Use of combination of FGF-2 and flavokawain c in preparation of drug for treating uterine adhesions

Abstract

Provided in the present invention is use of a combination of FGF-2 and Flavokawain C in the preparation of a drug for treating uterine adhesions. In the present invention, a gel prepared using FGF-2 and Flavokawain C has a therapeutic effect on uterine adhesions, and can repair the morphology and function of the damaged uterus.

Description

Application of FGF-2 and kava piperine C in the preparation of drugs for treating intrauterine adhesions Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of the combined use of FGF-2 and kavapiperine C in the preparation of drugs for treating intrauterine adhesions. Background Technology

[0002] Over the past five years, the total number of induced abortions has hovered around 9.5 million annually, with an intrauterine adhesion rate as high as 30% to 40%, a very high incidence. In 2022, the incidence of intrauterine adhesions even reached 55%. For the past decade, the gold standard for treating intrauterine adhesions in clinical practice has been hysteroscopic adhesiolysis, followed by adjuvant hormone maintenance therapy or non-degradable balloons, intrauterine devices, and anti-adhesion agents. This has significantly improved the quality of life for patients with mild intrauterine adhesions. However, for patients with moderate to severe intrauterine adhesions, the above standard treatment regimen has little effect, with a postoperative re-adhesion probability as high as 62.5%.

[0003] Due to the increase in intrauterine surgeries in clinical practice, intrauterine adhesions have become one of the main causes of secondary infertility. Among infertile women undergoing hysterosalpingography (HSG) evaluation, 1.5% have this condition; among women with recurrent miscarriages, 5%–39% have it. Clinically, more than 48% of patients with intrauterine adhesions are diagnosed with moderate to severe intrauterine adhesions.

[0004] Current treatment methods are not ideal and have significant side effects. For example, hysteroscopic adhesiolysis has poor applicability and, due to limitations in operation and equipment, easily causes postoperative complications such as cervical laceration, perforation, bleeding, infection, and cervical insufficiency. Estrogen or progesterone therapy currently lacks internationally unified standards; excessively high dosages increase the likelihood of side effects, and the body easily develops tolerance. Non-degradable balloons and IUDs cause pain and discomfort to patients, resulting in poor patient compliance. Anti-adhesion agents currently used clinically are expensive and have generally limited efficacy. Therefore, there is an urgent clinical need for a highly effective, patient-compliant, and safe intrauterine treatment product. Summary of the Invention

[0005] The purpose of this invention is to provide the application of FGF-2 and kavapiperine C in the preparation of a drug for treating intrauterine adhesions.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of FGF-2 and kavapiperine C in the preparation of drugs for treating intrauterine adhesions.

[0008] Preferably, the mass ratio of FGF-2 to kavapiperine C is 1-5:5-1.

[0009] The present invention also provides a drug for treating intrauterine adhesions, comprising FGF-2 and kavapiperidine C.

[0010] Preferably, the formulation type of the drug includes tablets, capsules, powders, granules, creams, suppositories, or gels.

[0011] The present invention also provides a method for preparing the drug for treating intrauterine adhesions, wherein FGF-2, kava piperine C, gel solution and photoinitiator are mixed and solidified to obtain the drug for treating intrauterine adhesions.

[0012] Preferably, the ratio of FGF-2 to the gel solution is 0.5–1.5 mg: 0.09–1 mL;

[0013] Preferably, the ratio of kava piperine C to the gel solution is 0.5–1.5 mg: 0.09–1 mL.

[0014] Preferably, the mass ratio of the gel solution to the photoinitiator is 1:3 to 12.

[0015] Preferably, the gel solution is a methacrylamide hyaluronic acid hydrogel solution or a silk fibroin gel solution.

[0016] Preferably, the mass concentration of the gel solution is 0.8% to 1.2%.

[0017] Preferably, the curing method is: irradiation with 365nm ultraviolet light for 12-18s.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The kava piperine C (FKC) used in this invention is an active ingredient extracted from kava (Piper methysticum) and has significant anti-inflammatory effects. Fibroblast growth factors (FGFs) comprise 23 family members, making them the largest subfamily of cell growth factors. Among them, basic fibroblast growth factor (FGF2) is a recognized regenerative mechanism and has achieved remarkable success in the field of wound repair. However, whether FGF2 can play a role in the repair of endometrial trauma is unknown. Through preliminary literature review, the applicant discovered that FGF2 also has anti-inflammatory effects. Given the heterogeneous proliferation and strong inflammatory response of intrauterine adhesion wounds, we combined FGF2, FKC, and hyaluronic acid (HA) using photocrosslinking technology to provide an FGF2 composite gel suitable for intrauterine adhesions. Data verification showed that the gel prepared using FGF-2 and FKC in this invention has excellent therapeutic effects on intrauterine adhesions, effectively repairing the morphology and function of the damaged uterus and restoring fertility. In the composite gel prepared in this invention, FGF2 plays a role in endometrial repair and inhibits scar hyperplasia, FKC plays an anti-inflammatory role, and hyaluronic acid or silk fibroin plays a role in drug carrier and mechanical support-physical blockade.

[0020] The synergistic mechanism between FGF2 and FKC in this invention is as follows: Excessive inflammation caused by intrauterine adhesions leads to abnormal fibrosis and scar tissue formation. Especially during wound healing, the persistent infiltration of inflammatory cells and the release of cytokines stimulate fibroblast proliferation and excessive collagen deposition, resulting in scar hyperplasia. Therefore, controlling the degree and duration of the inflammatory response plays an important auxiliary role in enhancing FGF2's promotion of endometrial remodeling.

[0021] FGF2 can stimulate the proliferation and migration of fibroblasts and keratinocytes, accelerating wound healing and scar repair. During this process, FGF2 can also regulate the expression of various cytokines, such as inhibiting the release of pro-inflammatory factors and reducing local inflammatory responses, thereby mitigating the adverse effects of inflammation. Simultaneously, FGF2 has a regulatory effect on the function of immune cells, inhibiting excessive immune responses and reducing the risk of chronic inflammation. Therefore, the gel prepared by the method of this invention can promote the repair of the endometrium, restore the shape and physiological function of the uterus, and has broad application prospects. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 shows the good physical properties and sustained drug release properties of the FGF2-FKC-HAMA gel. (A) Changes in elastic modulus G' and viscous modulus G” with frequency. (B) Changes in elastic modulus G' and viscous modulus G” with time. (C) Shear strain. (D) Changes in elastic modulus G' and viscous modulus G” with cycling. (E) Changes in the mass of the gel in PBS with time. (F) Changes in the mass of the gel at 37°C with time. (G) Changes in the concentrations of FGF2 and FKC loaded in the gel in PBS with time.

[0024] Figure 2 shows the therapeutic effects of FKC-HAMA gel containing kava piperine and SAK-HAMA gel containing sakura extract on intrauterine adhesions. (A) Eosin-hematoxylin (H&E) staining experiment; (B) Quantitative analysis results.

[0025] Figure 3 shows that FGF2-FKC-HAMA significantly improved the endometrial morphology in rats with intrauterine adhesions (the basic shape of the uterus in each group was examined on the 14th day after surgery).

[0026] Figure 4 shows that FGF2-FKC-HAMA significantly alleviated the degree of uterine inflammation in rats with intrauterine adhesions. (A) Immunohistochemical experiment diagram; (B) Quantitative statistical diagram of IL-6 expression level; (C) Quantitative statistical diagram of TNF-α expression level.

[0027] Figure 5 shows the increased number of endometrial glands, decreased fibrosis, and significantly improved endometrial morphology in rats with intrauterine adhesions after FGF2-FKC-HAMA administration. Endometrial sections from the control group, IUA model group, HAMA gel group, FKC-HAMA gel group, FGF2-HAMA gel group, and FGF2-FKC-HAMA gel group were analyzed using H&E staining. Masson staining was also used to analyze the endometrial sections from the same groups.

[0028] Figure 6 shows the excellent biocompatibility of FGF2-FKC-HAMA gel. (A) Chicken embryo experiment: The control group, HAMA gel group, NaOH group, bFGF2-HAMA gel group, FKC-HAMA gel group, and bFGF2-FKC-HAMA gel group were tested for induced vascular rupture in chicken embryos. (B) Hemolysis experiment: The hemolysis of each group was tested. The control group was treated with 0.9% physiological saline, while the others were treated with HAMA gel, NaOH (1M), bFGF2-HAMA gel, FKC-HAMA gel, and bFGF2-FKC-HAMA gel, respectively. Hemolysis experiments were conducted, and the corresponding hemolysis rates were measured to obtain the hemolysis rates under different formulations. Detailed Implementation

[0029] This invention provides the application of FGF-2 and kavapiperine C in the preparation of drugs for treating intrauterine adhesions.

[0030] In this invention, the mass ratio of FGF-2 to kavapiperine C is 1-5:5-1; preferably 2-4:5-1; more preferably 2-4:2-4; and even more preferably 1:1.

[0031] The present invention also provides a drug for treating intrauterine adhesions, comprising FGF-2 and kavapiperidine C.

[0032] In this invention, the formulation type of the drug includes tablets, capsules, powders, granules, creams, suppositories, or gels; preferably, gels.

[0033] The present invention also provides a method for preparing the drug for treating intrauterine adhesions, wherein FGF-2, kava piperine C, gel solution and photoinitiator are mixed and solidified to obtain the drug for treating intrauterine adhesions.

[0034] In this invention, the ratio of FGF-2 to gel solution is 0.5-1.5 mg: 0.09-1 mL; preferably 0.7-1.3 mg: 1 mL; more preferably 0.9-1.1 mg: 1 mL; and even more preferably 1 mg: 1 mL.

[0035] In this invention, the ratio of kava piperine C to the gel solution is 0.5–1.5 mg: 0.09–1 mL; preferably 0.7–1.3 mg: 1 mL; more preferably 0.9–1.1 mg: 1 mL; and even more preferably 1 mg: 1 mL.

[0036] In this invention, the mass ratio of the gel solution to the photoinitiator is 1:3 to 12; preferably 1:9 to 11; and more preferably 1:10.

[0037] In this invention, the gel solution is a methacrylamide hyaluronic acid hydrogel solution or a silk fibroin gel solution; preferably, it is a methacrylamide hyaluronic acid hydrogel solution.

[0038] In this invention, the mass concentration of the gel solution is 0.8–1.2%; preferably 0.9–1.1%; more preferably 1%.

[0039] In this invention, the curing method is as follows: irradiation with 365nm ultraviolet light for 12-18s; preferably 13-17s; more preferably 14-16s; and more preferably 15s.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Example 1

[0042] (1) Preparation of methacrylamide hyaluronic acid (HAMA) gel: 1 g of hyaluronic acid was added to 100 mL of sterile triple-distilled water and stirred to dissolve. Then, 3 mL of methacrylic anhydride (MA) was added and the mixture was stirred at room temperature for 8 h. During this period, the pH of the solution was maintained at 8.5 using sodium hydroxide solution. After the reaction was completed, the solution was placed in a dialysis bag (8000-12000 Da) and dialyzed for 3 days, with the water changed 4 times a day. After dialysis, the HAMA solution was obtained. The obtained HAMA solution was placed in an oven and concentrated to a concentration of 1.1%, which is the desired HAMA.

[0043] (2) Take 90 μL of 1.1% HAMA solution, 10 mg of FGF2 solution and 30 mg of kava piperine C into an EP tube, add 20 μL of photoinitiator LAP, stir and mix well, and irradiate with a 396 nm ultraviolet light for 15 s to obtain FGF2-FKC-HAMA composite gel.

[0044] Example 2

[0045] Preparation of silk fibroin (SFMA) gel:

[0046] 1) Heat a beaker containing 2L of water in an oil bath, add 4.24g of anhydrous sodium carbonate, stir and mix well, then weigh 2g of silkworm cocoons and cut them into small pieces. When the water temperature in the beaker reaches above 90℃, add the cut silkworm cocoon pieces and continue heating for 1 hour, stirring occasionally.

[0047] 2) After heating is complete, remove the beaker, filter out the silk fibroin (SF), and wash it with water. Repeat step 1) with the filtered silk fibroin and filter it again. Then place the silk fibroin in 2L of pure water and heat it at 100℃ for 1 hour. After heating, wash it with pure water and dry it.

[0048] 3) Weigh 1.4 g of dried silk fibroin, shred it, pack it into a glass bottle, add 7 mL of pure water and 5.65 g of lithium bromide, and heat in an oil bath at 65 °C for 1 h to dissolve. After dissolution, filter the solution, and add 700 μL of methacrylic anhydride (GMA) to the filtered solution. Stir the reaction at 60 °C for 3 h. After the reaction is complete, dialyze for 3 days, then dry and concentrate to obtain SFMA.

[0049] 4) FGF2 and kava piperine C (FKC) were mixed with 1% SFMA solution at a concentration of 1 mg / mL, and 4% LAP (photoinitiator) was added at a ratio of LAP:SFMA = 1:10. After mixing, the mixture was irradiated with 365 nm ultraviolet light for 15 s to obtain FGF2-FKC-SFMA composite gel.

[0050] Experimental Example 1

[0051] Gel rheological property testing: FGF2-FKC-HAMA composite gel (prepared in Example 1) was prepared into cylindrical samples with a diameter of 8 mm and a height of 5 mm. Its rheological properties were evaluated by performing frequency, duration, strain, and cycle scanning tests on a rheometer. An 8 mm diameter conical plate was used for the experiment, and the sample was placed on the platform, aligned with the conical plate. The temperature was kept constant at 25 °C, and the tests were performed at a fixed tension of 1% for 0.1–100 rad / s. -1 Dynamic frequency scanning within the range of 0 to 150 s and dynamic time scanning within the range of 0 to 150 s were performed. At a frequency of 0.1 Hz, the shear strain response rheological behavior of the hydrogel was studied, the critical point of the gel state was obtained, and dynamic cyclic scanning was obtained through the critical point.

[0052] Rheological experiments showed that FGF2-FKC-HAMA remained relatively stable under different pressures with varying time and frequency (Figures 1A and 1B), and exhibited good self-healing ability under different pressures (Figures 1C and 1D). Furthermore, FGF2-FKC-HAMA demonstrated good swelling and anti-swelling properties, indicating good water absorption (Figures 1E and 1F). Our drug release experiments confirmed that FGF2-FKC-HAMA has a sustained-release effect on drugs, with a sustained-release time of up to 24 hours for FGF2 and FKC, demonstrating excellent sustained-release efficacy (Figure 1G). In summary, the gel exhibits good stability and self-healing properties, excellent water absorption, and superior sustained-release effect.

[0053] Experiment Example 2

[0054] Construction and administration of a rat model of endometrial injury and adhesion: SPF-grade female SD rats weighing 250–300 g were used. Vaginal smears were observed daily. Female rats in the interestrus phase were selected for model construction. After anesthesia, the lower abdomen of the rats was disinfected, and the skin was incised 1 cm above the urethra to enter the abdominal cavity and expose the uterus. An incision was made at the bifurcation of the Y-shaped uterus, and the endometrium was scraped away using a curette, closely simulating intrauterine adhesions (IUA) induced by multiple abortions in clinical practice.

[0055] After curettage (i.e., successful IUA modeling), rats were divided into the following 6 groups (control group, model group, HAMA gel group (compared to Example 1, except without FGF2 and kava piperine C), FKC-HAMA gel group (compared to Example 1, except without kava piperine C), bFGF2-HAMA gel group (compared to Example 1, except without kava piperine C), and bFGF2-FKC-HAMA gel group). A composite gel containing various components was injected using a syringe (approximately 2 mL of gel was sufficient to fill one side of the rat uterus). The wound was sutured under sterile conditions, and after two weeks of continuous drug administration, the rats were suddenly euthanized. Their uteruses were then harvested for various pathological and inflammatory marker tests.

[0056] (1) Selection of kava piperine C (FKC)

[0057] To screen for the most effective natural product active ingredients for treating intrauterine adhesions, we further determined the in vivo therapeutic effects of two natural products, kava piperine C (FKC) and sakura extract (SAK), obtained through high-throughput screening of their in vitro anti-inflammatory activities, on intrauterine adhesions. After successfully establishing an intrauterine adhesion (IUA) model in rats via curettage, the rats were divided into five groups (control group, model group, HAMA gel group, FKC-HAMA gel group, and SAK-HAMA gel group (compared to Example 1, kava piperine C was replaced with an equal amount of SAK)). A composite gel containing various components was injected using a syringe (approximately 2 mL of gel was sufficient to fill one side of the rat uterus). Two weeks after gel treatment, the rats died suddenly, and their uteruses were harvested for H&E staining experiments, and the number of glands in the pathological sections was counted.

[0058] The experimental results are shown in Figure 2A below. In the control group, the nucleoplasm distribution in the uterine cavity tissue was uniform. In the model group, severe nucleoplasm adhesions were observed in the uterine cavity tissue. The nucleoplasm adhesions in the HAMA gel treatment group were alleviated compared to the model group. The FKC-HAMA gel group significantly alleviated the nucleoplasm adhesions, restoring them to the level of the control group, indicating that FKC has a good therapeutic effect on uterine cavity adhesions. However, the SAK-HAMA gel group showed comparable relief of nucleoplasm adhesions to the HAMA monotherapy group, indicating that SAK cannot effectively alleviate nucleoplasm adhesions. Further statistical results on the number of glands are shown in Figure 2B: the control group had a higher number of glands on the slices, with nearly 18 glands per square centimeter of field of view, while the model group had a significantly reduced number of glands, with only 3 glands per square centimeter of field of view. Compared with the model group, HAMA gel alone could slightly increase the number of glands, but the effect was not significant. The FKC-HAMA gel treatment group could significantly increase the number of glands, basically restoring it to the level of the control group. However, the effect of SAK-HAMA gel on the number of glands was comparable to that of HAMA gel, indicating that SAK cannot effectively increase the number of glands.

[0059] In conclusion, FKC is the best natural active ingredient for preventing intrauterine adhesions.

[0060] (2) In order to investigate the therapeutic effect of FGF2-FKC-HAMA gel on intrauterine adhesions, we examined the recovery of the rat uterus on the 14th day after surgery. The results showed that the uterus of the rat in the normal group was regular and full, while the uterus of the rat in the model group had severe adhesions and could not maintain a good full shape. Although the adhesions of the rat uterus in the HAMA gel treatment group and the FKC-HAMA gel treatment group were improved, there were still many local adhesions. The uterus shape of the rat in the bFGF2-HAMA gel treatment group was better improved. The uterus shape of the rat in the bFGF2-FKC-HAMA gel treatment group recovered significantly better than that in the bFGF2-HAMA gel treatment group, and was basically no different from that in the normal group (Figure 3).

[0061] (3) FGF2-FKC-HAMA gel significantly reduced the inflammatory response of the endometrium in rats with intrauterine adhesions.

[0062] Since inflammation is a major contributing factor to the formation and re-adhesion of intrauterine adhesions, effective control of inflammation is crucial for the treatment of intrauterine adhesions. Therefore, we investigated the degree of inflammation in each group of samples using immunohistochemistry and quantitative fluorescence PCR. The results showed that the degree of endometrial inflammation in rats with intrauterine adhesions was very high. HAMA gel alone could not effectively alleviate endometrial inflammation. FKC and bFGF2 alone could alleviate the degree of endometrial inflammation to some extent, but the effect was very limited. However, the combined use of FKC and bFGF2 could significantly alleviate the degree of endometrial inflammation in rats, gradually restoring it to normal levels (Figure 4).

[0063] (4) FGF2-FKC-HAMA gel increased the number of endometrial glands and decreased the degree of fibrosis in rats with intrauterine adhesions.

[0064] To investigate the therapeutic effect of FGF2-FKC-HAMA gel on intrauterine adhesions in rats, we observed the structural changes of the endometrium from a morphological perspective. Hematoxylin and eosin (H&E) staining results showed that, although the number of endometrial glands and the uterine thickness were significantly lower in the FGF2-HAMA and FKC-HAMA gel treatment groups compared to the normal group, the number of endometrial glands and the endometrial thickness were significantly increased in the FGF2-HAMA and FKC-HAMA treatment groups compared to the IUA model group. However, the number of endometrial glands and the endometrial thickness in the bFGF2-FKC-HAMA gel treatment group had returned to normal levels. Endometrial fibrosis is a major histopathological feature of the progression of intrauterine adhesions; therefore, we performed Masson staining on rat endometrial sections to evaluate the improvement of endometrial fibrosis by FGF2-FKC-HAMA gel on rat endometrial fibrosis. Masson staining results showed that, compared with the IUA model group, endometrial fibrosis in the FGF2-HAMA gel and FKC-HAMA gel treatment groups was reversed to a certain extent, and the degree of fibrosis decreased; endometrial fibrosis in the bFGF2-FKC-HAMA gel treatment group was completely reversed to a level comparable to that of the normal group (Figure 5).

[0065] Experimental Example 3

[0066] Biosafety is a critical consideration in drug development. The chorioallantoic membrane (CAM) of chicken embryos is a highly vascularized, non-nervated embryonic outer membrane, and also a naturally dedeficient host. CAM has a long history of being used as an in vivo experimental platform for drug safety assessment. Therefore, we first investigated the biosafety of the prepared gel using chicken embryo experiments. The specific experimental method is as follows: Hatching eggs were purchased from Taobao and incubated at 37°C for 9–11 days. After examination with a light bulb, the air cell boundary was drawn. A mark was made approximately 1 mm above the air cell boundary, avoiding blood vessels; this is the injection point. The area around the point was disinfected with 2.5% iodine solution, then deiodized with 75% alcohol. A small hole was punched at the injection point, taking care not to damage the shell membrane. 0.2 mL of each type of gel was injected using a syringe, then sealed with melted paraffin wax, and the mixture was returned to the 37°C incubator for another 6 hours. The paraffin wax was removed, and the condition of the blood vessels was examined. The specific gel groups were as follows: the control group was given 0.9% physiological saline, and the others were HAMA gel group, NaOH group (concentration of 1M), bFGF2-HAMA gel group, FKC-HAMA gel group, and bFGF2-FKC-HAMA gel group, respectively.

[0067] Chicken embryo experiments showed that bFGF2-FKC-gel did not cause capillary rupture in chicken embryos (Figure 6A), indicating its very high biocompatibility. Simultaneously, hemolysis experiments showed that bFGF2-FKC-gel hardly caused hemolysis (Figure 6B). These experimental results fully demonstrate the high biocompatibility of bFGF2-FKC-gel.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of FGF-2 and kava piperine C in the preparation of drugs for treating intrauterine adhesions.

2. Use according to claim 1, characterized in that, The mass ratio of FGF-2 to kavapiperine C is 1-5:5-1.

3. A medicament for treating intrauterine adhesion, characterized by, Including FGF-2 and kavapiperidine C.

4. The medicament according to claim 3, characterized in that, The formulation types of the drug include tablets, capsules, powders, granules, creams, suppositories, or gels.

5. A method of preparing a medicament for the treatment of intrauterine adhesions according to claim 3 or 4, characterized in that, FGF-2, kava piperine C, gel solution and photoinitiator are mixed and cured to obtain a drug for treating intrauterine adhesions.

6. The production method according to claim 5, wherein The ratio of FGF-2 to the gel solution is 0.5–1.5 mg: 0.09–1 mL; the ratio of kavapiperidine C to the gel solution is 0.5–1.5 mg: 0.09–1 mL.

7. The preparation method according to claim 5, characterized in that, The mass ratio of the gel solution to the photoinitiator is 1:3 to 12.

8. The preparation method according to claim 5, characterized in that, The gel solution is a methacrylamide hyaluronic acid hydrogel solution or a silk fibroin gel solution.

9. The preparation method according to claim 5, characterized in that, The mass concentration of the gel solution is 0.8%–1.2%.

10. The method of claim 5, wherein, The curing method is as follows: irradiate with 365nm ultraviolet light for 12-18s.

Images