Preparation method and application of gefitinib-hesperetin nanococrystal

Abstract

The present invention discloses a preparation method and application of gefitinib-hesperetin nano-cocrystals, belonging to the field of pharmaceutical technology. The gefitinib-hesperetin co-crystals and a stabilizer are placed in a grinding jar, ultrapure water is added, and ultrasound is applied until the gefitinib-hesperetin co-crystals and the stabilizer are evenly dispersed. Grinding beads are added, the rotation speed is adjusted, and the gefitinib-hesperetin nano-cocrystals are obtained after grinding. The present invention takes gefitinib as the research object and hesperetin as the co-crystal former, and successfully obtains gefitinib-hesperetin nano-cocrystals by combining crystal engineering and nanotechnology. The structure and physicochemical properties of the gefitinib-hesperetin co-crystals are characterized by a series of solid characterization techniques, and its in vitro dissolution, in vitro anti-tumor activity, and in vivo bioavailability in rats are systematically evaluated. It is shown that the gefitinib-hesperetin nano-cocrystals not only effectively improve the solubility and bioavailability of gefitinib, but also enhance the anti-tumor activity of the drug.

Description

Technical Field

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

[0002] Gefitinib (Gefitinib, Gef, chemical structure as Figure 1 (middle right image) is the world's first developed epidermal growth factor receptor tyrosine kinase inhibitor. It achieves its anti-tumor effect by binding to the tyrosine kinase region in the cell membrane, blocking the EGFR signaling and preventing EGFR-dependent cell proliferation. 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 its 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 Hesperetin (middle left image) is a naturally occurring dihydroflavonoid compound with multiple pharmacological activities such as anti-inflammatory and anti-tumor. From a structural analysis, hesperetin 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 the quinazoline of gefitinib, the O atom on the morpholine, the N atom, as well as -NH- and -OCH3, thereby synthesizing drug cocrystals.

[0004] Drug-drug cocrystals are supramolecular complexes composed of an active pharmaceutical ingredient (API) and a cocrystal former (CCF) with independent pharmacological activity, bound together through non-covalent interactions such as hydrogen bonding, halogen bonding, and π-π stacking. Drug nanocrystals are carrier-free colloidal drug dispersions less than 1000 nm in size and containing a small amount of stabilizer. Their high drug loading and enhanced solubility make them highly effective dosage forms for entering or transcellular delivery, achieving therapeutic concentrations. Summary of the Invention

[0005] The present invention aims to provide a method for preparing gefitinib-hesperetin nanocrystals and their use to address the aforementioned problems of the prior art. The present invention provides a method for preparing and using gefitinib nanocrystals, which improves in vitro dissolution and in vivo bioavailability in rats, and exhibits excellent in vitro antitumor activity against human lung cancer A549 cells.

[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 the triclinic system. The space group of the molecule is 1.57kJ / cm2, 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 using 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-crystal.

[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 15-45 Hz, and the grinding time is 30-60 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 jar, 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 above-mentioned gefitinib-hesperetin cocrystal 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 nanococrystal prepared by the above 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 hesperetin as the cocrystal former, and successfully obtains gefitinib-hesperetin nano-cocrystals by combining crystal engineering and nanotechnology. The structure and physicochemical properties of gefitinib-hesperetin cocrystals were 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 were systematically evaluated. It shows that gefitinib-hesperetin nano-cocrystals not only effectively improve the solubility and bioavailability of gefitinib, but also 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 following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

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

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

[0028] Figure 3 Schematic diagram of the three-dimensional structure of Gef-Hes prepared in Example 1;

[0029] Figure 4 1 is a PXRD comparison chart 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] Figure 9 IR spectra of Gef, Hes and Gef-Hes prepared in Example 1;

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

[0036] Figure 11 is the dissolution curve of Gef, Gef-Hes and Gef-Hes-NPs in pure water;

[0037] Figure 12 The drug-time curves of Gef, Gef+Hes, Gef-Hes and Gef-Hes-NPs;

[0038] Figure 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents 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 described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be 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 hesperetin were weighed and placed in a 25 mL grinding jar containing agate grinding balls. 150 μL of isopropanol was added and ground in a ball mill at a rate of 30 Hz for 30 min to obtain gefitinib-hesperetin cocrystal powder, recorded as Gef-Hes. The solvent was slowly evaporated to obtain gefitinib-hesperetin cocrystals.

[0046] Example 2

[0047] 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 containing agate grinding balls. 150 μL of methanol was added and ground in a ball mill at a rate of 30 Hz for 30 min to obtain gefitinib-hesperetin cocrystal powder, recorded as Gef-Hes. The solvent was slowly evaporated to obtain gefitinib-hesperetin cocrystals.

[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 containing agate grinding balls. 150 μL of dichloromethane was added and ground in a ball mill at a rate of 30 Hz for 30 min to obtain gefitinib-hesperetin cocrystal powder, recorded as Gef-Hes. The solvent was slowly evaporated to obtain gefitinib-hesperetin cocrystals.

[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, ultrasonication 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 h, 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, ultrasonication 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 h, 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, ultrasonication 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 h, 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, ultrasonication 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 h, 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, ultrasonication was performed to fully disperse the gefitinib-hesperetin cocrystals and 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 h, a gefitinib-hesperetin nano-eutectic suspension was obtained, which was recorded as Gef-Hes-NPs.

[0060] The gefitinib-hesperetin nanococrystal suspension prepared in Examples 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 the successful preparation of the gefitinib-hesperetin nanococrystal.

[0061] 1. Taking Example 1 as an example, characterization experiment of the prepared gefitinib-hesperetin 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. The dotted lines represent hydrogen bonds. The results show that the crystals of gefitinib-hesperetin cocrystal crystallize in the triclinic system. The space group of the drug cocrystal is monoclinic, and its asymmetric unit contains one gefitinib molecule and one hesperetin molecule. 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 hesperetin molecules, namely, the hydrogen atoms on the methoxyl group of gefitinib molecule and the oxygen atoms on the ketone carbonyl group of hesperetin molecule. Between the hydrogen atom on the hydroxyl group of the hesperetin molecule and the nitrogen atom on the quinoline ring of the gefitinib molecule Between the hydrogen atom on the ketone carbonyl group of the hesperetin molecule and the oxygen atom on the methoxyl group of the gefitinib molecule Between the hydrogen atom on the ether group of the hesperetin 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 atom on the imino group and the oxygen atom 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 hesperetin molecule and the oxygen atom on the ether group of another hesperetin 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 hesperetin 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 using a Cu target Kα ray 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). The data were processed using OriginPro 9.0 software.

[0067] Figure 4 The following is a comparison of the PXRD patterns of gefitinib, hesperetin, and the gefitinib-hesperetin cocrystal of Example 1. The results show that the gefitinib-hesperetin cocrystal has characteristic peaks at positions (2θ) of 7.29°, 8.30°, 9.97°, 12.61°, 13.25°, 14.51°, and 16.10°, indicating the formation of a new crystalline phase. Furthermore, the measured PXRD pattern of the gefitinib-hesperetin cocrystal is essentially consistent with the PXRD pattern 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. The results were analyzed 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-hesperetin cocrystal 1 H NMR (DMSO-d6) spectrum; by comparison, it can be seen that the chemical shifts of the characteristic peaks of gefitinib are as follows: 1 H NMR (500 MHz, DMSO-d6) δ9.57 (s, 1H), 8.50 (s, 1H), 8.12 (dd, J = 6.9, 2.6 Hz, 1H), 7.85-7.75 (m, 2H), 7.45 (t, J = 9.1 Hz, 1H), 7.20 (s, 1H), 4.18 (t, J = 6.3 Hz, 2H), 3.94 (s, 3H), 3.58 (t, J = 4.6 Hz, 4H), 2.50-2.30 (m, 6H), 2.00 (p, J = 6.6 Hz, 2H). The characteristic peak chemical shifts of hesperetin are as follows: 1 H NMR (500 MHz, DMSO-d6) δ 12.14 (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-hesperetin 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 gefitinib-hesperetin cocrystal is 1:1. Figure 7 It can be seen that except for the characteristic peaks of gefitinib and hesperetin 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 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-hesperetin 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 following are the TG and DSC spectra of gefitinib, hesperetin, and gefitinib-hesperetin cocrystals. The TG curves show that none of the gefitinib, hesperetin, or gefitinib-hesperetin cocrystals exhibit solvent loss at 100°C, indicating that these three cocrystals are pure solvent-free samples. The DSC spectra show that the gefitinib-hesperetin cocrystal exhibits a melting peak at 181.1°C, which differs from that of gefitinib (194.7°C) and hesperetin (230.9°C), further confirming the formation of a new crystalline phase. Furthermore, no other endothermic or exothermic peaks were detected before the melting point of gefitinib, hesperetin, or the gefitinib-hesperetin cocrystals, indicating that no other thermodynamic behavior occurred before melting.

[0078] 1.5 Infrared spectroscopy analysis

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

[0080] Figure 9 The IR spectra of gefitinib, hesperetin and gefitinib-hesperetin cocrystal are shown in Figure 1. As can be seen from the figure, gefitinib has a peak at 3403 cm -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 is at 3501cm in the infrared spectrum. -1 and 1639cm -1 The characteristic peaks at 1639 cm-1 correspond to the stretching vibrations of OH and C=O, respectively. After the formation of gefitinib-hesperetin cocrystal, the C=N stretching vibration of gefitinib shifts to 1639 cm-1. -1 , while the OH stretching vibration of hesperetin shifts to 3457 cm -1 , it can be inferred that the C=N of gefitinib and the OH of Hes participate in the formation of supramolecular synthon, which is consistent with the single crystal results.

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

[0082] Gefitinib, the gefitinib-hesperetin cocrystal prepared in Example 1, and the gefitinib-hesperetin nanococrystal 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 was taken (the gefitinib content in the three suspensions was the same) and added to a beaker containing 25 mL of pure water. 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] Figure 10 Figure 2 is the dissolution curve of gefitinib and gefitinib-hesperetin cocrystal in pure water. It can be seen that after the formation of gefitinib-hesperetin cocrystal, the solubility of gefitinib shows a significant dissolution rate compared with the raw material drug, which is about 49 times higher. Figure 11 Figure 2 shows the dissolution curves of Gef, Gef-Hes, and Gef-Hes-NPs in pure water. Figure 2 shows the dissolution curves of gefitinib, gefitinib-hesperetin cocrystal, and gefitinib-hesperetin cocrystal nanosystem detected in pure water. After the gefitinib-hesperetin cocrystal nanosystem was prepared, the solubility of gefitinib was further improved, and both exhibited a "spring-parachute" phenomenon, which plays an important role in improving drug solubility and dissolution rate.

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

[0085] 3.1 Plasma sample pretreatment

[0086] Plasma samples were processed using a protein precipitation method. 100 μL of plasma sample was added to 400 μL of methanol and vortexed for 30 seconds to denature the protein. The sample was then centrifuged at 12,000 rpm at 4°C for 15 minutes. The supernatant was nitrogen-purged and reconstituted by ultrasonication in 200 μL of methanol. 100 μL of the supernatant was then analyzed by HPLC.

[0087] 3.2 Chromatographic conditions

[0088] Gefitinib concentration in plasma was determined using an Agilent 1260 Infinity HPLC column (4.5 mm × 250 mm, 6 μm). The mobile phase consisted of methanol:0.1% triethylamine in water (75:25) at a flow rate of 1 mL / min, a detection wavelength of 330 nm, a column oven at 35°C, and an injection volume of 10 μL.

[0089] 3.3 Establishment of standard curve

[0090] Accurately weigh 10 mg of gefitinib API and dissolve completely in chromatographic methanol. Then, dilute to volume in a 100 mL volumetric flask to obtain a 100 μg / mL gefitinib stock solution. Accurately pipette different volumes of gefitinib stock solution into 10 mL volumetric flasks and dilute to volume with chromatographic methanol to obtain standard solutions with varying concentration gradients. Add these standard solutions to blank plasma and process according to the method in "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 in "3.2." Perform linear regression with gefitinib concentration as the horizontal axis and the corresponding peak area as the vertical axis to create a gefitinib standard curve.

[0091] 3.4 Pharmacokinetic studies

[0092] Twenty-five male Sprague-Dawley rats were randomly divided into five groups of five. All rats were fasted for 12 hours prior to the experiment. Gefitinib and gefitinib-hesperetin cocrystals were prepared as suspensions using sodium hydroxymethylcellulose (CMC-Na). Gefitinib suspension, gefitinib-hesperetin cocrystal suspension, and gefitinib-hesperetin cocrystal nanosuspension were administered orally at a dose of 20 mg / kg (based on gefitinib). Approximately 0.5 mL of blood was collected from the rats' orbits at 5, 15, 30, 45, 60, 90, 120, 240, 480, 720, and 1440 minutes after oral administration. The blood was placed in sodium heparin anticoagulant tubes and centrifuged at 8000 rpm at 4°C for 10 minutes. Plasma was collected from clean centrifuge tubes and stored at -80°C until further use. Blood was collected from SD rats in the blank group to prepare blank plasma for later use. The plasma samples were processed according to the method under "3.1" and the gefitinib content in the plasma was determined by HPLC. The experimental data were processed and analyzed using OriginPro 9.0.

[0093] Figure 12The concentration-time curves of Gef, Gef+Hes, Gef-Hes, and Gef-Hes-NPs are shown. Pharmacokinetic results show that the active ingredient gefitinib reached its maximum plasma concentration at 60 minutes, with a maximum concentration of 1.108±0.2611 μg / mL and an AUC value of 424.11. Gefitinib in the gefitinib + hesperetin physical mixture (Gef+Hes) reached its maximum plasma concentration at 60 minutes, with a maximum concentration of 1.510±0.432 μg / mL and an AUC value of 502.02. Gefitinib in the gefitinib-hesperetin cocrystal reached its maximum plasma concentration at 45 minutes, with a maximum concentration of 2.401±0.374 μg / mL and an AUC value of 718.46. Gefitinib in the gefitinib-hesperetin cocrystal nanosuspension reached its maximum blood concentration at 45 minutes, 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 gefitinib + hesperetin physical mixture 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 the drug multi-component crystals, and that the nanosuspension formulation was beneficial for promoting the oral absorption of gefitinib.

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

[0095] 4.1 Solution preparation

[0096] Preparation of complete culture medium: DMEM / F12 culture medium, fetal bovine serum (FBS), and penicillin-streptomycin mixture (double antibody) are prepared in a ratio of 90:10:1, mixed thoroughly, and stored in a 4°C refrigerator until use.

[0097] To prepare MTT solution: Weigh 250 mg of MTT powder into a 50 mL centrifuge tube and add 50 mL of phosphate buffered saline (PBS). Sonicate until fully dissolved. Sterilize the solution by filtering through a 0.22 μm filter and store in a refrigerator at 4°C until ready for use. Protect from light during preparation and storage.

[0098] 4.2 Cell recovery

[0099] Sterilize all materials in a clean bench for 30 minutes. Remove the cryovial of A549 cells from the -80°C freezer and place in a 37°C water bath. Thaw with rapid shaking and centrifuge at 1500 rpm for 3 minutes. Wipe the outside of the cryovial with an alcohol swab and quickly place it in the clean bench. Discard the supernatant, resuspend in fresh complete culture medium, transfer to a T25 cell culture flask, and culture in a 37°C, 5% CO2 incubator.

[0100] 4.3 Cell culture medium replacement

[0101] Sterilize all materials in a clean bench for 30 minutes under UV light. Remove the culture flask from the incubator, sterilize with alcohol, and place it in the clean bench. Discard the old culture medium, wash three times with PBS, add fresh complete culture medium, and continue culturing in a 37°C, 5% CO2 incubator. Subculture cells when the cell density reaches 80% to 90%.

[0102] 4.4 Cell passaging

[0103] Sterilize all materials in a clean bench for 30 minutes under UV light. Remove cells from the incubator and observe them under an inverted microscope to ensure they are growing well and free of contamination. After sterilizing with alcohol, place the cells in a clean bench. Discard the old culture medium, rinse with 3 mL of PBS, discard, and digest with 1 mL of trypsin-EDTA solution. Incubate in the incubator for 3 minutes. Once cells are observed to be spherical and no longer adhere to the wall, add an appropriate amount of complete culture medium to terminate digestion. Transfer the cell suspension to a centrifuge tube and centrifuge at 1500 rpm for 3 minutes. Discard the supernatant, then resuspend the cells in fresh complete culture medium in the centrifuge tube. Transfer the cells to a new T75 cell culture flask and continue culturing in a 37°C, 5% CO2 incubator.

[0104] 4.5 Cell inoculation

[0105] Place the materials to be used in a clean bench and sterilize them with UV for 30 minutes. Discard the old culture medium, wash with PBS, add trypsin to digest the cells, stop the digestion, centrifuge and discard the supernatant, add complete culture medium to resuspend, mix well, aspirate 10 μL and count on a cell counter, then take the cell resuspension and add an appropriate amount of complete culture medium, gently pipette 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, the gefitinib-hesperetin cocrystals prepared in Example 1, a physical mixture (gefitinib and hesperetin mixed in a 1:1 molar ratio), and the gefitinib-hesperetin nanococrystals prepared in Example 4 were prepared in DMSO solution at various concentrations and added to a 96-well plate, 100 μL per well, to achieve a final drug concentration gradient of 3.125, 6.25, 12.5, 25, 50, and 100 μM (DMSO content <0.1%). The 96-well plate was then placed in a 37°C, 5% CO2 incubator for 48 hours. A 0.1% DMSO control group and PBS were used as blank controls.

[0108] 4.7 MTT assay

[0109] Add 10 μL of MTT solution to each well of the incubated 96-well plate and incubate in an incubator for 4 hours. Discard the supernatant from the 96-well plate and add 100 μL of DMSO to each well. Shake on a shaker in the dark for 10 minutes to fully dissolve the crystals. Detect the cells on a microplate reader at 490 nm. Calculate the cell inhibition rate based on the absorbance: Cell inhibition rate = [1 - (OD value of the treatment group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)] × 100%.

[0110] Figure 13 The figure shows the comparison of the inhibition rate of gefitinib, gefitinib-hesperetin cocrystal, physical mixture and gefitinib-hesperetin cocrystal nanosystem on A549 tumor cells at 48 hours. Figure 13 It can be seen that compared with the gefitinib and physical mixture (Gef+Hes) groups, the gefitinib-hesperetin 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-hesperetin cocrystal and gefitinib-hesperetin cocrystal nanosystem groups increased in a concentration-dependent manner. These results indicate that the gefitinib-hesperetin cocrystal and its gefitinib-hesperetin nanococrystal system have excellent anti-tumor activity against A549 lung cancer cells.

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

Claims

1. A gefitinib-hesperetin cocrystal, characterized in that: The crystals of the gefitinib-hesperetin cocrystal are crystallized in the triclinic system of P The space group of , whose asymmetric unit contains one gefitinib molecule and one hesperetin molecule; The unit cell parameters are: axis length a = 12.2043(4)Å, b = 12.2206(4)Å, c = 13.3256(4)Å, axis angle α = 66.456(3)°, β = 85.878(2)°, γ = 72.606(3)°; unit cell volume V = 1736.10(11)Å 3 .

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, adding a solvent dropwise, and grinding to obtain a gefitinib-hesperetin cocrystal.

4. The method for preparing gefitinib-hesperetin cocrystal according to claim 3, wherein: The molar ratio of gefitinib to hesperetin is 1:1; the solvent is selected from one or more of isopropyl alcohol, methanol and dichloromethane; the grinding rate is 30 Hz, and the grinding time is 30 to 60 minutes.

5. A method for preparing gefitinib-hesperetin nano-cocrystals, characterized in that: The gefitinib-hesperetin cocrystal and the stabilizer according to claim 1 are placed in a grinding jar, ultrapure water is added, and ultrasound is performed until the gefitinib-hesperetin cocrystal and the stabilizer are evenly dispersed. Grinding beads are added, the rotation speed is adjusted, and the gefitinib-hesperetin nanococrystal is 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, 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 cocrystal according to claim 1 in preparing a drug for treating A549 lung cancer.

9. Use of gefitinib-hesperetin nanococrystal 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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