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WO2011077086A2 - Agents ayant une activité de génération de tissu - Google Patents

Agents ayant une activité de génération de tissu Download PDF

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Publication number
WO2011077086A2
WO2011077086A2 PCT/GB2010/002310 GB2010002310W WO2011077086A2 WO 2011077086 A2 WO2011077086 A2 WO 2011077086A2 GB 2010002310 W GB2010002310 W GB 2010002310W WO 2011077086 A2 WO2011077086 A2 WO 2011077086A2
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WIPO (PCT)
Prior art keywords
fraction
emp
peptide
peptides
cells
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PCT/GB2010/002310
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WO2011077086A3 (fr
Inventor
Nikolaos Donos
Irwin Olsen
Harsh Devangbhai Amin
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UCL Business Ltd
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UCL Business Ltd
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Priority to EP10801674A priority Critical patent/EP2516622A2/fr
Publication of WO2011077086A2 publication Critical patent/WO2011077086A2/fr
Publication of WO2011077086A3 publication Critical patent/WO2011077086A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the present invention relates generally to methods and materials having utility in tissue generation or regeneration.
  • EMP enamel matrix proteins
  • EMP periodontal ligament
  • EMP sometimes inhibits bone-associated genes such as alkaline phosphatase (ALP) and osteocalcin (OC) as well as terminal differentiation of bone-forming cells, including PDL (Wada et al, 2008; Hama et al, 2008).
  • ALP alkaline phosphatase
  • OC osteocalcin
  • EMP appears to promote the formation of mature chondrocytes required for new cartilage (Narukawa et al, 2007) and also the angiogenic differentiation of primary human endothelial cells required for the formation of new blood vessels (Schlueter et al, 2007).
  • Enamel matrix proteins or derivatives have previously been described in the patent literature to be able to induce hard tissue formation (i.e. enamel formation, U.S. Pat. No. 4,672,032 (Slavkin)), binding between hard tissues (EP-B-0 337 967 and EP-B-0 263 086) and wound healing, such as of skin and mucosa (WO 99/43344).
  • hard tissue formation i.e. enamel formation, U.S. Pat. No. 4,672,032 (Slavkin)
  • binding between hard tissues EP-B-0 337 967 and EP-B-0 263 086
  • wound healing such as of skin and mucosa
  • EMP-derived Fraction C and certain of the individual natural components present in and synthetic components derived therefrom (eg C1 and C2), have been found to have a major impact on a number of essential cell differentiation pathways. These include (A) osteogenesis, (B) chondrogenesis, (C) vasculogenesis and angiogenesis and (D) neurogenesis, leading to the inhibition of new bone but stimulation of cartilage, blood vessels and neuronal tissues. It is thus believed that these materials have differential effects on the progenitor and stem cell populations in PDL and other tissues and can thus be used to control the differentiation of such cells into these different lineages (e.g. osteogenic, chondrogenic, angiogenic, neurogenic respectively.)
  • C-Dep EMP lacking Fraction C
  • chondrogenesis, and neurogenesis may be mediated via these processes.
  • Use of these materials to identify the specific cell receptors responsible, and use of agents to modulate those receptors form further aspects of the invention.
  • the invention provides a method for effecting the lineage-directed induction of stem cells which comprises contacting the stem cells with a bioactive composition effective to induce differentiation thereof into a lineage of choice, wherein the bioactive composition is a fraction of EMP, or a synthetic equivalent or variant thereof, and wherein the lineage is selected from the group consisting of osteogenic, chondrogenic, vasculogenic/angiogenic, and neurogenic.
  • stem cells is intended to encompass also “progenitor cells” and for brevity the term “stem cell” is used in place of "progenitor/stem cell”.
  • the method may be performed in vitro, ex vivo, or in vivo.
  • the cells are contacted with the bioactive composition in a rigid porous vessel which may (by way of non-limiting Example) be a ceramic cube, titanium reactor or implant, or any culture vessel known in the art.
  • the cells are contacted with the bioactive composition in an injectable liquid which may be used, for example, during or after surgery e.g. orthopaedic or dental surgery.
  • the bioactive composition comprises one or more factors or fractions derived from or otherwise based on Fractions C and C-Dep, or a synthetic equivalent or variant thereof,
  • the method of the invention further provides administering to an individual in need thereof stem cells and the bioactive composition effective to induce differentiation of such cells into a lineage of choice.
  • This may in one embodiment comprise administering the bioactive composition to an individual to whom a preparation comprising isolated human stem cells had been administered.
  • the invention provides a method for inducing the in vivo production of bone, blood vessels and neuronal tissues in a patient which comprises administering to the individual isolated stem cells and a bioactive composition effective to induce such cells to differentiate into the respective lineage descendant in such individual.
  • the stem cells and bioactive composition are administered together or they may alternatively be administered separately.
  • stem cells or bioactive composition for use in the above methods, or for use in the preparation of a medicament for the above methods, for further aspects of the invention.
  • a major contribution of the present invention is a method comprising administering to an individual in need thereof the bioactive composition (e.g. a fraction of EMP, or a synthetic equivalent or variant thereof) to enhance the generation or regeneration of damaged or diseased tissue.
  • the bioactive composition e.g. a fraction of EMP, or a synthetic equivalent or variant thereof
  • the invention provides a method of treating a mammal in need of tissue generation, said method comprising administering to said mammal a bioactive composition as described herein.
  • a bioactive composition as described herein.
  • Such methods may be used for example for regeneration of a range of diseased and damaged tissues.
  • Fraction C containing two TRAP isoforms, a 43- and 45-amino acid tyrosine-rich peptide derived from the N-terminal of amelogenin, has been found to inhibit bone gene expression and new bone formation but to strongly up-regulate cartilage, blood vessel and nerve-associated genes and the differentiation of the corresponding chondrocyte, vascular and nerve-like cells in vitro.
  • a fraction of EMP may preferably be used to promote new cells of the nervous system (e.g. neurons and glial cells). Such may be used in selectively building neurological tissue for the treatment of stroke, Alzheimer's and neuromuscular disorders, and Parkinson's disease.
  • Fraction C (and C1 and C2) was effective at forming endothelial-like tubes.
  • Fraction C (or a fraction thereof, or a synthetic equivalent or variant thereof) may be used to enhance the formation of endothelial-like blood vessel cells.
  • Fraction C (and C1 and C2) formed more endothelial-like cells than did EMP. This has utility in cardiac disease.
  • the same bioactive composition has also been found to regulate the function of epithelial cells, and therefore also be clinically useful in treating skin-related and other epithelial disorders.
  • C-Dep contains a number of proteins including amelogenin and LRAP, a 56-amino acid leucine-rich peptide derived biologically from amelogenin. C-Dep greatly enhances new bone formation but strongly inhibits chondrogenesis,
  • vasculogenesis/angiogenesis and neurogenesis in vitro are vasculogenesis/angiogenesis and neurogenesis in vitro.
  • the bioactive composition will comprise C-Dep, or a fraction or factor of C-Dep, or a synthetic equivalent or variant thereof as described herein.
  • C-Dep or a fraction of C-Dep, or a synthetic equivalent or variant thereof as described herein
  • C-Dep may be used to inhibit chondrogenesis, and/or angiogenesis and/or neurogenesis and/or vasculogenesis.
  • C-Dep (or a fraction of C-Dep, or a synthetic equivalent or variant thereof as described herein) may be used in the regeneration of periodontal (PDL) tissue.
  • PDL periodontal
  • such preparations can be superior to EMD e.g. in growing new bone such as alveolar bone and rebuilding PDL.
  • the composition may (by way of non-limiting example) be administered by continuous injection or bolus injection.
  • it comprises a pharmaceutically acceptable excipient.
  • bioactive composition for use in the above methods, or for use in the preparation of a medicament for the above methods, for further aspects of the invention.
  • Fraction C has an inhibitory effect on bone formation.
  • Another aspect of the invention provides a composition comprising isolated, culture- expanded human stem cells and a bioactive composition as described effective to induce differentiation of such cells into a lineage of choice as described above.
  • the composition further comprises a tissue culture medium.
  • the composition can comprise a medium suitable for administration to an animal particularly a human, in need thereof. This aspect of the invention also provides for specific
  • EMP EMP
  • Fraction C Fraction C
  • Fraction C Fraction C
  • a fraction of Fraction C or a synthetic equivalent or variant thereof as described herein, such as C1 or C2
  • C1 or C2 C1 or C2
  • EMP and Fraction C are internalized in specific cellular compartments in PDL stem cells.
  • the receptor may be modelled in 3 dimensions to produce EMP fraction mimetics.
  • it may be used directly e.g. as a binding partner (optionally in phage display) to screen for compounds.
  • the use of the receptor, and in particular agonists or antagonists thereof, for effecting osteogenesis, chondrogenesis, vasculogenesis/angiogenesis and neurogenesis forms a further aspect of the invention.
  • any human stem cells may be employed - for example which are available commercially or via publication to those skilled in the art.
  • human PDL stem cells were used (Singhatanadgit et al. 2009).
  • stem cells and stem cell lines
  • bm-MSCs autologous bone marrow derived mesenchymal stem cells
  • human alveolar bone cells which form part of the periodontium structure.
  • bioactive compositions are fractions of EMP, or a synthetic equivalent or variant thereof. Sources of EMPs are discussed hereinafter, as are fractionation techniques.
  • Fraction C or derived or analogous agents
  • C-Dep or derived or analogous agents
  • Fraction C which comprises components less than 6 kDa consisting mainly of a group of hydrophobic peptides derived from the amelogenin gene by alternative splicing and post- translational modifications. These include the 5.1 and 5.3 kDa tyrosine-rich (TRAP) proteins (Fincham et al, 1994, which is specifically incorporated herein by reference), which have previously been isolated and the amino acid sequences fully delineated (Fincham et al, 1994). These two components, C1 and C2, have been used in some of the experiments described here, as noted.
  • TRIP tyrosine-rich
  • the bioactive composition will typically include only proteins of less than 6 kDA present in EMPs, or synthetic equivalents or variants thereof as described herein.
  • Such embodiments may use one or both of C1 and C2 (TRAP proteins as defined) which likewise may be obtained from EMPs, or be synthetic.
  • the present invention specifically embraces peptide variants which are active portions or fragments of the EMP-derived bioactive agent, for example TRAP, or variants of such a portion e.g. showing 75% or greater homology with it. Furthermore, it embraces synthetic analogs of such peptides.
  • the agents is a fragment selected from the 43- and 45-amino acid TRAP proteins, derived from the N-terminal of amelogenin:
  • the agent comprises or consists of or consists essentially of at least 6, 7, 8, 9, or 10 amino acids of the 10-amino acid C terminal of the 43-amino acid TRAP protein:
  • the agent comprises or consists of or consists essentially of at least 6, 7, 8, 9, 10, 11 , or 12 amino acids of the 12-amino acid C terminal of the 45-amino acid TRAP protein: NH 2 - YTSYGYEP GGW - COOH (which may be termed "TCT2" herein)
  • the agents consist of, or consist essentially of, the TCT1 and/or TCT2 peptides.
  • the agent is a synthetic analog of such a peptide.
  • the examples herein show that both naturally-occurring and chemically-synthesized TRAP have the same biological activities as Fraction C.
  • the TCT1 and TCT2 peptides suppresse bone formation and also exhibit the chondrogenic, vasculogenic, angiogenic and neurogenic stimulatory activities of the 'parent' TRAP proteins,
  • C-Dep is a preparation of EMP depleted of Fraction C (as above) and comprising components over 6 kDa, mainly 6.9 and 8.1 kDa leucine-rich amelogenin peptides (LRAP), sheathlin proteins (1 1 , 13, 15 and 17 kDa) and full-length amelogenin (>17 kDa protein) (Swanson et al, 2006; Kanazashi et al, 2006; Fincham et al, 1994). These individual proteins have previously been isolated and their amino acid sequences reported (Kanazashi et al, 2006; Maycock et al, 2002; Hu et al, 1997). MALDI-TOF experiments in our laboratory have confirmed the presence of these proteins in C-Dep.
  • LRAP leucine-rich amelogenin peptides
  • compositions consisting of Fraction C, and C-Dep (or synthetic equivalents or variants thereof), and their use in the methods described herein, form further aspects of the invention.
  • the present invention specifically embraces peptide variants which are active portions or fragments of the EMP-derived bioactive agent, for example LRAP, or variants of such a portion e.g. showing 75% or greater homology with it. Furthermore, it embraces synthetic analogs of such peptides.
  • the agent is a fragment of the 56 amino acid LRAP protein derived from the N-terminal of amelogenin:
  • the agent comprises or consists of or consists essentially of at least 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22 or 23 amino acids of the 23 amino acid C terminal of the 56 amino acid LRAP protein:
  • the agent consists of, or consists essentially of, the LCT peptide.
  • the agent is a synthetic analog of such a peptide.
  • the examples herein show that both naturally-occurring and chemically-synthesized LRAP have the same biological activities as C-dep.
  • 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, January 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, tuftelins, tuft proteins, serum proteins, salivary proteins, ameloblastin, sheathlin, and derivatives thereof, and mixtures thereof.
  • enamel matrix proteins includes enamel matrix derivatives, or enamel matrix protein derivatives. These are derivatives of enamel matrix which include one or several enamel matrix proteins or parts 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 (recombinant DNA methods or cultivation of diploid cells, either plant or animal 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.
  • the term enamel matrix derivatives also encompasses synthetic analogous substances.
  • EMDOGAIN® (BIORA AB, S-205 12 Malmo, Sweden) contains 30 mg enamel matrix protein, 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 20, 14 and 5 kDa, respectively.
  • Enamel matrix is 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.
  • 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 (EMD) are prepared, e.g. by extraction with aqueous solution such as a buffer, a dilute acid or base or a water/solvent mixture.
  • the sequences can be incorporated into a vector having control sequences operably linked to the bioactive agent nucleic acid to control its expression.
  • the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the bioactive agent peptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the peptide produced in the host cell is secreted from the cell, bioactive agent peptide can then be obtained by transforming the vectors into host cells in which the vector is functional, culturing the host cells so that the peptide is produced and recovering the peptide from the host cells or the surrounding medium.
  • Prokaryotic and eukaryotic cells are used for this purpose in the art, including strains of E. coli, yeast, and eukaryotic cells such as COS or CHO cells.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate.
  • plasmids viral e.g. 'phage, or phagemid, as appropriate.
  • Cells and techniques may be selected such as to permit or enhance the folding and/or formation of disulphide bridges (see e.g. "Protein Folding” by R. Hermann, Pub. 1993, European Patent Office, The Hague, Netherlands, ISBN 90-9006173-8).
  • Peptides may be synthesized by any suitable method, such as by exclusively solid-phase techniques, by partial solid-phase techniques, by fragment condensation or by classical solution couplings.
  • the peptide chain can be prepared by a series of coupling reactions in which the constituent amino acids are added to the growing peptide chain in the desired sequence.
  • Variants can be produced by a mixture of conservative variation, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
  • conservative variation i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine.
  • altering the primary structure of a polypeptide by a conservative substitution may not significantly alter the activity of that peptide because the side-chain of the amino acid which is inserted into the sequence may be able to form similar bonds and contacts as the side chain of the amino acid which has been substituted out.
  • substitutions are in a region which is critical in determining the peptides conformation.
  • variants having non-conservative substitutions As is well known to those skilled in the art, substitutions to regions of a peptide which are not critical in determining its conformation may not greatly affect its activity because they do not greatly alter the peptide's three dimensional structure. In regions which are critical in determining the peptides conformation or activity such changes may confer advantageous properties on the polypeptide. Indeed, changes such as those described above may confer slightly advantageous properties on the peptide e.g. altered stability or specificity.
  • the present invention also includes variants which are active portions or fragments of the EMP-derived bioactive agents employed in the invention.
  • bioactive portion of bioactive agent peptide means a peptide which is less than said full length bioactive agent peptide, but which retains at least some (say, 50%, 60%, 70%, 80%, 90% or more) of its activity as assayed above - e.g. in respect of stem cell differentiation.
  • variant protein of the invention are amino acid variants of the naturally occurring proteins and peptides as described above or in the References referred to herein (including 5.1 and 5.3 kDa tyrosine-rich proteins; 6.9 and 8.1 kDa leucine-rich a elogenin peptides; sheathlin proteins (11 , 13, 15 and 17 kDa); full-length amelogenin (>17 kDa protein) etc.) and which share the relevant activity of those proteins and peptides as assayed above - e.g. in respect of stem cell differentiation.
  • variant sequences are at least 75% homologous to the wild-type sequence, more preferably at least 80% homologous, even more preferably at least 85%
  • homologous yet more preferably at least 90% homologous or most preferably at least 95% homologous to at least a portion of the reference protein.
  • the homology will be as high as 94, 95, 96, 97, 98, or 99%.
  • Homology in this context means sequence similarity or identity, with identity being preferred.
  • the candidate amino acid sequence and the reference amino acid sequence are first aligned using a standard computer programme such as are commercially available and widely used by those skilled in the art.
  • the NCBI BLAST method is used (http://www.ncbi.nlm.nih.gov/BLAST/). Once the two sequences have been aligned, a percent similarity score may be calculated.
  • proteins may share at least about 50%, 60%, 70%, 80%, 90% or more sequence identity with an authentic sequence.
  • sequence identity means strict amino acid identity between the sequences being compared.
  • Proteins and peptide agents according to the present invention may be subject to degradation by a number of means (such as protease activity at a target site). Such degradation may limit their bioavailability and hence therapeutic utility.
  • a derivative suitable for use according to the invention is more protease-resistant than the protein or peptide from which it is derived.
  • Protease- resistance of a peptide derivative and the protein or peptide from which it is derived may be evaluated by means of well-known protein degradation assays. The relative values of protease resistance for the peptide derivative and peptide may then be compared.
  • Peptoid derivatives of proteins and peptides according to the invention may be readily designed from knowledge of the primary sequences given herein. Commercially available software may be used to develop peptoid derivatives according to well-established protocols.
  • Retropeptoids (in which all amino acids are replaced by peptoid residues in reversed order) are also able to mimic proteins or peptides according to the invention.
  • retropeptoid is expected to bind in the opposite direction in the ligand-binding groove of a receptor or other binding partner, as compared to a peptide or peptoid-peptide hybrid containing one peptoid residue.
  • the side chains of the peptoid residues are able to point in the same direction as the side chains in the original peptide.
  • a further embodiment of a modified form of peptides or proteins according to the invention comprises D-amino acid forms.
  • the order of the amino acid residues is reversed.
  • the preparation of peptides using D-amino acids rather than L- amino acids greatly decreases any unwanted breakdown of such derivative by normal metabolic processes, decreasing the amounts of the derivative which needs to be administered, along with the frequency of its administration.
  • Derivatives of peptide agents used according to the invention include derivatives that increase the half-life of the agent in vivo.
  • Examples of derivatives capable of increasing the half-life of polypeptides according to the invention include peptoid derivatives, D- amino acid derivatives and peptide-peptoid hybrids.
  • amino acid comprises not only the residues of the natural amino acids (e.g., Ala, Arg, Asn, Asp, Cys, Glu, Gin, Gly, His, Hyl, Hyp, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form but also unnatural amino acids (e.g., phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline,
  • gamma-carboxyglutamate hippuric acid, octahydroindole-2-carboxylic acid, statine, 1 ,2,3,4,-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, citruline, a-methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert-butylglycine).
  • the term also includes natural and unnatural amino acids bearing a conventional amino protecting group (e.g., acetyl or benzyloxycarbonyl), as well as natural and unnatural amino acids protected at the carboxy terminus (e.g. as a (C
  • peptide when used herein, describes a sequence of usually 5 to 25 amino acids (e.g. as defined hereinabove) or peptidyl residues.
  • the sequence may be linear or cyclic.
  • a cyclic peptide can be prepared or may result from the formation of disulfide bridges between two cysteine residues in a sequence.
  • a peptide can be linked to another molecule through the carboxy terminus, the amino terminus, or through any other convenient point of attachment, such as, for example, through the sulfur of a cysteine.
  • a peptide comprises 10 to 25 amino acids.
  • Peptide derivatives can be prepared as disclosed in U.S. Patent Numbers 4,612,302; 4,853,371 ; and 4,684,620.
  • Mimetics of peptides Also embraced by the present invention are uses of agents which are functional mimetics of the peptides described herein, and which retain the essential biological activity of the peptides.
  • mimetics include chemical compounds which are modeled to resemble the three dimensional structure of the peptides described herein.
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled according to its physical properties, e.g. stereochemistry, bonding, size and/or charge, using data from a range of sources, e.g. spectroscopic techniques, X-ray diffraction data and N R.
  • a range of sources e.g. spectroscopic techniques, X-ray diffraction data and N R.
  • the three dimensional structure may be determined.
  • Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modeling process.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesise, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic is peptide based
  • further stability can be achieved by cyclising the peptide, increasing its rigidity.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimisation or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • bioactive agents and nucleic acids encoding them can be formulated into
  • compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be nontoxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • 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.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution (PBS is preferred), dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection.
  • the modulators may be included in a pharmaceutical composition for formulated for slow release, such as in microcapsules formed from biocompatible polymers or in liposomal carrier systems according to methods known in the art.
  • the protein or peptide may be covalently conjugated to a water soluble polymer, such as a polylactide or biodegradable hydrogel derived from an amphipathic block copolymer, as described in U.S. Pat. No. 5,320,840.
  • Collagen-based matrix implants such as described in U.S. Pat. No. 5,024,841 , are also useful for sustained delivery of peptide therapeutics.
  • a composition that includes a biodegradable polymer that is self-curing and that forms an implant in situ, after delivery in liquid form. Such a composition is described, for example in U.S. Pat. No. 5,278,202.
  • compositions may be formulated according to conventional pharmaceutical practice, see, e.g., "Remington: The science and practice of pharmacy” 20 th ed. Mack Publishing, Easton Pa., 2000 ISBN 0-912734-04-3 and "Encyclopaedia of Pharmaceutical
  • the present invention provides a pharmaceutical composition comprising a bioactive agent peptide-encoding nucleic acid molecule and its use in methods of therapy or diagnosis.
  • the present invention provides a pharmaceutical composition comprising one or more bioactive agents as defined above and its use in methods of therapy or diagnosis.
  • the present invention provides the above bioactive agents and nucleic acid molecules for use in the use of bioactive agent peptides in the preparation of medicaments for therapy.
  • compositions may include anti-oxidant effective to prevent methionine oxidation.
  • anti-oxidant effective to prevent methionine oxidation.
  • Figure 1 a shows the effects of Fraction C on terminal osteogenic differentiation in vitro
  • the numbers are the alizarin red staining intensity.
  • the number in ( ) is the % inhibition by Fraction C compared with EMP.
  • Figure 1 b shows the effects of Fraction C and C-Dep on chondrogenic gene expression and terminal differentiation in vitro.
  • the Figure is a representative RT-PCR gel showing chondrogenic gene expression of PDL cells cultured in CM with EMD/Fractions for 2 weeks.
  • Figure 1 c shows the dose effects of EMD and EMD Fractions on PDL chondrogenesis using Alcian blue staining at 2 weeks. Terminal chondrogenic differentiation was examined by Alcian blue staining of paraffin sections of PDL cell pellets. Red arrows show proteoglycans stained by Alcian blue; Nuclei stained purple with hematoxylin.
  • Figure 2 shows in vitro endothelial-like tube formation assay in the presence of EMP, and Fraction C.
  • the numbers are the angiogenic scores.
  • Number in ( ) is the % stimulation by Fraction C, compared with control cultures in growth medium (GM) and endothelial growth medium (EGM-2) alone.
  • Figure 3 shows the effects of Fraction C on blood vessel development in vitro.
  • Figure 4 shows the effects of EMP and Fraction C on terminal neural differentiation.
  • the numbers are the % of neuron-specific ⁇ tubulin positive cells.
  • the number in ( ) is the % stimulation by Fraction C, compared with EMP.
  • Figure 5 shows the effects of Fraction C on nerve cell differentiation in vitro.
  • Figure 6 shows the effects of C-Dep on terminal osteogenic differentiation of bone- forming cells in vitro.
  • Figure 7 shows that EMP and Fraction C bind to and are internalized into PDL stem cells.
  • Figure 8 shows that Fraction C binds to the cells (green-fluroscence) at 4°C for 4 h and is then internalized at 37°C (red-fluroscence). Black arrows show the binding and white arrows show internalization of Fraction C into cellular vesicles. Cell nuclei are stained blue using Hoechst dye.
  • Figure 9 shows fractionation of EMD by low pressure SEC (BioGel P-30 Fine, 100 cm*5.0 cm) (from Mumulidu 2007).
  • Figure 10 shows RP HPLC analysis of the Figure 9 "5 kDa component" using different buffer concentrations in the mobile phase (YMC-Pack ODS-A, 250mm*4.6 mm). Top: 100 mM, middle: 50 mM, and bottom: 10 mM (from Mumulidu 2007).
  • tissue was separated from the surface of the middle portion of the root and digested with 3 mg/ml of collagenase type I and 4 mg/ml of dispase for 1 h at 37'C.
  • Single-cell suspensions were obtained by passing the cells through a 70 m strainer and cultured in ct-Modified Eagle's Medium ( ⁇ -MEM), containing 10% fetal calf serum (FCS)
  • EMP in the form of the commercially available product Emdogain®; Institut Straumann
  • Emdogain® Institut Straumann
  • Fraction C and C-Dep obtained following fractionation of EMP as described previously (Mumulidu A et al, Journal of Chromatography B, 857 (2007) 210-218 this is specifically incorporated herein by reference) were prepared at 1 and 10 mg/ml in 0.1% acetic acid, respectively.
  • HPLC high performance liquid chromatography
  • the SEC used a 5 cm* 100 cm column including BioGel P-30 Fine, with 125mM formic acid, measurement at 280 nm, and ambient temperature. The following fractions and molecular weights were obtained:
  • Figure 10 shows RP HPLC analysis of the 5 kDa component using different buffer concentrations in the mobile phase (YMC-Pack ODS-A, 250mmx4.6 mm). Top: 100 mM, middle: 50 mM, and bottom: 10 mM (from Mumulidu 2007).
  • Fraction C includes only proteins up to 6 kDa (based on Maldi TOF analysis). This preparation thus includes the "TRAP” proteins or peptides referred to in Mumulidu 2007, and other proteins or peptides in this size range, but will exclude LRAPs between 6 and 9 kDa for example.
  • Fractions C1 and C2 Two purified fractions, each containing only one of these two TRAP proteins, are referred to herein as Fractions C1 and C2. Results have also been obtained with synthetic TRAP proteins with equivalent or improved effect (results not shown).
  • C Dep as used herein is prepared from EMD by subtraction of “Fraction C”. It includes proteins between 6 and 20 kDa, inter alia, the LRAPs between 6 and 9 kDa (usually 6.9 and 8.1 ) referred to by Mumulida.
  • EMP and C-Dep were used at a final concentration of 100 g/ml, and Fraction C was used at 10 pg/rnl for osteogenesis and chondrogenesis- related experiments and 30 pg/ml for the vasculogenesis/angiogenesis- and
  • Quantitative polymerase chain reaction analysis of lineage associated genes
  • first strand cDNA was synthesized using 1 g of total RNA, as previously described (Singhatanadgit et al, 2009), with primers obtained from Applied Biosystems.
  • Q-PCR analysis was carried out using the ABI Prism ® 7300 sequence detector, the Taqman ® Gene Expression Assay consisting of the unlabelled specific PCR primers and Taqman ® MGB probes with FAMTM dye labelling in a 96-well plate format. Thermal cycler conditions were used as recommended by the manufacturer and the data were collected and analyzed by the SDS 1.2 software. All PCR reactions were performed in triplicate and each of the gene cycle threshold (ct) values were normalized to the GAPDH ct value detected simultaneously on the same plate.
  • ct gene cycle threshold
  • PDL cells were plated into 24-well plates at a density of 2.5 x 10 4 cells/well and cultured in GM for 2-3 days, then osteogenic medium (OM) added, consisting of GM
  • CM serum-free chondrogenic medium
  • TGF-p3 transforming growth factor-p3
  • dexamethasone L-ascorbate2-phosphate
  • sodium pyruvate L-proline
  • RNA extracted for RT-PCR analysis of the late markers aggrecan, Col2a1 and COMP after 2 weeks, as previously described.
  • Alcian blue staining for acid mucopolysaccharides and glycosaminoglycans was also carried out, after 3 weeks, to assess terminal chondrogenic differentiation as follows.
  • Cell pellets were fixed in 10% formalin at 4 ° C for 24 h, dehydrated in an ascending series of ethanol and embedded in paraffin. Sections (3 pm) were cut, stained with 1 % alcian blue (pH 2.5) (Sigma) for 5 min. The deposition of mucopolysaccharides and
  • glycosaminoglycans was visualized as blue staining of the extracellular matrix (ECM). Nuclei were stained purple using Harris Hematoxylin.
  • PDL cells were cultured in GM as in a. above and endothelial cell growth medium-2 (EGM-2) then added.
  • EGM-2, EMP and Fraction C were changed every 3-4 days.
  • Total RNA was extracted to measure the early angiogenic marker gene Ang-1 (at week 1 ) and the late marker gene vWF (at week 2), as described previously (Gang et al, 2006).
  • Angiogenic differentiation of PDL cells was performed using an in vitro angiogenesis assay kit. Briefly, 10 4 cells were plated on ECMatrix gel coated 96-well plates and cultured in the presence of EGM-2 with and without EMP and Fraction C. After 6 h, digital images were obtained using bright-field microscopy and angiogenic tube formation scored from 0 to 5, as previously described (Cochran et al, 2007), based on the progressive appearance of morphological features associated with angiogenesis:
  • Immunostaining was also carried out as previously described (Singhatanadgit et al, 2008). Briefly, cells were fixed with 4% paraformaldehyde for 15 min at room temperature (RT) and permeabilised using 0.1% Triton X for 15 min at RT. They were then treated with a blocking solution containing 10% normal donkey serum (NDS) in PBS for 1 h and incubated for 1 h at RT with primary mouse monoclonal anti- ⁇ tubulin antibody diluted 1 :1000 in PBS containing 1% NDS. Incubation was then carried out with donkey anti- mouse Alexa Fluor secondary antibody diluted 1 :200 in PBS containing 2% NDS for 1 h at RT.
  • NDS normal donkey serum
  • the neuron-like cells were visualized as green fluorescent stained cells with long axonal projections. Nuclei were stained blue using Hoechst dye. The proportion of neuron-specific ⁇ tubulin-positive cells was determined by manual counting of 5 separate fields of each culture.
  • OP, OC and BSP gene expression, and the ALP activity of bone-forming cells were significantly down-regulated when cultured in the presence of Fraction C, compared to EMP.
  • Figure 2 shows that EMP and, most notably, Fraction C stimulated PDL cells to form complex tube-like structures when cultured in EGM-2 for 5 h, compared to control cultures GM and EGM-2 alone.
  • Figure 3 shows the formation of elongated blood vessel-like structures after culture in the presence of Fraction C, compared to EGM-2 alone, which contained less elongated and smaller structures.
  • Fraction C up-regulates the neural-associated genes MAP-2 and GFAP when PDL cells are cultured in NM, compared with control cultures in NM alone and with EMP.
  • Figure 4 shows that Fraction C stimulated PDL cells to form more green-fluroscent stained neuronal-like cells than in control cultures NM alone and EMP.
  • Figure 5 shows the morphology of nerve-like cells which are induced in cultures incubated with Fraction C.
  • Example 2 C-Dep enhances bone-forming cell differentiation and osteogenesis in vitro
  • This Table shows that OP, OC and BSP gene expression, and ALP activity of bone- forming cells, were significantly up-regulated when cultured in the presence of C-Dep, compared to EMP.
  • the numbers are the alizarin red staining intensity.
  • the number in ( ) is the % stimulation by C-Dep compared with EMP.
  • Figure 6 shows that terminal osteogenic differentiation of bone-forming cells was significantly stimulated in the presence of C-Dep, compared with EMP.
  • Example 3 EMP and Fraction C bind to and are internalized into PPL stem cells
  • Figure 7 shows that EMP (visualized by green fluorescent staining) binds to the PDL cell surface at 4°C, and is then transported to intra-cellular vesicles, possibly lysosomes, after 5 h of incubation at 37°C. Cell nuclei are stained blue using Hoechst dye.
  • Figure 8 shows that Fraction C binds to the cells (green-fluroscence) at 4°C for 4 h and is then internalized at 37°C (red-fluroscence). Black arrows show the binding and white arrows show internalization of Fraction C into cellular vesicles. Cell nuclei are stained blue using Hoechst dye.
  • This Example shows for the first time that at least some specific components of EMP and Fraction C are able to bind to the PDL stem cells and thereafter become internalized in specific cellular compartments.
  • the biological effects of the materials on osteogenesis, angiogenesis and neurogenesis are highly likely to be mediated via these processes.
  • the present findings provide for the characterisation (identification and isolation) of the specific receptors involved in EMP 'uptake' and determining the ultimate fate of these EMP proteins in the 'target' stem cells, especially in relation to their specific biological effects on cell differentiation and tissue regeneration.
  • the EMP derived product(s) and their intracellular transport can be adapted, thereby improving their activity and potential clinical efficacy.
  • the receptors may be modified, with consequent change in the activity of the EMP products, and improved efficacy of materials e.g. to regulate the formation of new bone, blood vessel and nerve cells.
  • the present inventors have compared the amino acid sequences of the amelogenin- derived TRAP and LRAP peptides and probed their active domains, as follows.
  • TRAP and LRAP have the identical 33 N-terminal amino acid sequence as in the parent amelogenin protein. Without wishing to be bound by theory, it is proposed that this common overlapping N-terminal peptide (ONT) does not contribute specifically to the biological activities of the parent proteins examined herein.
  • TCT1 (10 NH, - YTSYGYEPMG - COOH amino acids)
  • TCT1 and TCT2 The present inventors then obtained the synthetic 10- and 12-amino acid C-terminal (TCT1 and TCT2; PepCI and PepC2) peptide that is unique to TRAP.
  • TCT1 and TCT2 the synthetic 10- and 12-amino acid C-terminal peptide that is unique to TRAP.
  • TCT1 and TCT2 peptides which are chemically produced and does not itself occur as a natural product, is the critically important sequence of TRAP responsible for the range of tissue developmental activities we have studied.
  • the present inventors then obtained the synthetic 23-amino acid C-terminal (LCT; PepDep) peptide that is unique to LRAP. Results obtained indicated that this new LCT peptide is likely to be responsible for both bone-forming activity as well as for suppression of cartilage, blood vessel and nerve development.
  • LCT 23-amino acid C-terminal
  • Enamel matrix derivative stimulates chondrogenic differentiation of ATDC5 cells. Journal of Periodontal Research 42.131-137.

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Abstract

L'invention concerne des procédés d'inhibition, de réalisation ou de stimulation de l'induction de cellules souches dirigée par lignées, les procédés comprenant la mise en contact des cellules souches avec une composition bioactive efficace pour inhiber ou induire leur différenciation dans une lignée de choix, la composition bioactive étant l'un des éléments suivants (a) une fraction de protéine de la matrice amélaire (EMP), ladite fraction étant soit (i) la fraction C de l'EMP, ladite fraction C étant la fraction de l'EMP comprenant des composants d'une taille inférieure à 6 kDa et étant dérivée du gène de l'amélogénine par épissage alternatif et modification post-traductionnelle, soit (ii) la fraction C-dep de l'EMP, la fraction C-dep étant la fraction de l'EMP comprenant des composants d'une taille supérieure à 6 kDa et étant dérivée du gène de l'amélogénine par épissage alternatif et modifications post-traductionnelles, (b) un peptide isolé de ladite fraction, (c) une partie active ou un fragment dudit peptide de (b), (d) un variant de (b) ou (c) présentant une identité de 70 % ou plus avec ceux-ci, (e) un analogue de synthèse ou un dérivé de l'un quelconque des peptides (b) à (d), et la lignée étant choisie dans le groupe constitué par des cellules ostéogéniques, chondrogéniques, angiogéniques, et neurogéniques. La présente invention concerne également des peptides particuliers et des fragments ayant une ou plusieurs des activités pertinentes, et concerne des procédés et compositions apparentés.
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* Cited by examiner, † Cited by third party
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WO2012166626A1 (fr) * 2011-05-27 2012-12-06 University Of Washington Through Its Center For Commercialization Réactifs et procédés pour traiter une maladie dentaire
WO2014177602A1 (fr) * 2013-04-30 2014-11-06 Straumann Holding Ag Trap 63
WO2017056092A1 (fr) 2015-09-30 2017-04-06 Hadasit Medical Research Services And Development Ltd. Utilisation de l'amélogénine pleine longueur pour favoriser la croissance ou la régénération nerveuse

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012166626A1 (fr) * 2011-05-27 2012-12-06 University Of Washington Through Its Center For Commercialization Réactifs et procédés pour traiter une maladie dentaire
US9290555B2 (en) 2011-05-27 2016-03-22 University Of Washington Through Its Center For Commercialization Reagents and methods for treating dental disease
US9809633B2 (en) 2011-05-27 2017-11-07 University Of Washington Through Its Center For Commercialization Reagents and methods for treating dental disease
WO2014177602A1 (fr) * 2013-04-30 2014-11-06 Straumann Holding Ag Trap 63
US10730918B2 (en) 2013-04-30 2020-08-04 Straumann Holding Ag Trap 63
WO2017056092A1 (fr) 2015-09-30 2017-04-06 Hadasit Medical Research Services And Development Ltd. Utilisation de l'amélogénine pleine longueur pour favoriser la croissance ou la régénération nerveuse
US20180271950A1 (en) * 2015-09-30 2018-09-27 Hadasit Medical Research Services And Development Ltd. Use of full-length amelogenin for promoting nerve growth or regeneration
US10478476B2 (en) 2015-09-30 2019-11-19 Hadasit Medical Research Services And Development Ltd. Use of full-length amelogenin for promoting nerve growth or regeneration

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