EP1534737A2 - Antagonistes peptidiques des elements de la famille du tgf-beta et utilisations therapeutiques de ces antagonistes - Google Patents

Antagonistes peptidiques des elements de la famille du tgf-beta et utilisations therapeutiques de ces antagonistes

Info

Publication number
EP1534737A2
EP1534737A2 EP03728390A EP03728390A EP1534737A2 EP 1534737 A2 EP1534737 A2 EP 1534737A2 EP 03728390 A EP03728390 A EP 03728390A EP 03728390 A EP03728390 A EP 03728390A EP 1534737 A2 EP1534737 A2 EP 1534737A2
Authority
EP
European Patent Office
Prior art keywords
seq
tgf
naturally occurring
amino acid
wound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP03728390A
Other languages
German (de)
English (en)
Other versions
EP1534737A4 (fr
Inventor
Jung San Huang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
St Louis University
Original Assignee
St Louis University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by St Louis University filed Critical St Louis University
Publication of EP1534737A2 publication Critical patent/EP1534737A2/fr
Publication of EP1534737A4 publication Critical patent/EP1534737A4/fr
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki

Definitions

  • the invention relates generally to antagonists of TGF- ⁇ activity, particularly to peptide antagonists of TGF- ⁇ activity.
  • the invention also relates to methods of accelerating wound healing and preventing scarring by administering peptide antagonists of TGF- ⁇ activity to vertebrates.
  • Transforming growth factor ⁇ is a family of 25-kDa structurally homologous dimeric proteins containing one interchain disulfide bond and four intrachain disulfide bonds.
  • the TGF- ⁇ family is composed of three known members (TGF- ⁇ i, TGF- ⁇ 2 , and TGF- ⁇ 3 ) in mammalian species.
  • TGF- ⁇ is a bifunctional growth regulator: it is a growth inhibitor for epithelial cells, endothelial cells, T-cells, and other cell types and a mitogen for mesenchymal cells.
  • TGF- ⁇ also has other biological activities, including stimulation of collagen, fibronectin, and plasminogen activator inhibitor -1 (PAI-1) synthesis, stimulation of angiogenesis, and induction of differentiation in several cell lineages.
  • PAI-1 plasminogen activator inhibitor -1
  • TGF- ⁇ has been implicated in the pathogenesis of various diseases such as cancer, macular degeneration, intimal hyperplasia following angioplasty, tissue fibrosis (which includes integument scar tissue formation, liver cirrhosis, kidney fibrosis, lung fibrosis, heart fibrosis and others) and glomerulonephritis. It is known in the art that TGF- ⁇ plays an important role in scarring ofthe skin or organ fibrosis, which occurs as a result of injury or other fibrogenic stimulus.
  • TGF- ⁇ 's role in wound healing and scarring revolves around its activity as an important regulator of the extracellular matrix stimulating fibroplasia and collagen deposition and inhibiting extracellular matrix degradation by up-regulating the syntheses of protease inhibitors (see Roberts, 1995; Roberts and Sporn, 1996; and O'Kane and Ferguson ,1997).
  • Neutralizing antibodies to TGF- ⁇ have been used experimentally to reduce scarring of wounds, to prevent lung injury in adult respiratory distress syndrome (ARDS), and to block restenosis following angioplasty in animal models.
  • ARDS adult respiratory distress syndrome
  • TGF- ⁇ antagonists inhibitors that might be useful in inhibiting, ameliorating or reversing the effects of TGF- ⁇ and treating diseases.
  • practical applications have been limited by the large molecular size of the antibodies with resulting instability and poor tissue penetration (O'Kane and Ferguson, ibid;, Shah et al, 1994; Shah et al, 1995).
  • TGF- ⁇ peptide antagonists that block TGF- ⁇ binding to cell surface receptors and inhibit TGF- ⁇ -induced growth and transcriptional activation are described in copending U.S. Application No. 09/095,637 and Huang et al, J. Biol Chem. 272:2 '155-2 '160 (1997).
  • the effective concentrations (EC 50 ) of these peptide antagonists, with amino acid sequences corresponding to the 41st to 65th of TGF- ⁇ ] and TGF- ⁇ 2 range from ⁇ 60 nM to 1 ⁇ M, depending on the targeted TGF- ⁇ isoform.
  • the peptide antagonists are relatively stable, exert rapid inhibitory actions, and can be applied topically.
  • the inventor has discovered that specific peptide-based TGF- ⁇ antagonists are effective in accelerating wound healing and reducing scarring due to wounds, such as burns, scrapes, puncture wounds and lacerations.
  • the TGF- ⁇ antagonist peptides may comprise any one of amino acid sequences as set forth in SEQ ID NO:4-l 1.
  • the advantages to using the TGF- ⁇ antagonist peptides in the treatment of skin wounds and diseases mediated by TGF- ⁇ activity are the chemical stability of the peptides, ease of manufacturing the peptides, and small size of the peptides, which allows for rapid penetration into the wound relative to anti- TGF- ⁇ antibodies.
  • the invention is drawn to a non-naturally occurring peptide that comprises an amino acid sequence derived from TGF- ⁇ 1, TGF- ⁇ 2 or TGF- ⁇ 3, wherein the peptide is capable of binding to a TGF- ⁇ receptor, thereby rendering the TGF- ⁇ receptor unavailable for the binding of TGF- ⁇ molecules.
  • the peptide comprises a core stretch of amino acids as set forth in SEQ ID NO: 10 or SEQ ID NO: 11.
  • the preferred peptide comprises an amino acid sequence according to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.
  • the peptide may also comprise an amino acid sequence that is at least 68% identical to any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
  • the invention is also drawn to methods of treating diseases in a vertebrate that are mediated by TGF- ⁇ or TGF- ⁇ receptor activity, comprising the step of administering to the vertebrate a peptide that is a TGF- ⁇ antagonist.
  • Diseases that are mediated by TGF- ⁇ or TGF- ⁇ receptor activity include cancer (via reduced immune function or increased angiogenesis), morbid angiogenesis (which includes e.g. macular degeneration and tumor growth ), intimal hyperplasia, cancer, scarring, fibrosis (e.g., liver cirrhosis, kidney fibrosis lung fibrosis, cystic fibrosis, heart fibrosis), diseases of reduced immune function, glomerulonephritis, and respiratory distress syndrome.
  • the peptide comprises a core stretch of amino acids as set forth in SEQ ID NO: 10 or SEQ ID NO: 11.
  • the preferred peptide comprises an amino acid sequence according to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.
  • the peptide may also comprise an amino acid sequence that is at least 68% identical to any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
  • the invention is also drawn to methods of inhibiting the activity of TGF- ⁇ in a vertebrate, comprising the step of administering to the vertebrate a peptide that is a TGF- ⁇ antagonist.
  • “Inhibiting the activity of TGF- ⁇ ” means inhibiting, ameliorating or reversing the physiological effects mediated by TGF- ⁇ in biological systems. Those physiological effects include scar formation, deposition of collagen or other extracellular matrix proteins during wound healing, wound contraction, inhibition or slowing of re-epithelialization (the proliferation of epithelial cells, usually epidermal cells) during the process of healing, restenosis of a blood vessel after angioplasty and the development of some types of cancers.
  • the peptide comprises a core stretch of amino acids as set forth in SEQ ID NO: 10 or SEQ ID NO:l 1.
  • the preferred peptide comprises an amino acid sequence according to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.
  • the peptide may also comprise an amino acid sequence that is at least 68% identical to any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
  • the invention is further drawn to methods of treating wounds comprising the step of topically administering to a wound a composition comprising a vehicle and a peptide that is a TGF- ⁇ antagonist.
  • the method of wound treatment may have any of the following outcomes, which are relative to wounds that have not been treated with the composition: the reduction of scarring, the reduction of wound contraction, the reduction of the deposition of extracellular matrix components, such as adhesion proteins (fibronectin, laminin, and vitronectin are examples of adhesion proteins) and collagens (collagens are of several types, including type I, type II, type III, type rV, type V, type NI and type IX collagen), and the promotion of re-epithelialization during wound healing.
  • adhesion proteins fibronectin, laminin, and vitronectin are examples of adhesion proteins
  • collagens are of several types, including type I, type II, type III, type rV, type V, type NI and type IX collagen
  • the peptide comprises a core stretch of amino acids as set forth in SEQ ID NO: 10 or SEQ ID NO:l 1.
  • the preferred peptide comprises an amino acid sequence according to SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8 or SEQ ID NO:9.
  • the peptide may also comprise an amino acid sequence that is at least 68% identical to any one of SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
  • a preferred vehicle comprises a physiological buffer, such as phosphate buffered saline and a gel, which contains a modified carboxymethyl-cellulose polymer and propylene glycol, such as IntraSite® Gel Hydrogel Wound Dressing (Smith & Nephew, pic, London UK). Wounds include puncture wounds, pressure wounds, abrasions, lacerations and burns. Wounds may be in any vertebrate, including humans.
  • a physiological buffer such as phosphate buffered saline and a gel, which contains a modified carboxymethyl-cellulose polymer and propylene glycol, such as IntraSite® Gel Hydrogel Wound Dressing (Smith & Nephew, pic, London UK).
  • Wounds include puncture wounds, pressure wounds, abrasions, lacerations and burns. Wounds may be in any vertebrate, including humans.
  • compositions comprising a peptide that is a TGF- ⁇ antagonist in a pharmaceutically acceptable excipient.
  • FIGURE 1 Effect of various concentrations of pentacosapeptides, decapeptides, and their structural variants on TGF- ⁇ binding to TGF- ⁇ receptors in mink lung epithelial cells.
  • Cells were incubated with 125 I-TGF- ⁇ , (Panels A and D), 125 I-TGF- ⁇ 2 (Panel B), and 125 I-TGF- ⁇ 3 (Panel C) both with and without 100-fold excess of unlabeled TGF- ⁇ isoforms and various concentrations of peptides ⁇ , 25 (41-65), ⁇ 2 25 (41-65), and ⁇ 3 25 (41-65) (Panels A, B, and C) or of ⁇ 0 (49-58), ⁇ 2 10 (49- 58), ⁇ 3 10 (49-58), ⁇ , 10 (49-58) W52A, ⁇ 2 10 (49-58) S53A, ⁇ 2 10 (49-58) D55A, ⁇ , 25 (41-6
  • the specific binding of 125 I-labeled TGF- ⁇ , isoforms was then determined. The specific binding obtained in the absence of peptide antagonists was taken as 0% inhibition.
  • the specific binding (0% inhibition) of 125 I-TGF ⁇ ls 125 I-TGF ⁇ 2 , and 125 I-TGF ⁇ 3 were 3930 ⁇ 540 cpm/well, 4512 ⁇ 131 cpm/well, and 4219 ⁇ 125 cpm/well, respectively.
  • the error bars are means ⁇ S.D. of triplicate cultures.
  • FIGURE 2 125 I-TGF- ⁇ , -affinity labeling of cell-surface TGF- ⁇ receptors after incubation of mink lung epithelial cells with l25 I-TGF ⁇ in the presence of various concentrations of peptides ⁇ 25 (41-65) and ⁇ 3 2:> (41-65).
  • Cells were incubated with 125 I-TGF- ⁇ in the presence of 100-fold excess of unlabeled TGF- ⁇ i (lane 1) and of various concentrations of ⁇ t 25 (41-65) (lanes 8-13) and ⁇ 3 25 (41-65) (lanes 2-7).
  • the 125 I-TGF- ⁇ affinity labeling was carried out in the presence of DSS.
  • the 125 I-TGF- ⁇ affinity-labeled TGF- ⁇ receptors were analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography.
  • the arrow indicates the location of the 125 I-TGF- ⁇ ! affinity- labeled type N TGF- ⁇ receptor (T ⁇ R-N).
  • the brackets indicate the locations of the 125 I-TGF- ⁇ ! affinity-labeled type I, type II, and type III TGF- ⁇ receptors (T ⁇ R-I, T ⁇ R-II, and TPR-III).
  • FIGURE 3 Effect of peptide ⁇ i 25 (41-65) on TGF- ⁇ induced growth inhibition as measured by DNA synthesis, and TGF- ⁇ i, induced PAI-1 expression in mink lung epithelial cells.
  • Cells were incubated with various concentrations of TGF- ⁇ i in the presence of 18 uM peptide ⁇ ] 25 (41-65).
  • [Methyl- 3 H]thymidine incorporation into cellular D ⁇ A was then determined.
  • the [methyl- 3 H]thymidine incorporation into cellular D ⁇ A in cells treated with and without 10 pM TGF- ⁇ i, were taken as 100 and 0% inhibition.
  • the error bars are means ⁇ S.D. of triplicate cultures.
  • FIGURE 4 Effect of ⁇ 2S (41-65)-CA and ⁇ 25 (41-65)-BSA peptide conjugates on 125 I-TGF- ⁇ binding to TGF- ⁇ receptors in mink lung epithelial cells and on mink lung epithelial cell growth as measured by DNA synthesis.
  • Cells were incubated with 125 I-TGF- ⁇ ! in the presence and absence of 100-fold excess of unlabeled TGF- ⁇ i and various concentrations of ⁇ i 25 (41- 65)-CA peptide conjugate. The specific binding of 125 I-TGF- ⁇ was then determined. The specific binding of 125 I-TGF- ⁇ obtained in the absence of the conjugates was taken as 0% inhibition.
  • the error bars are means + S.D. of triplicate cultures.
  • Cells were treated with various concentrations of ⁇ i 25 (41-65)-CA or ⁇ , 25 (41-65)-BSA peptide conjugate.
  • [Methyl- 3 H]thymidine incorporation into cellular DNA was determined.
  • the [methyl- Hjthymidine incorporation into cellular DNA in cell treated with and without 10 pM TGF- ⁇ i were taken as 100 and 0% inhibition, respectively.
  • the error bars are means ⁇ S.D. of triplicate cultures.
  • FIGURE 5 Panel A shows the amino acid sequences of three TGF- ⁇ molecules and Panel B shows three peptides derived from the TGF- ⁇ molecules, extending from amino acid residue number 41 to 65.
  • FIGURE 6 Kinetics of re-epithelialization and contraction in pig burn wounds treated with a TGF- ⁇ peptantagonist (peptide TGF-beta antagonist). The rates of wound re-epithelization and contraction were measured as a percent of the original wound (panels A and B, respectively).
  • the burns treated with the TGF- ⁇ peptantagonist (peptide TGF-beta antagonist) healed faster than the control wounds after post-bum day 10 (p ⁇ 0.005).
  • the bums treated with the TGF- ⁇ peptantagonist contracted significantly after post-bu day 10 when compared with the control treated with vehicle only (p ⁇ 0.005).
  • FIGURE 7 Acceleration of wound healing and reduction of scarring by application of a TGF- ⁇ peptantagonist (peptide TGF-beta antagonist) to burn wounds of pigs.
  • Bum wounds treated with a TGF- ⁇ peptantagonist (peptide TGF-beta antagonist) or vehicle (gel without peptide) only in two animals (left and right) were photographed immediately after bum injury (left, panel A and B and right, panel A and B), post-bum day 23 (left, panel C and D), post-bum day 34 (right, panel C and D), post-bum day 35 (left, panel E and F) and post-bum day 41 (right, panel E and F).
  • FIGURE 8 Kinetics of contraction in pig excision wounds treated with TGF- ⁇ peptantagonist (peptide TGF-beta antagonist).
  • Excision wounds (3 x 3 cm) were treated with a TGF- ⁇ peptantagonist (peptide TGF-beta antagonist) and vehicle-only every two days for the first 10 days and twice a week for the next 30 days. The rate of wound contraction was determined as a percent of the original wound.
  • the TGF- ⁇ peptantagonist-treated wound contracted vertically less than the control wound.
  • FIGURE 9 Reduction of contraction in pig excision wounds treated with a TGF- ⁇ peptantagonist (peptide TGF-beta antagonist).
  • Excision wounds (3 3 cm) on the back of pig skin were treated with a TGF- ⁇ peptantagonist (peptide TGF-beta antagonist) (panels A and C) and vehicle-only (panels B and D) every two days for the first 10 days and twice a week for the next 30 days.
  • the wounds were photographed immediately after excision injury (panels A and B) and at post- wound day 34 (panels C and D).
  • the TGF- ⁇ peptantagonist (peptide TGF-beta antagonist)-treated wound exhibited less vertical (length of the healed wound) contraction when compared with the control wound.
  • FIGURE 10 Immunostaining for type I collagen and fibronectin of excision wounds in pigs.
  • Sections of pig excision wounds treated with a TGF- ⁇ peptantagonist (peptide TGF-beta antagonist) (panels A, C and E), which were harvested on post-excision day 28, were histologically evaluated using hematoxylin/eosin staining (panels A and B) and were immunostained for type I collagen and fibronectin (panels C, D and E, F, respectively).
  • the wound treated with a TGF- ⁇ peptantagonist (peptide TGF-beta antagonist) showed less intensity of staining for type I collagen and fibronectin than the control wound.
  • FIGURE 1 1 Reduction of scarring in rabbit ear excision wounds treated with TGF- ⁇ peptantagonist (peptide TGF-beta antagonist).
  • Excision wounds 0.5 x 1 cm
  • rabbit ears were treated with a TGF- ⁇ peptantagonist (peptide TGF-beta antagonist) (TGF-blocker), vehicle only (sham) and nothing (negative). These wounds were photographed immediately after excision injury (panel A) and at post-excision day 10.
  • the TGF- ⁇ -peptantagonist (peptide TGF-beta antagonist) treated wounds showed reduced scarring relative to the control wounds.
  • TGF- ⁇ antagonists or inhibitors that specifically bind to TGF- ⁇ receptors which include type I, type II, type III and type V receptors. It was discovered that three chemically synthesized peptides, which correspond in sequence to amino acid numbers 41-65 of TGF- ⁇ i (SEQ ID NO:4), TGF- ⁇ 2 (SEQ ID NO:5), and TGF- ⁇ 3 (SEQ ID NO:6), and which comprise a core amino acid sequence as set forth in SEQ ID NO: 10 or SEQ ID NO:l l, inhibit the binding of TGF- ⁇ TGF- ⁇ 2 , and TGF- ⁇ 3 , to TGF- ⁇ receptors in epithelial cells.
  • the peptides also block TGF- ⁇ - induced growth inhibition and TGF- ⁇ -induced expression of PAI-1 in epithelial cells. It was also discovered that the W/RXXD motif found within the peptide sequences determines the specificity of activity of the antagonist peptide. In view of these discoveries, peptides that comprise amino acid sequences corresponding to SEQ ID NO: 10 or SEQ ID NO: 11 are considered to be antagonists of TGF- ⁇ activity. It was also discovered that these TGF- ⁇ peptide antagonists can be converted to partial agonists (i.e., agent which mimics the effects of TGF- ⁇ ) by conjugation to carriers such as proteins or synthetic polymers.
  • partial agonists i.e., agent which mimics the effects of TGF- ⁇
  • SEQ ID NO:4 amino acids 41-65 of TGF- ⁇ 1
  • SEQ ID NO:5 amino acids 41-65 of TGF- ⁇ 2
  • SEQ ID NO:6 amino acids 41-65 of TGF- ⁇ 3
  • TGF- ⁇ peptide agonists may comprise an amino acid sequence that is at least 68% identical to any one of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and including the decapeptides of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.
  • Percent identity is intended to mean the percentage of the same amino acid residues between two sequences.
  • the reference sequence may be SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
  • the two sequences being compared are aligned using the Clustal method (Higgins et al, Cabios 5:189-191, 1992) of multiple sequence alignment in the Lasergene biocomputing software (DNASTAR, INC, Madison, WT). In this method, multiple alignments are carried out in a progressive manner, in which larger and larger alignment groups are assembled using similarity scores calculated from a series of pairwise alignments.
  • Optimal sequence alignments are obtained by finding the maximum alignment score, which is the average of all scores between the separate amino acid residues in the alignment, determined from a residue weight table representing the probability of a given amino acid change occurring in two related peptides over a given evolutionary interval. Penalties for opening and lengthening gaps in the alignment contribute to the score.
  • the residue weight table used for the alignment program is PAM250 (Dayhoff et al., in Atlas of Protein Sequence and Structure, Dayhoff, Ed., NBRF, Washington, Vol. 5, suppl. 3, p. 345, 1978).
  • non-naturally occurring peptides and modifications thereof, that antagonize TGF- ⁇ activity, and compositions comprising peptides that antagonize of TGF- ⁇ activity.
  • non-naturally occurring it is meant that the peptide is artificially produced by chemical synthesis, genetic recombinant methods or enzymatic digestion of isolated polypeptides, and that the peptide does not comprise a full length mature TGF- ⁇ polypeptide.
  • the non-naturally occurring peptide may be modified, wherein such modifications include glycosylation, lipidation, amidation, phosphorylation, acetylation, PEGylation (the addition of polyethylene glycol to stabilize the peptide) and albumination (the conjugation of an albumin moiety to increase the biological half-life of the peptide).
  • antagonization it is meant that the non-naturally occurring peptide ofthe instant invention binds to a TGF- ⁇ receptor and prevents the activation of that TGF- ⁇ receptor.
  • Antagonization may be complete or it may be partial, whereby some TGF- ⁇ receptor activation may occur in the vertebrate after administration of the non-naturally occurring peptide.
  • TGF- ⁇ receptors are intergral membrane proteins that bind TGF- ⁇ molecules.
  • TGF- ⁇ receptors generally comprise a type I receptor component and a type II receptor component.
  • type I receptors activin receptor-like kinases 1 to 6 (ALK1- ALK6)
  • type II receptors activin type II and type IIB receptor (ActRII/IIB), TGF- ⁇ type II receptor (T ⁇ RII), BMP type II receptor (BMPRII), and MIS type II receptor (MISRII).
  • TGF- ⁇ receptors in addition to type I and type II types, also include type III and type V receptors (Ref. 6).
  • TGF- ⁇ receptors are covered by the term "TGF- ⁇ receptor".
  • TGF- ⁇ receptor biology see Moustakas, et al, J. Cell Sci. 114:4359-4369, which is herein incorporated by reference.
  • the non-naturally occurring peptides bind to TGF- ⁇ receptors, thereby blocking the binding of active TGF- ⁇ receptor agonists to TGF- ⁇ receptors and "inhibiting the activity of TGF- ⁇ ".
  • the activities of TGF- ⁇ , mimetics of TGF- ⁇ or TGF- ⁇ receptor agonists include (a) both proliferation and anti-proliferation effects on certain cells and tissues, depending on the state and type of cell, (b) cell differentiation, cell death, cell migration, embryonic development, tumor growth and wound healing, and (c) promoting the production of cell-adhesion molecules, extracellular matrix molecules and other growth factors.
  • the non-naturally occurring peptide TGF- ⁇ antagonists of he present invention are useful in treating individuals suffering from diseases or conditions that are modulated at least in part by TGF- ⁇ .
  • Diseases and conditions which may be ameliorated by the administration of peptide TGF- ⁇ antagonists include carcinomas, such as breast cancer and pancreatic cancer (see Gold, L.I., [1999] "The role of transforming growth factor- ⁇ (TGF- ⁇ ) in human cancer", Crit. Rev. Oncol.
  • developmental defects such as neural tube defects, wounds, such as cutaneous bums, lacerations, punctures and abrasions, intimal hyperplasia (which results in blood vessel blockage by the thickening of arterial lining) and restenosis of blood vessels after angioplasty, angiogenesis that allows tumor growth, insufficient immune system function, angiogenesis (which is involved in e.g.
  • compositions which comprise a peptide TGF- ⁇ antagonist, useful in the treatment of diseases or conditions that are modulated at least in part by TGF- ⁇ .
  • a peptide TGF- ⁇ antagonist which comprises a sequence of SEQ ID NO: 10 or SEQ ID NO: 11 , (a) accelerates re-epithelialization of skin and reduces wound contraction and scarring during the healing of a bum injury and diminishes wound contraction and scarring, relative to untreated control wounds, in both the pig and rabbit excision injury models.
  • "Re-epithelialization” is the growth of the outer layer of skin or epidermis over the wound during the healing process.
  • Bum wound healing consists of epithelialization, contraction and formation of granulation and scar tissue (Refs. 8-12). TGF- ⁇ is believed to be involved in most of these events.
  • the peptide TGF- ⁇ antagonist of the present invention is thought to block or slow down the occurrence of these events.
  • the mechanism of enhanced re-epithelialization in wounds treated with the peptide TGF- ⁇ antagonist of the present invention may involve increased keratinocyte proliferation (transient inhibition of keratinocyte proliferation by TGF— ⁇ may be an integral component in the complex process of wound healing) coupled with a migration response stimulated by growth factors other than TGF- ⁇ (Refs. 18-21).
  • the peptide TGF- ⁇ antagonist of the present invention has been shown to block complex formation between ⁇ 2 -macroglobulin and growth factors, cytokines and hormones (see reference 15) and thus, may enhance activation of these substances or agents by blocking inhibition of their activities mediated by ⁇ 2 -macroglobulin.
  • Peptide TGF- ⁇ antagonists ofthe present invention comprise the amino acid motif W/RSXD, wherein X is any amino acid (SEQ ID NO: 10 and SEQ ID NO: 11).
  • W/RXXD motif was demonstrated to be an important site involved in the interaction of peptides with TGF- ⁇ receptors.
  • Preferred peptide TGF- ⁇ antagonists comprise any one of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8 or SEQ ID NO:9; or peptides that are at least 68% identical to these sequences.
  • compositions of the present invention may be administered by any . suitable route known in the art including for example via intraarterial catheterization, intravenous, subcutaneous, intramuscular, transdermal, intrathecal, intracerebral, oral or topical. Administration may be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation. For treating tissues in the central nervous system, administration may be by injection or infusion into the cerebrospinal fluid (CSF). When it is intended that a peptide TGF- ⁇ antagonist be administered to cells in the central nervous system, administration may be with one or more agents capable of promoting penetration of a peptide TGF- ⁇ antagonist across the blood-brain barrier.
  • CSF cerebrospinal fluid
  • the peptide antagonist may be administered via intraarterial catheterization during angioplasty procedures.
  • the peptide may also be applied on the surface ofthe stent that is left in place during angioplasty.
  • the peptide TGF- ⁇ antagonist may be subcutaneously injected into the area of the incision or healing wound.
  • a peptide TGF- ⁇ antagonist may also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties.
  • a peptide TGF- ⁇ antagonist may be coupled to any substance known in the art to promote penetration or transport across the blood-brain barrier such as an antibody to the transferrin receptor, and administered by intravenous injection.
  • a peptide TGF— ⁇ antagonist may be stably linked to a polymer such as polyethylene glycol or albumin to obtain desirable properties of solubility, stability, half-life and other pharmaceutically advantageous properties.
  • a polymer such as polyethylene glycol or albumin
  • compositions comprising peptide TGF- ⁇ antagonists are usually employed in the form of pharmaceutical preparations.
  • Such preparations are made in a manner well known in the pharmaceutical art.
  • One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. It may also be desirable that a suitable buffer be present in the composition.
  • Such solutions may, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection.
  • the primary solvent may be aqueous or alternatively non- aqueous.
  • a peptide TGF- ⁇ antagonist may also be incorporated into a solid or semi-solid biologically compatible matrix which may be implanted into tissues requiring treatment.
  • a peptide TGF- ⁇ antagonist may also be incorporated into a hydrogel wound dressing, such as an IntraSite® Gel Hydrogel Wound Dressing (Smith & Nephew, pic, London UK), which comprises a modified carboxymethyl-cellulose polymer and propylene glycol.
  • the carrier may also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across membranes or other barriers, such as the epidermis, the lining of the alimentary canal, the endothelium or the blood- brain barrier.
  • formulations containing a peptide TGF- ⁇ antagonist are to be administered orally.
  • Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms.
  • suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.
  • the formulations may additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
  • the compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art.
  • the formulations may also contain substances that diminish proteolytic degradation and promote absorption such as, for example, surface active agents.
  • the specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied.
  • the dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations may be made without undue experimentation by one skilled in the art in light of the activity of a peptide TGF- ⁇ antagonist.
  • the data showing activity of a peptide TGF- ⁇ antagonist are herein disclosed in the Examples and in copending application Serial Number 09/095,637, which is herein incorporated by reference.
  • the activity of a peptide TGF- ⁇ antagonist on a particular target cell type may be determined by routine experimentation.
  • Exact dosages are determined in conjunction with standard dose-response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response ofthe individual patient, the severity of the patient's symptoms, and the chosen route of administration.
  • the C44S/C48S versions had better stability in solution during storage, so they were used in most ofthe experiments.
  • the peptides were synthesized using tert-butoxycarbonyl chemistry on an Applied Biosystems Model 431 A peptide synthesizer and purified using Sephadex G-25 column chromatography and reverse-phase HPLC (C-8 column). The purity of the synthesized peptides were verified by automated Edman degradation on an Applied Biosystems Model 477A gas/liquid phase protein sequenator with an on-line Applied Biosystems Model 120A phenylthiohydantoin amino acid analyzer. The purity of all peptides was estimated to be > 95%.
  • the reaction mixture was mixed with 50 ⁇ l of 1 M ethanolamine HCl in 0.1 M NaHC0 3 ( ⁇ pH 9.0). After 2 hr at room temperature, the reaction mixture was dialyzed against 2 liters of 0.1 M NaHCO 3 ( ⁇ pH 9.0). After four changes ofthe dialysis solution, the sample was stored at 4°C prior to use. The molar ratio of peptide ⁇ i 25 (41-65)/carrier protein in the conjugate was determined by amino acid composition analysis.
  • TGF- ⁇ receptors [044] Specific binding of l2i 1-labeled TGF- ⁇ ,, TGF- ⁇ 2 , and TGF- ⁇ 3 ("'i-TGF- ⁇ j, ,25 I-TGF- ⁇ 2 , and l25 I-TGF- ⁇ 3 ) to TGF- ⁇ receptors in mink lung epithelial cells.
  • 125 I-TGF- ⁇ , 125 I-TGF- ⁇ 2 , and ⁇ 5 I-TGF- ⁇ 3 were prepared by iodination of TGF- ⁇ ,, TGF- ⁇ 2 , and TGF- ⁇ 3 with Na 125 I as described previously (Ref. 7).
  • the specific radioactivities of 125 I-TGF- ⁇ , 1 5 I-TGF- ⁇ 2 , and 125 I-TGF- ⁇ 3 were 1-3 x IO 5 cpm ng.
  • Mink lung epithelial cells were grown on 24-well clustered dishes to near confluence in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum. The epithelial cells were incubated with 0.1 nM 125 I-TGF- ⁇ b 125 I-TGF- ⁇ 2 , or 125 I-TGF- ⁇ 3 both with and without 100-fold excess of unlabeled TGF- ⁇ b TGF- ⁇ 2 , or TGF- ⁇ 3 in binding buffer (Ref. 7).
  • DMEM Dulbecco's modified Eagle medium
  • I23 I-TGF- / -affinity labeling of cell-surface TGF- ⁇ receptors in mink lung epithelial cells Mink lung epithelial cells grown on 60-mm Petri dishes were incubated with 0.1 nM 125 I in the presence of various concentrations of peptide ⁇ i 25 (41-65) or peptide ⁇ 3 25 (41-65) in binding buffer. After 2.5 hr at 0°C, 125 I-TGF- ⁇ r aff ⁇ nity labeling was carried out in the presence of DSS as described. The 125 I-TGF- ⁇ -affinity-labeled TGF- ⁇ receptors were analyzed by 5% SDS-polyacrylamide gel electrophoresis under reducing conditions and autoradiography.
  • the cells were then washed twice with 1 ml of 10% trichloroacetic acid and once with 0.5 ml of ethanohether (2: 1, v/v). The cells were then dissolved in 0.4 ml of 0.2 N NaOH and counted with a liquid scintillation counter.
  • RNA Analysis- Mink lung epithelial cells were grown overnight in 12-well clustered dishes in DMEM containing 10% fetal calf serum. The medium was then changed to DMEM containing 0.1%) fetal calf serum and the cells were incubated with 0.25 and 2.5 pM TGF- ⁇ i in the presence of various concentrations of peptide ⁇ i 25 (41-65) for 2.5 hr. Total cellular RNA was extracted using RNAzol B (Tel-Test Inc.) according to the manufacturer's protocol. RNA was electrophoresed in 1.2 % agarose- formaldehyde gel and transferred to Duralon-UV membranes using 10 x SSC.
  • the membranes were probed at 42°C with a random-primed, radiolabeled 1-kb fragment from the Hind III and Neol digests of PAI-1 cDNA and glyceraldehyde-3 -phosphate dehydrogenase ("GAPDH”) cDNA.
  • the blots were washed with 0.1 x SSC containing 0.1 % SDS at room temperature.
  • peptide antagonists of TGF- ⁇ seven pentacosapeptides (peptides containing 25 amino acids) were synthesized: peptide ⁇ , 25 (21-45), peptide ⁇ , 25 (31-55), peptide ⁇ , 25 (41-65) (SEQ ID NO:4), peptide ⁇ , 25 ( 51-75), peptide ⁇ , 25 (61-85), peptide ⁇ , 25 (71-95), and peptide ⁇ , 25 (81-105), whose amino acid sequences overlap one another and cover most of the human TGF- ⁇ i molecule, the monomer of which has 112 amino acid residues (SEQ ID NO: l) (ref. 1).
  • the antagonist activities of these peptides were first tested for their abilities to inhibit 125 I-labeled TGF- ⁇ i ( 125 I-TGF- ⁇ ) binding to cell-surface TGF- ⁇ receptors in mink lung epithelial cells, an art recognized model system for investigating TGF- ⁇ receptor types and TGF- ⁇ -induced cellular responses (ref. 2).
  • peptide ⁇ ] 25 (41-65) (SEQ ID NO:4) is a TGF- ⁇ inhibitor or antagonist.
  • TGF- ⁇ isoforms (TGF- ⁇ b TGF- ⁇ 2 , and TGF- ⁇ 3 ) have been shown to exhibit different potencies in inducing cellular responses in certain cell types or systems. There is -70% amino acid sequence homology at the 41st to 65th amino acid residues among these three TGF- ⁇ isoforms (Refs. 1-3) ( Figure 5A).
  • Peptide ⁇ 2 25 (41-65) also had an IC 5 o of -0.08 ⁇ M for inhibiting 125 I-TGF- ⁇ 3 binding to TGF- ⁇ receptors in these epithelial cells.
  • the region spanning residues 41-65 comprises a loop in the three-dimensional structure of TGF- ⁇ ] and TGF ⁇ 2 (Ref. 4, 5). This loop is accessible to solvent according to X-ray and NMR analyses (Ref. 4, 5). There are two reasons why a WSXD (for TGF- ⁇ , and TGF ⁇ 2 ; SEQ ID NO: 10) or RSXD (for TGF- ⁇ 3 ; SEQ ID NO: 11) motif in the loop is a good candidate site whereby these antagonist peptides and their parent molecules could interact with TGF- ⁇ receptors.
  • Peptide ⁇ 3 25 (41-65) possesses a distinct motif of RSXD (SEQ ID NO: 11) and is a weaker inhibitor (IC 50 of -20 ⁇ M).
  • the K d s for TGF- ⁇ , and TGF ⁇ 2 binding to the type V TGF- ⁇ receptor are identical (-0.4 nM), whereas the Kd of TGF- ⁇ 3 binding to the type V receptor is higher (-5 nM) (Ref. 6).
  • Figure ID shows that peptide ⁇ , 25 (41-65) W52A D55A and peptide ⁇ 3 25 (41-65) R52A/D55A did not inhibit 125 I-TGF- ⁇ , binding to TGF- ⁇ receptors, thereby supporting the conclusion that the motif W/RXXD is involved in the interactions ofthe instant peptide antagonists with TGF- ⁇ receptors.
  • Mink lung epithelial cells express all of the known TGF- ⁇ receptors (type I, type II, type III, and type V receptors) (see Ref. 6). To determine the relative sensitivities of TGF- ⁇ receptor types to inhibition by peptides ⁇ ] 25 (41-65) and ⁇ 3 25 (41-65) with respect to ligand binding, mink lung epithelial cell-surface TGF- ⁇ receptors were labeled with 125 I-TGF- ⁇ in the presence of various concentrations of peptides ⁇ , 25 (41-65) and ⁇ 3 25 (41-65).
  • TGF- ⁇ receptors type I, type II, type III, and type N receptors
  • Peptide ⁇ , 25 (41-65) appeared to inhibit the I25 I-TGF- ⁇ -affinity labeling of all TGF- ⁇ receptor types in a concentration-dependent manner (lanes 8-12).
  • ⁇ , 25 (41-65) inhibition ofthe ,25 I-TGF- ⁇ ]-affinity labeling of the type V TGF- ⁇ receptor was greater than its inhibition of other TGF- ⁇ receptor types.
  • peptides ⁇ , 25 (41-65), ⁇ 2 25 (41-65), and ⁇ 3 25 (41-65) are potent inhibitors for 125 I-TGF- ⁇ , binding to TGF- ⁇ receptors.
  • these peptides are shown to block a TGF- ⁇ -induced cellular response, i.e., growth inhibition.
  • the effect of peptide ⁇ , 25 (41-65) on TGF- ⁇ r induced growth inhibition was investigated by exposing mink lung epithelial cells to various concentrations of TGF- ⁇ ] in the presence of 18 ⁇ M peptide ⁇ ] 25 (41-65) and measuring cellular D ⁇ A synthesis.
  • TGF- ⁇ transcriptional activation of collagen, adhesion protein (i.e., fibronectin), and PAI-1.
  • adhesion protein i.e., fibronectin
  • PAI-1 protein-bindesion protein
  • TGF- ⁇ The ' dimeric structure of TGF- ⁇ has been shown to be required for its biological activities.
  • peptide ⁇ , 25 (41-65) contains the active site sequence involved in the interaction of TGF- ⁇ ] with TGF- ⁇ receptors, one may be able to convert its antagonist activity to agonist activity by conjugating peptide ⁇ ] 25 (41-65) to carrier proteins, such that the ⁇ , 25 (41-65)-protein conjugates would carry multiple valences of the putative active site.
  • peptide ⁇ ] 25 (41-65) was conjugated to carrier proteins CA (carbonic anhydrase) and BSA (bovine serum albumin) using the cross-linking agent DSS.
  • DSS mainly cross-links the ⁇ -amino group of peptide ⁇ , 25 (41-65) to the G-amino groups of the carrier proteins.
  • the ⁇ , 25 (41-65)-BSA and ⁇ , 25 (41-65)-CA conjugates contained -5-10 molecules of peptide ⁇ ] 25 (41-65) per molecule of carrier protein.
  • the ⁇ j 25 (41-65)-CA conjugate inhibited 125 I-TGF- ⁇ binding to TGF- ⁇ receptors in mink lung epithelial cells with an IC 50 of -0.05 ⁇ M.
  • the ⁇ s 25 (41-65)-BSA conjugate had a similar IC 50 of - -0.06 ⁇ M.
  • These IC 5 o are -20-fold lower than that of peptide ⁇ ] 25 (41-65) prior to conjugation.
  • both BSA and CA conjugated without peptides did not have inhibitory activity.
  • Example 2 Peptide TGF- ⁇ antagonist reduces scarring and promotes healing
  • IntraSite® gel containing TGF- ⁇ peptantagonist (peptide TGF-beta antagonist). 1 ml of sterile 6 M peptide ⁇ i 25 (41-65) in phosphate buffered saline or 1 ml of sterile phosphate buffered saline was vigorously mixed with 3 ml of IntraSite® gel using two 10 ml syringes connected with a three-way connector. The Intrasite® gel containing peptide ⁇ ] 25 (41-65) and Intrasite® gel containing buffer without peptide were stable at least for several weeks.
  • Burn wound model Four pigs weighing 20-25 kg were anesthetized by intramuscular injection of ketamine (5 mg/kg), strenil® (cazaporonum) (20 mg/kg) and atropine (5 mg/kg). Six uniform bum wounds (110°C, 30 sec) were then made symmetrically on the back of each pig using a modified soldering iron (Ref. 15) with a flat contact area of 20 cm 2 . The bum injury was equivalent to a full-thickness bum injury in humans and uniformly caused coagulation and necrosis of dermis. After wounding, a thin layer of Intrasite® gel containing either peptide ⁇ ] 25 (41-65), buffer or nothing else was applied to the wounds.
  • Intrasite® gel containing either peptide ⁇ ] 25 (41-65), buffer or nothing else was applied to the wounds.
  • Excision wound model Four pigs received intramuscular injection of ketamine, strenil® and atropine as described above. Six excision injuries were generated by removing full-thickness sections of skin (3 x 3 cm) from standardized sites on the back of each animal using a scapel. Three rabbits, were anesthetized by intramuscular injection of ketamine (5 mg/kg). Three excision injuries were produced in each by removing full-thickness sections of skin (0.5 x 1 cm) from each ear.
  • IntraSite® gel containing peptide ⁇ , 25 (41-65) was applied to alternating wounds on each animal and IntraSite® gel + buffer was applied to the other half.
  • the excision wounds were then dressed with a paraffin gauze.
  • the dressing was changed every two days for the first 10 days and twice a week for the next 30 days.
  • the dressing was changed for the first 3 days.
  • Gel containing peptide ⁇ ] 25 (41-65) or control gel was applied at each dressing change.
  • E rate of re-epithelialization in percent
  • An area enclosed by the normal hair bearing skin on a given post-bu day
  • Ao area of open wound on the same day as was measured. Wound contraction was calculated using the following formula:
  • C rate of wound contraction in percent
  • Al wound area as measured immediately following the bum or excision injury
  • An area enclosed by normal hair bearing skin.
  • the pig model is an art recognized model used in bum experiments because porcine skin is anatomically very similar to human skin (Ref. 15, 17). Pigs weighing about 20-25 kg were anesthetized by intramuscular injection of ketamine (5 mg/kg). A soldering iron with a flat contact area of -20 cm 2 was used to generate a full-thickness bum injury (110°C, 30 sec) on the skin of the back in four pigs. Six thermal bums (three on each side) were created on each pig. After wounding, two lesions were treated with a thin layer of a sterile IntraSite® gel containing peptide ⁇ ] 25 (41-65) (1.5 mM); two received gel alone and two received topical applications.
  • the term "vehicle” refers generally to any solvent, buffer, gel or carrier in which the active peptide may be dispersed or dissolved.
  • the preferred vehicle is a gel, such as the IntraSite® gel comprising modified carboxymethyl-cellulose polymer and propylene glycol. Each animal served as its own control.
  • the excision injury wound In contrast to the bu injury, the excision injury wound exhibited near complete horizontal (width of the healed wound) contraction by post-bum day 30 (Fig. 9).
  • the wound treated with peptide ⁇ , 25 (41-65) showed less vertical (length ofthe healed wound) contraction compared with that treated with vehicle only (Fig. 9C and D).
  • TGF- ⁇ is known to mediate the deposition of such extracellular matrix proteins by stimulating their biosynthesis and attenuating their degradation. Therefore, the content of type I collagen and fibronectin in excision-injury wounds (on post-excision day 30) in pigs was determined using immunohistochemistry. As shown in Fig. 10, peptide ⁇ ] 25 (41-65) treatment diminished the deposition of type I collagen and fibronectin (Fig. 10C vs 10D and 10E vs 10F, respectively).
  • the mechanism of enhanced re-epithelialization in wounds treated with the peptide ⁇ [ 25 (41-65) remains to be determined, but may involve increased keratinocyte proliferation (transient inhibition of keratinocyte proliferation by TGF- ⁇ may be an integral component in the complex process of wound healing) coupled with a migration response stimulated by growth factors other than TGF- ⁇ (Refs. 18-21).
  • the peptide ⁇ , 25 (41-65) which was recently shown to block complex formation between ⁇ 2 - macroglobulin and growth factors, cytokines and hormones (Ref. 22), may enhance activation of these substances or agents by blocking inhibition of their activities mediated by ⁇ 2 -macroglobulin.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Zoology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention se rapporte à des procédés d'utilisation d'antagonistes peptidiques du TGF-bêta dans le but de faciliter la cicatrisation de plaies cutanées, notamment de brûlures, de lacérations et d'écorchures. L'administration à des plaies d'antagonistes peptidiques du TGF-bêta permet de réduire la formation de cicatrices, la contraction des plaies et le dépôt de composants matriciels extracellulaires, et elle permet également d'accroître les taux de ré-épithélialisation lors de la cicatrisation des plaies.
EP03728390A 2002-04-29 2003-04-15 Antagonistes peptidiques des elements de la famille du tgf-beta et utilisations therapeutiques de ces antagonistes Ceased EP1534737A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US13594602A 2002-04-29 2002-04-29
US135946 2002-04-29
PCT/US2003/011437 WO2003093293A2 (fr) 2002-04-29 2003-04-15 Antagonistes peptidiques des elements de la famille du tgf-beta et utilisations therapeutiques de ces antagonistes

Publications (2)

Publication Number Publication Date
EP1534737A2 true EP1534737A2 (fr) 2005-06-01
EP1534737A4 EP1534737A4 (fr) 2007-10-31

Family

ID=29399231

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03728390A Ceased EP1534737A4 (fr) 2002-04-29 2003-04-15 Antagonistes peptidiques des elements de la famille du tgf-beta et utilisations therapeutiques de ces antagonistes

Country Status (4)

Country Link
EP (1) EP1534737A4 (fr)
AU (1) AU2003234734A1 (fr)
CA (1) CA2484994C (fr)
WO (1) WO2003093293A2 (fr)

Families Citing this family (19)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7723473B2 (en) 1997-06-19 2010-05-25 St. Louis University Peptide antagonists of TGF-beta family members and therapeutic uses thereof
CA2588292C (fr) * 2004-12-01 2019-01-15 Health Protection Agency Conjugues proteiques non cytotoxiques
US10059756B2 (en) 2006-11-02 2018-08-28 Acceleron Pharma Inc. Compositions comprising ALK1-ECD protein
KR20170012582A (ko) 2006-11-02 2017-02-02 악셀레론 파마 인코포레이티드 Ａｌｋ１ 수용체 및 리간드 길항제 및 그의 용도
US8642031B2 (en) 2006-11-02 2014-02-04 Acceleron Pharma, Inc. Antagonists of BMP9, BMP10, ALK1 and other ALK1 ligands, and uses thereof
KR100879239B1 (ko) 2007-04-19 2009-01-16 (주)케어젠 Ｔｇｆｐ－ｃａｐ 펩타이드 및 그의 용도
EP2192904B1 (fr) 2007-08-27 2016-08-17 Auxagen, Inc. PROCÉDÉS D'INHIBITION DE TGF-beta
AU2009241755B2 (en) 2008-05-02 2015-10-01 Acceleron Pharma Inc. Methods and compositions based on ALK1 antagonists for modulating angiogenesis and pericyte coverage
ES2337973B8 (es) 2008-05-16 2011-07-21 Proyecto De Biomedicina Cima, S.L. Adenovirus auxiliares auto-inactivantes para la produccion de adenovirus recombinantes de alta capacidad.
ES2330826B1 (es) 2008-06-04 2010-07-26 Proyecto De Biomedicina Cima, S.L. Sistema para empaquetamiento de adenovirus de alta capacidad.
WO2010033507A1 (fr) 2008-09-16 2010-03-25 St. Louis University Procédé d'amélioration de signalisation tgf-bêta
WO2010119997A1 (fr) * 2009-04-14 2010-10-21 (주)케어젠 Peptide ayant une activite de facteur de croissance de transformation-beta et son procede de production
BR112012010755A2 (pt) 2009-11-05 2015-09-22 Proyecto Biomedicina Cima Sl constructo de gene que permite a expressão hepato-específica induzível de um polinucleotídeo de interesse em resposta a um agente indutor, vetor, genoma viral recombinante, vírion, método in vitro para a expressão de um polinucleotídeo de interesse em uma célula de origem hepática, composição farmacêutica e operador-promotor bi-direcional induzível adequado para a expressão hepato-específica induzível de dois polinucleotídeos de interesse por um agente indutor
WO2012001196A2 (fr) 2010-06-28 2012-01-05 Proyecto De Biomedicina Cima, S.L. Vecteurs alphaviraux et leur utilisation pour l'expression de gènes hétérologues
EP2407534A1 (fr) 2010-07-14 2012-01-18 Neo Virnatech, S.L. Procédés et réactifs pour obtenir des particules virales actives du point de vue transcriptionnel et des virions recombinants
WO2013113755A1 (fr) 2012-01-30 2013-08-08 Fundació Institut D'investigació Biomèdica De Bellvitge (Idibell) Réactifs et méthodes utilisables en vue du traitement de maladies par inhibition de la voie de signalisation calcineurine-nfat
ES2523016B1 (es) 2013-05-20 2015-09-09 3P Biopharmaceuticals Vectores alfavirales y líneas celulares para la producción de proteínas recombinantes
EP2878674A1 (fr) 2013-11-28 2015-06-03 Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) Épisomes stables sur la base des vecteurs lentiviraux non intégratifs
WO2015101666A1 (fr) 2014-01-03 2015-07-09 Fundación Biofísica Bizkaia Vlp, procédés pour leur obtention et applications de ceux-ci

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1132403A1 (fr) * 1998-11-24 2001-09-12 Instituto Cientifico Y Tecnologico De Navarra, S.A. Peptides inhibiteurs de tgf g beta1

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4423037A (en) * 1982-05-13 1983-12-27 The General Hospital Corporation Inhibitors of peptide hormone action
US5571714A (en) * 1988-12-22 1996-11-05 Celtrix Pharmaceuticals, Inc. Monoclonal antibodies which bind both transforming growth factors β1 and β2 and methods of use

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1132403A1 (fr) * 1998-11-24 2001-09-12 Instituto Cientifico Y Tecnologico De Navarra, S.A. Peptides inhibiteurs de tgf g beta1

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO03093293A2 *

Also Published As

Publication number Publication date
WO2003093293A2 (fr) 2003-11-13
EP1534737A4 (fr) 2007-10-31
CA2484994A1 (fr) 2003-11-13
WO2003093293A3 (fr) 2005-04-07
CA2484994C (fr) 2016-06-14
AU2003234734A1 (en) 2003-11-17
AU2003234734A8 (en) 2003-11-17

Similar Documents

Publication Publication Date Title
CA2484994C (fr) Antagonistes peptidiques des elements de la famille du tgf-beta et utilisations therapeutiques de ces antagonistes
US20110008248A1 (en) Peptide Antagonists of TGF-Beta Family Members and Therapeutic Uses Thereof
EP0494264B1 (fr) Inhibition de facteur de croissance a transformation afin d'empecher l'accumulation de matrice extracellulaire
EP0527787B1 (fr) Procede destine a predisposer les mammiferes a la regeneration acceleree des tissus
CA2105652C (fr) Reduction de la cicatrisation en utilisant des anticorps a facteurs de croissance
AU772778B2 (en) Novel inhibitors of angiogenesis and tumor growth
Huang et al. Synthetic TGF‐β antagonist accelerates wound healing and reduces scarring
JP3854307B2 (ja) 細胞外マトリックスの蓄積を防止するためのトランスフォーミング増殖因子βの阻害
US6013628A (en) Method for treating conditions of the eye using polypeptides
JPH08510443A (ja) 生物学的に活性なＴＧＦ−β１およびＴＧＦ−β２ペプチド
US7060680B2 (en) Morphogen treatments for limiting proliferation of epithelial cells
CA2859412C (fr) Distribution d'agents therapeutiques employant des segments polypeptidesliant un collagene bacterien
JP2000517174A (ja) ケラチノサイト増殖因子―２（ｋｇｆ―２または線維芽細胞増殖因子―１２、ｆｇｆ―１２）
US10213488B2 (en) Delivery of therapeutic agents by a collagen binding protein
JPH09505555A (ja) ＴＧＦ−βの活性を刺激および阻害する方法および組成物
US7488719B1 (en) Wound and cutaneous injury healing with a nucleic acid encoding perlecan
Liu et al. The regulatory effects of cytokines on lymphatic angiogenesis
US6500920B1 (en) Inhibitor of transforming growth factor β and A method of inhibiting the biological effects of transforming growth factor
EP0680334B1 (fr) Traitement d'ulceres gastro-intestinaux avec des morphogenes
WO1993013794A1 (fr) Nouveaux procedes et compositions de traitement des maladies angiogeniques
US20060100156A1 (en) Cell nucleus-entering compositions
WO1995015176A1 (fr) Procedes activant la duree de vie et facilitant la differentiation des sous-classes de neurones cholinergiques et serotonergiques a l'aide du facteur 5 de croissance des fibroblastes
KR20230034167A (ko) Kai1 폴리펩타이드를 포함하는 간 섬유화 억제용 약학 조성물 및 이의 용도
WO2024050501A2 (fr) Compositions peptidiques tyr et procédés d'utilisation
Khan Chronic in vivo overexpression of TGF-B| 1 using a retroviral expression vector

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20041126

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

A4 Supplementary search report drawn up and despatched

Effective date: 20071004

17Q First examination report despatched

Effective date: 20080118

APBK Appeal reference recorded

Free format text: ORIGINAL CODE: EPIDOSNREFNE

APBN Date of receipt of notice of appeal recorded

Free format text: ORIGINAL CODE: EPIDOSNNOA2E

APBR Date of receipt of statement of grounds of appeal recorded

Free format text: ORIGINAL CODE: EPIDOSNNOA3E

APAF Appeal reference modified

Free format text: ORIGINAL CODE: EPIDOSCREFNE

APAF Appeal reference modified

Free format text: ORIGINAL CODE: EPIDOSCREFNE

APAF Appeal reference modified

Free format text: ORIGINAL CODE: EPIDOSCREFNE

REG Reference to a national code

Ref country code: DE

Ref legal event code: R003

APBT Appeal procedure closed

Free format text: ORIGINAL CODE: EPIDOSNNOA9E

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN REFUSED

18R Application refused

Effective date: 20141229