KR100740972B1 - A agent comprising GM-CSF for treatment of neuropathy - Google Patents

Abstract

The present invention relates to a therapeutic agent for neurological diseases containing GM-CSF as an active ingredient, and specifically, a nerve containing GM-CSF as an active ingredient that not only inhibits the death of bone marrow cells but also inhibits the death of nerve cells. It relates to a disease treatment agent. The neurological disease therapeutic agent containing GM-CSF of the present invention can be usefully used for promoting recovery of impaired nerve function such as the spine.

GM-CSF

Description

A disease comprising GM-CSF for treatment of neuropathy             

1 is a bar graph showing that apoptosis is induced by addition of staurosporin to N2A cells, which are neuroblastoma of the mouse, and then treated with GM-CSF to decrease neuronal cell death. ego,

Figure 2 is a bar graph showing that the effect of inhibiting neuronal cell death is reduced when treated with GM-CSF alone when the neutralizing antibody to GM-CSF and GM-CSF receptor in N2A cells,

Figure 3 is a line graph showing that the recovery of nerve function is promoted when behavioral tests using the Basso-Beattie-Bresnahan (BBB) locomotor rating system after GM-CSF administration in spinal cord injury rats.

Figure 4 shows that when treated with GM-CSF compared to the Sham control group that did not cause spinal cord injury, the control group administered PBS to the spinal cord injury rats, the experimental group administered GM-CSF to the spinal cord injury rats It is a bar graph showing that neuronal cell death is inhibited in the damaged area and surrounding tissue.

The present invention relates to a therapeutic agent for neurological diseases containing GM-CSF, which has a function of inhibiting neuronal cell death. The present invention not only suppresses the death of known bone marrow cells but also suppresses the death of nerve cells to promote nerve function recovery. It relates to a GM-CSF-containing neurological disease treatment.

Traumatic spinal cord injury is a serious neurologic defect that causes pathological necrosis and bleeding of spinal cord tissue after injury, and pathologic findings that widen the spinal cord injury due to subsequent secondary injury. Apoptosis is known to play an important role as a cause of secondary injury that causes worsening of spinal cord injury (Wingrave JM et al., J. Neurosci. Res. 73: 95-104, 2003; Li GL et al., Acta. Neuropathol. (Berl) 98: 473-480, 1999). Necrosis of the spinal cord injury was also observed in autopsy of patients who died after spinal cord injury. Inhibition of apoptosis after spinal cord injury has been recognized as an important therapeutic goal.

The development of hematopoietic and neural tissues has many similarities. In particular, gene transcription and cell differentiation of the two tissues have been reported to be closely related (Vescovi AL et al., Nat. Med. 8: 535, 2002; Zhao LR et al., Exp. Neurol. 174: 11). -20, 2002). Recently, stem cells found in adult organs are not only differentiated into the cells of the high whey cells derived from them, but also have been reported to be capable of transdifferentiation to other tissues and organ cells. There is increasing interest in the similarity in. Vescovi A.L. Et al. Demonstrated that neural stem cells found in the adult subventricular layer differentiate into blood cells as well as neurons (Vescovi A.L. et al., Nat. Med. 8: 535, 2002). In contrast, transplantation of hematopoietic stem cells into neural tissues confirmed the differentiation of transplanted hematopoietic stem cells into neurons. Taken together, we can infer that the nervous system and hematopoietic organs have at least similarities in the differentiation process. These similarities suggest that cytokines of the nervous system and hematopoietic organs may also have similarities. In part, however, hematopoietic cytokines have been reported to affect the division, differentiation and viability of neural stem cells (Kannan Y. et al., Neuroimmunomodulation 8: 132-141, 2000; Kamegai M. et al., Neuron 4). : 429-436, 1990) IL2 and IL4 promote the proliferation of rat oligodendrocyte precursor cells and the TGF-b superfamily is known to inhibit proliferation (Schuster N. et al. , Glia 40: 95-108, 2002; Nesic O. et al., J. Neurotrauma. 18: 947-956, 2001; Mehler MF et al., Nature 362: 62-6, 1993). IL5,7,9 and 11 have also been reported to promote differentiation of embryonic murine hippocampal progenitor cells (Mehler MF. Et al., Nature; 362: 62-65, 1993). Therefore, hematopoietic cytokines (cytokine) plays an important role throughout the development and differentiation of neurons.

Granulocyte macrophage colony stimulating factor (GM-CSF) is one of the representative hematopoietic cytokines and is known to be capable of promoting the differentiation and function of hematopoietic cells. In addition, GM-CSF promotes the proliferation of myeloid hematopoietic stem cells and inhibits apoptosis of leukocytes, thereby increasing leukocyte levels in peripheral blood. GM-CSF is mainly used to treat patients with weakened bone marrow function due to these hematopoietic cell stimulating effects. However, there is no clear report on how GM-CSF affects neural stem cell differentiation, division and apoptosis.

Thus, the present inventors have confirmed that GM-CSF not only promotes bone marrow function, but also has an action of inhibiting neuronal cell death, thereby restoring nerve damage and completed the present invention.

It is an object of the present invention to provide a therapeutic agent for neurological diseases containing GM-CSF which not only inhibits the death of bone marrow cells but also inhibits the death of nerve cells and has a function of neurological recovery.

In order to achieve the above object, the present invention provides a therapeutic agent for neurological diseases containing GM-CSF having a function of inhibiting neuronal cell death.

Hereinafter, the present invention will be described in detail.

The present invention provides a neurological disease treatment containing GM-CSF having a function of inhibiting neuronal cell death.

Colony-stimulating factor (CSF) is a cytokine that stimulates expansion and differentiation of bone marrow hepatocytes. Various CSFs act on myeloid cells at various stages of development and promote the formation of specific colonies of various strains. GM-CSF is a hematopoeic growth factor that promotes differentiation of bone marrow cells and is an active factor of mature bone marrow cells.

Recently, recombinant GM-CSF proteins have been studied to treat a variety of diseases including cancer and hematopoietic diseases (Buchsel PC et al., Clin. J. Oncol. Nurs. 6: 198-205, 2002). -CSF acts on bone marrow cells to increase inflammatory white blood cell populations including granulocytes and macrophages (Staynov DZ et al., Proc. Natl. Acad. Sci . 92: 3606-3610, 1995) and It acts as an in vitro macrophage activating element, stimulating and inducing an inflammatory response (Jarmin DI, Exp. Hematol . 27: 1735-1745, 1999; Bischof RJ et al., Clin. Exp. Immunol. 119: 361-367 2000). GM-CSF has previously been used to promote bone marrow recovery after cancer chemotherapy and to recover bone marrow stem cells for transplantation (Keller JR et al., Proc. Natl. Acad. Sci . 88: 7190-7194. 1991; Aglietta M. et al., Cancer 88: 454-460, 2000). There is an increasing trend in clinical studies for the treatment of various diseases, including infectious diseases, hematopoietic diseases and cancer using GM-CSF (Janik JE et al., Clin. Immunol. 93: 209-221, 1999), vaccines Potential as adjuvant is also shown (Kapoor D. et al., Am. J. Nephrol. 20: 53-56, 2000).

The present inventors first confirmed the ability of the neurons to inhibit apoptosis in order to confirm whether the GM-CSF of the present invention can inhibit neuronal damage in addition to the above functions. To this end, injecting staurosporin into N2A cells, a neuroblastoma of cultured mice, induced apoptosis, and then measured the number of cells in which apoptosis occurred by TUNEL staining. As the concentration was increased, the neuronal cell death was reduced, and even when only a low concentration (5 ng / ml) was treated, it was confirmed that cell death was suppressed to less than half (see FIG. 1 ).

In addition, the inventors of the present invention, in order to confirm how the inhibitory function of apoptosis changes when the GM-CSF of the present invention is treated with a neutralizing antibody, the neutralizing antibody to the GM-CSF receptor is 1 ug / ml After treatment with the concentration compared with GM-CSF alone. As a result, the neutralizing antibody was confirmed to lower the apoptosis inhibitory effect by interfering with the binding between GM-CSF and the receptor (see Fig. 2 ). The above results reaffirm the neuronal cell death inhibition function of GM-CSF.

In addition, the present inventors have found that sham without damaging the spinal cord in order to check whether GM-CSF of the present invention can restore impaired nerve function by inhibiting apoptosis in neurons of rats with spinal cord injury. The behavioral test of the control group in which PBS was administered to control rats and artificially injured spinal cord rats and the group treated with GM-CSF to artificially spinal cord injury rats did not. A higher BBB score was observed. This means that GM-CSF has the effect of restoring impaired nerve function (see FIG. 3 ).

In addition, the inventors of the present invention, in order to determine whether the GM-CSF of the present invention has a function of inhibiting apoptosis from the site where the spinal cord injury occurs, the damaged area and the surrounding tissues, after harvesting and fixing the spinal cord tissue 5 days after spinal cord injury, TUNEL staining Was carried out. As a result, it was confirmed that unlike GM-CSF Siamese or PBS control, the treatment of GM-CSF also inhibited neuronal cell death of damaged areas (center of spinal cord) and surrounding tissues (front and tail of spinal cord) (see FIG. 4 ). .

Differences in N2A cell death by staurosporin, N2A cell protection by GM-CSF, GM-CSF effect blockade by neutralizing antibodies, and apoptosis of spinal cord injury tissues and differences in behavioral test between GM animals after GM-CSF treatment A single student tee test (non-paired student t-test) was performed. Statistical significance was defined as P value < 0.05 and the result was mean standard error.

GM-CSF of the present invention can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical preparations.

That is, the GM-CSF of the present invention can be administered in various parenteral formulations during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used Is prepared using. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, Calcium carbonate, Sucrose (Lucose) or lactose (Lactose), gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The effective dose of GM-CSF of the present invention is 250ug / m ² body surface area, and can be administered once a day.

On the other hand, in order to determine the acute toxicity of GM-CSF was carried out the same experiment as in <Example 3> , as a result of the GM-CSF of the present invention does not show a toxicity change up to 1 g / kg in rats, the minimum lethal dose (LD50) was determined to be a safe substance of more than 1 g / kg.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1> Confirmation of apoptosis inhibitory effect at the cellular level of GM-CSF

<1-1> Cell Culture

The present inventors confirmed the effect of inhibiting apoptosis at the cellular level of the GM-CSF of the present invention through the following experiment in laboratory conditions (in vitro).

Mouse neuroblastoma N2A cells were purchased from the American Tissue Culture Collection (ATCC). The cells were dispensed at a concentration of 3 × 10 4 cells / cm ² in 24 well plates, and then 2 mM l-glutamine, penicillin (Dulbecco's modified Eagle's medium, Gibco BRL, Grand Island, NY, USA) medium 20 units / mL), streptomycin (20 mg / mL) and 10% fetal bovine serum (FBS; Gibco BRL) were added and cultured. Culture conditions were maintained at 37 ℃ under 5% CO ₂ supply.

Staurosporin (staurosporin, Sigma, St Louis, MO, USA) was treated for 24 hours to induce apoptosis, and various concentrations (0, 0.5, 1) were determined to determine the appropriate concentration of staurosporin for inducing apoptosis. , 1.5, 2 uM) of staurosporin.

(2) GM-CSF treatment

Human recombinant GM-CSF (LG Chemical Co, Seoul, Korea) was used. To determine the effective amount of GM-CSF for neuroprotective action, various concentrations of GM-CSF (0, 50, 500, 5000 ng / ml) were treated for 6 hours before 1 uM of staurosporin.

(3) Measurement of cell death (TUNEL staining)

The number of apoptotic cells was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeled (TUNEL) staining. Cells were fixed in 4% paraformaldehyde for 10 minutes at 4 ° C., and cell membrane permeability was increased with 0.1% Triton X-100 (Sigma). Fluorescein-labelled UTP (Fluorescein-labelled UTP) was added together with TdT (Roche, Germany), and then reacted at 37 ° C. for 1 hour, followed by laser scanning confocal microscopy and Bio-rad MRC 1024 laser scanning. confocal imaging system mounted onto a NIKON microscope, Bio-Rad, Hercules, CA). As a result, as the concentration of GM-CSF was increased, the neuronal cell death was reduced and the cell death was inhibited to less than half even when only a low concentration (5 ng / ml) was treated ( FIG. 1 ).

In addition, the neutralizing antibody that binds to and neutralizes the GM-CSF receptor was treated to confirm a change in apoptosis inhibitory function.

As a result, it was confirmed that the neutralizing antibody lowers the apoptosis inhibitory effect by interfering with the binding between GM-CSF and the receptor ( FIG. 2 ). The above results confirm once again that the GM-CSF of the present invention has a function of killing neurons.

Example 2 Confirmation of GM-CSF Treatment Effect in Spinal Cord Injured Animals

(1) spinal cord injury animal model production

The spinal cord injury model used adult male Sprague-Dawley rats, weighing 300 to 350 g, and anesthesia was performed by ketamine intraperitoneal injection. Posterior archectomy was performed from T8 to T10. The spinal cord excision site was incised enough to enter the vascular narrows (vascular clip, occlusion pressure, 50 g, Aesculap, Melsungen, Germany). It was.

(2) GM-CSF administration and behavioral tests in spinal cord injury model

Laboratory animal management and research guidelines were carried out with the permission and guidance of the Institutional Animal Management Committee. Five were sham control, 25 were PBS control and 20 were used as GM-CSF administration experimental group. Human recombinant GM-CSF was injected at 20 μg / day for 5 days from the day after injury, and PBS control was injected subcutaneously with phosphate buffered saline (pH 7.4). The Sham control did not cause spinal cord injury and did not perform any medication. Behavioral tests were measured weekly for 5 weeks using the Basso-Beattie-Bresnahan (BBB) locomotor rating system.

As a result, the degree of experimental one GM-CSF treatment than the control a PBS treatment as in the third it was confirmed that to facilitate the recovery nerve injury.

Example 3 TUNEL Staining on Spinal Cord Injury Tissues

TUNEL staining was performed in GM-CSF experimental group, PBS control group and Siamese control group (n = 5). Spinal cord tissue was removed 5 days after spinal cord injury, and paraffin embedding was performed after fixation with 4% paraformaldehyde.

The nuclei of all nucleated cells were counterstained with 4'6'diamidino-2-phenylindole (DAPI, 1 ug / ml) (Sigma) and laser scanning for blue (nuclei) and green (TUNEL) fluorescence. Specimens were performed by laser scanning confocal microscopy (LSCM).

As a result, it was also confirmed that, unlike the Siamese or PBS control, treatment with GM-CSF inhibited neuronal cell death of the damaged area and surrounding tissues ( FIG. 4 ).

Example 4 Oral Acute Toxicity in Rats

Acute toxicity test was performed using SPF SD rats at 6 weeks of age. Two animals per group were injected subcutaneously with GM-CSF five times at a dose of 10 ug / kg. After administration of the test substance, mortality, clinical symptoms, and changes in body weight were observed, and hematological and hematological examinations were performed. As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc. As a result, all of the GM-CSF of the present invention did not show a toxicity change in rats up to 1 g / kg and the minimum lethal dose (LD50) of oral administration was determined to be a safe substance of more than 1 g / kg.

As described above, the GM-CSF of the present invention can be usefully used as a therapeutic agent for neurological diseases for restoring nerve function by inhibiting the death of neurons as well as inhibiting the death of previously known bone marrow cells.

Claims (4)

A therapeutic agent for neurological disorders comprising granulocyte macrophage-colony stimulating factor (GM-CSF) as an active ingredient. According to claim 1, GM-CSF is a therapeutic agent for nerve injury disease, characterized in that to inhibit neuronal cell death. According to claim 1, GM-CSF is a therapeutic agent for nerve injury disease, characterized in that to promote the repair of the spinal nerve. According to claim 1, wherein GM-CSF is a human neuropathy disease therapeutic agent, characterized in that GM GM-CSF.

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