1,2,4-TRIOXANE COMPOUNDS AND COMPOSITIONS COMPRISING THEM FOR USE IN THE PREVENTION AND TREATMENT OF CANCER - Patent application

Abstract

The present invention relates to an anti-cancer agent comprising a 1,2,4-trioxane compound, and an anti-cancer agent comprising a combination comprising a 1,2,4-trioxane compound and chlorogenic acid. [Solution] The present invention further provides pharmaceutical compositions containing such anti-cancer agents, kits containing the same, and methods and treatment regimens using the anti-cancer agents, pharmaceutical compositions and kits in the treatment and prevention of cancer and for prolonging the survival of subjects with cancer, particularly ovarian cancer or lung cancer.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 033502, filed June 2, 2021, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to anticancer agents comprising 1,2,4-trioxane compounds, and combinations comprising 1,2,4-trioxane compounds and chlorogenic acid. The present invention also provides pharmaceutical compositions comprising such anticancer agents, kits comprising the same, and methods and treatment regimens using the anticancer agents, pharmaceutical compositions, and kits in the treatment and prevention of cancer, and for prolonging the survival of subjects with cancer, particularly ovarian cancer or lung cancer. [Background technology]

[0003] background Cancer remains a significant health threat despite enormous efforts in the search for treatments. For example, ovarian cancer poses a significant health problem for women worldwide and is the fourth leading cause of cancer deaths in the United States, accounting for 5–6% of all cancer-related mortality. The 5-year survival rate for early-stage patients is 80–90%, but only 40–50% for patients diagnosed at advanced stages of the disease. Unfortunately, most ovarian cancer patients present with advanced disease at the time of diagnosis. While ovarian cancer mortality rates have not changed significantly over the past few decades, patient survival has steadily improved, largely as a result of the clinical application of newer and more effective chemotherapeutic agents for adjuvant therapy after surgery. For example, paclitaxel and carboplatin are the most important chemotherapeutic agents used for the adjuvant treatment of primary ovarian cancer and metastatic disease. Paclitaxel blocks cells in the G2 / M phase of the cell cycle, preventing such cells from forming the normal mitotic apparatus, while carboplatin leads to the formation of DNA adducts, resulting in G2 cell cycle arrest and subsequent apoptosis. Although effective, paclitaxel and carboplatin induce side effects including myelosuppression, neurotoxicity, and nephrotoxicity. Furthermore, after completion of initial therapy, 70% of patients experience relapse within the first 2 years after diagnosis; therefore, alternative or improved treatments are urgently needed.

[0004] Human hepatocellular carcinoma (HCC) is also one of the leading causes of cancer-related deaths worldwide, with over 80% of liver cancer cases occurring in developing countries, such as China and Africa. HCC has a long latency period and is therefore often diagnosed at a late stage when tumors are highly malignant and rapidly progressive. These characteristics, combined with its high invasive potential, lead to a poor prognosis for patients diagnosed with the disease. Nonsurgical approaches are necessary because patients with large tumors or multiple lesions are typically not suitable for liver resection. Unfortunately, the activity of single chemotherapeutic agents is limited, and response rates are very low. Aggressive combination chemotherapy regimens have not led to significant improvement in response rates. In advanced HCC, cancer cells do not respond to the cytotoxic effects of most available chemotherapeutic agents. The same is true for some types of lung cancer, particularly non-small cell lung cancer (NSCLC). Components of the Kelch-like ECH-associated protein 1 (KEAP1) / nuclear factor erythroid 2-related factor 2 (NRF2) pathway regulate the cellular response to oxidative stress and are mutated in approximately 30% of non-small cell lung cancer (NSCLC) cases. Overexpression of wild-type, but not inactivating, KEAP1 in NSCLC cell lines results in reduced colony formation in soft agar, reduced cell migration, and reduced tumor growth in subcutaneous xenografts. Additionally, overexpression of wild-type KEAP1 reduces NRF2 protein expression and the expression of NRF2 transcriptional targets, including heme oxygenase-1 (HO-1) and NAD(P)H quinone dehydrogenase 1 (NQO-1). Transgenic mouse models also demonstrate that NRF2 activation or deletion of Keap1 inhibits Kras GI2DWe have demonstrated that mutations in the KEAP1 / NRF2 pathway promote lung tumorigenesis. Compared with individuals without mutations in this pathway, patients with mutations in the KEAP1 / NRF2 pathway have significantly shorter progression-free and overall survival, less benefit from epidermal growth factor receptor (EGFR) inhibitors, are insensitive to chemotherapy, and have increased metastasis. Inactivating KEAP1 mutations promote tumor growth and migration through activated NRF2-mediated transcription of antioxidant response genes, such as NQO1, and KEAP1 / NRF2 mutations are potential drivers of clinical treatment resistance to various drugs. Given the frequency and clinical significance of mutations in this pathway, effective treatment strategies for KEAP1 / NRF2-mutated cancers are needed.

[0005] Various other cancers are in a similar situation to HCC, lung cancer, such as non-small cell lung cancer (NSCLC), and ovarian cancer, i.e., although some progress has been made in treatment or prevention, more effective treatments remain needed. Thus, there remains a continuing need for new drugs that can be used alone or in combination with conventional agents.

[0006] Phytochemicals show promise in cancer therapy. Recently, several authors have demonstrated that the antimalarial drug artemisinin, its natural derivatives such as dihydroartemisinin, and semi-synthetic derivatives such as artesunate may have other potential uses in anticancer regimens ([1] Tsuda, K. et al. Mechanisms of the pH- and oxygen-dependent oxidation activities of artesunate. Biol Pharm Bull 41, pp. 555-563 (2018); [2] Wang, B., Hou, D., Liu, Q., Wu, T., Guo, H., Zhang, X., Zou, Y., Liu, Z., Liu, J., Wei, J., Gong Y., and Shao, C. Artesunate sensitizes ovarian cancer cells to cisplatin by downregulating RAD51. Cancer Biology & Therapy 16, pp. 1548-1556 (2015); [3] Chen, X., Wong, YK, Lim, TK, Lim, WH, Lin, Q., Wang, J. and Hua, Z. Artesunate activates the intrinsic apoptosis of HCT116 cells through the suppression of fatty acid synthesis and the NF-κB pathway. Molecules 22, 1272(2017); [4] Kumar, B., Kalvala, A., Chu, S., Rosen, S., Forman, SJ, Marcucci, G., Chen, CC and Pullarkat, V. Antileukemic activity and cellular effects of the antimalarial agent artesunate in acute myeloid leukemia. LeukRes 59, pp. 124-135 (2017); [5] Liu, Y., Gao, S., Zhu, J., Zheng, Y., Zhang, H and Sun H.Dihydroartemisinin induces apoptosis and inhibits proliferation, migration, and invasion in epithelial ovarian cancer via inhibition of the hedgehog signaling pathway. Cancer Med 7, 5704 - 5715 (2018); [6] Greenshields, A., Shepherd, T. and Hoskin, D. Contribution of reactive oxygen species to ovarian cancer cell growth arrest and killing by the anti-malarial drug artesunate. Molecular Carcinogenesis 56, 75 - 93 (2017), [7] Zhang, Z.Y.; Yu, S.Q.; Miao, L.Y.; Huang, X.Y.; Zhang, X.P.; Zhu, Y.P.; Xia, X.H.; Li, D.Q. Artesunate combined with vinorelbine plus cisplatin in treatment of advanced non-small cell lung cancer: A randomized controlled trial. Zhong Xi Yi Jie He Xue Bao 2008, 6, 134 - 138, doi:10.3736 / jcim20080206; [8] Srinivas, U.S.; Tan, B.W.Q.; Vellayappan, B.A.; Jeyasekharan, A.D. ROS and the DNA damage response in cancer. Redox Biol. 2019, 25, 101084, doi:10.1016 / j.redox.2018.101084; [9] Moloney, J.N.; Cotter, T.G. ROS signalling in the biology of cancer. Semin. Cell Dev. Biol.2018, 80, pp. 50 - 64, doi:10.1016 / j.semcdb.2017.05.023,

[10] Deeken, J.F.; Wang, H.; Hartley, M.; Cheema, A.K.; Smaglo, B.; Hwang, J.J.; He, A.R.; Weiner, L.M.; Marshall, J.L.; Giaccone, G. et al. A phase I study of intravenous artesunate in patients with advanced solid tumor malignancies. Cancer Chemother. Pharmacol. 2018, 81, pp. 587 - 596, doi:10.1007 / s00280-018-3533-8,

[11] Crespo-Ortiz, M. P. and Wei, M. Q. (2012). Antitumor activity of artemisinin and its derivatives: from a well-known antimalarial agent to a potential anticancer drug. J Biomed Biotechnol, 2012;

[12] Efferth, T. (2017). From ancient herb to modern drug: Artemisia annua and artemisinin for cancer therapy. Semin Cancer Biol, 46, pp. 65 - 83; Efferth, T., Sauerbrey, A., Olbrich, A., Gebhart, E., Rauch, P., Weber, H. O., Hengstler, J. G., Halatsch, M. E., Volm, M., Tew, K. D., Ross, D. D. and Funk, J. O. (2003). Molecular modes of action of artesunate in tumor cell lines. Mol Pharmacol, 64(2), pp. 382 - 394,

[13] Chen, H. H., Zhou, H. J., Wang, W. Q. and Wu, G. D. (2004).抗疟药双氢青蒿素也能抑制血管生成。《癌症化疗药理学》,第53卷第5期,第423 - 432页;

[14] Gao, P., Wang, L. L., Liu, J., Dong, F., Song, W., Liao, L., Wang, B., Zhang, W., Zhou, X., Xie, Q., Sun, R.及Liu, J. (2020). 双氢青蒿素通过抑制STAT3信号通路抑制内皮细胞管形成。《生命科学》,第242期;

[15] Konstat-Korzenny, E., Ascencio-Aragon, J. A., Niezen-Lugo, S.及Vazquez-Lopez, R. (2018). 青蒿素及其合成衍生物作为癌症的一种可能治疗方法。《医学科学(巴塞尔)》,第6卷第1期;

[16] Slezakova, S.及Ruda-Kucerova, J. (2017). 青蒿素及其衍生物的抗癌活性。《抗癌研究》,第37卷第11期,第5995 - 6003页。https: / / doi.org / 10.21873 / anticanres.12046;

[17] Tilaoui, M., Mouse, H. A., Jaafari, A.及Zyad, A. (2014). 青蒿素对癌细胞系的差异作用。《天然产物生物勘探》,第4卷第3期,第189 - 196页;

[18] Deeken, J. F., Wang, H., Hartley, M., Cheema, A. K., Smaglo, B., Hwang, J. J., He, A. R., Weiner, L. M., Marshall, J. L., Giaccone, G., Liu, S., Luecht, J., Spiegel, J. Y., and Pishvaian, M. J. (2018). A phase I study of intravenous artesunate in patients with advanced solid tumor malignancies. Cancer Chemother Pharmacol, 81(3), 587 - 596;

[19] Jiao, Y., Ge, C. M., Meng, Q. H., Cao, J. P., Tong, J., and Fan, S. J. (2007). Dihydroartemisinin is an inhibitor of ovarian cancer cell growth. Acta Pharmacol Sin, 28(7), 1045 - 1056;

[20] Sertel, S., Eichhorn, T., Simon, C. H., Plinkert, P. K., Johnson, S. W., and Efferth, T. (2010). Pharmacogenomic identification of c-Myc / Max-regulated genes associated with cytotoxicity of artesunate towards human colon, ovarian and lung cancer cell lines. Molecules, 15(4), 2886 - 2910;

[21] von Hagens, C., Walter-Sack, I., Goeckenjan, M., Storch-Hagenlocher, B., Sertel, S., Elsasser, M., Remppis, B. A., Munzinger, J., Edler, L., Efferth, T., Schneeweiss, A., and Strowitzki, T. (2019).在参与一项I期研究(ARTIC M33 / 2)后,对转移性乳腺癌患者进行口服青蒿琥酯的长期附加治疗(同情用药)。《植物医学》,第54卷,第140 - 148页;

[22] 柯尼希,M.、冯·哈根斯,C.、霍思,S.、鲍曼,I.、瓦尔特 - 萨克,I.、埃德勒,L.及塞尔特尔,S.(《研究青蒿琥酯作为转移性或局部晚期乳腺癌患者附加治疗的耳毒性:一项前瞻性、开放、非对照、单中心I期研究的新听力学结果》。《癌症化疗与药理学》,第77卷(2),第413 - 427页;

[23] 李,Q.、倪,W.、邓,Z.、刘,M.、佘,L.及谢,Q.(《青蒿琥酯通过抑制Akt / mTOR和诱导氧化应激靶向鼻咽癌》。《基础与临床药理学》,第31卷(3),第301 - 310页;

[24] 李,Y.、单, F.、吴,J.M.、吴,G.S.、丁,J.、肖,D.、杨,W.Y.、阿塔西,G.、莱昂斯,S.、凯尼亚德,D.H.及勒纳尔,P.(《靶向细胞周期G1期的新型抗肿瘤青蒿素衍生物》。Bioorg Med Chem Lett, 11(1), 5~8 ranges;

[25] Liu, L., Zuo, LF, Zuo, J. and Wang, J. (2015). Artesunate induces apoptosis and inhibits growth of Ecal09 and Ec9706 human esophageal cancer cell lines in vitro and in vivo. Mol Med Rep, 12(1), 1465~1472;

[26] Luo, J., Zhu, W., Tang, Y., Cao, H., Zhou, Y., Ji, R., Zhou, X., Lu, Z., Yang, H., Zhang, S., and Cao, J. (2014). The artemisinin derivative artesunate induces radiosensitivity in cervical cancer cells in vitro and in vivo. Radiat Oncol、9、84;

[27] Morrissey, C., Gallis, B., Solazzi, JW, Kim, BJ, Gulati, R., Vakar-Lopez, F., Goodlett, DR, Vessella, RL, and Sasaki, T. (2010). Effect of artemisinin derivatives on apoptosis and cell cycle in prostate cancer cells. Anticancer Drugs, 21(4), 423~432;

[28] Roh, JL, Kim, EH, Jang, H. and Shin, D. (2017). Nrf2 inhibition reverses the resistance of cisplatin-resistant head and neck cancer cells to artesunate-induced ferroptosis. Redox Biol, 11, 254~262;

[29] Sertel , S. , Eichhorn , T. , Sieber , S. , Sauer , A. , Weiss , J .Plinkert, PK, and Efferth, T. (2010). Factors determining sensitivity or resistance of tumor cell lines towards artesunate. Chem Biol Interact, 185(1), 42~52D;

[30] Zhang , J. , Sun , X. , Wang , L. , Wong , YK , Lee , YM , Zhou , C. , Wu , G. , Zhao , T. , Yang , L. , Lu , L. , Zhong , J. , Huang , D. and Wang , J . (2018). Artesunate-induced mitophagy alters cellular redox status. Redox Biol, 19, 263~273 range).

[0007]

[31] McDowell, A., Jr.; Hill , KS ; McCorkle , JR ; Gorski , J. ; Zhang, Y.; Salahuddin, A.; Ueland, F.; Kolesar, JM, Preclinical Evaluation of Artesunate as an Antineoplastic Agent in Ovarian Cancer Treatment, Diagnostics 2021, 11, 395

[32] Hill, KS; McDowell , A. ; McCorkle , R. ; Schuler , E. ; Ellingson , S. ; Plattner , R. ; Kolesar, JM “KEAP1 is Required for Artesunate Anticancer Activity in Non-Small-Cell Lung Cancer” Cancers 2021, 13, 1885.

[0008] Although many mechanisms have been proposed, including induction of apoptosis, inhibition of angiogenesis, inhibition of hypoxia-inducible factor-1α (HIF-1α) activation, and direct DNA damage, the generally postulated primary mode of action is the production of reactive oxygen species (ROS) in both the cytoplasm and mitochondria and mitochondrial-dependent apoptosis. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2004 / 078163 [Non-patent literature]

[0010] [Non-Patent Document 1] Tsuda, K. et al. Mechanisms of the pH- and oxygen-dependent oxidation activities of artesunate. Biol Pharm Bull 41, pp. 555-563 (2018) [Non-patent document 2] Wang, B., Hou, D., Liu, Q., Wu, T., Guo, H., Zhang, X., Zou, Y., Liu, Z., Liu, J., Wei, J, Gong Y. and Shao, C. Artesunate sensitizes ovarian cancer cells to cisplatin by downregulating RAD51. Cancer Biology & Therapy 16, pp. 1548-1556 (2015) [Non-patent document 3] Chen, X., Wong, Y.K., Lim, T.K., Lim, W.H., Lin, Q., Wang, J., & Hua, Z. Artesunate activates the intrinsic apoptosis of HCT116 cells through the suppression of fatty acid synthesis and the NF-κB pathway. Molecules 22, 1272 (2017)

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[0011] However, despite encouraging results, there remains a need to further understand the mechanisms of action to improve cancer treatment and prevention, particularly for cancers that still have high mortality rates, such as ovarian, liver, and lung cancer. [Means for solving the problem]

[0012] SUMMARY OF THE INVENTION The present invention provides at least one compound having at least one 1,2,4-trioxane moiety or a) at least one compound having at least one 1,2,4-trioxane moiety; and b) at least one chlorogenic acid The present invention relates to an anti-cancer agent comprising the combination of

[0013] The combinations exhibit more potent activity against cancer cells in vitro compared to compounds having at least one 1,2,4-trioxane moiety, such as artemisinin and its natural or synthetic derivatives alone. They also have a very good safety profile, thus offering the opportunity for widespread clinical use without severe side effects.

[0014] The present invention further provides pharmaceutical compositions comprising such anti-cancer agents, and methods of using said anti-cancer agents and pharmaceutical compositions to prevent or treat cancer, or to prolong the survival of a subject with cancer, including delaying or preventing the recurrence of cancer. Further objects of this invention are described herein below.

[0015] In another aspect, the present invention relates to a kit comprising an anti-cancer agent or pharmaceutical composition according to the present invention and at least one additional therapeutic agent. [Brief explanation of the drawings]

[0016] [Figure 1] Figure 1 shows artesunate sensitivity across three commercially available ovarian cancer cell lines (Caov-3, OVCAR-3, and UWB1.289) where cells were treated with serially diluted concentrations of artesunate for 72 hours. Percent viability was calculated from the proliferation of treated cells relative to vehicle-treated control cells using the CellTiter-Glo 2.0 Viability Assay (Promega). Graphs show the mean ± SD from three independent experiments, and IC50s were calculated using a variable slope nonlinear regression line. [Figure 2] DNA damage assay shown as mean nuclear pH2AX intensity. The mean nuclear intensity of pH2AX staining was quantified in Caov-3 cells treated for 48 hours with artesunate concentrations ranging from 5 to 100 μM, with 25 μM cisplatin treatment as a positive control. The mean signal ± SD was graphed and a one-tailed t-test was performed (*p=0.0486, **p=0.0034). [Figure 3] Figure 1 shows cell cycle analysis using propidium iodide staining. The percentage of Caov-3 and UWB1 cells in G1 after 24 h treatment with 0.1% DMSO (control) or 10 μM artesunate is graphed as the mean ± SD from three independent experiments. An unpaired one-tailed t-test revealed a statistically significant increase in cells in G1 in UWB1 cells treated with artesunate for 24 h (*p<0.05). [Figure 4]Figure 1 shows cell cycle analysis using propidium iodide staining. The percentage of Caov-3 and UWB1 cells in G1 after 48 h of treatment with 0.1% DMSO (control) or 10 μM artesunate is graphed as the mean ± SD from three independent experiments. An unpaired one-tailed t-test revealed a statistically significant increase in cells in G1 in UWB1 cells treated with artesunate for 48 h (*p<0.05) and Caov-3 cells treated with artesunate for 48 h (**p<0.01). [Figure 5] Figure 5 shows cell cycle analysis using propidium iodide staining. Simultaneous analysis of the percentage of cells in S phase after 24 hours (Figure 5) or 48 hours (Figure 6) of treatment with artesunate reveals a significant decrease in cells in S phase after 48 hours (*p<0.05; ***p<0.001). [Figure 6] Figure 5 shows cell cycle analysis using propidium iodide staining. Simultaneous analysis of the percentage of cells in S phase after 24 hours (Figure 5) or 48 hours (Figure 6) of treatment with artesunate reveals a significant decrease in cells in S phase after 48 hours (*p<0.05; ***p<0.001). [Figure 7]Figure 1 shows an anticancer drug administration sequence assay for artesunate, carboplatin, and paclitaxel. Cells were treated with artesunate on day 1 (D1A) or day 2 (D2A), carboplatin and paclitaxel on day 2 (D2C / T), carboplatin, paclitaxel, and artesunate on day 2 (D2C / T / A), or artesunate on day 1 followed by carboplatin and paclitaxel on day 2 (D1A;D2C / T). The 24-hour treatment concentrations of artesunate, carboplatin, and paclitaxel were 40 μM, 16 μM, and 32 μM, respectively. Using the CellTiter-Glo 2.0 viability assay, percent viability was calculated for Caov-3 cells after treatment with carboplatin / paclitaxel and / or artesunate, as shown, relative to DMSO (control)-treated cells, shown graphically as the mean ± SD. Statistical significance was assessed by one-way ANOVA (ns - not significant, and *p<0.05). In both cell lines, the addition of artesunate as a combination treatment with carboplatin / paclitaxel resulted in a significant decrease in viable cells. [Figure 8]Figure 1 shows an anticancer drug administration sequence assay for artesunate, carboplatin, and paclitaxel. Cells were treated with artesunate on day 1 (D1A) or day 2 (D2A), carboplatin and paclitaxel on day 2 (D2C / T), carboplatin, paclitaxel, and artesunate on day 2 (D2C / T / A), or artesunate on day 1 followed by carboplatin and paclitaxel on day 2 (D1A;D2C / T). The 24-hour treatment concentrations of artesunate, carboplatin, and paclitaxel were 40 μM, 16 μM, and 32 μM, respectively. Using the CellTiter-Glo 2.0 viability assay, percent viability was calculated for UWB1 cells after treatment with carboplatin / paclitaxel and / or artesunate, as shown, relative to DMSO (control)-treated cells, shown graphically as the mean ± SD. Statistical significance was assessed by one-way ANOVA (ns - not significant, and *p<0.05). In both cell lines, the addition of artesunate as a combination treatment with carboplatin / paclitaxel resulted in a significant decrease in viable cells. [Figure 9] Figure 1 shows artesunate sensitivity across a panel of non-small cell lung cancer (NSCLC) cell lines. Cells were treated with serially diluted concentrations of artesunate for 96 hours. Each cell line was normalized to cells treated with 0.1% DMSO (dimethyl sulfoxide) as a control. Each cell line is graphed as the mean ± SD, n=6. [Figure 10] Figure 1 shows artesunate sensitivity across a panel of non-small cell lung cancer (NSCLC) cell lines. The mean IC50 of artesunate (Art) in each cell line is graphed ± SD. p-values ​​were calculated using a two-tailed t-test (*p=0.0290 or **p=0.0007 compared to A549). [Figure 11]Figure 1 shows the dose-dependent induction of DNA damage by artesunate in NSCLC cell lines, as quantified by nuclear pH2AX staining. Graphs are presented as the mean fold change ± SD of nuclear fluorescence intensity signal normalized to 0.1% DMSO per cell. p values ​​are calculated by one-way ANOVA and Dunnett's multiple comparison test for each cell line compared to the corresponding 0.1% DMSO control (*p<0.05, ***p<0.001). [Figure 12] Figure 1 shows that artesunate treatment induces time-dependent changes in kelch-like ECH-associated protein 1 (KEAP1) / nuclear factor erythroid 2-related factor 2 (NRF2) pathway protein expression in A549 and H1299 NSCLC cell lines. Cells were treated with 10 μM artesunate for 0, 6, or 24 hours, and KEAP1 and NQO-1 (NAD(P)H quinone dehydrogenase 1) protein levels were assessed by Western blot. In both cell lines, KEAP1 protein levels are decreased after treatment with artesunate. [Figure 13] FIG. 1 shows Western blot analysis of KEAP1, NQO-1 and β-actin and the reduction of KEAP1 protein in siKEAP1-treated cells 48 hours after transfection. [Figure 14] Figure 1 shows artesunate dose-response curves after transfection of non-targeting (siNT - solid line) or KEAP1 (siKEAP1 - dashed line) siRNA. Data were normalized to 0.1% DMSO. Data are plotted as mean ± SD, n = 4. [Figure 15] 1 shows the mean IC50±SD of artesunate in each cell line transfected with non-targeting (siNT) siRNA (solid) or siKEAP1 (open). p-values ​​were calculated using a two-tailed t-test. [Figure 16]Figure 1 shows quantification of nuclear pH2AX staining in A549 cells after transfection with siNT or siKEAP1. DNA damage was assessed by nuclear pH2AX staining after 24 h of treatment with 0.1% DMSO (control), artesunate at the indicated concentrations, or 25 μM cisplatin. Nuclear pH2AX staining was normalized to cells transfected with siNT and treated with the DMSO control. Nuclear pH2AX staining is plotted as the mean fold change ± SD. p values ​​are calculated using a two-way ANOVA comparing each artesunate concentration to the corresponding control after normalization (*p<0.05; ***p<0.001). [Figure 17] Figure 1 shows quantification of nuclear pH2AX staining in H1299 cells after transfection with siNT or siKEAP1. DNA damage was assessed by nuclear pH2AX staining after 24 h of treatment with 0.1% DMSO (control), artesunate at the indicated concentrations, or 25 μM cisplatin. Nuclear pH2AX staining was normalized to cells transfected with siNT and treated with the DMSO control. Nuclear pH2AX staining is plotted as the mean fold change ± SD. p values ​​are calculated using a two-way ANOVA comparing each artesunate concentration to the corresponding control after normalization (*p<0.05; ***p<0.001). [Figure 18] Pharmacological inhibition of NRF2 sensitizes A549 cells to artesunate, whereby artesunate was added to medium containing 0.1% DMSO (solid line) or 5 μM ML385 (dashed line), and artesunate dose-response data were normalized to DMSO or 5 μM ML385 alone. Artesunate dose-response data are graphed as mean ± SD, n = 6. [Figure 19] Graph of the mean IC50±SD of artesunate in each cell line containing 0.1% DMSO (solid) or 5 μM ML385 (open). p-values ​​were calculated using a two-tailed t-test. [Figure 20]Figure 1 shows quantification of nuclear pH2AX staining in A549 cells pretreated with 0.03% DMSO or 5 μM ML385 for 24 hours, followed by the addition of the indicated concentrations of artesunate for another 24 hours. Graphs are presented as the mean fold change ± SD of nuclear fluorescence intensity signal normalized to 0.03% pretreated vehicle control cells. p-values ​​are calculated using one-way ANOVA and Dunnett's multiple comparison test comparing each artesunate concentration to the matched control after normalization (**p<0.01; ***p<0.001). [Figure 21] Figure 1 shows quantification of nuclear pH2AX staining in H1299 cells pretreated with 0.03% DMSO or 5 μM ML385 for 24 hours, followed by the addition of the indicated concentrations of artesunate for another 24 hours. Graphs are presented as the mean fold change ± SD of nuclear fluorescence intensity signal normalized to 0.03% pretreated vehicle control cells. p-values ​​are calculated using one-way ANOVA and Dunnett's multiple comparison test comparing each artesunate concentration to the matched control after normalization (**p<0.01; ***p<0.001). [Figure 22] 1 is a graphical representation of the Bliss-independent model of synergy scoring as calculated by using a 6x6 dose-response matrix in A549 cells. Red indicates synergy between the drug combinations tested, while green indicates antagonism. [Figure 23] 1 is a graphical representation of the Bliss-independent model of synergy scoring as calculated by using a 6x6 dose-response matrix in H1299 cells. Red indicates synergy between the drug combinations tested, while green indicates antagonism. DETAILED DESCRIPTION OF THE INVENTION

[0017] Detailed Description For the purposes of interpreting this specification, the following definitions shall apply and, whenever appropriate, terms used in the singular shall also include the plural.

[0018] As used herein, terms have the following meanings unless the context clearly indicates otherwise.

[0019] As used herein, the term "cancer" refers to a group of diseases involving abnormal cell growth that has the potential to invade or spread to other parts of the body, and includes melanoma; multiple myeloma; carcinomas, such as adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, transitional cell carcinoma, e.g., of the breast, colon, and prostate; sarcomas, such as osteosarcoma, leiomyosarcoma, Kaposi's sarcoma, malignant fibrous histiocytoma, liposarcoma, and dermatofibrosarcoma protuberans; leukemias, such as acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and hairy cell leukemia; lymphomas, such as Hodgkin's lymphoma and non-Hodgkin's lymphoma; brain and spinal cord tumors, germ cell tumors, neuroendocrine tumors, and carcinoid tumors.

[0020] The aforementioned cancer types include the following cancer types that are also referenced in the literature: colorectal cancer, breast cancer, lung cancer, ovarian cancer, cervical cancer, bladder cancer, prostate cancer, stomach cancer, liver cancer, pancreatic cancer and thyroid cancer.

[0021] Preferred cancers to be treated with the compositions according to the present invention include lung cancer, particularly non-small cell lung cancer (NSCLC), ovarian cancer and liver cancer.

[0022] As used herein, the term "subject" refers to an animal. In certain aspects, an animal is a mammal. A subject also refers to, for example, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, poultry, rabbit, rat, mouse, fish, bird, etc. In certain embodiments, a subject is a human. A "patient," as used herein, refers to a human subject.

[0023] As used herein, the term "inhibition" or "inhibiting" refers to the reduction or suppression of a given condition, symptom, or disorder, or disease, particularly cancer, or a significant decrease in the baseline activity of a biological activity or process.

[0024] As used herein, the term "prolonging survival" means extending the lifespan of a subject with cancer by at least one day compared to a subject with the same cancer who does not receive the anticancer agent, pharmaceutical composition or kit according to the present invention.

[0025] Prolonged survival includes increasing a subject's lifespan by at least: 1 week, 2 weeks, 3 weeks, 4 weeks or more, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more, or 1 year, 2 years, 3 years, 4 years, 5 years or more.

[0026] As used herein, the term "treating" cancer or "treatment" of cancer refers, in one embodiment, to causing cancer remission, i.e., slowing, arresting, or reducing the onset of cancer, at least one of its clinical symptoms. In another embodiment, "treating" or "treatment" refers to reducing or ameliorating at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating cancer either physically, e.g., stabilizing discernible symptoms, physiologically, e.g., stabilizing physical parameters, or both. In yet another embodiment, "treating" or "treatment" refers to preventing or delaying the onset, development, progression, or recurrence of cancer.

[0027] As used herein, the terms "administered," "administration," "co-administered," and "co-administration" refer to administering to a subject a combination of compounds contemplated herein, and optionally, together with at least one additional compound that can also treat cancer as described herein.

[0028] In one embodiment, the compounds are administered separately as part of a single therapeutic approach, in any particular order. In a preferred embodiment, the compounds of the combination according to the invention are co-administered in a joint formulation, e.g., as a pharmaceutical composition according to the invention.

[0029] As used herein, in the context of the present invention, and particularly in the context of the claims, the terms "a," "an," "the," and similar terms are to be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0030] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all examples provided herein, or the use of illustrative language, such as "for example," are intended merely to better illustrate the invention and do not pose limitations on the scope of the invention as otherwise claimed.

[0031] "Optionally substituted" or "substituted" means that, at any position in the referenced molecule or moiety, one or more hydrogen atoms may be replaced with any one or any combination of substituents, the number, arrangement and selection of which is understood to encompass only those substitutions that a skilled chemist would expect to be reasonably stable.

[0032] Various embodiments of the present invention are described herein, and it will be recognized that the features specified in each embodiment may be combined with other specified features to provide further embodiments.

[0033] The combinations according to the present invention comprise at least one compound comprising at least one 1,2,4-trioxane moiety, which compound comprises at least one 1,2,4-trioxane ring that is optionally, but preferably, substituted.

[0034] In one embodiment, the compound comprising at least one 1,2,4-trioxane moiety is represented by formulas (I) to (V): [ka] and, where applicable, pharmaceutically acceptable salts of said compounds of formula (I) and (II).

[0035] In formula (I), The arrow indicates residue R 1 shows the bond between the illustrated oxygen atoms to n is an integer greater than 1, preferably from 2 to 10, more preferably 2, 3 or 4, even more preferably 2 or 3; R 1 is a residue substituted n times by the residue shown in brackets, preferably C1 to C 18 -Alkyl or C2-C 18 -alkenyl or -(CO) n (R 3 ), where the carboyl group is the residue R 1 together with the oxygen bonded to R to form a carboxylic acid ester moiety, 3 is C1~C 18 Alkan-n-yl or C2-C 18 alkene-n-yl, Above C1~C 18 -Alkyl, C2-C 18 -Alkenyl, C1-C 18 -Alkan-n-yl, C2-C 18 -alkene-n-yl group uninterrupted, or -0-, -S-, -SO2-, -SO-, -SO2NR 4 -, NR 4 S02-, -NR 4 -, -CO-, -O(CO)-, (CO)O-, -0(C0)0-, -NR 4 (CO)NR 4 -, NR 4 (CO)-, -(CO)NR 4 -, -NR4 (C0)0-, -0(C0)NR 4 - and is interrupted one, two or more times by non-consecutive functional groups selected from the group consisting of: uninterrupted or additionally or alternatively interrupted once, twice or more than twice by a divalent residue selected from the group consisting of heterocyclo-diyl and aryldiyl, not replacing, or in addition to or instead of, Hydroxy, halogen, cyano, azide, C6-C 14 -aryl, C1-C8-alkoxy, C1-C8-alkylthio, -SO3M, -COOM, PO3M2, -PO(N(R 5 )2)2, P0(0R 5 )2, -S02N(R 4 )2, -N(R 4 )2, -C02N(R 5 )2, -COR 4 , -OCOR 4 , -NR 4 (CO)R 5 , -(CO)OR 4 , -NR 4 (CO)N(R 4 )2 and is substituted once, twice or more than twice with a substituent selected from the group consisting of:

[0036] In formula (II), R 2 is C1~C 18 -Alkyl or C2-C 18 -alkenyl or -(CO)R 3 where the carboyl group is the residue R 1 together with the oxygen bonded to R to form a carboxylic acid ester moiety. 3 is C1~C 18 -Alkyl or C2-C 18 alkenyl, whereby Above C1~C 18 -Alkyl and C2-C 18 The alkenyl group is uninterrupted, or -0-, -S-, -SO2-, -SO-, -SO2NR 4 -, NR 4 S02-, -NR 4 -, -CO-, -O(CO)-, (CO)O-, -0(C0)0-, -NR 4 (CO)NR 4 -, NR 4 (CO)-, -(CO)NR 4 -, -NR 4 (CO)0- or -0(CO)NR 4 - and is interrupted one, two or more times by non-consecutive functional groups selected from the group consisting of: uninterrupted or additionally or alternatively interrupted once, twice or more than twice by a divalent residue selected from the group consisting of heterocyclo-diyl and aryldiyl, not replacing, or in addition to or instead of, Hydroxy, halogen, cyano, azide, C6-C 14 -aryl, C1-C8-alkoxy, C1-C8-alkylthio, -SO3M, -COOM, PO3M2, -PO(N(R 5 )2)2, PO(OR 5 )2, -S02N(R 4 )2, -N(R 4 )2, -C02N(R 5 )2, -COR 4 , -OCOR 4 , -NR 4 (CO)R 5 , -(CO)OR 4 or -NR 4 (CO)N(R 4 )2 and is substituted once, twice or more than twice with a substituent selected from the group consisting of In all the above formulas, when used: R 4 is hydrogen, C1-C8 alkyl, C6-C 14 -independently selected from the group consisting of aryl and heterocyclyl, or N(R4 )2 as a whole is an N-containing heterocycle, R 5 is C1-C8-alkyl, C6-C 14 -independently selected from the group consisting of aryl and heterocyclyl, or N(R 5 )2 as a whole is an N-containing heterocycle, M is hydrogen or a q-valent metal ion equivalent to 1 / q, or an ammonium ion or a guanidinium ion, or a primary, secondary, tertiary or quaternary organic ammonium ion, in particular a cation of the formula [N(C1-C 18 -alkyl) s H t ] + where s is 1, 2, 3, or 4, and t is (4-s).

[0037] As used herein, and unless otherwise specifically stated, C1-C 18 -Alkyl, C1-C 18 -Alken-n-yl, C1-C8-alkyl, C1-C8-alkoxy and C1-C8-alkylthio are straight-chain or C3-C 18 or for C3-C8, including, in part or in whole, branched or unbranched alkyl, alkoxy and alkylthio substituents of the supercyclic group, having the given number of carbon atoms in such substituents.

[0038] As used herein, and unless otherwise specifically stated, C2-C 18 -Alkenyl is straight chain or C5-C 18 For example, the group ##STR00002## includes, in part or in whole, branched or unbranched alkenyl of that supercyclic group, having the given number of carbon atoms in such substituent.

[0039] As used herein, and unless otherwise specifically stated, C6-C 14 -Aryl, C6-C 14 -aryloxy and C6-C 14-arylthio is a carbocyclic aromatic substituent having 6 to 14 carbon atoms within such an aromatic system, i.e., without any carbon atoms of the substituent, preferably phenyl (C6), naphthyl (C 10 ), phenanthrenyl and anthracenyl (each C 14 ), whereby said carbocyclic aromatic substituents are unsubstituted or substituted with up to five identical or different substituents per ring. For example and preferentially, the substituents are fluoro, chloro, C1-C 18 -Alkyl, C1-C 18 -Alkoxy, C6-C 14 -aryl.

[0040] In a more preferred embodiment, the carbocyclic aromatic substituent is unsubstituted.

[0041] C1~C 18 Specific examples of -alkyl are methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, tert-pentyl, neopentyl, cyclohexyl, n-hexyl, n-heptyl, n-octyl and isooctyl, n-decyl, n-dodecyl, n-hexadecyl, n-octadecyl.

[0042] Illustrative examples of C1-C8 alkoxy-substituents are methoxy, ethoxy, isopropoxy, n-propoxy, n-butoxy, sec-butoxy, tert-butoxy and cyclohexyloxy.

[0043] Illustrative examples of C1-C8 alkylthio-substituents are methylthio and ethylthio.

[0044] C6~C 14 Specific examples of -aryl are phenyl, o-, m- and p-tolyl.

[0045] C6~C 14 A further example of an -aryl-substituent is phenoxy.

[0046] C6~C 14 A further example of an -aryl-substituent is phenylthio.

[0047] Preferred compounds of formula (II) are those of formula (IIa), artemether, and those of formula (IIb), artesunate, and pharmaceutically acceptable salts of artesunate.

[0048] [ka]

[0049] The compound of formula (III) is dihydroartemisin.

[0050] The compound of formula (IV) is artemisinin.

[0051] The compound of formula (V) is artemisitene.

[0052] In one embodiment of the present invention, the anticancer agent according to the present invention contains more than one compound comprising at least one 1,2,4-trioxane moiety, and preferably more than one compound selected from those of formulas (I) to (V) above, and, where applicable, pharmaceutically acceptable salts of such compounds.

[0053] In one preferred embodiment, the combination according to the invention comprises at least two, such as two, three, four or all, compounds selected from formulae (IIa), (IIb), (III), (IV) and (V).

[0054] Naturally occurring compounds comprising at least one 1,2,4-trioxane moiety and combinations of such compounds can be used for the purposes of this invention by using the plant Artemisia annua or parts thereof as such, or can be obtained from Artemisia annua via known extraction methods and, if desired, standard work-up methods, for example as published in Triemer et al., Angewandte Chemie, International Edition 57, (2018), pp. 5525-5528.

[0055] When Artemisia annua is extracted, this can occur using the whole plant or parts thereof, such as leaves or stems, whether dried or freshly harvested. Suitable solvents for extraction include hexane, cyclohexane, supercritical carbon dioxide, hydrofluorocarbon HFC-134a, ionic liquids, water, methanol, ethanol, 1-butanol, acetone, cyclohexanone, toluene, ethyl acetate, acetonitrile, tetrahydrofuran, or mixtures thereof.

[0056] For example, if it is desired to use a single compound comprising at least one 1,2,4-trioxane moiety of the present invention in the combination, the extract of Artemisia annua can be separated in a manner known per se to obtain the individual compounds, for example by partitioning between a multiphasic solvent mixture, recrystallization and / or chromatographic separation, for example on silica gel, or by medium pressure liquid chromatography, for example on a reversed-phase column, or by fractional crystallization.

[0057] In one embodiment, the Artemisia annua plant is the Apollon variety, commercially available from Mediplant, Conthey, Switzerland. See X. Simmonet et al., "Apollon, a new Artemisia annua variety with high artemisinin content," Planta Medica, 2011, 77(12).

[0058] Individual compounds containing at least one 1,2,4-trioxane moiety can be worked up and / or purified according to standard methods, for example using chromatographic methods, distribution methods, (re)crystallization, etc.

[0059] Synthetic or semi-synthetic compounds comprising at least one 1,2,4-trioxane moiety are prepared, for example, by preparation methods known to those skilled in the art, some of which are described, for example, in Reiter, C., Frdhlich, T., Gruber, F., Hutterer, C., Marschall, M., Voigtlander, C., Friedrich, O., Kappes, B., Efferth, T., Tsogoeva, S.B., 2015a, Highly potent artemisinin-derived diers and trimers: Synthesis and evaluation of their antimalarial, antileukemia and antiviral activities. Bioorg. Med. Chem. 23 (17), pp. 5452-5458; Reiter, C., Frdhlich, T., Zeino, M., Marschall, M., Bahsi, H., Leidenberger, M., Friedrich, O., Kappes, B., Hampel, F., Efferth, T., Tsogoeva, SB, 2015b, New efficient artemisinin derived agents against human leukemia cells, human cytomegalovirus and Plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids. Eur. J. Med. Chem. 97, pp. 164-172; Posner, GH, Ploypradith, P., Parker, MH, O'Dowd, H., Woo, SH, Northrop, J., Krasavin, M., Dolan, P., Kensler, TW, Xie, S., Shapiro, TA, 1999, Antimalarial, antiproliferative, and antitumor activities of artemisinin-derived, chemically robust, trioxane dimers, J. Med.Chem. 42 (21), pp. 4275-4280; Paik, I.H., Xie, S., Shapiro, T.A., Eabonte, T., Narducci Sarjeant, A.A., Baege, A.C., Posner, G.H., 2006, Second generation, orally active, antimalarial, artemisinin-derived trioxane dimers with high stability, efficacy, and anticancer activity, J. Med. Chem. 49 (9), pp. 2731-2734; Ei, Y., Zhu, Y.M., Jiang, H.J., Pan, J.P., Wu, G.S., Wu, J.M., Shi, Y.L., Yang, J.D., Wu, B.A., 2000, Synthesis and antimalarial activity of artemisinin derivatives containing an amino group, J. Med. Chem. 43 (8), pp. 1635-1640; Ren, Y., Yu, J., Kinghorn, A.D., 2016, Development of anticancer agents from plant-derived sesquiterpene lactones, Curr. Med. Chem. 23 (23), pp. 2397-2420; O'Neill, P.M., Searle, N.L., Kan, K.W., Storr, R.C., Maggs, J.L., Ward, S.A., Raynes, K., Park, B.K., 1999, Novel, potent, semisynthetic antimalarial carba analogues of the first-generation 1,2,4-trioxane artemether, J. Med. Chem. 42 (26), pp. 5487-5493, which are hereby incorporated by reference.

[0060] The anti-cancer agent according to the present invention may comprise a combination of at least one compound having at least one 1,2,4-trioxane moiety and at least one chlorogenic acid.

[0061] As used herein, the term "chlorogenic acid" or "chlorogenic acids" refers to compounds in which one or two hydroxyl groups of quinic acid are esterified with caffeic acid, ferulic acid, or p-coumaric acid.

[0062] Preferred examples of chlorogenic acids include 3-O-caffeoylquinic acid (formula VIa), 4-O-caffeoylquinic acid (formula VIb), 5-O-caffeoylquinic acid (formula VIc), 3-O-feloylquinic acid (formula VId), 4-O-feloylquinic acid (formula VIe), 5-O-feloylquinic acid (formula VIf), 3,4-dicaffeoylquinic acid (formula VIIa), 3,5-dicaffeoylquinic acid (formula V), Particularly preferred are 3-O-caffeoylquinic acid (formula VIa), 4-O-caffeoylquinic acid (formula VIb), 5-O-caffeoylquinic acid (formula VIc), 3,4-dicaffeoylquinic acid (formula VIIa), 3,5-dicaffeoylquinic acid (formula VIIb) and 4,5-dicaffeoylquinic acid (formula VIIc).

[0063] [ka]

[0064] In one embodiment, the molar ratio between the one or more compounds comprising at least one 1,2,4-trioxane moiety and the one or more chlorogenic acids present in the combination according to the invention is, for example, between 2 and 0.002, preferably between 0.8 and 0.005, more preferably between 0.5 and 0.01, and even more preferably between 0.1 and 0.01.

[0065] The chlorogenic acids can be used in their isolated form or as components of the whole plant, plant parts or extracts of the aforementioned. In the case of Artemisia annua, the chlorogenic acids can be separated in a manner known per se to obtain the individual compounds, for example by partitioning between multiphasic solvent mixtures, recrystallization and / or chromatographic separation, for example on silica gel, or by medium pressure liquid chromatography, for example on a reversed-phase column, or by fractional crystallization.

[0066] The individual chlorogenic acids can be worked up and / or purified according to standard methods, for example using chromatographic methods, distribution methods, (re)crystallization, etc.

[0067] Due to its high content of chlorogenic acid, coffee, especially roasted coffee, can be used as a valuable source of chlorogenic acid.

[0068] In one embodiment, the combination according to the invention can be obtained by co-extracting coffee, in particular roasted coffee, with Artemisia annua, in particular dried Artemisia annua leaves, for example with water, preferably at a temperature of 40° C. to 100° C., more preferably 50° C. to 100° C. The mass ratio of roasted coffee to Artemisia annua is, for example, 1 to 50, preferably 5 to 50. Coffee includes those of the robusta variety (coffea canephora) and arabica (coffea arabica).

[0069] Suitable extracts also include teas containing Artemisia annua, such as black tea and green tea, and teas containing licorice and cinnamon. Furthermore, the compounds of the combination of the present invention, including their salts, can also be obtained in the form of their hydrates, or other solvents used for their crystallization and / or extraction. The compounds of the combination of the present invention can inherently or by design form solvates with pharmaceutically acceptable solvents, including water; therefore, the present invention is intended to encompass both solvated and unsolvated forms. The term "solvate" refers to a molecular complex of a compound of the present invention, including its pharmaceutically acceptable salts, with one or more solvent molecules. Such solvent molecules are commonly used in the pharmaceutical arts and are known to be harmless to the recipient, such as water, ethanol, etc. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0070] Compounds having at least one 1,2,4-trioxane moiety contained in the anticancer agents or pharmaceutical compositions of the present invention, including salts, hydrates, and solvates thereof, can inherently or by design form polymorphs, and all such polymorphs are encompassed by this invention.

[0071] As used herein, the term "salt" or "salts" refers to acid addition salts or base addition salts of the compounds of the present invention. "Salt" particularly includes "pharmaceutically acceptable salts." The term "pharmaceutically acceptable salts" refers to salts that retain the biological effectiveness and properties of the compounds having at least one 1,2,4-trioxane moiety contained in the anticancer agents or pharmaceutical compositions of the present invention and are typically not biologically or otherwise undesirable. In some cases, the compounds having at least one 1,2,4-trioxane moiety contained in the anticancer agents or pharmaceutical compositions of the present invention can form acid and / or base salts due to the presence of amino and / or carboxyl groups or groups similar thereto, such as in artesunate.

[0072] Pharmaceutically acceptable acid addition salts can be formed with inorganic and organic acids, such as acetate, aspartate, benzoate, besylate, bromide / hydrobromide, bicarbonate / carbonate, bisulfate / sulfate, camphorsulfonate, chloride / hydrochloride, chlortheophyllonate, citrate, ethanedisulfonate, fumarate, gluceptate, gluconate, glucuronate, hippurate, hydroiodide / iodide, isethionate, lactate, lauryl ester, thiazolinone ... The salts may be octadecanoate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methylsulfate, naphthoate, napsylate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, palmoate, phosphate / hydrogen phosphate / dihydrogen phosphate, polygalacturonate, propionate, stearate, succinate, sulfosalicylate, tartrate, tosylate and trifluoroacetate.

[0073] Inorganic bases from which salts can be derived include, for example, ammonium salts and metal cations of columns I to XII of the periodic table. In certain embodiments, salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium, and magnesium salts.

[0074] Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, etc. Certain organic amines include isopropylamine, benzathine, cholinate, diethanolamine, diethylamine, lysine, meglumine, piperazine, and tromethamine.

[0075] The pharmaceutically acceptable salts of the present invention can be synthesized from basic or acidic moieties by conventional chemical methods. Generally, these salts can be prepared by reacting the free acid form of these compounds with a stoichiometric amount of a suitable base (e.g., Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, etc.), or by reacting the free base form of these compounds with a stoichiometric amount of a suitable acid. Such reactions are typically carried out in water or an organic solvent, or a mixture of the two. Generally, the use of non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile is desirable where practical.

[0076] Lists of additional suitable salts can be found, for example, in "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002).

[0077] Any formula given herein is intended to represent unlabeled forms as well as isotopically labeled forms of the compounds of the combinations of the invention with up to three atoms having non-natural isotope distributions, e.g., deuterium or C- or N-enriched moieties.

[0078] Compounds having at least one 1,2,4-trioxane moiety that form part of the anticancer agent or pharmaceutical composition of the present invention may contain groups capable of acting as donors and / or acceptors for hydrogen bonds and may be capable of forming co-crystals with suitable co-crystal formers. These co-crystals can be prepared from the compounds of the present invention by known co-crystal formation procedures. Such procedures include grinding, heating, co-sublimation, co-melting, or contacting a solution compound of the present invention with a co-crystal former under crystallization conditions and isolating the co-crystal formed thereby. Suitable co-crystal formers include those described in WO 2004 / 078163. Therefore, the present invention further provides co-crystals comprising compounds containing at least one 1,2,4-trioxane moiety.

[0079] The present invention further includes a pharmaceutical composition comprising the anti-cancer agent of any of the preceding embodiments.

[0080] The present invention also provides a method for preventing or treating cancer in a subject having cancer, the method comprising administering to the subject having cancer a therapeutically effective amount of an anti-cancer agent or pharmaceutical composition according to the present invention.

[0081] The present invention also provides a method for prolonging the survival of a subject with cancer, the method comprising administering to the subject with cancer a therapeutically effective amount of an anti-cancer agent or pharmaceutical composition according to the present invention.

[0082] In a further embodiment, the present invention provides a method for treating cancer in a subject, comprising simultaneously administering to a subject having cancer a therapeutically effective amount of an anticancer agent or pharmaceutical composition according to the present invention and at least one additional therapeutic agent. The anticancer agent or pharmaceutical composition of the present invention and the additional therapeutic agent can be administered together or separately, with partially overlapping or completely overlapping administration periods. The additional therapeutic agent can be selected from any agent known for this purpose, including, for example, an anti-neoplastic agent, and can be combined with the combination or pharmaceutical composition of the present invention to create a single pharmaceutical dosage form. Alternatively, these additional agents can be administered separately to the patient as part of multiple dosage forms, e.g., using a kit.

[0083] In yet a further embodiment, the present invention provides a method for treating cancer in a subject, comprising sequentially administering to a subject having cancer a therapeutically effective amount of an anticancer agent according to the present invention, particularly a combination or pharmaceutical composition according to the present invention, and at least one additional therapeutic agent. The anticancer agent or pharmaceutical composition of the present invention and the additional therapeutic agent can be administered in overlapping or non-overlapping administration periods. The additional therapeutic agent can be selected from any agent known for this purpose, including, for example, antineoplastic agents such as gemcitabine, cisplatin, carboplatin, and paclitaxel. It is an important finding of the present invention, as further outlined in the experimental part, that compounds comprising at least one 1,2,4-trioxane moiety, particularly artesunate, and their pharmaceutically acceptable salts, and therefore, the anticancer agent or pharmaceutical composition according to the present invention, significantly improve the efficacy of known standard treatments for cancer, particularly ovarian cancer and lung cancer.

[0084] Therefore, in a further embodiment, the present invention provides a method for treating or prolonging the survival of a subject with cancer, comprising simultaneously administering to the subject a therapeutically effective amount of an anticancer agent or pharmaceutical composition according to the present invention and at least one additional therapeutic agent. The anticancer agent or pharmaceutical composition of the present invention and the additional therapeutic agent can be administered together or separately, with partially overlapping or completely overlapping administration periods. The additional therapeutic agent can be selected from any agent known for this purpose, including, for example, antineoplastic agents such as gemcitabine, cisplatin, carboplatin, and paclitaxel.

[0085] In another embodiment, the present invention provides a method for treating or prolonging the survival of a subject with cancer, comprising sequentially administering to the subject a therapeutically effective amount of an anticancer agent or pharmaceutical composition according to the present invention and at least one additional therapeutic agent, The anticancer agent or pharmaceutical composition of the present invention and the additional therapeutic agent can be administered in overlapping or non-overlapping administration periods.

[0086] The additional therapeutic agent can be selected from any agent known for this purpose, including, for example, anti-neoplastic agents such as gemcitabine, cisplatin, carboplatin, and paclitaxel. In certain embodiments, the present invention provides a method for treating or prolonging the survival of a subject with cancer, comprising administering to a subject in need thereof at least (a) an anticancer agent or pharmaceutical composition according to the present invention, and (b)(i) gemcitabine and cisplatin, for example, 1250 mg / m 2 Gemcitabine at a dose of 75 mg / m 2 (ii) pemetrexed and cisplatin, e.g., at a dose of 500 mg / m 2 and pemetrexed at a dose of 75 mg / m 2 or (iii) paclitaxel and carboplatin, e.g., at a dose of 200 mg / m 2and a platinum-based doublet chemotherapy (PT-DC) that is a combination of paclitaxel at a dose of 0.05 mg / mL and carboplatin at a dose of 0.05 mg / mL. The method further includes administering

[0087] In a preferred embodiment, the additional anticancer agents are paclitaxel and carboplatin, e.g., at 200 mg / m 2 and carboplatin at a target area under the curve (AUC6) of 6 mg / ml / min.

[0088] PT-DC is typically administered in 3-week cycles for up to six cycles of chemotherapy. Chemotherapy treatment continues until disease progression, unacceptable toxicity, or completion of four to six cycles, whichever occurs first.

[0089] If the cancer is non-small cell lung cancer (NSCLC), the platinum-doublet chemotherapy regimen is typically dependent on the NSCLC histology. Subjects with mixed histology are classified according to the predominant histology.

[0090] Squamous histology subjects were treated with cisplatin (75 mg / m 2 ) along with gemcitabine (1250 mg / m 2 or carboplatin (AUC5) with gemcitabine (1000 mg / m 2 Gemcitabine is administered on days 1 and 8 of each cycle.

[0091] Non-squamous histology subjects received cisplatin (75 mg / m 2 ) along with pemetrexed (500 mg / m 2 or pemetrexed (500 mg / m ) with carboplatin (AUC6) administered on day 1 of each cycle. 2 ) can be received.

[0092] In a preferred embodiment, the anti-cancer agent or pharmaceutical composition according to the present invention is co-administered with PT-DC on the same day.

[0093] Thus, the present invention provides a kit for use in treating a subject with cancer, particularly ovarian cancer or lung cancer, comprising: (a) the dosage of the anticancer agent or pharmaceutical composition according to the present invention, preferably as disclosed above; and (b) the dose of the additional therapeutic agent, which is platinum-based doublet chemotherapy (PT-DC); and (c) Instructions for using the anticancer agent or pharmaceutical composition according to the present invention and other therapeutic agents. The present invention further includes a kit comprising:

[0094] It is a further finding of the present invention, as evidenced by the examples below, that anti-cancer agents, pharmaceutical compositions, in particular artesunate and its pharmaceutically acceptable salts, when applied together with an NRF2 inhibitor, such as ML385 (N-[4-[2,3-dihydro-1-(2-methylbenzoyl)-1H-indol-5-yl]-5-methyl-2-thiazolyl]-1,3-benzodioxole-5-acetamide), are more synergistically effective in the treatment of NSCLC with mutations in KEAP1 or NFE2L2 (the genes encoding NRF2).

[0095] In a particular embodiment, the present invention therefore provides a method for treating or prolonging the survival of a subject with cancer, particularly a subject with NSCLC, preferably a subject with a mutation in KEAP1 or NFE2L2, comprising administering to a subject in need thereof at least (a) an anticancer agent or pharmaceutical composition according to the present invention, and (b) an additional therapeutic agent selected from the group consisting of an NRF2 inhibitor, e.g., ML385 The method further includes administering

[0096] The present invention further encompasses an enhanced pharmaceutical composition or kit for use in treating a subject with cancer, particularly non-small cell lung cancer, comprising: (a) the dosage of the anticancer agent or pharmaceutical composition according to the present invention, preferably as disclosed above; and (b) Dosages for NRF2 inhibitors, and also for kits (c) Instructions for using the anticancer agent or pharmaceutical composition and NRF2 inhibitor according to the present invention Includes.

[0097] Additional aspects of this invention include an article of manufacture comprising an anti-cancer or pharmaceutical composition or kit, as described above, for treating or preventing cancer or prolonging survival of a subject with cancer; and packaging material comprising a label stating that the composition can be used to treat or prevent cancer or prolong survival of a subject with cancer.

[0098] The invention further encompasses anti-cancer agents and pharmaceutical compositions and kits as described herein for use as pharmaceuticals, particularly for treating or preventing cancer or for prolonging survival of a subject with cancer.

[0099] Yet another aspect of this invention relates to a method of destroying or inhibiting the growth of cancer cells, comprising exposing the cancer cells to an effective amount of an anti-cancer agent or pharmaceutical composition described herein, either with or without an additional therapeutic agent. This method can be practiced in vitro or in vivo.

[0100] The scope of the present invention further includes the use of anti-cancer agents or pharmaceutical compositions, either in combination with or without additional therapeutic agents, to prevent or inhibit cancer cell growth, cancer cell division, or cancer metastasis.

[0101] The applicable daily dose range of the anticancer agent or pharmaceutical composition of the present invention is, for example, 0.1 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, and even more preferably 0.5 to 50 mg / kg body weight, calculated relative to the total amount of the compound having at least one 1,2,4-trioxane moiety of the anticancer agent or pharmaceutical composition as described and defined herein.

[0102] The same applies to kits as defined above.

[0103] Each dosage unit can contain 5% to 95 wt-% of the anti-cancer agent or pharmaceutical composition of the present invention. Preferably, the pharmaceutical composition according to the present invention contains 20% to 80 wt-% of the anti-cancer agent according to the present invention.

[0104] In another embodiment, a pharmaceutical composition according to the present invention contains 20% to 80 wt-% of a compound having at least one 1,2,4-trioxane moiety.

[0105] The actual pharmaceutically effective amount or therapeutic dose will, of course, depend on factors known by those skilled in the art, such as the age and weight of the patient, the route of administration, and the severity of the disease. In any case, the combination will be administered in a dosage and manner that allows a pharmaceutically effective amount to be delivered based on the patient's individual condition.

[0106] When the pharmaceutical composition of this invention comprises an anti-cancer agent of the invention and one or more additional therapeutic agents, both the anti-cancer agent and the additional agents should be present at dosage levels of between about 10% and 100%, more preferably between about 10 and 80%, of the dosage normally administered in a monotherapy regimen.

[0107] Compounds of formulas (VIII) to (XVIII) below have been reported to be present in Artemisia annua extracts, see inter alia Czechowski et al., Frontiers in Plant Science, 2019, Vol. 10, Article 984; Zarelli et al., Phytochemical Analysis 2019, 30, pp. 564-571.

[0108] Therefore, the anticancer agent, pharmaceutical composition, and kit according to the present invention can further comprise at least one compound selected from the group consisting of those of formulae (VIII) to (XVIII), for example, one, two, three, four, five, six, seven, eight, nine, ten, or all of the compounds. [ka]

[0109] The compound of formula (VIII) is scopoletin.

[0110] The compound of formula (IX) is 1,8-cineole.

[0111] The compound of formula (X) is artemisinic acid.

[0112] The compound of formula (XI) is Arteannuin-B.

[0113] The compound of formula (XII) is dihydroartemisinic acid.

[0114] The compound of formula (XIII) is fisetin.

[0115] The compound of formula (XIV) is casticin.

[0116] The compound of formula (XIV) is artemetine.

[0117] The compound of formula (XVI) is chrysoprenetic acid.

[0118] The compound of formula (XVII) is chrysoprenol-D.

[0119] The compound of formula (XVIII) is circilsilineol.

[0120] The pharmaceutical compositions of the present invention can be administered by known methods, including orally, parenterally, by inhalation, etc. In certain embodiments, the compounds of the present invention are administered orally as a pill, lozenge, troche, capsule, solution or extract, such as a tea or other type of infused drink, particularly an extract of Artemisia annua, or suspension.

[0121] In other embodiments, the pharmaceutical compositions of the present invention are administered by injection or infusion. Infusion is typically administered intravenously, often over a time period between about 15 minutes and 4 hours. In other embodiments, the pharmaceutical compositions of the present invention are administered intranasally or by inhalation, with inhalation methods being particularly useful. Because the pharmaceutical compositions of the present invention exhibit oral bioavailability, oral administration is sometimes preferred.

[0122] An "effective amount" of a compound is the amount necessary or sufficient to treat or prevent cancer or to prolong the survival of a subject with cancer.

[0123] An effective amount can vary depending on factors such as the size and weight of the subject, the type and severity of the disease, or the particular compound of the present invention. For example, the choice of a combination or pharmaceutical composition according to the present invention can affect what constitutes an "effective amount." One of ordinary skill in the art can study the factors contained herein and make the determination regarding the effective amount of a combination or pharmaceutical composition of the present invention without undue experimentation.

[0124] The administration regimen can affect what constitutes an effective amount. The combination or pharmaceutical composition of the present invention can be administered to a subject either before or after the onset of cancer. Furthermore, several divided doses, as well as staggered doses, can be administered daily or sequentially, or the dose can be continuously infused or bolus injected. Furthermore, the dosage of the combination or pharmaceutical composition of the present invention can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0125] The combinations of the invention can be used in the treatment of conditions, disorders or diseases as described herein or for the manufacture of a pharmaceutical composition for use in the treatment of cancer. The invention provides methods of use of the combinations of the invention in the treatment of these diseases or for the preparation of pharmaceutical compositions comprising such combinations of the invention for the treatment of these diseases.

[0126] The language "pharmaceutical composition" includes preparations suitable for administration to mammals, e.g., humans. When the combinations of the present invention are administered as a medicine to a mammal, e.g., a human, they may be given on their own or as a pharmaceutical composition containing, for example, 0.1% to 99.5% (more preferably, 0.5% to 90%) of the combination of the present invention or any subgenus thereof as the active ingredient in a pharmaceutically acceptable carrier or, optionally, in combination with two or more pharmaceutically acceptable carriers.

[0127] The phrase "pharmaceutically acceptable carrier" is art-recognized and includes any pharmaceutically acceptable material, composition, or vehicle suitable for administering a compound of the combination of the present invention to a mammal. Carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials that are involved in carrying or transporting the subject agent from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solution; and other non-toxic, compatible substances used in pharmaceutical formulations. Typically, the pharmaceutically acceptable carrier is sterile and / or substantially pyrogen-free.

[0128] Wetting agents, emulsifying agents and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0129] Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0130] The formulations of the present invention include those suitable for oral, nasal, inhalation, topical, transdermal, buccal, sublingual, rectal, vaginal, and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of combination or pharmaceutical composition that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 1 percent to about 99 percent of the active ingredient, preferably from about 5 percent to about 80 percent.

[0131] Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a combination or pharmaceutical composition of the present invention with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0132] Formulations of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, for example, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base, for example, gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc., each containing a predetermined amount of a compound of the present invention as an active ingredient. The compounds of the present invention may also be administered as a bolus, electuary, or paste.

[0133] In solid dosage forms of the invention for oral administration, such as capsules, tablets, pills, dragees, powders, granules, etc., the combination or pharmaceutical composition may comprise one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; binders, such as carboxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; humectants, such as glycerol; disintegrating agents, such as PEG-1, PEG-2, PEG-3, PEG-4, PEG-5, PEG-6, PEG-7, PEG-8, PEG-9, PEG-10, PEG-11, PEG-12, PEG-13, PEG-14, PEG-15, PEG-16, PEG-17, PEG-18, PEG-19, PEG-20, PEG-21, PEG-22, PEG-23, PEG-24, PEG-25, PEG-26, PEG-27, PEG-28, PEG-29, PEG-30, PEG-31, PEG-32, PEG-33, PEG-44, PEG-45, PEG-46, PEG-47, PEG-48, PEG-49 ...50, PEG-51, PEG-52, PEG-53, PEG-54, PEG-55, PEG-55, PEG-55, PEG-56, PEG-5 The pharmaceutical composition may be mixed with any of the following: agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarders, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as cetyl alcohol and glycerol monostearate; absorbents, such as kaolin and bentonite clay; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical composition may also contain buffering agents. Solid compositions of a similar type can also be used as fillers in soft and hard-filled gelatin capsules using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols, etc.

[0134] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0135] Tablets and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills, and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide slow or controlled release of the active ingredient therein, using, for example, hydroxypropylmethylcellulose, in varying proportions to provide the desired release profile, other polymer matrices, liposomes, and / or microspheres. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions can optionally contain opacifying agents and can be compositions that release the active ingredient only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the excipients described above.

[0136] The liquid dosage form for oral administration of the compound of the present invention includes pharmaceutically acceptable emulsions, microemulsions, solutions, teas, coffees, suspensions, syrups and elixirs.In addition to the active ingredient, the liquid dosage form may contain an inert diluent commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0137] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0138] Suspensions may contain, in addition to the active compounds, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0139] Dosage forms for topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0140] Pharmaceutical compositions of this invention suitable for parenteral administration may comprise one or more compounds of the invention in combination with one or more pharmaceutically acceptable carriers, such as sterile isotonic aqueous or non-aqueous materials, for example, ethanolic solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, which may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0141] The preparations of the present invention may be given orally, pulmonary, parenterally, topically, or rectally.

[0142] They are, of course, provided in forms suitable for each administration route: for example, they are administered in the form of tablets or capsules, by injection, inhalation, eye lotion, ointment, suppository, etc., by injection, infusion or inhalation; topically by lotion or ointment; and rectally by suppositories.

[0143] The phrases "parenteral administration" and "administered parenterally," as used herein, mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intrathecal, and intrasternal injection and infusion.

[0144] Intravenous infusion is sometimes the preferred method of delivery for the compounds of the present invention. Infusion can be used to deliver a single daily dose or multiple doses. In some embodiments, the anticancer agent or pharmaceutical composition of the present invention is administered by infusion at intervals of 15 minutes to 4 hours, typically 0.5 to 3 hours. Such infusions can be used once per day, twice per day, or up to three times per day.

[0145] The phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered," as used herein, refer to the administration of a compound, drug, or other material not directly into the central nervous system, e.g., subcutaneous administration, so that it enters the patient's system and therefore undergoes metabolic and other similar processes.

[0146] The anti-cancer agents or pharmaceutical compositions can be administered to humans and other animals for treatment by any suitable route of administration, including orally, pulmonary, nasally, for example, by spray, rectally, vaginally, parenterally, intracisternally and topically, including bucally and sublingually, by powders, ointments or drops.

[0147] Regardless of the route of administration selected, the anticancer agent or pharmaceutical composition of the present invention, which may be used in a suitable hydrated form, and / or the pharmaceutical composition or kit of the present invention, is formulated into a pharmaceutically acceptable dosage form by conventional methods known to those skilled in the art.

[0148] Actual dosage levels of the active ingredients in the pharmaceutical compositions and kits of this invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0149] The selected dosage level will depend upon a variety of factors, including the activity of the particular compound of the invention being used, the route of administration, the time of administration, the rate of excretion of the particular compound being used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound being used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical arts.

[0150] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition or kit required. For example, the physician or veterinarian can start the dosage of the anticancer agent of the present invention used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0151] Generally, a suitable daily dose of a compound of the present invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.

[0152] If desired, the effective daily dose of the active compound can be administered as a single dose per day, or as two, three, four, five, six or more subdoses, optionally in unit dosage form, administered separately at appropriate intervals throughout the day. Compounds delivered orally or by inhalation are commonly administered in one to four doses per day. Compounds delivered by injection are typically administered once per day or once every other day. Compounds delivered by infusion are typically administered in one to three doses per day. When multiple doses are administered within a day, the doses can be administered at intervals of about 4 hours, about 6 hours, about 8 hours or about 12 hours.

[0153] While it is possible for the anticancer agents of the present invention to be administered alone, it is preferable to administer them as a pharmaceutical composition, such as those described herein. Thus, methods of using the anticancer agents of the present invention include administering the anticancer agent as a pharmaceutical composition, wherein at least one compound having at least one 1,2,4-trioxane moiety is admixed with a pharmaceutically acceptable carrier prior to administration.

[0154] The present invention further encompasses the use of the anti-cancer agent, pharmaceutical composition or kit of the present invention in combination with an immune modulator.

[0155] The combinations and pharmaceutical compositions described herein can be used or administered in combination with one or more therapeutic agents that act as immune modulators, e.g., activators of costimulatory molecules, or inhibitors of immune-inhibitory molecules, or vaccines.

[0156] By "in combination with," it is not intended to imply that the therapies or therapeutic agents must be administered simultaneously and / or formulated together for delivery, although these delivery methods are within the scope described herein. An immune modulator may be administered simultaneously with, before, or after one or more compounds of the invention, and optionally one or more additional therapies or therapeutic agents. Therapeutic agents in the combination may be administered in any order. Generally, each agent will be administered at a dose and / or at a time schedule determined for that agent. It will further be understood that the therapeutic agents utilized in the combination can be administered together in a single composition or can be administered separately in different compositions. In general, it is expected that each of the therapeutic agents utilized in the combination will be utilized at levels that do not exceed the levels at which they are utilized individually.

[0157] This invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references cited throughout this application are hereby expressly incorporated by reference. [Example]

[0158] A) Ovarian cancer cell lines Materials and Methods 1.1 Cell culture and artesunate Commercially available human ovarian cancer cell lines UWB1.289 (ATCC CRL-2945), Caov-3 (ATCC HTB-75), and OVCAR-3 (ATCC HTB-161) were obtained from ATCC. Cell lines were cultured as recommended by ATCC and maintained in cell line-specific complete growth medium. All cells were incubated at 37°C in 5% CO2. Artesunate was purchased from MedChem Express, dissolved in DMSO as a 200 mM stock solution, and stored at -80°C. Artesunate was serially diluted into DMSO and then culture medium to the desired concentration at each experimental time point.

[0159] 1.2 Cell viability assay 3 x 10 cells in 100 µL of growth medium in a white-walled 96-well microplate 3 Cells were seeded at 1000 kJ / well and incubated at 37°C under 5% CO2 for 24 hours. Growth medium was removed and replaced with fresh medium containing serially diluted drugs or the drug of interest. Each drug concentration was tested in duplicate, and vehicle (0.1% DMSO) control medium was tested in triplicate assays. Cells were treated with 12 dilutions of artesunate stock solution ranging from 0.0011 to 200 μM and incubated for 72 hours. Cell viability was assessed using the CellTiter-Glo 2.0 Viability Assay (Promega), and luminescence was measured using a Varioskan LUX multimode microplate reader (ThermoFisher Scientific). Percent viability was calculated by normalizing the relative luminescence signal of each treated well to the corresponding vehicle control. The percent viability calculated for each artesunate concentration was graphed, then a nonlinear regression line was fitted using a four-parameter log-logistic model and the IC50 was calculated for each cell line using GraphPad Prism 5.01.

[0160] 1.3 DNA damage assay Caov-3 cells were seeded at a density of 4000 cells / well in 100 μL of complete growth medium into black-walled μClear 96-well plates and allowed to adhere for 24 hours at 37°C under 5% CO2. The medium was removed and replaced with complete medium containing 5 μM, 10 μM, 50 μM, or 100 μM artesunate, 0.1% DMSO as a negative control, or 25 μM cisplatin as a positive control. Cells were incubated with the drugs for 48 hours and then fixed in 4% paraformaldehyde for 15 minutes at room temperature. Cells were permeabilized with 0.25% Triton X-100 for 15 minutes and then blocked in 0.1% bovine serum albumin (BSA) for 1 hour. DNA damage was assessed by immunofluorescence staining for phosphorylated histone H2AX (pH2AX) using the HCS DNA Damage Kit (Invitrogen). A Cell-Insight CX7 High Content Analysis Platform was used for imaging, and nuclear pH2AX signal was quantified using HCS Studio software (both from ThermoScientific). Statistical analysis of pH2AX signal was completed in GraphPad Prism (version 5.01).

[0161] 1.4 Flow cytometry analysis of the cell cycle UWB1 and Caov-3 cells were grown in culture flasks under standard cell culture conditions as outlined above. For the staining solution, 2 mg of DNase-free Rnase A (Sigma) and 200 μL of 1 mg / mL propidium iodide were added to 10 mL of 0.1% (v / v) Triton X-100 in PBS. On the day of treatment, treatment medium was prepared by serially diluting a stock 200 μM artesunate solution into cell line-specific medium. Once cell cultures reached confluence, we removed the growth medium from the treatment flasks and added artesunate treatment medium at a concentration of 10 μM. Cells were harvested after 24 or 48 hours, washed in PBS, resuspended in 0.5 mL of PBS, and transferred to a tube containing 4.5 mL of 70% EtOH for fixation. Cells were fixed at least overnight at -20°C. Fixed cells were washed in PBS and resuspended in 1 mL of staining solution. PI-stained cells were sorted using an LSR II cell analyzer. Analysis was performed using ModFit LT v3.3 software. Statistical analysis comparing the percentage of cells in each cell cycle phase (G1, S, or G2) was performed using GraphPad Prism (version 5.01).

[0162] 1.5 Drug Administration Sequence Assay Similar to the protocol described above for the cell viability assay, cells were seeded into standard 96-well plates at a cell density of 3,000 cells / 100 μL and incubated for 24 hours. Artesunate, carboplatin, and paclitaxel stock solutions were diluted with DMSO and medium to achieve final concentrations of 40 μM, 16 μM, and 32 μM, respectively. Each drug was added as indicated for 24 hours, after which the treatment medium was replaced with fresh medium. The drug administration sequence was artesunate on day 1 (D1A) or day 2 (D2A), followed by carboplatin and paclitaxel on day 2 (D2C / T), carboplatin, paclitaxel, and artesunate on day 2 (D2C / T / A), or artesunate on day 1 followed by carboplatin and paclitaxel on day 2 (D1A; D2C / T). Cells were incubated for a total of 72 hours under standard growth conditions. Viability measurements were determined using the CellTiter-Glo 2.0 Viability Assay (Promega). Luminescence was measured using a Varioskan LUX multimode microplate reader (ThermoFisher Scientific). Statistical analysis was performed using GraphPad Prism (v5.01).

[0163] result 1.6 Determination of Antineoplastic Activity To evaluate the antineoplastic activity of artesunate in ovarian cancer, we used the CellTiter-Glo 2.0 assay to determine the dose-dependent effect of artesunate on the viability of three epithelial ovarian cancer cell lines: Caov-3 (adenocarcinoma), UWB1.289 (high-grade serous carcinoma with a BRCA1 mutation), and OVCAR-3 (adenocarcinoma). The IC50 values ​​of artesunate in all three cell lines were in the low- to mid-micromolar range; specifically, the IC50 values ​​were 26.91 μM (95% confidence interval: 6.287-115.2 μM) in UWB1 cells, 15.17 μM (10.49-21.93 μM) in Caov-3 cells, and 4.67 μM (3.280-6.638 μM) in OVCAR-3 cells. One-way ANOVA analysis showed no significant differences in IC50 between these three cell lines (p>0.05). The IC50s detected for all three ovarian cancer cell lines tested are consistent with previously established findings (see [6] cited above) and are within the range of therapeutically achievable in vivo plasma concentrations (approximately 20 μM) (see

[14] cited above).

[0164] Detailed results are shown in Figure 1.

[0165] 1.7 Assessment of the primary mode of action Caov-3 cells were chosen due to their close correlation with previously published IC50 values ​​and their known platinum sensitivity, which is associated with the most aggressive serous ovarian cancer. To assess the ability of artesunate to induce DNA damage in Caov-3 cells, immunofluorescence staining of pH2AX, a marker of double-strand breaks, was quantified after 48 hours of treatment with artesunate concentrations ranging from 5 to 100 μM and 25 μM cisplatin as a positive control.

[0166] The results are shown in Figure 2.

[0167] Cells treated with 0.1% DMSO as a control had a mean pH2AX signal of 443.2 ± 76.38, while treatment with 25 μM cisplatin resulted in a mean signal of 2517 ± 230.4. The only artesunate treatment that significantly increased DNA damage was 100 μM artesunate, with a mean pH2AX signal of 617.0 ± 31.96. Treatment with 5, 10, or 50 μM resulted in pH2AX measurements of 382.8 ± 39.58, 370.2 ± 9.283, and 393.8 ± 58.79, respectively. Only the highest concentrations of artesunate (100 μM) and cisplatin resulted in a significant increase in DNA damage compared to vehicle control-treated cells, as assessed by unpaired one-tailed t-tests, p = 0.0486 (100 μM artesunate) and p = 0.0034 (25 μM cisplatin).

[0168] Thus, although artesunate was capable of inducing DNA damage, this effect was barely observed at clinically relevant concentrations in Caov-3 cells.

[0169] The secondary mechanism of action of artesunate investigated was the induction of cell cycle arrest. Cell cycle progression was assessed by propidium iodide staining and flow cytometry in Caov-3 and UWB1 cells after 24 and 48 h of treatment with 10 μM artesunate or 0.1% DMSO (vehicle control). The percentage of cells in G1 and S phase with and without 10 μM artesunate treatment was determined from three independent experiments. In Caov-3 cells, 48 ​​h of artesunate treatment resulted in an increase in the percentage of cells in G1 (78.98% ± 0.9546) compared to 61.23% ± 1.789 in vehicle-treated cells. UWB1 cells had 65.35% ± 0.0849 cells in G1 after artesunate treatment compared to 60.35% ± 2.418 in control cells.

[0170] The results are shown in Figures 3 and 4.

[0171] Both cell lines showed a significant increase in cells in G1, with Caov-3 (p = 0.0032) and UWB1 (p = 0.0499) (unpaired, one-tailed t-test). In further subgroup analysis, the increase in the percentage of Caov-3 and UWB1 cells in G1 phase after treatment with artesunate was accompanied by a significant decrease in cells in S phase (p = 0.0009 and p = 0.0497, respectively). In Caov-3 cells, 48-hour artesunate treatment resulted in a decrease in the percentage of cells in S phase (12.14% ± 0.1556) compared with 24.42% ± 0.7354 in vehicle-treated cells. UWB1 cells had 18.87% ± 2.779 cells in S phase after artesunate treatment compared with 26.25% ± 2.234 in control cells.

[0172] The results are shown in Figures 5 and 6.

[0173] These experiments demonstrate that clinically relevant concentrations of artesunate induce G1 arrest in the cell lines tested.

[0174] 1.8 Addition of artesunate to carboplatin and paclitaxel The current first-line treatment regimen for ovarian cancer is a platinum / taxane doublet, e.g., carboplatin and paclitaxel. Given its favorable side effect profile and effect on cell viability, the effect of adding artesunate to cells treated with both paclitaxel and carboplatin was evaluated. Because artesunate induces cell cycle arrest in the G1 phase, artesunate was evaluated either as a pretreatment (24 h prior) or simultaneously with carboplatin and paclitaxel. In both cell lines, treatment with artesunate alone on either day 1 or day 2 resulted in a decrease in cell viability from 54.60 to 63.43% compared to control vehicle-treated cells.

[0175] The results are shown in Figures 7 and 8.

[0176] In Caov-3 cells, treatment with carboplatin / paclitaxel resulted in a decrease in cell viability to 29.60% ± 7.780, which was significantly reduced when artesunate was added concomitantly (11.55% ± 5.917, p<0.05 one-way ANOVA), but not when cells were pretreated with artesunate (33.16% ± 3.349, p>0.005).

[0177] Similarly, in UWB1, cell viability decreased to 62.06% ± 9.389 when treated with carboplatin and paclitaxel, which further decreased to 39.65% ± 6.850 with the simultaneous addition of 40 μM artesunate (p<0.05, one-way ANOVA).

[0178] In these cells, pretreatment with artesunate resulted in a cell viability of 56.41% ± 2.148, which was not statistically significant when compared to cells treated with carboplatin and paclitaxel.

[0179] The main finding is that combination treatment of artesunate with carboplatin and paclitaxel shows a significant improvement in the efficacy of the standard regimen.

[0180] B) Lung cancer cell lines Materials and Methods 2.1. Cell lines and reagents A549 (ATCC: CCL-185), H1299 (ATCC: CRL-5803), and H1563 (ATCC: CRL-5875) NSCLC cell lines were purchased directly from ATCC. All cell lines were initially expanded and low-passage aliquots were frozen and refrigerated to ensure experiments were performed on cell lines with similar passage numbers. Cell lines were screened for mycoplasma at regular intervals, including when they were frozen and refrigerated. All cells were grown in RPMI 1640 (Lonza, Basel, Switzerland: 12-167F) containing 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA: F0926), penicillin / streptomycin (Gibco, Waltham, MA, USA: 15140-122), and 2 mM Glutamax (Gibco: 35050-061) and maintained at 37°C in a humidified incubator under 5% CO. Purified artesunate (HY-N0193) was purchased from MedChem Express (Monmouth Junction, NJ, USA), and ML385 (SML1833) was purchased from Sigma-Aldrich.

[0181] 2.2. Drug Response Assay Cells were seeded into white-walled 96-well plates at 2500 cells / well (A549 and H1299) or 4000 cells / well (H1563) in 100 μL of complete growth medium and allowed to adhere for 24 hours at 37°C under 5% CO2. After 24 hours, artesunate was serially diluted 1:3 in DMSO to obtain 12 drug stocks in 100% DMSO. Each stock was then diluted 1:1000 in complete medium, resulting in a final DMSO concentration of 0.1%. Growth medium was aspirated from the cells and replaced with medium containing diluted artesunate, and each drug concentration was tested in duplicate wells; in addition, triplicate wells received medium containing 0.1% DMSO alone to serve as untreated controls. After incubating cells with drugs for 96 hours, cell viability was assessed using CellTiter-Glo 2.0 (Promega, Madison, WI, USA: G9243). Data are presented as percent viability of treated cells normalized to 0.1% DMSO-treated control cells. GraphPad Prism (version 5.01) was used to fit dose-response curves (four-parameter log-logistic model) to the data and calculate IC50 values. For studies evaluating artesunate in combination with ML385, artesunate drug stock was diluted in medium containing 5 μM ML385, and corresponding control wells were treated with 5 μM ML385.

[0182] 2.3.DNA Damage Assay A549 and H1299 cells were seeded at a density of 3000 cells / well in 100 μL of complete growth medium into black-walled μClear 96-well plates (ThermoScientific, Waltham MA, USA: 165305) and allowed to adhere for 24 hours at 37°C under 5% CO2. The medium was then removed and replaced with complete medium containing 5 μM, 10 μM, 50 μM, or 100 μM artesunate, 0.1% DMSO as a negative control, or 25 μM cisplatin (Tocris Bioscience, Minneapolis MN, USA: 2251) as a positive control. Cells were incubated with the drugs for 48 hours and then fixed in 4% paraformaldehyde (Alfa Aesar, Tewksbury MA, USA: J61899) for 15 minutes at room temperature. After fixation, cells were permeabilized with 0.25% Triton X-100 (Alfa Aesar: A16046) for 15 minutes and blocked in 0.1% bovine serum albumin (BSA) for 1 hour. DNA damage was assessed by immunofluorescence staining of phosphorylated histone H2AX (pH2AX) using the HCS DNA Damage Kit (Invitrogen, Waltham, MA, USA: H10292). Cells were imaged using a CellInsight CX7 High Content Analysis Platform (ThermoScientific: CX7A1110), and quantification of nuclear pH2AX signal was performed using HCS Studio software (ThermoScientific). Statistical analysis of pH2AX signal was performed with GraphPad Prism (version 5.01). The effect of combined treatment with artesunate and ML385 on DNA damage was also assessed using the HCS DNA Damage Kit. The following day, cells were seeded as described above and treated with DMSO or 5 μM ML385 for 24 hours. The following day, the medium was aspirated, and fresh medium containing 10 μM, 50 μM, or 100 μM artesunate, 0.1% DMSO as a negative control, or 25 μM cisplatin with or without fresh 5 μM ML385 was added to the cells. After 24 h, the cells were fixed with 4% paraformaldehyde, and DNA damage was assessed as described above.

[0183] Western blotting A549 and H1299 cells were treated with 10 μM artesunate for 0, 6, or 24 h, then lysed in RIPA buffer (Pierce, Waltham, MA, USA, 89900) containing Halt Protease and Phosphatase Inhibitor Cocktail (ThermoScientific, 78441) and Benzonase Nuclease (Sigma, E1014), incubated on ice for 10 min, and cleared by centrifugation. Protein concentration was determined using a BCA Protein Assay (Pierce, 23227). 40 μg of total protein was loaded onto a NuPAGE 4-12% Bis-Tris Gel (Life Technologies, Waltham, MA, USA, NP0321BOX) for electrophoresis, followed by transfer and blotting onto a PVDF membrane (Invitrogen, LC2005). Antibodies against KEAP1 (Cell Signaling, Danvers MA, USA: 8047S) and NQO-1 (Cell Signaling: 3187S) were purchased from Cell Signaling, while antibodies against β-actin were purchased from R&D Systems (MAB8929). IRDye-conjugated secondary antibodies were purchased from LI-COR (925-32213 and 925-68070), and Western blots were imaged using a LI-COR Odyssey imaging system.

[0184] 2.5. siRNA knockdown 2 x 10 cells in two wells of a 6-well plate 5A549 or H1299 cells were seeded at 2500 cells / well and allowed to adhere overnight before transfecting with 5 nM siGENOME Control Pool Non-Targeting No. 2 (Dharmacon, Boulder, CO, USA: D-001206-14-5) or siGENOME SMARTpool siRNA targeting human KEAP1 (GGACAAACCGCCUUAAUUC; CAGCAGAACUGUACCUGUU; GGGCGUGGCUGUCCUCAAU; CGAAUGAUCACAGCAAUGA) purchased from Dharmacon (M-012453-00-0005). After 24 h, cells were seeded at 2500 cells / well in 96-well plates or 60 mm tissue culture plates to assess knockdown efficiency by Western blot. Then, 48 hours after the initial transfection, cells seeded in 96-well plates were treated as detailed above (drug response assay) to assess the effect of KEAP1 knockdown on drug sensitivity, while 60 mm plates were harvested in RIPA buffer for Western blotting as described above to determine knockdown efficiency. To determine whether KEAP1 knockdown altered the ability of artesunate to induce DNA damage in NSCLC cells, cells were transfected with 5 nM non-targeting control or siRNA targeting human KEAP1. The following day, 6000 cells / well were seeded in complete growth medium in black-walled, clear-bottom 96-well plates, and DNA damage assays were performed as described above (2.3. DNA Damage Assay).

[0185] 2.6. Synergism or Artesunate and NRF2 Inhibitors To assess drug interactions, drug response assays were performed similarly to the method described above; however, a 6x6 matrix design was used to assay pairs of drugs alone and in combination with five serially diluted concentrations of each drug. After 96 hours of treatment, cell viability was assessed using CellTiter-Glo 2.0. Each well was normalized to untreated control cells grown in medium containing 0.2% DMSO, and the percentage of viable cells was determined. R statistical software, specifically the synergyfinder package (version 1.10.4), was used to generate synergy scores using the Bliss independent model and the Zero Interaction Potency (ZIP) model (Bliss, CI The Toxicity of Poisons Applied Jointlyl. Ann Appl. Biol 1939, 26, pp. 585-615, doi:10.1111 / j.1744-7348.1939.tb06990.x; Yadav, B.; Wennerberg, K.; Aittokallio, T.; Tang, J. Searching for Drug Synergy in Complex Dose-Response Landscapes Using an Interaction Potency Model. Comput Struct. Biotechnol J. 2015, 13, pp. 504-513, doi:10.1016 / j.csbj.2015.09.001.)

[0186] 2.7.Statistical analysis To assess whether differences in artesunate IC50 values ​​were statistically significant, IC50 values ​​calculated from multiple independent experiments were graphed using GraphPad Prism 5 along with the standard error for each experiment. A two-tailed t-test was used to determine whether the mean IC50 values ​​between two different cell lines or between treated and control cells were statistically different. p-values ​​are reported in the figure legends, and results with p-values ​​less than 0.05 indicate statistically significant differences. Differences in DNA damage were determined for each cell line using one-way ANOVA followed by Dunnett's multiple comparison test, comparing each treatment to the 0.1% DMSO control for cells treated with artesunate alone or in combination with 5 μM ML385. Two-way ANOVA and Bonferroni post-hoc tests were used to assess DNA damage after 24 hours of artesunate treatment in siRNA-transfected cells.

[0187] result 2.8 Artesunate sensitivity We first investigated the effects of artesunate on three NSCLC cell lines (A549, H1299, and H1563) by assessing cell viability using the CellTiter-Glo 2.0 assay after 96 hours of treatment with increasing concentrations of artesunate. The cell lines were classified as either sensitive or resistant based on a 10-fold difference in the IC50 values ​​of artesunate. A549 cells were less sensitive to artesunate, with an average IC50 of 23.63 μM ± 8.886 μM from three independent experiments, while H1299 and H1563 cells were sensitive to artesunate, with average IC50s of 2.36 μM ± 1.275 μM and 3.43 μM ± 1.190 μM, respectively (see Figures 9 and 10).

[0188] 2.9 Production of reactive oxygen species The primary mechanism of action of artesunate against malaria is known to be the generation of reactive oxygen species (ROS) through a reaction between endoperoxide bridges in artesunate and the heme iron of the malaria parasite. In cancer cells, ROS production can cause DNA damage through double-strand breaks, which can be assessed by immunofluorescence staining of phosphorylated histone H2AX (pH2AX). Therefore, we assessed the ability of artesunate to induce DNA damage in a dose-dependent manner in A549 (less sensitive) and H1299 (sensitive) NSCLC cell lines. Treatment with 25 μM cisplatin was used as a positive control for DNA damage, while 0.1% DMSO was used as a negative / vehicle control. The mean nuclear pH2AX signal intensity for each treatment was normalized to the corresponding 0.1% DMSO control and plotted as the treatment / 0.1% DMSO (dimethyl sulfoxide) ratio ± SD (see Figure 11). In both A549 and H1299 cells, cisplatin induced pH2AX staining more than that observed in the corresponding 0.1% DMSO-treated control cells (p<0.001) as determined by one-way ANOVA and Dunnett's multiple comparison test for each cell line. Specifically, 25 μM cisplatin resulted in an 11.17±2.79-fold increase in pH2AX staining in A549 cells and a 14.45±1.82-fold increase in pH2AX staining in H1299 cells. In contrast, in A549 cells, increasing concentrations of artesuante did not cause a significant increase in DNA damage compared with 0.1% DMSO-treated controls, even at the highest concentrations of artesunate (5 μM: 1.19 ± 0.05, 10 μM: 1.35 ± 0.01, 50 μM: 1.71 ± 0.02, 100 μM: 3.27 ± 0.13). Alternatively, in artesunate-sensitive H1299 cells, significant DNA damage (p < 0.05) was observed after treatment with only 10 μM artesunate. Specifically, 5 μM artesunate resulted in a 3.14±0.09-fold increase in pH2AX staining compared to 0.1% DMSO control-treated cells, 10 μM artesunate produced a 4.47±0.26-fold increase, 50 μM produced an 8.25±1.08 increase, and 100 μM artesunate increased pH2AX staining by 17.97±0.79-fold.

[0189] 2.10 Dependence of artesunate sensitivity on KEAP1 in NSCLC A549 cells harbor a G333C mutation in KEAP1, whereas both H1299 and H1563 cells are wild-type for KEAP1 and NFE2L2 (the gene encoding NRF2), which may explain the higher resistance of A549 cells to artesunate. Because artesunate is known to increase cellular ROS and the KEAP1 / NRF2 pathway is a key regulator of the cellular response to ROS, we first assessed whether treatment of A549 (resistant) or H1299 (sensitive) cells with artesunate altered the protein expression of either KEAP1 or NQO-1. NQO-1 is a well-established transcriptional target of NRF2 and was used as a proxy to assess NRF2 transcriptional activity by Western blot. Cells were treated with 10 μM artesunate for 0, 6, or 24 hours, after which lysates were harvested and Western blots for KEAP1, NQO-1, and β-actin were performed. The KEAP1 antibody used for Western blotting was generated using a peptide present in both wild-type and G333C mutant KEAP1, allowing detection of both wild-type and mutant forms from the cell lines tested. As seen in Figure 12, A549 cells had decreased basal expression of KEAP1 and increased basal expression of NQO-1 compared to H1299 cells, as expected since A549 cells harbor an inactivating mutation in KEAP1. Treatment of both cell lines with artesunate for 24 hours resulted in decreased expression of KEAP1. In addition, a slight but detectable increase in NQO-1 protein was observed in H1299 cells after 24 hours of treatment with 10 μM artesunate.

[0190] Because KEAP1 expression is reduced in both cell lines by artesunate, we used siRNA to knock down KEAP1 in both H1299 (sensitive) and A549 (resistant) cells to determine whether KEAP1 expression controls sensitivity to artesunate. The siRNAs used for this experiment were a pool of four sequences that did not overlap with the mutation site found in A549 cells; therefore, they were predicted to knock down both wild-type and mutant KEAP1 mRNA and thus reduce KEAP1 protein levels. Knockdown of mutant KEAP1 in A549 cells did not affect NQO-1 expression or response to artesunate (Figure 13). However, knockdown of KEAP1 in H1299 cells resulted in increased NQO-1 expression, indicating activation of the NRF2 antioxidant transcription pathway. In addition, knockdown of KEAP1 in H1299 cells increased the mean IC50 of artesunate from 0.98 μM in siNT-transfected cells to 2.30 μM in siKEAP1-transfected cells (Figures 14 and 15). Next, we evaluated the effect of KEAP1 knockdown on artesunate-induced DNA damage. In cells (A549) with an inactivating KEAP1 mutation, knockdown of KEAP1 had no effect on artesunate-induced DNA damage. In these cells, treatment with 25 μM cisplatin alone resulted in a significant increase in DNA damage compared to matched control cells, with a fold change in pH2AX staining of 4.255 ± 0.759 in siNT-transfected cells and 3.791 ± 0.957 in siKEAP1-transfected cells (Figure 16). In H1299 cells, knockdown of KEAP1 resulted in a significant reduction in artesunate-induced DNA damage when cells were treated with 50 μM and 100 μM artesunate or 25 μM cisplatin compared to cells treated with non-targeting control siRNA (siNT) (Figure 17).Specifically, 50 μM artesunate resulted in a 2.203 ± 0.170-fold increase in DNA damage in siNT-transfected cells, whereas only a 1.440 ± 0.186-fold increase in DNA damage was observed in siKEAP1-transfected cells compared with corresponding control-treated cells. Treatment with 100 μM artesunate and 25 μM cisplatin also resulted in significantly less DNA damage in KEAP1-knockdown cells by nuclear pH2AX staining (1.502 ± 0.168-fold and 2.678 ± 0.580-fold, respectively) compared with 2.238 ± 0.251 and 3.599 ± 0.414 in siNT-transfected cells. Thus, dysregulation of the NRF2 pathway by reduced expression of KEAP1 or loss-of-function KEAP1 mutations leads to increased resistance to artesunate in NSCLC cell lines.

[0191] 2.11 NRF2 inhibition sensitizes resistant NSCLC cells (KEAP1 mutant) to artesunate Because activation of the NRF2 pathway occurs downstream of KEAP1 loss, we next investigated whether pharmacological inhibition of NRF2 could sensitize resistant A549 cells to artesunate. Using ML385 (N-[4-[2,3-dihydro-1-(2-methylbenzoyl)-1H-indol-5-yl]-5-methyl-2-thiazolyl]-1,3-benzodioxole-5-acetamide), a small molecule shown to bind NRF2 and inhibit its function as a transcription factor by preventing DNA binding, we tested the effect of NRF2 inhibition on artesunate sensitivity in both A549 (less sensitive) and H1299 (sensitive) cells. Before treating cells with a combination of artesunate and ML385, we first evaluated the effect of ML385 alone on A549 and H1299 cells. ML385 had minimal effects on cell viability as a single agent in either cell line, with no significant differences in IC50 observed between the two cell lines tested. A549 and H1299 cells were then treated with increasing concentrations of artesunate alone or in combination with 5 μM ML385. To adjust for any effects of adding 5 μM ML385 directly to the cells, the percent viability of cells treated with artesunate alone was normalized to the DMSO (vehicle) control, while cells treated with artesunate + 5 μM ML385 were normalized to cells treated with 5 μM ML385. The addition of 5 μM ML385 shifted the IC50 from 13.3 μM in A549 cells treated with artesunate + 0.1% DMSO to 5.60 μM in cells treated with artesunate + 5 μM ML385, indicating that NRF2 inhibition sensitized these cells to artesunate (see Figures 18 and 19). In the H1299 (artesunate-sensitive) cell line, there was also a slight leftward shift in IC50 (from 2.35 μM to 1.19 μM in combination with ML385); however, this change was not statistically significant (see Figure 19). Next, the ability of 5 μM ML385 to enhance artesunate-induced DNA damage in A549 and H1299 cells was evaluated. For this assay, cells were pretreated with 5 μM ML385 or 0.03% DMSO for 24 hours.The following day, fresh medium was prepared using 0.03% DMSO or 5 μM ML385 alone or with 10 μM, 50 μM, or 100 μM artesunate or 25 μM cisplatin, and the cells were incubated for an additional 24 hours. Nuclear pH2AX staining was quantified from three independent experiments and normalized to control cells pretreated with 0.03% DMSO (Figures 20 and 21). ML385 alone did not increase DNA damage in A549 (resistant) or H1299 (sensitive) cells. Cotreatment with ML385 and artesunate resulted in a statistically significant increase in DNA damage only in A549 (less sensitive) cells treated with the highest dose of 100 μM artesunate, compared with cells treated with 5 μM ML385 alone. Specifically, 100 μM artesunate plus 5 μM ML385 resulted in 1.390 ± 0.327-fold increased DNA damage compared to 0.806 ± 0.142 in cells treated with 5 μM ML385 alone. In H1299 cells, cotreatment with artesunate and ML385 resulted in a significant increase in DNA damage at both 50 μM and 100 μM artesunate (3.431 ± 0.685 and 4.309 ± 1.309, respectively), but not in cells treated with 10 μM artesunate (2.187 ± 0.401).

[0192] 2.12 Artesunate and NRF2 inhibition are synergistic To better understand the effectiveness of the combination of artesunate with the NRF2 inhibitor (ML385), we next tested whether this drug combination was synergistic. A synergistic drug interaction occurs when one or both of the drugs used improve the effectiveness of the partner drug, thus allowing patients to achieve clinical benefit using lower concentrations of each drug. To evaluate synergy, we used a 6x6 checkerboard method in conjunction with both the Bliss and ZIP models to evaluate drug combinations. The Bliss-independent model has been one of the standards for evaluating drug combinations since its introduction in 1933 (Bliss, CI The Toxicity of Poisons Applied Jointlyl. Ann Appl. Biol 1939, 26, 585-615, doi:10.1111 / j.1744-7348.1939.tb06990.x), while the ZIP model was developed in 2015 to address some of the limitations of the Loewe and Bliss model (Yadav, B.; Wennerberg, K.; Aittokallio, T.; Tang, J. Searching for Drug Synergy in Complex Dose-Response Landscapes Using an Interaction Potency Model. Comput Struct. Biotechnol J. 2015, 13, pp. 504-513, doi:10.1016 / j.csbj.2015.09.001).

[0193] The output of both of these models is a synergy score, where a negative score indicates antagonism, while a positive score indicates synergy. When artesunate and ML385 were combined in A549 (artesunate-resistant) cells, the Bliss score was 9.38 (Figure 22), while the ZIP score was 8.744. In H1299 (artesunate-sensitive) cells, the Bliss score was 15.07 (Figure 23), and the ZIP score was 16.593. This indicates that artesunate and ML385 are synergistic in NSCLC cell lines regardless of KEAP1 mutation status. In addition, synergy is observed at clinically achievable plasma concentrations of artesunate (0.1-0.5 μM).

Claims

1. at least one compound having at least one 1,2,4-trioxane moiety, or a) at least one compound having at least one 1,2,4-trioxane moiety, and b) at least one chlorogenic acid An anti-cancer agent comprising a combination of:

2. The compound comprising at least one 1,2,4-trioxane moiety is represented by formula (I) to (V): 【Chemistry 1】 and, where applicable, pharmaceutically acceptable salts of said compounds of formula (I) and (II). (In formula (I), The arrows indicate the oxygen atoms and residues R 1 indicates the bond between n is an integer greater than 1, preferably from 2 to 10, more preferably 2, 3 or 4, even more preferably 2 or 3; R 1 is a residue substituted n times by the residue depicted in brackets, preferably C 1 ~C 18 -Alkyl, C 2 ~C 18 -alkenyl or -(CO) n (R 3 ), where the carboyl group is a residue R 1 together with the oxygen bonded to R to form a carboxylic acid ester moiety. 3 is C 1 ~C 18 -alkane-n-yl or C 2 ~C 18 -alkene-n-yl, Said C 1 ~C 18 -Alkyl, C 2 ~C 18 -Alkenyl, C 1 ~C 18 -Alkan-n-yl, C 2 ~C 18 -alkene-n-yl group - Uninterrupted, or -0-、-S-、-SO 2 -、-SO-、-SO 2 NR 4 -、NR 4 S0 2 -、-NR 4 -、-CO-、-O(CO)-、(CO)O-、-0(C0)0-、-NR 4 (CO)NR 4 -、NR 4 (CO)-、-(CO)NR 4 -、-NR 4 (CO)0-、-0(CO)NR 4 - and is interrupted one, two or more times by non-consecutive functional groups selected from the group consisting of: uninterrupted or additionally or alternatively interrupted once, twice or more than twice by a divalent residue selected from the group consisting of heterocyclo-diyl and aryldiyl, - not replacing, or in addition or alternatively Hydroxy, halogen, cyano, azide, C 6 ~C 14 -aryl, C 1 ~C 8 -alkoxy, C 1 ~C 8 -Alkylthio, -SO 3 M, -COOM, PO 3 M 2 , -PO(N(R 5 ) 2 ) 2 , PO(OR 5 ) 2 , -S0 2 N(R 4 ) 2 , -N(R 4 ) 2 , -C0 2 N(R 5 ) 2 , -COR 4 , -OCOR 4 , -NR 4 (CO)R 5 , -(CO)OR 4 , -NR 4 (C0)N(R 4 ) 2 and is substituted once, twice or more than twice with a substituent selected from the group consisting of: In formula (II), R 2 is C 1 ~C 18 -alkyl or C 2 ~C 18 -alkenyl or -(CO)R 3 where the carboyl group is the residue R 1 together with the oxygen bonded to R to form a carboxylic acid ester moiety. 3 is C 1 ~C 18 -alkyl or C 2 ~C 18 is alkenyl, Said C 1 ~C 18 -alkyl and C 2 ~C 18 The alkenyl group is - Uninterrupted, or -0-, -S-, -SO 2 -, -SO-, -SO 2 NR 4 -, NR 4 S0 2 -, -NR 4 -, -CO-, -O(CO)-, (CO)O-, -0(C0)0-, -NR 4 (CO)NR 4 -, NR 4 (CO)-, -(CO)NR 4 -, -NR 4 (C0)0- or -0(CO)NR 4 - and is interrupted one, two or more times by non-consecutive functional groups selected from the group consisting of: uninterrupted or additionally or alternatively interrupted once, twice or more than twice by a divalent residue selected from the group consisting of heterocyclo-diyl and aryldiyl, - not replacing, or in addition or alternatively Hydroxy, halogen, cyano, azide, C 6 ~C 14 -aryl, C 1 ~C 8 -alkoxy, C 1 ~C 8 -Alkylthio, -SO 3 M, -COOM, PO 3 M 2 , -PO(N(R 5 ) 2 ) 2 , PO(OR 5 ) 2 , -S0 2 N(R 4 ) 2 , -N(R 4 ) 2 , -C0 2 N(R 5 ) 2 , -COR 4 , -OCOR 4 , -NR 4 (CO)R 5 , -(CO)OR 4 or -NR 4 (CO)N(R 4 ) 2 and is substituted once, twice or more than twice with a substituent selected from the group consisting of: In all the above formulas, when used: R 4 is hydrogen, C 1 ~C 8 -Alkyl, C 6 ~C 14 -independently selected from the group consisting of aryl, and heterocyclyl, or N(R 4 ) 2 is, as a whole, an N-containing heterocycle, R 5 is C 1 ~C 8 -Alkyl, C 6 ~C 14 -independently selected from the group consisting of aryl, and heterocyclyl, or N(R 5 ) 2 is, as a whole, an N-containing heterocycle, M is hydrogen or a q-valent metal ion equivalent to 1 / q, or an ammonium ion or a guanidinium ion, or a primary, secondary, tertiary or quaternary organic ammonium ion, in particular a cation of the formula [N(C 1 ~C 18 -alkyl) s H t ] + where s is 1, 2, 3 or 4 and t is (4-s).

3. The anticancer agent according to claim 1 or 2, wherein the compound comprising at least one 1,2,4-trioxane moiety is selected from the group consisting of those of formula (IIa), artemether, and those of formula (IIb), artesunate, and pharmaceutically acceptable salts of artesunate. 【Chemistry 2】

4. The anticancer agent according to any one of claims 1 to 3, wherein the compound comprising at least one 1,2,4-trioxane moiety is selected from artesunate and pharmaceutically acceptable salts of artesunate.

5. 5. The anticancer agent according to claim 1, wherein the at least one chlorogenic acid is selected from 3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid, 3-O-feluoylquinic acid, 4-O-feluoylquinic acid, 5-O-feluoylquinic acid, 3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid.

6. 6. The anticancer agent according to claim 1, wherein the molar ratio between the one or more compounds having at least one 1,2,4-trioxane moiety and the one or more chlorogenic acids present in the combination is between 2 and 0.002, preferably between 0.8 and 0.005, more preferably between 0.5 and 0.01, and even more preferably between 0.1 and 0.

01.

7. 7. The anticancer agent according to claim 1, wherein the compound comprising at least one 1,2,4-trioxane moiety is obtained via extraction of Artemisia annua.

8. 8. The anticancer agent according to claim 1, comprising an extract of Artemisia annua.

9. 9. The anticancer agent according to claim 1, wherein the at least one chlorogenic acid is obtained via extraction of coffee or tea.

10. 10. An anti-cancer agent according to any one of claims 1 to 9, comprising an extract obtained by co-extraction of Artemisia annua and coffee and / or tea with water, preferably at 40°C to 100°C.

11. 11. The anti-cancer agent according to any one of claims 1 to 10, obtained via co-extraction of Artemisia annua and coffee and / or tea with water, preferably at 40°C to 100°C.

12. 12. The anticancer agent of claim 7, 8, 10 or 11, wherein the Artemisia annua plant is of the Apollo variety.

13. 13. The anticancer agent according to any one of claims 1 to 12, wherein the molar ratio between the one or more compounds having at least one 1,2,4-trioxane moiety and the one or more chlorogenic acids present in the combination is between 2 and 0.002, preferably between 0.8 and 0.005, more preferably between 0.5 and 0.01, and even more preferably between 0.1 and 0.

01.

14. The anticancer agent according to any one of claims 1 to 13, further comprising at least one compound selected from the group consisting of scopoletin, 1,8-cineole, artemisinic acid, arteannuin-B, dihydroartemisinic acid, fisetin, casticin, artemetin, chrysoprenetin, chrysoprenol-D, and circilsilineol.

15. A pharmaceutical composition comprising the anticancer agent according to any one of claims 1 to 14.

16. 16. A pharmaceutical composition comprising the anticancer agent of any one of claims 1 to 15, further comprising at least one additional therapeutic agent.

17. 17. A pharmaceutical composition comprising the anticancer agent of claim 16, wherein the additional therapeutic agent is selected from the group consisting of gemtricitabine, cisplatin, carboplatin, pemetrexed or paclitaxel and / or an NRF2 inhibitor, such as ML385.

18. (a) the dosage of the anticancer agent or pharmaceutical composition according to any one of claims 1 to 15; and (b) the dose of the additional therapeutic agent, which is platinum-based doublet chemotherapy (PT-DC); and (c) Instructions for using the anticancer agent or pharmaceutical composition according to any one of claims 1 to 15 and the additional therapeutic agent. Kit including:

19. A kit comprising: (a) the dosage of the anticancer agent or pharmaceutical composition according to any one of claims 1 to 17; and (b) dosages of NRF2 inhibitors, as well as for the kits; (c) Instructions for using the anticancer agent or pharmaceutical composition according to the present invention and the NRF2 inhibitor Includes a kit.

20. An anticancer agent according to claims 1 to 14, or a pharmaceutical composition according to claims 15 to 17, or a kit according to claims 18 and 19, for use as a medicament, in particular a medicament for the treatment and / or prevention of cancer or for the prolongation of the life of a subject with cancer.

21. A method for preventing or treating cancer in a subject having cancer or for prolonging the survival of a subject having cancer, the method comprising administering to the subject having cancer a therapeutically effective amount of an anticancer agent described in claims 1 to 14, or a pharmaceutical composition described in claims 15 to 17, or a kit described in claims 18 and 19.

22. A method for treating cancer in a subject or prolonging survival of a subject with cancer, comprising simultaneously or sequentially administering to a subject with cancer a therapeutically effective amount of an anticancer agent described in claims 1 to 14, or a pharmaceutical composition described in claims 15 to 17, or a kit described in claims 18 and 19, and at least one additional therapeutic agent.

23. 23. The method according to claim 21 or 22, wherein the daily dose range of the anticancer agent or pharmaceutical composition is 0.01 to 100 mg / kg body weight, preferably 0.1 to 50 mg / kg body weight, and even more preferably 0.5 to 50 mg / kg body weight, calculated on the total amount of the compound having at least one 1,2,4-trioxane moiety.

24. 24. The method of any one of claims 21 to 23, wherein the cancer is selected from melanoma, multiple myeloma, carcinoma, sarcoma, leukemia, lymphoma, brain and spinal cord tumors, germ cell tumors, neuroendocrine tumors, and carcinoid tumors.

25. At least to those who need it (a) The anticancer agent or pharmaceutical composition according to any one of claims 1 to 17 (b)(i) gemcitabine and cisplatin, preferably 1250 mg / m 2 Gemcitabine at a dose of 75 mg / m 2 (ii) pemetrexed and cisplatin, preferably at a dose of 500 mg / m 2 and pemetrexed at a dose of 75 mg / m 2 or (iii) paclitaxel and carboplatin, preferably at a dose of 200 mg / m 2 and a platinum-based doublet chemotherapy (PT-DC) that is a combination of paclitaxel at a dose of 0.05 mg / mL and carboplatin at a dose of 0.05 mg / mL and a target area under the curve (AUC6) of 0.05 mg / mL.

22. The method of claim 21, comprising administering

26. The additional therapeutic agent is paclitaxel and carboplatin, preferably at 200 mg / m 2 26. The method of claim 25, wherein the combination of paclitaxel at a dose of 0.05 mg / ml / min and carboplatin at a target area under the curve (AUC6) of 0.05 mg / ml / min is 0.05 mg / ml / min.

27. 27. The method of claim 25 or 26, wherein components a) and b) are administered simultaneously.

28. 28. The method of any one of claims 25 to 27, wherein the cancer is ovarian cancer.

29. At least to those who need it (a) The anticancer agent or pharmaceutical composition according to any one of claims 1 to 17 (b) an additional therapeutic agent selected from the group consisting of NRF2 inhibitors, preferably ML385 (N-[4-[2,3-dihydro-1-(2-methylbenzoyl)-1H-indol-5-yl]-5-methyl-2-thiazolyl]-1,3-benzodioxole-5-acetamide) 22. The method of claim 21, comprising administering

30. 30. The method of claim 29, wherein the cancer is non-small cell lung cancer.

31. A method for destroying or inhibiting the growth of cancer cells, comprising exposing the cancer cells in vitro or in vivo to an effective amount of an anticancer agent, pharmaceutical composition or kit according to claims 1 to 19.

32. 32. The method of claim 31, wherein an additional therapeutic agent is used.

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