BZD-1 as a cancer chemosensitizer

Abstract

Provided herein is a method of enhancing the effect of an anti-cancer agent in a subject diagnosed with cancer, comprising administering to the subject an effective amount of 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1) and a combination therapy comprising an anti-cancer agent.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 196,459, filed June 3, 2021, and U.S. Provisional Patent Application No. 63 / 208,246, filed June 8, 2021, the entire contents of which are incorporated by reference into this specification.

[0002]

[0002] The present disclosure relates to the field of cancer treatment. In particular, the present disclosure relates to a method of enhancing anti-cancer therapeutic agents by co-administering a benzodiazepine analog compound, BZD-1. [Background technology]

[0003]

[0003] GABA receptor (GABA A R) is a pentameric chloride (Cl) complex composed of two α, two β, and γ subunits, most commonly encoded by the GABR genes GABRA, GABRB, and GABRG, respectively. - ) channels. A ClR plays a fundamental role in determining the excitation / inhibition balance in the central nervous system. - GABA is a receptor that mediates the flux. A R has the primary function of hyperpolarizing neurons after binding of its ligand, GABA (see Figure 1).

[0004]

[0004] GABA A R has been an important therapeutic target since the clinical introduction of benzodiazepines in the 1960s. A It binds to the γ-α interface of R, increasing the availability of GABA and inhibiting Cl - Increases flux (Figure 1). FDA-approved benzodiazepines most commonly consist of a diazepine ring fused to a benzene ring (1,4-benzodiazepine), such as diazepam (Valium®), and a phenol ring (5-phenyl-1H-benzo[e]).

[0005] Benzodiazepines have traditionally been used to treat nervous system disorders such as anxiety, insomnia, seizure disorders, convulsive disorders, and alcohol withdrawal. A R has been found to be expressed not only in cancers of the central nervous system but also in cancers throughout the body.

[0006]

[0006] Many cancers are very difficult to treat and / or result in local and / or distant recurrence, including brain metastasis, after primary treatment. Furthermore, many of the first-line anticancer treatments become ineffective or are poorly tolerated by patients after a period of use. Improved therapies are needed to enhance tumor control, inhibit metastasis, and improve survival while reducing side effects. Summary of the Invention

[0007]

[0007] Accordingly, provided herein are methods of potentiating anti-cancer therapeutic agents by co-administering the benzodiazepine analog compound BZD-1.

[0008] In one embodiment, a method is provided for enhancing the effect of an anti-cancer agent in a subject diagnosed with cancer, the method comprising administering to the subject a combination therapy comprising an effective amount of 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1), or a salt thereof; and an anti-cancer agent.

[0008]

[0009] In another embodiment, a method of treating glioblastoma in a subject in need thereof is provided, comprising administering to the subject an effective amount of a combination therapy comprising BZD-1 or a salt thereof; and temozolomide.

[0009]

[0010] In another embodiment, a method of treating lung cancer in a subject in need thereof is provided, comprising administering to the subject an effective amount of a combination therapy comprising BZD-1 or a salt thereof; and docetaxel.

[0010]

[0011] These and other objects, features, embodiments and advantages will become apparent to those skilled in the art from a reading of the following detailed description and the appended claims. [Brief description of the drawings]

[0011] [Figure 1]

[0012] Diagram of the chloride ion permeable pore of GABAARs: Diagram of GABAARs looking down on the chloride ion permeable pore and showing the structures of the α, β, and γ subunits (left); Diagram showing that binding of two GABA ligands opens the channel and binding of a benzodiazepine enhances chloride ion flux (center, showing the GABAAR pore closing, opening, and enhanced permeability); Structures of the classical benzodiazepines diazepam (Valium®) and BZD-1 are illustrated. [Diagram 2]

[0013] 1 is a table showing the cytotoxicity response of glioblastoma cell lines treated with BZD-1 plus temozolomide (TMZ). [Diagram 3]

[0014] FIG. 3A shows images of spheroids of the unmethylated GBM cell line BT142-GFP treated with TMZ, BZD-1, BZD-1+TMZ, or control.

[0015] FIG. 3B shows images of spheroids of the unmethylated GBM cell line G43 treated with TMZ, BZD-1, BZD-1+TMZ, or control. [Figure 4]

[0016] FIG. 1 shows that BZD-1 inhibits the proliferation of H1792 human lung cancer cells in vitro. In the MTS assay using H1792 cells, an IC50 of 4.5-6 μM for BZD-1 is obtained. [Diagram 5]

[0017] FIG. 1 shows that BZD-1 enhances docetaxel-mediated cytotoxicity and inhibits the proliferation of H1792 cells. [Figure 6]

[0018] 1 shows that BZD-1 and docetaxel synergistically inhibit H1792 cell proliferation. Combination therapy of BZD-1 and DTX was analyzed by calculating a combination index (CI) value of 0.6, indicating a synergistic effect of the activity of both drugs in inhibiting H1792 cell proliferation in vitro, embodied by a left shift in the dose-response graph for DTX. [Figure 7]

[0019] FIG. 1 shows H1792 cells treated with BZD-1 alone or in combination with 0.5 nM docetaxel (DTX). [Figure 8]

[0020] FIG. 1 shows that BZD-1 inhibits the growth of H1792 tumor cells in vivo. [Figure 9]

[0021] FIG. 1 shows that BZD-1 inhibits the growth of H1792 tumor cells in vivo. [Figure 10]

[0022] 1 shows that BZD-1 inhibits the growth of H1792 tumor cells in vivo.BZD-1 treatment inhibited tumor growth of H1792 xenografts in NOD-SCID mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012]

[0023] Detailed descriptions of embodiments of the presently disclosed subject matter are described herein. Modifications of the embodiments described herein, as well as other embodiments, will become apparent to those skilled in the art after reviewing the information presented herein.

[0013]

[0024] Although the following terms are believed to be well understood in the art, definitions are provided to facilitate description of the presently disclosed subject matter. 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 the presently disclosed subject matter belongs.

[0014]

[0025] Unless otherwise indicated, all numerical values ​​expressing properties such as quantities of ingredients, reaction conditions, and the like used in the specification and claims are understood to be modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations and may vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0015]

[0026] As used herein, the term "about" when referring to a value or amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations in some embodiments of ±20%, in some embodiments of ±10%, in some embodiments of ±5%, in some embodiments of ±1%, in some embodiments of ±0.5%, and in some embodiments of ±0.1% from the specified amount, as such variations are appropriate for performing the disclosed methods.

[0016]

[0027] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification includes every narrower numerical range that is subsumed within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0017]

[0028] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0029] A "pharmaceutically acceptable salt" is a cationic salt formed with any acidic (e.g., hydroxamic or carboxylic) group, or an anionic salt formed with any basic (e.g., amino) group. Many such salts are known in the art, as described in WO 87 / 05297, published September 11, 1987, by Johnston et al. Specific cationic salts include alkali metal salts (such as sodium and potassium), alkaline earth metal salts (such as magnesium and calcium), and organic salts. Specific anionic salts include halides (such as chloride salts), sulfonates, carboxylates, phosphates, and the like.

[0018]

[0030] The term "subject" as used herein means any mammalian subject, including a human. In certain embodiments, the subject is diagnosed with cancer, a tumor, a brain tumor, or a brain metastasis.

[0019]

[0031] The terms "treat," "treatment," and "treating," as used herein, refer to a method of alleviating or eliminating a disease, disorder, and / or its symptoms.

[0020]

[0032] "Effective amount," as used herein, refers to an amount of a substance (e.g., a therapeutic compound and / or composition) that induces a desired biological response. In some embodiments, an effective amount of a substance is an amount sufficient to treat, diagnose, prevent, and / or delay and / or alleviate one or more symptoms of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to the disease, disorder, and / or condition. As will be recognized by those skilled in the art, an effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance delivered, the target cell or tissue, and the like. For example, an effective amount of a composition for treating a disease, disorder, and / or condition is an amount that alleviates, ameliorates, relieves, inhibits, prevents, delays the onset of; reduces the severity of, and / or reduces the incidence of, one or more symptoms or characteristics of the disease, disorder, and / or condition. Furthermore, an effective amount can be administered via a single dose or multiple doses within a treatment regimen. In some embodiments, an individual dose or composition is considered to include an effective amount when it includes an effective amount as a dose in the context of the treatment regimen. One of ordinary skill in the art will understand that a dose or amount is considered effective if it has been demonstrated or is demonstrated to exhibit statistically significant efficacy when administered to a population of patients; it is not necessary for a particular result to be achieved in a particular individual patient in order for an amount to be considered effective as described herein.

[0021]

[0033] Glioblastoma multiforme (GBM) is an aggressive (grade IV) primary brain tumor. Standard treatment for GBM includes radiation therapy with the DNA alkylating agent temozolomide (TMZ). This approach shows some efficacy in GBM cells with sufficient MGMT promoter methylation (approximately 50% of GBM tumors), because the reduction of MGMT protein leads to a reduced ability to reverse TMZ-induced DNA damage. Histone deacetylase inhibitors have recently been used to improve the efficacy of TMZ, but unfortunately, they result in bone marrow toxicity without contributing to a sustained response. TMZ is also not without its own debilitating and life-threatening side effects, such as leukopenia. Tragically, this treatment regimen results in a survival time of only 12-15 months. There is an urgent need to increase the efficacy of TMZ against MGMT-methylated GBM and to identify effective treatment approaches for MGMT-unmethylated GBM and reduce the side effects of TMZ.

[0022]

[0034] GBM is one of the most lethal human cancers and is extremely difficult to treat. GBM tumor cells interact with a variety of cells in a complex microenvironment. Furthermore, the blood-brain barrier (BBB) ​​acts to limit drug bioavailability and promote immune evasion. Unfortunately, GBM cells frequently disrupt the physiological function of cerebrovascular tissues, transforming the BBB into an effective blood-tumor barrier (BTB) that can protect cancer tissues from drugs as well as systemic immunity. Despite increasing knowledge of the genetic and epigenetic alterations underlying GBM tumor initiation and growth, the prognosis of GBM remains poor.

[0023]

[0035] TMZ is used to treat all GBM. However, TMZ is only effective in about half of GBM with MGMT methylation. The present disclosure provides a brain-penetrating benzodiazepine analogue that synergistically enhances TMZ regardless of the methylation status of GBM.

[0024]

[0036] By targeting the unique electrochemical vulnerability of GBM with a non-toxic, brain-penetrant small molecule, GBM tumor cells can be sensitized to TMZ regardless of MGMT methylation status. Without wishing to be bound by theory, GBM cells, as well as tumor cells from a subgroup of patients with pediatric brain tumors medulloblastoma and melanoma, express functional GABA receptor agonists. A R, which is believed to be able to target BZD-1 to alter ion dynamics and induce apoptosis. This data indicates that co-administration of BZD-1 and TMZ enhances the anticancer activity of TMZ regardless of MGMT methylation status. The observed effects are dramatic and synergistic.

[0025]

[0037] BZD-1 is GABA A Cl via R - It promotes anion transport, thereby altering ion dynamics, inhibiting the drug efflux transporter P-glycoprotein, and inducing an apoptotic response. BZD-1-induced electrochemical changes in cancer cells significantly and synergistically enhance TMZ, regardless of the MGMT status of GBM cells.

[0026]

[0038] Non-small cell lung cancer (NSCLC) accounts for the majority of lung cancer cases (80%-85%). The most common histological subtype of NSCLC (40%-50%) is lung adenocarcinoma. The majority of NSCLC patients with advanced stages of disease face local and / or distant recurrence, including brain metastases, during the first 2 years after completion of primary treatment.

[0027]

[0039] GABA A R expression is present in both lung adenocarcinoma and squamous cell carcinoma subtypes. As disclosed herein, BZD-1 treatment induces apoptosis in patient-derived adenocarcinoma cells. Furthermore, BZD-1 synergistically enhances the chemotherapy drug docetaxel even at subtherapeutic doses when administered alone. A flank xenograft mouse model using patient-derived adenocarcinoma cells was used to evaluate the ability of BZD-1 to sensitize tumor cells to docetaxel, reducing the toxicity profile of docetaxel while retaining efficacy.

[0028]

[0040] BZD-1 is a benzodiazepine analogue with the following structure:

[0029] [ka]

[0030] 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one.

[0041] In one embodiment, a method is provided for enhancing the effect of an anti-cancer agent in a subject diagnosed with cancer, the method comprising co-administering to the subject an effective amount of 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1), or a salt thereof; and an anti-cancer agent.

[0031]

[0042] In some embodiments, the BZD-1 and the anti-cancer agent are co-administered. "Co-administration," as used herein, refers to administration of the BZD-1 and the anti-cancer agent such that both agents are capable of achieving a physiological effect, e.g., in a recipient subject, at the same time. However, the two agents need not be administered at the same time. In certain embodiments, administration of one agent can precede administration of the other agent. In some embodiments, co-administration typically results in both agents being present in the subject at the same time. Thus, in some embodiments, the BZD-1 and the anti-cancer agent can be administered simultaneously or sequentially.

[0032]

[0043] For sequential administration, the BZD-1 and the anti-cancer agent can be administered within 1 hour, 2 hours, 4 hours, 8 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, or within 1 week of each other. In some embodiments, the BZD-1 is administered first, followed by the anti-cancer agent. In some embodiments, the anti-cancer agent is administered first, followed by the BZD-1.

[0033]

[0044] In some embodiments, the cancer to be treated is selected from the group consisting of lung cancer, melanoma, liver cancer, breast cancer, pancreatic cancer, colorectal cancer, ovarian cancer, thyroid cancer, prostate cancer, glioblastoma, medulloblastoma, and neuroblastoma.

[0034]

[0045] In a specific embodiment, the cancer is a lung cancer selected from the group consisting of non-small cell lung cancer (NSCLC), adenocarcinoma, large cell lung carcinoma (LCLC), and squamous cell carcinoma.

[0046] In another specific embodiment, the cancer is a central nervous system cancer selected from the group consisting of glioblastoma, medulloblastoma, and neuroblastoma.

[0035]

[0047] A variety of anti-cancer agents are suitable for use in combination with BZD-1 in the present methods, hi some embodiments, the anti-cancer agent is selected from the group consisting of chemotherapeutic agents, immunotherapeutic agents, targeted therapeutic agents, and combinations thereof.

[0036]

[0048] In specific embodiments, the chemotherapeutic agent is selected from the group consisting of alkylating agents, antibacterial agents, antimetabolites, topoisomerase inhibitors, cytotoxic antibiotics, and combinations thereof.

[0037]

[0049] In specific embodiments, the anticancer agent is a chemotherapeutic agent selected from the group consisting of temozolomide, docetaxel, cyclophosphamide, methotrexate, 5-fluorouracil, vinorelbine, doxorubicin, bleomycin, vinblastine, dacarbazine, mustine, vincristine, procarbazine, prednisolone, etoposide, cisplatin, epirubicin, capecitabine, folinic acid, oxaliplatin, gemcitabine, ifosfamide, and combinations thereof.

[0038]

[0050] In a specific embodiment, the chemotherapeutic agent is TMZ and the cancer is glioblastoma.

[0051] In another specific embodiment, the chemotherapeutic agent is docetaxel and the cancer is lung cancer.

[0039]

[0052] In another embodiment, a method of treating glioblastoma in a subject in need thereof is provided, comprising administering to the subject an effective amount of a combination therapy comprising 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1) or a salt thereof; and temozolomide (TMZ). BZD-1 and temozolomide may be administered simultaneously or sequentially.

[0040]

[0053] In some embodiments, BZD-1 enhances TMZ regardless of the MGMT methylation status of the glioblastoma. In further embodiments, the dose of TMZ co-administered with BZD-1 is lower than the dose of TMZ that is effective as a monotherapy in treating glioblastoma.

[0041]

[0054] In another embodiment, a method of treating lung cancer in a subject in need thereof is provided, comprising administering to the subject an effective amount of a combination therapy comprising 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1) or a salt thereof; and docetaxel. BZD-1 and docetaxel may be administered simultaneously or sequentially.

[0042]

[0055] In some embodiments, the lung cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), adenocarcinoma, large cell lung cancer (LCLC), and squamous cell carcinoma. In a specific embodiment, the lung cancer is NSCLC.

[0043]

[0056] In some embodiments, the dose of docetaxel co-administered with BZD-1 is lower than the dose of docetaxel effective as monotherapy in the treatment of lung cancer.

[0057] The following examples are illustrative and not intended to limit the scope of the disclosure. EXAMPLES

[0044] Example 1. BZD-1 potentiates temozolomide (TMZ) regardless of MGMT methylation status

[0058] In vitro cytotoxicity studies of methylated and unmethylated GBM cells treated with BZD-1 and TMZ alone or in combination demonstrated that the combination therapy overcame TMZ resistance in unmethylated GBM cell lines and was significantly more potent than TMZ monotherapy in killing GBM cells, regardless of methylation status (Figure 2). The synergistic effect of the two-drug combination was quantified using the Chou-Talay combination index formula, and BZD-1 and TMZ were found to act synergistically.

[0045]

[0059] We performed spheroid assays using two unmethylated GBM lines, BT142-GFP (Figure 3A) and G43 (Figure 3B), to qualitatively evaluate the effect of BZD-1+TMZ. The assays revealed significant disorganization of aggregates in response to the combination therapy, suggesting that tumorigenic activity was abolished.

[0046] Example 2. Formulated BZD-1 is metabolically stable and rapidly penetrates and accumulates in the brain

[0060] Various approaches to formulate BZD-1 have been explored. A cosolvent-based injectable formulation (already US FDA approved for benzodiazepines) has been found to be satisfactory. In this cosolvent formulation, BZD-1 is highly soluble (10 mg / mL) and stable at room temperature for up to 6 months, with no visible side effects observed 12 hours after a single dose intraperitoneally administered to rats. Metabolic stability studies using human liver microsomes were also performed, and in contrast to midazolam, an FDA-approved benzodiazepine, no degradation products were observed within 1 hour. Pharmacokinetics of BZD-1 showed rapid brain penetration (within approximately 5 minutes) and significant accumulation of BZD-1 in brain extracellular fluid (161.3 ng / mL).

[0047] Example 3. BZD-1 inhibits proliferation of H1792 human lung cancer cells in vitro.

[0061] 103 H1792 cells / well were seeded in 100uL of medium in 96-well plates and allowed to adhere for 24 hours. The next day, BZD-1 (stock, 40mM in DMSO) was first diluted to 40μM in fresh cell culture medium, then serially diluted 2-fold. The medium above the cells was then carefully aspirated and replaced with 100μL of vehicle (DMSO) / drug-containing medium, replicated 5 times for each control / drug dilution. The cells were returned to the incubator for 48 hours.

[0048]

[0062] For the MTS assay, the medium was carefully aspirated and replaced with 100 μL of phenol red-free medium. Then, 20 μL of MTS reagent (Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay reagent-Promega) was added to each well. The plate was returned to the incubator for 1 hour, and then the OD at 490 nm was taken with a plate reader. The OD values ​​were then analyzed and graphed using GraphPad Prism.

[0049]

[0063] The results are shown in Figure 4 and demonstrate that BZD-1 inhibits the proliferation of H1792 human lung cancer cells in vitro.

[0050] Example 4. BZD-1 enhances docetaxel-mediated cytotoxicity and inhibits proliferation of H1792 cells

[0064] 10 3 H1792 cells / well were seeded in 100 μL of medium in 96-well plates and allowed to adhere for 24 hours. The next day, a large volume of fresh medium containing 2.5 μM BZD-1 was prepared from a 40 mM BZD-1 stock in DMSO. For dilutions of docetaxel (DTX), fresh medium was used for DTX only and 2.5 μM BZD-1-containing medium for DTX+2.5 μM BZD-1 treatment. The medium above the cells was then aspirated and replaced with 100 μL of vehicle (DMSO) / drug-containing medium, with five replicates for each control / drug dilution. The cells were returned to the incubator for 48 hours.

[0051]

[0065] For the MTS assay, the medium was aspirated and replaced with 100 μL of phenol red-free medium. Then, 20 μL of MTS reagent (Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay reagent-Promega) was added to each well. The plate was returned to the incubator for 1 hour, and then the OD at 490 nm was obtained with a plate reader. The OD values ​​were then analyzed and graphed using GraphPad Prism.

[0052]

[0066] The results are shown in Figure 5 and demonstrate that BZD-1 enhances the cytotoxic effect of docetaxel against H1792 human lung cancer cells in vitro.

[0053] Example 5. BZD-1 and docetaxel synergistically inhibit proliferation of H1792 cells

[0067] 10 3 H1792 cells / well were seeded in 100 μL of medium in 96-well plates and allowed to adhere for 24 hours. The next day, a large volume of fresh medium containing 2.5 μM BZD-1 was prepared from a 40 mM BZD-1 stock in DMSO. Fresh medium was used for single drug dilutions. For DTX+2.5 μM BZD-1, 2.5 μM BZD-1-containing medium was used. The medium above the cells was then aspirated and replaced with 100 μL of vehicle (DMSO) / drug-containing medium, with five replicates for each control / drug dilution. The cells were returned to the incubator for 48 hours.

[0054]

[0068] For the MTS assay, the medium was aspirated and replaced with 100 μL of phenol red-free medium. Then, 20 μL of MTS reagent (Cell Titer 96 Aqueous Non-Radioactive Cell Proliferation Assay reagent-Promega) was added to each well. The plate was returned to the incubator for 1 hour, and then the OD at 490 nm was obtained with a plate reader. The OD values ​​were then analyzed and graphed using GraphPad Prism.

[0055]

[0069] The results are shown in Figure 6 and demonstrate that BZD-1 and docetaxel synergistically inhibit the proliferation of H1792 lung cancer cells in vitro.

[0056] Example 6. Clonogenic assay showing that BZD-1 and docetaxel synergistically inhibit proliferation of H1792 cells

[0070] H1792 cells were seeded at 300 cells / well in 3 mL of medium in 6-well plates and allowed to adhere for 36 hours. A large volume of fresh medium containing 0.5 nM docetaxel (DTX) was prepared from a 1 mM stock in DMSO. For dilutions of BZD-1, either fresh medium or +0.5 nM DTX-containing medium was used. The medium on the cells was then aspirated and replaced with 3 mL of vehicle (DMSO) / drug-containing medium, with triplicates for each control / drug dilution. The cells were returned to the incubator for 12 days. For staining, the medium was aspirated and the cells were washed once with PBS and fixed with methanol for 15 minutes at room temperature. The methanol was then removed and crystal violet stain was added to the cells and incubated for 30 minutes at room temperature. The plates were then washed extensively with tap water, dried and photographed.

[0057]

[0071] The results are shown in Figure 7 and demonstrate that the combination of BZD-1 and docetaxel synergistically inhibits the proliferation of H1792 lung cancer cells in vitro.

[0058] Example 6. In vivo dose-response experiments

[0072] NOD-SCID mice (3 mice / group) were injected with 10 6 H1792 cells were implanted subcutaneously. When tumors became palpable (11 days after implantation), treatment was initiated with intraperitoneal injection of vehicle (DMSO) or BZD-1 drug dissolved in DMSO daily for 7 days. Tumor size was measured every other day using calipers. Tumor volume was calculated as follows:

[0059]

[0073] Volume = (L x W 2 ) / 2

[0074] On day 46 after tumor implantation, mice were euthanized according to institutional IACUC procedures. Tumors from both flanks were excised, weighed, and the mean tumor mass for each group was calculated, plotted, and photographed.

[0060]

[0075] As shown in Figures 8, 9 and 10, BZD-1 reduced tumor volume compared to vehicle. BZD-1 is most effective at a clinically relevant dose of 2.5 mg / kg. At higher doses, BZD-1 exhibits sedative effects in animal models.

[0061]

[0076] All cited documents are incorporated herein by reference in their entirety, and the citation of any document shall not be construed as an admission that it is prior art with respect to the present invention.

[0062]

[0077] Additionally, when the terms "comprising" and / or "including" are used in describing various embodiments, those of ordinary skill in the art will understand that in some instances, the embodiments may alternatively be described using the language "consisting essentially of" or "consisting of."

[0063]

[0078] The above description illustrates specific embodiments of the present invention, but is not meant to limit its implementation. Although specific embodiments have been shown and described, it will be apparent to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the present invention. It is therefore intended in the appended claims to cover all such changes and modifications that are within the scope of the present invention.

Claims

1. A composition comprising 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1) or a salt thereof for use in a method of enhancing the effect of an anticancer agent in a subject diagnosed with cancer, wherein the method comprises administering to the subject, an effective amount of 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1) or a salt thereof, and an anticancer agent, in a co-administration step, wherein the cancer is a lung cancer selected from the group consisting of non-small cell lung cancer (NSCLC), adenocarcinoma, large cell lung cancer (LCLC), and squamous cell carcinoma, said composition.

2. The composition according to claim 1, wherein the anticancer agent is selected from the group consisting of chemotherapeutic agents, immunotherapeutic agents, targeted therapeutic agents, and combinations thereof.

3. The composition according to claim 2, wherein the chemotherapeutic agent is selected from the group consisting of alkylating agents, antibacterial agents, antimetabolites, topoisomerase inhibitors, cytotoxic antibiotics, and combinations thereof.

4. The composition according to claim 1, wherein the anticancer agent is a chemotherapeutic agent selected from the group consisting of temozolomide, docetaxel, cyclophosphamide, methotrexate, 5-fluorouracil, vinorelbine, doxorubicin, bleomycin, vincristine, dacarbazine, mustine, vinblastine, procarbazine, prednisone, etoposide, cisplatin, epirubicin, capecitabine, folic acid, oxaliplatin, gemcitabine, ifosfamide, and combinations thereof.

5. The composition according to claim 4, wherein the chemotherapeutic agent is temozolomide.

6. The composition according to claim 4, wherein the chemotherapeutic agent is docetaxel.

7. The composition according to claim 1, wherein BZD-1 and the anticancer agent are co-administered simultaneously or sequentially.

8. A composition comprising 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1) or a salt thereof for use in a method of treating glioblastoma in a subject in need of treatment of glioblastoma, The method comprises administering to a subject a combination therapeutic agent comprising an effective amount of 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1) or a salt thereof, temozolomide, wherein, in the method, temozolomide is administered at a dose lower than the effective amount of temozolomide when administered as a single chemotherapeutic agent, the composition.

9. The composition according to claim 8, wherein in the method, BZD-1 and temozolomide are administered simultaneously or sequentially.

10. The composition according to claim 8, wherein BZD-1 enhances temozolomide regardless of the MGMT methylation status of glioblastoma.

11. Use in a method of treating lung cancer in a subject in need thereof, a composition comprising 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1) or a salt thereof, wherein the method comprises administering to a subject a combination therapeutic agent comprising an effective amount of 7-ethynyl-5-(2-fluorophenyl)-1-methyl-1,3-dihydro-2H-benzo[e][1,4]diazepin-2-one (BZD-1) or a salt thereof, docetaxel, wherein the cancer is a lung cancer selected from the group consisting of non-small cell lung cancer (NSCLC), adenocarcinoma, large cell lung cancer (LCLC), and squamous cell carcinoma, the composition.

12. The composition according to claim 11, wherein in the method, BZD-1 and docetaxel are administered simultaneously or sequentially.

13. The composition according to claim 11, wherein the lung cancer is non-small cell lung cancer (NSCLC).

14. The composition according to claim 11, wherein in the method, docetaxel is administered at a dose lower than the effective amount of docetaxel when administered as a single chemotherapeutic agent. ​ ​

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