Preparation method and application of temperature-sensitive injection for treating stress urinary incontinence

Abstract

The invention discloses a preparation method and application of a temperature-sensitive injection for treating stress urinary incontinence, hydrogel and adipose-derived stem cells are combined in the injection to enhance the strength of urethral tissues and regeneration of sphincter, and meanwhile, metal organic framework type nanoparticles are matched to promote urethral nerve repair. The phenomena of urinary incontinence, recurrence and the like caused by gradual inactivation of sphincter are reduced.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a preparation method of a temperature-sensitive injection for treating stress urinary incontinence, and the application of the prepared injection in the preparation of a drug for treating stress urinary incontinence. Background Art

[0002] The clinical treatment of stress urinary incontinence (SUI) mostly focuses on reconstructing the suburethral support, and there is no good therapeutic effect on the essential disease development nature and pathological mechanism of SUI, that is, the atrophy of local nerves and muscles of the urethral sphincter in patients.

[0003] At present, some researchers have combined biocompatible hydrogels with muscle cells to enhance the strength of the tissues around the urethra and sphincter regeneration, effectively improving the urination function. However, in addition to muscle atrophy, the atrophy of the nerves around the urethral sphincter is also an important cause of sphincter dysfunction leading to involuntary urination. Although only targeting sphincter regeneration can improve sphincter function and treat SUI, in terms of long-term efficacy, because the atrophy of nerves is largely unresolved, the gradual inactivation of the sphincter and recurrence of urinary incontinence may still occur and need to be improved. Summary of the Invention

[0004] To solve the above at least one technical defect, the present invention provides the following technical solutions:

[0005] The first aspect of this application discloses a preparation method of a temperature-sensitive injection for treating stress urinary incontinence, including the following steps:

[0006] A step of synthesizing a hydrogel, reacting carboxyl-modified temperature-sensitive poly(N-isopropylacrylamide) with leucine and acellular adipose-derived stem cell membrane extracellular matrix powder to obtain a hydrogel;

[0007] A step of synthesizing metal-organic framework type nanoparticles, doping Mg 2+ into polyethylene glycol-modified ZIF-8 to obtain the required nanoparticles;

[0008] A step of mixing the hydrogel with adipose-derived stem cells pre-phagocytosing metal-organic framework type nanoparticles to obtain the injection.

[0009] In the injection, the hydrogel and adipose-derived stem cells are combined to enhance the strength of the urethral tissue and the regeneration of the sphincter, and at the same time, metal-organic framework type nanoparticles are used to cooperate to promote urethral nerve repair and reduce phenomena such as the gradual inactivation of the sphincter leading to recurrence of urinary incontinence.

[0010] Among them, polyethylene glycol-modified ZIF-8 can reduce the degree of aggregation and improve the cell phagocytosis rate. The nanoparticles formed under magnesium doping can play a role in programming the promotion of neurogenesis in adipose-derived stem cells, promoting the repair and regeneration of urethral nerves.

[0011] Further, in the synthesis step of the hydrogel, the monomers N-isopropylacrylamide and tert-butyl acrylate are copolymerized and hydrolyzed under acidic conditions to obtain carboxyl-modified thermosensitive poly(N-isopropylacrylamide). The ester is hydrolyzed under acidic conditions while the amide is retained.

[0012] Further, the molar ratio of the monomers N-isopropylacrylamide and tert-butyl acrylate is 30:1 - 10.

[0013] Further, the preparation of the extracellular matrix powder of decellularized adipose-derived stem cell sheets is as follows: Subcutaneous adipose tissue is taken and incubated in a solution containing trypsin and collagenase. The adipose-derived stem cells obtained after centrifugation are cultured to form cell sheets. The cell sheets are incubated in a Tris buffer solution containing Triton X-100, EDTA, and aprotinin to obtain the extracellular matrix of decellularized adipose-derived stem cell sheets, which is then freeze-dried and ground into powder.

[0014] Further, the mass ratio of carboxyl-modified thermosensitive poly(N-isopropylacrylamide), leucine, and the extracellular matrix powder of decellularized adipose-derived stem cell sheets is 3 - 6:1.5 - 2.5:1.5 - 2.5.

[0015] Further, in the synthesis step of the metal-organic framework nanoparticles, zinc nitrate hexahydrate, methanol, and 2-methylimidazole are mixed. Then, polyethylene glycol is added to the mixture and stirred. After solid-liquid separation, polyethylene glycol-modified ZIF-8 is obtained. The polyethylene glycol-modified ZIF-8 is added to a magnesium chloride solution and stirred. After solid-liquid separation, the required nanoparticles are obtained. The whole process has the advantages of simplicity and easy operation.

[0016] Further, the metal-organic framework nanoparticles are co-cultured with adipose-derived stem cells at a concentration of 20 - 60 μg / mL for the adipose-derived stem cells to pre-phagocytose. The hydrogel and the adipose-derived stem cells that have phagocytosed the metal-organic framework nanoparticles are mixed at a ratio of 80 - 1.2 million per ml.

[0017] The second aspect of this application discloses the use of the thermosensitive injection prepared by the above-mentioned preparation method in the preparation of a drug for the treatment of stress urinary incontinence. This drug has a significant effect in the treatment of urinary incontinence.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. The injection of the present invention integrates a hydrogel, metal-organic framework nanoparticles, and adipose-derived stem cells, which can promote the enhancement of the strength of urethral tissue, the regeneration of the sphincter, and the repair of urethral nerves, etc., and reduce the recurrence of urinary incontinence and other phenomena. Description of the Drawings

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is a schematic diagram of the synthesis process of the hydrogel in this injection.

[0022] Figure 2 It is a staining diagram of the urethral sphincter.

[0023] Among them, Figure 2 In each small figure, the middle part of the illustration is an enlarged view of the frame in the upper figure, and the lower part of the illustration is an enlarged view of the frame in the middle part of the illustration. Specific embodiments

[0024] The following further illustrates the present invention in conjunction with the drawings and specific embodiments.

[0025] Embodiment 1

[0026] A preparation method of a thermosensitive injection for treating stress urinary incontinence, comprising the following steps:

[0027] First, the synthesis step of the hydrogel is as follows Figure 1 As shown, a carboxyl-modified thermosensitive poly(N-isopropylacrylamide) is reacted with leucine and acellular adipose-derived stem cell membrane extracellular matrix powder to obtain a hydrogel.

[0028] The specific preparation process is as follows:

[0029] The chain transfer agent 4-cyano-4-(phenylthiocarbonothioylthio)valeric acid (CPAD), the initiator azobisisobutyronitrile (AIBN), the monomer N-isopropylacrylamide (NIPAm) and tert-butyl acrylate (t-BA) are added to the solution 1,4-dioxane in a molar ratio of 1:0.5:30:5, degassed and deoxygenated, and reacted in an oil bath at 70 °C for 12 h. After the reaction, it is precipitated with anhydrous ether and dried in vacuo to obtain a copolymer.

[0030] 1.0 g of the above copolymer is dissolved in dichloromethane, and 0.05 mol of trifluoroacetic acid is added to the solution, and the reaction is stirred at room temperature for 48 h. Then the reaction solution is fully rotary evaporated to completely remove dichloromethane and trifluoroacetic acid, and finally a carboxyl-modified thermosensitive poly(N-isopropylacrylamide) is obtained, denoted as PNIPAm-C, where PNIPAm refers to thermosensitive poly(N-isopropylacrylamide).

[0031] We found by nuclear magnetic resonance spectroscopy (NMR) that compared with PNIPAm, a new peak appeared in the 1H NMR spectrum of PNIPAm-C at 10.1 ppm, indicating that PNIPAm-C is rich in carboxyl groups.

[0032] The preparation of acellular adipose-derived stem cell sheet extracellular matrix powder is as follows:

[0033] Take the subcutaneous adipose tissue at the groin of 4-week-old female SD rats. After thoroughly cutting it with scissors, add it to a solution containing 0.25% trypsin and 0.1% collagenase and incubate at 37 °C for 1 h. Seed the centrifuged adipose-derived stem cells (ADSCs) in a culture dish and culture them in a low-glucose medium containing 15% fetal bovine serum and 1% penicillin / streptomycin (the ratio of penicillin to streptomycin is 1:1). The medium is changed every two days. When the cells reach 80%-90% confluence, add vitamin C to the medium, and the addition amount of vitamin C is 10 mg / ml for forming cell sheets. After culturing for 10 days, rinse the obtained ADSCs sheets three times with phosphate buffer saline (PBS), then incubate and shake in 10 mM Tris buffer containing 1% Triton X-100, 0.02% EDTA, and 1 μg / mL aprotinin for 24 h, and wash with deionized water to finally obtain acellular adipose-derived stem cell sheet extracellular matrix (ADSCs sheets dECM). Finally, freeze-dry the ADSCs sheets dECM and grind it into powder, that is, acellular adipose-derived stem cell sheet extracellular matrix. After sterilization by irradiation with C060γ rays, it is sealed and stored at -80 °C.

[0034] Synthesis of PNIPAm-C / Leucine / dECM hydrogel:

[0035] Add carboxyl-modified thermosensitive poly(N-isopropylacrylamide) to 2% wt of leucine. The mass ratio of carboxyl-modified thermosensitive poly(N-isopropylacrylamide) to leucine is 5:2. Stir at room temperature for 1 h, then add acellular adipose-derived stem cell sheet extracellular matrix powder. The mass ratio of acellular adipose-derived stem cell sheet extracellular matrix powder to carboxyl-modified thermosensitive poly(N-isopropylacrylamide) is 2:5, and add EDC / NHS. The concentration of EDC: 1% wt, the concentration of NHS: 0.5% wt. Stir and react at room temperature for 12 h to obtain the required PNIPAm-C / Leucine / dECM hydrogel, where EDC refers to 1-ethyl-(3-dimethylaminopropyl)carbodiimide and NHS refers to N-hydroxysuccinimide.

[0036] Second, the synthesis steps of metal-organic framework nanoparticles. Add Mg 2+Doped into polyethylene glycol-modified ZIF-8 to obtain the desired nanoparticles.

[0037] The specific preparation process is as follows:

[0038] Preparation of ZIF-8 / PEG: Take 1.098 g of zinc nitrate hexahydrate (Zn(NO 3 ) 2 ·6H 2 O) and 36 ml of methanol, place them in conical flask one and stir for 10 min. Take 2.443 g of 2-methylimidazole (C 4 H 6 N 2 ) and 60 ml of methanol, place them in conical flask two and stir for 10 min. Slowly add the solution in conical flask one to conical flask two, and continue to stir for 1 h. Then add 60 mL of PEG-200, stir for 1 h, let stand for 24 h, centrifuge, and wash with methanol 3 times to obtain ZIF-8 / PEG, that is, polyethylene glycol-modified ZIF-8.

[0039] Take 2 g of anhydrous magnesium chloride, add it to 20 mL of water, and stir for 10 min. Add 0.5 g of the prepared ZIF-8 / PEG powder, stir for 1 h, centrifuge, and obtain ZIF-8 / PEG@Mg, that is, metal-organic framework type nanoparticles. It is found by X-ray diffractometer (XRD) detection that the 003 crystal plane characteristic peak of MgCl 2 appears in ZIF-8 / PEG200@Mg. Through lens and elemental mapping analysis and processing, it is found that magnesium elements are evenly distributed on the surface and inside of ZIF-8 / PEG200@Mg particles.

[0040] Third, the step of mixing the hydrogel with adipose stem cells pre-phagocytosing metal-organic framework type nanoparticles to obtain the injection.

[0041] The specific preparation process is as follows:

[0042] The metal-organic framework type nanoparticles (ZIF-8 / PEG@Mg) are co-cultured with adipose stem cells (ADSCs) at a concentration of 40 μg / mL for 48 h for adipose stem cells to pre-phagocytose. Mix the PNIPAm-C / Leucine / dECM hydrogel and adipose stem cells that have phagocytosed metal-organic framework type nanoparticles at a ratio of 1 million per ml, and gently stir for 30 s. Avoid too long time to cause cell death, and make the cells evenly distributed in the hydrogel to obtain the desired injection.

[0043] Detect the product obtained in Example 1 as follows:

[0044] First, detection of the effect of ZIF-8 / PEG200@Mg on the pro-neural programming of ADSCs

[0045] The cultured ADSCs were starved for 12 h and mixed with ZIF-8 / PEG200@Mg at concentrations of 5, 10, 20, 40, and 80 μg / mL according to the groups. Using ZIF-8 / PEG200 as a control in the same treatment method, for ADSCs phagocytosing ZIF-8 / PEG200@Mg at a concentration of 40 μg / mL, and for ADSCs phagocytosing ZIF-8 / PEG200 at a concentration of 40 μg / mL -1 transcriptome sequencing was performed. The results showed that the ADSCs treated with ZIF-8 / PEG200@Mg showed high expression in pathways such as nervous system regeneration, neurogenesis, and actin generation. The high expression of the mineral absorption, actin cytoskeleton regulation, and axon guidance signaling pathways further illustrated that ZIF-8 / PEG200@Mg achieved a pro-neurogenic programming effect on ADSCs. Through reverse transcription polymerase chain reaction (qRT-PCR) and Western blot (WB), it was found that the expression of key genes related to the extracellular matrix (Itga2 and Clasp2), nervous system (Atxn7, Kirrel3, Nptxr, Fez1, Ntn1, and Plxnb1), and actin (Wasf2 and Acta2) all increased significantly. However, the genes expressed in the ADSCs treated with ZIF-8 / PEG200 were mainly enriched in pathways such as extracellular matrix secretion, cell migration, and response to zinc ions. -1 concentration of ZIF-8 / PEG200@Mg, and for ADSCs phagocytosing ZIF-8 / PEG200 at a concentration of 40 μg / mL -1 concentration of ZIF-8 / PEG200 were subjected to transcriptome sequencing. The results showed that the ADSCs treated with ZIF-8 / PEG200@Mg showed high expression in pathways such as nervous system regeneration, neurogenesis, and actin generation. The high expression of the mineral absorption, actin cytoskeleton regulation, and axon guidance signaling pathways further illustrated that ZIF-8 / PEG200@Mg achieved a pro-neurogenic programming effect on ADSCs. Through reverse transcription polymerase chain reaction (qRT-PCR) and Western blot (WB), it was found that the expression of key genes related to the extracellular matrix (Itga2 and Clasp2), nervous system (Atxn7, Kirrel3, Nptxr, Fez1, Ntn1, and Plxnb1), and actin (Wasf2 and Acta2) all increased significantly. However, the genes expressed in the ADSCs treated with ZIF-8 / PEG200 were mainly enriched in pathways such as extracellular matrix secretion, cell migration, and response to zinc ions.

[0046] Second, detection of the ability of the obtained injection to restore the urethral sphincter function and promote muscle regeneration in SUI rats

[0047] (1) Establishment of an animal model of SUI rats

[0048] We selected female SD rats at 4 weeks of age and constructed an SUI model by vaginal balloon dilation and bilateral ovariectomy to simulate the causes of the vast majority of SUI patients in clinical practice. Rats 4 weeks after modeling were subjected to leak point pressure (LPP) detection to verify whether the modeling was successful. Briefly, a polyethylene-90 catheter was inserted from the bladder dome, and then the bladder was continuously filled with sterile normal saline containing methylene blue. At the same time, the increase in bladder pressure was recorded using a pressure sensor until urine leakage occurred; the pressure at the time of urine leakage was regarded as LPP.

[0049] (2) Injection treatment of the urethral sphincter

[0050] Forty SUI rats with successful modeling were divided into five groups. Different injection systems - saline (SUI group), PNIPAm-C (PC group), PC + leucine + ADSCs sheets dECM powder (PCdL group), PCdL + ADSCs + ZIF-8 / PEG200 (PCdL / ADSC(PEG) group) and PCdL + ADSCs + ZIF-8 / PEG200@Mg (PCdL / ADSC(Mg) group) were injected around the urethral sphincter of rats for treatment.

[0051] (3) Detection of LPP and urethral sphincter section staining in different treatment groups

[0052] Since muscle tissue regeneration, functional recovery and neurogenesis require a certain amount of time, LPP detection and histological staining evaluation of the urethral sphincter were selected at 4W and 8W after treatment. Four weeks after injection treatment, the LPP of the PCdL / ADSC(Mg) group recovered to near normal levels (P > 0.05), and was significantly higher than that of the SUI group. The LPP of the PC group was also slightly increased compared with the SUI group, which may be due to the swelling agent provided by the PC injection system. Eight weeks after injection treatment, the LPP of the PCdL / ADSC(Mg) group was still very high, and there was a slight increase compared with 4W. This may be because ADSCs programmed by ZIF-8 / PEG200@Mg can slowly promote local neurogenesis and NMJ recovery, and this phenomenon also confirms the long-term efficacy of the PCdL / ADSC(Mg) injection system.

[0053] Next, we evaluated the local tissue condition and muscle regeneration level of the urethral sphincter at the bladder neck through tissue section staining. HE staining showed that the urethral sphincters of rats in the SUI and PC groups were severely atrophied, the local tissue was loose and disorderly arranged, and the muscle fibers were thin and irregular ( Figure 2 A). In addition, the muscle staining was lighter and the color of the muscle fibers became lighter. Due to the leucine and rich cytokines contained in PCdL, muscle atrophy was improved, the muscle fibers were relatively large and neat, and the density was significantly increased. This result was consistent with the LPP detection result. Furthermore, muscle regeneration in the PCdL / ADSC(PEG) and PCdL / ADSC(Mg) groups was very obvious, and was basically the same as that of the normal group. The myofibrils were red or pink, the boundaries of the muscle fibers were clear, the structure was tight, and the morphology was regular. Compared with the staining results 4 weeks after treatment, more obvious muscle regeneration effects were shown in the urethral sphincter 8 weeks later. Similarly, the Masson staining results showed the same trend ( Figure 2B). The muscle fibers in the SUI and PC groups faded to light red, showing small, degenerated muscle fibers. The PCdL group showed obvious, darker muscle fibers, and the regeneration of smooth muscle was more obvious than that of striated muscle. The muscle fibers in the PCdL / ADSC(Mg) group were red, brighter in color, larger in diameter, and tightly arranged, with less connective tissue.

[0054] Immunofluorescence staining was used to track Desmin, α-SMA, Synaptophys in, TUBB3, and VEGF to evaluate muscle regeneration, NMJ formation, neurogenesis, and angiogenesis, respectively.

[0055] Among them, we performed immunofluorescence staining of muscle-related indicators on the urethral sphincter at 4 and 8 weeks after injection treatment in different groups. The results showed that Desmin and α-SMA expression was low in the SUI and PC groups, which was consistent with the results of HE and Masson staining, indicating severe muscle atrophy. Although the fluorescence expression in the PCdL group increased significantly, it was not as good as that in the PCdL / ADSC(PEG) and PCdL / ADSC(Mg) groups. This shows that ADSCs programmed by nanoparticle MOF can significantly promote the regeneration of smooth muscle and striated muscle of the urethral sphincter.

[0056] VEGF staining showed the ability of the injection to promote muscle angiogenesis, and the fluorescence intensity was the highest in the PCdL / ADSC(Mg) group, indicating that adipose stem cells programmed by nanoparticle MOF can promote the vascularization of muscle tissue in vivo.

[0057] Synaptophys in and TUBB3 staining results highlight the excellent neurogenesis-promoting effect of ZI F-8 / PEG200@Mg. The fluorescence intensity of the PCdL / ADSC(Mg) group was significantly higher than that of the PCdL / ADSC(PEG) group, and even close to the level of normal tissue. This is because the ADSCs programmed by ZI F-8 / PEG200@Mg played a powerful role in promoting neurogenesis and synaptic regeneration. The nerves and synaptic tissues repaired by the urethral sphincter provided nutrition and control functions to the muscles, further restoring the sphincter function.

[0058] We also stained Ache by immunohistochemistry. The neuromuscular junction (NMJ) is the contact point of the neuron axon terminal on the muscle fiber, which can cause muscle contraction and maintain a certain degree of muscle tension to inhibit muscle atrophy. Therefore, the regeneration and functional recovery of NMJ are crucial for the regeneration and functional recovery of the urethral sphincter. NMJ is a special chemical synapse, and its neurotransmitter is acetylcholine. As a key enzyme in biological nerve conduction, Ache can degrade acetylcholine between synapses and is used to assist in localizing NMJ. The results of AChe immunohistochemistry showed that there was almost no expression in the urethral sphincter of the SUI and PC groups. In the PCdL / ADSC(Mg) group, Ache had the highest expression in the tissue, showing local strong positivity, especially in the areas near the postsynaptic membrane and basement membrane, forming obvious granular or band-like staining.

[0059] In summary, this injection system can achieve the synergistic effect of urethral sphincter regeneration and neurogenesis, showing the role and mechanism of promoting neurogenesis. In vivo experiments confirmed that after PCdL / ADSC(Mg) injection treatment, the micturition ability of SUI rats can be significantly improved and the function of the urethral sphincter can be restored by promoting muscle regeneration, angiogenesis, neurogenesis and NMJ regeneration.

[0060] The above is only the preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the idea of the present invention belong to the protection scope of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a thermosensitive injection for treating stress urinary incontinence, characterized in that: The following steps are involved: The hydrogel synthesis step is to react carboxyl-modified thermosensitive poly (N-isopropylacrylamide) with leucine and decellularized adipose stem cell membrane extracellular matrix powder to obtain the hydrogel; The synthesis steps of metal organic framework nanoparticles are as follows: Mg 2+ Doped into polyethylene glycol-modified ZIF-8 to obtain the desired nanoparticles; The step of mixing the hydrogel with adipose stem cells that have pre-phagocytosed metal organic framework nanoparticles to obtain the injection.

2. The method for preparing the temperature-sensitive injection for treating stress urinary incontinence according to claim 1, characterized in that: In the synthesis step of the hydrogel, monomers N-isopropylacrylamide and tert-butyl acrylate are copolymerized and hydrolyzed under acidic conditions to obtain carboxyl-modified thermosensitive poly (N-isopropylacrylamide).

3. The method for preparing the temperature-sensitive injection for treating stress urinary incontinence according to claim 2, characterized in that: The molar ratio of the monomers N-isopropylacrylamide and tert-butyl acrylate is 30:1-10.

4. The method for preparing the thermosensitive injection for treating stress urinary incontinence according to claim 1, characterized in that: The preparation of the extracellular matrix powder of the decellularized adipose stem cell membrane sheet is as follows: subcutaneous adipose tissue is added to a solution containing pancreatin and collagenase for incubation, the adipose stem cells obtained after centrifugation are cultured to form a cell membrane sheet, the cell membrane sheet is added to a Tris buffer containing Triton X-100, EDTA and aprotinin for incubation to obtain the extracellular matrix of the decellularized adipose stem cell membrane sheet, and the extracellular matrix is ​​freeze-dried and ground into powder.

5. The method for preparing the thermosensitive injection for treating stress urinary incontinence according to claim 1, characterized in that: The mass ratio of carboxyl-modified thermosensitive poly (N-isopropylacrylamide) to leucine and decellularized adipose stem cell membrane extracellular matrix powder is 3-6:1.5-2.5:1.5-2.

5.

6. The method for preparing the thermosensitive injection for treating stress urinary incontinence according to claim 1, characterized in that: In the synthesis steps of metal organic framework nanoparticles, zinc nitrate hexahydrate, methanol and 2-methylimidazole are mixed, and then polyethylene glycol is added to the mixture and stirred. After solid-liquid separation, polyethylene glycol-modified ZIF-8 is obtained. The polyethylene glycol-modified ZIF-8 is added to a magnesium chloride solution and stirred, and the desired nanoparticles are obtained after solid-liquid separation.

7. The method for preparing the thermosensitive injection for treating stress urinary incontinence according to claim 1, characterized in that: The metal organic framework nanoparticles are co-cultured with adipose stem cells at a concentration of 20-60 μg / mL for pre-phagocytosis by the adipose stem cells. The hydrogel and the adipose stem cells that have phagocytosed the metal organic framework nanoparticles are mixed at a ratio of 800,000 to 1.2 million per ml.

8. Use of the thermosensitive injection prepared by the preparation method according to any one of claims 1 to 7 in the preparation of drugs for treating stress urinary incontinence.

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