Vascular endothelial growth factor receptor-1 (VEGFR-1) inhibitors for promoting myelination and neuroprotection

Abstract

The present invention relates to vascular endothelial growth factor receptor (VEGFR) inhibitors, in particular VEGFR-1 inhibitors, for use in promoting myelination and / or neuroprotection in a subject. The invention also relates to compositions, pharmaceutical compositions, medicaments and kits comprising VEGFR-1 inhibitors and their use for promoting myelination and / or neuroprotection in a subject.

Description

Field of the Invention

[0001] The present invention relates to vascular endothelial growth factor receptor (VEGFR) inhibitors, in particular VEGFR-1 inhibitors, for promoting myelination and neuroprotection in a subject. The present invention also relates to compositions, pharmaceutical compositions, drugs and kits comprising VEGFR-1 inhibitors and their use in promoting myelination and neuroprotection in a subject. Background of the Invention

[0003] Myelin is an essential component of the nervous system, ensuring its lifelong health and function. Indeed, the myelin sheath insulates axons and facilitates saltatory conduction, allowing for rapid conduction of action potentials. Many neurological diseases are characterized by the destruction of myelin, a process known as demyelination, which may ultimately lead to neuronal loss. To date, several causes of demyelination have been identified, including, for example, inflammatory processes, viral infections, acquired metabolic disorders, hypoxia-ischemia, and traumatic injuries.

[0004] Among demyelinating diseases, multiple sclerosis (MS) is the leading cause of non-traumatic disability in young adults. A recent study estimated that MS costs the United States approximately $5.1 billion per year in sick leave, premature retirement, and lost income. Therefore, there is an urgent need to develop new therapies for demyelinating central nervous system diseases.

[0005] The inventors have found that VEGFR inhibitors, especially specific and selective inhibitors of VEGFR-1, can promote myelination and neuroprotection, thus demonstrating the application prospects of VEGFR-1 inhibitors in remyelination therapy. SUMMARY OF THE INVENTION

[0007] The present invention relates to inhibitors of vascular endothelial growth factor receptor 1 (VEGFR-1) for use in promoting myelination and / or neuroprotection in a subject in need thereof.

[0008] In one embodiment, the inhibitor is selected from the group comprising or consisting of a small organic molecule, a peptide, an antibody, an antibody fragment, an antibody mimetic, and a nucleic acid.

[0009] In one embodiment, the inhibitor is an organic small molecule. In one embodiment, the organic small molecule is selected from the group comprising or consisting of ZM306416 (CB 676475), SU14813, and salts, derivatives, and combinations thereof.

[0010] In one embodiment, the inhibitor is a peptide. In one embodiment, the inhibitor is a peptide selected from the group comprising or consisting of peptides having SEQ ID NO: 6 to 10 and combinations thereof, more preferably, the inhibitor is a peptide having SEQ ID NO: 6.

[0011] In one embodiment, the inhibitor is a nucleic acid. In one embodiment, the inhibitor is a shRNA, preferably selected from the group comprising or consisting of shRNAs having SEQ ID NOs: 15 to 18 and combinations thereof.

[0012] In one embodiment, the subject is affected by or diagnosed with a demyelinating disease.

[0013] In one embodiment, the demyelinating disease is selected from the group comprising or consisting of multiple sclerosis, optic neuritis, cerebral ischemia, leukodystrophy, and traumatic brain injury (TBI).

[0014] In one embodiment, the demyelinating disease is multiple sclerosis. In one embodiment, multiple sclerosis is selected from the group consisting of clinically isolated syndrome (CIS), relapsing remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS) and secondary progressive multiple sclerosis (SPMS), preferably, multiple sclerosis is a progressive form of the disease.

[0015] In one embodiment, the subject has received, is receiving, or will receive an anti-inflammatory drug.

[0016] In one embodiment, the anti-inflammatory agent is an immunomodulator or an immunosuppressant. In one embodiment, the anti-inflammatory agent is selected from the group comprising or consisting of: interferon beta, Glatiramer acetate, Fingolimod, Dimethyl fumarate, Diroximel fumarate, Teriflunomide, Siponimod, Cladribine, Natalizumab, Ocrelizumab, Alemtuzumab, Cyclophosphamide, Azathioprine, Mitoxandrone, and combinations thereof.

[0017] The present invention also relates to a pharmaceutical composition comprising the VEGFR-1 inhibitor as described above and at least one pharmaceutically acceptable excipient, for promoting myelination and / or neuroprotection in a subject in need thereof.

[0018] The invention also relates to a kit comprising: in one part at least one inhibitor of VEGFR-1, and in a second part at least one anti-inflammatory drug.

[0019] In one embodiment, the kit is for promoting myelination and / or neuroprotection in a subject in need thereof.

[0020] definition

[0021] In the present invention, the following terms have the following meanings:

[0022] The word "about" before a numerical value means plus or minus 10% of the numerical value.

[0023] "Adnectin", also known as monobody, is well known in the art and refers to proteins designed to bind to antigens with high affinity and specificity. They belong to a class of molecules collectively referred to as "antibody mimics".

[0024] "Alphabody", also known as cell-penetrating Alphabody, is an antibody mimetic consisting of a small 10kDa protein that has been engineered to bind to a variety of antigens. Alphabody is able to reach and bind to protein targets within cells.

[0025] "Affibody" refers to an affinity protein based on a protein domain of 58 amino acid residues, derived from one of the IgG binding domains of Staphylococcal protein A (Frejd & Kim, 2017. Exp Mol Med. 49(3): e306; Patent US5,831,012).

[0026] "Affilin" refers to artificial proteins designed to selectively bind antigens. Their affinity and specificity for antigens are similar to antibodies, but their structure is different, which makes them an antibody mimetic.

[0027] "Affitin" refers to highly stable engineered affinity proteins originally derived from Sac7d and Sso7d, two 7 kDa DNA-binding polypeptides from the genus Sulfolobus.

[0028] As used herein, "antibody" and "immunoglobulin" are used interchangeably to refer to a protein having a combination of two heavy chains and two light chains, whether or not it has any relevant specific immunoreactivity. "Antibody" refers to an assembly with significant known specific immunoreactivity to a target antigen (e.g., VEGFR-1). The term "anti-VEGFR-1 antibody" is used herein to refer to an antibody that exhibits immunospecificity to human VEGFR-1. As explained elsewhere herein, "specificity" to human VEGFR-1 does not exclude cross-reactions with species homologs of VEGFR-1 (e.g., with monkey VEGFR-1). Antibodies and immunoglobulins include light chains and heavy chains, with or without interchain covalent bonds between them. The basic immunoglobulin structure in vertebrate systems is relatively well understood. The general term "immunoglobulin" includes five different antibody classes, which can be distinguished biochemically. Although the following discussion is generally directed to IgG class immunoglobulin molecules, all five classes of antibodies are within the scope of the present invention. Regarding IgG, immunoglobulins contain two identical light chain polypeptides with a molecular weight of about 23 kDa, and two identical heavy chains with a molecular weight of about 53-70 kDa. These four chains are connected in a "Y" configuration by disulfide bonds, where the light chain starts at the opening of the "Y" configuration and extends to the variable region to enclose the heavy chain in the middle. The light chains of antibodies are divided into kappa (κ) or lambda (λ). Each heavy chain category can be combined with a κ or λ light chain. In general, when immunoglobulins are produced by hybridomas, B cells or genetically engineered host cells, the light chain and the heavy chain are covalently bound to each other, and the "tail" regions of the two heavy chains are bound to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y configuration to the C-terminus at the bottom of each chain. It is understood by those skilled in the art that heavy chains are classified as gamma (γ), mu (μ), alpha (α), delta () or epsilon (ε), among which there are some subclasses (e.g., γ1-γ4). The nature of this chain determines that the "class" of the antibody is IgG, IgM, IgA IgD or IgE, respectively. Immunoglobulin subclasses or "isotypes" (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) have been fully characterized and are known to confer functional specialization. In view of the present disclosure, it is easy for those skilled in the art to distinguish modified versions of each of these classes and isotypes, and therefore, they are within the scope of the present invention. As described above, the variable region of an antibody allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the light chain variable domain (VL domain) and the heavy chain variable domain (VH domain) of an antibody combine to form a variable region, which defines a three-dimensional antigen binding site. This four-level antibody structure forms an antigen binding site at the end of each arm of a "Y" shape. More specifically, the antigen binding site is defined by three complementary determining regions (CDRs) on each VH and VL chain.

[0029] As used herein, "antibody fragment" refers to a portion or region of an antibody that contains fewer amino acid residues than the entire antibody. An "antibody fragment" binds to an antigen and / or competes for antigen binding with the entire antibody from which it is derived (e.g., specifically binds to VEGFR-1). Antibody fragments include, but are not limited to, single-chain antibodies, dimeric single-chain antibodies, Fv, scFv, Fab, Fab', Fab'-SH, F(ab)'2, Fd, defucosylated antibodies, diabodies, triabodies, and tetrabodies. It may also include unibodies, domain antibodies, and nanobodies.

[0030] "Anticalin" is an antibody mimetic technology in which the binding specificity is derived from lipocalin. Anticalin can also be formatted as a dual-targeting protein, called Duocalin.

[0031] "Anti-inflammatory drug" is a medicine or substance that reduces inflammation (redness, swelling, and pain) in the body.

[0032] As used herein, "Armadillo repeat protein-based scaffolds" refers to a class of antibody mimetics corresponding to artificial peptide binding scaffolds based on Armadillo repeat proteins. Armadillo repeat proteins are characterized by an Armadillo domain, consisting of tandem Armadillo repeat sequences of approximately 42 amino acids, which mediate interactions with peptides or proteins.

[0033] "Atrimer" refers to binding molecules against target proteins whose trimerization is a prerequisite for their biological activity. Compared with other antibody-mimetic scaffolds, they are relatively large.

[0034] "Avimer" is an antibody mimicking technology.

[0035] As used herein, "diabodies" refer to small antibody fragments prepared by constructing scFv fragments with short linkers (about 5-10 residues) between VH and VL to achieve interchain pairing of variable domains rather than intrachain pairing, thereby producing a bivalent fragment, i.e., a fragment with two antigen binding sites. Bispecific diabodies are heterodimers of two "cross" scFv fragments, in which the VH and VL of the two antibodies are present on different polypeptide chains. Diabodies are described in, for example, patent EP0404097 or patent application WO1993011161.

[0036] “Demyelination”: refers to a pathological process in the nervous system in which the myelin sheath is damaged. Damage to the myelin sheath impairs signal conduction in the affected nerves. As a result, the reduced ability to conduct can lead to sensory, motor, cognitive, and / or other functional deficits, depending on the nerves affected. Demyelination can be caused by genetics, infectious agents, autoimmune reactions, as well as unknown factors. Demyelination can affect the central nervous system (CNS) and / or the peripheral nervous system (PNS).

[0037] "Demyelinating disease" refers to diseases associated with demyelination (i.e., loss of myelin).

[0038] "Derivative" refers broadly to a modification or substitution of one or more chemical moieties on a parent compound, and may include positional isomers, tautomers, zwitterions, enantiomers, diastereomers, racemates, isosteres or stereochemical mixtures thereof.

[0039] "Domain antibody" refers to the smallest functional binding unit of an antibody, corresponding to the variable region of an antibody heavy or light chain.

[0040] "Domain Kunitz peptides" are antibody mimetics based on the active domains of proteins that inhibit the function of proteases.

[0041] "Evasin" refers to a class of chemokine binding proteins.

[0042] "Fab" refers to fragment antibodies produced by digesting whole IgG antibodies with papain to remove the entire Fc fragment (including the hinge region). These antibodies are monovalent and contain only a single antigen-binding site. In contrast, F(ab') 2 Fragment antibodies are produced by digesting whole IgG antibodies with pepsin to remove most of the Fc region while retaining part of the hinge region. 2 The fragment has two antigen-binding F(ab) parts linked together by a disulfide bond. The term "Fab'" refers to an antibody fragment with a molecular weight of about 50,000 and antigen-binding activity, which is obtained by cleaving the F(ab') 2 Fab'-SH is the designation herein for Fab' in which the cysteine ​​residues of the constant domains bear a free thiol group.

[0043] "Fynomers" refer to proteins that belong to the class of antibody mimics. They are attractive binding molecules due to their high thermal stability and reduced immunogenicity.

[0044] As used herein, "Fv" refers to the smallest antibody fragment containing a complete antigen recognition and binding site. The fragment consists of a dimer of a VH and a VL in a tight, non-covalent binding manner. The folding of these two domains produces six hypervariable loops (three loops each for the heavy chain and light chain), which contribute to antigen binding and give the antibody antigen binding specificity. However, even a single variable domain (or half an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigens, although the affinity is lower than that of the complete binding site.

[0045] "Immunomodulators" are substances that affect the function of the immune system.

[0046] "Immunosuppressants" refer to agents that suppress or prevent an immune response.

[0047] An "inhibitor of VEGFR-1" refers to a substance that induces inhibition or down-regulation of biological activities associated with VEGFR-1 activation, including any downstream biological effects resulting from binding of VEGFR-1 to its natural ligand.

[0048] "Knottin" (also known as cystine knot inhibitor) refers to an antibody mimetic that contains a protein structural motif containing three disulfide bridges.

[0049] "Multiple sclerosis" or "MS" refers to an inflammatory demyelinating disease of the central nervous system (CNS).

[0050] "Neuroprotection" refers to the preservation of neuronal integrity and / or function in the face of nervous system damage, including, for example, demyelination, trauma, hypoxia-ischemia, oxidative stress, and metabolic disturbances.

[0051] "Neuronary loss" refers to the loss of neuronal cells.

[0052] "Neuron" and "neuronal cell" are equivalent and interchangeable in this application.

[0053] "Nerve" and "nerve fiber" are equivalent and interchangeable in this application.

[0054] "Oligodendrocyte lineage cell": as used herein refers to oligodendrocytes and oligodendrocyte progenitor cells (OPC).

[0055] "Peptide": refers to a linear polymer of amino acids of less than 50 amino acids linked together by peptide bonds.

[0056] "Promoting myelination" or "promoting myelin regeneration" refers to the process of promoting myelin formation by promoting the survival, differentiation and / or maturation of oligodendrocyte lineage cells and / or Schwann cells. In the context of the present invention, promoting the myelination or myelin regeneration of neurons is equivalent to promoting the myelination or myelin regeneration of nerve fibers.

[0057] "Salt": refers to a compound composed of an ionic assembly of positively charged cations and negatively charged anions, the resulting compound having no net charge.

[0058] As used herein, "single-chain antibody" refers to any antibody or fragment thereof, which is a protein having a primary structure comprising or consisting of an uninterrupted sequence of consecutive amino acid residues, including but not limited to (1) a single-chain Fv molecule (scFv); (2) a single-chain protein containing only one light chain variable domain, or a fragment thereof containing the three CDRs of the light chain variable domain without the associated heavy chain portion; and (3) a single-chain protein containing only one heavy chain variable region, or a fragment thereof containing the three CDRs of the heavy chain variable region without the associated light chain portion.

[0059] "Single-chain Fv", also abbreviated as "sFv" or "scFv", refers to a fragment comprising V linked into a single amino acid chain. H and V L Preferably, the scFv amino acid sequence also includes V H and V L The peptide linker between the domains enables the scFv to form the desired structure for antigen binding.

[0060] "Selective" means that the affinity of a molecule (e.g., an inhibitor) for VEGFR-1 is at least 10-fold, 25-fold, 50-fold, 75-fold, 80-fold, 90-fold, 95-fold, 100-fold, 125-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or 500-fold greater than its affinity for other VEGFRs (particularly VEGFR-2 and VEGFR-3).

[0061] "Organic small molecule" refers to a molecule of size comparable to organic molecules commonly used in medicine. The term does not include biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred organic small molecules range in size from up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. Preferred organic small molecules range in size from up to about 5000 g / mol, more preferably up to 2000 g / mol, and most preferably up to about 1000 g / mol.

[0062] "Specificity" refers to the ability of a molecule (e.g., an inhibitor) to detectably bind to its target (e.g., VEGFR-1) while having relatively little detectable reactivity with non-VEGFR-1 proteins. Specificity can be determined relatively by binding or competitive binding assays, such as using a Biacore instrument. Specificity can be expressed, for example, as a ratio of affinity / avidity for binding to a specific target to nonspecific binding to other unrelated molecules of about 2:1, about 5:1, about 10:1, about 20:1, about 50:1, about 100:1, 10.000:1 or greater (in this case, the specific target is VEGFR-1).

[0063] "Subject" refers to a mammal, preferably a human. In one embodiment, the subject can be a "patient", i.e., a warm-blooded animal, more preferably a human, who is waiting to receive medical care or is receiving medical care or was / is / will be the subject of a medical procedure, or is monitoring the development of a disease. In one embodiment, the subject is an adult (e.g., a subject over the age of 18). In another embodiment, the subject is a child (e.g., a subject under the age of 18). In one embodiment, the subject is a male. In another embodiment, the subject is a female.

[0064] "Schwann cells (SC)" refer to the major glial cell type in the peripheral nervous system. They play a crucial role in the myelination, development, maintenance, function and regeneration of peripheral nerves.

[0065] "Therapeutically effective amount" refers to the level or amount of an agent that is intended to achieve the following purposes without causing significant negative or adverse side effects to the target: (1) delaying or preventing the onset of demyelinating disease; (2) slowing or stopping the progression, aggravation or worsening of one or more symptoms of demyelinating disease; (3) improving the symptoms of demyelinating disease; (4) reducing the severity or incidence of demyelinating disease; or (5) curing demyelinating disease. The therapeutically effective amount can be administered before the onset of demyelinating disease to play a preventive or prophylactic role. Alternatively or additionally, the therapeutically effective amount can be administered after the onset of demyelinating disease to play a therapeutic role or maintain a therapeutic role.

[0066] "Treatment" or "relief" refers to both therapeutic treatment and prophylactic or preventative measures; the purpose is to prevent or slow down (alleviate) the target pathological condition or disorder. Those in need of treatment include those already suffering from the disorder as well as those susceptible to the disorder or those in need of prevention of the disorder. The target pathological condition of the subject or mammal is successfully "treated" if, after receiving a therapeutic amount of the composition of the present invention, an observable effect is exhibited on one or more of the following: one or more symptoms associated with the specific disorder or disorder are alleviated to some extent; morbidity and mortality are reduced, and quality of life issues are improved. The above parameters for evaluating successful treatment and improvement of the disorder can be easily measured by routine procedures familiar to physicians.

[0067] "VEGFR" or "vascular endothelial growth factor receptor" refers to three related receptor tyrosine kinases that bind to vascular endothelial growth factor (VEG F): VEGFR-1 (also known as Flt1), VEGFR-2 (also known as KDR), and VEGFR-3 (also known as Flt4). VEGFR has seven extracellular immunoglobulin-like domains, a transmembrane domain, and an intracellular tyrosine kinase domain. The ligands of VEGFR-1 are VEGFA, VEGFB, and PLGF. VEGFR2 binds to VEGFA and proteolytically processed VEGFC and VEGFD, while VEGFR3 is activated by the binding of VEGFC and VEGFD. An example of the amino acid sequence of human VEGFR-1 is SEQ ID NO:1.

[0068] SEQ ID NO:1

[0069]

[0070] "VEGFR function" or "vascular endothelial growth factor receptor function" refers to the biological activity associated with VEGFR activation, including any downstream biological effects resulting from binding of VEGFR to its natural ligand. VEGFR-1, VEGFR-2 or VEGF-3 function refers to the biological activity associated with activation of VEGFR-1, VEGFR-2 or VEGFR-3, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0072] The present invention relates to inhibitors of vascular endothelial growth factor receptor 1 (VEGFR-1) for use in promoting myelination and / or neuroprotection in a subject in need thereof.

[0073] In one embodiment, the inhibitors of VEGFR-1 of the present invention promote myelination and / or neuroprotection in a subject in need thereof, and thus can be used to treat or prevent demyelinating diseases in a subject in need thereof. Therefore, the present invention also relates to inhibitors of vascular endothelial growth factor receptor 1 (VEGFR-1) for treating or preventing demyelinating diseases in a subject in need thereof.

[0074] In one embodiment, the inhibitor of VEGFR-1 of the present invention is a natural compound, ie a compound that can be found in nature. In one embodiment, the inhibitor of VEGFR-1 of the present invention is a synthetic compound.

[0075] In one embodiment, the VEGFR-1 inhibitor of the present invention directly binds to VEGFR-1. Thus, in one embodiment, the VEGFR-1 inhibitor of the present invention is a compound that is able to inhibit the function of VEGFR-1 by directly binding to the receptor.

[0076] In one embodiment, the term "inhibits VEGFR-1 function" refers to a decrease in function compared to a reference function induced by VEGFR-1 activation, e.g., a function less than or equal to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the reference function. In one embodiment, inhibition of VEGFR-1 function is measured after contact of a cell with a compound being tested for its effect on function, and the reference function corresponds to the function induced by VEGFR-1 activation measured in cells not contacted with the compound.

[0077] In one embodiment, the VEGFR-1 inhibitor of the present invention is selected from the group comprising or consisting of: small organic molecules, peptides, antibodies, antibody fragments and antibody mimetics. In one embodiment, the VEGFR-1 inhibitor is a small organic molecule, peptide or antibody or a fragment thereof.

[0078] In one embodiment, the VEGFR-1 inhibitor of the present invention is an antibody or an antibody fragment thereof against VEGFR-1.

[0079] The antibody or fragment thereof may, for example, weaken the binding of the ligand to VEGFR- 1. Thus, in one embodiment, the antibody or antibody fragment thereof against VEGFR- 1 is a blocking antibody or fragment thereof.

[0080] Examples of antibodies or antibody fragments that bind to VEGFR-1 include, but are not limited to, antibodies or fragments thereof produced by the following hybridomas: KM1730 (deposited as FERM BP-5697), KM1731 (deposited as FERM BP-5718), KM1732 (deposited as FERM BP-5698), KM1748 (deposited as FERM BP-5699), and KM1750 (deposited as FERM BP-5700) disclosed in International Applications WO98 / 22616 and WO99 / 59636.

[0081] Examples of other antibodies or antibody fragments include IMC-18F (icrucumab), D16F7, and MF1 antibodies.

[0082] The heavy chain and light chain of eleucumab are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0083] SEQ ID NO:2

[0084] QAQVVESGGGVVQSGRSLRLSCAASGFAFSSYGMHWVRQAPGKGLEWVAVIWYDGSNKYYADSVRGRFTISRDNSENTLYLQMNSLRAEDTAVYYCARDHYGSGVHHYFYYGLDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0085] SEQ ID NO:3

[0086] EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC

[0087] The variable regions of the heavy and light chains of D16F7 are shown in SEQ ID NO:4 and SEQ ID NO:5 respectively.

[0088] SEQ ID NO:4

[0089] MGWSSIILFLVATASGVHSQYQQQVGSVWKPGASYKLSCKASGYWTNYWMHWYKQRPGQGLEWIGEIYPRNGNTNYDEKFKGKATLTIDTSSSTAYMQLSSLTSEDSAVYYCATYLGDYWGQGTTLTVSS

[0090] SEQ ID NO:5

[0091] MKLPVRLLLVLMFWIPASSSDVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWFLQKPGQSPKLLIYKVSNRFSGIPDRFSGSGSGTDFTLKISRVEAEDLGVYFCFQGSHVPYTFGGGTKLEIK

[0092] Antibodies against VEGFR-1 can be obtained by administering appropriate antigens or epitopes to selected host animals such as pigs, cattle, horses, rabbits, goats, sheep and mice according to known methods. Various adjuvants known in the art can be used to enhance antibody production. Although the antibodies used to implement the present invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against VEGFR-1 can be prepared and isolated using any technology that can produce antibody molecules through cultured continuous cell lines. The technology used to produce and isolate includes, but is not limited to, the hybridoma technology originally described by Kohler and Milstein (1975); human B cell hybridoma technology (Cote et al., 1983); and EBV hybridoma technology (Cole et al., 1985). Alternatively, the technology for producing single-chain antibodies described (see, for example, U.S. Patent No. 4,946,778) can be adjusted to produce single-chain antibodies against VEGFR-1.

[0093] Examples of antibody fragments include, but are not limited to, F(ab')2 fragments (which can be produced by pepsin digestion of intact antibody molecules) and Fab fragments (which can be produced by reducing the disulfide bonds of F(ab')2 fragments). Other examples of antibody fragments include, but are not limited to, Fv, particularly scFv, Fd, defucosylated antibodies, diabodies, triabodies and tetrabodies, monobodies, domain antibodies and nanobodies. Alternatively, Fab and / or scFv expression libraries can be constructed to rapidly identify fragments with the desired specificity for VEGFR-1.

[0094] Humanized antibodies (or fragments thereof) for VEGFR-1 can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. For the most part, humanized antibodies are human immunoglobulins (receptor antibodies), wherein the hypervariable region (CDR) residues of the receptor are replaced by hypervariable region residues of non-human species (donor antibodies) such as mice, rats, rabbits, or non-human primates with desired specificity, affinity, and ability. In some cases, the framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in the receptor antibody or the donor antibody. These modifications are made in order to further improve antibody performance. In general, humanized antibodies will include substantially all of at least one and usually two variable domains, wherein all or substantially all hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all FRs are FRs of human immunoglobulin sequences. The humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.For example, Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397) describe methods for making humanized antibodies.

[0095] In one embodiment, the VEGFR-1 inhibitor of the present invention is an antibody mimetic. As used herein, an antibody mimetic is an organic compound that can specifically bind to an antigen like an antibody, but is not structurally related to an antibody.

[0096] Examples of antibody mimetics include, but are not limited to, affibodies, alphabodies, armadillo repeat protein-based scaffolds, knottins, domain kunitz peptides, affilins, affitins, adnectins, atrimers, evasins, DARPins, anticalins, avimers, fynomers, versabodies, or du ocalins.

[0097] In one embodiment, the VEGFR-1 inhibitor of the present invention is a small organic molecule.

[0098] In one embodiment, the size of the small organic molecule is at most about 5000 g / mol, more preferably at most 2000 g / mol, and more preferably at most about 1000 g / mol.

[0099] In one embodiment, the VEGFR-1 inhibitor of the present invention is ZM306416 (CB 676475, CAS No. 690206-97-4) or a salt or derivative thereof. The molecular formula of ZM306416 is as follows:

[0100]

[0101] ZM306416 can be purchased from the supplier’s website (https: / / www.selleckchem.com / products / zm-306416.html, https: / / www.tocris.com / products / zm-306416-hydrochloride_2499, https: / / www.abcam.com / zm306416-hydrochloride-vegfr12-tyrosine-kinase-activity-inhibitor-ab144576.html).

[0102] In one embodiment, the VEGFR-1 inhibitor of the present invention is SU14813 (CAS No. 627908-92-3), or a salt or derivative thereof. The molecular formula of SU14813 is as follows:

[0103]

[0104] SU14813 can be purchased from the supplier's website (https: / / www.medchemexpress.com / SU14813.html, https: / / www.selleckchem.com / products / su14813.html, https: / / www.apexbt.com / su14813.html).

[0105] In one embodiment, the inhibitor of VEGFR-1 of the present invention is a peptide.

[0106] Those skilled in the art are familiar with examples of peptides that bind to VEGFR-1, including but not limited to anti-VEGFR-1 peptides of sequence GNQ WFI (SEQ ID NO: 6), D (CLPRC) (SEQ ID NO: 7), peptide F56 of the sequence WHSDMEWWYLLG (SEQ ID NO: 8), peptide A4 of the sequence TEGRELVIPARVT (SEQ ID NO: 9), and peptide B3 of the sequence IPARVTS (SEQ ID NO: 10).

[0107] In one embodiment, the VEGFR-1 inhibitor of the present invention is a peptide selected from the group consisting of peptides having SEQ ID NOs: 6 to 10 and combinations thereof. In one embodiment, the VEGFR-1 inhibitor of the present invention is an anti-VEGFR-1 peptide of sequence GNQWFI (SEQ ID NO: 6).

[0108] In one embodiment, the VEGFR-1 inhibitor of the present invention is a nucleic acid, in particular a nucleic acid targeting a gene encoding VEGFR-1, thereby inhibiting the expression of VEGFR-1.

[0109] In one embodiment, the VEGFR-1 inhibitor of the present invention is a nucleic acid, in particular a nucleic acid that targets the VEGFR-1 encoding gene in oligodendrocyte cells and / or Schwann cells, thereby inhibiting the expression of VEGFR-1 in oligodendrocyte cells and / or Schwann cells.

[0110] In one embodiment, the term "inhibits the expression of VEGFR-1" refers to a decrease in expression level compared to a reference expression level of VEGFR-1, e.g., 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of the reference expression level. In one embodiment, the inhibition of VEGFR-1 expression is measured after contact of the cell with a compound being tested for its effect on expression, and the reference expression level corresponds to the VEGFR-1 expression level measured in cells not contacted with the compound.

[0111] Methods for determining gene expression levels are well known to those skilled in the art, including but not limited to determining transcriptome and / or proteome.

[0112] In one embodiment, expression levels are assessed at the transcriptional level (ie, at the mRNA level).

[0113] In vitro methods for assessing gene transcription levels are well known in the prior art. Examples of such methods include, but are not limited to, RT-PCR, RT-qPCR, Northern Blot, hybridization techniques (e.g., using microarrays) and combinations thereof, including, but not limited to, hybridization of amplicons obtained by RT-PCR, sequencing such as next generation DNA sequencing (NGS) or RNA-seq (also referred to as "full transcriptome shotgun sequencing"), etc.

[0114] In one embodiment, expression levels are assessed at the translation level (ie, at the protein level).

[0115] In vitro methods for assessing gene translation levels are well known in the art. Examples of such methods include, but are not limited to, immunohistochemistry, multiplex methods (Luminex), Western blotting, enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), flow cytometry (FACS), etc.

[0116] Examples of nucleic acids that can be used in the present invention include, but are not limited to, RNA interference molecules, oligonucleotide antisense (including, but not limited to, antisense RNA or DNA molecules), ribozymes, aptamers, and morpholinos.

[0117] As used herein, RNA interference (RNAi) is a gene silencing process that targets mRNA, thereby reducing protein expression. Examples of RNAi substrates that regulate this process include, but are not limited to, shRNA (short hairpin or small hairpin RNA), siRNA (small interfering RNA) and miRNA (microRNA).

[0118] Therefore, in one embodiment, the inhibitor of VEGFR-1 is an RNA interference molecule, preferably selected from the group comprising or consisting of shRNA, siRNA, miRNA and a combination thereof.

[0119] shRNA is an RNA containing a circular structure, which is processed into siRNA and also causes mRNA degradation in a sequence-specific manner that depends on complementary binding to the target mRNA.

[0120] Examples of shRNA targeting VEGFR-1 include, but are not limited to, the following shRNA:

[0121] 5'GGTCATAGAAGGAACCAATAAGACGTTA'3(SEQ ID NO:15),

[0122] 5'TCACTCTGAACCTTGTCATCAAGAATGTG'3(SEQ ID NO:16),

[0123] 5'CTCGGGTGACCTGCTTCAAGCCAATGTCCA'3 (SEQ ID NO:17),

[0124] 5'GAGCATCTATCAGGCAGCGGATTGACCAA'3 (SEQ ID NO: 18).

[0125] In one embodiment, the inhibitor of VEGFR-1 of the present invention is a shRNA, preferably a shRNA selected from the group comprising or consisting of shRNAs having SEQ ID NOs: 15 to 18 and combinations thereof.

[0126] siRNA is a double-stranded RNA, containing an antisense strand (or guide strand) and a sense strand (or passenger strand), which form a double strand of 19 to 25 bp in length with a dinucleotide 3' overhang. These siRNAs bind to helicase and nuclease molecules to form a large complex called the RNA-induced silencing complex, which can unwind the siRNA and guide the sequence-specific degradation of the mRNA.

[0127] Examples of siRNA targeting VEGFR-1 include, but are not limited to, the following siRNA:

[0128] Sense: 5′-GGCCAGCACAUAGGAGAGATT-3′ (SEQ ID NO: 11);

[0129] Antisense: 5′-UCUCUCCUAUGUGCUGGCCTT-3 (SEQ ID NO: 12)

[0130] Sense: 5′-BCU GAG UUU AAA AGG CAC CCT TB-3′ (SEQ ID NO: 13);

[0131] Antisense: 5′-GGG UGC CUU UUA AAC UCA GTST-3′ (SEQ ID NO: 14),

[0132] Where "T" represents an unpaired deoxythymidine, "S" represents a phosphorothioate linkage, and "B" represents two inverted 2′-deoxyabasic nucleotides.

[0133] In one embodiment, the inhibitor of VEGFR-1 of the present invention is siRNA, preferably siRNA selected from the group comprising or consisting of siRNAs having SEQ ID NOs: 11 to 14 and combinations thereof.

[0134] miRNAs are gene regulatory RNAs that are loaded onto the RNA-induced silencing complex (RISC) and interact with partially complementary targets on mRNA to inhibit protein expression. miRNAs are typically single-stranded, and when loaded onto RISC, the miRNA "guide" sequence (also called the seed region) is held on the surface of RISC, where it can interact with the target mRNA. The targets recognized by the miRNA guide sequence are most commonly found in the 3' untranslated region (UTR) of RNA. Binding can inhibit the assembly of the initiation complex on the 5' cap of the mRNA, because the mRNA binds into a circular shape at the start of translation, bringing the 3'-UTR and 5'-UTR close together.

[0135] hRNA, siRNA, and miRNA targeting VEGFR-1 can be purchased through the product supply website.

[0136] In one embodiment, the inhibitor of VEGFR-1 of the present invention is an antisense oligonucleotide. Antisense oligonucleotide, including antisense RNA molecules and antisense DNA molecules, will directly block its translation by binding VEGFR-1mRNA, thereby preventing protein translation or increasing mRNA degradation, reducing the level of VEGFR-1 in cells, and then reducing its activity. For example, antisense oligonucleotides complementary to the unique region of the mRNA transcription sequence encoding VEGFR-1 can be synthesized by conventional phosphodiester technology, and administered by, for example, intravenous injection or infusion. The method of using antisense technology to specifically inhibit the gene expression of known sequences is well known in the art (for example, see U.S. Patent Nos. 6,566,135, 6,566,131, 6,365,354, 6,410,323, 6,107,091, 6,046,321 and 5,981,732, which are incorporated herein by reference).

[0137] In one embodiment, the inhibitor of VEGFR-1 of the present invention is a ribozyme. Ribozyme is an enzymatic RNA molecule that can catalyze RNA specific cutting. The mechanism of action of ribozyme involves sequence-specific hybridization of ribozyme molecule and complementary target RNA, followed by endonucleolytic cutting. Therefore, engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cutting of VEGFR-1mRNA sequence are useful within the scope of the present invention. The specific ribozyme cleavage site in any potential RNA target is initially identified by scanning the ribozyme cleavage site in the target molecule, and these sites generally include the following sequences: GUA, GUU and GUC. Once determined, the predicted structural features of the short RNA sequence of about 15 to 20 ribonucleotides corresponding to the region of the target gene comprising the cleavage site can be assessed, such as secondary structure, which may cause the oligonucleotide sequence to be inappropriate. The suitability of the candidate target can also be assessed by testing the hybridization accessibility of the candidate target to the complementary oligonucleotide, such as by using ribonuclease protection assay.

[0138] In one embodiment, the inhibitor of VEGFR-1 of the present invention is an aptamer. Aptamers are short single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can selectively bind to specific targets, including proteins, peptides, carbohydrates, small molecules, toxins, and even living cells. Aptamer binding is determined by its tertiary structure, involving three-dimensional shape-dependent interactions as well as hydrophobic interactions, base stacking, and insertion.

[0139] In one embodiment, the inhibitor of VEGFR-1 of the present invention is morpholine. Morpholine is an oligomeric molecule (commonly known as oligonucleotide) used to modify gene expression in molecular biology. The molecular structure has a backbone consisting of a methylene morpholine ring and a phosphoramide connection. Morpholine can prevent other molecules from entering a small (about 25 bases) specific sequence on the surface of ribonucleic acid (RNA) base pairing.

[0140] In one embodiment, the inhibitors of VEGFR-1 of the present invention are specific for VEGFR-1.

[0141] In one embodiment, the VEGFR-1 inhibitor of the present invention does not bind to proteins other than VEGFR-1. In one embodiment, the VEGFR-1 inhibitor of the present invention does not inhibit the expression of proteins other than VEGFR-1.

[0142] In one embodiment, the VEGFR-1 inhibitor of the present invention is selective for VEGFR-1. In one embodiment, the VEGFR-1 inhibitor of the present invention is selective for VEGFR-1 over VEGFR-2 and VEGFR-3.

[0143] In one embodiment, the inhibitor of VEGFR-1 of the present invention does not bind to VEGFR-2 and / or VEG FR-3, or binds to VEGFR-2 and / or VEGFR-3 with a lower affinity than VEGFR-1.

[0144] In one embodiment, the inhibitors of VEGFR-1 of the invention bind to VEGFR-1 with an affinity that is at least 10-fold, 25-fold, 50-fold, 75-fold, 80-fold, 90-fold, 95-fold, 100-fold, 125-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, 400-fold, 450-fold, or 500-fold greater than that of other VEGFRs, particularly VEGFR-2 and VEGFR-3.

[0145] Affinity can be determined relatively by binding or competitive binding assays, for example using a Biacore instrument.

[0146] In one embodiment, the VEGFR-1 inhibitor of the present invention does not inhibit VEGFR-2 and / or VEG FR-3 function, or inhibits VEGFR-2 and / or VEGFR-3 function to a lesser extent than VEGFR-1 function.

[0147] In one embodiment, the VEGFR-1 inhibitor of the invention inhibits VEGFR-1 function 1.5-fold, 2-fold, 2.5-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold more than it inhibits VEGFR-2 and / or VEGFR-3 function.

[0148] In one embodiment, the VEGFR-1 inhibitor of the present invention does not inhibit the expression of VEGFR-2 and / or VEGFR-3, or inhibits the expression of VEGFR-2 and / or VEGFR-3 to a lesser extent than the expression of VEGFR-1.

[0149] In one embodiment, the VEGFR-1 inhibitor of the invention inhibits the expression of VEGFR-1 1.5-fold, 2-fold, 2.5-fold, 5-fold, 10-fold, 50-fold, 100-fold, 500-fold, or 1000-fold greater than the expression of VEGFR-2 and / or VEGFR-3.

[0150] In one embodiment, the inhibitor of VEGFR-1 of the present invention is the sole therapeutic agent used to promote myelination and neuroprotection or to treat or prevent a demyelinating disease in a subject in need thereof.

[0151] Thus, in one embodiment, an inhibitor of VEGFR-1 is used as a monotherapy.

[0152] In one embodiment, an inhibitor of VEGFR-1 of the invention is administered in combination with another therapeutic agent.

[0153] In one embodiment, the additional therapeutic agent is an anti-inflammatory drug.

[0154] In one embodiment, the anti-inflammatory drug is an immunomodulator or immunosuppressant.

[0155] Examples of anti-inflammatory drugs include, but are not limited to, interferon beta, glatiramer acetate (CAS147245-92-9); fingolimod (CAS162359-55-9), dimethyl fumarate (CAS 624-49-7), desloximel fumarate (CAS1577222-14-0), teriflunomide (CAS163451-81-8), siponimod (CAS1230487-00-9), cladribine (CAS 4291-63-8), natalizumab (CAS189261-10-7), ocrelizumab (CAS 637334-45-3), alemtuzumab (CAS216503-57-0), cyclophosphamide (CAS 50-18-0), azathioprine (CAS 446-86-6), and mitoxantrone (CAS 65271-80-9).

[0156] Some anti-inflammatory drugs have the following general formula:

[0157]

[0158]

[0159]

[0160] Therefore, in one embodiment, the VEGFR-1 inhibitor of the present invention is administered in combination with an anti-inflammatory drug selected from the group consisting of interferon beta, glatiramer acetate, fingolimod, dimethyl fumarate, desloximel fumarate, teriflunomide, siponimod, cladribine, natalizumab, ocrelizumab, alemtuzumab, cyclophosphamide, azathioprine, mitoxantrone and combinations thereof.

[0161] The present invention also relates to a kit comprising: in one part at least one inhibitor of VEGFR-1; and in a second part at least one anti-inflammatory drug as described above.

[0162] In one embodiment, a kit as described above is used to promote myelination and / or neuroprotection in a subject in need thereof.

[0163] In one embodiment, the kit as described above is used to treat or prevent a demyelinating disease in a subject in need thereof.

[0164] In one embodiment, the at least one inhibitor of VEGFR-1 and the at least one inflammatory drug are administered simultaneously, separately or sequentially.

[0165] In one embodiment, the at least one inhibitor of VEGFR-1 and the at least one inflammatory drug are administered by the same route of administration. In one embodiment, the at least one inhibitor of VEGFR-1 and the at least one inflammatory drug are administered by different routes of administration.

[0166] In one embodiment, the subject is affected by, preferably diagnosed with, a demyelinating disease.

[0167] In one embodiment, the demyelinating disease affects the central nervous system (CNS).Thus, in one embodiment, the subject is affected by, preferably diagnosed with, a demyelinating disease of the central nervous system (CNS).

[0168] As used herein, demyelinating diseases of the CNS can be divided into several categories based on their pathogenesis: demyelination caused by inflammatory processes, viral demyelination, demyelination caused by acquired metabolic dysfunction, hypoxic-ischemic forms of demyelination, and demyelination caused by local compression.

[0169] Symptoms of demyelinating disease include, but are not limited to, vision loss, muscle weakness, muscle stiffness, muscle cramps, bladder and bowel problems, and changes in sensation.

[0170] Examples of demyelinating diseases of the CNS include, but are not limited to, multiple sclerosis, neuromyelitis optica or Devic disease, acute disseminated encephalomyelitis (ADEM), optic neuritis, cerebral ischemia, leukodystrophy, traumatic brain injury (TBI), and transverse myelitis.

[0171] In one embodiment, the demyelinating disease is selected from the group comprising or consisting of multiple sclerosis, neuromyelitis optica or Devic's disease, acute disseminated encephalomyelitis (ADEM), optic neuritis, cerebral ischemia, leukodystrophy, traumatic brain injury (TBI) and transverse myelitis.

[0172] In one embodiment, the demyelinating disease is selected from the group comprising or consisting of multiple sclerosis, optic neuritis, cerebral ischemia, leukodystrophy, and traumatic brain injury (TBI).

[0173] In one embodiment, the subject does not have multiple sclerosis.

[0174] In one embodiment, the subject is affected by, preferably diagnosed with, multiple sclerosis.

[0175] Symptoms of MS are varied and include, but are not limited to, fatigue, vision problems, numbness and tingling, muscle spasms, stiffness and weakness, difficulty moving, pain, problems thinking, learning and planning, depression and anxiety, sexual problems, bladder problems, bowel problems and difficulty speaking and swallowing.

[0176] Tests for diagnosing demyelinating diseases such as multiple sclerosis are well known to those skilled in the art and include, but are not limited to, MRI scans of the brain and spinal cord, cerebrospinal fluid analysis (lumbar puncture), and evoked potential testing.

[0177] It is well known to those skilled in the art that there are various classifications of MS subtypes in the literature. According to the National MS Society, several forms of multiple sclerosis may be encountered: clinically isolated syndrome (CIS), relapsing-remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS), and primary progressive multiple sclerosis (PPMS). The National MS Society has defined these multiple sclerosis disease subtypes as follows.

[0178] In one embodiment, multiple sclerosis is selected from the group consisting of clinically isolated syndrome (CIS), relapsing remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS) and secondary progressive multiple sclerosis (SPMS).

[0179] In one embodiment, multiple sclerosis is a progressive form of the disease, such as PPMS and SPMS.

[0180] In one embodiment, multiple sclerosis is CIS. As used herein, CIS refers to the first onset of neurological symptoms caused by inflammation and demyelination of the central nervous system. An attack that must last at least 24 hours by definition is characteristic of multiple sclerosis, but does not yet meet the diagnostic criteria for multiple sclerosis. However, if a person with CIS also has specific findings on a brain MRI that can provide evidence of early onset of damage in different locations and indicate active inflammation in areas other than the area causing the current symptoms, multiple sclerosis may be diagnosed in that person.

[0181] In one embodiment, multiple sclerosis is RRMS. As used herein, RRMS is characterized by clearly defined new or increased neurological symptom attacks. These attacks are also referred to as relapses or exacerbations, followed by a partial or complete recovery (remission) phase. During remission, all symptoms may disappear, or some symptoms may persist and become permanent symptoms. However, during remission, there is no obvious progression of the disease. RRMS can be further characterized as active (with evidence of relapse and / or new MRI activity within a specific time period) or inactive, and exacerbation (determination of disability after relapse) or no exacerbation.

[0182] In one embodiment, the multiple sclerosis is progressive multiple sclerosis.

[0183] In one embodiment, multiple sclerosis is PPMS. As used herein, PPMS is characterized by worsening neurological function (accumulation of disability) from the onset of symptoms, without early relapses or remissions. PP MS can be further characterized as active (occasional relapses and / or evidence of new MRI activity within a specific time period) or inactive, and progressive (evidence of accumulation of disability over time, with or without relapses or new MRI activity) or non-progressive.

[0184] In one embodiment, multiple sclerosis is SPMS. As used herein, SPMS follows an initial relapsing-remitting process. Some people diagnosed with RRMS will eventually transition to a secondary progressive process in which neurological function gradually deteriorates over time (disability accumulation). SPMS can be further characterized as active (evidence of relapse and / or new MRI activity within a specific time period) or inactive, and progress (evidence of disability accumulation over time, with or without relapse or new MRI activity) or no progression.

[0185] In one embodiment, demyelinating disease is optic neuritis. As used herein, optic neuritis is inflammation of the optic nerve. Clinical features include retro-orbital pain (exacerbated by eye movement), loss of color vision and contrast sensitivity (may develop into severe vision loss), afferent pupillary defect (Marcus-Gunn pupil), and in some cases optic disc hyperemia and swelling. Inflammation may occur in the nerve part inside the eyeball (papillitis or anterior optic neuritis) or in the back of the eyeball (retrobulbar neuritis or posterior optic neuritis).

[0186] In one embodiment, the demyelinating disease is cerebral ischemia. As used herein, cerebral ischemia, also known as brain ischemia or cerebrovascular ischemia, is an acute brain injury caused by insufficient cerebral blood flow.

[0187] In one embodiment, the demyelinating disease is a leukodystrophy. As used herein, leukodystrophy is a group of rare, progressive, metabolic, inherited diseases that affect the brain, spinal cord, and peripheral nerves. Each type of leukodystrophy is caused by a specific genetic abnormality that causes abnormal development or destruction of the white matter (myelin sheath) of the CNS.

[0188] In one embodiment, the demyelinating disease is traumatic brain injury (TBI). As used herein, TBI is an acquired brain injury that occurs when a sudden trauma causes brain damage. TBI occurs when the head suddenly and violently hits an object or when an object penetrates the skull and enters brain tissue.

[0189] In one embodiment, the demyelinating disease affects the peripheral nervous system (PNS).Thus, in one embodiment, the subject is affected by, preferably diagnosed with, a demyelinating disease of the peripheral nervous system (PNS).

[0190] Examples of demyelinating diseases of the PNS include, but are not limited to, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, anti-MAG peripheral neuropathy, Charcot-Marie-Tooth disease, copper deficiency-related diseases (peripheral neuropathies, myelopathy, and rare optic neuropathies), and progressive inflammatory neuropathies.

[0191] In one embodiment, the subject is at risk of developing a demyelinating disease, and in particular, the subject is at risk of developing multiple sclerosis.

[0192] Risk factors for developing multiple sclerosis include, but are not limited to, age (between 20-40 years), sex (female), genetic factors, smoking, infections (including exposure to viruses such as Epstein-Barr virus (EBV)), mononucleosis, human herpesvirus 6 (HHV6) or Mycoplasma pneumoniae, vitamin D deficiency, vitamin B12 deficiency.

[0193] In one embodiment, the subject has received, is receiving, or will receive a demyelinating therapy. In one embodiment, the subject has received, is receiving, or will receive a therapy as described above for treating a demyelinating disease.

[0194] In one embodiment, the subject has received, is receiving, or will receive a therapy for treating multiple sclerosis.

[0195] Examples of treatments for multiple sclerosis include, but are not limited to, physical therapy, anti-inflammatory drugs, and muscle relaxant medications.

[0196] In one embodiment, the subject has received, is receiving, or will receive an anti-inflammatory drug. Examples of anti-inflammatory drugs are provided above.

[0197] In one embodiment, the subject has received, is receiving, or will receive an immunomodulatory or immunosuppressive agent.

[0198] The present invention also relates to a composition comprising, consisting essentially of or consisting of an inhibitor of VEGFR-1 as described above, preferably, wherein the composition is used for promoting myelination and / or neuroprotection in a subject in need thereof.

[0199] The present invention also relates to a composition comprising, consisting essentially of or consisting of an inhibitor of VEGFR-1 as described above, preferably, wherein the composition is for use in treating or preventing a demyelinating disease in a subject in need thereof.

[0200] As used herein, "consisting essentially of," with respect to a composition, means that the inhibitor of VEGFR-1 is the only therapeutic agent or biologically active agent in the composition.

[0201] In one embodiment, the composition further comprises an anti-inflammatory drug as described above.

[0202] The present invention also relates to a pharmaceutical composition comprising the VEGFR-1 inhibitor as described above and a pharmaceutically acceptable excipient, preferably, wherein the pharmaceutical composition is used to promote myelination and / or neuroprotection in a subject in need thereof.

[0203] The present invention also relates to a pharmaceutical composition comprising the VEGFR-1 inhibitor as described above and a pharmaceutically acceptable excipient, preferably, wherein the pharmaceutical composition is used for treating or preventing demyelinating diseases in a subject in need thereof.

[0204] In one embodiment, the pharmaceutical composition further comprises an anti-inflammatory drug as described above.

[0205] In the meaning of the present invention, the expression "pharmaceutical composition" refers to a composition comprising an active ingredient and a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition is used for therapeutic purposes and is related to health.

[0206] The term "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The excipients do not produce harmful, allergic or other adverse reactions when administered to animals (preferably humans). For human administration, the formulation should meet the sterility, pyrogenicity and general safety and purity standards required by regulatory agencies (e.g., FDA office or EMA).

[0207] Pharmaceutically acceptable excipients that can be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances (e.g., sodium carboxymethylcellulose), polyethylene glycol, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and lanolin.

[0208] The present invention also relates to a medicament comprising, consisting essentially of or consisting of an inhibitor of VEGFR-1 as described above, preferably, wherein the medicament is used for promoting myelination and / or neuroprotection in a subject in need thereof.

[0209] The present invention also relates to a medicament comprising, essentially consisting of or consisting of the inhibitor of VEGFR-1 as described above, preferably, wherein the medicament is used for treating or preventing demyelinating diseases in a subject in need thereof.

[0210] In one embodiment, the medicament further comprises an anti-inflammatory drug as described above.

[0211] The present invention also relates to the use of the above-mentioned VEGFR-1 inhibitor in the manufacture of a medicament for promoting myelination and / or neuroprotection in a subject in need thereof, or for treating or preventing demyelinating diseases such as multiple sclerosis.

[0212] The VEGFR-1 inhibitors, kit components, combinations, compositions, pharmaceutical compositions or medicaments of the present invention are formulated for administration to a subject.

[0213] The inhibitor of VEGFR-1, the component of the kit, the combination, the composition, the pharmaceutical composition or the medicament can be administered orally, by injection, topically, nasally, by inhalation, buccally, rectally, intratracheally, endoscopically, transmucosally or transdermally. The term injection as used herein includes subcutaneous, intravenous (IV), intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, perispinal and intracranial injection or infusion techniques.

[0214] In one embodiment, the inhibitor of VEGFR-1, component of the kit, combination, composition, pharmaceutical composition or medicament is administered orally, nasally or by injection.

[0215] Examples of forms suitable for injection include, but are not limited to, solutions, such as sterile aqueous solutions, gels, dispersions, emulsions, suspensions, solid forms suitable for preparation of solutions or suspensions by addition of liquids prior to use, such as powders, liposome forms, and the like.

[0216] Examples of forms suitable for oral administration include, but are not limited to, tablets (including sustained release tablets), hard gelatin capsules, powders, pills (including dragees), capsules (including soft gelatin capsules), oral suspensions, oral solutions and other similar forms.

[0217] Examples of forms suitable for nasal administration include, but are not limited to, sprays, nasal drops, nasal ointments, and nasal spray solutions.

[0218] In one embodiment, the VEGFR-1 inhibitor, kit component, combination, composition, pharmaceutical composition or medicament of the present invention is administered in a therapeutically effective amount.

[0219] In one embodiment, the dose range of the inhibitor of VEGFR-1 to be administered (preferably contained in the kit, combination, composition, pharmaceutical composition or medicament of the present invention) is about 0.1 to about 1000 mg / kg, preferably about 0.5 to about 100 mg / kg, more preferably about 1 to about 50 mg / kg.

[0220] It should be understood that the dosage of the VEGFR-1 inhibitor, the components of the kit or the combination of the present invention will be determined by the attending physician within the scope of reasonable medical judgment. The specific dosage of any particular subject will depend on a variety of factors, including the symptoms being treated and the severity of the symptoms; the activity of the specific compound used; the specific composition used, the age, weight, general health, sex and diet of the patient; the administration time, administration route and excretion rate of the specific compound used; the duration of treatment; the drugs used in combination or simultaneously with the specific compound used; and similar factors well known in the medical field. For example, it is well known to those skilled in the art that the starting dose level of the compound is lower than the dose level required to achieve the desired therapeutic effect, and the dose is gradually increased until the desired effect is achieved.

[0221] In one embodiment, the VEGFR-1 inhibitor, kit component, combination, composition, pharmaceutical composition or medicament of the present invention is administered once.

[0222] In one embodiment, the VEGFR-1 inhibitor, kit component, combination, composition, pharmaceutical composition or medicament of the present invention is administered several times.

[0223] In one embodiment, the inhibitors of VEGFR-1 of the invention, components of the kit, combinations, compositions, pharmaceutical compositions or medicaments will be administered once a day (i.e., once a day), once every three days, once every four days, once every five days, once every six days, once every seven days, once every eight days, once every nine days, once every ten days, once every eleven days, once every twelve days, once every thirteen days, once every fourteen days, once every fifteen days, once a month, twice a month, once a week, twice a week, at least once a day, twice a day, or three times over a period of time determined by one skilled in the art, for example, at least one week, at least one month, at least two months, at least one year, or as needed for the rest of the subject's life.

[0224] In one embodiment, the VEGFR-1 inhibitor, kit component, combination, composition, pharmaceutical composition or medicament of the present invention is administered until the subject is completely cured.

[0225] In one embodiment, the inhibitor, combination, kit, composition, pharmaceutical composition or drug of VEGFR-1 of the present invention promotes myelination or myelin regeneration of nerve fibers in a subject in need thereof. In one embodiment, the inhibitor, combination, kit, composition, pharmaceutical composition or drug of VEGFR-1 of the present invention promotes the survival, differentiation and / or maturation of oligodendrocyte lineage cells and / or Schwann cells.

[0226] Therefore, the present invention also relates to a method for promoting the survival, differentiation and / or maturation of oligodendrocyte lineage cells and / or Schwann cells in a subject in need thereof, comprising administering to the subject an inhibitor, combination, kit, composition, pharmaceutical composition or drug of VEGFR-1 as described above. The present invention also relates to an inhibitor, combination, kit, composition, pharmaceutical composition or drug of VEGFR-1 as described above, for promoting the survival, differentiation and / or maturation of oligodendrocyte lineage cells and / or Schwann cells in a subject in need thereof.

[0227] In one embodiment, the inhibitor, combination, kit, composition, pharmaceutical composition or medicament of VEGFR-1 as described above prevents demyelination and / or neuronal loss in a subject.

[0228] Therefore, the present invention also relates to a method for preventing demyelination and / or neuronal loss in a subject in need thereof, comprising administering to the subject an inhibitor, combination, kit, composition, pharmaceutical composition or drug of VEGFR-1 as described above. The present invention also relates to an inhibitor, combination, kit, composition, pharmaceutical composition or drug of VEGFR-1 as described above, for preventing demyelination and / or neuronal loss in a subject in need thereof.

[0229] The present invention also relates to a method for treating or preventing a demyelinating disease, such as multiple sclerosis, in a subject in need thereof, comprising administering to the subject an inhibitor, combination, kit, composition, pharmaceutical composition or medicament of VEGFR-1 as described above.

[0230] The present invention also relates to an in vitro or ex vivo method for preventing neuronal demyelination or promoting neuronal remyelination, comprising contacting the neuron with the above-mentioned inhibitor of VEGFR-1.

[0231] The present invention also relates to an in vitro or ex vivo method for preventing neuronal loss or promoting neuroprotection, comprising contacting said neurons with an inhibitor of VEGFR-1 as described above.

[0232] Those skilled in the art will appreciate that the inhibitors of VEGFR-1 of the present invention exhibit several of these properties, for example, promoting remyelination and / or neuroprotection. By acting on myelin regeneration and / or neuron protection, the inhibitors of VEGFR-1 of the present invention can be used for remyelination therapy.

[0233] In one embodiment, the subject shows signs of remyelination after being administered the inhibitor of VEGFR-1, component of the kit, combination, composition, pharmaceutical composition or medicament of the present invention.

[0234] In one embodiment, the subject shows reduced signs of demyelination after being administered the inhibitor of VEGFR-1, component of the kit, combination, composition, pharmaceutical composition or medicament of the invention.

[0235] Methods of assessing myelination levels are well known to those skilled in the art and include, for example, MRI and / or visual evoked potential analysis.

[0236] In one embodiment, the subject has at least one symptom of demyelinating disease alleviated after being administered the VEGFR-1 inhibitor, kit component, combination, composition, pharmaceutical composition or medicament of the present invention.

[0237] In one embodiment, the subject exhibits a reduction in neuronal loss after being administered the inhibitor of VEGFR-1, component of the kit, combination, composition, pharmaceutical composition or medicament of the invention.

[0238] In one embodiment, the subject has improved lifespan and / or healthspan after being administered the inhibitor of VEGFR-1, component of the kit, combination, composition, pharmaceutical composition or medicament of the present invention.

[0239] BRIEF DESCRIPTION OF THE DRAWINGS

[0240] FIG1 is a composite of fluorescence microscopy images showing that VEGFR-1 inhibitors promote neuroprotection of Purkinje cells in organotypic cerebellar slices from mice. Cerebellar slices prepared at postnatal day 0 (P0) were cultured in vitro for 10 days in (A) basal medium or in basal medium supplemented with (B) pan-VEGFR inhibitors (Lucitanib and MGCD-265), (C) VEGFR-2 / 3 specific inhibitors (Telatinib), or (D) VEGFR-1 selective and specific inhibitors (SU14813, ZM306416). All compounds were used at a concentration of 500 nM, except ZM306416, which was used at a concentration of 30 μM. Purkinje cells were immunostained for Calbindin (CaBP, gray).

[0241] Fig. 2 is a combination of fluorescence microscopy images and charts showing that inhibitors of VEGFR-1 effectively promote OPC differentiation in vitro. Rat primary OPCs were cultured in a single differentiation medium (A, basal), a basal medium containing 9cis-RA (used as a positive control, (B)), a basal medium containing pan-VEGFR inhibitors Lucitanib and MGCD-265 (C), a basal medium containing VEGFR-2 / 3 selective inhibitor Telatinib (D) or a basal medium containing selective VEGFR-1 inhibitor SU14813 (E). All compounds were tested at 500nM. (F) MBP+Sox10+ oligodendrocytes increased by multiples relative to basal conditions after 5 days in vitro. Data represent the mean ± SEM of N=3-4 experiments. Student's t test: *p<0.05, **p<0.01, ***p<0.001 relative to basal conditions. In the t-test comparison between the VEGFR-2 / 3 inhibitor Telatinib and the VEGFR-1 selective inhibitor SU14813, ≠p<0.05.

[0242] Figure 3 is a combination of fluorescence microscopy images and a combination of graphs showing the effects of pan-VEGFR, VEGFR-2 / 3 and VEGFR-1 specific inhibitors on myelination of mouse cerebellar slices. Cerebellar slices were treated with (A) basal medium alone, (B) 9cis-retinoic acid (9-cis-RA, used as a positive control), (C) pan-VEGFR inhibitor (Lucitanib), (D) VEGFR-2 / 3 selective inhibitor Telatinib or (E) VEGFR-1 specific inhibitor (ZM306416). MBP (white) of myelin and Calbindin (CaBP) (gray) of Purkinje cells. (F) Myelination index graph after treatment with pan-VEGFR inhibitors (Lucitanib and MGCD-265), VEGFR-2 / 3 inhibitors (Telatinib) and VEGFR-1 selective and specific inhibitors (SU14813 and ZM306416) relative to basal conditions. Student's t-test: ***p<0.001, ****p<0.0001 relative to basal conditions. t-test comparison of the VEGFR-2 / 3 inhibitor Telatinib with the VEGF R-1 selective inhibitor, ≠p<0.05. t-test comparison of the pan-VEGFR inhibitor with the VEGFR-1 selective inhibitor, ≠≠p<0.01, ≠≠≠p<0.001. VEGFR-1 inhibitors promote more efficient myelination compared to pan-VEGFR and VEGFR-2 / 3 antagonists.

[0243] Figure 4 is a combination of western blots and graphs showing detection of VEGFR-1 in rat primary OPC cultures. VEGFR-1 expression at the protein level decreases during oligodendrocyte differentiation in vitro. Figure 4 A: Western blot analysis of VEGFR-1 protein expression in proliferating, 3-day and 5-day rat primary OPC cultures (experiments repeated three times). Western blots were probed with anti-VEGFR-1 antibody, anti-MBP antibody and anti-GAPDH antibody (used as a loading control). Figure 4 B: Western blots were quantified by ImageLab BioRad. VEGFR-1 levels were normalized to GAPDH and presented as relative expression levels relative to proliferation conditions. Figure 4 C: MBP levels normalized to GAPDH loading control. Data represent mean ± SEM. N = 2 to 5. Student's t' test: *p ≤ 0.05; **p ≤ 0.01.

[0244] Figure 5is a combination of fluorescence microscopy images and graphs showing that shRNA knockdown of VEGFR-1 promotes oligodendrocyte differentiation. Figure 5 A: Transduction of rat primary OPC cultures with VEGFR-1 shRNA lentivirus resulted in a 2-fold decrease in VEGFR-1 mRNA expression in rat primary OPC cultures relative to scrambled shRNA control cultures. Figure 5 B: MBP immunostaining of control and VEGFR-1 shRNA-transduced rat primary OPC cultures in basal medium at day 5 of differentiation. VEGFR1-knockdown favors OPC differentiation into MBP+ mature oligodendrocytes. Figure 5 C: MBP labeling of rat primary OPC cultures in basal medium alone or in basal medium supplemented with VEGFR-1 blocking peptides. Pharmacological inhibition of VEGFR-1 with a selective blocking peptide promotes oligodendrocyte differentiation. Figure 5 D: Fold increase in MBP+Olig2+ oligodendrocytes after transduction with control or VEGFR-1-specific shRNA or with VEGFR-1-specific blocking peptide. Data represent mean ± SEM. N = 1 to 2. Scale bar: 100 μm.

[0245] Figure 6 Is a combination of fluorescence microscopy images showing that VEGFR-1 blocking peptides promote myelination in organotypic cerebellar slices. CaBP (grey) and MBP (white) immunostaining of organotypic cerebellar slices from mice treated with basal conditions or VEGFR-1 blocking peptides from day 5 to day 10 cultures. Treatment with VEGFR 1 blocking peptides enhances the density of MBP+CaBP+ myelinated axons. Example

[0246] The present invention is further illustrated by the following embodiments.

[0247] Materials and methods

[0248] Rat primary OPC culture and immunocytochemistry

[0249] Rat primary OPC cultures were taken from newborn pups (P0-P1). The brain was dissected and the cerebellar cortex and meninges were removed. Next, the brain tissue was separated mechanically and enzymatically. After filtration and centrifugation, a mixed cell of astrocytes, microglia and OPC was cultured in a poly-ornithine-coated bottle with DMEM medium. After 13-14 days of culture, microglia were removed by 2 hours of shaking. Next, OPCs were collected after 18 hours of shaking and then differential adhesion was performed. The purification of OPCs was verified by immunostaining of Olig2 and Sox10. To induce differentiation, cells were kept in proliferation medium (DMEM-F12, 1% PS, FGF, PDGF, 1% laminin, biotin, 2% B27, N1 mixture) for 24 hours and then switched to a differentiation medium with the same composition but without FGF and PDGF. The cells were incubated at 37°C (5% CO2) for 5 days, with the medium partially replaced every 2 days. The cells were then fixed in 2% paraformaldehyde and immunostained with Sox10 and MBP. The cells were incubated for 2 hours with primary antibodies Sox10 (goat 1 / 100) and MBP (rabbit 1 / 500) diluted in PBS 0.1M / 0.1% Triton containing 4% BSA. After washing, the cells were incubated with secondary antibodies (anti-goat Alexa647, anti-rabbit Alexa488 and Dapi) for 1 hour, and finally washed in PBS 0.1M and mounted in Fluoromount medium. Fluorescence imaging was performed using a confocal microscope NIKON A1R-HD25.

[0250] Organotypic sections and immunohistochemistry

[0251] Organotypic cerebellar slices were generated from the brains of postnatal day 0 (P0) and P7 mouse pups. In brief, the cerebellum was dissected and cut into 350 μm slices. Cerebellar slices of the cerebellar vermis were collected and then cultured on Millicell cell culture inserts (Millipore) in 6-well plates, and the culture medium consisted of 50% basal culture medium Eagle, 25% Hank solution (HBSS), 25% inactivated horse serum, 5mg / ml high glucose, 1mM L-glutamine, 0.2% penicillin-streptomycin. The slices were incubated at 37°C (5% CO2) for 10 days (slices from P0) or 3 days (slices from P7). The culture medium was changed every 2-3 days. For immunohistochemistry, the slices were fixed with 4% paraformaldehyde, and CaBP and MBP were immunostained. Briefly, the sections were incubated in 4% BSA / 0.2% triton for 1 hour and then incubated overnight with the following primary antibodies: rabbit anti-CaBP (dilution: 1 / 10000) and rat anti-MBP (dilution: 1 / 250). After washing several times in PBS 0.1M, the sections were incubated with secondary antibodies (anti-rabbit alexaFluor555, anti-mouse alexaFluor488) and Dapi for 2 hours, and finally washed in PBS 0.1M and mounted in Fluoromount medium. Fluorescence imaging was performed using a confocal microscope NIKON A1R-HD25.

[0252] Neuroprotection

[0253] The neuroprotective effect of the drug was evaluated by evaluating the density of Purkinje cells in basal medium alone and in basal medium supplemented with VEGFR inhibitors. Organotypic cerebellar slices obtained from P0 mice (as described above) were cultured in vitro for 10 days in basal medium alone or in basal medium supplemented with pan-VEGFR inhibitors, VEGFR-2 / 3 specific inhibitors (Telatinib) or VEGFR-1 selective and specific inhibitors (SU14813 and ZM306416). Except for ZM306416, which was used at a concentration of 30 μM, all compounds were used at a concentration of 500 nM. After treatment, the slices were immunostained for CaBP as described above.

[0254] Oligodendrocyte differentiation and myelination

[0255] By analyzing MBP + Sox10 + Oligodendrocyte differentiation was assessed by fold increase in cells under treated conditions relative to basal conditions. MBP was quantified after treatment with pan-VEGFR and selective VEGFR-1 antagonists. + Sox10 +The percentage of cells was measured and compared with basal medium. The results are shown as an increase in differentiation fold relative to basal conditions. Myelination was assessed using organotypic cerebellar slices generated from P7 mouse cerebellum, and the effects of VEGFR subtype antagonists on myelination were analyzed after immunostaining for Sox10 of oligodendrocytes, MBP (white) and calbindin (grey) of myelin. The slices were cultured in basal medium (control condition) or in basal medium supplemented with 9cis-RA (used as a positive control for myelination) or VEGFR-1 selective and specific inhibitors (SU14813 and ZM306416). All compounds were used at a concentration of 500nM, except for ZM306416, which was used at a concentration of 30μM. The myelination index under each condition was quantified as the percentage of MBP+CaBP+ axons to the total CaBP+ axon surface area and normalized to basal conditions.

[0256] Western blotting

[0257] Cell lysis and denaturation. Primary rat OPC cultures coated on B60 culture dishes were lysed using RIPA buffer (ThermoFisher Scientific), DTT (ThermoFisher Scientific) and protease and phosphatase inhibitors (PPI, ThermoFisher Scientific), lysed in proliferation medium after 48 hours or in differentiation medium after 3 and 5 days. The lysed cells were centrifuged at 13000rpm for 10 minutes at 4°C. The supernatant containing protein was recovered and denatured using Laemmli 4X (BioRad) and β-mercaptoethanol, and then heated at 95°C for 5 minutes. The cell lysate was stored at -80°C.

[0258] Gel electrophoresis and transfer. Equal amounts of protein for each sample were loaded into the wells of a 4-20% TGX gel (BioRad) and separated by SDS-PAGE at 100 V for 1 hour and 30 minutes. Proteins were transferred to a PVDF membrane (BioRad) using Trans-Blot Turbo. Ponceau staining on the membrane confirmed the transfer of the protein.

[0259] Protein detection. The membrane was blocked at room temperature for 15-20 minutes using EveryBlot blocking buffer (BioRad), and then incubated overnight on a shaker at 4°C with the following primary antibodies diluted in blocking buffer: anti-VEGFR-1 (Abcam ab32152, 1:2000 dilution), anti-GAPDH (Cell Signaling CST2118, 1:2000 dilution) and anti-MBP (Genetex GTX133108, 1:5000 dilution). The next day, the membrane was washed 4 times with 1X TBS + 0.1% Tween for 2 minutes each time, and then incubated with horseradish peroxidase (HRP)-conjugated antibody (Cell Signaling CST7074, 1:5000 dilution) at room temperature for one hour. The membrane was washed again with 1X TBST 4 times for 2 minutes each time and washed once with 1X TBS. Chemiluminescence detection of proteins was performed using ECL Clarity (BioRad). Band intensity was analyzed using BioRad's ImageLab software. For each sample, the intensity value of the GAPDH band was used to normalize the measured intensities of VEGFR1 and MBP.

[0260] shRNA lentiviral transduction

[0261] Primary rat OPC cultures were transduced with rat lentiviral particles at different MOIs (multiplicity of infection) containing 4 unique 29mer shRNAs (OriGene) targeting VEGFR-1 with the following sequences:

[0262] 5'GGTCATAGAAGGAACCAATAAGACGTTA'3(SEQ ID NO:15),

[0263] 5'TCACTCTGAACCTTGTCATCAAGAATGTG'3(SEQ ID NO:16),

[0264] 5'CTCGGGTGACCTGCTTCAAGCCAATGTCCA'3 (SEQ ID NO:17),

[0265] 5'GAGCATCTATCAGGCAGCGGATTGACCAA'3 (SEQ ID NO: 18).

[0266] For RT-qPCR, transduction was used at MOI 8 and MOI 2. Transfection efficiency was confirmed by GFP lentiviral expression. For immunocytochemistry, transfection was used at MOI 4 and labeled with Olig2 and MBP to quantify Olig2+MBP+ differentiated oligodendrocytes.

[0267] Drug treatment with VEGFR-1 blocking peptides

[0268] Mouse cerebellar slices were prepared as described above. The slices were treated in vitro for 5 to 10 days with basal medium alone or with basal medium containing a specific VEGFR-1 blocking peptide (sequence: GNQWFI (SEQ ID NO: 6)) at a concentration of 10 μM. The slices were fixed and stained with Calbindin (CaBP) to stain Purkinje cells and MBP to mark myelinated axons.

[0269] result

[0270] VEGFR-1 inhibition enhances neuroprotection in organotypic cerebellar slices

[0271] We tested the effects of VEGFR isoform inhibitors on neuroprotection using an ex vivo model of organotypic mouse cerebellar slice cultures prepared from postnatal day 0 (P0) mouse cerebellum. Interestingly, organotypic cerebellar slices treated for 10 days with the pan-VEGFR inhibitors Lucitanib or MGCD-265 (500 nM) (Figure 1B) showed an increase in the number of Purkinje cells compared to basal conditions (Figure 1A), strongly supporting the effect of VEGFR inhibitors on Purkinje cell survival. To determine whether VEGFR isoforms are selectively involved in Purkinje cell neuroprotection, we examined selective inhibitors of VEGFR-2 / 3 (Telatinib) and VEGF R-1 (SU14813 and ZM306416). We found that selective and specific inhibitors of VEGFR-1, SU14813 and ZM306416, but not a VEGFR-2 / 3 inhibitor (Telatini b), enhanced the neuroprotection of Purkinje cells in organotypic cerebellar slices (Fig. 1C and D), similar to the results observed using pan-VEGFR inhibitors (Lucitanib or MGCD-265). Together, these data suggest that inhibition of VEGFR-1 enhances the survival of Purkinje cells in organotypic cerebellar slices and protects them from apoptosis. Notably, Purkinje cells undergo extensive apoptosis from P1 to P5 during mouse cerebellar development.

[0272] VEGFR-1 inhibition promotes oligodendrocyte differentiation and Myelination

[0273] To investigate the role of VEGFR-1 in OPC differentiation and myelination in vitro and in vitro models, we first analyzed the effects of pan-VEGFR and selective VEGFR-2 / 3 or VEGFR-1 inhibitors on OPC differentiation and maturation. Data are shown for Lucitanib and MGCD-265 (pan-VEGFR inhibitors), Telatinib (VEGFR-2 / 3 inhibitor), and SU14813 (VEGFR-1 inhibitor). After 5 days of in vitro treatment of rat primary OPC cultures with all tested compounds, SOX10 + MBP + The number of differentiated oligodendrocytes was significantly increased (Figure 2A-2F). Interestingly, the increase in OPC differentiation induced by pan-VEGFR inhibitors and VEGFR-1 inhibitors was stronger than that induced by VEGFR-2 / 3 inhibitors (Figure 2A-2F). Figure 2F Notably, dose-response analysis of different concentrations (1 nM, 100 nM, 500 nM, and 1 μM) revealed that 100 nM had a significant effect on OPC differentiation, with all compounds reaching a peak at 500 nM (data not shown).

[0274] The latter concentrations were then used to further validate the effects of these compounds on myelination in organotypic cerebellar slices prepared from P7 mouse brains. The slices were treated with these compounds ex vivo for 3 days. Notably, we found abundant MBP in organotypic cerebellar slices treated with pan-VEGFR inhibitors (Lucitanib or MGCD265) or VEGFR-1 selective and specific inhibitors (SU14813 and ZM306416). + In addition, quantification of the myelination index showed a higher density of myelinated axons in cerebellar slices treated with both families of VEGFR-1 inhibitor compounds relative to basal conditions, relative to the positive control 9cis-RA, relative to VEGFR-2 / 3 inhibitors, and relative to pan-VEGFR inhibitors ( Figure 3F ). These data clearly demonstrate that the density of myelinated axons is significantly increased after treatment with a VEGFR-1 specific inhibitor and, importantly, show that VEGFR-1 inhibitors induce myelination more efficiently than pan-VEGFR inhibitors or VEGFR-2 / 3 inhibitors.

[0275] VEGFR-1 expression is reduced during oligodendrocyte differentiation in vitro

[0276] To verify the expression of VEGFR-1 during oligodendrocyte differentiation and maturation, we investigated changes in VEGFR-1 protein expression in rat primary OPC cultures. To this end, cells were lysed at D3 and D5 (3 and 5 days in differentiation medium) under proliferative conditions to extract proteins. Western blot analysis showed that the expression of full-length VEGFR-1 was reduced by 37% ( 2.3 % ) at D3 and D5 compared with proliferation. Figure 4 A, 4B), indicating that VEGFR-1 expression decreases during oligodendrocyte differentiation. MBP protein levels at D5 confirmed an increase in differentiated mature oligodendrocytes in culture compared to D3 and proliferation conditions ( Figure 4 C). Our data indicate that VEGFR-1 is expressed by oligodendrocytes, primarily at the OPC stage. Interestingly, we also found that VEGFR-1 expression is also downregulated during OPC differentiation and maturation.

[0277] Knockdown of VEGFR-1 using shRNA lentiviral transduction or VEGFR1 blocking peptide promoted oligodendrocyte differentiation

[0278] To determine the functional role of VEGFR-1 in oligodendrocyte lineage progression and myelination, we first used a shRNA-mediated VEGFR-1 knockdown strategy in primary OPC cultures. Primary rat OPC cultures were transfected with lentivirus expressing different shRNAs targeting VEGFR-1. Of the 4 shRNAs tested, 3 targeted exons 11-12, exons 12-13, and exon 7 encoding the full-length and soluble forms of the immunoglobulin-like loop, and 1 targeted exon 26 encoding the full-length form of the tyrosine kinase domain. A scrambled shRNA was used as a control.

[0279] Our data confirmed efficient transduction of lentivirus at a multiplicity of infection of 4 (MOI4) and knockdown of VEGFR-1 ( Figure 5 A). RT-qPCR also showed increased expression of MOG (myelin oligodendrocyte glycoprotein), which is mainly expressed by mature myelinating oligodendrocytes (data not shown). This result suggests that VEGFR-1 knockdown in oligodendrocytes promotes their differentiation. To further confirm the effect of VEG FR-1 inhibition on OPC differentiation, we used shRNA or a VEGFR-1-specific blocking peptide (sequence: GNQWFI) that binds to the ligand-binding site of this receptor, thereby limiting ligand-receptor interactions in rat primary OPC cultures. VEGFR-1-shRNA- and VEGFR-1-blocking peptide-treated cultures were immunolabeled and compared with shRNA control and control conditions, respectively. On day 5 of differentiation medium, cells were immunolabeled with Olig2 and MBP to assess the proportion of differentiated oligodendrocytes ( Figure 5B, 5C and 5D). To this end, we quantified the proportion of Olig2+MBP+ differentiated oligodendrocytes to total Olig2+ oligodendrocytes in each condition (ratio of Olig2+MBP+ cells / Olig2+ cells). This showed that the number of Olig2+MBP+ cells increased by 1.23±0.12-fold and 1.18±0.19-fold when transfected with VEGFR-1-specific shRNA or treated with VEGFR-1 blocking peptide compared with the control ( Figure 5 D) These results indicate that inhibition of VEGFR-1 promotes the differentiation of OPCs into MBP+ oligodendrocytes.

[0280] VEGFR-1 blocking peptide promotes oligodendrocyte maturation and myelination

[0281] To further evaluate the effects of VEGFR1 inhibition on oligodendrocyte maturation and myelination, we used organotypic cerebellar slices from mice that were treated or untreated with a VEGFR-1 blocking peptide (sequence: GNQWFI). We found that inhibition of VEGFR-1 with 10 μM VEGFR-1 blocking peptide enhanced myelination in vitro in organotypic cerebellar slices ( Figure 6 ).

[0282] Together, these results strongly support an emerging role for VEGFR-1 in oligodendrocyte differentiation, myelination, and neuroprotection and reveal the possibility of VEGFR-1 as a new pharmacological target for remyelination therapy.

Claims

1. An inhibitor of vascular endothelial growth factor receptor 1 (VEGFR-1) for use in promoting myelination and / or neuroprotection in a subject in need thereof.

2. The VEGFR-1 inhibitor for use according to claim 1, wherein the inhibitor is selected from the group comprising or consisting of small organic molecules, peptides, antibodies, antibody fragments, antibody mimetics and nucleic acids.

3. The VEGFR-1 inhibitor for use according to claim 2, wherein the inhibitor is an organic small molecule, preferably an organic small molecule selected from the group comprising or consisting of: ZM306416 (CB 676475), SU14813 and salts, derivatives and combinations thereof.

4. The VEGFR-1 inhibitor for use according to claim 2, wherein the inhibitor is a peptide, preferably a peptide selected from the group consisting of: peptides having SEQ ID NO: 6 to 10 and combinations thereof, more preferably, the inhibitor is a peptide having SEQ ID NO:

6.

5. The VEGFR-1 inhibitor for use according to claim 2, wherein the inhibitor is a nucleic acid, preferably a shRNA selected from the group comprising or consisting of shRNAs having SEQ ID NOs: 15 to 18 and combinations thereof.

6. A VEGFR-1 inhibitor for use according to any one of claims 1 to 5, wherein the subject is affected by or diagnosed with a demyelinating disease.

7. The VEGFR-1 inhibitor for use according to claim 6, wherein the demyelinating disease is selected from the group comprising or consisting of multiple sclerosis, optic neuritis, cerebral ischemia, leukodystrophy and traumatic brain injury (TBI).

8. The VEGFR-1 inhibitor for use according to claim 6, wherein the demyelinating disease is multiple sclerosis.

9. A VEGFR-1 inhibitor for use according to claim 8, wherein the multiple sclerosis is selected from the group consisting of: clinically isolated syndrome (CIS), relapsing-remitting multiple sclerosis (RRMS), primary progressive multiple sclerosis (PPMS) and secondary progressive multiple sclerosis (SPMS), preferably, wherein the multiple sclerosis is a progressive form of the disease.

10. A VEGFR-1 inhibitor for use according to any one of claims 1 to 9, wherein the subject has received, is receiving or will receive an anti-inflammatory drug.

11. The VEGFR-1 inhibitor for use according to claim 10, wherein the anti-inflammatory drug is an immunomodulator or an immunosuppressant.

12. The VEGFR-1 inhibitor for use according to claim 10 or 11, wherein the anti-inflammatory drug is selected from the group comprising or consisting of interferon beta, glatiramer acetate, fingolimod, dimethyl fumarate, desloximel fumarate, teriflunomide, siponimod, cladribine, natalizumab, ocrelizumab, alemtuzumab, cyclophosphamide, azathioprine, mitoxantrone and combinations thereof.

13. A pharmaceutical composition comprising the VEGFR-1 inhibitor according to any one of claims 1 to 5 and at least one pharmaceutically acceptable excipient for use in promoting myelination and / or neuroprotection in a subject in need thereof.

14. A kit comprising in one part at least one VEGFR-1 inhibitor and in a second part at least one anti-inflammatory drug.

15. The kit according to claim 14, for promoting myelination and / or neuroprotection in a subject in need thereof.

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