KR20180079231A - Pharmaceutical composition for treating brain disease using combination therapy comprising stem cells - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cerebral diseases using a combination method comprising stem cells. According to one aspect of the present invention, a pharmaceutical composition and a kit according to another aspect of the present invention improve the engraftment of stem cells in the brain through a cerebrovascular barrier with increased permeability , The paracrine effect of stem cell constituents or secretions regulates immune function, promotes blood vessel and nerve regeneration, and stimulates nerve regeneration, including stem cells, stem cell components, and stem cell secretions It is effective to inhibit the release of the cerebral blood vessel wall of the factor or to directly activate the nerve and vascular regeneration by the drug and to be useful for the treatment of brain diseases.

Description

[0001] The present invention relates to a pharmaceutical composition for treating brain diseases using a combination therapy comprising stem cells,

The present invention also relates to a pharmaceutical composition for treating brain diseases using a combination method comprising stem cells.

Stroke is the second leading cause of death worldwide, with ischemic stroke accounting for about 84% of these deaths. In Korea, stroke is the second most common cause of death following cancer, and it occurs more often than heart disease. In general, stem cell basal and clinical studies on acute cerebral infarction are actively underway, and there are many reports that stem cell therapy is efficacious in many studies. However, the basic studies on stem cell efficacy in chronic cerebral infarction show that the efficacy is significantly lower than that of acute cerebral infarction, and there is no report yet to prove its efficacy in patients with chronic cerebral infarction. In patients with stroke, many patients are not effective at initial treatment, or are not adequately treated at the beginning, and are often transferred to a chronic stroke patient, leaving severe postural problems such as limb paralysis. Nevertheless, to date, few drugs have been developed to ameliorate disability in patients with chronic stroke.

Mannitol, on the other hand, is currently used in clinical trials to reduce cerebral edema in stroke as an osmotic agent. In addition, studies have shown that mannitol is used to increase the engraftment rate of stem cells in a rodent animal model of stroke. Temozolomide has been approved for use as an anticancer agent in brain cancer and is now being used therapeutically in patients with brain tumors.

Under these circumstances, the present inventors have made efforts to increase the therapeutic effect of stem cells on brain diseases, particularly chronic strokes. As a result, it has been discovered that administration of mannitol and temozolomide in combination with stem cells can maximize the therapeutic effect Thereby completing the present invention.

One aspect is mannitol or a pharmaceutically acceptable salt thereof; And an effective amount of temozolomide or a pharmacologically acceptable salt thereof as an active ingredient to increase the permeability of the drug to the cerebral blood vessel barrier or to provide an inhibitor of the release of the drug from brain or the activation of direct nerve and blood vessel regeneration.

Another aspect is mannitol or a pharmaceutically acceptable salt thereof; And a composition for use in increasing the blood-brain barrier permeability, which comprises as an active ingredient temozolomide or a pharmaceutically acceptable salt thereof.

Another aspect is mannitol or a pharmaceutically acceptable salt thereof; Temozolomide or a pharmaceutically acceptable salt thereof; And stem cells, wherein said mannitol and temozolomide are administered in combination with stem cells. The present invention also provides a pharmaceutical composition for treating brain diseases.

Another aspect is a pharmaceutical composition comprising mannitol or a pharmaceutically acceptable salt thereof; Temozolomide or a pharmaceutically acceptable salt thereof; And a stem cell, wherein the mannitol and the temozolomide are administered in combination with a stem cell.

One aspect is mannitol or a pharmaceutically acceptable salt thereof; Or an effective amount of temozolomide or a pharmaceutically acceptable salt thereof as an active ingredient, to increase the permeability of the drug to the cerebral blood vessel barrier, or to provide an inhibitor of the release from the brain or the direct nerve and vascular regeneration.

Another aspect is mannitol or a pharmaceutically acceptable salt thereof; And a composition for use in increasing cerebral vascular barrier permeability, comprising as an active ingredient temozolomide or a pharmaceutically acceptable salt thereof.

In one embodiment, the composition may be a pharmaceutical composition for treating brain diseases by increasing the permeability of the drug to the cerebral blood vessel barrier or inhibiting the release of the drug from the brain.

Another aspect is mannitol or a pharmaceutically acceptable salt thereof; Temozolomide or a pharmaceutically acceptable salt thereof; And stem cells, wherein the mannitol and the temozolomide are administered in combination with a stem cell.

Another aspect is a pharmaceutical composition comprising mannitol or a pharmaceutically acceptable salt thereof; Temozolomide or a pharmaceutically acceptable salt thereof; And administering the stem cells to a subject in need thereof.

Refers to or includes the reduction, progression, or prevention of a disease, disorder or condition, or one or more symptoms thereof, wherein the "active ingredient" or "pharmaceutically effective amount" refers to a disease, disorder or condition, May refer to any amount of composition used in the practice of the invention provided herein to ameliorate, inhibit or prevent progression of one or more symptoms.

The terms "administering "," introducing "and" transplanting "are used interchangeably and refer to a method of delivering a composition according to one embodiment ≪ / RTI > may refer to the arrangement of the composition according to the invention. May be administered by any suitable route that delivers at least a portion of the cells or cellular components of the composition according to one embodiment to the desired location within the living individual. The survival period of the cells after administration of the individual may be several hours if short, for example, 24 hours to several days, or several years if long.

 The term "mannitol" is a kind of sugar core derived from exose, and may include D-mannitol or L-mannitol, and may be represented by, for example, The capacity of the mannitol may be from 1 g / kg to 3 g / kg, or from 0.5 M to 2.0 M.

[Chemical Formula 1]

The term " temozolomide "is used interchangeably with temodal and is a drug used in the treatment of brain tumors, for example, one represented by the following formula (2). The dosage of temozolomide may be from 75 mg / m ² to 200 mg / m ² or from 1 mg / kg to 50 mg / kg.

(2)

The term " blood brain barrier "refers to a type of barrier that prevents certain substances from entering the blood vessels and is a highly differentiated form of the brain capillaries that maintains the homeostasis of the nerve microenvironment. The cerebrovascular barrier is a structure formed to keep the movement of useless materials to a minimum by keeping the capillaries in the nerve tissue lower vascular permeability compared to other tissues. The main structure of the cerebrovascular barrier consists of a tight junction or zonula occluden in capillary endothelial cells. Since the cerebral blood vessel barrier prevents unauthorized entry of ions, proteins and other substances into the central nervous system environment, its role as a barrier protects the neurons from harmful components supplied by the blood, but many kinds of drugs and metabolites are involved in the blood vessels Since the barrier is not permeable, the proper regulation of the cerebral blood vessel barrier is an important part of administering the drug in the proper place in the treatment of various brain diseases.

According to one embodiment, the mannitol and temozolomide can increase the permeability of the cerebral vascular barrier of the drug, inhibit the release of useful substances in the drug or brain, or activate direct neuronal and vascular regeneration. Therefore, when mannitol and temozolomide are coadministered with stem cells, they increase the migration of stem cells to the brain to increase the engraftment rate of stem cells, or to increase the engraftment of stem cells or various factors (secretions) Movement can be increased. As a result, the above-mentioned mannitol and temozolomide can enhance the engraftment in the brain of stem cells or stimulate immune regulation, vascular and nerve regeneration due to the paracrine effect of stem cell components or secretions have. Stem cell, stem cell components, stem cell secretion, and other factors that contribute to nerve regeneration. Mannitol and temozolomide can also directly activate nerve and vascular regeneration. As a result, the mannitol and temozolomide can reduce the indirect or direct therapeutic efficacy by the cerebrovascular barrier, for example, inhibition of the therapeutic effect of stem cells.

The stem cells may be any cells having differentiation ability irrespective of their origins, and may be, for example, mesenchymal stem cells, embryonic stem cells, adult stem cells, induced pluripotent stem cells, or combinations thereof. In addition, the stem cell may be one obtained from the patient himself or another person whose phenotype matches. "Mesenchymal Stem Cells (MSCs)" may mean cells capable of maintaining self-renewal and stemness maintenance and differentiating into various mesenchymal tissues, For example, mesenchymal stem cells of animals including humans. The mesenchymal stem cells may be umbilical cord stem cells, umbilical cord blood stem cells, placenta-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, or a combination thereof. The umbilical cord may refer to a line connecting the mother and the embryo so that the mammalian embryo can grow in the placenta. In general, three umbilical vessels surrounded by Wharton's Jelly, namely two umbilical arteries and one umbilical vein . ≪ / RTI > Thus, in the present specification, the mesenchymal stem cells may be mesenchymal stem cells derived from umbilical cord or umbilical cord wholly jelly tissue. In addition, for example, the adipose-derived stem cells are derived from subcutaneous fat tissue, bone marrow fat tissue, mesenteric fat tissue, gastric fat tissue, or retroperitoneal fat tissue, and have the ability to differentiate into various tissue cells, And the like. Separation of mesenchymal stem cells can be carried out by methods known to those skilled in the art, for example, by Pittenger et al (Science 284: 143, 1997) and van et al. (J. Clin. Invest., 58: 699, 1976 ). ≪ / RTI >

Such conjoint administration can mean administration of mannitol, temozolomide, and stem cells simultaneously, sequentially or individually, and in any order. Specifically, the above-mentioned co-administration may be administration of mannitol, temozolomide and stem cells simultaneously, or administering mannitol and temozolomide followed by stem cells. Combination therapy in accordance with the present invention may be used, for example, in combination with one or more of the components of the combination therapy, such as the degree of response, rate of response, duration to disease progression, or survival period, More therapeutically superior can be defined as providing a synergistic effect. For example, the efficacy of the combination therapy is synergistic if the efficacy is superior to that obtained by using each of the above individually. In particular, it is more preferable to use each component in a conventional manner without causing harm to one or more of the reaction degree, reaction rate, duration of disease progression, and survival data, , A synergistic effect is considered to exist if the usual side effects of mannitol, temozolomide or stem cells can be reduced while the problematic side effects are reduced / decreased or decreased.

In one embodiment, the brain disease may be selected from the group consisting of stroke, cerebral hemorrhage, brain injury, myelitis, multiple sclerosis and degenerative brain diseases. Examples of degenerative brain diseases include Alzheimer's disease (senile dementia), dementia, Parkinson's disease, Lou Gehrig's disease, Pick's disease, Huntington's disease or tauopathy. The stroke may also be a chronic stroke.

In addition, the stem cells may be loaded on a biodegradable support. The biodegradable scaffold may be spontaneously and slowly decomposed after a certain period of time in a living body. The biodegradable scaffold may include a polymer having at least one of biocompatibility, blood affinity, anti-calcification characteristic, cell nutrition characteristic, . Such biodegradable scaffolds include fibrin, collagen, gelatin, chitosan, alginate, hyaluronic acid, dextran, polylactic acid, polyglycolic acid, PGA, poly (lactic-co-glycolic acid) polyglycolic acid, PLGA, poly-ε- (caprolactone), polyanhydride, polyorthoester, polyvinyl alcohol, polyethylene glycol, polyurethane, polyacrylic acid, poly- Poly (ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide) copolymers, copolymers thereof, mixtures thereof, and the like. At this time, the content of the biodegradable polymer in the composite support may be from 5 to 99% by weight in view of molding of the support or the loading of stem cells. The composite support may be prepared by a known method such as a solvent-casting and particle-leaching technique, a gas forming technique, a nonwoven fabric made of polymer fibers, A method of forming a biodegradable polymer by a method such as a fiber extrusion and fabric forming process, a thermally induced phase separation technique, an emulsion freeze drying method, a high pressure gas expansion, can do.

The pharmaceutical compositions according to one embodiment may be used in combination with other cells, tissues, tissue fragments, growth factors, biologically active or inactive compounds, reabsorbable plastic scaffolds, or other delivery, efficacy, tolerance, Can be applied in combination with additives. The cell population may also be modified by insertion or injection of DNA in cell culture via a method of altering, enhancing or supplementing cell function for induction for structural or therapeutic purposes. For example, gene delivery techniques for stem cells are described in Morizono et al., 2003; As described in Walther and Stein, 2000 and Athanasopoulos et al., 2000, which are well known to those skilled in the art as disclosed in Mosca et al., 2000, , And adeno-associated virus gene transfer techniques. Non-viral based techniques can also be performed as described in Muramatsu et al., 1998.

The dose (effective amount) of the stem cells according to one embodiment may be 1.0 x 10 ⁶ to 1.0 x 10 ⁹ . Lt; / RTI > The dose of mannitol and temozolomide is as described above. However, the dose may be variously prescribed depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate and responsiveness of the patient, These factors can be taken into account to appropriately adjust the dosage. The number of administrations may be one or two or more times within the range of clinically acceptable side effects, and the administration site may be administered at one site or two or more sites. For an animal other than a human, the dosage may be the same as that of a human per kg, or the dosage may be converted into a dose in terms of a volume ratio (for example, an average value) One dose may be administered. Possible routes of administration may include parenteral (e.g. subcutaneous, intramuscular, intraarterial, intraperitoneal, intrathecal, or intravenous), topical (including transdermal), and injection, or implantation of an implantable device or material have. Examples of animals to be treated in accordance with one embodiment include humans and other mammals for the purpose of administration. Specifically, humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, .

The pharmaceutical composition according to one embodiment may include mannitol, temozolomide, and stem cells as the active ingredient and a pharmaceutically acceptable carrier and / or additive. Examples thereof include sterilized water, physiological saline, buffering agents (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (such as ascorbic acid), surfactants, suspending agents, isotonic agents, can do. For the topical administration, organic substances such as biopolymers, and inorganic substances such as hydroxyapatite, specifically, collagen matrix, polylactic acid polymer or copolymer, polyethylene glycol polymer or copolymer, and chemical derivatives thereof . When the pharmaceutical composition according to one embodiment is prepared into a formulation suitable for injection, the stem cell or the like may be frozen in a solution state in which it is dissolved or dissolved in a pharmaceutically acceptable carrier.

According to one embodiment, mannitol or temozolomide may be formulated as a suspension, dissolution aids, stabilizers, isotonizing agents, preservatives, adsorption inhibitors, surfactants, diluents, excipients, pH adjusters, Buffers, reducing agents, antioxidants, and the like. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995. Mannitol or temozolomide according to one embodiment may be formulated by using 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. May be prepared in a dosage form or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oil or aqueous media, or in the form of powders, granules, tablets or capsules.

Another aspect is mannitol or a pharmaceutically acceptable salt thereof; Temozolomide or a pharmaceutically acceptable salt thereof; And stem cells, wherein said mannitol and temozolomide are administered in combination with stem cells.

The above-mentioned mannitol, temozolomide and stem cells are as described above.

According to the pharmaceutical composition and kit according to one aspect, the engraftment of the stem cells in the brain through the increased permeability of the cerebral blood vessel barrier is improved, or the paracrine effect due to stem cell components or secretions, Not only promotes blood vessel and nerve regeneration but also inhibits the release of the cerebral vascular barrier of various factors that contribute to nerve regeneration, including stem cells, stem cell components, and stem cell secretions, Nerve, and blood vessel regeneration, thereby being effective for the treatment of brain diseases.

1 is a graph showing the mechanism of action of a pharmaceutical composition according to one embodiment.
2A is a diagrammatic representation of an in vitro BBB model according to one embodiment.
Figure 2B is a graph showing the transmittance of a 4.4 kda fluorescence tracer in an in vitro BBB model according to one embodiment.
Figure 2C is a graph showing the transmittance of a 70 kda fluorescence tracer in an in vitro BBB model according to one embodiment.
FIG. 3A is a graph showing the results of Western blotting of the expression levels of tight junction markers in cells according to the combination treatment of mannitol and temozolomide according to one embodiment.
FIG. 3B is a graph quantitatively showing the relative expression amount of tight junction markers in cells according to the combination treatment of mannitol and temozolomide according to one embodiment. FIG.
FIG. 3C is a photomicrograph showing the results of immunohistochemical assay for the expression level of occludin, which is one of tight junction markers in cells according to the combination treatment of mannitol and temozolomide according to one embodiment.
Figure 4A is a photograph of brain tissue isolated from an animal model after administration of an Evans blue dye to an animal model.
4B is a graph showing the amount of Evans blue dye in brain tissue and the ratio of IpSi-Contra.
FIG. 5A is a graph showing the results of Western blotting of the expression levels of tight junction markers in cells of an animal model in combination with mannitol and temozolomide according to one embodiment. FIG.
FIG. 5B is a graph quantitatively showing the relative expression amount of close-coupled markers in cells of an animal model according to the combination administration of mannitol and temozolomide according to one embodiment.
FIG. 5C is a microphotograph showing immunohistochemical assay of the expression level of close-coupled markers in cells of an animal model according to the combination administration of mannitol and temozolomide according to one embodiment.
FIG. 6A is a graph showing the results of mNSS test in a stroke animal model according to one embodiment of the present invention. FIG.
FIG. 6B is a graph showing the results of stepping tests in an animal model of stroke according to one embodiment of the present invention.
FIG. 6C is a graph showing the result of a foot fault test in an animal model of stroke according to one embodiment of the present invention. FIG.
FIG. 7A is an immunofluorescence micrograph showing the inflow of microbeads in brain tissue according to one embodiment of the present invention. FIG.
FIG. 7B is a graph showing the proportion of CD63-positive cells permeated into brain tissue according to the concurrent administration according to one embodiment.
FIG. 8A is an immunofluorescence microphotograph showing BrdU / DCX / NeuN positive cells according to the concurrent administration according to one embodiment.
8B is a graph showing the proportion of BrdU-positive cells according to the combination administration according to one embodiment.
8C is a graph showing the proportion of DCX-positive cells according to the combination administration according to one embodiment.
8B is a graph showing the proportion of NeuN-positive cells according to the combination administration according to one embodiment.
9A is an immunofluorescence micrograph showing the neovascular density at the brain lesion site according to the concurrent administration according to one embodiment.
FIG. 9B is a graph quantitatively showing the neovascular density at the brain lesion site according to the concurrent administration according to one embodiment. FIG.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

1, it is generally known that mannitol has an effect of increasing the permeability of the cerebral blood vessel barrier. However, it is believed that the administration of mannitol and stem cells will have four effects. First, it is believed that increasing the migration of stem cells into the parenchyma ultimately increases the engraftment rate of stem cells in the brain. Secondly, various factors such as constituents generated by breakage of stem cells or a large amount of nerve and vascular regeneration secreted from stem cells are transferred to the brain via the cerebral blood vessel barrier, which is increased in increased permeability. It is considered. Third, it suppresses the expression of p-glycoprotein of the temozolomide cerebral blood vessel barrier and inhibits drug efflux through the blood-brain barrier. Fourth, mannitol and demozolomide can directly activate nerve and vascular regeneration. Therefore, when temozolomide and stem cells are co-administered, not only stem cells themselves but also various components such as constituents generated by breakage of stem cells or a large amount of nerve and blood vessel regeneration secreted from stem cells can be easily inserted into the brain, It can be inhibited from escaping out and the drug itself is thought to activate nerve and vascular regeneration. In addition, the combination of mannitol and temozolomide increased the permeability of the cerebrovascular barrier, indicating that temozolomide can also increase the migration of stem cells, stem cell components, and stem cell secretions into the brain There is an effect.

Example 1 Confirmatory activity of BBB permeability enhancement by the combination administration of mannitol and temozolomide

1.1. in vitro activity confirmation

In order to confirm the BBB permeability enhancement activity by the combination treatment of mannitol and temozolomide increasing the BBB permeability, plastic inserts and human brain microvessel endothelial cells (HBMECs) (purchased from the cell system, ACBRI 376 , Kirkland, WA, USA) was used.

Specifically, in order to investigate the mechanism of action of mannitol (Man), temozolomide (TMZ), and their combination treatment and combination treatment and the temozolomide inhibitor (LiCl, temozolomide) in the HBMECs of the chamber of FIG. 2A ). Mannitol was treated with 100 mM for 6 hours, and temozolomide was treated with 50 μM for 3 days. We then investigated the enhancement of permeability using a fluorescent tracer of different size, 4.4 kda / 70 kda. Next, the permeation capacity of the fluorescent tracer was confirmed, and the results are shown in Figs. 2B and 2C.

Protein expression of close junction markers such as Occludin and ZO-1, which are closely related to BBB permeability, was analyzed by Western blotting. Specifically, protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes. (1: 1000, Thermo Fisher Scientific) and anti-ZO-1 (1: 500, Abcam) followed by horseradish peroxidase conjugated secondary anti-mouse or rabbit antibody 1: 2000, Vector Laboratories). Band images were obtained using Image Lab software (BIO-Rad, Hercules, CA, USA). GAPDH (1: 5000, Santa Cruz Biotechnology, Dallas, TX, USA) was used as an internal control. Measurements of band intensities were performed using the NIH Image J program, and the experiments were performed 4 independent times on different days, and the results are shown in FIG.

2A is a diagrammatic representation of an in vitro BBB model according to one embodiment.

Figure 2B is a graph showing the transmittance of a 4.4 kda fluorescence tracer in an in vitro BBB model according to one embodiment.

Figure 2C is a graph showing the transmittance of a 70 kda fluorescence tracer in an in vitro BBB model according to one embodiment.

FIG. 3A is a graph showing the results of Western blotting of the expression levels of tight junction markers in cells according to the combination treatment of mannitol and temozolomide according to one embodiment.

FIG. 3B is a graph quantitatively showing the relative expression amount of tight junction markers in cells according to the combination treatment of mannitol and temozolomide according to one embodiment. FIG.

FIG. 3C is a photomicrograph showing the results of immunohistochemical assay for the expression level of occludin, which is one of tight junction markers in cells according to the combination treatment of mannitol and temozolomide according to one embodiment.

As shown in FIG. 2B and FIG. 2C, it was found that the combination treatment of mannitol and temozolomide according to one embodiment increased the BBB permeability synergistically in each of the in vitro BBB models. In particular, it was confirmed that the transmittance of a large-sized fluorescent tracer of 70 kda was remarkably increased.

As shown in Figs. 3A to 3C, it was confirmed that each drug alone hardly decreased the expression of the tight junction marker. However, it was confirmed that the combination treatment of mannitol and temozolomide according to one embodiment remarkably reduced the expression of the tight junction marker.

1.2. Identification of in vivo activity

Based on the in vitro experiments of Example 1.1. Above, the combined treatment of drugs was applied to an animal model of ischemic stroke stroke. Stroke animal models were prepared by inserting silicon rubber filaments into the right carotid artery of 8-week-old rats (Sprague dawley rat, Orient Bio) and occluding the midbrain artery (MCA) for 90 minutes.

Thereafter, 2.5 ml of mannitol (1.1 M) was intravenously administered to the animal model for 3 days, and temozolomide was orally administered at a dose of 20 mg / kg for 5 days. After 5 days, 4 mg / kg of Evans blue dye was administered into the tail vein of the animal model, and after 4 hours, the heart was perfused with PBS and the brain was removed. Rats with an infarct size of less than 20% were excluded from the experimental group. The brain was placed on the brain matrix and the brain tissue was cut to 2 mm thickness. The hemisphere of the brain was divided into the ipsilateral and ipsilateral contralateral contralateral contralateral contralateral regions, and each tube was filled with 500 μl of 50% TCA (Sigma-Aldrich) solution The tissue was homogenized. After overnight incubation at 4 占 폚, the tissue was centrifuged at 1000 x g for 30 minutes. After the supernatant was harvested, the absorbance of each sample was measured. The amount of EBD detected was calculated using a standard curve and the value was converted to μg of dye per gram of tissue, the results of which are shown in FIG.

In addition, the results of the expression amount of occludin, which is a tight junction marker for isolated brain tissue, are shown in FIG. 5 in the same manner as in Example 1.2.

Figure 4A is a photograph of brain tissue isolated from an animal model after administration of an Evans blue dye to an animal model.

4B is a graph showing the amount of Evans blue dye in brain tissue and the ratio of IpSi-Contra.

FIG. 5A is a graph showing the results of Western blotting of the expression levels of tight junction markers in cells of an animal model in combination with mannitol and temozolomide according to one embodiment. FIG.

FIG. 5B is a graph quantitatively showing the relative expression amount of close-coupled markers in cells of an animal model according to the combination administration of mannitol and temozolomide according to one embodiment.

FIG. 5C is a microphotograph showing immunohistochemical assay of the expression level of close-coupled markers in cells of an animal model according to the combination administration of mannitol and temozolomide according to one embodiment.

As shown in FIGS. 4A and 4B, it was confirmed that Evans blue dye was stained and dyed in an in vivo chronic stroke animal model in combination with mannitol and temozolomide according to one embodiment, And the BBB transmittance increased synergistically.

As shown in Figs. 5A to 5C, when the combination treatment of mannitol and temozolomide is performed more than the drug alone, the amount of Occludin, which is one of the close-contact markers, is relatively decreased in the lesion (stroke) Could know.

Example 2. Confirmatory effect of stroke treatment by administration of mannitol, temozolomide, and stem cells

2.1. Behavior improvement activity analysis

Triple therapy was applied to an animal model of chronic stroke in which mannitol (Sigma-Aldrich, USA), temozolomide (Sigma-Aldrich, USA) and stem cell from CHA Biotech (Republic of Korea) Respectively. The stroke animal model was prepared in the same manner as in Example 1.2.

The severity of the animal model was evaluated by using modified neurological severity score (MNSS) (Reglodi D et al, 2003), stepping test using treadmill (Olsson M et al, 1995), and foot fault test (KL Schaar, MM Brenneman, SI After the drug treatment, the patients were assessed using the following criteria: (1) Savitz, Functional assessments in the rodent stroke model, Experimental & translational stroke medicine 2 (1) (2010) Drug treatment was performed at the end of 4 weeks of stroke modeling (Day 0), followed by the pre-behavioral test, followed by the administration of 150 mg / m ² of temozolomide for 5 days (Day 0-4) And 2 g / kg of mannitol was administered for 3 days (Day 1-3), and 1.0 × 10 ⁶ cells of umbilical cord stem cells were administered twice (Day 1, Day 3). The above behavior analysis was performed on Day 1, Day 3, Day 7, Day 14, Day 21, and Day 28, and thereafter for a total of two weeks at intervals of one week, and the results are shown in FIG. (SCTG): stem cell alone combination drug treatment group (SC + CDTG): drug (mannitol + temozolomide) + Temozolomide) + Stem cell therapy group. As a control group, no treatment group was used.

FIG. 6A is a graph showing the results of mNSS test in a stroke animal model according to one embodiment of the present invention. FIG.

FIG. 6B is a graph showing the results of stepping tests in an animal model of stroke according to one embodiment of the present invention.

FIG. 6C is a graph showing the result of a foot fault test in an animal model of stroke according to one embodiment of the present invention. FIG.

As shown in FIG. 6, when the mannitol, temozolomide, and stem cells were co-administered in the mNSS test, which can confirm the somatosensory and motor ability evaluation, and the foot fault test and the stepping test, , Stem cell alone, drug alone, and untreated group. It was confirmed that the treatment effect of stem cell and mannitol - temozolomide was increased in the animal models of stroke, compared with the stem cell alone, the drug alone, and the untreated group.

2.2. Increased activity of intracerebral inflow of mesenchymal stem cell-derived micro-vesicles

The rats of Example 2.1 were euthanized at 4 weeks of age, and brain tissues were separated to analyze the intracavitary inflow of mesenchymal stem cell-derived microvessels in brain tissue.

Specifically, microbial inflow into the brain was analyzed using a human specific CD63 antibody and the results are shown in Figures 7A and 7B; CDTG: drug (mannitol + temozolomide) alone group, SCTG: stem cell alone group, SC + CDTG: drug (mannitol + temozolomide) + stem cell combination group.

FIG. 7A is an immunofluorescence micrograph showing the inflow of microbeads in brain tissue according to one embodiment of the present invention. FIG.

FIG. 7B is a graph showing the proportion of CD63-positive cells permeated into brain tissue according to the concurrent administration according to one embodiment.

As shown in Fig. 7, it was found that the inflow of microvesicles derived from mesenchymal stem cells into the brain was remarkably increased by the combined administration. This means that the combination therapy with mannitol-temozolomide significantly restored the disorder of the stroke by significantly increasing the intracerebral inflow of the microvesicles derived from mesenchymal stem cells in the animal model of stroke.

2.3. Analysis of neuronal and vascular regeneration activity

In order to confirm the improvement of endogenous environmental factors in the brain lesions of chronic stroke stroke, the number of BrdU / DCX / NeuN positive cells related to nerve regeneration in the SVZ region, which is a stem cell niche, The density was observed. Microvasculature of brain tissue was stained with Reca-1 for blood vessel determination in ischemic hemisphere lesions and cortical areas. Images were acquired by confocal microscopy. Using the VesSeg-Tool software (http://www.isip.uni-luebeck.de/index.php?id=150&L=0), the color of the dyed microvessels is converted to white and the captured image is white The covered area was calculated as a percentage. The number of BrdU / DCX / NeuN positive cells is shown in 8A to 8D. In addition, the neovascular density was confirmed with Reca-1 and the results are shown in Figures 9A and 9B; CDTG: drug (mannitol + temozolomide) alone group, SCTG: stem cell alone group, SC + CDTG: drug (mannitol + temozolomide) + stem cell combination group.

FIG. 8A is an immunofluorescence microphotograph showing BrdU / DCX / NeuN positive cells according to the concurrent administration according to one embodiment.

8B is a graph showing the proportion of BrdU-positive cells according to the combination administration according to one embodiment.

8C is a graph showing the proportion of DCX-positive cells according to the combination administration according to one embodiment.

8B is a graph showing the proportion of NeuN-positive cells according to the combination administration according to one embodiment.

9A is an immunofluorescence micrograph showing the neovascular density at the brain lesion site according to the concurrent administration according to one embodiment.

FIG. 9B is a graph quantitatively showing the neovascular density at the brain lesion site according to the concurrent administration according to one embodiment. FIG.

As shown in FIG. 8, it was confirmed that the number of BrdU / DCX / NeuN positive cells was markedly increased by administration of mannitol-temozolomide in combination. This means that when the mesenchymal stem cells are administered in combination with mannitol-temozolomide, the synergistic effect of synaptic regeneration increases synergistically, which is useful for treating brain diseases such as stroke.

Further, as shown in Fig. 9, it was confirmed that the density of the new blood vessel was markedly increased by administration of mannitol-temozolomide in combination. This means that when the mesenchymal stem cells are administered in combination with mannitol-temozolomide, the angiogenic effect is synergistically increased, and thus it can be effectively used for treating cerebrovascular diseases such as stroke.

Claims (15)

Mannitol or a pharmaceutically acceptable salt thereof; A pharmaceutical composition for treating cerebral diseases by inhibiting the permeability of cerebral blood vessel barrier or the release of the drug from the brain, which comprises a temozolomide or a pharmaceutically acceptable salt thereof as an active ingredient. Mannitol or a pharmaceutically acceptable salt thereof;
Temozolomide or a pharmaceutically acceptable salt thereof; And
Wherein the stem cell is administered in combination with stem cells, wherein the mannitol and the temozolomide are administered in combination with the stem cell. The pharmaceutical composition according to claim 2, wherein the mannitol and the temozolomide increase the engraftment rate of stem cells in the brain. The pharmaceutical composition according to claim 2, wherein the mannitol and the temozolomide increase the paracrine effect by the stem cell component or secretion in the brain. 3. The pharmaceutical composition according to claim 2, wherein the mannitol and the temozolomide directly activate nerve and blood vessel regeneration. 3. The pharmaceutical composition according to claim 2, wherein the mannitol and the temozolomide reduce the inhibition of the therapeutic effect by the blood-brain barrier. [Claim 2] The pharmaceutical composition according to claim 2, wherein the stem cell is a mesenchymal stem cell, an embryonic stem cell, an adult stem cell, an induced pluripotent stem cell, or a combination thereof. [Claim 7] The pharmaceutical composition according to claim 7, wherein the mesenchymal stem cells are umbilical cord stem cells, cord blood derived stem cells, placenta derived stem cells, adipose derived stem cells, bone marrow derived stem cells, or a combination thereof. [Claim 2] The pharmaceutical composition according to claim 2, wherein the combination administration comprises administering mannitol, temozolomide, and stem cells simultaneously, or administering mannitol and temozolomide, followed by stem cells. The pharmaceutical composition according to claim 2, wherein the brain disease is selected from the group consisting of stroke, cerebral hemorrhage, brain injury, myelitis, multiple sclerosis and degenerative brain diseases. 11. The pharmaceutical composition according to claim 10, wherein the stroke is chronic stroke. Mannitol or a pharmaceutically acceptable salt thereof;
Temozolomide or a pharmaceutically acceptable salt thereof; And
A kit for treating brain diseases comprising stem cells, wherein said mannitol and temozolomide are administered in combination with stem cells. [Claim 13] The kit according to claim 12, wherein the stem cells are umbilical cord stem cells, cord blood derived stem cells, placenta derived stem cells, adipose derived stem cells, bone marrow derived stem cells, or a combination thereof. [Claim 12] The kit according to claim 12, wherein the coadministered administration comprises administration of mannitol, temozolomide and stem cells simultaneously, or administration of mannitol and temozolomide followed by stem cells. The kit according to claim 12, wherein the brain disease is selected from the group consisting of stroke, cerebral hemorrhage, brain damage, myelitis, multiple sclerosis and degenerative brain diseases.

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