WO2012060517A1

WO2012060517A1 - Pharmaceutical composition for prevention and treatment of neurologic disorders containing cd45-expressed mesenchymal stem cells as active ingredient

Info

Publication number: WO2012060517A1
Application number: PCT/KR2011/000253
Authority: WO
Inventors: 이권행; 지보근; 진영준
Original assignee: 하이스템(주)
Priority date: 2010-11-05
Filing date: 2011-01-13
Publication date: 2012-05-10

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of neurologic disorders containing CD45-expressed mesenchymal stem cells as an active ingredient. The mesenchymal stem cell of the invention can be useful for the prevention and treatment of neurologic disorders, especially Alzheimer's disease which is caused by amyloid beta.

Description

【Specification】

[Name of invention]

Pharmaceutical composition for neurological disease prevention and treatment comprising CD45-expressing mesenchymal stem cells as an active ingredient

Technical Field

The present invention relates to a pharmaceutical composition for preventing and treating neurological diseases including mesenchymal stem cells.

Background Art

Alzheimer's disease is a disease caused by the accumulation of amyloid beta protein in the brain and the destruction of brain cells. It is a serious disease that causes language and cognitive impairment due to aging. Alzheimer's disease progresses step by step, gradually destroying memory, reasoning, judgment, language, and even the simplest of work abilities, and eventually moving from human life to animal life because it cannot control emotions. There is no cure for Alzheimer's disease yet. Only drugs that can help alleviate the symptoms are used in clinical practice. However, the effectiveness of these drugs in real patients is limited. About half of the patients are failing initial drug therapy, and even diarrhea treatments show only mild relief. Therefore, there is an urgent need for new breakthrough treatments to meet the medical needs that are not satisfied with current drug treatments, and the economic and social ramifications of the development of Alzheimer's disease drugs can be expected to be very large. As Alzheimer's disease progresses, it is known that there is almost no treatment because the cortex and hippocampus are lost.

The pathogenesis of Alzheimer's disease is known to produce amyloid vinbeta (Αβ) by secretase, and amyloid-beta accumulates in the brain as amyloid plaques. Brain areas where plaques accumulate typically have decreased numbers of synapses and impaired neurites, suggesting that amyloid-beta damages synapses and neurites. Therefore, studies on the development of beta-secretase and / or gamma-secretase inhibitors that produce amyloid beta protein for the treatment of Alzheimer's disease, proteolytic enzymes that degrade the accumulated amyloid beta protein, and the acetylcholine Research into the development of inhibitors of acetylcholine esterase, which is responsible for degradation, has been intensified.

<4> Mesenchymal stem cells (MSCs) are bone, cartilage, fat, and muscle cells It is a multipotent stem cell having the ability to differentiate into various ectoderm cells or ectoderm cells such as neurons. Recently, as mesenchymal stem cells are known to have the potential to differentiate into glial cells in the brain, development of a method of differentiating mesenchymal stem cells into neurons has been attempted.

Of the mesenchymal stem cells, bone marrow-derived mesenchymal stem cells can be obtained from the patient, and autologous transplantation has an advantage in clinical use because there is no problem with immune rejection reaction. However, the harvesting of bone marrow-derived mesenchymal stem cells requires several stages of treatment, and the procedure is complicated, and there is a problem in that time, mental, and physical pains and burdens are collected on the subjects. Umbilical cord blood-derived mesenchymal stem cells can be obtained from simple umbilical cords that are thrown away during the delivery process, and it is easy to obtain donors through the already established infrastructure because the umbilical cord storage industry is activated. It is easy to secure and mesenchymal stem cells obtained from umbilical cord blood derived from taga has the advantage of having immunological stability because it does not cause immune reaction after transplantation.

Therefore, the present inventors studied new treatments for neurological diseases, particularly Alzheimer's disease, and the mesenchymal stem cells expressing CD45, a positive surface marker of hematopoietic stem cells, were treated with amyloid beta, an Alzheimer's-derived hippocampal tissue. The present invention was completed by confirming suppression of necrosis of cells.

[Detailed Description of the Invention]

[Technical problem]

The present invention provides a pharmaceutical composition for preventing and treating neurological diseases including mesenchymal stem cells.

Technical Solution

<8> The pharmaceutical composition for preventing and treating neurological diseases of the present invention is characterized by including mesenchymal stem cells expressing CD45 as an active ingredient.

In the pharmaceutical composition for preventing and treating neurological diseases of the present invention, the mesenchymal stem cells are characterized by expressing CD13, CD14, CD29, CD44 and CD105 which are positive surface markers of mesenchymal stem cells.

In the pharmaceutical composition for preventing and treating neurological diseases of the present invention, the mesenchymal stem cells are characterized by not expressing CD31, CD34, CD90, and CD117, which are positive surface markers of hematopoietic stem cells.

<ιι> The mesenchymal stem cells in the pharmaceutical composition for preventing and treating neurological diseases of the present invention Is characterized in that the cord blood-derived mesenchymal stem cells.

The pharmaceutical composition for preventing and treating neurological diseases of the present invention is characterized by suppressing necrosis of neural tissues by amyloid beta.

In the pharmaceutical composition for preventing and treating neurological diseases of the present invention, the neurological disease is characterized in that any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis and mania.

Advantageous Effects

The pharmaceutical composition comprising the mesenchymal stem cells expressing CD45 of the present invention as an active ingredient is effective in the treatment of neurological diseases, especially Alzheimer's disease.

[Brief Description of Drawings]

1 is a colony photograph formed of a collection of human umbilical cord blood-derived mesenchymal stem cells. Figure 2 shows the immunological characteristics of CD31, CD34, CD90, CD117 and CD45, the positive surface markers of hematopoietic stem cells and negative surface markers of mesenchymal enjoyment cells.

Figure 3 shows the immunological properties of CD13, CD14, CD29, CD44 and CD105, the positive surface markers of mesenchymal stem cells.

Figure 4 is a PI staining picture that can confirm the necrosis inhibitory effect of the hippocampal tissue according to the administration of the mesenchymal stem cells of the present invention in the dementia model induced by necrosis by administering amyloid beta to the hippocampal tissue of the rat.

5 is a graph showing the effect of inhibiting hippocampal tissue necrosis caused by miloid beta according to the concentration of mesenchymal stem cells.

[Best form for implementation of the invention]

In the present invention, "mesenchymal stem cell (MSC)" is bone (bone), cartilage (cartilage), fat (fat), tendon (tendon), nerve tissue (nerve tissue), fibroblast (fibroblast) and Pluripotent progenitors before they are differentiated into cells of specific organs such as muscle cells. These mesenchymal stem cells can be isolated and purified from bone marrow, peripheral nerve blood, re-blood, periosteum, dermis and tissues of mesodermal origin.

In addition, in the present invention, the neurological disease may be selected from the group consisting of Alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis and mania as diseases caused by abnormalities of amyloid beta.

Amyloid beta (Αβ) in the present invention means a major component of amyloid plaques present in the brain of patients with Alzheimer's disease. Amyloid Beta (Αβ) May be a peptide consisting of amino acids derived from the C-terminus of the amyloid precursor protein (APP), a transmembrane glycoprotein. The Αβ may be produced by the continuous action of β- and Υ-setletase from ΑΡΡ. The amyloid beta (Αβ) may be derived from mammals. For example, it may be derived from human or mouse. <23> As a medium for culturing the mesenchymal stem cells of the present invention, DMEM (Dulbecco's

Modified Eagle's Medium (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, Q-MEM (Q -Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential) Medium, ISDM (Iscove's Modified Dulbecco's Medium) and MSCGM cell base media can be used, and further, insulin base (10 ng / mL) and hydrocollet zone (first line) ( ： 0 ^ 1301) (10110 ， EGF

(lOng / mL), a medium containing growth factors such as FGF, NGF, LIF, or serum essential for the growth of fetal bovine serum, horse serum, goat serum, human serum, umbilical cord serum, or the like may be used.

<24> A pharmaceutical product for preventing or treating a disease containing mesenchymal stem cells of the present invention may further include a pharmaceutically acceptable additive in addition to the active ingredient, according to a conventional method in the pharmaceutical field It may be formulated in a unit dosage form suitable for administration in the body. Formulations suitable for this purpose include parenteral or formulations for topical administration. For example, it can be used parenterally in the form of injections of sterile solutions or suspensions using water or other pharmaceutically acceptable solvents as necessary. For example, pharmaceutically acceptable carriers or media such as sterile water, saline, vegetable oils, emulsifiers, suspending agents, surfactant surfactants, stabilizers, excipients, vehicles, preservatives, binders, etc. In combination, which may be formulated by popularizing to a generally accepted unit dosage form.

The formulations may be administered parenterally according to conventional methods. Such parenteral administration includes topical or systemic administration. The local administration may be to administer directly to or around the lesion, for example, to the brain or spinal cord, which is the lesion, or directly to or around the site. Such systemic administration includes administration to spinal fluid, veins or arteries. The spinal fluid includes cerebrospinal fluid. The artery may be a site for supplying blood to the lesion. In addition, the administration can be made according to the method described, for example, in Douglas Kondziolka, Pittsburgh, Neurology, vol. 55, pp. 565-569, 2000. That is, two of the targets first The bone can be cut into peas of about i _{cm in} diameter and then infused with HBSS (Hank's balanced salt solution) and mesenchymal stem cell solution. At this time, the injection of the cell solution can be made using a syringe with a long needle, and (stereotactic frame) for inserting the desired cell solution into the coordinates inside the brain.

The daily dose of the mesenchymal stem cells may be lxlO ⁴ to lxlO ⁷ cells / kg body weight ᅳ may be administered once or divided into several times. However, the actual dose of mesenchymal stem cells of the present invention, for example, umbilical cord blood-derived mesenchymal stem cells, may be increased or decreased in consideration of various related factors such as the disease to be treated, the severity of the disease, the route of administration, and the weight, age, and sex of the patient. This is possible.

<27> Mesenchymal stem cells of the present invention are mesenchymal stem cell positive surface markers CD13, CD14,

CD29, CD44 and CD105 are expressed, and among CD31, CD34, CD90, CD117 and CD45, which are negative surface markers of mesenchymal stem cells and positive surface markers of hematopoietic stem cells, CD31, CD34, CD90 and CD117 are not expressed, Specifically, CD45 is expressed. Expression of positive surface markers in the cells of the present invention includes expression of at least 90%, preferably at least 95%, of positive surface markers in the cells, and the fact that negative surface markers are not expressed is 10% of negative surface markers. Hereinafter, the meaning including preferably expressed in 5% or less.

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.

[Form for implementation of invention]

Example 1 Isolation and Culture of Cord Blood-derived Mesenchymal Stem Cells

<3i> Blood samples from cord blood were collected from DMEM (Dulbecco's Modified Eagle Medium,

Wei GENE, Republic of Korea) in a 1: 1 ratio and then centrifuged at 300xg for 30 minutes on a Ficoll-paque (GE Healthcare, UK) at a ratio of 1: 2. After centrifugation, the buffy coat consisting of the mononuclear cell layer (mononuclear cell layer) was diluted with DMEM at a ratio of 1: 1, and CD45 positive stem cells were obtained using CD45 mAb (DIN0NA, Republic of Korea). After removing the supernatant, the precipitated cells were washed twice with DPBS (Dulbecco's Phosphate Buffered Saline, WelGENE).

The obtained mononuclear sedimentary cell layer was 20% in DMEM medium containing antibiotics (1000 penicillin, 1000 fg / mt streptomycin, WelGENE) and 2 mM glutamine (Glutamine, WelGENE). As a cell growth factor with fetal bovine serum (WBS), fetal bovine serum (FBS), M— CSF (Macr ophage-Co 1 ony Stimulating Factor) 10 ng /, G-CSF (Gr anu 1 ocy t e-Co 1 ony Stimulating Factor ) 10 ng / ml, GM_CSF (Granulocyte Macrophage-Colony Stimulating Factor) 10 ng / ιη ^, Ib1 1 a (Inter leukin-1 alpha) 10 ng / iirf, Ib 一 3 (Inter leukin-3) 10 ng / m £ and Stem Cell Factor 50 ng / i or GM_ CSF (Granulocyte Macrophage® Colonyy Stimulating Factor) 10 ng /, Ib I 3 (Inter leukin-3) 10 ng /, Stem Cell Factor (SCF) 50 ng / t and Flt3L (Fms-like Tyrosine kinase 3 Ligand) 50 ng / was added for 5 days to remove the floating cells, only adherent cells were incubated for 20 days with DMEM containing 20% fetal calf serum. <33> Figure 1 is a photograph showing the human cord blood-derived mesenchymal stem cells obtained by the above method.

<34>

Experimental Example 1 Identification of Cell Surface Antigen Characteristics of Mesenchymal Stem Cells

In order to investigate the cell surface antigen characteristics of mesenchymal stem cells (hereinafter, also referred to as 'histem cells') isolated and cultured in Example 1, the cell surface antigens were analyzed by fluorescence activated cell sorting (FACS). The results are shown in FIGS. 2 and 3, and the results are summarized in Table 1.

2 is a positive surface marker of hematopoietic stem cells and a negative surface marker of mesenchymal stem cells

The immunological characteristics of CD31, CD34, CD90, CD117, and CD45 are shown, showing positive response to CD45 among surface antigens.

3 shows the positive surface markers of mesenchymal stem cells CD13, CD14, CD29, CD44 and

The immunological characteristics of CD105 are shown, indicating positive reaction in all surface antigens, indicating that the cells of the present invention are mesenchymal stem cells.

<39>

<40> [Table 1]

Experimental Example 2 Inhibition of Necrosis of Hippocampal Tissue Cells by Amyloid Beta

<42> Hippocampal tissues of the rat brain are sliced and cultured, and amyloid beta, a dementia-inducing substance, is known to kill the hippocampal tissues of the brain. Inhibition of necrosis of hippocampal tissue cells by amyloid beta was carried out by modified methods such as Chung (Journal of biological chemistry vol 281, No.29 pp20315-20325, 2006). Amyloid beta (Αβ25-35) was added to culture medium containing hippocampal slices of rat brain at 0, 2.5, and 5 μΜ concentrations, and stained by ΡΙ staining to stain dead cells. It was confirmed that the color became stronger. However, administration of 1 x 10 ⁵ cells / ml of mesenchymal stem cells expressing CD45 of Example 1 together with amyloid beta almost eliminated the red color due to cell necrosis and suppressed necrosis of hippocampal tissue cells. It could be confirmed (Fig. 4).

5 is a graph showing the effect of inhibiting hippocampal tissue necrosis caused by amyloid beta according to the concentration of mesenchymal stem cells, the mesenchymal in which CD45 of Example 1 is expressed while the amyloid beta is treated with 5 μΜ. Stem cells treated with 1 × 10 ³ cells / ml were also shown to inhibit necrosis of hippocampal tissue cells by amyloid beta.

Claims

[Range of request]

[Claim 1]

Pharmaceutical composition for neurological disease prevention and treatment comprising CD45-expressing mesenchymal stem cells as an active ingredient.

[Claim 2]

The pharmaceutical composition for preventing and treating neurological diseases according to claim 1, wherein the mesenchymal stem cells express CD13, CD14, CD29, CD44, and CD105 which are positive surface markers of mesenchymal stem cells.

[Claim 3]

The pharmaceutical composition for preventing and treating neurological diseases according to claim 1 or 2, wherein the mesenchymal stem cells do not express CD31, CD34, CD90, and CD117, which are positive surface markers of hematopoietic stem cells.

[Claim 4]

4. The pharmaceutical composition for preventing and treating neurological diseases according to claim 3, wherein the mesenchymal stem cells are umbilical cord blood-derived mesenchymal stem cells.

[Claim 5]

The pharmaceutical composition for preventing and treating neurological diseases according to claim 4, wherein the necrosis of neural tissues by amyloid beta is suppressed.

[Claim 6]

The pharmaceutical composition of claim 5, wherein the neurological disease is any one selected from the group consisting of Alzheimer's disease, Parkinson's disease, depression, epilepsy, multiple sclerosis and mania.