WO1999059614A1 - Modulation de l'angiogenese et de la cicatrisation - Google Patents

Modulation de l'angiogenese et de la cicatrisation Download PDF

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WO1999059614A1
WO1999059614A1 PCT/US1999/011209 US9911209W WO9959614A1 WO 1999059614 A1 WO1999059614 A1 WO 1999059614A1 US 9911209 W US9911209 W US 9911209W WO 9959614 A1 WO9959614 A1 WO 9959614A1
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leptin
agent
receptor
leptin receptor
angiogenic
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PCT/US1999/011209
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WO1999059614A9 (fr
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Rocio M. Sierra-Honigmann
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Yale University
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Priority to US09/700,813 priority Critical patent/US7261881B1/en
Priority to AU46721/99A priority patent/AU4672199A/en
Publication of WO1999059614A1 publication Critical patent/WO1999059614A1/fr
Publication of WO1999059614A9 publication Critical patent/WO1999059614A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2264—Obesity-gene products, e.g. leptin

Definitions

  • the present invention generally relates to methods for modulating the angiogenic response of a subject to angiogenesis-inducing stimuli or angiogenesis-inhibiting stimuli, such as are released from tumor cells or cells in wounds or ischemic or injured tissues. Specifically, the invention relates to modulating the leptin and leptin-receptor mediated response of endothelial cells and smooth muscle cells to such angiogenic stimuli.
  • Applications of the present invention include enhancing wound healing and/or repair of ischemic tissue and inhibiting the vascularization of tumors and intraocular angiogenesis on the retina or other structures of the eye.
  • Leptin is produced from the obese (ob) gene and binds to the ob receptors (Ob-R).
  • the ob gene is expressed in various tissues such as placenta, ovaries, muscle and adipose tissue.
  • Leptin is produced in the adipocyte and in ovaries, and is a circulating 16 kDa protein (G.A. Bray, (1996) Lancet 348: 140; C. Liu et al, (1997) Endocrinology 138: 3548).
  • Defective production of leptin results in gross obesity and type 2 diabetes in the obese (ob/ob) mouse. In humans, the leptin protein levels have been correlated to the percentage of body fat and is elevated in obese patients (R.V.
  • Leptin has been detected in the plasma of normal individuals and individuals receiving hemodialysis and in renal transplant patients, in placental tissue from pregnant women, and in cord blood of newborns (Respectively, J. K. Howard et al, (1997) Clin. Sci. 93: 119; S.G. Hassink et al, (1997) Pediatrics 100: 123). It has been suggested that leptin concentrations in newborns cannot be explained by adiposity alone. In women, leptin deficiency has been postulated to be involved with delayed puberty, menstrual disturbances and anorexia nervosa (M. Schwartz et al, (1997) N. Engl. J. Med. 336: 1802).
  • Leptin is also believed to regulate lipid metabolism, glucose uptake, ⁇ -cell function, gonadotropin secretion, sympathetic tone, ovarian function and thermogenesis.
  • Glucocorticoids and insulin increase leptin production.
  • Administration of leptin reduces food intake, decreases insulin concentrations, and lowers blood glucose concentrations in the ob/ob mouse, but not in the db/db mouse (G.A. Bray, (1996) Lancet 348: 140).
  • the leptin receptor belongs to the cytokine superfamily of receptors. Several forms of the leptin receptor are expressed in humans and rodents (G.A. Bray, (1996) Lancet 348: 140).
  • the short form (Ob-R(S)) considered to have limited signaling capability, is detected in many organs and has 5 identified isoforms, Ob-Ra, Ob-Rc, Ob-Rd, Ob-Re, and r-Ob-Rf (M.-Y. Wang et al, (1996) FEBS Letters 392: 87).
  • Ob-R(S) has been identified in the choroid plexus and may be involved in the transport of leptin across the blood-brain barrier (J. Girard, (1997) Diabetes Metabol. 23S: 16).
  • Ob-R long form (Ob-R(L) also known as Ob-Rb) predominates in the hypothalamus and cerebellum (A. Savioz et al. (1997 ⁇ Neuroreport 8: 3123: J. G. Mercer et al. ( 996 ⁇ FEBS Letters 387: 113).
  • Ob-R(L) has also been detected at low concentrations in peripheral tissues (Y. Wang et al, (1997) J. Biol. Chem. 272: 16216), such as the brain (A. Heritier et al, (1997) Neurosci. Res. Commun. 21: 113), spleen, testes, kidney, liver, lung, adrenal (N. Hoggard et al, (1997) Biochem. Biophys. Res. Commun. 232: 383), and hematopoietic tissues (A.A. Mikhail et al, (1997) Blood 89: 1507).
  • peripheral tissues Y. Wang et al, (1997) J. Biol. Chem. 272: 16216
  • peripheral tissues such as the brain (A. Heritier et al, (1997) Neurosci. Res. Commun. 21: 113), spleen, testes, kidney, liver, lung, adrenal (N. Hoggard et al, (1997) Biochem. Biophys
  • Ob-R(L) has also been observed in the placenta, fetal cartilage/bone, and hair follicles, and therefore is believed to play a role in development (N. Hoggard et al, (1997) Proc. Nat'l Acad. Sci. USA 94: 11073).
  • Ob-R(L) has been demonstrated to transduce intracellular signaling in a manner analogous to that observed for interleukin (IL)-6 type-cytokine receptors.
  • Ob-R(L) transmits its information via the Janus kinases (JAK), specifically Jak2 (N. Ghilardi et al, (1997) Mol. Endocrinol. 11 : 393), which subsequently phosphorylate transcription factors of the STAT3 family (J. Girard (1997)).
  • Leptin sensitizing compounds have also been discloses. See, for example, PCT Application No. 98/02159.
  • Angiogenesis refers to the growth of new blood vessels, or "neovascularization,” and involves the growth of new blood vessels of relatively small caliber composed of endothelial cells.
  • Angiogenesis is an integral part of many important biological processes including cancer cell proliferation solid tumor formation, inflammation, wound healing, repair of injured ischemic tissue, myocardial revascularization and remodeling, ovarian follicle maturation, menstrual cycle, and fetal development.
  • New blood vessel formation is required for the development of any new tissue, whether normal or pathological, and thus represents a potential control point in regulating many disease states, as well as a therapeutic opportunity to encourage growth of normal tissue and "normal” angiogenesis.
  • angiogenesis is not entirely understood, but it is known to involve the endothelial cells of the capillaries in the following ways: (1) The attachment between the endothelial cells and the surrounding extracellular matrix (ECM) is altered, presumably mediated by proteases and glycosidases, which permit the destruction of the basement membrane surrounding the microvascular endothelial cells, thus allowing the endothelial cells to extend cytoplasmic buds in the direction of chemotactic factors;
  • ECM extracellular matrix
  • FGF fibroblast growth factors
  • aFGF acidic
  • bFGF basic
  • vascular endothelium growth factor VEGF
  • angiopoietin I and II vascular endothelium growth factor
  • prostaglandins El and E2 B. M. Spiegelman et al, 1992
  • ceruloplasmin monocyte derived monocytoangiotropin, placental angiogenic factor, glioma-derived endothelial cell growth factor, and a heparin-binding growth factor from adenocarcinoma of the kidney that is immunologically related to bFGF (R. B . Whitman et al, (1995) U.S. Patent No. 5,470,831).
  • Platelet-derived endothelial cell growth factor does not stimulate proliferation of fibroblasts in contrast to the FGFs, but has demonstrated in vitro angiogenic activity (see C-H. Heldin et al, (1993) U.S. Patent No. 5,227,302).
  • Factors are also known that are capable of inhibiting endothelial cell growth in vitro.
  • protamine is found only in sperm.
  • Platelet factor 4 (PF4) and major basic protein also have been demonstrated to have inhibitory effects on angiogenesis (T. Maione, (1992) U.S. Patent No. 5,112,946).
  • Oncostatin A which is similar to native PF4, has also been implicated as effecting the growth of tumors and therefore may act as an angiogenesis inhibitor (T. Maione, 1992). Antibodies have also been created possessing anti-angiogenic activity (see for example, C.R. Parish (1997) U.S. Patent No. 5,677,181). Gene therapy has also been contemplated as a means of promoting or inhibiting angiogenesis (T. J. Wickhane et al, (1996) J. Virol. 70: 6831).
  • Wounds are internal or external bodily injuries or lesions caused by physical means, such as mechanical, chemical, bacterial, or thermal means, which disrupt the normal continuity of structures.
  • Such bodily injuries include contusions, wounds in which the skin is unbroken, incisions, wounds in which the skin is broken by a cutting instrument, and lacerations, wounds in which the skin is broken by a dull or blunt instrument. Wounds may be caused by accidents or by surgical procedures.
  • Wound healing consists of a series of processes whereby injured tissue is repaired, specialized tissue is regenerated, and new tissue is reorganized. Wound healing is usually divided into three phases: the inflammatory phase, the proliferative phase, and the remodeling phase. Fibronectin has been reported to be involved in each stage of the wound healing process, particularly by creating a scaffold to which the invading cells can adhere. Initially, many mediators, such as fibronectin and fibrinogen, are released to the wound site. Thereafter, angiogenesis and re-epithelialization take place (A. Beauliu (1997) U.S. Patent No. 5,641,483). Repair of injured tissue due to ischemia is a form of wound healing which requires extensive remodeling and re-vascularization.
  • An infarct is, by definition, and area of tissue ischemic necrosis caused by occlusion of local blood circulation.
  • the resulting necrotic lesion leaves the affected tissue deprived of oxygen and nutrients.
  • obstruction of coronary circulation in particular, results in myocardial infarction.
  • the hypoxic microenvironment of the infected cardiac muscle induces the synthesis of angiogenic factors to attempt re-vascularization.
  • VEGF vascular endothelium growth factor
  • Ref infarction
  • ischemic injured tissue outside the heart also produce various angiogenic factors.
  • the ECM contains several macromolecules, including collagen, fibronectin, fibrin, proteoglycans, and elastin.
  • repair also entails the removal of cellular debris, and the laying down of a new ECM over which epidermal continuity can be reestablished. This process of repair and dermal matrix reorganization is manifested as scar formation and maturation.
  • TGF ⁇ transforming growth factor ⁇
  • TGF ⁇ can also upregulate cell surface expression of the integrins that act as receptors for fibronectin, collagen, laminin, and vitronectin thereby influencing cell adhesion and migration.
  • TGF ⁇ enhances the epithelial covering of exposed dermis and increases tensile strength in incision wounds. See J. W. Siebert et al, (1997) U.S. Patent No. 5,591,716) for additional discussion of growth factors that are involved in the process of wound healing and scarring.
  • Leptin and its associated receptor previously have not been associated with angiogenesis, repair of ischemic tissue or wound healing. Moreover, before the present invention, the leptin receptor has never previously been reported as expressed in vascular cells such as endothelial cells and vascular smooth muscle cells.
  • This invention relates to a novel method of modulating angiogenesis, repair of ischemic tissue and wound healing using leptin and leptin receptors, which have been demonstrated to play a role in angiogenesis and wound healing. Isolation of agents that modulate leptin or the leptin receptor can be utilized in methods to treat diseases or conditions that are mediated by angiogenesis and/or wound healing in subjects such as humans. Leptin or its analogs or its specific inhibitors or other agents that modulate the leptin receptor or agents that may induce leptin or leptin receptor synthesis can be administered to the subject in an amount effective to produce an angiogenic response.
  • reagents contemplated for use in modulating angiogenesis include leptin homologues, angiogenic peptide fragments of leptin, idiotypic antibodies that bind to the leptin binding site on the leptin receptor, leptin sensitizers, and an angiogenesis-inducing compound released by a tumor.
  • Another aspect of the invention relates to the use of one or more agents that regulate angiogenesis in combination with compounds which modulate leptin activity, leptin receptor activity and/or leptin receptor ligand activity.
  • the other agents to be used in combination include VEGF, FGF, PDGF, TGF- ⁇ , angiopoietin, TNF and leptin sensitizers.
  • the invention also discloses a method of identifying agents that modulate the angiogenic activity of the leptin receptor in vascular cells .
  • This method comprises the steps of (1) providing an agent that binds to the leptin receptor or fragment thereof; (2) contacting endothelial cells with the agent; and (3) determining whether the agent induced a morphological change in the endothelial cells consistent with an angiogenic or anti-angiogenic effect.
  • Another method contemplated comprises the steps of (1) contacting vascular cells with the agent; (2) determining whether the agent modulates leptin receptor rnRNA expression; and (3) determining whether the agent induces a morphological change in the vascular cells consistent with an angiogenic or anti-angiogenic effect.
  • Too much or too little angiogenesis may be undesired depending on the disease or condition involved.
  • Preferred method comprises the step of administering to the subject an effective amount of an agent that modulates leptin expression or leptin receptor activity sufficient to modulate the undesired angiogenesis.
  • Another aspect of this invention relates to antibodies that bind to the leptin receptor, wherein the binding of the antibody to the receptor modulates leptin receptor-mediated response by the cell to an angiogenesis-inducing stimulus.
  • compositions such as a wound dressing comprising at least leptin and a suitable carrier.
  • Other wound healing compositions contemplated include a topical composition comprising at least one agent that modulates a response in a subject to an angiogenesis-inducing stimulus, comprising an effective amount of an agent that modulates leptin or leptin receptor mediated angiogenic response to that stimulus, together with a pharmaceutically acceptable carrier.
  • the preferred leptin receptor contemplated is the long form, however other isoforms of the leptin receptor are also considered.
  • One method for promoting the formation, maintenance or repair of tissue which comprises the step of administering, to a subject in need thereof, an effective amount of an agent that induces a leptin or leptin receptor-mediated angiogenic response in the subject.
  • This response can affect vascular cells such as endothelial cells or vascular smooth muscle cells.
  • Preferred administration of agents is local, although systemic administration is also contemplated.
  • agents can be used in combination with other angiogenic agents such as VEGF, FGF, PDGF and leptin sensitizers.
  • VEGF vascular endothelial cells
  • FGF vascular smooth muscle cells
  • leptin sensitizers vascular smooth muscle cells.
  • One preferred example would be the administration of leptin and VEGF to enhance wound healing.
  • Other agents to be used in combination with leptin include TGF- ⁇ , angiopoietin, and TNF.
  • compositions disclosed for the treatment of skin wounds are based on a pharmaceutical composition comprising at least one agent that modulates leptin or leptin receptor activities and/or their synthesis or degradation.
  • such compositions may be applied directly, and are preferably applied first to a dressing material and then the impregnated dressing material is applied to wounded or traumatized skin.
  • the dressing material may also include at least one additive selected from the group comprising: keratolytics, surfactants, counterirritants, humectants, antiseptics, lubricants, astringents, emulsifiers, wetting agents, wound healing agents, adhesion/coating protectants, vasoconstrictors, antichlolinergics, corticosteroids, anesthetics and anti-inflammatory agents.
  • at least one additive selected from the group comprising: keratolytics, surfactants, counterirritants, humectants, antiseptics, lubricants, astringents, emulsifiers, wetting agents, wound healing agents, adhesion/coating protectants, vasoconstrictors, antichlolinergics, corticosteroids, anesthetics and anti-inflammatory agents.
  • Figure 1 A shows confocal immunofluorescence microscopy of human umbilical vein endothelial cells HUVECs that were previously permeabilized (panels 1, 2 and 4), or not (panel 3), by a brief treatment with 0.1% Triton X-100.
  • Figure ID is a histochemical analysis of frozen sections from normal human dermis immunostained with normal rabbit IgG (panel 1), anti-von Willebrand factor IgG (panel 2), or anti-IC-1 antibodies against residues 1148-1156 from the carboxy terminus of human OB-Rb (panel 3).
  • Figure 2A shows chemotaxis of bovine lung microvascular endothelial cells BLMVEC as determined by the number of cells migrating across a porous membrane in a 48-well Boyden chamber in response to increasing concentrations of human recombinant leptin. For comparison, the directional chemotactic effect elicited by 1 nM VEGF is also shown (crosshatched bar).
  • Figure 2B depicts tubule formation in Type I collagen gel cultures of BLMVEC after 6 days of treatment with no leptin (panels 1 and 2), 0.5 nM leptin (panels 3 and 4), and 5 nM leptin (panels 5 and 6).
  • Panels, 1, 3 and 5 are Varel contrast microscope images of the collagen gel cultures, whereas panels 2, 4 and 6 represent the corresponding histological sections of the formalin-fixed gels after staining with hematoxilin and eosin (H&E). Note the appearance of a tubular network in the cultures treated with leptin.
  • the inset in panel 4 represents a magnified view (6300X) of the region indicated by the arrows showing the existence of tubular structures with a lumen. Scale bar is 200 ⁇ m.
  • Figure 3 In Vivo Angiogenic Activity of Leptin.
  • Figure 3A shows corneal response 7 days after implanting a Hydron pellet containing PBS. Note the quiescent appearance of the limbus and absence of new vessels.
  • Figure 3B shows corneal response 7 days after implanting a Hydron pellet containing 10 ng of human recombinant leptin. Only occasional vessels can be seen extending from the limbus toward the implant (not visible).
  • Figure 3C shows vigorous neo vascular response 7 days after implanting a Hydron pellet containing 50 ng of leptin.
  • FIG. 4A Leptin Induced Cell Cluster Formation.
  • the graphs show the ability of leptin to induce cluster formation after 1 hour (Fig. 4B and C) and after 24 hours (Fig. 4D and E).
  • No leptin Control as depicted in Fig. 4A
  • 1 nM of leptin Fig.4B and D
  • 10 nM of leptin Fig. 4C and E
  • Figure 5 Stained Cells of Leptin Induced and Uninduced Cells.
  • Figure 5A shows HUVEC non-treated control cells which display normal morphology. Staining of nuclei with DAPI (blue), and cell surface with TRITC-labeled ulex europeous lectin (red).
  • Figure 5B are HUVEC cells treated with 10 nM human recombinant leptin for 24 hours. Cells become elongated and arrange into cord-like structures and closed circles. Double exposure photograph. Staining of nuclei with DAPI (blue), and cell surface with TRITC-labeled ulex europeous lectin (red).
  • Figure 5C shows HUVEC treated for 24 hours with 10 nM human recombinant leptin. Cells become elongated and arrange into cord-like structures and closed circles. Double exposure photograph. Staining of the nuclei was accomplished using DAPI (blue) and of the cell surface using ulex europeous lectin (red).
  • Figure 5D depicts HUVEC treated with 10 nM human recombinant leptin for 24 hours. Cells become elongated and arrange into cord-like structures and closed circles. Double exposure photograph. Staining was described in the previous photographs.
  • Figure 5E the immunofluorescence image was captured by a confocal microscope of HUVEC treated with 10 nM human recombinant leptin for 24 hours. Cells were stained with anti-OB-Rb (long form of leptin receptor) antibodies. The intracellular distribution of the receptor appears in large clusters or vesicles.
  • O-OB-Rb long form of leptin receptor
  • Figure 6 VEGF and Leptin Synergistically Enhance Angiogenesis.
  • Figure 6A shows control cells.
  • Figure 6B shows cells in the presence of 2 nM leptin.
  • Figure 6C shows the effect of 4 nM leptin on cells.
  • Figure 6D shows the synergistic effect of leptin and VEGF.
  • FIG. 7 Leptin Enhanced Wound Healing in SCID-beige mice. This figure shows the histology results of scalpel induced wounds in SCID-beige mice.
  • Figures 7A and B are samples of stained tissue from the leptin untreated wound.
  • Panels C and D are of the leptin treated wound.
  • Figure 8. Expression of Ob-R(L in Human Vascular Smooth Muscle Cells This figure shows that both HUVEC and vascular smooth muscle cells (VSMC) express the long form of the leptin receptor, Ob-R(L).
  • VSMC vascular smooth muscle cells
  • Figure 9A to 9D show HUVEC cultures under various conditions; Figure 9E shows expression of uncoupling protein 2.
  • Leptin is meant the leptin protein, a product of the ob gene, and its allelic variants and homologues as found (or as is believed to be found) in all vertebrate species, including human, bovine, avian, etc.
  • Leptin encoding nucleic acid molecules include allelic variants, mutants and nucleic acids that encode biologically active variants.
  • the "biologically active variants” are those leptin variants that can induce angiogenic activity and/or enhance wound healing.
  • Leptin nucleic acid molecules also encompass cDNAs, RNAs, recombinant RNAs and DNAs, and antisense molecules.
  • Leptin receptor is meant to include both the long form, Ob-R(L), and the short form, Ob-R(S) or Ob-Rb, as well as other leptin receptor isoforms.
  • Leptin receptor also includes allelic variants and homologues as found in most or all vertebrate species, including human, bovine, avian, etc.
  • Leptin receptor encoding nucleic acid molecules include allelic variants, mutants and nucleic acids that encode biologically active variants of the leptin receptor.
  • the "biologically active variants” are those leptin receptor variants that are involved in the leptin-mediated induction of angiogenic activity and/or leptin mediated enhancement of wound healing.
  • Leptin receptor nucleic acid molecules also encompass cDNAs, RNAs, recombinant RNAs and DNAs, and antisense molecules.
  • modulating is meant the ability to regulate a biological effect or process, such as repair of ischemic tissue, wound healing and or angiogenesis. Modulation can occur by “inhibiting”, “blocking”, “down-regulating” or “depressing” leptin and/or leptin receptor-mediated activity. Modulation also encompasses instances wherein leptin or leptin receptor activity is “induced”, “up-regulated”, “increased”, “promoted”, or “enhanced”.
  • anti-angiogenic effect is meant a morphological response that inhibits or blocks vascularization including neovascularization or revascularization.
  • An “anti-angiogenic effect” is one wherein vascularization and associated morphological changes in vascular cells, such as endothelial cells and vascular smooth muscle cells, does not occur or is inhibited.
  • angiogenic and angiogenesis refer to revascularization or neovascularization of tissue. Such neovascularization can result from the process of wound healing, repair of ischemic tissue or tissue growth.
  • An “angiogenic effect” can be one wherein vascularization occurs or morphological changes associated with angiogenesis are observed in vascular cells such as endothelial cells ("EC”) and vascular smooth muscle cells.
  • polypeptide fragments and “peptide fragments” are meant those portions of leptin and the leptin receptor capable of modulating angiogenesis and/or wound healing.
  • Antists include those agents, compounds, compositions, etc. which when administered can up regulate (increase, promote or otherwise elevate the level of) angiogenesis and/or wound healing by promoting leptin activity, leptin receptor activity, leptin/leptin receptor interaction, or a combination thereof.
  • Antagonists include those agents, compounds, compositions, etc. which when administered cause the down regulation (inhibition, prevention, reduction, etc.) of angiogenesis, wound healing and/or repair of ischemic tissue by inhibiting leptin activity, leptin receptor activity, leptin/leptin receptor interaction, or a combination thereof.
  • Peptides and polypeptide fragments of leptin or of the leptin receptor include those peptide agents capable of modulating angiogenic, wound healing and/or repair of ischemic tissue activity.
  • polypeptides, and derivatives or analogs thereof, as contemplated by the present invention are those that have the ability to inhibit angiogenesis, wound healing and/or repair of ischemic tissue, or to promote angiogenesis, wound healing and/or repair of ischemic tissue by affecting leptin receptor activity, leptin activity and/or leptin receptor ligand activity.
  • These polypeptides and peptides encompass derivatives, analogs and peptidomimetics (i.e., molecules having some structural and functional characteristic in common with peptides, but that do not contain peptide bonds).
  • One preferred embodiment includes leptin and fragments thereof that bind to the leptin receptor.
  • leptin polypeptides or "leptin receptor polypeptides” are fragments of these peptides comprising at least about 2, 3, 5, 10, 15, 20, 25, 30 or 50 consecutive amino acid residues.
  • isolated DNA, RNA, peptides, polypeptides, or proteins are DNA, RNA, peptides polypeptides or proteins that are isolated or purified relative to other DNA, RNA, peptides, polypeptides, or proteins in the source material.
  • isolated DNA that encodes leptin (which would include cDNA) refers to DNA purified relative to DNA which encodes polypeptides other than leptin.
  • Disease states and other conditions involving "angiogenic activity” include, but are not limited to myocardial conditions, trauma, tumors (benign and malignant) and tumor metastases, ischemia, tissue and graft transplantation, diabetic microangiopathy, neovascularization of adipose tissue and fat metabolism, revascularization of necrotic tissue, eye conditions (e.g., retinal neovascularization), growth of new hair and ovarian follicle maturation.
  • wound healing diseases and other conditions involving "wound healing" include: scarring and scar formation, ischemia, burns, myocardial injury, enhancement of vascularization in microvascular transplants, enhancement of revascularization in necrotic tissue and tissue and graft transplants. Also contemplated is enhancement of wound healing in subject with poor wound healing, as in diabetic individuals. These conditions may be mediated by modulation of leptin, leptin receptor, and leptin receptor ligands activity.
  • vascular cells is meant to include both “endothelial cells” (also referred to as “EC”) and “smooth muscle cells” and “vascular smooth muscle cells” (also referred to as “SMC”).
  • “Pharmaceutically acceptable” refers to molecular entities and compositions such as fillers and excipients that are physiologically tolerated and do not typically produce an allergic or toxic reaction, such as gastric upset, dizziness and the like when administered to a subject or a patient; the preferred subjects of the invention are vertebrates, mammals, and humans.
  • One embodiment of this invention relates to leptin and leptin receptor-related methods and compositions that modulate angiogenesis, wound healing and/or repair of ischemic tissue.
  • modulating angiogenesis, repair of ischemic tissue and wound healing is the administration of leptin to a subject, either systemically or locally or both.
  • Other agents that mediate leptin activity, leptin receptor activity, and leptin/leptin receptor interaction are also contemplated.
  • "recombinant" leptin and “recombinant” leptin receptor refers to leptin and the leptin receptor produced by recombinant expression of nucleic acid molecules encoding said proteins.
  • the production of a recombinant form of a leptin or the leptin receptor protein typically involves the following steps. Similar steps can be utilized for production of other forms of leptin receptor ligands.
  • a nucleic acid molecule is obtained that encodes a leptin or leptin receptor protein or polypeptide fragment thereof.
  • the leptin or leptin receptor encoding nucleic acid molecule is then preferably placed in operable linkage with suitable control sequences to form an expression unit containing the leptin or the leptin receptor encoding sequences.
  • the expression unit is used to transform a suitable host, and the transformed host is cultured under conditions that allow the production of the desired protein or polypeptide fragments thereof.
  • the leptin and the leptin receptor proteins may be isolated from the medium or from the cells and further purified; recovery and purification of the protein may not be necessary in some instances where some impurities may be tolerated.
  • agents contemplated include leptin, the leptin receptor, other leptin receptor ligands, allelic variants of leptin and the leptin receptor, and corresponding proteins in which conservative amino acid substitutions have been made such as fusion proteins.
  • leptin and leptin receptor nucleic acid molecules and corresponding protein sequences are disclosed as follows: GenBank
  • an "allelic variant” refers to a naturally occurring leptin or leptin receptor having a different amino acid sequence than those sequences listed above that specifically recited above.
  • the allelic variants of leptin or the leptin receptor though possessing a slightly different amino acid sequence, such as a conservative amino acid substitution, than those recited above, will still have the requisite biological activity to modulate angiogenesis, wound healing and/or repair of ischemic tissue.
  • a “conservative amino acid substitution” refers to alterations in the amino acid sequence of either leptin or the leptin receptor which do not adversely effect their ability to modulate angiogenesis, wound healing and/or repair of ischemic tissue.
  • a substitution is said to adversely affect leptin or leptin receptor when the altered sequence decreases the capacity of leptin or the leptin receptor to modulate angiogenesis, wound healing and/or repair of ischemic tissue.
  • Allelic variants, conservative substitution variants and related proteins utilized herein preferably will have an amino acid sequence having at least about 75% amino acid sequence identity with the published leptin or leptin receptor sequences disclosed above, more preferably at least about 80%), even more preferably at least about 90%, and most preferably at least about 95%.
  • the peptides, variants and related molecules that are the subject of or utilized in this invention include molecules having the sequences disclosed; fragments thereof having a consecutive sequence of at least about 3, 5, 10, 15, 20, 25, 30, 50 or more amino acid residues from the corresponding leptin or the leptin receptor and amino acid sequence variants of the disclosed leptin or the leptin receptor sequences, or their fragments as defined above, that have been conservatively substituted by another residues.
  • Leptin or leptin receptor proteins or polypeptide fragments thereof also can be expressed in cells or in hosts by transfecting the cells or hosts with viral vectors capable of expressing said proteins.
  • the viral vectors contemplated include adenoviral. (See for example D. Armentano et al, (1998) U.S. Patent No. 5,707,618 and T. J. Wickham et al, (1998) U.S. Patent No. 5,731,190), retroviral (V. K. Pathak et al, (1998) U.S. Patent No. 5,714,353; E. F. Vanin et al, (1998) U.S. Patent No. 5,710,037; D.A. Williams et al, (1997) U.S. Patent No.
  • transfected eukaryotic cells such as fibroblasts or bone marrow-derived stromal cells
  • transfected eukaryotic cells such as fibroblasts or bone marrow-derived stromal cells
  • fibroblasts or bone marrow-derived stromal cells can provide a different method of introducing leptin or the leptin receptor encoding DNAs in vivo into host cells.
  • polypeptide fragments, peptides, peptide mimetics, derivatives, and analogs of leptin, the leptin receptor, and other leptin receptor ligands are contemplated for use in modulating angiogenic, wound healing activity and/or repair of ischemic tissue These compounds can be obtained from a variety of sources.
  • leptin and the leptin receptor as a means of enhancing angiogenesis, repair of ischemic tissue and wound healing
  • other agents which modulate angiogenesis, wound healing, and/or repair of ischemic tissue are contemplated in the present invention.
  • the leptin/leptin receptor example can also be applied to other leptin receptor-ligands which are involved in angiogenesis, wound healing and/or repair of ischemic tissue and are expressly contemplated by the present invention.
  • leptin or leptin receptor or fragments thereof can be prepared using chemical peptide synthesis.
  • Techniques for chemical synthesis are well known in the art. For example, see Fields et al, (1990) Int. J. Pept. Protein. Res. 35: 161; and Stewart (1984) SOLID PHASE SYNTHESIS (2nd ed., Pierce Chemical Col, Rockford, 111.).
  • the preferred fragments of leptin or the leptin receptor to be utilized have about at least 3, 5, 7, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more consecutive amino acids.
  • the present invention provides methods for identifying agents that modulate the synthesis, degradation and activity of leptin and/or the leptin receptor.
  • angiogenesis By regulating the activity of leptin and/or the leptin receptor, angiogenesis, repair of ischemic tissue and wound healing may be modulated. Modulation can proceed by regulating leptin, the leptin receptor, the leptin/leptin receptor interaction, other leptin receptor-ligand interactions, or the signaling cascade that follows activation of the leptin receptor by leptin or other receptor agonists.
  • the present invention contemplates modulating the activities of leptin and other agents on the leptin receptor shown by the present invention to be expressed on vascular cells, such as endothelial cells and smooth muscle cells.
  • vascular cells such as endothelial cells and smooth muscle cells.
  • agents or protein-protein combinations can be substituted.
  • This invention relates to agents (or compounds) that modulate (regulate, inhibit or promote) angiogenesis, wound healing and/or repair of ischemic tissue by modulating (1) leptin activity, leptin synthesis and leptin degradation, (2) leptin receptor activity, synthesis and degradation, (3) leptin/leptin receptor interactions, and/or (4) the interaction of the leptin receptor with other ligands.
  • agents or compounds that modulate (regulate, inhibit or promote) angiogenesis, wound healing and/or repair of ischemic tissue by modulating (1) leptin activity, leptin synthesis and leptin degradation, (2) leptin receptor activity, synthesis and degradation, (3) leptin/leptin receptor interactions, and/or (4) the interaction of the leptin receptor with other ligands.
  • leptin and/or leptin receptor activity angiogenesis, wound healing and/or repair of ischemic tissue can also be modulated.
  • an agent is said to modulate angiogenesis, wound healing and/or repair of ischemic tissue when it enhances or inhibits one of the four activities recited immediately above.
  • Agents contemplated include agonists and antagonists of the long form of the leptin receptor.
  • the agents being screened as agonists or antagonists can be randomly selected or rationally selected or designed.
  • an agent is said to be randomly selected when the agent is chosen randomly without considering the specific protein domains or sequences involved in the modulation of leptin or leptin receptor-mediated angiogenesis, wound healing and/or repair of ischemic tissue.
  • An example of randomly selected agents is the use a chemical library or a peptide combinatorial library, or a growth broth of an organism.
  • an agent (reagent, compound, composition, etc.) is said to be rationally selected or designed when the agent is chosen on a non-random basis which rationally selected or designed when the agent is chosen on a non-random basis which takes into account the sequence of the target site and/or its conformation in connection with the agent's action.
  • the agents contemplated include those that modulate (1) leptin activity, its synthesis or degradation, (2) leptin receptor activity, synthesis or degradation, (3) leptin/leptin receptor interaction, or (4) other leptin receptor ligands. Also contemplated are agents that alter leptin or the leptin receptor conformationally, thereby changing the protein such that it cannot bind with its ligand-partner.
  • Agents can be, for example, rationally selected or rationally designed by utilizing the peptide sequences that make up the contact sites of the leptin-leptin receptor complex. Additional agents that modulate the interaction of the leptin receptor with ligand partners other than leptin can be screened in a similar manner.
  • agents of the present invention can be, as examples, peptides, small molecules, vitamin derivatives, as well as carbohydrates. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • One preferred class of agents of the present invention are peptide agents whose amino acid sequences are chosen based on the amino acid sequence of either leptin or the leptin receptor.
  • agents that can bind to the domains on leptin and the leptin receptor, polypeptide fragments thereof (e.g., at least 3 consecutive amino acid residues or more of leptin or the leptin receptor, peptides thereof, peptide mimetics, antibodies (e.g., polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies), antibody fragments, derivatives or analogs capable of modulating angiogenesis, wound healing and/or repair of ischemic tissue.
  • Such agents can be obtained from any source, e.g., by purification from natural sources, using recombinant DNA technology or by chemical synthesis.
  • the peptide agents of the invention can be prepared using standard solid phase (or solution phase) peptide synthesis methods, as is known in the art.
  • the DNAs encoding these peptides may be synthesized using commercially available oligonucleotide synthesis instrumentation and produced recombinantly using standard recombinant production systems. Solid phase peptide synthesis may be appropriate, if non-gene-encoded amino acids are involved.
  • Leptin is mixed with the leptin receptor, particularly the leptin receptor obtained from or derived from leptin receptor expressing endothelial cells or smooth muscle cells, particularly human endothelial cells and smooth muscle cells, in the presence and absence of an agent to be tested.
  • This assay can also be done using intact live cells as the source of the leptin receptor.
  • the two mixtures are analyzed and compared to determine if the agent modulated (e.g., enhanced or inhibited) the association of leptin with the leptin receptor.
  • An agent that blocks or reduces the association of leptin with the leptin receptor will be identified by its ability to decrease the amount complexed leptin with the leptin receptor present in the sample containing the tested agent.
  • These assays also can be performed by attaching leptin or leptin receptor proteins or polypeptide fragments thereof which contain the leptin-binding domain or the leptin receptor binding domain to a solid substrate (e.g., a column or ELISA plate).
  • a solid substrate e.g., a column or ELISA plate.
  • the putative binding agent is then brought in contact with the protein or polypeptide fragments bound to the solid substrate. After washing away free compounds, it can then be determined whether the binding agent bound to the proteins (e.g., leptin or leptin receptor or polypeptide fragments thereof), which are linked to the solid surface.
  • agents which can bind to leptin, the leptin receptor, leptin receptor ligands, or polypeptide fragments thereof, these agents can then be analyzed using known in vitro systems to determine whether the agent can modulate angiogenesis, wound healing or repair of ischemic tissue.
  • Identification of agents capable of inhibiting angiogenesis, wound healing and/or repair of ischemic tissue can be made using assays utilizing in vitro endothelial cell cultures as described in B.M. Spiegelman et al, (1992) U.S. Patent No. 5,137,734).
  • the assay can also be modified to use smooth muscle cells.
  • the overall angiogenic regulation of a test substance can be measured in vivo in model systems such as the chick chorioallantoic system (which measures angiogenic activity in an embryonic system), in the rabbit corneal pocket assay, and the hamster cheek pouch assay (which measures angiogenic activity in more mature systems), also as described in B.M. Spiegelman et al, (1992).
  • model systems such as the chick chorioallantoic system (which measures angiogenic activity in an embryonic system), in the rabbit corneal pocket assay, and the hamster cheek pouch assay (which measures angiogenic activity in more mature systems), also as described in B.M. Spiegelman et al, (1992).
  • assays using human endothelial cells or smooth muscle cells are utilized.
  • Leptin and the leptin receptor or fragments thereof, oligopeptides, polypeptides, mimetics, and other chemical compounds can be used for screening in any of a variety of drug screening techniques.
  • the fragment employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • the modulation of leptin synthesis and degradation activity, leptin receptor activity, and leptin/leptin receptor interaction resulting from the presence of the candidate agent may then be measured. Modulation of leptin or leptin receptor activity can result from interactions with an agent that induce changes in stability, maturation, integrity or secretion of leptin or the leptin receptor.
  • Purified leptin or Leptin receptor can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support (see P.R. Hawkins et al, (1998) U.S. Patent No. 5,712,115).
  • Another method of screening contemplated may involve labeling leptin or the leptin receptor polypeptides with any of a myriad of suitable markers, including radiolabels (e.g., 125 I or 32 P), various fluorescent labels and enzymes (e.g., glutathione-S-transferase, luciferase, and ⁇ -galactosidase).
  • radiolabels e.g., 125 I or 32 P
  • various fluorescent labels and enzymes e.g., glutathione-S-transferase, luciferase, and ⁇ -galactosidase.
  • the target polypeptide may be immobilized by standard techniques. For example, but not for limitation, such immobilization may be effected by linkage to a solid support, such as a chromatographic matrix, or by binding to a charged surface, such as a nylon membrane.
  • Binding assays generally take one of two forms: immobilized leptin or the leptin receptor polypeptides can be used to bind the leptin receptor or leptin polypeptides, respectively.
  • the labeled polypeptide is contacted with the immobilized polypeptide under aqueous conditions that permit specific binding of the polypeptide(s) to form a leptin/leptin receptor complex in the absence of added agent.
  • Particular aqueous conditions may be selected by the practitioner according to conventional methods.
  • additions, deletions, modifications (such as pH), and substitutions (such as KCl substituting for NaCI or buffer substitution) may be made to these basic conditions. Modifications can be made to the basic binding reaction conditions, so long as specific binding of leptin or leptin receptor polypeptides to the leptin receptor or leptin polypeptides occurs in the control reactions. Conditions that do not permit specific binding in control reactions (no agent included) are not suitable for use in performing the assays.
  • At least one polypeptide species is labeled with a detectable marker.
  • Suitable labeling includes, but is not limited to radiolabeling by incorporation of a radiolabeled amino acid (e.g., 14 C-Leucine, 3 H-Glycine, 35 S-methionine), radiolabeling by post-translational radioiodination with 125 I or 131 I (e.g., Bolton-Hunter reaction and chloramine T), labeling by post-translational phosphorylation with 32 P (e.g., phosphorylase and inorganic radiolabeled phosphate), or labeling by other conventional methods known in the art.
  • a radiolabeled amino acid e.g., 14 C-Leucine, 3 H-Glycine, 35 S-methionine
  • radiolabeling by post-translational radioiodination with 125 I or 131 I (e.g., Bolton-Hunter reaction and chloramine T)
  • the other polypeptide is generally labeled with a detectable marker.
  • yeast two hybrid screening methods refer to J. R. Bischoff et al, (1998) U.S. Patent No. 5,705,342 and S. Fields etal., (1994) U.S. Patent No. 5,283,173.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the leptin receptor or leptin specifically compete with a test compound for binding with leptin or the leptin receptor such that the leptin/leptin receptor complex cannot form.
  • the antibodies can be used to detect the presence of any peptide sharing one or more antigenic determinants with leptin and the leptin receptor.
  • Drug screening can also be performed using vascular cells such as endothelial cells or vascular smooth muscle cells. Both of these cell lines express large quantities of leptin receptor making them extremely beneficial for studying methods of modulating leptin and the leptin receptor.
  • One preferred method for screening regulatory agents utilizes STAT3, as JAK/STAT interacts with the leptin receptor.
  • a reporter system using STAT3 can be prepared utilizing the specific binding site in STAT3, called cAPRE (the nucleic acid sequence is TTCCCGAA). The constructs are prepared such that the sequence is a multimerized minimal STAT3 binding cite (TTCCCGAA) inserted upstream from a minimal promoter, such as tyrosine kinase (tk) (refer to J.
  • Reporter genes are then placed in include, but are not limited to, luciferase, green fluorescent proteins (GFP), ⁇ -galactosidase, etc.
  • a related method of drug screening utilizes promoter driven toxigenes. In these models, toxins such as ricin or diphtheria toxins are driven via the promoter induced by the given hormonal (e.g., leptin) stimulus. Therefore, cell ablation (cell death) can be induced when the cell receives the hormonal signal (see these articles for method details on ablation style reporting systems, P. L. Herrera et al, 1994 Proc. Nat'l Acad. Sci. USA 91: 12999-13003; A. Negro et al, 1996 Eur. J. Biochem. 241: 507-15). Cell death would be the equivalent of luciferase detection of Other methods are known and available in the art.
  • Another embodiment of this invention relates to creating antibodies and antibody fragments that modulate leptin and/or leptin receptor activity and the interaction between leptin and the leptin receptor.
  • An "epitope” refers generally to a specific recognition feature of a molecule, which depends on the topological orientation of functional groups of the molecule. According to the invention, a molecule contains an epitope, or shares an epitope of a second molecule, if the first molecule specifically binds or interacts competitively with the specific binding of the second molecule.
  • the present invention relates to antibodies that target or bind to one or to more than one epitope on either leptin or the leptin receptor.
  • antibody is meant a polyclonal or monoclonal antibody which is capable of binding to leptin, the leptin receptor, or a leptin receptor ligand and modulating thereby their angiogenic, wound healing and/or repair of ischemic tissue activity.
  • antibody therefore encompasses monoclonal and polyclonal antibodies and fragments thereof (e.g., Fv, scFv, Fab, Fab 1 , or F(ab') 2 fragments).
  • the antibodies contemplated also include different isotypes and isotype subclasses (e.g., IgG ⁇ IgG 2 , IgM, to name a few). These antibodies can be prepared by raising them in vertebrates, in hybridoma cell lines or other cell lines, or by recombinant means.
  • chimeric, human, and humanized antibodies and fragments thereof which will be less immunogenic in the subject in which they are administered (e.g., a human or humanized antibody administered to a human subject).
  • a human or humanized antibody administered to a human subject For references on how to prepare these antibodies, see D. Lane, ANTIBODIES: A
  • Sequences comprising domains on leptin, the leptin receptor or leptin receptor ligands which are immunogenic for purposes of creating antibodies can be determined using such algorithms as described by Hopp and Woods, Proc. Nat'l Acad. Sci. USA 78: 3824 (1981); and Gamier et al, J. Mol. Bio. 120: 97 (1978). Additional algorithms would be known to the skilled artisan and can be used to identify peptides suitable for anti-peptide antibody production.
  • leptin and/or leptin receptor proteins Use of leptin and/or leptin receptor proteins, the nucleic acid molecules encoding them or agents that modulate their expression in combination with other angiogenic or anti-angiogenic factors is also contemplated.
  • the agents to be administered in combination with leptin or other agents that modulate leptin or leptin receptor activity include, but are not limited to, those agents described in: N. Catsimpoolas et al, (1988) U.S. Patent No. 4,778,787; D'Amato (1998), G. S. Schultz et al, (1991) Eye 5: 170; B.M. Spiegelman et al, (1992) U.S. Patent No. 5,137,734 (angiogenic monoglycerides); T.
  • Agents of the present invention that modulate the activity of leptin and/or leptin receptor can be provided alone, or in combination with other agents that modulate a particular biological or pathological process.
  • leptin can be administered in combination with VEGF (or PDGF and FGFs, TNFa, IL-1 IL-11 or IL-6) to enhance angiogenesis.
  • VEGF or PDGF and FGFs, TNFa, IL-1 IL-11 or IL-6
  • combination therapy are specific to regulation of leptin and/or leptin receptor activity.
  • Other combination therapies involving leptin and leptin receptor ligands are also contemplated in the present invention.
  • the therapies described by enhanced angiogenesis spurred by leptin being only one example.
  • two agents are said to be administered in combination when the two agents are administered simultaneously or are administered independently in a fashion such that the agents will act at the same time.
  • Other embodiments include the administration of two or more agents that regulate leptin receptor activity, leptin activity, or both.
  • One illustration includes combinations of agents wherein two or more leptin or leptin receptor antagonists or two or more agonists are administered to a subject.
  • Typical dosages of an effective leptin or leptin receptor agonists or antagonists can be in the ranges recommended by the manufacturer where known therapeutic compounds are used, and also as indicated to the skilled artisan by the in vitro responses or responses in animal models.
  • Such dosages typically can be reduced by up to about one order of magnitude in concentration or amount without losing the relevant biological activity.
  • the actual dosage will depend upon the judgment of the physician, the condition of the patient, and the effectiveness of the therapeutic method based, for example, on the in vitro responsiveness of the relevant primary cultured cells or histocultured tissue sample, such as biopsied malignant tumors, or the responses observed in the appropriate animal models, as previously described.
  • reagents that modulate leptin and/or the leptin receptor By utilizing reagents that modulate leptin and/or the leptin receptor, diseases and/or conditions mediated by angiogenesis, or conditions associated with repair of ischemic tissue or wound healing can be regulated.
  • This section describes the diseases wherein reagents can be administered to a subject to enhance or inhibit angiogenesis, wound healing and/or repair of ischemic tissue.
  • the subjects contemplated include all vertebrate species. The more preferred embodiments are the methods of treating diseases in mammals, and the most preferred method is the treatment of humans.
  • the control of angiogenesis, wound healing and/or repair of ischemic tissue can alter the pathological damage associated with the disease or with abnormal angiogenesis.
  • "Abnormal angiogenesis” can be an irregular or abnormal level of neovascularization (e.g., enhanced or depressed neovascularization).
  • angiogenesis Should Be Inhibited Angiogenesis should be inhibited in diseases or conditions in which it is desirable to block or inhibit neovascularization.
  • the conditions and diseases where angiogenesis desirably may be inhibited include: scar formation, tumor metastasis and tumor growth, and tissue adhesions.
  • these conditions and diseases include ocular neovascular diseases (e.g., including diabetic retinopathy, diabetic microangiopathy, retinal neovascularization, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, and retrolental fibroplasia), other diseases associated with corneal neovascularization (e.g., include: epidemic keratoconjunctivitis, vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal degeneration
  • Chronic inflammation may also involve pathological angiogenesis.
  • Diseases with chronic inflammatory conditions considered for treatment using the methods of the present invention include: ulcerative colitis, Crohn's disease, rheumatoid arthritis, and Bartonellosis.
  • Neovascularization also occurs in both benign and malignant tumors, and the vascular endothelial cells and vascular smooth muscle cells in the vicinity of a tumors, particularly those cells within the range of tumor-produced angiogenic factors, therefore correspondingly are also contemplated as preferred targets for therapy.
  • tumor diseases that are contemplated as being appropriate for treatment by the methods of the present invention include, but are not limited to: systemic forms of hemangiomas, hemangiomatosis, Osier- Weber-Rendu diseases, hereditary hemorrhagic telangiectasia, rhabdomyosarcomas, retinoblastomas, Ewing sarcomas, neuroblastomas adenocarcinomas and osteosarcomas.
  • myocardial ischemic conditions e.g., myocardial infarction, revascularization of necrotic tissue, for example of the myocardium after an infarction or an angioplasty, angina, heart transplants, vascular grafts, and reopening vessels to improve vascularization, perfusion, collagenization and organization of said lesions
  • ovarian follicle maturation which may also require down regulation of angiogenesis
  • wound healing and tissue and organ transplantations (e.g., enhancement of autologous or heterologous microvascular transplantation).
  • Neovascularization of grafted or transplanted tissue is also contemplated, especially in subjects suffering from vascular insufficiency, such as diabetic patients.
  • the dynamic process of wound healing is a well regulated sequence of events which, under normal circumstances, results in the successful repair of injured tissues.
  • a cutaneous wound that cuts through the epidermis and dermis (full thickness), is accompanied by blood vessel rupture. Rapidly, clot formation occurs providing a provisional matrix to cover the wound.
  • the clot is a key component because it provides mechanical closure with fibrin and other matrix proteins, and it is the initial source of cytokines, growth factors and chemotactic agents released by platelet degranulation. This cocktail initiates the process of wound healing.
  • neutrophils move into the interstitum at the site of injury in response to bacterial products and other chemotactic agents.
  • fibroblasts that release chemical signals to attract fibroblasts.
  • the resident and infiltrating fibroblasts secrete cytokines such as PDFG-BB and bFGF and begin to deposit a new extracellular matrix that will be an essential component of the scar tissue.
  • cytokines such as PDFG-BB and bFGF
  • the process of reepithelialization begins on the borders of the wound where keratinocytes of the basal layer display new integrins to attach to a provisional matrix.
  • the epidermal migration continues until a monolayer of keratinocytes covers the wound.
  • Several known growth factors intervene in the reepithelialization of the skin e.g., EGF, TGFa and KGF 1 and 2.
  • VEGF secreted acutely by the keratinocytes is responsible in great part for the angiogenic response.
  • leptin angiogenic factors like basic fibroblast growth factor (bFGF) and transforming growth factor b (TGFb) are also present.
  • bFGF basic fibroblast growth factor
  • TGFb transforming growth factor b
  • leptin a protein produced in the underlying adipose tissue
  • Leptin may be present at relatively high concentrations because the dermal vasculature, both superficial and deep plexuses, derive from larger vessels that originate from the subcutaneous adipose layer.
  • leptin plays a role in normal wound healing. Leptin is present at the wound site a few hours after injury. Leptin also peaks in the circulation 12 hours after wounding.
  • the compounds of this invention may be utilized in compositions such as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions or suspensions for parenteral or intramuscular administration and the like.
  • compositions of this invention can be used in the form of a pharmaceutical preparation, for example, in solid, semi-solid or liquid form which contains one or more of the compounds of the present invention, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications.
  • the active ingredient may be compounded, for example, with the usual non-toxic pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use.
  • the carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid or liquid form and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used.
  • the active object compound e.g., an agent capable of modulating leptin, the leptin receptor and/or leptin receptor ligand activity that mediates their angiogenic and/or wound healing capability is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition (e.g., regulation of neovascularization) of the disease.
  • the principal active ingredient is mixed with a pharmaceutical carrier (e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums) and other pharmaceutical diluents (e.g., water) to form a solid preformulation composition containing a substantially homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof.
  • a pharmaceutical carrier e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums
  • other pharmaceutical diluents e.g., water
  • the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
  • This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from about 0.1 mg to about 500 mg of the active ingredient of the present invention.
  • the tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action.
  • the tablet or pill can comprise an inner dosage an outer dosage component, the latter being in the form of an envelope over the former.
  • the two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permits the inner component to pass intact into the duodenum or to be delayed in release.
  • enteric layers or coatings such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.
  • the liquid forms in which the novel composition of the present invention may be incorporated for administration orally or by injection, include aqueous solution, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil as well as elixirs and similar pharmaceutical vehicles.
  • Suitable dispersing or suspending agents for aqueous suspensions include synthetic natural gums, such as tragacanth, acacia, alginate, dextran, sodium carboxymethyl cellulose, methylcellulose, polyvinylpynolidone or gelatin.
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for reconstitution with water or other suitable vehicles before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); preservatives (e.g., methyl or propyl p-hydroxybenzoates or sorbic acid); and artificial or natural colors and/or sweeteners.
  • suspending agents e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily
  • the composition may take the form of tablets or lozenges formulated in conventional manners.
  • formulations may be made up with at least one agent that modulates leptin or leptin receptor activity or the activity of a leptin receptor ligand or provides leptin, such as leptin producing cells.
  • the active ingredient may further be combined in admixture with at least one other ingredient constituting an acceptable carrier, diluent or excipient in order to provide a composition, such as a cream, gel, solid, paste, salve, powder, lotion, liquid, aerosol treatment, or the like, which is most suitable for topical application.
  • a composition such as a cream, gel, solid, paste, salve, powder, lotion, liquid, aerosol treatment, or the like, which is most suitable for topical application.
  • Sterile distilled water alone and simple cream, ointment and gel bases may be employed as carriers of the active agents.
  • bases and suspending vehicles examples include FattibaseTM (acrylic polymer resin base), PolybaseTM (polyethylene glycol base) by Paddock Laboratories, Inc.
  • Additional therapeutic agents may be added to the formulations as medically indicated, selected from the classes of: keratolytics, surfactants, counter-irritants, humectants, antiseptics, lubricants, astringents, wound additional healing agents, emulsifiers, wetting agents, additional adhesion/coating protectants, additional anti-inflammatory agents, vasoconstrictors, vasodilators, anticholinergics, corticosteroids (e.g., glucocorticoids) and anesthetics. Preservatives and buffers may also be added.
  • compositions may be applied to a sterile dressing, biodegradable, absorbable patches or dressings for topical application, or to slow release implant systems with a high initial release decaying to slow release.
  • compositions When the compositions are administered to treat burns, they may be in the form of an irrigant, preferably in combination with a physiological saline solution.
  • Compositions can also be in the form of ointments or suspensions, preferably in combination with purified collagen.
  • the compositions may also be impregnated into transdermal patches, plasters, and bandages.
  • M. W. J. Ferguson et al (1997) U.S. Patent No. 5,662,904; J.D. Gallina (1997) U.S. Patent No. 5,679,655; and M. B. Sporn et al, (1998) U.S. Patent No. 5,705,477.
  • the active compounds may be formulated for parenteral administration by injection, which includes using conventional catheterization techniques or infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampules, or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulating agents such as suspending, stabilizing, and/or dispersing agents.
  • the active ingredients may be in powder form for reconstitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • Leptin the product of the ob gene, is a 15 kDa polypeptide hormone. Produced and secreted largely by adipocytes, leptin primarily regulates adiposity through effects on food intake and energy expenditure via receptors expressed in central and peripheral targets (L.A. Tartaglia et al, 1995 CeU 83: 1263-71; and J. S. Flier, 1998 Proc. Nat'l Acad. Sci. USA 94: 4242-5). Recently, leptin has been reported to directly cause depletion of stored triacylglycerol (TG) in peripheral tissues by a mechanism that seems to involve decreased TG synthesis and increased TG oxidation within cells (U.
  • TG triacylglycerol
  • leptin includes apparent effects upon the activity of acetyl Co A carboxylase, the rate limiting enzyme in fatty acid (FA) synthesis, and increased rates of FA oxidation (Y. Bai et al, 1996 J. Biol. Chem. 271: 13939-42). This is to be distinguished from the lipolytic response normally observed in the transition from the "fed” to the "starved” state. During this transition, blood levels of free fatty acids (FFA) and ketones are not increased, suggesting that peripheral lipid oxidation may be occuring in response to leptin (M. Shimabukuro et al, 1997 Proc.
  • FFA free fatty acids
  • leptin appears to directly influence fuel homeostasis through changes in the expression and/or activity of biochemical pathways involved in thermogenesis and lipid metabolism (Shimabukuro et al, 1997; D. M. Muoio et al, 1997 Diabetes 48: 1360-3; J. A. Tuominen et al, 1997 Microcirculation 4: 211-32).
  • the present inventor here describe findings that leptin, which is produced and secreted by adipocytes, as discussed above, exhibits a potent and concentration-dependent angiogenic activity in EC, both in vivo and in vitro. Moreover, EC express the intact and functional long form of the leptin receptor (Ob-Rb(L)Ob-Rb), which is capable of tranducing a signal through the JAK/STAT pathway. (M. Roci ⁇ Sierra-Honigmann et al., 1998 Science 281:1683-86.) Adipose tissue increases or decreases its mass depending on the demands for storing or utilization of lipid fuels in the body.
  • FIG. 1A depicts confocal immunofluorescence microscopy of human umbilical vein endothelial cells (HUVEC) that were previously permeabilized (panels 1, 2 and 4), or not (panel 3), by a brief treatment with 0.1 % Triton X-100. hnmunostaining was performed (M. R. Sierra-Honigmann et al, (1996) Lab. Invest. 74: 684) with normal rabbit IgG (panel 4), using ⁇ OB-R ⁇ antibodies against the extracellular region of OB-R (panels 2 and 3) and ⁇ and ⁇ OB-R, authority, antibodies against the intracellular region of OB-R (panel 1) described below. Scale bar is 5 ⁇ m.
  • Peptide antibodies were based on the sequence of the human leptin receptor (G. H. Lee et al, (1996) Nature 379: 632) corresponding to regions within the intracellular or the extracellular domain. These peptides were synthesized and coupled to EXH. The intracellular region peptides were (1) IC-1 for residues 1148-65 at the carboxy terminal end of the receptor (CSTQTHKIMENKMCDLTV), and (2) IC-2, for residues 1062-1078 (KLEGNFPEENNDKKSIY).
  • the extracellular region peptides were (1) EC-1, for residues 247-263 (ITDDGNLKISWSSPPLV), (2) EC-2, for residues 473-487 (CSDIPSIHPISEPKD), and (3) EC-3 for residues 753-67 (CVIVSWILSPSDYKL).
  • the KLH-peptide conjugates were used to generate polyclonal antibodies in rabbits, and IgG fractions prepared from bleeds with the highest ELISA titers.
  • antibodies against IC-1 and IC-2 were combined in equal amounts giving rise to ⁇ OB-R, n , antibodies directed against intracellular epitopes of the OB-R.
  • equal amounts of antibodies against EC- 1 , EC-2 and EC-3 were mixed giving rise to ⁇ -OB-R e -, antibodies directed against extracellular epitopes of the OB-R.
  • Figure IB depicts the immunoblotting of total HUVEC cell lysates with ⁇ OB-R, beautase (lane 1), ⁇ OB-R ⁇ IgG (lane 2), or normal rabbit IgG (lane 3).
  • Total HUVEC cell lysates and immunoblotting were performed essentially as described (Sierra-Honigmann et al, 1996).
  • cells in confluent monolayers were washed with ice-cold Ca 2+ - and Mg 2+ -free HBSS, detached with trypsin, centrifuged and resuspended in 500 ⁇ l of lysis buffer [10 mM Tris-HCl pH 7.8, 2 mM MgCl 2 , 1% NP-40, 1 mM Pefablock® (Boehringer), and 1 ⁇ g/ml each of leupeptin, antipain, chyostatin, pepstatin Al and 10 ⁇ g/ml benzamidine]. After a 3 minute incubation on ice, the cell suspension was diluted with 1 volume of cold, deionized water and incubated for 2 additional min.
  • lysis buffer 10 mM Tris-HCl pH 7.8, 2 mM MgCl 2 , 1% NP-40, 1 mM Pefablock® (Boehringer), and 1 ⁇ g/ml each of leupeptin, antipain,
  • the extract was centrifuged at 300xg for 6 min. at 4°C, and the supernate collected and saved as an NP-40 lysate. Proteins in the lysate samples were separated by SDS-PAGE (U.K. Laemmli, (1970) Nature 227:680) in 7%-15% gradient gels. Proteins were electrophoretically transferred to nitrocellulose membranes from the gels. The filters were then blocked with TBS (pH 8.0) in the presence of 0.05% Tween-20 and 5% non-fat dried milk for 1 hour at room temperature. The blot was incubated with the indicated antibodies at 1 : 1,000 dilution in the same solution at 4°C overnight.
  • TBS pH 8.0
  • blots were incubated with a 1 :2,000 dilution of affinity-purified horseradish peroxidase-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch) for 1 hr at room temperature. Proteins were visualized by chemiluminescence using the SuperSignal Western Blotting System according to the manufacturer's instructions (Pierce).
  • RT-PCR analysis of mRNA prepared from HUVEC or HeLa cells was performed using the PCR sense/antisense primer combinations as indicated.
  • the relative location of these primers within the cDNAs encoding the OB-Ra (short form) and OB-Rb (long form) forms of the leptin receptor is also shown. Whereas the combination Vz would detect both the long and short forms of the leptin receptor, 1/3, 1/5, and 4/5 are specific for the OB-Rb long form.
  • the predicted size of the corresponding PCR products in each case is also indicated. After PCR amplification, the resulting DNA products were analyzed by agarose gel electrophoresis where each lane corresponds to the PCR primer combination indicated at the bottom. The PCR products are shown with respect to the migration of DNA molecular weight markers (lane M).
  • Figure ID depicts the histochemical analysis of frozen sections from normal human dermis immunostained with normal rabbit IgG (panel 1), anti-von Willebrand factor IgG (panel 2), or anti-IC-1 antibodies against residues 1148-1156 from the carboxy terminus of human OB-Rb (panel 3). After incubation with primary antibodies, tissue sections were developed with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG using a Vectastain ® Elite ABC kit (Vector Labs). Scale bar is 50 ⁇ m.
  • ⁇ OB-R ⁇ antibodies would recognize epitopes common to both the long (OB-Rb) and short (OB-Ra) forms of the receptor, it is likely that the immunostaining detected in this case reflects the coexpression of short receptor variants in addition to OB-Rb, which is observed when ⁇ OB-R, jail, antibodies are used exclusively (Fig. 1 A, panel 1).
  • cellular proteins present in total extracts prepared from primary cultures of EC were first fractionated by gel electrophoresis followed by immunoblot analysis employing the antibodies described above. With ⁇ OB-R, ⁇ ( antibodies, a single band with an apparent molecular mass above 200 kDa (Fig. IB, lane 1 was detected).
  • RNA from HUVEC was subjected to the RT-PCR reactions in the presence of PCR primer pair combinations that would specifically detect expression of the OB-Rb transcript (pairs 1/3, 1/5 and 4/5, Fig. IC), amplicon products of the predicted size were detected, but not when RNA from human HeLa cells was used as a control.
  • the cells were grown to confluence and starved for 12 hours in complete medium without serum and supplemented with 2% BSA. Chemotaxis was assayed as described by V. Kundra et al, (1995) J. Cell Biol. 130: 725 using a 48-well Boyden chamber (Neuroprobe) equipped with 25x80 mm, 8 ⁇ m polyvinylpynolidone-free filters (Nucleopore), previously coated with 100 ⁇ g/ml collagen type I (Collaborative Research). Human recombinant leptin was diluted in DMEM/2% BSA and added to the lower wells at the indicated concentrations, while the upper wells received 1.5xl0 4 cells suspended in 50 ml of the same medium.
  • the chamber was then incubated at 37°C in a 5% CO 2 humidified atmosphere for 4 hours.
  • the migrating cells were fixed with methanol, and the filter stained with Giemsa (Fisher Scientific).
  • the chemotactic response was analyzed by counting the number of cells in a given microscopic field observed at 400X. All cells in 10 random fields were counted.
  • Three dimensional (3D) cultures of BLMVEC were established in gel matrices using rat tail Type I collagen. Collagen solutions were prepared by mixing the protein with an appropriate volume of 1 OX Ml 99 culture medium and neutralizing the pH by the addition of IN NaOH. BLMVEC were added immediately to a final concentration of 1.5-2 x 10 6 cells/ml collagen (final collagen concentration was 2 mg/ml).
  • BLMVEC were allowed to form tube-like structures for about 4 days, and were then fixed in buffered formalin before being embedded in paraffin and processed for microscopic observation.
  • An Axiovert 25 microscope (Carl Zeiss) equipped with a Varel contrast optical system was used. Images were captured with a 3CCD camera (DAGE-MTI) and the digital images were acquired using a Scion Image software program for the Power Macintosh.
  • VEGF vascular endothelial growth factor
  • tubules apparently represent permeable structures as judged by the existence of a lumen clearly discernable in transversal sections observed at higher magnification (Fig. 2B, panel 4, inset).
  • proliferation assay experiments using several types of human and bovine EC did not show consistent or significant mitogenic activity attributable to leptin (results not shown). Therefore, as in the case of several angiogenic polypeptides (J. Folkman (1995) N. End. J. Med. 333: 1757), leptin does not appear to act as a growth factor for EC. However, it clearly does play a role in the morphogenesis of higher order, capillary like tubules that arise from EC.
  • Example 3 Angiogenic Affect of Leptin In Vivo After the in vitro effects of leptin were demonstrated as shown in Figure 2, leptin was studied in vivo to determine whether it would enhance angiogenesis.
  • Quantitative data of cell-cluster formation in response to leptin treatment was determined as follows: Cells were plated sparsely on microscope slide-culture chambers coated with fibronectin to promote cell attachment to the glass surface. The plating was done so that most of the cells were not in contact with each other (approximately 1 x 10 5 cells/ml). After 12 h in culture in complete media, the cells were treated with leptin and incubated for different times.
  • the cells were fixed for 1 min with acetone at -20°C, washed with PBS and incubated for 30 min with ulex europeaus lectin labeled with TRITC (red fluorochrome to stain the plasma membrane of endothelial cells).
  • the slides were washed and mounted with coverslips in anti-fade media containing DAPI (blue fluorochrome to stain nuclei).
  • DAPI blue fluorochrome to stain nuclei.
  • the results were acquired as follows: a blinded subject was instructed to count 100 cells per slide. First, each field, magnified 100 times, was visualized with the filter that allowed to see only cell nuclei. Fields should have at least 5 nuclei to be counted and were chosen at random.
  • the number of nuclei equaled the number of cells per field. After the nuclei were counted, the filter was changed so as to allow the examiner to visualize the plasma membrane of the cells (stained in red). At this point the examiner was asked to record how many of the cells, in the same field where the nuclei were counted, were single or making cell-cell contact in clusters of 2, 3, 4, and 5 or more cells.
  • VEGF and Leptin Act Synergisticallv to Enhance Angiogenesis
  • FIG. 6 shows the results of one such experiment wherein cells were treated with no leptin (Figure 6A), 2 nM leptin ( Figure 6B), 4 nM leptin (Figure 6C) and leptin and VEGF ( Figure 6D).
  • a wound site becomes very highly enriched in VEGF produced by keratinocytes on days 2 to 4 after injury (L.F. Brown et al, (1992) J. Exp. Med. 176: 1375; B. Berse et al, (1992) Mol. Biol. Cell. 3: 211; H. F. Dvorak et al, (1992) Ann. NY Acad. Sci. 667: 101; and P. Martin (1997) Science 276: 76).
  • Two full thickness longitudinal wounds of approximately 5 mm in length were done using a sterile scalpel. Each wound was localized in the ventral sub-axillary region of the mouse. Prior to wounding, the mouse was anesthetized, shaved and the skin was wiped with a disinfectant solution. One side was injected with 3 ⁇ g of recombinant leptin in a volume of 50 ⁇ l at a 3 mm distance from each border of the wound. The contralateral wound received the same volume of sterile saline.
  • the wounds were allowed to dry and then they were covered with microporous surgical tape.
  • the mouse wounds were allowed to dry. Then they were covered with microporous surgical tape.
  • the mouse was euthanized by cervical dislocation and skin was recovered from the wound sites including peripheral normal skin for sectioning and staining with H and E.
  • the samples obtained were discarded fluids from surgery.
  • the patients had undergone vitrectomy for surgical repair of proliferative neovascularization (in the case of diabetics) or for repair of retinal detachment etc. in the case of the controls.
  • Leptin concentration was measured using a commercially available RIA kit that uses 125 I-leptin (LINCO Laboratories). The results presented are the average of duplicate samples. Although the number of samples is low, it is likely that the elevated vitreal leptin can be a very important factor in the pathogenesis of retinal neovascularization. Some patients have low concentrations but blood/vitreal ratios may be more significant than values alone. Retinal neovascularization in diabetics is the major cause of blindness in the US.
  • wound healing by first intention means that the borders of the wounded skin are near each other, such as a wound created by a scalpel incision. Typically these wounds heal within 5-7 days. Leptin is administered to the wounded area to determine whether leptin enhances the rate of wound healing.
  • VEGF and leptin To detect the presence of VEGF and leptin, independent immunohistochemical procedures are followed using serial sections from the same tissue blocks. Because rabbit polyclonal antibodies are available against murine VEGF and murine leptin, it will be possible to use a double staining immunohistochemical approach employing standard immunoperoxidase and alkaline phosphatase methods. This will allow detection and comparison of the presence of angiogenic factors in the context of active neovascularization sites. A polyclonal antibody that recognizes and can distinguish Ob-R(L) from other isoforms can also be utilized.
  • Quantitation of angiogenesis is made by visual counting of the number of vessels (CD31 positive immunostaining) at low magnification (100X) in treated and control skin sections. The same slides will be utilized for computer enhanced video imaging employing analytical software (NIH Image), which compares the number of vessels based on the color of the histochemical reaction. Skin wounds processed by hematoxylin and eosin staining will be evaluated by an individual blinded to the origin of the various specimens studied. Results. The mouse wound treated with leptin is expected to be substantially healed in 3 days time, whereas the untreated control mouse is expected to only begin to show signs of healing.
  • This specimen is carefully divided into two equal portions, one of which is fixed in buffered formalin and then embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). The remaining portion is frozen and used to prepare sections for immunohistochemistry. Control skin specimens from the same animals are used to provide a baseline reference for quantifying the angiogenic effect.
  • Lysates were made from primary cultures of human umbilical vein endothelial cells (HUVEC), simian epithelial cells (COS-7) and primary culture of human vascular smooth muscle cells derived from aorta (VSMC) as described. Proteins were separated on a 5% SDS-PAGE gel and transfened by electrotransference onto a nitrocellulose membrane. The nitrocellulose membrane was immunostained using rabbit polyclonal serum, anti-human Ob-R(L) and standard Western blotting procedures, as described in the examples above.
  • HUVEC human umbilical vein endothelial cells
  • COS-7 simian epithelial cells
  • VSMC primary culture of human vascular smooth muscle cells derived from aorta
  • Figures 9 A-9D are images of monolayers of HUVEC primary cultures scanned using electron microscopy. The images were processed in the conventional methods for imaging with a transmission electron microscope.
  • Figure 9A HUVECs were treated for 72 hours in the presence of 25 nM human recombinant leptin. The image is at 8000X magnification.
  • Figure 9B is the same as Figure 9A. The two cells are presented side by side. This image illustrates the increased number of mitochondria and rough endoplasmic reticulum (rER).
  • FIG 9C the cells are the same as above only without leptin treatment.
  • the cells in Figure 9C represent the negative control.
  • Figure 9D HUVEC cells were treated again with 25 nM recombinant human leptin as performed for Figure 9 A.
  • Figure 9D shows the very high number of mitochondria present, which is presumably due to the incubation with leptin.
  • the very large number of mitochondria in the endothelial cells (HUVECs in this experiment) after leptin incubation may indicate that EC can oxidize free fatty acids (FFA). Leptin therefore is shown to have increased the number of mitochondria.
  • FFA free fatty acids
  • the energy generated by the bond breakage is released as heat instead of being used to form ATP.
  • the vasculature wall where endothelial and smooth muscle cells are found, is the perfect site for this to occur because as heat is generated, it can be readily dissipated through the blood stream (i.e., blood serves as a coolant).
  • This theory may explain why leptin does not mobilize fat stores into the blood stream, but does significantly decrease the fat content of adipocytes.
  • leptin can induce secretion of a factor or factors from the vessel walls that will then induce the fatty acid (FA) oxidation directly inside the adipocyte.
  • FA fatty acid
  • modulation of EC and smooth muscle cell metabolism of lipids may result in a method of regulating adipocyte fat storage as well as angiogenesis.
  • RT-PCR was performed and shows the expression of uncoupling protein 2 (UCP-2) transcripts in endothelial cells.
  • UCP-2 uncoupling protein 2
  • Leptin deficiency in experimental animals leads to a complex phenotype of obesity, type II diabetes and severely impaired wound healing.
  • transiently increased leptin levels in the circulation of experimentally wounded mice including in a wound model where a SCID strain of mice are transplanted with a small fragment of human skin (from elective plastic surgery).
  • mice are wounded on the human graft and the serum leptin levels are measured at 12 hours after incision is made.
  • the increase in circulating leptin is human leptin.
  • the process of myocardial healing after ischemic injury has distinctive characteristics that differ from skin healing.
  • the angiogenic activity of leptin also enhances repair of injured tissue inespective of its location in the body.
  • the serum leptin concentrations of patients has been evaluated at different times following myocardial infraction. Data indicate that there is a marked increase in circulating leptin between 12 and 24 hours after a heart attack. The elevated leptin is sustained for another 24 hours and returns to normal values by 72 hours post-infarct.

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Abstract

La présente invention concerne des procédés permettant de réguler l'angiogenèse, les lésions ischémiques et/ou la cicatrisation en modulant l'activité de la leptine, en particulier celle dont le médiateur est le récepteur de la leptine, et/ou l'interaction entre la leptine et le récepteur de la leptine. Les procédés de l'invention peuvent par conséquent être utilisés pour traiter les maladies dont le médiateur est l'angiogenèse, y compris la cicatrisation, les tumeurs et les métastases tumorales, la microangiopathie diabétique, la néovascularisation rétinienne, la néovascularisation du tissu adipeux et le métabolisme des graisses, la revascularisation des tissus nécrotiques, l'amélioration de la vascularisation dans les greffes microvasculaires, et la maturation des follicules ovariens. L'invention concerne enfin des dosages permettant d'identifier des agents qui modulent la leptine et/ou l'angiogenèse dont le médiateur est le récepteur de la leptine et/ou la cicatrisation, et leur utilisation dans le traitement des maladies induites par l'angiogenèse ou d'états dans lesquels la cicatrisation intervient.
PCT/US1999/011209 1998-05-20 1999-05-20 Modulation de l'angiogenese et de la cicatrisation WO1999059614A1 (fr)

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Cited By (13)

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WO2001018040A2 (fr) * 1999-09-05 2001-03-15 Yeda Research And Development Co. Ltd. Utilisation de leptine en vue d'empecher la proliferation de la cellule endotheliale
WO2002094256A1 (fr) * 2001-05-23 2002-11-28 Debatosh Datta Lysine et/ou analogues et/ou polymeres de ces derniers destines a promouvoir la cicatrisation et l'angiogenese
US6750234B2 (en) * 2001-03-08 2004-06-15 University Of Kentucky Research Foundation Methods for increasing leptin levels using nicotinic acid compounds
WO2005049655A1 (fr) * 2003-11-17 2005-06-02 Klinikum Der Universität München Antagoniste de leptine et procede de mesure quantitative de leptine
US6943146B2 (en) 2000-05-08 2005-09-13 Biogen Idec Ma Inc. Method for promoting neovascularization
US7208151B2 (en) 2001-09-12 2007-04-24 Biogen Idec Ma Inc. Tweak receptor agonists as anti-angiogenic agents
US8043619B2 (en) * 2004-10-08 2011-10-25 Yaron Ilan Methods and uses of leptin in immune modulation
CN101287483B (zh) * 2003-08-07 2012-02-29 希尔洛有限公司 用于加速伤口愈合的药物组合物和方法
US8440189B2 (en) 1999-01-15 2013-05-14 Biogen Idec Ma Inc. Antagonists of TWEAK and of TWEAK receptor and their use to treat immunological disorders
US8506958B2 (en) 2002-04-09 2013-08-13 Biogen Idec Ma Inc. Methods for treating TWEAK-related conditions
US8728475B2 (en) 2005-05-10 2014-05-20 Biogen Idec Ma Inc. Methods for treating inflammatory bowel disease
US9730947B2 (en) 2005-06-13 2017-08-15 Biogen Ma Inc. Method of treating lupus nephritis
US9775899B2 (en) 2005-02-17 2017-10-03 Biogen Ma Inc. Treating neurological disorders

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WO1997012037A1 (fr) * 1995-09-26 1997-04-03 Amrad Operations Pty. Ltd. Nouveau recepteur de l'hematopoietine et sequences genetiques codant ledit recepteur
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WO1997027286A1 (fr) * 1996-01-23 1997-07-31 Progenitor, Inc. Procedes d'utilisation du gene obese et de son produit genique afin de stimuler le developpement des cellules hematopoïetiques

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8440189B2 (en) 1999-01-15 2013-05-14 Biogen Idec Ma Inc. Antagonists of TWEAK and of TWEAK receptor and their use to treat immunological disorders
WO2001018040A3 (fr) * 1999-09-05 2001-06-14 Yeda Res & Dev Utilisation de leptine en vue d'empecher la proliferation de la cellule endotheliale
WO2001018040A2 (fr) * 1999-09-05 2001-03-15 Yeda Research And Development Co. Ltd. Utilisation de leptine en vue d'empecher la proliferation de la cellule endotheliale
US6943146B2 (en) 2000-05-08 2005-09-13 Biogen Idec Ma Inc. Method for promoting neovascularization
US7731963B2 (en) 2000-09-14 2010-06-08 Biogen Idec Ma Inc. TWEAK receptor agonists as anti-angiogenic agents
US6750234B2 (en) * 2001-03-08 2004-06-15 University Of Kentucky Research Foundation Methods for increasing leptin levels using nicotinic acid compounds
WO2002094256A1 (fr) * 2001-05-23 2002-11-28 Debatosh Datta Lysine et/ou analogues et/ou polymeres de ces derniers destines a promouvoir la cicatrisation et l'angiogenese
US7208151B2 (en) 2001-09-12 2007-04-24 Biogen Idec Ma Inc. Tweak receptor agonists as anti-angiogenic agents
US9011859B2 (en) 2002-04-09 2015-04-21 Biogen Idec Ma Inc. Methods for treating TWEAK-related conditions
US8506958B2 (en) 2002-04-09 2013-08-13 Biogen Idec Ma Inc. Methods for treating TWEAK-related conditions
CN101287483B (zh) * 2003-08-07 2012-02-29 希尔洛有限公司 用于加速伤口愈合的药物组合物和方法
DE10353593A1 (de) * 2003-11-17 2005-06-23 Klinikum der Universität München Großhadern-Innenstadt Leptinantagonist und Verfahren zur quantitativen Messung von Leptin
US7807154B2 (en) * 2003-11-17 2010-10-05 Biofusion Licensing Limited Leptin antagonist and method for quantitative measurement of leptin
WO2005049655A1 (fr) * 2003-11-17 2005-06-02 Klinikum Der Universität München Antagoniste de leptine et procede de mesure quantitative de leptine
US8043619B2 (en) * 2004-10-08 2011-10-25 Yaron Ilan Methods and uses of leptin in immune modulation
US9775899B2 (en) 2005-02-17 2017-10-03 Biogen Ma Inc. Treating neurological disorders
US8728475B2 (en) 2005-05-10 2014-05-20 Biogen Idec Ma Inc. Methods for treating inflammatory bowel disease
US9730947B2 (en) 2005-06-13 2017-08-15 Biogen Ma Inc. Method of treating lupus nephritis

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