WO2009025651A1 - Peptide biologiquement actif et procédé d'utilisation correspondant - Google Patents

Peptide biologiquement actif et procédé d'utilisation correspondant Download PDF

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Publication number
WO2009025651A1
WO2009025651A1 PCT/US2007/023020 US2007023020W WO2009025651A1 WO 2009025651 A1 WO2009025651 A1 WO 2009025651A1 US 2007023020 W US2007023020 W US 2007023020W WO 2009025651 A1 WO2009025651 A1 WO 2009025651A1
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seq
peptide
amino acid
acid sequence
mammal
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PCT/US2007/023020
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English (en)
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Anja Nohe
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University Of Maine System Board Of Trustees
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the present invention relates to a peptide having a novel function. More particularly, the present invention relates to a peptide that is capable of regulating osteogenesis and a method for using the peptide to regulate osteogenesis. Even more particularly, the present invention relates to a peptide that is capable of promoting osteogenic cell proliferation and a method of using the peptide for promoting osteogenic cell proliferation.
  • the peptide and method of the present invention therefore are useful for treating bone disorders, such as osteoporosis and Paget's disease, for example.
  • BMPs Bone Morphogenetic Proteins
  • reagents that regulate BMP function and expression might be potent for treating bone disorders because BMPs influence multipotent mesenchymal cells to differentiate into chondrocytes, osteoblasts, myocytes, and adipocytes, and because BMPs are crucial for skeletal development and homeostasis, as they play a role in osteoblast and osteoclast differentiation. It is therefore not surprising that abnormally functioning BMPs and reduced BMP levels are characteristic of several bone disorders, including osteoporosis and Paget's disease.
  • reagents that effect normal or enhanced functioning and expression of BMPs likely would be therapeutic for disorders other than those that affect bone.
  • a reagent that promotes functioning of BMPs may prevent failure or correct abnormalities of these organs and their processes.
  • BMP signal transduction involves binding of the cognate ligand to a combination of BMP receptors (BMPRs) on the plasma membrane.
  • BMP receptors BMP receptors
  • BMP type-I (BRIa) and type-II (BRII) receptors which also are known as activin receptor-like kinases (ALKs), aggregate and cluster in specific domains on the cell surface, but that these receptors also must shuttle between distinct membrane domains in order to transduce their signals. Interdomain shuttling influences BMP signaling and regulates the early steps of osteoblast differentiation.
  • CK-2 casein kinase-2
  • Ser/Thr kinase that is typically found in tetrameric complexes consisting of two catalytic (alpha and/or alpha') subunits and two regulatory beta subunits
  • Canton et ai Cell Signal (2006), vol. 18, pp. 267-275
  • phosphorylation of tyrosine in in vitro studies
  • the present inventor has also determined that bone marrow stromal cell (BMSC) mineralization is enhanced when normal binding of CK-2 to BMPRs is altered.
  • BMSC bone marrow stromal cell
  • the present inventor further has determined that normal binding of CK-2 to BRIa can be altered by peptide. It is the alteration to normal binding of CK-2 to BMPRs that forms the basis of the present invention.
  • the present invention is a peptide having a novel function, which optionally may be combined in a composition with one or more other peptides and/or with other organic or inorganic compounds, and a method of using the peptide and/or composition to regulate a bodily process in a mammal, including a human.
  • the peptide of the present invention is capable of altering the normal interaction between CK-2 and BRIa. Therefore, the peptide may be used to regulate any bodily process that involves, but does not necessarily require, normal interaction between CK-2 and BRIa.
  • the peptide of the present invention is capable of altering the normal interaction of CK-2 with BRIa, and therefore the peptide is capable of enhancing BMSC mineralization and osteogenic cell proliferation.
  • the peptide of the present invention has an amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 as described herein.
  • the peptide has an amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 and an amino acid signal sequence linked thereto the sequence represented by SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
  • the peptide is a conservative variant of the amino acid sequence represented by SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
  • the peptide is a conservative variant of the amino acid sequence represented by SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 and an amino acid signal sequence linked thereto the sequence represented by SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
  • the method of the present invention may be used to regulate a bodily process of a mammal, such as for the purpose of treating that mammal, for example.
  • the method of the present invention is used to treat a human having a bone disorder, such as osteoporosis or Paget' s disease, for example, and the method includes administering to the human one or more therapeutically effective doses of the peptide of the present invention.
  • a bone disorder such as osteoporosis or Paget' s disease
  • FIG. 1 is a table showing the alignment of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, and SEQ ID NO: 13.
  • FIG. 2 is a graph showing the effects of three examples of the peptide (each one having sequence corresponding to either SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3) of the present invention on Fluorescence Resonance Energy Transfer (FRET) efficiency between BRIa and CK-2.
  • FRET Fluorescence Resonance Energy Transfer
  • FIG. 3 is a graph showing the effects that altering the normal interaction between CK-2 and BRIa by using three examples of the peptide (each one having sequence corresponding to either SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3) of the present invention has on BMP-2-independent signaling.
  • the "ALK portion" of the peptide of the present invention is any amino acid sequence that is homologous to a mammalian ALK protein amino acid sequence.
  • the ALK portions of the peptides represented by SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 are the amino acid sequences corresponding to SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.
  • amino acid signal sequence or, alternatively, “signal sequence” is any sequence of amino acids that allows a cell to take within its plasma membrane, including into its nucleus or cytoplasm, any peptide having that sequence that is outside the plasma membrane.
  • a “conservative variant” is any peptide that is able to alter the interaction between CK-2 and BRIa and that has a single amino acid substitution, or a plurality of amino acid substitutions, such as, for example, two or three amino acid substitutions, to the ALK portion of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 as described herein.
  • a "conservative variant” includes any peptide that is able to alter the interaction between CK-2 and BRIa and that has an amino acid sequence having a small insertion or deletion of amino acids, such as an insertion or deletion of one-to-three amino acids, for example, to the ALK portion of the amino acid sequence represented by SEQ ID NO: 1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
  • a conservative variant therefore may be a peptide having amino acid sequence corresponding to SEQ ID NO: 14 through and including SEQ ID NO:25, as described herein, and no other amino acid sequence corresponding to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
  • a "therapeutically effective dose” is any amount of the peptide that is sufficient to effect regulation of a bodily process of a mammal for the purpose of aiding the treatment of that mammal.
  • CK-2 as a protein that interacts with BMPRs, such as BRIa, for example, in mammal. Briefly, the present inventor has performed FRET assays (see the Example section included herein), immunoprecipitation assays, and Western assays, and, based on observations made in these studies, has determined that CK-2, including its ⁇ , of, and ⁇ isoforms, co-localizes and interacts with BRIa.
  • the present inventor further has performed a motif search using seven human ALK proteins (the amino acid sequences of which are shown in FIG. 1) using publicly available human protein sequence (available at https://www.ncbi.nlm.nih.gov).
  • these seven human ALK proteins are ALK-I , ALK-2, ALK-3, ALK-6, ALK-4, ALK-5, and ALK-7, the amino acid sequences of which correspond to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, and SEQ ID NO: 13, respectively.
  • the present inventor has identified a plurality of consensus CK-2 recognition sites responsible for CK-2 phosphorylation of BRIa (these sites are shown in the three boxed in regions of the ALK protein amino acid sequences of FIG. 1). Further, the present inventor has determined that the normal interaction between CK-2 and BRIa can be altered by peptide including one of these recognition sites (a specific example of a method for altering the interaction between CK-2 and BRIa by peptide is described in the Example section included herein).
  • the present invention includes one or more peptides capable of regulating a mammalian bodily process and a method for using these peptides to regulate a mammalian bodily process.
  • the peptide of the invention includes one of the CK-2 recognition sites (shown within the boxed areas of FIG. 1) of seven human ALK proteins, represented by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 1 1, SEQ ID NO: 12, and SEQ ID NO: 13.
  • the peptide of the present invention is capable of altering the normal interaction between CK-2 and BRIa for the purpose of regulating the mammalian bodily process.
  • the present invention therefore is useful for regulating, for example, osteogenesis.
  • the present invention is useful for regulating osteogenesis, it is useful for treating bone disorders, such as osteoporosis and Paget's disease.
  • bone disorders such as osteoporosis and Paget's disease.
  • the peptide and method of the present invention are not limited to being used to regulate osteogenesis or to treat bone disorders.
  • the peptide of the present invention includes at least four amino acid residues.
  • Amino acid residues are abbreviated as follows: Alanine is A or Ala; Arginine is R or Arg; Asparagine is N or Asn; Aspartic Acid is D or Asp; Cysteine is C or Cys; Glutamic Acid is E or GIu; Glutamine is Q or GIn; Glycine is G or GIy; Histidine is H or His; Isoleucine is I or He; Leucine is L or Leu; Lysine is K or Lys; Methionine is M or Met; Phenylalanine is F or Phe; Proline is P or Pro; Serine is S or Ser; Threonine is T or Thr; Tryptophan is W or Tip; Tyrosine is Y or Tyr; and Valine is V or VaI.
  • X or Xaa represents any amino acid.
  • Other relevant amino acids include, but are not limited to, 4-hydroxyproline and 5-hydroxylysine.
  • the amino acid sequence of the peptide of the present invention is presented in conventional form in that the left-most amino acid residue of the sequence is the N-terminal residue and the right-most amino acid residue of the sequence is the carboxy-terminal residue.
  • the peptide of the present invention may have one of the following three amino acid sequences, but is not limited to having one of the following three amino acid sequences corresponding to SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
  • Each one of the peptides having amino acid sequence corresponding to SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 contains the Antennapedia (AP) homeodomain signal sequence, RQIKIWFQNRRMKWKK, or what is also known as the "penetratin sequence".
  • This penetratin sequence allows the peptides having SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 to penetrate biological membranes, such as to allow uptake of the peptide including the penetratin sequence by a cell into its cytoplasm or nucleus, for example.
  • amino acid sequence following the penetratin sequence in each of SEQ ID NO: 1 and SEQ ID NO:2, YHEMGSLYD YLQL and YVPNDPSFEDM, respectively, corresponds to partial amino acid sequence from the human ALK-2 protein (SEQ ID NO: 8), and amino acid sequence following the penetratin sequence in SEQ ID NO:3, which specifically is IPVGESLKDLIDQ, corresponds to partial amino acid sequence from the human ALK-3 protein (SEQ ID NO:9).
  • the ALK portions of SEQ ID NO: 1 , SEQ ID NO:2, or SEQ ID NO:3 include one of a plurality of consensus CK-2 recognition sites that were identified in motif searches for such sequences. As shown in the boxed in regions of FIG.
  • the consensus CK-2 recognition sequences from the seven human ALK proteins reviewed in the motif search are TMLGD (SEQ ID NO: 14), STLAD (SEQ ID NO: 15), SLKD (SEQ ID NO: 16), SLRD (SEQ ID NO: 17), TLQD (SEQ ID NO: 18), TLKD (SEQ ID NO: 19), SLYD (SEQ ID NO:20), SLFD (SEQ ID NO:21), SFED (SEQ ID NO:22), SYED (SEQ ID NO:23), SIEE (SEQ ID NO:24), and SVEE (SEQ ID NO:25).
  • SEQ ID NO: 14 corresponds to amino acid sequence from SEQ ID NO:7;
  • SEQ ID NO: 15 corresponds to amino acid sequence from SEQ ID NO:8;
  • SEQ ID NO: 16 corresponds to amino acid sequence from SEQ ID NO:9;
  • SEQ ID NO: 17 corresponds to amino acid sequence from SEQ ID NO: 12;
  • SEQ ID NO: 18 corresponds to amino acid sequence from SEQ ID NO: 10;
  • SEQ ID NO: 19 corresponds to amino acid sequence from SEQ ID NO: 1 1 and SEQ ID NO: 13;
  • SEQ ID NO:20 corresponds to amino acid sequence from SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 12, and SEQ ID NO: 13;
  • SEQ ID NO:21 corresponds to amino acid sequence from SEQ ID NO: 10 and SEQ ID NO: 1 1 ;
  • SEQ ID NO:22 corresponds to amino acid sequence from SEQ ID NO:7 and SEQ ID NO:8;
  • the peptide of the present invention is not limited to having an amino acid sequence that is identical to either SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.
  • the peptide of the present invention therefore includes other peptides having ALK amino acid sequence that are capable of altering the normal interaction between CK-2 and one or more BMPRs.
  • the peptide may have only the amino acid sequence YHEMGSLYDYLQL (SEQ ID NO:4), YVPNDPSFEDM (SEQ ID NO:5), or IPVGESLKDLIDQ (SEQ ID NO:6). Further, the peptide may have the amino acid sequence corresponding to SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 and an amino acid signal sequence other than the penetratin sequence linked thereto for allowing the peptide to cross a biological membrane.
  • peptide signal sequences such as TAT, transportan and polyarginine, for example, exist and would be suitable for allowing any peptide, including the peptide having SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6, to penetrate a biological membrane. See, e.g., Deshayes, Cell MoI. Life ScL 62: 1839-49 (2005); and Jones, Br. J. Pharmacol. 145: 1093-1102 (2005); Saalik, Bioconjug. Chem. 15: 1246-53 (2004), each of which is incorporated herein by reference in its entirety.
  • the ALK portion of the peptide of the present invention also is not limited to including an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9.
  • the amino acid sequence of the peptide therefore, may correspond to an amino acid sequence from another ALK protein, such as those represented by SEQ ID NO:7, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13, for example.
  • the ALK portion of the peptide also may be only the amino acid sequence corresponding to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, which are the CK-2 recognition sites identified by the present inventor.
  • the peptide also may include the amino acid sequence corresponding to SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21 , SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, or SEQ ID NO:25, and additional amino acid that does not correspond to ALK sequence.
  • This additional amino acid sequence may be, but is not limited to being, cellular uptake signal sequence, such as the pentratin sequence, for example. [0411
  • the amino acid sequence of the peptide may be a conservative variant of the amino acid sequence represented by SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
  • the peptide having SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, or a conservative variation thereof may include additional amino acid sequence.
  • This sequence may be very long, and therefore may include up to hundreds of amino acids, and may lie between the signal sequence or the ALK portion of the peptide, or it may immediately flank either the signal sequence or the ALK sequence or both. Therefore, the peptide of the present invention may contain more than or fewer than the number of amino acids present in SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
  • the ALK portion of the sequence of the peptide may be selected based on the species of mammal in which the bodily process is to be regulated.
  • the peptide of the present invention may include all or part of the amino acid sequence of any of the mouse, rat, canine, feline, equine, bovine, chimpanzee, ape, or monkey homologs of the ALK protein sequences shown in FIG. 1. This would be useful, for example, where the peptide is to be used to alter the interaction of CK-2 and BRIa to regulate oseteogenesis in a pet mammal, such as a pet dog or cat, for example for the purpose of treating a bone disorder in that pet mammal. This also would be useful, for example, where the peptide is to be used to conduct studies in an experimental laboratory mammal, such as a mouse, rat, or chimpanzee, for example.
  • the peptide of the present invention may be naturally occurring or artificially synthesized.
  • Several methods for synthesizing peptides are presented in, e.g., Pennington and Dunn, Peptide Synthesis Protocols (Methods in Molecular Biology), Humana Press, Totowa, New Jersey (1994), which is entirely incorporated by reference herein.
  • the peptide may be artificially synthesized, for example, by using standard solid-phase peptide synthesis (SPPS) methods, such as those described by, e.g., Nelson et al., Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, Academic Press, New York (1997), which is entirely incorporated by reference herein.
  • SPPS solid-phase peptide synthesis
  • the peptide may be purified.
  • the peptide when the peptide is to be purified, the peptide may be purified to be at least 95% free of compounds that are not the peptide, and more preferably, at least 98% free of compounds that are not the peptide.
  • Methods of purifying peptides are well understood to those of ordinary skill in the art and are described in Pennington and Dunn, Peptide Synthesis Protocols (Methods in Molecular Biology), Humana Press, Totowa, New Jersey (1994) and in Nelson et al., Methods in Enzymology, Volume 289: Solid-Phase Peptide Synthesis, Academic Press, New York (1997).
  • the peptide alternatively may be artificially synthesized by introducing DNA which encodes the peptide into a cell, such as a mammalian cell, for example, in a manner that enables the peptide to be expressed in the cell. After being expressed in engineered cells, the peptide may be isolated from the cells and purified. Methods for artificially engineering a cell to express a peptide and for isolating and purifying such expressed peptides are well understood to those of ordinary skill in the art. See, e.g., S. C. Makrides, Gene Transfer and Expression in Mammalian Cells (New Comprehensive Biochemistry), Elsevier Science, Saint Louis, Missouri (2004).
  • the one or more peptides of the present invention may be part of a composition including one or more other compounds.
  • this composition may include the peptide of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or the peptide of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 which also includes a signal sequence that is not the penetratin sequence, and one or more other compounds.
  • the one or more other compounds preferably are biologically benign, and therefore may be an inert solid carrier or polymer matrix material which is capable of helping to deliver and localize the peptide to bone, for example. Such a solid carrier or polymer matrix material may, for example, allow for the timed-release of the peptide.
  • U.S. Pat. No. 4,536,489 which is entirely incorporated herein by reference, describes a biodegradable organic polymer delivery system for bone morphogenetic protein which is capable of inducing formation of new bone in viable tissue.
  • U.S. Pat. No. 5,286,763 which is entirely incorporated herein by reference, describes bioerodible polymers for drug delivery in bone, including polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, and copolymers thereof, that may be used for the delivery of bioactive agents, including antibiotics, chemotherapeutic agents, inhibitors of angiogenesis, and simulators of bone growth, directly into bone.
  • the peptide of the present invention may be included as part of these systems, or systems similar thereto, for delivery of the peptide to bone.
  • Other compounds which may be included as part of a matrix mixture for delivering the peptide to bone include celluloses, polysaccharides, starches, agarose, hyaluronic acid, hyaluron polyethylene glycol, fibrin, elastin, collagen, gelatin, vitronectin, fibronectin, laminin, dextrans, alginates, chitin, chitosan, and glycosaminoglycans.
  • the peptide of the present invention also may be included in a composition having one or more other compounds that have therapeutic properties. Therefore, the peptide may be included in a composition further containing a growth factor, such as, but not being limited to, transforming growth factor- ⁇ , transforming growth factor- ⁇ , interleukin-1, and/or nerve growth factor, for example. The peptide may also be included in a composition that also includes antibacterial and/or antifungal agents.
  • a growth factor such as, but not being limited to, transforming growth factor- ⁇ , transforming growth factor- ⁇ , interleukin-1, and/or nerve growth factor, for example.
  • the peptide may also be included in a composition that also includes antibacterial and/or antifungal agents.
  • the peptide of the present invention also may be included in a composition that has one or more inorganic compounds.
  • inorganic compounds include, but are not limited to, those that may be used as part of implantable grafts, such as ceramic materials, titanium mesh, and hydroxyapatite, for example.
  • composition may include two or more peptides, such as those having SEQ ID NO: 1 and SEQ ID NO:3, for example, and one or more compounds as described above, or it may consist of only two or more peptides, such as those having SEQ ID NO: 1 and SEQ ID NO:3, and no other compound.
  • the method of the present invention includes using the peptide or the composition of the present invention to regulate a bodily process in either a whole mammal, such as a human, for example, or a living sample obtained therefrom, such as a human cell line, for example.
  • the bodily process may be, for example, osteogenesis, but it is to be understood that it is not limited thereto.
  • the bodily process therefore may be any bodily process that is capable of being regulated, partially or wholly, by alteration of normal interaction between CK-2 and one or more BMPRs, such as BRIa, for example, by using the peptide of the present invention.
  • the bodily process is not limited to being used to regulate a bodily process associated with any particular bone or the skeletal system as a whole, the bodily process regulated by the method of the present invention may be associated with another organ system or region of a mammal's body.
  • the bodily process may be one which occurs in, and/or is associated with the brain, pituitary, thyroid, heart, liver, lung, pancreas, colon, adrenal glands, kidney, muscle, blood vessel, skin, testis and/or ovary.
  • the bodily process may also be an abnormal bodily process, such as cancer, for example.
  • the method may be used to alter interaction of CK- 2 with one or more BMPRs, such as BRIa, for example, in all types of cancerous cells in vivo or in vitro. It is to be understood, however, that these are meant only to be examples, and that the present invention is not limited to these examples.
  • the method is used to regulate osteogenesis in a mammal and includes the step of administering one or more therapeutically effective doses of the peptide or composition having the peptide of the present invention to the mammal.
  • the mammal may be, for example, a human patient having a bone disorder, such as osteoporosis or Paget's disease, for example.
  • the step of introducing the peptide into the mammal is variable.
  • the peptide may be artificially purified in vitro and then the therapeutically effective dose may be, for example, subcutaneously injected into a patient at or near the area in need of treatment.
  • the therapeutically effective dose may be administered to a human patient by adding the peptide to an inert solid carrier or polymer matrix material which would help localize the peptide to bone in need of repair.
  • this might be achieved by using the peptide in conjunction with the delivery systems described in U.S. Patent Nos. 4,536,489 and 5,286,763.
  • the solid carrier or polymer matrix material may allow for the timed release of the peptide.
  • Another means for administering the therapeutically effective dose to the human patient is to include the peptide as part of an implantable graft and then implanting the graft into the human patient at or near the area that is in need of treatment, such as a weakened bone, for example.
  • the graft including the peptide may be wrapped around bones of the spine after those bones have been subjected to a spinal fusion procedure.
  • the peptide further may be synthesized within the mammal that is to be treated by the peptide.
  • the peptide may be encoded for by a DNA construct and the DNA construct may be expressed in a human cell line.
  • the human cell line such as an osteoblast cell line, for example, then may be administered to a human patient having a bone disorder, such as osteoporosis, for example. Accordingly, the cell line administered to the human synthesizes the peptide in an amount sufficient to provide the therapeutically effective dose.
  • Example One observation of normal interaction between BRIa and CK-2 and the experimental alteration of said interaction is described.
  • C2C12 cells were subcloned from a myoblast line established from normal adult C3H mouse leg muscle referred to as C2C12 cells. These cells were cultured in Dulbecco's modified Eagle medium (DMEM) with the addition of 10% fetal bovine serum (FBS) in a humidified 5% CO 2 incubator at 37°C. Antibiotics penicillin (50 IU/mL) and streptomycin (100 ⁇ g/mL) were added to the media to control growth of bacterial and fungal contamination. Cells were subcultured approximately twice a week when the confluency was at 60-70% (they were split at a 1: 10 ratio at these times into 25 cm 2 tissue culture flasks). Prior to being used in the described experiments, these cells were seeded into tissue culture dishes (Falcon 3001 35 x 10mm) containing a 25 x 25 mm glass coverslip (Fisher Scientific) in 2 ml culture medium.
  • tissue culture dishes Falcon 3001 35 x 10mm
  • FBS
  • Each one of the amino acids sequences of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3 includes the AP homeodomain signal sequence for cellular uptake.
  • the AP homeodomain signal sequence is a 16-amino acid polypeptide that has been shown to be able to cross cellular membranes with attached biologically active compounds.
  • the AP homeodomain signal sequence has direct interaction with the membrane phosopholipids without any specific binding location identified, making it ideal for transport into the cell cytoplasm.
  • the AP homeodomain signal sequence is a functional vector that allows the direct transport of polypeptides across the membrane.
  • a goat monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), at a concentration of 50 ⁇ g/ml was used in conjunction with donkey anti-goat AF568 (Molecular Probes, Inc., Eugene, OR) at a concentration of 5 ⁇ g/ml.
  • Immunostaining of CK-2 was achieved using a rabbit monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), at a concentration of 50 ⁇ g/ml, in conjunction with goat anti-rabbit AF488 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). All antibodies were exposed to their protein of interest for 30 minutes.
  • FRET Fluorescence Reassisted laser desorption spectroscopy
  • Periasamy et al. Molecular Imaging: FRET Microscopy and Spectroscopy, Academic Press (2006); Goldman et al., Live Cell Imaging, Cold Spring Harbor Laboratory Press (2005); Spector et al., Basic Methods in Microscopy: Protocols And Concepts from Cells, a Laboratory Manual, Cold Spring Harbor Laboratory Press (2005), each one of which is incorporated by reference herein in its entirety.
  • FRET was conducted using a multi-line Argon (457nm, 488nm, 514nm) laser and Green Helium Neon (543nm) laser. All images were recorded using an Olympus Fluoview FV300 inverted laser scanning confocal microscope (Olympus, Center Valley, PA). Labeled cell imaging was performed using a 6OX objective viewing cells. An FITC filter was used for visualization of the green and TRITC filter for red fluorescence. Images were recorded using Fluoview 5.0 software (Olympus, Center Valley, PA) and cropped and assembled with ImageJ software (available at: https://rsb.info.nih.gov/ij/download.html). For each experimental coverslip, 38 images were taken in total.
  • Example Two effects on BMP independent signaling caused by the alteration of the normal interaction between CK-2 and BRIa is described.
  • C2C12 cells were cultured in DMEM with 10% FBS in a humidified 5% CO2 incubator at 37 0 C.
  • Antibiotics penicillin (50 IU/mL) and streptomycin (100 ⁇ g/mL) were added to the media to control growth of bacterial and fungal contamination.
  • Cells were subcultured approximately twice a week when the confluency was at 60-70% (they were split at a 1 : 10 ratio at these times into 25 cm 2 tissue culture flasks). Prior to being used in the reporter gene assays described below, these cells were seeded into tissue culture dishes (Falcon 3001 35 x 10mm) containing a 25 x 25 mm glass coverslip (Fisher Scientific) in 2 ml culture medium.
  • C2C12 cells were transfected with the reporter gene pSBE according to protocols described in A. Nohe et al., Journal of Cell Science 116: 3277-3284 (2003), which is incorporated herein by reference in its entirety.
  • Peptide having sequence corresponding to either SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 was added at a concentration of about 10 ⁇ M to separate populations of cells and allowed to co-incubate with the cells overnight at 37 °C.
  • Cells were then stimulated or not stimulated with about 20 nM BMP-2 in DMEM according to protocols described in A. Nohe et al., Journal of Cell Science 1 16: 3277-3284 (2003).
  • Fig. 3 The results of these reporter gene assays is shown in Fig. 3. Specifically, Fig.
  • FIG. 3 shows the amount of luciferase activity that was measured for the unstimulated or BMP-2 stimulated cell lines that either were not co-transfected with a peptide ("control") or were co-transfected with peptide having amino acid sequence corresponding to either SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.
  • BSMCs were isolated from 8-week old C57BL/6 (B6) mice and cultured for seven days.
  • a peptide having a sequence corresponding to either SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3 was "exposed to" the BSMCs by adding the peptide at a concentration of about 10 ⁇ M to separate cultures of BSMCs and allowing the peptide and BMSCs to co-incubate for 48 hours at 37 °C.
  • the peptide of the present invention is able to induce nodule formation in BMSCs within 48 hours. This indicates that the peptide of the present invention may be used to induce osteoblast differentiation of BSMCs by altering the normal interaction between CK-2 and BRIa.

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Abstract

La présente invention concerne un peptide, une composition comprenant ce peptide et un procédé d'utilisation du peptide et la composition le comprenant. La présente invention peut être utilisée pour réguler l'ostéogenèse ou autre processus corporel chez un mammifère tel que, par exemple, l'être humain. La présente invention peut par conséquent être utilisée chez un mammifère pour traiter une maladie des os telle que l'ostéoporose ou la maladie de Paget.
PCT/US2007/023020 2007-08-17 2007-10-30 Peptide biologiquement actif et procédé d'utilisation correspondant WO2009025651A1 (fr)

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