[go: up one dir, main page]

WO2025160462A1 - Methods for treatment of hepatocellular carcinoma - Google Patents

Methods for treatment of hepatocellular carcinoma

Info

Publication number
WO2025160462A1
WO2025160462A1 PCT/US2025/013033 US2025013033W WO2025160462A1 WO 2025160462 A1 WO2025160462 A1 WO 2025160462A1 US 2025013033 W US2025013033 W US 2025013033W WO 2025160462 A1 WO2025160462 A1 WO 2025160462A1
Authority
WO
WIPO (PCT)
Prior art keywords
tegavivint
dose
hcc
formulation
patient
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/013033
Other languages
French (fr)
Inventor
David STENEHJEM
Casey CUNNINGHAM
Flora Jean CHANG
Rahul Aras
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Iterion Therapeutics Inc
Original Assignee
Iterion Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Iterion Therapeutics Inc filed Critical Iterion Therapeutics Inc
Publication of WO2025160462A1 publication Critical patent/WO2025160462A1/en
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/4545—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • Beta-catenin ( ⁇ -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. ⁇ -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. Wnt/ ⁇ -catenin pathway has been shown to play a role in cancer. Aberrant ⁇ -catenin signaling plays an important role in tumorigenesis. In particular, hepatocellular carcinoma is estimated to have greater than 50% mutations in the ⁇ -catenin pathway, leading to unregulated oncogenic signaling.
  • Aberrant ⁇ -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 non- Hodgkin lymphomas, colorectal and acute myeloid leukemia (AML) cancers.
  • Wnt/ ⁇ -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 1 65518548 Attorney Docket No.40069/108; ITER-009/01WO including but not limited to idiopathic pulmonary fibrosis (IPF), Dupuytren's contracture, Nonalcoholic steatohepatitis (NASH), and others.
  • IPF idiopathic pulmonary fibrosis
  • NASH Nonalcoholic steatohepatitis
  • Tegavivint is useful for modulating the activity of the Wnt/ ⁇ -catenin signaling pathway, and serves to reduce ⁇ -catenin levels present in cells, such as cancer cells.
  • Tegavivint and related compounds are described, for example, in U.S. Patent No. 8,129,519. Tegavivint has the following structural formula: The The molecular mass of tegavivint is 588.20763 amu.
  • tegavivint can provide significant effects with respect to treating cancers associated with the Wnt/ ⁇ -catenin pathway and aberrant ⁇ -catenin signaling, such as but not limited to HCC.
  • the invention provides in various embodiments therapeutic doses of tegavivint for the treatment of cancer, such as but not limited to HCC.
  • the invention provides a method of treating advanced hepatocellular carcinoma (HCC) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of tegavivint or a pharmaceutically acceptable salt 2 65518548 Attorney Docket No.40069/108; ITER-009/01WO thereof, wherein the method prevents ⁇ -catenin from associating with transducin ⁇ -like protein 1 (TBL1) in the patient, and wherein the therapeutically effective dose is from about 0.0001 to about 1000 mg/kg/day.
  • the patient has shown disease progression after, or intolerance or contraindication to, at least one prior line of systemic therapy for advanced HCC.
  • the patient has one or more mutations in CTNNB1, APC, or AXIN1.
  • 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.
  • said administering is performed intravenously.
  • said administering is performed weekly on days 1, 8, 15, and 22 of a 28 day cycle.
  • the therapeutically effective dose is from about 1 to about 20 mg/kg weekly. In some embodiments, the therapeutically effective dose is about 3, about 5, about 6.5, about 8, about 10, about 12.5, or about 15.5 mg/kg weekly.
  • the tegavivint is in a formulation comprising a poloxamer and one or more stabilizers selected from the group consisting of sucrose, trehalose, and sorbitol. In some embodiments, the formulation comprises 25 mg/mL tegavivint, 0.625% by weight poloxamer 188, and 10% by weight sorbitol.
  • 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 ° 2 ⁇ angle 3 65518548 Attorney Docket No.40069/108; ITER-009/01WO values independently selected from the group consisting of 5.0+ ⁇ 0.2°; 7.5+ ⁇ 0.2°; 14.8+ ⁇ 0.2°; 15.2+ ⁇ 0.2°; 15.4+ ⁇ 0.2°; 20.0+ ⁇ 0.2°; and 22.2+ ⁇ 0.2°, and wherein the nanosuspension consists essentially of Form I of tegavivint.
  • XRPD X-ray powder diffraction pattern
  • the tegavivint is in a formulation comprising particles of Form I or Form IV of tegavivint or a pharmaceutically acceptable salt thereof, wherein 90% of the particles have a diameter of less than or equal to 0.2 ⁇ m when measured using laser diffraction, and wherein the formulation was prepared by high energy agitator milling at a temperature between about 40° C and about 60° C.
  • the formulation is lyophilized and reconstituted.
  • the patient has had prior treatment with a PD-1/PD-L1 inhibitor.
  • the method further comprises administering to the patient a therapeutically effective amount of an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti PD-L1 antibody.
  • the immune checkpoint inhibitor comprises at least one of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, and durvalumab.
  • the immune checkpoint inhibitor is pembrolizumab.
  • the tegavivint and the pembrolizumab are both administered by intravenous infusion, wherein the tegavivint is administered on days 1, 8, and 15 of a 21 day cycle, and the pembrolizumab is administered on day 1 of the 21 day cycle.
  • the pembrolizumab dose is about 200 mg.
  • the patient does not have fibrolamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC. Additional features and advantages of embodiments of the present invention are described further below. This summary section is meant merely to illustrate certain features of embodiments of the invention, and is not meant to limit the scope of the invention in any way.
  • FIG.1 shows results of experiments testing tegavivint for short term inhibition in hepatocellular ⁇ -catenin activation.
  • Tegavivint reduces Wnt target genes, tumor initiating clones, and proliferation in CTNNB1 exon3 mutant HCC.
  • FIG.2 shows results of experiments testing TBL inhibition monotherapy in early ⁇ - catenin dependent disease. Tegavivint reduces tumor progression in CTNNB1 exon3 mutant HCC.
  • FIG.3 shows results of experiments testing TBL inhibition monotherapy in established ⁇ - catenin dependent disease. Tegavivint reduces CD3+ tumor infiltrating leukocytes (TILs) and tumor burden in CTNNB1 exon3 mutant HCC.
  • A 120-day study timeline; B: Immunohistochemistry with CD3+ staining; images at x20 magnification; C: Images of tumor nodules in mouse livers; D: Immunohistochemistry with GS staining; images at 20x magnification; E: Quantification of tumor nodules. 5 65518548 Attorney Docket No.40069/108; ITER-009/01WO
  • FIG.4 shows results of experiments testing TBL as a target for sensitization to immunotherapy.
  • FIG.5 shows tumor growth curves in the treatment of the syngeneic model H22.
  • FIG.6 shows dose response curves for different cell lines plotted based on IC50 data.
  • FIG.7A shows Cycle 1, Day 1 infusion tegavivint concentration versus time profiles by dose level in BCI-001-DT17.
  • FIG.7B shows Cycle 1, Day 1 AUC0-168 hour by total dose (mg) of tegavivint administered.
  • FIG.8 shows objectives and related endpoints for a study of tegavivint alone and in combination with pembrolizumab according to various embodiments of the invention.
  • FIG.9 shows a scheme for tegavivint single agent dose optimization and expansion according to various embodiments of the invention.
  • FIG.10 shows a scheme for tegavivint plus pembrolizumab dose escalation and optimization according to various embodiments of the invention.
  • DETAILED DESCRIPTION Definitions 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 the invention.
  • the term “tegavivint” refers to a compound having the following structure:
  • 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.
  • 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.
  • 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.
  • the term “mammal” includes, but is not limited to, a human. 7 65518548 Attorney Docket No.40069/108; ITER-009/01WO
  • 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.
  • 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.
  • a parameter such as inflammation, pain and/or tumor size.
  • an 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/ ⁇ -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.
  • compositions 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.
  • 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.
  • compositions for oral administration can form solutions, suspensions, tablets, pills, capsules, sustained release formulations, oral rinses or powders.
  • composition refers to compositions wherein all, or almost all of the particles are less than 1000 nm.
  • lyophilized formulation refers to a formulation resulted from freeze-drying of an aqueous solution.
  • 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.
  • 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.
  • the solid composition is a lyophilized formulation.
  • an “injectable formulation” refers to a formulation that is suitable for parenteral administration, e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal administration.
  • 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.
  • AUClast refers to the area under the curve from the time of dosing to the time of the last measurable (positive) concentration.
  • Cmax 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.
  • Tmax refers to the time after administration of a drug when the maximum plasma concentration is reached.
  • 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.
  • 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.
  • 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.
  • the nanosuspension further undergoes the annealing process at or above 60° C. 10 65518548 Attorney Docket No.40069/108; ITER-009/01WO 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 I 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.
  • Form I 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.
  • 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. 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).
  • 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.
  • 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.
  • the microscopic images confirmed absence of any crystal growth for all final Form I formulations at the end of 3 months of storage.
  • the milling is performed at a temperature of between about 40° C and about 60° C, most preferably at about 60° C.
  • the milling process is done at 11 65518548 Attorney Docket No.40069/108; ITER-009/01WO temperature of less than about 60° C
  • the nanosuspension further undergoes the annealing process at or above 60° C.
  • 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.
  • the formulations of the invention are filtered to remove any large particles.
  • the filter is 10 micron.
  • 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 ( ⁇ m) when measured using laser diffraction. In one embodiment, the particles have an effective D90 of about 100 nm when measured using laser diffraction.
  • 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.
  • the formulation may be lyophilized.
  • the particles have an effective D90 of less than or equal to 0.2 micron when measured using laser diffraction.
  • the particles have an effective D50 of less than or equal to 0.12 micron when measured using laser diffraction.
  • the particles have an effective D10 of less than or equal to 0.1 micron when measured using laser diffraction.
  • 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.
  • 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 ⁇ m, it means that 90% of the particles are less than 1.0 ⁇ m in diameter.
  • 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 1
  • 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.
  • the provided formulations are stable for therapeutic utility.
  • the lyophilized formulations of the invention are anhydrous.
  • the formulations, including lyophilized formulations, of the invention are stored in a dry atmosphere.
  • the storage temperature for the formulations of the invention can be about ⁇ 20° C, about 5° C, or about 25° C.
  • 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 13 65518548 Attorney Docket No.40069/108; ITER-009/01WO of less than or equal to 1.0 micrometer ( ⁇ m) 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.
  • tegavivint concentration in a formulation is 2% (20 mg/mL).
  • tegavivint concentration in a formulation is 2.5% (25 mg/mL). In another embodiment, tegavivint concentration in a formulation is 5% (50 mg/mL).
  • the poloxamer is Poloxamer 188. In one embodiment, the poloxamer concentration in a formulation is 6 mg/mL. In another embodiment, the poloxamer concentration in a formulation is 12.5 mg/mL.
  • the sucrose concentration in a formulation is 100 mg/mL. 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.
  • 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 ( ⁇ m) 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.
  • tegavivint concentration in a pre-lyophilized formulation is 2% (20 mg/mL).
  • tegavivint concentration in a pre-lyophilized formulation is 2.5% (25 mg/mL). In another embodiment, tegavivint concentration in a pre-lyophilized formulation is 5% (50 mg/mL).
  • the poloxamer is Poloxamer 188. 14 65518548 Attorney Docket No.40069/108; ITER-009/01WO In one embodiment, the poloxamer concentration in a pre-lyophilized formulation is 0.6%. In another embodiment, the poloxamer concentration in a pre-lyophilized formulation is 1.25%. 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%.
  • the sorbitol concentration in a pre-lyophilized formulation is 5%.
  • the formulation is autoclaved prior to lyophilization.
  • 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.
  • the invention also includes pre-lyophilized formulations of tegavivint.
  • the pre-lyophilized formulation is prepared by a process comprising ball milling at a temperature of about 60° C.
  • the pre-lyophilized formulation is prepared by a process comprising high energy milling at a temperature of about 60° C.
  • the tegavivint in the pre-lyophilized formulation is Form I polymorph.
  • 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. In a preferred embodiment, the formulations of the invention exhibit long term stability.
  • the formulations of the invention 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 15 65518548 Attorney Docket No.40069/108; ITER-009/01WO 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).
  • the 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.
  • IV intravenous
  • the 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: AUClast 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.
  • the 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; 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.
  • the pharmaceutical formulations of the invention can further comprise one or more pharmaceutically acceptable excipients, carriers, or a combination thereof.
  • 16 65518548 Attorney Docket No.40069/108; ITER-009/01WO
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the method of preventing, treating or ameliorating cancer or tumor metastasis in a mammal in need thereof can include administering an additional anti- cancer agent and/or cancer therapy (for example, cancer vaccines, anti-cancer adoptive cell therapies and radio therapies).
  • an additional anti- cancer agent and/or cancer therapy for example, cancer vaccines, anti-cancer adoptive cell therapies and radio therapies.
  • 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 repair pathway inhibitors, anti-hormonal agents, anti-cancer antibodies, and other cytotoxic chemotherapy agents.
  • the additional anti-cancer agent is an immune checkpoint inhibitor.
  • Immune checkpoint inhibitors that may be used in combination therapies according to 17 65518548 Attorney Docket No.40069/108; ITER-009/01WO embodiments of the invention include monoclonal antibodies that target checkpoint proteins on immune cells, such as, but not limited to, anti-programmed cell death-1 (PD-1) antibodies and anti-programmed death-ligand 1 (PD-L1) antibodies.
  • PD-1 inhibitors include pembrolizumab (Keytruda), nivolumab (Opdivo), and cemiplimab (Libtayo).
  • PD- L1 inhibitors examples include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). Additional antibodies may also be used, including, for example, CTLA-4 inhibitors such as ipilimumab (Yervoy) and tremelimumab (Imjuno), and LAG-3 inhibitors such as relatlimab (which may be provided with nivolumab in a combination known as Opdualag).
  • CTLA-4 inhibitors such as ipilimumab (Yervoy) and tremelimumab (Imjuno)
  • LAG-3 inhibitors such as relatlimab (which may be provided with nivolumab in a combination known as Opdualag).
  • the immune checkpoint inhibitors 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.
  • 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.
  • the dosage is within the range of 0.1-10 mg/kg body weight.
  • 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.
  • tegavivint and immune checkpoint inhibitors 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 immune checkpoint inhibitors can be administered at the same frequency or at different frequencies.
  • the dosage and 18 65518548 Attorney Docket No.40069/108; ITER-009/01WO scheduling of each may change during a course of treatment. Treatment may continue until complete response or confirmed progressive disease.
  • the invention encompasses formulations including tegavivint and a pharmaceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof.
  • 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).
  • the compositions of the invention can further comprise one or more pharmaceutically acceptable excipients, carriers, or a combination thereof.
  • the pharmaceutically acceptable excipients used in the formulation of the present invention can act in more than one way.
  • the pharmaceutically acceptable excipients can be, for example, a dispersion medium, a dispersion emulsifier, a dispersion enhancer, or a combination thereof.
  • 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.
  • the dispersion medium can be, for example, ethanol, propylene glycol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, glycerin, a combination thereof, etc.
  • the dispersion emulsifier 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 19 65518548 Attorney Docket No.40069/108; ITER-009/01WO monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, poloxamer, a combination thereof, etc.
  • dispersion enhancers examples 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, polyoxylglycerides, pyrrolidone, sorbitan esters, stearic acid, vitamin E polyethylene glycol succinate, polyethylene glycol 1000, povidone, a combination thereof, etc.
  • 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.
  • 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.
  • excipients are known in the art.
  • filling agents are lactose monohydrate, lactose anhydrous, and various starches
  • binding agents are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCCTM).
  • 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.
  • sweeteners are any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame.
  • sweeteners are any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame.
  • flavoring agents are Magnasweet® (trademark of MAFCO), bubble gum flavor, and fruit flavors, and the like.
  • preservatives examples include 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.
  • Suitable diluents include pharmaceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and/or mixtures of any of the foregoing.
  • 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.
  • 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.
  • 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.
  • 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.
  • sodium bicarbonate component of the effervescent couple may be present.
  • Hepatocellular carcinoma 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.
  • HCC 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 atezolizumab and bevacizumab).
  • HCC remains a deadly disease with poor prognosis in patients with unresectable cancer.
  • up to 40% of HCC patients have tumors driven by activating ⁇ -catenin and canonical Wnt pathway mutations.
  • the most frequently mutated genes include CTNNB1, AXIN1, and APC.
  • Most mutations in ⁇ -catenin occur within exon 3, the critical region responsible for its proteasomal degradation. These exon 3 mutations render ⁇ -catenin resistant to degradation, resulting in nuclear accumulation dysregulated transcriptional activity.
  • TBL1 transducin beta-like protein 1
  • TBL1 binds to ⁇ -catenin, inhibiting its degradation, and forms an active transcriptional complex that binds to promoters to activate downstream cancer-related genes such c-MYC, Cyclin D1 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. Tegavivint is a first-in-class small molecule inhibitor of TBL1, a novel downstream Wnt- signaling pathway target. Tegavivint binds to TBL1 in the ⁇ -catenin pocket, disrupting the formation of the activation complex necessary for oncogenic activity.
  • Tegavivint also enables the degradation of free nuclear ⁇ -catenin. Increased expression of ⁇ -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 ⁇ -catenin pools necessary for normal cellular function, avoiding toxicities commonly associated with other inhibitors of the Wnt pathway.
  • tegavivint The safety, pharmacodynamics, pharmacokinetics, and clinical activity of tegavivint was demonstrated through a proof-of-concept study in desmoid patients with activating ⁇ -catenin mutations and is currently being studied through Investigator Initiated Trials (IITs) in acute myeloid leukemia (AML), pediatric solid tumors, non-small cell lung cancer (NSCLC), and lymphoma.
  • IITs Investigator Initiated Trials
  • AML acute myeloid leukemia
  • NSCLC non-small cell lung cancer
  • lymphoma lymphoma
  • C57BL/6J mice with Ctnnb1 exon3/Wt Rosa26 c-MYC/c-MYC alleles were either injected with a high dose of the AAV-Cre vector to test the efficacy of tegavivint in inhibiting canonical ⁇ -Catenin target genes (Figure 1A; acute oncogene activation model) or a low dose of 23 65518548 Attorney Docket No.40069/108; ITER-009/01WO the AAV-Cre vector to test tegavivint reduction of clonal liver tumors representative of human HCC ( Figure 2A, Figure 3A).
  • mice were randomized to receive tegavivint (1.5 mg i.v. twice weekly) in combination with an anti-PD-1 antibody (aPD1/anti-PD-1) or isotype control (200 ⁇ g i.p. twice weekly).
  • the study endopoint was 135 days ( Figure 4A).
  • Combination therapy comprising tegavivint and anti-PD-1 antibody reduced liver size (liver/body ratio %), number of tumors (macroscopic tumors/liver), and tumor volume (tumor burden mm 3 ) as compared to tegavivint and isotype control ( Figures 4B-4E).
  • 24 65518548 Attorney Docket No.40069/108; ITER-009/01WO
  • H22 cells from the HCC syngeneic tumor type, H22, were inoculated subcutaneously in the right lower flank region of BALB/c mice. I.p.
  • tegavivint 100 mg/kg or vehicle phosphate-buffer solution [PBS]
  • PBS phosphate-buffer solution
  • Body weight and tumor volumes were measured twice or three times during the study (including on day 1 and at sacrifice).
  • H22 mice were sacrificed on day 3.
  • Tumors were collected from nine models on the termination day. Each tumor was divided for fluorescence activated cell sorting (FACS) analysis, and formalin-fixed paraffin-embedded (FFPE) blocks.
  • FACS analysis employed side scatter-forward scatter (SSC-FSC) plots to determine tumor infiltrating lymphocytes (TILs). A Bartlett test was used to test homogeneity of variance and normality.
  • H22 tegavivint-treated tumors Increases in PD-1+ CD3+ and PD-1+ CD4+ lymphocytes and M2 macrophages were also observed in H22 tegavivint-treated tumors.
  • PBS vehicle
  • H22 syngeneic mouse model was used to evaluate the efficacy and tolerability of tegavivint alone and in combination with anti-PD-1 or anti-PD-L1.
  • H22 mouse liver tumor cells 25 65518548 Attorney Docket No.40069/108; ITER-009/01WO (1 x 10 6 ) in 0.1 mL PBS were inoculated subcutaneously in the right upper flank region of in female BALB/c mice for tumor development. Mice were enrolled and randomly allocated into six treatment groups of ten mice: 1. Vehicle 2. Tegavivint 50 mg/kg, alone 3. anti-PD-110 mg/kg, alone 4.
  • Tegavivint 50 mg/kg plus anti-PD-110 mg/kg 6. Tegavivint 50 mg/kg plus anti-PD-L110 mg/kg. All test articles were administered i.p. over 15 days. Tegavivint was administered five times a week (5 days on, 2 days off). Immune checkpoint inhibitors (ICIs) were administered twice weekly (BIW). The tumor growth inhibition (TGI) of treatment with tegavivint at 50 mg/kg (G2) group was 43.50% (Table 1). Anti-PD-110 mg/kg alone and anti-PD-L110 mg/kg alone both resulted in significant TGIs (Table 1).
  • tegavivint has shown anti-tumor and immunomodulatory activity in a ⁇ - catenin activating mutant mouse model of HCC. Tegavivint also reduced tumor volume and promoted an immunomodulatory response in the syngeneic HCC model, H22.
  • Tegavivint tumor response in HCC model H22 was reproduced in a separate study, in which the therapeutic efficacy and tolerability of tegavivint alone or in combination with either anti-PD-1 or anti-PD- L1 was tested in the treatment of the subcutaneous H22 mouse liver cancer tumor syngeneic model in female BALB/c mice.
  • In Vitro HCC Data The effect of tegavivint was investigated in nine HCC and hepatoblastoma cell lines in a 2D viability assay. The results were compared to cisplatin as a reference control. In order to calculate absolute IC50, a dose-response curve was fitted using nonlinear regression model with a sigmoidal dose response.
  • Surviving rate (%) (LumTest article-LumT0)/ (LumNone treated-LumT0) ⁇ 100% 28 65518548 Attorney Docket No.40069/108; ITER-009/01WO
  • tegavivint shows a distinctive distribution pattern with initial localization primarily in the lungs, followed by subsequent redistribution to the liver and spleen. The volume of distribution exceeds total body water across species, indicating extensive tissue distribution. The compound demonstrates relatively low plasma clearance compared to liver blood flow, suggesting minimal first-pass metabolism. Notably, tegavivint shows limited penetration 29 65518548 Attorney Docket No.40069/108; ITER-009/01WO across the blood-brain barrier, but exhibits favorable tumor distribution characteristics as demonstrated in mouse models.
  • the distribution pattern of the nanoparticle formulation aligns with established literature showing initial lung distribution followed by hepatic and splenic redistribution.
  • the metabolic profile of tegavivint is consistent across species.
  • the parent drug remains the predominant circulating compound, with CYP3A4 serving as the primary metabolizing enzyme.
  • CYP3A4 serving as the primary metabolizing enzyme.
  • human hepatocytes approximately 80% of the parent drug remains after 120 minutes of incubation.
  • the primary metabolic pathways include oxidation and glucuronidation, producing predictable metabolites. Importantly, no unique human metabolites have been identified, suggesting good translation of preclinical safety findings to humans.
  • UGT1A1 IC50: 29.8 nM recombinant/236 nM microsomes; maximum inhibition: 92% recombinant/75% microsomes
  • UGT1A3 IC50: 66.0 nM recombinant/169 nM microsomes; maximum inhibition: 87% recombinant/64% microsomes
  • UGT1A4 IC50: 23.6 nM recombinant/145 nM microsomes; maximum inhibition: 89% recombinant/70% microsomes.
  • Minimal to no inhibition was observed for UGT1A6, UGT1A9, and UGT2B7.
  • tegavivint showed selective inhibition of key hepatic transporters. Tegavivint inhibited OATP1B1-mediated transport with an IC50 of 0.81 ⁇ M and maximum inhibition of 82%. Significant inhibition was also observed for BSEP (IC501.63 ⁇ M, 86% maximum inhibition) and BCRP (IC503.94 ⁇ M, 60% maximum inhibition). Other evaluated transporters including MATE1/2-K, MRP1/3, NTCP, and OCT1/3 showed minimal to no inhibition. The concurrent inhibition of both metabolic enzymes and transporters integral to hepatic function, particularly UGT1A1 and the OATP1B1/BSEP pathway, indicates that tegavivint may influence bilirubin homeostasis and xenobiotic clearance.
  • GLP Toxicology studies Rat The GLP 4-week toxicology rat study was conducted with doses of 0 (Group 1), 10 (group 2), 30 (Group 3) and 60 mg/kg (Group 4) administered twice weekly for a total of 8 doses. Animals were given BC2059 or vehicle (5% dextrose) via continuous infusion over 24 hours (on Days 1, 4, 8, 11, 15, 18, 22, and 25). The highest dose, 60 mg/kg, exceeded the maximum tolerated dose as 4 female rats at this dose level were found dead on Day 5 or 6.
  • Cmax peak blood concentrations
  • AUC0-t overall systemic concentrations determined from the plasma concentration over time curves
  • Table 4 Systemic Exposures in the GLP Toxicology Study in Rats 65518548 Attorney Docket No.40069/108; ITER-009/01WO GLP Toxicology studies: Minipig In the GLP 4-week toxicology minipig study, animals were divided into five treatment groups that included a vehicle control (Group 1: 5% dextrose), 1 mg/kg (Group 2), 5 mg/kg (Group 3) and 15 mg/kg (Group 4) BC2059 administered over 24 h and a 4 h infusion group with 5 mg/kg BC2059 (Group 5). The infusions were administered on Days 1, 6, 8, 13, 15, 20, 22 and 27 for a total of 8 doses. There were no treatment-dependent clinical observations. There were no effects on body weight or food consumption.
  • the systemic exposures to BC2059 similarly do not reflect any marked difference in systemic exposures between the two infusion durations.
  • Table 6 when BC2059 was infused at 5 mg/kg over 4 hours, mean Cmax and AUClast were very much the same as determined with the 5 mg/kg dose infused over 24 hours.
  • Plasma concentrations over time for the two different infusion durations of 5 mg/kg are provided in Table 7. As anticipated, with the shorter 4 h infusion, higher plasma concentrations are initially observed at up to 8 h post initiation of infusion. However, peak plasma concentrations are no greater than 1.3-fold higher in the 4 h infusion compared to the 24 h infusion.
  • Table 5 Systemic Exposures in the GLP Toxicology Study in Miniature Swine 34 65518548 Attorney Docket No.40069/108; ITER-009/01WO Table 6: Estimates of Terminal Elimination Half-Life in the GLP Toxicology Study in Miniature Swine Table 7: Plasma Concentrations in Miniature Swine Administered 5 mg/kg over a 24 h and a 4 h Infusion Non-GLP Toxicity study: Minipig The results of the GLP toxicology study in minipigs defined dose levels at which no toxicities with BC2059 infusion were observed; that is, 15 mg/kg.
  • a total of four female Yucatan miniature swine were administered an IV infusion of the test article for 24-consecutive hours on Day 1 with a target dose level of 20 mg/kg/day and 40 mg/kg/day.
  • a second IV infusion of the test article was administered for 24-consecutive hours with a target dose level of 20 mg/kg/day and 35 mg/kg/day.
  • the high dose of 40 mg/kg was not well tolerated with clinical signs of lethargy and decreased food consumption and was lowered to 35 mg/kg for Day 5 dosing; one of two animals was 35 65518548 Attorney Docket No.40069/108; ITER-009/01WO not dosed for a second time.
  • RP2D dose level cohort
  • that dose level cohort (5 mg/kg) was expanded with 7 additional patients enrolled to collect further 36 65518548 Attorney Docket No.40069/108; ITER-009/01WO safety, PK, and PD data.
  • Dose expansion also included 6 patients enrolled at lower doses levels who were eligible for intrapatient dose escalation to the RP2D.
  • Symptomatic or progressive disease was defined as a 20% increase in tumor volume within 6 months, or recurrent disease within 1 year of surgery, or desmoid related symptoms and documentation that symptoms were related to desmoid and not prior therapies. Patients were also required to have bi-dimensionally measurable tumors by WHO criteria; be ⁇ 18 years of age; and have an ECOG performance score of 0-1.
  • Table 8 Estimation of Starting Clinical Dose Based on GLP Toxicology Studies According to ICH S9 Guidance 37 65518548 Attorney Docket No.40069/108; ITER-009/01WO
  • the starting clinical dose was defined to be 0.5 mg/kg for the initial dose finding study BCI- 001-DT17. This starting dose was based on the lowest starting dose determined across both the minipigs and the rats. Certainly, the minipig is the more relevant species in terms of similarity of infusion conditions to human. The infusion volume in minipigs and total volume is comparable to anticipated clinical volume.
  • the minipig also more closely resembles the human, and the safety of the nanoparticles in terms of size of the nanoparticles and size of the microvasculature in the two species. Indeed, in the rat, there were toxicities observed that were related to the infusion procedure itself, with findings in several control animals. However, the most conservative approach was to utilize the 30 mg/kg dose in rats for the HNSTD as opposed to the 5 mg/kg dose administered over 4 h in minipigs to support the original starting clinical dose of 0.5 mg/kg in BCI-001-DT17.
  • 15 38 65518548 Attorney Docket No.40069/108; ITER-009/01WO (63%) were White, 4 (17%) Asian, 3 (13%) Black, and 2 (8%) other races.
  • the median time from initial diagnosis of desmoid to Cycle 1, Day 1 was 3.1 years (range 0.3-19.7).
  • the median number of cycles of tegavivint completed across all patients was 10 (interquartile range: 4.1-20.9).
  • the median relative dose intensity was 97% (range: 83- 100) and was consistent across dose levels.
  • the median number of missed infusions for any reason was 0.5 (range 0-4).
  • One patient had a dose reduction from 4 mg/kg to 3 mg/kg for grade 3 anemia considered not related to study treatment and subsequently tolerated intrapatient dose escalation to 5 mg/kg with no dose reductions or interruptions in therapy.
  • the primary reasons for discontinuation of study treatment were: 5 (20.8%) patients completed treatment; 5 (20.8%) patients discontinued due to progressive disease; 1 (4.2%) patient for an adverse event considered not related to tegavivint; and 12 patients for withdrawal of consent or other reasons.
  • Figure 7A graphically presents the Cycle 1, Day 1 concentration time profile from the start of the 4-hour tegavivint infusion to 168 hours post dose in the six dose levels assessed. The number of patients per dose level are depicted and data are presented as medians of all patients at each dose level with the interquartile range (25%-75%). See 41 65518548 Attorney Docket No.40069/108; ITER-009/01WO Table 9 for Cycle 1, Day 1 summary pharmacokinetic parameters by dose level. The median half-life across all dose levels was 39.9 hours (IQR 33.8-48.9) supporting once weekly administration. Accumulation was present prior to the next weekly infusion especially at the higher dose levels (4 mg/kg and 5 mg/kg), indicating sustained exposure to tegavivint between doses.
  • Exposure was increased at the 4 mg/kg and 5 mg/kg cohorts compared with the lower dose cohorts. An increase in AUC was observed from the 4 mg/kg to 5 mg/kg cohorts in Cycle 1 despite similar total dose administered, stemming from lower median body weight in the 5 mg/kg cohort. AUC correlated with the dose (mg) of tegavivint administered ( Figure 7B). In one patient with intrapatient dose escalation from 4 mg/kg to 5 mg/kg demonstrated an increase in AUC at the 5 mg/kg dose level.
  • Table 9 Cycle 1, Day 1 infusion pharmacokinetics of tegavivint in BCI-001-DT17 by dose level
  • Clinical Pharmacodynamics of tegavivint in BCI-001-DT17 Clinical pharmacodynamic activity of tegavivint in humans with unresectable desmoid tumor in BCI-001-DT17 (NCT03459469) with an objective response (all partial response per WHO criteria) and serum available was analyzed for changes in serum protein biomarkers.
  • a set of six proteins were selected as candidate biomarkers 42 65518548 Attorney Docket No.40069/108; ITER-009/01WO based on both their cancer-related function and regulation by aberrant Wnt signaling.
  • Serum from the four patients with an objective response that had been administered tegavivint at doses of 1, 4, or 5 mg/kg were analyzed at five timepoints (Cycle 1, Day: 15 minutes pre-dose and 2 hours, 4 hours, and 24 hours after initiation of the infusion; Cycle 1, Day 8: 15 minutes pre-dose) were analyzed. Tegavivint caused substantial alterations in these beta-catenin responsive biomarkers up to the last measured collection timepoint of 192-hours post start of infusion. For several of these biomarkers a dose response trend was observed with patient 03-01 (1mg/kg) showing a less depth of change and shorter persistence than patient 04-08 (5mg/kg).
  • patient 03-04 One of the four patients analyzed (patient 03-04) had a highly variable response in multiple biomarkers in this assay and was found to have much higher plasma drug levels than other patients in this biomarker study.
  • HCC Clinical Studies As described above, ⁇ -catenin activating mutations and over-active Wnt transcriptional activity are prevalent in HCC. TBL1 inhibition via tegavivint has demonstrated encouraging efficacy in preclinical models of HCC.
  • Activating mutations are found in the Wnt/ ⁇ -catenin signaling pathway in a majority of these patients, with the most frequent being mutations in CTNNB1 (gene encoding ⁇ -catenin) found in 20-30% of patients, mutations in AXIN1 found in 8-10% of patient samples, and mutations in APC found in 5-8% of liver cancer patients (Schulze et al.
  • Tegavivint targets the ⁇ -catenin signaling pathway in the nuclear compartment, and acts with a mechanism that is independent of the mutational status of CTNNB1, APC and AXIN1.
  • patient selection using mutations in these genes provides an enrichment of tumors that have active ⁇ - catenin oncogenic signaling but do not affect the activity of tegavivint to inhibit ⁇ -catenin signaling.
  • the activation of Wnt/ ⁇ -catenin signaling in HCC is associated with changes in the immune environment (C. Xu et al.2022 Journal of Clinical Investigation 132(4):e154515).
  • high Wnt/ ⁇ -catenin signaling is associated with elevated proportions of immunosuppressive cells, and significantly lower proportions of CD8+ T cells compared to the low Wnt signaling tumors.
  • Wnt signaling high tumors have substantial enrichment of negative modulators of immune signaling, and in agreement with preclinical models of HCC, suggest that inhibition of ⁇ -catenin can enhance immune function in tumors and cooperate with immunotherapies such as checkpoint inhibitors (W. Xu et al.2022 Frontiers in Immunology 13:1010554). Objectives and related endpoints of the study are described in Figure 8.
  • AE adverse event
  • AUC area under the concentration-time curve
  • CBR clinical benefit rate
  • Cmax maximum concentration
  • Cmin minimum concentration
  • CR complete response
  • ctDNA circulating tumor DNA
  • DLT dose- limiting toxicity
  • DOCB duration of clinical benefit
  • DOR duration of response
  • ECG electrocardiogram
  • MAD maximum administered dose
  • MTD maximum tolerated dose
  • NCI-CTCAE National Cancer Institute Common Terminology Criteria for Adverse Events
  • ORR objective response rate
  • OS overall survival
  • PFS progression- free survival
  • PK pharmacokinetic(s)
  • PR partial response
  • qPCR quantitative polymerase chain reaction
  • RP2D recommended phase 2 dose
  • SAE serious adverse event
  • SD stable disease
  • T1/2 half-life
  • TBL-1 transducin ⁇ -like protein 1
  • TIL tumor-infiltrating lymphocyte
  • Tmax time of maximum concentration
  • TTR time to response.
  • the study comprises two parts.
  • the first part is a phase 1, open-label, single-agent dose escalation, optimization, and expansion study of tegavivint in patients with advanced HCC after failure of at least one line of prior systemic therapy.
  • the second part is an assessment of the combination of tegavivint plus pembrolizumab with a limited dose escalation followed by a randomized dose optimization.
  • Dose escalation follows a standard 3+3 design to assess the safety and MTD/MAD/RP2D of tegavivint administered intravenously on day 1, 8, 15, and 22 of a 28-day cycle (with a ⁇ 1 day window for administration after Cycle 1 Day 1). Dose selection optimization will expand the two dose levels to at least 10 patients before declaring the recommended phase 2 dose (RP2D).
  • the phase 1 dose escalation scheme is shown in Figure 9.
  • DLT dose-limiting toxicity
  • HCC hepatocellular carcinoma
  • IV intravenous(ly)
  • MTD maximum tolerated dose
  • PD pharmacodynamics
  • PK pharmacokinetics
  • RP2D recommended phase 2 dose.
  • patients must have mutations in CTNNB1, APC, or AXIN1 in dose optimization/expansion. If the DLT rate is exceeded at dose level 1, then lower dose levels of tegavivint may be assessed (i.e., 2 mg/kg IV weekly). Additional higher dose levels (i.e. > 15.5 mg/kg IV weekly), if needed, may continue in increments of 25% from the highest safe dose.
  • Part 2 This part determines the safety, tolerability, and MTD/MAD/RP2D of tegavivint administered in combination with pembrolizumab to patients with advanced HCC, which must include prior PD-1/PD-L1 inhibitor use in the first- or second-line setting.
  • patients must have mutations in CTNNB1, APC, or AXIN1 genes.
  • Tegavivint is administered via IV on day 1, 8, and 15 of a 21-day cycle with pembrolizumab 200 mg administered via a 30- minute IV infusion on day 1 of the 21-day cycle.
  • the tegavivint plus pembrolizumab dose escalation and optimization scheme is shown in Figure 10.
  • Tegavivint may be provided, for example, as an opaque, white to off-white sterile liquid suspension concentrate for infusion in an amber glass vial with butyl rubber stopper and aluminum crimp seal.
  • Each 10 mL glass vial contains 10 mL of a 25 mg/mL tegavivint (BC- 2059) suspension concentrate, 0.625% poloxamer 188 NF, 10% sorbitol NF, and water for injection USP.
  • Tegavivint may be administered by IV infusion diluted in dextrose 5% in water (D5W) and infused through a 15-micron filtered infusion over a 4-hour duration ( ⁇ 5 minutes) at ambient temperature. Once diluted in D5W, the diluted suspension may be held at room temperature in a non-DEHP PVC bag for up to 4 hours prior to administration to the patient (maximum time at room temperature preferably no more than 8 hours).
  • Pembrolizumab should be administered through a different IV administration set from the tegavivint.
  • Pembrolizumab (Keytruda) may be supplied as a 100 mg/4 mL (25 mg/mL) solution in a single-dose vial.
  • Measurable disease as defined by RECIST 1.1 with spiral computerized tomography (CT) scan or magnetic resonance imaging (MRI). Lesions situated in a previously irradiated area, or in an area subjected to other loco-regional therapy, may be considered measurable if progression has been demonstrated in such lesions.
  • CT computerized tomography
  • MRI magnetic resonance imaging
  • Normal organ and marrow function as defined below a. Absolute neutrophil count (ANC) ⁇ 1.2 x 10 9 /L 47 65518548 Attorney Docket No.40069/108; ITER-009/01WO b. Platelets ⁇ 60 x 10 9 /L; no transfusion within 7 days prior to the screening laboratory assessment c.
  • Hemoglobin ⁇ 9 g/dL red blood cell transfusion or growth factors support is not allowed in the 14 days prior to the screening laboratory assessment
  • i. Estimated creatinine clearance (CrCl) ⁇ 50 mL/min by the Cockcroft-Gault equation using actual body weight, or ii.
  • HCV peripheral sensory neuropathy
  • Participants with past HCV infection will be eligible for the study. The treated participants must have completed their treatment at least 1 month prior to starting study intervention and HCV viral load must be below the limit of quantification. 14. Participants with controlled HBV will be eligible if they meet the following criteria: a. Antiviral therapy for HBV must be given for at least 4 weeks and HBV viral load must be less than 500 IU/mL prior to first dose of study drug. Patients on active HBV therapy with viral loads under 100 IU/mL should stay on the same therapy throughout study intervention. b.
  • BP blood pressure
  • 49 65518548 Attorney Docket No.40069/108; ITER-009/01WO
  • Exclusion criteria for patient eligibility for treatment as described above may comprise one or more of the following: 1.
  • Known fibrolamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC 2. Patients receiving therapy with other anti-neoplastic or experimental agents 3. Not recovered to Grade 1 or baseline from adverse events related to prior therapy 4. Patients receiving concomitant strong inhibitors of CYP3A4/5 that cannot be discontinued 7 days or 5 half-lives (whichever is longer) prior to Cycle 1 Day 1 5. Patients receiving concomitant inducers of CYP3A4/5 that cannot be discontinued at least 14 days prior to Cycle 1 Day 1 6. Known central nervous system (CNS) involvement 7. Patients with known history of Gilbert’s syndrome or other genetic conditions affecting UGT1A1 function 8.
  • CNS central nervous system
  • Circulating tumor DNA obtained via blood samples at screening and at the end of treatment may be used to sequence Wnt/ ⁇ -catenin pathway mutations. Alterations may be used to correlate response to tegavivint and changes in mutational profile with treatment.
  • ctDNA may also be used to correlate genomic alterations with alterations identified with alterations identified on tumor next generation sequencing (NGS) analysis.
  • NGS next generation sequencing
  • Tumor tissue obtained via core or excisional biopsy pre- and post-treatment may be used correlate immune response via T-cell infiltration and activated ⁇ -catenin/canonical Wnt signaling with clinical response. Tumor tissue may also be used to correlate genomic alternations with those identified on ctDNA analysis.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Methods for treatment of hepatocellular carcinoma using formulations of tegavivint, alone or in combination with an immune checkpoint inhibitor.

Description

Attorney Docket No.40069/108; ITER-009/01WO METHODS FOR TREATMENT OF HEPATOCELLULAR CARCINOMA RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.63/624,744, filed January 24, 2024, and U.S. Provisional Application No.63/556,656, filed February 22, 2024, each of which is incorporated by reference herein in its entirety. BACKGROUND 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. Beta-catenin (β-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. β-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. Wnt/β-catenin pathway has been shown to play a role in cancer. Aberrant β-catenin signaling plays an important role in tumorigenesis. In particular, hepatocellular carcinoma is estimated to have greater than 50% mutations in the β-catenin pathway, leading to unregulated oncogenic signaling. Aberrant β-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 non- Hodgkin lymphomas, colorectal and acute myeloid leukemia (AML) cancers. Further, aberrant Wnt/β-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 1 65518548 Attorney Docket No.40069/108; ITER-009/01WO including but not limited to idiopathic pulmonary fibrosis (IPF), Dupuytren's contracture, Nonalcoholic steatohepatitis (NASH), and others. SUMMARY The invention recognizes that a need still exists for methods of treating cancers that are associated with the Wnt/β-catenin pathway and aberrant β-catenin signaling, such as but not limited to hepatocellular carcinoma (HCC). Tegavivint is useful for modulating the activity of the Wnt/β-catenin signaling pathway, and serves to reduce β-catenin levels present in cells, such as cancer cells. Tegavivint and related compounds are described, for example, in U.S. Patent No. 8,129,519. Tegavivint has the following structural formula: The The molecular mass of tegavivint is 588.20763 amu. It has been found that tegavivint can provide significant effects with respect to treating cancers associated with the Wnt/β-catenin pathway and aberrant β-catenin signaling, such as but not limited to HCC. In that manner, the invention provides in various embodiments therapeutic doses of tegavivint for the treatment of cancer, such as but not limited to HCC. In some embodiments, the invention provides a method of treating advanced hepatocellular carcinoma (HCC) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of tegavivint or a pharmaceutically acceptable salt 2 65518548 Attorney Docket No.40069/108; ITER-009/01WO thereof, wherein the method prevents β-catenin from associating with transducin β-like protein 1 (TBL1) in the patient, and wherein the therapeutically effective dose is from about 0.0001 to about 1000 mg/kg/day. In some embodiments, the patient has shown disease progression after, or intolerance or contraindication to, at least one prior line of systemic therapy for advanced HCC. In some embodiments, the patient has one or more mutations in CTNNB1, APC, or AXIN1. 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. In some embodiments, said administering is performed intravenously. In some embodiments, said administering is performed weekly on days 1, 8, 15, and 22 of a 28 day cycle. In some embodiments, the therapeutically effective dose is from about 1 to about 20 mg/kg weekly. In some embodiments, the therapeutically effective dose is about 3, about 5, about 6.5, about 8, about 10, about 12.5, or about 15.5 mg/kg weekly. In some embodiments, the tegavivint is in a formulation comprising a poloxamer and one or more stabilizers selected from the group consisting of sucrose, trehalose, and sorbitol. In some embodiments, the formulation comprises 25 mg/mL tegavivint, 0.625% by weight poloxamer 188, and 10% by weight sorbitol. 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 ° 2θ angle 3 65518548 Attorney Docket No.40069/108; ITER-009/01WO values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°, and wherein the nanosuspension consists essentially of Form I of tegavivint. In some embodiments, the tegavivint is in a formulation comprising particles of Form I or Form IV of tegavivint or a pharmaceutically acceptable salt thereof, wherein 90% of the particles have a diameter of less than or equal to 0.2 µm when measured using laser diffraction, and wherein the formulation was prepared by high energy agitator milling at a temperature between about 40° C and about 60° C. In some embodiments, the formulation is lyophilized and reconstituted. In some embodiments, the patient has had prior treatment with a PD-1/PD-L1 inhibitor. In some embodiments, the method further comprises administering to the patient a therapeutically effective amount of an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti PD-L1 antibody. In some embodiments, the immune checkpoint inhibitor comprises at least one of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, and durvalumab. In some embodiments, the immune checkpoint inhibitor is pembrolizumab. In some embodiments, the tegavivint and the pembrolizumab are both administered by intravenous infusion, wherein the tegavivint is administered on days 1, 8, and 15 of a 21 day cycle, and the pembrolizumab is administered on day 1 of the 21 day cycle. In some embodiments, the pembrolizumab dose is about 200 mg. In some embodiments, the patient does not have fibrolamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC. Additional features and advantages of embodiments of the present invention are described further below. This summary section is meant merely to illustrate certain features of embodiments of the invention, and is not meant to limit the scope of the invention in any way. The failure to discuss a specific feature or embodiment of the invention, or the inclusion of one 4 65518548 Attorney Docket No.40069/108; ITER-009/01WO or more features in this summary section, should not be construed to limit the invention as claimed. BRIEF DESCRIPTION OF THE DRAWINGS 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. FIG.1 shows results of experiments testing tegavivint for short term inhibition in hepatocellular β-catenin activation. Tegavivint reduces Wnt target genes, tumor initiating clones, and proliferation in CTNNB1exon3 mutant HCC. A: 9-day study timeline; B: Change in gene expression of Wnt target genes in tumor tissue with activated beta-catenin compared with tumor tissue without activated beta-catenin as assessed by quantitative RT-PCR; C, D, E: Immunohistochemistry images at 20x magnification. FIG.2 shows results of experiments testing TBL inhibition monotherapy in early β- catenin dependent disease. Tegavivint reduces tumor progression in CTNNB1exon3 mutant HCC. A: 28-day study timeline; B: Immunohistochemistry with GS staining; images at 20x magnification. FIG.3 shows results of experiments testing TBL inhibition monotherapy in established β- catenin dependent disease. Tegavivint reduces CD3+ tumor infiltrating leukocytes (TILs) and tumor burden in CTNNB1exon3 mutant HCC. A: 120-day study timeline; B: Immunohistochemistry with CD3+ staining; images at x20 magnification; C: Images of tumor nodules in mouse livers; D: Immunohistochemistry with GS staining; images at 20x magnification; E: Quantification of tumor nodules. 5 65518548 Attorney Docket No.40069/108; ITER-009/01WO FIG.4 shows results of experiments testing TBL as a target for sensitization to immunotherapy. A: 135-day study timeline; B: Quantification of liver size; C: Quantification of tumor number; D: Quantification of tumor volume; E: Images of tumor nodules in mouse livers. FIG.5 shows tumor growth curves in the treatment of the syngeneic model H22. FIG.6 shows dose response curves for different cell lines plotted based on IC50 data. FIG.7A shows Cycle 1, Day 1 infusion tegavivint concentration versus time profiles by dose level in BCI-001-DT17. FIG.7B shows Cycle 1, Day 1 AUC0-168 hour by total dose (mg) of tegavivint administered. FIG.8 shows objectives and related endpoints for a study of tegavivint alone and in combination with pembrolizumab according to various embodiments of the invention. FIG.9 shows a scheme for tegavivint single agent dose optimization and expansion according to various embodiments of the invention. FIG.10 shows a scheme for tegavivint plus pembrolizumab dose escalation and optimization according to various embodiments of the invention. DETAILED DESCRIPTION Definitions 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 the 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. 6 65518548 Attorney Docket No.40069/108; ITER-009/01WO 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. The term “tegavivint” refers to a compound having the following structure: The 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. 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 the 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. The term “mammal” includes, but is not limited to, a human. 7 65518548 Attorney Docket No.40069/108; ITER-009/01WO 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. 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. 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, an 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/β-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 8 65518548 Attorney Docket No.40069/108; ITER-009/01WO 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. 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. The term “nanoparticulate composition” refers to compositions wherein all, or almost all of the particles are less than 1000 nm. The term “lyophilized formulation” refers to a formulation resulted from freeze-drying of an aqueous solution. 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. 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. The term an “injectable formulation” refers to a formulation that is suitable for parenteral administration, e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal administration. 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. The term, “AUClast,” as used herein, refers to the area under the curve from the time of dosing to the time of the last measurable (positive) concentration. 9 65518548 Attorney Docket No.40069/108; ITER-009/01WO 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. The term, “Tmax,” as used herein, refers to the time after administration of a drug when the maximum plasma concentration is reached. Formulations of Tegavivint 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. 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. 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. 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. 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. 10 65518548 Attorney Docket No.40069/108; ITER-009/01WO 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 I 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. 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. 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. 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 stability 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. 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 11 65518548 Attorney Docket No.40069/108; ITER-009/01WO temperature of less than about 60° C, the nanosuspension further undergoes the annealing process at or above 60° C. 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. In one embodiment, the formulations of the invention, including but not limited to pre- lyophilized formulations, are filtered to remove any large particles. In one embodiment, the filter is 10 micron. 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 (μm) when measured using laser diffraction. In one embodiment, the particles have an effective D90 of about 100 nm when measured using laser diffraction. 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. In one embodiment, the formulation may be lyophilized. In one embodiment, the particles have an effective D90 of less than or equal to 0.2 micron when measured using laser diffraction. In one embodiment, the particles have an effective D50 of less than or equal to 0.12 micron when measured using laser diffraction. 12 65518548 Attorney Docket No.40069/108; ITER-009/01WO In one embodiment, the particles have an effective D10 of less than or equal to 0.1 micron when measured using laser diffraction. 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. 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 μm, it means that 90% of the particles are less than 1.0 μm in diameter. 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. 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. The provided formulations are stable for therapeutic utility. In one embodiment, the lyophilized formulations of the invention are anhydrous. In one embodiment, the formulations, including lyophilized formulations, of the invention are stored in a dry atmosphere. The storage temperature for the formulations of the invention can be about −20° C, about 5° C, or about 25° C. 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 13 65518548 Attorney Docket No.40069/108; ITER-009/01WO of less than or equal to 1.0 micrometer (μm) 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. In one embodiment, tegavivint concentration in a formulation is 2% (20 mg/mL). In one embodiment, tegavivint concentration in a formulation is 2.5% (25 mg/mL). In another embodiment, tegavivint concentration in a formulation is 5% (50 mg/mL). In one embodiment, the poloxamer is Poloxamer 188. In one embodiment, the poloxamer concentration in a formulation is 6 mg/mL. 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. 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. 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 (μm) 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. In one embodiment, tegavivint concentration in a pre-lyophilized formulation is 2% (20 mg/mL). In one embodiment, tegavivint concentration in a pre-lyophilized formulation is 2.5% (25 mg/mL). In another embodiment, tegavivint concentration in a pre-lyophilized formulation is 5% (50 mg/mL). In one embodiment, the poloxamer is Poloxamer 188. 14 65518548 Attorney Docket No.40069/108; ITER-009/01WO In one embodiment, the poloxamer concentration in a pre-lyophilized formulation is 0.6%. In another embodiment, the poloxamer concentration in a pre-lyophilized formulation is 1.25%. 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%. In one embodiment, the sorbitol concentration in a pre-lyophilized formulation is 5%. In one embodiment, the formulation is autoclaved prior to lyophilization. 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. The invention also includes pre-lyophilized formulations of tegavivint. In one embodiment, the pre-lyophilized formulation is prepared by a process comprising ball milling at a temperature of about 60° C. In one embodiment, the pre-lyophilized formulation is prepared by a process comprising high energy milling at a temperature of about 60° C. In one embodiment, the tegavivint in the pre-lyophilized formulation is Form I polymorph. 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. In a preferred embodiment, the formulations of the invention exhibit long term stability. 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 15 65518548 Attorney Docket No.40069/108; ITER-009/01WO 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). 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. 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: AUClast 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. 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; 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. The pharmaceutical formulations of the invention can further comprise one or more pharmaceutically acceptable excipients, carriers, or a combination thereof. 16 65518548 Attorney Docket No.40069/108; ITER-009/01WO 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. 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. 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. 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 anti- cancer agent and/or cancer therapy (for example, cancer vaccines, anti-cancer adoptive cell therapies and radio therapies). 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 repair pathway inhibitors, anti-hormonal agents, anti-cancer antibodies, and other cytotoxic chemotherapy agents. In some embodiments, the additional anti-cancer agent is an immune checkpoint inhibitor. Immune checkpoint inhibitors that may be used in combination therapies according to 17 65518548 Attorney Docket No.40069/108; ITER-009/01WO embodiments of the invention include monoclonal antibodies that target checkpoint proteins on immune cells, such as, but not limited to, anti-programmed cell death-1 (PD-1) antibodies and anti-programmed death-ligand 1 (PD-L1) antibodies. Examples of PD-1 inhibitors include pembrolizumab (Keytruda), nivolumab (Opdivo), and cemiplimab (Libtayo). Examples of PD- L1 inhibitors include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). Additional antibodies may also be used, including, for example, CTLA-4 inhibitors such as ipilimumab (Yervoy) and tremelimumab (Imjuno), and LAG-3 inhibitors such as relatlimab (which may be provided with nivolumab in a combination known as Opdualag). The immune checkpoint inhibitors 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. For administration of immune checkpoint inhibitors, 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 the 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. When used in combination therapies according to embodiments of the present invention, tegavivint and immune checkpoint inhibitors 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 immune checkpoint inhibitors can be administered at the same frequency or at different frequencies. The dosage and 18 65518548 Attorney Docket No.40069/108; ITER-009/01WO scheduling of each may change during a course of treatment. Treatment may continue until complete response or confirmed progressive disease. The invention encompasses formulations including tegavivint and a pharmaceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof. 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). The compositions of the invention can further comprise one or more pharmaceutically acceptable excipients, carriers, or a combination thereof. The pharmaceutically acceptable excipients used in the formulation of the present invention can act in more than one way. The pharmaceutically acceptable excipients can be, for example, a dispersion medium, a dispersion emulsifier, a dispersion enhancer, or a combination thereof. 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. The dispersion medium can be, for example, ethanol, propylene glycol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, glycerin, a combination thereof, etc. 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 19 65518548 Attorney Docket No.40069/108; ITER-009/01WO monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, poloxamer, a combination thereof, etc. 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, polyoxylglycerides, pyrrolidone, sorbitan esters, stearic acid, vitamin E polyethylene glycol succinate, polyethylene glycol 1000, povidone, a combination thereof, etc. 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. 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. 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® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC™). 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. 20 65518548 Attorney Docket No.40069/108; ITER-009/01WO 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. 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. Suitable diluents include pharmaceutically acceptable inert fillers, 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. 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. 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. 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. 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. 21 65518548 Attorney Docket No.40069/108; ITER-009/01WO Tegavivint in Hepatocellular Carcinoma 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. 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. 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 atezolizumab and bevacizumab). However, despite these achievements HCC remains a deadly disease with poor prognosis in patients with unresectable cancer. Up to 40% of HCC patients have tumors driven by activating β-catenin and canonical Wnt pathway mutations. The most frequently mutated genes include CTNNB1, AXIN1, and APC. Most mutations in β-catenin occur within exon 3, the critical region responsible for its proteasomal degradation. These exon 3 mutations render β-catenin resistant to degradation, resulting in nuclear accumulation dysregulated transcriptional activity. Activated β- catenin/canonical Wnt signaling is associated with primary resistance to standard of care immune checkpoint blockade mediated through immune cell excluded ‘cold’ tumor microenvironment. 22 65518548 Attorney Docket No.40069/108; ITER-009/01WO Recruitment of β-catenin to canonical-Wnt target genes requires transducin beta-like protein 1 (TBL1) to activate transcription. TBL1 has been demonstrated to be necessary for β-catenin oncogenic activity. During Wnt-signaling, TBL1 binds to β-catenin, inhibiting its degradation, and forms an active transcriptional complex that binds to promoters to activate downstream cancer-related genes such c-MYC, Cyclin D1 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. Tegavivint is a first-in-class small molecule inhibitor of TBL1, a novel downstream Wnt- signaling pathway target. Tegavivint binds to TBL1 in the β-catenin pocket, disrupting the formation of the activation complex necessary for oncogenic activity. Tegavivint also enables the degradation of free nuclear β-catenin. Increased expression of β-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 β-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 β-catenin mutations and is currently being studied through Investigator Initiated Trials (IITs) in acute myeloid leukemia (AML), pediatric solid tumors, non-small cell lung cancer (NSCLC), and lymphoma. In Vivo HCC Data The efficacy of tegavivint was investigated in a genetically engineered C57BL/6J mouse model (GEMM) of β-Cateninexon3 mutant HCC. At day 0, C57BL/6J mice with Ctnnb1exon3/Wt Rosa26c-MYC/c-MYC alleles were either injected with a high dose of the AAV-Cre vector to test the efficacy of tegavivint in inhibiting canonical β-Catenin target genes (Figure 1A; acute oncogene activation model) or a low dose of 23 65518548 Attorney Docket No.40069/108; ITER-009/01WO the AAV-Cre vector to test tegavivint reduction of clonal liver tumors representative of human HCC (Figure 2A, Figure 3A). At day 4 (Figure 2A), day 5 (Figure 1A), or day 90 (Figure 3A) following oncogene activation, mice (n = 5 per group) were randomized to receive either tegavivint or vehicle. Tegavivint was dosed via intraperitoneal (i.p.) injection at 1.5 mg per mouse daily, with 5 days per week on treatment and 2 days off treatment. Study endpoints were either at day 9 (Figure 1A), day 28 (Figure 2A) or day 120 (Figure 3A). Tegavivint treatment of mice with activated β-cateninexon3 signaling throughout the entire liver significantly inhibited overexpression of canonical Wnt target genes, Glul, Axin2, Notum in early-stage disease (Figure 1B). In intestinal tissue, which depends on canonical Wnt signaling to maintain tissue homeostasis, off target effects were not observed. Inhibition of TBL1 in early-stage disease significantly reduced the number of tumors initiating clones identified by markers of canonical Wnt activation (Glutamine Synthetase, GS) compared with vehicle controls at day 9 (Figure 1C) and day 28 (Figure 2B). At day 9, tegavivint also increased proliferation identified by Ki67 staining (Figure 1D). As early as day 9, tegavivint induced an immunomodulatory response via increased infiltration of T-lymphocytes identified by hematoxylin and eosin (Figure 1E) and then via CD3+ staining at day 120 (Figure 3B). Furthermore, treatment of established β-cateninexon3 activated tumors with tegavivint inhibited tumor growth and resulted in reduced tumor burden compared to vehicle at day 120 (Figure 3C-Figure 3E). The efficacy of tegavivint in combination with an immune checkpoint inhibitor was also investigated. At day 90 (Figure 4A) following oncogene activation, mice (n = 5 per group) were randomized to receive tegavivint (1.5 mg i.v. twice weekly) in combination with an anti-PD-1 antibody (aPD1/anti-PD-1) or isotype control (200 µg i.p. twice weekly). The study endopoint was 135 days (Figure 4A). Combination therapy comprising tegavivint and anti-PD-1 antibody reduced liver size (liver/body ratio %), number of tumors (macroscopic tumors/liver), and tumor volume (tumor burden mm3) as compared to tegavivint and isotype control (Figures 4B-4E). 24 65518548 Attorney Docket No.40069/108; ITER-009/01WO To evaluate the pharmacodynamic response to tegavivint, cells from the HCC syngeneic tumor type, H22, were inoculated subcutaneously in the right lower flank region of BALB/c mice. I.p. treatment with tegavivint 100 mg/kg or vehicle (phosphate-buffer solution [PBS]) was initiated when tumors reached a mean volume of approximately 115.42 mm3 (n=6 per group). Body weight and tumor volumes were measured twice or three times during the study (including on day 1 and at sacrifice). H22 mice were sacrificed on day 3. Tumors were collected from nine models on the termination day. Each tumor was divided for fluorescence activated cell sorting (FACS) analysis, and formalin-fixed paraffin-embedded (FFPE) blocks. FACS analysis employed side scatter-forward scatter (SSC-FSC) plots to determine tumor infiltrating lymphocytes (TILs). A Bartlett test was used to test homogeneity of variance and normality. Group means were compared using analysis of variance (ANOVA), Kruskal-Wallis, and Wilcoxon rank sum tests. If the p-value of Bartlett test was no less than 0.05, ANOVA and two sample t-tests were used to compare group means. If the p-value of Bartlett test was less than 0.05, Kruskal-Wallis test and Wilcoxon rank sum test were used to compare group means. FACS analysis demonstrated significant changes in TIL populations in H22 tegavivint-treated tumors. Decreases in CD45+ and increases in CD45- lymphocytes were observed, while reductions in mMDSC populations were observed in H22 tegavivint-treated tumors. Increases in PD-1+ CD3+ and PD-1+ CD4+ lymphocytes and M2 macrophages were also observed in H22 tegavivint-treated tumors. The efficacy and tolerability of tegavivint was assessed in HCC syngeneic mouse tumor model, H22. Tumor cells were inoculated subcutaneously in the right front or lower flank region of BALB/c mice. I.p. treatment with tegavivint 50 mg/kg or vehicle (PBS) (n=10 per arm) was initiated when tumors reached a mean volume of approximately 93.05 mm3. Mice were sacrificed when the mean tumor burden reached ≥2000 mm3. In this mouse model, tegavivint 50 mg/kg demonstrated statistically significant TGI of 50.11% in H22 (Figure 5). The H22 syngeneic mouse model was used to evaluate the efficacy and tolerability of tegavivint alone and in combination with anti-PD-1 or anti-PD-L1. H22 mouse liver tumor cells 25 65518548 Attorney Docket No.40069/108; ITER-009/01WO (1 x 106) in 0.1 mL PBS were inoculated subcutaneously in the right upper flank region of in female BALB/c mice for tumor development. Mice were enrolled and randomly allocated into six treatment groups of ten mice: 1. Vehicle 2. Tegavivint 50 mg/kg, alone 3. anti-PD-110 mg/kg, alone 4. anti-PD-L110 mg/kg alone 5. Tegavivint 50 mg/kg plus anti-PD-110 mg/kg 6. Tegavivint 50 mg/kg plus anti-PD-L110 mg/kg. All test articles were administered i.p. over 15 days. Tegavivint was administered five times a week (5 days on, 2 days off). Immune checkpoint inhibitors (ICIs) were administered twice weekly (BIW). The tumor growth inhibition (TGI) of treatment with tegavivint at 50 mg/kg (G2) group was 43.50% (Table 1). Anti-PD-110 mg/kg alone and anti-PD-L110 mg/kg alone both resulted in significant TGIs (Table 1). When performing comparisons between the combination treatment groups with single agent groups, tegavivint 50 mg/kg plus anti-PD-1 at 10 mg/kg and tegavivint 26 65518548 Attorney Docket No.40069/108; ITER-009/01WO 50 mg/kg plus anti-PD-L110 mg/kg gross antitumor efficacy was insignificant in comparison to tegavivint, anti-PD-1, and anti-PD-L1 single agent administration (Table 2). 27 65518548 Attorney Docket No.40069/108; ITER-009/01WO Tegavivint, alone or in combination with anti-PD-L1 or 1 anti-PD-1, was well tolerated with manageable body weight loss. Animals given tegavivint 50 mg/kg alone and in combination with anti-PD-110 mg/kg or anti-PD-L110 mg/kg exhibited treatment-related body weight loss of 10.27%, 5.98%, and -3.00%, respectively. Tegavivint administration schedule was modified to three times per week on day 8 to mitigate body weight loss. Animal body weight loss was also stabilized with the introduction of dietary supplemental gels. In summary, tegavivint has shown anti-tumor and immunomodulatory activity in a β- catenin activating mutant mouse model of HCC. Tegavivint also reduced tumor volume and promoted an immunomodulatory response in the syngeneic HCC model, H22. Tegavivint tumor response in HCC model, H22 was reproduced in a separate study, in which the therapeutic efficacy and tolerability of tegavivint alone or in combination with either anti-PD-1 or anti-PD- L1 was tested in the treatment of the subcutaneous H22 mouse liver cancer tumor syngeneic model in female BALB/c mice. In Vitro HCC Data The effect of tegavivint was investigated in nine HCC and hepatoblastoma cell lines in a 2D viability assay. The results were compared to cisplatin as a reference control. In order to calculate absolute IC50, a dose-response curve was fitted using nonlinear regression model with a sigmoidal dose response. The formula for calculating surviving rate is shown below: Surviving rate (%) = (LumTest article-LumT0)/ (LumNone treated-LumT0)×100% 28 65518548 Attorney Docket No.40069/108; ITER-009/01WO The data are summarized in Table 3 and Figure 6. Pharmacokinetics and Absorption/Distribution/Metabolism/Excretion (ADME) Studies Tegavivint demonstrates high bioavailability when administered intravenously, with complete systemic exposure. The compound exhibits high plasma protein binding exceeding 99% across all species tested, including mice, rats, dogs, minipigs, monkeys, and humans. This characteristic contributes to its prolonged circulation time and extended half-life, which ranges from 16.5 hours in mice to over 50 hours in larger animals such as minipigs (56 hours) and dogs (53 hours). Following intravenous administration, tegavivint shows a distinctive distribution pattern with initial localization primarily in the lungs, followed by subsequent redistribution to the liver and spleen. The volume of distribution exceeds total body water across species, indicating extensive tissue distribution. The compound demonstrates relatively low plasma clearance compared to liver blood flow, suggesting minimal first-pass metabolism. Notably, tegavivint shows limited penetration 29 65518548 Attorney Docket No.40069/108; ITER-009/01WO across the blood-brain barrier, but exhibits favorable tumor distribution characteristics as demonstrated in mouse models. The distribution pattern of the nanoparticle formulation aligns with established literature showing initial lung distribution followed by hepatic and splenic redistribution. The metabolic profile of tegavivint is consistent across species. The parent drug remains the predominant circulating compound, with CYP3A4 serving as the primary metabolizing enzyme. In human hepatocytes, approximately 80% of the parent drug remains after 120 minutes of incubation. The primary metabolic pathways include oxidation and glucuronidation, producing predictable metabolites. Importantly, no unique human metabolites have been identified, suggesting good translation of preclinical safety findings to humans. Drug interaction studies demonstrate moderate to high inhibition of CYP2C8, necessitating caution when co-administered with CYP2C8 substrates, though inhibition of other CYP enzymes is limited. Additional in vitro studies evaluated the potential of tegavivint to inhibit UDP- glucuronosyltransferases (UGTs) and drug transporters. Using both human liver microsomes and recombinant protein systems, tegavivint demonstrated potent inhibition of UGT1A1 (IC50: 29.8 nM recombinant/236 nM microsomes; maximum inhibition: 92% recombinant/75% microsomes), UGT1A3 (IC50: 66.0 nM recombinant/169 nM microsomes; maximum inhibition: 87% recombinant/64% microsomes), and UGT1A4 (IC50: 23.6 nM recombinant/145 nM microsomes; maximum inhibition: 89% recombinant/70% microsomes). Minimal to no inhibition was observed for UGT1A6, UGT1A9, and UGT2B7. In transporter studies, tegavivint showed selective inhibition of key hepatic transporters. Tegavivint inhibited OATP1B1-mediated transport with an IC50 of 0.81 μM and maximum inhibition of 82%. Significant inhibition was also observed for BSEP (IC501.63 μM, 86% maximum inhibition) and BCRP (IC503.94 μM, 60% maximum inhibition). Other evaluated transporters including MATE1/2-K, MRP1/3, NTCP, and OCT1/3 showed minimal to no inhibition. The concurrent inhibition of both metabolic enzymes and transporters integral to hepatic function, particularly UGT1A1 and the OATP1B1/BSEP pathway, indicates that tegavivint may influence bilirubin homeostasis and xenobiotic clearance. Therefore, caution is advised when co- 30 65518548 Attorney Docket No.40069/108; ITER-009/01WO administering tegavivint with UGT1A1, UGT1A3, or UGT1A4 substrates, particularly those metabolized by UGT1A1, as this could impact bilirubin clearance. Similarly, caution should be taken with OATP1B1, BSEP, and BCRP substrates due to potential effects on hepatic uptake and biliary excretion pathways. Elimination studies conducted in rats demonstrate that unchanged tegavivint is not recovered in urine, and less than 2% of the initial dose is recovered in feces. The compound's slow elimination from systemic circulation is likely due to a tissue depot effect, though the drug is eventually metabolized. The extended half-life observed across species, particularly in larger animals, combined with the tissue distribution and metabolism patterns, supports a once-weekly intravenous administration schedule in clinical applications. Preclinical Toxicology GLP toxicology studies of the clinical formulation of BC2059 (tegavivint) are described below. In rats, the pathologies observed were primarily related to the infusion itself, and manifested as local infusion site reactions, although lung edema was observed in female rats at the dose exceeding the Maximum Tolerated Dose (MTD). In minipigs, the kidney was identified as the main target organ of toxicity with mild tubular necrosis and increases in serum creatinine and urea nitrogen levels. Reversible thrombocytopenia was also occasionally noted. GLP Toxicology studies: Rat The GLP 4-week toxicology rat study was conducted with doses of 0 (Group 1), 10 (group 2), 30 (Group 3) and 60 mg/kg (Group 4) administered twice weekly for a total of 8 doses. Animals were given BC2059 or vehicle (5% dextrose) via continuous infusion over 24 hours (on Days 1, 4, 8, 11, 15, 18, 22, and 25). The highest dose, 60 mg/kg, exceeded the maximum tolerated dose as 4 female rats at this dose level were found dead on Day 5 or 6. Histologically, alveolar edema, fibrin, alveolar macrophages, and hemorrhage were observed in these 4 females as the cause of mortality and were attributed to the physical nature of the nanomilled drug and large number of particles being infused into a small animal species. The high dose was subsequently lowered to 40 mg/kg for doses 3 31 65518548 Attorney Docket No.40069/108; ITER-009/01WO through 8. Additional premature deaths were observed: 3 males at 60 m/kg, 1 female at 60 mg/kg, 1 female at 30 mg/kg and 1 female in the control group. However, the cause of mortality in each of these rats was attributed to infusion site infection/tissue reaction and catheter retraction and not a direct toxicity of BC2059 or the nanoparticle formulation. The lower dose of 40 mg/kg administered for Doses 3-8 was well tolerated for the remainder of the study and no mortality nor life threatening toxicities were observed at this dose level. BC2059 treatment resulted in non-life-threatening changes in the liver, spleen and adrenal glands at the 30 mg and 60/40 mg/kg dose groups but alterations observed microscopically were not of sufficient severity to alter clinical pathology profiles. Importantly, the findings observed in the lungs, liver, spleen and adrenal gland of rats given 30 or 60/40 mg/kg were reduced or absent in the recovery group animals. The peak blood concentrations (Cmax) and overall systemic concentrations determined from the plasma concentration over time curves (AUC0-t) are provided in Table 4. Systemic exposures were consistently lower in female rats compared to male rats and this was especially evident after Dose 8. There was no appreciable accumulation of BC2059 with repeated bi- weekly dose administration, evidenced by the similar Cmax and AUC0-t values, for Dose 1 and Dose 8. However, pre-dose concentrations of BC2059 at Dose 8, which reflect the plasma concentration at approximately 48 h after completion of the Dose 7 infusion, were present at meaningful levels indicating some dose-dependent persistence of the drug in the systemic circulation that did not translate into increased overall systemic exposures to the drug. Table 4: Systemic Exposures in the GLP Toxicology Study in Rats 65518548 Attorney Docket No.40069/108; ITER-009/01WO GLP Toxicology studies: Minipig In the GLP 4-week toxicology minipig study, animals were divided into five treatment groups that included a vehicle control (Group 1: 5% dextrose), 1 mg/kg (Group 2), 5 mg/kg (Group 3) and 15 mg/kg (Group 4) BC2059 administered over 24 h and a 4 h infusion group with 5 mg/kg BC2059 (Group 5). The infusions were administered on Days 1, 6, 8, 13, 15, 20, 22 and 27 for a total of 8 doses. There were no treatment-dependent clinical observations. There were no effects on body weight or food consumption. There were no findings in the electrocardiograms (ECGs) or ophthalmologic exams. Clinical chemistry and coagulation values did not differ between baseline and post-treatment. There was a significant decrease in platelet count values at the 15 mg/kg dose level, although the values remained in the normal range and the decrease resolved in those animals kept for observation after dosing ceased. However, given the observation of decrements in platelets at higher doses in minipigs (20, 40 and 50 mg/kg over 24-h) and dogs (15 mg/kg twice weekly over 4-h) in separate non-GLP studies, this finding might provide a monitorable drug-dependent effect. There were no treatment-related findings in gross anatomic pathology and no treatment related findings in microscopic examination. The only potential test article related histology findings were perivascular mononuclear cell infiltrates in the brain and spinal cord. Although this finding was observed in two female control animals and two low dose females (1 mg/kg), it was more frequently observed in animals from Groups 3 (5 mg/kg, 24 hour infusion), 4 (15 mg/kg), and 5 (5 mg/kg, 4 hour infusion) of both sexes. The underlying pathogenesis or significance of this infiltration of mononuclear cells is unclear, but it appears that Group 2 at 1 mg/kg/dose represented the no observed effect level. It was also apparent that 28 days are sufficient to allow full recovery. The systemic exposures to BC2059 are provided in Table 5 and the terminal elimination half- lives are presented in Table 6. There were no apparent differences between genders in PK parameters and plasma concentrations; therefore, the genders were combined. In all treatment groups, drug accumulation was evident as Cmax and AUC were increased 1.3 to 2.4-fold when comparing the systemic exposures after Dose 8 to those observed with Dose 1. Half-lives ranged from 40 to 70 h. This long half-life for the drug is reflected in the accumulation of drug with repeated bi-weekly doses 33 65518548 Attorney Docket No.40069/108; ITER-009/01WO of BC2059 and in the trough concentrations prior to Dose 8. At 120 h after initiation of Dose 7, or 96 h after completion of the infusion in Groups 2, 3 and 4 and 116 h after completion of the infusion in Group 5, plasma concentrations were still in the biologically active range for in vitro tumor cell killing by BC2059. A long half-life of 47 to 60 h was also observed in a non-GLP study in dogs where BC2059 was administered at 15 mg/kg over a 4-h infusion. On this basis, once weekly administration of BC2059 is likely to provide therapeutic concentrations for the entire week. In this GLP toxicology study in minipigs, there were no serious adverse toxicities at the 5 mg/kg dose, regardless of whether the length of the infusion was 4 h or 24 h. The systemic exposures to BC2059 similarly do not reflect any marked difference in systemic exposures between the two infusion durations. As shown in Table 6 when BC2059 was infused at 5 mg/kg over 4 hours, mean Cmax and AUClast were very much the same as determined with the 5 mg/kg dose infused over 24 hours. Plasma concentrations over time for the two different infusion durations of 5 mg/kg are provided in Table 7. As anticipated, with the shorter 4 h infusion, higher plasma concentrations are initially observed at up to 8 h post initiation of infusion. However, peak plasma concentrations are no greater than 1.3-fold higher in the 4 h infusion compared to the 24 h infusion. At the 24 h time point, there is no difference in plasma concentrations for minipigs in the 24 h infusion versus the 4 h infusion. Consequently, the infusion durations are considered to be comparable and supportive of a 4 h infusion duration in the clinical trial. Table 5: Systemic Exposures in the GLP Toxicology Study in Miniature Swine 34 65518548 Attorney Docket No.40069/108; ITER-009/01WO Table 6: Estimates of Terminal Elimination Half-Life in the GLP Toxicology Study in Miniature Swine Table 7: Plasma Concentrations in Miniature Swine Administered 5 mg/kg over a 24 h and a 4 h Infusion Non-GLP Toxicity study: Minipig The results of the GLP toxicology study in minipigs defined dose levels at which no toxicities with BC2059 infusion were observed; that is, 15 mg/kg. In a 2-dose study in minipigs, a total of four female Yucatan miniature swine were administered an IV infusion of the test article for 24-consecutive hours on Day 1 with a target dose level of 20 mg/kg/day and 40 mg/kg/day. On Day 5, a second IV infusion of the test article was administered for 24-consecutive hours with a target dose level of 20 mg/kg/day and 35 mg/kg/day. The high dose of 40 mg/kg was not well tolerated with clinical signs of lethargy and decreased food consumption and was lowered to 35 mg/kg for Day 5 dosing; one of two animals was 35 65518548 Attorney Docket No.40069/108; ITER-009/01WO not dosed for a second time. Platelet counts were markedly decreased for 3 of the animals, but increased in the pig that was not administered the second infusion on Day 5, indicating reversibility of this finding. In the kidneys, minimal tubular necrosis was observed in both Group 2 animals, and was associated with minimal to mild acute inflammation. These histopathological findings were accompanied by increases in serum blood urea nitrogen (BUN) and creatinine. In the lungs, acute alveolar inflammation involved all animals and was moderate in severity in both high-dose and one low-dose animal, but of minimal severity in the other low-dose animal alveolar inflammation. These changes were considered possibly related to treatment. To sum, this two dose study of BC2059 identified the kidney as the target organ for toxicity. The response was likely related to drug and not to the physical nature of the nanoparticles. Lung findings were not definitively linked to drug formulation in this minipig study, in contrast to the GLP toxicology study in rats. Importantly, the findings in the kidneys would suggest that in clinical trials with the drug, changes in the kidney are a more sensitive indicator of drug-induced adverse effects than changes related to the nanoparticle nature of the formulation. The results of this non-GLP study are not used for dose setting, but provide insight into potential monitorable toxicities in clinical development. Clinical Experience of Tegavivint BCI-001-DT17, Phase 1 Trial of BC2059 (Tegavivint) in Patients with Unresectable Desmoid Tumor: This study was a phase I, open-label, non-randomized study to evaluate safety of tegavivint administered intravenously to patients with proven primary or recurrent desmoid tumors that are unresectable and symptomatic or progressive (defined below). This study utilized single patient cohorts for the first two dose levels (0.5 mg/kg and 1 mg/kg) in order to minimize sub- optimal drug exposures, followed by a conventional 3+3 dose escalation phase to achieve the RP2D determined by pharmacokinetics and biologically relevant activity. Once the RP2D was determined, that dose level cohort (5 mg/kg) was expanded with 7 additional patients enrolled to collect further 36 65518548 Attorney Docket No.40069/108; ITER-009/01WO safety, PK, and PD data. Dose expansion also included 6 patients enrolled at lower doses levels who were eligible for intrapatient dose escalation to the RP2D. Symptomatic or progressive disease was defined as a 20% increase in tumor volume within 6 months, or recurrent disease within 1 year of surgery, or desmoid related symptoms and documentation that symptoms were related to desmoid and not prior therapies. Patients were also required to have bi-dimensionally measurable tumors by WHO criteria; be ≥18 years of age; and have an ECOG performance score of 0-1. Patients with familial adenomatous polyposis were excluded. Primary objectives were to assess safety, dose limiting toxicities (DLTs), MTD or maximum administered dose (MAD), RP2D of tegavivint, response rate via WHO criteria, and PFS rate at 9 months. Secondary objectives included determining the duration of response after objective response, pharmacokinetics, and evaluation of patient reported outcomes. Rationale for Starting Clinical Dose and Duration in BCI-001-DT17: The high dose of 60 mg/kg in the GLP toxicology study in rats exceeded the Severely Toxic in 10% of the animals Dose (STD10). The 30 mg/kg dose given over 24 h was tolerated with no deaths and no severe life- threatening histopathological findings and this dose is considered the STD10. In the GLP 4-week toxicology minipig study, doses of up to 15 mg/kg over a 24 h infusion and 5 mg/kg over a 4 h infusion were administered with no severe adverse toxicities in this study. The lower dose of 5 mg/kg over the 4 h infusion was considered the Highest Non-Severely Toxic Dose (HNSTD) for this infusion duration. On the basis of those GLP toxicology studies, the starting clinical dose was defined as outlined in the ICH S9 Guidance “Nonclinical Evaluation for Anticancer Pharmaceuticals” as is detailed in Table 8. Table 8: Estimation of Starting Clinical Dose Based on GLP Toxicology Studies According to ICH S9 Guidance 37 65518548 Attorney Docket No.40069/108; ITER-009/01WO The starting clinical dose was defined to be 0.5 mg/kg for the initial dose finding study BCI- 001-DT17. This starting dose was based on the lowest starting dose determined across both the minipigs and the rats. Certainly, the minipig is the more relevant species in terms of similarity of infusion conditions to human. The infusion volume in minipigs and total volume is comparable to anticipated clinical volume. Physiologically, the minipig also more closely resembles the human, and the safety of the nanoparticles in terms of size of the nanoparticles and size of the microvasculature in the two species. Indeed, in the rat, there were toxicities observed that were related to the infusion procedure itself, with findings in several control animals. However, the most conservative approach was to utilize the 30 mg/kg dose in rats for the HNSTD as opposed to the 5 mg/kg dose administered over 4 h in minipigs to support the original starting clinical dose of 0.5 mg/kg in BCI-001-DT17. Tegavivint was administered as a 4-hour infusion on day 1, 8, 15 of a 28 day cycle and assessed the following dose levels: 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg and 5 mg/kg. Patients were treated for 2 years or until disease progression, unacceptable adverse events, death, or withdrawal of consent. A total of 24 patients were enrolled and treated in BCI-001-DT17. All results presented include data from all patients enrolled and treated during the study period. Six dose levels were assessed: 0.5 mg/kg, n = 1; 1 mg/kg, n = 2; 2 mg/kg, n = 3; 3 mg/kg, n = 3; 4 mg/kg, n = 5; and 5 mg/kg, n = 10. The median age of all participants was 43 years (range 18-66), 96% (n = 23) were female, and 16 (66.7%) patients had an ECOG performance status of 0 with the rest of the patients with a score of 1 (n = 8). Of the 24 patients enrolled, 15 38 65518548 Attorney Docket No.40069/108; ITER-009/01WO (63%) were White, 4 (17%) Asian, 3 (13%) Black, and 2 (8%) other races. The median time from initial diagnosis of desmoid to Cycle 1, Day 1 was 3.1 years (range 0.3-19.7). Primary sites of tumor involvement included extremity or trunk (n = 13, 54%), abdomen or pelvis (n = 8, 33%); and spinal or nervous system (n = 3, 13%). The median number of prior systemic treatments was 1 (range 0-6). Seventy one percent of patients had received prior systemic treatment with either tyrosine kinase inhibitors (n = 12, 50%), cytotoxic chemotherapy (n =9, 38%), tamoxifen (n = 6, 25%), non-steroidal anti-inflammatory agents (n = 4; 17%); and investigational agents (n = 3; 13%). Tumor resection was previously performed in 10 (42%) patients. Radiation, cryoablation, or high intensity ultrasound was previously conducted in 4 (17%) patients. No dose-limiting toxicities were observed, and the recommended phase 2 dose (5 mg/kg) was determined based on achieving pharmacologically relevant plasma concentrations and preliminary efficacy. The median number of cycles of tegavivint completed across all patients was 10 (interquartile range: 4.1-20.9). The median relative dose intensity was 97% (range: 83- 100) and was consistent across dose levels. The median number of missed infusions for any reason was 0.5 (range 0-4). One patient had a dose reduction from 4 mg/kg to 3 mg/kg for grade 3 anemia considered not related to study treatment and subsequently tolerated intrapatient dose escalation to 5 mg/kg with no dose reductions or interruptions in therapy. The primary reasons for discontinuation of study treatment were: 5 (20.8%) patients completed treatment; 5 (20.8%) patients discontinued due to progressive disease; 1 (4.2%) patient for an adverse event considered not related to tegavivint; and 12 patients for withdrawal of consent or other reasons. No deaths occurred on study. Overall, a total of 566 AEs occurred in the study, with all patients experiencing at least one AE. Grade 1 AEs were observed in all patients and represented 72% of all AEs that occurred during the study. Grade 2 AEs were observed in 22 patients (91.7%) and represented 24.9% of all AEs occurring during the study. Grade 3 AEs occurred in 9 patients (37.5%) and represented 3% of all AEs occurring during the study. There were no grade 4 AEs. The most commonly 39 65518548 Attorney Docket No.40069/108; ITER-009/01WO occurring adverse events (≥ 20% of patients) of any grade included: fatigue (83.3%, n = 20), headache (62.5%, n = 15), nausea (45.8%, n = 11), diarrhea (37.5%, n = 9), constipation (33.3%, n = 8), arthralgia (33.3%, n = 8), dysgeusia (25%, n = 6), paresthesia (25%, n = 6), anemia (25%, n = 6), vomiting (20.8%, n = 5), myalgia (20.8%, n = 5), decreased appetite (20.8%, n = 5), and depression (20.8%, n = 5). Only one grade 3 event occurred in more than one patient: hypophosphatemia (8.3%, n = 2). Overall, a total of 227 treatment related AEs (considered at least possibility related to tegavivint) occurred on study in 23 patients (95.8%). Grade 1 treatment related AEs occurred in 21 patients (87.5%) and represented 73% of all treatment related AEs observed. Grade 2 treatment related AEs occurred in 16 patients (66.7%) and represented 24.7% of all treatment related AEs observed. Grade 3 treatment related AEs occurred in 5 patients (20.8%) and represented 2.2% of all treatment related AEs observed. No grade 4 treatment related AEs were observed. The most commonly occurring treatment related AEs (≥ 20% of patients) of any grade included: fatigue (70.8%, n = 17), headache (37.5%, n = 9), nausea (33.3%, n = 8), dysgeusia (20.8%, n = 5), constipation (20.8%, n = 5), and decreased appetite (20.8%, n = 5). Grade 3 treatment related AEs occurred in 5 separate patients. No grade 3 treatment related AE occurred in more than one patient and included the following AEs with the dose level indicated: hypophosphatemia (2 mg/kg); stomatitis (3 mg/kg); ALT increase (5 mg/kg); headache (5 mg/kg), and diarrhea (5 mg/kg). Infusion or injection site reactions occurred in three patients. One patient experienced a grade 2 infusion site extravasation that resulted in pain and inflammation at the site of infusion, but no tissue necrosis was observed. The event was classified as a serious adverse event due to hospitalization for observation and considered related to tegavivint. This patient was able to tolerate subsequent treatment with tegavivint. Two other patients reported infusion site reactions, both grade 1, that included erythema, pain and swelling. 40 65518548 Attorney Docket No.40069/108; ITER-009/01WO In addition to the infusion site extravasation described above, one additional serious adverse event of pulmonary mycobacterium avium complex infection was observed (Grade 3), which was considered not related to tegavivint. The overall type and frequency of AEs is not uncommon for a phase 1 trial, considering the duration of treatment (median of 10 cycles). No dose-limiting toxicities were observed. Higher grade AEs were infrequent and did not result in treatment discontinuation, dose reductions or interruptions. Common (>20%) treatment related adverse events include fatigue, headache, nausea, constipation, decreased appetite, and dysgeusia. Overall, a high relative dose intensity of 97% was observed over a median of 10 cycles indicating tolerability of tegavivint at doses of up to 5 mg/kg on day 1, 8, 15 of a 28 day schedule. No significant bone, cardiac, lung, ocular, renal, hepatic, or hematologic toxicity were observed. In BCI-001-DT17 efficacy was assessed via WHO and RECIST 1.1 criteria by CT scans and/or magnetic resonance imaging (MRI) every three cycles. During the study, 4 objective responses were observed (WHO or RECIST) resulting in an objective response rate of 17% across all dose levels and 25% at the RP2D (WHO or RECIST). An additional patient achieved a 49% reduction (WHO) and was then judged surgically resectable and taken off study. Median duration of response was 9.5 months (interquartile range 2.6 to 13.3 months), and all responses were ongoing at the end of the study. The 9-month progression free survival rate was 76.7% (95% CI: 54 - 90%) among all patients and 79.6% (95% CI: 51.7 – 93%) among those treated at RP2D. Overall, an objective response rate of 25% at the RP2D that was durable during the study demonstrates preliminary efficacy in progressive desmoid tumors. Clinical pharmacokinetics of tegavivint in BCI-001-DT17: In BCI-001-DT17, 24 patients were treated with tegavivint across six dose levels (0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, and 5 mg/kg) on Day 1, 8, and 15 of a 28 day cycle. Figure 7A graphically presents the Cycle 1, Day 1 concentration time profile from the start of the 4-hour tegavivint infusion to 168 hours post dose in the six dose levels assessed. The number of patients per dose level are depicted and data are presented as medians of all patients at each dose level with the interquartile range (25%-75%). See 41 65518548 Attorney Docket No.40069/108; ITER-009/01WO Table 9 for Cycle 1, Day 1 summary pharmacokinetic parameters by dose level. The median half-life across all dose levels was 39.9 hours (IQR 33.8-48.9) supporting once weekly administration. Accumulation was present prior to the next weekly infusion especially at the higher dose levels (4 mg/kg and 5 mg/kg), indicating sustained exposure to tegavivint between doses. Exposure (AUC) was increased at the 4 mg/kg and 5 mg/kg cohorts compared with the lower dose cohorts. An increase in AUC was observed from the 4 mg/kg to 5 mg/kg cohorts in Cycle 1 despite similar total dose administered, stemming from lower median body weight in the 5 mg/kg cohort. AUC correlated with the dose (mg) of tegavivint administered (Figure 7B). In one patient with intrapatient dose escalation from 4 mg/kg to 5 mg/kg demonstrated an increase in AUC at the 5 mg/kg dose level. Table 9: Cycle 1, Day 1 infusion pharmacokinetics of tegavivint in BCI-001-DT17 by dose level Clinical Pharmacodynamics of tegavivint in BCI-001-DT17: Clinical pharmacodynamic activity of tegavivint in humans with unresectable desmoid tumor in BCI-001-DT17 (NCT03459469) with an objective response (all partial response per WHO criteria) and serum available was analyzed for changes in serum protein biomarkers. A set of six proteins were selected as candidate biomarkers 42 65518548 Attorney Docket No.40069/108; ITER-009/01WO based on both their cancer-related function and regulation by aberrant Wnt signaling. Serum from the four patients with an objective response that had been administered tegavivint at doses of 1, 4, or 5 mg/kg were analyzed at five timepoints (Cycle 1, Day: 15 minutes pre-dose and 2 hours, 4 hours, and 24 hours after initiation of the infusion; Cycle 1, Day 8: 15 minutes pre-dose) were analyzed. Tegavivint caused substantial alterations in these beta-catenin responsive biomarkers up to the last measured collection timepoint of 192-hours post start of infusion. For several of these biomarkers a dose response trend was observed with patient 03-01 (1mg/kg) showing a less depth of change and shorter persistence than patient 04-08 (5mg/kg). One of the four patients analyzed (patient 03-04) had a highly variable response in multiple biomarkers in this assay and was found to have much higher plasma drug levels than other patients in this biomarker study. HCC Clinical Studies As described above, β-catenin activating mutations and over-active Wnt transcriptional activity are prevalent in HCC. TBL1 inhibition via tegavivint has demonstrated encouraging efficacy in preclinical models of HCC. Based on the demonstrated activity of checkpoint inhibitors in HCC and the mechanism of action of tegavivint, the tolerability of tegavivint with and without pembrolizumab in HCC, as well as the potential for this combination to circumvent innate resistance to immune checkpoint inhibition arising from activation of the Wnt/β-catenin pathway, were evaluated. Dysregulated Wnt/β-catenin signaling has been shown to play a significant role in the promotion and development of liver cancer. Between 40-70% of HCC tumor samples show activated β-catenin signaling either by the presence of strong nuclear β-catenin protein or a Wnt active transcriptional program. Activating mutations are found in the Wnt/β-catenin signaling pathway in a majority of these patients, with the most frequent being mutations in CTNNB1 (gene encoding β-catenin) found in 20-30% of patients, mutations in AXIN1 found in 8-10% of patient samples, and mutations in APC found in 5-8% of liver cancer patients (Schulze et al. 2015 Nature Genetics 47(5):505–511; Calderaro et al.2017 Journal of Hepatology 67(4):727–738; 43 65518548 Attorney Docket No.40069/108; ITER-009/01WO Harding et al.2018; Luke et al.2019 Clinical Cancer Research 25(10):3074–3083; Cowzer et al. 2023 ). While mutations in CTNNB1, APC, and AXIN1 can activate the Wnt/β-catenin pathway they do not change the ability of tegavivint to inhibit β-catenin’s oncogenic signaling. Tegavivint targets the β-catenin signaling pathway in the nuclear compartment, and acts with a mechanism that is independent of the mutational status of CTNNB1, APC and AXIN1. Thus, patient selection using mutations in these genes provides an enrichment of tumors that have active β- catenin oncogenic signaling but do not affect the activity of tegavivint to inhibit β-catenin signaling. In addition to β-catenin's direct activity on tumor growth, the activation of Wnt/β-catenin signaling in HCC is associated with changes in the immune environment (C. Xu et al.2022 Journal of Clinical Investigation 132(4):e154515). In particular, high Wnt/β-catenin signaling is associated with elevated proportions of immunosuppressive cells, and significantly lower proportions of CD8+ T cells compared to the low Wnt signaling tumors. Wnt signaling high tumors have substantial enrichment of negative modulators of immune signaling, and in agreement with preclinical models of HCC, suggest that inhibition of β-catenin can enhance immune function in tumors and cooperate with immunotherapies such as checkpoint inhibitors (W. Xu et al.2022 Frontiers in Immunology 13:1010554). Objectives and related endpoints of the study are described in Figure 8. Abbreviations: AE = adverse event; AUC = area under the concentration-time curve; CBR = clinical benefit rate; Cmax = maximum concentration; Cmin = minimum concentration; CR = complete response; ctDNA = circulating tumor DNA; DLT = dose- limiting toxicity; DOCB = duration of clinical benefit; DOR = duration of response; ECG = electrocardiogram; MAD = maximum administered dose; MTD = maximum tolerated dose; NCI-CTCAE = National Cancer Institute Common Terminology Criteria for Adverse Events; ORR = objective response rate; OS = overall survival; PFS = progression- free survival; PK = pharmacokinetic(s); PR = partial response; qPCR = quantitative polymerase chain reaction; RP2D = recommended phase 2 dose; SAE = serious adverse event; SD = stable disease; T1/2 = half-life; TBL-1 = transducin β-like protein 1; TIL = tumor-infiltrating lymphocyte; Tmax = time of maximum concentration; TTR = time to response. 44 65518548 Attorney Docket No.40069/108; ITER-009/01WO The study comprises two parts. The first part is a phase 1, open-label, single-agent dose escalation, optimization, and expansion study of tegavivint in patients with advanced HCC after failure of at least one line of prior systemic therapy. The second part is an assessment of the combination of tegavivint plus pembrolizumab with a limited dose escalation followed by a randomized dose optimization. Part 1: Dose escalation follows a standard 3+3 design to assess the safety and MTD/MAD/RP2D of tegavivint administered intravenously on day 1, 8, 15, and 22 of a 28-day cycle (with a ± 1 day window for administration after Cycle 1 Day 1). Dose selection optimization will expand the two dose levels to at least 10 patients before declaring the recommended phase 2 dose (RP2D). The phase 1 dose escalation scheme is shown in Figure 9. Abbreviations: DLT = dose-limiting toxicity; HCC = hepatocellular carcinoma; IV = intravenous(ly); MTD = maximum tolerated dose; PD = pharmacodynamics; PK = pharmacokinetics; RP2D = recommended phase 2 dose. In some embodiments, patients must have mutations in CTNNB1, APC, or AXIN1 in dose optimization/expansion. If the DLT rate is exceeded at dose level 1, then lower dose levels of tegavivint may be assessed (i.e., 2 mg/kg IV weekly). Additional higher dose levels (i.e. > 15.5 mg/kg IV weekly), if needed, may continue in increments of 25% from the highest safe dose. Part 2: This part determines the safety, tolerability, and MTD/MAD/RP2D of tegavivint administered in combination with pembrolizumab to patients with advanced HCC, which must include prior PD-1/PD-L1 inhibitor use in the first- or second-line setting. In some embodiments, patients must have mutations in CTNNB1, APC, or AXIN1 genes. Tegavivint is administered via IV on day 1, 8, and 15 of a 21-day cycle with pembrolizumab 200 mg administered via a 30- minute IV infusion on day 1 of the 21-day cycle. The tegavivint plus pembrolizumab dose escalation and optimization scheme is shown in Figure 10. Rationale for starting dose and dose levels: In BCI-001-DT17 the maximum tolerated dose was not declared, and dose escalation was stopped due achieving pharmacologically relevant serum concentrations based on exceeding Cmin on day 8 of the IC50 (60 ng/mL or 100 45 65518548 Attorney Docket No.40069/108; ITER-009/01WO nM) identified from in vitro desmoid models coupled with preliminary clinical efficacy. Given the tolerability observed in BCI-001-DT17, with no DLTs observed, low grade 3/4 adverse event rate, no dose reductions, high dose intensity, and no discontinuations due to toxicity the current study increases the frequency of administration of tegavivint to weekly administration. Weekly administration will ensure trough concentrations are maintained at pharmacologically relevant concentrations throughout the cycle, without a period of rest which could allow for rapid proliferation observed in metastatic HCC. Also, based on the safety profile observed in the desmoid trial and the possibility that higher concentrations may be needed for efficacy in HCC higher dose levels of tegavivint will be provided. The starting dose and frequency in the current study is 3 mg/kg on day 1, 8, 15, and 22 of a 28 day schedule since this dose level in BCI-001-DT17 demonstrated substantially lower exposure of tegavivint than the 4 mg/kg and 5 mg/kg dose levels (Figure 7A and Table 9) and will allow for a safety margin to explore weekly administration. Subsequent dose levels include 5 mg/kg (highest and safe dose explored in BCI-001-DT17) and 6.5 mg/kg and 8 mg/kg on a weekly schedule, which are 30% and 23% increases, respectively. Tegavivint may be provided, for example, as an opaque, white to off-white sterile liquid suspension concentrate for infusion in an amber glass vial with butyl rubber stopper and aluminum crimp seal. Each 10 mL glass vial contains 10 mL of a 25 mg/mL tegavivint (BC- 2059) suspension concentrate, 0.625% poloxamer 188 NF, 10% sorbitol NF, and water for injection USP. Tegavivint may be administered by IV infusion diluted in dextrose 5% in water (D5W) and infused through a 15-micron filtered infusion over a 4-hour duration (± 5 minutes) at ambient temperature. Once diluted in D5W, the diluted suspension may be held at room temperature in a non-DEHP PVC bag for up to 4 hours prior to administration to the patient (maximum time at room temperature preferably no more than 8 hours). Pembrolizumab should be administered through a different IV administration set from the tegavivint. Pembrolizumab (Keytruda) may be supplied as a 100 mg/4 mL (25 mg/mL) solution in a single-dose vial. 46 65518548 Attorney Docket No.40069/108; ITER-009/01WO Upon completion of the combination dose escalation design, the study will further assess additional safety, PK/PD, and preliminary efficacy of tegavivint combined with pembrolizumab at the two selected tegavivint dose levels identified from the combination dose escalation. This dose optimization will utilize a 1:1, open-label randomization of up to 40 patients. Inclusion criteria for patient eligibility for treatment as described above may comprise one or more of the following: 1. Age 18 years or older 2. Confirmed diagnosis of HCC by either: a. Histologically or cytologically documented HCC b. Clinically confirmed diagnosis of HCC according to American Association for the Study of Liver Diseases (AASLD) criteria, including cirrhosis of any etiology and/or chronic hepatitis B or C infection 3. Presence of AXIN1, APC, or CTNNB1 mutation (except in single agent dose escalation phase) 4. Barcelona Clinic Liver Cancer (BCLC) Stage C disease or BCLC Stage B disease not amenable to locoregional therapy or refractory to locoregional therapy, and not amenable to a curative treatment approach 5. Child-Pugh class A or ≤ 7 class B liver score (no hepatic encephalopathy) 6. Disease progression, intolerance or contraindication to at least one line of systemic therapy for advanced HCC 7. Measurable disease as defined by RECIST 1.1 with spiral computerized tomography (CT) scan or magnetic resonance imaging (MRI). Lesions situated in a previously irradiated area, or in an area subjected to other loco-regional therapy, may be considered measurable if progression has been demonstrated in such lesions. 8. Normal organ and marrow function as defined below a. Absolute neutrophil count (ANC) ≥ 1.2 x 109/L 47 65518548 Attorney Docket No.40069/108; ITER-009/01WO b. Platelets ≥ 60 x 109/L; no transfusion within 7 days prior to the screening laboratory assessment c. Hemoglobin ≥ 9 g/dL (red blood cell transfusion or growth factors support is not allowed in the 14 days prior to the screening laboratory assessment) d. Total bilirubin ≤ ULN e. AST and ALT ≤ 5 x ULN f. Renal function i. Estimated creatinine clearance (CrCl) ≥ 50 mL/min by the Cockcroft-Gault equation using actual body weight, or ii. Estimated Glomerular Filtration Rate (eGFR) ≥ 50 mL/min/1.73m2 by CKD-EPI Creatinine Equation, or iii. Measured creatinine clearance ≥ 50 mL/min iv. Note: If estimated CrCl or eGFR is abnormal, accurate measurement may be obtained by 24-hour urine collection to measure creatinine clearance. g. Albumin ≥ 2.8 g/dL h. International normalized ratio (INR) ≤ 1.7, unless the patient is receiving anticoagulant therapy as long as the patient is within therapeutic range of intended use of anticoagulants 9. Willingness and ability to provide tumor biopsies during screening and while on treatment (end of Cycle 2) 10. Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 prior to the first dose 11. Washout period prior to Day 1 of Cycle 1: a. At least 21 days from the last dose of prior systemic anticancer treatment 48 65518548 Attorney Docket No.40069/108; ITER-009/01WO b. At least 14 days from palliative radiotherapy (≤ 10 fractions or ≤30 gray [Gy] total dose or at least 28 days from radiotherapy > 30 Gy) to extrahepatic tumor lesions c. At least 28 days from local or loco-regional therapy of intrahepatic tumor lesions (e.g. surgery, radiation therapy, hepatic arterial embolization, chemoembolization, radiofrequency ablation, percutaneous ethanol injection, or cryoablation) 12. Grade ≤ 1 toxicity due to any previous cancer therapy according to the NCI- CTCAE, v.5. Grade 2 is allowed in case of alopecia and/or peripheral sensory neuropathy. 13. Participants with past HCV infection will be eligible for the study. The treated participants must have completed their treatment at least 1 month prior to starting study intervention and HCV viral load must be below the limit of quantification. 14. Participants with controlled HBV will be eligible if they meet the following criteria: a. Antiviral therapy for HBV must be given for at least 4 weeks and HBV viral load must be less than 500 IU/mL prior to first dose of study drug. Patients on active HBV therapy with viral loads under 100 IU/mL should stay on the same therapy throughout study intervention. b. Patients who are positive for anti-hepatitis B core antibody HBc, negative for hepatitis B surface antigen (HBsAg), and negative or positive for anti- hepatitis B surface antibody (HBs), and who have an HBV viral load under 100 IU/mL, do not require HBV antiviral prophylaxis. c. Patients must have adequately controlled blood pressure (BP) with or without antihypertensive medications, defined as BP ≤ 150/90 mm Hg at Screening and no change in antihypertensive medications within 1 week before Cycle 1 Day 1. 49 65518548 Attorney Docket No.40069/108; ITER-009/01WO Exclusion criteria for patient eligibility for treatment as described above may comprise one or more of the following: 1. Known fibrolamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC 2. Patients receiving therapy with other anti-neoplastic or experimental agents 3. Not recovered to Grade 1 or baseline from adverse events related to prior therapy 4. Patients receiving concomitant strong inhibitors of CYP3A4/5 that cannot be discontinued 7 days or 5 half-lives (whichever is longer) prior to Cycle 1 Day 1 5. Patients receiving concomitant inducers of CYP3A4/5 that cannot be discontinued at least 14 days prior to Cycle 1 Day 1 6. Known central nervous system (CNS) involvement 7. Patients with known history of Gilbert’s syndrome or other genetic conditions affecting UGT1A1 function 8. HIV-positive patients on combination antiretroviral therapy Circulating tumor DNA (ctDNA) obtained via blood samples at screening and at the end of treatment may be used to sequence Wnt/β-catenin pathway mutations. Alterations may be used to correlate response to tegavivint and changes in mutational profile with treatment. ctDNA may also be used to correlate genomic alterations with alterations identified with alterations identified on tumor next generation sequencing (NGS) analysis. Tumor tissue obtained via core or excisional biopsy pre- and post-treatment may be used correlate immune response via T-cell infiltration and activated β-catenin/canonical Wnt signaling with clinical response. Tumor tissue may also be used to correlate genomic alternations with those identified on ctDNA analysis. Biomarkers, Wnt and immune response proteins including RANTES/CCL5, MIG/CXCL9, DKK1, FGF-2, IL-4, MMP-1, MMP-7, NSE, PAI-1, PDGF-AA, PDGF-BB, 50 65518548 Attorney Docket No.40069/108; ITER-009/01WO sCD40L, and VEGF-A, may be obtained via serial blood samples throughout treatment to assess changes in Wnt and immune response proteins. Biomarkers may also be used to assess Wnt and immunooncology mechanisms of action of tegavivint. 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. 51 65518548

Claims

Attorney Docket No.40069/108; ITER-009/01WO CLAIMS 1. A method of treating advanced hepatocellular carcinoma (HCC) in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of tegavivint or a pharmaceutically acceptable salt thereof, wherein the method prevents β-catenin from associating with transducin β-like protein 1 (TBL1) in the patient, and wherein the therapeutically effective dose is from about 0.0001 to about 1000 mg/kg/day. 2. The method of claim 1, wherein the patient has shown disease progression after, or intolerance or contraindication to, at least one prior line of systemic therapy for advanced HCC. 3. The method of claim 1, wherein the patient has one or more mutations in CTNNB1, APC, or AXIN1. 4. 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. 5. The method of claim 1, wherein said administering is performed intravenously. 6. The method of claim 1, wherein said administering is performed weekly on days 1, 8, 15, and 22 of a 28 day cycle. 7. The method of claim 1, wherein the therapeutically effective dose is from about 1 to about 20 mg/kg weekly. 52 65518548 Attorney Docket No.40069/108; ITER-009/01WO 8. The method of claim 1, wherein the therapeutically effective dose is about 3, about 5, about 6.5, about 8, about 10, about 12.5, or about 15.5 mg/kg weekly. 9. The method of claim 1, wherein the tegavivint is in a formulation comprising a poloxamer and one or more stabilizers selected from the group consisting of sucrose, trehalose, and sorbitol. 10. The method of claim 9, wherein the formulation comprises 25 mg/mL tegavivint, 0.625% by weight poloxamer 188, and 10% by weight sorbitol. 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 ° 2θ angle values independently selected from the group consisting of 5.0+−0.2°; 7.5+−0.2°; 14.8+−0.2°; 15.2+−0.2°; 15.4+−0.2°; 20.0+−0.2°; and 22.2+−0.2°, and wherein the nanosuspension consists essentially of Form I of tegavivint. 12. The method of claim 1, wherein the tegavivint is in a formulation comprising particles of Form I or Form IV of tegavivint or a pharmaceutically acceptable salt thereof, wherein 90% of the particles have a diameter of less than or equal to 0.2 µm when measured using laser diffraction, and wherein the formulation was prepared by high energy agitator milling at a temperature between about 40° C and about 60° C. 13. The method of claim 12, wherein the formulation is lyophilized and reconstituted. 53 65518548 Attorney Docket No.40069/108; ITER-009/01WO 14. The method of claim 1, wherein the patient has had prior treatment with a PD-1/PD-L1 inhibitor. 15. The method of claim 1, further comprising administering to the patient a therapeutically effective amount of an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti PD-L1 antibody. 16. The method of claim 15, wherein the immune checkpoint inhibitor comprises at least one of pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, and durvalumab. 17. The method of claim 16, wherein the immune checkpoint inhibitor is pembrolizumab. 18. The method of claim 17, wherein the tegavivint and the pembrolizumab are both administered by intravenous infusion, wherein the tegavivint is administered on days 1, 8, and 15 of a 21 day cycle, and the pembrolizumab is administered on day 1 of the 21 day cycle. 19. The method of claim 18, wherein the pembrolizumab dose is about 200 mg. 20. The method of claim 1, wherein the patient does not have fibrolamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC. 54 65518548
PCT/US2025/013033 2024-01-24 2025-01-24 Methods for treatment of hepatocellular carcinoma Pending WO2025160462A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US202463624744P 2024-01-24 2024-01-24
US63/624,744 2024-01-24
US202463556656P 2024-02-22 2024-02-22
US63/556,656 2024-02-22

Publications (1)

Publication Number Publication Date
WO2025160462A1 true WO2025160462A1 (en) 2025-07-31

Family

ID=96545881

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/013033 Pending WO2025160462A1 (en) 2024-01-24 2025-01-24 Methods for treatment of hepatocellular carcinoma

Country Status (1)

Country Link
WO (1) WO2025160462A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210161943A1 (en) * 2018-04-16 2021-06-03 The Regents Of The University Of California Pak4 inhibitors and methods of use
US20220098173A1 (en) * 2020-09-29 2022-03-31 Iterion Therapeutics, Inc. Crystalline form of tegavivint, method of preparation, and use thereof
US20220409724A1 (en) * 2019-10-29 2022-12-29 Eisai R&D Management Co., Ltd. Combination of a pd-1 antagonist, a vegfr/fgfr/ret tyrosine kinase inhibitor and a cbp/beta-catenin inhibitor for treating cancer
US20230190659A1 (en) * 2020-11-24 2023-06-22 Iterion Therapeutics, Inc. Formulations of tegavivint
US20230250433A1 (en) * 2020-02-27 2023-08-10 Board Of Regents, The University Of Texas System Methods and compositions for treatment of apc-deficient cancer

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210161943A1 (en) * 2018-04-16 2021-06-03 The Regents Of The University Of California Pak4 inhibitors and methods of use
US20220409724A1 (en) * 2019-10-29 2022-12-29 Eisai R&D Management Co., Ltd. Combination of a pd-1 antagonist, a vegfr/fgfr/ret tyrosine kinase inhibitor and a cbp/beta-catenin inhibitor for treating cancer
US20230250433A1 (en) * 2020-02-27 2023-08-10 Board Of Regents, The University Of Texas System Methods and compositions for treatment of apc-deficient cancer
US20220098173A1 (en) * 2020-09-29 2022-03-31 Iterion Therapeutics, Inc. Crystalline form of tegavivint, method of preparation, and use thereof
US20230190659A1 (en) * 2020-11-24 2023-06-22 Iterion Therapeutics, Inc. Formulations of tegavivint

Similar Documents

Publication Publication Date Title
US12364691B2 (en) Methods for treating pancreatic cancer using combination therapies
US20220339172A1 (en) Methods for treating castration-resistant and castration-sensitive prostate cancer
JP2018521058A (en) Methods of treating solid tumors using nanoparticulate mTOR inhibitor combination therapy
JP2018521058A5 (en)
US20180353602A1 (en) Combination of hdac inhibitor and anti-pd-l1 antibody for treatment of cancer
US20230190694A1 (en) Compositions for and methods of precision cancer treatment
US20240366600A1 (en) Pharmaceutical compositions and use thereof
TW202423445A (en) Use of infigratinib in the treatment of gastric cancer and adenocarcinoma
CN115768416A (en) Tucatinib in combination with trastuzumab and oxaliplatin-based chemotherapy for HER2-positive cancers
JP2022553041A (en) Methods of treating HER2-positive breast cancer with tucatinib in combination with capecitabine and trastuzumab
US20250017927A1 (en) Pharmaceutical composition and use thereof
WO2021026454A1 (en) Method of treating cancer
US20250235451A1 (en) Methods of Delaying Pain Progression And Treating Prostate Cancer
US11419862B2 (en) Quinoline derivative for treatment of nasopharyngeal carcinoma
JP7571053B2 (en) Quinoline derivatives for combination therapy of soft tissue sarcomas - Patent Application 20070223333
US20230038138A1 (en) Combination therapy for treating cancer
WO2025160462A1 (en) Methods for treatment of hepatocellular carcinoma
WO2024061203A1 (en) Pharmaceutical composition and use thereof
EP2755655A1 (en) Use of 4-amino-5-fluoro-3-[6-(4-methylpiperazin-1-yl)-1h-benzimidazol-2-yl]-1h-quinolin-2-one in the treatment of cancer in moderate hepatic impaired patients
CN120676946A (en) DEBIO-0123 in combination with temozolomide and radiotherapy for the treatment of glioma
KR20250046322A (en) Compositions and methods for precision cancer therapy
KR20150003786A (en) Methods for treating cancer using pi3k inhibitor and mek inhibitor
US20250170146A1 (en) Niraparib and abiraterone acetate plus prednisone to improve clinical outcomes in patients with metastatic castration-resistant prostate cancer and hrr alterations
EP4582085A1 (en) Use of heteroaryloxynaphthalene compound
WO2024006207A1 (en) An n-terminal domain androgen receptor inhibitor and uses thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25745822

Country of ref document: EP

Kind code of ref document: A1