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WO2014202833A1 - Traitement des lésions neronales - Google Patents

Traitement des lésions neronales Download PDF

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Publication number
WO2014202833A1
WO2014202833A1 PCT/FI2014/050490 FI2014050490W WO2014202833A1 WO 2014202833 A1 WO2014202833 A1 WO 2014202833A1 FI 2014050490 W FI2014050490 W FI 2014050490W WO 2014202833 A1 WO2014202833 A1 WO 2014202833A1
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Prior art keywords
gam
injury
neuronal
neuron
spinal cord
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PCT/FI2014/050490
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English (en)
Inventor
Heikki Rauvala
Mikhail PAVELIEV
Juha Kuja-Panula
Mikhail KISLIN
Natalia Kulesskaya
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University Of Helsinki
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Publication of WO2014202833A1 publication Critical patent/WO2014202833A1/fr

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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators

Definitions

  • the present invention relates to treatment of neuronal injuries. Specifically, the present invention relates to novel mechanisms and means for promoting neurite outgrowth, neural regeneration and/or maintenance of neu- rites (axons and dendrites) in pathologies where chondroitin sulphate proteoglycans (CSPGs) display adverse effects, such as in CNS injuries.
  • CSPGs chondroitin sulphate proteoglycans
  • Nervous system injuries affect millions of people every year. As a result of this high incidence of neurological injuries, nerve regeneration and repair are becoming a rapidly growing field dedicated to the discovery of new ways to recover nerve functionality after injury.
  • the nervous system is divided into two parts: the central nervous system (CNS), which consists of the brain and spinal cord, and the peripheral nervous system (PNS), which consists of cranial and spinal nerves along with their associated ganglia.
  • CNS central nervous system
  • PNS peripheral nervous system
  • a brain injury or brain damage is the destruction or degeneration of brain cells in the brain of a living organism. Brain injuries can be classified along several dimensions. Primary and secondary brain injuries are ways to classify the injury processes that occur in brain injury.
  • Post traumatic regeneration of the brain and spinal cord is a major unsolved medical problem because the brain and spinal cord are not able to regenerate like the peripheral nervous system. While peripheral axons regenerate in patients after nerve injury, brain and spinal cord axons fail to regenerate due to glial scar formation and the inhibitory action of chondroitin sulphate proteoglycans, CSPGs, in the scar. In addition, those factors that promote peripheral nerve regeneration, for instance nerve growth factor, NGF fail to improve regeneration in the brain and spinal cord.
  • the central nervous system and peripheral nervous system are very different in their reactions to drug treatment and regeneration ability.
  • Traumatic brain injury can result from direct impacts to the head, such as from a fall, or from acceleration/deceleration injuries, such as those encountered in motor vehicle accidents.
  • TBI Traumatic brain injury
  • TBI is the leading cause of death and disability in the most active population ( ⁇ 45 years of age). Individuals under the age of 5 and over the age of 75 are also particularly at risk. Each year, in the US alone, it has been estimated that more than 1 .7 million people sustain TBI. In addition, there are >5 million people coping with TBI related disabilities at an estimated cost of more than $60 billion per year. In Europe, there are an estimated -775,500 new cases per year and a case fatality rate of 1 1 %. TBI is also an epigenetic risk factor for Alzheimer's and Parkinson's disease. Thus, TBI is a major health concern and a significant socioeconomic burden.
  • Neuroprotective treatments have been historically pursued for the treatment of TBI, but have failed to demonstrate clear efficacy in Phase III trials thus far. Instead, neurorestorative processes such as promotion of angiogene- sis, neurogenesis and axonal remodelling are now being developed, in order to enhance endogenous brain plasticity processes and to improve functional recovery after TBI (Xiong et ai, 2010).
  • Bone marrow stromal cells are a promising source of cell- based therapy for TBI.
  • the safety and feasibility of treatment with autologous MSCs has been assessed in patients with TBI, during which no toxicity related to the cell therapy was observed.
  • Neurological function was significantly improved 6 months after cell therapy, but is potentially difficult to interpret in the absence of control data.
  • Increased expression of erythropoietin (EPO), and its receptors, is found in neurons, neural progenitor cells and glial cells in re- sponse to injury.
  • Clinical trials to investigate the safety of EPO treatment in patients with severe TBI and, separately, to investigate the early administration of EPO to TBI patients are both ongoing.
  • SCI Spinal cord injury
  • Methylprednisolone which helps to reduce swelling in the spinal cord, is widely prescribed as an off-label drug, but does not serve most patients' needs.
  • therapies to alleviate, or repair, the incurred damage to the spinal cord.
  • Very few compounds are in late stage development with the limited examples including Lyrica (a calcium channel modulator, targets neuropathic pain), umbilical cord blood mononuclear cell transplants (aimed at improving functional recovery) and Procord (autologous activated macrophage therapy, aimed at facilitating neuroprotection and wound healing).
  • Lyrica a calcium channel modulator, targets neuropathic pain
  • umbilical cord blood mononuclear cell transplants aimed at improving functional recovery
  • Procord autologous activated macrophage therapy, aimed at facilitating neuroprotection and wound healing.
  • none of these molecules are expected to reach the market before 2017.
  • CSPGs chondroitin sulphate proteoglycans
  • CNS trauma Silver and Miller, 2004
  • CSPGs are also known to be part of the glial scar that forms post-injury, acting as a barrier to prevent axon extension and regrowth.
  • Levels of versican, neurocan, brevican and phosphacan (those CSPGs measured) have all been found to be upregulated after spinal cord injury (Jones et al., 2003).
  • WO201 1/022462 discloses the use of soluble fragments of RPTPs that bind CSPGs, thus acting as competitive inhibitors to prevent the CSPGs from binding RPTPs on the neuron.
  • the neural cell can be associated with an injury or neurodegenerative condition.
  • WO2012/1 12953 discloses methods for contacting a neuron with an agent that binds RPTPo, to thereby induce neuronal outgrowth of the neuron.
  • the agent may induce clustering of RPTPo and/or inhibit binding of CSPGs to RPTPo.
  • suitable agents are heparan sulfate proteoglycan, heparan sulfate, heparan sulfate oligosaccha- rides, or heparin oligosaccharides.
  • An object of the invention is thus to provide novel means and mechanisms for promoting neurite outgrowth and/or neural regeneration e.g. in nervous system injuries.
  • the nervous system injury is selected from a group consisting of neurodegenerative diseases, traumatic brain injury, spinal cord injury, multiple sclerosis (MS), Alzheimer's disease, amyotropic lateral sclerosis (ALS), stroke, Parkinson's disease, eye injury and skin burn.
  • a further object of the invention is to provide a method of promoting neurite outgrowth and/or neural regeneration.
  • HB-GAM heparin-binding growth associated molecule
  • HB-GAM pleiotro- phin
  • HB-GAM can be therefore used to reverse the CSPG ef- fects in various diseases where the CSPGs have adverse effects in terms of neuronal regeneration or maintenance.
  • HB-GAM promotes neurite outgrowth and/or neural regeneration by increasing the amount of chondroitin sulphate proteoglycan, CSPG, binding to its receptor RPTPo and clustering of this receptor.
  • HB-GAM would sequester the CSPGs from RPTPo, through interaction of its basic residues with the negatively charged sulphate side chains, but the present inventors found the reverse to be true.
  • HB-GAM can promote neurite outgrowth by modulating CSPG matrix that leads to its enhanced chondroitin suphate epitope binding to PTPsigma.
  • HB-GAM can promote dendrite regeneration in the cortex. It is known that the regenerative ability of dendrites is also very different from that of axons (Le Roux and Reh, 1996).
  • HB-GAM and the novel mechanism of action are advantageous and useful for the treatment of neuronal injuries.
  • HB-GAM promotes CNS regeneration by converting CSPG-enriched glial scar into permissive milieu for axon and dendrite growth in adult CNS. Moreover, dendrite regeneration has been an unexplored strategy for surviving dendrite damage.
  • the present invention provides now an agent, HB-GAM that promotes dendrite regeneration.
  • HB-GAM promotes neurite growth on CSPG-coated sub- strate in rat cortical neurons in vitro. Chondroitin sulphate proteoglycan aggre- can prevents attachment and neurite growth in embryonic rat cortical neurons. HB-GAM overcomes the inhibitory action of aggrecan, promoting neuronal attachment and neurite growth on the aggrecan-coated substrate in vitro.
  • HB-GAM overcomes the inhibitory effect of neurocan and promotes neuronal attachment and neurite growth.
  • Neurocan was precoated on plastic wells alone or co-precoated with HB-GAM under the same experimental conditions as in Figure 1 .
  • Neurocan precoated at 5 ⁇ g ml inhibits neurite growth and cell attachment in dissociated culture of rat cortical neurons.
  • HB-GAM precoated at 25 ⁇ g ml together with neurocan (5 ⁇ g ml) overcomes the inhibitory effect of neurocan and promotes neuronal attachment and neurite growth.
  • HB-GAM increases the amount of aggrecan binding to RPTPo. Mutant RPTPo with a defective CSPG-binding site was used as a negative control.
  • HB-GAM promotes dendrite regeneration after traumatic brain injury (TBI) in the adult mouse cortex.
  • TBI traumatic brain injury
  • TBI was applied to adult mice as a needle prick to the parietal lobe of the brain cortex.
  • Transgenic mice expressed YFP under Thy1 promoter in dendrites of neurons from cortical layers 4 and 5.
  • Chronic cranial window was used to follow dendrite regeneration us- ing two photon microscopy on live animals over 20 days after injury.
  • Immunoglobulin G was used in parallel as a negative control.
  • HB-GAM turns CSPG into a potent activator of neurite growth. Embryonic cortical neurons were cultured for 3.5 days on unprecoated plastic (A, C, E) or on aggrecan (precoated at 10 pg/ml) (B, D, E). HB-GAM (10 ⁇ g ml) was added to culture medium when plating cells in (C, D).
  • HB-GAM but not NGF overcomes the CSPG-dependent inhibition of neurite growth in PC12 cells.
  • Cells were plated on the substrate precoated with aggrecan (AGG) or with AGG + or with AGG + HB-GAM (HB).
  • Immunoglobulin G (IgG) was used for the unspecific control precoating. All samples were treated with NGF (100 ng/ml).
  • neurite length was measured in live cell images obtained 1 day after plating. The scale bar in (A) is 20 ⁇ .
  • HB-GAM enhances recovery of locomotor functions after spinal cord injury. Motor function was assessed in mice under control (vehicle) or HB-GAM treatment following cervical spinal cord hemisection. Starting from week 6 the animals treated with HB-GAM demonstrated significant improvement in frontlimb use as compared to the vehicle-treated controls (A). * p ⁇ 0.05, Mann-Whitney test. During the weeks 7-9 after trauma the vehicle-treated animals needed significantly more time to climb up on vertical screen as compared to the sham-operated and the HB-GAM-treated mice (J).
  • HB-GAM was injected within 30 min after injury at I mg/ml in 7 ⁇ injection volume. In sham animals laminectomy was performed but the spinal cord remained intact (B).
  • the present invention relates to a use of HB-GAM or a fragment thereof for treating injuries of the nervous system in a subject by increasing the interaction of chondroitin sulphate proteoglycan (CSPG) to the receptor protein tyrosine phosphatase sigma (RPTPo).
  • CSPG chondroitin sulphate proteoglycan
  • RPTPo receptor protein tyrosine phosphatase sigma
  • the present invention relates to a use of HB-GAM as anti-CSPG therapy in diseases in which the CSPGs restrict neuronal regeneration or maintenance.
  • HB-GAM promotes neurite outgrowth and neuronal attachment on GSPG-coated substrate by overcoming the inhibitory action of CSPG.
  • HB-binding growth-associated molecule HB-
  • GAM also known as pleiotrophin
  • pleiotrophin is an 18 kDa protein, which was first isolated from rat brain based on its neurite outgrowth promoting activity. Its expression in nervous tissue is developmentally regulated with the highest expression during the perinatal period (Rauvala, 1989). Neurite outgrowth induced by HB- GAM is dependent on the neuronal cell surface heparan sulphate proteoglycan N-syndecan, whilst the chondroitin sulfate proteoglycan RPTP beta/zeta (receptor-type tyrosine phosphatase beta/zeta) has also been implicated in the receptor mechanism of HB-GAM (Rauvala et ai, 2000).
  • HB-GAM contains two thrombospondin type 1 repeat (TSR) do- mains, which fold independently and do not interact with one another in solu- tion (Raulo et al., 2005).
  • TSR thrombospondin type 1 repeat
  • This TSR is found in a larger family of extracellular matrix-associated and cell surface proteins, such as thrombospondins 1 and 2, F-sponding, mindin and semaphorins F and G.
  • a common feature for this su- perfamily is that their function in cell surface and matrix binding is dependent on heparan-type polysaccharides.
  • HB-GAM contains a high proportion of basic amino acid residues, consisting of lysine clusters, at its N- and C- terminals. These lysine-rich tails have not been implicated in the binding interactions.
  • HB-GAM refers to any part of HB-GAM that is long enough to have the desired activity to reverse the inhibitory effects of the CSPGs.
  • HB-GAM or a fragment thereof is native, i.e. a protein in its natural state, unaltered by heat, chemicals, enzyme action, or the exigencies of extraction.
  • HB-GAM of the pre- sent invention is a recombinant protein. Recombinant proteins of the invention may be produced in a generally known manner.
  • a polynucleotide fragment comprising the HB-GAM gene is isolated, the gene is inserted under a strong promoter into an expression vector, the vector is transformed into suitable host cells and the host cells are cultivated under conditions provoking production of the enzyme.
  • Methods for protein production by recombinant technology in different host systems are well known in the art (Sambrook et al., 2001 ; Coen, 2001 ; Gellissen, 2005).
  • the recombinant proteins are produced in a baculovirus expression system.
  • Neurite growth or "neurite outgrowth in- eludes the process by which axons or dendrites extend from a neuron.
  • the outgrowth can result in a new neuritic projection or in the extension of a previ- ously existing cellular process.
  • Neurite outgrowth may include linear extension of an axonal process by five cell-diameters or more.
  • Central nervous system (CNS) neurons include the neurons of the brain, the cranial nerves and the spinal cord.
  • the invention relates not only to CNS neurons but also to periph- eral neurons that make projections (axons) in CNS, for instance dorsal root ganglion neurons.
  • HB-GAM is used in treating neuronal disorders, which include disease, disorder, or condition directly or indirectly affecting the normal functioning or anatomy of a subject's nervous sys- tern.
  • the disorder may be a neuronal injury, which can be acute or chronic. Examples of acute injury are those that results from surgery, trauma, compression, contusion, transection or other physical injury, vascular pharmacologic or other insults including hemorrhagic or ischemic damage.
  • Chronic neuronal injury may result from repetitive stress, inflammation/oxidative stress within a neural tissue caused by disease, neurodegenerative or other neurological diseases.
  • HB-GAM is beneficial in all diseases where the CSPG matrix is inhibitory for regeneration or maintenance of axons and dendrites.
  • the disease is a neuronal injury selected from a group consisting of neurodegenerative diseases, traumatic brain injury, spinal cord injury, multiple sclerosis (MS), amyo- tropic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, stroke, peripheral nerve injury, eye injury and skin burn
  • the neuronal injury is TBI or SCI.
  • TBI Traumatic brain injury, TBI as used herein includes the condition in which a traumatic blow to the head causes damage to the brain or connecting spinal cord, with or without penetrating the skull. It relates more specifically to the actual mechanical damage that occurs at the type of trauma, such as shearing, tearing and stretching of axons, neurons and blood vessels. Usually, the initial trauma can result in expanding hematoma, subarachnoid hemorrhage, cerebral edema, raised intracranial pressure, and cerebral hypoxia, which can, in turn, lead to severe secondary events due to low cerebral blood flow.
  • a spinal cord injury, SCI as used herein is damage to any part of the spinal cord or nerves at the end of the spinal canal. It often causes permanent changes in strength, sensation and other body functions below the site of the injury.
  • the spinal cord injury may be a complete severing of the spinal cord, a partial severing of the spinal cord, or a crushing or compression injury of the spinal cord.
  • Spinal cord injury SCI proceeds over minutes, hours, days and even months after the initial traumatic insult and can lead to significant expansion of the original damage. These secondary events are a consequence of delayed biochemical, metabolic and cellular changes, which are initiated by the primary injury, and includes inflammation, free radical induced cell death and glutamate excitotoxicity.
  • Axonal sprouting from surviving neurons, is associated with spon- taneous motor and sensory recovery following TBI and SCI.
  • CNS has a limited capacity to regenerate, spontaneous pericontusional axon sprouting does take place approximately 1-2 weeks after trauma.
  • this process typically fails due to an inhibitory axonal environment promoted by chon- Georgiain sulphate proteoglycans (CSPGs).
  • CSPGs chon- Harveyin sulphate proteoglycans
  • ChABC chondroitinase ABC
  • MS Multiple sclerosis
  • MS is a chronic immune-mediated disease that is characterized by demyelinating and degenerative processes within the cen- tral nervous system.
  • MS potentially requires symptomatic and disease- modifying therapies. Numerous symptoms such as fatigue, spasticity, depression, bowel and bladder dysfunction, pain, and impaired mobility are associated with the neurologic damage that results from MS.
  • therapies e.g. modafinil, dalfampridine, baclofen, diazepam, gabapentin, opioids are used for symptomatic treatment of disability and symptoms, but these do not improve disease outcome.
  • Intravenous corticosteroids are used in the management of MS exacerbations, but do not appear to affect the degree of improvement from acute exacerbations.
  • MS disease- modifying therapies but thus far no beneficial agent has been established in primary-progressive MS.
  • ALS Amyotrophic lateral sclerosis
  • Lou Gehrig's disease is the most common form of the motor neuron diseases.
  • the disorder is characterized by rapidly progressive weakness, muscle atrophy, twitching and spasticity, difficulty with speaking and swallowing and a decline in breathing ability.
  • the defining feature of ALS is the death of both upper and lower motor neurons in the motor cortex of the brain, the brain stem, and the spinal cord.
  • the disease has its onset usually in midlife and leads to death within 3-5 years from diagnosis, usually due to respiratory failure. Once diagnosed, only 10% of patients survive for longer than 10 years. In the US, there are approximately 30,000 ALS sufferers, with 5,000 new cases each year.
  • CSPGs Chodroitin sulphate proteoglycans, CSPGs
  • GAGs glycosaminoglycans
  • the GAG side chains are of different lengths, which partially define the different CSPGs e.g. aggrecan (CSPG1 ), versican (CSPG2), neurocan (CSPG3), brevican (CSPG7) and phosphacan.
  • CSPGs play an active role in the neural development of postnatal babies, acting as guidance cues for developing growth cones. Growing axons are found to avoid CSPG dense areas. Similarly, CSPGs found near and around embryonic roof plates inhibit axon elongation through the spinal cord and direct the axons in an alternative direction. CSPGs absent on roof plates were found to attract axonal elongation (Snow et ai, 1990).
  • HB-GAM changes the CSPG matrix from regeneration inhibiting to regeneration activating structure.
  • the CSPG matrix means the type of extracellular matrix that expresses chondroitin sulphate proteoglycans.
  • the extracellular matrix (ECM) provides a number of critical functions in the CNS, contributing both to the overall struc- tural organization of the CNS and to control of individual cells.
  • the ECM affects its functions by a wide range of mechanisms, including providing structural support to cells, regulating the activity of second messenger systems, and controlling the distribution and local concentration of growth and differentiation factors.
  • the brain extracellular matrix has trophic effects on neuronal cells and affects neurite outgrowth.
  • the GSPG-coated substrate is a simplified experimental model used in experimental studies. It can be made for example by coating a CSPG, such as e.g. aggregan or neurocan, on a tissue culture well.
  • RPTPo protein tyrosine phosphatase sigma
  • DRG dorsal root ganglion
  • mice Following a dorsal column crush injury, axonal extension into the lesion penumbra was significantly improved, compared to controls (Shen et al., 2009).
  • the ability of corticospinal tract (CST) axons to regenerate after spinal hemisection and contusion injury in ⁇ ' " mice has also been assessed. Damaged CST fibers, in ⁇ ' " mice, were found to regenerate and extend for long distances after injury to the thoracic spinal cord. In contrast, no long distance axon regeneration of CST fibers was seen after similar lesions in wild-type mice (Fry et ai, 2010).
  • RPTPa is also known to bind heparan sulphate proteoglycans (HSPGs), which are similar to the CSPGs, in that they contain a protein core with heavily sulphated, negatively charged, sugar side chains.
  • HSPGs heparan sulphate proteoglycans
  • CSPGs through RPTPa
  • HSPGs also acting through RPTPa
  • Both CSPGs and HSPGs bind to a common site on RPTPa and these differential effects were rationalised based on RPTPa oligomerisation status.
  • Heparan sulphate GAGs were found to induce oligomerisation of RPTPa fragments, but chondroitin sul- phate GAGs did not support clustering.
  • HSPGs and CSPGs differ in the composition of their GAG chains; sulphate groups are more evenly distributed in CSPGs, whereas HSPGs contain areas of high sulphation.
  • Receptor oligomerisation may cause microdomains with high phosphotyrosine levels and support neuronal extension, which CSPGs are able to disrupt and hence inhibit axon growth (Coles et al., 201 1 ).
  • the HB-GAM is used for treating neuronal injuries. Moreover, HB-GAM may be used for promoting dendrite regeneration. The present invention discloses that HB-GAM increases dendritic density (Figure 3) thus promoting dendritic regeneration. Treating and treatment refers to increasing, enhancing and promoting neuron regeneration and/or nerve growth in the presence of a neuronal injury. Treating and treatment encompass both therapeutic and prophylactic treatment regimens.
  • a subject is a human or and animal.
  • the present invention also relates to a method of promoting neuronal outgrowth and/or neural regeneration. This is achieved by contacting the neuron with a therapeutically effective amount of HB-GAM that increases CSPG binding to RPTPa, to thereby induce neuronal outgrowth.
  • the contacting can occur in vitro or in vivo to the neuronal cell.
  • HB-GAM can be added to a cell culture containing the neuron.
  • HB-GAM can be administered to a subject, such that an effective amount of it comes in contact with the neuron to thereby induce the neuronal outgrowth.
  • the neuron is a central nervous system neuron.
  • HB-GAM may be delivered according to any known method in the art. These include, without limitation, subcutaneous, intramuscular, transdermal, intravenous, oral, sublingual, nasal, rectal and topical administrations. PEGylation of HB-GAM may be used to modify pharmacokinetics of a drug and its in- teraction with living tissues. PEGylation of HB-GAM may enhance its ability to promote neurite growth on CSPG-substrate in cortical neurons.
  • HB-GAM can be administered directly to the nervous system (particularly to the site of injury), intracranially, intraspinally, intracerebroventricularly, intravenously, topically or intrathecally, e.g. into a chronic lesion of a neurodegenerative disease or at the site(s) of traumatic injury.
  • HB-GAM is administered via direct injection or HB- GAM-bound carrier (scaffold) material implantation or viral vector expressing HB-GAM.
  • the scaffolds may include natural components (aggrecan, neurocan, hyaluronic acid) or artificial materials.
  • the route of administration and the dosage regimen will be determined by skilled clinicians, based on factors such as the exact nature of the condition being treated, the severity of the condition, and the age and general physical condition of the patient.
  • administration is to thereby contact injured and/or non-injured neurons proximal to the injury site. In one embodiment, administration is such as to deliver the agent across the blood brain barrier.
  • the agent of the present invention may be formulated as part of pharmaceuti- cal compositions comprising one or more of the specific agents.
  • an effective amount refers to the amount of an active agent sufficient to induce a desired biological result e.g. promotion and/or restoration of neuronal regeneration and/or neurite growth. That result may be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • Neuronal outgrowth induced by the methods described herein can be determined by a variety of methods, such as by determination of the formation of axons (e.g., detecting the formation of neuronal branching microscopically or by showing cytoplasmic transport of dyes). Neuronal outgrowth can also be detected by determination of the formation of neural connectivity. Outgrowth can also be determined by an increase or a restoration of function of the neuron. Neuronal function can be measured by standard assays such as detection of action potential or nerve impulse conduction by standard assays.
  • Rat (Rattus norvegicus) HB-GAM sequence was used for baculovi- rus expression of recombinant HB-GAM protein.
  • Bovine aggrecan was from Sigma.
  • the DNA sequence (the template cDNA clone MGC:63375 IM- AGE:6834684) of the CSPG-binding domain of mouse RPTPo was used for Fc-tagged RPTPo protein production.
  • Cortical or hippocampal neurons from E17 rat embryos were plated at 50,000 cells/cm 2 on plastic cell culture plates. Plates were pre-coated with aggrecan or aggrecan+lgG (both proteins at 10 ⁇ g ml) or with neurocan (at 5 ⁇ g ml).
  • HB-GAM (10pg/nnl) was pre-coated together with aggrecan or added to culture medium when plating neurons. HB-GAM was precoated at 25 ⁇ g ml together with neurocan (5 ⁇ g ml).
  • Cells were cultured for 1 .5-3.5 days and after that images of cell cultures were taken using phase contrast microscope with x20 objective. Neurite length was quantified using ImagePro software. Mouse neurons were immunostained with anti-tubulin ⁇ antibodies and im- aged using fluorescent microscope.
  • the Fc-tagged extracellular domain of RPTPo was immobilized on protein G-coated plates. Binding of biotinylated aggrecan to RPTPo was quantified via colorimetric assay using streptavidin-conjugated horse radish perox- idise. Experiments were done by using protein G coated 96 well plates (Pierce, prod. #15133). Wells were first washed briefly with PBS, 0.05%Tween-20 solution. RPTPo wild type and mutant FC-fusion proteins were diluted into 2 ⁇ g ml solution in buffer containing PBS, 1 %BSA, 0.05% Tween-20. 150 ⁇ of protein solution was added into the wells and plates were left for shaking at +RT for 1 hour.
  • Streptavi- din-Peroxidase Polymer (SIGMA, S2438) was diluted 1/10 000 into PBS, 1 %BSA, 0.05% Tween-20 solution and 150 ⁇ was applied into the washed wells which were left for shaking +RT for 30 minutes. Wells were washed 3 times 5 minutes with 250 ⁇ of PBS, 0.05% Tween-20. Detection of bound Streptavidin-Peroxidase Polymer was done by adding 200 ⁇ of OPD (o- Phenylenediamine dihydrochloride, SIGMA P9187) substrate into the wells and was measured by reading the absorbance at 450 nm (A450).
  • OPD o- Phenylenediamine dihydrochloride
  • TBI model Cortical prick trauma model
  • TBI was applied in adult mice as a 2 mm deep needle prick to the parietal lobe of the brain cortex.
  • Transgenic mice expressed YFP under Thy1 promoter in dendrites of neurons from cortical layers 4 and 5.
  • Chronic cranial window was used to follow dendrite regeneration using two photon microscopy on live animals over 40 days after injury.
  • HB-GAM (1 .5 ⁇ , 1 mg/ml) was injected within 30 min after injury directly to the injury site.
  • Immunoglobulin G was used in parallel as a negative control.
  • Example 1 - HB-GAM promotes neurite outgrowth on aggrecan or on neurocan substrate in vitro
  • Neurocan precoated at 5 ⁇ g ml inhibits neurite growth and cell attachment in dissociated culture of rat cortical neurons.
  • HB-GAM precoated at 25 ⁇ g ml together with neurocan (5 ⁇ g ml) overcomes the inhibitory effect of neurocan and promotes neuronal attachment and neurite growth (Figure 1 B).
  • Example 2 - HB-GAM increases aggrecan binding to RPTPo
  • Example 3 cortical prick trauma model (TBI model) with HB-GAM treatment
  • TBI was applied in adult mice as a 2 mm deep needle prick to the parietal lobe of the brain cortex.
  • Transgenic mice expressed YFP under Thy1 promoter in dendrites of neurons from cortical layers 4 and 5.
  • Chronic cranial window was used to follow dendrite regeneration using two photon microscopy on live animals over 40 days after injury.
  • HB-GAM (1 .5 ⁇ , 1 mg/ml) was inject- ed within 30 min after injury directly to the injury site. Immunoglobulin G was used in parallel as a negative control.
  • HB-GAM increased dendritic density (i.e. dendritic regeneration) at the injury site at 20 days after injury, as compared to IgG control ( Figure 3).
  • Example 4 - HB-GAM turns CSPG into a potent activator of neurite growth
  • Example 5 - HB-GAM but not NGF overcomes the CSPG-dependent inhibition of neurite growth in PC12 cells
  • mice under control or HB- GAM treatment following cervical spinal cord hemisection (Figure 6).
  • the animals treated with HB-GAM demonstrated significant improvement in frontlimb use as compared to the vehicle-treated controls (A).
  • * p ⁇ 0.05 Mann-Whitney test.
  • the vehicle- treated animals needed significantly more time to climb up on vertical screen as compared to the sham-operated and the HB-GAM-treated mice (J).
  • HB-GAM was injected within 30 min after injury at 1 mg/ml in 7 ⁇ injection volume. In sham animals laminectomy was performed but the spinal cord remained intact (B).
  • ⁇ is a receptor for chondrotin sulphate proteoglycan, an inhibitor of neural regeneration. Science (2009) 326 592 - 596.
  • Heparan sulphate proteoglycans are ligands for receptor protein tyrosine phosphatase sigma. Molecular Cellular Biology (2002) 22 1881 - 1892.
  • Rauvala, H. An 18-kd heparin-binding protein of developing brain that is distinct from fibroblast growth factors. EMBO Journal (1989) 8 2933 - 2941 . Rauvala H. et al., Heparin-binding proteins HB-GAM (pleiotrophin) and amphoterin in the regulation of cell motility. Matrix Biology (2000) 19 377 - 387.

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Abstract

La présente invention concerne le traitement de lésions neuronales. Plus particulièrement, la présente invention concerne de nouveaux mécanismes et des moyens pour favoriser l'excroissance des neurites, la régénération neuronale et/ou la conservation des neurites (axones et dendrites) dans les pathologies où les protéoglycanes de sulfate de chondroïtine (CSPGs) présentent des effets indésirables, tel que dans des lésions du système nerveux central.
PCT/FI2014/050490 2013-06-18 2014-06-18 Traitement des lésions neronales WO2014202833A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0474979A1 (fr) * 1990-08-20 1992-03-18 American Cyanamid Company Séquence du gène du facteur neurotrohpique liant l'héparine
EP1057489A1 (fr) * 1997-09-26 2000-12-06 Meiji Milk Products Company Limited Agents preventifs ou remedes pour les maladies ischemiques
US20060122116A1 (en) * 2004-11-03 2006-06-08 The Johns Hopkins University Treatment for disorders of the peripheral nervous system
US20060193831A1 (en) * 2005-02-25 2006-08-31 Rush University Medical Center Use of pleiotrophin to promote neurogeneration
US20070254842A1 (en) * 2006-04-25 2007-11-01 The Regents Of The University Of California Administration of growth factors for the treatment of cns disorders
WO2012004291A1 (fr) * 2010-07-06 2012-01-12 Nanologica Ab Procédé amélioré pour la différenciation de cellules souches in vivo par l'administration de morphogènes avec de la silice mésoporeuse et principes actifs pharmaceutiques correspondants

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0474979A1 (fr) * 1990-08-20 1992-03-18 American Cyanamid Company Séquence du gène du facteur neurotrohpique liant l'héparine
EP1057489A1 (fr) * 1997-09-26 2000-12-06 Meiji Milk Products Company Limited Agents preventifs ou remedes pour les maladies ischemiques
US20060122116A1 (en) * 2004-11-03 2006-06-08 The Johns Hopkins University Treatment for disorders of the peripheral nervous system
US20060193831A1 (en) * 2005-02-25 2006-08-31 Rush University Medical Center Use of pleiotrophin to promote neurogeneration
US20070254842A1 (en) * 2006-04-25 2007-11-01 The Regents Of The University Of California Administration of growth factors for the treatment of cns disorders
WO2012004291A1 (fr) * 2010-07-06 2012-01-12 Nanologica Ab Procédé amélioré pour la différenciation de cellules souches in vivo par l'administration de morphogènes avec de la silice mésoporeuse et principes actifs pharmaceutiques correspondants

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG, YT. ET AL.: "Upregulation of heparin-binding growth-associated molecule after spinal cord injury in adult rats.", ACTA PHARMACOLOGICA SINICA, vol. 25, no. 5, 2004, pages 611 - 616 *

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