Progesterone composition, progesterone foam gel and preparation method thereof

Abstract

The present invention relates to a progesterone composition, a progesterone foam gel, and a preparation method thereof. The present invention finds that choline carbenoic acid can effectively increase the solubility of progesterone in water. The present invention uses an aqueous solution containing choline carbenoic acid to dissolve progesterone, and then prepares the progesterone foam gel with a poloxamer and a propellant. The foam gel has a high progesterone content and can fully cover the vaginal mucosa after administration, increasing the contact area between the drug and vaginal tissue. It also has temperature- and pH-responsive gelling properties, and can achieve a rapid transition from foam to gel after vaginal administration, adhering to the vaginal mucosa. The gel also has good self-healing properties and can resist the vaginal discharge mechanism, thereby achieving good adhesion and sustained action at the medication site. At the same time, the progesterone foam gel of the present invention can promote the osmotic absorption of progesterone through the vaginal mucosa, significantly improving the accumulation of progesterone in the uterus after vaginal administration, and avoiding systemic side effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a progesterone composition, a progesterone foam gel and a preparation method thereof. Background Art

[0002] As a technical means of treatment that is opposed to the natural reproductive process, assisted reproductive technology (ART) inevitably interferes with the normal physiological conditions of pregnancy during treatment. This is especially true of the gonadotropin-releasing hormone (GnRH) agonists or antagonists used in ART, which disrupt the mother's own physiological hormone levels. Alternatively, the loss of granulosa cells during oocyte retrieval can impair luteal function. Therefore, exogenous progesterone supplementation is necessary in the early stages of ART pregnancy to maintain a smooth pregnancy. Luteal support has become an essential step in improving the success rate of ART.

[0003] Progesterone is a progestogen secreted primarily by the corpus luteum of the ovary. By binding to specific receptors, it acts on the uterus, ovaries, breasts, and central nervous system. It is the preferred medication for luteal phase support, responsible for coordinating the luteal phase through secretory transformation of the endometrium, preventing menstruation, and creating an optimal environment for embryonic development in the uterus, facilitating embryo implantation. It stabilizes the endometrium and maintains pregnancy in early pregnancy, providing immune tolerance to the implanted embryo. It is an essential hormone for maintaining early pregnancy. Therefore, successful pregnancy in both natural conception and assisted reproductive technology cycles requires adequate progesterone to achieve a good pregnancy outcome.

[0004] As a female-specific organ, the vagina can serve as an effective route for drug delivery due to its large mucosal surface area, abundant blood flow, and good drug permeability. Compared with intramuscular injection, vaginal administration is more convenient and can be performed by oneself, avoiding injection pain, and has better compliance and limited side effects. Compared with oral administration, vaginal administration avoids the first-pass effect in the liver and can achieve drug "targeting" through the first-pass effect in the uterus (drugs are absorbed through the vaginal mucosa and directly transported to the uterus), resulting in higher bioavailability. Therefore, vaginal progesterone supplementation is the preferred method for luteal support.

[0005] However, the properties of progesterone itself and the design of the formulation largely determine its absorption and utilization after vaginal delivery. 21 H 30O2, chemically known as pregnen-4-ene-3,20-dione, is classified as Class IV in the biopharmaceutics classification system. It is a compound with poor solubility and low permeability, which poses certain limitations in formulation design. Furthermore, two key factors influence drug absorption through the vaginal mucosa: the distribution and retention of the drug delivery system within the vagina, and the release of the drug from the delivery system and its dissolution in vaginal fluid, which then permeates the vaginal mucosa. The vaginal wall is narrow, irregular, and generally contracted, making it difficult for vaginal drug delivery formulations to fully contact the vaginal lining. This poor coverage of the vaginal folds significantly impacts drug absorption through the vaginal mucosa. Furthermore, the vagina has an expulsion mechanism. When the patient adopts a standing position or when the muscles around the vagina engage in mechanical movement, the drug formulation is squeezed and expelled from the vagina, preventing it from retaining the drug for extended periods of time. Therefore, the distribution and retention of the drug within the vaginal folds after administration, as well as drug absorption through the vaginal mucosa, are primary considerations in vaginal drug formulation design.

[0006] As the world's largest country in assisted reproductive technology treatment, my country has extremely high clinical demand and a huge market gap in the field of luteal support vaginal drug delivery. It is urgent to develop a vaginal drug delivery system that combines the breadth of drug delivery and the length of retention time, and increases the solubility of progesterone, so as to achieve efficient vaginal delivery of progesterone. Summary of the Invention

[0007] Based on this, the purpose of the present invention is to provide a composition capable of improving the solubility of progesterone and a vaginal drug delivery system thereof, so as to improve the coverage, distribution and retention of the drug in the vagina and the penetration and absorption rate of the drug through the vaginal mucosa, thereby improving the bioavailability and therapeutic effect of the drug.

[0008] The technical solutions for achieving the above-mentioned purpose include the following.

[0009] In a first aspect, the present invention provides the use of a choline ionic liquid as a cosolvent in improving the solubility of progesterone; the choline ionic liquid is choline carbenoic acid.

[0010] In some embodiments, the carbon chain length of the anion in the choline carbenoic acid is 8-36.

[0011] In some embodiments, the carbon chain length of the anion in the choline carbenoic acid is 12-30.

[0012] In some embodiments, the carbon chain length of the anion in the choline carbenoic acid is 16-22.

[0013] In some embodiments, the choline carbenic acid is choline palmitoleic acid, choline erucic acid, or choline oleic acid.

[0014] In some embodiments, the choline carbenoic acid is obtained by reacting choline bicarbonate or choline hydroxide with carbenoic acid in a molar ratio of 1-5:5-1.

[0015] In some embodiments, the choline carbenoic acid is obtained by reacting choline bicarbonate or choline hydroxide with carbenoic acid in a molar ratio of 1-2:2-1.

[0016] In some embodiments, the choline carbenoic acid is obtained by reacting choline bicarbonate or choline hydroxide with carbenoic acid in a molar ratio of 1:1.

[0017] In some embodiments, the carbon chain length of the carbene acid is 8-36.

[0018] In some embodiments, the carbon chain length of the carbene acid is 12-30.

[0019] In some embodiments, the carbon chain length of the carbene acid is 16-22.

[0020] In some embodiments, the carboxylic acid is palmitoleic acid, erucic acid or oleic acid.

[0021] In some embodiments, the progesterone solubility refers to the solubility of progesterone in water.

[0022] In a second aspect, the present invention further provides a composition capable of improving the solubility of progesterone, comprising progesterone and choline carbenoic acid.

[0023] In some embodiments, the carbon chain length of the anion in the choline carbenoic acid is 8-36.

[0024] In some embodiments, the carbon chain length of the anion in the choline carbenoic acid is 12-30.

[0025] In some embodiments, the carbon chain length of the anion in the choline carbenoic acid is 16-22.

[0026] In some embodiments, the choline carbenic acid is choline palmitoleic acid, choline erucic acid or choline oleic acid.

[0027] In some embodiments, the choline carbenoic acid is obtained by reacting choline bicarbonate or choline hydroxide with carbenoic acid in a molar ratio of 1-5:5-1.

[0028] In some embodiments, the choline carbenoic acid is obtained by reacting choline bicarbonate or choline hydroxide with carbenoic acid in a molar ratio of 1-2:2-1.

[0029] In some embodiments, the choline carbenoic acid is obtained by reacting choline bicarbonate or choline hydroxide with carbenoic acid in a molar ratio of 1:1.

[0030] In some embodiments, the carbon chain length of the carbene acid is 8-36.

[0031] In some embodiments, the carbon chain length of the carbene acid is 12-30.

[0032] In some embodiments, the carbon chain length of the carbene acid is 16-22.

[0033] In some embodiments, the carboxylic acid is palmitoleic acid, erucic acid or oleic acid.

[0034] In some embodiments, the mass ratio of progesterone to choline carbenic acid is 1:1-10.

[0035] In some embodiments, the mass ratio of progesterone to choline carbenic acid is 1:2-8.

[0036] In some embodiments, the mass ratio of progesterone to choline carbenic acid is 1:3-6.

[0037] In some embodiments, the mass ratio of progesterone to choline carbenic acid is 1:4-5.

[0038] In some embodiments, the mass ratio of progesterone to choline carbenic acid is 1:4.6-4.9.

[0039] In a third aspect, the present invention further provides a progesterone foam gel, which is prepared from a gel precursor solution and a propellant, wherein the gel precursor solution is obtained by mixing the aqueous solution of the choline ionic liquid of the present invention, progesterone and poloxamer.

[0040] In some embodiments, the concentration of the choline ionic liquid in the aqueous solution of the choline ionic liquid is 30 mg / mL-50 mg / mL.

[0041] In some embodiments, the concentration of the choline ionic liquid in the aqueous solution of the choline ionic liquid is 35 mg / mL-48 mg / mL.

[0042] In some embodiments, the concentration of the choline ionic liquid in the aqueous solution of the choline ionic liquid is 37 mg / mL-45 mg / mL.

[0043] In some embodiments, the concentration of the choline ionic liquid in the aqueous solution of the choline ionic liquid is 41 mg / mL-42.5 mg / mL.

[0044] In some embodiments, the concentration of the choline ionic liquid in the aqueous solution of the choline ionic liquid is 42 mg / mL-42.5 mg / mL.

[0045] In some embodiments, the concentration of progesterone in the gel precursor solution is 7 mg / mL-10 mg / mL.

[0046] In some embodiments, the concentration of progesterone in the gel precursor solution is 8.7 mg / mL-9.1 mg / mL.

[0047] In some embodiments, the concentration of progesterone in the gel precursor solution is 9.0 mg / mL-9.1 mg / mL.

[0048] In some embodiments, the weight percentage of the poloxamer in the gel precursor solution is 15-30%.

[0049] In some embodiments, the weight percentage of the poloxamer in the gel precursor solution is 22-25%.

[0050] In some embodiments, the weight percentage of the poloxamer in the gel precursor solution is 22.5-23.5%.

[0051] In some embodiments, the weight percentage of the poloxamer in the gel precursor solution is 23.1-23.2%.

[0052] In some embodiments, the weight percentage of the poloxamer in the gel precursor solution is 23.15-23.2%.

[0053] In some embodiments, the weight percentage of the propellant in the progesterone foam gel is 2-6%.

[0054] In some embodiments, the weight percentage of the propellant in the progesterone foam gel is 3-5%.

[0055] In some embodiments, the weight percentage of the propellant in the progesterone foam gel is 3.5-4.5%.

[0056] In some embodiments, the poloxamer is poloxamer 407.

[0057] In some embodiments, the propellant is propane and / or butane.

[0058] In some embodiments, the propellant is propane and butane in a volume ratio of 1:2-3.

[0059] In some embodiments, the propellant is propane and butane in a volume ratio of 3:7.

[0060] In a fourth aspect, the present invention further provides a method for preparing the progesterone foam gel, comprising the following steps:

[0061] The progesterone is added to the aqueous solution of the choline ionic liquid, and then the poloxamer is added and fully swelled to obtain the gel precursor solution. The gel precursor solution is added to an aerosol bottle and filled with a propellant to obtain the foam gel.

[0062] The present invention has the following beneficial effects:

[0063] The present invention finds that choline carbenic acid can effectively increase the solubility of progesterone in water, and in particular choline oleic acid can increase the solubility of progesterone in water by more than 1000 times.

[0064] Furthermore, the present invention dissolves progesterone in an aqueous solution containing choline carbenic acid, and then prepares the progesterone foam gel with poloxamer and a propellant. The progesterone content in the foam gel is high. The progesterone foam gel of the present invention can maintain a low viscosity state at a relatively high shear rate, can ensure that the precursor solution is ejected through the valve to form foam, the foam has a uniform drug content during the ejection process and between different batches of foam gels, has good stability, and has a low foam density, is fine and dense, and the expansion duration meets the standard. After administration, the vaginal mucosa can be fully covered, and the contact area between the drug and the vaginal tissue can be increased; it has both temperature and pH response gelling properties, both of which are in line with the physiological environment of the vagina, and can achieve rapid conversion from foam to gel after vaginal administration, adhere to the vaginal mucosa, achieve good adhesion and sustained action at the medication site; after being converted into a gel in the vagina, the gel has good self-healing properties, fully fits the vaginal environment, can resist the vaginal discharge mechanism, and achieves long-term retention in the vagina to continuously release the drug, thereby achieving extensive and sustained drug delivery.

[0065] At the same time, the progesterone foam gel of the present invention can promote the permeation and absorption of progesterone through the vaginal mucosa, significantly improve the accumulation of progesterone in the uterus after vaginal administration, avoid systemic side effects, and have good in vitro and in vivo safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 These are the H-NMR and C-NMR spectra of cholineglycine.

[0067] Figure 2 The H-NMR and C-NMR spectra of choline palmitoleic acid

[0068] Figure 3 These are the H-NMR and C-NMR spectra of choline erucic acid.

[0069] Figure 4 The H NMR spectra of choline oleic acid at different ratios.

[0070] Figure 5 The figure shows the C-NMR spectrum of choline oleic acid with a molar ratio of choline to oleic acid of 1:1.

[0071] Figure 6 The figure shows the C-NMR spectrum of choline oleic acid with a molar ratio of choline to oleic acid of 1:2.

[0072] Figure 7 The figure shows the C-NMR spectrum of choline oleic acid with a molar ratio of choline to oleic acid of 1:3.

[0073] Figure 8 The figure shows the C-NMR spectrum of choline oleic acid with a molar ratio of choline to oleic acid of 1:4.

[0074] Figure 9 This is the C-NMR spectrum of choline oleic acid with a molar ratio of choline to oleic acid of 1:5.

[0075] Figure 10 This is the C-NMR spectrum of choline oleic acid with a molar ratio of choline to oleic acid of 2:1.

[0076] Figure 11 This is the C-NMR spectrum of choline oleic acid with a molar ratio of choline:oleic acid of 3:1.

[0077] Figure 12 This is the C-NMR spectrum of choline oleic acid with a molar ratio of choline to oleic acid of 4:1.

[0078] Figure 13 This is the C-NMR spectrum of choline oleic acid with a molar ratio of choline to oleic acid of 5:1.

[0079] Figure 14 This is the HPLC chromatogram of progesterone.

[0080] Figure 15 This is the standard curve of progesterone.

[0081] Figure 16 The solubilization ability of choline ionic liquids with different anions on progesterone.

[0082] Figure 17 The solubilization ability of choline ionic liquids with different anion chain lengths on progesterone.

[0083] Figure 18 The solubilization ability of choline oleic acid with different anion-cation ratios on progesterone.

[0084] Figure 19 The solubilization ability of choline oleic acid with different anion-cation ratios on progesterone.

[0085] Figure 20These are the thixotropy test results of the progesterone foam gel precursor solution.

[0086] Figure 21 The results are for the drug content uniformity of progesterone foam gel.

[0087] Figure 22 It is the foam form of progesterone foam gel.

[0088] Figure 23 The modulus change of the gel formed by the progesterone foam gel precursor solution in temperature response and pH response under cyclic strain state.

[0089] Figure 24 To investigate the distribution and retention of progesterone foam gel in a vaginal structural model.

[0090] Figure 25 The distribution and retention of progesterone foam gel in the rat vagina.

[0091] Figure 26 is the fluorescence penetration intensity of progesterone foam gel containing DID in the vaginal mucosa.

[0092] Figure 27 Figure 2 shows the standard curves of progesterone in uterine tissue samples (A) and plasma samples (B).

[0093] Figure 28 The results are for the uterine pharmacokinetics of progesterone foam gel.

[0094] Figure 29 These are the plasma pharmacokinetics results of progesterone foam gel. DETAILED DESCRIPTION

[0095] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0096] In the following examples, the experimental methods without specific conditions are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0097] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0098] Furthermore, as used herein, the term "or" is inclusive and equivalent to the term "and / or," unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for being based on other factors not described, unless the context clearly dictates otherwise. Furthermore, throughout this specification, the meanings of "a," "an," and "the" include plural referents. The meaning of "in" includes "in" and "on."

[0099] The present invention is further described in detail below with reference to specific embodiments.

[0100] Example 1: Synthesis of choline ionic liquid

[0101] 1.1 Synthesis of Choline-Glycine (1:1)

[0102] [HOCH2CH2N + (CH3)3][OH - ]+NH2CH2COOH=[HOCH2CH2N + (CH3)3][NH2CH2COO - ]+H2O

[0103] Take 15.6 mL of choline hydroxide methanol solution ([Ch][OH] / MeOH, 45%, 57.76 mmol), evaporate the methanol in the solution under vacuum at 40°C, add 50 mL of glycine (4.34 g, 57.79 mmol) aqueous solution, cool in an ice bath and stir for 12 hours, and remove water under vacuum at 50°C. Add acetonitrile / methanol (9:1, v / v) to precipitate excess glycine (Glycinate, [Gly]), filter, and remove the solvent by rotary evaporation at 40°C. The resulting choline glycine ([Ch][Gly]) is dried under vacuum at 60°C overnight and stored under moisture-free conditions for future use.

[0104] 1.2 Synthesis of choline palmitoleate (1:1)

[0105] [HOCH2CH2N + (CH3)3][HCO3 - ]+C 15 H 29 COOH=[HOCH2CH2N + (CH3)3][C 15 H 29 COO - ]+H2O+CO2

[0106] 1.00 g of palmitoleic acid ([Pal], 3.93 mmol) was dissolved in 20 mL of methanol and slowly added dropwise to 0.79 g of an 80% aqueous solution of choline bicarbonate (3.82 mmol). The reaction mixture was stirred at room temperature for 1 hour until CO2 evolution ceased. Methanol and water were then removed by rotary evaporation at 60°C in vacuo. The resulting choline palmitoleic acid ([Ch][Pal]) was vacuum dried at 60°C overnight and stored in a moisture-free environment until further use.

[0107] 1.3 Synthesis of choline erucic acid (1:1)

[0108] [HOCH2CH2N + (CH3)3][HCO3 - ]+C 21 H 41 COOH=[HOCH2CH2N + (CH3)3][C 21 H 41 COO - ]+H2O+CO2

[0109] Weigh 2.00 g of erucic acid (Erucate, [Eru], 5.91 mmol) and dissolve it in 20 mL of methanol. Slowly add the resulting mixture dropwise to 1.22 g of an 80% aqueous solution of choline bicarbonate (5.88 mmol). Stir the reaction mixture at room temperature for 1 hour until CO2 evolution ceases. Remove the methanol and water by rotary evaporation at 60°C in vacuo. Dry the resulting choline erucic acid ([Ch][Eru]) in a vacuum oven at 60°C overnight and store in a moisture-free environment until ready for use.

[0110] 1.4 Synthesis of choline oleic acid in different proportions

[0111] [HOCH2CH2N + (CH3)3][HCO3 - ]+C 17 H 33 COOH=[HOCH2CH2N + (CH3)3][C 17 H 33 COO - ]+H2O+CO2

[0112] Choline oleic acid ([Ch][Ole]) was synthesized in various proportions according to the component ratios listed in Table 1 using the following method: the prescribed amount of oleic acid ([Ole]) was dissolved in 20 mL of methanol and then slowly added dropwise to the corresponding proportion of 80% choline bicarbonate aqueous solution. The reaction mixture was stirred at room temperature for 1 hour until CO2 evolution ceased. The methanol and water were then removed by rotary evaporation at 60°C in vacuo. The resulting choline oleic acid ([Ch][Ole]) was vacuum-dried at 60°C overnight and stored in a moisture-free environment until further use.

[0113] Table 1 Choline oleic acid prescription

[0114]

[0115] The prepared ionic liquids were characterized by nuclear magnetic resonance spectroscopy. 1 H NMR with 13 C NMR spectroscopy identification (such as Figures 1-13 The structure and composition of the prepared ionic liquid were confirmed.

[0116] Example 2: Solubilization ability of different choline ionic liquids on progesterone

[0117] 1.1 HPLC method establishment

[0118] 1) Chromatographic conditions: Chromatographic column: Kinetex C18 (250×4.6 mm, 5 μm); mobile phase: A (acetonitrile)-B (0.1% phosphoric acid-water), gradient elution program as shown in Table 2; flow rate: 1 mL / min; column temperature: 30°C; injection volume: 20 μL; detection wavelength: 241 nm.

[0119] Table 2 Gradient elution program

[0120]

[0121] 2) Standard curve

[0122] A series of progesterone methanol solutions at concentrations of 5, 10, 25, 50, 75, 100, 250, and 500 μg / mL were prepared using the gradient dilution method. These solutions were injected and tested. The HPLC chromatograms for each concentration were recorded, and the peak areas at each concentration were integrated. A standard curve for progesterone was constructed using linear regression, with progesterone concentration as the abscissa (x) and peak area (y) as the ordinate.

[0123] 3) Results

[0124] Liquid phase spectrum Figure 14 As shown in Figure 2, the retention time of progesterone is about 11.078 min; the standard curve is as follows Figure 15 As shown, the concentration and peak area are linearly related (R2 =0.9998).

[0125] 1.2 Determination of the solubilization ability of ionic liquids for progesterone

[0126] Weigh appropriate amounts of ionic liquids of different compositions and dissolve them in water to obtain ionic liquid solutions with concentrations of 10 g / L, 15 g / L, 25 g / L, 40 g / L, and 50 g / L, respectively. Store them in the dark as stock solutions for future use.

[0127] Transfer 3 mL of the stock solution to an EP tube. Add sufficient progesterone to form a supersaturated solution while stirring. Vortex for 15 minutes. Transfer the EP tube to a constant-temperature air bath shaker at 25°C, 100 rpm, and equilibrate for 72 hours. Centrifuge at 4500 rpm for 20 minutes, let stand for 3 hours, collect the supernatant, filter it, and assay the progesterone content by HPLC.

[0128] 1) Solubilization ability of choline ionic liquids with different anions on progesterone

[0129] The solubility of progesterone in choline glycine and choline oleic acid solutions with different concentrations is shown in Figure 2. Figure 16 As shown in the figure, choline oleic acid has a good solubility for progesterone, and the solubility is proportional to the content of choline oleic acid; while no matter how much the choline glycine content is, it has almost no effect on the solubility of progesterone.

[0130] 2) Solubilization ability of choline ionic liquids with different anion chain lengths on progesterone

[0131] The solubility of progesterone in ionic liquids composed of different chain lengths of oleic acids (palmitoleic acid (9-hexadecenoic acid), oleic acid (9-octadecenoic acid) and erucic acid (docosa-13-enoic acid)) and choline was compared. Figure 17 As shown, ionic liquids of varying carbon chain lengths significantly enhance the solubility of progesterone, with this increase occurring with increasing ionic liquid content. Furthermore, increasing the anionic carbon chain length within a certain range enhances the solubility of choline carbenoic acid ionic liquids for progesterone, but this solubilization capacity does not consistently increase with increasing anionic carbon chain length. Choline oleic acid exhibits the strongest solubilization capacity.

[0132] 3) Solubilization ability of choline oleic acid with different anion-cation ratios on progesterone

[0133] The solubility of progesterone in choline oleic acid with different anion and cation ratios is as follows Figure 18 and Figure 19 As shown in the figure, when the molar ratio of oleic acid to choline is greater than 1, its solubilizing ability for progesterone decreases; as the proportion of choline gradually increases, its solubilizing ability for progesterone also gradually decreases, and the degree of decrease gradually decreases. The optimal molar ratio of choline to oleic acid is 1:1.

[0134] Therefore, the choline ionic liquid that has the best effect on improving the solubility of progesterone is: [Ch][Ole](1:1).

[0135] Example 3: Preparation and Characterization of Ionic Liquid-Progesterone Foam Gel

[0136] An appropriate amount of choline and oleic acid (1:1) was weighed to prepare aqueous solutions of a series of concentrations (as shown in Table 3). A sufficient amount of progesterone was added to form a supersaturated solution, and the solution was equilibrated in an air bath shaker at 25°C, 100 rpm, for 72 hours. Subsequently, the solution was centrifuged at 4500 rpm for 20 minutes. The supernatant was removed, and the amount of poloxamer 407 (P407) indicated in Table 3 was slowly added. After mixing, the solution was stored at 4°C overnight (12 hours) to allow for full swelling, yielding a clear, transparent gel precursor solution (the percentage of P407 in Table 3 refers to the weight percentage of P407 in the gel precursor solution). 15 mL was transferred to an aluminum aerosol bottle, sealed with a capping machine, and filled with a propellant (propane:butane = 3:7) at a 4% (w / w) ratio to yield a foam gel (IL-FOAM).

[0137] 1) Determination of gelation temperature

[0138] The gelation temperature of the gel was determined using the test tube inversion method. 1 mL of the precursor solution was placed in a vial and placed in a water bath, maintaining the water bath level above the precursor solution. A thermometer was placed in the precursor solution, and the water bath temperature was controlled to rise at a rate of 0.5°C / min. Every 30 seconds, the vial was removed and the fluidity of the precursor solution was observed by tilting the vial. The gelation temperature was recorded as the temperature at which the precursor solution stopped flowing.

[0139] 2) Gel time determination

[0140] Preheat a water bath to 37°C and set the magnetic stirring speed to 20 rpm. Place 1 mL of the precursor solution into a vial containing a magnetic rotor. Start timing from the moment the vial is placed in the water bath (keeping the solution level below the water bath level). Record the time when the magnetic rotor stops rotating, which is the gelation time.

[0141] 3) pH responsiveness investigation

[0142] The normal vaginal pH ranges from 3.5 to 5.5, but decreases to 3.8 to 4.4 during pregnancy. Therefore, an intermediate pH of 4 was selected to verify the pH responsiveness of the prepared ionic liquid gel system. An acid solution with a pH of 4 was prepared with an appropriate amount of hydrochloric acid and stored for later use. 1 mL of each precursor solution was placed in a centrifuge tube and 0.2 mL of the pre-prepared acid solution (pH 4) was slowly added while stirring. The gel transition of the precursor solution was observed, and the time taken for the pH-responsive gel transition was recorded, starting from the addition of the acid solution.

[0143] The specific formulations and results are shown in Table 3. All formulations had gelation temperatures within the range of 31–35°C, meeting the vaginal temperature requirements. Their gelation times were all <90 seconds, allowing them to rapidly transform into gels upon contact with the vaginal surface and adhere to the vaginal lining. Formulation F6 had the highest gelation temperature within the expected range, minimizing the effects of ambient temperature. It also had the shortest gelation time and pH response time, ensuring a more rapid gel transformation and subsequent adhesion after vaginal administration. Therefore, F6 was the optimal formulation, increasing the solubility of progesterone to 9.08 ± 0.23 g / L.

[0144] Table 3 Response gelling properties of foam gels prepared by various prescriptions

[0145]

[0146] 4) Thixotropy

[0147] The thixotropy of the precursor solution prepared by the F6 formula was measured using an Anton Paar rotational rheometer, simulating the process of the precursor solution being ejected through a valve. The measuring fixture model was PP40, the temperature was set at 25°C, and the flow rate was 0.1s. -1 The low shear rate was continuously sheared for 120s, and the shear rate was increased to 100s -1 , maintain for 10 s, and restore the shear rate to 0.1 s -1 , continue shearing for 120s, and record the change of viscosity with shear rate. Figure 20 As shown, when the low shear rate is continued for 120s, the shear rate is increased from 0.1s -1 Rapidly increased to 100s -1 , the viscosity drops sharply and maintains a low viscosity state at a higher shear rate, which can ensure that the precursor solution is sprayed out through the valve.

[0148] 5) Content uniformity

[0149] Prepare the foam gel with F6 formulation, shake it evenly, discard the foam that sprays out within the first 3 seconds, and spray it onto a clean glass plate at a constant speed until the foam is completely sprayed out. Weigh the front, middle, and back sections of the foam, dissolve them in methanol to the same mass concentration, filter them, and analyze the progesterone content by HPLC to analyze the uniformity of the spraying process. Randomly prepare another foam gel, shake it evenly, discard the foam that sprays out within the first 3 seconds, spray an appropriate amount of foam into a centrifuge tube, weigh it, dissolve it in methanol to the same mass concentration, filter it, and analyze the progesterone content by HPLC to analyze the uniformity of content between different batches.

[0150] The results of drug content uniformity of foam gel are as follows Figure 21As shown, during the foam spraying process, there is no difference in the drug content in the front, middle and back sections of the foam, and there is no difference in the drug content between different batches of foam gels. The foam gel prepared by the present invention has a uniform content and is relatively stable.

[0151] 6) Foam form

[0152] After shaking the foam gel prepared with formula F6 evenly, discard the foam sprayed in the first 3 seconds. Evenly spray an appropriate amount of foam onto a culture dish and a glass slide preheated to 37°C (soak the surface with a pH = 4 acid solution before use). Maintain the ambient temperature at 37°C. Record the changes in foam morphology within 5 minutes using a camera and an upright microscope, respectively.

[0153] Comparative Example: Using Tween 80 (T80) instead of P407, a foam gel was prepared in the same manner, and the changes in foam morphology within 5 minutes were recorded in the same manner.

[0154] The results are as follows Figure 22 As shown, when the foam is first ejected, the P407-based foam gel exhibits a finer, denser appearance and a microstructure characterized by fewer air bubbles and voids compared to the foam gel prepared with T80. Furthermore, the foam formed with T80 maintains a typical "gas-liquid" structure, reflecting the normal decay process of foam: large bubbles continuously merge with smaller ones, followed by thinning and rupture of the surface layer of the large bubbles, and eventually disappearing. The P407-based foam, on the other hand, exhibits some expansion in the first two minutes but does not decay. Starting in the third minute, it transitions from a foam to a gel structure, exhibiting a network of voids similar to the typical three-dimensional network structure of a gel. This demonstrates that the P407-based foam gel prepared in this invention can achieve drug delivery in a foam form, leveraging the foam's expansion to increase the drug-tissue contact area. Furthermore, it directly transforms from foam to gel in response to the temperature and pH at the application site, achieving good adhesion and sustained action at the application site.

[0155] 7) Gel self-healing

[0156] The self-healing properties of the gels formed in response to temperature (37°C) and pH (4) were measured using an Anton Paar rotational rheometer. The measuring fixture model was PP 40, the temperature was set to 37°C, the frequency was constant at 1 Hz, and the strain was applied in a cycle of 0.5% → 100% → 0.5%. The cycle was repeated 3 times, and each strain value was maintained for 100 s. The modulus changes of the gels formed in response to the two conditions were recorded.

[0157] The results are as follows Figure 23As shown, the changes in modulus of the temperature-responsive and pH-responsive gels under cyclic strain are essentially the same: when the strain increases from 0.5% to 100%, the gel modulus exhibits dynamic changes and recovery, demonstrating good self-healing properties. This property ensures that the foam gel, after transforming into a gel in the vagina, can resist vaginal discharge mechanisms.

[0158] Example 4: Distribution and retention of simulated foam gel in vitro

[0159] A 3D-printed model of the human vaginal structure was used to simulate the distribution of the formulation in the vagina using a small animal in vivo imaging system. A P407-based foam gel and a T80-based foam gel were prepared as described in Example 3 (based on prescription 6 in Example 3, 10 μL of a 10 mg / mL DID ethanol solution was added to the gel precursor solution before filling and mixed). After filling and shaking, the foam ejected in the first 3 seconds was discarded and sprayed into the bottom of the vaginal model until the foam filled the entire vaginal cavity. The model was placed upright in an air bath shaker at 37°C and 100 rpm. 0.2 mL of SVF was slowly added from the top of the model every 2 hours to simulate a vaginal discharge environment. A small animal in vivo imaging system was used to observe the distribution of the foam gel in the vaginal model at 0, 1, 2, 4, 8, 12, and 24 hours to study the retention capacity of the formulation in the vagina.

[0160] The results are as follows Figure 24 As shown in the 0-hour image, due to the foam's expansion properties, the foam gel can quickly and evenly fill the entire vaginal cavity after administration, maximizing the contact area between the drug and the vaginal wall. The T80-based foam system completely breaks down and turns into liquid outflow within 1 hour, and the fluorescence dissipates accordingly. The P407-based foam gel, after administration, triggers the vaginal physiological environment and transforms from foam to gel, adhering to the vaginal wall. This increases the contact time between the drug and the vagina and ensures prolonged retention of the drug at the administration site, thus achieving sustained action. The 24-hour image shows that the gel can be maintained in the vagina for up to 24 hours.

[0161] Example 5: In vivo retention of foam gel

[0162] IL-FOAM containing Cy5.5 was prepared according to the method of Example 3. (Based on the formulation 6 of Example 3, 100 μL of a 10 mg / mL Cy5.5 aqueous solution was added to the gel precursor solution before filling and mixed thoroughly.) After filling and shaking, the foam ejected within the first 3 seconds was discarded. A blunt needle was inserted approximately 1 cm into the vagina of a female mouse and 200 μL of the drug was administered. The distribution of the foam gel in the vagina of the rats was observed using a small animal in vivo imaging system at 0, 1, 2, 4, 8, 12, and 24 hours to investigate the retention of the formulation in the vagina.

[0163] The results are as follows Figure 25 As shown, consistent with the results of in vitro simulation experiments, IL-FOAM can quickly fill the vaginal cavity of rats after administration, showing strong fluorescence in a large area of ​​the vagina at 0 h; with the action of the vagina, the fluorescence area and intensity gradually decrease, until weak fluorescence remains at the vaginal opening of the rats at 24 h, and the retention time can last up to 24 h, which is expected to achieve sustained effect of the drug.

[0164] Example 6: Mucosal penetration strength of ionic liquid foam gel

[0165] According to the method of Example 3, an ionic liquid foam gel (IL-FOAM) and a foam gel without ionic liquid (FOAM) were prepared using the F6 formula (except for the absence of ionic liquid, the other components and contents were the same as those of the IL-FOAM group, and progesterone was in a suspended form). Before filling, 10 μL of a 10 mg / mL DID ethanol solution was added and mixed. After filling, the foam sprayed in the first 3 seconds was discarded after shaking. A blunt needle was inserted into the vagina of a female mouse about 1 cm and 200 μL of the drug was administered; an appropriate amount of DID and The mixture was mixed until the concentration was consistent with that of the previous two groups. A blunt needle was inserted approximately 1 cm into the vagina of a female mouse and 200 μL of the solution was administered. A timer was started after administration, and the female mouse was euthanized 15 minutes after administration. The vaginal tissue was removed, the fallopian tubes were cut, and the upper vaginal bladder and surrounding adipose tissue were carefully dissected. The excised vagina was frozen at -80°C and sectioned. After DAPI staining, the fluorescence distribution of DAPI and DID was observed using a confocal laser microscope at 405 nm and 646 nm, respectively. ImageJ software was used to analyze the fluorescence penetration intensity in the vaginal mucosa.

[0166] The results are as follows Figure 26 The IL-FOAM group based on [Ch][Ole] showed stronger and deeper fluorescence in the vaginal mucosa. The fluorescence intensity-distance curve in the range of 0-250μm was calculated and plotted. It can be seen from the curve that it is significantly different from the commercially available preparation. Compared with the two groups, the two foam preparations had stronger fluorescence in the deeper mucosal tissue (200-250μm), among which the fluorescence intensity of the IL-FOAM group was the highest, indicating that it had the best penetration effect. The area under the fluorescence intensity-distance curve (AUC) was calculated. In the range of 0-250μm, the AUC of the IL-FOAM group was The addition of [Ch][Ole] could significantly enhance the penetration of vaginal mucosa, which was 2.06 times of the control group and 2.16 times of the FOAM group.

[0167] Example 7: In vivo pharmacokinetics of ionic liquid-progesterone foam gel

[0168] 1) Grouping

[0169] 144 female SD rats were randomly divided into three groups and administered a progesterone injection (using soybean oil as the solvent), a foam gel (FOAM) without ionic liquid (except for the absence of ionic liquid, the other components and contents were the same as those of the IL-FOAM group, and progesterone was present in a suspended form), and an ionic liquid foam gel (IL-FOAM) prepared using the F6 formulation according to the method of Example 3, at a dose of 8.1 mg / kg.

[0170] 2) Sample collection

[0171] After administration, rats in each group were anesthetized with isoflurane at 30 min, 45 min, 60 min, 90 min, 180 min, 360 min, and 720 min, respectively. Blood was collected from the abdominal aorta with a 10 mL syringe, and the blood was centrifuged at 5000 rpm for 10 min at 4°C. The upper plasma was collected and stored in a -20°C refrigerator.

[0172] After blood collection, the abdominal cavity was further opened to expose the reproductive system. The upper bladder and surrounding fat tissue were carefully peeled off, the fallopian tube and vaginal tissue at the lower end of the uterine opening were cut off, the blood on the surface of the uterus was cleaned with normal saline, the moisture was wiped off, and the uterus was stored in a -20℃ refrigerator.

[0173] 3) Biological sample processing

[0174] Blood sample processing: The collected rat blood samples were centrifuged at 4°C and 5000rpm for 10 minutes, and the supernatant (plasma) was taken for later use. 100μL of the centrifuged plasma was placed in a 2mL centrifuge tube, and 50μL of progesterone standard solution / methanol solution and internal standard solution were added in sequence. After vortexing for 5 minutes, 1mL of acetonitrile was added, and the mixture was vortexed for 10 minutes. After standing for 10 minutes, the mixture was vortexed again for 10 minutes, and centrifuged at 4°C and 11000rpm for 10 minutes. The supernatant was placed in another centrifuge tube and vacuum dried. After the organic phase was evaporated, 200μL of mobile phase was added, vortexed for 10 minutes, stood for 10 minutes, and vortexed again for 10 minutes. Subsequently, the mixture was centrifuged at 11000rpm for 10 minutes. The supernatant was filtered through a 0.22μm filter membrane and injected for detection.

[0175] Uterine Sample Processing: Remove the rat uterus, accurately weigh it, and dilute to 1 mL with pre-chilled PBS for tissue homogenization. Add 100 μL of the rat uterine tissue homogenate to 50 μL of the progesterone standard solution / methanol solution and the internal standard solution, vortexing for 5 minutes. Add 1 mL of acetonitrile and continue vortexing for 10 minutes. Let it stand for 10 minutes, then vortex again for 10 minutes. Centrifuge at 11,000 rpm for 10 minutes at 4°C. Transfer the supernatant to a separate centrifuge tube and vacuum dry. After evaporating the organic phase, add 200 μL of mobile phase, vortex for 10 minutes, let it stand for 10 minutes, then vortex again for 10 minutes. Centrifuge at 11,000 rpm for 10 minutes. Pass the supernatant through a 0.22 μm filter before sampling and analysis.

[0176] 4) Analysis conditions

[0177] Chromatographic conditions: The chromatographic column was an ACQUITY UPLC BEH C18 column (2.1×50 mm, 1.7 μm; Waters, USA); the mobile phase was A (0.1% formic acid in water)-B (acetonitrile); the gradient elution program was as shown in Table 4; the flow rate was 0.3 mL / min; the column temperature was 35° C.; and the injection volume was 10 μL.

[0178] Mass spectrometry conditions included an electrospray ionization (ESI) source in positive ion mode, multiple reaction monitoring (MRM) scanning, an ion spray voltage of 4000 V, and a nebulizer temperature of 300°C. The parent ion / daughter ion pairs for the analyte progesterone were m / z: 315.1→97.0, 315.1→109.1, with a collision energy of 30 eV; the parent ion / daughter ion pairs for the internal standard megestrol acetate (MA) were m / z: 385.1→325.2, 385.1→267.2, with a collision energy of 22 eV.

[0179] Table 4 Gradient elution program

[0180]

[0181] 5) Solution preparation

[0182] Reference solution: Accurately weigh 0.0200 g of progesterone standard, dissolve it in methanol, and dilute to 50 mL to prepare the reference stock solution. Store at 4°C until needed. Accurately pipette an appropriate amount of progesterone stock solution and dilute with methanol to obtain a series of reference solutions with concentrations of 1000 ng / mL, 750 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, 2 ng / mL, and 1 ng / mL, respectively. Store in a refrigerator at 4°C until needed.

[0183] Internal standard solution: Accurately weigh 0.0200 g of MA standard into a 50 mL volumetric flask, dissolve it in methanol, and dilute to volume. This will be the MA stock solution. Store at 4°C until needed. Dilute an appropriate amount of the stock solution to 500 ng / mL and use as the MA internal standard solution. Store at 4°C until needed.

[0184] 6) Linear relationship and standard curve

[0185] The progesterone reference solution with different concentrations was treated with the plasma sample treatment method and the uterine sample treatment method, and then the prepared plasma samples and uterine samples were analyzed according to the analytical conditions. The progesterone concentration was used as the horizontal axis and the relative peak area (the ratio of the progesterone peak area to the MA peak area) was used as the vertical axis. The weighted (1 / x 2 )Linear regression.

[0186] The results are as follows Figure 27 As shown in Figure 2, the progesterone levels in uterine tissue samples and plasma samples were in the range of 1 ng / mL to 500 ng / mL (R 2 =0.9997) and in the range of 2.5ng / mL to 750ng / mL (R 2 =0.9995) has a good linear relationship.

[0187] 7) Uterine Pharmacokinetics

[0188] The results are as follows Figure 28 As shown in Table 5, the time for progesterone to reach peak in the uterus (T max ) were 0.75h, reaching the peak sooner than the Injection group (1h), and the peak concentration (C max ) were 35.132±9.443μg / g and 19.054±1.159μg / g, respectively, significantly higher than the injection group (5.104±1.234μg / g), indicating that vaginal delivery of progesterone can be absorbed faster and more effectively into the uterus. Furthermore, the area under the curve of the IL-FOAM and FOAM groups during uterine administration increased by 11 to 17 times compared to the injection group, showing a significant difference. The uterine targeting efficiency of the two preparations was 85.96% and 81.95%, respectively, which can achieve greater accumulation of progesterone in the uterus, thereby better exerting its efficacy.

[0189] Table 5 Pharmacokinetic results in uterus (n=6)

[0190]

[0191] * p<0.05vs.FOAM; # p<0.05 vs. Injection

[0192] The overall trend of drug concentration changes in the uterus was similar between the IL-FOAM and FOAM groups, but the C max The drug concentration in the uterus of the IL-FOAM group was significantly higher than that of the FOAM group, and at different time points, the drug concentration in the uterus of the IL-FOAM group always maintained a high level. The area under the uterine drug-time curve was about 1.52 times that of the FOAM group, indicating that the presence of [Ch][Ole] in IL-FOAM can not only increase the solubility of progesterone to increase tissue absorption and utilization, but also promote the penetration and absorption of progesterone through the vaginal mucosa.

[0193] 8) Plasma pharmacokinetics

[0194] The results are as follows Figure 29 As shown in Table 6, the T max reduced to 0.5h, with the highest C max and AUC 0-12h , which were 70.936±14.628μg / L and 2987.791±24.083μg / L, respectively. max There was no change. The drug concentration-time curves in plasma of the two groups were always below those of the injection group, indicating that vaginal administration of progesterone can achieve a stable and high drug concentration locally in the uterus with the help of the first-pass effect in the uterus, and at the same time can be absorbed into the blood circulation by the capillaries, thus avoiding the first-pass effect in the liver. However, the proportion of progesterone absorbed into the blood is low, only 16% of that in the injection group, which can effectively reduce the risk of adverse reactions and systemic toxic side effects.

[0195] Table 6 Pharmacokinetic results in plasma (n=6)

[0196]

[0197] * p<0.05 vs. FOAM

[0198] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A progesterone foam gel, characterized in that: The gel precursor solution is prepared from a gel precursor solution and a propellant, wherein the gel precursor solution is obtained by mixing an aqueous solution of a choline ionic liquid, progesterone, and poloxamer; the choline ionic liquid is choline oleic acid, which is obtained by reacting choline bicarbonate or choline hydroxide with oleic acid in a molar ratio of 1:1; The concentration of the choline ionic liquid in the aqueous solution of the choline ionic liquid is 42 mg / mL-42.5 mg / mL; The concentration of progesterone in the gel precursor solution is 8.7 mg / mL-9.1 mg / mL; The weight percentage of the poloxamer in the gel precursor solution is 23.15-23.2%; The poloxamer is poloxamer 407.

2. The progesterone foam gel according to claim 1, characterized in that The concentration of the progesterone in the gel precursor solution is 9.0 mg / mL-9.1 mg / mL.

3. The progesterone foam gel according to claim 1, characterized in that The concentration of the choline ionic liquid in the aqueous solution of the choline ionic liquid is 42.27 mg / mL; The concentration of progesterone in the gel precursor solution is 9.08 mg / mL; The weight percentage of the poloxamer in the gel precursor solution is 23.17%.

4. The progesterone foam gel according to claim 1, characterized in that The propellant is propane and / or butane.

5. The progesterone foam gel according to any one of claims 1 to 4, characterized in that The weight percentage of the propellant in the progesterone foam gel is 2-6%.

6. The progesterone foam gel according to claim 5, characterized in that The weight percentage of the propellant in the progesterone foam gel is 3-5%.

7. The progesterone foam gel according to claim 6, characterized in that The weight percentage of the propellant in the progesterone foam gel is 3.5-4.5%.

8. The progesterone foam gel according to any one of claims 1 to 4, characterized in that The propellant is propane and butane in a volume ratio of 1:2-3.

9. The progesterone foam gel according to claim 8, characterized in that The propellant is propane and butane in a volume ratio of 3:

7.

10. A method for preparing the progesterone foam gel according to any one of claims 1 to 9, characterized in that: The steps include: The progesterone is added to the aqueous solution of the choline ionic liquid, and then the poloxamer is added and fully swelled to obtain the gel precursor solution. The gel precursor solution is added to an aerosol bottle and filled with a propellant to obtain the foam gel.

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