Microparticles, drug for preventing or treating erectile dysfunction, and method for improving erectile dysfunction

Abstract

To provide a novel microparticle capable of controlling the expression of a protein and / or gene related to erectile dysfunction.SOLUTION: Provided are: a microparticle that includes miRNA for controlling the production of nitric oxide and that is an agent for promoting the production of nitric oxide; a drug for the prevention or treatment of erectile dysfunction; and a method for mitigating erectile dysfunction.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to microparticles, a preventive or therapeutic agent for erectile dysfunction, and a method for improving erectile dysfunction. [Background technology]

[0002] It is widely known that erectile dysfunction (ED) increases with age. Due to the diversification of lifestyle-related diseases and increased social stress, it is estimated that one in three Japanese men over the age of 40 suffers from ED. More than 80% of ED cases have organic etiologies, the most common of which are vasculogenic disorders, which may involve impaired arterial inflow and abnormalities in venous outflow (cavernous systemic venous obstruction). Phosphodiesterase type 5 inhibitors (PDE5Is) are known as oral medications for ED, but they can rarely cause serious side effects such as myocardial infarction, so the development of new treatments is being considered.

[0003] For example, Non-Patent Document 1 describes that human umbilical cord blood stem cells have a beneficial effect on erectile function when administered to the penis of men with severe type 2 diabetes.

[0004] Meanwhile, research into microRNA (miRNA) has progressed, and the functions and target genes of miRNAs related to nitric oxide (NO) and nitric oxide synthase (NOS) have become clearer (see Non-Patent Documents 2 to 5).

[0005] Non-Patent Document 2 describes that miR-16 promotes inducible nitric oxide synthase (NOS2 or iNOS) activity and increases NO production, which is necessary for maintaining an anti-tumor microenvironment.

[0006] Non-Patent Document 3 describes that iNOS was strongly induced by the introduction of miR-155, but was alleviated by the inhibition of miR-155, indicating that miR-155 increases iNOS.

[0007] Non-Patent Document 4 describes that the nuclear accumulation of Nrf2 due to overexpression of miR-101 increases with HO-1 induction, VEGF expression, and endothelial nitric oxide synthase (eNOS)-derived NO production.

[0008] Non-Patent Document 5 describes that overexpression of miR-455-3p in human umbilical vein endothelial cells (HUVEC) increases the amount of eNOS protein. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Exp Clin Transplant. (2010) 8(2):150-160 [Non-patent document 2] Int. J. Mol. Sci. (2021) 22(12): 6264 [Non-patent document 3] Biochem Biophys Res Commun. (2018) 503(2): 452-458 [Non-patent document 4] Antioxid. Redox Signal. (2014) 21(18): 2469-2482 [Non-Patent Document 5] Sci. Rep. (2017) 7: 44807 Summary of the Invention [Problem to be solved by the invention]

[0010] Patent Document 1 does not mention miRNA. Non-Patent Documents 1 to 4 describe the functions and target genes of miRNAs related to nitric oxide (NO) and nitric oxide synthase (NOS), but do not specify how to administer these miRNAs as a treatment for erectile dysfunction.

[0011] The problem to be solved by the present invention is to provide a novel compound capable of controlling (suppressing, inhibiting, etc.) the expression of proteins and / or genes associated with erectile dysfunction. Drugs used to treat erectile dysfunction The purpose is to provide [Means for solving the problem]

[0012] The present inventors have found that microparticles containing specific microRNAs can regulate (suppress, inhibit, etc.) the expression of proteins and / or genes associated with erectile dysfunction.

[0013] Specifically, the present invention and preferred configurations thereof are as follows. [1] Contains miRNAs that regulate nitric oxide production. Microparticles that promote the production of nitric oxide. [2] The microparticles according to [1], wherein the microparticles are exosomes. [3] miRNAs that control the production of nitric oxide include miRNAs that target genes involved in the expression of nitric oxide synthase. The microparticles according to [1], which are promoters of nitric oxide synthase expression. [4] The microparticles according to [3], which contain at least one of the following NOS-related miRNAs as the miRNA targeting a gene involved in the expression of nitric oxide synthase: NOS-related miRNAs: hsa-miR-101-3p, has-miR-101-5p, has-miR-155-5p, hsa-miR-16-2-3p, has-miR-16-5p, hsa-miR-455-3p. [5] The microparticles according to [1], which are microparticles purified and isolated from the culture supernatant of dental pulp-derived stem cells. [6] The microparticles described in [5] do not contain components other than exosomes from the culture supernatant of dental pulp-derived stem cells. [7] The microparticles described in [1], which contain miRNA that controls the production of nitric oxide at a concentration higher than that of the culture supernatant of dental pulp-derived stem cells. [8] The microparticles described in [1] are intended to be administered to a subject who has developed erectile dysfunction, and whose International Institute for Erectile Function Score (IIEF-5) is 10 or less. [9] A preventive or therapeutic drug for erectile dysfunction, comprising the microparticles described in any one of [1] to [8]. [10-0] A method for improving erectile dysfunction, comprising administering an effective amount of the microparticles according to any one of [1] to [8] to a subject suffering from erectile dysfunction.

[10] A method for improving erectile dysfunction, comprising administering an effective amount of the microparticles according to any one of [1] to [8] to a non-human animal suffering from erectile dysfunction. [Effects of the Invention]

[0014] According to the present invention, novel microparticles capable of regulating the expression of proteins and / or genes associated with erectile dysfunction can be provided. [Brief explanation of the drawings]

[0015] [Figure 1] Figure 1 is a heat map showing the expression levels of miRNAs expressed in exosomes (microparticles of Example 1) purified from the culture supernatant of dental pulp-derived stem cells, which target genes related to the expression of nitric oxide synthase. [Figure 2] Figure 2(A) is a graph showing the average IIEF-5 values for 42 patients before treatment (pre) and after treatment (3rd). Figure 2(B) is a graph showing the average rigidity values for 42 patients before treatment (pre) and after treatment (post). Figure 2(C) is a graph showing the average Grade values for 42 patients before treatment (pre) and after treatment (post). [Figure 3] Figure 3 is a bar graph showing the relationship between the severity of ED based on IIEF-5 values before treatment and the rate of improvement in IIEF-5 values; and a line graph showing the relationship between the severity of ED based on IIEF-5 values before treatment and age. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below. The following description of the constituent elements may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values before and after "to" as the lower and upper limits.

[0017] [Small particles] The microparticles of the present invention contain miRNA that controls the production of nitric oxide and are agents that promote the production of nitric oxide. The microparticles of the present invention can regulate the expression of proteins and / or genes associated with erectile dysfunction, and as a result, the microparticles of the present invention can preferably improve, and more preferably prevent or treat, erectile dysfunction. Preferred embodiments of the microparticles of the present invention will now be described.

[0018] <Nitric oxide> Nitric oxide is produced in the body and has a vasodilatory effect. Nitric oxide is involved in signal transduction by activating soluble guanylate cyclase in cells to synthesize cyclic GMP (cGMP). The vascular endothelium uses nitric oxide as a signal to relax the surrounding smooth muscle, thereby dilating the arteries and increasing blood flow. Nitric oxide also plays a role in penile erection. Nitric oxide is released from the vascular endothelium of the corpus cavernosum of the penis, producing cGMP, which then relaxes the smooth muscle of the corpus cavernosum, dilating blood vessels and promoting erection.

[0019] (nitric oxide synthase) In vivo, nitric oxide is synthesized from arginine and oxygen by nitric oxide synthase (NOS). There are three types of nitric oxide synthases: neuronal NOS (nNOS) and endothelial NOS (eNOS), which produce low levels of nitric oxide for signaling, and inducible NOS (NOS2 or iNOS), which acts to combat pathogens.

[0020] <Erectile dysfunction> Erectile dysfunction is the persistent or recurrent inability to obtain and / or maintain an erection sufficient for satisfactory sexual intercourse (ED Treatment Guidelines, 3rd Edition, edited by the Japanese Society of Sexual Function / Japanese Urological Association). According to the 3rd edition of the ED Treatment Guidelines, ED is classified into three types: organic, psychogenic, and mixed. In Japanese men, 12 risk factors for erectile dysfunction are listed: aging, diabetes, obesity / lack of exercise, cardiovascular disease / high blood pressure, smoking, low testosterone, chronic kidney disease / lower urinary tract symptoms, neurological disease, surgery / trauma, psychological factors such as depression, medication, and sleep apnea. In non-Japanese men, dyslipidemia may also be a risk factor.

[0021] (Mechanism of ED) There are various risk factors for ED, and the mechanisms of its development are correspondingly diverse. The general mechanism of ED is due to impaired relaxation of the smooth muscle of the corpus cavernosum due to a decrease in local production of nitric oxide. The mechanisms of ED related to the main risk factors are as follows: The mechanism of erectile dysfunction caused by diabetes is that factors such as vascular disorders, nerve disorders, hypogonadism caused by diabetes, local infection of the penis, and the occurrence of Peyronie's disease, as well as psychological factors, reduce the local production of nitric oxide, which impairs the relaxation of the smooth muscle of the corpus cavernosum, resulting in ED. It is known that the reason why hypertensive patients suffer from ED is due to an imbalance in the nervous, hemodynamic, and physiologically active substances that are essential for maintaining both blood pressure homeostasis and erectile function. The mechanisms by which smoking causes erectile dysfunction include vascular endothelial damage, impaired blood flow to the penis, and sympathetic nerve stimulation. The mechanism of ED caused by chronic kidney disease is thought to be multifactorial, including blood flow disorders, nerve disorders, hormonal abnormalities, renal anemia, accumulation of endogenous NO synthesis inhibitors (asymmetrical dimethylarginine; ADMA), drug-induced disorders, and depression. Possible common mechanisms underlying both ED and lower urinary tract symptoms include overactivity of the sympathetic nervous system, ischemia of the pelvic vascular bed, decreased NOS / NO, and up-regulation of Rho kinase. The mechanism of ED caused by surgery / trauma is thought to be mainly due to damage to the cavernous nerve. Even if the nerve itself is not damaged by surgery, heat during surgery, local ischemia, and inflammation can damage the nerve.

[0022] (ED treatment methods) (1) PDE5I Phosphodiesterase 5 inhibitors (PDE5Is) are known as oral medications for ED. PDE5 is an enzyme that breaks down cyclic GMP (cGMP), an intracellular second messenger of nitric oxide, and is abundant in the corpus cavernosum of the penis. PDE5Is competitively inhibit the action of PDE5, increasing the concentration of cGMP in the smooth muscle cells of the corpus cavernosum, thereby relaxing the smooth muscle and promoting erection. Examples of PDE5 inhibitors include sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis). In addition, in cases of low testosterone levels, a treatment that combines PDE5I with TRT (testosterone replacement therapy) is also known.

[0023] (2) Vacuum erection aid A vacuum erection device (VED) applies negative pressure to the penis to draw blood into it, then wraps a rubber band around the base of the penis to retain the blood and create a pseudo-erect state.

[0024] (3) Intracavernous injection of prostaglandin E1 Prostaglandin E1 (PGE1) is injected into the penile cavernosal cavity. Both the 2015 EAU and ICSM guidelines recommend it as a second-line treatment when PDE5I is ineffective or contraindicated. Poor response to PGE1 is suspected to be due to the presence of vascular factors, with diabetes and metabolic syndrome being particularly poor responses. On the other hand, neurogenic ED responds well to PGE1, and is highly effective after spinal cord injury and radical prostatectomy.

[0025] (4) Low-intensity extracorporeal shock wave therapy In the EAU guidelines, low-intensity extracorporeal shock wave therapy (LI-ESWT) is adopted as a first-line treatment for patients with refractory or reduced efficacy of PDE5 inhibitors, alongside VED. The therapeutic mechanism is that low-intensity extracorporeal shock waves promote angiogenesis, which is expected to improve vascular endothelial function and hemodynamics. From this perspective, LI-ESWT is indicated for vascular ED.

[0026] (5) Components derived from mesenchymal stem cells Human umbilical cord blood stem cells have beneficial effects on erectile function when administered to the penis of men with severe type 2 diabetes (Exp Clin Transplant. (2010) 8(2):150-160). The paper does not provide details on the mechanism of action for AD treatment. On the other hand, the microparticles of the present invention contain miRNA that controls the production of nitric oxide and are therefore nitric oxide production promoters. The microparticles of the present invention can promote NO production, thereby causing relaxation of the smooth muscle of the corpus cavernosum of the penis, promoting erection, and are used to improve erectile dysfunction. In a preferred embodiment of the microparticles of the present invention, the miRNAs that target genes involved in the expression of nitric oxide synthase include an NOS-related miRNA group, and therefore can enhance the expression of nitric oxide synthase and increase NO production. The microparticles of the present invention may restore the vasodilatory activity of the corpus cavernosum through a mechanism of action that repairs vascular endothelial cells, which is different from the mechanism of action that promotes the expression of nitric oxide synthase.The microparticles of the present invention may significantly promote NO production from vascular endothelium through the synergistic effect of the repair of vascular endothelial cells and the promotion of nitric oxide synthase expression by NOS-related miRNAs.

[0027] <Details of microparticles> The miRNA, which is the 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 exuded, released, or shed into a 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, microparticles derived from the culture supernatant of dental pulp-derived stem cells do not necessarily have to be obtained from the culture supernatant of dental pulp-derived stem cells. For example, even if microparticles isolated from the interior of dental pulp-derived stem cells by any method are the same as microparticles that can be isolated from the culture supernatant of dental pulp-derived stem cells, they can be said to be microparticles derived from the culture supernatant of dental pulp-derived stem cells. The microparticles derived from the culture supernatant of dental pulp-derived stem cells or the like may be used in a state contained in the culture supernatant or in a state purified from the culture supernatant. The microparticles are preferably microparticles purified from the culture supernatant. The origin of the microparticles can be determined by known methods. For example, the method described in J Stem Cell Res Ther (2018) 8:2 can be used to determine whether the microparticles are derived from dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord-derived stem cells, or other stem cells. Specifically, the origin of each microparticle can be determined based on the miRNA pattern of the microparticles.

[0028] (miRNA) In the present invention, the microparticles contain miRNA that regulates the production of nitric oxide. In the present invention, miRNA (MicroRNAs) are RNA molecules of, for example, 21 to 25 bases (nucleotides). miRNA can regulate gene expression by degrading target gene (target) mRNA or suppressing it at the decoding stage. In the present invention, miRNA may be, for example, single-stranded (monomer) or double-stranded (dimer). Furthermore, in the present invention, miRNA is preferably mature miRNA cleaved by a ribonuclease such as Dicer.

[0029] The sequences of miRNAs described herein, such as has-miR-16-5p, are registered in publicly known databases (e.g., the miRBase database) with associated accession numbers, allowing those skilled in the art to unambiguously determine the sequence. For example, the accession number for has-miR-16-5p is MI0000070, and the sequence is registered in the miRBase database. Hereinafter, the accession numbers for each miRNA will be omitted. However, the term "miRNA" as used herein also includes variants that differ by approximately one to five bases from the mature miRNA, such as has-miR-16-5p. Furthermore, each miRNA as used herein includes a polynucleotide consisting of a nucleotide sequence identical to that of the miRNA (e.g., has-miR-16-5p) or a polynucleotide consisting of a complementary nucleotide sequence thereof, which has the function of the miRNA of the present invention. "Identity" refers to the degree of identity when the compared sequences are appropriately aligned, and refers to the percentage of exact amino acid matches between the sequences. Alignment can be performed using any algorithm, such as BLAST. Identity is, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or approximately 99%. A polynucleotide consisting of an identical nucleotide sequence may have, for example, point mutations, deletions, and / or additions in the nucleotide sequence of the miRNA. The number of bases of the point mutations, etc., is, for example, 1 to 5, 1 to 3, 1 to 2, or 1. Furthermore, a polynucleotide consisting of a complementary base sequence is, for example, a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a miRNA base sequence, and includes a polynucleotide having the function of the miRNA of the present invention. Stringent conditions are not particularly limited, but include, for example, the conditions described in

[0028] of JP 2017-184642 A, the contents of which are incorporated herein by reference.

[0030] In the present invention, the microparticles preferably contain miRNA that controls nitric oxide production at a concentration higher than that of the culture supernatant of dental pulp-derived stem cells. Preferred embodiments of the miRNA contained in the microparticles are described below.

[0031] In the present invention, the miRNA that controls nitric oxide production preferably contains a miRNA that targets a gene involved in the expression of nitric oxide synthase. In this case, the microparticles of the present invention are preferably agents that promote the expression of nitric oxide synthase.

[0032] miRNAs that control nitric oxide production, especially those that target genes involved in the expression of nitric oxide synthase, are effective as treatments for erectile dysfunction. The following miRNAs are known to be involved in NO production and NOS expression: According to Antioxid. Redox Signal. (2014) 21(18): 2469-2482, overexpression of miR-101 increases Nrf2 nuclear accumulation, accompanied by HO-1 induction, VEGF expression, and endothelial nitric oxide synthase (eNOS)-derived NO production. According to Int. J. Mol. Sci. (2021) 22(12): 6264, miR-155 negatively targets FGF-2 in esophageal adenocarcinoma cells (EAC) and promotes NOS2 in tumor-associated macrophages (TAM), thereby regulating proliferation, migration, invasion, and angiogenesis. Furthermore, according to Biochem Biophys Res Commun. (2018) 503(2): 452-458, TNF-α, IL-12, and iNOS are strongly induced by miR-155 introduction and are reduced by miR-155 inhibition. In other words, miR-155 increases iNOS. According to Int. J. Mol. Sci. (2021) 22(12): 6264, miR-16 promotes inducible nitric oxide synthase (NOS2 or iNOS) activity, increasing NO production, which is necessary for maintaining an antitumor microenvironment. miR-16 also targets PD-L1, reducing immunosuppression. According to Sci. Rep. (2017) 7: 44807, H2S regulates eNOS stability by promoting the expression of miR-455-3p. Moreover, overexpression of miR-455-3p in human umbilical vein endothelial cells (HUVECs) increases the amount of eNOS protein, whereas inhibition of miR-455-3p decreases it. These miRNAs related to NO production and NOS expression (miR-101, miR-155, miR-16, and miR-455-3p) are effective as therapeutic agents for erectile dysfunction.

[0033] Of these miRNAs related to NO production and NOS expression (miR-101, miR-155, miR-16, and miR-455-3p), it is more preferable that the microparticles of the present invention contain at least one type of NOS-related miRNA listed below as a miRNA targeting a gene related to the expression of nitric oxide synthase. NOS-related miRNAs: hsa-miR-101-3p, has-miR-101-5p, has-miR-155-5p, hsa-miR-16-2-3p, has-miR-16-5p, hsa-miR-455-3p. In this case, it is preferable that the microparticles contain at least one of has-miR-101-5p, has-miR-155-5p, hsa-miR-16-2-3p, has-miR-16-5p, and hsa-miR-455-3p, more preferably at least one of has-miR-101-5p, has-miR-155-5p, hsa-miR-16-2-3p, and has-miR-16-5p, particularly preferably at least one of has-miR-101-5p, has-miR-155-5p, and has-miR-16-5p, and even more particularly preferably they contain all of has-miR-101-5p, has-miR-155-5p, and has-miR-16-5p.

[0034] The microparticles preferably contain at least one type of miRNA targeting a gene involved in the expression of nitric oxide synthase, with a Log2Ratio of the read count obtained by analysis using IMOTA of 4.0 or more, more preferably 6.0 or more, particularly preferably 8.0 or more, and even more particularly preferably 10.0 or more. The microparticles preferably contain hsa-miR-101-3p, has-miR-101-5p, has-miR-155-5p, hsa-miR-16-2-3p, has-miR-16-5p, and hsa-miR-455-3p with a Log2Ratio of the read count obtained by analysis using IMOTA of 4.0 or more, and the microparticles preferably contain has-miR-101-5p, has-miR-155-5p, hsa-miR-16-2-3p, has-miR-16-5p, and hsa-miR-455-3p with a Log2Ratio of the read count obtained by analysis using IMOTA of 4.0 or more. It is more preferable that has-miR-16-5p and hsa-miR-455-3p all contain a value of 6.0 or higher, it is particularly preferable that has-miR-101-5p, has-miR-155-5p, hsa-miR-16-2-3p, and has-miR-16-5p all contain a value of 8.0 or higher, and it is even more particularly preferable that has-miR-101-5p, has-miR-155-5p, and has-miR-16-5p all contain a value of 10.0 or higher. It is more preferable that the microparticles contain both has-miR-155-5p and has-miR-16-5p in a Log2Ratio of 12.0 or more of the read counts obtained by analysis using IMOTA, and it is even more preferable that they contain has-miR-16-5p in a Log2Ratio of 14.0 or more.

[0035] The microparticles of the present invention preferably have an expression level of NOS-related miRNAs (preferably one of has-miR-101-5p, has-miR-155-5p, and has-miR-16-5p; more preferably has-miR-16-5p) that is 1.1 times or more, more preferably 1.5 times or more, and particularly preferably 2 times or more, compared to exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells.

[0036] The microparticles of the present invention are agents for promoting the production of nitric oxide, and are capable of promoting the production of nitric oxide in any cells (preferably penile vascular endothelial cells) to preferably 1.2 times or less the normal level (compared to untreated cells), more preferably 2.0 times or more, and particularly preferably 2.5 times or more. When the microparticles of the present invention are agents for promoting the expression of nitric oxide synthase, they can preferably promote the expression of the nitric oxide synthase gene in any cells (preferably penile vascular endothelial cells) to 1.2 times or less the normal level (as in untreated cells), more preferably to 2.0 times or more, and particularly preferably to 2.5 times or more.

[0037] (Type of miRNA) Here, microparticles derived from the culture supernatant of dental pulp-derived stem cells contain approximately 2,600 types of small RNA. Of these, approximately 1,800 types are miRNA. Of these miRNAs, 180 to 200 types are abundant. The miRNAs abundant in microparticles derived from dental pulp-derived stem cells are characterized by the fact that they contain many microRNAs related to the treatment of cranial nerve diseases and erectile dysfunction, a finding that was previously unknown and newly discovered by the present inventors. This characteristic is significantly different from the types of miRNAs abundant in other microparticles of mesenchymal stem cells. For example, the miRNAs abundant in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells contain almost no microRNAs related to the treatment of cranial nerve diseases and erectile dysfunction.

[0038] The microparticles preferably contain two or more types of microRNAs (hereinafter also referred to as erectile dysfunction-related microRNAs) that can control the expression of proteins and / or genes related to erectile dysfunction, more preferably three or more types, even more preferably four or more types, particularly preferably five or more types, and even more particularly preferably six or more types.

[0039] (Types of microparticles) The microparticles are preferably at least one type selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, and exovesicles, or microvesicles, and are more preferably exosomes. The diameter of the microparticles is preferably 10 to 1000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm. Furthermore, it is desirable that the surface of the microparticles contains tetraspanin molecules such as CD9, CD63, and CD81, and this may be CD9 alone, CD63 alone, or CD81 alone, or any combination of two or three of these. Hereinafter, a preferred embodiment in which exosomes are used as microparticles will be described, but the microparticles used in the present invention are not limited to exosomes.

[0040] Preferably, exosomes are extracellular vesicles that are 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 exosomes preferably contain 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, and they are easily traceable and can be targeted to specific regions.

[0041] (Fine particle content) The content of the microparticles in the microparticle composition is not particularly limited. 8 It is preferable to contain more than 1.0 × 10 8 It is more preferable to include 2.0 × 10 8 It is particularly preferable that the number of atoms contained is 2.5 × 10 or more.8 It is more particularly preferred that the number of atoms contained is 1.0 × 10 or more. 9 It is even more particularly preferred that the number of the hydroxyl groups contained is 1 or more. The concentration of the microparticles in the microparticle composition is not particularly limited. 8 It is preferable to contain more than 2.0 × 10 8 It is more preferable to have more than 4.0 × 10 8 It is particularly preferable that the content is 5.0 × 10 8 It is more particularly preferable that the content is 2.0 × 10 9 It is even more particularly preferred that the concentration is 1 / mL or more. A preferred embodiment of the microparticles of the present invention contains such a large amount or high concentration of microparticles, thereby maintaining a high amount of miRNA that controls nitric oxide production or miRNA used in the treatment of erectile dysfunction.

[0042] <Other ingredients> In addition to the microparticles, the microparticle composition may contain other components depending on the type of animal to be administered and the purpose, as long as the effects of the present invention are not impaired. Examples of other components include nutritional components, antibiotics, cytokines, protective agents, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, etc. Examples of nutritional components include fatty acids and vitamins. Examples of antibiotics include penicillin, streptomycin, and gentamicin. Carriers 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, etc. The purpose of the pharmaceutical composition is to facilitate the administration of the microparticles to a subject.

[0043] The pharmaceutically acceptable carrier is preferably a carrier (including a diluent) that does not cause significant irritation to the subject to be administered and does not suppress the biological activity and properties of the administered compound. Examples of the carrier include propylene glycol; (physiological) saline; emulsion; buffer solution; culture medium, such as DMEM or RPMI; and cryopreservation medium containing components that scavenge free radicals.

[0044] The microparticle composition may contain an active ingredient of a conventionally known therapeutic drug for erectile dysfunction, and those skilled in the art can appropriately modify the composition depending on the intended use, the subject of administration, etc.

[0045] On the other hand, it is preferred that the microparticle composition does not contain any predetermined substances. For example, the microparticle composition preferably does not include dental pulp-derived stem cells. Furthermore, the microparticle composition preferably does not contain MCP-1. However, the microparticle composition may contain cytokines other than MCP-1. Examples of other cytokines include those described in paragraphs

[0014] to

[0020] of JP 2018-023343 A. Furthermore, the microparticle composition preferably does not contain Siglec 9. However, it may contain other sialic acid-binding immunoglobulin-like lectins other than Siglec 9. It is preferable that the microparticle composition is substantially free of serum (such as fetal bovine serum, human serum, or sheep serum).It is also preferable that the microparticle composition is substantially free of conventional serum substitutes such as knockout serum replacement (KSR). In the microparticle composition, the contents (solid contents) of the other components described above are preferably 1% by mass or less, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

[0046] <Method of manufacturing microparticles> The method for producing the microparticles is not particularly limited. The microparticles of the present invention may be prepared by preparing a culture supernatant of dental pulp-derived stem cells or the like, and subsequently 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 may be prepared by obtaining a composition containing the culture supernatant of dental pulp-derived stem cells that had been discarded (or by appropriately purifying this composition), and purifying microparticles therefrom.

[0047] (Method for preparing culture supernatant of dental pulp-derived stem cells, etc.) The culture supernatant of dental pulp-derived stem cells and the like is not particularly limited. The culture supernatant of dental pulp-derived stem cells, etc. is preferably substantially free of serum. For example, the serum content of the culture supernatant of dental pulp-derived stem cells, etc. is preferably 1% by mass or less, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

[0048] Dental pulp-derived stem cells may be derived from humans or non-human animals, including the same animals (species) as those to which the microparticles of the present invention are administered, as described below, and preferably mammals.

[0049] There are no particular limitations on the dental pulp-derived stem cells used in the culture supernatant. Stem cells from exfoliated deciduous teeth, stem cells from deciduous teeth obtained by other methods, and stem cells from permanent teeth (DPSCs) can be used. In addition to human deciduous tooth pulp stem cells and human permanent tooth pulp stem cells, stem cells derived from dental pulp of animals other than humans, such as porcine deciduous tooth pulp stem cells, can also be used. In addition to exosomes, dental pulp-derived stem cells can produce various cytokines, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β)-1 and -3, TGF-α, KGF, HBEGF, SPARC, other growth factors, and chemokines, as well as many other physiologically active substances. In the present invention, it is particularly preferred that the dental pulp-derived stem cells used in the dental pulp-derived stem cell culture supernatant are dental pulp-derived stem cells that contain a large amount of miRNA that regulates the production of nitric oxide, and it is preferable to use deciduous dental pulp stem cells. That is, in the present invention, it is preferred to use the deciduous dental pulp stem cell culture supernatant.

[0050] The dental pulp-derived stem cells used in the present invention may be natural or genetically modified, as long as they can achieve the intended treatment. In particular, the present invention can use immortalized stem cells derived from dental pulp. By using immortalized stem cells that can proliferate virtually indefinitely, the amount and composition of biological factors contained in the stem cell culture supernatant can be stabilized over a long period of time. There are no particular limitations on the immortalized stem cells derived from dental pulp. The immortalized stem cells are preferably non-cancerous immortalized stem cells. Immortalized stem cells derived from dental pulp 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 an effect of inhibiting the function of transforming growth factor (TGF) β receptor, and examples thereof include 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-pyridine, and the like. Examples of suitable pyrazoles include 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (all manufactured by Merck), and SB431542 (Sigma-Aldrich). A-83-01 is preferred. The ROCK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of Rho-associated kinase. Examples of ROCK inhibitors include GSK269962A (Axonmedchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (all Fujifilm Wako Pure Chemical Industries, Ltd.). Y-27632 is preferred. The GSK3 inhibitor is not particularly limited as long as it inhibits GSK-3 (Glycogen synthase kinase 3), and examples include A 1070722, BIO, and BIO-acetoxime (all manufactured by TOCRIS). MEK inhibitors are not particularly limited as long as they have the effect of inhibiting the function of MEK (MAP kinase-ERK kinase), and examples include AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86-9766), SL327, U0126-EtOH (all from Selleck), PD98059, U0124, U0125 (all from Cosmo Bio Co., Ltd.), etc.

[0051] When the microparticles of the present invention are used in regenerative medicine, in accordance with the requirements of the Act on Safety Assurance of Regenerative Medicine, the culture supernatant of dental pulp-derived stem cells or these immortalized stem cells, or a composition containing microparticles derived therefrom, should be free of somatic stem cells other than dental pulp-derived stem cells, etc. The microparticle composition may contain mesenchymal stem cells or other somatic stem cells other than dental pulp-derived stem cells, etc., but preferably does not 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 somatic stem cells from the dermal system include epithelial stem cells, hair follicle stem cells, etc. Examples of somatic stem cells from the digestive system include pancreatic (general) stem cells, hepatic stem cells, etc. Examples of somatic stem cells from the bone marrow system (other than mesenchymal stem cells) include hematopoietic stem cells, etc. Examples of somatic stem cells from the nervous system include neural stem cells, retinal stem cells, etc. The microparticle composition may contain, but preferably does not contain, stem cells other than somatic stem cells, including embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).

[0052] 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. The culture supernatant of dental pulp-derived stem cells, etc., is a culture medium obtained by culturing dental pulp-derived stem cells. For example, a culture supernatant usable in the present invention can be obtained by separating and removing cellular components after culturing dental pulp-derived stem cells. Culture supernatants that have been appropriately subjected to various treatments (e.g., centrifugation, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) may also be used.

[0053] Dental pulp-derived stem cells for obtaining the conditioned medium can be selected by conventional methods based on cell size or morphology, or as adhesive cells. Adhesive cells or their subcultured cells can be selected from dental pulp cells collected from shed deciduous or permanent teeth. The conditioned medium for dental pulp-derived stem cells can be obtained by culturing selected stem cells.

[0054] It is preferable that the "culture supernatant of dental pulp-derived stem cells, etc." is a culture medium 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 cells (regardless of cell type) as a whole. This characteristic clearly distinguishes the composition of this embodiment from various compositions that contain dental pulp-derived stem cells, as well as dental pulp-derived stem cells themselves. A typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and is composed only of the culture supernatant of dental pulp-derived stem cells. The dental pulp-derived stem cell culture supernatant used in the present invention may contain the culture supernatant of both deciduous dental pulp-derived stem cells and adult dental pulp-derived stem cells. The dental pulp-derived stem cell culture supernatant used in the present invention preferably contains the deciduous dental pulp-derived stem cell culture supernatant as an active ingredient, more preferably 50% by mass or more, and preferably 90% by mass or more. It is particularly preferable that the dental pulp-derived stem cell culture supernatant used in the present invention is a composition composed solely of the deciduous dental pulp-derived stem cell culture supernatant.

[0055] The culture medium for dental pulp-derived stem cells to obtain the culture supernatant can be a basal medium or a basal medium supplemented with serum, etc. Examples of basal media that can be used include Dulbecco's Modified Eagle's Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO, etc.), Ham's F12 Medium (HamF12) (Sigma, GIBCO, etc.), and RPMI 1640 medium. Examples of ingredients that can be added to the 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. However, to prepare serum-free "dental pulp-derived stem cell culture supernatant," it is recommended to use serum-free medium throughout the entire process or for the final or penultimate few subcultures. For example, serum-free dental pulp-derived stem cell culture supernatant can be prepared by culturing dental pulp-derived stem cells in serum-free medium. Serum-free dental pulp-derived stem cell culture supernatant can also be obtained by performing one or more subcultures and culturing the final or penultimate few subcultures in serum-free medium. Alternatively, serum-free dental pulp-derived stem cell culture supernatant can also be obtained by removing serum from the collected culture supernatant using dialysis, solvent replacement using a column, or other methods.

[0056] The conditions commonly used for culturing dental pulp-derived stem cells to obtain a culture supernatant can be applied as is. 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 steps of isolating and selecting stem cells 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 depending on the type of stem cells. In addition, special conditions may be applied to the culture of dental pulp-derived stem cells to produce large amounts of microparticles such as exosomes, such as low temperature, low oxygen, and microgravity conditions, or co-culture with some kind of stimuli.

[0057] The culture supernatant of dental pulp-derived stem cells used in the present invention for preparing microparticles such as exosomes may contain other components in addition to the culture supernatant of dental pulp-derived stem cells, but it is preferable that it is substantially free of other components. However, each type of additive used in preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.

[0058] (Preparation of Microparticles) The microparticles can be prepared by purifying them from the culture supernatant of dental pulp-derived stem cells or the like.

[0059] 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 non-associated components based on a property of the microparticle, for example, they 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 (e.g., precipitate) rich in microparticles obtained by centrifuging the culture supernatant of dental pulp-derived stem cells. Unnecessary components (insoluble components) in fractions other than the specified fraction may be removed. The removal of the solvent, dispersion medium, and unnecessary components from the microparticle composition does not have to be complete. Centrifugation conditions include 100 to 20,000 g for 1 to 30 minutes. In the present invention, microparticles can be purified by filtering the culture supernatant of dental pulp-derived stem cells or a centrifuged product thereof. Unnecessary components can be removed by filtration. Furthermore, by using a filtration membrane with an appropriate pore size, removal of unnecessary components and sterilization can be performed simultaneously. The material and pore size of the filtration membrane used for filtration are not particularly limited. Filtration can be performed using a filtration membrane with an appropriate molecular weight or size cutoff using a known method. From the viewpoint of facilitating the separation of exosomes, the pore size of the filtration membrane is preferably 10 to 1,000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm. In the present invention, the culture supernatant of dental pulp-derived stem cells, its centrifuged product, or its filtered product can be further separated using a separation method such as column 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 stage. For example, light scattering, refractive index, dynamic light scattering, or UV-visible light detectors can be used to track the microparticles. Alternatively, specific enzyme activity, etc. can be used to track activity in each fraction. As a method for purifying microparticles, the method described in

[0034] to

[0064] of JP-A No. 2019-524824 may be used, the contents of which are incorporated herein by reference.

[0060] The final form of the microparticle composition is not particularly limited. For example, the microparticle composition may be in the form of microparticles packed in a container together with a solvent or dispersion medium; microparticles gelled with a gel and packed in a container; or microparticles solidified by freezing and / or drying, formulated, or packed in a container. Examples of the container include tubes, centrifuge tubes, bags, etc. suitable for cryopreservation. The freezing temperature can be, for example, -20°C to -196°C.

[0061] Compared with conventional compositions that can be used as therapeutic or preventive agents for erectile dysfunction, the microparticles of the present invention have advantages such as ease of mass production, the ability to utilize stem cell culture medium that was previously discarded as industrial waste, and reduced disposal costs for stem cell culture medium. In particular, when the culture supernatant of dental pulp-derived stem cells is a culture supernatant of human dental pulp-derived stem cells, there are also advantages in that the microparticles of the present invention are highly safe from immunological and other standpoints and pose fewer ethical issues when applied to humans. When the culture supernatant of dental pulp-derived stem cells is a culture supernatant of dental pulp-derived stem cells from a patient with erectile dysfunction, the safety of the microparticles of the present invention when applied to that patient will be enhanced and ethical issues will be reduced. 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 the culture supernatant of dental pulp-derived stem cells, etc., are preferably used in restorative medicine. It is known that in regenerative medicine based on stem cell transplantation, stem cells are not the main players in regeneration, but rather the liquid components produced by stem cells, together with the patient's own stem cells, repair organs. This solves the difficult problems associated with conventional stem cell transplantation, such as carcinogenesis, standardization, administration method, storage, and culture method, and makes restorative medicine possible using a composition using the culture supernatant of dental pulp-derived stem cells or microparticles derived therefrom. Compared to stem cell transplantation, the use of the microparticles of the present invention is safer because tumorigenesis is less likely to occur due to the lack of cell transplantation. Furthermore, the microparticles of the present invention have the advantage of being of consistently standardized quality. Mass production and efficient administration methods can be selected, allowing for low-cost use.

[0062] [Preventive or therapeutic drugs for erectile dysfunction] The prophylactic or therapeutic agent for erectile dysfunction of the present invention comprises the microparticles of the present invention. As used herein, "prevention" refers to preventing the onset of a disease (herein, erectile dysfunction). Also, as used herein, "treatment" refers to alleviating, suppressing, or preventing the progression of symptoms of an onset disease, and ameliorating the symptoms. The prophylactic or therapeutic agent for erectile dysfunction of the present invention is preferably a therapeutic agent for erectile dysfunction.

[0063] [How to improve erectile dysfunction] The method for improving erectile dysfunction of the present invention comprises administering an effective amount of the microparticles of the present invention or an effective amount of the drug for preventing or treating erectile dysfunction of the present invention to a subject who has developed erectile dysfunction.

[0064] There are no particular limitations on the step of administering the microparticles of the present invention to a subject suffering from erectile dysfunction. Examples of administration methods include spraying or inhalation into the oral cavity, nasal cavity, or respiratory tract, infusion, topical administration, and nasal drops, with minimal invasiveness being preferred. A preferred local administration method is injection. Electroporation is also preferred, which applies a voltage (electric pulse) to the skin surface to temporarily create minute holes in the cell membrane, allowing the active ingredient to penetrate into the dermis layer, which is difficult to reach with conventional care. Examples of local administration include intracavernous administration, intravenous administration, intraarterial administration, intraportal vein administration, intradermal administration, subcutaneous administration, intramuscular administration, and intraperitoneal administration, with intracavernous administration being more preferred. The administration method in the present invention is particularly preferably intracavernous injection (ICI). In addition, various formulation techniques can be used to change the in vivo distribution of microparticles.Many methods for changing in vivo distribution are known to those skilled in the art.Examples of such methods include, for example, protecting exosomes in vesicles composed of substances such as proteins, lipids (e.g., liposomes), carbohydrates or synthetic polymers. The microparticles of the present invention administered to a subject suffering from erectile dysfunction may circulate within the subject's body and reach a specific tissue. There are no particular limitations on the number of doses and the interval between doses. The number of doses can be one or more per month, preferably 1 to 10 times per month, more preferably 2 to 6 times per month, and particularly preferably 4 times per month (once per week). The interval between doses is preferably 1 hour to 2 weeks, more preferably 1 to 10 days, and particularly preferably 6 to 8 days (approximately 1 week). However, this can be adjusted appropriately depending on the target organism species and symptoms of the target. The microparticles of the present invention are preferably used for administering the microparticles to a subject suffering from erectile dysfunction at least once a week for an effective therapeutic period. When the subject is a human, the effective therapeutic period is preferably 1 to 10 weeks, more preferably 2 to 8 weeks, and particularly preferably 3 to 6 weeks. The effective therapeutic period is initially set at 3 to 4 weeks, and if the improvement rate in the evaluation of the therapeutic effect is low, it is preferably extended to 6 to 8 weeks. 2.0×10 9 When using a culture supernatant of dental pulp-derived stem cells at a concentration of 1 / ml, the amount is preferably 0.1 to 5 ml, more preferably 0.3 to 3 ml, and even more preferably 0.5 to 1 ml per mouse (approximately 25 g) in a mouse model.When the subject of administration is a human, the amount is preferably 0.1 to 10 ml, more preferably 0.5 to 5 ml, and even more preferably 1 to 3 ml per person. 0.1×10 8 When microparticles at a concentration of particles / μg are used, in a mouse model, the amount is preferably 1 to 50 μg, more preferably 3 to 30 μg, and even more preferably 5 to 25 μg per mouse (approximately 25 g).When the subject of administration is a human, the amount is preferably 1 to 100 μg, more preferably 5 to 50 μg, and even more preferably 10 to 30 μg per person. The preferred range of the dose per body weight for other animals can be calculated proportionally from the dose per body weight (approximately 25 g) for the model mouse, but can be adjusted appropriately depending on the symptoms of the subject.

[0065] There are no particular limitations on the animals (biological 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, quails, ducks, etc.), or fish (salmon, trout, tuna, bonito, etc.). The mammals may be humans or non-human mammals, with humans being particularly preferred. The non-human mammals are more preferably cows, pigs, horses, goats, sheep, monkeys, dogs, cats, mice, rats, guinea pigs, or hamsters.

[0066] The microparticles of the present invention may be used in combination with conventionally known therapeutic drugs for erectile dysfunction, specifically, for example, conventionally known PDE5Is. [Example]

[0067] The features of the present invention will be explained in more detail below with reference to examples, comparative examples, and reference examples. The materials, amounts used, ratios, treatment contents, treatment 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 construed as being limited by the specific examples shown below.

[0068] [Example 1] <Preparation of culture supernatant of dental pulp-derived stem cells> Culture supernatant of human deciduous dental pulp stem cells was prepared and collected according to the method described in Example 6 of Patent No. 6296622, except that DMEM medium was used instead of the DMEM / HamF12 mixed medium. Primary culture was performed with the addition of fetal bovine serum (FBS), and subculture was performed using the primary culture medium. The supernatant of the subculture medium was then collected so that it did not contain FBS, and the culture supernatant of deciduous dental pulp stem cells was prepared. Note that DMEM is Dulbecco's modified Eagle's medium, and F12 is Ham's F12 medium.

[0069] <Exosome preparation> Dental pulp-derived stem cell exosomes were purified from the culture supernatant of the obtained dental pulp-derived stem cells using the following method. The culture supernatant (100 mL) of primary dental pulp stem cells was filtered through a 0.22-micrometer pore size filter, and the solution was centrifuged at 100,000 × g for 60 minutes at 4°C. The supernatant was decanted, and the exosome-enriched pellet was resuspended in phosphate-buffered saline (PBS). The resuspended sample was centrifuged at 100,000 × g for 60 minutes. The pellet was again collected from the bottom of the centrifuge tube (approximately 100 μl) as the concentrated sample. Protein concentration was determined using a microBSA protein assay kit (Pierce, Rockford, IL). The exosome-containing composition (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.

[0070] The average particle size and concentration of the microparticles contained in the microparticle composition of Example 1 were evaluated. 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 is 1.0 x 10 9 It is a highly concentrated exosome solution of 2.0 x 10 9 It was a highly concentrated exosome solution with 100 cells / ml. Furthermore, the components of the obtained microparticle composition of Example 1 were analyzed by known methods. As a result, it was found that the microparticle composition of Example 1 did not contain stem cells derived from dental pulp, MCP-1, or Siglec 9. Therefore, it was found that the active ingredient of the microparticle composition of Example 1 was an active ingredient different from MCP-1 and Siglec 9, which are active ingredients in the culture supernatant of mesenchymal stem cells, and their analogs.

[0071] [Test Example 1]: MicroRNA expressed in exosomes The small RNAs contained in the microparticle composition of Example 1 were analyzed by next-generation sequencing (NGS). NGS analysis identified 1,787 miRNAs contained in the microparticle composition of Example 1 (exosomes from dental pulp-derived stem cells). The results are shown in Table 1 below.

[0072] [Table 1]

[0073] [Test Case 2]: Search for disease-related microRNAs We searched for disease-related microRNAs. Disease-related miRNAs were extracted using IMOTA (Interactive Multi-Omics-Tissue Atlas). IMOTA is an interactive multi-omics atlas that allows researchers to investigate the interactions and expression levels of miRNAs, mRNAs, and proteins in various tissues and cells (Nucleic Acids Research, Volume 46, Issue D1, January 4, 2018, Pages D770-D775, "IMOTA: an interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions"). We searched for microRNAs that regulate proteins and / or genes related to erectile dysfunction, or that regulate proteins and / or genes targeted by therapeutic drugs. In this Test Example 2, we searched for miRNAs that target genes related to the expression of nitric oxide synthase, a protein related to erectile dysfunction.

[0074] The miRNAs expressed in exosomes purified from the culture supernatant of dental pulp-derived stem cells, which target genes related to the expression of nitric oxide synthase, were the following NOS-related miRNA group (6 types). NOS-related miRNAs: hsa-miR-101-3p, has-miR-101-5p, has-miR-155-5p, hsa-miR-16-2-3p, has-miR-16-5p, hsa-miR-455-3p. The expression level of hsa-miR-101-3p was 11.84 as the Log2 ratio of the read counts obtained by analysis using IMOTA, which was the 54th highest of the 1,787 microRNAs contained in the microparticle composition of Example 1 (exosomes from dental pulp-derived stem cells) shown in Table 1. The expression level of hsa-miR-101-5p was 5.13 in terms of the Log 2 Ratio of the read counts, and was the 601st among the miRNAs contained in the microparticle composition of Example 1. The expression level of hsa-miR-155-5p was 12.17 in terms of Log 2 Ratio of read counts, ranking 44th among the miRNAs contained in the microparticle composition of Example 1. The expression level of hsa-miR-16-2-3p was 8.49 in terms of Log 2 Ratio of read counts, ranking it 162nd among the miRNAs contained in the microparticle composition of Example 1. The expression level of hsa-miR-16-5p was 17.69 in terms of Log 2 Ratio of read counts, which was the highest among the miRNAs contained in the microparticle composition of Example 1. The expression level of hsa-miR-455-3p was 7.73 in terms of Log 2 Ratio of read counts, ranking it 196th among the miRNAs contained in the microparticle composition of Example 1. Therefore, it was found that the microparticles of the present invention can be used as an agent for controlling the expression of nitric oxide synthase.

[0075] The results of comparing the expression levels of six miRNAs included in the NOS-related miRNA group are shown in the heat map in Figure 1. The density in the heat map is shown as the log ratio of the read count values.

[0076] 1, it was found that the microparticles of the present invention contain a high concentration of miRNA that controls the production of nitric oxide (miRNA that targets a gene involved in the expression of nitric oxide synthase), as a miRNA related to the improvement of erectile dysfunction. Therefore, the microparticles of the present invention can control the expression of nitric oxide synthase, thereby controlling the production of nitric oxide, i.e., can control the expression of proteins and / or genes related to erectile dysfunction. As a result, the microparticles of the present invention are useful for the improvement, prevention, or treatment of erectile dysfunction.

[0077] [Test Case 3]: Treatment of erectile dysfunction The culture supernatant prepared in Example 1 containing the microparticles of the present invention was administered to 42 patients with erectile dysfunction to be treated and evaluated. Note that these patients were not receiving ED treatment with other drugs such as PDE5I. The culture supernatant prepared in Example 1 was administered by direct injection into the patient's penis. Specifically, a small, soft hair band was loosely attached to the base of the penis, and 2 ml of culture supernatant was directly injected into each of the left and right corpora cavernosa using a fine needle. Each patient received a total of three culture supernatant injections at weekly intervals.

[0078] The therapeutic effect was evaluated for patients before (pre) and after the start of treatment using the IIEF-5, Rigidity and Grade therapeutic effect assessment methods.

[0079] (1) International Institute of Erectile Function (IIEF-5) Score (Japanese version): This is a type of erectile function questionnaire that derives a patient's IIEF-5 score based on the results of the questionnaire. The IIEF-5 is commonly used for screening for ED and assessing the effectiveness of ED treatment. In addition, the IIEF-5 value can also be used to determine the severity of ED. Severe ED: 5-7 Moderate ED: 8-11 Mild to moderate ED: 12-16 Mild ED: 17-21 Non-ED: 22-25

[0080] (2) Rigidity: This is a type of erectile function questionnaire, and the rigidity value is calculated as a percentage of the current erectile rigidity value (questionnaire results), assuming that the erectile rigidity at age 20 is 100%.

[0081] (3) Grade: This is a type of erectile function questionnaire that is simpler than the IIEF-5, and the Grade value is calculated based on the following erection hardness scale (Japanese version Erection Hardness Score; EHS). How do you rate your erection hardness? Grade 1: The penis enlarges but does not become firm. Grade 2: The penis is firm but not firm enough for insertion. Grade 3: The penis is firm enough for insertion but not completely firm. Grade 4: The penis is completely firm and erect.

[0082] <Comparison of Treatment Effect Judgment Methods> Figure 2(A) is a graph showing the average values of the IIEF-5 scores for 42 patients before treatment (pre) and after the end of treatment (3rd). Figure 2(B) is a graph showing the average values of the Rigidity scores for 42 patients before treatment (pre) and after the end of treatment (post). Figure 2(C) is a graph showing the average values of the Grade scores for 42 patients before treatment (pre) and after the end of treatment (post). From Figure 2(A), Figure 2(B) and Figure 2(C), it was found that the improvement rates before and after treatment showed significant improvement results (P<0.0001) for all treatment effect judgment methods.

[0083] There was a significantly positive correlation (P = 0.003) between the improvement rate of the IIEF-5 score (3rd IIEF-5 score / pre IIEF-5 score) and the improvement rate of the Rigidity score (3rd Rigidity score / pre Rigidity score). There was a significantly positive correlation (P<0.0001) between the improvement rate of the IIEF-5 score (3rd IIEF-5 score / pre IIEF-5 score) and the improvement rate of the Grade score (3rd Grade score / pre Grade score). From the above, it was found that there was a significantly positive correlation between the treatment effect judgment methods.

[0084] <Comparison of Treatment Effects for Each ED Severity> Figure 3 is a bar graph showing the relationship between the ED severity based on the IIEF-5 score before treatment and the improvement rate of the IIEF-5 score (3rd IIEF-5 score / pre IIEF-5 score); and a line graph showing the relationship between the ED severity based on the IIEF-5 score before treatment and age. From the bar graph of Fig. 3, it was found that the higher the severity of ED before treatment, the higher the improvement rate of the IIEF-5 value before and after treatment. That is, in the severity classification of ED before treatment based on the IIEF-5, the worse the severity, the more significant the treatment effect was obtained. In particular, it was found that the microparticles of the present invention can obtain a significant treatment effect when used for administration to subjects with an international erectile function score IIEF-5 of 10 or less. From the line graph of Fig. 3, no significant correlation was found between the severity of ED before treatment and age.

[0085] <Factors for predicting responder cases before treatment> Twenty-two cases with an improvement rate of the IIEF-5 value (3rd IIEF-5 value / pre IIEF-5 value) of 135% (the median of all 42 cases) or more were defined as Responders, and 20 cases less than 135% were defined as Non-Responders and analyzed. The obtained results are shown in Table 2 below. Note that 1st is the patient after the injection of the first culture supernatant, 2nd is the patient after the injection of the second culture supernatant, and 3rd is the patient after the injection of the third culture supernatant (i.e., after the end of treatment). From Table 2 below, significant differences were found in age and the IIEF-5 value (pre) between the two groups of Responders and Non-Responders.

[0086]

Table 2

[0087] Furthermore, all 42 cases were stratified into the following three groups according to the two factors of "age < 70" and "pre IIEF-5 value < 10". Score 0 ("age ≧ 70" and "pre IIEF-5 value ≧ 10"): Among 12 cases, the Responder was 1 case (8.3%) Score 1 ("age < 70" or "pre IIEF-5 value < 10"): Among 20 cases, the Responder was 12 cases (60.0%) Score 2 ("age < 70" and "pre-IIEF-5 score < 10"): 9 out of 10 cases (90.0%) were responders From the above, it was found that these two factors were significant predictors before treatment (p=0.0001). In the remaining case with a score of 2, after the third injection of culture supernatant, extended administration was performed by three more injections at weekly intervals. As a result, the improvement rate of IIEF-5 values after six injections (6th IIEF-5 value / pre IIEF-5 value) was 222%. In other words, with extended administration, all 10 of the 10 cases with a score of 2 ultimately became responders. From the above, it was found that the microparticles of the present invention can achieve significant therapeutic effects when administered to subjects under 70 years of age with severe ED, particularly subjects under 70 years of age with an International Institute of Erectile Function Scores (IIEF-5) of 10 or less.

[0088] <Predictive factors in other treatment efficacy assessment methods> First, 20 cases with an improvement rate of rigidity (post-rigidity / pre-rigidity) of 155% or more (median of all 42 cases) were defined as responders, and 20 cases with an improvement rate of less than 155% were defined as non-responders. The results are shown in Table 3 below. As shown in Table 3 below, a significant difference was observed in the rigidity value before the start of treatment (rigidity pre) between the two groups, Responder and Non-Responder.

[0089] [Table 3]

[0090] Next, 22 cases with a Grade improvement rate (Grade post / Grade pre) of 150% or more (median of all 42 cases) were defined as Responders, and 19 cases with an improvement rate of less than 150% were defined as Non-Responders. The results are shown in Table 4 below. As shown in Table 4 below, a significant difference was observed in the Grade values before the start of treatment (Grade pre) between the Responder and Non-Responder groups.

[0091] [Table 4]

[0092] From the above, it was found that there were significant differences in the rigidity and grade before the start of treatment, regardless of age. It was found that the microparticles of the present invention can achieve significant therapeutic effects when administered to subjects with severe ED, particularly subjects with a rigidity value of less than 40% (preferably less than 30%) or a grade value of less than 2.0.

[0093] Furthermore, erectile dysfunction can be further improved by using the exosomes prepared in Example 1, which contain a higher concentration of miRNA related to the miRNA that controls nitric oxide production, instead of the culture supernatant prepared in Example 1.

Claims

1. A therapeutic agent for erectile dysfunction, comprising a culture supernatant of dental pulp-derived stem cells, the culture supernatant of the dental pulp-derived stem cells contains microparticles, the microparticles contain miRNA that controls nitric oxide production; the microparticles are nitric oxide production promoters, It is intended for administration to a subject suffering from erectile dysfunction, A therapeutic agent for erectile dysfunction, wherein the subject is a human patient under 70 years of age with an International International Physiological Score (IIEF-5) of 11 or less.

2. The drug for treating erectile dysfunction according to claim 1, wherein the microparticles are exosomes.

3. the microparticles contain, as the miRNA that controls the production of nitric oxide, miRNA that targets a gene involved in the expression of nitric oxide synthase; The drug for treating erectile dysfunction according to claim 1, wherein the microparticles are agents that promote the expression of nitric oxide synthase.

4. The therapeutic agent for erectile dysfunction according to claim 3, wherein the microparticles contain at least one type of NOS-related miRNA as a target gene of the miRNA that targets a gene involved in the expression of nitric oxide synthase: NOS-related miRNAs: hsa-miR-101-3p, has-miR-101-5p, has-miR-155-5p, hsa-miR-16-2-3p, has-miR-16-5p, hsa-miR-455-3p.

5. The therapeutic agent for erectile dysfunction according to claim 1, which is to be administered to a subject who has developed erectile dysfunction, and whose International Institute of Erectile Function (IIEF-5) score is 10 or less.

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