Progesterone composition, progesterone foam gel and preparation method of progesterone foam gel

Abstract

The invention relates to a progesterone composition, progesterone foam gel and a preparation method thereof. The invention finds that choline carbenoic acid can effectively improve the solubility of progesterone in water. According to the progesterone foam gel, progesterone is dissolved in an aqueous solution containing choline carbenoic acid, then the progesterone foam gel, poloxamer and a propellant are prepared into the progesterone foam gel, the progesterone content in the progesterone foam gel is high, the progesterone foam gel can fully cover vaginal mucosa after administration, the contact area between a drug and vaginal tissue is increased, and the progesterone foam gel has temperature response gelling performance and pH response gelling performance at the same time; after vaginal administration, the gel can be quickly converted from foam to gel and is adhered to vaginal mucosa, and the gel is good in self-healing performance and can resist a vaginal discharge mechanism, so that relatively good adhesion and continuous action at a medication part are realized. Meanwhile, the progesterone foam gel can promote permeation and absorption of progesterone through vaginal mucosa, enrichment of progesterone in the uterus after vaginal administration is remarkably improved, and side effects of the whole body are avoided.

Description

Technical Field

[0001] The 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 that is opposite to the natural reproductive process, assisted reproductive technology will inevitably be misaligned with the normal physiological conditions of pregnancy during the treatment process, especially the gonadotropin-releasing hormone agonists or antagonists used in the assisted reproductive technology process, which disrupt the mother's own physiological hormone levels, or the loss of granulosa cells during egg retrieval, which damages the corpus luteum function. Therefore, in the early stages of assisted reproductive technology pregnancy, exogenous progesterone needs to be supplemented in time to maintain the smooth progress of pregnancy. Luteal support has become an indispensable part of improving the success rate of assisted reproductive technology.

[0003] Progesterone is a progestogen secreted mainly by the corpus luteum of the ovary. It acts on the uterus, ovaries, breasts and central nervous system by binding to specific receptors. It is the first choice for luteal support. It is responsible for coordinating the luteal phase through the secretion transformation of the endometrium, preventing the occurrence of menstruation, and creating the best environment for the development of the embryo in the uterus, which is conducive to embryo implantation. It plays a role in stabilizing the endometrium and preserving the pregnancy in early pregnancy, and provides immune tolerance for the embryo implanted in the uterus. It is a hormone necessary for maintaining early pregnancy. Therefore, successful pregnancy in both natural conception and assisted reproductive technology cycles requires sufficient progesterone to obtain a good pregnancy outcome.

[0004] As a tissue organ unique to women, the vagina can be used as an effective route for drug delivery because of its advantages of huge mucosal surface area, rich blood flow and good drug permeability. Compared with intramuscular injection, vaginal administration is more convenient and can be done by oneself, avoiding injection pain, and has better compliance and limited side effects; compared with oral administration, vaginal administration not only avoids the first-pass effect of the liver, but also can achieve "targeting" of the drug with the help of the first-pass effect of the uterus (after the drug is absorbed through the vaginal mucosa, it is directly transported to the uterus), and has 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 pregnane-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, and has certain limitations in formulation design. In addition, there are two important factors affecting the absorption of drugs through the vaginal mucosa: the distribution and retention of the drug delivery system in the vagina and the release of the drug from the drug delivery system and dissolution in the vaginal fluid to penetrate the vaginal mucosa. The folds of the vaginal wall are narrow and irregular in shape and are generally in a contracted state, making it difficult for vaginal drug delivery preparations to fully contact the inner wall of the vagina. The poor coverage of vaginal folds seriously affects the penetration and absorption efficiency of drugs through the vaginal mucosa; and the vagina has a discharge mechanism, that is, when the patient takes a standing position or the muscles around the vagina move in conjunction with the mechanical movement of the vagina, the drug preparation in the vagina will be squeezed and discharged, and it is impossible to achieve long-term retention in the vagina for continuous delivery of drugs. Therefore, the distribution and retention of the folds in the vagina after the preparation is administered and the penetration and absorption of drugs through the vaginal mucosa are the primary considerations in the design of vaginal drug preparations.

[0006] As the world's largest country in assisted reproductive technology treatment, my country has extremely high clinical needs 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 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 object 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 carbenic acid.

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

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

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

[0013] In some embodiments, the choline carboxylic 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 also provides a composition capable of improving the solubility of progesterone, which is composed of progesterone and choline carbenic acid.

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

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

[0025] In some embodiments, the carbon chain length of the anion in the choline carbenic 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 of the 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 described in 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 of the embodiments, the propellant is propane and butane in a volume ratio of 1:2-3.

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

[0060] In a fourth aspect, the present invention also 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 to fully swell to obtain the gel precursor solution. The gel precursor solution is added to an aerosol bottle, and the propellant is filled 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] Further, the present invention dissolves progesterone with an aqueous solution containing choline carbolic acid, and then prepares it into a progesterone foam gel with poloxamer and a propellant, and the content of progesterone 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, and can ensure that the precursor solution is ejected through a valve to form foam, and the foam has a uniform drug content during the ejection process and between different batches of foam gels, good stability, and a small foam density, fine and dense, and the expansion duration meets the standard, and can fully cover the vaginal mucosa after administration, and can increase the contact area between the drug and the vaginal tissue; it has two responsive gelling properties of temperature and pH, which are in line with the physiological environment of the vagina, and can achieve a rapid transition from foam to gel after vaginal administration, adhere to the vaginal mucosa, and achieve good adhesion and continuous action at the medication site; after being converted into a gel in the vagina, its gel self-healing performance is good, fully fits the vaginal environment, can resist the vaginal discharge mechanism, and achieves long-term retention in the vagina to continuously release the drug, and achieves extensive and continuous delivery of the drug.

[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 enrichment 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 carbon NMR spectrum of choline oleic acid with a molar ratio of choline:oleic acid of 1:1.

[0071] Figure 6 The carbon NMR spectrum of choline oleic acid with a molar ratio of choline:oleic acid of 1:2.

[0072] Figure 7 This is the C NMR spectrum of choline oleic acid with a molar ratio of choline:oleic acid of 1:3.

[0073] Figure 8 The carbon NMR spectrum of choline oleic acid with a molar ratio of choline:oleic acid of 1:4.

[0074] Fig. 9 This is the carbon NMR spectrum of choline oleic acid with a molar ratio of choline:oleic acid of 1:5.

[0075] Fig.10 The carbon NMR spectrum of choline oleic acid with a molar ratio of choline:oleic acid of 2:1.

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

[0077] Fig.12 This is the carbon NMR spectrum of choline oleic acid with a molar ratio of choline:oleic acid of 4:1.

[0078] Fig.13 This is the carbon NMR spectrum of choline oleic acid with a molar ratio of choline:oleic acid of 5:1.

[0079] Fig.14 It is the HPLC chromatogram of progesterone.

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

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

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

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

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

[0085] Fig. 20The thixotropy test results of the progesterone foam gel precursor solution are shown in FIG.

[0086] Fig.21 These are the drug content uniformity results for progesterone foam gel.

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

[0088] Fig.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] Fig.24 To investigate the distribution and retention of progesterone foam gel in a vaginal structural model.

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

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

[0092] Fig. 27 The standard curves of progesterone in uterine tissue samples (A) and plasma samples (B).

[0093] Fig.28 The uterine pharmacokinetics results of progesterone foam gel.

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

[0095] In order to facilitate the 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 disclosure of the present invention more thorough and comprehensive.

[0096] The experimental methods in the following examples without specifying specific conditions are usually 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 in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0098] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is 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. In addition, throughout the 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 15.6mL of choline hydroxide methanol solution ([Ch][OH] / MeOH, 45%, 57.76mmol), evaporate the methanol in the solution under vacuum at 40℃, add 50mL of glycine (4.34g, 57.79mmol) aqueous solution, cool in an ice bath and stir for 12h, remove water under vacuum at 50℃. Add acetonitrile / methanol (9:1, v / v) to precipitate excess glycine (Glycinate, [Gly]), filter, and remove the solvent by rotary evaporation at 40℃. The obtained choline glycine ([Ch][Gly]) is vacuum dried at 60℃ 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 (Palm Oleate, [Pal], 3.93 mmol) was weighed and dissolved in 20 mL of methanol, and then slowly added dropwise to 0.79 g of 80% choline bicarbonate (3.82 mmol) aqueous solution. The reaction mixture was stirred at room temperature for 1 h until CO2 was no longer released, and methanol and water were removed by vacuum rotary evaporation at 60°C. The obtained choline palmitoleic acid ([Ch][Pal]) was vacuum dried at 60°C overnight and stored in a moisture-free condition for later 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, then slowly drip it into 1.22 g of 80% choline bicarbonate (5.88 mmol) aqueous solution. Stir the reaction mixture at room temperature for 1 hour until CO2 is no longer released, and remove methanol and water by vacuum rotary evaporation at 60°C. The obtained choline erucic acid ([Ch][Eru]) is vacuum dried at 60°C overnight and stored in a moisture-free condition for later 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] According to the proportions of the components in Table 1, different proportions of choline oleic acid were synthesized by the following method: the prescribed amount of oleic acid (Oleate, [Ole]) was weighed and dissolved in 20 mL of methanol, and then slowly dripped into a corresponding proportion of 80% choline bicarbonate aqueous solution. The above reaction mixture was stirred at room temperature for 1 hour until CO2 was no longer released, and methanol and water were removed by vacuum rotary evaporation at 60°C. The obtained choline oleic acid ([Ch][Ole]) was vacuum dried at 60°C overnight and stored under moisture-free conditions for later use.

[0113] Table 1 Choline oleic acid prescription

[0114]

[0115] The prepared ionic liquids were characterized by nuclear magnetic resonance. 1 H NMR and 13 C NMR spectroscopy identification (such as Figure 1-Figure 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 with concentrations of 5, 10, 25, 50, 75, 100, 250 and 500 μg / mL were prepared by gradient dilution method, and the progesterone methanol solutions with different concentrations were injected and tested respectively. The HPLC chromatograms of the samples of each concentration were recorded and the peak areas at each concentration were calculated by integrating the sample peaks. The standard curve of progesterone was drawn by linear regression method with progesterone concentration as the abscissa (x) and peak area (y) as the ordinate.

[0123] 3) Results

[0124] Liquid phase spectrum Fig.14 As shown in Figure 2, the retention time of progesterone is about 11.078 min; the standard curve is as follows Fig.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 on progesterone

[0126] Weigh appropriate amounts of ionic liquids of different compositions, 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, and store them in the dark as reserve solutions for later use.

[0127] Take 3mL of the stock solution in an EP tube, add enough progesterone under stirring to form a supersaturated solution, vortex for 15 minutes, transfer the EP tube to a constant temperature air bath oscillating box, 25°C, 100rpm, balance for 72 hours. Centrifuge at 4500rpm for 20 minutes, let stand for 3 hours, take the supernatant and filter it, and detect its progesterone content by HPLC.

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

[0129] The solubility of choline glycine and choline oleic acid solutions at different concentrations on progesterone Fig.16 As shown, 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 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 carbolic acids (palmitoleic acid (9-hexadecenoic acid), oleic acid (9-octadecenoic acid) and erucic acid (docosa-13-enoic acid)) and choline was compared. Fig.17 As shown: Ionic liquids with different carbon chain lengths have significantly improved the solubility of progesterone, and the solubility increases with the increase of ionic liquid content. In addition, the increase of anionic carbon chain length within a certain range can increase the solubility of choline carbenic acid ionic liquid for progesterone, but its solubilization ability does not continue to increase with the increase of anionic carbon chain length. Among them, choline oleic acid has the strongest solubilization ability.

[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 Fig.18 and Fig.19 As shown in the figure, when the molar ratio of oleic acid / 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 and 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] Weigh an appropriate amount of choline oleic acid (1:1) to prepare a series of aqueous solutions (concentrations shown in Table 3), add a sufficient amount of progesterone to form a supersaturated solution, and balance in an air bath shaker at 25°C and 100rpm for 72h. Then centrifuge at 4500rpm for 20min, take the supernatant, slowly add poloxamer 407 (P407) in the amount prescribed in Table 3, mix well, and store at 4°C overnight (12h) to fully swell to obtain a clear and transparent gel precursor solution (the proportion of P407 in Table 3 refers to the weight percentage of P407 in the gel precursor solution), take 15mL into an aluminum aerosol bottle, seal it with a capping machine, and fill it with a propellant (propane: butane = 3:7) at a content ratio of 4% (w / w) to obtain a foam gel (IL-FOAM).

[0137] 1) Determination of gelation temperature

[0138] The gelation temperature of the gel was determined by the test tube inversion method. 1 mL of the precursor solution was placed in a vial and placed in a water bath, with the water bath level kept higher than the level of 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 taken out and the fluidity of the precursor solution was observed by tilting the vial. The temperature at which the precursor solution no longer flowed was recorded as its gelation temperature.

[0139] 2) Determination of gel time

[0140] Preheat the water bath to 37°C and set the magnetic stirring speed to 20 rpm. Take 1 mL of the precursor solution into a vial with a magnetic rotor in advance, start timing from the time it is placed in the water bath (keep the solution level below the water bath level), and record the time when the magnetic rotor stops rotating, which is the gelation time.

[0141] 3) pH responsiveness study

[0142] Normal vaginal pH is between 3.5 and 5.5, but changes to 3.8 to 4.4 during pregnancy. Therefore, the intermediate value pH = 4 was selected to verify the pH responsiveness of the prepared ionic liquid gel system. An appropriate amount of hydrochloric acid was used to prepare an acid solution of pH = 4 and stored for later use. Take 1 mL of the precursor solution in a centrifuge tube and slowly add 0.2 mL of the pre-prepared acid solution (pH = 4) under stirring. Start timing from the addition of the acid solution, observe the gel transformation of the precursor solution, and record the time taken to transform into gel through pH response.

[0143] The specific prescriptions and results are shown in Table 3. The gelation temperatures of all prescriptions are within the range of 31-35°C, which meets the vaginal temperature environment. The gelation time is <90s, and they can quickly turn into gel when in contact with the vaginal surface and adhere to the vaginal wall. Among them, the gelation temperature of F6 prescription is the highest within the expected range, which can avoid the influence of ambient temperature; the gelation time and pH response time are the shortest, which can ensure that it can turn into gel faster after vaginal administration to achieve adhesion retention. Therefore, F6 is the optimal prescription, and the solubility of progesterone can be increased to 9.08±0.23g / 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 the Anton Paar rotational rheometer to simulate 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. Fig. 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] Take the foam gel prepared by F6 prescription, shake it evenly, discard the foam sprayed in the first 3 seconds, move at a constant speed and spray it on a clean glass plate until the foam is completely sprayed. Take the front, middle and back foams respectively, weigh them, dissolve them in methanol to the same mass concentration, filter them, and detect their progesterone content by HPLC to analyze the uniformity of the spraying process; take another randomly prepared foam gel, shake it evenly, discard the foam sprayed in the first 3 seconds, spray an appropriate amount of foam into a centrifuge tube, weigh them, dissolve them in methanol to the same mass concentration, filter them, and detect their 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 Fig.21As shown, during the foam spraying process, there is no difference in the drug content in the front section, middle section and back section 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 by prescription F6 evenly, discard the foam sprayed in the first 3 seconds, and spray an appropriate amount of foam evenly on a culture dish and a glass slide preheated to 37°C (the surface is soaked with an acid solution of pH = 4 before use), maintain the ambient temperature at 37°C, and record the changes in foam morphology within 5 minutes with a camera and an upright microscope respectively.

[0153] Comparative Example: Using Tween 80 (T80) instead of P407 to prepare foam gel in the same way, and recording the changes in foam morphology within 5 minutes in the same way.

[0154] The results are as follows Fig. 22 As shown, when the foam is just sprayed out, compared with the foam formed by the foam gel prepared with T80, the foam of the foam gel based on P407 has a more delicate and dense appearance, and the microstructure shows fewer bubble gaps. In addition, the foam formed by T80 maintains a typical "gas-liquid structure", which is the normal decay process of the foam: large bubbles continuously merge with small bubbles, and then the surface of the large bubbles becomes thinner and breaks, and finally disappears. The foam based on P407 has a certain expansion in the first 2 minutes and does not decay; it starts to change from foam to gel structure from the 3rd minute, showing a mesh gap structure similar to the typical three-dimensional mesh structure of gel. It shows that the foam gel based on P407 prepared by the present invention can achieve drug administration in the form of foam, use the expansion advantage of foam to increase the contact area between the drug and the tissue, and directly transform from foam to gel under the temperature and pH response of the medication site, so as to achieve good adhesion and sustained action at the medication site.

[0155] 7) Gel self-healing

[0156] The self-healing properties of the gels formed in response to temperature (37°C) and pH (4), respectively, 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 strain was applied in a cycle of 0.5% → 100% → 0.5%. The cycle was repeated 3 times, with each strain value 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 Fig.23As shown in the figure, the changes in modulus of the gel formed by temperature response and pH response under cyclic strain are basically the same: when the strain increases from 0.5% to 100%, the gel modulus shows dynamic changes and recovery, with good self-healing properties. This feature can ensure that the foam gel can resist the vaginal discharge mechanism after it is transformed into a gel in the vagina.

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

[0159] A 3D printing simulation model of human vaginal structure was used, and the distribution of the preparation in the vagina was observed by a small animal in vivo imaging system. The foam gel based on P407 and the foam gel based on T80 were prepared respectively according to the method of Example 3 (based on the prescription 6 of Example 3, 10 μL of DID ethanol solution with a concentration of 10 mg / mL was added to the gel precursor solution before filling and mixed), and the foam sprayed in the first 3s was discarded after filling and shaking, and sprayed through the bottom of the vaginal model to fill the entire vaginal cavity with foam. The model was placed upright in an air bath shock box at 37°C, 100r / min, and 0.2mL of SVF was slowly added from the top of the model every 2h to simulate the vaginal discharge environment. The distribution of foam gel in the vaginal model at 0h, 1h, 2h, 4h, 8h, 12h and 24h was observed by a small animal in vivo imaging system to study the retention capacity of the preparation in the vagina.

[0160] The results are as follows Fig.24 As shown in the figure, it can be seen from the image at 0h that, based on the expansion characteristics of the foam, the foam gel can quickly and evenly fill the entire vaginal cavity after administration, maximizing the contact area between the drug and the inner wall of the vagina. The foam system based on T80 completely breaks down and turns into liquid outflow in 1h, and the fluorescence dissipates accordingly; the foam gel based on P407, after administration, is triggered by the vaginal physiological environment, and turns from foam to gel and adheres to the inner wall of the vagina, increasing the contact time between the preparation and the vagina, achieving a long-term retention of the preparation at the administration site, thereby achieving a sustained effect. The image at 24h shows that the gel can be maintained in the vagina for up to 24h.

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

[0162] According to the method of Example 3, IL-FOAM containing Cy5.5 was prepared (based on the prescription 6 of Example 3, 100 μL of 10 mg / mL Cy5.5 aqueous solution was added to the gel precursor solution before filling and mixed), the foam sprayed in the first 3 seconds was discarded after filling and shaking, and a blunt needle was inserted into the female mouse vagina about 1 cm, and 200 μL was administered. The distribution of foam gel in the rat vagina at 0h, 1h, 2h, 4h, 8h, 12h and 24h was observed using a small animal in vivo imaging system to study the retention capacity of the preparation in the vagina.

[0163] The results are as follows Fig.25 As shown, consistent with the results of the in vitro simulation experiment, IL-FOAM can quickly fill the vaginal cavity of rats after administration, showing a large area of ​​strong fluorescence in the vagina at 0h; with the action of the vagina, the fluorescence area and intensity gradually decreased until 24h when weak fluorescence remained at the vaginal opening of the rats. The retention time can last up to 24h, which is expected to achieve a 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 (FOAM) without ionic liquid were prepared with the F6 formula (except for the absence of ionic liquid, the other components and contents were consistent with the IL-FOAM group, and progesterone existed in a suspended form). Before filling, 10 μL of 10 mg / mL DID ethanol solution was added and mixed. After filling, the foam sprayed in the first 3 seconds was discarded, and a blunt needle was inserted into the vagina of the female mouse for about 1 cm, and 200 μL of the drug was administered; an appropriate amount of DID and Mix evenly until the concentration is consistent with the first two groups, insert a blunt needle into the female mouse vagina about 1 cm, and administer 200 μL. Start timing after administration, euthanize the female mouse at 15 minutes, remove the rat vaginal tissue, cut off the fallopian tube, carefully peel off the upper vaginal bladder and surrounding fat tissue, freeze the removed vagina at -80℃ and slice, and observe the fluorescence distribution of DAPI and DID at 405nm and 646nm fluorescence channels with laser confocal microscope after DAPI staining, and analyze the fluorescence penetration intensity in the vaginal mucosa using ImageJ software.

[0166] The results are as follows Fig.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 similar to the commercially available preparations. Compared with the two groups, both groups of 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 3 groups, and each group was given a progesterone injection (Injection) (with soybean oil as solvent), a foam gel (FOAM) without ionic liquid (except for the absence of ionic liquid, the other components and contents were consistent with the IL-FOAM group, and progesterone existed in a suspended form) and an ionic liquid foam gel (IL-FOAM) prepared by the method of Example 3 with the F6 prescription, 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 through the abdominal aorta with a 10 mL syringe, centrifuged at 4 ° C, 5000 rpm for 10 min, and the upper plasma was 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 saline, the water 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, 5000rpm for 10min, and the supernatant (plasma) was taken for later use. Take 100μL of the centrifuged plasma in a 2mL centrifuge tube, add 50μL of progesterone standard solution / methanol solution and internal standard solution in turn, vortex for 5min, add 1mL of acetonitrile, continue vortexing for 10min, let stand for 10min, vortex again for 10min, and centrifuge at 4°C, 11000rpm for 10min. Take the supernatant in another centrifuge tube and vacuum dry. After the organic phase evaporates, add 200μL of mobile phase, vortex for 10min, let stand for 10min, vortex again for 10min, then centrifuge at 11000rpm for 10min, take the supernatant and filter through a 0.22μm filter membrane for sampling and detection.

[0175] Uterine sample processing: Remove the rat uterus, weigh it accurately, and then use pre-cooled PBS to make the volume to 1mL for tissue homogenization. Take 100μL of rat uterine tissue homogenate, add 50μL of progesterone standard solution / methanol solution and internal standard solution in turn, vortex for 5min, add 1mL of acetonitrile, continue to vortex for 10min, let it stand for 10min, vortex again for 10min, centrifuge at 4℃, 11000rpm for 10min. Take the supernatant in another centrifuge tube and vacuum dry. After the organic phase evaporates, add 200μL of mobile phase, vortex for 10min, let it stand for 10min, vortex again for 10min, then centrifuge at 11000rpm for 10min, take the supernatant and filter it through a 0.22μm filter membrane for sampling and detection.

[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: electrospray ion source (ESI), positive ion mode detection, multiple reaction monitoring mode (MRM), ion spray voltage of 4000V, nebulizer temperature of 300°C. The parent ion / daughter ion pairs of the analyte progesterone (Progesterone) are m / z: 315.1→97.0, 315.1→109.1, and the collision energy is 30eV; the parent ion / daughter ion pairs of the internal standard megestrol acetate (MA) are m / z: 385.1→325.2, 385.1→267.2, and the collision energy is 22eV.

[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 make up to 50 mL as the reference stock solution, and store it at 4°C for later use. Accurately pipette an appropriate amount of progesterone stock solution, dilute it 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, and store it in a refrigerator at 4°C for later use.

[0183] Internal standard solution: Accurately weigh 0.0200 g of MA standard into a 50 mL volumetric flask, dissolve it in methanol and make up to volume, use it as MA stock solution, and store it at 4 °C for later use. Take an appropriate amount of the stock solution and dilute it to 500 ng / mL, use it as MA internal standard solution, and store it at 4 °C for later use.

[0184] 6) Linear relationship and standard curve

[0185] The progesterone reference solution with different concentrations was treated by the plasma sample treatment method and the uterine sample treatment method, respectively. 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 (ratio of the progesterone peak area to the MA peak area) was used as the vertical axis. The weighting (1 / x 2 )Linear regression.

[0186] The results are as follows Fig. 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 Fig.28 As shown in Table 5, the peak time of progesterone 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, which were significantly higher than the Injection group (5.104±1.234μg / g), indicating that vaginal delivery of progesterone can be absorbed faster and more into the uterus. In addition, the area under the curve of the IL-FOAM group and the FOAM group in the uterus was 11 to 17 times higher than that of the Injection group, which was significantly different. The uterine targeting efficiency of the two groups of preparations was 85.96% and 81.95%, respectively, which can achieve more enrichment of progesterone in the uterus to better exert its efficacy.

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

[0190]

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

[0192] The overall trend of uterine drug concentration changes in the IL-FOAM and FOAM groups was similar, but the C max The results showed that [Ch][Ole] in IL-FOAM group was significantly higher than that in FOAM group, and the drug concentration in the uterus of IL-FOAM group remained at a high level at different time points. The area under the uterine drug-time curve was about 1.52 times that of 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 Fig.29 As shown in Table 6, the T max Reduced to 0.5h, with the highest C max and AUC 0-12h , 70.936±14.628μg / L and 2987.791±24.083μg / L, respectively. T max There was no change. The drug concentration-time curves of the two groups in plasma were always below those of the Injection group, indicating that vaginal administration of progesterone can achieve a relatively high and stable drug concentration locally in the uterus with the help of the first-pass effect in the uterus, and can also be absorbed into the blood circulation by capillaries, thus avoiding the first-pass effect in the liver. However, the proportion of progesterone absorbed into the blood was relatively 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<0.05vs.FOAM

[0198] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of choline ionic liquid as a cosolvent in improving the solubility of progesterone; the choline ionic liquid is choline carbenic acid.

2. The use according to claim 1, characterized in that: The carbon chain length of the anion in the choline carbenic acid is 8-36, preferably 12-30, more preferably 16-22; Preferably, the choline carbenic acid is choline palmitoleic acid, choline erucic acid or choline oleic acid.

3. The use according to claim 1, characterized in that: 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; Preferably, the choline carbene acid is obtained by reacting choline bicarbonate or choline hydroxide with carbene acid in a molar ratio of 1-2:2-1; Preferably, the choline carbene acid is obtained by reacting choline bicarbonate or choline hydroxide with carbene acid in a molar ratio of 1:1; Preferably, the carbon chain length of the carbene acid is 8-36, preferably 12-30, more preferably 16-22; Preferably, the carboxylic acid is palmitoleic acid, erucic acid or oleic acid.

4. The use according to any one of claims 1 to 3, characterized in that: The progesterone solubility refers to the solubility of progesterone in water.

5. A composition capable of improving the solubility of progesterone, characterized in that: It is composed of progesterone and choline carboxylic acid.

6. The composition capable of improving the solubility of progesterone according to claim 5, characterized in that: The carbon chain length of the anion in the choline carbenic acid is 8-36, preferably 12-30, more preferably 16-22; Preferably, the choline carbenic acid is choline palmitoleic acid, choline erucic acid or choline oleic acid; Preferably, the mass ratio of progesterone to choline carbenic acid is 1:1-10, preferably 1:2-8, preferably 1:3-6, preferably 1:4-5, and more preferably 1:4.6-4.

9.

7. The composition capable of improving the solubility of progesterone according to claim 5, characterized in that: 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; Preferably, the choline carbene acid is obtained by reacting choline bicarbonate or choline hydroxide with carbene acid in a molar ratio of 1-2:2-1; Preferably, the choline carbene acid is obtained by reacting choline bicarbonate or choline hydroxide with carbene acid in a molar ratio of 1:1; Preferably, the carbon chain length of the carbene acid is 8-36, preferably 12-30, more preferably 16-22; Preferably, the carboxylic acid is palmitoleic acid, erucic acid or oleic acid.

8. 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 as described in any one of claims 1 to 3.

9. The progesterone foam gel according to claim 8, characterized in that The concentration of the choline ionic liquid in the aqueous solution of the choline ionic liquid is 30 mg / mL-50 mg / mL; and / or, The concentration of progesterone in the gel precursor solution is 7 mg / mL-10 mg / mL; and / or, The weight percentage of the poloxamer in the gel precursor solution is 15-30%; and / or, The weight percentage of the propellant in the progesterone foam gel is 2-6%; and / or, The poloxamer is poloxamer 407; and / or, The propellant is propane and / or butane; Preferably, the concentration of the choline ionic liquid in the aqueous solution of the choline ionic liquid is 35 mg / mL-48 mg / mL, more preferably 37 mg / mL-45 mg / mL, more preferably 41 mg / mL-42.5 mg / mL, more preferably 42 mg / mL-42.5 mg / mL; Preferably, the concentration of progesterone in the gel precursor solution is 8.7 mg / mL-9.1 mg / mL, more preferably 9.0 mg / mL-9.1 mg / mL; Preferably, the weight percentage of the poloxamer in the gel precursor solution is 22-25%, more preferably 22.5-23.5%, more preferably 23.1-23.2%, more preferably 23.15-23.2%; Preferably, the weight percentage of the propellant in the progesterone foam gel is 3-5%, more preferably 3.5-4.5%; Preferably, the propellant is propane and butane in a volume ratio of 1:2-3.

10. A method for preparing the progesterone foam gel according to claim 8 or 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 to fully swell to obtain the gel precursor solution. The gel precursor solution is added to an aerosol bottle, and the propellant is filled to obtain the foam gel.

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