Compounds for treatment of melanoma

Abstract

The invention provides a compound for treating melanoma. The compound T31 provided by the invention has application in preparation of drugs for preventing or treating melanoma. The tumor nano preparation (HPPN-T31-gp100) with active targeting and photo-thermal-chemical combined treatment effects is obtained by loading the Anti-GP100 modified hollow Prussian blue nano particles with the compound T31, the tumor nano preparation has better targeting performance on melanoma cells (C918), the accuracy and efficiency of drug delivery can be improved, the treatment effect is enhanced, and the tumor nano preparation has good application prospects. Tumogenesis is inhibited through multiple mechanisms (including photothermal effect, immunoregulation, cell apoptosis and the like), meanwhile, no tissue damage is found after the nanoparticles are delivered to mice, and good biological safety is achieved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a compound in preparing a drug for preventing or treating melanoma. Background Art

[0002] Melanoma is a malignant tumor derived from melanocytes, with high metastasis, recurrence and mortality rates. According to data from the Global Cancer Database, there were approximately 325,000 new cases worldwide in 2020, accounting for 1.7% of the total number of new tumors that year. The age-standardized incidence rate was 3.8 / 100,000 for men and 3.0 / 100,000 for women (Arnold M, Singh D, Laversanne M, et al. Global burden of cutaneous melanoma in 2020 and projections to 2040 [J]. JAMADermatol, 2022, 158(5): 495-503). In 2017, the number of new cases of melanoma in my country was estimated to be 16,073, with an age-standardized incidence rate of 0.9 / 100 000. The total number of patients that year was estimated to be 109,316, and the number of fatal cases was estimated to be 5,088, with an age-standardized mortality rate of 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 neural crest-derived melanocytes, which are responsible for producing melanin that protects the eye from UV damage. 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 is highly malignant and lethal, and is prone to metastasis. Most patients have already developed local metastasis of the tumor when diagnosed. The average survival after diagnosis of choroidal melanoma is 9.8 years, while the average survival 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 the development of the tumor, visual function is often severely impaired. In addition, 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 therapy must be combined with radiotherapy. Existing conservative treatments can neither improve the survival rate of patients nor have obvious side effects. At present, 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 solution:

[0007] Use of compound T31 and its pharmaceutical composition in the preparation of a drug for preventing or treating melanoma, wherein the compound T31 has the following structural formula:

[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 hollow Prussian blue nanoparticles modified with Anti-GP100 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 surface of the nanoparticles is connected to the anti-gP100 antibodies to obtain hollow Prussian blue nanoparticles modified with Anti-GP100.

[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 ferrocyanide (K 3 [Fe(CN) 6 ]) is dissolved in a hydrochloric acid solution, and the solution is continuously stirred until the solution becomes a yellow clear liquid. The reaction is carried out at 70-100° C. in a reactor for 10-30 hours, and the temperature is cooled to room temperature to obtain a blue liquid, which is washed with ultrapure water and ethanol, and then centrifuged at 10000-15000 rpm for 8-15 minutes, and 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 dispersed evenly, 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 10000-15000 rpm for 8-15 minutes, and 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 the targeting of the drug and reduce the side effects of the drug, the present invention obtains a tumor nanoformulation (HPPN-T31-gp100) with active targeting and photothermal-chemical combined therapeutic effects by loading the hollow Prussian blue nanoparticles modified with Anti-GP100 with the compound T31, and HPPN-T31-gp100 is characterized by electron microscopy, particle size, and potential, and the drug loading is evaluated. The structure, morphology, and size of the nanoformulation are observed, and the photothermal performance is evaluated, and then the cell uptake, cell ROS response, and cytotoxicity detection are performed. 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, immunomodulation, cell apoptosis, etc.). At the same time, no tissue damage was observed after the nanoparticles were delivered to 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 nanoparticle HPPN-T31-gp100;

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

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

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

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

[0022] Figure 6 is the UV spectrum analysis diagram 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] Fig. 9 This is a photothermal effect diagram of nanoparticles detected by 808nm infrared laser;

[0026] Fig.10 This is a diagram showing the killing effect of HPPN-T31-gp100 on the survival of tumor cells verified by CCK8 method;

[0027] Fig.11 This is a picture showing that the CCK8 method verifies that HPPN-T31-gp100 has no killing effect on retinal pigment epithelial cells;

[0028] Fig.12 is the antioxidant effect diagram of HPPN-T31-gp100 in detecting intracellular ROS;

[0029] Fig.13 is the targeted phagocytosis of HPPN-T31-gp100 by melanoma cells (C918);

[0030] Fig.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 illustrated by specific embodiments below. It should be understood that these embodiments are only helpful for understanding 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 parts by weight, and the percentages are all mass percentages.

[0032] The raw materials and reagents used in the present invention are all commercially available products, among which compound T31 was purchased from Taoshu Biotechnology, polyvinyl pyrrolidone (PVP, K30), potassium ferrocyanide (K3[Fe(CN)6]), polyacrylic acid (PAA), and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid powder (ABTS) were all purchased from Aladdin Reagent Company, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was purchased from Sigma, gp100 antibody was purchased from Wuhan Mitaka Company, and CCK8 reagent was purchased from Solebao Reagent Company.

[0033] Example 1

[0034] Preparation of Anti-GP100 Modified Hollow Prussian Blue Nanoparticles Loaded with Compound T31 Nanoformulation (1) Preparation of Hollow Mesoporous Prussian Blue Nanoparticles

[0035] 1) Weigh 3 g polyvinyl pyrrolidone (PVP, K30) and 113 mg potassium ferrocyanide (K 3 [Fe(CN) 6 ]), dissolved in 40 ml of 0.01 M hydrochloric acid solution;

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

[0037] 3) The yellow clear solution was transferred to a reaction vessel and heated in an oven at 80°C for 20 hours;

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

[0039] 5) Take 20 mg of dried solid Prussian blue nanoparticles and 100 mg of PVP and dissolve them in 20 ml of 1.0 M hydrochloric acid solution;

[0040] 6) Ultrasonic dispersion was uniform, and stirring was performed with a magnetic stirrer for 2 hours;

[0041] 7) The stirred solution was transferred to a reaction vessel and heated in an oven at 140° C. for 4 hours;

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

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

[0044] 1) Select a polymer with a carboxyl group, polyacrylic acid (PAA), and dissolve it 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 the polymer solution, and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was used as a crosslinking agent to further modify the surface of these hollow Prussian blue nanoparticles to ensure that the nanoparticles were evenly dispersed;

[0046] 3) adding 60ul 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 (same concentration 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 with 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 about 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 has no scratches; according to the Lambert-Beer law (A=εbc), where A is the absorbance, ε is the molar absorptivity, b is the optical path length, and c is the sample concentration, analyze the relationship between absorbance and concentration. The relationship between absorbance and wavelength is as follows Figure 6 As shown, the absorbance of nanoparticles HPPN-T31-gp100 increases with increasing concentration, and the maximum absorption peak can be seen at 700 nm.

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

[0052] 808 infrared laser detects the photothermal effect of nanozymes, the results are as follows Fig. 9 As shown in the figure, after 808nm laser irradiation, the temperature rise was positively correlated with the nanoparticle concentration. At 8min, the temperature rise trend gradually stabilized, while the temperature of the group without nanoparticles did not increase significantly. When the concentration of HPPN-T31-gp100 nanoparticles was 250ug / ml, 2W / cm 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 replicate wells 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 37°C, 5% CO 2 Pre-culture in a cell culture incubator for a period of time. Adherent cells need to be cultured for about 2-4 hours. Add 10 μL of CCK-8 solution to each well (if the initial culture volume is 200 μL, add 20 μL of CCK-8 solution). Continue to incubate the culture plate in the incubator for 1-4 hours. Use an enzyme marker to measure the OD value at a wavelength of 450 nm, which can indirectly reflect the number of living cells. Calculate and draw the cell proliferation curve based on the measured OD value, and use GraphPad software for data analysis. CCK8 detects the toxicity of HPPN-T31-gp100 to cells. The results are as follows Fig.10 , Fig.11 As shown. Compared with the normal group, HPPN-T31-gp100 has a certain killing effect on tumor cells. CCK8 biocompatibility experiment: Nanoparticles of different concentrations have no obvious cytotoxicity to normal human retinal pigment epithelial (RPE) cells. When the concentration of nanoparticles rises to 500ug / ml, the survival rate of RPE cells is still greater than 85%. There is no obvious cytotoxicity to normal eye tissues, and it has 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 stand at room temperature for 30 minutes to allow ABTS to react with hydrogen peroxide to generate free radicals; prepare standard curve 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 sample group to be tested, the negative control group (no sample, only PBS) and the positive control group (solution of known antioxidant substances) were set up; 10 μL of the sample to be tested or the 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 for incubation 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 sample to be tested was calculated according to the standard curve, and statistical analysis was performed. The antioxidant capacity results of the samples are shown in the figure. Fig.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. The cells were seeded in a 6-well plate, shaken evenly, and placed in a 5% CO 2 The cells were cultured in a 37°C incubator overnight; the cells were co-incubated with HPPN-T31-gp100 for 24 hours; the illumination group was irradiated with 808nm laser (2W / cm^2) for 10 minutes, and washed with PBS three times after 1 hour; the cells were digested with trypsin without ethylenediaminetetraacetic acid (EDTA) for 6-7 minutes, complete medium was added to terminate the digestion and blow the cells, the digested cells were washed with PBS and centrifuged (1000rpm, 3 minutes) three times and transferred to a flow tube; Annexin-FITC / PI was added and incubated at room temperature for 15 minutes and then analyzed by flow cytometry. The results of flow cytometry detection of cell apoptosis 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, indicating 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 that in Example 1;

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

[0059] (3) After the cells are fully attached, 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 culture dish was removed and the culture medium was discarded. The dish was then washed three times with pre-cooled PBS, and then fixed with 4% paraformaldehyde solution for 15 minutes. After fixation, the dish was washed three times with PBS. A small amount of DAPI dye was then added to the glass circle of the confocal dish and stained for 10 minutes. After staining, the dish was washed three times with PBS.

[0062] (6) Finally, the culture dish was placed under a laser scanning confocal microscope for observation. Fig.13 As shown, HPPN-T31-gp100 has a stronger targeting effect than HPPN-T31 in the process of melanoma cells (C918) phagocytosing nanoparticles, 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, cell apoptosis, etc.).

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

[0064] Wild-type C57 mice were selected and injected with PBS, HPPN, HPPN-T31, and HPPN-T31-gp100 through the tail vein. After 21 days, the following tissues were obtained: heart, liver, spleen, lung, and kidney. The tissues were fixed and sent for HE staining to verify the in vivo biosafety of the nanoparticles. Fig.14 As shown, no tissue damage was observed after the nanoparticles were delivered to mice, indicating that the nanoparticles (HPPN-T31-gp100) have good biosafety.

Claims

1. 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:

2. The use according to claim 1, characterized in that: The melanoma is choroidal melanoma.

3. The use according to claim 1 or 2, characterized in that: The pharmaceutical composition of compound T31 is a nano preparation.

4. The use according to claim 3, characterized in that: The nano preparation is a nano preparation with active targeting.

5. The use according to claim 4, characterized in that: The nanoformulation with active targeting is a nanoformulation of hollow Prussian blue nanoparticles modified with Anti-GP100 and loaded with compound T31.

6. The use according to claim 5, characterized in that The method for preparing hollow Prussian blue nanoparticles modified with Anti-GP100 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 surface of the nanoparticles is connected with the anti-gP100 antibodies, thereby obtaining hollow Prussian blue nanoparticles modified with Anti-GP100.

7. The use according to claim 5 or 6, characterized in that 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, and the solution is continuously stirred until the solution becomes a yellow clear liquid, reacted at 70-100°C for 10-30 hours in a reactor, cooled to room temperature to obtain a blue liquid, washed with ultrapure water and ethanol, and then centrifuged at 10000-15000rpm 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 dispersed uniformly, 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, the obtained liquid is washed with ultrapure water and ethanol, and then centrifuged at 10000-15000rpm for 8-15 minutes, precipitated and dried to obtain hollow Prussian blue nanoparticles.

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