Spinal cord regeneration therapy

Abstract

The present invention relates to spinal cord regeneration therapy. In one aspect, the present disclosure provides administration of an effective amount of at least four herb components, Polygalae (Polygala tenuifolia Willd.), Astragali (Astragalus membranaceus (Fisch.) Bunge), Ligusticum Chuanxiong, and Angelica sinensis (Oliv.), their roots or rhizomes, or extracts thereof, to a subject having a spinal cord injury. In other aspects, the present disclosure provides in vitro and in vivo methods for determining the effectiveness of drugs and drug combinations, and the effective amount thereof necessary for treating spinal cord injury.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 355,376, filed on June 24, 2022, the entire disclosure of which is incorporated herein by reference for all purposes.

[0002] The present invention relates to a method for promoting spinal cord regeneration, and more specifically, to inducing and / or accelerating recovery from spinal cord injury by administering to a subject a herbal composition comprising a combination of traditional Chinese medicines or extracts thereof.

Background Art

[0003] Spinal cord injury (SCI) is a serious medical problem. In severe cases, damage to the spinal cord disrupts the connection between the brain and the spinal cord, leading to paralysis of the lower limbs, quadriplegia, and even death. SCI not only impairs motor, sensory, and autonomic functions, but often causes devastating neurological disorders, such as loss of bladder and bowel control, respiratory problems, chronic pain, and an increased susceptibility to infections. As a result, the quality of life of SCI patients deteriorates, and the economic burden increases.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Despite SCI being a major cause of death and physical disability, there is a lack of effective and safe therapeutic agents for treating patients after SCI. Therefore, there is an urgent need for new treatment methods for treating SCI and its associated neurological disorders. More specifically, there is a need for new treatment methods that promote spinal cord regeneration and enable damaged or diseased nerves to function again.

Means for Solving the Problems

[0005] The following summary is provided to facilitate understanding of some of the innovative features specific to the present disclosure. A thorough understanding of the various aspects of the present disclosure can be obtained by considering the specification, claims, drawings, and summary as a whole.

[0006] According to a first aspect, there is provided a method of treating a subject to induce and / or accelerate recovery from spinal cord injury, comprising administering to the subject an effective amount of a herbal composition comprising at least four herbal components, Polygalae (Polygala tenuifolia Willd.), Astragali (Astragalus membranaceus (Fisch.) Bunge), Ligusticum Chuanxiong, and Angelica sinensis (Oliv.), their roots or rhizomes, or extracts thereof.

[0007] In one embodiment of the first aspect, the herbal composition of the method further comprises at least one additional herbal component selected from the group consisting of Salviae Miltiorrhizae (Salvia miltiorrhiza Bunge), Paeoniae Rubra (Paeonia veitchii Lynch), Carthamus Tinctorius (Carthamus tinctorius L.), Persicae seeds (Prunus persica (L.) Batsch seeds), and Acori Tatarinowii (Acorus tatarinowii Schott), their roots or rhizomes, or extracts thereof.

[0008] In one embodiment, the herbal composition of the method consists essentially of at least four herbal components, Polygalae (Polygala tenuifolia Willd.), Astragali (Astragalus membranaceus (Fisch.) Bunge), Ligusticum Chuanxiong, Angelica sinensis (Oliv.), their roots or rhizomes, or extracts thereof.

[0009] In one embodiment, the herbal composition of the method consists essentially of four herbal ingredients Polygalae (Polygalae), Astragali (Astragalus membranaceus), Ligusticum Chuanxiong, and Angelica sinensis (Angelica sinensis), their roots or rhizomes, or extracts thereof.

[0010] In one embodiment, the herbal composition of the method consists essentially of at least nine herbal ingredients Polygalae (Prunus persica), Astragali (Astragalus membranaceus), Ligusticum Chuanxiong (Cnidium rhizome), Angelica sinensis (Angelica sinensis), Salviae Miltiorrhizae (Red sage), Paeoniae Rubra (Red peony), Carthamus Tinctorius (Safflower), Prunus Persica (Peach seed), and Acori Tatarinowii (Acorus tartarius), their roots or rhizomes, or extracts thereof.

[0011] In one embodiment, the herbal composition of the method comprises a combination of extracts of nine herbal ingredients, Radix Polygalae Root, Radix Astragali Root, Rizome Ligusticum Chuanxiong, and Radix Angelica Sinensis Root, Radix et Rhizome Salviae Miltiorrhizae, Radix Paeoniae Rubra, Carthamus Tinctorius, Peach Seed, and Rhizome Acori Tatarinowii.

[0012] In one embodiment, the herbal composition of the method is a pharmaceutical composition. The pharmaceutical composition may also include one or more pharma- ceutically acceptable carriers or excipients.

[0013] In certain embodiments, a pharmaceutical composition for use in the methods of the present invention comprises a combination of extracts of nine herb components, Radix Polygalae, Radix Astragali, Rhizoma Ligustici Chuanxiong, and Radix Angelicae Sinensis, Radix et Rhizoma Salviae Miltiorrhizae, Radix Paeoniae Rubrae, Carthamus Tinctorius, Semen Persicae, and Rhizoma Acori Tatarinowii, together with one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment, the pharmaceutical composition is MLC901 (also known as NeuroAiD II™).

[0014] In another specific embodiment, a pharmaceutical composition for use in the methods of the present invention comprises a combination of extracts of four herb components, Radix Polygalae, Radix Astragali, Rhizoma Ligustici Chuanxiong, and Radix Angelicae Sinensis, together with one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment, the pharmaceutical composition is MLC1501.

[0015] In one embodiment, recovery from spinal cord injury is induced and / or accelerated by the regeneration of nerve cells.

[0016] In one embodiment, the herb composition of the method promotes the growth of nerve cells.

[0017] In one embodiment, the nerve cells are vertebrate nerve cells.

[0018] In one embodiment, the nerve cells of the vertebrate are human nerve cells.

[0019] In one embodiment, the nerve cells are derived from cortical nerve cells.

[0020] In one embodiment, administration of the herb composition results in spinal cord regeneration and / or regeneration of the connection between the brain and the spinal cord and / or regeneration of damaged nerve cell tissue or cells.

[0021] In one embodiment, administration of the herb composition promotes recovery of limb movement in a paralyzed subject.

[0022] In one embodiment, an effective amount of the herb composition of about 1 mg / kg to about 100 mg / kg is provided.

[0023] In one embodiment, a method is provided that includes administering a second agent for use in treating a subject having a spinal cord injury.

[0024] In one embodiment, the second agent is a pharmaceutical effective to inhibit pain and / or muscle spasm.

[0025] The accompanying drawings are included to more clearly illustrate specific embodiments of the present disclosure and related technologies. The drawings included herein provide a further understanding of the present disclosure, are incorporated into the present disclosure, and constitute a part of the present disclosure. It should be understood that the drawings illustrate embodiments of the present disclosure and, together with the remainder of the present disclosure, serve to explain the principles of the present disclosure. The drawings exemplify embodiments of the present disclosure, and it is clear that those skilled in the art can easily understand other embodiments from the figures described herein.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0027] Term Explanation This section is intended to provide guidance regarding the interpretation of the words and phrases shown below, and (where appropriate) their grammatical variations.

[0028] The term "neurite outgrowth" as used herein relates to the generally directed outward growth of axons and dendrites. Neurite outgrowth is important for synapse formation and development.

[0029] Unless otherwise stated, the terms "comprising" and "comprise" and their grammatical variations are intended to represent "open-ended" or "inclusive" language, including the recited elements and enabling the inclusion of additional elements not recited.

[0030] As used herein in the context of numerical values, the term "about" means, for example, about ±30%, about ±20%, about ±10%, about ±5%, or about ±1% of the numerical value. Where appropriate, the term "about" may be omitted from the definition of the invention.

[0031] The words "a", "an", and "the" are used to describe elements and components of the present invention. This is for convenience only and also to convey the general meaning of the invention. This description is to be interpreted as including one or at least one, and the singular form includes the plural form as well, unless the context clearly dictates otherwise. Thus, for example, the term "agent" includes references to not only a single agent but also multiple agents (including mixtures of agents). It should also be noted that the term "or" is used in the sense of "and / or" unless an exception is specified in the context.

[0032] As used herein, the term "in vivo" includes the use of an entire living organism, which is in contrast to the term "in vitro" which does not involve the use of the entire living organism. The term "in vitro" is understood to include, inter alia, "ex vivo" uses that do not form part of the whole living organism, such as the use of cells, tissues, etc. (e.g., cell or tissue culture, biopsy, cells or tissues from a dead organism, etc.). Further non-limiting examples of "in vitro" relate to the use of cell extracts or lysates.

[0033] As used herein, the term "extraction" includes reference to a separation method in which plant material (e.g., chopped portions of fresh or dried plants) is contacted with a liquid solvent to transfer one or more components of the plant material into the solvent.

[0034] The terms "patient" and "subject" are used interchangeably herein and include reference to any human or non-human animal (preferably a mammal) for which it is desirable to use the present invention for treatment. However, it will be understood that "patient" or "subject" does not mean that a symptom is present. As used herein, the term "mammal" includes, but is not limited to, humans and non-human primates such as chimpanzees, monkeys, etc.; domestic animals such as cows, sheep, pigs, goats and horses; pet / companion animals such as dogs and cats; and laboratory animals such as rabbits and rodents such as mice, rats and guinea pigs. Preferably, the mammal is a human.

[0035] The term "treatment" includes any use that treats a disease state or symptom, prevents the onset of a disease, or in some way prevents, impedes, delays or reverses the progression of a disease or other undesirable symptom. Thus, "treatment" includes prophylactic treatment and therapeutic treatment.

[0036] Throughout this disclosure, certain embodiments may be presented in a range format. The description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the disclosed range. Accordingly, a description of a range should be considered to have specifically disclosed all the individual numerical values and all the sub-ranges within the range. For example, a range description such as from 1 to 6 should be considered to have disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, as well as the individual numbers within the range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0037] Unless otherwise specified in the context, the terms "disease", "disorder", and "symptom" may be used interchangeably herein.

[0038] RA means retinoic acid.

[0039] PGE2 means prostaglandin E2.

[0040] DAPI means 4’,6-diamidino-2-phenylindole.

[0041] The BBB scale means the Basso, Beattie, and Bresnahan locomotor rating scale and is widely used to test the behavioral effects of spinal cord injury (SCI) in rats.

[0042] SEP examination means somatosensory evoked potential examination. SEP examination examines the transmission of physical sensations to the brain and how the brain receives those sensations.

[0043] H&E staining means hematoxylin and eosin staining and is one of the major histological stains used in histology.

[0044] ICC staining means immunocytochemical staining and refers to staining isolated or cultured intact cells. The sample may be derived from a tissue culture cell line and may be adherent or suspended.

[0045] ImageJ software is open-source software for processing and analyzing scientific images.

[0046] MTT assay means mean transit time assay and is used to measure the metabolic activity of cells as an indicator of cell viability, proliferation, and cytotoxicity.

[0047] DPBS means Dulbecco's phosphate buffered saline.

[0048] DMEM / F12 means Dulbecco's modified Eagle's medium / nutrient mixture F-12.

[0049] FBS means fetal bovine serum.

[0050] AA means amino acid.

[0051] NEAA means non-essential amino acid.

[0052] KA means kainic acid.

[0053] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention pertains.

[0054] Detailed embodiments and implementations of the claimed subject matter are disclosed in detail herein, along with technical matters, structural features, achieved objectives, and effects, with reference to the accompanying drawings. It is understood that the disclosed embodiments and implementations may be embodied in various forms, but are merely illustrative of the claimed subject matter. However, the present disclosure may be embodied in many different forms and should not be construed as limited to the exemplary embodiments and implementations described herein. Rather, these exemplary embodiments and implementations are provided so that the description of the present disclosure is thorough and complete and so that the scope of the present disclosure is fully conveyed to those skilled in the art. Specifically, the terms in the embodiments of the present invention are for the purpose of explaining the objectives of specific embodiments and do not limit the present disclosure. In the following description, details of well-known features and techniques may be omitted in order to avoid unnecessarily obscuring the presented embodiments and implementations.

[0055] The configurations described in the following description are changeable non-limiting examples and are merely cited to illustrate at least one embodiment and are not intended to limit the scope thereof.

[0056] In one embodiment, there is provided a method of treating a subject to induce and / or accelerate recovery from spinal cord injury, comprising administering to the subject an effective amount of a herbal composition comprising at least four herbal components, Radix Polygalae (root of Polygala tenuifolia), Radix Astragali (root of Astragalus membranaceus), Rhizoma Ligustici Chuanxiong, and Radix Angelica sinensis (root of Angelica sinensis) or extracts thereof.

[0057] In one embodiment, the herbal composition of the method may further comprise at least one herbal component selected from the group consisting of Radix et Rhizome Salviae Miltiorrhizae (Red Sage Root), Radix Paeoniae Rubra (Red Peony Root), Carthamus Tinctorius (Safflower), Semen Persicae (Prunus Persica Seed), and Rhizome Acori Tatarinowii (Acorus Tatarinowii Rhizome), or an extract thereof.

[0058] In one embodiment, the herbal composition of the method consists essentially of at least four herbal components, Radix Polygalae (Milkwort Root), Radix Astragali (Astragalus Root), Rhizome Ligusticum Chuanxiong, and Radix Angelica sinensis (Chinese Angelica Root), or an extract thereof.

[0059] In one embodiment, the herbal composition of the method may consist essentially of at least nine herbal components, Radix Polygalae (Milkwort Root), Radix Astragali (Astragalus Root), Rhizome Ligusticum Chuanxiong, Radix Angelica sinensis (Chinese Angelica Root), Radix et Rhizome Salviae Miltiorrhizae (Red Sage Root), Radix Paeoniae Rubra (Red Peony Root), Carthamus Tinctorius (Safflower), Semen Persicae (Prunus Persica Seed), and Rhizome Acori Tatarinowii (Acorus Tatarinowii Rhizome), or an extract thereof.

[0060] In one embodiment, the herbal composition of the method consists of a combination of extracts of nine herbal components, Radix Polygalae, Radix Astragali, Rhizoma Ligustici Chuanxiong, Radix Angelicae Sinensis, Radix et Rhizoma Salviae Miltiorrhizae, Radix Paeoniae Rubra, Carthamus Tinctorius, Semen Persicae, and Rhizoma Acori Tatarinowii.

[0061] In one embodiment, the herbal composition of the method is a pharmaceutical composition. The pharmaceutical composition may also contain one or more pharmaceutically acceptable carriers or excipients.

[0062] In certain embodiments, the pharmaceutical composition for use in the methods of the present invention comprises a combination of extracts of nine herbal components, Radix Polygalae, Radix Astragali, Rhizoma Ligustici Chuanxiong, Radix Angelicae Sinensis, Radix et Rhizoma Salviae Miltiorrhizae, Radix Paeoniae Rubra, Carthamus Tinctorius, Semen Persicae, and Rhizoma Acori Tatarinowii, and one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment, the pharmaceutical composition is MLC901 (also known as NeuroAiD II™).

[0063] In another specific embodiment, the pharmaceutical composition for use in the method of the present invention comprises a combination of one or more pharmaceutically acceptable carriers or excipients and extracts of four herb components, Radix Polygalae, Radix Astragali, Rhizoma Ligusticum Chuanxiong, and Radix Angelica sinensis. In a specific embodiment, the pharmaceutical composition is MLC1501.

[0064] In one embodiment, recovery from spinal cord injury is induced and / or accelerated by the regeneration of nerve cells.

[0065] In one embodiment, the herb composition of the method promotes the growth of nerve cells.

[0066] In one embodiment, the nerve cells are nerve cells of a vertebrate. The vertebrate nerve cells are human nerve cells. The nerve cells are derived from cortical nerve cells.

[0067] In one embodiment, administration of the herb composition results in the regeneration of the spinal cord and / or the regeneration of the connection between the brain and the spinal cord and / or the regeneration of damaged nerve tissue or cells.

[0068] In one embodiment, administration of the herb composition promotes the recovery of limb movement in a paralyzed subject.

[0069] In one embodiment, the effective amount of the herb composition is from about 1 mg / kg to about 100 mg / kg.

[0070] In one embodiment, a method is provided that includes administering a second agent for use in treating a subject having a spinal cord injury.

[0071] In one embodiment, the second agent is a pharmaceutical effective to suppress pain and / or muscle spasm.

[0072] NeuroAid II (trademark), referred to as "MLC901" in this specification, is a composition containing extracts of nine herb components (Radix Astragali, Radix et Rhizome Salviae Miltiorrhizae, Radix Paeoniae Rubra, Rhizoma Ligusticum Chuanxiong, Radix Angelica sinensis, Carthamus Tinctorius, Prunus persica, Radix Polygalae, Rhizoma Acori Tatarinowii). It is currently sold as an oral therapeutic agent to support recovery after stroke.

[0073] MLC1501 is a composition containing extracts of four herb components (Radix Astragali, Rhizoma Ligusticum Chuanxiong, Radix Polygalae, and Radix Angelica sinensis). Currently, human clinical trials are being conducted to evaluate its effectiveness in stroke recovery.

[0074] MLC901 and MLC1501, and their preparations, are described in the published PCT application International Publication No. WO 2017 / 048191 A1 pamphlet, the content of which is incorporated herein by reference in its entirety.

[0075] In one embodiment, the herb composition of the method of the present invention consists essentially of 1, 2, 3, 4, or 5 herb components, Radix Salviae Miltiorrhizae, Radix Paeoniae Rubra, Carthamus Tinctorius, Prunus Persica, and Rhizome Acori Tatarinowii, or extracts thereof, or 4 herb components, Radix Polygalae, Radix Astragali, Rhizome Ligusticum Chuanxiong, and Radix Angelica sinensis, or extracts thereof, in combination. Such herb compositions and their preparation are described in the published PCT application International Publication No. WO 2007 / 106049A1, International Publication No. WO 2010 / 053456A1, International Publication No. WO 2010 / 110755A1, and International Publication No. WO 2013 / 141818A1, the contents of which are incorporated herein by reference in their entirety.

[0076] The pharmaceutical composition of the present invention may optionally comprise one or more pharmaceutically acceptable additives, carriers, and / or diluents. Examples of pharmaceutically acceptable additives include pharmaceutically acceptable excipients, buffers, adjuvants, stabilizers, diluents, fillers, preservatives, lubricants, or other pharmaceutically acceptable substances known to those skilled in the art or described herein. Examples of suitable pharmaceutical carriers or diluents include phosphate buffered saline, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, and the like. Examples of excipients that can be used include, for example, saccharides, starches, celluloses, gums, proteins, dextrins, and maltodextrins. Various formulations are generally known and are described in detail in the latest edition of Remington’s Pharmaceutical Sciences (Maack Publishing, Easton PA). In at least some embodiments, the pharmaceutical composition described herein comprises an excipient such as dextrin or maldextrin.

[0077] In one embodiment, the composition of the present disclosure (e.g., a pharmaceutical composition) may be included within a kit. The kit may also include instructions for use in addition to the herbal component. The kit may be promoted, distributed, and / or sold as a unit for performing one of the aspects of the present disclosure.

[0078] Generally, the pharmaceutical compositions of the present disclosure may be prepared according to methods known to those skilled in the art.

[0079] The composition may be, for example, a solution, suspension, liquid, minced herb, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0080] The compositions of the present disclosure (e.g., pharmaceutical compositions) may be in liquid, semi-liquid, or solid form and may be administered by any suitable route such as oral, parenteral, intravenous, subcutaneous, intradermal, intraperitoneal, or topical, and are formulated by methods suitable for each route of administration. The term "administering", and variations of that term including "administer" and "administration", include contacting, applying, delivering, or providing the compositions of the present disclosure to a living being or surface by any suitable means.

[0081] The herbal components may be administered in a therapeutically effective amount (as part of a single or multiple administrations). The term "effective amount" or "therapeutically effective amount" means the amount administered to achieve a physiological significance. A drug is physiologically significant if it is present in an amount that produces a physiologically detectable change in the recipient patient such that a beneficial or desired result is achieved.

[0082] The exact amount required varies for each subject depending on factors such as the species being treated, the age, weight, general health of the subject, the medical condition being treated and its severity, the mode of administration, the sex of the subject, diet, the time and frequency of administration, combination of drugs, tolerance / response to treatment, etc.

[0083] In one embodiment, the compositions of the present disclosure (e.g., pharmaceutical compositions) are administered as capsules taken one or more times a day (e.g., 1, 2, 3, or 4 times). In certain embodiments, MLC901 is administered in the form of 2 capsules and taken 3 times a day. In certain embodiments, MLC1501 is administered in the form of 4 capsules and taken 2 times a day. For patients with difficulty swallowing, the capsules may be opened and the powder diluted with water and either drunk as is or infused through a gastrostomy tube.

[0084] The treatment period is usually three months or more, but can be adapted according to the patient's condition. In one embodiment, the daily dose for the patient is about 500 mg to about 8 g, or about 1 g to about 8 g (for example, about 1 g, 2 g, 3 g, 4 g, 5 g, 6 g, 7 g or 8 g). The "daily dose" can be a single unit dose (such as a tablet, capsule, etc.) taken on a particular day, or multiple unit doses. However, it should be understood that the dosage may vary depending on the patient's requirements and the severity of the medical condition being treated, etc.

[0085] In one embodiment, the treatment lasts for about 12 weeks. In another embodiment, the treatment lasts for about 24 weeks. In another embodiment, the treatment lasts for about 36 weeks. In another embodiment, the treatment lasts for about 48 weeks. In another embodiment, the treatment lasts longer than about 48 weeks.

[0086] In one embodiment, the herbal composition of the present disclosure (e.g., MLC901 or MLC1501) may be administered to a subject having SCI in combination with one or more additional active agents, such as other known SCI treatments, that are effective in controlling pain and / or muscle spasm. The one or more additional active agents may be administered simultaneously (e.g., together) or at different times (e.g., sequentially) and over different periods, which may be separate from or overlap with each other. In one embodiment, there may be a synergistic effect. The one or more additional active agents may be administered by the same route or a different route than the herbal composition of the present disclosure. The one or more additional active agents, and the appropriate route of administration and dosage levels, are known to those of ordinary skill in the art or can be readily determined by those of ordinary skill in the art. Typically, as is well known in the medical field, the dosing regimen may depend on various factors including the patient's physique, body surface area, age, the particular compound being administered, gender, time and route of administration, general health status, and other drugs being administered simultaneously. Individual needs vary, but it is within the skill of the art to determine the optimal range of effective amounts of each component. Typically, the dosage of the one or more additional active agents is the same as or similar to the amount that would be administered if the agent were used without the herbal composition of the present disclosure. When the herbal composition of the present disclosure is administered with one or more additional active agents, the one or more additional active agents may be provided in a composition or kit that includes the herbal composition of the present disclosure, or the one or more additional active agents may be provided separately (i.e., not part of the composition or kit that provides the herbal composition of the present disclosure).

[0087] In addition to the foregoing embodiments, the present disclosure includes the following specific embodiments. Embodiment 1: A herbal composition for use in the treatment of a subject for inducing and / or accelerating recovery from spinal cord injury, comprising at least four herbal components, Radix Polygalae (root of Polygala japonica Houtt.), Radix Astragali (root of Astragalus membranaceus (Fisch.) Bunge), Rhizoma Ligustici Chuanxiong, and Radix Angelicae Sinensis (root of Angelica sinensis (Oliv.) Diels) or extracts thereof. Embodiment 2: The herbal composition according to Embodiment 1 for use as claimed, further comprising at least one herbal component selected from the group consisting of Radix et Rhizoma Salviae Miltiorrhizae (root and rhizome of Salvia miltiorrhiza Bunge), Radix Paeoniae Rubra (root of Paeonia veitchii Lynch), Carthamus Tinctorius (safflower), Semen Persicae (seed of Prunus persica (L.) Batsch), and Rhizoma Acori Tatarinowii (rhizome of Acorus tatarinowii Schott), or extracts thereof. Embodiment 3: The herbal composition according to Embodiment 1 for use as claimed, consisting essentially of at least four herbal components, Radix Polygalae (root of Polygala japonica Houtt.), Radix Astragali (root of Astragalus membranaceus (Fisch.) Bunge), Rhizoma Ligustici Chuanxiong, and Radix Angelicae Sinensis (root of Angelica sinensis (Oliv.) Diels) or extracts thereof. Embodiment 4: A herb composition for use according to Embodiment 1, which consists essentially of at least nine herb components, Radix Polygalae (root of Polygala tenuifolia Willd.), Radix Astragali (root of Astragalus membranaceus (Fisch.) Bunge), Rhizoma Ligustici Chuanxiong, Radix Angelicae Sinensis (root of Angelica sinensis (Oliv.) Diels), Radix et Rhizoma Salviae Miltiorrhizae (red sage root), Radix Paeoniae Rubra (root of Paeonia veitchii Lynch), flowers of Carthamus tinctorius L., Semen Persicae (seeds of Prunus persica (L.) Batsch), and Rhizoma Acori Tatarinowii (rhizome of Acorus tatarinowii Schott) or extracts thereof. Embodiment 5: A herb composition for use according to any one of Embodiments 1 to 4, wherein extracts of each herb component are used in the composition. Embodiment 6: A herb composition for use according to any one of Embodiments 1 to 5, which is a pharmaceutical composition further comprising one or more pharmaceutically acceptable carriers or excipients. Embodiment 7: A herb composition for use according to Embodiment 6, wherein the pharmaceutical composition is MLC901. Embodiment 8: A herb composition for use according to Embodiment 6, wherein the pharmaceutical composition is MLC1501. Embodiment 9: A herb composition for use according to any one of Embodiments 1 to 8, wherein recovery from spinal cord injury is induced and / or accelerated by regeneration of nerve cells. Embodiment 10: A herb composition for use according to any one of Embodiments 1 to 9, wherein the herb composition promotes the growth of nerve cells. Embodiment 11: A herb composition for use according to Embodiment 9 or Embodiment 10, wherein the nerve cells are nerve cells of vertebrates. Embodiment 12: A herb composition for use according to Embodiment 11, wherein the vertebrate nerve cells are human nerve cells. Embodiment 13: A herb composition for use according to any one of Embodiments 9 to 12, wherein the nerve cells are derived from cortical nerve cells. Embodiment 14: A herb composition for use according to any one of Embodiments 1 to 13, wherein administration of the herb composition results in spinal cord regeneration. Embodiment 15: A herb composition for use according to any one of Embodiments 1 to 14, wherein administration of the herb composition results in regeneration of the connection between the brain and the spinal cord. Embodiment 16: A herb composition for use according to any one of Embodiments 1 to 15, wherein administration of the herb composition results in regeneration of damaged nerve tissue or cells. Embodiment 17: A herb composition for use according to any one of Embodiments 1 to 16, wherein administration of the herb composition promotes recovery of limb movement in a paralyzed subject. Embodiment 18: A herb composition for use according to any one of Embodiments 1 to 17, wherein the effective amount of the herb composition is from about 1 mg / kg to about 100 mg / kg. Embodiment 19: A herb composition for use according to any one of Embodiments 1 to 18, comprising administering a second agent for treating a subject having a spinal cord injury. Embodiment 20: A herb composition for use according to Embodiment 19, wherein the second agent is a pharmaceutical effective for suppressing pain and / or muscle spasm. Embodiment 21: A herb composition comprising at least four herb components, Radix Polygalae (root of milkwort), Radix Astragali (root of membranous milkvetch), Rhizoma Ligustici Chuanxiong, and Radix Angelicae Sinensis (root of Chinese angelica) or extracts thereof, for use in the manufacture of a pharmaceutical for treating a subject to induce and / or accelerate recovery from a spinal cord injury. Embodiment 22: The herb composition according to Embodiment 21 for use, further comprising at least one herb component selected from the group consisting of Radix et Rhizome Salviae Miltiorrhizae (Red Sage root), Radix Paeoniae Rubra (Red Peony root), Carthamus Tinctorius (Safflower), Semen Persicae (Prunus Persica seed), and Rhizome Acori Tatarinowii (Rhizome of Acorus tatarinowii), or an extract thereof. Embodiment 23: The herb composition according to Embodiment 21 for use, consisting essentially of at least four herb components, Radix Polygalae (root of Polygala tenuifolia), Radix Astragali (root of Astragalus membranaceus), Rhizome Ligusticum Chuanxiong, and Radix Angelica sinensis (root of Angelica sinensis), or an extract thereof. Embodiment 24: The herb composition according to Embodiment 21 for use, consisting essentially of at least nine herb components, Radix Polygalae (root of Polygala tenuifolia), Radix Astragali (root of Astragalus membranaceus), Rhizome Ligusticum Chuanxiong, Radix Angelica sinensis (root of Angelica sinensis), Radix et Rhizome Salviae Miltiorrhizae (Red Sage root), Radix Paeoniae Rubra (Red Peony root), flower of Carthamus Tinctorius (Safflower), Semen Persicae (Prunus Persica seed), and Rhizome Acori Tatarinowii (Rhizome of Acorus tatarinowii), or an extract thereof. Embodiment 25: A herb composition for use according to any one of Embodiments 21 to 24, wherein extracts of each herb component are used in the composition. Embodiment 26: A herb composition for use according to any one of Embodiments 21 to 25, wherein the herb composition is also a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers or excipients. Embodiment 27: A herb composition for use according to Embodiment 26, wherein the pharmaceutical composition is MLC901. Embodiment 28: A herb composition for use according to Embodiment 26, wherein the pharmaceutical composition is MLC1501. Embodiment 29: A herb composition for use according to any one of Embodiments 21 to 28, wherein recovery from spinal cord injury is induced and / or accelerated by regeneration of nerve cells. Embodiment 30: A herb composition for use according to any one of Embodiments 21 to 29, wherein the herb composition promotes the growth of nerve cells. Embodiment 31: A herb composition for use according to Embodiment 29 or Embodiment 30, wherein the nerve cells are nerve cells of a vertebrate. Embodiment 32: A herb composition for use according to Embodiment 31, wherein the vertebrate nerve cells are human nerve cells. Embodiment 33: A herb composition for use according to any one of Embodiments 29 to 32, wherein the nerve cells are derived from cortical nerve cells. Embodiment 34: A herb composition for use according to any one of Embodiments 21 to 33, wherein administration of the herb composition results in spinal cord regeneration. Embodiment 35: A herb composition for use according to any one of Embodiments 21 to 34, wherein administration of the herb composition results in regeneration of the connection between the brain and the spinal cord. Embodiment 36: A herb composition for use according to any one of Embodiments 21 to 35, wherein administration of the herb composition results in regeneration of damaged nerve tissue or cells. Embodiment 37: A herb composition for use according to any one of Embodiments 21 to 36, wherein administration of the herb composition promotes recovery of limb movement in a paralyzed subject. Embodiment 38: A herbal composition for use according to any one of Embodiments 1 to 37, wherein the effective amount of the herbal composition is from about 1 mg / kg to about 100 mg / kg. Embodiment 39: A herbal composition for use according to any one of Embodiments 21 to 38, wherein the method comprises a method of administering a second agent used for treating a subject having a spinal cord injury. Embodiment 40: A herbal composition for use according to Embodiment 39, wherein the second agent is a pharmaceutical effective for suppressing pain and / or muscle spasm.

[0088] In a further embodiment, the present disclosure provides an in vitro injury model of SCI that damages NSC34 cell lines using mechanical or chemical methods.

[0089] In a further embodiment, the in vitro mechanical injury method is shown in FIGS. 1 and 2.

[0090] In yet another embodiment, the present disclosure has the following objectives. 1. Examine the neurotoxic effect of the composition of the present disclosure on NSC-34 cells. 2. Create an in vitro SCI injury model using NSC-34 cell line culture. 3. Use time-lapse imaging and immunofluorescence labeling to determine the effect of the composition of the present disclosure on the regeneration of neurites after injury in the in vitro SCI injury model. 4. Study the underlying protein signal transduction pathways in the differentiation and regeneration of nerve cells when the composition of the present disclosure is supplemented.

Example

[0091] In vitro mechanical injury model Objective 1: Examine the neurotoxic effect of the composition of the present disclosure on NSC-34 cells Method: The NSC-34 cell line was cultured in DMEM / F12 (1:1) + 10% FBS + 1% AA, allowed to grow until reaching a 90% confluence density, and then treated with a differentiation medium consisting of DMEM / F12 (1:1) + 1% FBS + 1% NEAA + 1% AA and 1 μM of RA, 10 μM of RA, or 30 μM of PGE2 to differentiate into mature motor neurons. Differentiation was determined by estimating neurite outgrowth from day 0 to day 14 using ImageJ and ICC staining with the differentiation markers βIII-tubulin and DAPI. The differentiated motor neurons were seeded at a rate of 5000 cells / well in 96-well plates and allowed to adhere for 24 hours. Next, MLC901 at different concentrations (ranging from 25 μg / mL to 2000 μg / mL) was added to the wells and incubated for 24 hours and 48 hours. The neurotoxicity of MLC901 was estimated using the MTT salt assay and a plate reader at a wavelength of 595 nm.

[0092] Results: The differentiation of NSC-34 cells shown in Figure 10 reflects mature motor neurons on day 10 and day 14 of differentiation using 1 μM of RA (magnification 10x). The differentiation of NSC-34 cells shown in Figure 11 reflects mature motor neurons on day 3 and day 5 of differentiation using 30 μM of PGE2 (magnification 10x). Figure 12 shows a bar graph reflecting the viability of differentiated NSC-34 cells against different MLC901 concentrations. The neurotoxicity results of MLC901 show the survival percentage of differentiated NSC-34 cells against MLC901 in the concentration range of 25 μg / ml to 2 mg / ml by the MTT assay, where the IC50 values at 24 hours and 48 hours were 1178 μg / ml and 1251 μg / ml, respectively (n = 6; results are shown as mean ± SD). Figure 13 shows the NSC-34 cell differentiation study on day 7. In this study, βIII-tubulin and DAPI were used with 1 μM of RA, 10 μM of RA, and 30 μM of PGE2. More cells with longer neurites were observed with 10 μM of RA and 30 μM of PGE2 (n = 3).

[0093] Investigation: In the neurotoxicity study using MLC901, the IC50 value of differentiated NSC34 cells was 1251 μg / mL. Therefore, for further research, a treatment concentration range of 800 μg / mL to 1400 μg / mL was selected. In the differentiation study using different differentiation conditions of 1 μM or 10 μM RA and 30 μM PGE2, it was found that 1 μM RA differentiated NSC-34 cells in 14 days, 10 μM RA in 7 days, and 30 μM PGE2 in 3 to 5 days. Therefore, 30 μM PGE2 was used for differentiation in the in vitro mechanical injury study.

[0094] Objective 2: Establishment of an in vitro spinal cord injury model using NSC-34 cell line culture Method: Mechanical scratch of NSC-34 cell culture was performed using a pipette tip to create two parallel vertical scratches. Next, the cells were washed three times with DPBS, and the damaged neurons in the scratch area were examined by microscopic observation. The damaged cells were treated with MLC901 at concentrations of 800, 1000, 1200, and 1400 μg / mL for 3 days, and neurite regeneration was estimated from measurements of neurite outgrowth and the number of neurites in the damaged area by ImageJ, time-lapse assay, and ICC staining. Figure 5 shows a bar graph of neurite outgrowth at different MLC901 concentrations on day 0 and day 3. Figure 6 shows a bar graph of the number of neurites at different MLC901 concentrations on day 0 and day 3. In these experiments, MLC901 at concentrations of 1000 μg / mL and 1200 μg / mL showed longer neurite outgrowth than at concentrations of 800 μg / mL and 1400 μg / mL (p > 0.05) (n = 6). Figure 9 shows a bar graph of neurite growth after time-lapse imaging on day 0, day 1, day 2, and day 3 using different MLC901 concentrations. Neurite outgrowth is shown by ImageJ software for untreated cells and cells treated with MLC901 at concentrations of 800 μg / mL, 1000 μg / mL, 1200 μg / mL, and 1400 μg / mL. In all treatment groups, neurite outgrowth was longer on day 3 compared to day 0 (p > 0.05), and significant (p < 0.01) neurite outgrowth was seen at 800 μg / mL and 1000 μg / mL compared to UT cells and cells treated at 1200 μg / mL (p < 0.001, n = 6). In Figures 5, 6, and 9, * is p < 0.05, ** is p < 0.01, *** indicates p < 0.01.

[0095] Results: Figure 4 shows the mechanical scratch models on day 0 and day 3 after treatment with different concentrations of MLC901: a) control cells with only medium, b) 800 μg / mL of MLC901, c) 1000 μg / mL of MLC901, d) 1200 μg / mL of MLC901, and e) 1400 μg / mL of MLC901. The lines depicted in Figure 4 indicate the mechanically damaged areas, the arrows indicate neurite outgrowth, and a 10-fold magnification of the selected area and a 100-μm scale bar are included. On day 3 of treatment, higher neurite outgrowth was shown compared to the untreated group, and at concentrations of 1000 and 1200 μg / mL of MLC901, the neurite outgrowth in the damaged area was the highest (n = 6). Figure 15 shows a bar graph reflecting the percentage of cell survival for different MLC901 concentrations compared to untreated cells. Here, MLC administered at 1200 μg / mL showed excellent cell viability compared to untreated cells and cells treated with 800 μg / mL and 1400 μg / mL of MLC901 (p > 0.05) (n = 3).

[0096] Discussion: These results indicate that MLC901 administration promotes neurite outgrowth in the damaged area, particularly at concentrations of 1000 μg / mL and 1200 μg / mL. It was also observed that neurite outgrowth was better in the treated group than in the untreated group. Therefore, this result clearly shows that MLC901 promotes neurite outgrowth in both the culture of healthy and damaged differentiated NSC34 cells. The MTT assay showed that damaged cells in the MLC901-treated group had an excellent survival percentage compared to the untreated group in the damaged area with or without treatment.

[0097] Objective 3: To determine the effect of the composition of the present disclosure on post-injury neurite regeneration in an in vitro SCI injury model using time-lapse imaging and immunofluorescence labeling methods Method: Mechanical scratches of NSC-34 cell cultures were performed as described above. Next, the cells were treated with different concentrations of MLC901 (i.e., 800 μg / mL, 1000 μg / mL, 1200 μg / mL, and 1400 μg / mL), placed in medium, and put into an incubator at 37°C. Neurite regeneration was observed over 72 hours using a camera that took pictures every 30 minutes. The regenerative ability of MLC901 in the damaged area was compared with that of untreated cells at different time points during the observation period. After 3 days of treatment and time-lapse imaging, as shown in Figure 8, the cells were stained with βIII-tubulin and DAPI, and neurite regeneration was further observed. The length of neurite outgrowth was measured at a magnification of 20x and statistically analyzed.

[0098] Results: Figure 7 shows time-lapse imaging demonstrating that MLC901 promotes neurite outgrowth and the number of neurites at the damaged site. At 1000 μg / mL and 1200 μg / mL, neurite outgrowth at the damaged site was greater than at other concentrations (n = 3). Neurite growth was quantified using ImageJ software. Figure 8 shows time-lapse imaging of neurite outgrowth in damaged NSC34 cells on day 0 and day 3. Figure 14 shows ICC staining using DAPI and βIII-tubulin on day 3 after treatment of NSC-34 cells with different concentrations of MLC901 (i.e., 800 μg / mL, 1000 μg / mL, 1200 μg / mL, and 1400 μg / mL). The line drawn in Figure 4 indicates the mechanically damaged area, the arrows indicate neurite outgrowth, and a 10x magnification of the selected area and a 100 μm scale bar are included. On day 3 of treatment, higher neurite outgrowth was shown compared to the untreated group, and at concentrations of 1000 and 1200 μg / mL of MLC901, neurite outgrowth in the damaged area was the highest (n = 6).

[0099] Investigation: By time-lapse imaging, MLC901 was shown to promote neurite outgrowth compared to untreated cells, and when MLC901 was administered at concentrations of 1000 μg / mL and 1200 μg / mL, neurite outgrowth at the injury site was shown to be longer. ICC staining after time-lapse imaging also showed that when MLC901 was administered at concentrations of 1000 μg / mL and 1200 μg / mL, more βIII tubulin was expressed, suggesting that these two concentrations are optimal for neurite regeneration.

[0100] Objective 4: To study the underlying protein signaling pathways in neural differentiation and regeneration when the compositions of the present disclosure are supplemented Methods and Results: As shown in Figure 16, the phosphoinositide 3-kinase (PI3K) / AKT signaling pathway contributes to various processes and mediates many aspects of cell function, such as nutrient uptake, assimilation, cell growth, proliferation, and survival. The expression of different markers of this pathway suggests nerve regeneration. For example, upregulation of p-ACT (Thr308) suggests regeneration. Downregulation of p-GSKβ3 (Ser9) reflects regeneration. Similarly, increased expression of p53, ATF3, GAP43, and elF2B (ser535) suggests nerve regeneration. Figure 17 shows Western blot analysis of the expression of protein markers, phospho-ACT (Thr308), phospho-GSKβ3 (Ser9), ATF-3, GAP43, elF2β (ser535), and p-53 in untreated (UT) damaged NSC34 cells and damaged NSC34 cells treated with MLC901 at concentrations of 1000 μg / mL and 1200 μg / mL. Since 1200 μg / mL of MLC901 generated more protein marker bands than 1000 μg / mL of MLC901 and the expression of phospho-GSKβ3 (Ser9) was less in 1200 μg / mL of MLC901 than in 1000 μg / mL of MLC901, it was suggested that 1200 μg / mL of MLC901 brought about excellent nerve regeneration. Figure 18 shows Western blot analysis of the expression of proteins involved in the PI3K / ACT nerve regeneration pathway in damaged NSC34 cells. The cells tested were untreated cells and cells treated with MLC901 at concentrations of 1000 μg / mL and 1200 μg / mL. Cells treated with MLC901 at a concentration of 1200 μg / mL showed the highest expression of nerve regeneration proteins. Figure 19 is a bar graph reflecting the expression of PI3K / AKT pathway proteins, a) phospho-Akt (Thr308), b) phospho-GSK3β (Ser9), and c) ATF-3, in different MLC901 concentration groups (i.e., untreated control, 1000 μg / mL, 1200 μg / mL) over 72 hours. Compared with untreated cells, Phospho-Akt (Thr308) and ATF-3 showed upregulation, while phospho-GSK3β (Ser9) showed statistically significant downregulation, suggesting nerve regeneration.Figure 20 is a bar graph reflecting the expression of PI3K / AKT pathway proteins, d) GAP-43, e) p53, and f) elF2β(ser535) in different MLC901 concentration groups (i.e., untreated control, 1000 μg / mL, 1200 μg / mL) over 72 hours. elF2β(ser535) showed statistically significant (p>0.01) upregulation at 1000 μg / mL and 1200 μg / mL compared to untreated cells, suggesting regeneration.

[0101] Discussion: The high expression of markers at 1200 μg / mL of MLC901 indicates that neurite regeneration is progressing compared to untreated cells. Similarly, the decrease in the expression of phospho-GSK3β(Ser9) at 1200 μg / mL also indicates nerve regeneration. These results support the use of MLC901 as a nerve regeneration drug for the treatment of SCI.

[0102] In a further embodiment, the present disclosure provides an in vivo injury model of SCI using mechanical and chemical methods in rats.

[0103] In a further embodiment, the in vivo SCI injury method is shown in FIG. 3a or 3b.

[0104] In yet another embodiment, the present disclosure has the following objectives. 1. Establish an in vivo mechanical SCI injury model in adult rats. 2. Investigate the effectiveness of the compositions of the present invention on nerve regeneration in rats with spinal cord injury by mechanical means (e.g., compression). 3. Establish an in vivo chemical SCI model in adult rats. 4. Investigate the effectiveness of the compositions of the present invention on nerve regeneration in rats with spinal cord injury by chemical means (e.g., use of kainic acid).

[0105] The in vivo spinal cord injury (SCI) model is considered irreplaceable because it can be associated with similar pathophysiological states in humans. The SCI model helps in understanding the injury mechanism and analyzing the benefits of advanced therapeutic interventions. Since the functional, morphological, and electrophysiological effects of SCI in humans are similar, the animals most commonly used in the study of neuropathological states are rodents such as rats and mice.

[0106] SCI is classified into either complete injury or incomplete injury. Complete SCI refers to a state where the injury site and the underlying sensory and muscle functions are completely lost. Incomplete SCI refers to the loss of function below the injury level. The degree of injury is another extremely important aspect: SCI in the cervical and upper thoracic regions can cause irregularities in the breathing pattern and may be life - threatening. Injuries in the lower thoracic or lumbar regions are preferred as SCI models because they cause only lower limb paralysis without changing respiratory and cardiac functions. SCI models are classified based on the injury mechanism: mechanical or chemical. Mechanical injury is caused by mechanical means such as an impactor, forceps, clips, balloons, or scissors, while chemical injury is caused by injecting chemicals such as glutamate, aspartic acid, N - methyl - D - aspartic acid (NMDA), superoxide, hydroxyl radical, peroxynitrate, heavy metals, ethidium bromide, or kainate.

[0107] There are various mechanical injury models, such as contusion, compression, detachment, dislocation, or transection, to produce complete and incomplete SCI. Mechanical injury models are advantageous for evaluating axonal regeneration and subsequent functional recovery. Chemical injury models are useful for investigating axonal and neuronal degeneration, molecular mechanisms, and the effects of various therapies on specific pathways. Excitotoxic chemical injury models are becoming increasingly popular because they are useful for studying secondary injury mechanism events such as the degeneration of neurons and axons caused by glutamate excitotoxicity. Neurodegeneration is described as the progressive loss of the structure and function of neurons, axons, and nerve cells. Chronic neurodegenerative diseases such as Parkinson's disease, Huntington's disease, Alzheimer's disease, temporal lobe epilepsy, and amyotrophic lateral sclerosis (ALS) are caused by chemical excitotoxicity. In transgenic ALS rats, 6-hydroxydopamine (6-OHDA) causes neurotoxicity that induces Parkinson's disease, the G93A mutation causes hydroxyl radical generation, and it is further suggested that oxidative damage leads to the onset of ALS. Exposure to other chemicals such as heavy metals (e.g., aluminum) in the brain and spinal cord causes cognitive impairment and generally damages the nervous system, while scopolamine causes dementia, colchicine induces symptoms of Alzheimer's disease, kainic acid (KA) causes temporal lobe epilepsy by intracerebral or intra-amygdala administration, or causes SCI by intraspinal administration.

[0108] Kainic acid (KA) is an agonist of ion channel-type glutamate receptors, induces neuropathological changes both in vivo and in vitro, and is commonly used in the study of the mechanism of excitatory-induced neuronal apoptosis. Excitotoxicity plays a fundamental role in many nervous system diseases, including cerebral and spinal cord ischemia, trauma, and other neurodegenerative disorders. L-Glutamate is the main excitatory neurotransmitter present in the nervous system. It acts as a synaptic neurotransmitter, bringing about long-term changes in synaptic organization, neuronal migration, and neuronal excitability during the developmental stage, and ensuring the viability of neurons. The overactivation of glutamate receptors by KA alleviates the influx of intracellular calcium ions, controls the generation of free radicals, namely ROS (reactive oxygen species) and RNS (reactive nitrogen species), and together with ATPase, triggers the further influx of harmful ions and chemicals, causing neuronal death. The administration of KA induces excitotoxicity via glutamate, causing neuronal death and neurodegeneration. The mechanism of action of KA-induced excitotoxicity is shown in Fig. 37.

[0109] The use of the KA-induced neuronal excitotoxicity model is a method useful for screening potential therapeutic agents for nerve regeneration in SCI. The KA-induced SCI model in Sprague-Dawley (SD) rats will be described below. In this study, the kinematic, electrophysiological, neurological, and histological changes that occur when MLC901 is administered after KA is administered intraspinally were measured, and the nerve regeneration-inducing ability of MLC901 in SCI was evaluated.

[0110] The tests used in the in vivo study are as follows: Motor activity (a) Open field test The open field test (OFT) was performed to evaluate the changes in the motor activity of rats. The rats were placed in the central part of an acrylic box, and the motor activity of each rat was observed for 5 minutes by two blinded observers. Next, the rats were scored based on the Basso, Beattie, and Bresnahan (BBB) scale. The floor of the BBB scale was divided into three different parts. The inner square was composed of a distance of 20 cm, the middle square was 40 cm, and the outer square was 60 cm. A 10 cm × 10 cm box was marked on the floor of the open field, and the number of boxes crossed by the entire group during the 5-minute evaluation was recorded and compared on the 3rd, 7th, 14th, 21st, and 28th days after injury.

[0111] (b) Rotarod test Wheel running is one of the most widely used tests to evaluate the motor deficits in rodents with brain and spinal cord injuries. In this test, rats were placed in a wheel and the wheel was forced to rotate at 90 degrees. The rats used the coordinated movement of their forelimbs and hindlimbs to grasp the wheel and tried to restrain the movement of the wheel. The time until the rats completely restrained the wheel and stopped was recorded for each rat in all groups. The scale used was as follows (0 - 4).

[0112] [Table 1]

[0113] (c) Grid walk test In this test, a raised metal square grid (40x60 cm) was used [see Figure 33c]. The grid device was installed inside the aforementioned open field device. The rats were placed at one end of the grid and were allowed to walk to reach the other end of the grid. The walking behaviors such as the number of errors, the total number of steps, and the time required for grid walking were recorded and analyzed by an experimenter who was blinded to the experimental design.

[0114] (d) Inverted grid test The inverted grid test is a test for estimating the muscle strength of the forelimbs and hindlimbs. Usually, rats can grasp the grid in an inverted position for 30 to 40 seconds. Therefore, to evaluate the muscle strength after injury, the rats were placed upside down on a grid about 20 cm above the ground and a timer was set. The time each rat grasped the grid (gird) guard in an inverted position was recorded.

[0115] Sensory function test (a) Hot spatula method Using the hot spatula test, the changes in the sensory nociception of rats were evaluated. The reference temperature was determined by applying a hot spatula to the tail of the control rats, slowly increasing the temperature, and stopping when the rats began to lick their tails. The average temperature calculated for all control rats was used as the reference temperature. The latency of temperature sensation (licking the tail) was recorded and compared for each group. An increase in latency suggests a loss of sensory nociception accompanied by an increase in the threshold of response to temperature rise. To avoid injury due to burns, the duration of the stimulus was kept within 20 seconds. This process was repeated 3 to 5 times, and the average value was adopted as the threshold. The baseline for normal rats is about 12 seconds. Based on the behavior of the rats, a scale of 0 to 3 was used as follows:

[0116]

Table 2

[0117] (b) Cold sensation test The cold sensation test is also useful for the sensory function test. Anhydrous alcohol (98%) is mainly used for the cold sensation test of rats. First, the skin of the target part of the rats was shaved. Then, anhydrous alcohol was sprayed on the shaved part, and the reaction was recorded and evaluated based on the same scale as above.

[0118] Electrophysiological evaluation Somatosensory evoked potential (SEP) measurement SEP is the response of the brain and spinal cord induced by motor and electrical stimulation of the peripheral nerves. The commonly used stimulation sites in rats are the sciatic nerve at the wrist and the posterior tibial nerve at the ankle. SEP was measured in all rats of each group using Nicolet® Viking Quest™. The rats were anesthetized and the stimulating electrodes were fixed to the hind limbs. The recording electrodes were placed in the hind limb cortical sensory area between the coronal suture and the sagittal suture. The reference electrode was placed 0.5 cm behind the recording electrode, and a direct current rectangular wave electrical stimulation with an intensity of 10 - 30 mA, a pulse width of 0.1 ms, and a frequency of 1 Hz was generated and superimposed 50 - 60 times. The latency, duration, and amplitude of SEP were recorded, and the electrical physiological recovery of the nerves was observed in all groups.

[0119] Histological analysis after spinal cord extraction Four weeks after surgery and treatment, the rats were sacrificed. The spinal cord tissue (T12 - T13) was dissected, fixed overnight with 4% paraformaldehyde and then 30% sucrose, and then cut into sagittal and parasagittal sections with a thickness of 15 mm using a cryostat. Hematoxylin and eosin (H&E) staining was performed for general histological examination under microscopic observation.

[0120] Immunohistochemical analysis Using specific antibodies, immunohistochemical examination was performed on 5 - μm - thick sections of spinal cord tissue. The sections were incubated overnight at 4°C in a humidified chamber in monoclonal GAP43 (D9C8) rabbit antibody (1:200 cell signalling, USA) and GFAP monoclonal antibody (1:200 ThermoFisher Scientific), and then stained with anti - rabbit IgG 488 and 594 secondary antibodies (1:400, Santa Cruz Biotechnology, Santa Cruz, California, USA) at 37°C for 2 hours. Nuclear staining was performed using DAPI (1;15000) in DPBS solution and incubated at room temperature in the dark for 30 - 40 minutes. Finally, the slides were mounted and observed with a confocal microscope (Nikon A1R).

[0121] In - vivo injury model Objective 1: Establishment of an in vivo mechanical spinal cord injury model in adult rats (calibrated forceps compression method) Method: The calibrated forceps compression method was performed by compressing the T12 vertebra with forceps (Dumont #5) for 15 seconds. The arms of the forceps were placed on the adjacent side of the exposed spinal cord, and compression was applied until the bottoms of the tips of the forceps came into contact with each other. The forceps were held in this position for 15 seconds to cause a moderate injury. Figure 21 shows the compression method, where a) - h) represent the following respectively: a) sublime animals, b) marking of the T10, T12, and T13 vertebrae, c) subcutaneous incision, d) removal of muscle, e) exposure of the spinal cord, f) removal of the T12 vertebra, g) 15 - second compression of the spinal cord, h) closure of the wound (suturing of tissue and skin).

[0122] Objective 2: Investigate the effectiveness of the composition of the present invention on nerve regeneration in rats with spinal cord injury caused by mechanical means (e.g., compression) Method: Thirty minutes after mechanically inducing spinal cord injury in rats, MLC901 was administered subcutaneously at a single - dose of 10 mg / kg in physiological saline, and then orally at 10 mg / kg / day in drinking water for 4 weeks. When animals did not ingest the drug orally, oral administration (20 - 16G, 3.8 - 10 cm) technique was used. The oral administration needle was slowly advanced along the hard palate to reach the esophagus. The tube should pass easily through the esophagus. Once proper placement was confirmed, the drug was slowly administered with a syringe attached to the tip of the injection needle. After administration, the needle was gently withdrawn at the same angle as when inserted. During treatment, motor function was evaluated using the Basso - Beattie - Bresnahan (BBB) open - field locomotor scale. Other tests included the running wheel, grid - walking, and inverted grid tests as discussed above. After treatment, the rats were sacrificed (28 days later), and the spinal cord was removed for further evaluation. Post - sacrifice evaluations may include electrophysiological examinations (SEP and / or MEP), histological examinations, immunohistochemical examinations, and PI3K / Akt pathway neurite regeneration protein marker examinations.

[0123] Results: Plugged-dory rats were divided into three groups: (i) MLC901-treated SCI rats, (ii) untreated SCI rats, and (iii) healthy rats. The number of rats in each group was five. After injury by the above method, animals were administered MLC901 at 10 mg / kg / day in drinking water. If the animals did not ingest the drug with water, they were gavaged with 16G by forced oral administration. The administration period was 28 days. During these four weeks, as shown in Figures 23 - 27, the motor behavior of the animals was evaluated by running wheel, inverted grid, and electrophysiological analysis. The open field analysis was performed by two "blinded observers" and scored using the BBB scale shown in Figure 22, reflecting subscores of joint movement, foot placement, and toe clearance. Figure 23 shows a line graph of the BBB scores and subscores of control (healthy rats), untreated rats, and treated rats at 3, 7, 14, 21, and 28 days after mechanical spinal cord injury. As demonstrated, the treated rats showed better BBB scores (p < 0.05) regarding jaw movement, foot placement, and toe clearance than the untreated rats. Figure 24 is a line graph showing the running wheel restraint of control (healthy rats), untreated rats, and treated rats at 3, 7, 14, 21, and 28 days after mechanical spinal cord injury on a scale of 0 - 4, where 0 indicates no restraint and 4 indicates complete restraint. As demonstrated, the treated rats showed better restraint (p < 0.05) than the untreated rats (n = 5). Figure 25 is a line graph of grid walking of control (healthy rats), untreated rats, and treated rats after mechanical spinal cord injury at 3, 7, 14, 21, and 28 days after injury, where a) shows the time taken to walk on a 0.5 m grid and b) shows the distance (number of crossed lines) of the grid moved within 60 seconds. As demonstrated, the treated group showed improved grid walking and crossing more lines compared to the untreated rats (p < 0.05).

[0124] Table 1 below shows the results of the grid walking test, including the total number of limb steps per minute, the number of impairments in the forelimbs and hindlimbs, and the proportion of the total number of impairments, of treated and untreated rats after mechanical spinal cord injury, at pre-injury, 3 days, 7 days, 14 days, 21 days, and 28 days after injury. As demonstrated, on each test day after injury, the treated group showed statistically significant improvement compared to the untreated group (p<0.05 * ).

[0125]

Table 3

[0126] Figure 26 is a line graph of the reverse grid test conducted on the control group, untreated rats after mechanical spinal cord injury, and treated rats, at pre-injury and 3 days, 7 days, 14 days, 21 days, and 28 days after injury, where a longer time indicates the ability and strength of the forelimbs and hindlimbs. As demonstrated, the treated group was able to maintain an inverted posture for a longer time than the untreated rats (p<0.05).

[0127] Table 2 below shows the results of the sensory function evaluation of cold and warm reflexes. In treated and untreated rats, sensory coordination was tested using a cold / warm sensation scale of 0 - 3, where 0 indicates no response, 1 indicates a local response, 2 indicates a transient cry, and 3 indicates a persistent cry.

[0128]

Table 4

[0129] Figure 27 is a bar graph reflecting the electrophysiological SEP showing the latency, duration, and amplitude of the treated and untreated groups. In both the treated and untreated groups, the latency and duration increased, and the amplitude decreased. No significant difference was observed between the two groups.

[0130] Figure 28 shows the morphology of the spinal cord after excision. The spinal cords of untreated rats, treated rats, and healthy rats are shown in Figure 28. Figures 29 and 30 are histological analyses of spinal cord lesions stained with H&E.

[0131] Investigation: The mechanical compression injury method successfully induced hindlimb paralysis in rats with moderate and incomplete SCI. The calibrated clamp compression method is a convenient and highly reproducible experimental method that can control the intensity of injury according to the duration of compression. Using this method, gait analyses were created in various tests of the MLC901 treatment group, untreated group, and healthy rats. After the test, the MLC901 treatment group recovered motor ability within 2 weeks, while the untreated group took 4 weeks to recover motor ability. The results of the running wheel, grid walk, reverse grid running wheel, grid walk, and reverse grid tests also showed that rats administered MLC901 had significantly better scores than non-administered rats, indicating that MLC901 can restore the lost connection between the brain and spinal cord. The SEP results suggest shorter latency and higher amplitude, indicating interruption of potential transmission.

[0132] Objective 3: Establishment of an in vivo chemical spinal cord injury model in adults Methods: Fifteen adult Sprague-Dawley (SD) rats (body weight 300 - 400 g) were housed in clean cages under a bio-bubble air control system and bred under a 12-hour light-dark cycle, allowing free access to food and water. The rats were acclimated to the environment 7 days before the experiment. The rats were anesthetized by intramuscular injection of a ketamine-xylazine (9:1) solution at a rate of 0.1 mL / 100 g body weight of the animal. After anesthetizing the rats, ointment was applied to the eyes to prevent dehydration, and the rats were placed on a heating pad. The dorsal surface was shaved, and the vertebrae were marked at the position of T11 - T13 about 1 cm around the planned incision site. The T13 vertebra was identified by palpating the 13th rib externally, and then the vertebra was visualized with forceps. The forceps were gently hooked under the rib to determine which vertebra the rib was attached to, i.e., whether the T13 vertebra moved in response to the movement of the rib. The T11 vertebra was identified by counting from above. The incision site was disinfected 3 times with 70% isopropyl alcohol and then with iodine solution. Figure 38 shows the anatomical position of the T13 thoracic spinal cord segment in a view of the back of an SD rat from above. The skin was opened to expose the spinal column from T12 - L3. T13 is adjacent to the 13th rib.

[0133] Before making the incision, to confirm that the rat was properly anesthetized, the reflex nerves were checked using the method of pinching the toes or the tail. Next, an incision was made along the backbone, and the rat's reflex was examined again. A line was drawn along the T11 - T13 vertebrae, and the skin was incised by approximately 1.5 cm. A retractor was inserted to hold the skin, and the tissues on both sides of the spinal cord were removed to identify the position of the T13 vertebra. With appropriate lighting, the space between the T12 and T13 vertebrae was determined. The needle was slowly inserted between the vertebrae, and the reflexes of the lower limbs and the tail were checked to confirm that it was inserted at the appropriate position.

[0134] KA (1 mL, 0.1 mM) was diluted with 1 mL of physiological saline to a final concentration of 0.05 mM KA, and then 40 μL was injected at a rate of 0.01 mL (10 μL) / min until the syringe was empty. Immediately after the injection, rigidity of the lower limbs and the tail occurred, indicating that injury induction was successful. After the injection, the rat was placed in an upright position for 3 - 5 minutes to prevent drug perfusion into the brain, and then slowly placed in a supine position on a heating pad. Thereafter, while taking care not to apply pressure to the spinal cord, it was gently compressed with a surgical sponge to stop the bleeding.

[0135] Taking care not to damage or compress the spinal cord, the muscle layer was carefully sutured over the spinal cord. The skin of the wound was closed with sutures, and the rats were kept on a ventilator and a heating pad until they regained consciousness (60 - 90 minutes). Thereafter, to prevent dehydration due to the surgery, 3 - 4 mL of lactated Ringer's solution (preheated to 37°C) per 300 - 400 g of body weight was injected. Tramadol stock solution (0.4 mg / 100 g, 50 mg / mL) was administered 5 minutes after wound closure. Povidone and a topical antibacterial ointment were applied to the wound. The rats were carefully observed until they regained consciousness and then transferred to a normal clean cage with a comfortable and clean bedding. Figure 40 provides a schematic representation of KA-induced spinal cord injury, where A) shows the marking of T11 - T13 vertebrae; B) shows the subcutaneous incision; C) shows the exposure of the muscle and the spine; D) shows the removal of the muscle layer; E) shows the insertion of a 26-G syringe into the intervertebral space between the T12 and T13 vertebrae; F) shows the syringe placed at a 45° angle; G) shows the slow perfusion of KA (0.1 μL / min); H) shows the wound closure.

[0136] After spinal cord injury, to relieve the pain symptoms, tramadol 0.4 mg / 100 g was administered subcutaneously twice a day for 3 - 5 days. Soft food and autoclaved clean water were prepared nearby so that the rats could easily access them. The daily food and water intake was carefully observed, and for rats that did not appropriately consume food and water, lactated Ringer's solution (1 mL / 100 g of body weight) was administered subcutaneously on the 3rd to 5th day after injury. Urinary retention typically occurs due to lower limb paralysis. To avoid this, the bladder was massaged manually twice a day to promote urination: the abdomen was gently palpated to confirm the position of the bladder, and it was gently compressed downward until the bladder was emptied. If there was hematuria, the antibiotic Baytril (100 mg / mL) was injected subcutaneously at 50 mg / 100 g of body weight.

[0137] Two of the three groups of rats were induced to be injured. After injury, the rats were divided into a treatment group (T), an untreated group (UT), and a healthy group (C). The experimental time frames for various evaluation procedures are shown in Figure 31. All rats received pre-evaluations on the 1st, 3rd, and 7th days during the one-week period before surgery. After surgery, electrophysiological evaluations were performed on the 0th, 14th, and 28th days, and motor tests were performed on the 3rd, 7th, 14th, 21st, and 28th days. Sensory function tests were performed on the 7th and 21st days. The animals were sacrificed on the 28th day, and spinal cord specimens were collected for histological and pathological studies.

[0138] Objective 4: To investigate the effectiveness of the composition of the present invention on nerve regeneration in rats with spinal cord injury by chemical means Method: The experimental time frames for various evaluations are shown in Figure 31. All rats received pre-evaluations (one week before surgery) on the 1st, 3rd, and 7th days. After surgery, electrophysiological evaluations were performed on the 0th, 14th, and 28th days, and motor tests were performed on the 3rd, 7th, 14th, 21st, and 28th days. Sensory function tests were performed on the 7th and 21st days. The animals were sacrificed on the 28th day, and spinal cord specimens were collected for histological and pathological studies.

[0139] Results: Motor activity evaluation After KA injury, the rats showed complete lower body paralysis, with immobile hind limbs or tails, and urinary dysfunction without fecal dysfunction. In both groups (T and UT), the retraction of the puncture site began to heal at 1 week after injury. In the T group, slight movement of the hind limbs was observed at 1 week after injury and became more obvious 3 weeks later. However, in the UT group, slight coordinated movement was observed only after day 14. Complete coordinated movement was not seen in either group after day 28. In the T group, after day 28, the coordinated movement of the hind limbs showed slight improvement, but in the UT group, there was little improvement [Figure 32a]. Urinary function recovered partially 3 days later, but residual urine remained in the bladder and was completely resolved 7 - 10 days later in both injury groups. The BBB scale consists of 8 categories that can individually evaluate the forelimbs and hind limbs (joint movement of the affected limbs, weight support, finger position, foot placement, direction and movement during walking, limb coordination, tail position). When the total maximum score is 21 points, it indicates normal movement or complete functional recovery, and when it is 0 points, it indicates no movement at all. The BBB scores of the C, T, and UT groups were scored by 2 blinded observers. The results showed that the UT injury group had significantly lower BBB scores than the T group on days 7, 14, 21, and 28 after injury [Figure 32a)].

[0140] To examine spontaneous motor activity, the movement distance was observed in both pre - SCI and post - SCI rats [Figure 32b)]. Recording and scoring were performed during a 5 - minute walk in the open - field test. The UT - injured rats had a significantly reduced movement distance compared to the T group. The calculated movement distances of UT rats and T rats on days 3, 7, and 14 were 10.5 ± 0.707 cm and 17.5 ± 0.76 cm (p < 0.05 * )), 17.5 ± 0.707 cm and 25 ± 2.707 cm (p < 0.05 * ), and 32.5 ± 2.12 cm and 39.2 ± 3.53 cm (p < 0.05 * ). The movement distances between the UT group and the T group on days 21 and 28 (p < 0.01 **) were 35 ± 2.82 cm and 47 ± 1.12 cm, and 41 ± 2.207 and 55 ± 2.432 cm, respectively. The decrease in the movement distance of the UT rats was consistent with hindlimb paraplegia and dyssynergia [Figs. 32a) and 32b)].

[0141] Gait analysis was also estimated in the open field. The movements of the KA-lesioned rats were compared with those of the control (healthy) rats and scored using the BBB scale by two blinded observers at 7, 14, 21, and 28 days after injury and before injury. The scores for jaw movement, jaw position, and toe clearance are shown in Fig. 39.

[0142] The running wheel evaluations of the C, UT, and T groups before injury were estimated and compared. The UT rats and T rats completely lost the restraint force after injury and showed a significantly decreased restraint force compared with the C group (p < 0.001 *** ). However, on days 3 and 14 (p < 0.05), and days 21 and 28 (p < 0.01), the T group showed a greater restraint force than the UT group [Fig. 33a]. Similarly, in the grid walk and reverse grid evaluations, both injured groups (UT and T groups) showed a significantly decreased ability to hold the grid in an inverted posture due to hindlimb paralysis, and it was demonstrated that they could not walk on the grid without observing a large number of foot disorders [(Figs. 33b), 33c), and 33d)]. However, as the number of treatment days progressed, the T group showed improvement (p < 0.05 and p < 0.01), which was demonstrated by the longer holding time and grid distance movement than the UT group. This was statistically significant at 7, 14, 21, and 28 days after injury (Fig. 33).

[0143] Sensory function test The sensory function was examined on the 7th and 21st days after KA injury. In the warm and cold sensation tests, after injury, the rats in the UT group and the T group showed no response on the 3rd day. However, on the 7th day, the UT rats were evaluated as 0 points by both observers, while the rats in the T group showed a slow withdrawal and were evaluated as 1 point (see Table 3 below). The results showed a delay in response and impairment of sensory nerve conduction. However, the rats before injury and the rats in the C group were evaluated as score 3 throughout the experiment and withdrew immediately. The latency of the withdrawal response (time from stimulation to withdrawal of the hind paw) in the KA-injured rats was shorter than that in the rats of the C group. The latency of the withdrawal response observed in the T rats was better than that in the UT rats from the 7th day (see Table 3).

[0144]

Table 5

[0145] Electrophysiological evaluation As a result of SEP examination, it was shown that the waveforms disappeared immediately after KA-induced SCI in both injured groups. The electrophysiological results correlated with the results of motor evaluation. The healthy group did not show significant changes in the SEP waveforms over time. In contrast, in the KA injury groups (UT and T), the SEP waveforms disappeared immediately after SCI, and no waveforms were observed even 30 minutes after injury. Figures 34a), b), c), and d) show the waveforms, mean onset amplitudes, durations, and latencies of the UT group and the T group on the 14th and 28th days, respectively. In the UT and T injury groups, a decrease in amplitude was observed compared to before injury and the C group. The mean onset amplitudes of the KA injury groups were 19.56 ± 1.52 mV on the 14th day and 22.3 ± 0.81 mV on the 28th day. The mean onset amplitudes of the C group on the 0th, 14th, and 28th days were 25.63 ± 0.81 mV, 25.67 ± 0.50 mV, and 26.02 ± 1.14 mV, respectively [(Figure 34b)]. The UT injury group showed a statistically significant (p < 0.05) decrease in amplitude on the 14th day compared to the T group and a statistically significant (p < 0.01) decrease in amplitude on the 14th day compared to the C group [(Figure 34b)]. The duration of nerve conduction indicates the nerve conduction velocity. In the UT group, it was observed that the duration was significantly prolonged (p < 0.05) (1.32 ± 0.11 ms) compared to the T group (0.42 ± 0.17 ms) on the 14th day [(Figures 34a) and (c)]. Similarly, in the UT group, a statistically significant (p < 0.05) increase in latency was observed after the 14th day after injury compared to the T group. On the 0th day immediately after injury, no waveforms of somatosensory evoked potentials were observed. However, after the 14th day, the hindlimbs of UT rats showed severe motor dysfunction, the amplitude decreased rapidly, and the duration and latency increased significantly compared to the T group [(Figures 34a), b), c), d)].

[0146] Histological analysis Tissue damage was observed in spinal cord transverse sections of KA-lesioned (UT and T) rats. Histopathological examination using HE staining of spinal cord tissue showed lesions at the KA injection site. The most severe damage was at the epicenter, where the ventral horn of the gray matter and the lateral funiculus of the white matter were lost (see Fig. 35). As a result of HE staining, both the UT and T groups showed severe structural damage and a decrease in the number of neurons compared with the C group. Hemorrhagic foci were observed in the central part of the gray matter. Necrosis and cavitation were progressing in the central lesion area (gray matter). In both UT rats and T rats, a large number of red blood cells and neutrophils appeared at the primary lesion site. UT rats showed larger hemorrhagic foci than T rats, indicating greater damage and neurodegeneration. In spinal cord sections of UT and T KA-lesioned rats, significant extravascular leakage of albumin was observed. However, no extravascular leakage of albumin was observed in the C group. The extravascular leakage of albumin in KA-lesioned rats was mainly due to neurodegeneration and glutamate excitotoxicity. This co-localization effect of albumin extravascular leakage in neurons indicated that UT rats had more neurotoxicity and neuronal death than T rats (see Fig. 35).

[0147] Immunohistochemical analysis Upon macroscopic evaluation of the dissected spinal cord, hemorrhage and tissue damage at the lesion site were prominent in the KA-lesioned group. Immunohistochemistry showed systematic expression of GAP-43 and GFAP in both the UT and T groups. The highly decreased expression of GAP43 in the UT and T groups revealed that lesion formation and neuronal degeneration were adjacent (see Fig. 36). GAP-43 positive nerve fibers were observed in the gray matter of the C group. However, the expression of green fluorescence in the UT group was lower compared with the T group, which reflected neurodegeneration. Double staining with GFAP also showed the presence of astrocytes in the C, UT, and T groups. The decreased GFAP expression also indicated the loss of astrocytes. Confocal images reflected the significant differences in the expression of GAP-43 and GFAP among the C, UT, and T groups (see Fig. 36).

[0148] Observation: After intraspinal administration of KA, male Sprague-Dawley rats showed severe signs of motor dysfunction and sensory impairment. In KA-lesioned rats, behavioral changes such as restlessness and a tendency to continuously sit in the corner of the cage were observed, which were basically due to glutamate excitotoxicity. Rats that remained stationary for longer than 15 - 20 seconds were gently tapped or scratched on the side of the open field to force movement. If the animal still did not respond to these actions, the animal was lifted and placed in the center of the open field apparatus and moved towards the opposite side. The observation period was sometimes extended beyond 5 minutes to more accurately evaluate toe clearance, foot position, and the coordination of the forelimbs and hindlimbs.

[0149] The BBB scale is an excellent tool for evaluating motor activity. The lowest score, "0, no movement of the hindlimbs (HL) is observed", is usually given to a severe SCI state. The UT rats in this study showed a minimum BBB score of 3 - 4 points, indicating moderate SCI. In the T rat group, it reached 19 points 28 days after injury. Two blinded observers evaluated the subtle behaviors of locomotor movement, from the individual joint movements of the hindlimbs, to plantar walking, coordinated walking, and finally foot position, trunk stability, tail position, etc. The highest score on the BBB scale is 21 points, indicating "stable plantar walking and coordinated walking", showing complete recovery and balanced walking. The rats in the UT group and the T group obtained a maximum of 13 points and 19 points (on the 28th day after injury), reflecting motor dysfunction. Only the C group and the pre-injured rats obtained 21 points on the BBB scale. It was also observed that there was little improvement in the score between the 21st and 28th days after injury in UT rats. The rats in the T group showed a short delay period in the first 3 days after injury, followed by a more rapid recovery period between the 4th and 13th days, and a functional plateau between the 15th and 28th days. The BBB score is important in interpreting changes in motor activity after spinal cord injury and is used in this specification to detect the variation in results between the UT group and the T group.

[0150] Rats placed inside the running wheel tend to firmly grasp the wheel with their front and hind legs, restraining and stopping it. Therefore, the running wheel test is considered valuable for evaluating the muscle strength and coordination of rats after SCI. In all three groups, the BBB score and the distance traveled (cm) were statistically significantly lower (p < 0.05 * and p < 0.01 ** ) in the KA-lesioned rats, reflecting the successful induction of moderate paralysis. This result also shows that the T rats had better BBB scores and distance traveled than the UT group, highlighting the value of this model for studying the regenerative capacity of therapeutic agents under laboratory conditions.

[0151] Similarly, the grid walking test is an important laboratory test for evaluating and comparing the walking of different rat groups and for studying the regenerative capacity of therapeutic agents under laboratory conditions. In the grid walking test, the rat needs to accurately place its four limbs in specific positions. If a rat's foot falls through, this is considered a foot impairment. Errors and defects are counted while the rat is walking on the grid, and the more defects there are, the greater the defect in motor coordination is reflected.

[0152] The results of the running wheel and grid walking tests showed that the rats in the T group had significantly better motor coordination than the rats in the UT group. This result indicates that the motor control of the T-group rats was significantly improved compared to that of the UT-group rats (see Figures 33a), b), c), d)). In the inverted grid test, the rats in the T group showed significantly better strength than the rats in the UT group.

[0153] The results demonstrated that the coordination movement disorder and dyskinesia associated with KA injury in rats persisted until the 28th day after KA injury. These motor dysfunctions did not change until the 3rd day after injury in both the UT rat group and the T rat group. However, after that, the T rat group began to show improvement compared to the UT rats until the 28th day, indicating that the T rat group has the ability of nerve regeneration. The results obtained from all motor tests showed that MLC901 improved the motor recovery of the injured rats, which may also be related to the increase in GAP43 expression (nerve marker) and the decrease in demyelination (HE). The higher GAP43 expression in T rats than in UT rats indicates that MLC901 weakens the improvement of motor activity through the repair of lost connections and nerve regeneration.

[0154] Electrophysiological evaluation is reflected as a functional means to evaluate the integrity of various aspects of the nervous system, including the spinal cord. The clinical applications of electrophysiology, particularly evoked potentials such as somatosensory evoked potentials and motor evoked potentials, are used in the diagnosis of damage to the peripheral or central nervous system. In particular, somatosensory evoked potentials (SEP) that monitor the integrity of the dorsal columns of the spinal cord are used [Cruccu et al., Clinical Neurophysiology, 119(8), pages 1705 - 1719. (2008)]. SEP has reproducibility of axonal conduction in both the normal and damaged spinal cords in the descending sensory and motor pathways and provides a non-invasive and objective evaluation [Malhotra & Shaffrey, Spine, 35(25), pages 2167 - 2179(2010)]. Pulse conduction through nerve cells can be measured by stimulating an electrical signal from the nerve itself or the muscle innervated by that nerve. Latency is measured by the time from the stimulation by the stimulating electrode until the deflection of the signal by the recording electrode is recorded. The time it takes for the electrical impulse to travel from the stimulation site to the recording site is called latency. The time required for conduction is called duration and is measured in milliseconds (ms). The magnitude of the response of impulse conduction in motor and sensory neurons is known as amplitude and is measured in millivolts (mv). The signal is recorded as nerve conduction passing under the recording electrode, and the conduction speed (meters per second) can be estimated by dividing the latency by the distance. The measured values can be described from the perspectives of latency until the response appears, the amplitude of the response, and the conduction speed (duration). In this study, electrodes were placed on the sciatic nerve near the knee of rats, and the SEP waveforms of each group were observed from the perspectives of latency, amplitude, and duration. The increase in latency and duration in both injury groups (T and UT) reflected both demyelination and nerve loss of the peripheral nerve. The loss of nerve cells affects the amplitude of the nerve conduction velocity. Demyelination of nerve cells causes a delay in latency. In this study, both the UT and T injury groups did not show waveforms immediately after injury (within 30 minutes), but after 14 days post-injury, the latency in the T group increased significantly (p < 0.05) compared to the UT group. This demonstrates the improvement of nerve conduction after treatment.The decrease in amplitude is directly related to the decline (neurodegeneration) of motor neurons involved in impulse conduction and the presence of demyelinating lesions. When the amplitude is small and the latency and duration are long, the nerve conduction velocity (NCV) is slow, which indicates demyelinating lesions, while a small amplitude value reflects axonal degeneration. In this study, the inventors found that NCV was not observed immediately after injury (30 minutes). However, on the 14th day, the rats in the T group showed better NCV than those in the UT group.

[0155] In the evaluation of neurodegenerative diseases, morphological and pathological interventions are also important. Routine HE staining shows each structure of the spinal cord after injury, and demyelination of nerves in spinal cord sections after spinal cord injury can also be detected. HE-stained sections after KA injection showed the presence of bleeding foci, especially in the gray and white matter. Progressive necrosis and cavitation were also observed in UT and TKA-injured rats (see Figure 35). However, bleeding and cavitation in the UT group were more prominent than those in the T group, and cavitation decreased after administration of MLC901 (Figure 35). Inflammatory cells such as red blood cells and neutrophils were more frequently observed in the UT and T groups than in the C group, indicating neurodegeneration due to KA excitotoxicity (Figure 35).

[0156] To confirm the neuronal mass in the entire population (C, T, and UT), the expression of GAP43 and GFAP was determined. Growth-associated protein 43 (GAP43) is an activity-dependent plasticity protein rich in axons and neurons, which promotes actin polymerization and axonal regeneration. Glial fibrillary acidic protein (GFAP) is the main structural protein of filaments in the cytoskeleton of astrocytes and functions as a marker for mature astrocytes. The results obtained suggested that GAP43 was highly expressed in group C but not so much in the injury groups. In this study, it has been demonstrated that GAP43-positive fibers significantly increased in group T (see Figure 36), suggesting that treatment with MLC901 may have supported the regeneration of neurons and axons. Also, it was observed that GFAP expression was at a higher level in group T than in group UT, supporting the fact that treatment with MLC901 could protect against the disappearance of astrocytes (Figure 36). However, since the expression of GFAP staining in the gray matter was higher, it was suggested that gliosis of glial cells and astrocytes was present in both group UT and group T (Figure 36).

[0157] In summary, the neurodegenerative results of KA-induced injury and the nerve regeneration of MLC901-administered rats were observed by motor function evaluation (open field test, running wheel, grid walk, inverted grid) and evaluated by sensory function evaluation and SEP. The presence or absence of pathological features was evaluated based on the results of HE staining and IHC staining. All these results prove that the KA excitotoxicity model was successful as an in vivo model for creating and studying SCI. Furthermore, the nerve regeneration ability of MLC901 was demonstrated by the improvement of all motor activity scores, sensory, electrophysiological, and histological evaluations.

[0158] Conclusion The in vivo KA-induced excitotoxicity model provides a practical, simple, and efficient approach for inducing SCI in experimental animals. All the important findings of this study support the existence of the most prominent physiological and pathological consequences of SCI. Intraspinal administration of KA reduces the adverse effects of injuries that cause incomplete paraplegia in rats. The main mechanism of action of KA excitotoxicity is regulated by the activation of glutamate receptors, leading to increased Ca +2 influx into neurons, resulting in the formation of ROS and RNS, and ultimately stimulating nerve demyelination and neurodegeneration. Importantly, this study demonstrated that treating KA-lesioned rats with MLC901 improved the functional recovery process of SCI compared to untreated (UT) rats.

[0159] Incorporation by reference All references, articles, publications, patents, patent publications, and patent applications (the "References") cited in this document are hereby incorporated by reference in their entirety for all purposes. However, the descriptions of the References do not admit, nor do they suggest, that they are valid prior art or form part of the common general knowledge in any country in the world. The References include the following. a) Pamphlet of International Publication No. WO 2017 / 048191 A1 b) Pamphlet of International Publication No. WO 2007 / 106049 A1 c) Pamphlet of International Publication No. WO 2010 / 053456 A1 d) Pamphlet of International Publication No. WO 2010 / 110755 A1 e) Pamphlet of International Publication No. WO 2013 / 141818 A1 f) Cruccu et al., Clinical Neurophysiology, 119(8), pages 1705-1719. (2008) g) Malhotra & Shaffrey, Spine, 35(25), pages 2167-2179 (2010)

Claims

1. A composition or kit for use in the treatment of a subject to induce and / or accelerate recovery from spinal cord injury, comprising at least four herbal ingredients: Radix polygalae root, Radix Astragalus root, Rizome Ligusticum Chuanxiong rhizome, and Radix Angelica sinensis root, or extracts thereof.

2. The composition or kit according to claim 1, further comprising at least one herbal component selected from the group consisting of sage root and rhizome (Radix et Rhizome Salviae Miltiorrhizae) (red sage root), peony root (Radix Paeoniae Rubra) (red peony root), safflower (Carthamus Tinctorius) (safflower), peach seed (Semen Persicae) (peach (Prunus Persica) seed), and sweet flag rhizome (Rhizome Acori Tatarinowii) (sweet flag rhizome), or an extract thereof.

3. The composition or kit according to claim 1, wherein the composition comprises four herbal components: Radix polygalae root, Radix Astragalus root, Rizome Ligusticum Chuanxiong rhizome, and Radix Angelica sinensis root or extracts thereof.

4. The composition contains nine herbal ingredients: Radix Polygalae root, Radix Astragalus root, Rizome Ligusticum Chuanxiong rhizome, Radix Angelica sinensis root, Radix et Rhizome Salviae Miltiorrhizae root and rhizome, Radix Paeoniae Rubra root, and Carthamus The composition or kit according to claim 1, comprising the flowers of Tinctorius (safflower), peach seeds (Semen Persicae), and sweet flag rhizome (Rhizome Acori Tatarinowii) or extracts thereof.

5. The composition or kit according to claim 1, wherein the composition is a pharmaceutical composition and optionally comprises a pharmaceutically acceptable carrier or excipient.

6. The composition or kit according to claim 5, wherein the pharmaceutical composition is composition MLC901.

7. The composition or kit according to claim 5, wherein the pharmaceutical composition is composition MLC1501.

8. The composition or kit according to any one of claims 1 to 5, wherein recovery from spinal cord injury is induced and / or accelerated by the regeneration of nerve cells.

9. The composition or kit according to any one of claims 1 to 5, wherein the composition promotes the growth of nerve cells, vertebrate nerve cells, and optionally the vertebrate nerve cells are human nerve cells.

10. The composition or kit according to claim 9, wherein the nerve cells are derived from cortical nerve cells.

11. The composition or kit according to any one of claims 1 to 5, wherein the use of the composition or kit results in spinal cord regeneration.

12. The composition or kit according to any one of claims 1 to 5, wherein the use of the composition or kit results in the regeneration of the connection between the brain and the spinal cord.

13. The composition or kit according to any one of claims 1 to 5, wherein the use of the composition or kit results in the regeneration of damaged nerve tissue or cells.

14. The composition or kit according to any one of claims 1 to 5, wherein the use of the composition or kit promotes the recovery of limb movement in a paralyzed subject.

15. A composition or kit according to any one of claims 1 to 5, for use in conjunction with a second agent used to treat a subject having a spinal cord injury.

16. The composition or kit according to claim 15, wherein the second agent is a pharmaceutical product effective in suppressing pain and / or muscle spasms.

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