Application of statin in preparation of product for treating skin T cell lymphoma

Abstract

The invention discloses application of statin in preparation of a product for treating skin T cell lymphoma. The invention provides application of statins in preparation of products with any one of the following functions: 1) treating skin T cell lymphoma; 2) promoting apoptosis of skin T cell lymphoma cells; 3) reducing the activity of T cell lymphoma cells of the skin; 4) reducing the tumor formation ability of skin T cell lymphoma cells; and 5) inhibiting the growth of skin T cell lymphoma cells. The invention provides a new application of simvastatin in treating CTCL (China Type Culture Collection Language), and simvastatin and bexarotene are combined for treatment to achieve a synergistic treatment effect. Meanwhile, simvastatin and bexarotene with lower concentration are applied, so that the side effect of the medicine can be prevented, and the effect of effectively inhibiting CTCL tumor growth can be achieved. A certain basis is provided for treating a patient with CTCL in a progressive stage by combining the statin, the retinoic acid and the analogue thereof in future clinical research, and clinical transformation of'new use of old medicine 'of the statin is facilitated to be promoted.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of statins in preparing products for treating cutaneous T-cell lymphoma. Background Art

[0002] Simvastatin, chemical name [1S-[1α, 3α, 7β, 8β, (2S*, 4S*) 8αβ]]-1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-[2-(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1-naphthyl-2,2-dimethylbutyrate. The molecular formula is C 25 H 38 O5, with a molecular weight of 418.57. Simvastatin is a selective HMG-CoA reductase inhibitor. It reduces very low-density lipoprotein cholesterol (VLDLC) concentrations by inhibiting HMG-CoA reductase and cholesterol biosynthesis in the liver. It also induces low-density lipoprotein (LDL) receptors, leading to a decrease in LDL cholesterol and increased LDL-C catabolism, thereby lowering normal and elevated LDL-C concentrations. Simvastatin can also increase high-density lipoprotein cholesterol (HDL-C) concentrations and lower plasma triglycerides (TG). These factors can lead to lowering of total cholesterol / HDL-C and LDL-C / HDL-C ratios. Its clinical indications include hyperlipidemia, coronary artery disease with hypercholesterolemia, and children with heterozygous familial hypercholesterolemia. Combined with dietary control, this drug can be used to lower total cholesterol, LDL-C, apolipoprotein B, and triglycerides.

[0003] Cutaneous T-cell lymphoma (CTCL) is a group of non-Hodgkin's lymphomas caused by the malignant clonal expansion of skin-homing T cells. Its incidence has been increasing annually, rising to the second highest position among extranodal non-Hodgkin's lymphomas. Mycosis fungoides (MF) is the most common type of CTCL, accounting for approximately 60% of CTCL cases. Taking MF as an example, clinical manifestations include patches, plaques, and tumors, accompanied by varying degrees of pruritus, and the disease often affects non-sun-exposed areas. Early-stage MF (stages I-IIA) progresses slowly, presenting as patchy or plaque-like lesions with an indolent course and a favorable prognosis. Advanced-stage MF (stages IIB-IVB) presents as widespread skin tumors or erythrodermic lesions, which may involve extracutaneous organs. The disease course is aggressive, resistant to conventional treatment, and has a poor prognosis. However, the mechanisms of CTCL progression remain unclear, and effective management strategies for advanced disease are lacking. Currently, systemic therapies, including interferon, methotrexate, retinoic acid, and its analogs (bexarotene, acitretin, etc.), are commonly used to treat early-stage refractory or some advanced cases. Most patients with advanced disease have poor skin barrier function, and multidrug combinations increase the risk of secondary infection and the incidence of adverse drug reactions. Furthermore, patients with advanced disease are more likely to develop drug resistance, ultimately requiring chemotherapy or bone marrow transplantation, significantly impairing their quality of life.

[0004] Therefore, a safe and effective treatment plan that controls disease progression is an important issue that needs to be urgently addressed in clinical practice. Summary of the Invention

[0005] The present invention aims to solve the problem of difficult clinical treatment of cutaneous T-cell lymphoma, and explores new uses of simvastatin, an old clinical lipid-lowering drug, for treating cutaneous T-cell lymphoma, or combining it with bexarotene, a conventional drug for cutaneous T-cell lymphoma, for the synergistic treatment of cutaneous T-cell lymphoma.

[0006] In a first aspect, the present invention provides the use of statins in the preparation of products having any of the following functions:

[0007] 1) Treatment of cutaneous T-cell lymphoma;

[0008] 2) Promote apoptosis of cutaneous T-cell lymphoma cells;

[0009] 3) Reduce the viability of cutaneous T-cell lymphoma cells;

[0010] 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells;

[0011] 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

[0012] In another aspect, the present invention provides the use of statins and drugs or reagents required for systemic treatment of cutaneous T-cell lymphoma in the preparation of a product having any of the following functions:

[0013] 1) Treatment of cutaneous T-cell lymphoma;

[0014] 2) Promote apoptosis of cutaneous T-cell lymphoma cells;

[0015] 3) Reduce the viability of cutaneous T-cell lymphoma cells;

[0016] 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells;

[0017] 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

[0018] In the above application, the drug or reagent required for the systemic treatment of cutaneous T-cell lymphoma is retinoic acid or its analogues.

[0019] Furthermore, the retinoic acid or its analogue is bexarotene.

[0020] In the above application, the statin is simvastatin and / or atorvastatin.

[0021] In the above, the cutaneous T-cell lymphoma cells are in vitro cultured CTCL cell lines or primary CTCL cells.

[0022] In the above, the cutaneous T-cell lymphoma is progressive cutaneous T-cell lymphoma, specifically progressive cutaneous T-cell lymphoma requiring systemic treatment.

[0023] In the applications described above, the product is a drug, a kit or other biological product.

[0024] In a second aspect, the present invention provides a drug comprising the statin described in the first aspect and any other substance that can serve as a drug carrier;

[0025] The drug has any of the following functions:

[0026] 1) Treatment of cutaneous T-cell lymphoma;

[0027] 2) Promote apoptosis of cutaneous T-cell lymphoma cells;

[0028] 3) Reduce the viability of cutaneous T-cell lymphoma cells;

[0029] 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells;

[0030] 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

[0031] In a third aspect, the present invention provides a drug comprising the statin described in the first aspect, the drug or reagent required for systemic treatment of cutaneous T-cell lymphoma, and any other substance that can serve as a drug carrier;

[0032] The drug has any of the following functions:

[0033] 1) Treatment of cutaneous T-cell lymphoma;

[0034] 2) Promote apoptosis of cutaneous T-cell lymphoma cells;

[0035] 3) Reduce the activity of cutaneous T-cell lymphoma cells;

[0036] 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells;

[0037] 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

[0038] In a fourth aspect, the present invention provides the use of the statins described in the first aspect and any other substance that can serve as a drug carrier in the preparation of a drug having any of the following functions:

[0039] 1) Treatment of cutaneous T-cell lymphoma;

[0040] 2) Promote apoptosis of cutaneous T-cell lymphoma cells;

[0041] 3) Reduce the activity of cutaneous T-cell lymphoma cells;

[0042] 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells;

[0043] 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

[0044] In a fifth aspect, the present invention provides the use of the statin described in the second aspect, the drug or reagent required for the systemic treatment of cutaneous T-cell lymphoma, and any other substance that can serve as a drug carrier in the preparation of a drug having any of the following functions:

[0045] 1) Treatment of cutaneous T-cell lymphoma;

[0046] 2) Promote apoptosis of cutaneous T-cell lymphoma cells;

[0047] 3) Reduce the activity of cutaneous T-cell lymphoma cells;

[0048] 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells;

[0049] 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

[0050] This study reveals for the first time the potential therapeutic value of statins in CTCL. In primary cells, it was found that tumorous CD4+ T cells were more sensitive to statins than normal CD4+ T cells. Simvastatin, administered by gavage to CTCL mice, was found to effectively inhibit CTCL tumor growth without affecting mouse body weight or general condition. These results suggest that statins at this concentration can be a relatively safe and specific treatment for CTCL. Simvastatin is also proposed as a new use for the treatment of CTCL, and combination therapy with bexarotene can achieve a synergistic therapeutic effect. The most common side effect of retinoic acid and its analogs (bexarotene, acitretin, etc.), systemic CTCL treatments, is abnormal lipid metabolism, requiring statin lipid-lowering therapy in severe cases. Studies have shown that statins can synergistically kill CTCL cells with bexarotene. Therefore, the simultaneous use of low concentrations of statins and retinoic acid and its analogs can not only prevent drug side effects but also effectively inhibit CTCL tumor growth. These results may provide a certain basis for the combined treatment of patients with advanced CTCL with statins and retinoic acid and its analogues in future clinical studies, and help promote the clinical transformation of statins in the field of CTCL treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The CTCL cell lines Hut78 and Myla were treated with different concentrations of simvastatin, and the cell viability was detected by MTS (A, D), the degree of Caspase 3 cleavage was detected by protein immunoblotting to evaluate the cell apoptosis pathway (B, E), and the apoptosis and necrosis were analyzed by flow cytometry (C, F).

[0052] Figure 2 PBMCs from MF patients with peripheral blood involvement were treated with simvastatin and then flow cytometry was used to analyze the apoptosis and necrosis of CD3+CD4+CD7- T cells and CD3+CD4+CD7+ T cells.

[0053] Figure 3 CD4+T cells derived from peripheral blood PBMC of healthy volunteers were treated with different concentrations of simvastatin and then analyzed for apoptosis and necrosis by flow cytometry.

[0054] Figure 4 Relative cell viability (AC) and drug synergy analysis (DF) of Myla, Hut78, and HH cell lines after treatment with different concentrations of simvastatin and bexarotene alone or in combination.

[0055] Figure 5 Relative cell viability (A, B) and drug synergistic effect analysis (C, D) of Myla and Hut78 cell lines after treatment with different concentrations of atorvastatin and bexarotene alone or in combination.

[0056] Figure 6 To measure the changes in tumor volume (A) and mouse body weight (B) of CTCL xenograft mice in the control and simvastatin-treated groups over the treatment days. DETAILED DESCRIPTION

[0057] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0058] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0059] The human cutaneous T-cell lymphoma cell lines used in the following examples are as follows: HH and Myla cell lines: derived from human cutaneous T-cell lymphoma (MF) patients. Hut78 cell line: derived from a patient with Sézary syndrome (SS). RPMI-1640 basal medium supplemented with 10% fetal bovine serum and 1% penicillin (100 U / ml)-streptomycin (0.1 mg / ml) was used for culture in a cell culture incubator maintained at 37°C, 100% relative humidity, and 5% CO2. All CTCL cell lines were grown in suspension, with culture medium exchanged approximately every 18-24 hours depending on growth. Experiments were performed using cells in the logarithmic growth phase.

[0060] The experimental reagents and consumables in the following examples are shown in Table 1:

[0061] Table 1 shows the experimental reagents and consumables

[0062]

[0063]

[0064] The cell culture method in the following examples is as follows: CTCL cell lines and primary cells were cultured using RPMI-1640 basal medium + 10% fetal bovine serum + 1% penicillin (100 U / ml)-streptomycin sulfate (0.1 mg / ml) double-antibody culture medium in a cell culture incubator at a constant temperature of 37°C, a relative humidity of 100%, and 5% CO2.

[0065] Peripheral blood PBMC extraction in the following examples:

[0066] (1) 8 mL of EDTA-anticoagulated peripheral blood from CTCL patients with peripheral blood involvement or healthy controls was mixed with 8 mL of calcium- and magnesium-free phosphate buffered saline (PBS) to make a total of 16 mL;

[0067] (2) Add 4 ml of human peripheral blood lymphocyte separation medium (Tianjin Haoyang, LTS1077) to each of four 15 ml centrifuge tubes;

[0068] (3) Tilt the centrifuge tube and slowly add 4 ml of mixed peripheral blood to the centrifuge tube in a 1:1 ratio using a Pasteur pipette. Pay attention to the buffer and do not break the liquid surface.

[0069] (4) Centrifugation: 400 rcf, 30 min, room temperature, acceleration 1, deceleration 0. After centrifugation, the liquid in the tube is divided into four layers from top to bottom: plasma layer, circular milky white lymphocyte layer, transparent separation fluid layer, and red blood cell layer;

[0070] (5) Aspirate the second layer, which is the peripheral blood mononuclear cells, and transfer it to a new 15 mL centrifuge tube. Add 10 mL of PBS, mix well, and centrifuge at 1500 rpm for 10 minutes at room temperature. Wash and count the cells.

[0071] (6) The PBMCs isolated from MF patients with blood involvement were treated with drugs and subsequently stained with fluorescently labeled flow cytometry antibodies for CD3, CD4, and CD7; the PBMCs isolated from healthy controls were subjected to CD4+ T cell sorting (Miltenyi, 130-096-533) to obtain highly pure CD4+ T cells for subsequent drug treatment experiments.

[0072] In the following examples, MTS was used to detect cell viability:

[0073] This experiment used CellTiter AQueous One Solution Cell Proliferation Assay kit (Promega) was used to detect the number of viable cells using a colorimetric assay. 1 × 10 5 Cells were plated at a density of 100 μL / well / mL of culture medium. Cells were treated with various concentrations of simvastatin / atorvastatin / bexarotene (added as a solution in dimethyl sulfoxide (DMSO)) or blank control (no drug, but an equal volume of drug solvent) at 0, 24, and 48 hours after treatment. Cell viability was assessed by adding 100 μL of cell suspension and 20 μL of MTS reagent to each well of a 96-well plate. After incubation in a 37°C incubator for 2 hours, the absorbance at 490 nm (OD) was measured. Data were standardized using Excel, and relative cell viability values were calculated at each time point.

[0074] The calculation formula is as follows:

[0075] ODAμMBh refers to the change in OD value after treatment with drug at AμM concentration for B hours;

[0076] ODAμMBh = original OD of the well - OD of blank control treatment;

[0077] Relative cell viability after B hours of drug treatment at AμM concentration = ODAμM Bh / (ODAμM0h×OD0μM Bh / OD0μM0h)

[0078] In the following examples, protein levels of cellular caspase-3 and cleaved caspase-3 were detected by Western blot (WB):

[0079] 1. Whole protein extraction from suspended cells:

[0080] (1) Precool the centrifuge to 4°C.

[0081] (2) After counting, transfer the suspended cultured cell solution to a 5 mL EP tube, wash the cell plate with 1 mL of pre-cooled PBS, and transfer it to the EP tube. Centrifuge at 1500 rcf at 4°C for 5 min. Discard the supernatant after centrifugation and place the EP tube on ice.

[0082] (3) Prepare protein lysis buffer (operate on ice): prepare RIPA lysis buffer (Radio Immunoprecipitation Assay Lysis buffer): protease inhibitor (Beyotime P1045): phosphatase inhibitor (Beyotime P1045) at a ratio of 50:1:1, shake to mix, and store on ice until use.

[0083] (4) Add the prepared lysis buffer to the cells, approximately 80 μL per tube.

[0084] (5) Shake vigorously for 5 seconds, place on ice for 5 minutes, and repeat 3 times.

[0085] (6) After centrifugation at 14,000 rpm for 20 minutes at 4°C, carefully aspirate the supernatant and transfer it to a new EP tube.

[0086] 2. Protein concentration detection

[0087] This experiment was performed according to Pierce TM The procedure was performed according to the instructions of BCA Protein Assay Kit (23227).

[0088] (1) Prepare BCA working solution (WR): Mix 50 volumes of BCA Reagent A with 1 volume of BCA Regent B to obtain WR. Mix well and add 200 μL WR to each well of a 96-well plate.

[0089] (2) Add bovine serum albumin (BSA) standard (1 μg / μL): add 1 μL, 2 μL, 4 μL, and 8 μL respectively to construct a concentration gradient.

[0090] (3) Add protein sample, 1 μL per well, and set up 2 replicate wells for each sample.

[0091] (4) Mix well on a horizontal shaker and incubate at 37°C in the dark for 30 minutes.

[0092] (5) Cool to room temperature, vibrate the plate at low speed for 10 seconds, and then measure the absorbance at a wavelength of 595 nm using a microplate reader.

[0093] (6) Use Excel to create a linear graph using the OD value and absorbance of the standard and calculate the functional equation: y = ax + b (x: OD value, y: protein concentration (ng / μL)). Substitute the OD value of the sample to calculate its protein concentration.

[0094] (7) Based on the calculated sample concentration, prepare the protein stock solution to 2 μg / μL using 5x buffer (CWBIO) and RIPA.

[0095] (8) Boil the egg whites in boiling water for 10 minutes and store for later use.

[0096] 3. Western Blot

[0097] (1) Clean the glass plates used to prepare the gel, rinse them with deionized water, and let them dry naturally. Fix them on the gel-making device, add enough deionized water to the glass interlayer, let it stand for 5 minutes to observe whether there is any leakage. After confirming that there is no leakage, pour out the deionized water in the interlayer and absorb the remaining water with absorbent paper.

[0098] (2) Prepare SDS-PAGE gel: Prepare the separation gel first, then the stacking gel. When ready to cast the gel, add TMEMD in the final step and mix thoroughly to prevent the gel from solidifying prematurely.

[0099] Table 2 is the SDS-PAGE gel formula

[0100]

[0101] (3) Use a 1 mL pipette to pour separation gel (about 7 mL) into the plate, and then inject 1 mL of anhydrous ethanol from one side of the plate onto the liquid surface to press the gel and seal it.

[0102] (4) Let the separation gel stand for about 20 minutes to solidify. Determine whether the separation gel in the plate has completely solidified based on the state of the separation gel left in the centrifuge tube.

[0103] (5) After the separation gel solidifies, pour out the anhydrous ethanol and absorb any remaining water with absorbent paper. Add stacking gel (approximately 3 mL) until full, insert the sample comb vertically, and let it sit at room temperature for about 10 minutes until the stacking gel solidifies.

[0104] (6) Place the glass plate with gel in the electrophoresis tank and fix it (the short side of the glass plate is inward and the long side is outward). Add electrophoresis liquid (made of Servicebio fast electrophoresis dry powder, 1 pack of dry powder + 1L distilled water, item number: G2081-1L) into the electrophoresis tank. After checking for leakage, carefully pull out the sample comb and remove the bubbles on the liquid surface.

[0105] (7) Loading: Load 5 μL of protein marker and 10 μL of sample. Record the loading order.

[0106] (8) Electrophoresis: Connect the electrodes, paying attention to the positive and negative poles (red to red, black to black), turn on the power supply, and set the voltage to a constant voltage of 60V. When the sample reaches the junction of the stacking gel and the separation gel (about 20-30 minutes), increase the voltage to 120V and continue electrophoresis. End the electrophoresis when the Loading Buffer indicator line reaches the bottom.

[0107] (9) Room temperature transfer (wet method): Soak the PVDF membrane in anhydrous ethanol for 1 minute to activate it, then wash it once with transfer solution. Wash the square plate and add the rapid transfer solution (made of Servicebio rapid transfer powder, 1 pack of dry powder + 800mL distilled water + 200mL anhydrous ethanol, item number: G2028-1L) to the plate. Then put in the following order: negative electrode of the transfer splint (blackboard), sponge pad, filter paper, sample glue, PVDF membrane, filter paper, sponge pad, positive electrode of the transfer splint (transparent plate). Note that bubbles must be removed after covering the membrane. Close the splint in the transfer solution and place it in the electrotransfer tank, paying attention to the direction of the positive and negative electrodes. Pour the rapid transfer solution into the electrotransfer tank quickly, add an ice box so that the liquid level can completely cover the splint, turn on the power, and transfer the membrane at a constant current of 0.4A for 30 minutes.

[0108] (10) Blocking: Wet the inner layer of a transparent film glove with TBST, place the membrane inside the film glove with the protein side facing up, mark the kD size corresponding to the marker with a marker pen, and then cut the membrane according to the position of the target protein and the internal reference band. Then, carefully pick up the cut membrane with tweezers and place it in a container containing rapid blocking solution (Yazyme Protein-Free Rapid Blocking Solution (1x) Product No.: PS108P) and shake it slowly on a shaker for 10 minutes.

[0109] (11) Antibody incubation: Place each nitrocellulose membrane in a container containing TBST and wash on a fast shaker for 3 minutes. Then place the membrane in a centrifuge tube containing the corresponding primary antibody and shake it slowly in a refrigerator at 4°C overnight. The next day, after recovering the primary antibody, place the nitrocellulose membrane in a container containing TBST and wash on a fast shaker for 5 minutes, for a total of 3 times. Then add the corresponding secondary antibody, incubate on a slow shaker at room temperature for 1 hour, and then wash with TBST for 10 minutes, for a total of 3 times.

[0110] (12) Color development and imaging: Use ECL luminescent solution (Immobilon Western HRP, Merck Millipore), mix solution A and solution B in a 1:1 ratio, and evenly cover the PVDF membrane. Use SYNGENE gel image analysis system for imaging.

[0111] (13) Membrane regeneration: When the two protein bands to be detected are too close to be suitable for cropping, the bands can be washed with TBST for 5 minutes for a total of 3 times after exposure. Then, membrane regeneration solution is added. After incubation at room temperature for 1 hour, TBST is used to wash again for 5 minutes for a total of 3 times, and then blocking and antibody incubation are performed again.

[0112] Apoptosis detection in the following examples:

[0113] This experiment used a flow cytometer (FACS Calibur, BD Biosciences, San Diego, CA) to detect cell apoptosis. Cells were stained using the Annexin V-FITC or PE combined with the 7-AAD Detection Kit. In apoptotic cells, cell membrane molecules are exposed and recognized and bound by Annexin V. FITC or PE are fluorescent dyes coupled to Annexin V. Flow cytometry detected FITC- or PE-positive cell populations as apoptotic cells. 7-AAD was used to stain late-stage apoptotic and necrotic cells. This allowed comparison of the apoptosis levels of CTCL cells under different treatments.

[0114] The drug-specific apoptosis rate was calculated using the following formula:

[0115] Drug-induced specific apoptosis rate = (percentage of apoptotic cells during drug treatment - percentage of natural apoptosis) / (100 - percentage of natural apoptosis) × 100%

[0116] The data in the following examples were analyzed as follows: Graphpad Prism 8 was used for statistical analysis, and the data were expressed as mean ± SEM. Comparisons between two groups were performed using Student's t-test, and comparisons between three or more groups were performed using ANOVA. Differences were considered statistically significant when p value < 0.05.

[0117] Example 1: Application of statins in the treatment of CTCL

[0118] 1. Phenotype of CTCL-derived cell lines after statin treatment

[0119] Hut78 and Myla are recognized CTCL cell lines. Cells were seeded in well plates, and different concentrations of simvastatin (additional solution in DMSO) were added to each well. Cell viability was assessed at 0, 24, and 48 hours of simvastatin treatment using the previously described cell viability assay. Cells were harvested 48 hours after treatment, and protein was extracted as described above for caspase-3 and cleaved caspase-3 protein levels. Simvastatin-mediated apoptosis was assessed by measuring the extent of caspase-3 cleavage. Furthermore, the previously described apoptosis assay was used to assess the levels of early apoptosis (Annexin V-FITC single positive), late apoptosis (Annexin V-FITC and 7AAD double positive), and necrosis (7AAD single positive) in cells treated with simvastatin for 48 hours, and the drug-specific apoptosis rate was calculated.

[0120] The results are as follows Figure 1 As shown in the figure, A shows that the cell viability level of Hut78 cells after treatment with different concentrations of simvastatin gradually decreased, showing a concentration gradient dependence; B shows that the cleavage level of Caspase 3 of Hut78 cells after treatment with different concentrations of simvastatin gradually increased, showing a concentration gradient dependence; C shows that the cell apoptosis and necrosis levels of Hut78 cells after treatment with different concentrations of simvastatin significantly increased; D shows that the cell viability level of Myla cells after treatment with different concentrations of simvastatin gradually decreased, showing a concentration gradient dependence; E shows that the cleavage level of Caspase 3 of Myla cells after treatment with different concentrations of simvastatin gradually increased, showing a concentration gradient dependence; F shows that the cell apoptosis level of Myla cells after treatment with different concentrations of simvastatin gradually increased, showing a concentration gradient dependence.

[0121] The above results indicate that simvastatin can reduce the viability of CTCL cells and promote the apoptosis of CTCL cells.

[0122] Therefore, simvastatin can treat CTCL or kill CTCL cells.

[0123] 2. Phenotype of tumor T cells in patients with hematologic involvement after statin treatment

[0124] Peripheral blood mononuclear cells (PBMCs) were extracted from MF patients with blood involvement. Considering the characteristic that CTCL cells lose cell membrane surface markers, flow cytometry staining was used to distinguish tumor T cells (CD3+CD4+CD7-, i.e., CTCL primary cells) and reactive T cells (CD3+CD4+CD7+).

[0125] Different concentrations of simvastatin (added in the form of a solution, the solvent was DMSO) were then added to the tumor T cells (CD3+CD4+CD7-T) and reactive T cells (CD3+CD4+CD7+T) and cultured for 48 hours. The levels of early apoptosis (Annexin V-PE single positive), late apoptosis (Annexin V-PE and 7AAD double positive) and necrosis (7AAD single positive) were detected according to the previous cell apoptosis detection method.

[0126] The results are as follows Figure 2 As shown, the left side is a flow cytometry analysis diagram, in which lymphocytes, single cells, and CD3+CD4+T cells are circled in turn for analysis. CD3+CD4+CD7-T represents tumor T cells, and CD3+CD4+CD7+T represents reactive T cells; the right side is a drug-specific apoptosis and necrosis statistical diagram of these two groups of cells. It can be seen that after treatment with different concentrations of simvastatin, the overall apoptosis level of tumor T cells is significantly higher than that of reactive T cells; the efficacy of statins is tested in human primary cells. At a low concentration of 2.5μM, simvastatin has a strong killing effect on tumor T cells and a weak killing effect on normal reactive T cells, indicating that this concentration has a therapeutic effect without obvious toxic side effects.

[0127] The results showed that compared with non-tumor T cells, CTCL tumor cells were more sensitive to statin treatment; simvastatin had a high killing effect on tumor cells and was concentration-dependent.

[0128] 3. Phenotype of CD4+ T cells from healthy volunteers after statin treatment

[0129] According to the previous method, PBMCs of healthy volunteers were harvested and CD4+ T cells were separated and inoculated into well plates. Different concentrations of simvastatin (added in the form of a solution, the solvent was DMSO) were added to each well of the well plate and cultured for 48 hours. The levels of cell apoptosis and necrosis were detected according to the previous cell apoptosis detection method.

[0130] The results are as follows Figure 3As shown, the left side is the flow cytometry graph, and the right side is the statistical graph of drug-specific apoptosis and necrosis; it can be seen that the apoptosis and necrosis rates of CD4+ T cells (denoted as NCD4+ in the figure) from healthy volunteers only increase slightly with the increase of drug concentration.

[0131] The above results show that at the same concentration level, simvastatin has no obvious inhibitory effect on CD4+T cells of normal controls.

[0132] Therefore, the killing effect of simvastatin on normal control CD4+T cells is far weaker than that on tumor CTCL cells, indicating that simvastatin is more specific and safe in killing CTCL cells.

[0133] Example 2: Statins and bexarotene synergistically inhibit CTCL cell viability

[0134] In order to evaluate whether statins can synergistically kill CTCL cells with traditional CTCL treatment drugs such as Bexarotene,

[0135] Three CTCL cell lines, Myla, Hut78, and HH, were treated with statins (simvastatin or atorvastatin) alone, bexarotene alone, and combinations of various statins and bexarotene at varying concentrations (final system concentration). The specific methods are as follows:

[0136] 1. Simvastatin alone or in combination with bexarotene

[0137] 1. Cell viability assay

[0138] CTCL cell lines Myla, Hut78 and HH were each treated with 1×10 5 The cells were seeded at a density of 100 μg / mL in a 24-well plate, and different concentration gradients of drugs (added in the form of solution, the solvent was DMSO; simvastatin alone, bexarotene alone, or simvastatin + bexarotene combined) were added to treat the cells for 48 hours, and cell viability was detected by MTS.

[0139] The results are as follows Figure 4As shown in Figure A-4C, the horizontal and vertical scales 2.5, 5, 10, and 20 correspond to different concentrations of simvastatin after single-agent addition; 2.5, 5, 10, and 20 correspond to different concentrations of bexarotene after single-agent addition; and 2.5+2.5, 5+5, 10+10, and 20+20 correspond to different concentrations of simvastatin and bexarotene after combined addition (in μM). It can be seen that in the three CTCL cell lines, the combined drug effect is better than that of either drug alone, achieving the goal of inhibiting CTCL cell viability.

[0140] 2. Analysis of drug synergy

[0141] After evaluating the relative viability of cells after drug treatment, the combined effect of the two drugs was evaluated using Compusyn software, which calculates the Combination Index (CI) based on the Chou-Talalay model for quantitative analysis of multidrug dose-effect relationships.

[0142] After the MTS assay for cell viability, the drug-induced decrease in cell viability (i.e., relative cell viability after 48 hours of treatment at a 1-μM concentration of drug) was calculated as the drug effect fraction (Fa). The drug concentration and the corresponding Fa value were entered into Compusyn software to obtain the corresponding CI value. A CI of 1 indicates an additive effect between the two drugs; a CI of <1 indicates a synergistic effect between the two drugs; and a CI of >1 indicates an antagonistic effect between the two drugs.

[0143] When CI = 1, it indicates that the drugs produce an additive effect; when CI < 1, it indicates that the drugs produce a synergistic effect; when CI > 1, it indicates that the drugs produce an antagonistic effect.

[0144] The results are as follows Figure 4 As shown in Figures D-4F, simvastatin and bexarotene both produced a synergistic effect in inhibiting CTCL cell viability in all three CTCL cell lines. A higher inhibitory effect and a lower synergy index indicate a better effect.

[0145] 2. Atorvastatin combined with bexarotene

[0146] 1. Cell viability assay

[0147] The detection method is the same as that of the above-mentioned step 1, except that simvastatin is replaced by atorvastatin of different concentrations.

[0148] Myla's results are as follows Figure 5As shown in Figure A, the horizontal and vertical scales 10, 20, 40, and 80 correspond to different concentrations of atorvastatin after single-drug addition; 5, 10, 20, and 40 correspond to the concentrations of bexarotene after single-drug addition; 10+5, 20+10, 40+20, and 80+40 correspond to different concentrations of atorvastatin and bexarotene after combined addition (in μM). It can be seen that in the CTCL cell line Myla, the effect of combined drug use is better than that of single drug use.

[0149] Hut78's results are as follows Figure 5 As shown in Figure B, the horizontal and vertical scales 1.25, 2.5, 5, and 10 correspond to different concentrations of atorvastatin after single drug addition; 2.5, 5, 10, and 20 correspond to different concentrations of bexarotene after single drug addition; 1.25+2.5, 2.5+5, 5+10, and 10+20 correspond to different concentrations of atorvastatin and bexarotene after combined addition (in μM). It can be seen that in the CTCL cell line Hut78, the combined drug effect is better than that of either drug alone, thereby inhibiting the viability of CTCL cells.

[0150] 2. Analysis of drug synergy

[0151] The detection method is the same as that in step 1 above.

[0152] The results are as follows Figure 5 As shown in Figures 5C and 5D, it can be seen that in the two CTCL cell lines, atorvastatin and bexarotene were observed to produce a synergistic effect. The higher the inhibitory effect and the lower the synergistic index, the better the effect.

[0153] These results indicate that statins can synergistically inhibit the viability of CTCL cells with bexarotene.

[0154] Example 3: Low-concentration statin treatment of CTCL in vivo

[0155] 1. Mouse xenograft model and in vivo tumor efficacy experiments

[0156] This experiment used the NSG (NOD / scid interleukin-2 receptor γ-chain–deficient) interleukin-2 receptor γ-chain-deficient non-obese diabetic / severe combined immunodeficiency mouse model, which was purchased from Ruiye Animal Company.

[0157] (1) Resuspend the corresponding Myla cells in PBS at a concentration of 5×10 6 The concentration of 100 μL was inoculated subcutaneously in the right flank of NSG mice. 3At the same time, mice with moderate tumor volumes were selected as mouse xenograft models. The animals were randomly assigned to experimental groups according to tumor volume, with 3 mice in each group. Drug administration began on the day of grouping. The specific dosing schedule is shown in Table 3 below:

[0158] Table 3 shows the dosage regimen.

[0159]

[0160] Note: The solvent used to dilute the drug was a mixture of 50% PEG300 and 50% normal saline. The administration volume was calculated based on the weight of the experimental animals at 15 μL / g.

[0161] (2) Observation of experimental animals after drug withdrawal

[0162] Following the final dose, the animal's body weight and tumor growth were observed for an additional three days. Tumor volume and animal weight were measured and recorded three times weekly during the observation period. The experiment was terminated upon completion of the observation period.

[0163] (3) Humane endpoints for experimental animals

[0164] During the experiment, animals should be euthanized if any one or more of the following conditions occur:

[0165] When the tumor volume of a single animal exceeds 3000 mm 3 , or the average tumor volume of the whole group exceeds 2000mm 3 ;

[0166] Tumor ulceration, necrosis, or infection;

[0167] The animal exhibits abnormal movements or becomes paralyzed;

[0168] Animals lost more than 20% of their body weight at the start of drug treatment.

[0169] (4) Euthanasia

[0170] At the end of the experiment or when a humane end point is reached, animals are euthanized using CO2 inhalation.

[0171] (5) Detection indicators

[0172] 1) Tumor volume:

[0173] Tumor volume was measured three times a week using a vernier caliper to measure the long diameter (L) and short diameter (W) of the tumor. The volume was calculated using the following formula: tumor volume = 0.5 × L × W 2 .

[0174] 2) Weight test:

[0175] The animals were weighed before inoculation, grouping (i.e., before the first administration), three times a week during the administration / observation period, and before euthanasia.

[0176] 3) General observations:

[0177] During the adaptive feeding period and the experimental period, the animals were observed once a day. The observation contents included the rupture of tumor nodules, the mental state of the animals, and the diet.

[0178] 4) Tumor weighing and photographing:

[0179] At the end of the experiment, the animals were euthanized and the tumors were photographed and recorded. The tumors of the mice were removed, weighed, and photographed and recorded.

[0180] The results are as follows Figure 6 As shown, A is the tumor volume and B is the mouse weight. It can be seen that in the in vivo mouse xenograft model, simvastatin can significantly reduce the tumor formation ability of CTCL cells ( Figure 6 A) The average tumor volume of the control group gradually increased, while the tumor growth of the simvastatin gavage group was inhibited, indicating that simvastatin has a significant anti-tumor effect at a dose of 60 mg / kg. The animals were active and ate well during the administration period. There was no significant difference in the average body weight between the simvastatin gavage group and the control group ( Figure 6 B), indicating that simvastatin produces relatively low toxic side effects in vivo and is well tolerated by mice.

Claims

1. The use of statins in the preparation of products having any of the following functions: 1) Treatment of cutaneous T-cell lymphoma; 2) Promote apoptosis of cutaneous T-cell lymphoma cells; 3) Reduce the activity of cutaneous T-cell lymphoma cells; 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells; 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

2. The use of statins and drugs or reagents required for the systemic treatment of cutaneous T-cell lymphoma in the preparation of products with any of the following functions: 1) Treatment of cutaneous T-cell lymphoma; 2) Promote apoptosis of cutaneous T-cell lymphoma cells; 3) Reduce the activity of cutaneous T-cell lymphoma cells; 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells; 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

3. The use according to claim 2, characterized in that: The drug or reagent required for the systemic treatment of cutaneous T-cell lymphoma is retinoic acid or its analogues; And / or, the retinoic acid or its analogue is bexarotene.

4. The use according to any one of claims 1 to 3, characterized in that: The statin is simvastatin and / or atorvastatin.

5. The use according to any one of claims 1 to 4, characterized in that: The product is a drug, a kit or other biological product.

6. A drug comprising the statin according to any one of claims 1 to 5 and any other substance that can serve as a drug carrier; The drug has any of the following functions: 1) Treatment of cutaneous T-cell lymphoma; 2) Promote apoptosis of cutaneous T-cell lymphoma cells; 3) Reduce the activity of cutaneous T-cell lymphoma cells; 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells; 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

7. A drug comprising the statin according to any one of claims 2 to 5, the drug or reagent required for systemic treatment of cutaneous T-cell lymphoma, and any other substance that can serve as a drug carrier; The drug has any of the following functions: 1) Treatment of cutaneous T-cell lymphoma; 2) Promote apoptosis of cutaneous T-cell lymphoma cells; 3) Reduce the activity of cutaneous T-cell lymphoma cells; 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells; 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

8. Use of the statin according to any one of claims 1 to 5 and any other substance that can be used as a drug carrier in the preparation of a drug having any of the following functions: 1) Treatment of cutaneous T-cell lymphoma; 2) Promote apoptosis of cutaneous T-cell lymphoma cells; 3) Reduce the activity of cutaneous T-cell lymphoma cells; 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells; 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

9. Use of the statin according to any one of claims 2 to 5, the drug or reagent required for systemic treatment of cutaneous T-cell lymphoma, and any other substance that can serve as a drug carrier in the preparation of a drug having any of the following functions: 1) Treatment of cutaneous T-cell lymphoma; 2) Promote apoptosis of cutaneous T-cell lymphoma cells; 3) Reduce the activity of cutaneous T-cell lymphoma cells; 4) Reduce the tumor-forming ability of cutaneous T-cell lymphoma cells; 5) Inhibit the growth of cutaneous T-cell lymphoma cells.

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