Preparation and use of an anti-endometritis promoting flora modulating gel

Abstract

The application belongs to the technical field of medicine, and particularly relates to preparation and application of an anti-endometritis and probiotic regulating gel. The preparation method comprises the following steps: adding poloxamer and inulin into a solvent, and stirring and reacting to obtain a gel solution. The temperature-sensitive gel has stable gel structure and good rheological property, can be precisely delivered in the uterus, can realize slow release of the drug after loading the drug, fully plays the anti-inflammatory effect, and can regulate the flora. The application has simple preparation process, good stability, good biocompatibility, and good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the preparation and application of a gel that promotes the regulation of endometrial flora and combats endometritis. Background Technology

[0002] Currently, the routine clinical treatment for endometritis mainly involves systemic antibiotic administration, such as oral doxycycline or intravenous levofloxacin. However, this treatment regimen has two main limitations: first, it requires increasing systemic blood drug concentrations to maintain effective drug concentrations within the uterine cavity; second, systemic administration easily causes gastrointestinal discomfort and other toxic side effects. For more severe cases, clinical trials have attempted local treatment via intrauterine instillation of drug solutions, but the instillation solution is prone to leakage from the vagina due to gravity, making it difficult to maintain effective drug concentrations within the uterine cavity for extended periods, and repeated procedures increase patient discomfort. In recent years, the rapid development of drug delivery carrier technology has provided new solutions for improving uterine drug delivery.

[0003] With the deepening of microbiome research, methods for preventing and treating diseases by regulating the microbial community are receiving increasing attention. Recent studies have shown that uterine flora imbalance is closely related to the occurrence and development of endometritis. Under normal circumstances, the uterine cavity is not a sterile environment; the uterine flora within it constitutes a symbiotic organic whole, jointly maintaining the stability of the body's microecology. Once the composition or proportion of the uterine microbiota changes, it may lead to decreased endometrial receptivity, thereby inducing endometritis and adversely affecting female fertility.

[0004] Poloxamer (trade name: Pluronic) is a pharmaceutical excipient approved by the US FDA. Due to its excellent biodegradability and biocompatibility, this material has been widely used in drug delivery systems. Poloxamer-based polymeric gels not only prolong drug treatment but also have a sustained-release effect, making them one of the ideal materials for treating endometritis. On the other hand, inulin, a natural polysaccharide derived from plants, composed of oligomers and linear fructose polymers, also exhibits good biocompatibility. Inulin gel systems not only prolong the residence time of drugs at the site of action but also regulate the balance of the gut microbiota, promoting the growth of beneficial bacteria and inhibiting the proliferation of harmful bacteria. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide an in-situ gel that promotes uterine flora regulation and combats endometritis. This thermosensitive gel can achieve in-situ gelation within the uterus, prolonging drug treatment, alleviating uterine inflammation, and regulating uterine flora, thus effectively treating endometritis.

[0006] To achieve the above objectives, a first aspect of the present invention provides a uterine in situ gel.

[0007] Further, appropriate amounts of poloxamer and inulin were dissolved in a solvent, and the mixture was stirred thoroughly on a magnetic stirrer until all components were dissolved. After mixing, the mixture was placed at 4-8℃ for 2-24 h to swell, thus obtaining a polymer hydrogel solution. The drug was slowly added dropwise to the gel solution and incubated to obtain a drug-loaded gel solution.

[0008] Furthermore, the poloxamer is a polymer material such as poloxamer 124 (P124), poloxamer 188 (P188), poloxamer 237 (P237), poloxamer 388 (P388), poloxamer 407 (P407), and mixtures thereof.

[0009] Furthermore, the concentration of poloxamer is 1-50% (w / v), and the concentration of inulin is 1-30% (w / v).

[0010] Furthermore, the solvent is water, phosphate buffer, physiological saline, citrate buffer, Hank's balanced salt solution, HEPES buffer, and Tris buffer, etc.

[0011] Furthermore, the drugs mentioned include ceftriaxone, cefoxitin, metronidazole, tinidazole, levofloxacin, moxifloxacin, ciprofloxacin, clindamycin, gentamicin, doxycycline, dydrogesterone, progesterone, ibuprofen, naproxen, etc.

[0012] Furthermore, the gel solution obtains a network gel structure in a gel state at temperatures above 26°C.

[0013] Furthermore, the gel solution remains liquid below 26°C and transforms into a gel state above this temperature, enabling intrauterine delivery and long-term retention.

[0014] Furthermore, the gel can achieve long-term drug release within the uterus, fully exerting its anti-inflammatory and flora-regulating effects.

[0015] A second aspect of the invention provides the use of the in situ gel described in the first aspect in endometritis-related diseases.

[0016] Furthermore, the endometritis-related diseases include one or more of the following: acute endometritis, chronic endometritis, endometritis-induced premature birth, endometritis-induced miscarriage, pelvic peritonitis, and intrauterine adhesions.

[0017] The beneficial effects of this invention: This invention provides a simple and large-scale preparation method for an in-situ uterine gel. By forming a thermosensitive gel using poloxamer and inulin, precise delivery to the uterus is achieved, while prolonging drug retention and slow release to fully exert its anti-inflammatory effect, while simultaneously regulating the gut microbiota. This in-situ uterine gel can be used to treat endometritis and related diseases. Attached Figure Description

[0018] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings, wherein:

[0019] Figure 1 This is an image of the gel obtained in Example 1 of the present invention; Figure 2 This is an electron micrograph of the gel obtained in Example 1 of the present invention; Figure 3 The rheological diagram is shown for the gel obtained in Example 1 of this invention. Figure 4 The following is a diagram showing the adhesion behavior of the fluorescent gel obtained in Example 21 of the present invention: where A. retention of coumarin aqueous solution on the pig uterus; B. retention of coumarin-labeled gel on the pig uterus; Figure 5 This is an electron micrograph of the drug-loaded gel obtained in Example 23 of the present invention; Figure 6 The rheological diagram of the gel obtained in Example 23 of this invention; Figure 7 This is an in vitro release diagram of the drug-loaded gel obtained in Example 23 of the present invention; Figure 8 The following is an in vivo pharmacodynamic diagram of the drug-loaded gel obtained in Example 23 of the present invention: A. Relative expression of TNF-α mRNA in uterine tissue; B. Relative expression of IL-6 mRNA in uterine tissue; C. Relative expression of TLR-4 mRNA in uterine tissue; D. H&E section of uterine tissue. Detailed Implementation

[0020] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. The present invention will be further described in detail below with reference to embodiments, but those skilled in the art should understand that the present invention is not limited to these embodiments and the preparation methods used. Moreover, any modifications and substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0021] Example 1: Preparation of an in-situ gel for anti-endometritis and promoting gut microbiota regulation Suitable amounts of poloxamer (P407, 35%; P188, 10%) and inulin (10%) were dissolved in a solvent. The mixture was stirred thoroughly on a magnetic stirrer until all components were dissolved. After mixing, the mixture was placed at 4°C for 12 h to swell, thus obtaining a polymer hydrogel solution.

[0022] Example 2: Preparation of an in-situ gel for anti-endometritis and promoting gut microbiota regulation This embodiment is another exemplary illustration of the method for preparing an in-situ gel for promoting gut microbiota regulation and combating endometritis according to the present invention.

[0023] The preparation methods of Examples 2-17 are the same as those of Example 1, except as shown in Table 1.

[0024] Table 1. Preparation of in-situ gels for anti-endometritis and promoting gut microbiota regulation in Examples 2-17

[0025] Example 18: Appearance of an in-situ gel for anti-endometritis and promoting flora regulation The gel was prepared according to Example 1 and photographed at different temperatures.

[0026] like Figure 1 As shown, the prepared gel remains liquid at 4°C, but becomes gelled when the temperature exceeds 26°C (37°C in the figure).

[0027] Example 19: Microstructural observation of an in-situ gel for anti-endometritis and promoting gut microbiota regulation The gel was prepared according to Example 1. To examine the morphology of the gel, it was pre-frozen in liquid nitrogen for 40 min, freeze-dried for 36 h, and a suitable amount of sample was gold-plated. Finally, it was observed under an accelerating voltage of 15.0 kV.

[0028] Figure 2 This indicates that the prepared gel has a network structure.

[0029] Example 20: Rheological Properties of an In-situ Gel for Anti-Endometritis and Promoting Microbial Regulation The gel was prepared according to Example 1, and temperature scanning was performed on the gel within the range of 5-50°C with a gap of 1 mm. The strain and frequency were set to 1% and 2 Hz, respectively. The heating rate was maintained at 1°C / min. The storage modulus (G'), loss modulus (G''), and complex viscosity (η) were recorded.

[0030] Figure 3The results show that before 26℃, the difference between G' and G'' is not significant, indicating that the gel has low mechanical strength and is in a solution state, which does not affect injectability. However, after 26℃, G' increases significantly, and the difference between G'' and G'' gradually increases, reaching approximately 17000 Pa at 37℃, suggesting that the gel has high mechanical strength at this point and has completed the sol-gel phase transition. Viscosity measurements also show that the gel viscosity increases significantly around 26℃, indicating a sol-gel phase transition.

[0031] Example 21: Preparation of a fluorescent anti-endometritis in situ gel that promotes gut microbiota regulation Suitable amounts of poloxamer (P407, 35%; P188, 10%) and inulin (10%) were dissolved in a solvent. The mixture was stirred thoroughly on a magnetic stirrer until all components were dissolved. After mixing, the mixture was placed at 4°C for 12 h to swell, resulting in a polymer hydrogel solution. Coumarin 6 solution was slowly added dropwise to the gel solution, and after incubation, a coumarin 6-loaded gel was obtained.

[0032] Example 22: Adhesion behavior of a drug-loaded in-situ gel for treating endometritis and promoting gut microbiota regulation. Fluorescent gels were prepared according to Example 21. Equal amounts of free coumarin 6 and coumarin 6 gels were dropped onto porcine uterine tissue, and the changes in liquid morphology over time were observed.

[0033] like Figure 4 As shown, the gel group retained well on the surface of the porcine endometrium at 37°C and did not undergo morphological changes within 60 min.

[0034] Example 23: Preparation of a drug-loaded in-situ gel for treating endometritis and promoting gut microbiota regulation Suitable amounts of poloxamer (P407, 35%; P188, 10%) and inulin (10%) were dissolved in a solvent. The mixture was stirred thoroughly on a magnetic stirrer until all components were dissolved. After mixing, the mixture was placed at 4°C for 12 h to swell, resulting in a polymer hydrogel solution. Ciprofloxacin solution was slowly added dropwise to the gel solution, and after incubation, a drug-loaded gel was obtained.

[0035] Example 24: Microstructural observation of a drug-loaded in-situ gel for treating endometritis and promoting gut microbiota regulation. The gel was prepared according to Example 23. To examine the morphology of the gel, it was pre-frozen in liquid nitrogen for 40 min, freeze-dried for 36 h, and a suitable amount of sample was plated with gold. Finally, it was observed under an accelerating voltage of 15.0 kV.

[0036] Figure 5 This indicates that the prepared drug-loaded gel has a network structure.

[0037] Example 25: Rheological Properties of a Drug-Loaded Anti-Endometritis and Microbiome Regulation In-Situ Gel The gel was prepared according to Example 23, and a temperature scan was performed on the gel in the range of 5-50°C with a gap of 1 mm. The strain and frequency were set to 1% and 2 Hz, respectively. The heating rate was maintained at 1°C / min. The storage modulus (G'), loss modulus (G''), and complex viscosity (η) were recorded.

[0038] Figure 6 This indicates that the drug-loaded gel can undergo a sol-gel phase transition as the temperature increases.

[0039] Example 26: Investigation on the release behavior of a drug-loaded in-situ gel for treating endometritis and promoting gut microbiota regulation. Drug-loaded gels were prepared according to Example 23, and the in vitro release rate of ciprofloxacin gels was determined using the dialysis bag method. Both the control group (free ciprofloxacin) and the ciprofloxacin-loaded gel were added to dialysis bags with a molecular weight cutoff of 12000 Da and placed in 250 mL of PBS solution. Samples were taken at 1 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. Drug concentrations were determined using HPLC.

[0040] like Figure 7 As shown, drug-loaded gels enable the slow release of drugs.

[0041] Example 27: Evaluation of the therapeutic effect of a drug-loaded in-situ gel for treating endometritis and promoting gut microbiota regulation in an acute endometritis model. Drug-loaded gels were prepared according to Example 23. Rats were randomly divided into a normal group, a model group, a free drug group, and a drug-loaded gel group. Before the experiment, one side of the uterus of each rat was ligated to ensure that the same side of the uterus was perfused each time. The normal group received no treatment. Other groups were perfused with a mixed bacterial solution of *E. coli* and *Streptococcus* for three consecutive days. On the fourth day, the free drug group and the drug-loaded gel group were administered free ciprofloxacin and ciprofloxacin-loaded gel (2 mg / kg) to the uterus. On the ninth day, the rats were sacrificed, and the uterus was collected and stained with hematoxylin and eosin (H&E) to prepare pathological sections. Simultaneously, the uterus was collected, and total RNA was extracted from 100 mg of uterine tissue according to the instructions of the RNA extraction kit. The purity and concentration of the RNA were then determined. Using the extracted total RNA as a template, reverse transcription was performed according to the instructions of the reverse transcription kit to synthesize cDNA. The expression of inflammation-related genes (TNF-α, IL-6, TLR-4) was measured. Figure 8The results showed that, compared with free ciprofloxacin, treatment with the ciprofloxacin-loaded gel of this invention significantly reduced inflammation levels, indicating that the gel encapsulation allows the drug to exert its efficacy better. H&E results showed that after uterine administration of the ciprofloxacin-loaded gel to rats, necrosis and inflammation were significantly reduced, indicating that it can alleviate uterine damage caused by acute endometritis and effectively treat acute endometritis.

Claims

1. A gel for combating endometritis and promoting gut microbiota regulation, characterized in that, Prepare according to the following steps: Dissolve appropriate amounts of poloxamer and inulin in a solvent, stir thoroughly on a magnetic stirrer until all components are dissolved, and place the mixture at 4-8℃ for 2-24 h to swell, thereby obtaining a polymer hydrogel solution; slowly add the drug dropwise to the gel solution and incubate to obtain a drug-loaded gel solution.

2. The anti-endometritis and gut microbiota-regulating gel according to claim 1, characterized in that, The poloxamer is one or more of poloxamer 124 (P124), poloxamer 188 (P188), poloxamer 237 (P237), poloxamer 388 (P388), and poloxamer 407 (P407).

3. The anti-endometritis and gut microbiota-regulating gel according to claim 1, characterized in that, The concentration of poloxamer is 1-50% (w / v), and the concentration of inulin is 1-30% (w / v).

4. The anti-endometritis and gut microbiota-regulating gel according to claim 1, characterized in that, The solvents include water, phosphate buffer, physiological saline, citrate buffer, Hank's balanced salt solution, HEPES buffer, and Tris buffer, etc.

5. The anti-endometritis and gut microbiota-regulating gel according to claim 1, characterized in that, The drug is one or more of the following: ceftriaxone, cefoxitin, metronidazole, tinidazole, levofloxacin, moxifloxacin, ciprofloxacin, clindamycin, gentamicin, doxycycline, dydrogesterone, progesterone, ibuprofen, and naproxen.

6. The method described in claims 1-5 yields an anti-endometritis, probiotic-regulating gel, characterized in that... The gel solution remains liquid below 26°C and transforms into a gel state above this temperature, exhibiting a network gel structure in the gel state.

7. The application of the anti-endometritis and gut microbiota-regulating gel according to claim 6, characterized in that, The gel is used to treat one or more of the following: acute endometritis, chronic endometritis, endometritis-induced premature birth, endometritis-induced miscarriage, pelvic peritonitis, and intrauterine adhesions.

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