Treatment of nervous system conditions

Abstract

Provided is 4-methylumbelliferone, derivatives and salts thereof, or the same for use in treating a nervous system condition in a subject. Preferably, the condition is associated with scarring, such as glial scarring. Typically, the nervous system condition is selected from the group including trauma, injury, infection, degeneration, structural defects, tumors, and conditions resulting from interrupted blood flow. Nervous system and other injuries are made permeable and repairable.

Description

[Technical Field]

[0001] This invention relates to the treatment of the nervous system condition of a subject. In particular, this invention relates to the treatment of spinal cord injury in a subject. This invention also relates to the treatment of lesions related to the nervous system condition. [Background technology]

[0002] The spinal cord is a long, tubular structure containing nerve tissue that primarily transmits nerve signals or messages, enabling communication between the body and the brain. It also regulates reflexes. The spinal cord is enclosed within the vertebral column. Spinal nerves are located in the space between the vertebrae and the nerve roots that emerge from the spinal cord on either side of each vertebra. The spinal cord is divided into segments, and most bodily functions correspond to specific segments. There are 31 spinal nerve segments in the human spinal cord.

[0003] Spinal cord injury (SCI) involves damage to the spinal cord that results in changes to its function. These changes may be temporary or permanent. The injury can occur at any level of the spinal cord and may be complete or incomplete. The loss of function, both motor and sensory, depends on the location and extent of the injury.

[0004] Dislocation or fracture of the vertebrae due to traumatic injury can cause contusion or transection of the spinal cord. This primary mechanical injury initially damages the spinal cord, disrupting blood supply and damaging cells and nerves. Secondary responses follow, leading to the breakdown of the central portion of the spinal cord. Loss of myelin leads to loss of signaling. Glial cells migrate to the injured area of ​​the spinal cord in an attempt to repair the damage. Glial scars consist of two components: cellular and biochemical. Following injury to the spinal cord, astroglial hyperplasia and microglial activation occur. These cause the secretion of excess molecules, including chondroitin sulfate proteoglycans.

[0005] There are over 2 million cases of spinal cord contusion injuries. There are over 6 million cases of stroke. There are over 700,000 cases of cerebral palsy. There are over 1 million cases of Parkinson's disease. There are over 400,000 cases of multiple sclerosis.

[0006] Current treatments for spinal cord injury involve intravenously administering a steroid called methylprednisolone within eight hours of the injury; however, this is limited, and the therapeutic effect has been found to be ineffective.

[0007] Neuroplasticity is the way the central nervous system (CNS) adapts to changes from the external environment through the reorganization of synaptic connections and circuits. It is crucial for successful functional recovery after spinal cord injury. Perineuronal networks (PNNs) are dense pericellular extracellular matrix structures found throughout the CNS (Kwok et al., 2011), and their formation has been associated with the termination of developmental plasticity (Carulli et al., 2010; Pizzorusso et al., 2002). PNNs surround the nerve surface with holes where synapses exist (Figure 7). This means that these nerve cells will not have synapses for functional recovery. This limits synapse formation and, therefore, limits neuroplasticity.

[0008] Chondroitin sulfate proteoglycans (CSPGs) are a family of glycans that inhibit neurite outgrowth and, consequently, remodeling and plasticity (Kwok et al., 2011; Silver and Miller, 2004). CSPGs have been found to be present in PNNs after spinal cord injury and to increase in glial scars.

[0009] Glial scarring via CSPG removal in PNNs and chondroitinase ABC (ChABC) injection has been shown to open up opportunities for plasticity and reconstitution that promote post-SCI recovery in both acute and chronic injury models up to 18 months (Bradbury et al., 2002; Wang et al., 2011; Warren et al., 2018). ChABC has been shown to be beneficial for recovery in conjunction with other therapies, including rehabilitation (Garcia-Alias ​​et al., 2011). However, this approach presents significant obstacles to its translation into human applications (e.g., limited stability of the enzyme, thus requiring continuous injection, which is invasive, and the potential progression of immune responses due to prolonged exposure to bacterial proteins).

[0010] 4-Methylumbelliferone is a commonly used compound in biliary therapy. In Europe, it is available under the name: hymechromone. This drug has been used in this field for many years and has a good safety profile. This compound was first used in vitro in 1995 by Nakamura et al. to suppress HA synthesis in dermal fibroblasts (Nakamura et al., 1995).

[0011] Fontaine et al. conducted toxicological and teratological studies of 4-methylumbelliferone (Fontaine et al., 1968). They reported the results of studies on acute toxicity, chronic toxicity, local tolerance, and experimental teratogenesis in several species. In the chronic toxicity study, the maximum tolerable dose in oral proportions was found to be equal to 6000 mg / kg in rats. In the chronic study, rats were given 200 mg / kg / day and 40 mg / kg / day for 3 months. No deaths were reported, and there were no effects on the rats' appetite behavior or appearance. The authors report that, overall, under these conditions, the tolerable dose in rats can be determined to be at least 200 mg / kg / day, which is 10 times the daily dose expected in humans. The authors also report that the drug is well retained locally. The product does not appear to have teratogenic effects in the three species studied: rats, mice, and rabbits, even at very high doses. In particular, the drug was found to be well-retained even at a dose of 1200 mg / kg / day in pregnant rats. Furthermore, it had no effect on the development of young rats.

[0012] This invention helps to address the problems of the prior art and provides agents for use in the treatment of nervous system conditions. In particular, this invention provides agents for use in the treatment of spinal cord injury.

[0013] Adult central nervous system axons do not retain the ability to reconstitute. After injury, the extracellular matrix plays diverse roles in exacerbating poor regeneration. Chondroitin sulfate proteoglycans are a family of extracellular matrix molecules that increase in glial scars after spinal cord injury and inhibit neurite outgrowth, thus suppressing reconstitution. Furthermore, chondroitin sulfate proteoglycans are also present in certain structures called PNNs, which limit plasticity for potential functional recovery. Immunohistochemical examination of the spinal cord showed a decrease in PNNs around motor neurons, attributed to hyaluronic acid synthesis suppressed by hymechromone. In addition, we also observed a marked decrease in the staining intensity of chondroitin sulfate proteoglycans in the spinal cord.

Summary of the Invention

[0014] One embodiment of the present invention provides 4-methylumbelliferone (hereinafter referred to as "the PNN inhibitor (PNNi) of the present invention"), its derivatives or salts for use in treating the condition of the nervous system.

[0015] In one embodiment, the condition of the nervous system is related to the formation of lesions. In one embodiment, the lesion is a glial scar. In one embodiment, the lesion is a plaque resulting from the accumulation of toxic protein aggregates. One specific example is an amyloid scar.

[0016] In another further embodiment, the condition of the nervous system may be selected from the group including trauma, injury, infection, degeneration, structural defects, tumors, and conditions caused by blood flow interruption.

[0017] This condition may be selected from the group including stroke, transient ischemic attack, myelopathy, hemorrhage, meningitis, encephalitis, Bell palsy, brain or spinal cord tumors, Parkinson's disease, Huntington's disease, and Alzheimer's disease. It may be cerebral palsy.

[0018] In one embodiment, the condition of the nervous system may be damage to the nervous system. Preferably, the condition of the nervous system is spinal cord injury.

[0019] One embodiment of the present invention provides 4-methylumbelliferone, its derivatives or salts, that is, the PNN inhibitor (PNNi) of the present invention for use in treating lesions associated with the condition of the nervous system.

[0020] One embodiment of the present invention provides a method for treating the condition of the nervous system of a subject, the method comprising administering 4-methylumbelliferone (hereinafter referred to as "the PNN inhibitor (PNNi) of the present invention"), its derivatives or salts to the subject, and this condition may be as disclosed herein.

[0021] In further embodiments, the present invention provides a method for treating lesions related to a nervous system condition in a subject. This method involves administering 4-methylumbelliferone (hereinafter referred to herein as "the PNN inhibitor of the present invention (PNNi)"), its derivatives, or salts to the subject. This condition may be as disclosed herein.

[0022] (definition) All publications, patents, patent applications, and other references described herein, as well as their variations for all purposes, and the entirety of their cited content, are incorporated herein by reference, as each individual publication, patent, or patent application is identified and incorporated by reference individually.

[0023] As used herein, and unless otherwise specifically indicated herein, the following terms are intended to have the following meanings in addition to the broad (or narrow) meanings of terms enjoyed in the art:

[0024] Unless required by context, the use of the singular in this specification should be read as including the plural, and vice versa. The terms "a" or "an" used in relation to existence should be read as referring to one or more existences. The terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this specification.

[0025] As used herein, the term “comprise” or its variations such as “comprises” or “comprising” includes any cited integer (e.g., feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g., feature, element, characteristic, property, method / process step or limitation) and does not exclude any other integer or group of integers. Therefore, as used herein, the term “comprising” is inclusive or unrestrictive and does not exclude any additional unquoted integer or method / process step.

[0026] As used herein, the term “nervous system” or “human nervous system” means the part of the body that coordinates movement and transmits signals or messages between parts of the body. The nervous system comprises the central nervous system or CNS (brain and spinal cord) and the peripheral nervous system or PNS.

[0027] As used herein, the term “condition of the nervous system” may mean any disease, disorder, or condition that affects the normal functioning of the nervous system. This condition may be injury or damage. This condition may be selected from, but is not limited to, those resulting from trauma, injury, infection, degeneration, structural defects, tumors, and disruption of blood flow and autoimmune disorders. This condition may be selected from, but is not limited to, stroke, transient ischemic attack, myelopathy, hemorrhage, meningitis, encephalitis, Bell's palsy, brain or spinal cord tumors, Parkinson’s disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Huntington’s disease, and Alzheimer’s disease.

[0028] As used herein, the term “spinal cord injury” means injury or damage to the spinal cord or any part thereof that results in alteration of its function. It may be any site or segment of the spinal cord, and the damage may be at any level. There may be one or more sites of injury. The injury includes subcone cone injuries, including peripheral nerves. To avoid doubt, this term also includes injuries that open, close, and penetrate the spinal cord. This includes complete and incomplete lesions, partial and complete amputations, central spinal cord syndromes, Brown-Séquard syndrome, cauda equina syndrome, and myelopathy, as well as any degree or type of nerve root injury.

[0029] As used herein, the term “condition” is used to define any abnormal condition that impairs physiological function and is associated with a particular symptom. The term is used broadly to encompass any disease, disorder, illness, abnormality, condition, disease, state, or syndrome in which physiological function is impaired, regardless of its pathological nature (or whether a pathological basis for the disease is indeed established). Thus, it includes infection, trauma, injury, surgery, radiation cutting, poisoning, or malnutrition.

[0030] As used herein, the terms “treatment” or “to treat” mean an intervention (e.g., administering a drug to a subject) that cures, improves or reduces the symptoms of a condition or disease, or eliminates (or reduces the severity of) its cause. In this case, the term “treatment” may also include promoting recovery. In this case, the term is used synonymously with “therapy.” Additionally, the terms “treatment” or “to treat” mean an intervention (e.g., administering a drug to a subject) that prevents or delays the onset or progression of a disease, or reduces (eradicates) its occurrence within a treated population. The term “treatment” is used synonymously with the term “prevention.”

[0031] As used herein, the “effective dose” or “therapeutic dose” of a drug means an amount that is sufficient to provide the desired effect (e.g., a treatment or prevention that results in permanent or temporary improvement of the subject’s condition) in proportion to a reasonable benefit / risk ratio, without excessive toxicity, inflammation, allergic reaction, or other problems or situations. This dose will vary from subject to subject, depending on age and individual general condition, mode of administration, and other factors. Therefore, it is not possible to determine an exact effective dose, but those skilled in the art will be able to determine a suitable “effective” dose in any individual case using usual experiments and background general knowledge. In this context, the outcome of treatment includes the elimination or reduction of symptoms, pain, or discomfort, long-term survival, improved mobility, and other markers of medical improvement. The outcome of treatment does not necessarily have to be a complete cure.

[0032] As defined above, in the context of treatment and effective dose, the term “subject” (to be read as including “individual,” “animal,” “patient,” or “mammal,” where the context allows) refers to the subject, in particular a mammalian subject, to whom the treatment is indicated. In a preferred embodiment, the subject is human. In one embodiment, the subject is an adult. In one embodiment, the subject is, for example, a pediatric age subject under 21 years of age. The subject may be of any sex.

[0033] As used herein, the term “composition” should be understood to mean a product manufactured by human hands and not to include naturally occurring compositions. Compositions may be composed in unit dosage form, i.e., in the form of a single unit dosage or in the form of separate parts including multiple or subunit dosages of a single unit.

[0034] As used herein, the term “pharmaceutical composition” refers to the PNNi of the present invention or a mixture of the composition of the present invention with one or more pharmaceutically acceptable carriers, diluents, or excipients. Whether administered alone or in a mixture with acceptable carriers, excipients, or diluents, the PNNi of the present invention will generally be administered, particularly for human treatment. The pharmaceutical composition may also be for human or animal use in human and veterinary medicines. Specific examples of such suitable excipients for various different forms of the pharmaceutical composition described herein can be found in “Handbook of Pharmaceutical Excipients, 8th Edition, Edited by A Wade and PJ Weller. In particular, American Pharmaceutical Review “Opportunities and Challenges in Biologic Drug Discovery (Hooven, 2017),” and compositions for topical delivery are described in “Topical drug delivery formulations edited by David Osborne and Antonio Aman, Taylor & Francis,” all of which are incorporated herein by reference.

[0035] Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985). Specific examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol. Specific examples of suitable diluents include ethanol, glycerol, and water. The selection of pharmaceutically acceptable carriers, excipients, or diluents can be made in relation to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may include, or in addition to, any binders, lubricants, suspending agents, coating agents, or solvents as carriers, excipients, or diluents. Suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, β-lactose, corn sweeteners, natural and synthetic gums such as acacia and tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Specific examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride. Preservatives, stabilizers, colorants, and fragrances may also be provided in the pharmaceutical composition. Specific examples of preservatives include sodium benzoate, sorbic acid, and hydroxybenzoic acid esters. Antioxidants and suspending agents may also be used.

[0036] In certain embodiments, the term “medically acceptable” means that it is approved by a federal or state regulatory agency, or listed by the U.S. Drug Administration or any other generally accepted drug administration for use in animals, and more particularly for use in humans.

[0037] As used herein, the term “derivative” means a compound derived from a modified 4-methylumbelliferone that, for example, by a chemical reaction, retains the ability to remove PNNs, i.e., treats the nervous system conditions described herein. The method for determining PNN removal may be as described herein. Derivatives may have substitutions with one or more alkyl, allyl, acyl, hydroxyl, hydroxymethyl, methoxy, methyl and / or sulfonyl compounds compared to 4-methylumbelliferone. This term may be used interchangeably with functional derivative. 4-methylumbelliferone derivatives are well known in the art. Specific examples can be found in US2019269647 or US2018201640, all of which are incorporated herein by reference. All such derivatives are considered to be within the scope of this disclosure.

[0038] Derivatives or pharmaceutically acceptable salts may be used for the treatment methods of the present invention. The PNN inhibitor or compound of the present invention may be in the form of a salt, but those skilled in the art of medicinal chemistry will understand that the choice of salt is not important, and other pharmaceutically acceptable salts can be prepared by well known methods. The PNN inhibitor or compound of the present invention may also be in the form of a metabolite or prodrug.

[0039] As used herein, the term “lesion” means an abnormal change in an organ or part thereof due to injury or disease. It may include scars or plaques.

[0040] The term "sustained release" is used in its traditional sense in relation to the delivery system of a compound or activator, meaning that the compound or activator is released gradually over a period of time, or, if not necessarily, released at a relatively constant release level over that period.

[0041] Himechromone is a hyaluronic acid synthesis inhibitor. It is (4-methylumbelliferone) (Andreichenko et al., 2019). Our data showed that 10 days of non-invasive oral administration reduced both chondroitin sulfate and hyaluronic acid in the spinal cord. This makes Himechromone a first-line candidate for reducing inhibitory chondroitin sulfate proteoglycans after spinal cord injury.

[0042] The involvement of PNNs in limiting plasticity is a physiological event. Even when PNNs are removed under normal physiological conditions, activation of plasticity can be observed. Chronic spinal cord injury exhibits similar characteristics of PNNs, as in normal physiology, and similarly responds sensitively to beneficial conditions.

[0043] "Alkyl" refers to a linear or branched alkyl group with a specified number of carbon atoms (e.g., C1-C4 alkyl) or any number within this range (methyl, ethyl, propyl, i-propyl, butyl, i-butyl, t-butyl, etc.). 18 , C1-C 12 Alternatively, C1-C6 would also be acceptable.

[0044] "Allyl" is any monovalent organic radical derived from the removal of a hydrogen atom from an aromatic hydrocarbon. For example, a simple allyl group is the benzene-derived group, phenyl (chemical formula, C6H5).

[0045] An "acyl" is a group of atoms consisting of a carbonyl group bonded to an R group. An acyl group is a functional group with the chemical formula RCO-, where R is an alkyl group bonded to a carbon atom by a single bond. Typically, acyl groups are attached to larger molecules, where the carbon and oxygen atoms are linked by a double bond. Although acyl groups are hardly discussed in organic chemistry, they can originate from inorganic compounds, such as phosphonic acids and sulfonic acids. Esters, ketones, aldehydes, and amides all contain acyl groups. Specific examples include acetyl chloride (CH3COCl) and benzoyl chloride (C6H5COCl).

[0046] The present invention will be better understood from the following description of embodiments of the invention, provided for illustrative purposes only and with reference to the accompanying drawings. [Brief explanation of the drawing]

[0047] [Figure 1] Figure 1 shows the absence of aggrecan (ACAN) staining (PNN marker) in PNNs and in the extracellular matrix, particularly in the anterior horn, observed after treatment with PNNi according to the present invention (AB); (CD) shows that PNNi treatment surrounded parvalbumin (PV)-positive neurons and induced a decrease in hyaluronic acid-binding protein (HABP) in the extracellular matrix. [Figure 2] Figure 2 shows the Basso, Beattie, and Bresnahan (BBB) ​​hindlimb gait movement open field test apparatus (Basso et al., 1995). (A) A flat open field apparatus with a diameter of approximately 1 m. (B) Rats are placed in the open field apparatus for 4 minutes each week to evaluate their hindlimb gait movement ability. [Figure 3]Figure 3 shows the apparatus for the mechanosensory evaluation; von Frey assay. Animals were placed in a complete base assembly for plantar stimulation (A) with a wire mesh bottom (B) and acclimatized for approximately 20 minutes. Logarithmically thickened Von Frey hair (C) was pressed through the wire mesh bottom and then pressed perpendicularly against a flat surface of the left or right hind limb, and the 50% avoidance threshold was determined using the Dixon up-down method. [Figure 4] Figure 4 shows the timeline of a specific example of the research presented in Figures 2-5. [Figure 5] Figure 5 shows the contusion force analysis (A) in rats. All rats in all experimental groups received similar contusion strengths: (B) Basso, Beattie, and Bresnahan (BBB) ​​scores were obtained. The results showed better functional recovery in PNNi-treated rats (daily treatment) after moderate T9 contusion injury. Rats treated with excipients reached a score of ~10 5 weeks after injury, while the 4-MU-treated group reached a score of ~15; (C) Von Frey hair test results. Rats from different treatment groups showed no difference in tactile sensitivity using the Von Frey hair test. n=11 in all groups. [Figure 6] Figure 6 shows the calculation of the daily dosage of PNNi according to the present invention. [Figure 7] Figure 7 shows perineuronal networks (PNNs). (A) Schematic diagram of PNNs (green) on the surface of a neuron, (B) Synaptic vesicles (red) appear to be clustered within the pores of the PNNs (green) (de Winter et al., 2016; Vo et al., 2013). [Figure 8]Figure 8 shows representative images of the presence of PNNs on alpha motor neurons in the spinal cord (Galtrey et al., 2008; Irvine and Kwok, 2018). Aggrecan (ACAN)-positive PNNs surround most alpha motor neurons (Mns) (NeuN), and ChAT co-localization indicates Mns. (A) Ratio of Mns in the abdominal motor pool surrounded by NeuN, ACAN-positive PNNs, and their co-localization (ACAN+ / NeuN+). Confocal images in the spinal cord showing ACAN-positive PNNs (B), surrounding NeuN-positive (C), and ChAT-positive Mns (D), respectively. Error bars ± SD; n=3. Statistics: One-way ANOVA; significance level: *p<0.05. Scale bar, 100 μm. [Figure 9-1] Figure 9-1 (AI) shows an analysis of the efficacy of PNNi in vitro (AI) for removing PNNs. Untreated PNN+HEK cells showed clear signaling of WFA-positive PNNs (B), while 2-day PNNi treatment (0.5 mM or 1.0 mM) of PNN+HEK cells removed 86.4 ± 4.47% of WFA-positive staining (F3.66 = 73.60, p < 0.0001 compared to all treatment time points vs. untreated). Staining intensity partially recovered to 43.6 ± 14.6% of baseline lectin binding within 3 days post-treatment (p < 0.0001 compared to post-treatment at 3 and 5 days vs. during treatment, Figure CI). [Figure 9-2] Figure 9-2 (JO) shows that both methods of PNNi were sufficient to reduce WFA-positive binding throughout the CNS compared to untreated animals, based on histology from animals that completed the treatment period after 10 days (JO). [Figure 9-3] Figure 9-3(PS) shows that quantification in the spinal cord's dorsal horn indicated that 10 days of oral PNNi administration induced partial removal of WFA-positive areas to 71.0 ± 7.20% of baseline ECM levels (t(3)=5.15, p=0.0142; Figure 9P). Short-term PNNi was sufficient to induce sensory changes, but not sufficient to induce motor function in untreated rats (QS). [Figure 10-1]Figure 10-1(A) shows that the perineuronal net inhibitor (PNNi) induces reconstruction of the sensorimotor map (M1) in intact rats. Intracortical microstimulation (ICMS) was used to map HL and FL cortical motor representations to examine the functional tissue of M1 in intact / Siamese animals after long-term treatment with the promising plasticity enhancer, PNNi. ICMS was performed in (A) 11-week-old midthoracic laminectomy (Siamese) or age-controlled intact rats in a right hemisphere craniotomy, with a stereotactic fixation of 5 mm axially to the above section (B). [Figure 10-2] Figure 10-2(BD) shows a representative heatmap per group, where individual ICMS maps were combined to show the proportion of animals [in which hindlimb (HL;BD) movement could be induced] for each stereotactic fixation coordinate. Baseline HL cortical maps were created for Lister-food rats (B; dotted line in BD) to compare functional plasticity between the sham surgery group (C) and the long-term PNNi administration group (D). [Figure 10-3] In Figure 10-3(EH), measurements for HL analysis (FG) are shown in E). H) The mean area (mm²) of HL expression was reduced with PNNi treatment. PNNi treatment reduced the proportion of intact HL centers that induced HL (F), but did not reduce the proportion of total induced HL areas per group among intact HL centers (G). [Figure 10-4] Figure 10-4(IJ) demonstrates that double electric field potential stimulation for both short-term stimulation intervals (20-40 ms) and long-term stimulation intervals (150-250 ms) does not substitute for the short-term or long-term double stimulation ratio observed between groups (IJ). [Figure 10-5] Figure 10-5(KM) shows a representative heatmap per group, where individual ICMS maps were combined to show the proportion of animals [in which forelimb (FL;KM) movement could be induced] for each stereotactic fixation coordinate. Baseline FL cortical maps were created for Lister-food rats (enclosed by the dotted line in K;KM) to compare functional plasticity between the sham surgery group (L) and the long-term PNNi administration group (M). [Figure 10-6]In Figure 10-6(NS), measurements of FL analysis (PS) are shown in O). After PNNi treatment, FL movement was induced in areas not associated with FL or HL movement (see the right of the baseline HL map; enclosed by the white dotted line KM). The total surface area inducing FL did not change significantly (N), while PNNi treatment of Siamese animals reduced the area in which FL was induced within intact FL centers (P), but did not produce a corresponding increase in FL movement induced within intact HL areas (R). The ratio of total FL within intact FL centers (Q) or intact HL centers (S) did not change with Siamese surgery or PNNi treatment. For all ICMS groups, there were intact n=4, Siamese n=4, and Siamese / PNNi n=5. For the double stimulation group, there were intact n=32, Siamese n=15, and Siamese / PNNi n=19. Statistics, one-way ANOVA; significance levels: *p<0.05, **p<0.01, ***p<0.001. [Figure 11-1] Figure 11-1(A) shows the forelimb (FL) shift into the hindlimb (HL) area of ​​the sensorimotor cortex (M1) after the combination of perineuronal net inhibitor (PNNi) and spinal cord injury (SCI). PNNi and / or injury independently enhance cortical plasticity in spinal cord injury rats. Intracortical microstimulation (ICMS) was performed 11 weeks after injury in the right hemisphere craniotomy, in a stereotactic fixed coordinate 5 mm axially to the previous item (B). [Figure 11-2] Figure 11-2(BE) shows representative heatmaps for each group, illustrating the ratio of animals to each stereotactic fixation coordinate that can induce HL (BC) or FL (DE) movement. Lister-food rat baseline HL and FL cortical maps (outlined in BE) were compared between the mid-thoracic SCI (B,D) group and / or the long-term PNNi administration (C,E) group to evaluate structural plasticity. HL movement could not be induced after SCI (BC). [Figure 11-3]Figure 11-3(FI) shows a plot of mean electric field potential amplitude vs. stimulation intensity for each group (G). Synaptic responses were induced in cortical layers II / III by electrical stimulation via bipolar electrodes placed in cortical layers V / IV. Recordings were performed on cortical slices in the axial direction, with the following coordinates: ML: 2-3 mm and AP: 1.40-1.8 mm. The combination of injury and PNNi treatment enhanced synaptic transmission (G). Double electric field potential stimulation for both short (20-40 ms) and long (150-250 ms) intervals did not substituted for the prolonged double stimulation ratio (PPR) observed between groups (I). However, a lower short-term PPR (p=0.052;H) was observed with injury alone. Statistical analysis was performed using one-way ANOVA; for each group, sham n=4, excipient n=3, and PNNi n=3. G: Data are shown as mean ± SEM, and n represents cortical slices. Statistics: Two-way repeated measures analysis of variance (ANOVA). H-I: Statistics, one-way ANOVA for each group. [Figure 11-4] In Figure 11-4 (JN), measurements for FL analysis (KN) are shown in J. After PNNi and / or SCI, FL motion was induced in the area of ​​previously induced HL motion (see the right of DE enclosed by the white dotted line). Compared to the sham control (p=0.197, M; p<0.05, N), the total surface area inducing FL did not change significantly (F), and the ratio of FL in intact FL centers also did not change (KL), but it increased in the FL area associated with intact HL centers after post-injury PNNi treatment. [Figure 12-1] Figure 12-1(AB) shows that limiting PNNi administration with sustained rehabilitation further enables hindlimb (HL) motor recovery. Further HL improvement was observed in animals that continued rehabilitation training when PNNi treatment was terminated 2-3 weeks before the end of an experiment (8 weeks of PNNi treatment) to induce PNN reshaping (A). The bar graph shows the percentage of animals that were able to achieve forelimb-hindlimb (FL-HL) coordination at the end of PNNi administration at 9 weeks post-injury (WPI), 10, and 12 WPI (B). [Figure 12-2]Figure 12-2(CE) shows the classification of (C) HL and (D) forelimb (FL) steps on horizontal stepping platforms 9 and 12WPI using a stacked bar graph [using the old ladder scoring system (Metz and Whishaw, 2009), with scores of [Hit: 3-6, Slip: 1-2, and Miss: 0]. E) The mean left-right (LR) 50% release threshold for the hind limb plantar, determined by the Von Frey assay performed on 9 and 12WPI, did not indicate hyperalgesia. Strengthening of PNNs after completion of PNNi treatment did not induce sensory changes. (For the groups, n=10 and 9 for 8 weeks PNNi and 8 weeks PNNi+T, respectively. Statistics, A,CE: Two-way mixed-factor ANOVA; significance levels: *p<0.05, **p<0.01, ***p<0.001, A,C,D: Error bars ±SEM) [Figure 13-1] Figure 13-1(AE) shows that 8 weeks of PNNi with sustained rehabilitation partially restores cortical reconstruction of the FL area to an intact configuration. Individual ICMS maps were combined to give representative heatmaps for each group showing the proportion of animals [capable of inducing forelimb (FL:DE) movement, but not hindlimb (HL:BC)] to each stereotactic fixation coordinate. The dotted box shows the intact baseline areas for HL(BC) and FL(DE), comparing functional plasticity between the 8-week PNNi administration group (B,D) and the 8-week PNNi administration group with sustained treadmill training (C,E). [Figure 13-2]Figure 13-2(FK) shows that F) the mean area (mm2) of FL expression decreased to normal / intact levels only with sustained training and limited PNNi treatment, and measurements for analysis (GH,JK) are shown in I). After spinal cord injury, FL movements were induced in the areas where HL movements were previously induced (baseline HL map shown in the white dotted box DE). G) FL movements could be induced almost entirely within the intact FL area with a slight decreasing trend with 8 weeks of PNNi+T (p=0.243). H) The proportion of total FL area induced within the intact FL area decreased with 8 weeks of PNNi treatment, showing a trend only with sustained training (p=0.112). J) Following injury, with 8 weeks of PNNi treatment, FL movements could be induced within the HL area. However, partial separation of FL from the HL area was observed only with sustained training (p=0.514). K) The proportion of induced FL areas within intact HL areas appears to be increased compared to undamaged controls (p=0.114 8-week PNNi and p=0.177 week PNNi+T). [Figure 14] Figure 14 shows that JD009 and JD013 attenuate PNN formation. The efficiency of PNNi, JD009, and JD013 in reducing PNN formation was analyzed using immunocytochemistry. Staining intensity was measured using N-acetylgalactosamine-conjugated lectin WFA against labeled PNNs. 1 mM and 2 mM PNNi were insufficient to produce substantial changes in PNN morphology and expression in cells compared to untreated cells. In contrast, both JD009 and JD013 treatments altered PNN expression in cells at concentrations of 0.5 mM and 1 mM. [Figure 15-1]Figure 15-1(AL) shows that long-term PNNi treatment selectively reduces the expression of perineuronal networks (PNNs) labeled with key PNN components in the anterior horn (VH). Rat spinal cord segments (T4-6) obtained 12 weeks after injury were stained, and their intensity was analyzed in VH. Confocal images show spherical ACAN (AF) and WFA (GL) expression in VH with injury (AC,GI). Siamese animals treated with PNNi show altered overall PNN expression (D,J). Injured animals treated with PNNi alone (E,K) or in combination with PNNi and training (F,L) showed reduced WFA expression in VH. Scale bar, 100 μm. Error bars ± SD for all graphs; n=3 for treatment groups. Statistics, one-way ANOVA; significance levels: *p<0.05, **p<0.01, ***p<0.001. [Figure 15-2] Figure 15-2(MN) shows that PNNi, as labeled with aggrecan (ACAN:M) and Noda Fuji agglutinin (WFA:N), partially reduces the number of PNNs, especially after damage. [Modes for carrying out the invention]

[0048] To our surprise, the inventors have found that 4-methylumbelliferone (hereinafter referred to as "the PNN inhibitor of the present invention (PNNi)") can be used to treat conditions of the nervous system.

[0049] 4-methylumbelliferone is a small molecule and has the following chemical structure.

[0050] [ka]

[0051] Derivatives of 4-methylumbelliferone can be used for the use and treatment methods of the present invention. Derivatives of 4-methylumbelliferone may also be modified forms of 4-methylumbelliferone.

[0052] In one embodiment, a derivative of 4-methylumbelliferone is a compound having the following structure.

[0053] [ka]

[0054] The numbers in red indicate the carbon positions. Here, substitutions with one or more alkyl, allyl, acyl, dimethylamino, hydroxyl, hydroxymethyl, methoxy, methyl, morpholino, and sulfonyl compounds may be added. These substitutions may occur at any position.

[0055] The substitution may be at position C4. The substitution may be with one or more alkyl, allyl, acyl, dimethylamino, hydroxyl, hydroxymethyl, methoxy, methyl, morpholino, and sulfonyl groups. In one or more embodiments, NR1R2 may be added to a methyl group (CH3) at C4, and R1 and / or R2 may each be independently H, alkyl, allyl, acyl, and sulfonyl groups. Preferably, R1 and R2 are alkyl groups. The alkyl group is C1-C 18 It may be, for example, C1-C6.

[0056] Such derivatives have the following structure:

[0057] [ka]

[0058] In one embodiment, OR1 may be C4 and may be added to a methyl group (CH3), and R1 may be alkyl, allyl, or acyl. Preferably, R1 is hydroxyethyl.

[0059] Such derivatives have the following structure:

[0060] [ka]

[0061] The substitution may be at position C3. The substitution may be with one or more alkyl, allyl, acyl, dimethylamino, hydroxyl, hydroxymethyl, methoxy, methyl, morpholino, and sulfonyl molecules.

[0062] The substitution may be at position C5. The substitution may be with one or more alkyl, allyl, acyl, dimethylamino, hydroxyl, hydroxymethyl, methoxy, methyl, morpholino, and sulfonyl molecules.

[0063] The substitution may be at position C6. The substitution may also be with one or more alkyl, allyl, acyl, dimethylamino, hydroxyl, hydroxymethyl, methoxy, methyl, morpholino, and sulfonyl compounds.

[0064] The substitution may be at position C8. The substitution may be with one or more alkyl, allyl, acyl, dimethylamino, hydroxyl, hydroxymethyl, methoxy, methyl, morpholino, and sulfonyl compounds.

[0065] In one embodiment, the substitution may be at position C1. The substitution may also be by one or more alkyl, allyl, acyl, dimethylamino, hydroxyl, hydroxymethyl, methoxy, methyl, morpholino, and sulfonyl compounds. Preferably, the derivative is a compound with the following chemical formula.

[0066] [ka]

[0067] Here, O is substituted with NR1 at position 1. R1 may be allyl or acyl. Preferably, R1 is alkyl.

[0068] In particular, the derivatives in the embodiments of the present invention are molecules or compounds having the following structure. JD01-009:

[0069] [ka]

[0070] IUPAC name: 4-[(dimethylamino)methyl-7-hydroxy-2H-1-benzopyran-2-one] JD01-013:

[0071] [ka]

[0072] IUPAC name: 7-hydroxy-4-[(morphin-4-yl)methyl]-2H-1-benzopyran-2-one

[0073] In one embodiment, PNNi is Himechromon (C 10 It is H8O3.

[0074] It will be understood that pharmaceutically acceptable salts, metabolites, or prodrugs of 4-methylumbelliferone, or derivatives thereof, may be used in the uses and treatment methods of the present invention. With respect to PNNi of the present invention, the features, uses and methods may also be applied to derivatives, salts, metabolites, and prodrugs of 4-methylumbelliferone, as disclosed herein.

[0075] Further embodiments of the present invention provide derivatives of 4-methylumbelliferone or salts thereof, and compositions comprising such derivatives. The derivatives are those disclosed herein.

[0076] Perineuronal networks (PNNs) are dense pericellular extracellular matrix structures found throughout the central nervous system. PNNs surround the surface of neurons. Populations of neurons surrounded by PNNs are particularly found in the spinal cord. In the spinal cord, PNNs surround most (~97%) alpha motor neurons (Mns). PNNs are aggrecan (ACAN) / CSPG positive. After spinal cord injury, for example, PNNs decrease at the lesion site and remain intact in areas distant from the injury. PNNs decrease at the lesion site, while inhibitory CSPG increases in the fragmented extracellular matrix.

[0077] The inventors have surprisingly found that 4-methylumbelliferone, i.e., PNNi of the present invention, reduces hyaluronic acid and CPSGs, and therefore removes PNNs in the central nervous system. Removal of PNNs opens up opportunities for plasticity in the subject and promotes remodeling.

[0078] As shown in the attached examples, PNNi of the present invention induces a reduction in hyaluronic acid, as indicated by the decrease in hyaluronic acid-binding protein (HABP) intensity surrounding parvabulbin (PV)-positive neurons.

[0079] The inventors have also found that the PNNi of the present invention functions to suppress CSPG synthesis in the central nervous system. Reducing CSPGs promotes plasticity.

[0080] In this regard, the PNNi of the present invention can be used to treat conditions of the nervous system by promoting plasticity and remodeling. This action promotes recovery.

[0081] The nervous system condition may be the central or peripheral nervous system condition of the subject. The nervous system condition may be any condition associated with the formation of at least one lesion. The lesion is due to CSPG. CSPG may be reduced compared to the subject without the condition. The lesion may be a glial scar. The lesion may be a plaque. The lesion may be in or near the spinal cord. The lesion may be in the brain.

[0082] The neurological conditions may be selected from a group including trauma, injury, infection, degeneration, structural defects, tumors, blood flow disruption, and autoimmune diseases. This group may include, but is not limited to, stroke, transient ischemic attack, myelopathy, hemorrhage, meningitis, encephalitis, Bell's palsy, brain or spinal cord tumors, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, and cerebral palsy.

[0083] The nervous system condition may be a spinal cord injury. The injury may be to any part of the spinal cord. The spinal cord injury may be of any type, and all of these should be understood to be included herein.

[0084] When used in the context of treating spinal cord injury, the PNNi of the present invention and its derivatives inhibit PNN formation and remove HA and CSPGs from PNNs and the formed glial scars. This opens up opportunities for plasticity and reconstruction after spinal cord injury and promotes recovery.

[0085] The PNNi of the present invention may be administered at any time following a spinal cord injury. It may be administered immediately after the injury, within one hour after the injury, within two to twelve hours after the injury, or at any time within the first seven days after the injury. When the spinal cord injury is chronic, administration may occur at any time after the injury. A subject may receive the first dose of PNNi of the present invention as described above, and thereafter may optionally experience longer-term administration. This may be a continuous daily treatment or a periodic treatment. Administration may last for any number of months or years, typically about one month to about 36 months or six months to 12 or 24 months. The pattern and duration of administration will depend on the severity of the injury.

[0086] The PNNi of the present invention may be administered to a subject in combination with rehabilitation. Rehabilitation may be performed before, during, or after administration of the PNNi of the present invention, i.e., simultaneously with or after administration. The PNNi of the present invention may be the composition of the present invention. Suitable rehabilitation methods are well known to those skilled in the art and are all assumed herein. Specific examples assumed for use in the present invention include those disclosed in Garcia-Alias ​​et al. and Wang et al. (Garcia-Alias ​​et al., 2009; Wang et al., 2011).

[0087] The PNNi of the present invention may be administered in combination with electrical stimulation. The electrical stimulation may be performed before the administration of the PNNi of the present invention, during administration, i.e., simultaneously, after administration, or a combination thereof. The PNNi of the present invention may be the composition of the present invention. Suitable methods of electrical stimulation are well known to those skilled in the art and are all assumed herein. Specific examples include those disclosed in US62 / 800,817 or US16 / 781,696.

[0088] The PNNi of the present invention may be administered to a subject in combination with other treatments to maximize functional recovery. These other treatments may be treatments of the spinal cord, such treatments are well known in the art. These treatments may be ISP peptides or denatured ISP peptides.

[0089] The PNNi of the present invention may also be a pharmaceutical composition. In this regard, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of 4-methylumbelliferone or a derivative thereof. The pharmaceutical composition is used for the treatment disclosed herein.

[0090] The present invention also provides PNNi for use in the treatment of lesions of the nervous system. The lesion may be any lesion or scar in the central or peripheral nervous system of the subject.

[0091] In one embodiment, the lesion is a glial scar formed after spinal cord injury. In this respect, the present invention will be understood to provide PNNi for use during treatment of spinal cord injury. Treatment may enhance recovery after spinal cord injury. The lesion may be a glial scar. The lesion may be a protein-accumulating plaque. Other specific examples include one or more amyloid lesions, tau accumulations, and Lewy bodies.

[0092] Treatment of the lesion may be complete or partial removal. Treatment may be resolution of the lesion. Treatment may be restoration of normal function of the affected part or parts.

[0093] The methods for introducing or administering the PNNi or compositions of the present invention are not limited, but include percutaneous, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intracerebral, transrectal, and oral routes. It may also be administered by sublingual infusion. The PNNi or compositions of the present invention may be administered by any convenient route, e.g., infusion or bolus infusion, absorption through the epithelium or the underside of the skin mucosa (e.g., oral mucosa, rectal and intestinal mucosa). Administration may be tissue or topical. Furthermore, it may be desirable to introduce the composition or composition of the present invention into the central nervous system by any suitable route, including intraventricular and intrathecal infusion. Intraventricular infusion may be facilitated, for example, by an intraventricular catheter attached to a container. The PNNi or compositions of the present invention may be formulated for slow or sustained release.

[0094] The PNNi or composition of the present invention may be composed according to conventional procedures. The PNNi of the present invention may be composed in a composition suitable for its administration. Typically, the PNNi or composition of the present invention may be composed for oral delivery. The PNNi or composition of the present invention may be composed for injection. In the context of spinal cord injury, the injection may be administered directly into the spinal cord. The injection may be administered directly into glial scar tissue.

[0095] The PNNi or composition of the present invention may be composed to be released from a medical device. The medical device may be an implantable device such as a patch or stent.

[0096] The PNNi of the present invention and compositions comprising the PNNi of the present invention may be prepared / composed and / or administered in a variety of suitable forms. Such forms include, but are not limited to, liquid solutions (e.g., injectable and injectable solutions), dispersions or suspensions, emulsions, microemulsions, tablets, pills, powders, liposomes, dendrimers and other nanoparticles, microparticles and liquid, semi-solid and solid administration forms such as suppositories. The form may be understood to depend on the intended mode of administration.

[0097] In the context of spinal cord injury, the PNNi or composition of the present invention may be administered as the first intravenous dose, depending on the subject's condition. Subsequent doses may be administered orally and / or intravenously. The method of administration depends on the patient's condition, the extent and / or location of the injury.

[0098] The administration of PNNi according to the present invention should be understood to depend, as well as on the subject's condition and the severity of the condition being treated. This will depend on a variety of factors, including the activity of the compound applied, the metabolic stability and duration of its activity, age, body weight, general health status, sex, diet, mode and timing of administration, excretion rate, drug combination, the severity of the particular condition, and the treatment the individual is receiving. Of course, high or low doses will naturally occur in individual cases, but such may also fall within the scope of the present invention. For example, the composition may be administered at doses of 5 to 60 mg / kg body weight / day, preferably 17 to 42 mg / kg body weight / day, such as 10 to 50 mg / kg body weight / day. In one embodiment, the subject is administered 1000 to 3000 mg / day, preferably 1000 to 2000 mg / day, and preferably 1200 to 1300 mg / day. The amount and frequency should be appropriate for the purpose. The frequency of application and administration can vary considerably depending on the needs of each subject, with recommendations for application or administration ranging from once a month to up to 10 times a day, preferably once a week to four times a day, more preferably three times a week to three times a day, and even more preferably once or twice a day. The duration of treatment can also vary considerably depending on the needs of the subject. In preferred embodiments, repeated use is provided.

[0099] The administration method may be one suitable for use in children.

[0100] In one embodiment, PNNi is formulated in tablet form for oral administration. The tablet may contain more than 400 mg, preferably 500 mg to 600 mg, of the PNNi of the present invention. Each tablet is a single dose, and the frequency of administration may be twice or three times per day.

[0101] In one embodiment, a subject may receive, for example, tablets two to three times a day for a period of several months, such as two to six months or three to four months, and optionally have a period of, for example, two to six months or three to four months without administration. This form may be repeated thereafter. This administration may be as described herein. [Examples]

[0102] (Example 1) Oral administration of PNNi enhanced functional recovery of spinal cord injury in an animal model of acute contusion spinal cord injury.

[0103] (methodology) Adult female Lester-fed rats (200-250g) were purchased from Charles River Laboratories (Canterbury, UK). The rats were reared in pairs at Central Biomedical Services (University of Leeds, UK) under temperature-controlled conditions (20±1℃) and a 12-hour light-dark cycle (lights on at 7:00). All procedures and experiments were conducted in compliance with the UK Animals (Scientific Procedures) Act 1986.

[0104] Figure 4 shows an overview of the research timeline.

[0105] (Laminectomy and contusion (Cx) injury) Isoflurane was used as an anesthetic (5% in O2 during induction and 1-2% in O2 during interoperatively), the animals were depilated, and disinfected. Vertebral segments T7-13 were exposed, and a laminectomy of the spine was performed at T8. Vertebral levels T7 and T8 were stabilized while a 200 kdyn Cx (moderate injury) was administered at the T9 level using an amorphous horizon impactor (Precision Systems and Instrumentation, LLC, Fairfax Station, VA). Muscles were sutured, and the skin was closed with autoclips. Analgesics (veterinary buprenorphine; 0.015 mg / kg; Henry Schein Animal Health, Dumfries, UK) and antibiotics (Varitril enrofloxine; 2.5 mg / kg; Henry Schein Animal Health, Dumfries, UK) were administered subcutaneously immediately after injury and for three days postoperatively.

[0106] (treatment) Following surgical Cx injury, animals were grouped according to a treatment paradigm, and the efficacy of small molecule PNN inhibitors (PNNi), namely 4-methylumbelliferone, in enhancing post-SCI recovery was tested.

[0107] Due to the importance of rehabilitation as a treatment for SCI, a combination group of PNNi and rehabilitation training was included to identify compatibility issues.

[0108] (Pharmacological administration) Pharmacological treatment was initiated on the day of injury (PNNi; 2 g / kg from a stock solution of 0.2 g / ml). This dosage was higher than the permitted dose of PNNi for the treatment of non-CNS-related diseases and had been established for preliminary use in in vitro experiments. Oral administration was performed by injection, completing two doses daily, as opposed to forced administration.

[0109] Since PNNi is an oral compound, the duration of administration is controllable. Firstly, the drug was administered for an extended period from the time of injury / surgery to the day of treatment.

[0110] (Rehabilitation) The training consisted of assigned exercise quadruped interval treadmill training, providing task-specific rehabilitation. The first session began 7 days after injury (DPI) and was followed by the gait motor behavior test described below. Daily training consisted of 10 minutes on the treadmill, followed by a 10-minute break before the final 10-minute session on the treadmill. Rats were trained five times a week on the treadmill at the maximum speed that allowed them to maintain continuous walking for each 10-minute session.

[0111] (Gait movement assessment) Behavioral and functional aspects of hind limb (HL) function were evaluated throughout the entire study.

[0112] (Basso, Beattie, and Bresnahan (BBB) ​​HL Walking Exercise Open Field Test) HL gait motor ability was assessed at various time points throughout the acute SCI paradigm using the BBB HL gait motor scale. The BBB test was performed using an open gait motor field (custom-made Perspex O-ring: 80 cm in diameter, 30 cm in height), where the animal was placed for 4 minutes (Figure 2). Each BBB test was assessed simultaneously by two individuals. The obtained scores were pooled and averaged for objectivity. The BBB test assesses HL motor function using a ranking scale of 0-21. Based on their BBB scores, animals were ranked into three broad categories: an early phase showing little or no limb movement (scores 0-7); an intermediate stage with the occurrence of awkward gait (scores 8-13); and a late stage showing coordination and stability of FL and HL (scores 14-21) (Basso et al., 1995). Following injury, BBBs were then performed at 1 DPI to confirm the injury, and then weekly from 7 DPI. If the animals also underwent rehabilitation training, BBBs were performed beforehand.

[0113] (Von Frey's rating) Changes in HL sensory function were assessed using the Von Frey methodology to look for hyperalgesia and neuropathic pain. Four animals were simultaneously acclimatized to Perspex cages with wire mesh bottoms (Figure 3) for approximately 15-20 minutes before testing, until general exercise and grooming were completed. Von Frey filaments (Touch Test® Sensory Evaluator Kit of 20; #39337500; Leica Biosystems, Milton Keynes, England, Figure 3C) were pressed down through the wire mesh bottom against the more sensitive plantar arch of the HL footpad, where limb withdrawal was counted as a positive result. Walking was an ambiguous response requiring retesting after a suitable delay, but recoiling was also seen as a positive response. The tests were performed sequentially on the left and right hind limbs of all animals, with sufficient intervals between stimuli. The sensory testing procedure and analysis were performed using the Dixon up-down method (Dixon, 1980) as described by Chaplan et al. (1994) to determine the 50% escape threshold for each HL.

[0114] (Histology) (tissue preparation) The animals were deeply anesthetized without cardiac arrest by an overdose of pentobarbital sodium (Pentoject; Henry Schein; 200 mg / kg; intraperitoneal injection). Subsequently, transcardiac perfusion (Gage et al., 2012) was performed using phosphate buffer (PB; 0.12 M monosodium phosphate; 0.1 M NaOH; pH 7.4), and then 4% paraformaldehyde (PFA; in PB; pH 7.4) for tissue fixation. The brain and spinal cord were severed and then fixed overnight in PFA (4%; 4°C), and then frozen in 30% sucrose solution (30% v / w sucrose in PB; 4°C) until the tissue was saturated. The left cerebral hemisphere and appropriate spinal cord segments were excised and stored in a suitable temperature-controlled medium (OCT; Leica FSC 22 Frozen Section Media; Leica Biosystem) before being stored at -80°C until dissection. Tissue dissection was performed using a cryostat (Leica CM1850; Leica Biosystems) to obtain 40 μm cross-sections for floating sections, and collected in 48-well plates containing physiological buffer (PBS; 0.13 M sodium chloride, 0.7 M disodium phosphate, 0.003 M monosodium phosphate; pH 7.4). The OCT was removed before transferring to a 30% sucrose solution for storage at 4°C.

[0115] (immunohistochemical technique) At room temperature (RT), the sections were washed three times for 5 minutes each with Tris-buffered saline (TBS; 0.1 M Tris-based, 0.15 M NaCl; pH 7.4) to remove sucrose residue. The tissue was then blocked for 2 hours in 0.3% TBST (1x TBS solution and 0.3% v / v Triton X-100) and 3% standard donkey serum (NDS; v / v). The sections were then transferred to a blocking buffer containing primary antibody (3% NDS in 0.3% TBST; pH 7.4) and co-cultured at 4°C.

[0116] Following primary antibody or lectin culture, the sections were washed three times using TBS (10 min; RT). To visualize each primary antibody stain, the tissues were then co-cultured in a darkroom with a fluorescently conjugated secondary antibody (1:500; 2 hours; RT) against the primary antibody species. Subsequently, the tissues were washed three times in TBS (10 min; RT) while protected from light. A final wash in Tris non-physiological saline (TNS; 0.5 M Tris, pH 7.6) was performed to reduce precipitation before air drying. The tissues were placed on Superfrost Plus slides, air dried, and then covers with a coverslip on the mounting medium FluorSave® Reagent (EMD Millipore).

[0117] The primary antibody is a CSPG component containing ACAN, BCAN, and NCAN. Lectins: Biotin-labeled Fuji agglutinin (Bio-WFA), biotin-labeled hyaluronic acid-binding protein (bHABP) The total number of neurons and the number of neurons surrounded by PNNs were quantified by two independent researchers without informing them of the study. The results were analyzed by one-way ANOVA for statistical significance.

[0118] (Determination of the mechanism and pharmacokinetics of PNNi) <Objective (1) Mechanism of how PNNi crosses the blood-brain barrier (BBB)> Background: Our preliminary results have clearly shown that oral administration of PNNi leads to a reduction in PNNs in the central nervous system (CNS). However, the mechanism by which PNNi induces such a lasting effect is unknown. Does PNNi cross the blood-brain barrier (BBB) ​​and reduce PNNi in situ? Or does PNNi systematically reduce the substrate pool, thereby allowing fewer substrates to cross the BBB for PNN synthesis? Methods: Adult rats were orally administered PNNi for 10 consecutive days. Blood, urine, and cerebrospinal fluid were collected from the rats on days 0, 5, and 10 to analyze the concentration of PNNi in the samples. The presence of PNNi was measured using a fluorescence spectrometer. Number of animals: 8 rats

[0119] <Objective (2) Re-establishment of PNNi pharmacokinetics> Background: Previously published pharmacokinetic data indicate that the oral lethal doses in mice and rats are 7.5 g / kg and 6.2 g / kg of animal body weight, respectively. However, in our pilot experiments in mice, we administered 6.8–12.8 g / kg to mice continuously for 6 months, and the mice showed no deaths or adverse signs. This raises questions about whether our previous pharmacokinetics were accurately established. Therefore, we aim to re-establish the pharmacokinetics of PNNi in living rats and mice. Methods: A comprehensive pharmacokinetic study will inevitably cover the doses that lead to animal death. Therefore, this cannot be done under any individual project license in the UK. We will use the services of the company Charlies River (a legal animal provider) to conduct these experiments. Related information can be found on this website. https: / / www.criver.com / products-services / safety-assessment / dmpk / pharmacokinetics-toxicokinetics?region=3696

[0120] (Results and conclusions) Walking motor function was assessed weekly using Basso, Beattie, and Bresnahan (BBB). Subsequently, neural pathways were examined in both retrograde and antegrade directions using cholera poisoning B and biotin-labeled dextran.

[0121] (Results from BBB and Von Frey tests) As shown in Figure 5(A), all rats received similar bruising intensity in all experimental groups. (Center) The Basso, Beattie, and Bresnahan (BBB) ​​score showed good functional recovery in PNNi-treated rats (daily treatment) after moderate T9 bruising injury. Excipient-treated rats scored ~10 five weeks after injury, while the 4-MU-treated group reached ~15. (Right) Rats from different treatment groups showed no differences in tactile sensitivity using the Von Frey hair test. All groups n=11. These results indicate that oral administration using PNNi enhances functional recovery.

[0122] (Immunochemistry results) As shown in Figure 15, long-term PNNi treatment reduces the expression of PNNs labeled with key PNN components in the anterior horn (VH). Rat spinal cord sections (T4-6) obtained 12 weeks after injury were stained and their intensity in VH was analyzed. PNNi partially reduces the number of PNNs, particularly after injury, as they are labeled with aggrecan (ACAN) and Noda Fuji agglutinin (WFA).

[0123] (Example 2) Removal of PNNs by PNNi in rats (Results and conclusions) As shown in Figure 1, PNNs were reduced in intact rats 10 days after oral administration of PNNi. Figures 1A and 1B show the absence of ACAN staining (CSPG; PNN marker), which was observed particularly in the anterior horn after PNNi treatment. Figures C and D show that PNNi treatment induces a reduction in hyaluronic acid-binding protein (HABP) surrounding parvalbumin (PV)-positive neurons. This also suggests that PNNi is effective in removing CSPGs in the spinal cord.

[0124] (Example 3) In this embodiment, the present inventors introduce a non-invasive compound, a PNN inhibitor (PNNi), to reversibly remove PNNs and enhance plasticity, thereby removing PNNs through the breakdown of PNN formation and enhancing recovery after acute spinal cord injury.

[0125] (PNNs surround the majority (~97%) of alpha motor neurons (Mns) in the spinal cord.) Most PNNs research has been performed in brain samples, and the inventors are attempting to identify populations of neurons wrapped in PNNs in the spinal cord. Figure 8 is a representative image of the presence of PNNs on alpha motor neurons in the spinal cord (Galtrey et al., 2008; Irvine and Kwok, 2018).

[0126] (PNNi dramatically eliminates PNNs in vitro and in vivo.) PNN-like structures around cells (PNN) + The efficiency of PNNi in removing PNNs in vitro (Figure 9A-I) was investigated using a previously developed human embryonic kidney 293T (HEK) cell model modified to express essential ECM components (hyaluronic acid synthase 3 (HAS-3) and HAPLN-1) required to induce HEK cell formation (Kwok et al., 2010). Untreated PNNs + HEK cells show clear signaling of WFA-positive PNNs (Figure 9B), PNNs + Two-day PNN treatment (0.5 mM or 1.0 mM) administered to HEK cells resulted in 86.4 ± 4.47% of WFA-positive staining (F for all treatment time points vs. untreated cells). 3.66 Staining intensity was partially restored to 43.6 ± 14.6% of baseline lectin binding within 3 days post-treatment (3 and 5 days post-treatment vs. in between, p < 0.0001, Figure 9C-I). These results indicate that PNNi-mediated removal of PNNs is functional and reversible.

[0127] (Oral PNNi administration removes PNNs from the spinal cord.) PNNi treatment was investigated in vivo for a short period (10 days) via either oral administration twice daily or intraperitoneal (ip) infusion. Histology from animals after 10 days of treatment revealed that both methods of PNNi administration were sufficient to reduce WFA-positive binding throughout the CNS compared to untreated animals (Figure 1M-O). Interestingly, PNNi appeared to reduce lectin binding more efficiently in the spinal cord compared to the cortex (Figure 9J-O). Importantly, this indicates that PNNi, or its metabolites, can cross the blood-brain barrier and affect the ECM in the CNS. Quantification in the spinal cord's dorsal horn showed that 10 days of oral PNNi administration induced partial removal of WFA-positive portions to 71.0±7.20% of baseline ECM levels (t(3)=5.15, p=0.0142; Figure 9P). Short-term oral PNNi treatment reduced staining with HA-binding protein to 64.0±5.84 (HABP; t(3)=7.69, p=0.00456), and reduced HAPLN-1 to 68.3±8.09% of baseline levels in the posterior horn (t(3)=5.01, p=0.0153). Oral PNNi treatment is non-invasive and produces sufficient efficacy on the neuronal ECM in vivo; therefore, the following PNNi administration experiments using Lester-fed rats were used as the administration method.

[0128] (Partial removal of PNNs by acute short-term treatment with PNNi showed no side effects.) First, the inventors questioned whether the removal of PNN throughout the CNS would affect normal sensory and motor functions. To investigate this, adult female Lester-fed rats (n=11) were orally treated with PNNi for 10 days and then subjected to normal behavioral tests. These results showed that short-term PNNi was sufficient to induce sensory alterations in the treated, unharmed rats, but not alter motor functions (Figure 9Q-S). Compared to a pre-treatment baseline (7.9±2.73g) in similar rats, short-term PNNi administration reduced the escape threshold to 5.5±2.03, showing an approximately 30% increase in sensitivity (t(10)=2.76, p=0.02, Figure 9Q). In the open-field walking exercise test, all animals achieved a top score of 21 on the Basso, Beattie, and Bresnahan hindlimb (HL) scale (Basso et al., 1995) (ns:p=1; data not shown) after short-term PNNi treatment, with no significant differences observed. When evaluating gait activity using a more skill-intensive walking task, HL gait activity on a horizontal step showed that accurate walking (green) accounted for approximately 92.7±0.82% (ns, t(16)=0.259, p=0.799) of the time for all steps, while slips (yellow) and mistakes (red) increased to 5.43±0.70% (ns, t(16)=-0.978, p=0.343) and 1.92±0.41% (ns, t(16)=1.15, p=0.268), respectively, and these results appeared to be consistent between PNNi-treated and untreated animals (Figure 9R). Similarly, the performance of the forelimb (FL) was also unaffected by short-term PNNi treatment, with errors of 1.13±0.24% (ns,t(16)=-1.64,p=0.121), slips of 4.35±0.54% (ns,t(16)=1.97,p=0.0667), and accurate walking of 94.4±0.56% (ns,t(16)=-1.34,p=0.197).

[0129] Regardless of treatment, the HL and FL functions were similarly executed with a low rate of walking errors. Overall, acute removal of PNNs in normal adult rats, with the same treatment paradigm, decreased PNN components in the dorsal horn while slightly increasing the sensation of the tested limb, but did not affect walking motor function. No other behavioral differences were observed.

[0130] The sensorimotor cortex (M1) contains a highly organized local representation of movement that is affected by sensorimotor learning and responds to nerve injury with structural and functional plasticity.

[0131] Using intracortical microstimulation (ICMS), HL and FL cortical motor mapping was used to examine the functional organization of M1 in naïve / sham animals after long-term treatment with the promising plasticity enhancer, PNNi, by comparing it to the intact baseline HL and FL representations or “centers” (see dotted lines; Figure 10). PNNi treatment reduced the total area capable of eliciting HL movement (F 2、12 = 10.7, p = 0.00764 for sham / PNNi vs naïve, p = 0.00914 for sham / PNNi vs sham), which was due to some of the intact HL centers no longer being able to elicit HL movement (F 2、12 = 18.6, p = 0.00411 for sham vs sham / PNNi, p = 0.001 for naïve vs sham / PNNi; Figure 10A-H). A paired-pulse stimulation protocol (Gigout et al., 2013; Luhmann et al., 1995) was used as an index to study possible modifications of extracortical GABAergic inhibition simultaneously using in vitro sensorimotor cortex slices, where we were able to examine GABA A and, GABA B receptor-mediated inhibition, respectively. Healthy control slices showed significant paired-pulse inhibition (PRP: ~0.5), however, this was not modified by either sham surgery or PNNi treatment (n.s., F 2、65=1.52, p=0.227, Figure 10I;F 2、65 =2.35, p=0.0960, Figure 10J). This suggests that the PNNi-induced decrease observed in the HL area was due to structural reorganization and not to alteration of cortical inhibition (evidence of neural plasticity).

[0132] As observed in HL representation, PNNi induces a functional reconstruction of FL representation in Siamese animals (Figure 10K-S), even though the total surface area causing FL motion does not change (nsF). 2、12 =0.249, p=0.784, Figure 2N), but these events decreased in intact FL centers (F 2、12 =5.61, p=0.0226, Figure 2P), however, it did not appear to intrude into the HL area (nsF 2、12 =0.900, p=0.437, Figure 2R;F 2、12 (=2.38, p=0.142, Figure 10S). PNNi treatment did not produce significant changes in the cortical stimulation thresholds required to elicit HL and FL movements. Since short-term PNNi treatment enhanced tactile sensation, and long-term PNNi treatment induced structural reorganization, we can conclude that partial removal of PNNs with PNNi is sufficient to create a favorable environment for improving functional plasticity at multiple levels of the CNS, as shown above. These plasticity changes have been observed in treated, intact animals, so this may be a result of improper connections.

[0133] (PNNi and / or injury independently enhance cortical plasticity in spinal cord injury rats.) Following mid-thoracic SCI, FL movement could be induced in both intact FL and HL areas (Figure 11D-E), but HL movement could not be induced by ICMS despite evidence of open-field HL gait movement (Figure 11B-C). Injury and / or treatment did not alter the minimum or mean cortical stimulation threshold required to induce FL movement (Supplemental Table 1). Injury and / or long-term PNNi treatment did not result in overall changes in the surface area inducing FL movement (nsF 2、9 =0.403, p=0.683, Figure 5F), and there was no change in the area associated with the intact FL center (nsF 2、9 =0.891, p=0.452, Figure 5K;F 2、9 =0.0685, p=0.934, Figure 5L). However, the combination of injury and prolonged PNNi treatment resulted in a larger proportion of FL movement (4.27±1.56%~23.9±8.01%), which was associated with areas that had previously induced HL movement, suggesting that, compared to the sham control, it shifted the representative FL area of ​​the group, rather than dilating, towards the intact HL center as a whole (sham vs SCI / PNNi, F 2、9 =0.258, p=0.197, Figure 5M; for Sham vs SCI / PNNi, F 2、9 =5.23, p=0.0432, Figure 11N).

[0134] Using the Boltzmann equation, parallel experiments demonstrating local excitation of in vitro cortical slices with individual input-output curves of the fitted field potential magnitude (Figure 11G) revealed PSP max , I 50 Furthermore, a gradient factor was obtained. Importantly, this revealed that the combination of SCI and PNNi treatment yielded significantly different results from all other experimental groups (Greenhouse-Geisser, all groups vs SCI / PNNi, F 1.49、13.4(=1.61, p<0.001, Figure 11G), suggesting that the combination of PNNi and injury may contribute to the M1 reconstruction observed in this group, particularly at higher stimulation intensities, by creating an enhanced intracortical neurotransmission environment. The double-stimulation protocol, rather than the combination with SCI / PNNi, used injury alone, with short stimulation intervals rather than long ones, and PPR of 0.42±0.26%~0.63±0.24% (sham vs. SCI / excipients, F 2、57 =3.09, p=0.052, Figure 5H-I) Increased, affecting the double-stimulation protocol, SCI is GABA A The study also revealed that it suggests the induction of a reduction in receptor-mediated signaling. In summary, in M1, despite the ability of both damage and PNNi to independently induce a reduction in CSPG content, only long-term PNNi administration was able to sufficiently enhance plasticity to facilitate the reconstruction of the cortical motor map, suggesting that additional mechanisms, such as the observed increase in focal intracortical excitation, may underlie these functional changes.

[0135] (By limiting PNNi treatment in conjunction with sustained rehabilitation, motor recovery becomes possible.) To enhance plasticity for effective functional recovery, we administered PNNi for 8 weeks in combination with 11 weeks of rehabilitation. Continuing rehabilitation while limiting PNNi administration enabled further hindlimb (HL) movement, but did not result in sensory recovery (Figure 12). PNNi treatment was terminated 2-3 weeks before the end of the experiment (8 weeks of PNNi treatment) to enable PNN reconstruction, and further HL improvement was observed in animals that continued rehabilitation (A). The bar graph shows the percentage of animals that achieved forelimb-hindlimb (FL-HL) coordination at 9 weeks post-injury (WPI), at the end of PNNi administration, and at 12 WPI (B). Furthermore, animals that continued rehabilitation training (8 weeks of PNNi + T) showed an increased percentage of FL-HL coordination between 9 WPI and 12 WPI (A and B). The stacked bar graphs show the classification of (C) HL and (D) forelimb (FL) steps on horizontal step platforms 9 and 12 WPI, where scores of 3-6 for hits, 1-2 for slips, and 0 for misses (Metz and Whishaw, 2009) are shown on the classical step scoring system. HL performance on the step platform improved only with limited and sustained PNNi treatment and rehabilitation. Overall error decreased (black), but limited PNNi treatment and rehabilitation training particularly reduced the number of HL step misses (red). FL performance also improved with limited PNNi treatment and sustained training. The E) left-right (LR) mean 50% avoidance threshold for hindlimb soles, determined from the Von Frey assay performed on 9 and 12 WPI, did not indicate hyperalgesia. After the completion of PNNi treatment, strengthening of PNNs did not induce sensory changes (for the 8-week PNNi and PNNi+T groups, n=10 and n=9, respectively. Statistical analysis, A,CE: two-way mixed factorial ANOVA; significance level: * p<0.05, ** p<0.01, and, *** p<0.001. A, C, D: Error basis is ±SEM.

[0136] (8 weeks of PNNi with sustained rehabilitation partially restores cortical reconstruction in the FL area to intact tissue.) Intracortical microstimulation (ICMS) was performed in stereotactic fixed coordinates within a 5 mm craniotomy above and below Bregma in the right hemisphere (A) approximately 15 weeks after central thoracic contusion injury (Figure 13). Individual ICMS maps, combined, provide representative heatmaps for each group showing the ratio of animals to each stereotactic fixed coordinate where forelimb (FL;DE) movement could be induced, but not hindlimb (HL;BC). The dotted box shows the intact baseline area for HL (BC) and FL (DE), comparing 8 weeks of PNNi administration (B,D) with 8 weeks of PNNi administration with sustained treadmill training (C,E). F) Mean area of ​​FL display (mm 2 ) decreased to normal / intact levels with limited PNNi treatment accompanied by sustained training only. Measurements for analysis (GH, JK) are shown in I). After spinal cord injury, FL movements were induced in the areas where HL movements (baseline HL map shown in white dotted box DE) had previously been induced. G) FL movements could mostly be induced within intact FL areas with a slight decreasing trend with 8 weeks of PNNi+T (p=0.243). H) The proportion of total FL areas induced within intact FL areas decreased with 8 weeks of PNNi treatment, with a trend only when accompanied by sustained training (p=0.112). J) After injury, FL movements could be induced within HL areas with 8 weeks of PNNi treatment. However, partial FL separation from HL areas was observed only with sustained training (p=0.514). K) The proportion of induced FL areas within intact HL areas appeared to be increased compared to undamaged controls (p=0.114, 8-week PNNi and p=0.177, 7-week PNNi+T).

[0137] This suggests that limited PNNi, combined with sustained rehabilitation, limits maladaptive plasticity and strengthens newly established bonds from rehabilitation for functional restoration.

[0138] (Preliminary results for PNN analog) JD009 and JD013 may reduce PNN formation at lower concentrations than PNNi. Since PNNi is almost insoluble in aqueous solution (Nagy et al., 2015), derivatives of PNNi with improved solubility (JD009 and JD013) have been developed. The inventors hypothesize that, when designing the PNN analogs, the hydroxyl functional groups necessary for activity have not been altered, and therefore they should retain their ability to potently reduce PNNs.

[0139] Immunocytochemistry was performed using PNN-labeled N-acetylgalactosamine-conjugated lectin WFA to compare the efficiency of PNNi, JD009, and JD013 in reducing PNN formation. PNN-HEK293 cells were exposed in vitro to 0.5 mM and 1 mM compounds for 3 days. A DMSO excipient control was also included, and the amount used did not exceed 0.1% of the total volume. Cell-specific WFA fluorescence intensity is attributable to the matched region of interest (ROI), normalizing the overall WFA fluorescence intensity relative to the cell number. The following figures show that 0.5 and 1 mM administration of PNNi was insufficient to produce substantial changes in PNN morphology and expression in cells compared to untreated cells. In contrast, JD009 and JD013 treatment altered PNN expression in cells at concentrations of 0.5 mM and 1 mM.

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Claims

1. A pharmaceutical composition containing a therapeutically effective amount of 4-methylumbelliferone or its derivatives or salts for use in treating spinal cord injury in a subject, A pharmaceutical composition in which a derivative of 4-methylumbelliferone is a compound of the following formula (II). 【Chemistry 1】 (Here, R 2 , R 4 , R 5 And, R 7 is H or hydroxyl, and R 8 is N(CH 3 ) 2 (and selected from morpholino)

2. A pharmaceutical composition containing a therapeutically effective amount of 4-methylumbelliferone or its derivatives or salts for use in treating spinal cord injury in a subject, A pharmaceutical composition in which a derivative of 4-methylumbelliferone is a compound of the following formula (II). 【Chemistry 2】 (Here, R 2 , R 4 , R 5 and R 7 is H or hydroxyl, and R 8 is NR 9 R 10 where R9 and R10 are each independently selected from the group consisting of ethyl, propyl, i-propyl, butyl, i-butyl, and t-butyl).

3. The pharmaceutical composition for use according to claim 1 or 2, wherein the treatment includes treatment of glial scars.

4. A pharmaceutical composition for use according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered at any time after injury.

5. A pharmaceutical composition for use according to any one of claims 1 to 3, wherein the pharmaceutical composition is administered immediately after injury and / or within the first seven days of injury.

6. The pharmaceutical composition for use according to claim 4 or 5, administered in a continuous or stepwise manner.

7. A pharmaceutical composition for use according to any one of claims 1 to 6, wherein the pharmaceutical composition is provided in combination with rehabilitation or electrical stimulation or a combination thereof.

8. A pharmaceutical composition for use according to any one of claims 1 to 7, wherein the pharmaceutical composition is composed for oral delivery.

9. A pharmaceutical composition for use according to any one of claims 1 to 7, wherein the pharmaceutical composition is formulated for injection.

10. A pharmaceutical composition for use according to claim 9, which is formulated to be injected directly into the spinal cord or scar tissue of a subject.

11. The pharmaceutical composition is administered at a dose of 5 to 60 mg / kg body weight / day, and is a pharmaceutical composition for use according to any one of claims 1 to 10.

12. The pharmaceutical composition for use according to claim 11, wherein the dosage is approximately 500 to 2000 mg / kg at least twice a day.

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