Combinations of artemisinins, BCL-2 inhibitors, and kinase inhibitors for cancer treatment

Abstract

Methods of treating cancer, such as leukemia, via administration of therapeutically effective amounts of artemisinins and one or more additional therapeutic agents are detailed herein. The artemisinins include ART714. The one or more additional therapeutic agents include BCL-2 inhibitors such as ABT-199, ABT-263, and ABT-737; kinase inhibitors such as lestaurtinib, midostaurin, sorafenib, and gilteritinib; and anti-neoplastic agents such as cytarabine, doxorubicin, etoposide, cyclophosphamide, triplotide, vinorelbine, cisplatin, and rituximab.

Description

COMBINATIONS OF ARTEMISININS, BCL-2 INHIBITORS, AND KINASE INHIBITORS FOR CANCER TREATMENTCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 466,803, filed on May 16, 2023. The content of this earlier filed application is hereby incorporated by reference herein in its entirety.STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

[0002] This invention was made with government support under the Grant Numbers CA006973, RR026824, GM066691 and TR003098 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF INVENTION

[0003] Acute myeloid leukemia (AML) disproportionately affects the elderly, who often cannot tolerate intensive chemotherapy [1]. Less intensive combination of the BCL-2 inhibitor venetoclax (VEN) plus low dose cytarabine (LDAC) or hypomethylating agents (HMAs) has revolutionized treatment of older and unfit patients with AML [2,3]. However, the response to currently approved VEN-based combinations is heterogeneous across cytogenetic and molecular subgroups [4,5], reducing ability to use these biomarkers to predict efficacy, and most patients who initially respond will eventually relapse, at which point outcomes are dismal [6] .

[0004] To address these challenges, much research has focused on improving the efficacy of VEN-based combination therapy in AML [7], by substituting or adding various agents, while simultaneously attempting to minimize toxicity. As a recent example, in a synergism- focused high-throughput screen of 64 antileukemic drugs, the FMS-like tyrosine kinase 3 (FLT3) inhibitor gilteritinib (GILT) had the highest degree of synergy with VEN against FLT3 wild-type AMLs [6]. This VEN plus GILT combination was active in a high risk patient-derived xenograft (PDX) model containing a TP53 mutation, as well as in several heavily pretreated FLT3 wild-type AML patients. The proposed mechanism of this synergy involved indirect reduction of MCL-1 levels by GILT. Additional preclinical studies have demonstrated MCL- 1 dependent synergy between GILT and VEN in FLT3 mutated AMLs[8-11]. Further studies have reported higher rates of response and MRD negativity with VEN- based 3-drug combinations in FLT3 mutated and other high risk AML subgroups [12,13].However, there is often a trade-off between efficacy and toxicity, particularly myelosuppression, and it is unclear whether the higher response rates will translate into increased survival [14,15]. Thus, there is a need for novel VEN-based treatment paradigms with increased therapeutic efficacy across AML subgroups and low toxicity.

[0005] Artemisinins (ARTs) have extremely low toxicity in worldwide use for malaria treatment. There is now mounting evidence that semi-synthetic ART derivatives have antileukemic activity [16-19] and synergize with current antileukemic drugs [20,16,21]. For example, dihydroartemisinin (DHA) synergized with the BCL-2 inhibitor navitoclax in acute lymphoblastic leukemias harboring the BCR-ABL chromosomal rearrangement, and the identified mechanism involved DHA-induced indirect downregulation of MCL-1 levels [6]. Other studies indicated MCL-1 dependent synergy among the three drugs of a novel “SAV” combination regimen, comprising the 3 -carbon- linked ART-derived dimer (3C-ART) ART838, the kinase inhibitor sorafenib (SOR), and VEN

[0020] .

[0006] Good drug-like pharmacology and low toxicity was recently reported from preclinical data for a novel class of 2-carbon- linked ART-derived dimers (2C-ARTs) with potent antileukemic activity

[0021] . Similar to ART838

[0020] , the first-in-class 2C-ART ART631 cooperated with VEN and SOR both in vitro and in vivo. Impressively, the ART631 containing SAV regimen was more active than the other tested 2- and 3-drug combinations (including ART838 containing SAV) in a relapsed, refractory AML PDX model

[0021] . The present application reports on the results of experimental investigations into combination therapies using ART631 and methods for treating cancer based thereon, along with other important matters.BRIEF SUMMARY OF INVENTION

[0007] Provided herein are novel drug combinations comprising artemisinins (ARTs) and one, two or three additional therapeutic agents, wherein the additional therapeutic agents are BCL-2 inhibitors, kinase inhibitors, and anti-neoplastic agents. These combinations can be used in the treatment of cancers, such as leukemia, in particular acute myeloid leukemia (AML), and in prolonging survival of subjects having cancer.

[0008] In a first embodiment, the present invention is drawn to methods of treating cancer in a subject, comprising administering therapeutically effective amounts of an artemisinin and one additional therapeutic agent to a subject having cancer. The artemisinin and the one additional therapeutic agent may be administered in either order, alone or in combination, sequentially or concurrently, with overlapping or non-overlapping periods of administration. The one additional therapeutic agent may be a BCL-2 inhibitor, a kinase inhibitor, or an anti- neoplastic agent. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0009] In a second embodiment, the present invention is drawn to methods of treating cancer in a subject, comprising concurrently administering therapeutically effective amounts of an artemisinin and one additional therapeutic agent to a subject having cancer. The artemisinin and the one additional therapeutic agent may be administered together or separately, with partially overlapping or fully overlapping periods of administration. The one additional therapeutic agent may be a BCL-2 inhibitor, a kinase inhibitor, or an anti- neoplastic agent. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0010] In a third embodiment, the present invention is drawn to methods of treating cancer in a subject, comprising sequentially administering therapeutically effective amounts of an artemisinin and one additional therapeutic agent to a subject having cancer. The artemisinin and the one additional therapeutic agent may be administered with partially overlapping or non-overlapping periods of administration. The one additional therapeutic agent may be a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0011] In a fourth embodiment, the present invention is drawn to methods of treating cancer in a subject, comprising administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer. The artemisinin and first and second additional agents may be administered in any order, alone or in any combination, sequentially or concurrently, with overlapping or non-overlapping periods of administration. The first and second additional agents may independently be a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent. In one aspect, the first additional therapeutic agent is a BCL-2 inhibitor and the secondadditional therapeutic agent is a kinase inhibitor. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0012] In a fifth embodiment, the present invention is drawn to methods of treating cancer in a subject, comprising administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin and one or both of the additional agents are administered concurrently to the subject. The artemisinin and one or both of the additional agents may be administered together or separately, with partially overlapping or fully overlapping periods of administration. The first and second additional agents may independently be a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent. In one aspect, the first additional therapeutic agent is a BCL-2 inhibitor and the second additional therapeutic agent is a kinase inhibitor. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0013] In a sixth embodiment, the present invention is drawn to methods of treating cancer in a subject, comprising administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin and one or both of the additional agents are administered sequentially, in any order, to the subject. The artemisinin and one or both of the additional agents may be administered with partially overlapping or non-overlapping periods of administration. The first and second additional agents may independently be a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent. In one aspect, the first additional therapeutic agent is a BCL-2 inhibitor and the second additional therapeutic agent is a kinase inhibitor. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0014] As suggested above, the novel drug combinations provided herein can also be used in methods of prolonging survival of subjects having cancer. Therefore, and in a seventh embodiment, the present invention is drawn to methods of prolonging survival of a subject having cancer, comprising administering therapeutically effective amounts of an artemisinin and one additional therapeutic agent to a subject having cancer. The artemisinin and the one additional therapeutic agent may be administered in either order, alone or in combination, sequentially or concurrently, with overlapping or non-overlapping periods of administration. The one additional therapeutic agent may be a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0015] In an eighth embodiment, the present invention is drawn to methods of prolonging survival of a subject having cancer, comprising concurrently administering therapeutically effective amounts of an artemisinin and one additional therapeutic agent to a subject having cancer. The artemisinin and the one additional therapeutic agent may be administered together or separately, with partially overlapping or fully overlapping periods of administration. The one additional therapeutic agent may be a BCL-2 inhibitor, a kinase inhibitor, or an anti -neoplastic agent. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0016] In a ninth embodiment, the present invention is drawn to methods of prolonging survival of a subject having cancer, comprising sequentially administering therapeutically effective amounts of an artemisinin and one additional therapeutic agent to a subject having cancer. The artemisinin and the one additional therapeutic agent may be administered with partially overlapping or non-overlapping periods of administration. The one additional therapeutic agent may be a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0017] In a tenth embodiment, the present invention is drawn to methods of prolonging survival of a subject having cancer, comprising administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer. The artemisinin and first and second additional agents may be administered in any order, alone or in any combination, sequentially or concurrently, with overlapping or non-overlapping periods of administration. The first and second additional agents may independently be a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent. In one aspect, the first additional therapeutic agent is a BCL-2 inhibitor and the second additional therapeutic agent is a kinase inhibitor. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0018] In an eleventh embodiment, the present invention is drawn to methods of prolonging survival of a subject having cancer, comprising administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin and one or both of the additional agents are administered concurrently to the subject. The artemisininand one or both of the additional agents may be administered together or separately, with partially overlapping or fully overlapping periods of administration. The first and second additional agents may independently be a BCL-2 inhibitor, a kinase inhibitor, or an anti- neoplastic agent. In one aspect, the first additional therapeutic agent is a BCL-2 inhibitor and the second additional therapeutic agent is a kinase inhibitor. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0019] In a twelfth embodiment, the present invention is drawn to methods of prolonging survival of a subject having cancer, comprising administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin and one or both of the additional agents are administered sequentially, in any order, to the subject. The artemisinin and one or both of the additional agents may be administered with partially overlapping or non-overlapping periods of administration. The first and second additional agents may independently be a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent. In one aspect, the first additional therapeutic agent is a BCL-2 inhibitor and the second additional therapeutic agent is a kinase inhibitor. In one aspect of the invention, the artemisinin is ART714 or a metabolite thereof.

[0020] In each embodiment and aspect of the invention, the artemisinin is a 2-carbon- linked artemisinin-derived dimer (2C-ART). As specific examples, the artemisinin may be, but is not limited to, one or more of ART578-S, ART591, ART605, ART629, ART631, ART662, ART662-N, ART668, ART671, ART685, ART694, ART699, ART712, ART714, ART716, ART732, ART733, ART733-M, ART733-O, ART742, ART750, and ART757, or a metabolite thereof.

[0021] In each embodiment and aspect of the invention, the BCL-2 inhibitor may be, but is not limited to, one or more of ABT-199 (venetoclax (VEN)), ABT-263, and ABT-737.

[0022] In each embodiment and aspect of the invention, the kinase inhibitor may be, but is not limited to, one or more of lestaurtinib (LES), midostaurin (MID), sorafenib (SOR) and gilteritinib (GILT).

[0023] In each embodiment and aspect of the invention, the anti-neoplastic agent may be, but is not limited to, one or more of cytarabine (ARA), doxorubicin (DOX), etoposide (ETO), cyclophosphamide, triplotide, vinorelbine, cisplatin, and rituximab.

[0024] In each embodiment and aspect of the invention, the artemisinin and the additional therapeutic agents (i.e., the drugs) may be formulated in pharmaceutical compositions comprising one of the drugs, or combinations of two, three or more of the drugs, and a pharmaceutically acceptable carrier or diluent.

[0025] In each embodiment and aspect of the invention, the artemisinin and the additional therapeutic agent(s) may be administered via the same or different modes of administration, and combinations thereof when there are more than two drugs being administered to a subject.

[0026] In certain aspects of each embodiment, the combination of the artemisinin and the additional therapeutic agent(s) has an additive therapeutic effect on the cancer. In other aspects of each embodiment, the combination of the artemisinin and the additional therapeutic agent(s) has a synergistic therapeutic effect on the cancer.

[0027] In certain aspects of each embodiment, the artemisinin and the additional therapeutic agent(s) are administered to the subject in the same pharmaceutical composition and via the same mode of administration.

[0028] It will also be apparent that a number of different combinations of an artemisinin and one or more additional therapeutic agents may be used when practicing the various embodiments and aspects of the invention. Such combinations include, but are not limited to: the artemisinin is ART714 or a metabolite thereof and the one additional therapeutic agent is ABT-737; the artemisinin is ART714 or a metabolite thereof and the one additional therapeutic agent is ABT-199; the artemisinin is ART714 or a metabolite thereof and the one additional therapeutic agent is ABT-263; the artemisinin is ART714 or a metabolite thereof and the one additional therapeutic agent is ARA; the artemisinin is ART714 or a metabolite thereof and the one additional therapeutic agent is DOX; the artemisinin is ART714 or a metabolite thereof and the one additional therapeutic agent is ETO; the artemisinin is ART714 or a metabolite thereof and the one additional therapeutic agent is MID; the artemisinin is ART714 or a metabolite thereof and the one additional therapeutic agent is LES; the artemisinin is ART714 or a metabolite thereof and the one additional therapeutic agent is SOR; and the artemisinin is ART714 or a metabolite thereof and the one additional therapeutic agent is GILT.

[0029] Additional combinations include, but are not limited to: the artemisinin is ART714 or a metabolite thereof, the first additional therapeutic agent is a BCL-2 inhibitor,and the second additional therapeutic agent is a kinase inhibitor; the artemisinin is ART714 or a metabolite thereof, the first additional therapeutic agent is ABT- 199, and the second additional therapeutic agent is SOR; the artemisinin is ART714 or a metabolite thereof, the first additional therapeutic agent is ABT- 199, and the second additional therapeutic agent is GILT.

[0030] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising concurrently administering therapeutically effective amounts of an artemisinin and one additional agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the one additional agent is ABT-737, ABT- 199, ABT-263, ARA, DOX, ETO, MID, LES, SOR or GILT, and wherein the combination of the artemisinin and the one additional agent has an additive therapeutic effect on the cancer.

[0031] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising concurrently administering therapeutically effective amounts of an artemisinin and one additional agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the one additional agent is ABT-737, ABT- 199, ABT-263, ARA, DOX, ETO, MID, LES, SOR or GILT, and wherein the combination of the artemisinin and the one additional agent has a synergistic therapeutic effect on the cancer.

[0032] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising sequentially administering therapeutically effective amounts of an artemisinin and one additional agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the one additional agent is ABT-737, ABT- 199, ABT-263, ARA, DOX, ETO, MID, LES, SOR or GILT, and wherein the combination of the artemisinin and the one additional agent has an additive therapeutic effect on the cancer.

[0033] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising sequentially administering therapeutically effective amounts of an artemisinin and one additional agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the one additional agent is ABT-737, ABT- 199, ABT-263, ARA, DOX, ETO, MID, LES, SOR or GILT, and wherein the combination of the artemisinin and the one additional agent has a synergistic therapeutic effect on the cancer.

[0034] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising concurrently administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to asubject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT- 199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has an additive therapeutic effect on the cancer.

[0035] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising concurrently administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT- 199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has a synergistic therapeutic effect on the cancer.

[0036] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising sequentially administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT- 199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has an additive therapeutic effect on the cancer.

[0037] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising sequentially administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT- 199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has a synergistic therapeutic effect on the cancer.

[0038] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising concurrently administering therapeutically effective amounts of an artemisinin and one additional agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the one additional agent is ABT-737, ABT- 199, ABT-263, ARA, DOX, ETO, MID, LES, SOR or GILT, and wherein the combination of the artemisinin and the one additional agent has an additive therapeutic effect on the cancer.

[0039] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising concurrently administering therapeutically effective amounts of an artemisinin and one additional agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the one additional agent is ABT-737, ABT- 199, ABT-263, ARA, DOX, ETO, MID, LES, SOR or GILT, and wherein the combination of the artemisinin and the one additional agent has a synergistic therapeutic effect on the cancer.

[0040] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising sequentially administering therapeutically effective amounts of an artemisinin and one additional agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the one additional agent is ABT-737, ABT- 199, ABT-263, ARA, DOX, ETO, MID, LES, SOR or GILT, and wherein the combination of the artemisinin and the one additional agent has an additive therapeutic effect on the cancer.

[0041] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising sequentially administering therapeutically effective amounts of an artemisinin and one additional agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the one additional agent is ABT-737, ABT- 199, ABT-263, ARA, DOX, ETO, MID, LES, SOR or GILT, and wherein the combination of the artemisinin and the one additional agent has a synergistic therapeutic effect on the cancer.

[0042] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising concurrently administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT-199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has an additive therapeutic effect on the cancer.

[0043] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising concurrently administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additionaltherapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT-199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has a synergistic therapeutic effect on the cancer.

[0044] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising sequentially administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT-199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has an additive therapeutic effect on the cancer.

[0045] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising sequentially administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT-199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has a synergistic therapeutic effect on the cancer.

[0046] In each embodiment and aspect of the invention, the therapeutically effective amount of the artemisinin is between about 0.1 and 100 mg / kg body weight of the subject.

[0047] In each embodiment and aspect of the invention, the therapeutically effective amount of the BCL-2 inhibitor agent is between about 0. 1 and 50 mg / kg body weight of the subject.

[0048] In each embodiment and aspect of the invention, the therapeutically effective amount of the kinase inhibitor agent is between about 0. 1 and 100 mg / kg body weight of the subject.

[0049] In each embodiment and aspect of the invention, the therapeutically effective amount of the anti-neoplastic agent is between about 0. 1 and 100 mg / kg body weight of the subject.

[0050] In each embodiment and aspect of the invention, the cancer may be, but is not limited to, a cancer overexpressing a BCL-2 family member (e.g., Bcl-2, Bcl-x(L), Bcl-w, Mcl-1, Bcl-b (Al)) or a cancer overexpressing a kinase or having an increased or constitutively active kinase (e.g., tyrosine kinases, serine / threonine kinases). Examples of cancers exhibiting overexpression of BCL-2 include, but are not limited to, hematologic malignancies (including leukemia, lymphoma, and multiple myeloma) and solid tumors (including prostate, breast, small cell and non-small cell lung cancers, ovarian, neuroblastoma, bladder, colorectal, and head and neck cancers. Examples of cancers overexpressing a kinase or having an increased or constitutively active kinase include, but are not limited to, hematologic malignancies (including leukemia, lymphoma, and multiple myeloma) and solid tumors (including prostate, breast, small cell and non-small cell lung cancers, ovarian, neuroblastoma, bladder, colorectal, and head and neck cancers.

[0051] In each embodiment and aspect of the invention, the cancer may be, but is not limited to, one or more of acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), B cell ALL (B-ALL), T cell ALL (T-ALL), chronic myeloid leukemia (CML), chronic lymphoid leukemia (CLL); lymphoma; myeloma; myelodysplastic syndrome; non-small cell lung cancer; pancreatic cancer; gastric cancer; Kaposi’s sarcoma; hepatocellular carcinoma; osteosarcoma; laryngeal squamous cell carcinoma; metastatic uveal melanoma; lung and splenic metastases; advanced non-small cell lung cancer; cervical carcinoma; colorectal cancer; breast cancer; prostate cancer; and other hematologic malignancies and solid cancers including but not limited to brain cancers.

[0052] The present invention is also directed to the following chemical compounds, each of which is a metabolite of ART714.

[0053] The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described herein, which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that any conception and specific embodiment disclosed herein may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that any description, figure, example, etc. is provided for the purpose of illustration and description only and is by no means intended to define the limits the invention.BRIEF DESCRIPTION OF DRAWINGS

[0054] FIG. 1. ART714 and ART733 potently inhibited the growth of M0LM14 AML cells, with activity comparable to ART838. M0LM14 AML cells were cultured with a concise range of concentrations of ART838, ART714, and ART733 in triplicate for 48h.Graph indicates growth inhibition relative to vehicle (DMSO)-treated cells by alamarBlue metabolic assays

[0055] FIGS. 2A and 2B. ART714, ART733, and ART838-based combination treatments reduced cell viability in AML cell lines via reactive oxygen species (ROS)- mediated apoptosis. Fig. 2A. M0LM14 AML cells were treated for 18h with vehicle (DMSO) control, ART838 alone (200 nM), ART714 alone (200 nM), ART733 alone (200 nM), SOR alone (5 pM), VEN alone (50 nM), and 2- and 3-drug combinations, each with and without a 30-45 min pre -incubation with 10 nM pan-caspase inhibitor QVD-OPh (QVD). Treated cells were stained with 2.5 pg / mL 7-aminoactinomycin D (7-AAD) and 0.5 pg / mL allophycocyanin (APC)-conjugated Annexin V in Annexin V binding buffer and analyzed by flow cytometry. Graphs indicate % cell viability based on Annexin V / 7-AAD dual staining. The ART-SAV combinations were assessed in the +QVD experiments. Fig. 2B. MOLM14 AML cells were treated for 18h with vehicle (DMSO), ART838 (200 nM), ART714 (200 nM), ART733 (200 nM), or SOR (5 pM). Protein was isolated and western blotted for CHOP, MCL1, BCL2, and (3-actin.

[0056] FIG. 3. Concentration-time profiles of ART714 in NRG mice (n=3) treated with single or multiple doses of 60 mg / kg PO (MTD). Plasma was obtained over 12h, with ART714 concentration determined by LC / MS-MS. Dashed line represents the highest in vitro IC50 of ART714 (22 nM). Data points and error bars represent mean and SD of 3 replicates, respectively.

[0057] FIG. 4. Both VEN plus GILT and VEN plus GILT plus ART714 prolonged survival in M0LM14 AML xenografts substantially. (A) NRG mice were transplanted IV with Luc-labeled M0LM14 cells on day -10. After Xenogen quantification of baseline total body luminescence on day 0, mice were placed into balanced experimental groups prior to drug administration via gavage, then treated per our previously established MTD PO x 5 day schedule. Mice were treated with 60 mg / kg / d ART714 as monotherapy, 150 mg / kg / d VEN as monotherapy, 30 mg / kg / d GILT as monotherapy; 2-drug combinations of ART714 plus VEN, ART714 plus GILT, or VEN plus GILT; or with ART714 plus VEN plus GILT. Mice received a single 5 -day treatment cycle. (B) shows corresponding Kaplan-Meier survival curves. Mouse deaths empirically defined to have occurred for reasons other than leukemia (i.e., deaths of mice with luminescence at or below average background luminescence (5.7 x 105photons / mouse based on intensity values from non-leukemia bearing mice) werecensored. Comparisons between treatment groups were calculated by log-rank (Mantel-Cox) test. (C) shows % body weight change (+SEM) in each treatment group.

[0058] FIG. 5. Structure of Artemisinins.DETAILED DESCRIPTION OF THE INVENTIONI. Definitions

[0059] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found, for example, in Benjamin Lewin, Genes VII, published by Oxford University Press, 2000 (ISBN 019879276X);Kendrew et al. (eds.); The Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar technical references.

[0060] As used herein, “a” or “an” may mean one or more. As used herein when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one. As used herein “another” may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.

[0061] As used herein, “about” refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term “about” generally refers to a range of numerical values (e.g., + / - 5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term “about” may include numerical values that are rounded to the nearest significant figure.II. The Present Invention

[0062] Pursuing clinical development of 2-carbon-linked artemisinin (ART)-derived dimer (2C-ART)-containing combinations to treat acute myeloid leukemia (AML), the stability and solubility properties of ART631, a 2C-ART molecule previously reported, was rigorously assessed. As previously reported, ART631 had somewhat limited in vitro stability

[0021] . It was found that ART631 was not fully soluble in physiologic phosphate -buffered saline solutions, thus indicating that it was likely not an optimal lead 2C-ART. As a result,and as reported herein, 22 additional 2C-ARTs with published in vivo antimalarial activity

[0022] were assessed in an effort to identify a best-in-class 2C-ART lead compound. As also reported herein, the strategy involved culling out 2C-ARTs inferior to ART631 with respect to potency, stability, and solubility, and then validating in vivo pharmacokinetics (PK), pharmacodynamics (PD), and efficacy of the selected 2C-ART compounds. As a result of these studies, ART714 was identified as a lead compound. Additional studies demonstrated that ART714 may be used as monotherapy and in combinations with VEN (ABT- 199) and / or the FLT3 inhibitor GILT in a new 3 -drug regimen herein referred to as “FAV.”

[0063] Combinations of artemisinins and these additional therapeutic agents form the basis of the present invention and they may be used in methods of treating subjects having various forms of cancer and methods of prolonging survival in a subject having cancer, as detailed below.

[0064] The invention is generally directed to methods of treating cancer or prolonging survival in a subject by administering therapeutically effective amounts of the artemisinins and the additional therapeutic agents detailed herein to a subject having cancer. As will be evident from the present disclosure, there are a number of different artemisinins that show activity against cancer cells that may be used in these methods. Similarly, there are several different classes of additional therapeutic agents, e.g., BCL-2 inhibitors, kinase inhibitors, and anti-neoplastic agents, that can be used in the combination therapies. Further, the manner in which the drugs (i.e., the artemisinins and the additional therapeutic agents) are administered to a subject may vary, and include administration of the drugs in either order, together in various combinations or separately, sequentially or concurrently, with overlapping or non- overlapping periods of administration. It will thus be clear to the skilled person that the methods of the present invention can be practiced with wide latitude and that the scope of the claims is not narrow.Artemisinins

[0065] Artemisinins (ARTs) are rapidly acting antimalarial drugs that are effective against asexual life stages of the Plasmodium falciparum malaria parasite

[0029] . Artemisinin (FIG. 5) was first identified in 1972 as the active component of the sweet wormwood plant (Artemisia annua L.), used in traditional Chinese medicine to treat fevers (likely caused by malaria)

[0025] . A 1979 publication by the Qinghaosu Antimalarial Coordinating Research Group demonstrated that artemisinin is a sesquiterpene lactone with an endoperoxidepharmacophore [25,26]. While best known for their antimalarial activity, artemisinins also inhibit growth of a broad range of microbes and cancer cells, especially leukemias [30,44- 54]. Akin to the efficacy of the artemisinin artesunate (AS) in multidrug-resistant malaria, AS (FIG. 5) was as effective against chemotherapy (doxorubicin, hydroxyurea, methotrexate, or vincristine)-resistant CEM (T-ALL) sub-lines as against the parental line

[0044] . The proven safety profile of the drug in humans makes it an attractive candidate for further study in the treatment of cancer.

[0066] Due to the poor solubility and bioavailability of artemisinin, numerous semi- and fully-synthetic derivatives, referred to collectively as “artemisinins” have been developed with improved pharmacological properties

[0027] . The first generation of artemisinin derivatives includes dihydroartemisinin (DHA, the active metabolite of artemisinin; FIG. 5), artesunate (AS), artemether, arteether, and artelinate

[0028] . ART-838 (FIG. 5) is a second- generation artemisinin derivative, which includes dimers and trimers of the named drugs. The dimers and trimers were developed to increase stability, bioavailability, half-life, and efficacy. Trioxane dimers are one subset of semi-synthetic dimeric artemisinin derivatives, characterized by two artemisinin monomers coupled by a linker of varying length, flexibility, and composition, with enhanced antimalarial, antimicrobial, and antineoplastic activity over first-generation artemisinins [30-41]. ART- 838 (artemisinin-derived trioxane diphenylphosphate dimer 838; FIG. 5) exhibits enhanced antineoplastic activity over the artemisinin monomer or the monomeric artemisinin derivative, AS [39,42]. Other phosphate ester derivatives of artemisinin have demonstrated enhanced anti-leukemic activity

[0043] . Allyloxime-631 (ART-631; FIG. 5) is a novel dimeric artemisinin analog that has been recently developed

[0062] ,

[0067] The artemisinins that may be used in the methods of the present invention are 2- carbon- linked artemisinin-derived dimers (2C-ART) and metabolites thereof. The 2C-ARTs of the present invention include, but are not limited to, each of ART578-S, ART591, ART605, ART629, ART631, ART662, ART662-N, ART668, ART671, ART685, ART694, ART699, ART712, ART714, ART716, ART732, ART733, ART733-M, ART733-O, ART742, ART750, and ART757 shown below, and metabolites of each of these compounds.ART629 ART733-orthoART671 ART750ART699 ART662

[0068] Particular 2C-ART metabolites encompassed by the invention include metabolites of ART714. Suitable metabolites of ART714 include, but are not limited to, the following metabolites.Additional Therapeutic Agents

[0069] As indicated above, the methods of the present invention are practiced using combinations of an artemisinin and one or more additional therapeutic agents. As artemisinins exhibit therapeutic activity when administered to a subject having cancer, the agents discussed in the following paragraphs are termed “additional” therapeutic agents because they are administered in conjunction with one or more artemisinins. The additional therapeutic agents may be any drug, toxin, poison, agent, etc. that has the ability to inhibit, block, kill, etc. a cancer cell, either directly or indirectly. Suitable additional therapeutic agents include several different classes of agents, e.g., BCL-2 inhibitors, kinase inhibitors, inhibitors of other enzymes and pathways involving activities crucial to cancer, and anti- neoplastic agents including biological molecules as well as small molecule drugs.

[0070] As used herein, reference to a “BCL-2 inhibitor” means an inhibitor of any member of the BCL-2 family of proteins. Such proteins include, but are not limited to, the BCL-2 protein itself, the BCL-XL protein, the MCL-1 protein and other structurally similar proteins well-known to comprise the BCL-2 family of proteins. The BCL-2 inhibitors include, but are not limited to, ABT- 199 (venetoclax; VEN), ABT-263 (navitoclax), and ABT-737.

[0071] As used herein, reference to a “kinase inhibitor” means an inhibitor of any kinase associated with uncontrolled, i.e. neoplastic, cell growth. Such kinases include, but arelimited to, tyrosine kinases and serine / threonine kinases. The kinase inhibitors include, but are not limited to, lestaurtinib (LES), midostaurin (MID), and sorafenib (SOR), each of which target multiple kinases, including FLT3, as well as the FLT3 -selective FMS-like tyrosine kinase 3 inhibitor gilteritinib (GILT). Sorafenib inhibits multiple kinases which may mediate its antileukemic activity, with the importance of the targets varying from case to case; e.g.FLT3 is an important target in many AMLs. In addition, several reports have found that SOR reduces MCL1 protein stability and translation [60,61].

[0072] As used herein, reference to an “anti-neoplastic agent” means an agent that has activity against uncontrolled, i.e. neoplastic, cell growth. The anti-neoplastic agents include, but are not limited to, cytarabine (ARA), doxorubicin (DOX) [46,56], etoposide (ETO), cyclophosphamide

[0057] , triplotide

[0058] , vinorelbine, cisplatin

[0055] , and rituximab

[0059] .

[0073] When referring to “the additional therapeutic agent” or “an additional therapeutic agent”, it should be understood that these terms refer to both a single additional therapeutic agent as well as two or more additional therapeutic agents. When referring to “the additional agent” or “an additional agent” as a short-hand, it should be understood that these terms refer to additional therapeutic agents.Combinations

[0074] As summarized above, there are a number of different combinations of an artemisinin and the one or more additional therapeutic agents that may be used when practicing the various embodiments and aspects of the invention. Such combinations include, but are not limited to, (i) an artemisinin and a BCL-2 inhibitor; (ii) an artemisinin and a kinase inhibitor; (iii) an artemisinin and an anti-neoplastic agent; (iv) an artemisinin, a BCL-2 inhibitor and a kinase inhibitor; (v) an artemisinin, a kinase inhibitor and an anti-neoplastic agent; (vi) an artemisinin, a BCL-2 inhibitor, and an anti-neoplastic agent; and (vii) an artemisinin, a BCL-2 inhibitor, a kinase inhibitor, and an anti-neoplastic agent.

[0075] In particular examples of these combinations, the artemisinin is ART714 or a metabolite thereof, the BCL-2 inhibitor is ABT-737, ABT- 199 or ABT-263, the kinase inhibitor is SOR, and the anti-neoplastic agent is ARA, DOX, ETO, MID or LES. Additional combinations include, but are not limited to, those where the artemisinin is ART714 or a metabolite thereof, the BCL-2 inhibitor is ABT-737, ABT- 199 or ABT-263, the kinase inhibitor is GILT, and the anti-neoplastic agent is ARA, DOX, ETO, MID or LES.

[0076] Further combinations include, but are not limited to, those where the artemisinin is ART714 or a metabolite thereof, the BCL-2 inhibitor is ABT- 199, and the kinase inhibitor is SOR, those where the artemisinin is ART714 or a metabolite thereof, the BCL-2 inhibitor is ABT- 199, and the kinase inhibitor is GILT, those where the artemisinin is ART714 or a metabolite thereof, the BCL-2 inhibitor is ABT- 199, the kinase inhibitor is SOR, and the anti- neoplastic agent is ARA, DOX, ETO, MID or LES, and those where the artemisinin is ART714 or a metabolite thereof, the BCL-2 inhibitor is ABT- 199, the kinase inhibitor is GILT, and the anti-neoplastic agent is ARA, DOX, ETO, MID or LES.Cancer

[0077] The methods of the present invention can be used in the treatment of a variety of cancers and in prolonging survival in subjects having a variety of cancers. It will be apparent that different combinations of the drugs can be used in the treatment of different types of cancer and prolonging survival of subjects having different types of cancer. It will also be apparent that particular combinations of drugs may differ in effectiveness depending on the type of cancer, the stage and grade of a particular cancer, the physical location of the cancer within the subject, the molecular abnormalities in the cancer, and available means for administering the drugs, among other factors.

[0078] Exemplary cancers that may be treated via the methods of the invention include cancers overexpressing a BCL-2 family member (e.g., Bcl-2, Bcl-x(L), Bcl-w, Mcl-1, Bcl-b (Al)) and cancers overexpressing a kinase or having an increased or constitutively active kinase (e.g., tyrosine kinases, serine / threonine kinases). Examples of cancers exhibiting overexpression of BCL-2 include, but are not limited to, hematologic malignancies (including leukemia, lymphoma, and multiple myeloma) and solid tumors (including prostate, breast, small cell and non-small cell lung cancers, ovarian, neuroblastoma, bladder, colorectal, and head and neck cancers. Examples of cancers overexpressing a kinase or having an increased or constitutively active kinase include, but are not limited to, hematologic malignancies (including leukemia, lymphoma, and multiple myeloma) and solid tumors (including prostate, breast, small cell and non-small cell lung cancers, ovarian, neuroblastoma, bladder, colorectal, and head and neck cancers.

[0079] Exemplary cancers that may be treated via the methods of the invention also include, but are not limited to, leukemias including acute leukemias, such as acute myeloidleukemia (AML) and acute lymphoid leukemia (ALL; including B cell ALL (B-ALL) and T cell ALL (T-ALL)), chronic leukemias, such as chronic myeloid leukemia (CML) and chronic lymphoid leukemia (CLL); lymphoma; myeloma; myelodysplastic syndrome; nonsmall cell lung cancer; pancreatic cancer; gastric cancer; Kaposi’s sarcoma; hepatocellular carcinoma; osteosarcoma; laryngeal squamous cell carcinoma; metastatic uveal melanoma; lung and splenic metastases; advanced non-small cell lung cancer; cervical carcinoma; colorectal cancer; breast cancer; prostate cancer; and other hematologic malignancies and solid cancers including but not limited to brain cancer.Pharmaceutical Compositions

[0080] The artemisinins and the additional therapeutic agents used in the methods of the invention may be formulated in pharmaceutical compositions comprising pharmaceutically acceptable carriers, excipients and / or diluents. It will be apparent that depending on the identity of the particular drugs being used in the combinations, a suitable pharmaceutical composition may comprise a single drug or type of drug (i.e., one or more artemisinin) or combinations comprising two or more drugs or types of drug (i.e., one or more artemisinin and one or more additional therapeutic agent in the same pharmaceutical composition). As used herein, the terms “drug” and “drugs” mean the artemisinins, BCL-2 inhibitors, kinase inhibitors and anti-neoplastic agents as defined herein.

[0081] The pharmaceutical compositions may be formulated, for example, for oral, sublingual, intranasal, intraocular, rectal, transdermal, mucosal, pulmonary, topical or parenteral administration. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), intra-arterial, intramedulary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of drug formulations can be used to effect such administration. In preferred aspects of each of the embodiments on the invention, the pharmaceutical composition is administered to the subject as an oral formulation.

[0082] Pharmaceutically acceptable carriers, excipients and diluents are those compounds, solutions, substances or materials that can be used to produce formulations of the drugs that are suitable to be administered to a subject, such as a human. In particular, carriers, excipients and diluents of the present invention are those useful in preparing a pharmaceuticalcomposition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and that may present pharmacologically favorable profiles, and includes carriers and diluents that are acceptable for veterinary use as well as human pharmaceutical use. Suitable pharmaceutically acceptable carriers, excipients and diluents are well known in art and can be determined by those of skill in the art as the clinical situation warrants. Examples of suitable carriers and diluents include dextrose, water, glycerol, ethanol, propylene glycol, polysorbate 80 (Tween-80™), poly(ethylene)glycol 300 and 400 (PEG 300 and 400), PEGylated castor oil (e.g. Cremophor EL), poloxamer 407 and 188, a cyclodextrin or a cyclodextrin derivative (including HPCD ((2-hydroxypropyl)-cyclodextrin) and (2- hydroxyethyl)-cyclodextrin), hydrophilic and hydrophobic carriers, and combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, PEGylated phospholipids, polymer matrices, biocompatible polymers, lipospheres, vesicles, particles, and liposomes. The terms specifically exclude cell culture medium. More particularly: (1) 5% (w / v) dextrose, or (2) water (e.g., sterile water; Water-For-Injection), may be used as a pharmaceutically acceptable carrier. Pharmaceutically acceptable diluents also include tonicity agents that make the composition compatible with blood. Tonicity agents are particularly desirable in injectable formulations.

[0083] Excipients included in a formulation have different purposes depending, for example on the nature of the drug, and the mode of administration. Examples of generally used excipients include, without limitation: stabilizing agents, solubilizing agents and surfactants, buffers, antioxidants and preservatives, tonicity agents, bulking agents, lubricating agents, emulsifiers, suspending or viscosity agents, inert diluents, fillers, disintegrating agents, binding agents, wetting agents, lubricating agents, antibacterials, chelating agents, sweeteners, perfuming agents, flavoring agents, coloring agents, administration aids, and combinations thereof.

[0084] The pharmaceutical compositions may contain common carriers and excipients, such as cornstarch or gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, croscarmellose sodium, and sodium starch glycolate.

[0085] The particular carrier, diluent or excipient used will depend upon the means and purpose for which the active ingredient is being applied.

[0086] Acceptable methods for preparing the pharmaceutical compositions according to the invention are known to those skilled in the art. For example, pharmaceutical compositions may be prepared following conventional techniques of the pharmaceutical chemist involving steps such as mixing, granulating, and compressing when necessary for tablet forms, or mixing, filling, and dissolving the ingredients as appropriate, to give the desired products for various routes of administration.Modes of Administration

[0087] The pharmaceutical compositions and the drugs of the present invention may be formulated, for example, for oral, enteral, sublingual, intranasal, intraocular, rectal, intravaginal, transdermal, mucosal, topical or parenteral administration. Parenteral modes of administration include without limitation, intradermal, subcutaneous (s.c., s.q., sub-Q, Hypo), intramuscular (i.m.), intravenous (i.v.), intraperitoneal (i.p.), intra-arterial, intramedullary, intracardiac, intra-articular (joint), intrasynovial (joint fluid area), intracranial, intraspinal, and intrathecal (spinal fluids). Any known device useful for parenteral injection or infusion of drug formulations can be used to effect such administration. In certain aspects of each of the embodiments of the invention, the pharmaceutical compositions are administered to the subject intravenously.

[0088] Formulations for parenteral administration can be in the form of aqueous or nonaqueous isotonic sterile solutions, suspensions or fat emulsions. The unit dosage of these solutions or suspensions can be in a concentrated liquid, powder or granular form for ex tempore reconstitution in the appropriate pharmaceutically acceptable carrier, such as sterile water, at the time of delivery. In addition to the above-mentioned excipients, powder forms optionally include bulking agents (e.g. mannitol, glycine, lactose, sucrose, trehalose, dextran, hydroxyethyl starch, ficoll and gelatin), and cryo or lyoprotectants. In an alternative embodiment, the parenteral unit dosage form of pharmaceutical compositions and the antibacterial agents of the present invention can be a ready-to-use solution of the pharmaceutical compositions and the antibacterial agents in a suitable carrier in sterile, hermetically sealed ampoules or in sterile pre-loaded syringes. The suitable carrier optionally comprises any of the above-mentioned excipients. The parenteral form used for injection must be fluid to the extent that easy syringability exists.

[0089] Excipients used in parenteral preparations may also include, without limitation, stabilizing agents (e.g. carbohydrates, amino acids and polysorbates, such as 5% dextrose), solubilizing agents (e.g. cetrimide, sodium docusate, glyceryl monooleate, polyvinylpyrolidone (PVP) and polyethylene glycol (PEG)), surfactants (e.g., polysorbates, tocopherol PEG succinate, poloxamer and Cremophor™), buffers (e.g. acetates, citrates, phosphates, tartrates, lactates, succinates, amino acids and the like), antioxidants and preservatives (e.g., BHA, BHT, gentisic acids, vitamin E, ascorbic acid, sodium ascorbate and sulfur containing agents such as sulfites, bisulfites, metabisulfites, thioglycerols, thioglycolates and the like), tonicity agents (for adjusting physiological compatibility), suspending or viscosity agents, antibacterials (e.g., thimersol, benzethonium chloride, benzalkonium chloride, phenol, cresol and chlorobutanol), chelating agents, and administration aids (e.g. local anesthetics, anti-inflammatory agents, anti-clotting agents, vaso-constrictors for prolongation and agents that increase tissue permeability), and combinations thereof.

[0090] Parenteral formulations using hydrophobic carriers include, for example, fat emulsions and formulations containing lipids, lipospheres, vesicles, particles and liposomes. Fat emulsions include in addition to the above-mentioned excipients, a lipid and an aqueous phase, and additives such as emulsifiers (e.g. phospholipids, poloxamers, polysorbates, and polyoxyethylene castor oil), and osmotic agents (e.g. sodium chloride, glycerol, sorbitol, xylitol and glucose). Liposomes include natural or derived phospholipids and optionally stabilizing agents such as cholesterol.

[0091] In intravenous (IV) use, a sterile formulation of the pharmaceutical compositions of the present invention and optionally one or more additives, including solubilizers or surfactants, can be dissolved or suspended in any of the commonly used intravenous fluids and administered by infusion. Intravenous fluids include 5% dextrose in water.

[0092] In intramuscular preparations, a sterile formulation of the pharmaceutical compositions of the present invention can be dissolved and administered in a pharmaceutical diluent such as Water-for-Injection (WFI) or 5% dextrose in water. A suitable insoluble form of the pharmaceutical compositions may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.

[0093] When formulated into separate pharmaceutical compositions, the pharmaceutical compositions may be administered via the same or different modes of administration. In certain aspects of each invention, the artemisinin and one or more of the additional therapeutic agents are administered to the subject in the same pharmaceutical composition and via the same mode of administration.

[0094] The timing of administration used in the methods of the invention will vary depending on a number of factors, including whether there is concurrent or sequential administration, the identity of the drugs, the identity of the cancer, the physical characteristics of the subject, the severity of the subject’s symptoms, and the formulation and the means used to administer the drugs, among other factors. However, administration frequencies of the drugs will generally include 4, 3, 2 or once daily, every other day, every third day, every fourth day, every fifth day, every sixth day, once weekly, every eight days, every nine days, every ten days, bi-weekly, monthly and bi-monthly, whether the drugs are administered alone or in combination, concurrently or sequentially. In certain aspects, the concurrent or sequential administration is administration once daily. The duration of treatment will be based on the cancer being treated and will be best determined by the attending physician. Under some conditions, treatment will be continued for a number of days, weeks, or months. Under other conditions, complete treatment will be achieve through administering one, two or three doses of the combinations over the entire course of treatment.Methods of Treatment

[0095] As discussed in the summary of the invention above, methods of the invention include methods of treating cancer in a subject comprising administering therapeutically effective amounts of an artemisinin and one or more additional therapeutic agents to a subject having cancer. The artemisinin and the one or more additional therapeutic agents may be administered in any order, separately or in combination (two drugs per combination, or three drugs per combination, or four or more drugs per combination), sequentially or concurrently, with overlapping or non-overlapping periods of administration. The methods of the invention thus include methods of treating cancer in a subject, comprising concurrently administering therapeutically effective amounts of an artemisinin and one or more additional therapeutic agents to a subject having cancer. The methods of the invention thus also include methods of treating cancer in a subject, comprising sequentially administering therapeutically effectiveamounts of an artemisinin and one or more additional therapeutic agents to a subject having cancer.

[0096] The terms “treating” and “treatment” mean at least the mitigation of cancer, or a disease condition or symptom associated with cancer in a subject that is achieved by a reduction of growth, replication, and / or propagation, or death or destruction of cancer and / or cancer cells, on or in the subject. The terms “treating” and “treatment” include curing, healing, inhibiting, relieving from, improving and / or alleviating, in whole or in part, the cancer or associated disease condition or symptom. The mitigation of cancer or associated disease condition or symptom may be about 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% in the subject, versus a subject to which the combinations taught herein have not been administered. In one aspect, treating means reducing the population of cancer cells causing the cancer in the subject to an undetectable level, where detection is by any conventional means, such assay a blood sample in the laboratory. In another aspect, treating means complete healing of the cancer, shown by an absence of clinical symptoms associated with the cancer. In a further aspect of the invention, treating means the mitigation of cancer or an associated disease condition or symptom by at least about 90% in the subject. In an additional aspect, treating means the mitigation of cancer or an associated disease condition or symptom by at least about 95% in the subject.

[0097] The methods of the invention include also methods of prolonging survival of a subject having cancer comprising administering therapeutically effective amounts of an artemisinin and one or more additional therapeutic agents to a subject having cancer. The artemisinin and the one or more additional therapeutic agents may be administered in any order, separately or in combination (two drugs per combination, or three drugs per combination, or four or more drugs per combination), sequentially or concurrently, with overlapping or non-overlapping periods of administration. The methods of the invention thus include methods of prolonging survival of a subject having cancer, comprising concurrently administering therapeutically effective amounts of an artemisinin and one or more additional therapeutic agents to a subject having cancer. The methods of the invention thus also include methods of prolonging survival of a subject having cancer, comprising sequentially administering therapeutically effective amounts of an artemisinin and one or more additional therapeutic agents to a subject having cancer.

[0098] The term “prolonging survival” means extending the life span of a subject having cancer by at least one day versus a subject having the same cancer that does not receive the artemisinin and one or more additional therapeutic agents. Prolonged survival includes increasing the life span of the subject by at least: 1, 2, 3, 4 or more weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months, or 1, 2, 3, 4, 5, or more years.

[0099] The amount of the drugs sufficient to have an effect on cancer (additive, synergistic or otherwise) in a subject will vary, for example, in view of the identity of the drugs being used in the combination, the physical characteristics of the subject, the severity of the subject’s symptoms, the form of the cancer, the identity of the cancer, the formulations and means used to administer the drugs, and the method being practiced. The specific dose for a given subject is usually set by the judgment of the attending physician. However, in each dose comprising an artemisinin is typically between about 0. 1 and about 100 mg / kg body weight. In some aspects, about 0.5-50 mg / kg, about 0.5-40 mg / kg, about 0.5-30 mg / kg, about 0.5-20 mg / kg, about 1-20 mg / kg, about 2-20 mg / kg, about 2-10 mg / kg, about 2-8 mg / kg, about 2-6 mg / kg, about 5-100 mg / kg , or about 10-100 mg / kg body weight.

[0100] In each dose comprising a BCL-2 inhibitor, the amount of the drug is typically between about 0.1 and about 50 mg / kg body weight. In some aspects, about 0.1-40 mg / kg, about 0.1-30 mg / kg, about 0.1-20 mg / kg, about 0.1-25 mg / kg, about 0.1-10 mg / kg, about 1- 50 mg / kg, about 2-50 mg / kg, about 2.5-50 mg / kg, about 2.5-40 mg / kg, about 2.5-30 mg / kg , or about 2.5-20 mg / kg body weight.

[0101] In each dose comprising a kinase inhibitor, the amount of the drug will vary due to the size of this class of agents. However, it will typically be between about 0.1 and about 100 mg / kg body weight.

[0102] In each dose comprising an anti-neoplastic agent, the amount of the drug will vary due to the size of this class of agents. However, it will typically be between about 0. 1 and about 100 mg / kg body weight.

[0103] Depending on the means of administration, the dose may be administered all at once, such as with an oral formulation in a capsule, or slowly over a period of time, such as with an intravenous administration. For slower means of administration, the administering period can be a matter of minutes, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120 or more minutes, or a period of hours, such as about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 or more hours. The administration of the dose maybe interrupted, such as where the dose is administered via intravenous infusion and the dose is divided into two or more infusion bags. Under such circumstances, the administration of the dose may be interrupted while the infusion bags are changed.

[0104] As used herein, the terms “dose”, “unit dose”, “dosage”, “effective dose” and related terms refer to physically discrete units that contain a predetermined quantity of active ingredient or therapeutic agent (drug) calculated to produce a desired therapeutic effect. A single dose is thus a predetermined quantity of an artemisinin and / or one or more additional therapeutic agent that is administered to a subject.

[0105] As used herein, a “subject” is a human, a non-human primate, bird, horse, cow, goat, sheep, a companion animal, such as a dog, cat or rodent, or other mammal.Additive and Synergistic Effects

[0106] In many instances, the combinations of drugs taught herein, i.e., the combinations of artemisinins and one or more additional therapeutic agents, target two or more different aspects of a cancer cell, such as two or more different structures, or two or more different pathways, or one or more structures on one hand and one or more pathways on the other. As a result, while the combinations of artemisinins and one or more additional therapeutic agents may have an additive therapeutic effect on a cancer, the combinations may also or alternatively have a synergistic therapeutic effect on the cancer. Synergistic therapeutic effects are those that are substantially greater than what is seen when cancer cells are treated with either drug alone.

[0107] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising concurrently administering therapeutically effective amounts of an artemisinin and an additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the additional therapeutic agent is a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent, and wherein the combination of the artemisinin and the additional therapeutic agent has an additive therapeutic effect on the cancer. In certain aspects, the additional therapeutic agent is ABT-737, ABT- 199, ARA, DOX, ETO, MID, LES, SOR or GILT.

[0108] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising concurrently administering therapeutically effective amounts of an artemisinin and an additional therapeutic agent to a subject having cancer, wherein theartemisinin is ART714 or a metabolite thereof, wherein the additional therapeutic agent is a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent, and wherein the combination of the artemisinin and the additional therapeutic agent has a synergistic therapeutic effect on the cancer. In certain aspects, the additional therapeutic agent is ABT-737, ABT- 199, ARA, DOX, ETO, MID, LES, SOR or GILT.

[0109] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising sequentially administering therapeutically effective amounts of an artemisinin and an additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the additional therapeutic agent is a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent, and wherein the combination of the artemisinin and the additional therapeutic agent has an additive therapeutic effect on the cancer. In certain aspects, the additional therapeutic agent is ABT-737, ABT- 199, ARA, DOX, ETO, MID, LES, SOR or GILT.

[0110] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising sequentially administering therapeutically effective amounts of an artemisinin and an additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the additional therapeutic agent is a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent, and wherein the combination of the artemisinin and the additional therapeutic agent has a synergistic therapeutic effect on the cancer. In certain aspects, the additional therapeutic agent is ABT-737, ABT- 199, ARA, DOX, ETO, MID, LES, SOR or GILT.

[0111] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising concurrently administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT- 199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has an additive therapeutic effect on the cancer.

[0112] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising concurrently administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, whereinthe first additional therapeutic agent is ABT-737, ABT- 199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has a synergistic therapeutic effect on the cancer.

[0113] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising sequentially administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT- 199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has an additive therapeutic effect on the cancer.

[0114] In a particular aspect, the invention is directed to a method of treating cancer in a subject, comprising sequentially administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT- 199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has a synergistic therapeutic effect on the cancer.

[0115] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising concurrently administering therapeutically effective amounts of an artemisinin and an additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the additional therapeutic agent is a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent, and wherein the combination of the artemisinin and the additional therapeutic agent has an additive therapeutic effect on the cancer. In certain aspects, the additional therapeutic agent is ABT- 737, ABT- 199, ARA, DOX, ETO, MID, LES, SOR or GILT.

[0116] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising concurrently administering therapeutically effective amounts of an artemisinin and an additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the additional therapeutic agent is a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent, and wherein the combination of the artemisinin and the additional therapeutic agent has a synergistictherapeutic effect on the cancer. In certain aspects, the additional therapeutic agent is ABT- 737, ABT- 199, ARA, DOX, ETO, MID, LES, SOR or GILT.

[0117] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising sequentially administering therapeutically effective amounts of an artemisinin and an additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the additional therapeutic agent is a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent, and wherein the combination of the artemisinin and the additional therapeutic agent has an additive therapeutic effect on the cancer. In certain aspects, the additional therapeutic agent is ABT- 737, ABT- 199, ARA, DOX, ETO, MID, LES, SOR or GILT.

[0118] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising sequentially administering therapeutically effective amounts of an artemisinin and an additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the additional therapeutic agent is a BCL-2 inhibitor, a kinase inhibitor, or an anti-neoplastic agent, and wherein the combination of the artemisinin and the additional therapeutic agent has a synergistic therapeutic effect on the cancer. In certain aspects, the additional therapeutic agent is ABT- 737, ABT- 199, ARA, DOX, ETO, MID, LES, SOR or GILT.

[0119] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising concurrently administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT-199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has an additive therapeutic effect on the cancer.

[0120] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising concurrently administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT-199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein thecombination of the artemisinin and the additional therapeutic agents has a synergistic therapeutic effect on the cancer.

[0121] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising sequentially administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT-199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has an additive therapeutic effect on the cancer.

[0122] In a particular aspect, the invention is directed to a method of prolonging survival of a subject having cancer, comprising sequentially administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the artemisinin is ART714 or a metabolite thereof, wherein the first additional therapeutic agent is ABT-737, ABT-199, or ABT-263, and the second additional therapeutic agent is SOR or GILT, and wherein the combination of the artemisinin and the additional therapeutic agents has a synergistic therapeutic effect on the cancer.III. ExamplesMaterials and MethodsReagents

[0123] The reagents used were previously described

[0021] ,Animals

[0124] Immunodeficient NOD- Gg / '"'7' IL2rg,ul1(NRG) mice were purchased from Jackson Laboratory (Bar Harbor, ME), bred and housed at the University of Maryland Baltimore. The animal study was reviewed and approved by Animal Care and Use Program, University of Maryland Baltimore.Cells

[0125] MOLM14, KGla, and U937 human AML lines used for in vitro antileukemic activity assays were as detailed in Table 1 of Kagan et al

[0021] . Lentiviral-transduced luciferase (luc)-labeled M0LM14 AML cells were previously described

[0020] .Cytotoxicity

[0126] Cytotoxic antileukemic activity of 2C-ARTs was assessed in triplicate using alamarBlue metabolic assays with 48hr IC50 values determined over a range of drug concentrations (InM-luM) (Life Technologies, Grand Island, NY) and Annexin V77- aminoactinomycin D (7AAD) immunostaining (BioLegend, San Diego, CA) assays per manufacturer’s guidelines.Solubility

[0127] Solubility of 2C-ARTs was assessed by visual observation at 10 mM in DMSO, with warming to 37°C, shaking, and dilution to 5 mM as needed. 2C-ARTs were then diluted from their respective 5 mM or 10 mM DMSO stock solutions to 1 pM in PBS and solubility determined by the appearance of particles that could be detected via dynamic laser light scattering using a Malvern Zetasizer (Malvern, PA) over a period of approximately 15 minutes in a single determination. This method was used because the solubility was below the available analytical method detection limit. Thus, the 2C-ART derivative was added to PBS at concentrations around the estimated solubility, and then the solutions were measured to see if particles were present at concentrations greater than the background count. The presence of particles indicated that the tested concentration is above the solubility. Thus, by using different concentrations and the presence of particles, the solubility could be bracketed.Metabolic Stability

[0128] Stability of 2C-ARTs was assessed in PBS and human and mouse plasma and liver microsomes as previously described

[0021] . Briefly, for phase I microsomal stability assays, 2C-ARTs (10 pM in <1% DMSO) were incubated in PBS containing liver microsomes, with or without cofactors. Microsomal controls (without cofactors) were used to determine non-enzymatic degradation. The experiments were incubated at 37°C for 2 time points (0.5h and Ih) and performed in duplicate. The results of the 0.5h assessments (notshown) were utilized as a confirmation of the direction and magnitude of the definitive Ih assessments, on which stability decisions were made. If there was discordance between the 0.5h and Ih experiment, the experiment was repeated.Western Analysis

[0129] Western blotting to assess molecular markers of apoptosis was performed as described

[0020] . Cellular protein was extracted, quantitated, electrophoresed, and western blotted with monoclonal antibodies. Antibody-specific band intensities were quantitated by densitometry (ChemiDOC XRS+and Image Laboratory System; BioRad, Hercules, CA) and normalized to (3-actin.Pharmacokinetics (PK)

[0130] Mice were dosed orally with ART714 at 60 mg / kg as a single dose or multiple doses (once daily x5). Plasma was obtained over 12h, and ART714 concentrations determined using our previously described LC-MS / MS method, with the lower limit of quantitation being 5 ng / mL

[0021] . Pharmacokinetic parameters were calculated from mean concentration-time data, using non-compartmental methods in Phoenix WinNonlin version 8.3 (Certara, Princeton, NJ)

[0021] . The Method of Bailer was used to estimate the variance of AUC given the calculated variance of the mean concentration at each time point

[0023] . A pairwise comparison utilizing Z test was used to determine whether there was a significant difference between ART714 exposures as expressed by AUCiast

[0024] , In each case, p < 0.05 was considered statistically significant.Biodistribution

[0131] Mice (n=3 / time point) were dosed orally with ART714 at the MTD as single or multiple doses and were euthanized at 2, 3, 4, and 6 h after dosing. ART714 was quantified in plasma as described above, as well as in bone marrow, kidney, spleen, liver, and brain (ng / g tissue). The ratio of ng / g tissue to plasma concentration was then determined. If at least one sample had a detectable specimen, then 50% of the lower limit of quantitation (2.5 ng / mL) was utilized to calculate the average and standard deviation.Metabolites

[0132] Mice (n=3 / time point) were dosed orally with ART714 at the MTD as single or multiple doses and were euthanized at 1, 1.5, and 3 h after dosing was completed. LC- MS / MS was used to scan for proposed metabolites of ART714 in plasma, bone marrow, kidney, spleen, liver, and brain, based on molecular weight.Mutagenicity

[0133] The Ames mutagenicity test was conducted by Cyprotex (Watertown, MA). The maximum concentration tested was 200 pg / mL (280. 1 pM ), which is based on the maximum solubility under test conditions.Arrhythmogenicity

[0134] Testing of the arrhythmogenic potential (hERG channel) assay was conducted at 25 pM by Aptuit (Greenwich, CT).AML xenograft assays

[0135] M0LM14 xenograft assays were performed as described previously [20,21], using 4 mice per treatment group; mice were treated with a single 5-day course of drugs. The doses for the experiment were ART714 60 mg / kg / d, VEN 150 mg / kg / d PO, or GILT 30 mg / kg / d PO, alone or in combination.RESULTSEleven 2C-ARTs were soluble in physiologic solutions and had activity profiles similar to ART838

[0136] In an initial alamarBlue screening assay, the 22 tested 2C-ARTs had antileukemic cytotoxic activity against M0LM14 and U937, with IC50 values ranging from 6-176 nM and 0.5-151 nM, respectively (Table 1). Similar to ART838, the 2C-ARTs were less potent against KG la. Most of the 22 2C-ARTs were readily soluble visually at 10 mM in DMSO. However, three 2C-ARTs (ART685, ART694, ART750) required warming to 37°C and vigorous shaking for dissolution at 10 mM in DMSO, and four 2C-ARTs (ART591, ART671, ART732, ART733-M) required dilution to 5 mM to fully dissolve in DMSO. Eleven of the 22 2C-ARTs were objectively soluble at 1 pM in PBS when diluted from their respective 5 or10 mM DMSO stock solutions, as measured by laser light scattering diffraction analysis. ART733-O was partially insoluble in PBS, but was included in further assessment of pharmacological properties because it was the most potent antileukemic 2C-ART in preliminary testing.ART714 and ART733 had superior in vitro stability

[0137] The in vitro stability was assessed for the eleven 2C-ARTs that were objectively soluble in PBS plus ART733-O. Using the pre-determined criterion of >50% of the parent compound remaining after Ih incubation, ART714 was consistently stable under the tested conditions (Table 1). The stability profile of ART714 closely approximated that of ART838

[0020] . Both ART733 and ART733-O were also stable, with >50% of the parent compound remaining after Ih incubation under the tested conditions for ART733-O and in all but one category for ART733 (41% of parent compound remaining after Ih incubation in mouse plasma). Taking the antileukemic activity, solubility and stability results together, ART714 and ART733 were selected for further testing, while ART733-O was deprioritized based on its suboptimal solubility.Table 1: In vitro IC50 values, and solubility and stability data for 2C-ARTs.AML cell lines were treated with a range of concentrations of the 22 2C-ART derivatives for 48h. IC50S were calculated based on observed growth inhibition relative to vehicle (DMSO)-treated cells in one experiment performed in triplicate using alamarBlue absorbance assays. For solubility in DMSO, white represents compounds that were soluble instantly or upon shaking, orange represents compounds that required vigorous shaking or heating to 37°C for dissolution, and black represents compounds that required dilution to 5 mM for dissolution. Solubility in PBS at 1 pM was determined objectively using laser light scattering diffraction analysis as detailed in Methods; green represents compounds with a clean peak comparable to ART838, gray represents compounds with larger particle size or a broader peak compared with ART838, and red represents compounds with multiple peaks indicating precipitation and / or poor solubility. The values listed in the Stability section represent % compound remaining after Ih incubation at 37°C. A second time point (30 min) was assessed but data are not shown. “ND” indicates “not done.”ART714 and ART733 had similar antileukemic potencies as ART838

[0138] More precise determinations of the 48hr IC50 values for the top two selected compounds (ART714 and ART733) were performed in MOLM14 AML cells in triplicate wells for each data point. In these repeat alamarBlue assays using a focused concentration range centered around the IC50 values determined in Table 1, ART714 and ART733 were potent against M0LM14, with calculated IC50 values of 22 and 32 nM, respectively (FIG. 1).The antileukemic potency of ART714 was virtually identical to that of ART838 across the concentrations tested (FIG. 1), as were their calculated IC50S (21 nM for ART838).ART714 and ART733 induced apoptotic cell death and cooperated with VEN and SOR

[0139] Annexin / 7AAD assays confirmed that ART714 and ART733, like ART838 and ART631

[0021] , each induced apoptotic cell death of M0LM14 cells (FIG. 2A). ART plus VEN combinations had greater activity than ART plus SOR combinations, as was noted previously [20,21], The VEN plus SOR combination was also highly effective. The 3-drug “SAV” combinations induced maximal apoptotic killing in these 18h assays, inhibited by the caspase inhibitor QVD-Oph (QVD).ART714 and ART733 reduced MCL1 and increased CHOP levels in M0LM14 cells

[0140] Like ART838 and ART631, in previous experiments

[0021] , ART714 and ART733 each induced C / EBP homologous protein (CHOP), a protein known to be upregulated and play a role in the cellular stress response to reactive oxygen species (ROS)

[0016] (FIG. 2B). As observed previously for other ARTs, ART714 and ART733 decreased expression levels of the anti-apoptotic protein MCL-1, but not the anti-apoptotic protein BCL-2 (FIG. 2B).Maximum tolerated dose (MTD) andPK of ART714

[0141] ART714 was selected for further PK characterization and in vivo studies given its slightly superior solubility, stability, and antileukemic efficacy, as compared with ART733. In pilot experiments, the MTD of ART714 was determined to be 60 mg / kg / d PO x 5d, in non- leukemia-bearing NRG mice.

[0142] Plasma concentration-time profiles following single and multiple 60 mg (MTD) doses of ART714 are presented in FIG. 3. Maximum plasma concentrations (CmaX) of 5635 and 5458 nM occurred at Ih and 1.5h following single and multiple doses, respectively, with mono-exponential decline. The concentration of ART714 exceeded the in vitro IC50 in M0LM14 cells (22 nM) for at least 8h following administration as a single dose and for at least lOh following administration of multiple doses (FIG. 3). The terminal half-life (ti / 2), apparent clearance (CPF), and apparent volume of distribution (V / F) were 1.63h and 1.84h, 9. 1 and 7.9 L / h / kg, and 21 and 19.5 L / kg following single and multiple doses, respectively. Using the Method of Bailer, the AUCi2h was similar following multiple doses vs single doseof ART714 (10636 vs 9265 h*nmol / L). The AUCi2h following multiple doses of ART714 was significantly higher than that for the previously reported multiple doses of ART631, either alone or in the SAV combination (10636 vs 2821 and 3837 h*nmol / L, respectively)

[0021] -Biodistribution and metabolites of ART714

[0143] The biodistribution of ART714 in plasma and various organs (brain, spleen, kidney, liver, bone marrow) at 2h, 3h, 4h, and 6h following a single dose and multiple doses of 60 mg / kg is presented in Table 2. After a single dose of ART714, a small amount of accumulation was noted in the kidney at 3h, 4h, and 6h (Table 2). More accumulation in the kidney was noted in the mice and at the time points tested after multiple doses of ART714 (Table 2). ART714 was also detected in brain, liver, and spleen of 1 of 3 mice following multiple doses, to a lesser degree (with the notable exception of accumulation in the spleen at the 6h mark, which was skewed by a single mouse with 46481 ng / g ART714 in spleen). Interestingly, ART714 was not detected in bone marrow following either single or multiple doses.

[0144] Given these results indicating that ART714 was minimally detectable in tissues, plasma and tissue samples were scanned for metabolites predicted based on functional group reactivity and plausible metabolic mechanisms. The structure of predicted metabolites are shown above. Metabolites were identified with molecular weights (MW) of 562.7, 710.9, and 730.9 in most plasma samples tested, as well as in select tissues (Table 3). Each of these proposed metabolites retain the dual endoperoxide pharmacophores common to the 2C-ARTs and would thus be expected to retain antileukemic activity. Notably, the MW 562.7 metabolite was detected in bone marrow of 3 of 3 mice treated with multiple doses of ART714. A MW 546.8 metabolite, though present in control samples, was found in much higher levels in the ART714-treated samples.Table 2. Biodistribution of ART714 following single and multiple doses.Calibration range: 5-1000 ng / mL, BLQ (below limit of quantitation) < 5 ng / mL. There were 3 mice for each time point, for both single and multiple dose groups. Values are presented as the averages ± standard deviation from each group of 3 mice. For groups where there was a combination of BLQ samples and within range samples, 50% of the lower limit of quantitation (LLQ) was imputed for BLQ samples to calculate the average.Table 3. Biodistribution of proposed metabolites following multiple doses of ART714.Black indicates that the metabolite was present in 3 of 3 mice, medium gray indicates that the metabolite was present in 2 of 3 mice, and light gray indicates that the metabolite was present in 1 of 3 mice. White indicates that the metabolite was not detected.Mutagenicity and arrhythmogenicity of ART714

[0145] ART714 produced no detectable mutagenicity in a standard AMES mutagenicity screen at the maximum concentration tested (200 pg / mL (280,000 nM), the maximal concentration at which ART714 was fully visually soluble under test conditions). In astandard arrhythmogenicity screen, no concentration-dependent hERG channel inhibition was observed at 25 pM ART714.Both ART714-containing FA V and the VEN plus GILT combination were highly effective against M0LM14 xenografts

[0146] The antileukemic efficacy of ART714 was evaluated in vivo using the highly sensitive M0LM14 AML xenograft model that had previously been utilized to evaluate ART838

[0020] and ART631

[0021] . In these experiments, the newer, more potent, and more selective FLT3 inhibitor GILT was substituted for SOR. Experimental groups received 5-day treatment cycles of vehicle, ART714 60 mg / kg / d PO alone, VEN 150 mg / kg / d PO alone, GILT 30 mg / kg / d PO alone, or combinations of ART714 plus VEN, ART714 plus GILT, VEN plus GILT, or ART714 plus VEN plus GILT (i.e., ART714-containing FAV). In contrast to prior publications, mice were treated with a single 5-day cycle of drugs, in an attempt to more feasibly obtain comparative in vivo responses to each antileukemic regimen.

[0147] GILT was the monotherapy with detected activity, prolonging median survival by several days. Despite their in vitro activity against M0LM14 and other human leukemia cell lines (e.g., FIGS. 1, 2A), neither ART714 nor VEN monotherapy demonstrated substantial in vivo activity against M0LM14 xenografts in this in vivo experiment (FIG. 4). Of the 2-drug combinations, ART714 plus VEN was more effective than either ART714 or VEN monotherapy; and VEN plus GILT showed enhanced efficacy compared with GILT as a single agent, with 2 of the 4 mice in the VEN plus GILT treatment group surviving longterm. In this experiment, VEN plus GILT was somewhat more effective than the 3-drug FAV combination (FIG. 4). Notably however, ART714-FAV more than doubled survival compared to the other tested monotherapies and 2-drug combinations except for VEN plus GILT.DISCUSSION

[0148] Though classically used as antimalarial drugs, accumulating data indicates that semi-synthetic ARTs are effective as antineoplastics including antileukemics [16-19], and synergize with currently used antileukemic drugs [20,16,21], The inventors have previously presented compelling in vitro and in vivo data for the 3C-ART analog ART838

[0020] as well as, more recently, for a new class of 2C-ARTs which have potential for clinical developmentto use in novel combination regimens for leukemias

[0021] . The inventors have identified ART631 as a potential lead 2C-ART which was impressively efficacious, in combination with VEN and SOR, in a relapsed, refractory AML PDX model.

[0149] As presented herein, rigorous lead optimization studies, including assessments of the solubility, stability, and in vitro potency of 22 additional 2C-ARTs, led to a focus on ART714. While the data for ART733 was overall quite similar, ART714 was pursued herein for a more detailed assessments of pharmacokinetics, biodistribution, and metabolism, as well as xenograft experiments, because of its slightly superior stability and in vitro efficacy as a single agent. ART714 was soluble and stable under the conditions tested, closely approximating the stability properties of ART838

[0020] . ART714 had similar in vitro pro- apoptotic activity against M0LM14 AML cells as both ART631

[0021] and ART838

[0020] , as well as similar cooperativity with VEN and SOR in vitro.

[0150] Additional pharmacokinetic and toxicology assessments further confirmed the drug-like pharmacology and favorable therapeutic window of ART714. In particular, systemic exposure, as measured by AUCi2h, following multiple doses of ART714 was significantly higher than that following multiple doses of ART631 or ART838 (10636 vs 2821 and 5142 h*nmol / L

[0020] ), respectively), but did not result in clinical toxicity. Lurthermore, neither genotoxicity nor concentration-dependent hERG channel inhibition were noted on mutagenicity and arrhythmogenicity testing, respectively.

[0151] Similar to prior AML xenograft experiments with both ART838

[0020] and ART631

[0021] , ART714 was not active against M0LM14 xenografts as monotherapy and modestly more active with VEN among 2-agent combinations versus the single agents, despite consistently demonstrated contributions to VEN and / or FLT3 inhibitor cytotoxicity in vitro. The 2-drug combination of VEN plus GILT was highly effective against M0LM14 xenografts, even exceeding the 3-drug FAV combination of ART714, VEN, and GILT. This result is similar to previous results for VEN plus GILT versus the 3-drug SAV combination of ART838, VEN, and SOR in M0LM14 xenografts

[0020] , and is perhaps not surprising given the known exquisite sensitivity of M0LM14 cells and xenografts to VEN plus GILT.

[0152] While the invention has been described with reference to certain particular embodiments thereof, those skilled in the art will appreciate that various modifications may be made without departing from the spirit and scope of the invention. The scope of the appended claims is not to be limited to the specific embodiments described.REFERENCES

[0153] The patents and publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which the invention pertains. Each cited patent and publication is incorporated herein by reference in its entirety. The following references have been cited in this application:1. Sasaki K, Ravandi F, Kadia TM, DiNardo CD, Short NJ, Borthakur G, Jabbour E, Kantarjian HM (2021) De novo acute myeloid leukemia: A population-based study of outcome in the United States based on the Surveillance, Epidemiology, and End Results (SEER) database, 1980 to 2017. Cancer 127 (12):2049-2061.Doi:10.1002 / cncr.334582. 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Claims

WHAT IS CLAIMED IS:

1. A method of treating cancer in a subject or prolonging survival of a subject having cancer, comprising administering therapeutically effective amounts of an artemisinin, a first additional therapeutic agent, and a second additional therapeutic agent to a subject having cancer, wherein the first additional therapeutic agent is a BCL-2 inhibitor, and wherein the second additional therapeutic agent is a kinase inhibitor.

2. The method of claim 1, wherein the artemisinin and the additional therapeutic agents are administered in any order, alone or in any combination, sequentially or concurrently, with overlapping or non-overlapping periods of administration.

3. The method of claim 1 or 2, comprising concurrently administering therapeutically effective amounts of the artemisinin and at least one of the additional therapeutic agents.

4. The method of any one of claims 1-3, comprising concurrently administering therapeutically effective amounts of the artemisinin and both of the additional therapeutic agents.

5. The method of claim 1 or 2, comprising sequentially administering therapeutically effective amounts of the artemisinin and at least one of the additional therapeutic agents, in any order.

6. The method of any one of claims 1, 2 or 5, comprising sequentially administering therapeutically effective amounts of the artemisinin, the first additional therapeutic agent, and the second additional therapeutic agent, in any order.

7. The method of any one of claims 1 -6, wherein the artemisinin and the additional therapeutic agents are formulated, separately or together, in any combination, in pharmaceutical compositions comprising a pharmaceutically acceptable carrier or diluent.

8. The method of any one of claims 1-7, wherein the combination of the artemisinin and the additional therapeutic agents has an additive therapeutic effect on the cancer.

9. The method of any one of claims 1-8, wherein the combination of the artemisinin and the additional therapeutic agents has a synergistic therapeutic effect on the cancer.

10. The method of any one of claims 1-9, wherein the artemisinin is one or more artemisinins selected from the group consisting of ART578-S, ART591, ART605, ART629,ART631, ART662, ART662-N, ART668, ART671, ART685, ART694, ART699, ART712, ART714, ART716, ART732, ART733, ART733-M, ART733-O, ART742, ART750, and ART757, and metabolites thereof.

11. The method of any one of claims 1-10, wherein the artemisinin is ART714 or a metabolite thereof.

12. The method of any one of claims 1-11, wherein the therapeutically effective amount of the artemisinin is between about 0.1 and 100 mg / kg body weight of the subject.

13. The method of any one of claims 1-12, wherein the BCL-2 inhibitor selected from the group consisting of ABT- 199, ABT-263, and ABT-737.

14. The method of any one of claims 1-13, wherein the kinase inhibitor selected from the group consisting of lestaurtinib (LES), midostaurin (MID), sorafenib (SOR) and gilteritinib (GILT).

15. The method of any one of claims 1-14, wherein the therapeutically effective amount of the BCL-2 inhibitor is between about 0. 1 and 50 mg / kg body weight of the subject.

16. The method of any one of claims 1-15, wherein the therapeutically effective amount of the kinase inhibitor is between about 0. 1 and 100 mg / kg body weight of the subject.

17. The method of any one of claims 1-16, wherein the artemisinin is ART714 or a metabolite thereof, the first additional therapeutic agent is ABT- 199, ABT-737 or ABT- 263, and the second additional therapeutic agent is SOR or GILT.

18. The method of any one of claims 1-17, wherein the artemisinin is ART714 or a metabolite thereof, the first additional therapeutic agent is ABT- 199, and the second additional therapeutic agent is SOR.

19. The method of any one of claims 1-17, wherein the artemisinin is ART714 or a metabolite thereof, the first additional therapeutic agent is ABT- 199, and the second additional therapeutic agent is GILT.

20. The method of any one of claims 1-19, wherein the cancer is a cancer exhibiting overexpression of a BCL-2 family member.

21. The method of any one of claims 1-20, wherein the cancer is a cancer exhibiting overexpression of a kinase or having an increased or constitutively active kinase.

22. The method of any one of claims 1-21, wherein the cancer is one or more cancers selected from the consisting of acute myeloid leukemia (AML), acute lymphoidleukemia (ALL), B cell ALL (B-ALL), T cell ALL (T-ALL), chronic myeloid leukemia (CML), chronic lymphoid leukemia (CLL); lymphoma; myeloma; myelodysplastic syndrome; non-small cell lung cancer; pancreatic cancer; gastric cancer; Kaposi’s sarcoma; hepatocellular carcinoma; osteosarcoma; laryngeal squamous cell carcinoma; metastatic uveal melanoma; lung and splenic metastases; advanced non-small cell lung cancer; cervical carcinoma; colorectal cancer; breast cancer; prostate cancer; and other hematologic malignancies and solid cancers including but not limited to brain cancer.

23. A compound having one of the following structures:

Images