- or TNF ⁇ -induced formation of osteoclast like cells from bone marrow cell cultures may be assayed.
  • the bone marrow cultures used are preferably from mouse, rat, monkey, donkey or human, though other bone marrow cultures may be used.
  • a preferred system for assaying the ability of a putative peptide to modulate osteoclast formation has been previously described (Udagawa et al. Endocrinology 125, 1805-1813 (1989), Proc. Natl. Acad. Sci. USA 87, 7260-7264 (1990)) and employs a combination of bone marrow cells and cells from bone marrow stromal cell lines.
  • a description of a modified assay has been previously published (Lacey et al. Endocrinology 136, 2367-2376 (1995)).
  • bone marrow cells flushed from the femurs and tibiae of mice, are cultured overnight in culture media ( ⁇ -MEM with 10% heat inactivated fetal bovine serum) supplemented with 500 U/ml CSF-1 (colony stimulating factor 1 , also called M-CSF), a hematopoietic growth factor specific for cells of the monocyte/macrophage family lineage.
  • CSF-1 colony stimulating factor 1 , also called M-CSF
  • the non-adherent cells are collected, subjected to gradient purification, and then cocultured with cells from the bone marrow cell line ST2 (1x106 non-adherent cells: 1x105 ST2 cells/ml media).
  • the media is supplemented with dexamethasone (100 nM) and the biologically-active metabolite of vitamin D3 known as 1 ,25 dihydroxyvitamin D3 (1 ,25 (OH)2 D3, 10 nM).
  • dexamethasone 100 nM
  • the biologically-active metabolite of vitamin D3 known as 1 ,25 dihydroxyvitamin D3 (1 ,25 (OH)2 D3, 10 nM
  • prostaglandin E2 250 nM
  • the coculture period usually ranges from 8-10 days and the media, with all of the supplements freshly added, is renewed every 3-4 days.
  • the cultures are assessed for the presence of tartrate acid phosphatase (TRAP) using either a histochemical stain (Sigma Kit #387A, Sigma, St. Louis, Mo.) or TRAP solution assay.
  • TRAP tartrate acid phosphatase
  • the TRAP histochemical method allows for the identification of osteoclasts phenotypically which are multinucleated cells that are also TRAP+.
  • the solution assay involves lysing the osteoclast-containing cultures in a citrate buffer (100 mM, pH 5.0) containing 0.1 % Triton X-100. Tartrate resistant acid phosphatase activity is then measured based on the conversion of p-nitrophenylphosphate (20 nM) to p-nitrophenol in the presence of 80 mM sodium tartrate which occurs during a 3-5 minute incubation at room temperature. The reaction is terminated by the addition of NaOH to a final concentration of 0.5 M. The optical density at 405 nm is measured and the results are plotted.
  • Lacey et al. supra employs bone marrow macrophages as osteoclast precursors.
  • the osteoclast precursors are derived by taking the nonadherent bone marrow cells after an overnight incubation in CSF-1/M-CSF, and culturing the cells for an additional 4 days with 1 ,000-2,000 U/ml CSF-1. Following 4 days of culture, termed the growth phase, the non-adherent cells are removed.
  • the adherent cells, which are bone marrow macrophages can then be exposed for up to 2 days to various treatments in the presence of 1 ,000-2,000 U/ml CSF-1. This 2 day period is called the intermediate differentiation period.
  • ST-2 cells (1x105 cell/ml), dexamethasone (100 nM) and 1 ,25 (OH)2 D3 (10 nM) are added for the last 8 days for what is termed the terminal differentiation period.
  • Putative RDP58 compositions can be added during this terminal period as well. Acquisition of phenotypic markers of osteoclast differentiation are acquired during this terminal period.
  • Peptides useful in RDP58 compositions may also be identified by their ability to modulate bone resorption in vivo.
  • IL1 increases bone resorption both systemically and locally when injected subcutaneously over the calvaria of mice (Boyce et al., Endocrinology 125, 1142-1150 (1989)).
  • the systemic effects can be assessed by the degree of hypercalcemia and the local effects histologically by assessing the relative magnitude of the osteoclast-mediated response.
  • These assays may be used to determine if putative peptides can modify the local and/or systemic actions of IL1 when injected subcutaneously over the same region of the calvaria as IL1.
  • the subject RDP58 peptides comprise one or more of the cytomodulating peptides disclosed in co-pending U.S. Patent Applications U.S.S.N 09/028,083 & U.S.S.N. 08/838,916 as well as corresponding International application WO 98/46633, the disclosures of which are expressly incorporated herein by reference.
  • the RDP58 peptide comprises the core sequence Arg-nL-nL-nL-Arg-nL-nL-nL-Gly-Tyr, where nL is norleucine and all amino acids other than glycine are the D-stereoisomer.
  • the core sequence of the RDP58 peptide desirably comprises two basic amino acids separated by from three to four hydrophobic amino acids, particularly three hydrophobic amino acids, and particularly where the N-terminus is a basic amino acid. More desirably, the C-terminal amino acid is an aromatic amino acid, particularly tyrosine. Of particular interest is where at least one of the oligopeptide core terminal amino acids is an oligopeptide terminal amino acid, which may be in the monomeric or oligomeric form of the compound.
  • the preferred RDP58 peptides for use in the compositions and methods of the present invention comprise oligopeptides having the core sequence B-X-X-X-B-X-X-X-J-Tyr, where B is a basic amino acid, preferably Lys or Arg, particularly Arg on at least one position, preferably at both positions; J is Gly, B or an aliphatic hydrophobic amino acid of from 5 to 6 carbon atoms, particularly Gly or B; and X is an aliphatic or aromatic amino acid.
  • At least three X amino acid residues are the same non-polar aliphatic amino acid, preferably at least four are the same non-polar aliphatic amino acid, more preferably at least five are the same non-polar aliphatic amino acid, and most preferably, all are the same non-polar aliphatic amino acid.
  • the non-polar aliphatic amino acids are of from 5 to 6 carbon atoms, particularly 6 carbon atoms, particularly the non-polar aliphatic amino acids Val, lie, Leu, and nL.
  • X is any amino acid other than a charged aliphatic amino acid, and preferably any amino acid other than a polar aliphatic amino acid.
  • aliphatic amino acids indicated by X in the B-X-X-X-B-X-X-X-J-Tyr peptide sequence preferably at least 3 are aliphatic amino acids of from 5 to 6 carbon atoms, more preferably at least 4 are aliphatic amino acids of from 5 to 6 carbon atoms, most preferably at least 5 are aliphatic amino acids of 5-6 carbon atoms, more particularly 6 carbon atoms.
  • the aliphatic amino acids are non-polar aliphatic amino acids of from 5 to 6 carbon atoms, particularly Val, lie, Leu, and nL.
  • the other amino acids may be other uncharged aliphatic amino acids, particularly non-polar aliphatic amino acids or aromatic amino acids.
  • compositions of particular interest will include an RDP58 peptide having the core sequence:
  • U which comprises an uncharged aliphatic amino acid or aromatic amino acid, particularly a non-polar aliphatic amino acid or aromatic amino acid.
  • the amino acids may be naturally occurring amino acids or D- isomers thereof.
  • the peptides may have one or more D-stereoisomer amino acids, up to all of the amino acids. Additionally, the peptides may comprise oligomers of the subject peptides, particularly dimers thereof, or comprise a cyclic peptide, that is a ring structure, as further described below.
  • amino acids for the most part natural amino acids or their D-stereoisomers
  • RDP58 peptides are disclosed in PCT application serial number PCT/US98/07231 , filed 10 April 1998, US Patent Application Serial No. 08/838,916, filed 11 April 1997, and US Patent Application Serial No. 09/028,083 filed 23 February 1998, each being expressly incorporated herein in its entirety by reference.
  • the term "RD-58 peptide" as used herein is meant to encompass all of the foregoing peptide compounds.
  • HLA peptides and TCR peptides may be alternatively or additionally used in the subject invention as components of the subject RDP58 compositions.
  • HLA-B ⁇ 1 -domain particularly the amino acids from 75 to 84 and variations of this sequence where not more than 2 amino acids are replaced (see, e.g., WO 95/13288 and Buelow et al., expressly incorporated herein by reference).
  • sequences based on the human TCR- ⁇ transmembrane region consisting of that sequence and sequences having not more than 2 mutations from that sequence (see Australian Application Nos.
  • PN 0589 and PN 0590 filed January 16, 1995, expressly incorporated herein by reference).
  • These sequences include 2 basic amino acids, where the 2 basic amino acids are separated by 4 aliphatic hydrophobic amino acids, although the application indicates that from 3 to 5 hydrophobic amino acids may be present.
  • mutation is intended each substitution of one amino acid for another or an insertion or deletion, each being counted as one mutation.
  • peptide as used herein is meant to encompass all of the foregoing peptide compounds, as well as analogs, derivatives, fusion proteins and the like.
  • the subject peptides may be modified in a variety of conventional ways well known to the skilled artisan.
  • one or both, usually one terminus of the peptide may be substituted with a lipophilic group, usually aliphatic or aralkyl, of from 8 to 36, usually 8 to 24 carbon atoms and fewer than two heteroatoms in the aliphatic chain, the heteroatoms usually being oxygen, nitrogen and sulfur.
  • the chain may be saturated or unsaturated, desirably having not more than 3 sites, usually not more than 2 sites of aliphatic unsaturation.
  • aliphatic fatty acids, alcohols and amines may be used, such as caprylic acid, capric acid, lauric acid, myristic acid and myristyl alcohol, palmitic acid, palmitoleic acid, stearic acid and stearyl amine, oleic acid, linoleic acid, docosahexaenoic acid, etc. (see U.S. Patent No. 6,225,444, hereby incorporated by reference).
  • Preferred are unbranched, naturally occurring fatty acids between 14-22 carbon atoms in length.
  • Other lipohilic molecules include glyceryl lipids and sterols, such as cholesterol.
  • the lipophilic groups may be reacted with the appropriate functional group on the oligopeptide in accordance with conventional methods, frequently during the synthesis on a support, depending on the site of attachment of the oligopeptide to the support.
  • Lipid attachment is useful where oligopeptides may be introduced into the lumen of the liposome, along with other therapeutic agents (e.g., BMPs) for administering the peptides and agents into a host.
  • BMPs therapeutic agents
  • Increasing lipophilicity is also known to increase transport of compounds across endothelial cells and therefore useful in promoting uptake of such compounds from the intestine or blood stream into surrounding tissues.
  • the terminal amino group or carboxyl group of the peptide may be modified by alkylation, amidation, or acylation to provide esters, amides or substituted amino groups, where the alkyl or acyl group may be of from about 1 to 30, usually 1 to 24, preferably either 1 to 3 or 8 to 24, particularly 12 to 18 carbon atoms. This is done using conventional chemical synthetic methods.
  • the peptide or derivatives thereof may also be modified by acetylation or methylation to alter the chemical properties, for example lipophilicity. Methods for acylating, and specifically for acetylating the free amino group at the N-terminus are well known in the art.
  • the carboxyl group may be modified by esterification with alcohols or amidated to form -CONH2, CONHR, or CONR, wherein each R is a hybroxycarbyl (1-6 carbons).
  • Methods of esterification and amidation are done using well known techniques. Other modifications include deamination of glutamyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of serine or threonine; and methylation of amino groups of lysine, arginine, and histidine side chains (see T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co. San Francisco, CA, 1983).
  • either or both the N- and C-terminus of the peptide may be extended by not more than a total of about 100, usually not more than a total of about 30, more usually not more than about 20 amino acids, often not more than about 9 amino acids, where the amino acids will have fewer than 25%, more usually fewer than 20% polar amino acids, more particularly, fewer than 20% which are charged amino acids.
  • extensions of the above core sequences in either direction are mainly done with lipophilic, uncharged amino acids, particularly non- polar aliphatic amino acids and aromatic amino acids.
  • the peptides may comprise L-amino acids, D- amino acids, or mixtures of D and L amino acids. Exceptions to the number of amino acid extensions are contemplated when the oligopeptides are expressed as fusion or chimeric proteins, as described below.
  • the peptides may also be in the form of oligomers, particularly dimers of the peptides, which may be head to head, tail to tail, or head to tail, there being not more than about 6 repeats of the peptide.
  • the oligomer may contain one or more D-stereoisomer amino acids, up to all of the amino acids.
  • the oligomers may or may not include linker sequences between the peptides.
  • suitable linkers include those comprising uncharged amino acids and (Gly)n, where n is 1-7, Gly-Ser (e.g., (GS)n, (GSGGS)n and (GGGS)n, where n is at least 1), Gly-Ala, Ala- Ser, or other flexible linkers, as known in the art.
  • Linkers of Gly or Gly-Ser may be used since these amino acids are relatively unstructured, which allows interaction of individual peptides with cellular target molecules and limits structural perturbations between peptides of the oligomer.
  • Peptides may also be in a structurally constrained form such as cyclic peptides of from about 9-50, usually 12 to 36 amino acids, where amino acids other than the specified amino acids may be present as a bridge.
  • cyclic peptides of from about 9-50, usually 12 to 36 amino acids, where amino acids other than the specified amino acids may be present as a bridge.
  • addition of terminal cysteines allows formation of disulfide bridges to form a ring peptide.
  • one may use other than amino acids to cyclize the peptide.
  • Bifunctional crosslinking agents are useful in linking two or more amino acids of the peptide. Other methods for ring formation are described in Chen et al., Proc. Natl. Acad. Sci.
  • the subject peptides may also be modified by attachment to other compounds for the purposes of incorporation into carrier molecules, changing peptide bioavailability, extend or shorten half-life, control distribution to various tissues or the blood stream, diminish or enhance binding to blood components, and the like.
  • the subject peptides may be bound to these other components by linkers which are cleavable or non-cleavable in the physiological environment such as blood, cerebrospinal fluid, digestive fluids, etc.
  • the peptides may be joined at any point of the peptide where a functional group is present, such as hydroxyl, thiol, carboxyl, amino, or the like.
  • modification will be at either the N-terminus or the C-terminus.
  • the subject peptides may be modified by covalently attaching polymers, such as polyethylene glycol, polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidine, polyproline, poly(divinyl-ether-co-maleic anhydride), poly(styrene-c- maleic anhydride), etc.
  • Water soluble polymers such a polyethylene glycol and polyvinylpyrrolidine are known to decrease clearance of attached compounds from the blood stream as compared to unmodified compounds.
  • the modifications can also increase solubility in aqueous media and reduce aggregation of the peptides.
  • the RDP58 peptide or other useful peptide is preferably conjugated to one or more small molecules for detection and isolation of the peptide, and to target or transport the peptide into specific cells, tissues, and organs.
  • Small molecule conjugates include haptens, which are substances that do not initiate an immune response when introduced by themselves into an animal. Generally, haptens are small molecules of molecular weight less than about 2 kD, and more preferably less that about 1 kD.
  • Haptens include small organic molecules (e.g., p-nitrophenol, digoxin, heroin, cocaine, morphine, mescaline, lysergic acid, tetrahydrocannabinol, cannabinol, steroids, pentamidine, biotin, etc.). Binding to the hapten, for example for purposes of detection or purification, are done with hapten specific antibodies or specific binding partners, such as avidin which binds biotin.
  • hapten specific antibodies or specific binding partners such as avidin which binds biotin.
  • the peptides may have attached various label moieties such as radioactive labels and fluorescent labels for detection and tracing.
  • Fluorescent labels include, but are not limited to, fluorescein, eosin, Alexa Fluor, Oregon Green, rhodamine Green, tetramethylrhodamine, rhodamine Red, Texas Red, coumarin and NBD fluorophores, the QSY 7, dabcyl and dabsyl chromophores, BIODIPY, Cy5, etc.
  • the peptides are joined to a wide variety of other peptides or proteins for a variety of purposes.
  • the peptides may be linked to other peptides or proteins to provide convenient functionalities for bonding, such as amino groups for amide or substituted amine formation, e.g., reductive amination; thiol groups for thioether or disulfide formation; carboxyl groups for amide formation; and the like.
  • peptides of at least 2, more usually 3, and not more than about 60 lysine groups, particularly polylysines of from about 4 to 20, usually 6 to 18 lysine units referred to as multiple antigenic peptide system (MAPS), where the subject peptides are bonded to the lysine amino groups, generally at least about 20%, more usually at least about 50%, of available amino groups, to provide a multipeptide product
  • MAPS multiple antigenic peptide system
  • other naturally occurring or synthetic peptides and proteins may be used to provide a carrier immunogen for generating antibodies to the subject peptides, where the antibodies serve as reagents for detecting the peptides or for identifying other peptides having a comparable conformation.
  • Suitable carriers for generating antibodies include, among others, hemocyanins (e.g., Keyhole Limpet hemocyanin - KLH); albumins (e.g., bovine serum albumin, ovalbumin, human serum albumin, etc.); immunoglobulins; thyroglobulins (e.g., bovine thyroglobulin); toxins (e.g., diptheria toxoid, tetanus toxoid); and polypeptides such as polylysine, as described above, or polyalanine- lysine.
  • hemocyanins e.g., Keyhole Limpet hemocyanin - KLH
  • albumins e.g., bovine serum albumin, ovalbumin, human serum albumin, etc.
  • immunoglobulins e.g., bovine thyroglobulin
  • toxins e.g., diptheria toxoid, tetanus toxoid
  • proteins are preferred carriers
  • other carriers preferably high molecular weight compounds, including carbohydrates, polysaccharides, lipopolysaccharides, nucleic acids, and the like of sufficient size and immunogenicity.
  • the resulting antibodies may be used to prepare anti-idiotypic antibodies which may compete with the subject peptides for binding to a target site. These anti-idiotypic antibodies are useful for identifying proteins to which the subject peptides bind.
  • the peptides are conjugated to other peptides or proteins for targeting the peptide to cells and tissues, or adding additional functionalities to the peptides.
  • the protein or peptide used for conjugation will be selected based on the cell or tissue being targeted for therapy (Lee, R. et al. Arthritis. Rheum. 46: 2109-2120 (2002); Pasqualini, R. Q. J. Nucl. Med. 43: 159-62 (1999); Pasgualini, R. Nature 380: 364-366 (1996); hereby incorporated by reference).
  • the proteins may also compromise poly-amino acids including, but not limited to, polyarginine; and polylysine, polyaspartic acid, etc. , which may be incorporated into other polymers, such as polyethylene glycol, for preparation of vesicles or particles containing the conjugated peptides.
  • the subject peptides may be conjugated to tumor-targeting moieties.
  • Targeting to tumors may be done using techniques well known in the art. For example, antibodies with some selectivity for tumor cells, relative to normal cells, are known and may be used by coupling to the subject peptides (see Kyriakos et al., Cancer Res., 52: 835 (1992)).
  • the subject peptides may be expressed in conjunction with other peptides or proteins, so as to be a portion of the polypeptide chain, either internal, or at the N- or C- terminus to form chimeric proteins or fusion proteins.
  • fusion polypeptide or "fusion protein” or “chimeric protein” herein is meant a protein composed of a plurality of protein components that, while typically joined in the native state, are joined by the respective amino and carboxy termini through a peptide linkage to form a continuous polypeptide.
  • Plurality in this context means at least two, and preferred embodiments generally three to twelve components, although more may be used. It will be appreciated that the protein components can be joined directly or joined through a peptide linker/spacer as outlined below.
  • Fusion polypeptides may be made to a variety of other peptides or proteins to display the subject peptides in a conformationally restricted form, for targeting to cells and tissues, for targeting to intracellular compartments, tracking the fusion protein in a cell or an organism, and screening for other molecules that bind the peptides.
  • Proteins useful for generating fusion proteins include various reporter proteins, structural proteins, cell surface receptors, receptor ligands, toxins, and enzymes.
  • Exemplary proteins include fluorescent proteins (e.g., Aequoria victoria GFP, Renilla reniformis GFP, Renilla muelleri GFP, luciferases, etc., and variants thereof); ?-galactosidase; alkaline phosphatase; E. coli. maltose binding protein; coat proteins of filamentous bacteriophage (e.g., minor coat protein, pill, or the major coat protein, pVIII, for purposes of phage display).
  • fluorescent proteins e.g., Aequoria victoria GFP, Renilla reniformis GFP, Renilla muelleri GFP, luciferases, etc., and variants thereof
  • ?-galactosidase alkaline phosphatase
  • E. coli. maltose binding protein e.g., coat proteins of filamentous bacteriophage (e.g., minor coat protein, pill, or the major coat protein,
  • Fusion proteins also encompass fusions with fragments of proteins or other peptides, either alone or as part of a larger protein sequence.
  • the fusion polypeptides may comprise fusion partners.
  • fusion partners herein is meant a sequence that is associated with the peptide that confers all members of the proteins in that class a common function or ability. Fusion partners can be heterologous (i.e., not native to the host cell) or synthetic (i.e., not native to any cell).
  • the fusion partners include, but are not limited to, a) presentation structures, which provide the subject peptides in a conformationally restricted or stable form; b) targeting sequences, which allow localization of the peptide to a subcellular or extracellular compartment; c) stability sequences, which affects stability or protection from degradation to the peptide or the nucleic acid encoding it; d) linker sequences, which conformationally decouples the oligopeptide from the fusion partner; and e) any combination of the above.
  • the fusion partner is a presentation structure.
  • presentation structure as used herein is meant a sequence that when fused to the subject peptides presents the peptides in a conformationally restricted form.
  • Preferred presentation structures enhance binding interactions with other binding partners by presenting a peptide on a solvent exposed exterior surface, such as a loop.
  • presentation structures comprise a first portion joined to the N-terminus of the peptide and a second portion joined to the C-terminal end of the subject peptide. That is, the peptide of the present invention is inserted into the presentation structures.
  • the presentation structures are selected or designed to have minimal biological activity when expressed in the target cells.
  • the presentation structures maximize accessibility to the peptides by displaying or presenting the peptide on an exterior loop.
  • Suitable presentation structures include, but are not limited to, coiled coil stem structures, minibody structures, loops on Murns, dimerization sequences, cysteine linked structures, transglutaminase linked structures, cyclic peptides, helical barrels, leucine zipper motifs, etc.
  • the presentation structure is a coiled-coil structure, which allows presentation of the subject peptide on an exterior loop (see Myszka et al. Biochemistry 33: 2362-2373 (1994)), such as a coiled-coil leucine zipper domain (see Martin et al. EMBO J. 13: 5303-5309 (1994)).
  • the presentation structure may also comprise minibody structures, which is essentially comprised of a minimal antibody complementarity region.
  • the minibody structure generally provides two peptide regions that are presented along a single face of the tertiary structure in the folded protein (see Bianchi et al. J. Mol. Biol. 236: 649-659 (1994); Tramontano et al. J. Mol. Recognit. 7: 9-24 (1994)).
  • the presentation structure comprises two dimerization sequences.
  • the dimerization sequences which can be same or different, associate non-covalently with sufficient affinity under physiological conditions to structurally constrain the displayed peptide; that is, if a dimerization sequence is used at each terminus of the subject oligopeptide, the resulting structure can display the subject peptide in a structurally limited form.
  • a variety of sequences are suitable as dimerization sequences (see for example, WO 99/51625; incorporated by reference). Any number of protein-protein interaction sequences known in the art are useful.
  • the presentation sequence confers the ability to bind metal ions to generate a conformationally restricted secondary structure.
  • C2H2 zinc finger sequences are used.
  • C2H2 sequences have two cysteines and two histidines placed such that a zinc ion is chelated.
  • Zinc finger domains are known to occur independently in multiple zinc-finger peptides to form structurally independent, flexibly linked domains (see Nakaseko, Y. et al. J. Mol. Biol. 228: 619-636 (1992)).
  • a general consensus sequence is (5 amino acids)-C-(2 to 3 amino acids)-C-(4 to 12 amino acids)-H-(3 amino acids)-H-(5 amino acids).
  • a preferred example would be -FQCEEC-random peptide of 3 to 20 amino acids-HIRSHTG.
  • CCHC boxes having a consensus sequence -C- (2 amino acids)-C-(4 to 20 random peptide)-H-(4 amino acids)-C- can be used, (see Bavoso, A. et al. Biochem. Biophys. Res. Commun. 242: 385-389 (1998)).
  • Other examples include (1 ) -VKCFNC-4 to 20 random amino acids-HTARNCR-, based on the nucleocapsid protein P2; (2) a sequence modified from that of the naturally occurring zinc-binding peptide of the Lasp-1 LIM domain (Hammarstrom, A. et al. Biochemistry 35: 12723-32 (1996)); and (3) -MNPNCARCG-4 to 20 random amino acids- HKACF-, based on the NMR structural ensemble 1ZFP (Hammarstrom et al., supra).
  • the presentation structure is a sequence that comprises two or more cysteine residues, such that a disulfide bond may be formed, resulting in a conformationally constrained structure. That is, use of cysteine containing peptide sequences at each terminus of the subject peptides results in cyclic peptide structures, as described above.
  • a cyclic structure reduces susceptibility of the presented peptide to proteolysis and increases accessibility to its target molecules.
  • this particular embodiment is particularly suited when secretory targeting sequences are used to direct the peptide to the extracellular space.
  • the fusion partner is a targeting sequence.
  • Targeting sequences comprise binding sequences capable of causing binding of the expressed product to a predeterimed molecule or class of molecules while retaining bioactivity of the expression product; sequences signaling selective degradation of the fusion protein or binding partners; and sequences capable of constitutively localizing peptides to a predetermined cellular locale.
  • Typical cellular locations include subcellular locations (e.g, Golgi, endoplasmic recticulum, nucleus, nucleoli, nuclear membrane, mitochondria, secretory vesicles, lysosomes) and extracellular locations by use of secretory signals.
  • NLSs nuclear localization signals
  • Typical NLS sequences include the single basic NLS of SV40 large T antigen (Kalderon et al. Cell 39: 499-509 (1984)); human retinoic acid receptor-;-? nuclear localization signal (NF-kB p50 and p65 (Ghosh et al. Cell 62: 1019-1029 (1990)); Nolan et al.
  • the targeting sequences are membrane anchoring sequences. Peptides are directed to the membrane via signal sequences and stably incorporated in the membrane through a hydrophobic transmembrane domain (designated as TM). The TM segment is positioned appropriately on the expressed fusion protein to display the subject peptide either intracellularly or extracellularly, as is known in the art.
  • Membrane anchoring sequences and signal sequences include, but are not limited to, those derived from (a) class I integral membrane proteins such as IL-2 receptor?-chain; Hatekeyama et al. Science 244: 551-556 (1989)) and inuslin receptors-chain (Hetekayama et al, supra); (b) class II integral membrane proteins such as neutral endopeptidase (Malfroy et al Biochem. Biophys. Res. Commun. 144: 59-66 (1987)); and (c) type III proteins such as human cytochrome P450 NF25 (Hetekayama et al, supra); and those from CD8, ICAM-2, IL-8R, and LFA-1.
  • class I integral membrane proteins such as IL-2 receptor?-chain; Hatekeyama et al. Science 244: 551-556 (1989)) and inuslin receptors-chain (Hetekayama et al, supra)
  • class II integral membrane proteins such as neutral endo
  • Membrane anchoring sequences also include the GPI anchor, which results in covalent bond formation between the GPI anchor sequence and the lipid bilayer via a glycosyl-phosphatidylinositol.
  • GPI anchor sequences are found in various proteins, including Thy-1 and DAF (see Homans et al. Nature 333: 269-272 (1988)).
  • acylation sequences allow for attachment of lipid moieties, e.g., isoprenylation (i.e., famesyl and geranyl-geranyl; see Farnsworth et al. Proc. Natl. Acad. Sci. USA 91 : 11963-11967 (1994) and Aronheim et al.
  • the subject peptide will be bound to a lipid group at a terminus, so as to be able to be bound to a lipid membrane, such as that of a liposome.
  • lysozomal targeting sequences e.g., sequences in LAMP-1 and LAMP-2; Uthayakumar et al. Cell Mol. Biol. Res. 41 : 405-420 (1995) and Konecki et al. Biochem. Biophys. Res. Comm. 205: 1-5 (1994)
  • mitochondrial localization sequences e.g., mitochondrial matrix sequences, mitochondrial inner membrane sequences, mitochondrial intermembrance sequences, or mitochondrial outer membrane sequences; see Shatz, G. Eur. J. Biochem. 165: 1-6 (1987)
  • endoplasmic recticulum localization sequences e.g., calreticulin, Pelham, H. R.
  • the targeting sequence is a secretory signal sequence which effects secretion of the peptide.
  • secretory signal sequence A large number of secretory sequences are known to direct secretion of a peptide into the extracellular space when placed at the amino end relative to the peptide of interest, particularly for secretion of a peptide by cells, including transplanted cells. Suitable secretory signals included those found in IL-2 (Villinger et al. J. Immuno. 155: 3946-3954 (1995)), growth hormone (Roskam et al. Nucleic Acids Res. 7: 305-320 (1979)), preproinsulin, and influenza HA protein.
  • the fusion partner may further comprise a stability sequence, which confers stability to the fusion protein or the nucleic acid encoding it.
  • a stability sequence which confers stability to the fusion protein or the nucleic acid encoding it.
  • incorporation of glycines after the initiating methionine e.g., MG or MGG
  • MGG methionine
  • Additional amino acids may be added for tagging the peptide for purposes of detection or purification.
  • These sequences may comprise epitopes recognized by antibodies (e.g., flag tags) or sequences that bind ligands, such a metals ions.
  • tag sequences and ligand binding sequences are well known in the art. These include, but are not limited to, poly-histidine (e.g., 6xHis tags, which are recognized by antibodies but also bind divalent metal ions); poly-histidine-glycine (poly-his-gly) tags; flu HA tag polypeptide; c-myc tag; Flag peptide (Hopp et al. BioTechnoIogy 6: 1204-1210 (1988)); KT3 epitope peptide; tubulin epitope peptide (Skinner et al. J. Biol. Chem. 266: 15163-12166 (1991 )); and T7 gene 10 protein peptide tag (Lutz-Freyermuth et al. Proc. Natl. Acad. Sci. USA 87: 6363-6397 (1990)).
  • poly-histidine e.g., 6xHis tags, which are recognized by antibodies but also bind divalent metal
  • Fusion partners include linker or tethering sequences for linking the peptides and for presenting the peptides in an unhindered structure.
  • useful linkers include glycine polymers (G)n where n is 1 to about 7, glycine-serine polymers (e.g., (GS)n, (GSGGS)n and (GGGS)n, where n is at least 1 ), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
  • the linkers are glycine or glycine-serine polymers since these amino acids are relatively unstructured, hydrophilic, and are effective for joining segments of proteins and peptides.
  • cysteines can be used to make thioethers or cyclic peptides, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.
  • cysteine residues are introduced for cyclizing the peptide, formation of disulfide bonds are conducted in the presence of mild oxidizing agents.
  • Chemical oxidants may be used, or the cysteine bearing peptides are exposed to oxygen to form the linkages, typically in a suitable solution such as a aqueous buffer containing DMSO.
  • lipids may be attached either chemically or by use of appropriate lipidation sequences in the expressed peptide.
  • conjugating various molecules to the peptides of the present invention functional groups on the peptides and the other molecule are reacted in the presence of an appropriate conjugating (e.g., crosslinking) agent.
  • conjugating or crosslinking agent used will depend on the functional groups, such as primary amines, sulfhydryls, carbonyls, carbohydrates and carboxylic acids being used.
  • Agents may be fixatives and crosslinking agents, which may be homobifunctional, heterobifunctional, or trifunctional crosslinking agents (Pierce Endogen, Chicago, IL).
  • fixatives and crosslinking agents include formaldehyde, glutaraldehyde, 1 ,1-bis(diazoacetyl)-2- phenylethane, N-hydroxysuccinimide esters, dissuccimidyl esters, maleimides (e.g., bis-N-maleimido- 1-8-octane), and carbodiimides (e.g., N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide; dicyclohexylcarbodiimide.
  • Spacer molecules comprising alkyl or substituted alkyl chains with lengths of 2 - 20 carbons may be used to separate conjugates.
  • protecting group is a molecule bound to a specific functional group which is selectively removable to reexpose the functional group (see Greene, T.W. and Wuts, P.G.M. Protective Groups in Organic Synthesis (3rd ed.), John Wiley & Sons, Inc., New York, 1999).
  • the peptides may be synthesized with protected amino acid precursors or reacted with protecting groups following synthesis but before reacting with crosslinking agent. Conjugations may also be indirect, for example by attaching a biotin moiety, which can be contacted with a compound or molecule which is coupled to streptavidin or avidin.
  • the linkage between the peptides and the conjugated compound is chosen to be sufficiently labile to result in cleavage under desired conditions, for example after transport to desired cells or tissues.
  • Biologically labile covalent bonds e.g., imimo bonds and esters, are well known in the art (see U.S. Patent No. 5,108,921 , hereby incorporated by reference). These modifications permit administration of the peptides in potentially a less active form, which is then activated by cleavage of the labile bond.
  • combinations of fusion partners may be used. Any number of combinations of presentation structures, targeting sequences, rescue sequences, tag sequences and stability sequences may be used with or without linker sequences.
  • RDP58, TCR, or HLA peptides of the present invention may be prepared in a number of ways. Chemical synthesis of peptides are well known in the art. Solid phase synthesis is commonly used and various commercial synthetic apparatuses are available, for example automated synthesizers by Applied Biosystems Inc., Foster City, CA; Beckman; etc. Solution phase synthetic methods may also be used, although it is less convenient. By using these standard techniques, naturally occurring amino acids may be substituted with Unnatural amino acids, particularly D- stereoisomers, and also with amino acids with side chains having different lengths or functionalities.
  • Functional groups for conjugating to small molecules, label moieties, peptides, or proteins, or for purposes of forming cyclized peptides may be introduced into the molecule during chemical synthesis.
  • small molecules and label moieties may be attached during the synthetic process.
  • introduction of the functional groups and conjugation to other molecules minimally affects the structure and function of the subject peptide.
  • the peptides of the present invention may be present in the form of a salt, generally in a salt form which is pharmaceutically acceptable. These include inorganic salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and the like. Various organic salts of the peptide may also be made with, including, but not limited to, acetic acid, propionic acid, pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid, benozic acid, cinnamic acid, salicylic acid, etc.
  • Synthesis of the peptides and derivatives thereof may also be carried out by using recombinant techniques.
  • a nucleic acid sequence which encodes a single oligopeptide or preferably a plurality of the subject peptides in tandem with an intervening amino acid or sequence, which allows for cleavage to the single peptide or head to tail dimers.
  • methionine or tryptophane is absent, an intervening methionine or tryptophane may be incorporated, which allows for single amino acid cleavage using CNBr or BNPS-Skatole (2-(2- nitrophenylsulfenyl)-3-methyl-3-bromoindolenine), respectively.
  • cleavage is accomplished by use of sequences that are recognized by particular proteases for enzymatic cleavage or sequences that act as self-cleaving sites (e.g., 2A sequences of apthoviruses and cardioviruses; Donnelly, M.L. J. Gen. Virol. 78: 13-21 .(1997); Donnelly, M.L. J. Gen. Virol. 82: 1027- 41 (2001), hereby incorporated by reference).
  • the subject peptide may also be made as part of a larger peptide, which can be isolated and the oligopeptide obtained by proteolytic cleavage or chemical cleavage.
  • a gene encoding a particular peptide, protein, or fusion protein is joined to a DNA sequence encoding the peptides of the present invention to form a fusion nucleic acid, which is introduced into an expression vector.
  • Expression of the fusion nucleic acid is under the control of a suitable promoter and other control sequences, as defined below, for expression in a particular host cell or organism (see, Sambrook et al., Molecular Biology: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 2001 ; Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, 1988, updates up to 2002; incorporated by reference).
  • nucleic acids When the synthesis or delivery of the peptides is via nucleic acids encoding the subject peptides, the nucleic acids are cloned into expression vectors and introduced into cells or a host.
  • the expression vectors are either self-replicating extrachromosomal vectors or vectors that integrate into the host chromosome, for example vectors based on retroviruses, vectors with site specific recombination sequences, or by homologous recombination.
  • these vectors include control sequences operably linked to the nucleic acids encoding the peptides.
  • control sequences is meant nucleic acid sequences necessary for expression of the subject peptides in a particular host organism.
  • control sequences include sequences required for transcription and translation of the nucleic acids, including, but not limited to, promoter sequences, enhancer or transcriptional activator sequences, ribosomal binding sites, transcriptional start and stop sequences; polyadenylation signals; etc.
  • promoters are useful in expressing the peptides of the present invention.
  • the promoters may be constitutive, inducible, and/or cell specific and may comprise natural promoters, synthetic promoters (e.g. tTA tetracycline inducible promoters), or hybrids of various promoters. Promoters are chosen based on, among others, the cell or organism in which the proteins are to be expressed, the level of desired expression, and regulation of expression.
  • Suitable promoters are bacterial promoters (e.g., pL I phage promoter, tac promoter, lac lac promoter, etc.); yeast based promoters (e.g., GAL4 promoter, alcohol dehydrogenase promoter, tryptophane synthase promoter, copper inducible CUPI promoter, etc.), plant promoters (e.g., CaMV S35, nopoline synthase promoter, tobacco mosaic virus promoter, etc), insect promoters (e.g., Autographa nuclear polyhedrosis virus, Aedes DNV viral p& and p61 , hsp70, etc.), and promoters for expression mammalian cells (e.g., ubiquitin gene promoter, ribosomal gene promoter, ⁇ -globin promoter, thymidine kinase promoter, heat shock protein promoters, and ribosomal gene promoters, etc.), and particularly viral promoters, such
  • operably linked herein is meant that a nucleic acid is placed into a functional relationship with another nucleic acid.
  • operably linked means that the control sequences are positioned relative to the nucleic acid sequence encoding the subject peptides in such a manner that expression of the encoded peptide occurs.
  • the vectors may comprise plasmids or comprise viral vectors, for example retroviral vectors, which are useful delivery systems if the cells are dividing cells, or lentiviral and adenoviral vectors if the cells are non-dividing cells.
  • retroviral vectors which have inactivated viral promoters at the 3'-LTR, thereby permiting control of expression of heterologous genes by use of non-viral promoters inserted into the viral vector
  • SIN vectors self- inactivating retroviral vectors
  • modifications of the system by pseudotyping allows use of retroviral vectors for all eukaryotic cells, particularly for higher eukaryotes (Morgan, R.A. et al. J. Virol. 67: 4712-21 (1993); Yang, Y. et al. Hum. Gene Ther. 6: 1203-13 (1995)).
  • the expression vectors also contain a selectable marker gene to allow selection of transformed host cells.
  • the selection will confer a detectable phenotype that enriches for cells containing the expression vector and further permits differentiation between cells that express and do not express the selection gene.
  • Selection genes are well known in the art and will vary with the host cell used. Suitable selection genes included genes that render the cell resistant to a drug, genes that permit growth in nutritionally deficient media, and reporter genes (e.g. ?-galactosidase, fluorescent proteins, glucouronidase, etc.), all of which are well known in the art and available to the skilled artisan.
  • nucleic acids there are a variety of techniques available for introducing nucleic acids into viable cells.
  • introduction into herein is meant that the nucleic acid enters the cells in a manner suitable for subsequent expression of the nucleic acid.
  • Techniques for introducing the nucleic acids will vary depending on whether the nucleic acid is transferred in vitro into cultured cells or in vivo into the cells of the intended host organism and the type of host organism.
  • Exemplary techniques for introducing the nucleic acids in vitro include the use of liposomes, Lipofectin®, electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, and biolistic particle bombardment.
  • Techniques for transfer in vivo include direct introduction of the nucleic acid, use of viral vectors, typically retroviral vectors, and liposome mediated transfection, such as viral coated liposome mediated transfection.
  • the nucleic acids expressing the peptides of the present invention may exist transiently or stably in the cytoplasm or stably integrate into the chromosome of the host (i.e., through use of standard regulatory sequences, selection markers, etc.). Suitable selection genes and marker genes are used in the expression vectors of the present invention.
  • an agent that targets the target cells or tissues such as an antibody specific for a cell surface protein or the target cell, a ligand for a receptor on the target cell, a lipid component on the cell membrane, or a carbohydrate on the cell surface.
  • proteins that bind a cell surface protein which is endocytosed may be used for targeting and/or facilitating uptake. These include as non-limiting examples, capsid proteins or fragments thereof tropic for a particular cell types, antibodies for proteins which undergo internalization (see Wu et al. J. Biol. Chem. 262: 4429-4432 (1987); Wagner et al. Proc. Natl. Acad. Sci. USA 87: 3410-3414 (1990)), and proteins that direct localization (e.g., antibody to transferrin receptor for targeting to brain) or enhance in vivo half-life.
  • Expression is done in a wide range of host cells that span prokaryotes and eukaryotes, including bacteria, yeast, plants, insects, and animals.
  • the peptides of the present invention may be expressed in, among others, E. coli., Saccharomyces cerevisiae, Saccharomyces pombe, Tobacco or Arabidopsis plants, insect Schneider cells, and mammalian cells, such as COS, CHO, HeLa, and the like, either intracellularly or in a secreted form by fusing the peptides to an appropriate signal peptide.
  • Secretion from the host cell may be done by fusing the DNA encoding the peptide and a DNA encoding a signal peptide.
  • Nucleic acids expressing the peptides may be inserted into cells, for example stem cells for tissue expression or bacteria for gut expression, and the cells transplanted into the host to provide an in vivo source of the peptides.
  • the RDP58, TCR and HLA peptides of the present invention may be purified or isolated after synthesis or expression.
  • purified or “isolated” is meant free from the environment in which the peptide is synthesized or expressed and in a form where it can be practically used.
  • purified or isolated is meant that the peptide or its derivative is substantially pure, i.e., more than 90% pure, preferably more than 95% pure, and preferably more than 99% pure.
  • the peptides and derivatives thereof may be purified and isolated by way known to those skilled in the art, depending on other components present in the sample.
  • Standard purification methods include electrophoretic, immunological, and chromatographic techniques, including ion exchange, hydrophobic, affinity, size exclusion, reverse phase HPLC, and chromatofocusing.
  • the proteins may also be purified by selective solubility, for instance in the presence of salts or organic solvents. The degree of purification necessary will vary depending on use of the subject peptides. Thus, in some instances no purification will be necessary.
  • compositions used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and usually at least about 99.5% by weight, relative to contaminants related to the method of product preparation, the purification procedure, and its intended use, for example with a pharmaceutical carrier for the purposes of therapeutic treatment. Usually, the percentages will be based upon total protein.
  • Pharmaceutical Formulations, Dosage Forms, Dosages, and Methods of Administration will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and usually at least about 99.5% by weight, relative to contaminants related to the method of product preparation, the purification procedure, and its intended use, for example with a pharmaceutical carrier for the purposes of therapeutic treatment. Usually, the percentages will be based upon total protein.
  • compositions may be used in vitro, ex vivo, and in vivo depending on the particular application.
  • the present invention provides for administering a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a pharmacologically effective amount of one or more of the subject peptides, or suitable salts thereof.
  • the pharmaceutical composition may be formulated as powders, granules, solutions, suspensions, aerosols, solids, pills, tablets, capsules, gels, topical cremes, suppositories, transdermal patches, etc.
  • pharmaceutically acceptable salts of the peptides is intended to include any art recognized pharmaceutically acceptable salts including organic and inorganic acids and/or bases.
  • examples of salts include sodium, potassium, lithium, ammonium, calcium, as well as primary, secondary, and tertiary amines, esters of lower hydrocarbons, such as methyl, ethyl, and propyl.
  • Other salts include organic acids, such as acetic acid, propionic acid, pyruvic acid, maleic acid, succinic acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, salicylic acid, etc.
  • pharmaceutically acceptable carrier comprises any of standard pharmaceutically accepted carriers known to those of ordinary skill in the art in formulating pharmaceutical compositions.
  • the subject peptides by themselves, such as being present as pharmaceutically acceptable salts, or as conjugates, or nucleic acid vehicles encoding such peptides, may be prepared as formulations in pharmaceutically acceptable diluents; for example, saline, phosphate buffer saline (PBS), aqueous ethanol, or solutions of glucose, mannitol, dextran, propylene glycol, oils (e.g., vegetable oils, animal oils, synthetic oils, etc.), microcrystalline cellulose, carboxymethyl cellulose, hydroxylpropyl methyl cellulose, magnesium stearate, calcium phosphate, gelatin, polysorbate 80 or the like, or as solid formulations in appropriate excipients.
  • PBS phosphate buffer saline
  • oils e.g., vegetable oils, animal oils, synthetic oils, etc.
  • microcrystalline cellulose carboxymethyl cellulose,
  • the formulations may include bactericidal agents, stabilizers, buffers, emulsifiers, preservatives, sweetening agents, lubricants, or the like. If administration is by oral route, the oligopeptides may be protected from degradation by using a suitable enteric coating, or by other suitable protective means, for example internment in a polymer matrix such as microparticles or pH sensitive hydrogels.
  • Suitable formulations may be found in, among others, Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Co., Philadelphia, PA, 1985 and Handbook of Pharmceutical Excipients, 3rd Ed, Kibbe, A.H. ed., Washington DC, American Pharmaceutical Association, 2000; hereby incorporated by reference in their entirety.
  • the pharmaceutical compositions described herein can be made in a manner well known to those skilled in the art (e.g., by means conventional in the art, including mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes).
  • the peptides may also be introduced or encapsulated into the lumen of liposomes for delivery and for extending life time of the peptide formulations ex vivo or in vivo.
  • liposomes can be categorized into various types: multilamellar (MLV), stable plurilamellar (SPLV), small unilamellar (SUV) or large unilamellar (LUV) vesicles.
  • Liposomes can be prepared from various lipid compounds, which may be synthetic or naturally occurring, including phosphatidyl ethers and esters, such as phosphotidylserine, phosphotidylcholine, phosphatidyl ethanolamine, phosphatidylinositol, dimyristoylphosphatidylcholine; steroids such as cholesterol; cerebrosides; sphingomyelin; glycerolipids; and other lipids (see for example, U.S. Patent No. 5,833,948).
  • phosphatidyl ethers and esters such as phosphotidylserine, phosphotidylcholine, phosphatidyl ethanolamine, phosphatidylinositol, dimyristoylphosphatidylcholine; steroids such as cholesterol; cerebrosides; sphingomyelin; glycerolipids; and other lipids (see for example, U.
  • Cationic lipids are also suitable for forming liposomes.
  • the cationic lipids have a net positive charge and have a lipophilic portion, such as a sterol or an acyl or diacyl side chain.
  • the head group is positively charged.
  • Typical cationic lipids include 1 ,2-dioleyloxy-3- (trimethylamino)propane; N-[1-(2,3,-ditetradecycloxy)propyl]-N,N-dimethyl-N-N- hydroxyethylammonium bromide; N-[1-(2,3-dioleyloxy)propyl]-N,N-dimethyl-N-hydroxy ethylammonium bromide; N-[1-(2,3-dioleyloxy) propyl]-N,N,N-trimethylammonium chloride; 3-[N- (N'N'-dimethylaminoethane) carbamoyl] cholesterol; and dimethyldioctadecylammonium.
  • fusogenic liposomes which are characterized by their ability to fuse with a cell membrane upon appropriate change in physiological condition or by presence of fusogenic component, particularly a fusogenic peptide or protein.
  • the fusogenic liposomes are pH and temperature sensitive in that fusion with a cell membrane is affected by change in temperature and/or pH (see for example, U.S. Patent No. 4,789,633 and 4,873,089).
  • pH sensitive liposomes are acid sensitive.
  • fusion is enhanced in physiological environments where the pH is mildly acidic, for example the environment of a lysosome, endosome and inflammatory tissues. This property allows direct release of the liposome contents into the intracellular environment following endocytosis of liposomes (see Mizoue, T. Int. J. Pharm. 237: 129-137 (2002)).
  • fusogenic liposomes comprise liposomes that contain a fusion enhancing agent. That is, when incorporated into the liposome or attached to the lipids, the agents enhance fusion of the liposome with other cellular membranes, thus resulting in delivery of the liposome contents into the cell.
  • the agents may be fusion enhancing peptides or proteins, including hemaggulutinin HA2 of influenza virus (Schoen, P. Gene Ther. 6: 823-832 (1999)); Sendai virus envelope glycoproteins (Mizuguchi, H. Biochem. Biophys. Res. Commun.
  • VSV-G vesicular stomatitis virus envelope glycoproteins
  • ADe vesicular stomatitis virus envelope glycoproteins
  • peptide segments or mimics of fusion enhancing proteins and synthetic fusion enhancing peptides (Kono, K. et al. Biochim. Biophys. Acta. 1164: 81-90 (1993); Pecheur, E.I. Biochemistry 37: 2361-71 (1998); U.S. Patent No. 6,372,720).
  • Liposomes also include vesicles derivatized with a hydrophilic polymer, as provided in U.S. Patent No. 5,013,556 and 5,395,619, hereby incorporated by reference, (see also, Kono, K. et al. J. Controlled Release 68: 225-35 (2000); Zalipsky, S. et al. Bioconjug. Chem. 6: 705-708 (1995)) to extend the circulation lifetime in vivo.
  • Hydrophilic polymers for coating or derivation of the liposomes include polyethylene glycol, polyvinylpyrrolidone, polyvinylmethyl ether, polyaspartamide, hydroxymethyl cellulose, hydroxyethyl cellulose, and the like.
  • attaching proteins that bind a cell surface protein which is endocytosed e.g., capsid proteins or fragments thereof tropic for a particular cell types and antibodies for cell surface proteins which undergo intern alization (see Wu et al, supra; Wagner et al., supra), may be used for targeting and/or facilitating uptake of the liposomes to specific cells or tissues.
  • Liposomes are prepared by ways well known in the art (see for example, Szoka, F. et al. Ann. Rev. Biophys. Bioeng. 9: 467-508 (1980)).
  • One typical method is the lipid film hydration technique in which lipid components are mixed in an organic solvent followed by evaporation of the solvent to generate a lipid film. Hydration of the film in aqueous buffer solution, preferably containing the subject peptide or nucleic acid, results in an emulsion, which is sonicated or extruded to reduce the size and polydispersity.
  • Other methods include reverse-phase evaporation (see Pidgeon, C. et al. Biochemistry 26: 17-29 (1987); Duzgunes, N. et al. Biochim. Biophys. Acta. 732: 289-99 (1983)), freezing and thawing of phospholipid mixtures, and ether infusion.
  • the carriers are in the form of microparticles, microcapsules, micropheres and nanoparticles, which may be biodegradable or non-biodegradable (see for example, Microencapsulates: Methods and Industrial Applications, Drugs and Phamaceutical Sciences, Vol 73, Benita, S. ed, Marcel Dekker Inc., New York, 1996; incorporated by reference).
  • microparticles, microspheres, microcapsules and nanoparticles mean a particle, which is typically a solid, containing the substance to be delivered. The substance is within the core of the particle or attached to the particle's polymer network.
  • microparticles or microcapsules or microspheres
  • nanoparticles are one of size.
  • microparticles have a particle size range of about 1 to about >1000 microns.
  • Nanoparticles have a particle size range of about 10 to about 1000 nm.
  • Non-biodegradable microcapsules and microparticles include, but not limited to, those made of polysulfones, poly(acrylonitrile-co-vinyl chloride), ethylene-vinyl acetate, hydroxyethylmethacrylate-methyl-methacrylate copolymers. These are useful for implantation purposes where the encapsulated peptide diffuses out from the capsules.
  • the microcapsules and microparticles are based on biodegradable polymers, preferably those that display low toxicity and are well tolerated by the immune system.
  • Biodegradable synthetic polymers for encapsulating may comprise polymers such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly(caprolactone), polydioxanone trimethylene carbonate, polyhybroxyalkonates (e.g., poly( ?-hydroxybutyrate)), poly( -ethy!
  • PLA polylactide
  • PGA polyglycolide
  • PLGA poly(lactide-co-glycolide)
  • poly(caprolactone) polydioxanone trimethylene carbonate
  • polyhybroxyalkonates e.g., poly( ?-hydroxybutyrate)
  • microparticles containing the subject compositions include solvent removal process (see for example, U.S. Patent No. 4,389,330); emulsification and evaporation (Maysinger, D. et al. Exp. Neuro. 141 : 47-56 (1996); Jeffrey, H. et al. Pharm. Res. 10: 362-68 (1993)), spray drying, and extrusion methods.
  • nanoparticles which are generally suitable for intravenous administrations.
  • Submicron and nanoparticles are generally made from amphiphilic diblock, triblock, or multiblock copolymers as is known in the art.
  • Polymers useful in forming nanoparticles include, but are limited to, poly(lactic acid) (PLA; see Zambaux et al., J.
  • Nanoparticles may be also be made from poly(alkylcyanoacrylate), for example poly(butylcyanoacrylate), in which the peptide is absorbed onto the nanoparticles and coated with surfactants (e.g., polysorbate 80).
  • surfactants e.g., polysorbate 80.
  • Methods for making nanoparticles are similar to those for making microparticles and include, among others, emulsion polymerization in continuous aqueous phase, emulsification-evaporation, solvent displacement, and emulsification-diffusion techniques (see Kreuter, J. Nano-particle Preparation and Applications, In Microcapsules and nanoparticles in medicine and pharmacy," (M. Donbrow, ed.), pg. 125-148, CRC Press, Boca Rotan, FL, 1991 ; incorporated by reference).
  • Hydrogels are also useful in delivering the subject agents into a host.
  • hydrogels are crosslinked, hydrophilic polymer networks permeable to a wide variety of drug compounds, including peptides.
  • Hydrogels have the advantage of selective trigger of polymer swelling, which results in controlled release of the entrapped drug compound.
  • swelling and subsequent release may be triggered by a variety of stimuli, including pH, ionic strength, thermal, electrical, ultrasound, and enzyme activities.
  • Non-limiting examples of polymers useful in hydrogel compositions include, among others, those formed from polymers of poly(lactide- co-glycolide), poly(N-isopropylacrylamide); poly(methacrylic acid-g-polyethylene glycol); polyacrylic acid and poly(oxypropylene-co-oxyethylene) glycol; and natural compounds such as chrondroitan sulfate, chitosan, gelatin, or mixtures of synthetic and natural polymers, for example chitosan-poly(ethylene oxide).
  • the polymers are crosslinked reversibly or irreversibly to form gels embedded with the oligopeptides of the present invention (see for example, U.S. Patent No.
  • the gel polymers are acrylic acid polymers, preferably carbomers (e.g., carboxypolymethylene), such as Carbopol (e.g., Carbopol 420-430, 475, 488, 493, 910, 934P, 974P, and the like; Brock et al., Pharmacotherapy 14: 430-437 (1994)), which are nonlinear polymers of acrylic acid crosslinked with polyalkenyl polyether.
  • carbomers include acrylic acids crosslinked with polyfunctional compounds, such as polyallysucrose.
  • carbomer gels are mucoadhesive.
  • concentrations of the peptides or nucleic acid encoding therefore will be determined empirically in accordance with conventional procedures for the particular purpose.
  • the subject formulations are given at a pharmacologically effective dose.
  • pharmaceutically effective amount or “pharmacologically effective dose” is an amount sufficient to produce the desired physiological effect or amount capable of achieving the desired result, particularly for treating the disorder or disease condition, including reducing or eliminating one or more symptoms of the disorder or disease.
  • the amount administered to the host will vary depending upon what is being administered, the purpose of the administration, such as prophylaxis or therapy, the state of the host, the manner of administration, the number of administrations, interval between administrations, and the like. These can be determined empirically by those skilled in the art and may be adjusted for the extent of the therapeutic response. Factors to consider in determining an appropriate dose include, but are not limited to, size and weight of the subject, the age and sex of the subject, the severity of the symptom, the stage of the disease, method of delivery of the agent, half-life of the agents, and efficacy of the agents. Stage of the disease to consider include whether the disease is acute or chronic, relapsing or remitting phase, and the progressiveness of the disease. Determining the dosages and times of administration for a therapeutically effective amount are well within the skill of the ordinary person in the art.
  • the toxicity and therapeutic efficacy are generally determined by cell culture assays and/or experimental animals, typically by determining a LD50 (lethal dose to 50% of the test population) and ED50 (therapeutically effectiveness in 50% of the test population).
  • the dose ratio of toxicity and therapeutic effectiveness is the therapeutic index.
  • Preferred are compositions, individually or in combination, exhibiting high therapeutic indices. Determination of the effective amount is well within the skill of those in the art, particularly given the detailed disclosure provided herein.
  • the present invention provides for a bolus or infusion of the subject composition that will be administered in the range of about 0.1-50, more usually from about 1-25 mg/kg body weight of host.
  • the amount will generally be adjusted depending upon the half-life of the peptide where the half life will generally be at least one minute, more usually at least about 10 min, desirably in the range of about 10 min to 12 h. Short half-lives are acceptable, so long as efficacy can be achieved with individual dosages, continuous infusion, or repetitive dosages.
  • Formulations for administration may be presented in unit a dosage form, e.g., in ampules, capsules, pills, or in multidose containers or injectables.
  • Dosages in the lower portion of the range and even lower dosages may be employed, where the peptide has an enhanced half-life or is provided as a depot, such as a slow release composition comprising particles, a polymer matrix which maintains the peptide over an extended period of time (e.g., a collagen matrix, carbomer, etc.), use of a pump which continuously infuses the peptide over an extended period of time with a substantially continuous rate, or the like.
  • the host or subject may be any mammal including domestic animals, pets, laboratory animals, primates, particularly humans subjects.
  • nucleic acid molecules (DNA or RNA) encoding the subject peptides may also be administered thereto, thereby providing an effective source of the subject peptides for the application desired.
  • nucleic acid molecules encoding the subject peptides may be cloned into any of a number of well known expression plasmids (see Sambrook et al., supra) and/or viral vectors, preferably adenoviral or retroviral vectors (see for example, Jacobs et al., J. Virol.
  • nucleic acid-based vehicles may be administered directly to the cells or tissues ex vivo (e.g., ex vivo viral infection of cells for transplant of peptide producing cells) or to a desired site in vivo, e.g. by injection, catheter, orally (e.g., hybrogels), and the like, or, in the case of viral-based vectors, by systemic administration.
  • ex vivo e.g., ex vivo viral infection of cells for transplant of peptide producing cells
  • a desired site in vivo e.g. by injection, catheter, orally (e.g., hybrogels), and the like, or, in the case of viral-based vectors, by systemic administration.
  • Tissue specific promoters may optionally be employed, assuring that the peptide of interest is expressed only in a particular tissue or cell type of choice.
  • Methods for recombinantly preparing such nucleic acid-based vehicles are well known in the art, as are techniques for administering nucleic acid-based vehicles for peptide production.
  • the methods of administration is chosen depending on the condition being treated, the form of the subject compositions, and the pharmaceutical composition.
  • Administration of the oligopeptides can be done in a variety of ways, including, but not limited to, cutaneously, subcutaneously, intravenously, orally, topically, transdermally, intraperitoneally, intramuscularly, nasally, and rectally (e.g., colonic administration).
  • microparticle, microsphere, and microencapsulate formulations are useful for oral, intramuscular, or subcutaneous administrations.
  • Liposomes and nanoparticles are additionally suitable for intravenous administrations.
  • Administration of the pharmaceutical compositions may be through a single route or concurrently by several routes. For instance, oral administration can be accompanied by rectal or topical administration to the affected area. Alternatively, oral administration is used in conjunction with intravenous or parenteral injections.
  • the delivery systems also include sustained release or long term delivery methods, which are well known to those skilled in the art.
  • sustained release or long term release
  • long term release systems may comprise implantable solids or gels containing the subject peptide, such as biodegradable polymers described above; pumps, including peristaltic pumps and fluorocarbon propellant pumps; osmotic and mini-osmotic pumps; and the like.
  • Peristaltic pumps deliver a set amount of drug with each activation of the pump, and the reservoir can be refilled, preferably percutaneously through a port.
  • a controller sets the dosage and can also provides a readout on dosage delivered, dosage remaining, and frequency of delivery.
  • Fluorocarbon propellant pumps utilize a fluorocarbon liquid to operate the pump. The fluorocarbon liquid exerts a vapor pressure above atmospheric pressure and compresses a chamber containing the drug to release the drug.
  • Osmotic pumps (and mini-osmotic pumps) utilize osmotic pressure to release the drug at a constant rate. The drug is contained in an impermeable diaphragm, which is surrounded by the osmotic agent.
  • a semipermeable membrane contains the osmotic agent, and the entire pump is housed in a casing. Diffusion of water through the semipermeable membrane squeezes the diaphragm holding the drug, forcing the drug into bloodstream, organ, or tissue.
  • systemic e.g., intravenous or subcutaneous
  • localized doses e.g., intracerebroventricular
  • the method of administration is by oral delivery, in the form of a powder, tablet, pill, or capsule.
  • Pharmaceutical formulations for oral administration may be made by combining one or more peptide with suitable excipients, such as sugars (e.g., lactose, sucrose, mannitol, or sorbitol), cellulose (e.g., starch, methyl cellulose, hydroxylmethyl cellulose, carbonxymethyl cellulose, etc.), gelatin, glycine, saccharin, magnesium carbonate, calcium carbonate, polymers such as polyethylene glycol or polyvinylpyrrolidone, and the like.
  • suitable excipients such as sugars (e.g., lactose, sucrose, mannitol, or sorbitol), cellulose (e.g., starch, methyl cellulose, hydroxylmethyl cellulose, carbonxymethyl cellulose, etc.), gelatin, glycine, saccharin, magnesium carbonate, calcium carbonate, polymers such as poly
  • the pills, tablets, or capsules may have an enteric coating, which remains intact in the stomach but dissolves in the intestine.
  • enteric coating are known in the art, a number of which are commercially available, including, but not limited to, methacrylic acid-methacrylic acid ester copolymers, polymer cellulose ether, cellulose acetate phathalate, polyvinyl acetate phthalate, hydroxypropyl methyl cellulose phthalate, and the like.
  • oral formulations of the peptides are in prepared in a suitable diluent.
  • Suitable diluents include various liquid form (e.g., syrups, slurries, suspensions, etc.) in aqueous diluents such as water, saline, phosphate buffered saline, aqueous ethanol, solutions of sugars (e.g. sucrose, mannitol, or sorbitol), glycerol, aqueous suspensions of gelatin, methyl cellulose, hydroxylmethyl cellulose, cyclodextrins, and the like.
  • diluent or aqueous solutions also include infant formula.
  • lipohilic solvents are used, including oils, for instance vegetable oils, peanut oil, sesame oil, olive oil, corn oil, safflower oil, soybean oil, etc.); fatty acid esters, such as oleates, triglycerides, etc.; cholesterol derivatives, including cholesterol oleate, cholesterol linoleate, cholesterol myristilate, etc.; liposomes; and the like.
  • oils for instance vegetable oils, peanut oil, sesame oil, olive oil, corn oil, safflower oil, soybean oil, etc.
  • fatty acid esters such as oleates, triglycerides, etc.
  • cholesterol derivatives including cholesterol oleate, cholesterol linoleate, cholesterol myristilate, etc.
  • liposomes and the like.
  • administration is done rectally.
  • This may use formulations suitable for topical application in the form of salves, tinctures, cremes, or for application into the lumen of the intestine by use of compositions in the form of suppositories, enemas, foams, etc.
  • Suppositories may contain conventional suppository bases such as cocoa butter, carbowaxes, polyethylene glycols, or glycerides, which are solid or semi-solid at room temperature but liquid at body temperature.
  • the administration is carried out cutaneously, subcutaneously, intraperitonealy, intramuscularly or intravenously.
  • these are in the form of peptides dissolved or suspended in suitable aqueous medium, as discussed above.
  • the pharmaceutical compositions for injection may be prepared in lipophilic solvents, which include, but is not limited to, oils, such as vegetable oils, olive oil, peanut oil, palm oil soybean oil, safflower oil, etc; synthetic fatty acid esters, such as ethyl oleate or triglycerides; cholesterol derivatives, including cholesterol oleate, cholesterol linoleate, cholesterol myristilate, etc.; or liposomes, as described above.
  • compositions may be prepared directly in the lipophilic solvent or preferably, as oil/water emulsions, (see for example, Liu, F. et al. Pharm. Res. 12: 1060-1064 (1995); Prankerd, R.J. J. Parent. Sci. Tech. 44: 139-49 (1990); U.S. Patent No. 5,651 ,991 ).
  • the delivery systems also include sustained release or long term delivery methods, which are well known to those skilled in the art.
  • sustained release or long term release
  • long term release systems may comprise implantable solids or gels containing the subject peptide, such as biodegradable polymers described above; pumps, including peristaltic pumps and fluorocarbon propellant pumps; osmotic and mini-osmotic pumps; and the like.
  • Peristaltic pumps deliver a set amount of drug with each activation of the pump, and the reservoir can be refilled, preferably percutaneously through a port.
  • a controller sets the dosage and can also provides a readout on dosage delivered, dosage remaining, and frequency of delivery.
  • Fluorocarbon propellant pumps utilize a fluorocarbon liquid to operate the pump. The fluorocarbon liquid exerts a vapor pressure above atmospheric pressure and compresses a chamber containing the drug to release the drug.
  • Osmotic pumps (and mini-osmotic pumps) utilize osmotic pressure to release the drug at a constant rate. The drug is contained in an impermeable diaphragm, which is surrounded by the osmotic agent.
  • a semipermeable membrane contains the osmotic agent, and the entire pump is housed in a casing. Diffusion of water through the semipermeable membrane squeezes the diaphragm holding the drug, forcing the drug into bloodstream, organ, or tissue.
  • These and other such implants are particularly useful in treating an inflammatory disease condition, especially those manifesting recurring episodes or which are progressive in nature, by delivering the oligopeptides of the invention via systemic (e.g., intravenous or subcutaneous) or localized doses in a sustained, long term manner.
  • kits or packaged formulations as used herein includes one or more dosages of a subject peptide, and salts thereof, in a container holding the dosages together with instructions for simultaneous or sequential administration to a patient.
  • the package may contain the peptides along with a pharmaceutical carrier combined in the form of a powder for mixing in an aqueous solution, which can be ingested by the afflicted subject.
  • a pharmaceutical carrier combined in the form of a powder for mixing in an aqueous solution, which can be ingested by the afflicted subject.
  • Another example of packaged drug is a preloaded pressure syringe, so that the compositions may be delivered colonically.
  • the package or kit includes appropriate instructions, which encompasses diagrams, recordings (e.g., audio, video, compact disc), and computer programs providing directions for use of the combination therapy.
  • appropriate instructions which encompasses diagrams, recordings (e.g., audio, video, compact disc), and computer programs providing directions for use of the combination therapy.
  • GROUP 1 not treated
  • GROUP 2 5 ⁇ l of RDP58 (10mg/ml dissolved in water) was applied directly into the gingival sulcus of both left and right second maxillary molars once at the time of the ligature.
  • GROUP 3 5 ⁇ l of RDP58 (10mg/ml dissolved in 50% DMSO) was applied directly into the gingival sulcus of both left and right second maxillary molars once at the time of ligature.
  • GROUP 4 5 ⁇ l of RDP58 (10mg/ml dissolved in water) was applied directly into the gingival sulcus of both left and right second maxillary molars once per week.
  • GROUP 5 5 ⁇ l of RDP58 (10mg/ml dissolved in 50% DMSO) was applied directly into the gingival sulcus of both left and right second maxillary molars once per week.
  • GROUP 6 5 ⁇ l of RDP58 (10mg/ml dissolved in water) was applied directly into the gingival sulcus of both left and right second maxillary molars twice per week.
  • GROUP 7 5 ⁇ l of RDP58 (10mg/ml dissolved in 50% DMSO) was applied directly into the gingival sulcus of both left and right second maxillary molars twice per week.
  • Bone resorption was also quantified at 7 days and 28 days post ligature application.
  • TUNEL assay was also done on samples taken 3 days and 7 days post ligature application to examine apoptosis.
  • Anti-rat MMP9 antibody was also used with samples taken 3 days and 7 days post ligature application to examine MMP9 expression in tissue.
  • Staining with anti-CD45 antibody demonstrated a lack of leukocyte infiltration at the surface of the alveolar bone in untreated healthy rat periodontium. Dense infiltration both at the surface of the alveolar bone and throughout the bone was observed after application of the LPS-soaked ligature for 48 hours. RDP58 administration reduced leukocyte infiltration induced by the LPS-soaked ligature.
  • Bone loss as measured at day 7 and day 28 after LPS-ligature application. The bone loss was determined as the distance between the cemento-enamel junction and the alveolar bone. Statistically significant bone loss (p ⁇ 0.001) was observed 28 days post LPS treatment. Significant inhibition of bone loss with RDP58 was observed 28 days post LPS treatment.
  • TUNEL assay was done on sections taken on day 3 and day 7 post application of the LPS ligature.
  • LPS dramatically increased apoptosis as compared to control RDP58 reduced LPS- induced apoptosis to near control level.
  • PBMCs peripheral blood mononuclear cells
  • RAW cells murine macrophage cell line
  • PBMCs treated with LPS resorbed more bone that untreated PBMCs.
  • PBMCs treated with LPS resorbed less bone in the presence of RDP58 than in the absence of RDP58.
  • PBMCs treated with TNF- ⁇ resorbed more bone that untreated PBMCs.
  • Untreated RAW cells resorbed less bone in the presence of RDP58 than in the absence of RDP58.
  • RAW cells treated with LPS resorbed more bone that untreated RAW cells.
  • RAW cells treated with LPS resorbed less bone in the presence of RDP58 than in the absence of RDP58.
  • RAW cells treated with TNF- ⁇ resorbed more bone that untreated RAW cells.
  • RAW cells treated with RANKL resorbed more bone that untreated RAW cells.
  • RAW cells treated with RANKL resorbed less bone in the presence of RDP58 than in the absence of RDP58.
  • THP1 cells human monocyte cell line
  • THP1 cells matured to macrophages with 100 pg/ml IFNj/ for 24 hours after which the resultant cells were incubated with LPS, TNF ⁇ , or RNAKL in the presence or absence of RDP58 for 7 days.
  • Cells were then stained for Tartrate Resistant Acid Phosphatase (TRAP) activity using a TRAP Assay kit from Sigma.
  • the TRAP positive cells are mature osteoclasts.
  • the TRAP assay measures the conversion of p-nitrophenylphosphate to p-nitrophenol in the presence of 80 mM sodium tartrate which occurs during a 3-5 minute incubation at room temperature. The reaction is terminated by the addition of NaOH to a final concentration of 0.5 M.
  • Increased numbers of TRAP positive cells were generated in RANKL-treated THP1 cell cultures as compared to untreated THP1 cell cultures. In cultures of RANKL-treated THP1 cells, increased numbers of TRAP positive cells were generated in the absence of RDP58 as compared to the presence of RDP58.
  • PBMCs were incubated for 14 days in the presence or absence of RDP58 after which the cells were stained for alkaline phosphatase (AP) activity using an AP Assay kit from Sigma. AP activity served as an osteoblast marker.
  • AP activity served as an osteoblast marker.
  • Increased numbers of AP positive cells were generated in vitamin D3-treated PBMC cultures as compared to untreated PBMC cultures. In cultures of vitamin D3-treated PBMCs, increased numbers of AP positive cells were generated in the presence of RDP58 as compared to the absence of RDP58.
  • Increased numbers of AP positive cells were generated in vitamin D3-treated THP1 cell cultures as compared to untreated THP1 cell cultures. In cultures of vitamin D3-treated THP1 cells, increased numbers of AP positive cells were generated in the presence of RDP58 as compared to the absence of RDP58.
  • PBMCs were incubated on slides for 14 days in the presence or absence RDP58 after which the slides were stained for bone deposition using AgN0 3 using the von Kossa method.
  • Murine macrophage cell line, RAW264.7, and human macrophage cell line, THP1 were maintained in DMEM (Invitrogen Corporation, Carlsbad, CA) and RPMI respectively (ATCC, Manassas, VA). Cells were seeded at 5 X 10 6 in 20 ml media per 75cm 2 tissue culture flask and cultured overnight. Cells were stimulated with LPS (1 mg/ml; Sigma-Aldrich, St. Louis, MO) and/or RDP58 (50 ⁇ M) for 4 hours. After stimulation, cell culture media was collected for the measurement of TNF- ⁇ .
  • LPS 1 mg/ml; Sigma-Aldrich, St. Louis, MO
  • RDP58 50 ⁇ M
  • Microarray analysis was performed by the Genomic Core Laboratory at the J. David Gladstone Institute, San Francisco, CA, under a laboratory services contract. Briefly, the quality of the RNA was evaluated on Bioanlyzer (Agilent Technologies, Palo Alto, CA), and double stranded cDNA was prepared followed by an in vitro transcription using T7 RNA polymerase. The resulting cRNA was fragmented and hybridized initially to Affymetrix TestChips (Affimatrix Inc. Santa Clara, CA) to assess the quality of the cRNA and then to Affymetrix U74A GeneChips (Affimatrix Inc. Santa Clara, CA). Data analysis was performed in-house using GeneSpring software (Silicon Genetics, Redwood City, CA).
  • RNA (5 ⁇ g) was treated with DNasel (Invitrogen Corporation, Carlsbad, CA), and cDNA synthesis was performed using Superscript II RT kit (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer's recommendations.
  • Primers for PCR were designed using Primer Express software (Applied Biosystems, Foster City, CA).
  • Real-time PCR was performed using Sybergreen PCR Master Mix (Applied Biosystems, Foster City, CA) and GeneAmp 5700 sequence detection system (Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation for 10 min at 94°C followed by 40 cycles consisting of 15 sec at 94°C and 1 min at 60°C. To achieve the relative abundance, BMP2 and CBFA1 mRNA levels were normalized to that of HPRT.
  • Equal amounts of total protein from THP1 cell lysates were separated on 10% Tris-Glycine gels (Invitrogen Corporation, Carlsbad, CA), transferred to Immobilon-P (Millipore, Bedford, MA) and immunoblotted with anti-BMP2A antibodies (Aviva Antibody Corporation, San Diego, CA; Santa Cruz Biotechnology Inc., Santa Cruz, CA) according to standard protocols.
  • RDP58 increased steady state mRNA levels of multiple bone morphogenetic proteins (BMPs).
  • BMPs bone morphogenetic proteins
  • Murine macrophage cell line, RAW264.7 was stimulated with LPS and/or RDP58 for 4 and 24 hours.
  • Cellular RNA was isolated and mRNA levels of BMPs were analyzed by cDNA microarray.
  • steady state mRNA levels of BMP2A and BMP11 were about 7-fold and about 3- fold higher in RDP58 treated cells respectively.
  • RDP58 by itself caused about a 20-fold and about a 4.6-fold increase in BMP2A mRNA levels in RAW264J and THP1 cells respectively.
  • LPS stimulation also caused a comparable increase in BMP2A mRNA levels and at least in RAW264.7 cells, LPS and RDP58 appeared to synergize.
  • RDP58 treatment also resulted in an increase of mRNA levels of BMP4/2B, BMP6, BMP8B, and BMP10 by 1.6-fold or more.
  • RDP58 increased BMP2A protein levels.
  • Human macrophage cell line, THP1 was stimulated with LPS and/or RDP58 for 4 and 24 hours. Cells were lysed in NP40 lysis buffer and analyzed by western blotting with anti-BMP2 antibodies. BMP2A protein was not detectable in control THP1 cells. Although LPS induced the steady state levels of BMP2A mRNA, the protein levels remained undetectable at 4 and 24 hours. In contrast, RDP58 by itself increased the steady state levels of BMP2A protein in THP1 cells consistent with the RDP58-mediated increase in BMP2A mRNA levels in the same cells.
  • RDP58 increased mRNA steady state levels of multiple BMP2A/2B responsive genes including the osteoblast-specific transcription factor, Cbfal .
  • cDNA microaray analysis revealed that RDP58 induced the steady state mRNA levels of a number of known BMP2A responsive genes by about 2-fold or more in RAW264J cells. These included Dlx3, Akp2, Osteocalcin, Orm2, and the osteobast-specific transcription factor, Cbfal .
  • RDP58 also induced the mRNA levels of BMP4/2B responsive gene, Msx1 by about 9-fold in RAW264.7 cells.
  • Cbfal mRNA levels were about 6-fold higher in RDP58 treated RAW264.7 cells as detected by real-time PCR.
  • NF- ⁇ B- and AP1-luciferase reporter constructs were used in hFOB cells (human pre- osteoblast cells) to assay NF- ⁇ B transcriptional activity and AP1 transcriptional activity, respectively. Assays were done for untreated cells, cells in mineralization medium, cells treated with vitamin D3, cells treated with varied amounts of RDP58, and cells in mineralization medium treated with varied amounts of RDP58. RDP58 treated cultures, at all RDP58 concentrations used, exhibited lower AP1 transcriptional activity and NF- ⁇ B transcriptional activity than untreated cultures, vitamin D3-treated cultures, and cultures in mineralization medium.
  • RDP58 was used at concentrations of 6.25 ⁇ M, 12.5 ⁇ M, 25 ⁇ M, and 50 ⁇ M.
  • hFOB Human pre-osteoblast cells
  • RDP58 at concentrations of 50 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, or 6.5 ⁇ M for 14 days.
  • cells were stained for alkaline phosphatase activity (marker of osteoblast differentiation) or with alizarin red (to visualize bone deposition).
  • Mineralization medium containing ascorbic acid and glycerophosphate
  • vitamin D3 stimulation of osteoblast differentiation were used as positive controls.
  • An Alzet osmotic pump was implanted subcutaneously in the back and delivered the treatment of choice (saline; RDP58, O. ⁇ mg.ml; RDP58, 1 mg/ml; RDP58, 2mg/ml) at the site of the defect for two weeks.
  • the rats were sacrificed at two weeks and histology was done on calvaria samples.
  • Samples were embedded in plastic (without decalcification), cut, and stained with H&E (to assess bone organization), Mason's trichrome (to distinguish new bone formation from cartilage and connective tissue filling the defects), and von Kossa (to assess the mineralization status of new bone).
  • the saline treated control animals showed the beginning of bone formation within the connective tissue filling the defect.
  • the new bone formed pockets within the connective tissue and was disorganized and filled less than 50% of the defects.
  • Animals treated with RDP58 at 0.5mg/ml demonstrated new bone formation comparable to the saline treated group.
  • Animals treated with RDP58 at 1.Omg/ml and 2.0mg/ml demonstrated enhanced new bone formation, with more organized lamellar bone filling more than 80% of the defects.
  • von Kossa staining revealed the new bone of animals treated with RDP58 at 1.Omg/ml and 2.0mg/ml was well mineralized.
  • RDP58 inhibits induction of MMP9 and MMP2.
  • Tumor cell lines are cultured in MMP9 and/or MMP2 inducing media in the presence or absence of RDP58.
  • Protein samples are prepared from the cultures and run for western blot analysis with anti-MMP9 and anti- MMP2 antibodies. Results show that RDP58 inhibits MMP9 and MMP2 induction in tumor cells.

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Abstract

L'invention porte sur des compositions et sur des procédés permettant de réduire l'activité des ostéoclastes et d'augmenter celle des ostéoblastes, ceux-ci étant utiles dans le traitement de diverses pathologies liées à la perte osseuse, ces procédés visant également à favoriser la régénération osseuse.
PCT/US2004/015490 2003-05-15 2004-05-17 Compositions rdp58 et procedes de modulation de l'activite des osteoclastes et des osteoblastes WO2005001075A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104086626A (zh) * 2014-07-14 2014-10-08 上海苏豪逸明制药有限公司 一种固相多肽合成抗炎十肽的制备方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046633A1 (fr) * 1997-04-11 1998-10-22 Sangstat Medical Corporation Cytomodulation de peptides lipophiles aux fins d'une modulation de l'activite du systeme immunitaire et de la suppression de l'inflammation
WO2002046364A2 (fr) * 2000-12-04 2002-06-13 Quark Biotech, Inc. Genes associes au stress mecanique, produits d'expression de ces genes et utilisation de ces genes et de leurs produits d'expression

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998046633A1 (fr) * 1997-04-11 1998-10-22 Sangstat Medical Corporation Cytomodulation de peptides lipophiles aux fins d'une modulation de l'activite du systeme immunitaire et de la suppression de l'inflammation
WO2002046364A2 (fr) * 2000-12-04 2002-06-13 Quark Biotech, Inc. Genes associes au stress mecanique, produits d'expression de ces genes et utilisation de ces genes et de leurs produits d'expression

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BOURREILLE A ET AL: "RDP58, a novel immunomodulatory peptide with anti-inflammatory effects. A pharmacological study in trinitrobenzene sulphonic acid colitis and Crohn disease", SCANDINAVIAN JOURNAL OF GASTROENTEROLOGY 01 MAY 2003 NORWAY, vol. 38, no. 5, 1 May 2003 (2003-05-01), pages 526 - 532, XP009038147, ISSN: 0036-5521 *
IYER SUHASINI ET AL: "Inhibition of tumor necrosis factor mRNA translation by a rationally designed immunomodulatory peptide", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 275, no. 22, 2 June 2000 (2000-06-02), pages 17051 - 17057, XP002300806, ISSN: 0021-9258 *
IYER SUHASINI ET AL: "Rational design and development of RDP58", CURRENT PHARMACEUTICAL DESIGN, vol. 8, no. 24, 2002, pages 2217 - 2229, XP009038144, ISSN: 1381-6128 *
LAZAROV M ET AL: "RDP58, a rationally designed peptide, inhibits inflammation and bone resorption in a rat model of periodontitis.", INFLAMMATION RESEARCH, vol. 52, no. Supplement 2, July 2003 (2003-07-01), & 6TH WORLD CONGRESS ON INFLAMMATION; VANCOUVER, BRITISH COLUMBIA, CANADA; AUGUST 02-06, 2003, pages S 157, XP009038148, ISSN: 1023-3830 *
SANGSTAT: "RDP58, A Rationally Designed Peptide", 15 April 2003 (2003-04-15), XP002300612, Retrieved from the Internet <URL:www.sangstat.com/docs/4-15-03_RDP_Event_3.pdf> [retrieved on 20041013] *
THEILL LARS E ET AL: "RANK-L and RANK: T cells, bone loss, and mammalian evolution.", ANNUAL REVIEW OF IMMUNOLOGY. 2002, vol. 20, 2002, pages 795 - 823, XP002300613, ISSN: 0732-0582 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104086626A (zh) * 2014-07-14 2014-10-08 上海苏豪逸明制药有限公司 一种固相多肽合成抗炎十肽的制备方法
CN104086626B (zh) * 2014-07-14 2016-10-19 上海苏豪逸明制药有限公司 一种固相多肽合成抗炎十肽的制备方法

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