Methods for treating hepatocellular carcinoma

Abstract

The present invention provides methods for treating hepatocellular carcinoma using formulations of tegavivint alone or in combination with an immune checkpoint inhibitor.

Description

[0001] Cross-citation of related applications

[0002] This application claims the benefits 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 herein by reference in its entirety. Background Technology

[0003] In the United States, cancer is the second leading cause of death. Cancer presents complex challenges for developing new therapies. Cancer is characterized by the abnormal growth of malignant cells that have undergone a series of genetic changes that lead to the growth and metastatic nature of tumor masses.

[0004] β-catenin is part of a protein complex that makes up adhesion junctions (AJs). AJs are essential for the generation and maintenance of the epithelial cell layer by regulating cell growth and cell-to-cell adhesion. β-catenin also anchors to the actin cytoskeleton and is responsible for firing contact inhibition signals that cause cells to stop dividing once the epithelial sheet is formed.

[0005] The Wnt / β-catenin pathway has been shown to play a role in cancer. Aberrant β-catenin signaling plays a crucial role in tumorigenesis. Specifically, it is estimated that more than 50% of hepatocellular carcinomas have 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 cancer, lung cancer, colon cancer, liver cancer, gastric cancer, myeloma, multiple myeloma, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell non-Hodgkin lymphoma, colorectal cancer, and acute myeloid leukemia (AML). Furthermore, abnormal Wnt / β-catenin signaling has been found in a large number of other conditions, including osteoporosis, osteoarthritis, polycystic kidney disease, diabetes, schizophrenia, vascular disease, heart disease, hyperplastic disorders, neurodegenerative diseases, and fibrotic diseases, including but not limited to idiopathic pulmonary fibrosis (IPF), Dupuytren's contracture, and nonalcoholic steatohepatitis (NASH). Summary of the Invention

[0006] This invention recognizes the continued need for methods to treat cancers (such as, but not limited to, hepatocellular carcinoma (HCC)) associated with the Wnt / β-catenin pathway and aberrant β-catenin signaling. Tagaravine can be used to modulate the activity of the Wnt / β-catenin signaling pathway and to reduce the levels of β-catenin present in cells (such as cancer cells). Tagaravine and related compounds are described, for example, in U.S. Patent No. 8,129,519. Tagaravine has the following structural formula:

[0007] .

[0008] The molecular formula of tegavine is C 28 H 36 N4O6S2.

[0009] The molecular weight of tegavine is 588.20763 amu.

[0010] It has been found that tarvasin can provide significant efficacy in treating cancers associated with the Wnt / β-catenin pathway and aberrant β-catenin signaling, such as, but not limited to, HCC. In this way, the present invention provides therapeutic doses of tarvasin for treating cancers (such as, but not limited to, HCC) in various embodiments.

[0011] In some embodiments, the present invention provides a method for treating advanced hepatocellular carcinoma (HCC) in a patient in need, the method comprising administering to the patient a therapeutically effective amount of degavidin or a pharmaceutically acceptable salt thereof, wherein the method prevents β-catenin in the patient from associating with transductionin β-like protein 1 (TBL1), and wherein the therapeutically effective dose is from about 0.0001 mg / kg / day to about 1000 mg / kg / day.

[0012] In some embodiments, the patient has shown disease progression after at least one prior line of systemic therapy for advanced HCC, or is intolerant to or has contraindications to the prior line of systemic therapy.

[0013] In some embodiments, the patient has one or more mutations in CTNNB1, APC, or AXIN1.

[0014] In some embodiments, the administration is performed by one or more of the following methods: intravenous, parenteral, oral, inhalation (including nebulized delivery), buccal, intranasal, rectal, intraperitoneal, intradermal, percutaneous, subcutaneous, intraarterial, intracardiac, intracranial, intratracheal, intrathecal, intrathecal, intramuscular, intravitreal, and local application.

[0015] In some embodiments, the administration is performed intravenously.

[0016] In some embodiments, the application is performed weekly on days 1, 8, 15, and 22 of a 28-day cycle.

[0017] In some embodiments, the effective dose for treatment is about 1 mg / kg to about 20 mg / kg per week.

[0018] In some embodiments, the effective dose for treatment is about 3 mg / kg, about 5 mg / kg, about 6.5 mg / kg, about 8 mg / kg, about 10 mg / kg, about 12.5 mg / kg, or about 15.5 mg / kg per week.

[0019] In some embodiments, the tergavisin is in a formulation comprising poloxamer and one or more stabilizers selected from the group consisting of sucrose, trehalose, and sorbitol.

[0020] In some embodiments, the formulation comprises 25 mg / mL of tergavisin, 0.625% by weight of poloxamer 188, and 10% by weight of sorbitol.

[0021] In some embodiments, the tagawiine is in a nanosuspension prepared by a method comprising: using a crystal form of tagawiine designated as form IV as a starting material, and milling form IV at a temperature of about 40°C to about 60°C, wherein form IV has an X-ray powder diffraction (XRPD) pattern comprising the following diffraction peaks, the °2θ angle values ​​of which are 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 is substantially composed of form I of tagawiine.

[0022] In some embodiments, the tergavisin is in a formulation comprising particles of tergavisin form I or IV or a pharmaceutically acceptable salt thereof, wherein 90% of these particles have a diameter less than or equal to 0.2 μm when measured using laser diffraction, and wherein the formulation is prepared by high-energy stirring milling at a temperature of about 40°C to about 60°C.

[0023] In some embodiments, the formulation is lyophilized and reconstituted.

[0024] In some embodiments, the patient has previously been treated with PD-1 / PD-L1 inhibitors.

[0025] In some embodiments, the method further comprises administering a therapeutically effective amount of an immune checkpoint inhibitor to the patient, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0026] In some embodiments, the immune checkpoint inhibitor comprises at least one of the following: pembrolizumab, nivolumab, cemiplimab, atezolizumab, avelumab, and durvalumab.

[0027] In some embodiments, the immune checkpoint inhibitor is pembrolizumab.

[0028] In some embodiments, both tergavisin and pembrolizumab are administered via intravenous infusion, wherein tergavisin is administered on days 1, 8, and 15 of a 21-day cycle, and pembrolizumab is administered on day 1 of a 21-day cycle.

[0029] In some embodiments, the dose of pembrolizumab is about 200 mg.

[0030] In some embodiments, the patient does not have fibrolamellar HCC, sarcomatoid HCC, or a combination of cholangiocarcinoma and HCC.

[0031] Further features and advantages of embodiments of the invention will be described below. This summary section is intended to illustrate certain features of embodiments of the invention only and is not intended to limit the scope of the invention in any way. The omission of specific features or embodiments of the invention, or the inclusion of one or more features in this summary section, should not be construed as limiting the claimed invention. Attached Figure Description

[0032] The invention and the following detailed description can be better understood when read in conjunction with the accompanying drawings. Certain embodiments are shown in the drawings for the purpose of illustrating the systems and methods of this application. However, it should be understood that this application is not limited to the precise arrangements and means shown.

[0033] Figure 1 The experimental results demonstrating the short-term inhibition of hepatocyte β-catenin activation by tegavine are shown. Tegavine reduces CTNNB1. 外显子3 Wnt target genes, tumor initiation clones, and proliferation in mutant HCC. A: 9-day study timeline; B: Changes in Wnt target gene expression in tumor tissues with activated β-catenin compared to tumor tissues without activated β-catenin, as assessed by quantitative RT-PCR; C, D, E: Immunohistochemical images at 20x magnification.

[0034] Figure 2 This study presents experimental results testing TBL inhibition monotherapy in early β-catenin-dependent disease. Tergavid reduces CTNNB1. 外显子3 Tumor progression of mutant HCC. A: 28-day study timeline; B: Immunohistochemistry using GS staining; images at 20x magnification.

[0035] Figure 3 This study presents experimental results testing TBL inhibition monotherapy in established β-catenin-dependent diseases. Tergavid lowers CTNNB1. 外显子3 CD3+ tumor-infiltrating leukocytes (TILs) and tumor burden in mutant HCC. A: 120-day study timeline; B: Immunohistochemistry using CD3+ staining; image at 20x magnification; C: Image of tumor nodules in mouse liver; D: Immunohistochemistry using GS staining; image at 20x magnification; E: Quantification of tumor nodules.

[0036] Figure 4 The experimental results testing TBL as a sensitization target for immunotherapy are shown. A: 135-day study timeline; B: Quantification of liver size; C: Quantification of tumor number; D: Quantification of tumor volume; E: Image of tumor nodules in mouse liver.

[0037] Figure 5 Tumor growth curves are shown in the treatment with the syngeneic model H22.

[0038] Figure 6 The dose-response curves for different cell lines are shown, plotted based on IC50 data.

[0039] Figure 7 A shows the tergavir concentration versus time curves for day 1 of cycle 1 in BCI-001-DT17 at dose levels.

[0040] Figure 7 B shows the AUC based on the total dose (mg) of tergavir administered, day 1 of cycle 1. 0-168小时 .

[0041] Figure 8 The objectives and relevant endpoints of studies on tergavisin alone and combinations of tergavisin and pembrolizumab according to various embodiments of the invention are illustrated.

[0042] Figure 9 Schemes for optimizing and expanding the dosage of tergavir monotherapy according to various embodiments of the present invention are illustrated.

[0043] Figure 10Schemes for dose escalation and optimization of tergavein papembrolizumab according to various embodiments of the present invention are illustrated. Detailed Implementation

[0044] definition

[0045] Within the context of this invention and in the specific context in which each term is used, the terms used herein generally have their common meaning in the art. Certain terms used to describe the invention are discussed below or elsewhere in this specification to provide additional guidance to those skilled in the art regarding the description of the invention. Synonyms for certain terms are provided. A detailed description of one or more synonyms does not preclude the use of other synonyms. The use of instances anywhere in this specification (including instances of any term discussed herein) is illustrative only and is not intended to limit the scope and meaning of the invention or any of the illustrative terms. The invention is not limited to the various embodiments given in this specification.

[0046] 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 relates. In the event of any conflict, this document (including the definitions) shall prevail.

[0047] The term "tergawiine" refers to compounds having the following structure:

[0048] .

[0049] The term “BC-2059” is used interchangeably with “Tegavin”.

[0050] The terms “long-term storage” or “long-term stability” are understood to mean that the pharmaceutical composition can be stored for three months or more, 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 room temperature 15-25°C, or at any temperature between 2-8°C and 15-25°C.

[0051] The terms "stable" or "stabilized" in relation to long-term storage should be understood to mean that, relative to the activity of the composition at the start of storage, the loss of the active ingredient contained in the pharmaceutical composition does not exceed 20% of its activity, or more preferably 15%, or even more preferably 10%, and most preferably 5%. Furthermore, for the purposes of this invention, the long-term physical stability of the lyophilized formulation includes maintaining good filter cake integrity, the ability to be easily resuspended upon reconstitution with a suitable diluent, and the absence of any significant crystal growth that can be determined by optical microscopy during storage.

[0052] The term "mammal" includes, but is not limited to, humans.

[0053] The term "pharmaceutically acceptable carrier" refers to any conventional type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation adjuvant, or excipient. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dosage and concentration used and is compatible with other components of the formulation.

[0054] The term “treatment” refers to any application or treatment of a disease in a mammal, including suppressing the disease, halting its progression, alleviating the disease (e.g., by inducing degeneration, restoring or repairing lost, missing or defective functions), or stimulating an inefficient process. The term includes any treatment that achieves the desired pharmacological and / or physiological effect and covers any treatment of a pathological condition or symptom in a mammal. The effect may be preventative in relation to the complete or partial prevention of the symptom or its symptoms, and / or therapeutic in relation to the partial or complete cure of the symptom and / or side effects attributable to the symptom. This includes (1) preventing the occurrence or recurrence of the symptom in a subject who may be susceptible to the symptom but does not yet have symptoms, (2) suppressing the symptom, e.g., preventing its progression, (3) stopping or terminating the symptom or at least its associated symptoms, such that the host is no longer suffering from the symptom or its symptoms, such as causing the remission of the symptom or its symptoms, e.g., by restoring or repairing lost, missing or defective functions or stimulating an inefficient process, or (4) alleviating, relieving or improving the symptom or its associated symptoms, wherein improvement is used broadly to refer to a reduction in at least the magnitude of a parameter, such as inflammation, pain, and / or tumor size.

[0055] The term "therapeutic effective amount" refers to the amount that, when administered to a live subject, achieves the desired effect in that live subject. For example, the effective amount of the compositions of the present invention for live administration is an amount for the prevention and / or treatment of any disease mediated via the Wnt / β-catenin pathway. The exact amount will depend on the therapeutic purpose and will be determined by a person skilled in the art using known techniques. As is known in the art, adjustments may be necessary for systemic versus local delivery, age, weight, general health, sex, diet, timing of administration, drug interactions, and severity of the condition, and these can be determined by a person skilled in the art using routine experiments.

[0056] The terms "composition" or "formulation" refer to mixtures of carriers or excipients that typically contain a carrier, such as those conventionally available in the art and suitable for administration to a subject for therapeutic, diagnostic, or preventative purposes. For example, compositions for oral administration may be in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, oral washes, or powders. The terms "composition," "pharmaceutical composition," and "formulation" are used interchangeably.

[0057] The term "nanoparticle composition" refers to a composition in which all, or almost all, of the particles are less than 1000 nm.

[0058] The term "lyophilized formulation" refers to a formulation obtained by freeze-drying an aqueous solution.

[0059] The term "pre-lyophilized formulation" refers to the formulation of the present invention prior to any lyophilization process. This term also encompasses formulations that do not undergo any lyophilization at all.

[0060] The term "reconstituted formulation" refers to a formulation obtained by adding an amount of water (e.g., sterile water) or an aqueous solvent to a solid composition to dissolve the composition. In one embodiment, the solid composition is a lyophilized formulation.

[0061] The term "injectable formulation" refers to a formulation suitable for parenteral administration (e.g., subcutaneous, intravenous, intramuscular, or intraperitoneal administration).

[0062] The term "stable therapeutic efficacy" means a tadalafil preparation that remains suitable for treating conditions treatable with tadalafil for at least 6 months, and preferably at least 18 months.

[0063] As used in this article, the term "AUC" last "" refers to the area under the curve from the time of drug administration to the time when the final measurable (positive) concentration is reached.

[0064] As used in this article, the term "C" max "This refers to the maximum concentration of a drug in a designated tissue after the initial administration of the drug and before the administration of a second dose."

[0065] As used in this article, the term "T" max "This refers to the time it takes to reach the highest plasma concentration after drug administration."

[0066] Tergavir formulations

[0067] Developing a stable tadalafil formulation is extremely challenging and difficult. Numerous formulations have been developed and tested; however, they exhibit poor bioavailability and / or prove unstable during storage, and / or become highly toxic. Obtaining a formulation that maintains good filter cake integrity, is easily resuspended, and demonstrates chemical stability over time is challenging and difficult.

[0068] The inventors have unexpectedly and surprisingly discovered that when using polymorphs of tagavitine, form I or form IV, as starting materials, stable formulations can be prepared when the starting materials are subjected to high-energy stirring and milling at a temperature of at least 40°C to 60°C, preferably at about 60°C. Polymorphs of forms I and IV are described in U.S. Patent No. 11,136,307, the contents of which are hereby incorporated by reference in their entirety.

[0069] In one embodiment, the present invention provides a nanosuspension of tegavine, wherein the nanosuspension is prepared by a method comprising: using form I or form IV as a starting material and performing the process at a temperature of about 40°C to about 60°C, most preferably by high-energy stirring and milling at about 60°C.

[0070] In one embodiment, the stabilizing formulation may be generated from any polymorphic form of tergavisin (e.g., any one of form I, form II, form III, or form IV), provided that form I and / or form IV polymorphs are generated at some point in the generation process.

[0071] In one embodiment, if the grinding process is carried out at a temperature below about 60°C, the nanosuspension undergoes a further annealing process at 60°C or above.

[0072] Both polymorphs, Form I and Form IV, were used as starting materials in a controlled-temperature high-energy milling process. Milling was performed using 200 mg / ml tegavine and 5% poloxamer 188 stabilizer. Microscopic imaging and DSC post-milling analysis revealed that batches using Form I as the starting material contained only Form I in the final product of the milling process. In contrast, a conversion of Form IV to Form I was observed in batches using Form IV as the starting material. It was assumed that milling Form I at 60°C or higher prevented the formation of unwanted Form IV seed crystals, resulting in a highly crystalline milled Form I material that was annealed and free of high-energy particles and amorphous material. This invention confirms this finding.

[0073] The final product obtained by milling at 60°C using either Form I or Form IV as the starting material is a nano-suspension of Form I, because Form IV is converted to Form I during the 120-minute milling process.

[0074] Therefore, in one embodiment, the present invention allows the use of form I or form IV as the starting material for milling at a high temperature (40-60°C, preferably 60°C) to obtain the final desired form (form I). However, the advantage of using form IV as the starting material for milling at 60°C is that the system undergoes a completely solvent-mediated recrystallization from form IV to form I. Crystals of form I grow "bottom-up" during milling, thus significantly reducing the chance of obtaining any large, unmilled crystals, thereby enhancing the quality of the suspension.

[0075] In fact, the nanosuspension obtained by grinding form IV at 60°C was found to be uniform and well-dispersed, exhibiting a narrow unimodal particle size distribution. Stability analysis under ambient laboratory conditions at 5°C showed no significant change in particle size distribution. Microscopic images confirmed that no crystal growth occurred in any of the final form I formulations at the end of 3 months of storage.

[0076] In one embodiment, the grinding is performed at a temperature of about 40°C to about 60°C, most preferably at about 60°C. Preferably, if the grinding process is performed at a temperature below about 60°C, the nano-suspension further undergoes an annealing process at 60°C or above 60°C.

[0077] Then, a nano-suspension, using Form I as the starting material and poloxamer 188 as the dispersant and milled at high temperature (60°C), was used for formulation optimization. Additionally, four batches prepared at the original test scale were milled and compounded to provide materials for formulation optimization studies.

[0078] In one embodiment, the formulations of the present invention are filtered, including but not limited to pre-lyophilized formulations, to remove any large particles. In one embodiment, the filter is 10 micrometers.

[0079] In one embodiment, the present invention provides a stable lyophilized formulation comprising particles of tergavisin or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; wherein, when measured using laser diffraction, the effective D50 of these particles is less than or equal to 500 nm, and the D90 is less than or equal to 1.0 micrometer (μm).

[0080] In one embodiment, when measured using laser diffraction, the effective D90 of the particle is approximately 100 nm.

[0081] In one embodiment, the present invention provides a formulation comprising particles of a polymorph of form I or form IV of tergavisin or a pharmaceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; wherein the effective D90 of these particles is less than or equal to 0.2 micrometers when measured using laser diffraction, and wherein the formulation is prepared by high-energy stirring and milling at a temperature of about 40°C to about 60°C.

[0082] In one embodiment, the formulation may be lyophilized.

[0083] In one embodiment, when laser diffraction is used for measurement, the effective D90 of the particle is less than or equal to 0.2 micrometers.

[0084] In one embodiment, when laser diffraction is used for measurement, the effective D50 of the particle is less than or equal to 0.12 micrometers.

[0085] In one embodiment, when laser diffraction is used for measurement, the effective D10 of the particle is less than or equal to 0.1 micrometers.

[0086] D50 is also known as the median diameter of the particle size distribution. It refers to the particle size at 50% of the cumulative distribution. In other words, when the D50 value is less than or equal to 500 nm, it means that 50% of the particles have a diameter of less than 500 nm.

[0087] D90 refers to the percentage of particles at the reported particle size. In other words, when the D90 value is less than or equal to 1.0 μm, it means that 90% of the particles have a diameter of less than 1.0 μm.

[0088] In some embodiments, the effective average particle size of tegavine is approximately 4900 nm, approximately 4800 nm, approximately 4700 nm, approximately 4600 nm, approximately 4500 nm, approximately 4400 nm, approximately 4300 nm, approximately 4200 nm, approximately 4100 nm, approximately 4 micrometers, approximately 3900 nm, approximately 3800 nm, approximately 3700 nm, approximately 3600 nm, approximately 3500 nm, approximately 3400 nm, approximately 3300 nm, approximately 3200 nm, approximately 3100 nm, approximately 3 micrometers, approximately 2900 nm, approximately 2800 nm, approximately 2700 nm, approximately 2600 nm, approximately 2500 nm, approximately 2400 nm, approximately 2300 nm, approximately 2200 nm, approximately 2100 nm, approximately 2000 nm, approximately 1900 nm, approximately 1800 nm, approximately 1700 nm. nm, approximately 1600 nm, approximately 1500 nm, approximately 1400 nm, approximately 1300 nm, approximately 1200 nm, approximately 1100 nm, approximately 1000 nm, approximately 900 nm, approximately 800 nm, approximately 700 nm, approximately 600 nm, approximately 500 nm, approximately 400 nm, or approximately 300 nm.

[0089] Furthermore, in some embodiments, the effective average particle size of the compound is less than 900 nm, more preferably less than 500 nm, and even more preferably less than 300 nm.

[0090] The provided formulation has stable therapeutic efficacy.

[0091] In one embodiment, the lyophilized formulation of the present invention is anhydrous.

[0092] In one embodiment, the formulation of the present invention includes a lyophilized formulation stored in a dry atmosphere.

[0093] The storage temperature of the formulation of the present invention can be about -20°C, about 5°C or about 25°C.

[0094] In one embodiment, the present invention provides a stable formulation comprising particles of tergavisin; wherein, when measured using laser diffraction, the effective D50 of the particles is less than or equal to 500 nm, and the D90 is less than or equal to 1.0 micrometer (μm), wherein the formulation comprises tergavisin, poloxamer, and one or more stabilizers selected from the group consisting of sucrose, trehalose, and sorbitol, and wherein the formulation is prepared by high-energy stirring at a temperature of about 40°C to about 60°C.

[0095] In one embodiment, the concentration of tergavisin in the formulation is 2% (20 mg / mL).

[0096] In one embodiment, the concentration of tergavisin in the formulation is 2.5% (25 mg / mL).

[0097] In another embodiment, the concentration of tergavisin in the formulation is 5% (50 mg / mL).

[0098] In one embodiment, poloxamer is poloxamer 188.

[0099] In one embodiment, the concentration of poloxamer in the formulation is 6 mg / mL.

[0100] In another embodiment, the concentration of poloxamer in the formulation is 12.5 mg / mL.

[0101] In one embodiment, the sucrose concentration in the formulation is 100 mg / mL.

[0102] In another embodiment, the concentration of trehalose in the formulation is 100 mg / mL.

[0103] In one embodiment, the concentration of sorbitol in the formulation is 50 mg / mL.

[0104] In one embodiment, the present invention provides a stable lyophilized formulation comprising particles of tergavisin; wherein, when measured using laser diffraction, the effective D50 of these particles is less than or equal to 500 nm, and the D90 is less than or equal to 1.0 micrometer (μm), wherein the formulation is a lyophilized product of a pre-lyophilized formulation comprising tergavisin, poloxamer, and one or more stabilizers selected from the group consisting of sucrose, trehalose, and sorbitol.

[0105] In one embodiment, the concentration of tergavir in the pre-lyophilized formulation is 2% (20 mg / mL).

[0106] In one embodiment, the concentration of tergavir in the pre-lyophilized formulation is 2.5% (25 mg / mL).

[0107] In another embodiment, the concentration of tergavir in the pre-lyophilized formulation is 5% (50 mg / mL).

[0108] In one embodiment, poloxamer is poloxamer 188.

[0109] In one embodiment, the concentration of poloxamer in the pre-lyophilized formulation is 0.6%.

[0110] In another embodiment, the concentration of poloxamer in the pre-lyophilized formulation is 1.25%.

[0111] In one embodiment, the sucrose concentration in the pre-lyophilized formulation is 10%.

[0112] In another embodiment, the trehalose concentration in the pre-lyophilized formulation is 10%.

[0113] In one embodiment, the concentration of sorbitol in the pre-lyophilized formulation is 5%.

[0114] In one embodiment, the formulation is autoclaved before lyophilization.

[0115] In one embodiment, the freeze-drying process includes freezing the formulation to approximately -40°C, performing a first drying step at -30°C, and performing a second drying step at approximately -10°C.

[0116] The present invention also includes pre-lyophilized formulations of tergavir.

[0117] In one embodiment, the pre-lyophilized formulation is prepared by ball milling at a temperature of about 60°C.

[0118] In one embodiment, the pre-lyophilized formulation is prepared by a method involving high-energy milling at a temperature of about 60°C.

[0119] In one embodiment, the tergavir in the pre-lyophilized formulation is a form I polymorph.

[0120] In one embodiment, the formulation of the present invention is stable when stored at a temperature of 5°C to 25°C for three months, six months, twelve months, or eighteen months. In a preferred embodiment, the formulation of the present invention exhibits long-term stability.

[0121] In one embodiment, when reconstituted, the formulations of the present invention, including but not limited to the lyophilized formulations of the present invention, can be formulated into: (a) a dosage form selected from the group consisting of tablets and capsules; (b) a dosage form selected from the group consisting of controlled-release formulations, melt-on-render formulations, delayed-release formulations, extended-release formulations, pulsatile-release formulations, and mixtures of immediate-release and controlled-release formulations; (c) a dosage form suitable for inhalation or parenteral administration (including intramuscular, subcutaneous, intravenous, and intradermal injection); and (d) any combination of (a), (b), and (c).

[0122] In one embodiment, the formulations of the present invention, including but not limited to the lyophilized formulations of the present invention, when administered to rats via intravenous (IV) infusion at a concentration of 15.5 mg / mL, produce one or more of the following values: at least about 50,000 ng / mL in plasma; at least about 200 ng / g in brain; at least about 2,900 ng / g in heart; at least about 3,700 ng / g in kidney; at least about 35,000 ng / g in lung; at least about 400 ng / g in pectoral and hamstring muscles; at least about 360,000 ng / g in spleen; at least about 470 ng / g in visceral fat; and at least about 237,000 ng / g of C in liver. max .

[0123] In another embodiment, the formulations of the present invention, including but not limited to the lyophilized formulations of the present invention, when administered to rats via intravenous infusion at a concentration of 15.5 mg / mL, produce one or more of the following values: at least about 68,000 hr*ng / mL in plasma; at least about 1,800 hr*ng / g in brain; at least about 45,000 hr*ng / g in heart; at least about 58,000 hr*ng / g in kidney; at least about 450,000 hr*ng / g in lung; at least about 5,700 hr*ng / g in pectoral and hamstring muscles; at least about 4,800,000 hr*ng / g in spleen; at least about 4,000 hr*ng / g in visceral fat; and at least about 3,600,000 hr*ng / g in liver. last .

[0124] In another embodiment, the formulations of the present invention, including but not limited to the lyophilized formulations of the present invention, when administered to rats via intravenous infusion at a concentration of 15.5 mg / mL, produce one or more of the following values: about 0.08 hours or less in plasma; about 0.5 hours or less in the brain; about 1 hour or less in the heart; about 0.5 hours or less in the kidneys; about 0.5 hours or less in the lungs; about 0.5 hours or less in the pectoral and hamstring muscles; about 0.5 hours or less in the spleen; about 1 hour or less in visceral fat; and about 1 hour or less in the liver. max .

[0125] The pharmaceutical formulations of the present invention may further comprise one or more pharmaceutically acceptable excipients, carriers, or combinations thereof.

[0126] In another embodiment, the present invention provides a method for preventing, treating, or improving cancer or tumor metastasis in a mammal in need, the method comprising administering an effective amount of the formulation of the present invention to the mammal.

[0127] The method of administration is not limited to any specific route of administration, and includes, but is not limited to, intravenous, parenteral, oral, inhalation (including nebulized delivery), oral, intranasal, rectal, intraperitoneal, intradermal, percutaneous, subcutaneous, intra-arterial, intracardiac, intracranial, intratracheal, intrathecal, intrathecal, intramuscular, intravitreal, and topical application.

[0128] The total daily dose of the formulations of the present invention administered to humans or lower animals can range from about 0.0001 mg / kg / day to about 1000 mg / kg / day. In some embodiments, the dose is in the range of about 0.001 mg / kg of subject body weight to about 100 mg / kg of subject body weight or about 0.05 mg / kg of subject body weight to about 50 mg / kg of subject body weight. In some embodiments, the dose is in the range of 0.5-50 mg / kg of body weight. If desired, the effective daily dose can be divided into multiple doses for administration purposes; therefore, a single-dose composition may contain such amounts or approximations thereof to constitute a daily dose.

[0129] In another embodiment, methods for preventing, treating, or improving cancer or tumor metastasis in mammals in need may include administering additional anticancer agents and / or cancer therapies (e.g., cancer vaccines, adoptive cell therapy, and radiation therapy).

[0130] In one embodiment, the additional anticancer agent is selected from the group consisting of: antimitotic agents, antimetabolites, HDAC inhibitors, protein body inhibitors, immunotherapeutic agents, FLT-3, EGFR, MEK, PI3K and other protein kinase inhibitors, epigenetic targeting inhibitors and WNT pathway inhibitors, alkylating agents and DNA repair pathway inhibitors, antihormonal agents, anticancer antibodies and other cytotoxic chemotherapeutic agents.

[0131] In some embodiments, additional anticancer agents are immune checkpoint inhibitors. Immune checkpoint inhibitors that can be used in combination therapies according to 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 cimiprimab (Libtayo). Examples of PD-L1 inhibitors include atezolizumab (Tecentriq), avelumab (Bavencio), and durvalumab (Imfinzi). Other 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 can be provided in combination with nivolumab in a formula known as Opdualag).

[0132] Immune checkpoint inhibitors can be administered via one or more of the following methods: intravenous, parenteral, oral, inhalation (including nebulized delivery), buccal, intranasal, rectal, intraperitoneal, intradermal, percutaneous, subcutaneous, intraarterial, intracardiac, intracranial, intratracheal, intrathecal, intrathecal, intramuscular, intravitreal, and local administration.

[0133] For the administration of immune checkpoint inhibitors, the dosage ranges from about 0.0001 mg / kg to about 100 mg / kg. In some embodiments, the dosage ranges from about 0.001 mg / kg of subject body weight to about 20 mg / kg of subject body weight or from about 0.01 mg / kg of subject body weight to about 10 mg / kg of subject body weight. In some embodiments, the dosage ranges from 0.1 to 10 mg / kg of body weight. If desired, the effective daily dose may be divided into multiple doses for administration purposes; therefore, a single-dose composition may contain such amounts or approximations thereof to constitute a daily dose.

[0134] When used in combination therapy according to embodiments of the invention, degavidin and the immune checkpoint inhibitor can be administered alone or together, sequentially (in any order), or simultaneously, using the same or different administration methods. For example, administration may be daily, every other day, once or twice a 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. Degavidin and the immune checkpoint inhibitor can be administered at the same frequency or at different frequencies. The dosage and timing of each may be varied during treatment. Treatment may continue until a complete response or confirmed disease progression.

[0135] This invention covers formulations including tergavisin and its pharmaceutically acceptable salts, esters, amides, stereoisomers or geometric isomers.

[0136] In one embodiment, the composition of the present invention can be formulated upon reconstitution into: (a) a dosage form selected from the group consisting of tablets and capsules; (b) a dosage form selected from the group consisting of controlled-release formulations, melt-on-render formulations, delayed-release formulations, extended-release formulations, pulsatile-release formulations, and mixtures of immediate-release and controlled-release formulations; (c) a dosage form suitable for inhalation or parenteral administration (including intramuscular, subcutaneous, intravenous, and intradermal injection); and (d) any combination of (a), (b), and (c).

[0137] The compositions of the present invention may further comprise one or more pharmaceutically acceptable excipients, carriers, or combinations thereof.

[0138] Pharmaceutically acceptable excipients used in the formulations of this invention can act in more than one manner.

[0139] Pharmaceutically acceptable excipients may be, for example, dispersion media, dispersion emulsifiers, dispersion enhancers, or combinations thereof.

[0140] Examples of propellants include, but are not limited to, HFA-134a (1,1,1,2-tetrafluoroethane), HFA-227 (1,1,1,2,3,3,3-heptafluoropropane), and combinations thereof.

[0141] The dispersion medium can be, for example, ethanol, propylene glycol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, glycerol, or combinations thereof.

[0142] The dispersing emulsifier (enhancer) can be, for example, water, oleic acid, sodium lauryl sulfate, polyethylene glycol 1000, ammonium alginate, potassium alginate, calcium stearate, glyceryl monooleate, polyoxyethylene stearate, emulsifying wax, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, poloxamer, and combinations thereof.

[0143] Examples of dispersing enhancers include, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, ethylene glycol stearate, sorbitol monolaurate, sorbitol monooleate, sorbitol monopalmitate, sorbitol monostearate, sorbitol sesquioleate, sorbitol trioleate, glyceryl monostearate, lecithin, meglumine, poloxamer, polyoxyethylene alkyl ether, polyoxyethylene 35 castor oil, polyoxyethylene stearate, polyoxyethylene glycerol ester, pyrrolidone, sorbitol ester, stearic acid, vitamin E polyethylene glycol succinate, polyethylene glycol 1000, povidone, and combinations thereof.

[0144] The compositions of the present invention are suitable for all routes of administration, including but not limited to intravenous, parenteral, oral, inhalation (including nebulized delivery), buccal, intranasal, rectal, intraperitoneal, intradermal, percutaneous, subcutaneous, intra-arterial, intracardiac, intracranial, intratracheal, intrathecal, intrathecal, intramuscular, intravitreal, and local application methods.

[0145] The pharmaceutical compositions according to the invention may further comprise one or more binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients. Such excipients are known in the art.

[0146] Examples of fillers include lactose monohydrate, anhydrous lactose, and various starches; examples of binders include various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolv SMCC). TM ).

[0147] Suitable lubricants, including agents that affect the flowability of the powder to be pressed, are colloidal silica, such as Aerosil® 200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.

[0148] Examples of sweeteners are any natural or artificial sweeteners, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acesulfame potassium. Examples of flavorings are Magnasweet® (a trademark of MAFCO), chewing gum flavors, and fruit flavors, etc.

[0149] Examples of preservatives include potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of benzoic acid such as butylparaben, alcohols such as ethanol or benzyl alcohol, phenolic compounds such as phenol, or quaternary ammonium compounds such as benzalkonium chloride.

[0150] Suitable diluents include pharmaceutically acceptable inert fillers such as microcrystalline cellulose, lactose, calcium diphosphate, sugars, 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, anhydrous lactose, and Pharmatose® DCL21; calcium hydrogen phosphate such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0151] Suitable disintegrants include mildly cross-linked polyvinylpyrrolidone, corn starch, potato starch, corn starch and modified starch, croscarmellose sodium, croscarmellose, sodium carboxymethyl starch and mixtures thereof.

[0152] Examples of effervescent agents are effervescent combinations, such as organic acids and carbonates or bicarbonates. Suitable organic acids include, for example, citric acid, tartaric acid, malic acid, fumaric acid, adipic acid, succinic acid, and alginic acid, as well as acid anhydrides and acid salts.

[0153] 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 combination may be present.

[0154] The invention will be described in more detail in the examples below, which are intended to be illustrative only, as many modifications and variations therein will be apparent to those skilled in the art. In the following examples, it should be understood that the weight percentages of the various components are expressed as w / v percentages.

[0155] Tadalafil is used for hepatocellular carcinoma

[0156] Primary liver cancer was the sixth most frequently diagnosed cancer globally in 2020 and the third leading cause of cancer death (Rumgay 2022 Journal of Hepatology 7(66): 1598-1606). Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. HCC most commonly occurs 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, consumption of aflatoxin-contaminated food, diabetes, and obesity. Clinical signs and symptoms of cirrhosis are often present in patients with HCC, and these signs and symptoms often mask the presence of underlying early-stage HCC. Symptoms and signs of cirrhosis are often the only manifestation of the disease. Therefore, patients affected by HCC are usually in the late stages of the disease.

[0157] Cytotoxic chemotherapy has moderate efficacy in HCC patients, and the benefits are generally limited in duration. Furthermore, HCC is resistant to radiotherapy, and treatment with systemic radiation therapy plays only a secondary role in HCC cases. For HCC, the only proven potential cure remains surgery, hepatectomy, or liver transplantation. Significant breakthroughs have been made in the management of HCC in recent years, particularly with drug therapies for advanced disease (such as the combination of atezolizumab and bevacizumab). However, despite these achievements, HCC remains a fatal disease with a poor prognosis for patients with unresectable cancer.

[0158] Up to 40% of HCC patients have tumors driven by mutations in activated β-catenin and typical Wnt pathways. The most common mutated genes include CTNNB1, AXIN1, and APC. Most mutations in β-catenin occur in exon 3, a key region responsible for its proteasome degradation. These exon 3 mutations make β-catenin resistant to degradation, leading to nuclear accumulation and dysregulated transcriptional activity. Activated β-catenin / typical Wnt signaling is associated with primary resistance to standard-of-care immune checkpoint blockade, mediated by a 'cold' tumor microenvironment excluded by immune cells. β-catenin recruitment to typical Wnt target genes requires transductionin β-like protein 1 (TBL1) to activate transcription.

[0159] TBL1 has been shown to be essential for the oncogenic activity of β-catenin. During Wnt signaling, TBL1 binds to β-catenin, inhibiting its degradation and forming an active transcription complex that binds to promoters to activate downstream cancer-related genes such as c-MYC and cyclin D1. Mutations and translocations of TBL1 have also been observed in various cancer types. In HCC, high expression of TBL1 is associated with poor prognosis and unfavorable characteristics.

[0160] Targavisin is a first-in-class small-molecule inhibitor of TBL1, a novel downstream target of the Wnt signaling pathway. Targavisin binds to TBL1 in the β-catenin pouch, disrupting the formation of the activation complex essential for oncogenic activity. Targavisin also degrades free nuclear β-catenin. Increased expression of β-catenin and TBL1 is associated with metastasis and poor prognosis in various cancers. Importantly, targavisin's targeting of TBL1 does not affect the membrane-bound and cytoplasmic β-catenin pools essential for normal cellular function, avoiding the toxicities typically associated with other inhibitors of the Wnt pathway. The safety, pharmacodynamics, pharmacokinetics, and clinical activity of targavisin have been demonstrated in a proof-of-concept study in patients with desmoidoma containing activating β-catenin mutations and are currently being investigated in acute myeloid leukemia (AML), pediatric solid tumors, non-small cell lung cancer (NSCLC), and lymphoma through investigator-initiated trials (IITs).

[0161] In vivo HCC data

[0162] In β-catenin 外显子3 The efficacy of tegavine was investigated in a genetically engineered C57BL / 6J mouse model (GEMM) of mutant HCC.

[0163] On day 0, give Ctnnb1 外显子3 / Wt Rosa26 c-MYC / c-MYC C57BL / 6J mice with the allele were injected with a high dose of AAV-Cre vector to test the efficacy of tegavine in inhibiting typical β-catenin target genes. Figure 1 A; acute oncogene activation model) or low-dose AAV-Cre vector, to test degavein reduction in clonal liver tumors representing human HCC (A .... Figure 2 A, Figure 3 A). On the 4th day after oncogene activation ( Figure 2 A) Day 5 ( Figure 1 A) or 90th day ( Figure 3Mice (n = 5 per group) were randomly assigned to receive either tegavine or a carrier. Tegavine was administered at a dose of 1.5 mg per mouse per day via intraperitoneal (ip) injection, 5 days a week, with a 2-day treatment break. The study endpoint was reached on day 9 (…). Figure 1 A) Day 28 ( Figure 2 A) or day 120 ( Figure 3 A). Activated β-catenin throughout the liver. 外显子3 Signal transduction in mice treated with tegafur significantly suppressed the overexpression of typical Wnt target genes Glul, Axin2, and Notum in early disease stages. Figure 1 B). No off-target effects were observed in intestinal tissue that relies on typical Wnt signaling to maintain tissue homeostasis. Compared with day 9 ( Figure 1 C) and day 28 ( Figure 2 Compared to the mediator control (B), inhibition of TBL1 in early-stage disease significantly reduced the number of tumor-initiating clones identified by markers of typical Wnt activation (glutamine synthase, GS). On day 9, tegavine also increased proliferation, as identified by Ki67 staining. Figure 1 D).

[0164] As early as day 9, T lymphocyte infiltration, as identified by hematoxylin and eosin, increased ( Figure 1 E), and then through CD3+ staining on day 120 ( Figure 3 (B) Tergarine induced an immunomodulatory response. Furthermore, on day 120, treatment with tergarine on established β-catenin levels showed better efficacy compared to the mediator. 外显子3 Activated tumor cells inhibit tumor growth and reduce tumor burden. Figure 3 C- Figure 3 E).

[0165] The efficacy of combining degavitin with immune checkpoint inhibitors was also investigated. On day 90 after oncogene activation ( Figure 4 In study A), mice (n = 5 per group) were randomly assigned to receive either degavidin (1.5 mg, IV twice weekly) or a combination of anti-PD-1 antibody (aPD1 / anti-PD-1) or an allotype control (200 μg, IP twice weekly). The study endpoint was 135 days. Figure 4 A). Compared with tecavir and the allotype control, combination therapy including tecavir and an anti-PD-1 antibody reduced liver size (liver / body ratio %), tumor number (gross tumor / liver), and tumor volume (tumor burden mm). 3 () Figure 4 B-4E).

[0166] To evaluate the pharmacodynamic response to degavitin, cells from the HCC syngeneic tumor type H22 were subcutaneously inoculated into the right lower flank of BALB / c mice. When the tumor reached approximately 115.42 mm... 3 When the average volume was reached (n = 6 per group), the mice were treated with 100 mg / kg degavidin or a mediator (phosphate-buffered saline [PBS]) by intubation. Body weight and tumor volume were measured two or three times during the study period (including day 1 and at sacrifice). H22 mice were sacrificed on day 3. Tumors were collected from nine models on the termination day.

[0167] Each tumor was divided into fluorescence-activated cell sorting (FACS) analysis and formalin-fixed paraffin-embedded (FFPE) blocks. FACS analysis used side-scattered-forward-scattered (SSC-FSC) plots to determine tumor-infiltrating lymphocytes (TILs). Barthel's test was used to test for variance homogeneity and normality. Group means were compared using analysis of variance (ANOVA), the Kruskal-Wallis test, and the Wilcoxon rank-sum test. If the p-value of the Barthel's test was not less than 0.05, ANOVA and a two-sample t-test were used to compare group means. If the p-value of the Barthel's test was less than 0.05, the Kruskal-Wallis test and the Wilcoxon rank-sum test were used to compare group means. FACS analysis demonstrated a significant change in the TIL population in H22 tegavine-treated tumors. A decrease in CD45+ lymphocytes and an increase in CD45- lymphocytes were observed, while a decrease in the mMDSC population was observed in H22 tegavine-treated tumors. An increase in PD-1+CD3+ and PD-1+CD4+ lymphocytes and M2 macrophages was also observed in tumors treated with H22 tegavein.

[0168] The efficacy and tolerability of degavidin were evaluated in the H22 mouse model of HCC syngeneia. Tumor cells were subcutaneously inoculated into the right anterior or lower flank region of BALB / c mice. When the tumor reached approximately 93.05 mm... 3 When the average tumor volume reaches ≥ 2000 mm, begin intraperitoneal (IP) treatment with 50 mg / kg tergavir or PBS (n = 10 per group). 3 Mice were then sacrificed. In this mouse model, in H22, 50 mg / kg degavidin showed a statistically significant TGI of 50.11%. Figure 5 ).

[0169] The H22 syngeneic mouse model was used to evaluate the efficacy and tolerability of tecavir alone and in combination with anti-PD-1 or anti-PD-L1. H22 mouse liver tumor cells (1 × 10⁻⁶) were added to 0.1 mL PBS. 6Subcutaneous injection was administered to the right upper flank of female BALB / c mice to promote tumor development. Mice were enrolled and randomly assigned to six treatment groups, with ten mice in each group:

[0170] 1. Mediator

[0171] 2. Tergavir alone 50 mg / kg

[0172] 3. Anti-PD-1 alone 10 mg / kg

[0173] 4. Anti-PD-L1 alone 10 mg / kg

[0174] 5. Tergavir 50 mg / kg plus anti-PD-1 10 mg / kg

[0175] 6. Tergavir 50 mg / kg plus anti-PD-L1 10 mg / kg.

[0176] All test drugs were administered intraperitoneally (IP) over 15 days. Tergavir was administered five times a week (5 days of dosing, 2 days of rest). Immune checkpoint inhibitors (ICIs) were administered twice a week (BIW).

[0177] The tumor growth inhibition (TGI) in the group treated with 50 mg / kg (G2) tecavir was 43.50% (Table 1). Both 10 mg / kg anti-PD-1 and 10 mg / kg anti-PD-L1 alone produced significant TGIs (Table 1). When comparing the combination treatment groups with the single-agent groups, the total antitumor efficacy of 50 mg / kg tecavir plus 10 mg / kg anti-PD-1 and 50 mg / kg tecavir plus 10 mg / kg anti-PD-L1 was not significant compared to tecavir, anti-PD-1, and anti-PD-L1 single-agent administration (Table 2).

[0178] Table 1: Tumor growth inhibition and T on day 15 i / C i calculate

[0179]

[0180] Note: Barthel's test is used to test for homogeneity of variance and normality; P = 0.72. No significant one-way ANOVA was performed to compare the groups. P < 0.05 was considered statistically significant.

[0181] Table 2: Multiple comparisons of tumor growth inhibition among groups

[0182]

[0183] Note: ns = nonsignificant

[0184] Tagarivein alone or in combination with anti-PD-L1 or one anti-PD-1 was well tolerated, and weight loss was manageable. Animals administered 50 mg / kg tagarivein alone and in combination with tagarivein or 10 mg / kg anti-PD-L1 showed treatment-related weight loss of 10.27%, 5.98%, and -3.00%, respectively. On day 8, the tagarivein administration schedule was modified to three times weekly to reduce weight loss. The introduction of a dietary supplement gel also stabilized weight loss in the animals.

[0185] In summary, tagavitin demonstrated antitumor and immunomodulatory activities in a mouse model of HCC with β-catenin activating mutations. In the syngeneic HCC model H22, tagavitin also reduced tumor volume and promoted immunomodulatory responses. The tagavitin tumor response in the HCC model H22 was reproduced in separate studies, specifically in the treatment of subcutaneous H22 mouse hepatocellular carcinoma tumor syngeneic models in female BALB / c mice, testing the efficacy and tolerability of tagavitin alone or in combination with anti-PD-1 or anti-PD-L1.

[0186] In vitro HCC data

[0187] The effects of tadalafil were investigated in nine HCC and hepatoblastoma cell lines in a 2D viability assay. Results were compared with cisplatin as a reference control.

[0188] To calculate the absolute IC50, a nonlinear regression model with an sigmoid dose-response curve was used to fit the dose-response curve. The formula used to calculate survival is shown below:

[0189] Survival rate (%) = (LumTest product LumT0) / (treated LumNone - LumT0) × 100%

[0190] The data is summarized in Table 3 and Figure 6 middle.

[0191] Table 3: Summary of IC50 and maximum inhibition of the tested compounds on selected cells

[0192]

[0193] Pharmacokinetic and absorption / distribution / metabolism / excretion (ADME) studies

[0194] Tergavisin exhibits high bioavailability under complete systemic exposure when administered intravenously. The compound showed high plasma protein binding of over 99% in all tested species, including mice, rats, dogs, miniature pigs, monkeys, and humans. This property contributes to prolonged circulation time and extended half-life, ranging from 16.5 hours in mice to over 50 hours in larger animals such as miniature pigs (56 hours) and dogs (53 hours).

[0195] Following intravenous administration, targavein exhibited a unique distribution pattern, initially localizing primarily in the lungs, followed by redistribution to the liver and spleen. The volume of distribution across species exceeded total body water, indicating extensive tissue distribution. Compared to hepatic blood flow, the compound exhibited relatively low plasma clearance, suggesting minimal first-pass metabolism. Notably, targavein showed limited permeability across the blood-brain barrier, but demonstrated favorable tumor distribution characteristics, as shown in mouse models. The distribution pattern of the nanoparticle formulation was consistent with established literature, which showed initial lung distribution followed by redistribution to the liver and spleen.

[0196] The metabolic profile of tergaviscon is consistent across species. The parent drug remains the major circulating compound, with CYP3A4 acting as the primary metabolic enzyme. In human hepatocytes, approximately 80% of the parent drug remains after 120 minutes of incubation. Major metabolic pathways include oxidation and glucuronidation, producing predictable metabolites. Importantly, no unique human metabolites were identified, indicating that preclinical safety studies translate well to humans. Drug interaction studies demonstrated limited inhibition of other CYP enzymes, but moderate to high inhibition of CYP2C8 when co-administered with CYP2C8 substrates, thus requiring caution.

[0197] Further in vitro studies evaluated the potential of tarvasin to inhibit UDP-glucuronyl transferase (UGT) and drug transporters. Using human liver microsomes and a recombinant protein system, tarvasin demonstrated inhibition of UGT1A1 (IC50). 50 : 29.8 nM recombinant / 236 nM microsomes; maximum inhibition: 92% recombinant / 75% microsomes), UGT1A3 (IC 50 : 66.0 nM recombinant / 169 nM microsomes; maximum inhibition: 87% recombinant / 64% microsomes) and UGT1A4 (IC 50 Effective inhibition was observed at 23.6 nM recombinant / 145 nM microsomes; maximum inhibition was 89% recombinant / 70% microsomes. Minimal inhibition to no inhibition was observed for UGT1A6, UGT1A9, and UGT2B7.

[0198] In transporter studies, tarvasin showed selective inhibition of key hepatic transporters. Tarvasin inhibits OATP1B1-mediated transport, in which IC50... 50 The concentration was 0.81 μM, and the maximum inhibition was 82%. For BSEP (IC 50 1.63 μM, maximum inhibition 86%) and BCRP (IC50) 50 Significant inhibition was also observed at 3.94 μM (maximum inhibition of 60%). Other transporters evaluated, including MATE1 / 2-K, MRP1 / 3, NTCP, and OCT1 / 3, showed minimal to no inhibition.

[0199] Simultaneous inhibition of both essential hepatic metabolic enzymes and transporters (particularly the UGT1A1 and OATP1B1 / BSEP pathways) suggests that tadalafil may affect bilirubin homeostasis and xenobiotic clearance. Therefore, caution is advised when tadalafil is co-administered with UGT1A1, UGT1A3, or UGT1A4 substrates (especially those metabolized via UGT1A1), as this may affect bilirubin clearance. Similarly, OATP1B1, BSEP, and BCRP substrates should be used with caution due to their potential effects on hepatic uptake and bile excretion pathways.

[0200] Elimination studies in rats demonstrated that unaltered taravirin was not recovered in urine, and less than 2% of the initial dose was recovered in feces. The slow elimination of the compound from systemic circulation is likely due to tissue storage effects, although the drug is eventually metabolized. The prolonged half-life observed across species, particularly in larger animals, combined with tissue distribution and metabolic profiles, supports a weekly intravenous administration schedule for clinical use.

[0201] Preclinical Toxicology

[0202] The GLP toxicology studies of the clinical formulation of BC2059 (tergaviscon) are described below. In rats, the pathology observed was primarily related to the infusion itself and manifested as local infusion site reactions, although pulmonary edema was observed in female rats at doses exceeding the maximum tolerated dose (MTD). In miniature pigs, the kidneys were identified as the primary target organ for toxicity, with mild tubular necrosis and elevated serum creatinine and blood urea nitrogen levels. Reversible thrombocytopenia was also occasionally noted.

[0203] GLP toxicology studies: rats

[0204] A 4-week toxicology study of GLP in rats was conducted using doses of 0 mg / kg (Group 1), 10 mg / kg (Group 2), 30 mg / kg (Group 3), and 60 mg / kg (Group 4), administered twice weekly for a total of eight doses. Animals were given BC2059 or a carrier (5% glucose) via continuous infusion over 24 hours (days 1, 4, 8, 11, 15, 18, 22, and 25). The highest dose of 60 mg / kg exceeded the maximum tolerated dose, as four female rats died on day 5 or 6 at this dose level. Histologically, alveolar edema, fibrinogen, alveolar macrophages, and hemorrhage were observed in these four females as the cause of death, attributed to the physical properties of the nano-ground drug and the large number of particles infused into the small animal species. For doses 3 through 8, the high dose was subsequently reduced to 40 mg / kg.

[0205] Additional premature deaths were observed: three males in the 60 mg / kg group, one female in the 60 mg / kg group, one female in the 30 mg / kg group, and one female in the control group. However, the cause of death in each of these rats was attributed to infusion site infection / tissue reaction and catheter retraction, rather than direct toxicity of BC2059 or the nanoparticle formulation. In the remainder of the study, lower doses of 40 mg / kg administered at doses 3–8 were well tolerated, and no fatal or life-threatening toxicities were observed at this dose level.

[0206] In the 30 mg and 60 / 40 mg / kg dose groups, BC2059 treatment produced non-life-threatening changes in the liver, spleen, and adrenal glands, but the changes observed microscopically were not severe enough to alter the clinicopathological profile. Importantly, the results observed in the lungs, liver, spleen, and adrenal glands of rats administered 30 mg / kg or 60 / 40 mg / kg were reduced or absent in the recovery group animals. (See AUC curves for plasma concentration changes over time.) 0-t The peak blood concentration (C) determined max The total systemic concentrations are provided in Table 4. Systemic exposure in female rats was consistently lower than in male rats, and this was particularly pronounced after dose 8. Repeated two-week dose administration did not result in significant BC2059 accumulation, unlike the similar C values ​​at doses 1 and 8. max and AUC 0-t The values ​​provided are as follows. However, the pre-dose concentration of BC2059 at dose 8 (which reflects the plasma concentration approximately 48 hours after the infusion of dose 7) was at a meaningful level, indicating that the drug has some dose-dependent persistence in systemic circulation, which does not translate into an increase in overall systemic exposure to the drug.

[0207] Table 4: Systemic exposure in GLP toxicology studies in rats

[0208]

[0209] GLP Toxicology Study: Miniature Pigs

[0210] In a 4-week GLP toxicology study of miniature pigs, animals were divided into five treatment groups: a mediator control administered over 24 hours (Group 1: 5% glucose), 1 mg / kg (Group 2), 5 mg / kg (Group 3), and 15 mg / kg (Group 4) of BC2059, and a 4-hour infusion group using 5 mg / kg of BC2059 (Group 5). Infusions were administered on days 1, 6, 8, 13, 15, 20, 22, and 27, for a total of eight doses. No treatment-dependent clinical observations were observed. There was no effect on body weight or food consumption. No findings were found on electrocardiogram (ECG) or ophthalmological examination. Clinical chemistry and coagulation values ​​showed no difference from baseline to post-treatment. At the 15 mg / kg dose level, platelet counts were significantly reduced, although these values ​​remained within the normal range, and the reduction in these observed animals resolved upon discontinuation of administration. However, given the observed platelet reduction at higher doses in miniature pigs (20 mg / kg, 40 mg / kg, and 50 mg / kg over 24 hours) and dogs (15 mg / kg twice weekly over 4 hours) in separate non-GLP studies, this finding may provide a monitorable drug-dependent effect. No treatment-related results were found in gross anatomical pathology or microscopic examination. The only possible histological finding related to the test product was perivascular mononuclear cell infiltration in the brain and spinal cord. While this was observed in two female control animals and two low-dose female animals (1 mg / kg), it was more frequently observed in males and females in groups 3 (5 mg / kg, 24-hour infusion), 4 (15 mg / kg), and 5 (5 mg / kg, 4-hour infusion). The underlying pathogenesis or significance of this mononuclear cell infiltration is unclear, but it appears that group 2 at 1 mg / kg / dose represented an unobserved level of effect. It is also evident that 28 days was sufficient to allow for complete recovery.

[0211] Table 5 provides the systemic exposures of BC2059, and Table 6 presents the terminal elimination half-life. PK parameters and plasma concentrations did not differ significantly between sexes; therefore, sex was grouped. In all treatment groups, when comparing systemic exposures after dose 8 with those observed at dose 1, C... maxThe AUC increased by 1.3 to 2.4 times, resulting in significant drug accumulation. The half-life ranged from 40 to 70 hours. This long half-life was reflected in drug accumulation after repeated administration of BC2059 every two weeks and in the trough concentration before dose 8. Plasma concentrations remained within the range of BC2059's in vitro tumor-killing bioactivity at 120 hours after dose 7 initiation, or at 96 hours after completion of infusion in groups 2, 3, and 4, and at 116 hours after completion of infusion in group 5. A long half-life of 47 to 60 hours was also observed in a non-GLP study in dogs where BC2059 was administered at 15 mg / kg via 4-hour infusion. Based on this, weekly administration of BC2059 could provide therapeutic concentrations throughout the week.

[0212] In this GLP toxicology study in miniature pigs, no serious adverse toxicities were observed at a dose of 5 mg / kg, regardless of whether the infusion time was 4 hours or 24 hours. Similarly, systemic exposure to BC2059 did not reflect any significant difference in systemic exposure between the two infusion durations. As shown in Table 6, when BC2059 was infused at 5 mg / kg over 4 hours, the mean C max and AUC last The results were very similar to those determined by infusion of 5 mg / kg over 24 hours. Table 7 shows the changes in plasma concentrations over time for two different infusion durations of 5 mg / kg. As expected, higher plasma concentrations were initially observed at most 8 hours after infusion in the shorter 4-hour infusion. However, the peak plasma concentrations in the 4-hour infusion were not more than 1.3 times higher than those in the 24-hour infusion. At the 24-hour time point, there was no difference in plasma concentrations between the 24-hour and 4-hour infusions in miniature pigs. Therefore, in clinical trials, the infusion duration was considered comparable to the 4-hour infusion duration and supports the 4-hour infusion duration.

[0213] Table 5: Systemic exposure in GLP toxicology studies in miniature pigs

[0214]

[0215] Table 6: Estimates of terminal elimination half-life in GLP toxicology studies in miniature pigs

[0216]

[0217] Table 7: Plasma concentrations in miniature pigs administered with 5 mg / kg over 24 hours and 4 hours.

[0218]

[0219] Non-GLP toxicity studies: miniature pigs

[0220] Results of GLP toxicology studies in miniature pigs defined the dose level at which no BC2059 infusion toxicity was observed; that is, 15 mg / kg.

[0221] In the two-dose study in miniature pigs, on day 1, a total of four female yucatan miniature pigs were administered IV infusions of the test product at target dose levels of 20 mg / kg / day and 40 mg / kg / day, continuously for 24 hours. On day 5, a second IV infusion of the test product was administered at target dose levels of 20 mg / kg / day and 35 mg / kg / day, continuously for 24 hours.

[0222] The high dose of 40 mg / kg was not well tolerated as clinical signs of lethargy and reduced food consumption, and was reduced to 35 mg / kg on day 5; one of the two animals was not given a second dose.

[0223] Three of the animals showed a significant decrease in platelet count, but the platelet count increased in the pigs that did not receive a second transfusion on day 5, indicating the reversibility of this finding.

[0224] In the kidneys, minimal tubular necrosis was observed in all animals in group 2, 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 was present in all animals, with moderate severity in one high-dose animal and one low-dose animal, but minimal severity in another low-dose animal. These changes are thought to be potentially treatment-related.

[0225] In summary, this two-dose study of BC2059 identified the kidney as a target organ for toxicity.

[0226] The response may be drug-related, but not related to the physical properties of the nanoparticles. In this miniature pig study, lung findings showed no clear association with the drug formulation, compared to GLP toxicology studies in rats. Importantly, the kidney findings suggest that changes in the kidneys are a more sensitive indicator of drug-induced side effects in clinical trials, rather than changes related to the nanoparticle properties of the formulation. The results of this non-GLP study were not used for dose setting but provide insights into potential monitorable toxicities in clinical development.

[0227] Clinical experience with tegavine

[0228] A phase 1 trial of BCI-001-DT17, BC2059 (tergavisin) in patients with unresectable desmoid tumors:This study was a phase I, open-label, non-randomized trial evaluating the safety of intravenous dagavein in patients with confirmed unresectable and symptomatic or progressive (as defined below) primary or recurrent desmoidoma. The study used a single patient cohort at the first two dose levels (0.5 mg / kg and 1 mg / kg) to minimize suboptimal drug exposure, followed by a standard 3+3 dose escalation phase to achieve the RP2D determined by pharmacokinetic and biologically relevant activities. Once the RP2D was established, this dose level cohort (5 mg / kg) was expanded with an additional 7 patients to collect further safety, PK, and PD data. The dose expansion also included 6 patients enrolled at lower dose levels who were eligible for in-patient dose escalation of the RP2D.

[0229] Symptomatic or progressive disease was defined as a 20% increase in tumor volume within 6 months, recurrence of disease within 1 year after surgery, or symptoms associated with desmoidoma and documented symptoms related to desmoidoma rather than previous treatment. Patients were also required to have a two-dimensionally measurable tumor, be ≥18 years old, and have an ECOG performance score of 0–1, according to WHO criteria. Patients with familial adenomatous polyposis were excluded.

[0230] The primary objectives were to assess safety, dose-limiting toxicities (DLT), MTD or maximum administered dose (MAD), RP2D of tadalafil, response rate according to WHO criteria, and PFS rate at 9 months. Secondary objectives included determining the duration of response after objective response, pharmacokinetics, and evaluating patient-reported outcomes.

[0231] The basic principles of starting clinical dose and duration of action in BCI-001-DT17: In GLP toxicology studies in rats, a high dose of 60 mg / kg exceeded the 10% serious toxic dose (STD10) in animals. A dose of 30 mg / kg administered over 24 hours was tolerable, with no deaths or life-threatening histopathological findings, and this dose was considered the STD10. In a 4-week GLP toxicology study in miniature pigs, doses up to 15 mg / kg were administered over 24 hours and up to 5 mg / kg over 4 hours without serious adverse toxicity. The lower dose of 5 mg / kg over 4 hours was considered the highest non-serious toxic dose (HNSTD) for this infusion duration.

[0232] Based on these GLP toxicology studies, the definition of the starting clinical dose is outlined in the ICH S9 guideline “Nonclinical evaluation of anticancer drugs”, as detailed in Table 8.

[0233] Table 8: Estimated starting clinical dose based on GLP toxicology studies according to ICH S9 guidelines

[0234]

[0235] For the initial dose discovery study BCI-001-DT17, the initial clinical dose was defined as 0.5 mg / kg. This starting dose was based on the lowest starting dose determined across miniature pigs and rats. Of course, miniature pigs are a more relevant species in terms of the similarity of infusion conditions to humans. The infusion volume and total volume in miniature pigs were comparable to the expected clinical volume. Physiologically, miniature pigs are also more similar to humans, and the nanoparticles are safe in both species in terms of nanoparticle size and microvascular size. In fact, toxicities related to the infusion procedure itself were observed in rats, and also found in several control animals. However, for HNSTD, the most conservative approach is to use a dose of 30 mg / kg in rats, rather than the dose of 5 mg / kg administered over 4 hours in miniature pigs, to support the initial clinical dose of 0.5 mg / kg in BCI-001-DT17.

[0236] Tegavine was administered via 4-hour infusion on days 1, 8, and 15 of a 28-day cycle, and the following dose levels were evaluated: 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.

[0237] A total of 24 patients were enrolled and treated in BCI-001-DT17. All results presented include data from all enrolled and treated patients during the study period.

[0238] Six dose levels were evaluated: 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 patients (66.7%) had an ECOG performance status of 0, while the remaining patients had a score of 1 (n = 8). Of the 24 patients included, 15 (63%) were white, 4 (17%) were Asian, 3 (13%) were Black, and 2 (8%) were of other races. The median time from initial diagnosis of desmoidoma to day 1 of cycle 1 was 3.1 years (range 0.3–19.7). The main sites of tumor involvement included the extremities or trunk (n = 13, 54%), abdomen or pelvis (n = 8, 33%), and spinal cord or nervous system (n = 3, 13%). The median prior systemic treatment was 1 (range 0–6). Seventy-one percent of patients had previously received systemic therapy with tyrosine kinase inhibitors (n = 12, 50%), cytotoxic chemotherapy (n = 9, 38%), tamoxifen (n = 6, 25%), nonsteroidal anti-inflammatory drugs (n = 4; 17%), and investigational drugs (n = 3; 13%). Ten (42%) patients had previously undergone tumor resection. Four (17%) patients had previously undergone radiation, cryoablation, or high-intensity ultrasound.

[0239] 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 completed dagavit cycles 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 infusion errors for any reason was 0.5 (range: 0–4). One patient had their dose reduced from 4 mg / kg to 3 mg / kg due to grade 3 anemia deemed unrelated to study treatment, and subsequently underwent in-patient dose escalation to 5 mg / kg without dose reduction or discontinuation of therapy. The main reasons for discontinuation of study treatment were: completion of treatment in 5 patients (20.8%); discontinuation due to disease progression in 5 patients (20.8%); adverse events deemed unrelated to dagavit in 1 patient (4.2%); and withdrawal of consent or other reasons in 12 patients. No deaths occurred in the study.

[0240] Overall, a total of 566 adverse events (AEs) occurred in the study, with all patients experiencing at least one AE. Grade 1 AEs were observed in all patients, representing 72% of all AEs occurring during the study. Grade 2 AEs were observed in 22 patients (91.7%), representing 24.9% of all AEs occurring during the study. Grade 3 AEs occurred in 9 patients (37.5%), representing 3% of all AEs occurring during the study. No Grade 4 AEs were observed. The most common adverse events of any grade (≥ 20% of patients) 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).

[0241] Overall, a total of 227 treatment-related adverse events (AEs) occurred in 23 patients (95.8%) during the study (considered at least possibly related to degavisin). Grade 1 treatment-related AEs occurred in 21 patients (87.5%), representing 73% of all observed treatment-related AEs. Grade 2 treatment-related AEs occurred in 16 patients (66.7%), representing 24.7% of all observed treatment-related AEs. Grade 3 treatment-related AEs occurred in 5 patients (20.8%), representing 2.2% of all observed treatment-related AEs. No Grade 4 treatment-related AEs were observed. The most frequently occurring treatment-related AEs of any grade (≥ 20% of patients) included: fatigue (70.8%, n = 17), headache (37.5%, n = 9), nausea (33.3%, n = 8), taste disturbance (20.8%, n = 5), constipation (20.8%, n = 5), and decreased appetite (20.8%, n = 5).

[0242] Grade 3 treatment-related adverse events (AEs) occurred in 5 individual patients. No Grade 3 treatment-related AEs occurred in more than one patient, and these included AEs at the following indicated dose levels: hypophosphatemia (2 mg / kg); stomatitis (3 mg / kg); elevated ALT (5 mg / kg); headache (5 mg / kg); and diarrhea (5 mg / kg).

[0243] Infusion or injection site reactions occurred in three patients. One patient experienced Grade 2 infusion site extravasation, which resulted in pain and inflammation at the infusion site, but no tissue necrosis was observed. Due to hospitalization, the event was classified as a serious adverse event and considered to be related to tergavisin. This patient tolerated subsequent treatment with tergavisin. The other two patients reported infusion site reactions, both Grade 1, including erythema, pain, and swelling.

[0244] In addition to the extravasation at the infusion site described above, another serious adverse event (grade 3) of pulmonary Mycobacterium avium co-infection was observed, which was considered unrelated to tegavine.

[0245] Considering the duration of treatment (median of 10 cycles), the overall type and frequency of adverse events (AEs) were not uncommon in Phase 1 trials. No dose-limiting toxicities were observed. Higher-grade AEs were uncommon and did not lead to treatment discontinuation, dose reduction, or interruption. Common (>20%) treatment-related adverse events included fatigue, headache, nausea, constipation, decreased appetite, and dysgeusia. Overall, a high relative dose intensity of 97% was observed within the median of 10 cycles, indicating that tecavir at doses up to 5 mg / kg was tolerable on days 1, 8, and 15 of a 28-day timetable. No significant bone, cardiac, pulmonary, ocular, renal, hepatic, or hematological toxicities were observed.

[0246] In BCI-001-DT17, efficacy was assessed every three cycles by CT scans and / or magnetic resonance imaging (MRI) according to WHO and RECIST 1.1 criteria. Four objective responses (WHO or RECIST) were observed during the study, resulting in an objective response rate of 17% across all dose levels and 25% at RP2D (WHO or RECIST). Another patient achieved a 49% reduction (WHO) and was subsequently deemed surgically resectable and withdrew from the study.

[0247] The median duration of response was 9.5 months (interquartile range 2.6 to 13.3 months), and all responses were sustained at the end of the study. The 9-month progression-free survival rate was 76.7% (95% CI: 54–90%) for all patients and 79.6% (95% CI: 51.7–93%) for patients treated with RP2D. Overall, the objective response rate of 25% with RP2D during the study period demonstrates preliminary efficacy in progressive desmoidoma.

[0248] Clinical pharmacokinetics of tergavir in BCI-001-DT17:In BCI-001-DT17, 24 patients were treated with six dose levels (0.5 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg and 5 mg / kg) of tergavisin on days 1, 8 and 15 of a 28-day cycle. Figure 7 A is presented graphically as concentration-time curves from the start of a 4-hour tarvasin infusion to Day 1 of Cycle 1, 168 hours post-dose, across the six dose levels evaluated. The number of patients at each dose level is plotted, and data are presented as the median and quartile range (25%–75%) for all patients at each dose level. Pharmacokinetic parameters summarized by dose level for Day 1 of Cycle 1 are shown in Table 9. The median half-life across all dose levels was 39.9 hours (IQR 33.8–48.9), supporting weekly administration. Accumulation was observed prior to the next weekly infusion, particularly at higher dose levels (4 mg / kg and 5 mg / kg), indicating sustained exposure to tarvasin between doses. Increased exposure (AUC) was observed in the 4 mg / kg and 5 mg / kg groups compared to the low-dose group. Despite similar total doses administered, an increase in AUC was observed in the 4 mg / kg group from the 5 mg / kg group during Cycle 1, likely due to the lower median body weight in the 5 mg / kg group. AUC is related to the dose (mg) of tegafur administered. Figure 7 B). An increase in AUC was demonstrated at the 5 mg / kg dose level in a patient with dose escalation from 4 mg / kg to 5 mg / kg.

[0249] Table 9: Pharmacokinetics of tergavisin infusion at dose levels on day 1 of cycle 1 in BCI-001-DT17

[0250]

[0251] Clinical pharmacodynamics of tergavir in BCI-001-DT17:In BCI-001-DT17 (NCT03459469), the clinical pharmacodynamic activity of targavisin in patients with unresectable desmoid tumors was analyzed, where objective responses (all partial responses according to WHO criteria) and serum were available to obtain changes in serum protein biomarkers. A group of six proteins were selected as candidate biomarkers based on their cancer-related functions and regulation of aberrant Wnt signaling. Serum from four patients with objective responses who received targavisin at doses of 1 mg / kg, 4 mg / kg, or 5 mg / kg was analyzed at five time points (Cycle 1, Day 8: 15 minutes before administration and 2, 4, and 24 hours after infusion initiation; Cycle 1, Day 8: 15 minutes before administration). Targavisin caused significant changes in these β-catenin responsiveness biomarkers up to the last measurement collection time point of 192 hours after infusion initiation. For several of these biomarkers, dose-response trends were observed in patients 03-01 (1 mg / kg), showing less depth of variation and shorter duration of duration compared to patients 04-08 (5 mg / kg). One of the four patients analyzed (patients 03-04) had highly variable responses across multiple biomarkers in this assay and was found to have significantly higher plasma drug levels than other patients in this biomarker study.

[0252] HCC Clinical Research

[0253] As described above, activating mutations in β-catenin and overactive Wnt transcriptional activity are prevalent in HCC. In preclinical models of HCC, inhibition of TBL1 by tarvasin has demonstrated encouraging efficacy. Based on the demonstrated activity of checkpoint inhibitors in HCC and the mechanism of action of tarvasin, the tolerability of tarvasin in HCC with and without pembrolizumab was evaluated, as well as the potential for innate resistance to this combination of drugs to immune checkpoint inhibition resulting from Wnt / β-catenin pathway activation.

[0254] Dysregulation of Wnt / β-catenin signaling has been shown to play a significant role in the promotion and development of hepatocellular carcinoma (HCC). 40–70% of HCC tumor samples show activated β-catenin signaling via a transcriptional program involving strong nuclear β-catenin or Wnt activity. Activating mutations in the Wnt / β-catenin signaling pathway were found in most of these patients, most commonly CTNNB1 (the gene encoding β-catenin) mutations found in 20-30% of patients, AXIN1 mutations found in 8-10% of patient samples, and APC mutations found in 5-8% of hepatocellular carcinoma patients (Schulze et al. 2015 Nature Genetics 47(5):505 511; Calderaro et al. 2017 Journal of Hepatology 67(4):727-738; Harding et al. 2018; Luke et al. 2019 Clinical Cancer Research 25(10):3074-3083; Cowzer et al. 2023).

[0255] While mutations in CTNNB1, APC, and AXIN1 can activate the Wnt / β-catenin pathway, they do not alter tectarin's ability to inhibit β-catenin oncogenic signaling. Tegavitin targets the β-catenin signaling pathway in the nuclear compartment, and its mechanism of action is independent of the mutational status of CTNNB1, APC, and AXIN1. Therefore, patient selection using mutations in these genes can provide enrichment for tumors with active β-catenin oncogenic signaling without affecting tectarin's inhibitory activity against β-catenin signaling.

[0256] In addition to the direct activity of β-catenin on tumor growth, 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). Specifically, compared with tumors with low Wnt signaling, high Wnt / β-catenin signaling is associated with an increased proportion of immunosuppressive cells and a significantly reduced proportion of CD8+ T cells. Tumors with high Wnt signaling have a large number of enriched negative regulators of immune signaling, consistent with preclinical models of HCC, suggesting that inhibition of β-catenin can enhance immune function in tumors and can be combined with immunotherapies such as checkpoint inhibitors (W. Xu et al., 2022, Frontiers in Immunology, 13:1010554).

[0257] The research objectives and relevant endpoints are as follows: Figure 8 Description in Chinese. Abbreviations: AE = Adverse Events; AUC = Area Under the Concentration-Time Curve; CBR = Clinical Benefit Rate; C max = Maximum concentration; C min = 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 for Adverse Events; ORR = Objective response rate; OS = Overall survival; PFS = Progression-free survival; PK = Pharmacokinetics; PR = Partial response; qPCR = Quantitative polymerase chain reaction; RP2D = Recommended phase 2 dose; SAE = Serious adverse event; SD = Stable disease; T 1 / 2 = Half-life; TBL-1 = transductionin β-like protein 1; TIL = tumor-infiltrating lymphocytes; T max = Time to maximum concentration; TTR = Response time.

[0258] The study comprises two parts. The first part is a phase 1, open-label, single-dose dose-escalation, optimization, and expansion study of tergavisin in patients with advanced HCC following failure of at least one line of prior systemic therapy. The second part is an evaluation of the combination of tergavisin and pembrolizumab with limited dose escalation, followed by randomized dose optimization.

[0259] Part 1 Dose escalation followed a standard 3+3 design to evaluate the safety and MTD / MAD / RP2D of intravenous tedaviin administered on days 1, 8, 15, and 22 of a 28-day cycle (using a ±1-day window for administration after day 1 of cycle 1). Dose selection optimization will extend both dose levels to at least 10 patients before announcing the recommended phase 2 dose (RP2D). Figure 9The Phase 1 dose escalation regimen is shown. Abbreviations: DLT = Dose-limiting toxicity; HCC = Hepatocellular carcinoma; IV = Intravenous; MTD = Maximum tolerated dose; PD = Pharmacodynamics; PK = Pharmacokinetics; RP2D = Recommended Phase 2 dose. In some embodiments, during dose optimization / expansion, the patient must have a mutation in CTNNB1, APC, or AXIN1. If the DLT rate is exceeded at dose level 1, a lower dose level of tecavir (i.e., 2 mg / kg IV weekly) can be evaluated. If necessary, additional higher dose levels can be continued in 25% increments based on the highest safe dose (i.e., >15.5 mg / kg IV weekly).

[0260] Part 2 This section defines the safety, tolerability, and MTD / MAD / RP2D of tergavisin in combination with pembrolizumab for the treatment of patients with advanced HCC, which must include prior use of a PD-1 / PD-L1 inhibitor as first- or second-line therapy. In some embodiments, the patient must have a mutation in the CTNNB1, APC, or AXIN1 gene. Tergavisin is administered intravenously on days 1, 8, and 15 of a 21-day cycle, and pembrolizumab is administered via 30-minute intravenous infusion on day 1 of a 21-day cycle. Figure 10 The dosage escalation and optimization scheme for tergavid papembrolizumab is shown in the figure.

[0261] The basic principles of starting dose and dose level: In BCI-001-DT17, no maximum tolerated dose was announced, and the pharmacology-related serum concentration (based on the IC50 (60 ng / mL or 100 nM) identified from an in vitro desmoidoma model) was exceeded on day 8. min Based on preliminary clinical efficacy, dose escalation was discontinued. Given the tolerability observed in BCI-001-DT17, no observed DLT, low incidence of grade 3 / 4 adverse events, no dose reduction, high dose intensity, and failure to discontinue due to toxicity, the current study increases the frequency of tadalafil administration to weekly. Weekly administration will ensure that trough concentrations remain at pharmacologically relevant levels throughout the cycle without rest periods, which may allow for the rapid proliferation observed in metastatic HCC. Furthermore, based on the safety profile observed in the desmoidoma trial, and the possibility that higher drug concentrations may be required for efficacy in HCC, higher dose levels of tadalafil will be provided.

[0262] Because this dose level in BCI-001-DT17 demonstrated a substantially reduced exposure to tergavir compared to dose levels of 4 mg / kg and 5 mg / kg. Figure 7(A and Table 9), therefore the starting dose and frequency in the current study changed to 3 mg / kg on days 1, 8, 15, and 22 of a 28-day timeframe, allowing for a safety margin for exploring weekly administration. Subsequent dose levels included 5 mg / kg (the highest and safest dose explored in BCI-001-DT17) on the weekly schedule, as well as 6.5 mg / kg and 8 mg / kg, representing increases of 30% and 23%, respectively.

[0263] For example, tergavisin can be provided as an opaque, white to off-white sterile liquid suspension concentrate for infusion in amber glass vials with butyl rubber stoppers and aluminum crimp seals. Each 10 mL vial contains 10 mL of 25 mg / mL tergavisin (BC-2059) suspension concentrate, 0.625% poloxamer 188 NF, 10% sorbitol NF, and water for injection (USP). Tergavisin can be diluted in a 5% glucose solution (D5W) for intravenous infusion and administered at ambient temperature via a 15-micron filter over a duration of 4 hours (± 5 minutes). Once diluted in D5W, the diluted suspension can be stored at room temperature in a non-DEHP PVC bag for up to 4 hours before administration to the patient (preferably not exceeding 8 hours at room temperature).

[0264] Pembrolizumab should be administered via IV, unlike tergaviscon. Pembrolizumab (Keytruda) is available as a 100 mg / 4 mL (25 mg / mL) solution in single-dose vials.

[0265] Following the completion of the combination dose escalation design, the study will further evaluate the additional safety, PK / PD, and preliminary efficacy of the tergavisin + pembrolizumab combination at two selected tergavisin dose levels identified from the combination dose escalation. This dose optimization will utilize 1:1 open-label randomization to up to 40 patients.

[0266] The inclusion criteria for patient eligibility as described above may include one or more of the following:

[0267] 1. Must be 18 years of age or older

[0268] 2. HCC can be diagnosed through any of the following:

[0269] a. HCC recorded histologically or cytologically

[0270] b. Clinically diagnosed HCC according to the American Association for the Study of Liver Diseases (AASLD) criteria, including cirrhosis of any etiology and / or chronic hepatitis B or C infection.

[0271] 3. Presence of AXIN1, APC, or CTNNB1 mutations (except during single-dose escalation phases)

[0272] 4. Stage C or Stage B BCLC patients in the Barcelona Clinical Trial are not suitable for localized therapy or are refractory to localized therapy and are not suitable for curative treatment.

[0273] 5. Child-Pugh grade A or ≤ 7 (B liver score) (without hepatic encephalopathy)

[0274] 6. Disease progression, intolerance, or contraindications to at least one line of systemic therapy for advanced HCC.

[0275] 7. Diseases measurable by spiral computed tomography (CT) or magnetic resonance imaging (MRI) as defined by RECIST 1.1. Lesions located in previously irradiated areas or areas that have undergone other localized therapies can be considered measurable if they have shown progression.

[0276] 8. Normal organ and bone marrow function is defined as follows:

[0277] a. Absolute neutrophil count (ANC) ≥ 1.2 × 10⁻⁶ 9 / L

[0278] b. Platelet count ≥ 60 × 10 9 / L; No blood transfusions within 7 days prior to screening laboratory assessment.

[0279] c. Hemoglobin ≥ 9 g / dL (Red blood cell transfusions or growth factor support are not permitted within 14 days prior to screening laboratory assessment)

[0280] d. Total bilirubin ≤ ULN

[0281] e. AST and ALT ≤ 5 × ULN

[0282] f. Kidney function

[0283] i. Creatinine clearance (CrCl) estimated using actual body weight via the Cockcroft-Gault equation ≥ 50 mL / min, or

[0284] ii. Glomerular filtration rate (eGFR) estimated by the CKD-EPI creatinine equation ≥ 50 mL / min / 1.73 m2, or

[0285] iii. The measured creatinine clearance rate is ≥ 50 mL / min

[0286] iv. Note: If the estimated CrCl or eGFR is abnormal, creatinine clearance can be measured by collecting urine over 24 hours to obtain an accurate measurement.

[0287] g. Albumin ≥ 2.8 g / dL

[0288] h. International Normalized Ratio (INR) ≤ 1.7, unless the patient is receiving anticoagulant therapy, provided the patient is within the expected range of anticoagulant use.

[0289] 9. Willing and able to provide a tumor biopsy during screening and treatment (end of cycle 2).

[0290] 10. Prior to the first dose, the Eastern Cooperative Oncology Group (ECOG) performance status was 0 or 1.

[0291] 11. Wash-off period before day 1 of cycle 1:

[0292] a. At least 21 days from the last dose of previous systemic anticancer therapy.

[0293] b. From palliative radiotherapy (≤ 10 minutes or ≤ 30 Gy total dose or from radiotherapy > 30 Gy for at least 28 days) to extrahepatic tumor lesions for at least 14 days.

[0294] c. At least 28 days after local or regional therapy for intrahepatic tumor lesions (e.g., surgery, radiation therapy, hepatic artery embolization, chemoembolization, radiofrequency ablation, percutaneous ethanol injection, or cryoablation).

[0295] 12. According to NCI-CTCAE, v.5, any prior cancer therapy resulting in ≤ Grade 1 toxicity. Grade 2 is permitted in the case of alopecia and / or peripheral sensory neuropathy.

[0296] 13. Participants with a history of HCV infection will be eligible to participate in the study. Treated participants must have completed their treatment at least one month prior to the start of the study intervention, and their HCV viral load must be below the limit of quantification.

[0297] 14. Participants with controlled HBV will be eligible if they meet the following criteria:

[0298] a. Antiviral therapy for HBV must be administered for at least 4 weeks, and the HBV viral load must be below 500 IU / mL prior to the first dose of the study drug. Patients receiving active HBV therapy with a viral load below 100 IU / mL should maintain the same therapy throughout the study intervention.

[0299] b. Patients who are positive for hepatitis B core antibody (HBc), negative for hepatitis B surface antigen (HBsAg), negative or positive for hepatitis B surface antibody (HBs), or have an HBV viral load of less than 100 IU / mL do not require HBV antiviral prophylaxis.

[0300] c. Regardless of whether the patient is taking antihypertensive medication, their blood pressure (BP) must be adequately controlled, defined as BP ≤ 150 / 90 mm Hg at screening, and the antihypertensive medication dosage must not have changed in the week prior to day 1 of cycle 1.

[0301] The exclusion criteria for patient eligibility described above may include one or more of the following:

[0302] 1. Known fibrolamellar HCC, sarcomatoid HCC, or mixed cholangiocarcinoma and HCC

[0303] 2. Patients receiving therapy with other antitumor agents or experimental drugs.

[0304] 3. Failure to recover to Grade 1 or baseline from previously treated adverse events.

[0305] 4. Patients receiving concomitant strong CYP3A4 / 5 inhibitors must not discontinue treatment within 7 days before day 1 of cycle 1 or within 5 half-lives (whichever is longer).

[0306] 5. Patients receiving CYP3A4 / 5 inducers must not discontinue treatment for at least 14 days prior to day 1 of cycle 1.

[0307] 6. Known involvement of the central nervous system (CNS)

[0308] 7. Patients known to have Gilbert syndrome or other genetic disorders affecting UGT1A1 function

[0309] 8. HIV-positive patients receiving combination antiretroviral therapy

[0310] Circulating tumor DNA (ctDNA) obtained from blood samples during screening and at the end of treatment can be used to sequence mutations in the Wnt / β-catenin pathway. These alterations can be used to correlate responses to tagavirin with changes in the mutational profile under treatment. ctDNA can also be used to correlate genomic alterations with changes identified in next-generation sequencing (NGS) analyses of tumors.

[0311] Tumor tissue obtained through pre- and post-treatment core or excisional biopsies can be used to correlate immune responses resulting from T-cell infiltration and activated β-catenin / typical Wnt signaling with clinical responses. Tumor tissue can also be used to correlate genomic alterations with those identified through ctDNA analysis.

[0312] 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, sCD40L, and VEGF-A, can be obtained from serial blood samples throughout treatment to assess changes in Wnt and immune response proteins. Biomarkers can also be used to evaluate the Wnt and immuno-oncological mechanisms of action of tadalafil.

[0313] While the essential novel features of the invention applied to preferred and illustrative embodiments have been shown and described, it will be understood that those skilled in the art can omit, substitute, and change the form and details of the disclosed invention without departing from the spirit of the invention. Furthermore, it will be apparent to those skilled in the art that many modifications and variations will readily occur to them. For example, various features and structures of the different embodiments discussed herein can be combined and interchanged. Therefore, it is not intended to limit the invention to the exact constructions and operations shown and described, and thus all suitable equivalent modifications fall within the scope of the claimed invention. Therefore, it is intended to be limited only as indicated by the scope of the appended claims.

Claims

1. A method for treating advanced hepatocellular carcinoma (HCC) in a patient in need, the method comprising administering to the patient a therapeutically effective amount of tegavivint or a pharmaceutically acceptable salt thereof, wherein the method prevents β-catenin in the patient from associating with transductionin β-like protein 1 (TBL1), and wherein the therapeutically effective dose is from about 0.0001 mg / kg / day to about 1000 mg / kg / day.

2. The method of claim 1, wherein the patient has shown disease progression after at least one prior line of systemic therapy for advanced HCC, or is intolerant to or has contraindications to the at least one prior line of systemic therapy.

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 the administration is performed by one or more of the following methods: intravenous, parenteral, oral, inhalation (including nebulized delivery), buccal, intranasal, rectal, intraperitoneal, intradermal, percutaneous, subcutaneous, intraarterial, intracardiac, intracranial, intratracheal, intrathecal, intrathecal, intramuscular, intravitreal, and local application.

5. The method of claim 1, wherein the administration is performed intravenously.

6. The method of claim 1, wherein the application is performed weekly on days 1, 8, 15, and 22 of a 28-day cycle.

7. The method of claim 1, wherein the effective therapeutic dose is about 1 mg / kg to about 20 mg / kg per week.

8. The method of claim 1, wherein the effective therapeutic dose is about 3 mg / kg, about 5 mg / kg, about 6.5 mg / kg, about 8 mg / kg, about 10 mg / kg, about 12.5 mg / kg, or about 15.5 mg / kg per week.

9. The method of claim 1, wherein the tergavisin is in a formulation comprising 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 of tergavid, 0.625% by weight of poloxamer 188 and 10% by weight of sorbitol.

11. The method of claim 1, wherein the tagawiine is in a nanosuspension prepared by means of: using a crystal form of tagawiine designated as form IV as a starting material, and grinding form IV at a temperature of about 40°C to about 60°C, wherein form IV has an X-ray powder diffraction (XRPD) pattern comprising the following diffraction peaks, the °2θ angle values ​​of which are 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 is substantially composed of form I of tagawiine.

12. The method of claim 1, wherein the tergavisin is in a formulation comprising particles of tergavisin in form I or form IV or a pharmaceutically acceptable salt thereof, wherein 90% of the particles have a diameter less than or equal to 0.2 μm when measured using laser diffraction, and wherein the formulation is prepared by high-energy stirring at a temperature of about 40°C to about 60°C.

13. The method of claim 12, wherein the formulation is lyophilized and reconstituted.

14. The method of claim 1, wherein the patient has previously been treated with a PD-1 / PD-L1 inhibitor.

15. The method of claim 1, further comprising administering a therapeutically effective amount of an immune checkpoint inhibitor to the patient, 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 the following: 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 both tergavisin and pembrolizumab are administered by intravenous infusion, wherein tergavisin is administered on days 1, 8, and 15 of a 21-day cycle, and pembrolizumab is administered on day 1 of the 21-day cycle.

19. The method of claim 18, wherein the dose of said pembrolizumab 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.

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