A cyclodextrin structure and griseofulvin complex and preparation method and application thereof

Abstract

The present invention relates to a complex of a cyclodextrin structure and griseofulvin, which partially or completely includes the griseofulvin, or is complexed with the griseofulvin; and also relates to a preparation method and application of the complex. The technical means of the present invention improves the water solubility of griseofulvin by designing an inclusion complex formed by griseofulvin and cyclodextrin. The complex structure of the cyclodextrin structure and griseofulvin is disclosed. The product prepared in the atmosphere of supercritical carbon dioxide has a water solubility 477 times higher than that of the original drug, releases 90% of griseofulvin within 10 minutes, and the AUC of the in vivo pharmacokinetic study is increased by 178%, which is the best result in the study of griseofulvin water solubility so far.

Description

Technical Field

[0001] The invention belongs to the field of drug preparation, and in particular relates to a complex of a cyclodextrin structure and griseofulvin, a preparation method and application thereof. Background Art

[0002] Griseofulvin, as a non-polyene antifungal antibiotic, can strongly inhibit fungal cell mitosis and interfere with fungal DNA synthesis, bind to tubulin to prevent fungal cell division, and has been widely used to treat fungal infections of the skin and stratum corneum since 1958. The low water solubility and high permeability of griseofulvin make it a Class II in the biopharmaceutics classification system, and its low dissolution rate in the gastrointestinal tract reduces its bioavailability and clinical efficacy. Therefore, how to improve the solubility and dissolution rate of griseofulvin has become the focus of clinical practice.

[0003] Griseofulvin has been processed into 3-5 μm particles by coprecipitation, freeze drying or solid dispersion, and its water solubility has been improved. When its particle size is reduced, the solubility increases. Therefore, many methods have been developed to transfer griseofulvin powder to nanocrystals in the range of 10-1000 nm. Griseofulvin nanoparticles with a particle size of 145 nm were obtained by grinding in just 2 minutes and showed 97% drug release. Griseofulvin nanoparticles with a particle size of 60 nm were prepared by high-pressure homogenization and spray drying in the presence of sugar and stabilizers. Griseofulvin nanoparticles with a size of 16 nm made from its acetone and aqueous solution increased the water solubility by nearly 24 times and achieved almost 100% drug release in 60 minutes.

[0004] Through supercritical fluid technology, griseofulvin nanoparticles of 200-500nm were obtained, and 80% drug release was shown within 90min. Through freeze-drying process, phospholipid griseofulvin nanoparticles were obtained, and the drug release curve increased by 7 times within 5h; the diffusion method of 85nm griseofulvin nanoparticles from emulsion solvent showed a 6-fold increase in water solubility and achieved 85% drug release within 1min.

[0005] However, even with great efforts in preparing griseofulvin nanoparticles, the water solubility of griseofulvin has not been significantly improved.

[0006] Since 1988, cyclodextrins have been used to form complexes with griseofulvin to increase its water solubility. When α-cyclodextrin, β-cyclodextrin, dimethyl-β-cyclodextrin and γ-cyclodextrin were used to form complexes with griseofulvin by kneading, spraying, physical mixing or precipitation, γ-cyclodextrin was found to have the best effect on improving water solubility; the solubility, dissolution rate and bioavailability of griseofulvin in the complex were affected by temperature, surfactant and the ratio of cyclodextrin to drug. During the formation of the complex, HP-β-cyclodextrin can effectively inhibit particle growth and stabilize the suspension, with 72% of griseofulvin forming a precipitate and the rest forming an inclusion complex. When the griseofulvin solution was treated with β-cyclodextrin in a mixture of acetone and ethanol, griseofulvin nanoparticles of 600-900 nm were obtained, and the dissolution rate was significantly improved. Nevertheless, these results are obviously not optimal.

[0007] β-cyclodextrin grafted polymer poloxamer-hydroxyethylcellulose-α-cyclodextrin gel, polyvinyl alcohol-styrylpyridinium / β-cyclodextrin nanofibers and β-cyclodextrin-based nanosponges were complexed with griseofulvin to improve its dissolution and oral bioavailability. Griseofulvin encapsulated in a phospholipid bilayer showed a two-fold increase in bioavailability in rats compared with griseofulvin suspension alone, however, the use of polymers will lead to scale-up and quality control problems, which will limit their application.

[0008] Griseofulvin loaded on silica particles or oxidized mesoporous silica nanoparticles showed improved drug release, but the accumulation of silica nanoparticles in organs is also a problem that cannot be ignored.

[0009] Based on this, a technical solution is needed to solve the above technical problems. Summary of the invention

[0010] One object of the present invention is to disclose a cyclodextrin structure and griseofulvin complex, which partially or completely includes the griseofulvin or is complexed with the griseofulvin.

[0011] The above-mentioned cyclodextrin structure and griseofulvin can interact in any one of the two ways, or they can coexist.

[0012] Specifically, the molecular structure diagram of the complex of cyclodextrin structure and griseofulvin disclosed in the present invention is as follows: Figure 1 shown.

[0013] Furthermore, the cyclodextrin structure is selected from one or more of 2-hydroxypropyl-β-cyclodextrin, γ-cyclodextrin or HP-γ-cyclodextrin.

[0014] Furthermore, the cyclodextrin structure is a structure composed of 2-1000 cyclodextrin molecules and / or cyclodextrin complexes.

[0015] Another object of the present invention is to disclose a method for preparing the above-mentioned complex of cyclodextrin and griseofulvin, characterized in that the method for preparing the complex of cyclodextrin and griseofulvin comprises the following steps:

[0016] S1, preparing the griseofulvin into solution 1, adding solution 2 containing the cyclodextrin structure and acetonitrile, heating and stirring;

[0017] S2. Then, heat and stir under a carbon dioxide pressure atmosphere, release the pressure, remove the solvent, filter and freeze-dry to obtain a complex of the cyclodextrin structure and griseofulvin.

[0018] There are many methods for preparing the inclusion complex of griseofulvin and cyclodextrin, such as spray drying, grinding, ultrasonic method, microwave reaction method, autoclave reactor method, aqueous solution method, etc. The method assisted by supercritical carbon dioxide is more effective than other methods.

[0019] The inventors of the present invention designed the following orthogonal test: under the inclusion conditions of griseofulvin and HP-γ-cyclodextrin at a molar ratio of 1:1, 1:2 or 1:3, respectively; at 40°C, 50°C or 60°C, respectively, stirring for 10h, 15h or 20h, respectively, and the rotation speed is 800r / min, 1000r / min, or 1200r / min, respectively. Then, the inclusion complexes obtained under each condition were filtered and freeze-dried for HPLC analysis.

[0020] The best inclusion was obtained when griseofulvin and HP-γ-cyclodextrin were in a 1:1 molar ratio at 50°C, 1200 r / min, and 20 h. The solid stirring speed was 81% and the inclusion rate was 2%. The water solubility of griseofulvin in the complex was 2.95 mg / mL, which was 343 times that of griseofulvin (0.0086 mg / mL).

[0021] Furthermore, the method for preparing the complex of the cyclodextrin structure and griseofulvin also includes the following steps:

[0022] S0. Adding a water-soluble substance to the solution 1 or the solution 2.

[0023] Furthermore, in step S1 or step S2, the heating temperature is independently 70-90°C.

[0024] Furthermore, the water-soluble substance is selected from one or more of methyl cellulose, sodium carboxymethyl cellulose, polyvinyl pyrrolidone, polyethylene glycol, sodium lauryl sulfate, hexamethylenedimethylamine bromide, tris(hydroxymethyl)aminomethane, sodium acetate and benzalkonium chloride.

[0025] Cyclodextrins self-associate with each other to form aggregates or micellar structures composed of two to hundreds of cyclodextrin molecules and / or cyclodextrin complexes, and cyclodextrin aggregates can solubilize lipophilic water-insoluble drugs through non-inclusion complexation. However, larger cyclodextrin aggregates limit their water solubility, and water-soluble substances (mainly high molecular surfactants, anionic substances or cationic substances) can stabilize various aggregates and micellar structures as well as drug / cyclodextrin complexes.

[0026] Furthermore, in step S2, the pressure of the carbon dioxide is 8-10 MPa.

[0027] Another object of the present invention is to disclose the application of the complex of the cyclodextrin structure and griseofulvin in medicine.

[0028] The present invention has the following beneficial effects:

[0029] The technical means of the present invention improves the water solubility of griseofulvin by designing an inclusion complex formed between griseofulvin and cyclodextrin. The technical means discloses the complex structure of cyclodextrin and griseofulvin. The product prepared in a supercritical carbon dioxide atmosphere has a water solubility 477 times higher than that of the original drug, releases 90% of griseofulvin within 10 minutes, and the AUC of the in vivo pharmacokinetic study is increased by 178%, which is the best result in the study of griseofulvin water solubility so far. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A schematic diagram showing the molecular structure of the complex of cyclodextrin structure and griseofulvin.

[0031] Figure 2 The phase solubility curves of HP-γ-CD, γ-CD, and HP-β-CD for griseofulvin are shown.

[0032] Figure 3 (a) shows the Fourier transform infrared spectrum of griseofulvin;

[0033] Figure 3 (b) shows the Fourier transform infrared spectrum of HP-γ-cyclodextrin;

[0034] Figure 3 (c) shows the Fourier transform infrared spectrum of Example 1-1;

[0035] Figure 3 (d) shows the Fourier transform infrared spectrum of the physical mixture of HP-γ-cyclodextrin and griseofulvin.

[0036] Figure 4 (a) shows the D2O H NMR spectrum of γ-cyclodextrin;

[0037] Figure 4 (b) shows the H NMR spectrum of D2O of Example 1-1;

[0038] Figure 4 (c) shows the DMSOd6 H NMR spectrum of griseofulvin;

[0039] Figure 4 (d) shows the H-NMR spectrum of DMSOd6 of Example 1-1.

[0040] Figure 5 The X-ray diffraction patterns of HP-γ-cyclodextrin, griseofulvin, and the complex of HP-γ-cyclodextrin and griseofulvin of Example 1-1 are shown.

[0041] Figure 6 (a) shows a scanning electron micrograph of a HP-γ-cyclodextrin sample;

[0042] Figure 6 (b) shows a scanning electron micrograph of a griseofulvin sample;

[0043] Figure 6 (c) shows a scanning electron micrograph of a sample after physical mixing of HP-γ-cyclodextrin and griseofulvin;

[0044] Figure 6 (d) shows a scanning electron micrograph of a sample of the complex of HP-γ-cyclodextrin and griseofulvin.

[0045] Figure 7 The dissolution profiles of griseofulvin, a physical mixture of HP-γ-cyclodextrin and griseofulvin, and a complex of HP-γ-cyclodextrin and griseofulvin are shown.

[0046] Figure 8 A graph of the canine blank plasma griseofulvin standard curve is shown.

[0047] Fig. 9 (a) shows the HPLC profile of griseofulvin;

[0048] Fig. 9 (b) shows the HPLC profile of blank dog plasma;

[0049] Fig. 9 (c) shows the HPLC profile of dog plasma administered griseofulvin;

[0050] Fig. 9 (d) shows the HPLC profile of dog plasma administered with griseofulvin.

[0051] Fig.10 A graph showing the concentration of griseofulvin in the plasma of dogs administered griseofulvin or its inclusion complex is shown. DETAILED DESCRIPTION

[0052] The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.

[0053] Materials and testing equipment used:

[0054] Griseofulvin with a purity of 98% was purchased from Beijing Suobo Technology Co., Ltd.;

[0055] 2-Hydroxypropyl-β-cyclodextrin, γ-cyclodextrin or HP-γ-cyclodextrin was purchased from Shanghai Yien Chemical Technology Co., Ltd.;

[0056] Analytical grade ethanol was purchased from Guangdong Chemical Reagent Engineering Technology Research and Development Center;

[0057] Anhydrous sodium acetate was purchased from Xilong Technology Co., Ltd.;

[0058] Hydroxypropyl methylcellulose, benzalkonium chloride, sodium hydroxymethylcellulose, and hexamethylenediamine bromide were purchased from Shanghai McLean Biochemical Technology Co., Ltd.;

[0059] Sodium alkyl (12 carbon) sulfate was purchased from Tianjin Best Chemical Co., Ltd.;

[0060] Polyvinylpyrrolidone was purchased from Tianjin Tianxin Fine Chemical Development Center;

[0061] Polyethylene glycol 4000 was purchased from Tianjin Yongda Chemical Reagent Co., Ltd.;

[0062] Sodium salicylate was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.;

[0063] The supercritical carbon dioxide reactor BZ-100ML / S0-L was purchased from Shanghai Baikal Lake Intelligent Technology Co., Ltd.

[0064] 1 H NMR spectra were recorded on a Bruker spectrometer (400 MHz) with D2O and DMSOd6 as solvents;

[0065] The UV spectra were recorded using a UV-visible spectrophotometer instrument J51903001 from Shanghai Jinghua Technology Instrument Co., Ltd.;

[0066] HPLC analysis was performed using an LC-15C high performance liquid chromatograph from Shimadzu Enterprise Management (China) Co., Ltd. using an Inertsil ODS-3C18 (250 mm × 4.6 mm) column;

[0067] The dissolution test was conducted on an RC-3 dissolution apparatus from Tianjin Xintianguang Analytical Instrument Technology Co., Ltd.

[0068] The stability test was conducted on the Lhh-sg stability test chamber of Shanghai Yiheng Technology Co., Ltd.;

[0069] X-ray analysis was performed on a D8 Advance (Bruker) X-ray multicrystal diffractometer;

[0070] FTIR spectra were recorded on a Fourier transform infrared spectrometer vertex 70 (Bruker);

[0071] Scanning electron microscopy images were performed on a Merlin (Zeiss) high-resolution field emission scanning electron microscope;

[0072] The puppies used in the experiment were purchased from Guangdong Medical Experimental Animal Center, and blank dog plasma was purchased from Guangzhou Ruite Co., Ltd.;

[0073] Disposable blood collection needles and 5 mL anticoagulant tubes were purchased from Shenzhen Kangzhijian Medical Device Biotechnology Co., Ltd.;

[0074] 2mL centrifuge tubes were taken from BIOFIL clean centrifuge tubes;

[0075] 0.22 μm organic microporous filter was purchased from Tianjin Jinteng Experimental Equipment Co., Ltd.;

[0076] The nitrogen blower was purchased from Hangzhou Miaoou Instrument Co., Ltd.;

[0077] Low-fat dog food was purchased from Shanghai Jibaichuan Industrial Co., Ltd.

[0078] Example 1-1

[0079] The invention discloses a complex of HP-γ-cyclodextrin and griseofulvin, wherein HP-γ-cyclodextrin completely includes griseofulvin.

[0080] The preparation method of the above-mentioned complex of HP-γ-cyclodextrin and griseofulvin is as follows:

[0081] S1. In a closed system, griseofulvin (105 mg) was dissolved in acetonitrile (2 mL) to prepare solution 1 (concentration of 10 wt%), and pure water (8 mL) solution 2 containing HP-γ-cyclodextrin (1060 mg, 0.6 mmol) and acetonitrile (8 mL) were added, and the mixture was heated to 75 ° C and stirred for 2 h;

[0082] S2. Then, liquid carbon dioxide is pumped into the above-mentioned closed system, the internal pressure is maintained at 8 MPa, heated to 75°C and stirred for 12 hours, and then the pressure is slowly released, the solvent is distilled off under reduced pressure, filtered and freeze-dried to obtain the HP-γ-cyclodextrin and griseofulvin complex, and stored in a refrigerator under a nitrogen atmosphere.

[0083] Example 1-2

[0084] The raw materials, raw material amounts and operation methods used in this example are the same as those in Example 1-1, except that the amount of HP-γ-cyclodextrin and other substances is replaced by γ-cyclodextrin.

[0085] Examples 1-3

[0086] The raw materials, raw material amounts and operation methods used in this example are the same as those in Example 1-1, except that the amount of substances such as HP-γ-cyclodextrin is replaced by 2-hydroxypropyl-β-cyclodextrin.

[0087] Example 2

[0088] The invention discloses a complex of HP-γ-cyclodextrin and griseofulvin, wherein HP-γ-cyclodextrin completely includes griseofulvin.

[0089] The preparation method of the above-mentioned complex of HP-γ-cyclodextrin and griseofulvin is as follows:

[0090] S0. A composition of a compounded water-soluble substance, wherein the specific component is a mixture of methyl cellulose, sodium carboxymethyl cellulose and polyethylene glycol, and the weight ratio of the three is 1:1:1.

[0091] S1. In a closed system, the griseofulvin (105 mg) was dissolved in acetonitrile (2 mL) to prepare solution 1 (concentration of 10 wt%), and a pure water (8 mL) solution 2 containing the HP-γ-cyclodextrin (1060 mg, 0.6 mmol) and acetonitrile (8 mL) were added, and the above-mentioned water-soluble substance composition was added, and the mixture was heated to 75 ° C and stirred for 2 h;

[0092] S2. Then, liquid carbon dioxide is pumped into the above-mentioned closed system, the internal pressure is maintained at 8 MPa, heated to 75°C and stirred for 12 hours, and then the pressure is slowly released, the solvent is distilled off under reduced pressure, filtered and freeze-dried to obtain the HP-γ-cyclodextrin and griseofulvin complex, and stored in a refrigerator under a nitrogen atmosphere.

[0093] Comparative Example 1

[0094] In Comparative Example 1, HP-γ-cyclodextrin and griseofulvin were physically blended in a substance amount ratio of (1:10) to obtain a physical blend.

[0095] Test Case

[0096] A series of characterization tests were performed on the complexes of the cyclodextrin structures and griseofulvin of the above Examples 1-1 to 1-3, and one or more of the above Comparative Examples. Among them:

[0097] Fourier transform infrared spectroscopy: The FTIR spectra of the physical mixture of griseofulvin, HP-γ-cyclodextrin and KBr pellets of the inclusion complex were analyzed by Fourier transform infrared spectroscopy, and the necessary data were obtained. The data obtained were in the range of 400-3800cm -1 Collect between.

[0098] Proton NMR studies: The solutions of the obtained HP-γ-cyclodextrin and griseofulvin inclusion complex in D2O and the solutions of griseofulvin and its inclusion complex in DMSO-d6 were respectively carried out at room temperature. 1 D NMR studies at a frequency of 400 MHz.

[0099] X-ray diffraction analysis: Griseofulvin, HP-γ-cyclodextrin and its inclusion complex were analyzed by X-ray diffraction using Cu-Kα emission, lynxeye array detector, at a voltage of 40 kV and a current of 40 mA (scanning angle range 3-60°, scanning speed 0.01 / s).

[0100] The surface morphology of griseofulvin, HP-γ-cyclodextrin, griseofulvin, comparative example 1, and example 1-1 was studied using a scanning electron microscope at an accelerating voltage of 5 kV under high vacuum. Each solid sample was placed on a magnetic block, and after being treated with gold spraying, the sample was loaded onto a sample rod for scanning. The image was magnified 500 times, and the image type was a secondary electron image.

[0101] Determination of griseofulvin content: The inclusion complex (100 mg) was dissolved in deionized water (1 mL), and the solution was diluted to the range of 8-500 μg / mL. HPLC analysis was performed on it, and the griseofulvin content in the complex was obtained by regression equation.

[0102] Determination of water solubility of griseofulvin in the complex: The inclusion complex was added to water (1 mL) to obtain a saturated aqueous solution thereof. After filtration, the solution was analyzed by HPLC to obtain the water solubility results of griseofulvin in the complex.

[0103] Determination of inclusion rate and inclusion yield: The inclusion rate and yield of the complex of HP-γ-cyclodextrin and griseofulvin can be calculated according to the following formula:

[0104] Yield (%) = [complex of HP-γ-cyclodextrin and griseofulvin (mg) / (griseofulvin (mg)+HP-γ-cyclodextrin (mg))]×100%.

[0105] Inclusion rate (%) = [Griseofulvin in the complex of HP-γ-cyclodextrin and griseofulvin (mg) / griseofulvin (mg)] × 100%.

[0106] Dissolution determination: The dissolution of griseofulvin in the complex was determined by the pulp method. Griseofulvin (100 mg) and the complex of HP-γ-cyclodextrin and griseofulvin (containing 100 mg griseofulvin) were tested respectively. Degassed ultrapure water (900 mL) was used as the medium, and the experimental conditions were 37°C and 100 r / min. Samples (3 mL each) were collected at 2, 5, 10, 15, 20, 30, 45 and 60 minutes respectively. 3 mL of ultrapure water was added after each collection.

[0107] In vivo pharmacokinetic study: 12 healthy dogs weighing about 5 kg were randomly divided into a griseofulvin group and two groups of Example 1-1, each group containing three male and three female dogs. After fasting for 12 hours, water was withheld for 1 hour before administration. A single oral dose of griseofulvin or Example 1-1 was administered to each dog at a dose of 15 mg / kg in 10 mL of water. After administration, the dogs fasted for 4 hours and deprived of water for 1 hour, and then blood samples (2 mL) were collected using a disposable EDTA anticoagulant syringe at 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 8, 12, 24, 36, 48 and 72 hours, respectively. After centrifugation at 4000 r / min for 10 minutes, the plasma samples were stored in a -80°C refrigerator.

[0108] The plasma sample (0.5 mL) dissolved in 1 mL of acetonitrile was vortexed for 3 min in a 2 mL centrifuge tube, centrifuged at 13000 r / min for 10 min, and the supernatant was collected in a glass tube. The precipitate was extracted twice with 0.5 mL of acetonitrile, the combined supernatant was centrifuged and dried under nitrogen at 37°C, 0.5 mL of mobile phase was added, vortexed for 4 min; and filtered through a 0.22 μm microporous organic filter for HPLC analysis (mobile phase: 60:40 acetonitrile / water and 0.1% acetic acid; flow rate: 1.0 mL / min.; chromatographic column: Inertsil ODS-3C18, 250 mm × 4.6 mm; detector: UV at 290 nm; temperature: 30°C). The recovery rate of griseofulvin in plasma was measured to be 92.32±0.43%~108.75±6.12%; the intra-batch coefficient of variation was 1.11~3.73%; and the inter-batch coefficient of variation was 0.07~5.63%.

[0109] Statistical analysis: GraphPad Prism 5.0 software (GraphPad Software, USA) was used for statistical analysis, including data processing and graphical representation. In each case, a two-tailed t-test was used to assess the significance of the differences in PK parameter values ​​between dogs taking pure griseofulvin and dogs taking the inclusion complex. An Ap value of 0.05 was considered significant for statistical analysis.

[0110] Test results:

[0111] The cyclodextrin raw materials of Example 1-1, Example 1-2 and Example 1-3 ( Figure 2 The solubility of griseofulvin at room temperature was studied by phase dissolution method. We found that as the concentration of cyclodextrin derivatives increased, the HP-γ-cyclodextrin of Example 1-1 had the greatest effect on the solubility of griseofulvin and was selected to form an inclusion complex with griseofulvin. The phase solubility curve is shown in Figure 2 The phase solubility curve is shown as A L , typed according to Higuchi description.

[0112] After post-treatment and HPLC analysis, it was found that hydroxypropyl methylcellulose increased the water solubility of griseofulvin to 4.105 mg / mL by complexing with HP-γ-cyclodextrin, which is 477 times higher than that of griseofulvin. From the perspective of water solubility, this result is the best so far. This sample was used for its structural confirmation and in vitro and in vivo PK studies.

[0113] Fourier transform infrared spectroscopy was used to confirm the formation of griseofulvin inclusion complexes. Figure 3 As shown in (a), the FTIR spectrum of griseofulvin is at 1444.02 cm -1 There is a significant peak of CN stretching at 1682.16 cm -1 There is a significant peak at 1085.35cm -1 There is a significant peak at 3301.77cm -1 The peak appears at 1630.68 cm -1 There is a peak at 2799.45 cm -1 A peak appears at . In the inclusion complex, the non-covalent interactions such as hydrophobic interaction, van der Waals interaction, and hydrogen bonding between griseofulvin and HP-γ-cyclodextrin result in lower energy of the included part of griseofulvin, which reduces the peak intensity of griseofulvin. The corresponding frequency. Accordingly, if the infrared absorption peak intensity of griseofulvin decreases, shifts, or disappears, it proves that griseofulvin and HP-γ-cyclodextrin have an inclusion complex.

[0114] The peak shape of HP-γ-cyclodextrin is not changed compared with Example 1-1, which proves that the structure of HP-γ-cyclodextrin in the inclusion complex has not changed substantially ( Figure 3 b and 3c). However, compared with griseofulvin alone, the peak shape, peak position and peak intensity corresponding to griseofulvin in Example 1-1 changed significantly, indicating that griseofulvin formed a complex with HP-γ-cyclodextrin. The infrared spectrum of the physical mixture (Comparative Example 1) looks like the superposition of the spectra of griseofulvin and HP-γ-cyclodextrin (3d).

[0115] The proton NMR spectra of griseofulvin in DMSOd6, HP-γ-cyclodextrin in D2O, and inclusion complex in D2O and DMSOd6 are recorded as follows: Figure 4 As shown in (a)-(d). The proton NMR spectrum of the inclusion complex in D2O shows that the signals of griseofulvin are at 6.50 (1H, s), 5.73 (1H, s), 2.90-2.78 (m, 2H), 2.63 (1H, dd), 1.25 (as shown in Figures (a)-(d), OCH3), and the three OCH3 signals overlap with the signal from HP-γ-cyclodextrin in the range of 4.00-3.50 ppm. The proton NMR spectrum of griseofulvin in DMSOd6 showed signals of 6.51 (1H, s), 5.61 (1H, s), 4.05 (3H, s, OCH3), 4.03 (3H, s, OCH3), 3.63 (3H, s, OCH3), 2.86 (1H, m), 2.79 (1H, dd, J1 = 12.0 Hz, J2 = 16.0 Hz), 2.36 ( 1H, dd, J1 = 4.0 Hz, J2 = 16.0 Hz), 0.81 (3H, d, J = 8.0 Hz, OCH3); the proton NMR data of griseofulvin in the DMSOd6 complex showed signals at 6.45 (1H, s), 4.02 (3H, s, OCH3), 3.92 (3H, s, OCH3), 2.76 (1H, m), 2.65 (1H, dd), 2 .33 (1H, dd), 0.785 (3H, d), the chemical shifts of the two methoxy groups on the aromatic ring changed from 4.05ppm and 4.03ppm to 4.02ppm and 3.92ppm, and the shift of the chemical aromatic protons changed from 6.51ppm to 6.45ppm, indicating that the aromatic part of griseofulvin interacted with HP-γ-cyclodextrin, and the chemical shifts of the methyl, CH and CH2 groups of the cyclohexanone part of griseofulvin changed from 0.81ppm, 2.36ppm, 2.79ppm and 2.86ppm to 0.785ppm, 2.33ppm, 2.65ppm and 2.76ppm, indicating that the cyclohexanone part of griseofulvin interacted with HP-γ-cyclodextrin. Based on these, we assume that in the complex, two HP-γ-cyclodextrin molecules are complexed with one griseofulvin molecule, as shown in the attached figure Figure 1 shown.

[0116] The X-ray diffraction patterns of HP-γ-cyclodextrin, griseofulvin, and the complex of HP-γ-cyclodextrin and griseofulvin of Example 1-1 are shown in Figure 5As can be seen from the figure, griseofulvin is a crystalline compound with sharp peaks, and HP-γ-cyclodextrin is a crystalline compound with specific multiple sharp crystalline peaks. However, in the X-ray diffraction pattern of the product of Example 1-1, the characteristic peaks of HP-γ-cyclodextrin and griseofulvin completely disappeared, indicating that the product does not contain pure griseofulvin and pure HP-γ-cyclodextrin.

[0117] The surface morphology changes of HP-γ-cyclodextrin, griseofulvin, the physical mixture of HP-γ-cyclodextrin and griseofulvin of Comparative Example 1-1, and the composite of HP-γ-cyclodextrin and griseofulvin of Example 1-1 were studied by scanning electron microscopy. The micrographs are shown in Figure 6 (a)-(d). As can be seen from the figure, when the sample is magnified to 20μm, the HP-γ-cyclodextrin sample is in an uneven block shape (such as Figure 6 (a)), griseofulvin samples are amorphous (as shown in Figure 6 (b)), in Comparative Example 1, griseofulvin is distributed around HP-γ-cyclodextrin (as shown in Figure 6 (c)), while Example 1-1 presents irregular fragment shapes (such as Figure 6 The differences between the four micrographs directly confirmed the formation of the HP-γ-cyclodextrin-griseofulvin complex.

[0118] The dissolution rates of griseofulvin, Comparative Example 1, and the complex of HP-γ-cyclodextrin and griseofulvin of Example 1-1 were tested. Figure 7 As shown. After 10 minutes, the cumulative dissolution rate of griseofulvin in water is only close to 2%, the cumulative dissolution rate of the physical mixture of griseofulvin and HP-γ-cyclodextrin is about 18%, and the cumulative dissolution rate of the complex of HP-γ-cyclodextrin and griseofulvin in Example 1 is more than 90%, which is much better than using griseofulvin alone, and at the same time avoids the sudden release of griseofulvin in the complex of HP-γ-cyclodextrin and griseofulvin.

[0119] In the complex of HP-γ-cyclodextrin and griseofulvin, griseofulvin is encapsulated in HP-γ-cyclodextrin cylinders, inhibiting the direct contact between griseofulvin and taste sensors, thereby eliminating the unpleasant taste of griseofulvin.

[0120] The pharmacokinetic properties of griseofulvin change after complexation with HP-γ-cyclodextrin. In order to understand the effect of complexation of griseofulvin with HP-γ-cyclodextrin, the pharmacokinetic study of the complex in dogs was conducted. Therefore, griseofulvin and the complex of HP-γ-cyclodextrin and griseofulvin were orally administered to two groups of dogs (6 dogs in each group) at a single dose of 15 mg / kg, and blood samples were collected at 0, 0.25, 0.5, 0.75, 1, 2, 3, 4, 6, 8, 12, 24, 36, 48 and 72 h after administration.

[0121] A series of solutions were prepared by adding griseofulvin in acetonitrile (0.1 mg / mL) to blank dog plasma and analyzed by HPLC. Figure 8 The standard working curve is shown. When the concentration of griseofulvin in plasma is in the range of 0.05-20 μg / mL, the HPLC peak area is linearly related to the concentration of griseofulvin in the solution, and the standard working curve equation is: Y = 38.716X + 0.961 (R2 = 0.9994, Y: peak area, X: concentration). S / N ≥ 3: LLOD is 0.05 μg / mL, S / N ≥ 10, and LLOQ is 0.01 μg / mL.

[0122] HPLC analysis of griseofulvin, blank dog plasma, dog plasma given griseofulvin, and dog plasma given griseofulvin Fig. 9 (a)-(d) are shown. The HPLC results show that blank dog plasma has no interference with the detection of griseofulvin, and griseofulvin in dog plasma can be determined by HPLC.

[0123] The recovery rate, intra-batch coefficient of variation and inter-batch coefficient of variation of griseofulvin standard solution in blank plasma at 0.1 μg / mL, 1 μg / mL and 10 μg / mL were tested. The recovery rate of plasma griseofulvin content was in the range of 92.32±0.43% to 108.75±6.12%, the intra-batch coefficient of variation was in the range of 0.07% to 5.63%, and the inter-batch coefficient of variation was in the range of 1.11% to 3.73%.

[0124] The collected plasma samples were analyzed by HPLC, and the results were as follows: Fig.10 The experimental data of dog plasma administered with griseofulvin or its inclusion complex were analyzed using DAS 2.0 pharmacokinetic software. The relationship between the concentration and time of griseofulvin in dog plasma conformed to the first-order absorption two-compartment model, and the pharmacokinetic parameters (mean ± standard deviation) are summarized in Table 1.

[0125] Table 1 Pharmacokinetic parameters of griseofulvin and its inclusion complex

[0126]

[0127] After oral administration, the Cmax and Tmax of griseofulvin in dog plasma were 0.52μg / mL at 1.75h in the griseofulvin group, 0.72μg / mL at 2.0h in the griseofulvin inclusion complex group, AUC 0-120 was 1.55μg·h / mL in the griseofulvin group, and 2.749μg·h / mL in the HP-γ-cyclodextrin and griseofulvin complex group. Through complexation, the exposure level of griseofulvin increased by 177%, the clearance rate of griseofulvin changed from 51.78 to 24.16, and the half-life changed from 0.81h to 1.56h, indicating that less drug is needed to achieve the same therapeutic effect using the griseofulvin complex.

[0128] The complex of HP-γ-cyclodextrin and griseofulvin in Example 1-1 can increase the water solubility (477 times), which is the most ideal result in the study of griseofulvin water solubility so far. This is also the reason why 90% of the drug was released within 10 minutes, and 177% AUC 0-12 Increased, with shorter clearance and longer half-life.

[0129] In summary, a complex of HP-γ-cyclodextrin and griseofulvin was prepared by supercritical carbon dioxide-assisted complexation and the addition of a large amount of water-soluble polymers, and the drug-drug ratio was confirmed by FTIR, NMR, X-ray and ESM. The cyclodextrin in the complex was determined to be 1:2 based on NMR data. By complexing with HP-γ-cyclodextrin, the water solubility of griseofulvin was increased 477 times compared to griseofulvin alone, which is the best result of griseofulvin water solubility research to date. The complex showed that more than 90% of griseofulvin was released in vitro within 20 minutes. Its in vivo PK study showed that the pharmacokinetic properties were significantly improved. Compared with griseofulvin, C max AUC increased from 0.52 to 0.72 0-12 The expression of griseofulvin increased from 1.55 to 2.749, the clearance increased from 51.78 to 24.16, and the half-life increased from 0.81 to 1.56, indicating that the griseofulvin complex is more effective than griseofulvin alone.

[0130] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0131] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A complex of a cyclodextrin structure and griseofulvin, characterized in that: The cyclodextrin includes the griseofulvin, or is complexed with the griseofulvin; The cyclodextrin structure is selected from HP-γ-cyclodextrin; The preparation method of the complex of cyclodextrin structure and griseofulvin comprises the following steps: S1, preparing the griseofulvin into solution 1, adding solution 2 containing the cyclodextrin structure and acetonitrile, heating and stirring; S2, then heating and stirring under a carbon dioxide pressure atmosphere, then releasing the pressure, removing the solvent, filtering and freeze-drying to obtain the cyclodextrin and griseofulvin complex, The mass ratio of griseofulvin to HP-γ-cyclodextrin is 1:10; The heating temperature is independently 70-90°C; The pressure of the carbon dioxide is 8-10 MPa.

2. The complex of cyclodextrin structure and griseofulvin according to claim 1, characterized in that: The cyclodextrin structure is composed of 2 to 1000 cyclodextrin molecules.

3. The complex of cyclodextrin structure and griseofulvin according to claim 1, characterized in that: The preparation method of the complex of cyclodextrin structure and griseofulvin also includes the following steps: A water-soluble substance is added to the solution 1 or the solution 2.

4. The complex of cyclodextrin structure and griseofulvin according to claim 3, characterized in that: The water-soluble substance is selected from one or more of methylcellulose, sodium carboxymethylcellulose, polyvinyl pyrrolidone, polyethylene glycol, sodium lauryl sulfate, hexamethylenedimethylamine bromide, tris(hydroxymethyl)aminomethane, sodium acetate and benzalkonium chloride.

5. Use of the complex of the cyclodextrin structure and griseofulvin according to claim 1 in the preparation of antifungal drugs.

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