EP1296702A1 - Peptides neuroprotecteurs - Google Patents

Peptides neuroprotecteurs

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Publication number
EP1296702A1
EP1296702A1 EP01941562A EP01941562A EP1296702A1 EP 1296702 A1 EP1296702 A1 EP 1296702A1 EP 01941562 A EP01941562 A EP 01941562A EP 01941562 A EP01941562 A EP 01941562A EP 1296702 A1 EP1296702 A1 EP 1296702A1
Authority
EP
European Patent Office
Prior art keywords
seq
emp
aka
amino acids
antagonist
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01941562A
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German (de)
English (en)
Other versions
EP1296702A4 (fr
Inventor
Virginia Smith-Swintosky
Michael Renzi
Carlos Plata-Salaman
Linda Jolliffe
Francis Farrell
Dana L. Johnson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Pharmaceuticals Inc
Original Assignee
Ortho McNeil Pharmaceutical Inc
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Application filed by Ortho McNeil Pharmaceutical Inc filed Critical Ortho McNeil Pharmaceutical Inc
Publication of EP1296702A1 publication Critical patent/EP1296702A1/fr
Publication of EP1296702A4 publication Critical patent/EP1296702A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is directed to methods of treating diseases and conditions involving the nervous system by administration of compositions having the therapeutic activity of human erythropoietin.
  • compositions having the therapeutic activity of human erythropoietin.
  • therapeutic agents such as peptides, peptide dimers, polypeptides, and proteins that have the full range of biological activity of human erythropoietin or only certain biological activities of erythropoietin.
  • the present invention also provides improved therapeutic regimens wherein the therapeutic agent is administered at concentrations below those required to stimulate hematopoiesis.
  • Erythropoietin is a glycoprotein hormone produced by the kidney in response to tissue hypoxia that stimulates red blood cell production in the bone marrow.
  • the gene for erythropoietin has been cloned and expressed in Chinese hamster ovary (CHO) cells as described in United States Patent No. 4,703,008.
  • Recombinant human erythropoietin r- HuEPO or Epoetin alfa
  • r- HuEPO or Epoetin alfa has an amino acid sequence identical to that of human urinary erythropoietin, and the two are indistinguishable in chemical, physical and immunological tests.
  • Recombinant human erythropoietin acts by increasing the number of cells capable of differentiating into mature erythrocytes, triggering their differentiation and augmenting hemoglobin synthesis in developing erythroblasts (Krantz SB. Blood (1991) 77: 419-434, Beckman BS, Mason-Garcia M. The Faseb Journal (1991) 5: 2958-2964).
  • Epoetin alfa has been well tolerated in studies conducted to date. Hypertensive encephalopathy and seizures have occasionally been described in dialysis patients treated with Epoetin alfa, particularly during the early phase of therapy when hematocrit is rising.
  • Epoetin alfa Skin rashes and urticaria have been observed rarely and when reported have been mild and transient in nature, but these events suggest allergic hypersensitivity to some components of the Epoetin alfa formulation.
  • Epoetin alfa is approved for sale in many countries for the treatment of anemia in chronic renal failure (dialysis and predialysis), anemia in zidovudine treated HIV positive patients (US), anemia in cancer patients receiving platinum-based chemotherapy, as a facilitator of autologous blood pre-donation, and as a peri-surgical adjuvant to reduce the likelihood of requiring allogeneic blood transfusions in patients undergoing orthopedic surgery.
  • EPO influences neuronal stem cells, likely during embryonic development, and possibly during in vitro experiments of differentiation.
  • neonates and infants that suffer CNS injury via hypoxia, meningitis, and intraventricular hemorrhage show an
  • EPO helps prevent apoptosis of neural tissue in cases of injury that create hypoxia. This may be the result of EPO produced locally by astrocytes (Morishita et al Neuroscience
  • EPO induces biological effects of PC 12 cells, including changes in Ca 2+ , changes in membrane potential, and promotion of neuronal survival. This has been interpreted that EPO can stimulate neural function and viability (Koshimura et al J. Neurochem (1999) 72(6) 2565-2572. Tabria et al Int J Dev Neurosci (1995) 13(3/4) 241 - 252.).
  • EPO Erythropoietin receptor
  • NS20Y NS20Y
  • SK-N-MC SK-N-MC
  • PC 12 cells which includes sprouting, differentiation and neuroprotection.
  • this peptide does not promote proliferation of hematologic cells, thus it appears inactive in cell lines well understood for their sensitivity to EPO activity.
  • EPO may influence neuronal stem cell commitment to drive differentiation of neurons as opposed to astrocytes or oligodendrocytes. This is compared to a similar activity of EPO, where it functions to drive commitment of hematopoietic stem cells to produce red blood cells (RBCs).
  • RBCs red blood cells
  • the present invention is directed to methods of treating diseases and conditions involving the nervous system by administration of compositions having the neurological therapeutic activity of human erythropoietin.
  • the present invention is directed to a method for treating a patient having a disorder characterized by neurotoxicity, neurodegeneration or neurological damage, comprising administering to said patient a therapeutically effective amount of a peptide comprising one or more monomeric peptides of 8 to about 40 amino acids in length that bind to EPO receptor, each monomeric peptide comprising a sequence of amino acids X 4 X 5 X a X b X 6 X 0 X d X 7 (SEQ ID NO: 47), wherein
  • X a is G or A
  • X b is P or A
  • X c is T or A
  • X d is selected from W, A, and F;
  • X 4 is selected from R, H, Y, L, and W, or X 4 is nonexistent;
  • X 5 is selected from F, M, and I;
  • X 6 is independently selected from the 20 genetically coded L-amino acids or the stereoisomeric D-amino acids;
  • X 7 is selected from D, V, E, I, and L.
  • said sequence is X 3 X 4 X 5 GPX 6 TWX 7 X 8 (SEQ ID NO: 1), wherein
  • X 3 is selected from C, E, A, ⁇ -amino- ⁇ -bromobutyric acid, and homocysteine (Hoc); and
  • X 8 is selected from C, K, A, ⁇ -amino- ⁇ -bromobutyric acid, and homocysteine (Hoc).
  • the present invention is directed to peptides which behave as cell-surface receptor agonists and antagonists, as well as dimers and multimers of such peptides which exhibit binding and signal initiation of growth factor-type receptors.
  • the present invention provides peptides which behave as EPO agonists.
  • peptides may be dimers or multimers of such peptides, preferably 14 to about 20 residues in length, comprising a core amino acid sequence of X 3 X 4 X 5 GPX 6 TWX 7 X 8 (SEQ ID NO: 1) wherein each amino acid is indicated by standard one letter abbreviation;
  • X 3 can be C, E, A, ⁇ -amino- ⁇ -bromobutyric acid, or Hoc, where Hoc is homocysteine;
  • X 4 can be R, H, Y, L, or W, or X 4 is nonexistent;
  • X 5 can be M, F, or I;
  • X 6 is independently any one of the 20 genetically coded L-amino acids or the stereoisomeric D-amino acids;
  • X 7 can be D, E, I, L, or V; and
  • X g can be C, K, A, ⁇ - amino- ⁇ -bromobutyric acid, or Hoc, where
  • the monomeric peptide unit of the dimer or multimer comprises a core sequence of amino acids YX 2 X 3 X 4 X 5 GPX 6 TWX 7 X 8 (SEQ ID NO: 2), wherein each of X 2 and X 6 is independently any one of the 20 genetically coded L-amino acids; X 3 is C; and X 8 is C.
  • the monomeric peptide unit of the dimer comprises a core sequence of amino acids X J YX 2 X 3 X 4 X 5 GPX 6 TWX 7 X 3 X 9 X J0 X, ! (SEQ ID NO: 3), wherein each of X tract X 2 , X 6 , X 9 , X 10 , and X n is independently selected from the 20 genetically coded L-amino acids.
  • both X 3 and X 8 are C and thus, the monomeric peptide unit of the dimer comprises a core sequence of amino acids X, YX 2 CX 4 X 5 GPX 6 TWX 7 CX 9 X 10 X ⁇ (SEQ ID NO: 4).
  • the monomeric peptide unit comprises a core sequence of amino acids X j YX 2 CX 4 X 5 GPX 6 TWX 7 CX 9 X 10 X n (SEQ ID NO: 5), where X 4 can be R or H; X 5 can be F or M; X 6 can be I, L, T, M, or V; X 7 is D or V; X 9 can be G, K, L, Q, R, S, or T; and X 10 can be A, G, P, R, or Y.
  • the monomeric peptide unit of the dimer will comprise a core sequence of amino acids X, YX 2 CX 4 X 5 GPX 6 TWX 7 CX 9 X 10 X ⁇ (SEQ ID NO: 6), where X, can be D, E, L, N, S, T, or V; X 2 can be A, H, K, L, M, S, or T; X 4 is R or H; X 9 can be K, R, S, or T; and X 10 is P.
  • the monomeric peptide unit of the dimer will comprise a core sequence of amino acids X, YX 2 CX 4 X 5 GPX 6 TWX 7 CX 9 X 10 X n (SEQ ID NO: 6), where X ] can be D, E, L, N, S, T, or V; X 2 can be A, H, K, L, M, S, or T; X 4 is R or H; X 9 can be K, R, S, or T; and X 10 is P.
  • X can be D, E, L, N, S, T, or V;
  • X 2 can be A, H, K, L, M, S, or T;
  • X 4 is R or H;
  • X 9 can be K, R, S, or T; and X 10 is P.
  • Particularly preferred monomeric peptide units of the dimers include:
  • GGTYSCHFGPLTWVCKPQGG (aka EMP-1) (SEQ ID NO:8);
  • GGVYACRMGPITWVCSPLGG (SEQ ID NO: 11);
  • GGTYSCHFGPLTWVCKPQ (aka EMP-16) (SEQ ID NO: 13)
  • GGLYACHMGPMTWVCQPLRG (aka EMP-36) (SEQ ID NO: 14)
  • YSCHFGPLTWVCK (aka EMP-20 (SEQ ID NO: 16)
  • YCHFGPLTWVC (aka EMP-23) (SEQ ID NO: 17)
  • GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO: 19);
  • GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO:20);
  • TYSCHFGPLTWVCKPQGG (aka EMP-17) (SEQ ID NO:22)
  • TYSCHFGPLTWVCKPQ (aka EMP-18) (SEQ ID NO:23)
  • YSCHFGPLTWVC (aka EMP-21) (SEQ ID NO:25)
  • GGCRIGPITWVCGG (aka EMP-25) (SEQ ID NO:27)
  • HFGPLTWV (aka EMP-26) (SEQ ID NO:28)
  • GGTTSCHFGPLTWVCKPQGG (aka EMP-7) (SEQ ID NO:29);
  • GGTFSCHFGPLTWVCKPQGG (aka EMP-8) (SEQ ID NO:30);
  • GGTYSCHFGALTWVCKPQGG (aka EMP-10) (SEQ ID NO:31);
  • GGTYSCHFGPLAWVCKPQGG (aka EMP-12) (SEQ ID NO:33);
  • GGTYSCHFGPLTWVCKAQGG (aka EMP-15) (SEQ ID NO:36);
  • monomeric peptide units of the dimers include:
  • GGTYSCHFGPLTWVCKPQGG (aka EMP-1) (SEQ ID NO:8); GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO: 19); GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO:20); and YCHFGPLTWVC (aka EMP-23) (SEQ ID NO: 17).
  • EMP-1 SEQ ID NO:8
  • GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO: 19)
  • GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO:20); and YCHFGPLTWVC (aka EMP-23) (SEQ ID NO: 17).
  • the monomeric units of the dimers can be the same or different.
  • polyethylene glycol may be employed as a linker to form the dimeric peptides of the present invention through a covalent bond.
  • the present invention is directed to pharmaceutical compositions comprising at least one peptide of the invention and a pharmaceutical carrier to be used in a method of treating or preventing neurotoxicity.
  • the present invention provides a method for therapeutically treating a mammal having a disease or condition resulting from a neurotoxic or neurodegenerative or neuro-damaging event by administration of at least one of the peptides of the present invention.
  • a method for therapeutically treating a mammal having a neurotoxic, neuro-damaging or neurodegenerative condition which may be modulated by EPO by using at least one of the peptides of the present invention is provided.
  • FIG. 1 Panel A and Panel B show that EPO receptor is expressed in rat hippocampal and cortical cultures.
  • Figure 2 shows that EPO receptor is expressed in neuronal cell lines: PC12 and SK- N-MC cells.
  • FIG. 3 shows EPO induced gene expression in PC 12 cells.
  • Top Total RNA isolated from PC-12 cells treated with 1 run EPO for 24 hr was subject to RT-PCR to quantify the changes in gene expression of specific BCL family members. Pre-treatment with EPO resulted in a 6 fold increase in the expression of the anti-apoptotic gene BCL ⁇ and a greater than 5 fold decrease in the expression of the pro-apoptotic Bak. These results are consistent with the gene chip results suggesting a possible mechanism for EPO's protective effects.
  • Bottom Agarose gels showing RT-PCR products representing the regulation of Bcl ⁇ L and Bak. m - markers, 1 - RT-PCR negative control, lane 2 - No Treatment, lane 3 - 50 ng/ml NGF, lane 4 - EPO 1 nm.
  • Figure 4 shows that rhEPO protects rat cerebral cortical neurons against glutamate toxicity.
  • Figure 5 shows that rhEPO protects rat PC 12 cells against glutamate-induced cell death.
  • 7 day cultures of PC- 12 cells were treated with erythropoietin for 24 hours before being exposed to a toxic concentration of glutamate (200 um). Cultures were allowed to recover for 24 hours and cell survival was determined using a Trypan Blue exclusion assay. 1 to 10 pm Erythropoietin given 24 hours prior to a 15 minute exposure to glutamate significantly increased cell survival (p ⁇ .001, student's t-test. The protective activity of EPO decreased at higher doses.
  • Figure 6 shows that rhEPO protects rat PC 12 cells against NGF withdrawal-induced cell death.
  • Cultures of PC- 12 cells were grown in the presence of NGF for 7 days and then treated with EPO for 24 hours before they were switched to media without NGF.
  • Cell survival was determined by counting the number of viable cells immediately following the removal of NGF and comparing it to the number of viable cells at 12 hr, 24 hr, 48 hr and 72 hr following growth factor withdrawal. Cell viability was determined based on morphological characteristics including phase brightness, presence of axons and absence of membrane blebbing. Treatment with EPO increased the number of viable cells at each time point following growth factor withdrawal with an optimum concentration of 10 pm.
  • Figure 7 shows that rhEPO promotes neurite outgrowth in rat cerebral cultures.
  • Figure 8 shows that rhEPO promotes neurite outgrowth in rat hippocampal cultures.
  • Figure 9 shows that EMP-1 promotes neurite outgrowth in rat cerebral cortical cultures.
  • Figure 10 shows that EMP-1 promotes neurite outgrowth in rat hippocampal cultures.
  • Figure 11 shows that EMP-6 promotes neurite outgrowth in rat cerebral cortical cultures.
  • Figure 12 shows that EMP-6 promotes neurite outgrowth in rat hippocampal cultures.
  • Figure 13 shows that EMP-9 promotes neurite outgrowth in rat cerebral cortical cultures.
  • Figure 14 shows that EMP-9 promotes neurite outgrowth in rat hippocampal cultures.
  • Figure 15 shows that EMP-23 promotes neurite outgrowth in rat cerebral cortical cultures.
  • Figure 16 shows that EMP-23 promotes neurite outgrowth in rat hippocampal cultures.
  • Figure 17 shows that EMP-27 promotes neurite outgrowth in rat cerebral cortical cultures.
  • Figure 18 shows that EMP-27 promotes neurite outgrowth in rat hippocampal cultures.
  • Figure 19 shows that EPO protects against ischemic injury: study I continuous iv infusion via osmotic mini-pump.
  • Figure 20 shows plasma determinations for study I.
  • Figure 21 shows that EPO does not protect against ischemic injury: study II single iv bolus dose.
  • Figure 22 shows plasma determinations for study II.
  • Figure 23 shows that EPO protects against ischemic injury: study III repeat iv bolus dosing.
  • Figure 24 shows plasma determinations: study III.
  • Erythropoietin used herein includes those peptides, peptide dimers, polypeptides, and proteins that have the full range of biological activity (for example, hematopoietic and neurological activities) of human erythropoietin or only certain biological activities (for example, hematopoietic or neurological activities only) of erythropoietin, as well as erythropoietin analogs, erythropoietin isoforms, erythropoietin mimetics, erythropoietin fragments, hybrid erythropoietin proteins, fusion proteins, oligomers and multimers of the above, homologues of the above, glycosylation pattern variants of the above, and muteins of the above, regardless of the biological activity of same, and further regardless of the method of synthesis or manufacture thereof including, but not limited to, recombinant, whether produced from cDNA or genomic DNA,
  • erythropoietin examples include, Epoetin alfa (EPREX ® , ERYPO ® , PROCRIT ® ), NEORECORMON, Novel erythropoiesis stimulating protein (NESP or ARANESP, a hyperglycosylated analog of recombinant human erythropoietin (Epoetin) described in European patent application EP640619), human erythropoietin analog - human serum albumin fusion proteins such as those described in the international patent application WO 99/66054, erythropoietin mutants such as those described in the international patent application WO 99/38890, erythropoietin omega, which may be produced from an Apa I restriction fragment of the human erythropoietin gene described in United States Patent No.
  • EPO altered glycosylated human erythropoietin such as those described in the international patent application WO 99/11781, PEG conjugated erythropoietin analogs such as those described in WO 98/05363 or United States Patent No. 5,643,575.
  • PEG conjugated erythropoietin analogs such as those described in WO 98/05363 or United States Patent No. 5,643,575.
  • Specific examples of cell lines modified for expression of endogenous human erythropoietin are described in international patent applications WO 99/05268 and WO 94/12650.
  • the generally preferred form of EPO is purified, recombinant human EPO (rhEPO), currently formulated and distributed under the trademarks of EPREX ® , ERYPO ® , or PROCRIT ® .
  • EPO peptide mimetics of EPO, particularly certain peptides described in United States Patent No.'s 5,767,078 and 5,773,569.
  • the present invention is directed to methods of treating neuronal cells with a pharmaceutical composition comprising a therapeutically active peptide that behaves as cell-surface receptor agonists as well as dimers and multimers of such peptides that exhibit binding and signal initiation of growth factor-type receptors.
  • a pharmaceutical composition comprising a therapeutically active peptide that behaves as cell-surface receptor agonists as well as dimers and multimers of such peptides that exhibit binding and signal initiation of growth factor-type receptors.
  • the present invention provides peptides that behave as EPO agonists.
  • these peptides may be dimers or multimers that have two 'monomeric' peptide units of 8 to 40 or more amino acids, preferably 14 to about 20 residues in length, comprising a core amino acid sequence of X 3 X 4 X 5 GPX 6 TWX 7 X 8 (SEQ ID NO: 1) where each amino acid is indicated by standard one letter abbreviation;
  • X 3 can be C, E, A, ⁇ -amino- ⁇ - bromobutyric acid, or Hoc, where Hoc is homocysteine;
  • X 4 can be R, H, Y, L, or W, or X 4 is nonexistent;
  • X 5 can be M, F, or I;
  • X 6 is independently any one of the 20 genetically coded L- amino acids or the stereoisomeric D-amino acids;
  • X 7 can be D, E, I, L, or V; and
  • X 8 can be C, K, A, ⁇ -amino- ⁇
  • the monomeric peptide unit of the dimer or multimer comprises a core sequence YX 2 X 3 X 4 X 5 GPX 6 TWX 7 X 8 (SEQ ID NO: 2) where each amino acid is indicated by standard one letter abbreviation; each X 1 ⁇ X 2 , X 6 , X 9 , X 10 , and X n is independently selected from the 20 genetically coded L-amino acids; X 3 can be C, E, A, ⁇ - amino- ⁇ -bromobutyric acid, or Hoc, where Hoc is homocysteine; X 4 can be R, H, Y, L, or W, or X 4 is nonexistent; X 5 can be M, F, or I; X 7 can be D, E, I, L, or V; and X 8 can be C, K, A, ⁇ -amino- ⁇ -bromobutyric acid, or Hoc, where Hoc is homocysteine. More preferably
  • the monomeric peptide unit of the dimer or multimer comprises a core sequence of amino acids YX 2 X 3 X 4 X 5 GPX 6 TWX 7 X 8 (SEQ ID NO: 2), wherein each of X 2 and X 6 is independently any one of the 20 genetically coded L-amino acids; X 3 is C; and X 8 is C.
  • the monomeric peptide unit of the dimer comprises a core sequence of amino acids X 1 YX 2 X 3 X 4 X 5 GPX 6 TWX 7 X ⁇ X 9 X 10 X 11 (SEQ ID NO: 3), wherein each of X l5 X 2 , X 6 , X 9 , X 10 , and X n is independently selected from the 20 genetically coded L-amino acids.
  • both X 3 and X 8 are C and thus, the monomeric peptide unit of the dimer comprises a core sequence of amino acids Xj YX 2 CX 4 X 5 GPX 6 TWX 7 CX 9 X 10 X n (SEQ ID NO: 4).
  • the monomeric peptide unit comprises a core sequence of amino acids X t YX 2 CX 4 X 5 GPX 6 TWX 7 CX 9 X 10 X ⁇ (SEQ ID NO: 5), where X 4 can be R or H; X 5 can be F or M; X 6 can be I, L, T, M, or V; X 7 is D or V; X 9 can be G, K, L, Q, R, S, or T; and X 10 can be A, G, P, R, or Y.
  • the monomeric peptide unit of the dimer will comprise a core sequence of amino acids X, YX 2 CX 4 X 5 GPX 6 TWX 7 CX 9 X 10 Xrigg (SEQ ID NO: 6), where X x can be D, E, L, N, S, T, or V; X 2 can be A, H, K, L, M, S, or T; X 4 is R or H; X 9 can be K, R, S, or T; and X 10 is P.
  • the monomeric peptide unit of the dimer will comprise a core sequence of amino acids X x YX 2 CX 4 X 5 GPX 6 TWX 7 CX 9 X 10 Xrigg (SEQ ID NO: 6), where X ! can be D, E, L, N, S, T, or V; X 2 can be A, H, K, L, M, S, or T; X 4 is R or H; X 9 can be K, R, S, or T; and X I0 is P.
  • Particularly preferred monomeric peptide units of the dimers include:
  • GGTYSCHFGPLTWVCKPQ (aka EMP-16) (SEQ ID NO:13)
  • GGLYACHMGPMTWVCQPLRG (aka EMP ⁇ 36) (SEQ ID NO:14)
  • YSCHFGPLTWVCK (aka EMP ⁇ 20 (SEQ ID NO:16)
  • YCHFGPLTWVC (aka EMP-23) (SEQ ID NO:17)
  • GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO:19);
  • GGTYSCHFAPLTWVCKPQGG (aka EMP-9)(SEQIDNO:20);
  • TYSCHFGPLTWVCKPQGG (aka EMP-17)(SEQIDNO:22)
  • TYSCHFGPLTWVCKPQ (aka EMP-18)(SEQIDNO:23)
  • YSCHFGPLTWVC (aka EMP-21)(SEQIDNO:25)
  • GGCRIGPITWVCGG (aka EMP-25) (SEQ ID NO:27)
  • GGTTSCHFGPLTWVCKPQGG (aka EMP-7)(SEQIDNO:29);
  • GGTYSCHFGPLTWVCKAQGG (aka EMP-15)(SEQIDNO:36)
  • monomeric peptide units of the dimers include:
  • GGTYSCHFGPLTWVCKPQGG (aka EMP-1) (SEQ ID NO:8); GGTASCHFGPLTWVCKPQGG (aka EMP-6) (SEQ ID NO: 19); GGTYSCHFAPLTWVCKPQGG (aka EMP-9) (SEQ ID NO:20); and YCHFGPLTWVC (aka EMP-23) (SEQ ID NO: 17).
  • EPO is administered by any suitable means as appropriate for the particular patient being treated, as would be apparent to one skilled in the art.
  • the phrase "therapeutically effective" as used herein will vary from patient-to-patient, and depending on the particular range of biological activities possessed by the EPO molecule being administered.
  • a therapeutically effective amount will be from about 1 to 500 I.U./kg body weight and more preferably from 50 to 300 I.U./kg body weight especially when erythropoietin is administered via subcutaneously.
  • the therapeutically effective dose may be more or less that an EPO molecule having hematopoietic activity.
  • the preferred methods of administration are intravenous (iv) and subcutaneous (sc), with subcutaneous being generally preferred.
  • Hematopoietically active EPO is administered within the range of about 50 - 1000 U/kg per dose, one to five times per week.
  • the EPO composition is administrated directly to the nervous system.
  • This administration route includes, but is not limited to, the intracerebral, intraventricular, intracerebroventricular, intrathecal, intracisternal, intraspinal and/or peri-spinal routes of administration, which can employ intracranial and intravertebral needles, and catheters with or without pump devices.
  • Infusion doses can range, for example, from about 1.0 to 50,000 U/kg/min of EPO composition over a period ranging from several minutes to several days. Hematopoietically active EPO administration is delayed or withheld if the patient, male or female, exhibits a hemoglobin level in excess of about 15 g/dL.
  • the present invention provides in one embodiment a method to treat acute and chronic neurodegenerative disorders comprising administration of EPO or analogs thereof.
  • Acute neurodegenerative disorders include, but are not limited to, various types of acute neurodegenerative disorders associated with neuronal cell death or compromise including cerebrovascular insufficiency, focal or diffuse brain trauma, diffuse brain damage, and spinal cord injury.
  • Examples of acute neurodegenerative disorders are: cerebral ischemia or infarction including embolic occlusion and thrombotic occlusion, reperfusion following acute ischemia, perinatal hypoxic-ischemic injury, cardiac arrest, as well as intracranial hemorrhage of any type (such as epidural, subdural, subarachnoid and intracerebral), and intracranial and intravertebral lesions (such as contusion, penetration, shear, compression and laceration), and whiplash shaken infant syndrome.
  • Chronic neurodegenerative disorders that can be treated with one or more methods of the present invention include, but are not limited to, Alzheimer's disease, Pick's disease, diffuse Lewy body disease, progressive supranuclear palsy (Steel-Richardson syndrome), multisystem degeneration (Shy-Drager syndrome), chronic epileptic conditions associated with neurodegeneration, motor neuron diseases including amyotrophic lateral sclerosis, degenerative ataxias, cortical basal degeneration, ALS-Parkinson's-Dementia complex of Guam, subacute sclerosing panencephalitis, Huntington's disease, Parkinson's disease, synucleinopathies (including multiple system atrophy), primary progressive aphasia, striatonigral degeneration, Machado- Joseph disease / spinocerebellar ataxia type 3 and olivopontocerebellar degenerations, Gilles De La Tourette's disease, bulbar and pseudobulbar palsy, spinal and spinobulbar muscular atrophy (K
  • other clinical conditions can be treated with one or more methods of the present invention include treating and / or preventing the neurological (including, but not limited to, cognitive) and psychiatric (including, but not limited to, psychopathology, depression, or anxiety), manifestations associated with peripheral diseases including, but not limited to, EPO deficiency (e.g., renal disease), blood loss of any kind (including, but not limited to, hemodialysis, peritoneal dialysis, diagnostic sampling, occult gastrointestinal bleeding), renal failure and end-stage renal disease, renal transplantation, and other conditions associated with anemia and neurological and neuropsychiatric manifestations, including, but not limited to, hematological and non-hematological malignancies / cancer, symptoms or complications in patients receiving chemotherapy (including, but not limited to, cisplatin) and other drugs (including, but not limited to, zidovudine), other hematological disorders (including, but not limited to, sickle
  • Also included in the present invention are the treatment and / or prevention of neurological and neuropsychiatric manifestations resulting from chemical, toxic, infectious and radiation injury of the nervous system and as a result of prematurity, as well as the treatment and / or prevention of neurological and neuropsychiatric consequences of encephalopathies including, but not limited to, those of anoxic-ischemia, hepatic, glycemic, uremic, electrolyte and endocrine origin.
  • this molecule can also be applicable for the treatment and / or prevention of plexopathies (including plexus palsies), multifocal neuropathies, sensory neuropathies, motor neuropathies, sensory-motor neuropathies, infections neuropathies, autonomic neuropathies, sensory-autonomic neuropathies, demyelinating neuropathies (including, but not limited to, Guillain-Barre syndrome and chronic inflammatory demyelinating polyradiculoneuropathy), other inflammatory and immune neuropathies, neuropathies induced by drugs, neuropathies induced by pharmacological treatments, neuropathies induced by toxins, traumatic neuropathies (including, but not limited to, compression, crush, laceration and segmentation neuropathies), metabolic neuropathies, endocrine and paraneoplastic neuropathies, and other neuropathies such as Charcot-Marie-Tooth disease (type la, lb, 2, 4a,l-X linked
  • EXAMPLE 1 rhEPO is expressed in primary rat neuronal cultures and in neuronal cell lines
  • fetuses were removed via cesarean section from pregnant moms (Sprague-Dawley) anesthetized with halothane according to the AVMA Panel on Euthanasia. Pups were decapitated and the brains were removed and placed in HEPES-buffered Hank's Balanced Salt solution (HBSS; Gibco). The hippocampi and cortices were dissected out and pooled according to tissue-type.
  • Tissue was trypsinized for 15 min (lmg/ml trypsin-HBSS; Worthington), rinsed with fresh HBSS, incubated in trypsin inhibitor (lmg/ml; Sigma) for 5 min, rinsed again with fresh HBSS and then triturated in 1ml fresh HBSS with a fire-polished glass pipette. Dissociated cells were seeded at 30,000 cells/well onto poly-D-lysine coated 96-well plates (Collaborative BioScience).
  • Each well contained lOO ⁇ l of Eagle's Minimal Essential Media (MEM; Gibco) supplemented with 26mM NaHCO 3 (Sigma), 56mM glucose (Sigma), 15mM KC1 (Sigma), lmM sodium pyruvate (Sigma), l.lmM L-glutamine (Sigma), 10% (v/v) heat- inactivated fetal bovine serum (Hyclone), and 0.001% gentamicin sulfate (Sigma) (pH 7.4). Cells were allowed to attach for 24h in a humidified 37°C 5% CO 2 incubator before experimental treatment. The culture media was aspirated and exchanged with fresh media every three days. Immunocytochemistry
  • PC-12 cells can be reversibly induced to the neuronal phenotype in the presence of nerve growth factor (NGF).
  • NGF nerve growth factor
  • SK-N-MC cells were cultured in minimal essential media supplemented with 1.0 mM Sodium pyruvate, 1.5 g/L sodium bicarbonate, 2mM glutamine and 10% FBS for 4 days.
  • PC- 12 and SK-N-MC cells were cultured in a 96 well plate from Greiner, conducive for microscopy. On the day of the experiment cells were fixed in 10% Formalin containing 10% sucrose and incubated in blocking buffer (40 mM Tris HCL, Ph 8.0, 27mM NaCl, and 0.2%Tween 20).
  • Receptors for erythropoietin were detected by incubating the cells with a rabbit polyclonal, anti-erythropoietin receptor antibody (C-20 from Santa Cruz) and a FITC conjugated secondary antibody. Labeled cells were visualized using a fluorescent microscope (ATTO).
  • PC- 12 cells (from a rat Pheochromocytoma) were cultured on poly-D-Lysine coated tissue culture plastic in DMEM containing 10 % FBS and 5% Horse serum. To induce the neuronal phenotype in PC- 12 cells, serum was removed and the cells were treated with NGF (50 ng/ml). Cells were grown for 7 days in the presence of the NGF then used for experiments.
  • NGF 50 ng/ml
  • PC- 12 cells were cultured as described in Example 1 in a 10 cm poly-D-lysine coated tissue culture dish. Cells were incubated in the presence of lU/ml of EPO for 24 hr. Total RNA was then isolated using a Qiagen RNAeasy mini prep kit and used for RT-PCR.
  • fetuses were removed via cesarean section from pregnant moms (Sprague-Dawley) anesthetized with halothane according to the AVMA Panel on Euthanasia. Pups were decapitated and the brains were removed and placed in HEPES-buffered Hank's Balanced Salt solution (HBSS; Gibco). The hippocampi and cortices were dissected out and pooled according to tissue-type.
  • Tissue was trypsinized for 15 min (lmg/ml trypsin-HBSS; Worthington), rinsed with fresh HBSS, incubated in trypsin inhibitor (lmg/ml; Sigma) for 5 min, rinsed again with fresh HBSS and then triturated in 1ml fresh HBSS with a fire-polished glass pipette. Dissociated cells were seeded at 30,000 cells/well onto poly-D-lysine coated 96-well plates (Collaborative BioScience).
  • Each well contained lOO ⁇ l of Eagle's Minimal Essential Media (MEM; Gibco) supplemented with 26mM NaHCO 3 (Sigma), 56mM glucose (Sigma), 15mM KC1 (Sigma), lmM sodium pyruvate (Sigma), l.lmM L-glutamine (Sigma), 10% (v/v) heat- inactivated fetal bovine serum (Hyclone), and 0.001% gentamicin sulfate (Sigma) (pH 7.4). Cells were allowed to attach for 24h in a humidified 37°C 5% CO 2 incubator before experimental treatment. The culture media was aspirated and exchanged with fresh media every three days.
  • MEM Eagle's Minimal Essential Media
  • Cortical cells were seeded at 200,000 cells/dish onto polyethylenimine-coated 35mm culture dishes. Each dish contained 1.5ml MEM supplemented as described above. On the seventh day in culture, four fields per pre-marked dish were visualized with a Nikon Diaphot inverted microscope (lOx magnification) and photographed prior to experimental treatment.
  • rhEPO human erythropoietin
  • DPBS Dulbecco's phosphate buffered saline
  • BSA bovine serum albumin
  • PC- 12 cells (from a rat Pheochromocytoma) were cultured on poly-D-Lysine coated tissue culture plastic in DMEM containing 10 % FBS and 5% Horse serum. To induce the neuronal phenotype in PC- 12 cells, serum was removed and the cells were treated with NGF (50 ng/ml). Cells were grown for 7 days in the presence of the NGF then used for experiments.
  • NGF 50 ng/ml
  • PC- 12 cells were cultured as described above. 24 hr prior to insult, cells were treated with rhEPO at concentrations ranging from 1 pm to 1 nm. On the day of the experiment, cells were exposed to 200 ⁇ M glutamate for 30 min. Cells were then washed 2 times with fresh media to remove the glutamate and cultured in fresh media containing NGF but no EPO. After 24 hr cells were assayed for viability using a trypan blue exclusion assay. Briefly, media was removed and the cells were incubated in 0.4% Trypan Blue for 5 min. Cells were then washed gently with PBS, then fixed with 10% formalin. Cell viability was determined by counting the total number of cells vs. the number of trypan blue positive (dead) cells.
  • BDNF brain derived neurotrophic factor
  • GDNF glial- derived neurotrophic factor
  • NGF nerve growth factor
  • bFGF basic fibroblast growth factor
  • IGF-1 insulin-like growth factor -1
  • CNTF ciliary neurotrophic factor
  • EGF epidermal growth factor
  • VEGF vascular endothelial growth factor
  • Each treatment condition was run in quadruplicate or octuplicate.
  • the media was aspirated off and replaced with fresh media and test compound.
  • the cells were fixed with 10% phosphate-buffered formalin for 15min, then rinsed with DPBS (Sigma) and placed in blocking serum for 30 min (horse serum; 1:50 dilution in DPBS; Vector Labs).
  • MAP-2 microtubule-associated protein-2
  • FITC fluorescein for 1 hr
  • Neuroprotection study with primary neuronal cultures Pretreatment of cultures with rhEPO for 24h prior to glutamate administration resulted in a significant increase in neuronal survival (Figure 4). Cell survival was maximally increased approximately 200% over parallel cultures treated with glutamate alone. The neuroprotective effect of rhEPO was concentration-dependent, with the greatest effects observed at pM concentrations in which cell survival was greater than or equal to vehicle (no glutamate) treated cultures.
  • Neurite outgrowth study with primary neuronal cultures Cultures treated with rhEPO resulted in a significant increase in neurite outgrowth as measured by MAP2-FITC immunofluorescence.
  • the neurite outgrowth promoting effect was concentration dependent with maximal activity observed at pM levels ( Figures 7 and 8).
  • the results indicate that rhEPO treatment induced a larger outgrowth response in the hippocampal cultures (12-44% over control) than in the cortical cultures (15-29% over control).
  • a comparison between rhEPO and known growth factors indicates that they exhibit regional differences in their neurite outgrowth promoting abilities. rhEPO's ability to increase neurite outgrowth in cortical cultures is greater than or equal to that of known growth factors.
  • rhEPO promotes neurite outgrowth in primary mammalian neural cells.
  • the effect was robust for hippocampal and cortical cells.
  • the effect was potent with efficacy observed at sub-picomolar concentrations, far more potent than any previous EPO related observation.
  • rhEPO was superior in inducing neurite outgrowth relative to the maj ority of known growth factors .
  • rhEPO promotes neuroprotection and neurite outgrowth in cerebral cortical neurons.
  • neural cells can be in different stages of the process. Some may be stressed, others experience significant neurite retraction and loss of synaptic input, and eventually all affected cells will succumb to death.
  • a therapeutic agent that can intervene in this process at multiple levels can be of great benefit to the recovery of the neural cells and eventually neural function.
  • the present data support that rhEPO accomplishes this task by protecting the cells, by enhancing their survival, by promoting re-establishment of synaptic contacts and connections, and by stabilizing the neuronal and neural circuitry.
  • fetuses were removed via cesarean section from pregnant moms (Sprague-Dawley) anesthetized with halothane according to the AVMA Panel on Euthanasia. Pups were decapitated and the brains were removed and placed in HEPES-buffered Hank's Balanced Salt solution (HBSS; Gibco). The hippocampi and cortices were dissected out and pooled according to tissue-type.
  • Tissue was trypsinized for 15 min (lmg/ml trypsin-HBSS; Worthington), rinsed with fresh HBSS, incubated in trypsin inhibitor (lmg/ml; Sigma) for 5 min, rinsed again with fresh HBSS and then triturated in 1ml fresh HBSS with a fire-polished glass pipette. Dissociated cells were seeded at 30,000 cells/well onto poly-D-lysine coated 96-well plates (Collaborative BioScience).
  • Each well contained lOO ⁇ l of Eagle's Minimal Essential Media (MEM; Gibco) supplemented with 26mM NaHCO 3 (Sigma), 56mM glucose (Sigma), 15mM KC1 (Sigma), lmM sodium pyruvate (Sigma), l.lmM L-glutamine (Sigma), 10% (v/v) heat- inactivated fetal bovine serum (Hyclone), and 0.001% gentamicin sulfate (Sigma) (pH 7.4). Cells were allowed to attach for 24h in a humidified 37°C 5% CO 2 incubator before experimental treatment. The culture media was aspirated and exchanged with fresh media every 3 days.
  • MEM Eagle's Minimal Essential Media
  • MAP-2 microtubule- associated protein-2
  • Negative control wells were incubated in antibody diluent alone. Background signal was determined by blank wells (cell-free) incubated with or without antibody. Cultures were rinsed again with DPBS and then placed in fluorescein for 1 hr (FITC; anti-mouse IgG; rat adsorbed; 1:50 dilution in DPBS; Vector Labs). Cultures were rinsed a final time with DPBS and then the plates were read on a Cytofluor 4000 fluorescence plate reader. Neurite outgrowth was expressed as percent change from control (vehicle; mean fluorescence + SEM).
  • EMP-23 had a modest response to EPO in hippocampal cultures with peak activity observed between 30pM-lnM that led to a 43-46% increase over vehicle response.
  • EMP-1, 9 and 27 exhibited response profiles similar to most known growth factors - a flat response overall with maximal activity occurring between 30-300pM reaching 32-40% above the vehicle response.
  • EMP-6 and 23 displayed typical bell-shaped dose-response profiles with peak activity observed between 30-300pM resulting in a 68-87% increase in outgrowth over vehicle response levels.
  • EMP-6 promoted robust neurite outgrowth activity in both hippocampal and cortical cultures; whereas, EMP-1 showed selective effects in hippocampal cultures over cortical cultures and EMP-23 effects were greater in cortical cultures than hippocampal cells. EMP-9 and 27 neurite outgrowth responses were less impressive overall.
  • EMP's promote neurite outgrowth in mammalian cells.
  • the effect was robust for hippocampal and cortical cells.
  • the neurite outgrowth promoting effect was superior to that of various growth factors.
  • the effect was potent with efficacy observed at picomolar concentrations.
  • mice Male spontaneous hypertensive rats (Charles River) weighing between 250-300g were weighed and then anesthetized with ketamine (100mg/ml)/xylazine (20mg/ml) cocktail (1.2ml kg; i.p.). The level of anesthetic was assessed by corneal reflex (air puff to eye) and leg jerk in response to tail or foot pinch. Once the rat was anesthetized, it was placed on a small animal surgical board and restrained during the surgical procedure. The rat's body temperature was monitored continuously with a rectal probe and maintained at 37°C with a homeostatic heating pad.
  • Rats were rendered ischemic by tandem occlusion of the left common carotid artery and the left middle cerebral artery for 2h followed by 22h of reperfusion using a modification of the technique described by Brint and co-workers (J. Cereb Blood Flow Metab 8:474-485, 1988). Specifically, the left CCA was isolated through an incision in the ventral surface of the neck. For isolation of the ipsilateral MCA, a second incision was made between the lateral canthus of the left eye and the corresponding external auditory canal to bare the underlying skull.
  • the MCA was exposed through a 5mm burrhole drilled 2-3mm rostral to the fusion of the zygomatic arch and the squamosal bone under direct visualization with a Zeiss operating microscope.
  • the dura was opened with a sterile 26g needle and a platinum alloy wire (0.1mm diameter) was inserted beneath the MCA just superior to the inferior cortical vein.
  • the MCA was temporarily occluded by elevation and compression of the vessel across the alloy wire, as described by Aronowski and colleagues (Stroke, 25:2235-2240, 1994).
  • the CCA was occluded with an aneurysm clip.
  • the duration of occlusion of the CCA and the MCA was 2h. At the end of this period, the wire and the clip were carefully removed to allow reperfusion of the vessels and the incision area was sutured shut.
  • the rat was placed in an isolation cage to recover before returning to his home cage. . Study Design
  • Rats were divided into five groups: (1) sham-operated vehicle-treated, (2) ischemic vehicle treated, (3) ischemic 1.32U/day EPO treated, (4) ischemic 132U/day EPO treated and (5) ischemic 1321U/day EPO treated.
  • rats were rendered ischemic as described above. Twenty-two hours later, the rats were evaluated for behavioral performance, a blood sample was collected for terminal plasma levels of EPO and then the rat was euthanized, brain removed, sectioned and stained for histological analysis.
  • Rats were rendered ischemic as described above. Rats were divided into four groups: (1) ischemic vehicle treated, (2) ischemic lOOOU/kg EPO treated, (3) ischemic 2500U/kg EPO treated and (4) 5000U/kg EPO treated. Fifteen minutes post-occlusion, the rats received vehicle or EPO as an intravenous bolus injection. Twenty-two hours later, the rats were evaluated for behavioral performance, a blood sample was collected for terminal plasma levels of EPO and then the rat was euthanized, brain removed, sectioned and stained for histological analysis.
  • Rats On Day One, the rats were rendered ischemic as described above. Rats were divided into two groups: (1) ischemic vehicle treated and (2) ischemic 2500U/kg EPO treated.
  • Drug was administered as an intravenous bolus at 15min, 2h, 4h and 6h post- occlusion for a total dose of 10,000U/kg. Twenty-two hours later, the rats were evaluated for behavioral performance, a blood sample was collected for terminal plasma levels of EPO and then the rat was euthanized, brain removed, sectioned and stained for histological analysis. Outcome measures
  • Plasma Determinations Blood samples were collected from each rat via the orbital sinus at the time of sacrifice. Plasma was separated out, frozen and analyzed by EPO ELISA for determination of plasma concentration (U/ml).
  • Infarct Volume Brains were removed, blocked into 1mm slabs and stained with 2,3,5-triphenyl tetrazolium chloride dye (TTC; Sigma) for 15 min at room temperature. Stained sections were stored in 10% buffered formalin at 4oC. Sections were visualized by a Nikon SMZ-U microdissecting scope. Images of each brain section were captured with a CCD camera and processed using Image Pro Phase III software in order to calculate infarct volume. Results
  • Patent application WO 95/03821 O'Brien, J.S., Kishimoto, Y. and Altman, D.E. Title: Prosaposin and cytokine-derived peptides as therapeutic agents.
  • Patent application WO 98-CA991 Weiss, S and Sorokan, S.T. Title: Erythropoietin- mediated neurogenesis.
  • Patent application WO 95/03821, O'Brien, J.S., Kishimoto, Y. and Altman, D.E. Title: Prosaposin and cytokine-derived peptides as therapeutic agents.
  • Protease-activated receptor 2 (PAR-2) is present in the rat hippocampus and is associated with neurodegeneration. J. Neurochem., 69: 1890-1896.

Abstract

L'invention concerne des procédés destinés au traitement de maladies du système nerveux par administration de compositions possédant l'activité thérapeutique et neurologique de l'érythropoïétine humaine. Ces compositions contiennent des agents thérapeutiques, tels que des peptides, des dimères peptidiques, des polypeptides, et des protéines possédant la gamme complète des activités biologiques de l'érythropoïétine humaine ou seulement certaines d'entre elles. L'invention porte également sur des posologies thérapeutiques améliorées dans lesquelles l'érythropoïétine est administrée dans des concentrations inférieures à celles requises dans la stimulation de l'hématopoïèse.
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JP2003534384A (ja) 2003-11-18
AU2001274904B2 (en) 2006-08-31
WO2001091780A1 (fr) 2001-12-06
ZA200210304B (en) 2004-03-19
BR0111182A (pt) 2004-02-25
CN1318084C (zh) 2007-05-30
IL153079A0 (en) 2003-06-24
MXPA02011727A (es) 2003-10-24
EP1296702A4 (fr) 2005-05-11
NZ522924A (en) 2004-12-24
AU7490401A (en) 2001-12-11
CA2410453A1 (fr) 2001-12-06
CN1452492A (zh) 2003-10-29
HK1058484A1 (en) 2004-05-21

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