Mebendazole HP-beta-cyclodextrin inclusion compound and preparation method thereof

Abstract

The invention provides a mebendazole HP-beta-cyclodextrin clathrate compound. The mebendazole HP-beta-cyclodextrin clathrate compound is prepared from mebendazole and HP-beta-cyclodextrin, wherein the mebendazole HP-beta-cyclodextrin clathrate compound is prepared from the mebendazole and the HP-beta-cyclodextrin; wherein the mebendazole is positioned in a cavity of the HP-beta-cyclodextrin. According to the mebendazole HP-beta-cyclodextrin inclusion compound provided by the invention, the mebendazole and the HP-beta-cyclodextrin are prepared into the inclusion compound, so that the water solubility of the mebendazole is improved by 18333 times and reaches 6.05 mg / mL, which is the best result obtained so far. In addition, the in-vitro PK research shows that the inclusion compound releases 80% of mebendazole within 5 min, while the pure drug only releases 20% under the same condition; in the in-vivo PK research, the bioavailability is almost doubled, which indicates that the prepared toluene imidazole HP-beta-cyclodextrin inclusion compound can be clinically applied as a potential anti-cancer drug.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceuticals, and mainly relates to a mebendazole HP-β-cyclodextrin inclusion complex and a preparation method thereof. Background Art

[0002] Mebendazole is a synthetic benzimidazole carbamate that was approved by the FDA in 1974 for the treatment of intestinal parasite infections. It can inhibit the formation of the parasite cell microtubule system, arrest the cell cycle at the G2 / M phase, affect cell mitosis, inhibit the uptake of glucose by parasites, cause glycogen depletion in parasites, and reduce the production of adenosine triphosphate, interfering with energy metabolism, making the parasites unable to survive, and is used to treat human geohelminth infections caused by at least one nematode (roundworm, hookworm or whipworm) infection.

[0003] Recent studies have shown that mebendazole has preclinical efficacy in various cancers, including glioblastoma, medulloblastoma, colon cancer, breast cancer, pancreatic cancer, and thyroid cancer. It is an excellent candidate for the treatment of brain tumors as a single agent or in combination with other treatments. However, due to its poor water solubility (0.33±0.02 μg / mL), this limits its efficacy and greatly restricts its clinical application. Cancer drugs administered by IP or IV should have a water solubility in the range of 5 mg / mL to 10 mg / mL to achieve the required therapeutic dose. Therefore, there is an urgent need to find ways to improve the water solubility of mebendazole.

[0004] Many methods, such as particle size reduction, solid dispersions, nanosuspensions, and salt formation, have been used to improve the water solubility of mebendazole. The inclusion complexes formed by mebendazole and cyclodextrins (such as α, β, γ, β-sulfonated, and HP-β-cyclodextrin) showed a 16- to 31-fold increase in water solubility. When mebendazole was heated with HP-β-cyclodextrin and citric acid or tartaric acid at 95°C for 60 min, the water solubility of mebendazole increased to 0.68 mg / mL. Formulations of β-cyclodextrin, chitosan-based microcrystals, polyvinyl alcohol, and polysorbate 80-based nanoparticles were able to increase the solubility of mebendazole by 4-fold. Inclusion complexes with β-cyclodextrin and permethylated-β-cyclodextrin were able to increase the solubility by 35-fold and 4700-fold. A solid dispersion could increase the solubility of mebendazole by 15982-fold, but the solubility was measured in acidic aqueous solution (0.1 M HCl), so the result was not accurate. Despite great efforts, the research aimed at improving the water solubility of mebendazole has not achieved a significant solubility increase.

[0005] In addition, although various methods (such as Fourier transform infrared spectroscopy, differential scanning calorimetry, thermogravimetric analysis, X-ray diffraction, scanning electron microscopy, and mass spectrometry, etc.) can be used to verify the formation of the inclusion complex of mebendazole and cyclodextrin derivatives, these methods all have the problems of insufficient accuracy and weak correlation with the water solubility of mebendazole. Generally speaking, the core purpose of preparing the mebendazole inclusion complex is to improve its water solubility. As the most powerful characterization tool, nuclear magnetic resonance (NMR) spectroscopy can not only confirm the interaction between mebendazole and cyclodextrin derivatives in the inclusion complex, but also indirectly reflect the water solubility of mebendazole in the inclusion complex when deuterated water (D2O) is used as the solvent.

[0006] Research in 2007 showed that mebendazole can form inclusion complexes with α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, and permethylated β-cyclodextrin (PM-β-cyclodextrin) in an acetone / acetonitrile-water system (with or without formic acid). Among them, permethylated β-cyclodextrin can increase the water solubility of mebendazole by 4700 times. Research in 2008 further confirmed this result and defined PM-β-cyclodextrin as partially methylated β-cyclodextrin. Although the mass spectrum of the inclusion complex was recorded, no mass spectral peaks of the mebendazole and permethylated β-cyclodextrin inclusion complex were detected, and its NMR spectrum was not measured in D2O.

[0007] Research in 1999 reported the nuclear Overhauser effect spectroscopy (Noesy) signals of mebendazole and γ-cyclodextrin or α-cyclodextrin, confirming their interaction. However, this NMR spectrum was measured in deuterated dimethyl sulfoxide (DMSO-d6), indicating that the inclusion complex was formed in the dimethyl sulfoxide system, which has limited relevance to the actual water solubility of the drug. The proton nuclear magnetic resonance spectrum and Roesy spectrum obtained using D2O as the solvent can not only confirm the formation of the inclusion complex, but also indirectly reflect its water solubility characteristics.

[0008] Some research prepared the inclusion complex of mebendazole and citric acid-β-cyclodextrin in a formic acid-water system, but only increased the water solubility of mebendazole by 4 times. As mentioned above, the reported range of solubility improvement of cyclodextrin inclusion complexes in the literature is generally 4 - 2020 times, and none of them have verified the inclusion complex and its solubility through D2O NMR spectroscopy.

[0009] Therefore, there is an urgent need to develop a new technical solution to solve the problems existing in the prior art. Summary of the Invention

[0010] Based on the above background, the present invention provides a mebendazole HP-β-cyclodextrin inclusion complex. By preparing a mebendazole inclusion complex with HP-β-cyclodextrin, the water solubility of mebendazole is increased by 18,333 times, reaching 6.05 mg / mL, which is the best result obtained so far. In addition, in vitro PK studies have shown that 80% of mebendazole is released from the inclusion complex within 5 minutes, while only 20% of the pure drug is released under the same conditions; in in vivo PK studies, the bioavailability is almost doubled, indicating that the prepared mebendazole HP-β-cyclodextrin inclusion complex can be used as a potential anti-cancer drug in clinical applications.

[0011] An object of the present invention is to provide a mebendazole HP-β-cyclodextrin inclusion complex, which is composed of mebendazole and HP-β-cyclodextrin;

[0012] Among them,

[0013] the mebendazole is located in the cavity of HP-β-cyclodextrin.

[0014] Furthermore, the inclusion ratio of the mebendazole HP-β-cyclodextrin inclusion complex is 20-40%.

[0015] Another object of the present invention is to provide a preparation method of the above-mentioned mebendazole HP-β-cyclodextrin inclusion complex, which includes the following steps:

[0016] S1. Add mebendazole to a solvent, stir and dissolve it to obtain a mebendazole solution;

[0017] S2. Add HP-β-cyclodextrin to the mebendazole solution, heat and stir for reaction, then refrigerate at low temperature, purify and dry to obtain the mebendazole HP-β-cyclodextrin inclusion complex.

[0018] Furthermore, in step S1, the solvent is formic acid.

[0019] Furthermore, the mass ratio of mebendazole to HP-β-cyclodextrin is 1:4-6.

[0020] Furthermore, in step S2, the heating temperature is 40-60°C.

[0021] Furthermore, in step S2, the stirring speed is 400-600 r / min.

[0022] Furthermore, in step S2, the reaction time is 2-5 h.

[0023] The present invention has the following beneficial effects:

[0024] The present invention provides a mebendazole HP-β-cyclodextrin inclusion complex. By preparing a mebendazole HP-β-cyclodextrin inclusion complex, the water solubility of mebendazole is increased by 18,333 times, reaching 6.05 mg / mL, which is the best result obtained so far. In addition, in vitro PK studies have shown that 80% of mebendazole is released from the inclusion complex within 5 minutes, while only 20% of the pure drug is released under the same conditions; in in vivo PK studies, the bioavailability is almost doubled, indicating that the prepared mebendazole HP-β-cyclodextrin inclusion complex can be used as a potential anti-cancer drug in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shows the structures of mebendazole and the mebendazole HP-β-cyclodextrin inclusion complex.

[0026] Figure 2 Shows the standard curve and regression equation of mebendazole.

[0027] Figure 3 Shows the solubility of mebendazole in aqueous solutions of different concentrations of β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, HP-β-cyclodextrin, and HP-γ-cyclodextrin.

[0028] Figure 4 Shows the scanning electron microscope photos of mebendazole, the physical mixture of mebendazole HP-β-cyclodextrin, and the mebendazole HP-β-cyclodextrin inclusion complex;

[0029] Wherein,

[0030] Figure 4 (a) Shows the scanning electron microscope photo of mebendazole;

[0031] Figure 4 (b) Shows the scanning electron microscope photo of the physical mixture of mebendazole HP-β-cyclodextrin;

[0032] Figure 4 (c) Shows the scanning electron microscope photo of the mebendazole HP-β-cyclodextrin inclusion complex.

[0033] Figure 5 Shows the proton nuclear magnetic resonance spectrum and Roesy spectrum of the mebendazole HP-β-cyclodextrin inclusion complex in D2O;

[0034] Wherein,

[0035] Figure 5 (a) Shows the proton nuclear magnetic resonance spectrum of the mebendazole HP-β-cyclodextrin inclusion complex in D2O;

[0036] Figure 5 (b) Shows the Roesy spectrum of the mebendazole HP-β-cyclodextrin inclusion complex in D2O.

[0037] Figure 6 The dissolution rate curves of mebendazole, the physical mixture of mebendazole and HP-β-cyclodextrin, and the mebendazole HP-β-cyclodextrin inclusion complex are shown.

[0038] Figure 7 The standard curve and regression equation of mebendazole in dog blank plasma are shown.

[0039] Figure 8 The mebendazole concentration curves in the plasma of dogs administered with mebendazole or the mebendazole HP-β-cyclodextrin inclusion complex are shown. Detailed implementation manners

[0040] In order to more clearly illustrate the technical solutions of the present invention, the following examples are listed. The raw materials, reactions, and post-treatment means that appear in the examples are all common raw materials on the market and technical means well-known to those skilled in the art, unless otherwise specified.

[0041] The terms "preferred", "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain cases. However, in the same or other cases, other embodiments may also be preferred. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not available, nor is it intended to exclude other embodiments from the scope of the present invention.

[0042] It should be understood that, except in any operating example or otherwise indicated, all numbers representing the amounts of ingredients used in the specification and claims, should be understood to be modified in all cases by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that vary depending upon the desired properties sought to be obtained by the present invention.

[0043] In the examples, comparative examples, and test examples, the following substances are used:

[0044] Methyl-β-cyclodextrin, hydroxypropyl-β-cyclodextrin (HP-β-CD), hydroxypropyl-γ-cyclodextrin (HP-γ-CD), β-cyclodextrin, and γ-cyclodextrin were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China).

[0045] Mebendazole (purity ≥ 99%) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

[0046] Formic acid, acetonitrile, and methanol were purchased from Xilong Scientific Co., Ltd. (Guangzhou, China).

[0047] The experimental dogs were purchased from the Guangdong Provincial Medical Experimental Animal Center.

[0048] In the embodiments of the present invention, unless otherwise specified, the water is ultrapure water.

[0049] In the test examples of the present invention, the statistical analysis method is as follows: all experiments are repeated three times, and the data is subjected to one-way ANOVA and significance difference test using Prism 9 software (P≤0.05 indicates that the difference is statistically significant).

[0050] Example

[0051] A mebendazole HP-β-cyclodextrin inclusion complex, which is composed of mebendazole and HP-β-cyclodextrin;

[0052] Among them,

[0053] The mebendazole is located in the cavity of HP-β-cyclodextrin.

[0054] The preparation method of the above-mentioned mebendazole HP-β-cyclodextrin inclusion complex includes the following steps:

[0055] S1. Add 70 mg of mebendazole to 8 mL of formic acid, stir to dissolve to obtain a mebendazole solution;

[0056] S2. Slowly add 365.4 mg of HP-β-cyclodextrin to the mebendazole solution, stir at 50 °C and 500 r / min for 3 h, refrigerate at 4 °C for 8 h, evaporate to remove formic acid (until there is no sour smell), add 50 mL of water to dissolve, filter through a 0.22 μm filter membrane, pre-freeze at -20 °C overnight, and then lyophilize under reduced pressure to obtain a solid powder of mebendazole HP-β-cyclodextrin inclusion complex.

[0057] Figure 1 The structures of mebendazole and mebendazole HP-β-cyclodextrin inclusion complex are shown.

[0058] Comparative example

[0059] A mebendazole HP-β-cyclodextrin physical mixture, and its preparation method includes the following steps:

[0060] Mix 70 mg of ground mebendazole with 365.4 mg of HP-β-cyclodextrin evenly, and pass through an 80-mesh sieve to obtain a mebendazole HP-β-cyclodextrin physical mixture.

[0061] Test example 1

[0062] 1. Ultraviolet spectral analysis of mebendazole standard solution

[0063] Take mebendazole (10 mg) and place it in a 10 mL volumetric flask. Add a small amount of formic acid (1 mL), vortex to mix evenly, and then make up the volume to 10 mL with water to obtain a mebendazole standard solution with a concentration of 1 mg / mL. After filtration through a membrane filter, transfer it to a cuvette and scan it with a UV-visible spectrophotometer from Shanghai Jingke at room temperature in the wavelength range of 200 - 400 nm.

[0064] The above test results show that the maximum absorption wavelength of mebendazole is at 234 nm, which can be used for HPLC analysis.

[0065] 2. Establishment of HPLC standard curve and regression equation

[0066] Dissolve mebendazole (1 mg) in formic acid (1 mL), and dilute it with water to obtain a series of solutions with concentrations of 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mg / mL for HPLC analysis. Use a Shimadzu LC-15C high-performance liquid chromatography system equipped with a UV detector (wavelength 234 nm), a Shimpack VP ODS C18 chromatographic column (250×4.6 mm, 30 °C), with the mobile phase being acetonitrile - water (50:50, v / v) and the flow rate being 1.0 mL / min. Draw a standard curve based on the HPLC peak area and the mebendazole concentration and establish a linear regression equation.

[0067] The results are as Figure 2 shown.

[0068] Figure 2 The standard curve and regression equation of mebendazole are shown.

[0069] The above test results show that in the range of 0.01 - 0.1 mg / mL, the peak area and the concentration show a linear relationship, and the linear regression equation is Y = 13547X - 24.476 (determination coefficient R 2 : 0.9981, Y: absorbance peak area, X: concentration).

[0070] 3. Phase solubility study

[0071] Perform phase solubility analysis to select the cyclodextrin derivative that complexes with mebendazole.

[0072] Dissolve β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, HP-β-cyclodextrin, and HP-γ-cyclodextrin in ultrapure water (10 mL) respectively to prepare solutions with concentrations of 5, 10, 15, 20, 25, and 30 mmol / L. Add an excess of mebendazole and shake at 200 r / min in the dark at 25 °C for 48 h. After filtration, take 1 mL of the reaction solution for HPLC analysis to obtain phase solubility data.

[0073] Cyclodextrin derivatives with different concentrations were selected for phase solubility studies according to the solubility of cyclodextrin. Each reaction mixture (1 mL) was filtered and analyzed by HPLC. According to the regression equation and standard curve, the content of mebendazole in each solution was obtained.

[0074] The test results are as Figure 3 shown.

[0075] Figure 3 The solubility of mebendazole in aqueous solutions of different concentrations of β-cyclodextrin, γ-cyclodextrin, methyl-β-cyclodextrin, HP-β-cyclodextrin and HP-γ-cyclodextrin is shown.

[0076] The above test results show that in the range of cyclodextrin concentration of 5 - 25 mmol / L, the amount of mebendazole dissolved in aqueous solutions of HP-β-cyclodextrin and methyl-β-cyclodextrin increases linearly, and then the dissolved mebendazole concentration reaches a plateau, independent of the cyclodextrin concentration. The solubility curve of HP-β-cyclodextrin shows an AN type graph, indicating that the cavity of HP-β-cyclodextrin has a better affinity for mebendazole. Therefore, HP-β-cyclodextrin was selected to form an inclusion complex with mebendazole.

[0077] 4. Determination of the water solubility of mebendazole in the inclusion complex

[0078] An excessive amount of the mebendazole HP-β-cyclodextrin inclusion complex prepared in the example was added to water (1 mL), shaken for 2 h and then filtered, and the solubility of mebendazole in the inclusion complex was determined by HPLC analysis.

[0079] The results show that the solubility of mebendazole in the mebendazole HP-β-cyclodextrin inclusion complex prepared in the example is 6.05 mg / mL;

[0080] 5. Determination of the optimal inclusion conditions

[0081] The inclusion conditions (such as reaction time, reaction temperature, stirring speed and the ratio of mebendazole to HP-β-cyclodextrin) will affect the inclusion effect. Therefore, various complexation conditions were attempted according to the single factor and orthogonal strategies, and the water solubility was used as the standard to judge the inclusion effect.

[0082] (1) Single factor experiment

[0083] The single factor experiments were carried out according to the following conditions:

[0084] ① Rotation speed: 100 rpm, 500 rpm, 1000 rpm;

[0085] ② Temperature: 30 °C, 50 °C, 80 °C;

[0086] ③ Time: 1 h, 3 h, 5 h, 12 h;

[0087] ④ Molar ratio of mebendazole to HP-β-cyclodextrin: 1:1, 1:2, 1:3, 2:1.

[0088] The stirred solution was left standing in a 4 °C refrigerator for several hours and then rotary evaporated to remove formic acid (washed until there was no smell of formic acid). The dried product was dissolved in 10 ml of pure water, filtered through a 0.22 μm filter membrane, frozen overnight in a -20 °C refrigerator, and then freeze-dried under reduced pressure to obtain the inclusion complex solid powder. When detecting, the sample was added to 1 ml of ultrapure water, made into a saturated solution and then filtered, and detected by HPLC.

[0089] The results are shown in Table 1.

[0090] Table 1 Results of single factor experiments

[0091]

[0092]

[0093] (2) Orthogonal experiment

[0094] The orthogonal experiment table is shown in Table 2:

[0095] Table 2 Orthogonal experiment table

[0096] Temperature (°C) Rotation speed (rpm) Time (h) 40 400 2 40 500 4 40 600 3 50 400 4 50 500 3 50 600 2 60 400 3 60 500 2 60 600 4

[0097] Orthogonal experiments were carried out according to the orthogonal experiment table. The stirred solution was left standing in a 4 °C refrigerator for several hours and then rotary evaporated to remove formic acid (washed until there was no smell of formic acid). The dried product was dissolved in 10 ml of pure water, filtered through a 0.22 μm filter membrane, frozen overnight in a -20 °C refrigerator, and then freeze-dried under reduced pressure to obtain the inclusion complex solid powder. When detecting, the sample was added to 1 ml of ultrapure water, made into a saturated solution and then filtered, and detected by HPLC.

[0098] Calculate the inclusion rate and inclusion ratio of the inclusion complex according to the following formula:

[0099] Inclusion rate (%) = [mass of inclusion complex (mg) / (mass of mebendazole (mg) + mass of cyclodextrin (mg))] × 100%;

[0100] Inclusion ratio (%) = [mass of mebendazole in the inclusion complex (mg) / dosage of mebendazole (mg)] × 100%;

[0101] According to the above test results, the mebendazole HP-β-cyclodextrin inclusion complex was obtained with an inclusion ratio of 28% and an inclusion rate of 90%, and showed the best water solubility of 6.05 mg / mL so far.

[0102] 6. Scanning electron microscopy (SEM) analysis

[0103] The mebendazole raw material drug, physical mixture and inclusion complex were sputter-coated with gold and placed on the sample stage, and a German Zeiss Merlin scanning electron microscope (acceleration voltage 5 kV) was used for morphology observation.

[0104] The results are as Figure 4 shown.

[0105] Figure 4 The scanning electron microscope photos of mebendazole, the physical mixture of mebendazole HP-β-cyclodextrin, and the inclusion complex of mebendazole HP-β-cyclodextrin are shown;

[0106] Among them,

[0107] Figure 4 (a) shows the scanning electron microscope photo of mebendazole;

[0108] Figure 4 (b) shows the scanning electron microscope photo of the physical mixture of mebendazole HP-β-cyclodextrin;

[0109] Figure 4 (c) shows the scanning electron microscope photo of the inclusion complex of mebendazole HP-β-cyclodextrin.

[0110] The above test results show that mebendazole presents fine crystals ( Figure 4 (a)), while the physical mixture of mebendazole and HP-β-cyclodextrin ( Figure 4 (b)) is significantly different from the microscopic image of the inclusion complex ( Figure 4 (c)). The particle size and morphology of the inclusion complex are significantly different from those of mebendazole crystals, indicating the formation of a new phase.

[0111] 7. Nuclear magnetic resonance (NMR) study

[0112] NMR spectroscopy was used as the most effective method to confirm the formation of the mebendazole inclusion complex and the interaction between mebendazole and HP-β-cyclodextrin in the inclusion complex.

[0113] The proton nuclear magnetic spectrum ( 1 1H NMR) of the mebendazole-HP-β-cyclodextrin inclusion complex in D2O was recorded using a German Bruker 400 MHz nuclear magnetic resonance spectrometer, and the rotating frame nuclear Overhauser effect spectroscopy (ROESY) was recorded using a Bruker 600 MHz nuclear magnetic resonance spectrometer.

[0114] The test results are as Figure 5 shown.

[0115] Figure 5 The proton nuclear magnetic resonance spectrum and ROESY spectrum of the mebendazole HP-β-cyclodextrin inclusion complex in D2O are shown;

[0116] Among them,

[0117] Figure 5 (a) shows the proton nuclear magnetic resonance spectrum of mebendazole HP-β-cyclodextrin inclusion complex in D2O;

[0118] Figure 5 (b) shows the Roesy spectrum of mebendazole HP-β-cyclodextrin inclusion complex in D2O.

[0119] The above test results show that in the proton NMR spectrum, the signals at 7.97 ppm (1H, s), 7.74 ppm (1H, d), 7.63 ppm (2H, m), 7.50 ppm (3H, m), and 7.40 ppm (1H, d) indicate that the inclusion complex contains mebendazole molecules. The Roesy spectrum shows that there is an interaction between the protons at 7.97 ppm, 7.74 ppm, 7.63 ppm, 7.50 ppm, and 7.40 ppm of mebendazole and the proton at 3.80 ppm of HP-β-cyclodextrin, indicating that the mebendazole molecule is located inside the cavity of HP-β-cyclodextrin.

[0120] The present invention successfully verified the formation of the mebendazole-HP-β-cyclodextrin inclusion complex through the proton nuclear magnetic resonance spectrum and Roesy spectrum in D2O, and double-verified the interaction and dissolution properties of mebendazole and HP-β-cyclodextrin in the inclusion complex. These methods have high precision and confirm a strong correlation between the inclusion complex and the water solubility of mebendazole.

[0121] 8. In vitro pharmacokinetic (PK) study

[0122] Referring to the paddle method in the 2010 edition of the Chinese Veterinary Pharmacopoeia, ultrapure water (900 mL, degassed by ultrasonic) was placed in a dissolution apparatus, and stirred at 37 ± 0.5 °C and 100 r / min. Mebendazole raw material drug (50 mg), the mebendazole-HP-β-cyclodextrin inclusion complex prepared in the example (containing 50 mg of mebendazole), and the mebendazole-HP-β-cyclodextrin physical mixture prepared in the comparative example (containing 50 mg of mebendazole) were respectively added to the dissolution cups. Samples (1 mL) were taken at 1, 3, 5, 15, 30, 45, 60, and 75 min, filtered through a 0.22 μm microporous membrane within 30 s, and analyzed by HPLC. At the same time, 1 mL of isothermal dissolution medium was replenished. According to the HPLC results, a time-cumulative dissolution curve was plotted, and the experiment was repeated three times.

[0123] The results are as Figure 6 shown.

[0124] Figure 6 shows the dissolution rate curves of mebendazole, mebendazole HP-β-cyclodextrin physical mixture, and mebendazole HP-β-cyclodextrin inclusion complex.

[0125] The above test results showed that under the same conditions, the 15-minute release rate of mebendazole in the inclusion complex reached 80%, while that of the physical mixture was only 30%, and the release rate of the pure drug was even lower at 20%.

[0126] Test Example 2 (In Vivo Pharmacokinetics (PK) Study)

[0127] 1. Establishment of the standard curve of mebendazole in blank dog plasma

[0128] Dissolve mebendazole (10 mg) in chromatographically pure acetonitrile (2 mL), and after gradient dilution with methanol, add it to a test tube containing 0.2 mL of blank dog plasma to prepare solutions with concentrations of 0.015, 20, 40, 60, and 80 μg / mL respectively. Using potassium dihydrogen phosphate-acetonitrile gradient elution as the mobile phase (acetonitrile: 0.3% phosphoric acid solution, the pH of phosphoric acid was adjusted to 4.0 with triethylamine), the detection wavelength was 289 nm, the column temperature was 35 °C, the flow rate was 1 mL / min, the injection volume was 50 μL (gradient program: 25% acetonitrile from 0 - 14 min, 40% acetonitrile from 14 - 18 min, 25% acetonitrile from 18 - 25 min), and HPLC analysis was performed to establish the standard curve and regression equation of mebendazole in plasma.

[0129] The results were as Figure 7 shown.

[0130] Figure 7 The standard curve and regression equation of mebendazole in blank dog plasma are shown.

[0131] The above test results showed that in the range of 0.015 - 80 μg / mL, the HPLC peak area was linearly related to the concentration of mebendazole in blank dog plasma. Dog plasma did not interfere with the detection of mebendazole, and the standard curve and regression equation of mebendazole in dog plasma were obtained as Y = 7.0429X + 23.194 (determination coefficient R 2 : 0.9911, Y: absorbance peak area, X: concentration). At the same time, the lowest limit of detection (LLOD) was 0.005 μg / mL, and the lowest limit of quantification (LLOQ) was 0.015 μg / mL.

[0132] 2. Recovery, intra-day and inter-day precision and accuracy of mebendazole in plasma

[0133] Add mebendazole standard solution to 500 μL of blank dog plasma to prepare samples with concentrations of 10, 1, and 0.1 μg / mL respectively. After extraction and filtration, HPLC analysis was performed. Five replicates were prepared for each concentration, and the intra-day coefficient of variation was calculated by measuring the samples intra-day three times, and the inter-day coefficient of variation was calculated by performing experiments for three consecutive days.

[0134] The results showed that the recovery rate was 90.8% ± 0.27% to 110% ± 0.25%, the within-day coefficient of variation was 1.14% - 5.67%, and the between-day coefficient of variation was 1.4% - 3.5%.

[0135] 3. In vivo pharmacokinetic study

[0136] (1) Blood sample collection

[0137] Twelve healthy adult dogs (weight 5 ± 0.1 kg, half male and half female) were randomly divided into the mebendazole raw material drug group and the inclusion complex group. After numbering, they were adaptively raised in a temperature-controlled and ventilated environment for one week. They were fasted before dosing and orally administered mebendazole or the mebendazole-HP-β-cyclodextrin inclusion complex prepared in the example at a dose of 5 mg / kg. During the sampling period, they were fed a low-fat dog food and drank water normally. Forelimb venous blood (2 mL) was collected at 0.25, 0.5, 0.75, 1, 2, 4, 6, 10, 12, and 24 h after dosing, immediately transferred to a heparinized test tube, centrifuged at 3500 r / min for 10 min, and the plasma was stored at -20°C.

[0138] (2) Blood sample analysis

[0139] Take 0.5 mL of the plasma sample thawed to room temperature and place it in a 2 mL centrifuge tube. Add dichloromethane-methanol (1:1, 1 mL), vortex for 2 min, centrifuge at 13000 r / min for 10 min, and transfer the supernatant. Extract twice repeatedly, combine the supernatants, and evaporate to dryness under nitrogen at 40°C. Re-dissolve with acetonitrile (0.5 mL), vortex for 5 min, centrifuge at 13000 r / min for 10 min, and filter through a 0.22 μm filter membrane for HPLC analysis.

[0140] By using the same HPLC instrument and C-18 chromatographic column, the plasma samples were analyzed rapidly, and mebendazole in the standard sample was checked before and after each HPLC analysis to avoid systematic errors. By analyzing the plasma samples by HPLC, based on the standard curve and regression equation, the relationship between the concentration of mebendazole or its inclusion complex in dog plasma and the sampling time was obtained, and the drug concentration-time curve was obtained, as Figure 8 shown. The data was processed by WinNolin software, and the main pharmacokinetic parameters were obtained, as shown in Table 3.

[0141] Figure 8 The mebendazole concentration curves in the plasma of dogs administered mebendazole or mebendazole HP-β-cyclodextrin inclusion complex are shown.

[0142] Table 3 Pharmacokinetic parameters of mebendazole and mebendazole HP-β-cyclodextrin inclusion complex

[0143] Parameter Unit Mebendazole Mebendazole HP-β-cyclodextrin inclusion complex <![CDATA[T max > h 12.00±0.5 10±0.5 <![CDATA[C max > μg / mL 8.96±0.15 17.34±2.02 <![CDATA[AUC 0-24 > μg·h / mL 151.32±5.92 289.02±15.83 <![CDATA[t 1 / 2 > h 5.81±0.36 10.01±2.07 MRT h 13.35±0.3 16.85±0.22

[0144] The above test results show that after forming an inclusion complex with HP-β-cyclodextrin, the C of mebendazole max increased from 8.96 ± 0.15 μg / mL to 17.34 ± 2.02 μg / mL; the T max decreased from 12.00 ± 0.5 h to 10 ± 0.5 h; the half-life increased from 5.81 ± 0.36 h to 10.01 ± 2.07 h; the AUC 0-24 increased from 151.32 ± 5.92 μg·h / mL to 289.02 ± 15.83 μg·h / mL; the bioavailability nearly doubled.

[0145] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

[0146] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

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Claims

1. A mebendazole HP-β-cyclodextrin inclusion complex, characterized in that, The mebendazole HP-β-cyclodextrin inclusion complex is composed of mebendazole and HP-β-cyclodextrin; wherein, the mebendazole is located in the cavity of HP-β-cyclodextrin.

2. The mebendazole HP-β-cyclodextrin clathrate according to claim 1, wherein The inclusion ratio of the mebendazole HP-β-cyclodextrin inclusion complex is 20-40%.

3. The preparation method of the mebendazole HP-β-cyclodextrin inclusion complex according to any one of claims 1-2, characterized in that, It includes the following steps: S1. Add mebendazole into a solvent, stir and dissolve it to obtain a mebendazole solution; S2. Add HP-β-cyclodextrin into the mebendazole solution, heat and stir for reaction, then refrigerate at low temperature, purify and dry to obtain the mebendazole HP-β-cyclodextrin inclusion complex.

4. The preparation method of the mebendazole HP-β-cyclodextrin inclusion complex according to claim 3, characterized in that, In step S1, the solvent is formic acid.

5. The preparation method of the mebendazole HP-β-cyclodextrin inclusion complex according to claim 3, characterized in that, The mass ratio of mebendazole to HP-β-cyclodextrin is 1:4-6.

6. The preparation method of mebendazole HP-β-cyclodextrin inclusion complex according to claim 3, wherein, In step S2, the heating temperature is 40-60 °C.

7. The preparation method of mebendazole HP-β-cyclodextrin inclusion complex according to claim 3, characterized in that, In step S2, the stirring speed is 400-600 r / min.

8. The preparation method of mebendazole HP-β-cyclodextrin inclusion complex according to claim 3, wherein, In step S2, the reaction time is 2-5 h.

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