Preparation method and application of gefitinib-hesperetin nano eutectic

Abstract

The invention discloses a preparation method and application of a gefitinib-hesperetin nanometer eutectic crystal, and belongs to the technical field of pharmacy, the preparation method comprises the following steps: placing a gefitinib-hesperetin eutectic crystal and a stabilizer in a grinding tank, adding ultrapure water, carrying out ultrasonic treatment until the gefitinib-hesperetin eutectic crystal and the stabilizer are uniformly dispersed, adding grinding beads, adjusting the rotating speed, and carrying out ultrasonic treatment to obtain the gefitinib-hesperetin nanometer eutectic crystal. And grinding to obtain the gefitinib-hesperetin nano eutectic crystal. The gefitinib-hesperetin nano eutectic is successfully obtained by taking gefitinib as a research object and hesperetin as a eutectic forming substance and combining crystal engineering and a nanotechnology. The structure and physicochemical properties of the gefitinib-hesperetin eutectic crystal are characterized by a series of solid characterization technologies, and the in-vitro dissolution rate and the in-vitro antitumor activity of the gefitinib-hesperetin eutectic crystal and the in-vivo bioavailability of rats are systematically evaluated. The gefitinib-hesperetin nano eutectic crystal not only effectively improves the solubility and bioavailability of gefitinib, but also improves the antitumor activity of the medicine.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmacy and relates to a preparation method of gefitinib-hesperidin nano cocrystal and application thereof. Background Art

[0002] Gefitinib (Gefitinib, Gef, chemical structure as Figure 1 (middle right) is the world's first developed epidermal growth factor receptor tyrosine kinase inhibitor. It binds to the tyrosine kinase region in the cell membrane, blocks the EGFR signaling, and prevents EGFR-dependent cell proliferation to achieve anti-tumor effects. However, with the widespread use of gefitinib in clinical practice, it has also been found that some patients develop side effects such as drug resistance, which greatly weakens the efficacy. In addition, gefitinib belongs to Class II in the Biopharmaceutics Classification System (BCS), with low solubility and poor oral bioavailability. Therefore, it is necessary to develop new preparation technologies to improve its water solubility and lay the foundation for the development of gefitinib solid preparations with high solubility and high oral bioavailability.

[0003] Hesperetin (Hesperetin, Hes, chemical structure as Figure 1 Hesperidin (middle left picture) is a naturally occurring dihydroflavonoid compound with multiple pharmacological activities such as anti-inflammatory and anti-tumor. From a structural analysis, hesperidin contains functional groups such as hydroxyl, carbonyl, and methoxy, and has abundant hydrogen bond acceptors and donors. It is easy to form intermolecular hydrogen bonds with the N atom on quinazoline, the O atom on morpholine, the N atom, as well as -NH- and -OCH3 on gefitinib, thereby synthesizing drug cocrystals.

[0004] Drug-drug cocrystals are supramolecular complexes composed of active pharmaceutical ingredients (API) and cocrystal formers (CCF) with independent pharmacological activity through non-covalent interactions (hydrogen bonds, halogen bonds, and π-π stacking, etc.). Drug nanocrystals are carrier-free colloidal drug dispersion systems with a size of less than 1000nm and containing a small amount of stabilizers. The high drug loading and improved drug solubility make them efficient drug dosage forms that enter cells or cross cells, thereby achieving therapeutic concentrations. Summary of the invention

[0005] The purpose of the present invention is to provide a preparation method and application of gefitinib-hesperidin nano-cocrystal to solve the problems existing in the above-mentioned prior art. The present invention provides a preparation method and application of gefitinib nano-cocrystal, which can improve in vitro dissolution and in vivo bioavailability in rats, and also has excellent in vitro anti-tumor activity against human lung cancer cells A549.

[0006] One of the technical solutions provided by the present invention:

[0007] A gefitinib-hesperetin co-crystal, wherein the gefitinib-hesperetin co-crystal is crystallized in a triclinic system. The space group of the present invention is , and its asymmetric unit contains one gefitinib molecule and one hesperetin molecule.

[0008] The second technical solution provided by the present invention is:

[0009] A method for preparing the gefitinib-hesperetin co-crystal comprises taking gefitinib and hesperetin as raw materials and preparing the gefitinib-hesperetin co-crystal by a solvent-assisted grinding method.

[0010] Preferably, gefitinib and hesperetin are added to a grinding jar, a solvent is added dropwise, and the mixture is ground to obtain gefitinib-hesperetin co-crystals.

[0011] More preferably, the molar ratio of gefitinib to hesperetin is 1:1; the solvent is selected from one or more of isopropanol, methanol and dichloromethane; the grinding power is 15 to 45 Hz, and the grinding time is 30 to 60 min.

[0012] The third technical solution provided by the present invention is:

[0013] A preparation method of gefitinib-hesperetin nano cocrystals comprises placing the gefitinib-hesperetin cocrystals and a stabilizer in a grinding tank, adding ultrapure water, performing ultrasound until the gefitinib-hesperetin cocrystals and the stabilizer are evenly dispersed, adding grinding beads, adjusting the rotation speed, and grinding to obtain the gefitinib-hesperetin nano cocrystals.

[0014] Preferably, the stabilizer is selected from one or more of PVPK30, poloxamer, TGPS, sodium lauryl sulfate, and lecithin.

[0015] Preferably, the concentration ratio of the gefitinib-hesperetin cocrystal to the stabilizer is (5-25): (1-5); and / or

[0016] The volume ratio of the grinding beads to the grinding jar is (5-30):50; and / or,

[0017] The rotation speed is 400-1200 rpm.

[0018] The fourth technical solution provided by the present invention is:

[0019] An application of the gefitinib-hesperetin co-crystal in preparing a drug for treating A549 lung cancer.

[0020] The fifth technical solution provided by the present invention is:

[0021] Application of gefitinib-hesperetin nano co-crystal prepared by the preparation method in the preparation of lung cancer drugs.

[0022] Preferably, the lung cancer drug is a nanosuspension preparation.

[0023] Compared with the prior art, the present invention has the following advantages and technical effects:

[0024] The present invention uses gefitinib as the research object and hesperidin as the cocrystal former, and successfully obtains gefitinib-hesperidin nano cocrystal by combining crystal engineering and nanotechnology. The structure and physicochemical properties of gefitinib-hesperidin cocrystal are characterized by a series of solid characterization techniques (single crystal X-ray diffraction, nuclear magnetic resonance analysis, powder X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, etc.), and its in vitro dissolution, in vitro antitumor activity and in vivo bioavailability in rats are systematically evaluated. It shows that gefitinib-hesperidin nano cocrystal not only effectively improves the solubility and bioavailability of gefitinib, but also can enhance the antitumor activity of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0026] Figure 1 The left picture in the middle shows the chemical structure of Gef, and the right picture shows the chemical structure of Hes;

[0027] Figure 2 Schematic diagram of the Gef-Hes asymmetric unit prepared in Example 1;

[0028] Figure 3 This is a schematic diagram of the three-dimensional structure of Gef-Hes prepared in Example 1;

[0029] Figure 4 PXRD comparison diagram of Hes, Gef and Gef-Hes of Example 1;

[0030] Figure 5 For Gef 1H NMR (DMSO-d6) spectrum;

[0031] Figure 6 For Hes 1 H NMR (DMSO-d6) spectrum;

[0032] Figure 7 The Gef-Hes prepared in Example 1 1 H NMR (DMSO-d6) spectrum;

[0033] Figure 8 TG and DSC spectra of Gef, Hes and Gef-Hes prepared in Example 1;

[0034] Fig. 9 IR spectra of Gef, Hes and Gef-Hes prepared in Example 1;

[0035] Fig.10 is the dissolution curve of Gef and Gef-Hes in pure water;

[0036] Fig.11 are the dissolution curves of Gef, Gef-Hes and Gef-Hes-NPs in pure water;

[0037] Fig.12 Drug-time curves of Gef, Gef+Hes, Gef-Hes and Gef-Hes-NPs;

[0038] Fig.13 This is a comparison of the inhibition rates of Gef, Gef+Hes, Gef-Hes and Gef-Hes-NPs on human lung cancer cells A549 at 48 hours. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] Example 1

[0045] 446.90 mg (1 mmol) of gefitinib and 302.28 mg (1 mmol) of hesperidin were weighed and placed in a 25 mL grinding jar equipped with agate grinding balls, 150 μL of isopropanol was added, and the mixture was ground in a ball mill at a rate of 30 Hz for 30 min to obtain gefitinib-hesperidin eutectic powder, recorded as Gef-Hes, and the solvent was slowly evaporated to obtain gefitinib-hesperidin eutectic.

[0046] Example 2

[0047] 446.90 mg (1 mmol) of gefitinib and 302.28 mg (1 mmol) of hesperidin were weighed and placed in a 25 mL grinding jar equipped with agate grinding balls, 150 μL of methanol was added, and the mixture was ground in a ball mill at a rate of 30 Hz for 30 min to obtain gefitinib-hesperidin eutectic powder, recorded as Gef-Hes, and the solvent was slowly evaporated to obtain gefitinib-hesperidin eutectic.

[0048] Example 3

[0049] 446.90 mg (1 mmol) of gefitinib and 302.28 mg (1 mmol) of hesperetin were weighed and placed in a 25 mL grinding jar equipped with agate grinding balls, 150 μL of dichloromethane was added, and the mixture was ground in a ball mill at a rate of 30 Hz for 30 min to obtain gefitinib-hesperetin eutectic powder, recorded as Gef-Hes, and the solvent was slowly evaporated to obtain gefitinib-hesperetin eutectic.

[0050] Example 4

[0051] 225 mg of the gefitinib-hesperetin cocrystals prepared in Example 1 and 45 mg of the stabilizer (PVPK30) were weighed and placed in a 50 mL grinding jar. After adding 15 mL of ultrapure water, ultrasound was performed to fully disperse the gefitinib-hesperetin cocrystals and the stabilizer. Then, 25 mL of zirconium oxide (ZrO2) grinding beads were added and installed on a nano grinder. The speed was adjusted to 530 rpm and the grinder was operated. After grinding for 5 hours, a gefitinib-hesperetin nano eutectic suspension was obtained, which was recorded as Gef-Hes-NPs.

[0052] Example 5

[0053] 225 mg of the gefitinib-hesperetin cocrystals prepared in Example 1 and 30 mg of the stabilizer (PVPK30) were weighed and placed in a 50 mL grinding jar. After adding 15 mL of ultrapure water, ultrasound was performed to fully disperse the gefitinib-hesperetin cocrystals and the stabilizer. Then, 25 mL of zirconium oxide (ZrO2) grinding beads were added and installed on a nano grinder. The speed was adjusted to 530 rpm and the grinder was operated. After grinding for 5 hours, a gefitinib-hesperetin nano eutectic suspension was obtained, which was recorded as Gef-Hes-NPs.

[0054] Example 6

[0055] 225 mg of the gefitinib-hesperetin cocrystals prepared in Example 1 and 15 mg of the stabilizer (PVPK30) were weighed and placed in a 50 mL grinding jar. After adding 15 mL of ultrapure water, ultrasound was performed to fully disperse the gefitinib-hesperetin cocrystals and the stabilizer. Then, 25 mL of zirconium oxide (ZrO2) grinding beads were added and installed on a nano grinder. The speed was adjusted to 530 rpm and the grinder was operated. After grinding for 5 hours, a gefitinib-hesperetin nano eutectic suspension was obtained, which was recorded as Gef-Hes-NPs.

[0056] Example 7

[0057] 225 mg of the gefitinib-hesperetin cocrystals prepared in Example 1 and 30 mg of the stabilizer (PVPK30) were weighed and placed in a 50 mL grinding jar. After adding 15 mL of ultrapure water, ultrasound was performed to fully disperse the gefitinib-hesperetin cocrystals and the stabilizer. Then, 20 mL of zirconium oxide (ZrO2) grinding beads were added and installed on a nano grinder. The speed was adjusted to 530 rpm and the grinder was operated. After grinding for 5 hours, a gefitinib-hesperetin nano eutectic suspension was obtained, which was recorded as Gef-Hes-NPs.

[0058] Example 8

[0059] 225 mg of the gefitinib-hesperetin cocrystals prepared in Example 1 and 30 mg of the stabilizer (PVPK30) were weighed and placed in a 50 mL grinding jar. After adding 15 mL of ultrapure water, ultrasound was performed to fully disperse the gefitinib-hesperetin cocrystals and the stabilizer. Then, 15 mL of zirconium oxide (ZrO2) grinding beads were added and installed on a nano grinder. The speed was adjusted to 530 rpm and the grinder was operated. After grinding for 5 hours, a gefitinib-hesperetin nano eutectic suspension was obtained, which was recorded as Gef-Hes-NPs.

[0060] The gefitinib-hesperetin nano-eutectic suspension prepared in Example 4-8 was diluted with an appropriate amount of ultrapure water, and its particle size was measured using a Zetasizer NANO ZS90 particle size analyzer. The results showed that the particle size was between 160 and 175 nm, and the PDI (Polydispersity Index) was between 0.13 and 0.16, indicating that the gefitinib-hesperetin nano-eutectic was successfully prepared.

[0061] 1. Taking Example 1 as an example, characterization experiment of the prepared gefitinib-hesperidin cocrystal

[0062] 1.1 Single crystal X-ray diffraction analysis

[0063] The gefitinib-hesperetin cocrystal single crystal prepared in Example 1 was tested using a Rigaku X-ray single crystal diffractometer.

[0064] Figure 2 is a schematic diagram of the asymmetric unit of Gef-Hes in Example 1, Figure 3 The three-dimensional structure of Gef-Hes in Example 1 is shown in FIG. 1 , and the dotted lines represent hydrogen bonds. The results show that the crystals of gefitinib-hesperidin cocrystals are crystallized in the triclinic system. The space group of the drug cocrystal is monoclinic. Space group, unit cell parameters are: axis length Axis angle α=66.456(3)°, β=85.878(2)°, γ=72.606(3)°, There are four hydrogen bonds between gefitinib and hesperidin molecules, namely, the hydrogen atoms on the methoxyl group of gefitinib molecule and the oxygen atoms on the keto carbonyl group of hesperidin molecule. Between the hydrogen atom on the hydroxyl group of the hesperidin molecule and the nitrogen atom on the quinoline ring of the gefitinib molecule Between the hydrogen atom on the keto carbonyl group of the hesperidin molecule and the oxygen atom on the methoxyl group of the gefitinib molecule Between the hydrogen atom on the ether group of the hesperidin molecule and the oxygen atom on the propoxy group of the gefitinib molecule There are two types of intermolecular hydrogen bonds between gefitinib molecules, one between the hydrogen atoms on the imino group and the oxygen atoms on the morpholine ring. Between the hydrogen atom on the quinazoline ring and the oxygen atom on the morpholine ring In addition, there is an intermolecular hydrogen bond between the hydrogen atom on the hydroxyl group of a hesperidin molecule and the oxygen atom on the ether group of another hesperidin molecule. The hydrogen on the hydroxyl group of the hesperidin molecule forms an intramolecular hydrogen bond with the oxygen atom on the ketone carbonyl group. Depend on Figure 3 It can be seen that the above multiple weak interactions form a three-dimensional structure in which gefitinib molecules and hesperidin molecules alternate with each other.

[0065] 1.2 X-ray powder diffraction analysis

[0066] The gefitinib-hesperetin cocrystal powder and its raw materials (gefitinib and hesperetin) prepared in Example 1 were tested on a Rigaku MiniFlex 600 diffractometer, with Cu target Kα rays as the light source, a voltage of 40 kV, a current of 15 mA, a scanning range of 3-40° (2θ), a scanning speed of 5° / min, and a step size of 0.02° (2θ), and the obtained data were processed using the software OriginPro9.0.

[0067] Figure 4 The PXRD comparison diagram of gefitinib, hesperetin and gefitinib-hesperetin cocrystal of Example 1. The results show that the gefitinib-hesperetin cocrystal has characteristic peaks at positions (2θ) 7.29°, 8.30°, 9.97°, 12.61°, 13.25°, 14.51°, 16.10°, etc., indicating that a new crystalline phase is generated. In addition, the measured PXRD of gefitinib-hesperetin cocrystal is basically consistent with the PXRD spectrum of the single crystal simulation, indicating that the prepared powder sample is consistent with the single crystal phase and has a high purity.

[0068] 1.3 Nuclear magnetic resonance analysis

[0069] 5 mg of the gefitinib-hesperetin cocrystal powder and its raw materials (gefitinib and hesperetin) prepared in Example 1 were weighed and dissolved in deuterated dimethyl sulfoxide (DMSO-d6). About 0.5 mL of the sample solution was added to a nuclear magnetic resonance tube with an outer diameter of 5 mm and a length of 178 mm, and the spectrometer was used for spectroscopy on a Bruker Ascend 500 MHz nuclear magnetic resonance spectrometer. 1 H NMR analysis, chemical shift of deuterated dimethyl sulfoxide solvent peak: 2.50 ppm.

[0070] Figure 5For gefitinib 1 H NMR (DMSO-d6) spectrum, Figure 6 Hesperidin 1 H NMR (DMSO-d6) spectrum, Figure 7 Gefitinib-hesperidin cocrystal 1 H NMR (DMSO-d6) spectrum; by comparison, it can be seen that the chemical shift of the characteristic peak of gefitinib is as follows: 1 H NMR (500MHz, DMSO-d6) δ9.57 (s, 1H), 8.50 (s, 1H), 8.12 (dd, J = 6.9, 2.6Hz, 1H), 7.85-7.75 (m, 2H), 7.45 (t, J = 9.1Hz, 1H), 7.20 (s, 1H), 4.18 (t, J = 6.3Hz, 2H), 3.94 (s, 3H), 3.58 (t, J = 4.6Hz, 4H), 2.50-2.30 (m, 6H), 2.00 (p, J = 6.6Hz, 2H). The characteristic peak chemical shifts of hesperidin are as follows: 1 H NMR (500MHz, DMSO-d6) δ12.14 (s, 1H), 10.82 (s, 1H), 9.12 (s, 1H), 6.98-6.90 (m, 2H), 6.87 (dd, J = 8.2, 2.2 Hz, 1H), 5.98-5.80 (m, 2H), 5.43 (dd, J = 12.4, 3.1 Hz, 1H), 3.77 (s, 3H), 3.20 (dd, J = 17.1, 12.4 Hz, 1H), 2.71 (dd, J = 17.1, 3.2 Hz, 1H). The chemical shifts of the characteristic peaks of gefitinib-hesperidin cocrystal are as follows: 1H NMR (500MHz, DMSO-d6) δ12.14(s,1H),10.83(s,1H),9.58(d,J=3.2Hz,1H),9.13(s,1H),8.50(s,1H),8.12(dd,J= 6.8,2.6Hz,1H),7.92-7.68(m,2H),7.45(t,J=9.1Hz,1H),7.21(s,1H),7.06-6.90(m,2H),6.87(dd,J=8.3,2.1Hz, 1H),5.98-5.82(m,2H),5.43(dd,J=12.4,3.1Hz,1H),4.18(t,J=6.4Hz,2H),3.94(s,3H),3.77(s,3H),3.58(t,J= 4.6Hz, 4H), 3.20 (dd, J = 17.1, 12.4Hz, 1H), 2.70 (dd, J = 17.1, 3.1Hz, 1H), 2.50-2.32 (m, 6H), 2.00 (p, J = 6.7Hz, 2H).

[0071] According to the integration results, the stoichiometric ratio of the two in the gefitinib-hesperidin cocrystal is 1:1. In addition, Figure 7 It can be seen that except for the characteristic peaks of gefitinib and hesperidin and the solvent peaks of water and dimethyl sulfoxide, no other impurity peaks and solvent peaks appear, indicating that the prepared co-crystal sample has high purity and does not contain other impurities. The solvent peak containing water may be due to the trace amount of water contained in the deuterated dimethyl sulfoxide detection reagent.

[0072] 1.4 Thermal analysis

[0073] Differential Scanning Calorimetry (DSC)

[0074] The gefitinib-hesperetin cocrystal prepared in Example 1 and its raw materials (gefitinib and hesperetin) were tested using a NETZSCH STA 449F5 synchronous thermal analyzer with nitrogen as the protective gas, a heating temperature range of 26-500° C., and a heating rate of 5° C. / min.

[0075] Thermogravimetric analysis (TG)

[0076] The gefitinib-hesperidin cocrystal prepared in Example 1 and its raw materials were tested using a NETZSCH STA 449F5 synchronous thermal analyzer with nitrogen as the protective gas, a heating temperature range of 26-800° C., and a heating rate of 10° C. / min.

[0077] Figure 8The TG and DSC spectra of gefitinib, hesperidin and gefitinib-hesperidin cocrystals. It can be seen from the TG curve that gefitinib, hesperidin and gefitinib-hesperidin cocrystals do not show solvent weight loss curves at a temperature of 100°C, indicating that gefitinib, hesperidin and gefitinib-hesperidin cocrystal samples are all pure samples without solvent. From the DSC spectrum, it can be seen that the gefitinib-hesperidin cocrystal has a melting peak at 181.1°C, which is different from gefitinib (194.7°C) and hesperidin (230.9°C), further verifying the formation of a new crystal phase. In addition, no other endothermic or exothermic peaks were detected before the melting point of gefitinib, hesperidin and gefitinib-hesperidin cocrystals, indicating that no other thermodynamic behaviors occurred before melting.

[0078] 1.5 Infrared spectroscopy analysis

[0079] The gefitinib-hesperidin cocrystal and its raw material prepared in Example 1 were tested using a Nicolet Nexus 470 infrared spectrometer. The sample and the dried potassium bromide were fully ground and mixed under an infrared lamp. An appropriate amount was placed in a mold, the sample was pressed into a transparent sheet, and then placed in an infrared spectrometer for scanning. The scanning range was: 4000-400cm -1 , average scan 64 times, spectral resolution 2cm -1 .

[0080] Fig. 9 IR spectra of gefitinib, hesperidin and gefitinib-hesperidin cocrystal. -1 The characteristic peak at 1624 cm represents the NH stretching vibration in the structure, while the peak at 1624 cm -1 The characteristic peak at 3501cm corresponds to the C=N stretching vibration in the structure; hesperidin in the infrared spectrum is 3501cm -1 and 1639cm -1 The characteristic peaks at 1639 cm-1 and 1640 cm-2 correspond to the stretching vibrations of OH and C=O, respectively. After the formation of gefitinib-hesperidin cocrystal, the C=N stretching vibration of gefitinib shifts to 1639 cm-1. -1 , while the OH stretching vibration of hesperidin shifts to 3457 cm -1 , it can be inferred that the C=N of gefitinib and the OH of Hes participated in the formation of supramolecular synthons, which is consistent with the single crystal results.

[0081] 2. In vitro dissolution determination of gefitinib-hesperidin nanococrystals

[0082] Gefitinib, the gefitinib-hesperetin cocrystal prepared in Example 1, and the gefitinib-hesperetin nano cocrystal prepared in Example 4 were respectively formulated into suspensions to obtain gefitinib suspension, gefitinib-hesperetin cocrystal suspension and gefitinib-hesperetin cocrystal nanosuspension. 5 mL of each (the gefitinib content in the three suspensions was the same) was taken and added to beakers containing 25 mL of pure water, respectively. The mixture was sealed and placed on a magnetic stirrer at 37° C. and 100 rpm. The timing was started and samples were taken at time points of 5, 15, 30, 60, 120, 240, 480, 720 and 1440 min. 3 mL of solution was taken each time and the same volume of dissolution medium was added at the same time. The taken solution was filtered through a 0.22 μm microporous membrane and gefitinib was quantitatively analyzed at 249 nm on a UV spectrophotometer.

[0083] Fig.10 The dissolution curves of gefitinib and gefitinib-hesperidin cocrystal in pure water show that after the formation of gefitinib-hesperidin cocrystal, the dissolution performance of gefitinib shows a significant dissolution rate compared with the raw material drug, which is about 49 times higher; Fig.11 Figure 2 is the dissolution curves of Gef, Gef-Hes and Gef-Hes-NPs in pure water. The dissolution curves of gefitinib, gefitinib-hesperetin cocrystal and gefitinib-hesperetin cocrystal nanosystem detected in pure aqueous solution. After the gefitinib-hesperetin cocrystal nanosystem was prepared, the solubility of gefitinib was further improved, and all showed the "spring-parachute" phenomenon, which plays an important role in improving drug solubility and dissolution rate.

[0084] 3. Bioavailability Determination of Gefitinib-Hesperidin Nanococrystals

[0085] 3.1 Plasma sample pretreatment

[0086] The plasma samples were treated by protein precipitation method. 100 μL of plasma sample was taken, 400 μL of methanol was added, vortexed for 30 seconds to denature the protein, and then centrifuged at 12000 rpm and 4°C for 15 minutes. The supernatant was nitrogen blown, 200 μL of methanol was added and ultrasonically re-dissolved, and 100 μL of the supernatant was taken for detection on HPLC.

[0087] 3.2 Chromatographic conditions

[0088] Agilent 1260 Infinity was used to measure the concentration of gefitinib in plasma. The chromatographic column used was an Agilent C18 column (4.5 mm×250 mm, 6 μm), the mobile phase was methanol: 0.1% triethylamine water (75:25), and the measurement was performed under the conditions of a flow rate of 1 mL / min, a detection wavelength of 330 nm, a column oven of 35° C., and an injection volume of 10 μL.

[0089] 3.3 Establishment of standard curve

[0090] Weigh 10 mg of gefitinib API accurately, add chromatographic methanol to completely dissolve, and then place in a 100 mL volumetric flask to make up to volume to obtain 100 μg / mL gefitinib stock solution. Accurately pipette different volumes of gefitinib stock solution into a 10 mL volumetric flask and make up to volume with chromatographic methanol to obtain standard solutions with different concentration gradients. Add the above series of standard solutions to blank plasma and treat according to the method under "3.1" to obtain plasma standard solutions with concentrations of 0.5, 1, 5, 10, and 20 μg / mL. Determine the peak area according to the chromatographic conditions under "3.2". Take the concentration of gefitinib as the horizontal coordinate and the corresponding peak area as the vertical coordinate, perform linear regression, and draw a standard curve for gefitinib.

[0091] 3.4 Pharmacokinetic studies

[0092] 25 SD male rats were randomly divided into 5 groups with 5 rats in each group. They were fasted for 12 hours before the experiment but not water. Gefitinib and gefitinib-hesperetin cocrystal were made into suspensions with sodium hydroxymethylcellulose (CMC-Na). Gefitinib suspension, gefitinib-hesperetin cocrystal suspension and gefitinib-hesperetin cocrystal nanosuspension were given by gavage at a dose of 20 mg / kg (in terms of gefitinib). About 0.5 mL of blood was collected from the rat eye sockets at 5, 15, 30, 45, 60, 90, 120, 240, 480, 720, and 1440 min after oral administration, and placed in a sodium heparin anticoagulant tube, then centrifuged at 8000 rpm and 4 ° C for 10 min, and plasma was collected in a clean centrifuge tube and stored at -80 ° C for standby use. Blood was collected from SD rats in the blank group to prepare blank plasma for standby use. The plasma samples were processed according to the method under "3.1" and the content of gefitinib in plasma was determined by HPLC. The experimental data were processed and analyzed by OriginPro 9.0.

[0093] Fig.12The drug-time curves of Gef, Gef+Hes, Gef-Hes and Gef-Hes-NPs are shown in Figure 2. The pharmacokinetic results show that the API gefitinib reaches the maximum blood concentration at 60 min, with a maximum concentration of 1.108±0.2611μg / mL and an AUC value of 424.11. The gefitinib in the physical mixture of gefitinib + hesperidin (Gef+Hes) reaches the maximum blood concentration at 60 min, with a maximum concentration of 1.510±0.432μg / mL and an AUC value of 502.02. The gefitinib in the gefitinib-hesperidin cocrystal reaches the maximum blood concentration at 45 min, with a maximum concentration of 2.401±0.374μg / mL and an AUC value of 718.46. Gefitinib in the gefitinib-hesperetin cocrystal nanosuspension reached the maximum blood drug concentration at 45min, with a maximum concentration of 2.230±0.215μg / mL and an AUC value of 1013.18. By comparison, it was found that the relative bioavailability of the physical mixture of gefitinib + hesperetin was 1.18 times that of the raw material gefitinib, the relative bioavailability of the gefitinib-hesperetin cocrystal was 1.69 times that of the raw material gefitinib, and the relative bioavailability of the gefitinib-hesperetin cocrystal nanosuspension was 2.39 times that of the raw material gefitinib, indicating that the bioavailability of gefitinib was significantly improved after the formation of multi-component drug crystals, and the nanosuspension preparation was conducive to promoting the oral absorption of gefitinib.

[0094] 4. Study on the antitumor activity of gefitinib-hesperidin nanococrystals in vitro

[0095] 4.1 Solution preparation

[0096] Preparation of complete culture medium: Prepare DMEM / F12 culture medium, fetal bovine serum (FBS), and penicillin-streptomycin mixture (double antibody) in a ratio of 90:10:1, mix thoroughly, and store in a 4°C refrigerator for later use.

[0097] Preparation of MTT solution: Weigh 250 mg of MTT powder and dissolve it in a 50 mL centrifuge tube. Add 50 mL of phosphate buffer (PBS), sonicate until fully dissolved, filter with a 0.22 μm filter membrane for sterilization, and store in a 4°C refrigerator for later use. During preparation and storage, keep away from light.

[0098] 4.2 Cell recovery

[0099] Place the materials to be used in the clean bench for UV sterilization for 30 minutes in advance. Take out the cryopreserved tube of A549 cells from the -80℃ refrigerator, place it in a 37℃ water bath, shake it quickly to thaw, centrifuge it at 1500rpm for 3 minutes, wipe the outside of the cryopreserved tube with an alcohol cotton ball, quickly place it in the clean bench, discard the supernatant, add new complete culture medium to resuspend, transfer it to a T25 cell culture bottle, and culture it in an incubator at 37℃ and 5% CO2.

[0100] 4.3 Cell culture medium replacement

[0101] Place the materials to be used in the clean bench for UV sterilization for 30 minutes in advance. Take out the culture bottle from the incubator, sterilize it with alcohol and place it in the clean bench, discard the old culture medium, wash it three times with PBS, add new complete culture medium, and continue to culture in the incubator at 37°C and 5% CO2. However, subculture is performed when the cell density reaches 80% to 90%.

[0102] 4.4 Cell passaging

[0103] Place the materials to be used in the clean bench for UV sterilization for 30 minutes in advance. Take out the cells from the incubator and observe the cell status under an inverted microscope. After ensuring that the growth status is good and there is no contamination, sterilize with alcohol and place them in the clean bench, discard the old culture medium, wash with 3mL PBS, discard, add 1mL trypsin protein-EDTA digestion solution for digestion, and then place in the incubator for incubation for 3 minutes. When the cells are observed to be spherical and not attached to the wall under the microscope, add an appropriate amount of complete culture medium to terminate the digestion. Transfer the cell suspension to a centrifuge tube, centrifuge at 1500rpm for 3 minutes, discard the supernatant, and then add new complete culture medium to the centrifuge tube to resuspend the cells, transfer to a new T 75 cell culture flask, and continue to culture in an incubator at 37℃ and 5% CO2.

[0104] 4.5 Cell inoculation

[0105] Place the materials to be used in the ultra-clean workbench in advance and sterilize with UV for 30 minutes. Discard the old culture medium, wash with PBS, add trypsin to digest the cells, terminate the digestion, centrifuge and discard the supernatant, add complete culture medium to resuspend, mix evenly, draw 10 μL to count on the cell counter, then take the cell resuspension and add an appropriate amount of complete culture medium, gently blow and mix evenly, so that the cell concentration is 5×10 4 / mL, then inoculate the cell solution into a 96-well plate (100 μL per well), and place the inoculated cell culture plate in a 37°C, 5% CO2 incubator until the cell monolayer covers the bottom of the well, and then administer the drug.

[0106] 4.6 Cellular drug delivery

[0107] Gefitinib, gefitinib-hesperetin cocrystal prepared in Example 1, physical mixture (gefitinib and hesperetin mixed in a molar ratio of 1:1) and gefitinib-hesperetin nanococrystal prepared in Example 4 were prepared with DMSO solution at different concentrations and added to a 96-well plate, 100 μL per well, so that the final drug concentration gradient was 3.125, 6.25, 12.5, 25, 50, 100 μM (DMSO content <0.1%), and then the 96-well plate was placed in an incubator at 37° C. and 5% CO2 for 48 hours. 0.1% DMSO was used as a control group and PBS was used as a blank group.

[0108] 4.7 MTT assay

[0109] Add 10 μL of MTT solution to each well of the cultured 96-well plate, and then place it in an incubator for 4 hours. Discard the supernatant of the 96-well plate, add 100 μL of DMSO to each well, shake on a shaker in the dark for 10 minutes to fully dissolve the crystals, and then detect at 490 nm on an ELISA reader. Calculate the cell inhibition rate based on the absorbance value: cell inhibition rate = [1-(OD value of the drug group-OD value of the blank group) / (OD value of the control group-OD value of the blank group)] × 100%.

[0110] Fig.13 The figure is a comparison of the inhibition rate of gefitinib, gefitinib-hesperidin cocrystal, physical mixture and gefitinib-hesperidin cocrystal nanosystem on A549 tumor cells at 48 hours. Fig.13 It can be seen that compared with gefitinib and physical mixture (Gef+Hes) group, gefitinib-hesperidin cocrystal (Gef-Hes) and its nanosystem (Gef-Hes-NPs) showed excellent cell inhibition at different dosage concentrations. In addition, with the increase of dosage concentration, the cell inhibition rate of gefitinib, physical mixture, gefitinib-hesperidin cocrystal and gefitinib-hesperidin cocrystal nanosystem group increased in a concentration-dependent manner. These results indicate that gefitinib-hesperidin cocrystal and its gefitinib-hesperidin nanococrystal system have excellent anti-tumor activity against A549 lung cancer cells.

[0111] The above are only preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A gefitinib-hesperetin cocrystal, characterized in that: The crystals of the gefitinib-hesperidin cocrystal are crystallized in the triclinic system. _ The space group of 1, its asymmetric unit contains one gefitinib molecule and one hesperetin molecule.

2. A method for preparing the gefitinib-hesperetin cocrystal according to claim 1, characterized in that: The gefitinib-hesperetin cocrystal is prepared by using gefitinib and hesperetin as raw materials through a solvent-assisted grinding method.

3. The preparation method according to claim 2, characterized in that: The specific steps of the solvent-assisted grinding method include: adding gefitinib and hesperetin into a grinding jar, dripping a solvent, and grinding to obtain gefitinib-hesperetin cocrystals.

4. The method for preparing gefitinib-hesperetin cocrystal according to claim 3, characterized in that: The molar ratio of gefitinib to hesperetin is 1:1; the solvent is selected from one or more of isopropanol, methanol and dichloromethane; the grinding power is 15-45 Hz, and the grinding time is 30-60 min.

5. A method for preparing gefitinib-hesperidin nano-cocrystal, characterized in that: The gefitinib-hesperetin cocrystal and the stabilizer according to claim 1 are placed in a grinding jar, ultrapure water is added, ultrasound is performed until the gefitinib-hesperetin cocrystal and the stabilizer are evenly dispersed, grinding beads are added, the rotation speed is adjusted, and gefitinib-hesperetin nano cocrystals are obtained after grinding.

6. The method for preparing gefitinib-hesperetin nano-cocrystal according to claim 5, characterized in that: The stabilizer is selected from one or more of PVPK30, poloxamer, TGPS, sodium lauryl sulfate, and lecithin.

7. The method for preparing gefitinib-hesperetin nano-cocrystal according to claim 5, characterized in that: The concentration ratio of the gefitinib-hesperetin cocrystal to the stabilizer is (5-25): (1-5); and / or The volume ratio of the grinding beads to the grinding jar is (5-30):50; and / or, The rotation speed is 400-1200 rpm.

8. Use of the gefitinib-hesperetin co-crystal according to claim 1 in preparing a drug for treating A549 lung cancer.

9. Use of gefitinib-hesperetin nano-cocrystal prepared by the preparation method according to any one of claims 5 to 7 in the preparation of lung cancer drugs.

10. The use according to claim 9, characterized in that: The lung cancer drug is a nanosuspension preparation.

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