Microtubule-targeting agents (MTAs) are among the most effective chemotherapeutics used in the treatment of cancer. However, the clinical utility of current MTAs, such as the microtubule-targeting vinca alkaloids (e.g. vinblastine and vincristine) and taxanes (paclitaxel and docetaxel), are often limited due to adverse side effects or multidrug resistance (MDR). Previous studies have established that broad-based resistance to these drugs, arises in large part from the overexpression of P-glycoprotein (P-gp), to which paclitaxel, vinblastine, vincristine, docetaxel, and others, are substrates. See e.g., Gottesman et al. (2002), Nat Rev Cancer 2 48-58 and Mol Pharmacol. 2009 January; 75(1): 92-100. This presents major challenges to cancer chemotherapy, particularly in managing patients with metastatic cancers that are resistant to traditional MTA therapies.
To overcome P-gp-mediated MDR, many small molecule drugs which modulate the activity of P-gp have been tested. See e.g., Darby et al., Curr. Drug. Metab 2011, 12, 722-731. Most of these programs, however, have failed clinical trials due to lack of potency and/or toxicity issues. The need therefore exists for novel microtubule interactors, particularly those which are not substrates for P-gp.
Evidence now suggests that 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, herein referenced to as Compound 1, inhibits the formation of microtubule assembly. See e.g.,
Provided herein, therefore, are methods of treating a cancer responsive to the modulation of microtubule assembly using 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, or a pharmaceutically acceptable salt thereof. Such cancers include e.g., prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer.
Evidence also shows that Compound 1 is not a substrate for P-gp, the overexpression of which leads to P-gp-mediated multi-drug resistance with certain vinca alkaloids and taxane chemotherapeutics. Therefore, also provided are methods of treating P-gp-mediated resistant cancers using Compound 1, or a pharmaceutically acceptable salt thereof.
In certain aspects, the disclosure relates to a method of treating a cancer responsive to the modulation of microtubule assembly comprising administering to a subject in need thereof, a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the cancer is selected from prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). In some embodiments, the cancer is endometrial cancer. In some embodiments, the cancer is glioblastoma multiforme. In some embodiments, the cancer is sarcoma.
In certain aspects, the disclosure relates to a method of treating a P-gp-mediated resistant cancer comprising administering to a subject in need thereof, a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, or a pharmaceutically acceptable salt thereof. In some embodiments, the cancer is a taxane resistant cancer. In some embodiments, the cancer is resistant to paclitaxel, vinblastine, vincristine, or docetaxel. In some embodiments, the cancer is resistant to paclitaxel. In some embodiments, the cancer is selected from ovarian, prostate, breast, bladder, head and neck, and lung cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is a vinca alkaloid resistant cancer. In some embodiments, the cancer is resistant to vinblastine or vincristine. In some embodiments, the cancer is selected from lymphoma, acute lymphocytic leukemia (ALL), and solid tumors.
In certain aspects, the disclosure relates to a method of treating a cancer responsive to the modulation of microtubule assembly comprising administering to a subject in need thereof, a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, or a pharmaceutically acceptable salt thereof. The term “cancer responsive to the modulation of microtubule assembly” as used herein refers to a cancer that is responsive to a microtubule-targeting agent, such as a vinca alkaloid (e.g., vinblastine or vincristine) or a taxane (e.g., paclitaxel and docetaxel). In some embodiments, the cancer that is responsive to the modulation of microtubule assembly is selected from the group consisting of prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer.
In one aspect, provided is a method of treating a cancer selected from prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer in a subject comprising administering to the subject a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, or a pharmaceutically acceptable salt thereof. Also provided is a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, or a pharmaceutically acceptable salt thereof, for treating a cancer selected from prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer. Further provided is the use of a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating a cancer selected from prostate cancer, head and neck cancer, endometrial cancer, glioblastoma multiforme, sarcoma, and lung cancer. In one aspect, the cancer treated by the aforementioned method is prostate cancer. In another aspect, the cancer treated by the aforementioned method is head and neck cancer. In yet another aspect, the cancer treated by the aforementioned method is lung cancer such as small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). In yet another aspect, the cancer treated by the aforementioned method is endometrial cancer. In yet another aspect, the cancer treated by the aforementioned method is glioblastoma multiforme. In yet another aspect, the cancer treated by the aforementioned method is a sarcoma.
In one aspect, provided is a method of treating a P-glycoprotein (P-gp)-mediated resistant cancer in a subject comprising administering to the subject a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, or a pharmaceutically acceptable salt thereof. Also provided is a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, or a pharmaceutically acceptable salt thereof, for treating a P-gp-mediated resistant cancer. Further provided is the use of a therapeutically effective amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating a P-glycoprotein (P-gp)-mediated resistant cancer.
In one aspect, the P-gp-mediated resistant cancer described herein is a taxane resistant cancer. In another aspect, the P-gp-mediated resistant cancer described herein is a vinca alkaloid resistant cancer. In some embodiments, the P-gp-mediated resistant cancer described herein is a paclitaxel, vinblastine, vincristine, and/or docetaxel resistant cancer.
As used herein, “P-glycoprotein mediated resistant cancer” or “Pg-p mediated resistant cancer”, used interchangeably herein, refers to a cancer which is resistant to treatment with one or more anticancer agents which are substrates for P-glycoprotein (Pg-p). Such anticancer agents include, but are not limited to, paclitaxel, vinblastine, vincristine, and docetaxel.
The term “resistance”, in the context of a resistant cancer, means that the cancer no longer responds to treatment. This includes cancers which are unresponsive or demonstrate a worsening of the disease while on a given treatment. In one aspect, a resistant cancer refers to a cancer which has become resistant during the course of treatment, i.e., the cancer initially responded, but no longer responded to treatment after a certain period of time.
As used herein, “taxane resistant cancer” refers to a cancer which is resistant to treatment with taxane anticancer agents. Taxanes are known in the art and include small molecules comprising tetradecahydro-6,10-methanobenzo[10]annulene and derivatives thereof as a central core. Taxane resistant cancers can include e.g., ovarian, prostate, breast, bladder, head and neck, and lung cancers.
As used herein, “vinca alkaloid resistant cancer” refers to a cancer which is resistant to treatment with vinca alkaloid anticancer agents. Vinca alkaloids are known in the art and include small molecules comprised of two multi-ringed units, an indole nucleus, and a dihydroindole nucleus, joined together with other complex systems. Vinca alkaloid resistant cancers include e.g., lymphomas, acute lymphocytic leukemia (ALL), and solid tumors.
For use in medicines, the pharmaceutically acceptable salt salts described herein refer to non-toxic “pharmaceutically acceptable salts.” Pharmaceutically acceptable salt forms include pharmaceutically acceptable basic/cationic salts.
The terms “subject” and “patient” may be used interchangeably, and refer to a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). Typically, the subject is a human in need of treatment.
As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a described cancer. In some aspects, treatment may be administered after one or more symptoms have developed, i.e., therapeutic treatment. In other aspects, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of exposure to a particular organism, or other susceptibility factors), i.e., prophylactic treatment. Treatment may also be continued after symptoms have resolved, for example to delay their recurrence.
The term “effective amount” or “therapeutically effective amount” refers to an amount of 2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide that will elicit a biological or medical response of a subject e.g., a dosage of between 0.01-100 mg/kg body weight/day. In one aspect, the effective amount of Compound 1 ranges from about 50 mg/kg to about 250 mg/kg. In one aspect, the effective amount of Compound 1 is about 75 mg/kg, about 100 mg/kg, about 150 mg/kg or about 200 mg/kg.
In some aspects, Compound 1 may be administered as part of a pharmaceutical composition. Pharmaceutical compositions may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, or via an implanted reservoir. The term “parenteral” as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. In some embodiments, the compositions are administered orally, intraperitoneally or intravenously. In some embodiments, the compositions are administered orally. Sterile injectable forms of the compositions described herein may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated.
2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide (Compound 1) has the chemical structure shown below and may be synthesized according to the procedures described for compound 126 in U.S. Pat. No. 11,091,447, the entire contents of which are incorporated herein by reference.
2-(difluoromethoxy)-N-[[5-(2-methoxyphenyl)-1H-1,2,4-triazol-3-yl]methyl]benzamide may exist in various tautomeric forms, each of which are expressly included as part of the invention.
In vitro tubulin polymerization assays (using >99% Pure Tubulin, OD Based—Porcine (BK006P) from Cytoskeleton Inc.) demonstrated that certain anti-cancer effects from Compound 1 are a result of cytoskeleton targeting to inhibit microtubule assembly.
The protocol followed for tubulin polymerization is according to the manufacturer's protocol. The following components were diluted as follows:
The half area 96-well plate provided by kit manufacturer was allowed to warm up to 37° C. for 30 mins in the Molecular Devices M5 plate reader. The Compound 1 and nocodazole were diluted in DMSO to a concentration of 2 mM, followed by dilution of these working stocks in the General Tubulin Buffer (GTB-provided in the kit) to a 10× the final well concentration and final well volume of 10 μl. Same was done with the 2 mM Paclitaxel stock. Once the plate was warmed up, the compounds were added in duplicates to the wells. In the control wells (no ligand control) 10 μl of GTB was added. The plate was placed at 37° C. in the plate reader for 3 minutes. Flash frozen tubulin was diluted as follows to have a final concentration of 10% glycerol and 1 mM GTP:
The values were normalized to the first read by designating the first read as ‘0’ subtracting the value of the first read from the following reads.
Microtubule polymerization, as a readout of absorbance at 340 nm, under standard conditions (no ligand control) and in the presence of 10 μM paclitaxel, a microtubule polymerization agent, as well has high (10 μM) and low (20 nM) concentrations of Compound 1 and positive control nocodazole are shown with their respective Vmax values in
MIA PaCa2 cells were cultured in DMEM+10% FBS and 1% P/S. Cells were plated at 1.5-2×106 in 60 mm dishes. For drug treatments, cells were treated the day after plating with 0.1% DMSO or the compounds tested at the concentrations indicated in
Significance in difference of treated MIA PaCa-2 cells to 0.1% DMSO treated cells were tested by Student t test (* p<0.05; ** p<0.01; *** p<0.001).
As shown in
The HCT116 cells were cultured using DMEM media supplemented with 10% FBS and 1% Pen/Strep. Cells were maintained to a maximum of 80% confluency before being split and plated into coverslips in 12-well plates.
1. HCT116 cells were plated on Poly-D-Lysine German Glass Coverslips (#1.5, 18 mm)
2. After 24h cells were treated with 300 nM of Compound 1 and Nocodozole.
3. After 24h cells were washed briefly with PBS and fixed with warm 4% paraformaldehyde at room temperature for 10 min.
4. Cells were permeabilized and blocked in 10% goat serum, 0.5% Triton-X in PBS for 1 hr.
5. Cells were incubated with Primary antibodies overnight.
6. Washed 3× with PBS for 10 min
7. Incubated with secondary antibodies at room temperature for 1 hr.
8. Washed 3× with PBS for 10 min
9. Coverslips were mounted with ProLong Gold Anti-Fade Mount with DAPI and imaged with Olympus FV1200 MPE Microscope.
10. Images were collected with identical confocal settings for all of the samples and Z-stacked images were projected with maximal projection mode using Fluor View Software.
1. Selected cells of interest using the drawing/selection tools (i.e. rectangle, circle, polygon or freeform).
2. From the Analyze menu selected “Measure.”
3. Selected region with no fluorescence to be my background.
4. Repeated these steps for other cells in the field of view
5. Used the CTCF formula to calculate Mad1 expression:
CTCF=Integrated Density−(Area of selected cell×Mean fluorescence of background readings)
Micrographs were imaged using Olympus FV1200 MPE confocal microscope at 63× oil. White arrows indicate dividing cells. Representative images of N=3 biological replicates. Significance in difference of treated HCT116 cells to untreated cells was tested by Student t test (** p<0.01).
As shown in
As shown in
Potency of Compound 1 was evaluated in a panel of patient-derived organoids (PDOs) representative of multiple tumor types.
Test article: Test articles were prepared fresh weekly.
The vehicle for Compound 1 and Paclitaxel was DMSO.
Male Sprague-Dawley Rats (total of 6 animals) 6 to 8 weeks of age (210-227 g) was used in this biodistribution study. Compound 1 was dosed at 75 mg/kg to all animals through oral route of administration at a 10 ml/kg dose volume. Compound 1 formulation was freshly prepared in Tween-80+0.5% methyl cellulose and maintained at room temperature until dosing. Dose was administered in all animals under fasted conditions. Two rats were sacrificed at 0.25 hrs, 1 hr and 8 hrs respectively. Heart, lung, liver, kidney, stomach, small intestine, large intestine, skeletal muscle, brain, spleen, pancreas and adipose were harvested while blood was processed to plasma for bioanalytical quantification of Compound 1.
Compound 1 bioanalytical assay was developed and validated using the SCIEX 6500 LC-MS/MS. A mobile phase (isocratic flow) comprised of Acetonitrile and 0.2% formic acid in MilliQ water in a Atlantis dC18-HPLC column (50×4.6 mm, 3 uM), with a flow rate of 0.9 mL/min and a run time of 2.20 min. Mass transition (m/z) of 375.040 and 188.100 was used for detection of Compound 1 at an LLOQ of 2.14 ng/mL.
Results are shown below in Table 1-3 and represented graphically by
brain barrier penetration observed at 8 hours.
Preparation of Donor Solution a) 0.2 mM working solution was prepared by diluting 10 mM stock solution with DMSO. b) 10 μM donor solution (5% DMSO) was prepared by diluting 20 μL of working solution with 380 μL PBS. 2) 150 μL of 10 μM donor solutions to each well of the donor plate, whose PVDF membrane was precoated with 5 μL of 1% brain polar lipid extract (Porcine)/dodecane mixture. Duplicates were prepared. 3) 300 μL of PBS was added to each well of the PTFE acceptor plate. 4) The donor plate and acceptor plate were combined together and incubated for 4h at room temperature with shaking at 300 rpm. 5) Preparation of TO sample: 20 μL donor solution was transferred to new well followed by the addition of 250 μL PBS (DF: 13.5), 130 μL ACN (containing internal standard) as TO sample. 6) Preparation of acceptor sample: The plate was removed from incubator. 270 μL solution was transferred from each acceptor well and mixed with 130 μL ACN (containing internal standard) as acceptor sample. 7) Preparation of donor sample: μL solution was transferred from each donor well and mixed with 250 μL PBS (DF: 13.5), 130 μL ACN (containing internal standard) as donor sample. 8) Acceptor samples and donor samples were all analyzed by LC/MS/MS. 9) The equation used to determine permeability rates (Pe) was displayed as follow.
As shown by the results in Table 4, Compound 1 is highly permeable across the blood brain barrier.
Although taxane resistance can be derived in vitro, this approach has limitations, as it does not faithfully recapitulate all aspects of taxane resistance observed in patients. Hence, a study designed to test Compound 1 efficacy in patient-derived organoids (PDO) ex vivo models of taxane resistant cancers was performed. CTG-1520 and CTG-0896 were derived from Triple Negative Breast Cancer (TNBC) patients post-treatment, and progression on a taxane-based therapy and the models' resistance to paclitaxel was confirmed by testing in vivo PDX (Patient-derived xenograft) tumor growth. CTG-1017 was derived from the same patient as CTG-1520 although at an earlier point in the treatment course, and has been shown to be sensitive to paclitaxel in vivo (CRO historical data). Compound 1 and paclitaxel were tested head-to-head in each model in a dose-curve setting. 10% DMSO was used as the positive control for induction of 100% cell cytotoxicity.
In a next step, the ability of Compound 1 to inhibit the growth of CTG-1520 PDX in nude mice was assessed. CTG-1520 was implanted subcutaneously in nude mice and tumors were allowed to grow to a volume of 210 mm3 followed by randomization of animals into three (3) groups (vehicle control; 75 mg/kg, and 150 mg/kg Compound 1) with 12 animals per group (see
As shown in
Anti-tumor efficacy of Compound 1 was evaluated against three (3) different preclinical oncology mice models of colorectal cancer, prostate cancer, and lung adenocarcinoma (see Table 5 below). For this purpose, an oral formulation of Compound 1 was used.
Human Colorectal Cancer Model—COLO205
A previously performed screen of 102 cancer cell lines revealed that colorectal cancer cell lines were among the cancer indications with highest percentage of response and lowest IC50 for Compound 1. One of the most responsive cell lines was COLO205, which demonstrated a maximum effect of 95% and low IC50 of 67 nM (
DU145 is a human prostate cancer cell line that responded well to Compound 1 in vitro with an IC50 response of 77 nM and maximum effect of 91% (
A549 is a human lung adenocarcinoma cell line isolated from the lung tissue of their origin. A549 cells demonstrated a biphasic response to Compound 1 in vitro with an IC50 response of 53 nM and maximum effect of 52% in first phase followed by 100% at highest concentration tested (
As shown in
As shown in
The data from the human lung adenocarcinoma (A549) study demonstrated that treatment with Compound 1 decreased the average tumor growth at all doses evaluated (
Compound 1 demonstrated anti-tumor efficacy in various pre-clinical solid tumor models including colorectal, prostate and lung.
Glioblastoma (GBM) is the most common and highly malignant form of primary brain tumor with a poor prognosis despite advances in treatments. Therapy resistance and relapse remain a significant clinical challenge necessitating the development of novel therapeutic approaches that are not limited by the blood-tumor barrier. Accordingly, the in vitro potency of Compound 1 was examined in four different glioblastoma cell lines as evaluated by Realtime-Glo™ assay (C6, and U87 cells) or ATPlite 1Step™ Perkin Elmer (performed by NTRC Oncolines; T98G and A-172 cells). An inactive compound, BRG396, was used as a negative control, and nocodazole was used as the positive control. Nocodazole is known to be a mitotic agent that induces cell death. The rat C6 glioma cell line, in particular, is an intensively studied model used to evaluate therapeutic efficacy of a variety of modalities. Therefore, the in vivo tumor activity of Compound 1 was next assessed in Sprague Dawley rats implanted with C6 rat glioma cells into the right entorhinal cortex/subiculum region (AP: −7 mm; ML: 4.5 mm, DV: 5 mm). 14 days post implantation magnet resonance imaging (MRI) was performed to assure successful implantation and animals were randomized to receive either vehicle or Compound 1 treatment (5, 10, and 20 mg/kg groups) twice daily by oral gavage for 16 days (Day 30 post implantation). An overview of the study design is shown in
The anti-cancer activity of Compound 1 in the in vitro models of GBM was assessed to be in double digit nanomolar range (ranging from 42 nM to 89 nM) potency in four different cell lines tested:C6 cells: 42 nM, U87 cells: 89 nM, T98G cells: 39 nM, A-172 cells: 48 nM (
Compound 1 demonstrated efficacy in treating glioma in an orthotopic syngeneic glioma model.
While we have described a number of embodiments, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.
The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.
This application claims priority to U.S. Provisional Patent Application No. 63/525,377 filed on Jul. 7, 2023, and U.S. Provisional Patent Application No. 63/459,173 filed on Apr. 13, 2023, the contents of each of which are incorporated herein in their entirety.
Number | Date | Country | |
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63525377 | Jul 2023 | US | |
63459173 | Apr 2023 | US |