HK1148906B - Compositions and methods for cancer treatment - Google Patents

Description

Compositions and methods for cancer treatment

Reference to related applications

This application claims priority and benefit from U.S. provisional patent application nos. 60/971,144 and 61/013,372, filed on.9-10 and.12-13 of 2007, respectively, which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to compositions and methods for treating cancer and other disorders using a combination of Stat3 pathway inhibitors and cancer stem cell inhibitors.

Background

Cancer Stem Cells (CSC)

In recent years, a new tumor hairThe generative model is widely accepted, in which the hypothesis is put forward that only a small fraction of the total tumor mass is responsible for the tumorigenic activity within the tumor, whereas the old model or the clonal genetic model assumes that all mutated tumor cells contribute equally to this tumorigenic activity. According to a new model, this small fraction of tumorigenic cells are transformed cells with stem cell-like properties and are called "cancer stem cells" (CSCs). In the 90 s of the 20 th century, Bonnet and Dick first demonstrated the presence of CSCs in Acute Myeloid Leukemia (AML) in vivo. Their data indicate that only a small subpopulation of human AML cells have the ability to transfer AML when transplanted into immunodeficient mice, while other AML cells are not able to induce leukemia. Later, these CSCs were shown to have the same cellular signature CD34 as the original hematopoietic stem cells⁺/CD38^-[1]. Since then, researchers have conclusively discovered CSCs in various types of tumors, including tumors of the brain, breast, skin, prostate, and the like.

The CSC model of tumorigenesis can explain why tens or hundreds of thousands of tumor cells need to be injected into experimental animals in order to establish tumor transplants. In human AML, the frequency of these CSC cells is less than one ten thousandth [2 ]. Although CSCs are rare in a given tumor cell population, there is increasing evidence that such cells are present in almost all tumor types. However, since cancer cell lines are selected from a subpopulation of cancer cells specifically adapted to grow in tissue culture, the biological and functional properties of cancer cell lines may undergo significant changes. Thus, not all cancer cell lines contain CSCs.

Cancer stem cells share many similar traits as normal stem cells. For example, CSCs have the ability to self-renew, i.e., the ability to produce other tumorigenic cancer stem cells, typically at a lower rate than other dividing tumor cells, but not a limited number of divisions. CSCs also have the ability to differentiate into multiple cell types, which explains the histological phenomenon that many tumors not only contain multiple cell types native to the host organ, but also often retain heterogeneity in tumor metastasis. CSCs have been demonstrated to be fundamentally responsible for tumorigenesis, cancer metastasis and cancer recurrence. CSCs are also known as tumor initiating cells, cancer stem-like cells, stem-like cancer cells, highly tumorigenic cells, tumor stem cells, solid tumor stem cells, or super malignant cells.

The presence of cancer stem cells is of fundamental significance for future cancer treatments and therapies. The efficacy of current cancer treatments is often measured in the initial stages of testing as a reduction in tumor size, i.e., the amount of tumor material removed. Since CSCs constitute a very small fraction of tumors and have biological characteristics that are significantly different from their more differentiated progeny, measurement of tumor mass may not necessarily enable the selection of drugs that specifically act on such stem cells. In fact, cancer stem cells appear resistant to radiation therapy (XRT), and also appear refractory to chemotherapeutic agents and targeted drugs [3-5 ]. Normal somatic stem cells are naturally resistant to chemotherapeutic agents-they have multiple pumps (e.g., MDR) to pump drugs out and efficient DNA repair mechanisms. Moreover, they also have a slow cell renewal rate, while chemotherapeutic agents target rapidly replicating cells. Cancer stem cells are the mutated counterparts of normal stem cells and may also have similar mechanisms to survive drug therapy and radiation therapy. In other words, traditional chemotherapy and radiotherapy kill differentiated or differentiating cells that cannot regenerate a tumor, which make up the majority of the tumor. On the other hand, the cancer stem cell population that produces the differentiated or differentiating cells may remain intact and cause disease recurrence. Another risk with traditional anti-cancer therapies is that the treatment (e.g., chemotherapy) may result in leaving only cancer stem cells that are resistant to chemotherapy, thereby making it likely that the subsequent recurrent tumor is also resistant to chemotherapy.

Inclusion of anti-CSC strategies in anti-cancer therapies is absolutely necessary because surviving cancer stem cells can repopulate tumors and cause relapse (see fig. 1). This is similar to cutting grass requiring root removal [6 ]. By selectively targeting cancer stem cells, patients with aggressive unresectable tumors and refractory or recurrent cancer can be treated, as well as preventing tumor metastasis and recurrence. Therefore, the development of specific therapies targeting cancer stem cells offers the promise of survival and improving the quality of life of cancer patients, particularly metastatic cancer patients. The key to opening this untapped potential is the identification and validation of pathways of selective importance for cancer stem cell self-renewal and survival. Although a number of pathways behind the self-renewal of embryonic or adult stem cells or tumorigenesis of cancer have been described in the past, pathways for cancer stem cell self-renewal and survival have not been identified and validated.

There are also many studies to identify and isolate cancer stem cells. The methods used primarily exploit the ability of CSCs to efflux drugs or are based on the expression of cancer stem cell-associated surface markers.

For example, because CSCs are resistant to many chemotherapeutic agents, it is not surprising that CSCs almost universally overexpress drug efflux pumps such as ABCG2(BCRP-1) [7-11] and other ATP-binding cassette (ABC) superfamily members [12, 13 ]. Thus, the Side Population (SP) technique originally used to enrich for hematopoietic and leukemic stem cells was also employed to identify and isolate CSCs [14 ]. This technique was first described by Goodell et al, using differential ABC transporter-dependent efflux of fluorescent dyes such as Hoechst 33342 to identify and isolate CSC-rich cell populations [10, 15 ]. Specifically, the SP was revealed by blocking drug efflux with verapamil, where the dye was no longer able to pump out the SP.

Researchers have also focused on finding specific markers that distinguish cancer stem cells from most tumors. The most commonly expressed CSC surface markers include CD44, CD133 and CD166[16-24 ]. Sorting of tumor cells based primarily on differential expression of these surface markers has led to the majority of the highly tumorigenic CSCs described to date. Thus, these surface markers are well validated for the identification and isolation of cancer stem cells from cancer cell lines and large tumor tissues.

Stat3 pathway

There are many different genetic defects in mammalian or human cancer cells, and many have been investigated in search of cure for cancer. For example, the p53 tumor repressor protein has been found to be defective or completely non-existent in more than half of human cancers. The STAT (signal transducer and activator of transcription) protein family are latent transcription factors that are activated in response to cytokines/growth factors to promote proliferation, survival and other biological processes. Among them, Stat3 is activated by phosphorylation of a key tyrosine residue mediated by a growth factor receptor tyrosine kinase, Janus kinase, Src family kinase, or the like. These kinases include, but are not limited to, EGFR, JAK, Abl, KDR, c-Met, Src and Her2[25 ]. Upon tyrosine phosphorylation, Stat3 forms a homodimer, translocates to the nucleus, binds to specific DNA response elements in the promoter region of target genes, inducing gene expression [26] (see fig. 2).

In normal cells, Stat3 activation is transient, tightly regulated, lasting from 30 minutes to several hours. However, Stat3 was found to be abnormally activated in a wide variety of human cancers, including all major cancers as well as some hematological tumors. Stat3 plays multiple roles in cancer progression. As a potent transcriptional regulator, it targets genes involved in many important cellular functions, such as Bcl-xl, c-Myc, cyclin D1, Vegf, MMP-2, and survivin [27-32 ]. It is also a key negative regulator of tumor immune surveillance and immune cell recruitment [33-35 ].

Certain cancer cell lines or tumors can be inhibited in vitro and/or in vivo by antisense, siRNA, dominant negative forms of Stat3 and/or blocking tyrosine kinases, abrogating Stat3signaling [26, 28, 36, 37 ]. But a clear association between Stat3 and cancer stem cell function has not been provided empirically. Researchers have also not found potent Stat3 pathway inhibitors to develop potential therapeutic applications against cancers that have been found to contain cancer stem cells. As explained earlier, Cancer Stem Cells (CSCs) have recently been shown to be fundamentally responsible for tumorigenesis, cancer metastasis and cancer recurrence, and thus they should be taken into account when designing any curative therapy that targets tumors known to have these cells, regardless of how small a proportion of the tumor mass CSCs may account for.

Over-activation of Stat3 by various cytokines, such as interleukin 6(IL6), has been demonstrated in a number of autoimmune and inflammatory diseases, in diseases other than cancer [38 ]. It has recently been revealed that the Stat3 pathway also promotes pathological immune responses through its fundamental role in generating TH17T cellular responses [39 ]. In addition, inflammation mediated by the IL6-Stat3 pathway has been found to be a common cause of atherosclerosis, peripheral vascular disease, coronary artery disease, hypertension, osteoporosis, type 2 diabetes, and dementia.

Summary of The Invention

The present invention is based in part on empirical evidence provided herein that Stat3 plays a key role in both the survival and self-renewal capacity of Cancer Stem Cells (CSCs) in a broad spectrum of cancers. The present invention also provides data confirming that certain compounds may act as Stat3 pathway inhibitors and that they are effective in inhibiting CSCs both in vitro and in vivo.

Accordingly, a first aspect of the invention provides a method of treating a subject suffering from a disorder associated with aberrant Stat3 pathway activity, the method comprising the steps of: (a) administering to the subject a first amount of a first agent to inhibit at least some aberrant Stat3 pathway activity; and (b) administering to the subject a second amount of a second agent comprising a signal transduction inhibitor.

The first agent may inhibit Stat3 pathway activity by at least one of the following actions: substantially inhibiting phosphorylation of the Stat3 protein, substantially inhibiting dimerization of the Stat3 protein, substantially inhibiting nuclear translocation (translocation) of the Stat3 protein, substantially inhibiting DNA binding activity of the Stat3 protein, and substantially inhibiting transcriptional activity of the Stat3 protein.

In one embodiment, the first agent is selected from: 2- (1-hydroxyethyl) -naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl-7-chloro-naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl-7-fluoro-naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl naphtho [2, 3-b ] furan-4, 9-dione, 2-ethyl-naphtho [2, 3-b ] furan-4, 9-dione, an enantiomer, a diastereomer, a tautomer, a salt or a solvate thereof (hereinafter referred to as "the compound of the present invention").

Non-cancer disorders treatable by the method of the first aspect of the invention include, but are not limited to: autoimmune diseases, inflammatory bowel diseases, arthritis, asthma, and systemic lupus erythematosus, autoimmune demyelinating diseases, Alzheimer's disease, stroke, ischemia reperfusion injury, and multiple sclerosis. Cancers that can be treated by this method include, but are not limited to: breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumors, gastric cancer, multiple myeloma, leukemia, and lymphoma. These non-cancerous and cancerous disorders are known to be associated with aberrant Stat3 pathway activity.

In one feature, the second agent is a targeting agent, which may be a growth factor receptor targeting agent, a kinase targeting agent, or an angiogenesis inhibitor.

In a second aspect, the invention provides a method of treating a subject having a cancer associated with aberrant Stat3 pathway activity, the method comprising the steps of: (a) administering to the subject a first amount of a first agent to inhibit at least some aberrant Stat3 pathway activity; and (b) administering to the subject a second amount of a second anti-cancer agent.

The features relating to the first agent may be similar to those described in relation to the first aspect of the invention, while the second anti-cancer agent may be a cytotoxic agent or a chemotherapeutic agent. In one embodiment, the second agent is a standard first line therapy for at least one cancer.

In one feature, the anti-cancer agent is a DNA damaging agent, an antimitotic agent, and/or an antimetabolite. For example, the DNA damaging agent can be an alkylating agent, a topoisomerase inhibitor, or a DNA intercalating agent. In one embodiment, the second agent is one of carboplatin, doxorubicin, gemcitabine, docetaxel, or etoposide.

Cancers that can be treated by the methods of the second aspect of the invention include those known to be associated with aberrant Stat3 pathway activity, which have been listed above and are not repeated here.

Since cancer stem cells are generally resistant to radiation therapy and traditional chemotherapy, drugs targeting cancer stem cells should have synergistic effects when used in combination with other anti-cancer therapies. Thus, according to a third aspect of the invention, a method of treating cancer in a subject comprises the steps of: (a) administering to the subject a first amount of a first anti-cancer agent to inhibit a population of Cancer Stem Cells (CSCs); and (b) administering to the subject a second amount of a second anti-cancer agent to inhibit a majority of common cancer cells.

In various embodiments, step (a) of the method inhibits self-renewal, and/or kills, of the at least one CSC. In one embodiment, the first amount of the first anti-cancer agent also kills a majority of common cancer cells. In one embodiment, step (a) inhibits at least some Stat3 pathway activity in the cancer stem cells. The first anti-cancer agent may share the same features and characteristics as the first agent in the method according to the first aspect of the invention, as the invention has provided evidence that Stat3 pathway inhibitors are able to effectively inhibit CSCs. The shared features may include, for example, multiple Stat3 pathway steps that the first anti-cancer agent described herein is capable of targeting. In various embodiments, the first anti-cancer agent can be a small molecule Stat3 inhibitor, an RNAi agent to Stat3, an antisense agent to Stat3, a peptidomimetic Stat3 inhibitor, or a G quadruplet oligodeoxynucleotide Stat3 inhibitor.

Cancers that can be treated by this method are preferably those known or confirmed to contain CSCs, including but not limited to: breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, multiple myeloma, colorectal cancer, prostate cancer, melanoma, kaposi's sarcoma, ewing's sarcoma, liver cancer, stomach cancer, medulloblastoma, brain tumor, and leukemia.

The "second anti-cancer agent" in the method according to the third aspect of the invention may be the same as the "second anti-cancer agent" in the method according to the second aspect of the invention, so all shared features are not repeated here.

In one embodiment, the second agent is a standard first line therapy for at least one cancer. The second agent can be a cytotoxic agent. In one feature, the anti-cancer agent is a DNA damaging agent, an antimitotic agent, and/or an antimetabolite. For example, the DNA damaging agent can be an alkylating agent, a topoisomerase inhibitor, or a DNA intercalating agent. In one embodiment, the second agent is one of carboplatin, doxorubicin, gemcitabine, docetaxel, or etoposide.

According to a fourth aspect of the present invention there is provided a method of treating cancer in a subject, comprising the steps of: (a) administering to the subject a first amount of a first cancer stem cell inhibitor to inhibit Stat3 pathway activity; and (b) administering to the subject a second amount of a second cancer stem cell inhibitor to inhibit the activity of a different pathway.

In one embodiment, the second cancer stem cell inhibitor is lapatinib (lapatinib). In some embodiments, the second amount of the second anti-cancer agent is not itself therapeutically effective on the population of cancer stem cells. Cancers that can be treated by this method are preferably those known or confirmed to contain CSCs, some examples of which are listed above. In various embodiments, the cancer is metastatic, refractory to standard first-line cancer therapy, or recurrent.

According to a fifth aspect of the present invention there is provided a method of treating cancer in a subject, comprising the steps of: (a) administering to the subject a therapeutically effective amount of a first anti-cancer agent selected from the group consisting of: 2- (1-hydroxyethyl) -naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl-7-chloro-naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl-7-fluoro-naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl naphtho [2, 3-b ] furan-4, 9-dione, 2-ethyl-naphtho [2, 3-b ] furan-4, 9-dione, a pharmaceutically acceptable salt or solvate thereof; and (b) administering a second anti-cancer agent not selected from the same group.

The second anticancer agent may be any agent described in other aspects of the invention, including any cytotoxic or chemotherapeutic agent and any targeting agent.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a first anti-cancer agent selected from the group consisting of: 2- (1-hydroxyethyl) -naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl-7-chloro-naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl-7-fluoro-naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl naphtho [2, 3-b ] furan-4, 9-dione, 2-ethyl-naphtho [2, 3-b ] furan-4, 9-dione, a pharmaceutically acceptable salt or solvate thereof; and a second anti-cancer therapy selected from: cytotoxic agents, targeting agents, radiotherapeutic agents, biological agents (biologicals agents), hormonal agents, HDAC inhibitors, retinoids (retinoids), checkpoint activators (checkpoint activators), proteasome inhibitors, adjuvants (adjuvant agents) or adjuvant agents (adjuvant agents).

In one embodiment, the composition further comprises a pharmaceutically acceptable excipient, carrier or diluent.

Other aspects and embodiments of the invention, including all compositions and kits relating to the methods described herein, are set forth in, or will be apparent from, the following detailed description.

Brief description of the drawings

Figure 1 illustrates the differences between cancer stem cell-specific cancer therapy and traditional cancer therapy.

Figure 2 shows the Stat3 pathway in cancer.

FIG. 3A shows that Stat3 is constitutively activated in Hoechst-lateral population cells.

FIG. 3B shows Stat3 on CD133⁺Constitutively activated in the cell.

Figures 4A and 4B show that Stat3 knockdown (knockdown) in cancer stem cells induces apoptosis.

Figure 5 shows that Stat3 knockdown in cancer stem cells inhibits cancer stem cell spheroid formation.

Figure 6 shows that compound 401 inhibits the transcriptional activation activity of Stat3.

Figure 7A shows that compound 401 inhibits the DNA binding activity of Stat3 in nuclear extracts.

Figure 7B shows that compounds 401, 416, and 418 inhibit the DNA binding activity of Stat3 in nuclear extracts.

Figure 8A shows that compound 401 inhibits the DNA binding activity of Stat3 in xenograft tumor tissues.

Figure 8B shows that compound 401 inhibits the expression level of downstream effectors of Stat3 in xenograft tumor tissues.

FIG. 9A shows sorting and analysis of Hoechst side populations.

FIG. 9B shows that the Hoechst side population is as sensitive as compound 401 to the non-side population.

Figure 10A shows that compound 401 causes apoptosis of Hoechst-side population cells.

FIG. 10B shows Compound 401 rendering CD133⁺And (4) apoptosis.

FIG. 11 shows that Compound 401 blocks CD44^highSpheres are formed.

Figure 12 shows that in vivo compound 401 treatment reduced spheroid formation of xenograft tumor cells.

Figure 13 shows that compound 401 inhibits metastasis in the ISMS model.

Figure 14 shows that compound 401 has a synergistic effect with sorafenib (sorafenib) in a549 human lung cancer cells.

Figure 15 shows that compound 401 has a synergistic effect with erlotinib (erlotinib) in a549 human lung cancer cells.

Figure 16 shows that compound 401 has a synergistic effect with lapatinib in a549 human lung cancer cells.

Figure 17 shows that compound 401 has a synergistic effect with sotentan (patent) in a549 human lung cancer cells.

Figure 18 shows that compound 401 has a synergistic effect with gemcitabine in the Paca-2 human pancreas xenograft model.

Detailed Description

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "cell" includes a plurality of cells, including mixtures thereof.

The term "isolated" or "purified" as used herein means a material that is substantially or essentially free of components with which it normally accompanies in its natural state. Purity and homogeneity can generally be determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography.

As used herein, the terms "cancer stem cell" and "CSC" are interchangeable. CSCs are mammalian and in preferred embodiments, these CSCs are of human origin, although they are not intended to be so limited. Cancer stem cells are defined as, and functionally characterized as: a population of cells derived from a solid tumor which: (1) has wide proliferation ability; 2) capable of undergoing asymmetric cell division to produce one or more types of differentiated progeny having reduced proliferative or developmental potential; and (3) capable of symmetric cell division for self-renewal or self-maintenance. Other common methods of characterizing CSCs include morphology and examination of cell surface markers, transcriptional profiles, and drug responses. CSCs are also known in the research literature as tumor/cancer initiating cells, cancer stem-like cells, stem-like cancer cells, homotumorigenic cells, tumor stem cells, solid tumor stem cells, drug-surviving cells (DSCs), drug-resistant cells (DRCs), or super malignant cells.

As used herein, the term "self-renewal" refers to the ability of cancer stem cells to produce new tumorigenic cancer stem cells to supplement or increase their numbers.

As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterized by uncontrolled cell growth. As used herein, "cancer cells" and "tumor cells" refer to the total cell population derived from a tumor, including both non-tumorigenic cells, which make up the majority of the tumor cell population, and tumorigenic stem cells (cancer stem cells). Examples of cancer include, but are not limited to: carcinomas (carcinoma), lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, liver cancer, and various types of head and neck cancer.

As used herein, "tumor" refers to any tissue mass resulting from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancerous), including precancerous lesions.

As used herein, "metastasis" (metastasis) refers to the process by which cancer spreads from the site of origin or metastasizes to other areas of the body, forming similar cancerous lesions at new locations. "metastatic" or "metastatic" cells are cells that lose adhesive contact with neighboring cells and migrate from the primary site of disease via the bloodstream or lymph to infiltrate neighboring body structures.

As used herein, the term "subject" refers to any animal (e.g., mammal) that will be the recipient of a particular treatment, including, but not limited to, humans, non-human primates, rodents, and the like. In general, the terms "subject" and "patient" are used interchangeably herein with respect to a human subject.

As used herein, terms such as "treating" or "ameliorating" refer to both 1) therapeutic measures that cure, alleviate, reduce the symptoms of, and/or interrupt the progression of a diagnosed condition (condition) or disorder, and 2) preventative or prophylactic measures that prevent or slow the development of the targeted condition or disorder. Thus, those in need of treatment include those already suffering from the disorder; those prone to disorders; and those in which the disorder is to be prevented. A subject is successfully "treated" by the methods of the invention if the subject exhibits one or more of the following conditions: a reduction in the number of cancer cells or the complete absence of cancer cells; a reduction in tumor size; inhibition or absence of infiltration of cancer cells into peripheral organs, including spread of cancer into soft tissue and bone; inhibition or absence of tumor metastasis; inhibition or absence of tumor growth; reduction of one or more symptoms associated with a particular cancer; reduced morbidity and mortality; and improvement of quality of life.

As used herein, the term "inhibit" and its grammatical equivalents, when used in the context of biological activity, refers to a down-regulation of biological activity that can reduce or eliminate a targeted function, such as production of a protein or phosphorylation of a molecule. In particular embodiments, inhibition may refer to a reduction of about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the targeted activity. When used in the context of a disorder or disease, the term refers to the success of preventing the onset of symptoms, alleviating symptoms, or eliminating the disease, condition, or disorder.

As used herein, the singular or plural form of "common cancer cell" refers to a cancer cell that is not a cancer stem cell.

As used herein, "combination" therapy or treatment refers to the administration of at least two different treatments to treat a disorder, condition, or symptom, such as a cancer condition. Such combination therapy may include administration of one treatment before, during, and/or after administration of another treatment. Administration of these treatments can be separated in time by up to several weeks, but most commonly within 48 hours, and most commonly within 24 hours.

The term "pharmaceutically acceptable excipient, carrier or diluent" as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material involved in carrying or transporting a subject pharmaceutically active agent from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; astragalus membranaceus gel powder; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; ringer's solution; ethanol; phosphate buffer; and other non-toxic compatible materials employed in pharmaceutical formulations. Wetting agents, emulsifying agents, and lubricating agents (e.g., sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymers), as well as coloring, releasing, coating, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

The compounds of the present invention may form salts and this is also within the scope of the present invention. References herein to compounds of the invention are to be understood as including references to salts thereof unless otherwise indicated. The term "salt", as used herein, denotes acid addition salts and/or base addition salts formed with inorganic and/or organic acids and bases. In addition, when a compound of the present invention contains both a basic moiety (such as, but not limited to, a pyridine or imidazole) and an acidic moiety (such as, but not limited to, a carboxylic acid), zwitterions ("inner salts") may be formed and are included within the term "salt(s)" as used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful, e.g., in isolation or purification steps that may be employed during preparation. Salts of the compounds of the invention can be formed, for example, by reacting compound I, II or III with an amount (e.g., equivalent amount) of an acid or base in a medium (e.g., a medium in which the salt precipitates or in an aqueous medium) and then lyophilizing.

Solvates of the compounds of the invention are also contemplated herein. Solvates of the compounds of the invention include, for example, hydrates.

Targeting Stat3 pathway

The present invention provides compounds that are potent inhibitors of the Stat3 pathway activity (example 2). Since the Stat3 pathway is a latent transcription factor that is activated to promote proliferation, survival, and many other biological processes, it has been implicated in a wide variety of human cancers as well as non-cancer disorders, including many autoimmune and inflammatory diseases (table 1). Thus, a first aspect of the invention provides a combination treatment of a disorder associated with aberrant (e.g. overexpressed) Stat3 pathway activity. In particular, a first amount of a first agent is administered to a patient subject to inhibit at least some aberrant Stat3 pathway activity, and a second amount of a second agent comprising a signal transduction inhibitor is also administered. In various embodiments, some (e.g., 20%, 30%, 40%), most (about 50% or more), or substantially all (e.g., 60%, 70%, 80%, 90%, 95%, or 100%) of the aberrant Stat3 pathway activity is inhibited. One or both of the first amount and the second amount may be a therapeutically effective amount of the respective agent prior to use of the combination (i.e. when used by itself against a disorder), or less than that amount due to a pronounced synergistic effect of the combination. The first agent may target one or more steps in the Stat3 pathway. In one embodiment, the first agent is a compound of the invention.

TABLE 1 activation of STAT3 pathway in human diseases

The second agent, a signal transduction inhibitor, may be used to target a different pathway, a related pathway, or a different step in the same Stat3 pathway than the pathway inhibited by the first agent. Generally, when two classes of agents in a combination therapy target the same pathway (even at different steps), the amount of synergy that is expected is limited. However, the data provided in example 5 below shows a surprisingly high amount of synergy between the compounds of the invention and a second agent (e.g., a tyrosine kinase and a GFR targeting agent) that is supposed to target other steps in the same pathway, suggesting that an unexpected mechanism of inhibition is working.

Specifically, Stat3 is activated by phosphorylation of key tyrosine residues mediated by growth factor receptor tyrosine kinases, Janus kinases, Src family kinases, or the like; upon tyrosine phosphorylation, Stat3 forms a homodimer, translocates to the nucleus, binds to specific DNA response elements in the promoter region of target genes, inducing gene expression. Example 2 of the invention shows that the inhibitory effect of the compounds of the invention is evident in the Stat3 pathway through this DNA binding step. Furthermore, since such effects are seen in constitutively activated Stat3, it is likely that the compounds of the invention inhibit dimerization and/or nuclear translocation of the Stat3 protein. Thus, the amount of synergy observed when combined with Tyrosine Kinases (TKIs) and GFR targeting agents that also target the same Stat3 pathway is surprisingly high. For example, when compound 401 was combined with TKI sorafenib, 100% inhibition of cells from pancreatic cancer cell lines was achieved, whereas when compound 401 and sorafenib were administered alone, both were only able to achieve 66% inhibition of the same cell line, respectively — pancreatic cancer was known to be involved in overexpression of Stat3 [44 ]. In fact, all four TKIs tested in combination with compound 401 showed significant synergy. In preferred embodiments of the invention, the combination therapy achieves greater than about 50%, or 70%, or 90% inhibition of cancer cells.

The method according to the first aspect of the invention may be applied to the treatment of cancer or non-cancer disorders, preferably those known to be associated with aberrant Stat3 pathway activity. Examples of non-cancer disorders associated with aberrant Stat3 pathway activity include, but are not limited to: autoimmune diseases, inflammatory bowel diseases, arthritis, asthma, and systemic lupus erythematosus, autoimmune demyelinating diseases, Alzheimer's disease, stroke, ischemia reperfusion injury, and multiple sclerosis. Examples of cancers associated with aberrant Stat3 pathway activity include, but are not limited to: breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumors, gastric cancer, multiple myeloma, leukemia, and lymphoma.

The second agent according to the first aspect of the invention may be a targeting agent, such as a growth factor receptor targeting agent (see data for erlotinib (tarceva) in example 5), a kinase targeting agent (see data for lapatinib, erlotinib, sunitinib and sorafenib in example 5) or an angiogenesis inhibitor (see data for sunitinib and sorafenib in example 5).

In one embodiment, the second agent is a growth factor receptor targeting agent, such as an antibody that targets a growth factor receptor associated with a kinase [ e.g., Epidermal Growth Factor Receptor (EGFR) or Vascular Endothelial Growth Factor Receptor (VEGFR) ]. For example, the targeting agent may be gefitinib (ereshit) erlotinib (tarceva), PD153035, cetuximab (alloxan), avastin, panitumumab, trastuzumab, and an anti-c-Met antibody.

In one embodiment, the second agent is a kinase-targeting agent, which may be a kinase inhibitor, such as a Tyrosine Kinase Inhibitor (TKI). For example, the TKI may be erlotinib (erlotinib) (Tarceva), sot (Sutent) (sunitinib), lapatinib (lapatinib), sorafenib (sorafenib) (polygex (nexavar)), vandetanib (vandetanib), axitinib (axitinib), bosutinib (bosutinib), cedinaib (cedivanib), dasatinib (dasatinib) (sprycel), gefitinib (gefitinib) (irressa), imatinib (imatinib) (gleevec), lestatinib (lestaurtinib), and/or ARQ 197.

In various embodiments, the kinase-targeting agent is one of: gefitinib (aprezemib), ZD6474(AZD6474), EMD-72000 (matuzumab), panitumumab (panitumumab) (ABX-EGF), ICR-62, CI-1033(PD183805), lapatinib (lapatinib) (tykerb), AEE788 (pyrrolopyrimidine), EKB-569, EXEL7647/EXEL 0999, erlotinib (Tarcek), imatinib (gleevec), sorafenib (Dogeimel), sunitinib (Sotanib), dasatinib (Prader Rasait), vandetanib (ZACTIMA), temsirolimus (Tourethricil), PTK787 (Watitanib (vatalanib)), pazopanib (pazopanib), AZD2171, Everolimus (Everolimus), AZD6474, AZD 35272, Eszechiib-5933, AMD-5933, PKF-GCK, PKC-5944, PKC (Tykerb), PKC-GCE), AEK 788 (PKC), Geranix-75, Geranib), Geranilic Acid (ATC) and Geranilic acid B) and PSK-33, Geranilic acid, AZD0530, enzastaurin, MLN-518 and ARQ 197.

In one embodiment, the second agent is an angiogenesis inhibitor, which may be one of the following: CM101, IFN-alpha, IL-12, platelet factor-4, suramin (suramin), SU5416, thrombospondin, VEGFR antagonist, angiostatic sterols (angiostatic sterols) + heparin, chondrogenic angiogenesis inhibitor, matrix metalloproteinase inhibitor, batimastat (batimastat), marimastat (marimastat), angiostatin (angiostatin), endostatin (endostatin), 2-methoxyestradiol, tegolan (tecogalan), thrombospondin, alpha V beta 3 inhibitor, linoamine (linomide), and ADH-1.

In a related second aspect, the invention provides a method of treating a cancer subject associated with aberrant Stat3 pathway activity, the method comprising the steps of: (a) administering to the subject a first amount of a first agent to inhibit at least some aberrant Stat3 pathway activity; and (b) administering to the subject a second amount of a second anti-cancer agent. Cancers that may be treated by this method include those known to be associated with (e.g. caused at least in part by) abnormal pathway activity, a list of which is provided above in relation to the first aspect of the invention.

The features relating to the first agent may be similar to those described in relation to the first aspect of the invention, while the second anti-cancer agent may be a cytotoxic agent or a chemotherapeutic agent. In one embodiment, the second agent is a standard first line therapy for at least one cancer. The amount of the first agent and the second agent used in the method may be at or below the therapeutically effective amount of the respective agents prior to combined use.

In one feature, the anti-cancer agent is a DNA damaging agent, an antimitotic agent, and/or an antimetabolite. The DNA damaging agent can be an alkylating agent, a topoisomerase inhibitor, and/or a DNA intercalating agent. As shown in example 5, compound 401 of the present invention was added to Paca2 pancreatic cancer cells, respectively, along with each of the following: carboplatin (DNA alkylating agent), etoposide (topoisomerase II inhibitor), doxorubicin (DNA intercalator), docetaxel (antimitotic agent) and gemcitabine (Gemzar)/gemcitabine (antimetabolite). Significant amounts of synergy were found in each combination. For example, compound 401 in combination with the alternative/gemcitabine achieved 96% inhibition of pancreatic cancer cells, whereas compound 401 and alternative achieved only 66% and 36% inhibition of the same cell line, respectively, when administered alone.

The alkylating agent may be one of: chlorambucil (chlorambucil), cyclophosphamide (cyclophosphamide), ifosfamide (ifosfamide), mechlorethamine (mechlorethamine), melphalan (melphalan), uracil mustard (uracil mustard), thiotepa (thiotepa), busulfan (busufan), carmustine (carmustine), lomustine (lomustine), streptozocin (streptozocin), carboplatin (carboplatin), cisplatin (cilatin), satraplatin (saroplatin), oxaliplatin (oxaliplatin), altretamine (alttrametin), ET-743, XL119 (carbonaracin), dacarbazine (dacarbazine), carmustine (bendamustine), trothiotepine (carmustine), carboplatin (carmustine), carmustine (carmustine), carmustine (carmustine), carmustine (carmusti, Triethylenemelamine (triethyleneamine), and procarbazine (and procarbazin).

The topoisomerase inhibitor may be one of: doxorubicin (doxorubicin), daunorubicin (daunorubicin), epirubicin (epirubicin), idarubicin (idarubicin), dithianedione (anthrenedione, norstrin (novantrone)), mitoxantrone (mitoxantrone), mitomycin c (mitomycin c), bleomycin (bleomycin), actinomycin d (dactinomycin), plicamycin, irinotecan (irinotecan) (exploitation (camptosar)), camptothecin (camptothecin), rubitecan (rubitecan), belotecan (belotecan), etoposide (etoposide), teniposide (teniposide), and topotecan (and methicone).

The DNA intercalator may be proflavine (proflavine), doxorubicin (adriamycin), daunorubicin, actinomycin D andthalidomide(thalidomide)。

The antimitotic agent may be one of the following: paclitaxel (abraxane)/taxol (taxol), docetaxel (docetaxel), BMS-275183, polyglutamic acid paclitaxel (xyotax), tocosal, vincristine (vincristine), vinblastine (vinblastine), vindesine (vindesine), vinzolidine (vinzolidine), etoposide (VP-16), teniposide (VM-26), ixabepilone (ixabepilone), larotaxel, ortataxel, tesetaxel and ispinesis (ispinesib).

The antimetabolite may be one of: fluorouracil (5-FU), fluorouridine (5-FUdR), methotrexate, Hiroda (xeloda), alen (araranon), leucovorin (leucovorin), hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine, pentostatin (pentostatin), fludarabine phosphate, cladribine (2-CDA), asparaginase (asparaginase), gemcitabine, pemetrexed (pemetrexed), bortezomib (bortezomib), aminopterin (aminopterin), raltitrexed (raltitrexed), clofarabine (clofarabine), enocitabine (enocitabine), sapacitabine and azacitidine (azacitidine).

In one embodiment, the second anti-cancer agent is one of: carboplatin, doxorubicin, gemcitabine, docetaxel, and etoposide. Since this approach inhibits the Stat3 pathway, which is demonstrated herein to be critical for both self-renewal and survival of CSCs (see data in example 1), and CSCs have been found to be fundamentally responsible for drug resistance, tumor recurrence and metastasis, in preferred embodiments, this approach is used to treat or prevent refractory, recurrent, and/or metastatic cancers.

Further discussion of anti-Cancer chemotherapies and biotherapies and examples of suitable treatment regimens can be found in, for example, Cancer chemotherapeutics and biotherapies: principles and Practice, third edition (2001), Chabner and Longo editions, and Handbook of Cancer chemotherapeutics, sixth edition (2003), Skaet editions (both from Lippincott Williams)&Wilkins, philiadelphia, Pa., u.s.a.); also, regimens for anti-Cancer treatment, particularly chemotherapy, can be found, for example, in the american Cancer Institute (National Cancer Institute,www.cancer.gov) The American Society for Clinical Oncology (American Society for Clinical Oncology,www.asco.org) And the National Comprehensive Cancer Network (National Comprehensive Cancer Network,www.nccn.org) Those websites that are maintained.

Targeting cancer stem cells

The invention also provides in vitro and in vivo data for compounds of the invention to inhibit self-renewal and apoptosis of CSCs (example 3). In addition, the present inventors empirically confirmed the efficacy of the compounds of the present invention against metastatic cancer (example 4).

Cancer therapy (anti-cancer therapy) aims to prevent cancer cells from proliferating, infiltrating, metastasizing and eventually killing their host organism, such as a human or other mammal. Since cell proliferation is characteristic of many normal as well as cancer cells, most existing anticancer therapies have toxic effects on normal cells as well, especially on those with rapid turnover rates, such as bone marrow and mucosal cells. Thus, effective cancer therapy requires significant growth inhibitory or control effects on cancer cells while exerting minimal toxic effects on normal cells of the host.

Cancer recurrence and drug resistance remain some of the biggest problems in cancer treatment since the first effective anticancer compounds entered clinical trials in the 40 s of the 20 th century. Often, a resolution of symptoms can be achieved, but the response is often partial and of only short duration, and recurrent cancers tend to be resistant to the original drug. This can now be explained by the presence of Cancer Stem Cells (CSCs). As described above, this small population of cells in the total tumor mass is able to evade effective drug and radiation therapy for the remaining cancer cells because CSCs presumably share the same type of biological mechanisms as normal somatic stem cells, which have natural resistance to most, if not all, chemotherapeutic agents. As a true source of tumorigenic activity in cancer material, CSCs can re-support regeneration of cancer or cause metastasis if left untreated. Since the initial treatment leaves only drug-resistant cancer stem cells, the chances that the entire regenerated or metastatic tumor becomes resistant to the initial "effective" therapy are significantly increased.

Currently, anticancer therapies are used in combination for several reasons. First, treatment with two or more therapies that do not have cross-resistance can prevent the formation of resistant clones in tumors. Resistance to one anticancer drug (e.g., a platinum anticancer compound such as cisplatin) is often accompanied by cross-resistance to other drugs of the same class (e.g., other platinum compounds). Also, there is multidrug resistance, also known as pleiotropic resistance, that is, treatment with one drug imparts resistance not only to that drug and to other drugs of its class, but also to unrelated agents. Second, the combination of two or more therapies active on cells in different growth phases can kill slowly dividing cells as well as actively dividing cells, and/or recruit cells into a more actively dividing state, making them more sensitive to multiple anti-cancer therapies. Third, combination therapy can create biochemical enhancing effects by affecting different pathways or different steps in a single biochemical pathway.

These basic theories of combined anti-cancer therapy do not take into account recent advances in the identification and characterization of cancer stem cells. Failure to incorporate CSC-specific therapy in combination therapy can explain why current combination therapies fail to cure common cancers such as metastatic colon cancer and prostate cancer. Given the data provided herein that confirm the efficacy of the compounds of the invention against CSCs (example 3), the present invention enables the design of cancer treatment methods that combine CSC-targeting agents and other agents that target common cancer cells. Furthermore, while not wishing to be bound by a particular theory, the present invention provides a drug regimen that supersedes the situation where some untreated or undertreated common cancer cells may revert to or produce CSCs when the original CSCs are depleted as a result of a monotherapy that targets only CSCs (which is currently supported by some preliminary data).

Since cancer stem cells are generally resistant to radiation therapy and traditional chemotherapy, drugs targeting cancer stem cells should have synergistic effects when used in combination with other anti-cancer therapies. Accordingly, the present invention provides a method of treating cancer in a subject comprising the steps of: (a) administering to the subject a first amount of a first anti-cancer agent to inhibit the population of cancer stem cells; and (b) administering to the subject a second amount of a second anti-cancer agent to inhibit a majority of common cancer cells.

In various embodiments, some (e.g., 20%, 30%, 40%), most (about 50% or more), or substantially all (e.g., 60%, 70%, 80%, 90%, 95%, or 100%) of CSCs are inhibited. One or both of the first amount and the second amount may be a therapeutically effective amount of the respective agent prior to use of the combination (i.e. when applied against cancer by itself), or less than that amount due to a pronounced synergistic effect of the combination. In one embodiment, the first agent is a compound of the present invention, i.e., 2- (1-hydroxyethyl) -naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl-7-chloro-naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl-7-fluoro-naphtho [2, 3-b ] furan-4, 9-dione, 2-acetyl naphtho [2, 3-b ] furan-4, 9-dione, 2-ethyl-naphtho [2, 3-b ] furan-4, 9-dione, and pharmaceutically acceptable salts or solvates thereof.

The "second anti-cancer agent" herein may be the same "second anti-cancer agent" as in the above-described method, so all shared features are not repeated here. In one feature, the second anti-cancer agent is a DNA damaging agent, an antimitotic agent, and/or an antimetabolite. For example, the DNA damaging agent can be an alkylating agent, a topoisomerase inhibitor, or a DNA intercalating agent. Suitable alkylating agents, topoisomerase inhibitors, DNA intercalators, antimitotic agents and antimetabolites are listed above and are not repeated here. Significant synergy was observed in cancer inhibition experiments with the compounds of the present invention in combination with each of the above categories of chemotherapeutic agents (see example 5). In one embodiment, the second agent is one of carboplatin, doxorubicin, gemcitabine, docetaxel, and etoposide.

In another feature, the second anti-cancer agent is a targeting agent, such as a growth factor receptor targeting agent (see data for erlotinib (tarceva) in example 5), a kinase targeting agent (see data for lapatinib, erlotinib, sunitinib, and sorafenib in example 5), or an angiogenesis inhibitor (see data for sunitinib and sorafenib in example 5). Significant synergy was observed in cancer inhibition experiments with the use of the compounds of the present invention in combination with each of the above categories of targeting agents. Suitable growth factor receptor targeting agents, kinase targeting agents (especially TKIs), and angiogenesis inhibitors are listed above and are not repeated here.

Since this method employs therapeutic agents that specifically target CSC cells in the tumor, which are fundamentally responsible for drug resistance, tumor recurrence and metastasis, in preferred embodiments, this method is used to treat or prevent refractory, recurrent, and/or metastatic cancers.

In targeting CSCs in combination therapy, one strategy should be aimed at targeting more than one pathway involved in key biological functions of CSCs (such as self-renewal and survival). To this end, the present invention provides a method of treating cancer in a subject, comprising the steps of: (a) administering to the subject a first amount of a first cancer stem cell inhibitor to inhibit Stat3 pathway activity; and (b) administering to the subject a second amount of a second cancer stem cell inhibitor to inhibit the activity of a different pathway. In one embodiment, the amount of the first and/or second anti-cancer agent in this method is not therapeutically effective on its own for the population of cancer stem cells-but lower amounts can be applied in this method to elicit a response in the patient due to the significant synergy achieved by the combination.

In one embodiment, the second anti-cancer stem cell agent is lapatinib (INN) or lapatinib ditosylate (USAN) -approved by the FDA in 2007 for patients with advanced metastatic breast cancer. Lapatinib is an ATP competitive Epidermal Growth Factor Receptor (EGFR) and HER2/neu (ErbB-2) dual tyrosine kinase inhibitor. It inhibits autophosphorylation and activation of the receptor by binding to the ATP-binding pocket of the EGFR/HER2 protein kinase domain. The data presented in example 5 below shows that a significant synergy was achieved for Paca2 pancreatic cancer cells: the inhibition rates for compound 401 and lapatinib, before combination, were 32% and 27%, respectively, while the inhibition rate after combination rose to 74%, which is higher than the sum of the two inhibition rates. Since this method is additionally concerned with CSCs, which are fundamentally responsible for drug resistance, tumor recurrence and metastasis, in preferred embodiments, this method is used to treat or prevent refractory, recurrent, and/or metastatic cancers.

The formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the mammal to be treated and the particular mode of administration. The amount of active ingredient that may be combined with a carrier material to produce a single dosage form is generally that amount of compound which produces a therapeutic effect. Generally, the amount ranges from, for example, about 1% to about 99% active ingredient, about 5% to about 70%, about 10% to about 30% in 100%.

Materials and methods

Biological assay

The compounds of the invention can be examined according to the protocol described above. Table 2 shows a list of compounds described in this scheme.

TABLE 2

Name of Compound	Compound code
		2- (1-hydroxyethyl) -naphtho [2, 3-b]Furan-4, 9-diones	301
2-acetyl-7-chloro-naphtho [2, 3-b]Furan-4, 9-diones	416
		2-acetyl-7-fluoro-naphtho [2, 3-b]Furan-4, 9-diones	418
2-acetyl naphtho [2, 3-b ]]Furan-4, 9-diones	401
		2-ethyl-naphtho [2, 3-b ]]Furan-4, 9-diones	101

Cell culture: HeLa, DU145, H1299, DLD1, SW480, A549, MCF7, LN18, HCT116, HepG2, Paca2, Panc1, LNcap, FaDu, HT29 and PC3 cells (ATCC, Manassas, VA) were maintained in Dulbecco Modified Eagle Medium (DMEM) (Invitrogen, Carlsbad, Calif.) supplemented with 10% Fetal Bovine Serum (FBS) (Gemini Bio-Products, West Sacramento, Calif.) and 5% penicillin/streptomycin/amphotericin B (Invitrogen).

Hoechst side population: to identify and isolate the Side Population (SP) and non-SP fractions, SW480 cells were removed from the plates with trypsin and EDTA, pelleted by centrifugation, washed with Phosphate Buffered Saline (PBS), and resuspended in Dulbecco's Modified Eagle Medium (DMEM) containing 2% FBS and 1mM HEPES at 37 ℃. Cells were then labeled with Hoechst 33342(Invitrogen) at a concentration of 5. mu.g/mL. Labeled cells were incubated for 120 min at 37 ℃ alone or with 50. mu.M verapamil (Sigma-Aldrich, St. Louis). After staining, cells were suspended in Hanks balanced salt solution (HBSS; Invitrogen) containing 2% FBS and 1mM HEPES, passed through a 40 μm sieve, and maintained at 4 ℃ until flow cytometry analysis was performed. The Hoechst dye was excited at 350nm and its fluorescence was measured at two wavelengths using 450DF10(450/20nm bandpass filter) and 675LP (675nm long wavelength bandpass cut-off filter) optical filters. Gating forward and side scattered light is not critical, only excluding debris [15 ].

CSC separation with surface markers: by sorting tumor cells based primarily on differential expression of surface markers (e.g., CD44 or CD133), most of the highly tumorigenic CSCs described to date have been generated. CD133 is based on a slightly modified RicMethod of ci-Vitiani et al [21]And (5) separating. CD133⁺Cells are separated by Fluorescence Activated Cell Sorting (FACS) or magnetic nanoparticle based separation methods. Briefly, for FACS-based cell sorting, 10 was labeled with CD133/1(AC133) -PE⁷cell/mL; or for magnetic field-based separations, usingBiotin selection kit (Miltenyi Biotec) the CD133/1(AC133) -biotin (Miltenyi Biotec, Auburn, CA) was labelled 10 according to the manufacturer's recommendations⁷cells/mL. Non-specific labeling was blocked with the provided FcR blocking reagent, while antibody incubation (1: 11) was performed on ice for 15 min in PBS with 2% FBS and 1mM EDTA. For theThe separation was performed 5 washes, while the cells were pelleted at 400 Xg for 5 minutes and resuspended to 2X 10 before FACS sorting⁷/mL。

CD44^highCells were according to the slightly modified method described by Ponti et al [81 ]]Separation was by FACS. Briefly, after trypsinization and 30 min cell recovery in 37 ℃ growth medium, cells were pelleted at 400 Xg and resuspended to 1X 10 in PBS with 2% FBS and 1mM EDTA⁶cells/mL. The cells were then incubated with CD44-FITC (BD biosciences, san Diego, Calif.) at a 1: 100 dilution for 15 minutes on ice. Alternatively, negative selection was performed using CD24-PE (BD biosciences, San Diego, Calif.) (1: 100). After 3 washes, the cells were resuspended to 2X 10⁶mL, and passed through a 40 μm sieve, followed by sorting.

Sphere determination test: one reliable method to measure the ability of a cell population to self-renew is the ability to be cultured as spheres in the absence of serum or adherence. CD44^highFaDu or Hoechst side population cancer stem cells were cultured in cancer stem cell medium (DMEM/F12, B27Neurobasal supplement, 20ng/ml EGF, 10ng/ml FGF, 4. mu.g/ml insulin and 0.4% BSA) in ultra-low attachment plates to allow spheroids to form. Generally, 10 to 14 days after the cultureSphere formation was assessed by microscopy and spheres with > 50 cells were recorded.

Luciferase reporter assay: HeLa cells were co-transfected with Stat 3-luciferase (Stat3-Luc) reporter vector (Panomics, Fremont, CA) and Renilla luciferase (Promega, Madison, Wis.) by Lipofectamine 2000 as described by the manufacturer (Invitrogen). After transfection, cells were maintained in medium containing 0.5% FBS for 24 hours. The cells were then treated with the indicated compounds for 30 minutes, after which 25ng/ml oncostatin M (OSM) (R & D Systems, Minneapolis, MN) was added to the medium. Cells were harvested 6 hours after OSM addition and levels of firefly and renilla luciferases were measured using the Dual-Glo luciferase assay system as described by the manufacturer (Promega).

Apoptosis analysis: cells with or without compound treatment were harvested 5 hours after treatment and stained for annexin-V. The harvested cells were washed with PBS, resuspended in annexin-V-FITC containing buffer, and stained according to the manufacturer's (Roche) instructions. annexin-V positive cells were determined by flow cytometry.

STAT3DNA binding assay: electrophoretic Mobility Shift Analysis (EMSA) was performed as described by the manufacturer (Li-Cor Biosciences, Lincoln, NE). Briefly, nuclear extracts were prepared from HeLa cells using the NucBuster protein extraction kit as described by the manufacturer (EMD Biosciences, San Diego, CA). Mu.g of nuclear extract were preincubated for 30 minutes with the indicated compounds at the indicated doses, followed by 15 minutes with IR 700-labeled consensus Stat3 oligonucleotide. The samples were then electrophoresed on polyacrylamide gels and scanned directly using the Odyssey infrared imaging system (Li-Cor Biosciences). For enzyme-linked immunosorbent assay (ELISA), 5. mu.g of nuclear extract was preincubated with the indicated compounds at the indicated concentrations for 30 minutes, after which biotinylated oligomer (5 '-biotin-GATCCTTCTGGGAATTCCTAGATC-3' SEQ ID NO.1) was added. The Stat3-DNA complex was then captured on a streptavidin-coated 96-well plate (Pierce, Rockford, IL). The bound complexes were then incubated with Stat3 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA), followed by anti-rabbit HRP conjugated secondary antibody (GE Healthcare, Pittsburgh, PA). The bound antibody was then observed by adding TMB substrate (Pierce) and measuring the absorbance at 450 nm.

Cell viability determination: for 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium (MTT) (Sigma-Aldrich, st. louis, MO) assay, cells were seeded at 10,000 cells/well in 96-well plates. 24 hours after plating, compounds were added to the cells at the indicated doses. 22 hours after compound addition, MTT (0.5mg/ml final concentration) was added to each well and the plates were incubated for an additional 2 hours at 37 ℃. The medium was then aspirated and the formazan product was dissolved in 100 μ l isopropanol. The absorbance of each well was measured at 570nm using a microplate reader.

Immunofluorescence: cells treated with the indicated compounds for the indicated times were fixed in 4% formaldehyde or cold methanol for detection of annexin V, cleaved caspase 3(cleaved caspase 3) or stat3, respectively. The coverslip was air dried and rehydrated in PBS for 10 minutes at room temperature. The samples were then incubated in blocking buffer (PBS, 5% FBS) in a humidified chamber for 10 minutes at room temperature. Cells were incubated with primary antibody overnight at 4 ℃. After washing, cells were incubated with FITC conjugated anti-rabbit antibody at 1: 500 dilution for 1 hour at room temperature. Images were captured with a Nikon TE200 microscope equipped with epi-fluorescence and SPOT-mounted CCD cameras. Polyclonal anti-cleavage caspase 3 antibody (1: 100) was obtained from Cell Signaling Technology, Danvers, MA.. annexin-V-FITC was obtained from Roche (Roche) of Penzberg, Germany. Polyclonal anti-Stat 3 antibody was obtained from Santa Cruz corporation.

By passingTechnical knock-down of genes:(therapeutic pathway identification and validation) technology (Boston biomedical Inc., Norwood, MA, USA) provides plasmids that can be used to transfect bacteria first, for uptake by mammalian subjects. After lysis of the bacteria, fromdsRNA encoded by the plasmid and processed by the bacterium is released into the cytoplasm of mammalian cells, effecting knock-down of the targeted gene.The technology is described in commonly owned PCT patent application PCT/US08/68866, filed on 30.6.2008, which is incorporated herein by reference in its entirety. In particular, encoding an effective siRNA sequence against Stat3Plasmids Stat3 plasmid, purchased from Origene Technologies (Rockville, Md., USA), was cloned by PCR using the following primers: TPIV-Stat3(300bp insert)

Primer:

Stat3TPIV For 5’-GGATCTAGAATCAGCTACAGCAGC(SEQ ID NO.2)

Stat3TPIV Rev 5’-TCCTCTAGAGGGCAATCTCCATTG(SEQ ID NO.3)

control plasmids were constructed using pGL2 plasmid from Promega (Madison, Wis., USA). TPIV-GL2(300bp insert)

Primer:

GL2TPIV For 5’-CCCTCTAGATGGTTCCTGGAAC(SEQ ID NO.4)

GL2TPIV Rev 5’-GCTCTAGAAACCCCTTTTTGG(SEQ ID NO.5)

chemically competent Escherichia coli (E. coli) BL21(DE3) pLYSe bacteria (50-100 μ l) with control or 100ng targeting Stat3The plasmids were transformed according to the instructions of the manufacturer (Stratagene). Then, a single colony was inoculated into BHI medium containing 100. mu.g/ml ampicillinAnd grown overnight at 37 ℃. The following day, 5ml of each overnight culture were diluted 1: 40 into fresh BHI medium containing 100. mu.g/ml ampicillin and grown for an additional 2-4 hours (until OD₆₀₀0.5). Each culture was then treated with IPTG (1mM final concentration) for 2-4 hours to induce transcription of long double stranded RNA, which would be processed by the bacteria as mixed siRNA. After IPTG induction, by measuring OD₆₀₀The total number of bacteria in each culture was calculated (8X 10)⁸Bacteria/ml culture having OD₆₀₀1). The number of bacteria used for cell treatment was then calculated based on the degree of cell confluence and the required multiplicity of infection (MOI; trying a range of bacteria to cells from 20: 1 to 2000: 1) in the appropriate reaction volume. As a rule of thumb, the reaction volume should be chosen so as to result in a value of 3X 10 for a MOI of 1000: 1⁸And/ml. The desired volume of bacterial culture was then centrifuged at 2500g for 10 min at 4 ℃, the pellet washed once with serum-free medium (cells for bacterial infection, plus 100. mu.g/ml ampicillin and 1mM IPTG) and resuspended in the same medium at the density required for bacterial infection (bactofunction).

Simultaneously, cancer cells or cancer stem cells were isolated and 30 minutes prior to bacterial infection, the cell culture medium was replaced with 2ml of fresh serum-free medium containing 100. mu.g/ml ampicillin and 1mM IPTG. The bacteria prepared above were then added to the cells at the desired MOI and infected at 37 ℃ for 2 hours.

After the infection period, the cells were washed 3 times with serum-free cell culture medium. The cells were then incubated with 2ml of fresh complete cell culture medium containing 100. mu.g/ml ampicillin and 150. mu.g/ml gentamicin for 2 hours to kill any remaining extracellular bacteria. After ampicillin and gentamicin treatment, cells were incubated with 3ml of fresh complete RPMI 1640 medium containing 10. mu.g/ml ofloxacin to kill any intracellular bacteria. The cells are then harvested or analyzed at various time points to assess the degree of silencing of the target gene and the resulting phenotype.

Evaluation of living body (in life evaluation): daily checks of the health status of each animal were also made. Body weight was checked every three days. Institutional animal managementThe method provides food and water daily. Treatments that cause > 20% mortality and/or > 20% net weight loss are considered toxic. Results are expressed as mean tumor volume (mm)³) Plus or minus SE. P values < 0.05 were considered statistically relevant.

Animal management: male or female athymic nude mice (Charles River Laboratories, Wilmington, MA.) of 4-5 weeks of age were acclimatized to animal housing facilities for at least one week prior to study initiation. All experimental methods utilized are in accordance with guidelines given by the American Society of Physiology (American Physiology Society) and the guidelines for the Care and Use of Laboratory Animals (Guide for the Care and Use of Laboratory Animals), and are also approved by the institute of Animal Care and Use Committee (Institutional Animal Care Committee) of Boston biomedical Inc. Animals 4 were housed in groups of wood chip bedding cages and placed in a room with controlled temperature (68-72F), light (12 hours light-dark cycle) and humidity (45-55%). During the experiment the animals had free access to water and food.

Splenic nude mouse model system (ISMS model): female nude mice were anesthetized and placed under sterile conditions, and were dissected in the left flank to expose the spleen. 100 million human colon cancer HT29 cells in 0.1ml PBS were injected subpial to the spleen using a 27 gauge needle. The spleen was replaced in the peritoneal cavity and the incision was closed. Treatment was started the day after transplantation until the day of examination. The treatment strategy was an intraperitoneal injection once a day (5qd/wk) 5 days per week. Mice were sacrificed at sacrifice or 30 days post injection. Spleens and livers were removed for examination and tumor disease variables were recorded.

Example 1

Identification of Stat 3as an anti-cancer stem cell target

Knock-down of Stat3 in CSCs can induce apoptosis. To determine whether cancer stem cells express Stat3, and whether Stat3 is constitutively activated, we performed immunofluorescence microscopy, which not only allowed for analysis of rare cell populations, but also provided additional information about protein localization, and was able to correlate staining with phenotype (i.e., apoptosis). After immunofluorescence detection of p-Stat3 and Stat3 in NSP and SP cells isolated from SW480 colon cancer cells by FACS, we determined that Stat3 is indeed present in SP cells and moderately enriched in the nucleus (fig. 3A). In addition, we also observed increased p-Stat3 staining in SP cells compared to NSP cells, suggesting that SP cell survival may be heavily dependent on Stat3.

CD133 isolated from FaDu human head and neck cancer cells and LN18 human glioblastoma cells was also evaluated⁺Stat3 status in cells. As shown in fig. 3B, Stat3 is also constitutively active in these cells. Taken together, these data suggest that Stat3 is a particularly important target for cancer stem cells.

We next utilizeThe effect of Stat3 knockdown in CSCs was examined. Immunofluorescence analysis revealed that significant depletion of Stat3 could be achieved within 24 hours of infection on freshly isolated csc (sp) (fig. 4A) and found to target Stat3Most cells treated with plasmid underwent apoptosis within 24 hours of infection, while controlsThe plasmid did not induce levels of apoptosis above control uninfected cells (fig. 4B). These data demonstrate that cancer stem cells are dependent on Stat3 for survival.

Knock-down of Stat3 in CSCs inhibits CSC sphere formation. Isolation of CD44 by FACS^high/CD24^lowFaDu or Hoeschst side groups of cancer stem cells and cultured in cancer stem cell medium (DMEM/F12, B27Neurobasal supplement, 20ng/mL EGF, 10ng/mL FGF, 4. mu.g/mL insulin and 0.4% BSA) in ultra-low attachment plates to allow spheroids to form. Collecting primary spheres (prima)ry spheres) disaggregated with trypsin and inBefore treatment were dispensed into 96-well ultra-low attachment plates. Bacteria were applied at an MOI of 1000 for two hours, after which antibiotic mixtures (penicillin-streptomycin mixture (penta), gentamicin, ofloxacin) were added. Sphere formation was assessed after 10-14 days of culture. Representative sphere images were taken before (fig. 5, top left) or after (fig. 5, bottom left) trypan blue addition to identify dead cells. The opposing spheres form the right hand figure shown in figure 5. The data clearly show that Stat3 knockdown inhibits spheroid formation in cancer stem cells, demonstrating that Stat3 is a key self-renewal factor in cancer stem cells.

Example 2

Identification of Compounds that inhibit Stat3 pathway Activity

Inhibition of Stat3 transcriptional activity. The ability of compounds to inhibit Stat3 transcriptional activation activity in cells was examined using the Stat 3-luciferase (Stat3-luc) reporter construct. Cells transfected with Stat3-luc were cultured in reduced serum medium, followed by incubation for 30 min with the indicated compounds. The cells were then stimulated with 25ng/ml oncostatin M (OSM) for 6 hours before testing for Stat3-luc reporter activity. Incubation of cells with compound 401 inhibited the OSM-stimulated Stat3 reporter activity (figure 6, left panel). AG490, a known Jak-Stat pathway inhibitor, was included as a positive control for Stat3 inhibition. Etoposide was included as a genotoxic activity control that showed little or no Stat3 inhibition. Compound 1001 is naphthalene and not naphthoquinone, a compound of the invention, which does not inhibit the Stat3 reporter activity stimulated by OSM even at much higher concentrations (figure 6, right panel).

Other compounds were tested in the Stat3 luciferase reporter assay and the results are summarized in table 3.

TABLE 3

Compound #	IC in Stat3-Luc assay50
		401	～0.25μM
416	～0.75μM
		418	～0.75μM
301	～2μM

Inhibition of Stat3DNA binding activity. Stat3EMSA was performed using nuclear extracts from HeLa cells (containing constitutively activated Stat 3as detected by phosphorylation of tyrosine 705 residues) to monitor Stat3DNA binding activity. Nuclear extracts were incubated with the indicated compounds followed by incubation with IR700 labeled Stat3 consensus oligonucleotide. Binding of Stat3 to the oligonucleotide was monitored by gel electrophoresis and detected using a LiCor Odyssey infrared scanner. A Stat3 hysteresis band was identified and confirmed by hyper-migration of anti-Stat 3 antibody (fig. 7A, left panel) and dose-dependent inhibition of Stat3 peptide (fig. 7A, middle panel). Dose-dependent inhibition of Stat3DNA binding was observed after incubation of the labeled probe with compound 401 (fig. 7A, right panel).

Other compounds were tested in the EMSA assay. As shown in fig. 7B, compounds 401, 416 and 418 were able to inhibit the DNA binding activity of Stat3.

Inhibition of Stat3 downstream effectors in xenograft tumor tissues. Extracts were prepared from xenograft Paca2 tumors treated with compound 401 or vehicle control 4 hours prior to harvest. Samples were analyzed by western blot and EMSA to assess Stat3 downstream effector expression levels and Stat3DNA binding activity. Compound 401 treated sample (T) showed a decrease in Stat3DNA binding activity compared to control (V) (fig. 8A). In addition, compound 401 treatment resulted in a decrease in the expression levels of effector cyclin D1 and survivin downstream of Stat3 (fig. 8B).

Example 3

Identifying compounds targeting cancer stem cells

Identifying a compound that causes apoptosis of cancer stem cells. Since cancer stem cells have been demonstrated to be actively efflux Hoechst, SW480 cells were stained with Hoechst and the side population (the region enclosed by the left panel, as shown in fig. 9A) was sorted to enrich for cancer stem cells. To confirm that this side population was enriched for cancer stem cells, SW480 cells of the control group were first treated with verapamil (an ABC transporter inhibitor) and then stained with Hoechst. As shown in the right panel of fig. 9A, verapamil treatment resulted in lateral group loss.

IC of Compound 401 on the Hoechst side population was evaluated in the MTT assay₅₀And to the IC of the non-side group₅₀A comparison was made. The results show that the side population is as sensitive to compound 401 as the non-side population (fig. 9B, right panel). However, the lateral group was much more resistant to doxorubicin than the non-lateral group (fig. 9B, left panel), consistent with previous disclosure [7, 82]. These data suggest that compound 401 kills cancer stem cells.

Hoechst side population cells were treated with compound 401 and the cell death pattern was assessed by annexin V (an early marker of apoptosis) staining. The results showed that dying cells were positive for annexin V (fig. 10A), demonstrating that compound 401 caused cancer stem cells to apoptosis.

Alternatively, we performed CD133 (one of the common cancer stem cell surface markers) antibody magnetic bead sedimentation to enrich cancer stem cells. CD133⁺The cells were then treated with compound 401 followed by staining with an antibody against cleaved caspase 3 (apoptosis marker). As shown in FIG. 10B, a plurality of CDs 133⁺Cells became positive for lytic caspase 3 after compound 401 treatment, confirming that compound 401 caused cancer stem cells to apoptosis.

Identifying a compound that inhibits CSC sphere formation in vitro. One of the hallmarks of cancer stem cells is their ability to self-renew. One reliable method to measure the self-renewal capacity of a cell population is its ability to grow into spheres in the absence of serum or attachment. To compare this ability of compound 401 to other targeting and chemotherapeutic agents, FACS-isolated CD44^highCSCs were cultured as spheres for 72 hours, followed by challenge with a panel of therapeutic agents. Of the agents tested, only compound 401 was effective in preventing spheroid proliferation (fig. 11). Note that although the amount of doxorubicin and docetaxel applied was approximately ten times greater than its IC's that caused cell death in a similar assay₅₀Concentration, but the spheres are resistant. The addition of Tarceva (Tarceva), Sutent (Sutent) and Gleevec (Gleevec) was approximately three times their reported therapeutic concentrations. This demonstrates that compound 401 is highly effective in inhibiting the growth of cancer stem cells, although they are resistant to traditional chemotherapeutic and targeting agents.

Identifying a compound that inhibits CSC sphere formation in vivo. Six week old female athymic nu/nu mice were obtained from Charles River Labs (Wilmington, MA). Mice were injected subcutaneously at the flank with 6X 10 in 0.2mL serum-free DMEM⁶FaDu or Paca2 cancer cells. The size of the xenograft reaches-200 mm³Thereafter, animals bearing the Paca2 xenograft tumor were administered vehicle, gemcitabine (120mg/kg twice a week) or compound 401(20mg/kg) intraperitoneally for one week, while animals bearing the FaDu xenograft tumor were administered vehicle, daily, intraperitoneally,Carboplatin (30mg/kg) or Compound 401(20mg/kg) for two weeks before sacrifice. Tumors were then harvested against Paca2 and FaDu cells, respectively. Single cell suspensions were prepared after animals were sacrificed and tumors removed aseptically. Briefly, tumors were cut to 0.1mm with a sterile scalpel³After which digestion was continued with shaking in 1mg/mL collagenase/HBSS for 15-30 minutes. After passing through a 40 μm screen, the cell suspension was placed on 1mL Histopaque and the interface layer was collected after centrifugation at 1440 × g for 30 minutes to remove RBCs, dead cells and cell debris. Viable cells were then counted and used to measure their ability to form spheres. Cells were distributed at a density of 100 cells/well into cancer stem cell medium in ultra-low attachment 96-well plates (DMEM/F12, B27Neurobasal supplement, 20ng/mL EGF, 10ng/mL FGF, 4. mu.g/mL insulin and 0.4% BSA). Fresh medium was added every three days and spheroid formation was determined after 10-14 days of culture. Spheres with > 50 cells were recorded. At the end of the experiment, trypan blue was added to identify dead cells. As shown in figure 12, standard chemotherapies gemcitabine (upper panel) and carboplatin (lower panel) enriched cancer stem cells as evidenced by increased spheroid formation. Conversely, compound 401 treatment reduced cancer stem cells as evidenced by reduced sphere formation.

Example 4

Anti-metastatic effect

Compound 401 was also tested for its ability to inhibit metastasis in an ISMS model. The splenic nude mouse model system (ISMS model) is suitable for studying malignant behavior of colorectal cancer, as this technique is capable of generating experimental metastases in the liver. In this model, 100 million HT29 cells in 0.1ml PBS were injected subcapsulally into the spleen of nude mice. The spleen was replaced in the peritoneal cavity and the incision was closed. Mice were sacrificed at sacrifice or 30 days post injection. Spleens and livers were removed for examination and tumor disease variables were recorded (number of tomoles). The mice were divided into 2 groups, the control group was given vehicle (n-4) and the other group received 20mg/kg of compound 401 (n-4). Drugs were administered intraperitoneally from day 2 to day 30, 5 days/week after i.s. injection. Microscopy estimates the number of primary and metastatic liver tumors. A representative photograph is shown in fig. 13. In the vehicle control group, the spleen had a heavy primary tumor (fig. 13, top left panel). A large number of spontaneous liver metastases were also observed (fig. 13, upper right panel). Compound 401 treatment significantly reduced the number of primary tumor foci and the number of spontaneous liver metastases (figure 13, bottom panel).

Example 5

Combined activity

Paca2 human pancreatic cancer cells, A549 human lung cancer cells and HepG2 human hepatoma cells (American type culture Collection) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 units/mL penicillin, 100. mu.g/mL streptomycin and 2mM L-glutamine. Compound 401 and sotan were synthesized by Boston biomedicalal, inc. Carboplatin, doxorubicin, docetaxel, etoposide were obtained from Sigma (st. louis, MO) and dissolved at 10mM in water or DMSO. Erlotinib is from American Custom Chemicals (San Diego, Calif.). Gemcitabine is available from Eli Lilly (Indianapolis, IN) IN the form of an aqueous 20mM stock. Sorafenib was purchased from LKT (st. paul, MN). Lapatinib is from LC Laboratories (Woburn, MA). Unless otherwise indicated, all compounds were dissolved in DMSO at 10mM and aliquots of Paca2 pancreatic cancer cells stored exponentially at-20 ℃ were seeded at 1,000 cells/well in 6-well plates and allowed to adhere for 24 hours. The medium is then supplemented with increasing concentrations of the individual drugs and combination drugs for an additional 24 hours. After 24 hours of contact, the drug was removed and fresh medium was added for the next 10-14 days, allowing colonies to form. Cells were fixed and stained with giemsa (gibco brl). Colonies of greater than 50 cells were recorded as survivors and normalized for percent cell survival to untreated controls. Results are the average of duplicate experiments. Alternatively, MTT assays were performed 72 hours post-treatment in a549 and HepG2 cells.

Our data demonstrate that compound 401 has beneficial effects when combined with all compounds tested. Among them, the most excellent results were shown in combination with Tyrosine Kinase Inhibitors (TKIs). For example, as shown in figure 14, compound 401 in combination with sorafenib had a synergistic effect in human lung a549 cells for 72 hours. Similarly, figures 15 to 17 show that compound 401 is associated with erlotinib, lapatinib and sunitinib, respectivelyThe combination also had a synergistic effect in human lung a549 cells for 72 hours. The remaining data are summarized in table 4, demonstrating that compound 401 exhibits beneficial effects when combined with all of the test drugs.

TABLE 4

Furthermore, we also tested the combined effect of compound 401 and gemcitabine in a human pancreatic cancer xenograft model. Briefly, athymic female nude mice (Ncr) were inoculated subcutaneously with 8X 10⁶MIA PaCa-2 human pancreatic cancer cells and tumor growth to about 150mm³The size of (2). Animals were randomized into 4 groups of 6 animals each, treated with vehicle controls, orally administered compound 401 as 100mg/kg daily clinical formulation (20% Gelucire), intraperitoneally injected every three days with 120mg/kg (in PBS) gemcitabineOr both after administration. Mice received a total of two weeks of treatment and the mean volume of the tumor was analyzed.

As shown in FIG. 18, treatment with either Compound 401(100mg/kg) or gemcitabine (120mg/kg) alone delayed tumor growth to a similar extent during treatment. Animals treated with compound 401(100mg/kg) in combination with gemcitabine (120mg/kg) showed a synergistic effect on tumor growth. No significant toxicity was observed for any of the treatment strategies. Our data suggest that compound 401 in combination with gemcitabine has clinical benefit in the treatment of pancreatic cancer.

All references cited herein are incorporated by reference in their entirety to the extent allowed by applicable law and for all purposes to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to substitute and/or take precedence over any such contradictory material.

As used in the specification and claims, the numerical quantities expressing ingredients, reaction conditions, assay results, and so forth, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. Each numerical parameter should be construed in light of the number of significant digits and ordinary rounding techniques, and should not be construed as limiting the scope of the claims in any way.

It will be apparent to those skilled in the art that modifications and variations can be made to the present invention without departing from the spirit and scope thereof. The specific embodiments described herein are offered by way of example only and are not intended to be limiting in any way. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

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Claims (6)

1. Use of a composition comprising 2-acetylnaphtho [2, 3-b ] furan-4, 9-dione, or a pharmaceutically acceptable salt thereof, and a second agent in the manufacture of a medicament for treating cancer in a subject, wherein the composition is formulated for oral administration and the second agent is selected from: paclitaxel/taxol, erlotinib, sunitinib, lapatinib, sorafenib, carboplatin, doxorubicin, docetaxel, gemcitabine, and etoposide.

2. The use of claim 1, wherein the cancer is metastatic, refractory to a standard first-line cancer treatment, or recurrent.

3. The use of claim 2, wherein the cancer is selected from the group consisting of: breast cancer, head and neck cancer, lung cancer, ovarian cancer, pancreatic cancer, colorectal cancer, prostate cancer, renal cell carcinoma, melanoma, hepatocellular carcinoma, cervical cancer, sarcoma, brain tumors, gastric cancer, gastrointestinal cancer, multiple myeloma, leukemia, and lymphoma.

4. The use of claim 1, wherein the cancer is gastric cancer.

5. The use of claim 1, wherein the cancer is gastrointestinal cancer.

6. The use of claim 1, wherein the cancer is colorectal cancer.