Progesterone cyclodextrin-metal organic framework inclusion compound and preparation method thereof

Abstract

The embodiment of the invention discloses a cyclodextrin-metal organic framework inclusion compound for improving the solubility of progesterone. The inclusion compound comprises a cyclodextrin-metal organic framework material, and the progesterone is entrapped in the cyclodextrin-metal organic framework material. According to the inclusion compound disclosed by the invention, the drug-loaded inclusion compound is prepared by a simple impregnation method, so that the solubility and the dissolution rate of the progesterone can be effectively improved, rapid dissolution and rapid release of the progesterone in water can be realized, and the problems of poor water solubility, low oral bioavailability and the like of the progesterone are solved. The materials of the clathrate compound are cheap, easy to obtain, green and safe. And a new reference can be provided for research and development of oral preparations of the insoluble drug progesterone.

Description

Technical Field

[0001] The invention relates to a progesterone cyclodextrin-metal organic framework inclusion compound and a preparation method thereof. Background Art

[0002] Progesterone, also known as progesterone, 4-progesterone-3,20-dione, is a progestogen with a molecular formula of C 21 H 30 O2 is a white or off-white crystalline powder, odorless and tasteless; it dissolves in ethanol, ether or vegetable oil, but is insoluble in water. Progesterone has low solubility and poor absorption in the body. At the same time, oral administration also has a first-pass effect in the liver, is rapidly metabolized, has a low blood concentration, and has a bioavailability of less than 10%. Progesterone is relatively unstable to acid, alkali, and light.

[0003] Progesterone has many physiological effects. Clinically, progesterone is mainly used for menstrual disorders, amenorrhea, threatened abortion, luteal insufficiency, premenstrual syndrome and hormone replacement therapy. Progesterone is a BCS IV drug. Its low solubility, poor absorption in the body and first-pass effect in the liver lead to low oral bioavailability and poor clinical application.

[0004] In the field of new drug research and development, about 40% of the drugs on the market and nearly 90% of the compounds under development are poorly soluble drugs. The solubility and dissolution rate of drugs are important factors affecting the bioavailability of raw materials. The solubility and dissolution rate of drugs are also one of the key parameters that need to be considered in pre-drug prescription research. At present, a variety of strategies have been reported to improve the solubility and dissolution rate of progesterone, such as cyclodextrin inclusion, solid dispersion, proliposome, nanomicelle, emulsion injection, liposome, microsphere, etc. However, these traditional strategies often have problems such as low drug loading and loading efficiency. Therefore, the art needs to develop a formulation process that can effectively improve the solubility, drug release and oral bioavailability of progesterone. Summary of the invention

[0005] The embodiment of the present invention provides a method for preparing a progesterone-cyclodextrin metal organic framework inclusion complex to solve the problems of low solubility, low dissolution, and low bioavailability of progesterone.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides an inclusion compound, which comprises a cyclodextrin-metal organic framework and progesterone loaded in the cyclodextrin metal organic framework. The preparation method of the cyclodextrin metal organic framework is:

[0008] Anhydrous potassium carbonate and gamma-cyclodextrin are added to ultrapure water, heated and stirred at 50-70°C for 5-30 minutes, the reaction liquid is collected, the reaction liquid is placed in an ice bath at 0-10°C and stirred, acetone is added dropwise, flaky crystals are generated in the reaction liquid, the stirring is stopped after reacting for 10-30 minutes, the precipitate is collected by centrifugation, washed with isopropanol, and the precipitate is vacuum dried to obtain the cyclodextrin-metal organic framework.

[0009] According to the second aspect of the embodiment of the present invention, the embodiment of the present invention provides a method for preparing the progesterone-cyclodextrin metal organic framework inclusion complex:

[0010] dissolving progesterone in anhydrous ethanol by ultrasonication to obtain a progesterone ethanol solution;

[0011] Adding cyclodextrin metal organic framework to the progesterone ethanol solution to carry out drug loading reaction, centrifuging to remove the supernatant, washing with anhydrous ethanol, and vacuum drying to obtain a powder which is the progesterone-cyclodextrin metal organic framework inclusion complex;

[0012] Furthermore, the concentration of the progesterone ethanol solution is 0.5 mg-10 mg mL -1 .

[0013] Furthermore, the molar ratio of the cyclodextrin-metal organic framework to progesterone is 1:0.5-1:16.

[0014] Furthermore, the drug loading reaction time is 1-24h.

[0015] Further, the progesterone-cyclodextrin metal organic framework inclusion complex has one or more of the following characteristics:

[0016] (1) The average particle size of the cyclodextrin-metal organic framework material is 0.5-5 μm;

[0017] (2) The drug loading of the cyclodextrin-metal organic framework material is 8%-25%;

[0018] The inclusion compound of the present application has an effect of increasing the solubility of progesterone, and increases the solubility of progesterone in ultrapure water by 1-100 times, preferably 10-80 times, and further preferably 20-50 times.

[0019] The inclusion compound of the present application has an effect of increasing the dissolution of progesterone, and increases the solubility of progesterone in ultrapure water by 20%-60%.

[0020] According to pharmacokinetic data (AUC 0-t , C max ) and other statistical analyses showed that the progesterone-cyclodextrin metal organic framework inclusion complex can significantly improve the oral bioavailability of progesterone.

[0021] The progesterone-cyclodextrin metal organic framework inclusion compound of the present invention uses the cyclodextrin metal organic framework as a drug carrier, can effectively improve the water solubility, dissolution and oral bioavailability of the progesterone raw material, and can provide a new idea for the research and development of oral preparations of the poorly soluble drug progesterone. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0023] Figure 1 This is a SEM micrograph of the cyclodextrin metal organic framework provided in an embodiment of the present invention.

[0024] Figure 2 TEM micrograph of the cyclodextrin metal organic framework according to an embodiment of the present invention.

[0025] Figure 3 This is an infrared spectrum of the cyclodextrin metal-organic framework provided in an embodiment of the present invention.

[0026] Figure 4 This is a solubility diagram of the progesterone-cyclodextrin metal organic framework inclusion complex provided in an embodiment of the present invention.

[0027] Figure 5 This is a dissolution diagram of the progesterone-cyclodextrin metal organic framework inclusion complex provided in an embodiment of the present invention.

[0028] Figure 6 This is a graph showing the pharmacokinetic results of the progesterone-cyclodextrin metal organic framework inclusion complex provided in an embodiment of the present invention.

[0029] Table 1 shows the pharmacokinetic parameters of the progesterone-cyclodextrin metal organic framework inclusion complex provided in the examples of the present invention. DETAILED DESCRIPTION

[0030] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] As used herein, the terms "progesterone-cyclodextrin metal-organic framework inclusion complex", "progesterone-cyclodextrin metal-organic framework inclusion complex", "progesterone-CD-MOFs inclusion complex", "progesterone-CD-MOFs inclusion complex", "HTT-CD-MOFs inclusion complex", "CD-MOFs loaded progesterone", and "CD-MOFs loaded HTT" can be used interchangeably, and the above terms all represent samples obtained by drug loading reaction of progesterone and cyclodextrin metal-organic framework.

[0032] General Methods

[0033] Progesterone content determination conditions: (1) UV spectrophotometry: refer to the dissolution determination method of progesterone injection in the second part of the 2020 edition of the Chinese Pharmacopoeia, and the detection wavelength is 241nm. (2) Ultra-high performance liquid chromatography (UPLC) method: refer to the content determination method of progesterone injection in the second part of the 2020 edition of the Chinese Pharmacopoeia. The chromatographic column is ACQUITY UPLC BEH C18 (100mm×2.1mm, 1.7μm); mobile phase: methanol: 0.1% formic acid water = 75:25; flow rate 0.3ml / min; column temperature 40℃; detection wavelength 241nm; injection volume 1μl.

[0034] Method for determination of progesterone content in plasma: (1) Chromatographic conditions: Chromatographic column: Waters ACQUITY UPLC BEHC18 column (50 mm × 2.1 mm, 1.7 μm); flow rate: 0.3 mL / min; injection volume: 1 μL; column temperature: 35 °C; mobile phase: 0.1% formic acid water (A)-acetonitrile (B); gradient elution. (2) Mass spectrometry conditions: ion source: electrospray ionization source (ESI); multiple reaction monitoring mode (MRM); ion polarity: positive ion; ion reaction: progesterone m / z 315.0→96.9, cone voltage 44 V, collision energy 20 eV; desolvation gas temperature: 450 °C; source temperature: 150 °C; desolvation gas flow rate: 800 L / h; cone gas: 150 L / h; capillary voltage 3000 V.

[0035] Progesterone solubility determination method: Weigh an excess of progesterone-cyclodextrin metal organic framework inclusion complex, disperse it in ultrapure water, shake it at a constant temperature for 24 hours, collect the supernatant by centrifugation, filter it with a 0.22μm filter membrane, and determine the progesterone content in the solution according to the above "progesterone content determination method". Record it as the water solubility of the sample to be tested at 25°C.

[0036] Solubilization factor: the ratio of the solubility of the sample to be compared to the solubility of progesterone raw material in ultrapure water (25°C).

[0037] Determination and calculation method of drug loading and drug loading molar ratio: Accurately weigh 10 mg (m1) of the sample to be tested, place it in a 25 ml brown volumetric flask, dilute it to the scale with 50% methanol aqueous solution, and determine the progesterone content according to the "progesterone content determination conditions", and calculate the progesterone mass (m2) in the sample to be tested.

[0038] Drug loading (%) = m2 / m1×100%;

[0039] The molar ratio of the carrier (the cyclodextrin-metal organic framework) to progesterone is: ((m1-m2) / 1297.12) / (m2 / 314.46), wherein 1297.12 is the molecular weight of the cyclodextrin-metal organic framework, and 314.46 is the molecular weight of the progesterone.

[0040] Example 1 Preparation of Cyclodextrin Metal-Organic Framework

[0041] 100 ml of ultrapure water was measured and placed in a beaker, 25.6 g of γ-cyclodextrin and 10.9 g of anhydrous potassium carbonate were added and dissolved in the ultrapure water, and ultrasonically dissolved for 5 min, and heated in a water bath at 60 ° C for 5 min. After the reaction was completed, the reaction solution was collected and cooled at 0 ° C for 30 min. After a white turbid precipitate was precipitated in the reaction solution, the reaction solution was placed at 0 ° C and reacted with magnetic stirring (300 rpm, 10 min), and acetone (20 ml) was added dropwise during the reaction. After the acetone was added, the reaction was continued to stir and the reaction was stopped after the reaction solution became a white viscous liquid. The reaction solution was collected in a centrifuge tube, anhydrous ethanol was added and centrifuged for washing three times, and the precipitate after washing was collected. At this time, the precipitate was white powder. The white powder was placed in a vacuum drying oven at 50 ° C and dried for 6 h to obtain a micron-sized cyclodextrin metal organic framework with a particle size of 1-5 μm.

[0042] Test Example 1 Characterization of CD-MOFs

[0043] Scanning electron microscopy (SEM)

[0044] The CD-MOFs prepared in Example 1 were characterized by SEM micromorphology.

[0045] Method: Take a small amount of CD-MOFs powder and place it on the conductive glue. Place the conductive glue in a metal short stake for sampling and observation. After magnification at an appropriate multiple, obtain the SEM result image, and use Image J software to measure the particle size.

[0046] like Figure 1 As shown, SEM results show that CD-MOFs were successfully prepared with a particle size of 1-5 μm.

[0047] Transmission Electron Microscopy (TEM)

[0048] The CD-MOFs prepared in Example 1 were characterized by TEM microstructure.

[0049] Method: Take a small amount of CD-MOFs powder and place it in anhydrous ethanol solution, disperse it by ultrasonic for 5 minutes, and use a rubber-tipped dropper to draw 2 ml and drop it on the copper grid for dispersion. After natural air drying, the copper grid is tested and the TEM result image is obtained after magnification at an appropriate multiple.

[0050] like Figure 2 As shown, TEM results show that the microscopic morphology of CD-MOFs is rectangular flakes with uniform particle size.

[0051] Test Example Preparation and Content Calculation of Progesterone-CD-MOFs Inclusion Complex

[0052] The progesterone-CD-MOFs inclusion complex was prepared by the impregnation method, and the optimal drug loading conditions were screened from three factors: the initial concentration of the progesterone anhydrous ethanol solution, the molar ratio of progesterone to the carrier CD-MOFs, and the reaction time, in order to obtain the optimal drug loading.

[0053] Methods: Progesterone was dissolved in anhydrous ethanol solution by ultrasonication and prepared into 0.5, 1.0, 2.0, 4.0, and 8.0 mg·mL -1 The progesterone anhydrous ethanol solution was prepared, and the molar ratios of progesterone to CD-MOFs were 0.5:1, 1:1, and 2:1, respectively. The CD-MOFs were placed in the progesterone anhydrous ethanol solution at room temperature, and the drug loading reaction was carried out under constant temperature magnetic stirring conditions. The reaction time was set to 0.5h, 2h, 8h, and 12h. The progesterone CD-MOFs inclusion complex under different drug loading reaction conditions was collected, washed with anhydrous ethanol to remove unreacted impurities (progesterone, γ-cyclodextrin, etc.) on the surface, and the white powder precipitate was collected and vacuum dried at 50°C for 6h to obtain the progesterone-CD-MOFs inclusion complex.

[0054] Figure 3 The Fourier transform infrared spectra of progesterone API, progesterone-cyclodextrin metal organic framework inclusion complex and physical mixture (progesterone API and cyclodextrin metal organic framework were ground and mixed in a ratio of 1:1). Progesterone is at 1699cm -1 There is a sharp absorption peak at 1662cm, which is the C=O stretching vibration absorption peak; -1 The sharp absorption peak at 1662cm is the α-β unsaturated ketone. Due to the conjugation effect, the C=O stretching vibration absorption peak has blue shifted. -1 The C=O stretching vibration absorption peak at 1654 cm-1 in progesterone-CD-MOFs -1It indicates that the carbonyl group of progesterone may form intermolecular hydrogen bonds with CD-MOFs, and progesterone is successfully loaded into CD-MOFs. At the same time, compared with the physical mixture, the characteristic peaks of progesterone raw materials and CD-MOFs in the physical mixture exist at the same time, indicating that simple physical mixing does not cause the two to interact.

[0055] Calculation of inclusion compound content

[0056] By comparing different concentrations of progesterone anhydrous ethanol solutions, different molar ratios of progesterone to CD-MOFs, and different reaction times, it was found that the optimal preparation condition for progesterone-CD-MOFs inclusion complex was a progesterone anhydrous ethanol concentration of 8.0 mg mL -1 The optimal drug loading conditions were a molar ratio of progesterone to CD-MOFs of 2:1 and an inclusion time of 8 h, with a maximum drug loading of 17.85%.

[0057] Example 3 Solubility and dissolution determination of progesterone-CD-MOFs inclusion complex

[0058] Solubility determination: The excess progesterone API and progesterone-CD-MOFs inclusion complex were dissolved and diluted in 5 ml ultrapure water at room temperature, and the dissolved sample solution was placed in a constant temperature oscillating agitator (200 rpm) for 24 hours. After the reaction, the supernatant was aspirated, filtered with a 0.22 μm filter membrane, and the content of progesterone in the filtrate was measured by injection. The solubility determination results are shown in the figure. The water solubility of the progesterone-CD-MOFs inclusion complex is 708.47 mg / L, and the water solubility of the progesterone API is 25.70 mg / L. Compared with the API, the water solubility of progesterone encapsulated in CD-MOFs is increased by 27.56 times, indicating that CD-MOFs can be used as a delivery carrier for poorly soluble drugs and can significantly improve the water solubility of poorly soluble drugs.

[0059] Dissolution determination: Weigh 10 mg of progesterone raw material and progesterone-CD-MOFs inclusion complex (calculated as progesterone), set the in vitro release medium to ultrapure water and PBS, temperature 37 ° C, speed 150 rpm conditions, at 0h, 0.08h, 0.17h, 0.25h, 0.33h, 0.5h, 0.75h, 1h, 2h, 8h and 12h, aspirate 5 ml of release medium, filter with 0.22μm filter membrane and then perform sample determination.

[0060] The dissolution results showed that the progesterone-CD-MOFs inclusion complex could significantly improve the cumulative dissolution and dissolution rate of the progesterone API. Compared with the progesterone API, the progesterone-CD-MOFs inclusion complex dissolved nearly 80% within 0.5 h, and the dissolution rate was also significantly improved.

[0061] Pharmacokinetic Determination of Progesterone-CD-MOFs Inclusion Complex in SD Rats in Example 4

[0062] Ten healthy male SD rats were purchased from Henan Spex Biotechnology Co., Ltd., batch number NO. SCXK(Yu)2020-0005. The body weight was about 200±20 g and they were randomly divided into two groups, the progesterone raw material group and the progesterone-CD-MOFs group, with 5 rats in each group. The progesterone raw material group and the progesterone-CD-MOFs group were respectively given oral gavage at a dose of 18 mg / kg (calculated as progesterone). Blood samples of 0.3 ml were taken from the orbital cavity at 0 h, 0.08 h, 0.16 h, 0.33 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, and 12 h. After protein precipitation and protein recovery of the plasma samples, the content determination was carried out according to the item of "Determination of Progesterone Content in Plasma". The bioavailability of progesterone-CD-MOFs and progesterone raw material was evaluated in vivo.

[0063] The content of progesterone in plasma was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) analysis method. The pharmacokinetic results of SD rats were processed and analyzed by DAS 2.0 software. The results are as Figure 6 shown in Table 1. Compared with the progesterone raw material group, the AUC 0-∞ of the progesterone-CD-MOFs inclusion complex was 2.28 times that of the progesterone raw material. The AUC 0-∞ of progesterone-CD-MOFs was significantly increased, indicating that the oral bioavailability of progesterone could be significantly improved after being loaded inside CD-MOFs.

[0064] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A progesterone cyclodextrin-metal organic framework inclusion complex, the inclusion complex comprising (a) progesterone; (b) cyclodextrin-metal organic framework. The preparation method of the cyclodextrin-metal organic framework is: Anhydrous potassium carbonate and gamma-cyclodextrin are added to ultrapure water, heated and stirred at 50-70°C for 5-30 minutes, the reaction liquid is collected, the reaction liquid is placed in an ice bath at 0-10°C and stirred, acetone is added dropwise, flaky crystals are generated in the reaction liquid, the stirring is stopped after reacting for 10-30 minutes, the precipitate is collected by centrifugation, washed with isopropanol, and the precipitate is vacuum dried to obtain the cyclodextrin-metal organic framework.

2. The inclusion compound according to claim 1, characterized in that The inclusion compound has one or more characteristics selected from the group consisting of: (1) The progesterone in the inclusion compound is a fast-release inclusion compound (2) The cyclodextrin-metal organic framework in the inclusion complex is a crystal, preferably a rectangular plate-like crystal; (3) The particle size of the cyclodextrin-metal organic framework in the inclusion complex is 0.5-5 μm, for example, 0.5, 1, 2, 3, 4, 5 μm; (4) The combination of (a) progesterone and (b) cyclodextrin metal organic framework in the inclusion complex is such that progesterone enters the cavity of the cyclodextrin-metal organic framework and / or progesterone enters the cyclodextrin cavity in the cyclodextrin-metal organic framework; (5) The drug loading amount (wt%) of progesterone in the inclusion compound is 8%-25%, preferably 8%-18%, for example 8%, 10%, 15%, 18%; (6) The solubility of the inclusion compound in water (25°C) is 10-50 times higher than that of the API; (7) The cumulative dissolution rate of the inclusion compound in water (37°C) is increased by 20%-60% compared with the raw drug; (8) The oral bioavailability of the inclusion compound is increased by 1-3 times compared to the progesterone raw material; 3. The method for preparing the inclusion compound according to claim 1, characterized in that: The preparation method comprises the following steps: dissolving progesterone in anhydrous ethanol by ultrasonication to obtain a progesterone solution; Adding cyclodextrin-metal organic framework to the progesterone solution to carry out drug loading reaction, centrifuging to remove the supernatant, washing with anhydrous ethanol, and vacuum drying to obtain the progesterone cyclodextrin-metal organic framework inclusion complex; 4. The method for preparing the inclusion compound according to claim 3, characterized in that: The progesterone anhydrous ethanol concentration is 0.5-10 mg·mL -1 ; 5. The method for preparing the inclusion compound according to claim 3, characterized in that: The molar ratio of the cyclodextrin metal organic framework to progesterone in the inclusion complex is 1:0.5-1:

16.

6. The method for preparing the inclusion compound according to claim 3, characterized in that: The drug loading reaction time is 0.5-12h.

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