KR102064545B1 - A compositions for treating spinal cord injury comprising stem cell treated with novel compound - Google Patents

Abstract

The present invention relates to a composition for treating spinal cord injury, and provides a composition for treating spinal cord injury comprising a stem cell treated and cultured with a specific compound as an active ingredient.

Description

A composition for treating spinal cord injury comprising stem cells treated with a novel compound {A COMPOSITIONS FOR TREATING SPINAL CORD INJURY COMPRISING STEM CELL TREATED WITH NOVEL COMPOUND}

The present invention relates to a composition for treating spinal cord injury comprising stem cells treated with the novel compounds and cultured as an active ingredient.

Bones cause paralysis of human function when the spinal nerve is damaged by trauma. Spinal nerve tissue has a simple structure compared to the brain, but regeneration is not easy.

Pathological phenomena after spinal cord injury are divided into primary and secondary injuries over time.

Primary damage is a phenomenon that occurs within minutes after the injury, mainly due to necrosis of the cells at the wound site, which is almost impossible to treat with pharmacological treatment because the cells are so rapidly destroyed.

Secondary damage, which progresses slowly from hours to days, begins with apoptosis in the surrounding neurons and oligodendrocytes, rather than only the cells at the wound site.

Apoptosis proceeds continuously around the wound, which eventually widens the damage within the spinal cord.

In addition, degeneration of myelin sheaths, which help the function of axons and axons, which are the pathways of neural signals, occurs, eventually forming an empty space in the spinal cord, and further neural signal transmission cannot be achieved, resulting in permanent loss of function.

For a long time, studies on the cause and regeneration of pathological effects after spinal nerve injury have been conducted to suppress or alleviate permanent paralysis due to spinal cord injury.

Since the early mechanism of spinal cord injury progresses so rapidly that it is difficult to treat with pharmacological treatment, it is important to develop a pharmacological strategy that deals with secondary mechanisms.

To date, various potential pharmacological treatments have been attempted, including steroids, antioxidants, glutamate receptor inhibitors, ion channel inhibitors, gangliosides, antibodies against axon regeneration inhibitors, anti-inflammatory agents, and neurotrophic factors.

However, methylprednisolone is used as the only therapeutic agent after spinal cord injury, and nevertheless has many problems such as unclear therapeutic effect and side effects due to excessive dosage.

Therefore, the treatment of spinal cord injury using medicine or physiotherapy has reached its limit, and many experimental studies focusing on treatment by transplantation of cells have been conducted.

Stem cells are cells that have the ability to differentiate throughout the life of an organism for indeterminate periods, and treatment methods using them have been studied, but the method has not been established to date.

Transplantation of differentiated cells into the damaged area after spinal cord injury has been used as a therapeutic strategy to replace permanently lost nerve cells, providing new nerve cells to the damaged area, thereby promoting nerve regeneration.

The present inventors have found that the treatment of spinal cord injury can be significantly improved through the treatment of certain compounds in stem cell transplantation, and the present inventors have tried to prepare a reference point that can speed up the rapid application of clinical trials.

Korea Patent Publication No. 10-2008-0007800

The present invention aims to provide a stem cell-based spinal cord injury therapeutic agent in order to solve the above problems of the prior art.

According to one aspect of the present invention, there is provided a composition for treating spinal cord injury, comprising a stem cell treated with the compound of Formula 1 as an active ingredient.

[Formula 1]

In one embodiment, the spinal cord injury may be caused by trauma or inflammation.

In one embodiment, the inflammation is at least one selected from the group consisting of acute transverse myelitis, acute disseminated myelitis, myelopathy, non-Hodgkin's lymphoma, hydrocephalus, hereditary ataxia, neurosyphilis, Minamata disease, Lou Gehrig's disease, and multiple dysplasia. Can be caused by.

In one embodiment, the stem cells may be mesenchymal stem cells.

In one embodiment, the stem cells may be incubated for 96 to 144 hours after the compound treatment.

In one embodiment, the stem cells may be differentiated into migrating neuroblasts.

According to another aspect of the invention, (a) treating the stem cells with the compound of formula (1); And (b) incubating the compound-treated stem cells for 96 to 144 hours. There is provided a method for preparing a stem cell for treating spinal cord injury.

[Formula 1]

According to the present invention, since the composition for treating spinal cord injury uses stem cells differentiated at a predetermined stage, the neuronal regeneration effect is remarkably superior to that of the conventional stem cell-based therapeutics.

It is to be understood that the effects of the present invention are not limited to the above effects, and include all effects deduced from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a diagram illustrating a culture method of stem cells according to an embodiment of the present invention.
Figure 2 is a schematic diagram illustrating a method for evaluating motor function recovery using stem cells according to an embodiment of the present invention.
3 shows neuronal lineage markers according to stem cell differentiation.
Figure 4 confirms the differentiation stage of stem cells using a neuronal lineage marker.
5 shows behavioral evaluation results of animals.

Hereinafter, with reference to the accompanying drawings will be described the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. When a part is said to "include" a certain component, this means that it may further include other components, without excluding other components, unless specifically stated otherwise.

Unless defined otherwise, molecular biology, microbiology, protein purification, protein engineering, and DNA sequencing can be performed by conventional techniques commonly used in the field of recombinant DNA within the capabilities of those skilled in the art. These techniques are known to those skilled in the art and are described in many standardized textbooks and reference books.

Various scientific dictionaries that include the terms included herein are well known and available in the art. While any methods and materials similar or equivalent to those described herein are found to be used in the practice or testing herein, some methods and materials have been described. Depending on the context used by those skilled in the art, the present invention is not limited to specific methodologies, protocols, and reagents, because it can be used in various ways.

As used herein, the singular encompasses the plural objects unless the context clearly dictates otherwise. Also, unless otherwise indicated, nucleic acids are written from left to right, 5 'to 3', respectively, and amino acid sequences are written from left to right, amino to carboxyl directions. Hereinafter, the present invention will be described in more detail.

According to one aspect of the present invention, there is provided a composition for treating spinal cord injury, comprising a stem cell treated with the compound of Formula 1 as an active ingredient.

[Formula 1]

Name of the compound is N - (5- chloro-benzo [d] oxazol-2-yl) - N - (4- ethyl-phenyl) -2-methoxy-acetamide (N - (5-Chlorobenzo [ d] oxazol- 2-yl) - N - (may be a 4-ethylphenyl) -2-methoxyacetamide) , it can be prepared by chemical synthesis.

The present inventors confirmed that the spinal cord injury can be effectively improved by injecting the stem cells treated with the compound into the spinal cord.

The compound may be used in the form of a pharmaceutically acceptable salt, and as the salt, acid addition salts formed by pharmaceutically acceptable free acid may be useful.

The compounds may form pharmaceutically acceptable acid addition salts according to methods conventional in the art.

The free acid may be an organic acid and an inorganic acid. For example, the inorganic acid may be hydrochloric acid, bromic acid, sulfuric acid, or phosphoric acid. The organic acid may be citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid. (FμMaric acid), formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, benzenesulfonic acid, salicylic acid, It may be, but is not limited to, nicotinic acid, isnicotinic acid, picolinic acid, glutamic acid, or aspartic acid.

The spinal cord injury (SCI) refers to a clinical condition in which the spinal cord parenchyma is damaged by various factors such as physical shock, and thus paralysis of peripheral motor muscles, sensory and autonomic nervous systems under the damaged area.

The spinal cord injury may be caused by trauma or inflammation, and may be, for example, a traumatic spinal cord injury such as a traffic accident, sports, or industrial injury, or a non-traumatic spinal cord injury such as inflammation, bleeding, tumor, or spinal deformity.

The inflammation may be caused by one or more selected from the group consisting of acute transverse myelitis, acute disseminated myelitis, myelopathy, non-Hodgkin's lymphoma, hydrocephalus, hereditary ataxia, neurosyphilis, Minamata's disease, Lou Gehrig's disease, and multiple atherosclerosis, Inflammation can cause spinal cord injury without any particular limitation.

The term "treatment" refers to ameliorating the symptoms of spinal cord injury, or preventing the progression of symptoms, and may encompass the meaning of commonly used therapies.

The stem cells may be mesenchymal stem cells, mesenchymal stem cells derived from human umbilical cord blood stem cells.

The "stem cell" is a cell capable of differentiating into various cells due to the characteristics of multi potency when a proper signal is given under the influence of the environment in which the cell is located, as well as the self-replicating ability. to be.

The stem cells are included in fat, bone marrow, umbilical cord blood and placenta, and can be used to treat various cell damage diseases such as spinal cord injury, myocardial infarction, cerebral infarction, degenerative arthritis and fracture. The stem cells may be autologous or allogeneic stem cells, and may be derived from any type of animal, including humans and non-human mammals.

The "mesenchymal stem cells" are cells of stromal origin and have the characteristics of self-renewal and can differentiate into bone, cartilage, adipose tissue, muscle, tendon, ligament and neural tissue. Can be utilized.

The mesenchymal stem cells can be obtained from bone marrow, but the mesenchymal stem cells present in the bone marrow have a limited application range due to limited differentiation and proliferative capacity, and the antigenic phenotypes are matched through comparison of histocompatibility antigens for bone marrow transplantation. There is a problem in finding donors that do not exhibit graft-versus-host reactions.

On the other hand, the umbilical cord can be obtained through a simple procedure in the delivery process, and because it contains a large number of hematopoietic stem cells and stem cells, it is possible to separate a large amount of stem cells from the umbilical cord, placenta, umbilical cord blood and the like.

Methods of isolating the mesenchymal stem cells are well known in the art. For example, human cord, Barton's jelly, cord blood, placenta, fat, bone marrow, tonsils, human yolk sac, cord, skin, peripheral blood, muscle, liver, nerve tissue, periosteum, fetus Membrane, synovial membrane, synovial fluid, amniotic membrane, meniscus, trans cruciate ligament, articular chondrocyte, indwelling, perivascular cells, striatum, subpatellar mass, spleen and thymus and the like.

Treating the stem cells with the compound; And (b) incubating the compound-treated stem cells for 96 to 144 hours. The stem cells may be differentiated into migrating neuroblasts.

Referring to FIGS. 3 and 4, MSCs were cultured in a medium containing the compound for 5 days and the differentiation state was confirmed using neural lineage markers. As a result, Nestin and GFAP were hardly expressed, and about 90% of cells were present. Tuj1-positive cell was confirmed.

That is, after incubation for 96 to 144 hours after the compound treatment, the stem cells may be differentiated into mobile neuroblasts, and spinal cord injury may be effectively improved by injecting the differentiated cells in vivo.

The mesenchymal stem cells may be injected in vivo, either alone or in culture in a culture medium, such as Lindbal (1989, Arch. Neurol. 46: 615-31) or Douglas Kondziolka, Pittsburgh, 1998. ) Clinical methods are available.

The composition for treating spinal cord injury may include a pharmaceutically acceptable conventional carrier other than mesenchymal stem cells, and in the case of injection, a preservative, analgesic, solubilizing or stabilizing agent, and a base, excipient in the case of topical administration. , Lubricants or preservatives.

The mesenchymal stem cells may be administered 10 ⁴ to 10 ¹⁰ cells / times, preferably 10 ⁵ to 10 ⁹ cells / times, most preferably 1x10 ⁶ cells / times, but symptoms It may be different depending on the condition of the patient.

Through the following examples, the present invention will be described in more detail, but the present invention is not limited by the following examples.

Experiment method

Compound manufacture

AlCl ₃ (0.73 mmol, 2eq) was added to 5-chloro- N- (4-ethylphenyl) benzo [ d ] oxazol-2-amine (0.37 mmol, 1eq) dissolved in 10 mL of dichloromethane under nitrogen gas substitution. Stir at room temperature (15-30 ° C.) for 10 minutes.

In a ice bath, methoxymethylcarbonylchloride (0.73 mmol, 2eq) was added and stirred at room temperature (15-30 ° C.) for 5 hours.

10 mL of distilled water was added and extracted three times with 10 mL of EtOAc. The organic layer was separated, washed twice with 10% aqueous HCl solution (10 mL), twice with saturated aqueous NaCl solution (10 ml), dried over MgSO _4, and the solvent was concentrated under reduced pressure.

The precipitate was filtered under reduced pressure with n-hexane: EtOAc: ether = 5: 2: 1 (v / v / v) to obtain a compound of formula 1.

White powder (28%), mp 121.6-123 ° C .; ¹ H NMR (Acetone- d ₆ , 400 MHz) δ 7.653 (d, J = 6.0 Hz, 1H), 7.533 (d, J = 8.8 Hz, 1H), 7.357 (m, 4H), 4.611 (s, 2H) , 3.367 (s, 3H), 2.728 (q, J = 7.6 Hz, 2H), 1.271 (t, J = 7.6 Hz, 3H). HR-FABMS Calcd for C ₁₈ H ₁₈ ClN ₂ O ₃ (M ⁺ + H): 345.1006, Found: 345.1004.

In the experiments below, this compound was referred to as L2.

Spinal Cord Injury Animal Model Manufacturing

Adult male Sprague Dawley rats (Sampako, Korea) were anesthetized with chloral hydrate (500 mg / kg) and then anesthetized, and the thoracic vertebrae 8-10 were exposed.

A spinal cord trauma animal model was performed using a NYU impactor (Routes, Cytec Korea) designed to give a trauma similar to human spinal cord injury by dropping a 10 g weight at a constant height and to quantify the trauma intensity on a computer.

The wound site was closed after confirming that a certain damage was applied within a predetermined error range through data on the damage intensity displayed on the computer.

The wound was sterilized with povidin solution, and then reared in cages, and then artificially massaged bladder three times a day to promote urination.

Cell culture

Passage 3 MSCs from Kangstem Biotech were maintained in KSB3 basal medium containing 10% FBS (Gibco) and supplements.

After one passage, the MSCs from Passage 4 made stock and stored in liquid nitrogen.

Stem Cell Differentiation

Referring to FIG. 1, 1 mL per well was seeded into 12 wells of MSC of Passage 5 diluted to 1.1 × 10 ⁴ / mL.

After 4 hours, 40 μM of L1, L3, or 30 μM of L2 (Formula 1) were treated.

After 3 days, the medium was changed to fresh medium, and the compounds were treated in the same manner, followed by further incubation for 2 days.

Stem Cell Transplantation

Referring to Figure 2, 6 weeks after the spinal cord injury was induced to randomly divided mice into three groups.

(1) Control group 1 (Saline group): Injecting only saline to the lesion site instead of stem cells (2) Control group 2 (MSC group): Transplanting only undifferentiated mesenchymal stem cells (3) Experimental group (MSC + L2 group): Low molecular weight compound Tuj1-positive neural stem cell transplantation differentiated by phosphorus L2

After spinal cord injury rats were infused with chloral hydrate (500 mg / kg) intraperitoneally, the surgical site was dissected, and 10 μL of the sample was injected into the lesion epicenter using a glass pipette. The number of transplanted cells in the experimental group injected with stem cells was 1 x 10 ⁶ .

Experiment result

Behavioral evaluation

The following three behavioral assessments were performed for 8 weeks after transplantation (once a week).

(1) BBB locomotor score: A method of scoring 0-21 points of hind paw motor function in open files. 0 points: no movement, 21 points: normal

(2) Grid walk test: Calculates the number of times the foot falls out when the mouse walks on the ladder

(3) Foot print analysis: A method of analyzing the pattern of the sole printed on the floor by applying ink to the rat's foot (forefoot: red, hind foot: blue) and walking a long box.

BBB Open field score

The BBB test is a complex assessment of the recovery of various nerve bundles that control motor function.

By dividing the score from 0 to 21, the paws are not used at all and 0 points are given, and normal rats are given 21 points, which means that the motor function is improved as the score is increased.

Three days before spinal cord trauma, the rats were placed in open field boxes (90 cm x 90 cm x 7 cm in height) once per day for 4 minutes per animal and allowed to move freely in the box.

Three days after the spinal cord trauma, three trained experimenters observed the hind paw movement of each rat and scored a BBB score, and the average of these three BBB scores was determined as the final BBB score of the rat.

In order to rule out individual biases in animal experiments, test subjects were not informed about rats, and each rat was observed for 4 minutes to calculate a score.

Immediately after the spinal cord trauma, all rats showed no movement of the hind paws and the lower body was dragged depending on the front paws.

One week after spinal cord injury, spontaneous recovery was observed, and after 4 weeks, a plateau was maintained, indicating no recovery of motor function, and a chronic spinal cord injury model was established.

After 6 weeks of spinal cord injury, MSBs that had been differentiated for 5 days by either Saline or undifferentiated MSCs and L2 had higher BBB scores in the MSC and L2-treated MSCs group than in the saline-only group. The BBB score remained high until 8 weeks after transplantation.

After 14 weeks of spinal cord trauma (8 weeks after transplantation), the BBB score was about 9 points for the Saline rats (Control 1), which only supported the weight when standing still, and continued footing when walking. He showed a degree of recovery from his back.

On the other hand, rats treated with MSC (Control 2) or L2-treated MSC (Experimental Group) had a BBB score of about 12 points, and the recovery level at which the forefoot and hind legs intersect at about 50% while walking while supporting the weight. When compared with the control 1 showed a significant motor function recovery effect was confirmed (Fig. 5A).

In the case of transplanting MSC and L2-treated MSC, the BBB score was slightly higher than that of MSC group when transplanting L2 treated MSC, but no significant difference was found.

Grid walk test

We assessed the ability of the forefoot and hind legs to rhythmically intersect and to accurately support the hind feet.

The frequency of feet falling below the lattice was indicated as a percentage by passing the rat through a 1 m long metal lattice plate.

Adapted to the experimental environment by passing three times a day for each rat through a 1-meter metal grid three days before the test began, and the first test was performed just six weeks after spinal cord injury.

When passing the rat through the metal lattice plate, the middle stop was not counted, but spontaneously rested and passed through the grid three times by counting only if it passed to the end.

The rats were recorded with a video camera and analyzed by two experimenters who did not know which group each rat belongs to.

The experimental results show that the ratio of the number of steps that the hind foot falls under the grid from the total number of steps that the rat walks is expressed as a percentage (%), and the average value of each group is compared.

Referring to FIG. 5B, all groups of mice showed an error rate of about 70% before transplantation.

At 1 week after transplantation, the error rate of control group 2 and experimental group was lower than that of control group 1.

In particular, the experimental group decreased the error rate rapidly within 1 to 2 weeks of transplantation compared to the control group 2, and showed the lowest error rate for 8 weeks after transplantation.

The results suggest that mice implanted with L2-treated MSCs in three groups more accurately hind paws and increased ability to walk.

Foot print analysis

Footprint analysis was performed as a criterion for evaluating the recovery of hind-motor function in rats after spinal cord injury.

Rats were covered with red ink on their paws, blue ink on their hind paws, and their footprints were observed by passing them through a narrow passage of white paper (1m long and 7cm wide).

If rats did not pass or stop in the passage within 5 seconds, they were excluded from the analysis, and eight tests were performed per animal.

Coordination with the forefoot, distance, degree of rotation of the back foot, stride length between the back foot, distance between the feet, and toe drag were used as indicators of the function of the back foot.

Adapted to the experimental environment by passing the passage three times a day three days before the test began.

Referring to Figure 5C, after the spinal nerve trauma, the rat's hind paw is generally paralyzed and the trailing hind paw appears.

Six weeks after the injury, all groups of rats were unable to support their legs when walking, so that they did not get their back foot (blue) footsteps and were constantly dragged.

At 8 weeks after transplantation, Control 2 had improved recovery and had some toe drag, but recovered enough to walk on the sole of the foot. .

On the other hand, in the experimental group (L2-treated MSC transplantation), there was little bleeding, and the gap between the forefoot and hind foot was narrowed, thereby improving limb coordination.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.

The scope of the invention is indicated by the following claims, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the invention.

Claims (7)

A composition for treating spinal cord injury comprising a stem cell treated with the compound of Formula 1 as an active ingredient.
[Formula 1]

The method of claim 1,
The spinal cord injury is a composition for treating spinal cord injury caused by trauma or inflammation. The method of claim 2,
The inflammation is treatment of spinal cord injury caused by one or more selected from the group consisting of acute transverse myelitis, acute disseminated myelitis, myelopathy, non-Hodgkin's lymphoma, hydrocephalus, hereditary ataxia, neurosyphilis, Minamata disease, Lou Gehrig's disease and multiple symptomosis Composition. The method of claim 1,
The stem cells are mesenchymal stem cells (mesenchymal stem cell) composition for treatment of spinal cord injury. The method of claim 4, wherein
The stem cells are spinal cord injury treatment composition cultured for 96 to 144 hours after the compound treatment. The method of claim 1,
The stem cell is a composition for treating spinal cord injury that is differentiated into a migrating neuroblast (migrating neuroblast). (a) treating the stem cell with a compound of Formula 1; And
(B) culturing the stem cells treated with the compound 96 to 144 hours; spinal cord injury treatment stem cell manufacturing method comprising a.
[Formula 1]

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