WO2000057895A1 - Methode de stimulation de l'angiogenese regulee par peptides pr-39 - Google Patents

Methode de stimulation de l'angiogenese regulee par peptides pr-39 Download PDF

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WO2000057895A1
WO2000057895A1 PCT/US2000/007050 US0007050W WO0057895A1 WO 2000057895 A1 WO2000057895 A1 WO 2000057895A1 US 0007050 W US0007050 W US 0007050W WO 0057895 A1 WO0057895 A1 WO 0057895A1
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cells
pro
arg
peptide
proteasomes
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PCT/US2000/007050
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English (en)
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Michael Simons
Youhe Gao
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Beth Israel Deaconess Medical Center
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Priority claimed from US09/276,868 external-priority patent/US7202217B1/en
Application filed by Beth Israel Deaconess Medical Center filed Critical Beth Israel Deaconess Medical Center
Priority to AU40132/00A priority Critical patent/AU4013200A/en
Priority to CA002368638A priority patent/CA2368638A1/fr
Priority to EP00919442A priority patent/EP1165111A4/fr
Publication of WO2000057895A1 publication Critical patent/WO2000057895A1/fr

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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
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is concerned generally with the induction of angiogenesis within viable cells comprising living tissues and organs; and is particularly directed to mechanisms regulated by PR-39 peptides which result in a stimulation of angiogenesis on-demand and may be used as a controlled therapeutic treatment.
  • Angiogenesis by definition, is the formation of new capillaries and blood vessels within living tissues; and is a complex process first recognized in studies of wound healing and then within investigations of experimental tumors.
  • Angiogenesis is thus a dynamic process which involves extracellular matrix remodeling, endothelial cell migration and proliferation, and functional maturation of endothelial cells into mature blood vessels [Brier, G. and K. Alitalo, Trends Cell Biology 6: 454-456 (1996)].
  • the process of angiogenesis is a normal host response to injury; and as such, is an integral part of the host body's homeostatic mechanisms.
  • angiogenesis represents an important component part of tissue response to ischemia, or tissue wounding, or tumor-initiated neovascularization
  • relatively little new blood vessel formation or growth takes place in most living tissues and organs of mature adults (such as the myocardium of the living heart) [Folkman, and Y. Shing, L Biol. Chem. 267: 10931-10934 (1992); Folkman, J. , Nat Med. 1: 27-31 (1995); Ware, J.A. and M. Simons, Nature Med. 3: 158-164 (1997)].
  • heparin-binding growth factors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF); and thus the regulation of angiogenesis is believed today to involve matrix components such as extracellular heparin sulfate and core proteins such as syndecans which are found at the surface of endothelial cells.
  • bFGF basic fibroblast growth factor
  • VEGF vascular endothelial growth factor
  • heparin binding growth factors including VEGF, FGFl and FGF2
  • VEGF vascular endothelial growth factor
  • FGFl heparin binding growth factor 2
  • FGF2 heparin binding growth factor 2
  • a first aspect provides a method for stimulating angiogenesis within a targeted collection of viable cells in- situ, said method comprising the steps of: identifying a collection of cells comprising viable cells in-situ as a target for stimulation of angiogenesis; providing means for effecting an introduction of at least one member selected from the group consisting of the PR-39 oligopeptide collective to the cytoplasm of said targeted collection of cells; introducing at least one member of the PR-39 oligopeptide collective to the cytoplasm of said targeted collection of cells using said effecting means; allowing said introduced PR-39 oligopeptide collective member to interact with such proteasomes as are present within the cytoplasm of said targeted collection of cells whereby (a) at least some of the proteasomes interact with said PR-39 oligopeptide collective member, and (b) at least a part of the proteolytic activity mediated by said interacting proteasomes becomes selectively altered, and
  • a second aspect of the invention provides a method for selective inhibition of proteasome-mediated degradation of peptides in-situ within a collection of viable cells, said method comprising the steps of: identifying a collection of cells comprising viable cells in-situ as a target; providing means for effecting an introduction of at least one member selected from the group consisting of the PR-39 oligopeptide collective to the cytoplasm of said targeted collection of cells; introducing at least one member of the PR-39 oligopeptide collective to the cytoplasm of said targeted collection of cells using said effecting means; allowing said introduced PR-39 oligopeptide collective member to interact with such proteasomes as are present within the cytoplasm of said targeted collection of cells whereby
  • FIGS. 1A-1D are presentations of empirical data showing the direct interaction between PR-39 peptide and the 7 subunit of proteasomes intracellularly;
  • Figs. 2A-2D are presentations of empirical data showing the effect of PR- 39 peptide upon proteasome activity in-vivo;
  • Figs. 3A-3D are graphs demonstrating the results of in-vitro proteasome activity assays
  • Figs. 4A-4C are presentations of empirical data showing the in-vivo effects of PR-39 peptide expression
  • Figs. 5A-5C are photographs of representative sections showing differences in vascularity among control, PR-39 peptide and FGF2 impregnated Matri el pellets;
  • Fig. 6 is a graph providing a quantitative analysis of vascularity for the representative sections of Fig. 5; and Fig. 7 is a graph showing the induction of angiogenesis in-vivo using PR-39 peptide and short-length PR 11 peptide impregnated Matrigel pellets.
  • the present invention is a method for stimulating angiogenesis via the purposeful introduction of native PR-39 peptide or a member of the PR-39 derived oligopeptide family to the cytoplasm of viable cells in-situ.
  • the PR-39 peptide or the derived member of the family will interact with such proteasomes as are present intracellularly; and the consequence of PR-39 peptide/proteasome interaction is the selective inactivation of proteasomes such that intracellular degradation of proteins such as HIF-l ⁇ and I ⁇ B ⁇ is diminished and a marked stimulation of angiogenesis in-situ consequently results.
  • the present invention provides an in-situ stimulation of angiogenesis.
  • angiogenesis By definition, therefore, both in-vivo and in-vitro circumstances of use and application are envisioned and expected.
  • the viable cells which are the location of PR-39 peptide and proteasome interaction, alternatively may be isolated cells; be part of living tissues comprising a variety of different cells such as endothelial cells, fibrocytes and muscle cells; and may also comprise part of specific organs in the body of a living human or animal subject.
  • the present invention provides the opportunity to stimulate angiogenesis in tissues and organs in a living subject which has suffered defects or has undergone anoxia or infarction.
  • a common clinical instance is the myocardial infarction or chronic myocardial ischemia of heart tissue in various zones or areas of a living human subject.
  • the present invention thus provides opportunity and means for specific site stimulation and inducement of angiogenesis under controlled conditions.
  • the present invention also has major research value for research investigators in furthering the quality and quantity of knowledge regarding the mechanisms controlling angiogenesis under a variety of different conditions and circumstances. 3.
  • the present invention envisions and permits a diverse range of means for introducing native PR-39 peptide or a shorter-length peptide of the oligopeptide family to a specific location, site, tissue, organ, or system in the living body.
  • a variety of different routes of administration are available to the practitioner; and a wide and useful choice of delivery systems are conventionally available, and in accordance with good medical practice are adaptable directly for use.
  • PR-39 peptide introduction under the control of the user, but also the manner of localized application and the mode of limiting the area of peptide introduction can be chosen and controlled.
  • the present invention utilizes and relies upon novel and previously unknown direct and indirect mechanisms of interaction between PR-39 peptide (or its shorter-length homologs) and proteasomes in-situ as the basis for stimulation of angiogenesis in cells, living tissues, and organs.
  • Evidence of such multiple intracellular interactions is provided by the experiments and empirical data described hereinafter.
  • Such interactions between proteasomes (and its ⁇ 7 subunit in particular) and the PR-39 peptides collectively (of any size) are previously unknown; in fact, no meaningful relationship or interaction between any peptide whatsoever and intracellular proteasome function has ever been proposed or envisioned before the present invention was conceived or demonstrated empirically.
  • the PR-39 peptide when introduced into the cytoplasm of viable cells will interact, directly and indirectly, with proteasomes.
  • the interaction between the collective of PR-39 oligopeptides and the proteasome is direct and a direct binding with the ⁇ 7 subunit of the proteasome occurs.
  • no intermediaries or cofactors are involved in the binding reaction; and such direct ⁇ 7 subunit binding interactions result in a selective inactivation and inhibition of proteasome function intracellularly such that expression of certain proteins such as HIF-l ⁇ is increased and stimulation of angiogenesis subsequently occurs.
  • the experiments and empirical data presented hereinafter demonstrate that: the PR-39 peptide acts as a selective inhibitor of tumor necrosis factor-a induced I ⁇ B ⁇ degradation by proteasomes; and that PR-39 peptide inhibition of I ⁇ B ⁇ degradation is rapidly reversible (unlike the action of known inhibitory compounds such as lactacysin); and that even in the presence of PR-39 peptide, the intracellular expression of other proteasome-regulated proteins such as pl05 and p50 NFKB was unchanged.
  • These empirical findings are meaningful evidence of, harmonious with, and more consistent as demonstrating indirect mechanisms of interaction (rather than any direct action effect).
  • the methodology and means provided by the present invention for selectively inhibiting proteolysis and stimulating angiogenesis within viable cells is therefore directed at and focused upon the intracellular degradation capability and functional activity of proteasomes.
  • Such selective inhibition and/or disruption of proteasome-mediated degradation is achieved via the introduction of native PR-39 peptide or a member of the shorter-length PR-39 derived oligopeptide family in a therapeutic regimen of treatment.
  • the proteasome is a component of the ubiquitin-proteasome-dependent proteolysis system. This system plays a major role in the turnover of intracellular proteins, of misfolded proteins, and in the selective degradation of key proteins. Controlled protein degradation is an important and efficient way to remove nonfunctional proteins and/or to regulate the activity of key proteins.
  • Target proteins are selectively recognized by the ubiquitin system and subsequently marked by covalent linkage of multiple molecules of ubiquitin, a small conserved protein.
  • the polyubiquitinated proteins are degraded by 26S proteasome. This complex, however, is composed of two large subcomplexes: the 20S proteasome constituting the proteolytic core and the 19S regulatory complex which confers polyubiquitin binding and energy dependence.
  • a simplified scheme of the ubiquitin pathway is depicted by Flow Scheme A below.
  • Conversion of a protein into a substrate for ubiquuination can in certain ca.es occur after posttranslational modification or association with ancillary factors. Proteins can also be recognized by an E3 ubiquitin liease without prior modification or asso ⁇ a-
  • the degrading component in ubiquitin-dependent protealysis is the 26S proteasome.
  • the catalytic core of this complex is the 20S proteasome, which is highly conserved and can be found in eukaryotes, archaebacteria, and some eubacteria. In eukaryotes, the amount of proteasomes can constitute up to 1 % of the cell content, depending on the average protein breakdown rates of the organ.
  • Proteasomes are localized in the nucleus and the cytosol, sometimes colocalizing or associating with the cytosketon. [See for example: Hilt, W. and D.H. Wolf,
  • proteasomes were not restricted to eukaryotic cells.
  • a compositionally simpler, but structurally strikingly similar proteolytic complex was found in the archaeon Thermoplasma acidophilum, which later took a pivotal role in elucidating the structure and enzymatic mechanism of the proteasome.
  • the 20S proteasome is the major cytosolic protease in eukaryotic cells and is the proteolytic component of the ubiquitin-dependent degradative pathway. Proteasomes are also found in some, but not all, archaebacteria and eubacteria, and in eukaryotes. True proteasomes are composed of 28 subunits, 14 each of two different classes - non-catalytic alpha ( ⁇ ) and catalytically-active beta ( ⁇ ) subunits. The subunits are arranged in rings of seven subunits, all of a single type.
  • the 20S proteasome is a stack of four rings, two inner beta rings flanked by the alpha rings.
  • the junction between the beta rings produces a remarkable structural feature of proteasomes - an interior aqueous cavity large enough to accommodate about 70 kDa of protein and accessible only through narrow axial channels in the rings.
  • the catalytic sites are located on the beta subunits within the aqueous cavity. Isolation of the catalytic sites in this way, and the limited access via narrow channels, serves to compartmentalize proteolysis, allowing degradation of only those proteins that can be actively translocated into the interior of the proteasome.
  • the 20S proteasome has a cylindrical or barrel-like structure, typically 14.8 nm in length and 11.3 nm in diameter. It is composed of 28 subunits and arranged in four stacked rings, resulting in a molecular mass of about 700 kDa.
  • Table 2 shows some characteristics and alternative names of the subunits of the human and yeast 20S proteasome using the older and the new nomenclature proposed by Groll and coworkers [Groll et al.. Nature 386:
  • These three proteasomal activities refer to peptide bond cleavage at the carboxyl side of basic, hydrophobic and acidic amino acid residues, respectively. They were identified using short synthetic peptide substrates and are believed to be catalyzed at independent sites - in part because the different proteolytic activities respond differentially to various activators and inhibitors.
  • proteasome degrades a protein substrate all the way to small peptides, before attacking another protein substrate [Akopian et al.. Biol. Chem. 272: 1791-1798 (1997)]. Because the proteasome 's multiple active sites are located in its central chamber and because diffusion of a peptide substrate into this compartment must be a slow process, these particles function in a highly processive fashion; i.e. , they have mechanisms of action to bind tightly protein substrates and to make multiple cleavages in the polypeptide before releasing the peptide products.
  • the ratio of new peptides generated to the number of substrate molecules consumed is constant during the reaction. In other words, as peptides accumulated, they were not hydrolyzed further, even during prolonged incubations, where up to half of the substrate molecules were consumed. Equally important, the disappearance of these substrate molecules coincided exactly with the appearance of small peptide products [Goldberg et al.. Biol. Chem. 378: 131-140 (1997)].
  • proteasomes are able to cleave behind most amino acids in a protein.
  • the 20S proteasome is in fact a nonspecific endopeptidase.
  • the generated (degraded) peptides fall into a rather narrow size range of 6 to 10 amino acids in length, demonstrating the existence of a kind of 'molecular ruler' .
  • the average length of the degradation products is typically 7 to 8 amino acids; this finding is in agreement with the distance between the active sites in the proteasome. Similar nonspecific endopeptidase activity and size distribution of degration products from whole proteins was observed for proteasomes generally and by proteasomes of human origin in particular.
  • Native PR-39 peptide is a substance belonging to the cathelin family of proteins; the mature peptide is 39 amino acids in length in the naturally occurring state; and the peptide is able to exert a variety of activities and cause different cellular outcomes.
  • this peptide was subsequently isolated from wounds where it could simultaneously reduce infection and influence the action of growth factors, matrix components, and other cellular effectors involved in wound repair [Gallo et al.. Proc. Natl. Acad. Sci.
  • the native PR-39 peptide was shown to possess a syndecan- inducing activity in furtherance of its wound healing capabilities; and while renamed a "synducin", was shown to induce cellular production of two specific proteoglycans, syndecan-1 and syndecan-4, within living mesenchymal cells [U.S. Patent No. 5,654,273].
  • native PR-39 peptide has been shown to play a role in several inflammatory events including wound healing and myocardial infarction [Gallo et al.. Proc. Natl. Acad. Sci. USA 9 .: 11035-11039 (1994); Li et al.. Circ. Res.
  • the PR-39 peptide grouping Native PR-39 peptide is composed of the 39 amino acid sequence shown below (and also by Table 4).
  • the specific peptide can be substituted using conservative substitutions of amino acids having the same or functionally equivalent charge and structure, except for the required amino acid sequence "Arg-Arg-Arg" at the N-terminus and the intermediate amino acid sequences "Pro-Pro-X-X-Pro-Pro-X-X-Pro" and "Pro- Pro-X-X-X-Pro-Pro-X-X-Pro” where X can be substituted freely using any amino acid.
  • all of the preferred substituted amino acid sequences are of about the same size and each differ from the native PR-39 peptide sequence only by substitutions in the intermediate portions of the structure.
  • PR-39 derived oligopeptide structures In addition to the conventionally known native PR-39 peptide amino acid residue sequence and its readily recognizable substituted forms as described above, an entirely novel and unforeseen family of PR-39 derived oligopeptide structures is provided by the present invention for use.
  • This previously unknown family of PR- 39 derived oligopeptides is constituted of members which individually will cause a selective inhibition of proteasome-mediated degradation of peptides in-situ after introduction intracellularly to a viable cell.
  • Each member of this PR-39 derived oligopeptide family presents characteristics and properties which are commonly shared among the entire membership. These include the following:
  • each peptide sequence is less than 39 amino acid residues in length in every embodiment, and preferably is less than 20 residues in size in the best mode;
  • each short-length peptide sequence is at least partially homologous (or analogous) with the N-terminal amino acid residues of the native PR-39 peptide, and preferably is completely identical or markedly similar to the N-terminal end residues of the native PR-39 peptide;
  • each short-length peptide is able to interact in-situ with at least the ⁇ 7 subunit of such proteasomes as are present within the cytoplasm of the cell; and (iv) each short-length peptide sequence is able to alter markedly the proteolytic activity of proteasomes with an interacting ⁇ 7 subunit such that a selective increased expression of specific proteins (such as I ⁇ B ⁇ and HIF-l ⁇ ) occurs in-situ.
  • specific proteins such as I ⁇ B ⁇ and HIF-l ⁇
  • the members comprising 15, 11 and 8 amino acid residues respectively in length are presented below as the PR 15, PRl l, and PR8 entities respectively.
  • the complete amino acid sequence of the native PR-39 peptide is presented as well.
  • PR-39 peptides grouping includes by definition the native PR-39 structure and all substituted forms conventionally known of the naturally occurring 39 length amino acid sequence.
  • PR-39 derived oligopeptide family and its members includes by definition all the previously unknown shorter-length homologs and analogs of the native PR-39 structure as described above.
  • PR-39 oligopeptide collective includes by definition both the 'PR-39 peptide grouping' as well as the 'PR-39 derived oligopeptide family' members, and identifies any and all individual structures falling into either of the two subset categories. Table 4:
  • the PR-39 peptide can be synthesized using standard amino acid synthetic techniques.
  • An example is the conventionally used solid phase synthesis [Merrifield, J. , A Chem. Soc. 85: 2149 (1964)] described in U.S. Patent No. 4,244,946, wherein a protected alpha-amino acid is coupled to a suitable resin, to initiate synthesis of a peptide starting from the C-terminus of the peptide.
  • Other methods of peptide synthesis are described in U.S. Patent Nos. 4,305,872 and 4,316,891 , the teachings of which are incorporated herein.
  • PR-39 can also be commercially obtained from Magainin, Inc. (Plymouth Meeting, PA).
  • the PR-39 peptides (as a family of homologs and analogs with substituted amino acid residues) can be introduced as a peptide- containing preparation in a pharmaceutically acceptable format.
  • the PR-39 can be administered and introduced in-vivo systemically, topically, or locally.
  • the peptide can be administered as the peptide or as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with an inorganic acid (such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid); or with an organic acid (such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid); or by reaction with an inorganic base (such as sodium hydroxide, ammonium hydroxide, potassium hydroxide); or with an organic base (such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines).
  • an inorganic acid such as hydrochloric acid, hydrobromic acid, perchloric acid,
  • PR-39 peptide and any of the PR-39 derived oligopeptide family members may also be conjugated to sugars, lipids, other polypeptides, nucleic acids and PNA; and function in-situ as a conjugate or be released locally after reaching a targeted tissue or organ.
  • the PR-39 family of peptides may also be linked to targeting compounds for attachment in-situ to a specific cell type, tissue or organ.
  • PR-39 peptide or a derived oligopeptide family member
  • One desirable means uses a prepared DNA sequence fragment encoding the PR-39 peptide (or a shorter-length homolog) in a suitable vector as the means of introduction to the intended target in-situ.
  • These means for delivery envision and include in-vivo use circumstances; ex-vivo specimens and conditions; and in- vitro cultures.
  • the present invention intends and expects that the prepared DNA sequence fragment coding for PR-39 peptide (or shorter-length homologs) has been inserted in a suitable expression vector and will be used in a route of administration for delivery to living tissues comprising endothelial cells, and typically vascular endothelial cells which constitute the basal layer of cells within capillaries and blood vessels generally.
  • the cell recipients themselves are thus eukarytoic in origin, typically mammalian cells from human and animal sources; and most typically would include the higher orders of mammals such as humans and domesticated mammalian animals kept as pets or sources of food intended for future consumption.
  • the range of animals includes all domesticated varieties involved in nutrition including cattle, sheep, pigs and the like; as well as those animals typically used as pets or raised for commercial purposes including horses, dogs, cats, and other living mammals typically living with and around humans.
  • the expression vectors must be suitable for transfection of endothelial cells in living tissues of mammalian origin and thus be compatible with that type and condition of cells under both in-vivo and/or in-vitro conditions.
  • the expression vectors thus typically include plasmids and viruses as expression vectors.
  • both the plasmid based vectors and the viral expression vectors constitute conventionally known means and methods of introduction which are conventionally recognized today as "gene therapy" modes of delivery.
  • this overall approach is not the only means and method of delivery available for the present invention.
  • PR-39 peptide or an oligopeptide family member can be introduced directly as a synthesized compound to living cells and tissues via a range of different delivery means. These include the following.
  • Intracoronary delivery is accomplished using catheter-based deliveries of synthesized PR-39 peptide (or homolog member) suspended in a suitable buffer
  • a suitable local delivery catheter such as a 10mm InfusaSleeve catheter (Local Med, Palo Alto, CA) loaded over a 3.0mm x 20mm angioplasty balloon, delivered over a 0.014 inch angioplasty guidewire. Delivery is typically accomplished by first inflating the angioplasty balloon to 30 psi, and then delivering the protein through the local delivery catheter at 80 psi over 30 seconds (this can be modified to suit the delivery catheter).
  • a suitable local delivery catheter such as a 10mm InfusaSleeve catheter (Local Med, Palo Alto, CA) loaded over a 3.0mm x 20mm angioplasty balloon, delivered over a 0.014 inch angioplasty guidewire. Delivery is typically accomplished by first inflating the angioplasty balloon to 30 psi, and then delivering the protein through the local delivery catheter at 80 psi over 30 seconds (this can be modified to suit the delivery catheter).
  • Intracoronary bolus infusion of PR-39 peptide (or a short-length homolog) synthesized previously can be accomplished by a manual injection of the substance through an Ultrafuse-X dual lumen catheter (SciMed, Minneapolis, MN) or another suitable device into proximal orifices of coronary arteries over 10 minutes.
  • Pericardial delivery of synthesized PR-39 peptide is typically accomplished by instillation of the peptide-containing solution into the pericardial sac.
  • the pericardium is accessed via a right atrial puncture, transthoracic puncture or via a direct surgical approach. Once the access is established, the peptide material is infused into the pericardial cavity and the catheter is withdrawn. Alternatively, the delivery is accomplished via the aid of slow-release polymers such as heparin-alginate or ethylene vinyl acetate (EVAc).
  • the desired amount of PR-39/polymer is inserted under the epicardial fat or secured to the myocardial surface using, for example, sutures.
  • the PR-39/ polymer can be positioned along the adventitial surface of coronary vessels. 4.
  • Intramyocardial delivery of synthesized PR-39 peptide (or a shorter-length homolog) can be accomplished either under direct vision following thoracotomy or using thoracoscope or via a catheter. In either case, the peptide containing solution is injected using a syringe or other suitable device directly into the myocardium.
  • Up to 2 cc of volume can be injected into any given spot and multiple locations (up to 30 injections) can be done in each patient.
  • Catheter-based injections are carried out under fluoroscopic, ultrasound or Biosense NOG A guidance. In all cases after catheter introduction into the left ventricle the desired area of the myocardium is injected using a catheter that allows for controlled local delivery of the material.
  • a range of suitable pharmaceutical carriers and vehicles are known conventionally to those skilled in the art.
  • the compound will typically be dissolved or suspended in sterile water or saline.
  • the PR-39 peptide or homologous oligopeptide of choice will be typically incorporated into an inert carrier in tablet, liquid, or capsular form.
  • suitable carriers are starches and sugars; and often include lubricants, flavorings, binders, and other materials desirable in tablet making procedures.
  • the PR-39 peptide and oligopeptide family of compounds can also be administered topically by application of a solution, cream, gel, or polymeric material (for example, a PluronicTM, BASF).
  • a solution, cream, gel, or polymeric material for example, a PluronicTM, BASF.
  • the chosen peptide can be administered in liposomes or microspheres (or microparticles), which can be injected for local or systemic delivery.
  • Methods for preparing liposomes and microspheres for administration to a patient are conventionally known to those skilled in the art.
  • U.S. Patent No. 4,789,734 describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary. See also, G. Gregoriadis,
  • Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the bloodstream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted from days to months. See, for example, U.S. Patent Nos. 4,906,474; 4,925,673; and 3,625,214.
  • Direct myocardial injection direct vision-epicardial-open chest or under thorascope guidance
  • peripheral vascular disease intramuscular injection intraarterial injection and/or infusion.
  • proteasome-dependent degradation Proteolytic degradation in mammalian cells is known to proceed via two distinct pathways: lysosome-dependent degradation and proteasome-dependent.
  • the proteasome in its pathway plays a key role in proteolysis of intracellular proteins which are marked for degradation by the ubiquitin system.
  • the multienzyme complex involved in these events, the 26S proteasome consists of a 20S catalytic proteasome "core" and two 19S caps that bind uniquitylated proteins, as has been described in detail previously herein.
  • Proteasome-mediated proteolysis is a principal event quantitatively controlling intracellular levels of a number of different proteins including hypoxia-inducing factor (HIF)-l ⁇ , heat shock protein HSP70, protooncogenes c-Fos, c-Jun and c-Mos, NFKB inhibitor I ⁇ B ⁇ , and various cyclins.
  • HIF hypoxia-inducing factor
  • HSP70 heat shock protein
  • protooncogenes c-Fos protooncogenes
  • c-Jun and c-Mos protooncogenes
  • NFKB inhibitor I ⁇ B ⁇ NFKB inhibitor
  • various cyclins NFKB inhibitor
  • MHC major histocompatibility complex
  • the PR-39 peptide belonging to the cathelin family of proteins, plays an important role in several inflammatory events including wound healing and myocardial infarction.
  • the PR-39 peptide typically is rapidly taken up by a number of different cell types including endothelial cells; and prolonged treatment with PR-39 peptide leads to increased cell growth and angiogenesis.
  • the mechanism of action for this peptide activity has yet to be understood or defined.
  • the experiments and data presented below reveal for the first time the nature and detailed intracellular actions exerted by the PR-39 peptide.
  • Two-hybrid screening was carried out using MATCHMAKER GAL4 System 2 (Clontech) with exon 4 of the porcine PR-39 gene as a bait to screen the mouse embryo 3T3 cDNA library in yeast CGI 945.
  • U937 cells (ATCC) grown in RPMI medium 1640 with 10% FBS (Gibco- BRL) and ECV cells were treated with synthetic PR-39, lactacystin (CalBiochem, 426100) or MG132 (CalBiochem, 474790) at concentration indicated in the presence of 100 mM cyclohexamide 20 mM chloroquine [Merin et al., Biol. Chem. 273: 6373-6379 (1995)]. After 45 min. of incubation, TNF ⁇ (1 ng/ml) was added. After 5 min of 37°C incubation, the cells were lysed in SDS-PAGE loading buffer.
  • I ⁇ B- ⁇ and NF- ⁇ B pl05 p50 expressions were determined by Western blotting with anti-human antibodies (Santa Cruz, sc-203, scl l4G).
  • ECV cells were cultured in a hypoxia chamber (5% CO 2 /95 % N 2 ) at 37°C for 16 hr.
  • HIF-l ⁇ was immunoprecipitated with anti-HIF-l ⁇ mAb (OZ12 1 :5) in RIPA buffer and Western blotting with anti-HIF-l ⁇ mAb (OZ15 1 : 10) (courtesy of
  • VEGF expression was shown by Western blotting of hypoxia treated ECV cell lysate with anti-human VEGF antibody (Santa Cruz, sc-152).
  • HSP70 expression U937 was treated for 3 hr, harvest with SDS- PAGE loading buffer, Western blotting with anti-human HSP70 polyclonal antibody (Santa Cruz, sc-1060).
  • porcine PR-39 gene sequence was used to generate a rabbit polyclonal antibody RPE4.
  • Full length porcine cDNA (containing leader sequence) and a sequence corresponding to the 4th exon of porcine PR-39 gene were cloned into eukaryotic expression vector pGRE5-2 (USB). These expression constructs were then used to stably transfect an immortalized human endothelial cell line (ECV304, ATCC).
  • ECV full length PR-39
  • ECV-E4 exon 4 PR39 and exon 4 PR39
  • FBS fetal bovine serum
  • ECV-E4 exon 4 PR39 and exon 4 PR39
  • FBS fetal bovine serum
  • Cells were lysed with RIPA buffer; immunoprecipitated with 10 ⁇ g affinity purified rabbit anti-PR39 antibody; and following Protein A-Sepharose purification and SDS-PAGE, subjected to immunoblotting with 1: 1000 mouse anti-HC8 mAb (Affiniti Research Products Limited UK, PW8110).
  • Figs. 1A-1D show the interactions of PR-39 peptide and the ⁇ 7 subunit of proteasomes.
  • Fig. IA recites the cDNA sequence of cloned mouse ⁇ 7 subunit (top; GeneBank accession number AF055983) and corresponding human HC8 subunit of 20S proteasome.
  • Fig. IB shows the sequence alignment of C-terminal tails mouse ⁇ subunits of 20S proteasome.
  • Fig. 1C shows a deletion analysis of C.7-PR39 binding. Deletion mutants of the mouse ⁇ 7 subunit were cloned into an yeast-two hybrid vector and the extent of growth of lacZ + colonies on selective medium following co-transformation with PR-39 construct in the yeast CGI 945 was determined. It is noted that only full length ⁇ 7 construct was able to bind to PR-39.
  • Fig. ID shows the co-immunoprecipitation of PR-39 and ⁇ 7 subunit in ECV cells.
  • Fig. 1 represents the evidence of four clones growing on selective media and demonstrating lacZ staining. All four clones encoded overlapping identical cDNA sequences highly homologous to the human sequence of ⁇ 7 (HC8) subunit of proteasome (Fig. IA). Similar to all ⁇ subunits of the 20S proteasome, the cloned mouse protein possesses a highly conserved N-terminal region; in addition it demonstrated the presence of 16 amino acid long C-terminal sequence found in some but not all ⁇ subunits (Fig. IB). Deletion analysis showed that the presence of both C-terminal as well as N-terminal amino acids sequences was required for PR-39 binding (Fig. 1C).
  • anti-PR39 antibody was used to immunoprecipitate PR39 protein from ECV-PR39, ECV-E4 and mock-transfected ECV cells.
  • Western blotting of the immunoprecipitate from ECV-PR39 and ECV- E4 but not wild type ECV cells with anti- ⁇ 7 subunit antibody demonstrated the presence of a 29kDa band corresponding to the known size of ⁇ 7 subunit protein (Fig. ID). The evidence therefore reveals that PR39 peptide interacts with ⁇ 7 subunit of proteasome in ECV cells.
  • Fig. 2A shows a Western analysis of I ⁇ B ⁇ expression in ECV cells.
  • the results show that pretreatment of cultured ECV cells with lactacystin (10 ⁇ M, 4th lane) or stable expression of full length (ECV-PR39) or PR39 exon 4 (ECV-E4) constructs inhibited TNF- ⁇ -induced degradation of I ⁇ B.
  • tumor necrosis factor (TNF)- ⁇ induces rapid degradation of I ⁇ B ⁇ - a function that is blocked by the proteasome inhibitor lactacystin.
  • Western analysis of I ⁇ B ⁇ levels after TNF- ⁇ treatment demonstrated comparable levels of I ⁇ B ⁇ expression in both ECV-PR39 and ECV-E4 cells to that seen in ECV cells pre-treated with lactacystin.
  • Fig. 2B shows the effect of PR39, MG132 and lactacystin pretreatment on I ⁇ B ⁇ expression in U937 cells following TNF- ⁇ treatment. Note similar extent of inhibition of I ⁇ B ⁇ degradation by TNF- ⁇ following pretreatment with PR39, MG132 or lactacystin.
  • pretreatment of U937 cells with PR39 blocked TNF- ⁇ induced I ⁇ B ⁇ degradation in a manner that was similar to the degree of inhibition seen with MG132 and lactacystin.
  • Fig. 2C demonstrates the reversibility of PR39 inhibition of proteasome activity.
  • U937 cells were pretreated with PR39, MG132 or lactacystin for 45 min. After that time, the cells were extensively washed with fresh medium. 45 min later TNF- ⁇ (1 ng/ml) was added to the medium and the extent of I ⁇ B ⁇ degradation was determined 10 min later by Western blotting. Note preservation of I ⁇ B ⁇ in lactacystin-treated cells but not PR39-treated cells. Thus, unlike lactacystin but similar to MG132, PR-39 peptide mediated inhibition of I ⁇ B ⁇ degradation was rapidly reversible.
  • ECV cells were transiently transfected with a NF ⁇ B-Luc reporter construct containing a tandem of four NFKB binding sites in front of luciferase cDNA.
  • the results of Fig. 2D show that stimulation with TNF- ⁇ induced a significant increase in luciferase activity that was completely inhibited by pretreatment with PR39.
  • Figs. 3A-3D reveal that the PR-39 peptide inhibited, in a dose-dependent manner, degradation of all 4 peptides tested.
  • PR-39 peptide was as potent as lactacystin or MG132 in inhibiting degradation in three of the four peptides tested and was considerably more potent in inhibiting degradation of the Z-Leu-Leu-Glu- AMC peptide.
  • Figs. 4A-4C show the results of a Western blot analysis of HIF-l ⁇ , p50 and pl05 NFKB expression in wild type ECV cells and ECV-E4 and ECV-PR39 clones. It is noted that an increase in HIF-l ⁇ expression occurs (but not pi 05 or p50 NFKB expression) in ECV-E4 and ECV-PR 39 clones.
  • HIF-l ⁇ Since increased expression of HIF-l ⁇ is known to result in increased transcription of a number of angiogenesis-related molecules including VEGF, Northern analysis of VEGF mRNA levels in wild type and PR39 transfected ECV cells was performed. The results are shown by Fig. 4B. As expected, there was a significant increase in expression of both of these genes in ECV-PR39 and ECV-E4 cells compared to ECV controls.
  • mice matrigel assay system To demonstrate the stimulation of angiogenesis directly in living cells and tissues via the introduction of PR-39 peptide, a mice matrigel assay system was employed. Growth factor-depleted Matrigel pellets containing 5 ⁇ g of PR39, 50 ng of FGF2 or saline (control) were inserted intraperitoneally into C57BL/6 mice. Ten days later the pellets were removed, sectioned and stained with anti- CD31 antibody. The number of vessels was determined in multiple sections using a digital camera and Optimas 5.0 software. The results are shown by Figs. 5A-5C and Fig. 6 respectively.
  • Figs. 5A-5C are representative sections from control, PR39 and FGF2 impregnated Matrigel pellets and Fig. 6 provides a quantitative analysis of vascularity.
  • Figs. 5A-5C are representative sections from control, PR39 and FGF2 impregnated Matrigel pellets and Fig. 6 provides a quantitative analysis of vascularity.
  • the results of the representative sections and the graphic quantitative evaluations demonstrate that insertion of growth-factor depleted Matrigel pellet containing PR-39 peptide induced intense vessel growth that exceeded that seen with implantation of pellets containing 50 ng/ml of bFGF.
  • PRl l a novel peptide, composed of the first 11 amino acid residues [N-terminal end] of the native PR-39 sequence was purposely synthesized.
  • the amino acid sequence of PRl l is as follows:
  • a mouse Matrigel assay system was utilized.
  • 5 ⁇ g/ml of PRl l peptide or 5 ⁇ g/ml of native PR-39 peptide were individually placed into a growth factor-depleted Matrigel pellet; and then each prepared Matrigel pellet was inserted into the peritoneal cavity of a mouse. After 14 days intraperitoneal placement, each pellet was removed from its living host; and each pellet was examined for evidence of new vascularity.
  • the results are graphically presented by Fig. 7. Note that the bar graph of Fig. 7 shows the number of blood vessels [mean ⁇ SD] per 10 high power fields (HPF).
  • PR-39 peptide has the ability to selectively alter activity of 20S proteasome in human endothelial cells by interacting with proteasomes in a reversible manner. This interaction leads to suppression of I ⁇ B and HIF-l ⁇ degradation while not affecting expression of other proteasome-dependent proteins such as pl05 NFKB or HSP70. Unlike other proteasome inhibitors, treatment with PR39 is not associated with any cellular cytotoxicity. Thus, PR39 and its related peptides provide a unique and unforeseen means of regulating cellular function and stimulating angiogenesis. (2) Several observations also set PR39 apart from the conventionally known proteasome inhibitors.
  • PR39-mediated inhibition of I ⁇ B ⁇ degradation is demonstrably reversible, unlike that of lactacystin.
  • PR-39 peptide modulation of proteasome activity plays a functional role since the observed increased expression of HIF-l ⁇ was directly associated with an increased expression of its target genes, VEGF and flt-1.

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Abstract

La présente invention concerne à la fois une méthode et un moyen de régulation de l'angiogénèse dans des cellules vivantes, des tissus ainsi que des organes in situ. La régulation est exécutée à l'aide d'un peptide PR-39 natif ou d'un de ses homologues de longueur plus courte, afin d'avoir une interaction avec des protéasomes tels que ceux présents dans le cytoplasme de cellules viables. Le résultat de l'interaction du peptide PR-39 avec les protéasomes est une diminution de la dégradation intracellulaire de peptides actifs tels que HIF-1α et par conséquent une stimulation de l'angiogénèse in situ.
PCT/US2000/007050 1999-03-26 2000-03-16 Methode de stimulation de l'angiogenese regulee par peptides pr-39 WO2000057895A1 (fr)

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Cited By (10)

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EP1242107A1 (fr) * 1999-12-29 2002-09-25 Beth Israel Deaconess Medical Center Procede d'inhibition selective de la degradation de la proteine ikappabalpha induite par le peptide pr-39
US6676679B1 (en) 1999-11-05 2004-01-13 Boston Scientific Corporation Method and apparatus for recurrent demand injury in stimulating angiogenesis
US6695808B2 (en) 2000-03-23 2004-02-24 Scimed Life Systems, Inc. Pressure sensor for therapeutic delivery device and method
US6748258B1 (en) 1999-11-05 2004-06-08 Scimed Life Systems, Inc. Method and devices for heart treatment
WO2004067025A1 (fr) * 2003-01-29 2004-08-12 Lipopeptide Ab Utilisation de la cathelicidine ll-37 et de derives associes pour une cicatrisation de plaie
WO2006042661A3 (fr) * 2004-10-18 2006-07-27 Cognis France Sas Oligopeptides et utilisations
WO2006119183A2 (fr) * 2005-05-03 2006-11-09 Trustees Of Dartmouth College Procede pouvant augmenter la masse et la performance cardiaques
US7214223B2 (en) 2000-03-24 2007-05-08 Boston Scientific Scimed, Inc. Photoatherolytic catheter apparatus and method
US7382964B2 (en) 2001-11-14 2008-06-03 Thomson Licensing Digital video recorder and methods for digital recording
US7588554B2 (en) 2000-06-26 2009-09-15 Boston Scientific Scimed, Inc. Method and apparatus for treating ischemic tissue

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WO1996009322A2 (fr) * 1994-09-22 1996-03-28 Children's Medical Center Corporation Modulation de la reparation des tissus induite par des synducines

Non-Patent Citations (2)

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Title
LI JIAN ET AL.: "Cardiac-specific overexpression of PR-39 induces angiogenesis, myocardial hypertrophy and increased microvascular reactivity", CIRCULATION,, vol. 98, no. 17, 27 October 1998 (1998-10-27), pages 1794, SEE ABSTRACT NO. 4163, XP002929855 *
See also references of EP1165111A4 *

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6676679B1 (en) 1999-11-05 2004-01-13 Boston Scientific Corporation Method and apparatus for recurrent demand injury in stimulating angiogenesis
US7392077B2 (en) 1999-11-05 2008-06-24 Boston Scientific Scimed, Inc. Method for treating a patient at risk of loss of cardiac function by cardiac ischemia
US6748258B1 (en) 1999-11-05 2004-06-08 Scimed Life Systems, Inc. Method and devices for heart treatment
EP1242107A4 (fr) * 1999-12-29 2004-10-06 Beth Israel Hospital Procede d'inhibition selective de la degradation de la proteine ikappabalpha induite par le peptide pr-39
EP1242107A1 (fr) * 1999-12-29 2002-09-25 Beth Israel Deaconess Medical Center Procede d'inhibition selective de la degradation de la proteine ikappabalpha induite par le peptide pr-39
US7211063B2 (en) 2000-03-23 2007-05-01 Boston Scientific Scimed, Inc. Pressure sensor for therapeutic delivery device and method
US6695808B2 (en) 2000-03-23 2004-02-24 Scimed Life Systems, Inc. Pressure sensor for therapeutic delivery device and method
US7214223B2 (en) 2000-03-24 2007-05-08 Boston Scientific Scimed, Inc. Photoatherolytic catheter apparatus and method
US7588554B2 (en) 2000-06-26 2009-09-15 Boston Scientific Scimed, Inc. Method and apparatus for treating ischemic tissue
US7382964B2 (en) 2001-11-14 2008-06-03 Thomson Licensing Digital video recorder and methods for digital recording
US9125875B2 (en) 2003-01-29 2015-09-08 Lipopeptide Ab Use of the cathelicidin LL-37 and derivatives thereof for wound healing
US8506994B2 (en) 2003-01-29 2013-08-13 Lipopeptide Ab Use of the cathelicidin LL-37 and derivatives thereof for wound healing
WO2004067025A1 (fr) * 2003-01-29 2004-08-12 Lipopeptide Ab Utilisation de la cathelicidine ll-37 et de derives associes pour une cicatrisation de plaie
US7452864B2 (en) 2003-01-29 2008-11-18 Lipopeptide Ab Use of the cathelicidin LL-37 and derivatives thereof for wound healing
US8936807B2 (en) 2003-01-29 2015-01-20 Lipopeptide Ab Use of the cathelicidin LL-37 and derivatives thereof for wound healing
US8012933B2 (en) 2003-01-29 2011-09-06 Lipopeptide Ab Use of the cathelicidin LL-37 and derivatives therof for wound healing
WO2006042661A3 (fr) * 2004-10-18 2006-07-27 Cognis France Sas Oligopeptides et utilisations
US8044028B2 (en) 2004-10-18 2011-10-25 Cognis France S.A.S. Oligopeptides and their use in cosmetics
WO2006119183A3 (fr) * 2005-05-03 2007-06-14 Dartmouth College Procede pouvant augmenter la masse et la performance cardiaques
US7939499B2 (en) 2005-05-03 2011-05-10 Trustees Of Dartmouth College Method for increasing cardiac mass and performance
WO2006119183A2 (fr) * 2005-05-03 2006-11-09 Trustees Of Dartmouth College Procede pouvant augmenter la masse et la performance cardiaques

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CA2368638A1 (fr) 2000-10-05

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