阅读说明：本技术 治疗黑色素瘤的方法 (Method for treating melanoma ) 是由 卡罗·马切蒂 查尔斯·A·迪纳雷罗 于 2019-03-18 设计创作，主要内容包括：本发明涉及预防和/或治疗黑色素瘤(例如浅表扩散性黑色素瘤,结节性黑色素瘤,恶性雀斑样痣黑色素瘤,和肢端雀斑痣性黑色素瘤)的方法。该方法包括向有此需要的受试者施用有效量的达泮舒腈。该方法任选地包括将抗PD-1抗体与达泮舒腈共同施用。优选的施用途径是口服施用。(The present invention relates to a method for preventing and/or treating melanoma (e.g., superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentigo melanoma). The method comprises administering to a subject in need thereof an effective amount of dapzepine. The method optionally comprises co-administering an anti-PD-1 antibody with dapzepine. The preferred route of administration is oral.)

1. A method of preventing and/or treating melanoma in a subject, comprising the steps of:

Administering to the subject having the melanoma an effective amount of dapzepine, or a pharmaceutically acceptable solvate thereof.

2. The method of claim 1, wherein the melanoma is selected from the group consisting of: superficial spreading melanoma, nodular melanoma, malignant lentigo melanoma, and acral lentigo melanoma.

3. The method of claim 1, wherein the darpam sultamitrile is administered by systemic administration.

4. The method of claim 3, wherein the diazepam is administered by oral administration.

5. The method of claim 1, wherein the method treats melanoma by reducing the size of the melanoma.

6. The method of claim 1, further comprising administering to the subject an anti-PD-1 antibody.

Technical Field

The present invention relates to a method of treating melanoma by administering an effective amount of dapansulonitrile (dapansultril).

Background

Tumorigenesis is initiated by genomic alterations including point mutations, gene deletions, chromosomal rearrangements leading to cell transformation, self-sufficient (self-sufficient) proliferation, insensitivity against proliferative signals, circumventing apoptosis and unlimited replication potential, ultimately leading to tissue infiltration and metastasis. However, expansion of tumor cells (expansion) is associated with a complex network of events involving both cancer and non-cancer cells. Chronic inflammation is a typical example of such a promoting disorder (1, 2).

The proinflammatory cytokine IL-1 beta is a potent mediator of many chronic inflammatory diseases (3). Consistent with the link between cancer and chronic inflammation, IL-1 β has been shown to be overexpressed in several tumors and to act as an inducer of tumor-promoting mechanisms, including angiogenesis, immunosuppression, recruitment of tumor-associated macrophages (TAMs), and metastasis (4-6).

Melanoma develops when damage to skin cells by unrepaired deoxyribonucleic acid ("DNA") triggers mutations that result in proliferation of skin cells, eventually leading to the formation of malignant tumors. These tumors originate from melanocytes located in the basal layer of the epidermis. Melanoma is often caused by Ultraviolet (UV) exposure and is responsible for over 70,000 deaths annually in the united states.

There are four types of melanoma: superficial spreading melanoma, malignant lentigo melanoma, acral lentigo melanoma, and nodular melanoma. Superficial diffuse melanoma is most common and grows along the top layers of the skin and then penetrates deeper into the skin. Malignant lentigo melanoma is similar to superficial diffuse melanoma and is most common in the elderly, occurring in damaged skin that is exposed to sunlight for a long period of time. Compared to superficial diffuse melanoma and malignant lentigo melanoma, acral lentigo melanoma also spreads superficially before penetrating deeper and tends to progress to malignancy more frequently. At the initial diagnosis, nodular melanoma is most often invasive.

Melanoma is divided in stages, which refers to thickness, depth of penetration, and extent to which the melanoma has spread. Early stage melanoma (stages 0 and I) is usually localized. Stage 0 tumors are usually non-invasive and often do not penetrate beneath the epidermis. Stage I tumors often invade the dermis, are small in size, and have a low risk of metastasis. Stage II tumors are localized, more voluminous, and have a high risk of metastasis. Once a melanoma tumor has metastasized, it is classified as stage III or IV melanoma depending on the degree of metastasis.

NLRP3 (NOD-like receptor family, containing the thermolin (pyrin) domain 3), also known as NALP3 or cryptothermin (cryopyrin), is one of the sensors of the inflammasome, a macromolecular structure involved in the processing of interleukin 1 β (IL-1 β) and IL-18. NLRP3 senses intracellular risks during intracellular infection (bacterial and viral proteins) or tissue damage (ischemia). Activation of NLRP3 results in the recruitment of ASCs (apoptosis-associated speckled proteins containing a carboxy-terminal caspase-recruiting domain) and caspase-1, leading to the formation of inflammatory bodies and ultimately to cell death.

There is a need for methods of treating melanoma. The method should be effective and without significant side effects.

Brief description of the drawings

FIGS. 1A-1E illustrate OLT1177^TM(darpam sultanitrile) reduces tumor volume and inflammation associated with melanoma. (1A) Using standard or OLT1177^TMTumor size of diet-fed mice (N ═ 15). (1B) Using standard or OLT1177^TMPlasma in diet-fed tumor-bearing miceMean ± SEM of IL-6 (N ═ 6 per group). (1C) Using standard or OLT1177^TMMean ± SEM of G-CSF in plasma of diet-fed mice (N ═ 4-5 per group). (1D) For standard or OLT1177 ^TMIntracellular cytokine staining was performed on L-22 from splenic-derived (splenic-derived) cells from diet-fed mice. (1E) For standard or OLT1177^TMIntracellular cytokine staining was performed on IL-17 from splenogenic cells of mice fed with diet. P < 0.01, p < 0.05.

Figures 2A-2C show that dapzepine decreases endothelial function and angiogenesis. (2A) Using standard or OLT1177^TMMean ± SEM of plasma VEGF in diet-fed tumor-bearing mice (N ═ 4-5 per group). (2B) Representative images (indicated by arrows) of endothelial cell activation in matrix plugs (matrigel plug) stained for von willebrand Factor (von willebrand Factor) reflect the use of standard or OLT1177^TMFormation of new blood vessels in diet-fed mice (each panel represents one mouse). (2C) In the presence and absence of OLT1177^TMIn cases (3), mean ± SEM of tubular structure of HUVEC after stimulation with Melanoma Conditioned Medium (MCM). P < 0.05.

Figures 3A-3B show that dapzepine reduces tissue infiltration and metastasis in the lung and liver. (3A) Accept standard or OLT1177^TMB16F10-GFP in lungs of diet tumor-bearing mice⁺Mean ± SEM of cell counts/field area (whole chip field) was evaluated by an blinded microscopist (N ═ 3 per group). P < 0.0001, p < 0.01, p < 0.05. (3B) Accept standard or OLT1177 ^TMBlinded (blanked) GFP in the liver of a diet tumor-bearing mouse⁺Mean ± SEM of cell counts (N ═ 3 per group). P < 0.0001.

FIGS. 4A-4F show that dapzepine reduces MDSC amplification. And Standard (Standard) or OLT1177^TMPMN-MDSC (CD11 b) in non-tumor bearing mice (no tumor) compared to diet-fed tumor bearing mice⁺Ly6G⁺Ly6C^lo) Bone marrow, (4A), spleen, and (4C) lymph node levels of (4A). And acceptance Standard (Standard) or OLT1177^TMM-MDSC (CD11 b) in non-tumor bearing mice (no tumor) compared to tumor bearing mice on diet⁺Ly6G^-Ly6C^hi) Bone marrow, (4E) spleen, and (4F) lymph node levels of (4D). Data are expressed as percentage MDSC changes set to 100 in non-tumor bearing mice (no tumor). (each group N is 8-10). P < 0.001, p < 0.05.

Figures 5A-5D show that dapaglucone enhances the efficacy of anti-PD-1 blockade. (5A) Vehicle (vehicle), anti-PD-1, and anti-PD-1 + OLT1177^TMTumor size of diet-treated tumor-bearing mice (N ═ 13). (5B) Mean ± SEM of plasma IL-6 in tumor-bearing mice indicated by a (N ═ 8-9). (5C) Mean ± SEM of MPO whole blood lysates in tumor-bearing mice indicated by a (N ═ 11). (5D) NK T cells in Primary tumors in mice denoted A (CD 3)⁺CD8-CD161⁺CD335⁺) And (5) infiltrating. P < 0.0001, p < 0.001, p < 0.01, p < 0.05.

Detailed Description

Activation of the NLRP3 inflammasome amplifies the inflammatory response to tissue injury and mediates further damage. Dapzepine is a selective NLRP3 inflammasome inhibitor. Dapzepine reduces inflammation by preventing activation of NLRP3 inflammasome. Dapzepine inhibits the production of mature IL-1 β and IL-18 in mouse and human cells in vitro. By this mechanism of action, diazepam prevents the production and/or release of IL-1 β in animal and human subjects and inhibits the formation of NLRP3 inflammasome.

The inventors found that by preventing IL-1 β production, darpam-sultanitrile provides the following effects: reducing angiogenesis, reducing the production of IL-1 dependent Vascular Endothelial Growth Factor (VEGF), limiting the production of bone Marrow Derived Suppressor Cells (MDSCs), preventing elevated levels of IL-8, inhibiting the migration of endothelial precursor cells into tumors, reducing the levels of IL-6 and other matrix factors, reducing the accumulation of neutrophils at the tumor site, reducing the production of growth factors (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), FGF, and IL-1), reducing the expression of Matrix Metalloproteinases (MMP) and the production of cyclooxygenase. By reducing the production of IL-1 β, dapzepine reduces the effects induced by IL-1.

MDSCs are heterogeneous populations of immune cells from the myeloid lineage (a cell family derived from bone marrow stem cells). MDSCs rapidly expand in pathological conditions such as chronic infections and cancer due to altered hematopoiesis. MDSCs are distinguished from other myeloid cell types, which have strong immunosuppressive rather than immunostimulatory properties. The expansion of cells of myeloid origin (MDSCs) is often associated with chronic inflammation (10, 11), and MDSCs have been shown to play a major role in cancer-mediated immunosuppression (12). In melanoma patients, high levels of MDSCs (PMN-and M-MDSCs) were associated with stage, metastasis and poor prognosis compared to subjects with lower numbers of MDSC (PMN-and M-MDSC) cells (13).

The inventors have demonstrated that dapzepine reduces the tumor volume of melanoma in mice and maintains MDSC levels in mice with melanoma compared to those observed in tumor-free wild-type. This occurs because dapzepine prevents MDSC expansion and restores physiological levels of these cells.

The inventors have demonstrated that melanoma tumor-bearing mice fed a diet rich in diazepam nitrile show reduced circulating levels of IL-6, G-CSF, and VEGF compared to tumor-bearing mice fed a standard diet.

The mechanism of metastasis involves complex, multi-step processes of detachment from the primary tumor site, endosmosis into the circulation, survival in the circulation, extravasation from the circulation, attachment at secondary sites (secondary sites), and development at secondary tumor sites, each involving mediators induced by IL-1 β (23, 24). The inventors have demonstrated that tumor-bearing mice treated with dapzepine show reduced metastasis in both lung and liver.

Angiogenesis is a hallmark of tumor growth and is associated with the induction of a large number of infiltrating immune cells and pro-angiogenic factors (such as VEGF), linking chronic inflammation to angiogenesis. The inventors have demonstrated that dapzepine reduces inflammatory events associated with angiogenesis, lowers circulating VEGF plasma levels, and reduces tumor angiogenesis.

Immunotherapy has made significant progress in the treatment of advanced melanoma and is becoming the standard of care (standard of care). Combination immunotherapy against PD-1 (nivolumab) and CTLA-4 (ipilimumab) can lead to tumor regression with response rates exceeding 50% (7). However, immunotherapy is often associated with toxicity, such as immunotherapy-associated adverse effects (irAE) (8), and the number of non-responders and relapsing cases remains an important and unmet clinical need in melanoma treatment. The inventors have demonstrated that combination therapy of an anti-PD-1 antibody and dapzepine provides enhanced therapeutic efficacy in reducing tumor growth compared to anti-PD-1 monotherapy.

The inventors believe that dapzepine is effective in preventing melanoma growth by blocking the assembly of NLRP3 inflammasome and preventing the production and/or release of IL-1 β. By preventing IL-1 β processing in melanoma cells, dapam sultanitrile provides a new therapy for melanoma and immunotherapy-resistant cancers. Diazepam reduces a number of cancer markers: tumor growth, immunosuppression, inflammation, metastasis, and angiogenesis, and thus it provides a new cancer therapy.

The present invention relates to methods of treating melanoma, such as superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentigo melanoma.

Compound (I)

The present invention uses a purified compound of dapzepine dinitrile (3-methanesulfonyl-propionitrile), or a pharmaceutically acceptable salt or solvate thereof:

dapzepine is a synthetic small molecule of β -sulfonyl nitrile, which has been shown to selectively inhibit NLRP3 inflammasome and is safe when administered orally to healthy subjects (9).

As used herein, a "pharmaceutically acceptable salt" is a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects. Pharmaceutically acceptable salt forms include multiple crystalline polymorphs of different salts as well as amorphous forms. The pharmaceutically acceptable salt may be With metal or organic counterions and include, but are not limited to, alkali metal salts, such as sodium or potassium; alkaline earth metal salts, such as magnesium or calcium; and ammonium or tetraalkylammonium salts, i.e. NX₄+ (wherein X is C)_1-4)。

As used herein, a "solvate" is an addition complex in which a compound is combined with an acceptable co-solvent in some fixed ratio. Co-solvents include, but are not limited to, water, acetic acid, ethanol, and other suitable organic solvents.

Pharmaceutical compositions

The active compound, dapzepine, or a pharmaceutically acceptable salt or solvate thereof, in the pharmaceutical composition is typically present in an amount of about 0.1-5% for injectable formulations, about 1-90% for tablet formulations, about 1-100% for capsule formulations, about 0.01-20% for topical (topical) formulations, 0.05-20%, 0.1-20%, 0.2-15%, 0.5-10%, or 1-5% (w/w) and about 0.1-5% for patch formulations.

As used herein, "about" refers to ± 10% of the stated value.

Pharmaceutically acceptable carriers as inactive ingredients may be selected by those skilled in the art using conventional criteria. Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous based solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments (ointments). Pharmaceutically acceptable carriers may also comprise ingredients including, but not limited to, saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and glucose; pH adjusting and buffering agents, such as salts of hydroxides, phosphates, citrates, acetates, borates; and triethanolamine; antioxidants, for example salts, acids and/or bases of bisulfite (bisufite), sulfite, metabisulfite, thiosulfite, ascorbic acid, acetylcysteine, cysteine, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherol, and ascorbyl palmitate; surfactants such as lecithin, phospholipids including but not limited to phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol; poloxamers and poloxamines; polysorbates, such as polysorbate 80, polysorbate 60, and polysorbate 20, polyethers, such as polyethylene glycol and polypropylene glycol; polyethylene, such as polyvinyl alcohol and povidone; cellulose derivatives such as methyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose and hydroxypropyl methyl cellulose and salts thereof; petroleum derivatives such as mineral oil and white petrolatum; fats, such as lanolin, peanut oil, palm oil, soybean oil; mono-, di-, and triglycerides; polymers of acrylic acid, such as carboxypolymethylene gel, and hydrophobically modified cross-linked acrylate copolymers; polysaccharides, such as dextran, and glycosaminoglycans (glycosaminoglycans), such as sodium hyaluronate. Such pharmaceutically acceptable carriers can be preserved against bacterial contamination using well known preservatives, including, but not limited to, benzalkonium chloride (benzalkonium chloride), ethylenediaminetetraacetic acid and salts thereof, benzethonium chloride, chlorhexidine (chlorohexidine), chlorobutanol, methylparaben (methylparaben), thimerosal (thimerosal), and phenylethyl alcohol, or can be formulated as a single or multiple use non-preserved (non-preserved) formulation.

For example, a tablet or capsule formulation of dapzepine may contain other excipients that are not biologically active and do not react with the active compound. The excipients of a tablet may include fillers, binders, lubricants and glidants, disintegrants, wetting agents, and release rate modifiers. Binders promote the adhesion of the formulation particles and are important for tablet formulations. Examples of binders include, but are not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropyl methylcellulose, carrageenan, starch, and tragacanth, polyacrylic acid, and polyvinylpyrrolidone.

For example, a patch preparation of darunavir may contain some inactive ingredients, such as 1, 3-butylene glycol, dihydroxyaluminum aminoacetate (dihydroxyaluminum aminoacetate), disodium ethylenediaminetetraacetate, D-sorbitol, gelatin, kaolin, methyl paraben, polysorbate 80, povidone, propylene glycol, propyl paraben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. Patch formulations may also contain skin permeability enhancers such as lactate esters (e.g., lauryl lactate) or diethylene glycol monoethyl ether.

The external preparation comprising dapzepine may be in the form of a gel, cream, lotion, liquid, emulsion, ointment, spray, solution, and suspension. Inactive ingredients in the external preparations include, for example, but are not limited to, lauryl lactate (emollient/penetration enhancer), diethylene glycol monoethyl ether (emollient/penetration enhancer), DMSO (solubility enhancer), silicone elastomer (rheology/texture modifier), caprylic/capric triglyceride, (emollient), caprylate, (emollient/uv filter), silicone oil (emollient/diluent), squalene (emollient), sunflower oil (emollient), and silicone oxide (silicone oxide) (thickener). In one embodiment, diethylene glycol monoethyl ether is included in the topical gel formulation.

Application method

By inhibiting the assembly of NLRP3 inflammasome, dapzepanzepine prevents the production and/or release of the pro-inflammatory cytokines IL-1 β and IL-22, and ultimately reduces the growth of melanoma in mice.

In addition, dapzepine inhibits the processing and release of IL-1 β and IL-18, but does not inhibit the synthesis of IL-1 β precursors and other inflammasome components, including NLRP3 and ASC. Dapzepine also inhibits caspase-1 activation. In addition, dapzepine maintains immune surveillance of the body by not inhibiting other inflammatory bodies, such as NLRC4 and AIM2, constitutive cytokines (constitutive cytokines), and preventing cell death.

The present invention relates to a method for preventing and/or treating melanoma, such as superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentigo melanoma. Melanoma of the above type has an inflammatory component which is the cause of the disease or the result of an event. The method comprises the step of administering to a subject in need thereof an effective amount of dapzepine. As used herein, an "effective amount" is an amount effective to treat a disease by ameliorating (ameliorating) a pathological condition, and/or alleviating, ameliorating, and/or eliminating symptoms of the disease. For example, an effective amount is an amount that reduces melanoma growth (reduces tumor size).

Immunotherapy significantly improved the standard of treatment for melanoma patients; however, the number of non-responders, and relapsing patients remains high. Thus, combination therapies that enhance the efficacy of checkpoint inhibitors represent an important clinical benefit. In one embodiment, the invention relates to a combination therapy for treating melanoma by binding to dapzepine and a checkpoint inhibitor (e.g., an anti-PD-1 antibody). The method comprises administering to a subject in need thereof an effective amount of dapzepine and an effective amount of an anti-PD-1 antibody. The dapzepine and the anti-PD-1 antibody may be administered simultaneously or sequentially. The administration of dapzepine in combination with an anti-PD-1 antibody is advantageous because it enhances the therapeutic efficacy of anti-PD-1 and because it has safe pharmaceutical properties. The combined administration may also reduce the required anti-PD-1 antibody dose, thereby reducing adverse effects associated with immunotherapy.

Combination therapy of dapzepine and anti-PD-1 can simultaneously suppress tumor-induced immunosuppression and increase T cell activity. In addition, increases in inflammatory cytokines (e.g., IL-6) are associated with the pathophysiology of iraE. Dapzepine enhances the anti-PD-1 effect and further reduces circulating levels of IL-6, a marker of poor melanoma prognosis. The combination therapy may also enhance tumor-specific Th1 responses, suggesting that less tumor-induced immunosuppression and more T cell activation result in a stronger anti-tumor response. Thus, treatment with dapzepine in addition to anti-PD-1 enhances the effect of monotherapy, creating an alternative to therapy-resistant cancers.

The pharmaceutical compositions of the present invention may be applied by systemic administration or topical administration. Systemic administration includes, but is not limited to, oral, parenteral (e.g., intravenous, intramuscular, subcutaneous, or rectal), and inhalation administration. In systemic administration, the active compound first reaches the plasma and then is distributed to the target tissue. Oral administration is a preferred route of administration for the present invention. Local administration (local administration) includes topical administration (topical administration).

The dosage of the composition may vary depending on the extent of melanoma in the subject and the individual response of each patient. For systemic administration, the plasma concentration of the active compound delivered may vary; but is typically 1x10 ^-10-1x10^-4Mol/l, preferably 1X1^-8-1x10^-5Mol/l.

In one embodiment, the pharmaceutical composition is administered to the subject orally. The dose for oral administration is generally 0.1-100, 0.1-20, or 1-100 mg/kg/day, depending on the age and condition of the subject. For example, for human subjects, the dose administered orally is 0.1-10, 0.5-10, 1-10, 1-5, 5-50, or 5-100 mg/kg/day. In one embodiment, the active compound is administered orally to a human subject in an amount of 10-100, 10-500, 20-2000, 50-2000, or 100-2500mg per dose, 1-4 times per day, depending on the age and condition of the subject.

In one embodiment, the pharmaceutical composition is administered to the subject intravenously. The dose of an intravenous bolus or intravenous infusion is typically 0.03 to 5 or 0.03 to 1 mg/kg/day.

In one embodiment, the pharmaceutical composition is administered to the subject subcutaneously. The dosage for subcutaneous administration is usually 0.3-20, 0.3-3, or 0.1-1 mg/kg/day.

In one embodiment, the composition is administered topically (topically). The composition is administered topically at least 1 or 2 times a day, or 3 to 4 times a day, depending on medical problems and disease pathology. Generally, the topical composition comprises about 0.01-20%, or 0.05-20%, or 0.1-20%, or 0.2-15%, 0.5-10, or 1-5% (w/w) of the active compound. Typically, 0.2-10mL of the topical composition is administered to an individual per dose.

Those skilled in the art will recognize that a wide variety of delivery mechanisms are equally applicable to the present invention.

The invention is useful for treating mammalian subjects, such as humans, horses, dogs, and cats. The invention is particularly useful in the treatment of humans.

The following examples further illustrate the invention. These examples are intended only to illustrate the invention and should not be construed as limiting.

Examples

The following protocol was used in the experiments described below.

Abbreviations. IL-1 β (Interleukin 1 β), IL-6 (Interleukin 6), G-CSF (granulocyte colony stimulating factor), VEGF (vascular endothelial growth factor), IL-22 (Interleukin 22), IL-17 (Interleukin 17), PMN-MDSC (polymorphonuclear MDSC), M-MDSC (mononuclear MDSC), PD-1 (programmed cell death protein 1), MCM (melanoma conditioned Medium), HUVEC (human umbilical vein endothelial cells), PBMC (peripheral blood mononuclear cells), VWF (von Willebrand factor).

And (5) culturing the cells. 1205Lu human melanoma cells were cultured in RPMI. Each was supplemented with 10% FBS, 100 units/mL penicillin, 0.1mg/mL streptomycin. Cells were kept humidified at 37 ℃ with 5% CO₂In the environment. Human metastatic melanoma cell line 1205Lu at 2.5x10 in 24-well plates ⁵RPMI was seeded per well and allowed to adhere overnight. The following day, the medium was replaced with fresh RPMI 10% FBS (with or without OLT 1177T)^M(darpam-sultanitrile)). IL-1 α (20ng/ml) was used to induce cytokine production. OLT1177 was added 30 minutes before stimulation was performed^TM. The supernatant was collected under non-stimulated and stimulated conditions for 24 hours.

1205Lu NLRP3 siRNA. 1205Lu cells (2x 10)⁵) Incubation with siRNA or scrambled (scrambled) siRNA targeting NLRP3 for non-specific gene silencing (santa cruz biotechnology). Transfection of siRNA duplexes (2nM) was performed using siRNA transfection medium according to the manufacturer's instructions. After 24 hours, the medium was replaced with RPMI 10% FBS (500 μ l) and the cells were incubated for another 24 hours. Supernatants were collected to measure IL-1 β levels by ELISA. The effect of NLRP3 silencing was determined by immunoblotting (western blotting) in cell lysates.

And (4) measuring the cell factors. Cytokines were measured in supernatants and cell lysates by specific ELISA according to the manufacturer's instructions (DuoSet, R & D system, minneapolis, MN).

Melanoma conditioned medium experiments. From the agreed key according to COMIRBPBMC were isolated from healthy donors and plated out at each well (5X 10) ⁵) Seeded into 96-well plates. Then will come from via OLT1177^TMSupernatant of treated 1205Lu cells was added to PBMCs (1: 2) and cells were incubated for 72 hours. NLRP 3-deficient THP-1 cells (1X 10)⁵) All were plated in 96-well plates and activated with 10ug/mL LPS for 3 hours. MCM (1: 2) was then added to the wells as a stimulus. Cells were incubated for 3 days and supernatants were assayed for cytokine secretion.

Angiogenesis assay (HUVEC). HUVEC cells were seeded overnight on growth factor-free medium. Cells were plated in 24-well plates at 8X 10 per well⁴The cells were plated onto matrigel (corning) -coated wells. The cells were then cultured in HUVEC complete medium (control), MCM or with OLT1177^TMIncubation in the presence of MCM treated 1205Lu cells for 5 hours. MCM was added without dilution. The medium was then removed and the matrigel remained in PFA 4%. Pictures were taken at 40 x and the branch points were counted using the cross method.

A combination therapy model. B16F10 cells were injected as described. Four days after instillation of Matrigel plugs (plug), start with OLT1177^TMThe mice were fed diet or continued on a standard diet and on day 7, were injected intraperitoneally with neutralizing antibodies against PD-1 (200 ug/mouse; BioXCell, West Lebanon, NH). Mice were sacrificed 15 days after B16F10 instillation.

Tumor angiogenesis models. In use standard or OLT1177^TMInterscapular regions of diet-fed mice were injected subcutaneously with Matrigel and B16F10(2x 10)⁵) A mixture of (a). 7 days after implantation, the plugs were removed, fixed in 4% paraformaldehyde, embedded in paraffin and sectioned (4 μm). Subsequently, the sections were deparaffinized, hydrated, and stained with hematoxylin/eosin. The divided sections were subjected to heat-induced antigen retrieval at 95 ℃ for 15 minutes (10mM citrate 0.05% Tween 20-pH 6.0). The sections were then placed in a moist slide chamber, blocked in 10% normal donkey serum (Jackson immunology) for 1 hour, and immunostained using antibodies against von Willebrand factor (1: 100, Millipore-Sigma, Burlington, Mass.) at 4 ℃ and overnight to identify new blood vesselsAnd (4) forming. An anti-rabbit horseradish peroxidase (HRP) -conjugated antibody (1: 100, Jackson immuno research Laboratories, WestGrove, Pa.) was used as the secondary antibody for 2 hours at room temperature. The sections were then incubated with HRP substrate for 5-10 minutes according to the manufacturer's instructions (NovaRED substrate, Vector laboratories, Burlingame, CA). Nuclear counterstaining was achieved using Mayer's hematoxylin counterstaining (semer feishell scientific, MA).

And transferring the model. For standard or OLT1177^TMMice fed diet were injected with B16F10-GFP (1X 10) in the tail vein (i.v.)⁶) The formation of metastases was determined after the cells. Before injection, B16F10-GFP was subjected to flow cytometry⁺Cells were sorted and only the brightest 10% cells were injected. Mice were sacrificed 21 days after cell injection, lungs and liver were isolated and prepared for histology. In a previous separation, lungs were inflated with a solution containing 0.5% low melting agarose to avoid tissue collapse. GFP positive cells were present in the lung and liver of tumor-bearing mice by fluorescence microscopy. Tissue sections were stained with Alexa Fluor conjugated WGA for cell membrane detection and DAPI for nuclear staining. Images were acquired blindly and randomly across the tissue sections to obtain 7-10 images from each tissue section. Count GFP positive cells in each image and report the results as GFP⁺Number of cells/field area (full chip field).

And (5) carrying out statistical analysis. Statistical significance of differences was assessed by two-tailed student's t-test using Prism version 7.0 Software (GraphPad Software, La Jolla, CA, USA). Statistical significance was set at p < 0.05.