Temperature-sensitive hydroxybutyl polyvinyl alcohol, slow-release hydrogel, preparation method and application

Abstract

The invention discloses temperature-sensitive hydroxybutyl polyvinyl alcohol, sustained-release hydrogel and a preparation method and application thereof, and belongs to the field of pharmaceutical preparations. Temperature-sensitive hydroxybutyl polyvinyl alcohol is synthesized by grafting modified polyvinyl alcohol in an alkaline aqueous solution through epoxybutane, no organic solvent is used in the preparation process, and the preparation method is simple, economical and convenient. When the grafting substitution degree of the hydroxybutyl polyvinyl alcohol is 20%-60%, the hydroxybutyl polyvinyl alcohol is dissolved in deionized water or normal saline at a low temperature, progesterone microcrystals are added and uniformly stirred to obtain a drug-loaded gel precursor dispersion liquid, gel transformation is performed in a use environment at a gel transformation temperature or above, and the progesterone vaginal sustained-release hydrogel is formed. The gel is soluble in water at a low temperature, has good fluidity, can be conveniently filled into tissues, and forms gel at a body temperature, and has good tissue mucosa retention. As a progesterone vaginal sustained-release carrier material, the hydrogel disclosed by the invention is expected to be applied to development of products for preventing or treating gynecological diseases or assisting fertility.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations and relates to a sustained-release pharmaceutical agent, specifically to a thermosensitive hydroxybutyl polyvinyl alcohol, a sustained-release hydrogel, its preparation method, and its application. The thermosensitive modified polyvinyl alcohol is used as a pharmaceutical excipient to prepare a progesterone vaginal sustained-release hydrogel. Background Technology

[0002] Progesterone is a natural progestin secreted by the ovaries, placenta, and adrenal glands. It is an important component in regulating the female reproductive system, primarily acting on the uterus, ovaries, breasts, and central nervous system. Endogenous progesterone can transform the endometrium from the proliferative phase to the secretory phase, maintaining normal physiological changes. Clinically, progesterone is mainly used to treat threatened miscarriage, luteal insufficiency, and for assisted reproduction after embryo transfer. Progesterone is also often used in combination with estrogen as hormone replacement therapy, which can effectively avoid the adverse reactions of estrogen, prevent endometrial hyperplasia caused by long-term estrogen use, and reduce the risk of endometrial cancer. Currently, clinically used dosage forms include capsules, oil injections, vaginal suppositories, and vaginal gels. However, due to water insolubility and the first-pass effect of the liver, oral absorption of progesterone is poor, with a bioavailability of less than 10%. Oil injections are prone to causing adverse reactions such as local irritation, pain, local redness, swelling, induration, sterile cysts, inflammation, or allergies after injection, and frequent administration is inconvenient for patients, reducing patient compliance and having significant limitations. Vaginal gels can successfully bypass the first-pass effect, avoiding irritation and other adverse reactions. Furthermore, because the administration site is close to the target organ, the uterus, it has a certain degree of targeting, making it one of the better formulations currently on the market. This product was approved for entry into China in 2008 under the brand name Crinone, with a strength of 8% (90 mg). As of October 30, 2024, all marketing applications for progesterone vaginal extended-release gel in China, at least eight in total, have been rejected, meaning this product has been definitively rejected. Besides domestic and international markets, no generic versions of progesterone vaginal extended-release gel have yet been approved by the FDA.

[0003] Studies have shown that the preparation of these types of progesterone vaginal sustained-release gels suffers from poor mixing and dispersion uniformity, easily leading to progesterone raw materials agglomerating and failing to disperse, thus failing to guarantee the expected sustained-release effect. Therefore, it is necessary to first effectively solve the problem of mixing uniformity, and secondly, to improve the gel's ability to spread sufficiently and uniformly on the vaginal mucosa and adhere tightly (vaginal mucosal adhesion) to effectively prolong drug retention time, control drug release, and improve drug bioavailability. The main challenge here lies in using suitable excipients. Of course, existing progesterone vaginal sustained-release gels also have drawbacks such as vaginal leakage, irritation, itching, discharge, bleeding, difficulty or pain during intercourse, breast tenderness, and inaccurate absorption dosage. Therefore, the research and development of progesterone vaginal sustained-release carrier materials with simple preparation methods, the use of more environmentally friendly and low-toxicity excipients, convenient and uniform application, and better vaginal mucosal adhesion is highly anticipated. Summary of the Invention

[0004] One objective of this invention is to prepare a thermosensitive hydroxybutyl polyvinyl alcohol. By modifying polyvinyl alcohol with epoxide, hydroxybutyl polyvinyl alcohol is obtained. The synthesis process is simple, without organic solvents, and has good biocompatibility. It can gel at body temperature and transform into a stable hydrogel, making it an excellent sustained-release drug carrier.

[0005] Another objective of this invention is to prepare a progesterone vaginal sustained-release hydrogel based on the above-mentioned thermosensitive hydroxybutyl polyvinyl alcohol. The prepared progesterone vaginal sustained-release hydrogel has good biocompatibility, excellent adhesion and sustained-release ability.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: On one hand, the present invention provides a thermosensitive hydroxybutyl polyvinyl alcohol having the following general structural formula:

[0007] Where n is the degree of polymerization of polyvinyl alcohol, m is the number of units grafted with hydroxybutyl groups, and n, m and a are all positive integers. Thermosensitive hydroxybutyl polyvinyl alcohol is soluble in water or physiological saline at low temperatures, and its gelation transition temperature is lower than body temperature.

[0008] Preferably, the degree of polymerization of the polyvinyl alcohol is 500-7000; that is, the value of n is in the range of 500-7000. If the value is too small, the gel strength is poor, the sustained release ability is insufficient, and there is no temperature sensitivity; while if the value is too large, it is difficult to dissolve when loaded with drugs.

[0009] Preferably, the degree of polymerization of the polyvinyl alcohol is 1500-4500; that is, the value of n is in the range of 1500-4500.

[0010] Preferably, the hydroxybutyl grafting rate of the thermosensitive hydroxybutyl polyvinyl alcohol is 10%-70%, and its gelation transition temperature is 1-36℃. The grafting rate has a direct impact on the gelation transition temperature. If the gelation transition temperature is too low, it will cause difficulty in dissolving the drug during loading and the sustained release time after gel formation will be too long. If the gelation transition temperature is too high, it will not be able to form a stable hydrogel under body temperature conditions, and the sustained release time will be too short. Therefore, it is necessary to maintain a suitable gelation temperature transition range through an appropriate grafting rate.

[0011] Preferably, the hydroxybutyl grafting rate of the thermosensitive hydroxybutyl polyvinyl alcohol is 15%-60%, and its gelation transition temperature is 15-30℃.

[0012] On the other hand, the present invention provides a method for preparing thermosensitive hydroxybutyl polyvinyl alcohol, the specific steps of which are as follows: Step 1: Dissolve polyvinyl alcohol in water to obtain a polyvinyl alcohol aqueous solution; Step 2: Add epoxide and an alkaline catalyst to carry out the grafting reaction; Step 3: After the reaction, acid is added for neutralization, and the product is purified and dried to obtain hydroxybutyl polyvinyl alcohol.

[0013] Preferably, in step 1, the mass concentration of the polyvinyl alcohol aqueous solution is 1-10%.

[0014] Preferably, in step 1, the polyvinyl alcohol is dissolved at a temperature of 70-90°C, and then cooled to the grafting reaction temperature after dissolution.

[0015] More preferably, in step 1, the mass concentration of the polyvinyl alcohol aqueous solution is 2-8%.

[0016] Preferably, in step 2, the molar ratio of the structural units of polyvinyl alcohol to the added epoxide is 1:5-40.

[0017] More preferably, in step 2, the molar ratio of the structural units of polyvinyl alcohol to the added epoxide is 1:10-30.

[0018] Preferably, in step 2, the grafting reaction temperature is 20-60℃.

[0019] More preferably, in step 2, the grafting reaction temperature is 20-40℃.

[0020] Preferably, in step 2, the alkaline catalyst is any one or more of sodium hydroxide, potassium hydroxide, and calcium hydroxide.

[0021] More preferably, in step 2, the alkaline catalyst is sodium hydroxide, and the NaOH mass concentration is 2-15%.

[0022] More preferably, in step 2, the alkaline catalyst is sodium hydroxide, and the NaOH mass concentration is 5-10%.

[0023] Preferably, in step 3, the acid added after the reaction is any one or a combination of dilute hydrochloric acid, dilute sulfuric acid, and dilute nitric acid, and the pH value after neutralization is about 6-7.

[0024] Preferably, in step 3, the purification is dialysis, through which unreacted small molecules are removed. More preferably, the molecular weight cutoff of the dialysis bag is Mw 8000-14000.

[0025] Preferably, in step 3, the drying is freeze-drying.

[0026] On the other hand, the present invention provides an application of thermosensitive hydroxybutyl polyvinyl alcohol for the preparation of progesterone vaginal sustained-release hydrogel.

[0027] On the other hand, the present invention provides a method for preparing a progesterone vaginal sustained-release hydrogel, comprising the following steps: S1. Dissolve hydroxybutyl polyvinyl alcohol (obtained by lyophilization) in deionized water or physiological saline to obtain an aqueous polymer solution; S2. Add progesterone microcrystals and stir well to obtain a drug-loaded gel precursor dispersion that can be converted into a gel above the gel transition temperature.

[0028] The drug-loaded gel precursor dispersion was placed in an environment above the gel transition temperature to gel and transform into a progesterone vaginal sustained-release hydrogel.

[0029] Preferably, in step S1, the mass concentration of the polymer aqueous solution is 5%-30%.

[0030] More preferably, in step S1, the mass concentration of the polymer aqueous solution is 15%-22%.

[0031] Preferably, in step S2, the drug loading concentration in the drug-loaded gel precursor dispersion is 4-12%.

[0032] More preferably, in step S2, the drug loading concentration in the drug-loaded gel precursor dispersion is 8-10%.

[0033] Preferably, in step S2, the particle size of the progesterone microcrystals is 2-15 micrometers, more preferably 6-10 micrometers.

[0034] In use, the drug-loaded gel precursor dispersion is added to or injected into the environment where it is used. The temperature of the environment is above the gel transition temperature. After injection, the drug-loaded gel precursor dispersion is converted into a progesterone vaginal sustained-release hydrogel.

[0035] On the other hand, the present invention provides an application of progesterone vaginal sustained-release hydrogel for the preparation of products for the prevention or treatment of gynecological diseases or for assisted reproduction.

[0036] Compared with the prior art, the present invention has the following beneficial effects: 1. Because the hydroxybutyl polyvinyl alcohol of the present invention is thermosensitive, it dissolves in water at low temperatures and has good fluidity, which can be easily filled into tissues. At body temperature, it forms a gel and has good tissue and mucosal retention, which makes it easy to load drugs evenly and achieve a long-acting vaginal sustained-release effect.

[0037] 2. The preparation method of this invention is simple, synthesizing thermosensitive hydroxybutyl polyvinyl alcohol by grafting butane onto polyvinyl alcohol in an alkaline aqueous solution. The preparation process does not use organic solvents, making it simple, economical, and convenient. Drug loading also uses only an aqueous phase system, avoiding the use of an oil phase system, which is convenient, safe, and has commercial application potential. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in this 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 some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1a The above is the 1H NMR spectrum of hydroxybutyl polyvinyl alcohol HBPVA-24 with a hydroxybutyl substitution degree of 24% in deuterated DMSO in Example 1 of the present invention. Figure 1b The image shows the 1H NMR spectrum of hydroxypropyl polyvinyl alcohol HPPVA-21 with a hydroxypropyl substitution degree of 21% in deuterated DMSO in Comparative Example 1 of this invention.

[0040] Figure 2 This is an image showing the thermosensitive transition of hydroxybutyl polyvinyl alcohol (HBPVA-52) synthesized in Example 1 of this invention at a concentration of 20 wt% in physiological saline.

[0041] Figure 3 The ease of use test of the hydrogel (20 wt%) in Example 2 of the present invention: a) PVA, b) hydroxybutyl polyvinyl alcohol HBPVA-53, and c) F127 hydrogel.

[0042] Figure 4Adhesion tests of hydrogel F127 (20 wt%) and hydroxybutyl polyvinyl alcohol HBPVA-53 (20 wt%) in Example 2: Photographs of hydrogels adhering to cellophane (a) and adhesion time (b), hydrogels adhering to glass slides (c) and adhesion time (d), hydrogels adhering to vaginal tissue before and after rinsing (e), and residual mass of the sample in vaginal tissue (f).

[0043] Figure 5 The drug release curves of hydroxybutyl polyvinyl alcohol HBPVA-53 hydrogels loaded with different concentrations of progesterone in Example 3 are shown.

[0044] Figure 6A The images show fluorescence images of a fluorescent hydroxybutyl polyvinyl alcohol polymer solution / gel (20 wt%, right) with a fluorescein grafting rate of 0.13% and a non-fluorescein-grafted hydroxybutyl polyvinyl alcohol polymer solution / gel (20 wt%, left, HBPVA-53) at different temperatures in Example 4. Figure 6B The images show fluorescence images of a fluorescent hydroxybutyl polyvinyl alcohol polymer solution / gel (20 wt%) with a fluorescein grafting rate of 0.13% and small molecule fluorescein sodium carboxylate saline in rat vaginal sites, used in Example 4 to investigate the retention time of these solutions.

[0045] Figure 7 Images of the uterus after endometrial hyperplasia in rats from the untreated group (a), the oily intramuscular injection group (b), and the vaginal gel group (c) in Example 5.

[0046] Figure 8 The wet weight of the uterus of rats with endometrial hyperplasia in the untreated group, the oily intramuscular injection group, and the vaginal gel group in Example 5 is shown below. Figure 9 The uterine index of rats with endometrial hyperplasia in the untreated group, the oily intramuscular injection group, and the vaginal gel group in Example 5 is shown below. Figure 10 HE staining and magnified images of uterine sections from rats with endometrial hyperplasia in the untreated group, the oil-based intramuscular injection group, and the vaginal gel group in Example 5. Figure 11 The endometrial thickness of rats with endometrial hyperplasia in the untreated group, the oily intramuscular injection group, and the vaginal gel group in Example 5 is shown below. Figure 12 The thickness of the uterine epithelium in rats with endometrial hyperplasia after treatment in the untreated group, the oily intramuscular injection group, and the vaginal gel group in Example 5. Detailed Implementation To make the present invention easier to understand, specific embodiments of the present invention will be further described below.

[0047] The present invention will be further described below with reference to embodiments and accompanying drawings, the purpose of which is to help to better understand the content of the present invention, but these specific embodiments do not limit the scope of protection of the present invention in any way.

[0048] Example 1: Synthesis and Characterization of Thermosensitive Hydroxybutyl Polyvinyl Alcohol Accurately weigh 1.0 g of polyvinyl alcohol (PVA, type 1799, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and add it to 29 g of deionized water. Stir at room temperature for 30 minutes, then heat to 70-90℃ and continue stirring for 1 h until completely dissolved to obtain a PVA solution (3.33 wt%). Cool to room temperature, then add 20 g of 20% NaOH aqueous solution dropwise and stir until well mixed to obtain a 2% PVA solution containing 8% NaOH. Add 1,2-epoxybutane (BO) at a molar ratio of 1:10 to the PVA structural unit, and react at 25℃ for 24 h. Adjust the pH of the reaction system to 7 with 3 mol / L hydrochloric acid to terminate the reaction. Dialyze and freeze-dry to obtain a white sponge-like hydroxybutyl polyvinyl alcohol (HBPVA). Dissolve 5 mg of the hydroxybutyl polyvinyl alcohol solid sponge in 500 μL of deuterated DMSO and test the 1H NMR spectrum. Figure 1a The image shows HBPVA-24 with a unit feed ratio of 10:1 for epoxide and polyvinyl alcohol. 1 The ¹H NMR spectrum shows chemical shifts (f1) on the horizontal axis. Peaks 0.96-1.09 ppm (4 peaks) correspond to the methyl groups after ring-opening of butane oxide; peak 1.11-1.87 ppm (1 peak) corresponds to the methylene groups in the polyvinyl alcohol main chain and the methylene groups directly attached to the methyl groups after ring-opening of butane oxide; peak 2.25-3.92 ppm (2 peaks) corresponds to the methine groups in the polyvinyl alcohol main chain and the methylene and methine groups directly attached to the oxygen atoms after ring-opening of butane oxide; peak 3.16-5.17 ppm (3 peaks) corresponds to the branched hydroxyl groups and the terminal hydroxyl groups of the grafted butane oxide. The disappearance of the terminal methyl groups of the branched acetyl groups in the PVA raw material, indicating that the original branched acetyl groups were hydrolyzed after 24 h of reaction under alkaline conditions, demonstrates that butane oxide was successfully grafted onto the hydroxyl groups of the polyvinyl alcohol side chain. Based on the integral area A4 of peak 4 and the integral area A1 of peak 1, its degree of hydroxybutyl substitution (degree of hydroxybutyl substitution = (A4 / 3) / [(A1 - 2×A4 / 3) / 2]) can be calculated to be approximately 24%, denoted as HBPVA-24.

[0049] The feed ratios of butane oxide and polyvinyl alcohol (PVA) units were adjusted as shown in Table 1. With increasing butane oxide to PVA unit feed ratio, the grafting rate of the resulting hydroxybutyl polyvinyl alcohol (HBPVA) increased. These polymers are soluble in water at low temperatures, but their viscosity increases significantly with increasing temperature, even forming a non-flowing gel, exhibiting temperature sensitivity. When the butane oxide to PVA unit feed ratio was 30:1, and the reaction was carried out at 40℃ for 24 h, a hydroxybutyl polyvinyl alcohol (HBPVA-52) with a grafting rate of 51.5% was obtained using PVA1799. Rheological testing showed that the temperature at which the tangents of the viscosity-temperature change curve intersected at a polymer concentration of 20 wt% corresponded to its gelation transition temperature: 26.5℃.

[0050] Table 1 Grafting rate of HBPVA synthesized with different feed ratios

[0051] Note: The reaction time is fixed at 24 hours. The degree of grafting substitution (DS) is measured by hydrogen nuclear magnetic resonance spectroscopy.

[0052] Weigh approximately 200 mg of hydroxybutyl polyvinyl alcohol (HBPVA-52) and add it to approximately 800 mg of 0.15 mol / L phosphate buffer. Stir well at 4°C, then immediately remove the sample vial and tilt it to take a picture. Heat the sample solution to 37°C and incubate for 60 seconds, then invert the sample vial and take another picture. Figure 2 The figure shows that the 20% aqueous solution maintains good fluidity at 4°C. However, when heated to 37°C, the fluidity decreases and the solution solidifies at the bottom of the bottle.

[0053] Using PVA1788 instead of PVA1799, with all acetyl groups hydrolyzed, hydroxybutyl polyvinyl alcohol (HBO) with different grafting rates can also be obtained (with the same feed ratio and grafting rate). Using a larger molecular weight PVA (M146000-186000, degree of hydrolysis 99.3-100.0%) instead of PVA1799, with a butane oxide to PBO unit feed ratio of 30:1, and reacting at 25℃ for 24 hours, HBO HBPVA-25 with a grafting rate of 25% can also be obtained. Reacting at 40℃ for 24 hours yields HBO HBPVA-53 with a grafting rate of 53.1%. Rheological testing shows that the temperature at which the tangents of the viscosity-temperature change curve intersect at a polymer concentration of 20 wt% corresponds to its gelation transition temperature: 26.7℃.

[0054] Comparative Example 1: Synthesis and Characterization of Hydroxypropyl Polyvinyl Alcohol Accurately weigh 1.0 g of polyvinyl alcohol (PVA, type 1799, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) and add it to 29 g of deionized water. Stir at room temperature for 30 minutes, then heat to 70-90℃ and continue stirring for 1 h until completely dissolved to obtain a PVA solution (3.33 wt%). Cool to room temperature, then add 20 g of 20% NaOH aqueous solution dropwise and stir until well mixed to obtain a 2% PVA solution containing 8% NaOH. Add propylene oxide at a molar ratio of 1:10 to the PVA structural unit and react at 25℃ for 24 h. Adjust the pH of the reaction system to 7 with 3 mol / L hydrochloric acid to terminate the reaction. Dialyze and freeze-dry to obtain a white, sponge-like hydroxypropyl polyvinyl alcohol (HPPVA). Dissolve 5 mg of the hydroxypropyl polyvinyl alcohol solid sponge in 500 μL of deuterated DMSO and test the 1H NMR spectrum as shown below. Figure 1b As shown, its 1 In the 1H NMR spectrum, chemical shifts of 0.96–1.09 ppm correspond to the methyl groups after ring-opening of propylene oxide; chemical shifts of 1.11–1.87 ppm correspond to the methylene groups in the main chain of polyvinyl alcohol; chemical shifts of 3.25–3.92 ppm correspond to the methine groups in the main chain of polyvinyl alcohol and the methylene and methine groups directly attached to oxygen atoms after ring-opening of propylene oxide; and chemical shifts of 1.88–2.03 ppm correspond to the disappearance of the terminal methyl groups of the branched acetyl groups in the raw material PVA, indicating that the original branched acetyl groups were hydrolyzed after 24 h of reaction under alkaline conditions. This indicates that propylene oxide was successfully grafted onto the hydroxyl groups of the polyvinyl alcohol side chain, and based on the integrated area of ​​the peaks, its hydroxypropyl substitution degree can be calculated to be approximately 20.7%, denoted as HPPVA-21. Similarly, the feeding ratio of propylene oxide and polyvinyl alcohol units can be adjusted. As the feeding ratio increases, the grafting rate of the resulting hydroxypropyl polyvinyl alcohol (HPPVA) increases. For example, adding propylene oxide at a molar ratio of 1:20 to PVA structural units and reacting at 25°C for 24 hours yields hydroxypropyl polyvinyl alcohol with a grafting rate of 44%. Using PVA1788 instead of PVA1799, where all acetyl groups are hydrolyzed, also yields hydroxypropyl polyvinyl alcohols with different grafting rates. Using PVA with a larger molecular weight (M146000-186000, degree of hydrolysis 99.3-100.0%) instead of PVA1799 also yields hydroxypropyl polyvinyl alcohols with different grafting rates (from 15% to 60%). Tests showed that although all these hydroxypropyl polyvinyl alcohols are soluble in water, they did not exhibit significant changes upon heating, demonstrating no temperature dependence.

[0055] Example 2: Ease of use and adhesion of hydroxybutyl polyvinyl alcohol After cutting the pig vaginal tissue into a suitable shape, it was laid flat on two glass slides. The vaginal tissue was folded between the slides to form a groove, and then incubated at 37°C for 10 min. Phosphate-buffered aqueous solution of HBPVA-53 prepared in Example 1 (0.15 M PBS, 20 wt% HBPVA-53), phosphate-buffered aqueous solution of PVA (M146000-186000, degree of hydrolysis 99.3-100.0%) (0.15 M PBS, 20 wt% PVA), and phosphate-buffered aqueous solution of commercially available thermosensitive polyoxyethylene-polyoxypropylene-polyoxyethylene F127 (Sigma, catalog number P2443-250G) (0.15 M PBS, 20 wt%) were placed in a refrigerator for 1 hour. A small amount of methylene blue was then added to each and mixed thoroughly to observe the distribution of the viscous polymer, resulting in a low-temperature viscous liquid. Using a needleless syringe, inject 0.5 ml of the above-mentioned low-temperature viscous liquid onto the vaginal tissue surface incubated at 37°C. Take photos at the beginning, during, and end of the injection. Figure 3 This shows the distribution of different samples during continuous injection. Figure 3 It can be seen that the HBPVA-53 aqueous solution ( Figure 3 (b) and F127 aqueous solution ( Figure 3 (c) Both can quickly and evenly cover the grooves of vaginal tissue during injection, demonstrating good fluidity and ease of use in complex tissues, and are expected to be used as a carrier for vaginal preparations; while PVA ( Figure 3 a) Even after injection, it cannot completely fill the grooves in the vaginal tissue, and the efficiency of drug delivery to the target site is limited, making it unsuitable as a carrier for vaginal preparations.

[0056] Using a needleless syringe, approximately 0.2 mL each of 20% HBPVA-53 and F127 aqueous solutions (4°C) containing a small amount of methylene blue were placed on cellophane at 37°C and stabilized for 10 min to allow the samples to adhere to the cellophane. These polymer-loaded cellophane sheets were then slowly inverted into 50 mL centrifuge tubes containing 40 mL of 0.15 mol / L PBS aqueous solution (simulating vaginal pH 4.00), ensuring the hydrogel was completely submerged in the aqueous medium. The tubes were then shaken at 100 rpm at 37°C. The time it took for the samples to completely disappear or detach from the cellophane was recorded. Figure 4 Figures a and 4b show that the average adhesion time of F127 gel on cellophane is approximately 24.3 min, while the adhesion time of HBPVA is approximately 81.7 min, indicating that HBPVA has better adhesion than F127.

[0057] Similarly, the adhesion of the above samples to the glass slide surface was investigated: approximately 0.2 mL each of 20% HBPVA-53 and F127 aqueous solutions (4℃) containing a small amount of methylene blue were taken using a needleless syringe and placed on the surface of a glass slide at a constant temperature of 37℃. After stabilizing for 10 min, the samples adhered to the glass slide. These glass slides containing polymers were then placed vertically in 50 mL centrifuge tubes containing 40 mL of 0.15 mol / L PBS aqueous solution (simulating vaginal pH 4.00), ensuring that the hydrogel was completely immersed in the aqueous solution, and the tubes were shaken at 100 rpm at 37℃. The time it took for the samples to completely disappear or completely detach from the glass slide was recorded. Figure 4 c and 4d show that the average adhesion time of F127 gel on the slide was approximately 84.3 min, while the adhesion time of HBPVA was approximately 626.7 min, which also indicates that HBPVA has better adhesion than F127.

[0058] To further evaluate the retention properties of these hydrogel samples in the vagina, vaginal tissue was cut into suitable shapes and laid flat on glass slides. Approximately 0.5 ml each of 20% HBPVA-53 and F127 aqueous solutions (4°C) containing a small amount of methylene blue were added to the vaginal tissue at a constant temperature of 37°C using a needle-free syringe and stabilized for 10 min to allow the samples to adhere to the vaginal tissue. These polymer-loaded vaginal tissue slides were then fixed at a 60° angle in a 37°C oven, and the samples were rinsed with a single 1 mL volume of PBS aqueous solution (pH 4.00) before their retention in a simulated vaginal environment was recorded. Figure 4 The results showed that at 37°C, after rinsing with 50 mL of PBS aqueous solution, the HBPVA hydroxybutyl polyvinyl alcohol hydrogel showed no significant changes except for a lighter color, while the F127 hydrogel was mostly rinsed out of the vaginal tissue, with only a small amount of sample remaining at the bottom of the vagina. Figure 4 The results showed that after rinsing with 50 mL of PBS solution, the average residual amount of F127 gel in vaginal tissue was approximately 0.092 g, while the average residual amount of HBPVA gel in vaginal tissue was approximately 0.451 g. This indicates that hydroxybutyl polyvinyl alcohol hydrogel exhibits better vaginal adhesion compared to F127 hydrogel.

[0059] Example 3 In vitro drug release test Weigh 200 mg of hydroxybutyl polyvinyl alcohol (HBPVA-53) prepared in Example 1 and add it to 800 mg of 0.15 mol / L phosphate buffer (PBS). Stir at 4°C for 6 h until the solution is homogeneous. Weigh approximately 40, 80, and 120 mg of progesterone crystals (Hubei Gedian Renfu Pharmaceutical Co., Ltd., batch number: HTTG230913M1) with a particle size of approximately 7 μm and add them to the polymer solution at low temperature. Continue stirring for 12 h until the drug is homogeneously dispersed to obtain drug-loaded gel precursor dispersions with different drug loading capacities.

[0060] Approximately 100 mg of each of the above-mentioned drug-loaded gel precursor dispersions were added to different dialysis bags (Mw 8000-14000) using a syringe. The ends of the dialysis bags were tied tightly. These bags were then placed in centrifuge tubes containing 30 mL of 0.15 mol / L PBS aqueous solution (pH 4.00) containing 0.5% SDS (sodium dodecyl sulfate) and sealed (at 37°C) to ensure complete immersion of the formulation in the release medium. Release studies were conducted at 100 rpm. 5 mL samples were taken at different time points (2, 4, 6, 12, 24, 48, 72, 96, 120, 144, and 168 h, and an equal volume of 37°C release medium was added. The samples were shaken well before sampling. The collected samples were filtered through a 0.45 μm filter and then analyzed by high-performance liquid chromatography (HPLC). Figure 5 The results showed that the drug-loaded hydroxybutyl polyvinyl alcohol hydrogel had a significant long-term sustained-release effect without any obvious burst release phenomenon. Furthermore, as the progesterone loading increased, its release rate and cumulative release rate gradually decreased, and there was still no obvious burst release phenomenon.

[0061] Example 4: Retention of Fluorescent Hydroxybutyl Polyvinyl Alcohol Hydrogel in the Vagina 200 mg of hydroxybutyl polyvinyl alcohol (HBPVA-53) prepared in Example 1 was weighed and dissolved in 10 mL of DMSO. Then, approximately 5 mg of 4-dimethylaminopyridine and 1.5 mg (or 3.0 mg) of carboxyfluorescein were added and stirred at room temperature until dissolved. The reaction was then carried out in the dark for 24 h. After the reaction, the mixture was dialyzed against a 20% ethanol aqueous solution until no absorbance was observed in the external solution at 300 nm-500 nm. The solution was then dialyzed against pure water and lyophilized to obtain fluorescent hydroxybutyl polyvinyl alcohol. The small molecule carboxyfluorescein and the fluorescently grafted hydroxybutyl polyvinyl alcohol were dissolved in a 20% DMSO aqueous solution, and the excitation and emission spectra of the samples were scanned at 400-600 nm. The excitation and emission wavelengths of the small molecule carboxyfluorescein were 493 nm and 520 nm, respectively, while the excitation and emission wavelengths of the fluorescein-grafted hydroxybutyl polyvinyl alcohol were 505 nm and 521 nm, respectively. The excitation wavelengths of the two molecules shifted by 12 nm, indicating successful bonding between the small molecule fluorescein and hydroxybutyl polyvinyl alcohol (HBPVA). Fluorescent HBPVA with molar substitution degrees of 0.13% and 0.25% was prepared by UV quantitative analysis. Under natural light, HBPVA appeared as a white, spongy solid, while the fluorescent HBPVA with different molar substitution degrees of 0.13% and 0.25% exhibited a pale yellow color. Under UV irradiation at 254 and 365 nm, HBPVA showed no fluorescence, while the fluorescent HBPVA with different molar substitution degrees of 0.13% and 0.25% displayed yellow-green fluorescence with relatively uniform fluorescence distribution. Irradiation of a 20% HBPVA and a fluorescein grafted with 0.13% fluorescent HBPVA polymer solution at 365 nm under different temperature conditions... Figure 6A No fluorescence was observed in the HBPVA aqueous solution (20 wt%), while the HBPVA aqueous solution (20 wt%) grafted with fluorescein (0.13%) exhibited strong yellow-green fluorescence under UV irradiation. Both samples showed good flowability at 4°C; however, at 37°C, both samples thickened and solidified at the bottom of the container due to increased viscosity. This indicates that introducing a small amount of fluorescein into HBPVA does not alter its macroscopic thermosensitive behavior.

[0062] Six SD rats were randomly divided into two groups after being fed for one week. The small molecule fluorescein sodium carboxylate solution group served as the control. The residence time of a fluorescein-grafted 0.13% fluorescent hydroxybutyl polyvinyl alcohol polymer solution in the vagina was investigated. In both groups, 0.15 mL of the sample was injected into the vagina via a syringe inserted 0.5-0.8 cm into the vagina. Fluorescence imaging of the rat vagina was performed at 0, 2, 6, 12, and 24 hours after administration using an in vivo imaging system at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The fluorescence images were then processed using Living Image analysis software. Figure 6BThe small molecule fluorescein solution group showed no fluorescence after 6 hours of administration, indicating that although the solution is easy to inject, it has poor retention at the vaginal site and cannot remain at the lesion site for a long time. The in-situ gel group of the fluorescein grafting rate of 0.13% fluorescent hydroxybutyl polyvinyl alcohol polymer solution maintained fluorescence for 24 hours, indicating that it retains the advantages of good fluidity at low temperatures for easy injection while remaining at the administration site for at least 12-24 hours, forming a reservoir for in-situ sustained-release drug.

[0063] Example 5 Pharmacodynamic Study Endometrial hyperplasia has a high clinical incidence, and its etiology and mechanism are complex. Therefore, this experiment also established a rat model by administering an overdose of estradiol. Six 6-8 week old female SD rats were randomly divided into a control group (n=3) and a model group (n=3). Twenty-four hours before the experiment, the rats in the model group had their hair removed from their right hind leg and were tame to avoid stress. Daily intramuscular injections of estradiol benzoate oil-soluble injection solution were administered at a dose of approximately 1.5 mg / kg (Ningbo No. 2 Factory Veterinary Estradiol Benzoate Injection, Veterinary Drug Permit No. 110252207, 2 mg / mL, 0.15 mL for a 200 g rat). On day 21 after injection, all rats in both the model and control groups were sacrificed. The uterus was removed, photographed, weighed, and the uterine index was calculated. Finally, histopathological examination was performed to observe pathological changes in the endometrium to confirm successful modeling. Fifteen female SD rats were used again to establish an endometrial hyperplasia model using the above method. They were randomly divided into three groups: an untreated group, an intramuscular injection group, and a vaginal gel group, with five rats in each group. The weight of the rats in each group was measured (170-210 g) to ensure that there were no rats with excessively large weight differences that would affect the pharmacodynamic evaluation. The untreated group was fed normally and sacrificed after 14 days. The rats in the intramuscular injection group were injected daily into the right hind leg muscle with a dose of progesterone oil-soluble injection solution of approximately 1.8 mg / kg (Shanghai Quanyu Biotechnology Pharmaceutical Co., Ltd. progesterone injection solution, veterinary drug license 120031439, 50 mg / mL, diluted with soybean oil to 2.5 mg / mL, 0.15 mL for a 208 g rat) for 14 consecutive days before sacrifice. The rats in the vaginal gel group wore Elizabethan collars 24 hours before the experiment to prevent them from licking the vaginal preparation after administration. The tightness of the Elizabethan collars around the neck was adjusted appropriately to prevent them from falling off without affecting their eating and drinking. After the start of the experiment, a self-made vaginal gel, hydroxybutylpolyvinyl alcohol (HBPVA-53) (20 wt% aqueous solution), containing approximately 10 mg / kg of progesterone (progesterone concentration: 80 mg / mL, 26 μL for a 208 g rat), was injected into the vagina daily at a depth of approximately 0.3-0.5 cm. The rats were then sacrificed. After sacrifice, the uterus was removed, photographed, weighed, and the uterine index was calculated. Finally, pathological examination was performed to observe the pathological changes in the uterine tissue.

[0064] The uterus was dissected and placed in a tissue fixation solution for 0.5 hours before being removed and photographed. Figure 7 The results showed that the untreated model group rats had more pronounced uterine edema and congestion, and the uterus was larger. The uterine edema and congestion in the intramuscular injection group were less severe than in the untreated group, and the uterus was smaller. In contrast, the vaginal gel group showed no obvious uterine edema under gross observation, and the uterus was the smallest among the three groups, indicating that its treatment effect on endometrial hyperplasia was more significant and effective than that of the intramuscular injection group.

[0065] The wet weight of the rat uterus, uterine index, and endometrial thickness were statistically analyzed. Figure 8 , 9 The results showed that the mean wet weight of the uterus in the untreated model group was 1.127 g, and the mean uterine index was 0.561%; compared with the untreated group, the intramuscular injection group ( Figure 8 , 9 Intramuscular injection group and vaginal gel group ( Figure 8 , 9 In rats treated with the vaginal gel formulation, both uterine weight and uterine index decreased, indicating that both formulations had a therapeutic effect on endometrial hyperplasia. Furthermore, the vaginal gel group showed a greater decrease in uterine weight and uterine index, suggesting a more significant and effective therapeutic effect compared to the intramuscular injection group. While there were significant individual differences in the untreated and intramuscular injection groups, the vaginal gel group showed relatively smaller individual differences, indicating that the hydroxybutyl polyvinyl alcohol (HBPVA-53) vaginal formulation can enhance the drug's targeting effect on the uterus while reducing its distribution in peripheral organs and tissues, thereby mitigating side effects.

[0066] HE staining results of sections are as follows Figure 10 As shown, the results indicated that compared to the untreated group after modeling, the endometrial thickness of rats in the oil-soluble injection and vaginal gel groups was reduced, and the number of endometrial glands was also significantly decreased. Subsequently, the pathological results of the sections were statistically analyzed. Measurements were taken at three different locations in the uterus for each section, and the average value of the three measurements was taken as the result after section processing. Figure 11 , 12 The results showed that, compared with the untreated group after modeling, both the oil-soluble injection group and the vaginal gel group reduced the endometrial thickness and uterine epithelial thickness in rats treated with these methods. The vaginal gel group showed a more significant reduction compared to the oil-soluble injection group, and the difference was statistically significant. This indicates that both formulations have a therapeutic effect on endometrial hyperplasia, with the vaginal gel group showing a more pronounced therapeutic effect than the intramuscular injection group.

[0067] In addition, using PVA1788 type, PVA (M146000-186000, 99.3-100.0%), low degree of polymerization PVA (25,000-35,000, degree of polymerization: 570-800) or ultra-high degree of polymerization (molecular weight 250,000-300,000, average degree of polymerization 6820) instead of polyvinyl alcohol PVA1799 can also produce thermosensitive hydroxybutyl polyvinyl alcohol hydrogels, which can also be used as progesterone vaginal sustained-release carrier materials.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A thermosensitive hydroxybutyl polyvinyl alcohol, characterized in that: It has the following general structural formula: Where n is the degree of polymerization of polyvinyl alcohol and m is the number of units grafted with hydroxybutyl groups, both of which are positive integers. Thermosensitive hydroxybutyl polyvinyl alcohol is soluble in water or physiological saline at low temperatures, and its gelation transition temperature is lower than body temperature.

2. The thermosensitive hydroxybutyl polyvinyl alcohol according to claim 1, characterized in that: The thermosensitive hydroxybutyl polyvinyl alcohol has a hydroxybutyl grafting rate of 10%-70% and a gelation transition temperature of 1-36℃.

3. The thermosensitive hydroxybutyl polyvinyl alcohol according to claim 2, characterized in that: The hydroxybutyl grafting rate of the thermosensitive hydroxybutyl polyvinyl alcohol is 15%-60%, and its gelation transition temperature is 15-30℃.

4. A method for preparing thermosensitive hydroxybutyl polyvinyl alcohol according to any one of claims 1-3, characterized in that, The specific steps are as follows: Polyvinyl alcohol is dissolved in water to obtain an aqueous solution of polyvinyl alcohol; A grafting reaction is carried out by adding epoxide and an alkaline catalyst; After the reaction, acid was added for neutralization, and the product was purified and dried to obtain hydroxybutyl polyvinyl alcohol.

5. A method for preparing thermosensitive hydroxybutyl polyvinyl alcohol according to claim 4, characterized in that, The polyvinyl alcohol aqueous solution has a mass concentration of 1-10%, the molar ratio of the polyvinyl alcohol structural unit to the added epoxide is 1:5-40, and the reaction temperature is 20-60℃.

6. A method for preparing thermosensitive hydroxybutyl polyvinyl alcohol according to claim 4, characterized in that, The degree of polymerization of the polyvinyl alcohol is 500-7000.

7. An application of the thermosensitive hydroxybutyl polyvinyl alcohol according to any one of claims 1-3, characterized in that, Used to prepare progesterone vaginal sustained-release hydrogel.

8. The application of the thermosensitive hydroxybutyl polyvinyl alcohol according to claim 7, characterized in that, The method for preparing progesterone vaginal sustained-release hydrogel is as follows: Hydroxybutyl polyvinyl alcohol is dissolved in deionized water or physiological saline to obtain an aqueous polymer solution; Add progesterone microcrystals and stir well to obtain a drug-loaded gel precursor dispersion that can be converted into a gel above the gel transition temperature.

9. The application of the thermosensitive hydroxybutyl polyvinyl alcohol according to claim 8, characterized in that, The mass concentration of the polymer aqueous solution is 5%-30%.

10. The application of the thermosensitive hydroxybutyl polyvinyl alcohol according to claim 8, characterized in that, The drug loading concentration in the drug-loaded gel precursor dispersion is 4-12%.

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