Ticagrelor clathrate compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of ticagrelor, in particular to a ticagrelor clathrate compound and a preparation method and application thereof. The ticagrelor clathrate compound comprises ticagrelor and a macrocyclic compound for clathrating the ticagrelor, and the macrocyclic compound is at least one of cucurbit [7] uril, beta-cyclodextrin and hydroxypropyl beta-cyclodextrin. According to the invention, the macrocyclic compound and the ticagrelor are clathrated to form a stable drug clathrate, the solubility and pharmacological action of the ticagrelor are enhanced, and a preparation with excellent chemical stability, low biotoxicity and good drug release performance is provided. Meanwhile, the novel application of the ticagrelor clathrate compound in preparation of the medicine for treating systemic lupus erythematosus is defined.

Description

Technical Field

[0001] This invention relates to the field of ticagrelor technology, specifically to ticagrelor inclusion complexes, their preparation methods, and applications. Background Technology

[0002] Ticagrelor, a highly effective P2Y12 receptor antagonist, has become an important drug for the treatment of acute coronary syndromes due to its rapid onset of action and reversible inhibition of platelet aggregation. Its chemical name is (1S,2S,3R,5S)-3-[7-{[(1R,2S)-2-(3,4-difluorophenyl)cyclopropyl]amino}-5-(propylthio)-3H-[1,2,3]triazolo[4,5-d]pyrimidin-3-yl]-5-(2-hydroxyethoxy)cyclopentane-1,2-diol. The hydrophobic groups in its molecular structure result in extremely low water solubility (<10 μg / mL), with an oral bioavailability of only about 36%. This inherent limitation severely restricts its clinical applicability—low solubility not only reduces the drug's dissolution rate and absorption efficiency, but also may lead to side effects such as bleeding risk due to dose escalation in chronic disease treatments requiring long-term administration.

[0003] Currently, the mainstream technologies for improving ticagrelor solubility are cocrystallization, solid dispersion, and nanostructured lipid carrier technologies, all of which have significant limitations. While cocrystallization can dynamically improve solubility by combining with forming agents such as succinic acid and nicotinamide, the screening process relies on high-throughput experiments, which are time-consuming and have low success rates, and lack a universal library of cocrystallizing agents. Furthermore, the stability of reported cocrystallization systems in the complex in vivo environment has not been verified, posing a high risk for clinical translation. Solid dispersion technologies use polymer carriers such as polyvinylpyrrolidone and hydroxypropyl methylcellulose, but high-temperature melting or solvent evaporation processes can easily lead to drug degradation, and the conversion of amorphous to crystalline states during storage further causes a sharp drop in solubility. In addition, the problem of residual organic solvents makes it difficult to meet the ICH Q3C guidelines for drug impurity control, resulting in high compliance risks for industrial production. Although nanostructured lipid carriers can be solubilized through a lipid matrix, the drug loading capacity is generally less than 10%, resulting in low drug loading efficiency. During long-term storage, particle size increases (e.g., from 150 nm to 500 nm) and drug leakage are prominent issues, and industrial production costs are high, making it difficult to meet the economic requirements for long-term drug use.

[0004] Therefore, it is crucial to explore new technologies to improve the solubility of ticagrelor. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a highly soluble ticagrelor inclusion complex, its preparation method, and its application.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is: a ticagrelor inclusion complex, comprising ticagrelor and a macrocyclic compound containing ticagrelor, wherein the macrocyclic compound is at least one of cucurbita[7]urea, β-cyclodextrin and hydroxypropyl betacyclodextrin.

[0007] Another technical solution adopted in this invention is: the preparation method of the above-mentioned ticagrelor inclusion complex includes the following steps: adding ticagrelor and a macrocyclic compound to an acidic aqueous alcohol solution for reaction to obtain the ticagrelor inclusion complex.

[0008] Another technical solution adopted in this invention is: the preparation method of the above-mentioned ticagrelor inclusion complex includes the following steps: mixing and ball milling ticagrelor with a macrocyclic compound to obtain the ticagrelor inclusion complex.

[0009] Another technical solution adopted in this invention is: a pharmaceutical composition comprising the above-mentioned ticagrelor inclusion complex and a carrier, wherein the concentration of ticagrelor in the pharmaceutical composition is 0.01~10 mg / mL.

[0010] Another technical solution adopted in this invention is the application of the above-mentioned ticagrelor inclusion complex in the preparation of a drug for treating systemic lupus erythematosus (SLE).

[0011] The beneficial effects of this invention are as follows: This invention uses a macrocyclic compound to include ticagrelor, forming a stable drug inclusion complex, which enhances the solubility and pharmacological effects of ticagrelor, providing a formulation with excellent chemical stability, low biotoxicity, and good drug release performance. It also clarifies a new application of ticagrelor inclusion complexes in the preparation of drugs for the treatment of systemic lupus erythematosus. Attached Figure Description

[0012] Figure 1 The above is a PXRD (powder X-ray diffraction) characterization diagram of the ticagrelor@CB[7] inclusion complex in the embodiments of the present invention; Figure 2 The following are the infrared FTIR (Fourier transform infrared spectra) of ticagrelor, CB[7], and ticagrelor@CB[7] inclusion complexes in the embodiments of the present invention; Figure 3 The diagram shows the phase solubility of the ticagrelor@CB[7] inclusion complex in the embodiments of the present invention at different contents of CB[7]. Figure 4 The NMR spectrum of the ticagrelor@CB[7] inclusion complex in the embodiments of the present invention is shown below; Figure 5 The above are PXRD (powder X-ray diffraction) characterization images of ticagrelor@β-CD inclusion complex and ticagrelor@HP-β-CD inclusion complex in the embodiments of the present invention; Figure 6The above are TG (thermogravimetric analysis) characterization diagrams of ticagrelor@β-CD inclusion complex and ticagrelor@HP-β-CD inclusion complex in the embodiments of the present invention; Figure 7 The above are DSG (differential scanning calorimetry) characterization diagrams of ticagrelor@β-CD inclusion complex and ticagrelor@HP-β-CD inclusion complex in the embodiments of the present invention; Figure 8 This is a dissolution curve of ticagrelor and ticagrelor@HP-β-CD in an embodiment of the present invention; Figure 9 This is a QPCR (quantitative real-time nucleic acid amplification detection system) diagram showing the anti-inflammatory effect of ticagrelor@CB[7] inclusion complex in the embodiments of the present invention; Figure 10 This is a flow cytometry diagram illustrating the anti-inflammatory effect of the ticagrelor@CB[7] inclusion complex in this embodiment of the invention. Figure 11 This is a comparison chart of qPCR results for inflammatory markers in mice from different groups during animal experiments according to embodiments of the present invention; Figure 12 These are flow cytometry images of spleen cells from different groups of mice in animal experiments according to embodiments of the present invention. Figure 13 These are immunohistochemical images of mouse kidneys from different groups in animal experiments according to embodiments of the present invention; Figure 14 This is a damage score chart of different tissues in the kidneys of mice in different groups during animal experiments according to embodiments of the present invention; Figure 15 This is a comparison chart of serum anti-double-stranded DNA antibody concentrations in mice from different groups during animal experiments according to embodiments of the present invention. Detailed Implementation

[0013] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments and accompanying drawings.

[0014] A ticagrelor inclusion complex comprising ticagrelor and a macrocyclic compound containing ticagrelor, wherein the macrocyclic compound is at least one of cucurbita[7]urea, β-cyclodextrin and hydroxypropyl betacyclodextrin.

[0015] As can be seen from the above description, the beneficial effects of the present invention are as follows: The present invention uses at least one macrocyclic compound selected from cucurbita[7]urea, β-cyclodextrin and hydroxypropyl betacyclodextrin to encapsulate ticagrelor, forming a stable drug inclusion complex. The inclusion complex is shown to have amorphous characteristics by X-ray powder diffraction, and its differential scanning calorimetry curve has no endothermic peak at the melting point of ticagrelor, thus achieving optimization of ticagrelor solubility, bioavailability, biotoxicity and drug release performance, which helps to solve the problem that ticagrelor is limited in the treatment of systemic lupus erythematosus due to its low solubility.

[0016] The macrocyclic compounds CB[7] (cucurbita[7]urea), β-CD (β-cyclodextrin) and HP-β-CD (hydroxypropyl betacyclodextrin) used in this invention have good water solubility, large cavities, and high affinity for a variety of organic and inorganic guests. They have potential application value in the field of drug delivery and provide suitable carriers for the preparation of ticagrelor inclusion complexes.

[0017] Furthermore, the macrocyclic compound is cucurbita[7]urea.

[0018] As can be seen from the above description, the cavity size of cucurbita[7]urea is similar to that of cyclodextrin, but its cavity contains high-energy water molecules and has a special hydrophobic cavity, exhibiting a hydrophobic driving force that exceeds that of cyclodextrin. Therefore, ticagrelor and CB[7] are bound by supramolecular forces. When bound to guest molecules, these water molecules are displaced, resulting in a host-guest binding constant that is significantly higher than that of cyclodextrin. The inclusion of ticagrelor-CB[7] further improves its solubility, bioavailability and anti-inflammatory effect.

[0019] Another technical solution adopted in this invention is: the preparation method of the above-mentioned ticagrelor inclusion complex includes the following steps: adding ticagrelor and a macrocyclic compound to an acidic aqueous alcohol solution for reaction to obtain the ticagrelor inclusion complex.

[0020] The acid-containing alcohol-water solution is a solution composed of a mixture of acid, alcohol and water.

[0021] As can be seen from the above description, the preparation method of the present invention does not require other polymer carriers, has a short preparation cycle, and has the advantage of clinical translation.

[0022] Furthermore, the molar ratio of ticagrelor to the macrocyclic compound is 1:0.5~10.

[0023] Preferably, the molar ratio of ticagrelor to the macrocyclic compound is 1:1 to 5.

[0024] Preferably, the molar ratio of ticagrelor to the macrocyclic compound is 1:1.

[0025] As described above, if the proportion of macrocyclic compounds decreases, ticagrelor will not be adequately included, leaving free drug residues in the product. Conversely, an excess of macrocyclic compounds will reduce the purity of the inclusion complex.

[0026] Furthermore, the acid in the acid-containing aqueous alcohol solution is hydrochloric acid or sulfuric acid.

[0027] Furthermore, the concentration of acid in the acid-containing alcohol-water solution is 0.05~0.15M.

[0028] Preferably, the concentration of acid in the acid-containing aqueous alcohol solution is 0.08~0.12M.

[0029] As can be seen from the above description, maintaining the acidity of the solution ensures the protonation of ticagrelor and enhances the ion-dipole interaction with the negative carbonyl port of the macrocyclic compound. If the acid concentration is too low, the drug protonation is insufficient and the inclusion driving force is weakened. If the acid concentration is too high, it may cause drug degradation.

[0030] Furthermore, the alcohol in the acidic aqueous solution is methanol or ethanol.

[0031] Furthermore, the volume concentration of alcohol in the acid-containing aqueous alcohol solution is 5-20%.

[0032] Preferably, the volume concentration of alcohol in the acid-containing aqueous alcohol solution is 8-12%.

[0033] As can be seen from the above description, alcohol is used to improve the solubility of hydrophobic ticagrelor. Too low an alcohol concentration will result in insufficient solubility of ticagrelor, while too high an alcohol concentration will reduce inclusion stability.

[0034] Furthermore, the ratio of ticagrelor to the acid-containing alcoholic aqueous solution is 2-4 mg: 1 mL.

[0035] Furthermore, the reaction conditions are: stirring at a rate of 300-500 rpm at 20-37°C.

[0036] Furthermore, the reaction conditions are: stirring at 300-500 rpm for 2-48 hours at 20-37°C.

[0037] Preferably, the reaction conditions are: stirring at 350~400 rpm at 37°C for 24~48 h.

[0038] As can be seen from the above description, the preparation method of the present invention is carried out at room temperature, has low requirements for process conditions, and is easy to promote.

[0039] Another technical solution adopted in this invention is: the preparation method of the above-mentioned ticagrelor inclusion complex includes the following steps: mixing and ball milling ticagrelor with a macrocyclic compound to obtain the ticagrelor inclusion complex.

[0040] As can be seen from the above description, the preparation method of the present invention does not require other polymer carriers, has a short preparation cycle, and has the advantage of clinical translation.

[0041] Furthermore, the molar ratio of ticagrelor to the macrocyclic compound is 1:0.5~10.

[0042] Preferably, the molar ratio of ticagrelor to the macrocyclic compound is 1:1 to 5.

[0043] Furthermore, the ball milling conditions are: treatment at a vibration frequency of 10~30Hz for 10~60 minutes.

[0044] Preferably, the ball milling conditions are: treatment with a vibration frequency of 20 Hz for 20 to 30 minutes.

[0045] As can be seen from the above description, if the vibration frequency of the ball mill is too low, it will easily lead to insufficient mixing, while if the vibration frequency is too high, it will lead to overheating and degradation of the inclusion complex.

[0046] Furthermore, it also includes a sieving step after ball milling, after which ticagrelor inclusion compound powder with a particle size ≤50μm is obtained.

[0047] Another technical solution adopted in this invention is: a pharmaceutical composition comprising the above-mentioned ticagrelor inclusion complex and a carrier, wherein the concentration of ticagrelor in the pharmaceutical composition is 0.01~10 mg / mL. The carrier can be any pharmaceutically acceptable carrier, such as solvents like water, alcohols, and oils; solids like sugars, gelatin, and starch; and semi-solid carriers like agar and carbomer.

[0048] Another technical solution adopted in this invention is: a formulation containing ticagrelor inclusion complex, comprising the above-mentioned pharmaceutical composition, wherein the formulation is one of the following: active pharmaceutical ingredient, tablet, capsule, powder, water-soluble formulation, or emulsion; the dosing regimen is: 0.5~100mg / kg equivalent ticagrelor dose daily, the route of administration being selected from oral, intravenous infusion or subcutaneous injection, and the treatment period is 4~24 weeks.

[0049] Another technical solution adopted in this invention is the application of the above-mentioned ticagrelor inclusion complex in the preparation of a drug for treating systemic lupus erythematosus.

[0050] As described above, systemic lupus erythematosus (SLE), an autoimmune disease affecting multiple organs, has a pathological mechanism closely related to abnormal platelet activation: activated platelets release pro-inflammatory factors such as CD40L and P-selectin, promoting the formation of extracellular neutrophil traps (NETs) and exacerbating tissue damage (such as lupus nephritis). Traditional treatments rely on glucocorticoids such as prednisone and immunosuppressants such as cyclophosphamide, but long-term use can easily lead to osteoporosis, secondary infections, and metabolic disorders.

[0051] The ticagrelor inclusion complex of the present invention exerts a therapeutic effect on systemic lupus erythematosus through the following mechanisms: (a) reducing the proportion of M1 macrophages by ≥20% compared to pre-administration levels; (b) reducing serum anti-double-stranded DNA antibody concentration by ≥20% compared to pre-administration levels; and (c) regulating T cell subsets by reducing the proportion of Th1 cells by ≥20% and the proportion of Th17 cells by ≥20% compared to pre-administration levels.

[0052] Furthermore, it achieves immune regulation through the following molecular mechanisms: a) downregulating the gene expression levels of IFN-γ, TNF-α, IL-6, and IL-17 by ≥20% compared to pre-drug administration; b) inhibiting TBK1 phosphorylation by ≥20% compared to pre-drug administration; c) blocking the activation of the JAK / STAT signaling pathway and reducing STAT tyrosine phosphorylation rate by ≥20% compared to pre-drug administration.

[0053] The therapeutic effects include: reducing the glomerular activity index score by ≥20%, reducing the area of ​​renal interstitial fibrosis by ≥20%, and reducing the number of renal vasculitis cells by ≥20%, providing a new and effective drug option for the treatment of systemic lupus erythematosus, and having important clinical application value and social benefits.

[0054] Example 1 of the present invention is: a method for preparing ticagrelor inclusion complex, the specific steps are as follows: 4 mg ticagrelor and 10.69 mg cucurbita[7]urea are added to a methanol aqueous solution containing 1.8 ml hydrochloric acid, the molar concentration of hydrochloric acid in the methanol aqueous solution containing hydrochloric acid is 0.1 M, and the volume concentration of methanol is 10%; then, after stirring at 400 rpm for 48 h at 37 °C, the mixture is freeze-dried to obtain ticagrelor@CB[7] inclusion complex.

[0055] (1) Determination of the properties of the above ticagrelor@CB[7] inclusion complex: Powder X-ray diffraction was used to scan the powders of ticagrelor, CB[7] and ticagrelor@CB[7] inclusion complex, such as Figure 1 As shown, it is amorphous. The infrared FTIR images of ticagrelor and ticagrelor@CB[7] inclusion complex were obtained by scanning with a Fourier transform infrared spectrometer, as shown in the figure. Figure 2 As shown.

[0056] (2) Determination of dissolution curves of ticagrelor@CB[7] inclusion complexes: 1 mg of ticagrelor was dissolved in 1 mL of 0.1 M methanol aqueous solution containing hydrochloric acid with 1, 2, 3, 5, 10, and 15 mM CB[7] respectively. The solution was heated at 37 °C for 48 h with a stirring heater to obtain a series of ticagrelor@CB[7] inclusion complexes with CB[7] content. The series of ticagrelor@CB[7] inclusion complexes with CB[7] content were detected by HPLC (high performance liquid chromatography). The HPLC conditions were acetonitrile:water = 0.6:0.3, flow rate 1.0 mL / min, detection wavelength 299 nm, and standard curves were prepared in the range of 0.1~100 uM and 100~5000 uM. Figure 3 As shown, Figure 3 The results showed that the dissolution curve was linear.

[0057] Example 2 of the present invention is: a method for preparing ticagrelor inclusion complex, the specific steps of which are as follows: ticagrelor and β-CD are mixed at a molar ratio of 1:1~10 and ball-milled (25Hz) for 30 min to obtain a series of ticagrelor@β-CD inclusion complex samples with β-CD content.

[0058] Example 3 of the present invention is: a method for preparing ticagrelor inclusion complex, the specific steps of which are as follows: ticagrelor and HP-β-CD are mixed at a molar ratio of 1:1~10 and ball-milled (25Hz) for 30 min respectively to obtain a series of ticagrelor@HP-β-CD inclusion complex samples with HP-β-CD content.

[0059] (1) Determination of the properties of the above ticagrelor@β-CD inclusion complex and ticagrelor@HP-β-CD inclusion complex: The above ticagrelor@β-CD inclusion complex and ticagrelor@HP-β-CD inclusion complex were dissolved in an organic solvent and subjected to nuclear magnetic resonance. The NMR spectra are shown in the figure. Figure 4 The results showed that the NMR shift of the drug did not change significantly after the addition of HP-β-CD and β-CD. The above ticagrelor@β-CD inclusion complex and ticagrelor@HP-β-CD inclusion complex were characterized by PXRD, TG, and DSG. Figures 5-7 The results indicate that all prepared samples were amorphous.

[0060] (2) Dissolution determination of ticagrelor@HP-β-CD inclusion complex: Dissolution tests were performed on ticagrelor active pharmaceutical ingredient and the above-mentioned ticagrelor@HP-β-CD inclusion complex at 37℃, 250 rpm, and 50 mL PBS (pH=6.8) (phosphate buffer solution). Samples were taken at different time points, and the supernatant was collected by centrifugation at 10000 rpm and injected by HPLC. The HPLC results were the same as in Example 1. Figure 8 This indicates that HP-β-CD inclusion has a certain solubilizing effect, with approximately 20-fold solubility in 2 hours, and a significant increase in the dissolution rate.

[0061] Example 4 of the present invention is: detection of ticagrelor@CB[7] inclusion complex (Example 1) reducing the level of inflammatory factors. The specific steps are as follows: Step 1, Culturing RAW264.7 cells: Seed the cells in a cell culture flask and culture them in DMEM containing 5% fetal bovine serum under normal conditions of 37°C, 5% CO2, and 80% humidity. When the cells reach 80% confluence with the bottom of the flask, they can be passaged.

[0062] Step 2, Stimulation of RAW264.7 cells: RAW264.7 cells were treated with 0.2 ng / ml LPS (lipopolysaccharide), 20 μM ticagrelor, and 20 μM CB7 for 24 h.

[0063] Step 3: Verify changes in inflammation levels: (1) Collect the processed RAW264.7 cell pellet, extract total RNA from the cultured cells using FreeZol Reagent reagent, and synthesize cDNA using a reverse transcription kit. Amplify the cDNA using Taq Pro Universal SYBR qPCR Master Mix according to the manufacturer's instructions. Normalize the data based on the GAPDH expression level in each sample, and analyze the relative mRNA expression level of INOS (inducible nitric oxide synthase) using the 2-ΔΔCt method. The results are as follows: Figure 9 As shown.

[0064] (2) Collect the processed RAW264.7 cell pellets, and use fluorophore-binding antibodies against surface markers (CD11b-FITC, LD / BV510) and cytoplasmic markers (PE-CD206, APC-INOS) to stain the cells surface and fix them for cell membrane perforation. The expression of the cytokine INOS in each group was obtained as follows: Figure 10 As shown.

[0065] Figure 9 and 10 The results showed that the INOS expression of the ticagrelor and ticagrelor@CB[7] inclusion complex group (0.58±0.06) was significantly lower than that of the LPS group (1.03±0.3), suggesting its potential role in inhibiting inflammation.

[0066] Example 5 of the present invention is as follows: Animal experiments were used to verify the therapeutic effect of ticagrelor@CB[7] inclusion complex (Example 1) on systemic lupus erythematosus. The specific steps are as follows: Step 1: Preparation of experimental animals: Healthy C57BL / 6 mice and MRL / lpr lupus mice were selected. Ten C57BL / 6 mice served as the normal control group, and 30 MRL / lpr lupus mice were randomly divided into three groups: the MRL / lpr group (n=10), the ticagrelor group (n=10), and the prednisone group (n=10). All mice were housed in a suitable environment with free access to food and water.

[0067] Step 2, Administration Procedure: The ticagrelor group was given ticagrelor@CB[7] inclusion complex (Example 1) (30 mg / kg / d), dissolved in sodium carboxymethyl cellulose (CMC-Na), and administered to mice daily by gavage. The prednisone group was given prednisone (9 mg / kg / d), also dissolved in a solvent and administered by gavage. The C57BL / 6 group and the MRL / lpr group were given an equal volume of physiological saline. The administration was continued for 2 months. During this period, the mice's diet, activity, mental state and other general conditions were observed daily, and their weight was measured regularly and recorded in detail.

[0068] Step 3, Sample Collection and Testing: After 2 months of drug administration, mice were euthanized using appropriate methods, and the spleen was quickly removed and ground in a suitable environment.

[0069] (1) Splenic cells were collected for qPCR experiments. In the qPCR experiments, the standard operating procedures were strictly followed, including RNA extraction, reverse transcription, and PCR (polymerase chain reaction) amplification, to verify the expression of indicators such as IL6 (interleukin-6), JAK3, TBK1, STAT1 (signal transduction and transcription activator 1), SYK (spleen tyrosine kinase), and IFNG (interferon-γ).

[0070] The experimental results are shown in Figure 11 Compared with the MRL / lpr group, both the ticagrelor and prednisone groups showed a decrease in inflammatory markers, with the ticagrelor group showing a more significant decrease. The IFN-γ (interferon-γ) level in the MRL / lpr group was 1.0±0.3, while that in the ticagrelor group was 0.3±0.4, a reduction of 70%. The IL-6 level in the MRL / lpr group was 1.1±0.4, while that in the ticagrelor group was 0.8±0.5, a reduction of 27%. The TBK1 phosphorylation level in the MRL / lpr group was 1.0±0.3, while that in the ticagrelor group was 0.4±0.3, a reduction of 60%. The STAT tyrosine phosphorylation level in the MRL / lpr group was 1.0±0.4, while that in the ticagrelor group was 0.4±0.3, a reduction of 60%. This further demonstrates the potential of ticagrelor inclusion complexes in the treatment of systemic lupus erythematosus.

[0071] (2) The spleen single-cell suspension was further processed and stained with fluorophore-binding antibodies targeting TH1 (helper T cell type 1) and TH17 (helper T cell type 17) cell-specific markers, and detected by flow cytometry.

[0072] Test results as follows Figure 12 The results showed that the proportion of TH1 cells in the MRL / lpr group was 38.8±6.1, and the proportion of TH17 cells was 3.3±0.9. In the ticagrelor group, the proportion of TH1 cells was 16.91±5.9, a decrease of 56.4%, and the proportion of TH17 cells was 1.0±0.6, a decrease of 68.9%, which was significantly lower than that in the MRL / lpr group. Compared with the prednisone group, the ticagrelor group was more effective in reducing the proportion of TH1 and TH17 cells, suggesting that the ticagrelor inclusion complex can effectively regulate the cellular immune imbalance in mice with systemic lupus erythematosus, inhibit the excessive activation of TH1 and TH17 cells, and exert an anti-inflammatory effect.

[0073] (3) After euthanizing the mice, the kidney tissue was quickly removed, fixed, dehydrated, and embedded, and then prepared into pathological sections. After staining with HE, Cooma, etc., the sections were observed under a microscope.

[0074] The results are as follows Figure 13 The results showed that mice in the MRL / lpr group exhibited significant mesangial cell proliferation and thickening of the glomerular basement membrane, with inflammatory cell infiltration in the tubulointerstitium, indicating severe inflammatory damage to the kidneys. Mice in the prednisone group still showed some glomerular lesions and inflammatory cell infiltration in their kidneys. However, the ticagrelor group showed significant improvement in kidney pathology, with a marked reduction in glomerular mesangial cell proliferation, alleviated glomerular basement membrane thickening, and a significant decrease in tubulointerstitial inflammatory cell infiltration. This further confirms the nephroprotective effect of ticagrelor inclusion complex in the treatment of systemic lupus erythematosus and its significant anti-inflammatory effect.

[0075] The above pathological sections were scored using a semi-quantitative scoring system, and the scoring results are as follows: Figure 14 The results showed that, compared with the untreated model group, the severity of kidney lesions in mice treated with the drug was significantly reduced (p<0.001). Specifically, based on the International Standard for Pathology (ISN / RPS classification) and animal model research literature (semi-quantitative method) (0-4 points), the glomerular score in the drug group decreased from 3.4 ± 0.8 in the model group to 1.8 ± 0.8, a reduction of 47%; the tubulointerstitial score decreased from 3.5 ± 0.7 in the model group to 1.7 ± 0.8; and the renal vascular score decreased from 2.8 ± 0.6 in the model group to 1.3 ± 0.5.

[0076] (5) Collect ocular blood from mice, centrifuge, and collect the supernatant. Use a DSDNA kit to detect the serum anti-double-stranded DNA antibody concentration. The results are as follows: Figure 15 The results showed that the ticagrelor concentration (16.32±5.9 ng / ml) was significantly lower than that of the MRL / lpr group (34.86±9.8 ng / ml), indicating that ticagrelor has the potential to treat systemic lupus erythematosus. These data, analyzed using univariate ANOVA and Tukey multiple comparison tests, confirmed that the drug effectively alleviated the pathological progression of lupus nephritis through a multi-target mechanism, providing crucial experimental evidence for subsequent clinical translation.

[0077] Example 6 of the present invention is a method for preparing ticagrelor inclusion complex, the specific steps of which are as follows: ticagrelor and β-cyclodextrin are added to an ethanol aqueous solution containing sulfuric acid at a molar ratio of 1:0.5, the concentration of sulfuric acid in the ethanol aqueous solution is 0.05M, the volume concentration of ethanol is 5%, and the ratio of ticagrelor to the acid-containing ethanol aqueous solution is 3mg:1mL; then, the mixture is stirred at 300rpm at 20°C for 40h and then freeze-dried to obtain the ticagrelor inclusion complex.

[0078] Example 7 of the present invention is a method for preparing ticagrelor inclusion complex, the specific steps of which are as follows: ticagrelor and hydroxypropyl betacyclodextrin are added to an ethanol aqueous solution containing hydrochloric acid at a molar ratio of 1:10. The concentration of hydrochloric acid in the ethanol is 0.15M, the volume concentration of ethanol is 20%, and the ratio of ticagrelor to the acid-containing ethanol aqueous solution is 2mg:1mL. Then, the mixture is stirred at 500rpm for 2h at 35℃ and then freeze-dried to obtain the ticagrelor inclusion complex.

[0079] Example 8 of the present invention is: a method for preparing ticagrelor inclusion complex, the specific steps of which are as follows: ticagrelor and cucurbita[7]urea are added to a methanol aqueous solution containing sulfuric acid at a molar ratio of 1:5. The concentration of sulfuric acid in the methanol aqueous solution containing sulfuric acid is 0.12M, the volume concentration of methanol is 15%, and the ratio of ticagrelor to the acid-containing alcohol aqueous solution is 4mg:1mL. Then, the mixture is stirred at 480rpm at 25°C for 24h and then freeze-dried to obtain the ticagrelor inclusion complex.

[0080] Example 9 of the present invention is: a method for preparing ticagrelor inclusion complex, the specific steps of which are as follows: ticagrelor and β-cyclodextrin are mixed at a molar ratio of 1:1, placed in a planetary ball mill and processed at a speed of 10 Hz for 10 min, and sieved to obtain ticagrelor inclusion complex with a particle size ≤50 μm.

[0081] Example 10 of the present invention is: a method for preparing ticagrelor inclusion complex, the specific steps of which are as follows: ticagrelor and hydroxypropyl betacyclodextrin are mixed at a molar ratio of 1:10, placed in a planetary ball mill and processed at a speed of 30 Hz for 60 min, and sieved to obtain ticagrelor inclusion complex with a particle size ≤50 μm.

[0082] Example 11 of the present invention is: a ticagrelor inclusion complex, comprising ticagrelor and cucurbitaureus [7] containing ticagrelor.

[0083] Example 12 of the present invention is: a pharmaceutical composition comprising the ticagrelor inclusion complex of Example 1 and agar, wherein the concentration of ticagrelor in the pharmaceutical composition is 0.01 mg / mL.

[0084] Example 13 of the present invention is: a pharmaceutical composition comprising the ticagrelor inclusion complex of Examples 1 and 2 and agar, wherein the total concentration of ticagrelor in the pharmaceutical composition is 5 mg / mL.

[0085] Example 14 of the present invention is: a pharmaceutical composition comprising the ticagrelor inclusion complex of Examples 1 to 3 and agar, wherein the total concentration of the ticagrelor inclusion complex in the pharmaceutical composition is 10 mg / mL.

[0086] Example 15 of the present invention is: a formulation comprising ticagrelor inclusion complex, comprising the pharmaceutical composition of Example 4, wherein the formulation is one of the following: active pharmaceutical ingredient, tablet, capsule, powder, water-soluble formulation, or emulsion.

[0087] Example 16 of the present invention is the application of the ticagrelor inclusion complex of Example 1 in the preparation of a medicament for the treatment of systemic lupus erythematosus.

[0088] In summary, the ticagrelor inclusion complex, its preparation method, and its applications provided by this invention have the following advantages: 1. The ticagrelor inclusion complex of the present invention uses a macrocyclic compound to include ticagrelor, forming a stable drug inclusion complex, thereby enhancing the solubility and pharmacological effects of ticagrelor.

[0089] 2. The ticagrelor inclusion complex of the present invention can be used to prepare formulations with excellent chemical stability, low biotoxicity, and good drug release performance.

[0090] 3. The present invention can specifically select cucurbita[7]urea as a macrocyclic compound. Compared with cyclodextrin, it has a different binding mode with ticagrelor, which improves solubility, bioavailability and anti-inflammatory effect.

[0091] 4. This invention has verified the therapeutic effect of ticagrelor on systemic lupus erythematosus through animal experiments, and further enhanced its therapeutic effect by forming inclusion complexes, providing a new and effective drug option for the treatment of systemic lupus erythematosus, and has important clinical application value and social benefits.

[0092] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification and drawings, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A ticagrelor inclusion complex, characterized in that, The inclusion of ticagrelor and macrocyclic compounds containing ticagrelor, wherein the macrocyclic compound is at least one of cucurbita[7]urea, β-cyclodextrin and hydroxypropyl betacyclodextrin.

2. The ticagrelor inclusion complex according to claim 1, characterized in that, The macrocyclic compound is cucurbita[7]urea.

3. The method for preparing the ticagrelor inclusion complex as described in claim 1 or 2, characterized in that, Includes the following steps: Ticargul is reacted with a macrocyclic compound in an acidic aqueous alcohol solution to obtain a ticagrelor inclusion complex.

4. The method for preparing the ticagrelor inclusion complex according to claim 3, characterized in that, The molar ratio of ticagrelor to the macrocyclic compound is 1:0.5~10.

5. The method for preparing the ticagrelor inclusion complex according to claim 3, characterized in that, The concentration of acid in the acid-containing aqueous alcohol solution is 0.05~0.15M.

6. The method for preparing the ticagrelor inclusion complex according to claim 3, characterized in that, The reaction conditions are: stirring at a rate of 300-500 rpm at 20-37°C.

7. The method for preparing the ticagrelor inclusion complex as described in claim 1 or 2, characterized in that, Includes the following steps: Ticargul was mixed with a macrocyclic compound and ball-milled to obtain ticagrelor inclusion complexes.

8. The method for preparing the ticagrelor inclusion complex according to claim 7, characterized in that, The molar ratio of ticagrelor to the macrocyclic compound is 1:0.5~10.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition includes the ticagrelor inclusion complex and carrier as described in claim 1 or 2, wherein the concentration of ticagrelor in the pharmaceutical composition is 0.01 to 10 mg / mL.

10. The use of the ticagrelor inclusion complex as described in claim 1 or 2 in the preparation of a medicament for the treatment of systemic lupus erythematosus.

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