Myogenic progenitor cells used in optimized methods for the prevention and treatment of anal incontinence

Abstract

The present invention relates to myogenic precursor cells (MPCs) for use in a method for the prevention and / or treatment of anal incontinence in a subject, said subject being at risk of developing anal incontinence or having suffered from anal incontinence for up to 20 years, more preferably from about 6 months to about 20 years or from about 6 months to about 10 years. The present invention also refers to a pharmaceutical composition comprising MPCs and a pharma- ceutical acceptable excipient and / or carrier for use in a method for the prevention and / or treatment of anal incontinence according to the present invention.

Description

Technical Field

[0001] Field of the Invention The present invention relates to myogenic progenitor cells (MPCs) for use in a method for the prevention and / or treatment of anal incontinence in a subject, said subject being at risk of developing anal incontinence or having suffered from anal incontinence for 20 years or less, more preferably about 6 months to about 20 years, or about 6 months to about 10 years. The present invention also relates to a pharmaceutical composition comprising MPCs and a pharmaceutically acceptable additive and / or carrier for use in a method for the prevention and / or treatment of anal incontinence according to the present invention.

Background Art

[0002] Background of the Invention Anal Incontinence and Its Epidemiology The ability to maintain continence is fundamental to our well-being as social beings. The loss of voluntary control of the anus results in physical, physiological, and social handicaps. In general, anal incontinence is mainly considered to affect the elderly and the disabled, but these symptoms can occur in all age groups. The range of anal incontinence, i.e., the inability to control the contents of the intestine, ranges from small fecal marks remaining on underwear to massive episodes of loss of flatus, uncontrollable defecation of soft or solid stools. The reasons can be multilayered and complex. Regardless of the extreme impairment of the quality of life of affected individuals, disorders of anal voluntary regulation are a cost factor that cannot be underestimated for public health systems. In the United States, more than $400 million per year is spent on anal incontinence support programs. Furthermore, anal incontinence is the second most common reason for admission to nursing homes (more common than dementia). One-third of the elderly in nursing homes and hospitals are fecally incontinent.

[0003] Anatomy and Physiology of Anal Incontinence The anatomical structures necessary for voluntary anal control have been studied in more detail over the past decade, thanks to the potential of intraanal imaging technology. This has also deepened our understanding of the mechanisms of voluntary anal control and the factors involved in maintaining it. Voluntary anal control requires the coordination of different anatomical structures with different physiological functions. Undamaged sensation ensures the recognition of rectal fullness and stool quality. Functional innervation allows specialized ring-shaped muscles (sphincters) to respond appropriately (spontaneously and involuntaryly) to the increasing "closure demands" of the anus. Finally, if the sphincters are intact, they completely occlude the anal canal until defecation is appropriate. Dysfunction of any of these structures leads to impaired voluntary anal control. The function of sphincter tissue is based on the involuntary resting pressure of the internal anal sphincter, which is made of smooth muscle tissue, and the involuntary resting pressure and voluntary squeezing pressure arising from the external anal sphincter. A constant baseline tension in the puborectalis muscle creates a "distortion" in the anorectal junction, leading to a 90° angle between the anal canal and the rectum. This anorectal angle also contributes to maintaining voluntary control of the anus. However, voluntary control cannot be maintained by the puborectalis muscle alone. Voluntary control of the anus is further brought about by the interaction of the internal and external anal sphincters. The rectal cushion of the anal canal mucosa, tightened by the sphincters, ultimately results in airtight occlusion. At rest, the anal canal is closed by constant rigid activity of the external anal sphincter and baseline resting pressure of the internal anal sphincter. The internal anal sphincter is an extension and expansion of the round smooth muscle layer of the colon and provides approximately 75-85% of the baseline pressure of a closed anal canal. The activity of this smooth muscle component is completely inhibited by rectal dilation, the so-called anorectal inhibitory reflex. This relaxation is accompanied by reflex contractions of the external anal sphincter and puborectalis muscle, which, if inappropriate, can hinder defecation. If defecation is convenient at that moment, the external anal sphincter and puborectalis muscle relax, triggering involuntary movement of the colon and rectum. As a result, rectal pressure becomes greater than anal pressure, and stool is subsequently expelled. If defecation is inconvenient when the urge arises, the puborectalis muscle and external anal sphincter can be contracted voluntarily to push the stool back into the rectum until defecation becomes convenient, thereby delaying defecation.

[0004] Causes of fecal incontinence Since the dysfunction of these structures leads to incontinence, the importance of the described structures related to voluntary regulation is emphasized. Most patients with fecal incontinence are diagnosed with abnormalities of the anal sphincter. These abnormalities can occur in the external anal sphincter, the internal anal sphincter, or both. Sphincter-related incontinence can be caused by (1) obstetric or accidental trauma, (2) iatrogenic trauma such as surgery or radiotherapy, (3) neurological diseases such as multiple sclerosis or diabetes, or (4) muscle atrophy associated with aging. Obstetric trauma is the main cause of female incontinence because up to 9% of vaginal deliveries can lead to rupture of the anal sphincter.

[0005] Treatment of fecal incontinence Patients are referred to invasive treatment when conservative treatments such as dietary changes, biofeedback exercise, and antidiarrheal medications fail, are inadequately successful, or cannot be performed due to severe anatomical, physiological, or neurological dysfunction. Conventional invasive treatments include surgical approaches such as anal sphincter repair and reconstruction (sphincteroplasty, colostomy sphincter reconstruction, membranoplasty) if there is a sphincter defect, or surgical approaches such as colostomy or antegrade inhibitory enemas if incontinence occurs due to complete spinal cord injury. However, conventional surgical approaches for sphincter repair have a high failure rate of 50%, and surgical approaches for creating new sphincters have a high morbidity. One less invasive treatment for incontinence is perianal injection of a volume expander thought to increase pressure in the anal canal. In a single randomized sham-controlled trial, injections of NASHA Dx (hyaluronic acid-stabilized dextranomer) were found to provide significantly better treatment outcomes than placebo injections. In detail, 52% of patients experienced at least a 50% reduction in weekly incontinence episodes compared to 32% of patients who received sham treatment. However, only 6% of patients were found to be fully voluntarily controlled, and the efficacy data available to date only covers short-term effects of up to 6 months. It was also noted that the impact of dose increasers on patients' quality of life was limited. Therefore, more effective treatments for anal incontinence are needed. Over the past 20 years, neuromodulation has attracted attention as a treatment option for incontinence, with sacral nerve stimulation (SNS) proving to be the most promising in terms of efficacy. SNS therapy is based on the implantation of medical devices (e.g., Interstim, Medtronic, USA) that use chronic low-voltage electrical stimulation of the sacral nerves to improve neuromuscular function, although the mode of action (MoA) is still unclear. The success rate was defined as the percentage of patients whose weekly incontinence frequency (IEF) decreased by at least 50% after social steroid therapy (SNS). This was aggregated from 61 trials and the median success rates for short-term (less than 12 months), medium-term (12–36 months), and long-term (>36 months), and was found to be 63%, 58%, and 54%, respectively.However, 13.1% of the screened patients did not respond to the test stimulus and therefore did not receive permanent implantation, and approximately 25% of the patients included in the long-term follow-up had their stimulators removed due to complications (technical or infectious) or loss of efficacy.

[0006] Since incontinence is primarily caused by a defect / weakness of rectal muscle tissue, animal models of muscle regeneration suggest that intramuscularly injected cells engraft within the host's muscle, and early clinical trials of treating stress urinary incontinence with injections of muscle-derived cells were considered promising. This led to the idea of ​​regenerating the anal sphincter muscle through cell therapy (Frudinger et al., 2009). However, for cell therapy for incontinence to be market-approved and subsequently widely applied, the treatment must be more effective than the placebo effect and clinically relevant in that it provides sufficient benefit and / or complete remission to the target population. An effect of at least a 50% reduction in incontinence episodes per week is considered clinically relevant. For the method to be useful, the proportion of patients receiving cell therapy must be significantly higher compared to those receiving placebo, and such a reduction is necessary for the method to be considered effective.

[0007] As outlined above, incontinence is a highly complex condition with many different causes, histories, and severity levels, so it is necessary to select the most appropriate patient population that will benefit from a particular approach. Each treatment method may target different patient populations to achieve significant and relevant therapeutic effects.

[0008] EP210976B1 discloses the use of myoblasts for the prevention and treatment of rectal incontinence by injecting myoblasts into the external anal sphincter of subjects with rectal injury. WO2014 / 044867A1 discloses a method of treating urinary and / or rectal incontinence, or women and men at risk of developing urinary or rectal incontinence, by administering skeletal muscle-derived cells. WO2019 / 115790 discloses skeletal muscle-derived cells for use in the treatment of incontinence. WO2020 / 193460 discloses induced smooth muscle cells for use in a method of treating a disease or disorder in a subject. Clinical studies have been conducted to test the potential efficacy of myoblasts in the treatment of fecal incontinence, but no superior effect of these methods compared to the placebo effect has been demonstrated. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] This invention has been made in view of the prior art described above. Accordingly, one object of this invention is to provide an effective method for using myogenic progenitor cells for the prevention and / or treatment of anal incontinence.

[0010] To our surprise, the inventors discovered that while injection of myogenic progenitor cells had little to no effect in some subjects, injection of myogenic progenitor cells significantly improved the condition of other subjects.

[0011] None of the above disclosures, within the broad range of indications for incontinence, select a narrow patient population that is ultimately eligible for the administration of myogenic progenitor cells for improved prevention / treatment of anal incontinence. Therefore, a fundamental objective of the present invention was to identify a group of subjects that can be effectively treated by injection of myogenic progenitor cells in the context of an individualized therapeutic approach. Another objective of the present invention is to provide an optimized method for the use of myogenic progenitor cells for the prevention and / or treatment of anal incontinence, resulting in a more effective treatment of anal incontinence.

[0012] None of the above disclosures select a patient population according to the subject's condition related to incontinence, the duration of incontinence, and / or the severity of incontinence. Therefore, another object of the present invention is to provide a method for using myogenic progenitor cells for the prevention and / or treatment of anal incontinence, which allows for the selection of a patient population according to the subject's condition related to incontinence, the duration of incontinence, and / or the severity of incontinence.

[0013] None of the above disclosures constitute a selection of a safe and effective cell count for the prevention and / or treatment of anal incontinence. Therefore, one object of the present invention is to select a specific cell dose in a method for using myogenic progenitor cells for the prevention and / or treatment of anal incontinence.

[0014] Furthermore, none of the above disclosures select an optimized stimulus to accompany a method of using myogenic progenitor cells for the prevention and / or treatment of anal incontinence. Therefore, another object of the present invention is to select an optimized accompanying stimulus in a method of using myogenic progenitor cells for the prevention and / or treatment of anal incontinence. [Means for solving the problem]

[0015] The object of the present invention is solved by the subject matter defined in the claims. [Brief explanation of the drawing]

[0016] The following figures form part of this specification and are included to further illustrate specific aspects of the invention. The invention may be better understood by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0017] [Figure 1]Figure 1 shows the change in incontinence episode frequency (IEF) per week from baseline (A, B) and the 50% response rate (C, D) over the duration of the study in Example 5 in the treatment groups (low cell count - LCC, high cell count - HCC, placebo - PBO) in the inclusive analysis (ITT) population. Here, episodes classified as trace are counted for analysis (A, C) or excluded (B, D). [Figure 2] Figure 2 shows the change in IEF per week from baseline to 12 months post-treatment (A, B) and the 50% response rate at 12 months post-treatment (C, D) according to Example 5, as is evident from the fact that each was counted as an incontinence episode, calculated by including or excluding (B, D) (A, C), respectively. Data were visualized between treatment groups (PBO, LCC, HCC) in different patient populations according to the subject selection of Example 6. The different patient population defined in Example 6, called TPP1, consists of patients with fecal incontinence for 10 years or less. TPP2 consists of patients with more than two episodes at baseline, classified as small or large. TPP3 consists of patients with fecal incontinence for 10 years or less and with more than two IEFs per week at baseline, classified as small or larger. NR1 consists of patients with fecal incontinence (FI) for 10 years or more, and NR2 consists of patients with two or fewer incontinence episodes (IE) classified as "small" or "large". The p-value for a two-sided Wilcoxon test (A, B) or Fisher's exact test (C, D) comparing PBO with LCC or HCC is always shown if the p-value reaches the significance threshold of p < 0.05. [Figure 3] Figure 3 shows phase-contrast microscopy images of myogenic progenitor cells isolated according to Example 1 and tested for differentiation potential according to Example 2. At 600x magnification, myotubes are visible and contain multiple nuclei (black arrows), thereby demonstrating the potential for fusion of mononuclear MPCs. [Figure 4]Figure 4 shows an overview of the clinical study conducted according to Example 5. The timeline of study visits and data collection, including the parameters evaluated, is shown in A. The treatment allocation is indicated by the number (N) in B. QoL = Quality of Life, V = Visits, VAS = Visual Analog Scale, WIE = Incontinence Episodes per Week, CGI = Overall Impression Scale. [Figure 5] Figure 5 shows Tukey's box plots summarizing the acetylcholinesterase (AChE) activity of LCC(A) and HCC(B) batches containing MPC, isolated according to Example 1, differentiated in vitro according to Example 2, and then measured for AChE activity according to Example 3. Activity is expressed as relative mU per 2 × 10⁵ cells. The LCC batch (n=83) was found to possess a mean ± SD AChE activity of 241.90 ± 151.90 in the range of 36 to 568. The HCC batch (n=75) was found to possess a mean ± SD AChE activity of 213 ± 137.40 in the range of 49 to 680. [Figure 6] Figure 6 shows the percentage of MPCs that were positive for the surface cell markers CD34, CD56, and CD90 in either the low-cell-count (LCC, n=83) (A) or high-cell-count (HCC, n=75) formulations prepared according to Example 1 and tested for surface markers according to Example 4. The data are visualized as Tukey's bar graphs (outliers are shown as points). All LCC and HCC batches were found to be CD34 negative (mean ±SD %CD34 positive at 1.29 ± 1.37 for LCC and 1.10 ± 0.90 for HCC). All LCC and HCC batches were found to be CD56 positive (mean ±SD %CD56 positive at 92.06 ± 6.73 for LCC and 89.86 ± 7.06 for HCC). All LCC and HCC batches were found to be CD90 positive (94.00±5.00 for LCC and 94.99±4.52% for HCC, with a mean ±SD of %CD90 positivity). [Figure 7]Figure 7 shows the change in ITF from baseline to 6 months post-treatment among treatment groups (PBO, LCC, HCC) in ITT sets of patients further subgrouped according to the increasing number of baseline IEFs (including traces). Data were visualized as mean ± SEM. The change in IEF from baseline to 6 months increased in all groups by successively excluding patients with low baseline IEFs, but this was more pronounced in LCC and HCC patients than in PBO patients. [Figure 8] Figure 8 shows the effect size of IEF (Cohen's d) (A, B) from baseline to 12 months post-treatment, and the odds ratio of the 50% responder rate at 12 months post-treatment (C, D), calculated by comparing LCC or HCC with PBO treatment, with or without considering traces of incontinence episodes (A, C) or not (B, D), in the patient populations of ITT, TPP1, TPP2, TPP3, NR1, and NR2. [Figure 9] Figure 9 shows the change in IEF (A, B) and 50% responder rate (C, D) from baseline to 12 months post-treatment between treatment groups (PBO, LCC, HCC) in TPP3 patients, further subgrouped to select only TPP3 set patients and patients with fecal incontinence associated with anal sphincter injury (TPP3_injury). Data are shown either with or without traces of incontinence episodes (A, C). Data are shown mean ± SEM. For the change in IEF, a two-sided Wilcoxon rank-sum test p-value of less than 0.05 alpha error was indicated for the change in IEF. A Fisher's exact test p-value of less than 0.05 was indicated for the response rates of LCC or HCC versus PBO treatment. [Figure 10]Figure 10 shows the effect size of IEF (Cohen's d) (A, B) from baseline to 12 months post-treatment, and the odds ratio of the 50% responder rate at 12 months post-treatment (C, D), calculated by comparing LCC or HCC with PBO treatment in the TPP3 and TPP3_injured patient populations, with or without considering traces of incontinence episodes (A, C) or (B, D). [Modes for carrying out the invention]

[0018] [Terms and Definitions] As used herein, the term “anal incontinence” refers to the undesirable loss of intestinal contents through the anus, such as flatulence, liquid or solid feces. The term comprises all three severity grades: Grade 1 is gas only, Grade 2 is liquid and loose stools, and Grade 3 is solid stool.

[0019] As used herein, the terms “anal sphincter” or “anal sphincter tissue” preferably refer to the levator ani muscle and the anal sphincter as part of the anal sphincter, and the puborectalis muscle. However, the pubococcygeus muscle, coccygeus muscle, iliococcygeus muscle and pudendal nerve may also be included.

[0020] As used herein, the term “fecal incontinence” refers to the undesirable loss of intestinal contents through the anus, preferably limited to liquid or solid feces. The term may include severity grades of “anal incontinence”: Grade 2 = liquid and loose stools, Grade 3 = solid, formed stools.

[0021] As used herein, the term “myogenic progenitor cells” or short for “MPC” preferably refers to cells having myogenic potential, which may be primary cells and / or in vitro cultured cells derived from muscle tissue, or other cells from which myogenic potential originates, such as adipose tissue or other stem cell-containing tissues, such as bone marrow, but is not limited to these. The term also includes cells that are pluripotent stem cells, or cells derived from pluripotent stem cells that can become muscle cells after administration, either when isolated and cultured, or within or adjacent to muscle tissue. Since myogenic progenitor cells are preferably obtained by isolation of muscle tissue, the term “myogenic progenitor cells” may also refer to a suspension of several different cell types, where at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% of the cells are “myogenic progenitor cells” as defined herein. Preferably, the suspension is a single-cell suspension in which at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or at least 99% of the single cells are homogeneous of the same cell type, i.e., myogenic progenitor cells. In addition to myogenic progenitor cells as defined herein, the suspension may also contain other cell types, such as cells of adipose tissue, chondrocytes, osteocytes, and / or fibroblasts. Such cells may be mesenchymal cells, adipocytes, chondrocytes, osteocytes, and / or process-related impurities, and / or any other cell type that can be isolated together with the myogenic progenitor cells. Such cells may constitute up to 1%, 2%, 5%, 10%, 20%, 30%, 40%, or 50% of the myogenic progenitor cell suspension.

[0022] The term "myogenic potential" preferably refers to the potential of a cell, cells, or cell population to develop, regenerate, and / or enhance muscle tissue. As used herein, myogenic potential may further relate to the potential of a cell, cells, or cell population to differentiate into multinucleated myotubules in vitro and / or express enzymatically active acetylcholinesterase.

[0023] As used herein, the term “penetration” preferably refers to the process of introducing an injection device, such as a needle, into body tissue without affecting the injection process.

[0024] As used herein, the term “injection” preferably refers to the release of an injectable solution from an injection device to a specific site in the human body, particularly within or adjacent to muscle tissue resulting in anal incontinence (e.g., anal sphincter tissue), which releases the above-mentioned cells. The injection process may, but is not limited to, a static injection; that is, the injection device remains in the position reached. The static injection process preferably refers to an injection process in which the injection device is stationary during the discharge of the injectable solution, i.e., does not move relative to the discharged injectable solution. Alternatively, the injection process may be dynamic. The dynamic injection process preferably refers to an injection process in which the injection device is dynamic during the discharge of the injectable solution, i.e., moves relative to the discharged injectable solution. A dynamic injection process may be performed, for example, by moving a hollow needle; the needle's trajectory is reversed within the external anal sphincter, thereby simultaneously discharging the injectable solution. Alternatively, the term “injection” as used herein preferably refers to any route of administration suitable for bringing the cells adjacent to the anal sphincter tissue. Such routes of administration preferably include oral, topical, intravenous, or intra-arterial administration.

[0025] As used herein, the term “injection site” preferably refers to, but is not limited to, a site within the human body where the injection process may be initiated, such as a site near rectal incontinence or a site near muscle tissue. The injection site may or may not be the same as the site where the injection process ends.

[0026] As used herein, the term “injection device” includes any device suitable for reaching the target injection site and penetrating human tissue to deliver a solution, particularly a solution containing myogenic progenitor cells, to the target injection site.

[0027] As used herein, the term “passive incontinence” preferably refers to the absence of sensory awareness of the loss of stool. This includes a low reference pressure value in the anus and a lack of sensory capacity of the mucous membranes of the anus and rectum.

[0028] As used herein, the terms “urge urinary incontinence” or “urge urgency” preferably refer to a lack of ability to delay defecation for five minutes or more after the recognition of the urge to defecate. Such patients must go to the toilet immediately and / or are unable to go to the toilet quickly enough to empty their bowel contents into the toilet, resulting in involuntary defecation.

[0029] As used herein, the terms “CD56+” or “CD56-positive” preferably refer to cells that express the cell marker CD56. The terms “CD56+” or “CD56-positive” may also be used for cell populations containing different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the CD56 cell marker.

[0030] As used herein, the terms “CD56-” or “CD56-negative” preferably refer to cells that do not express the cell marker CD56. The terms “CD56-” or “CD56-negative” may also be used for cell populations containing different cell types, preferably if at least 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent of the cell population expresses the cell marker CD56.

[0031] As used herein, the terms “A2B5+” or “A2B5-positive” preferably refer to cells that express the cell marker A2B5. The terms “A2B5+” or “A2B5-positive” may also be used for cell populations containing different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the cell marker A2B5.

[0032] As used herein, the terms “A2B5-” or “A2B5-negative” preferably refer to cells that do not express the cell marker A2B5. The terms “A2B5-” or “A2B5-negative” may also be used for cell populations containing different cell types, preferably if at least 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent of the cell population expresses the cell marker A2B5.

[0033] As used herein, the term “desmin-positive” preferably refers to cells that express the cell marker desmin. The term “desmin-positive” may also be used for cell populations containing different cell types, preferably if at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the cell marker desmin.

[0034] As used herein, the term “desmin-negative” preferably refers to cells that do not express the cell marker desmin. The term “desmin-negative” may also be used for cell populations containing different cell types, preferably when at least 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent of the cell population express the cell marker desmin.

[0035] As used herein, the terms “CD105+” or “CD105-positive” preferably refer to cells that express the cell marker CD105. The terms “CD105+” or “CD105-positive” may also be used for cell populations containing different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the cell marker CD105.

[0036] As used herein, the terms “CD105-” or “CD105-negative” preferably refer to cells that do not express the cell marker CD105. The terms “CD105-” or “CD105-negative” may also be used for cell populations containing different cell types, preferably if at least 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent of the cell population expresses the cell marker CD105.

[0037] As used herein, the terms “CD34+” or “CD34-positive” preferably refer to cells that express the cell marker CD34. The terms “CD34+” or “CD34-positive” may also be used for cell populations containing different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the cell marker CD34.

[0038] As used herein, the terms “CD34-” or “CD34-negative” preferably refer to cells that do not express the cell marker CD34. The terms “CD34-” or “CD34-negative” can also be used for cell populations containing different cell types, preferably when less than 50% of the cell population, or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent, express the cell marker CD34. In a particularly preferred embodiment, the terms “CD34-” or “CD34-negative” can also be used for cell populations containing different cells, preferably when at most 19, 10, 5, 4, 3, 2, 1, or 0 percent express the cell marker CD34.

[0039] As used herein, the terms “CD90+” or “CD90-positive” preferably refer to cells that express the cell marker CD90. The terms “CD90+” or “CD90-positive” may also be used for cell populations containing different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the cell marker CD90.

[0040] As used herein, the terms “CD90-” or “CD90-negative” preferably refer to cells that do not express the cell marker CD90. The terms “CD90-” or “CD90-negative” can also be used for cell populations containing different cell types, preferably when less than 50% of the cell population, or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent, express the cell marker CD90.

[0041] As used herein, the terms "Tuj1+" or "Tuj1-positive" preferably refer to cells that express the cell marker Tuj1. The terms "Tujl+" or "Tujl-positive" may also be used for cell populations containing different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the cell marker Tuj1.

[0042] As used herein, the terms "Tuj1-" or "Tuj1-negative" preferably refer to cells that do not express the cell marker Tuj1. The terms "Tujl-" or "Tujl-negative" can also be used for cell populations containing different cell types, preferably when less than 50% of the cell population, or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent, express the cell marker Tuj1.

[0043] As used herein, the terms “nestin+” or “nestin-positive” preferably refer to cells that express the cell marker nestin. The terms “nestin+” or “nestin-positive” may also be used for cell populations including different cell types, preferably if at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the cell marker nestin.

[0044] As used herein, the terms “nestin-” or “nestin-negative” preferably refer to cells that do not express the cell marker nestin. The terms “nestin-” or “nestin-negative” may also be used for cell populations containing different cell types, preferably if less than 50% of the cell population, or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent, express the cell marker nestin.

[0045] As used herein, the terms “Sca-1+” or “Sca-1 positive” preferably refer to cells that express the cell marker Sca-1. The terms “Sca-1+” or “Sca-1 positive” can also be used for cell populations containing different cell types, preferably when at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the cell marker Sca-1.

[0046] As used herein, the terms "Sca-1-" or "Sca-1 negative" preferably refer to cells that do not express the cell marker Sca-1. The terms "Sca-1-" or "Sca-1 negative" can also be used for cell populations containing different cell types, preferably if less than 50% of the cell population, or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent, express the cell marker Sca-1.

[0047] As used herein, the terms "MyoD+" or "MyoD-positive" preferably refer to cells that express the cell marker MyoD. The terms "MyoD+" or "MyoD-positive" may also be used for cell populations containing different cell types, preferably if at least 50, 60, 70, 80, 90, 95, 98, or 99 percent of the cell population express the cell marker MyoD.

[0048] As used herein, the terms "MyoD-" or "MyoD-negative" preferably refer to cells that do not express the cell marker MyoD. The terms "MyoD-" or "MyoD-negative" may also be used for cell populations containing different cell types, preferably when less than 50% of the cell population expresses MyoD, or at most 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, or 0 percent.

[0049] As used herein, the term “differentiation medium” preferably refers to a cell culture medium that induces fusion in multinuclear fusion-competent cells or myogenic progenitor cells, such as myoblasts. However, the term may also refer to a cell culture medium that does not contain substances necessary for inducing fusion, if multinuclear fusion-competent cells or myogenic cells can fuse without induction.

[0050] As used herein, the term “cell growth medium” preferably refers to any medium suitable for incubation of mammalian cells, such as myogenic progenitor cells, that allows the mammalian cells to adhere to the surface of an incubation container (flask or dish).

[0051] As used herein, the terms “incontinence episode” or “IE” preferably refer to an event in which liquid or solid feces passing through the rectum are lost uncontrollably, unexpectedly, and / or involuntarily. Incontinence episodes can be classified as “trace,” “small,” or “large.” This classification can be done by having the patient keep a diary, which allows tracking of the date and time of the incontinence event and, if possible, classification of the size and / or amount of incontinence. The diary is preferably kept for at least one week, more preferably about one to four weeks, about one to three weeks, or about one to two weeks.

[0052] As used herein, “weekly incontinence episode frequency” is also referred to as “weekly IE frequency” or “weekly IEF,” and preferably refers to the number of incontinence episodes that occurred during a 28-day period, normalized to a 7-day period and calculated as follows: IEF per week = (Number of incontinence episodes reported during the period / Number of days completed during the period) x 7

[0053] In this specification, episodes of incontinence classified as “trace” preferably correspond to episodes of anal incontinence in which liquid or solid feces unexpectedly leak from the rectum of the person concerned, and the amount of feces is so small that it appears as a stain or mark on the linen. Such “trace” may not require the patient to change their underwear because the amount is absorbed by the linen of the underwear.

[0054] As used herein, the term “responder” preferably refers to a patient whose weekly IEF, calculated by comparing two incontinence diaries, has decreased by at least 50%, one of which diaries was completed during a specific period prior to a particular intervention (e.g., a method for the use of myogenic progenitor cells according to the present invention), and the other was completed during a specific period after the particular intervention. Such periods prior to and / or after the intervention are preferably one week, more preferably two weeks, even more preferably three weeks, and even more preferably four weeks.

[0055] In this specification, an episode of incontinence classified as “small amount” preferably refers to an episode of anal incontinence in which liquid or solid stool unexpectedly leaks from the subject’s rectum, and the amount of stool is greater than what would appear as a stain or mark on the subject’s linen. However, the amount is only a small amount compared to the result of a normal bowel movement. In such episodes, the amount is not efficiently absorbed by the linen and causes discomfort, so the patient may need to change their underwear.

[0056] In this specification, an incontinence episode classified as “more voluminous” preferably refers to an episode of anal incontinence in which liquid or solid feces unexpectedly leak from the rectum in question, and the amount of feces may be greater than what would appear as a stain or mark on the linens in question. The amount may be comparable to the amount produced in a normal bowel movement. In such episodes, both underwear and secondary clothing are affected, leading to severe discomfort, and therefore preferably both need to be changed.

[0057] As used herein, the term “incremental stomping pressure” preferably corresponds to the result of subtracting the resting stomping pressure from the maximum stomping pressure.

[0058] As used herein, the term “incontinence-related conditions” preferably refers to physician’s findings obtained when examining a subject at risk of developing or having developed anal incontinence. Such subjects may be examined for sphincter tissue, for example, by ultrasound and / or pressure measurement, to define conditions of muscle damage and / or muscle atrophy. Furthermore, the examiner may consider whether conditions such as pelvic floor dysfunction, nerve damage, or loss of storage capacity are associated with the risk of developing or having developed anal incontinence. Other symptoms classified as incontinence-related include diarrhea and constipation.

[0059] As used herein, the term “pelvic floor dysfunction” preferably refers to a condition in which a person is unable to properly relax and regulate the muscles of the pelvic floor for urination or defecation. Pelvic floor organs affected by pelvic floor dysfunction include the bladder, uterus, vagina, prostate, and rectum. Dysuria, incontinence, or a persistent urge to urinate may be signs of pelvic floor dysfunction.

[0060] As used herein, the term “muscle injury” preferably corresponds to a loss of muscle in the anal sphincter tissue. Such muscle injuries may be detected by a physician when performing ultrasound or other useful diagnostic techniques to visualize the anal sphincter. Such muscle injuries may affect, for example, the external and / or internal anal sphincter. A typical example of muscle injury visible on ultrasound is scar formation after sphincter rupture during childbirth.

[0061] As used herein, the term “muscle atrophy” or alternatively “atrophy” preferably refers to a decrease in the muscle mass of the anal sphincter. Such a decrease in muscle mass may be accompanied by deposition of connective tissue and / or adipose tissue between muscle cells. Muscle atrophy can occur with aging or when muscles remain unused. A physician may be able to detect muscle atrophy of the anal sphincter tissue, for example, by ultrasound.

[0062] As used herein, the term “nerve damage” preferably corresponds to the loss of function of nerve tissue that normally functionally innervates and is part of the anal sphincter tissue. Such nerves may be the pudendal nerve and / or nerves leading to the pudendal nerve. The term may also correspond to the loss of nerve tissue that may be associated with incontinence.

[0063] As used herein, the term “loss of storage capacity” preferably corresponds to an abnormality in the rectal’s tolerable volume. Loss of storage capacity can occur when the rectum hardens and loses its normal elasticity due to surgery, inflammation, or radiation therapy. Loss of rectal storage capacity leads to a lack of rectal stretching (i.e., elongation) capacity, resulting in leakage of excess stool through the rectum.

[0064] As used herein, the term “duration of incontinence” preferably corresponds to the time elapsed from the onset date or diagnosis date of incontinence. The onset date of incontinence preferably coincides with the onset date of symptoms, i.e., the date of the first occurrence of the incontinence episode. The diagnosis date of incontinence preferably corresponds to the date on which the physician diagnosed the patient with incontinence. The diagnosis date of incontinence is preferably the same as the onset date of symptoms, or alternatively, later than the onset date of symptoms. In the case of fecal incontinence, the term “duration of incontinence” may also be referred to as “duration of FI” or “duration of incontinence.” When the duration of incontinence is measured from the diagnosis of incontinence, the term “duration of incontinence” may also refer to “time from the first diagnosis of FI.”

[0065] As used herein, the term “severity of incontinence” preferably corresponds to the burden on the individual who has developed anal incontinence. Such severity may be expressed by the number of incontinence episodes per week, the impact of incontinence on the patient’s life, a quality of life score for fecal incontinence, and / or a Wexner score. Preferably, the Wexner score corresponds to the scale for evaluating voluntary control of defecation and urination disclosed in Table 3 of Jorge et al., 1993.

[0066] As used herein, the term “stimulate” preferably corresponds to any action performed on the anal sphincter tissue that results in muscle contraction. Stimulation as used herein may include spontaneous contraction of the anal sphincter or may be performed by electrical stimulation. The latter may be performed by stimulating the nerves that innervate the anal sphincter or by directly stimulating the anal sphincter muscles, such as the external and / or internal anal sphincter.

[0067] As used herein, the term "comprising" should not be construed as being limited to the meaning of "consisting of" (i.e., excluding the presence of additional other substances). Rather, "comprising" means that additional substances may optionally be present. The term "comprising" encompasses, as particularly contemplated embodiments within its scope, "consisting of" (i.e., excluding the presence of additional other substances) and "comprising but not consisting of" (i.e., requiring the presence of additional other substances), with the former being more preferred.

[0068] As used herein, the term "AChE positive" or "AChE+" preferably refers to cells or cell populations that exhibit (i.e., are positive for) acetylcholinesterase enzyme (AChE) activity. In particular, such AChE activity is positive when 2 × 10 5 cells have an AChE activity of about 20 mU rel to about 1000 mU rel , more preferably about 30 mU rel to about 800 mU rel , even more preferably about 50 mU rel to about 700 mU rel . Preferably, the 2 × 10 5 cells are cultured, for example, in the skeletal muscle differentiation medium described in the examples herein. The unit "mU rel " refers to the relative "mU / ml" of 2 × 10 cells measured 60 minutes after the addition of acetylthiocholine iodide (ATI) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and refers to the relative "mU / ml" related to the linear equation obtained from a dilution series of the AChE stock solution in the range of 4 to 500 mU / ml under the same conditions, except that the OD of the diluted solution of the stock solution has been measured at 6 to 8 minutes, preferably at 6, 7, or 8 minutes after the addition of ATI and DTNB. The term "mU 5 " may also refer to the relative "mU / ml" of 2 × 10 rel cells measured and calculated in Example 3.

[0069] ​​As used herein, the terms "AChE-negative" or "AChE-" preferably refer to cells or cell populations that do not possess AChE activity (i.e., are negative). In particular, 2 × 10 5 Each cell is approximately 0 mU rel ~about 19mU rel If AChE activity is observed, such AChE activity is negative, and the cells are preferably cultured in, for example, skeletal muscle differentiation medium. This is described in Example 2 of this specification.

[0070] As used herein, the term "multipotency" preferably refers to the differentiation ability of mesenchymal cells characterized by in vitro differentiation ability into at least adipogenic, chondrogenic, and osteogenic lines.

[0071] As used herein, the term “oligopotent” refers to the differentiation potential of cells characterized by in vitro differentiation potential that is preferably limited to myogenic lineages such as smooth muscle, striated muscle, and / or cardiac muscle.

[0072] As used herein, the term “myogenic differentiation potential” preferably refers to the ability of cells to express one or more of the following markers, desmin and myosin, in detectable amounts, known to be expressed by myogenic cells in vivo. Tests for the expression of such markers in in vitro cultured cells are well known to those skilled in the art. Such tests preferably include flow cytometry and / or immunofluorescence staining and other immunocytochemical assays, including Western blotting. Alternatively, it is preferable to use the term myogenic differentiation potential to refer to the ability of cells (e.g., myogenic progenitor cells) to form multinucleated myotubes. Such myotubes are known to those skilled in the art as muscle cells, preferably containing at least three distinct nuclei. The formation of such myotubes by mononuclear cells originally occurs, for example, intracellularly. Therefore, differentiation media can be used as tests for myogenic differentiation potential.

[0073] As used herein, the term "myotube" preferably refers to a muscle cell containing at least three distinct nuclei. Preferably, such myotubes are formed by the fusion of mononuclear cells (e.g., myogenic progenitor cells).

[0074] As used herein, the terms “neuronal differentiation potential” or “neuronal differentiation potential” refer to, but are not limited to, a population of cells that express a neuronal marker such as one or more of the markers A2B5, TUJ1, NCAM, nestin, or equivalent markers. Preferably, the terms refer to a population of cells in which at least 60% of the cells express A2B5, more preferably at least 60% of the cells express A2B5 and TUJ1, or at least 60% of the cells express A2B5 and NCAM, or at least 60% of the cells express A2B5 and nestin, or at least 60% of the cells express TUJ1 and NCAM, or at least 60% of the cells express TUJ1 and nestin, or at least 60% of the cells express NCAM and nestin. More preferably, the terms refer to a cell population in which at least 60% of cells express A2B5, TUJ1, and NCAM, or at least 60% of cells express TUJ1, NCAM, and nestin, or at least 60% of cells express A2B5, NCAM, and nestin. Even more preferably, the terms refer to a cell population in which at least 60% of cells express A2B5, TUJ1, NCAM, and nestin. Tests for the expression of such markers in in vitro cultured cells are well known to those skilled in the art. Such tests preferably include flow cytometry and / or other immunocytochemical assays including immunofluorescence staining and Western blotting.

[0075] As used herein, the term “adjacent” refers, preferably, to the distance between a pre-identified location and the actually reached location when a pharmaceutical ingredient is injected into a patient. Preferably, the “adjacent” location is directly adjacent to the pre-identified location. Preferably, the term “adjacent” refers to any type of tissue that is indirectly, more preferably directly, attached to the muscle of the anal sphincter tissue, not limited to the mucosa, submucosa, muscularis mucosa, or anal canal epithelium, by direct adjacency of one type of tissue, i.e., by direct contact with one another. Alternatively, preferably, the term “adjacent” refers to the distance, preferably 0.3 to 15 mm, over which cells, preferably myogenic progenitor cells, can migrate from the injection site to the desired site within the target area. Alternatively, “adjacent” refers to the relative distance between the actual injection site and the desired location, preferably the two locations are not more than 5 cm apart, more preferably 1 cm apart, and even more preferably less than 0.5 cm apart.

[0076] [Description of Embodiments] The present invention relates to myogenic progenitor cells (MPCs) for use in a method for the prevention and / or treatment of anal incontinence in subjects, characterized in that the subjects have suffered from anal incontinence for 20 years or less, more preferably 6 months to about 20 years, or about 6 months to about 10 years.

[0077] Selection of target The inventors selected a broad patient population (ITT) from Example 5 and found that treating patients within this population with MPC according to the method of the present invention resulted in a significantly greater reduction in incontinence symptoms than placebo treatment in an equivalent patient population within the ITT population (Figure 1). However, statistical significance only indicates that the effect is not random (e.g., not due to a placebo effect or an effect unrelated to MPC). Secondly, the observed effect must be clinically appropriate. The higher the rate of reduction in weekly IEF and / or the higher the proportion of responders, the more clinically appropriate the outcome of the treatment in a particular patient population is. The applicant found that the treatment effect varied by selecting different subgroups of patients from the ITT, as described in Example 7.

[0078] When a subgroup of patients was selected from the ITT population (TPP1 population) of Example 5, defined as patients who had suffered from IE for 10 years or less, according to Example 7, it was observed that in these patients, LCC and / or HCC treatment resulted in a greater reduction in IEF per week and a higher responder rate than LCC or HCC treatment in ITT patients (Figure 2). By introducing such a narrow target population, the inventors found that the method of using MPC was more effective in terms of effect size and odds ratio compared to a broader patient population (Figure 8), and therefore the method of using MPC was advantageous compared to already known methods.

[0079] Accordingly, in a preferred embodiment of the present invention, the subject suffering from anal incontinence, more preferably fecal incontinence, suffers from the incontinence for a limited period of time. More preferably, such a period is limited to about 6 months to about 20 years, more preferably to about 6 months to about 10 years, more preferably to about 6 months to about 8 years, more preferably to about 6 months to about 6 years, more preferably to about 6 months to about 4 years, and even more preferably to about 6 months to about 2 years. The preferred starting point of the period is either the onset of incontinence or the diagnosis of incontinence.

[0080] In a preferred embodiment, the present invention provides an MPC for use in a method for preventing anal incontinence, preferably fecal incontinence, in particular, when the subject is healthy in terms of anal incontinence, preferably fecal incontinence, but is at risk of developing the incontinence due to histological and / or anatomical abnormalities of the sphincter tissue. The abnormality is caused by muscle injury (e.g., obstetric anal sphincter injury or anal intercourse), genetic disorders (e.g., Hirschsprung's disease, Duchenne muscular dystrophy), perianal fistula, or surgical procedure (e.g., Gracilplasty, sphincter overlap repair, cancer resection). Preferably, the abnormality is related to a rupture or laceration of the anal sphincter, such as the external and / or internal anal sphincter. More preferably, the rupture or laceration is at an angle of less than 180° of the total angle of 360° in which the anal sphincter is visible in the ultrasound image.

[0081] For prevention, it is desirable to start treatment at the point when there is a risk of developing anal incontinence, such as when muscle damage occurs or when a diagnosis of being at risk of developing anal incontinence is made.

[0082] Another preferred embodiment of the present invention provides MPC for use in a method for treatment in which the subject to be treated is suffering from anal incontinence, particularly fecal incontinence, more preferably urge fecal incontinence and / or passive fecal incontinence, more preferably urge fecal incontinence.

[0083] In a preferred embodiment of the present invention, the target anesthetic incontinence, preferably fecal incontinence, or the risk of developing such incontinence, is caused, for example, by muscle damage as described above. Preferably, the muscle damage exists as scarring within the external and / or internal anal sphincter, visualized by intraanal ultrasound. The muscle damage may occur in the external anal sphincter, the internal anal sphincter, or both. Causes include (1) obstetric or accidental trauma, (2) ectopic trauma such as surgery or radiation, (3) neurogenic diseases such as multiple sclerosis or diabetes, and (4) age-related muscle atrophy. Obstetric trauma is a major cause of incontinence in women, as up to 9% of vaginal deliveries result in anal sphincter rupture.

[0084] Preferably, the subject suffers from fecal incontinence to a well-defined degree of severity. Preferably, such severity is defined by suffering from a certain number of incontinence episodes. More preferably, such anal or fecal incontinence patient suffers from more than 2, more preferably more than 3, even more preferably more than 4, even more preferably more than 5, even more preferably more than 6, even more preferably more than 7, even more preferably more than 8, even more preferably more than 9, even more preferably more than 10, even more preferably more than 11, even more preferably more than 10 incontinence episodes per week, where all types of episodes classified as “trace,” “small,” and / or “large” are considered.

[0085] The inventors found that patients with increasing severity (i.e., increasing baseline IEF) were more responsive to MPC treatment (Figure 7). By selecting such a narrow subgroup of patients defined by having more than 6 weekly IEF prior to treatment (TPP2), in addition to the definition of TPP1, according to Example 7, the inventors found that in the aforementioned patient population after administration of MPC (LCC or HCC), the reduction in weekly IEF was increased, the response rate increased, and the effect size and odds ratio increased compared to those observed in the broader patient population (ITT, TPP1) (Figure 2). This is further highlighted by the increase in overall effect size and odds ratio (Figure 8).

[0086] Furthermore, the inventors found that the treatment of anal or fecal incontinence according to the present invention is most effective for episode types defined as "heavier" and "lighter" compared to "scarce" (Table 3). Therefore, preferably, the severity is defined by suffering from a specific amount of weekly incontinence episode frequency. In preferred embodiments, the episode frequency for evaluating severity is limited to incontinence episodes defined as "lighter" and "heavier," thereby excluding "scarce." The inventors found that a subgroup of TPP1 (TPP3), defined by pre-treatment incontinence severity as having more than two "lighter" or "heavier" incontinence episodes per week, was more responsive to treatment with LCC or HCC than the other patient groups (ITT, TPP1, and TPP2), as illustrated by the increase in effect size and odds ratio (Figure 8).

[0087] In an alternatively preferred embodiment, the MPC is for use in a method for treating anal incontinence in a subject, wherein the subject has a severity of incontinence defined as more than two, more preferably more than three, and even more preferably more than four, incontinence episodes classified as “small” or “large” per week prior to treatment. Prior to treatment preferably refers to treatment according to the present invention. Accordingly, preferably, weekly incontinence episodes are classified prior to the administration of the MPC described herein. Preferably, “prior to treatment” does not include attempts at treatment other than treatment according to the present invention, such as conservative treatment with loperamide or surgical treatment by graciloplasty or sphincteroplasty.

[0088] The inventors, in accordance with Example 7, found that selecting a subgroup of patients (TPP2) defined by having an IEF of more than 6 weeks prior to treatment, in addition to the definition of TPP1, resulted in an increased weekly reduction in IEF and an increased responder rate in this subgroup after administration of MPC (LCC or HCC) compared to what was observed in the broader patient population (ITT, TPP1) (Figure 2). This is further emphasized by the increase in overall effect size and odds ratio (Figure 8). Therefore, the selection of this narrow range of patient populations is advantageous compared to the selection of a broad range of patient populations already known in the art.

[0089] Therefore, in a very preferred embodiment of the present invention, the MPC is for use in a method for treating anal incontinence, more preferably fecal incontinence, in a subject, where the subject is (i) Having suffered from anal incontinence, more preferably fecal incontinence, for 20 years or less, more preferably for about 6 months to about 10 years, and (ii) Having had more than six episodes of incontinence per week prior to treatment, and / or (iii) Prior to treatment, the patient has a severity of incontinence of more than two, more than three, or more than four episodes of incontinence per week, classified as small or large.

[0090] The inventors found that the combination of a limited duration of incontinence and an increased severity of FI in terms of weekly incontinence episode frequency in which episodes classified as “trace” are excluded is a preferred selection process in Example 7 for selecting patients who would benefit far better from the method of treating fecal incontinence using MPC according to the present invention. By selecting such a narrow subgroup of patients following Example 7, defined, in addition to the definition of TPP1, by having more than two IEFs per week before treatment (TPP3) in which traces are not counted as incontinence episodes, the inventors found that in the said patient population after administration of MPC (LCC and / or HCC), the reduction in weekly IEF was greater and the responder rate was increased compared to that observed in the broader patient population (ITT, TPP1) (Figure 2). This is further emphasized by the increase in overall effect size and odds ratio (Figure 8). Thus, the selection of this narrow range of patient population is advantageous compared to the selection of a broad range of patient population already known in the art.

[0091] The inventors selected a subgroup of patients with fecal incontinence accompanied by muscle damage, who had been diagnosed with fecal incontinence less than 10 years prior to their first visit for fecal incontinence, and who experienced more than two episodes of involuntary fecal ejection (IEF) per week before treatment. This subgroup responded particularly well to the treatment of symptoms by the method of the present invention (Example 7, Figures 9 and 10).

[0092] In a preferred embodiment, the subjects are 18 years of age or older. In a further preferred embodiment, the subjects have suffered from fecal incontinence for 6 months or longer, which is confirmed at the time of screening by relevant medical history and rectal examination. In a further preferred embodiment, the subjects have a Wexner score greater than 9 and have at least 3 episodes of fecal incontinence per week measured in a bowel diary over a period of time, such as 2, 3, or 4 weeks. In a further preferred embodiment, the subjects have at least 3 non-gastric incontinence episodes per week measured using a diary as described above.

[0093] In a preferred embodiment, the subject has not undergone anal surgery within the six months prior to the day of administration of MPC. In a more preferred embodiment, the subject has not undergone one or more overlap repair surgeries in the past. As used herein, the term “overlap repair” preferably refers to primary overlap repair, as is commonly performed in the art to correct acute damage in cases of birth lacerations. Preferably, the term also refers to an equivalent treatment performed at a later stage (without acute damage) in a primary overlap repair, which may also be called “sphincteroplasty.” In a more preferred embodiment, the subject has not undergone more than two anal surgeries in total (e.g., primary repair after delivery and one subsequent overlap repair, or insertion and removal of a permanent nerve stimulation system). In a more preferred embodiment, the subject does not have overlap repair of the external anal sphincter and associated premature atrophy. In a more preferred embodiment, the subject has no history of colostomy sphincter (AAS) surgery. In a preferred embodiment, the subject has not received any transanal or perianal injection of any volume-enhancing product. In a preferred embodiment, the subject has not received radiotherapy of the intestines and pelvis. In a preferred embodiment, the subject has not received chemotherapy within the past five years prior to the day of administration of MPC. In a preferred embodiment, the subject does not have chemotherapy-related neuropathy of the intestines and pelvis. In a preferred embodiment, the subject has not received immunosuppressive therapy. In a preferred embodiment, the subject has not been diagnosed with chronic inflammatory bowel disease. In a preferred embodiment, the subject does not have a diagnosis of current or anal fistula disease. In a preferred embodiment, the subject does not have chronic diarrhea. In a preferred embodiment, the subject does not have obstetric trauma or other trauma, acute disc dehiscence, or acute anal sphincter injury, including neurological disorders (such as spinal cord injury, multiple sclerosis, Parkinson's disease, or stroke). In a preferred embodiment, the subject does not have uncontrolled type 1 or type 2 diabetes mellitus, or suffer from diabetic peripheral neuropathic pain.In a preferred embodiment, the subject is not diagnosed with human immunodeficiency virus (HIV), acute or chronic viral hepatitis HCV, acute or chronic viral hepatitis HBV, active syphilis, or HTLV (e.g., tested for based on a risk assessment by the principal investigator). In a preferred embodiment, the subject does not have any metal implants in the electrical stimulation treatment area. In a preferred embodiment, the subject does not have chronic constipation and / or overflow urinary incontinence.

[0094] In a preferred embodiment, the subject has a condition associated with incontinence due to muscle injury, pelvic floor dysfunction, nerve injury, loss of storage capacity, or atrophy. In a preferred embodiment, the fecal incontinence of the subject is passive incontinence, urge incontinence, or fecal leakage. In a very preferred embodiment, the subject has a condition associated with fecal incontinence due to muscle injury, atrophy, and / or urge fecal incontinence.

[0095] MPC for use in accordance with the present invention is preferably administered to the target anal sphincter tissue, preferably the external anal sphincter, the internal anal sphincter, and / or the pectineus muscle, or adjacent thereto, as further described herein. Administration is preferably carried out by injection. The injection process may be, but is not limited to, a static injection, i.e., an injection that remains at the location reached by the injection device. Alternatively, the injection process may be dynamic. Alternatively, the administration of MPC may include any administration route suitable for adjaculating the cells to the anal sphincter tissue. Such administration routes may include, for example, oral administration, topical administration, intravenous administration, or intra-arterial administration.

[0096] In any of the above preferred embodiments of this embodiment, the method further includes stimulating the anal sphincter tissue before and / or after administration of MPC. Preferably, the stimulation includes anal sphincter tissue stimulation for at least two weeks, more preferably at least four weeks, after administration of MPC, and more preferably includes anal sphincter tissue stimulation before administration of MPC and for at least two weeks, more preferably at least four weeks, after administration of MPC.

[0097] Preferably, the stimulation is performed by Kegel exercises and / or electrical stimulation, more preferably by Kegel exercises and / or transcutaneous electrical stimulation. The transcutaneous electrical stimulation is preferably performed daily, more preferably twice a day, and even more preferably three times a day. Preferably, any of the stimulations is performed for at least 10 minutes, more preferably at least 20 minutes. Also preferably, the stimulation is performed multiple times a day, more preferably 1 to 10 times, even more preferably 1 to 5 times, and even more preferably 3 times.

[0098] The electrical stimulation is preferably performed at specific times per day, for example, at least 5 minutes per session, preferably at least 15 minutes, and more preferably at least 20 minutes. Preferably, the patient receives stimulation three times a day, each session lasting about 1 to 60 minutes, preferably about 10 to 30 minutes, and more preferably about 20 minutes. Preferably, the electrical stimulation includes the use of a rectoanal probe attached to the stimulator. Preferably, the rectoanal probe is inserted into the rectum of the subject during stimulation. Preferably, the stimulator is programmed to transmit electrical pulses to the probe, and the pulses are preferably biphasic or monophasic. Preferably, the pulses are embedded in a sequence of pause, increase, equilibrium, decrease, pause. Preferably, the biphasic or monophasic pulses have a frequency of about 10 Hz to about 100 Hz, more preferably about 25 Hz to about 75 Hz, and more preferably about 50 Hz at equilibrium. Preferably, each equilibrium phase has the same duration as each increasing or decreasing phase. Preferably, each pause, increase, decrease, or equilibrium phase lasts for about 1 to 15 seconds, more preferably about 2 to 8 seconds, and more preferably about 4 seconds. Preferably, the current strength in the equilibrium phase is set to about 1 mA to 1000 mA, more preferably about 10 mA to 500 mA, more preferably about 50 mA to 250 mA, more preferably about 75 mA to 150 mA, and more preferably about 100 mA. Preferably, the two-phase or single-phase pulse has a width of about 50 μs to 500 μs, more preferably about 150 μs to 300 μs, more preferably about 200 μs to 300 μs, and more preferably about 250 μs.

[0099] Kegel exercises are preferably performed by voluntary contraction of the pelvic floor muscles, depending on the individual. Preferably, the contraction is held for about 1 to 10 seconds, more preferably 2 to 5 seconds, and more preferably 3 seconds. After contraction, the muscles are preferably relaxed for about 1 to 10 seconds, more preferably 2 to 5 seconds, and more preferably 3 seconds. Preferably, the contraction and relaxation sequence is repeated 1 to 20 times, more preferably 5 to 15 times, and more preferably about 10 times. Preferably, each set of the sequence is repeated 1 to 10 times, more preferably 3 to 5 times, and more preferably 3 times daily.

[0100] The inventors found that a combination of electrical stimulation and cell injection (e.g., HCC or LCC treatment according to Example 1) was superior to electrical stimulation and sham injection alone (e.g., PBO treatment according to Example 1) (Figure 1), suggesting that a combination of MPC and electrical stimulation is an effective treatment for incontinence.

[0101] Cell dose selection Preferably, MPC is for use in a method for the prevention and / or treatment of anal incontinence in a subject, the method comprising the administration of an effective amount of MPC, preferably about 1 million to about 200 million MPC, preferably about 4 million to about 60 million MPC, more preferably about 4 million to about 6 million MPC or about 40 million to about 60 million MPC.

[0102] The inventors found that in a broad patient population (ITT), after injecting 40 to 60 million MPCs (HCC), when traces were counted as incontinence episodes at 6 months post-treatment (Figure 1A), and when traces were excluded as incontinence episodes at 12 months post-treatment (Figure IB), the weekly incontinence episode frequency showed a significantly higher reduction compared to placebo injection (PBO). Throughout the entire study period of Example 5, regardless of whether traces were included or excluded as incontinence episodes, HCC tended to show a higher rate of reduction in weekly incontinence frequency than PBO (Figure 1). Furthermore, when traces were included in the ITT patient population (Figure 1C), consistently higher responder numbers were observed in the LCC and HCC groups compared to the PBO group (Figure ID). Preferably, according to this embodiment, the amount is 4 to 6 million cells per patient. The present applicant found that, compared to PBO therapy, treatment of ITT patients with 4 to 6 million cells (LCC) showed both a strong tendency towards a reduction in weekly IEF and an increase in responder rates (Figure 1). This trend was particularly pronounced when 4 to 6 million cells were used in patients with fecal incontinence who had not been incontinence for more than 10 years. The inventors found that LCC transplantation significantly reduced IEF from baseline to 12 months post-treatment in such patients (Figures 2A and B), and was clinically significant by reducing weekly IEF (excluding traces) by at least 50% in most patients (Figure 2D). The inventors further found that patients injected with 40 to 60 million cells showed a stronger tendency towards a reduction in fecal incontinence symptoms (weekly IEF) compared to patients injected with 4 to 6 million cells.

[0103] Injection procedure In a preferred embodiment of the present invention, MPC is injected into or adjacent to the anal sphincter tissue of the subject. Preferably, the cells are injected into or adjacent to the external anal sphincter, internal anal sphincter, and / or pectineus muscle. More preferably, the cells are injected into the external anal sphincter, internal anal sphincter, or pectineus muscle. Most preferably, the cells are injected into the external anal sphincter. The inventors found that injection of MPC into the external anal sphincter was effective compared to placebo treatment (Figure 1). The inventors found that cells isolated according to Example 1 were successfully injected into the desired subject according to Example 5. The injection site was the external anal sphincter, which belongs to the anal sphincter tissue.

[0104] In a preferred embodiment of the present invention, MPC is injected into a designated tissue or injury site, thereby providing the MPC with a therapeutically effective number of cells in the solution or suspension, for example, about 1 × 10⁶ cells. 6 ~about 2×10 8 The solution contains a number of cells. The number of cells for injection is preferably suspended in a solution of about 0.1 mL to about 100 mL, more preferably about 1 mL to about 10 mL, more preferably about 3 mL to about 6 mL, and even more preferably 6 mL. The injection solution is a physiologically acceptable medium, with or without serum. The physiologically acceptable medium may, in non-limiting examples, be physiological saline or phosphate buffer solution.

[0105] In a preferred embodiment of the present invention, MPC injection into anal sphincter tissue is performed as a treatment for anal incontinence to enhance, improve, and / or repair the external and / or internal anal sphincter. Preferably, MPC is injected into or adjacent to the external and / or internal anal sphincter to survive and differentiate into mature muscle cells, thereby enhancing the sphincter and / or improving sphincter function. The feasibility and long-term survival of MPC according to this embodiment have been previously demonstrated (Messner et al., 2021; Thumer et al., 2020).

[0106] In another preferred embodiment of the present invention, MPC injection is administered to the anal sphincter tissue to prevent anal incontinence by enhancing and / or strengthening existing incontinence tissue. The feasibility of MPC injection into healthy muscle tissue has already been demonstrated (Frudinger et al., 2015).

[0107] In a preferred embodiment of the present invention, the effective number of cells is administered in fractions (aliquots) of one or more cell suspensions. Preferably, multiple fractions are injected per patient, more preferably 2 to 50 aliquots, more preferably 2 to 20 aliquots, even more preferably 5 to 15 aliquots, preferably a total of 0.1 to 100 ml per patient, more preferably 1 to 10 ml, and even more preferably 3 to 6 ml. The inventors have found that such dispensing numbers and volumes are effective (i.e., superior to placebo) and clinically appropriate (i.e., most patients experience at least a 50% reduction in IEF per week) (Figures 1 and 2).

[0108] In preferred embodiments of the present invention, MPC is administered by one or more injections into and / or adjacent to the anal sphincter tissue of the subject, preferably by one or more injections into the external anal sphincter, internal anal sphincter and / or pectineus muscle. Preferably, MPC for use according to the present invention is injected into the internal anal sphincter of the subject, wherein the subject is further characterized by suffering from passive fecal incontinence or passive and urge fecal incontinence. Preferably, MPC for use according to the present invention is injected into the external anal sphincter and / or pectineus muscle of the subject, more preferably the external anal sphincter, wherein the subject is further characterized by suffering from urge fecal incontinence or urge and passive fecal incontinence. Preferably, the injection of MPC is performed at multiple locations throughout the anal sphincter tissue.

[0109] Preferably, the MPC is injected at one or more locations, more preferably at about 2 to about 20 locations, more preferably at about 6 to about 18 locations, more preferably at about 10 to about 15 locations, and even more preferably at about 12 locations. The inventors found that distributing the MPC injection of Example 1 to the patient according to Example 5 was effective and significantly more effective than placebo treatment (Figure 1).

[0110] MPC used in this invention The present invention provides MPCs for use in accordance with the present invention. In one embodiment of the present invention, the MPCs are characterized as “muscle-derived cells” as already disclosed in EP2120976B1. Non-limiting examples of such cells include myoblasts, fibroblasts, and muscle-derived stem cells present in muscle tissue. The present invention also intends to use cells with myogenic potential (e.g., from liposuctioned tissue 15, or other stem cell-containing tissue (bone marrow), or from adipose-derived cells) particularly for use in the repair of anal sphincter tissue. In particular, the cells used in the present invention can fuse in vitro and / or in vivo (forming a syncytium of at least three cells) to establish an oriented contractile cytoskeleton (actin-myosin sequence). In accordance with the present invention, MPCs comprising myoblasts may be primary cells or cultured cells. These may be histocompatible (autologous) or non-histocompatible (allogeneic) to recipients, including humans. Specific embodiments of the present invention include myoblasts and muscle-derived stem cells, including autologous myoblasts and muscle-derived stem cells that are not recognized as foreign to the recipient. In this regard, myoblasts can be matched to the major histocompatibility centers (MHC or HLA in humans). Such MHC or HLA-matched cells may be autologous cells, or they may be human cells with the same or similar MHC or HLA antigen profiles. Patients may also be tolerant to allogeneic MHC antigens, or their cells may be manipulated to lack MHC proteins, thereby making them immune to recipients with originally mismatched HLA.

[0111] In another embodiment of the present invention, the MPC lacks MHC class I and / or II antigens, as described in U.S. Patent No. 5,538,722.

[0112] In a very preferred embodiment, the MPC is a population of skeletal muscle-derived cells (SMDCs), where at least 60% of the cells are CD56-positive, at least 80% are CD90-positive, and at most 10% are CD34-positive. Preferably, at least 60% of the cells are also positive for A2B5 and CD105. Preferably, the SMDC cell population is Sca-1-negative, with at most 10% or 0% of the cells expressing Sca-1. Preferably, at least 60% of the cells are also positive for desmin. Preferably, the SMDC cell population is MyoD-negative, with at most 5% or 10% of the cells expressing MyoD.

[0113] In a more preferred embodiment, the MPC is a cell population of SMDCs, where approximately 64% to 99.9% of the cells are CD56-positive, approximately 80% to 99.9% of the cells are CD90-positive, and approximately 0% to 9% of the cells are CD34-positive. Preferably, at least 60% of the cells are positive for A2B5 and CD105. Preferably, the cell population of SMDCs is Sca-1-negative, in particular, at most 10% or 0% of the cells express Sca-1. Preferably, at least 60% of the cells are also positive for desmin. Preferably, the cell population of SMDCs is MyoD-negative, in particular, at most 5% or 10% of the cells express MyoD.

[0114] In another embodiment of the present invention, the MPC is characterized as “skeletal muscle-derived cells” (SMDCs) as disclosed in WO2019 / 115790 (Thumer et al., 2019, p. 115790). The SMDCs preferably exhibit a characteristic expression pattern. Preferably, about 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more of the SMDCs express CD56 and A2B5. Preferably, the SMDCs do not express CD34, Sca-1 and MyoD. Thus, the term “does not express” preferably means that less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 2% of the SMDCs express the markers. In a preferred embodiment, 0% of the SMDCs express Sca-1.

[0115] In a further preferred embodiment, the MPC is a cell population of SMDCs, which consists of at least 90% CD56-positive, at least 90% A2B2-positive, at least 90% CD105-positive, and at least 90% desmin-positive cells, with less than 10% of the population expressing CD34, Sca-1, and MyoD. In a preferred embodiment, 0% of the SMDCs express Sca-1.

[0116] The SMDC expression patterns described above can be used to determine myogenicity indicators of cell cultures without requiring differentiation. Therefore, the SMDC expression patterns can be used to determine whether skeletal muscle-derived cells can be used to treat muscle dysfunction, particularly incontinence such as urinary and / or anal incontinence.

[0117] In another embodiment of the present invention, the MPCs described herein are characterized as “myogenic progenitor cells” as disclosed in WO2020 / 193460. Preferably, these cells are characterized by positive expression of CD56 and CD90, and negative expression of CD34. In a more preferred embodiment of the present invention, the MPCs are gene-poor. The inventors have found that such cells are useful in the methods of the present invention (Figure 1).

[0118] In another embodiment of the present invention, the MPC described herein is characterized as a “mesenchymal stromal cell” or “MSC” as disclosed in WO2020 / 193460, characterized by positive expression of CD105 and CD73, and negative expression of CD34 and CD56. In a more preferred embodiment of the present invention, the MPC is an MSC characterized by negative expression of desmin and / or positive expression of CD90. In a more preferred embodiment of the present invention, the MPC is a pluripotent MSC.

[0119] In another embodiment of the present invention, the MPC described herein is characterized by positive expression of aSMA, CD49a, desmin, CD56, and CD146, and negative expression of CD34.

[0120] In another embodiment of the present invention, the MPC described herein is characterized by positive expression of aSMA, CD49a, and CD146, and negative expression of CD56.

[0121] In a very preferred embodiment, the MPC for use in the method according to the present invention is characterized by positive expression of the markers CD56 and CD90, in particular at least 60%, more preferably at least 80%, more preferably at least 95% of the MPC expressing CD56, and at least 60%, more preferably at least 80%, more preferably at least 95% of the myogenic progenitor cells expressing CD90. Preferably, about 60 to about 99.9% of the MPC express CD56, and about 80 to about 99.9% of the MPC express CD90. Preferably, the MPC is further characterized by negative expression of the markers CD34 and / or Sca-1, in particular up to 10%, more preferably up to 5%, and even more preferably up to 1% of the MPC expressing CD34 and / or Sca-1. Even more preferably, the MPC is further characterized by positive expression of the marker desmin, in particular at least 60%, more preferably at least 80%, and even more preferably at least 95% of the cells are desmin-positive.

[0122] In a preferred embodiment, the MPC for use in the method according to the present invention consists of a cell population having the following marker expression characteristics: Approximately 60 to 99.9% of the cell population (i.e., MPC) express the cell marker CD56, approximately 60 to 99.9% express the marker CD90, and approximately 0 to 15% express the marker CD34. More preferably, approximately 60 to 99.9% of the cell population express the cell marker CD56, approximately 70 to 99.9% express the cell marker CD90, and approximately 0 to 10% express the cell marker CD34. More preferably, approximately 80 to 99% of the cell population express the cell marker CD56, approximately 80 to 99.9% express the marker CD90, and approximately 0 to 5% express the marker CD34.

[0123] The MPC for use according to the present invention may further have the following marker expression characteristics: Approximately 60 to 100% of the cell population express the cell marker desmin, and / or approximately 60 to 100% of the cell population express the marker CD105. More preferably, approximately 60 to 99.9% of the cell population express the cell marker desmin, and approximately 90 to 100% of the cells express the cell marker CD105. Preferably, approximately 60 to 100% of the cell population further express the cell marker A2B5.

[0124] In a particularly preferred embodiment, the MPC has the following marker expression characteristics: Approximately 60-100% of the cell population expresses the cell marker CD56, approximately 60-100% express the cell marker CD90, approximately 0-10% express the cell marker CD34, approximately 60-100% express the cell marker desmin, approximately 60-100% express the cell marker A2B5, and approximately 60-100% express the cell marker CD105. Preferably, approximately 0-10% of the cell population expresses the cell marker Sca-1.

[0125] In more preferred embodiments of all the above embodiments relating to MPC, about 0 to about 10% of the MPC express MyoD and / or about 0 to about 10% of the MPC express Sca-1.

[0126] In a particular preferred embodiment, the MPC for use according to the present invention has the following marker expression characteristics: Approximately 60% to 100% of the cell population express the cell marker CD56, approximately 80% to 99.9% express the cell marker CD90, approximately 0% to 10% express the cell marker CD34, approximately 60% to 100% express the cell marker desmin, approximately 60% to 100% express the cell marker A2B5, approximately 60% to 100% express the cell marker CD105, approximately 0% to 10% express the cell marker MyoD and / or approximately 0% to 10% express the cell marker Sca-1.

[0127] In another preferred embodiment of the present invention, the MPC for use in the method of the present invention is characterized by its ability to differentiate into cell lineages. Preferably, the MPC is characterized by having myogenic differentiation ability in vitro and / or in vivo, thereby defined as the ability to increase, form, or become mature muscle tissue by the formation of multinucleated muscle fibers or by the electrophysiological coupling of multiple single-nucleated cells, i.e., the ability to increase, form, or become mature muscle tissue in vitro and / or in vivo. Preferably, the MPC can increase, form, or become mature muscle tissue in vitro and / or in vivo.

[0128] In another embodiment of the present invention, MPCs are characterized by having myogenic and neurogenic differentiation potential. Furthermore, MPCs may have further differentiation potential, namely oligo, pluripotency, or pluripotency. Preferably, MPCs include differentiation potential into all tissues adjacent to the injection site of the anesthetic target of the anesthetic incontinence to be desired. More preferably, such differentiation potential includes differentiation into skeletal tissue, smooth tissue, and / or nerve tissue. Preferably, the cells are pluripotent to enable the enlargement and regeneration of skeletal muscle and smooth muscle tissue of the anal sphincter tissue. More preferably, the cells are pluripotent to enable the enlargement and regeneration of skeletal muscle and smooth muscle tissue, as well as nerve tissue, of the anal sphincter tissue. Cells with myogenic and / or neurogenic potential can be isolated from muscle tissue, preferably by muscle biopsy. Preferably, cells with myogenic potential are isolated from muscle biopsy by obtaining skeletal muscle-derived cells (SMDCs).

[0129] Such cells can be tested for myogenic potential by methods known to those skilled in the art. To determine the myogenic and / or neurogenic potential of MPC according to the present invention, cells are tested positive for their AChE activity according to the examples of the present invention or other methods known to those skilled in the art (e.g., Thumer et al., 2018).

[0130] The MPC for use according to the present invention is preferably about 20 mU rel ~about 1000mU rel Comfortably at approximately 30 mU rel ~about 800mU rel More preferably about 50 to about 700 mU rel It has AChE activity, and each AChE activity is preferably 2x10 in a skeletal muscle differentiation medium cultured for about 5 to 7 days. 5 It is measured per cell.

[0131] As described above, the selected cell dose administered to the target is preferably about lx10 2 mU rel _ total ~about lx10 6 mU rel _total It exhibits total AChE activity, more preferably about lx10 3 mU rel _ total ~about 5x10 5 mU rel _ total More preferably about 7 x 10 4 mU rel _ total ~about 2x10 5 mU rel _ total Therefore, to determine the total AChE activity, 2 x 10 5 The AChE activity determined for individual cells can be extrapolated. For example, if a batch of cells is 2x10 5 50 mU per cell rel If the acetylcholinesterase enzyme activity is such that a cell suspension containing 50 million such cells totals 1.25 x 10⁶ 4 mU rel It can be extrapolated that it has total acetylcholinesterase enzyme activity. Therefore, the unit "mU" rel _ total " preferably refers to the extrapolated AChE activity of a certain number of cells, where AChE activity is mU rel It is linearly extrapolated from the relative AChE activity measured by . In a further preferred embodiment, mU rel _ total The total AChE activity given is the total AChE activity of the cell population or cell dose of MPC relative to an AChE standard as described herein. Therefore, the unit "mU" rel _ total " may also refer to the relative "mU / ml" of the cell dose of the cell population or MPC measured 60 minutes after the addition of ATI and DTNB, with respect to a linear equation obtained by a dilution series of AChE stock solutions in the range of 4-500 mU / ml, under the same conditions, except that the OD of the diluted stock solution is already measured 6-8 minutes, preferably 6, 7, or 8 minutes, after the addition of ATI and DTNB.

[0132] The inventors have determined that the MPC obtained according to the method disclosed herein yields 36 mU in the case of an LCC batch.rel ~568mU rel We found that it possesses AChE activity in the range of 241.90±SD (where the AChE activity is 2x10 for each of the following values). 5 (Measured per individual cell). The HCC batch (n=75) was 49 mU rel ~680mU rel Mean ±SD range of AChE activity: 213 ± 137.40 mU rel It was found that they possessed it.

[0133] Methods for obtaining myogenic progenitor cells The MPCs used in the method according to the present invention are preferably isolated cells from muscle tissue. These can be obtained by methods well known to those skilled in the art, for example, by isolation from muscle biopsy. Preferably, the cells can be isolated from muscle tissue, preferably by obtaining a muscle biopsy. Preferably, the muscle biopsy is obtained from a subject by surgical procedure. Preferably, the MPCs are isolated from a subject, more preferably from a human subject, even more preferably from a human subject in need, such as a subject suffering from incontinence. Preferably, the MPCs are isolated from a muscle biopsy of the subject, or instead from another tissue source, such as, but not limited to, adipose tissue or connective tissue. Preferably, the muscle biopsy is obtained from skeletal muscle, more preferably from the pectoralis major, biceps brachii, or latissimus dorsi muscle.

[0134] MPC is preferably obtained from a muscle biopsy obtained by a method comprising the following steps: (a) cooling the obtained muscle biopsy; (b) processing and cooling the sample; (c) resuspending the sample from step (b) in a medium containing serum with at least one enzyme and heating to 38°C for 1 to 20 hours; pelletizing the sample; and (d) resuspending the pellet of the sample from step (c) to provide a single-cell suspension from the sample from step (c), thereby obtaining the MPC. Step (a) comprises performing the muscle biopsy. Such a muscle biopsy, which will be the source of the MPC, can be obtained from the muscle at the site of injury or from another site that is more easily accessible to the clinical surgeon. In a further preferred embodiment, step (a) is performed at a temperature range lower than 16°C, preferably 1 to 16°C, preferably 4 to 10°C, and particularly preferably 7°C; and for a time range of up to 96 hours. Therefore, step (a) can be carried out in a temperature range of 1 to 16°C, or any intermediate temperature within this range, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15°C, or any intermediate temperature within this range, for example, 6 to 8°C. Alternatively, the temperature range is 4°C or less, preferably in the range of 1 to 3°C. Step (a) is preferably carried out for a time in the range of up to 96 hours, or any time within this range, for example, 12 to 96 hours, 12 to 72 hours, 12 to 48 hours, 24 to 96 hours, 24 to 72 hours, 24 to 48 hours, or any other intermediate time range. Preferably, the processing in step (b) includes the use of scissors, scalpels, tweezers, filters, ball mills, and centrifuges. The processing refers in particular to the mechanical destruction of the tissue sample. The processing of the sample is preferably carried out at room temperature. The cooling in step (b) is preferably carried out after processing the sample. The cooling in step (b) can be carried out at a temperature in the range of 1 to 16°C, or any temperature within this range, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15°C, or any intermediate temperature within this range, for example, in the range of 4 to 8°C or in the range of 1 to 3°C. Step (b), in particular the cooling, can be carried out within a clear time range such as 2 to 48 hours, 2 to 36 hours, or 2 to 24 hours.Step (c) preferably comprises enzymatic treatment using a solution containing one or more selected from the group consisting of trypsin, papain, elastase, hyaluronidase, collagenase, deoxyribonuclease, and DNAse. In a preferred embodiment, step (c) includes the use of collagenase. The resuspension in step (c) preferably includes centrifugation of the sample from step (b), discarding the supernatant, and resuspending the cell pellet in a serum-containing medium containing at least one enzyme such as trypsin. Step (c) may further include one or more washing steps, including vortexing the cells in a suitable solution such as a buffer. The sample from step (b) is preferably centrifugated after incubation time to pelletize the cells of the sample and discard the enzyme-containing supernatant. Step (c) can be carried out at a temperature in the range of 25 to 38°C, preferably 36 to 38°C. Preferably, step (d) includes a method selected from at least one of FACS sorting, centrifugation, electrokinetic sorting, acoustic sorting, bead-based cell sorting, and optical sorting. A suitable concentration method for obtaining a single-cell suspension is magnetically activated cell sorting (MACS®). In the MACS method, cells can be separated by incubating them with particles coated with antibodies against a specific surface antigen. The incubated cells are then transferred to a column placed in a magnetic field. In this step, cells that express the antigen and adhere to the nanoparticles remain on the column, while other cells that do not express the antigen pass through the column. In this method, cells are separated positively and / or negatively for a specific antigen. Another example of a preferred concentration method is fluorescence-activated cell sorting (FACS®), as described, for example, by Webster et al. (Exp Cell Res. 1988 Jan; 174(l):252-65). A further optional step (e) may be carried out after step (d), which includes incubation of the single-cell suspension obtained in step (d), the incubation in step (e) is preferably carried out at a temperature in the range of 25-38°C, preferably 36-38°C, and particularly preferably 37°C, thereby obtaining adherent MPCs.This additional optional incubation step allows the cells to grow and a larger quantity of MPC to be obtained. After step (e), optionally, a further step (f) may be performed, which includes discarding the non-adherent cells from step (e), preferably at least 6 hours to 4 days later. After step (f), optionally, a further step (g) may be performed to grow the adherent cells from step (e), the growth in step (h) consisting of subculturing the adherent cells to 70-80% confluence for 1-5 passages.

[0135] In another embodiment of the present invention, MPCs are obtained from any donor and any somatic cells that are cultureable and reprogrammable in vitro. Preferably, these somatic cells are first reprogrammed into induced pluripotent stem cells by ectopic expression of reprogramming factors consisting of Klf-4, Sox-2, Oct4, and Myc (Takahashi et al., 2007; Takahashii·Yamanaka, 2006; Yamanaka, 2008), and then differentiated to MPCs as already described in the prior art (Bajpai et al., 2012; Incitti et al., 2020; Miyagoe-Suzuki & Takeda, 2017; Xuan et al., 2021). More preferably, myogenic progenitor-derived cells are induced from somatic cells by direct reprogramming without pluripotent intermediates according to methods known in the art (Hirai et al., 2018; Ito et al., 2017).

[0136] The present invention also provides a method for preventing or treating anal incontinence in a subject, the method comprising: (a) selecting a subject at risk of developing anal incontinence, more preferably fecal incontinence, or suffering from anal incontinence, more preferably fecal incontinence, according to the subject's condition related to incontinence; (b) administering an effective amount of MPC into or adjacent to the anal sphincter tissue, preferably by injection; and (c) optionally stimulating the anal sphincter tissue before and / or after step (b).

[0137] Preferably, the method is the same as the method described above for the prevention and / or treatment of anal incontinence in subjects for whom MPC is provided for use. Accordingly, all the embodiments described above, including embodiments relating to the selection of subjects in terms of duration, severity and causality of incontinence, embodiments relating to the characteristics of MPC, embodiments relating to the injection of an effective amount of MPC in terms of cell dose, target tissue and distribution, and / or embodiments relating to stimulation in terms of electrical stimulation and Kegel movements, also represent embodiments of the method according to the present invention.

[0138] Step (a) of the method according to the present invention may comprise selecting subjects having muscle injury as a condition associated with incontinence. Alternatively or additionally, step (a) may comprise selecting subjects having a duration of anal incontinence of 20 years or less, preferably 10 years or less, or any of the further respective time ranges disclosed above. Alternatively or additionally, step (a) may comprise selecting subjects having a severity of incontinence defined as more than 6, preferably more than 7, preferably more than 8, more than 9, or more than 10 incontinence episodes per week prior to treatment. Alternatively or additionally, step (a) may comprise selecting subjects having a severity of incontinence defined as more than 2 incontinence episodes per week being classified as small or large prior to treatment. Step (c) may comprise anal sphincter tissue stimulation, preferably for at least 2 weeks, after or before and after injecting the MPC.

[0139] The present invention also provides a pharmaceutical composition comprising MPC and a pharmaceutically acceptable excipient and / or carrier for use in a method for the prevention and / or treatment of anal incontinence, preferably fecal incontinence, in a subject, wherein the subject is at risk of developing anal incontinence, preferably fecal incontinence, or is suffering from anal incontinence, preferably fecal incontinence, for a period of 20 years or less, more preferably about 6 months to about 20 years, or about 6 months to about 10 years. In particular, the present invention provides a pharmaceutical composition comprising (i) MPC for use in a method according to all embodiments of the present invention, and (ii) a pharmaceutically acceptable excipient and / or carrier.

[0140] The pharmaceutical compositions according to the present invention may further include one or more conventional additives. Examples of such additives include physiologically acceptable buffers, albumin, collagen, laminin, and dimethyl sulfoxide.

[0141] The present invention is further directed toward a process for preparing pharmaceutical compositions for use in the methods according to the present invention. Preferably, the process comprises mixing MPC with a pharmaceutically acceptable diluent, excipient, or carrier.

[0142] The present invention is further directed to a pharmaceutical pack comprising one or more compartments, wherein at least one compartment comprises an MPC for use in the method according to the present invention.

[0143] The present invention is further directed toward the use of MPC as a pharmaceutical for the prevention and / or treatment of anal incontinence, preferably fecal incontinence, in subjects such as those described herein.

[0144] The present invention is further directed toward the use of MPC in the manufacture of pharmaceuticals for the prevention and / or treatment of anal incontinence, preferably fecal incontinence, in subjects such as those described herein.

[0145] The target of treatment or prevention of disease development according to any of the embodiments described above is preferably a human or an animal, particularly a mammal, and most preferably a human.

[0146] The following examples illustrate the present invention, but are not intended to limit it. [Examples]

[0147] Example 1 - Isolation of skeletal muscle-derived myogenic cells (MPCs) In a clinical trial conducted by the applicant (EudraCT number: 2010-021463-32), MPCs were isolated from human fecal incontinence patients, as already disclosed in WO2019115790. While WO2019115790 involved the isolation of myogenic skeletal muscle progenitor cells from a small number of patient samples, the aforementioned clinical trial involved the isolation of skeletal muscle-derived myogenic progenitor cells from approximately 170 separate patient samples. Therefore, the quality of the samples used in this trial may have differed considerably from that used in WO2019115790, depending on the patient and the quality of the biopsies obtained. Specifically, skeletal muscle biopsies were taken from the pectoralis major or biceps brachii muscle of each incontinence patient treated according to Example 5. To perform the biopsy, the skin was first opened by making an incision over the muscle for approximately 1 cm until the fascia of the pectoralis major was reached. After opening the fascia, 1 cm 3 Muscle tissue (biopsy) was collected. The biopsy was directly transferred to a biopsy transport medium pre-cooled to approximately 4°C, consisting of Ham F10 basal medium supplemented with gentamicin (final concentration 1-5 μg / ml). The biopsy was stored in the biopsy transport medium at 1-11°C for approximately 26 hours. Next, the biopsy was transferred to a petri dish filled with lx PBS. The muscle tissue was separated from the connective tissue using sterile forceps and a scalpel. Next, the muscle tissue was transferred to another petri dish filled with lx PBS and cut 2-3 mm using a scalpel. 2The tissue was cut into pieces of the size described above. After the additional transfer steps described above, the tissue pieces were further cut into 1 mm cubes. Finally, these fragments were transferred to a centrifuge tube filled with lx PBS and centrifuged at 1300 rpm for 10 minutes. After centrifugation, the supernatant was removed and the muscle tissue was suspended in lx PBS supplemented with 8 μg / ml gentamicin. The muscle tissue suspension was then cooled to 2-8°C for 48 hours. After cooling, the muscle tissue suspension was centrifuged at 1300 rpm for 10 minutes, the supernatant was removed, and 2.5 ml of digestate prepared by dissolving 1-5 mg / ml collagenase, 2-4% v / v Hepes buffer, 0.1-10% v / v fetal calf serum, and 5-10 μg / ml gentamicin in Ham F10 was added. The muscle tissue suspension was incubated at 37°C in 5% CO2 for 6-20 hours. Next, the suspension was centrifuged at 1300 rpm for 10 minutes, the supernatant was removed, and the pellet was resuspended in Ham F10 medium containing 10-20% v / v FCS, 1-3 ng / ml bFGF, and 3-10 μg / ml gentamicin, and plated into cell culture flasks. MPC adhering to the bottom of the culture flasks was maintained by changing the medium every 3-4 days, detaching it after reaching confluence, and then performing subcultures. Subcultures were 1 x 10 6 ~8x10 7 The process was carried out until MPCs were reached. MPCs isolated according to this example were found to form multinucleated myotubes and be highly positive for AChE activity when cultured under differentiation conditions (Examples 2 and 3) (Figures 3 and 5). Furthermore, cell analysis according to Example 4 revealed that CD56 and CD90 were positive and CD34 was negative (Figure 6). In addition, when cultured under differentiation conditions according to Example 2, the cells were found to form multinucleated myotubes (Figure 3).

[0148] Example 2 - Differentiation ability of MPC 2x10 obtained according to Example 1 6Individual MPC cells were seeded into 24-well Nunclon® Delta Surface plastic plates. Approximately 2–4 days after seeding, the growth medium was replaced with skeletal muscle cell differentiation medium (500 mL, PromoCell GmbH, Germany), and 10 mL of skeletal muscle cell differentiation medium supplement pack (PromoCell GmbH, Germany) and 240 μL of gentamicin (8 mg / mL, Sandoz GmbH, Austria) were added to initiate skeletal muscle differentiation of the cells. The cells were cultured for approximately 5–7 days and analyzed by phase-contrast microscopy.

[0149] The MPCs obtained in Example 1 were tested for in vitro differentiation into the skeletal myogenic system under appropriate culture conditions. Successful differentiation was observed in all batches of MPCs used in the treatment in Example 5. Successful myogenic differentiation was determined by microscopic observation of muscle cells having at least one myotube, i.e., at least three separate nuclei formed by the fusion of multiple single-nucleus MPCs (Figure 3).

[0150] Example 3 - Measurement of AChE enzyme activity Cells isolated according to Example 1 were cultured for 5-7 days under differentiation conditions according to Example 2, and then AChE expression was analyzed.

[0151] Reagent preparation American Public Health Association (APHA) phosphate buffer, pH 7.2 (Sigma-Aldrich Co. LLC, Germany) was prepared according to the manufacturer's instructions. In summary, 17 g of the powder mixture (monopotassium phosphate 22.66 g / L and sodium carbonate 7.78 g / L) was added to 400 mL of distilled water. After adding 0.5 mL of Triton X-100, the mixture was dissolved on a magnetic stirrer at room temperature for 30 minutes. The final volume was made 500 mL in a graduated cylinder and used without further dilution. The buffer was stored at 4°C until use. Elmann's reagent (5,5'-dithiobis-2-nitrobenzoic acid, DTNB, 0.5 mM) was weighed at 2 mg into a 1.5 mL Eppendorf tube and freshly prepared for each AChE assay. This was dissolved in 1 mL of phosphate buffer (pH 7.2, 0.1% Triton X-100) by vortexing for 1-2 minutes. The final volume was reduced to 10 mL in a 15 mL Falcon tube containing phosphate buffer (pH 7.2, 0.1% Triton X-100), and the solution was stored at 4°C until use. Acetylthiocholine iodide (ATI, 5.76 mM) was weighed at 2 mg into a 1.5 mL Eppendorf tube and freshly prepared for each AChE assay. It was dissolved in 1.2 mL of distilled water by vortexing for 1-2 minutes and stored at 4°C until use.

[0152] Preparation and measurement of AChE standard enzymes: AChE standard dilutions were prepared in phosphate buffer (pH 7.2, 0.1% triton X-100) and used immediately. A readily available 50 U / mL AChE stock (derived from Electrophorus electrous) was purchased from AAT Bioquest® Inc., Sunnyvale, CA, USA. This AChE was diluted to prepare 1000 mU / mL AChE according to the manufacturer's instructions, and then further diluted in a 1:2 ratio to obtain eight different dilutions ranging from 4 to 500 mU / mL. 200 μL of each AChE standard enzyme dilution was mixed with 300 μL of 0.5 mM DTNB and 50 μL of 5.76 mM ATI. Furthermore, at least one blank reaction mix was prepared by mixing 0 mU / mL phosphate buffer with the above DTNB and ATI. Standards and blanks were incubated in the dark at 30°C for 6, 7, or 8 minutes, followed by OD measurement at 412 nm using an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK). The OD value of the blank reaction was subtracted from the OD value of the standard enzyme reaction. The correlation between the blank-corrected OD 412 nm value and the AChE concentration (mU / mL) of the standard dilution was visualized using GraphPad Prism software. A linear equation between AChE concentration and the corrected OD value was calculated.

[0153] Cell measurement To measure the AChE activity of the cells, cells cultured in skeletal muscle differentiation medium according to Example 2 were processed as follows: The differentiation medium was carefully removed from the 24-well plate, and 300 μL of 0.5 mM DTNB solution (prepared with phosphate buffer, pH 7.2, 0.1% triton X-100) was immediately added. After incubation at room temperature in the dark for 2 minutes, 50 μL of 5.76 mM ATI (prepared with distilled water) was added. Furthermore, at least one blank reaction mix was prepared by mixing phosphate buffer, DTNB, and ATI as described above. After incubation of all reaction mixes at 30°C in the dark for 60 minutes, OD measurements at 412 nm were performed using an Anthos Zenyth 340rt microplate reader (Biochrom Ltd., Cambridge, UK).

[0154] mU rel Calculation of AChE in: After 60 minutes of colorimetric measurement of the cells, the OD412nm value obtained as described above was corrected by subtracting the OD412nm value from the blank reaction. The corrected OD412nm value of the cells after 60 minutes was input into the linear equation generated from the AChE standard enzyme reaction (as outlined above, where the OD412nm was measured 6, 7, or 8 minutes after the addition of ATI and DTNB), and 2 x 10⁻¹⁰ values ​​relative to the AChE standard were obtained. 5 The AChE activity per cell was determined. Therefore, the unit of the determined AChE activity is 2 x 10⁻¹⁰ 5 mU represents the relative AChE activity of individual cells. rel It is given by.

[0155] The MPC manufactured according to Example 1 and used for treatment according to Example 5 measured 2 x 10⁻¹⁰ according to Example 3. 5 At least 36, and up to 680 mU per individual cell rel It was found that the patient had AChE (Figure 4).

[0156] Example 4 - Surface Marker Expression Cells isolated according to Example 1 and derived from approximately 170 different subjects were tested for the expression of surface markers CD34, CD56, and CD90. Flow cytometry was performed using a Guava easyCyte 6HT 2L flow cytometer (Merck Millipore, Darmstadt, Germany) to determine surface marker expression. Briefly, cells obtained according to Example 1 were collected by covering them with IX trypsin at 37°C for 5 minutes, and 400 *The cells were centrifuged at 1g and resuspended in 1x PBS supplemented with 1% FCS. 40,000 cells were suspended in 195 μl of 1x PBS, 5 μl of CD34-PE, CD56-PE, and CD90-PE (Beckman Coulter) were added, and the cells were incubated in 1.5 mL Eppendorf tubes for 20 minutes at 4°C in the dark. Next, 5 μl of viability dye 7-aminoactinomycin D (Beckman Coulter Inc., France) was added to each reaction, and the plates were incubated in the dark at room temperature for 10 minutes. Finally, cell events were acquired using Guava InCyte® v.2.3 software. Histograms and dot plots were generated with a minimum of 5000 events at a sample flow rate of 1.8 μL / mL. Positive staining was obtained by comparison with isotype controls set to at least 95% negative, or by comparison with control (negative) cells.

[0157] Furthermore, individual randomly and exemplary batches of cells obtained in Example 1 were tested for the expression of Sca-1, A2B5, and CD105 by flow cytometry. Flow cytometry analysis was performed using a Guava easyCyte 6HT 2L flow cytometer (Merck Millipore, Darmstadt, Germany). Briefly, cells were collected by trypsin at 37°C for 5 minutes, centrifuged at 400 rcf, and resuspended in lx PBS supplemented with 1% FCS. Cells at a reaction concentration of 40000 were incubated with 5 μL of IgG1-PE (Beckman Coulter), Isotype Alexa488 (Sigma), anti-CD105-PE (Beckman Coulter, France), or A2B5-Alexa488 antibody (Millipore) in 1.5 mL Eppendorf® tubes in the dark at 4°C for 15 minutes. Cells were washed with 1 mL of PBS, centrifuged at 400 rcf, and resuspended in 200 μL of 1x PBS for FACS analysis in a 96-well round-bottom plate. After washing and resuspending, 5 μL of the viability dye 7-aminoactinomycin D (Beckman Coulter Inc., France) was added to each reaction, and the plates were incubated at 4°C for 10 minutes. Cell events were acquired using Guava InCyte® v2.3 software. Histograms and dot plots were generated with a minimum of 3000 events at a sample flow rate of 1.8 μL / mL. Positive staining was obtained by comparison with an isotype control set to at least 99% negative.

[0158] MPC batches prepared according to Example 1 and used for treatment according to Example 5 were found to be CD56-positive in the range of 64.10% to 99.83%, CD90-positive in the range of 80.05% to 99.88%, and CD34-positive in the range of 0.04% to 8.33%. This expression profile was determined for MPC batches isolated from biopsies of approximately 170 different patients. Furthermore, individual batches randomly and exemplary selected from the aforementioned MPC batches were found to be Sca-1-negative, with at least 60% of cells per batch positively expressing A2B5 and CD105 for each marker, and at most 10% of cells positively expressing Sca-1.

[0159] Immunocytochemistry was performed to detect desmin and MyoD expression intracellularly in cells isolated according to Example 1. First, the supernatant of the cell culture dish was discarded and the cells were washed three times with PBS. Permeabilization was performed by covering the cells with 4% formaldehyde solution (v / v; diluted with PBS) and incubating at room temperature for 20 minutes. Subsequently, after each incubation step, the cells were washed three times with PBS. Then, the cells were covered with 500 μl of hydrogen peroxide block (Thermo Fisher Scientific) and incubated at room temperature for 5 minutes. Primary antibody (desmin or MyoD) at a final concentration of 40 μg pro ml (w / v) was pipetteed into the cells and incubated for at least 90 minutes (37°C, 5% CO2). Next, the cells were covered with 500 μl of biotin-labeled secondary antibody (goat anti-rabbit, polyclonal, Thermo Fisher Scientific) and incubated under the same conditions as the primary antibody, but for at least 60 minutes. To visualize antibody binding, 500 μl of horseradish streptoavidin peroxidase (Vectorlabs) was added at a final concentration of 2–5 μg / ml (diluted with PBS) and incubated at 37°C in 5% CO2 for 20 minutes. A final wash with PBS was performed before observing the results. Cells stained desmin-positive by immunocytochemistry were visualized as dark red.

[0160] As described above, individual batches randomly and exemplary selected from the MPC batch isolated according to Example 1 were found to be desmin-positive, with at least 60% of the cells being positive. Furthermore, individual batches randomly and exemplary selected from the MPC batch isolated according to Example 1 were found to be MyoD-negative, with at most 10% of the cells being MyoD-positive.

[0161] Example 5 - Treatment of fecal incontinence patients using MPC The applicant conducted a multinational, multicenter, randomized, double-blind, placebo-controlled, parallel-group clinical trial IIb to investigate the efficacy of myogenic progenitor cell therapy in a patient population reflecting the diversity actually encountered in clinical practice. The patient inclusion and exclusion criteria are shown below (Table 1). Figure 4 shows an overview of the examinations and tests performed and the patient flow.

[0162] Table 1: Inclusion and exclusion criteria for patients to be included in the ITT population of the clinical trial according to Example 5.

[0163] [Table 1-1]

[0164] [Table 1-2]

[0165] [Table 1-3]

[0166] Eligible patients were randomized to one of three treatment groups (see Supplemental Methods for randomization procedures) and received a low dose (LCC, 5±1 x 10¹) of isolated injected cells according to Example 1. 6 ) or high dose (HCC, 50±10x10 6Patients received cell therapy in combination with electrical stimulation for 4 weeks, or control therapy in combination with electrical stimulation and infusion of cell-free medium. MPC transplantation was performed by trained physicians on anesthetized patients as described above. Patients were placed in a supine position, and cells or placebo were injected directly under ultrasound guidance using a specially designed injection device. Each patient was given a fixed amount of frozen cells diluted with Ringer's lactate to an appropriate cell concentration. As a result, a total volume of 6 ml was injected in 12 depots (12 x 0.5 ml), with each depot arranged in a circular pattern directly in the external anal sphincter (EAS). Cell injection into the longitudinal muscle, internal anal sphincter, and subepithelial tissue was avoided. All patients were hospitalized for one day for this treatment. Cells isolated according to Example 1 and used for transplantation were analyzed for skeletal muscle differentiation potential (Example 2), AChE enzyme activity (Example 3), and surface marker expression (Example 4). All cell batches produced during the trial were found to be positive for the potential to form multinucleated myotubes (Figure 3). Furthermore, the cell populations used to treat both LCC and HCC were found to be 64.10%–99.83% positive for CD56, 80.05%–89.99% positive for CD90, and 0.04%–8.33% positive for CD34 (Figure 6). Pelvic floor muscle electrical stimulation therapy is considered the gold standard for conservative treatment of fecal incontinence because it is known to stimulate muscle growth and growth-related signaling. An overview of the trial is shown in Figure 4A. The patients selected for treatment in this trial (inclusive trial population, ITT) had the basic population statistical parameters shown in Table 2.

[0167] Table 2: Demographics, time since first FI visit, and associated sphincter abnormalities in the ITT patient population in clinical trials.

[0168] [Table 2-1]

[0169] [Table 2-2]

[0170] [Table 2-3]

[0171] Example 6 - Analysis of effectiveness Following the initial screening examination in Example 5, patients were instructed to keep an incontinence diary for two weeks before a muscle biopsy sample was taken from the pectoralis major at the next examination. Subsequently, four weeks of electrical stimulation therapy were initiated. After another four weeks of diary keeping, patients received either cell (LCC or HCC) or control (PBO) injections and received post-transplant control the following day. Patients received another four weeks of electrical stimulation therapy and kept diaries. Patients were surveyed at 3, 6, and 12 months post-injection and interviewed with the control group before keeping four weeks of diaries. In the diaries, patients were required to score the daily impact of fecal incontinence on their day using a visual analog scale (VAS), record fecal incontinence episodes, and categorize them into three categories: “trace,” “small,” and “large.” Further parameters, as outlined in the scheme, consisted of response rate assessment, anal manometry, ultrasound measurements, and the FI-QoL questionnaire. The primary endpoint was the change in incontinence episode frequency (IEF) over a 4-week period from baseline (VO) 4 weeks prior to injection to 6 months post-injection (V4 = 6 months post-treatment in Figure 1). Secondary endpoints included changes in VAS calculated from the mean values ​​over a 4-week diary period and quality of life. Furthermore, the proportion of patients with a ≥25%, ≥50%, ≥75%, and ≥90% reduction in IEF compared to baseline, as well as changes in anal manometry and ultrasound data over time, were evaluated. Further exploratory endpoints included changes in IEF and other parameters from baseline to 12 months post-injection, changes in various types of incontinence episodes (IEs), and incontinence-free days.

[0172] Anoscopy, ultrasound, and anal pressure measurements were performed according to the standards set by each participating facility. Evaluation items included anal canal length, resting pressure, and maximum squeezing pressure. Balloon deflation tests measured the amount of balloon volume required to reach the first sensation, the level of defecation urge, the level of defecation urgency, and the maximum tolerable volume.

[0173] During the study period, adverse events (AEs) and serious AEs were recorded, standard health checkups, hematological, blood chemistry, and urinalysis were performed, and concomitant medications were registered. This study was overseen by an independent data safety monitoring committee. Based on this evaluation, a total of 252 patients were randomly assigned to three treatment groups in a 1:1:1 ratio. For the primary endpoint analysis, a one-sided Wilcoxon rank-sum test was applied, with p-value < 0.025 considered significant. For continuous secondary endpoints, unpaired t-tests (when the compared study groups were normally distributed) or Wilcoxon rank-sum tests (when the compared study groups were not normally distributed) were used to compare study groups, with p-value < 0.05 considered significant. For discontinuous variables, chi-square tests or Fisher tests were used to compare study groups.

[0174] Analysis of treatment effectiveness for different types of incontinence episodes using PBO, LCC, and HCC in terms of the 50% responder rate at the fifth visit (12 months post-treatment) revealed that the responder rate was consistently lowest for episodes classified as "trace" (Table 3). Therefore, for further analysis of IEF changes and responder rates, calculations were performed both when all episode types were counted and when only "small" and "heavy" episode types were counted.

[0175] Table 3: Percentage of patients who experienced at least a 50% reduction in weekly incontinence episodes from baseline to 12 months post-treatment, by treatment group (LCC, HCC, PBO) and episode type (trace, small, large).

[0176] [Table 3]

[0177] Analysis of changes in IEF between treatment groups in the ITT patient population revealed a consistent decrease in IEF across all treatment groups over the post-transplant visit period, regardless of whether all episode types were counted or traces were excluded (Figure 1A, Figure IB, Table 4, Table 5). The decrease in IEF was highest in the HCC group across all post-treatment visits, followed by the LCC group, and finally the PBO group. In the ITT set, a two-sided Wilcoxon-Mann-Whitney test with an α level of 0.05 was performed between HCC vs. PBO and LCC vs. PBO. The change in IEF from baseline to post-transplant visits was significantly greater in HCC compared to PBO at 6 months post-treatment (p=0.035) with traces included, and at 12 months post-treatment (p=0.034) with traces excluded (Figure 1A and B), suggesting that high-cell-count MPC transplantation is superior to placebo treatment.

[0178] Table 4: Absolute change in weekly IEF (including trace values) from baseline (VO) during the study period of Example 5, by treatment group in the ITT patient population. PBO: Placebo. LCC: Low cell count. HCC: High cell count. SD: Standard deviation. N: Number of patients.

[0179] [Table 4]

[0180] Table 5: Absolute change in weekly IEF (excluding traces) from baseline (VO) during the study period of Example 5, by treatment group in the ITT patient population. PBO: Placebo. LCC: Low cell count. HCC: High cell count. SD: Standard deviation. N: Number of patients.

[0181] [Table 5]

[0182] Responder rate Consistent with other researchers (Rao, 2016), we considered a reduction of 50% or more in IEF to be a clinically significant improvement, and classified patients showing this degree of reduction as responders, distinguishing them from non-responders with smaller reductions. Therefore, we re-examined the above subgroups, evaluating data on the proportion of responders in each treatment group, including data at 1 and 3 months post-injection. We detected that in all defined groups and subgroups, the proportion of responders continued to increase up to the 6-month follow-up, cell counts were higher than the control group in all cases, and HCC was higher than LCC in most cases (proportion of patients in the ITT patient population, by treatment group, with at least a 50% reduction in IEF (including traces) per week from baseline to post-treatment visit according to Example 5). PBO, placebo. LCC, low cell count. HCC, high cell count (Table 6). Between 6 and 12 months, the responder rate remained stable or decreased in the control and LCC groups, but in the HCC group, it continued to increase to over 50% when trace cells were included (Table 6), and over 60% when trace cells were excluded (Table 7). Fisher's exact test, with a significance level of p=0.05, was performed on the 50% responder rate between LCC or HCC and PBO treatment in ITT patients. When trace cells were excluded from the analysis, the superiority of HCC treatment over PBO treatment was observed at 1 month (p=0.037) and 12 months (p=0.006) after treatment (Figure 1D, Table 7). This suggests that the application of high cell count myogenic progenitor cells is an effective and clinically appropriate treatment for incontinence.

[0183] Table 6: Percentage of patients in the ITT patient population, by treatment group, who experienced at least a 50% reduction in weekly IEF (including traces) from baseline to post-treatment visits according to Example 5. PBO: Placebo. LCC: Low cell count. HCC: High cell count.

[0184] [Table 6]

[0185] Table 7: Percentage of patients in the ITT patient population, by treatment group, who experienced at least a 50% reduction in IEF (excluding traces) per week from baseline to post-treatment visit, according to Example 5. PBO: Placebo. LCC: Low cell count. HCC: High cell count.

[0186] [Table 7]

[0187] Example 7 - Relationship between patient characteristics and treatment outcomes In the clinical trial outlined in Example 5, 288 patients were screened, 251 were randomly assigned, and 244 of them received the study drug. 218 women and 19 men completed the trial for at least 6 months of follow-up (96%) (Figure 4). The median age of participants was 63 years (interquartile range, IQR, 53.8–70), and the median duration of FI was 5.3 years (IQR, 2.7–9.9). Baseline demographic and clinical characteristics of ITT patients available for analysis of the primary endpoint are summarized in Table 2.

[0188] Subgroup-specific effects An exploratory post-hoc analysis was conducted to establish a patient group that responded more specifically to cell therapy than to placebo. For this purpose, the following hypothesis-driven approach was applied: It was hypothesized that injection of cells into or near existing muscle tissue would be effective in restoring EAS function. This is thought to be due to the limited migratory capacity of MPCs after transplantation. Therefore, muscle regeneration may be impaired in patients with thin muscle tissue due to persistent scarring of damaged EAS or time-dependent sarcopenia. Both conditions correlate with the duration of FI prior to the trial. Re-analysis of treatment effects, focusing only on patients with FI less than 10 years (73% of the ITT set; control group 1, TPP1), revealed that the decrease in IEF in the LCC and HCC groups ultimately significantly exceeded the decrease in IEF in the control group. Consequently, the change in IEF from baseline to 12 months was much higher than the respective changes in the ITT set, excluding traces (Figure 2B). Responder rates and within the TPP1 patient subgroup, after excluding trace cases, a significantly higher 50% responder rate was observed in HCC than in PBO at 12 months post-treatment (Figure 2B).

[0189] Another factor that diminished the apparent impact of cell therapy was the decrease in IEF even in the control group. Therefore, to clearly capture the effect of cell infusion, we hypothesized that a higher baseline IEF would lead to a greater decrease in IEF after treatment, resulting in a clear difference between the PBO group and the cell group (LCC, HCC). This hypothesis was tested by subgrouping ITT set patients according to their baseline IEF (including trace levels) and comparing the change in IEF from baseline to 6 months between the treatment group and the subgroup. The results showed that after excluding patients with low baseline IEF, the change in IEF increased in all groups. However, the difference between the PBO group and the cell group became increasingly pronounced as baseline IEF increased (Figure 7, Table 8). This suggests that patients with high baseline IEF benefit more from MPC-based treatment than patients with low baseline IEF. Since the difference between treatment groups widened rapidly when baseline IEF exceeded 6, we combined TPP1 with this feature to create TPP2 (less than 10 years from initial FI visit, baseline IEF greater than 6). When the treatment effect was re-analyzed focusing only on these patients, the decrease in IEF in the LCC and HCC groups was significantly greater than that in the control group. As a result, the change in IEF from baseline to 12 months was much larger compared to the changes in the ITT and TPP1 groups, respectively (Figure 2A). Regarding the responder rate in the TPP2 patient subgroup, a significantly higher 50% responder rate was observed in HCC than in PBO at 12 months post-treatment, and ultimately, the responder rate was higher in the TPP2 HCC group compared to the ITT patient HCC group (Figure 2C). This suggests that TPP2 cases are more responsive to MPC-based treatment than ITT cases.

[0190] Incontinence episodes classified as "trace" were the least responsive to treatment according to Example 5, and as demonstrated above, patients with high baseline IEF responded better to MPC-based treatment. Therefore, we analyzed TPP1 patients with a baseline IEF greater than 2 who were not likely to be classified as "trace." This patient group was called TPP3. When the treatment effect was re-analyzed focusing only on TPP3 patients, the decrease in IEF in the LCC and HCC groups significantly exceeded that of the control group, which did not count traces (Figure 2B). Ultimately, the change in IEF from baseline to 12 months was much greater compared to the changes in ITT, TPP1, and TPP2 respectively (Figures 2A and 2B). Regarding the response rate of the TPP3 patient subgroup, including traces, a significantly higher response rate of 50% was observed in HCC compared to PBO at 12 months post-treatment. When traces were not included in the analysis, a significantly higher responder rate was observed in both HCC and LCC compared to PBO. Ultimately, the TPP3 responder rate among HCC-treated patients was the highest compared to all other patient groups, regardless of whether traces were counted as episodes (Figure 2C, Figure 2D) (Table 11, Table 12). This suggests that TPP3 patients respond best to MPC-based treatment compared to ITT, TPP1, and TPP2 patients. Conversely, patients excluded by the TPP3 subgroup (i.e., "NR1" with an FI period of more than 10 years, or "NR2" with a baseline IEF of less than 2 (excluding traces)) had lower IEF change and response rates than TPP1, TPP2, and TPP3 patients. Patient group selection may affect not only how cell therapy with LCC or HCC affects IEF reduction and response rates, but also how placebo (PBO) treatment affects them. Therefore, effect sizes and odds ratios were further calculated for IEF change rate and responder rate, respectively. In detail, the treatment of LCC or HCC versus PBO was compared for patient subgroups according to Table 13, and Cohen's d and odd ratios were calculated. Regardless of whether traces were counted as episodes, TPP3 patients showed the highest treatment efficacy with LCC or HCC treatment (compared to PBO treatment, respectively) in terms of effect size and odd ratios (Figure 8).

[0191] Furthermore, we analyzed the change in IEF from baseline to 12 months post-treatment, the responder rate, and the associated effect size and odds ratio in all TPP3 patients compared with TPP3 patients with pre-treatment fecal incontinence-related external anal sphincter injury (TPP3 injury) (Tables 9, 10, 11, and 12). Comparing these patient groups, we found that TPP3 patients with FI due to muscle injury experienced a greater decrease in IEF after HCC treatment compared with TPP3 patients whose FI cause was not identified (Figures 9 A and B). This effect was observed regardless of whether traces were counted as FI. Also, in the TPP3 injury group, the decrease in IEF after HCC treatment was significantly higher compared to PBO treatment. Similarly, in the TPP3 injury group, the 50% responder rate was higher, regardless of whether traces were counted or not (Figures 9 C and D). In TPP3_injured patients, HCC treatment showed a significantly higher responder rate than PBO treatment. However, after excluding traces from the analysis, the final responder rate in HCC-treated patients was 87.5%, the highest over the entire period (Table 12). When analyzing the effect size and odds ratio of IEF change and responder rate with LCC or HCC compared to PBO treatment in TPP3_injured patients, HCC treatment showed a higher effect size than the TPP3 patient group, regardless of whether traces were considered incontinence episodes (Figure 10 A, B). The odds ratio for the 50% responder rate was also higher in the TPP3_injured patient group compared to the TPP3 patient group for both LCC and HCC treatment, regardless of whether traces were counted or excluded (Figure 10 C, D). These results suggest that patients who suffered from EAS injury before treatment experienced a greater reduction in incontinence episodes and greater changes in response when treated with a different number of MPCs compared to a broader patient population with multiple potential FI-related conditions (e.g., EAS atrophy, pelvic floor dysfunction, etc.).

[0192] Table 8: Change in IEF from baseline to 6 months post-treatment in ITT sets, further grouped according to baseline IEF among treatment groups (PBO, LCC, HCC). Mean, standard deviation (SD), and number of patients (N) are shown.

[0193] [Table 8]

[0194] Table 9: Changes in the patient population from baseline to 12 months post-treatment in IEF (including trace cases) treatment groups (PBO, LCC, HCC), and shown in mean, standard deviation (SD), and number of patients (N).

[0195] [Table 9]

[0196] Table 10: Changes in patient population from baseline to 12 months post-treatment by treatment group (PBO, LCC, HCC), including IEF (excluding traces), mean, standard deviation (SD), and number of patients (N).

[0197] [Table 10]

[0198] Table 11: Percentage of patients who experienced at least a 50% reduction in IEF (including traces) from baseline to 12 months post-treatment, by treatment group (PBO, LCC, HCC) and patient population.

[0199] [Table 11]

[0200] Table 12: Percentage of patients who experienced at least a 50% reduction in IEF (excluding traces) from baseline to 12 months post-treatment, by treatment group (PBO, LCC, HCC) and patient population. [Table 12]

[0201] Table 13: Summary of the patient population analyzed according to Example 7, by number of patients (N), severity of fecal incontinence, time since diagnosis of fecal incontinence, and status related to FL. MD = muscle injury, PFD = pelvic floor dysfunction, ND = nerve injury, LOSC = loss of storage capacity, AT = atrophy.

[0202] [Table 13]

[0203] References Bajpai, VK, Mistriotis, P., Loh, Y.-H., Daley, GQ, & Andreadis, ST (2012). Functional vascular smooth muscle cells derived from human induced pluripotent stem cells via mesenchymal stem cell intermediates. Cardiovascular Research, 96(3), 391-400. Frudinger, A., Kolle, D., Schwaiger, W., Pfeifer, J., Paede, J., & Halligan, S. (2009). Muscle-derived cell injection to treat anal incontinence due to obstetric trauma. Gut, 59(01), 55- Frudinger, A., Pfeifer, J., Paede, J., Kolovetsiou-Kreiner, V., Marksteiner, R., & Halligan, S. (2015). Autologous skeletal-muscle-derived cell injection for anal incontinence due to obstetric trauma: a 5-year follow-up of an initial study of 10 patients. Colorectal Disease: The Official Journal of the Association of Coloproctology of Great Britain and Ireland, 17(9), 794-801. Hirai, H., Yang, B., Garcia-Barrio, M. T., Rom, O., Ma, P. X., Zhang, J., & Chen, Y. E. (2018). Direct Reprogramming of Fibroblasts Into Smooth Muscle-Like Cells With Defined Transcription Factors-Brief Report. Arteriosclerosis, Thrombosis, and Vascular Biology, 38(9), 2191-2197. Incitti, T., Magli, A., Jenkins, A., Lin, K., Yamamoto, A., & Perlingeiro, R. C. R. (2020). Pluripotent stem cell-derived skeletal muscle fibers preferentially express myosin heavy chain isoforms associated with slow and oxidative muscles. Skeletal Muscle, 10(f), 17. Ito, N., Kii, I., Shimizu, N., Tanaka, H., & Takeda, S. (2017). Direct reprogramming of fibroblasts into skeletal muscle progenitor cells by transcription factors enriched in undifferentiated subpopulation of satellite cells. Scientific Reports, 7(1), 8097. Jorge, J. Marcio N., Wexner, Steven D. (1993). Etiology and Management of Fecal Incontinence. Dis Colon Rectum, Vol. 36, No.l, pp. 77-96. Messner, F., Thumer, M., Muller, J., Blumer, M., Hofmann, J., Marksteiner, R., Couillard- Despres, S., Troppmair, J., Ofiier, D., Schneeberger, S., & Hautz, T. (2021). Myogenic progenitor cell transplantation for muscle regeneration following hindlimb ischemia and reperfusion. Stem Cell Research & Therapy, 12(V), 146. Miyagoe-Suzuki, Y., & Takeda, S. (2017). Skeletal muscle generated from induced pluripotent stem cells - induction and application. World Journal of Stem Cells, 9(6), 89-97. Rando, T. A., & Blau, H. M. (1994). Primary mouse myoblast purification, characterization, and transplantation for cell-mediated gene therapy. The Journal of Cell Biology, 125(6), 1275-12M. Rao, S. S. C. (2016). Endpoints for therapeutic interventions in faecal incontinence: small step or game changer. In Neurogastroenterology and Motility (Vol. 28, Issue 8, pp. 1123-1133). Blackwell Publishing Ltd. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., & Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131(5), 861-872. Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), 663-676. Thumer, M., Deutsch, M., Janke, K., Messner, F., Kreutzer, C., Beyl, S., Couillard-Despres, S., Hering, S., Troppmair, J., & Marksteiner, R. (2020). Generation of myogenic progenitor cell-derived smooth muscle cells for sphincter regeneration. Stem Cell Research & Therapy, 11(1), 233. Xuan, W., Khan, M., & Ashraf, M. (2021). Pluripotent stem cell-induced skeletal muscle progenitor cells with givinostat promote myoangiogenesis and restore dystrophin in injured Duchenne dystrophic muscle. Stem Cell Research & Therapy, 72(1), 131. Yamanaka, S. (2008). Induction of pluripotent stem cells from mouse fibroblasts by four transcription factors. Cell Proliferation, 41 Suppl 1, 51-56.

Claims

1. A pharmaceutical composition comprising myoblasts for use in a method for treating anal incontinence in a subject, wherein the pharmaceutical composition is administered by one or more injections into and / or near the anal sphincter of the subject, the subject has suffered from anal incontinence for 6 months to 10 years, and has a severity of incontinence defined as more than two incontinence episodes per week prior to treatment, wherein the incontinence episodes result in the unintended leakage of liquid or solid stool from the subject's rectum, and incontinence episodes that appear only as traces in the form of stains or marks on linens are excluded.

2. A pharmaceutical composition according to claim 1, characterized in that, as a result of the incontinence episode, the amount of stool produced exceeds the amount that appears as a stain or mark on the subject's linen, and is comparable to the amount that results from normal defecation.

3. The pharmaceutical composition according to claim 1, characterized in that the anal incontinence of the subject is induced by muscle damage.

4. The pharmaceutical composition according to claim 1, characterized in that the subject has a severity of incontinence defined as more than six incontinence episodes per week prior to treatment.

5. The pharmaceutical composition according to claim 1, wherein the method comprises stimulating anal sphincter tissue before and / or after administration of myoblasts.

6. The pharmaceutical composition according to claim 5, characterized in that the stimulation includes stimulation of anal sphincter tissue for at least two weeks after administration of myoblasts.

7. The pharmaceutical composition according to claim 5, characterized in that the stimulation includes stimulation of anal sphincter tissue for at least two weeks before and after administration of myoblasts.

8. The pharmaceutical composition according to claim 5, characterized in that the stimulation is performed by Kegel exercises and / or transcutaneous electrical stimulation.

9. The pharmaceutical composition according to claim 8, wherein the transcutaneous electrical stimulation is performed at least twice or at least three times per day.

10. The pharmaceutical composition according to claim 5, characterized in that each stimulus lasts for at least 10 minutes.

11. The pharmaceutical composition according to claim 5, characterized in that each stimulus lasts for at least 20 minutes.

12. (i) By positive expression of the CD56 and CD90 markers, at least 60% of myoblasts express CD56 and at least 60% express CD90, and / or (ii) 20mU rel ~1000 mU rel It is measured by the AChE activity, and the AChE activity is 2 × 10 5 By being measured per cell The pharmaceutical composition according to claim 1, characterized as follows.

13. (ii) 30mU rel ~800mU rel It is measured by the AChE activity, and the AChE activity is 2 × 10 5 The pharmaceutical composition according to claim 12, characterized by being measured per cell.

14. (ii) 50-700mU rel It is measured by the AChE activity, and the AChE activity is 2 × 10 5 The pharmaceutical composition according to claim 12, characterized by being measured per cell.

15. The pharmaceutical composition according to claim 1, characterized in that 60-99.9% of myoblasts express the cell marker CD56, 80-99.9% of myoblasts express the cell marker CD90, and 0-10% of myoblasts express the cell marker CD34.

16. The method described above is (i) administration of 1 million to 200 million myoblasts, and / or (ii) 1 × 10 2 mU rel _ total ~1 × 10 6 mU rel _ total administration of myoblasts in an amount having a total AChE activity of The pharmaceutical composition according to claim 1, characterized by comprising the administration of an effective amount of [the substance].

17. The method described above is (ii) 1 x 10 3 mU rel _ total ~5 x 10 5 mU rel _ total Administration of myoblasts in an amount having total AChE activity The pharmaceutical composition according to claim 16, characterized by comprising the administration of an effective amount of myoblasts.

18. The method described above is (ii) 7 x 10 4 mU rel _ total ~2 x 10 5 mU rel _ total Administration of myoblasts with a total amount of AChE activity The pharmaceutical composition according to claim 16, characterized by comprising the administration of an effective amount of [the substance].

19. The pharmaceutical composition according to claim 1, characterized in that the myoblasts are administered by one or more injections into the external anal sphincter, internal anal sphincter and / or puborectalis muscle.

20. A pharmaceutical composition according to claim 1, characterized in that it comprises pharmaceutically acceptable additives and / or carriers.

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