WO2008024776A1 - L'inhibition du signal jnk favorise la régénération des axones du snc - Google Patents

L'inhibition du signal jnk favorise la régénération des axones du snc Download PDF

Info

Publication number
WO2008024776A1
WO2008024776A1 PCT/US2007/076423 US2007076423W WO2008024776A1 WO 2008024776 A1 WO2008024776 A1 WO 2008024776A1 US 2007076423 W US2007076423 W US 2007076423W WO 2008024776 A1 WO2008024776 A1 WO 2008024776A1
Authority
WO
WIPO (PCT)
Prior art keywords
axon
regeneration
inhibitor
jnk
injury
Prior art date
Application number
PCT/US2007/076423
Other languages
English (en)
Inventor
Zhigang He
Glenn Yiu
Original Assignee
Children's Medical Center Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Children's Medical Center Corporation filed Critical Children's Medical Center Corporation
Publication of WO2008024776A1 publication Critical patent/WO2008024776A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • 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
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • 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
    • 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

Definitions

  • the field of the invention is inhibition of cJun-N-terminal kinase (JNK) activity to promote regeneration of a lesioned CNS axon of a mature neuron.
  • JNK cJun-N-terminal kinase
  • Adult CNS injury often results in the exposure of severed axons to a variety of myelin-derived inhibitory molecules which can severely limit axon repair. Many of these molecules including Nogo-A, MAG, and OMgp [1-3] can bind to the neuronal receptor NgR [4-7], and trigger intracellular signals in neurons that ultimately result in their failure to regenerate.
  • RhoA-associated kinase (ROCK) [8-12].
  • Small GTPases of the Rho family such as RhoA, Racl, and Cdc42 are known regulators of the actin cytoskeleton [13].
  • RhoA activation can signal through LIM-kinase and Slingshot (SSH) phosphatase to regulate the actin depolymerization factor cofilin [14].
  • SSH Slingshot
  • NgR is a GPI-linked molecule and lacks an intracellular domain, it must rely on transmembrane co-receptors to transduce the inhibitory signals.
  • the identification of two TNFR family members, p75 [2, 15-17] and TROY [18, 19], as functionally homologous co- receptors for NgR suggests that intracellular domains shared by both molecules may be involved in triggering downstream signals like RhoA.
  • Rho-GDI Rho guanine dissociation inhibitor
  • GEF guanine nucleotide exchange factor
  • TNF receptors can be classified into two major groups. Some members have a cytoplasmic death domain which can trigger the caspase signaling cascade. Other members such as p75 and TROY contain intracellular motifs that interact with TNF-receptor associated factors (TRAFs). Receptor recruitment of these adaptor molecules leads to the activation of signaling mediators such as JNK and NF-KB [21-23].
  • TNF-receptor associated factors TNF-receptor associated factors
  • JNK activation has been implicated in a variety of signaling networks, and is associated with the phosphorylation of microtubule-associated proteins (MAPs) such as MAP2 [26], Tau [27], and doublecortin [28], as well as stress-related transcription factors like cJun [29].
  • MAPs microtubule-associated proteins
  • JNK is enriched in neuronal axons and can associate with motor proteins [30].
  • a number of reports have shown that nerve fiber transection triggers a characteristic axonal response that leads to dramatic changes in the transcription program of the injured neuron [31-33]. However, it is not known whether these effects result from the interruption of constitutive retrograde signals in the severed axons, or from positive electrical or molecular injury signals arising from the lesion site.
  • JNKs have been implicated in the initiation of this axonal response because they are rapidly activated following nerve injury and may be transported along microtubules through their association with motor proteins [30].
  • immediate-early transcription factors of the AP-I family including cJun are also highly induced in response to neuronal injury, and in part mediate the transcriptional response [34].
  • Current models postulate that cell stress and inflammatory signals trigger the activation and retrograde transport of JNK along axons to phosphorylate cJun at the cell body [35].
  • JNK and cJun have been linked to axotomy- induced cell death and axonal regeneration [36].
  • US Pat Publ No. 20060122179 to Zeldis et al. proposes treating or preventing a CNS injury, including axonal injury, by administering a therapeutically or prophylactically effective amount of a JNK inhibitor to a patient.
  • Zeldis et al used 10mg/kg dosages, which they translate to final plasma and brain concentrations of 7 and 65uM, respectively.
  • Zeldis elsewhere recites enormous dosage ranges of "about 1 mg to about 10,000 mg per day" (para 0248).
  • JNK inhibition has been reported to reduce neuronal apoptotic death in several neurodegenerative diseases and ischemic brain damage (see e.g. Yang et al, Proc. Natl. Acad. Sci. U. S. A. (1997) 94:3004- 3009; Yang et al., Nature (1997) 389:865- 870; Okuno et al, J. Neurosci. (2004) 24:7879-7887; Saporito et al, J Pharmacol Exp Ther (1999) 288:421-427; and Gao et al, J. Cereb. Blood Flow Metab. (2005) 25:694-712). [012] Yin et al (Neurobiol Dis.
  • JNK activation contributes to trauma-induced DP5 expression and subsequent apoptosis in spinal cord injury.
  • Yin suggests that the JNK signaling pathway may be a potential target for therapeutic interventions for spinal cord injury, and proposes further studying the effect of JNK inhibition after spinal cord injury.
  • Yin's rats received 15mg/kg dosages of SP600125.
  • One aspect of the invention is a method of promoting regeneration of a lesioned CNS axon of a mature neuron determined to be subject to regeneration inhibition by endogenous cJun-N-terminal kinase (JNK).
  • JNK endogenous cJun-N-terminal kinase
  • the method comprises the steps of: (a) contacting the neuron with an exogenous JNK signaling pathway inhibitor at a concentration sufficient to only partially inhibit the JNK signaling, and thereby promote a resultant regeneration of the axon; and (b) detecting the resultant regeneration of the axon.
  • the inhibitor is
  • the lesion results from a traumatic injury, an acute spinal cord injury, or CNS degeneration.
  • the lesioned axon is in the spinal cord of a patient, and the inhibitor is intrathecally administered to the patient.
  • the axon is a CNS axon of a sensory neuron, or a CNS axon of a cerebellar granule neuron.
  • the detecting step may be effected by an indirect or direct assay of axon regeneration.
  • JNK cJun-N-terminal kinase
  • TNFR tumor necrosis factor receptor
  • NgR Nogo-66 receptor-
  • the method comprises the steps of: contacting the neuron with an exogenous JNK signaling pathway inhibitor at a concentration sufficient to only partially inhibit the JNK signaling, and thereby promote a resultant regeneration of the axon; and detecting the resultant regeneration of the axon.
  • the lesioned CNS axon is subject to regeneration inhibition by JNK, which may be detected directly, indirectly, or inferred.
  • activated JNK in a neuron can be detected by antibody specific for phosphorylated JNK.
  • the presence of activated JNK in the neuron may be inferred where the lesioned axon is in contact with injured myelin.
  • the lesioned axon is a CNS axon of a dorsal root ganglion (DRG) sensory neuron.
  • the lesioned axon is a CNS axon of a cerebellar granule neuron.
  • DRG dorsal root ganglion
  • terminally-differentiated, non-embryonic neuron may be in vitro or in situ in a patient.
  • the patient is a mammal (e.g. human, companion animal, livestock animal, rodent or primate animal model for neurodegeneration or CNS injury, etc.).
  • the lesion can result from traumatic injury, optic nerve injury or disorder, brain injury, stroke, chronic neurodegeneration such as caused by neurotoxicity or a neurological disease or disorder (e.g. Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple system atrophy (MSA), etc.).
  • a neurological disease or disorder e.g. Huntington's disease, Parkinson's disease, Alzheimer's disease, multiple system atrophy (MSA), etc.
  • the inhibitor is used to treat an ocular injury or disorder (e.g. toxic amblyopia, optic atrophy, higher visual pathway lesions, disorders of ocular motility, third cranial nerve palsies, fourth cranial nerve palsies, sixth cranial nerve palsies, internuclear ophthalmoplegia, gaze palsies, eye damage from free radicals, etc.), or an optic neuropathy (e.g., an ocular injury or disorder (e.g. toxic amblyopia, optic atrophy, higher visual pathway lesions, disorders of ocular motility, third cranial nerve palsies, fourth cranial nerve palsies, sixth cranial nerve palsies, internuclear ophthalmoplegia, gaze palsies, eye damage from free radicals, etc.), or an optic neuropathy (e.g.
  • an optic neuropathy e.g.
  • ischemic optic neuropathies toxic optic neuropathies, ocular ischemic syndrome, optic nerve inflammation, infection of the optic nerve, optic neuritis, optic neuropathy, papilledema, papillitis, retrobulbar neuritis, commotio retinae, glaucoma, macular degeneration, retinitis pigmentosa, retinal detachment, retinal tears or holes, diabetic retinopathy, iatrogenic retinopathy, optic nerve drusen, etc.).
  • the lesion results from acute or traumatic injury such as caused by contusion, laceration, acute spinal cord injury, etc.
  • the lesioned CNS axon is in CNS white matter, particularly white matter that has been subjected to traumatic injury.
  • the contacting step is initiated within 96, 72, 48, 24, or 12 hours of formation of the lesion.
  • our methods allow treatment even after neuronal apoptosis; hence, treatment with a JNK inhibitor may be initiated or continued subsequent to cessation of apoptosis in indications that are associated with neuronal apoptosis.
  • the contacting step is initiated, and/or treatment is continued, more than 5, 7, 14, 30, or 60 days after formation of the lesion.
  • the inhibitor can be administered to the injured neuron in combination with, or prior or subsequent to, other treatments such as the use of anti-inflammatory or anti-scarring agents, growth or trophic factors, etc.
  • the lesion results from acute spinal cord injury and the method additionally comprises contacting the neuron with methylprednisolone sufficient to reduce inflammation of the spinal cord.
  • the inhibitor is administered in combination with trophic and/or growth factors such as NT-3 (Piantino et al, Exp Neurol. (2006) Jun 7; [Epub ahead of print]), inosine (Chen et al, Proc Natl Acad Sci U S A.
  • JNK inhibitors are well-known in the art (see e.g. US Pat No 6,987,184 to Sakata et al; US Pat No 7,084,159 to Cao et al; US Pat Publ No.
  • JNK pathway inhibitors include CEP-1347 (Maroney et al, J Neurosci. (1998) 18:104-11), SP600125 (Bennett et al, Proc Natl Acad Sci U S A. (2001) 98:13681-6), AS601245 (Carboni et al, J Pharmacol Exp Ther. (2004) 310:25-32), DJNKl (Manning and Davis, Nat Rev Drug Discov.
  • the inhibitor is siRNA targeted to a JNK pathway member (e.g. JNKl, JNK2, and JNK3).
  • the inhibitor is preferably used at a concentration that permits some basal level of JNK activity, i.e. the inhibitor only partially inhibits the JNK signaling and does not result in complete JNK inhibition.
  • Concentrations of a specific inhibitor that achieve partial inhibition of JNK signaling are readily determined using assays such as a neurite outgrowth assay (e.g. see Example 1).
  • Many inhibitors provide a bell-shaped titration curve beginning with a dose-dependent concentration range in which increasing inhibitor concentration results in increased neutralization of myelin- associated outgrowth inhibition and hence, increased axon regeneration, followed by an inversion wherein increasing inhibitor concentration results in decreasing degrees of axon regeneration until there is complete JNK inhibition and no detectable resultant axon regeneration.
  • Preferred inhibitors achieve partial inhibition of JNK signaling at nanomolar or micromolar concentrations.
  • the JNK inhibitor is SP600125 that contacts the neuron at nanomolar concentrations.
  • the inhibitor does not inhibit signaling of p38 MAPK.
  • the inhibitor is contacted with the neuron using a suitable drug delivery method and treatment protocol sufficient to promote regeneration of the axon.
  • the inhibitor is added to the culture medium, usually at nanomolar or micromolar concentrations.
  • the inhibitor can be administered orally, by intravenous (i.v.) bolus, by i.v. infusion, subcutaneously, intramuscularly, ocularly (intraocularly, periocularly, retrobulbarly, intravitreally, subconjunctivally, topically, by subtenon administration, etc.), intracranially, intraperitoneally, intraventricularly, intrathecally, by epidural, etc.
  • compositions may be administered in one or more dosage form(s) (e.g. liquid, ointment, solution, suspension, emulsion, tablet, capsule, caplet, lozenge, powder, granules, cachets, douche, suppository, cream, mist, eye drops, gel, inhalant, patch, implant, injectable, infusion, etc.).
  • dosage forms may include a variety of other ingredients, including binders, solvents, bulking agents, plasticizers etc.
  • the inhibitor is contacted with the neuron using an implantable device that contains the inhibitor and that is specifically adapted for delivery to a CNS axon of neuron.
  • Examples of devices include solid or semi-solid devices such as controlled release biodegradable matrices, fibers, pumps, stents, adsorbable gelatin (e.g. Gelfoam), etc.
  • the device may be loaded with premeasured, discrete and contained amounts of the inhibitor sufficient to promote regeneration of the axon.
  • the device provides continuous contact of the neuron with the inhibitor at nanomolar or micromolar concentrations, preferably for at least 2, 5, or 10 days.
  • the subject methods typically comprise the further step of detecting a resultant regeneration of the axon.
  • axonal regeneration may be detected by any routinely used method to assay axon regeneration such as a neurite outgrowth assay.
  • axonal regeneration can be detected directly using imaging methodologies such as MRI, or indirectly or inferentially, such as by neurological examination showing improvement in the targeted neural function.
  • the detecting step may occur at any time point after initiation of the treatment, e.g. at least one day, one week, one month, three months, six months, etc. after initiation of treatment.
  • the detecting step will comprise an initial neurological examination and a subsequent neurological examination conducted at least one day, week, or month after the initial exam. Improved neurological function at the subsequent exam compared to the initial exam indicates resultant axonal regeneration.
  • the specific detection and/or examination methods used will usually be based on the prevailing standard of medical care for the particular type of axonal lesion being evaluated (i.e.
  • the invention also provides inhibitor-eluting or inhibitor-impregnated CNS implantable solid or semi-solid devices.
  • CNS implantable devices include polymeric microspheres (e.g. see Benny et al., Clin Cancer Res. (2005) 11:768-76) or wafers (e.g. see Tan et al., J Pharm Sci. (2003) 4:773-89), biosynthetic implants used in tissue regeneration after spinal cord injury (reviewed by Novikova et al., Curr Opin Neurol. (2003) 6:711-5), biodegradable matrices (see e.g.
  • Preferred devices are particularly tailored, adapted, designed or designated for CNS implantation.
  • the implantable device may contain one or more additional agents used to promote or facilitate neural regeneration.
  • an implantable device used for treatment of acute spinal cord injury contains the inhibitor and methylprednisolone or other antiinflammatory agents.
  • the implantable device contains the inhibitor and a nerve growth factor, trophic factor, or hormone that promotes neural cell survival, growth, and/or differentiation, such as brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), inosine, oncomodulin, NT- 3, etc.
  • BDNF brain-derived neurotrophic factor
  • CNTF ciliary neurotrophic factor
  • NEF nerve growth factor
  • inosine inosine
  • oncomodulin NT- 3, etc.
  • JNK is a member of the mitogen-activated protein kinase (MAP kinase) family which is abundantly expressed in axons and growth cones, and has been implicated in numerous roles including cell stress and cell proliferation.
  • MAP kinase mitogen-activated protein kinase
  • rat DRGs were dissected, dissociated and plated onto immobilized substrates (AP (100 ng/cm 2 ) or AP-Nogo-66 (100 ng/cm 2 )).
  • AP 100 ng/cm 2
  • AP-Nogo-66 100 ng/cm 2
  • Cells were cultured for 24h before fixation with 4% paraformaldehyde and staining with a neuronal- specific anti- ⁇ -tubulin III antibody (Tuj-1, Covance).
  • Tuj-1 neuronal-specific anti- ⁇ -tubulin III antibody
  • the average lengths of the longest neurite in individual neurons were measured from at least 150 neurons per condition, from duplicate wells and from three independent experiments, and quantified as described previously [6, 15]. Immunohistochemistry was performed using antibodies against p75 (Chemicon).
  • Example 2 Partial inhibition of JNK activation promotes axonal regeneration after spinal injury in rats
  • This animal study demonstrates that in an animal model for spinal injury, axonal regeneration can be promoted by intrathecal or intravenous administration of SP600125. Methodology for this animal study was adapted from Nash et al (J. Neurosci (2002) 22:7111- 7120), Luo et al (Molecular Pain (2005) 1:29), and Obata et al (J. Neurosci. (2004) 24:10211- 22).
  • the apparatus is configured such that in order to retrieve a food pellet from a slot, a rat must extend a forelimb through a hole in the Plexiglas divider, and grasp the pellet and lift it over the gap and out of the slot. Jf the rat merely rakes the food in the slot towards the hole in the Plexiglas, the food will drop from the slot into the gap and fall to the floor of the minor compartment.
  • the floor of the minor compartment can be configured to allow a rat to retrieve food that drops or it can be lowered to prevent the rat from reaching dropped food.
  • the rats Prior to inducing spinal injury, the rats are food restricted, receiving ⁇ 3 gm food/ 100 gm body weight per day, before and throughout training and testing.
  • Weight is monitored to ensure that rats are reduced to no less than 80% of their original body weight at any time. All rats are given shaping periods for 2-3 d in the box to allow them to learn the task while they become familiar with the testing situation. Animals are trained twice per day for 5 d and then tested twice per day for 5 d, and presurgical DFR data is collected. During the testing period, rats are given 5 min to complete the task and are allowed to make as many attempts as they want during this time period. Rats are required to return to at least 95% of their original weight to ensure that they are healthy before undergoing surgery.
  • Rats are randomly assigned to control or experimental groups. Sham control rats undergo surgical procedure without lesioning, and with or without placement of a mini osmotic pump (Alzet type 2001; Durect, Cupertino, CA). Lesioned control rats receive no treatment, tail vein injection with vehicle only treatment, insertion of a mini osmotic pump only treatment, or insertion of a mini osmotic pump with vehicle only treatment. After anesthesia with isoflurane, the rats are placed on an operating board in such a way as to bend the cervical spinal cord for maximum exposure. A laminectomy is performed exposing the dorsum of the spinal cord between C2 and C4.
  • the dorsal columns are identified bilaterally, and, in all rats except for those in the sham group, a suture needle is passed through the spinal cord, isolating the dorsal funiculus. The suture thread is gently lifted, and a pair of iridectomy scissors is used to bilaterally transect the dorsal funiculus, thereby transecting the dorsal corticospinal tract (CST). Visualization of the dorsal horns and the central gray commissure confirms accuracy of the lesion borders.
  • Rats designated for SP600125 treatment or corresponding control treatment are implanted with mini osmotic pumps adjacent to the lesion site.
  • the pumps in the SP600125 treatment group operate at a rate of 1 ⁇ l/hr for a period of 7 days and are filled with SP600125 at a concentration of 500 ng/ ⁇ l.
  • the overlying muscles and skin are sutured, and the rats are placed on a heating pad to maintain body temperature.
  • Each rat receives a single dose of buprenorphine (0.1 mg/kg) immediately after surgery to alleviate pain.
  • the rats in the tail vein injection treatment group receive a bolus injection of 100 ⁇ g/kg SP600125 in a saline/DMSO vehicle. The treatment is repeated every 24 hours on days 1 through 7 post-lesion. Vehicle only control rats undergo the same treatment but are injected with an equal volume of saline/DMSO in a tail vein.
  • Rats are trained twice per week during weeks 2-5 after surgery. Some rats may be profoundly impaired such that they may not be able to grasp food in the DFR task in the early postsurgical period.
  • the apparatus can be configured to allow the rats to rake food into the main compartment that drops from the slot onto the floor of the minor compartment (see Nash et al., supra). This ensures that the reaching portion of the DFR task does not extinguish.
  • the severity of the grasping impairment decreases as the postsurgical period increases, and the configuration of the apparatus that does not permit food raking can be gradually reestablished. By the end of the postsurgical recovery period, all rats are able to successfully perform the DFR task, to some degree.
  • rats are tested twice per day for 5 d, and postsurgical DFR data is collected by a blinded investigator. Just as during the presurgery testing period, the rats are allowed 5 min to complete the task during the postsurgery testing period and are allowed to make as many attempts as they want during this time period. The data is collected in terms of total number of attempts and percentage of successful attempts.
  • An attempt is scored only when a rat reaches into a slot and displaces the pellet or drops it to the floor of the minor compartment. A successful attempt is scored when a rat grasps a pellet, lifts it over the gap and pulls it through the Plexiglas divider into the main portion of the testing apparatus.
  • Sham animals perform the DFR task as well postsurgically as they do presurgically, demonstrating that only the lesion, and no other portion of the surgical procedure, inhibits the rats' abilities to perform the DFR task.
  • the lesion and vehicle groups are the most impaired of all of the groups after surgery.
  • the lesion and vehicle groups are able to perform the DFR task with a success rate of only about 40%.
  • Significantly better performance by the SP600125-treated group demonstrates the effect of the treatment on functional recovery after spinal injury.
  • rats are prepared for injection of biotin dextran tetramethylrhodamine (BDT; Molecular Probes). This fluorescent anterograde tracer, injected into the primary motor cortex, is used to label CST axons caudal to the lesion site in the spinal cord. After anesthesia with isoflurane (5%), rats are placed in a stereotaxic instrument, and a total of six stereotaxically determined holes (0.9 mm diameter) are drilled in the skull over the primary motor cortices associated with the forelimbs.
  • BDT biotin dextran tetramethylrhodamine
  • the anteroposterior (AP) and mediolateral (ML) coordinates for these injections, from bregma, are as follows: ⁇ 0.5 AP and ⁇ 3.5 ML; ⁇ 1.5 A/P and ⁇ 2.5 ML; and ⁇ 2.5 AP and ⁇ 1.5 ML. All injections are delivered at a depth of 2.5mm from the surface of the skull.
  • a 10 ⁇ l Hamilton syringe is used to inject BDT bilaterally into layer V of the cortex.
  • Three injections into each cortical hemisphere are used to administer a total of 1.2 ⁇ l of the anterograde tracer.
  • Bone wax (Ethicon, Somerville, NJ) is used to seal the holes in the skull, the scalp is sutured, and a single dose of buprenorphine (0.1 mg/kg) is administered immediately after surgery to alleviate pain. Rats are killed 3 d after tracer injections.
  • rats are anesthetized with chloral hydrate (10 ml/kg) and perfused transcardially with 300 ml of PBS, pH 7.4, followed by 300 ml of 4% paraformaldehyde in 0.1 M phosphate buffer. After the animals are killed, all brains and spinal cords are removed and soaked overnight in 30% sucrose in a 0.1M phosphate buffer solution. The brains are cut coronally and the spinal cords are cut horizontally at a thickness of 20 ⁇ m with a freezing microtome and mounted on ProbeOn (Fisher Scientific, Pittsburgh, PA) coated slides.
  • ProbeOn ProbeOn
  • Brain and spinal cord sections are examined using a Nikon (Tokyo, Japan) Labophot fluorescent microscope, and images are captured using a digital still camera.
  • the forelimb representation of the primary motor cortex is identified based on the stereotaxic BDT injection sites.
  • the primary motor cortex is examined in all rats. Presence of Fluorogold-labeled neurons in layer V of the primary motor cortex, confirms that the dorsal CST axons were transected during the lesioning procedure. Because all CST axons located in the dorsal funiculus are transected during surgery and not just those in the forelimb representation, Fluorogold-labeled neurons are found throughout the primary motor cortex in layer V. The only exception to this labeling pattern is in the brains of the rats in the sham group whose brains have no Fluorogold label.
  • the spinal cord caudal to the lesion is examined, and the BDT-labeled axons occupying the region of the spinal cord normally occupied by the dorsal CST are counted. For each section, the number of BDT-labeled axons is counted at 3 mm intervals caudal to the lesion, beginning 1 mm distal to the injury (i.e., 1 mm, 4 mm, 7 mm, etc.) and ending 19 mm caudal to the lesion site. Innervation of the rat forepaw extends to Tl, a distance of 15.1 mm from the lesion at C3.
  • the mean number of axons is highest in the sham group, and, at each distance examined, the mean number of labeled axons in the sham group is significantly higher than in the other groups.
  • No significant difference is observed between the means of the lesion and vehicle groups at any distance examined. In these groups, axons are found only a short distance caudal to the injury, and, by 10 mm distal to the lesion to the farthest distance examined, all of the tissue is virtually devoid of axons.
  • Significantly more labeled axons at each distance in the SP600125-treated group compared to lesioned control rats demonstrates that this treatment promotes axonal regeneration after spinal injury.
  • Example 3 Improved neurological outcome following SP600125 treatment for acute spinal cord injury
  • NASCIS II National Acute Spinal Cord Injury Studies
  • MPSS methylprednisolone
  • SCI spinal cord injury
  • the placebo group has a loading dose of placebo and then 56 days of placebo.
  • the low dose SP600125 group has a 50-mg loading dose administered intravenously (i.v.) followed by 10 mg/day i.v. for 56 days.
  • the high dose SP600125 group has a 250-mg loading dose followed by 50 mg/day for 56 days.
  • the baseline neurologic assessment includes both the AIS and detailed American Spinal Injury Association (ASIA) motor and sensory examinations. Modified Benzel Classification and the ASIA motor and sensory examinations are performed at 4, 8, 16, 26, and 52 weeks after injury.
  • the Modified Benzel Classification is used for post-baseline measurement because it rates walking ability and, in effect, subdivides the broad D category of the AIS. Because most patients have an unstable spinal fracture at baseline, it is not possible to assess walking ability at that time; hence the use of different baseline and follow- up scales. Marked recovery is defined as at least a two-grade equivalent improvement in the Modified Benzel Classification from the baseline AIS.
  • the primary efficacy assessment is the proportion of patients with marked recovery at week 26.
  • the secondary efficacy assessments include the time course of marked recovery and other established measures of spinal cord function (the ASIA motor and sensory scores, relative and absolute sensory levels of impairment, and assessments of bladder and bowel function).
  • Rats are fasted overnight and anesthetized with 3.5% isoflurane in 100% oxygen in a vented anesthesia chamber. Following endotracheal intubation with a 16-gauge Teflon catheter, the rats are mechanically ventilated with 2% isoflurane in 100% oxygen for the surgical preparation and for the impact injury.
  • Intracranial pressure (ICP) is monitored by a 3F microsensor transducer (Codman & Schurtleff, Randolph, MA) inserted in the left frontal lobe, well away from the impact site. ICP is monitored during the impact injury as a measure of the severity of the injury. Rectal temperature is maintained at 36.5-37.5 0 C by a heating pad, which is controlled by rectal thermistor. Brain temperature is kept constant at 37 0 C with the help of a heating lamp directed at the head.
  • the rats that are to receive i.c.v. administration of AS601245 receive mini-pump implants using procedures described by Kitamura et al (J Pharmacol Sci (2006) 100:142-148). Briefly, the rats are fixed in a stereotaxic frame (David Kopf Instruments, Tujunja, CA). Guide cannulae are implanted into the left lateral ventricle (Bregma -0.8 mm, lateral 1.5 with a depth of 3.7 mm below the dura).
  • Each cannula is then connected by a catheter to an ALZET® mini-osmotic pump implanted subcutaneously in the scapular region and configured to continuously infuse the drug to achieve the specified daily dose of AS601245 (or vehicle only for control groups).
  • AS601245 or vehicle only for control groups.
  • each dose of AS601245 is dissolved in 1 ml of sterile 0.9% saline so that the volume delivered is the same for each group and only the dosage of AS601245 varies.
  • the first dose is administered within 1 hour following impact injury. And once daily thereafter for the assigned treatment duration.
  • the rats After removing all catheters and suturing the surgical wounds, the rats are allowed to awaken from anesthesia. For the first 3 days post injury, the rats are treated with butorphanol tartrate, 0.05 mg of i.m. every 12 h (twice a day), for analgesia and enrofloxacin 2.27%, 0.1 ml of EVI qd, to reduce the risk of postoperative infections.
  • the outcome measures are performed by investigators who are blinded to the treatment group. At 2 weeks after the impact, the animals are deeply anesthetized with a combination of ketamine/xylazine/acepromazine and perfused transcardially with 0.9% saline, followed by 10% phosphate buffered formaldehyde. The entire brain is removed and fixed in 4% formalin. The fixed brains are examined grossly for the presence of contusion, hematoma, and herniation. The brains are photographed, sectioned at 2-mm intervals, and then embedded in paraffin. Hematoxylin and eosin (H&E) stained 9- ⁇ m thick sections are prepared for histologic examination.
  • H&E Hematoxylin and eosin
  • the H&E-stained coronal sections are digitized using a Polaroid Sprint Scanner (Polaroid Corporation, Waltham, MA) equipped with a PathScan Enabler (Meyer Instruments, Houston, TX).
  • the injury volume is measured by determining the cross-sectional area of injury in each H&E-stained coronal image and multiplying by the thickness of the tissue between the slices.
  • This slab volume technique is implemented on the image processing program Optimas 5.2 (Optimas Corporation, Seattle, WA). Neurons in the middle 1-mm segments of the CAl and CA3 regions of the hippocampus are counted at a magnification of 200X.
  • Neurons are identified by nuclear and cytoplasmic morphology, and individual cells are counted whether normal or damaged. Neurons with cytoplasmic shrinkage, basophilia, or eosinophilia or with loss of nuclear detail are regarded as damaged. The regions measured are 1 mm long and 1 mm wide (0.5 mm on either side of the long axis of the segment). The total number of neurons and the number of neurons that appear normal are expressed as neurons per squared millimeter.
  • Optic nerves are cryosectioned at 10 ⁇ m and stained with an anti-GAP43 antibody (Chemicon) to detect regenerating axons [Fischer et al, supra]. Little regeneration is detected in DMSO-treated control mice. However, suppression of JNK activity by injury site application of SP600125 results in significant increases in axonal regrowth and the number of regenerating axons, measured 0.25 mm beyond the injury site, compared to control mice.
  • Example 6 JNK inhibition promotes neural regeneration in animal models of focal brain ischemia
  • Sprague-Dawley male rats weighing -250 g are anesthetized with pentobarbital [60 mg/kg body weight (BW)].
  • Body core temperature is thermostatically maintained at 37 0 C by using a water blanket and a rectal thermistor (Harvard Apparatus) for the duration of the anesthesia.
  • the carotid arteries are visualized, and the right carotid is occluded by two sutures and cut.
  • a burr hole adjacent and rostral to the right orbit allows visualization of the MCA, which is cauterized distal to the rhinal artery. Animals are then positioned on a stereotaxic frame.
  • the contralateral carotid artery is occluded for 1 h by using traction provided by a fine forceps.
  • 0.5 ml of a 1 ⁇ g/ml solution of SP600125 or vehicle control is administered at lhr, 1 day, 5 days, or 10 days from the onset of the reversible carotid occlusion.
  • the animals are killed after 15 days, the brains are removed, and serial 1-mm thick sections through the entire brain are cut by using a brain matrix device (Harvard Apparatus).
  • Each section is then incubated in a solution of 2% triphenyltetrazolium chloride (wt/vol) in 154 mM NaCl for 30 min at 37 0 C and stored in 4% paraformaldehyde until analysis.
  • Quantification of the extent of injury is determined by using a computerized image analysis system (MCID, Imaging Research, St. Catharine's, ON, Canada). To accomplish this, a digital image of each section is obtained and the area of injury delineated by outlining the region in which the tetrazolium salt is not reduced, i.e., nonviable tissue. For cases in which the necrosis is so severe that tissue is actually lost and therefore the borders can not be directly assessed, an outline of the contralateral side is used to estimate the volume of injured brain. Total volume of infarct is calculated by reconstruction of the serial 1-mm thick sections.

Landscapes

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

Abstract

Selon la présente invention, la régénération d'un axone lésé du SNC appartenant à un neurone mature, dont il été déterminé que la régénération est sujette à une inhibition par une cJun-N-terminal kinase (JNK) endogène, est favorisée par la mise en contact du neurone avec un inhibiteur exogène de la JNK à une concentration suffisante pour inhiber partiellement la JNK, et favoriser de la sorte une régénération consécutive de l'axone.
PCT/US2007/076423 2006-08-22 2007-08-21 L'inhibition du signal jnk favorise la régénération des axones du snc WO2008024776A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US83959506P 2006-08-22 2006-08-22
US60/839,595 2006-08-22

Publications (1)

Publication Number Publication Date
WO2008024776A1 true WO2008024776A1 (fr) 2008-02-28

Family

ID=39107139

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2007/076423 WO2008024776A1 (fr) 2006-08-22 2007-08-21 L'inhibition du signal jnk favorise la régénération des axones du snc

Country Status (1)

Country Link
WO (1) WO2008024776A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010151638A1 (fr) * 2009-06-25 2010-12-29 Medical College Of Georgia Research Institute, Inc. Inhibiteurs de jnk destinés à être utilisés dans le traitement de l'amyotrophie spinale
CN102307593A (zh) * 2008-10-22 2012-01-04 弗·哈夫曼-拉罗切有限公司 轴突变性的调节
US9777059B2 (en) 2007-11-30 2017-10-03 Genentech, Inc. Anti-VEGF antibodies
CN109303782A (zh) * 2018-10-24 2019-02-05 厦门大学 Jnk-in-8在制备干性年龄相关性黄斑变性的神经保护剂中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020058245A1 (en) * 1998-05-14 2002-05-16 Liu Ya Fang JNK inhibitors for the treatment of neurological disorders
US20040087642A1 (en) * 2002-10-24 2004-05-06 Zeldis Jerome B. Methods of using and compositions comprising a JNK inhibitor for the treatment, prevention, management and/or modification of pain

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020058245A1 (en) * 1998-05-14 2002-05-16 Liu Ya Fang JNK inhibitors for the treatment of neurological disorders
US20040087642A1 (en) * 2002-10-24 2004-05-06 Zeldis Jerome B. Methods of using and compositions comprising a JNK inhibitor for the treatment, prevention, management and/or modification of pain

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MARONEY A.C. ET AL.: "CEP-1347(KT7515), an inhibitor of JNK activation, rescues sympathetic neurons and neuronally differentiated PC12 cells from death evoked by three distinct insults", J. NEUROCHEM., vol. 73, no. 5, November 1999 (1999-11-01), pages 1901 - 1912 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9777059B2 (en) 2007-11-30 2017-10-03 Genentech, Inc. Anti-VEGF antibodies
CN102307593A (zh) * 2008-10-22 2012-01-04 弗·哈夫曼-拉罗切有限公司 轴突变性的调节
US9101645B2 (en) 2008-10-22 2015-08-11 Genentech, Inc. Modulation of axon degeneration
CN102307593B (zh) * 2008-10-22 2016-04-13 弗·哈夫曼-拉罗切有限公司 轴突变性的调节
US9937224B2 (en) 2008-10-22 2018-04-10 Genentech, Inc. Modulation of axon degeneration
WO2010151638A1 (fr) * 2009-06-25 2010-12-29 Medical College Of Georgia Research Institute, Inc. Inhibiteurs de jnk destinés à être utilisés dans le traitement de l'amyotrophie spinale
CN109303782A (zh) * 2018-10-24 2019-02-05 厦门大学 Jnk-in-8在制备干性年龄相关性黄斑变性的神经保护剂中的应用

Similar Documents

Publication Publication Date Title
Tweedie et al. Tumor necrosis factor-α synthesis inhibitor 3, 6′-dithiothalidomide attenuates markers of inflammation, Alzheimer pathology and behavioral deficits in animal models of neuroinflammation and Alzheimer’s disease
US8142782B2 (en) EGFR inhibitors promote axon regeneration
US5733871A (en) Methods for the treatment of neuronal damage associated with ischemia, hypoxia or neurodegeneration
US8728756B2 (en) Promoting axon regeneration in the adult CNS through control of protein translation
Cotton et al. Anticonvulsant effects of magnesium sulfate on hippocampal seizures: therapeutic implications in preeclampsia-eclampsia
US20080051319A1 (en) Inhibiting JNK Signaling Promotes CNS Axon Regeneration
WO2008024776A1 (fr) L'inhibition du signal jnk favorise la régénération des axones du snc
US20080260644A1 (en) Chloride transport upregulation for the treatment of traumatic brain injury
PT792160E (pt) Factor neurotrofico derivado das celulas gliais utilizado como agente neuroprotector
JP3623501B2 (ja) インターロイキン−１阻害化合物による神経病学的状態の治療
US7524640B2 (en) Inhibiting Smad2/3 signaling promotes neurite outgrowth in dorsal root ganglia
EP1655032A1 (fr) Composes destines au traitement de maladies demyelinisantes et auto-immunes
EP1795193B1 (fr) Histidine pour supprimer la necrose des tissus cerebraux attribues a une ischemie de longue duree
WO2001049313A1 (fr) Utilisation du facteur de croissance neuronale pour la production d'un medicament destine au traitement d'affections allergiques
AU2013248182A1 (en) Promoting axon regeneration in the adult cns through control of protein translation
Schwartz et al. Optic nerve disease and injury: Prospects for induction of regeneration
Ombrellino et al. Role of vagus nerve signaling in CNI-1493-mediated suppression of acute

Legal Events

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

Ref document number: 07841166

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

NENP Non-entry into the national phase

Ref country code: RU

122 Ep: pct application non-entry in european phase

Ref document number: 07841166

Country of ref document: EP

Kind code of ref document: A1