Diclofenac-methotrexate co-amorphous material, process for its preparation and use

Abstract

The present application relates to a kind of diclofenac-methotrexate co-amorphous substance and its preparation method and application, in the diclofenac-methotrexate co-amorphous substance, the molar ratio of diclofenac and methotrexate is 1:2, its X-ray powder diffraction pattern has no sharp diffraction peak, indicating the formation of co-amorphous form.The present application is obtained by using rotary evaporation method to obtain co-amorphous substance, and the results of pharmacokinetics and in vitro and in vivo pharmacodynamics experiments show that the performance indicators such as DIC-MTX co-amorphous system are superior to physical mixture in bioavailability, anti-inflammatory effect, etc., which embodies its potential application value, and shows that there is positive effect between the two drugs for treating rheumatoid arthritis, which provides a research basis for realizing the combination of DIC and MTX.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a dadiclofenac-methotrexate co-amorphous compound, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a common chronic systemic autoimmune disease characterized by synovial inflammation and the degradation and destruction of cartilage and bone tissue. Patients typically experience persistent joint pain, swelling, and stiffness, and in severe cases, it can lead to multi-organ damage, including the cardiovascular, pulmonary, and skeletal systems. RA has a high global incidence rate and can occur at any age. It is characterized by recurrent episodes and difficulty in complete cure, significantly impacting patients' daily lives and social activities. Current clinical treatment for RA primarily aims at anti-inflammatory and analgesic effects, prevention or improvement of joint lesions, and adheres to the principle of early detection and treatment. However, due to the complex etiology and pathogenesis of RA, studies have shown that it may be related to immune cells, inflammatory mediators, matrix metalloproteinases (MMPs), abnormal oxidative stress, genetic factors, and environmental factors. Therefore, existing drugs cannot completely cure RA, presenting limitations and posing significant challenges to patient treatment. Currently available drugs for treating rheumatoid arthritis (RA) are all single-target drugs, which can be divided into nonsteroidal anti-inflammatory drugs (NSAIDs), disease-modifying antirheumatic drugs (DMARDs), glucocorticoids (GCs), and biologics. However, the inherent limitations of monotherapy make it difficult to address the multiple objectives of anti-inflammatory and analgesic treatment, prevention of articular cartilage damage, and reduction of systemic complications. Therefore, combination therapy with multiple drugs is often used in clinical prescriptions. However, this method involves the physical mixing of different active pharmaceutical ingredients, which can easily lead to differences in the dosage ratio between different batches of drugs, poor patient compliance, and a heavy economic burden. Therefore, the development of monophasic multi-target drugs for the treatment of RA is of great significance.

[0003] In recent years, the discovery and application of supramolecular complex systems have become a hot topic in drug development. As a novel solid form of drug, it has shown great superiority and potential in improving the physicochemical properties and bioavailability of drugs, and also provides a new approach to formulation research, achieving synergistic effects of enhanced efficacy and reduced toxicity. This invention aims to explore new directions for the treatment strategy of rheumatoid arthritis (RA) by constructing a diclofenac-methotrexate dual-target supramolecular system. Summary of the Invention

[0004] The purpose of this invention is to provide a diclofenac-methotrexate co-amorphous compound (DIC-MTX) with good bioavailability and in vitro and in vivo anti-inflammatory activity, and to provide its preparation method and application.

[0005] The present invention provides a diclofenac-methopterin co-amorphous compound, characterized in that the co-amorphous compound comprises diclofenac and methotrexate, wherein the molar ratio of diclofenac and methotrexate is 1:2.

[0006] As a further improvement of the present invention, the co-amorphous material, when subjected to Cu-kα radiation, exhibits a distinct diffuse diffraction halo in X-ray powder diffraction patterns expressed as 2θ, with the disappearance of crystalline diffraction peaks. This preliminarily indicates the formation of the co-amorphous material. Both DSC and TG analyses confirm the formation of amorphous morphologies in each system of the DIC-MTX.

[0007] As a further improvement of the present invention, the X-ray powder diffraction pattern is as follows: Figure 1 As shown in D-M1:2.

[0008] As a further improvement of the present invention, the glass transition temperature of the co-amorphous material is 62.5±0.5℃.

[0009] As a further improvement of the present invention, the co-amorphous material is prepared by rotary evaporation or solvent-assisted grinding.

[0010] Another aspect of the present invention provides a method for preparing the above-mentioned diclofenac-methotrexate.

[0011] Rotary evaporation method was used: diclofenac and methotrexate were added to the solvent, the sample was ultrasonically treated with a solid-liquid ratio of 1~3 mg / mL for 10-15 min, and then dried under vacuum at 60±2℃ to obtain the co-amorphous product.

[0012] As a further improvement of the present invention, the solvent is composed of solvent A and solvent B, wherein solvent A is selected from methanol or ethanol, and solvent B is selected from N,N-dimethylformamide.

[0013] As a further improvement of the present invention, the volume ratio of solvent A to solvent B is 35-65:1.

[0014] In another aspect, the present invention provides the application of the above-mentioned preparation of diclofenac-methotrexate coamorphs in the preparation of medicaments for treating rheumatoid arthritis.

[0015] The beneficial effects of adopting the above technical solution are as follows:

[0016] This invention uses diclofenac and methotrexate as raw materials to obtain a co-amorphous compound by ultrasonic rotary evaporation. Experimental verification shows that the co-crystal has good bioavailability and stability.

[0017] The co-amorphous compounds provided by this invention exhibit enhanced inhibitory effects on endogenous ROS and improved anti-inflammatory effects, potentially demonstrating excellent ROS scavenging capabilities for RA treatment. The formation of the co-amorphous system helps macrophages polarize from the M1 phenotype to the M2 phenotype and reduces inflammation, making it a preferred candidate for RA treatment.

[0018] The co-amorphous compound provided by this invention has been verified through in vivo experiments. The co-amorphous compound can significantly reduce the expression levels of inflammatory factors such as TNF-α, IL-1β, IL-6, and NO, thus verifying the efficacy of CO in delaying or reversing the malignant behavior of RA (such as reducing joint damage and increasing arthritis scores) and improving ankle joint tissue structure (by relieving inflammation, improving synovial invasion, reducing cartilage damage, and preventing bone destruction). Attached Figure Description

[0019] Figure 1 These are the PXRD patterns of crystalline DIC, MTX, and DIC-MTX composites with different molar ratios prepared by rotary evaporation according to the present invention.

[0020] Figure 2 The DSC curves are for DIC crystalline state, MTX crystalline state, DIC-MTX physical mixture, and co-amorphous state;

[0021] Figure 3 TG(A) and DTG(B) curves of crystalline DIC, MTX, and DIC-MTX with amorphous forms;

[0022] Figure 4 PLM diagrams of crystalline DIC, MTX, and DIC-MTX with amorphous forms;

[0023] Figure 5 The infrared spectra of crystalline DIC, MTX, PM and DIC-MTX co-amorphous states are those of the present invention.

[0024] Figure 6 These are the Raman spectra of the DIC crystalline state, MTX crystalline state, and co-amorphous state of this invention;

[0025] Figure 7 This refers to the oil-water distribution coefficient between DIC and MTX in this invention;

[0026] Figure 8 The cumulative permeation-time curves for DIC, PM-DIC, and CO-DIC in this invention are shown below.

[0027] Figure 9The cumulative permeation-time curves of MTX, PM-MTX and CO-MTX of this invention are shown below.

[0028] Figure 10 PXRD pattern of the sample after 8 months of storage under dry conditions;

[0029] Figure 11 The blood concentration-time curves of DIC and MTX in this invention;

[0030] Figure 12 The cell survival rates of M0 and M1 macrophages of the present invention under different concentrations of DIC (A) and MTX (B);

[0031] Figure 13 The toxic effects of the DIC-MTX physical mixture and co-amorphous compounds of this invention on macrophages;

[0032] Figure 14 ROS immunofluorescence staining (green) and nuclear staining (blue) of the DCHF-DA probe of this invention. Scale bar = 50 μm. *P<0.05, **P<0.01, ***P<0.001;

[0033] Figure 15 Immunofluorescence staining of iNOS (M1 marker, red) and CD206 (M2 marker, green) of this invention, nuclear staining (blue) (A), and expression levels of iNOS (B) and CD206 (C) in RAW 264.7, scale bar = 100 μm;

[0034] Figure 16 The results show the immunofluorescence staining of COX-2 (M1 marker, red), nuclear staining (blue) (A), and the expression level of COX-2 (B) in RAW 264.7 macrophages, P<0.05, **P<0.01, ***P<0.001;

[0035] Figure 17 This is a graph showing the paw thickness and arthritis score of AIA rats according to the present invention;

[0036] Figure 18 The following are the iCro-CT imaging evaluation images of the present invention: (A) representative claw micro-CT image, (B) mineral density (BMD) of the joint, and (C) bone surface area / bone mass (BS / BV), *P<0.05, **P<0.01, ***P<0.001;

[0037] Figure 19 H&E, Masson, and Safranin O-Fast Green staining images of ankle joint tissues from various rat groups in this invention, scale bar = 100 μm;

[0038] Figure 20 The serum cytokine levels of rats in each group after drug treatment were (A) TNF-α, (B) IL-6, (C) IL-1β and (D) NO. *P<0.05, **P<0.01, ***P<0.001;

[0039] Figure 21 The effects of the drug of this invention on the cardiac, hepatic, and renal functions of rats;

[0040] Figure 22 The image shows the H&E staining results of rat organs in each group of the present invention. Scale bar = 100 μm.

[0041] Figure 23 The changes in body weight of rats in each group in this invention are shown. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the present invention clearer, the invention will be described clearly and completely below in conjunction with specific embodiments.

[0043] Example 1 Preparation of diclofenac-methotrexate (DIC-MTX) co-amorphous compound

[0044] Diclofenac (DIC) and methotrexate (MTX) were accurately weighed at a molar ratio of 1:2, with 22.21 mg of DIC and 68.17 mg of MTX. They were placed in 250 mL round-bottom flasks, and 50 mL of ethanol and 1 mL of N,N-dimethylformamide (DMF) were added. The mixture was sonicated at room temperature for 10 min to achieve complete dissolution. The solvent was then removed by rotary evaporation under reduced pressure at 60 °C (100 rpm) to obtain a yellow transparent solid product. The solid product was dried in a vacuum drying oven at 60 °C for 24 h to obtain the final product.

[0045] Comparative Example 1: Preparation of DIC-MTX (PM) physical mixture

[0046] Weigh appropriate amounts of DIC and MTX raw materials and add them to a 1.5 mL EP tube at a molar ratio of 1:2. Mix well to obtain the final product.

[0047] Comparative Example 2

[0048] DIC and MTX were accurately weighed at molar ratios of 1:1, 2:1, 2:3, and 3:2, respectively, and placed in 250 mL round-bottom flasks. 50 mL of ethanol and 1 mL of N,N-dimethylformamide (DMF) were added to each flask. The mixtures were sonicated at room temperature for 10 min to achieve complete dissolution. Subsequently, the solvent was removed by rotary evaporation under reduced pressure at 60 °C, yielding a yellow, transparent solid product. Finally, the solid was placed in a vacuum drying oven at 60 °C for 24 hours for later use.

[0049] Experimental Example 1: Characterization of Co-amorphous Structures

[0050] In this experimental example, the diclofenac-methotrexate co-amorphous compound obtained in Example 1 was used and named D-M1:2. The samples obtained in Comparative Example 2 were DM 1:1 (DIC:MTX=1:1), DM 2:1 (DIC:MTX=2:1), DM 2:3 (DIC:MTX=2:3), and DM 3:2 (DIC:MTX=3:2).

[0051] (1) X-ray powder diffraction detection

[0052] Appropriate amounts of diclofenac, methotrexate, and the physical mixture of Comparative Example 1 and the sample prepared in Comparative Example 2 were weighed onto the stage, and their X-ray diffraction patterns were determined using a Smart LAbSE diffractometer. The scanning range was 5 to 40° (2θ), the step size was 0.02°, the scanning speed was 10° / min, and the operating voltage and current were set to 40 kV and 50 mA, respectively. The PXRD patterns are shown below. Figure 1 As shown in the figure, the DIC active pharmaceutical ingredient exhibits characteristic diffraction peaks at 2θ values ​​of 10.79°, 13.31°, 15.10°, 18.82°, and 24.34°; the MTX active pharmaceutical ingredient shows characteristic diffraction peaks at 2θ values ​​of 9.37°, 11.56°, 12.99°, 27.08°, and 27.99°. The PXRD pattern of the DM 1:2 supramolecular system shows a distinct diffuse diffraction halo without sharp characteristic peaks, and the crystalline diffraction peaks disappear, preliminarily indicating the formation of a co-amorphous morphology. The figure shows that only when the DIC-MTX molar ratio is 1:2 does the crystalline diffraction peaks of the complex disappear, exhibiting an overall diffuse diffraction ring, preliminarily indicating the successful preparation of a co-amorphous morphology. When the molar ratio of DIC-MTX is 1:1, 2:1, 2:3, and 3:2, although the overall shape shows an amorphous outline, obvious sharp diffraction peaks can still be seen, indicating that there are crystals in the product. This shows that a co-amorphous system of the two cannot be obtained under these ratios.

[0053] (2) DSC detection

[0054] Quantitatively weigh 10 mg of DIC, MTX, DIC-MTX physical mixture and the co-amorphous sample from Example 1, seal them in an alumina crucible, and then use dry nitrogen gas at a rate of 40 mL / min as the purge gas to perform a heating scan in the temperature range of 0-250 °C at a heating rate of 10 K / min.

[0055] The results are as follows Figure 2 As shown, crystalline DIC exhibits a sharp endothermic melting peak at 180.62 °C, while the DSC spectrum of crystalline MTX shows two endothermic peaks, with 139.24 °C belonging to the lattice water desorption peak and 213.28 °C being the characteristic melting point peak. The DIC-MTX co-amorphous system prepared by rotary evaporation shows significant changes: the characteristic melting peaks of both DIC and MTX disappear, and a typical glass transition step appears at 62.5 °C.

[0056] (3) Thermogravimetric analysis (TG / DTG)

[0057] Quantitatively weigh 8 mg of DIC, MTX, a physical mixture of DIC-MTX, and the sample from Example 1 were amorphously sealed in an alumina crucible. TG analysis was performed at a temperature range of 35-700 °C, with a flow rate of 40 mL / min of dry nitrogen and a heating rate of 10 K / min.

[0058] The results are as follows Figure 3 As shown, (A) and (B) are the TG and DTG curves for DIC crystalline state, MTX crystalline state, and DIC-MTX co-amorphous state, respectively. In the TG spectrum, DIC begins to lose weight slowly from 155 °C and loses weight completely at 300 °C; MTX crystalline state undergoes a two-step weight loss process. The first step of weight loss occurs from 65 °C to 105 °C, which is due to the removal of free water by MTX during the endothermic process. The second step of significant weight loss begins at 215 °C, which is due to the decomposition of MTX itself; while the co-amorphous state begins to lose weight slowly from 200 °C, and its initial temperature is earlier than that of MTX crystalline state (215 °C), falling between DIC and MTX.

[0059] DTG analysis revealed distinct thermal decomposition characteristics in DIC, MTX, and their co-amorphous forms: both DIC and the co-amorphous form exhibited a single weight loss rate peak, while MTX showed two peaks. This difference indicates a fundamental difference in the decomposition behavior of the co-amorphous form compared to MTX. Combined with the thermogravimetric analysis results, no solvent evaporation stage was detected in the co-amorphous sample, confirming the successful preparation of a solvent-free co-amorphous system using rotary evaporation. Although both crystalline and co-amorphous DIC exhibited single-peak decomposition on DTG, their decomposition kinetics differed significantly. Crystalline DIC underwent a slow decomposition process before reaching the maximum weight loss rate; in contrast, the co-amorphous form exhibited rapid, single-stage decomposition, demonstrating its unique thermal decomposition pathway. Specifically, the maximum decomposition rate of crystalline MTX occurred at 252 °C (its peak temperature on the DTG curve). The characteristic decomposition peak of crystalline DIC was located at 272 °C, while the maximum decomposition rate temperature of the co-amorphous form fell between these two, at 260 °C. These significant differences revealed by the TG / DTG data further corroborate that the DIC-MTX composite prepared by rotary evaporation is a novel phase that is different from both the DIC crystalline state and the MTX crystalline state.

[0060] (4) Polarizing microscopy (PLM) analysis

[0061] An appropriate amount of DIC, MTX, and DIC-MTX co-amorphous powder from Example 1 were evenly spread on a glass slide and observed under a polarizing microscope.

[0062] Figure 4 PLM images of DIC and MTX monomers and their amorphous powder samples prepared at a 1:2 molar ratio are shown. Observations reveal that the DIC monomer exhibits a typical needle-like crystal morphology, while MTX displays a bulk crystal structure; both show significant birefringence. In contrast, the sample prepared by rotary evaporation appears as an irregular powder, and no birefringence is observed under polarized light.

[0063] (5) FT-IR detection

[0064] Measurements were performed using the KBr pellet method on an FT-IR-8400S (Shimadzu Corporation). Spectroscopically pure KBr was dried, thoroughly ground, and pelleted for background collection. Then, 2 mg of DIC, MTX, a physical mixture of DIC-MTX, and co-amorphous powder were mixed with 160 mg of spectroscopically pure KBr, thoroughly ground in a mortar, pelleted, and measured at 4000-400 cm⁻¹. -1 Scanning within the wavelength range, with a test resolution of 4 cm. -1 Use Spectrum software to record and process chromatograms.

[0065] The amorphous FT-IR spectra of DIC active pharmaceutical ingredient, MTX active pharmaceutical ingredient, PM, and DIC-MTX are as follows: Figure 5 As shown in the infrared spectrum of DIC, the characteristic absorption peak of -C=O is located at 1694 cm⁻¹, and the characteristic absorption peak of -OH is located at 3323 cm⁻¹. -1 The characteristic absorption peak of -NH is located at 1507 cm⁻¹. -1 This is consistent with reports in the literature; in MTX, 1643 cm -1 The characteristic absorption peak at -C=O is 3422 cm⁻¹. -1 There is only one single peak at this point, which is the characteristic absorption peak of -NH (secondary amide), consistent with the data in the literature.

[0066] The shift of vibrational peaks and changes in bandwidth are important characteristics of molecular interactions. The figure clearly shows that the infrared spectrum of the DIC and MTX physical mixture is a simple superposition of their infrared spectra, while the co-amorphous infrared spectrum differs from both active pharmaceutical ingredients and their physical mixture. In DIC, the carbonyl absorption peak at 1694 cm⁻¹ shifts to 1654 cm⁻¹. -1 Location; in MTX, 3422 cm -1 The amide absorption peak shifted to 3413 cm⁻¹ -1 Therefore, it is speculated that the -C=O bond of DIC and the -NH bond (secondary amide) on MTX may have undergone intermolecular interaction, forming a C=O…HN hydrogen bond.

[0067] (6) Raman spectroscopy analysis

[0068] Measurements were performed using an ATR3110-785nm laser (Aopu Tiancheng Optoelectronics Co., Ltd.). Appropriate amounts of DIC, MTX, a physical mixture of DIC-MTX, and co-amorphous powder were spread evenly on a clean glass slide for measurement. The excitation wavelength was 785 nm, the laser power was 200 mW, and the scanning range was 4000-400 cm⁻¹. -1 An average of 3 scans were performed, with a resolution of 4 cm. -1 The integration time is 5 s and the sampling interval is 1 s.

[0069] DIC, MTX raw materials, and co-amorphous Raman spectra, such as Figure 6 As shown, the DIC peak position is 1600.2 cm⁻¹. -1 and 1575.8cm -1 It is generated by the C=O and C=C and stretching vibrations of the benzene ring; 1289.3 cm -1 The peak is due to the stretching vibration of CN; 1235.3 cm -1 The peak is due to the stretching vibration of CO; 1092.4 cm⁻¹ -1The peak is generated by the C-Cl stretching vibration; 836.2 cm⁻¹ -1 The peak is due to the out-of-plane bending vibration of the CH group of the benzene ring. In MTX, the peak is 1640.7 cm⁻¹. -1 The peak at 1601.5 cm⁻¹ is generated by the C=O stretching vibration (amide I band). -1 It is the C=C vibration peak on the benzene ring, at 1548.9 cm⁻¹. -1 It is the -NH bending vibration peak (amide I band), 1335.6 cm⁻¹. -1 and 1240.7 cm -1 The peaks at these locations are generated by the stretching vibrations of CN and CO, respectively.

[0070] After DIC and MTX form a co-amorphous structure, 1575.8 cm⁻¹ in DIC -1 The C=C peak at this location merges with surrounding peaks, while the C=O peak decreases from 1600.2 cm⁻¹. -1 The location has moved to 1614.3 cm. -1 1548.9 cm in MTX -1 The -NH peak shifted to 1555.7 cm⁻¹. -1 The shifts in these peaks suggest a strong interaction between the C=O group of DIC and the NH group of MTX, resulting in changes in the bond lengths, linkages, and spatial conformations of the two molecules compared to the active pharmaceutical ingredient. Hydrogen bonding interactions have formed between them, consistent with the results of infrared analysis.

[0071] Experimental Example 3 Oil-water partition coefficient

[0072] Using the classic shake-flask method, n-octanol and water were mixed in a 1:1 volume ratio in a 250 mL stoppered conical flask, sealed tightly, and placed at 37 ℃ and 200 rpm·min. -1 Shake in a constant temperature shaker for 24 h until saturated. Accurately weigh DIC raw material, MTX raw material, PM and CO into a 10 mL EP tube, add 4 mL of water-saturated n-octanol solution and 4 mL of water-saturated n-octanol aqueous solution, seal and place the EP tube at 37 ℃ and 200 rpm·min. -1 Shake again in a constant-temperature shaker for 24 h until equilibrium is reached. Perform the experiment in triplicate. Filter the oil and aqueous phases through a 0.45 μm filter membrane, and determine the peak area using high-performance liquid chromatography (HPLC). Calculate the concentrations C of DIC and MTX in the oil phase, respectively. O and its concentration C in the aqueous phase W The oil-water partition coefficient log P of both components was calculated and a bar chart was plotted.

[0073] P=C O / C W

[0074] P: Apparent oil-water distribution coefficient; C O : Concentration of the sample in the oil phase (μg / mL); C W : Concentration of the sample in the aqueous phase (μg / mL); log P: Oil-water partition coefficient.

[0075] The results of the oil-water partition coefficients of DIC and MTX are shown in the figure. Figure 7 Tables 1 and 2 show that for DIC, the log P value of crystalline DIC is 1.93, and the log P value of CO-DIC is 1.74. DIC belongs to BSC Class II drugs, which are highly lipophilic and poorly water-soluble. This result indicates that the hydrophilicity of DIC is slightly improved after the formation of the co-amorphous form. For MTX, MTX has poor lipophilicity, which affects drug absorption and reduces bioavailability. In this experiment, the log P value of crystalline MTX is -1.73. After forming the co-amorphous form with DIC, the log P value increases to -0.28, indicating that the lipophilicity of MTX is greatly improved, which is more conducive to its absorption in the gastrointestinal tract.

[0076] Table 1. Oil-water partition coefficients of DIC crystalline and DIC in physical mixtures and co-amorphous forms.

[0077]

[0078] Table 2 Oil-water partition coefficients of MTX in crystalline and physical mixtures and co-amorphous forms

[0079]

[0080] Test Example 5 Permeability

[0081] The in vitro permeation characteristics of DIC, MTX, PM, and CO were evaluated using a Franz vertical diffusion cell system (effective diffusion area 1.327 cm², receiving chamber volume 16 mL). The experimental setup was as follows: 1 mL of an aqueous solution containing 20 μg·mL⁻¹ DIC and 61.38 μg·mL⁻¹ DIC were injected into the donor chamber, respectively. -1 An aqueous solution of MTX and aqueous solutions of PM and CO containing equal doses of API were prepared; the receiving chamber was filled with 16 mL of pure water. The entire apparatus was equilibrated in a constant temperature water bath at 37 ± 0.5 ℃, and the solution in the receiving chamber was continuously magnetically stirred at 300 rpm. An artificial membrane was installed between the donor and receiver chambers to remove air bubbles. 100 μL samples were taken at time intervals of 10, 20, 30, 45, 60, 90, 120, 180, and 240 min, and an equal volume of aqueous solution at the same temperature was added simultaneously. The experiment was performed in triplicate. The samples at each time point were filtered through a 0.45 μm filter, and the peak area of ​​the samples was measured by high performance liquid chromatography at a wavelength of 276 nm. The cumulative diffusion was calculated, and a cumulative permeability curve was plotted.

[0082]

[0083] Q n : Cumulative drug permeation per unit area in hour n (μg·cm³) -2 ); C n Concentration at hour n (μg·mL) -1 V: Volume of aqueous solution in the receiving cell (mL); A: Diffusion surface area (cm²) 2 ).

[0084] Permeability, influenced by the oil-water partition coefficient, is a key parameter for measuring the entry of APIs into the bloodstream and can predict the drug's absorption characteristics in the body. For example... Figure 8 , 9 The cumulative permeability curves for DIC and MTX are shown in the figure. As can be seen from the figure, the cumulative permeability of crystalline DIC, crystalline MTX, PM, and CO all showed an increasing trend during the 240-minute permeation experiment, gradually approaching equilibrium and reaching a peak at 4 hours. The cumulative permeability of crystalline DIC, crystalline MTX, DIC in PM and CO, and MTX in PM and CO were 71.37, 41.78, 70.07, 70.40, 49.56, and 103.70 μg·mL, respectively. -1 .

[0085] For DIC, the cumulative permeation of CO was higher than that of crystalline DIC within 40 minutes, but after 4 hours, there was no significant difference in the cumulative permeation of CO and crystalline DIC, indicating that the permeability of DIC did not increase after CO formation. For MTX, the permeation rate of MTX in CO was faster than that of crystalline MTX and PM-MTX within 2 hours, and the final cumulative permeation was also higher than both, increasing by about 2.48 times. In summary, the permeability of DIC in CO did not increase, while the permeability of MTX in CO increased, increasing by about 2.48 times, which is consistent with the results of the oil-water partition coefficient.

[0086] Test Example 7 Stability

[0087] The newly prepared DIC-MTX co-amorphous system was processed through an 80-mesh sieve and then placed under three stability test conditions: stored for 8 months in a desiccator containing color-changing silica gel at 25°C.

[0088] The PXRD patterns of the samples during the stability study of the DIC-MTX co-amorphous samples are as follows: Figure 10 As shown. After 8 months of room temperature drying storage, the PXRD pattern showed that the co-amorphous sample still exhibited diffuse diffraction rings, indicating that the DIC-MTX co-amorphous sample maintains good physical stability under drying conditions and is suitable for storage.

[0089] Experimental Example 8 Pharmacokinetic Determination of Diclofenac-Methotrexate

[0090] This experiment was approved by the Animal Welfare and Ethics Committee (approval number: IACUC-Hebmu-2024157). The experimental animals were healthy male SD rats, SPF grade, weighing 200 ± 10 g, provided by the Experimental Animal Center of Hebei Medical University (animal production license number: SCXK (Ji) 2022-001). The rats were housed at room temperature (25 ± 2) °C and humidity (50 ± 5) °C. They were adaptively housed for 1 week before the experiment, with a light cycle of 12 h light and dark alternation, and free access to water. They were fasted for 12 h before the experiment but had free access to water.

[0091] Grouping: 32 SD rats were randomly divided into 4 groups of 8 rats each, namely the DIC group, MTX group, PM group, and CO group, with 8 rats in each group.

[0092] Preparation of gavage solution: 0.5% sodium carboxymethylcellulose (CMC-Na) solution: Accurately weigh 1 g of CMC-Na, add 200 mL of double-distilled water, heat and stir on a magnetic stirrer at 50 °C until completely dissolved, and set aside for use.

[0093] Accurately weigh 36.00 mg of DIC crystalline, 110.48 mg of MTX crystalline, 146.48 mg of PM, and 146.48 mg of CO respectively, and dissolve them in 30 mL of 0.5% CMC-Na solution, and sonicate for 20 min to make the suspension homogeneous.

[0094] Administration: By gavage (fasted for 12 h before gavage but had free access to water, and weigh each rat and record its weight), each animal was given the corresponding dose of drug suspension according to its weight (equivalent to DIC 9.00 mg·kg-1, MTX 27.62 mg·kg-1). After each group of rats was administered, 0.1 mL of jugular vein blood was collected at the sampling time points of 0, 0.083, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 h. Then centrifuge at 3500 rpm and 4 °C for 10 min within 30 min, take the supernatant, and store it in a -80 °C refrigerator for later use.

[0095] Accurately measure 10 μL of the supernatant and inject it into the liquid chromatograph, recording the chromatogram. Construct an individual drug-time profile (Plasmaconcentration-time profile) with the sampling time point (h) as the x-axis and the measured blood drug concentration (μg·mL⁻¹) as the y-axis. Using the DAS 2.0 pharmacokinetic analysis system, calculate key parameters based on a non-compartmental analysis (NCA) model: time to peak concentration (Tmax), peak concentration (Cmax), elimination half-life (t¹ / ²), and areas under the plasma concentration-time curve (AUC0-t and AUC0-∞).

[0096] The concentrations of DIC and MTX were determined by HPLC after methodological validation. The drug-time curves are shown below. Figure 11 As shown in Tables 3 and 4, the pharmacokinetic parameters are as follows. For DIC, the AUC0-t of CO was 1.20 times higher than that of PM (P<0.05), and the Cmax was 1.47 times higher (P<0.05). These results indicate that the bioavailability of DIC is significantly improved after co-amorphization; the co-amorphous form of the drug is better absorbed than the crystalline form, and the more sufficient contact with the gastrointestinal tract facilitates absorption. For MTX, the AUC0-t of CO was higher than that of PM. 0-t Increased by 1.24 times ( P <0.05), AUC 0-∞ Increased by 1.26 times ( P <0.05), C max Increased by 1.33 times ( P <0.05), t 1 / 2 It was extended by 1.12 times ( P <0.05). The results indicate that the poor lipid solubility of MTX crystals leads to poor absorption, but the bioavailability is significantly improved after the formation of the co-amorphous form.

[0097] Table 3. Mean pharmacokinetic parameters of DIC in rats after administration ( n =6, x̅ ± sd)

[0098]

[0099] Note:* P <0.05, ns = no significance (vs. PM)

[0100] Table 4. Mean pharmacokinetic parameters of MTX in rats after administration (n=6, x̅ ± sd)

[0101]

[0102] Note:* P <0.05, ns = no significance (vs. PM)

[0103] Experimental Example 9: Study of a Macrophage Inflammation Model

[0104] 1. Cell culture and treatment:

[0105] RAW 264.7 cells were cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 100 U·mL⁻¹ penicillin, and incubated at 37 °C, 5% CO₂, and 95% humidity. Cells were passaged when they covered more than 80% of the bottom of the culture flask.

[0106] 2. Cell Culture

[0107] RAW 264.7 cells were cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 100 U·mL⁻¹ penicillin, and incubated at 37 °C, 5% CO₂, and 95% humidity. Cells were passaged when they covered more than 80% of the bottom of the culture flask.

[0108] 3.1. Cell viability assay

[0109] To evaluate the inhibitory effect of the drug on M0 and M1 macrophages and determine the safe drug concentration, RAW 264.7 cells were cultured at a concentration of 1 × 10⁻⁶. 5 Cells were seeded at a density of 10% FBS in 96-well plates of DMEM containing 10% FBS and cultured overnight at 37 °C for 24 h in a CO2 incubator. After cell adhesion, the cells were divided into two groups. One group was treated with 1 μg / mL DMEM. -1 One group was treated with 50 μL of LPS (lipopolysaccharide) for 12 h to polarize M0 macrophages into M1 macrophages before being treated with the drug. The other group was not polarized and was treated directly.

[0110] Control and experimental groups were set up. Control group: Control cells (with sample dissolution solvent) were grown in DMEM containing 10% FBS without treatment (no drugs added); Experimental group: Cells were treated with different concentrations of sample (DIC: 0, 0.032, 0.16, 0.32, 1.6, 3.2, 6.4, and 12.8 μg·mL⁻¹). -1 MTX: 0, 0.1, 0.5, 1, 5, 10, 20 and 40 μg·mL -1The final volume was 100 μL / well, and three copies were prepared for each treatment. The incubation was carried out at 37 ℃ and 5% CO2 for 24 h. After the treatment, 10 μL of CCK-8 was added to each well and incubated for another 2 h. The absorbance of the formed formazan was measured at 450 nm using an enzyme-linked immunosorbent assay (ELISA) spectrophotometer. The cell viability at different drug concentrations was calculated using the following formula, and the highest drug concentration with cell viability >80% was used for subsequent experiments.

[0111] CV (%) = [OD 490 (experimental group) / OD 540 (control group)] × 100%

[0112] The toxicity of the DIC-MTX physical mixture and co-amorphous mixture to M0 and M1 macrophages was then determined. RAW264.7 cells were cultured and adhered to the culture vessel as described above, with one group receiving LPS stimulation and the other receiving no stimulation. Test samples (PM and CO at the same maximum safe concentrations as DIC and MTX) were then added, and cell viability was calculated after 24 hours of culture as described above.

[0113] like Figure 12 The figure shows the survival rate of RAW 264.7 cells after treatment with different concentrations of DIC and MTX. As can be seen from the figure, when the DIC concentration is ≤ 1.6 μg / mL, the cell viability of RAW 264.7 cells can be maintained above 80%; when the MTX concentration is ≤ 0.5 μg / mL, the cell viability of RAW 264.7 cells can be maintained above 80%. Therefore, in subsequent experiments, the concentrations of DIC and MTX used should not exceed the concentrations required to maintain 80% cell viability.

[0114] Subsequently, the toxicity of DIC-MTX physical mixtures and co-amorphous compounds to M0 and M1 macrophages was determined, such as... Figure 13 As shown, the concentrations of PM and CO are the same as the maximum safe concentrations of DIC and MTX. After 24 h of culture, the cell viability was above 80%, indicating that the selected concentrations of PM and CO are safe and have no obvious toxicity to cells.

[0115] 3.2. ROS cleanup capability

[0116] Logarithmically growing M0 type RAW 264.7 cells were cultured in 24-well plates and divided into Control, LPS, DIC, MTX, PM, and CO groups. After 24 h of culture, except for the Control group, all cells were treated with a final concentration of 100 ng / mL. -1 Treatment with 50 μL of LPS for 12 h, followed by different drug groups (DIC, MTX, PM, CO, equivalent to a DIC concentration of 0.16 μg / mL and an MTX concentration of 0.5 μg / mL).-1 Cells were treated for 24 h, washed with PBS, and ROS fluorescent redox probe DCFH-DA (1:1000 dilution, 1 uL + 1 mL serum-free medium) was added to monitor intracellular ROS levels. Cells were then incubated with shaking at 37 ℃ for 30 min, washed with PBS, fixed with 4% paraformaldehyde for 20 min, and the cell nuclei were stained with DAPI for 20 min. Cells were then mounted and the staining results were observed under a fluorescence microscope.

[0117] like Figure 14 As shown in (A, B), DCFH-DA was used as a probe to monitor the total ROS level in cells. The results showed that RAW 264.7 macrophages without LPS activation showed the lowest ROS, while macrophages activated by LPS showed a large amount of ROS. Both DIC and MTX active pharmaceutical ingredients effectively scavenged some reactive oxygen species, while the CO group showed the weakest fluorescence intensity and effectively scavenged almost all reactive oxygen species. It is evident that DIC, MTX, and PM have a certain inhibitory effect on LPS-induced endogenous ROS in macrophages, and the DIC-MTX co-amorphic form has a stronger inhibitory effect on endogenous ROS. Therefore, it can be concluded that after DIC and MTX form a co-amorphic form, their inhibitory effect on endogenous ROS becomes stronger, and their anti-inflammatory effect is enhanced, potentially exhibiting excellent ROS scavenging ability for RA treatment.

[0118] 3.3. Cell smear immunofluorescence homologous double labeling

[0119] Immunofluorescence staining was used to stain iNOS, COX-2, and CD206 proteins in RAW 264.7 cells from the Control, LPS, DIC, MTX, PM, and CO groups to evaluate the inflammatory response. RAW 264.7 cells were seeded in 24-well plates (4 × 10⁻⁶). 4 In a well, cells were cultured for 24 h, ensuring cell attachment, and then mixed with LPS (100 ng / mL). -1 RAW 264.7 cells were incubated for 24 h to induce activation (Control group without LPS). Subsequently, RAW 264.7 cells were treated with DIC, MTX, PM, and CO for 24 h, respectively, fixed with 4% paraformaldehyde at room temperature for 20 min, infiltrated with 0.1% Triton X-100 at room temperature for 20 min, and blocked with 5% bovine serum albumin (BSA) at room temperature for 1 h. Cells were incubated overnight at 4 °C in the dark with primary antibodies against iNOS (1:50), COX-2 (1:50), and CD206 (1:1000), followed by incubation with fluorescently labeled secondary antibodies at room temperature in the dark for 1 h. Cell nuclei were stained with DAPI, and the staining results were observed using a fluorescence microscope. The expression and localization of iNOS, COX-2, and CD206 in RAW 264.7 cells were recorded and analyzed.

[0120] Such as Figure 15 (A, B, C) and Figure 16 (A, B) were used to study the immunomodulatory effect of the co-amorphous formed by DIC and MTX on RAW 264.7 polarized cells through immunofluorescence staining.

[0121] The phenotypes of macrophages were determined by immunofluorescence staining of specific cell surface markers iNOS, COX-2 (M1 marker) and CD 206 (M2 marker). Immunofluorescence staining images of iNOS, COX-2 and CD 206 showed that the expression levels of iNOS and COX-2 in LPS-activated macrophages were significantly up-regulated compared with those in unactivated macrophages. Compared with DIC alone, MTX crystals and their physical mixtures, the co-amorphous of DIC-MTX was more capable of enhancing the protein expression of CD 206-labeled M2 and reducing the protein expression of iNOS and COX-2-labeled M1.

[0122] The above results indicate that the formation of the co-amorphous system helps macrophages polarize from the M1 phenotype to the M2 phenotype and reduce inflammation, making it a preferred candidate for RA treatment.

[0123] Experimental Example 10 Evaluation of Anti-inflammatory Activity in Vivo

[0124] 1. Animals

[0125] This experiment has been approved by the Experimental Animal Welfare and Ethics Committee (Approval No.: IACUC-Hebmu-2024157). Healthy male SD rats, SPF grade, weighing 180 ± 10 g, were provided by the Experimental Animal Center of Hebei Medical University (Animal Production License No.: SCXK (Ji) 2022-001). The rats were housed in an environment with a room temperature of (25 ± 2) °C and a humidity of (50 ± 5) %, acclimated for 1 week before the experiment, with a light cycle of 12 h light and dark alternation, free access to water, and fasted for 3 h but not water before the experiment.

[0126] 2. Experimental Method

[0127] 2.1. Preparation of Gavage Solutions

[0128] 0.5% Sodium Carboxymethylcellulose (CMC-Na) Solution: Accurately weigh 1 g of CMC-Na, add 200 mL of double-distilled water, heat and stir on a magnetic stirrer at 50 °C until completely dissolved, and set aside.

[0129] Accurately weigh 2.00 mg of DIC, 6.00 mg of MTX, 8 mg of PM, and 8 mg of CO respectively into 20 mL of 0.5% CMC-Na solution, and ultrasonicate for 20 min to make them evenly suspended.

[0130] 2.2. Establishment and grouping treatment of adjuvant-induced arthritis (AIA) rat model

[0131] After one week of acclimatization, male SD rats were randomly divided into a normal control group (n=6) and a model group (n=30). Except for the normal control group (Normol), rats in the model group received a subcutaneous injection of 150 μL of complete Freund's adjuvant (whirled and mixed before injection) into the right hind paw, while rats in the normal control group received an equal volume of physiological saline at the same site. After model establishment, paw swelling was observed, and adequate food and water supply was ensured. Significant redness and swelling of the right hind paw and a significant decrease in the pain threshold were considered indicators of successful model establishment, indicating that the AIA rats were in the acute inflammatory phase.

[0132] Using the day of model establishment as d1, successfully established models were randomly divided into five groups on d2: AIA model group; DIC group; MTX group; DIC-MTX physical mixture (PM) group; and DIC-MTX co-amorphous (CO) group, with six animals in each group. According to the gavage solutions prepared in "2.3.2.1," the DIC group was administered DIC solution at 0.30 mg·kg⁻¹, the MTX group at 0.90 mg·kg⁻¹, and the PM group at 1.20 mg·kg⁻¹. -1 PM solution was administered by gavage; the CO group was administered at a dose of 1.20 mg / kg. -1 The control group received CO solution by gavage, while the normal group received only normal saline by gavage. All treatments started on day 2, with administration once a week for a total of four times over 28 days.

[0133] 2.3. Evaluation of changes in rat paw thickness and rat joint index

[0134] After successfully inducing an AIA rat model, the drug was administered at four-week time points, with untreated healthy rats serving as normal controls. Changes in rat paw thickness were continuously monitored over the next four weeks.

[0135] The AIA rat model enters the chronic inflammatory phase after approximately 10 days, at which point the arthritis index can be evaluated. Untreated healthy rats serve as normal controls. After approximately 10 days, the AIA rat model enters the chronic inflammatory phase, and arthritis index evaluation begins. Untreated healthy rats serve as normal controls, and arthritis index scoring is performed every 2 days starting from day 10, continuing for 10 days.

[0136] The Arthritis Index (AI) scoring criteria are as follows:

[0137] No clinical signs, normal activity, no redness or swelling of joints: 0 points; slight diffuse redness and swelling of toe joints: 1 point; swelling of toe joints and plantar edema: 2 points; swelling of plantar edema below the ankle joint: 3 points; swelling of the ankle joint and the entire foot, with inability to bend the toes: 4 points.

[0138] Results: The degree of redness and swelling of the rat paws was recorded over a period of one month, such as... Figure 17 As shown in (A), images of rat paws reveal that the AIA group consistently exhibited severe swelling of the paws and joints. In contrast, the groups treated with DIC, MTX, PM, and CO all showed some degree of relief of paw inflammation over a one-month period, with the CO-treated group showing the most significant improvement in paw and joint swelling.

[0139] Changes in paw thickness in rats after drug treatment are as follows: Figure 17 As shown in (B, C), quantitative assessment of paw thickness further confirmed the above results. On day 30, the paw thickness in the CO-treated group was comparable to that in the normal group, with no significant difference. The clinical arthritis scores of the rats during treatment are shown below. Figure 17 As shown in (D, E), except for the AIA group, all other treatment groups showed a trend of decreasing scores. P The CO treatment group showed the most effective treatment outcome, exhibiting a low score below 1 at day 30. These results indicate that the CO group was the most effective at alleviating inflammation in AIA rats among all treatment groups.

[0140] 2.4. Evaluation of drug therapeutic effects using micro-CT imaging

[0141] The status of bone erosion in rat paws was evaluated using micro-CT imaging after treatment. All treatment groups were evaluated, including the normal control group, DIC group, MTX group, PM group, and CO group. Rats were euthanized, and their harvested paws were fixed with 4% paraformaldehyde for subsequent analysis. 3D images of rat paws were generated using high-resolution micro-CT (80 kV, 0.06 mA, 35 μm scan resolution), and the original images were reconstructed using the 3D reconstruction software Recon. Furthermore, the target region ROI was analyzed using the data analysis software Avatar to examine the bone mineral density (BMD, g / cm³). 3 ) and bone surface area / bone volume (BS / BV, %).

[0142] like Figure 18 As shown in (A), micro-CT imaging of untreated AIA rats revealed severe bone erosion compared to the normal group. In contrast, the DIC, MTX, PM, and CO treatment groups showed improvement, with clear bone boundaries and fewer signs of erosion. The claw bone structure in the CO treatment group showed a very stable bone structure with minimal erosion, comparable to that of normal rats. This suggests that the DIC-MTX co-amorphic therapy can better inhibit the progression of RA and reduce articular cartilage damage.

[0143] like Figure 18As shown in (B), compared with the untreated AIA group, the CO treatment group showed a significant increase in BMD and a decrease in BS / BV, while other treatment groups also showed some improvement in BMD and BS / BV.

[0144] 2.5. Histopathological evaluation of rat ankle joint tissue

[0145] The bones on both sides of the right ankle joint of the rat were severed using surgical scissors, leaving approximately 0.5 cm of bone tissue on each side of the joint. The ankle joint was immediately fixed in 10% neutral formalin for 48 hours, and then placed in 15% EDTA neutral decalcification solution for decalcification. The decalcification solution was changed daily until the ankle joint tissue could be easily punctured with a needle. The ankle joint was then removed and dissected along the sagittal plane, followed by dehydration and paraffin embedding. Synovitis was assessed by hematoxylin and eosin (H&E) staining, Masson staining, and safranin-fast green staining of the right ankle joint. The morphology, structure, synovial hyperplasia, and inflammatory infiltration of the synovium were observed under an optical microscope, and images were acquired and analyzed.

[0146] H&E staining results of rat ankle joint tissue are as follows Figure 19 As shown, compared with normal rats, rats treated with CO exhibited clear and intact synovial structure with only a small amount of fibrous tissue hyperplasia and no other obvious pathology, consistent with literature reports. Conversely, H&E staining images of rat joints in the AIA model group and the DIC and MTX crystalline treatment groups showed symptoms of synovial hyperplasia, extensive fibrous tissue hyperplasia, and immune cell infiltration, which are typical features of RA. Rats treated with PM showed an intermediate disease state. Similarly, in Masson staining and Safranin-Fix-Green staining results, rats treated with CO showed uniform thickness of articular cartilage and intact tidal lines, similar to normal rats. In contrast, rats in the AIA model group or those treated with DIC and MTX raw materials showed varying cartilage thickness and incomplete tidal lines.

[0147] 2.6. Effects of Rat Serum Cytokines

[0148] Serum samples were randomly selected from three rats in each group to assess inflammation levels. The expression of cytokines TNF-α, IL-6, IL-1β, and NO in the serum was assessed using an ELISA kit according to the manufacturer's instructions.

[0149] like Figure 20Table 5 shows the serum cytokine (TNF-α, IL-6, IL-1β, NO) levels in rats one month after treatment. The serum TNF-α, IL-6, IL-1β, and NO cytokine levels in the model AIA group were significantly higher than those in the normal group (P<0.001). The serum TNF-α, IL-6, IL-1β, and NO cytokine levels in the CO treatment group were significantly lower than those in the model group (P<0.001), and showed the lowest cytokine levels compared to other groups, indicating levels close to those in the normal group. In conclusion, DIC-MTX co-amorphic therapy effectively alleviates RA symptoms and significantly enhances the therapeutic efficacy of RA treatment.

[0150] Table 5. Serum TNF-α, IL-6, IL-1β, and NO levels in rats of each group after treatment.

[0151]

[0152] Note: * P <0.05, ** P <0.01, *** P <0.001 (vs. PM group)

[0153] 2.7. Determination of biochemical indicators in rat heart, liver, and kidney

[0154] To assess the effects of DIC, MTX, PM, and CO on cardiac, hepatic, and renal function, serum was collected from each group of rats before sacrifice and tested using an automated blood biochemistry analyzer for six indicators: CKMB (creatine kinase isoenzyme), LDH (lactate dehydrogenase), ALT (alanine aminotransferase), AST (aspartate aminotransferase), UA (uric acid), and UREAL (urea).

[0155] The results are as follows Figure 21 As shown, the normal ranges for CKMB and LDH in rats were 58.24-530.38 and 456.51-1041.07 U / L, respectively; the normal ranges for ALT and AST were 21.53-61.75 U / L and 41.47-195.65 U / L, respectively; and the normal ranges for UA and UREAL in rats were 58.38-122.65 μmol / L and 3.5-8.11 mmol / L, respectively. All biochemical indicators related to cardiac, hepatic, and renal function in rats were within the normal range, and there were no statistically significant differences between groups (p>0.05), indicating that the drug had no effect on cardiac, hepatic, and renal function in rats.

[0156] 2.8. H&E staining of rat organs

[0157] To further evaluate the safety of the formulation, rats were dissected, and their hearts, livers, spleens, lungs, and kidneys were removed and fixed in 10% neutral formalin for 48 h for organ H&E staining to observe whether there were any pathological changes.

[0158] H&E staining results of organs as follows Figure 22 As shown, pathological observation of lung tissue in the model group revealed scattered inflammatory cell infiltration in some air-conducting regions, thickening of alveolar septa with mild fibrosis, and a reduction in capillaries and elastic fibers. Numerous lymphocytes were observed infiltrating the septa, exhibiting typical pathological features of interstitial lung inflammation. No such pathological changes were observed in the other experimental groups, suggesting this phenomenon is related to rheumatoid arthritis-induced interstitial lung disease (RA-ILD). No pathological abnormalities were observed in the sections of other organs in any group; the sections were smooth in color and normal in size, indicating that the drugs in each group had no significant toxicity to the organs.

[0159] 2.9. Safety Evaluation

[0160] Changes in body weight are one of the indicators for evaluating the toxicity of drugs in vivo. During the treatment of rats, the body weight of the rats was measured once a week.

[0161] The changes in body weight of rats in each group are as follows: Figure 23 As shown, with the increase of treatment time, the body weight of rats in each group showed a slow upward trend, but there was no statistically significant difference in body weight between the groups (p>0.05), which preliminarily indicates that the drug has no toxic effect on rats.

[0162] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A diclofenac-methotrexate co-amorphous compound, characterized in that, The co-amorphous compound comprises diclofenac and methotrexate, wherein the molar ratio of diclofenac and methotrexate is 1:2; The co-amorphous material, when subjected to Cu-kα radiation, exhibits X-ray powder diffraction spectra expressed in 2θ without sharp diffraction peaks. The glass transition temperature of the amorphous material is 62.5 ± 0.5 °C.

2. The diclofenac-methotrene co-amorphous compound according to claim 1, characterized in that, The co-amorphous compound was prepared by rotary evaporation.

3. A method for preparing the diclofenac-methotrexate co-amorphous compound as described in claim 1 or 2, characterized in that, Diclofenac and methotrexate were added to a solvent, and the sample was ultrasonically treated for 10-15 min at a solid-liquid ratio of 1-3 mg / mL. The sample was then dried under vacuum by rotary evaporation at 60±2℃ to obtain the co-amorphous product. The solvent is composed of solvent A and solvent B, wherein solvent A is selected from ethanol and solvent B is selected from N,N-dimethylformamide.

4. The method for preparing the diclofenac-methotrexate co-amorphous compound according to claim 3, characterized in that, The volume ratio of solvent A to solvent B is 35-65:

1.

5. The use of a diclofenac-methotrexate coamorphous compound as described in claim 1 or 2 in the preparation of a medicament for rheumatoid arthritis.

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