WO2008077234A1 - Effets synergiques des c3, de l'amp cyclique et du facteur neurotrophique ciliaire sur la survie neuronale et la régénération axonale - Google Patents

Effets synergiques des c3, de l'amp cyclique et du facteur neurotrophique ciliaire sur la survie neuronale et la régénération axonale Download PDF

Info

Publication number
WO2008077234A1
WO2008077234A1 PCT/CA2007/002241 CA2007002241W WO2008077234A1 WO 2008077234 A1 WO2008077234 A1 WO 2008077234A1 CA 2007002241 W CA2007002241 W CA 2007002241W WO 2008077234 A1 WO2008077234 A1 WO 2008077234A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
camp
pharmaceutical composition
active agent
cntf
Prior art date
Application number
PCT/CA2007/002241
Other languages
English (en)
Inventor
Alan Harvey
Original Assignee
Bioaxone Therapeutique Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bioaxone Therapeutique Inc. filed Critical Bioaxone Therapeutique Inc.
Publication of WO2008077234A1 publication Critical patent/WO2008077234A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • 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
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/363—Fibrinogen
    • 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
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue

Definitions

  • the invention in particular pertains to methods and compositions comprising C3 fusion proteins combined with CNTF and cAMP or analogues thereof that have synergistic effects on promoting neuronal survival and axonal regeneration.
  • RGCs retinal ganglion cells
  • BDNF brain-derived neurotrophic factor
  • NT-4/5 neurotrophin 4/5
  • CNTF ciliary neurotrophic factor
  • RGCs damage to RGCs also causes changes in receptor expression and may alter responsiveness to trophic signals. It is therefore important to ensure that responsiveness to such factors is maintained or even enhanced during the regenerative process. Further, raised intracellular cAMP can increase neurotrophin receptor levels in cell membrane and enhances neuronal responsiveness to diffusible growth factors.
  • axonal regrowth is inhibited by the glial scar, which contains reactive astrocytes and secreted chondroitin sulfate proteoglycans (CSPGs). Regrowth is also inhibited by myelin-associated factors such as oligodendrocyte myelin glycoprotein (OMgp), myelin-associated glycoprotein (MAG) and Nogo.
  • OMgp oligodendrocyte myelin glycoprotein
  • MAG myelin-associated glycoprotein
  • Rho GTPase signaling pathway Rho GTPase signaling pathway
  • Rho-kinase (ROCK) Rho-kinase
  • the enzyme C3 transferase inactivates Rho by ADP ribosylation.
  • Cell-permeable C3 fusion proteins stimulate neurite growth in tissue culture and axonal growth in vivo when applied to optic nerve and spinal cord lesion sites.
  • Rho antagonists as agents to stimulate regeneration of (cut) axons, i.e. nerve lesions; please see, for example, Canadian Patent application Nos 2,304,981 (McKerracher et al.) and 2,300,878 (Strittmatter).
  • Rho antagonists such as for example the chimeric C3 proteins as well as substances selected from among known trans-4-amino (alkyl)-l-pyridylcarbamoylcyclohexane compounds or Rho kinase inhibitors for use in the regeneration of axons.
  • C3 inactivates Rho by ADP- ribosylation and is fairly non-toxic to cells (Dillon and Feig, 1995, Methods in Enzymology: Small GTPases and their regulators Part. B, 256: 174-184).
  • Clostridium botulinum C3 exotransferase (hereinafter simply referred to as C3) can stimulate regeneration and sprouting of injured axons;
  • C3 is a toxin purified from Clostridium botulinum (Saito et al., 1995, FEBS Lett, 371 : 105-109; Wilde et al., 2000, J. Biol. Chem., 275: 16478).
  • Compounds of the C3 family from Clostridium botulinum inactivate Rho by ADP-ribosylation and thus act as antagonists of Rho effect or function (Rho antagonists).
  • C3 protein can promote regeneration, it has been noted that C3 does not easily penetrate into cells, and high doses must therefore be applied for it to be effective.
  • the high dose of recombinant C3 needed to promote functional recovery presents a practical constraint or limitation on the use of C3 in vivo to promote regeneration.
  • tissue culture studies it has been determined that the minimum amount of C3 that can be used to induce growth on inhibitory substrates is 25 ⁇ g/ml (Lehmann et al., 1999, J. Neurosci. 19: 7537-7547). If the cells are not triturated, even this dose is ineffective.
  • compositions and methods for promoting neuronal survival and axonal regeneration are provided.
  • the present invention relates to the synergistic effects of combined administration of a Rho antagonist, e.g. a C3 fusion protein, with a growth factor, e.g. CNTF, and cAMP or analogues or modulators thereof, which have synergistic effects on promoting neuronal, e.g. adult RGC, survival and axonal regeneration.
  • Compositions and therapeutic methods comprising administering the compositions of the invention to a subject in need thereof for e.g. promoting neuronal survival and axonal regeneration in a subject are provided.
  • Exemplary compositions of the invention comprise Rho antagonists, e.g. C3 proteins or C3-like proteins, growth factors, e.g. CNTF, and cAMP, or analogues or modulators thereof.
  • a Rho antagonist e.g. C3 protein or C3-like protein, a growth factor, e.g. CNTF, and cAMP or an analogue or modulator thereof.
  • the Rho antagonist e.g. C3 protein or C3-like protein
  • growth factor e.g. CNTF
  • cAMP or an analogue or modulator thereof
  • the invention relates to methods comprising concomitant administration of any two of these agents.
  • the invention relates to methods comprising concomitant administration of all three of these agents.
  • the invention relates to compositions comprising any two or any three of these agents.
  • compositions comprising a Rho antagonist, a growth factor, and cAMP or an analogue or modulator thereof are provided.
  • the Rho antagonist includes a polypeptide comprising an amino acid sequence of an active agent, wherein the sequence is ADP-ribosyl transferase C3 or a fragment thereof which retains ADP-ribosyl transferase activity.
  • the Rho antagonist also includes an amino acid sequence of a transport agent - A -
  • transport agent covalently linked to the amino acid sequence of the active agent (e.g. ADP-ribosyl transferase C3 or a fragment thereof).
  • the transport agent may facilitate uptake of the active agent by a receptor-independent or receptor-dependent mechanism.
  • transport agents for use in compositions and methods of the invention include, without limitation: SEQ ID NOs: 18 - 52; Silaproline conjugates, gamma-amino-L- proline oligomers; VP22 protein; b-FGF; SAP sweet arrow peptide; hCT(9-32)-br human calcitonin derived peptide; BagP peptide; Mycobacterium cell entry protein; Synthetic peptide YT A2; Synthetic peptide YT A4; TPlO; dynorphin A; and dynorphin B.
  • the pharmaceutical compositions and methods of the invention include an active agent having at least 90% sequence identity with SEQ ID NO: 2 and retaining ADP-ribosyl transferase activity.
  • the active agent is a polypeptide consisting of: SEQ ID NO: 2; SEQ ID NO: 4; a polypeptide consisting of SEQ ID NO: 4 which is truncated by 20 amino acids at its N-terminus; a polypeptide consisting of SEQ ID NO: 4 which is truncated by 10 amino acids at its C-terminus; and/or a polypeptide consisting of SEQ ID NO: 4 which is truncated by 20 amino acids at its N-terminus and 10 amino acids at its C-terminus.
  • compositions and methods of the invention include polypeptides which are PEGylated.
  • polypeptide consisting of SEQ ID NO: 4 may be PEGylated.
  • compositions and methods of the invention may include or be administered with a biological adhesive such as fibrin or a fibrin sealant.
  • compositions and methods including cAMP or a cAMP analogue are also provided.
  • the cAMP analogue is cell-permeant, e.g. chlorphenylthio-cAMP.
  • the growth factor used in the compositions and methods of the invention is a ciliary neurotrophic factor (CNTF), BDNF, NT-4/5, CNTF, GDNF, NGF, neurotrophin 3, neurotrophin 4, neurotrophin 5, IGF, MGF, PDGF, neurturin, FGFs, neuregulin type I and/or neuregulin type II.
  • a pharmaceutical composition which includes: a polypeptide having an amino acid sequence of a transport agent covalently linked to an amino acid sequence of an active agent, wherein the amino acid sequence of the active agent is ADP-ribosyl transferase C3 or a fragment thereof which retains ADP- ribosyl transferase activity; a ciliary neurotrophic factor (CNTF); and cAMP, or an analogue or modulator thereof.
  • CNTF ciliary neurotrophic factor
  • methods which include administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to the invention.
  • a method for preventing or inhibiting loss of retinal ganglion cells by administering a pharmaceutical composition as described herein is provided.
  • the loss of retinal ganglion cells may be associated, for example, with optical neuropathy, ageing or blindness caused by glaucoma.
  • Methods are also provided herein for preventing or inhibiting axon loss or for providing neuroprotection by administering a pharmaceutical composition of the invention.
  • Axon loss may be associated with a condition or disease such as, for example, spinal cord injury, immune neuropathy, peripheral neuropathy, multiple sclerosis, optic neuropathy, Parkinson's, Alzheimer's, Charcot-Marie-Tooth disease, Giant axonal neuropathy, branch/central retinal vein/artery occlusion, macular edema, angle-closure glaucoma, open-angle glaucoma, age related macular degeneration, retinitis pigmentosa, retinal detachments, damage associated with laser therapy, diabetic retinopathy, surgical light-induced iatrogenic retinopathy, macular degeneration, traumatic optic neuropathy, Stargardt disease, Lebers Congenital Amaurosis, Best disease, Choroideremia, Retinoschisis, Bardet- Biedl syndrome, Anterior ischemic optic neuropathy, Purtscher's retinopathy, Optic neuritis, Optic disc edema, Coats' disease and/or Leber's
  • methods are provided for promoting adult retinal ganglion cell survival and/or for promoting axonal regeneration by administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition of the invention.
  • the methods provided herein may further include the insertion of a nerve conduit.
  • the Rho antagonist, growth factor, and/or cAMP (or analogue or modulator thereof) may be administered simultaneously or sequentially.
  • a pharmaceutical composition of the invention in the manufacture of a medicament for preventing or treating traumatic or degenerative nerve injury, for promoting axonal regeneration, for preventing or inhibiting axon loss, and/or for promoting adult retinal ganglion cell survival.
  • the nerve injury may be a result of, for example, physical injury, surgery, cancer growth, cancer treatment chemotherapy and/or anesthesia.
  • the present invention relates to a kit for preventing or treating traumatic or degenerative nerve injury, promoting axonal regeneration, preventing or inhibiting axon loss, or promoting adult retinal ganglion cell survival in a subject in need thereof, the kit having: a polypeptide which includes an amino acid sequence of a transport agent covalently linked to an amino acid sequence of an active agent, wherein the active agent is ADP-ribosyl transferase C3 or a fragment thereof which retains ADP-ribosyl transferase activity; a growth factor such as, for example, ciliary neurotrophic factor (CNTF); and cAMP or an analogue or modulator thereof.
  • a polypeptide which includes an amino acid sequence of a transport agent covalently linked to an amino acid sequence of an active agent, wherein the active agent is ADP-ribosyl transferase C3 or a fragment thereof which retains ADP-ribosyl transferase activity
  • a growth factor such as, for
  • FIG. 1 illustrates fluorogold (FG) labeled regenerating (A, C) and viable ⁇ lll- tubulin positive (B, D) RGCs in rats that received saline (A and B, same field) or SEQ ID NO: 4 injections (C and D same field) and wherein scale bars represent 100 ⁇ m;
  • FIG. 2 illustrates the average number of surviving ( ⁇ lll-tubulin positive) and regenerating (fluorogold labeled) RGCs per retina after injection of SalineD, saline double injection; C3D, SEQ ID NO: 4 double injection; C3T, SEQ ID NO: 4 triple injection and wherein * p ⁇ 0.05, **p ⁇ 0.000 ⁇ ; unpaired student t test Welch's corrected; comparisons were made against the saline group respectively and error bars represent SEM;
  • FIG. 3 illustrates the average number of surviving ( ⁇ lll-tubulin positive) and regenerating (fluorogold labeled) RGCs per retina after various double injection (days 4 and 11 after peripheral nerve transplantation) protocols; the surviving RGC comparisons, **/> ⁇ 0.01 Dunnett's test; regenerating RGC comparisons, */? ⁇ 0.05 Kruskall-Wallis test; both comparisons were made against the respective saline group; and wherein **/? ⁇ 0.01 SEQ ID NO: 4 vs. CNTF/CPT-cAMP group, ***p ⁇ 0.001 SEQ ID NO: 4/CNTF/CPT-cAMP vs. CNTF/CPT-cAMP group, Bonferroni's test; and error bars represent SEM;
  • FIG. 4 illustrates the average number ( ⁇ SEM) of pan-neurofilament immunostained axons at various distances along peripheral nerve (PN) autograft in different eye injection groups, wherein the average number of pan-neurofilament positive axons per section across the PN is plotted against incremental distance from the PN-ON interface;
  • A illustrates saline and SEQ ID NO: 4 single injection groups
  • B SEQ ID NO: 4 double injection versus saline double injection groups
  • C repeated SEQ ID NO: 4 treatments
  • D comparison of pan-neurofilament counts in peripheral nerve grafts from four eye injection groups: SEQ ID NO: 4/CNTF/CPT-cAMP, SEQ ID NO: 4/CNTF, SEQ ID NO: 4/CPT-cAMP and CNTF/CPT-cAMP; and wherein in (D) the data from the double injection saline control group (plotted in B) is shown by the dotted line and
  • FIG. 5 illustrates RGC densities at different eccentricities from the optic nerve head (ONH) for six eye injection groups, wherein central, intermediate and peripheral values represent density measurements of surviving ( ⁇ lll-tubulin positive) and regenerating (fluorogold positive) RGCs sampled within concentric circles with radii of 0.1-1.4 mm, 1.4-2.8 mm, or over 2.8 mm from the ONH respectively, and wherein the proportion of viable RGCs that regenerated an axon into PN autografts is shown by the percentage values and error bars represent SEM.
  • the present invention relates to the synergistic effects of a combined administration of a Rho antagonist, e.g. a C3 fusion protein, with a growth factor, e.g. CNTF, and cAMP or analogues or modulators thereof, which have synergistic effects on promoting neuronal, e.g. adult RGC, survival and axonal regeneration.
  • a Rho antagonist e.g. a C3 fusion protein
  • a growth factor e.g. CNTF
  • cAMP or analogues or modulators thereof which have synergistic effects on promoting neuronal, e.g. adult RGC, survival and axonal regeneration.
  • the present invention relates therefore to compositions, and to methods for treating comprising administering the compositions of the invention to a subject in need thereof, for e.g. promoting neuronal, e.g. adult RGC, survival and axonal regeneration in a subject.
  • Exemplary compositions of the invention comprise Rh
  • the present invention also relates to methods comprising administering to a subject in need thereof a Rho antagonist, e.g. C3 protein or C3-like protein, a growth factor, e.g. CNTF, and cAMP or an analogue or modulator thereof.
  • a Rho antagonist e.g. C3 protein or C3-like protein
  • the Rho antagonist e.g. C3 protein or C3-like protein
  • growth factor e.g. CNTF
  • cAMP or an analogue or modulator thereof
  • the invention relates to methods comprising concomitant administration of any two of these agents.
  • the invention relates to methods comprising concomitant administration of all three of these agents.
  • subject is intended to include animals.
  • the subject is a mammal, e.g. a human or nonhuman primate, a dog, a cat, a horse, a cow or a rodent.
  • Rho antagonists includes, but is not restricted to, C3 proteins, including C3-like proteins, C3 chimeric proteins, etc.
  • C3 protein refers to ADP-ribosyl transferase C3 isolated from Clostridium botulinum, Bacillus cereus or Staphylococcus aureus or a recombinant ADP-ribosyl transferase.
  • C3-like protein refers to any protein (polypeptide) having a biological activity similar (e.g., the same, substantially similar), to ADP-ribosyl transferase C3.
  • ADP-ribosyl transferase C3 include, without limitation, SEQ ID NO: 2, 4, 6, 7-17, and PEGylated forms thereof.
  • Polypeptides of the present invention comprise, for example, biologically active mutants, variants, fragments, chimeras, and analogues thereof.
  • mutant, variant, fragment, chimera, and analogue are used interchangeably herein, and encompass, for example, polypeptide sequences having truncations of one or more amino acids, wherein the truncation may originate from the amino terminus (N- terminus), carboxy terminus (C-terminus), or from the interior of the protein; polypeptide sequences having an insertion or a substitution of one or more amino acids; and the like.
  • Variants, mutants, fragments, chimeras and analogues may have the biological properties of polypeptides of the present invention including, for example (without being restricted to the present examples), to act as a Rho antagonist, to facilitate neuronal axon growth, to suppress the inhibition of neuronal axon growth, to regenerate injured axons, to facilitate neurite growth, to inhibit apoptosis, and/or to treat nervous system and/or nerve injuries or disease.
  • polypeptides of the present invention may include a transport agent such as, for example, a subdomain of HIV Tat protein, or a homeodomain of Antennapedia.
  • the transport agent may be repeated more than one time in a polypeptide comprising the ADP-ribosyl transferase C3 or ADP-ribosyl transferase C3 analogues.
  • the transport agent region may be either at the amino-terminal region of an ADP-ribosyl transferase C3 or ADP-ribosyl transferase C3 analogues or at its carboxy-terminal region or at both regions.
  • the repetition of a transport agent region may affect (e.g., increase) the uptake of the ADP-ribosyl transferase C3 or ADP- ribosyl transferase C3 analogues by a desired cell.
  • a transport agent facilitates uptake of an active agent by a receptor-independent mechanism.
  • examples of transport sequences include, but are not limited to, SEQ ID NOs: 18-25.
  • Other transport agents encompassed by the present invention include, without limitation: the third helix of the homeodomain of Antennapedia protein (PenetratinTM; SEQ ID NO: 26); TAT (SEQ ID NO: 27); proline rich sequence (SEQ ID NO: 28); Silaproline conjugates; gamma-amino-L-proline oligomers; polyarginine (SEQ ID NO: 29); Transportan (SEQ ID NOs: 30-31); Pep-1 (SEQ ID NO: 32); S4 13 -PV (SEQ ID NO: 33); VP22 protein; MAO (Model sunthic peptide; SEQ ID NO: 34); SynBl (SEQ ID NO: 35); Syn B3 (SEQ ID NO: 36); Syn B5 (SEQ ID NO: 37); b-F
  • growth factor refers to neurotrophic factors, such as CNTF, BDNF, NT-4/5, CNTF, GDNF, NGF, neurotrophin 3, 4 and 5, IGF, MGF, PDGF, neurturin, FGFs such as bFGF, neuregulin type I (neu) and type II (glial growth factor).
  • neurotrophic factors such as CNTF, BDNF, NT-4/5, CNTF, GDNF, NGF, neurotrophin 3, 4 and 5, IGF, MGF, PDGF, neurturin, FGFs such as bFGF, neuregulin type I (neu) and type II (glial growth factor).
  • pharmaceutical composition means therapeutically effective amounts (dose) of an agent together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsii ⁇ ers, adjuvants and/or carriers.
  • dose pharmaceutically acceptable diluents, preservatives, solubilizers, emulsii ⁇ ers, adjuvants and/or carriers.
  • a “therapeutically effective amount” as used herein refers to that amount which provides a therapeutic effect for a given condition and administration regimen.
  • compositions may be liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, and detergents (e.g., TweenTM 20, TweenTM 80, Pluronic® F68, bile acid salts).
  • buffer content e.g., Tris-HCl, acetate, phosphate
  • pH and ionic strength e.g., Tris-HCl, acetate, phosphate
  • additives such as albumin or gelatin to prevent absorption to surfaces
  • detergents e.g., TweenTM 20, TweenTM 80, Pluronic® F68, bile acid salts.
  • compositions of the invention may also comprise solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, parabens), and bulking substances or tonicity modifiers (e.g., lactose, mannitol) which contribute to covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • solubilizing agents e.g., glycerol, polyethylene glycerol
  • anti-oxidants
  • Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention include particulate forms, protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral routes.
  • the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intracranially intratumorally or more preferably, directly at a central nervous system (CNS) lesion site or a peripheral nervous system (PNS) lesion site.
  • CNS central nervous system
  • PNS peripheral nervous system
  • pharmaceutically effective amount refers to an amount (dose) effective for treating a patient, having, for example, a nerve injury. It is also to be understood herein that a “pharmaceutically effective amount” may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route or taken alone or in combination with other therapeutic agents.
  • a "pharmaceutically effective amount” may be understood as an amount which may, for example, suppress (e.g., totally or partially) the inhibition of neuronal axon growth, facilitate axon growth, prevent cell apoptosis, decrease inflammation, suppress Rho activity, help regenerate injured axon, or which may help neurons to make new connections with other cells.
  • a therapeutically effective amount or dosage of an active agent may range from about 0.001 to 30 mg/kg body weight, with other ranges of the invention including about 0.01 to 25 mg/kg body weight, about 0.025 to 10 mg/kg body weight, about 0.3 to 20 mg/kg body weight, about 0.1 to 20 mg/kg body weight, about 1 to 10 mg/kg body weight, 2 to 9 mg/kg body weight, 3 to 8 mg/kg body weight, 4 to 7 mg/kg body weight, 5 to 6 mg/kg body weight, and 20 to 50 mg/kg body weight.
  • a therapeutically effective amount or dosage of an active agent may range from about 0.001 to 50 mg total, with other ranges of the invention including about 0.01 to 10 mg, about 0.3 to 3 mg, about 3 to 10 mg, about 6 mg, about 9 mg, about 10 to 20 mg, about 20-30 mg, about 30 to 40mg, and about 40 to 50 mg.
  • compositions of the present invention may also be formulated for intravenous or subcutaneous administration.
  • single-dose vials can be produced containing about 25, about 40, about 60, about 100, about 150, about 200, about 300, or about 500 micrograms of active agent.
  • treatment of a subject with a therapeutically effective amount of an active compound can include a single treatment or a series of treatments.
  • a subject is treated with an active compound in the range of between about 0.3 to 10 mg, one time per week for between about 1 to 10 weeks, alternatively between 2 to 8 weeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks.
  • the effective dosage of an active compound used for treatment may increase or decrease over the course of a particular treatment.
  • cAMP analogue includes non-degradable cell permeant cAMP analogues, for example chlorphenylthio-cAMP (CPT-cAMP).
  • CPT-cAMP chlorphenylthio-cAMP
  • the cAMP analogue is non-hydrolyzable cAMP analogue.
  • cAMP modulator includes any compound which has the ability to modulate the amount, production, concentration, activity or stability of cAMP in a cell, or to modulate the pharmacological activity of cellular cAMP.
  • cAMP modulators may act at the level of adenylate cyclase, upstream of adenylate cyclase, or downstream of adenylate cyclase, such as at the level of cAMP itself, in the signaling pathway that leads to the production or to the degradation of cAMP.
  • Modulators can be, but are not limited to, adenylate cyclase activators, macrophage activators, macrophage-derived factors that stimulate cAMP synthesis, calcium ionophores, membrane depolarization agents, phosphodiesterase inhibitors, beta2-adrenoreceptor inhibitors and vasoactive intestinal peptide.
  • Agents modulating adenylate cyclase include but are not limited to forskolin, NKH477, pituitary adenylyl cyclase-activating peptide (PACAP).
  • Cyclic AMP modulators include activators of adenylate cyclase such as forskolin; non- hydrolyzable analogues of cAMP including 8-bromo-cAMP, 8-chloro-cAMP, or dibutyryl cAMP (db-cAMP); isoprotenol; vasoactive intestinal peptide; calcium ionophores; membrane depolarization; macrophage-derived factors that stimulate cAMP; agents that stimulate macrophage activation such as zymosan or IFN- .
  • beta-2 adrenoreceptor agonists such as salbutamol.
  • beta-2 adrenoreceptor agonists are known in the art and include salmeterol, fenoterol and isoproterenol.
  • cAMP modulators or analogues encompassed by the present invention are Sp or Rp-cAMP (adenosine-3',5'-cyclic monophosphorothioate Sp or Rp-isomer), dibutyryl cAMP, dioctanoyl cAMP, 2'-O-Me-cAMP; , 8- ⁇ CPT-2'-O-Me- cAMP; , 8-pMeOPT-2'-O-Me-cAMP, N6-Bnz-cAMP, Sp or Rp-8-Br-cAMPS (8- Bromoadenosine-3', 5'-cyclic monophosphorothioate, Sp or Rp-isomer) , CPT-cAMP (8-(4-chlorophenylthio)-cAMP), 8-NH2-cAMP, 8-bromo-cAMP, N6-O2'-dibutyryl- cAMP, 8- and 2-BDB-TcAMP (8
  • diseases or conditions such as optical neuropathy, ageing causing loss of RGC and blindness caused by glaucoma can be treated by promoting adult RGC survival.
  • the present invention provides methods and compositions for preventing and/or treating damage to the retina and optic nerve, including damage resulting from ischemic or hypoxic stress, excess intraocular pressure, or injury.
  • the methods and compositions can be used specifically to treat damage associated with vascular occlusion or anterior ischemic optic neuropathy.
  • the methods and compositions are also useful for treating damage arising from the presence of cytotoxins or neurotoxins, such as glutamate or other excitatory amino acids or peptides, excess intracellular calcium, and free radicals.
  • cytotoxins or neurotoxins such as glutamate or other excitatory amino acids or peptides, excess intracellular calcium, and free radicals.
  • the methods and compositions can be useful in treating damage associated with branch and central vein/artery occlusion, trauma, macular edema, angle-closure glaucoma, open-angle glaucoma, age related macular degeneration, retinitis pigmentosa, retinal detachments, damage associated with laser therapy (including photodynamic therapy), diabetic retinopathy, and surgical light-induced iatrogenic retinopathy.
  • the methods and compositions of the invention are used to treat damage associated with macular degeneration, traumatic optic neuropathy, Stargardt disease, Lebers Congenital Amaurosis, Best disease, Choroideremia, Retinoschisis, Bardet-Biedl syndrome, Anterior ischemic optic neuropathy, Purtscher's retinopathy, Optic neuritis, Optic disc edema, Coats' disease and/or Leber's miliary aneurysm.
  • the optic nerve functional disorder in the present invention encompasses, for example, an optic nerve functional disorder caused by pressing an optic nerve due to fracture of an optic fasciculus duct or an intraorbital abscess and arteriovenous aneurysm; an intracranial disease; a cerebral tumor; a pituitary tumor, an optic nerve functional disorder caused by hemorrhage, interruption of blood circulation or infarction, a glaucomatous optic nerve disease and the like.
  • Glaucoma is a group of diseases of the optic nerve involving loss of retinal ganglion cells in a characteristic pattern of optic neuropathy. Although raised intraocular pressure is a significant risk factor for developing glaucoma, there is no set threshold for intraocular pressure that causes glaucoma. One person may develop nerve damage at a relatively low pressure, while another person may have high eye pressures for years and yet never develop damage. Untreated glaucoma leads to permanent damage of the optic nerve and resultant visual field loss, which can progress to blindness.
  • glaucoma encompasses low tension glaucoma (normal tension glaucoma), high tension glaucoma (open-angle glaucoma or narrow-angle glaucoma (acute inflammatory glaucoma), congenital glaucoma and secondary glaucoma) and the like, but the present invention is not limited thereby, and the effect of the present invention exhibits in all kinds of optic nerve functional disorders other than the above, particularly glaucoma.
  • Macular degeneration is a group of diseases that affect the central retina, or macula. There are two basic types of macular degeneration: “wet” and “dry”. In wet macular degeneration, there is an abnormal growth of new blood vessels. These new blood vessels break and leak fluid, causing damage to the central retina. This form of macular degeneration is often associated with aging. Approximately 90% of macular degeneration cases are dry macular degeneration. Vision loss can result from the accumulation of deposits in the retina called drusen, and from the death of photoreceptor cells. This process can lead to thinning and drying of the retina.
  • Retinitis pigmentosa is a retinal degeneration disease which manifests as night blindness, progressive loss of visual field and peripheral vision, eventually leading to total blindness; ophthalmoscopic changes can consist of dark mosaic-like retinal pigmentation, attenuation of the retinal vessels, waxy pallor of the optic disc, and in the advanced forms, macular degeneration. In some cases there can be a lack of pigmentation.
  • This disease is hereditary and the degeneration of retinal photoreceptor cells proceeds with increasingly narrow retinochoroidal blood vessels and circulatory disorders.
  • Diabetic retinopathy a leading cause of blindness in type 1 and type 2 diabetics, is a complication of diabetes which produces damage to the blood vessels inside the retina.
  • Diabetic retinopathy can have four stages: (1) mild nonproliferative retinopathy, wherein microaneurysms in the retina's blood vessels occur; (2) moderate nonproliferative retinopathy, wherein some blood vessels feeding the retina become blocked; (3) severe nonproliferative retinopathy, wherein many blood vessels to the retina are blocked, depriving several areas of the retina with their blood supply; and (4) proliferative retinopathy, wherein new, abnormal, thin- and fragile-walled blood vessels grow to supply blood to the retina, but these new blood vessels may leak blood to produce severe vision loss and blindness.
  • Hemorrhages can occur more than once, often during sleep. Fluid can also leak into the center of the macula at any stage of diabetic retinopathy and cause macular edema and blurred vision. About 40 to 45 percent of Americans diagnosed with diabetes have some stage of diabetic retinopathy, and about half of the people with proliferative retinopathy also have macular edema.
  • Methods and pharmaceutical compositions of the present invention can also be used to treat various neuropathological states such as those arising within the central nervous system and affecting the brain or the spinal cord as well as those affecting peripheral nerves (e.g. optic, sciatic, facial, lingual, penile, maxillary, intestinal) which damage can take place following physical injury, surgery, cancer growth, cancer treatment chemotherapy (e.g., by vinca alkaloids and doxorubicin) or other type of treatments, anasthesia, disease (e.g., diabetic neuropathy).
  • various neuropathological states such as those arising within the central nervous system and affecting the brain or the spinal cord as well as those affecting peripheral nerves (e.g. optic, sciatic, facial, lingual, penile, maxillary, intestinal) which damage can take place following physical injury, surgery, cancer growth, cancer treatment chemotherapy (e.g., by vinca alkaloids and doxorubicin) or other type of treatments, anasthesia, disease (e.g., diabet
  • the methods and pharmaceutical compositions of the present invention can also be used to treat diseases or conditions such as spinal cord injury, immune and peripheral neuropathy, multiple sclerosis, Parkinson's, amyotrophic lateral sclerosis, Alzheimer's, Charcot-Marie-Tooth disease, Giant axonal neuropathy, trigeminal neuralgia, glossopharyngeal neuralgia, Bell's palsy, myasthenia gravis, muscular dystrophy, progressive muscular atrophy, progressive bulbar inherited muscular atrophy, herniated, ruptured or prolapsed vertebral disk syndromes, cervical spondylosis, plexus disorders, thoracic outlet destruction syndromes, acrylamides, gamma-diketones (glue-sniffer's neuropathy), carbon disulfide, dapsone, ticks, porphyria, Gullain-Barre syndrome, Huntington's chorea and other diseases associated with axonal loss and retraction, such as stroke, human immunode
  • the methods and pharmaceutical compositions of the invention may be used to promote neuronal regeneration of the peripheral nervous system.
  • the peripheral nervous system consists of the nerves and neurons that reside or extend outside the central nervous system (the brain and spinal cord) to serve the limbs and organs. Unlike the central nervous system, however, the PNS is not protected by bone or the blood-brain barrier, leaving it exposed to toxins and mechanical injuries.
  • the peripheral nervous system is divided into the somatic nervous system and the autonomic nervous system.
  • the methods and pharmaceutical compositions of the invention may be used to promote recovery following the transplantation of a nerve graft or stem cell graft.
  • a nerve graft or stem cell graft For example, after an allograft is performed, the transected ends of the peripheral nerve or spinal cord may be filled with a non-cellular gap-filling material as known in the art, e.g. collagen, fibrin, methyl cellulose, etc., and a nerve cell growth promoting factor, such as the compositions of the invention.
  • Nerve conduits are typically tubular in shape and may be made of various materials. They have been used to guide nerve regeneration following injury in the nervous system. Most nerve regeneration conduits are restricted to guidance of axon elongation of peripheral nerves. Nerve conduits have been described; see, for example, Doolabh et al., 1996 Rev. Neurosci. 7(l):47-84.
  • the pharmaceutical compositions described herein may have the Rho antagonist (e.g. C3 protein or C3-like protein), growth factor (e.g. CNTF), and cAMP (or an analogue or modulator thereof) in the same pharmaceutically acceptable carrier or in a different pharmaceutically acceptable carrier for each described embodiment.
  • the Rho antagonist e.g. C3 protein or C3-like protein
  • growth factor e.g. CNTF
  • cAMP or an analogue or modulator thereof
  • two of these agents e.g. the Rho antagonist (e.g. C3 protein or C3-like protein) and the growth factor (e.g.
  • the Rho antagonist and cAMP may be administered simultaneously, and the third agent (e.g. the cAMP or the growth factor) may be administered before or after the first two agents.
  • the methods of the invention include administration of these three agents in various combinations and orders.
  • the methods of the invention include co-administration of the Rho antagonist (e.g. C3 protein or C3-like protein) and the growth factor (e.g. CNTF), which for example may be dissolved or intermixed in the same pharmaceutically acceptable carrier, or administration of the Rho antagonist, followed by the growth factor, or administration of the growth factor, followed by the Rho antagonist, and/or then the cAMP, and so on.
  • Concomitant as in the phrase “concomitant administration” includes administering an agent, e.g. a Rho antagonist, in the presence of a second agent, e.g. a growth factor.
  • Concomitant administration includes methods in which the first, second, third, or additional agents are co-administered. It also includes methods in which the first or additional agents are administered in the presence of a second or additional agent, wherein the second or additional agents, for example, may have been previously administered. Concomitant administration may be executed step-wise by different actors.
  • one actor may administer to a subject a first agent and a second actor may administer to the subject a second agent, and the administering steps may be executed at the same time, or nearly the same time, or at distant times, so long as the first agent (and additional agents) are after administration in the presence of the second agent (and additional agents).
  • Concomitant administration according to the methods and pharmaceutical compositions described herein may have a therapeutic additive or synergistic effect on the condition(s) or disease(s) targeted for treatment.
  • the combination of agents used within the methods or pharmaceutical compositions described herein also may reduce a detrimental effect associated with at least one of the agents when administered alone or without the other agent(s). For example, the toxicity of side effects of one agent may be attenuated by another agent of the composition, thus allowing for example a higher dosage.
  • a compound can be confirmed as a Rho antagonist in one of the following ways: a. Cells are cultured on a growth inhibitory substrate as above, and exposed to the candidate Rho antagonist; b. Cells of step a) are homogenized and a pull-down assay is performed. This assay is based on the capability of GST-Rhotektin to bind to GTP -bound Rho. Recombinant GST-Rhotektin or GST rhotektin binding domain (GST-RBD) is added to the cell homogenate made from cells cultured as in a). It has been found that inhibitory substrates activate Rho, and that this activated Rho is pulled down by GST-RBD.
  • GST-RBD GST rhotektin binding domain
  • Rho antagonists will block activation of Rho, and therefore, an effective Rho antagonist will block the detection of Rho when cells are cultured as described by a) above; or c.
  • An alternate method for this pull-down assay would be to use the GTPase activating protein, Rho-GAP as bait in the assay to pull down activated Rho, as described (Diekmann and Hall, 1995, In Methods in Enzymology, 256 part B, 207-215).
  • Rho antagonists when added to living cells antagonists that inactivate Rho by ADP-ribosylation of the effector domain can be identified by detecting a molecular weight shift in Rho (Lehmann et al, 1999 supra). The molecular weight shift can be detected after treatment of cells with Rho antagonist by homogenizing the cells, separating the proteins in the cellular homogenate by SDS polyacrylamide gel electrophoresis. The proteins are transferred to nitrocellulose paper, and then Rho is detected with Rho- specific antibodies by a Western blotting technique.
  • adult (8-10 weeks old) female Fischer 344 (F344) rats can be used.
  • animals are anesthetized with an intraperitoneal injection of 1 ml/kg body weight of an equal volume mixture of xylazine (20 mg/ml) and ketamine (100 mg/ml).
  • Rats also receive a subcutaneous injection of the analgesic buprenorphine (0.02mg/kg, Temgesic; Reckitt & Colman, Hull, UK) and intramuscular injection of Benacillin (0.1 ml, Troy Laboratories Pty. Ltd. Australia).
  • the left optical nerve is exposed intraorbitally and transected about 1.5mm behind the optical nerve head.
  • a 1.5 cm segment of peroneal nerve is dissected from the left leg of the same animal and sutured onto the proximal stump of the cut optical nerve using 10/0 suture (Ethilon; Johnson & Johnson, Australia).
  • the distal part of the peripheral nerve is placed over the skull, the free end tied with 6/0 suture and secured to connective tissue.
  • Peripheral nerve grafted animals are allocated to different experimental groups.
  • One group receives no intravitreal injections; the second and third groups receive a single saline injection at 4 days, or a double saline injection at 4 and 11 days after PN-ON (peripheral nerve-optical nerve) surgery respectively.
  • These three groups serve as controls.
  • Another 3 groups receive single, double or triple injections of SEQ ID NO: 4 at 4 days, 4 and 11 days, or 4, 11 and 18 days after PN-ON surgery respectively.
  • the remaining animals receive intravitreal injections of SEQ ID NO: 4, the cell-permeable cAMP analog 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) (0.1 mM; Sigma, St.
  • the fastest regenerating RGC axons grow in peripheral nerve grafts at a rate of about 2 mm/day after an initial delay period of 4-5 days.
  • the number of regenerating RGCs reaches a peak at 3-4 weeks after PN-ON transplantation.
  • the number of adult axotomized RGCs regrowing axons into peripheral nerve grafts 4 weeks after PN-ON surgery is counted.
  • retinas are removed from the slides, washed in phosphate buffered saline (PBS) and immunostained with an antibody to ⁇ lll-tubulin, (TUJl; BabCO, Richmond, CA, USA).
  • PBS phosphate buffered saline
  • TUJl an antibody to ⁇ lll-tubulin
  • TUJl antibody 1 :500
  • retinas are incubated with Cy3 -conjugated goat anti -mouse secondary antibody (Sigma) overnight at 4°C.
  • Cy3 -conjugated goat anti -mouse secondary antibody Sigma
  • peripheral nerve grafts are cut from the back of the operated eye and are cryoprotected in a 30% sucrose solution overnight.
  • Frozen cryostat sections (16 ⁇ m thickness) are cut longitudinally and collected on gelatin coated slides. Parallel series of sections are taken, collecting every fifth section from each peripheral nerve such that each slide contains a series of sections representing the whole nerve.
  • pan-neurofilament antibodies that recognize three types of neurofilament proteins (68kD, 16OkD and 20OkD) present in the perikarya and axons of neurons throughout the CNS and PNS.
  • Cryosections are washed in PBS and blocked for 1 hour in antibody diluent. Sections are incubated overnight at 4°C in pan-neurofilament antibodies (1 :400; Zymed, San Francisco, USA). Control IgG immunoglobulin or omission of primary antibody are used as negative controls. After washes in PBS, appropriate secondary fluorescent antibody (Jackson ImmunoResearch Labs or Sigma) is added for 1 hour at room temperature and after washes sections are coverslipped in Citifluor.
  • SEQ ID NO: 4 enhances regeneration of RGC axons in peripheral nerve grafts.
  • the present invention provides the effect of the Rho GTPase inhibitor SEQ ID NO: 4 on adult RGC survival and axonal regeneration after optical nerve transection and autologous peripheral nerve transplantation.
  • a single intravitreal SEQ ID NO: 4 injection 4 days after peripheral nerve grafting increases RGC viability for at least 4 weeks, and repeated injections also increase the amount of RGC axonal regrowth.
  • RGC survival and long-distance axonal regeneration into peripheral nerve grafts are significantly and substantially increased when SEQ ID NO: 4 is co-injected with the neurotrophic factor CNTF and the cell-permeant cAMP analogue CPT-cAMP.
  • the present invention further provides new data demonstrating interactive effects between Rho inhibition, CNTF and raised cAMP levels in mature RGCs.
  • the effects on neuronal survival and axonal regeneration are synergic suggesting at least some convergence in the signaling pathways activated by these various factors. It is known for example that cAMP-induced activation of protein kinase A phosphorylates and thus inhibits Rho signaling pathways (Dong et al, 1998, J Biol Chem, 273: 22554-22562; Lang et al, 1996, EMBO J, 15: 510-519).
  • CNTF combined with other neurotrophic factors appears to initiate a series of events that results in Rho-A inactivation in RGCs, and CNTF-induced growth of adult retinal neurites in vitro is enhanced when rho-A is inactivated (Ahmed et al, 2006, Brain, 129: 1517-1533).
  • a more independent and complementary route by which cAMP elevation overcomes myelin inhibitors such as MAG is by activation of cAMP response element binding protein (CREB) and upregulation of Arginase I and polyamines (Cai et al, 2002, Neuron, 35: 711-719; Gao et al, 2004, Neuron, 44: 609- 621).
  • CREB cAMP response element binding protein
  • Growth inhibition elicited by myelin and CSPGs may also be mediated by an NgR-and Ca 2+ -dependent mechanism that activates epidermal growth factor receptor and as yet unknown downstream pathways (Ahmed et al, 2004; Koprivica et al, 2005, Science, 310: 106-110).
  • RGCs in central retina are more vulnerable to optical nerve axotomy (Hou et al , 2004, Invest Ophthalm Vis Sci 45: 662-667; Klocker et al, 1997, Neuroreport, 8: 3439-3442), because the length of the axon within the retina and associated trophic support from adjacent fibers and glia influences the kinetics of RGC death (Isenmann et al, 2003, Prog Retin Eye Res, 22, 483-543).
  • Intraocular injections of neuroprotective factors have variously been described as enhancing greater adult RGC survival in central retina (Hou et al. , 2004, Invest Ophthalmol Vis Sci, 45: 662-667; Isenmann et al., 1998, Eur J Neurosci, 10: 2751-2756) or in peripheral retina (Klocker et al., 1997, Neuroreport, 8: 3439-3442; Klocker et al., 1998, J Neurosci, 1038-1046).
  • RGC survival ( ⁇ lll-tubulin positive) and axonal regeneration (FG labeled) are at three different retinal eccentricities (0.1-1.4 mm, 1.4-2.8 mm, or over 2.8 mm from the optical nerve head).
  • RGC density shows a similar centro-peripheral gradient to normal rats.
  • About 10% of RGCs are viable across the whole retina, but the proportion of surviving RGCs that regenerate an axon is higher in cells located close to the optic disk.
  • Overall trends are similar in the SEQ ID NO: 4 and SEQ ID NO: 4/CPT-cAMP groups although the total number of viable RGCs is higher.
  • a survival gradient is less evident in SEQ ID NO: 4/CNTF/CPT-cAMP injected rats, and in this group there is also an increase in the proportion of RGCs that regenerate an axon.
  • RGCs that regenerate an axon.
  • 86% of surviving RGCs in central retina regenerate an axon 1-1.5mm into a peripheral nerve graft, while in peripheral retina this proportion is increased to about 50%.
  • RGCs in central retina are closer to trophic factors emanating from the grafted peripheral nerve tissue, yet the observed regenerative bias towards central retina is observed even though the repeated intraocular injections would presumably affect all surviving RGCs, irrespective of their retinal location.
  • the present experiment highlights that the closer a neuron is to the site of axotomy the more likely it is that the neuron will die, but if the neuron survives it has a much greater capacity to regenerate its axon.
  • the data disclosed hereinafter demonstrate that delivery of neurotrophic factors and growth inhibitor antagonists to neurons has differential effects on survival versus axonal regeneration in vivo.
  • Rho GTPase antagonist SEQ ID NO: 4 on RGC survival and the regeneration of RGC axons into the relatively permissive regenerative environment of a peripheral nerve graft. These effects are significantly enhanced by combining Rho inactivation with provision of exogenous CNTF and intraretinal elevation of cAMP, showing the potential power of combinatorial pharmacotherapeutic and transplant strategies in the treatment of neuro trauma.
  • C3 variants include, but are not limited to, C3-11 (BA-210; SEQ ID NO: 3 and 4) which is a further derivative of C3APLT (SEQ ID NO: 5 and 6).
  • Successive groups of amino acid residues from either the N- or C-terminus, not including the transport peptide sequence, of SEQ ID NO: 4 are disclosed in Table 1.
  • Variants of SEQ ID NO: 4 include also the minimal functional size of N-terminally truncated by 212 amino acids, and that of C-terminally truncated by 224 amino acids. The expected double-deletion of amino acids from both termini would produce a functional variant of just over 204 amino acids; this variant of SEQ ID NO: 6 is also encompassed by the invention.
  • SEQ ID NO: 4 molecule can be modified by chemically coupling it to polyethylene glycol moieties to enhance the biological residence properties and/or reduce their immunogenicity.
  • Methods of PEGylation and purification of PEGylated BA-210 variants are well known in the art.
  • the present invention provides a kit for preventing or treating traumatic or degenerative nerve injury, promoting axonal regeneration, preventing or inhibiting axon loss, preventing or inhibiting RGC loss or axon loss, or promoting adult RGC survival.
  • the kit includes the combination of a Rho antagonist inhibitor, e.g. C3 or a C3-like protein; a growth factor, e.g. CNTF; and cAMP or an analogue or modulator thereof.
  • the components of the kit may be administered concomitantly, simultaneously or sequentially to a subject in need thereof.
  • the components of the kit may be provided separately for contemporaneous administration or may be formulated together for ease of administration.
  • C3-11 SEQ ID NO: 4
  • Treatment with C3-11 stimulates RGC survival and axonal regeneration into peripheral nerve grafts.
  • Rho antagonists promote RGC survival and axonal regeneration into a more permissive growth environment by injecting the cell- permeable Rho antagonist SEQ ID NO: 4 into the eye of young adult Fischer F344 rats at 4, 11, 18 days after optical nerve transection and autologous peripheral nerve transplantation.
  • the number of surviving RGCs is assessed in retinal wholemounts using ⁇ lll-tubulin immunohistochemistry.
  • RGCs with regenerating axons are retrogradely labeled with fluorogold (FG) after injection of the tracer into the distal end of each peripheral nerve graft.
  • FG fluorogold
  • Fig. IA, B and 1C, D Examples of ⁇ lll-tubulin and FG label in retinal wholemounts from saline and SEQ ID NO: 4 injected eyes are shown in Fig. IA, B and 1C, D respectively.
  • RGC densities in different treatment groups are disclosed at various eccentricities from the optical nerve head (Fig. 5). There is a gradual decrease in the density of surviving RGCs with increasing distance from the optical nerve head in saline control groups (Fig. 5A). This largely reflects similar changes in RGC density at increasing eccentricities in normal rat retina and suggests that, in peripheral nerve grafted rats, without additional intervention there is proportionate loss of about 90% of RGCs at all retinal eccentricities. In the SEQ ID NO: 4 and SEQ ID NO: 4/CPT- cAMP injection groups a decreasing gradient is also evident although less obvious than in the control group (Fig. 5B, C).
  • RGC densities are similar at all eccentricities, especially in the SEQ ID NO: 4/CNTF and C3/CNTF/CPT-cAMP groups (Fig. 5D, F), suggesting that the neuroprotective effects of Rho inhibition on axotomized RGCs are relatively greater in peripheral retina.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Hematology (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Inorganic Chemistry (AREA)
  • Psychology (AREA)
  • Ophthalmology & Optometry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne en particulier des procédés et des compositions comprenant des protéines de fusion C3 combinées au CNTF et à l'AMPc ou à des analogues de ces composés qui ont des effets synergiques sur la promotion de la survie neuronale et la régénération axonale. La multithérapie impliquant l'inactivation de Rho, l'élévation de l'AMPc et l'approvisionnement du facteur neurotrophique ciliaire (CNTF) (i) a augmenté la survie des cellules ganglionnaires rétiniennes (RGC) adultes axotomisées et (ii) a favorisé la régénération axonale dans le nerf périphérique (NP) autogreffé sur le nerf optique coupé. Les yeux greffés avec le NP se sont vus injectés des combinaisons d'une enzyme C3 transférase désactivant Rho (SEQ ID No.4), du CNTF et d'un analogue d'AMPc perméable aux cellules (CPT-AMPc). Quatre semaines après la transplantation du NP, la survie des RGC a été quantifiée en utilisant l'immunohistochimie de la β-III tubuline. La régénération a été évaluée en utilisant le traçage rétrograde par le Fluorogold et l'immunocoloration des pan-neurofilaments des greffes. Le traitement avec la SEQ ID No.4 a augmenté la survie des RGC mais une co-injection avec la CPT-AMPc, le CNTF ou la combinaison CNTF/CPT-AMPc n'a pas amélioré davantage la viabilité des RGC. De plus grands nombres de RGC régénératrices ont été obtenus après de multiple injections de la SEQ ID No.4 et la régénération a été en outre et de manière significative augmentée après des injections intravitréennes de l'ensemble des trois facteurs. Dans le groupe de traitement ayant reçu la combinaison SEQ ID No.4/CNTF/CPT-AMPc, environ 15 % des RGC sont demeurées viables parmi lesquelles plus de la moitié ont régénéré un axone.
PCT/CA2007/002241 2006-12-22 2007-12-11 Effets synergiques des c3, de l'amp cyclique et du facteur neurotrophique ciliaire sur la survie neuronale et la régénération axonale WO2008077234A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US87650906P 2006-12-22 2006-12-22
US60/876,509 2006-12-22

Publications (1)

Publication Number Publication Date
WO2008077234A1 true WO2008077234A1 (fr) 2008-07-03

Family

ID=39562048

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CA2007/002241 WO2008077234A1 (fr) 2006-12-22 2007-12-11 Effets synergiques des c3, de l'amp cyclique et du facteur neurotrophique ciliaire sur la survie neuronale et la régénération axonale

Country Status (1)

Country Link
WO (1) WO2008077234A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2236516A1 (fr) * 2009-03-31 2010-10-06 Charité-Universitätsmedizin Berlin (Charité) Polypeptides et utilisation associée pour le traitement d'une lésion neuronale traumatique ou dégénérative
WO2018222723A1 (fr) * 2017-05-30 2018-12-06 Vertex Pharmaceuticals Incorporated Protéine de fusion c3 et méthodes de fabrication et d'utilisation de celle-ci
WO2020142349A1 (fr) * 2019-01-04 2020-07-09 The Regents Of The University Of California Compositions et procédés pour favoriser l'angiogenèse dans l'œil
CN114917367A (zh) * 2021-06-02 2022-08-19 中国科学院动物研究所 Lhx2在促进中枢系统神经元的损伤修复再生中的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HU Y. ET AL.: "Interactive effects of C3, cyclic AMP and ciliary neurotrophic factor on adult retinal ganglion cell survival and axonal regeneration", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 34, 27 November 2006 (2006-11-27), pages 88 - 98, XP005751268, DOI: doi:10.1016/j.mcn.2006.10.005 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2236516A1 (fr) * 2009-03-31 2010-10-06 Charité-Universitätsmedizin Berlin (Charité) Polypeptides et utilisation associée pour le traitement d'une lésion neuronale traumatique ou dégénérative
WO2010112556A1 (fr) * 2009-03-31 2010-10-07 Charité - Universitätsmedizin Berlin Polypeptides et leur utilisation pour traiter une lésion neuronale d'origine traumatique ou dégénérative
WO2018222723A1 (fr) * 2017-05-30 2018-12-06 Vertex Pharmaceuticals Incorporated Protéine de fusion c3 et méthodes de fabrication et d'utilisation de celle-ci
US11324802B2 (en) 2017-05-30 2022-05-10 BioAxone BioSciences, Inc. C3 fusion protein and methods of making and using thereof
WO2020142349A1 (fr) * 2019-01-04 2020-07-09 The Regents Of The University Of California Compositions et procédés pour favoriser l'angiogenèse dans l'œil
CN114917367A (zh) * 2021-06-02 2022-08-19 中国科学院动物研究所 Lhx2在促进中枢系统神经元的损伤修复再生中的应用

Similar Documents

Publication Publication Date Title
US7767643B2 (en) Protection, restoration, and enhancement of erythropoietin-responsive cells, tissues and organs
EP1406922B1 (fr) Protection, restauration et amelioration de tissus, d'organes et de cellules sensibles a l'erythropoietine
Tanaka et al. Cytoplasmic p21Cip1/WAF1 enhances axonal regeneration and functional recovery after spinal cord injury in rats
Sapieha et al. Fibroblast growth factor-2 gene delivery stimulates axon growth by adult retinal ganglion cells after acute optic nerve injury
Berry et al. Regeneration of axons in the visual system
US20030072737A1 (en) Tissue protective cytokines for the protection, restoration, and enhancement of responsive cells, tissues and organs
Wang et al. Axonal regeneration induced by blockade of glial inhibitors coupled with activation of intrinsic neuronal growth pathways
EP2616094B1 (fr) Traitement des lésions provoquées par une pénétration du cerveau
US8673594B2 (en) Methods for stimulating nervous system regeneration and repair by regulating arginase I and polyamine synthesis
Hu et al. Interactive effects of C3, cyclic AMP and ciliary neurotrophic factor on adult retinal ganglion cell survival and axonal regeneration
EA010200B1 (ru) Рекомбинантные тканезащитные цитокины и кодирующие их нуклеиновые кислоты для защиты, восстановления и усиления чувствительных клеток, тканей и органов
KR20170095367A (ko) 조직 석회화의 치료 방법
BRPI0910338A2 (pt) uso de variantes peguiladas de igf-i para o tratamento de distúbios neuromusculares
WO2008077234A1 (fr) Effets synergiques des c3, de l'amp cyclique et du facteur neurotrophique ciliaire sur la survie neuronale et la régénération axonale
Tomita et al. Myelin‐associated glycoprotein reduces axonal branching and enhances functional recovery after sciatic nerve transection in rats
AU2001259453A1 (en) Methods for stimulating nervous system regeneration and repair by regulating arginase 1 and polyamine synthesis
US20210347821A1 (en) Inhibitors of pick1 and uses thereof
JP2001504123A (ja) Ｃｎｓ障害に対して保護するための成長因子を用いた予備治療
JP2020527132A (ja) ミエリン障害を治療するための組成物および方法
JP2024514204A (ja) 再発寛解型多発性硬化症を治療するためのエリスロポエチン由来ペプチド
AU2010202384A1 (en) Protection, restoration, and enhancement of erythropoietin-responsive cells, tissues and organs
Hollis II Intrinsic and extrinsic mechanisms of corticospinal axon regeneration
Iannotti Neuroregenerative and neuroprotective effects of glial cell line-derived neurotrophic factor (GDNF) in repair of spinal cord injury

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07855523

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC, EPO FORM 1205A OF 12.10.2009

122 Ep: pct application non-entry in european phase

Ref document number: 07855523

Country of ref document: EP

Kind code of ref document: A1