Combined use of nitroxoline and its analogues with chemotherapy and immunotherapy in cancer treatment

Abstract

Describes a novel combination therapy comprising nitroxoline, its analogs or pharmaceutically acceptable salts thereof and at least one additional anti-cancer chemotherapeutic or immunotherapeutic agent. Also describes related kits, pharmaceutical compositions or methods for their production.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 315,774, filed Mar. 31, 2016, the disclosure of which is incorporated herein by reference in its entirety. Field of the invention

[0003] The present invention relates to a combination therapy for treating cancer, particularly to a combination of niridazole or its analogs or pharmaceutically acceptable salts thereof, with at least one additional anti - cancer chemotherapy or immunotherapeutic agent, wherein the combination therapy exhibits a synergistic anti - cancer effect. Background of the invention

[0005] Combination therapies for treating cancer help to minimize the likelihood that cancer will become tolerant to any one anti - cancer therapy, and they allow the use of lower doses of the individual anti - cancer therapies, which results in reduced toxicity. Depending on the nature of the drug - drug interaction, the combination of two active ingredients can produce a synergistic, additive, or antagonistic effect.

[0006] Niridazole (NIT) is an antimicrobial agent that has been on the market for a long time for the treatment of urinary tract infections. It has recently been found that niridazole also has activities of inhibiting angiogenesis (4) and inhibiting cancer growth and invasion (5, 6). NIT and its analogs, such as hydroxyquinoline, chloroiodoquine, and diiodoquinoline, have a common clinical use in the treatment of infectious diseases. It has also been reported that NIT analogs have anti - cancer cytotoxicity (7). However, to the knowledge of the present inventors, the combination of these drugs with other anti - cancer drugs has not been studied, and the drug - drug interaction of NIT or its analogs with any other cancer therapy has not been reported.

[0007] There is a need in the art for novel combination therapies for treating cancer with enhanced anti - cancer effects or reduced toxicity.

[0008] Summary of the invention

[0009] The present invention meets these needs by providing a novel combination therapy comprising niridazole, its analogs, or pharmaceutically acceptable salts thereof, with at least one additional anti - cancer chemotherapy or immunotherapeutic agent. The combination therapy shows a synergistic effect on inhibiting cancer cell growth.

[0010] In a general aspect, the present invention provides a method for treating cancer by administering niridazole, its analogs, or pharmaceutically acceptable salts thereof, in combination with chemotherapy to a subject in need thereof. The chemotherapy is preferably selected from the group consisting of microtubule depolymerization inhibitors, agents that cross - link DNA, and platinum compounds.

[0011] In another general aspect, the present invention provides a method for treating cancer by administering nitroxoline, its analogs, or pharmaceutically acceptable salts thereof in combination with an immunotherapeutic agent to a subject in need thereof. The immunotherapeutic agent is preferably an agent that can stimulate an effective immune response and inhibit immunosuppression. More preferably, the immunotherapeutic agent is an inhibitor or modulator of regulatory T cells or myeloid-derived suppressor cells.

[0012] Other aspects, features, and advantages of the present invention will be apparent from the following disclosure, which includes a detailed description of the invention, its preferred embodiments, and the appended claims.

[0013] Brief Description of the Drawings

[0014] The above summary, as well as the following detailed description of the invention, can be better understood when read in conjunction with the accompanying drawings. For purposes of exemplifying the invention, it is shown in the presently preferred embodiments of the drawings. However, it is to be understood that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0015] Figure 1 Showing the structure of nitroxoline (NIT) and examples of its analogs;

[0016] Figure 2 Showing the combination index (CI) plot of inhibiting the growth of human bladder cancer cell line 5637 by the combination of NIT and carboplatin;

[0017] Figure 3 Showing the CI plot of inhibiting the growth of human bladder cancer cell line 5637 by the combination of NIT and paclitaxel;

[0018] Figure 4 Showing the CI plot of inhibiting the growth of human hepatocellular carcinoma cell line HepG2 by the combination of NIT and carboplatin;

[0019] Figure 5 Showing the CI plot of inhibiting the growth of human hepatocellular carcinoma cell line HepG2 by the combination of NIT and paclitaxel;

[0020] Figure 6 Showing the CI plot of inhibiting the growth of human bladder cancer cell line 5637 by the combination of NIT and mitomycin C;

[0021] Figure 7 Showing the CI plot of inhibiting the growth of human bladder cancer cell line 5637 by the combination of NIT and epirubicin;

[0022] Figure 8 Showing the CI plot of inhibiting the growth of human bladder cancer cell line 5637 by the combination of NIT and pirarubicin;

[0023] Figure 9Shows the inhibition of tumor growth by the combination of oral NIT and mitomycin C in an orthotopic murine MBT-2-Luc bladder cancer model;

[0024] Figure 10 Shows the inhibition of tumor growth by the combination of oral NIT and intravesical BCG in an orthotopic murine MBT-2-Luc bladder cancer model;

[0025] Figure 11 Shows the inhibition of tumor growth by the combination of oral NIT and anti-PD-1 antibody in an orthotopic murine MBT-2-Luc bladder cancer model; and

[0026] Figure 12 Shows the inhibition of tumor growth by the combination of oral NIT and anti-PD-1 antibody in an orthotopic murine RM-9-Luc prostate cancer model. DETAILED DESCRIPTION OF THE INVENTION

[0028] Numerous publications, articles, and patents are cited or described in the Background and throughout the specification; each of these references is hereby incorporated by reference in its entirety. The discussion of documents, acts, materials, devices, articles, etc. contained in this specification is provided to give context to the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention disclosed or claimed herein.

[0029] DEFINITIONS

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Otherwise, certain terms used herein have the meanings set forth in the specification. All patents, published patent applications, and publications cited herein are hereby incorporated by reference as if fully set forth herein. It must be noted that, as used herein and in the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural references.

[0031] Unless otherwise indicated, any numerical value, such as a concentration or concentration range described herein, should be understood to be modified in all instances by the term "about". Thus, numerical values generally include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). Unless the context clearly indicates otherwise, the use of numerical ranges as used herein expressly includes all possible sub-ranges, all individual values within the range (including integers and fractions of these values within the ranges).

[0032] As used herein, the terms "treat", "treating", and "treatment" are each intended to mean improving or reversing at least one measurable physical parameter associated with cancer, which need not be discernible in a subject, but may be discernible in a subject. The terms "treat", "treating", and "treatment" can also refer to causing regression, arresting progression, or at least slowing the progression of cancer. In one particular embodiment, "treat", "treating", and "treatment" mean alleviating, preventing development or onset, or reducing the duration of one or more symptoms associated with the cancer. In one particular embodiment, "treat", "treating", and "treatment" mean preventing recurrence of cancer. In one particular embodiment, "treat", "treating", and "treatment" mean increasing the survival rate of a subject having cancer. In one particular embodiment, "treat", "treating", and "treatment" mean eliminating the cancer in the subject.

[0033] As used herein, the term "subject" refers to an animal, preferably a mammal. According to a particular embodiment, the subject is a mammal including a non - primate (e.g., camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig, or mouse) or a primate (e.g., monkey, chimpanzee, or human). In a particular embodiment, the subject is a human.

[0034] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of an active ingredient or component that elicits a desired biological or pharmacological response in a subject. In certain embodiments, the effective amount is the amount of an active ingredient or compound that is effective to achieve a synergistic effect with another active ingredient or compound. As used herein, "synergistic effect" refers to an effect that is greater than the additive effect of two separate active ingredients or compounds for cancer treatment. A therapeutically effective amount can be determined empirically and in a conventional manner for the stated purpose. For example, in vitro assays can optionally be used to assist in determining the optimal dosage range. A person skilled in the art can determine the selection of a particular effective dose (e.g., through clinical trials) based on consideration of several factors, including the disease to be treated or prevented, the symptoms involved, the weight of the patient, the immune status of the patient, and other factors known to a person skilled in the art. The exact dose used in a formulation also depends on the route of administration and the severity of the disease and should be decided according to the judgment of a practicing physician and the circumstances of each patient. An effective dose can be extrapolated from the dose-response curve obtained from in vitro or animal model test systems. For example, a mathematical algorithm based on the Loewe additivity model can be used to evaluate a drug combination. In this model, the combination index (CI) value is calculated. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive, and antagonistic interactions, respectively.

[0035] As used herein, the term "pharmaceutically acceptable salt" refers to a safe and effective salt form of nitroxoline or one of its analogs. Non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, pear salts, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, mesylate, esylate, benzenesulfonate, and p-toluenesulfonate. Preferably, the pharmaceutically acceptable salt of nitroxoline is a base addition salt of nitroxoline, such as those described in International Patent Application No. PCT / US14 / 30532, the entire content of which is incorporated herein by reference.

[0036] As used herein, the term "chemotherapeutic agent" refers to any chemical substance that acts as an anticancer drug.

[0037] As used herein, the term "immunizing agent" or "immunomodulatory agent" refers to any agent that is capable of stimulating an immune response and / or inhibiting immunosuppression.

[0038] As used herein, the term "combination", in the context of administering two or more therapies to a subject, refers to the use of more than one therapy. The use of the term "combination" does not limit the order in which the therapies are administered to the subject. For example, a first therapy (such as an effective amount of nitroxoline or an analogue or pharmaceutically acceptable salt thereof described herein) can be administered before (such as 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks before), simultaneously with, or after (such as 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks after) a second therapy (such as an effective amount of an immunotherapeutic agent or an effective amount of a chemotherapeutic agent).

[0039] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation or other material well known in the art for pharmaceutical formulations. It is understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for the particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the effectiveness of the composition according to the invention or the biological activity of the composition according to the invention. According to particular embodiments, in view of the present disclosure, any pharmaceutically acceptable carrier suitable for nitroxoline-based pharmaceutical compositions can be used in the present invention. Non-limiting examples of carriers include saline and water.

[0040] Nitroxoline (NIT) and its analogues

[0041] As Figure 1 shown, NIT and its analogues, such as hydroxyquinoline, chloroiodoquine and diiodoquinoline, have a common clinical use in the treatment of infectious diseases. NIT is a broad-spectrum antibiotic that has been on the market since the 1960s for the treatment of urinary tract infections. Its mechanism of action in inhibiting bacterial growth remains uncharacterized. It has been found that NIT inhibits the adhesion of uropathogenic Escherichia coli (E. coli) to uroepithelial cells and catheters under sub-MIC of NIT, and it is thought that NIT promotes the disintegration of the bacterial outer membrane by chelating divalent ions Mg 2+ and Mn 2+ (1). Hydroxyquinoline (Oxine) is an antibacterial agent with mild antifungal, antibacterial, anthelmintic and amoebicidal activities. Hydroxyquinoline promotes the disintegration of the bacterial outer membrane by substantially depriving Mn 2+ and Mg 2+The enzyme can inhibit isolated Escherichia coli RNA polymerase without directly contacting the enzyme (2). Hydroxyquinoline is also used as a metal chelator and thus as a carrier for radioactive indium for diagnostic purposes. Clioquinol has long been used as a topical anti-infective, an intestinal amebicide, and a vaginal trichomonacide. Oral formulations of clioquinol have been shown to cause subacute myelopathic optic neuropathy and are thus banned worldwide. Clioquinol has been found to prevent cognitive decline in Alzheimer's disease in animal models, possibly because it has the ability to act as a chelator for Cu 2+ and Zn 2+ (3). Diiodohydroxyquinoline is another halogenated 8-hydroxyquinoline widely used as an intestinal antibacterial agent, particularly as an amebicide. It can also be used topically for other infections and may cause CNS and eye damage. Its mechanism of action remains unknown.

[0042] It has been shown that NIT and its analogs have anticancer activity. The results showed that NIT is a MetAP2 inhibitor and can thus block cancer angiogenesis by inhibiting the proliferation of human umbilical vein endothelial cells (HUVECs) (4). It has also been demonstrated that NIT can induce apoptosis in prostate cancer cells (5), and its cytotoxicity to cancer cells is more obvious when administered in combination with Cu 2+ (6). NIT analogs, such as clioquinol, hydroxyquinoline, and diiodohydroxyquinoline, have also been reported to have anticancer cytotoxicity (7).

[0043] Chemotherapy

[0044] Cancer is the uncontrolled growth of cells accompanied by malignant behavior, including invasion and metastasis, among other characteristics. It is caused by the interaction between genetic susceptibility and environmental factors. These factors lead to the accumulation of gene mutations in oncogenes, genes that control the rate of cell growth, and tumor suppressor genes, genes that help prevent cancer, which endow cancer cells with malignant characteristics such as uncontrolled growth. Chemotherapy is a cancer treatment strategy that uses chemicals, particularly one or more anticancer drugs. Broadly speaking, most chemotherapeutic drugs act by reducing mitosis (cell division), which effectively targets rapidly dividing cells. Since these drugs cause damage to cells, they are called cytotoxic. Anticancer drugs prevent mitosis through various mechanisms, including damaging DNA and inhibiting cellular machinery involved in cell division. One theory regarding why these drugs kill cancer cells is that they induce a form of programmed cell death called apoptosis. There are many clinically available cytotoxic chemotherapies for treating cancer, and they are classified into different categories based on their chemical structural characteristics. See, for example, Table 1 below and www.en.wikipedia.org / wiki / List_ of_antineoplastic_agents .

[0045] Table 1. List of Anticancer Chemotherapies

[0046]

[0047]

[0048]

[0049] Traditional chemotherapeutic agents are cytotoxic, and they act by killing cells that divide rapidly, which is one of the fundamental properties of most cancer cells. This targeting of rapidly dividing cells means that chemotherapy also harms cells that normally divide rapidly, such as cells in the bone marrow, digestive tract, and hair follicles, which results in the most common side effects of chemotherapy, including myelosuppression (decreased production of blood cells and thus also immunosuppression), mucositis (inflammation of the inner lining of the digestive tract), and alopecia (hair loss). Combination therapy involves treating a patient with multiple different drugs simultaneously. Because the mechanisms and side effects of these drugs are different, combination therapy can minimize the probability of developing resistance to any one agent, and thus it can be used to treat drug-resistant cancers or at lower doses to reduce toxicity. However, due to the unpredictable nature of drug-drug interactions, known combinations of treatments can also lead to reduced efficacy or increased side effects.

[0050] Cancer immunotherapy

[0051] Cancer immunotherapy attempts to stimulate the immune system to reject and destroy tumors. It has been found that tumors can evade host immunity by manipulating the tumor microenvironment and driving immunosuppression, such that although the components necessary to generate an effective anti-tumor immune response are present in cancer patients, the host generally cannot arrest tumor progression (8). Many molecular and cellular mechanisms have been proposed in the past to explain this counterintuitive situation, which has mainly focused on the "immune escape" of tumors and implied that tumors have the ability to avoid tumor-specific immune responses generated by the host, or that they completely abrogate the host's anti-tumor immunity. This process, known as "tumor-induced immunosuppression", has been recognized in recent years and is the subject of intensive research. Tumors appear to be able to interfere with all components of the immune system and affect all stages of the anti-tumor immune response.

[0052] As tumors progress from premalignant to metastatic phenotypes, the molecular alterations that occur in tumor cells are the result of genetic instability, which is now considered a hallmark of all tumors. Genetic changes can be detected in the early stages of tumorigenesis, become more prominent as tumors progress, and are greatest in metastatic cells, and are the cause of tumor heterogeneity and the altered antigenic epitope profiles of tumor cells. It has been proposed that the immune system may drive these antigenic changes via "immune editing" by eliminating those malignant cells that are sensitive to immune intervention and allowing the selection and survival of immune-tolerant variants. The ultimate result of immune editing is tumor escape from the host immune system. Regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) are two cell types used by tumors to achieve immune escape. Protecting immune cells from the adverse effects of Tregs, MDSCs, or inhibitory factors, thereby enhancing their effector functions, can restore effective anti-tumor immunity in cancer patients.

[0053] CD4 + CD25 high FOXP3 + Treg cells accumulate in the peripheral circulation of human tumors and cancer patients. It is unclear whether these cells migrate into tumors or expand in situ. Since tumor-associated antigens (TAAs) are self-antigens, Treg accumulation may be a response to enforce immune tolerance. Treg cells downregulate the immune activity of effector T cells through multiple mechanisms, including the production of IL-10 and TGF-β1, the enzymatic degradation of ATP to immunosuppressive adenosine, or the engagement of the Fas / FasL and granzyme / perforin pathways. Tumors benefit from Treg-mediated immunosuppression.

[0054] Myeloid-derived immature myeloid cells MDSCs (CD34 + CD33 + CD13 + CD15-) are present at elevated frequencies in the peripheral circulation and tumors of almost all cancer patients. They are recruited by tumor-derived soluble factors (such as TGF-β1, IL-10, VEGF, GMCSF, IL-6, PGE2). They promote tumor growth by inhibiting T cell responses through several mechanisms, including the production of arginase-1, an enzyme involved in L-arginine metabolism, and the activation of inducible nitric oxide synthase (iNOS). They also control the production of tumor indoleamine-2,2-dioxygenase (IDO), which is involved in the catabolism of tryptophan, an amino acid essential for T cell differentiation.

[0055] BCG immunotherapy for early (non-invasive) bladder cancer utilizes the instillation of attenuated live bacteria into the bladder and is effective in preventing recurrence in up to two-thirds of cases. This immune response to BCG can be summarized as follows (9): Infection of urothelial and bladder tumor cells by BCG leads to BCG internalization, which increases the expression of antigen-presenting molecules. This induces an immune response through the release of cytokines, such as Th1 cytokines (IL-2, tumor necrosis factor, L-12, and IFN-γ) and Th2 cytokines (IL-4, IL-5, IL-6, and IL-10), along with IL-8 and IL-17. This complex immune cascade induces anti-tumor activity mediated by cytotoxic T lymphocytes, natural killer cells, neutrophils, and macrophages. In another BCG study in an in situ murine bladder tumor model, the MDSCs population was significantly downregulated after high-dose BCG therapy compared to low-dose therapy. In the same study, CD4 + / Foxp3 + Tregs also showed the same change. After BCG treatment, the population of CD4 + / Foxp3 + Tregs in the blood decreased. These inhibitory effects on immunosuppressive factors can explain the potent anti-tumor therapeutic effect of BCG therapy (10).

[0056] Immune checkpoint regulators (which can be both co-stimulatory and co-inhibitory molecules) regulate the immune system. The balance between these checkpoint signals regulates lymphocyte activation and thus the immune response. Tumors can use these checkpoint regulators to protect themselves from the immune system. Immune checkpoint therapy can enhance the proliferation, migration, persistence, and / or cytotoxic activity of T cells in a subject, particularly by increasing the number of tumor-infiltrating T cells (11). The best-characterized immune checkpoint receptors are cytotoxic T lymphocyte-associated antigen 4 (CTLA-4; also known as CD152), programmed cell death protein 1 (PD-1; also known as CD279), and indoleamine 2,3-dioxygenase (IDO), and agents targeting these molecules have been approved or are being widely tested in clinical trials for the treatment of various solid or hematological cancers.

[0057] CTLA-4, an inhibitory receptor, is a global immune checkpoint regulator that participates in initiating immune responses by downregulating the initial phase of T cell activation. CTLA-4 was the first clinically validated checkpoint pathway target. CTLA-4 is homologous to the T cell co-stimulatory protein CD28, and both molecules bind to CD80 and CD86 on antigen-presenting cells. CTLA-4 binds CD80 and CD86 with significantly higher affinity and avidity than CD28, enabling CTLA-4 to outcompete CD28 for ligand binding and thereby effectively inhibit T cell activation (12). Interfering with this mechanism by blocking CTLA-4 activity with antagonistic antibodies thus maintains T cell activity. Currently, Bristol-Myers Squibb's anti-CTLA-4 mAb, ipilimumab, has been approved by the US Food and Drug Administration (FDA) for patients with metastatic melanoma. In addition, AstraZeneca's anti-CTLA-4 mAb, tremelimumab, has been granted orphan drug status by the FDA for the treatment of patients with malignant mesothelioma.

[0058] PD-1 is another inhibitory receptor expressed on activated T and B cells, and it functions to suppress immune responses (13). PD-1 acts as an immune checkpoint regulator, and upon binding to one of its ligands, PD-L1 (B7-H1, CD274) or PD-L2 (B7-DC, CD273), PD-1 inhibits T cell proliferation and cytokine production. Overexpression of PD-L1 or PD-L2 in the tumor microenvironment leads to inhibition of the intratumoral immune response (14). Inhibiting the interaction between PD-1 and PD-L1 with anti-PD-1 / PD-L1 antibodies can inhibit T cell inactivation and thus enhance the anti-tumor response, delay tumor growth, and promote tumor rejection (15). Two anti-PD-1 mAbs, Bristol-Myers Squibb's nivolumab and Merck's pembrolizumab, have been approved by the US FDA for patients with metastatic melanoma and non-small cell lung cancer. Recently, nivolumab was approved by the FDA for the treatment of patients with metastatic renal cell carcinoma. In 2016, Roche's anti-PD-L1 mAb, atezolizumab, was approved by the FDA for the treatment of metastatic urothelial carcinoma and non-small cell lung cancer. Other anti-PD-L1 mAbs, such as AstraZeneca's durvalumab and Pfizer's avelumab, are in late-stage clinical trials.

[0059] Indoleamine 2,3-dioxygenase (IDO) is an enzyme that catalyzes the oxidative cleavage of tryptophan (16). IDO plays a role in suppressing the immune system because T cells undergoing antigen-dependent activation require tryptophan for cell proliferation and survival (17). IDO is overexpressed in most tumors and / or tumor-draining lymph nodes and plays an important role in helping tumors evade attacks from the immune system. IDO inhibitors block the IDO enzyme, which reduces tryptophan consumption and can ultimately help promote an enhanced immune response against tumors (18). Currently, many clinical trials are underway to evaluate IDO inhibitors for monotherapy and combination cancer therapies.

[0060] In addition to PD-1, CTLA-4, and IDO, other immune checkpoints are also involved in the occurrence and progression of malignancies, including T cell membrane protein-3 (TIM-3), LAG3, T cell immunoreceptor with Ig and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT), BTLA, inducible T cell co-stimulator (ICOS), killer inhibitory receptor (KIR), and V-domain Ig-containing inhibitor of T cell activation (VISTA). Similar to PD-1, CTLA-4, and IDO, these immune checkpoints inhibit lymphocyte activity and / or induce lymphocyte dysfunction, and thus are ideal targets for cancer immunotherapy. Blocking antibodies against these immune checkpoints have shown specific anti-tumor activity in animal models, and some are being tested in clinical trials.

[0061] Therapeutic methods

[0062] In some aspects, the present invention encompasses a therapeutic method using a combination of an effective amount of nitrohydroxyquinoline or its analog or a pharmaceutically acceptable salt and an effective amount of at least one second agent (including any chemotherapeutic or immunotherapeutic agent for treating various types of cancer). The present invention provides a method of combining an effective amount of nitrohydroxyquinoline or its analog or a pharmaceutically acceptable salt with an effective amount of an immunotherapeutic or chemotherapeutic agent to produce a significantly enhanced anti-cancer effect, which is preferably synergistic.

[0063] The cancer can be any cancer. In some embodiments, the cancer is melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial cancer, bladder cancer, non-small cell lung cancer, small cell lung cancer, sarcoma, colorectal adenocarcinoma, gastrointestinal stromal tumor, gastroesophageal cancer, colorectal cancer, pancreatic cancer, kidney cancer, hepatocellular carcinoma, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasma cell tumor, Wilm's tumor, glioblastoma, retinoblastoma, or hepatocellular carcinoma. In certain embodiments, the cancer is bladder cancer, prostate cancer, kidney cancer, urothelial cancer, testicular cancer, non-small cell lung cancer, breast cancer, or hepatocellular carcinoma. In more certain embodiments, the cancer is bladder cancer, prostate cancer, kidney cancer, or urothelial cancer. In more certain embodiments, the cancer is bladder cancer or prostate cancer.

[0064] The second agent can be any chemotherapeutic agent that enhances the anti-cancer effect of nitroxoline or its analogs or pharmaceutically acceptable salts. In some embodiments, the second agent is a microtubule depolymerization inhibitor, an agent that crosslinks DNA, an agent that intercalates into DNA base pairs, or a platinum compound. In some embodiments, the second agent is a microtubule depolymerization inhibitor, such as a taxane, such as paclitaxel, docetaxel, or cabazitaxel, or a taxane mixed or conjugated with a protein or antibody. In some embodiments, the second agent is an agent that crosslinks DNA, such as mitomycin C. In some embodiments, the second agent is an agent that intercalates into DNA base pairs, such as an anthracycline, such as daunorubicin, doxorubicin, epirubicin, pirarubicin, idarubicin, mitoxantrone, or valrubicin. In some embodiments, the second agent is a platinum compound, such as carboplatin, cisplatin, nedaplatin, or oxaliplatin. In certain embodiments, the second agent is selected from the group consisting of paclitaxel, mitomycin C, epirubicin, pirarubicin, cisplatin, and carboplatin.

[0065] The second agent can also be any immunotherapeutic agent that enhances the anti-cancer effect of nioxynil or its analogs or pharmaceutically acceptable salts. In some embodiments, the second agent is an immune agent that can stimulate an effective immune response and / or inhibit immunosuppression. In some embodiments, the second inhibitor is an agent that modulates, particularly inhibits and downregulates immunosuppressive factors such as regulatory T cells and MDSCs, which includes any inhibitor or antibody of the programmed cell death 1 ligand 1 (PD-L1) / programmed cell death protein 1 (PD-1) pathway. In some embodiments, the second inhibitor is any effective inhibitor / antibody capable of modulating the activity of immune cells, including but not limited to targeting cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), CD20, CD19, IL-17a, CD25, arginase 1 (ARG1), indoleamine-2,3-dioxygenase (IDO) or tryptophan 2,3-dioxygenase (TDO2). In some embodiments, the second inhibitor is BCG. In certain embodiments, the second agent is an antibody against PD-1 or an antigen-binding fragment thereof. In other certain embodiments, the second agent is an antibody against PD-L1 or an antigen-binding fragment thereof. In other certain embodiments, the second agent is an antibody against CTLA-4 or an antigen-binding fragment thereof. In other certain embodiments, the second agent is BCG therapy, preferably attenuated BCG therapy, more preferably mycobacterial cell wall fragments, and most preferably mycobacterial cell wall fragments (fragment) with biologically active nucleic acids derived from Mycobacterium phlei.

[0066] The dose of the compound or agent is selected according to the particular mode of administration chosen, such as based on the desired dose, fluid volume, viscosity, etc., to preferably achieve synergy. In some embodiments, the nioxynil or its analogs or its pharmaceutically acceptable salts are administered at a dose of about 100 mg / day to about 1600 mg / day, such as about 100 mg / day, 200 mg / day, 300 mg / day, 400 mg / day, 500 mg / day, 600 mg / day, 700 mg / day, 800 mg / day, 900 mg / day, 1000 mg / day, 1100 mg / day, 1200 mg / day, 1300 mg / day, 1400 mg / day, 1500 mg / day and 1600 mg / day, preferably orally. Preferably, the nioxynil or its analogs or its pharmaceutically acceptable salts are administered at a dose of about 600 mg / day to about 1600 mg / day, preferably orally.

[0067] In some embodiments, the second agent is an anti-PD-1 antibody or an antigen-binding fragment thereof, which is preferably administered at a dose of about 0.1 mg / kg to about 20 mg / kg, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg / kg, preferably by intravenous or intramuscular injection, once every 2, 3 or 4 weeks. More preferably, the anti-PD-1 antibody or an antigen-binding fragment thereof is preferably administered by intravenous or intramuscular injection at a total dose of about 2 mg / kg to about 15 mg / kg over a period of about 3 weeks, and the treatment is optionally repeated one or more times.

[0068] In some embodiments, the second agent is an anti-PD-L1 antibody or an antigen-binding fragment thereof, which is preferably administered at a dose of about 1 mg / kg to about 40 mg / kg, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 mg / kg, preferably by intravenous or intramuscular injection, once every 2, 3 or 4 weeks. More preferably, the anti-PD-L1 antibody or an antigen-binding fragment thereof is preferably administered by intravenous or intramuscular injection at a total dose of about 10 mg / kg to about 30 mg / kg over a period of about 4 weeks, and the treatment is optionally repeated one or more times.

[0069] In some embodiments, the second agent is an anti-CTLA-4 antibody or an antigen-binding fragment thereof, which is preferably administered at a dose of about 0.1 mg / kg to about 6 mg / kg, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5 or 6 mg / kg, preferably by intravenous or intramuscular injection, once every 3, 4 or 5 weeks. More preferably, the anti-CTLA-4 antibody or an antigen-binding fragment thereof is preferably administered by intravenous or intramuscular injection at a total dose of about 1 mg / kg to about 4 mg / kg over a period of about 4 weeks, and the treatment is optionally repeated one or more times.

[0070] In some embodiments, the second agent is BCG therapy, which is preferably at about 0.5 x 10 8 to about 50 x 10 8The dose of colony-forming units (CFU), such as about 0.5x, 1x, 2x, 4x, 6x, 8x, 10x, 12x, 14x, 16x, 18x, 20x, 22x, 24x, 26x, 28x, 30x, 32x, 34x, 36x, 38x, 40x, 42x, 44x, 46x, 48x or 50x 10 8 CFU, preferably administered intravesically, once every 1, 2, 3, 4 or 5 weeks. More preferably, the BCG therapy is preferably administered intravesically, once a week at a dose of about 1x10 8 to about 8x 10 8 CFU, and the treatment is optionally repeated one or more times.

[0071] In some embodiments, the second agent is a chemotherapeutic agent, which is preferably administered intravesically at a concentration of about 0.1 mg / kg or higher (up to the maximum solubility of the agent in water or physiological saline). In some embodiments, the chemotherapeutic agent is a DNA cross-linking or intercalating agent, such as mitomycin C, anthracyclines (such as epirubicin, pirarubicin, etc.), platinum-based anti-tumor agents (such as carboplatin, cisplatin, oxaliplatin, nedaplatin), at a concentration of about 0.1 mg / ml to about 5 mg / ml, such as about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mg / ml, preferably administered intravesically in a bladder instillation solution, once every 1, 2, 3, 4 or 5 weeks. More preferably, the chemotherapeutic agent is preferably administered intravesically in a bladder instillation solution at a concentration of about 0.5 mg / mL to 2 mg / mL once a week, and the treatment is optionally repeated one or more times.

[0072] In some embodiments, the chemotherapeutic agent is a taxane drug targeting tubulin, such as paclitaxel, docetaxel, cabazitaxel, conjugates thereof with proteins or antibodies, etc., which is at a concentration of about 0.5 mg / ml to about 10 mg / ml of the taxane drug, such as about 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg / ml, preferably administered intravesically in a bladder instillation solution, once every 1, 2, 3, 4 or 5 weeks. More preferably, the taxane drug is preferably administered intravesically in a bladder instillation solution at a concentration of about 1 mg / mL to 5 mg / mL once a week, and the treatment is optionally repeated one or more times.

[0073] The compound or agent can be administered by any acceptable route. In some embodiments, the compound is administered orally, intralipally, intraarterially, intraarticularly, intracranially, intradermally, intralesionally, intramuscularly, intranasally, intraocularally, intrapericardially, intraperitoneally, intrapleurally, intraprostatically, rectally, intrathecally, intratracheally, intratumorally, intraumbilically, vaginally, intravenously, intravesically, intravitreally, via liposomes, locally, mucosally, orally, parenterally, rectally, subconjunctivally, subcutaneously, sublingually, topically, buccally, transdermally, vaginally, as a cream, as a lipid composition, via a catheter, via lavage, via continuous infusion, via infusion, via inhalation, via injection, via local delivery, via local perfusion, by direct immersion of target cells, or any combination thereof. In some embodiments, the nitrohydroxyquinoline or an analogue thereof or a pharmaceutically acceptable salt thereof is administered orally or by injection, and preferably orally. In some embodiments, the immunotherapeutic agent is an antibody administered by injection. In some embodiments, the immunotherapeutic agent is BCG therapy administered intravesically or by injection, and preferably intravesically. In some embodiments, the chemotherapeutic agent is administered intravesically or by injection, and preferably intravesically.

[0074] In some embodiments, the compound or agent is administered once daily. In other embodiments, the compound or agent is administered twice daily. In other embodiments, the compound is administered multiple times a day, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every two months, once every three months, once every four months, once every five months, once every six months, or once a year. The dosing regimen of the nitrohydroxyquinoline or an analogue thereof or a pharmaceutically acceptable salt thereof can be different from that of the immunotherapeutic agent or the chemotherapeutic agent.

[0075] The compound or agent can be administered for one day, two days, three days, four days, five days, six days, seven days, two weeks, three weeks, four weeks, two months, three months, four months, five months, six months, one year, two years, three years, four years, five years, ten years, or fifteen years.

[0076] The methods of the present invention can be used in combination with additional cancer therapies. In some embodiments, the additional cancer therapies include surgery, radiotherapy, chemotherapy, toxin therapy, immunotherapy, cryotherapy, or gene therapy. In some embodiments, the cancer is a chemotherapy-resistant, immunotherapy-resistant, or radiotherapy-resistant cancer.

[0077] The combination therapy of the present invention according to the embodiments of the present invention can be achieved by administering the individual components of the therapy simultaneously, sequentially, or separately. The treatment method in the present invention can be applied in any animal model or clinic in combination with an effective amount of nioxynil or its analog or a pharmaceutically acceptable salt thereof and an effective amount of an immunotherapeutic agent or an effective amount of a chemotherapeutic agent.

[0078] Composition / Kit

[0079] Aspects of the present invention also relate to a pharmaceutical composition or a kit, which comprises an effective amount of nioxynil or its analog or a pharmaceutically acceptable salt thereof, an effective amount of an immunotherapeutic agent or a chemotherapeutic agent, and a pharmaceutically acceptable carrier. The present invention also relates to a method for preparing the pharmaceutical composition or the kit by combining the nioxynil or its analog or a pharmaceutically acceptable salt thereof, the at least one additional anti-cancer immunotherapeutic agent or chemotherapeutic agent, and the pharmaceutically acceptable carrier based on the disclosure by using methods known in the art.

[0080] According to an embodiment of the present invention, the kit comprises one or more pharmaceutical compositions having an effective amount of nioxynil or its analog or a pharmaceutically acceptable salt thereof and an effective amount of an immunotherapeutic agent. According to other embodiments of the present invention, the kit comprises one or more pharmaceutical compositions having an effective amount of nioxynil or its analog or a pharmaceutically acceptable salt thereof and an effective amount of a chemotherapeutic agent. The effective amount of nioxynil or its analog or a pharmaceutically acceptable salt thereof and the effective amount of an immunotherapeutic agent or a chemotherapeutic agent can be present in one pharmaceutical composition. They can also be present in separate pharmaceutical compositions. The kit further contains instructions for using the combination of the effective amount of nioxynil or its analog or a pharmaceutically acceptable salt thereof and the effective amount of an immunotherapeutic agent or a chemotherapeutic agent for treating cancer.

[0081] The composition or kit according to the embodiments of the present invention can be prepared by using methods known in the art based on the present disclosure.

[0082] According to certain embodiments, an effective amount is the amount of each active ingredient or compound that effectively achieves a synergistic effect of one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of cancer to be treated or symptoms associated therewith; (ii) reducing the duration of cancer to be treated or symptoms associated therewith; (iii) preventing the progression of cancer to be treated or symptoms associated therewith; (iv) causing regression of cancer to be treated or symptoms associated therewith; (v) preventing the progression or onset of cancer to be treated or symptoms associated therewith; (vi) preventing the recurrence of cancer to be treated or symptoms associated therewith; (vii) reducing hospitalization of a subject having cancer to be treated or symptoms associated therewith; (viii) reducing the length of hospitalization of a subject having cancer to be treated or symptoms associated therewith; (ix) increasing the survival rate of a subject having cancer to be treated or symptoms associated therewith; (xi) inhibiting or reducing cancer to be treated or symptoms associated therewith in a subject; and / or (xii) enhancing or improving the prophylactic or therapeutic effect of another therapy.

[0083] Embodiment

[0084] Embodiment 1 is a method of treating cancer, comprising administering to a subject in need thereof an effective amount of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and an effective amount of an immunotherapeutic agent. Preferably, the immunotherapeutic agent is an inhibitor and modulator of regulatory T cells or myeloid-derived suppressor cells, and the combination of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and the immunotherapeutic agent causes a synergistic effect.

[0085] Embodiment 2 is the method of Embodiment 1, wherein the immunotherapeutic agent is an inhibitor of the PD-L1 / PD-1 pathway or an inhibitor of the CTLA-4 pathway. Preferably, the immunotherapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or an antigen-binding fragment thereof. More preferably, the antibody or fragment thereof is administered by intravenous or intramuscular injection.

[0086] Embodiment 3 is the method of Embodiment 1, wherein the immunotherapeutic agent is BCG (Bacillus Calmette-Guérin) therapy, preferably attenuated BCG therapy, more preferably mycobacterial cell wall fragments, and most preferably mycobacterial cell wall fragments having biologically active nucleic acids derived from Mycobacterium phlei. More preferably, the BCG therapy is administered intravesically.

[0087] Embodiment 4 is the method of Embodiment 3, wherein nioxynil, its analogue, or a pharmaceutically acceptable salt is not administered within 24 hours of the intravesical instillation of BCG therapy.

[0088] Embodiment 5 is the method of any one of Embodiments 1 to 4, wherein the cancer is selected from the group consisting of: melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial cancer, bladder cancer, non-small cell lung cancer, small cell lung cancer, sarcoma, colorectal adenocarcinoma, gastrointestinal stromal tumor, gastroesophageal cancer, colorectal cancer, pancreatic cancer, kidney cancer, hepatocellular carcinoma, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasma cell tumor, nephroblastoma, glioblastoma, retinoblastoma, and hepatocellular carcinoma.

[0089] Embodiment 6 is the method of Embodiment 5, wherein the cancer is bladder cancer.

[0090] Embodiment 7 is the method of Embodiment 5, wherein the cancer is prostate cancer.

[0091] Embodiment 8 is the method of any one of Embodiments 1 to 7, wherein the effective amount of nioxynil or its analog or pharmaceutically acceptable salt is administered orally or by intravenous or intramuscular injection.

[0092] Embodiment 9 is a method of treating cancer, preferably bladder cancer, liver cancer or prostate cancer, which comprises administering to a subject in need thereof an effective amount of nioxynil or its analog or pharmaceutically acceptable salt and an effective amount of a chemotherapeutic agent, wherein the combination of nioxynil or its analog or pharmaceutically acceptable salt and the chemotherapeutic agent causes a synergistic effect.

[0093] Embodiment 10 is the method of Embodiment 9, wherein the chemotherapeutic agent is a taxane drug targeting tubulin, preferably paclitaxel, docetaxel, cabazitaxel; or the chemotherapeutic agent is a DNA cross-linking or intercalating agent, preferably mitomycin C, anthracycline drugs preferably epirubicin, pirarubicin, or platinum-based anti-tumor agents preferably carboplatin, cisplatin, oxaliplatin or nedaplatin.

[0094] Embodiment 11 is the method of Embodiment 9 or 10, wherein the chemotherapeutic agent is administered intravesically once a week, or once every 2, 3, 4 or 5 weeks.

[0095] Embodiment 12 is the method of any one of Embodiments 1 to 11, wherein the nioxynil analog is selected from the group consisting of hydroxyquinoline, clioquinol and diiodohydroxyquinoline.

[0096] Embodiment 13 is the method of any one of Embodiments 1 to 12, wherein the effective amount of nioxynil or its analog or pharmaceutically acceptable salt is administered orally at 100 mg to 1600 mg per day, preferably orally at 600 mg to 1600 mg per day.

[0097] Embodiment 14 is a method for treating cancer, preferably bladder cancer or prostate cancer, which comprises administering to a subject in need thereof an effective amount of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and an effective amount of an anti-PD-1 antibody or an antigen-binding fragment thereof, wherein the nioxynil or an analogue thereof or a pharmaceutically acceptable salt is administered orally at a daily dose of 100 mg to 1600 mg, preferably 600 mg to 1600 mg per day, and the anti-PD-1 antibody or an antigen-binding fragment thereof is administered by intravenous or intramuscular injection at a dose of 0.1 mg / kg to 20 mg / kg once every 1 to 4 weeks, preferably at a total dose of 2 mg / kg to 15 mg / kg over a period of 3 weeks, and the combination of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and the anti-PD-1 antibody or an antigen-binding fragment thereof results in a synergistic effect.

[0098] Embodiment 15 is a method for treating cancer, preferably bladder cancer or prostate cancer, which comprises administering to a subject in need thereof an effective amount of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and an effective amount of an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the nioxynil or an analogue thereof or a pharmaceutically acceptable salt is administered orally at a daily dose of 100 mg to 1600 mg, preferably 600 mg to 1600 mg per day, and the anti-PD-L1 antibody or an antigen-binding fragment thereof is administered by intravenous or intramuscular injection at a dose of 1 mg / kg to 40 mg / kg once every 2 to 4 weeks, preferably at a total dose of 10 mg / kg to 30 mg / kg over a period of 4 weeks, and the combination of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and the anti-PD-L1 antibody or an antigen-binding fragment thereof results in a synergistic effect.

[0099] Embodiment 16 is a method for treating cancer, preferably bladder cancer or prostate cancer, which comprises administering to a subject in need thereof an effective amount of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and an effective amount of an anti-CTLA-4 antibody or an antigen-binding fragment thereof, wherein the nioxynil or an analogue thereof or a pharmaceutically acceptable salt is administered orally at a daily dose of 100 mg to 1600 mg, preferably 600 mg to 1600 mg per day, and the anti-CTLA-4 antibody or an antigen-binding fragment thereof is administered by intravenous or intramuscular injection at a dose of 0.1 mg / kg to 6 mg / kg once every 3 to 5 weeks, preferably at a total dose of 1 mg / kg to 4 mg / kg over a period of 4 weeks, and the combination of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and the anti-CTLA-4 antibody or an antigen-binding fragment thereof results in a synergistic effect.

[0100] Embodiment 17 is a method for treating cancer, which is preferably bladder cancer or prostate cancer, and comprises administering to a subject in need thereof an effective amount of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and an effective amount of BCG therapy, wherein the nioxynil or an analogue thereof or a pharmaceutically acceptable salt is orally administered at a dose of 100 mg to 1600 mg per day, preferably 600 mg to 1600 mg per day, and the BCG therapy is intravesically administered at a dose of 0.5 x 10 8 to 50 x 10 8 colony forming units (CFU) once every 1 to 5 weeks, preferably 1 x 10 8 to 8 x 10 8 CFU once a week, and the combination of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and the BCG therapy causes a synergistic effect.

[0101] Embodiment 18 is a method for treating cancer, which is preferably bladder cancer, liver cancer or prostate cancer, and comprises administering to a subject in need thereof an effective amount of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and an effective amount of a chemotherapeutic agent, wherein the nioxynil or an analogue thereof or a pharmaceutically acceptable salt is orally administered at a dose of 100 mg to 1600 mg per day, preferably 600 mg to 1600 mg per day, and the effective amount of the chemotherapeutic agent is administered intravesically or by intravenous or intramuscular injection, and the combination of nioxynil or an analogue thereof or a pharmaceutically acceptable salt and the chemotherapeutic agent causes a synergistic effect.

[0102] Embodiment 19 is the method of Embodiment 18, wherein the chemotherapeutic agent is a DNA cross-linking or intercalating agent selected from the group consisting of mitomycin C, epirubicin, pirarubicin, carboplatin, cisplatin, oxaliplatin and nedaplatin, and the chemotherapeutic agent is intravesically administered at a concentration of 0.1 mg / ml to 5 mg / ml in a bladder instillation solution once every 1 - 5 weeks, preferably 0.5 mg / mL to 2 mg / mL.

[0103] Embodiment 20 is the method of Embodiment 18, wherein the chemotherapeutic agent is a taxane drug selected from the group consisting of paclitaxel, docetaxel and cabazitaxel, and the chemotherapeutic agent is intravesically administered at a concentration of 0.5 to 10 mg / mL in a bladder instillation solution, preferably 1 mg / mL to 5 mg / mL.

[0104] Embodiment 21 is the method of any one of Embodiments 1 to 20, wherein the nioxynil or a pharmaceutically acceptable salt thereof is orally or by injection administered to a subject in need thereof.

[0105] Embodiment 22 is the method of any one of Embodiments 1 to 20, wherein a nitroxoline analogue selected from the group consisting of hydroxyquinoline, clioquinol, and diiodohydroxyquinoline or a pharmaceutically acceptable salt thereof is administered orally or by injection to a subject in need thereof.

[0106] Embodiment 23 is a kit, which comprises an effective amount of nitroxoline or an analogue thereof or a pharmaceutically acceptable salt thereof, an effective amount of an immunotherapeutic agent or a chemotherapeutic agent, and one or more pharmaceutically acceptable carriers, wherein the effective amount of nitroxoline or an analogue thereof or a pharmaceutically acceptable salt thereof and the effective amount of the immunotherapeutic agent or the chemotherapeutic agent are present in the same pharmaceutical composition or in separate pharmaceutical compositions.

[0107] Embodiment 24 is the kit of Embodiment 23, wherein the kit comprises a pharmaceutical composition containing 100 mg to 1600 mg, preferably 600 mg to 1600 mg, of nitroxoline or an analogue thereof or a pharmaceutically acceptable salt thereof per dosage form.

[0108] Embodiment 25 is the kit of Embodiment 23 or 24, wherein the kit comprises a pharmaceutical composition containing an immunotherapeutic agent selected from the group consisting of: an anti-PD-1 antibody or an antigen-binding fragment thereof; an anti-PD-L1 antibody or an antigen-binding fragment thereof; an anti-CTLA-4 antibody or an antigen-binding fragment thereof; and BCG therapy.

[0109] Embodiment 26 is the kit of Embodiment 23 or 24, wherein the kit comprises a pharmaceutical composition containing a chemotherapeutic agent selected from the group consisting of: mitomycin C, epirubicin, pirarubicin, carboplatin, cisplatin, oxaliplatin, nedaplatin, paclitaxel, docetaxel, and cabazitaxel.

[0110] Embodiment 27 is the kit of any one of Embodiments 23 to 26, wherein the kit comprises a pharmaceutical composition containing nitroxoline or a pharmaceutically acceptable salt thereof.

[0111] Embodiment 28 is the kit of any one of Embodiments 23 to 26, wherein the kit comprises a pharmaceutical composition containing a nitroxoline analogue selected from hydroxyquinoline, clioquinol, and diiodohydroxyquinoline or a pharmaceutically acceptable salt thereof.

[0112] Embodiment 29 is a method for preparing the kit of any one of Embodiments 23 to 28, which comprises combining the nitroxoline, its analogue or a pharmaceutically acceptable salt thereof, the immunotherapeutic agent or the chemotherapeutic agent with one or more pharmaceutically acceptable carriers in the kit.

[0113] Embodiment 30 is a method of preparing a kit according to any one of embodiments 23 to 28, which includes obtaining a first pharmaceutical composition comprising the effective amount of nitroxoline, its analogs or its pharmaceutically acceptable salts, obtaining a second pharmaceutical composition comprising the effective amount of an immunotherapeutic agent or a chemotherapeutic agent, and combining the first and second pharmaceutical compositions in the kit.

[0114] Embodiment 31 is the use of a kit according to any one of embodiments 23 to 28 in the preparation of a medicament for the treatment of cancer, and the combined use of nitroxoline or its analogs or pharmaceutically acceptable salts with the immunotherapeutic agent or chemotherapeutic agent causes a synergistic effect.

[0115] Embodiment 32 is the use according to embodiment 31, wherein the cancer is selected from melanoma, cervical cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, urothelial cancer, bladder cancer, non-small cell lung cancer, small cell lung cancer, sarcoma, colorectal adenocarcinoma, gastrointestinal stromal tumor, gastroesophageal cancer, colorectal cancer, pancreatic cancer, kidney cancer, hepatocellular carcinoma, malignant mesothelioma, leukemia, lymphoma, myelodysplastic syndrome, multiple myeloma, transitional cell carcinoma, neuroblastoma, plasma cell tumor, nephroblastoma, glioblastoma, retinoblastoma, and hepatocellular carcinoma.

[0116] Embodiment 33 is the use according to embodiment 32, wherein the cancer is bladder cancer.

[0117] Embodiment 34 is the use according to embodiment 32, wherein the cancer is prostate cancer.

[0118] Embodiment 35 is the use according to embodiment 32, wherein the cancer is liver cancer. Examples

[0119] The following examples of the present invention are for further illustrating the nature of the present invention. It should be understood that the following examples do not limit the present invention, and the scope of the present invention is determined by the appended claims.

[0120] Example 1: Synergistic inhibition of tumor growth by the combination of NIT and one or more chemotherapies

[0121] The growth inhibitory effects of NIT in combination with a panel of chemotherapies, including carboplatin, paclitaxel, mitomycin C, epirubicin, and pirarubicin, were tested on two human cancer cell lines. As shown in Tables 2 and 3, NIT inhibited the growth of the human bladder cancer cell line 5637 and the human liver cancer cell line HepG2.

[0122] Table 2. Incubation of NIT with the human bladder cancer cell line 5637 for 120 hours resulted in the inhibition of cell growth

[0123]

[0124] Table 3. Incubation of NIT with human hepatocellular carcinoma cell line HepG2 for 120 hours resulted in inhibition of cell growth

[0125]

[0126] As shown in Tables 4 and 5, carboplatin inhibited the growth of human bladder cancer cell line 5637 and human hepatocellular carcinoma cell line HepG2.

[0127] Table 4. Incubation of carboplatin with human bladder cancer cell line 5637 for 120 hours resulted in inhibition of cell growth.

[0128]

[0129] Table 5. Incubation of carboplatin with human hepatocellular carcinoma cell line HepG2 for 120 hours resulted in inhibition of cell growth

[0130]

[0131] As shown in Tables 6 and 7, paclitaxel inhibited the growth of human bladder cancer cell line 5637 and human hepatocellular carcinoma cell line HepG2.

[0132] Table 6. Incubation of paclitaxel with human bladder cancer cell line 5637 for 112 hours resulted in inhibition of cell growth

[0133]

[0134] Table 7. Incubation of paclitaxel with human hepatocellular carcinoma cell line HepG2 for 112 hours resulted in inhibition of cell growth

[0135]

[0136] The IC 50 values of the cell growth inhibition of these compounds are summarized in Table 8.

[0137] Table 8. IC 50 values of the cell growth inhibition of NIT, carboplatin and paclitaxel on human bladder cancer cell line 5637 and human hepatocellular carcinoma cell line HepG2

[0138]

[0139] Similarly, the inhibition of the growth of bladder cancer cell line 5637 by mitomycin C, epirubicin and pirarubicin was tested, and their IC 25 , IC 50 and IC 75 values are summarized in Table 9.

[0140] Table 9. IC25 , IC 50 and IC 75 value.

[0141] Drug <![CDATA[IC 25 (ng / ml)]]> <![CDATA[IC 50 (ng / ml)]]> <![CDATA[IC 75 (ng / ml)]]> Mitomycin C 48.36 152.53 536.62 Epirubicin 34.97 45.21 63.93 Pirarubicin 96.58 142.57 219.58

[0142] The combination study of NIT and chemotherapy was carried out by mixing NIT with each of 5 to 7 concentration levels of chemotherapy (where the medium concentration level is close to their IC 50 value). The combination index (CI) was calculated and plotted according to the method of Ting-Chao Chou (19) to evaluate the combination effect, which includes synergism, additivity, and antagonism. As shown in Tables 10 and 11, NIT combined with carboplatin and paclitaxel inhibited the growth of human bladder cancer 5637, and the CI values of these studies were calculated and plotted separately in Figure 2 and Figure 3 respectively.

[0143] Table 10. Incubation of the combination of NIT and carboplatin with human bladder cancer cell line 5637 for 120 hours resulted in inhibition of cell growth.

[0144]

[0145] Table 11. Incubation of the combination of NIT and paclitaxel with human bladder cancer cell line 5637 for 96 hours resulted in inhibition of cell growth.

[0146]

[0147]

[0148] As shown in Tables 12 and 13, NIT combined with carboplatin and paclitaxel inhibited the growth of human hepatocellular carcinoma cell line HepG2, and the CI values of these studies were plotted separately in Figure 4 and Figure 5 respectively.

[0149] Table 12. Incubation of the combination of NIT and carboplatin with human hepatocellular carcinoma cell line HepG2 for 120 hours resulted in inhibition of cell growth.

[0150]

[0151] Table 13. Incubation of the combination of NIT and paclitaxel with human hepatocellular carcinoma cell line HepG2 for 120 hours resulted in inhibition of cell growth.

[0152]

[0153] Similarly, NIT was combined with mitomycin C, epirubicin, and pirarubicin to inhibit the growth of the bladder cancer cell line 5637, and their combined inhibition results and CI plots are shown in Tables 14-17 and Figures 6 - 8 below.

[0154] Table 14. Incubation of the combination of NIT and mitomycin C with human bladder cancer cell line 5637 for 96 hours resulted in inhibition of cell growth.

[0155]

[0156]

[0157] Table 15. CI values of the combination of NIT and mitomycin C when incubated with human bladder cancer cell line 5637 for 96 hours.

[0158]

[0159] Table 16. CI values of the combination of NIT and epirubicin when incubated with human bladder cancer cell line 5637 for 96 hours.

[0160]

[0161] Table 17. CI values of the combination of NIT and pirarubicin when incubated with human bladder cancer cell line 5637 for 96 hours.

[0162]

[0163]

[0164] According to Chou (19), a CI value below 0.9 indicates a synergistic combination of two drugs, and values of 0.1-0.3, 0.3-0.7, 0.7-0.85, and 0.85-0.9 indicate strong synergy, synergy, moderate synergy, and slight synergy, respectively. Thus, Figure 2 and 4 the CI plots in Figure 3 support the conclusion that NIT and carboplatin inhibit the growth of bladder cancer cell line 5637 and hepatocellular carcinoma cell line HepG2 in a manner classified as synergistic or strongly synergistic. Figure 5 The CI plot in Figure 6 shows that the combination of NIT and paclitaxel exhibits slight synergy in inhibiting the growth of bladder cancer cell line 5637, and 50The concentration of the value has this partial synergistic effect on inhibiting the bladder cancer cell line 5637. Tables 16 and 17, and Figure 7 and 8 indicate that the combination of NIT with epirubicin or pirarubicin partially synergistically inhibits the growth of the bladder cancer cell line 5637 at certain concentrations.

[0165] The synergistic tumor growth inhibition exhibited by the combination of NIT with mitomycin C (MMC) was further evaluated in an orthotopic xenograft of murine bladder cancer. Figure 9 The inhibitory effect of the combination of orally administered NIT and intravesical MMC on tumor progression in the MBT-2-luc orthotopic murine bladder cancer model was shown. After the indicated treatment, tumor volume was analyzed by the Xenogen IVIS200 system. Figure 9 A shows representative IVIS images of each treatment group. Tumor volume per mouse was determined by analysis of the region of interest of total photons per second. Six mice were analyzed in each group. As Figure 9 shown in B, the mean tumor volume of the group with the combination of 15 mg / kg Bid orally administered NIT and 1 mg / ml intravesical mitomycin C Q7d (NIT+MMC) was significantly different from the mean tumor volumes of the single NIT or single MMC treatment groups (p<0.0001 and p = 0.0001, respectively), indicating that treatment with the combination of NIT and MMC significantly enhanced tumor growth inhibition. Kaplan-Meier analysis was also performed to evaluate the survival rate of mice for each treatment and is summarized in Figure 9 C. Consistently, the survival status of the NIT plus MMC group was significantly improved compared to the control group and the single-drug treatment groups.

[0166] The results of tumor growth inhibition, survival ratio, and calculation by the Bliss independence model of additivity are summarized in Table 18.

[0167] Table 18: Calculation by the Bliss independence model of additivity of the combination of orally administered NIT and MMC in the orthotopic murine MBT-2-Luc bladder cancer model.

[0168]

[0169] NIT administered orally at a dose of 15 mg / kg (Bid) inhibited tumor growth at a rate of 0.33, and the proportion of surviving mice at the end of the experiment (45 days) was 0.33. The tumor inhibition rate and the proportion of surviving mice treated with intravesical MMC were 0.56 and 0.57, respectively. When orally administered NIT was co-administered with MMC, the observed tumor inhibition rate and the proportion of surviving mice increased significantly to 0.71 and 1, respectively, which were greater than their expected Bliss additive values, indicating a synergistic effect of the combination of NIT and MMC on tumor inhibition.

[0170] The discovery that NIT has varying degrees of synergy with different classes of chemotherapy can be used for combination therapy of various solid tumors and hematological malignancies.

[0171] Example 2: Synergistic inhibition of tumor growth by combination of NIT and immunotherapy

[0172] Test the inhibition of tumor growth in an orthotopic xenograft model of murine bladder and prostate cancer by NIT in combination with a panel of immunotherapies, including BCG and anti-PD-1 antibody.

[0173] Figure 10 The effect of the combination of orally administered NIT and intravesical instillation of BCG on inhibiting tumor progression was shown in the MBT-2-luc orthotopic murine bladder cancer model. After the indicated treatment, tumor volume was analyzed by the Xenogen IVIS200 system, and Figure 10 A shows representative IVIS images of each treatment group of the indicated treatment. Tumor volume per mouse was determined by analysis of the region of interest of total photons per second. Eight mice were analyzed in each group. As Figure 10 shown in B, 30 mg / kg NIT administered orally either continuously or intermittently for 48 h inhibited cancer growth, slightly higher or similar compared to the activity induced by intravesical administration of BCG. When orally administered NIT (administered continuously or intermittently for 48 h) was co-administered with BCG, the inhibition rate increased significantly compared to single NIT or BCG treatment (all comparisons p < 0.0001). Kaplan-Meier analysis was also performed to evaluate the survival rate of mice in each treatment, and the results are summarized in Figure 10 C. The survival status of the group treated with orally administered NIT (administered continuously or intermittently for 48 h) in combination with BCG was significantly improved compared to the single-drug treatment group.

[0174] The results calculated by the Bliss independence model for additivity of tumor growth inhibition and survival fraction are summarized in Table 19 (continuous NIT) and Table 20 (intermittent NIT for 48 h).

[0175] Table 19. Calculation by the Bliss independence model for additivity of orally administered NIT (continuous) in combination with intravesical BCG in an orthotopic murine MBT-2-Luc bladder cancer model.

[0176]

[0177] Table 20. Calculation by the Bliss independence model for additivity of orally administered NIT (intermittent for 48 h) in combination with intravesical BCG in an orthotopic murine MBT-2-Luc bladder cancer model.

[0178]

[0179] Whether administered continuously or intermittently for 48 hours, when oral NIT is combined with intravesical BCG, a significantly increased tumor inhibition rate and survival proportion are observed compared to single NIT and BCG treatments. The actual tumor inhibition rate and survival proportion of the NIT and BCG combination are much higher than their expected Bliss additive values, indicating a strong synergistic effect of the NIT and BCG combination on tumor inhibition.

[0180] To evaluate the synergistic tumor inhibitory effect of the combination of NIT and anti-PD-1, the inhibitory effect of this combination on tumor growth in orthotopic xenograft models of murine bladder cancer and prostate cancer was tested.

[0181] Figure 11 The inhibitory effect of the combination of orally administered NIT and intraperitoneal anti-PD-1 antibody on tumor growth in the MBT-2-luc orthotopic murine bladder cancer model is shown. Figure 11 A shows representative IVIS images of each indicated treatment group. The tumor volume of each mouse was determined by analysis of the region of interest of total photons per second. Five mice were analyzed in each group. As Figure 11 shown in B, 10 mg / kg of intraperitoneal anti-PD-1 strongly inhibited tumor growth at a rate of 80%, while orally administered 15 mg / kg of NIT showed a 31% tumor inhibition rate. When orally administered NIT was combined with anti-PD-1, the inhibition rate was significantly increased (90%) compared to single NIT or anti-PD-1 treatment (p < 0.0001 and p = 0.039, respectively). Kaplan-Meier analysis was also performed and summarized in Figure 11 C. The survival status of the NIT plus anti-PD-1 group or the single anti-PD-1 group was significantly higher than that of the single NIT group.

[0182] The calculated results of the Bliss independence model for the additivity of tumor growth inhibition are summarized in Table 21. When NIT is combined with anti-PD-1, the actually observed tumor inhibition rate is greater than the expected Bliss additive value, indicating a synergistic effect on tumor inhibition.

[0183] Table 21. Calculation of the Bliss independence model for the additivity of the combination of orally administered NIT and anti-PD-1 antibody in the orthotopic murine MBT-2-Luc bladder cancer model.

[0184]

[0185] Figure 12 The inhibitory effect of the combination of orally administered NIT and intraperitoneal anti-PD-1 antibody on tumor growth in the RM-9-luc orthotopic murine prostate cancer model is shown. Figure 12Typical IVIS images of each indicated treatment group are shown in A. Tumor volume of each mouse was determined by analysis of the region of interest of total photons per second. Eight mice were analyzed in each group. As Figure 12 shown in B, 10 mg / kg intraperitoneal anti-PD-1 showed strong tumor growth inhibition (76%), while orally administered 15 mg / kg NIT showed a 52% tumor inhibition rate. When NIT was combined with anti-PD-1, the tumor inhibition rate was significantly enhanced to 96% compared to single NIT and anti-PD-1 treatments, with p-values of 0.017 and 0.027, respectively. Figure 12 C shows images of tumors collected from each group. The tumor weight of each mouse was measured and summarized in Figure 12 D. Analysis of the tumor weight data showed that the combination of NIT and anti-PD-1 led to a significant enhancement of tumor inhibition (74%) compared to single NIT and anti-PD-1 treatments (37% and 56%, respectively), with p-values of 0.0006 and 0.0111, respectively.

[0186] The results of the Bliss independence model calculation of the additivity of tumor volume and weight are summarized in Table 22. When NIT was administered in combination with anti-PD-1, the actually observed tumor volume and weight inhibition rates were 0.96 and 0.74, respectively, which were greater than their expected Bliss additive values (0.89 and 0.72, respectively), indicating a synergistic effect of tumor inhibition.

[0187] Table 22. Calculation of the Bliss independence model of additivity of oral NIT and anti-PD-1 antibody combination in the in situ mouse RM-9-Luc prostate cancer model.

[0188]

[0189]

[0190] The finding that NIT has varying degrees of synergistic effects with different immunotherapies can be used for combination therapy of various solid tumors and hematological tumors.

[0191] Example 3: Experimental procedures

[0192] 3.1 Research materials

[0193] Compounds: NIT, carboplatin, paclitaxel, epirubicin, and pirarubicin were purchased from suppliers and dissolved in DMSO to form stock solutions, which were stored at -20 °C. Before use, the stock solutions were diluted to working solutions at different concentrations. The DMSO concentration in the working solutions was less than 1%. All cancer cell lines were purchased from suppliers.

[0194] Cell lines and reagents: The murine bladder cancer cell lines MBT-2 and RM-9 were obtained from the American Type Culture Collection (Rockville, MD, USA). All cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. The cells were cultured at 37 °C in a 5% CO2 atmosphere and routinely passaged by trypsin-EDTA treatment in 100-cm2 flasks containing BCG (81 mg; Connaught substrain, ImmuCyst, Nihou Kayaku, Inc., Tokyo, Japan) and phosphate-buffered saline (PBS) for in vivo studies.

[0195] By using -3T3 transfection kit (Mirus Bio LLC, Madison, WI, USA), MBT-2 and RM-9 cells were transfected with pGL3-Luc plasmid to generate MBT-2 and RM-9 cells stably expressing luciferase (MBT-2-Luc and RM-9-Luc; luciferase L4899 obtained from Sigma-Aldrich Japan GK). Cells stably expressing luciferase were obtained by screening with 500 μg / mL G418 for two weeks. After G418 screening, the luciferase activity of the growth medium from MBT-2-Luc and RM-9-Luc was tested to confirm luciferase expression and secretion into the cell culture medium.

[0196] Rat anti-mouse PD-1 mAb (RMP1-14; IgG2a) was purchased from BioXCell (West Lebanon, NH, USA).

[0197] Animals: Eight-week-old female C3H / HeN and C57 / BL6 mice were obtained from a supplier. The mice were maintained in an animal facility in a specific pathogen-free environment with free access to food and water.

[0198] 3.2 In vitro analysis

[0199] The MTT assay was used as the test method. An appropriate amount of cells (2×10 3(100 μL) were seeded into 96-well plates and incubated overnight at 37 °C in a CO₂ incubator for cell adhesion and adaptation. Cancer cells were also seeded in separate 96-well plates (T0 plates), 12 wells per cell line, to determine the OD values at time zero (T0). After overnight adaptation, 20 μL of MTT reagent (final concentration 0.5 mg / mL) was added to each well of the T0 plates and incubated at 37 °C for 4 hours. The supernatant medium was removed with a pipette, and approximately 150 μL of DMSO was added to each well. Using 550 nm as the test wavelength and 630 nm as the reference wavelength, the plates were read on a plate reader to obtain the OD values at time zero (T0). After overnight adaptation, the test compounds and vehicle were added to the plates to treat the cells. The test compound was tested at 6 concentrations in triplicate wells. The plates were incubated at 37 °C in a CO₂ incubator for 48 hours. Longer incubation times can be used. 20 μL of MTT reagent was added to each well and incubated at 37 °C for 4 hours. The supernatant medium was removed with a pipette, and approximately 150 μL of DMSO was added to each well. Using approximately 550 nM as the test wavelength and using approximately 630 nM as the reference wavelength, the plates were read on a plate reader (TECAN, Infinite M200). Cell viability (%) was calculated in two ways:

[0200] % Viability = [(T - B) / (C - B)] × 100%

[0201] % Viability = [(T - C0) / (C - C0)] × 100%

[0202] T: Mean absorbance of treatment at different times;

[0203] C: Mean absorbance of negative control at different times;

[0204] B: Mean absorbance of blank wells (media only) at different times;

[0205] C0: Mean absorbance of negative control at 0 h.

[0206] Results were expressed as mean ± SD. IC was calculated by XLfit software 50 . Each compound was tested independently in duplicate.

[0207] 3.3 In vivo effects on mouse bladder and prostate cancer models.

[0208] To establish orthotopic bladder cancer tumors, mice were anesthetized by intraperitoneal (ip) administration of a dose of ketamine / xylazine solution (K113; Sigma-Aldrich Japan GK, Tokyo, Japan) at 0.1 ml / 10 g body weight. Subsequently, a 24-gauge polytetrafluoroethylene intravenous catheter was inserted through the urethra into the bladder or prostate using an inert lubricant. To prepare the bladder for tumor implantation, brief acid exposure, followed by alkaline neutralization, was performed to promote chemical injury on the bladder wall by instilling 8 μl of 1 MOI silver nitrate into the bladder. This resulted in the formation of sufficient and controlled diffuse bladder wall injury. After 15 seconds, the contents were washed out by transurethral infusion of PBS. The first catheter was removed, and a new 24-gauge catheter was inserted into the urethra for intravesical instillation of MBT-2-Luc cells (5 x 10 4 cells mixed with 0.1 ml PBS), and it was maintained for 1.5 h by suture. Every 10 days, after i.p. administration of luciferin, tumor imaging was performed using bioluminescence technology (Xenogen IVIS200 system; Xenogen Corporation, Hopkinton, MA, USA).

[0209] For the NIT and BCG combination, mice bearing orthotopic bladder cancer tumors were randomly divided into six groups: control (PBS), BCG, continuously administered NIT, NIT administered continuously except for a 24-hour interruption before and after weekly BCG instillation (48-hour interruption group), combination of NIT (continuously administered) and BCG, and combination of NIT (48-hour interruption) and BCG. The detailed description of drug administration is shown in Table 23. According to the tumor imaging results, as determined by luciferase expression, BCG (1 x 10 5 CFU / 100 μL) was instilled into the bladder once a week for three weeks. NIT was administered continuously or with a 48-hour interruption.

[0210] Table 23. Schematic diagram of drug administration

[0211]

[0212] ○: Intravesical administration of PBS, ●: Intravesical administration of BCG, △: CMC p.o, ▲: NIT p.o, *: 48-hour interruption of NIT administration

[0213] For the NIT and MMC combination, mice bearing orthotopic bladder cancer tumors were randomly divided into four groups: control (PBS), NIT, MMC, and combination of NIT and MMC. The detailed description of drug administration is shown in Table 24.

[0214] Table 24. Schematic diagram of drug administration

[0215]

[0216]

[0217] ○: Intravesical administration of PBS, ●: Intravesical administration of MMC, △: CMC p.o, ▲: NIT p.o

[0218] For the NIT and anti-PD-1 combination, mice bearing orthotopic bladder cancer tumors were randomly divided into four groups: control (PBS), NIT, anti-PD-1, and the combination of NIT and anti-PD-1. The detailed description of drug administration is shown in Table 25.

[0219] Table 25. Schematic diagram of drug administration

[0220]

[0221] ○: Intravesical administration of PBS, ●: Intraperitoneal administration of anti-PD-1, △: CMC p.o, ▲: NIT p.o

[0222] To establish orthotopic prostate cancer tumors, C57 / BL6 mice were anesthetized intraperitoneally with a dose of ketamine / xylazine solution (K113; Sigma-Aldrich Japan GK, Tokyo, Japan) at 0.1 ml / 10 g body weight. A transverse incision was made in the lower abdomen to expose the bilateral dorsal lobes of the prostate. After trypsin digestion of RM-9-Luc cells, 5.0×10 3 cells in 10 μl of Hanks balanced salt solution were directly injected into the right dorsal lobe of the prostate using a new 24-gauge catheter. An obvious and recognizable blister in the injected prostate lobe was considered a sign of a technically satisfactory injection. The abdominal wound was closed with a stainless steel clip (Autoclip; Becton Dickinson Co., Sparks, MD). One week after RM-9-Luc cell injection, when the tumor diameter reached 5 mm, mice bearing orthotopic prostate cancer tumors were randomly divided into four groups: control (PBS), NIT, anti-PD-1, and the combination of NIT and anti-PD-1. The detailed description of drug administration is shown in Table 26.

[0223] Table 26. Schematic diagram of drug administration

[0224]

[0225] ○: Intraperitoneal administration of PBS, ●: Intraperitoneal administration of anti-PD-1, △: CMC p.o, ▲: NIT p.o

[0226] Mice received an i.p. injection of luciferin, and luciferase expression in the tumors was measured by the Xenogen IVIS200 system. The mice were killed for euthanasia using CO2 according to the animal euthanasia guidelines (2013 version).

[0227] 3.4 Statistical analysis

[0228] Data from all quantitative analyses were expressed as mean ± standard deviation and were statistically analyzed using one-way analysis of variance (ANOVA) and independent-samples t-test. Statistical calculations were performed using GraphPad Prism 5. A P-value of less than 0.05 was considered statistically significant.

[0229] Additivity (E xy = E x + E y -(E x E y )) was determined using the fractional product concept or the Bliss independence model, where E XY is the additive effect of two compounds x and y, which is calculated from the product of the individual effects E x and E y of these two compounds. Synergy was established when the actually observed tumor inhibition value was greater than the expected tumor inhibition value determined by the Bliss independence model (20).

[0230] References

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Claims

1. Use of a combination of an effective amount of nitroxoline or a pharmaceutically acceptable salt thereof and an effective amount of an immunotherapeutic agent in the preparation of a medicament for treating cancer, wherein the combination of the nitroxoline or a pharmaceutically acceptable salt thereof and the immunotherapeutic agent causes a synergistic effect, and wherein the immunotherapeutic agent is an anti-PD-1 antibody or an antigen-binding fragment thereof, and the cancer is bladder cancer or prostate cancer.

2. A kit comprising an effective amount of nitroxoline or a pharmaceutically acceptable salt thereof, an effective amount of an immunotherapeutic agent, one or more pharmaceutically acceptable carriers, and instructions for using the combination of the effective amount of nitroxoline or a pharmaceutically acceptable salt thereof and the effective amount of the immunotherapeutic agent for treating cancer in a subject in need thereof, wherein the immunotherapeutic agent is an anti-PD-1 antibody or an antigen-binding fragment thereof, and the cancer is bladder cancer or prostate cancer.

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