Compounds for the treatment of melanoma

Abstract

The present application provides a kind of compound for treating melanoma.The compound T31 provided by the present application has the use in preparing the drug for preventing or treating melanoma.The present application is loaded with compound T31 by hollow prussian blue nanoparticles modified by Anti-GP100 to obtain tumor nano-preparation (HPPN-T31-gp100) with active targeting and photo-thermal-chemical combined therapy effect, which is more targeted to melanoma cells (C918), can improve the accuracy and efficiency of drug delivery, enhance the therapeutic effect, and inhibit tumor growth through multiple mechanisms (including photo-thermal effect, immunomodulation, apoptosis, etc.), while no tissue damage is found after delivering nanoparticles to mice, and has good biological safety.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a use of a compound in preparation of a drug for preventing or treating melanoma. BACKGROUND

[0002] Melanoma is a malignant tumor derived from melanocytes, and has high metastasis rate, recurrence rate and mortality rate. Data from the Global Cancer Database shows that there were about 325,000 new cases worldwide in 2020, accounting for 1.7% of the total number of new tumors that year. The male age-standardized incidence rate was 3.8 / 100,000, and the female was 3.0 / 100,000 (Arnold M, Singh D, Laversanne M, et al. Global burden of cutaneous melanoma in 2020 and projections to 2040 [J]. JAMA Dermatol, 2022, 158(5): 495-503). In 2017, the number of new cases of melanoma in China was estimated to be 16,073, and the age-standardized incidence rate was 0.9 / 100,000. The total number of patients with the disease was estimated to be 109,316 that year, and the number of deaths was estimated to be 5,088, and the age-standardized mortality rate was 0.3 / 100,000 (Wu Y, Wang Y, Wang L, et al. Burden of melanoma in China, 1990-2017: findings from the 2017 global burden of disease study [J]. Int J Cancer, 2020, 147(3): 692-701).

[0003] Choroidal melanoma originates from melanocytes derived from the neural crest, which are responsible for producing melanin that protects the eye from ultraviolet light. Choroidal melanoma is the second largest subtype of malignant melanoma and the most common intraocular malignancy in adults, accounting for about 70% of intraocular tumors. Choroidal melanoma has a high degree of malignancy and mortality, and is prone to metastasis. Most patients have already developed local metastasis of the tumor by the time of diagnosis. The average survival period after diagnosis of choroidal melanoma is 9.8 years, and the average survival period after tumor metastasis is only 35.2 months.

[0004] Currently, the most common treatments for choroidal melanoma include enucleation, local tumor resection, radiotherapy, laser therapy, and other therapies. Although tumor resection combined with radiotherapy can effectively control tumor development, visual function is often severely impaired. Moreover, the side effects of radiotherapy last from a few hours to several years. Transpupillary thermotherapy cannot kill tumor cells in the scleral tissue, so this treatment must be combined with radiotherapy. Existing conservative treatments can neither improve patient survival rates nor have significant side effects. Currently, there are very few chemotherapy drugs that are toxic to tumor cells but do not damage normal cells and tissues. The dilemma of improving the survival rate of melanoma patients remains an important research topic, and systemic chemotherapy combined with multimodal therapy is imminent in order to provide new ideas for the treatment of choroidal melanoma. Summary of the Invention

[0005] In order to solve the problems in the prior art, the present invention provides a use of a compound in preparing a drug for preventing or treating melanoma.

[0006] The present invention adopts the following technical solutions:

[0007] Use of compound T31 and its pharmaceutical composition in the preparation of a drug for preventing or treating melanoma. The structural formula of compound T31 is as follows:

[0008]

[0009] Compound T31.

[0010] According to one embodiment of the present invention, the melanoma is choroidal melanoma.

[0011] According to one embodiment of the present invention, the pharmaceutical composition of compound T31 is a nanoformulation.

[0012] According to one embodiment of the present invention, the nanoformulation is a nanoformulation with active targeting; further, the nanoformulation with active targeting is a nanoformulation of hollow Prussian blue nanoparticles modified with Anti-GP100 and loaded with compound T31.

[0013] According to one embodiment of the present invention, the method for preparing the above-mentioned Anti-GP100-modified hollow Prussian blue nanoparticles is as follows: polyacrylic acid is dissolved in deionized water to prepare a uniform polymer solution, a cross-linking agent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is added, and then gp100 antibodies are added for co-incubation, so that the anti-gP100 antibodies are connected to the surface of the nanoparticles to obtain Anti-GP100-modified hollow Prussian blue nanoparticles.

[0014] According to one embodiment of the present invention, the preparation method of the hollow Prussian blue nanoparticles is as follows: polyvinyl pyrrolidone and potassium ferricyanide (K3[Fe(CN)6]) are dissolved in a hydrochloric acid solution, continuously stirred until the solution becomes a yellow clear liquid, reacted in a reactor at 70-100°C for 10-30 hours, cooled to room temperature to obtain a blue liquid, washed with ultrapure water and ethanol, and then centrifuged at 10,000-15,000 rpm for 8-15 minutes, precipitated and dried to obtain solid Prussian blue nanoparticles; then the dried solid Prussian blue nanoparticles and PVP are dissolved in a hydrochloric acid solution and evenly dispersed, stirred for 1-3 hours, and then the stirred solution is transferred to a reactor, reacted at 120-150°C for 3-6 hours, cooled to room temperature, and the obtained liquid is washed with ultrapure water and ethanol, and then centrifuged at 10,000-15,000 rpm for 8-15 minutes, precipitated and dried to obtain hollow Prussian blue nanoparticles.

[0015] Beneficial effects:

[0016] The present invention provides a compound T31 for treating melanoma. The compound T31 provided by the present invention has the use in preparing a drug for preventing or treating melanoma. In order to improve drug targeting and reduce drug side effects, the present invention obtains a tumor nanoformulation (HPPN-T31-gp100) with active targeting and photothermal-chemotherapy effects by loading compound T31 onto hollow Prussian blue nanoparticles modified with Anti-GP100. HPPN-T31-gp100 is characterized by electron microscopy, particle size, and potential, and drug loading is evaluated. The structure, morphology, and size of the nanoformulation are observed, and its photothermal performance is evaluated. Cell uptake, cellular ROS response, and cytotoxicity are then tested. The in vivo biosafety of HPPN-T31-gp100 was evaluated using a choroidal melanoma subcutaneous tumor mouse model. The results showed that it has a targeted phagocytic effect on melanoma cells (C918), can improve the accuracy and efficiency of drug delivery, enhance the therapeutic effect, and inhibit tumor growth through multiple mechanisms (including photothermal effect, immune regulation, cell apoptosis, etc.). At the same time, no tissue damage was observed after the nanoparticles were delivered to the mice, indicating that the nanoparticles (HPPN-T31-gp100) prepared by the present invention have good biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a scanning electron micrograph of nanoparticles HPPN-T31-gp100;

[0018] Figure 2 is a transmission electron microscopy image of nanoparticles HPPN-T31-gp100;

[0019] Figure 3 is the energy spectrum analysis diagram of nanoparticle HPPN-T31-gp100;

[0020] Figure 4 This is the effective particle size frequency diagram of nanoparticles HPPN-T31-gp100;

[0021] Figure 5 This is a characterization analysis of the zeta potential of nanoparticles HPPN-T31-gp100;

[0022] Figure 6 is the UV spectrum analysis of nanoparticles HPPN-T31-gp100;

[0023] Figure 7 This is a HPLC standard curve chart made based on different concentrations of T31 drug;

[0024] Figure 8 is a HPLC chromatogram analyzing the efficiency of T31 drug delivery to nanoparticles;

[0025] Figure 9 This is a photothermal effect diagram of nanoparticles detected by 808nm infrared laser;

[0026] Figure 10 This is a diagram showing the CCK8 assay used to verify that HPPN-T31-gp100 has a killing effect on tumor cell survival;

[0027] Figure 11 This is a diagram showing that the CCK8 assay verifies that HPPN-T31-gp100 has no killing effect on retinal pigment epithelial cells;

[0028] Figure 12 This is a graph showing the antioxidant effect of HPPN-T31-gp100 in detecting intracellular ROS;

[0029] Figure 13 is a graph of targeted phagocytosis of HPPN-T31-gp100 by melanoma cells (C918);

[0030] Figure 14 This is a safety verification diagram of nanoparticles (HPPN-T31-gp100) in animal organs. DETAILED DESCRIPTION

[0031] In order to further illustrate the present invention and its advantages, the technical scheme of the present invention is further described below by specific embodiments. It should be understood that these embodiments are only for the purpose of helping to understand the present invention and should not be regarded as specific limitations of the present invention. Unless otherwise specified, the parts described in the present invention are all by weight and the percentages are all by mass.

[0032] The raw materials and reagents used in the application are all commercially available products, wherein compound T31 is purchased from Tao Shu Biology, polyvinylpyrrolidone (PVP, K30), potassium ferricyanide (K3[Fe(CN)6]), polyacrylic acid (PAA), 2,2'-azobis-3-ethylbenzothiazoline-6-sulfonic acid powder (ABTS) are all purchased from Aladdin Reagent Company, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is purchased from Sigma Company, gp100 antibody is purchased from Wuhan Sanying Company, and CCK8 reagent is purchased from Solabio Reagent Company.

[0033] Example 1

[0034] Preparation of Anti-GP100 modified hollow prussian blue nanoparticle loaded compound T31 nanoformulation (1), preparation of hollow mesoporous prussian blue nanoparticles

[0035] 1) 3 grams of polyvinylpyrrolidone (PVP, K30) and 113 milligrams of potassium ferricyanide (K3[Fe(CN)6]) were weighed and dissolved in 40 milliliters of 0.01M hydrochloric acid solution;

[0036] 2) Stir with a magnetic stirrer for 30 minutes until the solution becomes a yellow clear liquid;

[0037] 3) Transfer the yellow clear solution to the reaction kettle and heat in the 80℃ oven for 20 hours;

[0038] 4) After the reaction is completed, the reaction kettle is cooled to room temperature, and the obtained blue liquid is washed with ultrapure water and ethanol, then centrifuged at 12000 rpm for 10 minutes and dried to obtain solid prussian blue nanoparticles;

[0039] 5) Take 20 milligrams of dried solid prussian blue nanoparticles and 100 milligrams of PVP, and dissolve in 20 milliliters of 1.0M hydrochloric acid solution;

[0040] 6) Ultrasonic dispersion and magnetic stirrer stirring for 2 hours;

[0041] 7) Transfer the stirred solution to the reaction kettle and heat in the 140℃ oven for 4 hours;

[0042] 8) After the reaction is completed, the reaction kettle is cooled to room temperature, and the obtained liquid is washed with ultrapure water and ethanol, then centrifuged (12000 rpm, 10 minutes) and dried to obtain hollow prussian blue nanoparticles;

[0043] (2) Anti-GP100 modified hollow prussian blue nanoparticles

[0044] 1) Select a polymer with carboxyl group-polyacrylic acid (PAA) and dissolve in deionized water to prepare a uniform polymer solution;

[0045] 2) 1 ml (1 mg / ml) of the synthesized hollow Prussian blue nanoparticles were suspended in a polymer solution and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used as a crosslinker to further modify the surface of these hollow Prussian blue nanoparticles to ensure uniform dispersion of the nanoparticles.

[0046] 3) Add 60 μl of gp100 antibody for co-incubation, so that the surface of the modified nanoparticles is connected to the anti-gP100 antibody to form the final nanostructure;

[0047] (3) Anti-GP100-modified hollow Prussian blue nanoparticles were loaded with compound T31. Anti-GP100-HPPN (concentration was the same as before) was incubated with 500ul 25ug / ml compound T31 in the dark with stirring for 24h. At this time, the T31 compound was loaded into the hollow Prussian blue nanoparticles to obtain a nanoformulation (HPPN-T31-gp100) with active targeting of melanoma.

[0048] Scanning electron microscope images of nanoparticles Figure 1 As shown, the transmission electron microscope images of nanoparticles are shown in Figure 2 The energy spectrum analysis results are shown in Figure 3 Scanning electron microscopy and transmission electron microscopy showed that the diameter of the nanoparticles (HPPN-T31-gp100) was about 110 nm; the structure was a hollow cube, and energy spectrum analysis showed that the nanoparticles were rich in iron.

[0049] The particle size analysis results are as follows Figure 4 As shown in Figure 2, the effective particle size frequency shows that the diameter of the nanoparticles (HPPN-T31-gp100) is around 110 nm. Zeta potential characterization analysis is shown in Figure 2. Figure 5 As shown, the zeta potential results showed that the nanoparticles (HPPN-T31-gp100) had good dispersibility and stability.

[0050] The inventors used ultraviolet spectroscopy to analyze the relationship between the absorbance and wavelength of nanoparticles (HPPN-T31-gp100) at different concentrations. Prepare sample solutions of different concentrations: 31.25ug / ml, 62.5ug / ml, 125ug / ml, 250ug / ml, 500ug / ml and 1000ug / ml sample solutions, use a spectrophotometer to conduct experiments, and calibrate the instrument with standard samples or solutions of known concentrations to ensure its accuracy; measure the absorbance at the wavelength: set the wavelength range of the spectrophotometer, place the sample in a cuvette, use the blank solution as a control, and measure the absorbance at different wavelengths. Make sure the cuvette is clean and free of scratches; analyze the relationship between absorbance and concentration according to the Lambert-Beer law (A=εbc), where A is absorbance, ε is the molar absorptivity, b is the optical path length, and c is the sample concentration. The relationship between absorbance and wavelength is as follows: Figure 6 As shown in the figure, the absorbance of nanoparticles HPPN-T31-gp100 increases with increasing concentration, and the maximum absorption peak can be seen at 700 nm.

[0051] The efficiency / encapsulation rate of drug delivery of the T31 compound into the nanoparticles was analyzed by high-performance liquid chromatography. The detector type was UV-Vis, and the column type and dimensions were, for example, C18, 250 mm × 4.6 mm, 5 μm. Mobile phase A was water: acetonitrile = 60:40, containing 0.1% trifluoroacetic acid (TFA); mobile phase B was pure acetonitrile, and the sample solution was dissolved in mobile phase A to 1 mg / mL. The flow rate was 1.0 mL / min, the detection wavelength was 254 nm, the column temperature was room temperature, and the injection volume was 10 μL. Drug encapsulation rate = (mass of encapsulated drug / mass of total sample) × 100%. The results of the high-performance liquid chromatography analysis are shown in FIG. Figure 7 、 Figure 8 As shown, the results showed that the efficiency / encapsulation rate of drug delivery of T31 compound into nanoparticles was 97.2% by HPLC analysis.

[0052] 808 infrared laser detection of the photothermal effect of nanozymes, the results are as follows Figure 9 As shown in the figure, after 808nm laser irradiation, the temperature rise is positively correlated with the nanoparticle concentration. After 8min, the temperature rise trend gradually stabilizes, while the temperature of the group without nanoparticles does not increase significantly. When the concentration of HPPN-T31-gp100 nanoparticles is 250ug / ml ... 2 After 10 minutes of laser irradiation, the temperature rose from 20°C to 46°C, and the nanoparticles showed good photothermal conversion efficiency, which is beneficial for thermal treatment of tumors.

[0053] CCK8 detects the toxicity of HPPN-T31-gp100 on cells. First, the cells to be tested need to be digested with trypsin and dispersed in complete culture medium by centrifugation; the cell suspension is inoculated into a 96-well plate, about 100 μL per well, different concentration groups are set according to the experimental design, and 3-6 replicates are designed for each concentration group, and a blank group and a control group are set at the same time; the 96-well plate is placed in a cell culture incubator at 37°C and 5% CO2 for pre-culture for a period of time, and the adherent cells need to be cultured for about 2-4 hours; 10 μL of CCK-8 solution is added to each well (if the initial culture volume is 200 μL, 20 μL of CCK-8 solution needs to be added); the culture plate is continued to incubate in the incubator for 1-4 hours; the OD value is measured at a wavelength of 450 nm using an enzyme reader, which can indirectly reflect the number of living cells; based on the measured OD value, the cell proliferation curve is calculated and drawn, and the data is analyzed using the software GraphPad. CCK8 detects the toxicity of HPPN-T31-gp100 on cells, and the results are as follows Figure 10 、 Figure 11 Compared with the normal control group, HPPN-T31-gp100 showed a moderate killing effect on tumor cells. CCK8 biocompatibility experiments showed that nanoparticles at different concentrations showed no significant cytotoxicity to normal human retinal pigment epithelial (RPE) cells. When the nanoparticle concentration was increased to 500 μg / ml, the RPE cell survival rate remained above 85%. There was no significant cytotoxicity to normal ocular tissue, demonstrating high biocompatibility.

[0054] The antioxidant capacity of the sample was determined by the ABTS method to evaluate its scavenging effect on reactive oxygen species (ROS). Weigh an appropriate amount of ABTS powder and add an appropriate amount of phosphate buffer, dissolve and dilute to the required concentration (usually 7mM), add the same volume of hydrogen peroxide solution (usually 2.45mM), mix well and let it stand at room temperature for 30 minutes to allow ABTS and hydrogen peroxide to react and generate free radicals; prepare a standard curve for determination and prepare sample pretreatment, and add 10mM Trolox standard solution was diluted to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5 mM; 200 μL of ABTS free radical solution was added to each well of a 96-well plate, and different concentrations of the test sample group, negative control group (no sample, only PBS) and positive control group (solution of known antioxidant substances) were set up; 10 μL of the test sample or control solution was added to each well, and after gently mixing, the 96-well plate was placed in a 37°C constant temperature water bath and incubated for 30 minutes; after the incubation, the absorbance of each well was measured at a wavelength of 734 nm using a spectrophotometer, and the data was recorded as a quantitative indicator of the antioxidant capacity of the sample; the antioxidant capacity of the test sample was calculated according to the standard curve, and statistical analysis was performed. The antioxidant capacity results of the samples are shown in Figure 2. Figure 12As shown, intracellular ROS detection demonstrated the scavenging effect of nanoparticles (HPPN-T31-gp100) on reactive oxygen species (ROS), and had good antioxidant capacity.

[0055] Cell apoptosis was detected by flow cytometry. Cells were seeded in a 6-well plate, shaken, and cultured overnight in a 37°C incubator with 5% CO2. Cells were incubated with HPPN-T31-gp100 for 24 hours. The illumination group was irradiated with an 808 nm laser (2 W / cm²) for 10 minutes and washed three times with PBS after 1 hour. The cells were then digested with ethylenediaminetetraacetic acid (EDTA)-free trypsin for 6-7 minutes. Complete medium was added to terminate the digestion and the cells were pipetted. The digested cells were washed with PBS, centrifuged three times (1000 rpm, 3 minutes), and transferred to a flow cytometer. Annexin-FITC / PI was added and incubated at room temperature for 15 minutes before analysis by flow cytometry. The results of cell apoptosis detection by flow cytometry showed that the apoptosis rate of the HPPN-T31-gp100 group was higher than that of the control group, proving that nanoparticles can effectively promote cell apoptosis to a certain extent; the apoptosis rate of the HPPN-T31-gp100+808 laser group was significantly higher than that of the blank control group, with significant differences, suggesting that the effect of combined photothermal and chemodynamic therapy is stronger than that of single chemodynamic therapy.

[0056] Example 2

[0057] Analysis of the homologous targeting of nanoparticles (HPPN-T31-gp100) to melanoma cells (C918) (1) First, HPPN-T31 and HPPN-T31-gp100 nanoparticles labeled with CY5.5 red fluorescent dye were prepared. The specific preparation method was the same as in Example 1;

[0058] (2) C918 cells were seeded in confocal culture dishes (5×10 4 cells, a total of 1 mL), and placed in a cell culture incubator for 12 hours;

[0059] (3) After the cells have fully adhered, discard the original culture medium and add serum-free culture medium according to the different experimental groups, and incubate in the incubator for 2 hours;

[0060] (4) HPPN-T31 and HPPN-T31-gp100 nanoparticles were then added, and the dose of nanoparticles added was equal (2 mg / mL);

[0061] (5) The removed culture dish is first discarded of the culture solution, then washed with pre-cooled PBS three times, then fixed with 4% paraformaldehyde solution for 15 minutes, after fixation, washed with PBS three times, then add a little DAPI dye in the co-focal culture dish glass circle, stain for 10 minutes, after staining, wash with PBS three times;

[0062] (6) Finally, the culture dish is placed under the laser scanning confocal microscope for observation, the results are shown in Figure 13 , melanoma cells (C918) have stronger targeting effect in the process of phagocytosis of nanoparticles than HPPN-T31-gp100, which can improve the accuracy and efficiency of drug delivery, enhance the therapeutic effect, and inhibit tumor growth through multiple mechanisms (including photothermal effect, immune regulation, apoptosis, etc.).

[0063] Example 3: In vitro verification of the biological safety of nanoparticles (HPPN-T31-gp100)

[0064] Select wild type C57 mice, inject PBS; HPPN; HPPN-T31; HPPN-T31-gp100 into the tail vein respectively, dissection of tissues: heart, liver, spleen, lung, kidney after 21 days, tissue fixation; send sample HE staining to verify the in vivo biological safety of nanoparticles. The results are shown in Figure 14 , after delivering nanoparticles to mice, no tissue damage was observed, indicating that the nanoparticles (HPPN-T31-gp100) have good biological safety.

Claims

1. Use of a pharmaceutical composition of compound T31 in the preparation of a drug for treating melanoma. The structural formula of compound T31 is as follows: Compound T31; The melanoma is choroidal melanoma; the pharmaceutical composition of compound T31 is a nanoformulation; the nanoformulation is a nanoformulation with active targeting; the nanoformulation with active targeting is a nanoformulation of hollow Prussian blue nanoparticles modified with Anti-GP100 and loaded with compound T31.

2. The use according to claim 1, characterized in that The method for preparing hollow Prussian blue nanoparticles modified with anti-GP100 comprises dissolving polyacrylic acid in deionized water to prepare a uniform polymer solution, adding a crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and then adding an anti-gp100 antibody for co-incubation, so that the anti-gP100 antibody is connected to the surface of the nanoparticles to obtain hollow Prussian blue nanoparticles modified with anti-GP100.

3. The use according to claim 1 or 2, characterized in that The preparation method of the hollow Prussian blue nanoparticles comprises: dissolving polyvinyl pyrrolidone and potassium ferricyanide in a hydrochloric acid solution, continuously stirring until the solution becomes a yellow clear liquid, reacting at 70-100°C in a reactor for 10-30 hours, cooling to room temperature to obtain a blue liquid, washing with ultrapure water and ethanol, and then centrifuging at 10,000-15,000 rpm for 8-15 minutes, precipitating and drying to obtain solid Prussian blue nanoparticles; then dissolving the dried solid Prussian blue nanoparticles and PVP in a hydrochloric acid solution and uniformly dispersing them, stirring for 1-3 hours, and then transferring the stirred solution to a reactor, reacting at 120-150°C for 3-6 hours, cooling to room temperature, washing the obtained liquid with ultrapure water and ethanol, and then centrifuging at 10,000-15,000 rpm for 8-15 minutes, and precipitating and drying to obtain hollow Prussian blue nanoparticles.

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