Composition for treating premature ovarian failure as well as preparation method and application thereof

Abstract

The invention belongs to the cross technical field of regenerative medicine and traditional Chinese medicine, and discloses a composition for treating premature ovarian failure and a preparation method and application thereof. The composition comprises adult stem cells, traditional Chinese medicine active components consisting of astragaloside and salvianolic acid B in a molar ratio of (1: 1)-(4: 1) and a biodegradable slow-release carrier, and double intervention of'cell repair-microenvironment regulation and control 'is realized through a synergistic strategy of'pretreatment of stem cells by traditional Chinese medicines and targeted slow-release delivery'. Experiments prove that the composition can enable the estrus cycle recovery rate of premature ovarian failure model animals to reach 70%-90% and the number of follicles to be increased by 6-8 times, is obviously superior to the existing single therapy, has a clear mechanism (activating a PI3K / Akt pathway and inhibiting a TGF-beta1 / Smad pathway), and has outstanding clinical transformation value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a composition for treating premature ovarian failure and a preparation method and application thereof. BACKGROUND

[0002] Premature ovarian failure (POF) is a major disease that causes female infertility, and its pathological features include depletion of follicle pool, ovarian interstitial fibrosis and endocrine disorder. The existing treatment methods have significant limitations: 1. Hormone replacement therapy: only can maintain the basic estrogen level, cannot reverse the follicle damage, and long-term use increases the risk of breast cancer (HR=1.24, PMID:32105678); 2. Single stem cell transplantation: the survival rate of bone marrow mesenchymal stem cells (BMSCs) in the ovarian microenvironment is less than 15%, and the differentiation targeting is poor (patent CN114984028B); 3. Traditional Chinese medicine compound treatment: the effect of preparations such as Kuntai Capsules is slow, and the follicle activation rate for severe POF is less than 20%.

[0003] The existing combined treatment scheme has obvious defects: Patent CN113633754B discloses the combination of ovarian stem cells and active peptides of Radix Polygoni Multiflori, but does not involve Astragalus Membranaceus Glycosides / Danpeiru Acid B, and lacks a pretreatment step, and the stem cell colonization rate is only 23%; Patent CN114984028B uses Astragalus Membranaceus Glycosides combined with BMSCs, but does not combine the anti-fibrosis effect of Danpeiru Acid B, and the improvement rate of ovarian fibrosis is less than 30%.

[0004] The existing technologies do not disclose the "pretreatment-slow release" synergistic mechanism, and cannot solve the synchronization problem of stem cell survival and microenvironment optimization.

[0005] 1. The survival efficiency of stem cells in the ischemic and hypoxic microenvironment of the ovary is low (<7 days), and the differentiation targeting is poor; 2. The active ingredients of traditional Chinese medicine have a short action time (half-life <6 hours), and cannot continuously improve the ovarian fibrosis; 3. The existing combined therapy lacks a clear synergistic mechanism, and the curative effect repeatability is poor (intra-group difference CV>25%). SUMMARY

[0006] The purpose of the present application is to provide a composition for treating premature ovarian failure and a preparation method and application thereof, through the synergistic strategy of "pretreatment of traditional Chinese medicine stem cells + targeted slow release delivery", realizing the double intervention of "cell repair-microenvironment regulation", so that the estrus cycle recovery rate of premature ovarian failure model animals reaches 70%-90%, and the follicle number is increased by 6-8 times.

[0007] To achieve the above object, the application provides a composition for treating premature ovarian failure, which comprises adult stem cells pretreated by traditional Chinese medicine active components and PLGA microspheres loaded with traditional Chinese medicine active components, wherein the traditional Chinese medicine active components comprise astragaloside IV and salvianolic acid B.

[0008] Further, the molar ratio of astragaloside IV to salvianolic acid B is (1-4):1.

[0009] Further, the adult stem cells comprise one or more of ovary-derived adult stem cells, bone marrow mesenchymal stem cells and umbilical cord mesenchymal stem cells.

[0010] Further, the carrier microspheres are PLGA microspheres, chitosan microspheres or gelatin microspheres. The particle size of the carrier microspheres is 400-600 μm, and the loading amount of the traditional Chinese medicine active components in the carrier microspheres is 10-15 wt%. The microspheres are targeted and released by hydrophobic interaction (astragaloside IV and salvianolic acid B are both fat-soluble components) and pore adsorption to fix the drugs, rather than chemical bonding. The particle size of the microspheres is 400-600 μm (better than the tissue retention of nanoparticles), which is suitable for local slow release after ovarian interstitial injection.

[0011] Further, the use ratio of the adult stem cells pretreated by traditional Chinese medicine active components to the PLGA microspheres loaded with traditional Chinese medicine active components is (1-9) x 10 6 mg: 10 mg.

[0012] A preparation method of the composition for treating premature ovarian failure comprises the following steps: Mixing and incubating an aqueous solution containing traditional Chinese medicine active components with adult stem cells for 1-30 h to obtain pretreated adult stem cells; Dissolving astragaloside IV, salvianolic acid B and PLGA in an organic solvent to obtain an oil phase, dropping the oil phase into an aqueous phase, stirring to form an emulsion, and then volatilizing the organic solvent, washing with water and drying to obtain PLGA microspheres loaded with traditional Chinese medicine active components.

[0013] Further, the concentration of astragaloside IV in the aqueous solution containing traditional Chinese medicine active components is 10-25 μM, and the concentration of salvianolic acid B is 5-10 μM.

[0014] Further, the adult stem cells used are P3-P5 generation adult stem cells cultured, and the incubation time is 15-30 h.

[0015] Further, the organic solvent is dichloromethane, and the aqueous phase further contains polyvinyl alcohol.

[0016] The application of the composition in the preparation of a drug for improving ovarian fibrosis and improving estrogen levels.

[0017] The method for using the composition comprises mixing the pretreated stem cells with the carrier microspheres, and administering by injection into the ovarian interstitium, once every 2 weeks, for 3 times in succession.

[0018] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages: 1. The present application realizes double intervention of "cell repair-microenvironment regulation" through the synergistic strategy of "traditional Chinese medicine pretreated stem cells + targeted sustained release delivery". Experiments have proved that the composition can restore the estrous cycle of the premature ovarian failure model animal by 70%-90%, and increase the number of follicles by 6-8 times, which is significantly better than the existing single therapy, and has clear mechanism (activating the PI3K / Akt pathway and inhibiting the TGF-beta1 / Smad pathway), and has outstanding clinical transformation value.

[0019] 2. The synergistic mechanism is clear: Astragaloside B activates the PI3K / Akt pathway by binding with Akt protein (molecular docking energy-8.2 kcal / mol), reduces the apoptosis rate of stem cells from 35% to 12%; Salvianolic acid B targets and inhibits TGF-beta1 (binding energy-7.9 kcal / mol), reduces the expression of alpha-SMA by 42%, and the synergistic effect of the two makes the stem cell colonization rate increase to 65% (only 28% in the single stem cell group).

[0020] 3. The ratio range is scientific: the ratio of 1:1 to 4:1 is verified by orthogonal experiment, and the effect of the 2:1 group is the best (the number of follicles is 25.6±3.5), and the cross-species experiment (rat-rhesus monkey) shows that the effect is stable (CV<15%).

[0021] 4. The delivery system is optimized: the PLGA microspheres realize 7-day sustained release (cumulative release rate 85%), which is 3 times longer than the traditional injection in action time, and the biocompatibility score reaches 92 points (ISO10993 standard). DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present application clearer and more apparent, the present application will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and not to limit the present application. In addition, the technical features involved in each embodiment of the present application described below can be combined with each other as long as they do not conflict with each other.

[0023] The loading capacity of the PLGA (poly-lactic-co-glycolic acid) microspheres of the present application can be quantified as follows: Drug loading range: the drug loading of the PLGA microspheres is determined to be 10-15%, i.e. 10-15 mg of active components of traditional Chinese medicine per 100 mg of microspheres.

[0024] Single dose loading calculation: The single dose is "10 mg microspheres per mouse" (in the technical solution part), and the traditional Chinese medicine loading capacity in each 10 mg microspheres is: 10 mg x 10% = 1 mg (lower limit) to 10 mg x 15% = 1.5 mg (upper limit).

[0025] Example 1 A preparation method of the composition for treating premature ovarian failure, comprising: (1) Adult stem cell pretreatment Stem cell separation and culture: OSCs were digested with 0.2% collagenase for 30 min, and CD117+ cells were screened; BMSCs were separated by density gradient centrifugation; and UC-MSCs were digested with collagenase, and all were cultured with the corresponding medium containing 10% FBS to P3-P5 generation; the pretreatment system contains 15 μM astragaloside and 7.5 μM salvianolic acid B, which can up-regulate the expression of p-Akt protein by ≥2 times.

[0026] The adult stem cells and the active components of traditional Chinese medicine (astragaloside (purity ≥98%) and salvianolic acid B (purity ≥98%) with a molar ratio of 2:1) are co-incubated at 37°C and 5% CO2 for 24 hours, wherein the final concentration of astragaloside is 15 μM and the final concentration of salvianolic acid B is 7.5 μM. The adult stem cells are selected from ovary-derived adult stem cells (OSCs), bone marrow mesenchymal stem cells (BMSCs), or umbilical cord mesenchymal stem cells (UC-MSCs), all of which are CD117+ / CD44+ / CD34- phenotypes, with a passage number of P3-P5 and a cell density of 1 x 10 6 -5 x 10 6 cells / mL; (2) Preparation of PLGA microspheres Oil phase preparation: astragaloside, salvianolic acid B (molar ratio 2:1) and PLGA are co-dissolved in dichloromethane and other organic solvents to form a drug-polymer mixed solution.

[0027] Emulsification: The oil phase is dropped into the water phase containing polyvinyl alcohol (PVA), and high-speed homogenization (such as 10,000-15,000 r / min) is performed to form an O / W emulsion. The organic solvent is volatilized by stirring to solidify the PLGA into spheres, and the drug is wrapped inside the microspheres.

[0028] Separation and purification: The microspheres are collected by centrifugation, the PVA is removed by water washing, and the PLGA microspheres loaded with traditional Chinese medicine are obtained by freeze-drying.

[0029] The particle size of the PLGA microspheres is 400-600 μm, the drug loading capacity is 15%, and the 7-day cumulative release rate is ≥80%.

[0030] (3) A method for treating premature ovarian failure, comprising: Targeted delivery: mixing pretreated stem cells with PLGA microspheres loaded with traditional Chinese medicine components, and administering by intraperitoneal injection, once every 2 weeks, for 3 times in succession, with a single dose of 2 x 10 6 cells / only stem cells + 10 mg microspheres / only.

[0031] Stem cell identification supplement: flow cytometry verified that the positive rate of CD117⁺ / CD44⁺ / CD34⁻ phenotype was >95%, and the expression amount of OV-6 marker of OSCs was 2.3 times higher than that of BMSCs (qPCR data).

[0032] Molecular docking experiment: AutoDockVina software was used for molecular docking, and the binding site of Astragaloside IV and Akt was Asp292, and the binding site of Salvianolic Acid B and TGF-β was Arg25, which were verified by mutation experiment (the binding energy decreased ≥3 kcal / mol after mutation).

[0033] Sustained-release carrier comparison: supplement the release curve of different carriers, and the release rate of PLGA microspheres is 85% in 7 days, which is significantly higher than that of chitosan nanoparticles (62%) and gelatin microspheres (58%), and has no effect on the activity of stem cells (survival rate >90%).

[0034] Long-term safety data: continuous administration for 28 days, there is no difference in the levels of ALT, AST and Cr in rats compared with the normal group (P>0.05), and there is no pathological abnormality in ovarian tissue.

[0035] Synergistic mechanism data: Western Blot showed that the p-Akt / Akt ratio in the 2:1 group (2.1±0.3) was significantly higher than that in the single traditional Chinese medicine group (1.2±0.2) and the single stem cell group (0.8±0.1); the single traditional Chinese medicine group refers to Astragaloside IV and Salvianolic Acid B with a molar ratio of 2:1. The single stem cell group refers to only using adult stem cells.

[0036] RNA-seq analysis showed that 127 differential genes in the combined group of Example 1 were enriched in "cell survival" and "fibrosis regulation" pathways (FDR<0.01).

[0037] Table 1 Verification of the ratio gradient of Astragaloside IV and Salvianolic Acid B

[0038] Table 1 shows that the fibrosis area of the 2:1 group is reduced to 12.1%, which is significantly better than other ratios, and high drug loading is more conducive to anti-fibrosis (Dan-physcion B needs to continuously inhibit TGF-β1). For the preparation of a drug for treating premature ovarian failure, it can significantly improve the serum E2 level (≥50 pg / mL), reduce FSH (≤40 IU / L), and reduce the ovarian fibrosis area to ≤20%.

[0039] The PLGA microspheres loaded with traditional Chinese medicine components wrap astragaloside and salvianolic acid B in the inside of the microspheres by the emulsion-solvent evaporation method, and realize drug release by using the hydrophobic skeleton of PLGA. For "10 mg microspheres", the traditional Chinese medicine loading amount is 1-1.5 mg (drug loading amount 10-15%), and the optimal ratio (2:1) corresponds to about 1.5 mg, which ensures the synergistic effect of stem cell colonization (65%) and improvement of ovarian fibrosis (≤20%). The present application breaks through the bottleneck of the prior art by screening the optimal ratio of active components of traditional Chinese medicine, optimizing the stem cell pretreatment process and developing a targeted slow-release system, and realizes a breakthrough improvement in the treatment effect of POF.

[0040] Those skilled in the art will readily understand that the above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A composition for treating premature ovarian failure, characterized by, The active components of traditional Chinese medicine include Astragaloside IV and Salvianolic acid B.

2. The composition for treating premature ovarian failure according to claim 1, characterized by, The molar ratio of Astragaloside IV and Salvianolic acid B is (1-4):

1.

3. The composition for treating premature ovarian failure according to claim 1, wherein The adult stem cells include one or more of ovary-derived adult stem cells, bone marrow mesenchymal stem cells and umbilical cord mesenchymal stem cells.

4. The composition for treating premature ovarian failure according to claim 1, wherein The carrier microspheres are PLGA microspheres, chitosan microspheres or gelatin microspheres. The particle size of the carrier microspheres is 400-600 μm, and the loading amount of the active components of traditional Chinese medicine in the carrier microspheres is 10-15 wt%.

5. The composition for treating premature ovarian failure according to any one of claims 1 to 4, wherein The amount ratio of the adult stem cells pretreated by the active components of traditional Chinese medicine and the PLGA microspheres loaded with the active components of traditional Chinese medicine is (1-9) × 10 6 mg: 10 mg.

6. A method of preparing a composition for the treatment of premature ovarian failure according to any one of claims 1 to 5, characterized in that, The method comprises: incubating an aqueous solution containing the active components of traditional Chinese medicine with the adult stem cells for 1-30 h to obtain the pretreated adult stem cells; dissolving Astragaloside IV, Salvianolic acid B and PLGA in an organic solvent to obtain an oil phase, dropping the oil phase into an aqueous phase, stirring to form an emulsion, and then evaporating the organic solvent, washing with water and drying to obtain the PLGA microspheres loaded with the active components of traditional Chinese medicine.

7. The method of claim 6, wherein the composition is prepared by mixing the active ingredient and the pharmaceutically acceptable carrier. The concentration of Astragaloside IV in the aqueous solution containing the active components of traditional Chinese medicine is 10-25 μM, and the concentration of Salvianolic acid B is 5-10 μM.

8. The method of claim 6, wherein the composition is prepared by mixing the active ingredient and the pharmaceutically acceptable carrier. The adult stem cells used are P3-P5 generation adult stem cells, and the incubation time is 15-30 h.

9. The method for preparing the composition for treating premature ovarian failure according to claim 6, characterized in that, The organic solvent is dichloromethane, and the aqueous phase further contains polyvinyl alcohol.

10. Use of the composition of any one of claims 1-5 in the preparation of a drug for improving ovarian fibrosis and increasing estrogen levels.

Images