WO2023190267A1 - Fine particle, prophylactic drug or therapeutic drug for parkinson's disease, and method for improving parkinson's disease - Google Patents

Abstract

Provided are, comprising miRNA downregulated in Parkinson's disease or miRNA used for the treatment of Parkinson's disease: a fine particle containing a prescribed miRNA of interest as a therapeutic means for Parkinson's disease; a prophylactic drug or therapeutic drug for Parkinson's disease; and a method for improving Parkinson's disease.

Description

Microparticles, preventive or therapeutic drugs for Parkinson's disease, and methods for improving Parkinson's disease

The present invention relates to microparticles, preventive or therapeutic agents for Parkinson's disease, and methods for improving Parkinson's disease.

Parkinson's disease is a neurodegenerative disease that occurs frequently around the world, and further development of treatments and prevention methods for Parkinson's disease is desired. Research to date has made progress in elucidating the pathogenesis of Parkinson's disease.

Non-Patent Document 1 states that among sirtuins (SIRTs), inhibition of SIRT2 exerts a neuroprotective effect in Parkinson's disease (PD), that SIRT2 protein level is highly increased in PD models, and that it is negatively affected by miR-212-5p. It is stated that it is controlled by Based on the direct link between miR-212-5p and SIRT2-mediated p53-dependent programmed cell death in the pathogenesis of PD, SIRT2 inhibitors have been described as a new therapeutic tool for PD. There is.
Non-Patent Document 2 states that degeneration of dopaminergic (DA) neurons occurs in PD, and that glial cell line-derived neurotrophic factor (GDNF) increases the survival of DA neurons in PD and increases the survival of primary mesencephalic neurons (PMNs). ) has been described to specifically increase the expression of miR-182-5p and miR-183-5p. It has also been described that miR-182-5p and miR-183-5p serve as new therapeutic means for PD.

On the other hand, Non-Patent Document 3 describes a review of miRNAs and new therapeutic targets in Parkinson's disease, and Figure 1 on p1957 lists 41 types of miRNAs as miRNAs that are down-regulated in the brain tissue of Parkinson's disease. Are listed.

However, Non-Patent Documents 1 to 3 do not describe the use of microparticles such as exosomes as a means for introducing or transfecting the miRNA described in these documents into living organisms.

The problem to be solved by the present invention is to provide microparticles containing a specific miRNA that is attracting attention as a means of treating Parkinson's disease.

The present inventors have discovered that the above problems can be solved by microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease.

Specifically, the present invention and preferred configurations of the present invention are as follows.
[1] Microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used in the treatment of Parkinson's disease.
[2] The microparticle according to [1], wherein the microparticle is an exosome.
[3] The microparticle according to [1], wherein the microparticle contains miRNA that targets a gene related to Parkinson's disease at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
[4] The microparticle according to [1], wherein the microparticle contains at least one type of the following Parkinson's disease-related miRNA group;
Parkinson's disease-related miRNA group:
hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-212-5p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa- miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543.
[5] The microparticles contain hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p among the Parkinson's disease-related miRNA group, [4 The microparticles described in ].
[6] The microparticle according to [4], wherein the microparticle is a SIRT2 inhibitor and contains hsa-miR-212-5p from the Parkinson's disease-related miRNA group.
[7] The agent according to [4], which is an inhibitor of dopaminergic neuron degeneration or apoptosis and includes hsa-miR-182-5p or hsa-miR-183-5p from the Parkinson's disease-related miRNA group. Microparticles.
[8] The microparticles are microparticles purified and isolated from the culture supernatant of dental pulp-derived stem cells,
The microparticle according to [1], wherein the microparticle does not contain components other than the culture supernatant of dental pulp-derived stem cells except for exosomes.
[9] A prophylactic or therapeutic agent for Parkinson's disease, comprising the microparticle according to [1].
[10] Parkinson's disease, comprising administering an effective amount of the microparticles according to [1] or an effective amount of the prophylactic or therapeutic agent for Parkinson's disease according to [9] to a subject who has developed Parkinson's disease. How to improve.

According to the present invention, it is possible to provide microparticles containing a specific miRNA that is attracting attention as a means of treating Parkinson's disease.

FIG. 1 is a heat map showing the expression level of miRNA expressed in exosomes (microparticles of Example 1) purified from the culture supernatant of dental pulp-derived stem cells. FIG. 2 is a heat map showing the expression level of each miRNA shown in FIG. 1 in the cerebral cortex.

The present invention will be explained in detail below. Although the constituent elements described below may be explained based on typical embodiments and specific examples, the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "~" means a range that includes the numerical values written before and after the "~" as lower and upper limits.

[Microparticles]
The microparticles of the present invention are microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease.
The microparticles of the present invention contain specific miRNAs that are attracting attention as a means of treating Parkinson's disease.
Hereinafter, preferred embodiments of the microparticles of the present invention will be explained.

<Definition of Parkinson's disease>
Parkinson's disease (PD) is caused by the formation of abnormal proteinaceous inclusions within certain nerve cells (neurons) or glial cells in the central nervous system, which gradually attack the nerve cells, causing them to decline and disappear. It is a type of progressive neurological disease (neurodegenerative disease).
From an anatomical and pathological point of view, Parkinson's disease is a selective disease of the substantia nigra pars compacta (SNc; a neuronal nucleus composed of melanin-containing dopaminergic neurons that sends their own projections to the striatum). Characterized by degeneration.
Parkinson's disease (PD) symptoms include motor disorders such as the four major symptoms (resting tremor, bradykinesia/akinesia, muscle rigidity, postural reflex disorder), and non-motor symptoms (autonomic nervous symptoms, sensory disorders, mental disorders). symptoms, sleep disturbances, etc.), impulse control disorders (such as pathological gambling).
Pathological features mainly include dopaminergic neuron degeneration and the presence of Lewy bodies in the substantia nigra pars compacta (SNc), as well as the pallial cortex, hypothalamus, cerebral cortex, basal ganglia, and central and peripheral autonomic nervous system. Degeneration of other strains including component parts has also been reported. Parkinson's disease symptoms are often seen when there is approximately 80% loss of dopaminergic neurons in the substantia nigra pars compacta.

<Mechanism of Parkinson's disease>
An overview of the pathogenesis of Parkinson's disease is as follows.
(1) Progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta occurs.
(2) Due to a decrease in dopaminergic neurons in the substantia nigra pars compacta, the amount of dopamine produced decreases.
(3) Defects in dopaminergic neurons in the striatum disrupt basal function.
(4) Bring about the manifestation of Parkinson's disease symptoms.
In other words, under normal conditions, dopamine produced in the substantia nigra acts as a liaison with various parts of the brain in the striatum of the cerebrum and regulates body movements and functions, but when Parkinson's disease develops, dopamine becomes insufficient. The patient becomes unable to control body movements and functions, and motor and non-motor symptoms appear.

Lewy bodies are known to be one of the causes of progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta. Then, α-synuclein protein aggregates and accumulates in dopaminergic neurons in the substantia nigra pars compacta, causing the formation of Lewy bodies.
The disease in which Lewy bodies appear is called "Lewy body disease," and in addition to Parkinson's disease, there is Lewy body dementia.

<Parkinson's disease treatment drugs and mechanism of action>
Preferably, the microparticles of the present invention can improve Parkinson's disease. Hereinafter, the therapeutic agent for Parkinson's disease and its mechanism of action will be explained together with the mechanism of action of the microparticles of the present invention.
The following are examples of Parkinson's disease therapeutic drugs and their mechanisms of action:

(a) SIRT2 inhibitor Front. Mol. Neurosci. vol. 11, 381 (2018) states that among sirtuins (SIRTs), inhibition of SIRT2 exerts neuroprotective effects in Parkinson's disease (PD), that SIRT2 protein levels are highly increased in PD models, and that miR-212-5p It has been described that it is negatively controlled by And based on the direct link between miR-212-5p and SIRT2-mediated p53-dependent programmed cell death in the pathogenesis of PD, SIRT2 inhibitors represent a new therapeutic tool for PD. Specifically, miR-212-5p transfection reduces SIRT2 expression, inhibits SIRT2 activity, and prevents dopaminergic neuron loss and DAT decline. That is, miR-212-5p exhibits neuroprotective effects in PD. The mechanism of action is that nuclear acetylated p53 is upregulated by p53, which is the major deacetylation substrate of SIRT2. Furthermore, reduction of cytoplasmic p53 promotes autophagy in PD models. In contrast, miR-212-5p attenuates apoptosis in PD models.
When the microparticles of the present invention contain miR-212-5p, they function as SIRT2 inhibitors.

(b) Inhibitor of degeneration or apoptosis of dopaminergic neurons Molecular Therapy: Nucleic Acids, Vol. 11 p9-22 (2018) states that degeneration of dopaminergic neurons occurs in PD and that glial cell line-derived neurotrophic factor (GDNF) increases the survival of dopaminergic neurons in PD and improves primary mesencephalic neurons. It has been described that the expression of miR-182-5p and miR-183-5p is specifically increased in (PMN). And it has been described that transfection of miR-182-5p and miR-183-5p mimics results in increased neurite outgrowth and mediates neuroprotection of dopaminergic neurons, mimicking the GDNF effect. Furthermore, it has been described that inhibition of endogenous miR-182-5p or miR-183-5p in PMNs treated with GDNF attenuated the pro-DAergic effects of GDNF. Based on these facts, miR-182-5p and miR-183-5p serve as new therapeutic means for PD.
When the microparticles of the present invention contain miR-182-5p or miR-183-5p, they function as an inhibitor of dopaminergic neuron degeneration or apoptosis.

(c) Supplementation of miRNA whose expression is suppressed in Parkinson's disease Neural Regen. Res. , 12, (12), p1945-1959 (2017) describes miRNAs that are downregulated in brain tissue of Parkinson's disease. Supplementing such miRNAs provides a new means of treatment for PD.
The microparticles of the present invention function as a supplement for miRNAs that are downregulated in Parkinson's disease brain tissue. In particular, the microparticles of the present invention include hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p , hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa -If it contains at least one of miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543, Parkinson's It functions as a supplement for miRNAs that are down-regulated in diseased brain tissue.

(d) Replenishment of L-DOPA and dopamine agonists: By taking L-DOPA (levodopa), a precursor of dopamine, it enters the brain from the blood and replenishes the dopamine that is deficient in the brain in patients with Parkinson's disease. is the treatment (dopamine does not enter the brain from the blood). In addition, taking a dopamine agonist (dopamine receptor stimulant), which has a longer action time than L-DOPA, can be used as a similar treatment.
Note that supplementation with L-DOPA and dopamine agonists has little relevance to the microparticles of the present invention.

(e) Anticholinergic Drugs In Parkinson's disease, as dopamine decreases, acetylcholine, another neurotransmitter, becomes relatively excessive, so methods to reduce the effects of acetylcholine are a means of treatment.
Note that anticholinergic drugs have little relevance to the microparticles of the present invention.

(f) Amantadine Hydrochloride Amantadine hydrochloride has the effect of promoting dopamine release in the striatum, and is also known to have the effect of suppressing L-DOPA-induced involuntary movements (dyskinesia) in which the body moves on its own.
Note that amantadine hydrochloride has little relevance to the microparticles of the present invention.

(g) Medications used in combination with L-DOPA Zonisamide (which improves Parkinson's symptoms) is known to be effective in improving wear-off and tremors when the effects of L-DOPA wear off when used in combination with L-DOPA. (The mechanism of action has not been completely elucidated), and adenosine receptor antagonists are known to be effective in improving wear-off when the effects of L-DOPA wear off. There are also drugs known to inhibit dopa decarboxylase (DDC), an enzyme in the blood that breaks down L-DOPA, and inhibitors of catechol-O-methyltransferase (COMT), an enzyme in the blood that breaks down L-DOPA. It is being
Note that the drugs used in combination with L-DOPA as exemplified above have little relevance to the microparticles of the present invention.

(h) MAO-B inhibitor Selegiline hydrochloride, an MAO-B inhibitor, reduces the activity of MAO and suppresses the decomposition of dopamine, so taking it serves as a therapeutic means. MAO-B inhibitors also inhibit the breakdown of other neurotransmitters such as norepinephrine and serotonin.
Note that MAO-B inhibitors have little relevance to the microparticles of the present invention.

(i) Droxidopa Donorepinephrine is a different neurotransmitter than dopamine. However, norepinephrine is synthesized from dopamine by β-hydroxylase, so it becomes insufficient when dopamine decreases. By taking droxidopa, which is a norepinephrine precursor, norepinephrine can be supplemented and the stiffness of the legs caused by norepinephrine deficiency can be improved.
Note that droxidopa has little relevance to the microparticles of the present invention.

<Details of microparticles>
miRNA, which is an active ingredient of the microparticles of the present invention, is contained in the microparticles.
The microparticles of the present invention are derived from dental pulp-derived stem cells, for example, by secretion, budding, or dispersion from mesenchymal stem cells such as dental pulp-derived stem cells, and are leached, released, or shed into the cell culture medium. The microparticles are preferably contained in the culture supernatant of dental pulp-derived stem cells, and more preferably are microparticles derived from the culture supernatant of dental pulp-derived stem cells. However, the microparticles derived from the culture supernatant of dental pulp-derived stem cells do not necessarily need to be obtained from the culture supernatant of dental pulp-derived stem cells. For example, even if the microparticles inside dental pulp-derived stem cells are isolated by any method, if they are the same as the microparticles that can be isolated from the culture supernatant of dental pulp-derived stem cells, then It can be said that they are microparticles derived from water.
Microparticles derived from a culture supernatant such as dental pulp-derived stem cells may be used in a state contained in the culture supernatant, or may be used in a state purified from the culture supernatant. Preferably, the microparticles are microparticles purified from culture supernatant.
The origin of the microparticles can be determined by a known method. For example, it can be determined whether the microparticles are derived from stem cells such as dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, or umbilical cord-derived stem cells using the method described in J Stem Cell Res Ther (2018) 8:2. I can do it. Specifically, the origin of each microparticle can be determined based on the miRNA pattern of the microparticle.

(miRNA)
In the present invention, the microparticles contain miRNA whose expression is suppressed in Parkinson's disease or miRNA used in the treatment of Parkinson's disease.
In the present invention, miRNA (MicroRNAs) is, for example, an RNA molecule of 21 to 25 bases (nucleotides). miRNAs can regulate gene expression by suppressing target gene (target) mRNA degradation or decoding steps.
In the present invention, miRNA may be single-stranded (monomer) or double-stranded (dimer). Furthermore, in the present invention, the miRNA is preferably a mature miRNA that has been cleaved with a ribonuclease such as Dicer.

Note that the sequences of miRNAs described herein, such as hsa-miR-127-5p, are registered in known databases (e.g., miRBase database) in association with accession numbers, and those skilled in the art will be able to identify the sequences. It can be defined uniquely. For example, the accession number of hsa-miR-127-5p is MI0000472, and the sequence is registered in miRBase database. Hereinafter, the accession number of each miRNA will be omitted.
However, miRNA in this specification also includes variants that differ by about 1 to 5 bases from mature miRNA such as hsa-miR-127-5p. In addition, each miRNA in this specification refers to a polynucleotide consisting of a base sequence having identity with the base sequence of each miRNA (for example, hsa-miR-127-5p), or a polynucleotide consisting of a complementary base sequence thereof. It includes a polynucleotide having the function of miRNA in the present invention. "Identity" refers to the degree of identity when the sequences to be compared are properly aligned, and refers to the incidence (%) of exact amino acid matches between the sequences. Alignment can be performed, for example, by using any algorithm such as BLAST. Identity is, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99%. Polynucleotides consisting of identical base sequences may have, for example, point mutations, deletions, and/or additions in the miRNA base sequence. The number of bases in the point mutation, etc. is, for example, 1 to 5, 1 to 3, 1 to 2, or 1. In addition, the polynucleotide consisting of a complementary base sequence is, for example, a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence of miRNA, and includes a polynucleotide having the function of miRNA in the present invention. . Stringent conditions are not particularly limited, and include, for example, the conditions described in [0028] of JP-A-2017-184642, the contents of which are incorporated herein by reference.

In the present invention, it is preferable that the microparticles contain miRNA targeting a gene related to Parkinson's disease at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
Hereinafter, preferred embodiments of miRNA contained in the microparticles will be described.

In the present invention, it is more preferable that the microparticles contain at least one type of the following Parkinson's disease-related miRNA group;
Parkinson's disease-related miRNA group:
hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-212-5p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa- miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543.

(1) SIRT2 inhibitor In the present invention, it is preferable that the microparticle is a SIRT2 inhibitor and contains hsa-miR-212-5p from the Parkinson's disease-related miRNA group.
The microparticles preferably contain hsa-miR-212-5p as Log2Ratio of read counts obtained by analysis using IMOTA of 0.1 or more, more preferably 0.5 or more, and 1.0 It is particularly preferable to include the above.

The microparticles of the present invention express 1.1 times more hsa-miR-212-5p than exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. It is preferably at least 1.5 times, more preferably at least 1.5 times, particularly preferably at least 2 times.

When the microparticles of the present invention are SIRT2 inhibitors, it is preferable that the expression of the SIRT2 gene in any cell can be suppressed to 0.8 times or less, and preferably 0.6 times or less compared to normal (in untreated cells). More preferably, it can be suppressed to 0.4 times or less, and particularly preferably to 0.4 times or less.

(2) Inhibitor of degeneration or apoptosis of dopaminergic neurons In the present invention, the microparticles are inhibitors of degeneration or apoptosis of dopaminergic neurons, and hsa-miR-182 among Parkinson's disease-related miRNA group -5p or hsa-miR-183-5p, and more preferably both hsa-miR-182-5p and hsa-miR-183-5p.
It is preferable that the microparticles independently contain hsa-miR-182-5p or hsa-miR-183-5p as Log2Ratio of read counts obtained by analysis using IMOTA of 4.0 or more, and 10. It is more preferable to contain 0 or more, and it is particularly preferable to contain 15.0 or more.

In the present invention, the expression level of hsa-miR-182-5p or hsa-miR-183-5p is compared with exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. is preferably 1.1 times or more, more preferably 1.5 times or more, particularly preferably 2 times or more.

(3) Supplementation of miRNAs whose expression is suppressed in Parkinson's disease If microparticles function as a supplement for miRNAs whose expression is downregulated in Parkinson's disease brain tissue, hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330- 5p, hsa-miR-337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR- It is preferable that at least one of 485-5p, hsa-miR-542-3p, and hsa-miR-543 is included. Furthermore, among the Parkinson's disease-related miRNA group, it may contain any one of hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p. More preferably, it is particularly preferred to contain at least hsa-miR-199a-3p.

The microparticle preferably contains at least one type of miRNA that is down-regulated in Parkinson's disease brain tissue as a Log2Ratio of read counts obtained by analysis using IMOTA of 4.0 or more, and preferably 10.0 or more. It is more preferable to include 15.0 or more, particularly preferably 15.0 or more. Microparticles are hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p as Log2 Ratio of read count number obtained by analysis using IMOTA. It is preferable that each of them contains 4.0 or more, and it is particularly preferable that hsa-miR-199a-3p contains 15.0 or more.

The microparticles of the present invention express 1.1 times more hsa-miR-199a-3p than exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. It is preferably at least 1.5 times, more preferably at least 1.5 times, particularly preferably at least 2 times.

(Type of miRNA)
Here, the microparticles derived from the culture supernatant of dental pulp-derived stem cells contain about 2600 types of small RNA. Approximately 1800 of these are miRNAs. Among these miRNAs, there are 180 to 200 types of miRNAs that are abundant. The high content of miRNA in microparticles derived from dental pulp-derived stem cells is characterized by the fact that there are many microRNAs related to treatment of cranial nerve diseases and eye diseases. This is my knowledge. This feature is significantly different from the types of miRNA that are contained in large amounts in other mesenchymal stem cell microparticles. For example, miRNAs that are abundant in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells contain almost no microRNAs related to treatment of cranial nerve diseases or eye diseases.

The microparticles preferably contain 5 or more types of miRNAs whose expression is suppressed in Parkinson's disease or miRNAs used for the treatment of Parkinson's disease, more preferably 10 or more types, and especially 15 or more types. Preferably, it is particularly preferable that 20 or more types are included. Furthermore, the microparticles preferably contain 5 or more types of the aforementioned Parkinson's disease-related miRNA group, more preferably 10 or more types, particularly preferably 15 or more types, and 20 or more types. is particularly preferred.

(Type of microparticles)
The microparticles are preferably at least one type selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, exovesicles, or microvesicles. , more preferably exosomes.
The diameter of the microparticles is preferably 10 to 1000 nm, more preferably 30 to 500 nm, particularly preferably 50 to 150 nm.
Furthermore, it is desirable that molecules called tetraspanins such as CD9, CD63, and CD81 exist on the surface of the microparticles, and it may be CD9 alone, CD63 alone, CD81 alone, or any combination of two or three thereof. good.
Hereinafter, preferred embodiments of the case where exosomes are used as microparticles may be described, but the microparticles used in the present invention are not limited to exosomes.

Preferably, exosomes are extracellular vesicles released from cells upon fusion of multivesicular bodies with the plasma membrane.
The surface of the exosome preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells.
The interior of the exosome preferably contains intracellular substances of dental pulp-derived stem cells, such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins.
Exosomes are known to be used for cell-to-cell communication by transporting genetic information from one cell to another. Exosomes are easily traceable and can be targeted to specific regions.

(Content of fine particles)
The content of microparticles in the microparticle composition is not particularly limited. The microparticle composition preferably contains 0.5 x 10 ⁸ or more microparticles, more preferably 1.0 x 10 ⁸ or more, particularly preferably 2.0 x 10 ⁸ or more, It is particularly preferable to contain 2.5×10 ⁸ or more, and even more particularly preferably to contain 1.0×10 ⁹ or more.
Further, there is no particular restriction on the concentration of fine particles contained in the fine particle composition. The microparticle composition preferably contains 1.0×10 ⁸ particles/mL or more, more preferably 2.0×10 ⁸ particles/mL or more, and 4.0×10 ⁸ particles/mL or more. It is particularly preferable to contain 5.0×10 ⁸ pieces/mL or more, and it is even more particularly preferable to contain 2.0×10 ⁹ pieces/mL or more.
A preferred embodiment of the microparticles of the present invention can maintain a high amount of miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease by containing the microparticles in such a large amount or at a high concentration.

<Other ingredients>
The microparticle composition may contain other components in addition to the microparticles, depending on the type of animal to be administered and the purpose, to the extent that the effects of the present invention are not impaired. Other ingredients include nutritional ingredients, antibiotics, cytokines, protective agents, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, etc. It will be done.
Examples of nutritional components include fatty acids, vitamins, and the like.
Examples of antibiotics include penicillin, streptomycin, and gentamicin.
The carrier may include materials known as pharmaceutically acceptable carriers.
The microparticle composition may be the culture supernatant of dental pulp-derived stem cells itself or the microparticles themselves, or may be a pharmaceutical composition further containing a pharmaceutically acceptable carrier, excipient, and the like. The purpose of the pharmaceutical composition is to facilitate administration of microparticles to the recipient.

Preferably, the pharmaceutically acceptable carrier is a carrier (including diluents) that does not cause significant irritation to the administered subject and does not inhibit the biological activity and properties of the administered compound. Examples of carriers are propylene glycol; (physiological) saline; emulsions; buffers; media, such as DMEM or RPMI; cryopreservation media containing components that scavenge free radicals.

The microparticle composition may contain an active ingredient of a conventionally known therapeutic agent for Parkinson's disease. Those skilled in the art can make appropriate changes depending on the intended use, administration target, etc.

On the other hand, it is preferable that the microparticle composition does not contain the predetermined substance.
For example, the microparticle composition preferably does not contain dental pulp-derived stem cells.
Furthermore, the microparticle composition preferably does not contain MCP-1. However, it may contain cytokines other than MCP-1. Other cytokines include those described in [0014] to [0020] of JP 2018-023343A.
Moreover, it is preferable that the microparticle composition does not contain Siglec-9. However, it may contain other sialic acid-binding immunoglobulin-like lectins other than Siglec-9.
Note that the microparticle composition preferably does not substantially contain serum (fetal bovine serum, human serum, sheep serum, etc.). It is also preferred that the microparticle composition is substantially free of conventional serum replacements, such as Knockout serum replacement (KSR).
In the microparticle composition, the content (solid content) of the other components described above is preferably 1% by mass or less, more preferably 0.1% by mass or less, and 0.01% by mass or less. It is particularly preferable that

<Method for manufacturing microparticles>
There are no particular limitations on the method for producing microparticles.
The microparticles of the present invention may be prepared by preparing a culture supernatant of dental pulp-derived stem cells, etc., and then purifying microparticles from the culture supernatant of dental pulp-derived stem cells. Alternatively, the microparticles of the present invention may be prepared by purifying microparticles from the culture supernatant of commercially purchased dental pulp-derived stem cells. Furthermore, the microparticles of the present invention are prepared by receiving a composition containing the culture supernatant of dental pulp-derived stem cells that had been disposed of (or purifying the composition as appropriate) and refining the microparticles therefrom. You may.

(Method for preparing culture supernatant of dental pulp-derived stem cells, etc.)
There are no particular limitations on the culture supernatant of dental pulp-derived stem cells or the like.
It is preferable that the culture supernatant of dental pulp-derived stem cells and the like is substantially free of serum. For example, the serum content of the culture supernatant of dental pulp-derived stem cells is preferably 1% by mass or less, more preferably 0.1% by mass or less, and preferably 0.01% by mass or less. Particularly preferred.

Dental pulp-derived stem cells may be derived from humans or animals other than humans. Examples of animals other than humans include those similar to the animals (species) to which the microparticles of the present invention are administered, which will be described later, and mammals are preferred.

There are no particular limitations on the dental pulp-derived stem cells used in the culture supernatant. Stem cells from exfoliated deciduous teeth, dental pulp stem cells from deciduous teeth obtained by other methods, and dental pulp stem cells (DPSCs) from permanent teeth can be used. In addition to human deciduous tooth pulp stem cells and human permanent tooth pulp stem cells, dental pulp-derived stem cells derived from animals other than humans, such as pig deciduous tooth pulp stem cells, can be used.
In addition to exosomes, dental pulp-derived stem cells contain vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-). β)-1 and -3, TGF-α, KGF, HBEGF, SPARC, other growth factors, and chemokines. It can also produce many other physiologically active substances.
In the present invention, it is particularly preferable that the dental pulp-derived stem cells used in the culture supernatant of dental pulp-derived stem cells are dental pulp-derived stem cells that contain many proteins, and it is preferable to use dental pulp stem cells of deciduous teeth. That is, in the present invention, it is preferable to use a culture supernatant of deciduous tooth pulp stem cells.

The dental pulp-derived stem cells used in the present invention may be natural or genetically modified, as long as the desired treatment can be achieved.
In particular, in the present invention, immortalized stem cells derived from dental pulp can be used. By using immortalized stem cells that can proliferate virtually indefinitely, the amount and composition of biological factors contained in the culture supernatant of stem cells can be stabilized over a long period of time. There are no particular restrictions on the immortalized stem cells derived from dental pulp. The immortalized stem cells are preferably immortalized stem cells that have not become cancerous. Immortalized stem cells of dental pulp-derived stem cells can be prepared by adding the following low molecular weight compounds (inhibitors) alone or in combination to dental pulp-derived stem cells and culturing them.
The TGFβ receptor inhibitor is not particularly limited as long as it has the effect of inhibiting the function of the transforming growth factor (TGF)β receptor, and for example, 2-(5-benzo[1,3 ] Dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1- Phenylthiocarbamoyl-1H-pyrazole (A-83-01), 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridine -2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine( Merck & Co.), SB431542 (Sigma-Aldrich), and the like. Preferred is A-83-01.
The ROCK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of Rho-binding kinase. Examples of ROCK inhibitors include GSK269962A (Axonmedchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (Fujifilm Wako Pure Chemical Industries, Ltd.). Preferred is Y-27632.
GSK3 inhibitors are not particularly limited as long as they inhibit GSK-3 (Glycogen Synthase Kinase 3), and include A 1070722, BIO, BIO-acetoxime (TOCRIS), etc. can be mentioned.
MEK inhibitors are not particularly limited as long as they have the effect of inhibiting the function of MEK (MAP kinase-ERK kinase), such as AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86 -9766), SL327, U0126-EtOH (all sold by Selleck), PD98059, U0124, and U0125 (all sold by Cosmo Bio Inc.).

When using the microparticles of the present invention in regenerative medicine, in accordance with the requirements of the Regenerative Medicine Safety Act, dental pulp-derived stem cells, culture supernatants of these immortalized stem cells, and compositions containing microparticles derived therefrom must be This embodiment does not contain any other somatic stem cells other than derived stem cells. Although the microparticle composition may contain mesenchymal stem cells and other somatic stem cells other than dental pulp-derived stem cells, it is preferable not to contain them.
Examples of somatic stem cells other than mesenchymal stem cells include, but are not limited to, stem cells derived from the dermal system, digestive system, bone marrow system, nervous system, etc. Examples of dermal somatic stem cells include epithelial stem cells, hair follicle stem cells, and the like. Examples of somatic stem cells of the digestive system include pancreatic (general) stem cells, hepatic stem cells, and the like. Examples of myeloid somatic stem cells (other than mesenchymal stem cells) include hematopoietic stem cells and the like. Examples of somatic stem cells of the nervous system include neural stem cells, retinal stem cells, and the like.
The microparticle composition may contain stem cells other than somatic stem cells, but preferably does not contain them. Stem cells other than somatic stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).

There are no particular limitations on the method for preparing the culture supernatant of dental pulp-derived stem cells or immortalized stem cells, and conventional methods can be used.
A culture supernatant such as dental pulp-derived stem cells is a culture solution obtained by culturing dental pulp-derived stem cells. For example, a culture supernatant that can be used in the present invention can be obtained by separating and removing cell components after culturing dental pulp-derived stem cells. A culture supernatant that has been appropriately subjected to various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) may be used.

Dental pulp-derived stem cells for obtaining a culture supernatant of dental pulp-derived stem cells can be selected by conventional methods, and can be selected based on cell size and morphology, or as adherent cells. Dental pulp cells collected from fallen baby teeth or permanent teeth can be sorted as adherent cells or their successor cells. As the culture supernatant of dental pulp-derived stem cells, a culture supernatant obtained by culturing selected stem cells can be used.

The "culture supernatant of dental pulp-derived stem cells, etc." is preferably a culture solution that does not contain the cells themselves obtained by culturing dental pulp-derived stem cells, etc. In one embodiment, the culture supernatant of dental pulp-derived stem cells used in the present invention preferably does not contain any cells (regardless of the type of cells) as a whole. Due to this feature, the composition of this embodiment is clearly distinguished from dental pulp-derived stem cells themselves as well as from various compositions containing dental pulp-derived stem cells. A typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and is composed only of a culture supernatant of dental pulp-derived stem cells.
The culture supernatant of dental pulp-derived stem cells used in the present invention may contain culture supernatants of both deciduous dental pulp-derived stem cells and adult dental pulp-derived stem cells. The culture supernatant of dental pulp-derived stem cells used in the present invention preferably contains the culture supernatant of deciduous dental pulp-derived stem cells as an active ingredient, more preferably 50% by mass or more, and preferably 90% by mass or more. It is particularly preferable that the culture supernatant of dental pulp-derived stem cells used in the present invention is a composition composed only of the culture supernatant of dental pulp-derived stem cells of deciduous teeth.

As a culture solution for dental pulp-derived stem cells to obtain a culture supernatant, a basic medium or a basic medium to which serum or the like is added can be used. In addition to Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM) (GIBCO, etc.), Ham F12 medium (HamF12, SIGMA, GIBCO, etc.), RPMI1640 medium, etc. may be used as the basic medium. I can do it. In addition, examples of components that can be added to the culture medium include serum (fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various Vitamins and various minerals can be mentioned.
However, in order to prepare a "culture supernatant of dental pulp-derived stem cells" that does not contain serum, a serum-free medium may be used throughout the entire process or during the last or several subcultures from the end. For example, by culturing dental pulp-derived stem cells in a serum-free medium (serum-free medium), a serum-free culture supernatant of dental pulp-derived stem cells can be prepared. It is also possible to obtain serum-free culture supernatant of dental pulp-derived stem cells, etc. by performing one or more subcultures and culturing the last or several subcultures in a serum-free medium. I can do it. On the other hand, a serum-free culture supernatant of dental pulp-derived stem cells can also be obtained by removing serum from the collected culture supernatant using dialysis, solvent replacement using a column, or the like.

For culturing dental pulp-derived stem cells to obtain a culture supernatant, commonly used conditions can be applied as they are. The method for preparing the culture supernatant of dental pulp-derived stem cells may be the same as the cell culture method described below, except that the stem cell isolation and selection steps are appropriately adjusted depending on the type of stem cells. Those skilled in the art can appropriately isolate and select dental pulp-derived stem cells according to the type of the dental pulp-derived stem cells.
Furthermore, special conditions may be applied to culturing dental pulp-derived stem cells in order to produce a large amount of microparticles such as exosomes. Examples of special conditions include conditions for co-cultivation with some stimulus, such as low temperature conditions, hypoxic conditions, microgravity conditions, and the like.

The culture supernatant of dental pulp-derived stem cells used in the preparation of microparticles such as exosomes in the present invention may contain other components in addition to the culture supernatant of dental pulp-derived stem cells, but it does not substantially contain other components. Preferably not.
However, various types of additives used for preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.

(Preparation of microparticles)
Microparticles can be prepared by purifying microparticles from culture supernatants such as dental pulp-derived stem cells.

Purification of microparticles is preferably separation of a fraction containing microparticles from the culture supernatant of dental pulp-derived stem cells, and more preferably isolation of microparticles.
Microparticles can be isolated by separating them from unassociated components based on the properties of the microparticles. For example, microparticles can be isolated based on molecular weight, size, morphology, composition, or biological activity.
In the present invention, microparticles can be purified by separating a specific fraction (eg, precipitate) containing many microparticles obtained by centrifuging the culture supernatant of dental pulp-derived stem cells. Unnecessary components (insoluble components) in fractions other than the predetermined fractions may be removed. Removal of solvents and dispersion media and unnecessary components from the microparticle composition may not be complete. Examples of conditions for centrifugation are 100 to 20,000 g and 1 to 30 minutes.
In the present invention, microparticles can be purified by filtering a culture supernatant of dental pulp-derived stem cells or a centrifuged product thereof. Unnecessary components can be removed by filtration treatment. Furthermore, by using a filtration membrane with an appropriate pore size, removal of unnecessary components and sterilization can be performed at the same time. The material, pore size, etc. of the filtration membrane used for the filtration treatment are not particularly limited. Filtration can be carried out using filtration membranes of appropriate molecular weight or size cut-off using known methods. The pore size of the filtration membrane is preferably from 10 to 1000 nm, more preferably from 30 to 500 nm, and particularly preferably from 50 to 150 nm, from the viewpoint of easy separation of exosomes.
In the present invention, a culture supernatant of dental pulp-derived stem cells, a centrifuged product thereof, or a filtered product thereof can be separated using an additional separation means such as lamb chromatography. For example, high performance liquid chromatography (HPLC) using various columns can be used. The column can be a size exclusion column or a binding column.
One or more properties or biological activities of the microparticles can be used to track the microparticles (or their activity) in each fraction at each processing step. For example, light scattering, refractive index, dynamic light scattering or UV-visible light detectors can be used to track microparticles. Alternatively, specific enzyme activities, etc. can be used to track the activity in each fraction.
As a method for purifying microparticles, the method described in [0034] to [0064] of Japanese Patent Publication No. 2019-524824 may be used, and the contents of this publication are incorporated herein by reference.

The final form of the microparticle composition is not particularly limited. For example, the microparticle composition can be formed by filling a container with microparticles together with a solvent or dispersion medium; by gelling the microparticles together with a gel and filling the container; by freezing and/or drying the microparticles. For example, it may be solidified into a formulation or filled into a container. Examples of containers include tubes, centrifuge tubes, bags, etc. suitable for cryopreservation. The freezing temperature can be, for example, -20°C to -196°C.

The microparticles of the present invention can be easily mass-produced compared to conventional compositions that can be used as therapeutic or preventive drugs for Parkinson's disease, and can be used in stem cell culture fluids that were previously discarded as industrial waste. It has advantages such as being able to be used for various purposes and reducing the cost of disposing of stem cell culture fluid. In particular, when the culture supernatant of dental pulp-derived stem cells is the culture supernatant of human dental pulp-derived stem cells, the microparticles of the present invention are highly safe from an immunological standpoint when applied to humans. Another advantage is that there are fewer ethical issues. If the culture supernatant of dental pulp-derived stem cells is a culture supernatant of dental pulp-derived stem cells from a patient with dementia, the microparticles of the present invention will be safer and more ethical when applied to that patient. There will be fewer problems.
When the microparticles of the present invention are derived from the culture supernatant of dental pulp-derived stem cells, they can also be used in restorative medicine. In particular, compositions containing microparticles derived from culture supernatants such as dental pulp-derived stem cells are preferably used for restorative medical applications. In regenerative medicine based on stem cell transplantation, it is known that stem cells are not the main actors in regeneration, but rather that the humoral components produced by stem cells repair organs together with one's own stem cells. Difficult issues associated with conventional stem cell transplantation, such as canceration, standardization, administration methods, storage stability, and culture methods, have been resolved, and restorations can be achieved using a composition using the culture supernatant of dental pulp-derived stem cells or microparticles derived therefrom. Medical care becomes possible. Compared to stem cell transplantation, when using the microparticles of the present invention, tumor formation is less likely to occur because cells are not transplanted, and it can be said to be safer. Furthermore, the microparticles of the present invention have the advantage of being of uniformly standardized quality. Since mass production and efficient administration methods can be selected, it can be used at low cost.

[Prophylactic or therapeutic drug for Parkinson's disease]
The prophylactic or therapeutic agent for Parkinson's disease of the present invention contains the microparticles of the present invention.
As used herein, "prevention" refers to preventing the onset of a disease (neurodegenerative disease in this specification). In addition, as used herein, "treatment" refers to alleviating, suppressing, or preventing the progression of symptoms of a disease that has developed, and improving symptoms.

[How to improve Parkinson's disease]
The method for improving Parkinson's disease of the present invention includes administering an effective amount of the microparticles of the present invention or an effective amount of the prophylactic or therapeutic agent for Parkinson's disease of the present invention to a subject who has developed Parkinson's disease.

There are no particular restrictions on the process of administering the microparticles of the present invention to a subject who has developed Parkinson's disease.
Administration methods include spraying or inhalation into the oral cavity, nasal cavity, or respiratory tract, dripping, local administration, nasal spray, etc., and minimally invasive methods are preferred. Injection is a preferred method of local administration. Also preferred is electroporation, which temporarily creates minute holes in cell membranes by applying voltage (electrical pulses) to the skin surface, allowing active ingredients to penetrate into the dermal layer, which cannot be reached with normal skin care. In the case of local administration, examples include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, etc.; intraarterial administration, intravenous administration, subcutaneous administration or Intraperitoneal administration is more preferred.
Also, various formulation techniques can be used to alter the in vivo distribution of microparticles. Many methods of altering in vivo distribution are known to those skilled in the art. Examples of such methods include protection of exosomes in vesicles composed of materials such as proteins, lipids (eg, liposomes), carbohydrates or synthetic polymers.
The microparticles of the present invention administered to a subject who has developed Parkinson's disease may circulate within the subject's body and reach a predetermined tissue.
There are no particular restrictions on the number of administrations or the interval between administrations. The frequency of administration can be one or more times per week, preferably five or more times, more preferably six or more times, particularly preferably seven or more times. The administration interval is preferably 1 hour to 1 week, more preferably half a day to 1 week, and particularly preferably 1 day (once a day). However, it can be adjusted as appropriate depending on the species to be administered and the symptoms of the subject.
The microparticles of the present invention are preferably used to administer the microparticles to a subject who has developed dementia at least once a week over a therapeutically effective period. When the subject to be administered is a human, the number of administrations per week is preferably large, preferably 5 or more times per week over the effective therapeutic period, and preferably daily.
When using a culture supernatant of dental pulp-derived stem cells at a concentration of 2.0 × 10 ⁹ cells/ml, in a mouse model, the amount is preferably 0.1 to 5 ml per mouse (about 25 g), and 0.3 to 5 ml per mouse (approximately 25 g). The amount is more preferably 3 ml, and particularly preferably 0.5 to 1 ml.
When using microparticles at a concentration of 0.1×10 ⁸ particles/μg, in mouse models, the amount is preferably 1 to 50 μg, more preferably 3 to 30 μg, and more preferably 5 to 50 μg per mouse (approximately 25 g). More particularly preferred is ~25 μg.
The preferred range of dosage per body weight for other animals can be calculated from the dosage per body weight (about 25 g) for model mice using a proportional relationship. However, it can be adjusted as appropriate depending on the symptoms of the subject.

There are no particular restrictions on the animal (species) to which the microparticles of the present invention are administered. The animals to which the microparticles of the present invention are administered are preferably mammals, birds (chickens, quail, ducks, etc.), and fish (salmon, trout, tuna, bonito, etc.). The mammal may be a human or a non-human mammal, but humans are particularly preferred. More preferably, the non-human mammal is a cow, pig, horse, goat, sheep, monkey, dog, cat, mouse, rat, guinea pig, or hamster.

The microparticles of the present invention may be used in combination with conventionally known therapeutic agents for Parkinson's disease. Specifically, it may be used in combination with, for example, L-DOPA (levodopa).

The features of the present invention will be explained in more detail below with reference to Examples and Comparative Examples or Reference Examples. The materials, usage amounts, proportions, processing details, processing procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be interpreted as being limited by the specific examples shown below.

[Example 1]
<Preparation of culture supernatant of dental pulp-derived stem cells>
Using DMEM medium instead of the DMEM/HamF12 mixed medium and otherwise following the method described in Example 6 of Patent No. 6296622, a culture supernatant of human deciduous dental pulp stem cells was prepared, and the culture supernatant was fractionated. did. In primary culture, fetal bovine serum (FBS) is added to the culture, and in subculture, the supernatant of the subculture medium is cultured using the primary culture medium, and the supernatant of the subculture medium is separated so that it does not contain FBS. culture supernatant was prepared. Note that DMEM is Dulbecco's modified Eagle's medium, and F12 is Ham's F12 medium.

<Preparation of exosomes>
From the obtained culture supernatant of dental pulp-derived stem cells, exosomes of dental pulp-derived stem cells were purified and collected by the following ultracentrifugation method.
After filtering the culture supernatant (100 mL) of deciduous dental pulp stem cells through a filter with a pore size of 0.22 micrometers, the solution was centrifuged at 100,000×g at 4° C. for 60 minutes. The supernatant was decanted and the exosome enriched pellet was resuspended in phosphate buffered saline (PBS). The resuspended sample was centrifuged at 100,000 xg for 60 minutes. The pellet was again collected from the bottom of the centrifuge tube as a concentrated sample (approximately 100 μl). Protein concentration was determined by microBSA protein assay kit (Pierce, Rockford, IL). The composition containing exosomes (concentrated solution) was stored at -80°C.
A composition containing exosomes purified from the culture supernatant of dental pulp-derived stem cells was used as the microparticle composition sample of Example 1.

The average particle diameter and concentration of the microparticles contained in the microparticle composition of Example 1 were evaluated using a nanoparticle analysis system NanoSight (manufactured by Nippon Quantum Design Co., Ltd.).
The average particle size of the microparticles contained in the microparticle composition of Example 1 was 50 to 150 nm.
The microparticle composition of Example 1 was a high concentration exosome solution of 1.0×10 ⁹ particles/ml or more, specifically, a high concentration exosome solution of 2.0×10 ⁹ particles/ml.
In addition, the components of the obtained microparticle composition of Example 1 were analyzed by a known method. As a result, it was found that the microparticles of Example 1 did not contain stem cells of dental pulp-derived stem cells, did not contain MCP-1, and did not contain Siglec-9. Therefore, it was found that an active ingredient different from MCP-1 and Siglec-9, which are the active ingredients of the culture supernatant of mesenchymal stem cells, and their analogs, was the active ingredient of the microparticle composition of Example 1. .

[Test Example 1]: MicroRNA expressed in exosomes
Small RNA contained in the microparticle composition of Example 1 was analyzed by next generation sequencing (NGS) analysis. NGS analysis identified 1787 miRNAs contained in the microparticle composition (dental pulp-derived stem cell exosomes) of Example 1. The results obtained are shown in Table 1 below.

[Test Example 2]: Comparison with miRNA in cerebral cortex IMOTA (Interactive Multi-Omics-Tissue Atlas) was used for microRNA analysis.
MicroRNAs that control proteins were searched for using IMOTA, focusing on the cerebral cortex. IMOTA is an interactive multi-omics atlas that allows you to investigate interactions and expression levels of miRNAs, mRNAs, and proteins in each tissue and cell (Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018, Pages D770- D775, "IMOTA: an interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions"). In the cerebral cortex, the Omic counts were 3071 proteins, 14182 mRNAs, and 1124 miRNAs. Furthermore, the counts of Omics with a high expression rate in the cerebral cortex were 1119 for proteins, 1110 for mRNA, and 145 for miRNA.

A common difference between 1124 miRNAs expressed in the cerebral cortex and 1787 miRNAs expressed in exosomes (microparticle composition of Example 1; SGF exosome) purified from the culture supernatant of dental pulp-derived stem cells. There were 859 miRNAs expressed. That is, among the miRNAs expressed in the microparticle composition of Example 1 (exosomes of dental pulp-derived stem cells), the proportion of miRNAs also expressed in the cerebral cortex was 48.1%, which was very high.

Additionally, there were 122 miRNAs expressed in the microparticle composition of Example 1 (exosomes of dental pulp-derived stem cells) and highly expressed in the cerebral cortex as shown in Tables 2 and 3 below.

[Test Example 3]: Search for microRNAs involved in diseases Based on the obtained analysis results, microRNAs involved in diseases were searched. Extraction of disease-related miRNAs was performed using IMOTA. Here, we investigated whether microRNAs that suppress proteins related to Parkinson's disease are contained, or microRNAs that control proteins that are targets of therapeutic drugs. In this example, microRNA, which is a gene related to Parkinson's disease, was searched. Non-patent document 1 (Front. Mol. Neurosci. vol. 11, 381 (2018)) and non-patent document 2 (Molecular Therapy: Nucleic Acids, Vol. 11 p9-), which describe microRNAs that contribute to the treatment of Parkinson's disease. 22 (2018)), the expression levels of microRNAs expressed in the microparticle composition (SGF exosome) of Example 1 are shown in FIG.
Furthermore, among the miRNAs that are down-regulated in the brain tissue of Parkinson's disease described in Non-Patent Document 3 (Neural Regen. Res. 12 (12) p1945-1959 (2017)), the microparticle composition of Example 1 (SGF The expression levels of microRNAs expressed in exosomes are also shown in Figure 1.
Additionally, the expression level of miRNA expressed in the microparticle composition of Example 1 in the cerebral cortex is shown in FIG. 2. Blank rows in FIG. 2 indicate hsa-miR-182-5p and hsa-miR-183 among the miRNAs of the cerebral cortex registered in IMOTA that are expressed in the microparticle composition of Example 1. -5p, hsa-miR-330-5p, and hsa-miR-429 were absent.

From FIG. 1, the microparticle composition of Example 1 contains hsa-, which is described in Non-Patent Document 1 (Front. Mol. Neurosci. vol. 11, 381 (2018)) as a microRNA that contributes to the treatment of Parkinson's disease. miR-212-5p, as well as hsa-miR-182-5p and hsa-miR-183-5p described in Non-Patent Document 2 (Molecular Therapy: Nucleic Acids, Vol. 11 p9-22 (2018)) are expressed. I found out that there is.
Moreover, from FIG. 1, the microparticle composition of Example 1 has the effect of inhibiting Parkinson's disease brain cells ( hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155- among the 41 types of microRNAs that are down-regulated (down-regulated), i.e., hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155- 5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR- 337-5p, hsa-miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa- It was found that 18 types of miR-542-3p and hsa-miR-543 were expressed.

From the above, it was found that the microparticles of the present invention contain miRNAs whose expression is suppressed in Parkinson's disease or miRNAs used in the treatment of Parkinson's disease.

Claims (10)

1. Microparticles containing miRNA whose expression is suppressed in Parkinson's disease or miRNA used for the treatment of Parkinson's disease.
2. The microparticle according to claim 1, wherein the microparticle is an exosome.
3. The microparticle according to claim 1, wherein the microparticle contains miRNA that targets the gene related to Parkinson's disease at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
4. The microparticle according to claim 1, wherein the microparticle includes at least one type of the following Parkinson's disease-related miRNA group;
   Parkinson's disease-related miRNA group:
   hsa-miR-127-5p, hsa-miR-133b, hsa-miR-155-5p, hsa-miR-182-5p, hsa-miR-183-5p, hsa-miR-184, hsa-miR-198, hsa-miR-199a-3p, hsa-miR-212-5p, hsa-miR-299-5p, hsa-miR-3200-3p, hsa-miR-330-5p, hsa-miR-337-5p, hsa- miR-339-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-423-5p, hsa-miR-429, hsa-miR-485-5p, hsa-miR-542-3p, hsa-miR-543.
5. 4. The microparticles include hsa-miR-155-5p, hsa-miR-199a-3p, hsa-miR-382-5p, and hsa-miR-423-5p among the Parkinson's disease-related miRNA group. Microparticles described in .
6. The microparticle according to claim 4, wherein the microparticle is a SIRT2 inhibitor and includes hsa-miR-212-5p among the Parkinson's disease-related miRNA group.
7. The microparticle according to claim 4, which is an inhibitor of dopaminergic neuron degeneration or apoptosis and contains hsa-miR-182-5p or hsa-miR-183-5p from the Parkinson's disease-related miRNA group. .
8. The microparticles are microparticles purified and isolated from the culture supernatant of dental pulp-derived stem cells,
   The microparticle according to claim 1, wherein the microparticle does not contain any components other than the exosomes from the culture supernatant of the dental pulp-derived stem cells.
9. A prophylactic or therapeutic agent for Parkinson's disease, comprising the microparticles according to claim 1.
10. A method for improving Parkinson's disease, which comprises administering an effective amount of the microparticles according to claim 1 or an effective amount of the preventive or therapeutic agent for Parkinson's disease according to claim 9 to a subject who has developed Parkinson's disease. .
    
    

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