A pharmaceutical co-crystal of cytarabine and 5-fluorouracil and a preparation method thereof

Abstract

The application relates to a pharmaceutical cocrystal of cytarabine and 5-fluorouracil and a preparation method thereof, and relates to the technical field of pharmaceutical cocrystals. The pharmaceutical cocrystal is composed of one cytarabine molecule and one 5-fluorouracil molecule as a basic structural unit, and has a chemical formula of [C9H 13 N3O5.C4H3N2O2F]; the pharmaceutical cocrystal belongs to an orthorhombic system and has a space group of P212121. The pharmaceutical cocrystal prepared by a solvent evaporation method and a cooling method improves the permeability of cytarabine, appropriately reduces the solubility of cytarabine, and significantly improves the antitumor activity of cytarabine; through the complementary advantages of the properties and the pharmacodynamic effects of two components, the synergistic antitumor effects of cytarabine and 5-fluorouracil are realized, and a new idea is provided for developing synergistic antitumor pharmaceutical cocrystals. The pharmaceutical cocrystal can keep the skeleton structure of the crystal unchanged after long-term placement at room temperature, the preparation method is simple and easy to implement, the cost is low, and the pharmaceutical cocrystal is convenient for large-scale production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical cocrystals, and particularly relates to a pharmaceutical cocrystal of cytarabine and 5-fluorouracil and a preparation method thereof. BACKGROUND

[0002] With the development of economy and the improvement of living standards, the incidence and mortality of cancer are rising, which has become the second major disease threatening human health. In order to cope with these challenges, more and more researchers are committed to the creation of anti-tumor drugs and the modification of existing drugs. However, so far, the demand for anti-tumor drugs in the clinic cannot be met. It is well known that due to the particularity of marine ecological environment, marine organisms have rich species diversity, novel chemical structures and diverse biological activities, which have become an important resource for finding new drugs or lead compounds with high activity. Therefore, the research and development of marine anti-tumor drugs has become a hot field of drug research in recent decades. Cytarabine (ARC) as the first marine anti-tumor drug is one of the milestones in the research of marine anti-tumor drugs. In 1969, ARC was approved by the FDA for the treatment of acute lymphoblastic leukemia, acute granulocytic leukemia, etc. Subsequently, it was found that ARC also had certain curative effect on malignant lymphoma, lung cancer, colon cancer, head and neck cancer. However, ARC has large molecular polarity, poor lipid solubility, and is not easy to transport across the membrane, which hinders the exertion of curative effect to a certain extent; in addition, the short plasma elimination half-life of ARC limits its retention time in the body, and frequent large-dose administration is required to maintain the curative effect, resulting in poor patient compliance; more seriously, it is easily metabolized by deaminase to become inactive arabinosyluridine, which makes its bioavailability very low. Therefore, domestic and foreign scholars have developed various strategies to improve the anti-cancer activity of ARC, including pharmaceutical methods, chemical structure modification, etc. Although these strategies have made certain research progress in improving the physicochemical properties and efficacy of ARC, the complex process technology, high cost and uncontrollable by-products limit their practical application. With the continuous in-depth research, domestic and foreign scholars focus on fixed-dose combination (FDC), which combines ARC with other drugs (such as methotrexate, romidepsin, mitoxantrone, etc.) to resist tumor cells, so as to improve the curative effect. However, due to the simple physical mixing of FDC to mix the related components together, the compatibility of the physicochemical properties of the combined drugs is poor, and it is difficult to exert the ideal effect of combined therapy, which hinders the further application and development to a certain extent. Therefore, it is of outstanding theoretical significance and practical value to introduce new drug research concepts and technical means, to find a new method to improve the physicochemical properties of ARC without changing its covalent structure, and to realize the combination at the molecular level to achieve synergistic effect.

[0003] Drug co-crystal technology, as an important branch of crystal engineering, is widely considered as one of the effective ways to improve the drug. Compared with the traditional drug modification technology, the drug co-crystal technology can improve the physical and chemical properties, efficacy and toxicity of the drug without changing the covalent structure and pharmacological behavior of the drug. With its unique advantages, the design, preparation and application of drug co-crystals in the field of pharmacy have attracted widespread attention from academia, industry and drug regulatory authorities. At present, in order to improve the physical and chemical properties of the raw material, most of the researches focus on the self-assembly of the active pharmaceutical ingredient (API) and the pharmacologically acceptable co-crystal former (CCF). With the in-depth research, researchers found that API can also be used as CCF to form "drug-drug" co-crystals with other APIs, which not only can adjust the pharmaceutical properties of API, but also can provide a new perspective for molecular level combination therapy. However, the research of ARC drug-drug co-crystals seems extremely difficult, and so far only a few ARC molecular salts have been reported. The research of ARC co-crystals with clear structure has been blank. The reason may be that ARC molecules are weakly basic and more likely to be salted with acidic substances, and the sugar ring contained in the ARC structure has multiple conformations, which to some extent hinders the co-crystallization process with other drugs. Therefore, it is of great practical significance to design and prepare drug co-crystals by co-crystallization technology, which combines drugs with complementary structures and the same pharmacological activity in the same crystal lattice to achieve synergistic therapy at the molecular level. 5-fluorouracil (FU) is a commonly used antimetabolite drug, which has therapeutic effect on various tumors such as colon cancer, lung cancer and leukemia, making it an excellent "partner" for ARC to enhance the anti-tumor effect. Structurally, FU molecule has a pyrimidine ring similar to ARC, containing C=O and N-H as hydrogen bond donor / acceptor groups, which has the ability to form complementary hydrogen bonds with ARC molecules. Secondly, compared with ARC, FU has the advantages of relatively low solubility and higher permeability, such as being able to direct the assembly of the two, which is expected to reduce the solubility and enhance the permeability of ARC. More importantly, by co-crystallizing ARC and FU with similar anti-tumor activity together through co-crystallization technology, it ensures that cancer cells can be exposed to their damage at the same time, which is conducive to exerting synergistic anti-tumor effect. Therefore, by co-crystallizing ARC and FU through co-crystallization technology, preparing ARC-FU co-crystals can not only retain the anti-tumor effect of ARC drug itself, but also moderately down-regulate the solubility and enhance the permeability of ARC, which has important clinical value and broad application prospect for realizing the synergistic anti-tumor effect of the two at the molecular level and producing synergistic effect.

[0004] In view of the above background, in view of the poor ARC physicochemical properties and poor efficacy, etc., the unique advantages of the co-crystallization technology in improving the properties of drugs are exerted, and the present application creatively proposes and implements the co-crystallization research and development strategy of ARC and FU synergistic antitumor drugs. The strategy is guided by the principles of supramolecular chemistry and crystal engineering technology, and the solubility of ARC is moderately reduced while the permeability is significantly enhanced, realizing the molecular level combination of drugs, thereby enhancing the antitumor efficacy, and laying a foundation for overcoming drug resistance and improving patient compliance.

[0005] Currently, there is no public report on the co-crystal of cytarabine and 5-fluorouracil. SUMMARY

[0006] The present application aims to provide a co-crystal of cytarabine and 5-fluorouracil and a preparation method thereof. The co-crystal of the present application has a clear structure, which is assembled by cytarabine and 5-fluorouracil molecules through hydrogen bonds and other non-covalent interactions. The co-crystal realizes the synergistic effect of cytarabine and 5-fluorouracil at the molecular level, and significantly improves the antitumor activity of cytarabine.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a co-crystal of cytarabine and 5-fluorouracil, which is composed of one cytarabine molecule and one 5-fluorouracil molecule as a basic structural unit, and the chemical formula of the co-crystal is [C9H 13 N3O5·C4H3N2O2F].

[0008] Further, the co-crystal belongs to the orthorhombic system, the space group is P212121, and the cell parameters are: α = 90.00°, β = 90.00°, γ = 90.00°.

[0009] Further, the characteristic diffraction peaks of the X-ray powder diffraction (PXRD) of the co-crystal appear at 7.68°±0.2, 11.68°±0.2, 15.38°±0.2, 17.94°±0.2, 27.60°±0.2, 28.76°±0.2; preferably, the characteristic diffraction peaks of the PXRD thereof appear at 7.68°±0.1, 11.68°±0.1, 15.38°±0.1, 17.94°±0.1, 27.60°±0.1, 28.76°±0.1; most preferably, the characteristic diffraction peaks of the PXRD thereof appear at 7.68°, 11.68°, 15.38°, 17.94°, 27.60°, 28.76°.

[0010] The present application also provides a preparation method of the co-crystal of cytarabine and 5-fluorouracil, which comprises the following steps:

[0011] (1) Cytarabine and 5-fluorouracil are added to a mortar in a molar ratio of 1:1 and mixed, and isopropanol solvent is added to assist grinding to obtain a white powder;

[0012] (2) The white powder is transferred to a round-bottom flask, dissolved with a solvent, stirred at room temperature for 3-5 h, filtered, and left to volatilize for 2-5 days to obtain colorless columnar crystals.

[0013] As preferred, the organic solvent in step (2) is one or a mixture of several of methanol, ethanol, isopropanol, acetonitrile, ethyl acetate, and n-butanol, and further preferably a mixed solvent of n-butanol and methanol.

[0014] As preferred, the volume ratio of n-butanol to methanol in the organic solvent in step (2) is 1:1-6:1, further preferably 2:1-5:1, more further preferably 3:1-4:1, and most preferably 3:1.

[0015] The columnar crystals obtained by the preparation method can be used for X-ray single crystal diffraction structure determination.

[0016] The application also provides a preparation method of the cytarabine and 5-fluorouracil pharmaceutical co-crystal, comprising the following steps: Cytarabine and 5-fluorouracil are added to a mortar in a molar ratio of 1:1 and mixed, and isopropanol solvent is added to assist grinding, and then the obtained solid powder is transferred to a round-bottom flask, dissolved with a solvent, stirred in a water bath at 70°C for 2-3 h, filtered while hot, and left to cool to room temperature to precipitate white crystalline solids, collect the white solids, and vacuum dry to obtain the cytarabine and 5-fluorouracil pharmaceutical co-crystal.

[0017] As preferred, the organic solvent is one or a mixture of several of methanol, ethanol, isopropanol, acetonitrile, ethyl acetate, and n-butanol, and further preferably a mixed solvent of n-butanol and methanol.

[0018] Further, the cytarabine and 5-fluorouracil pharmaceutical co-crystal can be used in the application of resisting tumors.

[0019] Compared with the prior art, the present application has the beneficial effects that: (1) the present application first prepares a drug cocrystal of cytarabine and 5-fluorouracil, and determines the accurate crystal structure thereof. The drug cocrystal has high purity and crystallinity, and can maintain the skeleton structure of the crystal after long-term storage at room temperature; (2) the drug cocrystal of cytarabine and 5-fluorouracil significantly enhances the permeability and improves the antitumor activity while appropriately down-regulating the solubility of cytarabine, thereby laying a foundation for good antitumor effect at a low dose; (3) the drug cocrystal of cytarabine and 5-fluorouracil realizes the combination of drugs at the molecular level through the complementary advantages of the two components in properties and efficacy, thereby providing a new idea for developing synergistic antitumor drugs; (4) the preparation method of the drug cocrystal is simple and easy to operate, has mild reaction conditions, high yield and low cost, and is suitable for large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 FIG. 1 is a crystal structure diagram of the drug cocrystal of cytarabine and 5-fluorouracil according to the present application.

[0021] Figure 2 FIG. 2 is a two-dimensional hydrogen bond connection mode diagram of the drug cocrystal of cytarabine and 5-fluorouracil according to the present application.

[0022] Figure 3 FIG. 3 is a three-dimensional supramolecular packing diagram of the drug cocrystal of cytarabine and 5-fluorouracil according to the present application.

[0023] Figure 4 FIG. 4 is a comparison diagram of the PXRD spectrum of cytarabine and 5-fluorouracil raw materials and the PXRD spectrum of the drug cocrystal of cytarabine and 5-fluorouracil.

[0024] Figure 5 FIG. 5 is a comparison of the simulated software PXRD spectrum and the PXRD spectrum of the drug cocrystal of cytarabine and 5-fluorouracil.

[0025] Figure 6 FIG. 6 is a TG-DSC curve of the drug cocrystal of cytarabine and 5-fluorouracil.

[0026] Figure 7 FIG. 7 is a comparison of the solubility of the drug cocrystal of cytarabine and 5-fluorouracil and cytarabine raw materials in a buffer solution.

[0027] Figure 8 FIG. 8 is a comparison of the cumulative permeation amount of the drug cocrystal of cytarabine and 5-fluorouracil and cytarabine raw materials in a buffer solution.

[0028] Figure 9 FIG. 9 is a comparison of the pharmacokinetic properties of the drug cocrystal of cytarabine and 5-fluorouracil and cytarabine raw materials. DETAILED DESCRIPTION

[0029] The technical solutions of the present application will be described in detail below with specific examples.

[0030] Example 1: The preparation method of the drug cocrystal of cytarabine and 5-fluorouracil is implemented according to the following steps:

[0031] Cytarabine (48.60 mg) and 5-fluorouracil (26.00 mg) are mixed in a mortar at a molar ratio of 1:1, 90 μL of isopropyl alcohol is added for rapid grinding until dry, and the white powder obtained after 1 h of continuous grinding is transferred to a round-bottom flask and dissolved in 6 mL of a mixed solvent of n-butanol and 2 mL of methanol (volume ratio of 3:1). After stirring at room temperature for 5 h, the filtrate is filtered and left to volatilize for 3 days to obtain colorless columnar crystals.

[0032] The drug cocrystal of cytarabine and 5-fluorouracil prepared by solvent evaporation in this example is a colorless columnar crystal, and a single crystal of appropriate size and high quality is selected therefrom for X-ray single crystal diffraction determination. A STADIVARI diffractometer of Germany STOE Company is used for scanning by Cu-Kα rays monochromatized by a multi-layer film focusing mirror, and the mode is ω scanning. The required current voltage is set to 0.6 mA and 50 kV in the diffraction experiment. The results show that the drug cocrystal of cytarabine and 5-fluorouracil in this example belongs to an orthogonal crystal system, the space group is P212121, and the cell parameters are: α = 90.00°, β = 90.00°, γ = 90.00°, and the chemical formula of the drug cocrystal is [C9H 13 N3O5·C4H3N2O2F]; the PXRD characteristic diffraction peaks thereof obtained by simulation with Mercury software are at 7.68°, 11.68°, 15.38°, 17.94°, 27.60°, and 28.76°.

[0033] Figure 1 and Figure 2 The crystal structure unit and the two-dimensional hydrogen bond connection mode of the drug cocrystal prepared in this example are respectively shown. Figure 1 It can be seen that the drug cocrystal of cytarabine and 5-fluorouracil is composed of one cytarabine molecule and one 5-fluorouracil molecule. In the asymmetric unit, the D-furanose ring of cytarabine is in a distorted configuration (endo-C7, exo-C8), and forms an R2 2 (8) a planar "pyrimidine-pyrimidine" cyclic hydrogen bond system. It can be seen from Figure 2 It can be seen that the cytarabine molecules in the crystal are connected through one hydroxyl group (O3) of the arabinose and the carbonyl oxygen atom (O1 iv) form O-H···O hydrogen bonds, constituting a 21 -ara-C hydrogen bond helix running along the a axis Figure 2 ); fluorouracil can be considered to contain two amide groups, one containing atoms O6 and N4 and the other containing O7 and N5, in addition to the former and ara-C to form the above "pyrimidine-pyrimidine" cyclic hydrogen bonds, the latter also forms R2 with the arabinose of another molecule of ara-C located at x-1 / 2, 1 / 2-y, 1-z (ii) 2 (9) "pyrimidine-sugar" cyclic hydrogen bonds. Thus, the 5-fluorouracil molecule assembles the ara-C hydrogen bond helix into a two-dimensional hydrogen bond network parallel to the a0b plane. In addition, there is also a partially overlapping aromatic ring stacking interaction between adjacent "pyrimidine-pyrimidine" units, i.e., between 5-fluorouracil and cytosine located at symmetry positions (ii) and x+1 / 2, 1 / 2-y, 1-z (iii), and vice versa. The vertical distances of the overlapping atoms to the corresponding pyrimidine ring are: 3.312(2), 3.302(2), 3.186(3), 3.212(2), 3.222(2), 3.338(3) (C2 ii ,C3 ii ,C4 ii and C2 iii ,C3 iii ,C4 iii to fluorouracil); 3.290(2), 3.271(2), 3.340(2), 3.324(2), 3.218(2), 3.237(2), 3.168(2) (N5 ii ,C10 ii ,O6 ii and N4 iii ,N5 iii ,C10 iii ,O6 iii to cytosine). Figure 3 is a three-dimensional supramolecular packing diagram of the pharmaceutical co-crystal prepared in this example. From Figure 3 it can be seen that in the crystal structure, the "pyrimidine-sugar" hydrogen bond units form chains parallel to the b axis through hydrogen bonds N3-H3A···O4 i (i = x, y-1, z) between the amino group of the ara-C cytosine and the hydroxyl group of the sugar of another molecule, while the chains between two adjacent layers can be further connected to each other through hydrogen bonds O4-H4A···O7 vi (v = 1 / 2-x, 1-y, z+1 / 2) between the hydroxyl groups of the sugars of two adjacent molecules, forming a complete three-dimensional hydrogen bond supramolecular structure.

[0034] Example 2: Preparation method of ara-C and 5-fluorouracil pharmaceutical co-crystal was carried out according to the following steps:

[0035] The difference between this embodiment and Example 1 is that the volume ratio of n-butanol to methanol in the system is 1:1, while the other steps and parameters are the same as in Example 1.

[0036] Example 3: The preparation method of cytarabine and 5-fluorouracil cocrystal is carried out according to the following steps:

[0037] The difference between this embodiment and Example 1 is that the volume ratio of n-butanol to methanol in the system is 6:1, while the other steps and parameters are the same as in Example 1.

[0038] Example 4: The preparation method of cytarabine and 5-fluorouracil cocrystal is carried out according to the following steps:

[0039] Cytarabine (486.0 mg) and 5-fluorouracil (260.0 mg) were mixed in a mortar at a molar ratio of 1:1. 270 μL of isopropanol was added, and the mixture was rapidly ground until dry. After grinding for 1 hour, the resulting solid was transferred to a round-bottom flask, and 60 mL of acetonitrile and 10 mL of methanol were added. The mixture was stirred in a water bath at 70 °C for 3 hours. The mixture was filtered while hot, and the filtrate was allowed to stand and cool to room temperature. A white solid precipitated. The white solid was collected, and after vacuum drying, a cocrystal of cytarabine and 5-fluorouracil was obtained with a yield of 98.2%.

[0040] The cytarabine and 5-fluorouracil drug cocrystal prepared by the cooling method in this embodiment is a white crystalline powder with good chemical stability and no deterioration after long-term storage at room temperature.

[0041] Example 5: PXRD determination of cytarabine and 5-fluorouracil cocrystal

[0042] PXRD diffraction experiments were performed on the powder sample obtained by the cooling method in Example 4. The PXRD diffraction data were obtained using a BRUKER D8 X-ray diffractometer (Germany). Test conditions: Cu-Kα target tube voltage 40 kV, tube current 10 mA, scan rate 2° / min. Figure 4 As shown, by comparing the obtained cytarabine / 5-fluorouracil cocrystal (hereinafter referred to as ARC-FU) with the corresponding starting materials, it can be seen that its characteristic PXRD diffraction peaks appear at 7.68°±0.2, 11.68°±0.2, 15.38°±0.2, 17.94°±0.2, 27.60°±0.2, and 28.76°±0.2. The positions and intensities of the characteristic diffraction peaks have changed significantly, indicating the formation of a new phase. Furthermore, the PXRD spectrum of the measured ARC-FU sample was compared with the theoretical spectrum of ARC-FU simulated using Mercury software based on crystal data obtained from X-ray single-crystal diffraction (see...). Figure 5), the powder diffraction peaks of the co-crystal ARC-FU sample prepared in Example 4 are sharp and consistent with the diffraction peaks of the theoretical simulation spectrum thereof, proving that the prepared co-crystal ARC-FU has high crystallinity and purity.

[0043] Example 6: Thermal property determination of the drug co-crystal of cytarabine and 5-fluorouracil

[0044] The co-crystal ARC-FU obtained in Example 4 was subjected to thermal analysis using a Netzsch STA409PC simultaneous thermal analyzer, and the thermogravimetric-differential scanning calorimetry (TG-DSC) thereof is shown in FIG. 2. Figure 6 As can be seen from the figure, a sharp endothermic peak appears at 231.7℃, which corresponds to the melting point of the co-crystal ARC-FU, and it can be inferred that the obtained co-crystal is a homogeneous phase; in addition, the TG curve does not have obvious weight loss behavior before the co-crystal melts, proving that the drug co-crystal does not contain solvent molecules, which is consistent with the X-ray single crystal diffraction result.

[0045] Experimental Example 7: Solubility determination of the drug co-crystal of cytarabine and 5-fluorouracil

[0046] The solubilities of the raw material drug ARC and the drug co-crystal ARC-FU sample were determined in pH = 1.2, 4.0, 6.8 buffer media, respectively, and the results are shown in FIG. 3. Figure 7 As can be seen from the figure, in the pH 6.8, 4.0 and 1.2 buffer solutions, the solubility of ARC in the drug co-crystal ARC-FU is reduced by 43-61% compared with that of pure ARC. It indicates that the drug co-crystal of the present application realizes the reduction of the solubility of ARC; the research results suggest that the drug co-crystal not only lays a foundation for realizing the reduction of the polarity and the improvement of the liposolubility of ARC, but also creates favorable conditions for improving the therapeutic effect of ARC.

[0047] Experimental Example 8: Cumulative permeation amount determination of the drug co-crystal of cytarabine and 5-fluorouracil

[0048] The permeabilities of ARC and the drug co-crystal ARC-FU sample were determined in pH = 6.8 buffer medium by Franz diffusion cell method, and the results are shown in FIG. 4. Figure 8 As can be seen from the figure, the cumulative permeation amount of ARC in the drug co-crystal ARC-FU is 219.9 nmol·cm -2 , which is 1.65 times higher than that of pure ARC. It indicates that the drug co-crystal of the present application realizes the improvement of the permeation ability of ARC; the research results suggest that the drug co-crystal not only lays a foundation for realizing the improvement of the liposolubility and the bioavailability of ARC, but also provides a guarantee for exerting the synergistic antitumor effect and enhancing the antitumor effect of both.

[0049] Experimental Example 9: In vitro antitumor activity determination of the drug co-crystal of cytarabine and 5-fluorouracil

[0050] For simplicity, ARC+FU in the following description refers to a physical mixture of cytarabine and 5-fluorouracil in a molar ratio of 1:1.

[0051] The anti-tumor activities of ARC-FU, ARC, FU and ARC+FU were determined by SRB method. The test samples were dissolved in phosphate buffer solution (PBS) containing 0.1% DMSO and pre-diluted into different working concentrations. Human pro-myelocytic leukemia cells (HL-60), human chronic myeloid leukemia cells (K562) and human colon cancer cells (HT-29) were cultured in 96-well plates. After the above-mentioned cells were cultured in a 37°C incubator containing 5% CO2for 24 h, 100 μL of the test sample solution was added and the culture was continued in the incubator for 24 h and 48 h. After the culture was completed, the cells were stained with 0.4% (w / v) SRB staining solution (1% acetic acid) for 30 min. After staining, the unbound dye was removed by washing with 1% (v / v) acetic acid, and the dye bound to the cell protein was dissolved with 100 μL of non-buffered Tris-base alkali solution (10 mM, pH = 10.5) on a horizontal shaker for 20 min. The absorbance of the liquid in each well of the 96-well plate was measured at 540 nm using a microplate reader, and the drug concentration at which half of the cells were inhibited, i.e. the IC 50 value, was calculated. The results are shown in Table 1. To further evaluate the synergy between the two components in the drug co-crystal, the combination index (CI) of the two drugs in the co-crystal was calculated by the Chou-Talalay combination index method, and the results are shown in Table 1.

[0052] Table 1 IC 50 (μM) and synergy index (CI) values of test samples on different tumor cell lines

[0053]

[0054] Note: The CI values of ARC-FU on HL-60, K562 and HT-29 cell lines at 24 h were 0.52, 0.86 and 0.93, respectively; the CI values of ARC-FU on HL-60, K562 and HT-29 cell lines at 48 h were 0.53, 0.83 and 0.86, respectively, *p < 0.05.

[0055] As can be seen from the data in Table 1, the test samples showed different inhibitory effects on the three tumor cells. Among them, the IC 50 value of the drug co-crystal ARC-FU was the lowest. As observed for the ARC group, ARC showed a smaller IC 50 value on HL-60 and K562 cells, showing a good anti-leukemia effect, and its IC 50The relatively high values ​​suggest that ARC has a good inhibitory effect on the proliferation of leukemia cells, while its inhibitory effect on solid tumors is significantly reduced. Observation of the FU group showed that FU significantly reduced the IC50 value of HT-29 cells. 50 The value was lower than ARC, indicating better inhibitory effect on solid tumors than ARC. In the physical combination ARC+FU group, ARC+FU showed a lower IC50 value for HL-60. 50 The value is approximately the same as ARC, while the ICs for K529 and HT-29 are similar. 50 The value is closer to FU, indicating that a simple physical mixture of the two components can only achieve the level of the one with stronger antitumor activity, and there is no synergistic effect between them. This may be due to the significant differences in their properties. In contrast, the IC50 value of the drug cocrystal ARC-FU is much higher. 50 The values ​​were significantly lower than those in the drug-only group and the physical mixture group (p<0.05), indicating a significant enhancement in its antitumor activity. More importantly, at 24 h, the CI values ​​of ARC and FU in the drug cocrystal ARC-FU against HL-60, K562, and HT-29 tumor cells were 0.52, 0.86, and 0.93, respectively; at 48 h, the CI values ​​were 0.53, 0.83, and 0.86, respectively. It is evident that the CI values ​​were all less than 1, suggesting a synergistic effect between the two components in the drug cocrystal ARC-FU, which is crucial for enhancing the antitumor effect of the marine antitumor drug cytarabine. Through the above comparative analysis, it can be confirmed that the drug cocrystal ARC-FU achieves a synergistic enhancement of the two components in terms of antitumor activity, laying the foundation for the further development of this drug cocrystal.

[0056] Experimental Example 10: In vivo pharmacokinetic properties of cytarabine and 5-fluorouracil cocrystal

[0057] Wistar rats were randomly divided into two groups (n=5), receiving oral administration of ARC aqueous solution and oral administration of cocrystal ARC-FU (10 mg / kg of ARC). Blood samples (~500 μL) were collected at regular time intervals after treatment, placed in heparinized tubes, centrifuged at 10000 rpm for 5 min, and plasma was separated and stored at -20℃ for analysis. The blood concentration of ARC was determined by high-performance liquid chromatography (HPLC), and the results are shown in the appendix. Figure 9 The corresponding pharmacokinetic parameters are shown in Table 2.

[0058] from Figure 9 It can be seen that, in the ARC group, the plasma drug concentration increased sharply within 1 hour, reaching its maximum value (C). MAX The concentration was 549.34 ng·mL. -1 Subsequently, the blood drug concentration began to decrease, becoming almost undetectable after 10 hours. However, in the cocrystallized ARC-FU group, the blood drug concentration showed an upward trend within 4 hours, reaching a maximum of C... MAXFar higher than the ARC group, up to 1273.25 ng·mL -1 Moreover, trace amounts of plasma drug concentrations can still be detected after 12 hours, showing better absorption capacity. To better illustrate this point, from Table 2, the half-life (T 1 / 2 ) of the drug co-crystal ARC-FU is 5.38 times higher than that of ARC, which is more conducive to circumvent rapid clearance and maintain a persistent therapeutic effect in vivo. The area under the curve (AUC) of the drug co-crystal ARC-FU is 4.22 times higher than that of ARC, i.e., the relative bioavailability F REL = 4.22, showing better absorption effect than ARC, which is of great significance for improving its oral absorption and efficacy.

[0059] Table 2 Pharmacokinetic parameters of test samples

[0060]

[0061] * p < 0.05

[0062] The above is only the preferred specific embodiment of the present application, but the protection scope of the present application is not limited to this. Any skilled person in the art, according to the technical solution and the inventive concept of the present application, makes equivalent replacement or change within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A pharmaceutical co-crystal of cytarabine and 5-fluorouracil, characterized by: The pharmaceutical co-crystal consists of one cytarabine molecule and one 5-fluorouracil molecule as a basic structural unit, and the chemical formula of the pharmaceutical co-crystal is [C9H 13 N3O5·C4H3N2O2F]; The arabinoside and 5-fluorouracil drug co-crystal belongs to an orthogonal crystal system, and the space group is P2(1)2(1)2(1) P2 1 2 1 2 1 The cell parameters are: a = 6.32~6.72 Å, b = 9.84~10.24 Å, c = 22.82~23.22 Å, α = 90.00°, β = 90.00°, γ = 90.00°; The PXRD characteristic diffraction peaks of the cocrystal of cytarabine and 5-fluorouracil drug appear at 7.68°±0.1, 11.68°±0.1, 15.38°±0.1, 17.94°±0.1, 27.60°±0.1, 28.76°±0.

1.

2. The pharmaceutical co-crystal of cytarabine and 5-fluorouracil as claimed in claim 1, wherein, The PXRD characteristic diffraction peaks of the cocrystal of cytarabine and 5-fluorouracil drug appear at 7.68°±0.1, 11.68°±0.1, 15.38°±0.1, 17.94°±0.1, 27.60°±0.1, 28.76°±0.

1.

3. The pharmaceutical co-crystal of cytarabine and 5-fluorouracil as claimed in claim 1, wherein, The PXRD characteristic diffraction peaks of the cocrystal of cytarabine and 5-fluorouracil drug appear at 7.68°, 11.68°, 15.38°, 17.94°, 27.60°, 28.76°.

4. The process for the preparation of Cytarabine and 5-Fluorouracil pharmaceutical co-crystal as claimed in claim 1, wherein, The preparation method comprises the following steps: (1) cytarabine and 5-fluorouracil are added to a mortar in a molar ratio of 1:1, mixed, and isopropanol solvent is added to assist grinding to obtain white crystalline powder; (2) the obtained powder is transferred to a round-bottom flask, dissolved by adding a solvent, stirred at room temperature for 3-5 h, filtered, and the filtrate is left to volatilize for 2-5 days to obtain colorless columnar crystals.

5. A process for the preparation of a pharmaceutical co-crystal of cytarabine and 5- fluorouracil as claimed in claim 4, wherein, The solvent in step (2) is one or a mixture of several of methanol, ethanol, isopropanol, acetonitrile, ethyl acetate, and n-butanol.

6. The process for the preparation of Cytarabine and 5-Fluorouracil pharmaceutical co-crystal as claimed in claim 4, wherein, The solvent in step (2) is a mixed solvent of n-butanol and methanol in a volume ratio of 1:1 to 6:

1.

7. A process for the preparation of a pharmaceutical co-crystal of Cytarabine and 5- Fluorouracil as claimed in claim 1, wherein, The preparation method comprises the following steps: Cytarabine and 5-fluorouracil are added to a mortar in a molar ratio of 1:1, mixed, and isopropanol solvent is added to assist grinding to obtain white crystalline powder; 8. A process for the preparation of a pharmaceutical co-crystal of cytarabine and 5- fluorouracil as claimed in claim 7, wherein, The solvent is a mixed solvent of acetonitrile and methanol.

9. Use of the cocrystal of cytarabine and 5-fluorouracil drug according to claim 1 in the preparation of a drug for treating leukemia and colon cancer.

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