KR20150059671A - Pharmaceutical Compositions for Preventing or Treating Parkinsonism Comprising Mesenchymal Stem Cells Derived from Human Bone-Marrow - Google Patents

Abstract

In the present invention, provided is a pharmaceutical composition for preventing or treating Parkinson′s syndrome including mesenchymal stem cell derived from human bone marrow as active ingredients wherein the mesenchymal stem cell derived from human bone marrow is allowed to protect neurons from apoptosis and accordingly has effects of preventing or treating multiple system atrophy.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition for preventing or treating Parkinson's syndrome comprising human bone marrow-derived mesenchymal stem cells as an active ingredient. The pharmaceutical composition for preventing or treating Parkinson's syndrome includes human bone marrow-derived mesenchymal stem cells,

The present invention relates to a pharmaceutical composition for preventing or treating Parkinson's syndrome comprising human bone marrow-derived mesenchymal stem cells as an active ingredient.

Multisystemic atrophy is a rare progressive neurological disorder characterized by the combination of various symptoms. People with this disorder may experience symptoms similar to Parkinson's disease, such as cerebellar ataxia and autonomic failure such as heart rate, blood pressure, sweating, bowel and bladder control . The exact cause of multi-system atrophy is not yet known, and Shy-Drager syndrome, Striatonigral degeneration, sporadic olivopontocerebellar atrophy are included in multi-system atrophy . Multiple system atrophy affects men and women equally. Some papers report that men are slightly more affected than women. The onset age of multiple system atrophy is approximately between 52 and 55 years, and the incidence of this disease in the United States is 4.4 cases per 100,000. In the United States, multi-system atrophy is estimated at 25,000-100,000, which is difficult to distinguish from Parkinson's and other diseases, making it difficult to determine the actual frequency of multi-system atrophy in the general population. In one study, 10% of people diagnosed with Parkinson's disease were later found to have multiple system atrophy.

The exact cause of multi-system atrophy is unknown, but some researchers suggest that environmental or genetic factors play an important role in this disease. However, environmental associations and family cases have not been identified and more research is needed. Multidirectional atrophy is caused by the progressive loss of neurons (neurons), which are various structures of the brain. Striatonigral degeneration is the destruction of communication between nerve cells in the two structures of the brain known as the striatum ≪ / RTI > These two structures are involved in exercise and balance. Olivopontocerebellar atrophy is caused by specific structures of the brain known as cerebellum, pons, and inferior olives. The cerebellum plays an important role in maintaining posture and balance as well as coordinating autonomous movements. . The brain is part of the brainstem, which contains important nerve pathways between the cerebrum, cerebellum and spinal cord. The lexicon acts as a message relay point between these structures. The bottom olive is the two circular structures that contain the nucleus needed to balance and balance the movement. Shy-Drager syndrome is associated with nerve loss in the medulla and spinal cord intermedolateral cell columns and Onuf nucleus.

There is no special treatment for multiple system atrophy. Treatment targets the symptom control of the disease. Medicines used for the treatment of people with Parkinson's disease may be prescribed for individuals with multiple system atrophy. However, the usefulness of these drugs is very diverse among patients. The Levodopa / carbidopa combination, known as Sinemet, can be used to treat individuals with multiple system atrophy. Orthostatic hypotension may be treated alone with increased salt intake, or may be used in combination with fludrocortisone. This drug should be carefully observed by a specialist and used with caution because of the potential for side effects. Midodrine hydrochloride has been approved by the FDA for the treatment of low blood pressure, sometimes associated with multiple system atrophy. In addition to levodopa, other medications used to treat Parkinson's disease can be used to treat individuals with multiple system atrophy. This includes dopamine agonists including Pergolide and antiviral agents known as Amantadine. Adrenergic drugs such as Ephedrine can be used to treat low blood pressure. Urinary incontinence can be treated with drugs such as catheterization or Oxybutynin, and constipation can be improved by increased dietary fiber intake or laxatives. Continuous positive pressure ventilation can be used to treat sleep apnea, and anticonvulsants such as Clonazepam can be used to treat REM sleep disorders. In the later stages of multi-system atrophy, a Gastrostomy tube can be inserted directly into the stomach for dietary and nutritional management.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop stem cells capable of effectively treating Parkinson's syndrome and safely applicable to human body, and as a result, it has been found that mesenchymal stem cells obtained from human bone marrow are very effective for preventing or treating Parkinson's syndrome The present invention has been completed.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating Parkinson's syndrome.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention and claims.

According to one aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating Parkinsonism comprising human bone marrow-derived mesenchymal stem cells as an active ingredient, wherein the human bone marrow-derived mesenchymal stem cell comprises (i) CD29 , ≪ / RTI > CD44, CD49C, CD73 and CD105; And (ii) at least one surface antigen selected from the group consisting of CD34, CD45 and HLA-DR.

The present inventors have made extensive efforts to develop stem cells capable of effectively treating Parkinson's syndrome and safely applicable to human body, and as a result, it has been found that mesenchymal stem cells obtained from human bone marrow are very effective for preventing or treating Parkinson's syndrome Respectively.

Parkinson's syndrome is a clinical syndrome in which the following six important symptoms are combined: Resting tremor, rigidity, bradykinesia, postural instability, flexed posture, and freezing, among others. In general, Parkinson's syndrome, except idiopathic Parkinson's disease, has a relatively rapid rate of disease progression and tends to fall easily because it does not maintain posture at the initial stage of disease. In addition, no sustained response is observed, even if there is no response to treatment with levodopa or an initial response. In addition, various clinical symptoms such as autonomic dysfunction, cerebellar ataxia, ocular motility disorder, pyramidal sign, and cerebral sensory dysfunction are additionally observed.

Parkinson's syndrome is divided into primary and secondary causes depending on the cause. The primary cause is idiopathic Parkinson's disease and atypical Parkinson's syndrome. Atypical Parkinson's syndrome is a sporadic disease with dementia with lewy body, multiple system atrophy, progressive supranuclear palsy and corticobasal degeneration. Families with family history include FTDP-17, SCAs, Huntington's disease, and Wilson's disease.

Multiple System Atrophy (MSA) is a neurodegenerative disorder that manifests autonomic dysfunction and motor impairment due to L-dopa-unresponsive parkinsonism, cerebellar ataxia, and vertebral atrophy. Histologically, neurons are lost in the striatum, black granules, cerebellum, gonads, lower olives, and the medial lateral aspect of the spinal cord.

The human bone marrow-derived mesenchymal stem cells of the present invention exhibit a neuronal cell protection effect by inhibiting apoptosis in brain tissue, and can prevent or treat Parkinson's syndrome through this effect.

The human bone marrow-derived mesenchymal stem cells of the present invention comprise at least one, preferably at least two, more preferably at least three, selected from the group consisting of CD29, CD44, CD49C, CD73 and CD105, , Even more preferably at least four, and most preferably five, surface antigens. According to one embodiment of the present invention, the human bone marrow-derived MSCs express 60-100%, more preferably 80-100%, of the surface surface antigens CD29, CD44, CD49C, CD73 and CD105 , And most preferably 95-99%.

Meanwhile, the human bone marrow-derived mesenchymal stem cells of the present invention are at least one kind selected from the group consisting of CD34, CD45, and HLA-DR, preferably at least two, more preferably three types of hematopoietic stem cell surface antigen Lt; RTI ID = 0.0 > of < / RTI > surface antigens. According to one embodiment of the present invention, the human bone marrow-derived mesenchymal stem cells express 0-20% of CD34, CD45 and HLA-DR which are hematopoietic stem cell surface surface antigens, more preferably 0.1-10 %, And most preferably 0.1-5%.

The term "% " used in reference to the expression rate of the surface antigen means the ratio of cells expressing surface antigens in the cells to be analyzed. For example, the expression rate of the CD29 surface antigen is 100%, which means that all the cells express the CD29 surface antigen in the cell sample to be analyzed.

According to one embodiment of the present invention, the pharmaceutical composition for preventing or treating Parkinson's syndrome of the present invention is a composition for parenteral administration, particularly for intravascular administration, wherein when the composition is administered into a blood vessel, Stem cells protect or treat Parkinsonian syndrome by protecting nerve cells.

The composition of the present invention comprises (a) a pharmaceutically effective amount of the aforementioned human bone marrow-derived mesenchymal stem cells; And (b) a pharmaceutically acceptable carrier. As used herein, the term " pharmaceutically effective amount " means an amount sufficient to achieve the therapeutic efficacy or activity of the human bone marrow derived mesenchymal stem cells described above.

When the composition of the present invention is manufactured from a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, or a medium, but are not limited thereto.

The human bone marrow-derived mesenchymal stem cells of the present invention are used by suspending cells in a pharmaceutically acceptable carrier and filling the cells into a vial, a vinyl bag, a syringe or the like.

The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally, topically (buccal, sublingual, skin or ocular), transdermal or parenteral (subcutaneous, intradermal, intramuscular, intravascular or intraarticular) Parenteral administration, more preferably intravenous administration.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . Typical dosages of the pharmaceutical compositions of this invention are 10 < ² > -10 < ¹⁰ > cells per adult basis.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides a pharmaceutical composition for preventing or treating Parkinson's syndrome comprising human bone marrow-derived mesenchymal stem cells as an active ingredient.

(Ii) The human bone marrow-derived mesenchymal stem cells of the present invention exhibit preventive or therapeutic efficacy against Parkinson's syndrome by protecting nerve cells from apoptosis.

FIG. 1 shows the result of confirming the neuroprotective effect of mesenchymal stem cells using immunostaining.
Figure 2 shows the results of the rotational behavior test induced by the excipient (Apomorphin).
Vertical axis: rotation rates / min, blue bars: contral rotation, red bars: ipsil rotation rates
FIG. 3 shows the results of comparing cellular morphology according to number of passages in the passage culture of bone marrow-derived mesenchymal stem cells from patients suffering from multi-system atrophy. A. Passage 2 (P2), B. Passage 3 (P3), C. Passage 4 (P4), D. Passage 5 (P5), E. Passage 6 (P6), F. Passage 7 (P7), G. Substance 8 (P8), H. Substance 9 (P9), I. Substance 10 (P10), J. Substance 11 (P11)
FIG. 4 is a graph showing the cell proliferation rate of the bone marrow-derived mesenchymal stem cells of the multi-system atrophy patients according to the passage time of the cells.
FIG. 5 is a graph showing the expression of mesenchymal stem cell surface markers in bone marrow-derived mesenchymal stem cells cultured in multi-system atrophy patients.
FIG. 6 is a schematic diagram of possible therapeutic efficacy of mesenchymal stem cells expected for treatment of multi-system atrophy.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Construction of multi-system atrophy rat model using double toxin

Multidirectional atrophy rat models were prepared by administering 6-hydroxydopamine (6-OHDA), Sigma and quinolinic acid (QA), tocris to adult Sprague Dawley rats (SD rats, male, 225-250 g) . 6-OHDA was administered to the medial forebrain bundle (MFB) at 8 μg / μl in the track AP -2.2, ML +1.5, and DV-8.0 using the stereotaxic method. 4 μl total was administered, and 1 μl was administered per minute at the time of administration.

After 3 weeks of 6-OHDA administration, 90 nM QA was applied to the right stratum of the SD rat brain in two tracks (Track 1: AP +1.2, ML +3.2, DV .5.2 / 4.2, Track 2: AP 0.4, ML +4.0, DV 5.2 / 4.0). A total of 2 μl was administered to each track at a rate of 0.5 μl every 60 seconds, and 1 μl was administered per minute at the time of administration.

After 12 weeks of 6-OHDA administration, brain tissue was extracted and immunostained. After 12 weeks, the animal model was perfused, brain tissue was extracted and left in 40% sucrose for 2-3 days. After brain sections were sectioned at 20 μm, they were stained using immunostaining. The primary antibodies used for immunostaining were TH (Tyrosine Hydroxylase, Pel-Freez Biologicals) and Neun (millpore), and the cytoplasm of brain tissue and apoptotic cell death were confirmed (Fig. 1).

Example 2: Evaluation of therapeutic efficacy of mesenchymal stem cells in a multi-system atrophic disease model

Analysis of lethal dose and neuroprotective effect concentration of mesenchymal stem cells administered

To confirm the effect of mesenchymal stem cell treatment in animal models, stem cells (2 x 10 ⁶ / head) were administered into the arteries of animal models.

After mesenchymal stem cells (2 × 10 ⁶ / head) were administered, the cytoplasm of midbrain and cell death of striatum were confirmed by immunohistochemistry. The primary antibodies used for immunostaining were TH (Tyrosine Hydroxylase) and Neun, and it was confirmed that the cell death of the substantia nigra and the striatum was significantly reduced in the brain tissue of the experimental group treated with mesenchymal stem cells (Fig. 1).

The behavioral changes of mesenchymal stem cells with 2 × 10 ⁶ / head injections were analyzed. After the injection of apomorphine into the double toxin disease model, rotational behavior test was performed. After administering 2 mg / kg of apomorphine to each individual, the direction and number of rotations for 30 minutes were recorded and the results were obtained. Rotational behavior in the disease model was significantly increased from 3 weeks after the induction of double lesion, and gradually decreased with time thereafter (FIG. 2). In the disease model group treated with mesenchymal stem cells, the rotational behavior was remarkably improved compared with the disease model group, which was almost similar to that of the normal control group (FIG. 2).

Example 3: Comparison of cellular characteristics of cultured bone marrow-derived mesenchymal stem cells

The patient's bone marrow-derived stem cells were thawed at the semi-product storage stage, and cultured from p2 to p13 by subculture. The cells were observed under a microscope to maintain fibroblast-like characteristics characteristic of stem cells in each passage (Fig. 3). The characteristics and proliferation of cells were observed under an optical microscope (Nikon, Japan). As shown in FIG. 3, it was confirmed that the cells were uniformly proliferated in the form of a fibroblast-like spindle, as observed from the passage 2 (P2) to the passage 9 (P11).

Example 4: Comparative analysis of mesenchymal stem cell surface markers expression in bone marrow-derived mesenchymal stem cells

(FACS) was used to determine the expression characteristics of stem cell surface antigen on cells. The mesenchymal stem cells isolated from the bone marrow were cultured with 2 x 10 ⁶ cells in 2% fetal bovine serum (PBS) at passage 3 (P3), passage 5 (p5), passage 7 (P7), and passage 9 The cells were washed three times with DPBS (Gibco, USA) added thereto, suspended at a concentration of 1 × 10 ⁵ cells / 100 μl, and then were divided into test tubes and analyzed by FACS caliber (Becton Dickinson, NJ, USA). PE-CD90, PE-CD44, PE-CD73 and PE-CD105, and PE-CD34 and PE-CD45, hematopoietic stem cell markers, were used as anti-human stem cell markers, (Immunoglobulin isotype IgG1). All antibodies were purchased from Becton Dickinson, USA. The antibody used was reacted with cells on ice for 30 minutes under the dark condition, and the extra antibody was washed with DPBS (Gibco, USA) and analyzed by flow cytometry. More than 80% of the cells showed positive immunological characteristics for the stem cell surface surface antigens CD29, CD44, CD49C, CD73 and CD105, and all of the hematopoietic stem cell surface antigen CD34 and CD45 were less than 5% Respectively. As a result, it was confirmed that the separated cells had characteristics of stem cells in the subculture (Tables 1 and 2, FIG. 5).

Identification of mesenchymal stem cell marker expression Patient B PE-NC (%) CD29 CD44 CD73 CD105 CD90 P3 0.4 93.6 98.1 99.7 100 99,9 P5 1.7 89.8 92.8 97.2 99.9 100 P7 0.2 100 99.6 99.7 100 100 P9 1.9 99.3 96.5 99.8 99.9 100

Identification of hematopoietic stem cell markers and HLA-DR (MHC class II) expression Patient B PE-NC (%) CD34 CD45 HLA-DR P3 0.4 1.4 0.1 0.0 P5 1.7 3.0 0.4 0.1 P7 0.2 1.3 0.3 0.2 P9 1.9 3.0 0.2 0.0

Example 5: Comparison of karyotypes for the comparison of the stability of bone marrow-derived mesenchymal stem cells

P3, p5, p7, p7, p7, p7, p7, p7, p7, p6, p7, p7, The mesenchymal stem cells of p9 stage were analyzed by Samkwang Medical Laboratories (Korea).

Karyotype analysis of autologous bone marrow mesenchymal stem cell cultures in patients with multiple system atrophy number product gender P3 P5 P9 One BM-MSC M N N - 2 BM-MSC M N N - 3 BM-MSC M N N N

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

The present invention relates to a pharmaceutical composition for preventing or treating parkinsonism comprising human bone marrow-derived mesenchymal stem cells as an active ingredient, wherein said human bone marrow-derived mesenchymal stem cells are selected from the group consisting of (i) CD29, CD44, CD49C, CD73 and CD105 A positive immunological characteristic for at least one surface antigen selected from the group consisting of; And (ii) negative immunological properties against CD34 or CD45.
The method of claim 1, wherein the Parkinson's syndrome is selected from the group consisting of Parkinson's disease, dementia with lewy body, multiple system atrophy, progressive supranuclear palsy, corticobasal degeneration, Huntington ' s disease or Wilson ' s disease.
3. The pharmaceutical composition according to claim 2, wherein the Parkinson's syndrome is multiple system atrophy.
The composition according to claim 1, wherein the bone marrow-derived mesenchymal stem cells exhibit an effect of protecting nerve cells from apoptosis.
2. The pharmaceutical composition according to claim 1, wherein the composition is for parenteral administration.
6. The pharmaceutical composition according to claim 5, wherein the parenteral administration is intravascular or intracerebroventricular administration.

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