JP7525840B2 - Treatment of lower urinary tract dysfunction - Google Patents

Description

The present invention relates to a therapeutic agent for lower urinary tract dysfunction and its use.

Lower urinary tract dysfunction presents symptoms such as frequent urination, urgency, urinary incontinence, difficulty in urination, residual urine, urinary retention, and bladder pain, and reduces the patient's quality of life (QOL). Lower urinary tract dysfunction also occurs as a complication after radical pelvic surgery for colorectal cancer, prostate cancer, and bladder cancer (see Non-Patent Document 1). Although there are drugs and treatments for lower urinary tract dysfunction, many cases are ineffective or ineffective. In addition, intermittent catheterization is generally performed to treat overflow incontinence, which indicates a chronic state of urinary retention, but this places a heavy burden (degree of pain) on the patient. On the other hand, interstitial cystitis (IC), one of the lower urinary tract dysfunctions, is designated as an intractable disease accompanied by symptoms such as bladder pain, urgency, and frequent urination, but an effective treatment has not yet been found (see Non-Patent Document 2). Attempts have been made to treat interstitial cystitis with stem cell transplantation, and it has been reported that adipose-derived stem cells themselves improve urination intervals when injected into the tail vein, directly into the bladder tissue, or administered transurethrally to IC model rats (see Non-Patent Document 3).

Chian-Shiung Lin, et al. urol sci. Volume 29, Issue 5, Page 237-242, 2018 Heesakkers J ,et al. Eur Urol. 2017 71(6);936-944 Chung JW, et al. ProS One. 14(22)e0226390.2019.

In addition to existing therapies, therapies using stem cells have also been attempted, but there remains a great need for new, highly effective therapies. Although therapies using stem cells are promising in terms of therapeutic effects, there are concerns about the difficulty of preserving and managing the transplanted cells, as well as side effects (e.g., the risk of cells becoming cancerous). In light of these circumstances, the objective of the present invention is to provide a novel treatment strategy for lower urinary tract dysfunction.

In order to solve the above problems, the present inventors focused on adult stem cells (also called tissue stem cells or somatic stem cells) and investigated their effectiveness in detail. As a result, in animal models of lower urinary tract dysfunction (interstitial cystitis model and overflow incontinence model), the "filtrate" obtained by filtering the homogenate of adipose tissue-derived stem cells (Adipose-derived stem cells: ADSC, ASC, Adipose-derived regeneration cells: ADRC, Adipose-derived mesenchymal stem cells: AT-MSC, AD-MSC, etc.) or bone marrow-derived stem cells (Bone marrow-derived mesenchymal stem cells: BM-MSC, bone marrow-derived stem cells: BMSC, etc.) showed excellent therapeutic effects. This result shows that the filtrate (filtered cell homogenate) prepared from ADSC or BM-MSC is effective as a therapeutic agent for lower urinary tract dysfunction, and also shows its wide range of application, i.e., high versatility. In addition, the fact that the filtrate prepared from the cell lysate, rather than the cells themselves, showed medicinal properties is of great significance in terms of clinical advantages, such as the fact that there is no need to wait until the cell culture is timed for use, the ease of preparation and handling, the fact that the materials (i.e., ADSCs or BM-MSCs) can be prepared in advance, shortening the preparation time at the time of use, and the fact that treatment with fewer side effects is possible. The filtrate used by the present inventors was obtained by centrifuging the cells (ADSCs or BM-MSCs) after disruption, and filtering the resulting supernatant through a filter, and one of its features is that cell debris and other impurities are more reliably removed by the filter treatment.

Based on the above results and considerations, the following invention is provided.
[1] A therapeutic agent for lower urinary tract dysfunction for treating interstitial cystitis or overflow urinary incontinence, comprising a filtrate obtained by filtering a homogenate of adipose tissue-derived stem cells or bone marrow-derived stem cells.
[2] The therapeutic agent for lower urinary tract dysfunction according to [1], wherein the homogenate is centrifuged before the filtration, and the resulting supernatant is subjected to the filtration.
[3] The therapeutic agent for lower urinary tract dysfunction according to [1] or [2], wherein the homogenate is obtained by a freeze-thaw treatment or an ultrasonic treatment.
[4] A method for producing a therapeutic agent for lower urinary tract dysfunction for treating interstitial cystitis or overflow urinary incontinence , comprising the following steps (1) to (3):
(1) disrupting adipose tissue-derived stem cells or bone marrow-derived stem cells;
(2) filtering the disruption solution obtained in step (1) or a supernatant obtained by centrifuging the disruption solution to obtain a filtrate; and (3) formulating the filtrate obtained in step (2).
[5] The method according to [ 4 ], wherein step (1) is carried out by freeze-thaw treatment or ultrasonic treatment.

Effect of stem cell filtrate on interstitial cystitis model. A chart of cystometric pressure measurement is shown. Comparison was made between the Sham+vehicle group (PBS injection on the day after saline injection treatment), the HCl+vehicle group (PBS injection on the day after HCl injection treatment), and the HCl+FLADSC group (ADSC filtrate injection on the day after HCl injection treatment). The figure shows the cystometric curves on the 7th day after saline or HCl treatment. Effect of stem cell filtrate on interstitial cystitis model. Micturition interval (left) and maximum intravesical pressure (right) were compared among the Sham + vehicle group, the HCl + vehicle group, and the HCl + FLADSC group. n = 9-12. Statistics were analyzed by ANOVA and Bonferroni multiple t-test. ** p < 0.01 Effect of stem cell filtrate on interstitial cystitis model. Bladder weight/body weight ratio was compared among the Sham+vehicle group, HCl+vehicle group, and HCl+FLADSC group. n=9-12. Statistics were analyzed by ANOVA and Bonferroni multiple t-test. Effect of stem cell filtrate on overflow urinary incontinence model. Cystometric chart. Comparison was made between Sham+vehicle group (PBS administration after Sham surgery), HGNI+vehicle group (PBS injection after hypogastric nerve injury), and HGNI+FLBM-MSC group (BM-MSC filtrate injection after hypogastric nerve injury). Effect of stem cell filtrate on overflow urinary incontinence model. Micturition interval (left) and maximum intravesical pressure (right) were compared between the Sham+vehicle group, the HGNI+vehicle group, and the HGNI+FLBM-MSC group. n=3-7. Statistics were analyzed by ANOVA and Bonferroni multiple t-test. ** p<0.01, * p<0.05 Effect of stem cell filtrate on overflow urinary incontinence model. Bladder weight/body weight ratio was compared among the Sham+vehicle group, HGNI+vehicle group, and HGNI+FLBM-MSC group. n=7-10. Statistics were analyzed by ANOVA and Bonferroni multiple t-test. **p<0.01

The present invention relates to a therapeutic agent for lower urinary tract dysfunction (hereinafter, also referred to as the "therapeutic agent of the present invention"). The therapeutic agent of the present invention is used for the treatment or prevention of lower urinary tract dysfunction. The "therapeutic agent" refers to a medicine that exhibits a therapeutic or preventive effect against a target disease (lower urinary tract dysfunction). The therapeutic effect includes alleviating (alleviating) symptoms (pathological condition) or associated symptoms characteristic of the target disease, and preventing or delaying the worsening of symptoms. The latter can be considered as a preventive effect in that it prevents the disease from becoming severe. Thus, the therapeutic effect and the preventive effect are partially overlapping concepts, and it is difficult to clearly distinguish them, and there is little practical benefit in doing so. A typical example of a preventive effect is preventing or delaying the recurrence of symptoms characteristic of the target disease. In addition, as long as a therapeutic agent exhibits some therapeutic effect or preventive effect, or both, against the target disease, it corresponds to a therapeutic agent for the target disease.

Lower urinary tract dysfunction is a condition in which the bladder or urinary tract is damaged, causing a decrease or impairment in urine storage or voiding function, and symptoms include frequent urination, urgency, urinary incontinence, difficulty in urination, residual urine, urinary retention, and bladder pain. For example, lower urinary tract dysfunction occurs due to diseases such as interstitial cystitis, hypertension, diabetes, kidney disorders, arteriosclerosis, spinal cord injury, and postoperative conditions, or inflammation, ischemia, and neurological disorders. Lower urinary tract dysfunction also causes overflow urinary incontinence, underactive bladder, overactive bladder, neurogenic bladder, stress urinary incontinence, prostatic hyperplasia, and the like. Therefore, the therapeutic agent of the present invention can be used to treat the above-mentioned disease groups (interstitial cystitis, underactive bladder, overactive bladder, neurogenic bladder, overflow urinary incontinence, stress urinary incontinence, prostatic hyperplasia, and the like). However, the cause of lower urinary tract dysfunction is not particularly limited. Examples of causes of lower urinary tract dysfunction include prostate disease, high blood pressure, pelvic organ prolapse, diabetes, kidney disorders, arteriosclerosis, spinal disease, pelvic surgery, ischemia, and nerve disorders.

In the therapeutic agent of the present invention, a filtrate obtained by filtering the lysate of adipose tissue-derived stem cells (ADSC) or bone marrow-derived stem cells (BM-MSC) (in other words, an extract obtained by filtering the cell lysate of ADSC or BM-MSC) is used, and the components contained therein have a unique action and effect, namely, improvement of lower urinary tract function.

Typically, the therapeutic agent of the present invention contains a filtrate obtained by filtering the lysate obtained by lysing adipose tissue-derived stem cells (ADSCs) or bone marrow-derived stem cells (BM-MSCs). However, the lysate may be centrifuged before filtering to remove insoluble components. That is, the supernatant obtained by centrifugation of the cell lysate may be filtered (filtrate) and used. An example of the centrifugation conditions is 200-300 g for 5-10 minutes.

For example, a cell suspension prepared at a concentration of 1×10 ⁶ cells/ml to 1×10 ⁷ cells/ml is used for the disruption treatment. To obtain a disrupted solution of ADSCs or BM-MSCs, ADSCs or BM-MSCs may be subjected to a disruption treatment, such as a freeze-thaw treatment (a treatment in which cells are frozen and then thawed), ultrasonic treatment, or treatment with a French press or homogenizer. Cells may be disrupted by a non-physical treatment. In addition, the cells to be subjected to the disruption treatment may not be limited to live cells, but may also be dead cells or damaged cells. Among various disruption treatments, the freeze-thaw treatment is particularly preferable because it is simple and easy, and can avoid contamination due to contact between the instrument and the cells, and is hygienic. The freeze-thaw treatment may be repeated multiple times (for example, 2 to 5 times). The freezing conditions in the freeze-thaw treatment are not particularly limited, but may be, for example, frozen at -20°C to -196°C. The thawing conditions are also not particularly limited. For example, melting in a water bath (eg, 35° C. to 40° C.) or at room temperature can be adopted.

On the other hand, an example of ultrasonic treatment conditions would be 30 minutes of treatment at an output of 200W to 300W (repeat with 10 seconds of crushing and 20 seconds of rest).

Unnecessary components are removed by filtering. Furthermore, if a filter with an appropriate pore size is used, the removal of unnecessary components and sterilization can be performed simultaneously. There are no particular limitations on the material or pore size of the filter used in filtering. However, cellulose acetate is an example of a preferred material. Metal filters may also be used. An example of the pore size is 0.2 μm to 0.45 μm.

The therapeutic agent of the present invention may contain other pharma- ceutical acceptable ingredients, such as carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, and physiological saline.

The origin, i.e., the biological species, of the ADSCs or BM-MSCs used in the therapeutic agent of the present invention is not limited, but considering issues such as immune rejection, it is preferable to use human cells.

As is clear from the above explanation, the therapeutic agent of the present invention can be produced by the following steps (1) to (3).
(1) disrupting adipose tissue-derived stem cells or bone marrow-derived stem cells; (2) filtering the disrupted solution obtained in step (1) or the supernatant obtained by centrifuging the disrupted solution to obtain a filtrate; and (3) formulating the filtrate obtained in step (2).

The cells (ADSC or BM-MSC) used in step (1) may be prepared according to standard methods. ADSC and BM-MSC are widely used for various purposes, and those skilled in the art can easily prepare them by referring to literature and textbooks. Cells provided from public cell banks or commercially available cells may also be used. Below, an example of a method for preparing ADSC is described.

<Preparation of ADSCs>
In the present invention, "adipose tissue-derived stem cells (ADSCs)" refer to somatic stem cells contained in adipose tissue, but as long as they maintain pluripotency, cells obtained by culturing (including subculture) such somatic stem cells are also considered to be "adipose tissue-derived stem cells (ADSCs)". Usually, ADSCs are prepared in an "isolated state" as cells constituting a cell population (including cells other than ADSCs derived from adipose tissue) using adipose tissue separated from a living body as a starting material. Here, "isolated state" means a state in which the cells are taken out of their original environment (i.e., a state in which they constitute a part of a living body), that is, a state in which they exist in a state different from their original state due to artificial manipulation. In addition, ADSCs are also called ASCs (Adipose-derived stem cells), AT-MSCs (Adipose-derived mesenchymal stem cells), AD-MSCs (Adipose-derived mesenchymal stem cells), etc. In this specification, the following terms, i.e., adipose tissue-derived stem cells, ADSCs, ASCs, ADRCs, AT-MSCs, and AD-MSCs, are used interchangeably.

ADSCs are prepared through steps such as isolating, washing, concentrating, and culturing stem cells from adipose matrix. The method for preparing ADSCs is not particularly limited. For example, ADSCs can be prepared according to known methods (Fraser JK et al. (2006), Fat tissue: an underappreciated source of stem cells for biotechnology. Trends in Biotechnology; Apr;24(4):150-4. Epub 2006 Feb 20. Review.; Zuk PA et al. (2002), Human adipose tissue is a source of multipotent stem cells. Molecular Biology of the Cell; Dec;13(12):4279-95.; Zuk PA et al. (2001), Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Engineering; Apr;7(2):211-28., etc. are useful references). In addition, devices for preparing ADSCs from adipose tissue (e.g., Celution (registered trademark) device (Cytori Therapeutics, San Diego, USA)) are also commercially available, and ADSCs may be prepared using such devices. Using such devices, a cell population containing ADSCs can be isolated from adipose tissue (K. Lin. et al. Cytotherapy (2008) Vol. 10, No. 4, 417-426). A specific example of a method for preparing ADSCs is shown below.

(1) Preparation of cell population from adipose tissue Adipose tissue is collected from animals by means of resection, suction, or the like. The term "animal" as used herein includes humans and non-human mammals (including pet animals, livestock, and laboratory animals, specifically, for example, monkeys, pigs, cows, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, hamsters, etc.). In order to avoid the problem of immune rejection, it is preferable to collect adipose tissue (autologous adipose tissue) from a patient (recipient) to which the therapeutic agent of the present invention is to be applied. However, this does not preclude the use of adipose tissue from the same species of animal (allogeneic) or adipose tissue from a different species of animal.

Examples of adipose tissue include subcutaneous fat, visceral fat, intramuscular fat, and intermuscular fat. Among these, subcutaneous fat is a preferred cell source because it can be collected very easily under local anesthesia and places less of a burden on the donor during collection. Usually, one type of adipose tissue is used, but two or more types of adipose tissue can be used in combination. In addition, adipose tissue collected in multiple batches (not necessarily the same type of adipose tissue) may be mixed and used for subsequent operations. The amount of adipose tissue collected can be determined taking into consideration the type of donor, the type of tissue, or the amount of ADSC required, and is, for example, about 0.5 to 500 g. However, taking into consideration the burden on the donor, it is preferable to collect an amount of about 10 to 20 g or less at one time. The collected adipose tissue is subjected to the removal of blood components attached thereto and to fragmentation as necessary, and then subjected to the following enzymatic treatment. Blood components can be removed by washing the adipose tissue in an appropriate buffer solution or culture medium.

The enzyme treatment is carried out by digesting the adipose tissue with enzymes such as collagenase, trypsin, and dispase. Such enzyme treatment may be carried out by methods and conditions known to those skilled in the art (see, for example, R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, 4th Edition, A John Wiley & Sones Inc., Publication). The cell population obtained by the above enzyme treatment includes pluripotent stem cells, endothelial cells, interstitial cells, blood cells, and/or their precursor cells. The types and ratios of cells constituting the cell population depend on the origin and type of the adipose tissue used.

(2) Obtaining sedimented cell population (SVF fraction: stromal vascular fractions) The cell population is then subjected to centrifugation. The sediment from the centrifugation is collected as a sedimented cell population (also referred to as "SVF fraction" in this specification). The conditions for centrifugation vary depending on the type and amount of cells, but are, for example, 1 to 10 minutes and 800 to 1500 rpm. Prior to centrifugation, it is preferable to filter the cell population after the enzyme treatment to remove tissues that are not digested by the enzyme.

The "SVF fraction" obtained here contains ADSC. The types and ratios of cells that make up the SVF fraction depend on the origin and type of adipose tissue used, the conditions of enzyme treatment, etc. The characteristics of the SVF fraction are also shown in the pamphlet of International Publication No. WO 2006/006692 A1.

(3) Selective culture of adherent cells (ADSC) and cell recovery
The SVF fraction contains other cellular components (endothelial cells, stromal cells, blood cells, their precursor cells, etc.) in addition to ADSCs. Therefore, in one embodiment of the present invention, the following selective culture is performed to remove unnecessary cellular components from the SVF fraction. The cells obtained as a result are used in the present invention as ADSCs.

First, the SVF fraction is suspended in an appropriate medium, then seeded on a culture dish and cultured overnight. Floating cells (non-adherent cells) are removed by changing the medium. Then, culture is continued while changing the medium as appropriate (for example, once every 2 to 4 days). Subculture is performed as necessary. The number of passages is not particularly limited, but from the viewpoint of maintaining pluripotency and proliferation ability, excessive repeated passage is not preferable (it is preferable to limit it to about 5 passages). In addition, a medium for normal animal cell culture can be used as the culture medium. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., Ltd., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Institute), etc. can be used. A medium supplemented with serum (fetal bovine serum, human serum, sheep serum, etc.) or a serum replacement (Knockout serum replacement (KSR), etc.) may be used. The amount of serum or serum replacement added can be set within the range of 5% (v/v) to 30% (v/v), for example.

The above procedure allows the adherent cells to selectively survive and proliferate. The proliferated cells are then harvested. The harvesting procedure can be performed according to standard methods, and for example, the cells can be easily harvested by detaching them with a cell scraper or pipette after enzyme treatment (trypsin or dispase treatment). In addition, when sheet culture is performed using commercially available temperature-sensitive culture dishes, it is possible to harvest the cells in sheet form without enzyme treatment. By using the cells (ADSC) harvested in this way, a cell population containing ADSC at high purity can be prepared.

(4) Low-serum culture (selective culture in low-serum medium) and cell recovery In one embodiment of the present invention, the following low-serum culture is performed instead of or after the procedure (3) above. The cells obtained as a result are used in the present invention as ADSCs.

In low-serum culture, the SVF fraction (if this step is performed after (3), the cells collected in (3) are used) is cultured under low-serum conditions to selectively proliferate the desired pluripotent stem cells (i.e., ADSCs). Since only a small amount of serum is used in the low-serum culture method, it is possible to use the serum of the subject (patient) to whom the therapeutic agent obtained by the method of the present invention is applied. In other words, culture using autologous serum is possible. Here, "under low-serum conditions" refers to conditions in which the medium contains 5% or less serum. Preferably, the cells are cultured in a culture medium containing 2% (V/V) or less serum. More preferably, the cells are cultured in a culture medium containing 2% (V/V) or less serum and 1 to 100 ng/ml fibroblast growth factor-2 (bFGF).

The serum is not limited to fetal bovine serum, and human serum, sheep serum, etc. can also be used. When the therapeutic agent obtained by the method of the present invention is used for human treatment, human serum is preferably used, and serum from the subject to be treated (i.e., autologous serum) is more preferably used.

As for the medium, any medium for normal animal cell culture can be used, provided that it contains a low amount of serum when used. For example, Dulbecco's modified Eagle's Medium (DMEM) (Nissui Pharmaceutical Co., Ltd., etc.), α-MEM (Dainippon Pharmaceutical Co., Ltd., etc.), DMEM:Ham's F12 mixed medium (1:1) (Dainippon Pharmaceutical Co., Ltd., etc.), Ham's F12 medium (Dainippon Pharmaceutical Co., Ltd., etc.), MCDB201 medium (Functional Peptide Institute), etc. can be used.

By culturing using the above method, it is possible to selectively proliferate pluripotent stem cells (ADSCs). In addition, since pluripotent stem cells (ADSCs) proliferated under the above culture conditions have high proliferation activity, the number of cells required for the present invention can be easily prepared by subculture. In addition, the pamphlet of International Publication No. 2006/006692A1 shows the characteristics of cells that selectively proliferate when the SVF fraction is cultured in low serum.

Next, the cells selectively proliferated by the above-mentioned low-serum culture are collected. The collection procedure can be carried out in the same manner as in (3) above. By using the collected cells (ADSC), a cell population containing ADSC at a high purity can be obtained.

In the above method, the cells proliferated by culturing the SVF fraction in low serum are used, but the cells proliferated by directly culturing a cell population obtained from adipose tissue in low serum (without going through centrifugation to obtain the SVF fraction) may also be used as ADSCs. That is, in one aspect of the present invention, the cells proliferated when a cell population obtained from adipose tissue is cultured in low serum are used as ADSCs. Also, instead of pluripotent stem cells obtained by selective culture (above (3) and (4)), the SVF fraction (containing adipose tissue-derived mesenchymal stem cells) may be used as is. Note that "using as is" here means using in the present invention without going through selective culture.

<Applicable diseases and administration methods>
The therapeutic agent of the present invention is used for the treatment and prevention of lower urinary tract dysfunction. Therefore, the therapeutic agent of the present invention is usually administered to patients with lower urinary tract dysfunction. However, the therapeutic agent of the present invention can also be used for experimental or research purposes such as confirming and verifying its effects.

The therapeutic agent of the present invention is preferably administered by local injection into the affected area. The injection site is typically intravesical, intravenous, or into the bladder tissue. It may be administered to two or more injection sites simultaneously or at intervals.

As an example of the dosage (injection amount) of the therapeutic agent of the present invention, for local administration to the bladder, for example, about 0.5 ml to 10 ml (preferably 1 ml to 5 ml) may be administered. For intravesical injection, the dosage may be determined according to the administration route, such as filling the bladder with about 50 ml to 100 ml. It is advisable to administer the entire amount in a single injection, by shifting the injection site and administering it in multiple portions.

The administration schedule may be created taking into consideration the sex, age, weight, and condition of the subject (patient). In addition to a single administration, the drug may be administered multiple times continuously or periodically. The administration interval when administering multiple doses is not particularly limited, and may be, for example, one day to one month. The number of administrations is also not particularly limited. An example of the number of administrations is 2 to 10 times.

When applying the therapeutic agent of the present invention, an existing drug may be administered in combination. In other words, the therapeutic agent of the present invention may be administered in combination with an existing drug. Such a combination is expected to increase the therapeutic effect.

1. Preparation of stem cell filtrate (1) Preparation of ADSC filtrate Rat ADSCs were prepared from subcutaneous fat by the usual method, and after adjusting the concentration (1 x ¹⁰⁶ cells/ml PBS), they were stored at -30°C for more than one night (if not used immediately, store at -80°C). The cell solution was thawed in a 38°C water bath or at room temperature. After the cells were disrupted in this way, they were centrifuged (1200 rpm, 5 minutes) and the supernatant was collected. Next, the supernatant was filtered through a cellulose acetate membrane filter (pore size 0.2 μm) to obtain ADSC filtrate (FLADSC).

(2) Preparation of BM-MSC filtrate Rat bone marrow-derived stem cells (BM-MSCs) that had been prepared by standard methods and cryopreserved were thawed in a water bath at 38°C or at room temperature, and then centrifuged (1200 rpm, 5 min). The supernatant was filtered through a cellulose acetate membrane filter (pore size 0.2 μm) to obtain the BM-MSC filtrate (FLBM-MSCs).

2. Therapeutic Effects of Stem Cell Lysate (FSCL) 2-1. Effects on Interstitial Cystitis Model (1) Methods Rats were divided into three groups: Sham + vehicle group (n = 10), HCl + vehicle group (n = 12), and HCl + FLADSC group (n = 9). On Day 0, saline was injected into the bladder of the Sham + vehicle group, and HCl (1M) was injected into the bladder of the remaining two groups via the urethral route. Saline or HCl was left in the bladder for 1 minute, and then the bladder was washed twice with saline. On the following day, PBS was injected into the bladder of the vehicle group, and ADSC filtrate was injected into the FLADSC group via the urethral route, and the bladder was held for 1 hour. On Day 7, cystometric measurements were performed to measure the micturition interval and maximum bladder pressure during micturition.

(2) Results
The HCl+vehicle group showed a shorter micturition interval than the Sham+vehicle group (Fig. 1, Fig. 2, left). The HCl+FLADSC group showed a longer micturition interval than the HCl+vehicle group, and frequent urination symptoms improved (Fig. 2, left). There was no significant difference in maximum bladder pressure during micturition between the groups (Fig. 2, right). There was no significant difference in bladder weight between the groups (Fig. 3).

(3) Observations
ADSC filtrate improved the frequent urination symptom of interstitial cystitis. It has been shown that in the HCl-induced interstitial cystitis model, an increase in inflammatory cytokines in the bladder epithelium and fibrosis of the bladder tissue occur (Phil Hyun Song, et al. Int Neurourol J. 2017 Sep; 21(3): 163-170.). Therefore, it is expected that the ADSC filtrate exerted its effect through its anti-inflammatory action. In addition, since the HCl-induced interstitial cystitis model also causes damage to the bladder epithelial layer, it is expected that the effect may be due to tissue repair of the bladder epithelium.

2-2. Effect of FSCL administration on overflow urinary incontinence model (1) Method Rats were divided into three groups: sham + vehicle group (n = 7), HGNI + vehicle group (n = 10), and HGNI + FLBM-MSC group (n = 10). Immediately after sham surgery or HGNI, vehicle or FLBM-MSC was administered via the tail vein. One week later, bladder function was evaluated by cystometry.

(2) Results
Overflow incontinence was observed in the HGNI+vehicle group. Three of the 10 rats had urination peaks, while seven rats had no peaks and were constantly incontinent (Fig. 4). The urination intervals of the three rats in which urination peaks were observed were significantly longer than those of the sham group rats (Fig. 5, left). On the other hand, in the HGNI+FLBM-MSC group, seven of the 10 rats had urination peaks, while three did not (Fig. 4). The urination intervals of the seven rats in which urination peaks were observed were significantly shorter than those of the three rats in the HGNI+vehicle group, indicating improvement (Fig. 5, left). No significant differences were observed in the maximum intravesical pressure and baseline between the groups (Fig. 5, right). The bladder weight/body weight ratio was significantly increased in the HGNI+vehicle group compared to the sham+vehicle group, but the increase was suppressed in the HGNI+FLBM-MSC group (Fig. 6).

(3) Observations
Administration of BM-MSC filtrate reduced the proportion of individuals with symptoms of overflow urinary incontinence. Evaluation of individuals whose urination patterns were observed showed that administration of BM-MSC filtrate improved bladder weight and urination interval. Thus, it was suggested that BM-MSC filtrate is effective in treating overflow urinary incontinence.

3. Summary As described above, it has been demonstrated that stem cell filtrate is extremely useful as a preventive or therapeutic drug for lower urinary tract function. The use of stem cell filtrate, a non-cellular preparation, instead of stem cells themselves, allows for treatment with significantly higher safety than previously reported stem cell therapies. In particular, if stem cell filtrate is administered locally (e.g., injected into the bladder or bladder tissue), the risk of systemic side effects is greatly reduced.

The therapeutic agent of the present invention is used for the treatment and prevention of lower urinary tract dysfunction. The therapeutic agent of the present invention contains a specific stem cell filtrate (obtained by filtering cell lysate) as an active ingredient, and exerts its efficacy through a unique mechanism of action. Therefore, it is expected to be effective in treating patients who have not responded to conventional treatments.

This invention is not limited in any way to the above-mentioned embodiments and examples. This invention also includes various modifications within the scope of the claims and within the scope that can be easily conceived by a person skilled in the art. The contents of papers, published patent publications, patent publications, etc. explicitly stated in this specification are hereby cited by reference in their entirety.

Claims (5)

A therapeutic agent for treating lower urinary tract dysfunction for treating interstitial cystitis or overflow urinary incontinence, comprising a filtrate obtained by filtering a homogenate of adipose tissue-derived stem cells or bone marrow-derived stem cells. The lower urinary tract dysfunction treatment agent according to claim 1, wherein the homogenate is centrifuged before the filtration, and the resulting supernatant is subjected to the filtration. The lower urinary tract dysfunction treatment agent according to claim 1 or 2, wherein the homogenate is obtained by freeze-thawing or ultrasonic treatment. A method for producing a therapeutic agent for treating lower urinary tract dysfunction for treating interstitial cystitis or overflow urinary incontinence, comprising the following steps (1) to (3):
(1) disrupting adipose tissue-derived stem cells or bone marrow-derived stem cells;
(2) filtering the disruption solution obtained in step (1) or a supernatant obtained by centrifuging the disruption solution to obtain a filtrate; and (3) formulating the filtrate obtained in step (2). The method according to claim 4 , wherein step (1) is carried out by a freeze-thaw treatment or an ultrasonic treatment.

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