An amh-regulated ovarian repair gel and methods of making the same

Abstract

The application discloses an ovary repair gel based on AMH regulation and a preparation method thereof, and belongs to the technical field of biological medicine. The gel comprises an AMH receptor 2 binding peptide (AMHR2BP), a temperature-sensitive PEG-PLGA hydrogel matrix and AMHR2BP-loaded PLGA nanoparticles, the content of the AMHR2BP in the gel is 0.01%-0.5% (w / w), the particle size of the nanoparticles is 100-300 nm, and the encapsulation rate is 65%-85%. The gel is an injection type gel and is used for local injection administration of an ovary. The application further discloses a preparation method of the gel. The gel directly combines the AMHR2BP with the AMH receptor 2 to regulate the AMH signal pathway, and combines the PLGA nanoparticle encapsulation and the temperature-sensitive hydrogel sustained release, so that long-acting protection of the ovary is realized, and the gel is used for preparing a drug for treating reduced ovarian reserve function and chemotherapy-induced ovarian damage.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and reproductive medicine technology, specifically relating to an ovarian repair gel based on AMH regulation and its preparation method, for the treatment of ovarian dysfunction-related diseases such as decreased ovarian reserve and ovarian damage caused by chemotherapy. Background Technology

[0002] Anti-Müllerian hormone (AMH) is a diglycoprotein secreted by ovarian granulosa cells and belongs to the transforming growth factor-β (TGF-β) superfamily. AMH activates the downstream SMAD signaling pathway by binding to its specific receptor, AMH receptor 2 (AMHR2), thereby regulating the PI3K / AKT / FOXO3a pathway, maintaining the dormant state of primordial follicles, and inhibiting premature recruitment and depletion of primordial follicles. AMH is continuously secreted by the granulosa cells of small antral follicles, and its serum level is positively correlated with the number of follicles. It is not subject to feedback regulation by the hypothalamus-pituitary-ovarian axis and exhibits relatively small fluctuations during the menstrual cycle, making it one of the most accurate biomarkers for evaluating ovarian reserve. When AMH levels are significantly lower than the age-appropriate normal range, it indicates diminished ovarian reserve (DOR); while abnormally elevated AMH levels are closely associated with polycystic ovary syndrome (PCOS).

[0003] Decreased ovarian reserve (DOR) is a pathological condition characterized by a reduction in the number and quality of oocytes in the ovary. Ovarian damage induced by chemotherapy drugs (especially alkylating agents) is a significant cause of DOR. Currently, clinical interventions for DOR are limited, and no drugs or medical devices that can fundamentally protect follicular reserve have been approved for marketing. Directly regulating the AMH signaling pathway has become an important strategy for protecting ovarian reserve. AMH receptor 2 binding peptide (AMHR2BP) is a bioactive polypeptide that mimics the natural AMH-AMHR2 binding interface. It can specifically bind to AMHR2 and activate the downstream SMAD signaling pathway, inhibiting the excessive conversion of primordial follicles. Studies have confirmed that in animal models of chemotherapy-induced ovarian damage, administration of AMHR2BP can effectively prevent follicular loss and maintain ovarian reserve.

[0004] Chinese patent application CN116966138A discloses the application of a gel in the preparation of drugs that increase AMH levels. This gel uses kudzu root extract, polygonatum extract, ampelopsis root extract, alpinia oxyphylla extract, and clove extract as a complex plant raw material. The active ingredient is a complex plant fermentation concentrate obtained through fermentation with *Lactobacillus plantarum*, *Lactobacillus casei*, and *Lactobacillus acidophilus*, combined with surfactants such as poloxamer 407 to prepare a vaginal delivery gel. However, this prior art has the following shortcomings:

[0005] First, the mechanism of action is unclear, and the active ingredients cannot be precisely quantified. The active ingredient in this gel is a complex plant fermentation concentrate, containing secondary metabolites produced by microbial fermentation of various plant extracts and lactobacillus fermentation lysates. The composition is extremely complex, and it is unclear which specific substance plays a core role or through which molecular target it achieves ovarian function repair. The gel's effect of increasing AMH levels is broadly attributed to "nutritional repair" of ovarian cells, rather than direct regulation of the AMH signaling pathway, representing a crude intervention with an unclear mechanism of action. Furthermore, because the active ingredient originates from a bio-fermentation process, batch-to-batch consistency is difficult to guarantee, making precise quality control and dosage standardization of the active ingredient impossible.

[0006] Secondly, the administration method results in low bioavailability and requires frequent dosing. This gel is a vaginal delivery formulation; the active ingredient needs to cross the vaginal epithelial barrier and matrix layer to enter the systemic circulation before being distributed to the ovaries via the bloodstream. This involves multiple biological barriers, leading to low local accumulation efficiency in the ovaries. The requirement for daily dosing for 21 consecutive days directly reflects insufficient bioavailability. Furthermore, the gel matrix is ​​a common surfactant-based gel, lacking sustained-release capabilities and failing to achieve long-term exposure of the active ingredient to the target tissue.

[0007] In view of the shortcomings of the existing technology, there is an urgent need in the field to develop an ovarian repair gel with a clearly defined active ingredient, a clear mechanism of action, and the ability to achieve precise local drug delivery and long-lasting sustained release to the ovary. Summary of the Invention

[0008] To address the shortcomings of existing ovarian repair gels, such as unclear active ingredients, ambiguous mechanisms of action, low bioavailability after vaginal administration, and the need for frequent dosing, this invention provides an ovarian repair gel based on AMH regulation and its preparation method.

[0009] The purpose of this invention is to provide an injectable thermosensitive gel formulation with clearly defined active ingredients that achieves ovarian function repair by directly regulating the AMH signaling pathway.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned gel, which is stable and suitable for industrial production.

[0011] Another object of the present invention is to provide the use of the above-mentioned gel in the preparation of a medicament for treating ovarian reserve deficiency and / or ovarian damage caused by chemotherapy.

[0012] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0013] An ovarian repair gel based on AMH regulation, comprising the following components:

[0014] (1) AMH signaling pathway regulates active ingredients

[0015] The active ingredient is an AMH receptor 2 binding peptide (AMHR2BP), whose amino acid sequence is shown in SEQ ID NO:1 (VGSNAYNQFKVGSVAL). This sequence is based on the reported AMHR2BP sequence shown in SEQ ID NO:2 (VGSNAYNQFRVGSVALS, Detti et al., US12662542B2), and is an AMHR2BP variant designed by site-specific substitution (replacing the 10th D-Arg with Lys) and C-terminal truncation (deleting the 17th Ser). This variant uses a full L-amino acid composition, which simplifies the synthesis process while retaining the ability to bind to AMH receptor 2 (AMHR2). AMHR2BP can specifically bind to AMH receptor 2 (AMHR2), activate the downstream SMAD signaling pathway, inhibit premature recruitment and depletion of primordial follicles, thereby protecting ovarian reserve function.

[0016] The AMHR2BP content in the gel is 0.01%-0.5% (w / w), preferably 0.05%-0.2% (w / w).

[0017] (2) Thermosensitive injectable hydrogel matrix

[0018] The hydrogel matrix is ​​composed of a polyethylene glycol-polylactic acid-glycolic acid copolymer (PEG-PLGA) block copolymer, wherein the molecular weight of PEG is 1000-2000 Da, the molecular weight of PLGA is 1000-3000 Da, preferably PEG and PLGA are both 1500 Da, and the molar ratio of PEG to PLGA is 1:2. The concentration of the PEG-PLGA block copolymer in phosphate buffer is 15%-25% (w / v).

[0019] The hydrogel exhibits thermosensitive properties, remaining a liquid sol at room temperature (20-25℃) with good needle penetration; it transforms into a solid gel at body temperature (37℃), forming a drug reservoir locally in the ovary after injection, achieving targeted and sustained release of the active ingredient. The sol-gel transition temperature of the hydrogel is 30-37℃.

[0020] The biodegradation half-life of the hydrogel is 2-6 weeks, and the degradation products are non-toxic, metabolizable natural products.

[0021] (3) PLGA nanoparticles loaded with AMHR2BP

[0022] The AMHR2BP is encapsulated in polylactic-co-glycolic acid (PLGA) nanoparticles. The nanoparticles have a particle size of 100-300 nm, an encapsulation efficiency of 65%-85% for AMHR2BP, and a drug loading of 1.5%-5.0% (w / w). The nanoparticles are dispersed in the hydrogel matrix.

[0023] PLGA nanoparticles can protect AMHR2BP peptides from enzymatic degradation and immune clearance in vivo, prolonging their in vivo half-life. The in vitro drug release duration of the nanoparticles is 2-6 weeks.

[0024] Preferably, the PLGA nanoparticles are coupled with an ovarian targeting ligand, which is follicle-stimulating hormone (FSH) or its receptor-binding fragment. The targeting ligand is coupled to the nanoparticle surface via a carbodiimide method (EDC / NHS activation), with a coupling efficiency of 60%-85%. The FSH targeting ligand can specifically bind to the follicle-stimulating hormone receptor (FSHR) highly expressed on the surface of ovarian granulosa cells, achieving active targeted enrichment of the nanoparticles in the ovary.

[0025] Preferably, the hydrogel matrix further comprises decellularized ovarian extracellular matrix (OvaECM), with a final concentration of 1-5 mg / mL. The OvaECM is obtained from porcine or human ovarian tissue through repeated freeze-thaw cycles, nuclease digestion, and detergent treatment followed by freeze-drying. OvaECM can mimic the natural microenvironment of the ovary and promote ovarian niche regeneration.

[0026] (4) Pharmaceutically acceptable excipients

[0027] The gel also contains pharmaceutically acceptable excipients selected from one or more of phosphate buffer, isotonic adjusters, and preservatives. The pH of the gel is 6.8-7.4.

[0028] The gel is an injectable gel used for local ovarian injection under ultrasound guidance.

[0029] This invention also provides a method for preparing the above-mentioned AMH-based ovarian repair gel, comprising the following steps:

[0030] Step 1: Preparation of AMHR2BP

[0031] AMHR2BP (VGSNAYNQFKVGSVAL), with the amino acid sequence shown in SEQ ID NO:1, was synthesized using a solid-phase peptide synthesis method (Fmoc chemical strategy). The synthesized crude peptide was purified to a purity of ≥95% by preparative high-performance liquid chromatography, lyophilized, and stored at -20℃ for later use.

[0032] Step 2: Nano-encapsulation

[0033] The AMHR2BP obtained in step one was encapsulated in PLGA nanoparticles using a double emulsion method (W / O / W). Specific conditions were as follows: PLGA (molecular weight 5000-15000 Da) was dissolved in dichloromethane as the oil phase, with a PLGA concentration of 3%-8% (w / v); AMHR2BP was dissolved in the inner aqueous phase (containing 0.5%-2% (w / v) PVA), with a volume ratio of the inner aqueous phase to the oil phase of 1:5 to 1:10; after ultrasonic emulsification (power 100-300W, time 30-120 seconds) to form a primary emulsion, an outer aqueous phase (containing 1%-3% (w / v) PVA as an emulsifier) ​​was added, followed by re-emulsification and stirring to evaporate the organic solvent. The nanoparticles were collected by high-speed centrifugation (10000-20000 rpm, 10-30 minutes), washed three times with deionized water, and then freeze-dried. The nanoparticle size was controlled within the range of 100-300 nm by adjusting the polymer concentration, oil-water ratio, and stirring speed.

[0034] Step 3: Targeted ligand conjugation

[0035] FSH peptides were coupled to the surface of the nanoparticles obtained in step two using the carbodiimide method. Specific conditions were as follows: nanoparticles were dispersed in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.0-6.5), EDC and NHS were added (molar ratio 1:1-1:3), and the mixture was activated at room temperature for 30-60 minutes. Then, the FSH peptides were added (FSH to nanoparticle mass ratio 1:10-1:50), the pH was adjusted to 6.5-7.5, and the reaction was carried out at room temperature for 2-4 hours. After the reaction, uncoupled FSH was removed by centrifugation and washing, and the nanoparticles were lyophilized for storage.

[0036] Step 4: Preparation of Thermosensitive Hydrogel

[0037] The PEG-PLGA block copolymer was dissolved in phosphate buffer (pH 7.4) at 4°C at a concentration of 15%-25% (w / v), and magnetically stirred at 2-8°C for 12-24 hours until completely dissolved to form a homogeneous polymer solution.

[0038] Preferably, decellularized ovarian extracellular matrix (OvaECM) powder is added to the polymer solution to a final concentration of 1-5 mg / mL, and stirring is continued until the mixture is evenly dispersed.

[0039] Step 5: Gel Assembly

[0040] The drug-loaded targeted nanoparticles obtained in step three were dispersed in the hydrogel solution obtained in step four, so that the content of AMHR2BP in the final gel was 0.01%-0.5% (w / w). The mixture was stirred at 2-8℃ for 30-60 minutes until uniformly dispersed, the pH was adjusted to 6.8-7.4, and the mixture was sterilized by filtration through a 0.22μm filter membrane, dispensed into pre-filled syringes, and stored at 2-8℃.

[0041] All steps two through five above are performed under aseptic conditions.

[0042] The ovarian repair gel of this invention is an injectable formulation, and its usage method is as follows:

[0043] (1) Remove the gel from storage conditions at 2-8℃ and allow it to return to room temperature (20-25℃) for about 15-30 minutes. The gel will be in a liquid sol state.

[0044] (2) Under ultrasound guidance, the gel is injected into the subcortical or hilar region of the ovary using an 18-22G injection needle. The injection volume is determined according to the volume of the ovary, generally 0.5-2.0 mL / side.

[0045] (3) After injection, the gel transforms into a solid gel within 30-120 seconds under the influence of body temperature, thereby achieving localized sustained release of the active ingredients in the ovary;

[0046] (4) A single injection can continuously release the active ingredient for 2-6 weeks. The injection can be repeated every 2-6 weeks as needed.

[0047] Compared with the prior art, the present invention has the following advantages:

[0048] This invention uses the sequence-defined AMHR2BP as the active ingredient, achieving precise repair of ovarian reserve function by directly binding to AMH receptor 2 and activating the downstream SMAD signaling pathway. This overcomes the shortcomings of existing technologies, such as complex and unclear active ingredients and vague mechanisms of action. Simultaneously, this invention uses an injectable, thermosensitive PEG-PLGA hydrogel as a carrier, combined with PLGA nanoparticle encapsulation for protection, achieving localized sustained release of AMHR2BP in the ovary (a single injection can last for 2-6 weeks), overcoming the shortcomings of existing technologies such as low bioavailability and the need for daily administration via vaginal delivery. In the preferred formulation, the FSH-targeting ligand coupled to the nanoparticle surface further achieves active enrichment in the ovary, significantly increasing the target organ concentration of the active ingredient. Experiments show that the gel of this invention can increase serum AMH by 72.9% in DOR model rats, significantly restore the number of follicles at all stages, and exhibits good biocompatibility, providing a novel formulation for the treatment of diminished ovarian reserve and related diseases. Attached Figure Description

[0049] Figure 1 This is a flowchart illustrating the preparation method of the ovarian repair gel of the present invention;

[0050] Figure 2 The in vitro cumulative release curve of AMHR2BP in the gel of Example 4 of this invention is shown, and compared with Comparative Example 1 (free peptide gel) and Comparative Example 2 (nanoparticle suspension, non-thermosensitive gel) (n=3, mean±SD).

[0051] Figure 3 The bar chart shows the serum AMH levels of rats in each group (n=10, mean±SD). ##P<0.01 vs normal group; *P<0.05, **P<0.01 vs model group;

[0052] Figure 4 The bar chart shows the follicle counts at each stage in rats of this invention (n=10, mean±SD). Detailed Implementation

[0053] The present invention will be further described in detail below with reference to embodiments and comparative examples. Any improvements and modifications made by those skilled in the art without departing from the spirit of the invention fall within the protection scope of the present invention.

[0054] Glossary

[0055] AMH: Anti-Müllerian Hormone, a diglycoprotein secreted by ovarian granulosa cells, is the most accurate biomarker for evaluating ovarian reserve function.

[0056] AMHR2: AMH Receptor Type 2, a specific receptor for AMH.

[0057] AMHR2BP: AMHR2 Binding Peptide, the polypeptide shown in SEQ ID NO:1 of this invention.

[0058] DOR: Diminished Ovarian Reserve.

[0059] Thermosensitive hydrogels: Hydrogel materials with temperature-sensitive phase transition properties, which are liquid sols at room temperature and transform into solid gels at body temperature.

[0060] PLGA: Polylactic acid-glycolic acid copolymer.

[0061]

[0062] Example 1:

[0063] Preparation of AMHR2BP-PLGA nanoparticles

[0064] 1.1 Obtaining AMHR2BP

[0065] The AMHR2BP peptide used in this embodiment was synthesized by Jier Biochemical (Shanghai) Co., Ltd. using a solid-phase peptide synthesis method (Fmoc chemical strategy). Its amino acid sequence is Val-Gly-Ser-Asn-Ala-Tyr-Asn-Gln-Phe-Lys-Val-Gly-Ser-Val-Ala-Leu (VGSNAYNQFKVGSVAL, SEQ ID NO:1). After purification by preparative high-performance liquid chromatography (HPLC), the purity was determined to be 95.8%. The molecular weight, identified by electrospray ionization mass spectrometry (ESI-MS), was consistent with the theoretical value (theoretical value 1640.9 Da, measured value 1641.2 Da). The lyophilized powder was stored at -20℃ for later use.

[0066] 1.2 Preparation of AMHR2BP-PLGA nanoparticles

[0067] Prepared using the double emulsion method (W / O / W), such as Figure 1 The process, as shown, includes the following steps:

[0068] (1) Oil phase preparation: PLGA 200mg was dissolved in 4mL of dichloromethane (5% w / v).

[0069] (2) Preparation of internal aqueous phase: 10 mg AMHR2BP is dissolved in 1 mL of ultrapure water containing 1% PVA.

[0070] (3) Preparation of colostrum: The aqueous phase is slowly added dropwise to the oil phase (volume ratio 1:4), and ultrasonic emulsification is performed for 60 seconds in an ice-water bath using a probe-type ultrasonic homogenizer (Sonics, 200W) (5 seconds working / 5 seconds intermittent).

[0071] (4) Preparation of re-emulsion: Add 20 mL of external aqueous phase (containing 2% PVA) to the colostrum, stir magnetically (IKA, 800 rpm) for 2 minutes, and then emulsify with probe-type ultrasonic (150W, ice water bath) for 90 seconds (5 seconds working / 5 seconds intermittent).

[0072] (5) Solvent evaporation and nanoparticle collection: The re-emulsion was magnetically stirred (600 rpm) overnight (12-16 hours) in a fume hood to allow the dichloromethane to evaporate completely. The nanoparticle suspension was centrifuged at 12,000 rpm for 20 minutes (4°C), washed three times with ultrapure water, and 5% sucrose freeze-drying protectant was added. After pre-freezing at -80°C, it was freeze-dried for 48 hours and stored at -20°C.

[0073] 1.3 Characterization of Nanoparticles

[0074] The average particle size was 185.3±25.6 nm, PDI was 0.087±0.021, and Zeta potential was -12.5±2.3 mV, as determined by a dynamic light scattering particle size analyzer (Malvern Zetasizer Nano ZS).

[0075] Drug loading and encapsulation efficiency: 5 mg of lyophilized nanoparticle powder was accurately weighed, dissolved in 1 mL of dichloromethane, extracted with 1 mL of ultrapure water, and the AMHR2BP content in the aqueous phase was determined by BCA method; another 10 mg was completely dissolved in DMSO / water (1:1) to determine the total amount. The encapsulation efficiency was 78.6% ± 4.2%, and the drug loading was 3.1% ± 0.4%.

[0076] Example 2:

[0077] Preparation of FSH-targeted AMHR2BP nanoparticles.

[0078] Example 1: 100 mg of lyophilized nanoparticle powder was dispersed in 10 mL of MES buffer (0.1 M, pH 6.0), 5 mg of EDC and 8 mg of NHS were added, and the mixture was activated at room temperature for 30 minutes. The mixture was centrifuged at 10,000 rpm for 10 minutes, and the precipitate was resuspended in PBS. 2 mg of recombinant human FSH peptide was added, and the mixture was reacted at room temperature for 3 hours at pH 7.0. The mixture was then centrifuged at 12,000 rpm for 15 minutes, washed three times with PBS, and lyophilized.

[0079] The coupling efficiency was determined by ELISA: the FSH content was determined by collecting the washed supernatant, and the coupling efficiency was 72.5% ± 5.1%.

[0080] Example 3

[0081] Preparation of PEG-PLGA thermosensitive hydrogel

[0082] 20g of PEG-PLGA block copolymer (PEG 1500Da-PLGA 1500Da, molar ratio 1:2) was dissolved in 100mL PBS (0.01M, pH 7.4) at a concentration of 20% (w / v) and magnetically stirred at 4℃ for 24 hours.

[0083] The sol-gel transition temperature was determined using the inverted test tube method: 32.5 ± 1.5℃. At 4℃, it is a transparent liquid sol; within 2 minutes in a 37℃ water bath, it transforms into a milky white, opaque solid gel.

[0084] Example 4

[0085] Preparation of thermosensitive hydrogels loaded with FSH-targeted AMHR2BP nanoparticles

[0086] At 4°C, the lyophilized nanoparticle powder of Example 2 was dispersed in a small amount of PBS at 4°C and dispersed by sonication (100W, 30 seconds). It was then slowly added dropwise to the polymer solution of Example 3 and stirred at low speed (300 rpm) for 40 minutes at 4°C. The final AMHR2BP content was 0.1% (w / w), the pH was adjusted to 7.2, sterilized by filtration through a 0.22 μm filter membrane, and dispensed into 1 mL pre-filled syringes. The solution was stored at 4°C protected from light.

[0087] Example 5

[0088] Preparation of gels with different AMHR2BP contents

[0089] Referring to Example 4, the amount of nanoparticles added was adjusted so that the AMHR2BP content was 0.01% (w / w), 0.05% (w / w), 0.2% (w / w), and 0.5% (w / w), respectively, and these were numbered Examples 5-1 to 5-4.

[0090] Comparative Example 1

[0091] It does not contain nano-encapsulation.

[0092] Free AMHR2BP (without nano-encapsulation) was dissolved in PBS at 4°C and a PEG-PLGA polymer solution was added. The AMHR2BP content was 0.1% (w / w), and the rest was the same as in Example 4.

[0093] Comparative Example 2

[0094] It does not contain a thermosensitive hydrogel matrix.

[0095] FSH-targeted AMHR2BP nanoparticle lyophilized powder was dispersed in PBS (0.01M, pH 7.2), with an AMHR2BP content of 0.1% (w / w), free of PEG-PLGA matrix, and stored at 4°C.

[0096] Test Example 1

[0097] In vitro properties characterization of the gel.

[0098] 1.1 Sol-gel phase transition behavior

[0099] Example 4: The gel transition temperature was 32.8±1.2℃, which was not significantly different from the blank matrix (32.5±1.5℃). It was liquid at 25℃ and could be injected through a 22G needle; it completely gelled in a 37℃ water bath in 60-90 seconds.

[0100] 1.2 Rheological properties

[0101] Rotational rheometer (TA Instruments, AR2000), frequency 1Hz, strain 1%. Viscosity at 4℃: 0.15-0.25 Pa·s; storage modulus G' = 850-1200 Pa at 37℃, loss modulus G" = 120-180 Pa.

[0102] 1.3 In vitro drug release

[0103] Dialysis bag method (MWCO 100kDa): In Example 4, 1g of gel (containing 0.1% AMHR2BP) was placed in a dialysis bag and pre-gelled at 37°C for 30 minutes. It was then placed in 50mL of release medium (PBS, 0.01M, pH 7.4, 0.02% NaN3) and shaken at 37°C (100rpm). At preset time points, 5mL of medium was collected and replenished, and the concentration was determined by ELISA. Comparative Example 1 (free peptide gel) and Comparative Example 2 (nanoparticle suspension, no gel) were used as controls. The results are as follows: Figure 2 As shown in Table 2.

[0104] Table 2: Cumulative in vitro release rate of AMHR2BP in different formulations (%, n=3, mean±SD)

[0105]

[0106] Test Example 2

[0107] Pharmacodynamic evaluation of the DOR rat model.

[0108] 2.1 Establishment of animal models

[0109] Twelve-week-old SPF-grade female SD rats, weighing 220-250g, were acclimatized for one week. Vaginal smears were taken daily, and rats with regular estrous cycles were selected for inclusion in the group.

[0110] A dorsal regurgitation (DOR) model was induced by a single intraperitoneal injection of cyclophosphamide (CTX, 75 mg / kg). CTX was prepared at a concentration of 20 mg / mL using 0.9% sodium chloride solution and used immediately. The control group received the same volume of physiological saline. Vaginal smears were taken daily after modeling, and successful modeling was defined as persistent estrous cycle irregularities lasting for more than 2 weeks.

[0111] 2.2 Animal grouping and administration

[0112] Rats that successfully established the model were randomly divided into 7 groups of 10 rats each:

[0113] Normal group: No model was created; the same amount of PBS was injected during surgery.

[0114] Model group: DOR model was injected with an equal volume of PBS after modeling;

[0115] Example 4 group: DOR + Example 4 gel (0.1%), 50 μL injected into each ovary;

[0116] Example 5-1 group: DOR + Example 5-1 gel (0.01%), 50 μL per side;

[0117] Example 5-3 group: DOR + Example 5-3 gel (0.2%), 50 μL per side;

[0118] Comparative Example 1: DOR + Comparative Example 1 gel (free peptide), 50 μL per side;

[0119] Comparative Example 2: DOR+ Comparative Example 2 nanoparticle suspension (without gel), 50 μL per side.

[0120] Drug administration was performed on the 7th day after modeling. Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (1 mL / kg), and the ovaries were exposed by incisions below the costal margins on both sides of the back. Insulin was slowly injected under the ovarian capsule with an insulin syringe (29G needle), and the needle was left in place for 30 seconds before suturing.

[0121] On day 28 after administration, the animals were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.6 mL / animal), blood was collected from the abdominal aorta, allowed to stand at room temperature for 1 hour, centrifuged at 3000 r / min for 15 minutes, and the serum was collected and stored at -80℃.

[0122] 2.3 Serum AMH Level Measurement

[0123] The rat AMH ELISA kit (Shanghai Keaibo Biotechnology, CB10627-Ra) was used for detection, and the results were measured at 450 nm using a microplate reader (BioTek ELx808).

[0124] The results are as follows Figure 3 As shown in Table 3.

[0125] Table 3: Serum AMH levels in each group (ng / mL, n=10, mean±SD)

[0126]

[0127] Note: Compared with the normal group, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0128] 2.4 Serum FSH and E2 Level Measurement

[0129] ELISA method for detecting FSH and estradiol (E2).

[0130] Table 4: Serum FSH and E2 levels in each group (n=10, mean±SD)

[0131]

[0132] Note: Compared with the normal group, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0133] 2.5 Ovarian histological observation and follicle counting

[0134] After blood collection, both ovaries were dissected and their wet weight was measured. The left ovary was fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and serially sectioned (5 μm). Five sections were taken from each rat, one section every five sections, and stained with hematoxylin and eosin (HE). The number of primordial follicles, primary follicles, secondary follicles, and antral follicles was counted under an optical microscope (Olympus BX53), and the average value was taken. The results are as follows: Figure 4 As shown in Table 5.

[0135] Ovarian index = bilateral ovarian wet weight (mg) / rat body weight (g) × 100%.

[0136] Table 5: Ovarian index and follicle count for each group (n=10, mean±SD)

[0137]

[0138] Note: Compared with the normal group, ##P<0.01; compared with the model group, *P<0.05, **P<0.01.

[0139] Test Example 3

[0140] 3.1 Local irritation at the injection site

[0141] Twelve SD rats were used, and the gel from Example 4 was injected bilaterally into each ovary, 50 μL per ovary, following the administration method described in Test Example 2. Three rats were harvested on days 1, 3, 7, 14, and 28 post-administration for dissection and observation of congestion, edema, adhesions, and necrosis in the ovary and surrounding tissues at the injection site. Scoring was based on the following criteria: 0 points = no response; 1 point = mild congestion; 2 points = moderate congestion with slight edema; 3 points = severe congestion, edema, and adhesions; 4 points = necrosis or ulceration.

[0142] Table 6: Local irritation response score at the injection site (n=3, mean±SD)

[0143]

[0144] No obvious congestion, edema, adhesion or necrosis was observed at any time point. Only mild congestion of the needle tract was occasionally observed on the first day, which completely subsided within 3 days.

[0145] 3.2 Major organ coefficients

[0146] Blood was collected 28 days after drug administration. At the same time, the heart, liver, spleen, lungs and kidneys of rats were separated, and their wet weight was measured. The organ coefficient (wet weight of organ / body weight × 100%) was calculated.

[0147] Table 7: Major organ coefficients for each group (%, n=10, mean±SD)

[0148]

[0149] There were no significant differences in the coefficients of each organ among the groups (P>0.05).

[0150] 3.3 Complete Blood Count Indicators

[0151] Blood was collected 28 days after administration of the drug, and the whole blood was sent for complete blood count testing.

[0152]

[0153] All blood routine indicators in each group were within the normal physiological range, and there were no significant differences between the groups (P>0.05).

[0154] 3.4 Liver and kidney function indicators

[0155] Blood was collected 28 days after administration, and serum was separated to detect liver function (ALT, AST) and kidney function (BUN, Cr) indicators.

[0156] Table 9: Liver and kidney function indicators for each group (n=10, mean±SD)

[0157]

[0158] The levels of ALT, AST, BUN, and Cr in each group were all within the normal physiological range, and there were no significant differences between the groups (P>0.05).

[0159] Based on the above test examples 1-3, the main experimental results of this invention are as follows:

[0160] (1) In vitro release behavior (test example 1)

[0161] In Example 4, AMHR2BP in the gel exhibited a continuous and stable release, with a cumulative release rate of 84.6% over 28 days; Comparative Example 1 (free peptide) released approximately 70% in one day, showing a significant burst release; Comparative Example 2 (nanoparticle suspension, without thermosensitive gel) had a release rate between the two. The dual sustained-release system composed of PLGA nanoparticles and thermosensitive hydrogel achieved long-term release.

[0162] (2) Pharmacodynamics of DOR model rats (test case 2)

[0163] After 28 days of administration of the gels in Examples 4 (0.1%) and 5-3 (0.2%), the serum AMH levels increased by 72.9% and 70.6%, respectively, recovering to near-normal levels, with no significant difference between the two groups (P>0.05). The low-dose Example 5-1 (0.01%) showed an increase of 36.4% (P<0.05). The increase rates in Comparative Example 1 (free peptide) were 9.6% and 21.7% (non-thermosensitive gel), both significantly lower than those in Example 4 (P<0.01). The trends in FSH and E2 levels were consistent with those of AMH. Ovarian histology showed a significant recovery in the number of follicles at each stage in the Example 4 group, with the number of primordial follicles increasing from 12.5±2.8 to 25.8±3.2.

[0164] (3) Biocompatibility (Test Example 3)

[0165] Mild congestion was only occasionally observed at the injection site on the first day, and subsided within 3 days. There were no significant differences in organ coefficients, blood routine and liver and kidney function indicators between the drug administration groups and the normal group (P>0.05), and all were within the normal physiological range.

[0166] The above embodiments, comparative examples, and test cases fully illustrate the technical solution of the present invention and its beneficial effects. Those skilled in the art can make modifications or variations to the present invention based on the above teachings, but all such modifications or variations fall within the protection scope of the present invention.

Claims

1. An ovarian repair gel based on AMH regulation, characterized in that, The gel comprises the following components: (1) An active ingredient regulating the AMH signaling pathway, wherein the active ingredient is the AMH receptor 2 binding peptide AMHR2BP, the amino acid sequence of which is shown in SEQ ID NO:1, and the content of AMHR2BP in the gel is 0.01%-0.5% (w / w). (2) A thermosensitive injectable hydrogel matrix, wherein the hydrogel matrix is ​​composed of polyethylene glycol-polylactic acid-glycolic acid copolymer PEG-PLGA block copolymer, wherein the molecular weight of PEG is 1000-2000 Da and the molecular weight of PLGA is 1000-3000 Da, and the hydrogel is a liquid sol at room temperature of 20-25℃ and transforms into a solid gel at body temperature of 37℃; (3) Polylactic acid-glycolic acid copolymer (PLGA) nanoparticles loaded with AMHR2BP, wherein an ovarian targeting ligand, namely follicle-stimulating hormone (FSH), is coupled to the surface of the PLGA nanoparticles via a carbodiimide method with a coupling efficiency of 60%-85%, the nanoparticles are dispersed in the hydrogel matrix, the particle size of the nanoparticles is 100-300 nm, and the encapsulation efficiency of AMHR2BP is 65%-85%; The gel is an injectable gel used for local injection into the ovary.

2. The gel according to claim 1, characterized in that, The AMHR2BP content in the gel is 0.05%-0.2% (w / w).

3. The gel according to claim 1, characterized in that, The PEG-PLGA block copolymer has a molecular weight of 1500 Da for both PEG and PLGA, and the molar ratio of PEG to PLGA is 1:

2. The concentration of the PEG-PLGA block copolymer in phosphate buffer is 15%-25% (w / v).

4. The gel according to claim 1, characterized in that, The hydrogel matrix also contains decellularized ovarian extracellular matrix (OvaECM), with a final concentration of 1-5 mg / mL. The OvaECM is obtained by decellularizing porcine ovarian tissue through repeated freeze-thaw cycles, nuclease digestion, and detergent treatment, followed by freeze-drying.

5. The gel according to claim 1, characterized in that, The nanoparticles contain 1.5%-5.0% (w / w) of AMHR2BP, and the in vitro drug release duration of the nanoparticles is 2-6 weeks.

6. The gel according to claim 1, characterized in that, The gel has a pH of 6.8-7.4 and also contains phosphate buffer as a pharmaceutically acceptable excipient.

7. The method for preparing the AMH-regulated ovarian repair gel as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Preparation of AMHR2BP: AMHR2BP with the amino acid sequence shown in SEQ ID NO:1 was synthesized by solid-phase peptide synthesis and purified by high performance liquid chromatography to a purity of ≥95%; (2) Nanoencapsulation: AMHR2BP obtained in step (1) is encapsulated in PLGA nanoparticles using a double emulsion method (W / O / W). The specific conditions are as follows: PLGA is dissolved in dichloromethane as the oil phase, with a PLGA concentration of 3%-8% (w / v). AMHR2BP is dissolved in the inner aqueous phase, with a volume ratio of the inner aqueous phase to the oil phase of 1:5 to 1:

10. After ultrasonic emulsification, an outer aqueous phase is added, which contains 1%-3% (w / v) PVA as an emulsifier. After emulsification again, the organic solvent is evaporated by stirring. The nanoparticles are collected by centrifugation, and the nanoparticle size is controlled at 100-300 nm. (3) Targeted ligand coupling: FSH peptides were coupled to the surface of the nanoparticles obtained in step (2) by EDC / NHS activation using the carbodiimide method. The reaction conditions were pH 6.5-7.5 and room temperature reaction for 2-4 hours. (4) Preparation of thermosensitive hydrogel: Dissolve PEG-PLGA block copolymer at a concentration of 15%-25% (w / v) in phosphate buffer at 4℃, and stir magnetically at 2-8℃ for 12-24 hours until completely dissolved to form a homogeneous polymer solution. (5) Gel assembly: Disperse the drug-loaded targeted nanoparticles obtained in step (3) in the hydrogel solution obtained in step (4) so ​​that the content of AMHR2BP in the final gel is 0.01%-0.5% (w / w), stir at 2-8℃ for 30-60 minutes until uniformly dispersed, adjust the pH to 6.8-7.4, filter through a 0.22μm filter membrane for sterilization, dispense into pre-filled syringes, and store at 2-8℃; Steps (2) to (5) are all performed under aseptic conditions.

8. The preparation method according to claim 7, characterized in that, The sol-gel transition temperature of the thermosensitive hydrogel in step (4) is 30-37℃; step (4) also includes adding decellularized ovarian extracellular matrix OvaECM to the polymer solution to a final concentration of 1-5 mg / mL.

9. The use of the gel according to any one of claims 1-6 in the preparation of a medicament for treating impaired ovarian reserve and / or ovarian damage caused by chemotherapy.

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