CN111454221B

CN111454221B - Gefitinib and bumetanide drug cocrystal and preparation method thereof

Info

Publication number: CN111454221B
Application number: CN202010315091.3A
Authority: CN
Inventors: 江燕斌; 蒋云鹏; 吴艳婷; 宋阳
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2020-04-21
Filing date: 2020-04-21
Publication date: 2023-01-06
Anticipated expiration: 2040-04-21
Also published as: CN111454221A

Abstract

The invention discloses a gefitinib and bumetanide drug cocrystal and a preparation method thereof; the molar ratio of gefitinib to bumetanide in the drug cocrystal is 1, and an X-ray diffraction spectrogram expressed by 2 theta has characteristic peaks at 5.08 +/-0.2 degrees, 8.88 +/-0.2 degrees, 10.26 +/-0.2 degrees, 10.56 +/-0.2 degrees, 17.78 +/-0.2 degrees, 19.29 +/-0.2 degrees, 19.68 +/-0.2 degrees, 20.04 +/-0.2 degrees, 21.47 +/-0.2 degrees and 22.70 +/-0.2 degrees; compared with gefitinib active compound, the gefitinib active compound has better storage stability, solubility and dissolution rate. The result of cytotoxicity experiment shows that the cocrystal has better inhibition effect on cancer cells, and the toxic and side effect on normal cells is obviously reduced. The preparation method of the eutectic is environment-friendly, stable in yield and easy to industrialize.

Description

Gefitinib and bumetanide drug cocrystal and preparation method thereof

Technical Field

The invention relates to a gefitinib medicament, in particular to a gefitinib and bumetanide medicament cocrystal and a preparation method thereof, belonging to the technical field of medicinal chemistry.

Background

Gefitinib (GFB), also known as Iressa, was developed by Astrazeneca, USA, and a biologica classification system belonging to BCS class II with high permeability and low solubility, molecular formula C ₂₂ H ₂₄ ClFN ₄ O ₃ Molecular weight 446.9, chemical structure shown in formula 1 below. Gefitinib, a selective epidermal growth factor tyrosine kinase (EGFR-TK) inhibitor, inhibits EGFR-TK, blocks tumor growth, metastasis and angiogenesis, promotes apoptosis of tumor cells, and is therefore commonly used for treating locally advanced or metastatic non-small cell lung cancer (NSCLC) that has been previously treated with chemotherapy or is otherwise not suitable for chemotherapy. Meanwhile, the gefitinib also has the defects of slow absorption, poor targeting property and low bioavailability when being used for anti-tumor treatment,the side effects such as edema, nephrotoxicity, hypertension and the like are caused by excessive administration.

Bumetanide (BUM), also known as butylamine furosemide, of formula: c ₁₇ H ₂₀ N ₂ O ₅ S, the molecular weight is 361.4, and the chemical structure is shown in formula 2. Is insoluble in water, and can be dissolved in organic solvent such as ethanol and acetone. Bumetanide is a powerful quick-acting diuretic, is mainly used for clinically treating heart failure, liver diseases, renal disease edema including various refractory edema and acute pulmonary edema, and is particularly suitable for patients with acute renal failure.

Pharmaceutical Co-crystals are crystals in which an Active Pharmaceutical Ingredient (API) and a Co-crystal former (CCF) are bonded by hydrogen bonding or other non-covalent bonds (van der waals force, pi \8230; pi stacking effect, halogen bond, etc.). The drug cocrystal is designed and identified corresponding supramolecular synthons based on the principle of crystal engineering to form an expected structure, and the interaction between molecules in the drug cocrystal can be changed through the design of the crystal engineering, so that non-covalent bonds between the molecules are opened and generated, and the lattice accumulation mode of the solid drug is changed. The pharmaceutical co-crystal is a novel pharmaceutical solid form, and can improve the physicochemical properties of the drug, such as stability, melting point, solubility, hygroscopicity, metabolic stability, dispersion rate, dissolution rate, drug sustained release, mechanical properties, bioavailability and the like. The maximum application value of the cocrystal in pharmacy is that the physicochemical property of the cocrystal can be changed without changing the covalent structure of a drug. The co-crystal is a potential solid form, and has profound effects on pre-prescription research and dosage form design of the medicine.

Gefitinib belongs to a BCS II drug of a biological preparation classification system, has lower solubility, and causes lower bioavailability. As an anti-tumor chemotherapeutic drug, gefitinib taken for a long time is easy to generate serious side effects such as edema, nephrotoxicity, hypertension and the like. Based on the reasons, in order to improve the solubility and bioavailability of gefitinib, reduce the side effect of the gefitinib on a patient, the gefitinib multi-drug cocrystal is screened and prepared, so that the stability of the gefitinib can be enhanced, the bioavailability of the gefitinib is improved, and the side effect caused by long-term administration of the gefitinib is reduced.

Currently, in US20050209229A1, forms 1 crystal Form of gefitinib, forms 2 ZD1839MeOH solvate, forms 3 ZD1839 DMSO solvate, forms 5 ZD1839 trihydrate, powder diffraction (PXRD), integrated thermal analysis (DSC & TGA) and fourier transform infrared spectroscopy (FTIR) spectra of four crystal forms (Form 1, form2, form3, form5 for short) are disclosed, and preparation methods and mutual conversion methods of the four crystal forms are disclosed. The Chinese patent application CN110283131A discloses a co-crystal methanol solvate of gefitinib and vanillic acid and a preparation method thereof, although the co-crystal solvate improves the physicochemical property of a medicament to a certain extent, the vanillic acid as a pesticide intermediate has no actual medicinal value, and simultaneously, a methanol organic solvent also exists in the product. International patent W2014016848A2 discloses a preparation method of cocrystals of gefitinib and Imatinib (Imatinib) with caffeic acid, fumaric acid and the like, but there is no data indicating that these cocrystals can improve the physicochemical properties of drugs.

In summary, the existing reports mainly relate to researches on gefitinib polymorphic forms and solvates and on the aspect of improving drug solubility, and no research report on gefitinib multi-drug cocrystal exists.

Disclosure of Invention

The invention aims to provide a gefitinib and bumetanide drug cocrystal and a preparation method thereof.

The invention aims at the problems of low solubility, poor stability, poor reproducibility in the preparation process and excessive organic solvent residue existing in the BCS II medicament of the existing gefitinib crystal form belonging to a biological preparation classification system, and simultaneously has larger side effect when being taken too much as a novel antitumor medicament. The gefitinib and bumetanide co-crystal designed by the invention can replace the existing crystal forms, the co-crystal has better chemical stability, and the phenomenon that the crystal forms of the existing crystal forms are transformed or solvate is formed is effectively avoided through the formation of the co-crystal; meanwhile, gefitinib and a diuretic bumetanide are combined to form a eutectic, so that the safety of each component in the medicament is ensured, the effect of drug combination can be achieved, the formation of the eutectic improves the solubility of the medicament, and in-vitro anti-tumor experiments show that the eutectic has better cancer cell inhibition effect compared with bulk drugs. Especially has unexpected effect on relieving the toxic and side effect of the medicine.

The purpose of the invention is realized by the following technical scheme:

a gefitinib and bumetanide drug cocrystal, wherein the molar ratio of gefitinib to bumetanide in the cocrystal is 1; an X-ray diffraction spectrum expressed by 2 theta by using Cu-Kalpha radiation has characteristic peaks at 5.08 +/-0.2 degrees, 8.88 +/-0.2 degrees, 10.26 +/-0.2 degrees, 10.56 +/-0.2 degrees, 17.78 +/-0.2 degrees, 19.29 +/-0.2 degrees, 19.68 +/-0.2 degrees, 20.04 +/-0.2 degrees, 21.47 +/-0.2 degrees and 22.70 +/-0.2 degrees.

A DSC curve of the gefitinib and bumetanide drug cocrystal is measured by differential scanning calorimetry, an endothermic peak exists, the corresponding temperature range is 135.9-195.4 ℃, and the peak value is 168.4 ℃.

The preparation method of the gefitinib and bumetanide co-crystal comprises the following steps: uniformly mixing gefitinib and bumetanide according to a molar ratio of 1.

To further achieve the object of the present invention, preferably, the organic solvent is selected from one or more of ethanol, acetonitrile, methanol, acetone and ethyl acetate.

Preferably, the mass-to-volume ratio of the gefitinib to the added organic solvent A is 50.

Preferably, the grinding time is 30-60 min, and the water bath temperature is 50-70 ℃.

Preferably, the step of uniformly mixing is to add gefitinib and bumetanide into a centrifuge tube and mix the gefitinib and bumetanide in a vortex mixer until uniform.

Compared with the prior art, the invention has the following advantages

1) The invention combines the anti-tumor drug gefitinib and the diuretic bumetanide to form the drug eutectic, can achieve the effect of drug combination, reduces the toxic and side effect of the drug obviously better than the scheme of stopping the drug or reducing the drug dosage, and simultaneously the eutectic better inhibits cancer cells. 2) The gefitinib and bumetanide medicine cocrystal disclosed by the invention overcomes the problems of poor reproducibility and excessive organic solvent residue in the medicine preparation process.

3) The gefitinib and bumetanide drug cocrystal disclosed by the invention improves the solubility and dissolution rate of the bumetanide bulk drug.

4) The gefitinib and bumetanide co-crystal disclosed by the invention avoids the problem that a single drug crystal form is subjected to crystal form conversion or solvate and hydrate are formed through the form of the co-crystal.

5) The preparation method of the technical scheme of the invention has simple and convenient operation, the raw materials and the used reagents are commercially available, the environmental pollution is less, and the industrial production is easy.

Drawings

Figure 1 is an ORTEP plot of gefitinib and bumetanide co-crystals.

Fig. 2 shows PXRD patterns measured and simulated by gefitinib and bumetanide co-crystal experiments, respectively.

Figure 3 is a DSC profile of gefitinib and bumetanide co-crystal.

Figure 4 is a DSC profile of gefitinib, bumetanide and gefitinib and bumetanide co-crystals.

Figure 5 is an FT-IR spectrum of gefitinib, bumetanide, and co-crystals of gefitinib and bumetanide.

Fig. 6 PXRD patterns of gefitinib and bumetanide co-crystal before and after one month of placement in a constant temperature and humidity chamber at a temperature of 40 ℃ and a relative humidity of 75%.

Fig. 7 is a graph of the release profiles of gefitinib, gefitinib and bumetanide co-crystal in hydrochloric acid solution PH = 3.0.

Fig. 8 is a release profile of bumetanide from bumetanide gefitinib and bumetanide co-crystal in hydrochloric acid solution PH = 3.0.

FIG. 9 is a graph of the results of toxicity tests on HACAT normal cells of gefitinib drug substance and gefitinib and bumetanide co-crystal.

Fig. 10 is a graph of the results of a toxicity test of gefitinib drug substance and gefitinib and bumetanide co-crystal on a549 carcinoma cells.

Detailed Description

For better understanding of the present invention, the present invention will be further described with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto.

Example 1

Preparing gefitinib and bumetanide pharmaceutical co-crystal: accurately weighing 89.4mg of gefitinib and 72.3mg of bumetanide in a 4mL centrifuge tube, placing the gefitinib and the bumetanide in a vortex mixer for mixing for 10min, placing the mixed sample in a mortar, dropwise adding 1mL of ethanol, grinding the mixture to white powder, adding 10mL of ethanol, transferring the solution into a 25mL beaker, heating the beaker in a 70 ℃ water bath for 10min to fully dissolve the solution, filtering the solution by using a microporous filter membrane with the aperture of 2.5 mu m, culturing and standing the filtrate to volatilize a solvent to separate out crystals for 72h, filtering and drying the crystals to obtain a massive transparent crystal product meeting the single crystal diffraction requirement, and carrying out a single crystal diffraction experiment on the massive transparent crystal product to determine the crystal structure of the product.

Gefitinib and bumetanide co-crystals were measured using an Agilent Technology Supernova, dual, cu at home/near, atlas 2 diffractometer at a crystal test temperature of 295 (1) K with Cu/Ka radiation. The structure was solved with Olex2 and refined using least squares.

FIG. 1 is an ORTEP diagram of a gefitinib and bumetanide co-crystal, and the solved crystal data is imported into Platon software, and a proper ellipsoid degree is selected, and then the ORTEP diagram of the crystal is exported. The type and number and relative position of molecules in the asymmetric unit of the crystal can be visually shown in figure 1, and the conformation of the molecules in the crystal can be displayed at the same time, and the figure can more visually display the crystal structure.

The crystallographic data of the obtained gefitinib and bumetanide co-crystal are analyzed as shown in table 1, and the main crystallographic parameters are as follows: monoclinic system, chiral space group is P-1, unit cell parameters are

b =12.2067 (5), c =17.3665 (12), α =93.522 (4) °, β =92.265 (5) °, γ =110.630 (4) °, and the crystal volume is

The crystal size is 0.28X 0.26X 0.06mm ³ 。

TABLE 1 Primary crystallographic data for Gefitinib and bumetanide drug cocrystals

The characteristic peaks in the corresponding X-ray diffraction patterns (Cu/Ka) of the gefitinib and bumetanide cocrystal are detailed in the following table 2 and the attached figure 2.

TABLE 2 PXRD peaks for Gefitinib and bumetanide drug cocrystals

The gefitinib and bumetanide pharmaceutical co-crystal obtained in example 1 is characterized by X-ray powder diffraction (PXRD), an instrument is Bruker D8 Advance in Germany, and an obtained PXRD spectrum is shown in an attached figure 2, wherein a is the PXRD spectrum obtained in the sample experiment in example 1, b is the PXRD spectrum simulated by solving the obtained crystal structure, and the positions of characteristic peaks of the a spectrum and the b spectrum are basically consistent by comparing the a spectrum and the b spectrum, which proves that the obtained product is the gefitinib and bumetanide pharmaceutical co-crystal.

Figure 3 is a DSC profile of gefitinib and bumetanide co-crystal. The graph is measured by a TA DSC250 differential scanning calorimeter, the differential scanning calorimetry analysis technology is to explore the change of a physical quantity Delta H with temperature under the programmed temperature control, and the DSC technology is to measure the energy difference required by maintaining a sample and a reference substance at the same temperature and reflect the change of enthalpy of the sample. By carrying out thermal analysis on the sample, the melting point of the crystal can be determined, so that different crystal forms of the crystal can be distinguished.

Figure 4 is a DSC profile of gefitinib, bumetanide and gefitinib and bumetanide co-crystals. In the figure, a and b represent a technical gefitinib and a technical bumetanide respectively, and c is a gefitinib and bumetanide pharmaceutical co-crystal prepared by the invention. Under the test conditions of a nitrogen atmosphere, the melting endothermic peak temperature of gefitinib is 195.7 ℃, the melting endothermic peak temperature of bumetanide is 235.7 ℃, the eutectic endothermic peak temperature of gefitinib and bumetanide physically mixed is 153.9 ℃, and the melting endothermic peak temperature of gefitinib and bumetanide eutectic is 168.4 ℃. The formation of a new phase can be demonstrated from the difference in melting peaks, demonstrating the formation of co-crystals of gefitinib and bumetanide drug.

Infrared spectroscopy (FT-IR) was performed on the gefitinib and bumetanide co-crystal obtained in example 1 using Bruker TENSOR 27 from Germany to obtain an infrared absorption spectrum as shown in FIG. 5. In the figure, a and b represent the original gefitinib and the original bumetanide respectively, and c is the gefitinib and bumetanide pharmaceutical co-crystal prepared in example 1. Comparing the corresponding infrared absorption spectrograms to find that the infrared spectrogram of two original drugs after physical mixing is the superposition of the infrared spectrograms of two single original drugs and the red of the eutecticThe external spectrum and two single original drug infrared spectra are in 1850-1600cm ^-1 C = O double bond stretching vibration region and 3650-3400cm ^-1 The obvious difference exists in the hydrogen bond stretching vibration area, which shows that hydrogen bonds are formed between the original drug gefitinib and the original drug bumetanide, so that the infrared spectrum absorption is correspondingly changed, and the formation of the eutectic of the gefitinib and the bumetanide drug is proved.

Fig. 6 PXRD patterns of a gefitinib and bumetanide co-crystal before and after one month's storage in a constant temperature and humidity chamber at a temperature of 40 ℃ and a relative humidity of 75%. Weighing a certain amount of sample to be tested, placing the sample to be tested in a constant temperature and humidity box with the temperature of 40 ℃ and the humidity of 75%, storing for 30 days, and comparing the crystal form change before and after the sample through a PXRD test result. The stability of the crystal product can be obtained by comparing PXRD spectrums of the test samples before and after the experiment.

Example 2

Preparing gefitinib and bumetanide pharmaceutical co-crystal: accurately weighing 89.4mg of gefitinib and 72.3mg of bumetanide in a 4mL centrifuge tube, placing the gefitinib and the bumetanide in a vortex mixer for mixing for 10min, placing the mixed sample in a mortar, dropwise adding 1mL of methanol, grinding the mixture to white powder, adding 10mL of methanol, transferring the solution into a 25mL beaker, heating the beaker in a water bath at 70 ℃ for 10min for fully dissolving, filtering the solution by using a microporous filter membrane with the aperture of 2.5 mu m, culturing the filtrate, standing the solvent, volatilizing the solvent to separate out crystals for 72h, filtering and drying to obtain an irregular crystal product.

As shown in the PXRD pattern of fig. 2, the PXRD diffraction peaks obtained in example 2 and example 1 are substantially consistent, which indicates that both the crystal products obtained in example 2 and example 1 are the same cocrystal, as shown in the DSC pattern of fig. 4 and the FT-IR pattern of fig. 5, the solid obtained in example 2 and example 1 have similar endothermic peak positions and infrared spectrum characteristics, and further indicates that the solid obtained in this example is also a gefitinib and bumetanide pharmaceutical cocrystal.

Example 3

As shown in the PXRD pattern of fig. 2, the PXRD diffraction peaks obtained in example 3 and example 1 are substantially consistent, which indicates that both the crystal products obtained in example 3 and example 1 are the same cocrystal, as shown in the DSC pattern of fig. 4 and the FT-IR pattern of fig. 5, the example 3 and example 1 have similar endothermic peak positions and infrared spectrum characteristics, and also indicates that the solid obtained in example 3 is a gefitinib and bumetanide pharmaceutical cocrystal.

Stability test

The stability is an important index of the pharmaceutical preparation, provides scientific basis for the production, packaging, storage and transportation conditions of the medicine, and can establish the validity period of the medicine according to the test. In the experiment, the gefitinib and bumetanide eutectic high-quality crystal product obtained in the embodiment 1 is taken for stability experiment, and the specific test method is as follows: 40mg of gefitinib and bumetanide co-crystal product obtained in example 1 is weighed and placed in a constant temperature and humidity chamber with the temperature of 40 ℃ and the humidity of 75% for one month, and then PXRD is used for representing whether the sample has crystal form change or not. Fig. 6 shows PXRD patterns before and after stability experiments of gefitinib and bumetanide eutectic samples in example 1 under different conditions, and the results show that after the stability experiments, the characteristic peak intensity of the PXRD pattern of the sample is not obviously changed after the sample is placed in a constant temperature and humidity chamber with the temperature of 40 ℃ and the relative humidity of 75% for one month, which indicates that the prepared gefitinib and bumetanide co-crystal drug are not subjected to crystal transformation and have excellent stability.

Solubility test

Experimental determination of the solubility of the high quality gefitinib and bumetanide co-crystal product obtained in example 1 in hydrochloric acid solution PH =3.0 was determined as follows: (1) Taking an excess of gefitinib and bumetanide co-crystal sample of example 1 and a 15ml test tube, adding 5ml of buffer solution; (2) Placing the test tube into a constant-temperature oscillation box with the preset temperature of 37 ℃ and the rotating speed of 100 rpm; (3) After shaking for 24h, 2ml of the solution was filtered through a 0.22 μm membrane and quantified by HPLC. The solubility of the gefitinib active drug in a sodium acetate-acetic acid buffer solution with the pH =5.0 is 258 mug/ml through experiment; bumetanide is 9 mug/ml; the solubility of gefitinib in the gemfibrozil co-crystal of gefitinib and bumetanide is 187 mug/ml, and the solubility of bumetanide is 16 mug/ml, which shows that the solubility of the gemfibrozil co-crystal of gefitinib and bumetanide is obviously enhanced.

Dissolution Rate test

A fixed amount of the gefitinib and bumetanide co-crystal product obtained in example 1 was weighed into a dialysis bag containing 3ml of hydrochloric acid solution (P = 3.0), and the dialysis bag was placed into a brown sealed bottle containing 27ml of buffer solution, the brown bottle was placed in a constant temperature shaking chamber set at a predetermined rotation speed of 100rpm and a temperature of 37 ℃, and then 2ml of release medium was taken out of the container at fixed time intervals, and 2ml of buffer solution was replenished, and after filtration with a 0.22 μm filter, quantitative analysis was performed by liquid chromatography. From fig. 7, curve a is the release profile of gefitinib bulk drug substance, and curve b is the release profile of gefitinib in the co-crystal of gefitinib and bumetanide obtained in example 1; in fig. 8, the a-curve is the release curve of bumetanide bulk drug, and the b-curve is the release curve of bumetanide in co-crystal of gefitinib and bumetanide obtained in example 1; it is evident from fig. 8 that the gefitinib drug substance has slightly higher solubility and dissolution rate than gefitinib and bumetanide co-crystal, probably because the interaction force between the co-crystal molecules formed is stronger than the interaction force between gefitinib molecules, resulting in decreased gefitinib solubility and slower dissolution rate in the co-crystal. In contrast, it can be seen from fig. 8 that the bumetanide drug substance has a lower solubility than the co-crystal, since the hydrogen bonding between bumetanide molecules is stronger than the co-crystal, resulting in a faster solubility and dissolution rate of the co-crystal. The dissolution rate experiment shows that the solubility and dissolution rate of gefitinib in the gefitinib and bumetanide co-crystal are slightly reduced, and the gefitinib and bumetanide co-crystal can be used for preparing a new controlled-release medicament dosage form. Meanwhile, the solubility of bumetanide is improved, and the bumetanide can be used for preparing better and faster bumetanide medicament dosage forms.

Cytotoxicity test

The gefitinib and bumetanide CO-crystal product obtained in example 1 was subjected to cytotoxicity test using HACAT normal cells and A549 cancer cells, and the cells were put in RPMI1640 medium and added with 10% (v/v) fetal bovine serum and penicillin (100U/mL), streptomycin (100. Mu.g/mL), and then put in a humidified incubator at 37 ℃ with CO maintained ₂ The concentration is 5%, then the cells are inoculated into 96 well plates with the concentration of 5 × 104/mL, the 96 well plates are placed into an incubator to be cultured for 24h, the cultured cells are washed clean by fresh media, the gefitinib and the bumetanide cocrystal with the same amount and different concentrations and the gefitinib serving as the raw material are added into each well, the mixture is placed into the incubator to be cultured for 48h, finally 10 μ LWST-8 is added into each well of the 96 well plates, the mixture is placed into the incubator to be cultured for two hours, the absorbance of each well is measured at 450nm by a microplate reader, the background value at 630nm is subtracted, and the relative survival rate of the cells is measured by an absorbance method. As can be seen from fig. 9, the HACAT normal cells cultured by the gefitinib and bumetanide co-crystal product have higher cell viability rate than the gefitinib raw material drug, and the normal cell viability rate is above 95%, which indicates that the damage of the gefitinib and bumetanide co-crystal to the HACAT normal cells is less than that of the gefitinib raw material drug. This may be due to the formation of gefitinib co-crystals that cause a change in the structure of the drug, resulting in a slower release of the drug and thus improved side effects of the drug. Meanwhile, the introduction of bumetanide accelerates the drug metabolism, and the damage of the drug to cells is reduced.

At present, the incidence of lung cancer in malignant tumors is much higher than that of other cancers, and the incidence rate accounts for about 20% of all cancers. Chemotherapy is still the main means for treating cancer, chemotherapeutic drugs also injure normal cells of a host while killing tumor cells, and the current scheme for relieving the toxic effect of the drugs mainly adopts a method of stopping the drugs or reducing the dosage of the drugs, which undoubtedly reduces the antitumor activity of the drugs. The toxic and side effects of the gefitinib and bumetanide co-crystal on normal HACAT cells are reduced by about 5 percent, so that the problem that the toxic effect of the gefitinib serving as a chemotherapeutic drug for treating lung cancer on normal cells is large and the patient is deeply influenced by the side effects of the drug after long-term administration is solved to a great extent. The article "liver toxicity and side effects of antitumor drugs and treatment strategy" published by Zhaolin et al in the academic journal of cancer progression on 2009, 1, 7, volume 1, indicates that the side effects of antitumor drugs on liver damage are firstly drug withdrawal or reduction, and then the next treatment strategy is determined according to the degree of liver damage. According to the graph 9, the drug concentration is reduced, the toxicity of the gefitinib raw drug substance and the co-crystal product to HACAT cells is obviously reduced, and the cell survival rate is obviously improved, but the comparison shows that the cell survival rate of the gefitinib and bumetanide co-crystal is always better than that of the gefitinib raw drug substance, especially the cell survival rate of the gefitinib and bumetanide co-crystal drug concentration of 100 mug/mL is equivalent to that of the gefitinib raw drug substance of 10 mug/mL. The normal use dose of the gefitinib is 250mg per day, under the condition of ensuring the curative effect of the medicament, the effect of reducing the medicament dose obviously cannot achieve the effect of relieving the toxic and side effect of the gefitinib raw material medicament and the cocrystal on HACAT, and under the condition of not reducing the medicament dose of taking 250mg of the medicament per day, the toxic and side effect of the cocrystal is far lower than that of the gefitinib raw material medicament. The invention has particularly obvious effect on relieving the toxic and side effect of the medicine.

It is obvious from fig. 10 that a549 cancer cells cultured by gefitinib and bumetanide co-crystal have lower cell survival rate compared with gefitinib bulk drug, when the drug concentration is 0, the cell survival rate of cancer cells proliferated faster reaches 106%, and when the co-crystal concentration reaches 100 μ g/mL, the cell survival rate of cancer cells is less than 50%. This indicates that the inhibitory effect of gefitinib and bumetanide co-crystal on cancer cells is more significant. The reason is probably that the formation of the co-crystal changes the crystal form of the drug, and the co-crystal has better controlled and sustained release effect, so that the co-crystal has better inhibition effect on cancer cells.

In conclusion, the gefitinib and bumetanide co-crystal has a better inhibition effect on lung cancer cells A549, simultaneously reduces the toxic and side effect on human normal cells by more than 5%, enhances the antitumor activity of the medicament, improves the toxic and side effect of chemotherapeutic medicaments, and has wider application in lung cancer chemotherapy schemes.

The tests of the figures are all performed on the gefitinib and bumetanide co-crystal obtained in example 1 and the gefitinib and bumetanide technical substances, and the test results of the gefitinib and bumetanide co-crystal obtained in examples 2 and 3 are basically consistent and are not necessarily illustrated.

The above-described embodiments are intended to illustrate rather than to limit the invention, and any insubstantial modifications and variations of the invention within the spirit of the invention and the scope of the claims are intended to fall within the scope of the invention.

Claims

1. A gefitinib and bumetanide drug cocrystal, wherein the molar ratio of gefitinib to bumetanide in the cocrystal is 1; an X-ray diffraction spectrum expressed by 2 theta by using Cu-Kalpha radiation has characteristic peaks at 5.08 +/-0.2 degrees, 8.88 +/-0.2 degrees, 10.26 +/-0.2 degrees, 10.56 +/-0.2 degrees, 17.78 +/-0.2 degrees, 19.29 +/-0.2 degrees, 19.68 +/-0.2 degrees, 20.04 +/-0.2 degrees, 21.47 +/-0.2 degrees and 22.70 +/-0.2 degrees.

2. The gefitinib and bumetanide drug cocrystal of claim 1, wherein differential scanning calorimetry is used to determine the DSC curve for the gefitinib and bumetanide drug cocrystal, which has an endothermic peak at a temperature in the range of 135.9-195.4 ℃ and a peak at 168.4 ℃.

3. The preparation method of gefitinib and bumetanide co-crystal as claimed in claim 1 or 2, wherein gefitinib and bumetanide are uniformly mixed according to molar ratio of 1.

4. The method according to claim 3, wherein the organic solvent is one or more selected from the group consisting of ethanol, acetonitrile, methanol, acetone and ethyl acetate.

5. The preparation method according to claim 3, wherein the mass-to-volume ratio of gefitinib to the added organic solvent A is 50.

6. The method of claim 3, wherein the milling time is 30 to 60min and the water bath temperature is 50 to 70 ℃.

7. The method of claim 3, wherein the step of mixing is performed by adding gefitinib and bumetanide to a centrifuge tube and mixing in a vortex mixer until homogeneous.