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WO2002062387A1 - Traitement de pathologies necessitant l'inhibition de gsk-3 - Google Patents

Traitement de pathologies necessitant l'inhibition de gsk-3 Download PDF

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Publication number
WO2002062387A1
WO2002062387A1 PCT/GB2002/000542 GB0200542W WO02062387A1 WO 2002062387 A1 WO2002062387 A1 WO 2002062387A1 GB 0200542 W GB0200542 W GB 0200542W WO 02062387 A1 WO02062387 A1 WO 02062387A1
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Prior art keywords
gsk
pharmaceutically acceptable
inhibitor
disease
axonal
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PCT/GB2002/000542
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English (en)
Inventor
Patrick Doherty
Britta Johanna Eickholt
Stephen Drake Skaper
Frank Sinclair Walsh
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SmithKline Beecham Ltd
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SmithKline Beecham Ltd
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Publication of WO2002062387A1 publication Critical patent/WO2002062387A1/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

Definitions

  • GSK-3 is a serine/threonine protein kinase composed of two iso forms ( ⁇ and ⁇ ) which are encoded by distinct genes.
  • GSK-3 is one of several protein kinases which phosphorylates glycogen synthase (GS) (Embi et al Eur. J. Biochem. (107) 519-527 (1980)).
  • GS glycogen synthase
  • the ⁇ and ⁇ iso forms have a monomeric structure and are both found in mammalian cells.
  • Type II diabetes (or Non-Insulin Dependent Diabetes Mellitus, NTDDM) is a multifactorial disease.
  • Hyperglycaemia is due to insulin resistance in the liver, muscle and other tissues coupled with inadequate or defective secretion of insulin from pancreatic islets.
  • Skeletal muscle is the major site for insulin-stimulated glucose uptake and in this tissue, glucose removed from the circulation is either metabolised through glycolysis and the TCA cycle, or stored as glycogen.
  • Muscle glycogen deposition plays the more important role in glucose homeostasis and Type II diabetic subjects have defective muscle glycogen storage.
  • the stimulation of glycogen synthesis by insulin in skeletal muscle results from the dephosphorylation and activation of glycogen synthase (Villar-Palasi C. and Larner J. Biochim.
  • GSK-3 is responsible for phosphorylation and deactivation of GS, while glycogen bound protein phosphatase 1 (PP1G) dephosphorylates and activates GS. Insulin both inactivates GSK-3 and activates PP1G (Srivastava A K and Pandey S K Mol. and Cellular Biochem. (182) 135-141 (1998)).
  • GSK-3 ⁇ and constitutively active GSK-3 ⁇ (S9A, S9E) mutants in HEK-293 cells resulted in suppression of glycogen synthase activity (Eldar-Finkelman et al, PNAS (93) 10228-10233 (1996)) and overexpression of GSK-3 ⁇ in CHO cells, expressing both insulin receptor and insulin receptor substrate 1 (IRS-1), resulted in an impairment of insulin action (Eldar-Finkelman and Krebs PNAS (94) 9660-9664 (1997)).
  • GSK-3 activity is subject to inhibitory phosphorylation by PI 3 kinase-mediated or Wnt- 1 class-mediated signals that can be mimicked by treatment with lithium, a low mM inhibitor of GSK-3 (Stambolic V., Ruel L. and Woodgett J.R. Curr. Biol. 1996 6(12): 1664-8).
  • GSK-3 inhibitors may be of value as neuroprotectants in treatment of acute stroke and other neurotraumatic injuries. Roles for PI 3-kinase signalling through PKB/akt to promote neuronal cell survival are well established, and GSK-3 is one of a number of PKB/akt substrates to be identified that can contribute to the inhibition of apoptosis via this pathway (Pap & Cooper, (1998) J. Biol. Chem. 273: 19929-19932). Evidence suggests that astrocytic glycogen can provide an alternative energy source to facilitate neuronal survival under conditions of glucose deprivation (for example see Ransom, B.R. and Fern, R. (1997) Glia 21: 134-141 and references therein).
  • Lithium is known to protect cerebellar granule neurons from death (D'Mello et al., (1994) Exp. Cell Res. 211: 332-338 and Volonte et al (1994) Neurosci. Letts. 172: 6-10) and chronic lithium treatment has demonstrable efficacy in the middle cerebral artery occlusion model of stroke in rodents (Nonaka and Chuang, (1998) Neuroreport 9(9): 2081-2084). Wnt- induced axonal spreading and branching in neuronal culture models has been shown to correlate with GSK-3 inhibition (Lucas & Salinas, (1997) Dev. Biol. 192: 31-44) suggesting additional value of GSK-3 inhibitors in promoting neuronal regeneration following neurotraumatic insult.
  • Tau and ⁇ -catenin, two known in vivo substrates of GSK-3, are of direct relevance in consideration of further aspects of the value of GSK-3 inhibitors in relation to treatment of chronic neurodegenerative conditions.
  • Tau hyperphosphorylation is an early event in neurodegenerative conditions such as Alzheimer's disease (AD), and is postulated to promote microtubule disassembly.
  • AD Alzheimer's disease
  • Lithium has been reported to reduce the phosphorylation of tau, enhance the binding of tau to microtubules, and promote microtubule assembly through direct and reversible inhibition of glycogen synthase kinase-3 (Hong M., Chen D.C., Klein P.S. and Lee V.M. J.Biol. Chem. 1997 272(40) 25326-32).
  • ⁇ -catenin is phosphorylated by GSK-3 as part of a tripartite complex with axin, resulting in ⁇ -catenin being targetted for degradation (Dceda et al., (1998) EMBO J. 17: 1371-1384). Inhibition of GSK-3 activity is a key mechanism by which cytosolic levels of catenin are stabilised and hence promote ⁇ -catenin-LEF-1/TCF transcriptional activity (Eastman, Grosschedl (1999) Curr. Opin. Cell Biol. 11: 233). Rapid onset AD mutations in presenilin-1 (PS-1) have been shown to decrease the cytosolic ⁇ -catenin pool in transgenic mice.
  • PS-1 presenilin-1
  • fibroblasts from the GSK-3 ⁇ knockout mouse support a role for this kinase in positively regulating the activity of NFkB.
  • This transcription factor mediates cellular responses to a number of inflammatory stimuli. Therefore, pharmacologic inhibition of GSK-3 may be of use in treating inflammatory disorders through the negative regulation of NFkB activity.
  • Semaphorin 3 A a repulsive axon guidance molecule, contributes to the correct wiring of the nervous system by channelling the growth of axons away from inappropriate territories (Nakamura et al., J. Neurobiol, 2000, 44, 219). Following injury, the same molecule has been postulated to inhibit nerve regeneration via growth cone collapse (Pasterkamp et al., Mol. Cell. Neurosci., 1999, 13, 143).
  • GSK-3 inhibitors may be useful in the promotion of nerve regeneration, for example, axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse in cases of acute neuronal injury, such as crush injury, acute stroke, ischaemia, neurotraumatic insult, spinal cord injury and neurotrauma and chronic neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, neurodegenerative diseases, Lou Gehrig's disease, and multiple sclerosis.
  • acute neuronal injury such as crush injury, acute stroke, ischaemia, neurotraumatic insult, spinal cord injury and neurotrauma
  • chronic neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, neurodegenerative diseases, Lou Gehrig's disease, and multiple sclerosis.
  • the present invention provides a method of treatment for the promotion of nerve regeneration, including axonal regrowth, axonal outgrowth, and prevention of growth cone collapse, in cases of acute neuronal injury, such as crush injury, acute stroke, ischaemia, neurotraumatic insult, spinal cord injury and neurotrauma in humans or non-human mammals, which method comprises the administration of an effective, non-toxic and pharmaceutically acceptable amount of a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof.
  • the invention also provides a method of treatment for the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of chronic neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, neurodegenerative diseases, Lou Gehrig's disease, and multiple sclerosis, in humans or non-human mammals, which method comprises the administration of an effective, non-toxic and pharmaceutically acceptable amount of a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof.
  • the present invention provides a method of treatment for the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of Parkinson's Disease, Lou Gehrig's disease and multiple sclerosis in humans or non-human mammals, which method comprises the administration of an effective, non-toxic and pharmaceutically acceptable amount of a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof.
  • said GSK-3 inhibitor is a compound described in WO 00/21927, WO 00/38675, WO 01/09106 and WO 01/74771 or a pharmaceutically acceptable derivative thereof.
  • the promotion of nerve regeneration according to the present invention is a consequence of the prevention of growth cone collapse response induced by Semaphorin 3A.
  • the promotion of nerve regeneration according to the present invention is a consequence of stimulation of axonal regeneration into otherwise inhibitory Semaphorin 3A territory.
  • a suitable neurodegenerative condition for treatment in accordance with the present invention is Alzheimer's disease.
  • a suitable neurodegenerative condition for treatment in accordance with the present invention is Parkinson's disease.
  • a suitable condition for treatment according to the present invention is multiple sclerosis.
  • a suitable condition for treatment according to the present invention is Lou Gehrig's disease.
  • Suitable GSK-3 inhibitors include a compound of formula (IA), or a derivative thereof, wherein a compound of formula (IA) is defined as being a compound of formula (I) as defined in WO 00/21927.
  • the suitable and preferred compounds of formula (IA) are those compounds of formula (I) defined as suitable and preferred in WO 00/21927. Particularly preferred compounds of formula (IA) are disclosed on page 50, at Example 116 ("SB-331371") and at page 67, Example A558 ("SB-415286") of WO 00/21927.
  • Suitable GSK-3 inhibitors include a compound of formula (LB), or a derivative thereof, wherein a compound of formula (IB) is defined as being a "Compound of Group (I)" as defined in WO 00/38675.
  • Suitable and preferred compounds of formula (LB) are those "Compound of Group (I)" defined as suitable and preferred in WO 00/38675.
  • Suitable GSK-3 inhibitors include a compound of formula (LB'), or a derivative thereof, wherein a compound of formula (IB') is defined as being a "Compound of Group (II)" as defined in WO 00/38675.
  • Suitable and preferred compounds of formula (LB') are those "Compound of Group (II)" defined as suitable and preferred in WO 00/38675.
  • Suitable GSK-3 inhibitors include a compound of formula (IC), or a derivative thereof, wherein a compound of formula (IC) is defined as being a compound of formula (I) as defined in WO 01/09106.
  • the suitable and preferred compounds of formula (IC) are those compounds of formula (I) defined as suitable and preferred in WO 01/09106.
  • Suitable GSK-3 inhibitors include a compound of formula (LD), or a derivative thereof, wherein a compound of formula (LD) is defined as being a compound of formula (I) as defined in WO 01/74771.
  • the suitable and preferred compounds of formula (LD) are those compounds of formula (I) defined as suitable and preferred in WO 01/74771.
  • GSK-3 inhibitor for use in the present invention is described in Coghlan et al., Chemistry & Biology, September 2000, 7, 793, at page 795 as "SB- 216763".
  • GSK-3 inhibitor contains a chiral carbon atom and hence exists in one or more stereoisomeric forms, or where one or more geometric isomers exist
  • the method of the present invention encompasses all of the said forms of the GSK-3 inhibitor whether as individual isomers or as mixtures of isomers, including racemates.
  • Certain of the compounds of formulae (IA), (IB), (LB'), (IC) and (LD) may contain chiral atoms and/or multiple bonds, and hence may exist in one or more stereoisomeric forms.
  • the present invention encompasses all of the isomeric forms of the compounds of formulae (IA), (IB), (LB'), (IC) and (LD) whether as individual isomers or as mixtures of isomers, including geometric isomers and racemic modifications.
  • Suitable derivatives of a GSK-3 inhibitor are pharmaceutically acceptable derivatives, for example salts and solvates.
  • Suitable derivatives of any particular GSK-3 inhibitor include those disclosed in the above mentioned publications.
  • Suitable pharmaceutically acceptable salts include salts of salts derived from appropriate acids, such as acid addition salts, or bases.
  • Suitable pharmaceutically acceptable salts include metal salts, such as for example aluminium, alkali metal salts such as lithium, sodium or potassium, alkaline earth metal salts such as calcium or magnesium and ammonium or substituted ammonium salts, for example those with lower alkylamines such as triethylamine, hydroxy alkylamines such as 2-hydroxyethylamine, bis-(2-hydroxyethyl)-amine or tri-(2-hydroxyethyl)-amine, cycloalkylamines such as bicyclohexylamine, or with procaine, dibenzylpiperidine, N-benzyl-b-phenethylamine, dehydroabietylamine, N,N'-bisdehydroabietylamine, glucamine, N-methylglucamine or bases of the pyridine type such as pyridine, collidine, quinine or quinoline.
  • metal salts such as for example aluminium, alkali metal salts such as lithium, sodium or potassium,
  • Suitable acid addition salts include pharmaceutically acceptable inorganic salts such as the sulphate, nitrate, phosphate, borate, hydrochloride and hydrobromide and pharmaceutically acceptable organic acid addition salts such as acetate, tartrate, maleate, citrate, succinate, benzoate, ascorbate, methane-sulphonate, a-keto glutarate and a- glycerophosphate, especially the maleate salt.
  • pharmaceutically acceptable inorganic salts such as the sulphate, nitrate, phosphate, borate, hydrochloride and hydrobromide
  • pharmaceutically acceptable organic acid addition salts such as acetate, tartrate, maleate, citrate, succinate, benzoate, ascorbate, methane-sulphonate, a-keto glutarate and a- glycerophosphate, especially the maleate salt.
  • Suitable pharmaceutically acceptable salts of a compound of formula (IA) are as disclosed in WO 00/21927.
  • Suitable pharmaceutically acceptable salts of a compound of formula (LB) are as disclosed in WO 00/38675.
  • Suitable pharmaceutically acceptable salts of a compound of formula (LB') are as disclosed in WO 00/38675.
  • Suitable pharmaceutically acceptable salts of a compound of formula (IC) are as disclosed in WO 01/09106.
  • Suitable pharmaceutically acceptable salts of a compound of formula (LD) are as disclosed in WO 01/74771.
  • Suitable pharmaceutically acceptable solvates include hydrates.
  • GSK-3 inhibitors referred to herein are conveniently prepared according to the methods disclosed in the above mentioned patent publications in which they are disclosed.
  • a compound of formula (IA), and/or a derivative thereof may be prepared using the processes described in WO 00/21927;
  • a compound of formula (LB), and/or a derivative thereof may be prepared using the processes described in WO 00/38675,
  • a compound of formula (IB'), and/or a derivative thereof may be prepared using the processes described in WO 00/38675,
  • a compound of formula (IC), and/or a derivative thereof may be prepared using the processes described in WO 01/09106 and a compound of formula (ID), and/or a derivative thereof, may be prepared using the processes described in WO 01/74771.
  • the present invention also provides a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof, for use in the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of acute neuronal injury, such as crush injury, acute stroke, ischaemia, neurotraumatic insult, spinal cord injury and neurotrauma.
  • a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof for use in the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of acute neuronal injury, such as crush injury, acute stroke, ischaemia, neurotraumatic insult, spinal cord injury and neurotrauma.
  • the present invention also provides a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof, for use in the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of chronic neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, neurodegenerative diseases, Lou Gehrig's disease, and multiple sclerosis.
  • the present invention also provides a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof, for use in the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of Parkinson's Disease, Lou Gehrig's disease and multiple sclerosis.
  • said GSK-3 inhibitor is a compound described in WO 00/21927, WO 00/38675, WO 01/09106 and WO 01/74771 or a pharmaceutically acceptable derivative thereof.
  • the present invention also provides a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof, for use in the manufacture of a medicament for the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of acute neuronal injury, such as crush injury, acute stroke, ischaemia, neurotraumatic insult, spinal cord injury and neurotrauma.
  • a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof for use in the manufacture of a medicament for the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of acute neuronal injury, such as crush injury, acute stroke, ischaemia, neurotraumatic insult, spinal cord injury and neurotrauma.
  • the present invention also provides a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof, for use in the manufacture of a medicament for the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of chronic neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, neurodegenerative diseases, Lou Gehrig's disease, and multiple sclerosis.
  • a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof for use in the manufacture of a medicament for the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of chronic neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, neurodegenerative diseases, Lou Gehrig's disease, and multiple sclerosis.
  • the present invention also provides a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof, for use in manufacture of a medicament for the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of Parkinson's Disease, Lou Gehrig's disease and multiple sclerosis.
  • said GSK-3 inhibitor is a compound described in WO 00/21927, WO 00/38675, WO 01/09106 and WO 01/74771 or a pharmaceutically acceptable derivative thereof.
  • the GSK-3 inhibitor may be administered per se or, preferably, as a pharmaceutical composition also comprising a pharmaceutically acceptable carrier.
  • the GSK-3 inhibitor mentioned herein is formulated and administered in accordance with the methods disclosed in the above mentioned patent applications and patents.
  • the present invention also provides a pharmaceutical composition for the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of acute neuronal injury, such as crush injury, acute stroke, ischaemia, neurotraumatic insult, spinal cord injury and neurotrauma, which composition comprises a GSK-3 inhibitor, or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier therefor.
  • the present invention also provides a pharmaceutical composition for the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of chronic neurodegenerative conditions such as Alzheimer's disease, Parkinson's disease, neurodegenerative diseases, Lou Gehrig's disease, and multiple sclerosis, which composition comprises a GSK-3 inhibitor, or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier therefor.
  • the present invention also provides a pharmaceutical composition for the promotion of nerve regeneration, for example axonal regrowth, axonal outgrowth, and the prevention of growth cone collapse, in cases of Parkinson's Disease, Lou Gehrig's disease and multiple sclerosis, which composition comprises a GSK-3 inhibitor or a pharmaceutically acceptable derivative thereof, and a pharmaceutically acceptable carrier therefor.
  • said GSK-3 inhibitor is a compound described in WO 00/21927, WO 00/38675, WO 01/09106 and WO 01/74771 or a pharmaceutically acceptable derivative thereof.
  • pharmaceutically acceptable embraces compounds, compositions and ingredients for both human and veterinary use: for example the term pharmaceutically acceptable salt' embraces a veterinarily acceptable salt.
  • compositions of the present invention may, if desired, be in the form of a pack accompanied by written or printed instructions for use.
  • pharmaceutical compositions of the present invention will be adapted for oral administration, although compositions for administration by other routes, such as by injection and percutaneous absorption are also envisaged.
  • Particularly suitable compositions for oral administration are unit dosage forms such as tablets and capsules.
  • Other fixed unit dosage forms, such as powders presented in sachets, may also be used.
  • the carrier may comprise a diluent, filler, disintegrant, wetting agent, lubricant, colourant, flavourant or other conventional adjuvant.
  • Typical carriers include, for example, microcrystalline cellulose, starch, sodium starch glycollate, polyvinylpyrrolidone, polyvinylpolypy ⁇ olidone, magnesium stearate, sodium lauryl sulphate or sucrose.
  • Suitable dosages of the GSK-3 inhibitor include the known doses for these compounds as described or refe ⁇ ed to in reference texts such as the British and US Pharmacopoeias, Remington's Pharmaceutical Sciences (Mack Publishing Co.), Martindale The Extra Pharmacopoeia (London, The Pharmaceutical Press) (for example see the 31st Edition page 341 and pages cited therein) or the above mentioned publications or doses which can be determined by standard procedures.
  • Suitable dosages of the compound of formula (IA) include those disclosed in WO 00/21927.
  • Suitable dosages of the compound of formula (IB) include those disclosed in WO 00/38675.
  • Suitable dosages of the compound of formula (IB') include those disclosed in
  • Suitable dosages of the compound of formula (IC) include those disclosed in WO 01/09106.
  • Suitable dosages of the compound of formula (LD) include those disclosed in WO 01/74771.
  • composition of the invention may be administered from 1 to 6 times a day, but most preferably 1 or 2 times per day.
  • the solid oral compositions may be prepared by conventional methods of blending, filling or tabletting. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are of course conventional in the art.
  • the tablets may be coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.
  • Oral liquid preparations may be in the form of, for example, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminium stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and if desired conventional flavouring or colouring agents.
  • suspending agents for example sorbitol, syrup, methyl cellulose,
  • fluid unit dosage forms are prepared utilizing the compound and a sterile vehicle, and, depending on the concentration used, can be either suspended or dissolved in the vehicle.
  • the compound can be dissolved in water for injection and filter sterilized before filling into a suitable vial or ampoule and sealing.
  • adjuvants such as a local anaesthetic, a preservative and buffering agents can be dissolved in the vehicle.
  • the composition can be frozen after filling into the vial and the water removed under vacuum.
  • Parenteral suspensions are prepared in substantially the same manner, except that the compound is suspended in the vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration.
  • the compound can be sterilized by exposure to ethylene oxide before suspending in the sterile vehicle.
  • a surfactant or wetting agent is included in the composition to facilitate uniform distribution of the compound.
  • Compositions may contain from 0.1% to 99% by weight, preferably from 10-60% by weight, of the active material, depending upon the method of administration. Compositions may, if desired, be in the form of a pack accompanied by written or printed instructions for use.
  • compositions are formulated according to conventional methods, such as those disclosed in standard reference texts, for example the British and US Pharmacopoeias, Remington's Pharmaceutical Sciences (Mack Publishing Co.),
  • Mouse monoclonal anti-GSK-3 (clone 4G-1E) and anti-P-(Y279/216)-GSK-3 (clone 5G-2F) antibodies were obtained from UBI.
  • Rabbit polyclonal antibody against P- (Ser9)-GSK-3 ⁇ was purchased from Biosource.
  • Mouse monoclonal GSK-3 ⁇ antibody was obtained from Transduction Labs.
  • Polyclonal sheep antibodies recognizing GSK-3 ⁇ and P-(Ser21)-GSK-3 ⁇ were purchased from UBI.
  • Mouse monoclonal neurofilament antibodies recognising the 68 kDa, 160 kDa, and the 200 kDa forms were obtained from Zymed and Sigma, and the monoclonal anti- ⁇ -tubulin antibody was obtained from Sigma.
  • Fertile eggs were obtained from a local supplier (Needle farm). DRG explants cultures from E7 chick embryos were prepared as previously described (Eickholt et al., Mol Cell. Neurosci., 1997, 9, 358). For immunohistochemistry DRG explants were plated onto poly- ' Lysine (20 ⁇ g/ml)/laminin (20 ⁇ g/ml) coated glass coverslips, and cultures were incubated for 20 hours in DMEM/10% FCS/PenStrep, supplemented with 20ng/ml NGF and 1% penicillin/streptomycin before fixation in 4% PFA/10% sucrose.
  • DRGs were cultured for 24 hours on 20 ⁇ g/ml laminin coated Labtec chamberslides (Nunc). LiCl (Sigma) and specific GSK-3 inhibitors ("SB-216763" and “SB-415286”) were applied at given concentrations and incubated for 1 hour before the Sema 3A-Fc was applied (at l ⁇ g/ml). After 30 minutes, the cultures were carefully fixed in 4% paraformaldehyde/10% sucrose.
  • PFA fixed DRG explants were washed twice with PBS, permeablized in PBS/1% Triton and blocked in blocking buffer (PBS/0.5% triton/2% BSA).
  • Primary antibody was applied (all antibodies were diluted 1 :50 in blocking buffer, except anti-P(Y /Y )- GSK-3 antibody that was diluted 1:100) and incubated overnight at 4°C with agitation. Bound antibody was visualized using FITC conjugated secondary antibodies (Sigma, anti-sheep antibody was from DAKO). The distribution of filamentous actin was visualized using Texas-Red conjugated Phalloidin (Molecular Probes). All samples were analyzed using volume deconvolution.
  • Cos-7 cells were transfected using Lipofectamine plus reagents after the manufacturer's protocol. Transfected cells were washed with icecold PBS and lysed in lysis buffer (20mM Hepes, 150mM NaCl, 1% Triton, 5mM CaCl 2 , ImM MgCl 2> protease inhibitors). Brain lysate was prepared from E9 chick brains using same lysis conditions. After removing insoluble material by centrifugation, protein extracts were separated by SDS-PAGE (10%) and transferred onto nitrocellulose. Bound proteins were detected by western blotting. All primary antibodies were used at 1 :1000. Secondary antibodies were purchased from Vector and used at 1 :5000.
  • DRG explants were cultured in the absence and presence of SB415286 for 20 hours on substrates consisting of pure laminin or on a mixture of laminin and affinity captured Sema 3A-Fc (see biological protocol).
  • substrates consisting of pure laminin or on a mixture of laminin and affinity captured Sema 3A-Fc (see biological protocol).
  • Sema 3 A the length of the 20 longest neurites that extended from isolated DRG explants cultured under the given culture conditions were measured (n>5).
  • DRG explant shows profuse axonal growth when cultured on a permissive laminin substrate.
  • a permissive laminin substrate On a substrate consisting of a mixture of laminin and affinity captured Sema 3 A-Fc, DRG axons remain in close proximity to the explant (middle).
  • neurons Following inhibition of GSK-3 with SB415286 at 30 ⁇ M, neurons acquire an ability to extend onto the inhibitory laminin/Sema 3A substrate.
  • Fluorescence micrographs show explants stained with an anti- ⁇ -tubulin antibody. Scale bar, 250 ⁇ m.
  • Example 2 The results obtained from Example 2 were found to be consistent with those detailed above for Example 1.

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Abstract

L'invention concerne un procédé de traitement visant à favoriser la régénérescence nerveuse, y compris la régénérescence axonale, la croissance axonale, et la prévention de l'effondrement du cône de croissance, en cas de lésion neuronale aiguë du type lésion par écrasement, attaque aiguë, ischémie, lésion neurologique, lésion médullaire et traumatisme neurologique chez des mammifères humains ou non humains. Le procédé consiste à administrer une quantité efficace, non toxique et pharmaceutiquement acceptable d'inhibiteur de GSK-3 ou bien de dérivé pharmaceutiquement acceptable de cet inhibiteur.
PCT/GB2002/000542 2001-02-07 2002-02-07 Traitement de pathologies necessitant l'inhibition de gsk-3 Ceased WO2002062387A1 (fr)

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GB0103031A GB0103031D0 (en) 2001-02-07 2001-02-07 Novel treatment
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004091663A1 (fr) * 2003-04-18 2004-10-28 Kyowa Hakko Kogyo Co., Ltd. Medicament pour la regeneration des nerfs
WO2006077824A1 (fr) * 2005-01-18 2006-07-27 Kyoto University Medicaments pour regeneration des cellules nerveuses
WO2006117212A2 (fr) 2005-05-04 2006-11-09 Develogen Aktiengesellschaft Utilisation des inhibiteurs gsk-3 dans la prevention et le traitement des maladies auto-immunes pancreatiques
EP1889619A4 (fr) * 2005-02-10 2009-12-23 Neuropharma Sa Utilisation de composes derives de tiadazolidines comme agents neurogeniques

Citations (4)

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WO2000021927A2 (fr) * 1998-10-08 2000-04-20 Smithkline Beecham Plc Procede et composes
WO2000038675A1 (fr) * 1998-12-23 2000-07-06 Smithkline Beecham Plc Traitement d'affections necessitant une inhibition de gsk-3
WO2001009106A1 (fr) * 1999-08-02 2001-02-08 Smithkline Beecham P.L.C. Composes diamino-1,2,4-triazole-acide carboxylique et derives de ces derniers utiles en tant qu'inhibiteurs de gsk-3
WO2001074771A1 (fr) * 2000-04-04 2001-10-11 Smithkline Beecham P.L.C. Derives de pyrrole-2, 5-dione destines au traitement du diabete

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000021927A2 (fr) * 1998-10-08 2000-04-20 Smithkline Beecham Plc Procede et composes
WO2000038675A1 (fr) * 1998-12-23 2000-07-06 Smithkline Beecham Plc Traitement d'affections necessitant une inhibition de gsk-3
WO2001009106A1 (fr) * 1999-08-02 2001-02-08 Smithkline Beecham P.L.C. Composes diamino-1,2,4-triazole-acide carboxylique et derives de ces derniers utiles en tant qu'inhibiteurs de gsk-3
WO2001074771A1 (fr) * 2000-04-04 2001-10-11 Smithkline Beecham P.L.C. Derives de pyrrole-2, 5-dione destines au traitement du diabete

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004091663A1 (fr) * 2003-04-18 2004-10-28 Kyowa Hakko Kogyo Co., Ltd. Medicament pour la regeneration des nerfs
WO2006077824A1 (fr) * 2005-01-18 2006-07-27 Kyoto University Medicaments pour regeneration des cellules nerveuses
EP1889619A4 (fr) * 2005-02-10 2009-12-23 Neuropharma Sa Utilisation de composes derives de tiadazolidines comme agents neurogeniques
WO2006117212A2 (fr) 2005-05-04 2006-11-09 Develogen Aktiengesellschaft Utilisation des inhibiteurs gsk-3 dans la prevention et le traitement des maladies auto-immunes pancreatiques

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