WO2001003692A1 - Compositions et procedes activant la regeneration nerveuse - Google Patents

Compositions et procedes activant la regeneration nerveuse Download PDF

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WO2001003692A1
WO2001003692A1 PCT/US2000/018539 US0018539W WO0103692A1 WO 2001003692 A1 WO2001003692 A1 WO 2001003692A1 US 0018539 W US0018539 W US 0018539W WO 0103692 A1 WO0103692 A1 WO 0103692A1
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group
bastadin
agent
analogs
complex
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Bruce G. Gold
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Oregon Health Sciences University
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Priority to JP2001508972A priority Critical patent/JP2003504330A/ja
Priority to AU60748/00A priority patent/AU777997B2/en
Priority to EP00947081A priority patent/EP1200078A4/fr
Priority to US10/030,904 priority patent/US6734211B1/en
Priority to CA002377918A priority patent/CA2377918A1/fr
Publication of WO2001003692A1 publication Critical patent/WO2001003692A1/fr

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    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • 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
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • 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
    • 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
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F7/00—Heating or cooling appliances for medical or therapeutic treatment of the human body
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • This invention concerns neurotrophic compounds and agents useful in the treatment of neurological injury and disease.
  • axonal regeneration often ensues, resulting in functional recovery.
  • the rate of axonal elongation (3-4 mm/day) is slow, and sometimes does not result in recovery of full neurological function. If neurological function is restored, recovery usually occurs in weeks or months, depending upon the distance between the site of injury and the target tissue. Therapies that speed regeneration over long distances would be highly beneficial to patients and would significantly reduce health care costs.
  • the immunosuppressant drug FK506 (USAN tacrolimus; Prograf ® ) induces immunosuppression by binding the immunophilin FKBP-12. This binding prevents calcineurin from dephosphorylating the transcription factor NF/AT (nuclear factor of activated T-cells), which blocks translocation of calcineurin into the nucleus, and prevents a receptor-mediated increase in the synthesis and secretion of cytokines, such as interleukin-2 (IL-2), which are required for T-cell proliferation.
  • IL-2 interleukin-2
  • U.S. Patent No. 5,654,332 discusses immunosuppressive FK506 analogs that bind FKBP-12, and are said to stimulate neurite outgrowth in the presence of NGF.
  • the neurotrophic activity of these FKBP-12 binding compounds was said to be "directly related to their affinity for FKBP-12 and their ability to inhibit FKBP-12 rotamase activity" (id. at col. 7, lines 47-50).
  • Rotamase activity measures peptidylisomerase cis-trans isomerization, and inhibition of this activity has been accepted as an indication of the immunosuppresant and neurotrophic activity of therapeutic agents. See U.S. Patent No. 5,614,547 (Hamilton et al.).
  • the present invention takes advantage of the surprising discovery that nerve growth stimulation is promoted by disruption of the mature steroid receptor complex, and not by interaction with FKBP-12, as was previously thought.
  • Disruption of the complex can include inhibition of physical assembly, promotion of disassembly, or functional interference with the steroid receptor complex, for example the mature steroid receptor complex, or a less mature form of the complex that is a predecessor to the mature complex.
  • MEK MAP kinase/kinase
  • assays have been developed for selecting new compounds that may have activity in promoting nerve growth.
  • Such assays may include determining if a test compound, other than a steroid ligand such as an androgen or an estrogen, disrupts assembly of the steroid receptor complex, and selecting a compound that disrupts assembly of the steroid receptor complex.
  • the assay may include determining the ability of test compounds to stimulate MEK activity, and selecting compounds on this basis.
  • Examples of specific classes of compounds that can be screened include geldanamycin and its structural analogs, rapamycin and its structural analogs, and FK506 and its structural analogs, radicicol and its analogs and bastadins and their analogs.
  • Compounds selected by this assay for further investigation may be tested in additional assays to measure actual neurite outgrowth induced by the compound. In this way neurotrophic compounds have been identified by the assay for disruption of the steroid receptor complex or stimulation of MEK.
  • Methods have been designed for stimulating nerve cell growth in a subject by administering to the subject a compound (including a compound discovered by the assay) that disrupts assembly or function of the steroid receptor complex, for example of the mature steroid receptor complex, (for example by inhibiting association or promoting dissociation), or stimulates MEK activity, wherein the compound is other than a ligand for the steroid hormone bmding portion of the steroid receptor complex (such as an androgen or an estrogen), and in some specific embodiments does not bind with high affinity to FKBP-12
  • a therapeutic amount of heat may be administered m combination with nerve growth stimulating compounds
  • the compound is administered to disrupt association of a p23 component of the steroid receptor complex with an hsp-90 component or disrupt association of FKBP-52 with hsp-90
  • the compound is administered to competitively bind with ATP at an ammo terminal ATP binding site of hsp-90, for example at a geldanamycin bindm
  • the method is useful in the treatment of animals (including mammals such as humans) having a neurological condition associated with neuronal dysfunction caused by disease or injury to neurons in either the central or peripheral nervous systems
  • Compounds or compositions are administered, with or without heat, to the animal in a therapeutically effective neurotrophic amount to bind to the mature steroid receptor complex (for example at a geldanamycin binding site of hsp-90) to disrupt association of the mature steroid receptor complex or stimulate MEK activity, and promote neurite outgrowth from neurons
  • the method can also be used in association with procedures such as a surgical nerve graft, or other implantation of neurological tissue, to promote healing of the graft or implant, and promote incorporation of the graft or implant into adjacent tissue
  • Certain pharmaceutical compounds that are not a ligand for the steroid hormone binding portion of the steroid receptor complex can disrupt assembly of a steroid receptor complex
  • These compounds can be geldanamycin and its structural analogs, rapamycin and its structural analogs, FK506 and its structural analogs, radicicol or a structural analog or mimetic thereof, or a bastadin or a structural analog or mimetic thereof, but more particular embodiments of the compound may have low rotamase inhibition activity, may be other than an FK506 or rapamycin analog, may not bmd with high affinity to FKBP-12, or are not immunosuppressive
  • the compound specifically disrupts formation of the steroid receptor complex (for example the mature steroid receptor complex) either by inhibiting association or promoting dissociation of the steroid receptor complex, for example by disrupting association of a p23 component of the steroid receptor complex with an hsp-90 component, or disrupting association of FKBP-52 with hsp
  • the compound competitively bind with ATP at an amino terminal ATP binding site of hsp-90, which is also the binding site for geldanamycin binding to the steroid receptor complex.
  • the compound is radicicol or a radicicol analog that binds to a geldanamycin binding site of hsp-90.
  • the compound is an anti- FKBP-52 antibody, or another agent that specifically causes FKBP-52 to dissociate from hsp-90 of the steroid receptor complex.
  • the compound can be incorporated into a pharmaceutical composition, which can also include another neurotrophic factor, such as NGF, IGF-1, ⁇ FGF, ⁇ FGF, PDGF, BDNF,
  • another neurotrophic factor such as NGF, IGF-1, ⁇ FGF, ⁇ FGF, PDGF, BDNF,
  • CNTF GDNF, NT-3, NT 4/5, and mixtures thereof, or a steroid hormone that is a ligand of the steroid receptor complex (such as an estrogen, an androgen or a corticosteroid such as dexamethasone).
  • a steroid hormone that is a ligand of the steroid receptor complex such as an estrogen, an androgen or a corticosteroid such as dexamethasone
  • the compound is a nerve growth stimulating amount of an agent that binds to a polypeptide of a steroid receptor complex other than a steroid hormone binding portion of the complex, the agent being selected from the group consisting of an FK506 analog having low binding affinity for FKBP-12 and low rotamase activity, for example a benzoquinone ansamycin and structural analogs thereof, a peptide comprising a sequence of a selected polypeptide component of the complex at a site of interaction between the selected component and another polypeptide component of the complex, an antibody, and combinations thereof, wherein the agent disrupts assembly or interferes with function of the steroid receptor complex by causing p23 or FKBP-52 dissociation from the complex, or inhibiting p23 or FKBP-52 association with the complex, or inhibiting interaction of p23, FKBP-52 or hsp-90 with the complex.
  • an FK506 analog having low binding affinity for FKBP-12 and low rotamase activity
  • Compounds of the present invention need not have significant calcineurin inhibition or rotamase inhibition.
  • the compounds may have an IC 50 for rotamase inhibtion of greater than 1 nM, for example greater than 10 nM, 25 nM, or even 50-100 nM.
  • Nerve cell growth can be stimulated in a subject by administering to the subject a compound that stimulates nerve cell growth, wherein the compound is one or more of radicicol or its analogs; a bastadin or its analogs; or an agent that stimulates MAP kinase/kinase activity.
  • the compound is a radicicol analog, or a bastadin such as bastadin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, particularly bastadin 10, or an analog of a bastadin.
  • the method also includes administering radicicol or its analogs, or the bastadin or its analogs, in combination with a neurotrophic factor other than the compound that disrupts association of the mature steroid receptor complex or stimulates MAP kinase/kinase activity.
  • the neurotrophic factor may be, for example, NGF, IGF-1, ⁇ -FGF, ⁇ -FGF, PDGF, BDNF, CNTF, GDNF, NT-3, NT 4/5, and mixtures thereof.
  • Another aspect is screening for agents that stimulate nerve cell growth, by detecting agents that stimulate MAP kinase/kinase activity, such as radicicol analogs, or platelet derived growth factor BB (PDGFBB) or analogs thereof.
  • agents that stimulate MAP kinase/kinase activity such as radicicol analogs, or platelet derived growth factor BB (PDGFBB) or analogs thereof.
  • PDGFBB platelet derived growth factor BB
  • the method also includes applying a sufficient amount of heat to an area where nerve cell growth is desired, for example along a normal anatomic pathway, or in an anatomic region, of a transected, partially transected or otherwise damaged nerve.
  • the body temperature of a subject can be systemically elevated, for example by inducing a fever or placing the body in a heated environment.
  • the invention can also include providing a template in an area where nerve growth is desired, for example a tubular member that defines an anatomical pathway along which nerve growth is desired. If desired, a therapeutically effective amount of the neurotrophic compound may be provided in association with the template to promote nerve growth.
  • the template may be placed between opposing ends of a transected or partially transected nerve. Heat can be applied to the template in a therapeutically sufficient amount, effective to enhance nerve growth.
  • the template along the desired anatomical path can be impregnated with the neurotrophic compound, or the impregnated template can be heated.
  • the radicicol compound or its analogs are of the formula:
  • the neurotrophic compound is a complete bastadin or bastadin analog, such as,
  • each R is independently selected from the group consisting of H, Cl-8 alkyl, or sulfato
  • W is selected from the group consisting of H, OH, or Cl-8 alkoxy
  • X, Y, and Z are independently selected from the group consisting of hydrogen, halogen, hydroxyl, or Cl-8 alkoxy
  • a and B are carbon atoms that are joined by a single or a double bond.
  • the compound is a bastadin subunit such as
  • A, B, R, W, and X are defined as above for the complete bastadins.
  • a specifice example of a bastadin subunit is shown in Example 15.
  • FIG. 1 is a schematic diagram illustrating receptor-hsp-90 heterocomplex assembly of the steroid receptor complex.
  • FIG. 2 is a schematic diagram illustrating the mature steroid receptor complex, and the binding sites of some of the agents of the present invention that promote nerve growth.
  • FIG. 3 is a cumulative histogram showing neurite outgrowth lengths after72 hours for untreated hippocamapal cells and for hippocampal cells treated with either NGF or radicicol at the concentrations indicated.
  • FIG. 4 is a cumulative histogram showing neurite outgrowth lengths after 72 hours for untreated hippocampal cells and for hippocampal cells treated with heat, NGF, or a combination of heat and NGF.
  • FIG. 5 is a cumulative histogram showing neurite outgrowth lengths after 72 hours for untreated hippocampal cells and for hippocampal cells treated with NGF, a combination of heat and NGF, a combination of heat and Hsp-90 antibodies (Ab), and a combination of heat, NGF, and Hsp-90 antibodies.
  • FIG. 3 is a cumulative histogram showing neurite outgrowth lengths after72 hours for untreated hippocamapal cells and for hippocampal cells treated with either NGF or radicicol at the concentrations indicated.
  • FIG. 4 is a cumulative his
  • FIG. 6 is a histogram showing the mean neurite length after 72 hours for untreated hippocampal cells and for hippocampal cells treated with NGF, FK506, combinations of NGF and the MAP kinase/kinase inhibitor PD 098059 (2 concentrations), and combinations of FK506 and PD 098059 (2 concentrations).
  • FIG. 7 is a histogram showing the mean neurite length after 72 hours for untreated hippocampal cells and for hippocampal cells treated with NGF and various combinations of radicicol (2 concentrations) and PD 098059 (2 concentrations).
  • FIG. 8 is a cumulative histogram showing neurite outgrowth lengths after 72 hours for hippocampal cells treated with NGF, radicicol and two combinations of radicicol and the MAP Kinase/Kinase inhibitor PD 098059.
  • FIGS. 9A, 9B,and 9C are light micrographs showing hippocampal cells after 72 hours for untreated cells, cells treated with radicicol, and cells treated with a bastadin 10 analog, respectively.
  • FIG. 10 is a cumulative histogram of neurite outgrowth lengths for hippocamapal cells after 72 hours when untreated and treated with either radicicol or a bastadin 10 analog.
  • Steroid receptors are part of a superfamily of molecules that regulate gene expression by direct interaction with the upstream region of specific structural genes. It is essential to hormone action that a receptor must be able to assume both an active and an inactive state. This regulation is accomplished by association of the receptor (the steroid ligand binding component) with a multimeric complex of chaperone proteins, such as heat shock proteins (hsp-90), p23 and FKBP-52, which form the steroid receptor complex (SRC). When the steroid receptor binds its ligand, the receptor is activated, the chaperone proteins of the SRC are dissociated, and a DNA binding domain of the receptor is exposed for interaction with gene regulatory sequences.
  • chaperone proteins such as heat shock proteins (hsp-90), p23 and FKBP-52
  • steroid receptor family that are regulated in this fashion include mineralocorticoids (such as aldosterone), glucocorticoids (such as dexamethasone), progestins (such as progesterone), androgens (such as testosterone), and estrogens (including estrogen, ⁇ -estriol and ⁇ -estradiol).
  • mineralocorticoids such as aldosterone
  • glucocorticoids such as dexamethasone
  • progestins such as progesterone
  • androgens such as testosterone
  • estrogens including estrogen, ⁇ -estriol and ⁇ -estradiol
  • TPR domain proteins such as the immunophilins (e.g. FKBP-52 and CyP40)
  • FKBP-52 and CyP40 the nearly mature, metastable SRC is formed (SR/hsp-90/p23/FKBP-52).
  • the TPR domain is indicated by the solid black crescent to which FKBP-52 is bound.
  • Assembly of the mature steroid receptor complex is the last step shown in FIG. 1, in which the complex of chaperone proteins hsp-90/p23/FKBP-52 is assembled with the steroid receptor SR.
  • the immunophilins are a highly conserved family of chaperone proteins that are known to be mediators of immunosuppressant drug activity.
  • the best characterized immunophilin is FKBP-12, which interacts with the immunosuppressant drug FK-506 in T lymphocytes, to prevent calcineurin from dephosphorylating the nuclear factor of activated T-cells (NF/AT), thereby blocking synthesis and secretion of cytokines required for immune function.
  • Immunophilins have peptidylisomerase (PPIase) activity, and inhibitors of this activity can be detected with a rotamase assay which measures inhibition of cis-trans isomerization of the peptidylprolyl.
  • PPIase peptidylisomerase
  • FKBP-12 immunosupression is not mediated by an ability to inhibit rotamase activity.
  • Rotamase activity has nonetheless been accepted as an indication of immunosuppressant activity of immunophilins, even though it does not measure the dephosphorylation of calcineurin activity by which immunosuppression is actually mediated.
  • Previous researchers had taken advantage of the rotamase assay to look for FKBP-12 binding drugs that would promote nerve regeneration (as FK506 had been found to do).
  • the present invention takes advantage of the surprising finding that disrupting assembly of the SRC, and not binding to FKBP-12, is what promotes nerve regeneration. Hence previous efforts to find FKBP-12 analogs that promote nerve regeneration by measuring rotamase or immunosuppressive activity was misdirected, and was likely to find drugs that had unwanted side effects (such as immunosuppression and cardiomyopathy).
  • the present invention has taken advantage of the discovery of the actual biological mechanism by which nerve regeneration is promoted to provide a superior assay for finding new neurotrophic drugs that are superior to those in the prior art.
  • geldanamycin a benzoquinone ansamycin antibiotic, which binds in a pharmacologically specific manner to hsp-90 (Whitesell et al., Proc. Nat I. Acad. Sci. USA 91:8324-8328, 1994) and inhibits association of the ⁇ 23 component of the heterocomplex assembly system with hsp-90 (Johnson and Toft, Mol. Endocrinol. 9:670-678, 1995).
  • Geldanamycin and radicicol thereby promote dissociation of a steroid receptor complex, and block reassembly of the hormone-responsive form of the complex, preventing hormone activation and ultimately resulting in the degradation of the hormone receptor.
  • Geldanamycin blocks assembly of the progesterone receptor (PR) complex (Smith et al., Mol. Cell. Biol. 15:6804-6812, 1995) and of the glucocorticoid receptor (GR) complex (Czar et al., Biochem. 36:7776-7785, 1997) at an intermediate stage of assembly where the hormone binding domain is not properly folded and therefore cannot bind steroid with high affinity (for example, does not bind steroid ligand that is present in concentrations of less than about 10 nM).
  • PR progesterone receptor
  • GR glucocorticoid receptor
  • Another class of neurotrophic agents that have been found in accordance with the invention are those that act at the FKBP-52 component of the SRC. It has surprisingly been found that the action of the neurotrophic immunosuppressant FK506 is via an interaction with FKBP-52, which induces a conformational change in hsp-90, enabling dissociation of p23 from hsp-90, thereby interfering with assembly of the mature SRC.
  • the present invention also includes bastadins that may disrupt the SRC by binding to FKBP-52. Mack et al. have shown that bastadin 5 stimulates [ 3 H]ryanodine binding to ryanodine receptors, but that such stimulation is antagonized by FK506.
  • FIG. 2 of the present application shows how FK506 binds to FKBP-52. By competing with bastadin 5 for this binding site or by inducing changes in the conformation of FKBP-52, FK506 may block the action of bastadin 5.
  • bastadin 10 stimulates neurite outgrowth.
  • An anti-FKBP-52 antibody also inhibits assembly of the mature SRC. Binding of FK506 to GR-associated FKBP-52 causes increased nuclear translocation of GR in response to dexamethasone and potentiation of GR-mediated gene expression (Sanchez and Ning, METHODS: A Companion to Meth. Enzymol. 9:188-200, 1996).]
  • CyP40 is an example of a protein targeted by cyclosporin A (CsA) and its analogs. These immunophilins bind hsp-90 in a mutually exclusive fashion, leading to the formation of separate CyP40-hsp-90 and FKBP-52-hsp-90 complexes (Ratajczak and Carrello, /. Biol. Chem. 271:2961-2965, 1996).
  • CsA cyclosporin A
  • Immunophilins such as FKBP-52, CyP40 and PP5 and non-immunophilin proteins such as p60 and Mas70p, have one or more tetratricopeptide repeat (TPR) domains (Ratajczak et al., J. Biol. Chem. 268:13187-13192, 1993) that bind to the TPR- binding domain of hsp-90.
  • TPR tetratricopeptide repeat
  • An increased number of TPR domains in a protein appears to correlate with increased hsp-90-binding affinity.
  • peptides having one or more TPR domains would be expected to have increased hsp-90 binding affinity, and would interfere with FKBP-52 association with hsp-90, which is required for assembly of the mature steroid receptor complex.
  • binding of both FKBP-52 and CyP40 to hsp-90 is competitively inhibited by a purified fragment of human CyP40 comprising its three TPR domains, and by a fragment of rat PP5 comprising its four TPR domains (reviewed in Pratt and Toft, Endocrine Rev. 18:306-360, 1997).
  • Such purified fragments, or other peptides such as PP5, p60 and Mas70, containing one or more TPR domains (particularly two, three or more TPR domains) are therefore suitable for interfering with assembly or function of the steroid receptor complex, and are included within the scope of this invention.
  • radicicol, radicicol analogs, bastadins, bastadin analogs, geldanamycin and the other neurotrophic agents that do not bind directly to the steroid binding domain of the steroid receptor complex are believed to result from binding of these compounds to components of steroid receptor complexes, causing the dissociation of hsp-90 from the steroid receptor complex either directly (by binding to hsp-90 or interfering with the binding of hsp-90 to the steroid receptor) or indirectly y binding to a polypeptide such as FKBP-52 that itself binds to hsp-90, or a polypeptide that binds to p23), or alternatively by preventing association of hsp-90 or p23 with the steroid receptor complex.
  • a polypeptide such as FKBP-52 that itself binds to hsp-90, or a polypeptide that binds to p23
  • Interference with the ability of hsp-90 to complex with and perform its chaperone function for other hsp-90 substrate proteins is believed to also be responsible for or contribute to the observed stimulation of nerve regeneration by FK506 and/or geldanamycin.
  • Any agent that interferes with the function of the mature steroid receptor complex (including interference with a precursor of the mature complex, such as the nearly mature complex or foldosome) is also included in the scope of this invention.
  • Also included in the scope of this invention are compounds that stimulate MAP kinase/kinase.
  • neurotrophic compounds of this invention can be substantially free of calcineurin inhibition, and can have low rotamase inhibition, for example an IC 50 of greater than about 1 nM, or even 5 or 10 nM.
  • MEK MAP kinase/kinase
  • AR Androgen Receptor ER: Estrogen receptor GR: Glucocorticoid receptor PR: Progesterone receptor SRC: Steroid receptor complex.
  • a multiprotein complex associated with any steroid receptor including, but not limited to, the progesterone receptor, glucocorticoid receptor, estrogen receptor, androgen receptor, and mineralocorticoid receptor.
  • TPR Tetratricopeptide repeats are Domain III of FKBP-52; TPRs were first identified by Sikorski et al., Cell 60:307-317, 1990, as degenerate consensus sequences of 34 amino acids.
  • Mimetic A biological compound (such as a peptide) that mimics the effect of a pharmaceutical, for example a peptide that mimics the effect of a benzoquinone ansamycin by binding to a geldanamycin binding site on hsp-90.
  • Ligand for the steroid hormone bmding portion of the steroid receptor complex An already recognized ligand for the receptor subtype. For example, dexamethasone for the GR, estrogen for the ER, testosterone for the AR, progesterone for the PR.
  • Immunophilins A highly conserved family of chaperone proteins that have PPIase activity, producing cis-trans isomerization.
  • the immunophilins are divided into low molecular weight (less than 40kD) and high molecular weight (40-65 kD) immunophilins.
  • the high molecular weight immunophilins e.g., FKBP-52
  • FKBP-12 in contrast to FKBP-12, contain three or more tetratricopeptide repeats (TPRs) which mediate binding to hsp-90.
  • the immunophilins may be subdivided into two classes on the basis of their abilty to bind either cyclosporin A (cyclophilins) or FK506 and rapamycin (the FK binding proteins, FKBPs).
  • FKBP family of immunophilins include FKBP-12, FKBP-13, FKBP-25, FKBP-52 (also referred to as FKBP-59), and FKBP-65.
  • PP5 is also considered a member of this family, because it binds FK506 weakly, as reported by Silverstein et al., /. Biol. Chem. 272:16224-16230, 1997.
  • NGPA Nerve growth promoting agent.
  • a "nerve growth promoting agent” or NGPA is defined as a substance that binds to a polypeptide component of a steroid receptor complex, such components including but not limited to hsp-90 and FKBP-52, and promotes nerve regeneration, without limitation to a particular mechanism of action.
  • the NGPA does not bind FKBP-12 (or binds it with low affinity, with a Kd of greater than 1 ⁇ M), has low rotamase inhibitory activity (an apparent Ki of more than 2500 nM), binds with low affinity to calcineurin (requires concentrations greater than 30 ⁇ M to bind), or is non- immunosuppressive, as measured by the substantial absence of a drop in total blood lymphocyte counts in subjects to whom the agent is administered.
  • NGPAs include, but are not limited to, non- FKBP12-binding ("non-binding") or low affinity FKBP-12 binding analogs of FK506; benzoquinone ansamycins, including geldanamycin, naturally occurring analogs of geldanamycin, including, but not limited to, herbimycin A and macbecin (DeBoer et al., J. Antibiot. (Tokyo) 23:442-447, 1970; Omura et al., J. Antibiot. (Tokyo) 32:255-261, 1979; Ono et al., Gann.
  • geldampicin a particular polypeptide component of a steroid receptor complex at a site of interaction between that component and another component of the complex (such as the TPR domain), which interferes or competitvely binds with the component of the SRC
  • peptides including an amino acid sequence of a particular polypeptide component of a steroid receptor complex at a site of interaction between that component and another component of the complex (such as the TPR domain), which interferes or competitvely binds with the component of the SRC
  • antibodies that bind specifically to polypeptide components of steroid receptor complexes e.g., anti-hsp-90, anti-FKBP-52, etc.
  • the neurotrophic agents include compounds that either physically disrupt association of the mature SRC (either by inhibiting association or promoting dissociation of the SRC), or inhibit interaction of components (such as p23, FKBP-52 or hsp-90) of the SRC. Further, the neurotrophic agents include compounds which stimulate MAP kinase/kinase (MEK) activity. Heat applied for physiologically tolerable periods to boost a mammal's body temperature to a physiologically tolerable temperature in combination with administering compounds which stimulate nerve growth is yet another neurothophic agent. Neurotrophic: Promoting nerve growth.
  • Transformation conversion of the 9S non-DNA-binding form of a steroid receptor complex to the 4S DNA-binding form.
  • activation refers to the conversion of a steroid receptor from a form that does not bind steroid ligand to a steroid-ligand-binding form.
  • Rotamase activity can be determined, for example, as in WO 92/04370, and can be expressed as a Ki.
  • the cis-trans isomerization of the alanine- proline peptide bond in a model substrate, N-succinyl-Ala-Ala-Pro-Phe-4-nitroanalide may be monitored spectrophotemetrically in a coupled assay with chymotrypsin, which releases 4- nitroanalide from the trans form of the substrate.
  • the inhibitory effect upon the addition of different concentrations of inhibitor on the extent of the reaction is determined, and analysis of the change in the first order rate constant as a function of inhibitor concentration yields an estimate of the apparent K,.
  • rotamase activity has been found to be unrelated to the neurotrophic activity of the compounds, and need not be determined.
  • "Known" or "recognized” compounds such as FKBP-12 binding compounds or
  • FK506 analogs are those that have previously been reported in patents or publications, or that otherwise qualify as prior art.
  • Assays for Identifying Nerve Growth Promoting Agents There are a number of well-known methods for assaying compounds that bind to hsp-90, FKBP-52, and other polypeptide components of a steroid receptor complex that can be used as an initial screen for candidate compounds that stimulate nerve regeneration. Compounds can subsequently be tested in vitro or in vivo for activity in stimulating nerve regeneration.
  • Examples include assays for the binding of a test compound to a polypeptide that is a component of a steroid receptor complex.
  • An assay for detecting bmding to hsp-90 is described, for example, by Whitesell et al. (Proc. Natl. Acad. Sci. USA 91:8324-8328, 1994).
  • hsp-90 StressGen Biotechnologies, Victoria, BC
  • TNESV buffer 50 mM Tris-HCl, pH 7.4/1 % Nonidet P-40/2 mM EDTA/100 mM NaCl/1 mM orthovanadate/1 mM phenylmethylsulfonyl fluoride/20 ⁇ g leupeptin per mL/20 ⁇ g of aprotinin per ml
  • the test compound are incubated for 45 min at 4°C with geldanamycin immobilized on a conventional solid support, e.g. , geldanamycin-coupled agarose beads (Whitesell et al., Proc. Natl.
  • the beads are then washed with TNESV buffer and bound hsp-90 is eluted by heating in reducing loading buffer, and can be analyzed by SDS/PAGE and silver staining (Bio-Rad).
  • the assay can be performed for the bound label instead of the free label. Test compounds that compete with geldanamycin for binding to hsp-90 inhibit the binding of solubilized hsp-90 to the beads.
  • Binding to FKBP-52 can be assayed using recombinant FKBP-52 (Peattie et al., Proc. Natl. Acad. Sci. USA 89: 10974-10978, 1992). Binding to ⁇ 23 can be assayed using recombinant human p23 (Johnson et al. , Mol. Cell. Biol. 14: 1956-1963, 1994) and immobilized hsp-90. Purified hsp70 and recombinant ⁇ 60 (Dittmar et al. , /. Biol. Chem. 271: 12833-12839, 1996) are also available for use in such binding assays.
  • Immunoassays can also be performed using conventional immunoassay methodologies and antibodies that are specific for steroid receptor complex components, e.g., antibodies against FKBP-52 (Tai et al., Biochem. 25:5269-5275, 1986), hsp-90 (Sanchez et al., J. Biol. Chem. 260: 12398-12401, 1985; Catelli et al., EMBO J. 4:3131-3135, 1985; Schuh et al., J. Biol. Chem.
  • hsp70 a serum that also recognizes hsp-90
  • p23 Johnson et al. , Mol. Cell. Biol. 14: 1956-1963, 1994.
  • a well-accepted qualitative assay for receptor transformation which involves dissociation of hsp-90 from the receptor complex, is conversion of a receptor complex to a state that binds polyanions such as phosphocellulose (Kalimi et al., /. Biol. Chem. 250: 1080-1086, 1975; Atger and Milgrom, Biochem.
  • Example 2 An in vitro assay for nerve cell growth (neurite outgrowth) is provided in Example 2, and in Gold et al., Exp. Neurol. 147:269-278, 1997.
  • In vivo assays for nerve regeneration are discussed in, for example, Gold et al., Restor. Neurol. Neurosci. 6:287-296, 1994; Gold et al., J. Neurosci. 15:7505-7516, 1995; Wang et al. , /. Pharmacol. Exp. Therapeutics 282:1084-1093, 1997; Gold et al., Exp. Neurol. 147:269-278, 1997 and Gold et al., Soc. Neurosci. Abst.
  • Nerve regeneration is also assessed by sampling tissues from the sciatic nerve at known (0.5 cm) distances from the crush site and counting the number of myelinated fibers by light microscopy. The size of axons is calculated by electron microscopy. Axonal areas of both myelinated and unmyelinated fibers are determined by tracing the axolemma using a digitizing tablet connected to a computer with appropriate software. Cumulative histograms are constructed from these data and mean values and standard errors are calculated to assess the effect of administration of the test compound on axonal areas.
  • This Example illustrates that FK506 and geldanamycin promote nerve regeneration by a common mechanism.
  • SH-SY5Y human neuroblastoma cells were maintained in DMEM medium (GIBCO) supplemented with 10% fetal calf serum (SIGMA), 50 IU/mL penicillin, and 50 mg/mL streptomycin (GIBCO) at 37°C in 7% C0 2 .
  • Cells were plated in six- well plates at 1 x 10 6 cells/well and treated with 0.4 mM aphidicolin (SIGMA).
  • Geldanamycin and FK506 each stimulate neurite outgrowth in a concentration dependent manner.
  • the similar neurotrophic effects of geldanamycin and FK506, their additive effects at very low concentrations (e.g. 0.1 nM; data not shown), and their inhibitory effects at high concentrations (like high concentrations of either compound alone) demonstrate that the two compounds act on nerve cells to promote nerve outgrowth by a common mechanism.
  • that mechanism has now been found to involve an interaction of both compounds with components of the steroid receptor complex.
  • FKBP12 does not appear to have a role in the stimulation of neurite outgrowth by either geldanamycin or FK506.
  • Cell lines other than the SH-SY5Y human neuroblastoma cells can be used in the nerve growth assays.
  • suitable other cell lines include PC-12 (rat pheochromocytoma), LA-N-5 cells (human neuroblastoma cells less differentiated than SY5Y cells), and Neuro-2a and NS20Y cells (mouse neuroblastoma).
  • FKBP-12 knockout mice (Shou, et al., Nature 391:489, 1998) were used to test whether FKBP-12 is necessary for FK506's ability to increase nerve elongation. Such mice usually die from severe cardiomyopathy between embryonic day 14.5 (E14.5) and birth, consistent with the known association between FKBP-12 and calcium release channels. No gross pathology has been noted in brains of these mice. Primary neuronal hippocampal cultures were prepared from El 8.5 homozygote FKBP-12 knockout and wild-type mice. No difference was found in FK506's regenerative-promoting response of neurons in FKBP-12 knockout and wild-type mice.
  • neuronal cells from FKBP-12 knockout mice retain their responsiveness to the neurite outgrowth-promoting property of FK506.
  • FKBP-12 is therefore not required for FK506 to promote neurite outgrowth in vitro.
  • FKBP-52 Blocks FK506 Neurotrophic Activity Neuroblastoma
  • SH-SY5Y cells do not extend processes in the absence of exogenous nerve growth factor (NGF), with optimal neurotrophic activity at 10 ng/ml NGF.
  • NGF nerve growth factor
  • FKBP-52 The involvement of FKBP-52 was demonstrated using a mouse monoclonal antibody, FKBP-52 Ab (StressGen Biotechnologies Corp. , British Columbia, Canada) that does not interact with FKBP-12.
  • FKBP-52 Ab StressGen Biotechnologies Corp. , British Columbia, Canada
  • SH-SY5Y cells were permeabilized with saponin (30 ⁇ g/ ⁇ l) for 10 min in the presence of the antibody; preliminary experiments showed that saponin treatment did not alter the response of the cells to NGF alone.
  • ⁇ -Estradiol 50 nM
  • ⁇ -Estradiol 50 nM
  • dexamethasone 50 nM
  • This is supported by the data also showing that the combination of ⁇ -estradiol and FK506 did not produce a further significant [Mann- Whitney U test ( ⁇ 0.05)] increase in neurite outgrowth, indicating that these compounds act at the same steroid receptor sub-type.
  • this conformational change may be blocked, thereby preventing release of p23, when FKBP-52 is bound to FK506, because FK506 does not dissociate FKBP-52 from the complex; a similar interaction may occur in the presence of steroid hormones to prevent the conformational change in hsp-90.
  • This model indicates that the FKBP-52 antibody dissociates FKBP-52 from the complex, perhaps by altering its degree of phosphorylation and thereby reducing its binding to hsp-90, and leads to a conformational change in hsp-90 that results in release of p23.
  • the combination of geldanamycin and the FKBP-52 antibody are additive (not inhibitory) because dissociation of FKBP-52 from hsp-90 would not prevent the geldanamycin- induced conformational change that releases p23.
  • This Example shows that prevention of the dissociation of the steroid receptor complex inhibits neurite outgrowth, as predicted by the model shown in FIG. 2.
  • SH-SY5Y cells were treated with sodium molybdate, a transition metal oxyanion, that at a concentration of 20 mM prevents dissociation of the complex in intact cells.
  • inhibition of neurotrophic activity by adding molybdate to an assay can constitute a test for determining whether a neurotrophic agent is structurally or functionally disrupting the SRC.
  • Some embodiments of the present invention have low or absent inhibition of peptidyl-prolyl isomerase (rotamase) activity. Inhibition of this activity can be evaluated by techniques known in the art, such as that described in U.S. Patent No. 5,614,547. Inhibition is expressed as an apparent Ki for cis-trans isomerization of an alanine-proline bond in a model substrate, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, which is monitored spectrophometrically in a chymotrypsin-coupled assay, which releases para-nitroanilide from the trans form of the substrate. The inhibition of this reaction caused by the addition of different concentrations of inhibitor is determined, and the data is analyzed as a change in the first-order rate constant as a function of inhibitor concentration to yield the apparent Ki values.
  • a plastic cuvette In a plastic cuvette are added 950 ml of ice cold assay buffer (25 mM HEPES, pH 7.8, lOOnM NaCl), 10 ml of FKBP (2.5 mM in 10 mM Tris-Cl pH j7.5, 100 mM NaCl, 1 mM dithiothreitol), 25 ml of chymotrypsin (50 mg/ml in 1 mM HC1) and 10 ml of test compound at various concentrations in dimethyl sulfoxide.
  • the reaction is initiated by the addition of 5 ml of substrate (succinyl-Ala-Phe-Pro-Phe-para-nitroanilide, 5 mg/ml in 2.35 mM LiCl in trifluroethanol).
  • Detection of disruption of the SRC can be assessed by additional assays.
  • Disruption of the SRC by release of p23 can be assessed by the techniques disclosed in Whitesell and Cook, 1996, where benzoquinone ansamycin binding to hsp-90 was shown to result in complete loss of p23 protein from glucocorticoid receptor immunoprecipitates, which was associated with a rapid, noncompetitive loss of dexamethasone binding activity, and a slower (2-8 hours) marked decline in the cellular level of glucocorticoid receptor protein.
  • drug treatment did not disrupt coprecipitation of hsp-90 with glucocorticoid receptor, and a complete loss of detectable p23 from glucocorticoid receptor precipitates.
  • Immnoprecipitation from cell lysates is performed using a specific monoclonal antibody BuGR-2, and protein G Sepharose beads (Pharmacia).
  • BuGR-2 protein G Sepharose beads
  • cells are lysed in detergent-free hypotonic buffer with 10 mM sodium molbydate.
  • Immunoblot detection of proteins in total cells lysates, geldanamycin affinity precipitates, and glucocorticoid receptor immunoprecipitates are performed after SDS-PAGE and electrophoretic transfer of proteins to nitrocellulose.
  • BuGR-2 hybridoma supernatant (1 :40) is used for detection of rodent derived GR while a peptide-derived rabbit polyclonal antibody (1:250; PA1-512, Affinity Bioreagents; Golden, CO) is used for the human GR.
  • Hsp-90 and hsp-70 are detected with antibodies AC88 and N27F3- 4 respectively (1:5000; StressGen; Victoria, BC, Canada).
  • Ascites containing antibody JJ3 (1:1000) is used to blot for p23.
  • Polyclonal rabbit anti-ubiquitin antiserum (1:500; Sigma Chemcial Co.) is used to detect ubiquitinated proteins after blots are autoclaved for 20 minutes to fully denature ubiquitinated proteins and enhance their detection. Detection is achieved using appropriate peroxidase-conjugated secondary antibodies (1:20,000) and chemiluminescent substrate (Kierkegaard and Perry Laboratories, Gaithersburg, MD).
  • Loss of dexamethasone binding activity can be determined with a binding assay in which HeLa cells (2 x lOVwell, 24-well plate) are treated with various concentrations of geldanamycin for varying periods of time in complete medium at 37 degrees C. At the end of the treatment interval, medium is aspirated and monolayers washed twice with ice-cold PBS containing 1 % BSA and 0.1 % sodium azide (binding buffer). Monolayers are then incubated for 60 minutes on ice with 1.0 ⁇ Ci/well (48 nM) [ 3 H]dexamethasone (Amersham, 82 Ci/mmol) in binding buffer with or without 5mM non-radioactive dexamethasone.
  • EXAMPLE 11 Preparation of Antibodies
  • the present invention also contemplates the preparation of antibodies against components of the SRC.
  • the components of the SRC can be purified by techniques known in the art, such as immunoprecipitation.
  • Monoclonal or polyclonal antibodies may be produced to either the SRC component proteins, peptide fragments, or mutant forms of these proteins.
  • antibodies raised against the protein will specifically detect the protein. That is, antibodies raised against the protein would recognize and bind the protein and would not substantially recognize or bind to other proteins found in human cells.
  • the determination that an antibody specifically detects a protein is made by any one of a number of standard immunoassay methods; for instance, the Western blotting technique (Sambrook et al., 1989).
  • total cellular protein is extracted from human cells (for example, lymphocytes) and electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel.
  • the proteins are then transferred to a membrane (for example, nitrocellulose) by Western blotting, and the antibody preparation is incubated with the membrane.
  • Antibodies which specifically detect the protein will, by this technique, be shown to bind to the protein band (which will be localized at a given position on the gel determined by its molecular weight). Non-specific binding of the antibody to other proteins may occur and may be detectable as a weak signal on the Western blot. The non-specific nature of this binding will be recognized by one skilled in the art by the weak signal obtained on the Western blot relative to the strong primary signal arising from the specific protein binding.
  • Antibodies that specifically bind to a protein component of the SRC belong to a class of molecules that are referred to herein as "specific binding agents. " Specific binding agents that are capable of specifically binding to the SRC protein may include polyclonal antibodies, monoclonal antibodies (including humanized monoclonal antibodies) and fragments of monoclonal antibodies such as Fab, F(ab')2 and Fv fragments, as well as any other agent capable of specifically binding to a protein component of the SRC.
  • Monoclonal antibody to epitopes of the SRC protein components identified and isolated as described can be prepared from murine hybridomas according to the classical method of Kohler and Milstein (1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the selected protein over a period of a few weeks. The mouse is then sacrificed, and the antibody-producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media).
  • HAT media aminopterin
  • the successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued.
  • Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Harlow and Lane (1988). In addition, protocols for producing humanized forms of monoclonal antibodies (for therapeutic applications) and fragments of monoclonal antibodies are known in the art.
  • Polyclonal antiserum containing antibodies to heterogeneous epitopes of a single protein can be prepared by immunizing suitable animals with the expressed protein, which can be unmodified or modified to enhance immunogenicity.
  • Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant.
  • host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable.
  • An effective immunization protocol for rabbits can be found in Vaitukaitis et al. (1971).
  • Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony et al. (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum (about 12 ⁇ M). Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (1980).
  • a third approach to raising antibodies against the SRC proteins is to use synthetic peptides synthesized on a commercially available peptide synthesizer based upon the predicted amino acid sequence of the protein components of the SRC.
  • geldanamycin derivative refers to compounds that are structurally analogous to geldanamycin in their ability to stimulate neurite outgrowth.
  • Geldanamycin consists of a closed ansa ring with a planar benzoquinone embedded in it.
  • the ansa ring is sterically hindered because its backbone includes a planar amide and three carbon-carbon double bonds (two of them arranged in a 1,3-diene), and nine of its sixteen backbone atoms carry nonhydrogen substitutents such as a carbonyl, a carbamate (-OC(0)NH 2 ), a hydroxyl, two methoxy and four methyl groups.
  • the crystal structure of geldanamycin has been set forth, and the structure/activity relationships of the benzoquinone ansamycins have been described in Stebbins et al., Cell 89:239-250, 1997; Schnur et al., J. Med. Chem.
  • Geldanamycin derivatives may have the carbamate group and ansa ring of geldanamycin (Schur et al., J. Med. Chem. 38:3806-3812, 1995), and/or have modifications at functional groups such as the C23 methoxy and C22 methyl groups (Stebbins et al., Cell 89:239-250, 1997).
  • Geldanamycin derivatives are also discussed in U.S. Patent No. 5,3877,584, 4,261,989, and 3,987,035, and in Japanese Patent Applications 88041885, 56100766, and 89002593, for example.
  • Tables 3 and 4 Additional benzoquinone ansamycin analogs of geldanamycin are shown in Tables 3 and 4.
  • Table 3 illustrates several synthesis schemes for gledanamycin derivatives, while Table 4 sets forth substitutions for the derivatives, as described more fully in Schnur, et al., /. Med. Chem. 38:3813-3820, 1995.
  • Analogs can also be modified by appending appropriate functionalities by well-known methods to enhance selected biological properties, including increasing penetration of the analogs into a given cellular compartment (e.g., blood, lymphatic system, central nervous system, etc.), increase oral availability, increase solubility to permit administration by injection, alter metabolism, and alter rate of excretion.
  • a given cellular compartment e.g., blood, lymphatic system, central nervous system, etc.
  • analogs have a molecular weight below about 750 atomic mass units (a.m.u.) (as the parent compound, although the salts of such compounds can have higher molecular weights).
  • FK506 analogs refers to compounds that are structurally analogous to FK506 in their ability to stimulate neuritic outgrowth. Some FK506 analogs, such as V-10,367, retain the FKBP12 binding domain but lack the structural components of the effector domain that interacts with calcineurin. The FK506 analogs may bind FKBP12 with low or high affinity. V- 10,367, for example, binds FKBP12 with high affinity (K d ⁇ 1 nM) (Armistead et al., Acta Crystallogr. 51:522-528, 1995).
  • FK506 analogs have a wide range of binding affinities for FKBP-12.
  • the mechanism for neurotrophic activity of FK506 presented herein indicates that the effectiveness of FK506 and FK506 analogs in stimulating nerve cell growth is unrelated to their ability to bind FKBP-12. Instead, their effectiveness in stimulating nerve cell growth relates to ability of such compounds to physically or functionally disrupt the steroid receptor complex, for example by interfering with the interaction of FKBP-52 and hsp-90 in a steroid receptor complex, or by promoting dissociation of p23 from the complex.
  • non-binding FK506 analog is defined as an FK506 analog that does not substantially bind to FKBP-12.
  • An FK506 analog with low affinity for binding FKBP-12 refers to an analog that binds FKBP12 with an apparent K,, of greater than 10 ⁇ M as measured using well- known assays, and preferably greater than 30 ⁇ M, and more preferably greater than 100 ⁇ M.
  • Values for the apparent Kj can be determined, for example, by a competitive LH-20 binding assay performed as described, for example, in Harding et al., Nature 341:758-760, 1989 (using 32-[l- 14 C]-benzoyl FK506 as a reporting ligand; Siekierka et al., Nature 341:755-757, 1989, using [ 3 H]dihydro-FK506 as a reporting ligand); and U.S. Patent No. 5,654,332.
  • the analog may be one that does not significantly inhibit FKBP-12 rotamase activity when administered to a patient at dosage levels of about 0.01 to about 100 mg/kg body weight/day. Assays for inhibition of FKBP12 rotamase activity are described in Example 9.
  • Non-binding FK506 analogs are non-immunosuppressive, as can be demonstrated by well-known assays, e.g., as discussed in U.S. Patent No. 5,516,797, WO 92/21313, WO 92/19593, and WO 92/04370.
  • Rapamycin analogs include compounds structurally similar to rapamycin, for example WAY-124,466 shown in Ocain et al. , Biochem. Biophys. Res. Commun. 192-1340-1346, 1993. This analog is identical to rapamycin, except that it has been modified in the triene region, and has a Ki for PPIase activity of 12.5 nM, as determined by the methods shown in that reference. It is also non-immunosuppressive, as determined in that reference by an inability to inhibit the proliferation of murine thymocytes.
  • Other rapamycin analogs include other macrocyclic trienes, mono- and diacylated derivatives esterified at the 31 and 42 positions (U.S. Patent No.
  • Radicicol analogs refers to compounds structurally similar to radicicol, for example those shown in U.S. Patent No. 5,731,343, U.S. Patent No. 5,650,430, U.S. Patent No. 4,228,079, and published international patent applications W096/33989 and WO98/18780 which are all incorporated by reference. Particular examples of such compounds are those shown in the following structural formula:
  • R,, R 2 , and R 3 are independently selected from the group consisting of H, C1-C8 alkyl or COR 5 .
  • R 5 is chosen from the group consisting of hydrogen, substituted alkyl, alkoxy, alkenyl, substituted alkenyl, alkenyloxy, alkynyl, substituted alkynyl, aryl with 6 to 14 ring atoms, arlyoxy with 6 to 14 ring atoms, heterocyclic groups with 5 or 6 ring atoms, heterocylic groups with 5 or 6 ring atoms fused to an aryl group, cycloalkyl, cycloalkenyl, and cycloalkyl fused to an aryl group.
  • COR 5 is given in Table 7; any of the groups appearing in Table 7 can be used for R,, R 2 , and R 3 .
  • X is halogen.
  • Y is chosen from the group consisting of O, and N-0-R 4 , wherein R 4 is chosen from the group consisting of hydrogen and Cl-8 alkyl.
  • Z is halogen. Alternatively, Z can be combined with R 3 to form an epoxide ring.
  • “Bastadins” refer to any bastadin presently known, or discovered in the future.
  • a compound from the bastadin family refers to subunits of the bastadins, such as halogenated tyrosines, including bromotyrosines and the 3-bromotyramine amide of oxalic acid amide.
  • a bastadin also includes the hemibastadins, which represent bromotyrosine dimers.
  • the hemibastadins include hemibastadins 1, 2, and 3 as well as hemibastadinols 1,2, and 3.
  • the methods of the present invention include the use of any bastadin or any member of the bastadin family.or their analogs in any aspect of the invention (including their use in assays to search for other neurotrophic compounds).
  • bastadins and other members of the bastadin family include those shown in Pettit et al., J. Nat. Prod., 59(10): 927-34, 1996; Franklin et al., J. Nat. Prod. ,
  • bastadins and their analogs include bastadins of the formula where the identity and substitution patterns of some particular bastadins and bastadin analogs are given in Table 8 below.
  • bastadins include compounds of the structure, where the identity and substitution patterns of some particular bastadins and bastadin analogs are given in Table 9 below.
  • Another example of a compound in the bastadin family is the 3-bromotyramine amide of oxalic acid amid which has the following structure, where R can for example be H or COCH 3 .
  • the bastadins also include the hemibastadins, some of which can be described by the following structure, where the identities and substimtion patterns are defined in Table 10.
  • the bastadins also include the hemibastadinols, some of which have the following structure.
  • the identity and substitution patterns of several examples are given below in Table 11.
  • FIG. 4 shows that heat in combination with NGF treatment is more effective than either heat or NGF treatment alone in stimulating neurite outgrowth for the hippocampal cells tested.
  • FIG. 5 shows that heat treatment is more effective when administered in combination with NGF than when administered in combination with an Hsp-90 Antibodies.
  • FIG. 5 also illustrates that a combination of heat treatment and treatment with both NGF, and Hsp-90 antibodies is not as effective as the combination of heat and NGF treatment. Involvment of Hsp-90 as a mediator of the heat shock effect is demonstrated by the ability of the Hsp-90 antibody to inhibit the increase in neurite outgrowth.
  • MAP Kinase/Kinase The role of MAP Kinase/Kinase was investigated utilizing the in vitro assay described in Example 2. Hippocampal cells were treated with the MAP kinase/kinase inhibitor PD 098059 in combination with NGF, FK506, and radicicol to determine if there is down-stream involvement of the MAP kinase pathway in the action of each of these compounds.
  • the selective MAP kinase/kinase (MEK) inhibitor blocks neurite outgrowth by NGF and FK-506 in a concentration dependent fashion.
  • FIG. 7 illustrates that PD 098059 also blocks the action of radicicol in a concentration dependent fashion and the highest concentration almost completely blocks activity.
  • FIG 9 A is a light micrograph of untreated hippocampal cells after 72 hours.
  • FIG 9B another light micrograph, illustrates the effect radicicol has on neurite outgrowth and 9C demonstrates the same for a bastadin 10 analog (Davis B-10, bastadin).
  • FIGS 9 and 10 illustrate the ability of bastadin to stimulate neurite outgrowth.
  • the neurotrophic compounds of the invention are admimstered in an effective amount sufficient to stimulate nerve growth or regeneration compared to a control.
  • Suitable local concentrations for nerve cell growth or nerve regeneration can be readily assessed using an in vitro assay, e.g., the assay described in Example 1.
  • nerve cell growth or regeneration can be determined by an in vivo assay, or by direct or indirect signs of nerve cell growth and regeneration in a subject (for example a restoration of motor and/or sensory function in the hand in the area of innervation of a previously transected median nerve).
  • the increase in nerve cell growth or regeneration rate is at least 10%, preferably at least 30%, and most preferably 50% or more compared to a control.
  • Preferred dosage levels are between about 0.1 to about 400 mg/kg per day of the FK506 analog for subcutaneous delivery.
  • dosage level examples are between about 0.01 to about 40 mg/kg/day.
  • the dose can be sufficient to achieve tissue concentrations that have been shown to be neurotrophic in vitro.
  • compositions according to the invention can be periodically administered to a mammalian subject (e.g., a human patient), in need of such treatment, to promote neuronal regeneration and functional recovery and to stimulate neurite outgrowth and thereby to treat various neuropathological states, including damage to peripheral nerves and the central nervous system caused by physical injury (e.g., spinal cord injury and trauma, sciatic or facial nerve lesion or injury, limb transplantation following amputation), disease (e.g., diabetic neuropathy), cancer chemotherapy (e.g., neuropathy induced by acrylamide, taxol, vinca alkaloids and doxorubicin), brain damage associated with stroke and ischemia, and neurological disorders including, but not limited to, various peripheral neuropathic and neurological disorders related to neurodegeneration including, but not limited to: trigeminal neuralgia, glossopharyngeal neuralgia, Bell's palsy, myasthema gravis, muscular dystrophy, amyotrophic lateral sclerosis, progressive muscular atrophy, progressive bulb
  • compositions according to the present invention display a wide range of other therapeutic or prophylactic properties, including, treatment of stroke (see, e.g., Sharkey and Butcher, Nature 371:336-339, 1994, Vagita et al., Life Sciences 59:1643- 1650, 1996; Tokime et al., Neurosci. Lett. 206:81-84, 1996; Drake et al., Acta. Physiol. Scand. 158:155-159, 1996; and Kuroda et al., Neurosci. Res. Comm. 19:83-90, 1996), AIDS dementia (see, e.g., Dawson and Dawson, Adv. Neuroimmunol.
  • a transection of a peripheral nerve or a spinal cord injury can be treated by administering a nerve growth stimulating amount of the agent to the mammal and grafting to the peripheral nerve or spinal cord a nerve graft such as an allograft (Osawa et al., J. Neurocytol. 19:833-849, 1990; Buttemeyer et al., Ann. Plastic Surgery 35:396-401, 1995) or an artificial nerve graft (Madison and Archibald, Exp. Neurol. 128:266-275, 1994; Wells et al., Exp. Neurol. 146:395-402, 1997).
  • a nerve graft such as an allograft (Osawa et al., J. Neurocytol. 19:833-849, 1990; Buttemeyer et al., Ann. Plastic Surgery 35:396-401, 1995) or an artificial nerve graft (Madison and Archibald, Exp. Neurol. 128:266-275
  • the space between the transected ends of the peripheral nerve or spinal cord is preferably filled with a non-cellular gap-filling material such as collagen, methyl cellulose, etc., or cell suspensions that promote nerve cell growth, such as Schwann cells (Xu et al., /. Neurocytol. 26:1-16, 1997), olfactory cells, and sheathing cells (Li et al. Science 277:2000-2002, 1997).
  • the nerve growth promoting agent can be included together with such cellular or non-cellular gap-filling materials, or admimstered systemically before, during or after the nerve graft procedure.
  • compositions according to the present invention encompass formulations that include an amount (for example, a unit dosage) of the neurotrophic agent together with one or more non-toxic pharmaceutically acceptable excipients, including carriers, diluents, and/or adjuvants, and optionally other biologically active ingredients.
  • Standard pharmaceutical formulation techniques are used, such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (19th Edition).
  • a pharmaceutical formulation according to the invention includes one or more of the neurotrophic agents of the present invention, and can also include, for example, one or more other biologically active ingredients, such as FK506 or an FKBP12-binding FK506 analog, NGF, IGF-1, ⁇ -FGF, ⁇ -FGF, PDGF, BDNF, CNTF, GDNF, NT-3, and NT 4/5.
  • FK506 or an FKBP12-binding FK506 analog such as FK506 or an FKBP12-binding FK506 analog, NGF, IGF-1, ⁇ -FGF, ⁇ -FGF, PDGF, BDNF, CNTF, GDNF, NT-3, and NT 4/5.
  • the first agent may be geldanamycin (which promotes dissociation of p23) and the second agent may be FKBP52-Ab (which interferes with association of FKBP-52 to hsp-90).
  • the composition includes NGF, or another agent that stimulates nerve growth in combination with the SRC disrupting complex, or the neurotrophic action of which is augmented by administration in combination with the SRC disrupting agent.
  • the dosage of the combined biologically active agents is sufficient to achieve tissue concentrations at the site of action that are similar to those that are shown to achieve in vivo nerve regeneration.
  • Pharmaceutical formulations may include, for example, an amount of a NGF, such that the subject receives a dosage of between about 0.01 to 100 ⁇ g/kg body weight/day.
  • the NGF (or other adjuvant) can be admimstered separately, concurrently, consecutively, or within less than about five hours of each other.
  • the compositions can be in the form of tablets, capsules, powders, granules, lozenges, liquid or gel preparations, such as oral, topical, or sterile parenteral solutions or suspensions (e.g., eye or ear drops, throat or nasal sprays, etc.), transdermal patches, and other forms known in the art.
  • Such pharmaceutical compositions can be administered systemically or locally in any manner appropriate to the treatment of a given condition, including orally, parenterally, rectally, nasally, buccally, vaginally, topically, optically, by inhalation spray, or via an implanted reservoir.
  • parenterally as used herein includes, but is not limited to subcutaneous, intravenous, intramuscular, intrasternal, intrasynovial, intrathecal, intrahepatic, intralesional, and intracranial administration, for example, by injection or infusion.
  • the pharmaceutical compositions preferably readily penetrate the blood-brain barrier when peripherally administered or are administered intraventricularly.
  • Pharmaceutically acceptable carriers include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffers (such as phosphates), glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffers such as phosphates
  • glycine glycine
  • sorbic acid
  • Tablets and capsules for oral administration can be in a form suitable for unit dose presentation and can contain conventional pharmaceutically acceptable excipients.
  • binding agents such as syrup, acacia, gelatin, sorbitol, tragacanth, and polyvinylpyrrolidone
  • fillers such as lactose, sugar, corn starch, calcium phosphate, sorbitol, or glycine
  • tableting lubricants such as magnesium stearate, talc, polyethylene glycol, or silica
  • disintegrants such as potato starch
  • dispersing or wetting agents such as sodium lauryl sulfate.
  • Oral liquid preparations can be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or can be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • compositions can also be administered parenterally in a sterile aqueous or oleaginous medium.
  • the composition can be dissolved or suspended in a non-toxic parenterally-acceptable diluent or solvent, e.g., as a solution in 1,3-butanediol.
  • a non-toxic parenterally-acceptable diluent or solvent e.g., as a solution in 1,3-butanediol.
  • Commonly used vehicles and solvents include water, physiological saline, Hank's solution, Ringer's solution, and sterile, fixed oils, including synthetic mono- or di-glycerides, etc.
  • the drug may be made up into a solution, suspension, cream, lotion, or ointment in a suitable aqueous or non-aqueous vehicle.
  • Additives may also be included, e.g., buffers such as sodium metabisulphite or disodium edeate; preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents, such as hypromellose.
  • buffers such as sodium metabisulphite or disodium edeate
  • preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride or chlorhexidine, and thickening agents, such as hypromellose.
  • the dosage unit involved depends, for example, on the condition treated, nature of the formulation, nature of the condition, embodiment of the claimed pharmaceutical compositions, mode of administration, and condition and weight of the patient. Dosage levels are typically sufficient to achieve a tissue concentration at the site of action that is at least the same as a concentrations that has been shown to be neurotrophic in vitro. For example, a dosage of about 0.1 to about 400 mg/kg per day of the active ingredient may be useful in the treatment of the conditions listed above.
  • the compounds can be used in the form of salts preferably derived from inorganic or organic acids and bases, including, but not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate,
  • Base salts include, but are not limited to, ammonium salts, alkali metal salts (such as sodium and potassium salts), alkaline earth metal salts (such as calcium and magnesium salts), salts with organic bases (such as dicyclohexylamine salts), N-methyl-D-glucamine, and salts with amino acids (such as arginine, lysine, etc.).
  • Basic nitrogen-containing groups can be quatemized, e.g., with such agents as Cl-8 alkyl halides (such as methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (such as dimethyl, diethyl, dibutyl, an diamyl sulfates), long-chain halides (such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), aralkyl halides (such as benzyl and phenethyl bromides), etc. Water or oil-soluble or dispersible products are produced thereby.
  • Pharmaceutical compositions can be included in a kit accompanied by instructions for intended use, for example instructions required by a pharmaceutical regulatory agency, such as the Food and Drug Administration in the United States.
  • FK506 neuroimmunophilin ligands
  • steroid hormones are mediated by physical or functional disruption of steroid receptor complexes.
  • Some of the components of the complex that can act as targets for disruption include FKBP-52, hsp-90 and p23, which are all present together in mature steroid receptor complexes, which can be disrupted by geldanamycin. Since FKBP-52 can associate with microtubules and dynein, and via its TPR motifs also associate with kinesin, it can also have a direct role in the movement (axonal transport) of cytoskeletal elements and, consequently, axonal elongation.

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Abstract

La formation d'axones et la régénération nerveuse sont activées par dissociation du complexe de récepteurs stéroïdiens et par stimulation de l'activité de MAP kinase/kinase. Cette dissociation peut prendre la forme d'une dissociation de l'assemblage physique ou de la fonction du complexe de récepteurs stéroïdiens, tel que le complexe mature ou un précurseur du complexe mature nécessaire à l'assemblage du complexe mature. La geldanamycine et ses analogues, la bastadine et des membres de la famille des bastadines, le radicicol et ses analogues, ainsi que l'anticorps FKBP-52 dissocient le complexe et activent la croissance nerveuse. L'invention concerne des techniques de recherche de composés neurotrophiques, les composés trouvés à l'aide de ces techniques, les compositions pharmaceutiques dans lesquels ils sont incorporés, et des méthodes de traitement de sujets présentant un dysfonctionnement neuronal provoqué par une lésion ou par une maladie. N'importe lequel de ces composés peut être utilisé en combinaison avec une quantité thérapeutiquement efficace de chaleur, telle que de la chaleur appliquée localement sur une zone où on veut obtenir une croissance nerveuse, ou de manière systémique dans un organisme dans lequel on veut obtenir une croissance d'axones. Dans un autre mode de réalisation, ces composés peuvent être utilisés en association avec un modèle tel qu'un élément tubulaire définissant une voie anatomique le long de laquelle on veut obtenir une régénération nerveuse (notamment autour d'un nerf sectionné ou partiellement sectionné).
PCT/US2000/018539 1999-07-09 2000-07-07 Compositions et procedes activant la regeneration nerveuse WO2001003692A1 (fr)

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AU60748/00A AU777997B2 (en) 1999-07-09 2000-07-07 Compositions and methods for promoting nerve regeneration
EP00947081A EP1200078A4 (fr) 1999-07-09 2000-07-07 Compositions et procedes activant la regeneration nerveuse
US10/030,904 US6734211B1 (en) 1999-07-09 2000-07-07 Compositions and methods for promoting nerve regeneration
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WO2002094259A1 (fr) * 2001-05-03 2002-11-28 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Composes inhibant hsp90 et stimulant hsp70 et hsp40, utiles pour prevenir ou traiter des maladies associees a l'agregation de proteines et a la formation d'amyloides
US6734211B1 (en) 1999-07-09 2004-05-11 Oregon Health & Sciences University Compositions and methods for promoting nerve regeneration
JP2004257862A (ja) * 2003-02-26 2004-09-16 Akihiko Yano 重症筋無力症の検査方法及びそのための検査薬
WO2008150302A1 (fr) * 2007-06-04 2008-12-11 Nexgenix Pharmaceuticals Traitement de la neurofibromatose avec du radicicol et ses dérivés
US7648996B2 (en) 2005-03-11 2010-01-19 Biotica Technology Limited 39-desmethoxy derivatives of rapamycin
US7803808B2 (en) 2004-08-11 2010-09-28 Wyeth Llc Production of polyketides and other natural products
EP2277898A2 (fr) 2002-07-16 2011-01-26 Biotica Technology Limited Analogues de Rapamycine
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US5968921A (en) * 1997-10-24 1999-10-19 Orgegon Health Sciences University Compositions and methods for promoting nerve regeneration
WO1999051223A1 (fr) * 1998-04-03 1999-10-14 University Of Pittsburgh Of The Commonwealth System Of Higher Education Ansamycines benzoquinoides pour le traitement d'une crise ou d'un arret cardiaque

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6734211B1 (en) 1999-07-09 2004-05-11 Oregon Health & Sciences University Compositions and methods for promoting nerve regeneration
EP1196171A2 (fr) * 1999-07-13 2002-04-17 Medicure Inc. Traitement du diabete et des pathologies associees
WO2002094259A1 (fr) * 2001-05-03 2002-11-28 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Composes inhibant hsp90 et stimulant hsp70 et hsp40, utiles pour prevenir ou traiter des maladies associees a l'agregation de proteines et a la formation d'amyloides
EP2277898A2 (fr) 2002-07-16 2011-01-26 Biotica Technology Limited Analogues de Rapamycine
JP2004257862A (ja) * 2003-02-26 2004-09-16 Akihiko Yano 重症筋無力症の検査方法及びそのための検査薬
US7803808B2 (en) 2004-08-11 2010-09-28 Wyeth Llc Production of polyketides and other natural products
US7648996B2 (en) 2005-03-11 2010-01-19 Biotica Technology Limited 39-desmethoxy derivatives of rapamycin
US8008318B2 (en) 2005-03-11 2011-08-30 Biotica Technology Limited 39-desmethoxy derivatives of rapamycin
US8329683B2 (en) 2006-06-02 2012-12-11 Nexgenix Pharmaceuticals, Llc Treatment of neurofibromatosis with radicicol and its derivatives
WO2008150302A1 (fr) * 2007-06-04 2008-12-11 Nexgenix Pharmaceuticals Traitement de la neurofibromatose avec du radicicol et ses dérivés

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