Combinations of tegavivint with tyrosine kinase inhibitors

Abstract

Formulations of tegavivint, methods of making such formulations, and methods of treatment of cancer by administering the formulations in combination with tyrosine kinase inhibitors.

Description

[0001] Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0002] COMBINATIONS OF TEGAVIVINT WITH TYROSINE KINASE INHIBITORS

[0003] BACKGROUND

[0004] Cancer is the second leading cause of death in the United States. It presents complex challenges for the development of new therapies. Cancer is characterized by the abnormal growth of malignant cells that have undergone a series of genetic changes that lead to growth of tumor mass and metastatic properties.

[0005] Beta-catenin (P-catenin) is part of a complex of proteins that constitute adherens junctions (AJs). AJs are necessary for the creation and maintenance of epithelial cell layers by regulating cell growth and adhesion between cells. P-catenin also anchors the actin cytoskeleton and may be responsible for transmitting the contact inhibition signal that causes cells to stop dividing once the epithelial sheet is complete.

[0006] Wnt / p-catenin pathway has been shown to play a role in cancer. Aberrant P-catenin signaling plays an important role in tumorigenesis. In particular, hepatocellular carcinoma is estimated to have greater than 50% mutations in the P-catenin pathway, leading to unregulated oncogenic signaling. Aberrant P-catenin signaling has been shown to be involved in various cancer types, including but not limited to, melanoma, breast, lung, colon, liver, gastric, myeloma, multiple myeloma, chronic myelogenous leukemia, chronic lymphocytic leukemia, T-cell nonHodgkin lymphomas, colorectal and acute myeloid leukemia (AML) cancers. Further, aberrant Wnt / p-catenin signaling has been found in a large number of other disorders, including osteoporosis, osteoarthritis, polycystic kidney disease, diabetes, schizophrenia, vascular disease, cardiac disease, hyperproliferative disorders, neurodegenerative diseases, and fibrotic diseases including but not limited to idiopathic pulmonary fibrosis (IPF), Dupuytren's contracture, Nonalcoholic steatohepatitis (NASH), and others.Attorney Docket No. ITER-011 / 01 WO 40069 / 123

[0007] SUMMARY

[0008] The invention recognizes that a need still exists for methods of treating cancers that are associated with the Wnt / p-catenin pathway and aberrant P-catenin signaling, such as but not limited to hepatocellular carcinoma (HCC). Tegavivint is useful for modulating the activity of the Wnt / p-catenin signaling pathway, and serves to reduce P-catenin levels present in cells, such as cancer cells. Tegavivint and related compounds are described, for example, in U.S. Patent No.

[0009] 8,129,519. Tegavivint has the following structural formula:

[0010]

[0011] The molecular formula of tegavivint is C28H36N4O6S2.

[0012] The molecular mass of tegavivint is 588.20763 amu.

[0013] It has unexpectedly been found that combining tegavivint and tyrosine kinase inhibitors provides additive and synergistic effects with respect to treating cancers associated with the Wnt / p-catenin pathway and aberrant P-catenin signaling, such as but not limited to hepatocellular carcinoma (HCC). It was found that the combination of tegavivint and tyrosine kinase inhibitors provides additive and synergistic growth inhibitory effects directly on cancer cells indicating a novel combinatorial mechanism based on cancer cell tyrosine kinase inhibition independent of any activity of the tyrosine kinase inhibitors on VEGF activity. Moreover, it has been unexpectedly discovered that the combinatorial effectiveness of tegavivint and tyrosine kinase inhibitors is influenced by the CTNNB1 mutational status of the cell line. In that manner, theAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0014] invention provides in various embodiments combinations of tegavivint and tyrosine kinase inhibitors for the treatment of cancer, such as but not limited to hepatocellular carcinoma (HCC).

[0015] In some embodiments, the invention provides a method for treating a cancer or tumor metastasis in a mammal in need thereof, comprising administering to the mammal a combination of: an effective amount of tegavivint; and an effective amount of a tyrosine kinase inhibitor.

[0016] In some embodiments, the cancer or tumor metastasis comprises hepatocellular carcinoma (HCC).

[0017] In some embodiments, the HCC cells have wild-type CTNNBl.

[0018] In some embodiments, the HCC cells have mutations in CTNNBl or AXIN1.

[0019] In some embodiments, the mutations in CTNNBl comprise deletion of exon 3 or one or more point mutations in exon 3.

[0020] In some embodiments, the tyrosine kinase inhibitor is a therapeutic that inhibits tyrosine kinases.

[0021] In some embodiments, the tyrosine kinase inhibitor is a small molecule that inhibits the activity of a protein expressed by the cancer cell.

[0022] In some embodiments, the tyrosine kinase inhibitor is a small molecule that directly inhibits a kinase expressed by the cancer cell.

[0023] In some embodiments, the tyrosine kinase inhibitor directly inhibits one or more tyrosine kinases selected from the group consisting of PDGFR, FGFR, c-Kit, c-Met, c-RET, AXL, MER, VEGF-R1, and VEGF-R2.

[0024] In some embodiments, the tyrosine kinase inhibitor is selected from the group consisting of axitinib, nintedanib, pazopanib, vandetanib, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, gefitinib, erlotinib, sorafenib, sunitinib, afatinib, canertinib, osimertinib, acalabrutinib, regorafenib, cabozantinib, lenvatinib, and zanzalitinib.

[0025] In some embodiments, the tegavivint is in a nanosuspension prepared by a process comprising using a crystalline form of tegavivint designated as Form IV as the starting material and milling Form IV at a temperature between about 40° C and about 60° C, wherein Form IVAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0026] has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 20 angle values independently selected from the group consisting of 5.0+-0.20; 7.5+-0.20; 14.83 — 0.2°; 15.23 — 0.2°; 15.43 — 0.2°; 20.03 — 0.2°; and 22.23 — 0.2°, wherein the nanosuspension consists of Form I of tegavivint.

[0027] In some embodiments, the tegavivint is in a reconstituted formulation prepared from a lyophilized formulation comprising particles of tegavivint or a pharmaceutically acceptable salt thereof; wherein the particles have a D50 of less than or equal to 500 nm and a D90 of less than or equal to 1.0 micron when measured using laser diffraction; wherein the lyophilized formulation comprises a poloxamer and one or more stabilizers selected from the group consisting of sucrose, trehalose, and sorbitol; and wherein a starting material to produce the lyophilized formulation is Form I polymorph of tegavivint or Form IV polymorph of tegavivint.

[0028] In some embodiments, the tegavivint in the reconstituted formulation consists of Form I polymorph of tegavivint.

[0029] In some embodiments, the tegavivint is in a formulation comprising a poloxamer and sorbitol; wherein the tegavivint is in the form of a nanosuspension comprising particles of tegavivint or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; wherein the particles have a D50 of less than or equal to 500 nm and a D90 of less than or equal to 1.0 micron when measured using laser diffraction.

[0030] In some embodiments, the formulation comprises 0.25 mg / ml tegavivint, 0.625% by weight poloxamer 188, and 10% by weight sorbitol.

[0031] In some embodiments, said administering is performed through one or more of intravenous, parenteral, oral, inhalation (including aerosolized delivery), buccal, intranasal, rectal, intra-lesional intraperitoneal, intradermal, transdermal, subcutaneous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, intravitreous injection, and topical application methods.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0032] In some embodiments, the invention provides a combination therapy for treating a cancer or tumor metastasis in a patient in need thereof, comprising: an effective amount of tegavivint; and an effective amount of a tyrosine kinase inhibitor.

[0033] In some embodiments, the cancer or tumor metastasis comprises hepatocellular carcinoma (HCC).

[0034] In some embodiments, the HCC cells have wild-type CTNNB1.

[0035] In some embodiments, the HCC cells have mutations in CTNNB1 or AXIN1.

[0036] In some embodiments, the mutations in CTNNB1 comprise deletion of exon 3 or one or more point mutations in exon 3.

[0037] In some embodiments, the tyrosine kinase inhibitor is a therapeutic that inhibits tyrosine kinases.

[0038] In some embodiments, the tyrosine kinase inhibitor is a small molecule that inhibits the activity of a protein expressed by the cancer cell.

[0039] In some embodiments, the tyrosine kinase inhibitor is a small molecule that directly inhibits a kinase expressed by the cancer cell.

[0040] In some embodiments, the tyrosine kinase inhibitor directly inhibits one or more tyrosine kinases selected from the group consisting of PDGFR, FGFR, c-Kit, c-Met, c-RET, AXL, MER, VEGF-R1, and VEGF-R2.

[0041] In some embodiments, the tyrosine kinase inhibitor is selected from the group consisting of axitinib, nintedanib, pazopanib, vandetanib, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, gefitinib, erlotinib, sorafenib, sunitinib, afatinib, canertinib, osimertinib, acalabrutinib, regorafenib, cabozantinib, lenvatinib, and zanzalitinib.

[0042] In some embodiments, the tegavivint is in a nanosuspension prepared by a process comprising using a crystalline form of tegavivint designated as Form IV as the starting material and milling Form IV at a temperature between about 40° C and about 60° C, wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 20 angle values independently selected from the group consisting of 5.0+-0.20; 7.5+-O.20; 14.83 — 0.2°;Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0043] 15.23 — 0.2°; 15.4+-0.20; 20.03—0.2°; and 22.23—0.2°, wherein the nanosuspension consists of Form I of tegavivint.

[0044] In some embodiments, the tegavivint is in a reconstituted formulation prepared from a lyophilized formulation comprising particles of tegavivint or a pharmaceutically acceptable salt thereof; wherein the particles have a D50 of less than or equal to 500 nm and a D90 of less than or equal to 1.0 micron when measured using laser diffraction; wherein the lyophilized formulation comprises a poloxamer and one or more stabilizers selected from the group consisting of sucrose, trehalose, and sorbitol; and wherein a starting material to produce the lyophilized formulation is Form 1 polymorph of tegavivint or Form IV polymorph of tegavivint.

[0045] In some embodiments, the tegavivint in the reconstituted formulation consists of Form I polymorph of tegavivint.

[0046] In some embodiments, the tegavivint is in a formulation comprising a poloxamer and sorbitol; wherein the tegavivint is in the form of a nanosuspension comprising particles of tegavivint or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; wherein the particles have a D50 of less than or equal to 500 nm and a D90 of less than or equal to 1.0 micron when measured using laser diffraction.

[0047] In some embodiments, the formulation comprises 0.25 mg / ml tegavivint, 0.625% by weight poloxamer 188, and 10% by weight sorbitol.

[0048] In some embodiments, the formulation is provided in a dosage form (a) selected from the group consisting of tablets, and capsules; (b) selected from the group consisting of controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; (c) suitable for inhalation or parenteral administration; or (d) any combination of (a), (b), and (c).Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The foregoing summary, as well as the following detailed description, will be better understood when read in conjunction with the appended drawings. For the purposes of illustrating the systems and methods of the present application, there are shown in the drawings certain embodiments. It should be understood, however, that the application is not limited to the precise arrangements and instrumentalities shown.

[0051] FIG. 1 shows a synergy and antagonism contour map and matrix for tegavivint and regorafenib in HepG2 cells.

[0052] FIG. 2 shows a synergy and antagonism contour map and matrix for tegavivint and regorafenib in SNU_398 cells.

[0053] FIG. 3A and FIG. 3B show exemplary plate layouts and raw data for tegavivint and regorafenib in SNU_398 cells.

[0054] FIG. 4A shows a synergy and antagonism contour map and matrix for tegavivint and sorafenib in HepG2 cells.

[0055] FIG. 4B shows a synergy and antagonism contour map and matrix for tegavivint and regorafenib in HepG2 cells.

[0056] FIG. 4C shows a synergy and antagonism contour map and matrix for tegavivint and cabozantinib in HepG2, SNU_878, and SNU_398 cells.

[0057] FIG. 4D shows a synergy and antagonism contour map and matrix for tegavivint and lenvatinib in HepG2, HUH 7, and SNU 398 cells.

[0058] DETAILED DESCRIPTION

[0059] Definitions

[0060] The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terms that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of theAttorney Docket No. ITER-011 / 01 WO 40069 / 123

[0061] invention. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. The invention is not limited to the various embodiments given in this specification.

[0062] 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 pertains. In the case of conflict, the present document, including definitions will control.

[0063] The term “tegavivinf ’ refers to a compound having the following structure:

[0064]

[0065] The term “BC-2059” is used interchangeably with “tegavivint.”

[0066] The term “long-term storage” or “long-term stability” is understood to mean that the pharmaceutical composition can be stored for three months or more, for six months or more, twelve months or more, and eighteen months or more. Long term storage is also understood to mean that the pharmaceutical composition is stored at 2-8° C or at room temperature 15-25° C, or at any temperature between 2-8° C and 15-25° C.

[0067] The term “stable” or “stabilized” with respect to long-term storage is understood to mean that active ingredient contained in the pharmaceutical compositions does not lose more than 20%, or more preferably 15%, or even more preferably 10%, and most preferably 5% of its activity relative to activity of the composition at the beginning of storage. Furthermore, for theAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0068] purposes of this invention, the long-term physical stability of lyophilized formulation includes maintenance of good cake integrity, ability to readily re-suspend upon reconstitution with the respective diluent, and the absence of any significant crystal growth upon storage that can be determined by optical microscopy.

[0069] The term “mammal” includes, but is not limited to, a human.

[0070] The term “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material, formulation auxiliary, or excipient of any conventional type. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.

[0071] The term “treatment” refers to any administration or application of remedies for disease in a mammal and includes inhibiting the disease, arresting its development, relieving the disease (for example, by causing regression, or restoring or repairing a lost, missing, or defective function) or stimulating an inefficient process. The term includes obtaining a desired pharmacologic and / or physiologic effect and covering any treatment of a pathological condition or disorder in a mammal. The effect may be prophylactic in terms of completely or partially preventing a disorder or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disorder and / or adverse effect attributable to the disorder. It includes (1) preventing the disorder from occurring or recurring in a subject who may be predisposed to the disorder but is not yet symptomatic, (2) inhibiting the disorder, such as arresting its development, (3) stopping or terminating the disorder or at least its associated symptoms, so that the host no longer suffers from the disorder or its symptoms, such as causing regression of the disorder or its symptoms, for example, by restoring or repairing a lost, missing or defective function, or stimulating an inefficient process, or (4) relieving, alleviating or ameliorating the disorder, or symptoms associated therewith, where ameliorating is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, such as inflammation, pain and / or tumor size.

[0072] The term “therapeutically effective amount” refers to an amount which, when administered to a living subject, achieves a desired effect on the living subject. For example, anAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0073] effective amount of the compositions of the invention for administration to the living subject is an amount that prevents and / or treats any of the diseases mediated via the Wnt / p-catenin pathway. The exact amount will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.

[0074] The term “composition” or “formulation” refers to a mixture that usually contains a carrier, such as a pharmaceutically acceptable carrier or excipient that is conventional in the art and which is suitable for administration into a subject for therapeutic, diagnostic, or prophylactic purposes. For example, compositions for oral administration can form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses or powders. The terms “composition,” “pharmaceutical composition” and “formulation” are used interchangeably.

[0075] The term “nanoparticulate composition” refers to compositions wherein all, or almost all of the particles are less than 1000 nm.

[0076] The term “lyophilized formulation” refers to a formulation resulted from freeze-drying of an aqueous solution.

[0077] The term “pre-lyophilized formulation” refers to a formulation of the invention before any lyophilization takes place. The term encompasses formulations that will not undergo any lyophilization at all.

[0078] The term a “reconstituted formulation” refers to a formulation resulted from adding water (for example, sterile water) or an aqueous solvent to a solid composition in an amount to dissolve the composition. In one embodiment, the solid composition is a lyophilized formulation.

[0079] The term an “injectable formulation” refers to a formulation that is suitable for parenteral administration, e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal administration.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0080] The term “stable for therapeutic utility” refers to a tegavivint formulation that over a period of at least 6 months and preferably at least 18 months remains suitable for treatment of conditions treatable with tegavivint.

[0081] The term, “AUCiast,” as used herein, refers to the area under the curve from the time of dosing to the time of the last measurable (positive) concentration.

[0082] The term, “Cmax,” as used herein, refers to the maximum concentration that a drug achieves in a specified tissue after the drug has been administered and before the administration of a second dose.

[0083] The term, “Tmax,” as used herein, refers to the time after administration of a drug when the maximum plasma concentration is reached.

[0084] Formulations of Tegavivint

[0085] It has been very challenging and difficult to develop a stable formulation of tegavivint. A large number of formulations were developed and tested; however, they had poor bioavailability and / or proved unstable upon storage, and / or turned to be highly toxic. It was challenging and difficult to obtain formulations that retained good cake integrity, re-suspended readily and demonstrated chemical stability over time.

[0086] The inventors have unexpectedly and surprisingly discovered that a stable formulation can be made when it is produced utilizing Form I or Form IV polymorphs of tegavivint as starting material, when the starting material is subjected to high energy agitator milling at an elevated temperature of at least 40° C-60° C, and preferably at about 60° C. Form I and Form IV polymorphs are described in detail in U.S. Patent No. 11,136,307, the contents of which are hereby incorporated by reference in their entirety.

[0087] In one embodiment, the invention provides a nanosuspension of tegavivint wherein the nanosuspension was prepared by a process comprising using either Form I or Form IV as the starting material and high energy agitator milling performed at a temperature of between about 40° C and about 60° C, most preferably at about 60° C.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0088] In one embodiment, the stable formulation can be produced starting with any of the polymorph forms of tegavivint (e.g., any of Form I, Form II, Form III or Form IV), as long as at some point within the production process, Form I and / or Form IV polymorphs are produced.

[0089] In one embodiment, if the milling process is done at temperature of less than about 60° C, the nanosuspension further undergoes the annealing process at or above 60° C.

[0090] Both polymorphic forms, Form I and Form IV, were utilized as starting materials for the controlled temperature high energy milling process. The milling used the concentration of tegavivint of 200 mg / ml and concentration of the stabilizer of 5% Poloxamer 188. Microscopic imaging and DSC post milling analysis found that batches utilizing Form 1 as the starting material contained only Form I at the end product of the milling process. In contrast, the conversion of Form IV to Form I was seen in batches that utilized Form IV as the starting material. The milling of Form I at 60° C or higher was hypothesized to prevent formation crystal seeds for the undesirable Form IV and result in a highly crystalline milled Form I material that is annealed and free of high energy particles and free of amorphous material. This finding was confirmed by the invention.

[0091] The end product obtained from milling at 60° C using either Form I or Form IV as starting material was a nanosuspension of Form I, since Form IV was converted to Form I during milling over 120 min.

[0092] Consequently, in one embodiment, the invention allows to utilize either Form I or Form IV as the starting material for milling at an elevated temperature (40-60° C, preferably 60° C) to obtain the final desired form (Form I). However, the advantage of using Form IV as a starting material for milling at 60° C is that the system undergoes a full solvent mediated recrystallization from Form IV to Form I. The crystals for Form I grow “bottom-up” as they are milled, so the chance of getting any un-milled larger crystals is significantly diminished, thereby enhancing the quality of the suspension.

[0093] Indeed, the nanosuspension from milling Form IV at 60° C was found to be a uniform, well-dispersed Form I with a narrow unimodal particulate size distribution. Upon stabilityAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0094] analysis at 5° C and ambient laboratory conditions, there were no significant changes in particle size distribution. The microscopic images confirmed absence of any crystal growth for all final Form I formulations at the end of 3 months of storage.

[0095] In one embodiment, the milling is performed at a temperature of between about 40° C and about 60° C, most preferably at about 60° C. Preferably, if the milling process is done at temperature of less than about 60° C, the nanosuspension further undergoes the annealing process at or above 60° C.

[0096] Nanosuspension from the batch that utilized Form I as the starting material with Poloxamer 188 as the dispersant milled at elevated temperatures (60° C) was then taken forward for the formulation optimization. Additionally, four batches, prepared at the original test scale, were milled and composited to provide the material for the formulation optimization study.

[0097] In one embodiment, the formulations of the invention, including but not limited to prelyophilized formulations, are filtered to remove any large particles. In one embodiment, the filter is 10 micron.

[0098] In one embodiment, the invention provides a stable lyophilized formulation comprising particles of tegavivint or a pharmaceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof; wherein the particles have an effective D50 of less than or equal to 500 nm and D90 of less than or equal to 1.0 micrometer (pm) when measured using laser diffraction.

[0099] In one embodiment, the particles have an effective D90 of about 100 nm when measured using laser diffraction.

[0100] In one embodiment, the invention provides a formulation comprising particles of Form I or Form IV polymorph of tegavivint or a pharmaceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof; wherein the particles have an effective D90 of less than or equal to 0.2 micron when measured using laser diffraction, and wherein the formulation was prepared by high energy agitator milling at a temperature of between about 40° C and about 60° C.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0101] In one embodiment, the formulation may be lyophilized.

[0102] In one embodiment, the particles have an effective D90 of less than or equal to 0.2 micron when measured using laser diffraction.

[0103] In one embodiment, the particles have an effective D50 of less than or equal to 0.12 micron when measured using laser diffraction.

[0104] In one embodiment, the particles have an effective D10 of less than or equal to 0.1 micron when measured using laser diffraction.

[0105] D50 is also known as median diameter of particle size distribution. It refers to the value of the particle diameter at 50% in the cumulative distribution. In other words, when D50 value is less than or equal to 500 nm, it means that 50% of the particles are less than 500 nm in diameter.

[0106] D90 refers to the percentage of the particles under the reported particle size. In other words, when D90 value is less than or equal to 1.0 pm, it means that 90% of the particles are less than 1.0 pm in diameter.

[0107] In some embodiments, the effective average particle size of tegavivint is about 4900 nm, about 4800 nm, about 4700 nm, about 4600 nm, about 4500 nm, about 4400 nm, about 4300 mm, about 4200 nm, about 4100 nm, about 4 microns, about 3900 nm, about 3800 nm, about 3700 nm, about 3600 nm, about 3500 nm, about 3400 mm, about 3300 nm, about 3200 nm, about 3100 nm, about 3 microns, about 2900 mm, about 2800 nm, about 2700 nm, about 2600 nm, about 2500 nm, about 2400 nm, about 2300 nm, about 2200 nm, about 2100 nm, about 2000 nm, about 1900 nm, about 1800 nm, about 1700 nm, about 1600 nm, about 1500 nm, about 1400 nm, about 1300 nm, about 1200 nm, about 1100 nm, about 1000 nm, about 900 nm, about 800 nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, or about 300 nm.

[0108] Further, in some embodiments, the effective average particle size of the compounds is less than 900 nm, more preferably less than 500 nm, and even more preferably, less than 300 nm.

[0109] The provided formulations are stable for therapeutic utility.

[0110] In one embodiment, the lyophilized formulations of the invention are anhydrous.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0111] In one embodiment, the formulations, including lyophilized formulations, of the invention are stored in a dry atmosphere.

[0112] The storage temperature for the formulations of the invention can be about -20° C, about 5° C, or about 25° C.

[0113] In one embodiment, the invention provides a stable formulation comprising particles of tegavivint; wherein the particles have an effective D50 of less than or equal to 500 nm and D90 of less than or equal to 1.0 micrometer (pm) when measured using laser diffraction, wherein the formulation comprises tegavivint, a poloxamer, and one or more stabilizer selected from the group consisting of sucrose, trehalose and sorbitol, and wherein the formulation is prepared by high energy agitator milling at a temperature of between about 40° C and about 60° C.

[0114] In one embodiment, tegavivint concentration in a formulation is 2% (20 mg / mL).

[0115] In one embodiment, tegavivint concentration in a formulation is 2.5% (25 mg / mL).

[0116] In another embodiment, tegavivint concentration in a formulation is 5% (50 mg / mL). In one embodiment, the poloxamer is Poloxamer 188.

[0117] In one embodiment, the poloxamer concentration in a formulation is 6 mg / mL.

[0118] In another embodiment, the poloxamer concentration in a formulation is 12.5 mg / mL. In one embodiment, the sucrose concentration in a formulation is 100 mg / mL.

[0119] In another embodiment, the trehalose concentration in a formulation is 100 mg / mL. In one embodiment, the sorbitol concentration in a formulation is 50 mg / mL.

[0120] In one embodiment, the invention provides a stable lyophilized formulation comprising particles of tegavivint; wherein the particles have an effective D50 of less than or equal to 500 nm and D90 of less than or equal to 1.0 micrometer (pm) when measured using laser diffraction, wherein the formulation is a product of lyophilization of a pre-lyophilized formulation comprising tegavivint, a poloxamer, and one or more stabilizer selected from the group consisting of sucrose, trehalose and sorbitol.

[0121] In one embodiment, tegavivint concentration in a pre-lyophilized formulation is 2% (20 mg / mL).Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0122] In one embodiment, tegavivint concentration in a pre-lyophilized formulation is 2.5% (25 mg / mL).

[0123] In another embodiment, tegavivint concentration in a pre-lyophilized formulation is 5% (50 mg / mL).

[0124] In one embodiment, the poloxamer is Poloxamer 188.

[0125] In one embodiment, the poloxamer concentration in a pre-lyophilized formulation is 0.6%.

[0126] In another embodiment, the poloxamer concentration in a pre-lyophilized formulation is 1.25%.

[0127] In one embodiment, the sucrose concentration in a pre-lyophilized formulation is 10%. In another embodiment, the trehalose concentration in a pre-lyophilized formulation is 10%.

[0128] In one embodiment, the sorbitol concentration in a pre-lyophilized formulation is 5%. In one embodiment, the formulation is autoclaved prior to lyophilization.

[0129] In one embodiment, the lyophilization process comprises freezing the formulation to about -40° C, a primary drying step at -30° C, and a secondary drying step at about -10° C.

[0130] The invention also includes pre-lyophilized formulations of tegavivint.

[0131] In one embodiment, the pre-lyophilized formulation is prepared by a process comprising ball milling at a temperature of about 60° C.

[0132] In one embodiment, the pre-lyophilized formulation is prepared by a process comprising high energy milling at a temperature of about 60° C.

[0133] In one embodiment, the tegavivint in the pre-lyophilized formulation is Form I polymorph.

[0134] In one embodiment, the formulations of the invention are stable for three months, six months, twelve months or eighteen months at storage at a temperature of between 5° C and 25° C.

[0135] In a preferred embodiment, the formulations of the invention exhibit long term stability.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0136] In one embodiment, the formulations of the invention, including but not limited to lyophilized formulations of the invention, when reconstituted, may be formulated: (a) into a dosage form selected from the group consisting of tablets, and capsules; (b) into a dosage form selected from the group consisting of controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; (c) into a dosage form suitable for inhalation or parenteral administration, including intramuscular, subcutaneous, intravenous and intradermal injection; (d) any combination of (a), (b) and (c).

[0137] In one embodiment, the formulations of the invention, including but not limited to the lyophilized formulations of the invention, when administered to rats via intravenous (IV) infusion at 15.5 mg / mL concentration, result in one or more of the following values: Cmax of at least about 50,000 ng / mL in plasma; at least about 200 ng / g in brain; at least about 2900 ng / g in heart; at least about 3700 ng / g in kidney; at least about 35000 ng / g in lungs; at least about 400 ng / g in pectoral thigh muscle; at least about 360000 ng / g in spleen; at least about 470 ng / g in visceral fat; and at least about 237,000 ng / g in liver.

[0138] In another embodiment, the formulations of the invention, including but not limited to the lyophilized formulations of the invention, when administered to rats via IV infusion at 15.5 mg / mL concentration, result in one or more of the following values: AUCiast of at least about 68,000 hr*ng / mL in plasma; at least about 1800 hr*ng / g in brain; at least about 45000 hr*ng / g in heart; at least about 58000 hr*ng / g in kidney; at least about 450000 hr*ng / g in lungs; at least about 5700 hr*ng / g in pectoral thigh muscle; at least about 4800000 hr*ng / g in spleen; at least about 4000 hr*ng / g in visceral fat; and at least about 3,600,000 hr*ng / g in liver.

[0139] In another embodiment, the formulations of the invention, including but not limited to the lyophilized formulations of the invention, when administered to rats via IV infusion at 15.5 mg / mL concentration, result in one or more of the following values: Tmax of about 0.08 hr or less in plasma; about 0.5 hr or less in brain; about 1 hr or less in heart; about 0.5 hr or less in kidney;Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0140] about 0.5 hr or less in lungs; about 0.5 hr or less in pectoral thigh muscle; about 0.5 hr or less in spleen; about 1 hr or less in visceral fat; and about 1 hr or less in liver.

[0141] The pharmaceutical formulations of the invention can further comprise one or more pharmaceutically acceptable excipients, carriers, or a combination thereof.

[0142] In another embodiment, the invention provides a method of preventing, treating or ameliorating cancer or tumor metastasis in a mammal in need thereof comprising administering to said mammal an effective amount of the formulations of the invention.

[0143] The method of administering is not limited to any specific route of administration, and includes, but is not limited to, intravenous, parenteral, oral, inhalation (including aerosolized delivery), buccal, intranasal, rectal, intra-lesional intraperitoneal, intradermal, transdermal, subcutaneous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, intravitreous injection, and topical application methods.

[0144] The total daily dose of the formulations of the invention administered to a human or lower animal may range from about 0.0001 to about 1000 mg / kg / day. In some embodiments, the dosage ranges from about 0.001 to about 100 mg / kg, or from about 0.05 to about 50 mg / kg, of the subject's body weight. In some embodiments, the dosage is within the range of 0.5-50 mg / kg body weight. If desired, the effective daily dose can be divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.

[0145] In another embodiment, the method of preventing, treating or ameliorating cancer or tumor metastasis in a mammal in need thereof can include administering an additional anticancer agent and / or cancer therapy (for example, cancer vaccines, anti-cancer adoptive cell therapies and radio therapies).

[0146] In one embodiment, the additional anti-cancer agent is selected from the group consisting of antimitotic agents, antimetabolite agents, HDAC inhibitors, proteosome inhibitors, immunotherapeutic agents, FLT-3, EGFR, MEK, PI3K and other protein kinase inhibitors, epigenetic targeted inhibitors, and WNT pathway inhibitors, alkylating agents and DNA repairAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0147] pathway inhibitors, anti-hormonal agents, anti-cancer antibodies, and other cytotoxic chemotherapy agents.

[0148] In some embodiments, the additional anti-cancer agent is a kinase inhibitor. In some embodiments, the kinase inhibitor is a tyrosine kinase inhibitor (TKI). TKIs that may be used in combination therapies according to embodiments of the invention can be any therapeutic that inhibits tyrosine kinases, including, but not limited to, antisense, PROTAC (proteolysis targeting chimera), and peptide therapeutics (currently known or developed in the future) as well as monoclonal antibodies and small molecules, and can target oncogenic receptor tyrosine kinase (RTK) and optionally additional protein kinases. TKIs that may be used in combination therapies according to embodiments of the invention include small molecule vascular endothelial growth factor receptor (VEGFR) TKIs. In some embodiments, the RTK is expressed in the cancer cell and directly affects the cancer cell growth. In some embodiments, the TKI is a small molecule that directly inhibits a kinase expressed by the cancer cell. In some embodiments, the TKI directly inhibits one or more tyrosine kinases selected from the group consisting of PDGFR, FGFR, c-Kit, c-Met, c-RET, AXL, MER, VEGF-R1, and VEGFR2. Specific TKIs that may be used in combination therapies according to embodiments of the invention include, but are not limited to, axitinib, nintedanib, pazopanib, vandetanib, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, gefitinib, erlotinib, sorafenib, sunitinib, afatinib, canertinib, osimertinib, acalabrutinib, regorafenib, cabozantinib, lenvatinib, and zanzalitinib.

[0149] The TKIs may be administered through one or more of intravenous, parenteral, oral, inhalation (including aerosolized delivery), buccal, intranasal, rectal, intra-lesional intraperitoneal, intradermal, transdermal, subcutaneous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, intravitreous injection, and topical application methods.

[0150] For administration of TKIs, the dosage ranges from about 0.0001 to about 100 mg / kg. In some embodiments, the dosage ranges from about 0.001 to about 20 mg / kg, or from about 0.01 to about 10 mg / kg, of the subject's body weight. In some embodiments, the dosage is within theAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0151] range of 0.1-10 mg / kg body weight. If desired, the effective daily dose can be divided into multiple doses for purposes of administration; consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose.

[0152] When used in combination therapies according to embodiments of the present invention, tegavivint and TKIs can be administered separately or together, sequentially (in either order) or simultaneously, by the same method or by different methods of administration. Administration may be, for example, daily, every other day, once or twice per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every three months or once every three to six months. Tegavivint and TKIs can be administered at the same frequency or at different frequencies. The dosage and scheduling of each may change during a course of treatment. Treatment may continue until complete response or confirmed progressive disease.

[0153] The invention encompasses formulations including tegavivint and a pharmaceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof.

[0154] In one embodiment, the compositions of the invention, when reconstituted, may be formulated: (a) into a dosage form selected from the group consisting of tablets, and capsules; (b) into a dosage form selected from the group consisting of controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; (c) into a dosage form suitable for inhalation or parenteral administration, including intramuscular, subcutaneous, intravenous and intradermal injection; (d) any combination of (a), (b) and (c).

[0155] The compositions of the invention can further comprise one or more pharmaceutically acceptable excipients, carriers, or a combination thereof.

[0156] The pharmaceutically acceptable excipients used in the formulation of the present invention can act in more than one way.

[0157] The pharmaceutically acceptable excipients can be, for example, a dispersion medium, a dispersion emulsifier, a dispersion enhancer, or a combination thereof.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0158] Examples of the propellant include, but not limited to, HFA-134a (1, 1, 1, 2-tetrafluoroethane), HFA-227 (1,1,1,2,3,3,3-heptafluoropropane), a combination thereof, etc.

[0159] The dispersion medium can be, for example, ethanol, propylene glycol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, glycerin, a combination thereof, etc.

[0160] The dispersion emulsifier (enhancer) can be, for example, water, oleic acid, sodium lauryl sulfate, polyethylene glycol 1000, ammonium alginate, potassium alginate, calcium stearate, glyceryl monooleate, polyoxyethylene stearates, emulsifying wax, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, poloxamer, a combination thereof, etc.

[0161] Examples of the dispersion enhancers include, but not limited to, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, carboxymethylcellulose sodium, hypromellose, ethylene glycol stearates, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, glyceryl monostearate, lecithin, meglumine, poloxamer, polyoxyethylene alkyl ethers, polyoxyl 35 castor oil, polyoxyethylene stearates, poly oxylglyceri des, pyrrolidone, sorbitan esters, stearic acid, vitamin E polyethylene glycol succinate, polyethylene glycol 1000, povidone, a combination thereof, etc.

[0162] The compositions of the invention can be suitable for all routes of administration, including but not limited to, intravenous, parenteral, oral, inhalation (including aerosolized delivery), buccal, intranasal, rectal, intra-lesional intraperitoneal, intradermal, transdermal, subcutaneous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, intravitreous injection, and topical application methods.

[0163] Pharmaceutical compositions according to the invention may also comprise one or more binding agents, filling agents, lubricating agents, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0164] Examples of filling agents are lactose monohydrate, lactose anhydrous, and various starches; examples of binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PHI 01 and Avicel® PHI 02, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™).

[0165] Suitable lubricants, including agents that act on the flowability of the powder to be compressed, are colloidal silicon dioxide, such as Aerosil® 200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.

[0166] Examples of sweeteners are any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame. Examples of flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like.

[0167] Examples of preservatives are potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quarternary compounds such as benzalkonium chloride.

[0168] Suitable diluents include pharmaceutically acceptable inert fdlers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and / or mixtures of any of the foregoing. Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose such as lactose monohydrate, lactose anhydrous, and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0169] Suitable disintegrants include lightly crosslinked polyvinyl pyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, cross-povidone, sodium starch glycolate, and mixtures thereof.

[0170] Examples of effervescent agents are effervescent couples such as an organic acid and a carbonate or bicarbonate. Suitable organic acids include, for example, citric, tartaric, malic, fumaric, adipic, succinic, and alginic acids and anhydrides and acid salts.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0171] Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate. Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0172] The present invention is more particularly described in the following examples that are intended as illustrative only, since many modifications and variations therein will be apparent to those skilled in the art. In the following examples it should be understood that weight percentages of various ingredients are expressed as w / v percentages.

[0173] Tegavivint in Hepatocellular Carcinoma

[0174] Primary liver cancer was the sixth most commonly diagnosed cancer and the third leading cause of cancer deaths globally in 2020 (Rumgay 2022 Journal of Hepatology 7(66): 1598-1606). Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. HCC occurs most often in people with underlying liver diseases, such as cirrhosis caused by hepatitis B or hepatitis C infection. Other risk factors for developing HCC include alcoholic liver disease, non-alcoholic steatohepatitis, intake of aflatoxin-contaminated food, diabetes, and obesity.

[0175] Clinical signs and symptoms of hepatic cirrhosis, which is often present in patients with HCC, usually mask the presence of an underlying early HCC. Symptoms and signs of cirrhosis are often the only expression of the disease. Because of this, patients affected by HCC usually present at an advanced stage of the disease.

[0176] The efficacy of cytotoxic chemotherapy is modest in patients with HCC, and in general, the duration of benefit is limited. In addition, HCC is radiotherapy-resistant and treatment with systemic radiotherapy plays only a minor role in HCC cases. The only proven potentially curative therapy for HCC remains surgical, either hepatic resection or liver transplantation. Major breakthroughs have been made over the past few years in the management of HCC, especially in medical therapies for advanced disease (such as the combination of atezolizumabAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0177] and bevacizumab). However, despite these achievements HCC remains a deadly disease with poor prognosis in patients with unresectable cancer.

[0178] Up to 40% of HCC patients have tumors driven by activating P-catenin and canonical Wnt pathway mutations. Most mutations in P-catenin occur within exon 3, the critical region responsible for its proteasomal degradation. These exon 3 mutations render P-catenin resistant to degradation, resulting in nuclear accumulation dysregulated transcriptional activity. Activated P-catenin / canonical Wnt signaling is associated with primary resistance to standard of care immune checkpoint blockade mediated through immune cell excluded ‘cold’ tumor microenvironment. Recruitment of P-catenin to canonical-Wnt target genes requires transducin beta-like protein 1 (TBL1) to activate transcription.

[0179] TBL1 has been demonstrated to be necessary for P-catenin oncogenic activity. During Wnt-signaling, TBL1 binds to P-catenin, inhibiting its degradation, and forms an active transcriptional complex that binds to promoters to activate downstream cancer-related genes such c-MYC, Cyclin DI and others. Mutations and translocations of TBL1 have also been observed in multiple types of cancers. High expression of TBL1 is associated with poor prognosis and unfavorable characteristics in HCC.

[0180] Tegavivint is a first-in-class small molecule inhibitor of TBL1, a novel downstream Wnt-signaling pathway target. Tegavivint binds to TBL1 in the P-catenin pocket, disrupting the formation of the activation complex necessary for oncogenic activity. Tegavivint also enables the degradation of free nuclear P-catenin. Increased expression of P-catenin and TBL1 are associated with metastasis and poor prognosis in a broad range of cancers. Importantly, tegavivint’ s targeting of TBL1 does not affect membrane-bound and cytoplasmic P-catenin pools necessary for normal cellular function, avoiding toxicities commonly associated with other inhibitors of the Wnt pathway. The safety, pharmacodynamics, pharmacokinetics, and clinical activity of tegavivint was demonstrated through a proof-of-concept study in desmoid patients with activating P-catenin mutations and is currently being studied through Investigator Initiated TrialsAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0181] (IITs) in acute myeloid leukemia (AML), pediatric solid tumors, non-small cell lung cancer (NSCLC), and lymphoma.

[0182] Tegavivint and Tyrosine Kinase Inhibitors (TKIs)

[0183] Example 1:

[0184] The effect of tegavivint in combination with regorafenib in HepG2 hepatocellular carcinoma cells following 72h of treatment

[0185] In this Example, the growth inhibitory effect of tegavivint (BC-2059) was evaluated alone and in combination with regorafenib (BAY-734506) on the CTNNB1 exon 3 deleted hepatocellular carcinoma cell line, HepG2.

[0186] In summary, synergy in cell growth inhibition was observed when 12.5 pM BAY-734506 was combined with BC-2059 at concentrations ranging from 31 nM - 500nM for 72 hours. Slight antagonism was observed when 3.125 or 6.25 pM BAY-734506 was combined with BC-2059 at 31, 62, and 125 nM. A tendency toward synergy was generally when 1.56 pM - 25 pM BAY-734506 was combined with 250, 500, or 1000 nM BC-2059.

[0187] Materials: Table 1 lists the materials used in this Example.

[0188] Table 1

[0189]

[0190] Methods: The exemplary protocol used in this Example is as follows.

[0191] 1. Seed 25 x 103HepG2 cells in a 96-well plate in 80 pL media. Incubate at 37°C overnight. 2. Prepare serial dilutions of BC-2059 and BAY-734506 in 96-well plate as outlined below.

[0192] BC-2059

[0193] 1. Prepare a 3X serial dilutionAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0194] (a) In Al, add 120 pL of 3 tnM BC-2059 stock solution

[0195] (b) In A2-A9 add 80 pL of DMSO

[0196] (c) Transfer 40 / L from Al to A2, mix and then transfer 40 pL into A3, continue this down to A9

[0197] (d) Add 120 pL of 100% DMSO in A10

[0198] 2. Create a 5x dilution into Row B

[0199] (a) Add 160 pL of RPMI into Row B, transfer 40 pL from each well in Row A into Row B

[0200] (b) Mix Row B 5 times

[0201] 3. Make 1200 |TL of a 1 OX dilution

[0202] (a) Add 1080 / L of media into a 1.5 mb tube; transfer 120 |TL from each well of Row B into the tube

[0203] (b) Mix contents in tube 5 times

[0204] BAY-734506

[0205] 1. Prepare a 2X serial dilution

[0206] (a) In Al, add 120 pL of 20 mM regorafenib stock solution

[0207] (b) In A2-A9 add 80 pL of DMSO

[0208] (c) Transfer 40 pL from Al to A2, mix and then transfer 40 pL into A3, continue this down to A9

[0209] (d) Add 120 pL of 100% DMSO in A10

[0210] 2. Create a 5x dilution into Row B

[0211] (a) Add 160 pL of RPMI into Row B, transfer 40 pL from each well in Row A into Row B

[0212] (b) Mix Row B 5 times

[0213] 3. Make 1200 pL of a 1 Ox dilution

[0214] (a) Add 1080 pL of media into a 1.5mL tube; transfer 120 pL from each well of Row B into the tubeAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0215] (b) Mix contents in tube 5 times

[0216] 3. Treat cells with 5 |1L of 20x compound for a final lx dilution dose of each compound.

[0217] Incubate at 37C for 72h.

[0218] 4. Allow CTG reagent and plate to equilibrate to room temperature for 10-15 minutes.

[0219] 5. Add 50 p.L CTG reagent to each well.

[0220] 6. Nutate plates for 2 minutes to induce cell lysis.

[0221] 7. Incubate plate at room temperature for 15 minutes to stabilize luminescent signal.

[0222] 8. Measure luminescence using Spectromax M5 machine.

[0223] 9. Record luminescence signal as an indicator of cell viability.

[0224] Table 2 shows an exemplary plate layout and raw data for this Example.

[0225] Table 2

[0226]

[0227]

[0228] Data Analysis: Combenefit software (v2.021) was used to evaluate the effects of BC-2059 and BAY-734506 in combination. Dose-response curves for each agent alone and in combination were generated based on concentration gradients to determine IC50 values. Combenefit was used to calculate synergy scores across a range of doses, using the Highest Single Agent (HSA) model. The synergy scores indicate the degree of synergism, additivity, or antagonism between compounds. Results were visualized as heatmaps, providing a comprehensive view of the interaction effects across different dose combinations, allowing for a quantitative assessment of potential therapeutic benefits. A heatmap of the synergy scores as well as a graph of the synergy score graphed to the dose response curves is shown in FIG. 1.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0229] Results: Synergy between BC-2059 (tegavivint) and BAY-734506 (regorafenib) in HepG2 cell growth inhibition was observed for concentrations between 1,560 nM and 25,000 nM BAY-734506 when combined with BC-2059 at concentrations ranging from 31 nM - 500 nM for 72hrs. At low concentrations of BC-2059 where minimal growth inhibition was observed slight antagonism was observed when 3.125 or 6.25 pM BAY-734506 was combined with BC-2059 at concentrations up to 125 nM. A tendency toward synergy was generally observed when 1.56 pM - 25 pM BAY-734506 was combined with 250, 500, or 1000 nM BC-2059.

[0230] Example 2:

[0231] The effect of tegavivint in combination with regorafenib in SNU-398 hepatocellular carcinoma cells following 72h of treatment

[0232] In this Example, the growth inhibitory effect of tegavivint (BC-2059) was evaluated alone and in combination with regorafenib (BAY-734506) on the hepatocellular carcinoma cell line SNU_398, which possesses a point mutation on Serine 37 of the beta-catenin gene (CTNNB1 S37C).

[0233] In summary, synergy in cell growth inhibition was observed when SNU_398 cells were treated with BAY-734506 at concentrations ranging from 205.76 - 617.28 nM in combination with BC-2059 at concentrations ranging from 0.457 nM - 37.04 nM. BC-2059 at 37.04 nM in combination withl6.6 pM BAY-734506 also demonstrated synergy in growth inhibition at 72 hours. Slight antagonism was observed when 68.59 nM BAY-734506 was combined with BC-2059 at concentrations ranging from 0.4 nM - 111 nM BC-2059 and when 111 nM BC-2059 was combined with 68.58 nM - 1.85 pM BAY-734506.

[0234] Materials: Table 3 lists the materials used in this Example.Attorney Docket No. ITER-011 / 01 WO 40069 / 123

[0235] Table 3

[0236]

[0237] Methods: The exemplary protocol used in this Example is as follows.

[0238] Seed 25 x 103SNU_398 cells in a 384-well plate in 45 pL media. Incubate at 37°C overnight. Prepare serial dilutions of BC-2059 and BAY-734506 in 96-well plate as outlined below.

[0239] BC-2059

[0240] 1. Prepare a 3X serial dilution

[0241] (a) In Al, add 60 pL of 3 mM BC-2059 stock solution

[0242] (b) In A2-A9 add 40 pL of DMSO

[0243] (c) Transfer 20 pL from Al to A2, mix and then transfer 20 pL into A3, continue this down to A9

[0244] (d) Add 60 pL of 100% DMSO in A10

[0245] 2. Create a 5x dilution into Row B

[0246] (a) Add 80 pL of RPMI into Row B, transfer 20 pL from each well in Row A into Row B

[0247] (b) Mix Row B 5 times

[0248] 3. Make 600 pL of a 1 Ox dilution

[0249] (a) Add 540 pL of media into a 1.5 m tube; transfer 60 pL from each well of Row B into the tube

[0250] (b) Mix contents in tube 5 timesAtorney Docket No. ITER-011 / 01 WO 40069 / 123

[0251] BAY-734506

[0252] 1. Prepare a 2X serial dilution

[0253] (a) In Al, add 60 pL of 20 mM regorafenib stock solution

[0254] (b) In A2-A9 add 40 pL of DMSO

[0255] (c) Transfer 20 pL from Al to A2, mix and then transfer 20 pL into A3, continue this down to A9

[0256] (d) Add 60 pL of 100% DMSO in A10

[0257] 2. Create a 5x dilution into Row B

[0258] (a) Add 80 pL of RPM1 into Row B, transfer 20 pL from each well in Row A into Row B

[0259] (b) Mix Row B 5 times

[0260] 3. Make 600 pL of a 1 Ox dilution

[0261] (a) Add 540 pL of media into a 1.5 mL tube; transfer 60 pL from each well of Row B into the tube

[0262] (b) Mix contents in tube 5 times

[0263] 3. Treat cells with 2.5 pL of 20x compound for a final lx dilution dose of each compound.

[0264] Incubate at 37C for 72h.

[0265] 4. Allow CTG reagent and plate to equilibrate to room temperature for 10-15 minutes.

[0266] 5. Add 50 pL CTG reagent to each well.

[0267] 6. Nutate plates for 2 minutes to induce cell lysis.

[0268] 7. Incubate plate at room temperature for 15 minutes to stabilize luminescent signal.

[0269] 8. Measure luminescence using Promega GLOMax machine.

[0270] 9. Record luminescence signal as an indicator of cell viability.

[0271] Data Analysis: Combenefit software (v2.021) was used to evaluate the effects of BC-2059 and BAY-734506 in combination. Dose-response curves for each agent alone and in combination were generated based on concentration gradients to determine IC50 values. Combenefit was used to calculate synergy scores across a range of doses, based on the Bliss reference models including Bliss.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0272] Calculated synergy scores indicate the degree of synergism, additivity, or antagonism between compounds. Results were visualized as heatmaps, providing a comprehensive view of the interaction effects across different dose combinations, allowing for a quantitative assessment of potential therapeutic benefits. The resulting heatmap and matrix of synergy scores is shown in FIG. 2. Plate layout and raw data are shown in FIGS. 3A and 3B.

[0273] Results: Synergy in cell growth inhibition was observed when SNU_398 cells were treated with BAY-734506 at concentrations ranging from 205.76 - 617.28 nM in combination with BC-2059 at concentrations ranging from 0.457 nM - 37.04 nM. BC-2059 at 37.04 nM in combination withl6.6 pM BAY-734506 also demonstrated synergy in growth inhibition at 72 hours. Slight antagonism was observed when 68.59 nM BAY-734506 was combined with BC-2059 at concentrations ranging from 0.4 nM - 111 nM BC-2059 and when 111 nM BC-2059 was combined with 68.58 nM - 1.85 pM BAY-734506.

[0274] Example 3 :

[0275] In vitro growth inhibitory effects of tegavivint (BC-2059) in combination with tyrosine kinase inhibitors (TKIs) in hepatocellular carcinoma cells possessing different CTNNBI mutational states

[0276] To investigate the potential of BC-2059 to enhance TKI activity in vitro a non-GLP study was conducted to assess growth inhibitory effects of BC-2059 in combination with cabozantinib, lenvatinib, sorafenib, and regorafenib in hepatocellular carcinoma cell lines following 72 hours of treatment. HepG2 CTNNBI Ex3del), SNU_878 CTNNBI WT), SNU_398 (CTNNBI S37C), and HUH_7 (CTNNBI WT) cells were treated with BC-2059 at concentrations ranging from 1 nM to 23 pM and with concentrations of TKIs ranging from 78 nM to 100 pM based on single agent activity (780 nM - 100 pM: regorafenib, lenvatenib, and cabozantinib, 78 nM - 40 pM: sorafenib). Combenefit software (v2.021) was used to evaluate the effects of BC-2059 and regorafenib in combination.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0277] Sorafenib (FIG. 4A): Synergy in HepG2 cell growth inhibition was observed when 5 pM sorafenib was combined with BC-2059 at 125 nM to 250 nM, when 10 pM sorafenib was combined with BC-2059 at 62 nM to 250 nM, or when 2.5 pM sorafenib was combined with BC-2059 at 250 nM for 72 hours. Moderate antagonism was observed when 625 nM, 1.25 pM, or 2.5 pM sorafenib was combined with BC-2059 at 31 and 62 nM.

[0278] Regorafenib (FIG. 4B): Synergy in HepG2 cell growth inhibition was observed when 12.5 pM regorafenib was combined with BC-2059 at concentrations ranging from 31 nM to 500 nM for 72 hours. Slight antagonism was observed when 3.125 or 6.25 pM regorafenib was combined with BC-2059 at 31, 62, and 125 nM. A tendency toward synergy was generally observed when 1.56 pM to 25 pM regorafenib was combined with 250, 500, or 1000 nM of BC-2059. The combination of BC-2059 with regorafenib demonstrates synergistic effects in inhibiting HepG2 cell growth.

[0279] Cabozantinib (FIG. 4C): Synergy in SNU_878 cell growth inhibition was observed when concentrations ranging from 45 nM to 33.33 nM cabozantinib were combined with 37 nM or 111 nM tegavivint and when 1.2 pM or 3.7 pM cabozantinib was combined with tegavivnt at concentrations ranging from 12 nM to 333 nM. In SNU_398 slight synergy in cell growth inhibition was observed when concentrations ranging from 39 nM to 50 pM lenvatinib were combined with tegavivint at concentrations ranging from 333 nM to 3 pM. The combination of cabozantinib to tegavivint in HepG2 cells was neutral.

[0280] Lenvatinib (FIG. 4D): Additivity to synergy in HUH_7 cell growth inhibition was observed when concentrations ranging from 50 nM to 33.33 nM lenavtinib were combined with tegavivint concentrations ranging from 12 nM to 3 pM. Similarly, additivity to synergy in SNU_398 cell growth inhibition was observed when concentrations ranging from 15 nM to 33.33 nM lenvatinib were combined with tegavivint at concentrations ranging from 37 nM to 3 pM. The combination of lenvatinib to tegavivint in HepG2 cells demonstrated additivity when lenvatinib at concentrations of 780 pM to 100 pM was combined with BC-2059 at concentrations of 31 nM to 2 pM.Atorney Docket No. ITER-011 / 01 WO 40069 / 123

[0281] The combination of BC-2059 with VEGFR-TKIs exhibits additive to synergistic effects in inhibiting the growth of hepatocellular carcinoma cell lines in vitro, with outcomes influenced by CTNNBJ mutational status. When tegavivint was combined with cabozantinib or lenvatinib in CTNNB1 wild-type cells (HUH_7 or SNU_398), exon 3 point-mutated cells (SNU_398), or exon 3-deleted cells (HepG2), the inventors unexpectedly observed the greatest potency in wild-type cells, followed by exon 3 point-mutant cells. In contrast, the combination showed additive effects in exon 3-deleted HepG2 cells. These findings suggest that CTNNB1 mutational status may influence cellular susceptibility to tegavivint in combination with TKIs.

[0282] While there have been shown and described fundamental novel features of the invention as applied to the preferred and illustrative embodiments thereof, it will be understood that omissions and substitutions and changes in the form and details of the disclosed invention may be made by those skilled in the art without departing from the spirit of the invention. Moreover, as is readily apparent, numerous modifications and changes may readily occur to those skilled in the art. For example, various features and structures of the different embodiments discussed herein may be combined and interchanged. Hence, it is not desired to limit the invention to the exact construction and operation shown and described and, accordingly, all suitable modification equivalents may be resorted to falling within the scope of the invention as claimed. It is the intention, therefore, to be limited only as indicated by the scope of the claims appended hereto.

Claims

Atorney Docket No. ITER-011 / 01 WO 40069 / 123CLAIMS1. A method for treating a cancer or tumor metastasis in a mammal in need thereof, comprising administering to the mammal a combination of: an effective amount of tegavivint; and an effective amount of a tyrosine kinase inhibitor.

2. The method of claim 1, wherein the cancer or tumor metastasis comprises hepatocellular carcinoma (HCC).

3. The method of claim 2, wherein the HCC cells have wild-type CTNNBJ.

4. The method of claim 2, wherein the HCC cells have mutations in CTNNBJ or AXIN1.

5. The method of claim 4, wherein the mutations in CTNNBJ comprise deletion of exon 3 or one or more point mutations in exon 3.

6. The method of claim 1, wherein the tyrosine kinase inhibitor is a therapeutic that inhibits tyrosine kinases.

7. The method of claim 1, wherein the tyrosine kinase inhibitor is a small molecule that inhibits the activity of a protein expressed by the cancer cell.

8. The method of claim 1, wherein the tyrosine kinase inhibitor is a small molecule that directly inhibits a kinase expressed by the cancer cell.Atorney Docket No. ITER-011 / 01 WO 40069 / 1239. The method of claim 8, wherein the tyrosine kinase inhibitor directly inhibits one or more tyrosine kinases selected from the group consisting of PDGFR, FGFR, c-Kit, c-Met, c-RET, AXL, MER, VEGF-R1, and VEGF-R2.

10. The method of claim 1, wherein the tyrosine kinase inhibitor is selected from the group consisting of axitinib, nintedanib, pazopanib, vandetanib, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, gefitinib, erlotinib, sorafenib, sunitinib, afatinib, canertinib, osimertinib, acalabrutinib, regorafenib, cabozantinib, lenvatinib, and zanzalitinib.

11. The method of claim 1, wherein the tegavivint is in a nanosuspension prepared by a process comprising using a crystalline form of tegavivint designated as Form IV as the starting material and milling Form IV at a temperature between about 40° C and about 60° C, wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 20 angle values independently selected from the group consisting of 5.0+-0.20; 7.5+-0.20; 14.8+-0.20; 15.23 — 0.2°; 15.43 — 0.2°; 20.03 — 0.2°; and 22.23 — 0.2°, wherein the nanosuspension consists of Form I of tegavivint.

12. The method of claim 1, wherein the tegavivint is in a reconstituted formulation prepared from a lyophilized formulation comprising particles of tegavivint or a pharmaceutically acceptable salt thereof; wherein the particles have a D50 of less than or equal to 500 nm and a D90 of less than or equal to 1.0 micron when measured using laser diffraction; wherein the lyophilized formulation comprises a poloxamer and one or more stabilizers selected from the group consisting of sucrose, trehalose, and sorbitol; and wherein a starting material to produce the lyophilized formulation is Form I polymorph of tegavivint or Form IV polymorph of tegavivint.Atorney Docket No. ITER-011 / 01 WO 40069 / 12313. The method of claim 12, wherein the tegavivint in the reconstituted formulation consists of Form I polymorph of tegavivint.

14. The method of claim 1, wherein the tegavivint is in a formulation comprising a poloxamer and sorbitol; wherein the tegavivint is in the form of a nanosuspension comprising particles of tegavivint or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; wherein the particles have a D50 of less than or equal to 500 nm and a D90 of less than or equal to 1.0 micron when measured using laser diffraction.

15. The method of claim 14, wherein the formulation comprises 0.25 mg / ml tegavivint, 0.625% by weight poloxamer 188, and 10% by weight sorbitol.

16. The method of claim 1, wherein said administering is performed through one or more of intravenous, parenteral, oral, inhalation (including aerosolized delivery), buccal, intranasal, rectal, intra-lesional intraperitoneal, intradermal, transdermal, subcutaneous, intra-arterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, intravitreous injection, and topical application methods.

17. A combination therapy for treating a cancer or tumor metastasis in a patient in need thereof, comprising: an effective amount of tegavivint; and an effective amount of a tyrosine kinase inhibitor.

18. The combination therapy of claim 17, wherein the cancer or tumor metastasis comprises hepatocellular carcinoma (HCC).

19. The combination therapy of claim 18, wherein the HCC cells have wild-type CTNNB1.Atorney Docket No. ITER-011 / 01 WO 40069 / 12320. The combination therapy of claim 18, wherein the HCC cells have mutations in CTNNB1 or AXIN1.

21. The combination therapy of claim 20, wherein the mutations in CTNNB1 comprise deletion of exon 3 or one or more point mutations in exon 3.

22. The combination therapy of claim 17, wherein the tyrosine kinase inhibitor is a therapeutic that inhibits tyrosine kinases.

23. The combination therapy of claim 17, wherein the tyrosine kinase inhibitor is a small molecule that inhibits the activity of a protein expressed by the cancer cell.

24. The combination therapy of claim 17, the tyrosine kinase inhibitor is a small molecule that directly inhibits a kinase expressed by the cancer cell.

25. The combination therapy of claim 24, wherein the tyrosine kinase inhibitor directly inhibits one or more tyrosine kinases selected from the group consisting of PDGFR, FGFR, c-Kit, c-Met, c-RET, AXL, MER, VEGF-R1, and VEGF-R2.

26. The combination therapy of claim 17, wherein the tyrosine kinase inhibitor is selected from the group consisting of axitinib, nintedanib, pazopanib, vandetanib, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, gefitinib, erlotinib, sorafenib, sunitinib, afatinib, canertinib, osimertinib, acalabrutinib, regorafenib, cabozantinib, lenvatinib, and zanzalitinib.

27. The combination therapy of claim 17, wherein the tegavivint is in a nanosuspension prepared by a process comprising using a crystalline form of tegavivint designated as Form IV as the starting material and milling Form IV at a temperature between about 40° C and about 60° C,Atorney Docket No. ITER-011 / 01 WO 40069 / 123wherein Form IV has an X-ray powder diffraction pattern (XRPD) comprising diffraction peaks having ° 29 angle values independently selected from the group consisting of 5.0+-0.20;7.5+-O.20; 14.8+-O.20; 15.2+-O.20; 15.4+-0.20; 20.0+-0.20; and 22.2+-O.20, wherein the nanosuspension consists of Form I of tegavivint.

28. The combination therapy of claim 17, wherein the tegavivint is in a reconstituted formulation prepared from a lyophilized formulation comprising particles of tegavivint or a pharmaceutically acceptable salt thereof; wherein the particles have a D50 of less than or equal to 500 nm and a D90 of less than or equal to 1.0 micron when measured using laser diffraction; wherein the lyophilized formulation comprises a poloxamer and one or more stabilizers selected from the group consisting of sucrose, trehalose, and sorbitol; and wherein a starting material to produce the lyophilized formulation is Form I polymorph of tegavivint or Form IV polymorph of tegavivint.

29. The combination therapy of claim 28, wherein the tegavivint in the reconstituted formulation consists of Form I polymorph of tegavivint.

30. The combination therapy of claim 17, wherein the tegavivint is in a formulation comprising a poloxamer and sorbitol; wherein the tegavivint is in the form of a nanosuspension comprising particles of tegavivint or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; wherein the particles have a D50 of less than or equal to 500 nm and a D90 of less than or equal to 1.0 micron when measured using laser diffraction.

31. The combination therapy of claim 30, wherein the formulation comprises 0.25 mg / ml tegavivint, 0.625% by weight poloxamer 188, and 10% by weight sorbitol.Attorney Docket No. ITER-011 / 01 WO 40069 / 12332. The combination therapy of claim 17, wherein the combination therapy is provided in a dosage form (a) selected from the group consisting of tablets, and capsules; (b) selected from the group consisting of controlled release formulations, fast melt formulations, delayed release formulations, extended release formulations, pulsatile release formulations, and mixed immediate release and controlled release formulations; (c) suitable for inhalation or parenteral administration; or (d) any combination of (a), (b), and (c).