Icaritin and tripterine composition, preparation method and application thereof

Abstract

The application belongs to the field of medicine, and particularly relates to a combination of icariin and celastrol, a preparation method and application thereof. The preparation method comprises the following steps: mixing icariin solution and celastrol solution, adding water, ultrasonicating, dialyzing, and collecting the remaining solution, and the combination is obtained. The icariin and celastrol are prepared into self-assembled nanomedicine, the preparation process is simpler, the drug delivery efficiency is higher, and the combination is applied to the treatment of liver cancer, and the combination has a significant synergistic effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicine, and particularly relates to a composition of icariin and tripterine, a preparation method and application thereof. BACKGROUND

[0002] Hepatocellular carcinoma (HCC) is the most common type of liver cancer, and the main causes of HCC include HBV, HCV, alcohol-related liver disease and metabolic dysfunction-related fatty liver disease. The treatment of liver cancer, including the application of targeted drugs, immunotherapy and comprehensive treatment strategies, significantly prolongs the survival of patients at each stage. However, some HCC patients are diagnosed at an advanced stage and have lost the opportunity for radical treatment such as surgery and liver transplantation. Therefore, it is extremely critical to find an effective treatment for advanced HCC.

[0003] At present, significant progress has been made in the field of systemic therapy for advanced hepatocellular carcinoma, especially with the widespread use of tyrosine kinase inhibitors (such as sorafenib and lenvatinib) and immune checkpoint inhibitors (such as atezolizumab and bevacizumab), the overall survival and quality of life of patients have been improved to some extent. However, there are still many severe challenges in clinical practice. First, hepatocellular carcinoma has high heterogeneity and is prone to drug resistance. There are significant differences in driver genes among patients, and multiple pathogenic factors promote the development of HCC through different mechanisms, which leads to the easy occurrence of primary and secondary drug resistance when using single drug treatment, especially single targeted drug. Second, existing targeted and immune drugs and their combination regimens are often accompanied by significant side effects, such as hypertension, thrombosis, immune hepatitis and hand-foot skin reactions, etc. These adverse reactions not only limit the application of drugs, but also can seriously affect the quality of life of patients. More notably, different causes shape the immunosuppressive microenvironment of tumors through their own unique mechanisms. For example, in HBV-related liver cancer, the continuous stimulation of HBV viral antigens (such as HBsAg) can lead to the exhaustion of effector CD8+ T cells, while recruiting regulatory T cells (Treg), regulatory B cells (Breg) and granulocyte-like myeloid-derived suppressor cells (gMDSC), etc., forming a tolerogenic immune microenvironment. This complex immunosuppressive state significantly weakens the efficacy of immunotherapy drugs. Therefore, developing new multi-target synergistic, efficient and low-toxicity drugs is still an important direction for future research.

[0004] Traditional Chinese medicine (TCM) has shown unique therapeutic advantages in the field of cancer treatment, with its multi-pathway, multi-target, small side effects and significant efficacy, etc. providing an important supplement to modern tumor treatment. From the molecular mechanism, traditional Chinese medicine can exert anti-tumor effects through multiple dimensional action pathways, including inducing tumor cell apoptosis, inhibiting tumor cell proliferation and cycle arrest, inducing tumor immunogenic death, inhibiting angiogenesis and remodeling the immune microenvironment, etc. Therefore, in-depth study of the anti-tumor efficacy of traditional Chinese medicine has important clinical value.

[0005] Celastrol, a pentacyclic triterpenoid derived from the traditional medicinal plant Tripterygium hookeri (T. hookeri) Hook. f., is considered to have special potential as a cancer treatment drug. It has a wide range of pharmacological activities, including significant anti-inflammatory, anti-obesity, and anti-tumor effects. Multiple lines of evidence show that Celastrol exhibits significant proliferation-inhibiting activity against a variety of tumor cell lines. Icaritin is a bioactive compound derived from the traditional Chinese medicine Epimedium brevicornum Maxim. Icaritin has a variety of biological activities, not only directly killing tumors by inducing cell death and autophagy, but also regulating the tumor immune microenvironment and promoting anti-tumor immune responses, showing a wide range of anti-tumor effects in various solid tumors, including colorectal cancer, prostate cancer, and urothelial cancer. Some basic and clinical studies have shown that Icaritin has good biological safety in patients with advanced HCC, significantly improving survival. In 2022, Icaritin soft capsules were approved by the National Medical Products Administration (NMPA) for the immunotherapy of advanced HCC. Tripterygium wilfordii Celastrol, a pentacyclic triterpenoid derived from the traditional medicinal plant Tripterygium hookeri (T. hookeri) Hook. f., is considered to have special potential as a cancer treatment drug. It has a wide range of pharmacological activities, including significant anti-inflammatory, anti-obesity, and anti-tumor effects. Multiple lines of evidence show that Celastrol exhibits significant proliferation-inhibiting activity against a variety of tumor cell lines. Icaritin is a bioactive compound derived from the traditional Chinese medicine Epimedium brevicornum Maxim. Icaritin has a variety of biological activities, not only directly killing tumors by inducing cell death and autophagy, but also regulating the tumor immune microenvironment and promoting anti-tumor immune responses, showing a wide range of anti-tumor effects in various solid tumors, including colorectal cancer, prostate cancer, and urothelial cancer. Some basic and clinical studies have shown that Icaritin has good biological safety in patients with advanced HCC, significantly improving survival. In 2022, Icaritin soft capsules were approved by the National Medical Products Administration (NMPA) for the immunotherapy of advanced HCC. Epimedium brevicornu

[0006] Although Icaritin soft capsules have been used in the immunotherapy of advanced hepatocellular carcinoma and have shown certain therapeutic potential in clinical practice, their application still has many limitations. First, Icaritin has poor water solubility and exhibits low intestinal mucosa and cell membrane permeability in vivo, severely limiting its absorption efficiency. At the same time, Icaritin undergoes significant first-pass effect when passing through the liver after oral administration, resulting in a large amount of drug being metabolized and inactivated, thus making the oral bioavailability extremely low. In addition, Icaritin lacks an active targeting mechanism, and it is easily recognized by the reticuloendothelial system (RES) and rapidly cleared in vivo, resulting in short blood circulation time, limited tumor tissue accumulation, and difficulty in achieving effective therapeutic concentrations in the lesion area, with overall low drug delivery efficiency. Clinical treatment of advanced HCC usually employs a combination drug strategy, such as the combined use of immunotherapy with anti-angiogenic drugs, chemotherapy drugs, or other targeted preparations. However, existing preparations often fail to achieve the simultaneous arrival of two or more drugs at the tumor site in terms of time and space.

[0007] ​Nano-DDS has been widely studied in the field of cancer treatment delivery. Nano-DDS can prolong the blood circulation time of drugs and achieve tumor-specific distribution through the enhanced permeability and retention (EPR) effect. In addition, specific surface functionalization can enable nano-DDS to respond specifically to the physiological microenvironment at the tumor site (such as excess enzymes, hypoxia, redox, and acidic conditions). Pure drug self-delivery nano-systems (PDANS) assembled from single or multiple pure drug molecules are of great interest due to their simpler preparation process and higher drug delivery efficiency. PDANS is completely composed of pharmacologically active compounds, further reducing the biological toxicity of the carrier. Compared with traditional nanocrystalline delivery systems, PDANS has the advantages of high drug loading efficiency and simple preparation process.

[0008] Therefore, a nano-drug self-assembled from icariin and celastrol is provided, and the related mechanism of the key pharmacodynamic components of the drug in the process of renal fibrosis is analyzed in depth, which can provide material basis and transformation value for clinical treatment of liver cancer. SUMMARY

[0009] In view of the deficiencies of the prior art, the present application provides a composition of icariin and celastrol, and a preparation method and application thereof.

[0010] In order to achieve the above-mentioned purpose of the present application, the specific technical scheme adopted by the present application is as follows:

[0011] A preparation method of a composition of icariin and celastrol, comprising the following steps:

[0012] Mixing icariin (ICT) solution and celastrol (CEL) solution, adding water, ultrasonic treatment, dialysis, and collecting the remaining solution to obtain the composition of icariin and celastrol (CAI).

[0013] Preferably, the solvent of the icariin solution and the celastrol solution is dimethyl sulfoxide (DMSO).

[0014] Preferably, the mass concentration of the icariin solution and the celastrol solution is 8-12 mg / mL, preferably 10 mg / mL.

[0015] Preferably, the volume ratio of the icariin solution and the celastrol solution is 1-3:5, preferably 3:5.

[0016] Preferably, the added amount of water is 15-20 times, preferably 18 times, the volume of the celastrol solution.

[0017] Preferably, the power of the ultrasonic treatment is 35-45 kHz, and the ultrasonic treatment time is 5-10 min.

[0018] Preferably, the molecular weight cutoff for dialysis is 3000-5000 Da, and more preferably 3500 Da.

[0019] More preferably, dialysis is performed using dialysis tubing.

[0020] The present invention also relates to a composition of icariin and tripterygium wilfordii obtained by the above preparation method.

[0021] The present invention also relates to the use of the icariin and tripterygium oleoresin composition prepared by the above preparation method in the preparation of a drug for treating liver cancer.

[0022] Preferably, the drug further comprises a pharmaceutically acceptable carrier selected from one or more of emulsifiers, fillers, binders, wetting agents, disintegrants, absorption enhancers, flavoring agents, coloring agents, and solubilizers.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) In this invention, triptolide (CEL) and icariin (ICT) are prepared into self-assembled nanomedicines, which have a simpler preparation process and higher drug delivery efficiency.

[0025] (2) The self-assembled nanomedicines of triptolide and icariin in this invention are applied to the treatment of liver cancer, and there is a significant synergistic effect between the two. Attached Figure Description

[0026] Figure 1 This is a hydrated particle size distribution diagram of CAI;

[0027] Figure 2 This is a graph showing the changes in particle size and PDI of CAI over seven days;

[0028] Figure 3 This is a transmission electron microscope image of CAI;

[0029] Figure 4 These are the infrared spectra of CEL, ICT, and CAI.

[0030] Figure 5 These are the UV-Vis absorption spectra of CAI and CEL in water and CAI in DMSO solution;

[0031] Figure 6 These are the UV-Vis absorption spectra of CAI, 0.2% SDS solution, and CAI in 0.2% SDS solution;

[0032] Figure 7 These are the UV-Vis absorption spectra of CAI, 0.5M NaCl solution of CAI, and 1M NaCl solution of CAI.

[0033] Figure 8 is a graph of the change in the body weight of mice administered with PBS, CAI, ICT and CEL;

[0034] Figure 9 is a graph of the change in the tumor volume of mice administered with PBS, CAI, ICT and CEL, wherein, compared with the PBS group, & P<0.05, && P<0.01; compared with the CAI group, # P<0.05, ## P<0.01;

[0035] Figure 10 is a graph of the average tumor weight of mice in each group on the 15th day of administration, wherein, compared with the PBS group, && P<0.01, &&& P<0.001; compared with the CAI group, ## P<0.01, ### P<0.001;

[0036] Figure 11 is an image of the tumor tissue in each group ex vivo. DETAILED DESCRIPTION

[0037] The present application will be further described below in conjunction with specific examples. The following examples are not intended to limit the present application, but merely serve to illustrate the present application. Unless otherwise specified, the experimental methods used in the following examples are generally performed according to conventional conditions. Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available.

[0038] Example 1

[0039] The preparation method of the composition of icariin and celastrol (CAI) is as follows:

[0040] (1) Celastrol (CEL) and icariin (ICT) were first configured into a stock solution of 10 mg / mL with DMSO;

[0041] (2) CEL (50 μL) and ICT (30 μL) were mixed in ultrapure water (900 μL), and after ultrasonic treatment at 40 kHz for 5 min, the solution was transferred into a dialysis tube (MWCO 3500 Da) for dialysis for 12 h, and the remaining solution was collected, thereby obtaining the composition of icariin and celastrol (CAI).

[0042] Example 2

[0043] The preparation method of the composition of icariin and celastrol (CAI) is as follows:

[0044] (1) Prepare celastrol (CEL) and icariin (ICT) into 10 mg / mL stock solution with DMSO;

[0045] (2) Mix CEL (50 μL) and ICT (20 μL) in ultrapure water (900 μL), after 10 min of 35 kHz ultrasonic, transfer the solution into dialysis tube (MWCO 3500 Da) for dialysis for 12 h, collect the remaining solution, and obtain it.

[0046] Example 3

[0047] The preparation method of icariin and celastrol composition (CAI) is as follows:

[0048] (1) Prepare celastrol (CEL) and icariin (ICT) into 10 mg / mL stock solution with DMSO;

[0049] (2) Mix CEL (50 μL) and ICT (10 μL) in ultrapure water (900 μL), after 15 min of 40 kHz ultrasonic, transfer the solution into dialysis tube (MWCO 3500 Da) for dialysis for 12 h, collect the remaining solution, and obtain it.

[0050] Effect test

[0051] The icariin and celastrol composition (CAI) prepared in Example 1 is tested as follows.

[0052] Test Example 1 Particle size and stability test

[0053] Under ultrasonic conditions (40 kHz, 5 min), dilute the CAI in Example 1 with ultrapure water by 10 times, take 1 mL to a particle size cup, use Malvern particle size instrument for dynamic light scattering analysis (DLS), obtain the particle size and dispersion index (PDI) of CAI, and continuously measure for 7 days to observe the stability of CAI.

[0054] The results are shown in Figure 1 The dynamic light scattering hydration particle size of icariin and celastrol composition (CAI) is about 160.72 nm, and the PDI is 0.109.

[0055] The stability results are shown in Figure 2 The PDI and particle size of CAI do not change significantly within 7 days, and CAI has good stability in ultrapure water.

[0056] Test Example 2 Transmission electron microscopy test

[0057] Using a pipette, 10 μL of the CAI dilution solution (20-fold dilution of the CAI in Example 1 with ultrapure water) was dropped onto a 100-mesh copper mesh, and after natural air-drying, the CAI morphology was observed using TEM. As shown in the TEM image in Figure 3 , the CAI was uniformly dispersed as round-shaped nanoparticles.

[0058] Test Example 3 Fourier Transform Infrared Spectroscopy Test

[0059] The CAI prepared in Example 1 was freeze-dried. The infrared spectra of CEL, ICT and CAI were detected using the total reflection infrared spectroscopy method (ATR method) in the range of 4000-600 cm -1 .

[0060] As shown in Figure 4 , the infrared spectroscopy analysis showed that the ICT and CAI exhibited a significant ether bond characteristic absorption peak near 1147 cm -1 , while the CEL and CAI showed a carboxyl C=0 bond characteristic absorption peak near 1704 cm -1 , indicating that the CAI contained both CEL and ICT components. In addition, the absorption peak of the CAI near 3309 cm -1 changed significantly, which was probably due to the conversion of free O-H groups into bound OH groups during the self-assembly process.

[0061] Test Example 4 UV-vis Characterization

[0062] Preparation of the solution:

[0063] The following preparation of the CEL stock solution and the ICT stock solution was consistent with step (1) in Example 1.

[0064] CAI solution: 50 μL of the CEL stock solution and 30 μL of the ICT stock solution were mixed with 900 μL of ultrapure water to obtain the solution;

[0065] CEL aqueous solution: 50 μL of the CEL stock solution was mixed with 900 μL of ultrapure water to obtain the solution;

[0066] CAI DMSO solution: 50 μL of the CEL stock solution and 30 μL of the ICT stock solution were mixed with 900 μL of DMSO to obtain the solution;

[0067] 0.2% sodium dodecyl sulfate (SDS) solution: 2 g of SDS was dissolved in 1000 mL of water to obtain the solution;

[0068] CAI 0.2% SDS solution: 50 μL of the CEL stock solution and 30 μL of the ICT stock solution were mixed with 900 μL of the 0.2% SDS solution to obtain the solution;

[0069] 0.5M NaCl solution of CAI: 50 μL of CEL stock solution and 30 μL of ICT stock solution were mixed with 900 μL of 0.5M NaCl solution;

[0070] 1M NaCl solution of CAI: 50 μL of CEL stock solution and 30 μL of ICT stock solution were mixed with 900 μL of 1M NaCl solution.

[0071] UV-vis spectra of CAI in different solutions were detected using UV-vis detector, and the detection wavelength range was 300-800 nm. The results are shown in Figure 5 to Figure 7 .

[0072] Compared with water, CAI showed a significant blue shift phenomenon in DMSO Figure 5 , indicating that CAI decomposed in DMSO, suggesting the existence of π-π stacking interaction. Subsequently, after adding sodium dodecyl sulfate (SDS) to the CAI aqueous solution, the absorption peak also showed a significant blue shift, indicating that intermolecular hydrophobic interaction was involved in the self-assembly process Figure 6 . However, when CAI was dispersed in different concentrations of sodium chloride solution, its characteristic absorption peak did not change significantly, indicating the absence of hydrogen bond interaction Figure 7 , which is consistent with the infrared spectrum results.

[0073] Test Example 5 In vivo tumor inhibition test

[0074] To detect the in vivo anti-tumor behavior, H22 cells resuspended with PBS were subcutaneously injected into female BALB / c mice (purchased from Guangzhou Rige Biotechnology Co., Ltd., 5 in each group, a total of 20) (1.5 × 10 6 per mouse). When the tumor volume reached about 50mm 3 , the mice were randomly divided into 4 groups, and tail vein administration of PBS (pH 7.2), CEL (1.875 mg / kg), ICT (1.125 mg / kg) and CAI (3 mg / kg) was performed, and tail vein administration was performed on days 1, 3, 4, 6, 8, 9 and 11, and samples were taken on day 15. During the treatment, the tumor size and body weight were measured every two days with a vernier caliper and a balance. After the end of the treatment, all mice were euthanized, and the tumors were collected for photography and weighing.

[0075] According to the body weight change graph in Figure 8 , compared with the PBS group, the body weight did not change significantly after ICT administration, and the body weight decreased after CAI and CEL administration, but the body weight recovered significantly after stopping administration.

[0076] According to the tumor volume change graph in Figure 9It can be seen that the tumor growth of the PBS group and the ICT group was rapid throughout the treatment period, while the tumor growth of the CEL group and the CAI group was slightly slower, and the CAI group showed the strongest tumor inhibition effect. In addition, all tumor-bearing mice were sacrificed at the end of the treatment, and the results of tumor weight showed that CAI had a significant anti-tumor effect ( Figure 10 ), and the actual tumor size of different groups was basically consistent with the tumor volume curve ( Figure 11 ).

[0077] The above detailed description is a specific description of one of the possible embodiments of the present application, which is not intended to limit the patent scope of the present application, and any equivalent implementation or change without departing from the present application shall be included in the scope of the technical solutions of the present application.

Claims

1. A method for preparing a composition of icariin and tripterygium wilfordii, characterized in that, Includes the following steps: Mix the icariin solution and tripterygium wilfordii solution, add water, sonicate, dialyze, and collect the remaining solution to obtain the product; The solvent for both the icariin solution and the triptolide solution is dimethyl sulfoxide, the mass concentration of both the icariin solution and the triptolide solution is 8-12 mg / mL, and the volume ratio of the icariin solution and the triptolide solution is 1-3:

5.

2. The preparation method according to claim 1, characterized in that, The amount of water added is 15-20 times the volume of the triptolide solution.

3. The preparation method according to claim 1, characterized in that, The ultrasonic power is 35-45kHz, and the ultrasonic time is 5-10min.

4. The preparation method according to claim 1, characterized in that, The molecular weight cutoff for dialysis is 3000-5000 Da.

5. A composition of icariin and tripterygium wilfordii prepared by the preparation method according to any one of claims 1-4.

6. The use of a composition of icariin and tripterygium wilfordii prepared by any one of claims 1-4 in the preparation of a medicament for treating liver cancer.

7. The application according to claim 6, characterized in that, The drug further comprises a pharmaceutically acceptable carrier selected from one or more emulsifiers, fillers, binders, humectants, disintegrants, absorption enhancers, flavoring agents, colorants, and solubilizers.

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