Pharmaceutical composition for treating urinary incontinence and preparation method and application thereof

Abstract

The invention relates to the technical field of stem cell treatment, in particular to a pharmaceutical composition for treating urinary incontinence and a preparation method and application thereof. The invention provides a pharmaceutical composition for treating urinary incontinence. The pharmaceutical composition is prepared from the following components: adipose tissue-derived stem cell exosome, platelet-rich plasma, rutin, astragaloside, fructus alpiniae oxyphyllae extract, human serum albumin, sodium hydrogen sulfite, sorbitol and the balance of water for injection. The PRP and the adipose tissue-derived stromal cell exosome are applied to treatment of urethral sphincter injury, clinical application experiments show that the PRP and the adipose tissue-derived stromal cell exosome have a good repairing effect in connective tissue and muscle tissue, symptoms of patients can be greatly improved, the patients can recover more quickly, and the curative effect of the patients on urethral sphincter injury is improved. The urinary incontinence state of a patient can be greatly improved, and a basis is provided for clinical application of a cell technology.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell therapy, and in particular to a pharmaceutical composition for treating urinary incontinence, and a preparation method and application thereof. Background Art

[0002] With the improvement of living standards, the incidence of prostate cancer is getting higher and higher. The ultimate means of treating prostate cancer is still surgery. Due to the special location of the prostate, it is difficult to avoid urethral sphincter damage, paraprostatic neurovascular bundle damage, etc. when removing cancerous tissue during surgery, leading to urinary incontinence and sexual dysfunction. In particular, urethral sphincter damage can cause urinary incontinence, which brings difficulties to patients' life and social life.

[0003] Currently, the treatment methods for urinary incontinence on the market are divided into two methods: Western medicine and traditional Chinese medicine. For patients with moderate to severe urinary incontinence, surgical treatment is often used, including vaginal tension-free mid-urethral suspension, which has significant therapeutic effects, but the cost of surgery is high and complications often occur after surgery; Western medicine treatment often uses midodrine hydrochloride tablets, which have an efficacy of 50% to 60% for mild patients, but the treatment effect is not ideal for moderate to severe patients. Traditional Chinese medicine mainly treats stress urinary incontinence with oral Chinese medicine, acupuncture, massage and other methods, but the treatment cycle is long, generally taking several months or a year.

[0004] Platelets are a type of anucleated cell that exists in the blood. They are small in size and are divided into a static phase and a functional phase. Static phase platelets have no function, but under the stimulation of extravascular collagen, they quickly transform from the static phase to the functional phase, extend pseudopodia, form a network to prevent the escape of blood cells, and play a hemostatic role. After that, the platelets in the tissue can also release a large amount of cytokines, promote endothelial proliferation, and promote tissue repair. The lifespan of normal platelets in the blood is about 1 to 2 weeks. PRP (platelet-rich plasma therapy) technology is to draw whole blood from the vein, centrifuge it under sterile conditions, and collect the platelets therein to obtain platelet-rich plasma, which is locally injected to promote injury repair. Therefore, how to reasonably utilize the repair ability of platelet-rich plasma for injuries and provide a pharmaceutical composition that effectively treats urinary incontinence is a problem that technicians in this field urgently need to solve. Summary of the invention

[0005] The purpose of the present invention is to provide a pharmaceutical composition for treating urinary incontinence, and its preparation method and application. The purpose of the present invention is to use PRP to promote the reconstruction of local capillary beds and promote the repair of local injuries, thereby repairing and regenerating urethral sphincter cells after radical prostatectomy to achieve the purpose of treating urinary incontinence.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a pharmaceutical composition for treating urinary incontinence, comprising the following components: 3×10 10 ~5×10 10 / mL, platelet-rich plasma 0.03-0.05mL / mL, rutin 7-11mg / mL, astragaloside 7-11mg / mL, Alpinia oxyphylla extract 12-16mg / mL, human serum albumin 30-40mg / mL, sodium bisulfite 3-5mg / mL, sorbitol 15-25mg / mL, and the rest is water for injection.

[0008] Preferably, the water for injection is 0.9% sodium chloride water for injection.

[0009] Preferably, the preparation method of the Alpinia oxyphylla extract is:

[0010] (1) grinding Alpinia oxyphylla seeds to obtain material 1;

[0011] (2) The material 1 is mixed with an extractant for extraction to obtain an extractant layer, the solvent is removed by reduced pressure distillation, and the extract is freeze-dried to obtain an Alpinia oxyphylla extract.

[0012] Preferably, the extracting agent in step (2) is acetone and ethyl acetate, and the volume ratio is 8-12:6-10; the volume concentration of the acetone is 25-30%; and the volume concentration of the ethyl acetate is 15-20%.

[0013] Preferably, the extraction time in step (2) is 40 to 50 seconds.

[0014] Preferably, in step (2), the material 1 is mixed with the extractant in a mass volume ratio of 1 g: 6-8 mL.

[0015] Preferably, the freeze-drying temperature in step (2) is -35 to -45°C, and the freeze-drying time is 24 to 34 hours.

[0016] Preferably, the pharmaceutical composition further contains 2-3 mg / mL of urapidil and 4-6 mg / mL of tolterodine tartrate.

[0017] The present invention also provides a method for preparing the pharmaceutical composition, comprising mixing water for injection, sodium bisulfite, sorbitol, rutin, astragaloside, Alpinia oxyphylla extract, urapidil, and tolterodine tartrate, and then sequentially adding human serum albumin, platelet-rich plasma, and adipose-derived mesenchymal stem cell exosomes and mixing.

[0018] The present invention also provides the use of the pharmaceutical composition or the pharmaceutical composition prepared by the preparation method in preparing a drug for treating urinary incontinence.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention mainly restores the physiological function of the urethra from the cellular level through two components, adipose mesenchymal stem cell exosomes and platelet-rich plasma, and increases the unit function of the urethral sphincter. The rutin added by the present invention has the effect of inhibiting platelet aggregation, which can open blood vessels, dilute blood and promote circulation. The present invention uses rutin in combination with platelet-rich plasma, and the addition of rutin helps to promote the platelet-rich plasma to better play a repairing role. The addition of astragaloside can reduce the frequency and amplitude of the contraction of bladder smooth muscle strips. The use of Alpinia oxyphylla extract has the effect of inhibiting the overactivity of the bladder detrusor.

[0021] The urapidil added in the present invention is an M receptor blocker. The addition of this component can reduce detrusor contraction, and the use of tolterodine tartrate can increase sphincter tension. The addition of the two components can further improve the patient's urinary incontinence-related symptoms from the two aspects of reducing detrusor contraction and increasing sphincter tension. Therefore, the components in the pharmaceutical composition of the present invention are used in combination, which can not only effectively control the excessive contraction of bladder smooth muscle, detrusor muscle, etc., improve the tension of sphincter, etc., but also can repair the unit function of the relevant urethral sphincter from the cellular level, thereby achieving the purpose of effectively treating urinary incontinence.

[0022] In the pharmaceutical composition provided by the present invention, the addition of human serum albumin can improve the antioxidant capacity of the composition, sodium bisulfite as an antioxidant can also prevent the drug from oxidative deterioration and improve the stability of the injection, and the addition of sorbitol can balance body fluids to accelerate the body's absorption of the drug.

[0023] The present invention discloses the therapeutic application of autologous platelet-enriched plasma to urethral sphincter injury and provides a medicine for treating urethral sphincter injury. The present invention applies PRP and adipose-derived mesenchymal stem cell exosomes to the treatment of urethral sphincter injury. Clinical application experiments show that the use of PRP and adipose-derived mesenchymal stem cell exosomes have a good repair effect in connective tissue and muscle tissue, which can greatly improve the patient's symptoms, facilitate the patient's faster recovery, and can greatly improve the patient's urinary incontinence state, providing a basis for the clinical application of cell technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0025] Figure 1 This is the frequency diagram of urinary incontinence in Experimental Example 1.

[0026] Figure 2 This is the urinary incontinence score chart of Experimental Example 2. DETAILED DESCRIPTION

[0027] The present invention provides a pharmaceutical composition for treating urinary incontinence, comprising the following components: 3×10 10 ~5×10 10 / mL, platelet-rich plasma 0.03-0.05mL / mL, rutin 7-11mg / mL, astragaloside 7-11mg / mL, Alpinia oxyphylla extract 12-16mg / mL, human serum albumin 30-40mg / mL, sodium bisulfite 3-5mg / mL, sorbitol 15-25mg / mL, and the rest is water for injection;

[0028] The preferred components include: adipose-derived mesenchymal stem cell exosomes 4×10 10 / mL, platelet-rich plasma 0.04mL / mL, rutin 8-10mg / mL, astragaloside 8-10mg / mL, Alpinia oxyphylla extract 13-15mg / mL, human serum albumin 32-38mg / mL, sodium bisulfite 4mg / mL, sorbitol 17-23mg / mL, and the rest is water for injection;

[0029] The following components are further preferably included: adipose-derived mesenchymal stem cell exosomes 4×10 10 / mL, platelet-rich plasma 0.04mL / mL, rutin 9mg / mL, astragaloside 9mg / mL, Alpinia oxyphylla extract 14mg / mL, human serum albumin 34-36mg / mL, sodium bisulfite 4mg / mL, sorbitol 19-21mg / mL, and the rest is water for injection;

[0030] More preferably, the following components are included: adipose-derived mesenchymal stem cell exosomes 4×10 10 / mL, platelet-rich plasma 0.04mL / mL, rutin 9mg / mL, astragaloside 9mg / mL, Alpinia oxyphylla extract 14mg / mL, human serum albumin 35mg / mL, sodium bisulfite 4mg / mL, sorbitol 20mg / mL, and the rest is water for injection.

[0031] In the present invention, the water for injection is 0.9% sodium chloride water for injection.

[0032] In the present invention, the preparation method of the Alpinia oxyphylla extract is:

[0033] (1) grinding Alpinia oxyphylla seeds to obtain material 1;

[0034] (2) The material 1 is mixed with an extractant for extraction to obtain an extractant layer, the solvent is removed by reduced pressure distillation, and the extract is freeze-dried to obtain an Alpinia oxyphylla extract.

[0035] In the present invention, the extracting agent in step (2) is acetone and ethyl acetate, and the volume ratio is 8-12:6-10; preferably 9-11:7-9; more preferably 10:7-9; more preferably 10:8.

[0036] In the present invention, the volume concentration of acetone in step (2) is 25-30%; preferably 26-29%; more preferably 27-28%; and more preferably 28%.

[0037] In the present invention, the volume concentration of ethyl acetate in step (2) is 15-20%; preferably 16-19%; more preferably 17-18%; and more preferably 18%.

[0038] In the present invention, the extraction time in step (2) is 40 to 50 seconds, preferably 42 to 48 seconds, more preferably 44 to 46 seconds, and more preferably 45 seconds.

[0039] In the present invention, the material 1 in step (2) is mixed with the extractant in a mass volume ratio of 1 g:6-8 mL; preferably 1 g:7 mL.

[0040] In the present invention, the freeze-drying temperature of step (2) is -35 to -45°C, and the freeze-drying time is 24 to 34 hours; preferably, the freeze-drying temperature is -37 to -43°C, and the freeze-drying time is 26 to 32 hours; further preferably, the freeze-drying temperature is -39 to -41°C, and the freeze-drying time is 28 to 30 hours; more preferably, the freeze-drying temperature is -40°C, and the freeze-drying time is 29 hours.

[0041] In the present invention, the pharmaceutical composition further contains 2-3 mg / mL of urapidil and 4-6 mg / mL of tolterodine tartrate; preferably, the pharmaceutical composition further contains 2.5 mg / mL of urapidil and 5 mg / mL of tolterodine tartrate.

[0042] The present invention also provides a method for preparing the pharmaceutical composition, comprising mixing water for injection, sodium bisulfite, sorbitol, rutin, astragaloside, Alpinia oxyphylla extract, urapidil, and tolterodine tartrate, and then sequentially adding human serum albumin, platelet-rich plasma, and adipose-derived mesenchymal stem cell exosomes and mixing.

[0043] The present invention also provides the use of the pharmaceutical composition or the pharmaceutical composition prepared by the preparation method in preparing a drug for treating urinary incontinence.

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] A method for preparing adipose-derived mesenchymal stem cell exosomes, comprising the following steps:

[0047] (1) Extracting and isolating primary adipose-derived mesenchymal stem cells from the patient’s autologous fat samples:

[0048] (2) The primary adipose-derived mesenchymal stem cells obtained in step (1) were inoculated in a T75 culture flask at a density of 4200 cells / cm 2 , primary culture was carried out after supplementing the culture medium; the culture temperature was 36°C and the carbon dioxide concentration was 4%.

[0049] The culture medium is composed of the following components: 0.3 g / L L-isoleucine, 1 g / L L-valine, 1 g / L glycine, 7 mM β-glycerophosphate, 7 g / L glutathione, 4 ng / mL platelet-derived growth factor, 4 ng / mL EGF, 9 ng / mL bFGF, 4 ng / mL fibroblast growth factor, and the rest is DMEM-F12 culture medium;

[0050] (3) When the cell density in step (2) reaches 70%, the cell culture medium is collected and centrifuged to obtain adipose stem cell exosomes;

[0051] The centrifugation method is as follows: centrifuging the cell culture medium at 200×g for 12 minutes, 1900×g for 5 minutes, and 9000×g for 25 minutes in sequence to remove cells and cell debris; continuing to centrifuge at 99000×g for 65 minutes, resuspending and washing the sediment at the bottom, and finally centrifuging at 99000×g for 65 minutes to remove impurities to obtain adipose mesenchymal stem cell exosomes.

[0052] Example 2

[0053] A method for preparing adipose-derived mesenchymal stem cell exosomes, comprising the following steps:

[0054] (1) Extracting and isolating primary adipose-derived mesenchymal stem cells from the patient’s autologous fat samples:

[0055] (2) The primary adipose-derived mesenchymal stem cells obtained in step (1) were inoculated in a T75 culture flask at a density of 4400 cells / cm 2 , primary culture was carried out after supplementing the culture medium; the culture temperature was 38°C and the carbon dioxide concentration was 6%.

[0056] The culture medium is composed of the following components: 0.5 g / L L-isoleucine, 2 g / L L-valine, 2 g / L glycine, 9 mM β-glycerophosphate, 9 g / L glutathione, 8 ng / mL platelet-derived growth factor, 6 ng / mL EGF, 11 ng / mL bFGF, 8 ng / mL fibroblast growth factor, and the rest is DMEM-F12 culture medium;

[0057] (3) When the cell density in step (2) reaches 80%, the cell culture medium is collected and centrifuged to obtain adipose stem cell exosomes;

[0058] The centrifugation method is as follows: centrifuging the cell culture medium at 250×g for 16 min, 2100×g for 10 min, and 11000×g for 35 min to remove cells and cell debris; continuing to centrifuge at 100000×g for 75 min, resuspending and washing the sediment at the bottom, and finally centrifuging at 10000×g for 75 min to remove impurities to obtain adipose mesenchymal stem cell exosomes.

[0059] Example 3

[0060] A method for preparing adipose-derived mesenchymal stem cell exosomes, comprising the following steps:

[0061] (1) Extracting and isolating primary adipose-derived mesenchymal stem cells from the patient’s autologous fat samples:

[0062] (2) The primary adipose-derived mesenchymal stem cells obtained in step (1) were inoculated into a T75 culture flask at a density of 4300 cells / cm 2 , primary culture was carried out after supplementing the culture medium; the culture temperature was 37°C and the carbon dioxide concentration was 5%.

[0063] The culture medium is composed of the following components: 0.4 g / L L-isoleucine, 1.5 g / L L-valine, 1.5 g / L glycine, 8 mM β-glycerophosphate, 8 g / L glutathione, 6 ng / mL platelet-derived growth factor, 5 ng / mL EGF, 10 ng / mL bFGF, 6 ng / mL fibroblast growth factor, and the rest is DMEM-F12 culture medium;

[0064] (3) When the cell density in step (2) reaches 75%, the cell culture medium is collected and centrifuged to obtain adipose stem cell exosomes;

[0065] The centrifugation method is as follows: centrifuging the cell culture medium at 230×g for 14 min, 2000×g for 8 min, and 10000×g for 30 min to remove cells and cell debris; continuing to centrifuge at 100000×g for 70 min, resuspending and washing the sediment at the bottom, and finally centrifuging at 10000×g for 70 min to remove impurities to obtain adipose mesenchymal stem cell exosomes.

[0066] Example 4

[0067] A pharmaceutical composition for treating urinary incontinence, prepared from the following components: 3×10 adipose-derived mesenchymal stem cell exosomes prepared in Example 1 10 / mL, platelet-rich plasma 0.03mL / mL, rutin 7mg / mL, astragaloside 7mg / mL, Alpinia oxyphylla extract 12mg / mL, human albumin 30mg / mL, sodium bisulfite 3mg / mL, sorbitol 15mg / mL, and the rest is 0.9% sodium chloride water for injection;

[0068] The preparation method of the Alpinia oxyphylla extract is as follows:

[0069] (1) Grinding Alpinia oxyphylla seeds into 40 meshes to obtain material 1;

[0070] (2) Acetone with a volume concentration of 25% and ethyl acetate with a volume concentration of 15% are mixed in a volume ratio of 8:6 to obtain an extractant.

[0071] (2) The material 1 and the extractant were mixed and extracted at a mass volume ratio of 1 g:6 mL for 40 seconds to obtain an extractant layer, and the solvent was removed by reduced pressure distillation. The extract was freeze-dried at -35°C for 24 hours to obtain an Alpinia oxyphylla extract.

[0072] The preparation method of the pharmaceutical composition comprises mixing water for injection, sodium bisulfite, sorbitol, rutin, astragaloside and Alpinia oxyphylla extract, and then sequentially adding human serum albumin, platelet-rich plasma and adipose-derived mesenchymal stem cell exosomes for mixing.

[0073] Example 5

[0074] A pharmaceutical composition for treating urinary incontinence, prepared from the following components: 5×10 adipose-derived mesenchymal stem cell exosomes prepared in Example 2 10 / mL, platelet-rich plasma 0.05mL / mL, rutin 11mg / mL, astragaloside 11mg / mL, Alpinia oxyphylla extract 16mg / mL, human albumin 40mg / mL, sodium bisulfite 5mg / mL, sorbitol 25mg / mL, and the rest is 0.9% sodium chloride water for injection;

[0075] The preparation method of the Alpinia oxyphylla extract is as follows:

[0076] (1) grinding Alpinia oxyphylla seeds into 50 meshes to obtain material 1;

[0077] (2) Acetone with a volume concentration of 30% and ethyl acetate with a volume concentration of 20% are mixed in a volume ratio of 12:10 to obtain an extractant.

[0078] (2) The material 1 and the extractant were mixed and extracted at a mass volume ratio of 1 g: 8 mL for 50 seconds to obtain an extractant layer, and the solvent was removed by reduced pressure distillation, and the extract was freeze-dried at -45°C for 34 hours to obtain an Alpinia oxyphylla extract.

[0079] The preparation method of the pharmaceutical composition comprises mixing water for injection, sodium bisulfite, sorbitol, rutin, astragaloside and Alpinia oxyphylla extract, and then sequentially adding human serum albumin, platelet-rich plasma and adipose-derived mesenchymal stem cell exosomes for mixing.

[0080] Example 6

[0081] A pharmaceutical composition for treating urinary incontinence, prepared from the following components: 4×10 adipose-derived mesenchymal stem cell exosomes prepared in Example 3 10 / mL, platelet-rich plasma 0.04mL / mL, rutin 9mg / mL, astragaloside 9mg / mL, Alpinia oxyphylla extract 14mg / mL, human albumin 35mg / mL, sodium bisulfite 4mg / mL, sorbitol 20mg / mL, and the rest is 0.9% sodium chloride water for injection;

[0082] The preparation method of the Alpinia oxyphylla extract is as follows:

[0083] (1) Grinding Alpinia oxyphylla seeds into 45 meshes to obtain material 1;

[0084] (2) Acetone with a volume concentration of 28% and ethyl acetate with a volume concentration of 18% are mixed in a volume ratio of 10:8 to obtain an extractant.

[0085] (2) The material 1 and the extractant were mixed at a mass volume ratio of 1 g:7 mL and extracted for 45 seconds to obtain an extractant layer, and the solvent was removed by reduced pressure distillation, and the mixture was freeze-dried at -40°C for 29 hours to obtain an Alpinia oxyphylla extract.

[0086] The preparation method of the pharmaceutical composition comprises mixing water for injection, sodium bisulfite, sorbitol, rutin, astragaloside and Alpinia oxyphylla extract, and then sequentially adding human serum albumin, platelet-rich plasma and adipose-derived mesenchymal stem cell exosomes for mixing.

[0087] Example 7

[0088] A pharmaceutical composition for treating urinary incontinence, prepared from the following components: 3×10 adipose-derived mesenchymal stem cell exosomes prepared in Example 110 / mL, platelet-rich plasma 0.03mL / mL, rutin 7mg / mL, astragaloside 7mg / mL, Alpinia oxyphylla extract 12mg / mL, human albumin 30mg / mL, sodium bisulfite 3mg / mL, sorbitol 15mg / mL, and the rest is 0.9% sodium chloride water for injection;

[0089] The preparation method of the Alpinia oxyphylla extract is as follows:

[0090] (1) Grinding Alpinia oxyphylla seeds into 40 meshes to obtain material 1;

[0091] (2) Acetone with a volume concentration of 25% and ethyl acetate with a volume concentration of 15% are mixed in a volume ratio of 8:6 to obtain an extractant.

[0092] (2) The material 1 and the extractant were mixed and extracted at a mass volume ratio of 1 g:6 mL for 40 seconds to obtain an extractant layer, and the solvent was removed by reduced pressure distillation. The extract was freeze-dried at -35°C for 24 hours to obtain an Alpinia oxyphylla extract.

[0093] The preparation method of the pharmaceutical composition comprises mixing water for injection, sodium bisulfite, sorbitol, rutin, astragaloside, Alpinia oxyphylla extract, urapidil and tolterodine tartrate, and then sequentially adding human serum albumin, platelet-rich plasma and adipose-derived mesenchymal stem cell exosomes and mixing the mixture.

[0094] Example 8

[0095] A pharmaceutical composition for treating urinary incontinence, prepared from the following components: 5×10 adipose-derived mesenchymal stem cell exosomes prepared in Example 2 10 / mL, platelet-rich plasma 0.05mL / mL, rutin 11mg / mL, astragaloside 11mg / mL, Alpinia oxyphylla extract 16mg / mL, human serum albumin 40mg / mL, sodium bisulfite 5mg / mL, sorbitol 25mg / mL, urapidil 3mg / mL, tolterodine tartrate 6mg / mL, and the rest is 0.9% sodium chloride water for injection;

[0096] The preparation method of the Alpinia oxyphylla extract is as follows:

[0097] (1) grinding Alpinia oxyphylla seeds into 50 meshes to obtain material 1;

[0098] (2) Acetone with a volume concentration of 30% and ethyl acetate with a volume concentration of 20% are mixed in a volume ratio of 12:10 to obtain an extractant.

[0099] (2) The material 1 and the extractant were mixed and extracted at a mass volume ratio of 1 g: 8 mL for 50 seconds to obtain an extractant layer, and the solvent was removed by reduced pressure distillation, and the extract was freeze-dried at -45°C for 34 hours to obtain an Alpinia oxyphylla extract.

[0100] The preparation method of the pharmaceutical composition comprises mixing water for injection, sodium bisulfite, sorbitol, rutin, astragaloside, Alpinia oxyphylla extract, urapidil and tolterodine tartrate, and then sequentially adding human serum albumin, platelet-rich plasma and adipose-derived mesenchymal stem cell exosomes and mixing the mixture.

[0101] Example 9

[0102] A pharmaceutical composition for treating urinary incontinence, prepared from the following components: 4×10 adipose-derived mesenchymal stem cell exosomes prepared in Example 3 10 / mL, platelet-rich plasma 0.04mL / mL, rutin 9mg / mL, astragaloside 9mg / mL, Alpinia oxyphylla extract 14mg / mL, human serum albumin 35mg / mL, sodium bisulfite 4mg / mL, sorbitol 20mg / mL, urapidil 2.5mg / mL, tolterodine tartrate 5mg / mL, and the rest is 0.9% sodium chloride water for injection;

[0103] The preparation method of the Alpinia oxyphylla extract is as follows:

[0104] (1) Grinding Alpinia oxyphylla seeds into 45 meshes to obtain material 1;

[0105] (2) Acetone with a volume concentration of 28% and ethyl acetate with a volume concentration of 18% are mixed in a volume ratio of 10:8 to obtain an extractant.

[0106] (2) The material 1 and the extractant were mixed at a mass volume ratio of 1 g:7 mL and extracted for 45 seconds to obtain an extractant layer, and the solvent was removed by reduced pressure distillation, and the mixture was freeze-dried at -40°C for 29 hours to obtain an Alpinia oxyphylla extract.

[0107] The preparation method of the pharmaceutical composition comprises mixing water for injection, sodium bisulfite, sorbitol, rutin, astragaloside, Alpinia oxyphylla extract, urapidil and tolterodine tartrate, and then sequentially adding human serum albumin, platelet-rich plasma and adipose-derived mesenchymal stem cell exosomes and mixing the mixture.

[0108] Comparative Example 1

[0109] The other methods were the same as those in Example 9, except that no rutin was added.

[0110] Comparative Example 2

[0111] The other methods are the same as those in Example 9, except that, during the preparation of the Alpinia oxyphylla extract, the extractant is replaced with an equal amount of ethyl acetate having a volume concentration of 18%, and no acetone is added.

[0112] Comparative Example 3

[0113] The other methods were the same as those in Example 9, except that adipose-derived mesenchymal stem cell exosomes were not added.

[0114] Experimental Example 1

[0115] Animal experiment: C57BL / 6 female nulliparous rats, 8-11 weeks old, 18-22 g were selected to prepare the urinary incontinence model.

[0116] Preparation of urinary incontinence model: Intramuscular injection of botulinum toxin A and subcutaneous electrical stimulation of the abdominal area of ​​female rats were used to increase abdominal pressure, thereby expressing urinary incontinence. A total of 48 rats with successful urinary incontinence model were prepared.

[0117] The experimental groups are as follows: the product obtained in Example 6 is used as experimental group 1; the product obtained in Example 9 is used as experimental group 2; the products obtained in comparative examples 1 to 3 are used as control groups 1 to 3. The specific injection dose is: 0.3 mL / 100 g body weight.

[0118] Model group: injected with an equal volume of normal saline.

[0119] Rats in the blank control group did not receive any treatment.

[0120] The frequency of urinary incontinence in each group was counted before administration, 30 minutes after administration, and 2 hours after administration. The results are shown in Table 1.

[0121] Table 1 Frequency of urinary incontinence

[0122] Group Before administration 30min 120min Experimental Group 1 8 5 4 Experimental Group 2 8 4 1 Control group 1 8 5 3 Control group 2 8 4 2 Control group 3 8 5 3 Blank control group 0 0 0 Model Group 8 8 8

[0123] It can be seen from the contents recorded in Table 2 that after the pharmaceutical composition of the present invention was administered to rats, the frequency of urinary incontinence in rats was significantly reduced. It can be seen from the data of the control group that only by adopting the complete formulation of the present invention can the best use effect be achieved, and abandoning a certain component will significantly affect the therapeutic effect of the drug.

[0124] Example 2

[0125] Clinical application experiment of autologous PRP in the treatment of urethral sphincter injury.

[0126] A total of 60 patients with urinary incontinence caused by prostate cancer surgery diagnosed by clinicians from March 2022 to December 2024 were selected and randomly divided into 6 groups.

[0127] Selection criteria for experimental subjects: Physical examination, urine pad test, cystourethroscopy, and urodynamic examination were used to determine whether the patients had urinary incontinence caused by prostate cancer surgery.

[0128] Exclusion criteria for experimental subjects: patients with prostate cancer who have received medical endocrine therapy; patients with imaging evidence showing multiple metastases of prostate cancer throughout the body.

[0129] Among them, the product obtained in Example 6 is used as experimental group 1; the product obtained in Example 9 is used as experimental group 2; and the products obtained in comparative examples 1 to 3 are used as control groups 1 to 3.

[0130] Patients with postoperative urinary incontinence were given a single perineal subcutaneous injection at six points within the anatomical position of the urethral sphincter, with 1 mL injected into each point once a day for a total of 21 days.

[0131] Prior art group: taking tolterodine tartrate, 2 mg once, twice a day.

[0132] On the 0th, 7th, 14th and 21st days of treatment, the scores of different patients in different experimental groups were counted using the International Consultative Committee on Incontinence Questionnaire (ICI-Q-SF), and the average values ​​were taken. The results are shown in Table 2.

[0133] Table 2 Urinary incontinence scores

[0134] Group 0d 7d 14d 21d Experimental Group 1 8.35 6.84 5.62 4.35 Experimental Group 2 8.56 5.73 3.54 2.86 Control group 1 8.21 6.19 4.58 4.17 Control group 2 8.34 5.94 3.95 3.52 Control group 3 8.63 6.55 4.77 4.26 Existing Technology Group 8.44 5.91 3.75 3.33

[0135] It can be seen from the data recorded in Table 2 that the pharmaceutical composition provided by the present invention can significantly reduce the evaluation score of patients with urinary incontinence, and the evaluation score is significantly better than the drugs in the prior art group, which shows that the composition has an excellent technical effect in treating urinary incontinence.

[0136] It can be seen from the data of the control group that if some of the components are discarded or the Alpinia oxyphylla extract is not prepared according to the uninventive preparation method, it will have a significant impact on the therapeutic efficacy of the pharmaceutical composition.

[0137] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating urinary incontinence, characterized in that: Includes the following components: Adipose-derived mesenchymal stem cell exosomes 3×10 10 ~5×10 10 / mL, platelet-rich plasma 0.03-0.05mL / mL, rutin 7-11mg / mL, astragaloside 7-11mg / mL, Alpinia oxyphylla extract 12-16mg / mL, human serum albumin 30-40mg / mL, sodium bisulfite 3-5mg / mL, sorbitol 15-25mg / mL, and the rest is water for injection.

2. A pharmaceutical composition for treating urinary incontinence according to claim 1, characterized in that: The water for injection is 0.9% sodium chloride water for injection.

3. A pharmaceutical composition for treating urinary incontinence according to claim 1 or 2, characterized in that: The preparation method of the Alpinia oxyphylla extract is as follows: (1) grinding Alpinia oxyphylla seeds to obtain material 1; (2) The material 1 is mixed with an extractant for extraction to obtain an extractant layer, the solvent is removed by reduced pressure distillation, and the extract is freeze-dried to obtain an Alpinia oxyphylla extract.

4. A pharmaceutical composition for treating urinary incontinence according to claim 3, characterized in that: The extracting agents in step (2) are acetone and ethyl acetate, and the volume ratio is 8-12:6-10; the volume concentration of the acetone is 25-30%; and the volume concentration of the ethyl acetate is 15-20%.

5. A pharmaceutical composition for treating urinary incontinence according to claim 3, characterized in that: The extraction time in step (2) is 40 to 50 seconds.

6. A pharmaceutical composition for treating urinary incontinence according to claim 3, characterized in that: In step (2), the material 1 is mixed with the extractant in a mass volume ratio of 1 g: 6-8 mL.

7. A pharmaceutical composition for treating urinary incontinence according to claim 3, characterized in that: The freeze-drying temperature of step (2) is -35 to -45°C, and the freeze-drying time is 24 to 34 hours.

8. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition further contains 2-3 mg / mL of urapidil and 4-6 mg / mL of tolterodine tartrate.

9. A method for preparing a pharmaceutical composition for treating urinary incontinence according to any one of claims 1 to 8, characterized in that: Mix water for injection, sodium bisulfite, sorbitol, rutin, astragaloside, Alpinia oxyphylla extract, urapidil, and tolterodine tartrate, and then add human serum albumin, platelet-rich plasma, and adipose-derived mesenchymal stem cell exosomes in sequence and mix.

10. Use of the pharmaceutical composition according to any one of claims 1 to 8 or the pharmaceutical composition prepared by the preparation method according to claim 9 in the preparation of drugs for treating urinary incontinence.

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