Formulations of tegavivint and related compounds

Abstract

To provide:stable, readily bioavailable formulations of tegavivint and related compounds; and methods for treating cancers.SOLUTION: A composition includes particles of tegavivint or a pharmaceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof, and a surfactant. The particles have an effective D50 of less than or equal to 500 nm and D90 of less than or equal to 1.0 micrometer (μm), measured using laser diffraction.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates generally to formulations of tegavivint and related compounds, methods of making such formulations, and methods of utilizing such formulations to treat various conditions. [Background technology]

[0002] Cancer is the second leading cause of death in the United States. It presents complex challenges in the development of new treatments. Cancer is characterized by the abnormal growth and metastatic properties of malignant cells that have undergone a series of genetic changes that result in the proliferation of a tumor mass.

[0003] Beta-catenin (β-catenin) is part of a complex of proteins that constitutes adherens junctions (AJs). AJs are required for the formation and maintenance of epithelial cell layers by regulating cell growth and cell-cell adhesion. β-catenin may also be involved in the transmission of contact inhibitory signals that anchor the actin cytoskeleton and cause the cessation of cell division when the epithelial sheet is completed.

[0004] The Wnt / β-catenin pathway has been shown to play a role in cancer. Aberrant β-catenin signaling plays a key role in tumorigenesis. In particular, colorectal cancer is estimated to have more than 80% mutations in the β-catenin pathway, resulting in deregulated oncogenic signaling. Aberrant β-catenin signaling has been shown to be involved in various types of cancer, including but not limited to melanoma, breast cancer, lung cancer, colon cancer, liver cancer, gastric cancer, myeloma, multiple myeloma, chronic myelogenous leukemia, chronic lymphocytic leukemia, T-cell non-Hodgkin's lymphoma, colorectal and acute myeloid leukemia (AML) cancer. In addition, aberrant Wnt / β-catenin signaling has been found in many other disorders, including osteoporosis, osteoarthritis, polycystic kidney disease, diabetes, schizophrenia, vascular disease, cardiac disease, hyperproliferative disorders, neurodegenerative diseases, and fibrotic diseases, including but not limited to idiopathic pulmonary fibrosis (IPF), Dupuytren's contracture, nonalcoholic steatohepatitis (NASH), etc. Myeloproliferative neoplasms (MPNs) are a group of closely related hematological malignancies in which the bone marrow cells that produce the body's blood cells develop and function abnormally. The three major myeloproliferative neoplasms are polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Genetic mutations in JAK2 are found in most PV patients and in 50% of ET and PMF patients. The beta-catenin pathway is often activated in MPNs and is necessary for the survival of these cells.

[0005] Tegavivint and related compounds are described, for example, in U.S. Patent No. 8,129,519. Tegavivint has the following structural formula: [ka]

[0006] The molecular formula for Tegavivin is C 28 H 36 It is N4O6S2.

[0007] The molecular weight of Tegavivint is 588.20763 amu.

[0008] There is a need in the art to provide stable, readily bioavailable formulations of tegavivint and related compounds, which formulations are stable such that they allow for administration via different routes of administration, including but not limited to parenteral and inhalation, and are suitable for clinical trials and treatment of a variety of diseases treatable with tegavivint. Summary of the Invention

[0009] Developing a stable, non-toxic formulation of tegavivint has been very challenging and difficult. Numerous formulations have been developed and tested; however, they have proven to have low bioavailability and / or are unstable on storage and / or have proven to be highly toxic. These formulations include microemulsions, solid suspensions, liposome-based formulations, various oral formulations, and IV formulations.

[0010] The inventors have unexpectedly and surprisingly discovered that a nanosuspension of tegavivint is effective, the nanosuspension including a surfactant, and the particles of tegavivint have an effective D50 of 500 nm or less and a D90 of 1.0 micrometer (μm) or less, as measured using laser diffraction. It has also been discovered that a particularly preferred concentration of tegavivint is 10-25 mg / ml, most preferably 25 mg / ml. The preferred surfactant is a poloxamer surfactant (preferably poloxamer 188), preferably at a concentration of 0.625%, and the nanosuspension should preferably include a polyol, more preferably sorbitol.

[0011] Thus, the most preferred formulation is a composition comprising 25 mg / ml tegavivint, 0.625% poloxamer 188, and 10% sorbitol, wherein the tegavivint is in the form of a nanosuspension comprising particles of tegavivint, the particles having an effective D50 of 500 nm or less and a D90 of 1.0 micrometers (μm) or less, as measured using laser diffraction.

[0012] Thus, in one embodiment, the present invention provides a method for producing a composition comprising: a) Formula I [ka] (In the formula, R A is hydrogen, R7 and R8 are independently selected from H and SO2NR3R4, one of R7 and R8 is hydrogen, and NR1R2 and NR3R4 are independently a 6-15 membered heterocycloalkyl containing one nitrogen in the ring, or a pharma- ceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; b) a surfactant, A composition is provided in which the particles have an effective D50 of 500 nm or less and a D90 of 1.0 micrometers (μm) or less, as measured using laser diffraction.

[0013] In some embodiments, the effective average particle size of the compound is about 4900 nm, about 4800 nm, about 4700 nm, about 4600 nm, about 4500 nm, about 4400 nm, about 4300 mm, about 4200 nm, about 4100 nm, about 4 microns, about 3900 nm, about 3800 nm, about 3700 nm, about 3600 nm, about 3500 nm, about 3400 mm, about 3300 nm, about 3200 nm, about 3100 nm, about 3 microns, about 2900 mm, about 2800 nm, about 2700 nm, about 2600 nm, about 2500 nm, about 2400 nm, about 2300 nm, about 2200 nm, about 2100 nm, about 2000 nm, about 1900 nm, about 1800 nm, about 1700 nm, about 1600 nm, about 1500 nm, about 1400 nm, about 1300 nm, about 1200 nm, about 1100 nm, about 1000 nm, about 900 nm, about 800 nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, or about 300 nm.

[0014] Further, 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.

[0015] In a preferred embodiment, the surfactant is a poloxamer surfactant.

[0016] In another preferred embodiment, the poloxamer surfactant is poloxamer 188.

[0017] In a preferred embodiment, the particle composition further comprises a stabilizer.

[0018] In preferred embodiments, the stabilizer is selected from the group consisting of sugars, polyols, polysorbate surfactants, and polyvinylpyrrolidone (PVP).

[0019] In another preferred embodiment, the sugar is selected from the group consisting of sucrose and / or trehalose.

[0020] In a preferred embodiment, the polyol comprises sorbitol and / or mannitol.

[0021] In one embodiment, the concentration of the compound in the provided compositions is from about 1 mg / ml to about 100 mg / ml, more preferably from about 10 mg / ml to about 50 mg / ml, more preferably from about 10 mg / ml to about 25 mg / ml, and even more preferably about 25 mg / ml.

[0022] In one embodiment, the compositions of the present invention are prepared by grinding.

[0023] In another embodiment, the composition of the present invention is prepared by Lyocell technology. U.S. Patent No. 7,713,440 describes Lyocell technology. The contents of U.S. Patent No. 7,713,440 are incorporated herein by reference in their entirety.

[0024] In another embodiment, the compositions of the present invention can be prepared by dry milling techniques as described in U.S. Patent No. 8,808,751, the contents of which are incorporated herein by reference in their entirety. By appropriately selecting the milling media and suitable abrasive compounds, it is possible to produce nanoparticle compositions from conventional bulk drug particles and prevent agglomeration of the small particles produced in dry milling equipment.

[0025] In yet another embodiment, the composition of the present invention can be prepared by a process that utilizes human serum albumin as a carrier, such as the process described in U.S. Patent No. 6,537,579. The contents of U.S. Patent No. 6,537,579 are incorporated herein by reference in their entirety. This process may be particularly suitable for making nanoparticle compositions of compounds with low water solubility. The composition made by such a process may allow for the effective administration of biologically active compounds with low water solubility.

[0026] In another embodiment, nanoparticle compositions containing polymers such as poly(DL-lactide-co-glycolide) can deliver biologically active compounds that are poorly water soluble. As shown in U.S. Patent No. 5,543,158, these compositions can be designed as long-acting vehicles. The contents of U.S. Patent No. 5,543,158 are incorporated herein by reference in their entirety.

[0027] In another embodiment, the compositions of the invention can be formulated as polymeric micelles that have been successful in improving the solubility of biologically active compounds. A commercial product using this technology, Genexol-PM, incorporates the anticancer drug paclitaxel and was approved in Korea in 2007.

[0028] In one embodiment, the present invention provides a process for preparing a composition comprising the steps of: a) Formula I [ka] (In the formula, R A is hydrogen, R7 and R8 are independently selected from H and SO2NR3R4, one of R7 and R8 is hydrogen, and NR1R2 and NR3R4 are independently 6-15 membered heterocycloalkyl containing one nitrogen in the ring, or or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof, mixing with a surfactant and an acceptable carrier to form a suspension; b) milling the suspension of step (a) using a roller mill or a high energy mill; and c) adding a polyol to the particles of step (b).

[0029] In one embodiment, the acceptable carrier is a liquid carrier (eg, water).

[0030] In one embodiment, the suspension is an aqueous suspension.

[0031] In another embodiment, the process for preparing the composition comprises the following steps (a) to (b): a) Formula I [ka] (In the formula, R A is hydrogen, R7 and R8 are independently selected from H and SO2NR3R4, one of R7 and R8 is hydrogen, and NR1R2 and NR3R4 are independently 6-15 membered heterocycloalkyl containing one nitrogen in the ring, or or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof, mixing with a surfactant, a polyol, and an acceptable carrier to form a suspension; and b) milling the suspension of step (a) using a roller mill or a high energy mill.

[0032] In one embodiment, the acceptable carrier is a liquid carrier (eg, water).

[0033] In one embodiment, the suspension is an aqueous suspension.

[0034] In preferred embodiments, the compositions of the present invention exhibit long-term stability.

[0035] In a preferred embodiment, the compositions of the present invention are nanoparticle compositions.

[0036] In a preferred embodiment, the compound of formula I in the composition of the invention is It has the following structure: [ka] , or a pharma- ceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof.

[0037] The compound having the above formula is also known as tegavivint (BC2059).

[0038] In one embodiment, the compositions of the present invention may 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, fast dissolve, delayed release, sustained release, pulsatile release, and mixed immediate and controlled release formulations; (c) a dosage form suitable for inhaled or parenteral administration, including intramuscular, subcutaneous, intravenous, and intradermal injection; and (d) any combination of (a), (b), and (c).

[0039] The compositions of the present invention may further comprise one or more pharma- ceutically acceptable excipients, carriers, or combinations thereof.

[0040] In another embodiment, the present invention provides a method of preventing, treating or ameliorating cancer or tumor metastasis in a mammal in need thereof, comprising administering to said mammal an effective amount of a composition of the present invention.

[0041] Methods of administration are not limited to any particular route of administration and include, but are not limited to, intravenous, parenteral, oral, inhalation (including aerosolized delivery), buccal, intranasal, rectal, intralesional, intraperitoneal, intradermal, transdermal, subcutaneous, intraarterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, intravitreal injection, and topical application.

[0042] In another embodiment, the method of preventing, treating or ameliorating cancer or tumor metastasis in a mammal in need thereof may include administering additional anti-cancer agents and / or cancer therapies (e.g., cancer vaccines, anti-cancer adoptive cell therapy and radiation therapy).

[0043] In one embodiment, the additional anti-cancer agent is selected from the group consisting of anti-mitotic agents, antimetabolites, HDAC inhibitors, proteosome inhibitors, immunotherapeutic agents, FLT-3 EGFR, MEK, PI3K and other protein kinase inhibitors, LSD1 inhibitors, and WNT pathway inhibitors, alkylating agents and DNA repair pathway inhibitors, anti-hormones, anti-cancer antibodies, and other cytotoxic chemotherapeutic agents.

[0044] In another embodiment, the present invention provides a method for treating and / or preventing a fibrotic disease in a mammal in need thereof, comprising administering to said mammal an effective amount of a composition of the present invention.

[0045] In one embodiment, the fibrotic disease is selected from the group consisting of pulmonary fibrosis, Dupuytren's contracture, scleroderma, systemic sclerosis, scleroderma-like disorders, scleroderma without skin sclerosis, liver cirrhosis, interstitial pulmonary fibrosis, keloids, chronic kidney disease, chronic transplant rejection and other scarring / wound healing disorders, post-surgical adhesions, and reactive fibrosis.

[0046] In one embodiment, the method of treating and / or preventing a fibrotic disease in a mammal in need thereof may comprise administering an additional anti-fibrotic agent. [Brief description of the drawings]

[0047] [Figure 1]1 is a graph of the particle size distribution (PSD) of one of the formulations of the present invention. [Diagram 2] 1 is a graph of the PSD of another one of the formulations of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] definition The terms used herein generally have their ordinary meanings in the art, within the context of the present invention, and in the specific context in which each term is used. Certain terms used to describe the present invention are described below or elsewhere in this specification to provide further guidance to the practitioner regarding the description of the present invention. Synonyms of certain terms are provided. The listing of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification, including examples of any term discussed herein, is illustrative only and does not limit the scope and meaning of the present invention or any exemplified term in any way. The present invention is not limited to the various embodiments described herein.

[0049] 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 belongs. In case of conflict, the present specification, including definitions, shall control.

[0050] The term "tegavivint" refers to a compound having the following structure: [ka] . The term "BC2059" is used interchangeably with "TegaVivint."

[0051] The term "long-term storage" or "long-term stability" is understood to mean that the pharmaceutical composition can be stored for 3 months or more, 6 months or more, and preferably 1 year or more. Long-term storage is also understood to mean that the pharmaceutical composition is stored at 2-8°C or at room temperature of 15-25°C.

[0052] The term "stable" or "stabilization" with respect to long-term storage should be understood to mean that the active ingredient contained in the pharmaceutical composition does not lose more than 20%, more preferably 15%, even more preferably 10%, and most preferably 5% of its activity compared to the activity of the composition at the start of storage.

[0053] The term "mammal" includes, but is not limited to, humans.

[0054] The term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material, formulation aid, or excipient of any conventional type. Pharmaceutically acceptable carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation.

[0055] The term "treatment" refers to any administration or application of a therapeutic agent against a disease in a mammal, including suppressing the disease, arresting its onset, alleviating the disease (e.g., by causing regression or restoring or repairing lost, missing or defective function), or stimulating an inefficient process. The term includes obtaining a desired pharmacological and / or physiological effect in a mammal, as well as covering any treatment of a pathology or disorder. The effect may be prophylactic, in that the disorder or its symptoms are completely or partially prevented, and / or therapeutic, in that the disorder and / or adverse effects resulting from the disorder are partially or completely cured. This includes (1) preventing the onset or recurrence of the disorder in a subject who may be predisposed to the disorder but who is not yet symptomatic; (2) inhibiting the disorder, such as arresting onset; (3) halting or terminating the disorder or at least its associated symptoms such that the host is no longer afflicted by the disorder or its symptoms, e.g., by restoring or repairing a lost, missing, or defective function or stimulating an inefficient process, or (4) reducing, alleviating, or ameliorating the disorder or its associated symptoms, where amelioration is used broadly to refer to at least a decrease in the magnitude of parameters such as inflammation, pain, and / or tumor size.

[0056] The term "therapeutically effective amount" refers to an amount that achieves a desired effect on an organism when administered to the organism. For example, the effective amount of the composition of the present invention for administration to an organism is an amount that prevents and / or treats any disease mediated through the Wnt / β-catenin pathway. The exact amount depends on the purpose of treatment and can be ascertained by the skilled artisan using known techniques. As known in the art, adjustments may be required for systemic versus local delivery, age, weight, overall health, sex, diet, duration of administration, drug interactions, and severity of condition, and can be ascertained by the skilled artisan using routine experimentation.

[0057] The term "composition" or "formulation" is conventional in the art and refers to a mixture that typically includes a carrier, such as a pharma- ceutically acceptable carrier or excipient, suitable for administration to a subject for therapeutic, diagnostic, or prophylactic purposes. For example, a composition for oral administration can form a solution, suspension, tablet, pill, capsule, sustained release formulation, oral rinse, or powder. The terms "composition," "pharmaceutical composition," and "formulation" are used interchangeably.

[0058] The term "nanoparticle composition" refers to a composition in which all or almost all of the particles are less than 1000 nM.

[0059] Compositions of the Invention In one embodiment, the present invention provides a) Formula I [ka] (In the formula, R a is hydrogen, R7 and R8 are independently selected from H and SO2NR3R4, one of R7 and R8 is hydrogen, and NR1R2 and NR3R4 are independently 6-15 membered heterocycloalkyl containing one nitrogen in the ring, or or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof; b) a surfactant, A composition is provided in which the particles have an effective D50 of 500 nm or less and a D90 of 1.0 micrometers (μm) or less, as measured using laser diffraction.

[0060] D50 refers to the particle size value at 50% of the cumulative distribution, also known as the median size of the particle size distribution. In other words, a D50 value of 500 nm or less means that 50% of the particles have a diameter less than 500 nm.

[0061] D90 refers to the percentage of particles below the reported particle size. In other words, a D90 value of 1.0 μm or less means that 90% of the particles are less than 1.0 μm in diameter.

[0062] In some embodiments, the effective average particle size of the compound is about 4900 nm, about 4800 nm, about 4700 nm, about 4600 nm, about 4500 nm, about 4400 nm, about 4300 mm, about 4200 nm, about 4100 nm, about 4 microns, about 3900 nm, about 3800 nm, about 3700 nm, about 3600 nm, about 3500 nm, about 3400 mm, about 3300 nm, about 3200 nm, about 3100 nm, about 3 microns, about 2900 mm, about 2800 nm, about 2700 nm, about 2600 nm, about 2500 nm, about 2400 nm, about 2300 nm, about 2200 nm, about 2100 nm, about 2000 nm, about 1900 nm, about 1800 nm, about 1700 nm, about 1600 nm, about 1500 nm, about 1400 nm, about 1300 nm, about 1200 nm, about 1100 nm, about 1000 nm, about 900 nm, about 800 nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, or about 300 nm.

[0063] Further, 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.

[0064] In a preferred embodiment, the surfactant is a poloxamer surfactant.

[0065] In another preferred embodiment, the poloxamer surfactant is poloxamer 188.

[0066] In a preferred embodiment, the composition further comprises a stabilizer.

[0067] In preferred embodiments, the stabilizer is selected from the group consisting of sugars, polyols, polysorbate surfactants, and polyvinylpyrrolidone (PVP).

[0068] In another preferred embodiment, the sugar is selected from the group consisting of sucrose and / or trehalose.

[0069] In a preferred embodiment, the polyols include sorbitol and mannitol.

[0070] In one embodiment, the concentration of the compound in the provided compositions is from about 1 mg / ml to about 100 mg / ml, more preferably from about 10 mg / ml to about 50 mg / ml, more preferably from about 10 mg / ml to about 25 mg / ml, and even more preferably about 25 mg / ml.

[0071] A particularly preferred concentration of tegavivint is 10-25 mg / ml, most preferably 25 mg / ml, the preferred surfactant is a poloxamer surfactant (preferably poloxamer 188), preferably at a concentration of 0.625%, and the nanosuspension preferably comprises a polyol, more preferably sorbitol.

[0072] Therefore, the most preferred formulation is a nanosuspension containing 25 mg / ml tegavivint, 0.625% poloxamer 188, and 10% sorbitol.

[0073] In one embodiment, the compositions of the present invention are prepared by milling, preferably wet milling.

[0074] In one embodiment, the present invention provides a process for preparing a composition comprising the steps of: a) Formula I [ka] (In the formula, R A is hydrogen, R7 and R8 are independently selected from H and SO2NR3R4, one of R7 and R8 is hydrogen, and NR1R2 and NR3R4 are independently 6-15 membered heterocycloalkyl containing one nitrogen in the ring, or or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof, mixing with a surfactant and an acceptable carrier to form a suspension; b) milling the suspension of step (a) using a roller mill or a high energy mill; and c) adding a polyol to the particles of step (b).

[0075] In one embodiment, the acceptable carrier is a liquid carrier (eg, water).

[0076] In one embodiment, the suspension is an aqueous suspension.

[0077] In another embodiment, the process for preparing the composition comprises the following steps (a) to (b): a) Formula I [ka] (In the formula, R a is hydrogen, R7 and R8 are independently selected from H and SO2NR3R4, one of R7 and R8 is hydrogen, and NR1R2 and NR3R4 are independently 6-15 membered heterocycloalkyl containing one nitrogen in the ring, or or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof, mixing with a surfactant, a polyol, and an acceptable carrier to form a suspension; and b) milling the suspension of step (a) using a roller mill or a high energy mill.

[0078] In one embodiment, the acceptable carrier is a liquid carrier (eg, water).

[0079] In one embodiment, the suspension is an aqueous suspension.

[0080] In another embodiment, the composition of the present invention is prepared by Lyocell technology. U.S. Patent No. 7,713,440 describes Lyocell technology. The contents of U.S. Patent No. 7,713,440 are incorporated herein by reference in their entirety.

[0081] In another embodiment, the compositions of the present invention can be prepared by dry milling techniques as described in U.S. Patent No. 8,808,751, the contents of which are incorporated herein by reference in their entirety. By appropriately selecting the milling media and suitable abrasive compounds, it is possible to produce nanoparticle compositions from conventional bulk drug particles and prevent agglomeration of the small particles produced in dry milling equipment.

[0082] In yet another embodiment, the composition of the present invention can be prepared by a process that utilizes human serum albumin as a carrier, such as the process described in U.S. Patent No. 6,537,579. The contents of U.S. Patent No. 6,537,579 are incorporated herein by reference in their entirety. This process may be particularly suitable for making nanoparticle compositions of compounds with low water solubility. The composition made by such a process may allow for the effective administration of biologically active compounds with low water solubility.

[0083] In another embodiment, nanoparticle compositions containing polymers such as poly(DL-lactide-co-glycolide) can deliver biologically active compounds that are poorly water soluble. As shown in U.S. Patent No. 5,543,158, these compositions can be designed as long-acting vehicles. The contents of U.S. Patent No. 5,543,158 are incorporated herein by reference in their entirety.

[0084] In another embodiment, the compositions of the invention can be formulated as polymeric micelles that have been successful in improving the solubility of biologically active compounds. A commercial product using this technology, Genexol-PM, incorporates the anticancer drug paclitaxel and was approved in Korea in 2007.

[0085] In preferred embodiments, the compositions of the present invention exhibit long-term stability.

[0086] In one embodiment, the compositions of the invention are nanoparticle compositions.

[0087] In a preferred embodiment, the compound of formula I in the composition of the invention has the following structure: [ka] , or a pharma- ceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof.

[0088] This compound is also known as tegavivint.

[0089] The present invention encompasses formulations comprising tegavivint and its pharma- ceutically acceptable salts, esters, amides, stereoisomers or geometric isomers.

[0090] The aqueous solubility of tegavivint has been measured over a pH range of 2 to 10 and was found to be <0.25 mcg / mL over that range.

[0091] In organic solvents, tegavivint has solubilities as shown: DMSO (334 μg / mL), ethanol (260 μg / mL), methanol (299 μg / mL), acetone (1 mcg / mL), dichloromethane:ethanol (1:4) (1 mg / mL).

[0092] In one embodiment, the compositions of the present invention may 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, fast dissolve, delayed release, sustained release, pulsatile release, and mixed immediate and controlled release formulations; (c) a dosage form suitable for inhaled or parenteral administration, including intramuscular, subcutaneous, intravenous, and intradermal injection; and (d) any combination of (a), (b), and (c).

[0093] The compositions of the present invention may further comprise one or more pharma- ceutically acceptable excipients, carriers, or combinations thereof.

[0094] The pharma- ceutically acceptable excipients used in the formulation of the present invention can act in more than one way.For example, the role of dispersing agent is mainly to keep individual particles separate, i.e., to minimize agglomeration.However, this component can also, for example, change the surface tension of the formulation, and can act to reduce viscosity.

[0095] The pharma- ceutically acceptable excipient can be, for example, a dispersion medium, a dispersion-emulsifying agent, a dispersion enhancer, or a combination thereof.

[0096] 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), combinations thereof, and the like.

[0097] The dispersion medium can be, for example, ethanol, propylene glycol, polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400, glycerin, combinations thereof, and the like.

[0098] The dispersing emulsifier (enhancing agent) can be, for example, HO, 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, combinations thereof, and the like.

[0099] Examples of dispersion enhancers include, but are not limited to, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sodium carboxymethylcellulose, hypromellose, ethylene glycol stearate, sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate, glyceryl monostearate, lecithin, meglumine, poloxamer, polyoxyethylene alkyl ethers, polyoxyl 35 castor oil, polyoxyethylene stearate, polyoxyglycerides, pyrrolidone, sorbitan esters, stearic acid, vitamin E polyethylene glycol succinate, polyethylene glycol 1000, povidone, combinations thereof, and the like.

[0100] The compositions of the present invention may be suitable for all routes of administration, including, but not limited to, intravenous, parenteral, oral, inhalation (including aerosolized delivery), buccal, intranasal, rectal, intralesional, intraperitoneal, intradermal, transdermal, subcutaneous, intraarterial, intracardiac, intraventricular, intracranial, intratracheal, intrathecal administration, intramuscular injection, intravitreal injection, and topical application.

[0101] Pharmaceutical compositions according to the invention may also include one or more binders, fillers, lubricants, suspending agents, sweeteners, flavoring agents, preservatives, buffers, wetting agents, disintegrants, effervescent agents, and other excipients, such excipients being known in the art.

[0102] Examples of fillers are lactose monohydrate, anhydrous lactose, and various starches, and examples of binders are various celluloses and cross-linked polyvinylpyrrolidone, microcrystalline cellulose such as Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, and silicified microcrystalline cellulose (ProSolvSMCC™).

[0103] Suitable lubricants, including agents that act on the flowability of the powder to be compressed, are colloidal silicon dioxide such as Aerosil® 200, talc, stearic acid, magnesium stearate, calcium stearate, and silica gel.

[0104] Examples of sweeteners are any natural or artificial sweetener, such as sucrose, xylitol, sodium saccharin, cyclamate, aspartame, and axulfame, etc. Examples of flavoring agents are Magnasweet® (a trademark of MAFCO), bubble gum flavor, fruit flavors, etc.

[0105] Examples of preservatives are potassium sorbate, methylparaben, propylparaben, benzoic acid and its salts, other esters of parahydroxybenzoic acid such as butylparaben, alcohols such as ethyl or benzyl alcohol, phenolic compounds such as phenol, or quaternary compounds such as benzalkonium chloride.

[0106] Suitable diluents include pharma- ceutically acceptable inert fillers, such as microcrystalline cellulose, lactose, dibasic calcium phosphate, saccharides, and / or any mixture of the above.Examples of diluents include microcrystalline cellulose, such as Avicel® PH101 and Avicel® PH102; lactose, such as lactose monohydrate, anhydrous lactose, and Pharmatose® DCL21; dibasic calcium phosphate, such as Emcompress®; mannitol; starch; sorbitol; sucrose; and glucose.

[0107] Suitable disintegrants include lightly cross-linked polyvinylpyrrolidone, corn starch, potato starch, maize starch, and modified starches, croscarmellose sodium, crospovidone, sodium starch glycolate, and mixtures thereof.

[0108] Examples of effervescent agents are effervescent couples, 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 anhydrides and acid salts.Suitable carbonates and bicarbonates include, for example, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, and arginine carbonate.Alternatively, only the sodium bicarbonate component of the effervescent couple may be present.

[0109] In a preferred embodiment, the compound of formula I has the following structure: [ka] or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof.

[0110] In another embodiment, the present invention provides a method of preventing, treating or ameliorating cancer or tumor metastasis in a mammal in need thereof, comprising administering to said mammal an effective amount of a composition of the present invention.

[0111] In another embodiment, the method of preventing, treating or ameliorating cancer or tumor metastasis in a mammal in need thereof may include administering additional anti-cancer agents and / or cancer therapies (e.g., cancer vaccines, anti-cancer adoptive cell therapy and radiation therapy).

[0112] In one embodiment, the additional anti-cancer agent is selected from the group consisting of anti-mitotic agents, antimetabolites, HDAC inhibitors, proteosome inhibitors, immunotherapeutic agents, FLT-3 EGFR, MEK, PI3K and other protein kinase inhibitors, LSD1 inhibitors, and WNT pathway inhibitors, alkylating agents and DNA repair pathway inhibitors, anti-hormones, anti-cancer antibodies, and other cytotoxic chemotherapeutic agents.

[0113] In another embodiment, the present invention provides a method for treating and / or preventing a fibrotic disease in a mammal in need thereof, comprising administering to said mammal an effective amount of a nanoparticle composition of the present invention.

[0114] In one embodiment, the fibrotic disease is selected from the group consisting of pulmonary fibrosis, Dupuytren's contracture, scleroderma, systemic sclerosis, scleroderma-like disorders, scleroderma without skin sclerosis, liver cirrhosis, interstitial pulmonary fibrosis, keloids, chronic kidney disease, chronic transplant rejection and other scarring / wound healing disorders, post-surgical adhesions, reactive fibrosis.

[0115] The present invention is more particularly described in the following examples, which are intended to be illustrative only, since numerous modifications and variations thereof will be apparent to those skilled in the art, and it should be understood that in the following examples the weight percentages of the various components are expressed as w / v percentages. EXAMPLES

[0116] Arriving at an effective, ie, stable and non-toxic, formulation of tegavivint has been extremely challenging and difficult.

[0117] The formulation that proved effective was a nanosuspension of tegavivint, the nanosuspension including a surfactant, where the particles of tegavivint were found to have an effective D50 of 500 nm or less and a D90 of 1.0 micrometer (μm) or less, as measured using laser diffraction. It was also discovered that a particularly preferred concentration of tegavivint is 10-25 mg / ml, most preferably 25 mg / ml. The preferred surfactant is a poloxamer surfactant (preferably poloxamer 188), preferably at a concentration of 0.625%, and the nanosuspension should preferably include a polyol, more preferably sorbitol.

[0118] In the example section, first, several experiments for formulating tegavivint are described, which ultimately fail for various reasons.Then, this section describes the feasibility experiment of milling, which demonstrates that tegavivint can be roller milled when suspended in aqueous solution with various dispersants.However, even when roller milled, many formulations of tegavivint are still unsuccessful.

[0119] Finally, successful experiments involving the claimed nanosuspension of tegavivint are described.

[0120] A failed experiment Example 1 The microemulsion formulation of Tegavivint was highly toxic A microemulsion formulation of tegavivint was developed that contained 20 mg / ml BC2059, 10% Tween (polysorbate 80), 30% ethanol, 50% propylene glycol (PG) and 10% D-α-tocopherol polyethylene glycol 1000 succinate.

[0121] Although good stability of the formulation was observed, the formulation was highly toxic to rodents and therefore was not pursued further.

[0122] Example 2 Liposome-based formulations were unstable Based on preliminary studies, two liposomal formulations of BC2059 were selected as leads for scale-up and stability evaluation at 100 ml.

[0123] The first was a 100% ePC formulation with a lipid to drug ratio of 15:1.

[0124] The other lead formulation contained 80:20% ePC:LysoPC liposomes with a lipid to drug ratio of 10:1.

[0125] Both of these formulations proved to be unstable (precipitation was observed) when stored at 5° C. Additionally, the formulations were also unstable when frozen.

[0126] Example 3 Oral and IV formulations were unsuccessful An oral formulation of tegavivint containing soy lecithin, PEG200, PEG400, PG, and TPGS was initially selected as the lead oral formulation, however, this lead oral formulation was not pursued due to poor bioavailability in dog studies.

[0127] After further screening, an IV-based formulation was selected as the next lead. This IV formulation consisted of an oil phase (vegetable oil and polysorbate 80 (PS80) as a solubilizer) and soy lecithin as an emulsifier. The formulation contained the following ingredients (all numbers are by weight):

[0128] BC2059: 1%, PS80: 10%, Miglyol812: 12%, soy lecithin (LIPOID S-100): 12%, propylene glycol (PG): 50%, deionized water: to taste.

[0129] This formulation exhibited good physical and chemical stability while also demonstrating the potential to be 0.2 micron filterable with minimal losses, however, it was not pursued further due to high toxicity in rodent studies.

[0130] Experiments with Tegavivint nanosuspension Example 4 Feasibility of grinding First, we determined whether milling was possible in principle. Experiments showed that it was.

[0131] Milling feasibility experiments were initiated by roller milling a laboratory-scale batch of tegavivint suspended at 5% (50 mg / mL) in aqueous solution with the following dispersants, selected for their suitability for intravenous administration: Polysorbate 20 (0.5%) Poloxamer 188 (0.5%) Polyvinylpyrrolidone, K17 (1%) Polyvinylpyrrolidone, K17 (1%) and sodium deoxycholate (0.25%) Lecithin (1%)

[0132] Five mL of each test suspension was milled using 0.5 mm diameter yttria-stabilized zirconia (YTZ) grinding media at a media load of approximately 10 mL and sampled periodically for particle size distribution analysis by laser diffraction. After 12 hours of milling, only the poloxamer and polyvinylpyrrolidone suspensions showed production of uniform nanoparticle dispersions, the lecithin suspension showed no obvious size reduction, the polysorbate suspension showed solidification of BC2059 in the milling vessel, and the polyvinylpyrrolidone / sodium deoxycholate suspension showed high aspect ratio crystals. For informal particle size stability, the terminated test suspensions were left at uncontrolled ambient conditions for 4 days. All suspensions tested showed some degree of particle growth, with particle elongation similar to that initially seen with the polyvinylpyrrolidone / sodium deoxycholate suspension.

[0133] To prevent crystal growth, preparations were made with 5% (50 mg / mL) tegavivint using both poloxamer and polyvinylpyrrolidone (PVP) as dispersing agents, incorporating sucrose, sorbitol, and trehalose at 10% each. Milling and storage were performed under similar conditions as in the initial feasibility experiments. All preparations were milled into nanosuspensions, but none of the additives appeared to have a discernible effect on inhibiting crystal growth. Poloxamer 188 was selected as the primary dispersing agent for further experiments.

[0134] Additional material was milled at 5% (50 mg / mL) in Poloxamer 188. To facilitate scale-up, the poloxamer content was increased from 1% to 1.5% to ensure a uniform nanosuspension. The milled nanosuspension was diluted to produce a 2% (20 mg / mL) BC2059 / 0.6% poloxamer / 0.9% sodium chloride formulation for use in initial pharmacokinetic studies conducted by a third party. The remaining milled concentrated material was saved to test the effectiveness of lyophilization in preventing apparent crystal growth.

[0135] This formulation was then tested in the experiments described in Example 5.

[0136] Example 5 Feasibility of freeze-drying This experiment was to determine whether lyophilization is feasible in principle. It has been shown that, in principle, tegavivint can be lyophilized.

[0137] A 5% (50 mg / mL) tegavivin poloxamer aqueous suspension was diluted with various potential cryoprotectant-containing diluents to give 2% (20 mg / mL) tegavivin, 0.6% poloxamer, and the following final concentrations: Sucrose (10%) Mannitol (5%) Sucrose (5%) and mannitol (2.5%) Sorbitol (10%) Sorbitol (5%) and mannitol (2.5%) Trehalose (10%) Trehalose (5%) and Mannitol (2.5%)

[0138] 5 mL serum vials were filled to 2 mL of each preparation and lyophilized at -40°C and 100 mTorr pressure. Dried vials were resuspended in purified water and analyzed for particle size distribution. Of the systems tested, only the 10% sorbitol and 10% trehalose resuspensions returned to a particle size distribution comparable to the suspension before lyophilization. Additional nanosuspensions were milled and the component concentrations were increased to 10% (100 mg / mL) BC2059 and 3% poloxamer to increase milling efficiency and facilitate larger batch production.

[0139] The following suspensions were prepared from the ground material for low temperature differential scanning calorimetry (DSC) analysis: 2% (20 mg / mL) BC2059 with 0.6% polysorbate and 10% sorbitol · 2% (20mg / mL) BC2059 with 0.6% polysorbate and 10% trehalose.

[0140] DSC analysis performed from 25° C. to −40° C. and then back to 25° C. at a rate of 1° C. / min gave the following glass transition values ​​for the suspensions: Sorbitol suspension: -18℃ Trehalose suspension: -33℃

[0141] The suspension was lyophilized, filling 2 mL into 5 mL vials, and dried primarily at -30 °C / 150 mTorr and secondarily at -16 °C / 550 mTorr. Informally, the lyophilized samples were shown to have a reproducible, uniform size distribution and to be physically stable for up to 1 week at ambient laboratory conditions. For subsequent experiments, sorbitol was chosen rather than trehalose because of the former's higher glass transition temperature and the greater availability of historical toxicity data.

[0142] Test batches were milled and lyophilized with primary drying at -24°C / 250mTorr and secondary drying at -16°C / 500mTorr to provide material for animal studies. Milling was performed with 20% (200mg / mL) TegaVivint with 5% poloxamer content to facilitate larger batch sizes and increase milling efficiency. The dried formulation contained approximately 1% water by Karl Fischer method and showed adequate particle size stability after 24 hours when reconstituted with purified water.

[0143] These formulations were then tested in the experiments described in Example 6.

[0144] Example 6 Nonclinical Toxicology / Pharmacokinetics, Batch Production The purpose of this study was to test a lyophilized formulation of tegavivint.

[0145] Four successively prepared sub-batches of suspension, each representing 15 g of tegavivint, were milled at increasing loads and extracted from the milling media using a diluent of 11.43% aqueous sorbitol to increase product yield, resulting in a suspension of 2% (20 mg / mL) tegavivint / 0.5% poloxamer / 10% sorbitol.

[0146] 5 mL vials were filled in 2 mL sub-batches and lyophilized under previously optimized conditions. Although some vials showed signs of meltback, likely due to batch scale up, the dried material was easily resuspended into a uniform nanosuspension. Intermediate assay, PSD, and water results for each sub-batch of vials showed acceptable batch-to-batch agreement, so the four sets of vials were combined and processed as a single batch for use in stability and animal studies. See Table 1 below. [Table 1]

[0147] One of the sub-batches failed due to the steady production of air bubbles during milling, which prevented further milling and caused permanent particle agglomeration. It was discarded and another batch was made to replace it. In the failed batch production, 250 mL serum bottles were used instead of media bottles to facilitate PSD sampling during milling. The foaming was due to differences in bottle dimensions, which apparently allowed air entrapment, resulting in batch failure. During extraction of all batches, dark insoluble particles separated from the milling media. This material was later analyzed by XRPD and found to be fused aggregates of Tega Vivint.

[0148] At the one-month stability time point, the composite batch showed a significant increase in particle size due to poloxamer aggregation rather than API ripening or crystal growth. An attempt was made to determine a suitable route for experimentation whereby the test article could be reused. Samples were reconstituted, resealed, and heated to 50°C for up to 3 hours without any reduction in aggregates. Resuspended vials were autoclaved at 121°C for 10 minutes using a slow release liquid cycle and cooled to ambient conditions, returning to an acceptable particle size distribution.

[0149] This type of treatment did not indicate a suitable route forward so it was decided to reformulate the product.

[0150] Example 7 Reformulation and lyophilization of the composition ultimately failed, but a liquid suspension containing poloxamer seemed promising. In this study, further reformulated compositions of Tegavivint were tested. Finally, lyophilization was unsuccessful, but a liquid suspension (nanosuspension) showed promising results.

[0151] Feasible scale batches were made for some of the dispersions initially tested, but with an increased tegavivint concentration of 20% (200 mg / mL). This change may have made dispersions that were not promising at 5% (5 mg / mL) feasible. The following dispersions were tested along with a 5% poloxamer 188 control: Polysorbate 20 (2%) Polyvinylpyrrolidone (2%) Polyvinylpyrrolidone (2%) and sodium deoxycholate (1%) Polyvinylpyrrolidone (2%) and Polysorbate 20 (2%) Partially hydrolyzed polyvinyl alcohol (5%)

[0152] After 12 hours of milling, the polysorbate preparation showed more rapid milling than the control with good uniformity. The polyvinylpyrrolidone preparation showed the presence of non-crystalline particles, likely aggregates or remnants of polyvinylpyrrolidone, which did not significantly affect the particle size distribution measurements but were visible under an optical microscope. The polyvinyl alcohol preparation did not result in significant size reduction, likely due to the viscosity of the dispersant. Although the binary polyvinylpyrrolidone preparation showed significant aggregates, it was determined that the polyvinylpyrrolidone / sodium deoxycholate preparation might prove useful in further development.

[0153] Polyvinylpyrrolidone and polysorbate 20 preparations, as well as modified polyvinylpyrrolidone (1%) and sodium deoxycholate (0.5%) suspensions, were used in the following freeze-drying development experiments for cryoprotectants. Sorbitol (10%) Sucrose (10%) Trehalose (10%) Mannitol (5% Mannitol (5%) Sorbitol (5%) and mannitol (2.5%) Sucrose (5%) and mannitol (2.5%)

[0154] Lyophilization was performed at -36°C / 100mTorr and -15°C / 500mTorr with a -15°C annealing step. Upon resuspension, only the 10% sucrose preparation showed proper recovery of particle size. Additional suspensions were milled using a 1% polyvinylpyrrolidone / 0.5% sodium deoxycholate preparation, but contained citrate buffer to maintain the pH at approximately 7.0. The milled suspension preparation reversibly gelled on standing, which was combined with the following cryoprotectants: Sucrose (15%) 25mg / mL sucrose (10%) BC2059 Sorbitol (10%) Lactose (5%) Sucrose (5%) and sorbitol (5%)

[0155] High concentrations of sucrose compared to the API were shown to provide the best particle size protection, and although the formulation appeared to be susceptible to meltback, accelerated stability studies conducted at 25°C / 60% RH and 40°C / 75% RH showed that the formulation had good physical stability over a 4-week period.

[0156] However, in dilution studies, the formulation was found to aggregate in the saline diluent used for administration, and the pharmacokinetic release of the polyvinylpyrrolidone / sodium deoxycholate formulation was significantly lower than that of the poloxamer formulation originally tested.

[0157] Nanosuspension containing Poloxamer 188 200mg / mL (20%) BC2059 was milled in poloxamer 188 and submitted to a third party for lyophilization optimization. The suspension was combined with a series of cryoprotectants listed in Table 2 below that were used in the lyophilization experiments. Initially, the 2.5% dextran / 2.5% sorbitol formulation showed the most promising particle size retention upon reconstitution, however, after 1 month at 40°C / 75% RH, the only formulation that retained the nanosuspension was the undried control.

[0158] Thus, these tests indicated that freeze-drying was not successful under the conditions evaluated. The findings also surprisingly indicated that the liquid suspension was more stable than previously observed. It was determined that the initial particle elongation was an immediate, limited phenomenon, likely the result of initial supersaturation of the dispersant causing slight reprecipitation after milling was stopped. See Table 2 below. [Table 2]

[0159] Example 8 Feasibility of irradiation Both of the developed formulation dispersion systems (polyvinylpyrrolidone / sodium deoxycholate and poloxamer) were used to determine the feasibility of terminal sterilization by irradiation. Both samples were prepared for both irradiation feasibility. Samples of both formulations were provided for parallel pharmacokinetic (PK) studies in laboratory animals, for which a diluent containing poloxamer 188 was also provided to be used with the polyvinylpyrrolidone / sodium deoxycholate formulation to determine whether the bioavailability of the drug was related to the poloxamer. The results of the PK study showed that the bioavailability was surprisingly correlated with the poloxamer content.

[0160] Frozen vials of both formulations were sent for irradiation. Both gamma and electron beam irradiation were tested, at both 15 and 25 kGy. The vials were processed under frozen conditions, but also at 5°C as a worst case scenario to simulate potential thawing during irradiation. Degradation was independent of temperature, but appeared to be correlated with dose, regardless of the type of radiation.

[0161] However, initial particle size testing, supported by subsequent stability data, showed extensive particle aggregation. Based on previously successful freeze / thaw testing, the aggregation was attributed to irradiation. Subsequent freeze / thaw cycles on a separate suspension, however, showed similar aggregation.

[0162] Frozen storage resulted in unpredictable aggregation and was determined not to be an option for moving the GLP batch forward. The vials were stored in a -20°C freezer prior to irradiation, which may have resulted in different freezing rates between vials in different locations on the shelf.

[0163] Example 9 Preclinical generation The production of the 25 mg / mL tegavivint nanosuspension was performed using the cleanest conditions, i.e., although sterility was not guaranteed, various controls and precautions were taken to minimize microbial contamination.

[0164] Sterile water for injection was used to make the formulations to minimize microbial as well as pyrogenic contamination. All excipients were USP / NF grade. All product contact items were sterilized by autoclaving or disinfected with 70% isopropanol if not suitable for heating. All exposure preparations were performed in an ISO5 quality laminar flow hood using aseptic processing techniques. The API used in manufacturing the preclinical batches was gamma irradiated at 30 kGy prior to use.

[0165] Example 10 Preparation of rat test articles A 1,600 gram (nominal) batch of tegavivint suspension was prepared for administration in the rat toxicity study.

[0166] The production started with a 200 gram batch of concentrated (200 mg / mL) BC2059 nanosuspension. 10 g of poloxamer 188 was dissolved in 150 grams of water in a 250 mL serum bottle. 200 grams of YTZ grinding media was added and the bottle was stoppered and sealed. Given the small batch size, the entire assembly and the poloxamer solution preparation were autoclaved at 121°C for 15 minutes to minimize bioburden. 40 grams of irradiated API was added and the bottle was again stoppered and sealed. The preparation was rolled on a roller mill until the break angle of the cascading media was approximately 45 degrees as determined visually.

[0167] It was noted that the amount of grinding media used was approximately half the amount normally used to process a 200 gram batch of suspension, as air entrapment can be a tendency for formulations to fail. The bottles used were also smaller than normal to minimize head space. The grinding was allowed to proceed over the weekend, and the suspension was sampled by hypodermic needle through a septum.

[0168] The D90 of the particle size distribution was 0.23 microns and was deemed sufficient to proceed to extraction. Extraction was performed using an autoclaved solution of 160 grams of sorbitol in 1240 grams of water and a glass pressure funnel containing a 60 micron sintered glass frit. The extracted suspension was mixed and filled into autoclaved 10 mL glass vials using a positive displacement pipette set at 5.00 mL. 295 vials were filled, stoppered and sealed, representing a 92% yield. The batch was stored at 5°C until use.

[0169] The nanosuspension was considered ready to be administered to rats.

[0170] Example 11 Preparation of porcine test articles A 10,400 gram (nominal) batch of Tegavivint was prepared for administration in the swine toxicity study. Production began with a 1,300 gram batch of concentrated (200 mg / mL) BC2059 nanosuspension. 65 grams of poloxamer 188 was dissolved in 975 grams of water in a 2000 mL media bottle. 1000 grams of YTZ grinding media was rinsed and bagged for sterilization. The media and solution were autoclaved separately and combined with 260 grams of sterile API in a media bottle. This preparation was rolled on a roller mill such that the break angle of the cascading media was approximately 45 degrees as determined visually. Milling was allowed to proceed for a total of approximately 3 days until the D90 of the particle size distribution was 0.33 and deemed sufficient to proceed to extraction. Two aliquots of sorbitol solution, made by dissolving 520 g of sorbitol in 4030 g of water, were autoclaved and used to extract the milled suspensions as was done for the rat study batches.

[0171] Difficulties in extraction were noted as apparently unmilled or larger particle size API clogged the 60 micron filter frit, necessitating removal of the media and rinsing of the frit. The extracted suspension was mixed and filled into autoclaved 10 mL glass vials using a positive displacement pipette set at 10.0 mL. 970 vials were filled, stoppered and sealed, representing a 93% yield. The batch was stored at 5°C until use.

[0172] The nanosuspension was considered ready for administration to pigs.

[0173] Example 12 Autoclaving had no significant effect on degradation. In conjunction with the generation of test article for preclinical testing, two batches of tegavivint suspension were prepared. One batch was made with sorbitol and the other without sorbitol. Stability evaluation of these suspensions stored at 5°C, 25°C / 60% RH, and 40°C / 75% RH showed that the suspensions were reasonably stable under all conditions.

[0174] A portion of the vials from the porcine study batches were autoclaved at 121° C. for 20 minutes using a liquid cycle and the formulations designated batch 515-76 and FID5910.

[0175] Stability data showed that autoclaving did not significantly affect degradation, but appeared to increase particle size.

[0176] Example 13 Engineering Testing of Nanosuspensions Although several engineering batches of TegaVivint nanosuspension have been prepared in anticipation of clinical production, certain modifications to the process were necessary to maintain compliance and minimize loss and contamination: Partly for safety reasons and partly to incorporate a larger surface area filter for extraction purposes, the glass pressure funnel used for extraction was replaced with a stainless steel in-line filter housing (Pall) fitted with a 55 μm stainless steel filter element.

[0177] Pressurization of the extraction equipment, previously using nitrogen, was accomplished using a peristaltic pump as this pump was incorporated into the process as a means to sterile filter both the diluent and dispersion rather than autoclaving. A metered peristaltic pump unit was used to fill the vials. Given the tendency of autoclaving to increase particle size of the drug product, the first engineering batch (RD4050-5) was submitted for gamma irradiation.

[0178] Stability results showed minimal degradation and stability similar to previous batches. [Table 3]

[0179] Based on preliminary particle testing, the formulation contained some larger particles when processed, which did not appear to be packed tightly enough to significantly affect the laser diffraction particle size measurements, but were in accordance with the USP <788> The results were significant enough to affect the test. To mitigate particulates, "polished" filters were proposed, with porosity small enough to retain larger particles, but not so small as to affect the Tega Vivint assay. However, previous attempts to filter the suspension had resulted in significant assay loss. Therefore, a contract was signed with Pall Corporation to evaluate several of their membranes for suitability for filtering the Tega Vivint nanosuspension.

[0180] Some of the least clean suspensions were filtered using a variety of 47mm membranes available from Pall, with a pressure feedback pump system used to determine how much material the filter could process before clogging and failure. The following membrane types were used and the filtrates were tested for PSD and assays: [Table 4]

[0181] The 6 micron HDCII membrane was selected as the best candidate as it showed no discernible effect on either the assay values ​​or particle size distribution of the nanosuspension. However, the relatively long lead time of the Pall filters was a limiting factor in the timely production of clinical material. Therefore, an alternative was sought and Sartorius 8 micron polypropylene filters were used in the manufacture of two engineering batches to be used in determining bioburden for sterility validation purposes.

[0182] Unfortunately, the assay values ​​of two batches were adversely affected by filtration (80.9% LC and 91.9% LC, respectively). Because they did not represent the final clinical batch profile, the batches were discarded and another engineering batch was processed using Pall HDC membrane filters. This batch was within the 90-110% LC, and Pall HDC filters were incorporated into the final step of the manufacturing process prior to filling vials.

[0183] Example 14 Pharmacokinetic study of tegavivin following slow intravenous bolus administration to female Sprague-Dawley rats The objective of this study was to determine the pharmacokinetics of tegavivint following a single slow bolus intravenous dose of tegavivint in female Sprague-Dawley rats.

[0184] The study was conducted using a parallel group design (n=4 / group) with sequential sampling, as summarized in Table 5. [Table 5] Source: Sprague-Dawley rats used in this study were obtained from the in-house animal resource facility of Advinus Therapeutics Ltd. (Bengaluru, India). Animals were approximately 10-11 weeks old on the day of dosing.

[0185] Identification: Each animal was identified by a unique identification number on a cage card and by turmeric solution markings on the animal's body. The cage card identified each cage by study number, identification number, species and strain, dose, and sex.

[0186] Housing and environment: Rats were acclimated to the conditions of the test area for 3 days prior to dosing. Animals were housed in polypropylene cages (one per cage) and maintained in controlled environmental conditions with a 12-h light and 12-h dark cycle. Room temperature and humidity were maintained at 22 ± 3°C and 40-70%, respectively. The room was subjected to 10-15 fresh air ventilation cycles per hour.

[0187] Food and water: Experimental animals were fed ad libitum with a standard pelleted diet (Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet: pelleted rodent food manufactured by Harlan Laboratories BV, Maasheseweg 87c PO Box 553, 5800, AN Venray, The Netherlands).

[0188] Dose preparation and administration: A stock formulation (25 mg / mL) was provided. Precisely, 600 μL of the dosing formulation (stock, 25 mg / mL) was transferred to a labeled glass container. To this, 2.4 mL of 5% dextrose solution was added, vortex mixed, and sonicated to obtain a homogenous suspension at a strength of 5 mg / mL. Animals were dosed under fed conditions. Rats were administered a single dose of 10 mg / kg tegavivint via a slow intravenous bolus (over 1.5 min) jugular vein catheter using a 1 mL BD syringe guided by a 23 G blunt needle, with a dosing volume of 2 mL / kg. The syringe used for dosing was weighed before and after dosing to calculate the actual dose administered.

[0189] Sample collection and processing: Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours after dosing. At each time point, approximately 0.25 mL of blood was collected from the cannulated jugular vein of rats and transferred to a labeled microtube containing 200 mM K2EDTA (20 μL per mL of blood). After sampling, an equal volume of heparinized saline was substituted into the catheter. Blood samples were kept on wet ice at all times immediately after collection, and plasma was separated by centrifugation at 5000 g for 5 min at 4 ± 2 °C. Plasma samples were separated within 1 hour of scheduled time and stored below -60 °C until bioanalysis.

[0190] Bioanalysis: Bioanalysis was performed using an adapted LC-MS / MS method for the quantification of BC2059 in rat plasma samples. The calibration curve (CC) of the method consisted of at least six non-zero constituent standards, as well as a blank and a blank containing an internal standard sample with a lower limit of quantification (LLOQ) of 0.050 μg / mL. Test samples were analyzed along with three sets of quality control samples (nine QC samples: low, medium, and high QC samples in triplicate).

[0191] Pharmacokinetic Data Analysis: Pharmacokinetic parameters of tegavivint were calculated using the non-compartmental analysis tool (extravascular) of the validated Phoenix® WinNonlin® software (version 6.3). Areas under the concentration-time curves (AUCIast and AUCinf) were calculated by the linear trapezoidal method. C0 (back-extrapolated concentration at time zero) was estimated after administration of an intravenous bolus dose by back-extrapolating the first two concentration values. Total plasma clearance (CL) and volume of distribution at steady state (Vss) were estimates. Elimination rate constant values ​​(k) were calculated by linear regression of the log-linear terminal phase of the concentration-time profile using at least three decreasing concentrations in the terminal phase with a correlation coefficient >0.8. Terminal half-life values ​​(T1 / 2) were calculated using the formula 0.693 / k. Alpha and beta half-lives were calculated and reported.

[0192] Experimental Results After a single slow intravenous bolus administration of tegavivint (dose: 10 mg / kg) to rats, the mean plasma clearance (CL) was estimated to be 9.92 mL / min / kg, approximately 5.5-fold lower than the normal rat hepatic blood flow of 55 mL / min / kg. The mean plasma distribution volume (Vss) at steady state was found to be nearly 9.34-fold higher than the normal body water of 0.7 L / kg, which may suggest a wide distribution to tissue compartments. Semi-logarithmic plasma concentration-time plots show that BC2059 exhibited a biexponential elimination pattern with a rapid distribution half-life (T1 / 2α) of 0.546 hours and a long terminal plasma half-life (T1 / 2β) of 13.8 hours. [Table 6]

[0193] To calculate the elimination rate constants, turning points of 0.5, 1, and 2 h were selected for the α phase, and 6, 8, 12, and 24 h for the terminal β phase.

[0194] Example 15 An ascending dose intravenous infusion study of tegavivint in male beagle dogs Test animals Four male non-naïve beagle dogs were released for the study from Xenometrics on April 20, 2017. Animals were fed Harlan Teklad® Global 25% Protein Certified Dog Diet 2025C ad libitum throughout the study (except for brief periods between in-life procedures). dosage: Animals were dosed by intravenous (IV) infusion (via the jugular vein) for 4 hours (h) [± 5 minutes (min)]. [Table 7]

[0195] Pharmacokinetic (PK) Blood Sampling: Blood samples were collected on the last dosing day (dose 6; 15 mg / kg) before the start of the infusion, and at 4, 12, 24, 36, 48, and 72 hours after the start of the infusion. All blood samples were collected within 10 minutes of the target time and processed per protocol. Bioanalysis results showed that tegavivint was present in all plasma samples. [Table 8]

[0196] Pharmacokinetic parameters were determined for BC-2059 following a final 15 mg / kg infusion of drug over 4 hours. Mean values ​​are presented above in Table 8. The data show a half-life of 53.0 hours and an overall AUC0-120h of 73480 ng×hour / mL. [Table 9]

[0197] Example 16 Nebulized delivery of tegavivint formulations The purpose of this experiment was to test the nebulized delivery of a nanosuspension, and the experiment showed that nebulized delivery was successful.

[0198] Tegavivint particles suspended in poloxamer 188 / sorbitol at a concentration of 25 mg / mL were used.

[0199] These formulations were applied to mice in the form of aerosols by whole body exposure. Mice were placed in a plastic box. The box was sealed, one side of which was connected to the outlet of a nebulizer device, and the other side was connected to a closed water system. The entire procedure was carried out in the fume hood of the animal room.

[0200] For the first experiment, a nebulizer kit from SATER LABS was used. This device uses an injection system. For each group of 5 mice, 5 ml of the drug, i.e. 125 mg of Tega Vivint (BC2059), was loaded into the device, and then the device was connected to a power source for nebulization. The energy was provided by a DeVilbiss compressor model 646, which allows a pressure of 5 to 7 pounds, and a flow rate of 6 to 8 liters per minute. For the second experiment, the device used was an ultrasonic nebulizer Altera.

[0201] For the two experiments, 10 male bcat-Ex3 mice were used for each set. These mice were divided into two groups of 5 mice each. The first group was administered the drug every day for 5 consecutive days. The second group was administered the drug only once (on the 5th day). On the 5th day, all mice were sacrificed, the lungs were harvested, and the samples were stored at -30 degrees in two labeled nylon bags, each containing 5 samples from each group. [Table 10]

[0202] Example 17 Studies in pigs using a liquid formulation of tegavivint and a nanosuspension of tegavivint liquid suspension have shown It was poorly tolerated by pigs Tegavivint was administered intravenously to minipigs in a series of pharmacokinetic studies to determine a formulation suitable for GLP toxicology studies in terms of both systemic exposure and formulation tolerability, all of which were conducted at Sinclair Research (Auxvasse, MO).

[0203] In the first study, the drug was obtained in a formulation consisting of Tween 80, ethanol, polyethylene glycol (PEG), and Vitamin E TGPS (d-alpha tocopheryl polyethylene glycol 1000 succinate). This stock formulation was diluted in 20% Intralipid® (phospholipid stabilized soybean oil) to the final dosing concentration. Two pigs were dosed at 1.7 mg / kg over 6 hours and two pigs were dosed at 2.2 mg / kg over 24 hours. The formulation provided good systemic exposure.

[0204] Comparing dose-normalized values ​​for the shorter 6-hour duration versus the 24-hour duration, there was a higher peak concentration (C max However, the overall systemic exposure over time (AUC / dose) was similar between the two infusion times, i.e., it was possible to obtain similar overall systemic exposure while avoiding higher peak plasma concentrations with the longer infusion time.

[0205] However, while good systemic exposure was observed with this formulation, significant infusion reactions were observed and the formulation was not tolerated by minipigs over either the 6- or 24-h infusion period. It was hypothesized that the Tween / ethanol / PEG / vitamin E / lipid-based excipients and likely drug precipitation, rather than tegavivint itself, were responsible for the infusion reactions. Indeed, tegavivint does not cause hemolysis of red blood cells, either in nanoparticle form or when dissolved in DMSO.

[0206] A freeze-dried nanosuspension of Tegavivint was better tolerated but was ultimately discarded due to stability issues.

[0207] In a subsequent study, tegavivint was milled to nanoparticle size and a non-solvent formulation was used. In this study, Study B01-109, a lyophilized form of tegavivint was obtained and reconstituted in water to obtain a stock formulation consisting of a suspension of 10 mg / mL tegavivint, 2.5 mg / mL poloxamer 188, and 5 mg / mL sorbitol. This stock solution was diluted with saline to obtain the final concentration required for intravenous administration. Two pigs were infused with 2.9 mg / kg and two pigs were infused with 12.3 mg / kg over a 4-hour infusion period. One pig in the 12.1 mg / kg dose group showed very high systemic exposure. Even in this pig, the dose-normalized AUC and, to a lesser extent, C max was linear across doses of 2.8 to 12.1 mg / kg given over the same time period. With the exception of one pig in the 12.1 mg / kg dose group, dose-normalized exposure was lower with this lyophilized form of nanomilled tegavivint compared to the Tween / ethanol / PEG / vitamin E / lipid solvent-based formulation used in Study B01-107.

[0208] Nonetheless, the formulation was well tolerated by minipigs, with no infusion reactions observed in study B01-107, and the lyophilization process was scaled up for future experiments. However, the scale-up process did not yield a lyophilized product that exhibited sufficient stability, necessitating the need for an alternative lyophilized formulation of tegavivint.

[0209] The TXPK-006-2059-24h study used a lyophilized formulation of milled tegavivint. The lyophilized formulation used was reconstituted in water to a final concentration of 25 mg / mL BC2059, 0.125% polyvinylpyrrolidone (PVP), 0.0625% sodium deoxycholate (NaDOC), and 10% sucrose. This bulk solution was diluted in saline to the required concentration to dose two pigs per treatment group at 12.3 or 49.2 mg / kg for a 24-h infusion. Syringes were agitated throughout the 24-h infusion period because clumping of the test article was observed in the syringes. Nevertheless, systemic exposure was very low compared to studies B01-107 and B01-109. Subsequent formulation experiments showed that saline with this lyophilized formulation caused aggregation of the test article, making an ionic (saline) diluent unusable.

[0210] Frozen liquid nanosuspension of Tegavivin showed efficacy At this point, lyophilization was stopped and a frozen liquid formulation of milled tegavivint was examined. In study TXPK-001-2059-pig 24h PK, three minipigs were assigned to one of three groups, one per group, and the formulation was administered over 24 hours. Two cryo-milled suspensions were provided by Particle Sciences. BC2059-1 was 25mg / mL BC2059, 0.125% PVP, 0.0625% NaDOC, 10% sucrose, suspension (batch no. 515-10), and BC2059-2 was 25mg / mL BC2059, 0.625% poloxamer 188, 10% sorbitol, suspension (batch no. 515-13). For both of these formulations, the diluent was D5W. The third group was BC2059-1, a poloxamer 188 / saline diluent PVP / NaDOC / sucrose frozen formulation, to investigate the possible role of poloxamer 188 in systemic exposure.

[0211] Of these three frozen test article formulations, the nanosuspension diluted in 25 mg / mL tegavivint, 0.625% poloxamer 188, 10% sorbitol, and D5W demonstrated the highest systemic exposure. The dose-normalized AUC for this formulation was somewhat less than the dose-normalized exposure observed with the 24-hour infusion in Study B01-107, but not significantly less. The dose-normalized exposure was significantly higher than that observed in three of four pigs in Study B01-109, suggesting that the saline diluent in this study may have influenced systemic exposure with the lyophilized poloxamer 188 formulation of BC2059, albeit to a much lesser extent than observed with the PVP / NaDOC formulation of tegavivint. [Table 11]

[0212] a BC2059 at 20 mg / mL in 30% ethanol, 50% PG, 10% Tween 80, and 10% D-alpha tocopherol polyethylene glycol 1000 succinate (Lot P492-01): Values ​​reported for 2 minipigs b 10 mg / mL BC2059, 2.5 mg / mL poloxamer 188 and 5 mg / mL sorbitol (Lot No. BET 1213-001-29): Values ​​reported for 2 minipigs c 25 mg / mL BC2059, 0.125% PVP, 0.0625% NaDOC and 10% sucrose (Lot No. BET 1213-001-49): Values ​​reported for 2 minipigs d 25 mg / mL BC2059, nanosuspension diluted to a final concentration of 2 mg / mL in 0.125% PVP, 0.0625% NaDOC, 10% sucrose, 5% dextrose (Lot 515-10): 1 minipig e25 mg / mL BC2059, 0.625% poloxamer 188, 10% sorbitol, 5% dextrose diluted nanosuspension to a final concentration of 2 mg / mL (Lot 515-13): 1 minipig f Nanosuspension (Lot 515-10): 25 mg / mL BC2059, 0.125% PVP, 0.0625% NaDOC, 10% sucrose, diluted with poloxamer 188 / saline to a final concentration of 2 mg / mL for a final concentration of 0.05% poloxamer.

[0213] Based on the results of these single-dose infusion studies in minipigs, the formulation selected for the repeated two-dose toxicity studies was a frozen formulation of 25 mg / mL BC2059 containing 0.625% poloxamer 188, 10% sorbitol (nanosuspension) and diluted in D5W.

[0214] During the conduct of a two-dose non-GLP study to support dose selection for an IND-enabling GLP toxicology study, it was found that in the scale-up process and production of Particle Sciences batch number 515-33, freezing the formulation caused aggregation of tegavivint in the vial.

[0215] Given this aggregation due to freezing, it was subsequently decided to pursue 25 mg / mL nanomilled BC2059 in a 0.625% poloxamer 188, 10% sorbitol formulation held at 2-4 °C. No aggregation was observed in multiple lots of milled BC2059 suspension unless the formulation was frozen. The poloxamer / sorbitol formulation refrigerated at 2-4 °C was used for the IND-enabling GLP toxicology study and the non-GLP beagle dog study.

[0216] Example 18 Efficacy of nebulized tegavivint in a mouse model of idiopathic pulmonary fibrosis The purpose of this study was to examine tegavivint nanosuspension in a mouse model of bleomycin-induced idiopathic pulmonary fibrosis (IPF).

[0217] Tegavivint (BC2059) in a 25 mg / mL nanomilled suspension in 0.625% poloxamer 188 and 10% sorbitol. The test articles were refrigerated at approximately 4°C.

[0218] The nebulization device was an Altera ultrasonic nebulizer eRapid machine (model number 678G1002).

[0219] Animals were 8-12 week old C57BL / 6 male mice (Jackson Lab, Bar Harbor, ME).

[0220] Experimental procedure [Table 12]

[0221] A mouse model of pulmonary fibrosis was induced by intratracheal (IT) injection of bleomycin (APP Pharmaceuticals, Schaumburg, Ill.). One dose of 0.025 U of bleomycin in 50 microliters of saline 0.9% or PBS was administered to each animal on day 0 as a control.

[0222] Tegavivint nanosuspension was applied to group 3 by whole body exposure method in the form of aerosol. Mice were placed in a plastic box. The box was sealed and one of its sides was connected to the outlet of a nebulizer device and the other side to a closed water system. The whole procedure was carried out in the fume hood of the animal room. In each treatment session, each group of 4-5 mice in the chamber was nebulized with 5 ml of 25 mg / ml tegavivint (125 mg) over 15 min. To increase the exposure of the mice to the aerosol, the tegavivint that had settled in the aerosol chamber was collected with a syringe and re-nebulized a second and third time. Mice were nebulized twice a day between days 5 and 21 after administration of bleomycin. Groups 1 and 2 were nebulized with 5 ml of vehicle in a similar manner.

[0223] Animal weights were recorded on days 0, 5, 8, 12, 16, 19, and 21.

[0224] Pulmonary function measurements were performed on day 21 as previously described (Morales-Nebreda L et al., AJRCMB 2015) using a FlexiVent mouse ventilator (Scireq, Montreal, PQ, Canada) following protocols established by Scireq. Standard ventilation histories were obtained for each mouse with a total of three lung volume manipulations prior to forced oscillatory and quasi-static pressure-volume curve protocols that were used to calculate airway resistance, dynamic and quasi-static tissue compliance, and elastance.

[0225] On day 21, all animals were sacrificed and lungs were harvested. Total lung collagen content was assessed using a hydroxyproline assay as previously described (Morales-Nebreda L et al., AJRCMB 2015). Briefly, mouse lungs were harvested, suspended in 1 ml of 0.5 M acetic acid, and then homogenized using 15 strokes, first in a tissue homogenizer (60 s on ice) and then in a Dounce homogenizer (on ice). The resulting homogenate was spun (12,000 × g) for 10 min, and the supernatant was used for subsequent analysis. Collagen standards were prepared in 0.5 M acetic acid using rat tail collagen (Sigma-Aldrich, St. Louis, MO). Picrosirius red dye was prepared by mixing 0.2 g of Sirius Red F3B (Sigma-Aldrich) with 200 ml of water. One ml of picrosirius red dye was added to 100 μl of collagen standard or lung homogenate and then mixed continuously at room temperature for 30 min on an orbital shaker. The precipitated collagen was then pelleted and washed once with 0.5 M acetic acid (12,000 × g for 15 min each). The resulting pellet was resuspended in 1 ml of 0.5 M NaOH and Sirius red staining was quantified spectrophotometrically (540 nm) using a colorimetric plate reader (Bio-Rad, Hercules, CA).

[0226] result Group 2 showed statistically significant weight loss after bleomycin treatment, one indicator of IPF induction. In contrast, inhaled tegavivint treatment in group 3 reversed the weight loss caused by bleomycin-induced lung injury. [Table 13]

[0227] Furthermore, bleomycin induced a decrease in lung compliance in Group 2, indicative of induction of fibrosis. Inhaled tegavivint treatment after bleomycin injury in Group 3 reversed compliance values ​​to near those of sham-treated controls in Group 1. [Table 14]

[0228] Furthermore, total lung collagen content, measured by hydroxyproline assay, showed a marked increase in group 2, suggesting active fibrosis after bleomycin injury. In contrast, inhaled tegavivint treatment after bleomycin injury in group 3 reversed this change, with collagen levels approaching those of sham-treated controls in group 1. [Table 15]

[0229] Thus, this study demonstrated that tegavivint has great potential for treating IPF.

[0230] Example 19 Evaluation of an aerosolized tegavivin formulation To determine the most efficient aerosol generation method, a series of BC-2059 (Tegavivint) formulations were aerosolized using vibrating mesh and compressed air nebulizers. Aerosols were characterized for aerosol concentration and particle size distribution in a rodent nose exposure chamber. Each formulation had different variables adjusted to assess their impact on aerosol performance. These included API particle size reduction, excipient profile, and nebulizer used.

[0231] The objective of this study was to determine how the test article BC-2059 could be aerosolized for inhalation studies in rodents.

[0232] The test article, BC-2059, was suspended in 0.1% Tween 80 in purified water at a concentration of 15 mg / mL. The suspension was sonicated using a Covaris S220x Ultrasonicator (Covaris, Boston MA) and then mixed by vortexing for 1 min. The sonication and vortex mixing were repeated a total of 15 times.

[0233] The remaining bulk BC-2059 powder was milled in a planetary ball mill (Retsch, Germany) using a 12 mL ball mill jar and three metal balls at 150 RPM for 10 minutes. The milled BC-2059 powder was suspended in 0.1% Tween 80 in purified water at a concentration of 15 mg / mL. The suspension was sonicated using the procedure outlined above.

[0234] The remaining bulk BC-2059 powder was milled in a planetary ball mill (Retsch, Germany) using a 12 mL ball mill jar and three metal balls at 300 RPM for 60 minutes. The milled BC-2059 powder was suspended in 0.1% Tween 80 in purified water and 10% PEG400 in purified water at a concentration of 15 mg / mL. The suspension was sonicated using the procedure outlined above. An additional 15 mg / mL BC-2059 suspension was prepared using 10% ethanol in purified water. The suspension was sonicated for 10 minutes using a VWR ultrasonicator (VWR, Radnor PA) and mixed for 4 minutes using a vortex mixer.

[0235] An additional formulation (a nanomilled suspension of 25 mg / mL BC-2059 in 0.625% poloxamer 188 and 10% sorbitol) was used as received without further modification.

[0236] Aerosols were generated from a range of surfactant formulations in four separate nebulizers (Aeroneb Solo (Aerogen, Ireland), Pari LC Plus (Pari Respiratory Equipment Inc. Midlothian VA), Hospitak Up Mist, Hospitak Inc. Farmdale, NY), and Hudson Micro-Mist (Teleflex Inc. Research Triangle Park, NC) and transferred to a two-layer flow-past rodent exposure system.

[0237] The total aerosol concentration in the exposure atmosphere was determined by analysis of filter samples (GF / A 47 mm filters). Filter samples were collected at a nominal flow rate of 0.3 L / min. Filter samples collected throughout the study were gravimetrically analyzed and submitted for HPLC analysis to determine the total aerosol concentration.

[0238] Filters containing the test substances were extracted in 1:1 acetonitrile:methanol and analyzed by HPLC-UV assay.

[0239] Particle size distribution (PSD) of the test articles was measured in the respirable zone using a Mercer style cascade impactor In Tox.

[0240] result The aerosol concentrations (weight and chemical concentrations) are shown in Table 16 below. [Table 16]

[0241] Particle sizes for the test atmospheres were measured using an In-Tox cascade impactor for suspensions prepared in 0.1% Tween 80 in ultrapure water, and the sponsor provided the poloxamer suspension using a compressed air nebulizer. The mass median aerodynamic diameter and geometric standard deviation for each formulation are shown below in Table 17. Particle size distributions are shown in Figures 1 and 2. [Table 17]

[0242] conclusion Formulations of BC-2059 were nebulized and introduced into a nasal inhalation exposure chamber. The exposure atmosphere was characterized for aerosol concentration using gravimetric and HPLC assays. The highest gravimetric aerosol concentration was measured at 2.47 mg / L for the poloxamer formulation, which corresponds to 0.48 mg / L of active test article. The particle size distribution of this formulation was measured by cascade impactor with an MMAD of 2.46 μm and a geometric standard deviation of 1.45 μm.

[0243] Considering the results of the poloxamer formulation against previous results, an aerosol concentration of 0.484 mg / L of BC2059 would result in a lung-deposited dose of 1.5 mg / kg (10% DF) in a 30 gram mouse in 30 minutes. Based on a standard mouse lung weight, this would translate to approximately 0.2 mg / g of lung tissue. Previous studies yielded an assay concentration of approximately 0.02 mg / g.

[0244] Thus, this nebulized BC2059 nanomilled suspension demonstrated optimal concentration in the aerosol compared to other BC2059 formulations.

Claims

1. a) a compound of formula I [Formula 1] (In the formula, R Ａ is hydrogen, R ７ and R ８ are independently H and SO ２ N.R. ３ R ４ is selected from R ７ and R ８ one of the groups is hydrogen, and NR １ R ２ and N.R. ３ R ４ is independently a 6-15 membered heterocycloalkyl containing one nitrogen in the ring), or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof; b) a surfactant, A composition, wherein the particles have an effective D50 of 500 nm or less and a D90 of 1.0 micrometers (μm) or less, as measured using laser diffraction.

2. 2. The composition of claim 1, wherein the compound of formula I is tegavivint or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof.

3. The composition of claim 1 , wherein the composition is a nanoparticle composition.

4. The composition of claim 1 , wherein the surfactant is a poloxamer surfactant.

5. 2. The composition of claim 1, wherein the poloxamer surfactant is poloxamer 188.

6. The composition of claim 1 , wherein the composition further comprises a stabilizer.

7. 7. The composition of claim 6, wherein the stabilizer is selected from the group consisting of sugars, polyols, polysorbate surfactants and polyvinylpyrrolidone (PVP).

8. 8. The composition of claim 7, wherein the sugar is selected from the group consisting of sucrose and trehalose.

9. The composition of claim 7, wherein the polyol comprises sorbitol and mannitol.

10. The composition of claim 1, wherein the concentration of the compound is from about 10 mg / ml to about 25 mg / ml.

11. The composition of claim 1 , wherein the concentration of the compound is about 25 mg / ml.

12. a. 10-25 mg / ml of tegavivint or a pharma- ceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof; b. Poloxamer 188, C. sorbitol, The composition, wherein the tegavivint or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof is in the form of a nanosuspension comprising particles of the tegavivint or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof, the particles having an effective D50 of 500 nm or less and a D90 of 1.0 micrometer (μm) or less as measured using laser diffraction.

13. the amount of tegavivint or a pharma- ceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof is 25 mg / ml; The amount of poloxamer 188 was 0.625%; 13. The composition of claim 12, wherein the amount of sorbitol is 10%, the percentages being by weight of the composition.

14. The composition of claim 1 , wherein the composition is prepared by grinding.

15. The composition of claim 1 , wherein the composition is prepared by lyocell technology.

16. A process for preparing a composition comprising: a) a compound of formula I [chemical 2] (In the formula, R Ａ is hydrogen, R ７ and R ８ are independently H and SO ２ N.R. ３ R ４ is selected from R ７ and R ８ one of the groups is hydrogen, and NR １ R ２ and N.R. ３ R ４ is independently a 6-15 membered heterocycloalkyl containing one nitrogen in the ring), or a pharma- ceutically acceptable salt, ester, amide, stereoisomer or geometric isomer thereof, mixing with a surfactant and an acceptable carrier to form a suspension; b) milling the suspension of step (a) by roller milling or using a high energy mill; c) adding a polyol to the particles of step (b).

17. 17. The composition of claim 16, wherein the composition exhibits long-term stability.

18. 17. The composition of claim 16, wherein the compound of formula I is tegavivint, or a pharma- ceutically acceptable salt, ester, amide, stereoisomer, or geometric isomer thereof.

19. The composition comprises: (a) a dosage form selected from the group consisting of tablets and capsules; (b) a dosage form selected from the group consisting of a controlled release formulation, a fast dissolving formulation, a delayed release formulation, a sustained release formulation, a pulsatile release formulation, and a mixed immediate release and controlled release formulation; (c) a dosage form suitable for inhaled or parenteral administration, including intramuscular, subcutaneous, intravenous and intradermal injection, or (d) The composition of claim 1, formulated in any combination of (a), (b), and (c).

20. 13. A method for preventing, treating or ameliorating cancer or tumor metastasis in a mammal, comprising administering to said mammal in need thereof an effective amount of the composition of claim 1.

21. 1. A method for treating cancer, comprising: 1) a pharma- ceutical effective amount of the nanoparticle composition of claim 1; and 2) a pharma- tically effective amount of at least one additional anti-cancer agent, in combination with said compound, to a subject in need thereof.

22. 22. The method of claim 21, wherein the additional anti-cancer agent is selected from the group consisting of anti-mitotic agents, antimetabolites, HDAC inhibitors, proteosome inhibitors, immunotherapeutic agents, FLT-3 EGFR, MEK, PI3K and other protein kinase inhibitors, LSD1 inhibitors, and WNT pathway inhibitors, alkylating agents and DNA repair pathway inhibitors, anti-hormones, anti-cancer antibodies, and other cytotoxic chemotherapeutic agents.

23. 13. A method for treating and / or preventing a fibrotic disease in a mammal, comprising administering to said mammal in need thereof an effective amount of the composition of claim 1.

24. 24. The method of claim 23, wherein the fibrotic disease is selected from the group consisting of pulmonary fibrosis, Dupuytren's contracture, scleroderma, systemic sclerosis, scleroderma-like disorders, scleroderma without skin sclerosis, liver cirrhosis, interstitial pulmonary fibrosis, keloids, chronic kidney disease, chronic transplant rejection and other scar / wound healing disorders, post-surgical adhesions, reactive fibrosis.

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