Use of butylidenephthalide in dopamine precursor cell transplantation therapy

Abstract

An application of using n-butylidenephthalide (BP) in dopamine precursor cell transplantation treatment, including using n-butylidenephthalide (BP) to improve the treatment efficacy of dopamine precursor cell transplantation treatment and using n-butylidenephthalide (BP) and dopamine precursor cells treated by n-butylidenephthalide (BP) in dopamine precursor cell transplantation treatment. The foregoing application is particularly related to using n-butylidenephthalide (BP) to improve the efficacy of dopamine precursor cell transplantation treatment in treating Parkinson's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the use of butylphthalide (BP) in cell transplantation therapy, in particular the use of butylphthalide (BP) in dopamine neural precursor cell transplantation therapy, including the use of butylphthalide (BP) to enhance the therapeutic efficacy of dopamine neural precursor cell transplantation therapy, and the use of butylphthalide (BP) and butylphthalide (BP)-treated dopamine neural precursor cells in dopamine neural precursor cell transplantation therapy. The aforementioned uses are particularly related to the use of butylphthalide (BP) to enhance the efficacy of dopamine neural precursor cell transplantation therapy in treating Parkinson's disease. BACKGROUND

[0002] Parkinson's disease is a common central nervous system degenerative disease, mainly caused by the decrease in dopamine secretion due to the degeneration and / or death of dopamine neurons, which leads to the loss of motor control in patients. Therefore, the symptoms of Parkinson's disease are mainly motor impairment. Currently, the drugs used to treat Parkinson's disease in clinical practice, such as L-dopa, are used to control the disease by increasing the amount of dopamine in the body. However, as the disease progresses, when the number of dopamine neurons in the patient's body reaches a certain level, the therapeutic effect of L-dopa or more invasive stimulation therapy is quite limited.

[0003] Therefore, the industry and related research units have been continuously committed to developing drugs or methods that can effectively treat Parkinson's disease. In recent years, dopamine neural precursor cell transplantation therapy has brought new opportunities for the treatment of Parkinson's disease patients. So-called "dopamine neural precursor cell transplantation therapy" is to transplant dopamine neural precursor cells into the brain of a patient, so that the dopamine neural precursor cells differentiate into dopamine neurons in the patient's brain, thereby achieving the purpose of supplementing the number of dopamine neurons in the patient's body to increase neurite outgrowth. However, it was found that when dopamine neural precursor cells are transplanted into the brain of a patient in the form of cell clusters, although they can differentiate into dopamine neurons, most of the dopamine neurons still concentrate in the cell clusters and cannot move outside the cell clusters, so they cannot effectively establish new neural networks, and the therapeutic effect achieved is still limited.

[0004] To solve the above problems, the present inventors have found that, in the process of differentiating dopamine neural precursor cells into dopamine neurons, adding butylphthalide (BP) to the cell culture environment can induce the movement of dopamine neurons, promote the movement of dopamine neurons outside the cell mass, and help to quickly establish neural connections. Therefore, butylphthalide (BP) can be used in the treatment of dopamine neural precursor cell transplantation to promote the movement of dopamine neural precursor cells outside the cell mass after differentiation into dopamine neurons, help to quickly establish neural connections, and has the effect of improving the therapeutic efficacy of dopamine neural precursor cell transplantation treatment. SUMMARY

[0005] One object of the present application is to provide a method for improving the therapeutic efficacy of dopamine neural precursor cell transplantation treatment, which comprises using a dopamine neural precursor cell culture solution containing an active ingredient to treat the dopamine neural precursor cells, wherein the active ingredient is butylphthalide (BP) and / or a pharmaceutically acceptable salt thereof. Preferably, the content of the active ingredient in the culture solution is 0.5 to 20 μg of butylphthalide (BP) per ml of culture solution. For example, the aforementioned method can improve the efficacy of dopamine neural precursor cell transplantation treatment in treating Parkinson's disease.

[0006] Another object of the present application is to provide a combination comprising the following components: (1) a conditional medium comprising a basal medium and a neural induction factor; and (2) butylphthalide (BP) and / or a pharmaceutically acceptable salt thereof. Preferably, the neural induction factor is selected from the group consisting of fibroblast growth factor, transforming growth factor inhibitor, glycogen synthase kinase inhibitor, purmorphamine, and combinations thereof, and wherein the fibroblast growth factor is at least one of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor-8b (FGF-8b), the transforming growth factor inhibitor is SB-431542, and the glycogen synthase kinase inhibitor is BIO. More preferably, the neural induction factor is fibroblast growth factor-8b (FGF-8b) and purmorphamine.

[0007] Yet another object of the present application is to provide a use of an active ingredient in the preparation of a medicament, wherein the active ingredient is butylidenephthalide (BP) and / or a pharmaceutically acceptable salt thereof, and the medicament is for use in cell transplantation therapy with dopamine neural precursor cells treated with butylidenephthalide (BP) and / or a pharmaceutically acceptable salt thereof. The treatment is preferably performed with a dopamine neural precursor cell culture medium containing butylidenephthalide (BP) and / or a pharmaceutically acceptable salt thereof, and preferably the content of butylidenephthalide (BP) and / or a pharmaceutically acceptable salt thereof in the culture medium is 0.5 to 20 μg / mL of the culture medium. Preferably, the medicament is administered in at least one manner selected from the group consisting of oral administration, nasal administration, intracortical injection, intrathecal injection, intracerebral injection, intravenous injection, intraperitoneal injection, and subcutaneous injection; and the treated dopamine neural precursor cells are administered in at least one manner selected from the group consisting of intracortical injection, intrathecal injection, intracerebral injection, intravenous injection, intraperitoneal injection, and subcutaneous injection. For example, the medicament and the treated dopamine neural precursor cells are used in cell transplantation therapy for treating Parkinson's disease.

[0008] Yet another object of the present application is to provide a method of dopamine neural precursor cell transplantation therapy, which comprises administering to a subject in need thereof an effective amount of a first part and an effective amount of a second part, wherein the first part comprises butylidenephthalide (BP) and / or a pharmaceutically acceptable salt thereof, and the second part comprises dopamine neural precursor cells treated with butylidenephthalide (BP) and / or a pharmaceutically acceptable salt thereof. Preferably, the first part is administered in at least one manner selected from the group consisting of oral administration, nasal administration, intracortical injection, intrathecal injection, intracerebral injection, intravenous injection, intraperitoneal injection, and subcutaneous injection; and the second part is administered in at least one manner selected from the group consisting of intracortical injection, intrathecal injection, intracerebral injection, intravenous injection, intraperitoneal injection, and subcutaneous injection. For example, the method of dopamine neural precursor cell transplantation therapy of the present application can be used for treating Parkinson's disease.

[0009] The detailed technical content and some specific embodiments of the present application will be described in the following content, so that those skilled in the art of the present application can understand the features of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0010] The present application will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0011] Figure 1 is a result graph showing the Corin expression on the cell surface detected by Influx cell sorter;

[0012] Figure 2is a photograph showing the differentiation process of dopamine neural precursor cells without butylidene phatalide (BP) treatment, continuously photographed by JuLI™ Br cell imager;

[0013] Figure 3 is a photograph showing each group of cells observed by inverted microscope, wherein the control group is dopamine neural precursor cells cultured in conditioned medium without butylidene phatalide (BP) for 6 days, the "BP (5)" group, the "BP (10)" group, the "BP (20)" group, the "BP (50)" group, and the "BP (100)" group are dopamine neural precursor cells cultured in conditioned medium with butylidene phatalide (BP) at concentrations of 5, 10, 20, 50, and 100 μM, respectively, for 6 days; and

[0014] Figure 4 is a photograph showing each group of cells observed by upright fluorescence microscope, wherein the control group is dopamine neural precursor cells cultured in conditioned medium without butylidene phatalide (BP) for 10 days, the "BP (5)" group, the "BP (10)" group, the "BP (20)" group, the "BP (50)" group, and the "BP (100)" group are dopamine neural precursor cells cultured in conditioned medium with butylidene phatalide (BP) at concentrations of 5, 10, 20, 50, and 100 μM, respectively, for 10 days, and wherein the green fluorescence represents dopamine neural precursor cells, the red fluorescence represents dopamine neurons, and the blue fluorescence represents cell nuclei. DETAILED DESCRIPTION

[0015] Particular embodiments according to the present application will be described below; however, the present application can be practiced in a variety of different forms and should not be construed as limited to the embodiments set forth in the specification. Additionally, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" includes a single compound as well as two or more such compounds and the like.

[0016] Numerical ranges as used herein (for example 5 to 100) are to be understood as being inclusive of the numerical values within the range and any rational values within the range, unless the context clearly dictates otherwise. Numerical ranges as used herein are inclusive of the endpoints and all the values including integers within the range.

[0017] As used herein, the term "pharmaceutically acceptable salt" includes "pharmaceutically acceptable base addition salts" of a "compound containing an acidic functionality" with "organic or inorganic bases" and "pharmaceutically acceptable acid addition salts" of a "compound containing a basic functionality" with "organic or inorganic acids".

[0018] Examples of the "pharmaceutically acceptable base addition salt" formed with an inorganic base include, but are not limited to, alkali metal salts (such as sodium salt, potassium salt), alkaline earth metal salts (such as calcium salt, magnesium salt), transition metal salts (such as iron salt, zinc salt, copper salt, manganese salt and aluminum salt) and ammonium salts.

[0019] Examples of the "pharmaceutically acceptable base addition salt" formed with an organic base include, but are not limited to, salts with methylamine, dimethylamine, trimethylamine, ethylamine, diethylamine, triethylamine, isopropylamine, tripropylamine, tributylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucosamine, theobromine, purines, piperidine, N-ethylpiperidine, tetramethylammonium compound, tetraethylammonium compound, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, benzhydrylamine, N,N-dibenzhydrylphenethylamine, 1-ephenamine, N,N-dibenzhydryl ethylenediamine, polyamine resin and the like.

[0020] Examples of the "pharmaceutically acceptable acid addition salt" formed with an inorganic acid include, but are not limited to, salts with hydrobromic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid and the like.

[0021] Examples of the "pharmaceutically acceptable acid addition salt" formed with an organic acid include, but are not limited to, salts with sulfonic acids (such as p-toluenesulfonic acid, benzene sulfonic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, naphthalenesulfonic acid), carboxylic acids (such as acetic acid, propionic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, succinic acid), anionic amino acids (such as glutamic acid, aspartic acid), hydroxy acids (such as citric acid, lactic acid, tartaric acid, glycolic acid, malic acid), fatty acids (such as hexanoic acid, octanoic acid, decanoic acid, oleic acid, stearic acid), pamoic acid, resin acid and the like.

[0022] In the present context, by "in terms of butylphthalide (BP)", it is meant that when a pharmaceutically acceptable salt of BP is used as the active ingredient, the amount of the salt of BP used is calculated on the basis of the amount of BP that the salt of BP is able to provide.

[0023] Parkinson's disease is mainly caused by the decrease of dopamine secretion due to the degeneration and / or death of dopamine neuron cells, which further causes the patient to lose the ability to control movement. The transplantation of dopamine precursor cells has brought new opportunities for the treatment of Parkinson's disease. Currently, the dopamine precursor cells used for cell transplantation therapy are mostly derived from induced embryonic stem cells. For example, embryonic stem cells are cultured in a basal medium, and nerve induction factors such as fibroblast growth factor (e.g., FGF-2, FGF-8b), transforming growth factor inhibitor (e.g., SB-431542), liver glycogen synthase kinase inhibitor (e.g., BIO), and / or purmorphamine are added to the culture medium to induce the differentiation of embryonic stem cells into dopamine precursor cells.

[0024] However, when the dopamine precursor cells are transplanted into the brain of a patient in the form of cell clusters, although they can differentiate into dopamine neurons, most of the dopamine neurons are still concentrated in the cell clusters and cannot move outside the cell clusters, so they cannot effectively establish new neural networks, and the treatment effect is limited. The foregoing can be found in, for example, Human iPS cell-derived dopaminergic neurons function in a primate Parkinson's disease model. Nature 548, 592-596 (2017) and Predictive Markers Guide Differentiation to Improve Graft Outcome in Clinical Translation of hESC-Based Therapy for Parkinson's Disease. Cell stem cell 20, 135-148, (2017), the entire contents of which are hereby incorporated by reference.

[0025] The present inventors have found that, during the differentiation of dopamine precursor cells into dopamine neurons, the addition of butylidene phthalide (BP) to the cell culture environment can induce the movement of dopamine neurons, promote the movement of dopamine neurons outside the cell clusters, and help quickly establish neural connections. Therefore, butylidene phthalide (BP) can be used in the transplantation of dopamine precursor cells to promote the movement of dopamine precursor cells outside the cell clusters after differentiation into dopamine neurons, help quickly establish neural connections, and improve the therapeutic efficacy of dopamine precursor cell transplantation therapy.

[0026] Therefore, the present application relates to butylidenephthalide (BP) for improving the therapeutic efficacy of dopamine precursor cell transplantation therapy and applications, in particular, for improving the efficacy of dopamine precursor cell transplantation therapy in treating Parkinson's disease, including methods and combinations for improving the therapeutic efficacy of dopamine precursor cell transplantation therapy. The method comprises using a dopamine precursor cell culture solution containing butylidenephthalide (BP) and / or its pharmaceutically acceptable salt to treat the dopamine precursor cells, and the combination comprises (1) a conditioned medium containing a basal medium and neural induction factors, and (2) butylidenephthalide (BP) and / or its pharmaceutically acceptable salt.

[0027] In the method of the present application for improving the therapeutic efficacy of dopamine precursor cell transplantation therapy, "using a dopamine precursor cell culture solution containing butylidenephthalide (BP) and / or its pharmaceutically acceptable salt to treat the dopamine precursor cells" means that the dopamine precursor cells are present in the dopamine precursor cell culture solution during the treatment. In addition, the dopamine precursor cell culture solution used according to the method of the present application comprises a basal medium and neural induction factors, wherein the basal medium provides the necessary components for the growth of dopamine precursor cells, such as pH value, etc. Generally, examples of basal medium that can be used include, but are not limited to, DMEM / F12 medium (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) supplemented with N2 supplement and neural basal medium supplemented with N2 supplement. For example, DMEM / F12 medium supplemented with N2 supplement can be used as a basal medium for the treatment of dopamine precursor cells.

[0028] The content of butylidenephthalide (BP) and / or its pharmaceutically acceptable salt in the culture solution used to treat dopamine precursor cells is generally 0.5 to 20 μg / mL of culture solution based on butylidenephthalide (BP); preferably 2 to 15 μg / mL of culture solution based on butylidenephthalide (BP); more preferably 3 to 12 μg / mL of culture solution based on butylidenephthalide (BP). For example, as shown in the following examples, when 0.9 to 19 μg / mL of butylidenephthalide (BP) is used per mL of culture solution (equivalent to using a concentration of 5 to 100 μM of butylidenephthalide (BP)), it can effectively induce dopamine neurons to move and promote dopamine neurons to move out of cell clumps.

[0029] The combination according to the present application comprises: (1) a conditional medium comprising a basal medium and a neural induction factor; and (2) butylphthalide (BP) and / or a pharmaceutically acceptable salt thereof. The basal medium and the amount of butylphthalide (BP) and / or a pharmaceutically acceptable salt thereof in the combination are as described above.

[0030] In addition to the basal medium, the conditional medium of the combination according to the present application can further comprise any neural induction factor that can help induce stem cells to differentiate into dopaminergic neural precursor cells, such as, but not limited to, fibroblast growth factor, transforming growth factor inhibitor, glycogen synthase kinase inhibitor, Purmorphamine, and a combination thereof. Preferably, the fibroblast growth factor is at least one of fibroblast growth factor-2 (FGF-2) and fibroblast growth factor-8b (FGF-8b), the transforming growth factor inhibitor is SB-431542, and the glycogen synthase kinase inhibitor is BIO. More preferably, the neural induction factor is fibroblast growth factor-8b (FGF-8b) and Purmorphamine.

[0031] The combination according to the present application can be a kit or a composition. When the combination according to the present application is a kit, the conditional medium of component (1) and butylphthalide (BP) and / or a pharmaceutically acceptable salt thereof of component (2) are usually packaged separately, stored in different storage spaces (such as plastic bags, plastic bottles, glass bottles, ampoules), and can be shipped or sold separately or combined together for delivery and sale. In addition, the sub-components of the conditional medium of component (1) can also be packaged separately and stored individually. The kit can further comprise an instruction manual, so that the user can mix the components on site according to the procedures and processes described therein to carry out cell culture, treatment and administration.

[0032] For example, when the sub-components of the conditioned medium of component (1) and the butylphthalide (BP) and / or pharmaceutically acceptable salt thereof of component (2) in the kit according to the present application are packaged separately, stored in different storage spaces, and shipped or sold separately, the neural induction factors (e.g., FGF-2, FGF-8b, SB-431542, and BIO, etc.), and the butylphthalide (BP) and / or pharmaceutically acceptable salt thereof can be stored in the dark at -20°C or below; and the basal medium can be stored at -20°C. For another example, when the components of the kit according to the present application are combined into a kit for distribution and sale, the aforementioned neural induction factors and butylphthalide (BP) can be contained in a light-proof container with an internal temperature of -20°C, and the basal medium can be contained in a container with an internal temperature of -20°C. The shape and size of each container are not particularly limited, as long as the storage space of each container has a function of isolating the internal temperature from the external temperature, so that the storage temperature of each component does not affect each other when the components are distributed and sold together.

[0033] In the use of the kit according to the present application, the order of mixing and preparation of each component is not particularly limited. When the sub-components of the conditioned medium are packaged separately, for example, the conditioned medium can be prepared first, and then mixed with the butylphthalide (BP) and / or pharmaceutically acceptable salt thereof; or the butylphthalide (BP) and / or pharmaceutically acceptable salt thereof can be mixed with the basal medium first, and then mixed with other sub-components; or each sub-component of the conditioned medium can be mixed with the butylphthalide (BP) and / or pharmaceutically acceptable salt thereof at the same time. In addition, the butylphthalide (BP) and / or pharmaceutically acceptable salt thereof can be directly mixed with the conditioned medium or the basal medium; or the butylphthalide (BP) and / or pharmaceutically acceptable salt thereof can be dissolved in a solvent to provide a butylphthalide (BP) solution, and then the butylphthalide (BP) solution is mixed with the conditioned medium or the basal medium. Examples of the solvent that can be used to dissolve the butylphthalide (BP) and / or pharmaceutically acceptable salt thereof include, but are not limited to, dimethyl sulfoxide (DMSO), ethanol, and vegetable oil.

[0034] When the combination according to the present application is a composition, the conditioned medium of component (1) and the butylphthalide (BP) and / or pharmaceutically acceptable salt thereof of component (2) are usually mixed and stored in the same storage space (e.g., plastic bag, plastic bottle, glass bottle, ampoule).

[0035] According to the present application, the combination provided by the present application is used in the treatment of dopamine neural precursor cell transplantation, which can induce the movement of dopamine neurons, promote the movement of dopamine neurons outside the cell mass, and help to quickly establish neural connections, thereby achieving the effect of improving the therapeutic efficacy of dopamine neural precursor cell transplantation. For example, but not limited to, when the combination of the present application is used in the treatment of dopamine neural precursor cell transplantation, a stem cell can be first cultured in a conditioned medium containing a basal medium and nerve induction factors such as FGF-2, FGF-8b, SB-431542, BIO, Purmorphamine, etc. to induce the stem cell to differentiate into dopamine neural precursor cells; then, the aforementioned conditioned medium is replaced with a conditioned medium containing a basal medium and butylidene phatalide (BP) and / or a pharmaceutically acceptable salt thereof, and the dopamine neural precursor cells are cultured for 8 to 12 days; finally, the obtained dopamine neural precursor cells are transplanted into an individual in need thereof.

[0036] In the use of butylidene phatalide (BP) and / or a pharmaceutically acceptable salt thereof for the preparation of a medicament according to the present application, the medicament is used with dopamine neural precursor cells treated with butylidene phatalide (BP) and / or a pharmaceutically acceptable salt thereof and used in cell transplantation therapy. The treatment is carried out with a dopamine neural precursor cell culture medium containing butylidene phatalide (BP) and / or a pharmaceutically acceptable salt thereof, and the content of butylidene phatalide (BP) and / or a pharmaceutically acceptable salt thereof in the culture medium is usually 0.5 to 20 micrograms per milliliter of culture medium in terms of butylidene phatalide (BP); preferably 2 to 15 micrograms per milliliter of culture medium in terms of butylidene phatalide (BP); more preferably 3 to 12 micrograms per milliliter of culture medium in terms of butylidene phatalide (BP).

[0037] The medicament provided by the present application can be in any convenient form and is not particularly limited, and is in a corresponding appropriate dosage form according to the required use. For example, but not limited to, the medicament can be administered to an individual in an oral or non-oral (e.g., nasal administration, intraspinal injection, intrathecal injection, intracerebral injection, intravenous injection, intraperitoneal injection, and subcutaneous injection) manner. Depending on the form of use and purpose, a suitable carrier can be selected to provide the medicament, wherein the carrier includes excipients, diluents, adjuvants, stabilizers, absorption delaying agents, disintegrants, solubilizers, emulsifiers, antioxidants, binders, binding agents, tackifiers, dispersants, suspending agents, lubricants, hygroscopic agents, etc.

[0038] For example, in the pharmaceutical preparation according to the present application, any pharmaceutically acceptable carrier which does not adversely affect the desired effects of the active ingredients (i.e., butylidene phthalide (BP) and / or pharmaceutically acceptable salts thereof) can be contained, such as water, saline, dextrose, glycerol, ethanol or the like, cellulose, starch, sugar bentonite, and combinations of the foregoing. The pharmaceutical preparation can be provided in a dosage form suitable for oral administration by any suitable method, such as tablets (e.g., sugar-coated tablets), pills, capsules, granules, powders, liquid extracts, solutions, syrups, suspensions, tinctures, and the like.

[0039] For example, in the pharmaceutical preparation according to the present application, any pharmaceutically acceptable carrier which does not adversely affect the desired effects of the active ingredients (i.e., butylidene phthalide (BP) and / or pharmaceutically acceptable salts thereof) can be contained, such as water, saline, dextrose, glycerol, ethanol or the like, cellulose, starch, sugar bentonite, and combinations of the foregoing. The pharmaceutical preparation can be provided in a dosage form suitable for oral administration by any suitable method, such as tablets (e.g., sugar-coated tablets), pills, capsules, granules, powders, liquid extracts, solutions, syrups, suspensions, tinctures, and the like.

[0040] If necessary, the pharmaceutical preparation according to the present application can further contain an additive in an appropriate amount, such as a flavoring agent, a colorant, a coloring agent, and the like, which can improve the taste and visual appearance of the pharmaceutical preparation when taken, and a buffer, a preservative, an antiseptic, an antifungal agent, and the like, which can improve the stability and storage of the pharmaceutical preparation. In addition, the pharmaceutical preparation can further contain one or more other active ingredients, or be used in combination with a pharmaceutical containing the one or more other active ingredients, as necessary, to further enhance the effects of the pharmaceutical preparation or increase the flexibility and adjustability of the formulation, as long as the other active ingredients do not adversely affect the effects of the active ingredients (i.e., butylidene phthalide (BP) and / or pharmaceutically acceptable salts thereof) of the present application.

[0041] In the use according to the present application, in addition to the administration of the medicament provided by the present application, the subject in need is also required to be administered with dopamine neural precursor cells treated with butylphthalide (BP) and / or pharmaceutically acceptable salts thereof, which can be administered simultaneously or separately with the administration of the medicament. The administration mode of the treated dopamine neural precursor cells is not particularly limited, and is only correspondingly appropriate dosage form according to the required use. For example, but not limited to, the treated dopamine neural precursor cells can be in the form of injections, cell infusion agents, etc., and can be administered to the subject by intracortical injection, intrathecal injection, intracerebral injection, intravenous injection, intraperitoneal injection, subcutaneous injection, etc. When the treated dopamine neural precursor cells are in the form of injections, cell infusion agents, etc., one or more pharmaceutically acceptable carriers, such as physiological saline, can be additionally contained in these dosage forms.

[0042] In addition, in the use according to the present application, the medicament and the dopamine neural precursor cells treated with butylphthalide (BP) and / or pharmaceutically acceptable salts thereof can be administered at different frequencies, such as once a day, multiple times a day, once every few days, or once every few weeks, etc., according to the needs, age, weight and health status of the subject to be administered. In the medicament, the content of the active ingredient (i.e., butylphthalide (BP) and / or pharmaceutically acceptable salts thereof) in the medicament can be adjusted according to the actual application needs. For example, when the medicament is administered orally twice a day, and the dopamine neural precursor cells treated with butylphthalide (BP) and / or pharmaceutically acceptable salts thereof are administered to a subject by intracerebral injection once every two weeks to treat Parkinson's disease and / or delay its onset, the dosage of the medicament, calculated as butylphthalide (BP), is usually 30 mg / kg to 2,000 mg / kg, preferably 50 mg / kg to 1,000 mg / kg, and more preferably 100 mg / kg to 500 mg / kg, per administration, wherein the unit "mg / kg" refers to the amount of administration required per kg of the subject. In addition, the amount of dopamine neural precursor cells used is usually 1x10 5 to 5x10 6 preferably 1x10 6 to 2x10 6 cells per administration.

[0043] The present application further relates to a method for treating a subject in need of dopamine neuron regeneration, comprising administering to the subject an effective amount of a first part and an effective amount of a second part, wherein the first part comprises butylphthalide (BP) and / or a pharmaceutically acceptable salt thereof, and the second part comprises dopamine neural precursor cells treated with butylphthalide (BP) and / or a pharmaceutically acceptable salt thereof. The subject in need of dopamine neuron regeneration refers to a subject with dopamine neuron degeneration, dopamine neuron degeneration and death, and / or insufficient dopamine secretion. The dosage range and usage conditions of the first part and the second part are as described above.

[0044] The present application is further illustrated by the following examples. These examples are provided only to illustrate and not to limit the scope of the present application. The scope of the present application is shown in the claims. Examples

[0045] Preparation Examples

[0046] A. Preparation of conditioned medium

[0047] A-1. DMEM / F12 medium (Dulbecco's Modified Eagle Medium / Nutrient Mixture F-12; purchased from Gibco, product number: 11320033) with N2 supplement (purchased from Gibco, product number: 17502048) was used as the base medium, and the following neural induction factors were further added to the base medium: BIO (purchased from Sigma-Aldrich, product number: B1686), SB-431542 (purchased from Sigma-Aldrich, product number: S4317), FGF-2 (purchased from Peprotech, product number: 100-18B), Purmorphamine (purchased from Cayman Chemical, product number: 10009634), and FGF-8b (purchased from R&D System, product number: 423-F8), to provide a final concentration of BIO (0.5 µM), SB-431542 (10 µM), FGF-2 (10 ng / mL), Purmorphamine (1 µM), and FGF-8b (50 ng / mL) in the base medium.

[0048] Prepared according to [Preparation Example A-1], but further adding Purmorphamine and FGF-8b in the basal medium only, and the final concentrations of these neural induction factors in the basal medium were Purmorphamine (1 microM) and FGF-8b (50 ng / ml), respectively, to provide another conditioned medium.

[0049] Preparation of dopaminergic precursor cells

[0050] Pre-culture of embryonic stem cells

[0051] Embryonic stem cells (provided by Taiwan MaxM Medical Center) were cultured in DMEM / F12 medium containing 20% serum replacement (KnockOut Serum Replacement, KSR; purchased from Gibco, Cat. No. 10828028) for 2 days to make the cells into floating spheres.

[0052] Differentiation of embryonic stem cells

[0053] The floating spheres of embryonic stem cells provided in [Preparation Example B-1] were cultured in the conditioned medium provided in [Preparation Example A-1] for 2 days. Then, the medium was removed and the cells were further cultured in the conditioned medium provided in [Preparation Example A-2] for 6 days to obtain a cell suspension.

[0054] Since Corin is known to be a specific surface protein of the dopaminergic precursor cells in the ventral midbrain, Corin antibody (purchased from R&D System, Cat. No. MAB2209) was added to the cell suspension obtained above and allowed to react for 15 minutes. The cells were then washed once with PBS, followed by the addition of fluorescent-labeled secondary antibody (purchased from Invitrogen, Cat. No. A21208) and allowed to react for 15 minutes. The cells were then washed once with PBS and finally suspended in PBS. The cell suspension was subjected to fluorescence detection (as shown in Figure 1 Figure 2) using an Influx cell sorter (purchased from BD) and the cells with fluorescent signals were sorted out, which were the dopaminergic precursor cells in the ventral midbrain.

[0055] Treatment of dopaminergic precursor cells

[0056] The dopaminergic precursor cells provided in [Preparation Example B-2] were cultured in the conditioned medium provided in [Preparation Example A-2] at 37°C under 5% carbon dioxide for 24 hours. Then, the cells were divided into six groups and treated as follows:

[0057] (1) Control group: Cells were cultured in the conditioned medium provided in [Preparation Example A-2] (i.e., cells were cultured in the medium without butylidene phatalide (BP)) for 10 days.

[0058] (2) "BP (5)" group, "BP (10)" group, "BP (20)" group, "BP (50)" group, and "BP (100)" group: The conditions were the same as those of the control group, but butylidene phatalide (BP) (purchased from Sigma-Aldrich, product number: W333301) was further added to each group of medium, so that the final concentration of butylidene phatalide (BP) in each group of medium was 5, 10, 20, 50, and 100 μM, respectively.

[0059] Example 1: Effect of butylidene phatalide (BP) on the differentiation ability of dopamine neural precursor cells

[0060] To understand the effect of butylidene phatalide (BP) on the differentiation of dopamine neural precursor cells into dopamine neurons, JuLI TM Stromal Cell Video Analyzer (purchased from NanoEnTek) was used to continuously take pictures of the differentiation process of the control group cells of [Preparation Example C] (results shown in Figure 2 ), and when each group of cells of [Preparation Example C] was cultured for 6 days, an inverted microscope (purchased from Nikon) was used to take pictures and record the morphology of each group of cells (results shown in Figure 3 ).

[0061] As shown in Figure 2 , the control group cells (i.e., dopamine neural precursor cells without butylidene phatalide (BP) treatment) showed signs of neural differentiation when cultured for 5 days, and when cultured for 10 days, the cells were differentiated into dopamine neurons with neural axon morphology. In addition, as shown in Figure 3 , the control group, "BP (5)" group, "BP (10)" group, "BP (20)" group, "BP (50)" group, and "BP (100)" group all showed the formation of neural fibers. These results show that butylidene phatalide (BP) treatment does not affect the normal neural differentiation of dopamine neural precursor cells.

[0062] Example 2: Effect of butylidene phatalide (BP) on promoting the movement of dopamine neurons

[0063] To understand the effect of butylidenephthalide (BP) on dopamine neurons, each group of cells of [Preparation Example C] was fixed at 10 days of culture, and each group of cells was subjected to fluorescence staining with antibodies of Sox-1 (purchased from Santa Cruz, product number: SC-17318), TH (tyrosine hydroxylase; purchased from Millipore, product number: MAB152), and DAPI (diamidino-2-phenylindole; purchased from ThermoFisher Scientific, product number: D1306) (in which Sox-1 is expressed in dopamine neural precursor cells, TH is expressed in dopamine neurons, and DAPI is a nuclear stain), and finally, the dopamine neural precursor cells (green fluorescence), dopamine neurons (red fluorescence), and cell nuclei (blue fluorescence) were observed by upright fluorescence microscopy (purchased from Nikon). Figure 4 .

[0064] As shown in Figure 4 , compared with the control group, the "BP (5)" group, the "BP (10)" group, the "BP (20)" group, the "BP (50)" group, and the "BP (100)" group all showed that dopamine neurons moved out of the cell mass, and the "BP (50)" group showed the most significant movement of dopamine neurons. The foregoing results show that butylidenephthalide (BP) can effectively induce dopamine neurons to move, and thus can be used in dopamine neural precursor cell transplantation therapy to promote the movement of dopamine neurons after differentiation from dopamine neural precursor cells, help to quickly establish neural connections, and improve the therapeutic efficacy of dopamine neural precursor cell transplantation therapy.

Claims

1. A combination comprising, It comprises the following components: (1) a conditional medium comprising a basal medium and a neural induction factor; and (2) butylphthalide (BP) and / or a pharmaceutically acceptable salt thereof, wherein the neural induction factor is a combination of fibroblast growth factor, transforming growth factor inhibitor, glycogen synthase kinase inhibitor and Purmorphamine, and wherein the fibroblast growth factor is fibroblast growth factor-2 (FGF-2) and fibroblast growth factor-8b (FGF-8b), the transforming growth factor inhibitor is SB-431542, and the glycogen synthase kinase inhibitor is BIO.

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