Salinomycin derivative liposome as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly provides a salinomycin derivative sal-098 targeted liposome as well as a preparation method and application thereof. The membrane is prepared from a membrane material, a long circulating substance, a stabilizer, a salinomycin derivative and a tumor targeting substance through a membrane hydration method. The salinomycin derivative targeted liposome disclosed by the invention can target colorectal cancer tissues, more efficiently kill tumor cells and reduce the toxicity of salinomycin derivatives at the same time.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical formulation technology. Specifically, this application provides a salinomycin derivative (sal-098) targeted liposome, its preparation method, and its application. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors. Despite significant improvements in diagnosis and treatment, it remains the third leading cause of cancer death worldwide. If colorectal cancer does not metastasize, the 5-year survival rate is close to 90%, but once metastasis occurs, the 5-year survival rate drops sharply to 12%. Increasing research indicates that cancer stem cells are a key factor driving tumor growth and metastasis. Cancer stem cells (CSCs) are a small population of cells with stem cell-like properties found in tumor tissue. They self-renew and replicate rapidly, producing differentiated daughter cells and promoting the growth of heterogeneous tumor tissue. Traditional cancer treatments primarily target differentiated, ordinary tumor cells, while CSCs, due to their G0 phase and high expression of drug resistance proteins, can survive conventional treatments. After a period of proliferation and differentiation, they can re-form new tumors, leading to tumor recurrence.

[0003] There is an urgent clinical need for new drugs with anti-tumor stem cell activity to address issues such as tumor recurrence and metastasis, and to meet the growing demand for precision medicine. Currently, only Roche's vemoderavir, an anti-tumor stem cell drug, was approved by the FDA in 2012. It works by inhibiting the Hedgehog signaling pathway and is used to treat adult patients with symptomatic metastatic basal cell carcinoma (BCC) or locally advanced BCC who are unsuitable for surgery or radiotherapy. Anti-tumor stem cell drugs are urgently needed in clinical practice, and their development needs to be accelerated.

[0004] Salinomycin (SAL) was extracted from the culture medium of *Streptomyces albopictus* by Miyazaki et al. in the 1970s, and has since been used as an antibiotic in poultry. In 2009, Gupta et al. obtained breast stem cells from human mammary epithelial cells that could form spheroids in vitro and were resistant to chemotherapy drugs such as paclitaxel and doxorubicin through epithelial-mesenchymal transition (EMT). They then conducted high-throughput screening of more than 16,000 compounds to test their cytotoxic effects on breast stem cells. They ultimately found that salinomycin (SAL) could highly and selectively inhibit breast cancer stem cells, with an efficacy more than 100 times that of the traditional chemotherapy drug paclitaxel. Since then, the anti-tumor stem cell activity of SAL has attracted increasing attention. Currently, the inhibitory effects of SAL on tumor stem cells of colorectal cancer, acute myeloid leukemia, lung cancer, prostate cancer, and gastric cancer have been reported in the literature. However, salinomycin itself still has some problems; its in vivo activity needs further improvement, and it also has certain toxic side effects.

[0005] The novel compound SAL-098 is a salinomycin derivative (Wu Song, Zhou Qi, Zhang Wenxuan, et al. Salinomycin oxime and oxime ether derivatives, their preparation methods and antitumor uses: CN107417699B[P]), a highly active antitumor stem cell drug. Its in vitro activity is more than 100 times higher than that of salinomycin, and it has shown certain anti-colorectal cancer activity in mice, while also inhibiting tumor metastasis in mice, indicating potential clinical value. Despite these advantages, SAL-098's poor water solubility, high lipid solubility, easy crossing of the blood-brain barrier, significant neurotoxicity, and narrow therapeutic window limit its clinical application.

[0006] Liposomes possess high biocompatibility and safety, can improve drug solubility, and exhibit some passive targeting activity. Active targeting can be enhanced by modifying liposomes with antibodies, hormones, sugar residues, and receptor ligands. Therefore, liposomes were chosen as the drug delivery carrier for SAL-098, and they were modified with hyaluronic acid (HA) to target CD44, which is overexpressed in colorectal cancer cells, as the receptor, thereby improving the active targeting effect. Summary of the Invention

[0007] To address the technical problems existing in the prior art, this invention provides a sal-098 targeted liposome and its preparation method. Specifically, this is achieved through the following technical solution:

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing sal-098-targeting liposomes includes the following steps:

[0010] Step 1: Dissolve the lipid material, long-circulating substance, targeting material, and sal-098 in an organic solvent, remove the organic solvent by rotary evaporation to obtain a uniform oil film, and add phosphate buffer solution at pH 7.4 for hydration.

[0011] Step 2: The hydrated solution is ultrasonically broken to obtain sal-098 targeted liposomes.

[0012] Furthermore, in step 1, the targeting material is hyaluronic acid (HA) and dioleoylphosphatidylethanolamine (DOPE) obtained by condensation reaction to obtain HA-DOPE.

[0013] Furthermore, in step 1, the molecular weight of HA is 1,000-200,000, preferably 10,000-20,000.

[0014] Furthermore, the lipid material in step 1 is phospholipid and cholesterol.

[0015] Furthermore, in step 1, the raw materials and excipients include: phospholipids, cholesterol, DSPE-mPEG, and target HA-DOPE.

[0016] The mass ratio of sal-098 is 20:2:2:1.

[0017] Furthermore, the organic solvent in step 1 is chloroform.

[0018] Furthermore, in step 1, the temperature of the rotary evaporation water bath is 40-50℃, preferably 45℃.

[0019] Furthermore, the hydration method in step 1 is hydration on a shaker at 37°C with a shaker speed of 100 rpm.

[0020] Furthermore, in step 2, the ultrasonic fragmentation is probe ultrasound.

[0021] The sal-098 liposomes prepared using the above method can be used for the treatment of colorectal cancer.

[0022] The sal-098 liposomes prepared in this invention have a targeting effect on colorectal cancer cells. They can deliver the drug to the colorectal cancer site via the bloodstream, increasing the drug concentration at the tumor site and reducing the toxicity of sal-098. Attached Figure Description

[0023] Figure 1 Particle size distribution of sal-098-targeting liposomes

[0024] Figure 2 Zeta potential distribution map of sal-098 targeted liposomes

[0025] Figure 3 Scanning electron microscopy image of sal-098-targeting liposomes

[0026] Figure 4 Schematic diagram of cell culture plate

[0027] Figure 5 In vitro inhibitory effect of sal-098-targeting liposomes on CT26 cells

[0028] Figure 6 The targeting effect of sal-098-targeting liposomes in mice (from top to bottom: DIR, DIR-lips, HA@DIR-lips).

[0029] Figure 7 The targeting effects of sal-098 liposomes on in vitro tumors and tissues (from top to bottom: DIR, DIR-lips, HA@DIR-lips)

[0030] Figure 8In vivo pharmacodynamics of different drug groups

[0031] Figure 9 To assess the in vivo efficacy of different drug groups in tumor recurrence

[0032] Figure 10 Displaying tumor size for different drug groups

[0033] Figure 11 Changes in body weight of mice in different drug groups

[0034] Figure 12 Weights of various organs in mice from different drug groups Detailed Implementation

[0035] The present invention will now be described in detail with reference to specific embodiments. These embodiments are for illustrative purposes only, and the scope of protection of the present invention is defined by the claims and is not limited to these embodiments.

[0036] Example 1: Preparation of HA-DOPE

[0037] Weigh 90 mg of HA and dissolve it in 30 mL of distilled water to obtain an HA solution. Weigh 172.8 mg of EDC and 195 mg of NHS, dissolve them in 1 mL of distilled water, and add them to the HA solution. Adjust the pH of the reaction solution to 7.5 using 0.1 M NaOH solution and activate it in a 37°C water bath for 3 hours. Weigh 216 mg of DOPE, dissolve it in 30 mL of tetrahydrofuran, and add it to the HA reaction solution. Maintain the pH of the reaction solution at 7.5 and react it in a 37°C water bath for 24 hours. Dialyze the reaction solution into 2000 mL of distilled water. After lyophilization, HA-DOPE is obtained.

[0038] Example 2: Preparation of blank liposomes (kb-lips)

[0039] Dissolve 100mg egg yolk lecithin, 10mg cholesterol, 10mg DSPE-mPEG2000, and 10mg HA-DOPE in 10ml chloroform, and evaporate the organic solvent in a water bath at 40-50℃ to form a uniform film.

[0040] After adding PBS at pH 7.4 and hydrating in a shaker at 37°C for 2 hours, the sal-098 liposomes were obtained by sonication for 15 minutes.

[0041] Example 2: Preparation of sal-098 liposomes (sal-lips)

[0042] Dissolve 100mg egg yolk lecithin, 10mg cholesterol, 10mg DSPE-mPEG2000, and 5mg sal-098 in 10ml chloroform, and evaporate the organic solvent in a water bath at 40-50℃ to form a uniform film.

[0043] After adding PBS at pH 7.4 and hydrating in a shaker at 37°C for 2 hours, the sal-098 liposomes were obtained by sonication for 15 minutes.

[0044] Example 3: Preparation of sal-098-targeted liposomes (HA@sal-lips)

[0045] Take 100mg egg yolk lecithin, 10mg cholesterol, 10mg DSPE-mPEG2000, 10mg HA-DOPE, and 5mg sal-098 and dissolve them in 10ml chloroform. Evaporate the organic solvent in a water bath at 40-50℃ to form a uniform film.

[0046] After adding PBS at pH 7.4 and hydrating in a shaker at 37°C for 2 hours, the sal-098-targeted liposomes (HA@sal-lips) were obtained by sonication for 15 minutes.

[0047] Experimental Example 1: Investigation of the basic properties of sal-098-targeted liposomes

[0048] The physicochemical properties and in vitro stability of the sal-098-targeting liposomes prepared in Examples 2 and 3 were investigated. The results are as follows: Figure 1-3 As shown: Particle size and potential diagrams indicate that the prepared liposomes have a uniform particle size distribution; the average particle size is 98 nm, and the potential is -18.7 mV, indicating that they are not prone to aggregation; scanning electron microscopy images show that the obtained liposomes are spherical. In vitro stability tests show that the particle size and potential of the prepared targeted liposomes do not change significantly within 30 days, indicating good liposome stability.

[0049] Experimental Example 2: In vitro toxicity of CT26 cells

[0050] The kb-lips, sal-lips, and HA@sal-lips solutions prepared in Examples 1-3, as well as the sal-098 solution, were used to investigate their toxicity to CT26 cells using the CCK-8 assay. Based on the amount of sal-098 contained, the molar concentration was calculated, and seven experimental groups were set up with concentrations of 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 μM.

[0051] Take CT26 cells in the logarithmic growth phase, gently pipette to the bottom of the culture flask to completely detach the cells, centrifuge the evenly dispersed cells at 800 rpm for 4 min, discard the supernatant, and add 1640 complete culture medium to adjust the cell concentration to 1×10⁻⁶. 6Cells / mL were added to each well of a 96-well cell culture plate, and the plate was then incubated in a cell culture incubator (37°C, 5% CO2, and saturated humidity) for 24 hours. After incubation, the supernatant was discarded. Each 96-well plate was divided into three parts (e.g., ...). Figure 4 Add a series of solutions of different sal-098 concentrations diluted with serum-free 1640 medium, and continue culturing in a cell culture incubator for 72 hours. Discard the medium in the 96-well cell culture plate.

[0052] Add 20 μL of MTT to each well and incubate for 4 hours in a cell culture incubator. Then discard the culture medium and add 150 μL of dimethyl sulfoxide to each well. After this, place the plate in a 37°C constant temperature shaker and shake at 50 rpm for 10 minutes in the dark to ensure complete dissolution. Using blank culture medium as a control, measure the absorbance (OD) of each group at 490 nm using a microplate reader, and calculate the inhibitory effects of different concentrations of sal-098 solution, kb-lips, sal-lips, and HA@sal-lips on cells.

[0053] like Figure 5 As shown, sal-098 solution, sal-lips, and HA@sal-lips all showed inhibitory effects on CT26 cells, and these effects were concentration-dependent. However, kb-lips did not have an inhibitory effect on CT26 cells, indicating that the excipients used in the preparation of sal-098 targeted liposomes were non-toxic to cells.

[0054] Experimental Example 3: In vivo targeting study

[0055] CT26 cells in the logarithmic growth phase were centrifuged at 800 rpm for 4 min, the supernatant was discarded, and then sterile PBS was added to adjust the cell suspension concentration to 2.5 × 10⁻⁶. 6 Cells were counted at a density of [number] per mL, and the cell suspension was stored on ice for later use. Under aseptic conditions, the cells were subcutaneously injected into the right side of BALB / c mice to establish a tumor-bearing mouse model. The length and width of the tumor were measured using calipers, and the tumor volume was calculated using the following formula.

[0056] Volume = (length × width) 2 ) / 2

[0057] When the tumor grows to 800mm 3 Then, they were randomly divided into 3 groups of 6 each.

[0058] Liposomes were prepared by using a DIR-treated fluorescent substance instead of sal-098, and DIR solutions were prepared accordingly.

[0059] DIR-lips and HA@DIR-lips formulations were administered to mice via tail vein injection. Fluorescence distribution was observed in vivo at 648 / 680 nm wavelengths, as shown in the figure. Mice injected with DIR-lips and HA@DIR-lips showed detectable fluorescent material targeting the tumor site, with the HA@DIR-lips group exhibiting significantly higher levels of fluorescence on the tumor surface. DIR solution alone showed the lowest fluorescence on the tumor, with most of the fluorescence being metabolized by the liver.

[0060] Experimental Example 4: In vivo pharmacodynamic study

[0061] Following the method described in Example 3, mice with successfully induced sal-098 were randomly divided into a control group, a free sal-098 group, a kb-lips group, a sal-lips group, and an HA@sal-lips group. The control and experimental groups were injected via tail vein with PBS solution, 200 μL of kb-lips, free sal-098 solution, sal-lips, or HA@sal-lips, respectively.

[0062] (sal-098, 2 mg / kg). Mice were treated with the drug on days 3, 6, 9, 12, 15, 18, and 21. Tumor size and body weight changes were recorded during treatment. After treatment, tumors were surgically removed, collected, weighed, and photographed. Various tissues were collected and weighed. Safety was assessed.

Claims

1. A salinomycin derivative targeting liposomes, characterized in that... The salinomycin derivative liposomes described above are prepared from membrane materials, long-circulating substances, stabilizers, salinomycin derivatives as shown in sal-098, and tumor-targeting substances; wherein the active pharmaceutical ingredient, salinomycin derivative, is as follows.

2. The salinomycin derivative targeted liposome according to claim 1, wherein the salinomycin derivative targeted liposome is prepared from 1-200 parts of phospholipid membrane material, 1-20 parts of long-circulating substance, 1-20 parts of stabilizer, 1-10 parts of salinomycin derivative, and 0.1-20 parts of tumor-targeting substance.

3. The salinomycin derivative targeted liposome according to claim 2, wherein the salinomycin derivative targeted liposome is prepared from 50-150 parts of phospholipid membrane material, 5-15 parts of long-circulating substance, 5-15 parts of stabilizer, 1-10 parts of salinomycin derivative, and 5-15 parts of tumor-targeting substance.

4. The salinomycin derivative targeted liposome according to any one of claims 2 or 3, wherein the phospholipid membrane material is selected from one or more of soybean lecithin, egg yolk lecithin, and hydrogenated soybean lecithin; the long-circulating substance is methoxy-polyethylene glycol-phosphatidylethanolamine mPEG-DSPE; the stabilizer is selected from one or more of sodium cholesterol sulfate, cholesterol, and vitamin E; and the antitumor targeting substance is a composition of hyaluronic acid HA and dioleoylphosphatidylethanolamine DOPE.

5. The salinomycin derivative targeted liposome according to claim 4, wherein the tumor-targeting substance is obtained by HA-DOPE through an amino and carboxyl condensation reaction of HA and DOPE.

6. The salinomycin derivative targeted liposome according to claim 5, wherein the amount of hyaluronic acid molecule (HA) is 1,000 to 200,000, preferably 10,000 to 20,000.

7. The salinomycin derivative targeted liposomes according to any one of claims 1-5, wherein the salinomycin derivative targeted liposomes are prepared by the following method: (1) Dissolve phospholipid membrane material, long-cycle substance, stabilizer, salinomycin derivative and targeting material in chloroform and mix evenly; evaporate organic solvent by water bath rotary evaporation to form a thin film; (2) Add PBS, hydrate in a shaker at 37°C, and then use a probe to sonicate to obtain salinomycin derivative-targeted liposomes.

8. The salinomycin derivative liposomes according to any one of claims 1-7, wherein the salinomycin derivative targeting liposomes are prepared by the following method: (1) Take 100mg egg yolk lecithin, 10mg cholesterol, and 10mg DSPE-mPEG2000. 10 mg HA-DOPE and 5 mg sal-098 were dissolved in 10 ml chloroform and the organic solvent was removed by rotary evaporation in a water bath at 40-50 °C to form a uniform film. (2) Add PBS at pH 7.4, hydrate in a shaker at 37°C for 2 hours, and then sonicate with a probe for 15 minutes to obtain sal-098 targeted liposomes.

9. The use of the salinomycin derivative targeting liposomes according to any one of claims 1-5 in the preparation of a medicament for treating cancer.

10. The application according to claim 9, wherein the cancer is colorectal cancer.

11. The application according to claim 10, wherein the treatment has a targeted effect on colorectal cancer tissue.

12. The application according to any one of claims 9-10, wherein the toxicity of the salinomycin derivative sal-098 is reduced during the treatment.

Images