Methods for treating melanoma metastasis to liver and lungs

Abstract

The present invention is directed to a method for preventing and / or treating metastasis to the lungs or to the liver in a subject. The method comprises administering to a subject in need thereof an effective amount of dapansutrile. The method optionally comprises co-administering anti-PD-1 antibody and / or anti-PD-L1 with dapansutrile. A preferred route of administration of dapansutrile is oral administration.

Description

[0001] METHODS FOR TREATING MELANOMA METASTASIS TO LIVER AND LUNGS

[0002] TECHNICAL FIELD

[0003] The present invention relates to methods for treating melanoma metastasis to liver or lungs by administering an effective amount of dapansutrile.

[0004] BACKGROUND

[0005] Melanoma develops when unrepaired deoxyribonucleic acid (“DNA”) damage to skin cells triggers mutations which cause skin cells to proliferate, ultimately forming malignant tumors. These tumors originate in melanocytes, which are located within the basal layer of the epidermis. Melanomas are often caused by ultraviolet (UV) exposure and is the cause-of- death for more than 70,000 people in the United States, per year.

[0006] There are four types of melanomas: superficial spreading melanoma, lentigo maligna melanoma, acral lentiginous melanoma, and nodular melanoma. Superficial spreading melanoma is the most common and grows along the top layer of the skin before penetrating more deeply into the skin. Lentigo maligna melanoma is similar to the superficial spreading melanoma and occurs most often in the elderly, arising on chronically sun-exposed, damaged skin. Acral lentiginous melanoma also spreads superficially before penetrating more deeply and tends to advance into malignancy more frequently than superficial spreading melanoma and lentigo maligna melanoma. Nodular melanoma is most often invasive when first diagnosed.

[0007] Melanomas are classified in stages, which refer to the thickness, depth of penetration, and the degree to which the melanoma has spread. Early melanomas (stages 0 and I) are generally localized. Stage 0 tumors are generally non-invasive and often have not penetrated below the epidermis. Stage I tumors have often invaded into the dermis, are small, and are a low risk for metastasis. Stage II tumors are localized, larger, and are at a high risk for metastasis. Once the melanoma tumor has metastasized, it is classified as a stage III or IV melanoma depending in on the degree of metastasis.

[0008] Metastasis refers to the spread of cancer cells from the place where they first formed to another part of the body. In metastasis, cancer cells break away from the original (primary) tumor, travel through the blood or lymph system, and form a new tumor in other organs or tissues of the body. Once a melanoma metastatic cell colonizes a new tissue such as lung and

[0009] 1

[0010] 182910194 1 liver, the tumor microenvironment (TME) of the new organ is significantly different from the primary tumor.

[0011] The mechanisms of metastasis involve a complex multi-step process of detachment from the primary tumor site, intravasation into circulation, survival in the circulation, extravasation from circulation, attachment at a secondary site, and development of secondary tumor sites.

[0012] During the metastasis process, tumor cells, from the primary tumor undergo significant biological changes that allow tumor cells to leave the primary tumors (i.e. Epithelial-to-mesenchymal transition (EMT)). In view of the intrinsic changes that the tumor cells undergo to become metastatic, metastasis mass may be considered different tumors compared to the primary masses and therefore treatment used for primary tumors may not be effective in treating metastatic masses.

[0013] NLRP3 (NOD-like receptor family, pyrin domain containing 3), also known as NALP3 or cryopyrin, is one of the sensors of the inflammasome, a macromolecular structure involved in interleukin- 113 (IL-1 P) and IL- 18 processing. NLRP3 senses intracellular danger during intracellular infections (bacterial and viral proteins) or tissue injury (ischemia). NLRP3 activation leads to recruitment of ASC (apoptosis-associated speck-like protein containing carboxyterminal caspase recruitment domain) and caspase- 1 leading to inflammasome formation and ultimately cell death.

[0014] There is a need for a method for treating melanoma metastasis to the liver and the lungs. The method should be effective and have no significant side effects.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows mean ± SEM of GFP + cell counts / field area (full chip field) in the lungs of mice treated with standard diet vs. standard diet plus dapansutrile; N=3 per mice group. The cell counts are assessed by a microscopist without knowledge of the treatments to prevent possible bias. ****p<0.0001.

[0017] FIG. 2 shows mean ± SEM of GFP + cell counts / field area (full chip field) in the liver of mice treated with standard diet vs. standard diet plus dapansutrile; N=3 per mice group. The cell counts are assessed by a microscopist. ****p<0.0001.

[0018] 2

[0019] 182910194 1 DETAILED DESCRIPTION

[0020] Activation of the NLRP3 inflammasome amplifies the inflammatory response to tissue injury and mediates further damage. Dapansutrile is a selective NLRP3 inhibitor that prevents the assembly of the NLRP3 inflammasome; dapansutrile reduces inflammation by preventing activation of the NLRP3 inflammasome. Dapansutrile inhibits the production of mature IL- 1 P and IL- 18 in mice and in human cells. Through this mechanism of action, dapansutrile prevents production and / or release of IL- 1 and inhibits the formation of NLRP3 inflammasome in animals and human subjects.

[0021] The mechanisms of metastasis involve a complex multi-step process of detachment from the primary tumor site, intravasation into circulation, survival in the circulation, extravasation from circulation, attachment at a secondary site, and development of secondary tumor sites, each of which involves mediators induced by IL- 1 p.

[0022] The inventors discover that dapansutrile is effective to prevent and treat melanoma metastasis by blocking the assembly of the NLRP3 inflammasome and preventing the production and / or release of IL- 1 p.

[0023] The present invention is directed to methods of preventing and / or treating melanoma metastasis to the lungs and / or the liver by administering dapansutrile to a subject in need thereof.

[0024] Compound

[0025] The present invention uses a purified compound of dapansutrile (3-methanesulfonyl- propionitrile), or the pharmaceutically acceptable solvate thereof:

[0026] Dapansutrile is a small, synthetic molecule of P-sulfonyl nitrile which has been demonstrated to selectively inhibit the NLRP3 inflammasome and be safe when orally administered to healthy subjects (9).

[0027] “Pharmaceutically acceptable solvates,” as used herein, are solvates that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects.

[0028] “Solvates,” as used herein, are addition complexes in which the compound is combined with an acceptable co-solvent in some fixed proportion. Co-solvents include, but are not limited

[0029] 3

[0030] 182910194 1 to, water, acetic acid, ethanol, and other appropriate organic solvents.

[0031] Pharmaceutical Compositions

[0032] The active compound dapansutrile, or its pharmaceutically acceptable salt or solvate in the pharmaceutical compositions in general is in an amount of about 0.1-5% for an injectable formulation, about 1-90% for a tablet formulation, 1-100% for a capsule formulation, about 0.01-20%, 0.05-20%, 0. 1-20%, 0.2-15%, 0.5-10%, or 1-5% (w / w) for a topical formulation, and about 0.1-5% for a patch formulation.

[0033] “About” as used in this application, refers to ± 10% of the recited value.

[0034] Pharmaceutically acceptable carriers, which are inactive ingredients, can 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. The pharmaceutically acceptable carriers may also contain ingredients that include, but are not limited to, saline and aqueous electrolyte solutions; ionic and nonionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and dextrose; pH adjusters and buffers such as salts of hydroxide, phosphate, citrate, acetate, borate; and trolamine; antioxidants such as salts, acids and / or bases of bisulfite, sulfite, metabisulfite, thiosulfite, ascorbic acid, acetyl cysteine, cystein, glutathione, butylated hydroxyanisole, butylated hydroxytoluene, tocopherols, and ascorbyl palmitate; surfactants such as lecithin, phospholipids, including but not limited to phosphatidylcholine, phosphatidylethanolamine and phosphatidyl inositiol; poloxamers and ploxamines, polysorbates such as polysorbate 80, polysorbate 60, and polysorbate 20, polyethers such as polyethylene glycols and polypropylene glycols; polyvinyls such as polyvinyl alcohol and povidone; cellulose derivatives such as methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, carboxymethyl cellulose and hydroxypropyl methylcellulose and their salts; 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 copolymer; polysaccharides such as dextrans and glycosaminoglycans such as sodium hyaluronate. Such pharmaceutically acceptable carriers may be preserved against bacterial contamination using well-known preservatives, these include, but are not limited to, benzalkonium chloride, ethylene diamine tetra-acetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben,

[0035] 4

[0036] 182910194 1 thimerosal, and phenylethyl alcohol, or may be formulated as a non-preserved formulation for either single or multiple use.

[0037] A pharmaceutical composition comprising dapansutrile can be in a form of a tablet, a capsule, a patch, a topical formulation, an injectable formulation, an inhalation formulation, and other formulations.

[0038] For example, a tablet formulation or a capsule formulation of dapansutrile may contain other excipients that have no bioactivity and no reaction with the active compound. Excipients of a tablet may include fillers, binders, lubricants and glidants, disintegrators, wetting agents, and release rate modifiers. Binders promote the adhesion of particles of the formulation and are important for a tablet formulation. Examples of binders include, but not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, starch, and tragacanth gum, poly(acrylic acid), and polyvinylpyrrolidone.

[0039] For example, a patch formulation of dapansutrile may comprise some inactive ingredients such as 1,3-butylene glycol, dihydroxyaluminum aminoacetate, disodium edetate, D- sorbitol, gelatin, kaolin, methylparaben, polysorbate 80, povidone, propylene glycol, propylparaben, sodium carboxymethylcellulose, sodium polyacrylate, tartaric acid, titanium dioxide, and purified water. A patch formulation may also contain skin permeability enhancer such as lactate esters (e.g., lauryl lactate) or diethylene glycol monoethylether.

[0040] For example, a topical formulations including dapansutrile can be in a form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution, and suspension. The inactive ingredients in the topical formulations for example include, but not limited to, lauryl lactate (emollient / permeation enhancer), diethylene glycol monoethylether (emollient / permeation enhancer), DMSO (solubility enhancer), silicone elastomer (rheology / texture modifier), capiylic / capric triglyceride, (emollient), octisalate, (emollient / UV filter), silicone fluid (emollient / diluent), squalene (emollient), sunflower oil (emollient), and silicone dioxide (thickening agent). In one embodiment, diethylene glycol monoethylether is included in the topical gel formulation.

[0041] Method of Use

[0042] By inhibiting assembly of the NLRP3 inflammasome, dapansutrile prevents the production and / or release of proinflammatory cytokines IL- 1 and IL- 18, and reduces melanoma metastasis to the lungs and the liver.

[0043] 5

[0044] 182910194 1 The present invention is directed to methods of treating melanoma metastasis to the lungs and the liver. In one embodiment, the primary melanoma includes superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, and acral lentiginous melanoma. The present method comprises the step of administering to a subject in need thereof an effective amount of dapansutrile. “An effective amount,’’ as used herein, is the amount effective to treat a disease by ameliorating the pathological condition, and / or reducing, improving, and / or eliminating the symptoms of the disease. For example, an effective amount is an amount that reduces the growth of metastatic melanoma (reducing tumor size) in the lung or liver.

[0045] In one embodiment, dapansutrile treats melanoma metastasis to the lungs by reducing size of existing lung lesions, stopping new pulmonary lesions, or reducing the total number of pulmonary lesions.

[0046] In one embodiment, dapansutrile treats melanoma metastasis to the liver by reducing size of existing liver lesions, stopping new liver lesion, or reducing the total number of liver lesions.

[0047] The present method optionally includes a step of administering an effective amount of a checkpoint inhibitor such as anti-PD-1 antibody and / or anti-PD-Ll for treating melanoma metastasis to the lungs or to the liver. The method comprises administering an effective amount of dapansutrile and an effective amount of anti-PD-1 and / or anti-PD-Ll antibody to a subject in need thereof. Dapansutrile and anti-PD-1 antibody / anti-PD-Ll antibody can be administered simultaneously or sequentially. It is advantageous to co-administer dapansutrile with anti-PD- 1 / anti-PD-Ll antibody because dapansutrile improves the efficacy of anti-PD-1 / anti-PD-Ll antibody. The co-administration may also reduce the required dosage of anti-PD-1 / anti-PD-Ll antibody, which reduces immunotherapy-related adverse events. The treatment with dapansutrile in addition to anti-PD-1 or anti-PD-Ll potentiates the effect of a single therapy, creating an alternative for therapy-resistant cancers.

[0048] The pharmaceutical composition of the present invention can be applied by systemic administration or local administration. Systemic administration includes, but is not limited to oral, parenteral (such as intravenous, intramuscular, subcutaneous or rectal), and inhaled administration. In systemic administration, the active compound first reaches plasma and then distributes into target tissues. Oral administration is a preferred route of administration for the present invention. Local administration includes topical administration.

[0049] 6

[0050] 182910194 1 Dosing of the composition can vary based on the extent of the subject’s melanoma and each patient’s individual response. For systemic administration, plasma concentrations of the active compound delivered can vary; but are generally lxlO’lo-lxlO’4moles / liter, and preferably 1x1 O’8- 1x1 O'5moles / liter.

[0051] In one embodiment, the pharmaceutical composition of dapansutrile is administrated orally to a subject. The dosage for oral administration is generally at least 1 mg / kg / day and less than 100 mg / kg / day, preferably 5-100 mg / kg / day, depending on the subject’s age and condition. For example, the dosage for oral administration is 1-10, or 1-50, or 1-100, or 5-50, or 5-100, or 10-50, or 10-100 mg / kg / day for a human subject. For example, the dosage for oral administration is 100-10,000 mg / day, and preferably 100-2500, 500-2500, 500-4000, 1000-5000, 2000-5000, 2000-6000, or 2000-8000 mg / day for a human subject. The drug can be orally taken once, twice, three times, or four times a day. The patient is treated daily for 14 days up to 1 month, 2 months, or 3 months or for lifespan.

[0052] In one embodiment, the pharmaceutical composition is administrated intravenously to a subject. The dosage for intravenous bolus injection or intravenous infusion is generally 0.03 to 5 or 0.03 to 1 mg / kg / day.

[0053] In one embodiment, the pharmaceutical composition is administrated subcutaneously to the subject. The dosage for subcutaneous administration is generally 0.3-20, 0.3-3, or 0.1- 1 mg / kg / day.

[0054] In one embodiment, the composition is applied topically. The composition is topically applied at least 1 or 2 times a day, or 3 to 4 times per day, depending on the medical issue and the disease pathology. In general, 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, 0.2-10 mL of the topical composition is applied to the individual per dose.

[0055] Those of skill in the art will recognize that a wide variety of delivery mechanisms are also suitable for the present invention.

[0056] The present invention is useful in treating a mammal subject, such as humans, horses, dogs and cats. The present invention is particularly useful in treating humans.

[0057] The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.

[0058] 7

[0059] 182910194 1 EXAMPLES

[0060] Example 1. Dapansutrile Reduced Tissue Invasion and Metastasis in Lung and Liver Caused by Injection of Melanoma Cells in Mice

[0061] To determine whether dapansutrile reduces lung and liver metastasis from melanoma, melanoma cells were injected in mice fed with standard diets or dapansutrile diets. Mice were fed ad libitum with either a standard diet or a diet containing dapansutrile at a dose of 7.5 g per Kg of food, starting the day of injection of melanoma cells. The dosage is approximately 1200 mg dapansutrile / kg / day based on feed concentrations of 7.5 g / kg and food consumption of 4g / day. These diets are continued after tumor implantation. N=3 per mice group.

[0062] Lung and liver metastasis formation were determined following tail intravenous (i.v.) injection of B16F10-GFP ( lx 106) cells in mice fed standard or dapansutrile diet. B16-F10 is a cell line exhibiting a morphology of spindle-shaped and epithelial-like cells that was isolated from skin tissue of a mouse with melanoma. B16F10-GFP cell line expresses green fluorescent protein (GFP) gene sequences as free cytoplasmatic proteins.

[0063] Before injection, the B16F10-GFP+ cells were sorted by flow cytometry and only the top brightest 10% cells were injected. Mice were sacrificed after 21 days from the cell injection and lung and liver were isolated and prepared for histology. Prior to isolation, lungs were inflated with a solution containing 0.5% low melting agarose to avoid the tissue from collapsing. The presence of GFP-positive cell in the lung and liver of tumor bearing mice were performed by fluorescent microscopy. Tissue sections were stained with Alexa Fluor conjugated wheat germ agglutinin (WGA) for membrane detection, and stained with 4', 6- diamidino-2-phenylindole (DAPI) for nuclear stain. Images were acquired blindly and randomly across the tissue sections to obtain 7-10 images per tissue section. GFP positive cells were counted in each image and the results reported as number of GFP+ cells / field area (full chip field).

[0064] Immunofluorescence analysis of the lungs shows a reduced number of GFP+cells in mice that received dapansutrile compared to the standard diet (Pictures not shown). In FIG. 1, the number of GFP1cells in the lungs treated with dapansutrile were reduced by 66% (pO.OOOI).

[0065] Immunofluorescence analysis of the liver shows a reduced number of GFP+cells in mice that received dapansutrile compared to the standard diet (Pictures not shown). In FIG. 2, the number of GFP+cells in the liver treated with dapansutrile were reduced by 60% (pO.OOOI).

[0066] 8

[0067] 182910194 1 The reductions in the number of metastatic cells in the lung and liver show that dapansutrile reduced tissue invasion and reduced melanoma metastasis to the lung and the liver.

[0068] Example 2. Clinical Study of Orally Administered Dapansutrile for Treating Melanoma Metastasis to the Lungs and the Liver

[0069] Methodology

[0070] The study is to evaluate safety and efficacy of orally administered dapansutrile for treating melanoma metastasis to the lungs and to the liver.

[0071] Subject Section

[0072] 1. Has histologically or cyto logically confirmed melanoma

[0073] 2. Has unresectable Stage III or Stage IV melanoma, including melanoma metastasis to the lungs or the liver, per AJCC 8th Edition Staging Criteria, not amenable to local therapy.

[0074] 3. Measurable disease based on RECIST v. 1.1. Lesions situated in a previously irradiated area are considered measurable if progression has been demonstrated in such lesions a. Multiple target lesions are allowed and are selected based on standard RECIST criteria. b. The following cutaneous lesions are considered measurable lesions: lesions > 10 mm in longest diameter or multiple melanoma lesions which in aggregate have the longest diameter of > 10 mm, when measured by caliper.

[0075] Drug Administration

[0076] For monotherapy, dapansutrile is dispensed orally on day 1 of every cycle and administered at 500 mg BID or 1000 mg BID, throughout the treatment period.

[0077] For combination therapy, in addition to dapansutrile administration as described in monotherapy, pembrolizumab 200 mg is administered in an outpatient setting as a 30-minute IV infusion on Day 1 of each cycle. A treatment window of + / - 3 days of day 1 is allowed for cycle 2 and all subsequent cycles.

[0078] 9

[0079] 182910194 1 Treatment Period

[0080] 1. Monotherapy of dapansutrile

[0081] Dapansutrile is administered daily by oral administration throughout the Treatment Period, at 500 mg QD (once a day), 500 mg BID (twice a day), or 1000 mg BID.

[0082] 2. Combination therapy of dapansutrile and pembrolizumab

[0083] During the Treatment Period, subjects receive a dapansutrile monotherapy lead-in for the first 14 days of Cycle 1. Following the monotherapy lead-in, each subject receives pembrolizumab on C1D15 and on day 1 of every subsequent cycle. Cycle 1 is 35 days. Cycle 2 and all subsequent cycles are 21 days in length. Dapansutrile is administered daily throughout the Treatment Period. The Treatment Period lasts until: 1) disease progression; 2) the occurrence of unacceptable treatment-attributed toxicity; or 3) other reason(s) for subject discontinuation.

[0084] Melanoma Tumor Assessments

[0085] Melanoma tumor response is assessed using RECIST v. 1. 1 and iRECIST. Radiographic imaging is performed with CT scan of chest / abdomen / pelvis with and without contrast and / or MRI scan of abdomen / pelvis every 4 cycles (i.e., at week 14 and eveiy 12 weeks thereafter). The same method for tumor assessment is employed at every assessment.

[0086] Primary Endpoint of Treating Melanoma Metastasis to the Lungs

[0087] The primary end point is the objective response rate (ORR) in the lung lesion(s).

[0088] The primary end point of metastasis to the lungs can be assessed by modified Response Evaluation Criteria in Solid Tumors (RECIST). ORR is the percentage of participants in the study who have a partial response or complete response to the treatment per RECIST criteria in the lungs.

[0089] Secondary Endpoint of Treating Melanoma Metastasis to the Lungs

[0090] The secondary end points are 6 and 12 month progression-free survival (PFS) and overall survival (OS).

[0091] Primary Endpoint of Treating Melanoma Metastasis to the Liver

[0092] The primary end point is ORR in the liver lesion(s).

[0093] 10

[0094] 182910194 1 The primary end point of metastasis to the liver can be assessed by modified Response Evaluation Criteria in Solid Tumors (RECIST). ORR is the percentage of participants in the study who have a partial response or complete response to the treatment per RECIST criteria in the liver.

[0095] Secondary Endpoint of Treating Melanoma Metastasis to the Liver

[0096] The secondary end points are 6 and 12 month progression-free survival (PFS) and overall survival (OS). It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims.

[0097] 11

[0098] 182910194.1

Claims

WHAT IS CLAIMED IS:

1. A method of treating melanoma metastasis to the lungs or to the liver in a subject, comprising the step of: administering to the subject suffering from melanoma metastasis to the lungs or to the liver an effective amount of dapansutrile, or a pharmaceutically acceptable solvate thereof.

2. The method according to claim 1, where the subject suffers melanoma metastasis to the lungs.

3. The method according to claim 1, where the subject suffers melanoma metastasis to the liver.

4. The method according to any one of claims 1-3, wherein dapansutrile is administered by systemic administration.

5. The method according to claim 4, wherein the dapansutrile is administered by oral administration.

6. The method according to claim 2, where the method reduces the size of melanoma or reduces the total number of lesions in the lungs.

7. The method according to claim 3, where the method reduces the size of melanoma or reduces the total number of lesions in the liver.

8. The method according to claim 1, further comprising administering anti-PD-1 and / or anti-PD-Ll antibody to the subject.12182910194.1

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