TWI706781B - The composition for treating multiple system atrophy - Google Patents

Abstract

The invention discloses a use for treating multiple system atrophy by exogenous mitochondria. Specifically, by administering an effective amount of the exogenous mitochondria of this invention or a composition comprising a pharmaceutically effective amount of the exogenous mitochondria of this invention to a subject who suffers multiple system atrophy, to achieve sustained and effective treatment of multiple systemic degenerative and/or the subject’s motion ability.

Description

Pharmaceutical composition for treating multiple system degeneration

The present invention belongs to the second use of a pharmaceutical composition, and particularly refers to a pharmaceutical composition for the treatment of multiple system degeneration.

According to the theory, multiple system atrophy (MSA) is a kind of neurodegenerative disease, and different from general neurodegenerative diseases, it usually has two or more neurodegenerative symptoms. Studies have shown that about 80% of patients with multiple system degenerative diseases will develop symptoms of disability after 5 years, and about 80% of patients will die within 12 years of onset.

However, the current research is still unable to understand the cause of multiple system degeneration. It is only known that the lesion area of multiple system degeneration is mainly the nerve cells in the cerebellum, and its nerve cell degeneration area is similar to other neurodegeneration. Diseases are different. However, since the cause of multiple system degeneration is still unknown, there is not only a lack of methods for judging multiple system degeneration in clinical practice, but there is no method to treat multiple system degeneration. Give medication to control symptoms.

The main purpose of the present invention is to provide a use of isolated exogenous mitochondria, which can be used to treat or prevent multiple system degenerative diseases and their symptoms. In other words, by administering the isolated exogenous mitochondria of the present invention or a composition containing an effective amount of the isolated exogenous mitochondria of the present invention to an individual suffering from multiple systemic degenerative diseases, It can achieve the effect of continuous and effective treatment of multiple system degeneration and/or individual exercise ability.

In the disclosed embodiments of the present invention, the isolated exogenous mitochondrial system can be used to prepare a composition for treating multiple system degeneration and its symptoms.

Among them, multiple system degenerative disease refers to a disease that includes at least two neurodegenerative symptoms.

Among them, the symptoms of multiple system degeneration include degeneration or loss of exercise capacity or degeneration of the substantia nigra striatum.

In the embodiment of the present invention, the composition contains at least 5 μ g of the separated exogenous mitochondria, wherein the composition again comprising 5 ~ 100 μ g of the separated exogenous mitochondria Those are better, such as 10 μ g, 15 μ g, 20 μ g, 30 μ g, 40 μ g, 50 μ g, 60 μ g, 70 μ g, 80 μ g, 90 μ g, 95 μ g, 100 μ g.

In an embodiment of the present invention, the isolated exogenous mitochondrial system is isolated from a cell, for example, the cell line is a stem cell, a somatic cell, or a cell with differentiation ability.

Wherein, the isolated exogenous mitochondrial system can be separated from the cells through external force or mitochondrial extraction reagents and other techniques known in the technical field of the present invention.

In another embodiment of the present invention, the donor of the cell or the donor system of the isolated exogenous mitochondria is different from the recipient of the isolated exogenous mitochondria.

Wherein, the isolated exogenous mitochondria or the donor of the cell and the recipient system of the isolated exogenous mitochondria belong to different species.

Figure 1 shows the statistical results of injection of 6-hydroxydopamine, 6-hydroxydopamine and pyridine-2,3-dicarboxylic acid, and apomorphine-induced rotation test in normal rats.

Figure 2A shows the statistical results of pedaling test and L-dopa reaction between 6-hydroxydopamine injected rats and normal SD rats.

Figure 2B shows the statistical results of pedaling test and L-dopa reaction of rats injected with 6-hydroxydopamine and pyridine-2,3-dicarboxylic acid solution and normal SD rats.

Figure 3 shows the results of the exercise performance evaluation test for each group of rats.

Figure 4 shows the results of the pedaling test for each group of rats.

The “exogenous mitochondria” referred to in the present invention refers to mitochondria that have a complete external structure and have been separated from cells, and the exogenous mitochondria can be isolated from the recipient’s own cells and non-receptor own cells, meaning The exogenous mitochondrial system has been isolated from the cell to which it belongs, and if the exogenous mitochondria come from a non-recipient, there is no restriction that the non-recipient species need to be the same as the recipient's species.

The term "isolated" in the present invention refers to the existence of the exogenous mitochondrial system disclosed in the present invention in an independent form, which means that the mitochondria have been separated or purified from the cells through well-known techniques in the technical field of the present invention, and The separation or purification of mitochondria includes the separation or purification of mitochondria from cells, the separation of mitochondria by centrifugation, and the separation of mitochondria by destruction of cell membranes and filtration.

The "donor" referred to in the present invention refers to an individual who provides cells as a source of extraction of exogenous mitochondria or as a source of extraction of exogenous mitochondria.

The "recipient" referred to in the present invention refers to an individual who has received exogenous mitochondrial transplantation.

The "extraction" referred to in the present invention refers to the technical means for separating the exogenous mitochondria disclosed in the present invention from the cells to which they belong. For example, the exogenous mitochondrial system disclosed in the present invention can be isolated from adipose stem cells or other Cells; and the extraction is only a means of isolating mitochondria from cells as exemplified in the specification of the present invention, and is not limiting the preparation process or production technology of exogenous mitochondria disclosed in the present invention.

The "composition" referred to in the present invention contains an effective amount of the required active ingredient to produce a specific effect, and at least one carrier. As understood by those with ordinary knowledge in the technical field to which the present invention belongs, the form of the composition may vary depending on the route of administration that is intended to cause a specific effect, and the carrier may also depend on the form of the composition. Change patterns.

Hereinafter, several examples and figures will be given to further illustrate the effects of the technical features disclosed in the present invention.

Example 1: Establish an animal model for multiple system degeneration

Take 7-week-old SD rats, weighing about 175~200 grams, and prepare a double-toxin double-lesion animal model of multiple system degeneration by chemical induction, which means that this example is based on the previous literature ( Fernagut et al., 2012; Stefanova et al., 2015) use a stereotaxic instrument (David Kopf Instruments, Califonia, USA) to locate the injection site and inject pyridine-2,3-dicarboxylic acid (quinolinic acid) and 6-hydroxydopamine (6-hydroxydopamine) method to establish the animal model of multiple system degeneration.

In detail, 4 μL of 6-hydroxydopamine solution (the 6-hydroxydopamine solution was prepared to a concentration of 2 μg/μl with normal saline containing 0.1% L-ascorbic acid) was injected into the right brain of a 7-week-old SD rat. In the previous medial forebrain bundle, the stereotactic parameters of the injection point were AP (anteroposterior) -2.2, ML (mediolateral) +1.5, and DV (dorsoventral) -8.0. One month after the injection of 6-hydroxydopamine solution, 0.5μL of pyridine-2,3-dicarboxylic acid solution (concentration of 0.12M, prepared in 0.1M phosphate buffered saline) was injected at 4 injection points, in which, Stereotactic positioning of the injection point The parameters are AP+1.2, ML+2.9, DV-5.2; AP+1.2, ML+2.9, DV-4.2; AP-0.4, ML+3.7, DV-5.2; AP-0.4, ML+3.7, DV-4.2 .

Apomorphine induction rotation test was performed at the 3rd week after the injection of 6-hydroxydopamine solution and the 2nd week after the injection of pyridine-2,3-dicarboxylic acid solution. Among them, apomorphine was administered by subcutaneous injection to the greatest extent. Rats, and the dose of apomorphine is 0.25mg/kg, and it is modulated with 0.2% L-ascorbic acid-saline; the number of rotations is tested every five minutes, and the number of rotations per minute is calculated (contralateral -The same side), the result is shown in Figure 1. From the results in Figure 1, it can be seen that after injection of 6-hydroxydopamine solution and administration of apomorphine, the number of spins in rats increased, and the rats were treated with 6-hydroxydopamine solution and pyridine-2,3-dicarboxylic acid solution successively. Administration of apomorphine did not increase the number of spins in rats. It can be seen that rats injected with 6-hydroxydopamine solution alone can increase the number of rotations by administering apomorphine, while those injected with 6-hydroxydopamine solution and pyridine-2,3-dicarboxylic acid solution can be administered by apomorphine. Morphine reduces the number of rotations, showing that injection of 6-hydroxydopamine and pyridine-2,3-dicarboxylic acid can be used to induce multiple systemic degeneration rats (hereinafter referred to as MSA model rats).

In the fourth week after the injection of 6-hydroxydopamine solution (not yet injected with pyridine-2,3-dicarboxylic acid) and the fourth week after injection of 6-hydroxydopamine solution and pyridine-2,3-dicarboxylic acid solution, respectively In the test, the rat L-dopa reaction was detected at the same time (the detailed steps are as described in Example 4 below). The results are shown in Figure 2-A and Figure 2-B. The results in Figure 2-A show that injection 6- The administration of L-dopa after hydroxydopamine solution can improve the exercise status of rats, and Figure 2-B shows that after 6-hydroxydopamine solution and pyridine-2,3-dicarboxylic acid solution, administration of L-dopa can not improve The movement state of the rat.

Based on previous studies, L-dopa is effective for the treatment of Parkinson’s disease. Therefore, the results of Figure 1 and Figure 2-A can confirm that injection of 6-hydroxydopamine solution can be used to construct an animal model of Parkinson’s disease. Administration of L-dopa to rats induced by injection of 6-hydroxydopamine solution and pyridine-2,3-dicarboxylic acid solution failed to improve their exercise ability and coordination ability, indicating that injection of 6-hydroxydopamine solution and pyridine-2,3 -The animal model of the disease induced by the dicarboxylic acid solution is not the animal model of Parkinson's disease, but the animal model of multiple system degeneration. In other words, it can be seen from the results of Figure 1 and Figure 2 that the injection of pyridine-2,3-dicarboxylic acid and 6-hydroxydopamine can indeed reduce the exercise coordination ability of rats and destroy the striatum. Ability to establish MSA model rats.

Example 2: Animal test

SD rats are trained on an animal treadmill (Rotarod) before inducing the establishment of multiple system degenerative syndrome models, and record the benchmark value of each experimental rat, each training or recording at least 24 hours.

The SD rats were divided into 4 groups according to different treatments. Among them: normal group: normal SD rats without MSA animal model establishment treatment; no treatment group: MSA model rats, 4 μl , 0.9% phosphate buffer Mitochondria treatment group: MSA model rats, transplanted exogenous mitochondria isolated from human adipose stem cells to the brain; stem cell treatment group: MSA model rats, transplanted human adipose stem cells to Brain.

The injection method of the no-treatment group, the transplantation method of the mitochondrial treatment group or the stem cell treatment group is as follows: Take the MSA model rat established in Example 1, remove the hair on the top of the head, and place it in a stereo On the locator, after scalping the rat's scalp, remove the subcutaneous tissue to expose the position of the Bregma and use it as the origin of the location. Use an electric bone drill at the position of AP+0.4, ML+3.3, DV-4.7 (this position is the position of the striatum), and use a micro syringe (Hamilton, modle 80920, USA) at a speed of 1 μl per minute Inject human adipose-derived stem cells (Adipose-Derived Stem Cell), remove exogenous mitochondria or 0.9% phosphate buffer from human adipose-derived stem cells, for 4 minutes, inject a total volume of 4 μl , of which, human adipose stem cells the number of transplantation 3.4x105cells; exogenous mitochondria by graft quantity based 3.4x105cells body of human adipose derived stem cells are extracted in an amount of about 10 μ g; human adipose stem cells or exogenous particles will float line system Inject in 4 μl , 0.9% phosphate buffer. After the injection, the needle is removed, the gap in the skull is filled with bone wax, and the scalp is sutured.

Example 3: Exercise ability evaluation test

Rotate the roller (about 7 cm in diameter) at a speed of 4 rpm. When the animal walks on the roller steadily, start timing and acceleration synchronously, so that the roller speed will reach 40 rpm from 4 rpm within 5 minutes. If the animal falls halfway, it will touch the sensor The timer interrupts the timing, and the number of seconds presented is the result of the animal in the test.

The rats in each group of Example 2 were evaluated for exercise capacity after transplantation treatment according to the above description. The results are shown in Figure 3, which shows that the exercise capacity of rats in the mitochondrial treatment group was significantly improved two weeks after mitochondrial transplantation, and Four weeks after transplantation, the exercise capacity of rats in the mitochondrial treatment group was better than that in the stem cell treatment group, and the administration of the isolated exogenous mitochondria could continue to maintain the therapeutic effect after transplantation. The exercise capacity of the rats in the mitochondrial treatment group continued to improve, while the rats transplanted with stem cells could not maintain the treatment effect continuously, and the treatment effect decreased after the third week of transplantation. It can be seen that by administering the exogenous mitochondria or the composition containing exogenous mitochondria of the present invention to an individual suffering from multiple system degeneration, the treatment of multiple system degeneration and It has the efficacy of symptoms and long-term treatment.

Example 4: pedaling test

In this example, the speed of the walking machine is 0.9m/5 seconds. Record the number of times each rat’s forepaw touches the walking machine within 5 seconds, and calculate the ratio (%) of the ipsilateral paw relative to the contralateral paw touch. 30 minutes before the pedaling test, 8 mg/kg of L-dopa (Sigma) and 15 mg/kg of 0.9% decarboxylase inhibitor (benserazide, Sigma) mixed saline were administered to the rats by intraperitoneal injection.

According to the above method, the rats in each group of Example 2 after transplantation were tested. The results are shown in Figure 4. It can be seen that the rats in the mitochondrial treatment group had a significant improvement in their movement ability one week after the mitochondria were transplanted. The improvement effect not only lasts four weeks after transplantation, but also makes the action ability of the rats similar to Rats in the normal group; moreover, compared with the rats in the stem cell treatment group, the rats in the mitochondrial treatment group have significantly better motor ability.

From the above results, it can be seen that for individuals suffering from multiple system degeneration, administering stem cell lines cannot effectively and continuously treat multiple system degeneration and its symptoms, while administering the exogenous mitochondria disclosed in the present invention or It is a composition containing exogenous mitochondria that can achieve effective and continuous treatment of multiple system degenerative diseases, and can simultaneously improve two or more neurodegeneration or damage symptoms.

Furthermore, because the mitochondria transplanted to the affected part of the individual are isolated from stem cells, and the amount of stem cells used to isolate the mitochondria is equivalent to the amount administered by direct transplantation of stem cells, which means that the transplanted mitochondria The total amount of mitochondria transplanted to the affected area should be the same or nearly the same between the group of stem cells and the group of stem cells transplanted. However, the results of the above examples can confirm that compared with the group of directly transplanted stem cells, transplantation The separated mitochondrial system can achieve significantly improved treatment and improvement effects for multiple system degeneration, which shows that the separated mitochondria can improve the degeneration symptoms of multiple parts of the brain. In other words, an effective amount of the isolated mitochondria disclosed in the present invention must be administered to achieve the effect of treating multiple system degenerations.

Further, although the amount of the above examples are transplanted to an affected area outside the endogenous mitochondrial of 10 μ g, but, the amount of the graft-based merely illustrative only and not to limit the scope of the case. For example, see FIGS. 5 and 6, degeneration of rat multiple system prepared according to the method of example before exposing, respectively, and the damaged administered different doses: than 10 μ g and 90 μ g endogenous Mitochondria, observe the status of each rat at the damaged striatum and its contralateral, substantia nigra damage and its contralateral, and statistically analyze the proportion of TH (tyrosine hydroxylase) cells in each rat. , The administered exogenous mitochondrial system has been isolated from its donor and exists independently; the results of Figures 5 and 6 show that when the administration of 10 μ g and 90 μ g of exogenous mitochondria occurs at most The affected area of an individual with degenerative sexual system can not only prevent the continued occurrence of nerve damage, but also effectively improve the situation of nerve damage and restore nerve function. As the dose increases, its treatment and improvement The effect is better. It can be seen from this that the exogenous mitochondria disclosed in the present invention or the composition containing the exogenous mitochondria disclosed in the present invention can indeed treat multiple system degeneration diseases, and those with ordinary knowledge in the technical field of the present invention found that administering separated based on their experience than endogenous mitochondrial dose of at least 5 μ g, again administered amount of 5 ~ 100 μ g is preferred.

Claims (6)

A use of isolated exogenous mitochondria for the preparation of a composition for the treatment of multiple system degeneration and its symptoms, wherein the multiple system degeneration symptoms are degeneration of exercise capacity, loss of exercise capacity or nigral striae Shape body degeneration. Patent application range based on the use of item 1, wherein the composition contains at least 5 μ g than the endogenous mitochondrial. Patent application range based on the use of item 2, wherein the composition contains 5 ~ 100 μ g of the separated exogenous mitochondria. Patent application range based on the use of item 2, wherein the composition comprises 10 ~ 90 μ g of the separated exogenous mitochondria. The use according to item 1 of the scope of patent application, wherein the donor system of the isolated exogenous mitochondria is different from the recipient of the isolated exogenous mitochondria. According to the use described in item 5 of the scope of patent application, wherein the donor of the isolated exogenous mitochondria and the recipient system of the isolated exogenous mitochondria belong to different species.

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