KR20070113088A - Compositions and methods for treating motor neuron diseases - Google Patents

Abstract

A method for treating motor neuron diseases is provided to improve clinical amyotrophic lateral sclerosis(ALS) symptoms and show significant therapeutic effects by administering mesenchymal stem cells in combination with erythropoietin(EPO) to ALS animal models or patients. A method for treating a motor neuron disease comprises a step of administering erythropoietin(EPO) or an active fragment or analogue thereof and mesenchymal stem cells simultaneously or sequentially. In the method, the mesenchymal stem cells express positive cell surface markers including CD105(SH2), CD29, CD44, CD73 and CD166 to 90% or more and express negative cells surface markers including CD34, CD45, HLA-DR, CD1a, CD14, CD31 and CD80 to 10% and less. The motor neuron disease is amyotrophic lateral sclerosis (ALS), progressive bulbar palsy(PBP), primary lateral sclerosis(PLS), and progressive muscular atrophy(PMA). A composition for the treating a motor neuron disease comprises EPO or an active fragment or analogue thereof, and mesenchymal stem cells.

Description

Compositions and methods for treating motor neuron diseases {Compositions and methods for treating motor neuron diseases}

1 is a combination of or isolated from autologous mesenchymal stem cells (MSCs) and recombinant rhEPO isolated from ALS patients in SOD1 mutant transgenic mice model animal model to confirm the effects of MSCs and rhEPO It is a graph showing the evaluation of exercise ability by rotarod after treatment (-■-: MSC + EPO treatment group;-▼-: control group (Con);-●-: MSCs treatment group;-▲-: rhEPO treatment) group).

Figure 2 is a hospitalization plan to check whether the treatment of ALS disease by administering autologous mesenchymal stem cells (MSCs) and rhEPO collected from bone marrow cultured to ALS patients.

Figure 3 is a graph showing the change of clinical symptoms after combined treatment of autologous mesenchymal stem cells (MSCs) and rhEPO obtained from bone marrow cultured in ALS patients.

4 is a functional rating scale, Appel score, Norris score (Norris) after combined treatment of autologous mesenchymal stem cells (MSCs) and rhEPO collected from bone marrow and rhEPO in ALS patients. scale) and neurophysiologic index to show changes in clinical symptoms of patients.

The present invention relates to a composition for treating motor neuron diseases (MND) and a method for treating motor neuron diseases using the same. Specifically, the present invention relates to a method and a composition for treating motor neuron disease by administering erythropoietin (EPO) and mesenchymal stem cells (MSC) simultaneously or sequentially to motor neuron disease patients.

Motor neuron disease (MND) is a disorder of motor neuron function due to loss of motor neuron source cells and degenerative changes in motor neuron pathways in the central nervous system. Parkinson's disease, Alzheimer's disease and cerebellar atrophy in which other neurons are damaged It is a disease that is fundamentally different from neurodegenerative diseases such as. These MNDs include primary lateral sclerosis (PLS), which involve only upper motor neurons, and progressive muscular dystrophy (PLS), in addition to Lou Gehrig's disease and amyotrophic lateral sclerosis (ALS). progressive muscular atrophy (PMA), progressive bulbar palsy (PBP) invading the brain's lower motor neurons.

Amyotrophic lateral sclerosis (ALS) is one of motor neuron disease (motoneuron disease, MND), but its pathogenesis is not clear, but it is a neurological disease that selectively invades only the motor nervous system. Haverkamp LJ et al., Validation of a scoring system and a model for survival prediction Brain 1995; 118: 707-19., Caroscio JT et al., Neurol Clin 1987; 5: 1-9). Pathologically, ALS causes loss of cerebral motor cortex, angiant betz cells, anterior spinal motor neurons, and brain stem motor neurons and degeneration to the vertebrae. It is a disease that dies within a few years because it shows signs of upper and lower movements and shows rapid clinical course after the onset. The incidence and prevalence of ALS in Korea is not yet clear, but according to the data so far, about 1,300 people have been affected in Korea, and in foreign countries, the incidence rate of 1 to 2 and 4 to 6 cases per 100,000 population In general, it is 1.5 times higher in men, but the ratio of men and women tends to be similar when they go to 50 to 60.

The only one treatment for ALS has been approved by the US Food and Drug Administration and proven to be safe. Only one drug, riluzole, which antagonizes glutamate, is insignificant. There is no clear treatment that can improve.

Therefore, in recent years, attempts have been made to use stem cells to treat neurological diseases including ALS. Stem cells are pluripotent cells with self-proliferation ability and differentiation potential into various functional cells, and can be classified into embryonic stem cells, fetal stem cells, and adult stem cells. The development of cell therapies using these cells is ongoing, especially adult stem cells have no ethical problems in cell acquisition compared to other cells, and stability has been proved based on long experiences in bone marrow transplantation, etc. to be.

For example, Korean Patent Laid-Open Publication No. 2005-0012208 discloses differentiation and proliferation of neurons by culturing mesenchymal stem cells in a medium containing epidermal growth factor and hepatocyte growth factor. The present invention discloses a method of transplanting it into a patient to treat neurological diseases. However, the method disclosed in the patent is a method for transplanting neurons derived from mesenchymal stem cells into a patient, which requires a complicated and long time of the differentiation induction process.

On the other hand, EPO is a glycoprotein associated with the production of red blood cells, and has been reported to be expressed in a large amount in brain neurons and nervous tissues (Marti et al., Kidney Int., 51: 416-418, 1997) and See Morishita et al. (Neuroscience, 76: 105-116, 1997). In addition, EPO, along with other cytokines and hormones, alone or in combination, may be effective in increasing cell survival or protecting nerves in ischemic lesions and animal models associated with neurological disorders such as Alzheimer's, Parkinson's disease, and Huntington's disease, as well as neurons (US Patent Publication No. US20060063705, US Patent Publication No. US20050197284, US Patent Publication No. US2004025907, US Patent Publication No. US20040018978), and there have been several reports of increased expression and release by hypoxia and other metabolic stress factors (Marti et al. Eur. J. eurosci., 8: 666-676, 1996, Masda et al., J. Biol. Chem., 268: 11208-11216, 1993 and Masda et al., J. Biol. Chem., 269: 19488-19493, 1994). International Publication No. WO 00/61164 also discloses a method of protecting or increasing the function of excitatory tissues, namely the central nervous system, heart and retina, by infusion of a receptor activity modulator of EPO into a mammal.

In this regard, the present inventors relieve the clinical course of ALS disease, which rapidly worsens when mesenchymal stem cells and erythropoietin (EPO) are administered to animal models and patients with ALS disease. By confirming that there is a significant therapeutic effect, the present invention was completed.

One object of the present invention is to provide a method for treating motor neuropathy by administering erythropoietin (EPO), an EPO active fragment or an analog thereof, and mesenchymal stem cells to a subject simultaneously or sequentially.

Another object of the present invention is to provide a composition for treating motor neuron disease, including erythropoietin (EPO), an EPO active fragment or an analog thereof, and mesenchymal stem cells.

In one embodiment, the present invention relates to a method for treating motor neuron disease by administering EPO, an EPO active fragment or analog thereof, and mesenchymal stem cells to a subject simultaneously or sequentially.

As used herein, the term “EPO” refers to a substance that binds to an EPO receptor in an individual to express and activate the EPO receptor, and is mediated therefrom to treat and / or prevent motor neuron disease. In general, EPO is produced primarily in the kidneys of an adult's kidney or fetus, and has a function associated with erythropoiesis that carries oxygen to regulate the formation of red blood cells, as well as to control the production of red blood cells. It is known in the brain as an inducible cytokine produced by the brain. In a preferred embodiment of the present invention, EPO has a non-erythropoietic activity that acts primarily for the treatment and prevention of motor neuron disease, as well as functions associated with erythropoiesis. EPO forms useful in the present invention are naturally occurring, synthesized or recombinant forms of erythropoietin (EPO), erythropoietin analogues, erythropoietin analogues, erythropoietin fragments, hybrid erythropoietin molecules, erythropoietin Receptor-binding molecules, glycoprotein hormones produced in the body to regulate blood cell production, and the like, and for convenience herein, "EPO" may be used as a term that includes all of the above useful EPO forms.

As used herein, the term "EPO active fragment" refers to a polypeptide comprising a minimal fragment that performs physiological functions by EPO. The EPO active fragment of the present invention may bind to the EPO receptor to express and activate an EPO receptor, and may be mediated therefrom to prevent and / or treat motor neuron disease. These EPO active fragments may have a higher binding capacity to EPO receptors than human native EPO.

As used herein, the term "EPO analogue" refers to a polypeptide capable of performing various physiological functions mediated through the EPO receptor while having one or more changes in the amino acid sequence encoding the EPO or EPO active fragment. Say. The EPO analogs of the invention may also include site directed mutants with additions, deletions or substitutions of amino acid residues that increase or change glycosylation sites that do not interfere with the secondary or tertiary structures required for biological activity. The EPO analogue may have a higher binding capacity to the EPO receptor than human native EPO.

EPO, EPO active fragment (analog) or analogues thereof of the present invention include, but are not limited to, various forms such as EPO dimers, EPO polymers, EPO isoforms, etc. .

EPO, EPO active fragments or analogs thereof of the present invention can be produced by cell culture, extraction and isolation from animals, including humans, chemically synthesized or genetically recombined vectors and the like. In addition, the EPO of the present invention is preferably human EPO, more preferably recombinant human EPO having high physiological activity.

As used herein, the term "mesenchymal stem cell (MSC)" refers to bone, cartilage, fat, tendon, nerve tissue, fibroblast These are pluripotent progenitors before they are differentiated into cells of specific organs such as fibroblast and muscle cells. These mesenchymal stem cells can be isolated and purified from bone marrow, peripheral nerve blood, umbilical cord blood, periosteum, dermis and tissues of mesodermal origin and the like. As a specific example, femoral head cancellous bone pieces plugs obtained from a patient with degenerative arthritis during hip or knee replacement, and from a normal donor or patient with bone marrow to be harvested for future bone marrow transplantation Inhaled bone marrow or the like. In a specific implementation, autologous mesenchymal stem cells obtained from bone marrow of ALS patients were used.

Mesenchymal stem cells are separated by a number of different mechanical separation methods depending on the source of bone marrow (ie bone fragments, the presence of peripheral blood, etc.) and such separation methods are known. The most critical step in the isolation of mesenchymal stem cells is to use a specially prepared medium. The medium is characterized in that it contains an agent capable of growing mesenchymal stem cells without differentiation and direct attachment of mesenchymal stem cells only to the plastic or glass surface of the culture dish. Mesenchymal stems from other cells present in the bone marrow (ie erythrocytes, white blood cells and other differentiated mesenchymal cells, etc.) by using a medium that allows selective attachment of mesenchymal stem cells present in extremely small amounts in the bone marrow sample. It is possible to isolate the cells.

As a medium for culturing the mesenchymal stem cells of the present invention, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α- Cell-based media such as α-Minimal Essential Medium (MEM), Glass's Minimal Essential Medium (G-MEM), Iscove's Modified Dulbecco's Medium (ISDM), and MSCGM can be used and further, insulin (10ng / mL) ), Growth factors such as hydrocortisone (10nM), EGF (10ng / mL), FGF, NGF, LIF or essential for growth of fetal bovine serum, horse serum, goat serum, human serum, umbilical cord serum, etc. A medium containing serum or the like can be used. In particular, in a specific embodiment of the present invention, a large amount of mesenchymal stem cells were obtained by culturing mesenchymal stem cells without differentiation using a culture medium containing human umbilical cord blood serum developed by the present inventors. Mesenchymal stem cells are, for example, 36 to 38 ℃, 3 to 10% CO ₂ in the medium It can be cultured in the culture conditions maintained.

In the present invention, the cultured mesenchymal stem cells have a mesenchymal stem cell-positive surface marker such as CD105 (or CD105 (SH2)), CD29, CD44, CD73, CD166 and the like at least 50%, preferably at least 90%, more preferably. CD29, CD44, CD73 and CD105 positive surface markers are expressed at least 95%, and mesenchymal stem cell negative surface markers such as CD34, CD45, HLA-DR, CD1a, CD14, CD31, CD80 and the like is 50% or less, preferably 10% or less, more preferably CD34, CD45 and HLA-DR Negative surface markers are characterized by being expressed at 5% or less.

In one specific embodiment, the present invention is a method for treating motor neuron disease by sequentially administering EPO, an EPO active fragment or an analog thereof, and mesenchymal stem cells to a subject. More specifically, EPO, EPO active fragments or analogues thereof are administered to the subject after administration of mesenchymal stem cells, or mesenchymal stem cells after administration of EPO, EPO active fragments or analogs thereof to the subject to exercise neuropathy disease It is about how to treat it.

In another specific embodiment, the present invention relates to a method for treating motor neuron disease by simultaneously administering to a subject a composition comprising EPO, an EPO active fragment or an analog thereof, and a mesenchymal stem cell.

As used herein, the term “individual” refers to any mammal in need of treatment of motor neuron diseases, including humans, primates and livestock, breeding, pet or sport animals such as dogs, horses, cats, sheep, pigs, cattle, and the like. Say In particular, the treatment of the present invention may be desirable in humans.

As used herein, the term "treatment" refers to therapeutic treatment and prophylactic or prophylactic means. Therefore, those in need of treatment include those who already have motor neuron disorder or motor neuron disease, as well as those who want to prevent motor neuron disorder or motor neuron disease.

EPO, EPO active fragments or analogues thereof can be oral, topical (including buccal, sublingual, skin and intraocular administration), parenteral (subcutaneous, intradermal, cerebrospinal, intraperitoneal, intramuscular, Administration via a variety of routes including intravascular and intraarticular administration) or transdermal administration, preferably parenteral administration, and more preferably intrathecal, intraperitoneal, intramuscular, intravascular Can be. For parenteral administration, for example, intravenous administration may be by needle or compatible means, and may contain a pharmaceutically acceptable carrier. The composition of the present invention is preferably administered via an intravenous route, in an appropriate dosage, and in liquid form. For oral administration, for example, the compositions of the present invention can be administered in a liquid or solid form with a suitable carrier.

Mesenchymal stem cells, or compositions containing EPO, EPO active fragments or analogues and mesenchymal stem cells, may be topical (including buccal, sublingual, dermal and intraocular), parenteral (subcutaneous). Administration, including intradermal, intramuscular, intravascular and intraarticular) or transdermal administration, preferably parenterally. The most preferred method of parenteral administration is to administer the mesenchymal stem cells or the mixed composition intrathecalally. In addition, the mesenchymal stem cells or the mixed composition can be administered to a subject by suspending in a suitable diluent, which is used as a use to protect and maintain the cells, and to facilitate the use when transplanted into the desired brain tissue. The diluent may include physiological saline, PBS, HBSS and the like buffer, plasma, cerebrospinal fluid or blood components, preferably saline (normal saline), PBS, HBSS, plasma or cerebrospinal fluid. The present inventors are very effective in the treatment of motor neuron diseases, especially ALS, when the spinal fluid is diluted with a mesenchymal stem cell, or a composition containing an EPO, an EPO active fragment or an analog thereof, and a mesenchymal stem cell. Found that. Injecting mesenchymal stem cells or the mixed composition into the spinal cavity can avoid complications such as the risk of infection and nerve damage caused by direct injection into the spinal cord, and can also be expected by using autologous cerebrospinal fluid. It has the advantage of minimizing side effects and enabling non-invasive treatment.

When sequentially administering EPO, an EPO active fragment or an analog thereof, and mesenchymal stem cells, 5 minutes to 15 hours after administration of either EPO, an EPO active fragment or an analog thereof, or mesenchymal stem cells, preferably 10 The other one may be administered after minutes to 12 hours, and preferably, mesenchymal stem cells may be administered after 5 minutes to 15 hours, preferably 10 minutes to 12 hours after administration of EPO. Such administration may be repeated as necessary. For example, administration may be repeated daily if clinically necessary, or after a suitable time interval, for example every 1-12 weeks, preferably every 3-8 weeks.

When simultaneously administering EPO, an EPO active fragment or an analog thereof, and mesenchymal stem cells, administration of EPO, an EPO active fragment or an analog thereof, and mesenchymal stem cells may be repeated as necessary. For example, administration may be repeated daily if clinically necessary, or after a suitable time interval, for example every 1-12 weeks, preferably every 3-8 weeks.

EPO, an EPO active fragment or analog thereof, and mesenchymal stem cells are administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease, and an effective dose level may include the severity of the disease; The age, body weight, health and sex of the patient; Sensitivity to the drug of the patient; Time of administration, route of administration, and rate of excretion; Duration of treatment; It may be determined according to factors including drugs which are used in combination or coincidental with the composition of the present invention and other factors well known in the medical field. In a specific embodiment, the dose of EPO of the invention is 200 to 10,000 units (IU) / weight (kg), preferably about 200 to 5,000 units / weight (kg), per dose for animals other than humans. Preferably from 200 to 2,000 units / kg body weight, most preferably from 500 to 1,000 units / kg body weight. It can also be administered in humans at 20,000 to 45,000 units, preferably 25,000 to 40,000 units, more preferably 30,000 to 40,000 units per dose, irrespective of body weight. The dose of mesenchymal stem cells varies over a wide range and depends on the individual requirements of each particular case. Generally, for parenteral administration, it is common to administer from about 0.01 to about 5 million cells per Kg of recipient body weight.

EPO, EPO active fragments or analogs thereof, and / or mesenchymal stem cells are administered to the individual simultaneously or sequentially, and then factors which promote differentiation and growth into neurons are further carried out by the method of administration described above. It can be administered at a concentration of / ml. Such additional factors include, for example, growth factors such as FGF, EGF, IGFs, TGFs, NGF, etc .; Neurotrophic factors such as BNF and CTF; Peptide radiation drugs; Antisense drugs; Antibodies to biologically active agents; Immunosuppressive agents such as cyclosporin; Cytokines such as interleukin, interferon and LIF; GSK (Glycogen Synthase Kinase 3 beta) inhibitors.

In addition, the present invention provides for the attachment of EPO, EPO active fragments or analogs thereof, and / or mesenchymal stem cells through the endothelial cell barrier before or simultaneously with administration of EPO, EPO active fragments or analogs thereof, and / or mesenchymal stem cells. 80-120 ml (20% manitol) can be administered by the administration method described above using mannitol, which is a substance known to facilitate cell protection.

As another aspect, the present invention relates to a composition for treating motor neuron disease, including EPO, EPO active fragment or analogue thereof, and mesenchymal stem cells.

In the present invention, "composition for the treatment of motor neuron disease" refers to a composition for treating a disease causing disorders in the function of motor neurons. The motor neuropathy disease refers to ALS, progressive bulbar palsy (PBP), primary lateral sclerosis (PLS), and progressive muscular atrophy (PMA). In particular, the composition of the present invention is preferably a composition for treating amyotrophic lateral sclerosis (ALS).

The composition of the present invention may be used in admixture with a pharmaceutically acceptable carrier or excipient or diluted with a diluent according to conventional methods. Examples of suitable carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, undecided Vaginal cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The composition may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like.

Compositions of the present invention may be formulated using methods well known in the art to provide rapid or delayed release of the active ingredient after administration to a mammal. The formulations may be in the form of tablets, powders, pills, sachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders and the like. The formulation will be convenient in a single dosage form. Most preferred in the formulations of the invention are injections.

The composition of the present invention has an excellent effect in the treatment of motor neuron diseases. That is, in the evaluation of the exercise capacity of the rats to which the composition of the present invention was administered, the exercise ability was improved by 100% or more, compared to the rats to which the composition of the present invention was not administered. Compared with 30% of athletic performance was improved. In clinical trials, respiratory, pronunciation, and swallowing ability was remarkably improved in patients administered the composition of the present invention, prolonged standing posture was maintained, and insomnia was reduced. The change lasted for some time. Therefore, the composition of the present invention can be effectively used for the treatment of motor neuron diseases.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely to aid the understanding of the present invention, and the scope of the present invention is not limited to the following examples.

Example  1: after the patient's bone marrow is taken Bone marrow Mesenchymal stem cells  Isolation and In Vitro Culture

1-1. ALS  Patient Screening Criteria

The effect of EPO treatment on ALS patients was assessed by Revised El Escorial Criteria in two patients who were clinically confirmed as ALS (Probable ALS and above). At the first hospital stay, the patient's overall pre-treatment status is assessed and screened using functional rating scale, appeal score, norris scale, and neurophysiologic index. It was.

1-2. Bone marrow Mesenchymal stem cells  Isolation and In Vitro Culture

30 mL of bone marrow was collected from a posterior superior iliac spine (PSIS) of the ALS patient selected in Example 1-1 using a Jamshidi needle, and then sterilized and treated with heparin. Transferred to the tube. The bone marrow contained in the tube was moved to a clean room and then the process was performed. Bone marrow containing tubes were centrifuged at 700 x g for 10 minutes at room temperature to store approximately 10 mL separately for stability testing. 20 mL of remaining bone marrow was diluted 1: 3 with DMEM (Dulbecco's Modified Eagle Medium, Gibco) and layered onto a previously prepared Histopaque (density 1.077 g / mL, sigma) layer and 30 to 400 × g. Centrifuged at room temperature for minutes. Monocytes (mononuclear cell) layer was separated and DMEM was added to harvest monocytes by centrifugation at room temperature for 5 minutes at 400 × g. The harvested monocytes were washed twice with DMEM by centrifugation at 400 × g for 5 minutes at room temperature. After washing, monocytes were inoculated into a culture flask (175 cm ² ) using DMEM / F12 (Gibco) containing 10 ng / mL of insulin, 10 nM of hydrocortisone, 10 ng / mL of EGF, and 5% human umbilical cord blood serum. And incubated at 37 ° C. for 5 days while maintaining 5% CO ₂ . After 5 days, the cells that did not adhere to the bottom of the flask were removed by replacing with fresh medium, and then the medium was changed, passaged and propagated every 3 days. Cells at the bottom of the flask were separated using 0.125% trypsin-EDTA (Gibco) and washed three times with D-PBS (Dulbecco's Phosphate-buffered Salines, Gibco), leaving only the cell layer.

Example  2 : Amyotrophic Lateral Sclerosis Model  Rat ( SOD1 mutant transgenic mice model ) In EPO Wow MSC Treatment of merging

Human EPO used in the experiment was purchased from Dong-A Pharmaceutical (rhEPO; Eporon Co .; 4,000 IU), and Amyotrophic Lateral Sclerosis model rat (B6SJL-Tg (SOD1-G93A) 1Gur / J mouse) was used in the Jackson laboratory. 4 weeks of age from Bar Harbor, ME, USA, and used to experiment after breeding up to 12 weeks of age. The experimental group was divided into control group administered with saline, EPO group administered with EPO alone, MSC group administered with MSC alone, and combined administration group administered with EPO and MSC, and consisted of 5 mice in each group. The EPO group was intraperitoneally administered 1 unit (IU) of EPO per gram of mouse weight, and the MSC group was injected 10 ³ MSCs in saline solution per mouse into the spinal cord. The EPO and MSC combination group injected MSC into the spinal cavity two hours after intraperitoneal administration of EPO. Each group was administered twice, two weeks apart, and the control group was intraperitoneally administered only saline. Each group compared and observed symptoms caused by amyotrophic lateral sclerosis such as limb tremor, muscle spasm, hyperreflection, limb muscle weakness, and limb muscle paralysis, and using Rotarod (Ugo Basile; Comerio-Varese, Italy). An exercise test was performed to compare the effects.

The earliest symptom of limb tremor, muscle spasms, and hyperreflection, the initial symptoms of amyotrophic lateral sclerosis, was measured at the time of onset of symptoms, and when the rats were unable to rise spontaneously from the ground within 30 seconds. After measuring the period until then, it was sacrificed. The change in weight was also measured periodically.

Changes in athletic performance using Rotarod were measured in a universal manner, and were trained using a Rotarod machine for a week before the dose. As the index to be measured, the holding time on the rotarod and the rotation speed when falling were used.

The results are shown in FIG. 1. In the evaluation of the motor performance by rotarod, rats treated with EPO and MSC were improved by 30% compared to the group treated with each other alone and over 100% compared to the saline-treated control group. Increased over 15 days (FIG. 1).

Example  3: ALS In the patient MSC  Treatment effect

3-1. For treating patients Origin of autologous bone marrow MSC Standards and tests

Mesenchymal stem cells (MSC) cells were isolated and cultured in the same manner as in Example 1. The stability criteria and tests were applied to the human body for therapeutic purposes as follows. The cultured cell appearance should show fibroblast-like morphology. Cell culture solution and cells obtained 72 hours prior to the final collection date of the cell was entrusted to Samkwang Medical Foundation (Seoul, Korea) to perform Gram staining and Ordinary culture in liquid medium to check the growth of bacteria. In addition, human immunodificency virus, Hepatitis B virus, Cytomegalovirus, and Parvo B-19 virus were tested for PCR (Polymerase chain reaction) and RT. -Cryptically confirmed by the Reverse Transcription-Polymerase Chain Reaction (PCR) assay, only those that were negative were used as cell therapy. Also, 72 hours before the final collection of cells, the cells were commissioned to the Samkwang Medical Foundation for chromosomal karyotype and only those identified as normal karyotype were used as cell therapy. To confirm the presence of mycoplasma infection, PCR was performed using the cell culture medium and cells obtained 72 hours prior to the final collection of cells, using the method described in the MycoSensor ^TM PCR assay kit (Stratagene). Only those used were used as cell therapy. To confirm the presence of endotoxin, the cell culture solution obtained 72 hours before the cell collection date was subjected to gelation by the method described in the Limulus Amebocyte Lysate Endosafe ^R kit (Charles river endosafe). Only cells identified as 0.2 Endotoxin Units (EU) or less per 1 mL of culture were used as cell therapy. Flow cytometry was performed to analyze markers using cells obtained 72 hours prior to cell collection. CD29 ⁺ (Serotec), CD44 ⁺ (Dako), CD73 ⁺ (BD Biosciences), CD105 ⁺ (Serotec), Only cells with at least 90% of CD34 ⁻ (Serotec), CD45 ⁻ (Dako), and HLA-DR ⁻ (Serotec) expression were used as cell therapy. Final harvest of cells satisfying the above conditions is carried out by Trypan blue exclusion assay, and the living cells should be 70% or more of the total cell number, and the whole cells using hemacytometer. When counting, only 2-6 × 10 ⁷ cells per vial were used as cell therapy. When all of these conditions were met, it was deemed suitable for the final product, a cell therapy product.

3-2. ALS In the patient MSC Wow EPO  Merged administration

Cells grown in Example 3-1 were washed three times with D-PBS (Dulbecco's Phosphate-buffered Salines, Gibco), leaving only the cell layer. After determining the individual cell dose according to body weight, 5 mL of cerebrospinal fluid (CSF) and rhEPO were mixed with the harvested MSCs, mounted in a sterile syringe, and then treated through the cerebrospinal cavity. The hospitalization plan and the treatment plan are shown in Table 1 and FIG. 2 below.

Treatment plan at hospitalization Admission Contents Person in charge One ^st Admission Day1 1. Consent Form 2. Neurological Examination 3. ALS Clinic Patient Sheet 4. Functional Scale Record 5. CBC, Electrolyte, U / A, EKG, Chest PA SMA, TFT, PFT, Neurophysiology Full-time doctor, Professor in charge Full-time doctor, Physician Specialist Nurse Specialist Nurse Physician Day2 1. Primary bone marrow collection 2. Observation of side effects after bone marrow collection Full-time, Professor Day3 1. Observation of side effects and discharge from bone marrow The attending physician 2 ^nd Admission Day1 1. Neurological examination 2. ALS Clinic Patient sheet recording 3. Functional scale sheet recording 4. CBC, Electrolyte, U / A, EKG, Chest PA SMA, TFT, PFT, Neurophysiological examination 5. Second bone marrow extraction Full-time Doctor, Specialist Nurse Specialist, Full-time Doctor Day2 1. Primary rhEPO treatment 2. Observation of side effects after treatment Full-time, Professor in charge Full-time, Professor in charge Day3 1. Primary MSCs treatment 2. Observation of side effects and discharge after treatment The attending physician 3 ^rd Admission Day1 1. Neurological examination 2. ALS Clinic Patient sheet recording 3. Functional Rating Scale sheet recording 4. CBC, Electrolyte, U / A, EKG, Chest PA, SMA, TFT, PFT, Neurophysiology Full-time, Physician Specialist Nurse Specialist Physician Physician Day2 1. Secondary rhEPO treatment 2. Side effects observed after treatment Full-time, Professor in charge Full-time, Professor in charge Day3 1. Secondary MSCs treatment 2. Observation of side effects and discharge after autologous bone marrow stem cell treatment The attending physician

Functional ratings during the first hospitalization (Functional rating scale), Appel scores (Appel score), Norris points (Norris scale), neurophysiological index (Neurophysiologic index) such as harvesting bone marrow after evaluating the overall treatment before the patient's condition to If it is determined that the bone marrow was harvested using the method specified above. After discharge for 1 to 2 days after checking the side effects of bone marrow extraction.

At the second hospitalization , the patient's condition is re-evaluated with functional rating scale, Appel score, Norris scale, Neurophysiologic index, etc. Was carried out. The day after bone marrow extraction, 35,000 IU of rhEPO (Dong-A Pharmaceutical, Eporon, 4,000 IU) was diluted in 50 ml of saline and injected intravenously for 30 minutes. The day after rhEPO treatment, the cerebrospinal fluid was secured by a cerebrospinal fluid puncture, and the MSCs prepared by the above-mentioned method were diluted in the autologous cerebrospinal fluid (5ml) secured in advance and then injected through a dural puncture. Mannitol immediately after the introduction of MSCs (Samsung Pharmaceutical, 20% Mannitol ™ 100ml / B) intravenous administration and position change were performed. The method is as follows.

* Mannitol  Dosing method

Foley catheter insertion, Frequent Input / Output

1) mannitol (overseas pharmaceutical, 20% Mannitol ™, 100ml / B) dose

(1) Primary capacity: Determine 20% mannitol amount by calculating 0.5g / kg. For example, for a 50 kg patient, a 25 g dose is required, so 125 cc of 20% mannitol is determined.

(2) Secondary capacity: 0.25g / kg

(3) 3rd capacity: 0.125g / kg

2) Intravenous infusion (20-30 minutes) was performed three times a total of 12 hours at the same dose.

* Posture variation law (Postprocedure position change )

1) position

(1) Lateral decubitus position with hip and leg held high (30 degrees)

(2) lateral decubitus position with hip and leg held high (30 degrees)

(3) supine position with hip and leg held high (30 degrees)

(4) supine position-flat position

2) Each posture was repeated three times with 30 minute intervals (total 6 hours).

After discharge for 2 to 3 days after confirming the side effects.

At the third hospitalization , the patient's condition was reevaluated with a functional rating scale, Appel score, Norris scale, and Neurophysiologic index. On day 2 of hospitalization, 35,000 IU of rhEPO (Dong-A Pharmaceutical, Eporon Co., 4,000 IU) was diluted in 50 ml of saline and injected intravenously for 30 minutes (same as treatment at the second hospitalization). The day after rhEPO treatment, the cerebrospinal fluid was secured by a cerebrospinal fluid puncture, and the MSCs prepared by the above-described method were diluted in the secured cerebrospinal fluid (5ml) and injected through a dural puncture. Immediately after the injection of MSCs, mannitol (Monoitol, 20% Mannitol ™, 100 ml / B) was administered intravenously and position change (same as treatment at the second hospitalization).

Afterwards, outpatient follow-up was conducted every month to measure and observe the clinical changes of the patients. The results are shown in FIGS. 3 and 4.

Symptoms of muscle weakness, such as dysphagia, which had been progressing since January 2005, improved after the first treatment. The improvement was maintained for about 2 to 3 months, and no adverse effects were observed. In addition, when compared with the predicted clinical course graph (dotted line) inferred from the past, the clinical course could be observed after HYNR-CS1 + rhEPO treatment, and thus the therapeutic effect was evaluated. In addition, various clinical improvements and changes such as decreased sputum and decreased saliva were observed in addition to the delayed effects of muscle weakness symptoms (FIGS. 3 and 4).

The composition of the present invention can be effectively used for the treatment of motor neuron diseases, in particular amyotrophic lateral sclerosis (ALS).

Claims (17)

A method of treating motor neuron disease by administering EPO, an EPO active fragment or an analog thereof, and mesenchymal stem cells to a subject simultaneously or sequentially. The method of claim 1, wherein the mesenchymal stem cells express at least 90% of CD105 (SH2), CD29, CD44, CD73, and CD166 positive surface markers. The method of claim 2, wherein the mesenchymal stem cells are CD29, CD44, Wherein at least 95% of the CD73 and CD105 positive surface markers are expressed. The method of claim 1, wherein the mesenchymal stem cells express less than 10% of the negative surface markers of CD34, CD45, HLA-DR, CD1a, CD14, CD31, and CD80. The method of claim 4, wherein the mesenchymal stem cells are CD34, CD45 and HLA-DR 5% or less expressing the negative surface marker. The method of claim 1, wherein the motor neuron disease is amyotrophic lateral sclerosis (ALS). The method of claim 1, wherein the motor neuropathy disease is progressive bulbar palsy (PBP), primary lateral sclerosis (PLS), and progressive muscular atrophy (PMA). EPO, EPO active fragment or analogues thereof, and a composition for treating motor neuron diseases, including mesenchymal stem cells. The composition of claim 8, wherein the mesenchymal stem cells express at least 90% of CD105 (SH2), CD29, CD44, CD73, and CD166 positive surface markers. The method of claim 9, wherein the mesenchymal stem cells are CD29, CD44, And at least 95% of the CD73 and CD105 positive surface markers. The composition of claim 8, wherein the mesenchymal stem cells express up to 10% of the negative surface markers of CD34, CD45, HLA-DR, CD1a, CD14, CD31, and CD80. The method of claim 11, wherein the mesenchymal stem cells are CD34, CD45 and HLA-DR A composition that expresses 5% or less of the negative surface marker. The composition of claim 8, wherein the erythropoietin, erythropoietin active fragment or analog thereof is comprised between 20,000 and 45,000 unity (IU) in the total composition. The composition of claim 13, wherein the erythropoietin, erythropoietin active fragment or analog thereof is comprised between 30,000 and 40,000 units (IU) in the total composition. The composition of claim 8, wherein the motor neuropathic disease is amyotrophic lateral sclerosis (ALS). The composition of claim 8, wherein the motor neuropathy disease is progressive bulbar palsy (PBP), primary lateral sclerosis (PLS), and progressive muscular atrophy (PMA). The composition of claim 8, wherein said composition is used as an injection for parenteral administration.

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