WO2011073447A1 - Emd c-dépleté - Google Patents

Emd c-dépleté Download PDF

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WO2011073447A1
WO2011073447A1 PCT/EP2010/070255 EP2010070255W WO2011073447A1 WO 2011073447 A1 WO2011073447 A1 WO 2011073447A1 EP 2010070255 W EP2010070255 W EP 2010070255W WO 2011073447 A1 WO2011073447 A1 WO 2011073447A1
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proteins
emd
tissue
pharmaceutical
dental
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PCT/EP2010/070255
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WO2011073447A9 (fr
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Aaldert Molenberg
Ruzica Ranevski
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Straumann Holding Ag
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Publication of WO2011073447A9 publication Critical patent/WO2011073447A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses

Definitions

  • the present invention relates to an improved pharmaceutical, dental and/or cosmetic composition
  • an improved pharmaceutical, dental and/or cosmetic composition comprising purified Enamel Matrix Derivative (EMD) proteins, which is substantially depleted of any Enamel Matrix Derivative (EMD) proteins below 6kDa.
  • the invention relates to an improved pharmaceutical, dental and/or cosmetic composition consisting of purified Enamel Matrix Derivative (EMD) proteins which have a molecular weight as determined by SDS PAGE electrophoresis substantially equal and/or above 6kDa, formulated in a suitable pharmaceutical carrier.
  • Said improved composition is herein preferably intended to be used for treating and/or preventing periodontitis, promoting and/or inducing regeneration of hard tissue, tissue mineralization, bone growth and/or bone regrowth, regeneration of dentin, cementogenesis, and/or binding between parts of living mineralized tissue, for bonding of a piece of living mineralized tissue to a bonding site on a piece of other living tissue, for endorsing binding between hard tissues, and/or for filling a mineralized wound cavity and/or tissue defect following from a procedure and/or trauma.
  • the present invention in particular relates to the use of said improved pharmaceutical, dental and/or cosmetic composition of purified Enamel Matrix Derivative (EMD) proteins which have a molecular weight as determined by SDS PAGE electrophoresis substantially equal and/or above 6kDa for promoting, and/or inducing mineralized tissue healing and bone differentiation
  • EMD Enamel Matrix Derivative
  • osteogenesis through regulating osteoblast differentiation.
  • the present invention further relates to the use of said pharmaceutical, dental and/or cosmetic composition of purified Enamel Matrix Derivative (EMD) proteins which have a molecular weight as determined by SDS PAGE electrophoresis substantially equal and/or above 6kDa as a medicament and/or for the manufacture of a pharmaceutical composition for a variety of different medical and/or dental indications, such as for promoting and/or inducing regeneration of hard tissue, tissue mineralization, bone growth and/or bone regrowth, regeneration of dentin, cementogenesis, and/or binding between parts of living mineralized tissue, for bonding of a piece of living mineralized tissue to a bonding site on a piece of other living tissue, for endorsing binding between hard tissues, and/or for filling a mineralized wound cavity and/or tissue defect following from a procedure and/or trauma.
  • EMD Enamel Matrix Derivative
  • Enamel matrix proteins, present in the enamel matrix, are most well-known as precursors to enamel. Prior to cementum formation, enamel matrix proteins are deposited on the root surface at the apical end of the developing tooth-root. There is evidence that the deposited enamel matrix is the initiating factor for the formation of cementum. Again, the formation of cementum in itself is associated with the development of the periodontal ligament and the alveolar bone. Enamel matrix proteins can therefore promote periodontal regeneration through mimicking the natural attachment development in the tooth (Gestrelius S, Lyngstadaas SP, Hammarstrom L. Emdogain - periodontal regeneration based on biomimicry. Clin Oral Invest 4: 120-125 (2000).
  • Isolated enamel matrix proteins are able to induce not only one, but an orchestrated cascade of factors, naturally found in tissues developing adjacent to the enamel matrix. They mimic the natural environment of a developing tissue and thus mimic a natural stimulation for tissue regeneration, cell differentiation and/or maturation.
  • EMD Enamel Matrix Derivative
  • HPLC High Pressure Liquid Chromatography
  • the fraction C typically has a molecular weight of between approximately 3.5 and 5kDa, such as approximately 5kDa, 4kDa and 3.5kDa, as determined by SDS PAGE electrophoresis.
  • the fraction A typically has a molecular weight of approximately 20kDa, as determined by SDS PAGE electrophoresis.
  • the fraction B typically has a molecular weight of between approximately 6kDa and 15kDa, such as approximately 15kDa, 12kDa, 10kDa and 6kDa, as determined by SDS PAGE electrophoresis.
  • Enamel matrix derivative in the form of a purified acid extract of proteins from pig enamel matrix, has previously been successfully employed to restore functional periodontal ligament, cementum and alveolar bone in patients with severe tooth attachment loss (Hammarstrom et al., 1997, Journal of Clinical Periodontology 24, 658-668).
  • EMD is composed of a number of proteins, such as amelogenins, enamelin, tuft protein, proteases, and albumin.
  • Amelogenins a major constituent of EMD, are a family of hydrophobic proteins derivable from a single gene by alternative splicing and controlled post secretory processing. They are highly conserved throughout vertebrate evolution and demonstrate a high overall level of sequence homology among all higher vertebrates examined (80%). In fact, the sequences of porcine and human amelogenin gene transcript differ only in 4% of the bases. Thus, enamel matrix proteins, although of porcine origin, are considered "self when encountered in the human body and can promote dental regeneration in humans without triggering allergic responses or other undesirable reactions.
  • enamel contains a complex of amelogenin proteins which includes components ranging in size from 3.5-25 kDa. This is due to the expression and secretion of a family of amelogenins derivable from multiple mRNAs generated by differential splicing from one or two copies of the amelogenin gene, located on the X and Y chromosome. What is more, subsequent to secretion, these proteins appear further to undergo extensive proteolytic processing.
  • LRAP leucine-rich amelogenin polypeptide
  • TRIP tyrosine-rich amelogenin polypeptide
  • TRIPs Two human tyrosine-rich amelogenin polypeptides (TRAPs) of approximately 5 kDa in size have prior been identified (see Fincham et al., 1989). These polypeptides were found to be of 42
  • TRAP-2 and 44 (TRAP-1 ) amino acid residues in length; two forms of TRAP molecules, differing only by cleavage of a carboxy-terminal dipeptide, which were described to be a general feature of human and other mammalian enamel proteins, probably being derived by postsecretory cleavage from the primary extracellular amelogenin.
  • the present invention for the first time presents an improved pharmaceutical, dental and/or cosmetic composition
  • an improved pharmaceutical, dental and/or cosmetic composition comprising purified Enamel Matrix Derivative (EMD) proteins, which is substantially depleted of any Enamel Matrix Derivative (EMD) proteins below 6kDa.
  • EMD Enamel Matrix Derivative
  • DISCLOSURE OF THE INVENTION The porcine Enamel Matrix Derivative (EMD) is used widely in clinical dentistry because of its ability to promote regeneration of soft and hard tissues and to reduce inflammation and infections. Previous studies have used indirect methods to explain its angiogenic and proliferative effect on cells associated with wound healing.
  • Emdogain® a product composed of an alginate carrier (Propylene Glycol Alginate) and porcine Enamel Matrix Derivative (EMD) is widely used in the treatment of periodontal diseases and has been shown to promote hard and soft tissue regeneration and decrease inflammation following periodontal surgery. Not surprisingly, it has been shown to contain a number of low molecular weight proteins (mainly amelogenins) which have been associated with cementogenesis and osteogenesis during tooth development.
  • EMD Enamel Matrix Derivative
  • amelogenin due to alternative splicing of the primary transcript and the following proteolytic processing of the secreted proteins, degrades into smaller pieces (fragments and polypeptide fragments), and these pieces are hypothesized to interact differentially with the surrounding tissue and promote serial steps in the development of the periodontal system.
  • fraction C is the component in EMD that acts on preosteoblasts and which has both osteogenic and cementogenic potential, thus suggesting that it is one of the active components of EMD and amelogenin with respect to bone and cementum regeneration.
  • the inventors have recently found that fraction C were upregulating activity of periodontal cells, and/or upregulating very early osteoblast differentiation and/or proliferation markers, as well as upregulating mesenchymal stem cell proliferation and/or reducing and/or inhibiting differentiation of mesenchymal stem cells.
  • fraction C was more strongly detected as a strong inducer or facilitator of proliferation of osteoblast or mesenchymal cells, whereas the effect on early differentiation, measured by the expression of marker genes in these cells, was rather negative.
  • Fraction C is thus found to be the earliest active fraction of EMD, comprising an assertion of components of EMD that will induce instant proliferating stimuli into the surrounding tissue, at an early stage prioritising the amassment of undifferentiated cells over the specification of them.
  • Fraction C clearly demonstrated a strong biological effect on the induction of proliferation in osteoblast like precursor cells as well as in PDL cells, and could even be shown to reduce the level of later differentiation markers.
  • EMD's long documented biological effect does on the other hand include not only induction of early proliferation stimuli, but is often shown to promote early, as well as terminal differentiation of bone cells.
  • fraction C has been shown to promote initial induction of osteoblast precursor proliferation
  • fraction A and B are likely candidates for signals inducing the terminal differentiation of the same cell population.
  • the present inventors have therefore isolated EMD which is substantially depleted of fraction C and shown that a combination of fraction A and B, comprising more or less only EMD proteins of at least 6kDa molecular weight, is more effective in promoting osteogenesis on its own than the complete EMD.
  • the present invention for the first time presents an improved pharmaceutical, dental and/or cosmetic composition
  • an improved pharmaceutical, dental and/or cosmetic composition comprising purified Enamel Matrix Derivative (EMD) proteins, which is substantially substantially depleted of any Enamel Matrix Derivative (EMD) proteins below 6kDa.
  • EMD Enamel Matrix Derivative
  • fraction A typically has a molecular weight of approximately 20kDa, as determined by SDS PAGE electrophoresis
  • fraction B typically has a molecular weight of between approximately 6kDa and 15kDa, such as approximately 15kDa, 12kDa, 10kDa and 6kDa, as determined by SDS PAGE electrophoresis.
  • the present invention relates to a pharmaceutical, dental and/or cosmetic composition
  • a pharmaceutical, dental and/or cosmetic composition comprising purified Enamel Matrix Derivative (EMD) proteins, wherein the weight ratio of the main protein peaks at 20/14/5 kDa of the enamel matrix proteins as determined by SDS PAGE electrophoresis is about x/y/z and wherein:
  • EMD Enamel Matrix Derivative
  • Another, equally preferred aspect relates to a pharmaceutical, dental and/or cosmetic composition consisting of a suitable pharmaceutical carrier and purified Enamel Matrix Derivative (EMD) proteins, wherein the weight ratio of the main protein peaks at 20/14/5 kDa of the enamel matrix proteins as determined by SDS PAGE electrophoresis is about x/y/z and wherein:
  • EMD Enamel Matrix Derivative
  • a pharmaceutical, dental and/or cosmetic composition according to the present invention is typically purified from porcine, rat, human, or mouse Enamel Matrix Derivative (EMD) proteins.
  • a pharmaceutical composition according to the present invention is envisioned to be usable as a medicament.
  • a pharmaceutical, dental and/or cosmetic composition according to the present invention provides an improved formulation for treating and/or preventing periodontitis, for promoting mineralized tissue healing and osteogenesis and/or for regulating osteoblast differentiation.
  • a pharmaceutical, dental and/or cosmetic composition provides an improved formulation for promoting and/or inducing regeneration of hard tissue, tissue mineralization, bone growth and/or bone regrowth, regeneration of dentin, cementogenesis, and/or binding between parts of living mineralized tissue, for bonding of a piece of living mineralized tissue to a bonding site on a piece of other living tissue, for endorsing binding between hard tissues, and/or for filling a mineralized wound cavity and/or tissue defect following from a procedure and/or trauma.
  • the present invention in one aspect also relates to the use of purified Enamel Matrix Derivative (EMD) proteins which have a molecular weight as determined by SDS PAGE electrophoresis substantially substantially equal and/or above 6kDa for the manufacture of a pharmaceutical, dental and/or cosmetic composition, for treating and/or preventing periodontitis, for promoting regeneration of hard tissues, for promoting and/or inducing regeneration of hard tissue, tissue mineralization, bone growth and/or bone regrowth, regeneration of dentin, cementogenesis, and/or binding between parts of living mineralized tissue, for bonding of a piece of living mineralized tissue to a bonding site on a piece of other living tissue, for endorsing binding between hard tissues, and/or for filling a mineralized wound cavity and/or tissue defect following from a procedure and/or trauma.
  • EMD Enamel Matrix Derivative
  • the invention in another aspect, relates to a method of promoting the formation and/or regeneration of dentin following dental procedures involving exposure of vital dental pulp tissue, the method comprising applying an effective amount of a pharmaceutical, dental and/or cosmetic composition according to the present invention.
  • a pharmaceutical, dental and/or cosmetic composition comprising purified Enamel Matrix Derivative (EMD) proteins, being consequently depleted of fraction C.
  • EMD Enamel Matrix Derivative
  • a pharmaceutical, dental and/or cosmetic composition according to the present invention is thus applied on exposed vital dental pulp tissue after dental procedures.
  • the present invention further relates to the use of a pharmaceutical, dental and/or cosmetic composition according to the present invention, for application on medical and/or dental implants or devices.
  • the invention also relates to medical and/or dental implants or devices on which a pharmaceutical, dental and/or cosmetic composition according to the present invention has been applied.
  • the implant or device may be any implant or device intended for use in the human or animal body, in particular in the dental area, gastrointestinal tract, urethra, bladder, pulmonary cavity, lungs, trachea, larynx, esophagus, joints, bone, skull, ears, sinuses, veins, arteries or abdominal cavity.
  • the implant or device may be used for fixation of complicated fractures, e.g. of the neck, legs or arms, or skull fractures, thus the implant or device may be a pin or screw conventionally used to immobilize (fix) fragments of fractured bone.
  • Such pins or screws typically comprise a portion that penetrates the skin of the patient at or near the site of the fracture.
  • Pins and screws for this purpose may conventionally be prepared from a metal such as titanium or steel, and may optionally be coated with a polymeric material which may typically be biodegradable or stabilized to facilitate soft tissue closure and sealing.
  • an implant may be an electrical conductor such as one used in, e.g., pacemakers, brain implants or biosensors.
  • the implant may also be an artificial tooth or a dental prosthesis, such as a screw and/or an abutment.
  • a pharmaceutical, dental and/or cosmetic composition according to the present invention may be admixed with other ingredients, e.g. pharmaceutically acceptable excipients to constitute a pharmaceutical composition, as discussed below, and coated onto the surface of the implant or device, e.g. by dipping the relevant portion of the implant or device in a solution or dispersion of the EMD proteins or by spraying a solution or dispersion of the EMD proteins onto the relevant surface of the implant or device followed, in both cases, by drying.
  • the pharmaceutical, dental and/or cosmetic composition according to the present invention is adsorbed to the surface of the implant or device and may be fixed thereon by means of conventional fixatives such as formaldehyde, glutaraldehyde or ethanol.
  • a pharmaceutical, dental and/or cosmetic composition according to the present invention may be applied on the relevant surface of the implant or device by cross-linking said EMD proteins, to a polymer component of the implant or device, e.g. by UV radiation or chemical treatment in a manner known per se, or by covalently binding to a suitable functional group of a polymeric component present on the surface of the implant or device.
  • the amount of a pharmaceutical, dental and/or cosmetic composition according to the present invention applied on the appropriate surface of the implant or device will normally result in an amount of total protein per cm 2 area of the implant or device corresponding to from about 0,005 mg/cm 2 to about 20 mg/cm 2 such as from about 0,01 mg/cm 2 to about 15 mg/cm 2 .
  • application of a pharmaceutical, dental and/or cosmetic composition according to the present invention according to the present invention on a surface of an implant or device for the present purpose may optionally be combined with application of other types of suitable biologically active substances, e.g.
  • osteoblast is a uninucleated cell that synthesizes both collagenous and noncollagenous bone proteins (the organic matrix, osteoid). They are responsible for mineralization and are derivable from a multipotent mesenchymal cell. The osteoblast is generally considered to differentiate through a precursor cell, the preosteoblast.
  • Soft tissues i.e.
  • non-mineralised tissues can in the present context be used interchangeably with gingival tissue, and may be defined as collagen or epithelium containing tissues, including skin and mucosa, muscle, blood and lymph vessels, nerve tissues, glands, tendons, eyes and cartilage.
  • hard-tissue formation in “mineralized tissue” may be summarized as the production by cells of an organic matrix capable of accepting mineral, with the activity of the enzyme alkaline phosphatase and a good blood supply prerequisites.
  • the cells which form part of the periodontal ligament are mainly fibroblasts.
  • the fibroblasts are characterized by an ability to achieve an exceptionally high rate of turnover of the extracellular compartment, in particular, collagen.
  • Ligament fibroblasts are aligned along the general direction of the fiber bundles and with extensive processes that wrap around the fiber bundles. Also epithelial cells and undifferentiated mesenchymal cells are constituents of the PDL.
  • peripheral cells refers to cells such as periodontal ligament cells (PDL), gingival cells, epithelial cells and/or bone cells, but is not limited thereto.
  • PDL periodontal ligament cells
  • gingival cells gingival cells
  • epithelial cells epithelial cells and/or bone cells
  • “Differentiation" of a cell refers to a process by which a cell undergoes a change to an overtly specialized cell type. Such a cell may be a stem cell differentiating into other specialized cell types during embryogenesis or later stages of development, or any other cell receiving instructions to do so.
  • a typical example for differentiation would in the present context e.g. be the differentiation of mesenchymal stem cells into osteoblasts.
  • Proliferation of a cell refers to a stage wherein the cell actively is growing and dividing to generate a cell population of a greater size. Such proliferation may be stimulated by external stimuli, such as growth factors etc.
  • Mesenchyme refers to an immature, unspecialized form of connective tissue in animals, consisting of cells embedded in a tenuous extracellular matrix. Embryonic connective tissue derivable from mesoderm is named mesenchyme.
  • mesenchymal stem cells are undifferentiated mesenchyme cells, such as bone marrow cells. In a presently preferred embodiment, said mesenchymal stem cells are differentiated into e.g. osteoblasts, osteoclasts, or any other bone cell.
  • the EMD proteins of a pharmaceutical, dental and/or cosmetic composition according to the present invention will typically originate from pig.
  • the presently isolated fractions A and B of enamel matrix (EMD) proteins, or the pharmaceutical preparation, which is depleted of fraction C comprises polypeptides and fragments of amelogenin, produced naturally by alternate splicing or processing, or by either enzymatic or chemical cleavage of a natural length protein, or by synthesis of polypeptides in vitro or in vivo (e.g. recombinant DNA methods and/or cultivation of diploid cells) will typically originate from porcine.
  • EMD enamel matrix
  • a pharmaceutical, dental and/or cosmetic composition according to the present invention is consequently employed to describe a pharmaceutical, dental and/or cosmetic composition comprising purified Enamel Matrix Derivative (EMD) proteins, which is substantially depleted of Enamel Matrix Derivative (EMD) proteins which have a molecular weight below 6kDa.
  • EMD Enamel Matrix Derivative
  • said pharmaceutical, dental and/or cosmetic composition according to the present invention thus consists of a suitable pharmaceutical carrier and purified Enamel Matrix Derivative (EMD) proteins, which have a molecular weight equal to and/or above 6kDa.
  • EMD Enamel Matrix Derivative
  • Enamel Matrix Derivative (EMD) proteins which have a molecular weight equal to and/or above 6kDa are well known in the field of the art and include amelogenins, proline-rich non-amelogenins, tuftelin, tuft proteins, serum proteins, salivary proteins, ameloblastic sheathlin, and derivatives thereof, and mixtures thereof, as well as amelins.
  • Figures 6, 7, 8 and 9 clearly illustrate molecular weights of the major protein peaks of the respective fractions A,B and C, whereas figures 10 and 11 list the amino acid sequences of the main components found in the major protein peaks of fraction B and C.
  • Polypeptides of the invention may be in a substantially isolated form. It will be understood that the peptides may be mixed with carriers or diluents, which will not interfere with the intended purpose of the peptide and still be regarded as substantially isolated.
  • a peptide of the invention may also be in a substantially purified form, in which case it will generally comprise the peptide or a fragment thereof in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the protein in the preparation is a peptide of the invention.
  • a polypeptide fragment as shown in any of figures 10 or 1 1 does include such a recombinantly produced, or chemically manufactured polypeptide which is at least 95%, such as 95%, 96%, 97%, 98%, 99% or 99,9% identical with said sequences, and which exerts the same biological effect.
  • EMD is processed as described in the experimental section.
  • enamel matrix means a precursor to enamel and may be obtained from any relevant natural source, i.e. a mammal in which teeth are under development.
  • a suitable source is developing teeth from slaughtered animals such as, e.g., calves, pigs or lambs.
  • Another source is e.g. fish skin.
  • an active enamel substance is used to encompass enamel matrix derivatives and/or enamel matrix proteins nondiscriminant of their source.
  • Enamel matrix can be prepared from developing teeth as described previously (EP-B-0 337 967 and EP-B-0 263 086).
  • the enamel matrix is scraped off and enamel matrix derivatives are prepared, e.g. by extraction with aqueous solution such as a buffer, a dilute acid or base or a water/solvent mixture, followed by size exclusion, desalting or other purification steps, alternatively followed by freeze-drying.
  • Enzymes may alternatively be deactivated by treatment with heat or solvents, in which case the derivatives may be stored in liquid form without freeze-drying.
  • enamel matrix derivatives or proteins may also use generally applicable synthetic routes, well known to a person skilled in the art, or use cultivated eukaryotic and/or prokaryotic cells modified by DNA-techniques.
  • the enamel matrix proteins may thus be of recombinant origin and alternatively genetically and/or chemically modified (see, e.g., Sambrook, J. et al.: Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).
  • enamel matrix derivatives are derivatives of enamel matrix which include one or several enamel matrix proteins or parts or fragments of such proteins, produced naturally by alternate splicing or processing, or by either enzymatic or chemical cleavage of a natural length protein, or by synthesis of polypeptides in vitro or in vivo (e.g. recombinant DNA methods and/or cultivation of diploid cells).
  • Enamel matrix protein derivatives also include enamel matrix related polypeptides or proteins.
  • the polypeptides or proteins may be bound to a suitable biodegradable carrier molecule, such as polyamine acids or polysaccharides, or combinations thereof.
  • enamel matrix derivatives also encompass synthetic analogous substances.
  • Proteins are biological macromolecules constituted by amino acid residues linked together by peptide bonds. Proteins, as linear polymers of amino acids, are also called polypeptides. Typically, proteins have 50-800 amino acid residues and hence have molecular weights in the range of from about 6,000 to about several hundred thousand Dalton or more. Small proteins are called peptides, oligopeptides or polypeptides.
  • polypeptide fragment for use in accordance with the present invention, refers to a polypeptide which may be, but is not limited to, being 1-50 amino acids in length, such as 5, 10, 15, 20, 25, 30, 35, 40, 41 , 42, 43, 44, 45, 46, 47, 47, 48, 49 or 50 amino acids. Such polypeptides may also be longer than 50 amino acids.
  • Enamel matrix proteins are proteins that normally are present in enamel matrix, i.e. the precursor for enamel (Ten Cate: Oral Histology, 1994; Robinson: Eur. J. Oral Science, Jan. 1998, 106 Suppl. 1 :282-91 ), or proteins which can be obtained by cleavage of such proteins. In general, such proteins have a molecular weight below 120,000 Dalton and include amelogenins, non- amelogenins, proline-rich non-amelogenins and tuftelins.
  • proteins for use according to the invention are amelogenins, proline-rich non- amelogenins, tuftelin, tuft proteins, serum proteins, salivary proteins, ameloblastin, sheathlin, and derivatives thereof, and mixtures thereof.
  • proteins for use according to the invention are found in the marketed product EMDOGAIN® (BIORA AB, Sweden).
  • EMDOGAIN® (BIORA AB, S-205 12 Malmo, Sweden) contains 30 mg enamel matrix protein (EMD), heated for 3 hours at about 80°C in order to inactivate residual proteases, and 1 ml Vehicle Solution (Propylene Glycol Alginate), which are mixed prior to application, unless the protein and the vehicle are tested separately.
  • the weight ratio is about 80/8/12 between the main protein peaks at approximately 20, 14 and 5 kDa, respectively, such as approximately 26, 12 and 5,5 kDa, or approximately 22, 1 1 and 4 kDa, being such as approximately about 80/8/12, 90/5/5, 81/8/1 1 , 82/8/10, 75/10/15, 80/10/10, or 88/5/7, respectively.
  • amelogenins the major proteins of an enamel matrix. They are markedly hydrophobic substances that under physiologically conditions form aggregates. They may carry or be carriers for other proteins or peptides.
  • the improved composition comprising EMD proteins found in fractions A and/or B, as disclosed by the present invention, may in the context of the present invention, be in a substantially isolated or purified form. It will be understood that the fractions, proteins, polypeptides, peptides and/or fragments thereof may be mixed with carriers or diluents or be comprised in a pharmaceutical composition, which will not interfere with the intended purpose of the proteins, polypeptides, peptides and/or fragments thereof and which will still be regarded as substantially isolated.
  • Such a substantially isolated and/or purified form will generally comprise proteins, polypeptides, peptides and/or fragments of the fraction A and/or B in a preparation in which more than 90%, e.g. 95%, 96%, 97%, 98% or 99% of the protein in the preparation is a proteins, polypeptides, peptides and/or fragments of the fraction A and/or B according to the invention.
  • a protein, polypeptide, peptide and/or fragment thereof having an amino acid sequence at least, for example 95% identical to a reference amino acid sequence is intended that the amino acid sequence of e.g. the polypeptide is identical to the reference sequence, except that the amino acid sequence may include up to 5 point mutations per each 100 amino acids of the reference amino acid sequence.
  • up to 5% of the amino acids in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acids in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the amino and/or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • a local algorithm program is best suited to determine identity.
  • Local algorithm programs (such as Smith-Waterman) compare a subsequence in one sequence with a subsequence in a second sequence, and find the combination of subsequences and the alignment of those subsequences, which yields the highest overall similarity score. Internal gaps, if allowed, are penalized. Local algorithms work well for comparing two multidomain proteins, which have a single domain or just a binding site in common.
  • Methods to determine identity and similarity are codified in publicly available programs.
  • Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J et al ( 994)) BLASTP, BLASTN, and FASTA (Altschul, S.F. et al (1990)).
  • the BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S.F. et al, Altschul, S.F. et al (1990)).
  • Each sequence analysis program has a default scoring matrix and default gap penalties. In general, a molecular biologist would be expected to use the default settings established by the software program used.
  • the proteins of an enamel matrix can typically be divided into a high molecular weight part and a low molecular weight part, which fraction contains acetic acid extractable proteins generally referred to as amelogenins (cf. EP-B-0 337 967 and EP-B-0 263 086).
  • the different molecular weight amelogenins can be purified.
  • the EMD proteins of fractions A and/or B for use according to the invention typically have a molecular weight of between approximately 6 and 20kDa, such as approximately 6, 10, 12, 15 and/or 20kDa, as determined by SDS PAGE electrophoresis.
  • the enamel matrix, enamel matrix derivatives and enamel matrix proteins are hydrophobic substances, i.e. less soluble in water, especially at increased temperatures.
  • these proteins are soluble at non-physiological pH values and at a low temperature such as about 4-20°C, while they will aggregate and precipitate at body temperature (35-37°C) and neutral pH.
  • a formulation for use according to the present invention thus comprises active enamel substances which at least partially are aggregated, and/or which after application in vivo are capable of forming aggregates.
  • the particle size of said aggregates being in a range of from about 1 ⁇ to about 20 nm, such as between 1 ⁇ and 20nm, 1 ⁇ and 10nm, 5 ⁇ and 10nm, 10 ⁇ and 1 nm, 100 ⁇ and 10nm, 100 ⁇ and 1 nm, 1 ⁇ and 1 nm, 1 ⁇ and 5nm, 1 ⁇ and 15nm.
  • the isolated fractions A and/or B may be used together with other active drug substances such as, e.g. anti-bacterial, anti-inflammatory, antiviral, antifungal substances or in combination with local chemotherapy, inducers of apoptosis, growth factors such as, e.g., TGF , PDGF, IGF, FGF, EGF, keratinocyte growth factor or peptide analogues thereof.
  • active drug substances such as, e.g. anti-bacterial, anti-inflammatory, antiviral, antifungal substances or in combination with local chemotherapy, inducers of apoptosis, growth factors such as, e.g., TGF , PDGF, IGF, FGF, EGF, keratinocyte growth factor or peptide analogues thereof.
  • Enzymes - either inherently present in the enamel matrix or preparation thereof or added - may also be used in combination with an enamel matrix fraction and/or polypeptide fragment according to the present invention, especially proteases.
  • a composition may be a pharmaceutical and/or a therapeutic and/or a dental and/or a cosmetic composition.
  • pharmaceutical, dental and/or cosmetic composition is also intended to embrace therapeutic compositions, as well as compositions belonging to the so-called grey area between pharmaceuticals and cosmetics, namely cosmeceuticals.
  • a pharmaceutical, dental and/or cosmetic composition according to the present invention serves as a drug delivery system.
  • drug delivery system denotes pharmaceutical, dental and/or cosmetic composition (a formulation, or a dosage form) that upon administration presents the active substance to the body of a human or an animal.
  • compositions according to the present invention and/or a preparation thereof are preferably formulated into a composition containing the EMD proteins and, optionally, one or more pharmaceutically acceptable excipients.
  • compositions according to the present invention to be administered may be adapted for administration by any suitable route, e.g. by systemic administration to a patient through a hose, syringe, spray or draining device.
  • compositions may be adapted to administration in connection with surgery, e.g. as a systemic administration by infusion into the blood, lymph, ascites, or spinal fluids, or by inhalation.
  • the compositions according to the invention may contain conventionally non-toxic pharmaceutically acceptable carriers and excipients according to the invention, including microspheres and liposomes.
  • Administration of a composition according to the present invention may also be performed via any other conventional administration route, such as, but not limited to, an oral, parenteral, intravenous, buccal, aural, rectal, vaginal, intraperitoneal, topical (dermal), or nasal route, or by the administration to a body cavity such as e.g. a tooth root or a tooth root canal.
  • the mucosa may be selected from oral, buccal, nasal, aural, rectal and vaginal mucosa.
  • the application may be directly on or onto a wound or other soft tissue injuries.
  • dental/odontologic area is also of great importance.
  • Relevant examples are application to periodontal (dental) pockets, to gingiva or to gingival wounds or other wounds located in the oral cavity, or in connection with oral surgery.
  • a composition for use in accordance with the present invention may be, but is not limited to, in the form of, e.g., a fluid, semi-solid or solid composition such as, but not limited to, dissolved transfusion liquids, such as sterile saline, Ringer's solution, glucose solutions, phosphate buffer saline, blood, plasma, water, powders, microcapsules, bioabsorbable patches, drenches, sheets, bandages, plasters, implants, pills, sprays, soaps, suppositories, vagitories, toothpaste, lotions, mouthwash, shampoo, microspheres, nanoparticles, sprays, aerosols, inhalation devices, solutions, dispersions, wetting agents, suspensions, emulsions, pastes, ointments, hydrophilic ointments, creams, gels, hydrogels (e.g.
  • a fluid, semi-solid or solid composition such as, but not limited to, dissolved transfusion liquids, such as ster
  • poly ethylene glycols poly ethylene glycols
  • dressings devices, templates, smart gels, grafts, solutions, emulsions, suspensions, powders, films, foams, pads, sponges (e.g. collagen sponges), transdermal delivery systems, granules, granulates, capsules, agarose or chitosan beads, tablets, microcapsules, freeze-dried powders, granules, granulates or pellets, and mixtures thereof.
  • Suitable dispersing or wetting agents for use in accordance with the invention may be naturally occurring phosphatides, e.g., lecithin, or soybean lecithin; condensation products of ethylene oxide with e.g. a fatty acid, a long chain aliphatic alcohol, or a partial ester derivable from fatty acids and a hexitol or a hexitol anhydride, e.g. polyoxyethylene stearate, poiyoxyethylene sorbitol monooleate, polyoxyethylene sorbitan monooleate, etc.
  • the invention is however not limited thereto.
  • Suitable suspending agents are, e.g., naturally occurring gums such as, e.g., gum acacia, xanthan gum, or gum tragacanth; celluloses such as, e.g., sodium carboxymethylcellulose, microcrystalline cellulose (e.g. Avicel® RC 591 , methylcellulose); alginates and chitosan such as, but not limited to, sodium alginate, etc.
  • naturally occurring gums such as, e.g., gum acacia, xanthan gum, or gum tragacanth
  • celluloses such as, e.g., sodium carboxymethylcellulose, microcrystalline cellulose (e.g. Avicel® RC 591 , methylcellulose)
  • alginates and chitosan such as, but not limited to, sodium alginate, etc.
  • a liquid composition for use in accordance with the present invention, may e.g. be, but is not limited to, a solution, dispersion or suspension for application on a surface of e.g. a medical implant or device. Once applied, the composition should preferably solidify, e.g. by drying, to a solid or at least highly viscous composition which does not dissolve on storage or when the implant or device is in use.
  • Such a composition is preferably applied under sterile conditions and/or sterilized after application by irradiation or exposure to ethylene oxide gas.
  • the composition may also be applied shortly before the medical implant or device is to be introduced into the body.
  • the composition may be applied on a surface of a tissue which is in contact with the implant or device, such as a tissue comprising a substantial proportion of epithelial cells as indicated above.
  • the composition may be applied on both the implant and/or device and on a tissue in contact therewith.
  • any other pharmaceutical composition as disclosed by the present invention may be used for the application on a surface of a medical implant or device.
  • composition according to the present invention may also, in addition to what already has been disclosed herein, be formulated according to conventional pharmaceutical practice, see, e.g., "Remington's Pharmaceutical Sciences” and “Encyclopedia of Pharmaceutical Technology", edited by Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988.
  • a pharmaceutically acceptable excipient is a substance which is substantially harmless to the individual to which the composition is to be administered.
  • An excipient is comprised in a pharmaceutical composition according to the invention.
  • Such an excipient normally fulfils the requirements given by the national health authorities.
  • Official pharmacopoeias such as e.g. the British Pharmacopoeia, the United States of America Pharmacopoeia and The European
  • Pharmacopoeia set standards for pharmaceutically acceptable excipients.
  • the choice of pharmaceutically acceptable excipient(s) in a composition, and the optimum concentration thereof, for use according to the invention, cannot generally be predicted and must be determined on the basis of an experimental evaluation of the final composition.
  • suitable excipients for the present purpose may be selected from such excipients that promote application of the pharmaceutical, dental and/or cosmetic composition according to the present invention on a surface of the implant or device, or that promote the adherence of the composition to the surface on application, or that prevent immediate dissolution of the composition or protract the release of the EMD proteins according to the present invention from the composition.
  • a person skilled in the art of pharmaceutical formulation can find guidance in e.g., "Remington's Pharmaceutical Sciences", 18th Edition, Mack Publishing Company, Easton, 1990.
  • Whether a pharmaceutically acceptable excipient is suitable for use in a pharmaceutical composition is generally dependent on which kind of dosage form is chosen for use for a particular kind of wound, and/or any other type of disorder and/or damage to a body.
  • the pharmaceutically acceptable excipients may include solvents, buffering agents, preservatives, humectants, chelating agents, antioxidants, stabilizers, emulsifying agents, suspending agents, gel forming agents, ointment bases, penetration enhancers, perfumes, powders and skin protective agents. It should however be emphasized that the invention is not limited thereto.
  • solvents for use in a composition in accordance with the present invention are water, alcohols, vegetable or marine oils (e.g. edible oils like almond oil, castor oil, cacao butter, coconut oil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil, peanut oil, poppy seed oil, rape seed oil, sesame oil, soybean oil, sunflower oil, and tea seed oil), mineral oils, fatty oils, liquid paraffin, polyethylene glycols, propylene glycols, glycerol, liquid polyalkylsiloxanes, or other hydrophilic or etheric solvents such as weak acids with a pH of about 5,5-6,0 facilitating the subsequent application of filling materials in the tooth, as well as mixtures thereof.
  • vegetable or marine oils e.g. edible oils like almond oil, castor oil, cacao butter, coconut oil, corn oil, cottonseed oil, linseed oil, olive oil, palm oil, peanut oil, poppy seed oil, rape seed oil, sesame oil, soybean oil,
  • buffering agents are citric acid, acetic acid, tartaric acid, lactic acid, hydrogen phosphoric acid, bicarbonates, phosphates, diethylamine etc.
  • Suitable examples of preservatives are parabens, such as methyl, ethyl, propyl p-hydroxybenzoate butylparaben, isobutylparaben, isopropylparaben, potassium sorbate, sorbic acid, benzoic acid, methyl benzoate, phenoxyethanol, bronopol, bronidox, MDM hydantoin, iodopropynyl
  • humectants examples include glycerin, propylene glycol, sorbitol, lactic acid, urea, and mixtures thereof.
  • Examples of chelating agents are sodium EDTA and citric acid.
  • antioxidants are butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, cysteine, and mixtures thereof.
  • examples of emulsifying agents are naturally occurring gums, e.g. gum acacia or gum tragacanth; naturally occurring phosphatides, e.g. soybean lecithin, sorbitan monooleate derivatives; wool fats; wool alcohols; sorbitan esters; monoglycerides; fatty alcohols; fatty acid esters (e.g. triglycerides of fatty acids); and mixtures thereof.
  • examples of suspending agents are e.g. celluloses and cellulose derivatives such as, e.g., carboxymethyl cellulose, hydroxyethylcellulose, hydroxypropylcellulose,
  • gel bases viscosity-increasing agents or components which are able to take up exudates from a wound are: liquid paraffin, polyethylene, fatty oils, colloidal silica or aluminum, zinc soaps, glycerol, propylene glycol, tragacanth, carboxyvinyl polymers, magnesium-aluminum silicates, Carbopol®, hydrophilic polymers such as, e.g.
  • starch or cellulose derivatives such as, e.g., carboxymethylcellulose, hydroxyethylcellulose and other cellulose derivatives, water-swellable hydrocolloids, carragenans, hyaluronates (e.g. hyaluronate gel optionally containing sodium chloride), collagen, gelatin, pectin, chitosans and alginates including propylene glycol alginate.
  • cellulose derivatives such as, e.g., carboxymethylcellulose, hydroxyethylcellulose and other cellulose derivatives, water-swellable hydrocolloids, carragenans, hyaluronates (e.g. hyaluronate gel optionally containing sodium chloride), collagen, gelatin, pectin, chitosans and alginates including propylene glycol alginate.
  • an EMD protein according to the present invention can be incorporated into a polymeric matrix so that it is released by degradation of the polymeric matrix, by enzymatic action and/or by diffusion.
  • Said polymeric matrix is either suitable for cellular in-growth, or cell- occlusive.
  • Comprised in the invention is thus in particular a pharmaceutical, dental and/or cosmetic composition according to the present invention at a low total concentration within the formulation, wherein a spatial and/or selective regulation of release of said active enamel substance permits a great percentage of the active enamel substance to be released at the time of appropriate cellular activity.
  • one aspect of the present invention relates to pharmaceutical, dental and/or cosmetic composition according to the present invention for administering, comprising a polymeric matrix, either suitable for cellular growth, in-growth and/or migration, or being cell-occlusive, and a fraction and/or polypeptide fragment, wherein said matrix is formed by a nucleophilic addition reaction between a strong nucleophile and a conjugated unsaturated bond, or a conjugated unsaturated group.
  • the conjugated unsaturated groups or conjugated unsaturated bonds are acrylates, vinylsulfones, methacrylates, acrylamides, methacrylamides, acrylonitriles, vinylsulfones, 2- or 4- vinylpyridinium, maleimides, or quinones.
  • ointment bases are e. g. beeswax, paraffin, cetanol, cetyl palmitate, vegetable oils, sorbitan esters of fatty acids (Span), polyethylene glycols, and condensation products between sorbitan esters of fatty acids and ethylene oxide, e. g. polyoxyethylene sorbitan monooleate (Tween).
  • hydrophobic or water-emulsifying ointment bases are paraffins, vegetable oils, animal fats, synthetic glycerides, waxes, lanolin, and liquid polyalkylsiloxanes.
  • hydrophilic ointment bases are solid macrogols (polyethylene glycols).
  • ointment bases are triethanolamine soaps, sulfated fatty alcohol and polysorbates.
  • powder components are: alginate, collagen, lactose, powder which is able to form a gel when applied to a wound (absorbs liquid/wound exudates). Normally, powders intended for application on large open wounds must be sterile and the particles present must be micronized.
  • excipients examples include polymers such as carmelose, sodium carmelose, and
  • hydroxypropylmethylcellulose hydroxyethylcellulose, hydroxypropylcellulose, methylcellulose, pectin, xanthan gum, locust bean gum, acacia gum, gelatin, carbomer, emulsifiers like vitamin E, glyceryl stearate, cetanyl glycoside, collagen, carrageenan, hyaluronates and alginates and chitosan.
  • diluents and disintegrating agents are but not limited to lactose, saccharose, and emdex, calcium phosphate materials, such as calcium phosphate substrates, calcium phosphate carriers (comprising hydroxyapatite, bi-phasic calcium phosphates, and tri-calcium phosphates), calcium carbonate, calcium sulfate, mannitol, starches and microcrystalline cellulose.
  • binding agents are, but not limited to, saccharose, sorbitol, gum acacia, sodium alginate, gelatin, starches, cellulose, sodium carboxymethylcellulose, methylcellulose,
  • compositions which have proved to be of importance in connection with topical application are those which have tixothropic properties, i.e. the viscosity of the composition is affected e.g. by shaking or stirring so that the viscosity of the composition at the time of administration can be reduced and when the composition has been applied, the viscosity increases so that the composition remains at the application site.
  • a pharmaceutically acceptable excipient may be employed in different dosage forms or compositions
  • the application of a particular pharmaceutically acceptable excipient is not limited to a particular dosage form or of a particular function of the excipient.
  • the EMD proteins in a pharmaceutical, dental and/or cosmetic composition according to the present invention may either be present in a dissolved state in a vehicle of slightly acid pH or as a dispersion in a vehicle of neutral pH. It is anticipated that a pharmaceutical, dental and/or cosmetic composition according to the present invention may form a protective layer on the surface of the teeth, thereby preventing the attachment of caries producing bacteria.
  • the pharmaceutical, dental and/or cosmetic composition may be formulated together with one or more other compounds which have a caries preventive effect, notably fluorine or another trace element such as vanadium or molybdenum. At neutral pH, the trace element is believed to be bound to (e.
  • the EMD proteins according to the present invention are generally present in a concentration ranging from about 0.01 % to about 99.9% w/w.
  • the amount of composition applied will normally result in an amount of total protein per cm 2 area of dental pulp corresponding to from about 0.005 mg/mm 2 to about 5 mg/mm 2 such as from about 0.01 mg/mm 2 to about 3 mg/mm 2 .
  • the concentration of the EMD protein(s) in the composition is in a range corresponding to from about 0.01 to about 50 mg/ml, e.g. from about 0.1 to about 30 mg/ml. Higher concentrations are in some cases desirable and can also be obtained such as a concentration of at least about 100 mg/ml.
  • Defect areas in dental pulp in humans typically have a size of about 5-10 x 2-4 x 5-10 mm corresponding to about 200 pi and normally at the most about 0.5-1 ml such as about 0.2-0.3 ml per tooth is applied of a composition having a concentration of about 1 -40 mg total protein/ml such as, e.g., 5-30 mg/ml is applied.
  • 0.2-0.3 mg/ml corresponds to about 6 mg protein per 25-100 mm 2 or about 0.1 mg/mm 2 if calculated only on root surface. Normally an excessive volume is applied to cover the affected surfaces adequately. Even a multilayer would only require a small fraction of the above-mentioned amounts.
  • FIG. 1 RT-PCR of cultured osteoblasts (one donor, NHO-3). A1 stimulates expression of the following gene products to a greater extend than A2, most prominently after 7 days: osteocalcin and leptin. Values represent the relative concentrations of each protein relative to tubulin and are shown as the means of the single results from duplicate experiments.
  • FIG. 1 RT-PCR of two osteosarcoma cell cultures (SaOS-2 and HOS) stimulated with fractions A1 and A2: Expression of ALP and OC increased, especially by treatment with A1. No effect was observed on the expression of Cbfa-1 and CD44 with A1 , whereas expression of CD44 was slightly reduced after treatment with A2.
  • n 3 values represent the relative concentrations of each protein relative to tubulin and are shown as the mixed means from the two cell lines results with +SD.
  • Figure 3 A (right panel) SDS PAGE and Western blot EMD vs. C12/ C 5kD (Tricine system), (left panel)To further characterize the fraction C 5kD from EMD, SDS PAGE with subsequent Western blotting was performed. Western blot of antibody raised against antigen code EP042543 in rabbit SK3184 (Peptide SYGYEPMGWLH corresponding to C terminal of TRAP). C 5k D showed mainly as a band at around 5 kDa.
  • FIG. 1 Size exclusion (SEC) method description.
  • Schema 1 Fraction C, size exclusion chromatography (SEC), TOSOH 3000SW column.
  • Reverse phase HPLC (YMC-C8, 250x20mm), in a linear gradient of 30% ACN/0.9% NaCI to 60% CAN/0.9% NaCI.
  • the components A including the two distinct components A1 and A2, both of which are >20kD and recognized by anti-amelogenin antibodies
  • B, B1 , B2, B3, C 5kD , C3, C4, D, D2 were showing as distinct peaks and could be separated by fractionating.
  • the last step was intended to remove ACN from the samples by using a Hl-Trap desalting column (HR 16/20). To determine concentrations of the fractions, a size exclusion HPLC column was utilized (same as in step 1 )) with PBS).
  • the mouse osteoblastic cell line MC3T3-E1 was obtained from Deutsche Sammlung von
  • the monoclonal osteosarcoma cell line, OHS was a kind gift from Dr. Bruland (The Norwegian Radium Hospital, Oslo, Norway).
  • the cells were cultured in RPMI 1640 (PAA) with 10% FCS (PAA), 50 lU/ml penicillin, and 50 g/ ml streptomycin.
  • Colony-forming-unit fibroblasts (Cfu- ⁇ , human mesenchymal stem cells which were separated and characterized by flow analysis cytometry (FACS) were obtained from the bone marrow of two voluntary donors (Radium hospital, Oslo) and treated in the same way as the osteoblasts
  • Real time RT PCR Cells were cultured after treatment with 5ug/ml peptides or C 5kD (50ug/ml EMD respectively) for 24h, 4d and 7d and messenger RNA was extracted using magnetic beads (Dynal AS. Oslo, Norway) and cDNA was synthesized from cell lysates according to the manufacturer's instructions (iScript One-Step RT-PCR with SYBR Green, BIORAD). Real time PCR has been performed according to the manufacturer's protocol (iCycler, BIORAD). Each reaction has been run in duplicate, and results represent the mean values of at least 2 independent cell donors.
  • Affymetrix gene array Primary osteoblasts were cultured in T75 culture flasks and cultured after treatment for 24h, 72h or 7d respectively and lysed in 7ml Trizol solution (Gibco, USA). Trizol lysates were kept at -70°C until RNA isolation according to the manufacturer's protocols and further processing (Dep. of Medical Biochemistry, Oslo University, Norway). Double stranded cDNA and biotin labeled cRNA probes were made from 5ug total RNA using the Superscript Choice system (Invitrogen) and the Enzo Bioarray respectively. Procedures were according to recommendations from Affymetrix.
  • This cRNA was hybridized to Hu-133A chips (Affymetrix) containing cDNA oligonucleotides representing more than 22 ⁇ 00 transcripts followed by washing and staining on the Gene Chips Fluidics Station 450 (Affymetrix) according to manufacturer's instructions. The chips were scanned on the Affymetrix Gene Array® 2500 scanner. The quality of the RNA and probe was controlled by an Affymetrix based test measuring the ratio between 5' and 3' mRNAs for n-actin and GAPDH and found to be highly satisfactory. The datasets were processed by the Affymetrix Mas5.0 software, and signal values representing the expression level of each transcript were generated. Each procedure has been done in twice in parallel and the resulting values indicate the mean of at least two donor's cells in duplicate experiments.
  • Cells were seeded in 48 wells plates at a density of 5000 cells / well/ ml medium.
  • n 3.
  • As controls served medium cells with medium and peptides in medium without cells (in case of background values).
  • the peptides were added immediately after cell seeding at a final concentration of 10pg/ml medium.
  • the medium from each well was transferred to Eppendorf tubes, centrifuged at 1 ⁇ 00 rpm for 5 min. Supernatants were kept at -20C until the assays were performed.
  • Cell layers were washed with 2 x 1 ml PBS. After addition of 0.5 ml of milliQwater to the different cell layers bended pipette tips were used to scrape of the cells.
  • Cell suspensions were transferred to Eppendorf tubes and lysed by the use of an ultrasonic water bath for 10 min and centrifuged at 1000 rpm for 5 min. The cell lysates were kept at -20C until the assays were performed. Changes of medium (with peptides included as for the initial seeding of cells) were made after 3 days.
  • Enzyme-linked immunosorbant assays were used.
  • FIG. 1 RT-PCR of cultured osteoblasts (one donor, NHO-3). A1 stimulates expression of the following gene products to a greater extend than A2, most prominently after 7 days: osteocalcin and leptin. Values represent the relative concentrations of each protein relative to tubulin and are shown as the means of the single results from duplicate experiments.
  • A1 and A2 have different effects on cultured osteosarcoma cells
  • A1 stimulates bone formation by up-regulation of ALP and osteocalcin
  • A2 reduced the expression of CD44, an osteocyte marker Protein analysis by ELISA
  • TOSOH 300 SW length: 25 cm, diameter: 1 cm.
  • SEC size exclusion column
  • GPC gel-filtration or gel-permeation chromatography
  • the SEC gel has to be swollen over night with the degassed. After 12 h it has to be degassed again and cast in the air lever straighten XK column. If the gel is dropped the column bed will be perched with eluent for around 30 mL (2 CV).
  • the liquid sample is separated and monitored. Monitoring is taken place with the JASCO HPLC and a TOSOH column.
  • EMD-C has retention time between 3.6 to 6 min. The characterization was done with ESI MS and sequencing procedures.
  • Spectrophotometer Spectra Max plus
  • EMD 31 13 (7x 30mg) and 31 15 (1 g) total: 1 ,2g, non-heat treated.
  • HPLC High performance liquid chromatography
  • JASCO HPLC 2 pumps (PU 1580), one detector (UV 1575) and one column oven (CO-2065Puls)).
  • SEC gel-filtration or gel-permeation chromatography
  • GPC gel-permeation chromatography
  • the SEC gel has to be swollen over night with the degassed. After 12 h it has to be degassed again and cast in the air lever straighten XK column. If the gel is dropped the column bed will be perched with eluent for around 2 CV.
  • the lyophilized EMD was redissolve in 3 mL 0.05 M formic acid.
  • the dissolved EMD was collected in a 50ml tube, the vials rinsed with some more formic acid.
  • the liquid sample was separated (flow rate: 60 mL/h, fraction size: 10 mL (10 Min) and monitored. Monitoring is taken place with the JASCO HPLC and a TOSOH column.
  • Frac A has retention time between 5 to 6 min. The characterization was done with 1 D-SDS page 10-20%.
  • SEC gel-filtration or gel-permeation chromatography
  • GPC gel-permeation chromatography
  • Fraction C has retention time from 6.2 to 7.4 with a slide tailing.
  • the characterization was done with ESI MS and sequencing procedures.
  • TGF-beta 1 transforming growth factor-beta 1
  • Fetuin/alpha2-HS glycoprotein is a transforming growth factor-beta type II receptor mimic and cytokine antagonist. J Biol Chem 1996; 271 : 12755-12761.
  • Campostrini N Areces LB, Rappsilber J, et al. Spot overlapping in two-dimensional maps: a serious problem ignored for much too long. Proteomics 2005; 5: 2385-2395.
  • emPAI Exponentially modified protein abundance index

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Abstract

La présente invention concerne une composition pharmaceutique, dentaire et/ou cosmétique comprenant des protéines dérivées de matrice d'émail (EMD) purifiées, qui sont sensiblement séparées de protéines dérivées de matrice d'émail (EMD) au-dessous de 6 kDa. Dans un mode de réalisation, l'invention concerne une composition pharmaceutique, dentaire et/ou cosmétique constituée de protéines dérivées de matrice d'émail (EMD) purifiées qui ont un poids moléculaire tel que déterminé par électrophorèse sur SDS PAGE sensiblement égal et/ou supérieur à 6 kDa, formulée dans un véhicule pharmaceutique adapté. Ladite composition améliorée est présentement de préférence destinée à être utilisée pour traiter et/ou prévenir la parodontite, stimuler et/ou induire la régénération de tissu dur, la minéralisation tissulaire, la croissance osseuse et/ou la recroissance osseuse, la régénération de dentine, la cémentogénèse, et/ou fixation entre des parties de tissu minéralisé vivant, pour fixer une pièce de tissu minéralisé vivant à un site de fixation sur une pièce d'autre tissu vivant, pour favoriser la fixation entre des tissus durs, et/ou pour remplir une cavité de plaie minéralisée et/ou un défaut tissulaire consécutif à une procédure et/ou un traumatisme.
PCT/EP2010/070255 2009-12-18 2010-12-20 Emd c-dépleté WO2011073447A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011051457A3 (fr) * 2009-11-02 2012-02-16 Straumann Holding Ag Composition de protéine emd purifiée
WO2011077086A3 (fr) * 2009-12-22 2012-04-05 Ucl Business Plc Agents ayant une activité de génération de tissu
JP2014517693A (ja) * 2011-05-27 2014-07-24 ザ ユニバーシティ オブ ワシントン スルー イッツ センター フォー コマーシャライゼーション 歯科疾患を治療するための試薬および方法
WO2014177602A1 (fr) * 2013-04-30 2014-11-06 Straumann Holding Ag Trap 63

Citations (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0042543A1 (fr) 1980-06-20 1981-12-30 Glob Ring AG Poudre à briques, son application et procédé d'utilisation en tant qu'explosif
US4672032A (en) 1983-11-09 1987-06-09 University Of Southern California Dental enamel production
WO1989008441A1 (fr) * 1988-03-17 1989-09-21 Biora Ab Composition produisant une adherence
EP0263086B1 (fr) 1986-09-25 1991-12-27 Bioventures N.V. Composition pour induire une liaison
WO2000053196A1 (fr) 1999-03-10 2000-09-14 Biora Bioex Ab Compositions de proteines de matrices destinees a induire l'apoptose
WO2000053197A1 (fr) 1999-03-10 2000-09-14 Biora Bioex Ab Compositions de proteines de matrices destinees au greffage
EP1105993A1 (fr) 1998-08-21 2001-06-13 Weblink Wireless, Inc. Systeme et procede pour la modelisation de l'etalement des temps de diffusion simultanee et l'optimisation des temps de lancement
EP1153610A1 (fr) * 1998-02-27 2001-11-14 Biora Bioex AB Compositions protéiniques matricielles pour des conditions inflammatoires et infectieuses
WO2001097834A1 (fr) 2000-06-20 2001-12-27 Biora Ab Compositions de proteines matricielles permettant la regeneration de dentine
EP1201915A2 (fr) 2000-10-23 2002-05-02 Toyota Jidosha Kabushiki Kaisha Corps de soupape d'injection de combustible pour moteur à combustion interne de type à injection directe
WO2002080994A1 (fr) 2001-02-23 2002-10-17 Biora Bioex Ab Compositions proteiques de matrice pour croissance guidee de tissu conjonctif
WO2003024479A1 (fr) 2001-09-14 2003-03-27 Biora Bioex Ab Compositions proteiques a substance matricielle a base d'email pour la modulation de reponse immunitaire
US6677306B1 (en) * 1998-07-29 2004-01-13 Northwestern University Chondrogenic and osteogenic inducing molecule
WO2006126096A2 (fr) * 2005-04-09 2006-11-30 Makoto Fukae Effet des proteines d'enveloppe porcines sur l'activite de regeneration du ligament parodontal
WO2009157869A1 (fr) * 2008-06-27 2009-12-30 Straumann Holding Ag Fraction c des dérivés de la matrice amélaire

Patent Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0042543A1 (fr) 1980-06-20 1981-12-30 Glob Ring AG Poudre à briques, son application et procédé d'utilisation en tant qu'explosif
US4672032A (en) 1983-11-09 1987-06-09 University Of Southern California Dental enamel production
EP0263086B1 (fr) 1986-09-25 1991-12-27 Bioventures N.V. Composition pour induire une liaison
WO1989008441A1 (fr) * 1988-03-17 1989-09-21 Biora Ab Composition produisant une adherence
EP0337967B1 (fr) 1988-03-17 1993-05-12 Bioventures N.V. Compositions induisant la liaison entre des parties de tissu vivant minéralisé
EP1153610A1 (fr) * 1998-02-27 2001-11-14 Biora Bioex AB Compositions protéiniques matricielles pour des conditions inflammatoires et infectieuses
EP1059934B1 (fr) 1998-02-27 2002-07-24 Biora Bioex AB Compositions proteiniques matricielles de cicatrisation
US6677306B1 (en) * 1998-07-29 2004-01-13 Northwestern University Chondrogenic and osteogenic inducing molecule
EP1105993A1 (fr) 1998-08-21 2001-06-13 Weblink Wireless, Inc. Systeme et procede pour la modelisation de l'etalement des temps de diffusion simultanee et l'optimisation des temps de lancement
WO2000053197A1 (fr) 1999-03-10 2000-09-14 Biora Bioex Ab Compositions de proteines de matrices destinees au greffage
WO2000053196A1 (fr) 1999-03-10 2000-09-14 Biora Bioex Ab Compositions de proteines de matrices destinees a induire l'apoptose
WO2001097834A1 (fr) 2000-06-20 2001-12-27 Biora Ab Compositions de proteines matricielles permettant la regeneration de dentine
EP1201915A2 (fr) 2000-10-23 2002-05-02 Toyota Jidosha Kabushiki Kaisha Corps de soupape d'injection de combustible pour moteur à combustion interne de type à injection directe
EP1201915B1 (fr) 2000-10-23 2005-08-31 Toyota Jidosha Kabushiki Kaisha Corps de soupape d'injection de combustible pour moteur à combustion interne de type à injection directe
WO2002080994A1 (fr) 2001-02-23 2002-10-17 Biora Bioex Ab Compositions proteiques de matrice pour croissance guidee de tissu conjonctif
WO2003024479A1 (fr) 2001-09-14 2003-03-27 Biora Bioex Ab Compositions proteiques a substance matricielle a base d'email pour la modulation de reponse immunitaire
WO2006126096A2 (fr) * 2005-04-09 2006-11-30 Makoto Fukae Effet des proteines d'enveloppe porcines sur l'activite de regeneration du ligament parodontal
WO2009157869A1 (fr) * 2008-06-27 2009-12-30 Straumann Holding Ag Fraction c des dérivés de la matrice amélaire

Non-Patent Citations (31)

* Cited by examiner, † Cited by third party
Title
"Encyclopedia of Pharmaceutical Technology", 1988, MARCEL DEKKER, INC.
"Remington's Pharmaceutical Sciences" and "Encyclopedia of Pharmaceutical Technology", 1988, MARCEL DEKKER, INC.
"Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING COMPANY
ALTSCHUL, S.F. ET AL., BLAST MANUAL, 1990
BOABAID F. ET AL., J. PERIODONTOL, vol. 75, no. 8, 2004
CAMPOSTRINI N; ARECES LB; RAPPSILBER J ET AL.: "Spot overlapping in two-dimensional maps: a serious problem ignored for much too long", PROTEOMICS, vol. 5, 2005, pages 2385 - 2395
COLCLASURE GC; LLOYD WS; LAMKIN M ET AL.: "Human serum alpha 2HS-glycoprotein modulates in vitro bone resorption", J CLIN ENDOCRINOL METAB, vol. 66, 1988, pages 187 - 192
DEMETRIOU M; BINKERT C; SUKHU B; TENENBAUM HC; DENNIS JW.: "Fetuin/alpha2-HS glycoprotein is a transforming growth factor-beta type II receptor mimic and cytokine antagonist", J BIOL CHEM, vol. 271, 1996, pages 12755 - 12761
GAO J; SYMONS AL; BARTOLD PM.: "Expression of transforming growth factor-beta 1 (TGF- beta1) in the developing periodontium of rats", J DENT RES, vol. 77, 1998, pages 1708 - 1716
GESTRELIUS S; LYNGSTADAAS SP; HAMMARSTROM L.: "Emdogain - periodontal regeneration based on biomimicry", CLIN ORAL INVEST, vol. 4, 2000, pages 120 - 125
GESTRELIUS, S.; ANDERSSON, C.; LIDSTROM, D.; HAMMARSTROM, L.; SOMERMAN, M.: "In vitro studies on periodontal ligament cells and enamel matrix derivative", J CLIN PERIODONTOL, vol. 24, 1997, pages 685 - 92
GODA S; INOUE H; KANESHITA Y ET AL.: "Emdogain stimulates matrix degradation by osteoblasts", J DENT RES, vol. 87, 2008, pages 782 - 787
GORG A; BOGUTH G; OBERMAIER C; WEISS W.: "Two-dimensional electrophoresis of proteins in an immobilized pH 4-12 gradient", ELECTROPHORESIS, vol. 19, 1998, pages 1516 - 1519
HAMMARSTROM ET AL., JOURNAL OF CLINICAL PERIODONTOLOGY, vol. 24, 1997, pages 658 - 668
ISHIHAMA Y; ODA Y; TABATA T ET AL.: "Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein", MOL CELL PROTEOMICS, vol. 4, 2005, pages 1265 - 1272
IWATA T; MOROTOME Y; TANABE T; FUKAE M; ISHIKAWA I; OIDA S.: "Noggin blocks osteoinductive activity of porcine enamel extracts", J DENT RES, vol. 81, 2002, pages 387 - 391
JAHNEN-DECHENT W; SCHAFER C; KETTELER M; MCKEE MD.: "Mineral chaperones: a role for fetuin-A and osteopontin in the inhibition and regression of pathologic calcification", J MOL MED, vol. 86, 2008, pages 379 - 389
LAEMMLI UK.: "Cleavage of structural proteins during the assembly of the head of bacteriophage T4", NATURE, vol. 227, 1970, pages 680 - 685
LYNGSTADAAS ET AL., JOURNAL OF CLINICAL PERIODONTOLOGY, vol. 28, 2001, pages 181 - 188
MAYCOCK J; WOOD SR; BROOKES SJ; SHORE RC; ROBINSON C; KIRKHAM J.: "Characterization of a porcine amelogenin preparation, EMDOGAIN, a biological treatment for periodontal disease", CONNECT TISSUE RES, vol. 43, 2002, pages 472 - 476
MUMULIDU ET AL: "Purification and analysis of a 5kDa component of enamel matrix derivative", JOURNAL OF CHROMATOGRAPHY B: BIOMEDICAL SCIENCES & APPLICATIONS, ELSEVIER, AMSTERDAM, NL, vol. 857, no. 2, 25 September 2007 (2007-09-25), pages 210 - 218, XP022266137, ISSN: 1570-0232, DOI: DOI:10.1016/J.JCHROMB.2007.07.017 *
NADRA I; MASON JC; PHILIPPIDIS P ET AL.: "Proinflammatory activation of macrophages by basic calcium phosphate crystals via protein kinase C and MAP kinase pathways: a vicious cycle of inflammation and arterial calcification", CIRC RES, vol. 96, 2005, pages 1248 - 1256
OKUDA K; MIYAZAKI A; MOMOSE M ET AL.: "Levels of tissue inhibitor of metalloproteinases-1 and matrix metalloproteinases-1 and -8 in gingival crevicular fluid following treatment with enamel matrix derivative (EMDOGAIN)", J PERIODONTAL RES, vol. 36, 2001, pages 309 - 316
ROBINSON, EUR. J. ORAL SCIENCE, JAN., vol. 106, no. 1, 1998, pages 282 - 91
ROBINSON, EUR. J. ORAL SCIENCE, vol. 106, no. 1, January 1998 (1998-01-01), pages 282 - 91
SAMBROOK, J. ET AL.: "Molecular Cloning", 1989, COLD SPRING HARBOR LABORATORY PRESS
SUZUKI S ET AL: "Enamel matrix derivative gel stimulates signal transduction of BMP and TGF-beta", JOURNAL OF DENTAL RESEARCH, vol. 84, no. 6, June 2005 (2005-06-01), pages 510 - 514, XP002625549, ISSN: 0022-0345 *
SUZUKI S; NAGANO T; YAMAKOSHI Y ET AL.: "Enamel matrix derivative gel stimulates signal transduction of BMP and TGF-{beta}", J DENT RES, vol. 84, 2005, pages 510 - 514
TEN CATE, ORAL HISTOLOGY, 1994
ZHANG H; LI XJ; MARTIN DB; AEBERSOLD R.: "Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry", NAT BIOTECHNOL, vol. 21, 2003, pages 660 - 666
ZHANG X; SHI L; SHU S ET AL.: "An improved method of sample preparation on AnchorChip targets for MALDI-MS and MS/MS and its application in the liver proteome project", PROTEOMICS, vol. 7, 2007, pages 2340 - 2349

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* Cited by examiner, † Cited by third party
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WO2011077086A3 (fr) * 2009-12-22 2012-04-05 Ucl Business Plc Agents ayant une activité de génération de tissu
JP2014517693A (ja) * 2011-05-27 2014-07-24 ザ ユニバーシティ オブ ワシントン スルー イッツ センター フォー コマーシャライゼーション 歯科疾患を治療するための試薬および方法
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