Treatments for breast cancer

Abstract

The present invention relates to a method for treating breast cancer. The method comprises administering an effective amount of dapamosulfuron or a pharmaceutically acceptable solvate thereof to a subject in need thereof. The method optionally comprises further administering an effective amount of a checkpoint inhibitor to the subject.

Description

Technical Field

[0001] The present invention relates to a method for treating breast cancer by administering an effective amount of dapansutrile. Background Art

[0002] Tumorigenesis is driven by genomic alterations, including point mutations, gene deletions, and chromosomal rearrangements that lead to cellular transformation, self-sustaining proliferation, insensitivity to antiproliferative signals, evasion of apoptosis, and unlimited replicative potential, ultimately leading to tissue invasion and metastasis. However, the expansion of tumor cells is associated with a complex network of events involving both cancerous and noncancerous cells. Chronic inflammation is a prime example of such a promoting condition (1,2).

[0003] The proinflammatory cytokine IL-1β is a potent mediator of many chronic inflammatory diseases (3). Consistent with the link between cancer and chronic inflammation, IL-1β 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 migration (4-6).

[0004] Types of breast cancer include ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), triple-negative breast cancer (TNBC), inflammatory breast cancer (IBC), metastatic breast cancer, and gestational breast cancer. Triple-negative breast cancer tumors are characterized by the absence of estrogen receptors (ER), progesterone receptors (PR), and elevated levels of human epidermal growth factor receptor 2 (HER2) protein (7).

[0005] NLRP3 (NOD-like receptor family, pyrin domain containing 3), also known as NALP3 or cryopyrin, is one of the sensors of the inflammasome and a macromolecular structure involved in the processing of interleukin-1β (IL-1β) and IL-18. NLRP3 senses intracellular danger during intracellular infection (bacterial and viral proteins) or tissue injury (ischemia). NLRP3 activation leads to the recruitment of ASC (apoptosis-associated speck-like protein containing a carboxyl-terminal caspase recruitment domain) and caspase-1, which leads to inflammasome formation and ultimately cell death.

[0006] Dapanoxanil is a small synthetic molecule of β-sulfonitrile that has been shown to selectively inhibit the NLRP3 inflammasome and is safe when administered orally to healthy subjects (8).

[0007] There is a need for a method for treating breast cancer that is effective and has no significant side effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figures 1A-1D . Figure 1ASchematic diagram depicting the experimental design. Figure 1B Depicts the use of dapamosulfuron Graph of tumor volume in treated mice compared to vehicle. Measurements were made after tumor resection. **p<0.01 (n=5). Figure 1C Depicted are growth curves showing tumor volumes in mice treated with dapamoate compared to tumors in mice fed a standard diet. Measurements were taken on live mice on the days indicated. Figure 1C (n=5). Figure 1D Depicted is a diagram showing feeding a diet rich in dapoxetine Graph of the survival percentage of mice on the diet. *p<0.05.

[0009] Figures 2A-2G . Figure 2A Schematic diagram depicting the experimental design. Figure 2B Depicted is a graph of tumor volume analysis showing the effect of dapamoate on Tumor volumes were reduced in mice treated with dapoxetine and in mice treated with dapoxetine and anti-IL1α. Figure 2C Depicted are growth curves showing tumors in mice treated with dapamoate compared to mice treated with vehicle control. Measurements were taken on live mice on the days indicated. Figure 2C (n=5). Figure 2D and 2E Depicted is a graph showing the expression of TSLP ( Figure 2D ) and its related receptors (TSLPR; Figure 2E ) relative mRNA expression levels. *p<0.05 (n=8, two independent experiments). Figure 2F and 2G Depicted is a method showing the use of TSLP in vitro ( Figure 2F ) and TSLPR( Figure 2G ). Cells were stimulated with IL-1α and treated with dapoxetine as indicated. *p<0.05 (n=8, two independent experiments).

[0010] Figures 3A-3E . Figure 3A Schematic diagram depicting the experimental design. Figure 3B Depicts the use of dapamosulfuron Graph of tumor volume in 4T1 TNBC mice treated with anti-PD-1, both anti-PD-1 and dapamoate, or vehicle. Measurements were taken after tumor resection. Figure 3B(Vehicle vs. anti-PD-1, p=0.0069; Vehicle vs. dapamoic acid, p=0.0041; Vehicle vs. dapamoic acid + anti-PD-1, p=0.0014) (n=5). Figure 3C Depicted are growth curves showing tumor volume over 15 days in mice treated with anti-PD-1, dapoxetine, both anti-PD-1 and dapoxetine, or vehicle. Measurements were taken on live mice on the days indicated. Figure 3C (n=5). Figure 3D Depicted is a graph showing relative PD-L1 mRNA expression levels in 4T1 tumors from mice treated with dapamoate compared to vehicle. *p<0.05 (n=8, two independent experiments). Figure 3E Depicted is a graph showing relative PD-L1 mRNA expression in vitro using murine triple-negative breast cancer cells (E0771). E0771 cells were stimulated with IL-1α and treated with vehicle or dapamoate at the indicated concentrations. *p<0.05 (n=3).

[0011] Figures 4A-4C . Figure 4A Schematic diagram depicting the experimental design. Figure 4B Depicts the quantitative use of dapamosulfuron Graph of IL-22 levels in splenocytes from mice treated with anti-PD-1, both anti-PD-1 and dapamoate, or vehicle. Figure 4B (Vehicle vs. dapoxetine, p = 0.037; Vehicle vs. dapoxetine + anti-PD-1, p = 0.048). Figure 4C Depicted are graphs quantifying INFγ levels in splenocytes from mice treated with dapamoate, anti-PD-1, both anti-PD-1 and dapamoate, or vehicle. Figure 4C (Vehicle vs. dapoxetine, p=0.044; Vehicle vs. dapoxetine + anti-PD-1, p=0.022) (n=5). DETAILED DESCRIPTION

[0012] Activation of the NLRP3 inflammasome amplifies the inflammatory response to tissue injury and mediates further damage. Dapamuxil is a selective NLRP3 inflammasome inhibitor; it reduces inflammation by preventing activation of the NLRP3 inflammasome. Dapamuxil inhibits the production of mature IL-1β and IL-18 in mouse and human cells in vitro. Through this mechanism of action, dapamuxil blocks the production and / or release of IL-1β and inhibits the formation of the NLRP3 inflammasome in animals and human subjects.

[0013] The present invention relates to a method for treating breast cancer by administering to a subject an effective amount of dapamosulfuron. Dapamosulfuron can reduce tumor volume and / or prevent further tumor growth by inhibiting IL-1β, a major driver of breast tumor progression.

[0014] Compound

[0015] The present invention uses a purified compound of dapamoic acid (3-methanesulfonyl-propionitrile) or a pharmaceutically acceptable salt or solvate thereof.

[0016]

[0017] As used herein, "pharmaceutically acceptable salts" are salts that retain the desired biological activity of the parent compound and do not produce undesired toxicological effects.

[0018] As used herein, a "pharmaceutically acceptable solvate" is a solvate that retains the desired biological activity of the parent compound and does not produce undesirable toxicological effects. As used herein, a "solvate" is an addition complex in which a compound is combined in a fixed ratio with an acceptable cosolvent. Cosolvents include, but are not limited to, water, acetic acid, ethanol, and other suitable organic solvents.

[0019] Pharmaceutical composition

[0020] The amount of the active compound dapamoxil or a pharmaceutically acceptable salt or solvate thereof in the pharmaceutical composition is generally about 0.1-5% for injectable formulations, about 1-90% for tablet formulations, about 1-100% for capsule formulations, about 0.01-20%, 0.05-20%, 0.1-20%, 0.2-15%, 0.5-10% or 1-5% (w / w) for topical formulations, and about 0.1-5% for patch formulations.

[0021] As used herein, "about" means ± 10% of the recited value.

[0022] Those skilled in the art can use conventional criteria to select pharmaceutically acceptable carriers as inactive ingredients. Pharmaceutically acceptable carriers include, but are not limited to, non-aqueous solutions, suspensions, emulsions, microemulsions, micellar solutions, gels, and ointments. Pharmaceutically acceptable carriers may also contain, but are not limited to, the following ingredients: saline and aqueous electrolyte solutions; ionic and non-ionic osmotic agents such as sodium chloride, potassium chloride, glycerol, and glucose; pH regulators and buffers such as hydroxide salts, phosphates, citrates, acetates, borates; and triethanolamine; antioxidants such as salts, acids, and / or bases of bisulfites, sulfites, metabisulfites, thiosulfites, 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 ( Examples of the present invention include polysorbates such as polysorbate 80, polysorbate 60, and polysorbate 20; polyethers such as polyethylene glycol and polypropylene glycol; polyethylenes such as polyvinyl alcohol and povidone; cellulose derivatives such as methylcellulose, hydroxypropylcellulose, hydroxyethylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose, and their salts; petroleum derivatives such as mineral oil and white petrolatum; fats such as lanolin, peanut oil, palm oil, and soybean oil; mono-, di-, and triglycerides; polymers of acrylic acid such as carboxypolymethylene gelatin and hydrophobically modified cross-linked acrylate copolymers; polysaccharides such as dextran; and glycosaminoglycans such as sodium hyaluronate. These pharmaceutically acceptable carriers may be protected from bacterial contamination with well-known preservatives including, but not limited to, benzalkonium chloride, ethylenediaminetetraacetic acid and its salts, benzethonium chloride, chlorhexidine, chlorobutanol, methylparaben, thimerosal, and phenylethyl alcohol, or they may be formulated as non-preservative preparations for single or multiple use.

[0023] For example, the tablet formulation or capsule formulation of dapanoxanil can contain other excipients that do not have biological activity and do not react with the active compound. The excipient of tablet can comprise filler, adhesive, lubricant and slip agent, disintegrant, wetting agent and release rate regulator. Adhesive promotes the adhesion of preparation particles and is very important to tablet formulation. The example of adhesive includes but is not limited to carboxymethyl cellulose, cellulose, ethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, karaya gum, starch, starch and tragacanth gum, poly (acrylic acid) and polyvinyl pyrrolidone.

[0024] For example, the patch preparation of dapanoxanil can include 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. The patch preparation can also contain a skin permeability enhancer, such as a lactate (e.g., lauryl lactate) or diethylene glycol monoethyl ether.

[0025] The topical preparation comprising dapanoxanil can be in the form of gel, cream, lotion, liquid, emulsion, ointment, spray, solution and suspension. The inactive ingredients in the topical preparation are, for example, including but 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), octisalate (emollient / UV filter), silicone oil (emollient / diluent), squalene (emollient), sunflower oil (emollient) and silicon dioxide (thickening agent). In one embodiment, diethylene glycol monoethyl ether is included in the topical gel preparation.

[0026] How to use

[0027] By inhibiting the assembly of the NLRP3 inflammasome, dapamoate prevents the production and / or release of the proinflammatory cytokines IL-1β and IL-22 and ultimately treats breast cancer tumor growth.

[0028] The present invention relates to a method for treating breast cancer. The method comprises administering an effective amount of dapamosulfuron to a subject in need thereof. As used herein, an "effective amount" is an amount effective to treat the disease by ameliorating the pathological condition and / or reducing, ameliorating, and / or eliminating the symptoms of the disease. For example, an effective amount is an amount that reduces the growth of breast cancer and / or reduces the size of a breast tumor.

[0029] Breast cancers suitable for treatment by this method include triple-negative breast cancer (TNBC), ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), inflammatory breast cancer (IBC), metastatic breast cancer, and gestational breast cancer, among other types.

[0030] Checkpoint inhibitor therapy is a form of cancer immunotherapy. This therapy targets immune checkpoints, key regulators of the immune system that, when stimulated, suppress the immune response to immune stimulation. Some cancers can protect themselves from attack by stimulating immune checkpoint targets.

[0031] Immunotherapy has significantly improved the standard of care for patients with breast cancer; however, the number of nonresponders and relapsed patients remains high. Therefore, combination therapies that augment the efficacy of checkpoint inhibitors represent an important clinical benefit.

[0032] In one embodiment, the present invention relates to a combination therapy for treating breast cancer by combining dapamidine and a checkpoint inhibitor. The method comprises administering an effective amount of dapamidine and an effective amount of a checkpoint inhibitor to a subject in need. Dapamidine and the checkpoint inhibitor can be administered simultaneously or sequentially. It is advantageous to administer dapamidine in combination with a checkpoint inhibitor because dapamidine can improve the efficacy of checkpoint inhibitors and dapamidine has a safe drug profile. Co-administration can also reduce the required dose of the checkpoint inhibitor, thereby reducing adverse events associated with immunotherapy.

[0033] Checkpoint inhibitors suitable for use with dapagliflozin in the treatment of breast cancer include cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), and programmed death ligand 1 (PD-L1).

[0034] PD-1 is present on the surface of T cells and is a receptor for PD-L1. PD-1 plays a role in downregulating immune responses by inhibiting the activity of inflammatory T cells. This mechanism helps the body prevent autoimmune diseases, but it can also prevent cancer cells from being killed (9).

[0035] In a preferred embodiment, the checkpoint inhibitor is an anti-PD-1 antibody. The method comprises administering an effective amount of daptomycin and an effective amount of an anti-PD-1 antibody to a subject in need. Daptomycin and the anti-PD-1 antibody can be administered simultaneously or sequentially. It is advantageous to administer daptomycin in combination with an anti-PD-1 antibody because daptomycin improves the efficacy of anti-PD-1 and daptomycin has a safe drug profile. Co-administration can also reduce the required dose of the anti-PD-1 antibody, thereby reducing adverse events associated with immunotherapy.

[0036] The inventors have demonstrated that dapamoate treatment reduced 4T1 TNBC tumor growth in mice compared to vehicle.

[0037] Furthermore, the present inventors have demonstrated that dapamoate reduces tumor-promoting IL-22 and increases INFγ in splenocytes of treated mice.

[0038] The pharmaceutical compositions of the present invention can be applied by systemic administration or topical administration. Systemic administration includes, but is not limited to, oral administration, parenteral administration (such as intravenous administration, intramuscular administration, subcutaneous administration or rectal administration) and inhalation administration. In systemic administration, the active compound first reaches the plasma and then distributes to the target tissue. Oral administration is the preferred route of administration of the present invention. Topical administration includes topical administration.

[0039] The dosage of the composition may vary depending on the extent of breast cancer in the subject and the individual response of each patient. For systemic administration, the plasma concentration of the delivered active compound may vary; but is generally 1x10 -10 -1x10 -4 mol / L, and preferably 1x10 -8 -1x10 -5 mol / L.

[0040] In one embodiment, the pharmaceutical composition is orally administered to the subject. The dosage for oral administration is generally at least 1mg / kg / days and less than 100mg / kg / days, preferably 5-100mg / kg / days, depending on the age and condition of the subject. For example, for human subjects, the dosage for oral administration is 1-10, or 1-50, or 1-100, or 5-50, or 5-100, or 10-50, or 10-100mg / kg / days. For example, for human subjects, the dosage for oral administration is 100-10,000mg / days, preferably 100-2500, 500-2500, 500-4000, 1000-5000, 2000-5000, 2000-6000 or 2000-8000mg / days. The medicine can be taken orally once, twice, three times or four times a day. Patients are treated daily for 14 days, up to 1, 2, or 3 months, or for life.

[0041] In one embodiment, the pharmaceutical composition is administered to the subject intravenously. The dose for intravenous bolus injection or intravenous infusion is typically 0.03 to 5 mg / kg / day or 0.03 to 1 mg / kg / day.

[0042] In one embodiment, the pharmaceutical composition is administered subcutaneously to the subject. The dosage for subcutaneous administration is typically 0.3-20 mg / kg / day, 0.3-3 mg / kg / day, or 0.1-1 mg / kg / day.

[0043] In one embodiment, the composition is applied topically. Depending on the medical problem and disease pathology, the composition is applied topically at least once or twice a day or three to four times a day. Typically, 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.

[0044] Those skilled in the art will recognize that a variety of delivery mechanisms are suitable for use with the present invention.

[0045] The present invention can be used to treat mammalian subjects, such as humans, horses, dogs and cats. The present invention is particularly effective in treating humans.

[0046] The present invention is further illustrated by the following examples. These examples are intended only to illustrate the present invention and should not be construed as limiting the present invention.

[0047] Example

[0048] The following materials and protocols were used in the examples described below.

[0049] Cell lines. Breast cancer cell lines 4T1, E0771, and MDA-468 were obtained from ATCC (Manassas, VA). Cells were cultured in DMEM supplemented with 10% FBS, 100 units / mL penicillin, and 0.1 mg / mL streptomycin. Cells were maintained at 37°C in a humidified 5% CO2 atmosphere.

[0050] In vitro. Tumor cell lines were allowed to adhere overnight at a concentration of 200,000 cells per well. The next day, dapoxetine was added or not. Human recombinant IL-1α or IL-1β (R&D Systems, Minneapolis, MN) was added and incubated for 24 h. The cells were then lysed using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA).

[0051] Gene Expression. RNA was then isolated using Trizol (Thermo Fisher Scientific) and synthesized into cDNA using SuperScript III First-Strand (Thermo Fisher Scientific). Quantitative PCR (qPCR) was performed on the cDNA using Power SYBR Green PCR master mix (Thermo Fisher Scientific) on a Biorad CFX96 real-time system. Gene expression of Tslp, Tslpr, and Pdcd-11L1 mRNA was assessed using the following primers as indicated: Tslp forward, 5'-TACTCTCAATCCTATCCCTGGCTG-3' (SEQ ID NO: 1); Tslp reverse, 5'-TGTGAGGTTTGATTCAGGCAGATG-3' (SEQ ID NO: 2); Tslpr forward, 5'-TGACGTCACGGGGTGATGTC-3' (SEQ ID NO: 3); Tslpr reverse, 5'-GAGGATGCACCCGGAAGTGA-3' (SEQ ID NO: 4); Pdcd1L1 forward, 5'-GCTCCAAAGGACTTGTACGTG3' (SEQ ID NO: 5); Pdcd1L1 reverse, 5'-TGATCTGAAGGGCAGCATTTC3' (SEQ ID NO: 6).

[0052] Tumor Model. Animal protocols were approved by the Animal Care and Use Committee of the University of Colorado Health Sciences Center. Female BALB / c mice (The Jackson Laboratory) aged 6-8 weeks were fed a standard or dapamoate diet. Diet (4), which was started on the day of 4T1 injection. 4T1 cells (2x10 5 ) was injected orally into the mammary fat pad. Mice treated with dapamoate were fed food pellets containing 7.5 g / kg dapamoate ad libitum, starting on the day of the 4T1 injection and continuing for 15 days. The mice typically consumed about 4 grams of food per day, resulting in a daily dose of about 0 mg / kg / day for the control group and about 1,000 mg / kg / day for the treated group. This concentration of food pellets in the mouse food (7.5 g / kg dapamoate in food) resulted in blood levels that were almost identical to those in humans who took an oral dose of 1,000 mg / d of dapamoate (40 μg / mL blood levels) (14). Control (“vehicle”) mice were fed control food pellets that did not contain dapamoate. The mice were sacrificed 15 days after the 4T1 injection.

[0053] Anti-PD-1 combination therapy. 4T1 cells were injected as described. After infusion of 4T1 cells, mice began to receive dapamoate. The mice were fed a standard diet or continued to eat a standard diet as described, and a neutralizing antibody against PD-1 (200 μg / mouse; BioXCell, West Lebanon, NH) was injected intraperitoneally on day 7. The mice were sacrificed 15 days after the B16F10 instillation.

[0054] Anti-IL-1α combination therapy. 4T1 cells were injected as described. After infusion of 4T1 cells, mice began to receive dapamoate. The mice were fed a standard diet or continued on a standard diet as described and received intraperitoneal injections of a neutralizing antibody against IL-1α (200 μg / mouse; XBioTech, Austin, TX) every three days. Mice were sacrificed 15 days after the B16F10 instillation.

[0055] Splenic cytokine secretion. 4T1 cells were injected as described. Spleens from tumor-bearing mice were mechanically dissociated and processed for cell culture. Cells were suspended in RPMI supplemented with 10% FBS, 100 units / mL penicillin, and 0.1 mg / mL streptomycin, plated at 5.0 e^5, and stimulated with 10 μg / mL LPS. After 72 hours, the supernatant was removed and cytokine levels were measured by ELISA (R&D Systems, Minneapolis, MN).

[0056] Example 1: Dapanoxanil reduces breast tumor volume in 4T1 TNBC mice

[0057] In this example, the inventors evaluated whether the oral NLRP3 inhibitor dapamosulfuron is effective in reducing tumor growth in the murine 4T1 TNBC model.

[0058] 4T1 TNBC mice were generated and fed a standard diet or a dapoxetine diet for 15 days. Tumors were measured on the indicated days to generate growth curves ( Figure 1C The final tumor volume was obtained after tumor resection ( Figure 1B Tumor-bearing mice fed the dapamoate diet showed significantly reduced tumor volume ( Figure 1B , **p<0.01). In the survival study, compared with mice fed a standard diet ( Figure 1D , n=10, *p<0.05), 4T1 TNBC mice treated with dapamoate showed significantly higher survival rates compared with controls, with half of the mice on the dapamoate diet surviving for more than 30 days.

[0059] In these experiments, mice treated with dapamoate showed a significant reduction in tumor volume compared to vehicle controls. Figures 1A-1DIt was shown that in a triple-negative breast cancer mouse model (4T1 TNBC mice), dapamoate reduced tumor volume and significantly improved survival rate.

[0060] Example 2: Dapanoxanil reduces breast tumor volume when administered alone and in combination with anti-IL-1α

[0061] Previous studies have shown that HER2 - Metastatic breast cancer patients downregulate components of the inflammatory signature in metastatic breast cancer patients (10). To further evaluate the role of IL-1α and IL-1β in driving 4T1 tumor progression, the inventors treated mice with anti-IL-1α (200 μg / mouse), a dapoxetine diet, and a combination of the two. 4T1 TNBC mice were generated and fed a standard diet or a dapoxetine diet for 15 days. Mice were injected with anti-IL1α every 3 days as indicated. Subsequently, tumor volume was analyzed. Figure 2B and 2C Tumor-bearing mice treated with dapamoate diet alone and in combination with IL-1α showed significantly reduced tumor volume when compared to vehicle (*p<0.05).

[0062] In these experiments, treatment of mice with dapamoate effectively reduced tumor volume, whereas anti-IL-1α monotherapy did not significantly reduce tumor volume. Together, these data suggest that IL-1β may have a more potent tumor-promoting effect than IL-1α in 4T1 TNBC.

[0063] Previous studies on tumor-bone marrow cell interactions in triple-negative breast cancer have shown that tumor-derived IL-1α promotes infiltrating bone marrow cells to secrete TSLP (thymic stromal lymphopoietin), which is in turn critical for the sites of primary and distant metastases (15). To further evaluate the role of blocking NLRP3 in 4T1 tumor progression, the inventors quantified the gene expression of TSLP and its cognate receptor TSLPR in primary 4T1 tumors by relative mRNA expression measurements. Figure 2D and 2E The expression of TSLP and TSLPR was significantly decreased in tumor-bearing mice treated with a dapoxetine diet compared to vehicle (*p<0.05). Figure 2B and 2C To confirm these findings in vitro, the inventors used the human TNBC cell line MDA-468. MDA-468 cells were stimulated with IL-1α (20 ng / mL) and incubated with dapamoate (10 μM) or without dapamoate (control) for 24 hours. Figure 2F and 2GFigure 3: IL-1α and IL-1β expression of TSLP and TSLPR in the tumour tissues.

[0064] Example 3: Dapanoxanil reduces breast tumor volume when administered alone and co-administered with anti-PD-1

[0065] In this example, the inventors studied the tumor reduction effect of dapamoate and anti-PD-1.

[0066] 4T1 TNBC mice were generated and fed a standard diet or a dapoxetine diet for 15 days. Mice receiving combination therapy were injected with anti-PD-1 ( Figure 3A ).

[0067] The results are shown in Figure 3B and 3C Tumor volume was measured in Figure 3C Tumors were obtained from mice in vivo on the indicated days. After sacrifice on day 15, the tumors were excised and the tumor volumes were measured. Figure 3B As shown. Compared with the vehicle, tumor-bearing mice treated with dapamoate monotherapy showed significant tumor reduction (**p = 0.0041). Mice receiving anti-PD-1 alone also showed significant tumor reduction (**p = 0.0069). Mice receiving dapamoate and anti-PD-1 combination showed even more significant tumor reduction (**p = 0.0014).

[0068] To examine the effect of dapamoate on PD-L1 gene expression levels, Figure 2B and 2C Tumors from 4T1 TNBC mice were further analyzed in vivo.

[0069] The results are as follows Figure 3D As shown, 4T1 tumors from mice fed the dapamoate diet expressed significantly lower PD-L1 gene expression, as quantified by relative mRNA expression, than those from mice fed a standard diet. Figure 3D (*p<0.05; n=8, two independent experiments). This finding suggests a possible mechanism for enhancing the efficacy of anti-PD-1 therapy.

[0070] In vitro analysis was performed on the murine triple-negative breast cancer cell line E0771 to examine the effects of dapamoate on PD-L1 expression levels following cell death signaling. IL-1α stimulation was used to mimic cell death signaling in the tumor microenvironment. Cells were stimulated with 10 ng / mL of IL-1α and treated with the indicated concentrations of dapamoate or left untreated (vehicle).

[0071] The results are as follows Figure 3E As shown in Figure 3, E0771 cells treated with dapamoate after IL-1α stimulation expressed significantly reduced PD-L1 (*p<0.05) (n=3).

[0072] Example 4: Dapanoxanil reduces IL-22 and increases INFγ in splenocytes

[0073] Recent studies on the cytokine IL-22 have revealed its pro-tumor role in breast cancer as it recruits immunosuppressive cells into the tumor microenvironment (11). Experiments were performed here to examine cytokine production in spleens from tumor-bearing mice following monotherapy and combination therapy with dapamoate and anti-PD-1.

[0074] In this example, 4T1 TNBC mice were generated and fed a standard diet or a dapoxetine diet for 15 days. Figure 4A As shown, mice were injected with anti-PD-1 on day 7 in the combination study.

[0075] In these experiments, dapamoate monotherapy and combination therapy with anti-PD-1 significantly reduced IL-22 levels (*p<0.05). However, anti-PD-1 therapy alone failed to reduce IL-22 levels compared to vehicle. Figure 4B These data suggest that IL-1β induction of IL-22 is ultimately suppressed in mice fed dapamoate. The results also showed that IFNγ levels increased in the dapamoate and combination groups, indicating increased tumoricidal NK cell activity (*p<0.05). Figure 4C .

[0076] References

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[0080] 4. RNApte, et al. Cancer Metastasis Rev 25, 387-408 (2006).

[0081] 5. CADinarello. Cancer Metastasis Rev 29, 317-329 (2010).

[0082] 6. B. Guo, et al. Sci Rep 6, 36107 (2016).

[0083] 7. KLLee, et al. Cancers 11(9), 1334(2019).

[0084] 8. C. Marchetti, et al. Proc Natl Acad Sci USA 115, E1530-E1539 (2018).

[0085] 9. NLSyn, et al. Lancet Oncology 18(12), PE731-E741(2017).

[0086] 10.TC Wu, et al., Cancer Res 78(18):5243-5258(2018).

[0087] 11. C. Voigt, et al. Proc Natl Acad Sci USA 114:12994-12999 (2017).

[0088] 12. PM Ridker et al. Lancet 390(10105):1833-1842(2017).

[0089] 13. I. Kaplanov, et al. Proc Natl Acad Sci USA 116(4):1361-1369(2019).

[0090] 14. C. Marchetti, et al. Arthritis Res Ther 20:169 (2018).

[0091] 15. ELKuan, et al. Nat Immunol 19, 366–374 (2018).

[0092] From the foregoing it will be appreciated that while specific embodiments of the present invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the present disclosure. Accordingly, the present invention is not to be limited by the appended claims. Sequence Listing <110> CA Dinarello I. Tangestal <120> Treatments for breast cancer <130> 076619-8041.WO01 <150> US 62 / 914,833 <151> 2019-10-14 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 1 tactctcaat cctatccctg gctg 24 <210> 2 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 2 tgtgaggttt gattcaggca gatg 24 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 3 tgacgtcacg gggtgatgtc 20 <210> 4 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 4 gaggatgcac ccggaagtga 20 <210> 5 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 5 gctccaaagg acttgtacgt g 21 <210> 6 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 6 tgatctgaag ggcagcattt c 21

Claims

1. Use of dapamuxil or a pharmaceutically acceptable solvate thereof in the preparation of a medicament for treating breast cancer in a subject, wherein dapamuxil or a pharmaceutically acceptable solvate thereof is administered to a subject in need thereof.

2. The method according to claim 1, wherein the breast cancer is selected from the group consisting of ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), triple-negative breast cancer (TNBC), and inflammatory breast cancer (IBC). The use according to claim 1 , wherein the breast cancer is metastatic breast cancer. The use according to claim 1 , wherein dapamoxil is for oral administration.

5. The use according to claim 1, wherein the drug for treating breast cancer and a checkpoint inhibitor are administered to the subject in combination. The method according to claim 5 , wherein the checkpoint inhibitor is an anti-PD-1 antibody.

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