Pharmaceutical preparations containing 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid

Abstract

Provided herein are pharmaceutical formulations of the KDM4 inhibitor 3-({[(4r)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2h-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 293,488, filed December 23, 2021, which is incorporated by reference in its entirety. [Background technology]

[0002] There is a need in the art for effective treatment of cancer and neoplastic disease.Provided herein is an improved pharmaceutical formulation of KDM4 inhibitor 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid. Summary of the Invention

[0003] One embodiment provides a pharmaceutical composition comprising the lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid is milled.

[0004] One embodiment provides a pharmaceutical composition comprising the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid is freeze-dried, or spray-dried, or a combination thereof. [Brief description of the drawings]

[0005] The features of the present invention are set forth with particularity in the appended claims and will be better understood by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0006] [Figure 1] FIG. 1 shows in vitro dissolution testing of nanomilled material before and after supplementation with PEG400. [Diagram 2] FIG. 13: In vitro dissolution study of nanomilled material after supplementation with PEG400. [Diagram 3] FIG. 1 shows an in vitro dissolution test of lyophilized formulation F13A. [Figure 4] FIG. 1 shows in vitro dissolution testing of hot melt formulations. [Diagram 5] FIG. 13 shows in vitro dissolution testing of additional formulations. [Figure 6] FIG. 13 shows in vitro dissolution testing of additional formulations. [Figure 7] FIG. 13 shows in vitro dissolution testing of additional formulations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] definition 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. All patents and publications mentioned herein are incorporated by reference.

[0008] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0009] The term "about" when referring to a numerical value or numerical range means that the numerical value or numerical range referred to is an approximation within experimental variation (or statistical experimental error), and thus the numerical value or numerical range may vary, in some cases, from 1% to 15% of the stated numerical value or numerical range.

[0010] Histone lysine demethylase Cancer is the second leading cause of death in the United States. It presents complex challenges for the development of new therapeutic approaches. Cancer is characterized by the abnormal proliferation of malignant cells that have undergone a series of genetic alterations that result in the growth of tumor masses and metastatic properties. Not only genetic alterations but also aberrant epigenetic regulation add to the complexity of cancer.

[0011] Epigenetic regulation causes the transition between transcriptionally inactive heterochromatin and transcriptionally active euchromatin through the action of enzymes that add or remove chemical marks from histones, such as histone acetyltransferases, deacetylases, methyltransferases, and histone demethylases, followed by chromatin remodeling (Dimitrova et al., 2015; Rotili & Mai, 2011). Epigenetic modifications control essential physiological functions, including nuclear functions such as programming development, activating or repressing transcription, timing and control of the cell cycle, and initiating DNA replication and repair. Genetic alterations and overexpression of epigenetic enzymes are common in cancer, suggesting that epigenetic dysregulation may be essential for cancer. Overexpression of histone demethylases has been associated with many cancer types (Black et al., 2010; Dimitrova et al., 2015; Gregory & Cheung, 2014; Rotili & Mai, 2011) and has been investigated as potential therapeutic targets.

[0012] The histone lysine demethylase known as KDM4 is an epigenetic regulator and key oncogenic promoter across multiple tumor types. KDM4 specifically removes methyl groups from di- or tri-methyl histone H3 lysine 9 (H3K9me2 / 3) (Cloos, 2006; Klose, 2006; Fodor, 2006), di- or tri-methyl histone H3 lysine 36 (H3K36me2 / 3) (Klose, 2006; Young, 2013; Cascante, 2014), and linker di- or tri-methyl histone H1.4 lysine 26 (H1.4K26me2 / 3) (Trojer, 2009). Overexpression of KDM4 has been reported in many tumor types, including breast, colon, brain, kidney, pancreatic, gastric, lung, testicular, prostate, and bladder cancers, melanoma, squamous cell carcinoma, and lymphoma, as it can lead to downregulation of its substrates, e.g., H3K9me3, and aberrant gene activation causing dysregulation of numerous pathways that may lead to malignant transformation (Berry et al., 2012; Cloos et al., 2006; Ding et al., 2013; Kogure et al., 2013; Liu et al., 2009; Shi et al., 2011; Shin & Janknecht, 2007; Wissmann et al., 2007; Yamamoto et al., 2013; Yang et al., 2000; Young & Hendzel, 2013).

[0013] Six distinct isoforms of KDM4 (AF) have been identified, with KDM4A-C being structurally similar. Several studies have documented the critical role of KDM4 isoforms in cancer progression, suggesting that KDM4 isoform-selective inhibitors may not be effective because other isoforms may compensate for the loss of function. To overcome these challenges, novel potent inhibitors that simultaneously target multiple isoforms of KDM4 are needed.

[0014] KDM4 inhibitors The heterocyclic KDM4 inhibitor described herein as Compound 1 refers to 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid. Compound 1 has the structure shown below and is also known as QC8222 or TACH101.

[0015] [ka]

[0016] Compound 1 has been disclosed in PCT patent publication WO2015 / 200709 and related patent applications, and in granted patents such as US9,242,968, which are incorporated by reference in their entirety. Compound 1 is a pan-inhibitor of KDM4 that simultaneously targets multiple isoforms of KDM4. Throughout this disclosure, when referring to heterocyclic KDM4 inhibitors, or their pharma-ceutically acceptable salts or solvates, the reference refers to compound 1.

[0017] There is a need in the art to provide stable, bioavailable formulations of Compound 1, which allow for administration by the oral route and are stable over extended periods of storage.

[0018] The lysine salt of compound 1 was selected for further study. However, it was found that the lysine salt of compound 1 has low permeability and low solubility (BCS class IV). BCS class IV compounds generally show the lowest oral bioavailability, the lowest solubility, and the lowest intestinal permeability of all drug classes. This class of drugs requires a more compatible and efficient delivery system. Therefore, it is generally recognized that BCS class IV compounds are difficult to develop formulations that are non-toxic and show good bioavailability.

[0019] Pharmaceutical Compositions One embodiment provides a pharmaceutical composition comprising the lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid is milled. Another embodiment provides a pharmaceutical composition, wherein the milling is performed using a ball mill. Another embodiment provides a pharmaceutical composition, wherein the milling is performed using a roller mill or a high-energy mill.

[0020] One embodiment provides a pharmaceutical composition in which the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid exhibits a particle size of less than 1000 nanometers. Another embodiment provides a pharmaceutical composition in which the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid exhibits a particle size of about 50 nanometers to about 1000 nanometers. Another embodiment provides a pharmaceutical composition, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid exhibits a particle size of about 50 nanometers to about 100 nanometers, about 100 nanometers to about 200 nanometers, about 200 nanometers to about 300 nanometers, about 300 nanometers to about 400 nanometers, about 400 nanometers to about 500 nanometers, about 500 nanometers to about 600 nanometers, about 700 nanometers to about 800 nanometers, about 800 nanometers to about 900 nanometers, or about 900 nanometers to about 1000 nanometers. Another embodiment provides a pharmaceutical composition, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid exhibits a particle size of about 150 nanometers to about 300 nanometers.

[0021] Another embodiment provides a pharmaceutical composition that does not increase in particle size upon storage. Another embodiment provides a pharmaceutical composition that does not increase in particle size by more than 5% upon storage. Another embodiment provides a pharmaceutical composition that does not increase in particle size by more than 10% upon storage. Another embodiment provides a pharmaceutical composition that does not increase in particle size by more than 15% upon storage.

[0022] Another embodiment provides a pharmaceutical composition, wherein at least one pharma- ceutically acceptable excipient is a solubilizer. Another embodiment provides a pharmaceutical composition, wherein the solubilizer is polyethylene glycol (PEG). Another embodiment provides a pharmaceutical composition, wherein the PEG is selected from PEG200, PEG300, PEG400, PEG500, or PEG600. Another embodiment provides a pharmaceutical composition, further comprising a stabilizer. Another embodiment provides a pharmaceutical composition, wherein the stabilizer is selected from the group consisting of hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), polyvinylpyrrolidone (PVP), or poloxamer.

[0023] One embodiment provides a pharmaceutical composition comprising the lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, the composition comprising: (a) about 0.1% (w / w) to about 0.2% (w / w) of the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid; (b) about 0.91% (w / w) to about 1.5% (w / w) hydroxypropylcellulose; (c) about 5.0% (w / w) to about 10.0% (w / w) PEG 400, and (d) about 89.3% (w / w) to about 94.4% (w / w) water Includes.

[0024] Another embodiment provides a pharmaceutical composition, the composition comprising: (a) 0.17% (w / w) of the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid; (b) 0.91% (w / w) hydroxypropyl cellulose; (c) 8.32% (w / w) PEG 400, and (d) 90.49% (w / w) water Includes.

[0025] Another embodiment provides a pharmaceutical composition in tablet or capsule form.Another embodiment provides a pharmaceutical composition that exhibits long term stability.

[0026] One embodiment provides a pharmaceutical composition comprising the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid is freeze-dried, or spray-dried, or a combination thereof.

[0027] Another embodiment provides a pharmaceutical composition, wherein at least one pharma- ceutically acceptable excipient is a solubilizer. Another embodiment provides a pharmaceutical composition, wherein the solubilizer is a polyethylene glycol (PEG) selected from PEG200, PEG300, PEG400, PEG500, or PEG600. Another embodiment provides a pharmaceutical composition, wherein the PEG is selected from PEG1000, PEG1500, or PEG2000.

[0028] Another embodiment provides a pharmaceutical composition, wherein at least one pharma- ceutically acceptable excipient is a stabilizer. Another embodiment provides a pharmaceutical composition, wherein the stabilizer is selected from copovidone, or Kollidon VA64.

[0029] Another embodiment provides a pharmaceutical composition, wherein at least one pharma- ceutically acceptable excipient is a disintegrant.Another embodiment provides a pharmaceutical composition, wherein the disintegrant is selected from crospovidone or Kollidon CL.

[0030] One embodiment provides a pharmaceutical composition comprising the lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, the composition comprising: (a) about 10% (w / w) to about 20% (w / w) of the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid; (b) about 70% (w / w) to about 80% (w / w) Kollidon VA64; (c) about 2% (w / w) to about 7% (w / w) PEG 1500, and (d) About 5% (w / w) to about 15% (w / w) of Kollidon CL Includes.

[0031] Another embodiment provides a pharmaceutical composition, the composition comprising: (a) 15% (w / w) of the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid; (b) 77% (w / w) Kollidon VA64; (c) 5% (w / w) PEG 1500, and (d) 10% (w / w) Kollidon CL Includes.

[0032] Another embodiment provides a pharmaceutical composition in tablet or capsule form.Another embodiment provides a pharmaceutical composition that exhibits long term stability.

[0033] The pharmaceutical composition is administered in a manner appropriate for the disease to be treated (or prevented). The appropriate dosage and the appropriate duration and frequency of administration are determined by factors such as the condition of the patient, the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration. In general, the appropriate dosage and treatment regimen provides a sufficient amount of the composition(s) to provide therapeutic and / or prophylactic benefits (e.g., improved clinical outcomes such as more frequent complete or partial remissions, or longer disease-free and / or overall survival, or reduced severity of symptoms). The optimal dosage is generally determined using experimental models and / or clinical trials. The optimal dosage depends on the patient's size, weight, or blood volume.

[0034] Oral dosages typically range from about 1.0 mg to about 1000 mg, one to four times per day, or more.

[0035] Treatment method One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a pharmaceutical composition comprising the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid is milled.

[0036] One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid is milled.

[0037] Another embodiment provides a method, wherein the cancer is selected from a hematological malignancy or a solid malignancy.

[0038] One embodiment provides a method of treating cancer in a patient in need thereof comprising administering to the patient a pharmaceutical composition comprising the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid is freeze-dried or spray-dried, or a combination thereof.

[0039] One embodiment provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition comprising the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid is freeze-dried or spray-dried, or a combination thereof.

[0040] Another embodiment provides a method, wherein the cancer is selected from a hematological malignancy or a solid malignancy. EXAMPLES

[0041] The present disclosure is further illustrated by the following examples, which should not be construed as limiting in any way. The experimental procedures for generating the data presented are discussed in more detail below. It is to be understood that the present disclosure has been described in an illustrative manner, and that the terms used are intended to be of a descriptive nature, rather than limiting.

[0042] It has been a challenge and difficulty to develop a stable and non-toxic formulation of the lysine salt of Compound 1. Many formulations have been developed and tested, but most have poor bioavailability and / or have been found to be unstable during storage and / or have been found to be highly toxic. It was an unexpected and surprising discovery that the formulations disclosed herein showed good bioavailability in animal PK studies.

[0043] Example 1: Nanosuspension by nanomilling Nanomilling reduces the particle size of the API to the nanometer range. The initial particle size before milling is ~6-15 μm in length. Nanomilling procedure

[0044] [Table 1]

[0045] [Table 2] procedure Preparation of slurry for grinding 1. Prepare 0.25 L of a 1% HPC solution in deionized water. 2. 2.0 g of API was weighed into a beaker and 1% HPC was added up to 200 g for a final slurry concentration of 10 mg / mL. The slurry contents were stirred to mix well. Ball Milling Process 1. Mill beads (140 mL of 0.4 mm zirconium oxide beads) were added to the grinding tank of the mill using a feed funnel. 2. The grinding tank was closed and the mill was set in a horizontal operating position. 3. The mill slurry was added to the collection vessel of the mill. 4. The overhead stirrer was turned on. 5. Open the cooling water cock and start the product pump at its minimum flow rate. 6. Once the product had exited the discharge port into the collection bin, the mill (Netzsch MiniCer Bead Mill) was started at minimum speed (10 Hz or 600 RPM) and then ramped up to 2400 RPM within one minute. 7. The slurry was milled in a Netzsch MiniCer at 2400 RPM. 8. The circulation speed of the grinding slurry was gradually increased. 9. Samples were taken intermittently and the grinding process was stopped and considered complete when particle size no longer decreased according to the PSD results (maximum grinding time observed was ~20 min). 10. The final formulation was prepared as described in Table 1A by adding PEG400 and sodium benzoate to the nanomillings and mixing well.

[0046] [Table 3]

[0047] Results: The particle size distribution of the lysine salt nanosuspension of Compound 1 after milling, as measured by dynamic light scattering, is shown in Table 1B below. The particle size distribution samples were prepared by diluting the nanomilled samples 100 times with deionized water. A Zetasizer was used for particle size distribution measurements. PdI represents the distribution of particle size populations within a given sample. It is a numerical range from 0.0 (for a completely homogeneous sample) to 1.0 (for a highly polydisperse sample with multiple particle size populations). The value D10 refers to the diameter of the particles in the sample at which 10% of the particles in the sample have a diameter smaller than this value (118.8 nm). The value D50 refers to the diameter of the particles in the sample at which 50% of the particles in the sample have a diameter smaller than this value (249.0 nm). The value D90 refers to the diameter of the particles in the sample at which 90% of the particles in the sample have a diameter smaller than this value (611 nm).

[0048] [Table 4]

[0049] The composition after milling is a homogeneous, yellow, translucent suspension. During storage (at 2-8°C), no particle aggregation or precipitation was observed, and a stable suspension was observed. The particle size of the API was reduced to 212 nm by ball milling technique using specific stabilizers, resulting in a homogeneous nanosuspension.

[0050] An in vitro dissolution test was performed on the nanomilled material before the addition of PEG400, and the results are shown in Figure 1. An in vitro dissolution test was performed on the nanomilled material after the addition of PEG400, and the results are shown in Figure 2. Comparison of the dissolution profiles of the nanomilled material before and after the addition of PEG400 shows a significant increase in the dissolution of the lysine salt of Compound 1, with the in vitro dissolution threshold reaching a maximum of 30% within 60 minutes for the nanomilled material before the addition of PEG400, whereas 100% was released within 40 minutes for the nanomilled material after the addition of PEG400.

[0051] The stability of the nanosuspension based on particle size distribution under refrigerated and room temperature conditions is shown in Table 2 below.

[0052] [Table 5]

[0053] The assay and impurity testing of the nanosuspension under stability testing conditions is shown in Table 3 below.

[0054] [Table 6]

[0055] Under room temperature storage conditions, particle growth was observed, possibly due to the Ostwald ripening phenomenon. Under test conditions of 2-8°C, no particle growth was observed over time.

[0056] The suspension formulation was found to be chemically stable over both storage conditions.

[0057] To facilitate administration of the formulation, the nanosuspension can be spray dried to convert it into a fine powder that can be filled into capsules or compressed into tablets for oral administration. Furthermore, when the nanosuspension is dried by spray drying, there is no concern of particle growth or Ostwald ripening because there is no moisture to promote particle growth or Ostwald ripening.

[0058] Example 2: Lyophilized formulation The use of surfactants and disintegrants was studied to determine improvements in bioavailability and dissolution rate.

[0059] [Table 7]

[0060] [Table 8]

[0061] [Table 9] procedure 1. Weigh appropriate amounts of API, Coridan VA64 and PEG 1500 into a lyophilization tube. 2. Deionized water was added to dissolve the powder, or in some cases, a combination of ethanol and deionized water was used to improve solubility. Then, the container was sealed and mixed. 3. The above solution was transferred into a glass container and placed in a -40°C freezer until all the solution solidified. 4. Freeze-dry overnight at -40℃. 5. The solid was crushed with a spatula and mixed with the required amount of Kollidon CL. 6. Fill an appropriate amount of the formulation into capsules (Manufacturer: Capsulgel, Size: 00, Lot # 7171508[2]) to obtain the required drug loading for oral administration in dog PK studies. Capsule Size 000 Size 000 capsules can be used to achieve the desired drug loading. 7. Stability assay and dissolution test were performed and the results are shown in Table 5 and Figure 3, respectively. The dissolution test conditions and procedures are shown in Example 3. 8. The stability of the formulation was monitored under controlled ambient conditions. As shown in Table 5, no degradation was observed.

[0062] [Table 10]

[0063] Example 3: Additional formulation studies Hot melt formulation The materials used in the hot melt compositions are shown in Table 6. The components of each hot melt composition are shown in Table 7.

[0064] [Table 11]

[0065] [Table 12] procedure 1. Preheat the oven to 160°C. 2. The ingredients were measured out into a mortar. 3. The ingredients were mixed thoroughly with a pestle. 4. The samples were placed in a preheated oven for 5 minutes. If the samples did not melt, they were heated for an additional 5 minutes. 5. The sample was cooled to room temperature. 6. Then, each formulation was pulverized in a jet mill to obtain fine granules.

[0066] Purity evaluation of each formulation by hot melt experiments showed that no impurities or degradation was observed compared to the purity of the active ingredient prior to formulation, indicating that the active ingredient is stable at the elevated temperatures utilized in the preparation of these hot melt formulations. The dissolution profiles of these formulations were evaluated according to the outlined procedure.

[0067] Dissolution and Assay Testing of API, F1, F7, F9, F10, F11 and F12

[0068] [Table 13] Sink Status 1. 5 mg of API was placed in a glass vial. 2. Add 0.2 mL of diluent (deionized water and SGF) to the vial, followed by sonication and vortexing to dissolve the API. If the solution is not clear after 3.5 minutes, add an additional 0.2 mL of diluent to the vial and sonicate and vortex to dissolve the API. 4. Repeat step 4 until all API is dissolved. 5. Calculate the final concentration and maximum solubility of the API used in the dissolution test. Dissolution Test 6. Transfer 500 mL of deionized water dissolution solvent to each vessel. 7. Equilibrate the dissolution medium to 37.0±0.5°C. 8. Transfer 600 mg of each formulation granule into a capsule. 9. Transfer capsules to basket and lower into position. Begin mixing at 100 RPM. 10. At the specified time, sample 1 mL of the solution using the autosampler. Assay sample preparation (0.3mg / mL) 11.Weigh 18 mg of granules into a 5 mL volumetric flask. 12. Dissolve and dilute to volume with diluent (50% ACN in H2O). 13. Mix well.

[0069] [Table 14]

[0070] Conclusion: The dissolution profile in deionized water is shown in Figure 4. API alone and F7 were not released at all after 120 minutes from the dissolution test. In contrast, F1, F9 and F10 showed faster dissolution rates. Among them, F9 showed about 40% release after 1 hour. The dissolution results of formulations F11 and F12 are shown in Figure 5. Although F11 and F12 formulations were jet milled and the granule size was about 5 microns, no improvement in dissolution rate was observed. F11 and F12 contained different amounts of polysorbate 80 (PB80), and the results suggested that it did not affect the dissolution rate.

[0071] Lyophilized preparations Objective: To prepare a fast-dissolving formulation by freeze-drying.

[0072] [Table 15] procedure 1. Compound 1 lysine salt and Kollidan VA64 were weighed into an appropriate glass vial. 2. tert-butanol was added to dissolve the powder, sealed and mixed. The mixture was heated if necessary. 3. The vial was placed in a -20°C freezer until all the solution was solid. 4. The vials were lyophilized at -20°C for 16 hours and at 30°C for 3 to 5 hours. 5. The final solid was crushed with a spatula.

[0073] Results: An off-white fluffy powder was obtained. The powder was filled into HPMC capsules and dissolution tested. Formulation F13 showed improved dissolution rate and dissolution percentage. The data is shown in Figure 5.

[0074] Clear Sol and SEDDS Technology Objective: To screen two additional compositions to improve the oral availability of the lysine salt of Compound 1. Clearsol Technology (F15) is an FDA-approved lipid-based proprietary solubilizer described in US Patent Application Publication 2020 / 0368159A1. Self-emulsifying drug delivery systems (SEDDS) (F16 and F17) were also investigated. material

[0075] [Table 16]

[0076] [Table 17]

[0077] [Table 18] procedure 1. The lysine salt of Compound 1 was weighed into a suitable container / vial. 2. Vehicle was added to each vial until the total weight was 2 g. 3. The contents were vortexed and sonicated to dissolve the API.

[0078] Results: The active ingredients were not completely miscible in formulations F15, F16 or F17. Therefore, the formulations containing Clearsol and the SEDDS-2 formulation were not investigated further. The SEDDS-1 formulation was investigated further with the addition of a co-solvent.

[0079] Modified Clearsol and SEDDS Technology Objective: To prepare F19 (Lysine salt of Compound 1 in a self-emulsifying vehicle), 10% Propylene Glycol (PG) in SEDDS V1.

[0080] [Table 19]

[0081] [Table 20]

[0082] [Table 21]

[0083] procedure Vehicle preparation: 10% PG in SEDDS V1 (2.7 g of SEDDS V1 was mixed with 0.3 g of PG). 1. 2 mg of the lysine salt of Compound 1 was weighed into a suitable container. 2. Vehicle was then added to the vial according to the recipe to bring the total weight to 1 g. 3. The contents were vortexed and sonicated to dissolve the API. 4. This formulation was then subjected to dissolution testing.

[0084] Results: A clear solution was observed indicating that the active ingredient was completely miscible with the vehicle. Formulation 19 had a lower release rate compared to F14_2 or F13A, as shown in Figure 6. Further modification of formulation F19 by pH adjustment was performed to determine if dissolution could be further increased.

[0085] pH-adjusted SEDDS formulation Objective: To optimize F19 (SEDDS formulation) and improve release by increasing the pH using ammonium hydroxide.

[0086] [Table 22]

[0087] [Table 23]

[0088] [Table 24]

[0089] procedure 1. 400 mL of formulation F19 was mixed with 3.2 mL of ammonium hydroxide (28-30%). 2. Assay and dissolution tests were performed on the pH-adjusted formulation.

[0090] Results: Dissolution data are shown in Figure 7. Compared to F19, F19C showed an improved initial release from 60% to 80%. Re-equilibration was observed after 15 min. However, the released drug may be absorbed into the biological system (in vivo study), and re-equilibration issues may not be observed in in vivo studies. As this formulation showed good solubility, it was subjected to another dog PK study along with two other formulations, F13A and F14_2 (which were subsequently concluded as lead formulations), to compare bioavailability.

[0091] Clearsol technology using nanomilled materials Objective: To evaluate whether formulation F14 (see Example 1) can be further optimized by incorporating a clearsol solubilizer into the post-nanomilled material.

[0092] [Table 25]

[0093] [Table 26]

[0094] procedure: 1. Weigh out ingredients into vials according to recipe. 2. The contents were then vortexed and sonicated.

[0095] Results: The dissolution data are shown in Figure 6. For formulation F25, there was no release upon dissolution. Therefore, formulation F25 was not investigated further.

[0096] Example 4: Pharmacokinetic and bioavailability studies A summary of the pharmacokinetic studies in dogs conducted with the formulations prepared in Examples 1-3 above is provided in Table 17.

[0097] [Table 27-1]

[0098] [Table 27-2]

[0099] Results: The relative bioavailability (%F) of F13 was calculated by comparing the exposure of F13 with that of the internal control PK using PEG / MC (oral administration).

[0100] Based on the above results, the study with F13 showed only about 48% bioavailability compared to PEG / MC as a control. Therefore, this formulation was further optimized to F13A by the addition of a solubilizer such as PEG, which resulted in a notable improvement in oral bioavailability (82% compared to IV). Similarly, good oral bioavailability was also observed for formulation F14_2 (87%), which is the API after nanomilling with HPC and PEG400 excipients.

[0101] Kollidon-based formulations such as F13 and F13A are max is low, T 1 / 2 These were long. max A high C is a favorable pharmacokinetic parameter because it increases the likelihood of off-target interactions. As a potent KMD4 inhibitor, a high C is required to inhibit KDM4. max It may not be necessary to have a longer T 1 / 2 may increase the time of target interaction.

[0102] Formulation F19C showed good in vitro solubility, but poor bioavailability was observed from PK studies in dogs.

[0103] PK studies in dogs showed that formulations F13A and F14_2 had high bioavailability, and these two are the lead formulations for clinical development.

[0104] A pharmacokinetic study was conducted in rats using formulation F13A, and the data are shown in Table 18.

[0105] [Table 28]

[0106] Results: F13A was dissolved in water and the F13A solution was administered. High bioavailability was observed in rats. Absolute dose-adjusted bioavailability was calculated based on the IV data generated in rats.

[0107] Example 5: Spray drying process Formulation F13A was further optimized for larger scale manufacturing. The improved process utilizes spray drying followed by freeze drying (for extended drying) instead of freeze drying throughout (as described above). The spray drying step followed by freeze drying improved the removal of processing solvents (ethyl alcohol and water) from the formulation to an acceptable level. On the other hand, the initial steps of freezing the solution, freeze drying the solution overnight, and grinding the resulting product are not the most optimal steps for commercial scale production. Therefore, a spray drying step followed by freeze drying (for extended drying) was optimized for larger scale manufacturing of this formulation.

[0108] Objective: To develop a spray drying process.

[0109] [Table 29]

[0110] [Table 30]

[0111] [Table 31]

[0112] [Table 32]

[0113] Procedure for spray drying solvent evaluation: A fast dissolving freeze dried / lyophilization formulation (15% Compound 1 lysine salt, 77% Kollidon VA64, and 5% polyethylene glycol 1500) was mixed with various diluents and evaluated for solution appearance and amount of formulation dissolved. The results are shown in Table 21.

[0114] [Table 33]

[0115] Results: Based on appearance and maximum concentration results from solvent evaluation, an 80% ethanol solution was selected as the lead spray drying solvent.

[0116] After selecting 80% ethanol solution as the lead spray drying solvent. The spray drying process was further optimized through evaluation of inlet temperature, atomization pressure, orifice pressure, and pump speed. Five further spray drying conditions were evaluated based on assay rate, recovery, and appearance. The spray drying conditions are shown in Table 22. Promising conditions obtained from the preliminary evaluation are shown in Table 23 for comparison.

[0117] Steps to optimize the spray drying process: The spray drying conditions shown in Table 22 were evaluated.

[0118] [Table 34]

[0119] [Table 35]

[0120] Long term drying (freeze drying) of the spray dried samples was performed in a preheated tray at 40 °C. The spray dried material was then weighed and transferred to the preheated tray. The vacuum was set to 40-150 mTorr and the material was allowed to dry for a minimum of 12 hours. After 12 hours, the shelf temperature was reduced to 25 °C and allowed to dry further for at least 1 hour.

[0121] The formulations were monitored for stability under controlled ambient conditions and, as shown in Table 24, no degradation was observed.

[0122] [Table 36]

[0123] Example 6: Evaluation of Compound 1 in a panel of cancer cell lines In a larger scale screening study, compound 1 was evaluated using an extensive cancer cell line panel (OncoPanel; HD Biosciences) consisting of 301 cancer cell lines of different tumor types.

[0124] Two-dimensional cell viability inhibition assays were performed in multi-well plates seeded with the panel of cells. After seeding, plates were cultured overnight in a humidified incubator at 37 °C to promote adhesion. The assay was initiated by adding either DMSO as a vehicle control, growth medium as a blank, or serially diluted Compound 1 (10 μM to 0.0005 μM in a 1:3 serial dilution) to individual wells. Cultures were incubated for 168 hours, after which the number of viable cells in each test well was assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. Luminescence measurements were performed using an EnVision® Multilabel Reader, and Compound 1 measurements were normalized to those of the DMSO control and expressed as a percentage of control. Outliers were excluded by visual inspection. Percentages of control were plotted against the corresponding Compound 1 concentrations, and absolute IC50 values ​​were determined using a four-parameter logistic nonlinear regression as the concentration at which inhibition was 50% of control. If maximal inhibition was less than 50%, absolute IC50 was reported as greater than 10. In addition to EC50 and IC50, AUC was determined and normalized to the area corresponding to theoretical non-inhibition (rectangular area defined by compound dose range and y 0–100). Emax was recorded as the minimum of compound dose range and y. For cell lines with Emax greater than 40, EC50 was manually set to 10 μM. Cellular titration data for compound 1 was visualized using a hierarchical clustering algorithm based on Euclidean distance between standardized input values ​​and the “Ward.D” method implemented in the hclust function in R. The susceptible / intermediate / resistant status of each cell line was determined by visual inspection of the resulting clustering dendrogram. All analyses were performed in R Core Team.

[0125] As a result, compound 1 potently inhibited the proliferation of cancer cell lines, with an absolute IC 50 Compound 1 was shown to exhibit a highly selective potency profile, with IC values ​​ranging from <500 pM to >10 μM. Of the 301 cell lines, 209 showed IC 50 89 strains were inhibited at IC values ​​below 1 μM.50 Values ​​above 10 μM were insensitive. Table 25 shows the results for all 301 cell lines.

[0126] [Table 37-1]

[0127] [Table 37-2]

[0128] [Table 37-3]

[0129] [Table 37-4]

[0130] [Table 37-5]

[0131] [Table 37-6]

[0132] [Table 37-7]

[0133] [Table 37-8]

[0134] [Table 37-9]

[0135] [Table 37-10]

[0136]

Table 37-11

[0137]

Table 37-12

[0138]

Table 37-13

[0139]

Table 37-14

[0140]

Table 37-15

[0141]

Table 37-16

[0142]

Table 37-17

[0143]

Table 37-18

[0144] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in implementing the present invention. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

Claims

1. 1. A pharmaceutical composition comprising the lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid is milled.

2. The pharmaceutical composition of claim 1 , wherein the grinding is carried out using a ball mill.

3. 2. The pharmaceutical composition of claim 1, wherein the milling is carried out using a roller mill or a high energy mill.

4. 4. The pharmaceutical composition of claim 1, 2 or 3, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid exhibits a particle size of less than 1000 nanometers.

5. 5. The pharmaceutical composition of any one of claims 1 to 4, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid exhibits a particle size of about 50 nanometers to about 1000 nanometers.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid exhibits a particle size of about 50 nanometers to about 100 nanometers, about 100 nanometers to about 200 nanometers, about 200 nanometers to about 300 nanometers, about 300 nanometers to about 400 nanometers, about 400 nanometers to about 500 nanometers, about 500 nanometers to about 600 nanometers, about 700 nanometers to about 800 nanometers, about 800 nanometers to about 900 nanometers, or about 900 nanometers to about 1000 nanometers.

7. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid exhibits a particle size of about 150 nanometers to about 300 nanometers.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the particle size does not increase upon storage.

9. The pharmaceutical composition according to any one of claims 1 to 7, wherein the particle size does not increase by more than 5% upon storage.

10. The pharmaceutical composition according to any one of claims 1 to 7, wherein the particle size does not increase by more than 10% upon storage.

11. The pharmaceutical composition according to any one of claims 1 to 7, wherein the particle size does not increase by more than 15% upon storage.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the at least one pharma- ceutically acceptable excipient is a solubilizing agent.

13. 13. The pharmaceutical composition of claim 12, wherein the solubilizing agent is polyethylene glycol (PEG).

14. The pharmaceutical composition of claim 13, wherein the PEG is selected from PEG 200, PEG 300, PEG 400, PEG 500, or PEG 600.

15. The pharmaceutical composition according to any one of claims 1 to 14, further comprising a stabilizer.

16. 16. The pharmaceutical composition of claim 15, wherein the stabilizer is selected from the group consisting of hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), polyvinylpyrrolidone (PVP) or poloxamer.

17. The pharmaceutical composition comprises: (a) from about 0.1% (w / w) to about 0.2% (w / w) of the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid; (b) from about 0.91% (w / w) to about 1.5% (w / w) hydroxypropylcellulose; (c) about 5.0% (w / w) to about 10.0% (w / w) PEG 400, and (d) about 89.3% (w / w) to about 94.4% (w / w) water The pharmaceutical composition according to any one of claims 1 to 16, comprising:

18. The pharmaceutical composition comprises: (a) 0.17% (w / w) of the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid; (b) 0.91% (w / w) hydroxypropyl cellulose; (c) 8.32% (w / w) PEG 400, and (d) 90.49% (w / w) water 20. The pharmaceutical composition of claim 17, comprising:

19. 19. The pharmaceutical composition of claim 17 or 18, wherein the pharmaceutical composition is in tablet or capsule form.

20. 1. A pharmaceutical composition comprising the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid and at least one pharma- ceutically acceptable excipient, wherein the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid has been freeze-dried or spray-dried, or a combination thereof.

21. 21. The pharmaceutical composition of claim 20, wherein the at least one pharma- ceutically acceptable excipient is a solubilizing agent.

22. 22. The pharmaceutical composition of claim 21, wherein the solubilizing agent is a polyethylene glycol (PEG) selected from PEG 200, PEG 300, PEG 400, PEG 500, or PEG 600.

23. 23. The pharmaceutical composition of claim 22, wherein the PEG is selected from PEG 1000, PEG 1500, or PEG 2000.

24. The pharmaceutical composition according to any one of claims 20 to 23, wherein the at least one pharma- ceutically acceptable excipient is a stabilizer.

25. 25. The pharmaceutical composition of claim 24, wherein the stabilizer is selected from copovidone, or Kollidon VA64.

26. The pharmaceutical composition according to any one of claims 20 to 25, wherein the at least one pharma- ceutically acceptable excipient is a disintegrant.

27. 27. The pharmaceutical composition of claim 26, wherein the disintegrant is selected from crospovidone, or Kollidon CL.

28. The pharmaceutical composition comprises: (a) from about 10% (w / w) to about 20% (w / w) of the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid; (b) about 70% (w / w) to about 80% (w / w) Kollidon VA 64; (c) about 2% (w / w) to about 7% (w / w) PEG 1500, and (d) about 5% (w / w) to about 15% (w / w) Kollidon CL The pharmaceutical composition according to any one of claims 20 to 27, comprising:

29. The pharmaceutical composition comprises: (a) 15% (w / w) of the L-lysine salt of 3-({[(4R)-7-{methyl[4-(propan-2-yl)phenyl]amino}-3,4-dihydro-2H-1-benzopyran-4-yl]methyl}amino)pyridine-4-carboxylic acid; (b) 77% (w / w) Kollidon VA 64; (c) 5% (w / w) PEG 1500, and (d) 10% (w / w) Kollidon CL 29. The pharmaceutical composition of claim 28, comprising:

30. The pharmaceutical composition according to any one of claims 20 to 29, wherein the pharmaceutical composition is in tablet or capsule form.

31. The pharmaceutical composition according to any one of claims 20 to 30, wherein the pharmaceutical composition exhibits long-term stability.

32. A method of treating cancer in a patient in need thereof comprising administering to the patient a pharmaceutical composition according to any one of claims 1 to 31.

33. A method of treating cancer in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition according to any one of claims 1 to 32.

34. 34. The method of claim 32 or 33, wherein the cancer is selected from a hematological malignancy or a solid malignancy.

35. 34. The method of claim 32 or 33, wherein the cancer is selected from the group consisting of colorectal cancer, esophageal cancer, triple-negative breast cancer, gastric cancer, lymphoma, gastric adenocarcinoma, diffuse large B-cell non-Hodgkin's lymphoma, acute T-cell leukemia, esophageal squamous cell carcinoma, multiple myeloma, acute myeloid leukemia, colon adenocarcinoma, colorectal carcinoma, pancreatic cancer, pancreatic carcinoma, breast cancer, and T-cell acute lymphoblastic leukemia.

36. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with colorectal cancer.

37. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with esophageal cancer.

38. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with triple negative breast cancer.

39. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with gastric cancer.

40. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with lymphoma.

41. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with gastric adenocarcinoma.

42. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with diffuse large B-cell non-Hodgkin's lymphoma.

43. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with acute T-cell leukemia.

44. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with esophageal squamous cell carcinoma.

45. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with multiple myeloma.

46. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with acute myeloid leukemia.

47. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with colorectal adenocarcinoma.

48. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with colorectal carcinoma.

49. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with pancreatic cancer.

50. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with pancreatic carcinoma.

51. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with breast cancer.

52. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with T-cell acute lymphoblastic leukemia.

53. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with a cancer selected from lung cancer, small cell lung cancer, non-small cell lung cancer, large cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, small cell lung carcinoma, large cell lung carcinoma, or bronchioloalveolar adenocarcinoma.

54. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with a cancer selected from acute lymphoblastic B-cell leukemia, mantle cell lymphoma, plasma cell myeloma, diffuse large B-cell lymphoma, B-cell lymphoma, Burkitt's lymphoma, acute phase chronic myelogenous leukemia.

55. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with a cancer selected from intestinal cancer, intestinal adenocarcinoma, and squamous cell carcinoma of the upper gastrointestinal tract.

56. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with a cancer selected from gastric cancer, gastric signet ring adenocarcinoma, gastric adenocarcinoma, or adenosquamous carcinoma.

57. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with a cancer selected from ovarian cancer, ovarian endometrioid cancer, ovarian clear cell carcinoma, ovarian adenocarcinoma, endometrial cancer, endometrial adenocarcinoma, prostate cancer, or prostate adenocarcinoma.

58. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with skin cancer.

59. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with a cancer selected from thyroid cancer or follicular thyroid cancer.

60. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with a cancer selected from breast cancer or ductal carcinoma.

61. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with a cancer selected from liver cancer or hepatocellular carcinoma.

62. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with a cancer selected from central nervous system cancer, astrocytoma grade IV, or gliosarcoma.

63. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with bone cancer.

64. 34. The method of claim 32 or 33, wherein the cancer patient has been diagnosed with a cancer selected from kidney cancer, clear cell renal cell carcinoma, renal cell carcinoma, urinary tract cancer, or urothelial transitional cell carcinoma.

65. 65. The method of any one of claims 34 to 64, wherein the cancer has recurred after previous treatment, is ineffective against previous treatment, or has acquired resistance to previous treatment.

Images