Combination therapy for treating cancer

Abstract

The present disclosure provides a method of treating cancer by administering to an individual in need thereof a pharmaceutical composition comprising a benzenesulfonamide derivative and at least one other therapeutic agent in a different class of anticancer agents.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 321,185, filed on March 18, 2022. The entire disclosure of the foregoing application is incorporated herein by reference.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is electronically submitted through the patent center in XML format, with the file name "211106US-sequence listing-jc-20230320.xml", created on March 20, 2023, and the file size is 7kb. The sequence listing submitted through the patent center is part of the specification and is incorporated herein by reference. Technical Field

[0005] The present disclosure relates to methods of treating proliferative diseases, and in particular to methods of treating cancer by combination therapy. Background Art

[0006] Description of related fields

[0007] In 2020, there are an estimated 18.1 million cases of cancer worldwide. By 2040, the global burden is expected to grow to 27.5 million new cancer cases and 16.3 million cancer deaths.

[0008] There are approximately more than 700 oncology drugs, and more are still under development. Chemotherapy is one of the common cancer treatments, which involves the interruption of cell metabolism and may be more effective than other treatment options, but due to individual genetic differences, especially in tumor somatic cells, drug resistance may occur. Moreover, once cancer drug resistance is acquired, the drug resistance may occur through different mechanisms, such as cell death inhibition and gene amplification. For example, cisplatin is a broad-spectrum anticancer chemotherapy agent. However, cisplatin resistance represents a treatment problem, which includes insufficient DNA binding, increased detoxification, increased DNA repair, dysregulated expression of transporters, and expression and activity changes of genes such as p53, Bcl-2 and Akt / mTOR involved in cell death pathways. Gemcitabine is another chemotherapy drug used to treat different types of cancer. However, most cancer patients who receive gemcitabine chemotherapy eventually develop resistance to gemcitabine, which includes changes in gemcitabine metabolism, reduced intracellular drug accumulation, inhibition of apoptotic pathways, and abnormal activity in signal transduction pathways that regulate cell cycle and apoptosis. Pemetrexed is another chemotherapy drug that improves overall survival of cancer patients; however, pemetrexed resistance also often emerges during therapy.

[0009] Other forms of therapy such as radiation, hormone therapy, and surgery often have challenging limitations such as damage to surrounding tissue, limited penetration of radiation to the target, fatigue, illness, increased risk of clotting, bleeding, infection, intestinal obstruction, etc.

[0010] As the arsenal of targeted anticancer drugs grows, despite promising results and initial high response rates observed in preclinical experiments, a large number of targeted drugs have failed to successfully provide reproducible improvements in cancer patient survival when used as single agents. Furthermore, the recognition of treatment-induced toxicity and the inability of many targeted therapies to be administered in continuous pharmacokinetic effective doses requires creative intermittent schedules.

[0011] As the treatment of cancer remains an unmet medical need, there is a clear and urgent medical need for additional cancer therapeutics for cancer patients. Summary of the invention

[0012] In view of the above problems in the technical field, the present disclosure provides an effective method for treating proliferative diseases such as cancer.

[0013] The present disclosure provides a method for treating cancer, comprising administering a therapeutically effective amount of a pharmaceutical composition and a chemotherapeutic agent to an individual in need thereof, wherein the pharmaceutical composition comprises a benzenesulfonamide derivative and a pharmaceutically acceptable carrier thereof, and the chemotherapeutic agent comprises at least one selected from the group consisting of an alkylating agent, an antimetabolite and a corticosteroid.

[0014] In at least one embodiment of the present disclosure, the benzenesulfonamide derivative is represented by the following formula (I):

[0015]

[0016] or a pharmaceutically acceptable salt thereof,

[0017] wherein R1 to R7 are independently selected from the group consisting of H, C1-C6 straight or branched alkyl, C1-C6 straight or branched alkoxy, C3-C6 cycloalkyl, C3-C6 cycloheteroalkyl, amino and halogen, or R6 and R7 are connected to each other to form a ring, and

[0018] wherein the alkyl, alkoxy, cycloalkyl, cycloheteroalkyl and ring in R1 to R7 are independently unsubstituted or substituted with one or more substituents.

[0019] In at least one embodiment of the present disclosure, the substituent is selected from the group consisting of phenyl, halo, oxo, ether, hydroxyl, carboxyl, amino, sulfo, and sulfonamide groups.

[0020] In at least one embodiment of the present disclosure, the benzenesulfonamide derivative or a pharmaceutically acceptable salt thereof is at least one selected from the group consisting of p-toluenesulfonamide, o-toluenesulfonamide, m-toluenesulfonamide, N-ethyl-o-toluenesulfonamide, N-ethyl-p-toluenesulfonamide, N-cyclohexyl-p-toluenesulfonamide,

[0021]

[0022]

[0023]

[0024] and

[0025] In at least one embodiment of the present disclosure, the alkylating agent is cisplatin.

[0026] In at least one embodiment of the present disclosure, the antimetabolite is gemcitabine or pemetrexed.

[0027] In at least one embodiment of the present disclosure, the cancer is melanoma or lung cancer.

[0028] In at least one embodiment of the present disclosure, the cancer is melanoma and the chemotherapeutic agent comprises cisplatin. In some embodiments, the ratio of the benzenesulfonamide derivative to cisplatin is in the range of 25: 1 to 2000: 1. In some embodiments, the benzenesulfonamide derivative is administered to the individual at a dose of about 100 mg / kg and cisplatin is administered to the individual at a dose of about 2 mg / kg.

[0029] In at least one embodiment of the present disclosure, the cancer is squamous cell carcinoma of the lung, and the chemotherapeutic agent includes gemcitabine and cisplatin. In some embodiments, the ratio of the benzenesulfonamide derivative to gemcitabine is in the range of 1.5: 1 to 100: 1, and the ratio of the benzenesulfonamide derivative to cisplatin is in the range of 25: 1 to 2000: 1. In some embodiments, the benzenesulfonamide derivative is administered to the individual at a dose of about 330 mg / kg, gemcitabine is administered to the individual at a dose of about 160 mg / kg, and cisplatin is administered to the individual at a dose of about 3 mg / kg.

[0030] In at least one embodiment of the present disclosure, the cancer is lung adenocarcinoma, and the chemotherapeutic agent includes pemetrexed and cisplatin. In some embodiments, the ratio of the benzenesulfonamide derivative to pemetrexed is in the range of 1.5:1 to 100:1, and the ratio of the benzenesulfonamide derivative to cisplatin is in the range of 25:1 to 2000:1. In some embodiments, the benzenesulfonamide derivative is administered to the individual at a dose of about 330 mg / kg, pemetrexed is administered to the individual at a dose of about 100 mg / kg, and cisplatin is administered to the individual at a dose of about 2 mg / kg.

[0031] In at least one embodiment of the present disclosure, the benzenesulfonamide derivative is administered to the subject at a dose of about 330 mg / kg, pemetrexed is administered to the subject at a dose of about 100 mg / kg, and cisplatin is administered to the subject at a dose of about 2 mg / kg.

[0032] In at least one embodiment of the present disclosure, the benzenesulfonamide derivative in the pharmaceutical composition is administered to a subject in an effective amount of about 3,300 mg to about 26,400 mg.

[0033] In at least one embodiment of the present disclosure, the benzenesulfonamide derivative in the pharmaceutical composition is administered to a subject in an effective amount of about 165 mg to about 6,600 mg per day.

[0034] In at least one embodiment of the present disclosure, the pharmaceutical composition or chemotherapeutic agent is administered to an individual once to three times a week. In some embodiments, the pharmaceutical composition is administered to an individual twice a week, and the chemotherapeutic agent is administered to an individual once a week.

[0035] In at least one embodiment of the present disclosure, the pharmaceutically acceptable carrier is selected from the group consisting of fillers, binders, preservatives, disintegrants, lubricants, suspending agents, wetting agents, fragrances, thickeners, acids, biocompatible solvents, surfactants, complexing agents, and any combination thereof.

[0036] In at least one embodiment of the present disclosure, the pharmaceutically acceptable carrier is selected from the group consisting of polyethylene glycol, alkylene glycol, propylene glycol, sebacic acid, dimethyl sulfoxide, ethanol, and any combination thereof.

[0037] In at least one embodiment of the present disclosure, the pharmaceutical composition is in a form selected from the group consisting of injection preparations, dry powders, tablets, oral solutions, wafers, films, lozenges, capsules, granules, pills, gels, lotions, ointments, emulsions, pastes, creams, eye drops and ointments.

[0038] In at least one embodiment of the present disclosure, the pharmaceutical composition or chemotherapeutic agent is administered to the subject intratumorally, intravenously, subcutaneously, intradermally, orally, intrathecally, intraperitoneally, intranasally, intramuscularly, intrapulmonary, topically, or by aerosolization.

[0039] In at least one embodiment of the present disclosure, the individual is a human, a dog, a cat, or a mouse. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] This patent or application publication contains at least one drawing printed in color. Copies of this patent or patent application publication in color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0041] The present disclosure will become more readily understood with reference to the following description in conjunction with the accompanying drawings.

[0042] Figures 1A to 1B Shown are the effects of cisplatin, PTS, and the combination of both on M5 melanoma tumor growth in BALB / c nude mice. Figure 1A The images show tumors excised from mice 28 days after tumor cell transplantation. Figure 1B Figure 2 is a graph showing tumor volume over time. Treatments were administered three times a week starting on day 7 (when tumors were detected) (except for controls, which were administered daily) as follows: saline (control); 2 mg / kg cisplatin (cisplatin); 100 mg / kg PTS (PTS); and 100 mg / kg PTS combined with 2 mg / kg cisplatin (cisplatin + PTS). Data presented are mean ± standard deviation (SD), n = 7 per group. ***p < 0.05, compared to control; # p < 0.05, compared with cisplatin; + p<0.05, compared with PTS.

[0043] Figures 2A to 2B Shown is a TUNEL / DAPI assay for apoptosis of M5 canine melanoma tumors implanted in BALB / c nude mice. Figure 2A One panel has color staining of DAPI, TUNEL, and a merge of both. Figure 2B The percentage of TUNEL-positive cells in tumors of control mice and those mice treated three times a week (except control, which was administered daily) starting on day 7 (when tumors were detected): saline (control); 2 mg / kg cisplatin (cisplatin); 100 mg / kg PTS (PTS); or 100 mg / kg PTS and 2 mg / kg cisplatin (cisplatin + PTS). Data are mean ± SD, n = 7 per group. **p < 0.01, *** p < 0.001, compared with control; ### p < 0.001, compared with cisplatin; +++p<0.001, compared with PTS. Scale bar: 50 μm; Magnification: 200x.

[0044] Figures 3A to 3D Shown are the results of Western blot analysis of apoptosis-related proteins in M5 canine melanoma tumors implanted in BALB / c nude mice. Figure 3A Representative Western blot images. Figure 3B Quantitative comparison of the expression of cleaved apoptosis proteinase 3. Figure 3C The figure is a quantitative comparison of the expression of phosphorylated Erk. Figure 3D Quantitative comparison of Bcl-2 expression. Mice were administered the following treatments three times a week (except for controls, which were administered daily): saline (control), 2 mg / kg cisplatin (cisplatin), 100 mg / kg PTS (PTS), or 100 mg / kg PTS and 2 mg / kg cisplatin (cisplatin + PTS). Data are mean ± SD, n = 7 per group. *p < 0.05, ** p < 0.01, and *** p < 0.001, compared with control; # p<0.05 and ## p < 0.01, compared with cisplatin; ++ p<0.01, compared with PTS.

[0045] Figures 4A to 4E Immunohistochemical (IHC) analysis of the expression of three inflammatory cytokines in M5 canine melanoma tumors implanted in BALB / c nude mice. Figure 4A A set of representative colored images showing IL-1 expression. Figure 4B A set of representative colored images showing TNF-α expression. Figure 4C For quantitative analysis of IL-1β expression by IHC. Figure 4D For quantitative analysis of TNF-α expression by IHC. Figure 4E For quantitative analysis of serum IL-6 expression by IHC. Mice were administered the following treatments three times a week (except for controls, which were administered daily): saline (control), 2 mg / kg cisplatin (cisplatin), 100 mg / kg PTS (PTS), or 100 mg / kg PTS and 2 mg / kg cisplatin (cisplatin + PTS). Data are mean ± SD, n = 7 per group. ** p < 0.01, and *** p < 0.001, compared with control; ## p<0.01, compared with cisplatin; and ++ p<0.01, compared with PTS. Scale bar; 50 μm.

[0046] Figures 5A to 5DShown is an analysis of factors associated with inflammation in M5 canine melanoma tumors implanted in BALB / c nude mice. Figure 5A Representative Western blot images of COX-2 are shown.

[0047] Figure 5B Quantitative comparison of COX-2 expression in four treatment groups (control, cisplatin, PTS, and cisplatin + PTS) is shown. Quantitative polymerase chain reaction (PCR) was performed to assess Figure 5C Relative mRNA expression levels of NF-κB and expression levels of lκBα in mice. Mice were administered the following treatments three times a week (except for controls, which were administered daily): saline (control), 2 mg / kg cisplatin (cisplatin), 100 mg / kg PTS (PTS), or 100 mg / kg PTS and 2 mg / kg cisplatin (cisplatin + PTS). Data are mean ± SD, n = 7 per group. ** p<0.01 and *** p < 0.001, compared with control; # p<0.05 and ## p < 0.01, compared with cisplatin; + p<0.05 and ++ p<0.01, compared with PTS.

[0048] Figures 6A to 6G Analysis of factors associated with metastasis in M5 canine melanoma tumors implanted in BALB / c nude mice is shown. Fig. 6A and 6B ) and TGF-β( Figure 6C and 6D A set of representative colored images of IHC analysis of Fig. 6A and 6C ) and quantitative comparison ( Figure 6B and 6D ). Figures 6E to 6G Shown from EGFR ( Fig. 6F ) and VEGF( Figure 6G Representative Western blot analysis of the relative expression of Fig. 6E Mice were administered the following treatments three times per week (except controls, which were administered daily): saline (control), 2 mg / kg cisplatin (Cisplatin), 100 mg / kg PTS (PTS), or 100 mg / kg PTS and 2 mg / kg cisplatin (Cisplatin+PTS). Data are mean ± SD, n = 7 per group. * p<0.05, ** p<0.01, and ** p < 0.001, compared with control; ## p < 0.01, compared with cisplatin; ++p<0.01, compared with PTS. Scale bar; 50 μm.

[0049] Figures 7A to 7C Western blot analysis of various aspects of the mTOR (mammalian TOR) signaling pathway in M5 canine melanoma tumors implanted in BALB / C nude mice. Fig. 7A Representative Western blots of mTOR and S6K1 expression. Figure 7B and 7C Shows mTOR phosphorylation ( Figure 7B ) and S6K1 phosphorylation ( Figure 7C ) quantification of the relative expression level. Specifically, the present disclosure shows that relative to the control group, the relative content of phosphorylated mTOR / mTOR in the cisplatin group, the PTS group, and the cisplatin+PTS group was reduced to less than that in the control group ( Figure 7B ). The present disclosure shows that the levels in the combined treatment group were significantly lower than those in the two single treatment groups. Relative to the control group, the relative levels of p-S6K1 / S6K1 in the cisplatin group, the PTS group, and the cisplatin+PTS group were reduced to less than that in the control group ( Figure 7C ). The present disclosure shows that the levels in the combination treatment group were significantly lower than the two single treatment groups.

[0050] Mice were administered the following treatments three times per week (except controls, which were administered daily): saline (control), 2 mg / kg cisplatin (Cisplatin), 100 mg / kg PTS (PTS), or 100 mg / kg PTS and 2 mg / kg cisplatin (Cisplatin+PTS). Data are mean ± SD, n = 7 per group. * p<0.05, ** p<0.01, and *** p < 0.001, compared with control; # p < 0.05, compared with cisplatin; + p<0.05, compared with PTS.

[0051] Figure 8 It is a bar graph showing serum levels of C-reactive protein (CRP) in BALB / cByJNarl mice transplanted with canine melanoma tumor cells, wherein CRP is an acute phase protein synthesized in response to proinflammatory cytokines during the inflammatory process. The following treatments were administered to mice three times a week (except for controls, which were administered daily): saline (control), 2 mg / kg cisplatin (cisplatin), 100 mg / kg PTS (PTS), or 100 mg / kg PTS and 2 mg / kg cisplatin (cisplatin + PTS). Data are mean ± SD, n = 7 per group. *** p < 0.001, compared with control; # p < 0.05, compared with cisplatin; +p<0.05, compared with PTS.

[0052] Figures 9A to 9B Shown is IHC analysis of Ki67 expression in M5 canine melanoma tumors implanted in BALB / c nude mice. Fig.9A A set of colorful representative IHC images indicating the expression of Ki67 protein, a cell marker for proliferation, which is present during all active phases of the cell cycle. Fig. 9B For quantitative comparison of Ki67 expression levels. Mice were administered the following treatments three times a week (except for controls, which were administered daily): saline (control), 2 mg / kg cisplatin (cisplatin), 100 mg / kg PTS (PTS), or 100 mg / kg PTS and 2 mg / kg cisplatin (cisplatin + PTS). Data are mean ± SD, n = 7 per group. * p<0.05 and *** p < 0.001, compared with control; ## p < 0.01, compared with cisplatin; ++ p<0.01, compared with PTS. Scale bar; 50 μm.

[0053] Fig.10 Bar graph showing quantitative PCR analysis of relative expression levels of ZEB1 mRNA in M5 canine melanoma tumors implanted in BALB / c nude mice, where ZEB1 acts as a transcriptional repressor that inhibits interleukin-2 (IL-2) gene expression and induces / accelerates / promotes cell invasion and migration. Mice were administered the following treatments three times per week (except for controls, which were administered daily): saline (control), 2 mg / kg cisplatin (cisplatin), 100 mg / kg PTS (PTS), or 100 mg / kg PTS and 2 mg / kg cisplatin (cisplatin + PTS). Data are mean ± SD, n = 7 per group. *** p < 0.001, compared with control; ## p < 0.01, compared with cisplatin; ++ p<0.01, compared with PTS.

[0054] Figures 11A to 11B IHC analysis of CD45 expression in M5 canine melanoma tumors implanted in BALB / c nude mice. Fig.11A A set of colorful representative IHC images indicating Ki67 expression. Fig. 11BFor quantitative comparison of Ki67 expression levels. Mice were administered the following treatments three times a week (except for controls, which were administered daily): saline (control), 2 mg / kg cisplatin (cisplatin), 100 mg / kg PTS (PTS), or 100 mg / kg PTS and 2 mg / kg cisplatin (cisplatin + PTS). Data are mean ± SD, n = 7 per group. * p<0.05 and ** p < 0.01, compared with control; # p < 0.05, compared with cisplatin; + p<0.05, compared with PTS. Fig.11A The scale bar is 50 μm.

[0055] Figures 12A to 12C In the first experiment, the effect of treatment with PTS combined with chemotherapy in lung squamous cell carcinoma in BALB / c nude mice was shown. Fig. 12A Shown are changes in tumor volume over time in four groups: control, PTS, gemcitabine + cisplatin (G+C), and gemcitabine + cisplatin + PTS (G+C+P). Fig. 12B Tumor weights in four groups are shown: control, PTS, GEM+CDDP, and GEM+CDDP+PTS. Fig. 12C The combination index (CI) graph is shown, and the combination values ​​are plotted as a function of Fa for a set of 3 drug combinations (G+C+P) in the first experiment.

[0056] Figures 13A to 13C Shown is the effect of treatment with PTS combined with chemotherapy in lung squamous cell carcinoma in BALB / c nude mice in a repeated (second) experiment. Fig.13A Shown are changes in tumor volume over time in four groups: control, PTS, gemcitabine + cisplatin (GEM + CDDP), and gemcitabine + cisplatin + PTS (GEM + CDDP + PTS). Fig. 13B Tumor weights in four groups are shown: control, PTS, GEM+CDDP, and GEM+CDDP+PTS. Fig. 13C The combination index (CI) graph is shown, and the combination values ​​are plotted as a function of Fa for a set of 3 drug combinations (G+C+P) in the first experiment.

[0057] Figures 14A to 14C The effect of treatment with PTS combined with chemotherapy in lung adenocarcinoma in BALB / c nude mice is shown. Fig.14A Shown are changes in tumor volume over time in four groups: control, PTS (330 mg / kg), cisplatin (2 mg / kg) + pemetrexed (100 mg / kg), and combination (PTS + cisplatin + pemetrexed). Fig. 14BShown are changes in tumor weight over time in control, PTS, CDDP+pemetrexed, and total (PTS+CDDP+pemetrexed). Fig. 14C The combination index (CI) graph is shown, and the combination values ​​are plotted as a function of Fa for a set of 3 drug combinations (PTS+cisplatin+pemetrexed). DETAILED DESCRIPTION

[0058] The following embodiments are provided to illustrate the present disclosure in detail. One of ordinary skill in the art can easily understand the advantages and effects of the present disclosure after reading the disclosure of this specification, and can also be implemented or applied in other different embodiments. Therefore, the following embodiments may be modified and / or changed to carry out the present disclosure without conflicting with its scope for different aspects and applications, and any element or method within the scope of the present disclosure disclosed herein may be used in conjunction with any other element or method disclosed in any embodiment of the present disclosure.

[0059] As used herein, the singular forms "a," "an," and "the" include plural referents unless expressly and unambiguously limited to one referent. The term "or" is used interchangeably with the term "and / or" unless the context clearly dictates otherwise.

[0060] As used herein, the term "tumor" refers to any malignant form of cancer caused by melanocytes. For example, melanoma may include, but is not limited to, mucosal melanoma, acral melanoma, ocular melanoma, cutaneous melanoma, superficial spreading melanoma, nodular melanoma, polypoid melanoma, lentigo malignant melanoma, desmoplastic melanoma, soft tissue melanoma, amelanotic melanoma, juvenile melanoma, Harding-Passey melanoma, subungual melanoma, Spitz nevus-like melanoma, blue nevus-like melanoma or any combination thereof, and melanoma may also include, but is not limited to, metastatic melanoma. Ultraviolet (UV) radiation status, epidemiology, histopathological features, genetics, prognosis and results may be different between the subtypes of these melanomas.

[0061] As used herein, the term "combination" means any combination of PTS with other chemotherapeutic agents, including, but not limited to, cisplatin (abbreviated as C or CDDP), gemcitabine (abbreviated as G or GEM), pemetrexed, or any type of alkylating agent (e.g., bendamustine, chlorambucil, cyclophosphamide, efamide, methyldichloroethylamine, melphalan, carmustine, lomustine, streptozotocin, busulfan, dacarbazine, temozolomide, hexamethylmelamine or thiotepa), or any type of antimetabolite (e.g., 6-mercaptopurine, fludarabine, 5-fluorouracil, gemcitabine, cytarabine, pemetrexed, methotrexate, or any type of corticosteroid.

[0062] The numerical ranges used herein are inclusive and combinable, and any numerical value falling within the numerical range herein will be taken as a maximum or minimum value to derive a subrange therefrom. For example, it should be understood that the numerical range "1% to 60%" includes any subrange between a minimum of 1% and a maximum of 60%, such as a subrange from 1% to 50%, a subrange from 10% to 60%, and a subrange from 20.5% to 40.5%.

[0063] As used herein, the term "about" when used in reference to a numerical value is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or ±0.1% from that numerical value. Such variations in numerical values ​​may occur, for example, due to experimental error; typical errors in measurements or processes used to prepare compounds, compositions, concentrates, or formulations; differences in the origin, manufacture, or purity of starting materials or ingredients used in the present disclosure; or similar considerations.

[0064] As used herein, "subject" may encompass any vertebrate, including, but not limited to, humans, mammals, reptiles, amphibians, and / or fish. However, advantageously, the subject is a mammal such as a human, or an animal mammal such as a domesticated mammal, e.g., a dog, cat, horse, etc., or a production mammal, e.g., a cow, sheep, pig, etc.

[0065] As used herein, the terms "comprises," "including," "having," "containing," and any other variations thereof are intended to cover a non-exclusive inclusion. For example, when describing an item "comprising" a limitation, unless otherwise specified, it may additionally include other ingredients, elements, components, structures, regions, parts, devices, systems, steps, or connections, etc., and other limitations should not be excluded.

[0066] As used herein, "melanoma" refers to any malignant form of cancer caused by melanocytes. For example, melanoma may include, but is not limited to, mucosal melanoma, acral melanoma, ocular melanoma, cutaneous melanoma, superficial spreading melanoma, nodular melanoma, polypoid melanoma, lentigo malignant melanoma, desmoplastic melanoma, soft tissue melanoma, amelanotic melanoma, juvenile melanoma, Harding-Passey melanoma, subungual melanoma, Spitz nevus melanoma, blue nevus melanoma or any combination thereof, and melanoma may also include, but is not limited to, metastatic melanoma. Ultraviolet (UV) radiation status, epidemiology, histopathological features, genetics, prognosis and outcome may be different between the subtypes of these melanomas.

[0067] Example

[0068] Exemplary embodiments of the present disclosure are further described in the following examples, which should not be construed as limiting the scope of the present disclosure.

[0069] Preparation Example

[0070] Pharmaceutical composition of benzenesulfonamide (PTS):

[0071]

[0072]

[0073] p-Toluenesulfonamide is an organic small molecule used as a local therapeutic drug injected directly into tumors, and has been shown to induce cancer cell death in hepatocellular carcinoma, non-small cell lung cancer, and tongue squamous cell carcinoma by initiating apoptosis and necrosis. The present disclosure also provides the use of PTS compositions as drugs for treating cancer.

[0074] The preparation of the PTS composition of the present disclosure includes the following process: adding and mixing a solvent and an adjuvant in a given ratio; heating the mixture to 80° C. to 110° C. under stirring to form a clear oily liquid; gradually adding the sulfonamide drug under stirring until it is completely dissolved; filtering and cooling the mixture to obtain the composition of the present disclosure in the form of an oily liquid.

[0075] The preparation of the PTS composition injection can be performed by some techniques known in the art, for example, adding an adjuvant and / or a solvent to adjust the mixture to an isotonic state, or filtering the mixture using a micro-mesh.

[0076] In the following experimental designs, drugs are used in combination to cause measurable tumor regression, each of these drugs may or may not exhibit different mechanisms of action when taken alone to minimize the development of resistance, the clinical toxicity of each compound does not overlap to allow their use at effective doses, and intensive intermittent treatment is superior to continuous low-dose therapy for enhancing tumor elimination.

[0077] Experimental Design I

[0078] Synergistic effect of PTS and cisplatin in the treatment of canine melanoma

[0079] Materials and Methods

[0080] Animals and cell lines

[0081] Six-week-old male BALB / cByJNarl mice were obtained from the Experimental Animal Breeding Research Center in Taipei, Taiwan. Two mice were housed in each cage and provided with sterile food and water. The mice were maintained at a constant temperature (22±2°C) and relative humidity (55±5%) with a 12:12 hour light-dark cycle. The Laboratory Animal Care and Use Committee (IACUC) of Chiayi University reviewed and approved the study protocol (IACUC approval number ll0003), in which the procedures were carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals" issued by the Taiwan Health and Welfare Affairs Department.

[0082] M5 canine melanocyte cell line was obtained from the College of Veterinary Medicine, National Taiwan University. Cells were cultured in 90% high glucose Dulbecco's modified Eagle's medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 5% fetal bovine serum, 50 IU / ml penicillin and 50 mg / ml streptomycin at 37°C. Cells were routinely passaged by removing the culture medium and covering the cell monolayer with 0.25% trypsin and 0.1% ethylenediaminetetraacetic acid.

[0083] Tumor vaccination and treatment

[0084] Experimental treatment began after the implanted M5 cells had formed detectable tumor masses, which were assessed on day 7 after transplantation. Briefly, mice were anesthetized via intraperitoneal injection of Zoletil (Virbac Taiwan, Taipei, China) and then 7100 μ l cell suspension of live M5 cells was subcutaneously injected into the hind leg. The development of tumor lesions was assessed on the 7th day, and animals showing different tumors of 4 to 5 mm in diameter were randomly divided into four groups (7 mice in each group). All treatments were administered via intraperitoneal injection. The control group received saline every day, and the three treatment groups received their corresponding treatment three times a week. The cisplatin group received 2 mg / kg cisplatin (Sigma; St.Louis, MO, USA). The PTS group was administered with 100 mg / kg PTS (Gongwin Biopharm Holding in Taipei, Taiwan, China). The cisplatin+PTS group received 100 mg / kg PTS and 2 mg / kg cisplatin. The dosage of cisplatin was selected so that the drug affected apoptosis, invasion, metastasis, angiogenesis and growth signal mechanisms in canine melanomas transplanted in mice. PTS doses are based on doses generally used to treat lung cancer, e.g., 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 400, 500, 600, 700, 800 mg / kg. Tumor growth was assessed every seven days by measuring the maximum and minimum diameters of the tumor and calculating its volume according to the following formula: V = 0.5 x a x b 2 , where a and b are the maximum and minimum diameters, respectively.

[0085] Clinical observation and histopathological analysis

[0086] The clinical signs of mice were observed every day, the mice were weighed every three days, and the mice were euthanized with an overdose of anesthetics and carbon dioxide one month after tumor cell transplantation. Blood of mice was collected under anesthesia for hematological assessment, and then the mice were sacrificed immediately. After euthanizing the mice, the tumors were immediately removed. The tumor specimens were divided into two groups. One group was treated with 10% formalin and the specimens were embedded in paraffin. These specimens were studied by the following: hematoxylin and eosin staining; terminal deoxyribonucleoside transferase (TdT)-mediated biotin-16-dUTP nick end labeling (TUNEL assay; APO-BrdUTUNEL assay kit, BD Pharmingen, San Diego, CA, USA); and IHC for IL-1β, TNF-α, TGF-β and CD44, using primary antibodies against these factors (Merck, Billerica, MA, USA). Protein expression was assessed via IHC using the TAlink mouse / rabbit polymer detection system produced by BioTnA in Kaohsiung, Taiwan, China. Images were captured and analyzed using a high-resolution digital microscope (Moticam 2300, Motic Instruments, Richmond, Canada) equipped with Motic Images Plus (version 2.0). Tumor specimens from other groups were first roughly chopped and homogenized, and then stored in a refrigerator at -80 °C. Western blotting was performed on the tumor specimens of these groups to analyze the expression of cleaved apoptotic protease 3, ERK, Bcl-2, COX-2, VEGF and EGFR.

[0087] Measurement of IL-6 serum levels

[0088] Serum levels of IL-6 in blood samples were measured using commercial mouse kits (ab213749 and ab100697; Abcam, Cambridge, MA, USA) according to the manufacturer's protocol. 50 μl of sample was added to each well in the 96-well plate coated with the antibody, and then incubated at room temperature for 2 hours. 50 μl of detection antibody solution was loaded into each well, and the plate was incubated at room temperature for another 1 hour. Afterwards, 50 μl of HRP-avidin solution (ab210901, Abcam) was added to each well, and the plate was incubated at room temperature for 1 hour again. Finally, 100 μl of tetramethylbenzidine substrate was added to each well, and the plate was incubated again at room temperature in the dark for 10 minutes. The reaction was terminated by adding 100 μl of stop solution. The absorption wavelength was 450 nm, and the results were expressed in pg / ml.

[0089] RNA extraction and real-time quantitative polymerase chain reaction (PCR)

[0090] TRI reagent (Sigma) was used to extract total RNA from tumor tissue. The concentration of RNA was quantified based on the absorption at 260 to 280 nm and 230 to 260 nm using a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). 1 μg of RNA was reverse transcribed into cDNA using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. Real-time PCR was performed using the cDNA and iTaq universal SYBR Green supermix (Bio-Rad) according to the manufacturer's protocol. The mRNA expression levels of NF-κB and IκBα were quantified using the CFX connected real-time PCR detection system (Bio-Rad). The following settings were used for the PCR: 40 cycles of: 30 s at 95°C; 15 s at 95°C and 30 s at 60°C; and finally 5 minutes at 72°C. We used the following primers for NF-κB, IκBα, and β-actin: NF-κB forward: 5′-ATGGCTTCTATGAGGCTGAG-3′, and reverse: 5′-GTTGTTGTTGGTCTGGATGC-3′; IκBα forward: 5′-GCCCTTGTCCCTGTCCCTA-3′, and reverse: 5′-GCAGAG-TATTTCCCTTTGGTTTGA-3′; and β-actin forward: 5′-ACTGGAAC-GGTGAAGGTGACA-3′, and reverse: 5′-ATGGCAAGGGACTTCCTGTAAC-3′. The expression levels of the two target genes were calculated relative to the β-actin level under investigation using 2-fold. ΔΔCt Methods represent them.

[0091] Western blotting

[0092] Western blot analysis was performed on the second group of tumor specimens according to standard protocols (Wu et al 2020). Antibodies to β-actin, Bcl-2, COX-2, VEGF, and EGFR (Sigma) and antibodies to cleaved apoptotic proteinase 3, phosphorylated ERK (threonine 202 / tyrosine 204), and ERK (Cell Signaling Technology, Beverly, MA, USA) were used. Immunoreactivity was assessed using enhanced chemiluminescence reagents (Thermo Scientific, Rockford, MA, USA), and signals were detected using UVP ChemStudio (Analytik Jena, Upland, CA, USA). Protein expression and phosphorylation were quantified using ImageJ (National Institutes of Health, Bethesda, MA, USA).

[0093] Statistical analysis

[0094] All data are expressed as mean ± SD. T-test was used to assess significant differences between multiple pairs of groups. When more than two groups were compared, analysis of variance (ANOVA) with post hoc Bonferroni correction was performed. P values ​​less than 0.05, 0.01, and 0.001 were considered significant, very significant, and extremely significant, respectively.

[0095] Limiting tumor growth through PTS

[0096] Treatment with cisplatin and PTS (either alone or in combination) resulted in smaller tumor size and volume relative to the control group ( Figure 1A Furthermore, time course analysis showed that tumor growth was significantly blocked by all three treatments relative to the control group (p<0.001) ( Figure 1B These effects were strongest when PTS was used with cisplatin: cisplatin alone reduced tumor mass by 48.7%, PTS alone reduced it by 44.2%, and cisplatin combined with PTS reduced tumor mass by at least 72.8% relative to control (Table 1). Thus, canine melanoma growth was strongly inhibited by treatment with both PTS and cisplatin.

[0097] Table 1. Tumor mass after resection from sacrificed BALB / cByJNarl mice

[0098]

[0099]

[0100] Data are mean ± SD. ***P < 0.001, compared with control;# p < 0.05, compared with cisplatin; + p<0.05, compared with PTS

[0101] The abundance of TUNEL-positive cells increases in response to PTS

[0102] In at least one embodiment of the present disclosure, an important aspect of the growth and survival of all organisms is apoptosis, that is, programmed cell death. In the late stages of apoptosis, the DNA of dying cells becomes substantially degraded, and cells with this degraded DNA can be detected using the TUNEL assay. The TUNEL assay showed that all three treatments resulted in a greater abundance of apoptotic cells: in the cisplatin group, TUNEL-positive counts were 3.9 times that of the control (p<0.01); in the PTS group, the ratio was 3.5 (p<0.01); and in the cisplatin+PTS group it was 8.6 (p<0.001; Figure 2A and 2B ). The difference between the PTS group and the cisplatin group was not significant, but the difference between the combination therapy group and the other two treatment groups was substantial and significant (p<0.001). Therefore, all three treatments enhanced apoptosis. Importantly, the combination therapy enhanced the anti-tumor activity of PTS in this model. This effect was most pronounced in the group that used PTS in addition to cisplatin, which was far more effective than either PTS or cisplatin alone.

[0103] Expression of apoptosis-related proteins is affected by PTS treatment

[0104] In the present disclosure, during the apoptotic process, specific enzymes are activated which dissolve the nuclear components of apoptotic cells, including protein components of both the nucleus and cytoplasm. To further assess the effect of treatment on apoptosis, Figure 3A As shown in , several proteins with relevant functions were analyzed: anti-apoptotic protein B-cell lymphoma 2 (Bcl-2), extracellular signal-regulated kinase (ERK), phosphorylated ERK, and cleaved apoptotic proteinase 3. According to Western blot analysis, Figure 3B and 3C Quantitative comparison of expression is shown, where cleaved apoptosis proteinase 3 and phosphorylated ERK expression in the cisplatin and PTS groups (all p<0.01) and the cisplatin + PTS group (both p<0.001) were significantly higher than the control group. The combined treatment group also had higher expression of these proteins than the two single treatment groups (for all except phosphorylated ERK in the cisplatin group, p<0.01; for phosphorylated ERK in the cisplatin group, p<0.05). Figure 3DAs shown in, the expression of Bcl-2, which is considered to suppress apoptosis, decreased in the single treatment group (p<0.05) and the combined treatment group (p<0.01) compared with the control group. There was no significant difference in Bcl-2 expression between the PTS group and the cisplatin group, but the expression in the cisplatin + PTS group was significantly lower than that in the two single treatment groups (p<0.05, compared with PTS; p<0.01, compared with cisplatin). Therefore, both PTS treatment and cisplatin + PTS treatment enhanced the expression of proteins associated with promoting apoptosis and reduced the expression of proteins that inhibit apoptosis, with the combined treatment showing the strongest effect.

[0105] Limiting cytokine production by PTS administration

[0106] In the present disclosure, there is a close relationship between inflammation and cancer, and anti-inflammatory can improve the efficacy of cancer prevention and therapy. One way the present disclosure achieves this is to suppress the expression of inflammatory cytokines. Immunohistochemistry (IHC) analysis was performed on the histopathology of tumors from mice, focusing on the inflammatory cytokine IL-1β ( Figure 4A ) and TNF-α( Figure 4B ). Compared with the control group, the activities of the two drugs were reduced by 29.7% and 77.4% in the cisplatin group, 26.5% and 73.0% in the PTS group, and 75.0% and 92.2% in the cisplatin + PTS group, respectively. Figure 4C and 4D ). There were no significant differences between the PTS and cisplatin groups for any cytokine, and the effect was also strongest in the combination treatment group. The reduction in the cisplatin + PTS group relative to the PTS group was 65.9% for IL-1β and 71.3% for TNF-α; and the reduction relative to the cisplatin group was 64.4% for IL-1β and 65.8% for TNF-α (all p < 0.01). This result shows that the combination treatment can strongly suppress the expression of these inflammatory cytokines in canine melanoma.

[0107] Enzyme-linked immunosorbent assay (ELISA) was performed to assess serum expression of IL-6, another inflammatory cytokine. A similar pattern to that of IL-1β and TNF-α was observed. Relative to the control group, the expression of IL-6 in the cisplatin group, the PTS group, and the cisplatin + PTS group was reduced by 62.8%, 57.5%, and 76.8%, respectively ( Figure 4E). The present disclosure shows that the difference between the cisplatin group and the PTS group was not significant, but the level in the combined treatment group was significantly lower than that of the two single treatment groups (37.6% and 45.4% lower, respectively; both p<0.01). These findings indicate that the combined administration of PTS and cisplatin strongly inhibits the expression of inflammatory cytokines IL-1β, TNF-α, and IL-6 in cancers not limited to non-small cell lung cancer, lung adenocarcinoma, and especially canine melanoma.

[0108] The expression of inflammation-related factors decreased in response to PTS administration

[0109] Western blot analysis was performed for factors associated with inflammation. The expression of cyclooxygenase-2 (COX-2) was lower in the cisplatin group and the PTS group (p<0.01) and the combination treatment group (p<0.001) than in the control group ( Figure 5A In at least one embodiment of the present disclosure, the difference between the cisplatin group and the PTS group was not significant, but the difference between the combination treatment group and the cisplatin group or the PTS group was significant (p<0.05; Figure 5B ).

[0110] In at least one embodiment of the present disclosure, when cells are stimulated with inflammatory cytokines, IκB kinase (IκBα) is degraded, resulting in inhibition of nuclear factor κB (NF-κB), which in turn accumulates and regulates the expression of specific genes. In at least one embodiment of the present disclosure, in the analysis of NF-κB and IκBα mRNA expression, such as Figure 5C As shown in Figure 2, NF-κB expression was found to be significantly lower in all three treatment groups (all p < 0.001) compared to the control group. As expected, the opposite was true for IκBα expression (p < 0.001 for all three experimental groups compared to the control; Figure 5D For both genes, there were no significant differences between the two single treatment groups, while each of them was significantly different between the combination treatment group and the cisplatin group or the PTS group (p<0.01; Figure 5C and 5D ). These findings indicate that PTS inhibits the expression and response of inflammation-related factors, although the effect is no different from that of cisplatin. When two anti-tumor agents (e.g., cisplatin + PTS) are combined, the inhibition of animal inflammatory responses is stronger than when the two are not combined.

[0111] Metastasis-related factors were reduced by PTS administration

[0112] In at least one embodiment of the present disclosure, the high morbidity and mortality associated with cancer is primarily due to metastasis. Therefore, the expression of TGF-β, CD44, EGFR, and VEGF analyzed in the present disclosure are all associated with metastasis. Fig. 6A and6C As shown in , TGF-β and CD44 expression were analyzed by IHC, while EGFR and VEGF were detected by Western blotting, as shown in Fig. 6E As shown in Figure 6B and 6D As shown in , the expression of both TGF-β and CD44 was decreased in the cisplatin and PTS groups (all p<0.01) and in the cisplatin+PTS group (both p<0.001) relative to the control group. Fig.6D As shown in , relative to the control group, the expression of TGF-β in the cisplatin group, the PTS group, and the cisplatin+PTS group was decreased by approximately 26.6%, 23.3%, and 62.6%, respectively. Figure 6B As shown in the results, CD44 expression decreased by approximately 40.9%, 34.4% and 61.6%, respectively. The difference between the cisplatin group and the PTS group was not significant, but there was a significant difference between the combined treatment group and the cisplatin group or the PTS group (all p < 0.01): relative to the cisplatin group and the PTS group, the expression in the cisplatin + PTS group decreased by 49.0% and 51.2% (TGF-β) and 35.1% and 41.5% (CD44), respectively.

[0113] like Fig. 6E As shown in the representative Western blot from the analysis of the relative expression of EGFR in FIG, the pattern for VEGF and EGFR was similar. Fig. 6F and 6G As shown in, the expression of the two factors was significantly lower in the single treatment group (all p < 0.05) and the combined treatment group (both p < 0.001) than in the control group. Although the relative levels of EGFR and VEGF were similar in the PTS group and the cisplatin group, the relative levels of EGFR and VEGF expression were significantly lower in the cisplatin + PTS combination group than in the other two treatment groups (all p < 0.01). This shows that PTS treatment can inhibit the expression of these factors related to metastasis, and combining PTS with cisplatin or adding cisplatin to PTS or adding PTS to cisplatin amplifies this effect.

[0114] In at least one embodiment of the present disclosure, the effects of treatment with PTS used alone and in combination with cisplatin on M5 canine melanoma tumors implanted in nude mice are studied. For example, mice have been used as an effective model to assess the efficacy of treatment for human cancer cells that have been xenografted ectopically. Tumor growth may be particularly advantageous in such mice because of the absence or defect of the thymus, which in turn leads to a complete or partial lack of T cells. Similar effects were observed when cisplatin was used to treat tumors, and just like PTS treatment, both cisplatin and PTS showed effects in reducing tumor size and weight. Even when the effects of PTS and cisplatin in treating tumors are similar, for example, in reducing tumor size and weight, when PTS and cisplatin are used in combination, for example, PTS is added to cisplatin or cisplatin is added to PTS as a combination therapy, they achieve a substantially stronger anti-tumor effect. In other words, in at least one embodiment of the present disclosure, the combination therapy promotes apoptosis by increasing the expression of cleaved apoptotic proteinase 3 and phosphorylated ERK and suppressing the expression of Bcl-2. Furthermore, the combination therapy reduces inflammation by limiting the production of IL-1β, TNF-α, IL-6, COX-2, and NF-κB. Finally, the combination therapy makes metastasis less likely by blocking TGF-β, CD44, VEGF, and EGFR.

[0115] In at least one embodiment of the present disclosure, the mTOR pathway is essential for regulating the cell cycle: the mTOR pathway is an important downstream signaling pathway that is activated by a variety of biological functions, and the mTOR pathway plays an important role in regulating autophagy. When the mTOR pathway is abnormally activated, it produces signals that promote tumor cell growth and metastasis and enable these cells to invade healthy tissues. In at least one embodiment of the present disclosure, AKT and mTOR and their downstream product p70S6K are present in canine melanoma cells and are active. In at least one embodiment of the present disclosure, activation of the mTOR pathway can be inhibited by rapamycin; treatment of melanoma cells with rapamycin reduces the survival of tumor cell fractions. In at least one embodiment of the present disclosure, inhibiting the mTOR pathway is an effective method for treating cancer. In at least one embodiment of the present disclosure, the proliferation of M5 canine melanoma is limited by PTS, which may be useful in clinical settings.

[0116] In at least one embodiment of the present disclosure, chemotherapy using a combination of an anticancer drug and a cell signal inhibitor is more effective than a single anticancer agent or antitumor agent. For example, two active ingredients or drugs of the present disclosure can be used in combination with other drugs for treating cancer, such as rapamycin and sorafenib (a multikinase inhibitor that inhibits Raf-1 and B-RAF), wherein the combination of the drug synergistically reduces melanoma cell proliferation, and the two types of signal transduction pathways can be used to treat melanoma more effectively than using either agent alone. Drug combinations in canine melanoma treatment are performed. The combination of MEK inhibitor trametinib and dual PI3K / mTOR inhibitor dactolisib synergistically reduces cell survival associated with apoptotic protease 3 / 7 activation, and changes the expression of cell cycle regulatory proteins and Bcl-2 family proteins. The inhibition and synergistic effect of combining cisplatin with PTS to treat mice transplanted with M5 canine melanoma cells were evaluated. As expected, although PTS alone showed an inhibitory effect, the combination strongly and synergistically inhibited tumor cell growth.

[0117] Factors related to apoptosis were examined. This is an important aspect of tumors because cancer cells are able to escape apoptosis and continue to replicate themselves despite abnormalities. In order to further explore chemotherapy methods that force tumor cells to undergo apoptosis by causing cell distress or DNA damage (which triggers cell death signals), TUNEL and DAPI assays were performed to study the extent of apoptosis that occurs in tumor cells in mice. As with tumor growth, PTS itself is beneficial in enhancing tumor cell apoptosis. However, in combination with cisplatin, treatment strongly promotes apoptosis. Therefore, the latter treatment reduces the ability of tumor cells to escape apoptosis, thereby limiting tumor growth. In mice administered with this combined treatment, the upregulation of cleaved apoptotic proteinase 3 and phosphorylated ERK (both of which are critical for apoptosis) was observed. Unexpectedly, anti-apoptotic Bcl-2 contributes to cancer formation and progression by promoting the survival of altered cells. In one embodiment according to the present disclosure, siRNA against the canine Bcl-2 gene reduced Bcl-2 mRNA expression and protein expression in a canine malignant oral melanoma cell line (MCM-N1), and resulted in both a decrease in the number of viable cells and an increase in the rate of apoptotic cells. These findings indicate an important role for Bcl-2 activity in inhibiting apoptosis in canine melanoma cells and reinforce the interest in Bcl-2 as a putative therapeutic target in tumors. In another embodiment according to the present disclosure, PTS triggers downregulation of S6K1 and mTOR ( Fig. 7A). In one embodiment, the relative content of phosphorylated mTOR / mTOR in the cisplatin group, the PTS group, and the cisplatin and PTS combination group is reduced to be lower than that in the control group. In another embodiment, the relative content of phosphorylated mTOR / mTOR in the cisplatin and PTS combination group is reduced to be lower than that in the cisplatin group alone or the PTS group or the control group. In one embodiment, the relative content of phosphorylated S6K1 / S6K1 in the cisplatin group, the PTS group, and the cisplatin and PTS combination group is reduced to be lower than that in the control group. ( Figure 7C ). In another embodiment, the relative content of phosphorylated S6K1 / S6K1 in the cisplatin combined with PTS group is reduced to a level lower than that of the cisplatin alone group or the PTS group or the control group. In at least one embodiment of the present disclosure, PTS must be able to repress the expression of mTOR, otherwise, the mTOR signaling pathway regulates autophagy and tumor cell apoptosis can promote tumor appearance and progression. Therefore, PTS alone and in combination with cisplatin caused a significant increase in the expression of cleaved apoptotic proteinase 3 and phosphorylated ERK relative to the control group. This may be caused by the inhibition of the mTOR pathway. As expected, the pattern of apoptosis was consistent with tumor size.

[0118] In another embodiment according to the present disclosure, PST treatment has an effect on various factors related to inflammation (IL-1β, TNF-α, IL-6, COX-2, NF-κB and IκBα). The administration of PTS reduces, partially or completely suppresses the expression of IL-1β, TNF-α, IL-6, COX-2 and NF-κB, as well as the expression of IκBα. Furthermore, CRP is mainly synthesized as a result of stimulation by proinflammatory cytokines, and a higher risk of lung cancer and tumor progression are associated with elevated CRP levels. Therefore, if Figure 8 As shown in , when PTS was used in combination with cisplatin, the CRP level in mouse serum was the lowest.

[0119] In at least one embodiment of the present disclosure, the combination therapy reduces inflammation by suppressing inflammatory cytokines and promoting anti-inflammatory mediators. This is another approach in which PTS contributes to the retardation of tumor growth. In the present invention, nuclear protein Ki67 can be a marker of proliferation at the site of inflammation and may be closely associated with tumor growth. Taking canine melanoma as an example, the relative abundance of Ki67-positive tumor cells is used as a prognostic factor. The Ki67 index can be used as a differential assessment, which is significant between malignant and benign melanocytic tumor formation in dogs and is negatively correlated with survival. Fig.9A and 9B As shown in, data have shown that PTS suppresses Ki67 expression when used alone or in combination with cisplatin. The present data have demonstrated that PTS can effectively inhibit tumor growth based on anti-apoptotic factors at least in canine melanoma cells by suppressing inflammation and tumor progression.

[0120] In at least one embodiment of the present disclosure, PTS can be used to treat cancer, including a method for reducing metastasis. In an embodiment of the present disclosure, the inventors evaluated metastasis in cancer prognosis. In one embodiment of the present disclosure, the proliferation and migration of severely transformed tumor cells are stimulated by TGF-β, resulting in metastasis and tumor progression. CD44 is a cell surface molecule that mediates cell adhesion and communication with the extracellular matrix. In the case of melanoma in the present disclosure, CD44 has been involved in cell migration and proliferation in vitro. Furthermore, CD44 is identified as a cancer stem cell marker in canine mammary tumors, and its expression is also increased in various types of cancers including but not limited to canine leukemia, melanoma and osteosarcoma. In the present disclosure, PTS, used alone or in combination with cisplatin, substantially suppresses TGF-β and CD44 expression, thereby reducing the metastatic potential of tumor cells. Suppression of TGF-β expression may also enhance the efficacy of drug therapy by assisting in inhibiting metastasis. Furthermore, VEGF is another factor in the present disclosure that strongly promotes angiogenesis, and its overexpression is associated with tumor progression and metastasis. Angiogenesis represents a fundamental step in the malignant growth and metastasis of tumors. Many pro-angiogenic and anti-angiogenic factors affect the formation of new blood vessels, but the core growth factor in this process is VEGF. In addition, in some malignant tumors, VEGF expression appears to be associated with tumor grade and prognosis. Overexpression of VEGF in human melanoma leads to a phenotype that has an increased malignant potential when compared to melanomas with low VEGF expression. EGFR has also shown potential as an important therapeutic target in human cancers because it may be involved in the progression of cutaneous melanoma, specifically and more generally acting as a signal transducer of a mitogen upstream of mTOR, which plays an important role in cancer pathogenicity and development. In addition, the expression of zinc finger E-box binding homeobox (ZEB1), a transcription factor that accelerates migration and invasion, indicates epithelial-mesenchymal transition (EMT) in canine melanoma cells. In at least one embodiment of the present disclosure, such as Fig.10 As shown in, data have shown that PTS, when used alone or in combination with cisplatin, has reduced the metastatic potential of M5 canine melanoma cells based on downregulation of ZEB1 mRNA. The present data have demonstrated that PTS can exert anti-tumor effects by repressing VEGF and EGFR expression, especially when administered in combination with cisplatin.

[0121] In at least one embodiment of the present disclosure, canine and human melanoma tumors are often resistant to chemotherapy and radiation. Currently, in the field of veterinary oncology, standard care for melanoma has not yet been established. In the present disclosure, treatment for dogs with melanoma may include surgery, with optional hypofractionated radiation therapy or definitive radiation therapy and platinum chemotherapy. Other treatment methods may include combining carboplatin, cisplatin and melphalan, but poor response rates and no improvement in survival time have been observed. This is especially true when chemotherapeutic drugs are tested as the sole treatment or as an adjuvant therapy after surgery or radiation. In embodiments of the present disclosure, the development and metastasis of tumors are closely related to the tumor microenvironment, which changes in structure and function. The tumor microenvironment can affect tumor progression, prognosis and the efficacy of immunotherapy, and it has been reported that tumor infiltrating lymphocytes are at the core of these effects. Preferred IHC staining is performed using CD45, a marker for these lymphocytes. As Fig.11A and 11B As shown in, the CD45 activity of the cisplatin group, PTS group, and cisplatin + PTS group was 1.4 times, 1.3 times, and 1.8 times higher than that of the control group, respectively. The data of the present invention have demonstrated that PTS can inhibit cancer, for example, canine melanoma. The inhibitory effect is strongest when PTS is used in combination with cisplatin. The combined therapy is determined to strongly inhibit the growth of M5 cells by promoting apoptosis, suppressing inflammation, and reducing the potential for metastasis.

[0122] Experimental Design II

[0123] Evaluation of adding PTS to gemcitabine and cisplatin versus gemcitabine and cisplatin in the treatment of squamous cell lung cancer

[0124] Materials and Methods

[0125] Male nude BALB / c mice aged six weeks were obtained from the Experimental Animal Breeding Research Center in Taipei, Taiwan. Mice were housed in cages and provided with sterile food and water. Mice were maintained at a constant temperature (22 ± 2 ° C) and relative humidity (55 ± 5%) with a 12:12 hour light-dark cycle. The Institutional Animal Care and Use Committee (IACUC) of Chiayi University (IACUC approval number ll0003) reviewed and approved the research protocol, wherein the procedure was carried out according to the "Guide to the Care and Use of Laboratory Animals" issued by the Taiwan Health and Welfare Affairs Department.

[0126] Tumor vaccination and treatment

[0127] Human non-small cell lung cancer cell line (H520) was purchased from ATCC Cell Bank. H520 cells were incubated in RPMI 1640 medium (Catalog No. 61870044; Gibco; Thermo Fisher, Inc) supplemented with 10% fetal bovine serum at 37°C in an environment containing 5% CO2. Experimental treatment began after the implanted H520 cells had formed detectable tumor masses, which were assessed on day 7 after transplantation. Briefly, mice were anesthetized via intraperitoneal injection of Zoletil (Virbac Taiwan, Taipei, China), and then 5*10 6 100 μl cell suspension of viable H520 cells was injected subcutaneously into the right hind leg. The development of tumor lesions was assessed on day 7 and will show an initial dose tumor volume of 100 mm 3 Animals were randomly divided into four groups (10 mice per group). All treatments were administered via intraperitoneal injection (IP) or intratumoral injection (IT). The control group (Group 1) received normal saline every day, while the other three treatment groups received BIW (twice a week) or QW (once a week) for their corresponding treatment. The PTS100 group (Group 2) received 330mg / kg of PTS100 (Gongwin Biopharm Holding, Taipei, Taiwan, China). The gemcitabine + cisplatin group (Group 3) received 160mg / kg of gemcitabine and 3mg / kg of cisplatin. Gemcitabine + cisplatin + PTS100 (Group 4) received 160mg / kg of gemcitabine, 3mg / kg of cisplatin and 300mg / kg of PTS100. In Group 4, when administered in combination, gemcitabine + cisplatin and PTS were given on different days, e.g., PTS was given on Mondays and Thursdays, while gemcitabine + cisplatin was given on Wednesdays. See Table 2 for the experimental design for the corresponding treatments, doses, injection sites, concentrations, volumes, number of individuals, and dosing frequency. The doses of cisplatin, gemcitabine, and PTS100 were selected so that these drugs affected apoptosis, invasion, metastasis, angiogenesis, and growth signaling mechanisms of canine melanoma implanted in mice. Tumor growth was assessed every seven days by measuring the maximum and minimum diameters of the tumor and calculating its volume according to the following formula: V = 0.5 × a × b2, where a and b are the maximum and minimum diameters, respectively.

[0128] Table 2

[0129]

[0130] a: Volume calculated based on 20g mouse BW, e.g. 20g x 10μL / g = 200μL b: BIW represents twice weekly dosing frequency

[0131] c: IT = intratumoral injection. The total volume was divided into 3 different injection sites.

[0132] d: PTS100 has a concentration of 330 mg / ml. For example, when the dose is 300 mg / kg, the volume is 20 g (mouse BW) x 1 μL / g = 20 μL

[0133] e: QW represents the frequency of administration once a week

[0134] f: When administered in combination, gemcitabine+cisplatin and PTS were given on different days.

[0135] Treatment with PTS as a single agent induced a slight reduction in tumor volume compared to controls, whereas the addition of gemcitabine + cisplatin to PTS resulted in a strong reduction in tumor burden, as shown in Figure 2. Fig. 12A Importantly, the addition of gemcitabine + cisplatin enhanced the antitumor activity of PTS in this model.

[0136] This effect was most pronounced in the group that received PTS first and then gemcitabine and cisplatin, such as on day 13 ( Fig. 12A In addition, the drug combination was more effective than single agent PTS in reducing tumor burden in large tumors. On day 13 of antitumor therapy, tumor volume in the control group increased significantly, while tumor volume in groups 2, 3, and 4 remained constant at low tumor volume. After about 32 days of treatment, the tumor volume of the control group exceeded 2500 cm 3 , while Groups 3 and 4 showed significantly enhanced antitumor activity. As shown in Table 3, tumor growth inhibition was 0% in the control group, 39.2% in the PTS group, 60.1% in the G+C group, and 80% in the G+C+P group. G+C+P showed significantly enhanced antitumor activity against squamous cell lung cancer compared to the control.

[0137] Table 3

[0138] Group inhibition Comparison 0.0％ PTS 39.2％ G+C 60.1％ G+C+P 80.0％

[0139] like Fig. 12B As seen in Table 4, the tumor weight of G+C+P was also the lowest compared to the tumor weight of the control. Table 4 shows references to the combination index and their descriptions, where the combination index method is based on those described by Chou and Talahay et al. (1984) and the computer software of Chou and Martin (2005). The ranges of CI were modified from those previously described by Chou (1991). In the present disclosure, CI<1, =1 and >1 indicate synergistic, additive and antagonistic effects, respectively. Fig. 12CAn effect fraction (Fa) of about 0.80 and a combination index (CI) of about 0.63 were highlighted, which respectively indicated the synergistic effect of such combination G+C+P and the G+C+P synergistic effect results capable of increasing the therapeutic effect against squamous cell lung cancer.

[0140] Table 4

[0141]

[0142] In repeated experiments, Fig.13A As shown in , mice receiving treatment with PTS as a single agent induced a slight reduction in tumor volume compared to controls, whereas the addition of gemcitabine + cisplatin to PTS resulted in a strong reduction in tumor burden, as shown in Fig. 13B Similar results were observed in Group 3 (G+C) and Group 4 (G+C+P), where the tumor volume in the control group increased significantly, while the tumor volume in Groups 2, 3, and 4 remained constant at a low tumor volume. Fig. 13C An effect fraction (Fa) of about 0.77 and a combination index (CI) of about 0.47 were highlighted, which respectively indicated a synergistic effect of such combination G+C+P and a synergistic effect result of G+C+P capable of increasing the therapeutic effect against lung squamous cell lung cancer.

[0143] The novel finding that adding gemcitabine + cisplatin to PTS reduced tumor growth rate in squamous cell lung cancer was unexpected.

[0144] Experimental Design III

[0145] Evaluation of combination therapy in the treatment of lung adenocarcinoma

[0146] Tumor vaccination and treatment

[0147] A549 cell line was purchased from ATCC Cell Bank and cultured in F-12K medium (Catalog No.: ATCC 30-2004) plus 10% fetal bovine serum at 37°C, 5% CO2. For experiments, cells were grown to 90% confluency. Experimental treatments were initiated after implanted A549 cells had formed detectable tumor masses, which were assessed on day 7 after transplantation. Briefly, mice were anesthetized via intraperitoneal injection of Virbac (Virbac Taiwan, Taipei, China) and then inoculated with 6*10 6 100 μl cell suspension of viable A549 cells was injected subcutaneously into the right hind leg. The development of tumor lesions was assessed on day 7 and will show an initial dose tumor volume of 100 mm 3Animals were randomly divided into four groups (10 mice per group). All treatments were administered via intraperitoneal injection (IP) or intratumoral injection (IT). The control group (Group 1) received normal saline every day, while the other three treatment groups BIW (twice a week) or QW (once a week) received their corresponding treatment. The PTS100 group (Group 2) received 330 mg / kg of PTS100 (Gongwin Biopharm Holding, Taipei, Taiwan, China). Normal saline + pemetrexed + cisplatin (Group 3) received 100 mg / kg of pemetrexed and 2 mg / kg of cisplatin. Pemetrexed + cisplatin + PTS100 (Group 4, also referred to as "combination") received 100 mg / kg of pemetrexed, 2 mg / kg of cisplatin and 330 mg / kg of PTS100. In Group 4, when administered in combination, pemetrexed, cisplatin, and PTS100 were given on different days, e.g., PTS was given on Mondays and Thursdays, while pemetrexed + cisplatin was given on Wednesdays. See Table 4 for the experimental design corresponding to treatment, dose, injection site, concentration, volume, number of individuals, and dosing frequency. The doses of pemetrexed, cisplatin, and PTS100 were selected so that these drugs affect apoptosis, invasion, metastasis, angiogenesis, and growth signaling mechanisms in canine melanoma implanted in mice. Tumor growth was assessed every seven days by measuring the maximum and minimum diameters of the tumor and calculating its volume according to the following formula: V = 0.5 × a × b 2 , where a and b are the maximum and minimum diameters, respectively.

[0148] Table 4

[0149]

[0150] a: Volume calculated based on 20g mouse BW, e.g. 20g x 10μL / g = 200μL b: BIW represents twice weekly dosing frequency

[0151] c: IT = intratumoral injection. The total volume was divided into 3 different injection sites.

[0152] d: PTS100 has a concentration of 330 mg / ml. For example, when the dose is 330 mg / kg, the volume is 20 g (mouse BW) x 1 μL / g = 20 μL

[0153] e: QW represents the frequency of administration once a week

[0154] f: When administered in combination, pemetrexed+cisplatin and PTS were given on different days.

[0155] Treatment with PTS as a single agent induced a slight reduction in tumor volume compared to control, whereas the addition of pemetrexed + cisplatin to PTS resulted in a strong reduction in tumor burden, as shown in Fig.14A Importantly, the addition of pemetrexed + cisplatin to PTS greatly enhanced the antitumor activity of PTS in this model, with tumor inhibition of 89.31%.

[0156] This effect was most pronounced in group 4, which received the combination (pemetrexed + cisplatin and PTS). Fig.14A ). In addition, Group 4 was more effective than Group 2 (in other words, this group contained cisplatin and pemetrexed without PTS) in reducing tumor burden in large tumors. After 35 days of antitumor therapy, the tumor volume in the control group increased significantly, while the tumor volume of Groups 2, 3, and 4 remained constant at a low tumor volume. In addition, after about 40 days of treatment, the tumor volume of the control group and Group 2 completely exceeded 1000 and 700 cm, respectively. 3 , while Groups 3 and 4 showed significantly enhanced antitumor activity. As shown in Table 5, tumor growth inhibition was 0% in the control group, 84.13% in the PTS group, 32.60% in the C+P group, and 95.20% in the G+C+P group. The group containing PTS100 showed significantly enhanced antitumor activity against squamous cell lung cancer compared to the control.

[0157] Table 5

[0158] Group inhibition(%) Comparison 0 PTS 84.13 Cisplatin + Pemetrexed (C+P) 32.60 PTS+cisplatin+pemetrexed (G+C+P) 95.20

[0159] like Fig. 14B As seen in Figure 3, the tumor weight of G+C+P was the lowest compared to that of the control. Moreover, the tumor weight of the PTS group also showed a significant reduction in tumor weight. In the present disclosure, CI<1, =1, and >1 indicate synergistic, additive, and antagonistic effects, respectively. Fig. 14C An effect score (Fa) of about 0.952 and a combination index (CI) of about 0.31 were highlighted, indicating the synergistic effect of these combinations in Group 4, and the G+C+P synergistic effect results capable of increasing the efficacy of the therapeutic effect against lung squamous cell lung adenocarcinoma, respectively.

[0160] The description of the above detailed embodiments is intended to illustrate the preferred embodiments according to the present disclosure, and is not intended to limit the scope of the present disclosure. Therefore, all modifications and changes completed by those skilled in the art should fall within the scope of the present disclosure defined by the appended claims.

Claims

1. A method for treating cancer, comprising administering to an individual in need thereof a therapeutically effective amount of a pharmaceutical composition and a chemotherapeutic agent, wherein: The pharmaceutical composition comprises a benzenesulfonamide derivative and a pharmaceutically acceptable carrier thereof, and the chemotherapeutic agent comprises at least one selected from the group consisting of an alkylating agent, an antimetabolite and a corticosteroid.

2. The method according to claim 1, wherein the benzenesulfonamide derivative is represented by the following formula (I): or a pharmaceutically acceptable salt thereof, in, R1 to R7 are independently selected from the group consisting of H, C1-C6 straight or branched alkyl, C1-C6 straight or branched alkoxy, C3-C6 cycloalkyl, C3-C6 cycloheteroalkyl, amino and halogen, or R6 and R7 are connected to each other to form a ring, and wherein the alkyl, alkoxy, cycloalkyl, cycloheteroalkyl and ring in R1 to R7 are independently unsubstituted or substituted with one or more substituents.

3. The method according to claim 2, wherein: The substituents are selected from the group consisting of phenyl, halo, oxo, ether, hydroxyl, carboxyl, amino, sulfo and sulfonamide groups.

4. The method according to claim 3, wherein: The benzenesulfonamide derivative or the pharmaceutically acceptable salt thereof is at least one selected from the group consisting of p-toluenesulfonamide, o-toluenesulfonamide, m-toluenesulfonamide, N-ethyl-o-toluenesulfonamide, N-ethyl-p-toluenesulfonamide, N-cyclohexyl-p-toluenesulfonamide, and 5. The method according to claim 1, wherein: The alkylating agent is cisplatin.

6. The method according to claim 1, wherein: The antimetabolite is gemcitabine or pemetrexed.

7. The method according to claim 1, wherein: The cancer is melanoma or lung cancer.

8. The method according to claim 7, wherein: The cancer is melanoma, and the chemotherapeutic agent comprises cisplatin.

9. The method according to claim 8, wherein: The ratio of the benzenesulfonamide derivative to the cisplatin is in the range of 25:1 to 2000:

1.

10. The method according to claim 9, wherein: The benzenesulfonamide derivative is administered to the individual at a dose of about 100 mg / kg, and the cisplatin is administered to the individual at a dose of about 2 mg / kg.

11. The method according to claim 7, wherein: The cancer is squamous cell carcinoma of the lung, and the chemotherapeutic agents include gemcitabine and cisplatin.

12. The method according to claim 11, wherein: The ratio of the benzenesulfonamide derivative to the gemcitabine is in the range of 1.5:1 to 100:1, and the ratio of the benzenesulfonamide derivative to the cisplatin is in the range of 25:1 to 2000:

1.

13. The method according to claim 12, wherein: The benzenesulfonamide derivative is administered to the individual at a dose of about 330 mg / kg, the gemcitabine is administered to the individual at a dose of about 160 mg / kg, and the cisplatin is administered to the individual at a dose of about 3 mg / kg.

14. The method according to claim 7, wherein: The cancer is lung adenocarcinoma, and the chemotherapeutic agents include pemetrexed and cisplatin.

15. The method according to claim 14, wherein: The ratio of the benzenesulfonamide derivative to the pemetrexed is in the range of 1.5:1 to 100:1, and the ratio of the benzenesulfonamide derivative to the cisplatin is in the range of 25:1 to 2000:

1.

16. The method according to claim 15, wherein: The benzenesulfonamide derivative is administered to the individual at a dose of about 330 mg / kg, the pemetrexed is administered to the individual at a dose of about 100 mg / kg, and the cisplatin is administered to the individual at a dose of about 2 mg / kg.

17. The method according to claim 1, wherein: The benzenesulfonamide derivative in the pharmaceutical composition is administered to the subject in an effective amount of about 3,300 mg to about 26,400 mg.

18. The method according to claim 1, wherein: The benzenesulfonamide derivative in the pharmaceutical composition is administered to the subject in an effective amount of about 165 mg to about 6,600 mg per day.

19. The method according to claim 1, wherein: The pharmaceutical composition or the chemotherapeutic agent is administered to the individual one to three times a week.

20. The method according to claim 19, wherein: The pharmaceutical composition is administered to the individual twice a week, and the chemotherapeutic agent is administered to the individual once a week.

21. The method according to claim 1, wherein: The pharmaceutically acceptable carrier is selected from the group consisting of fillers, binders, preservatives, disintegrants, lubricants, suspending agents, wetting agents, fragrances, thickeners, acids, biocompatible solvents, surfactants, complexing agents and any combination thereof.

22. The method according to claim 1, wherein: The pharmaceutically acceptable carrier is selected from the group consisting of polyethylene glycol, alkylene glycol, propylene glycol, sebacic acid, dimethyl sulfoxide, ethanol and any combination thereof.

23. The method according to claim 1, wherein: The pharmaceutical composition is in a form selected from the group consisting of injection preparations, dry powders, tablets, oral liquids, wafers, films, lozenges, capsules, granules, pills, gels, lotions, ointments, emulsions, pastes, creams, eye drops and ointments.

24. The method according to claim 1, wherein: The pharmaceutical composition or the chemotherapeutic agent is administered to the subject intratumorally, intravenously, subcutaneously, intradermally, orally, intrathecally, intraperitoneally, intranasally, intramuscularly, intrapulmonaryly, topically, or by aerosolization.

25. The method of claim 1, wherein: The individual is a human.

26. The method of claim 1, wherein: The subject is a dog, a cat or a mouse.

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